Phage Display: Methods and Protocols (Methods in Molecular Biology, 2702) [2 ed.] 1071633805, 9781071633809

Table of contents :
Preface
Contents
Contributors
Part I: Introduction
Chapter 1: Antibody Phage Display
1 Introduction
2 Antibody Structure and Function
2.1 Recombinant Formats
3 Antibody Phage Display Libraries
3.1 Immune Antibody Library
3.2 Naïve Antibody Library
3.3 Synthetic and Semisynthetic Antibody Library
4 Conclusion
References
Part II: Construction of Antibody Phage Display Libraries
Chapter 2: Construction of Human Immune and Naive scFv Phage Display Libraries
1 Introduction
2 Materials
2.1 Isolation of Lymphocytes
2.2 Sorting of B Lymphocytes/Plasma Cells (Optional)
2.3 cDNA Synthesis
2.4 First and Second Antibody Gene PCR
2.5 First Cloning Step - VL
2.6 Second Cloning Step - VH
2.7 Colony PCR
2.8 Library Packaging and scFv Phage Production
2.9 Phage Titration
3 Methods
3.1 Isolation of Lymphocytes (Peripheral Blood Mononuclear Cells (PBMC))
3.2 Fluorescence-Automated Sorting of B Lymphocytes/Plasma Cells
3.3 cDNA Synthesis
3.4 First Antibody Gene PCR
3.5 Second Antibody Gene PCR
3.6 First Cloning Step - VL
3.7 Second Cloning Step - VH
3.8 Colony PCR
3.9 Library Packaging and scFv Phage Production
3.10 Phage Titration
4 Notes
References
Chapter 3: Construction of Naïve and Immune Human Fab Phage Display Library
1 Introduction
2 Materials
2.1 Isolation of B Cells
2.2 First-Strand cDNA Synthesis
2.3 Amplification of HC and LC Fab Gene Repertoire
2.4 Two-Step Cloning
2.4.1 First Step Cloning (Fab HC)
2.4.2 Second Step Cloning (Fab LC)
2.5 Colony PCR
2.6 Fab Phage Library Packaging
2.7 Phage Titration
2.8 Fab Library Panning
2.8.1 Fab Selection
2.8.2 Polyclonal and Monoclonal Phage ELISA
3 Methods
3.1 Isolation of B Cells
3.2 First-Strand cDNA Synthesis
3.3 Amplification of HC and LC Fab Gene Repertoire
3.4 Two-Step Cloning
3.4.1 First Step Cloning (Fab HC)
3.4.2 Second Step Cloning (Fab LC)
3.4.3 Library Size Estimation
3.4.4 Preparation of Bacteria Library Stock
3.5 Colony PCR
3.6 Fab Phage Library Packaging
3.7 Phage Titration
3.8 Fab Library Panning
3.8.1 Fab Selection
3.8.2 Polyclonal and Monoclonal Phage ELISA
4 Notes
References
Chapter 4: Construction of Synthetic Antibody Phage Display Libraries
1 Introduction
2 Materials
3 Methods
3.1 Phagemid Design
3.2 Library Construction
3.2.1 Purification of dU-ssDNA Template
3.2.2 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Oligonucleotide Phosphorylation
3.2.3 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Annealing of Phosphorylated Oligonucleotides to the dU-ssDNA Template
3.2.4 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Enzymatic Synthesis of CCC-dsDNA
3.3 Conversion of CCC-dsDNA into a Phage-Displayed Antibody Library
4 Notes
References
Chapter 5: Construction of Chicken and Ostrich Antibody Libraries
1 Introduction
2 Materials (See Note 1)
2.1 RNA Isolation (See Note 2)
2.2 cDNA Synthesis, PCR, and Ligation
2.3 Electroporation and Growth of E. coli and Bacteriophage
3 Methods
3.1 RNA Isolation from Tissue
3.2 RNA Isolation from Blood
3.3 cDNA Synthesis and Amplification by PCR
3.4 Assembly of scFv Genes Based on Natural VH and VL
3.5 Assembly of scFv Genes Based on Natural VL and Synthetic, Randomized VH CDR3s
3.6 Restriction Digestion of the scFv Gene Construct and pHEN1 Vector
3.7 Ligation of the scFv Gene and Vector
3.8 Electroporation
3.9 Phage Rescue
3.10 PEG Precipitation of Phages
3.11 Determining Phage Titer
3.12 Crystal Violet Stained Gels (See Note 14)
4 Notes
References
Chapter 6: Construction of Rabbit Immune Antibody Libraries
1 Introduction
2 Materials
3 Method
3.1 Rabbit Immunization
3.2 Preparation of Total RNA from the Spleens of Immunized Rabbits
3.3 Synthesis of First-Strand cDNA
3.4 Assembly of Rabbit/Human Chimeric Fab Repertoire by PCR
3.4.1 First Round of PCR
3.4.2 Second Round of PCR
3.4.3 Third Round of PCR
3.5 Construction of Fab Library
3.6 Phage Antibody Library Rescue
4 Notes
References
Chapter 7: Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries
1 Introduction
2 Material
2.1 Antigen Preparation, Llama Immunization, and Immune Response Monitoring
2.2 VHH Library Construction
2.3 Phage Rescue from the Library
2.4 Biopanning of Phage-Displayed Library
2.5 Polyclonal and Monoclonal Phage ELISA Screening (See Note 10)
2.6 Sub-cloning of R3/R4 Phage Pool, In Vitro Translation and Screening of the Soluble VHHs (An Alternative to Monoclonal Phag...
2.7 VHH Sub-cloning, Soluble Expression, and Purification
2.8 Characterization of VHH Binders: Surface Plasmon Resonance (SPR)
2.9 Cell Binding of VHHs by Flow Cytometry
2.10 Immunoprecipitation of the Cell Surface Receptor with VHHs
3 Methods
3.1 Antigen Preparation, Llama Immunization, and Immune Response Monitoring
3.2 VHH Library Construction
3.3 Phage Rescue of the VHH Library
3.4 VHH Phage Library Biopanning
3.5 Polyclonal and Monoclonal Phage ELISA Screening
3.6 Sub-cloning of R3/R4 Phage Pool, In Vitro Translation and Screening of the Soluble VHHs (an Alternative to Monoclonal Phag...
3.6.1 Sub-cloning of R3/R4 Phage Pool
3.6.2 In Vitro Translation and Screening of the Soluble VHHs (an Alternative to Monoclonal Phage ELISA Screening)
3.7 VHH Sub-cloning, Soluble Expression, and Purification
3.7.1 Large-Scale VHH Expression in E. coli
3.7.2 Preparation of the Biotin Ligase BirA Extract
3.8 Characterization of VHH Binders by Surface Plasmon Resonance (SPR)
3.9 Cell Binding of VHHs by Flow Cytometry
3.10 Validating VHH Specificity by Immunoprecipitation of the Target Antigen
4 Notes
References
Chapter 8: Phagekines: Directed Evolution and Characterization of Functional Cytokines Displayed on Phages
1 Introduction
2 Materials
2.1 Displaying Cytokines on Filamentous Phages
2.2 Quantifying Cytokine Display Levels by ELISA
2.3 Screening Receptor Binding Properties of Phage-Displayed Cytokines by ELISA
2.4 Assessing CTLL-2 Proliferation Induced by Phage-Displayed IL-2 and IL-2 Variants
2.5 Measuring Trans-Signaling Induced by Phage-Displayed IL-6
2.6 Producing Single-Stranded DNA Templates for Cytokine Library Construction
2.7 Kunkel Mutagenesis Diversification Reactions
2.8 Preparation of Electrocompetent Cells and Electroporation with Mutagenesis Products to Construct Libraries of Cytokine Var...
2.9 Library Phage Production at a 300 mL Scale
2.10 Phage Panning and Amplification
2.11 Phage Production at 96-Well Scale
2.12 Clonal Screening by ELISA and Sequencing
3 Methods
3.1 Displaying Cytokines on Filamentous Phages
3.2 Quantifying Cytokine Display Levels by ELISA
3.3 Screening Receptor Binding Properties of Phage-Displayed Cytokines by ELISA
3.4 Assessing CTLL-2 Proliferation Induced by Phage-Displayed IL-2 and IL-2 Variants
3.5 Measuring Trans-Signaling Induced by Phage-Displayed IL-6
3.6 Producing Single-Stranded DNA Templates for Cytokine Library Construction
3.7 Kunkel Mutagenesis Diversification Reactions
3.8 Preparation of Electrocompetent Cells and Electroporation with Mutagenesis Products to Construct Libraries of Cytokine Var...
3.9 Library Phage Production at a 300 mL Scale
3.10 Phage Panning and Amplification
3.11 Phage Production at 96-Well Scale
3.12 Clonal Screening by ELISA and Sequencing
4 Notes
References
Chapter 9: Efficient Cloning of Inserts for Phage Display by Golden Gate Assembly
1 Introduction
2 Materials
2.1 Polymerase Chain Reactions (PCR)
2.2 1% Agarose Gel
2.3 Golden Gate Assembly Reactions
2.4 Transformation
2.5 Plasmid Extraction and Sequencing
2.6 Chemical Biotinylation of Proteins
2.7 Phage ELISA
3 Methods
3.1 PCR Amplification
3.2 Golden Gate Assembly
3.3 Transformation
3.4 Plating
3.5 Sequencing
3.6 Chemical Biotinylation of Proteins
3.7 Phage Amplification
3.8 Phage Enzyme-Linked Immunosorbent Assay (ELISA)
4 Notes
References
Chapter 10: Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification
1 Introduction
2 Materials
2.1 Preparation of Single-Stranded Circular DNA
2.2 1% Agarose Gel
2.3 Kunkel Mutagenesis
2.4 Rolling Circle Amplification (RCA)
2.5 Restriction Enzyme Digestion and Ligation
2.6 Transformation and Library Production
2.7 Plasmid Extraction and DNA Sequencing
2.8 Affinity Selection
2.9 Phage ELISA
3 Methods
3.1 Preparation of Single-Stranded Circular DNA
3.2 Kunkel Mutagenesis
3.3 Rolling Circle Amplification (RCA)
3.4 Ethanol Precipitation of Ligated DNA
3.5 Bacterial Transformation and Library Production
3.6 Sanger Sequencing of Library Clones
3.7 Next-Generation Sequencing (NGS)
3.8 Affinity Selection of the Library
3.9 Phage ELISA
3.10 Competition ELISA
4 Notes
References
Chapter 11: Construction of Semisynthetic Shark vNAR Yeast Surface Display Antibody Libraries
1 Introduction
2 Materials
2.1 Shark Handling and Blood Isolation
2.2 Preparation of Total RNA from Whole Blood
2.3 cDNA Synthesis and Gene-Specific Amplification of vNAR Regions as Template for Library Construction
2.4 Yeast Surface Display Library Construction
3 Methods
3.1 Blood Collection
3.2 Total RNA Preparation
3.3 cDNA Synthesis
3.4 Amplification of the Natural vNAR Repertoire
3.5 Generation of the CDR3-Randomized PCR Insert for Library Establishment Using Yeast Surface Display as Platform Technology
3.5.1 First PCR
3.5.2 Second PCR
3.5.3 Third PCR
3.6 Shark vNAR Library Generation for Yeast Surface Display
3.6.1 Digestion of pCT Plasmid
3.6.2 Yeast Transformation
3.7 Affinity Maturation by CDR1 Diversification and Sublibrary Establishment of Target-Enriched Binders
3.7.1 First PCR
3.7.2 Second PCR
3.7.3 Third PCR
4 Notes
References
Part III: Selection Strategies for Antibodies
Chapter 12: Antibody Selection via Phage Display in Microtiter Plates
1 Introduction
2 Materials
2.1 Coating of Microtiter Wells
2.2 Panning
2.3 Phage Titration
2.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
2.5 ELISA of Soluble Monoclonal Antibody Fragments
3 Methods
3.1 Coating of Microtiter Plate Wells
3.2 Panning
3.3 Phage Titration
3.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.5 ELISA of Soluble Monoclonal Antibody Fragments
3.6 Fast On-Rate Panning
4 Notes
References
Chapter 13: Antibody Selection in Solution Using Magnetic Beads
1 Introduction
2 Materials
2.1 Preparation of the Magnetic Beads
2.2 Panning
2.3 Phage Titration
2.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
2.5 ELISA of Soluble Monoclonal Antibody Fragments
3 Methods
3.1 Preparation of the Magnetic Beads
3.2 Panning
3.3 Phage Titration
3.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.5 ELISA of Soluble Monoclonal Antibody Fragments
4 Notes
References
Chapter 14: Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning
1 Introduction
2 Materials
2.1 Preparation of Phage Display Antibody Library
2.1.1 Phage Display Antibody Libraries and E. Coli Host Strains
2.1.2 Preparation of Antibody Library
2.2 Phage Display MSD Biopanning
2.3 Phage ELISA
2.4 DNA Sequencing
2.5 Soluble Antibody Fragment Detection
3 Methods
3.1 Preparation of Antibody Library Phage
3.2 Library Phage Packaging
3.3 Preparation of First-Generation Stock
4 Phage Display Biopanning
4.1 MSIA Streptavidin D.a.R.T´s Loading of Biotinylated Antigen
4.2 MSIA Streptavidin D.a.R.T´s Antibody Biopanning
5 Phage ELISA
5.1 Polyclonal Phage ELISA
5.2 Monoclonal Phage Propagation
5.3 Monoclonal ELISA
6 DNA Sequencing
7 Generation of Soluble Antibody Fragments
7.1 Expression and Extraction of Soluble Antibody
7.2 Soluble ELISA
8 Analysis
9 Notes
References
Chapter 15: Magnetic Nanoparticle-Based Semi-automated Panning for High-Throughput Antibody Selection
1 Introduction
2 Materials
2.1 Loading of Magnetic Beads
2.2 Semi-automated Panning Using a Magnetic Particle Processor
2.3 Packaging of Phagemids
2.4 Titration of Phage Particles
2.5 Magnetic Particle ELISA of Polyclonal Antibody Phage
2.6 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
2.7 ELISA of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3 Methods
3.1 Loading of Magnetic Beads
3.2 Semi-automated Panning on Magnetic Particle Processor
3.3 Packaging of Phage Particles
3.4 Titration of Phage Particles
3.5 ELISA of Polyclonal Antibody Phage
3.6 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.7 ELISA of Soluble Monoclonal Antibody Fragments in Microtiter Plates
4 Notes
References
Chapter 16: Antibody Selection on Cells Targeting Membrane Proteins
1 Introduction
2 Materials
2.1 Phage Display on Cells
2.2 Phage Titration
2.3 High-Throughput Screening of Binders by Flow Cytometry
3 Methods
3.1 Phage Display on Cells
3.1.1 Preparation of Cells
3.1.2 Depletion of Phage Display Library on Cells
3.1.3 Selection of scFv Binders on Target Expressing Cells
3.1.4 Amplification of Phage Particles
3.2 Phage Titration
3.3 Screening of scFv on Cells
3.3.1 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.3.2 Screening of scFv Binders by Flow Cytometry
4 Notes
References
Chapter 17: Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach
1 Introduction
2 Materials
2.1 Phage Panning via Dynabeads
2.2 Phage Panning via T2 Cells
2.3 Amplification
2.4 Clone Selection for Dynabeads Panning
2.5 Clone Selection for Cell Panning
2.6 Screening of Phage Clones via ELISA
2.7 Screening of Phage Clones via FACS
2.8 Large-Scale Expression
2.9 Fc Fusion Protein Cloning and Expression
3 Methods
3.1 Panning via Dynabeads
3.2 Panning via Cell Panning
3.3 Amplification
3.4 Clone Selection for Dynabeads Panning
3.5 Clone Selection for Cell Panning
3.6 ELISA Screening for pMHC Binding Phage Clones
3.7 FACS Screening for pMHC Binding Phage Clones
3.8 Large-Scale Expression
3.9 Generating Fc Fusion Proteins
4 Notes
References
Chapter 18: Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS
1 Introduction
2 Materials
2.1 Panning
2.2 Phage-Seq
3 Methods
3.1 Affinity Selection of scFv-Displaying Phages on Immobilized Antigen
3.1.1 Growth and Helper Phage Rescue of the Library (See Note 2)
3.1.2 Affinity Selection (Panning) on Immobilized Antigen
3.2 Identification of Antigen Binders
3.3 Phage-Seq
4 Notes
References
Chapter 19: Selection of Affibody Affinity Proteins from Phagemid Libraries
1 Introduction
2 Materials
3 Methods
3.1 Phage Stock Preparation
3.2 Target Antigen Preparation
3.3 Phage Display Selection
3.4 Phage-ELISA Screening
4 Notes
References
Part IV: Complementary Approaches for Antibody Phage Display Selections
Chapter 20: Antibody Affinity and Stability Maturation by Error-Prone PCR
1 Introduction
2 Materials
2.1 Error-Prone PCR
2.2 Library Construction
2.3 Library Validation
2.4 Library Packaging
2.5 Titration
2.6 Selection by Panning
2.7 Production of Soluble, Monoclonal Antibody Fragments
2.8 Monoclonal ELISA and Thermal Stability Screening
3 Methods
3.1 Error-Prone PCR
3.2 Library Construction
3.3 Library Validation
3.4 Library Packaging
3.5 Titration
3.6 Selection by Panning
3.7 Production of Soluble Monoclonal Antibody Fragments
3.8 ELISA of Soluble Monoclonal Antibody Fragments
3.9 Stability Screening of Soluble Monoclonal Antibody Fragments
4 Notes
References
Chapter 21: Antibody Batch Cloning
1 Introduction
2 Materials
2.1 Preparation of scFv-Encoding DNA After Panning
2.2 Ligation
2.3 Heat-Shock Transformation
2.4 Colony Identification and Inoculation
2.5 Plasmid Isolation
3 Methods
3.1 Preparation of scFv-Encoding DNA
3.2 Ligation
3.3 Heat-Shock Transformation
3.4 Colony Identification and Inoculation
3.5 Plasmid Isolation
4 Notes
References
Chapter 22: Deep Mining of Complex Antibody Phage Pools
1 Introduction
2 Materials
3 Methods
3.1 Protein Depletion Followed by Panning on Whole Cells
3.1.1 Coating of Polystyrene Beads
3.1.2 Binding to Streptavidin Beads
3.1.3 Coupling of Tosylactivated M-280 Beads
3.1.4 Depletion of Phage Pool
3.1.5 Panning on Cells
3.1.6 Elution of Phages Using Trypsin
3.2 Panning on Whole Cells with Protein Competition
3.2.1 Calculations of Protein Concentrations
3.2.2 Panning on Cells
3.2.3 Elution of Phages Using Trypsin
3.3 Panning on Whole Cells with Antibody Blocking
3.3.1 Calculations of Antibody Concentrations
3.3.2 Panning on Cells
3.3.3 Elution of Phages Using Trypsin
4 Notes
References
Chapter 23: High-Throughput IgG Reformatting and Expression Using Hybrid Secretion Signals and InTag Positive Selection Techno...
1 Introduction
2 Materials
2.1 General Reagents
2.2 Preparation of Linearized Vector and Adaptor for Cloning
2.3 PCR Amplification of LC and VH Regions
2.4 In-Fusion Cloning
2.5 Isolation of Plasmid DNA and Sequencing Analysis
2.6 Transient Transfection
3 Methods
3.1 Preparation of Linearized Vector and Adaptor
3.2 PCR Amplification of Antibody Light Chain and Variable Heavy Chain
3.3 In-Fusion Cloning (Also see Note 5)
3.4 Transformation
3.5 IgG Reformatting Using the Cut-Paste Method
3.6 Isolation of Plasmid DNA and Sequence Confirmation
3.7 Transient Transfection
4 Notes
References
Chapter 24: Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols
1 Introduction
2 Materials and Reagents
3 Methods
3.1 Construction of Sortase-Acceptor Plasmid
3.2 Preparation of MILKSHAKE Protein Inoculant
3.3 Expression of MILKSHAKE Protein
3.4 Purification of MILKSHAKE Protein
3.5 Modified Maltose Binding Protein Conjugation to Peptide
3.5.1 Conjugation Controls
3.5.2 Peptide Design
3.5.3 Modified Maltose Binding Protein Conjugation to Peptides
3.6 Ab Validation Using Western Blot
3.6.1 Protein Electrophoresis and Membrane Transfer
3.6.2 Developing and Exposing the Membrane
3.6.3 Interpretation of Data
3.7 Construction of Genetically Encoded Target Sequence Plasmid (Sundae)
3.8 Sundae Protocol-Preparation of Inoculant
3.9 Expression of Sundae Protein
3.10 Purification of Sundae Protein
3.11 Ab Validation Using Sundae ELISA
4 Notes
References
Chapter 25: Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display
1 Introduction
2 Materials
2.1 General
2.2 Biopanning Against Polyclonal Immunoglobulin G
2.3 Bioinformatic Analysis to Identify Enriched Peptides Specific for Infection
2.4 Confirmation of Peptide Specificity for Infection
3 Methods
3.1 Biopanning Against Polyclonal Immunoglobulin G
3.2 Bioinformatic Analysis to Identify Enriched Peptides Specific for Infection
3.3 Confirmation of Infection-Specific Diagnostic Peptides
4 Notes
References
Chapter 26: Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering
1 Introduction
2 Materials
2.1 Core NGS Workflow
2.1.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
2.1.2 RNA Extraction and cDNA Synthesis
2.1.3 Construction and Panning of Phage-Displayed sdAb Libraries
2.1.4 Illumina MiSeq Sequencing
2.1.5 Data Analysis
2.2 Identification of sdAbs Against Prespecified Epitopes
2.2.1 Isolation of PBMCs
2.2.2 RNA Extraction and cDNA Synthesis
2.2.3 Construction of Phage-Displayed sdAb Libraries
2.2.4 Panning of Phage-Displayed sdAb Libraries with Competitive Elution
2.2.5 Illumina MiSeq Sequencing
2.2.6 Data Analysis
2.3 Direct Selection of Antigen-Specific B Cells from PBMCs and Identification of Antigen-Specific sdAbs
2.3.1 Isolation of PBMCs
2.3.2 Preparation of MBP-Int277-Coupled Magnetic Beads
2.3.3 Positive Selection of MBP-Int277-Reactive B cells
2.3.4 RNA Extraction and cDNA Synthesis
2.3.5 Illumina MiSeq Sequencing
2.3.6 Data Analysis
2.4 Affinity Maturation of sdAbs Using NGS
2.4.1 Construction of Random sdAb Mutagenesis Libraries
2.4.2 Construction of Site-Saturating sdAb Mutagenesis Libraries
2.4.3 Panning of sdAb Mutagenesis Libraries
2.4.4 Illumina MiSeq Sequencing
2.4.5 Data Analysis
3 Methods
3.1 Core NGS Workflow
3.1.1 Isolation of PBMCs
3.1.2 RNA Extraction and cDNA Synthesis
3.1.3 Construction and Panning of Phage-Displayed Single-Domain Antibody Libraries
3.1.4 Illumina MiSeq Sequencing
3.1.5 Data Analysis
3.2 Identification of sdAbs Against Prespecified Epitopes
3.2.1 Isolation of PBMCs
3.2.2 RNA Extraction and cDNA Synthesis
3.2.3 Construction of Phage-Displayed sdAb Libraries
3.2.4 Panning of Phage-Displayed sdAb Libraries with Competitive Elution
3.2.5 Illumina MiSeq Sequencing
3.2.6 Data Analysis
3.3 Direct Selection of Antigen-Specific B Cells from PBMCs and Identification of Antigen-Specific sdAbs
3.3.1 Isolation of PBMCs
3.3.2 Preparation of MBP-Int277-Coupled Magnetic Beads
3.3.3 Positive Selection of MBP-Int277-Reactive B cells
3.3.4 RNA Extraction and cDNA Synthesis
3.3.5 Illumina MiSeq Sequencing
3.3.6 Data Analysis
3.4 Affinity Maturation of sdAbs Using NGS
3.4.1 Construction of Random sdAb Mutagenesis Libraries by Error-Prone PCR
3.4.2 Construction of Site-Saturating sdAb Mutagenesis Libraries
3.4.3 Panning of Random and Site-Saturating sdAb Mutagenesis Libraries
3.4.4 Illumina MiSeq Sequencing
3.4.5 Expression of sdAbs and Affinity Determination by Surface Plasmon Resonance
4 Notes
References
Part V: Epitope Mapping and Biomarker Discovery by Phage Display
Chapter 27: Biomarker Discovery by ORFeome Phage Display
1 Introduction
2 Materials
2.1 Isolation of Genomic DNA
2.2 Amplification of Genomic DNA
2.3 Fragmentation of DNA
2.4 DNA End Repair
2.5 Library Construction
2.6 Antigen Library Packaging
2.7 Colony PCR
2.8 Antigen Panning and Screening-ELISA
3 Methods
3.1 Isolation of Genomic DNA
3.2 Amplification of Genomic DNA (Optional)
3.3 DNA Fragmentation
3.4 Removal of Cohesive Ends
3.5 Phagemid-Fragment Ligation and Library Construction
3.6 Library Quality Control
3.7 Library Packaging and ORF Enrichment
3.8 Colony PCR
3.9 Antigen Panning
3.10 Monoclonal Phage Production and Screening ELISA
4 Notes
References
Chapter 28: Mapping Epitopes by Phage Display
1 Introduction
2 Materials
2.1 Antigen Library Construction
2.2 Antigen Panning and Screening
3 Methods
3.1 Gene Amplification, Fragmentation, and End-Repair
3.2 Phagemid-Fragment Ligation and Library Construction
3.3 Library Quality Control and Packaging
3.4 Antigen Panning
3.5 Monoclonal Phage Production and Screening
3.6 Selection and Sequencing of Positive Hits and Epitope Determination
4 Notes
References
Chapter 29: Epivolve: A Protocol for Site-Directed Antibodies
1 Introduction
2 Materials, Reagents, and Equipment
3 Methods
3.1 Peptide Design
3.2 Phage Display Library
3.3 Isolation of mod1-specific scFvs by Phage Display Biopanning.
3.4 Site-Specific scFv Protein Titration ELISA
3.5 Error-Prone PCR
3.6 Discovery Maturation (DisMat) and Affinity Maturation (AffMat) Biopanning
3.6.1 DisMat Modifications
3.6.2 AffMat Modifications
3.7 Converting scFv Hits into Full-Length IgG Proteins and Final Antibody Validation.
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2702

Michael Hust Theam Soon Lim  Editors

Phage Display Methods and Protocols Second Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Phage Display Methods and Protocols Second Edition

Edited by

Michael Hust Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universit€at Braunschweig, Braunschweig, Germany

Theam Soon Lim Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia

Editors Michael Hust Institut fu¨r Biochemie, Biotechnologie und Bioinformatik Technische Universit€at Braunschweig Braunschweig, Germany

Theam Soon Lim Institute for Research in Molecular Medicine (INFORMM) Universiti Sains Malaysia Penang, Malaysia

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3380-9 ISBN 978-1-0716-3381-6 (eBook) https://doi.org/10.1007/978-1-0716-3381-6 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Paul Ehrlich first coined the term magic bullet to describe antibodies which were initially described by his colleague Emil von Behring as “Antitoxine” (anti-toxins) about 125 years ago. The Emil von Behring got the first Nobel Prize for the anti-diphtheria treatment using polyclonal antisera. This concept of polyclonal antibodies was used for several applications as, e.g., anti-snake venom serum preparations, anti-botulinum sera, or as passive therapy against infectious diseases. Since the initial use of antibodies for therapy, the progress of antibodies as a means for therapy was slower compared to the influence antibodies played in the field of diagnostics. It was 15 years after the introduction of hybridoma technology that phage display technology was first reported, and not long after other new technologies were also introduced coinciding with the growing interest of adapting antibodies for therapy. The advancement in recombinant DNA technology and phage display allowed for the selection of fully human antibodies from antibody phage display libraries. The first fully human antibody Adalimumab was developed by guided selection using a human antibody phage display library. There has since been a significant number of different types of phage display antibody libraries been developed. The advent of machine learning and artificial intelligence has also impacted the field of antibody phage display in the number of clones it is able to sieve and allow more in silico-based methods to complement traditional affinity maturation strategies. The wide array of applications of phage display highlights the robustness and durability of the method for antibody generation and other biomolecules. This book is the second edition culminating from the success of the first edition. We were able to collate a collection of various examples of protocols leading to the generation of different forms of antibody libraries including libraries from different hosts as well as different antibody selection (“panning”) strategies. We are grateful as many renowned research groups internationally were willing to share their expertise and experience in this book making it an ideal companion for avid researchers in the field of antibody technology. A comprehensive list of different antibody libraries as well as novel approaches for antibody discovery is covered in this book. The chapters in this book are divided into four sections: the first focuses on the construction of antibody libraries, followed by selection strategies for antibodies, complementary approaches for antibody selection, and finally other phage display-related applications such as epitope mapping and biomarker identification. Although a comprehensive list of topics has been covered in this book, there are still many more chapters that can still be written considering so much activity in all the antibody laboratories in the world. We also included a chapter on yeast display to highlight the synonymous nature of other display technologies to phage display. The diverse author list showcases the ability for science to transcend borders allowing keen researchers from different parts of the world to carry out antibody development projects. The process to put this book together has helped strengthen our friendship and exchange between our laboratories not just on a research level but also more importantly on a personal level. Many new friendships and ideas have been developed over the course of this book that bolds well for cross-border collaborative initiatives. It is our hope that this book can provide technical assistance to new groups and researchers that are venturing into the field of antibody phage display. We also hope this book will help spur interest and ideas in the field while expanding our growing family of enthusiastic antibody researchers.

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Preface

We would like to thank all the authors whose contributions to this volume have allowed it to be a comprehensive guide to the processes involved in antibody phage display. We would also like to thank Prof. John M. Walker for his guidance and assistance throughout the editorial process. We would also like to express our gratitude to our great mentors Erhard Rhiel, Thomas Reinard, and Stefan Du¨bel and Zolta´n Konthur and Jo¨rn Glo¨kler who are great influences in our scientific careers. On a personal note, we would like to thank our families Poi Hong, Hayley, and Hayden and Dagmar, Noah Joris, and Lenja Marie for their patience and understanding while preparing this book and with our other commitments. Braunschweig, Germany Penang, Malaysia

Michael Hust Theam Soon Lim

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

INTRODUCTION

1 Antibody Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alia Nur, Maren Schubert, Jing Yi Lai, Michael Hust, Yee Siew Choong, Wan Yus Haniff Wan Isa, and Theam Soon Lim

PART II

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3

CONSTRUCTION OF ANTIBODY PHAGE DISPLAY LIBRARIES

2 Construction of Human Immune and Naive scFv Phage Display Libraries . . . . . ¨ hner, Maximilian Ruschig, Philip Alexander Heine, Viola Fu Kilian Johannes Karl Zilkens, Stephan Steinke, Maren Schubert, Federico Bertoglio, and Michael Hust 3 Construction of Naı¨ve and Immune Human Fab Phage Display Library . . . . . . . Jing Yi Lai and Theam Soon Lim 4 Construction of Synthetic Antibody Phage Display Libraries . . . . . . . . . . . . . . . . . Kim Anh Giang, Sachdev S. Sidhu, and Johan Nilvebrant 5 Construction of Chicken and Ostrich Antibody Libraries . . . . . . . . . . . . . . . . . . . . Jeanni Fehrsen, Susan Wemmer, and Wouter van Wyngaardt 6 Construction of Rabbit Immune Antibody Libraries . . . . . . . . . . . . . . . . . . . . . . . . Thi Thu Ha Nguyen, Jong Seo Lee, and Hyunbo Shim 7 Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Martin A. Rossotti, Frederic Trempe, Henk van Faassen, Greg Hussack, and Mehdi Arbabi-Ghahroudi 8 Phagekines: Directed Evolution and Characterization of Functional Cytokines Displayed on Phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gertrudis Rojas, Tania Carmenate, Gisela Garcı´a-Pe´rez, and Dayana Pe´rez-Martı´nez 9 Efficient Cloning of Inserts for Phage Display by Golden Gate Assembly . . . . . . Ashley K. Grahn, Grace L. Allen, and Brian K. Kay 10 Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renhua R. Huang, Michael Kierny, Veronica Volgina, Makio Iwashima, Christina Miller, and Brian K. Kay

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39 59 77 93

107

149

191

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Contents

Construction of Semisynthetic Shark vNAR Yeast Surface Display Antibody Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Harald Kolmar, Julius Grzeschik, Doreen Ko¨nning, Simon Krah, and Stefan Zielonka

PART III 12

13

14

15

16

17

18

19

Antibody Selection via Phage Display in Microtiter Plates. . . . . . . . . . . . . . . . . . . . Stephan Steinke, Kristian Daniel Ralph Roth, Maximilian Ruschig, Nora Langreder, Saskia Polten, Kai-Thomas Schneider, Rico Ballmann, Giulio Russo, Kilian Johannes Karl Zilkens, Maren Schubert, Federico Bertoglio, and Michael Hust Antibody Selection in Solution Using Magnetic Beads . . . . . . . . . . . . . . . . . . . . . . Philip Alexander Heine, Maximilian Ruschig, Nora Langreder, Esther Veronika Wenzel, Maren Schubert, Federico Bertoglio, and Michael Hust Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theam Soon Lim, Angela Chiew Wen Ch’ng, Brenda Pei Chui Song, and Jing Yi Lai Magnetic Nanoparticle-Based Semi-automated Panning for High-Throughput Antibody Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angela Chiew Wen Ch’ng, Zolta´n Konthur, and Theam Soon Lim Antibody Selection on Cells Targeting Membrane Proteins . . . . . . . . . . . . . . . . . . € mran Karsli-U € nal, Maike Hagedorn, Viktor Glaser, U and Tom Pieper Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zhiyuan Yang, Zhihao Wu, Brian H. Santich, Jingbao Liu, Cheng Liu, and Nai-Kong V. Cheung Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adi Amir, David Taussig, Almog Bitton, Limor Nahary, Anna Vaisman-Mentesh, Itai Benhar, and Yariv Wine Selection of Affibody Affinity Proteins from Phagemid Libraries . . . . . . . . . . . . . . Kim Anh Giang, Per-Åke Nygren, and Johan Nilvebrant

PART IV 20

SELECTION STRATEGIES FOR ANTIBODIES 247

261

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291

315

327

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373

COMPLEMENTARY APPROACHES FOR ANTIBODY PHAGE DISPLAY SELECTIONS

Antibody Affinity and Stability Maturation by Error-Prone PCR. . . . . . . . . . . . . . 395 Nora Langreder, Dorina Sch€ a ckermann, Tobias Unkauf, Maren Schubert, Andre´ Frenzel, Federico Bertoglio, and Michael Hust 21 Antibody Batch Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 Rico Ballmann, Kai-Thomas Schneider, ¨ bel Kristian Daniel Ralph Roth, and Stefan Du

Contents

22

Deep Mining of Complex Antibody Phage Pools . . . . . . . . . . . . . . . . . . . . . . . . . . . Tulika Tulika and Anne Ljungars 23 High-Throughput IgG Reformatting and Expression Using Hybrid Secretion Signals and InTag Positive Selection Technology . . . . . . . . . . . . . . . . . . Georgina Sansome, Veronika Rayzman, Irene Kiess, Michael J. Wilson, Con Panousis, and Chao-Guang Chen 24 Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mary R. Ferguson, Qiana M. Mendez, Felicity E. Acca, Cassandra D. Chapados, Holland A. Driscoll, Kezzia S. Jones, Gregory Mirando, Michael P. Weiner, and Xiaofeng Li 25 Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maria T. Tsoumpeli, Anitha Varghese, Jonathan P. Owen, Ben C. Maddison, Janet M. Daly, and Kevin C. Gough 26 Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael J. Lowden, Eric K. Lei, Greg Hussack, and Kevin A. Henry

PART V

ix

419

433

451

467

489

EPITOPE MAPPING AND BIOMARKER DISCOVERY BY PHAGE DISPLAY

27

Biomarker Discovery by ORFeome Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . 543 Philip Alexander Heine, Rico Ballmann, Praveen Thevarajah, Giulio Russo, Gustavo Marc¸al Schmidt Garcia Moreira, and Michael Hust 28 Mapping Epitopes by Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563 Stephan Steinke, Kristian Daniel Ralph Roth, Ruben Englick, ¨ hner, Nora Langreder, Rico Ballmann, Viola Fu Kilian Johannes Karl Zilkens, Gustavo Marc¸al Schmidt Garcia Moreira, Allan Koch, Filippo Azzali, Giulio Russo, Maren Schubert, Federico Bertoglio, Philip Alexander Heine, and Michael Hust 29 Epivolve: A Protocol for Site-Directed Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 587 Xiaofeng Li, Kezzia S. Jones, Felicity E. Acca, Cassandra D. Chapados, Holland A. Driscoll, Emily P. Fuller, Qiana M. Mendez, Gregory Mirando, Michael P. Weiner, and Mary R. Ferguson Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

603

Contributors FELICITY E. ACCA • Abbratech, Inc., Branford, CT, USA GRACE L. ALLEN • Tango Biosciences, Chicago, IL, USA ADI AMIR • The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel MEHDI ARBABI-GHAHROUDI • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada; Department of Biology, Carleton University, Ottawa, ON, Canada FILIPPO AZZALI • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Technische Universit€ at Braunschweig, Braunschweig, Germany RICO BALLMANN • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie und Medizinische Biotechnologie, Technische Universit€ at Braunschweig, Braunschweig, Germany ITAI BENHAR • The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel FEDERICO BERTOGLIO • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Technische Universit€ at Braunschweig, Braunschweig, Germany; Choose Life Biotech SA, Bellinzona, Switzerland ALMOG BITTON • Ukko LTD, Rehovot, Israel TANIA CARMENATE • Center of Molecular Immunology, La Habana, Cuba ANGELA CHIEW WEN CH’NG • Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia CASSANDRA D. CHAPADOS • Abbratech, Inc., Branford, CT, USA CHAO-GUANG CHEN • Research and Development, CSL Limited, Parkville, Australia NAI-KONG V. CHEUNG • Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, NY, USA YEE SIEW CHOONG • Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia JANET M. DALY • School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire, UK HOLLAND A. DRISCOLL • Abbratech, Inc., Branford, CT, USA STEFAN DU¨BEL • Institute of Biochemistry, Biotechnology and Bioinformatics, Technische Universit€ at Braunschweig, Braunschweig, Germany RUBEN ENGLICK • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Technische Universit€ at Braunschweig, Braunschweig, Germany HENK VAN FAASSEN • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada JEANNI FEHRSEN • ARC-Onderstepoort Veterinary Research, Pretoria, South Africa MARY R. FERGUSON • Abbratech, Inc., Branford, CT, USA ANDRE´ FRENZEL • YUMAB GmbH, Science Campus Braunschweig-Su¨d, Braunschweig, Germany

xi

xii

Contributors

VIOLA FU¨HNER • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie und Medizinische Biotechnologie, Technische Universit€ at Braunschweig, Braunschweig, Germany EMILY P. FULLER • Institute for Systems Genomics, University of Connecticut, Storrs, CT, USA; Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA GISELA GARCI´A-PE´REZ • Center of Molecular Immunology, La Habana, Cuba KIM ANH GIANG • Division of Protein Engineering, School of Chemistry, Biotechnology and Health, Royal Institute of Technology, Stockholm, Sweden VIKTOR GLASER • Department of Gastroenterology, Hepatology, Infectious Diseases and Endocrinology, Hannover Medical School, Hannover, Germany; Berlin Center for Advanced Therapies (BeCAT), Charite´ – Universit€ a tsmedizin Berlin, corporate member of Freie Universit€ a t Berlin, Humboldt-Universit€ at zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany KEVIN C. GOUGH • School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire, UK ASHLEY K. GRAHN • Tango Biosciences, Chicago, IL, USA JULIUS GRZESCHIK • Institute for Organic Chemistry and Biochemistry, Technische Universit€ at Darmstadt, Darmstadt, Germany MAIKE HAGEDORN • Department of Gastroenterology, Hepatology, Infectious Diseases and Endocrinology, Hannover Medical School, Hannover, Germany PHILIP ALEXANDER HEINE • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie und Medizinische Biotechnologie, Technische Universit€ at Braunschweig, Braunschweig, Germany KEVIN A. HENRY • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada RENHUA R. HUANG • MacroGenics, Rockville, MD, USA GREG HUSSACK • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada MICHAEL HUST • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ a t Braunschweig, Braunschweig, Germany WAN YUS HANIFF WAN ISA • School of Medical Sciences, Department of Medicine, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia MAKIO IWASHIMA • Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, USA KEZZIA S. JONES • Abbratech, Inc., Branford, CT, USA € MRAN KARSLI-U € NAL • Department of Gastroenterology, Hepatology, Infectious Diseases and U Endocrinology, Hannover Medical School, Hannover, Germany BRIAN K. KAY • Tango Biosciences, Chicago, IL, USA MICHAEL KIERNY • AstraZeneca, Gaithersburg, MD, USA IRENE KIESS • Research and Development, CSL Limited, Parkville, Australia ALLAN KOCH • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Technische Universit€ at Braunschweig, Braunschweig, Germany; Innovationszentrum Niedersachsen GmbH, startup.niedersachsen, Hannover, Germany HARALD KOLMAR • Institute for Organic Chemistry and Biochemistry, Technische Universit€ at Darmstadt, Darmstadt, Germany

Contributors

xiii

DOREEN KO¨NNING • Antibody-Drug Conjugates and Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany ZOLTA´N KONTHUR • Department of Analytical Chemistry, Reference Materials, Bundesanstalt fu¨r Materialforschung und -pru¨fung (BAM), Berlin, Germany SIMON KRAH • Antibody Discovery & Protein Engineering, Merck KGaA, Darmstadt, Germany JING YI LAI • Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia NORA LANGREDER • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ a t Braunschweig, Braunschweig, Germany JONG SEO LEE • AbClon Inc., Seoul, Korea ERIC K. LEI • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada XIAOFENG LI • Abbratech, Inc., Branford, CT, USA THEAM SOON LIM • Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia; Analytical Biochemistry Research Center, Universiti Sains Malaysia, Penang, Malaysia CHENG LIU • Eureka Therapeutics, Emeryville, CA, USA JINGBAO LIU • Eureka Therapeutics, Emeryville, CA, USA ANNE LJUNGARS • Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark MICHAEL J. LOWDEN • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada BEN C. MADDISON • RSK-ADAS Ltd, Beeston, Nottinghamshire, UK QIANA M. MENDEZ • Abbratech, Inc., Branford, CT, USA CHRISTINA MILLER • Tango Biosciences, Chicago, IL, USA GREGORY MIRANDO • Abbratech, Inc., Branford, CT, USA GUSTAVO MARC¸AL SCHMIDT GARCIA MOREIRA • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie und Medizinische Biotechnologie, Technische Universit€ at Braunschweig, Braunschweig, Germany; Tacalyx GmbH, Sector for Antibody and Protein Biochemistry, Berlin, Germany LIMOR NAHARY • The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel THI THU HA NGUYEN • Imvastech Inc., Seoul, Korea JOHAN NILVEBRANT • Division of Protein Engineering, School of Chemistry, Biotechnology and Health, Royal Institute of Technology, Stockholm, Sweden ALIA NUR • Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia PER-ÅKE NYGREN • Division of Protein Engineering, School of Chemistry, Biotechnology and Health, Royal Institute of Technology, Stockholm, Sweden; Science for Life Laboratory, Solna, Sweden JONATHAN P. OWEN • RSK-ADAS Ltd, Beeston, Nottinghamshire, UK CON PANOUSIS • Research and Development, CSL Limited, Parkville, Australia DAYANA PE´REZ-MARTI´NEZ • Center of Molecular Immunology, La Habana, Cuba TOM PIEPER • Department of Gastroenterology, Hepatology, Infectious Diseases and Endocrinology, Hannover Medical School, Hannover, Germany

xiv

Contributors

SASKIA POLTEN • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ a t Braunschweig, Braunschweig, Germany VERONIKA RAYZMAN • Research and Development, CSL Limited, Parkville, Australia GERTRUDIS ROJAS • Center of Molecular Immunology, La Habana, Cuba MARTIN A. ROSSOTTI • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada KRISTIAN DANIEL RALPH ROTH • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ at Braunschweig, Braunschweig, Germany MAXIMILIAN RUSCHIG • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ at Braunschweig, Braunschweig, Germany GIULIO RUSSO • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ a t Braunschweig, Braunschweig, Germany GEORGINA SANSOME • Research and Development, CSL Limited, Parkville, Australia BRIAN H. SANTICH • Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, NY, USA DORINA SCHA€ CKERMANN • Technische Universit€ a t Braunschweig, Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Braunschweig, Germany; Wirtschaftsgenossenschaft deutscher Tier€ a rzte eG (WDT), Garbsen, Germany KAI-THOMAS SCHNEIDER • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ at Braunschweig, Braunschweig, Germany MAREN SCHUBERT • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ a t Braunschweig, Braunschweig, Germany HYUNBO SHIM • Department of Life Sciences, Ewha Womans Univesity, Seoul, Korea SACHDEV S. SIDHU • School of Pharmacy, University of Waterloo, Kitchener, ON, Canada BRENDA PEI CHUI SONG • Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia STEPHAN STEINKE • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Departments Biotechnology and Medical Biotechnology, Technische Universit€ a t Braunschweig, Braunschweig, Germany DAVID TAUSSIG • The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel PRAVEEN THEVARAJAH • Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie und Medizinische Biotechnologie, Technische Universit€ at Braunschweig, Braunschweig, Germany FREDERIC TREMPE • Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada MARIA T. TSOUMPELI • School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire, UK TULIKA TULIKA • Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

Contributors

xv

TOBIAS UNKAUF • Technische Universit€ a t Braunschweig, Institut fu¨r Biochemie, Biotechnologie und Bioinformatik, Braunschweig, Germany; Bayer Consumer Care AG, Basel, Switzerland ANNA VAISMAN-MENTESH • 1E Therapeutics LTD, Rehovot, Israel ANITHA VARGHESE • School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire, UK VERONICA VOLGINA • Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, USA MICHAEL P. WEINER • Abbratech, Inc., Branford, CT, USA SUSAN WEMMER • ARC-Onderstepoort Veterinary Research, Pretoria, South Africa ESTHER VERONIKA WENZEL • Abcalis GmbH, Braunschweig, Germany MICHAEL J. WILSON • Research and Development, CSL Limited, Parkville, Australia YARIV WINE • The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel ZHIHAO WU • Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA WOUTER VAN WYNGAARDT • ARC-Onderstepoort Veterinary Research, Pretoria, South Africa ZHIYUAN YANG • Eureka Therapeutics, Emeryville, CA, USA STEFAN ZIELONKA • Institute for Organic Chemistry and Biochemistry, Technische Universit€ at Darmstadt, Darmstadt, Germany; Antibody Discovery & Protein Engineering, Merck KGaA, Darmstadt, Germany KILIAN JOHANNES KARL ZILKENS • Abcalis GmbH, Braunschweig, Germany

Part I Introduction

Chapter 1 Antibody Phage Display Alia Nur, Maren Schubert, Jing Yi Lai, Michael Hust, Yee Siew Choong, Wan Yus Haniff Wan Isa, and Theam Soon Lim Abstract The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities. Key words Antibody libraries, Biopanning, M13 bacteriophage, Monoclonal antibodies, Phage display

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Introduction Antibodies are one of the prominent products of the human immune system. They are produced by B cells as a response to encounters with any foreign antigen. B cells will proliferate and secrete soluble antibodies into circulation via the blood and lymphatic system when triggered by these foreign antigens [1]. The inherent ability of antibodies to confer protection to the human body against a plethora of pathogens is governed by the diverse repertoire of antibodies produced by the B cells through mutational processes. Germline level diversification of antibody genes functions to generate a diverse population of antibodies with different binding specificities. The binding specificities are refined through clonal selection to produce an affinity-rich population of

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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antibodies to bind specific target antigens from any pathogen [2, 3]. The increase in demand for antibodies especially monoclonal antibodies after the advent of hybridoma technology [4] has exposed some of the challenges associated with the method. This brought about innovations to produce monoclonal antibodies using different approaches to achieve fully human antibodies as well for other species at a faster rate. This was possible with the introduction of phage display as the first major display technique for antibody discovery [5–7] based on the work of G.P. Smith [8]. It changed the way monoclonal antibodies can be generated allowing the generation of fully human antibodies. The ideation of phage display is based on the possibility to clone antibody sequences as a fusion to the minor coat protein of the phage particle. Therefore, when phage proteins are produced, the antibody fused to the minor coat protein would also be expressed simultaneously by the host cell. During virion packaging in the periplasm, phage particles would be produced presenting the particular antibody fragment on its surface. The presented antibody molecule on the surface of the phage particle is attached to the minor coat protein of the mature phage particles with the genetic information being encapsulated in the phage particle allowing for easy clone identification [9–11]. A directed evolution selection process is carried out by selecting clones with the highest specificity and affinity by selective elimination through several enrichment cycles. This entire select and concentrate cycle makes for the basis of phage display panning for monoclonal antibody selection [12, 13].

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Antibody Structure and Function The general architecture of an antibody resembles a typical Y-shaped structure that is typically made up of two identical pairs of heavy chain (HC) and light chain (LC) [14]. The bivalency of these four polypeptide chains is formed via disulfide bonds between the two halves of the Y-shape structure [15]. The human antibody LC is unique where it can be classified as either kappa (κ) or lambda (λ) families [16, 17]. Each antibody chain is further divided into the constant (C) region and a highly varied variable (V) region. The sequence variability stems from three regions of hypervariability that are separated by four framework regions (FR). These hypervariable sites are commonly referred to as complementaritydetermining regions (CDRs). The 6 CDRs found throughout the HC and LC (each chain having 3 CDRs) accounts for the antibody variability and specificity in antigen binding. The heavy chain (VH) and light chain (VL) variable domains will fold to form the fragment variable region (Fv), which plays the pivotal role of target binding.

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Fig. 1 Structure of an antibody and various recombinant antibody formats (a) Typical structure of IgG comprises two identical heavy chains (HC) and two identical light chains (LC) interconnected through disulfide bonds to form a flexible Y-shaped molecule. VH and VL form the fragment variable region (Fv); Fv together with CH1 and CL form the fragment antigen-binding region (Fab); CH2 and CH3 form the fragment crystallizable region (Fc). (b) Some of the commonly used recombinant antibody formats includes the single-chain fragment variable (scFv), single-domain antibody (sdAb), Fab, single-chain Fab (scFab), scFv-CH3, and scFv-Fc

However, the VH and VL when fused to their respective constant domains (light chain constant region, CL and first heavy chain constant domain, CH1) will form the fragment antigen-binding region (Fab) giving rise to two identical arms of the “Y”-shaped structure [3, 18, 19]. The Fab structure makes up the upper half of the antibody Y shape structure. However, recombinant DNA technology has brought about various antibody domain configurations to yield a selection of different antibody formats in Fig. 1. The upper half of the antibody is then linked to the bottom half by a flexible linker. The bottom half of the antibody consist of the fragment crystallizable (Fc) region. The Fc domain is made up of the heavy chain constant domains 2 and 3 (CH2 and CH3). The Fc region is able to elicit an immune response by interacting with the Fc receptors (FcR) or C1q complement components to eliminate invading antigens either by antibody-dependent cellular cytotoxic (ADCC) or complement-dependent cytotoxic (CDC) mechanism [3, 20]. The sequence makeup of the C region is relatively constant with only slight variations occurring at specific sites responsible for the effector cell binding [3]. 2.1 Recombinant Formats

The main characteristic of antibodies is its ability to specifically bind and distinguish target antigens. As the variable region of the antibody molecules is responsible for antigen binding, a diverse

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collection of antibody fragments or formats focusing on variable domains have been engineered for display. Truncated antibody fragments can be produced either by enzymatic fragmentation or genetic modification. This can be achieved either by disulfide bond reduction or by protease digestion using papain and pepsin to yield Fab, F(ab)’2, and Fc formats from a single antibody molecule [21, 22]. However, genetic-based approaches allows for greater freedom to generate unique antibody formats. Of the many potential recombinant formats, the format that is most widely utilized is the single-chain variable fragment (scFv). The scFv construct consists of both the VH and a VL domain that is connected through a short flexible peptide linker [23]. The scFv format is a monomeric protein in general, but further genetic modification has also developed multimeric scFvs that consist of multiple scFv fragments that are joined together through a shorter peptide linker to form multivalent diabodies, triabodies, or tetrabodies. These multimeric scFv formats would also generate a molecule with a higher avidity [24, 25]. Additionally, fusing multiple scFv molecules with different target binding pockets can also help to achieve antibody fragments with multispecificities. Likewise, the freedom of genetic-based approaches brought about various combinations utilizing the VH, VL, CH1, CL, CH2, and CH3 domains. Some of the alternative formats achievable by genetic modification includes the Fab, scFab, scFv-CH3, and scFv-Fc [26–28] (Fig. 1). The smallest antibody fragment available is the single-domain antibody (sdAb) that consist of a single variable domain. sdAb, also termed as nanobody, is derived from heavy chain antibody (hcAb) such as VHH of camelids and VNAR of sharks [29–31]. Alternatively, sdAb can also be engineered using either the VH or VL of human or mouse IgG [32]. A major advantage of sdAb over conventional antibodies or other recombinant antibody formats is the rapid penetration to buried sites or cryptic epitopes due to its smaller size which are not accessible to larger antibodies [33, 34]. At the same time, its size is also a disadvantage as clearance from the body is faster and therefore resulting in a reduced halflife compared to an IgG.

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Antibody Phage Display Libraries The primary application of antibody phage display libraries is based on the fundamental ability of phage display to allow the recovery of the genotype (DNA sequence) based on the phenotypic (protein) characteristic [6, 7]. In order to achieve this coupling of phenotype and genotype, the antibody is fused to the pIII protein of M13 as a fusion. This way, the phenotype is now displayed on the phage particles encapsulating the genotype corresponding to the specific antibody being displayed. When constructing antibody phage

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display libraries, the genes encoding different antibody clones are cloned into a phagemid with the antibody sequence located upstream of pIII gene. The phagemid lacking other accessory proteins required would depend on helper phages that contains the remaining sequences required to regulate the packaging of phage particles. When phage particles are packaged, the antibody fragment is translated with the coat protein as a fusion protein permitting it to be anchored and displayed on the surface of the phage particle [6, 9, 10, 35]. A large collection of unique phage particles each encoding a unique antibody gene forms the basis for antibody phage display libraries. Antibody libraries are generally designed based on the choice of antibody format preferred with various antibody formats such as scFv [23], Fab, or sdAb [36] that are normally used for phage display. Another factor to be considered is the source of antibody genes which can be derived from humans or other species which includes rodents, camelids, non-cartilaginous fishes and nonhuman primates [37]. Even so, antibody phage display libraries can be categorized into three main categories based on the source of the V gene repertoire. They are commonly referred to as either immune libraries, naı¨ve libraries, or synthetic antibody libraries [38]. 3.1 Immune Antibody Library

Immune antibody libraries are constructed using B cells or plasma cells from either immunized animals or disease infected individuals. Immune libraries take advantage of the in vivo affinity maturation and V gene hypermutation process that shapes the antibody response upon exposure to an infection [39–41]. These processes help shape a skewed repertoire against the infection, therefore allowing the isolation of antibodies with higher affinities in the nanomolar range. Like naı¨ve antibody libraries, the antibody genes could also be isolated from PBMC, bone marrow, lymph nodes, or spleen [42]. The library quality can be improved by isolating donor plasma cells for library construction [43]. The antibody isotype that is typically used for the development of immune antibody libraries is IgG, while IgA and IgE repertoires are sometimes considered especially for mucosal-, parasitic-, and allergy-based libraries [39]. As the nature of immune libraries is somewhat focused in its response, the required repertoire size for immune antibody libraries is relatively smaller compared to naı¨ve antibody libraries. Even so, immune libraries should not be discounted from the presence of an extended repertoire due to the complexity generated by combinatorial cloning [44]. The general application of immune antibody libraries is mainly for the generation of antibodies against a specific set of antigens that are related to a specific infection given the nature of the skewed repertoire. Even so, it is also possible to isolate binders against closely related antigens using immune libraries [45, 46]. However, such a practice

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with immune libraries is not common as the general perception of a skewed repertoire would mean a focused application of immune libraries. Thus in general, new libraries would have to be constructed for each new antibody development campaign against a new target or disease which may not be cost effective and tedious. 3.2 Naı¨ve Antibody Library

A naı¨ve antibody library as the name depicts is naı¨ve in terms of its repertoire preference. Such a library is generated using B cells from either healthy donors or individuals with no known infection at the point of collection. The antibody genes can be retrieved by mRNA extraction of various source materials ranging from either peripheral blood mononuclear cells (PBMC), bone marrow, lymph nodes, or spleen. The mRNA is then reverse transcribed into cDNA using either oligo(dt) primers or random primers in order to preserve the large and diverse repertoire of the antibody population. After the first-strand cDNA synthesis, the specific V genes are amplified using a set of primers designed to provide a maximum coverage all known V gene families [42, 47, 48]. The gene coverage of the designed primer set is a critical aspect in antibody library construction to ensure good gene coverage during amplification for a diverse library repertoire [49]. Although the general repertoire of the antibody genes is very diverse, the inclusion of a combinatorial mixing step of the antibody genes during cloning can further expand the diversity of the library. During cloning, the VH genes are paired randomly with the VL gene repertoire giving rise to multiple combinations where some may not even exist naturally [42, 47]. Naı¨ve libraries would usually utilize the IgM repertoire for construction of the library repertoire as it targets the repertoire from naı¨ve B cells. The repertoire of naı¨ve antibody libraries must be large, generally in the vicinity of between 109 and 1011 independent clones. The diversification of the six CDRs would mathematically yield an infinite number of combinations that is sufficient to generate a diverse repertoire. However, the ability to replicate the possible repertoire in vitro is not possible in actual practice with the repertoire of a phage display library usually being limited to 1010– 1011 due to the limits of transformation efficiency of E. coli [24, 50]. Even so, the large repertoire of such libraries is sufficient to allow isolation of antibodies against more or less infinite number of targets. Naı¨ve antibody libraries can be used for selection of antibodies against a wide host of targets ranging from infectious diseases, autoimmune disease, cancer, self-antigens, and toxins [3, 42]. The acceptance is that antibodies derived from naı¨ve antibody libraries could have a lower affinity, but this is not always the case. The lower affinity clones derived from naive libraries is not a major hindrance as these clones could be improved through downstream refinement such as in vitro affinity maturation [51].

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3.3 Synthetic and Semisynthetic Antibody Library

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The third and fourth classes of antibody libraries are semisynthetic and synthetic antibody libraries. Semisynthetic libraries are composed of one antibody framework and randomization of at least the CDR-H3 [52]. This allows a for a hybrid of natural framework sequences with the synthetic randomization of the CDRs to generate a library of unique clones. Synthetic libraries utilize predesigned repertoires where the genetic makeup of the library is chemically synthesized and assembled in vitro with a level of control [53]. The approach allows the CDR and framework designs to be optimized and predefined using bioinformatic analysis. This allows the scaffolds used for synthetic antibody libraries to be pre-defined to promote various traits such as good expression levels in E. coli and proper folding for good solubility and antigen binding. The repertoire of synthetic libraries is thus defined by predetermined CDRs that are synthetically randomized to mimic either germline or rearranged V genes [54–57]. There are several different approaches that are applied for synthetic CDR randomization. One of the most common methods to generate randomization is through mutagenesis using degenerate codons such as NNN, NNS, and NNK, where N represents all four nucleotide bases, S represents guanine (G) and cytosine (C), and K represents G or thymine (T) [58]. Although the application of degenerate codons is capable of providing a high diversity, the process introduces a risk factor to the library quality. As the diversification is uncontrolled, it could yield codons that encode unnatural amino acids or stop codons that will disrupt the folding or translation of the antibody fragments as well as solubility. An improvement to the randomization process was introduced in the form of trinucleotide mutagenesis (TRIM) technology. TRIM is able to generate similar diversities devoid of the risk involved using standard degenerate primers. TRIM applies a series of trimers encoding for 20 amino acids instead of random combination of nucleotides to generate the diversity in the CDRs, thus providing a more precise and controlled manipulation process. This approach alleviates stop codons or unfavorable amino acid distribution or combination during diversity generation that could impede the library quality [11, 24]. The nature of the repertoire of synthetic antibody libraries makes it synonymous to naı¨ve libraries where a highly diverse repertoire and library size are required. This in turn permits the application of synthetic libraries for use in unbiased selection against a wide range of targets [54]. A close derivative of synthetic antibody libraries can be found in the form of semisynthetic libraries. Semisynthetic libraries are designed to exhibit characteristics that are a combination from both naı¨ve and synthetic library designs. In semisynthetic libraries, the framework sequence of the library is naturally obtained from naı¨ve B cells or a known antibody sequence [52]. The diversity of semisynthetic libraries is dependent on the prospect of at least one

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of the CDRs being randomized at positions responsible for antigen binding. In many cases, the HCDR3 is the most frequently used CDR for randomization given its major role in antigen binding [52, 56].

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Conclusion Phage display is a powerful technology for the development of monoclonal antibodies used in therapy or as diagnostic tools. Monoclonal human antibodies against more or less any target protein, peptide, or other target structure can be generated in a short amount of time. Immediate sequence information of the individual antibodies allows direct transfer into the desired format starting from the natural IgG format to multimeric and/or multispecific scFv formats. The different kinds of phage libraries (naı¨ve, immune, synthetic, and semisynthetic) offer different advantages for specific target proteins and applications. Up to 2022, antibody phage display yielded 14 approved antibodies against different diseases and several more in clinical trials highlighting the capabilities and importance of the method in the field of immunotherapy [9].

Acknowledgments This work was supported by a Universiti Sains Malaysia, Special (Matching) Short-Term Grant with Project No: 304/CIPPM/ 6315708. References 1. Hoffman W, Lakkis FG, Chalasani G et al (2016) B cells, antibodies, and more. Clin J Am Soc Nephrol 11:137 –154 2. Chi X, Li Y, Qiu X et al (2020) V(D)J recombination, somatic hypermutation and class switch recombination of immunoglobulins: mechanism and regulation. Immunology 160:233 –247 3. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, Wu HC et al (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27(1):1–30 4. Ko¨hler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495 –497 5. Barbas CF, Kang AS, Lerner RA, Benkovic SJ et al (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88:7978 6. Breitling F, Du¨bel S, Seehaus T, Klewinghaus I, Little M et al (1991) A surface

expression vector for antibody screening. Gene 104:147 –153 7. McCafferty J, Griffiths AD, Winter G, Chiswell DJ et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552 –554 8. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315 –1317 9. Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, Hashem AM et al (2020) Phage display derived monoclonal antibodies: from bench to bedside. Front Immunol 11:1986 10. Frenzel A, Schirrmann T, Hust M et al (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8: 1177 11. Wen J, Yuan K, Wen J, Yuan K et al (2021) Phage display technology, phage display

Antibody Phage Display system, antibody library, prospects and challenges. Adv Microbiol 11:181 –189 12. Lim CC, Chan SK, Lim YY, Ishikawa Y, Choong YS, Nagaoka Y, Lim TS et al (2021) Development and structural characterisation of human scFv targeting MDM2 spliced variant MDM215kDa. Mol Immunol 135:191 –203 13. Russo G, Meier D, Helmsing S, Wenzel E, Oberle F, Frenzel A, Hust M et al (2018) Parallelized antibody selection in microtiter plates. Methods Mol Biol 1701:273 –284 14. Porter RR (1963) Chemical structure of γ-globulin and antibodies. Br Med Bull 19: 197 –201 15. Alzari PM, Lascombe MB, Poljak RJ et al (1988) Three-dimensional structure of antibodies. Annu Rev Immunol 6:555 –580 16. Barbie´ V, Lefranc MP (1998) The human immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp Clin Immunogenet 15:171 –183 17. Malcolm S, Barton P, Murphy C, FergusonSmith MA, Bentley DL, Rabbitts TH et al (1982) Localization of human immunoglobulin kappa light chain variable region genes to the short arm of chromosome 2 by in situ hybridization. Proc Natl Acad Sci U S A 79: 4957 –4961 18. Chiu ML, Goulet DR, Teplyakov A, Gilliland GL et al (2019) Antibody structure and function: the basis for engineering therapeutics. Antibodies (Basel). https://doi.org/10. 3390/ANTIB8040055 19. Hanson QM, Barb AW (2015) A perspective on the structure and receptor binding properties of immunoglobulin G Fc. Biochemistry 54:2931 –2942 20. Bruhns P, Jo¨nsson F (2015) Mouse and human FcR effector functions. Immunol Rev 268: 25 –51 21. Power CA, Bates A (2019) David vs. Goliath: the structure, function, and clinical prospects of antibody fragments. Antibodies. https:// doi.org/10.3390/ANTIB8020028 22. Kinman AWL, Pompano RR (2019) Optimization of enzymatic antibody fragmentation for yield, efficiency, and binding affinity. Bioconjug Chem 30:800 –807 23. Huston JS, Levinson D, Mudgett-Hunter M et al (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci 85:5879 –5883 24. Almagro JC, Pedraza-Escalona M, Arrieta HI, Pe´rez-Tapia SM et al (2019) Phage display libraries for antibody therapeutic discovery

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and development. Antibodies (Basel). https:// doi.org/10.3390/ANTIB8030044 25. Kafil V, Saei AA, Tohidkia MR, Barar J, Omidi Y et al (2020) Immunotargeting and therapy of cancer by advanced multivalence antibody scaffolds. J Drug Target 28:1018 –1033. https:// doi.org/10.1080/1061186X20201772796 26. Steinwand M, Droste P, Frenzel A, Hust M, Du¨bel S, Schirrmann T et al (2014) The influence of antibody fragment format on phage display based affinity maturation of IgG. MAbs 6:204 –218 27. Omar N, Hamidon NH, Yunus MH, Noordin R, Choong YS, Lim TS et al (2018) Generation and selection of naı¨ve Fab library for parasitic antigen: anti-BmSXP antibodies for lymphatic filariasis. Biotechnol Appl Biochem 65:346 –354 28. Loh Q, Leong SW, Tye GJ, Choong YS, Lim TS et al (2015) Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system. Anal Biochem 477: 56 –61 29. Khodabakhsh F, Behdani M, Rami A, KazemiLomedasht F et al (2018) Single-domain antibodies or nanobodies: a class of nextgeneration antibodies. Int Rev Immunol 37: 316 –322 30. Muyldermans S, Atarhouch T, Saldanha J, Barbosa JARG, Hamers R et al (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7:1129 – 1135 31. Nuttall SD, Krishnan UV, Doughty L, Pearson K, Ryan MT, Hoogenraad NJ, Hattarki M, Carmichael JA, Irving RA, Hudson PJ et al (2003) Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70. Eur J Biochem 270:3543 –3554 32. Hairul Bahara NH, Chin ST, Choong YS, Lim TS et al (2016) Construction of a semisynthetic human VH single-domain antibody library and selection of domain antibodies against α-crystalline of mycobacterium tuberculosis. J Biomol Screen 21:35 –43 33. Be´langer K, Tanha J (2021) High-efficacy, high-manufacturability human VH domain antibody therapeutics from transgenic sources. Protein Eng Des Sel 34:1 –7 34. Rossotti MA, Be´langer K, Henry KA, Tanha J et al (2022) Immunogenicity and humanization of single-domain antibodies. FEBS J 289:4304 –4327 35. Ledsgaard L, Ljungars A, Rimbault C, Sørensen CV, Tulika T, Wade J, Wouters Y, McCafferty J, Laustsen AH et al (2022)

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Advances in antibody phage display technology. Drug Discov Today 27:2151 –2169 36. Nagano K, Tsutsumi Y (2021) Phage display technology as a powerful platform for antibody drug discovery. Viruses. https://doi.org/10. 3390/V13020178 37. Oreste U, Ametrano A, Coscia MR et al (2021) On origin and evolution of the antibody molecule. Biology (Basel) 10:1 –18 38. Hust M, Du¨bel S (2005) Phage display vectors for the in vitro generation of human antibody fragments. Methods Mol Biol 295:71 –96 39. Hamidon NH, Suraiya S, Sarmiento ME, Acosta A, Norazmi MN, Lim TS (2018) Immune TB antibody phage display library as a tool to study B cell immunity in TB infections. Appl Biochem Biotechnol 184:852 – 868 40. Rahumatullah A, Ahmad A, Noordin R, Lim TS (2015) Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library. Mol Immunol 67:512 –523 41. Rahumatullah A, Karim IZA, Noordin R, Lim TS (2017) Antibody-based protective immunity against helminth infections: antibody phage display derived antibodies against BmR1 antigen. Int J Mol Sci. https://doi. org/10.3390/IJMS18112376 42. Chan SK, Lim TS (2017) Immune human antibody libraries for infectious diseases. Adv Exp Med Biol 1053:61 –78 43. Wenzel EV, Bosnak M, Tierney R, Schubert M, Brown J, Du¨bel S, Efstratiou A, Sesardic D, Stickings P, Hust M (2020) Human antibodies neutralizing diphtheria toxin in vitro and in vivo. Sci Rep 10:1 –21 44. Lai JY, Lim TS (2020) Infectious disease antibodies for biomedical applications: a mini review of immune antibody phage library repertoire. Int J Biol Macromol 163:640 45. Rahumatullah A, Ahmad A, Noordin R, Lai JY, Baharudeen Z, Lim TS (2020) Applicability of Brugia malayi immune antibody library for the isolation of a human recombinant monoclonal antibody to Echinococcus granulosus antigen B. Exp Parasitol. https://doi.org/10.1016/J. EXPPARA.2020.108029 46. Rahumatullah A, Balachandra D, Noordin R, Baharudeen Z, Lim YY, Choong YS, Lim TS (2021) Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides. Sci Rep 11:2502 47. Ku¨gler J, Wilke S, Meier D et al (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol. https://doi.org/10.1186/ S12896-015-0125-0

48. Lim BN, Chin CF, Choong YS, Ismail A, Lim TS (2016) Generation of a naı¨ve human single chain variable fragment (scFv) library for the identification of monoclonal scFv against Salmonella Typhi Hemolysin E antigen. Toxicon 117:94 –101 49. Lim TS, Mollova S, Rubelt F, Sievert V, Du¨bel S, Lehrach H, Konthur Z (2010) V-gene amplification revisited – an optimised procedure for amplification of rearranged human antibody genes of different isotypes. Nat Biotechnol 27:108 –117 50. Ku¨gler J, Tomszak F, Frenzel A, Hust M (2018) Construction of human immune and naive scFv libraries. Methods Mol Biol 1701: 3 –24 51. Lim CC, Choong YS, Lim TS (2019) Cognizance of molecular methods for the generation of mutagenic phage display antibody libraries for affinity maturation. Int J Mol Sci 20:1861 52. de Wildt RMT, Mundy CR, Gorick BD, Tomlinson IM (2000) Antibody arrays for highthroughput screening of antibody-antigen interactions. Nat Biotechnol 18:989 –994 53. Hayashi N, Welschof M, Zewe M, Braunagel M, Dubel S, Breitling F, Little M (1994) Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR. Biotechniques 17:310 , 312, 314–5 54. Erasmus MF, D’Angelo S, Ferrara F, Naranjo L, Teixeira AA, Buonpane R, Stewart SM, Nastri HG, Bradbury ARM (2021) A single donor is sufficient to produce a highly functional in vitro antibody library. Commun Biol 4:1 –16 55. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wo¨lle J, Plu¨ckthun A, Virnek€as B (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57 –86 56. Kumar R, Parray HA, Shrivastava T, Sinha S, Luthra K (2019) Phage display antibody libraries: a robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol 135:907 –918 57. Tiller T, Schuster I, Deppe D et al (2013) A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. MAbs 5:445 58. Zadeh AS, Gr€asser A, Dinter H, Hermes M, Schindowski K (2019) Efficient construction and effective screening of synthetic domain antibody libraries. Methods Protoc 2:1 –19

Part II Construction of Antibody Phage Display Libraries

Chapter 2 Construction of Human Immune and Naive scFv Phage Display Libraries Maximilian Ruschig, Philip Alexander Heine, Viola Fu¨hner, Kilian Johannes Karl Zilkens, Stephan Steinke, Maren Schubert, Federico Bertoglio, and Michael Hust Abstract Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such highquality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications. Key words Antibody phage display, Single-chain fragment variable (scFv), Antibody gene library, Naive antibody library, Immune antibody library, Library generation

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Introduction Antibody phage display is a widely used technology for the generation of fully human recombinant antibodies for applications in the research field, for diagnostic purposes, and most importantly for therapeutic applications. As of January 2023, more than 110 antibodies and antibody-based therapeutics, including antibody-drug conjugates, are approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMEA, since 2009 EMA) with 19 additional candidates pending approval [1]. The market is rapidly growing with 115.2 billion US$ business volume in 2018 and is estimated to reach 300 billion US$ by 2025 [2], with an estimated annual growth rate of 13.2% from 2022 to 2028 [3]. Most prominent applications of therapeutic antibodies are cancer and autoimmune diseases [4] with 45% and 27% of all approved antibodies, respectively [5]. The first therapeutic antibody,

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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muronomab-CD3 (Orthoclone OKT3®), was approved in 1986 and is of murine origin applied for immunosuppression after organ transplantation [6] . Chimeric antibodies formed the next generation of therapeutic antibodies like the anti-tumor necrosis factor (TNF) antibody infliximab (Remicade®) for the treatment of rheumatic arthritis but also Crohn’s disease [7] or the anti-epidermal growth factor receptor (EGFR) antibody cetuximab (Erbitux®) for cancer therapy [8] . Subsequently, humanized antibodies were used for therapy, e.g., trastuzumab (Herceptin®) [9] . Finally adalimumab (Humira®), the first fully human antibody generated by phage display, was commercially available in 2002 [10]. Despite their lower risk of immunogenicity, human antibodies can have adverse effects in some cases [11, 12]. Currently, 14 FDA/EMA-approved antibodies were generated by phage display and more are still in clinical development [13]. The basis for the phage display technology are the studies of George P. Smith on the filamentous bacteriophage M13, which was awarded with a Noble Prize in 2018 [14]. Key to the technology is linking genotype and phenotype of displayed oligopeptides, by fusing the corresponding gene fragments encoded on the phagemid to the minor coat protein III gene of the phage. Thereby, the peptide::pIII fusion protein is expressed and displayed on the surface of the phage allowing the affinity selection of the peptide and its encoding gene. Likewise, antibody fragments fused to pIII can be presented on the surface of M13 phage particles. This technology was invented independently in parallel in Cambridge, Heidelberg, and La Jolla in 1990/1991 [15–20]. Because of limitations of the E. coli protein machinery, phage display is routinely only done with antibody fragments like scFv (single-chain fragment variable), Fab (fragment antigen binding), VHH (camel heavy chain variable domain), or dAbs (human heavy chain variable domain) [21– 23]. Due to the parallel invention, two different genetic systems have been established for the expression of the antibody::pIII fusion proteins. On one hand, the antibody genes can be directly integrated into the phage genome fused to the wild-type pIII gene [15]. Alternatively, antibody expression is uncoupled from phage amplification by providing the genes encoding the antibody::pIII fusion proteins on a phagemid. This system leads to more efficient antibody expression. The genes encoding the remaining structural proteins and the proteins needed for phage assembly are located on the phage genome. In addition to the antibody::gIII fusion, the phagemid carries a morphogenetic signal for encapsulation of the vector into the assembled phage particles [19]. Even though there are alternative in vitro display techniques like ribosomal display [24, 25], puromycin display [26], yeast surface display [27–29], or mammalian cell display [30–32], antibody phage display is the most commonly used selection technique for human antibodies.

Construction of Human Immune and Naive scFv Phage Display Libraries

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The choice between different types of antibody gene libraries depends on the scientific or medical applications. Immune libraries are constructed from antibody V genes isolated from IgG-secreting plasma cells of immunized donors [18, 33–36]. As immune libraries are usually enriched in in vivo affinity maturated antibody genes against certain antigens, they are usually used in medical applications, e.g., to obtain antibodies against a particular cell surface antigen of a pathogen or a tumor marker. In contrast to that, naı¨ve, semisynthetic, and synthetic libraries are designed to have a high diversity to select antibody fragments directed against nearly every possible antigen. Naive libraries are constructed from rearranged V genes from B cells (IgM) of non-immunized donors. Detailed description of antibody gene libraries and vectors needed for construction are shown in several reviews [21, 37, 38]. Various methods have been established to clone the genetic diversity of human antibody repertoires in vitro. After isolation of mRNA from peripheral blood mononuclear cells (PBMC), isolated B lymphocytes, or isolated plasma cells, the corresponding cDNA is synthesized. Immune libraries are usually constructed by a consecutive two-step cloning or by assembly PCR (see below). The heavy chain contributes more to antibody diversity, as a consequence of its highly variable CDRH3 [39]. Therefore, the amplified repertoire of light chain genes is cloned into the phage display vector first in the “two-step cloning strategy.” Subsequently, the repertoire of amplified heavy chain genes is cloned into the vectors containing the light chain gene repertoire [40–43]. Another commonly used method for the cloning of naive [44, 45] or immune [18] scFv phage display libraries is assembly PCR. Here the amplified VH and VL genes are fused directly by assembly PCR instead of consecutively cloning VH and VL genes into the vector. This approach can be combined with a randomization of the CDR3 regions and thereby introducing sequence variety, leading to semisynthetic libraries [39]. Oligonucleotide primers encoding various CDR3 and J gene segments can be used for the amplification of the V gene segments of human germlines [46]. Hoogenboom and Winter [47] as well as Nissim et al. [48] demonstrated a different approach by using degenerated CDRH3 oligonucleotide primers to create a semisynthetic heavy chain repertoire with human V gene germline origin. Subsequently, the semisynthetic heavy chain repertoire was combined with a known anti-BSA light chain. Similarly, the framework of a single well-known/robust antibody can be used as a backbone for the introduction of randomly created CDRH3 and CDRL3 [49, 50]. Amplification of all CDR regions derived from B cells and shuffling them into a robust antibody framework by assembly PCR was demonstrated by Jirholt et al. [46] and So¨derlind et al. [51]. A more recent approach for library constructions is Golden Gate cloning allowing a one-step construction of the library plasmid [52]. Full synthetic libraries were first described

18

Maximilian Ruschig et al.

by Knappik et al. [53] by using 49 framework regions, while the randomized CDR3s of the heavy and light chains were generated by trinucleotide synthesis. Rothe et al. used seven different VH and VL germline master frameworks to construct an entirely synthetic library and combined those with all six synthetically created CDR cassettes [54]. To ensure favorable antibody developability of selected antibodies, Tiller et al. [55] used 36 fixed VH and VL pairs that were selected for suitable biophysical properties as a basis to generate a fully synthetic human library. Bottleneck in construction of large naive and synthetic libraries is achieving genetic diversity due to the limited transformation efficiency of E. coli, e.g., 287 transformations were performed for the generation of the Hust/human antibody libraries (HAL) 9/10 to reach a combined size of 1.5 × 1010 independent clones [56]. The protocols described here demonstrate the generation of human naive or immune scFv antibody gene libraries by a two-step cloning strategy, which was already successfully applied previously for naive [43, 56] and immune libraries [33, 34, 36].

2

Materials

2.1 Isolation of Lymphocytes

1. Phosphate-buffered saline (PBS) pH 7.4: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4*2H2O, 0.24 g KH2PO4 in 1 L. 2. Lymphoprep™ (Serumwerk Bernburg AG). 3. mRNA Isolation Kit or TRIzol for total RNA.

2.2 Sorting of B Lymphocytes/Plasma Cells (Optional)

1. FACS buffer: PBS + 2% FCS/5% BSA + 1 mM EDTA. 2. Fluorescence-automated cell sorter. 3. Anti-human CD19 antibody, APC conjugated (MHCD1905), Thermo Fisher Scientific. 4. Anti-human CD138 antibody, FITC conjugated (352304), BioLegend. 5. mRNA Isolation Kit or TRIzol for total RNA.

2.3

cDNA Synthesis

1. Superscript IV Reverse buffer + 0.1 m DTT.

Transcriptase

+

5×

RT

2. RNAseOut Recombinant Ribonuclease Inhibitor. 3. Random hexamer oligonucleotide primer (dN6) or Oligo d (T)20 primer. 4. dNTP mix: 10 mM each. 2.4 First and Second Antibody Gene PCR

1. GoTaq2 Polymerase + 5× buffer. 2. dNTP mix: 10 mM each, 3. Oligonucleotide primer: see Table 1.

Construction of Human Immune and Naive scFv Phage Display Libraries

19

Table 1 Primers used for first and second PCR of antibody genes for antibody gene library construction using phagemids like pHAL14, pHAL30, pHAL35, pHAL51, or pHAL52. Restriction sites are underlined Primer

5′ to 3′ sequence

First antibody gene PCR VH MHVH1_f

cag gtb cag ctg gtg cag tct gg

MHVH1/7_f

car rts cag ctg gtr car tct gg

MHVH2_f

cag rtc acc ttg aag gag tct gg

JokVH3_f1

sag gtg cag ctg gtg gag tct gg

JokVH3_f2

gar gtg cag ctg ktg gag tct gg

MHVH4_f1

cag gtg car ctg cag gag tcg gg

JokVH4_f2

cag gtg cag cta car cag tgg gg

JokVH4_f3

cag ctg cag ctg cag gag tcs gg

MHVH5_f

gar gtg cag ctg gtg cag tct gg

MHVH6_f

cag gta cag ctg cag cag tca gg

MHIgMCH1_r

aag ggt tgg ggc gga tgc act

MHIgGCH1_r2

cgt tga cca ggc agc cca ggg

MHIgECH1_r

tgg gct ctg tgt gga gg

First antibody gene PCR kappa MHVK1_f1

gac atc cag atg acc cag tct cc

MHVK1_f2

gmc atc crg wtg acc cag tct cc

MHVK2_f

gat rtt gtg atg acy cag wct cc

MHVK3_f

gaa atw gtg wtg acr cag tct cc

MHVK4_f

gac atc gtg atg acc cag tct cc

MHVK5_f

gaa acg aca ctc acg cag tct cc

MHVK6_f

gaw rtt gtg mtg acw cag tct cc

MHkappaCL_r

aca ctc tcc cct gtt gaa gct ctt

First antibody gene PCR lambda MHVL1_f1

cag tct gtg ctg act cag cca cc

MHVL1_f2

cag tct gtg ytg acg cag ccg cc

MHVL2_f

cag tct gcc ctg act cag cct

MHVL3_f1

tcc tat gwg ctg acw cag cca cc

MHVL3_f2

tct tct gag ctg act cag gac cc

MHVL4_f1

ctg cct gtg ctg act cag ccc

MHVL4_f2

cag cyt gtg ctg act caa tcr yc (continued)

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Maximilian Ruschig et al.

Table 1 (continued) Primer

5′ to 3′ sequence

MHVL5_f

cag sct gtg ctg act cag cc

MHVL6_f

aat ttt atg ctg act cag ccc ca

MHVL7/8_f

cag rct gtg gtg acy cag gag cc

MHVL9/10_f

cag scw gkg ctg act cag cca cc

MHlambdaCL_r

tga aca ttc tgt agg ggc cac tg

MHlambdaCL_r2

tga aca ttc cgt agg ggc aac tg

Second antibody gene PCR VH MHVH1-NcoI_f

gtcctcgca cc atg gcc cag gtb cag ctg gtg cag tct gg

MHVH1/7-NcoI_f

gtcctcgca cc atg gcc car rts cag ctg gtr car tct gg

MHVH2-NcoI_f

gtcctcgca cc atg gcc cag rtc acc ttg aag gag tct gg

JokVH3-NcoI_f1

gtcctcgca cc atg gcc sag gtg cag ctg gtg gag tct gg

JokVH3-NcoI_f2

gtcctcgca cc atg gcc gar gtg cag ctg ktg gag tct gg

MHVH4-NcoI_f1

gtcctcgca cc atg gcc cag gtg car ctg cag gag tcg gg

JokVH4-NcoI_f2

gtcctcgca cc atg gcc cag gtg cag cta car cag tgg gg

JokVH4-NcoI_f3

gtcctcgca cc atg gcc cag ctg cag ctg cag gag tcs gg

MHVH5-NcoI_f

gtcctcgca cc atg gcc gar gtg cag ctg gtg cag tct gg

MHVH6-NcoI_f

gtcctcgca cc atg gcc cag gta cag ctg cag cag tca gg

MHIgMCH1scFv-HindIII_r

gtcctcgca aag ctt tgg ggc gga tgc act

MHIgGCH1scFv-HindIII_r

gtcctcgca aag ctt gac cga tgg gcc ctt ggt gga

MHIgECH1scFv-HindIII_r

gtcctcgca aag ctt tgg gct ctg tgt gga gg

Second antibody gene PCR kappa MHVK1-MluI_f1

accgcctcc a cgc gta gac atc cag atg acc cag tct cc

MHVK1-MluI_f2

accgcctcc a cgc gta gmc atc crg wtg acc cag tct cc

MHVK2-MluI_f

accgcctcc a cgc gta gat rtt gtg atg acy cag wct cc

MHVK3-MluI_f

accgcctcc a cgc gta gaa atw gtg wtg acr cag tct cc

MHVK4-MluI_f

accgcctcc a cgc gta gac atc gtg atg acc cag tct cc

MHVK5-MluI_f

accgcctcc a cgc gta gaa acg aca ctc acg cag tct cc

MHVK6-MluI_f

accgcctcc a cgc gta gaw rtt gtg mtg acw cag tct cc

MHkappaCLscFv-NotI_r

accgcctcc gc ggc cgc gaa gac aga tgg tgc agc cac agt

Second antibody gene PCR lambda MHVL1-MluI_f1

accgcctcc a cgc gta cag tct gtg ctg act cag cca cc (continued)

Construction of Human Immune and Naive scFv Phage Display Libraries

21

Table 1 (continued) Primer

5′ to 3′ sequence

MHVL1-MluI_f2

accgcctcc a cgc gta cag tct gtg ytg acg cag ccg cc

MHVL2-MluI_f

accgcctcc a cgc gta cag tct gcc ctg act cag cct

MHVL3-MluI_f1

accgcctcc a cgc gta tcc tat gwg ctg acw cag cca cc

MHVL3-MluI_f2

accgcctcc a cgc gta tct tct gag ctg act cag gac cc

MHVL4-MluI_f1

accgcctcc a cgc gta ctg cct gtg ctg act cag ccc

MHVL4-MluI_f2

accgcctcc a cgc gta cag cyt gtg ctg act caa tcr yc

MHVL5-MluI_f

accgcctcc a cgc gta cag sct gtg ctg act cag cc

MHVL6-MluI_f

accgcctcc a cgc gta aat ttt atg ctg act cag ccc ca

MHVL7/8-MluI_f

accgcctcc a cgc gta cag rct gtg gtg acy cag gag cc

MHVL9/10-MluI_f

accgcctcc a cgc gta cag scw gkg ctg act cag cca cc

MHLambdaCLscFv-NotI_r

accgcctcc gc ggc cgc aga gga sgg ygg gaa cag agt gac

Primer for colony PCR and sequencing MHLacZ-Pro_f

ggctcgtatgttgtgtgg

MHgIII_r

c taa agt ttt gtc gtc ttt cc

4. Agarose. 5. TAE-buffer 50×: 2 M TrisHCl, 1 M acetic acid, 0.05 M EDTA pH 8. 6. NucleoSpin Gel and PCR Clean-up. 2.5 First Cloning Step – VL

1. MluI-HF restriction enzyme (NEB). 2. NotI-HF restriction enzyme (NEB). 3. Cut Smart Buffer. 4. Calf intestine phosphatase (CIP). 5. T4 ligase (Promega). 6. Amicon Ultra Centrifugal Filters (30 K) (Millipore, Schwalbach, Germany). 7. E. coli XL1-Blue MRF‘(Agilent), genotype: Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lac IqZΔM15 Tn10 (Tetr)]. 8. Electroporator MicroPulser, 0.1 cm cuvettes. 9. 2 M glucose, sterile filtered 10. 2 M magnesium solution (autoclaved): 1 M MgCl2, 1 M MgSO4.

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11. SOC medium pH 7.0: 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05% (w/v) NaCl, 20 mM Mg solution, 20 mM glucose, sterilize magnesium and glucose separately, add solutions after autoclavation. 12. 2xYT-medium pH 7,0: 1,6% (w/v) tryptone, 1% (w/v) yeast extract, 0,5% (w/v) NaCl) 13. Ampicillin: 100 mg/mL stock. 14. 2xYT-GA: 2xYT + 100 mM glucose + 100 μg/mL ampicillin + 20 μg/mL tetracycline 15. Tetracycline: 10 mg/mL stock. 16. 9 cm Petri dishes 17. 25 cm square Petri dishes (“pizza plates”) 18. 2xYT-GA agar plates: 2xYT-GA, 1.5% (w/v) agar-agar 19. Nucleobond Extra Midi Kit. 2.6 Second Cloning Step – VH

1. NcoI-HF restriction enzyme (NEB). 2. HindIII-HF restriction enzyme (NEB). 3. AscI restriction enzyme (NEB). 4. Cut Smart Buffer. 5. E. coli ER2738 (Lucigen), genotype: [F′ proA + B+ lacIq Δ(lacZ)M15 zzf::Tn10 (tetr)] fhuA2 glnVΔ(lac-proAB) thi-1Δ(hsdS-mcrB)5. 6. Glycerol of 99.5%.

2.7

Colony PCR

2.8 Library Packaging and scFv Phage Production

1. Oligonucleotide primer: see Table 1. 1. 2xYT media pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl 2. 2xYT-GA: 2xYT, 100 mM glucose, 100 μg/mL ampicillin 3. M13K07 helper phage for monovalent display (Thermo Fisher Scientific, Waltham, USA). 4. Hyperphage for oligovalent display (Progen, Heidelberg, Germany). 5. 2xYT-AK: 2xYT + 100 μg/mL ampicillin +50 μg/mL kanamycin 6. Sorval Centrifuge RC5B Plus, rotor GS3 and SS34. 7. Polyethylenglycol (PEG) solution: 20% (w/v) PEG 6000, 2.5 M NaCl. 8. Phage dilution buffer: 10 mM TrisHCl pH 7.5, 20 mM NaCl, 2 mM EDTA.

Construction of Human Immune and Naive scFv Phage Display Libraries

23

9. Mouse α-pIII monoclonal antibody PSKAN3 (Mobitec, Go¨ttingen, Germany). 10. Goat α-mouse IgG alkaline phosphatase (AP) conjugate. 2.9

3

Phage Titration

1. 2xYT-GA agar plates: 2xYT-GA + 1.5% (w/v) agar-agar.

Methods

3.1 Isolation of Lymphocytes (Peripheral Blood Mononuclear Cells (PBMC))

1. Mix 20 mL fresh blood or EDTA/citric acid treated blood (~2 × 107 cells) of each donor with 20 mL PBS (see Note 1). 2. Fill 10 mL Lymphoprep™ in a 50 mL polypropylene tube. Carefully cover Lymphoprep™ with 40 mL of the diluted blood using a plastic pipette. 3. Centrifuge the blood with 500× g for 30 min at RT (without brake and with low acceleration!). 4. The lymphocytes form a distinct layer between the Lymphoprep™ and the medium, whereas the erythrocytes and granulocytes will be pelleted. Carefully aspirate the lymphocytes using a plastic pipette and transfer to a new 50 mL polypropylene tube. 5. Fill up with 50 mL PBS and pellet the lymphocytes with 280× g for 7 min at RT. Discard the supernatant (be careful, the lymphocyte pellet is not solid). 6. Repeat this washing step to remove most of the thrombocytes. 7. Resuspend the lymphocyte pellet in the supplied extraction buffer of the mRNA Isolation Kit according to the manufacturer’s instructions or use 0.5–1 mL TRIzol for total RNA isolation (see Note 2). After resuspension using the mRNA extraction buffer or TRIzol, the RNA can be stored at -80 °C.

3.2 FluorescenceAutomated Sorting of B Lymphocytes/ Plasma Cells

1. Isolate PBMC as described above. 2. Prepare 10 mL FACS buffer in 15 mL centrifugation tube and add PBMC fraction. 3. Centrifuge at 280× g for 7 min at RT. Discard the supernatant (be careful, the lymphocyte pellet is not solid). 4. Add 9 mL ddH2O and incubate 30 s at RT (lysis of remaining erythrocytes). 5. Add 1 mL 10× PBS and pellet cells with 280× g for 5 min at RT. 6. Repeat lysis of erythrocytes. 7. Resuspend cells in 1 mL FACS buffer. 8. Stain cells for CD19 and CD138 (1 h at 4 °C, in the dark!). 9. Wash cells with 10 mL FACS buffer twice, resuspend in 0.5–1 mL FACS buffer, and proceed with sorting.

24

3.3

Maximilian Ruschig et al.

cDNA Synthesis

1. Set up mixture for the first-strand cDNA synthesis: Solution or component

Volume

Final concentration

mRNA or total RNA

Up to 11 μL

50–250 ng (mRNA) or 2–20 μg (total RNA)

Random hexamer oligonucleotide primer (dN6) or oligo d(T)20 primer (50 μM)

1 μL

2.5 μM

dNTP-mix (10 mM each)

1 μL

500 μM

DEPC-treated or nuclease-free water To 13 μL

2. Denature the RNA for 5 min at 65 °C. Afterward chill on ice for at least 1 min. 3. Add the following components: Solution or component

Volume

Final concentration

SSIV buffer (5×)

4 μL

1×

0.1 M DTT

1 μL

10 mM

Superscript IV reverse transcriptase (200 U/μL)

1 μL

200 U

RNAseOut (200 U/μL)

1 μL

–

4. If using random hexamer, incubate the 20 μL mixture for 10 min at 23 °C for primer annealing. Afterward incubate 10 min at 50–55 °C for first strand synthesis If using Oligo d(T)20 primer directly, incubate 10 min at 50–55 °C for first-strand synthesis. 5. Inactivate the reaction by incubating for 10 min at 80 °C. Store at -20 °C or use directly for first antibody gene PCR. 3.4 First Antibody Gene PCR

1. The cDNA derived from 50–250 ng mRNA or 2–20 μg total RNA will be used as template to amplify VH and the light chain. Set up the PCR reactions as follows (30× master mix for 28 PCR reactions): Solution or component Volume Final concentration ddH2O

1130 μL –

Buffer with MgCL2 (5×)

300 μL

1×

dNTPs (10 mM each)

30 μL

200 μM each (continued)

Construction of Human Immune and Naive scFv Phage Display Libraries

25

Solution or component Volume Final concentration cDNA

20 μL

Complete first-strand synthesis reaction

GoTaq2 5 U/μL

7.5 μL

1.25 U

2. Divide the master mix in 500 μL for VH, 350 μL for kappa, and 550 μL for lambda. 3. Add to each of the three reactions the corresponding reverse primers (see also Table 1) as follows (use the IgM primer for naive antibody gene libraries or the IgG primer for immune antibody gene libraries. Also, IgE libraries are possible with the IgE primer set): Antibody gene

Final Volume concentration

Primer

VH

MHIgMCH1_r or MHIgGCH1_r2 or MHIgECH1_r (10 μM)

20 μL

0.4 μM

Kappa

MHkappaCL_r (10 μM)

14 μL

0.4 μM

Lambda

MHlambdaCL_r1/ _r2 mix ( 9:1 ) 22 μL (10 μM)

0.4 μM

4. Divide the mixture to 10 (VH), 7 (kappa) and 11 (lambda) PCR reactions each with 48 μL and add 2 μL (10 μM, 0.4 μM final concentration) of the subfamily specific forward primer (see also Table 1) VH: (1) MHVH1_f, (2) MHVH1/7_f, (3) MHVH2_f, (4) JokVH3_f1 (5) JokVH3_f2, (6) MHVH4_f1, (7) JokVH4_f2, (8) JokVH4_f3, (9) MHVH5_f, (10) MHVH6_f Vkappa: (11) MHVK1_f1, (12) MHVK1_f2, (13) MHVK2_f, (14) MHVK3_f, (15) MHVK4_f, (16) MHVK5_f, (17) MHVK6_f Vlambda: (18) MHVL1_f1, (19) MHVL1_f2, (20) MHVL2_f, (21) MHVL3_f1, (22) MHVL3_f2, (23) MHVL4_f1, (24) MHVL4_f2, (25) MHVL5_f, (26) MHVL6_f, (27) MHVL7/8_f, (28) MHVL9/10_f 5. Carry out the PCR using the following program: 95 °C

120 s

95 °C 55 °C 72 °C 72 °C

45 s 45 s 90 s 10 min

30×

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Maximilian Ruschig et al.

6. Separate PCR products by 1.5% TAE agarose gel electrophoresis, cut out the amplified antibody genes (VH: ~380 bp, kappa/ lambda: ~650 bp) (see Notes 3 and 4), and purify the PCR products using a gel extraction kit according to the manufacturer’s instructions. Pool all VH, kappa, and lambda subfamilies separately. Determine the DNA concentration. Store the three purified first PCR pools at -20 °C. 3.5 Second Antibody Gene PCR

1. In the second PCR, the restriction sites for library cloning will be added. Set up the PCR reactions as follows (30× master mix for 28 PCR reactions) (see Note 5): Solution or component

Volume

Final concentration

ddH2O

2200 μL

–

Buffer with MgCL2 (5×)

600 μL

1×

dNTPs (10 mM each)

60 μL

200 μM each

GoTaq 5 U/μL

15 μL

2.5 U

2. Divide the master mix in 1000 μL for VH, 700 μL for kappa, and 1100 μL for lambda. 3. Add to each of the three reactions the corresponding reverse primers (see also Table 1) as follows: Antibody gene Primer

Final Volume concentration

VH

MHIgMCH1scFv-HindIII_r or MHIgGCH1scFv-HindIII_r (10 μM)

20 μL

0.2 μM

Kappa

MHKappaCLscFv-NotI_r2 (10 μM)

14 μL

0.2 μM

Lambda

MHLambdaCLscFv-NotI_r (10 μM)

22 μL

0.2 μM

4. Add the corresponding PCR products of the first PCR as follows: VH

1000 ng

Kappa

700 ng

Lambda

1100 ng

5. Divide the solutions to 10 (VH), 7 (Kappa) and 11 (Lambda) PCR reactions, each with 98 μL and add 2 μL (10 μM, 0.2 μM final concentration) the subfamily specific forward primer (see also Table 1)

Construction of Human Immune and Naive scFv Phage Display Libraries

VH:

Vkappa:

Vlambda:

27

(1) MHVH1-NcoI_f, (2) MHVH2-NcoI_f, (3) MHVH1/7-NcoI_f, (4) JokVH3-NcoI_f1, (5) JokVH3-NcoI_f2, (6) MHVH4-NcoI_f1, (7) JokVH4NcoI_f2, (8) JokVH4-NcoI_f3, (9) MHVH5-NcoI_f, (10) MHVH6-NcoI_f (11) MHVK1-MluI_f1, (12) MHVK1-MluI_f2, (13) MHVK2-MluI_f, (14) MHVK3-MluI_f, (15) MHVK4MluI_f, (16) MHVK5-MluI_f, (17) MHVK6-MluI_f (18) MHVL1-MluI_f1, (19) MHVL1-MluI_f2, (20) MHVL2-MluI_f, (21) MHVL3-MluI_f1, (22) MHVL3-MluI_f2, (23) MHVL4-MluI_f1, (24) MHVL4-MluI_f2, (25) MHVL5-MluI_f, (26) MHVL6-MluI_f, (27) MHVL7/8-MluI_f, (28) MHVL9/10-MluI_f

6. Carry out the PCR using the following program: 95 °C

60 s

95 °C 57 °C 72 °C

45 s 45 s 60 s

72 °C

10 min

20×

7. Separate the PCR products by 1.5% TAE agarose gel electrophoresis, cut out the amplified antibody genes (VH: ~450 bp, kappa/lambda: ~450 bp), and purify the PCR products using a gel extraction kit according to the manufacturer’s instructions. Pool all VH, kappa, and lambda subfamilies separately. Determine the DNA concentration. Store the three purified second PCR pools at -20 °C. 3.6 First Cloning Step – VL

1. Prepare a plasmid preparation of pHAL52 vector for library cloning (see Note 6). 2. Digest the vector and the VL PCR products. Always perform additional single-enzyme digestions of the vector in parallel to check whether the digestion is complete (see also Note 7): Solution or component

Volume

Final concentration

ddH2O

87-x μL

–

pHAL52 or VL

x μL

5 μg or 2 μg

NEB cut smart buffer (10×)

10 μL

1×

NEB MluI-HF (20 U/μL)

1.5 μL

30 U

NEB NotI-HF (20 U/μL)

1.5 μL

30 U

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Maximilian Ruschig et al.

3. Incubate at 37 °C for 2 h. Control the digestion of the vector by using a 5 μL aliquot on 1.5% TAE agarose gel electrophoresis. If the vector is not fully digested, extend the incubation time. 4. Inactivate the enzymes at 65 °C for 10 min. 5. Add 0.5 μL CIP (1 U/μL) to the vector digest and incubate at 37 °C for 30 min. Repeat this step once. 6. Purify the vector and the PCR product using a PCR Purification Kit according to the manufacturer’s instructions and elute with 50 μL elution buffer or water. The short stuffer fragment containing multiple stop codons between MluI and NotI in pHAL52 will be removed. Determine the DNA concentration. 7. Ligate the vector pHAL52 (4258 bp) and VL (~380 bp) as follows (see Note 4): Solution or component

Volume

Final concentration

ddH2O

89-x-y μL

–

pHAL52

x μL

1000 ng

VL (kappa or lambda)

y μL

270 ng

T4 ligase buffer (10×)

10 μL

1×

T4 ligase (3 U/μL)

1 μL

3U

8. Incubate at 16 °C overnight. 9. Inactivate the ligation at 70 °C for 10 min. 10. Purify the ligation using an Amicon Ultra column. Add the ligation to the column and add water to 500 μL. Centrifuge at 10 min at 14,000× g. Discard the flow through and repeat the washing with 470 μL (about 30 μL will remain in the column) step three times. 11. For elution invert the column and elute the remaining DNA solution in a new cap for 3 min at 1000× g. 12. Add water to 35 μL. 13. Thaw 25 μL electrocompetent E. coli XL1-Blue MRF’ on ice and mix with the ligation reaction. 14. Transfer the 60 μL mix to a prechilled 0.1 cm cuvette. Dry the electrode of the cuvette with a tissue paper. 15. Perform a 1.7 kV pulse using an electroporator (see Note 8). Immediately, add 1 mL 37 °C prewarmed SOC medium carefully, transfer the suspension to a 2 mL cap, and shake for 1 h at 600 rpm and 37 °C.

Construction of Human Immune and Naive scFv Phage Display Libraries

29

16. To determine the amount of transformants, use 10 μL (=102 dilution) of the transformation and perform a dilution series down to 10-6 dilution. Plate out a 10-6 dilution on 2xYT-GA agar plates and incubate overnight at 37 °C. 17. Plate out the remaining 990 μL on 2xYT-GA agar “pizza plate” and incubate overnight at 37 °C. 18. Calculate the amount of transformants which should be around 1 × 106–2 × 108 cfu/mL. Control colonies for full size insert by colony PCR (see Subheading 3.8). 19. Scrape off the colonies on the “pizza plate” with 40 mL 2xYT medium using a Drigalski spatula. Pellet bacterial suspension, discard the medium, and use the whole pellet for midi plasmid preparation according to the manufacturer’s instructions. Determine the DNA concentration. 3.7 Second Cloning Step – VH

1. Digest the pHAL52-VL repertoire and the VH PCR products. Always perform additional single-enzyme digestions of the vector in parallel (see also Note 7): Solution or component

Volume

Final concentration

ddH2O

82-x μL

–

pHAL52-VL or VH

x μL

5 μg or 2 μg

NEB cut smart buffer (10×)

10 μL

1×

NEB NcoI-HF (20 U/μL)

1.5 μL

30 U

NEB HindIII-HF (20 U/μL)

1.5 μL

30 U

2. Incubate at 37 °C for 2 h (see Note 9). Control the digest of the vector by using a 5 μL aliquot on 1.5% agarose gel electrophoresis. 3. Inactivate the digestion at 80 °C for 20 min. 4. Add 0.5 μL CIP (1 U/μL) to the vector digest and incubate at 37 °C for 30 min. Repeat this step once. 5. Purify the vector and the PCR product using a PCR Purification Kit according to the manufacturer’s instructions and elute with 50 μL elution buffer or water. The short stuffer fragment between NcoI and HindIII in pHAL52 will be removed. Determine the DNA concentration. (See also Note 10). 6. Ligate the vector pHAL52-VL (~4640 bp) and VH (~380 bp) as follows (see Note 4):

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Maximilian Ruschig et al.

Solution or component

Volume

Final concentration

ddH2O

89-x-y μL

–

pHAL30

x μL

1000 ng

VH

y μL

250 ng

T4 ligase buffer (10×)

10 μL

1×

T4 ligase (3 U/μL)

1 μL

3U

7. Incubate at 16 °C overnight. 8. Inactivate the ligation at 65 °C for 10 min. 9. Purify the ligation using an Amicon Ultra column. Add the ligation to the column and add water to 500 μL. Centrifuge at 10 min at 14,000× g. Discard the flow through and repeat the washing with 470 μL (about 30 μL will remain in the column) step three times. 10. For elution invert the column and elute the remaining DNA solution in a new cap for 3 min at 1000× g. 11. Add water to 35 μL. 12. Thaw 25 μL electrocompetent E. coli ER2738 on ice and mix with the ligation reaction (see Note 11). 13. Transfer the 60 μL mix to a prechilled 0.1 cm cuvette. Dry the electrode of the cuvette with a tissue paper. 14. Perform a 1.8 kV pulse using an electroporator (see Note 8). Immediately, add 1 mL 37 °C prewarmed SOC medium (Lucigen) carefully, transfer to a 2 mL cap, and incubate for 1 h at 37 °C with 600 rpm. 15. To determine the amount of transformants, use 10 μL (=102 dilution) of the transformation and perform a dilution series down to 10-6 dilution. Plate out a 10-6 dilution on 2xYTGA agar plates and incubate overnight at 37 °C. 16. Plate out the remaining 990 μL on 2xYT-GA agar “pizza plate” and incubate overnight at 37 °C. 17. Calculate the amount of transformants (1 × 107–2 × 108 should be reached to be included into the final library). Control colonies for full size insert by colony PCR (see Subheading 3.8.). 18. Scrape off the colonies on the “pizza plate” with 25 mL 2xYT medium using a Drigalski spatula (~OD 20–25 = ~2 × 1010 cells/mL). Use 800 μL bacteria solution (~1 × 1010 bacteria) and 200 μL glycerol for glycerol stocks. Make 5–20 glycerol stocks per sublibrary, freeze in liquid nitrogen, and store at 80 °C.

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31

19. When all transformations are done, thaw one aliquot of each sublibrary on ice, mix all sublibraries, and make new aliquots for storage at -80 °C (see also Note 12). 3.8

Colony PCR

1. Choose 10–20 single colonies per transformation. Set up the 10 μL PCR reaction per colony as follows (see Table 1 for primer sequences): Solution or component

Volume

Final concentration

ddH2O

7.5 μL

GoTaq buffer (5×)

2 μL

1×

dNTPs (10 mM each)

0,2 μL

200 μM each

MHLacZPro_f 10 μM

0,1 μL

0,1 μM

MHgIII_r 10 μM

0,1 μL

0,1 μM

GoTag2 (5 U/ μL)

0,1 μL

0.5 U

Template

Picked colonies from dilution plate

2. Control the PCR by 1.5% TAE agarose gel electrophoresis. 3. The PCR products should be ~1100 bp when including VH and VL, ~750 bp when including only VL or VH, and 375 bp if the vector contains no insert. Each used sublibrary should have more than 80% full size inserts to be included into the final library. 3.9 Library Packaging and scFv Phage Production

1. To package the library, inoculate 400 mL 2xYT-GA in a 1 L Erlenmeyer flask with 1 mL antibody gene library stock. Grow at 250 rpm at 37 °C up to an O.D.600 nm ~ 0.5. 2. Infect 25 mL bacteria culture (~1.25*1010 cells) with 2.5*1011 colony-forming units (cfu) of the helper phage M13K07 or hyperphage according to a multiplicity of infection (moi) = 1: 20 (see Note 13). Incubate 30 min without shaking and the following 30 min with 250 rpm at 37 °C. 3. To remove the glucose that represses the lac promoter of pHAL52 and therefore the scFv::pIII fusion protein expression, harvest the cells by centrifugation for 10 min at 3200 xg in 50 mL polypropylene tubes. 4. Resuspend the pellet in 400 mL 2xYT-AK in a 1 L Erlenmeyer flask. Produce scFv-phage overnight at 250 rpm and 30 °C. 5. Pellet the bacteria by centrifugation for 10 min at 10,000× g in two GS3 centrifuge tubes. If the supernatant is not clear, centrifuge again to remove remaining bacteria.

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6. Precipitate the phage from the supernatant by adding 1/5 volume PEG solution in two GS3 tubes. Incubate for 1 h at 4 °C with gentle shaking, followed by centrifugation for 1 h at 10,000× g. 7. Discard the supernatant, resolve each pellet in 10 mL phage dilution buffer in SS34 centrifuge tubes, and add 1/5 volume PEG solution. 8. Incubate on ice for 20 min and pellet the phage by centrifugation for 30 min at 10,000× g. 9. Discard the supernatant and put the open tubes upside down on tissue paper. Let the viscous PEG solution move out completely. Resuspend the phage pellet in 1 mL phage dilution buffer. Titrate the phage preparation (see Subheading 3.9). Store the packaged antibody phage library at 4 °C. 10. The library packaging should be controlled by 10% SDS-PAGE, Western blot, and anti-pIII immunostain (mouse anti-pIII 1:2000 , goat anti-mouse IgG AP conjugate 1: 10000 ). Wild-type pIII has a calculated molecular mass of 42.5 kDa, but it runs at an apparent molecular mass of 65 kDa in SDS-PAGE. Accordingly, the scFv::pIII fusion protein runs at about 95 kDa. 3.10

Phage Titration

1. Inoculate 5 mL 2xYT-T in a 100 mL Erlenmeyer flask with E. coli XL1-Blue MRF’ and grow overnight at 37 °C and 250 rpm. 2. Inoculate 50 mL 2xYT-T with 500 μL overnight culture and grow at 250 rpm at 37 °C up to O.D.600 ~ 0.5 (see Note 14). 3. Make serial dilutions of the phage suspension in PBS. The package library phage preparation should have a titer of about 1011–1013 phage/mL. 4. Infect 50 μL bacteria with 10 μL phage dilution and incubate 30 min at 37 °C. 5. You can perform titrations in two different ways: (a) Plate the 60 μL infected bacteria on 2xYT-GA agar plates (9 cm Petri dishes). (b) Pipette 10 μL (in triplicate) on 2xYT-GA agar plates. Here, about 20 titrating spots can be placed on 1 9 cm Petri dish. Dry drops on workbench. 6. Incubate the plates overnight at 37 °C. 7. Count the colonies and calculate the cfu or cfu/mL titer according to the dilution.

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4

33

Notes 1. Be careful with human blood samples since they are potentially infectious (HIV, hepatitis, etc.). 2. Both methods, mRNA and total RNA isolation, work well. 3. The VH amplifications of VH subfamilies sometimes result also in longer PCR products. Cut out only the ~380 bp fragment. The amplifications of kappa subfamilies should always give a clear ~650 bp fragment (complete light chain). When amplifying lambda subfamilies, often other PCR products are generated; especially the amplification of the lambda2 subfamily results often in slushy bands. If some subfamilies are bad amplified and no clear ~650 bp fragment is detectable, use only the ~650 bp fragments from the well-amplified subfamilies. Additional comment: Since the first PCR amplifies the full LC, it can be used also to construct Fab or scFab [57] libraries from this material. 4. Even if after first gene amplification there is no clear PCR product visible in gel electrophoresis, it is possible to proceed to second gene amplification and cut the gel on expected size of PCR product. This can be the case especially when working with sorted B lymphocytes or plasma cells for library construction, as the total cell number will be most likely low. 5. For a very large naive antibody gene library perform as many PCRs as sufficient to perform 20 light chain ligations/transformations and about 100 VH ligations. For an immune library four light chain ligations/transformations and eight VH ligations are usually sufficient. Prepare and digest also adequate amounts of pHAL52 and VL for the first cloning step and pHAL52-VL library and VH for the second cloning step. To increase cloning efficiency, pHAL52 can be digested and purified twice before cloning of VL. Keep kappa and lambda libraries in all steps (cloning, packaging) separately and mix only after phage production before panning. 6. The vector pHAL52 is a modified version of pHAL51 [33, 56]. In pHAL52 there is a SalI-HF restriction site introduced in the stuffer additional to the AscI site introduced in pHAL51, in order to remove uncut vector backbone [34]. 7. Always perform single digests using only one enzyme in parallel, to control the success of the restriction reaction. Analyze the digestion by TAE agarose gel electophoresis by comparing with the undigested plasmid. Use only material where both single digests are successful and where no degradation is visible in the double digest.

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8. The pulse time should be between 4 and 5 ms for optimal electroporation efficiency. 9. Often the HindIII digestion is incomplete after 2 h. Proceed with inactivating the enzymes by heating up to 65 °C for 10 min, add additional 5 μL of HindIII, and incubate overnight. You can use also higher concentrated HindIII. Alternative: Perform the NcoI digest first for 2 h, inactive the digest, and afterward perform the HindIII digest. This problem only occurs when HindIII is used and not if HindIII-HF. 10. Keep aliquots of the light chain repertoire as plasmid but also – more convenient – the NheI/NotI digested VL chains for future light chains shuffling for affinity and/or stability maturation. For affinity maturation, use the VH and clone it into an empty pHAL52 vector, and subsequently clone the new light chain repertoire to combine it with the selected. This light chain shuffling library can be used for the panning under harsher conditions, competition, etc. to select improved antibodies [58]. 11. The E. coli ER2738 cells have a higher transformation efficiency compared to E. coli XL1-Blue MRF’. These ER2738 cells are used only for the second VH cloning step, because the quality of isolated plasmids from these cells is lower compared to XL1-Blue MRF’. The XL1-Blue MRF’ cells are used for the first cloning VL cloning step to get high-quality plasmids for the second digestion and VH cloning step. In the VL cloning step, the library size can be lower (1 × 107–1 × 108) because this repertoire will be combined with the VH repertoire in the second cloning step. 12. To minimize loss of diversity, avoid too many freeze and thaw steps, e.g., when constructing an immune library, make eight transformations in parallel and directly package the immune library. When making a big immune library, combine only a glycerin stock of each sublibrary, which corresponds to max. 2 × 109 independent clones to ensure that the library diversity can be kept when packaging 1 mL of mixed library glycerin stock. When the library size is bigger than 2 × 109 independent clones, do not package the library as complete library; package “blocks” of sublibraries. Combine the phage particles of each “block” before panning to get the final complete library. 13. The use of hyperphage as helper phage instead of M13K07 offers oligovalent phage display and facilitates the selection of specific binders in the first and most critical panning round by avidity effect [59–62]. The hyperphage should be only used for library packaging. For the following panning rounds, use M13K07 to enhance the stringency of the panning process.

Construction of Human Immune and Naive scFv Phage Display Libraries

35

14. If the bacteria have reached O.D.600 ~ 0.5 before they are needed, you can store the culture immediately on ice to maintain the F pili on the E. coli cells for several hours. M13K07 helper phage (kan+) or other scFv-phage (amp+) can be used as positive control to check the infectibility of the E. coli cells.

Acknowledgments This review is an updated and revised version of Frenzel et al. (2014) [63] and Ku¨gler et al. (2018) [64]. References 1. The Antibody Society (2023) Antibody therapeutics approved or in regulatory review in the EU or US – The Antibody Society. https:// w w w. a n t i b o d y s o c i e t y. o r g / r e s o u r c e s / approved-antibodies/. Accessed 20 Jan 2023 2. Lu R-M, Hwang Y-C, Liu I-J et al (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27:1 3. Global Market Insights Inc. (2023) Antibody therapy market share | Global statistics – 2028. https://www.gminsights.com/industry-analy sis/antibody-therapy-market. Accessed 20 Jan 2023 4. Du¨bel S (2007) Recombinant therapeutic antibodies. Appl Microbiol Biotechnol 74:723– 729 5. Kaplon H, Chenoweth A, Crescioli S et al (2022) Antibodies to watch in 2022. MAbs 14:2014296 6. Chatenoud L, Bluestone JA (2007) CD3-specific antibodies: a portal to the treatment of autoimmunity. Nat Rev Immunol 7: 622–632 7. Harriman G, Harper LK, Schaible TF (1999) Summary of clinical trials in rheumatoid arthritis using infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58(Suppl 1):I61–I64 8. Dalle S, Thieblemont C, Thomas L et al (2008) Monoclonal antibodies in clinical oncology. Anti Cancer Agents Med Chem 8:523–532 9. Jones SE (2008) Metastatic breast cancer: the treatment challenge. Clin Breast Cancer 8: 224–233 10. Osbourn J, Groves M, Vaughan T (2005) From rodent reagents to human therapeutics using antibody guided selection. Methods 36: 61–68 11. Harding FA, Stickler MM, Razo J et al (2010) The immunogenicity of humanized and fully

human antibodies: residual immunogenicity resides in the CDR regions. MAbs 2:256–265 12. Getts DR, Getts MT, McCarthy DP et al (2010) Have we overestimated the benefit of human(ized) antibodies. mAbs 2:682–694 13. Alfaleh MA, Alsaab HO, Mahmoud AB et al (2020) Phage display derived monoclonal antibodies: from bench to bedside. Front Immunol 11:1986 14. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317 15. McCafferty J, Griffiths AD, Winter G et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554 16. Marks JD, Hoogenboom HR, Bonnert TP et al (1991) By-passing immunization. J Mol Biol 222:581–597 17. Hoogenboom HR, Griffiths AD, Johnson KS et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19:4133–4137 18. Clackson T, Hoogenboom HR, Griffiths AD et al (1991) Making antibody fragments using phage display libraries. Nature 352:624–628 19. Breitling F, Du¨bel S, Seehaus T et al (1991) A surface expression vector for antibody screening. Gene 104:147–153 20. Barbas CF, Kang AS, Lerner RA et al (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88:7978–7982 21. Hust M, Du¨bel S (2005) Phage display vectors for the in vitro generation of human antibody fragments. Methods Mol Biol 295:71–96

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22. Holt LJ, Herring C, Jespers LS et al (2003) Domain antibodies: proteins for therapy. Trends Biotechnol 21:484–490 23. Hoet RM, Cohen EH, Kent RB et al (2005) Generation of high-affinity human antibodies by combining donor derived and synthetic complementarity-determining-region diversity. Nat Biotechnol 23:344–348 24. He M, Taussig MJ (1997) Antibody-ribosomemRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res 25:5132–5134 25. Hanes J, Plu¨ckthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A 94: 4937–4942 26. Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94:12297– 12302 27. Teymennet-Ramı´rez KV, Martı´nez-Morales F, Trejo-Herna´ndez MR (2021) Yeast surface display system: strategies for improvement and biotechnological applications. Front Bioeng Biotechnol 9:794742 28. Zhang C, Chen H, Zhu Y et al (2022) Saccharomyces cerevisiae cell surface display technology: strategies for improvement and applications. Front Bioeng Biotechnol 10: 1056804 29. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557 30. Jin Y-J, Yu D, Tian X-L et al (2022) A novel and effective approach to generate germlinelike monoclonal antibodies by integration of phage and mammalian cell display platforms. Acta Pharmacol Sin 43:954–962 31. Luo R, Qu B, An L et al (2022) Simultaneous maturation of single chain antibody stability and affinity by CHO cell display. Bioengineering (Basel, Switzerland) 9 32. King DJ, Bowers PM, Kehry MR et al (2014) Mammalian cell display and somatic hypermutation in vitro for human antibody discovery. Curr Drug Discov Technol 11:56–64 33. Wenzel EV, Bosnak M, Tierney R et al (2020) Human antibodies neutralizing diphtheria toxin in vitro and in vivo. Sci Rep 10:571 34. Bertoglio F, Fu¨hner V, Ruschig M et al (2021) A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2RBD interface and is tolerant to most known RBD mutations. Cell Rep 36:109433 35. Arakawa M, Yamashiro T, Uechi G et al (2007) Construction of human Fab (gamma1/kappa)

library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus. Microbiol Immunol 51:617–625 36. Trott M, Weiβ S, Antoni S et al (2014) Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody. PLoS One 9: e97478 37. Qi H, Lu H, Qiu H-J et al (2012) Phagemid vectors for phage display: properties, characteristics and construction. J Mol Biol 417:129– 143 38. Hust M, Du¨bel S (2004) Mating antibody phage display with proteomics. Trends Biotechnol 22:8–14 39. Shirai H, Kidera A, Nakamura H (1999) H3-rules: identification of CDR-H3 structures in antibodies. FEBS Lett 455:188–197 40. Welschof M, Terness P, Kipriyanov SM et al (1997) The antigen-binding domain of a human IgG-anti-F(ab’)2 autoantibody. Proc Natl Acad Sci U S A 94:1902–1907 41. Little M, Welschof M, Braunagel M et al (1999) Generation of a large complex antibody library from multiple donors. J Immunol Methods 231:3–9 42. Johansen LK, Albrechtsen B, Andersen HW et al (1995) pFab60: a new, efficient vector for expression of antibody Fab fragments displayed on phage. Protein Eng 8:1063–1067 43. Hust M, Meyer T, Voedisch B et al (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152:159–170 44. Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314 45. McCafferty J, Fitzgerald KJ, Earnshaw J et al (1994) Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display. Appl Biochem Biotechnol 47:157–171. discussion 171–3 46. Akamatsu Y, Cole MS, Tso JY, Tsurushita N (1993) Construction of a human Ig combinatorial library from genomic V segments and synthetic CDR3 fragments. J Immunol 151: 4651–4659 47. Hoogenboom HR, Winter G (1992) By-passing immunisation. J Mol Biol 227: 381–388 48. Nissim A, Hoogenboom HR, Tomlinson IM et al (1994) Antibody fragments from a ‘single

Construction of Human Immune and Naive scFv Phage Display Libraries pot’ phage display library as immunochemical reagents. EMBO J 13:692–698 49. Desiderio A, Franconi R, Lopez M et al (2001) A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a singleframework scaffold. J Mol Biol 310:603–615 50. Barbas CF, Bain JD, Hoekstra DM et al (1992) Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc Natl Acad Sci U S A 89:4457–4461 51. So¨derlind E, Strandberg L, Jirholt P et al (2000) Recombining germline-derived CDR sequences for creating diverse singleframework antibody libraries. Nat Biotechnol 18:852–856 52. Sellmann C, Pekar L, Bauer C et al (2020) A one-step process for the construction of phage display scFv and VHH libraries. Mol Biotechnol 62:228–239 53. Knappik A, Ge L, Honegger A et al (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57–86 54. Rothe C, Urlinger S, Lo¨hning C et al (2008) The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J Mol Biol 376:1182–1200 55. Tiller T, Schuster I, Deppe D et al (2013) A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. MAbs 5: 445–470

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56. Ku¨gler J, Wilke S, Meier D et al (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol 15:10 57. Hust M, Jostock T, Menzel C et al (2007) Single chain Fab (scFab) fragment. BMC Biotechnol 7:14 58. Steinwand M, Droste P, Frenzel A et al (2014) The influence of antibody fragment format on phage display based affinity maturation of IgG. MAbs 6:204–218 59. Kirsch MI, Hu¨lseweh B, Nacke C et al (2008) Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV). BMC Biotechnol 8:66 60. Thibaut Pelat M, Hust EL et al (2007) Highaffinity, human antibody-like antibody fragment (single-chain variable fragment) neutralizing the lethal factor (LF) of bacillus anthracis by inhibiting protective antigen-LF complex formation. Antimicrob Agents Chemother 61. Soltes G, Hust M, Ng KKY et al (2007) On the influence of vector design on antibody phage display. J Biotechnol 127:626–637 62. Rondot S, Koch J, Breitling F et al (2001) A helper phage to improve single-chain antibody presentation in phage display. Nat Biotechnol 19:75–78 63. Frenzel A, Ku¨gler J, Wilke S et al (2014) Construction of human antibody gene libraries and selection of antibodies by phage display. Methods Mol Biol 1060:215–243 64. Ku¨gler J, Tomszak F, Frenzel A et al (2018) Construction of human immune and naive scFv libraries. In: Phage display. Humana Press, New York, pp 3–24

Chapter 3 Construction of Naı¨ve and Immune Human Fab Phage Display Library Jing Yi Lai and Theam Soon Lim Abstract Phage display has been applied successfully for the rapid isolation of monoclonal antibodies against various targets including infectious diseases, autoantigens, cancer markers, and even small molecules. The main component in any phage display experiment is the availability of an antibody library to carry out the selection process of target-specific antibodies through an iterative process termed as biopanning. To generate human antibody libraries, the antibody repertoire can be obtained from human peripheral blood mononuclear cell (PBMC) or directly from cell-sorted B-cell populations. The choice of antibody isotype is dictated by the nature of the library. Naı¨ve libraries would utilize IgM repertoires, whereas the IgG repertoire is commonly used for immune libraries. Antibody genes are amplified through polymerase chain reaction (PCR) and paired in a combinatorial fashion to expand the diversity of the cloned library repertoire. The protocol here describes the use of a two-step cloning method that can be applied for the construction of either a naı¨ve or immune human antibody library in Fab format followed by the subsequent panning. Key words Naı¨ve antibody repertoires, Immune antibody repertoires, Antibody libraries, Combinatorial, Fab, Human, Phage display, Phagemid, Monoclonal antibodies

1

Introduction The introduction of phage display by George Smith in 1985 has led to a major evolution in monoclonal antibody (mAb) discovery [1]. The technique utilizes a unique feature that allows for a direct linkage to be established between the phenotype and genotype. The design encompasses the fusion of the gene of interest (antibody gene) to the gene of the phage coat protein. This results in a fusion protein that is anchored on the surface of the bacteriophage upon packaging [2, 3]. Smith’s initial study showed the successful display of peptides on bacteriophage [1]. Since its inception, phage display has progressed to include the display of other complex biomolecules such as enzymes [4, 5], scaffolds [6, 7], receptors [8], and antibodies [9–12], highlighting the robust and versatile nature of the method.

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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The main component required for antibody phage display campaigns is the availability of a high-quality antibody library. There are several types of antibody libraries, classified based on the source of the library repertoire. A naı¨ve library is generated from IgM repertoire of healthy individuals at the point of collection [13]. Theoretically the naı¨ve library repertoire is unbiased and diverse, but the influence of immune memory associated to IgM has to be considered [14]. A truly unbiased repertoire might not be possible as individuals may have experienced different infections throughout their lifetime and would also likely have received standard vaccinations throughout the course of their lives. Nonetheless, naı¨ve libraries have been shown to be competent in generating mAbs against different targets including disease-specific antigens with good affinities [15, 16]. On the contrary, an immune library is less diverse but is specific to a particular disease. Immune repertoires are collected from infected individuals, typically of IgG isotype in response to a foreign invasion resulting in a skewed preference. The mAbs generated from immune libraries are usually of higher affinity compared to mAbs derived from naı¨ve libraries due to affinity maturation of the repertoire [17, 18]. Phage display technology takes advantage of the bacterial system for protein expression and phage packaging. However, a fullsize IgG antibody is too big for it to be tolerated by the expression mechanism to allow efficient display on the surface of M13 bacteriophage. Consequently, antibodies in smaller recombinant formats such as single-domain antibody (sdAb), single-chain fragment variable (scFv), and fragment antigen-binding (Fab) were introduced to overcome the size restriction of phage packaging mechanisms [19]. sdAb is the smallest human antibody format with only a heavy chain (VH) or light chain (VL) variable domain although animal derived domain antibodies are also available [20]. scFv contains both the variable domains linked together by a short flexible linker (VH-VL) [15]. Fab includes the variable and constant domains of heavy chain (VH, CH1) and light chain (VL, CL), whereby the heavy chain and light chain are linked together by a disulfide bond [21]. The size of the Fab is relatively bigger and poses more of a challenge for phage presentation than scFv. However, the Fab antibody is advantageous in terms of their stability and easy reformatting to IgG molecules for downstream application [22, 23]. Another important aspect in antibody library construction is the cloning approach. mRNAs isolated from B lymphocytes are first reverse transcribed to cDNA through RT-PCR. The antibody repertoires are then amplified from the cDNA and cloned into a phagemid using different approaches such as PCR assembly [24], TOPO intermediate cloning [25], isothermal rolling circle amplification [26, 27], and Golden Gate cloning [28]. The conventional strategy is a two-step cloning method in which the antibody light chain is inserted into the phagemid to generate an intermediate

Construction of Naı¨ve and Immune Human Fab Phage Display Library

41

mini-library prior to the insertion of the heavy chain to generate the complete library repertoire [29, 30]. The cloning process plays a pivotal role to ensure the library repertoire is highly diverse while reducing sequence redundancy and cloning bias. The general consideration of a naı¨ve library size is the correlation of the estimated antibody affinities that are directly proportional to the library size [31]. In this protocol, we describe the use of a two-step cloning method to construct a diverse human Fab antibody phage display library. The protocol is suitable for the construction of both naı¨ve and immune libraries as the difference would depend on the choice of isotype-specific primers, which ultimately determines the repertoire being amplified. This protocol also describes the panning process for the generation of Fab monoclonal antibodies.

2

Materials

2.1 Isolation of B Cells

1. Ficoll-Paque™ PLUS (Cytiva, USA). 2. Phosphate-buffered saline (PBS), pH 7.4: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4·2H2O, 0.24 g KH2PO4 in 1 L dH2O, autoclave and store at room temperature. 3. QIAamp® RNA Blood Mini Kit (QIAGEN, Germany).

2.2 First-Strand cDNA Synthesis

1. 300–500 ng RNA

2.3 Amplification of HC and LC Fab Gene Repertoire

1. Vent® DNA polymerase (NEB, USA) (see Note 1).

2. SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, USA)

2. Pfu DNA polymerase (Thermo Scientific, USA) (see Note 1). 3. Forward and reverse primers for first amplification of HC and LC (see Table 1). 4. Forward and reverse primers for second amplification of HC and LC with restriction endonuclease sites (see Table 2). 5. 10 mM dNTP mixture. 6. Agarose. 7. 10× TBE buffer, pH 8.0: 108 g Tris, 55 g boric acid, and 7.4 g EDTA in 1 L dH2O, autoclave and store at room temperature. 8. QIAquick® Gel Extraction Kit (QIAGEN, Germany).

2.4 Two-Step Cloning

1. Antarctic phosphatase. 2. T4 DNA ligase. 3. 3 M sodium acetate, pH 5.2: sodium acetate in 800 mL dH2O, adjust pH to 5.2 with glacial acetic acid, top up to 1 L with dH2O, and store at room temperature.

42

Jing Yi Lai and Theam Soon Lim

Table 1 Primers for first amplification of Fab HC and LC gene repertoire Primer name

Primer sequence

Fab HC amplification VH1 Fw

5′ – CAG GTC CAG CTK GTR CAG TCT GG – 3′

VH157 Fw

5′ – CAG GTG CAG CTG GTG SAR TCT GG – 3′

VH2 Fw

5′ – CAG RTC ACC TTG AAG GAG TCT G – 3′

VH3 Fw

5′ – GAG GTG CAG CTG KTG GAG WCY – 3′

VH4 Fw

5′ – CAG GTG CAG CTG CAG GAG TCS G – 3′

VH4 DP63 Fw

5′ – CAG GTG CAG CTA CAG CAG TGG G – 3′

VH6 Fw

5′ – CAG GTA CAG CTG CAG CAG TCA – 3′

Human Fab IgM CH1 Rv

5′ – TGG AAG AGG CAC GTT CTT TTC TTT – 3′

Human Fab IgG CH1 Rv

5′ – TCT TGT CCA CCT TGG TGT TG – 3′

Fab LC amplification Vλ1 Fw

5′ – CAG TCT GTS BTG ACG CAG CCG CC – 3′

Vλ1459 Fw

5′ – CAG CCT GTG CTG ACT CAR YC – 3′

Vλ15910 Fw

5′ – CAG CCW GKG CTG ACT CAG CCM CC – 3′

Vλ2 Fw

5′ – CAG TCT GYY CTG AYT CAG CCT – 3′

Vλ3 Fw

5′ – TCC TAT GWG CTG ACW CAG CCA A – 3′

Vλ3 DPL16 Fw

5′ – TCC TCT GAG CTG AST CAG GAS CC – 3′

Vλ338 Fw

5′ – TCC TAT GAG CTG AYR CAG CYA CC – 3′

Vλ6 Fw

5′ – AAT TTT ATG CTG ACT CAG CCC C – 3′

Vκ1 Fw

5′ – GAC ATC CRG DTG ACC CAG TCT CC – 3′

Vκ246 Fw

5′ – GAT ATT GTG MTG ACB CAG WCT CC – 3′

Vκ3 Fw

5′ – GAA ATT GTR WTG ACR CAG TCT CC – 3′

Vκ5 Fw

5′ – GAA ACG ACA CTC ACG CAG TCT C – 3′

Fab Lambda CL1 Rv

5′ – TGA ACA TTC TGT AGG GGC CAC TG – 3′

Fab Lambda CL2 Rv

5′ – TGA ACA TTC CGT AGG GGC AAC TG – 3′

Fab Kappa CL Rv

5′ – ACA CTC TCC CCT GTT GAA GCT CTT – 3′

4. pLABEL-Fab phagemid vector. 5. QIAGEN Plasmid Maxi Kit (QIAGEN, Germany). 6. QIAquick®PCR Purification Kit (QIAGEN, Germany). 7. Ampicillin stock solution (50 mg/mL): 2.5 g ampicillin sodium salt in 50 mL of 50% (v/v) ethanol, filter sterilize and store at -20 °C.

Construction of Naı¨ve and Immune Human Fab Phage Display Library

43

Table 2 Primers with restriction endonuclease site for second amplification Primer name

Primer sequence

Fab HC amplification VH1 NcoI Fw

5′ – CCC AGC CGG CCA TGG CC CAG GTC CAG CTK GTR CAG TCT GG – 3′

VH157 NcoI Fw 5′ – CCC AGC CGG CCA TGG CC CAG GTG CAG CTG GTG SAR TCT GG – 3′ VH2 NcoI Fw

5′ – CCC AGC CGG CCA TGG CC CAG RTC ACC TTG AAG GAG TCT G – 3′

VH3 NcoI Fw

5′ – CCC AGC CGG CCA TGG CC GAG GTG CAG CTG KTG GAG WCY – 3′

VH4 NcoI Fw

5′ – CCC AGC CGG CCA TGG CC CAG GTG CAG CTG CAG GAG TCS G – 3′

VH4 DP63 NcoI Fw

5′ – CCC AGC CGG CCA TGG CC CAG GTG CAG CTA CAG CAG TGG G – 3′

VH6 NcoI Fw

5′ – CCC AGC CGG CCA TGG CC CAG GTA CAG CTG CAG CAG TCA – 3′

Human Fab IgM 5′ – ATG ACG CGT TGG AAG AGG CAC GTT CTT TTC TTT – 3′ CH1 MluI Rv Human Fab IgG CH1 MluI Rv

5′ – ATG ACG CGT TCT TGT CCA CCT TGG TGT TG – 3′

Fab LC amplification Vλ1 SalI Fw

5′ – TGT GAC AAA GTC GAC G CAG TCT GTS BTG ACG CAG CCG CC – 3′

Vλ1459 SalI Fw

5′ – TGT GAC AAA GTC GAC G CAG CCT GTG CTG ACT CAR YC – 3′

Vλ15910 SalI Fw 5′ – TGT GAC AAA GTC GAC G CAG CCW GKG CTG ACT CAG CCM CC – 3′ Vλ2 SalI Fw

5′ – TGT GAC AAA GTC GAC G CAG TCT GYY CTG AYT CAG CCT – 3′

Vλ3 SalI Fw

5′ – TGT GAC AAA GTC GAC G TCC TAT GWG CTG ACW CAG CCA A – 3′

Vλ3 DPL16 SalI Fw

5′ – TGT GAC AAA GTC GAC G TCC TCT GAG CTG AST CAG GAS CC – 3′

Vλ338 SalI Fw

5′ – TGT GAC AAA GTC GAC G TCC TAT GAG CTG AYR CAG CYA CC – 3′

Vλ6 SalI Fw

5′ – TGT GAC AAA GTC GAC G AAT TTT ATG CTG ACT CAG CCC C – 3′

Vκ1 SalI Fw

5′ – TGT GAC AAA GTC GAC G GAC ATC CRG DTG ACC CAG TCT CC – 3′

Vκ246 SalI Fw

5′ – TGT GAC AAA GTC GAC G GAT ATT GTG MTG ACB CAG WCT CC – 3′

Vκ3 SalI Fw

5′ – TGT GAC AAA GTC GAC G GAA ATT GTR WTG ACR CAG TCT CC – 3′

Vκ5 SalI Fw

5′ – TGT GAC AAA GTC GAC G GAA ACG ACA CTC ACG CAG TCT C – 3′

Fab Lambda CL1 5′ – CTT GCT AGC TTA TGA ACA TTC TGT AGG GGC CAC TG – 3′ NheI Rv Fab Lambda CL2 5′ – CTT GCT AGC TTA TGA ACA TTC CGT AGG GGC AAC TG – 3′ NheI Rv Fab Kappa CL NheI Rv

5′ – CTT GCT AGC TTA ACA CTC TCC CCT GTT GAA GCT CTT – 3′

44

Jing Yi Lai and Theam Soon Lim

8. 40% (w/v) glucose: 40 g D-(+)-glucose in 100 mL of dH2O, autoclave and store at 4 °C. 9. 2× YT medium: 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 1 L dH2O, autoclave and store at room temperature. 10. 2× YT agar: 16 g tryptone, 10 g yeast extract, 5 g NaCl, and 15.5 g agar in 1 L dH2O, autoclave. 11. 2× YT-amp medium: 2× YT medium, 0.1 mg/mL ampicillin, and 2% (v/v) glucose. 12. 2× YT-amp agar plate: 2× YT agar, 0.1 mg/mL ampicillin, and 2% (v/v) glucose. 13. 80% (v/v) glycerol: 40 mL glycerol in 10 mL dH2O, autoclave and store at room temperature. 14. Sterile petri dish, 90 mm. 15. Nunc™ Square Scientific, USA).

BioAssay

Dish,

25

mm

(Thermo

16. MicroPulserTM Electroporator (Bio-Rad, USA). 2.4.1 First Step Cloning (Fab HC)

1. NcoI 2. MluI 3. ElectroMAX™ DH10β cells (Invitrogen, USA), genotype: F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139Δ(ara, leu)7697 galU galK λ-rpsL nupG

2.4.2 Second Step Cloning (Fab LC)

1. SalI 2. NheI 3. XL1-Blue MRF’ competent cells (Agilent Technologies, USA), genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)]

2.5

Colony PCR

1. DreamTaq DNA polymerase (Thermo Scientific, USA) 2. Forward primer, LMB3: 5′ – CAG GAA ACA GCT ATG AC – 3′ 3. Reverse primer, pIII: 5′ – TTA GAT CGT TAC GCT AAC – 3′

2.6 Fab Phage Library Packaging

1. 2× YT medium: 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 1 L dH2O, autoclave and store at room temperature. 2. 2× YT agar: 16 g tryptone, 10 g yeast extract, 5 g NaCl, and 15.5 g agar in 1 L dH2O, autoclave. 3. Ampicillin stock solution (50 mg/mL): 2.5 g ampicillin sodium salt in 50 mL of 50% (v/v) ethanol, filter sterilize and store at -20 °C. 4. Kanamycin stock solution (30 mg/mL): 1.5 g kanamycin sulfate in 50 mL of dH2O, filter sterilize and store at -20 °C.

Construction of Naı¨ve and Immune Human Fab Phage Display Library

45

5. 40% (w/v) glucose: 40 g D-(+)-glucose in 100 mL of dH2O, autoclave and store at 4 °C. 6. 2× YT-amp medium: 2× YT medium, 0.1 mg/mL ampicillin, and 2% (v/v) glucose. 7. 2× YT-amp/kan medium: 2× YT medium, 0.1 mg/mL ampicillin, and 0.06 mg/mL kanamycin. 8. 2× YT-amp agar: 2× YT agar, 0.1 mg/mL ampicillin, and 2% (v/v) glucose. 9. 2× YT-kan agar: 2× YT agar and 0.06 mg/mL kanamycin. 10. M13K07 helper phage (Invitrogen, USA). 11. PEG/NaCl solution: 200 g polyethylene glycol 6000 (PEG 6000) and 146 g NaCl in 1 L dH2O, autoclave and store at room temperature. 12. 1 M isopropyl-β-D-thiogalactopyranoside (IPTG): 2.38 g IPTG in 10 mL dH2O, filter sterilize and store at -20 °C. 13. Phosphate-buffered saline (PBS), pH 7.4: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4·2H2O, 0.24 g KH2PO4 in 1 L dH2O, autoclave and store at room temperature. 2.7

Phage Titration

1. 2× YT medium: 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 1 L dH2O, autoclave and store at room temperature. 2. 2× YT agar: 16 g tryptone, 10 g yeast extract, 5 g NaCl, and 15.5 g agar in 1 L dH2O, autoclave. 3. 2× YT-amp agar: 2× YT agar, 0.1 mg/mL ampicillin, and 2% (v/v) glucose. 4. 2× YT-kan agar: 2× YT agar and 0.06 mg/mL kanamycin. 5. Phosphate-buffered saline (PBS), pH 7.4: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4·2H2O, 0.24 g KH2PO4 in 1 L dH2O, autoclave and store at room temperature. 6. TG1 electrocompetent cells (Lucigen, USA), genotype: supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK – mK -) [F´ traD36 proAB lacIqZ ΔM15].

2.8 Fab Library Panning

1. Corning® 96-well clear polystyrene high bind Stripwell™ microplate (Corning, USA).

2.8.1

2. Coating buffer, pH 9.6: 1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 L dH2O, store at 4 °C.

Fab Selection

3. Phosphate-buffered saline (PBS), pH 7.4: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4·2H2O, 0.24 g KH2PO4 in 1 L dH2O, autoclave and store at room temperature. 4. PBST: 1 mL Tween 20 in 1 L PBS. 5. PTM blocking buffer: 2 g skim milk in 100 mL PBST (see Note 2).

46

Jing Yi Lai and Theam Soon Lim

6. Trypsin (10 μg/mL): 10 μg trypsin in 1 mL dH2O, store at 20 °C. 7. Ampicillin stock solution (50 mg/mL): 2.5 g ampicillin sodium salt in 50 mL of 50% (v/v) ethanol, filter sterilize and store at -20 °C. 8. Kanamycin stock solution (30 mg/mL): 1.5 g kanamycin sulfate in 50 mL of dH2O, filter sterilize and store at -20 °C. 9. 40% (w/v) glucose: 40 g D-(+)-glucose in 100 mL of dH2O, autoclave and store at 4 °C. 10. 2× YT medium, pH 7.2: 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 1 L dH2O, autoclave and store at room temperature. 11. 10× amp: 1 mg/mL ampicillin and 20% (v/v) glucose in PBS. 12. 2× YT-amp: 2× YT medium with 0.1 mg/mL ampicillin and 2% (v/v) glucose. 13. 2× YT-amp/kan: 2× YT medium with 0.1 mg/mL ampicillin and 0.06 mg/mL kanamycin. 14. 1 M isopropyl-β-D-thiogalactopyranoside (IPTG): 2.38 g IPTG in 10 mL dH2O, filter sterilize and store at -20 °C. 15. XL1-Blue competent cells (Agilent Technologies, USA), genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)]. 2.8.2 Polyclonal and Monoclonal Phage ELISA

1. Bovine Serum Aldrich, USA).

Albumin

(BSA)

Fraction

V

(Sigma-

2. PBST: 1 mL Tween-20 in 1 L PBS. 3. PTM blocking buffer: 2 g skim milk in 100 mL PBST (see Note 2). 4. Anti-M13 horseradish peroxidase (HRP): Prepare at 1:5000 dilution in PTM blocking buffer. 5. 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic (ABTS) tablets (Roche, USA).

acid)

6. ABTS buffer (Roche, USA).

3

Methods

3.1 Isolation of B Cells

Blood samples are collected from healthy or disease-related donors for the generation of a human naı¨ve and immune library, respectively (see Note 3(a) and 3(b)). 1. Dilute EDTA-treated fresh blood with PBS at 1:1 ratio. 2. Carefully layer 10 mL of whole blood on top of 7.5 mL of Ficoll-PaqueTM PLUS (see Note 4) and centrifuge at 1200 ×g for 15 min at 18 °C.

Construction of Naı¨ve and Immune Human Fab Phage Display Library

47

3. Aspirate lymphocytes layered between Ficoll-PaqueTM PLUS and plasma with care and transfer to a new sterile 50 mL polypropylene tube. 4. Dilute the isolated lymphocytes with PBS up to 50 mL and centrifuge at 250 ×g for 10 min at 18 °C. 5. Remove the supernatant using a clean filter tip. 6. Repeat the PBS washing step once. 7. Subject the isolated lymphocytes to RNA extraction using a total RNA extraction kit based on the manufacturer’s protocols (see Note 5). 8. Keep extracted total RNA at -80 °C until use. 3.2 First-Strand cDNA Synthesis

1. Synthesize the first-strand cDNA using SuperScript™ II Reverse Transcriptase according to manufacturer’s procedure. 2. Store the cDNA at -20 °C until use.

3.3 Amplification of HC and LC Fab Gene Repertoire

The cDNA serves as the template for amplification of the Fab heavy chain (HC) and Fab light chain (LC) repertoire. Fab HC fragment is comprised of the VH and CH1, while Fab LC fragment includes the VL and CL regions. Depending on the type of library, different reverse primers are used for the heavy chain amplification. IgM CH1 Rv is used for naı¨ve library construction, whereas IgG CH1 Rv is used for immune library construction. The forward primers are the same for both libraries, which includes seven VH primers, eight Vλ primers, and four Vκ primers (see Table 1). LC family specific reverse primers are used in combination with the matching family V gene primers to amplify the LC lambda and kappa repertoires. 1. Set up a PCR reaction using 200 ng of cDNA, 200 μM dNTPs, 0.2 μM Fw and Rv primers (Table 1), 2 μL of Pfu buffer with MgSO4 and 0.5 U Pfu DNA polymerase. Top up the reaction to 20 μL using dH2O. 2. Carry out PCR using the following program: initial denaturation at 95 °C for 2 min, 30 cycles of amplification with denaturation at 95 °C for 30 s, annealing at 55 °C or 62 °C for 30 s, elongation at 72 °C for 45 s, and a final elongation at 72 °C for 5 min (see Note 6). 3. Separate the amplified PCR product on 1% TBE agarose gel using gel electrophoresis at 110 V for 50 min. Excise the band (~650–700 bp) and extract using QIAquick® Gel Extraction Kit according to the manufacturer’s protocol (see Note 7). 4. Determine the concentration of the extracted DNA and proceed to second amplification to introduce restriction

48

Jing Yi Lai and Theam Soon Lim

endonuclease sites with overhangs. Alternatively, store the DNA at -20 °C until use. 5. For second amplification, set up the PCR reaction using 20 μg of the amplified HC or LC Fab fragment, 200 μM dNTPs, 0.2 μM Fw and Rv primers (Table 2), 2 μL of ThermoPol® buffer, and 0.4 U Vent® DNA polymerase. Top up the reaction to 20 μL using dH2O. The Fab Lambda CL1 Rv and Fab Lambda CL2 Rv primers were used in equimolar ratio for lambda repertoire amplification. 6. Carry out PCR using the following program: initial denaturation at 95 °C for 2 min, 30 cycles of amplification with denaturation at 95 °C for 30 s, annealing at 55 °C or 62 °C for 30 s, elongation at 72 °C for 45 s, and a final elongation at 72 °C for 5 min (see Note 6). 7. Separate the amplified PCR product on 1% TBE agarose gel using gel electrophoresis at 110 V for 50 min. Excise the band (~650–700 bp) and extract using QIAquick® Gel Extraction Kit according to the manufacturer’s protocol. 8. Pool the DNA according to subfamilies for every group of five donors. Determine the concentration and store the DNA at 20 °C until use. 3.4 Two-Step Cloning

3.4.1 First Step Cloning (Fab HC)

The cloning is carried out using two-step cloning strategy. First cloning step generates a mini-library containing the Fab HC repertoire using DH10β cells, whereas the second cloning step introduces Fab LC (κ and λ) repertoire to the HC-mini-library to generate the final Fab library using XL1-Blue cells. The schematic diagram of the Fab library construction process is outlined in Fig. 1. 1. Double digest pLABEL-Fab vector and pooled Fab HC with NcoI and MluI overnight at 37 °C. Heat-inactivate the reaction at 65 °C for 20 min (see Note 8). 2. Add 5 U of alkaline phosphatase to double-digested pLABELFab vector and incubate at 37 °C for 1 h. Heat-inactivate the reaction at 80 °C for 2 min. 3. Separate the digested vector on 1% TBE agarose gel using gel electrophoresis at 110 V for 50 min. Excise the band and extract using QIAquick® Gel Extraction Kit according to the manufacturer’s protocol. 4. Purify the digested Fab HC using QIAquick®PCR Purification Kit according to the manufacturer’s protocol. 5. Ligate digested pLABEL-Fab and digested Fab HC pools at 1: 2 ratio using T4 DNA ligase (see Note 9). Incubate the ligation

Construction of Naı¨ve and Immune Human Fab Phage Display Library

49

Fig. 1 A schematic diagram of the Fab library construction process. B cells, layering in the peripheral blood mononuclear cell (PBMC) fraction, are isolated out for RNA extraction. The total RNA is subsequently reverse transcribed to cDNA. The Fab LC and Fab HC repertoires are amplified from the cDNA using designed primers, respectively. Fab HC are cloned into phagemid to generate the intermediate mini-library, and subsequently Fab LC are cloned into the intermediate phagemid to generate the final Fab library

reaction overnight at 16 °C. Heat-inactivate the reaction at 65 ° C for 10 min. 6. Precipitate the ligated DNA using 2.5 volume of ethanol and 0.1 volume of 3 M sodium acetate. Incubate in – 80 °C for 1 h and centrifuge for 20 min at 14,000× g. 7. Wash the DNA pellet with 500 μL of 70% (v/v) ethanol and centrifuge again at 14,000× g for 20 min. 8. Dissolve the DNA pellet with 4 μL dH2O per ligation reaction. 9. Thaw DH10β cells on ice for 2 min and mix with 2 μL DNA. 10.

Transfer the mixture to a pre-chilled 0.1 cm electroporation cuvette. Transform the mixture at 1.7 kV using an electroporator.

11. Resuspend the electroporated mixture with 1 mL of pre-warmed 2× YT medium and transfer the suspension to a 1.5 mL microcentrifuge tube. Incubate the cell suspension for 1 h at 37 °C and 700 rpm. 12. Pool the cell suspension according to subfamilies and take 10 μL of cells to determine the cloning efficiency. Dilute the 10 μL cells in 90 μL 2× YT-amp and plate out on 2× YT-amp agar plates in 90 mm petri dish. 13. Plate out the remaining pooled cell suspension on 30 BioAssay Dish with 2× YT-amp agar. Incubate the agar plates overnight at 37 °C.

50

Jing Yi Lai and Theam Soon Lim

14. Scrape the colonies on BioAssay Dish with 2× YT-amp. 15. Estimate the library diversity by titrating the scraped library stock (see Subheading 3.4.3). 16. Prepare glycerol stock of the Fab HC-mini-library (see Subheading 3.4.4). 17. Perform colony PCR using colonies on the 90 mm petri dish to confirm successful cloning of the Fab HC repertoire (see Subheading 3.5) (see Note 10). 3.4.2 Second Step Cloning (Fab LC)

1. Culture a tube of Fab HC-mini-library glycerol stock overnight in 500 mL of 2× YT-amp at 37 °C with shaking at 200 rpm. 2. Extract the plasmid using QIAGEN Plasmid Maxi Kit according to manufacturer’s protocol. 3. Double digest the Fab HC-mini-library and Fab LC (κ and λ) with SalI and NheI overnight at 37 °C. Heat-inactivate the reaction at 65 °C for 20 min (see Note 8). 4. Perform the digestion and ligation as in the first step cloning (see Subheading 3.4.1). 5. Transform the Fab library into XL1-Blue MRF’ cells. 6. Pool the cell suspension according to subfamilies combination and take 10 μL of cells to determine the cloning efficiency. Dilute the 10 μL cells in 90 μL 2× YT-amp and plate out on 2× YT-amp agar plates in 90 mm petri dish. 7. Plate out the remaining pooled cell suspension on 40 BioAssay Dish with 2× YT-amp agar. Incubate the agar plates overnight at 37 °C. 8. Scrape the colonies on BioAssay Dish with 2× YT-amp. 9. Estimate the library diversity by titrating the scraped library stock (see Subheading 3.4.3) (see Note 11). 10. Prepare glycerol Subheading 3.4.4).

stock

of

the

Fab

library

(see

11. Perform a colony PCR using colonies on the 90 mm petri dish to confirm successful cloning of the Fab LC repertoire (see Subheading 3.5) (see Note 10). 3.4.3 Library Size Estimation

1. Take 10 μL of cells from the scraped library suspension to perform a tenfold serial dilution down to 10-13. 2. Spot the dilution on the 2× YT-amp agar plate and incubate overnight at 37 °C. 3. Count the library size based on the formula: Library size =

number of colonies × dilution factor total volume

Construction of Naı¨ve and Immune Human Fab Phage Display Library 3.4.4 Preparation of Bacteria Library Stock

51

1. Pellet down the colonies scraped from BioAssay Dish at 4500× g, 10 min. 2. Resuspend the pellet with adequate amount of 2× YT-amp and add 80% (v/v) glycerol to the cell suspension to make a 20% library stock (see Note 12). 3. Aliquot the final library suspension into 2 mL cryogenic vial and keep in -80 °C until use.

3.5

Colony PCR

1. Pick single colonies randomly and resuspend in 10 μL dH2O. 2. Set up PCR reaction using 2 μL of colony, 200 μM dNTPs, 0.5 μM LMB3 Fw and pIII Rv primers, 1× DreamTaq buffer, and 0.5 U DreamTaq DNA Polymerase. Top up the reaction to 20 μL using dH2O. 3. Carry out PCR using the following program: initial denaturation at 95 °C for 5 min, 20 cycles of amplification with denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 2 min, and a final elongation at 72 °C for 5 min. 4. Separate the amplified PCR product on 1% TBE agarose gel using gel electrophoresis run at 110 V for 50 min (see Notes 10 and 13). 5. Send the colony with correct band size for sequencing. 6. Analyze the DNA sequence on IMGT/V-quest (www.imgt. org/IMGT_vquest/vquest) (see Note 14).

3.6 Fab Phage Library Packaging

1. Thaw a tube of the library glycerol stock and inoculate into 500 mL of 2× YT-amp. Culture at 37 °C with shaking at 200 rpm until OD600 ~0.5. 2. Infect 250 mL of the culture with 1012 M13KO7 helper phage, following the multiplicity of infection (MOI) of 1:20. Incubate the culture static at 37 °C for 30 min. 3. Pellet down the culture at 3500× g for 10 min. Remove the supernatant (see Note 15). 4. Resuspend the pellet with 300 mL 2× YT-amp/kan and 100 μM IPTG. Culture overnight at 30 °C, 200 rpm (see Note 16). 5. Pellet down the cells at 10,000× g for 10 min. Transfer the phage-containing supernatant to new 50 mL polypropylene tube. 6. Add 1/5 volume of PEG/NaCl solution to 4/5 volume of supernatant. Mix well and incubate on ice for 1 h (see Note 17). 7. Centrifuge the mixture at 10,000× g for 30 min at 12 °C, remove the supernatant, and resuspend the phage pellet in 8 mL PBS.

52

Jing Yi Lai and Theam Soon Lim

8. Add 2 mL PEG/NaCl solution, mix well, and incubate on ice for 20 min. 9. Centrifuge the mixture at 10,000× g for 30 min at 12 °C. Remove the supernatant, short spin briefly, and pipette out remaining PEG/NaCl solution. 10. Resuspend the phage pellet in 2 mL PBS. 11. Centrifuge at 10,000× g for 10 min to remove remaining bacterial cells from the phage precipitation. Repeat centrifugation until no pellet is observed. 12. Take 10 μL of the phage to check the titer of the packaged Fab library (see Subheading 3.7). 13. Store the Fab antibody library at 4 °C until use (see Note 18). 3.7

Phage Titration

1. Dilute 10 μL of Fab phage library in 90 μL of PBS and perform a tenfold serial dilution. 2. Add 100 μL of TG1 or XL1-Blue MRF’ cells at OD600 ~0.5 to the serial diluted phage. 3. Spot 10 μL of the infected cells on 2× YT-amp agar plate and 2× YT-kan agar plate. Incubate overnight at 37 °C (see Note 19). 4. Calculate the phage titer using formula: Phage titer ðcfu=mLÞ =

3.8 Fab Library Panning 3.8.1

Fab Selection

number of colonies × dilution factor total volume spotted

1. Coat a microtiter well with 1–10 μg of antigen using coating buffer at a final volume of 100 μL. Reduce the amount of antigen for subsequent panning rounds to increase the selection stringency. Incubate overnight at 4 °C (see Note 20). 2. Wash the antigen-coated well 3× with PBST using ELISA plate washer (see Note 21). 3. Block the antigen-coated well with 300 μL of PTM blocking buffer for 1 h at room temperature and then wash 3× with PBST. 4. Simultaneously, dilute 1011 phage particles in PTM blocking buffer at final volume of 200 μL and preincubate for 1 h on a blocked microtiter well (see Note 22). 5. Transfer the preincubated phage to antigen-coated well and incubate for 2 h at room temperature with constant shaking at 700 rpm. 6. Remove unbound and unspecific phages by washing 10× with PBST. Increase the wash steps by 10× for each subsequent panning round. 7. Elute the bound phages with 100 μL of 10 μg/mL trypsin and incubate static at 37 °C for 30 min (see Note 23).

Construction of Naı¨ve and Immune Human Fab Phage Display Library

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8. Transfer the eluted phages to a new 2 mL microcentrifuge tube and add 100 μL of XL1-Blue MRF’ cells at OD600 ~0.5. Incubate the cells static at 37 °C for 30 min followed by 30 min of incubation with shaking at 700 rpm. 9. Take 10 μL of infected culture to determine the rescued phage titer (see Subheading 3.7). 10. Add 20 μL of 10× amp to the remaining culture and culture overnight at 37 °C, 700 rpm. 11. Inoculate 190 μL of 2× YT-amp with 10 μL of the overnight culture. Culture at 37 °C with shaking at 700 rpm for 2.5 h. 12. Mix the remaining overnight culture with 65 μL of 80% (v/v) glycerol and store the glycerol stock in -80 °C. 13. Infect the culture with 1011 M13KO7 helper phage and incubate static at 37 °C for 30 min. 14. Pellet down the culture at 3500× g for 10 min. Resuspend the cell pellet with 230 μL of 2× YT-amp/kan supplemented with 100 μM IPTG. Culture overnight at 30 °C with shaking at 700 rpm. 15. Pellet down the overnight culture at 3500× g for 10 min. 16. Transfer the supernatant to a new clean tube. Take 100 μL of the phage for subsequent round of panning and keep the remaining phage at 4 °C for polyclonal ELISA. The amplified phage titer from each round of panning is determined (see Subheading 3.7) (see Note 24). 3.8.2 Polyclonal and Monoclonal Phage ELISA

For a successful panning, an obvious enrichment is observed in polyclonal ELISA as the panning round increases. It is critical to choose the best panning round in order to isolate different monoclonal antibodies with good specificity and diversity. 1. Coat appropriate number of microtiter wells with 1–10 μg of antigen using coating buffer at a final volume of 100 μL. Incubate overnight at 4 °C. Concurrently, coat equal number of empty wells with BSA at the same amount as the negative controls to observe nonspecific binders. 2. Wash the coated wells 3× with PBST using ELISA plate washer (see Note 21). 3. Block the coated wells with 300 μL of PTM blocking buffer for 1 h at room temperature and then wash 3× with PBST. 4. Dilute 50 μL phage with 50 μL PTM blocking buffer, and add the 100 μL mixture to corresponding antigen-coated wells and the control wells. Incubate for 2 h at room temperature with shaking at 700 rpm. 5. Wash the wells 3× with PBST.

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6. Add 100 μL of anti-M13-HRP to each antigen-coated wells and control wells. Incubate for 1 h at room temperature with shaking at 700 rpm. 7. Wash the wells 3× with PBST. 8. Develop the wells with 100 μL ABTS solution for 30 min in the dark. Measure the readings at 405 nm using a microtiter plate spectrophotometer. 9. After determining the panning round for monoclonal selection, take 10 μL of the remaining phage to perform a tenfold serial dilution and infect 100 μL of XL1-Blue MRF’ cells of OD600 ~ 0.5 at 37 °C, static. Plate out the infected cell on 2× YT-amp agar plate. 10. Pick 92 monoclonal antibody clones randomly to inoculate 200 μL of 2× YT-amp. Culture overnight at 37 °C, 1250 rpm. 11. Package the monoclonal Fab antibody as described in steps 11–15 in Subheading 3.8.1. 12. Perform monoclonal ELISA using procedures as described in steps 1–8 in this section. 13. Identify positive clones with good signal/noise ratio for colony PCR (see Subheading 3.5) and send for sequencing for DNA analysis.

4

Notes 1. Alternatively, other high-fidelity proofreading polymerase can be used. 2. Alternatively, other blocking agents such as bovine serum albumin (BSA), casein, ovalbumin, and gelatin can be used for blocking. However, the blocking solution needs to be prepared fresh to avoid contamination by microorganisms. 3. The sampling requirements includes: (a) Donors with family backgrounds of severe illnesses, suffering from autoimmune disorder, having ongoing infection, experiencing symptoms of infection such as fever, or on medication including antibiotics and immunosuppressors within a month from date of blood collection are not suitable for naı¨ve library sampling. (b) Donor’s medication history including types of treatment and state of illness should be recorded for immune library sampling. The type of treatment regimen is important to standardize the library generation process because the capability of the treatment to elicit donor’s immune response to the disease is critical to isolate effective repertoire. Donor that has undergone immunosuppressors is

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not suitable as the immune response is ineffective. However, the conditions may vary depending on the mechanism of disease. 4. Be careful that the blood layer must not mix with the FicollPaque™ PLUS solution because it causes aggregation of erythrocytes and reduces the yield of lymphocytes as lymphocytes trapped in the aggregate sediment with erythrocytes to the bottom of tube. 5. Integrity and concentration of the total RNA extracted from lymphocytes can be analyzed using Bioanalyzer Instrument (Agilent, USA) or other suitable instruments. 6. The HC and LC repertoire is amplified from each donor independently to avoid bias and loss of repertoire caused by sample pooling. This step ensures better repertoire diversity. 7. Fab HC and LC fragment is about 650–700 base pair (bp); however, the presence of bands with various sizes is expected due to the rearranged V genes. Excise the targeted band size with care. 8. Increase digestion reaction until sufficient amount of DNA is obtained for the following cloning procedure. A sequential digestion may be required if the pair of restriction endonucleases have incompatible reaction buffers. This is because digestion efficiency is affected by the salt content in the buffer. 9. Multiple ligation reactions may be required to obtain sufficient amount of DNA material for a highly diverse library. The library size for the heavy chain repertoire should be as high as possible because the variable heavy chain (VH) is the predominant region for antigen binding. The size should reach 107–109 or higher for naı¨ve library and 105–107 for immune library. 10. The expected band size is approximately 1200 bp for the Fab HC in the first step cloning and 1800 bp for the whole Fab in the second step cloning. 11. The final library size should range between 109–1012 or higher for a naı¨ve library and 106–108 for an immune library. 12. The library stocks are usually prepared to allow a starting OD600 ~ 0.1 when the tube of library stock is thawed into 500 mL of 2× YT-amp during library packaging. 13. A cloning efficiency of more than 80% inserts for Fab HC-minilibrary and full Fab library is preferred. 14. Alternatively, the library repertoire diversity can be determined by deep sequencing. 15. Excess glucose suppresses the expression of the HC::pIII fusion protein as well as the free LC. Therefore, it is necessary

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to pellet down the cells and remove medium containing glucose. 16. Addition of 100 μM IPTG during Fab phage packaging induces expression of the HC::pIII fusion protein and LC monomers. This will facilitate the natural formation of Fab antibody molecules on the phage particle. 17. The ice-cold incubation can be prolonged to 2 h to increase the amount of phage particles precipitated by the PEG/NaCl solution. 18. Avoid storing the phage preparation for more than 4 weeks at 4 °C. Package phage freshly to prevent loss of binding caused by proteolysis of the displayed antibodies. 19. The colony on ampicillin agar plate should be at least twofold higher in comparison with kanamycin plate. This indicates that less helper phage is being produced than the phage library. In addition, the colony can be used to quantify the phage library size prior to panning. 20. Prior to panning, evaluate purity of the antigens using SDS-PAGE and Western blot. The purity of the antigen used in panning increases the probability of isolating good quality Fab binders. 21. Alternatively, wash the wells manually with a squirt bottle. Fill the wells with PBST and shake the washing buffer off. Tap the wells on a stack of clean dry paper towels to remove remaining PBST. Care must be taken to ensure no bubbles are present and to avoid buffer carry over during the washing step. 22. The amount of phage used for the first panning round should be twofold higher than the library size. 23. There are other elution buffers or methods that can be employed at this stage. Competitive elution can also be carried out by competing with free antigens. Acid-based elution using glycine-HCL, pH2.2 can also be used. The best elution method would be dependent on the requirements and preference of each user. 24. The phage recovery of each panning round can be calculated by dividing the rescued phage titer with the input phage titer. Phage recovery should increase gradually over panning rounds, indicating enrichment of binding clones.

Acknowledgments This work was supported by a Universiti Sains Malaysia, Special (Matching) Short-Term Grant with Project No: 304/CIPPM/ 6315708.

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References 1. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317. https://doi.org/10. 1126/science.4001944 2. McCafferty J, Griffiths AD, Winter G et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554. https://doi.org/10. 1038/348552a0 3. Breitling F, Du¨bel S, Seehaus T et al (1991) A surface expression vector for antibody screening. Gene 104(2):147–153. https://doi.org/ 10.1016/0378-1119(91)90244-6 4. Soumillion P, Jespers L, Bouchet M et al (1994) Selection of beta-lactamase on filamentous bacteriophage by catalytic activity. J Mol Biol 237(4):415–422. https://doi.org/10. 1006/jmbi.1994.1244 5. Love KR, Swoboda JG, Noren CJ et al (2006) Enabling glycosyltransferase evolution: a facile substrate-attachment strategy for phagedisplay enzyme evolution. ChemBioChem 7(5):753–756. https://doi.org/10.1002/ cbic.200600018 6. Zhao N, Schmitt MA, Fisk JD (2016) Phage display selection of tight specific binding variants from a hyperthermostable sso7d scaffold protein library. FEBS J 283(7):1351–1367. https://doi.org/10.1111/febs.13674 7. Lawrie J, Waldrop S, Morozov A et al (2021) Engineering of a small protein scaffold to recognize sulfotyrosine with high specificity. ACS Chem Biol 16(8):1508–1517. https://doi. org/10.1021/acschembio.1c00382 8. Zuo S, Dai G, Wang L et al (2019) Suppression of angiogenesis and tumor growth by recombinant t4 phages displaying extracellular domain of vascular endothelial growth factor receptor 2. Arch Virol 164(1):69–82. https://doi.org/ 10.1007/s00705-018-4026-0 9. Hoogenboom HR, Griffiths AD, Johnson KS et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (fab) heavy and light chains. Nucleic Acids Res 19(15):4133–4137. https://doi.org/10.1093/nar/19.15.4133 10. Omar N, Hamidon NH, Yunus MH et al (2018) Generation and selection of naı¨ve fab library for parasitic antigen: anti-bmsxp antibodies for lymphatic filariasis. Biotechnol Appl Biochem 65(3):346–354. https://doi.org/ 10.1002/bab.1591 11. Pan Y, Du J, Liu J et al (2021) Screening of potent neutralizing antibodies against sars-cov-

2 using convalescent patients-derived phagedisplay libraries. Cell Discov 7(1):57. https:// doi.org/10.1038/s41421-021-00295-w 12. Dong S, Guan L, He K et al (2021) Screening of anti-idiotypic domain antibody from phage library for development of bt cry1a simulants. Int J Biol Macromol 183:1346–1351. https:// doi.org/10.1016/j.ijbiomac.2021.05.093 13. Chan SK, Rahumatullah A, Lai JY et al (2017) Naı¨ve human antibody libraries for infectious diseases. In: Lim TS (ed) Recombinant antibodies for infectious diseases. Springer, Cham, pp 35–59. https://doi.org/10.1007/978-3-31972077-7_3 14. Weill J-C, Reynaud C-A (2020) Igm memory b cells: specific effectors of innate-like and adaptive responses. Curr Opin Immunol 63:1–6. https://doi.org/10.1016/j.coi.2019.09.003 15. Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14(3):309–314. https://doi.org/10. 1038/nbt0396-309 16. Roth KDR, Wenzel EV, Ruschig M et al (2021) Developing recombinant antibodies by phage display against infectious diseases and toxins for diagnostics and therapy. Front Cell Infect Microbiol 11:617 17. Lai JY, Lim TS (2020) Infectious disease antibodies for biomedical applications: a mini review of immune antibody phage library repertoire. Int J Biol Macromol 163:640–648. https://doi.org/10.1016/j.ijbiomac.2020. 06.268 18. Almagro JC, Pedraza-Escalona M, Arrieta HI et al (2019) Phage display libraries for antibody therapeutic discovery and development. Antibodies 8(3):44 19. Shim H (2017) Antibody phage display. Adv Exp Med Biol 1053:21–34. https://doi.org/ 10.1007/978-3-319-72077-7_2 20. Feng M, Bian H, Wu X et al (2019) Construction and next-generation sequencing analysis of a large phage-displayed vnar single-domain antibody library from six naive nurse sharks. Antib Ther 2(1):1–11 21. Kang AS, Barbas CF, Janda KD et al (1991) Linkage of recognition and replication functions by assembling combinatorial antibody fab libraries along phage surfaces. Proc Natl Acad Sci U S A 88(10):4363–4366 22. Steinwand M, Droste P, Frenzel A et al (2014) The influence of antibody fragment format on phage display based affinity maturation of igG.

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407:26–34. https://doi.org/10.1016/j.jim. 2014.03.015 28. Chockalingam K, Peng Z, Vuong CN et al (2020) Golden gate assembly with a bi-directional promoter (gbid): a simple, scalable method for phage display fab library creation. Sci Rep 10(1):2888. https://doi.org/10. 1038/s41598-020-59745-2 29. Sheets MD, Amersdorfer P, Finnern R et al (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci U S A 95(11):6157–6162. https://doi.org/10. 1073/pnas.95.11.6157 30. de Haard HJ, van Neer N, Reurs A et al (1999) A large non-immunized human fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274(26):18218–18230. https:// doi.org/10.1074/jbc.274.26.18218 31. Erasmus MF, D’Angelo S, Ferrara F et al (2021) A single donor is sufficient to produce a highly functional in vitro antibody library. Commun Biol 4(1):350. https://doi.org/10. 1038/s42003-021-01881-0

Chapter 4 Construction of Synthetic Antibody Phage Display Libraries Kim Anh Giang, Sachdev S. Sidhu, and Johan Nilvebrant Abstract Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival natural antibodies and be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies targeting a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories. Key words Human antibody, Antibody fragment, Domain antibody, Phage display, Protein engineering, Degenerate oligonucleotide

1

Introduction Antibodies are well established as affinity reagents and therapeutic drugs. They can be generated with high affinity and specificity and are long-lived in serum and generally well tolerated as drugs. They are by far the most widely used group of specific protein detection reagents and the dominating class of agents on the biopharmaceutical market [1–3]. Monoclonal antibodies have traditionally been generated by hybridoma technology, where splenocytes derived from immunized animals are harvested and fused with an immortalized myeloma cell line [4]. Although effective, this approach is laborious and expensive and results in nonhuman and potentially immunogenic antibodies. Moreover, the natural immune system imposes restrictions that make it difficult to raise antibodies against certain antigens, including self-antigens and highly conserved proteins across species, toxic antigens, and antigens that are unstable in serum. Some

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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of these limitations are also shared by more recent strategies, including generation of human antibodies in transgenic mice engineered to produce a human immunoglobulin repertoire followed by hybridoma [5]. Antibody repertoires displayed on phage provide an attractive alternative to circumvent animal immunization and other caveats of hybridoma- or single-cell technology [6, 7]. In vitro selection methods can be used to target almost any protein and offer more precise control, high throughput, and adaptability to automation [8]. Phage display, originally introduced by George Smith in 1985 [9] and awarded one half of the 2018 Nobel Prize in Chemistry (with Gregory Winter), has become the most widely used selection method for human antibodies. Antibody phage display technology [10, 11] has been used to generate and improve a large number of antibodies for research and medical use [12–15]. Pools of billions of unique antibodies displayed on phage are subjected to selective pressure for antigen recognition. Binding clones are amplified by infection of an Escherichia coli host and used for additional rounds of selection. Eventually the population is dominated by antigenbinding clones, which can be screened and subjected to DNA sequencing to decode the sequences of the displayed antibodies. Antibody phage display is now an established method for robust generation of reliable antibody reagents in vitro. A key advantage is that the unique sequence of each antibody is encoded in the encapsulated phage DNA, which allows for facile downstream manipulation or reformatting to optimize antibody properties. Furthermore, use of well-characterized antibody reagents defined by their sequence is critical to help battle the much discussed “antibody reproducibility crisis” [16–18]. Phage-displayed antibody libraries commonly integrate diversity from immune or non-immune natural B-cell sources. However, this approach is limited by the diversity provided by the natural adaptive immune system of the host. Nowadays, molecular details of antibody structure and function are so well understood that defined diversity can be encoded in degenerate synthetic DNA and introduced in regions most likely to contribute to antigen binding in a defined framework. Use of human frameworks optimized for favorable biophysical properties can minimize the risk of immunogenicity and thus the need for humanization while ensuring high stability and protein production [19, 20]. Use of synthetic libraries allows control over both library design and selection conditions and facilitates the generation of antibodies with precisely engineered binding specificities. Immunoglobulin G is the most common antibody class in humans. It is composed of two heavy chains and two light chains. The antigen-binding site is formed by three hypervariable loops on each variable domain. Since the structure of the variable domains is

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only slightly influenced by the diversity in the complementarity-determining regions (CDRs) [21], a single framework can accommodate an array of CDR sequences and binding properties. Our work has shown that a single framework based on the highly stable therapeutic human 4D5 scaffold can support remarkably diverse antibody functions [20]. Furthermore, highly simplified library designs, which engage fewer CDRs and may encode as little as two possible residues per randomized position, have been shown to be capable of producing specific synthetic antibodies against a multitude of protein targets [22, 23]. Complex architecture, requirement of expression in mammalian cells, relatively poor tissue penetration, and, sometimes, undesired Fc-mediated effector functions have inspired progressive reduction of the size of the antibody molecule (Fig. 1a). Smaller fragments such as fragment antigen binding (Fab) and single-chain fragment variable (scFv) can retain the binding properties of the parental antibody while enabling high-yield production in prokaryotic expression systems. Domain antibodies (VH) consisting of a single variable domain represent the smallest antibody fragments capable of mediating antigen recognition [24, 25]. In recent years, camelid domain antibodies (VHH) have become an increasingly popular choice for synthetic library construction due to the versatility of their longer CDR loops compared to the human VH [26– 28]. Highly functional single framework libraries with various degrees of CDR diversity can be constructed using any of these smaller antibody fragments [29, 30]. This protocol describes the construction of highly diverse synthetic domain antibody libraries built on a single human VH domain (Fig. 1b) [31–33] cloned into a phagemid vector. Following bacterial transformation and infection with helper phage, a phage-displayed library containing billions of individual clones can be used for the rapid isolation of recombinant domain antibodies targeting virtually any protein antigen.

2

Materials Prepare all solutions using MilliQ water and analytical grade reagents. 1. 0.2 cm gap electroporation cuvette (BTX Harvard Apparatus, Holliston, MA). 2. 10 mM ATP. 3. 10x TM buffer: 0.1 M MgCl2, 0.5 M Tris, and pH 7.5. 4. 100 mM dithiothreitol (DTT). 5. 14 mL round-bottomed tube (Falcon 352059).

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Fig. 1 Autonomous domain antibody scaffold and diversification. (a) Structure of IgG1 (PDB 1IGT) with fragment antigen binding (Fab) and fragment crystallizable (Fc) indicated. IgG is a homotetramer consisting of two heavy chains each built from three-constant (CH1, CH2, and CH3) and one-variable (VH) domain and two light chains each comprising a constant (CL) and a variable (VL) domain. The backbone is shown as green or gray tubes for the VH or other domains, respectively. An engineered autonomous human VH domain (PDB 3B9V) is used as a scaffold to introduce diversity in three complementarity-determining regions shown in purple (CDRH1), yellow (CDRH2), and red (CDRH3). Spheres represent paratope positions that are diversified. The figure was generated using PyMOL (http://www.pymol.org/). (b) Sequence of the fusion protein designed to enable phage display of an autonomous VH domain (bold) fused to the truncated protein III [17]. The signal sequence stII (underlined) directs the VH domain-pIII fusion to the periplasm, and the dimerization domain (dashed box) is used to achieve bivalent display. (c) Amino acids encoded in three mutagenic oligonucleotide sets used to introduce synthetic antibody diversity. All oligonucleotides can be used simultaneously and are designed with at least 15 nucleotides complementary to the template sequence on either side of the region to be randomized. The distance between annealing oligonucleotides used in the same mutagenesis reaction should be at least 15 base pairs. Binary diversity is used in CDRH1 and CDRH2. In CDRH3, “X” denotes nine amino acids (Y, S, G, A, F, W, H, P, V in a ratio of 5:4:4:2:1:1:1:1:1) encoded by a custom trimer phosphoramidite mixture containing the indicated ratio of nine trimers. A mixture of oligonucleotides is used to introduce length diversity in CDRH3

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6. 2YT medium: 10 g bacto-yeast extract, 16 g bacto-tryptone, and 5 g NaCl. Add water to 1.0 L; adjust pH to 7.0 with NaOH and autoclave. 7. 2YT/carb/cmp medium: 2YT, 100 μg/mL carbenicillin, and 10 μg/mL chloramphenicol. 8. 2YT/carb/kan medium: 2YT, 100 μg/mL carbenicillin, and 25 μg/mL kanamycin. 9. 2YT/carb/kan/uridine medium: 2YT, 100 μg/mL carbenicillin, 25 μg/mL kanamycin, and 0.25 μg/mL uridine. 10. 2YT/tet medium: 2YT and 10 μg/mL tetracycline. 11. 3 M sodium acetate, pH 5.0. 12. 96-microwell round-bottom plate (Corning). 13. Baffled E-flasks (250 and 2000 mL). 14. 100 mg/mL carbenicillin: Dissolve 100 mg of carbenicillin in 1 mL of water and filter sterilize. 15. 10 mg/mL chloramphenicol: Dissolve 10 mg of chloramphenicol in 1 mL of ethanol and filter sterilize. 16. dNTP mix: solution containing 10 mM each of dATP, dCTP, dGTP, and dTTP. 17. ECM-630 electroporation system (BTX). 18. E. coli CJ236 (New England Biolabs, Ipswich, MA). 19. E. coli OmniMax 2 T1R (Invitrogen, Grand Island, NY). 20. E. coli SS320 (Lucigen, Middleton, WI). 21. 25 mg/mL kanamycin: Dissolve 25 mg of kanamycin in 1 mL of water and filter sterilize. 22. LB/carb plates: To 1 L of LB agar, add 1 mL of 100 mg/mL carbenicillin to achieve a final concentration of 100 μg/mL carbenicillin. 23. LB/kan plates: To 1 L of LB agar, add 1 mL of 25 mg/mL kanamycin to achieve a final concentration of 25 μg/mL kanamycin. 24. LB/tet plates: To 1 L of LB agar, add 2 mL of 10 mg/mL tetracycline to achieve a final concentration of 20 μg/mL tetracycline. 25. M13K07 helper phage (New England Biolabs). 26. MLB buffer: 1 M sodium perchlorate and 30% (v/v) isopropanol. 27. MP buffer: Dissolve 3.3 g citric acid monohydrate in 3 mL USP water at room temperature. Filter through a 0.2 μm syringe filter to give ca. 6 mL buffer.

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28. Phosphate-buffered saline (PBS): 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4. Adjust pH to 7.2 with HCl and autoclave. 29. PBST: PBS, 0.05% (v/v) Tween 20. 30. PEG/NaCl: 20% PEG-8000 (w/v), 2.5 M NaCl. Mix and filter sterilize. 31. Phenylmethanesulfonyl fluoride (PMSF): 100 mM in 96% ethanol. 32. QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). 33. QIAprep Spin Miniprep Kit (Qiagen). 34. SOC medium: 5 g bacto-yeast extract, 20 g bacto-tryptone, 0.5 g NaCl, and 0.2 g KCl. Add water to 1.0 L and adjust pH to 7.0 with NaOH and autoclave. Add 5.0 mL of autoclaved 2.0 M MgCl2 and 20 mL of filter-sterilized 1.0 M glucose. 35. Spectrophotometer. 36. SYBR Safe DNA gel stain (Invitrogen). 37. T4 DNA ligase (New England Biolabs). 38. T7 DNA polymerase (New England Biolabs). 39. T4 polynucleotide kinase (New England Biolabs). 40. TAE buffer: 40 mM Tris-acetate and 1.0 mM EDTA; adjust pH to 8.0; autoclave. 41. TAE/agarose gel: TAE buffer, 1.0% (w/v) agarose, 1:10000 (v/v) SYBR Safe DNA gel stain. 42. TE buffer: 10 mM Tris–HCl, 1 mM EDTA, and pH 8.0. 43. 10 mg/mL tetracycline: Dissolve 10 mg of tetracycline in 1 mL of 70% ethanol and filter sterilize. 44. Ultrapure glycerol. 45. Ultrapure irrigation US Pharmacopeia (USP) water (B. Braun Medical Inc., Bethlehem, PA). 46. Uridine: 0.25 mg/mL in water; filter sterilize.

3

Methods The following sections describe optimized protocols for the construction of phage-displayed libraries containing in excess of 1010 variants. This synthetic antibody diversity can rival or exceed that of the human periphery. A parental antibody framework is cloned into a phagemid vector to enable display on filamentous phage particles. The framework sequence in the phagemid is modified to introduce appropriate genetic diversity into the CDRs. Passing the genetic library through an E. coli host generates a phage-displayed antibody

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library that can be used for selections to isolate antigen-binding clones. This protocol uses an engineered human autonomous variable heavy domain (VH/dAb) as a library framework and incorporates diversity in three CDRs (Fig. 1b, c). This scaffold was generated by systematic mutagenesis of a VH3 domain derived from an approved therapeutic antibody to yield a variant with structurally compatible hydrophilic substitutions at the former light chain interface that promote autonomous behavior [31]. These libraries are routinely used to select human dAbs against a variety of targets with affinities in the nanomolar range. A protocol to engineer new alternative autonomous VH domain scaffolds is found in Tonikian and Sidhu [34]. 3.1

Phagemid Design

3.2 Library Construction

This protocol describes the construction of a synthetic dAb library in a phagemid vector. A phagemid (Fig. 2a) is a specialized vector with a double-stranded DNA origin of replication (dsDNA ori), which allows replication in E. coli, and a filamentous phage ori (f1 ori) to enable packaging of single-stranded DNA (ssDNA) into phage particles. Rather than inserting the antibody genes directly into the phage genome fused to a coat protein-encoding gene, antibody expression can thus be separated from phage propagation by providing the passenger antibody/coat protein fusion on a separate plasmid. Our phagemid has been used for the display of Fabs [35, 36], scFvs [37], VH domains [21, 33], peptides, and other polypeptides [38, 39]. Monomeric scFvs or VH domains are displayed by direct fusion to the N-terminus of the C-terminal domain of the truncated M13 bacteriophage minor coat protein 3 (pIII). Secretion signals direct the pIII-fusion to the periplasm. The isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter (Ptac) is used in the phagemid for display of VH domains. Incomplete repression of the promoter during phage production allows low expression of the VH-pIII fusion in the absence of limiting IPTG [40]. Display of heterodimeric Fabs requires bicistronic expression. Co-infection with helper phage such as M13K07 is used to provide additional components necessary to assemble new virions, which also contain phagemid-encoded pIII-fusions. This protocol is a scaled-up and optimized version based on the mutagenesis method of Kunkel et al. [41]. Mutagenic oligonucleotides are incorporated into heteroduplex covalently closed, circular, double-stranded DNA (CCC-dsDNA) by a three-step procedure (Fig. 2b). dU-ssDNA is used as a template for annealing of phosphorylated mutagenic oligonucleotides to prime the original strand for extension by T7 DNA polymerase followed by ligation by T4 DNA ligase. Upon transformation of the heteroduplex CCC-dsDNA into a dut+/ung+ host, the uracil-containing parental DNA strand is degraded, whereas the mutated strand is

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Fig. 2 Phagemid design and library construction workflow. (a) Phagemid vector designed for display of autonomous VH domain. The vector contains origins of single-stranded f1- (f1 ori) and a double-stranded DNA (dsDNA ori) replication and a selectable marker that confers resistance to carbenicillin (AmpR). An N-terminal stII signal sequence directs the VH-pIII fusion protein into the periplasm. (b) dU-ssDNA template is prepared from phage particles produced by CJ236 E. coli cells harboring the phagemid and superinfected with M13K07 helper phage. Mutations (*) are introduced through annealing of phosphorylated oligonucleotides on the dU-ssDNA template. Using T7 DNA polymerase and T4 DNA ligase, the oligonucleotides are extended and ligated to form heteroduplex covalently closed ds-DNA (CCC-dsDNA). Following transformation into the dut+/ ung+ E. coli SS320, the mutated strand is preferentially replicated, while the uracil-containing parental strand is degraded

preferentially replicated and propagated as a double-stranded plasmid. This procedure results in the formation of ca. 20 μg of highly pure product that can be electroporated into an E. coli host containing an F’ episome to enable M13 bacteriophage infection and propagation. This is sufficient to construct a library containing more than 1010 unique members. One of the major advantages of using this method is the ability to simultaneously mutate multiple CDRs in a single reaction without any need for restriction sites. Precise control over library design can be achieved by using mutagenic oligonucleotides than containing degenerate codons to introduce defined diversity at desired positions. Moreover, CDR length diversity can easily be introduced by using pools of degenerate oligonucleotides of varying lengths.

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Using a template with stop codons introduced in the CDRs intended for randomization can ensure display of only mutated antibodies. Templates with non-mutated CDRs will contain one or several stop codons that prevent expression of functional pIIIfusions and are thereby eliminated from the pool during binding selections. 3.2.1 Purification of dUssDNA Template

The use of highly pure dU-ssDNA is critical for successful library construction since mutagenesis efficiency depends on template purity. Template is prepared by using a modified Qiagen QIAprep spin M13 kit protocol. Twenty microgram is recommended for the construction of one library (see Note 1). 1. Transform the phagemid vector carrying the template sequence to be diversified into competent E. coli CJ236 (or analogous dut-/ung- strain). Plate on LB agar plate supplemented with appropriate antibiotic to select for the vector and grow overnight at 37 °C. 2. Pick a single colony of E. coli CJ236 containing the phagemid vector and inoculate 1 mL 2YT medium supplemented with appropriate antibiotics and M13K07 (1010 pfu/mL) in a 14 mL round-bottomed tube. For example, 2YT/carb/cmp medium contains carbenicillin to select for a phagemid carrying a β-lactamase gene and chloramphenicol to select for the F’ episome of E. coli CJ236. 3. Incubate at 37 °C with 200 rpm for 2 h before addition of kanamycin (25 μg/mL) to select for bacteria co-infected with helper phage M13K07. 4. Shake for 6 h at 37 °C. 5. Transfer the culture to a baffled 250 mL E-flask containing 30 mL 2YT/carb/kan/uridine medium and incubate at 37 °C and 200 rpm for 20 h. 6. Transfer cultures to 50 mL Falcon tubes and pellet bacteria by centrifuging at 27000× g for 10 min at 4 °C. 7. Transfer the phage-containing supernatant to a new tube containing 1/5 final volume of PEG/NaCl and incubate for 5 min on ice to precipitate phage. 8. Pellet precipitated phage by centrifugation at 27000× g for 20 min at 4 °C. Decant the supernatant. Centrifuge at 2000× g for 2 min and carefully aspirate the remaining supernatant. Always use filter tips when handling phage to avoid pipette contamination. 9. Use a pipette to resuspend the phage pellet in 0.5 mL PBS and transfer to a 1.5 mL microcentrifuge tube.

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10. Remove residual cell debris by centrifugation for 5 min at 15800× g in a benchtop microcentrifuge at room temperature (RT). All microcentrifuge steps are performed at RT. Transfer the supernatant to a fresh microcentrifuge tube. 11. Add 7 μL MP buffer and mix. Incubate at RT for at least 2 min. The solution should become cloudy when phages are precipitated in this step. 12. Apply the sample to a QIAprep spin column in a 2 mL collection tube. Spin for 30 s at 6000× g in a microcentrifuge and discard the flow-through. The phage particles remain bound to the column matrix. 13. Add 700 μL MLB buffer to the column. Spin for 30 s at 6000× g and discard the flow-through. 14. Add another 700 μL MLB buffer and incubate at RT for at least 1 min. 15. Spin for 30 s at 6000× g and discard the flow-through. The DNA is separated from the protein coat and remains adsorbed to the matrix. 16. Add 700 μL buffer PE (provided in QIAprep kit), centrifuge for 30 s at 6000× g, and discard the flow-through. 17. Add an additional 700 μL buffer PE (provided in QIAprep kit) and centrifuge for 30 s at 6000× g to remove residual proteins and salt. 18. Centrifuge the empty column for 30 s at 6000× g to remove residual PE buffer. 19. Discard the collection tube and transfer the QIAprep column to a fresh 1.5 mL microcentrifuge tube. Add 100 μL buffer EB (provided in QIAprep kit or 10 mM Tris–HCl, pH 8.5) to the center of the column membrane. Incubate at RT for 10 min. 20. Spin for 30 s at 6000× g and save the eluted purified dU-ssDNA. 21. Analyze the dU-ssDNA by electrophoresing 1 μL on a TAE/agarose gel. The DNA should appear as a predominant single band (Fig. 3). However, faint bands with lower electrophoretic mobility are often visible, which likely represent secondary structure in the dU-ssDNA. 22. Use absorbance at 260 nm to measure DNA concentration (A260 = 1.0 for 33 ng/μL of dU-ssDNA). Typical dU-ssDNA concentrations range from 200 to 500 ng/μL. 23. Aliquot 20 μg for each library to be prepared and store frozen.

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A B C

Fig. 3 Electrophoretic analysis of in vitro synthesis of heteroduplex covalently closed circular, double-stranded DNA (CCC-dsDNA). Lane 1: DNA markers; Lane 2: uracil-containing single-stranded DNA template (dU-ssDNA); Lane 3: product from the heteroduplex CCC-dsDNA synthesis reaction. The lower band (C) is correctly extended and ligated CCC-dsDNA, the middle band (B) is knicked dsDNA, and the upper band (A) is strand-displaced dsDNA 3.2.2 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Oligonucleotide Phosphorylation

1. For each mutagenic oligonucleotide, mix 0.6 μg oligonucleotide with 2 μL 10x TM buffer, 2 μL 10 mM ATP, and 1 μL 100 mM DTT in a 1.5 mL microcentrifuge tube. Add ultrapure irrigation water (USP water) to a total volume of 20 μL (see Note 2). 2. Add 20 U T4 polynucleotide kinase to each oligonucleotide and incubate for 1 h at 37 °C. Transfer reactions to ice and use as soon as possible for annealing (see Note 3).

3.2.3 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Annealing of Phosphorylated Oligonucleotides to the dUssDNA Template

1. To 20 μg dU-ssDNA template in a microcentrifuge tube, add 25 μL 10x TM buffer, 20 μL of each phosphorylated oligonucleotide (or oligonucleotide pool, see Note 4), and USP water to a final volume of 250 μL. Assuming a oligonucleotide to template length ratio of 1:100, these DNA quantities provide an oligonucleotide/template molar ratio of 3:1. 2. Incubate at 90 °C for 3 min, 50 °C for 3 min, and RT for 5 min using a thermocycler or dry block heaters.

3.2.4 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Enzymatic Synthesis of CCC-dsDNA

1. To the annealed oligonucleotide/template mixture, add 10 μL 10 mM ATP, 15 μL 100 mM DTT, 25 μL 10 mM dNTP mix, 30 Weiss units T4 DNA ligase (5 μL 400 U/μL), and 30 U (3 μL 10 U/μL) T7 DNA polymerase. 2. Mix and incubate at 20 °C overnight.

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3. Optional: Analyze 1 μL of the reaction mixture alongside the dU-ssDNA template on a TAE/agarose gel (see Step 11). 4. Purify and desalt the DNA using the QIAquick Gel Extraction Kit (Qiagen). Add 1 mL buffer QG (Qiagen) and mix (see Note 5). 5. Apply half of the sample to each of two QIAquick spin columns placed in 2 mL collection tubes. 6. Spin at 15800× g for 1 min in a microcentrifuge and discard the flow-through. 7. Add 750 μL buffer PE (Qiagen) to each column. Incubate 2–5 min at RT (see Note 6), centrifuge for 1 min at 15800× g, and discard the flow-through. 8. Spin the empty columns at 15800× g for 1 min to remove excess buffer PE. 9. Transfer the columns to fresh 1.5 mL microcentrifuge tubes and add 35 μL USP water to the center of each membrane. Incubate for 10 min at RT. 10. Spin at 15800× g for 1 min to elute the purified DNA. Combine the eluates from the two columns and determine DNA concentration by measuring absorbance at 260 nm (A260 = 1.0 for 50 ng/μL dsDNA). The total recovery should be at least 20 μg. The DNA can be used immediately for E. coli electroporation or stored frozen for later use. 11. Analyze 1 μL of the CCC-dsDNA by electrophoresis alongside the ssDNA template (Fig. 3). A successful reaction will result in near-complete conversion of ssDNA to dsDNA, which has lower electrophoretic mobility. Usually, two product bands are visible and no ssDNA should remain. The lower band with higher electrophoretic mobility represents the desired product: correctly extended and ligated CCC-dsDNA with a high mutation frequency (ca. 80%) and high E. coli transformation efficiency. The band with lower mobility is a stranddisplaced product, which results from undesirable activity of T7 DNA polymerase [42]. It provides a low (ca. 20%) mutation frequency and at least 30-fold lower transformation efficiency than CCC-dsDNA. A band with intermediate mobility between the other two product bands is sometimes visible. It represents correctly extended product but contains unligated dsDNA and may result from incomplete oligonucleotide phosphorylation or insufficient T4 DNA ligase activity. 3.3 Conversion of CCC-dsDNA into a Phage-Displayed Antibody Library

The final step of library preparation requires transformation of the heteroduplex CCC-dsDNA into an E. coli host containing the F′ episome to enable M13 bacteriophage infection and propagation. We use an E. coli strain (SS320) that is ideal for both high-efficiency

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electroporation and phage production [43]. Protocols to prepare M13K07 helper phage and electrocompetent E. coli SS320 pre-infected with M13K07 can be found in [34]. Once transformed with a phagemid, each cell will be able to produce phage particles without the need for further helper phage infection. 1. Chill the purified, desalted CCC-dsDNA (20 μg in a maximum volume of 100 μL) and a 0.2 cm gap electroporation cuvette on ice. 2. Pre-warm SOC medium in a water bath at 37 °C (2 × 1 mL in 1.5 mL microcentrifuge tubes and 25 mL in a 250 mL baffled E-flask). 3. Thaw a 350 μL aliquot of electrocompetent E. coli SS320 on ice. Add the cells to the DNA and mix gently by pipetting several times (avoid introducing air bubbles). 4. Transfer the mixture to the cuvette, wipe the outside with paper tissue, and electroporate according to the manufacturer’s instructions. We use a BTX ECM-630 electroporation system with the following settings: 2.5 kV field strength, 125 Ω resistance, and 50 μF capacitance. 5. Immediately rescue the electroporated cells by adding 1 mL pre-warmed SOC medium with a 1 mL sterile stripette, and transfer to 25 mL pre-warmed SOC medium in a 250 mL baffled E-flask. Rinse the cuvette with 1 mL SOC medium. 6. Incubate at 37 °C for 30 min with shaking at 200 rpm. 7. Prepare serial dilutions and plate on LB/carb plates to determine library diversity. Transfer 10 μL from the culture flask and make eight tenfold serial dilutions in 90 μL 2YT medium in a round-bottomed 96-microwell plate. Plate 5 μL of each dilution using a multi-pipette. Optional: Plate on LB/tet and/or LB/kan plates to determine total cell concentrations and titer of M13K07-infected cells, respectively (see Note 7). Incubate the plates at 37 °C overnight. 8. Transfer the culture to a 2-L baffled E-flask containing 500 mL 2YT/carb/kan medium for selection for phagemid and M13K07 helper phage, respectively. 9. Incubate at 37 °C and 200 rpm overnight. 10. After overnight incubation (ca. 18 h), transfer to two 1-L centrifuge bottles and pellet bacteria by centrifugation for 10 min at 16000× g at 4 °C. 11. Inoculate 25 mL 2YT/tet medium with a single colony of E. coli OmniMax 2 T1R from a fresh LB/tet plate. Grow at 37 °C and 200 rpm to mid-log phase (OD600 = 0.6–0.8), and use for phage titration (see Step 19).

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12. Transfer the supernatants to fresh centrifuge bottles containing 1/5 total volume of PEG/NaCl solution to precipitate phage. Incubate 20 min on ice. 13. Spin for 20 min at 16000× g at 4 °C to pellet precipitated phage. Decant the supernatant. Spin briefly (2 min 4000× g) and remove the remaining supernatant with a pipette. 14. Resuspend each phage pellet in 20 mL of pre-chilled TE buffer supplemented with 0.5 mM PMSF by gentle pipetting. 15. Combine the resuspended phage pellets and transfer to a clean 50 mL Falcon tube. 16. Pellet insoluble matter by centrifuging at 16000× g for 10 min at 4 °C. 17. Transfer supernatant to a clean tube containing 1/5 volume of PEG/NaCl. Incubate on ice for 20 min to precipitate phage. 18. Spin at 16000× g for 20 min at 4 °C and resuspend phage pellet in 4 mL PBST. 19. Determine phage concentration by infecting log-phase E. coli OmniMax 2 T1R cells with serial dilutions of phage: Dilute 10 μL in 90 μL 2YT and prepare 12 tenfold dilutions. Transfer 10 μL of each dilution to a 96-well round bottom plate and add 90 μL of log-phase E. coli OmniMax 2 T1R cells. Incubate still for 30 min at 37 °C and plate 5 μL of each dilution on LB/carb plates. Optional: Plate samples on LB/tet and LB/kan plates to determine cell number and helper phage concentration (see Note 8). 20. We recommend that the phage-displayed antibody library be used directly for selection experiments. Alternatively, it can be stored frozen at -80 °C following addition of glycerol to a final concentration of 10% and EDTA to a final concentration of 2 mM. Several protocols describing phage display selection strategies, screening, and expression of synthetic antibodies have been published [29, 35, 40, 44].

4

Notes 1. This protocol is based on the discontinued Qiagen QIAprep Spin M13 Kit for dU-ssDNA purification. The QIAprep Spin Miniprep Kit can be used with MP buffer prepared as described in the Materials section. Moreover, the MLB buffer originally provided in the QIAprep Spin M13 Kit was replaced with PB buffer, which resulted in lower yield of dU-ssDNA. We recommend using MLB buffer for lysis of phage particles to achieve comparable yield and quality.

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2. If length variation is desired, pools of oligonucleotides can be prepared and phosphorylated. It is recommended to test all oligonucleotides in small-scale annealing and enzymatic reactions using 1/20 of the volumes described above (Subheading 3.2.2) followed by gel electrophoresis to confirm efficient oligonucleotide-mediated conversion of ssDNA to dsDNA. 3. It is recommended to use phosphorylated oligonucleotides immediately for synthesis of CCC-dsDNA. However, they can be stored at -20 °C for up to a month without a significantly reduced performance. 4. If many different oligonucleotides are used to introduce length diversity in one CDR, they may be split into sub-pools during the mutagenesis reactions and pooled prior to electroporation (see (Chen, 2014 #7) for an example). 5. DNA adsorption to the QIAquick column is only efficient at pH below 7.5, under which the pH indicator in buffer QG is yellow. If the solution turns orange or violet upon addition of reaction mixture, adjust the pH by adding 10 μL 3 M sodium acetate (pH 5.0). 6. Incubation after addition of buffer PE helps remove salt in the DNA solution, which prevents potential electrical discharge during electroporation. 7. The titers from LB/tet and LB/kan plates should be approximately the same and carb ca. tenfold lower. Approximately 50% of cells survive after electroporation. 8. The expected phage concentration is 1012–1013 cfu/mL.

Acknowledgments Members of the Sidhu lab are acknowledged for input, particularly Alia Pavlenco and Wei Ye. We thank Frederic Fellouse for assistance with Fig. 3. References 1. Castelli MS, McGonigle P, Hornby PJ (2019) The pharmacology and therapeutic applications of monoclonal antibodies. Pharmacol Res Perspect 7(6):e00535. https://doi.org/ 10.1002/prp2.535 2. Kaplon H, Chenoweth A, Crescioli S, Reichert JM (2022) Antibodies to watch in 2022. MAbs 14(1):2014296. https://doi.org/10.1080/ 19420862.2021.2014296 3. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, Wu HC (2020) Development of therapeutic antibodies for the treatment of diseases.

J Biomed Sci 27(1):1. https://doi.org/10. 1186/s12929-019-0592-z 4. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517): 495–497 5. Tomszak F, Weber S, Zantow J, Schirrmann T, Hust M, Frenzel A (2016) Selection of recombinant human antibodies. Adv Exp Med Biol 917:23–54. https://doi.org/10.1007/978-3319-32805-8_3

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6. Bradbury ARM, Dubel S, Knappik A, Pluckthun A (2021) Animal- versus in vitroderived antibodies: avoiding the extremes. MAbs 13(1):1950265. https://doi.org/10. 1080/19420862.2021.1950265 7. Pedrioli A, Oxenius A (2021) Single B cell technologies for monoclonal antibody discovery. Trends Immunol 42(12):1143–1158. https://doi.org/10.1016/j.it.2021.10.008 8. Miersch S, Li Z, Hanna R, McLaughlin ME, Hornsby M, Matsuguchi T, Paduch M, Saaf A, Wells J, Koide S, Kossiakoff A, Sidhu SS (2015) Scalable high throughput selection from phage-displayed synthetic antibody libraries. J Vis Exp 95:51492. https://doi.org/10.3791/ 51492 9. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317 10. Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, Benkovic SJ, Lerner RA (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246(4935): 1275–1281 11. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554. https://doi. org/10.1038/348552a0 12. Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, Hashem AM (2020) Phage display derived monoclonal antibodies: from bench to bedside. Front Immunol 11:1986. https://doi.org/10. 3389/fimmu.2020.01986 13. Bradbury AR, Sidhu S, Dubel S, McCafferty J (2011) Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol 29(3):245–254. https://doi.org/10.1038/ nbt.1791 14. Carter PJ, Rajpal A (2022) Designing antibodies as therapeutics. Cell 185(15):2789–2805. https://doi.org/10.1016/j.cell.2022.05.029 15. Ledsgaard L, Ljungars A, Rimbault C, Sorensen CV, Tulika T, Wade J, Wouters Y, McCafferty J, Laustsen AH (2022) Advances in antibody phage display technology. Drug Discov Today 27(8):2151–2169. https://doi. org/10.1016/j.drudis.2022.05.002 16. Bradbury A, Pluckthun A (2015) Reproducibility: standardize antibodies used in research. Nature 518(7537):27–29. https://doi.org/ 10.1038/518027a 17. Gray A, Bradbury ARM, Knappik A, Pluckthun A, Borrebaeck CAK, Dubel S

(2020) Animal-free alternatives and the antibody iceberg. Nat Biotechnol 38(11): 1234–1239. https://doi.org/10.1038/ s41587-020-0687-9 18. Voskuil JLA, Bandrowski A, Begley CG, Bradbury ARM, Chalmers AD, Gomes AV, Hardcastle T, Lund-Johansen F, Pluckthun A, Roncador G, Solache A, Taussig MJ, Trimmer JS, Williams C, Goodman SL (2020) The antibody society’s antibody validation webinar series. MAbs 12(1):1794421. https://doi. org/10.1080/19420862.2020.1794421 19. Rouet R, Dudgeon K, Christie M, Langley D, Christ D (2015) Fully human VH single domains that rival the stability and cleft recognition of camelid antibodies. J Biol Chem 290(19):11905–11917. https://doi.org/10. 1074/jbc.M114.614842 20. Sidhu SS, Fellouse FA (2006) Synthetic therapeutic antibodies. Nat Chem Biol 2(12): 6 8 2 – 6 8 8 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nchembio843 21. Bond CJ, Wiesmann C, Marsters JC Jr, Sidhu SS (2005) A structure-based database of antibody variable domain diversity. J Mol Biol 348(3):699–709. https://doi.org/10.1016/j. jmb.2005.02.063 22. Fellouse FA, Li B, Compaan DM, Peden AA, Hymowitz SG, Sidhu SS (2005) Molecular recognition by a binary code. J Mol Biol 348(5): 1153–1162. https://doi.org/10.1016/j.jmb. 2005.03.041 23. Fellouse FA, Wiesmann C, Sidhu SS (2004) Synthetic antibodies from a four-aminoacid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci U S A 101(34):12467–12472. https://doi.org/10. 1073/pnas.0401786101 24. Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM (2003) Domain antibodies: proteins for therapy. Trends Biotechnol 21(11):484–490. https://doi.org/10.1016/j. tibtech.2003.08.007 25. Nilvebrant J, Tessier PM, Sidhu SS (2016) Engineered autonomous human variable domains. Curr Pharm Des 22(43):6527–6537 26. Moutel S, Bery N, Bernard V, Keller L, Lemesre E, de Marco A, Ligat L, Rain JC, Favre G, Olichon A, Perez F (2016) NaLiH1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. elife 5. https://doi. org/10.7554/eLife.16228 27. Yan J, Li G, Hu Y, Ou W, Wan Y (2014) Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic

Synthetic Antibody Libraries applications. J Transl Med 12:343. https://doi. org/10.1186/s12967-014-0343-6 28. Zimmermann I, Egloff P, Hutter CA, Arnold FM, Stohler P, Bocquet N, Hug MN, Huber S, Siegrist M, Hetemann L, Gera J, Gmur S, Spies P, Gygax D, Geertsma ER, Dawson RJ, Seeger MA (2018) Synthetic single domain antibodies for the conformational trapping of membrane proteins. elife 7. https://doi.org/ 10.7554/eLife.34317 29. Fellouse FA, Sidhu S (2013) Making antibodies in bacteria. In: Howard GC, Kase MR (eds) Making and using antibodies a practical handbook. CRC Press, pp 151–172 30. Nilvebrant J, Sidhu SS (2018) Construction of synthetic antibody phage-display libraries. Methods Mol Biol 1701:45–60. https://doi. org/10.1007/978-1-4939-7447-4_3 31. Barthelemy PA, Raab H, Appleton BA, Bond CJ, Wu P, Wiesmann C, Sidhu SS (2008) Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains. J Biol Chem 283(6): 3639–3654. https://doi.org/10.1074/jbc. M708536200 32. Ma X, Barthelemy PA, Rouge L, Wiesmann C, Sidhu SS (2013) Design of synthetic autonomous VH domain libraries and structural analysis of a VH domain bound to vascular endothelial growth factor. J Mol Biol 425(12):2247–2259. https://doi.org/10. 1016/j.jmb.2013.03.020 33. Nilvebrant J, Ereno-Orbea J, Gorelik M, Julian MC, Tessier PM, Julien JP, Sidhu SS (2021) Systematic engineering of optimized autonomous heavy-chain variable domains. J Mol Biol 433(21):167241. https://doi.org/10. 1016/j.jmb.2021.167241 34. Tonikian R, Sidhu SS (2012) Selecting and purifying autonomous human variable heavy (VH) domains. Methods Mol Biol 911:327– 353. https://doi.org/10.1007/978-161779-968-6_20 35. Lee CV, Liang WC, Dennis MS, Eigenbrot C, Sidhu SS, Fuh G (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 340(5):1073–1093. https://doi.org/10. 1016/j.jmb.2004.05.051 36. Persson H, Ye W, Wernimont A, Adams JJ, Koide A, Koide S, Lam R, Sidhu SS (2013)

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Chapter 5 Construction of Chicken and Ostrich Antibody Libraries Jeanni Fehrsen, Susan Wemmer, and Wouter van Wyngaardt Abstract Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage-displayed scFvs for use in veterinary diagnostics and research. To add diversity to the scFv repertoire, we expanded the library to include genes from the ostrich, indigenous to southern Africa. The libraries described in this chapter are based on the chicken and ostrich variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries. Key words Chicken antibody library, Ostrich antibody library, scFv, Naı¨ve, Immune, Phagemid, Recombinant antibody, Phage display

1

Introduction Recombinant antibody libraries derived from chicken or ostrich immunoglobulin genes can be used as a source of diagnostic and research reagents. Accessing their immunoglobulin repertoire is relatively easy since all the immunoglobulin variable heavy (VH) and all the variable light (VL) chain sequences are identical at their 5′ and 3′ ends. This implies that they can be amplified using only two sets of PCR primers [1–3]. Immunoglobulin diversity is generated in the bursa of Fabricius by gene conversion using pseudogene variable regions. This occurs in the first 4 months; therefore, the naı¨ve immunoglobulin repertoire can be accessed from avian bursal cells. Alternatively, chickens can be immunized with the antigens of interest and their spleens, blood lymphocytes, and/or bone marrow used as source of immunoglobulin mRNA [4–7]. The single-chain fragment variable (scFv) form of recombinant antibody described here consists of the immunoglobulin VH region and the VL region joined via a (Gly4Ser)3 flexible linker (see Fig. 1) [8]. The phagemid vector used enables the scFv to be either

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_5, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Flow diagram of the process to convert mRNA to the scFv gene construct using the two-component method. Primers are shown by solid lines and the arrows indicate the direction of extension. All chicken primers start with a “C” and those for ostrich start with “Os”

displayed as a fusion protein on the phage or alternatively expressed as a soluble scFv protein [9]. Diversity of the naı¨ve repertoire can be increased by using primers to randomize the complementaritydetermining regions (CDRs) [10, 11]. Shortening of the flexible linker between the VH and VL regions results in the formation of dimers or higher-order multimers and consequently the production of binding entities with higher avidity [5, 12]. Two strategies are described to link the variable domains of the heavy and light chains. In the first strategy, portions of the (Gly4Ser)3 linker are added to the 3′ end of the VH and the 5′

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Fig. 2 Expected translation of the scFv gene construct. Positions of primers (from Table 1 and Fig. 1) are indicated by the lines below. “X” indicates amino acids encoded by randomized codon (MNN). n number of (MNN) codons to encode the synthetic CDR3. Amino acids in gray are removed by the restriction enzymes before cloning into the vector

end of the VL using appropriate PCR primers. Splicing by overlap extension (SOE) [13] is then used to assemble the gene construct coding for the scFv (Fig. 1). This method is usually used for naı¨ve and immune libraries. The second option incorporates synthetically randomized VH CDR3s. The linker is amplified as a third component using primers complementary to the 3′ end of the VH chain and the 5′ end of the VL chain (Fig. 2). An extended primer complementary to the 3′ end of the VH chain adds the synthetic CDR3. The scFv gene construct is then assembled using a threecomponent SOE. Most of the methods described here, with some adjustments, are based on previously published work that has produced scFvs that have proven useful in veterinary diagnostics and research [5, 6, 11, 12, 14–18].

2

Materials (See Note 1)

2.1 RNA Isolation (See Note 2)

1. RNA as source of the VH and VL genes (see Note 3) 2. RNAlater™ stabilization solution 3. Diethylpyrocarbonate (DEPC) 4. RNase-free plasticware. Scalpels, syringes, Petri dishes, filter tips, and centrifuge tubes 5. Fine stainless steel sieve 6. TRI Reagent 7. 1-Bromo-3-chloro propane (BCP) 8. Isopropanol 9. 75% v/v ethanol in nuclease-free water 10. Nuclease-free water

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2.2 cDNA Synthesis, PCR, and Ligation

1. Thermocycler. 2. RT-PCR kit (TaKaRa RNA PCR Kit (AMV) Ver 3.0. TaKaRa Bio Inc., Shiga, Japan). 3. Primers (see Table 1).

Table 1 Nucleotide sequence of primers Primera

Nucleotide sequence (5′-3′)

Chicken variable heavy chain CVHFsfi

GTCCTCGCAACTGCGGCCCAGCCGGCCCTGATGGCGGCCGTGACG

CVHR

CCGCCTCCGGAGGAGACGATGACTTCG

CVHRlink

CCGCCAGAGCCACCTCCACCTGAACCGCCTCCACCGGAGGAGACGATGAC TTCGG

Chicken variable light chain CVLF

GACTCAGCCGTCCTCGGTGTCAG

CVLRnot

TGATGGTGGCGGCCGCATTGGGCTG

CVLFlink

TCAGGTGGAGGTGGCTCTGGCGGAGGCGGATCGGCGCTGACTCAGCCGTCC TCGG

Ostrich variable heavy chain OsVHF

GCCGTGCAGTTGGTGGAGTCCGG

OsVHR

TGAGGAGACGGTGACCGAGG

OsVHFsfi

GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGCCGTGCAGTTGGTGG

OsVHRlink CCGCCAGAGCCACCTCCACCTGAACCGCCTCCACCTGAGGAGACGGTGACCG Ostrich variable light chain OsVLFlink

CAGGTGGAGGTGGCTCTGGCGGAGGCGGATCGCAGCCAGCCTCGCTGT

OsVLRnot

TGATGGTGGCGGCCGCGGGCTGACCCAGGACG

Chicken primers for randomized synthetic heavy chain CDR3 RandVH

GACTTCGGTCCCGTGGCCCCATGCGTCGAT(MNN)n TTTGGCGCAGTAGTAGGTGCCGGTGTCCTC

Primers to amplify Gly-Ser GSfor

GGGGCCACGGGACCGAAGTC

GSrev

CGCTGACACCGAGGAC

Sequencing primers

a

OP52

CCCTCATAGTTAGCGTAACG

M13R

CAGGAAACAGCTATGAC

Primers were derived from [1–3, 11] for chicken and own sequencing data for ostrich. Restriction enzyme sites and extra bases to enable restriction digestion are underlined. M = A/C, N = A/C/G/T. n length of desired CDR3

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4. DNA polymerase. 5. High-fidelity DNA polymerase. 6. Agarose. 7. 50x TAE buffer: 2 M Tris, 1 M acetate, and 50 mM EDTA. Dissolve 242 g Tris in 700 mL ddH2O, add 57.1 mL glacial acetic acid and 100 mL 0.5 M Na2EDTA (pH 8), and make up to 1 L. 8. Crystal violet: 10 mg/mL stock solution. 9. Loading buffer (6x) for crystal violet gels: 2% Ficoll 400 and 0.002% xylene cyanol in ddH2O. 10. Gel extraction kit. 11. PCR Purification Kit. 12. Phage display vector; phagemid pHEN1 [9]. 13. SfiI and NotI restriction enzymes. 14. 2.5 M NaCl. 15. 1 M Tris (pH 8). 16. 10 mg/mL acetylated BSA. 17. Enzyme reaction purification kit. 18. T4 DNA ligase. 2.3 Electroporation and Growth of E. coli and Bacteriophage

1. Electroporator. 2. 0.1 cm electroporation cuvette. 3. Incubator at 37 °C and 30 °C. 4. E. coli TG1 electroporation-competent cells (Agilent Technologies, Santa Clara, USA, see Note 4) or chemically competent TG1 cells, e.g., Mix and Go cells (Zymo Research, Irvine, CA, USA). 5. SOC medium: 20 g tryptone, 5 g yeast extract, 0.5 g NaCl, and 0.186 g KCl in 970 mL ddH2O. Autoclave for 20 min. Add 10 mL 1 M MgCl2 and 20 mL 1 M glucose before use. 6. TYE agar: 10 g tryptone, 5 g yeast extract, 8 g NaCl, and 15 g agar in 900 mL ddH2O. Autoclave for 20 min and cool to 50 °C. Add 100 mL of 20% v/v glucose and 1 mL of 100 mg/mL ampicillin before pouring plates. 7. 2xTY: 16 g tryptone, 10 g yeast extract, and 5 g NaCl. Dissolve in ddH2O to obtain a final volume of 1 L. Autoclave for 20 min. 8. Helper phage M13K07. 9. Petri dishes, 9 cm and 15 cm. 10. 60% v/v glycerol.

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11. 20% w/v glucose: 20 g D-glucose, add glucose slowly to 70 mL ddH2O, when dissolved make up to 100 mL. Autoclave or filter sterilize. 12. PEG/NaCl: 20% w/v polyethylene glycol 6000 or 8000, 2.5 M NaCl. Autoclave for 20 min. 13. 10x PBS: 1.37 M NaCl, 27 mM KCl, 80 mM Na2HPO4.2H2O, and 18 mM KH2PO4. Dissolve 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4.2H2O, and 2.4 g KH2PO4 in 800 mL ddH2O and adjust to pH 7.4 and final volume of 1 L. Autoclave for 20 min. Alternatively use PBS tablets.

3

Methods

3.1 RNA Isolation from Tissue

Harvest lymphocytes from bursa, spleen, bone marrow, or blood (see Subheading 3.2) from chicken or ostrich. This is the source of the immunoglobulin genes. Total RNA is isolated and the VH and VL genes are amplified using specific primers after the mRNA in the pool is transcribed to cDNA. 1. Harvest tissue, cut into 0.5 cm pieces, and immediately place in RNAlater™ until ready to proceed. 2. Cut tissue into smaller pieces. 3. Place tissue fragments into 10 mL TRI Reagent® and homogenize into a Petri dish by forcing through a fine stainless steel sieve using the plunger of a 20 mL disposable syringe as a pestle. Add more TRI Reagent® if needed up to 30 mL. Transfer to a 50 mL disposable tube. 4. Centrifuge for 10 min at 1500× g to remove the cell debris. 5. Transfer supernatant fluid (SNF) to a clean 50 mL tube, add 3 mL BCP, vortex, and incubate at room temperature for 15 min. 6. Centrifuge at 2000× g for 15 min at 4 °C. 7. Transfer the clear aqueous phase to a clean centrifuge tube, add 15 mL isopropanol, vortex, and incubate at room temperature for 10 min to precipitate the RNA. 8. Centrifuge at 10000× g for 30 min at 4 °C to recover the RNA. 9. Discard the SNF, wash pellet with 75% ethanol, and centrifuge at 10000× g for 30 min at 4 °C. 10. Air-dry the pellet at room temperature for 10 min and dissolve in 500 μL nuclease-free water at 55–60 °C. 11. Remove 5 μL for analysis. Determine the concentration spectrophotometrically (OD of 1 at 260 nm = 40 μg/mL RNA). Store aliquots at -80 °C.

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1. Collect blood in anticoagulant and isolate lymphocytes from the blood. 2. Lyse approximately 107 cells 1 mL TRI Reagent® by pipetting and incubate at room temperature for 5 min. 3. Add 100 μL BCP/mL TRI Reagent, vortex, and incubate at room temperature for 15 min. 4. Centrifuge at 12000× g for 15 min at 4 °C. 5. Transfer the clear aqueous phase to a clean tube, add 500 μL isopropanol/mL TRI Reagent, vortex, and incubate at room temperature for 10 min to precipitate the RNA. 6. Centrifuge at 12000× g for 10 min at 4 °C. 7. Discard SNF, wash RNA pellet by adding 1 mL 75% ethanol, vortex, and then centrifuge at 12000× g for 5 min at 4 °C. 8. Air-dry the pellet at room temperature for 10 min and dissolve in 500 μL nuclease-free water at 55–60 °C. Analyze as above (see Subheading 3.1, step 11).

3.3 cDNA Synthesis and Amplification by PCR

1. Synthesize cDNA by reverse transcription. Prepare 20 separate 20 μL reactions to ensure a diverse mixture of mRNAs is amplified. 2. Each 20 μL reaction (using the TaKaRa RNA PCR Kit) consists of 5 mM MgCl2, 1x RT buffer, 1 mM dNTPs, 20 units RNase inhibitor, 5 units reverse transcriptase, 0.125 μM Oligo dT-Adapter primer, and 0.5–1 mg total RNA. Instead of Oligo dT-Adapter primer, a chain-specific primer can be used, e.g., OsVHR. 3. Synthesize cDNA in a thermocycler using the following conditions: 30 °C for 10 min, 42 °C for 1 h, 95 °C for 5 min, and 5 °C for 5 min. 4. The cDNA is then converted to dsDNA using primers specific for the VH and VL genes (see Table 1 and Note 5). Ten reverse transcription reactions (step 2) are used in conjunction with each set of primers. An individual reaction (using the TaKaRa RNA PCR kit) consists of 20 μL cDNA, 1x PCR buffer, 2.5 units TaKaRa Ex Taq HS, 0.4 μM of each primer (chicken: CVHFsfi and CVHR or CVLF and CVLRnot; ostrich: OsVHF and OsVHR and/or OsVLF and OsVLRnot), and ddH2O up to 100 μL (see Subheading 3.5 for primer options to generate synthetic randomized VH CDR3s for chicken). 5. Synthesize dsDNA in a thermocycler using the following conditions: 30 cycles: 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min, followed by a final extension at 72 °C for 3 min. 6. The VH and VL PCR products are analyzed by electrophoresis on a 1.5–2% agarose gel (Fig. 3). Usually there are additional

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Fig. 3 Agarose gel electrophoresis showing the expected sizes of the VH chain, VL chain, and the scFv gene construct. VH ≥ 400 bp, VL ≥ 300 bp and scFv ≥ 800 bp

amplicons present. These are removed by selectively purifying the desired VH or VL amplicons from the agarose gel. Crystal violet stained gels are used for this purpose as described below (also see Subheading 3.12). 7. Precipitate the VH and VL PCR products with 1 volume of isopropanol to concentrate the DNA. Centrifuge at 10000× g for 10 min. Wash the pellet with 70% ethanol, and spin at 10000× g for 10 min. Remove all the ethanol, and air-dry the pellet for 10 min. Resuspend the DNA pellet in approximately one-tenth volume ddH2O (this may take some time). 8. Load on a 2% agarose gel containing 10 μg/mL crystal violet (see Subheading 3.12). 9. Cut out the selected amplicons and purify the DNA fragments using an agarose gel extraction kit. 10. Quantify DNA spectrophotometrically. 3.4 Assembly of scFv Genes Based on Natural VH and VL

1. Segments of the (Gly4Ser)3 linker are added to the VH and VL gene fragments with primers CVHRlink, CVLFlink, OsVHRlink, and OsVLFlink (see Table 1). This results in overlapping regions on the 3′ end of the VH with the 5′ end of VL. 2. Each 100 μL reaction contains 1x PCR buffer, 0.8 mM dNTPs, 500 ng VH DNA, 500 ng VL DNA, 0.2 μM of each primer (CVHRlink and CVLFlink or OsVHRlink and OsVLFlink), 3 units Pfu polymerase enzyme, and 2.5 units TaKaRa Ex taq HS. Prepare two to four reactions.

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3. Use the following conditions: 94 °C for 2 min, then 15 cycles: 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 5 min. 4. Join the VH and VL chains via the overlapping regions by SOE. Primers CVHFsfi and CVLRnot or OsVHFsfi and OsVLRnot are added to complete and amplify the joined products. The amount of product from step 3 requires optimization for the SOE. Initially use 2 and 4 μL and evaluate the product on an agarose gel. Select the conditions that result in the most prominent amplicon at around 800 bp (Fig. 3) and repeat using 40 reactions. 5. Each reaction (using TaKaRa Ex Taq HS) consists of 1x PCR buffer, 0.8 mM dNTPs, 2 or 4 μL DNA from step 3, 0.2 μM of each primer (CVHFsfi/Os VHFsfi and CVLRnot/OsVLRnot), 2.5 units Ex taq HS, and ddH2O to a final volume of 100 μL. 6. Use the following conditions: 30 cycles of 94 °C for 1 min, 60 ° C for 1 min, and 72 °C for 1 min, followed by the final extension at 72 °C for 5 min. 7. The reaction mixtures are pooled and an aliquot is analyzed on a 1% agarose gel. If a single amplicon at ≥800 bp is observed, purify the product using a PCR Purification Kit. If multiple amplicons are present, concentrate the sample by precipitation and gel purify as described above (see Subheading 3.3, steps 7– 10). The scFv gene constructs are now ready for digestion with SfiI and NotI. Concurrently digest the vector (pHENI) using the same enzymes. 3.5 Assembly of scFv Genes Based on Natural VL and Synthetic, Randomized VH CDR3s

1. To construct a library containing synthetically randomized VH CDR3s, the linker, VH and VL genes are amplified separately prior to joining all three components by overlap extension. 2. Prepare the VL gene as described in Subheading 3.3. 3. The primer on the 5′ end of the VH gene (CVHFsfi) is combined with RandVH primers (see Note 6, Table 1). 4. Amplify the linker fragment with the primer set GSfor and GSrev (Table 1) using a clone containing the linker as template (see Note 7). 5. All the methods and conditions as in Subheadings 3.3 and 3.4 are used except Subheading 3.4, step 2 where the reactions contain 40 ng VL DNA, 40 ng VH DNA, and 54 ng linker. Here the primers, CVHRlink and CVLFlink, are omitted.

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3.6 Restriction Digestion of the scFv Gene Construct and pHEN1 Vector

1. Digest a total of 6 μg scFv “joined” product and 20 μg pHEN. Each 100 μL reaction (using Roche enzymes) should contain 2 μg scFv or vector DNA, 1x buffer M, 0.1 mg/mL acetylated BSA, and 40 U SfiI. Incubate overnight at 50 °C (see Note 8). 2. For the NotI digest, add to each 100 μL reaction from step 1: 3 μL of 2.5 M NaCl, 6 μL of 1 M Tris (pH 8), 0.5 μL of 10 mg/ mL acetylated BSA, 40 U NotI, and ddH2O to make a final volume of 150 μL. Incubate at 37 °C for at least 3 h or overnight. 3. Analyze the integrity of both digested products on a 1% agarose gel (see Note 9). 4. Purify the digested scFv construct with an enzyme reaction purification kit. 5. A stuffer fragment (+/-50 bp) is present between the SfiI and NotI sites of pHEN1 and should be removed. The pHEN1 restriction digest reactions are concentrated by precipitation, separated by crystal violet stained agarose gel electrophoresis, the vector excised from the gel and purified (see Subheadings 3.3, steps 7–10 and 3.12).

3.7 Ligation of the scFv Gene and Vector

1. The molar ratio of vector (4.5 kbp): insert (800 bp) should be 1:2 (approximately 100 ng vector: 40 ng insert). 2. Evaluate the reagents by preparing a small ligation reaction. This is followed by reactions containing a total of 3 μg digested vector and 1.2 μg digested scFv insert. Include a ligation reaction without insert as control to confirm the efficacy of the restriction digestions. 3. Each 50 μL ligation reaction contains 1 μg pHEN1, 0.4 μg scFv gene, 1x ligation buffer, and 3 U T4 DNA ligase (when using Roche enzymes) and ddH2O. 4. Incubate at 15 °C overnight. 5. Desalt the ligation before electroporation. This can be done by diffusion [19] with a DNA purification kit or by ethanol precipitation [20] and resuspension in a small volume of ddH2O (see Note 10). If using chemically competent cells, there is no need to desalt the ligation and just follow manufacturer’s instructions.

3.8

Electroporation

1. Electroporate a small aliquot to evaluate the efficacy of the ligation reaction (see Subheading 3.7) and to determine the amount of re-ligated vector. This ensures that competent cells are not wasted. 2. Use E. coli TG1 electroporation-competent cells.

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3. Each electroporation reaction consists of 2–4 μL salt-free ligation reaction and 40 μL freshly thawed electrocompetent cells. Premix and then transfer to a cold 0.1 cm electroporation cuvette. Pulse at 1700 V, 200 Ω, and 25 μF. 4. Transfer the cells immediately to 1 mL pre-warmed SOC medium and incubate at 37 °C with shaking for 1 h. 5. Plate tenfold dilutions (10-1 to 10-4) on TYE agar containing 100 μg/mL ampicillin and 2% glucose. 6. Pellet the rest of the cells at 2000× g, resuspend in a small volume of medium, and plate on a larger Petri dish (15 cm). 7. Based on the trial ligation, calculate the number of plates required for the remainder of the ligation reaction, as well as ligation reactions required to reach the predetermined library size (see Note 11). 8. From the titer plates, analyze a few clones to confirm the presence of the correct size insert. PCR the colonies using primers OP52 and M13R specific for pHEN1 (Table 1). The product should be around 1000 bp since these external primers add approximately 200 bp to the scFv gene construct. 9. Sequence a few clones with the same primers to confirm that the scFv gene constructs are correct (Fig. 2). 10. Scrape all the cells from the plates into 2x TY medium (see Note 12), add glycerol to final concentration of 15% v/v, aliquot, and freeze away at -70 °C. Make the aliquots of 500 μL or less since a small volume of cells is required per rescue, and once thawed the stock should rather not be used again. 11. Before freezing away, dilute a small aliquot 200x and determine OD600, in preparation for phage rescue (see Subheading 3.9). 3.9

Phage Rescue

1. For panning, phages are rescued from the bacterial stocks. An aliquot of the glycerol stock is taken and the number of cells should be at least 10x the library size (see Note 13). Example volumes are given in brackets for a small library of 107clones. OD600 of 1 = 8 × 108 bacteria/mL. 2. Inoculate the bacterial glycerol stock (80 μL) into 2xTY (200 mL) containing 100 μg/mL ampicillin and 2% glucose. The OD600 should be less than 0.05. Ensure that the number of cells is still 10× larger than the library size. 3. Incubate at 37 °C with shaking at 240 rpm for about 2 h until OD600 = 0.5. 4. Take enough to over-represent the library (40 mL) and add helper phage (e.g., M13KO7) at a ratio of 1:20 (bacteria: phage). Incubate at 37 °C without shaking for 30 min and 30 min with shaking at 100 rpm. Continue with step 6.

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5. The remainder of the culture (from step 3) can be centrifuged, resuspended in 1/100 volume of 2xTY containing 15% v/v glycerol, and stored at -70 °C. This serves as secondary glycerol stocks and is convenient if sub-libraries were pooled. 6. Centrifuge for 15 min at 3300× g and resuspend the cell pellet in 2xTY (200 mL) containing 100 μg/mL ampicillin and 25 μg/mL kanamycin but no glucose. 7. Grow overnight at 30 °C shaking at 240 rpm. 8. Centrifuge at 3300× g for 15 min and use the supernatant that contains the phages displaying the scFvs. The phages are concentrated by PEG precipitation (see Subheading 3.10). 3.10 PEG Precipitation of Phages

1. Add 1/5 volume PEG/NaCl to the supernatant containing the rescued phages (see Subheading 3.9). 2. Mix well and incubate on ice for at least 30 min. 3. Spin at ≥2000× g for 15 min. Discard the SNF. 4. Respin for 1 min to remove all of the PEG solution. 5. Resuspend in a small volume of PBS (5 mL). Titer the phage as described below (Subheading 3.11). 6. Freeze away aliquots in the presence of 15% v/v glycerol at 70 °C. 7. The phages are ready for affinity selection [21, 22]. Use 1000x or more phages than the primary library size for each panning.

3.11 Determining Phage Titer

1. Prepare “midlog” E. coli TG1 cells by inoculating 0.5 mL overnight TG1 culture into 50 mL 2xTY. 2. Incubate at 37 °C with shaking to an OD600 of 0.3–0.6. Use immediately. 3. Make tenfold dilutions of the phage stock in 2xTY or PBS (up to 10-10). 4. Mix 0.5 mL midlog TG1 with 0.5 mL phage dilution and incubate at 37 °C for 30 min without shaking. 5. Plate 100 μL on TYE plates containing 100 μg/mL ampicillin and 2% glucose. Incubate at 30 °C overnight. 6. Plate 100 μL of the “midlog” TG1 cells on an extra plate to ensure the cells are free of any contamination. 7. Determine the titer of the phage stock solution as follows: CFU=mL = number of colonies on plate × 1=dilution × 1=fraction plated × 2:

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1. Prepare agarose gel in 1x TAE. Don’t add usual stain such as ethidium bromide. 2. While cooling, add 10 mg/mL crystal violet solution to a final concentration of 10 μg/mL. 3. Add 6x loading buffer for crystal violet gels to DNA samples. 4. Electrophorese in the presence of 1x TAE containing 10 μg/ mL crystal violet. 5. The DNA bands can be observed as blue bands on a white light box.

4

Notes 1. Suppliers of chemicals and kits listed are used with success in our laboratory; these are naturally not the only options available. Where specified, the volumes apply to the kits/reagents. 2. When working with RNA, use disposable plasticware and RNase-free filter tips where possible. All other reagents and tools must be treated with DEPC in order to inactivate RNases. 3. To access the naı¨ve repertoire of chickens, isolate RNA from the bursae of 5-week-old chickens. For immune libraries, immunize chickens [24] and when the chickens have seroconverted, use the spleen. Lymphocytes from blood and bone marrow can also be used as source of RNA. Handling the chickens and harvesting the organs should be performed by a trained animal health technician and/or veterinarian according to your institute’s Animal Ethics Code. For the ostrich libraries, we used lymphocytes from blood, spleen, and bone marrow taken at the abattoir from 12-month-old birds, farmed for the food industry. 4. TG1 is an amber mutation suppressor strain to enable the pIIIscFv fusion protein production and has the F′ for phage transfection, both of which are essential. Overexpression with IPTG induction and/or alternative strains (HB2151) allows soluble scFv production. 5. In order to clone inserts in the pHEN1 vector [9], primers CVHFsfi and OSVHFsfi incorporate a SfiI site to the VH 5′ end and CVLRnot and OsVLRnot a NotI site to the VL 3′ end. 6. RandVH primers will vary according to the desired CDR3 length. Usually a sub-library is prepared for each CDR3 length. For the Nkuku® library, the synthetic VH CDR3s ranged from 6 to 14 amino acids [11]. 7. Alternatively, the same strategy can be followed as Subheading 3.3 by adding the linker sequence with a primer, but CVHRlink will have to be extended to create a longer overlap with the 3′ end of VH.

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8. The digestion at 50 °C is performed in a thermocycler (with a heated lid) to prevent condensation in the lid of the tube. 9. The agarose gel will not show a difference after restriction digestion of the scFv construct product or that the vector was digested by both enzymes, but we have had cases where there was possibly star activity or DNase contamination and the DNA reduced to smears. 10. Select a method that works best for you and results in the highest yield. We prefer ethanol precipitation. 11. By plating the primary library instead of growing it in liquid medium, all clones have an equal chance to grow. It is essential to titer the electroporated cells to determine the primary library size. You need to know that the library is large enough. Aim for at least 107 clones for an immune library and 109 for a naı¨ve or synthetic library. 12. Scrape cells into a small as possible volume. Use about 10 mL medium or less for three 15 cm plates. Add some medium to the first plate, scrape the colonies, and transfer to the second plate. Give the first plate a “wash” with fresh medium and transfer as before. Continue until all the plates are scraped, and add medium as needed. 13. Try to start with 10x the library size or more if possible. Do the calculations and determine the final volumes before you start to ensure that you have the capacity to process the calculated volumes. There are often multiple glycerol stocks from different ligations and these need to be mixed proportionally to allow equal representation of each sub-library. 14. Due to the lower sensitivity of the crystal violet stain, the gels must be “overloaded” to visualize the product. This is a convenient way to purify a large amount of DNA in a small volume.

Acknowledgments We thank Dr. Dion H du Plessis, our now retired research leader and mentor who started the phage display group. We are grateful to Dr. Marco Romito for the veterinary support, the good care of our chickens over the years, including taking them out for walks, and also to Mr. Rankala Isaac Palare for the daily care of the chickens. We thank Dr. Adrian Olivier (South African Ostrich Business Chamber, Oudtshoorn) for the donation of ostrich material and Prof. Celia Abolnik for the support to sequence the ostrich immunoglobulin genes. The Medical Research Council (Cambridge, United Kingdom) for the gift of pHENI vector. Funders, which made the work possible, include Agricultural Research Council-Onderstepoort Veterinary Research, Innovation Fund of the Department of Science and Technology, the National Department of Agriculture, and the Red Meat Research and Development, all from South Africa.

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References 1. Davies EL, Smith JS, Birkett CR, Manser JM, Anderson-Dear DV, Young JR (1995) Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes. J Immunol Methods 186:125–135 2. Yamanaka HI, Inoue T, Ikeda-Tanaka O (1996) Chicken monoclonal antibody isolated by a phage display system. J Immunol 157: 1156–1162 3. Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas CF III (2000) Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 242:159–181 4. Abi-Ghanem D, Waghela SD, David J, Caldwell DJ, Danforth HD, Berghman LR (2008) Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites. Vet Immunol Immunopathol 121:58–67 5. Chiliza TE, Wouter Van Wyngaardt W, Du Plessis DH (2008) Single-chain antibody fragments from a display library derived from chickens immunized with a mixture of parasite and viral antigens. Hybridoma 27:412–421. https://doi.org/10.1089/hyb.2008.0051 6. Abolnik CA, Fehrsen J, Olivier A, van Wyngaardt W, Fosgate G, Ellis C (2013) Serological investigation of highly pathogenic avian influenza (HPAI) H5N2 in ostriches (Struthio camelus). Avian Pathol. https://doi.org/10. 1080/03079457.2013.779637 7. Li B, Yea J, Lin Y, Wanga M, Zhua J (2014) Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9. Vet Immunol Immunopathol 161:258–264 8. Huston JS, Levinson D, Mudgett-Hunter M, Tai M-S, Novotny´ J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R, Oppermann H (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A 85:5879–5883 9. Hoogenboom HR, Griffiths AD, Johnson KS, David J, Chiswell DJ, Hudson P, Winter G (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucl Acids Res 19:4133–4137 10. Nissim A, Hoogenboom HR, Tomlinson IM, Flynn G, Midgley C, Lane D, Winter G (1994) Antibody fragments from a ‘single pot’ phage

display library as immunochemical reagents. EMBO J 13:692–698 11. Van Wyngaardt W, Malatji T, Mashau C, Fehrsen J, Jordaan F, Miltiadou DR, Du Plessis DH (2004) A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes. BMC Biotechnol 4:6 12. Sixholo J, van Wyngaardt W, Mashau C, Frischmuth J, Du Plessis DH, Fehrsen J (2011) Improving the characteristics of a mycobacterial 16kDa-specific chicken scFv. Biologicals 39:110–116 13. Clackson T, Hoogenboom HR, Griffiths A, Winter G (1991) Making antibody fragments using phage display libraries. Nature 352:624– 628 14. Fehrsen J, van Wyngaardt W, Mashau C, Potgieter C, Chaudhary VK, Gupta A, Jordaan F, du Plessis DH (2005) Serogroupreactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs. J Virol Methods 129(1): 31–39 15. Wemmer S, Mashau C, Fehrsen J, Wyngaardt W, du Plessis DH (2010) Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis. Biologicals 38:407–414 16. Rakabe M, Van Wyngaardt W, Fehrsen J (2011) Chicken single-chain antibody fragments directed against recombinant VP7 of bluetongue virus. Food Agric Immunol 22(3):283–295 17. Opperman PA, Maree FF, Van Wyngaardt W, Vosloo W, Theron J (2012) Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments. Virus Res 167:370–379 18. Van Wyngaardt W, Mashau C, Wright I, Fehrsen J (2013) Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs. J Vet Sci 14: 95–98 19. Atrazhef AM, Elliot JF (1996) Simplified desalting of ligation reactions immediately prior to electroporation into E. coli. BioTechniques 21:1024 20. Sambrook J, Russel DW (2001) Molecular cloning. A laboratory manual, Standard ethanol precipitation of DNA in microfuge tubes, vol 3, 3rd edn. Cold Spring Harbour Laboratory Press, New York, p A8.14

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21. Clackson T, Lowman HB (eds) (2004) Phage display. A practical approach. Oxford University Press, New York 22. Barbas CF III, Burton DR, Scott JK, Silverman GJ (eds) (2001) Phage display. A laboratory manual. Cold Spring Harbour Laboratory Press, New York 23. Rand KN (1996) Crystal violet can be used to visualize DNA bands during gel

electrophoresis and to improve cloning efficiency. TTO 1:23–24 24. Schade R, Staak C, Hendriksen C, Erhard M, Hugl H, Koch G, Larsson A, Pollmann W, van Regenmortel M, Rijke E, Spielmann H, Steinbusch, Straughan D (1996) The production of avian (egg yolk) antibodies: IgY. ATLA 24: 925–934

Chapter 6 Construction of Rabbit Immune Antibody Libraries Thi Thu Ha Nguyen, Jong Seo Lee, and Hyunbo Shim Abstract Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and wellestablished tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies. Key words Rabbit antibody, Antibody library, Phage display, Immune antibody library, Fab library, Rabbit monoclonal antibody

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Introduction Traditionally, most monoclonal antibodies have been generated by immunizing mice and preparing hybridomas [1]. Murine myeloma cell lines as fusion partners, as well as the protocols for fusion, cloning, and screening, have been well established. On the other hand, the generation of non-murine monoclonal antibodies by hybridoma technology has been hampered by the lack of suitable fusion partner cell lines and/or difficulty in the growth and cloning of the fused cells [2, 3]. Although some fusion partner cell lines and monoclonal antibodies are available from animals such as rats and rabbits [4–7], the technology is less well established than mouse

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_6, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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hybridoma and/or not widely accessible. Because these non-murine animals are considerably larger than mice and thus have a greater number and diversity of B cells, they may elicit stronger humoral immune response and produce higher-affinity antibodies against wider range of antigens or epitopes [4, 8]. Therefore alternative technologies to hybridoma that allow the production of monoclonal antibodies from non-murine sources are highly desirable. One of such technologies is antibody phage display, which enables rapid in vitro selection of target-specific antibody clones from a large pool of antibody fragments displayed on the surface of bacteriophage [9]. Through physical linkage of the antibody fragment and its gene on a same bacteriophage particle, target-binding antibody clones can be rapidly selected, amplified, and enriched from an antibody library by iterative rounds of biopanning. For successful antibody generation by phage display, it is important to prepare and use a large, high-quality antibody library. Antibodies can be isolated from either naı¨ve (unbiased) antibody libraries [9] or immune antibody libraries [10, 11]. The former can be constructed synthetically or from the B-cell repertoire of unimmunized animals and need to be highly diverse (>109 independent clones) in order to yield specific binders against a target antigen. The latter are made from the B cells of immunized animals, and because they are already enriched with binders produced by humoral immune response, their size may be much smaller; electroporation of E. coli routinely yields >108 transformants, which is often sufficient to overcome the loss of VH-VL pairing information during immune library construction process [12]. When an immune library is constructed from immunized laboratory animals such as mice or rabbits, the animals are sacrificed after the completion of immunization schedule, and their spleens are removed. Spleen contains a large number of B cells and plasma cells and hence is a good source of diverse antibody genes including those encoding target-specific antibodies. In this article, a detailed protocol for the construction of rabbit immune Fab library is provided. The PCR amplification and assembly protocols are based on Andris-Widhopf et al. [13], with modifications in primer sequences. Because the Fab format generally conserves the binding activity of the immunoglobulin antibodies better than scFv [14, 15], it may be the preferable format for immune library construction, although target-specific monoclonal antibodies have also been successfully isolated from immune scFv libraries. After construction, the phage-displayed antibody library can be panned against the immunization antigen for rapid isolation of multiple, high-affinity monoclonal antibody fragments.

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Materials Reagents and equipment suggested here can be substituted with equivalent products from different vendors. If not listed below, standard molecular biology laboratory equipment and molecular biology grade chemicals/reagents can be used. 1. Freund’s adjuvant: complete and incomplete (Sigma). 2. Maxi H minus First-Strand cDNA synthesis kit # K1651 (Thermo Scientific). 3. Nuclease-free water. 4. Oligonucleotide primers for polymerase chain reaction: See Table 1. 5. DNA polymerase: Taq polymerase (New England Biolabs, Ipswich, MA. USA) with vendor-provided reaction buffer. 6. dNTP mixture (10 mM each of four dNTPs, New England Biolabs). 7. T4 DNA ligase (New England Biolabs). 8. pCom3X-TT phagemid vector (Addgene, Cambridge, MA, USA. Plasmid #63891). 9. SfiI restriction endonuclease (New England Biolabs). 10. ER2537 electrocompetent cells: prepared according to [16]. 11. SB medium: Dissolve 20 g of yeast extract, 30 g of trypton, and 10 g of MOPS (3-(N-morpholino)propanesulfonic acid) in 1 L deionized water. Adjust pH to 7.2 and autoclave. Store at room temperature. 12. SOC medium: Dissolve 20 g of trypton, 5 g of yeast extract, and 0.5 g of NaCl in 950 mL of deionized water. Add 10 mL of 250 mM KCl. Adjust pH to 7.0 and add water to 1 L. Sterilize by autoclave, allow to cool down to 60 °C or less, and then add 10 mL of sterile 1 M MgCl2 and 20 mL of filter-sterilized 1 M glucose. 13. Agarose electrophoresis gel: for 1% agarose gel, use 1 g of agarose and 100 mL of TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA, pH 8.0). 14. QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). 15. LB-ampicillin glucose (LBAG) plate: Dissolve 10 g of trypton, 5 g of yeast extract, and 10 g of NaCl in 1 L deionized water. Add 18 g of bacteriological agar and autoclave. After the autoclaved solution cools down to 45–60 °C, add 50 mL of 40% (w/v) filter-sterilized glucose and 1 mL of filter-sterilized ampicillin (100 mg/mL). Mix gently and pour on 100-mmand 150-mm-diameter polystyrene Petri dishes (10 and 20 mL, respectively, per dish). Keep the plates at room temperature until agar solidifies, and store at 4 °C.

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Table 1 List of primers used in this protocol Name

Sequences

HIgCH1-f

GCCTCCACCAAGGGCCCA

Dpseq

AGAAGCGTAGTCCGGAACG

HKC-f

ACTGTGGCTGCACCATCTG

Lead-B

GGCCATGGCTGGTTGGGC

RVH1

GCCCAACCAGCCATG GCCCAG GAGCAGCTGAAGGAG

RVH2

GCCCAACCAGCCATG GCCCAGGAGCAG CTG RTG GAG

RVH3

GCCCAACCAGCCATGGCCCAGGAGCAGCTGGAGGAGTCC

RVH4

GCCCAACCAGCCATGGCCCAGTCGSTGGAGGAGTCC

RVH5

GCCCAACCAGCCATGGCCCAGTCGGTGAAGGAGTCC

RVH6

GCCCAACCAGCCATGGCCCAGCAGCTGGAGCAGTCC

RJH-b

TGGGCCCTTGGTGGAGGCTGARGAGAYGGTGACCAGGGT

RVK1

TAATTGGCCCAGGCGGCCGACCCTATGCTGACCCAG

RVK2

TAATTGGCCCAGGCGGCCGATGTCGTGATGACCCAG

RVK3

TAATTGGCCCAGGCGGCCGCAGCCGTGCTGACCCAG

RVK4

TAATTGGCCCAGGCGGCCGCCATCGATATGACCCAG

RVK5

TAATTGGCCCAGGCGGCCGCCCAAGTGCTGACCCAG

RVK6

TAATTGGCCCAGGCGGCCGCCCTTGTGATGACCCAG

RVK7

TAATTGGCCCAGGCGGCCGCTCAAGTGCTGACCCAG

RVK8

TAATTGGCCCAGGCGGCCTATGTCATGATGACCCAG

RJK1-b

AGATGGTGCAGCCACAGTTCGTTTGATTTCCACATTGGT

RJK2-b

AGATGGTGCAGCCACAGTTCGTTYGACSACCACCTYGGT

RJK3-b

AGATGGTGCAGCCACAGTTCGTAGGATCTCCAGCTCGGT

RJK4-b

AGATGGTGCAGCCACAGTTCGTTTGATYTCCASCTTGGT

RVL1

TAATTGGCCCAGGCGGCCCAGCCTGCCCTCACTCAG

RVL2

TAATTGGCCCAGGCGGCCTCCTATGAGCTGACACAG

RVL3

TAATTGGCCCAGGCGGCCTCCTTCGTGCTGACTCAG

RVL4

TAATTGGCCCAGGCGGCCCAGCCTGTGCTGACTCAG

RVL5

TAATTGGCCCAGGCGGCCAGCGTTGTGTTCACGCAG

RVL6

TAATTGGCCCAGGCGGCCCAGTTTGTGCTGACTCAG

RJL-b

AGATGGTGCAGCCACAGTTCGGCCTGTGACGGTCAGCTGGGT

Lead-VH

GCCCAACCAGCCATGGCC (continued)

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Table 1 (continued) Name

Sequences

RSC-SF

TAATTGGCCCAGGCGGCC

pC3X-f

GCACGACAGGTTTCCCGAC

pC3X-b

AACCATCGATAGCAGCACCG

Pelseq

ACCTATTGCCTACGGCAGCCG

16. Electroporation cuvette: 2 mm gap (Bio-Rad, Hercules, CA, USA). 17. Electroporator (Micropulser™, Bio-Rad). 18. VCSM13 helper phage (Agilent Technologies, Santa Clara, CA, USA). Helper phage can be prepared according to [16]. 19. Phosphate-buffered saline (PBS): Dissolve 80 g NaCl, 2.0 g KCl, 17 g Na2HPO4, and 1.63 g KH2PO4 in 0.95 L deionized water. Set pH to 7.4 with HCl, and add water to 1 L. 20. 5× PEG precipitation buffer: 20% (w/v) polyethyleneglycol8000, 15% (w/v) sodium chloride in deionized water. 21. Protease inhibitor cocktail: cOmplete™ EDTA-free (Roche).

3

Method

3.1 Rabbit Immunization

1. Prepare antigen (e.g., 350 μg of protein or 500 μg of keyhole limpet hemocyanin [KLH]-conjugated peptide/hapten) (see Note 1) in 500 μL of sterile PBS, and mix with 500 μL of Freund’s complete adjuvant, for a total of 1 mL antigen mixture per rabbit. 2. Subcutaneously inject the antigen mixture to New Zealand White rabbits (see Notes 2 and 3). It is recommended that two rabbits are immunized in order to improve the probability of success of antibody production. 3. Four weeks after the initial immunization, perform boost immunization. Subcutaneously inject the antigen (300 μg of protein or 400 μg of KLH-conjugated peptide/hapten) mixed 1:1 with Freund’s incomplete adjuvant (1 mL/animal). 4. One week after the first boost, take 1 mL blood from central auricular artery. Test the serum for the antigen-binding activity by indirect ELISA [17]. 5. Repeat two additional boost immunizations at 2-week interval. Perform bleeding and ELISA test as above.

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6. One week after the final (third) boost, collect the whole blood (>50 mL) by cardiac puncture (see Note 3). Place the blood stationary for 1 h at room temperature, centrifuge (500× g, 15 min), and store the serum at -20 °C for future use. 7. Surgically extract spleen from the rabbit, and proceed directly to Subheading 3.2. Alternatively, spleens can be frozen with liquid nitrogen and stored at -80 °C or kept submerged in RNA stabilization solution (e.g., RNAlater®) until use. 3.2 Preparation of Total RNA from the Spleens of Immunized Rabbits

1. Put the spleen removed from the immunized rabbit in a 50 mL conical polypropylene tube, and add 1 mL of TRI reagent per 100 mg of tissue. Homogenize the spleens using tissue homogenizer. 2. Allow the homogenized sample to stand for 5 min at room temperature. Centrifuge at 3000× g for 15 min, and transfer the supernatant to a fresh tube. 3. Add 0.1 mL of 1-bromo-3-chloropropane per mL of TRI reagent used, and shake vigorously for 20 s. Allow the sample to stand for 5 min at room temperature. 4. Centrifuge the sample at 12,000× g for 15 min at 4 °C. The mixture separates into three phases, of which the aqueous top layer contains RNA. 5. Carefully transfer only the top layer to a new centrifuge tube, and precipitate RNA by adding 0.5 mL of 2-propanol per 1 mL of TRI reagent used. Mix well and allow the mixture to stand for 5 min at room temperature. 6. Centrifuge the sample at 12,000× g for 10 min at 4 °C. Carefully remove supernatant and wash the RNA pellet with ice-cold 75% ethanol (1 mL per mL of TRI reagent used). Vortex the tube briefly, and centrifuge at 12,000× g for 10 min at 4 °C. 7. Remove supernatant and air-dry the pellet for about 5 min, taking care not to allow it to overdry. Dissolve the pellet in 0.5 mL of nuclease-free water. The RNA solution can be stored at -80 °C for several weeks.

3.3 Synthesis of First-Strand cDNA

1. To 5 μg RNA from Subheading 3.2, add 1 μL of 100 μM oligo (dT)18 primer, 1 μL of 10 mM dNTP mix, 4 μL of 5× RT buffer, and 1 μL Maxima H minus enzyme mix. Add nucleasefree water to a final volume of 20 μL. 2. The reaction mixture was incubated for 30 min at 50 °C and the reaction was terminated by heating the mixture at 85 °C for 5 min.

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3.4 Assembly of Rabbit/Human Chimeric Fab Repertoire by PCR

VH and VL repertoires were amplified from the rabbit spleen cDNA and assembled with the human constant domains (Cκ and CH1) into a Fab library in three steps.

3.4.1

In the first round of PCR, individual domains (variable and constant) are amplified. Variable domains are amplified from the cDNA, and constant domains are amplified from the pComb3XTT vector harboring genes for human Fab. Perform a separate reaction for each primer pair to maximally retain the diversity of the immunized repertoire.

First Round of PCR

1. For the amplification of rabbit VH and VL repertoires, add the following to nuclease-free water to make final volume of 100 μL in a PCR tube on ice: 1–3 μL cDNA (about 0.5 μg), dNTP mixture (0.2 mM final concentration of each dNTP), 10 μL of 10× standard Taq buffer, 0.5 μL of Taq polymerase, and 0.6 μM of forward and reverse primers. Primer sequences are shown in Table 1 and primer pairs for the amplification of rabbit VH and VL are shown in Table 2. 2. Perform PCR with the following thermal cycle: initial melting at 94 °C for 2 min; 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 7 min. 3. Electrophorese PCR products on 1% agarose gel with ethidium bromide, and inspect the gel under UV light. Excise gel bands for VH and VL at about 350 bp lengths and extract DNA from the excised band using the Gel Extraction Kit, according to the manufacturer’s protocol. 4. Add the following to nuclease-free water to make final volume of 100 μL in PCR tube on ice: 1 μg of pComb3X-TT vector DNA, dNTP mixture (0.2 mM final concentration of each dNTP), 10 μL of 10X standard Taq buffer, 0.5 μL of Taq polymerase, and 0.6 μM of forward and reverse primers. Sequences of primers are shown in Table 1 and primer pairs for the amplification of human CH1, CK are shown in Table 2. 5. Perform PCR and purify the amplified DNA by agarose gel extraction using the same protocol as step 3. 3.4.2 PCR

Second Round of

In the second round of PCR, Fd fragment (VH-CH1) and the light chain (LC; VL-CL) are generated by an overlap extension assembly of the rabbit variable domains and human constant domains from Subheading 3.4.1. VH, Vλ, and Vκ genes separately amplified in Subheading 3.4.1 can be combined at this stage, and only three PCRs (one each for VH, Vλ, and Vκ repertoires) are performed. 1. Use the following PCR mixture: 500 ng of template DNA, dNTP mixture (0.2 mM final concentration of each dNTP),

Table 2 Primer pairs for the amplification of VH, Vλ, Vκ, CH1, and Cκ Variable domain

Forward primer

Backward primer

VH1 VH2 VH3 VH4 VH5 VH6

RVH1 RVH2 RVH3 RVH4 RVH5 RVH6

RJH1

VK1

RVK1

RJK1-b RJK2-b RJK3-b RJK4-b

VK2

RVK2

RJK1-b RJK2-b RJK3-b RJK4-b

VK3

RVK3

RJK1-b RJK2-b RJK3-b RJK4-b

VK4

RVK4

RJK1-b RJK2-b RJK3-b RJK4-b

VK5

RVK5

RJK1-b RJK2-b RJK3-b RJK4-b

VK6

RVK6

RJK1-b RJK2-b RJK3-b RJK4-b

VK7

RVK7

RJK1-b RJK2-b RJK3-b RJK4-b

VK8

RVK8

RJK1-b RJK2-b RJK3-b RJK4-b

VL1 VL2 VL3 VL4 VL5 VL6

RVL1 RVL2 RVL3 RVL4 RVL5 RVL6

RJL-b

Human CH1

HIgCH1-f

Dpseq

Human Cκ

HKC-f

Leab-b

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Table 3 PCR scheme for the amplification of Fd chains and light chains by overlap extension PCR Template Template 1 (pooled)

Template 2

Forward primer

Reverse primer

Product name

VH1-VH6

CH1

Lead-VH

dpseq

Fd

VK1-VK8

Cκ

RSC-SF

Lead-B

LC (kappa)

VL1-VL6

Cκ

RSC-SF

Lead-B

LC (lambda)

10 μL of 10× standard Taq buffer, 0.5 μL of Taq polymerase, 0.6 μM of forward and reverse primers, and nuclease-free water to bring the final reaction volume to 100 μL. Primer sequences are shown in Table 1, and the primer pairs for the overlap extension PCR are shown in Table 3. 2. Perform the PCR under the following thermal cycles: initial melting at 94 °C for 2 min; 25 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min; final extension at 72 °C for 7 min. 3. Isolate the PCR products by 1% agarose gel electrophoresis as described above. The expected lengths for LC and Fd PCR products are around 750 bp. 3.4.3

Third Round of PCR

In the third round of PCR, the chimeric light chain products (kappa and lambda class) and the heavy chain (Fd) fragment are joined by second overlap extension PCR. Kappa and lambda LCs can be combined before this PCR. 1. Use PCR mixture: 500 ng of template DNA, dNTP mixture (0.2 mM final concentration of each dNTP), 10 μL of 10× standard Taq buffer, 0.5 μL of Taq polymerase, 0.6 μM of RSC-SF (forward) and dpseq (reverse) primers, and nucleasefree water to bring the final reaction volume to 100 μL. Perform eight 100 μL reactions in parallel. The primer sequences are provided in Table 1. 2. Perform the PCR under the following thermal cycle: initial melting at 94 °C for 2 min; 25 cycles of 94 °C for 30 s, 56 ° C for 30 s, and 72 °C for 1.5 min; final extension at 72 °C for 7 min. 3. Pool the PCR products (800 μL), and add 0.1 volume (80 μL) of 3 M sodium acetate (pH 5.2) and 2.5 volume (2 mL) of ethanol solution (70% final ethanol concentration). Mix well and incubate at -20 °C for over 1 h. Centrifuge the mixture at 14,000× g for 15 min, and wash the DNA pellet three times with ice-cold 70% ethanol. Air-dry the pellet briefly, and dissolve the DNA in 50 μL of nuclease-free water (see Note 4). Run the DNA on 1% agarose gel and extract Fab DNA from ~1500 bp band as described in Subheading 3.4.1, step 3.

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3.5 Construction of Fab Library

1. Digest the purified PCR product and pComb3X-TT phagemid vector with SfiI restriction enzyme. Incubate 9 μg of DNA and 40 units of SfiI in 50 μL reaction volume at 50 °C for 16 h (see Note 5). Separate DNA by electrophoresis and extract DNA bands (~1500 bp for Fab and ~3500 bp for the vector) from 1% agarose gel using DNA Gel Extraction Kit. 2. Mix 1 μg of the digested PCR product, 1.5 μg of the digested vector, 5 μL of 10× T4 ligase buffer, 2.5 μL of T4 DNA ligase (1000 units), and nuclease-free water to 50 μL final volume. Incubate the ligation reaction mixture overnight at room temperature. 3. Next morning, precipitate the ligated DNA as described in Subheading 3.4.3, step 3, and dissolve the pellet in 20 μL of 10% glycerol solution. 4. Mix the ligated DNA with 300 μL of ER2537 competent cells and add the mixture into an electroporation cuvette. Incubate the cuvette on ice for 1–2 min, and transform the bacteria by electroporation (a single 2.50 kV pulse). 5. After electroporation, immediately add 1 mL of warm (37 °C) SOC medium to the cuvette and resuspend the cells by pipetting up and down several times. Repeat the procedure twice again, and combine the bacterial suspensions (3 mL). Incubate the transformed cells at 37 °C for 1 h with shaking at 200 rpm. 6. For transformation titration, plate 10 μL and 100 μL of 10-3 dilutions of the transformed cells on LBAG agar plates (see Note 6). Centrifuge the remaining cells (3500× g, 15 min), and resuspend the pellet in 200 μL SOC medium. Plate the resuspended cell on a 150-mm-diameter LBAG agar plate and incubate overnight at 37 °C. 7. Next morning, add 5 mL of SB medium to the agar plates, and scrape the bacterial growth using flame-sterilized glass spreader. Add 0.5 volume of sterile 50% glycerol to the collected bacteria, mix well, and snap-freeze several 1 mL aliquots in liquid nitrogen. Store the frozen stocks at -80 °C or in a liquid nitrogen tank.

3.6 Phage Antibody Library Rescue

Phage-displayed antibody libraries can be rescued from the transformed E. coli by superinfection of the bacteria with helper phage. Helper phage is a derivative of M13 bacteriophage with a defective phage origin of replication that makes its production and packaging much slower than the phagemid DNA. As a result, when superinfected, the phage proteins inside their host E. coli preferentially package the phagemid DNA, ensuring the proper linkage of genotype with phenotype. Helper phages also have an antibiotic resistance gene, e.g., kanamycin, for the selection of superinfected

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bacteria. After helper phage superinfection, E. coli cells secrete antibody-displaying phage particles to culture medium. These phages can then be precipitated, resuspended in a small volume, and kept frozen until use. Biopanning protocols for the identification of target-specific clones are described elsewhere [16, 18]. 1. Thaw one 1 mL aliquot from Subheading 3.5, step 7, and add to 400 mL of SB medium supplemented with 100 μg/mL ampicillin and 2% (w/v) glucose (see Note 7). Grow at 37 °C with shaking at 200 rpm for 2~3 h, until OD600 reaches ~0.7. 2. Centrifuge the culture (3500× g, 15 min, 4 °C), and resuspend the pellet in 400 mL of SB medium with 100 μg/mL ampicillin and without glucose. Add VCSM13 helper phage (1012 pfu) (see Note 8), and superinfect the bacteria at 37 °C for 1 h with slow shaking (80 rpm). Add kanamycin to 70 μg/mL and incubate overnight at 30 °C with shaking at 200 rpm. 3. Next morning, centrifuge the culture (3500× g, 15 min, 4 °C). Transfer the supernatant to a clean centrifugation bottle, and add and dissolve 4% (w/v) PEG-8000 and 3% (w/v) NaCl. Keep the bottle in ice for >30 min to precipitate the phage. 4. Centrifuge the precipitated phage (3500× g, 15 min, 4 °C). Remove the supernatant, and dissolve the phage pellet in 10 mL of PBS. 5. Centrifuge again (12,000× g, 20 min, 4 °C) to remove insoluble cell debris, and transfer to a clean centrifugation tube. Add 0.25 volume of 5× PEG precipitation buffer, mix well, and keep the mixture in ice for >30 min. 6. Centrifuge the precipitated phage (12,000× g, 20 min, 4 °C), remove the supernatant, and dissolve the phage pellet in 2 mL PBS with protease inhibitor cocktail. The resulting phage solution is highly viscous. 7. Add 0.5 volume of 50% (v/v) glycerol, and mix well. Keep the mixture at 4 °C. 8. To measure phage titer, make 10-7 and 10-8 dilutions of the phage in SB medium, add 1 μL of the diluted phage to 50 μL of mid-log phase ER2537 E. coli cells and incubate for 30 min, and plate the infected bacteria on an LBAG plate. Incubate the plate overnight at 37 °C, and count the number of colonies next day to calculate the phage titer (see Note 9). 9. Aliquot 1012~1013 cfu of phage glycerol stock from step 7 into microcentrifuge tubes, and keep the aliquots frozen at -80 °C until use. Phage antibody library can be stored frozen for at least several months without affecting the antibody isolation capacity.

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Notes 1. Peptides are typically conjugated to KLH through amino, carboxylate, or thiol groups [19]. Conjugation site and chemistry need to be carefully chosen because linkage via a side chain within a desired epitope may interfere with antibody binding. For sequences that do not contain cysteines, thiol conjugation using maleimide chemistry may give best results. For haptens, functional groups may need to be introduced synthetically in order to enable the conjugation to a carrier protein away from the antigenic determinant. 2. New Zealand White (NZW) strain of rabbits are commonly used as an immunization host; however it has been reported that the Cys80 residue in the K1 kappa light chain isotype of NZW and many other rabbit strains may have a harmful effect on library diversity and functionality when existing as a free thiol (i.e., not in disulfide linkage to Cys171 of rabbit Cκ) and that the use of Basilea mutant or b9 wild-type rabbits yielded clones with greater diversity and affinity [20]. 3. For immunization, use 0.6 mL intramuscular injection of 1:1 mixture of Zoletil™ and Rompun™ for anesthesia. For terminal cardiac puncture, use 1 mL of the same mixture intramuscularly. Wait 15 min post-injection before beginning the procedure to ensure proper anesthesia of the animal. 4. Be careful not to overdry the pellet, since completely dry DNA pellet is difficult to re-dissolve. After removing supernatant, the tube can be briefly centrifuged, and the remaining liquid in the bottom of the microcentrifuge tube can be removed by pipetting. Remaining DNA pellet can then be immediately dissolved in water without brief air-drying. 5. When incubating the reaction mixture in a water bath at 50 °C, water evaporates and condenses underneath the cap of the microcentrifuge tube, effectively increasing the concentration of the mixture. To minimize this, the microcentrifuge tube containing the reaction mixture can be put into a 50 mL conical tube, which is then submerged in water bath. By doing this the temperature is kept homogeneous around the microcentrifuge tube and condensation can be prevented. 6. Transformation titer can be calculated by the following formula: ½No:of colonies × 2 ðmL recovery medium cultureÞ × 1000 ðμL=mLÞ= 10 or 100 ðμL platedÞ × dilution fold 10 - 3 7. Glucose is added to suppress lac promoter, which controls the transcription of Fab-pIII fusion gene in pComb3X vector.

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Because pComb3X phagemid vector has the N-terminally truncated form of pIII minor coat protein (pIII C-terminal domains), which does not inhibit the bacteriophage superinfection, glucose is not required for the helper phage superinfection. However, the cells harboring different antibody genes under the control of lac promoter grow more evenly in the presence of 2% glucose. 8. In 400 mL of E. coli culture at OD600 = 0.7, there are estimated to be ~1011E. coli cells [21]. 1012 pfu of helper phage is added to ensure near-complete superinfection of bacteria. 9. The phage titer is calculated as: ½No:of colonies × ðtotal volume of phage solution in μLÞ= dilution fold 10 - 7 or 10 - 8 × 1 ðμL of diluted phage added to ER2537Þ

Acknowledgments This work was supported in parts by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A1A10039823) and by Korea Drug Development Fund (KDDF) funded by Ministry of Science and ICT; Ministry of Trade, Industry, and Energy; and Ministry of Health and Welfare (HN21C0317, Republic of Korea) to H.S. References 1. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495 –497 2. Little M, Kipriyanov SM, Le Gall F, Moldenhauer G (2000) Of mice and men: hybridoma and recombinant antibodies. Immunol Today 21:364 –370 3. Shay JW (1985) Human hybridomas and monoclonal antibodies: the biology of cell fusion. In: Engleman EG, Foung SKH, Larrick JR, Raubitschek A (eds) Human hybridomas and monoclonal antibodies. Plenum Press, New York 4. Spieker-Polet H, Sethupathi P, Yam PC, Knight KL (1995) Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas. Proc Natl Acad Sci U S A 92:9348 –9352 5. Liguori MJ, Hoff-Velk JA, Ostrow DH (2001) Recombinant human interleukin-6 enhances the immunoglobulin secretion of a rabbitrabbit hybridoma. Hybridoma 20:189 –198

6. Keen MJ (1995) The culture of rat myeloma and rat hybridoma cells in a protein-free medium. Cytotechnology 17:193 –202 7. Digneffe C, Cormont F, Platteau B, Bazin H (1990) Fusion cell lines. In: Bazin H (ed) Rat hybridomas and rat monoclonal antibodies. CRC Press, New York 8. Bystryn JC, Jacobsen JS, Liu P, Heaney-Kieras J (1982) Comparison of cell-surface human melanoma-associated antigens identified by rabbit and murine antibodies. Hybridoma 1: 465 –472 9. Shim H (2016) Therapeutic antibodies by phage display. Curr Pharm Des 22:6538 10. Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, Benkovic SJ, Lerner RA (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246:1275 –1281 11. Barbas CF 3rd, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody

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libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88:7978 –7982 12. Hammers CM, Stanley JR (2014) Antibody phage display: technique and applications. J Invest Dermatol 134:1 –5 13. Andris-Widhopf J, Steinberger P, Fuller R, Rader C, Barbas CF 3rd (2001) Generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. In: Barbas CF 3rd, Burton DR, Scott JK, Silverman GJ (eds) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor 14. Steinwand M, Droste P, Frenzel A, Hust M, Dubel S, Schirrmann T (2014) The influence of antibody fragment format on phage display based affinity maturation of IgG. MAbs 6: 204 –218 15. Menzel C, Schirrmann T, Konthur Z, Jostock T, Dubel S (2008) Human antibody RNase fusion protein targeting CD30+ lymphomas. Blood 111:3830 –3837 16. Rader C, Steinberger P, Barbas CF 3rd (2001) Selection from antibody libraries. In: Barbas CF 3rd, Burton DR, Scott JK, Silverman GJ (eds) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

17. Andris-Widhopf J, Rader C, Barbas CF 3rd (2001) Generation of antibody libraries: immunization, RNA preparation, and cDNA synthesis. In: Barbas CF 3rd, Burton DR, Scott JK, Silverman GJ (eds) Phage display: a laboratory manual. Cold Spring Harbot Laboratory Press, Cold Spring Harbor 18. Yang HY, Kang KJ, Chung JE, Shim H (2009) Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Mol Cells 27:225 –235 19. Angeletti RH (1999) Design of useful peptide antigens. J Biomol Techniq 10:2 –10 20. Popkov M, Mage RG, Alexander CB, Thundivalappil S, Barbas CF 3rd, Rader C (2003) Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display. J Mol Biol 325: 325 –335 21. Tang X, Nakata Y, Li HO, Zhang M, Gao H, Fujita A, Sakatsume O, Ohta T, Yokoyama K (1994) The optimization of preparations of competent cells for transformation of E. coli. Nucleic Acids Res 22:2857 –2858

Chapter 7 Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries Martin A. Rossotti, Frederic Trempe, Henk van Faassen, Greg Hussack, and Mehdi Arbabi-Ghahroudi Abstract Naturally occurring heavy chain antibodies (HCAbs) in Camelidae species were a surprise discovery in 1993 by Hamers et al. Since that time, antibody fragments derived from HCAbs have garnered considerable attention by researchers and biotechnology companies. Due to their biophysico-chemical advantages over conventional antibody fragments, camelid single-domain antibodies (sdAbs, VHHs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple VHH-based therapeutic agents in different phases of clinical trials in various formats such as bi- and multivalent, biand multi-specific, CAR-T, and antibody-drug conjugates. The first approved VHH, a bivalent humanized VHH (caplacizumab), was approved for treating rare blood clotting disorders in 2018 by the EMA and the FDA in 2019. This was followed by the approval of an anti-BCMA VHH-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alphabased VHH drug (ozoralizumab; Nanozora®) in Japan for the treatment of rheumatoid arthritis. In this chapter we provide protocols describing the latest developments in isolating antigen-specific VHHs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble VHHs by ELISA, affinity measurements by surface plasmon resonance, functional cell binding by flow cytometry, and additional validation by immunoprecipitation. We present and discuss comprehensive, step-by-step methods for isolating and characterization of antigen-specific VHHs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated VHHs. To demonstrate the feasibility of the entire strategy, we present examples of VHHs previously isolated and characterized in our laboratory. Key words Single-domain antibody, VHH, Camelid, Library construction, Phage display, Panning, Site-specific biotinylation, Surface plasmon resonance, Flow cytometry

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Introduction Immunoglobulins or antibodies are generated through a complex mechanism of genetic recombination, allelic exclusion, and RNA splicing followed by translation, the assembly of the heavy and light chains, and display on the surface of B lymphocytes. Upon

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encountering the antigen, B cells proliferate and differentiate into plasma cells and secrete antibodies. Moreover, a group of activated B cells persist after the antigen has been eliminated and form immunological memory cells. Upon encountering the same antigen, these cells are quickly reactivated to generate a strong and effective immune response against the encountered antigen [1]. This is indeed the principle of vaccination practiced in humans and animals for decades to protect them against future viral and bacterial infections. Similarly, and for research purpose, animal immunizations take a similar path of exposing the animal to an antigen of interest followed by multiple rounds of boosting that can lead to high-affinity antibodies produced by B lymphocytes in the blood. These B cells are the source of the immune antibody repertoire, which can be used directly for generating hybridoma cells [2] capable of producing monoclonal antibodies (mAbs) or by using their mRNA transcripts to build antibody libraries to screen for antigen-specific antibody fragments [3]. Immunoglobulins are Y-shaped bifunctional glycoproteins that interact with antigen on the N-terminal variable domains and engage other cells and molecules of the immune system through C-terminal constant domains, and this paradigm has been relatively conserved in mammals, although Ig structure, in particular, in constant region varies among antibody isotypes across species. For instance, immunoglobulin G (IgG) is the most common isotype in humans and composed of two heavy and two light chains (H2L2). Each heavy chain has one variable domain (VH) and three constant domains (CH1, CH2, and CH3), and each light chain has one variable domain (VL) and one constant domain (CL) [1]. However, discovery of camelid heavy chain antibodies [4] followed by those found in cartilaginous fish [5] changed the canonical view of IgG where heavy chain-only antibodies (HCAbs; H2: VHH-Hinge-CH2-CH3) have complementary function to those of H2L2 molecules. Moreover, the first constant domain CH1 in heavy chain-only antibodies is spliced out by a single mutation in the exon-intron junction [6, 7]. This discovery coincided with the progress in the field of antibody engineering in the 1990s by which antibody fragments (Fab and scFv) derived from existing mAbs were expressed in simple bacterial system and shown to be functional in binding to their cognate antigens [8, 9]. Alternatively, these antibody fragments could also be cloned from the repertoire of an immunized donor to build an antibody library and then be displayed on the surface of filamentous phages for panning and screening [10, 11]. It was reasonable to assume that the same technology could be applied to the camelid HCAbs with an exceptional advantage that only single-domain (the VHH) cloning step would be required [12]. This straightforward cloning strategy for library construction and phage display bypasses a number of complex process, such as (1) genetically linking VH-linker-

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VL for scFvs or VH-CH1//VL-CL assembly for Fabs, (2) multidomain antibody library construction, (3) library stability, (4) the efficiency of displayed products on the phage surface, and (5) issues related to expression yield [13]. In contrast to scFv and Fab libraries, VHH libraries are also a true representation of the heavy chain repertoire generated in vivo. From the first successful report of VHH isolation against lysozyme and tetanus toxoid until today [11], there have been countless scientific reports on the isolation of VHHs against infectious agents, cancer biomarkers, and neurological targets. The VHHs are also ideal building blocks for bi- and multi-specific antibodies and CAR-T molecules [14, 15 McComb, S., 2019, 16]. Additionally, strategies have been developed to extend the short serum half-life of VHHs and reduce potential immunogenicity for human applications [17, 18]. In this chapter we provide detailed protocols for the construction of immune VHH libraries and isolation and preliminary characterization of antigen-specific VHHs. Specifically, we highlight (i) preparation and characterization of antigens, immunization of llamas, and analysis of polyclonal immune responses, (ii) phagedisplayed library construction using the amplified VHH repertoire, (iii) panning of the phage-displayed VHH library against recombinant protein targets, (iv) sub-cloning of panning-enriched VHHs into an expression vector and perform in vitro translation, (v) identification of positive VHH binders by protein ELISA, (vi) binding by SPR and flow cytometry, and (vii) antigen binding by immunoprecipitation. We provide a representative example of a VHH successfully isolated against a known cancer target and demonstrate binding in a variety of assays. The protocols presented have been used for a large panel of antigens, ranging from bacterial and viral surface proteins to cancer and neurological biomarkers and to intracellular proteins including the extracellular domain of membrane proteins [19, 20]. Alternative immunization methods including DNA and cell/VLP immunization coupled with carefully designed and more suitable selection strategies are required to successfully isolate functional VHH antibodies against multi-pass membrane proteins and intracellular proteins with therapeutic potential.

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Material Prepare all solutions using distilled and deionized water (ddH2O). ddH2O is prepared by purifying deionized water to attain a resistivity of 18 M Ω cm at 25 °C. Use analytical grade reagents to prepare buffers and other media solutions and store them at room temperature (RT) (unless indicated otherwise). Autoclave all media solutions and buffers on liquid cycle at 121 °C, 15 lbs./sq. for 20 min. Alternatively, sterilize autoclave-sensitive solutions by

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using 0.2 μm filter units. Follow diligently waste disposal regulations guidelines when disposing chemical waste materials and biohazard such as bacterial and viral cultures. 2.1 Antigen Preparation, Llama Immunization, and Immune Response Monitoring

1. Recombinant protein (antigen), commercially purchased or produced in-house and characterized, 0.5–1 mg (see Note 1) [19]. 2. Young male or female llama (Cedarlane, see Note 2). 3. Syringes (1 and 2 mL) with 21-G, 1–1.5-in.-long needles and 10–15 mL vacutainers for blood collection. 4. Heparin-coated tubes (Becton Dickinson). 5. Freund’s complete adjuvant (CFA) and incomplete adjuvants (IFA) (Sigma). 6. Lymphoprep® tubes (Progen, 1114544) or equivalent. 7. TRIzol (ThermoFisher, 15596026). 8. RPMI 1640 medium. 9. Pre-immune and immune sera (test and production bleeds). 10. 10× phosphate-buffered saline stock (PBS): Dissolve the following in 800 mL of MilliQ H2O: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, and 2.4 g KH2PO4, and adjust pH to 7.4 using 6 N HCl. Adjust volume to 1 L with additional MilliQ H2O. Sterilize by autoclaving. Dilute ten times to prepare 1x PBS (see Note 3). 11. PBST: PBS containing 0.05% (v/v) Tween-20. 12. Blocking buffer: StartingBlock™ (ThermoFisher). Alternatively, 1% casein, 1% BSA in PBS could be prepared in the lab. 13. Goat anti-llama IgG heavy and light chain antibody HRP conjugated (Bethyl Laboratories, A160-100P). ELISA substrate solution (Abcam, ab171523). 14. ELISA stop solution, 1 M H3PO4 or 1 M H2SO4. 15. 96-well microtiter plates (Nunc MaxiSorp, ThermoFisher, 44–2404-21 or equivalent). 16. Multiskan™ FC Microplate Photometer ELISA Reader (ThermoFisher). 17. Common laboratory consumables including 0.1-1000 μL filter tips, 0.5 and 1.5 mL Eppendorf tubes, 15 and 50 mL falcon tubes, and parafilm and paper towels from various supplies.

2.2 VHH Library Construction

1. Peripheral blood lymphocytes in TRIzol solution. 2. Invitrogen™ SuperScript™ VILO™ cDNA Synthesis Kit. (ThermoFisher, 11754050).

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3. Library primers (10 pmol/μL): MJ1: 5′-gcccagccggcc ATGGCC TGGAKTCTGGGGGA -3′

SMKGTGCAGCTGG

AQPAMA XVQLVXSGG (partial SfiI + MA to restore the pelB leader sequence) MJ2: 5′-gcccagccggcc ATGGCC AGGAGTCTGGGGGA -3′

CAGGTAAAGCTGG

AQPAMA QVKLEESGG MJ3: 5′-gcccagccggcc ATGGCC TGGAGTCT -3′

CAGGTACAGCTGG

AQPAMA QVQLVES (The VH/VHH N-terminal regions are in capital letter and underlined) CH2FORTA4: 5’- CGCCATCAAGGTACCAGTTGA -3′ CH2B3-F: 5′-GGGGTACCTGTCATCCACGGACCAGC TGA-3′ 4. VHH domain primers (10 pmol/μL) (see Note 4). MJ7: 5′- catgtgtagactcgcggcccagccggcc ATGGCC -3′. MJ8: 5′- catgtgtagattcctggccggcctggcc TGAGGAGACGGT GACCTGG -3′. (SfiI sites are underlined and coding regions are in capital letter) 5. Invitrogen™ Platinum™ polymerase (5 units/μL) and 10× PCR buffer containing MgCl2 (ThermoFisher, 10966026). 6. dNTP mix: 10 mM each of dTTP, dATP, dCTP, and dGTP. 7. 0.2 mL PCR tubes, strips, or plates (Diamed). 8. BioRad T100 Thermal Cycler (BioRad, Hercules) or equivalent. 9. pMED1 phagemid vector [21, 22]. 10. SfiI (10 units/μL) (ThermoFisher, ER1821). 11. FastDigest restriction endonucleases XhoI and PstI restriction endonucleases and their respective 10× buffers (ThermoFisher, FD0694 and FD0614). 12. Promega Wizard™ SV Gel and PCR Cleanup System (Promega, PR-A9281). 13. GeneJET Gel Extraction and DNA Cleanup Micro Kit (ThermoFisher, K0832). 14. 50× TAE buffer (per L): 242 g (2 M) Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA, pH 8, solution in ultrapure H2O. Store at RT. Dilute 1:50 in H2O to prepare 1× TAE buffer.

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15. 1% (w/v) agarose gel, prepared in 1× TAE buffer. 16. Agarose gel electrophoresis reagents and equipment. 17. NanoDrop™ One Microvolume UV-Vis spectrophotometer (NanoDrop Technologies) or a similar instrument (see Note 5). 18. T4 DNA ligase and 5× buffer (ThermoFisher, 15224041) (see Note 6). 19. Escherichia coli TG1 electrocompetent cells (Lucigen) (see Note 7). 20. Electroporation cuvettes 0.1 cm gap (BioRad). 21. MicroPulser™ electroporator (BioRad) or equivalent electroporation device. 22. Glucose 36% (w/v) solution: 360 g glucose per L dissolved in ultrapure ddH2O sterilized by passing through a 0.22 μm filter and store at RT. 23. SOC medium: 20 g tryptone, 5 g yeast extract, 0.58 g NaCl, and 0.19 g KCl, in 1 L of ddH2O. Autoclave, cool, and add 0.4% filter-sterilized glucose and 10 mM filter-sterilized MgCl2. Store at -20 °C in 1 mL aliquots. 24. Ampicillin stock solution: 100 mg/mL in ddH2O, filtersterilize and store at -20 °C in 0.5–1 mL aliquots. 25. 2 × YT medium: 16 g tryptone, 10 g yeast extract, and 5 g NaCl, in 1 L of ddH2O. Autoclave to sterilize. 26. LB medium (per L): 10 g tryptone, 5 g yeast extract, and 10 g NaCl dissolved in 1 L ddH2O. Autoclave to sterilize. 27. 2 × YT-Amp-medium: 2 × YT + 100 μg/mL ampicillin. 28. 2 × YT-Amp-Glu medium: 2 × YT + 100 μg/mL ampicillin +2% (w/v) filter-sterilized glucose. 29. LB-Amp plates: Add 15 g agar in 1 L of LB medium. Autoclave, cool to ~55 °C, add ampicillin to a final concentration of 100 μg/mL, pour plates, and store at 4 °C for up to a month. 30. 80% (v/v) glycerol. Autoclave to sterilize. 31. Shaker flasks. 32. Colony-PCR primers at 10 pmol/μL: M13 universal reverse primer (M13RP): 5′CAGCTATGAC -3′.

CAGGAAA-

-96gIII (PN2) primer: 5′- CCCTCATAGTTAGCGTAACG ATCT -3′ 33. Common laboratory consumables (see Subheading 2.1, step 18).

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34. Common laboratory equipment including shaker incubator, vortex, heat block, 37 °C incubator, Sorvall centrifuge, highspeed, swinging-bucket benchtop refrigerated centrifuge (4 °C), Thermo Micro 17 and 21R centrifuges, Thermo Scientific Sorvall RC-6 Plus centrifuge. 35. Access to Sanger DNA sequencing facility. 2.3 Phage Rescue from the Library

1. Frozen glycerol stock library cells (5 × 1010 cell/mL). 2. Ampicillin stock solution (see Subheading 2.2, step 24). 3. Kanamycin stock solution: 50 mg/mL in ddH2O, filtersterilize and store at -20 °C in 0.5–1 mL aliquots. 4. 2 × YT-Amp-medium (see Subheading 2.2, step 25). 5. M13KO7 helper phage (New England Biolabs, N0315S). 6. 0.22 μm Steriflip-GP sterile centrifuge tube top filter unit and 0.22 μm Stericup-GP sterile vacuum filtration system (MilliporeSCGP00525 and SCGPU02RE). 7. Sterile PEG/NaCl solution: 20% (v/v) PEG 8000, 2.5 M NaCl. Autoclave, store at RT. 8. E. coli TG1 cells (see Note 8). 9. LB-Amp plates (see Subheading 2.2, step 29). 10. Sterile 1× PBS (see Subheading 2.1, step 10). 11. Protease inhibitor tablets (Sigma, catalog number: S8820). 12. Common laboratory consumables (see Subheading 2.1, step 18). 13. Shaker flask. 14. Common laboratory equipment (see Subheading 2.2, step 34).

2.4 Biopanning of Phage-Displayed Library

1. Recombinant biotinylated antigen (see Note 9). 2. NUNC MaxiSorp™ plates (ThermoFisher) or equivalent. 3. Streptavidin (Jackson ImmunoResearch, 016–000-113). 4. StartingBlock (see Subheading 2.1, step 12). 5. Phage library (~1 × 1012 cfu/mL). 6. Sterile 1× PBS (see Subheading 2.1, step 10). 7. Wash buffer (PBS-T): Prepare PBS-T by adding 0.05% (v/v) Tween-20 to 1× PBS. 8. 0.22 micron syringe filters. 9. 10 mL syringe(s). 10. Sterile PEG solution (see Subheading 2.3, step 7). 11. Elution buffer: 100 mM triethylamine: Add 35 μL of 7.18 M triethylamine in 2.5 mL of MilliQ H2O, made fresh daily.

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12. Neutralization buffer: 1 M Tris–HCl pH 7.4. 13. LB-Amp/agar plates (see Subheading 2.2, step 29). 14. Small diameter plates for LB-agar. 15. 2 × YT-Amp-medium (see Subheading 2.2, step 27). 16. E. coli TG1 cells (see Subheading 2.3, step 8). 17. M13KO7 helper phage (see Subheading 2.3, step 5). 18. Kanamycin stock solution (see Subheading 2.3, step 3). 19. Common laboratory consumables (see Subheading 2.1, step 18). 20. Common laboratory equipment (see Subheading 2.2, step 34). 2.5 Polyclonal and Monoclonal Phage ELISA Screening (See Note 10)

1. NUNC MaxiSorp ™ plates. 2. Streptavidin (see Subheading 2.4, step 3). 3. Biotinylated antigen(s). 4. 1× PBS (see Subheading 2.1, step 10) 5. Wash buffer (PBS-T) (see Subheading 2.4, step 7). 6. StartingBlock (ThermoFisher) or alternative blocking buffers (see Subheading 2.1, step 12). 7. Phage library: phages rescued before and after each round of panning (see Subheading 3.4, steps 7 and 9). 8. ELISA substrate (see Subheading 2.1, step 14). 9. ELISA stop solution (see Subheading 2.1, step 15). 10. Multiskan FC microplate reader (ThermoFisher). 11. Bacterial colonies from round 3 or 4 seeded in LB-Amp plates. 12. 2 × YT-Amp-Glu medium: 2 × YT + 100 μg/mL ampicillin +0.1% (w/v) filter-sterilized glucose. 13. 96-well deep well plates (Cole-Palmer, 229572), or equivalent 14. Gas-permeable adhesive tape (VWR, 10141–844). 15. M13KO7 helper phage (see Subheading 2.3, step 5). 16. Mouse anti-M13 IgG conjugated to HRP (Sino Biological, 11973-MM05T-H). 17. Kanamycin stock solution (see Subheading 2.3, step 3). 18. Humid chamber for culture plate (see Note 12). 19. Common laboratory consumables (see Subheading 2.1, step 18). 20. Common laboratory equipment (see Subheading 2.2, step 34).

Isolation and Characterization of Single-Domain Antibodies from Immune. . .

2.6 Sub-cloning of R3/R4 Phage Pool, In Vitro Translation and Screening of the Soluble VHHs (An Alternative to Monoclonal Phage ELISA Screening; See Note 10)

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1. Eluted phages from R3/R4 (see Note 11). 2. E. coli TG1 cells (see Subheading 2.3, step 8). 3. Ampicillin stock solution (see Subheading 2.2, step 24). 4. M13KO7 helper phage (see Subheading 2.3, step 5). 5. Kanamycin stock solution (see Subheading 2.3, step 3). 6. Common laboratory consumables (see Subheading 2.1, step 18). 7. Common laboratory equipment (see Subheading 2.2, step 34). 8. M1 Buffer: 30% PEG 6000, 3 M NaCl, autoclave and store at 4 °C. 9. M2 Buffer: 1% Triton X-100, 500 mM guanidine-HCl, 10 mM MOPS, and pH 6.5, store at RT. 10. QIAGEN Plasmid Miniprep (QIAGEN). 11. 0.2 μm GP Express™ Plus Membrane filtration system (see Subheading 2.3, step 6). 12. MJ7 and MJ8 primers (see Subheading 2.2, step 4). 13. PCR reagents and equipment (see Subheading 2.2, steps 5–8). 14. GeneJET Gel Extraction and DNA Cleanup Micro Kit (see Subheading 2.2, step 13). 15. pMRO expression vector [19, 23] (pET28a + [pelB][sfiI]-VHH-[sfiI]-BAP.H6). 16. SfiI restriction endonucleases and 10× buffer (see Subheading 2.2, step 10). Alkaline phosphatase (1 unit/μL) and 10× buffer (ThermoFisher, EF0651). 17. T4 DNA ligase and 5× buffer (see Subheading 2.2, step 18). 18. Savant SpeedVac concentrator (ThermoScientific). 19. T7 promoter forward (Fw) primer (10 pmol/uL): 5′- TAATACGACTCACTATAGGG-3′. 20. T7 terminator reverse (rv) primer (10 pmol/ul) 5′- CCGCTGAGCAATAACTAGC-3′. 21. 80% (v/v) glycerol. 22. Sanger DNA sequencing facility (see Subheading 2.2, step 35). 23. Promega S30 T7 High-yield Expression System (Promega, L1110). 24. pMRO-VHH plasmid DNAs from individual clones. 25. LB-Kan: LB + 50 μg/mL kanamycin (see Subheading 2.3, step 3). 26. NUNC MaxiSorp™ plates. 27. Biotinylated antigen(s) or antigen fused to human IgG Fc domain.

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28. Streptavidin (see Subheading 2.4, step 3). 29. Blocking solution (see Subheading 2.1, step12). 30. Goat anti-llama IgG, VHH-HRP conjugated (Jackson ImmunoResearch, 128–035-230). 31. 1× PBS (see Subheading 2.1, step 10). 32. In vitro translated VHHs (see Subheading 3.6.2, step 3). 33. ELISA substrate solution (see Subheading 2.1, step 14). 34. ELISA stop solution (see Subheading 2.1, step 15). 35. Multiskan FC microplate photometer (see Subheading 2.1, step 17). 2.7 VHH Sub-cloning, Soluble Expression, and Purification

1. pMRO-VHH constructs (TWIST Bioscience; or from Subheading 2.6) (see Note 13). 2. E. coli BL21 (DE3) electrocompetent cells. 3. E. coli transformation reagents (see Subheading 2.2, steps 19–21). 4. E. coli AVB101 cells harboring the pACYC184 plasmid (Avidity LLC) [24]. 5. LB-Kan agar plates: Add 15 g agar in 1 L of LB medium. Autoclave, cool to ~55 °C, add kanamycin to a final concentration of 50 μg/mL, pour plates, store at 4 °C for up to a month. 6. Sterilized 500 mL baffled flasks. 7. Polypropylene centrifuge bottles (500 mL). 8. Protease inhibitor tablets (see Subheading 2.3, step 11). 9. Emulsiflex-C5 connected to a nitrogen tank (Avestin) or Ultrasonic Homogenizer to lyse the bacteria (OMNI International). 10. Sterile polypropylene centrifuge tubes. 11. Chloramphenicol stock solution (Sigma, C0378) (Cam) 1000× stock solution (35 mg/mL) is prepared in ethyl alcohol anhydrous under sterile condition. Store at -20 °C in the dark (see Note 14). 12. D-(+)-biotin solution (1000× stock at 100 mM) (VWR97061–446). Dissolve 1.52 g of D-biotin in NaOH 0.1 M, filter-sterilize (0.22 μM), and store at -20 °C. If necessary, add drops of 1 M NaOH until the powder dissolves completely. 13. ATP solution (100 mM) (Alfa Aesar, CAAAJ61125–09). Prepare 1000× stock solution by dissolving 2.75 g in 50 mL of PBS, and adjust pH to 7.4 with drops of NaOH 1 M. Filtersterilize throughout 0.22 μM and aliquot and store at -20 °C. 14. Superdex™ G75 Increase column (Cytiva Life Sciences, 29148721).

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15. Glucose solution (36%, w/v) (see Subheading 2.2, step 22). 16. LB medium (see Subheading 2.2, step 26). Supplement with 50 μg/mL Cam. 17. Common laboratory consumables (see Subheading 2.1, step 18). 18. Common laboratory equipment (see Subheading 2.2, step 34). 19. Isopropyl-β-D-thiogalactopyranoside (IPTG) solution (1 M). Dissolve 2.4 g of IPTG in 10 mL of ultrapure H2O. Sterilize by passing through a 0.22 μm filter. 20. Sterile dd H2O (1 L). 21. 0.22 μm Steriflip-GP sterile centrifuge tube top filter unit and 0.22 μm Stericup-GP sterile vacuum filtrations (see Subheading 2.3, step 6). 22. Dialysis tubing (3.5 kDa MWCO) or as alternative Amicon ultra-15 centrifugal devices (10 kDa cutoff) for buffer exchange. 23. NanoDrop™ One Microvolume UV-Vis spectrophotometer (see Subheading 2.2, step 17). 24. 1× PBS (see Subheading 2.1, step 10). 25. Molecular Imager® Gel Doc™ XR+ System (Bio-Rad). 26. 4–20% Mini-Protean® TGX™ precast gels (Bio-Rad). 2.8 Characterization of VHH Binders: Surface Plasmon Resonance (SPR)

¨ KTA FPLC purification system (Cytiva Life Sciences). 1. A 2. Superdex™ G75 Increase column (see Subheading 2.7, step 14). 3. NanoDrop spectrophotometer (see Subheading 2.2, step 17). 4. HBS-EP buffer: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20, and pH 7.4 (Cytiva, BR100188). This buffer can be purchased prepacked ready to use from Cytiva. If prepared in the lab, it should be thoroughly degassed with a vacuum pump system before use. 5. Filtered and degassed ddH2O. 6. 20% ethanol. 7. CM5 sensor chip series S (Cytiva, BR100530). 8. Amine coupling kit (Cytiva, BR100050). 9. Acetate buffer, 10 mM, pH 4.5 (Cytiva, BR100349). 10. SEC-purified VHHs ( see Subheading 3.8, step 1). 11. Appropriate reference protein as a negative control for SPR. 12. Biacore T200 instrument with BIAevaluation software 3.2 (Cytiva).

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2.9 Cell Binding of VHHs by Flow Cytometry

1. Tumor antigen-positive and tumor antigen-negative cell lines. 2. Growth medium: DMEM/RPMI supplemented with: GlutaMAX™ (ThermoFisher, 35050061), antibiotic-antimycotic solution (100×) (ThermoFisher, 15240112), and heatinactivated fetal bovine serum (ThermoFisher, A3840001). 3. Tissue culture T75-treated flasks (ThermoFisher, 156499) or equivalent. 4. Accutase™ cell A1110501).

dissociation

reagent

(ThermoFisher,

5. Cell counting slides for TC10™/TC20™ Cell Counter DualChamber 30 slides 60 counts (Bio-Rad, 1450015). 6. Humidified incubator at 37 °C with 5% CO2. 7. Cold 1× PBS supplemented with 1% BSA and 0.1% w/v NaN3 sodium azide (PBS-BA) (see Note 15). 8. Purified and biotinylated VHHs (see Subheading 3.7.1, step 12). 9. Streptavidin, R-phycoerythrin (Invitrogen, S866).

conjugate

(SAPE)

10. BD FACSCanto™ instrument (BD Biosciences), CytoFLEX S flow cytometer (Beckman Coulter) or equivalent instrument and associated buffers. 2.10 Immunoprecipitation of the Cell Surface Receptor with VHHs

1. Tumor antigen-positive cells. 2. Solubilization/wash buffer (1× PBS supplemented with 1% Triton X-100 and protease inhibitor) (see Note 16). 3. 10 mL syringe(s) and 0.22 μm syringe filters (Millipore). 4. High-capacity streptavidin agarose (ThermoFisher, 20357) or equivalent. 5. Purified biotinylated VHHs (see Subheading 3.7.1, step 12). 6. 4–20% Mini-Protean® TGX™ precast gels (Bio-Rad). 7. 8 M urea: Dissolve urea in 1× PBS. 8. Immobilon®-P PVDF membrane (SIGMA, IPVH08100). 9. mAb specific to the target antigen or equivalent mAb suitable for western blot. 10. HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 715–035-150) or equivalent. 11. Peroxidase substrate: colorimetric (3, 3′-diaminobenzidine, SIGMA, D0426) or chemiluminescent (ThermoFisher, 34579).

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Methods

3.1 Antigen Preparation, Llama Immunization, and Immune Response Monitoring

Llama immunization is outsourced and performed at the Cedarlane facility in Burlington, Ontario, Canada (http://www.cedarlane.ca), and is based on the protocol provided by NRC and following the guidelines set by the Canadian Council on Animal Care (CCAC). Being outbred, it is preferred to use more than one llama for protein immunization, as the quality of the immune response varies with individual animals. 1. Aliquot the protein antigen for immunization into five separate tubes and clearly label them for injections 1 through 5 (100 μg of protein in each). The antigen needs to be well characterized and it is recommended to run an SDS-PAGE and perform an ELISA to determine its purity and functionality, respectively, before proceeding with immunization. This is a critical step toward eliciting a robust immune response in the llama (see Note 17). 2. On day 1, conduct a pre-immune bleed (10 to 15 mL) and then immunize the llama by subcutaneous injection of antigen aliquot 1 in CFA in the lower back or intramuscular injection at the lower rump [21] (see Note 18). Store the collected test bleed overnight at 4 °C. Prepare the serum the next day by centrifuging 10 min at 2700× g, 4 °C, and store the sera at 4 °C for short-term use and at -20 °C for long-term use. 3. Four additional boosts (antigen aliquots 2–5, in IFA) are performed on days 21, 28, 35, and 42 by subcutaneous injection (Fig. 1a). 4. Collect blood (10 to 15 mL) into 50 mL falcon tubes on day 35 as a test bleed. Store the collected test bleed overnight at 4 ° C. Prepare the serum as described above (see Subheading 3.1, step 2). 5. Collect 60 mL of blood 7 days after the last boost, on day 49 (also called the production bleed) into heparin-coated (or other anticoagulant such as EDTA) tubes. Place the blood on ice immediately. Use 10 mL of the blood to prepare the production serum (terminal bleed) (see Subheading 3.1, step 2). 6. Isolate peripheral blood mononuclear cells (PBMCs) from the production bleed by diluting 50 mL blood at 1:1 ratio in RPMI medium or a physiological saline solution. 7. Centrifuge five 10 mL Lymphoprep™ tubes at 400 x g for 1 min to displace the liquid to the bottom of the tube before use.

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A

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Fig. 1 Llama immunization and serum response monitoring. (a) A llama was immunized with protein antigens at days 0, 21, 28, 35, and 42. Sera were collected before the first injection and at days 35 and 49. PBMCs were collected after a positive serum response on day 49 and used for VHH-phage display library construction. Usually after the third boost (day 35), one can observe a specific immune response against the immunogen. (b) Serum titration ELISA performed with pre-immune (day 0) and immune (days 35 and 49) sera demonstrated antigen-immunized llamas generated strong immune polyclonal antibody responses to the antigen. Sera did not react with nontarget antigen (blocker), demonstrating specificity of the immune response

8. Slowly add 20 mL diluted blood to each of Lymphoprep™ tube and centrifuge the tubes at 800× g for 20 min at 18° to 22 °C. 9. Collect the PBMCs that have formed an interface between the two layers using a Pasteur pipette or a syringe with 21G needle. 10. Dilute the harvested fraction with RPMI medium to reduce the density and harvest the cells by centrifugation at 250× g for 10 min and at 18° to 22 °C. 11. Count the cells and make aliquots of 5 × 106 to 1 × 107, frozen on dry ice, and then store at -80 °C until further use. We strongly recommend that at least half of the cells be lysed in 500 μL of TRIzol reagent according to the manufacturer’s instructions and then stored at -80 °C to prevent or minimize RNA degradation, helping to maintain the integrity of the RNA due to highly effective inhibition of RNase activity while disrupting cells and dissolving cell components during sample homogenization. 12. Perform an ELISA assay to analyze the llama polyclonal immune response as described in the next step.

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13. Coat a 96-well microtiter plate with 100 μL of 1 μg/mL of the antigen in PBS (rows A–F) (plate A). Use a second plate to coat with 100 μL PBS for (rows A–F) as a blank (plate B). Incubate the plate overnight at 4 °C. Label rows A–B as pre-immune, C– D as test bleed day 35, and E–F as production bleed. If using biotinylated antigen (hereafter referred to as BTA), first coat the wells with streptavidin at 1–10 μL/mL in PBS overnight at 4 °C. 14. The next day, rinse the wells twice with PBS, and then block all the wells with 200 μL/well of blocking buffer for 2 h at RT. Then wash the plates with PBS. For the BTA plate, add the BTA at 0.1–0.5 μg/mL in PBS and incubate at RT for 30 min (see Note 19). 15. Perform twofold serial dilutions of the sera (pre-immune, test bleed, and production bleed) giving a dilution range of 1/100–1/102,400 and add to the respective wells in duplicate in each plate (e.g., add diluted pre-immune 1/100–1/ 102,400 to wells A1–A11 and B1–B11, respectively, and PBS to wells A12 and B12. Repeat for the test bleed and production bleed for rows C–D and E–F). Incubate for 1.5 h at RT. 16. Wash the wells five times, each time with 200 μL PBST, empty the plate on a pad of tissue paper, and then add 100 μL/well of goat anti-llama IgG-HRP (diluted 1:10 ,000 in blocking buffer or PBST). Incubate 1 h at RT (see Note 20). 17. Wash the wells again as described in step 16, add HRP substrate (100 μL/well), and incubate at RT for ~10 min. Stop the reaction with 1 M H3PO4 (100 μL/well). After full color development, read the absorbance at 450 nm with a microtiter plate reader (Fig. 1b). 3.2 VHH Library Construction

The PBMCs collected on day 49 (see Subheading 3.1, step 11) are used as a source of mRNA for library construction. First, complementary DNA (cDNA) is synthesized from the extracted total RNA population and amplified by a two-step PCR. The VHH region is then amplified by gene-specific primers and cloned into a phagemid vector and transformed into E. coli cells, building the VHH library. The phagemid vector allows the VHH to be expressed as a fusion to the gene-III minor coat protein (gpIII) on the surface of filamentous phages when the bacterial cells in the library are superinfected with a helper phage. 1. Take 5 × 107 PBMCs from day 49 and isolate total RNA using the TRIzol reagent according to the manufacturer’s instructions. Measure the RNA concentration and purity at A260 nm and A280 nm with a spectrophotometer22. Typical RNA yields range from 20 to 50 μg/5 x 107 cells with an A260/280 ratio > 1.8 (see Note 21).

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2. Use a total of 2.5 μg of RNA in 20 μL of ddH2O to synthesize cDNA in a total reaction volume of 20 μL, using the SuperScript™ VILO™ cDNA Synthesis Kit and oligo(dT) or random hexamers, according to the manufacturer’s instructions (see Note 22). For the library construction, we perform four reactions, which results in a total volume of 80 μL cDNA and use 2 μL/PCR reaction. 3. For the amplification of the VHH encoding regions, perform test polymerase chain reactions (PCRs) using various amounts of the cDNA reaction mix from step 2 ranging in volume from 0.5 μL to 5 μL using an equimolar mix of framework 1-specific primers MJ1, MJ2, and MJ3 with either CH2FORTA4 (annealing to the DNA-encoding CH2 domain of both conventional and heavy chain antibodies) or CH2B3-F (annealing to the DNA-encoding CH2 domain of heavy chain antibodies) primer (see Subheading 2.2, step3). Set up the PCR reaction as follows: 10× buffer

5.0 μL

MJ1–3 primer mix (10 pmol/μL each)

0.5 μL

CH2FORTA4 or CH2B3-F primer

0.5 μL

dNTPs

1.0 μL

cDNA

2.0 μL

Taq DNA polymerase

0.5 μL

ddH2O

Add to 50.0 μL

Perform the PCR for 30 cycles using this program: preheating step at 94 °C for 3 min followed by 30 cycles of 94 °C for 60 s, 55 °C for 30 s, and 72 °C for 60 s and a final step of 72 °C for 7 min.

4. Analyze 5 μL of the PCR reaction on a 1% agarose gel [25]. Identify the cDNA volume that gives the best yield in terms of amplifying the VHH-CH2/VHH-CH2b3 fragments and repeat the PCR experiment for the remaining cDNA mixture under the same conditions. Separate the PCR fragments on 1% agarose, visualize products (see Note 23), excise the band corresponding to the VHHs, and extract the DNA using Promega Gel and PCR Cleanup Kit following manufacturer instructions. Finally measure the DNA concentration (see Subheading 3.2, step 1). 5. Re-amplify the purified product (20–30 ng of the amplified DNA/reaction tube) in a second nested PCR under the same conditions as above, using framework 1 (FR1) MJ7 and FR4-specific MJ8 primer, respectively. Perform a total of 40 PCR reactions. Analyze 5 μL of the PCR reaction on a 1% agarose gel, expecting to see relatively wide bands ranging from

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Fig. 2 Overview of library construction, phage panning, and screening for the isolation of antigen-specific VHHs. (a) Schematic representation of RNA extraction, cDNA synthesis, and PCR amplification of VHH-CH2/ CH2b3 fragments using MJ1-3:CH2 and MJ1-3:CH2b3 primer sets. The heavy chain immunoglobulin repertoire was gel-purified and used as a template to amplify the VHH fragments using SfiI-MJ7 and MJ8-SfiI primers. (b) A phagemid library was constructed and phage rescued using M13KO7 helper phage. (c) Phagedisplayed VHHs were panned against orientated and immobilized antigen by using streptavidin and biotinylated antigen. (d) After four rounds of selection, VHHs were screened by phage ELISA against the antigen presented in two formats: (i) passively adsorbed and (ii) orientated immobilization on streptavidin pre-coated wells. Specificity against the antigen was tested by applying the same phage supernatant on a plate coated with blocker only

400 to 450 bp, which correspond to the VHH fragments. Desalt the PCR products with Promega Gel and PCR Cleanup Kit and determine the concentration. Primers MJ7 and MJ8 will introduce the SfiI sites for subsequent cloning (Fig. 2a). 6. Digest the PCR products with SfiI (5 units/μg DNA) overnight at 50 °C and subsequently analyze a few microliters on a 1% agarose gel to ensure that it is of the proper size. Re-purify the digested DNA with Micro DNA Cleanup Micro Kit including the additional prewash step in order to remove the small fragments released upon the digestion. Measure the concentration. 7. Digest 30 μg of pMED1 phagemid vector with SfiI (5 units/μg DNA) overnight at 50 °C in a final volume of 150 μL. Then, purify the vector reaction mixture by using two columns from the Promega Kit and elute the DNA in 120 μL of ddH2O. Add

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15 μL of FastDigest Buffer (10×), 5 μL of XhoI and PstI enzymes (10 units/ μL), and 10 μL of FastAP alkaline phosphatase to reduce self-ligation of pMED1. After 2 h at 37 °C, column purify by the DNA Cleanup Micro Kit column and measure the concentration as described in step 4. 8. Examine 5 μL of the digested pMED1 (~1000 ng) on a 1% agarose gel, including the undigested control vector (1000 ng) to ensure that the vector is completely linearized. 9. Use the T4 DNA ligase to ligate the SfiI-digested VHH DNA with SfiI-digested pMED1 vector as follows: Digested vector

20.0 μg

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3.5 μg

T4 DNA ligase buffer (5×)

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T4 DNA ligase

5.0 μL

Sterile ddH2O

Add to 100.0 μL

Incubate at RT for 60 min (see Note 24).

10. Purify the ligated materials using DNA Cleanup Micro Kit using two spin columns and elute the DNA in a final volume of 35 μL of sterile ddH2O per column. Pool the eluted material and measure the concentration. 11. Transform 50 μL of electrocompetent TG1 E. coli cells with 3 μL of the purified ligated material using 0.1 cm cuvettes and a MicroPulser™ electroporator or an equivalent instrument. Add 1 mL of pre-warmed SOC and transfer the electroporated cells into a 50 mL conical tube and incubate for 1 h at 37 °C, 110 rpm. Repeat the transformation for the remaining DNA for a total of 20 transformations. Pool all the SOC cell suspensions in the same tube. 12. After the recovery time, take a small aliquot and carry out 103-, 104-, and 105-fold dilutions in LB. Spread 100 μL of the diluted cells on LB-Amp plates and incubate overnight at 32 °C. 13. Next day, count the colonies on the titer plates (see Subheading 3.2, step 12) and determine the total library size. 14. Amplify the library and generate a bank of cells by transferring the transformed bacteria into 500 mL of 2 × YT-Amp-Glu and incubating for 3–4 h at 220 rpm and 37 °C. 15. Centrifuge the cells at 5000× g for 20 min at 4 °C. Discard the supernatant and resuspend the cells in 50 mL of 2 × YT-AmpGlu. Make dilutions of the cells in 2 × YT, measure the A600 nm, and use this value to calculate the density of cells/mL. (An A600 nm = 1 corresponds to approximately 109 TG1

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E. coli cells). Add 50 mL of sterile 80% glycerol solution to the cell stock, make several aliquots of 1010 bacterial cells/vial, and freeze the cells at -80 °C. 16. Pick up 96 colonies from the dilution plates and grow in a 96-deep well culture plate in 200 μL of LB-Amp overnight at 37 °C, 110 rpm in a humid chamber. 17. The next day, dilute 2 μL of overnight culture into 100 μL of ddH2O and use 1 μL of diluted cells in colony PCR. Add 50 μL of 80% glycerol stock to the remaining culture plate and store it at -80 °C for the future use. 18. Perform colony PCR to determinate the insert ratio of the library. Prepare a master mix as follows enough for the analysis of 100 samples: 10× buffer

150.0 μL

dNTPs

32.0 μL

PN2 primer

16.0 μL

M13RP primer

16.0 μL

Platinum Taq DNA polymerase

16.0 μL

ddH2O

Add to 1500 μL

19. Dispense 15 μL in a 96-well PCR plate and add 1 μL of diluted culture to the corresponding well. Place the reaction plate in a thermal cycler and perform the PCR reaction following this condition: preheating step at 94 °C for 5 min followed by 30 cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 60 s and a final step of 72 °C for 7 min. 20. Analyze 5 μL of each PCR reaction on a 1% agarose gel to identify the clones with full inserts [400–450 bp (VHH) + 317 bp (flanking sequence)] ffi 750 bp band. Additionally, use 0.5 μL of the PCR mixture and 0.2 μL of M13RP (10 pmol/ μL) as primer to sequence the clones and identify those expressing VHH sequences (see Note 25 and Note 26). Determine the functional library size by multiplying the percentage of the 96 clones with VHH sequences by the total library size calculated in Step 13 (Fig. 2b). 3.3 Phage Rescue of the VHH Library

1. To produce the phage library, thaw 1–2 mL of frozen library cells (see Subheading 3.2, step 15) and grow in 200 mL of 2 × YT-Amp-Glu at 37 °C and 220 rpm shaking until the cell culture density reaches an A600 nm of 0.5 (2–3 h). Infect the cells with a 20× excess of M13KO7 helper phage (2 × 1012 plaque-forming units, pfu, kanamycin resistant) (see Note 27) for 30 min at 37 °C without agitation followed by 30 min at

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37 °C with agitation at 220 rpm. Pellet the infected cells by centrifugation at 5000× g for 10 min at 4 °C. Discard the supernatant and resuspend the bacterial pellet in 200 mL of 2 × YT-Amp-Kan and grow overnight at 37 °C and 220 rpm. This step is necessary to remove any traces of glucose from the growing medium, which acted as repressor of the expression of the VHH during the first step of library generation. 2. To purify the phage, harvest the overnight culture (10,000× g, 15 min, 4 °C), filter the 200 mL supernatant through a 0.22 μm filter unit, and then in order to precipitate the phage particles, add 1/5 the volume (40 mL) of 20% PEG/2.5 M NaCl. Incubate for 1 h on ice, centrifuge as above, and discard the supernatant. Resuspend the phage pellet in 1–5 mL of sterile PBS (see Note 28) supplemented with protease inhibitor and determine the titer of the phage library by serial dilution and infecting TG1 E. coli cells (see Notes 29 and 30). Store at -80 °C until further use. 3.4 VHH Phage Library Biopanning

The VHH library, displayed on the surface of the phages, is panned against the antigen (BTA) immobilized on streptavidin-coated wells. This approach potentially allows for improved display of the antigen and consequently may provide better recognition of conformationally sensitive epitopes by phage-displayed VHHs. Using a relatively constant number of input phages in each round, it is expected that the output (eluted) phages to be enriched, which will be a strong indication for the success of panning. Alternatively, one may send a sample of 20–30 colonies after each round for DNA sequencing and look for enriched sequences. After four rounds of selection, individual colonies are screened for their specificity against the BTA by monoclonal phage ELISA (Subheading 3.5) or by soluble protein ELISA (Subheading 3.6), where the binding of VHH-displaying phages or of soluble VHH to the BTA is detected colorimetrically by using anti-M13 antibody-HRP conjugate or anti-VHH-HRP/anti-His-HRP (for non-His-tagged antigen) followed by adding TMB substrate (Fig. 2c). 1. Coat an 8-well microwell strip or a 96-well Maxisorp™ plate with 100 μL of 4 μg/mL streptavidin in PBS/well (two wells/ antigen), seal the wells with parafilm, and incubate overnight at 4 °C (see Note 31). 2. Discard the contents of the coated wells the next day, wash with 250 μL of PBS, and block for 2 h at RT with 200 μL/well of blocking buffer. Start growing 10 mL of TG1 E. coli cells in a sterile 50 mL conical tube (see Note 8). 3. Add 50 μL of 2 μg/mL of the BTA in PBS to the antigen well. Seal and incubate both wells at RT for 1.5 h. Empty the antigen wells and wash three times with PBS.

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4. Add 100 μL of 1012/mL phage library (see Subheading 3.3, step 2) to the antigen-coated wells for 2 h at RT. Remove unbound phage, wash 5× with PBS-T and 5× with PBS (250 μL per wash), and elute the bound phage by incubating with freshly prepared 0.1 M triethylamine (100 μL/well) for 10 min at RT. Pipette the elution solution up and down several times in the well, transfer the contents to a 1.5 mL tube, and neutralize with 50 μL of 1 M Tris–HCl, pH 7.4. Keep the tube on ice (see Note 32). 5. Titrate the output by infection of TG1 E. coli cells. Keep 100 μL of the exponentially growing TG1 E. coli cells (see Subheading 3.4, step 2) as the titer negative control (see below) and infect the remaining cells from step 2 (~9.9 mL) with 100 μL of the eluted phage by incubating the mixture at 37 °C for 15 min without shaking followed by incubation with shaking at 220 rpm at 37 °C, 1 h (store the remaining phage at -80 °C for future reference). Titrate the infected cells, referred as the eluted/output phage (see Note 33). 6. Superinfect the infected bacterial cells (10 mL) with 1011 pfu of M13KO7 helper phage as described (see Subheading 3.3, step 1). Subsequently, add kanamycin to a final concentration of 50 μg/mL and incubate overnight at 37 °C and 220 rpm. 7. The next day, purify the phage by precipitation with PEG/NaCl, resuspend in 200 μL of PBS, and determine the phage titer as described (see Subheading 3.3, step 2). Use the purified phages as the input phage for the next round of selection. Keep an aliquot of the phages (50 μL of 1 × 1011 pfu/ml) for polyclonal phage ELISA. 8. To assess the progress of the panning, perform colony PCR and DNA sequencing on titer plate colonies of the eluted phages (see Subheading 3.2, steps 16–20) as described. We routinely sequence 15–20 clones in each of the first 2 rounds (R1 and R2), 25 clones in the third round (R3), and 50–100 clones in the fourth round (R4). 9. Repeat the panning process (see Subheading 3.4, steps 1 – 7) for three additional rounds using the amplified phage from the previous round as the input phage for the next round to generate R2-R4. We recommend to keep the amount of antigen constant in the first two rounds and reduce the amount of the BTA to half and a quarter in third and fourth rounds of panning (see Note 34). Additionally, increase the number of washes before elution of the phages or more stringent conditions during the selection (such as higher concentration of Tween-20) for the subsequent round of selection to promote enrichment of high-affinityVHHs.

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3.5 Polyclonal and Monoclonal Phage ELISA Screening

Panning is a lengthy process and its progress needs to be monitored during and after the panning. Before starting to analyze individual VHH colonies for binding to the BTA, it is recommended to perform polyclonal phage ELISA by using the original phage library (R0) and the enriched phages from each round (R1-R4) (Fig. 2d). 1. For polyclonal ELISA, start by coating a 96-well Maxisorp™ plate with 100 μL of 4 μg/mL streptavidin in PBS/well (two wells/antigen/round of panning), seal the wells with parafilm, and incubate overnight at 4 °C. Allocate a duplicate well for control phage (M13KO7). Perform a twin plate that will serve as negative control for the phages against streptavidin alone, without antigen. 2. Discard the contents of the coated wells the next day, wash with 250 μL of PBS, and block for 2 h at RT with 200 μL/well of blocking buffer. 3. Add 50 μL of 2 μg/mL of the BTA in PBS to the antigen well. Seal and incubate both the plate at RT for 30 min. Empty the antigen wells and wash it three times with PBS. 4. Add 100 μL of 109/mL original phage library (R0) and the same number of amplified phages from R1-R4 and control M13KO7 phage to the respective antigen-coated wells and twin plate and incubate for 1 h at RT. 5. Discard unbound phage particles in a plastic container containing 10% bleach and wash the wells with PBS-T (5 × 250 μL). Add 100 μL/well of anti-M13 IgG-HRP conjugate (previously diluted 1:5000 in blocking buffer) and incubate for 1 h at RT. 6. Remove the unbound conjugate, wash the plates 5× with PBS-T, and add 100 μL/well of TMB substrate (pre-warmed to RT). Incubate for 5–10 min until the color has developed and stop the reaction as described previously. 7. Read the absorbance as described before (see Subheading 3.1, step 17). 8. For monoclonal ELISA, start by amplification of monoclonal phages. In the morning prepare a 96-deep well culture plate with 200 μL/well of 2 × YT-Amp-0.2%Glu and inoculate 94 colonies from the titration plate from round four of panning (see Subheading 3.4, steps 5 and 9). Leave positions H11 and H12 for internal negative controls. Place the plate in a humid chamber box (e.g., an empty tip box with lid and containing wet tissue paper) and incubate at 37 °C and 110 rpm of shaking until the A600 nm of the cultures reaches 0.4 (4–5 h). Thereafter, infect the cells with 5 × 109 pfu of M13KO7 for 30 min without agitation. Then add kanamycin, place the plate in the humid chamber box, and continue growing the culture at 37 ° C overnight at 110 rpm.

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9. The next day spin down the plate at 3600× g for 15 min, 4 °C, using a benchtop centrifuge with a swinging plate bucket rotor. Transfer the supernatant to a new plate and keep it on ice or at 4 °C (master plate) to be used in step 14. Resuspend the pellet in the culture plate wells, using 50 μL of 80% glycerol stock and store it at -80 °C for future use. 10. Simultaneously with step 8, coat a 96-well Maxisorp™ plate with 100 μL of 4 μg/mL streptavidin in PBS, seal the plate with parafilm, and incubate overnight at 4 °C as described in the polyclonal phage screening above (step 1). Include a twin plate without the antigen, coated only with streptavidin followed by blocker to evaluate the specificity of monoclonal VHH-phage or antigen. 11. The next day, block the plate using either casein 1%, BSA 1%, or superblock buffer and incubate at RT for 2 h (see Subheading 3.5, step 1). 12. Discard the blocking buffer from both plates, wash with PBS-T (3 × 250 μL), discard the supernatant by hand, and tap the plate on a pad of tissue paper. 13. Add 50 μL of 0.5 μg/mL of the BTA in PBS to the antigen well. For the twin plate, add 50 μL of PBS. Seal and incubate both the plates at RT for 30 min. Then, wash the plates 3× with PBS. 14. Add 50 μL/well of phage supernatants (clones 1–94) from the master plate (step 9) to both plates. As a negative control, include ~109 M13KO7 helper phage diluted in PBS to wells 95 (H11) and 96 (H12). Incubate the plates for 1.5 h at RT. 15. To prepare a master plate glycerol stock, add 25 μL/well of sterile 80% glycerol to the bacterial culture plate (step 9), seal the lid with parafilm, label properly, and store at -80 °C. This plate can be used for colony PCR and plasmid sequencing (see Subheading 3.2, steps 16–20). 16. Discard unbound phage particles in a plastic container containing 10% bleach and wash the wells with PBS-T (5 × 250 μL). Add 100 μL/well of anti-M13 IgG-HRP conjugate (previously diluted 1:5000 in blocking buffer) and incubate for 1 h at RT. 17. Remove the unbound conjugate, wash the plate 5× with PBS-T, and add 100 μL/well of TMB substrate (pre-warmed to RT). Incubate for 5–10 min until the color has developed and stop the reaction as described previously. 18. Read the absorbance as described (see Subheading 3.5, step 7). 19. The next day, perform a colony PCR on positive clones from the plate prepared in step 9 as described (see Subheading 3.2,

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steps 16–20) for DNA sequencing and for the identification of unique sequences to identify VHHs for further characterization. Monoclonal phage ELISA screening is shown in Fig. 2d for 94 phage populations displaying VHHs that were isolated against a typical protein antigen. 3.6 Sub-cloning of R3/R4 Phage Pool, In Vitro Translation and Screening of the Soluble VHHs (an Alternative to Monoclonal Phage ELISA Screening) (See Note 9)

Sub-cloning and in vitro translation of the unique VHH sequences after 3–4 rounds of panning is a straightforward approach that bypasses gene synthesis and therefore reduces the cost and time required to obtain VHHs in an expression vector for large-scale protein purification and further characterization. We found this method quite efficient, as a side-by-side comparison of hundreds of VHH clones after round four of panning and after sub-cloning into pMRO expression vector [19, 23] showed that there is a high degree of sequence overlap between the two approaches, indicating no loss of diversity. Following the sub-cloning of the VHH pool from round 3 or round 4 of panning, plasmid DNAs are prepared from randomly selected individual colonies and used for in vitro translation using the Promega S30 T7 High-yield Expression System. The protein expression system includes T7 RNA polymerase for transcription and lacks E. coli outer membrane (OmpT) and intracellular (Lon) proteases, which led to greater stability of the target protein. Using this system, it is possible to express approximately 50–80 μg of VHH per reaction tube in under 1 h. This amount is sufficient for ELISA-based screening. Additionally, the VHH could be biotinylated and purified if multiple in vitro translation reactions are used and the VHH could then be biotinylated and purified for SPR and flow cytometry (Fig. 3a–c).

3.6.1 Sub-cloning of R3/ R4 Phage Pool

1. Amplify the eluted phage as described in Subheading 3.4, steps 5–6. 2. The next day, harvest the supernatant containing the phages, filter it through a 0.22 μm filter, and add 1/5 volume of chilled M1 buffer. Mix to precipitate the phage particles. Incubate on ice for 60 min. 3. Then centrifuge at 10,000× g for 10 min at RT. Discard the supernatant, resuspend the pellet in 1 mL of Buffer M2, and incubate at 80 °C for 20 min to lyse the phage particles. 4. Cool the solution to RT and purify the ssDNA using a QIAGEN Tip 20 (Plasmid Miniprep Kit) following manufacturer’s instructions and measure DNA concentration using a NanoDrop. 5. Set up the PCR pre-mix for 5× PCR of 50 μL/reaction for the amplification of VHH fragment from R3/R4 pool as follows:

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Fig. 3 En masse cloning into pMRo.BAP.H6 and screening using in vitro translated VHHs or bio-VHHs. (a) ssDNA was extracted from phages obtained after four rounds of selection and used as template for the amplification of the VHH repertoire before cloning en masse to high-yield expression vector pMRo.BAP.H6. (b) Screening of VHHs generated by in vitro translation against passively adsorbed or orientated immobilized antigen using an anti-VHH IgG conjugated to HRP. (c) The same screening in (b) can also be performed against the Fc-fused version of the antigen followed by detection of the VHH with anti-His6, streptavidin, or anti-VHH IgG conjugated to HRP

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6. Pool the five reactions, verify amplification by agarose 1% gel, and then use desalt using the Promega Gel and PCR Cleanup Kit, eluting in 40 μL of ddH2O. 7. Digest 2 μg PCR products with SfiI (5 units/μg DNA) for 4 h at 50 °C and purify the digested VHH fragment with DNA Cleanup Micro Kit and measure the concentration (see Subheading 3.2, steps 5–6).

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8. Digest 20 μg of pMRO expression vector with (232022) SfiI (5 units/μg DNA) overnight at 50 °C in a final volume of 100 μL. Then, gel-purify the vector reaction mixture by using two columns from the Promega Gel and PCR Cleanup Kit and elute the DNA in 80 μL of ddH2O. Add 10 μL of FastDigest Buffer (10×) and 10 μL of FastAP alkaline phosphatase and incubate for 2 h at 37 °C. Then, column-purify the vector DNA by the DNA Cleanup Micro Kit column and measure the concentration. 9. Use T4 DNA ligase to ligate the SfiI-digested VHH DNA with SfiI-digested pMRO vector as follows: Digested vector

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10. Purify the ligated materials using a DNA Cleanup Micro Kit using one spin column and elute the DNA in a final volume of 20 μL of sterile ddH2O and measure concentration. 11. Transform 2 × 50 μL of electrocompetent BL21(DE3) E. coli cells with 500 ng of the purified ligated material using 0.1 cm cuvettes and a MicroPulser™ electroporator or an equivalent instrument. Add 1 mL of pre-warmed SOC and transfer the electroporated cells into a 15 mL conical tube and incubate for 1 h at 37 °C, 110 rpm. After the recovery time, plate neat 101- to 104-fold dilutions in LB media. Spread 100 μL of the diluted cells on LB-Kan-plates and incubate overnight at 37 °C. 12. The next day, pick 96 colonies from the dilution plates and culture in a 96-deep well plate using 200 μL of LB-Kan overnight at 37 °C, 110 rpm, in a humid chamber. 13. Dilute 2 μL of the overnight culture into 100 μL of ddH2O and perform a colony PCR as described before (see Subheading 3.2, steps 16–20) using T7 Promoter Fw and T7 Terminator Rv primer. 14. Then add 40 μL of 80% glycerol to the remaining culture plate and store it at -80 °C for future use. 15. Identify the unique VHH clones for in vitro translation.

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1. Grow a 1 mL culture of the unique VHH clones (see Subheading 3.6.1, step 15) in LB-Kan and perform a plasmid miniprep to obtain 1–2 μg plasmid DNA. 2. Set up the in vitro translation reaction as follows: S30 Promega premix (stored at -80 °C)

20 μL

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3. Incubate the reaction mixture for 1 h at 37 °C and then store at 4 °C until further use. 4. Coat MaxiSorp plates with streptavidin at 2 μg/mL and incubate overnight at 4 °C using as many wells as the number of unique sequences identified, in duplicate. Include a twin plate coated with streptavidin to test the specificity of the VHH. 5. The next day, block the wells with 1% casein or 1% BSA and incubate 2 h at RT, and then rinse the plate 3× with PBST +2× with PBS. 6. To the antigen plate, add 100 μL BTA at 0.5 μg/mL in PBS and incubate for 30 min at RT. Keep the control twin plate in PBS. 7. Make 1:2 serial dilutions of each VHH clone from 5 μL of the non-purified 50 μL in vitro reaction (step 3). Incubate at RT for 1 h and then wash the plate 3× with PBST followed by 2× with PBS. 8. Add 100 μL of goat anti-llama IgG, VHH- HRP conjugate ( 1: 5000 ) in PBST and incubate at RT 1 h. Wash the plate 3× with PBS-T and 2× with PBS. 9. Add 100 μL of TMB substrate to each well and wait 5 min. 10. Add 100 μL of stop solution to each well to stop the reaction and analyze with a plate reader at 450 nm. 3.7 VHH Sub-cloning, Soluble Expression, and Purification

The VHH sub-cloning and expression follow directly from Subheading 3.5 and the unique VHH sequences obtained by phage ELISA can be gene-synthesized and sub-cloned by a provider, such as TWIST Bioscience. This will provide the option of codon optimization for expression in E. coli or other expression systems. The delivered plasmid constructs are transformed into BL21(DE3) E. coli as described before (see Subheading 3.6.1, step 11). If biotinylation of the VHH is required for a future detection and downstream applications, Subheading 3.7.2 could be performed in advance before starting VHH expression.

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3.7.1 Large-Scale VHH Expression in E. coli

1. Prepare individual starter cultures for each VHH by inoculating a single colony harboring pMRO-VHH in 10 mL of LB-Kan-1% glucose and grow overnight at 37 °C, 220 rpm. 2. The next day, transfer each overnight culture into 200 mL of LB-Kan in a 500 mL baffled flask and grow at 37 °C with continuous 220 rpm shaking until an A600 ~ 0.6–0.8 is reached (1.5–2 h). 3. Induce VHH expression by adding 1 μL of 1 M IPTG (final concentration 5 μM) and grow overnight at 28 °C with continuous shaking at 220 rpm (see Note 35). 4. The next day, transfer the bacterial culture to a 500 mL centrifuge bottle and harvest cells by centrifugation at ~4000× g 15 min at 4 °C. 5. Resuspend the pellet in 30 mL of sterilized 1× PBS and transfer each sample to a 50 mL conical tube, keeping the samples on ice. We recommend the addition of an appropriate volume of protease inhibitor cocktail at this stage (see Note 36). 6. Use the Emulsiflex-C5 or similar instrument to lyse the bacteria at a pressure of 20,000 psi. Sonication could be used as an alternative method. 7. Centrifuge at 17,000× g, 30 min at 4 °C and transfer the supernatant to a new tube. 8. To perform the BirA-AviTag site-specific biotinylation of VHHs, add 5 mL of BirA cleared bacterial extract (see Subheading 3.7.2, step 6) and supplement with 350 μL of 100 mM D-biotin and 350 μL of 100 mM ATP. Incubate for 2 h at 37 ° C (with low agitation or in a water bath with regular manual agitation). 9. Centrifuge the mixture at 17,000× g, 4 °C for 20 min. 10. Repeat step 9 to remove any carryover cell debris or precipitation and keep the supernatant. 11. To minimize impurities, add 1.8 mL of 500 mM imidazole and 4.5 mL of 5 M NaCl to reach 20 mM and 500 mM, respectively. Add PBS to give a volume of 45 mL. 12. Purify VHHs using a 5 mL HiTrap™ chelating HP column ¨ KTA FPLC system as described elsewhere connected to an A [26, 27]. Pool the fractions corresponding to the VHH peak and buffer exchange into PBS by dialysis or using an Amicon® Ultra-15 Centrifugal Filter Unit. Analyze the quality of the proteins by SDS-PAGE and size exclusion chromatography (SEC) on a Superdex G75 Increase column (Fig. 4a) as described before [26]. 13. Store the samples at -20 °C.

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Fig. 4 Characterization of antigen-specific VHHs. (a) Size exclusion chromatography demonstrating that the isolated VHHs are non-aggregating with a single monomeric peak was observed. (b) SPR sensorgrams showing single-cycle kinetics analysis of a representative VHH. The VHH specifically recognized the target antigen. Black lines show raw data points and red lines show fits to a 1:1 interaction model. (c) Binding profiles of three representative biotinylated VHHs and an isotype control to antigen-positive and antigennegative cell lines using flow cytometry. Binding of bio-VHHs was detected with streptavidin PE

3.7.2 Preparation of the Biotin Ligase BirA Extract

1. Prepare 10 mL of LB supplemented with 10 μg/mL of chloramphenicol, inoculate with one colony of E. coli AVB101 (see Note 37), and grow overnight at 37 °C and 220 rpm. 2. The next day, transfer 5 mL of the starter culture to a 2 L flask containing 500 mL of LB/Cam and continue growing under the same conditions until the A600 reaches 0.6–0.8. 3. Induce the expression of the biotin ligase by the addition of 500 μL of 1 M IPTG to reach 1 mM and incubate for 4 h at 37 °C and 220 rpm shaking. 4. Harvest the bacteria by centrifugation as described before. 5. Resuspend the pellet in 50 mL of PBS and lyse the bacteria using the Emulsiflex-C5 or by sonication as described in Subheading 3.7.1, step 6. 6. Centrifuge twice as in Subheading 3.7.1, step 9, filter through a 0.22 μm filter to remove any cell debris, and make 5 mL aliquots. Store at -80 °C.

3.8 Characterization of VHH Binders by Surface Plasmon Resonance (SPR)

Binding specificity of the isolated VHHs to BTA (or Fc fusion format of the same antigen) can be determined by ELISA as described in Subheading 3.6.2 (Fig. 3b–c). However, accurate binding affinities and kinetics can be derived from SPR analyses

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performed with a Biacore instrument (Fig. 4b). Before starting the SPR, the VHH antibodies need to be purified by SEC to collect the fractions of VHH monomers to eliminate any aggregates (Fig. 4a). 1. Use 300–500 μg of the IMAC-purified and buffer-exchanged VHH from Subheading 3.7.1, step 12. It is recommended to spin down at maximum speed to remove any precipitants generated after thawing the sample before performing SEC. 2. Start the Unicorn software (Cytiva) and prepare the Superdex G75. Increase column by washing with 50 mL of filtered and degassed ddH2O. Then equilibrate with 30 mL of filtered and degassed HBS-EP buffer at flow of 0.8 mL/min. 3. Inject the VHH sample onto a 1 mL loop, initiate the chromatography method file, and run for 35 mL at 0.8 mL/min. Collect fractions every 0.5 mL; monomeric fractions of VHHs usually elute at 12–14 mL. On the EVALUATION screen, obtain the chromatogram and export the data as a Microsoft Excel file. 4. Integrate monomeric and aggregate peaks to obtain % of monomer. 5. Select the peak fractions of the monomeric peak, measure its absorbance at 280 nm, and determinate concentration in mg/mL for SPR analysis. 6. Carry out SPR experiments at 25 °C using a Biacore T200 instrument with HBS-EP as the running buffer. 7. Immobilize streptavidin on a CM5 sensor chip to give maximum surface density of ~2500 response units (RUs). Briefly, activate the CM-dextran surface with a 7-min injection of a mixture of 50 mM NHS and 200 mM EDC at a flow rate of 5 μL/min. Inject 50 μg/mL streptavidin diluted in 10 mM acetate buffer, pH 4.5, for 7 min at a flow rate of 5 μL/min and block the surface with a 7-min injection of 1 M ethanolamine, pH 8.5. 8. Saturate the streptavidin surface with BTA (range = 300–500 RU) by injecting 35 μL of 200 nM biotinylated antigen at a flow rate of 5 μL/min. 9. Analyze the VHH interaction with the immobilized BTA using a streptavidin surface as a reference. Inject a range of five concentrations of VHH (this range is dependent on the affinity), in single-cycle kinetics mode, over both the streptavidin and streptavidin-antigen surfaces at a flow rate of 40 μL/min with 180 s of contact time and 300 s of dissociation time. 10. Analyze the reference flow cell subtracted sensograms and fit the data to a 1:1 interaction model using BIAevaluation software 4.1 (Cytiva) (see Fig. 4b).

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For the development of therapeutic reagents, it is often required that antibody molecules recognize cell-displayed receptors in their natural state and conformation. Because many steps leading to the identification of antibody candidates involve the use of recombinant antigens passively adsorbed or chemically conjugated to surfaces, it is necessary to evaluate the binding of the lead candidates on cells. Binding assays using conventional flow cytometry are used to analyze the cell-binding behavior of the isolated VHHs (Fig. 4c). The entire process is facilitated by the presence of a site-specific biotin moiety at the C-terminus of the VHHs. 1. Expand antigen-positive and antigen-negative cell lines in T75 flasks by culture in complete medium (see Subheading 2.9, step 2). Grow cells at 37 °C in a humidified 5% CO2 incubator until they reach 70–80% confluency. 2. For flow cytometry, detach cell monolayers using Accutase following manufacturer’s instructions. Transfer each cell suspension to two 15 mL conical tubes and spin down for 5 min at 300× g at RT. 3. Discard the supernatant, resuspend each in 10 mL of PBS-BA as a washing step, and resuspend the cells in the same buffer at 1 × 106 cells/mL. 4. Prepare one plate for each cell line. Dispense 50 μL/well (200,000 cells) in a Nunc MicroWell 96-well conical plate. 5. Next, prepare dilutions of each biotinylated VHH to be tested at 2 μg/mL in PBS-BA and transfer 50 μL to wells containing the tumor-positive and tumor-negative cell lines. Keep on ice for 1 h. 6. Centrifuge the plate for 5 min at 300× g at 4 °C and then with a multichannel remove the VHH solutions carefully without disturbing the cell pellet. 7. Wash once with 200 μL of PBS-BA and homogenize by pipetting up and down with a multichannel. Repeat the centrifugation step and discard the supernatant. 8. Add 50 μL of PE-conjugated streptavidin (1/500 from a stock of 1 mg/mL, final concentration of 2 μg/mL) and keep the plate on ice. 9. After an incubation of 1 h on ice, perform a washing step as described in step 6. Finally, resuspend the cell pellet in 100 μL of PBS-BA and acquire the binding data on a CytoFLEX S flow cytometer followed by data analysis using FlowJo software (FlowJo LLC). Alternatively, the cell suspension can be transferred to individual tubes and data can be collected on BD FACSCanto™ System or equivalent (Fig. 4c).

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3.10 Validating VHH Specificity by Immunoprecipitation of the Target Antigen

1. Preparation of cell extract: Expand cells as described in Subheading 3.9, step 1 and grow until confluency on T75 tissue culture flask. Harvest 50 × 106 target cells (see Subheading 3.9, step 2) (see Note 38) by Accutase treatment and wash twice 10 mL of PBS by centrifugation for 5 min at 300× g at 4 °C. 2. Resuspend the pellet in 10 mL of solubilization/wash buffer, vortex until homogenized, and incubate 1 h on ice. Next, spin down at 20.000× g for 10 min at 4 °C to remove insoluble material and filter through a 0.22 μm filter to remove any cell debris. 3. Preparation of resin/bio-VHH: For every antibody to test, take 50 μL of streptavidin sepharose and wash twice by adding 950 μL of PBS to equilibrate the resin. This is performed by cycles of centrifugation for 5 min at 14.000× g rpm followed by removal of the supernatant. 4. Resuspend in 100 μL of PBS and add 10 μg of biotinylated VHH. Incubate 30 min with rotation for the immobilization of the VHH. Then wash the resin twice as described in step 3 to remove any unbound VHH. Include two additional tests: (i) without bio-VHH and (ii) with an isotype control such as a VHH targeting an irrelevant antigen. We usually use A20.1, a VHH specific for Clostridioides difficile toxin A [28] as a negative control. 5. To each VHH-resin test sample, add 1 mL of the cell extract prepared in step 1. Incubate for 1 h at room temperature to allow the antigen/VHH interaction. 6. Then centrifuge for 5 min as described in step 3, resuspend the resin in 1 mL of solubilization/wash buffer, and incubate for 10 min in RT to wash the resin. Spin down for 5 min at 14.000× g, discard the supernatant, and repeat this process three additional times. 7. Next, discard the washing solution and resuspend the resin in 50 μL of 8 M urea to disrupt the antigen/VHH interaction by incubating for 5 min at room temperature. Next, add 50 μL of 2× SDS loading buffer and incubate 5 min at 95 °C in a heating block to completely denature the sample. Then, briefly vortex the samples and centrifuge 5 min at 14.000× g. Transfer the supernatant to a new tube. This material contains the streptavidin, bio-VHH, and the pulled-down antigen (Fig. 5). 8. Finally, separate 10 μL of each sample in a 4–20% SDS-PAGE and transfer the gel to PVDF membrane. Block the membrane overnight with 1% PBS-BSA.

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Fig. 5 Recognition and immunoprecipitation of the cell-displayed antigen from antigen-positive cells. Biotinylated VHHs were immobilized on streptavidin sepharose and used to pull down the antigen from antigen-positive cells solubilized with 1% Triton X-100. The resin was extensively washed with 1% Triton X-100 and the material was denatured with 8 M urea and SDS-loading buffer before separation by 4–20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane, and the presence of the antigen in the wells was detected by using a mAb specific for the antigen, followed by an anti-mouse IgG conjugated to HRP as a probe. Colorimetric HRP substrate was added to demonstrate the presence of the antigen in wells where the VHHs were able to pull down the target antigen

9. The next day, wash the membrane twice with PBS-T and incubate with a mAb specific for the target antigen that is suitable for western blot. It is recommended to use the detection mAb at 1 μg/mL diluted in 10 mL of PBST for 1 h at room temperature. 10. Wash the membrane twice with PBST and detect binding using an anti-mouse Fc conjugated to HRP (if the mAb in step 9 is from a species other than mouse, an alternative detection antibody is required) or equivalent. Incubate 1 h at room temperature. 11. Wash twice with PBS-T and develop using a colorimetric or chemiluminescent substrate (Fig. 5). Additionally, for the accurate identification of the target antigen, the pulled-down bands should be analyzed by mass spectrometry (MS-TOF). In this example, it is not essential because the identity of the antigen was known beforehand. The procedure described here using biotinylated VHHs is also useful for the identification of unknown receptors as described previously [28] and has benefits compared with the immunoprecipitation assay performed with conventional mAbs.

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Notes 1. This is considered the most critical step in the successful isolation of VHHs against the target of interest. Purity of the antigen in stable and functional format is key to trigger proper immune response and for the selection of good-quality antibodies by phage display. It is generally best to analyze the quality of the antigen by size exclusion chromatography and examine its interaction with benchmark monoclonal/polyclonal antibodies in ELISA. Impure and poorly prepared protein products could result in nonspecific immune response and may lead to a failure in obtaining antibodies. Before starting the selection campaigns, it is recommended to browse the availability of site-specific in vivo biotinylated antigens (AviTag) as its immobilization mediated by streptavidin allows for the use of lower amounts of the antigen during panning. This also has the advantage of displaying the antigen in a more natural and functional conformation instead of passive adsorption on an ELISA plate that, for some antigens, will have a direct impact in the quality of the selected antibodies and thus increasing the chance of obtaining more diverse set of VHHs. If the AviTag is not available, it is recommended to perform a mild chemical biotinylation of the antigen using NHS-biotin at a low ratio such as 1:1 [29, 30]. 2. There is no universal llama immunization protocol and various immunization schedules have been reported by different laboratories. Generally a short immunization protocol of 7–8 weeks is used for protein antigen and longer immunization regimen is used for DNA/cell immunization as described elsewhere. As reagents to stimulate and enhance the immune response, we use emulsions of purified antigen and Freund’s complete adjuvant (CFA) (first injection) and boosters with Freund’s incomplete adjuvant (IFA). 3. When using PBS as buffer on the recombinant proteins, it may be supplemented with 3 mM EDTA to chelate any residual Ni2+ and to prevent protein oxidation and degradation, as long as small amounts of EDTA will not adversely affect downstream assays. 4. The MJ7 primer is specifically designed to overlap with the MJ1–3 primers for the pMED1 vector with the specific SfiI sites (GGCCN5GGCC) and the pelB leader sequence. If another vector with a different signal peptide is used, then an overlapping primer with appropriate restriction enzyme is required. The MJ8 primer is complementary to the FR4 region of the VHH plus the SfiI restriction enzyme overhang.

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5. NanoDrop™ One Microvolume UV-Vis spectrophotometer (or instruments with similar technology) measures absorbance at very low volumes (1 μL) and the use of cuvettes is not required. 6. To avoid thawing and freezing the ligase buffer, aliquot and store at -20 or -80 °C for future use. 7. For library construction, it is recommended to purchase commercial electrocompetent cells as the efficiency is generally higher than what is achievable in the lab following conventional protocols. This has a big impact on the quality of the library as the diversity will be higher, allowing a better representation of the original diversity from the llama’s antibody repertoire of antibodies. 8. From the glycerol stock of E. coli TG1 cells, streak out M9 agar plate [25] supplemented with 0.01% of thiamine. Incubate the plate overnight at 37 °C for 24 h. The sealed plate could be kept at 4 °C for up to month. It is required that TG1 E. coli cells be grown in M9 minimal medium supplemented with thiamine to ensure that the F′ pilus (mediating phage infection) is maintained on the cells. 9. If biotinylated antigen is not available, one may use non-biotinylated antigen and proceed with the panning by passive absorption on Nunc maxisorp plate or alternatively performing gentle/mild chemical biotinylation as mentioned in Note 1. 10. Monoclonal phage ELISA is the traditional screening method practiced in the past several decades. However, we have occasionally observed an inconsistency between the VHH-phage ELISA results and the protein ELISA of the same soluble VHH (when not fused to the phage). The first method relies in the amplification of the signal by using a mAb/pAb against the minor capsid protein pVIII, which is represented in ~1500 copies, compared with detection using streptavidin or anti-Tag antibodies, which are present in only one copy in the monomeric VHH. Therefore, we suggest an alternative VHH protein screening procedure where the pool of R3/R4 phages are sub-cloned into an expression vector (pMRO) [19, 21] and then use the Promega S30 T7 High-yield In vitro Protein Expression System to produce soluble VHHs for ELISA screening. Moreover, this approach bypasses the time-consuming gene synthesis and sub-cloning steps after monoclonal phage ELISA. 11. Selecting phage from round 3 or 4 depends on the level of enrichment in sequences observed during the panning. If higher diversity is desired, then round 3 phages may be a better source of target-specific VHH binders.

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12. In order to prevent evaporation during the overnight culture of bacterial culture, the 96-well U-shaped bottom microtiter plate is placed in a humid box with a wet pad of tissue paper. 13. Unique VHH sequences obtained after monoclonal phage ELISA screening (Subheading 3.5) are cloned into the pMRO-BAP-H6 vector (using SfiI restriction enzyme sites) and used for large-scale expression. Alternatively, the VHHs could be synthetized and cloned by a commercial vendor such as TWIST Bioscience. Alternatively, unique and positive colonies after R3/R4 pool sub-cloning into pMRO (Subheading 3.6) are used for large-scale expression. 14. Dilute the Cam stock solution to 1 mg/mL in ultrapure H2O before use and store the working stock at 4 °C for up to 30 days. Protect from light to avoid photodegradation. 15. The use of azide and cold buffer is optional. Their use is suggested to prevent the internalization of the VHH during the staining steps that might reduce the amount of antibody bound to the surface of the cell. 16. The detergent at this percentage will be able to lyse cells, solubilize membrane proteins, and preserve them in native conditions. Do not use RIPA as it will cause protein denaturation and decrease protein-protein interactions. 17. This is a critical step in the isolation of high-affinity VHH antibody fragments. As adjuvants, we often use emulsions of purified immunogen and Freund’s complete (CFA)/incomplete (IFA) adjuvants, which results in its sustained presentation to the animal immune system, thereby stimulating and enhancing the immune response to the immunogen. Other complex materials such as DNA, mRNA, VLP, and fractionated membranes or even cell lines expressing antigen of interest could be also used for immunization [19, 21]. Also, 1–5 antigens with similar origin (mammalian vs. nonmammalian) could be combined in each llama immunization to save the cost. 18. We routinely use llamas (Lama glama) for immunization due to their availability; however, other camelid members such as alpacas, camels, or dromedaries can be used for this purpose. 19. If using biotinylated antigen in ELISA or in the panning experiment, it is very important to make sure that all reagents, buffers, and solutions used for panning are free of biotin in order to avoid or minimize blocking the binding capacity of streptavidin before the immobilization of the biotinylated antigen. 20. Alternatively, one could use non-biotinylated antigen or Fc-fused format of the antigen followed by using biotinylated

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mAbs specific to the camelid heavy chain antibodies (available at NRC-HHT; see Ref. [31] and detect the bound antibodies with a streptavidin-HRP conjugate. 21. Before starting the RNA work, it is strongly recommended that all the surfaces and instruments get thoroughly cleaned to minimize the presence of RNase before starting the RNA extraction. Commercially available RNase AWAY™ Surface Decontaminant or similar RNase blocking reagents are recommended for this purpose. 22. At times it may be required to optimize the amount of input RNA, but generally 3–5 μg total RNA per reaction results in a good yield of synthesized cDNA to be used as a template for PCR. 23. Three PCR fragments are obtained following RT-PCR: one with a size of ffi850 bp, which corresponds to conventional antibodies, and two intimately close smaller fragments with sizes in the range of 550–650 bp, which correspond to heavy chain antibodies and contain the VHH genes. The aim of optimizing the PCR reaction is to increase the intensity of the VHH-CH2/VHHCH2b bands relative to the conventional antibody band. However, differential intensities of the two VHH bands with respect to each other are routinely observed on agarose gels. 24. A small-scale ligation could be performed with different molar ratios of insert (VHH) to vector (pMED1). Typically a 1:3 ratio works best. Additionally, we suggest to perform colony PCR of the products to test the efficiency of ligation and percentage of VHH insert before going ahead with the largescale ligation and electroporation. A library size of close to the number of lymphocytes (5 × 107) used for RNA extraction is considered a good representation of the VHH repertoire, although in llamas less than half of the immunoglobulin repertoire is heavy chain antibody [4, 32]. 25. Generally a 0.5–1 μL aliquot of the PCR product can be used directly for DNA sequencing. If the data profile is not readable, purify the PCR product with any commercial kit before proceeding. Additionally, either upstream (M13R) or downstream primer (PN2) gives enough coverage to obtain a read covering the complete VHH fragments. 26. When analyzing the library sequences, bear in mind that VHHs are distinguished from contaminating VHs by examining the four amino acids at positions 37, 44, 45, and 47 (Kabat numbering system, see Ref. [33]. VHHs characteristically have Phe or Tyr at position 37; Glu at position 44; Gln, Arg, or Cys at position 45; and Gly, Ser, Leu, or Phe at 47, whereas VHs have, respectively, Val, Gly, Leu, and Trp at these four positions.

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27. The commercially available M13KO7 has a low titer (1 × 1011 cfu/mL). Before starting we expand and amplify the helper phage accordingly to Protocol 10.2 described in phage display: a laboratory manual [34]. In our laboratory we usually get a titer of 1013 cfu/mL, which is 100× more concentrated. 28. The volume of PBS to resuspend the phage pellet depends on the size of the pellet and the viscosity of the phage solution. Increase the volume accordingly. 29. To determine the titer of the phage, make 10-6, 10-8, 10-10, and 10-12 serial dilutions of phage in PBS and mix 10 μL of each dilution with 100 μL of the exponentially growing TG1 E. coli cells. Incubate the cells at RT for 15 min and subsequently plate them on LB-Amp medium. In the morning count the colonies and determine the titer. Phage titers are typically 5 × 1012–1 × 1013 colony-forming units/mL from a 100 mL culture. 30. It is extremely important to discard the phage contaminated tips, solutions, and waste properly, and the M13 phage could easily contaminate the bio-hood and working areas and pipette and cause quite a bit of headache afterward. Therefore, allocate a container with 10% bleach to discard all the contaminated tips, tubes, etc. Clean the working areas properly and leave the UV light on after completing your work. 31. If desired, one might do an alternative panning by passive absorption of the antigen to compare the outcome obtained from the panning against the oriented immobilized biotinylated antigen. We generally obtain a more diverse set of VHH binders using biotinylated antigen than the passively absorbed antigen panning, and the quality/functionality is frequently better. 32. From a freshly prepared TG1 E. coli plate (see Note 8), pick a single colony and inoculate a 2–3 mL of 2 × YT medium (no antibiotics) in a sterile 15 mL falcon tube and incubate at 37 °C in a rotary shaker at 220 rpm. Remove aliquots from the culture flask at different time intervals and measure the A600 nm in a spectrophotometer in disposable cuvettes using 2 × YT as the blank. Stop the incubation at A600 nm ≈ 0.3–0.4 (this usually takes 2–3 h but could take longer). 33. Serial dilutions (10-2–10-6) of the infected cells are performed in 2 × YT in 500 μL volumes. Spread 100 μL of each dilution on LB-Amp plates. Also plate 100 μL of the uninfected cells as a negative control. Incubate at 32 °C overnight. Keep the plates parafilm-sealed and stored at 4 °C for clonal analysis (colony PCR, sequencing, and phage ELISA).

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34. It is recommended to alternate between two or more blocking buffer to minimize any chance of obtaining phage binders against the blocking proteins (e.g., use PBS-1% casein in the second round and fourth round and PBS-1% BSA in the third). This step is essential for the panning of naı¨ve or synthetic libraries but less important when dealing with the panning of immune libraries. 35. A test run is recommended to be done when working with a new construct for protein expression using a small volume of culture (10 mL) and applying varying conditions: different IPTG concentrations (5 μM–800 μM), growth temperatures, (16 °C, 28 °C, 37 °C) and expression time (3 h vs. overnight). 36. Following the manufacturer’s recommendations, 10 mL of a 10× protease inhibitor cocktail is sufficient to inhibit proteases in 20 g of bacterial pellet; therefore, determine the mass of your pellet and add enough protease inhibitor. 37. Strain AVB101 is an E. coli B strain (hsdR, lon11, sulA1) harboring a plasmid encoding for the biotin ligase BirA, which is induced by the addition of IPTG. The enzyme will recognize the AviTag encoded at the C-terminus of the VHHs. For simplicity we prefer to perform the enzymatic biotinylation post-expression only when needed. 38. The number of cells used may need to be optimized depending on the expression level of the antigen of interest.

Acknowledgments This work was supported by the National Research Council of Canada, Human Health Therapeutic Research Center. We gratefully acknowledge the excellent assistance of Sonia Leclerc for DNA sequencing. Conflict of Interest Statement The authors have no conflicts of interest to declare.

References 1. Murphy KM, Weaver C (2016) Janeway’s immunobiology: ninth international, Student edn. Garland Science 2. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ et al (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27(1):1 3. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage

display technology. Annu Rev Immunol 12: 433 –455 C, 4. Hamers-Casterman Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363(6428):446–448 5. Flajnik MF, Kasahara M (2010) Origin and evolution of the adaptive immune system:

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genetic events and selective pressures. Nat Rev Genet 11(1):47–59 6. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775 –797 7. Nguyen VK, Hamers R, Wyns L, Muyldermans S (2000) Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire. EMBO J 19(5):921–930 8. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM et al (1988) Single-chain antigen-binding proteins. Science 242(4877):423–426 9. Skerra A, Pluckthun A (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240(4855): 1038–1041 10. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554 11. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414(3):521–526 12. Muyldermans S (2021) Applications of nanobodies. Annu Rev of Anim Biosci 9:21 13. Arbabi-Ghahroudi M (2017) Camelid singledomain antibodies: historical perspective and future outlook. Front Immunol 8:1589 14. Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M et al (2009) Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol 198(3):157–174 15. Holliger P, Winter G (1993) Engineering bispecific antibodies. Curr Opin Biotechnol 4(4): 446–449 16. McComb S, Nguyen T, Shepherd A, Henry KA, Bloemberg D, Marcil A et al (2022) Programmable attenuation of antigenic sensitivity for a Nanobody-based EGFR chimeric antigen receptor through hinge domain truncation. Front Immunol 13:864868 17. Henry KA, Tanha J, Hussack G (2015) Identification of cross-reactive single-domain antibodies against serum albumin using nextgeneration DNA sequencing. Protein Eng Des Sel 28(10):379–383 18. Chakravarty R, Goel S, Cai W (2014) Nanobody: the “magic bullet” for molecular imaging? Theranostics 4(4):386–398

19. Trempe F, Rossotti MA, Maqbool T, MacKenzie CR, Arbabi-Ghahroudi M (2022) Llama DNA immunization and isolation of functional single-domain antibody binders. Methods Mol Biol 2446:37 –70 20. Rossotti MA, van Faassen H, Tran AT, Sheff J, Sandhu JK, Duque D et al (2022) Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models. Commun Biol 5(1):933 21. Baral TN, MacKenzie R, Arbabi GM (2013) Single-domain antibodies and their utility. Curr Protoc Immunol 103:2 17 1-2 57 22. Arbabi-Ghahroudi M, To R, Gaudette N, Hirama T, Ding W, MacKenzie R et al (2009) Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points. Protein Eng Des Sel 22(2):59–66 23. Rossotti MA, Henry KA, van Faassen H, Tanha J, Callaghan D, Hussack G et al (2019) Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling. Biochem J 476(1):39–50 24. Li Y, Sousa R (2012) Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin. Biotechnol Lett 34(9):1629–1635 25. Maniatis T, Fritsch EF, Sambrook J, Sambrook J (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory 26. Hussack G, Arbabi-Ghahroudi M, Mackenzie CR, Tanha J (2012) Isolation and characterization of Clostridium difficile toxin-specific single-domain antibodies. Methods Mol Biol 911:211 –239 27. Arbabi-Ghahroudi M, Tanha J, MacKenzie R (2009) Isolation of monoclonal antibody fragments from phage display libraries. Methods Mol Biol 502:341 –364 28. Hussack G, Arbabi-Ghahroudi M, van Faassen H, Songer JG, Ng KK, MacKenzie R et al (2011) Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. J Biol Chem 286(11):8961–8976 29. Rossotti MA, Pirez M, Gonzalez-Techera A, Cui Y, Bever CS, Lee KS et al (2015) Method for sorting and pairwise selection of nanobodies for the development of highly sensitive sandwich immunoassays. Anal Chem 87(23): 11907–11914 30. Hussack G, Ryan S, van Faassen H, Rossotti M, MacKenzie CR, Tanha J (2018) Neutralization

Isolation and Characterization of Single-Domain Antibodies from Immune. . . of Clostridium difficile toxin B with VHH-fc fusions targeting the delivery and CROPs domains. PLoS One 13(12):e0208978 31. Henry KA, van Faassen H, Harcus D, Marcil A, Hill JJ, Muyldermans S et al (2019) Llama peripheral B-cell populations producing conventional and heavy chain-only IgG subtypes are phenotypically indistinguishable but immunogenetically distinct. Immunogenetics 71(4): 307–320 32. Wernery U (2001) Camelid immunoglobulins and their importance for the new-born—a

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Chapter 8 Phagekines: Directed Evolution and Characterization of Functional Cytokines Displayed on Phages Gertrudis Rojas, Tania Carmenate, Gisela Garcı´a-Pe´rez, and Dayana Pe´rez-Martı´nez Abstract The current chapter focuses on the use of filamentous phages to display and modify biologically active cytokines, with special emphasis on directed evolution of novel variants showing improved receptor binding. Cytokines are essential protein mediators involved in cell-to-cell communication. Their functional importance and the complexity of their interactions with multichain receptors make cytokine engineering a promising tool for the discovery and optimization of therapeutic molecules. Protocols used at the laboratory are illustrated through examples of manipulation of interleukin-2 and interleukin-6, two members of the family of alpha-helix-bundle cytokines playing pivotal roles in immunity and inflammation. Key words CTLL-2 proliferation assay, ELISA, Cytokine receptor, Interleukin-2, Interleukin-6, Kunkel mutagenesis, Library, Phage display, STAT3 phosphorylation, Western blot

1

Introduction Phage display, an invention awarded with the Nobel Prize in Chemistry in 2018 [1, 2], is frequently used to select antibody fragments [3] and short peptides [4] with the desired binding properties, from large single-pot libraries. Its impact goes beyond such common applications (extensively described here and in other volumes). Virtually every protein can be displayed on filamentous phage [5]. This chapter focuses on phage display of cytokines, an essential class of protein mediators involved in cell-to-cell communication. Their expression during immune and inflammatory responses is tightly regulated, and their binding to multi-subunit receptors results in activation, proliferation, differentiation, survival, or death of particular cell types. Phage display has been used to investigate structure-function relationships of cytokines and for their in vitro evolution [6–9].

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_8, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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The current chapter is an updated version of our previous publication about phagekines [10]. From a methodological point of view, we are including protocols for the construction and screening of large libraries of cytokine-derived variants made by Kunkel mutagenesis [11]. The discovery of mutated molecules with better developability profiles is mentioned [12]. Our previous experience with interleukin-2 (IL-2) has been extended to interleukin-6 (IL-6). Described techniques are illustrated with examples involving both cytokines. IL-2 is a key mediator controlling the balance between tolerance and immune responsiveness [13], used for therapy in diverse clinical scenarios [14, 15]. The first reports of phage-displayed IL-2 were published in 1997 [16, 17]. This platform was revisited and expanded in our laboratory for high-resolution epitope mapping of anti-IL-2 antibodies [18, 19] and characterization of IL-2-derived muteins [20]. More recently, the construction of large libraries allowed selecting novel variants of the molecule by virtue of their ability to bind to a selector receptor subunit. Two classes of mutations were identified: those increasing the affinity for the receptor and those improving display levels, global folding, and biological activity of the displayed protein [12]. Introduction of the latter in recombinant proteins (outside the phage context) gave rise to IL-2 variants with favorable developability profiles (highly stable, less aggregation-prone, and well expressed by mammalian cells). Such recent experiences incorporated phage display to the toolbox used to engineer IL-2-derived molecules with biased immunological functions and pharmaceutical potential, an area dominated up to now by yeast display and rational design [21–26]. IL-6, also displayed on phage long time ago [27, 28], is a central mediator of both acute and chronic inflammation. Beyond its known relevance in infections, autoimmunity, and cancer, IL-6 gained special attention in the last few years due to its crucial involvement in cytokine storm events behind the adverse effects of modern anticancer adoptive cell therapies and the pathogenesis of severe COVID-19 [29–31].

2

Materials

2.1 Displaying Cytokines on Filamentous Phages

1. Use XL-1 Blue Escherichia coli (E.coli) strain (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac F′ proAB lacIqZ_M15 Tn10 Tetr) to obtain plasmid DNA for cloning and sequencing. 2. Use TG1 E. coli strain (K12_(lac-pro), supE, thi, hsdD5/F′ traD36, proA+B+, lacIq, lacZ_M15), and M13KO7 to rescue phagemid-containing viral particles displaying cytokines.

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3. Prepare 2xTY by dissolving tryptone (16 g/L), yeast extract (10 g/L), and sodium chloride (5 g/L) in water. Sterilize by autoclaving during 20 min at 120 °C and 1 atm. 4. Add 1.5% (w/v) of bacteriological agar to liquid 2xTY to obtain solid 2xTY. Sterilize by autoclaving during 20 min at 120 °C and 1 atm. 5. Prepare 40% glucose (w/v) stock solution in sterilized water, and filter through 0.2 μm disposable filters. 6. Prepare 100 mg/mL ampicillin stock solution in sterilized water, and filter through 0.2 μm disposable filters. Keep aliquots at -20°C. 7. Prepare 70 mg/mL kanamycin stock solution in sterilized water, and filter through 0.2 μm disposable filters. Keep aliquots at -20°C. 8. Prepare 2xTY/AG by supplementing 2xTY with 100 μg/mL ampicillin and 2% glucose (w/v) before use. 9. Prepare 2xTY/AK by supplementing 2xTY with 100 μg/mL ampicillin and 70 μg/mL kanamycin before use. 10. Always use aerosol-resistant filter pipette tips for samples containing phages (M13KO7 and phagemid-containing viral particles). 11. Use disposable gamma-irradiated plastic materials for E. coli culture, DNA manipulation, and phage production/purification (pipette tips, aerosol-resistant filter pipette tips, 1.5 mL vials, 50 mL conical-bottom tubes, Petri dishes, 0.2 μm syringe filters, and 0.2 μm centrifugal filtering devices). 12. Leave all the glassware (Erlenmeyer flasks, bottles) that has been in contact with phage samples (M13KO7 or phagemidcontaining viral particles) in 2% bleach (v/v) overnight to decontaminate. 13. Sterilize decontaminated and washed glassware and materials during 20 min at 120 °C and 1 atm before use. 14. Prepare PEG/NaCl phage precipitation solution: 20% polyethylene glycol 6000 or 8000 (w/v) and 2.5 mol/L sodium chloride in water. Sterilize by autoclaving during 20 min at 120 °C and 1 atm. 15. Prepare 10× phosphate-buffered saline (PBS): 1.5 mol/L sodium chloride, 80 mmol/L dibasic sodium phosphate, and 20 mmol/L potassium monobasic phosphate, pH 7.2–7.4. Sterilize by autoclaving during 20 min at 120 °C and 1 atm. 16. Prepare PBS by diluting 100 mL of 10× PBS up to 1 L with sterilized water in a previously sterilized bottle. 17. Prepare 60% glycerol solution (v/v) in water. Sterilize by autoclaving during 20 min at 120 °C and 1 atm.

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2.2 Quantifying Cytokine Display Levels by ELISA

1. Use polyvinyl chloride (PVC) microtitration plates as the solid support. 2. Use 9E10 (anti-c-myc tag) as coating monoclonal antibody (mAb). 3. Prepare PBS as described in Subheading 2.1. 4. Always use aerosol-resistant filter pipette tips for phagecontaining samples. 5. Prepare blocking/diluting solution M-PBS: 4% skim powder milk (w/v) in PBS. 6. Prepare washing solution: 0.1% Tween 20 (v/v) in water. Use a washing bottle to dispense. 7. The protocol was optimized using the anti-M13/horseradish peroxidase (HRP) conjugate (1/5000 working dilution) provided by GE Healthcare to detect bound phages. This product has been discontinued and must be replaced by another HRP-conjugated antibody against M13 PVIII. 8. Prepare a solution of 200 mmol/L dibasic sodium phosphate. 9. Prepare a second solution of 100 mmol/L citric acid. 10. Prepare horseradish peroxidase (HRP) substrate buffer: Adjust the pH of the above described phosphate solution to 5.0 with the citric acid solution. Keep at 4 °C. 11. Prepare HRP substrate solution immediately before use by adding 5 mg of o-phenylenediamine and 5 μL of 30% hydrogen peroxide (v/v) to 10 mL of HRP substrate buffer. 12. Prepare stop solution: Dilute 100 mL of 37% HCl fuming up to 1 L with water.

2.3 Screening Receptor Binding Properties of PhageDisplayed Cytokines by ELISA

1. Recombinant extracellular domain (ECD) of cytokine receptor subunits can be either purchased from commercial sources or expressed and purified at the laboratory. 2. 9E10 anti-c-myc mAb and specific anti-cytokine mAbs can be used to confirm the presence and proper folding of phagedisplayed cytokines, respectively. 3. See the previous Subheading for the rest of materials and solutions required to perform the ELISA.

2.4 Assessing CTLL2 Proliferation Induced by Phage-Displayed IL-2 and IL-2 Variants

1. Use the mouse CTLL-2 cell line (ATCC® TIB-214™) to test in vitro biological activity of phage-displayed IL-2 and IL-2derived muteins. This cell line is derived from cytotoxic CD8+ T cells on a C57BL6 background, depends on IL-2 for growth, and responds to both human and mouse IL-2. 2. Recombinant IL-2 (34–8029-85) for CTLL-2 cell line culture can be purchased from eBioscience (USA). Other IL-2 sources can be used, as long as their specific activity is equal or higher than 106 IU/mg.

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3. Store the frozen cells on liquid nitrogen. 4. Use fetal bovine serum (FBS) supplemented with 10% dimethylsulfoxide (DMSO) (v/v) as freezing medium. 5. Use RPMI culture medium supplemented with additives: 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 10% FBS (v/v), 50 IU/mL penicillin, 50 μg/mL streptomycin, and 50 IU/mL of recombinant IL-2, for CTLL-2 culture. The latter component (IL-2) is not included during some steps of the protocol, specified in Subheading 3.4. 6. Use only disposable cell culture-treated gamma-irradiated plastic materials for CTLL-2 culture (pipette tips, vials, cryovials, 50 mL conical-bottom tubes, 6-well plates, 96-well plates, and flasks). 7. Use alamarBlue reagent DAL1025 (Invitrogen, USA) for detection of viable cells. Other suppliers are also available. 2.5 Measuring Trans-Signaling Induced by PhageDisplayed IL-6

1. Use human lung carcinoma A549 cell line (ATCC® CCL-185™). Alternatively, H125 cell line can be used. 2. Use DMEM F12 medium, supplemented with 10% FBS (v/v), for A549 culture. 3. Use disposable cell culture-treated gamma-irradiated plastic materials for A549 culture (6-well plates, 50 mL conicalbottom tubes, pipettes, pipette tips, microcentrifuge tubes, and scrapers). 4. Use a recombinant protein comprising the IL-6 receptor alpha subunit ECD. 5. Use recombinant IL-6 as positive control. 6. Prepare PBS as described in Subheading 2.1. 7. Prepare RIPA buffer, 50 mmol/L Tris–HCl buffer, pH 7.4 with additives: 1% NP-40 (v/v), 0.5% Na-deoxycholate (w/v), 0.1% sodium dodecyl sulfate (w/v), 150 mmol/L NaCl, 2 mmol/L EDTA. 8. Supplement RIPA buffer right before use with protease inhibitors: 1 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L NaF and 0.2 mmol/L Na3VO4, and with 1/100 dilutions of Sigma-Aldrich (USA) phosphatase inhibitor cocktails 1 (P2850), 2 (P5726), and 3 (P0044). 9. Use commercially available Laemmli reducing sample buffer, solutions for SDS/PAGE gel preparation and running, as well as a vertical electrophoretic chamber and gel casting devices. 10. Use Ponceau S red dye. 11. Use transfer buffer: 25 mmol/L Tris, 192 mmol/L glycine, 20% methanol (v/v), pH 8.3, Immun-Blot® PVDF Membrane

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(Bio-Rad, USA, 1620177) and Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad, USA) for protein transfer from gels. 12. Prepare Tris-buffered saline (TBS): 20 mmol/L Tris, 150 mmol/L NaCl, pH 7.5. 13. Prepare washing solution (TBS-T): 0.1% Tween 20 (v/v) in TBS. 14. Prepare B-TBS-T: 5% bovine serum albumin (w/v) in TBS-T. 15. Prepare M-TBS-T: 5% skim powder milk (w/v) in TBS-T. 16. The protocol has been optimized using the following primary antibodies: phospho-Stat3 (Tyr105) (DA37) XP® rabbit mAb #9145, Stat3 (79D7) rabbit mAb #4904, and β-actin (13E5) rabbit mAb #4970 from Cell Signaling Technology (USA). 17. Use a suitable secondary anti-IgG antibody recognizing the primary antibodies (conjugated to HRP). If rabbit primary antibodies were used (see above), the secondary antibody must be a conjugated anti-rabbit IgG. 18. Prepare 10% SDS (w/v). 19. Prepare 0.5 mol/L Tris–HCl, pH 6.8. 20. Prepare striping buffer by mixing: 20 mL of 10% (w/v) SDS, 12.5 mL of 0.5 mol/L Tris–HCl, pH 6.8, 67.5 mL of distilled water, and 0.8 mL ß-mercaptoethanol. 21. Use Luminol reagent sc-2048 (Santa Cruz Biotechnology, USA) or a similar HRP chemiluminiscent substrate and X-ray films for signal detection. 2.6 Producing Single-Stranded DNA Templates for Cytokine Library Construction

1. Use CJ236 E. coli strain (dut- ung- thi-1 relA1 spoT1 mcrA/ pCJ105 (F′ camr)) to obtain single-stranded DNA (ss-DNA) templates. 2. Use XL-1 Blue E. coli strain (see Subheading 2.1) as an alternative host to obtain ss-DNA templates. 3. Use M13KO7 helper phage to rescue template-containing viral particles. 4. Prepare the following media as described in Subheading 2.1: 2xTY, solid 2xTY, 2xTY/AG, and 2xTY/AK. 5. Prepare the stock solution of 5 mg/mL chloramphenicol in sterilized water, and filter through 0.2 μm disposable filters. Keep aliquots at -20 °C. 6. Prepare the stock solution of 0.25 mg/mL uridine in sterilized water, and filter through 0.2 μm disposable filters. Keep aliquots at -20 °C. 7. Prepare a stock solution of 5 mg/mL tetracycline in ethanol. Keep at 4 °C.

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8. Supplement 2xTY with the additives described above immediately before use (at the final concentrations described in Subheading 3.6). 9. Always use aerosol-resistant filter pipette tips for samples containing phages. 10. Use disposable gamma-irradiated plastic materials for E. coli culture, phage production/purification, and ss-DNA purification (pipette tips, aerosol-resistant filter pipette tips, vials, 50 mL conical-bottom tubes, and Petri dishes). 11. Decontaminate, wash, and sterilize all the glassware and non-disposable plastic materials as described in Subheading 2.1. 12. Prepare PEG/NaCl phage precipitation solution and PBS as described in Subheading 2.1. 13. Use QIAprep Spin M13 kit solutions and columns (Qiagen, Germany) to purify ss-DNA. 14. Use standard agarose gels with ethidium bromide for DNA electrophoresis. 2.7 Kunkel Mutagenesis Diversification Reactions

1. The protocol was optimized using the following DNA-modifying enzymes provided by New England Biolabs (USA): T4 polynucleotide kinase (10,000 units/mL), T7 DNA polymerase (unmodified) (10,000 units/mL), and T4 DNA ligase (400,000 units/mL). 2. Use solutions of ATP (10 mmol/L) and dNTPs (10 mmol/L each) also provided by New England Biolabs. 3. Prepare 10× TM buffer: 0.5 mol/L Tris, 0.1 mol/L magnesium chloride, pH 7.5. Sterilize by autoclaving during 20 min at 120 °C and 1 atm. 4. Prepare 100 mmol/L dithiothreitol (DTT) solution in sterilized water. Filter through 0.2 μm disposable filters. Keep aliquots at -20 °C. 5. Use sterilized water to dilute mutagenic oligonucleotides and to prepare the mutagenesis reactions. 6. Use disposable gamma-irradiated pipette tips and vials for sitedirected mutagenesis.

2.8 Preparation of Electrocompetent Cells and Electroporation with Mutagenesis Products to Construct Libraries of Cytokine Variants

1. Prepare 2xTY, 2xTY/AG, and solid 2xTY/AG as described in Subheading 2.1. 2. Use TG1 E. coli (see Subheading 2.1) to prepare electrocompetent cells. 3. Use disposable gamma-irradiated plastic materials for E. coli culture, cell manipulation, and electroporation: pipette tips,

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1.5 mL vials, 50 mL conical-bottom tubes, Petri dishes, 25 x 25cm2 square plates, 0.22 μm syringe filters, and 2 mm gap electroporation cuvettes. 4. Sterilize decontaminated and washed glassware (Erlenmeyer flasks) and non-disposable plastic materials (centrifuge bottles and bacterial spreaders) during 20 min at 120 °C and 1 atm. 5. Use low-conductivity distilled water (ideally purified with a milli-Q lab system). 6. Rinse a clean bottle extensively to remove salt traces before filling with water (500 mL). Sterilize during 20 min at 120 °C and 1 atm. Keep on ice until use. 7. Rinse another bottle as described, and fill with 100 mL of 15% glycerol solution (v/v). Sterilize during 20 min at 120 °C and 1 atm. Keep on ice until use. 8. Use Qiaquick PCR Purification Kit (Qiagen, Germany) to purify mutagenesis products before electroporation. 2.9 Library Phage Production at a 300 mL Scale

1. Prepare 2xTY, 2xTY/AG, and 2xTY/AK as described in Subheading 2.1. 2. Prepare PEG/NaCl phage precipitation solution, 60% glycerol solution, and PBS as described in Subheading 2.1. 3. Use M13KO7 helper phage to rescue phagemid-containing viral particles. 4. Use disposable gamma-irradiated plastic materials for E. coli culture and phage production/purification (pipette tips, aerosol-resistant filter pipette tips, 1.5 mL vials, 50 mL conical-bottom tubes, 0.2 μm syringe filters). 5. Leave all the glassware (Erlenmeyer flasks) and non-disposable plastic materials (centrifuge bottles) that have been in contact with phages (M13KO7 helper phage or phagemid-containing viral particles) in 2% bleach (v/v) overnight to decontaminate. 6. Sterilize decontaminated and washed glassware and non-disposable plastic materials during 20 min at 120 °C and 1 atm before use.

2.10 Phage Panning and Amplification

1. Use Immunotubes (Nunc, Denmark) as solid phase for phage panning. 2. Recombinant extracellular domains of cytokine receptor subunits (ECD) can be either purchased from commercial sources or expressed and purified at the laboratory. 3. Prepare 2xTY, 2xTY/AG, and 2xTY/AK as described in Subheading 2.1. 4. Prepare PEG/NaCl phage precipitation solution, 60% glycerol solution, and PBS as described in Subheading 2.1.

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5. Prepare M-PBS as described in Subheading 2.2. 6. Prepare PBS-T: 0.1% Tween 20 in PBS (v/v). 7. Use TG1 E. coli (see Subheading 2.1) for phage infection. 8. Use M13KO7 helper phage to rescue phagemid-containing viral particles. 9. Use disposable gamma-irradiated plastic materials for E. coli culture and phage production/purification/selection (pipette tips, aerosol-resistant filter pipette tips, 1.5 mL vials, 50 mL conical-bottom tubes, 0.2 μm syringe filters, medium-sized plates of 15 cm diameter, and Petri dishes). 10. Leave all the glassware (Erlenmeyer flasks) and non-disposable plastic materials (centrifuge bottles) that have been in contact with phages (M13KO7 helper phage or phagemid-containing viral particles) in 2% bleach (v/v) overnight to decontaminate. 11. Sterilize decontaminated and washed glassware and non-disposable plastic materials during 20 min at 120 °C and 1 atm before use. 12. Prepare TEA elution solution: 100 mmol/L triethylamine, right before use by adding 140 μL of TEA to 10 mL of sterilized water. 13. Prepare neutralization solution: 1 mol/L Tris, pH 7.5. Sterilize during 20 min at 120 °C and 1 atm. 2.11 Phage Production at 96-Well Scale

1. Prepare 2xTY, 2xTY/AG, and 2xTY/AK as described in Subheading 2.1. 2. Use TG1 E. coli (see Subheading 2.1) for phage infection. 3. Use M13KO7 helper phage to rescue phagemid-containing viral particles. 4. Use disposable gamma-irradiated plastic materials for E. coli culture and phage production (pipette tips, aerosol-resistant filter pipette tips, polypropylene 96 deep-well plates with capacity for 1–2 mL, gas-permeable lids).

2.12 Clonal Screening by ELISA and Sequencing

1. Use recombinant cytokine receptor or receptor subunit ECD and 9E10 anti-c-myc mAb, as coating molecules for ELISA. 2. See Subheading 2.2 for the rest of materials and solutions required to perform the ELISA. 3. Use XL-1 Blue E. coli (see Subheading 2.1) for plasmid DNA production. 4. Prepare 2xTY, 2xTY/AG, and solid 2xTY/AG as described in Subheading 2.1. 5. Prepare 5 mg/mL tetracycline stock as described in Subheading 2.6.

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6. Use disposable gamma-irradiated plastic materials for E. coli culture, phage infection, and DNA purification (pipette tips, aerosol-resistant filter pipette tips, 1.5 mL vials, U-bottom 96-well cell culture plates, 12-well cell culture plates, 50 mL conical-bottom tubes). 7. Sterilize decontaminated and washed glassware (Erlenmeyer flasks) during 20 min at 120 °C and 1 atm before use. 8. Use QIAprep Spin minikit (Qiagen, Germany) for plasmid DNA purification.

3

Methods

3.1 Displaying Cytokines on Filamentous Phages

1. Synthesize cytokine genes, flanked by ApaLI and NotI unique restriction sites. Optimize the DNA sequences according to E. coli codon usage. See Notes 1–3 for useful IL-2 gene modifications. Use mutated genes to display muteins instead of wild-type (wt) cytokines. 2. Clone cytokine genes between ApaLI and NotI restriction sites of pCSM phagemid vector (Fig. 1) using standard DNA cloning techniques (see Note 4). 3. Transform TG1 competent cells with the genetic constructs and grow transformed cells on solid 2xTY/AG during 16–20 h at 37 °C. 4. Pick isolated colonies and inoculate them into 50 mL tubes containing 10 mL of 2xTY/AG (see Note 5). Grow at 37 °C with shaking at 250 rpm for 16–20 h. 5. Dilute the cell suspension from step 4 (1/100) in a 50 mL tube containing 10 mL of fresh 2xTY/AG. Grow at 37 °C with shaking at 250 rpm until an absorbance at 600 nm in the range 0.4–0.8 is reached. 6. Add 1011 plaque-forming units (pfu) of M13KO7 helper phage (see Note 6) to grown bacteria and incubate at 37 °C during 30 min without shaking. 7. Centrifuge at 2000 g during 15 min. Remove the supernatant. 8. Resuspend the cell pellet in 40 mL of 2xTY/AK in a 250 mL Erlenmeyer flask. Grow the cells at 28 °C with shaking at 250 rpm during 16–20 h. 9. Centrifuge at 4000 g during 15 min at 4 °C. 10. Collect the supernatant and mix it with 10 mL of PEG/NaCl solution. Incubate during 1 h on ice to precipitate the phages (see Note 7). 11. Centrifuge at 4000 g during 15 min at 4 °C. Remove the supernatant.

Fig. 1 Genetic constructs for cytokine phage display. Schematic representation of the pCSM+ phagemid vector (a). The vector includes DNA sequences coding for DsbA signal peptide (MKKIWLALAGLVLAFSASA), the cytokine of interest, and hexahistidine and c-myc tags. Additional elements are shown: lac promoter, ribosomal binding site, amber stop codon, ApaLI and NotI restriction sites, M13 gene 3, transcription terminator sequence, E. coli and phage replication origins, and ampicillin resistance gene. Protein sequences deduced from the cloned genes coding for human IL-2 (b), mouse IL-2 (c), and human IL-6 (d) genes are also shown. Unpaired C125 (human IL-2) and C140 (mouse IL-2) are replaced by Ser (shaded in gray). Residue K35 (indicated by arrow) in human IL-2 can be optionally replaced by Glu, resulting in higher display levels and improved folding of the molecule

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12. Resuspend the phage pellet in 1 mL of PBS and transfer to a vial. 13. Centrifuge at 10,000 g during 10 min at 4 °C in a microcentrifuge to remove the remaining bacteria and cell debris. 14. Mix the supernatant with 250 μL of PEG/NaCl solution in a new vial (see Note 8). Incubate during 20 min on ice. 15. Centrifuge at 10,000 g during 10 min at 4 °C in a microcentrifuge. Remove the supernatant. 16. Gently resuspend the phage pellet in 1 mL of PBS (see Note 9). Add 500 μL of 60% glycerol solution to the vial and mix. 17. Filter purified phages through a 0.2 μm centrifugal filtering device (see Note 10) to sterilize them. 18. Keep purified phages at -20 °C to characterize their binding properties and biological activity as described in the next Subheadings. 3.2 Quantifying Cytokine Display Levels by ELISA

1. Coat a PVC ELISA microplate with 100 μL/well of 10 μg/mL anti-c-myc tag 9E10 mAb in PBS (see Note 11). Cover the plate with a lid and incubate 16–20 h at 4 °C. 2. Discard the coating solution by inverting the plate several times. 3. Add 200 μL/well of M-PBS to block the plate. Cover the plate with a lid and incubate 30 min at room temperature (RT). 4. During the incubation, dilute phage standard (see Note 12) and purified phage samples (see Note 13) in M-PBS. 5. Discard the blocking solution from the plate. 6. Add 100 μL/well of diluted phage standard or samples. Each dilution should be applied at least twice to obtain independent replicates for each data point. Add M-PBS alone to some coated/blocked wells to assess the background levels. Cover the plate with a lid and incubate 1 h at RT. 7. Discard the samples by inverting the plates several times. Wash the plate at least five times by filling the wells with washing solution and discarding it (see Note 14). 8. Add 100 μL/well of the anti-M13/HRP conjugate (appropriately diluted in M-PBS). Cover the plate with a lid and incubate 1 h at RT. 9. Wash the plate at least eight times as described in step 7. 10. Add 100 μL/well of HRP substrate solution. Incubate during 15 min at RT. A yellow/orange color should develop in the standard and sample wells, while the wells containing M-PBS should remain colorless (indicating low background levels). 11. Stop the reaction with 50 μL/well of the stop solution. Read the absorbances at 490 nm with a microplate reader.

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12. Calculate the mean absorbance values for replicated data points. 13. Use the absorbance values from diluted phage standard to obtain the best fitting curve (see Note 15). Assume a concentration of 100 arbitrary display units/mL of the displayed cytokine for the undiluted standard phage preparation. 14. Interpolate the absorbance value of each diluted phage sample on the standard curve to estimate its relative display level (see Note 16). Consider the dilution factors to calculate the concentrations (in display units/mL) of the undiluted samples (see Note 17). Use the calculated concentrations to normalize the amounts of phage-displayed cytokines in subsequent experiments. 3.3 Screening Receptor Binding Properties of PhageDisplayed Cytokines by ELISA

1. Coat a PVC ELISA microplate with 100 μL/well of recombinant cytokine receptor subunit(s) ECD at 5 μg/mL in PBS (see Note 18). 9E10 anti-c-myc mAb and anti-cytokine mAbs can be used to coat additional wells as controls of the presence and correct folding of the displayed cytokine(s)/cytokine variants. The use of an unrelated coating protein is also recommended to assess nonspecific binding of phage samples (see Note 19). Cover the plate with a lid, and incubate during 16–20 h at 4 °C. 2. Discard the coating solutions by inverting the plate several times. 3. Add 200 μL/well of M-PBS to block the plate. Cover the plate with a lid and incubate 30 min at RT. 4. Discard the blocking solution. Add 100 μL/well of phage samples, properly diluted in M-PBS, to wells coated with the different molecules (see Note 20). Dilutions should be selected to achieve the same concentrations (in display units/mL) for all the phage-displayed cytokines to be compared (see Note 21). Cover the plates with a lid and incubate during 1 h at RT. 5. Discard the samples, and wash at least five times, by filling the wells with washing solution and discarding it (see Note 14). 6. Add 100 μL/well of the anti-M13/HRP conjugate (properly diluted in M-PBS). Cover the plate and incubate 1 h at RT. 7. Wash the plate at least eight times. 8. Add 100 μL/well of HRP substrate solution. Incubate during 15 min at RT. A yellow/orange color should develop in some wells, while the wells containing no phage samples or a non-related coating molecule should remain colorless (indicating low background levels). 9. Stop the reaction with 50 μL/well of the stop solution. Read the absorbances at 490 nm with a microplate reader.

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Fig. 2 Assessment of receptor binding properties of phage-displayed cytokines by ELISA. Reactivity of phagedisplayed human IL-2 and its mutated variant containing V69A and Q74P replacements was evaluated on plates coated with human and mouse IL-2 receptor alpha chain extracellular domain (ECD) (a). Phagedisplayed IL-6 was similarly tested against immobilized human and mouse IL-6 receptor alpha chain ECD (b). Bound phages were detected with an anti-M13 antibody conjugated with horseradish peroxidase. All phagedisplayed cytokines were evaluated in parallel on plates coated with 9E10 anti-c-myc mAb (positive control) and an unrelated antibody (negative control). While phage-displayed hIL-2 binds to both human and mouse alpha receptor subunits, the double-mutated variant shows increased reactivity with the human receptor and negligible binding to its mouse counterpart. Phage-displayed hIL-6 only recognizes human IL-6 receptor alpha chain

10. Calculate the mean absorbance values for replicates and analyze the results. Figure 2 illustrates receptor binding assessment for phage-displayed cytokines. 3.4 Assessing CTLL2 Proliferation Induced by Phage-Displayed IL-2 and IL-2 Variants

1. Thaw one CTLL-2 cell vial and grow the cells on IL-2-containing supplemented RPMI medium, at 37 °C and 5% CO2. Keep cell concentration between 103 and 104 cells/mL. Use 6-well suspension culture plates or 25 cm2 flasks for suspension cultures (see Note 22). 2. Subculture the cells every 2 days. Centrifuge the cells at 300 g during 5 min, discard the supernatant, and resuspend the cells in fresh supplemented medium with IL-2. Subculture the cells until 95% of viability is reached. 3. To perform the cell proliferation assay, centrifuge the culture at 300 g during 5 min, and wash the cells three times with non-supplemented RPMI medium to remove IL-2 (see Note 23).

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4. Use supplemented RPMI medium without IL-2 to dilute each purified/filtered phage sample in order to obtain the same concentration of displayed IL-2 or its mutated variants (measured as display units/mL) (see Note 24). 5. Add 100 μL/well of diluted phage samples to a 96-well tissue culture plate. Use a phage sample displaying an unrelated protein as negative control and some wells with medium to assess the assay background. 6. Adjust the CTLL-2 cells to a concentration of 2 × 105 cells/mL and add 100 μL of the cell suspension to each well. 7. Incubate the plate at 37 °C and 5% CO2 for 48 h. 8. Add 20 μL/well of alamarBlue reagent and incubate the plate in the same conditions during 12 h. A pink color should appear in those wells with IL-2 or IL-2-derived functional muteins, while the original blue color should be kept in wells with the non-related phage samples or medium. 9. Read the absorbance at 540 nm and 620 nm with a microplate reader. 10. Calculate the difference between readings at both wavelengths and the mean value for replicates. Figure 3 shows an example of cell proliferation curves. 3.5 Measuring Trans-Signaling Induced by PhageDisplayed IL-6

1. Seed 5 × 105 A549 cells/well in 3 mL of DMEM F12 supplemented with 10% FBS (v/v) in six-well plates. Incubate 8 h at 37 °C and 5% CO2 to allow cell attachment.

Fig. 3 Assessment of the ability of human and mouse phage-displayed IL-2 to induce proliferation of CTLL2 cell line. Cells were incubated with diluted phages (previously normalized according to the levels of displayed cytokines), and their proliferation levels were assessed in a colorimetric assay with alamarBlue reagent. Phages displaying an unrelated protein (single chain Fv antibody fragment, scFv) were used as negative control

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2. Remove medium. Wash wells with 3 mL of medium without FBS. Incubate with 3 mL of medium without FBS per well, during 16–20 h at 37 °C and 5% CO2. 3. Dilute phage-displayed IL-6 or unrelated phages (negative control) in medium without FBS (see Note 25) containing 200 ng/mL of recombinant IL-6R alpha (see Note 26). Pre-incubate during 1 h at 37°C before adding to the cells. 4. Pre-incubate in parallel recombinant IL-6 (at 50 ng/mL in medium containing 200 ng/mL of recombinant IL-6R alpha), as positive control. 5. Completely remove medium and incubate the cells 1 h at 37 °C and 5% CO2, with 2 mL of medium containing either pre-incubated phage samples or pre-incubated recombinant IL-6. Leave some wells with medium alone (negative controls). 6. Remove the medium and place the plates on ice. Wash cells twice with ice-cold PBS. Completely remove PBS. 7. Add 100 μL/well of ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors. Incubate for 5 min on ice. 8. Scrape the cells off the dish using cell scrapers. Transfer the cell suspensions to microcentrifuge tubes. 9. Incubate tubes on ice for 10 min and sonicate each suspension at 40 kHz for 10 s. Repeat twice the cycle of cooling on ice and sonicating. 10. Centrifuge at 15000 g for 15 min at 4 °C. Collect supernatants and store at -20 °C, after taking a small sample to quantify protein concentration in each lysate. 11. Mix each cell lysate (50 μg of total proteins) with Laemmli sample buffer containing β-mercaptoethanol. Denature during 10 min at 95 °C. 12. Prepare a 10% SDS-polyacrylamide gel. Load denatured lysates on the gel, along with a suitable molecular weight marker (MW) (see Note 27). 13. Run the electrophoresis at 150 V for 2 h or until the dye front runs off the bottom of the gel. 14. Pre-wet PVDF membrane in methanol for 5 s and then submerge in transfer buffer, along with the gel and filter papers, for 5 min. 15. Prepare the transfer sandwich in the following order: two sheets of filter paper, gel, PVDF membrane, and two sheets of filter paper (see Note 28). 16. Place the transfer sandwich with the gel toward the cathode of the transfer device and the membrane toward the anode. Transfer at 200 mA for 1.5 h (see Note 29).

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17. Check transfer success by staining the membrane with Ponceau S red dye during 5 min. Rinse with distilled water to remove excess staining. Efficient transfer is shown by the presence of red bands in the lanes where protein samples were applied. 18. Label the bands corresponding to the MW marker with a pencil, without damaging the membrane. Wash away Ponceau S red dye with TBS-T before blocking. 19. Cut two horizontal pieces of the PVDF membrane, using labeled MW bands as a guide. One piece must comprise the STAT3 bands (~88 kDa) and the other one must contain the β-actin bands (~42 kDa). 20. Block both pieces during 1 h at RT with either B-TBS-T (for detection of phosphorylated STAT3) or M-TBS-T (for detection of β-actin), with gentle agitation. 21. Wash the membranes three times (5 min each) with TBS-T while gently shaking. 22. Incubate the membranes with the appropriate dilution of the primary antibodies (anti-phospho-STAT3 or anti-β-actin) in B-TBS-T, during 16–20 h while gently shaking at 4 °C. Wash the membranes as described in step 21. 23. Incubate the membranes with the appropriate dilution of the secondary anti-rabbit IgG antibody conjugated to HRP in B-TBS-T, during 1 h at RT, while gently shaking. Wash the membranes again. 24. Apply chemiluminescent HRP substrate following manufacturer’s instructions. 25. Capture the chemiluminescent signal in a dark room using X-ray films. 26. Analyze the intensity of the bands with an image analysis software. Check the loading control protein (β-actin) signals to assess the presence of equivalent protein amounts in each lysate. 27. Recover the membrane piece showing phospho-STAT3 bands in order to proceed with stripping and total STAT3 detection. 28. Wash the membrane and incubate with preheated stripping buffer (50°C) for 45 min in a container with a tight lid. 29. Discard stripping buffer and rinse the membrane with tap water during 30 min. 30. Wash the membrane and block with M-TBS-T as described in step 20. 31. Incubate the membrane with the appropriate dilution of the primary antibody (anti-STAT3 in B-TBS-T), during 16–20 h, while gently shaking at 4 °C. Wash the membrane.

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Fig. 4 Induction of trans-signaling by phage-displayed human IL-6. A549 cells were treated with either phagedisplayed human IL-6 or phage-displayed human IL-2 (negative control), pre-incubated with recombinant IL-6 receptor (IL-6R) alpha. Phage-displayed proteins were previously normalized according to their display levels, assessed with 9E10 mAb recognizing the c-myc tag fused to both of them. Soluble recombinant IL-6 (also pre-incubated with IL-6R alpha) was included as positive control. Treated cells were lysed and the content of phosphorylated STAT3 (pSTAT3), total STAT3, and β-actin was assessed by western blot with specific antibodies (a). Nontreated (NT) cells were also analyzed to rule out IL-6-independent basal phosphorylation of STAT3. The ratio between signals corresponding to pSTAT3 and total STAT3 (b) allows normalizing the results taking into account possible differences in STAT3 content of cell lysates

32. Repeat steps 23–25 to detect signals corresponding to total STAT3. 33. Analyze the intensity of the bands using an image analysis software. Use total STAT3 signals to normalize target protein (phospho-STAT3) signals obtained for each sample. Figure 4 illustrates the results of biological activity assessment by western blot. 3.6 Producing Single-Stranded DNA Templates for Cytokine Library Construction

1. Transform CJ236 E. coli competent cells with the genetic constructs obtained after step 2 of Subheading 3.1. Grow transformed cells in solid 2xTY/AG during 16–20 h at 37 °C (see Note 30). 2. Inoculate an isolated colony (see Note 31) in a 50 mL tube containing 10 mL of 2xTY/AG supplemented with 5 μg/mL chloramphenicol (see Note 32). Grow at 37 °C with shaking at 250 rpm during 16–20 h. 3. Dilute the cell suspension obtained after step 2 (1/100) in a 50 mL tube containing 10 mL of fresh 2xTY/AG supplemented with 5 μg/mL chloramphenicol. Grow at 37 °C with shaking at 250 rpm until the culture reaches an absorbance at 600 nm in the range 0.4–0.8. 4. Add 1011 pfu of M13KO7 helper phage (see Note 6) and incubate at 37 °C during 30 min without shaking.

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5. Centrifuge at 2000 g during 15 min. Remove the supernatant. 6. Resuspend the cell pellet in 40 mL of 2xTY/AK supplemented with 2% glucose and 0.25 μg/mL uridine, contained in a 250 mL Erlenmeyer flask (see Note 33). 7. Grow the cells at 37 °C during 20 h with shaking at 250 rpm (see Note 34). 8. Centrifuge at 4000 g during 15 min at 4 °C. 9. Collect the supernatant and mix it with 10 mL of PEG/NaCl phage precipitation solution. Incubate during 1 h on ice to precipitate phages (see Note 35). 10. Centrifuge at 4000 g during 15 min at 4 °C. Remove the supernatant. 11. Resuspend the phage pellet in 0.5 mL of PBS and transfer to a vial. 12. Centrifuge at 10,000 g during 10 min in a microcentrifuge to remove the remaining E. coli and cell debris. 13. Mix the supernatant in a new vial with 10 μL of MP solution from the Qiaprep Spin M13 kit. Incubate 2 min at RT (see Note 36). 14. Add the mixture to a QIA column. Centrifuge 1 min at 6000 g. Discard the flow-through. 15. Add 0.7 mL of MLB/PB solution from the Qiaprep Spin M13 kit to the column. Centrifuge 1 min at 6000 g. Discard the flow-through. 16. Add 0.7 mL of MLB/PB solution from the Qiaprep Spin M13 kit to the column. Incubate 1 min at RT. Centrifuge 1 min at 6000 g. Discard the flow-through. 17. Add 0.75 mL of PE solution from the Qiaprep Spin M13 kit (already containing ethanol as recommended by the manufacturer) to the column. Centrifuge 1 min at 6000 g. Discard the flow-through. 18. Repeat step 17. 19. Transfer the column to an empty vial and centrifuge again 1 min at 6000 g to remove residual PE. 20. Transfer the column to a new vial. Add 100 μL of EB solution from the Qiaprep Spin M13 kit to the center of the column and incubate 10 min at RT. Centrifuge 1 min at 6000 g. Collect the eluted DNA in the vial. 21. Visualize the eluted ss-DNA (1 μL) in an agarose gel (1%) electrophoresis in the presence of ethidium bromide (see Note 37). 22. Determine the ss-DNA concentration in a Nanodrop quantitation machine. Typical yields vary widely from 50 to 500 ng/μL.

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3.7 Kunkel Mutagenesis Diversification Reactions

1. Design and synthesize antisense mutagenic oligonucleotides (see Note 38), annealing with cytokine genes from 15 nucleotides before the beginning of the target region to be modified to 15 nucleotides after its end, and introducing the desired diversity (see Note 39). Targeted codons can be either contiguous or separated by invariant sequences (see Notes 40–41). Distant target sequences can be modified using several mutagenic oligonucleotides in the same reaction (see Note 42). Figure 5 illustrates mutagenic oligonucleotides’ design. 2. Prepare stocks of mutagenic oligonucleotides at 330 ng/μL in water. 3. Prepare a phosphorylation mix for each mutagenic oligonucleotide, with the following components (see Note 43): • 2 μL 10x TM buffer • 2 μL ATP (10 mmol/L) • 1 μL DTT (100 mmol/L) • 11 μL water • 2 μL polynucleotide kinase (10,000 units/mL) • 2 μL of the oligonucleotide stock (at 330 ng/μL) 4. Incubate the above-described mix 1 h at 37 °C. 5. Prepare the annealing reaction(s) as follows: • 25 μL of 10x TM buffer • x μL of ss-DNA (20 μg) • 20 μL of each phosphorylated oligonucleotide (obtained after step 4) • y μL of water to complete a total volume of 250 μL 6. Incubate the annealing reactions 3 min at 90 °C, 3 min at 50 ° C, and 5 min at RT. 7. Prepare a fill-in mix with the following components per each annealing reaction: • 10 μL ATP (10 mmol/L) • 25 μL dNTPs (10 mmol/L each) • 15 μL DTT (100 mmol/L) • 6 μL T4 DNA ligase (400,000 units/mL) • 4 μL T7 DNA polymerase (10,000 units/mL). 8. Add the above-described fill-in mix to each annealing reaction. Incubate during 16–20 h at RT (see Note 44).

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Fig. 5 Examples of Kunkel mutagenic oligonucleotides’ design to construct libraries derived from human IL-2. Two degenerate antisense oligonucleotides were designed for controlled diversification of solvent-exposed

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9. Visualize 5 μL of the products of each reaction in an agarose gel (1%) in the presence of ethidium bromide. Use 1 μL of the ss-DNA template as control. Figure 6 shows the typical electrophoretic pattern of reactions (see Note 45). 10. Keep mutagenesis products at -20°C until use (see the next Subheading).

Fig. 6 Typical results of agarose gel electrophoresis of Kunkel mutagenesis products obtained during library construction. Lanes a–d show a single band of template single-stranded DNA (ss-DNA). Lanes e–h correspond to Kunkel reaction products obtained with four different mutagenic oligonucleotides and exhibit the band corresponding to double-stranded mutated DNA (desired product) and a band with lower electrophoretic mobility, which should represent DNA derived from aberrant strand displacement synthesis (polymerization does not stop after the emerging strand reaches the double strand formed between the template and mutagenic oligonucleotide). Note the presence of non-migrating DNA in the wells where mutagenesis products were applied, presumably due to precipitation during the reaction. GeneRuler 1 kb Plus DNA ladder (MW) is included

ä Fig. 5 (continued) residues at the interface with IL-2 receptor alpha chain (segments 35–45 and 61–74). Annealing between the template gene and the mutagenic oligonucleotides is shown in a. Dots represent match between both. Targeted triplets in the template and modified positions in the antisense oligonucleotides are underlined. The resulting mutated sequence is represented in b. Modified triplets are underlined. Amino acids encoded by modified triplets appear in c. Two spiked antisense mutagenic oligonucleotides were designed for soft randomization of solvent-exposed residues at the interface with IL-2 receptor beta chain (segments 12–23 and 81–95). Annealing between the template and these oligonucleotides is shown in d. Targeted triplets in the template and modified positions in the antisense oligonucleotides are underlined. The composition of spiked oligonucleotides at these locations includes 90% of the original nucleotide (represented in the sequence) and 10% of the remaining three nucleotides. The resulting mutated sequence is shown in e. Soft-randomized triplets appear in italics and underlined

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3.8 Preparation of Electrocompetent Cells and Electroporation with Mutagenesis Products to Construct Libraries of Cytokine Variants

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1. Inoculate one colony of E. coli cells (TG1 strain) in 10 mL of 2xTY. Grow during 16–20 h at 37°C with shaking at 250 rpm. 2. Add 3 mL of the cell suspension obtained in the previous step to 300 mL of 2xTY (1:100 dilution). Grow at 37°C with shaking at 250 rpm, until absorbance at 600 nm reaches a value in the range 0.6–0.8. 3. Centrifuge the cell suspension at 4000 g during 15 min at 4°C. Discard the supernatant. 4. Gently resuspend the cell pellet in 300 mL (the original culture volume) of ice-cold sterilized water (see Note 46). Incubate 30 min on ice. 5. Centrifuge at 4000 g during 15 min at 4°C. Carefully discard supernatant (see Note 47). 6. Gently resuspend the cell pellet in 150 mL (half of the original culture volume) of ice-cold sterilized water. Incubate 30 min on ice. Repeat step 5 (centrifugation). 7. Gently resuspend the cell pellet in 80 mL of ice-cold sterilized 15% glycerol solution. Incubate 30 min on ice. Repeat step 5 (centrifugation). 8. Carefully remove the remaining liquid as much as possible. Resuspend the cell pellet in 2 mL of ice-cold sterilized 15% glycerol solution (see Note 48). 9. Distribute the electrocompetent cells into 1.5 mL vials at 400 μL/vial. You can either use them directly for electroporation (see below) or freeze immediately at -80°C until use. 10. Before electroporation, mutagenesis products obtained after step 10 of the previous Subheading must be purified and eluted in water using the PCR Purification Kit (Qiagen, Germany) according to manufacturer’s instructions with modifications (see Note 49). 11. Place 2 mm gap electroporation cuvettes and purified mutagenesis products on ice. 12. Defrost electrocompetent cells (one aliquot per electroporation cuvette and one control aliquot) on ice. 13. Mix purified eluted mutagenesis products from one Qiagen column with 400 μL of electrocompetent cells and keep on ice. 14. Add the mixture of cells and mutagenesis product to the 2 mm gap electroporation cuvette and keep on ice until electroporation. Add electrocompetent cells alone to another cuvette (negative control). 15. Electroporate the negative control cells at 2.5 kV using an electroporator. Check the value of time constant provided by the machine after the electroporation pulse (see Note 50).

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16. Electroporate the cells mixed with mutagenesis products at 2.5 kV. Check the values of time constant after each electroporation pulse (see Note 51). 17. Add 1 mL of 2xTY medium to the negative control electroporation cuvette, resuspend gently, and collect the cells into a vial. 18. Add 1 mL of 2xTY medium to each cuvette containing mutagenesis products, resuspend, and collect the cells in a new 50 mL conical-bottom tube. 19. Repeat step 18 until the cuvettes are clean. Mix all collected cells from the library in an Erlenmeyer flask with a total volume of 50 mL of 2xTY medium supplemented with 2% glucose (v/v). 20. Shake the Erlenmeyer flask containing electroporated cells and the negative control vial at 250 rpm and 37°C during 30 min. 21. Spread 100 μL of the negative control cells in a Petri dish containing solid 2xTY/AG. Incubate 16–20 h at 37°C. 22. Take a sample of 10 μL of the electroporated cell suspension from the Erlenmeyer and prepare 1/10 serial dilutions (from 10-1 to 10-8) with 90 μL of 2xTY. Always change the tip between dilutions. Seed 10 μL of each dilution in Petri dishes with solid 2xTY/AG (see Note 52). 23. Centrifuge the electroporated cells from the Erlenmeyer flask at 2000 g during 15 min at RT. Discard the supernatant. 24. Resuspend the cell pellet in the minimal volume of remaining liquid after discarding the supernatant and spread over one large square plate (25 × 25cm2) containing solid 2xTY/AG. 25. Incubate at 37°C during 16–20 h. 26. Check the negative control plate to confirm the absence of colonies (see Note 53). Count the colonies corresponding to each dilution of electroporated cells in the titration plates. 27. Use colony counts to calculate the total number of colonies in the library (according to each dilution) as follows (see Note 54): Total number of colonies in the library = colony count x dilution factor x 5000 28. Estimate the library size as the mean value of calculated numbers of colonies (obtained with all dilutions) (see Note 55). 29. Scrape bacteria from the large square plate containing the library formed by multiple colonies (usually a bacterial lawn where colonies cannot be distinguished), using a bacterial spreader and 2xTY/AG medium. Collect the cell suspension. 30. Repeat step 29 until solid medium looks clean. All the cells collected during scraping should be mixed as a single cell suspension.

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31. Determine the volume of cell suspension. Add half this volume of sterile 60% glycerol solution to the cell suspension (20% glycerol final concentration), and homogenize. 32. Distribute the cell suspension+glycerol in vials. Keep them at 80°C until use. 3.9 Library Phage Production at a 300 mL Scale

1. Use the cell suspension obtained after step 30 of the previous Subheading or defrost on ice one aliquot of the library glycerol stock from step 32 (kept at -80°C). 2. Add 10 μL of the cell suspension to 50 mL of 2xTY/AG in a 250 mL Erlenmeyer flask, take a small sample, and measure the absorbance at 600 nm. 3. Repeat step 2, adding more cell suspension to the flask, until absorbance at 600 nm reaches a value between 0.05 and 0.1. 4. Grow the inoculated cells at 37°C with shaking at 250 rpm until the absorbance at 600 nm is between 0.6 and 0.8. 5. Add 5 × 1011 plaque-forming units (pfu) of M13KO7 helper phage (see Note 56) to grown bacteria and incubate at 37 °C during 30 min without shaking. 6. Centrifuge at 2000 g during 15 min. Remove the supernatant. 7. Resuspend the cell pellet in 300 mL of 2xTY/AK contained in a 1000 mL Erlenmeyer flask. 8. Grow the cells at 28 °C during 16–20 h with shaking at 250 rpm. 9. Centrifuge at 4000 g during 15 min at 4 °C. 10. Collect the supernatant and mix it with 60 mL of PEG/NaCl phage precipitation solution. Incubate during 1 h on ice (see Note 7). 11. Centrifuge at 4000 g during 15 min at 4 °C. Remove the supernatant. 12. Resuspend the phage pellet in 20 mL of PBS and transfer to a 50 mL conical-bottom tube. 13. Centrifuge at 4000 g during 15 min at 4 °C to remove the remaining E. coli and cell debris. 14. Mix the supernatant with 5 mL of PEG/NaCl solution (see Note 8). Incubate during 20 min on ice. 15. Centrifuge at 4000 g during 10 min at 4 °C. Remove the supernatant. 16. Gently resuspend the phage pellet in 20 mL of PBS (see Note 9). Add 10 mL of 60% glycerol solution, homogenize, and distribute the phage mixture in 1.5 mL vials. Keep at -20°C until use.

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17. Phage preparations can be (optionally) titrated. Make serial 1/10 dilutions (10 μL of purified phages plus 90 μL of 2xTY), until 10-10. 18. Mix diluted phages with 90 μL of exponentially growing TG1 cells. Incubate 30 min at 37°C. 19. Seed 10 μL of infected cells (from each dilution) in 2xTY/AG solid medium (see Note 57). Seed uninfected TG1 cells as negative control. Grow the plates at 37°C during 16–20 h. 20. Confirm the absence of colonies in the negative control. Count colony numbers from each dilution. 21. Calculate the concentration of infective phages using data from each dilution as follows (see Note 58): c(phages) (cfu/mL) = number of colonies × dilution factor x 2 × 100 22. Estimate the phage titer (cfu/mL) as the average value between phage concentrations calculated for several dilutions (see Note 59). 3.10 Phage Panning and Amplification

1. Add diluted coating molecule (usually a recombinant protein comprising the ECD of a cytokine receptor or cytokine receptor subunit) at 10 μg/mL in 500 μL of PBS to an immunotube. Cover the tube with parafilm and incubate 16–20 h at 4°C (Note 60). 2. Dilute 250 μL of purified library phages obtained as described in the previous Subheading with 250 μL of M-PBS (see Note 61). Mix and incubate 1 h at RT for blocking. 3. Discard coating solution from the immunotube and fill it totally with M-PBS. Cover with parafilm and block during 1 h at RT. 4. Discard blocking solution from the tube, and add blocked phages (500 μL). Incubate 1 h at RT. 5. Discard the phages. Wash the empty tube by filling with PBS-T and discarding the solution. Repeat this washing step 20 times in total. 6. Wash the immunotube twice with PBS. 7. Add 500 μL of freshly prepared TEA elution solution to the tube. Cover with parafilm and incubate during 10 min at RT with eventual manual shaking. 8. Collect the eluted phages in a vial containing 500 μL of Tris 1 mol/L and pH 7.5 (neutralization solution). 9. Add 500 μL of TEA solution to the tube. Cover with parafilm and incubate during 10 min at RT with eventual manual shaking.

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10. Collect the second eluate in the same vial that contains the first eluate mixed with neutralization solution. Keep at -20°C until use. 11. Infect 10 mL of exponentially growing TG1 cells with 500 μL of the neutralized eluate. Incubate 30 min at 37°C. 12. Centrifuge at 2000 g during 15 min. Discard supernatant and resuspend the cell pellet in the minimal residual liquid volume. 13. Seed the infected cells in a medium-sized plate (15 cm diameter) with solid 2xTY/AG. Seed 100 μL of non-infected cell suspension in a Petri dish with the same medium, as negative control. 14. Check the absence of colonies in the negative control plate. If bacteria grow there, discard the plate containing the infected cells and repeat the infection procedure from step 11. 15. Scrape the medium-sized plate with a bacterial spreader and 2xTY/AG medium trying to collect all the cells. 16. Determine the volume of the collected cell suspension and add half of this volume of 60% glycerol solution. Homogenize, distribute in 1.5 mL vials, and keep at -80°C until use. 17. Take the cell suspension obtained after step 15 and use it immediately or defrost on ice one aliquot obtained after step 16 (kept at -80°C). 18. Add 10 μL of the cell suspension to 50 mL of 2xTY/AG in a 250 mL Erlenmeyer flask, take a small sample, and measure the absorbance at 600 nm. 19. Repeat step 18, adding more cell suspension to the flask, until absorbance at 600 nm reaches a value between 0.05 and 0.1. 20. Grow the inoculated cells at 37°C with shaking at 250 rpm until absorbance at 600 nm reaches a value between 0.6 and 0.8. 21. Add 1011 plaque-forming units (pfu) of M13KO7 helper phage (see Note 6) to 10 mL of grown E. coli and incubate at 37 °C during 30 min without shaking. Discard the remaining cell suspension. 22. Continue phage production/purification as described in steps 7–16 of Subheading 3.1. Keep purified phages at -20°C until use. 23. Go to step 1 of the current Subheading and use purified phages as the starting material for a next selection round (see Note 61). Three selection rounds should be performed in total. 24. Receptor reactivity of phage mixtures obtained after each selection round can be compared by ELISA with the one of unselected library phages as described in Subheading 3.3.

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3.11 Phage Production at 96-Well Scale

1. Infect exponentially growing TG1 cells with either the diluted phages from the unselected library or the neutralized eluates obtained after selection rounds (see Note 62). 2. Seed 100 μL of infected cells in plates containing solid 2xTY/ AG. Seed non-infected cells in another plate as negative control. Grow 16–20 h at 37°C. 3. Transform competent TG1 cells with the genetic construct encoding phage-displayed wt cytokine (reference molecule). Grow 16–20 h at 37°C. 4. Check the absence of colonies in the negative control plate. 5. Add 900 μL of 2xTY/AG to each well of a 96 deep-well polypropylene plate. Inoculate colonies obtained after step 2 in individual wells of the plate. Inoculate colonies from transformation of TG1 cells with the reference (non-mutated cytokine) genetic construct. Leave several non-inoculated wells as negative controls in order to detect any contamination. 6. Cover the plate with a gas-permeable lid, and grow the cells at 37 °C with shaking at 250 rpm during at least 4 h (see Note 63). 7. Add 100 μL/well of M13KO7 helper phage diluted in 2xTY, at a final concentration of 1011 pfu/mL (see Note 64). Incubate during 30 min at 37 °C without shaking. 8. Centrifuge the plate at 1000 g during 15 min. Quickly discard the supernatant by inverting the plate in a single movement. Be extremely careful to avoid cross-contamination between the wells (see Note 65). 9. Cover the plate with a new gas-permeable lid, and shake it at 250 rpm at least 20 min at 28 °C to disrupt the cell pellets (see Note 66). 10. Add 1 mL of 2xTY/AK to each well. Cover the plate with a new gas-permeable lid, and grow the cells during 16–20 h at 28 °C with shaking at 250 rpm.

3.12 Clonal Screening by ELISA and Sequencing

1. Prepare coating solutions of recombinant proteins comprising either the ECD of cytokine receptor or anti-c-myc tag 9E10 mAb, at 10 μg/mL in PBS. Coat different PVC ELISA microplates with 100 μL/well of each coating solution (see Note 18). Cover the plates with lids and incubate during 16–20 h at 4 °C (see Note 67). 2. Discard the coating solutions by inverting the plates several times. 3. Add 200 μL/well of M-PBS to block the plates. Cover the plates and incubate 30 min at RT.

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4. In parallel with the incubation, centrifuge the deep-well culture plates obtained as described in the previous Subheading during 15 min at 4000 g at 4 °C. Make sure that there is no cell pellet in the non-inoculated wells, indicating the absence of contamination. 5. Discard blocking solution from the plates and add 80 μL/well of M-PBS. 6. Transfer 20 μL of the phage-containing supernatants obtained after step 4 from each well of the deep-well plate to the equivalent well in the ELISA plates, coated with both recombinant receptor and 9E10 mAb. Transfer can be easily done with a multichannel pipette (see Note 68). Incubate 1 h at RT. 7. Cover the deep-well plates containing phage supernatants with a freezing-resistant lid and keep at -20°C until further use. 8. Discard the samples and wash the ELISA plates at least five times by filling the wells with washing solution and discarding it (see Note 14). 9. Add 100 μL/well of the anti-M13/HRP conjugate (properly diluted in M-PBS). Cover the plates with lids and incubate during 1 h at RT. 10. Wash the plates at least eight times as described. 11. Add 100 μL/well of HRP substrate solution. Incubate during 15 min at RT. A yellow/orange color should develop. The wells containing supernatants from the non-inoculated wells of the culture plate should remain colorless (indicating low background levels). 12. Stop the reaction with 50 μL/well of stop solution. Read the absorbances at 490 nm with a microplate reader. 13. Choose those samples producing positive signals (> 0.3) on 9E10-coated wells (cytokine-displaying clones). See Note 69. 14. Calculate the normalized reactivity for each clone by dividing the absorbance detected on the wells coated with cytokine receptor by the absorbance of the same clone on 9E10 mAb. Choose those clones producing the highest relative reactivities (see Note 70). 15. Grow XL-1 Blue E. coli cells in 2xTY containing 10 μg/mL of tetracycline at 37 °C with shaking at 250 rpm until the culture reaches an absorbance at 600 nm in the range 0.4–0.8. 16. Defrost the deep-well plates stored at step 7. 17. Add 150 μL/well of 2xTY to a tissue culture plate (U-bottom). 18. Add 6 μL of phage-containing supernatant from each well in the deep-well plate corresponding to a variant selected for sequencing (see steps 13–14) to one of the wells in lane A of the tissue culture plate (see the previous step).

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19. Make serial dilutions by transferring with a multichannel pipette 6 μL from each well in lane A to the corresponding well in lane B and repeating the procedure until lane H (see Note 71). 20. Add 60 μL/well of the suspension of grown XL-1 Blue cells derived from step 15 to each well of lane H (the one containing the highest phage dilutions) of the tissue culture plate. Incubate during 30 min at 37 °C without shaking. 21. Transfer 30 μL of infected cells from each well in lane H to a well of a 12-well tissue culture plate containing solid 2xTY/ AG. Add 100 μL of 2xTY to each well to guarantee that the liquid covers the well surface. Dry the plate, invert it, and grow at 37 °C during 16–20 h (see Note 72). 22. Pick isolated colonies (one from each selected variant) and inoculate each one in 5 mL of 2xTY/AG. Grow the cultures during 16–20 h at 37 °C with shaking at 250 rpm. 23. Purify plasmid DNA with QIAprep Spin minikit according to manufacturer’s instructions. 24. Send the plasmids for automated sequencing of the inserted cytokine genes. Deduce the protein sequences of the different variants. 25. Check the diversity pattern within targeted regions and the presence/absence of gross artifacts in the sample of cytokinedisplaying clones from the unselected library. This analysis provides an idea of global library correctness. 26. Identify repeated cytokine sequences, recurrent individual mutations, and shared physical-chemical features among receptor-selected variants. This information gives clues about the structural bases of increased receptor binding. 27. Selected variants, and new phage-displayed variants rationally designed by combining selection-driven mutations/motifs, can be rescued as described in Subheading 3.1 and characterized using protocols of Subheadings 3.2, 3.3, 3.4 and 3.5.

4

Notes 1. The first two residues of mature IL-2 (Ala-Pro) have been shown to interfere with IL-2 periplasmic secretion [17]. Exclude the corresponding codons from the genetic constructs for IL-2 display. 2. Unpaired Cys can cause the formation of non-natural disulfide bonds resulting in misfolding and/or intermolecular aggregation. Remove the unpaired Cys (C125 in human IL-2 and C140 in mouse IL-2), replacing the corresponding codons by a triplet coding for Ser.

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3. The replacement K35E has been shown to improve the quantity/quality of phage-displayed human IL-2 [12]. This modification can be introduced in the genetic constructs, replacing the K35 codon by a triplet encoding a Glu. 4. Most of our cytokine display experience is based on pCSM phagemid, but other vectors could be used. Genetic fusion of the displayed cytokine to a tag (like c-myc) recognized by a monoclonal antibody is crucial, as quantitation of the displayed cytokine variants is necessary to compare them. 5. Steps 5–18 of Subheading 3.1 refer to the production and purification of phages starting from a single colony. It is recommendable to obtain phages from several colonies, in order to characterize independent replicas. 6. M13KO7 helper phage stocks are identified with their titers (measured as plaque-forming units (pfu)/mL). 1011 pfu are usually contained in 10 μL of a typical stock obtained at the laboratory (1013 pfu/mL). The titer can vary and the required volume needs to be calculated. 7. During the incubation of phage-containing supernatants with PEG/NaCl, the formation of a white phage precipitate, accumulating at the bottom of the conical tube or flask, is usually observed. 8. In the second phage precipitation step, the formation of a white precipitate is immediately observed after mixing phages with PEG/NaCl. The mixture looks like milk. If this does not happen, indicating phage production failure, the samples should be discarded at this step. 9. Purified phage pellets (after removal of E. coli and cell debris) are very difficult to resuspend. Avoid vortexing. Add PBS to the pellet and shake slowly at room temperature until it can be disrupted. Resuspend by gently pipetting. 10. Purified phages can be filtered through 0.2 μm syringe filters, but the use of centrifugal filter devices is a better choice to recover most of the phage volume in the flow-through. This is an optional step. Contaminant bacteria do not interfere in ELISA, but filtration is strictly required to keep the sterility in cell culture-based assays. 11. PVC ELISA microplates can be replaced by polystyrene plates. 12. Take one preparation of purified phages (usually displaying the wt cytokine) as the standard to assess the relative display levels of the others. Make serial twofold dilutions (from 1/25 to 1/6400) in M-PBS, to prepare a standard curve. 13. Use several dilutions of samples with unknown cytokine display levels (from 1/100 to 1/1600). If they do not produce absorbances in the linear range of the standard curve, prepare other

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suitable dilutions. If the samples are going to be filtered, it is important to quantify their display levels after filtration, as this step can result in a moderate phage content decrease. 14. Make sure that all the wells are totally filled with washing solution, with no air bubbles inside. Effective washing guarantees low background and reproducibility of the results. 15. Multiple software are available to obtain the best fitting standard curve. Linear regression is often preferred, but four parameter logistic (4-PL) or logarithmic regression might result in an optimal fit. The highest and lowest standard concentrations can be removed to focus on the linear range of the curve. Fitting quality is assessed by checking the R2 value, which must be above 0.99. 16. Sample dilutions producing absorbances corresponding to the linear range of the standard curve are optimal to calculate the concentrations of the displayed cytokines. 17. If several dilutions of the same phage sample have been tested, its display level is calculated as the average of display levels obtained with each dilution. Sometimes the values obtained with the highest/lowest dilutions deviate from the rest. In these cases, such dilutions can be excluded from the analysis. 18. PVC microplates and PBS are suitable for coating with multiple proteins, but different coating conditions might be required in some cases. Polystyrene microplates can often be used. Coating concentrations may vary from 1 to 10 μg/mL, depending on the availability of the coating molecules and the absorbances obtained in the assay. 19. 9E10 mAb is useful to assess the presence of equivalent amounts of every cytokine/cytokine variant. Anti-cytokine mAbs, particularly those against conformational epitopes, can be used to check the proper folding of the displayed proteins. The use of an unrelated coating protein provides a negative control to assess the specificity. Highly concentrated phage samples can produce nonspecific background on unrelated coating molecules. If this happens, such dilutions should not be considered when analyzing results. 20. The volume of diluted phage samples must be enough to fill the wells containing all coating molecules (receptors, mAbs, and control protein). Each sample has to be applied twice on each coating molecule, to obtain replicates. 21. Comparison of the binding properties of cytokine variants depends on the use of equivalent amounts of them. Normalization of phage preparations according to the content of the displayed protein(s) is necessary (see Subheading 3.2). Calculate the required dilution for each phage in order to reach the same final concentration (in display units/mL).

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22. CTLL-2 cells grow in suspension, but tend to form clusters. It is important to keep cell concentration below 2 × 105 cells/ml. Otherwise IL-2 in the medium would be rapidly depleted and the cells would lose viability. Obtaining a properly growing culture could take up to 10 days; don’t give up. 23. All the IL-2 must be removed from the culture prior to performing the cell proliferation assay. Cells can be maintained without IL-2 up to 5 h. 24. Comparison of the biological activity of IL-2 variants strictly depends on the use of equivalent amounts of them. Normalization of the diverse phage preparations, as described in Subheading 3.2, is necessary. Calculate the required dilution for each phage in order to reach the same final concentration (in display units/mL). Do not use dilutions below 1/10, as too concentrated phage samples can interfere with the cell proliferation assay. 25. In order to compare biological activity of samples of phagedisplayed IL-6, IL-6-derived variants, and irrelevant proteins, they should be diluted to reach equivalent concentrations of the displayed proteins, according to the results of quantitation (see Subheading 3.2). Our experience suggests that 5- to 20-fold dilutions of a typical purified phage preparation (having around 1013 colony-forming units (cfu)/mL) work properly. Higher dilutions could be tested to detect differences between phage preparations. 26. Since A549 cells only express one of the IL-6R subunits (gp130), IL-6-mediated signaling requires the addition of external IL-6R alpha subunit. Signaling induced in that way is called trans-signaling. Pre-incubation of IL-6 (either soluble or phage displayed) with recombinant IL-6R alpha ECD is required to pre-assemble the complex between both before adding to the cells. 27. The molecular weight marker must allow the identification of bands of STAT3 and phospho-STAT3 (~88 kDa) and β-actin (~42 kDa). 28. Eliminate completely air bubbles between the layers of the transfer sandwich. 29. Transfer conditions may vary if using a transfer device different from Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad, USA). 30. CJ236 is the preferred E. coli strain to obtain ss-DNA template due to its ability to produce uracil-containing DNA. This feature allows the mutated DNA strand to replicate preferentially in a conventional E. coli host over the non-mutated uracil-containing template and increases mutagenesis efficiency

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(see Subheading 3.7). Despite this advantage, phage production using CJ236 is not robust. If the procedure described in Subheading 3.6 fails, replace CJ236 by XL-1 Blue E. coli strain, which renders higher amounts of ss-DNA, with acceptable mutagenesis efficiency. 31. Lack of robustness of phage production by CJ236 E. coli results in a failure to isolate ss-DNA from some colonies without any evident reason. This fact, together with requirement of several dozens of micrograms for library construction, makes it recommendable to start the procedure described in Subheading 3.6 with at least three colonies in parallel. Depending on the yield, it might be necessary to repeat the protocol with more colonies. 32. When using XL-1 Blue E. coli strain instead of CJ236 for ss-DNA production, chloramphenicol must be replaced by tetracycline at 10 μg/mL. 33. When using XL-1 Blue E. coli for ss-DNA isolation, uridine is not included. 34. When using XL-1 Blue E. coli for ss-DNA isolation, phage production (step 7) is performed at 28 °C. 35. Low phage production by CJ236 E. coli usually results in the absence of any visible precipitate during the incubation of phage-containing supernatants with PEG/NaCl, in contrast to what is observed with TG1 or XL-1 Blue E. coli. 36. During phage precipitation with MP, the formation of a white precipitate is sometimes visible. While it can be observed or not when using CJ236 E. coli (being useless to predict the subsequent success of ss-DNA isolation), precipitate formation must always be seen when using XL-1 Blue strain. 37. Successful ss-DNA purification results in a main band on an agarose gel electrophoresis, sometimes accompanied by minor bands with less electrophoretic mobility. 38. As pCSM+ phagemid is used in the current protocol, the presence of a phage replication origin results in packaging of sense strand into phages and isolation of sense ss-DNA template. Antisense mutagenic oligonucleotides anneal with this template. When using (-) phagemid versions, antisense template DNA is obtained, and sense mutagenic oligonucleotides are required. 39. Oligonucleotides having G/C at both 3′ and 5′ ends are better for annealing. Design slightly shorter or longer annealing regions with the template to fulfill this requirement. If the ends of the mutagenic oligonucleotide coincide with repeated triplets in the template, annealing can occur at the wrong

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position causing undesired insertions/deletions. Extend the mutagenic oligonucleotides a few triplets beyond the repetitive region to guarantee specific annealing. 40. Targeted regions are chosen according to their functional relevance. In order to obtain cytokine variants with higher receptor affinity, residues belonging to the binding interface, as well as other neighbor amino acids (aa), can be targeted. Structural knowledge and in silico modeling may aid in semi-rational library design, allowing conservation of critical residues and/or crucial physic-chemical features (hydrophobicity, polarity, positive/negative charge, aromaticity, or particular chemical moieties) at certain positions. Soft randomization is useful for an initial exploration of the interface. It can be accomplished through the use of spiked mutagenic oligonucleotides biased to the original nucleotide composition but introducing certain degree of diversity at each targeted position. The presence of 90% of the original nucleotide and 10% of the mixture of the three remaining nucleotides at a given position guarantees the inclusion of a few changes per molecule and all possible replacements in the whole library. Total randomization of selected positions is achieved through the introduction of degenerate NNK codons (coding for the 20 aa and avoiding the stop codons TAA and TGA). TAG amber stop codon, contained in NNK, is suppressed in E. coli strains commonly used for phage display. Other degenerate codons, coding for amino acid subsets, can be introduced: NTK codes for a mixture of hydrophobic aa (Ile, Leu, Met, Phe, Val), GAN codes for negatively charged Asp/Glu, ARR codes for positively charged Arg/Lys, and TWY codes for the aromatic residues Phe/Tyr. 41. Although multiple residues can be targeted within a library, the attainable library size, constrained by limitations in the transformation efficiency (see Subheading 3.8), should be considered. Libraries of up to 109 molecules are relatively easy to obtain, and optimal performance at every step allows construction of libraries containing 1010–1011 members. Theoretical library diversity should not exceed the expected library size, in order to achieve complete coverage of the explored sequence space. For full randomization of five residues (theoretical diversity = 3.2 × 106), a library size above this number must be obtained. As mutagenesis efficiency is below 100% (resulting in the presence of non-mutated template within the library), and some mutated variants can be under-/over-represented due to technical biases during library construction, library size should exceed by tenfold the theoretical diversity to guarantee full coverage. In the example above, the ideal library size would be at least 3.2 × 107 members.

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42. Distant sequences can be modified with two or more mutagenic oligonucleotides (each one targeting a given region) as long as there are more than 30 nucleotides between such target sequences, allowing the simultaneous annealing of the mutagenic oligonucleotides to the same template. 43. Several mutagenesis reactions might be necessary to obtain enough DNA to construct the library. If more than one reaction is required, prepare one phosphorylation reaction (20 μL final volume) of each mutagenic oligonucleotide per each mutagenesis reaction. 44. During overnight incubation, a white precipitate might appear. This does not interfere with subsequent work. 45. Typically, at least two bands are observed after the mutagenesis reaction (see Fig. 6). One of them has less electrophoretic mobility than the template DNA and corresponds to the hybrid double-stranded DNA formed by the template and the newly synthesized mutated strand (the desired product). A second band, with even lower electrophoretic mobility, comes from strand displacement, an artifact of fill-in reaction, when synthesis of the new strand does not stop after reaching the double strand formed between the template and the mutagenic oligonucleotide. Synthesis thus continues, displacing the mutagenic oligonucleotide and the newly formed strand and giving rise to high molecular weight aberrant DNA species. Sometimes a third weak band is observed between the two previously described bands. 46. Be very careful when resuspending the cells, in order to maintain their integrity. Avoid pipetting. Homogenize by repeatedly inverting closed bottle(s) containing the cells or by headto-head movement in a rotator. The whole process has to be done at 4°C. 47. Avoid losing cells while discarding the supernatant. The quality of electrocompetent cells depends upon their concentration. The cell pellet is becoming fluffy along the washing steps, so avoiding cell lost is going to be more difficult after subsequent washes. 48. At this step, homogenization has to be done by pipetting. Be extremely careful to minimize cell damage and to avoid contamination of the cells. 49. Split mutagenesis products of each reaction (synthesized from 20 μg of template DNA) into two QIA columns for purification. The washing step with PE should be done twice to guarantee removal of salts. Use only sterilized distilled water for columns’ elution.

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50. Time constant values vary depending upon the electroporation machine used. The general rule is: the higher the better. Values should be in the range 4.5–5.0 msec, or even more with some machines. Very low values can result in an electric arch, usually indicated in the display and by a brief (but clearly perceptible) noise corresponding to the “explosion” within the cuvette. Low time constant values and electric arch indicate an excess of residual salts in the electrocompetent cells and preclude their use. 51. Time constants should also be as high as possible in this step, although the addition of mutagenesis products often results in a moderate reduction of the values compared to negative control cells (see Note 50). If too low values (100 fold). 15. The yield of RCA DNA varied from 7.5- to 40-fold of the amount of starting UDG-treated cccDNA. For instance, for 116 μg of UDG-treated DNA used in this experiment, an amount of at least 900 μg of RCA DNA should be recovered. 16. To reduce the volume of the ligation reaction, it may be worth testing different concentrations of DNA in small volumes, in a pilot study. 17. DNA was diluted to 1.2 ng/μL to minimize intermolecular ligation. 18. The amount of the ligase used here can be further optimized to reduce the amount used. 19. No need to wash in 70% ethanol due to the extra step of PCR purification. 20. Dilute a small aliquot of recovered cells over a range (i.e., 103– 106) of concentrations and spread equal volumes (i.e., 200 μL) onto petri plates. We find sterile glass beads an effective way to distribute the liquid over the plate surface. Remove beads, let the liquid absorb into the plate medium, and incubate overnight at 30 °C. 21. Such cells can be frozen and stored to amplify the library. 22. When a large amount of DNA is used in electroporation, we find that many cells take up more than one DNA molecule. Sequence analysis showed (Fig. 3) that majority of the randomly selected colonies harbor more than one sequence (1–4 sequences), suggesting that the size of the final library can be as large as 2.5 × 1011. 23. The read lengths of Illumina’s MiniSeq and MiSeq machines are 151 and 251 nucleotides, respectively. Design the primers so that one is immediately adjacent to the first randomized region. If the entire randomized region is within 151 or 251 nucleotides, then only one strand need be sequenced. 24. The final compiled sequences showed that the distribution of amino acids of the mutagenic loops in the final library was mostly in line with the original design (Fig. 4b, c). However, there was an uneven distribution of mutants with different loop lengths; longer loops were under-presented from predicted (Fig. 4d).

Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis. . .

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A clone containing one sequence (29%)

A clone containing two sequences (29%)

A clone containing three sequences (31%)

A clone containing four sequences (11%)

Fig. 3 Sequencing trace files of different types of randomly picked bacterial colonies of the mutant library. Well-separated single colonies were randomly selected for Sanger sequencing. Representative sequencing trace files that contain one, two, three, and four mixed sequences in the loop area are shown here. The black straight line designates where the reading of mixed sequences starts at BC loop of FN3 monobody

25. The addition of biotin is to fill all the biotin-binding pockets, as certain short peptide sequences (i.e., HPQ/M) can bind to streptavidin. 26. One plate will be used as background (i.e., NeutrAvidin alone), and the other will carry the target (i.e., NeutrAvidin coated with captured biotinylated target). NeutrAvidin is used to coat microtiter plate wells and capture biotinylated target in the phage ELISA to avoid identification of phage clones that bind to streptavidin instead of the target. NeutrAvidin and streptavidin are only 30% similar in amino acid sequence. 27. A squeeze bottom containing PBS-0.1% Tween 20 is equally effective in quickly filling and washing many wells in a microtiter plate as a multichannel pipettor. 28. Background binding (i.e., NeutrAvidin-coated wells) is typically ~0.2 OD405nm units, whereas strong “binders” can have OD405nm values >1.0. “Sticky” binders will have equally strong signals on both “NeutrAvidin alone” and “NeutrAvidin and target” wells. Any clones that show selective binding to the target should be retested in triplicate to confirm binding, prior to the preparation of phagemid DNA and DNA sequencing.

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A.

B. BC loop

DE loop

FG loop

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT

ZZZ ZZZZ ZZZZZZ ZZZZZZZ ZZZZZZZZ

XXXXX XXXXXX XXXXXXX XXXXXXXX X= one of 20 residues except C and M

C.

Expected

Observed

BC loop

Expected

FG loop

Observed

D.

BC loop

Z= Y/S/G/W

DE loop

XXXXXXX XXXXXXXXX XXXXXXXXXX XXXXXXXXXXX XXXXXXXXXXXX XXXXXXXXXXXXX X= one of 20 residues except C and M FG loop

DE loop

Fig. 4 The design of the FN3 monobody library and the next-generation sequencing (NGS) analysis of the final library. (a) The cartoon structure of the FN3 scaffold with the mutagenic region of the three loops highlighted in three different colors: BC loop in blue, DE in yellow, and FG in orange. (b) The designed various lengths of the loops and the amino acid distribution of the FN3 library. For BC and FG loops, each position has 1 of the 20 amino acid residues except methionine and cysteine. DE loop residue contains one of the tyrosine, serine, glycine, or tryptophan residues. (c) Histograms comparing the designed percentage distribution of the amino acid residues with the observed distribution revealed by NGS analysis. (d) The observed distribution of various loop lengths of library mutants obtained by NGS analysis. It was designed to have equal representation for each individual length at all three loops, but as can be seen in panel D, shorter inserts are favored

29. Virions are incubated with different concentrations of non-biotinylated target to reach a binding equilibrium between the displayed monobody and target. The virions that are still free are detected by their subsequent binding to targetcoated wells. 30. Figure 5c presents data of various competition ELISA experiments for phage clone affinity selected from the library to COPS5, HRBL, and Ku70/80. In our experience, the IC50 value approximates the dissociation constant measured by surface plasmon resonance (SPR).

Acknowledgments The work was supported in part at the University of Illinois at Chicago, Loyola University, and Tango Biosciences by grant awards, U54DK093444, R21AI100565 and RO1AI100129, and 1R43AI157021, from the National Institutes of Health, respectively. The authors thank Dr. Leslyn Hanakahi (College of

Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis. . .

A.

Targets

Full name

IC50/ affinity (nM)

Methods

COPS5

COP9 signalosome complex subunit 5

15-32

ELISA

TGF-b1

Transforming growth factor beta1

9.5

SPR

HRBL

HIV-1 Rev binding protein-like

KU70/80 SARS-CoV-2 spike protein

X-ray repair cross-complementing 5/6 severe acute respiratory syndrome coronavirus 2 spike protein

225

B.

0.5-2

ELISA

13-15

ELISA

TGF-b1 binder

ka (1/Ms)

kd (1/s)

KD(M)

3-14

Octet

6-FC

1.4x104

2.8x10-4

9.5x10-9

C. B11

% Max Signal

100

E10

50

0 0.01

0.1

1

10

100

Free Ku70/80 (nM)

Fig. 5 Affinity and inhibition concentration-50 (IC50) binding values of FN3 mutants obtained by competition ELISA, Octet, and SPR analysis. (a) A table lists all the measured IC50 and affinity values of selected FN3 mutants. The affinity of the FN3 monobodies against SARS-CoV-2 spike protein was measured by bio-layer interferometry (BLI) with an Octet [13]. (b) The sensorgram of SPR analysis and the measured Kon and Koff values of a monobody binding to the TGF-β1. (c) Competition ELISAs, from which IC50 values of multiple phage clones were determined for COPS5, Ku70/80, and HRBL

Pharmacy, University of Illinois, Chicago, Rockford) for the gift of Ku70/80 protein. We thank Drs. Emily Delaney and Nigel Delaney for assistance in analyzing the next-generation sequencing data for the library. We also thank Ms. Ashley Grahn and Ms. Grace Allen for editorial assistance in preparing this manuscript. References 1. Smith GP (2019) Phage display: simple evolution in a petri dish (nobel lecture). Angew Chem Int Ed Engl 58:14428–14437. https://doi.org/10.1002/anie.201908308 2. Ling MM (2003) Large antibody display libraries for isolation of high-affinity antibodies. Comb Chem High Throughput Screen 6:421–432 3. Perelson AS, Oster GF (1979) Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination. J Theor Biol 81:645–670. https://doi.org/10.1016/0022-5193(79) 90275-3 4. Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson

KS (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314 5. Gru¨ndemann D, Scho¨mig E (1996) Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21:898–903. https:// doi.org/10.2144/96215rr02 6. Scholle MD, Kehoe JW, Kay BK (2005) Efficient construction of a large collection of phage-displayed combinatorial peptide libraries. Comb Chem High Throughput Screen 8:545–551 7. Weiss GA, Watanabe CK, Zhong A, Goddard A, Sidhu SS (2000) Rapid mapping of protein functional epitopes by combinatorial

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alanine scanning. Proc Natl Acad Sci U S A 97: 8950–8954. https://doi.org/10.1073/pnas. 160252097 8. Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382. https://doi.org/10.1016/ 0076-6879(87)54085-x 9. Firnberg E, Ostermeier M (2012) PFunkel: efficient, expansive, user-defined mutagenesis. PLoS One 7:e52031. https://doi.org/10. 1371/journal.pone.0052031 10. Holland EG, Acca FE, Belanger KM, Bylo ME, Kay BK, Weiner MP, Kiss MM (2015) In vivo elimination of parental clones in general and site-directed mutagenesis. J Immunol Methods 417:67–75. https://doi.org/10.1016/j.jim. 2014.12.008

11. Huovinen T, Brockmann EC, Akter S, PerezGamarra S, Yla-Pelto J, Liu Y, Lamminmaki U (2012) Primer extension mutagenesis powered by selective rolling circle amplification. PLoS One 7:e31817. https://doi.org/10.1371/ journal.pone.0031817 12. Koide A, Bailey CW, Huang X, Koide S (1998) The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol 284: 1141–1151. https://doi.org/10.1006/jmbi. 1998.2238 13. Miller CJ, McGinnis JE, Martinez MJ, Wang G, Zhou J, Simmons E, Amet T, Abdeen SJ, Van Huysse JW, Bowsher RR, Kay BK (2021) FN3-based monobodies selective for the receptor binding domain of the SARSCoV-2 spike protein. New Biotechnol 62:79– 85. https://doi.org/10.1016/j.nbt.2021. 01.010

Chapter 11 Construction of Semisynthetic Shark vNAR Yeast Surface Display Antibody Libraries Harald Kolmar, Julius Grzeschik, Doreen Ko¨nning, Simon Krah, and Stefan Zielonka Abstract The adaptive immune system of sharks comprises a unique heavy chain-only antibody isotype, termed immunoglobulin new antigen receptor (IgNAR), in which antigen binding is mediated by a single variable domain, referred to as vNAR. In recent years, efforts were made to harness these domains for biomedical and biotechnological applications particularly due to their high affinity and specificity combined with a small size and high stability. Herein, we describe protocols for the construction of semisynthetic, CDR3randomized vNAR libraries for the isolation of target-specific paratopes by yeast surface display. Additionally, we provide guidance for affinity maturation of a panel of antigen-enriched vNAR domains through CDR1 diversification of the FACS-selected, antigen-enriched population and sublibrary establishment. Key words Shark, IgNAR, vNAR, Yeast surface display, Antibody engineering, Protein engineering, Library generation, Affinity maturation, Semisynthetic antibody library, Single domain antibody

1

Introduction Of note, this is a slightly modified but updated version of a chapter that has been published before [1]. Similar to heavy chain-only antibodies found in camelids, sharks produce a unique antibody isotype that does not associate with light chains and is referred to as IgNAR [2, 3]. In this homodimer, each heavy chain consists of an N-terminal variable domain (vNAR, IgNAR V), which acts as an independent paratope, followed by five constant domains. vNAR domains display several remarkable features, clearly distinguishing them from camelidderived VHH domains. In this respect, a deletion can be found in the framework2-CDR2-region, resulting in only two complementarity determining regions (CDR1 and CDR3) for IgNAR V

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Structural depiction of a vNAR antibody fragment (based on pdb entry 2YWZ). CDR1 and CDR3 are highlighted in red. The non-canonical disulfide bond is not considered in the library generation strategy. Surface-exposed loop structures corresponding to HV2 and HV4 are represented in deep teal. (The figure was generated with PyMOL v0.99)

domains (Fig. 1) [4, 5]. However, at the CDR2 truncation site, the remaining surface-exposed loop forms a “belt-like” structure which, after antigen contact, undergoes somatic hypermutation. The same holds true for an additional loop which corresponds to the T-cell receptor HV4, to which vNAR domains share structural similarity [6]. Hence, these regions have been termed HV2 and HV4, respectively. Owing to their beneficial biophysical and structural properties, shark vNAR domains emerged as promising tools for biotechnological and biomedical applications. Based on the number and pattern of non-canonical disulfide bond motifs, which are typically not found in mammalian antibody domains, vNARs can be categorized into four groups [3]. Consequently, the different types of vNAR domains form a diverse set of additional disulfide bond patterns, resulting in an huge repertoire of different loop structures [7]. IgNAR V domains can be used to target recessed epitopes as well as sites which are inaccessible for classical antibodies with rather flat paratopes [8–10]. Further inherent attributes of the vNAR domain encompass its small size, which might be beneficial for tissue penetration [11], superior stability compared to conventional antibody domains, as well as tolerance to irreversible denaturation [3, 12–14]. Moreover, and similar to camelid-derived VHH domains [15–19], there are multiple opportunities to re-format vNAR domains as elegantly described by Ubah et al. [20].

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All of the aforementioned attributes enabled the generation and engineering of vNAR domains for a broad range of applications. For instance, vNAR domains targeting ICOSL or TNF-α were generated for the treatment of autoimmune diseases [21, 22]. Moreover, IgNAR V domains were constructed as blood-brain barrier shuttles by targeting the transferrin receptor 1 [23], or as anti-angiogenic agents [24]. Further, it has been shown by our group that anti-idiotypic vNAR domains can be readily isolated from semisynthetic libraries [25]. We were able to demonstrate that this might pave the way for the rapid generation of patient-specific anti-idiotypic vNARs targeting the B-cell receptor of lymphomas for personalized anticancer therapy [26]. In another approach, Ho and coworkers engineered PD-L1-targeting vNAR-based CAR-T cells [27]. In addition, shark IgNAR V domains that potently neutralize SARS-CoV-2 were constructed [28, 29], serving as one of many examples for the broad utility of vNARs as bioprocessing or diagnostic reagents [30, 31]. In this chapter, we provide a protocol for library establishment of semisynthetic CDR3-randomized shark vNAR antibody domain libraries for yeast surface display, based on the natural IgNAR repertoire of the bamboo shark (Chiloscyllium plagiosum). Yeast surface display has proven to be a versatile platform technology for the generation of a plethora of different antigen-specific antibody formats and scaffolds [32–34], including nonclassical antibodies such as camelid VHH domains [35–37], shark vNAR domains [38], or more recently, cattle-derived ultralong CDR-H3 antibodies [39, 40]. Furthermore, we also detail our methods for affinity maturation of target-specific clones based on sublibrary establishment using second-generation randomization of CDR1 to isolate molecules with substantially enhanced affinities.

2

Materials

2.1 Shark Handling and Blood Isolation

1. 0.1% (w/v) Tricaine methanesulfonate in artificial seawater. 2. 4% (w/v) trisodium citrate. 3. 23-gauge needle and 2 mL syringe. 4. TRI Reagent® BD (Sigma-Aldrich, Taufkirchen, Germany). 5. 5 N acetic acid.

2.2 Preparation of Total RNA from Whole Blood

1. 1-Bromo-3-chloropropane. 2. Isopropanol. 3. 75% (v/v) ethanol. 4. RNAse-free water or DEPC-treated water.

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2.3 cDNA Synthesis and Gene-Specific Amplification of vNAR Regions as Template for Library Construction

1. Omniscript® Reverse Transcriptase Kit (Qiagen, Hilden, Germany). 2. Oligo(dT)18 Primer. 3. RNase inhibitor, murine. 4. Taq DNA polymerase. 5. 10x Taq buffer. 6. dNTPs. 7. Nuclease-free water. 8. Thermocycler. 9. Device and reagents for agarose gel electrophoresis. 10. PCR Clean-Up System. 11. BioSpec (VWR) Nano or equivalent instrumentation.

2.4 Yeast Surface Display Library Construction

1. BamHI-HF. 2. NheI-HF. 3. 10x CutSmart buffer. 4. pCT plasmid [32]. 5. yeast strain: EBY100. 6. YPD media: 20 g/L D(+)-glucose, 20 g/L tryptone, 10 g/L yeast extract. 7. Electroporation buffer 1 M Sorbitol, 1 mM CaCl2. 8. LiAc buffer: 0.1 M LiAc, 10 mM DTT. 9. 1 M Sorbitol. 10. Electroporator Gene Pulser Xcell™ (Bio-Rad, Dreieich, Germany). 11. 0.2 cm Electroporation cuvettes (Bio-Rad). 12. Bacto™ Casamino acids (BD Biosciences, San Jose, USA). 13. SD-CAA media: 8.6 g/L NaH2PO4 × H2O, 5.4 g/L Na2HPO4, 20 g/L D(+)-glucose, 6.7 g/L yeast nitrogen base without amino acids and 5 g/L Bacto™ casamino acids. 14. SD-CAA agar plates: 8.6 g/L NaH2PO4 × H2O, 5.4 g/L Na2HPO4, 20 g/L D(+)-galactose, 6.7 g/L yeast nitrogen base without amino acids, 5 g/L Bacto™ casamino acids and 100 g/L polyethylene glycol 8000. 15. 9 cm Petri dishes. 16. Randomized trinucleotide primers (Table 1).

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Table 1 Randomized trinucleotide primers used for the construction of semisynthetic shark vNAR yeast display antibody libraries Name

Sequence (5′!3′)

Amplification of the natural vNAR repertoire as template for library construction bamboo/nat_up

ATGGCCSMACGGSTTGAACAAACACC

bamboo/nat_lo

WTTCACAGTCASARKGGTSCC

Generation of CDR3-randomized vNAR yeast surface display libraries FR1/CDR1/ Tyr_up

ACCATCAATTGCGTCCTAAAAGGTTCCRNMTATGBATTGGGTA NMACGTACTGGT

FR3_lo

CGCTTCACAGTGATATGTACC

FR1_up

ATGGCCGCACGGCTTGAACAAACACCGACAACGACAACAAAGGAGGCA GGCGAATCACTGACCATCAATTGCGTCCTAA

CDR3rand/ Fr4_lo

WTTCACAGTCASARKGGTSCCSCCNCCTTCAAT(X)12 CGCTTCACAGTGATATGTACC

GR_up

GTGGTGGTGGTTCTGCTAGCATGGCCGCACGGCTTGAACA

GR_lo

ATAAGCTTTTGTTCGGATCCWTTCACAGTCASARKGGTSCCSCCNCC

pCT_Seq_up

GCGGCGGTTCCAGACTACGCTCTGCAGGCT

pCT_Seq_lo

GCGCGCTAACGGAACGAAAAATAGAAA

CDR1 diversification for affinity maturation CDR1rand_up

ACCATCAATTGCGTCCTAAAA (X)5TTGGGTAGCACGTACTGGTATTTCACAAAGAAG

X: triplet codon for all natural amino acids w/o Cys

3

Methods Below we describe the establishment of a semisynthetic, CDR3randomized vNAR yeast surface display library based on the natural IgNAR V repertoire of the bamboo shark. At first, blood samples need to be harvested followed by the isolation of total RNA from whole blood samples. Subsequently, cDNA needs to be prepared, and a generic three-step PCR methodology needs to be exploited for library generation (Fig. 2). Alternatively, a single bamboo shark vNAR sequence can be utilized as template (see Note 1). To this end, we strongly recommend starting with a IgNAR V region that has proven to be well-behaved with respect to manufacturability. In general, we advise starting with a template pool of different vNAR scaffolds, since it is known that subtle sequence variations in the scaffold (e.g., in the framework regions of Ig domains) may have a

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Fig. 2 Schematic representation of the PCR-based library design. PCR-amplified vNAR fragments from the repertoire of a bamboo shark or, alternatively, a DNA fragment encoding a defined vNAR domain is used as template. In a first PCR, the vNAR framework is amplified up to CDR3. The cysteine residue in CDR1 is replaced by tyrosine, and a marginal diversity is introduced in this region using the forward primer. During the second PCR, CDR3 is fully diversified (without cysteine), and in the final PCR reaction, overhangs for gap repair cloning are added. (This figure was generated using www.biorender.com)

large impact on folding stability and protein solubility [41, 42]. These amino acid changes in the framework might contribute to the isolation of a large set of stable binders, as previously shown by our group [12, 43]. 3.1

Blood Collection

All procedures need to be conducted in accordance with national laws. For instance, the generation of CDR3-randomized vNAR libraries as described by our group was in accordance with the national laws } 4 Abs. 3 of the German Tierschutzgesetz (TierSchG, animal welfare act). Permission number: V 54–19 c 20 15 (1) Gl

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18/19 Nr. A 35/2011, Regierungspr€asidium Giessen, Germany (Regional council Giessen). Make sure that an experienced veterinarian performs blood collection. 1. Transfer the bamboo shark from its original tank into a smaller container prefilled with MS-222 working solution. Anesthetize by submersion. 2. Collect 1–2 mL of blood from the caudal vein using a 23-gauge needle (size depends on weight of the animal; adjust when needed). Syringe and needle should be prefilled with approximately 100 μL trisodium citrate solution in order to prevent coagulation. 3. Add approximately 200 μL collected blood to 750 μL TRI Reagent BD supplemented with 20 μL 5 N acetic acid. Vortex or shake thoroughly. Samples can be stored at –80 °C. (Caution: TRI Reagent BD is a mixture of phenol and guanidine thiocyanate. Take appropriate safety precautions.) 3.2 Total RNA Preparation

1. Incubate blood samples (in TRI Reagent BD) for at least 5 min at room temperature. 2. Add 100 μL 1-Bromo-3-chloropropane to each sample (i.e., per 200 μL blood). Shake or vortex for approx. 15 s and incubate at room temperature for 5 min. 3. Centrifuge for 15 min at 4 °C at minimum 12,000× g. 4. Transfer upper aqueous phase to a fresh tube prefilled with 500 μL isopropanol per 200 μL blood. Incubate for 10 min at room temperature. 5. Centrifuge for 10 min at 4 °C at minimum 12,000× g. 6. Remove the supernatant carefully and wash RNA by adding 1 mL 75% ethanol per 200 μL blood. Vortex samples and centrifuge for 10 min at 4 °C at minimum 12,000× g. 7. Remove the supernatant carefully and air-dry the RNA pellet for 5–10 min at room temperature. Dissolve RNA in RNAsefree water (approx. 100 μL per 200 μL blood sample. Volume of RNAse-free water depends on size of the pellet; see Note 2). 8. Calculate the concentration of isolated RNA by using a BioSpec Nano or equivalent instrumentation. An OD260 of 1 corresponds to 40 μg/mL total RNA. 9. Analyze RNA integrity by running a 1% (w/v) gel. Two clear and distinct bands representing the 28 s and 18 s rRNA should be visible. Otherwise, the isolated RNA might already be partially degraded (see Note 3). Keep RNA exclusively on ice or freeze at -20 °C or -80 °C.

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cDNA Synthesis

The protocol below describes the cDNA synthesis for one reaction. If more reactions should be performed, prepare a master mix. Reverse transcription is described based on the Omniscript® Reverse Transcription Kit (Qiagen). The total volume per reaction is set to 20 μL. 1. Place a nuclease-free PCR tube on ice and add approximately 2 μg of isolated total RNA. Add 2 μL of buffer RT (component of Omniscript RT Kit), 2 μL of dNTP mix (component of Omniscript RT Kit), 1 μL Oligo-dT-primer, 1 μL RNase inhibitor, and 1 μL Omniscript Reverse Transcriptase. Add RNasefree water to a final volume of 20 μL. 2. Mix by vortexing and centrifuge briefly. 3. Incubate for 60 min at 37 °C in a thermocycler. Use 5 μL of cDNA as template for the subsequent amplification (PCR) of the natural vNAR repertoire.

3.4 Amplification of the Natural vNAR Repertoire

For the construction of semisynthetic shark IgNAR V libraries, the natural framework repertoire of the bamboo shark is exploited as template. Therefore, a PCR is performed based on the synthesized cDNA. As described above, the randomization can alternatively be performed based on a single IgNAR V region template (see Note 1). From each cDNA reaction, 5 μL was used as template for subsequent PCR in a final volume of 50 μL. 1. Place a PCR tube on ice and add 36.75 μL nuclease-free water. Add 5 μL of cDNA reaction as template as well as 5 μL 10x Standard Taq Buffer, 1 μL bamboo/nat_up and 1 μL bamboo/ nat_lo (out of a 10 μM stock, primer sequences are listed in Table 1). Add 1 μL dNTP mixture (10 mM each) and 0.25 μL Taq DNA polymerase (we recommend scaling up a master mix for at least five reactions). 2. Carry out PCR using the following parameters: initial denaturation 95 °C for 2 min. 30 cycles of 30 s at 95 °C, 30 s at 55 °C and 40 s at 68 °C, followed by 72 °C for 7 min. 3. Analyze PCR products by 1–1.5% (w/v) agarose gel electrophoresis. Amplified vNAR genes should yield a distinct band at approx. 330–350 bp. Pool PCR products and purify using a PCR clean up kit according to the manufacturer’s instruction. PCR products might be stored at -20 °C.

3.5 Generation of the CDR3-Randomized PCR Insert for Library Establishment Using Yeast Surface Display as Platform Technology

The initial, CDR3-randomized library is constructed in three consecutive PCR steps (Fig. 2). In the first PCR, the forward primer FR1/CDR1/Tyr_up replaces the cysteine residue in CDR1 by tyrosine and introduces a marginal diversity within CDR1 that mimics the diversity found in the natural vNAR repertoire. The cysteine residue in CDR1 is replaced to avoid mispairing of disulfide bonds that might lead to a significant fraction of nonfunctional vNAR molecules in the final library, drastically complicating the selection of favored library candidates.

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The second PCR is performed to fully randomize the CDR3 using the degenerated primer CDR3rand/Fr4_lo. To this end, we recommend using trinucleotide phosphoramidite primers (see Note 4). In the last PCR, overlaps up- and downstream of the NheI and BamHI restriction sites of the pCT plasmid [32] are added to the vNAR fragment in order to establish a library using gap repair cloning. For all PCRs, the conditions are as follows.

3.5.1

First PCR

95 °C

2 min

95 °C 55 °C 68 °C 68 °C

30 s 30 s 40 s 7 min

35 cycles

1. Carry out approx. Five reactions in parallel. Prepare a master mix. Reagents per reaction in a final volume of 50 μL: approx. 100 ng vNAR PCR product of the natural vNAR repertoire (see Subheading 3.4). Add 1 μL FR1/CDR1/Tyr_up and 1 μL FR3_lo (out of a 10 μM stock, primer sequences are listed in Table 1). Add 1 μL dNTP mixture (10 mM each), 5 μL 10x Standard Taq Buffer, and 0.25 μL Taq DNA polymerase. Add nuclease-free water to a final volume of 50 μL. 2. Perform PCR in a thermocycler and separate PCR products on a 1–1.5% agarose gel. The amplified PCR product should be visible as a distinct band on the gel at a size of approx. 200 bp. Purify PCR products using a PCR cleanup kit according to the manufacturer’s instructions (see Note 5). Determine the DNA concentration. PCR products can be stored at -20 °C.

3.5.2

Second PCR

1. Carry out approx. Ten reactions in parallel. Prepare a master mix. Reagents per reaction in a final volume of 50 μL: approx. 100 ng first PCR product (see Subheading 3.5.1). Add 1 μL FR1_up and 1 μL CDR3rand/Fr4_lo (out of 10 μM stocks). Add 1 μL dNTP mixture (10 mM each), 5 μL 10x Standard Taq Buffer, and 0.25 μL Taq DNA polymerase. Add nucleasefree water to a final volume of 50 μL. 2. Perform PCR in a thermocycler and separate PCR products on a 1–1.5% agarose gel. The amplified PCR product should be visible as a distinct band on the gel at a size of 330 bp. Purify PCR products using a PCR cleanup kit according to the manufacturer’s instructions. Determine the DNA concentration. Primer CDR3rand/FR4_lo introduces a fully randomized CDR3 (without any cysteine residues) in a length of 12 amino acids. When a longer CDR3 is needed, the corresponding sequence of the oligonucleotide can be adjusted accordingly.

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1. Carry out as many reactions as needed to achieve an adequate library size. In general, we perform about ten transformation reactions for a yeast surface library with an estimated complexity of more than 108 unique clones. For each electroporation, we use approx. 6–8 μg of PCR product. Consequently, approx. 80 μg of insert DNA is needed (approx. 96 PCRs). Prepare a master mix. Reagents per reaction in a final volume of 50 μL: approx. 100–200 ng second PCR product (see Subheading 3.5.2). Add 1 μL GR_up and 1 μL GR_lo. Add 1 μL dNTP mixture (10 mM each), 5 μL 10x Standard Taq Buffer, and 0.25 μL Taq DNA polymerase. Add nuclease-free water to a final volume of 50 μL. 2. Perform PCR in a thermocycler and analyze PCR products on a 1–1.5% agarose gel. The amplified PCR product should be visible as a distinct band on the gel at a size of 370 bp. Purify PCR products using a PCR cleanup kit according to the manufacturer’s instructions. Determine the DNA concentration.

3.6 Shark vNAR Library Generation for Yeast Surface Display

The following protocol for the library establishment in S. cerevisiae is a modified version of the improved yeast transformation protocol by Benatuil and colleagues [44].

3.6.1 Digestion of pCT Plasmid

Libraries for yeast surface display are typically constructed by transformation of yeast in a homologous recombination-based process referred to as gap repair. To this end, the display vector needs to be digested as a first step. As mentioned above, we perform ten transformation reactions in general. For each electroporation, 1–2 μg NheI and BamHI digested plasmid are used. Hence, digestion is performed with 50 μg plasmid DNA. 1. The volume for the restriction enzyme double digest is set to 100 μL. Add 50 μg pCT plasmid, 60 U of NheI-HF®, 60 U BamHI-HF®, and 10 μL CutSmart buffer. Add nuclease-free water to a final volume of 100 μL. 2. Digest overnight at 37 °C. Analyze an aliquot of the digestion on a 1% agarose gel. Make sure that the double digest by both enzymes is complete. Purify the digested pCT plasmid using a PCR cleanup kit according to the manufacturer’s instructions. Since no ligation reaction is performed, there is no need for gel excision, because no re-ligation can occur. Determine the DNA concentration. PCR products might be stored at -20 °C.

3.6.2 Yeast Transformation

The protocol for the improved yeast electroporation can be found elsewhere [44]. In brief: 1. Incubate EBY100 overnight to stationary phase in YPD media at 180 rpm and 30 °C.

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2. Inoculate 100 mL fresh YPD media with the overnight culture to an OD600 of about 0.3. 3. Incubate cells at 30 °C and 180 rpm until OD600 reaches about 1.6. 4. Centrifuge cells at 4000× g for 3 min, remove supernatant. 5. Wash cells twice (by resuspending) using 50 mL ice-cold water followed by a wash step using 50 mL ice-cold electroporation buffer. 6. Incubate cells (after resuspending) in 20 mL LiAc-buffer for 30 min at 30 °C and 180 rpm. 7. Centrifuge cells, wash once with 50 mL ice-cold electroporation buffer. 8. Resuspend cell pellet in approx. 200 μL electroporation buffer to a final volume of approx. 1 mL. This gives two electroporation reactions with 400 μL electrocompetent EBY100 each. 9. Combine 1–2 μg digested pCT plasmid with 3–6 μg insert DNA (volume should not exceed 50 μL) and add mixture to 400 μL electrocompetent cells. 10. Transfer cell-DNA mix to ice-cold electroporation cuvette (0.2 cm). Electroporate at 2.500 V. Time constant should range from 3.0 to 4.5 ms. Transfer cells from each “shot” into 8–10 mL of a 1:1 mixture of YPD and 1 M sorbitol. Incubate for 1 h at 30 °C and 180 rpm. 11. Centrifuge cells and resuspend in 10 mL SD-CAA media. Calculate complexity of library by dilution plating (SD-CAA plates, estimate number of transformants after 72 h). Incubate library for at least 2 days at 30 °C and 180 rpm. 12. For long-term storage, centrifuge library and resuspend cells in 5% (v/v) glycerol and 0.67% (w/v) yeast nitrogen base. The final library is now ready to be screened via fluorescence activated cell sorting. To induce surface expression, cells need to be transferred into SG-CAA medium. Protocols for screening yeast surface display libraries can be found elsewhere [45]. To evaluate the quality of the final library, the authors recommend testing at least 96 colonies for presence of an insert with correct length and sequence (from dilution plating). For this, plasmid DNA from overnight cultures of single clones is extracted using a commercially available yeast plasmid miniprep kit or yeast DNA extraction kit according to the manufacturer’s instructions (see Note 6). PCR can be performed using the conditions found in 3.5 with pCT_Seq_up and pCT_Seq_lo as primer combination. This should result in a distinct band on a 1–1.5% agarose gel with a size of approx. 600 bp. Send out PCR positive clones for sequencing.

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3.7 Affinity Maturation by CDR1 Diversification and Sublibrary Establishment of Target-Enriched Binders

3.7.1

First PCR

We previously established a generic, two-step strategy for the isolation of high-affinity shark-derived antibody domains [12]. At first, CDR3 was randomized as described above. This library was subjected to screening for target-specific molecules, albeit with moderate affinities to their antigen. The DNA of the target-specific population was isolated after the last screening round, and CDR1 comprising five residues was fully diversified. Sublibraries were established and screened with significantly decreased target concentrations. This strategy proved to be useful to obtain vNARs with affinities in the nanomolar range. Interestingly, this in vitro method resembles the natural immune response in sharks to select clones from a primary and nearly entirely CDR3-based IgNAR repertoire, followed by affinity maturation of CDR1 and hypervariable loops after antigen exposure [46]. As shown in Fig. 3, CDR1 of the target-specific population (see Note 7) is randomized in a consecutive three-step PCR, similar to the initial diversification of CDR3. The starting point is isolated plasmid DNA from the last round of screening using yeast surface display in which target-specific clones have been significantly enriched. Plasmid DNA can be isolated using commercially available kits according to the manufacturer’s instructions. For all PCRs, the conditions are as follows. 95 °C

2 min

95 °C 55 °C 68 °C

30 s 30 s 40 s

68 °C

7 min

35 cycles

1. Prepare a master mix. Reagents per reaction in a final volume of 50 μL: approx. 100 ng isolated plasmid DNA (pCT) isolated after last round of screening. Primer combination: add 1 μL CDR1rand_up and 1 μL GR_lo (out of 10 μM stocks). Primer sequences are listed in Table 1. Add 1 μL dNTP mixture (10 mM each), 5 μL 10x Standard Taq Buffer, and 0.25 μL Taq DNA polymerase. Add nuclease-free water to a final volume of 50 μL. 2. Perform PCR in a thermocycler and separate PCR products on a 1–1.5% agarose gel. The amplified PCR product should be visible as a distinct band on the gel at a size of approx. 290 bp. Purify PCR products using a gel cleanup kit according to the manufacturer’s instructions. Determine the DNA concentration. PCR products might be stored at -20 °C.

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Fig. 3 Schematic depiction of PCR-based sublibrary design for affinity maturation. Plasmid DNA of FACSselected, target-enriched vNAR domains or, alternatively, of a single antigen-specific vNAR domain is used as template for sublibrary establishment. In a first PCR, five residues of CDR1 are fully randomized (without cysteine). Subsequent PCRs are performed to generate a complete IgNAR V domain molecule with gap repair overhangs as shown. (This figure is generated using www.biorender.com) 3.7.2

Second PCR

1. Carry out approximately ten reactions in parallel. Prepare a master mix. Reagents per reaction in a final volume of 50 μL: approx. 100 ng first PCR product (see Subheading 3.7.1). Primer combination: add 1 μL FR1_up and 1 μL GR_lo (out of 10 μM stocks). Add 1 μL dNTP mixture (10 mM each), 5 μL 10x Standard Taq Buffer, and 0.25 μL Taq DNA polymerase. Add nuclease-free water to a final volume of 50 μL. 2. Perform PCR in a thermocycler and separate PCR products on a 1–1.5% agarose gel. The amplified PCR product should be visible as a distinct band on the gel at a size of 330 bp. Purify PCR products using a gel cleanup kit according to the manufacturer’s instructions. Determine the DNA concentration using BioSpec-nano or equivalent equipment.

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1. Carry out as many reactions as needed to achieve an adequate library size. Prepare a master mix. Reagents per reaction in a final volume of 50 μL: approx. 100–200 ng second PCR product (see Subheading 3.7.2). Primer combination: add 1 μL GR_up and 1 μL GR_lo (out of 10 μM stocks). Add 1 μL dNTP mixture (10 mM each), 5 μL 10x Standard Taq Buffer, and 0.25 μL Taq DNA polymerase. Add nuclease-free water to a final volume of 50 μL. 2. Perform PCR in a thermocycler and separate PCR products on a 1–1.5% agarose gel. The amplified PCR product should be visible as a distinct band on the gel at a size of 370 bp. Purify PCR products using a gel cleanup kit according to the manufacturer’s instructions. Determine the DNA concentration.

4

Notes 1. Instead of constructing a semisynthetic vNAR library based on the natural repertoire of the bamboo shark, the construction of a fully synthetic vNAR library based on a single vNAR scaffold is possible. The following sequence can be used as template: vNAR template sequence: ATGGCCGCACGGCTTGAACAAACACCGACAACGACA ACAAAGGAGGCAGGCGAATCACTGACCATCAATT GCGTCCTAAAAGGTTCCAGATATGGATTGGGTAC AACGTACTGGTATTTCACAAAAAAGGGCGCAACA AAGAAGGCGAGCTTATCAACTGGCGGACGATACT CGGACACAAAGAATACGGCATCAAAGTCCTTTTC CTTGCGAATTAGTGACCTAAGAGTTGAAGACAGT GGTACATATCACTGTGAAGCGATGCTGGGCATTA ACCCATTTGGCTGGAAACGGCTGATTGAAGGAGG GGGCACCACTGTGACTGTGAAA. 2. For resuspension of the RNA pellet, we recommend starting with a small volume of RNase-free water. Add small aliquots of water, until the pellet is completely dissolved. This should ensure that RNA is concentrated as much as possible. 3. Since it is not necessary to harness the full diversity of the natural vNAR repertoire, working with partially degraded RNA might also work for semisynthetic library establishment. 4. Other diversification strategies, e.g., NNK or NNS randomization, might also work. However, these technologies typically result in the incorporation of unwanted stop codons, clearly impairing the quality of the library. Nowadays, also even more sophisticated library diversification procedures can be considered, such as incorporation of amino acids in predefined frequencies or strategies avoiding sequence liabilities such as hydrophobic patches or glycosylation sites.

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5. When unwanted side products appear on the gel, gel excision using a commercial kit according to the manufacturer’s instructions is possible. However, at least in the third PCR, we do not recommend gel excision since the yield of extracted DNA might be too low for library construction. Instead, try enhancing PCR stringency by increasing the annealing temperature. 6. Alternatively, a single clone can be picked with a sterile pipette tip and transferred into 10 μL 0.02 M NaOH. After a 10 min incubation at 99 °C, use 1 μL as template for colony PCR using pCT_Seq_up and pCT_Seq_lo. 7. Alternatively, affinity maturation using this methodology might be performed with a defined single clone, i.e., targetspecific molecule. However, success of affinity maturation depends on the structure of the paratope of the initially isolated vNAR. References 1. Grzeschik J, Ko¨nning D, Hinz SC et al (2018) Generation of semi-synthetic shark IgNAR single-domain antibody libraries. In: Hust M, Lim TS (eds) Phage display. Springer New York, New York, pp 147–167 2. Greenberg AS, Avila D, Hughes M et al (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168– 173. https://doi.org/10.1038/374168a0 3. Zielonka S, Empting M, Grzeschik J et al (2015) Structural insights and biomedical potential of IgNAR scaffolds from sharks. MAbs 7:15–25. https://doi.org/10.4161/ 19420862.2015.989032 4. Krah S, Schro¨ter C, Zielonka S et al (2016) Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol 38:21–28 . https://doi.org/10.3109/ 08923973.2015.1102934 5. Ko¨nning D, Zielonka S, Grzeschik J et al (2017) Camelid and shark single domain antibodies: structural features and therapeutic potential. Curr Opin Struct Biol 45:10–16. https://doi.org/10.1016/j.sbi.2016.10.019 6. Dooley H, Stanfield RL, Brady RA, Flajnik MF (2006) First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate. Proc Natl Acad Sci 103:1846– 1851. https://doi.org/10.1073/pnas. 0508341103 7. Simmons DP, Streltsov VA, Dolezal O et al (2008) Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structures. Proteins

Struct Funct Bioinf 71:119–130. https://doi. org/10.1002/prot.21663 8. Streltsov VA, Carmichael JA, Nuttall SD (2005) Structure of a shark IgNAR antibody variable domain and modeling of an earlydevelopmental isotype: structure of a shark ignar antibody variable domain and modeling of an early-developmental isotype. Protein Sci 14:2901–2909. https://doi.org/10.1110/ps. 051709505 9. Flajnik MF, Deschacht N, Muyldermans S (2011) A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels? PLoS Biol 9:e1001120. https://doi.org/10.1371/journal.pbio. 1001120 10. Henderson KA, Streltsov VA, Coley AM et al (2007) Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure 15:1452–1466. https://doi.org/10.1016/j. str.2007.09.011 11. Li Z, Krippendorff B-F, Sharma S et al (2016) Influence of molecular size on tissue distribution of antibody fragments. MAbs 8:113–119. https://doi.org/10.1080/19420862.2015. 1111497 12. Zielonka S, Weber N, Becker S et al (2014) Shark attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation. J Biotechnol 191: 236–245. https://doi.org/10.1016/j.jbiotec. 2014.04.023 13. Barelle C, Porter A (2015) VNARs: an ancient and unique repertoire of molecules that deliver

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small, soluble, stable and high affinity binders of proteins. Antibodies 4:240–258. https:// doi.org/10.3390/antib4030240 14. Kovaleva M, Ferguson L, Steven J et al (2014) Shark variable new antigen receptor biologics – a novel technology platform for therapeutic drug development. Expert Opin Biol Ther 14:1527–1539. https://doi.org/10.1517/ 14712598.2014.937701 15. Bannas P, Hambach J, Koch-Nolte F (2017) Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol 8. https://doi.org/10. 3389/fimmu.2017.01603 16. Chanier T, Chames P (2019) Nanobody engineering: toward next generation immunotherapies and immunoimaging of cancer. Antibodies 8:13. https://doi.org/10.3390/ antib8010013 17. Pekar L, Busch M, Valldorf B et al (2020) Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform. MAbs:1812210. https:// doi.org/10.1080/19420862.2020.1812210 18. Yanakieva D, Pekar L, Evers A et al (2022) Beyond bispecificity: controlled Fab arm exchange for the generation of antibodies with multiple specificities. MAbs 14. https:// doi.org/10.1080/19420862.2021.2018960 19. Lipinski B, Arras P, Pekar L et al (2023) NKP46 -specific single domain antibodies enable facile engineering of various potent NK cell engager formats. Protein Sci. https://doi.org/10. 1002/pro.4593 20. Ubah OC, Buschhaus MJ, Ferguson L et al (2018) Next-generation flexible formats of VNAR domains expand the drug platform’s utility and developability. Biochem Soc Trans 46:1559–1565. https://doi.org/10.1042/ BST20180177 21. Kovaleva M, Johnson K, Steven J et al (2017) Therapeutic potential of shark anti-ICOSL VNAR domains is exemplified in a murine model of autoimmune non-infectious uveitis. Front Immunol 8. https://doi.org/10.3389/ fimmu.2017.01121 22. Ubah OC, Steven J, Porter AJ, Barelle CJ (2019) An anti-hTNF-α variable new antigen receptor format demonstrates superior in vivo preclinical efficacy to Humira® in a transgenic mouse autoimmune polyarthritis disease model. Front Immunol 10. https://doi.org/ 10.3389/fimmu.2019.00526 23. Sehlin D, Stocki P, Gustavsson T et al (2020) Brain delivery of biologics using a cross-species reactive transferrin receptor 1 VNAR shuttle.

FASEB J 34:13272–13283. https://doi.org/ 10.1096/fj.202000610RR 24. Camacho-Villegas T, Mata-Gonza´lez M, Garcı´a-Ubbelohd W et al (2018) Intraocular penetration of a vNAR: in vivo and in vitro VEGF165 neutralization. Mar Drugs 16:113. https://doi.org/10.3390/md16040113 25. Ko¨nning D, Rhiel L, Empting M et al (2017) Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver antiidiotypic binders. Sci Rep 7. https://doi.org/ 10.1038/s41598-017-10513-9 26. Macarro´n Palacios A, Grzeschik J, Deweid L et al (2020) Specific targeting of lymphoma cells using semisynthetic anti-idiotype shark antibodies. Front Immunol 11. https://doi. org/10.3389/fimmu.2020.560244 27. Li D, English H, Hong J et al (2022) A novel PD-L1-targeted shark VNAR single-domainbased CAR-T cell strategy for treating breast cancer and liver cancer. Mol Ther Oncolytics 24:849–863. https://doi.org/10.1016/j. omto.2022.02.015 28. Ubah OC, Lake EW, Gunaratne GS et al (2021) Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography. Nat Commun 12. https://doi.org/10.1038/ s41467-021-27611-y 29. Gauhar A, Privezentzev CV, Demydchuk M et al (2021) Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro. FASEB J 35. https://doi.org/10.1096/fj. 202100986RR 30. Buschhaus MJ, Becker S, Porter AJ, Barelle CJ (2019) Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents. Protein Eng Des Sel 32:385–399. https://doi.org/ 10.1093/protein/gzaa002 31. Leow CH, Fischer K, Leow CY et al (2018) Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library. Malar J 17. https://doi.org/10.1186/ s12936-018-2531-y 32. Ko¨nning D, Kolmar H (2018) Beyond antibody engineering: directed evolution of alternative binding scaffolds and enzymes using yeast surface display. Microb Cell Factories 17. https://doi.org/10.1186/s12934-0180881-3 33. Pekar L, Klausz K, Busch M et al (2021) Affinity maturation of B7-H6 translates into enhanced NK cell–mediated tumor cell lysis

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Part III Selection Strategies for Antibodies

Chapter 12 Antibody Selection via Phage Display in Microtiter Plates Stephan Steinke, Kristian Daniel Ralph Roth, Maximilian Ruschig, Nora Langreder, Saskia Polten, Kai-Thomas Schneider, Rico Ballmann, Giulio Russo, Kilian Johannes Karl Zilkens, Maren Schubert, Federico Bertoglio, and Michael Hust Abstract The most common and robust in vitro technology to generate monoclonal human antibodies is phage display. This technology is a widely used and powerful key technology for recombinant antibody selection. Phage display-derived antibodies are used as research tools, in diagnostic assays, and by 2022, 14 phage display-derived therapeutic antibodies were approved. In this review, we describe a fast high-throughput antibody (scFv) selection procedure in 96-well microtiter plates. The given detailed protocol allows the antibody selection (“panning”), screening, and identification of monoclonal antibodies in less than 2 weeks. Furthermore, we describe an on-rate panning approach for the selection of monoclonal antibodies with fast on-rates. Key words Phage display, scFv, Antibody fragments, Panning, Antibody selection, Monoclonal antibodies, Antibody phage display, Microtiter plates (MTPs), On-rate panning

1

Introduction Antibody phage display is an in vitro technology to generate monoclonal antibodies, which is independent of the restrictions of the immune system. This in vitro isolation of antibody fragments is called “panning” in accordance to the gold washers [1]. In the panning procedure, the antigen is immobilized to a solid surface, such as either column matrixes [2], nitrocellulose [3], magnetic beads [4], or plastic surfaces with high protein binding capacities like polystyrene microtiter plates (MTPs) [5]. A further strategy is the selection of antibodies in solution using biotinylated antigens followed by a “pull-down” step with streptavidin beads [6]. However, this “panning” approach will be presented in more detail in the chapter “Antibody selection in solution using beads.” To gen-

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_12, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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erate antibodies against cell surface markers, e.g., cancer targets, the panning can be performed directly on cells [7, 8]; this will be described in the chapter “Cell panning.” For the selection in MTPs, the antibody phage is incubated with the surface-bound antigen, followed by stringent washing to remove the non-binding antibody phage. Subsequently, the bound antibody phage will be eluted and reamplified by infection of E. coli. The selection cycle will be completed by co-infection of the phagemid containing E. coli colonies derived from the previous panning round with a helper phage to produce new antibody phage. This phage preparation will be used for upcoming panning rounds until a significant enrichment of antigen-specific antibody phage is achieved. The fraction of antigen-specific antibody phage clones increases with every panning round, and usually after three to four panning rounds, a sufficient amount of binders was enriched. Screening of the monoclonal binders is performed in a 96-well microtiter plate format. Therefore, scFv antibody fragments are produced as soluble monoclonal fragments, or as monoclonal antibody phage, in microtiter plates and then identified by, e.g., ELISA [9], immunoblot [5], or flow cytometer [10]. Subsequently, the gene fragments encoding the antibody fragments can be subcloned into any other antibody format, e.g., scFv-Fc, Fab, or IgG [9, 11–13]. A schema of the selection procedure is given in Fig. 1. Panning can be performed with different types of libraries. Depending on the panning strategy patient-derived immune libraries [17–19] or naı¨ve libraries like the HAL libraries [12, 20, 21], the McCafferty library [22], Pfizer library [23], or the Tomlinson libraries [24], but also semisynthetic or synthetic libraries like the HuCAL libraries [25–27] can be used. Antibody phage display libraries are valuable sources for the generation of therapeutic antibodies. An overview of some phage display-derived therapeutic antibodies can be found in a review by Frenzel et al. [14]. Currently, 14 phage display-derived antibodies (initial selection, affinity maturation, or humanization by phage display) are FDA/EMA approved (Table 1). The following protocol describes the panning and the screening of scFv antibody fragments in microtiter plates (MTPs). This protocol is an improved and upgraded versions of the “classical panning” protocol published by Schirrmann et al. [28] and the “high throughput panning” protocol published by Russo et al. [16]. With this panning strategy, a successful and fast antibody selection against SARS-CoV2 could be achieved [19, 20]. Furthermore, an additional approach to select for on-rate binders is included. The antibody selection, screening, and identification of monoclonal antibodies can be performed within 2 weeks.

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Fig. 1 Antibody panning schema for three panning rounds and subsequent cloning into scFv-Fc format. (Modified figure from former publications [14–16])

2

Materials

2.1 Coating of Microtiter Wells

1. MaxiSorp microtiter plates or stripes (Nunc, Langenselbold, Germany) or other polystyrene microtiter plates. 2. PBS pH 7.4: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4*2H2O, 0.24 g KH2PO4 in 1 L. 3. Dimethyl sulfoxide (DMSO). 4. PBST (PBS + 0.05% (v/v) Tween 20). 5. MilliQ-T (ultrapure H2O + 0.05% (v/v) Tween 20).

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Table 1 Phage display-derived antibodies approved by FDA/EMA Name

Drug

Company

Target

Type

Approval

Adalimumab

Humira

Abbott

TNF- α

Human IgG1

2002 (USA) 2003 (EU)

Ranibizumab

Lucentis

Novartis

VEGF-A

Humanized IgG1-Fab

2006 (USA) 2007 (EU)

Belimumab

Benysta

GSK

B-lymphocyte stimulator

Human IgG1

2011 (USA/EU)

Raxibacumab

ABthrax

GSK

B. anthracis PA

Human IgG1

2012 (USA)

Ramucirumab

Cyramza

Lilly

VEGFR2

Human IgG1

2014 (USA/EU)

Necitumumab

Portrazza Lilly

EGFR

Human IgG1

2015 (USA) 2016 (EU)

Ixekizumab

Taltz

IL-17a

Humanized IgG4

2016 (USA/EU)

Atezolizumab

Tecentriq Roche

PD-L1

Human IgG1

2016 (USA) 2017 (EU)

Avelumab

Bavencio Merck KGaA

PD-L1

Human IgG1

2017 (USA/EU)

Guselkumab

Tremfya

IL-23

Human IgG1

2017 (USA/EU)

Lanadelumab

Takhzyro Dyax/Shire

Plasma kallikrein

Human IgG1

2018 (USA/EU)

Caplacizumab

Cablivi

VWF

Humanized nanobody

2018 (EU) 2019 (USA)

Moxetumomab pasudotox

Lumoxiti AstraZeneca CD22

Murine IgG1, dsFv

2018 (USA) 2021 (EU)

Emapalumab

Gamifant Novimmune Interferongamma (IFNγ)

Human IgG1

2018 (USA)

2.2

Panning

Lilly

MorphoSys

Ablynx

1. MPBST: 2% skim milk in PBST, prepare fresh (2% BSA in PBST for biotinylated targets). 2. Panning block solution: 1% (w/v) skim milk + 1% (w/v) BSA in PBST, prepare fresh. 3. 10 μg/mL Trypsin in PBS. 4. E. coli TG1 (supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rK-mK-) [F′ traD36 proAB lacIqZΔM15]). 5. M13K07 helperphage Waltham, USA).

(Thermo

Fisher

Scientific,

6. Round-bottom polypropylene (PP) Deepwell 96 MTPs (Greiner, Frickenhausen, Germany).

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7. Labnet VorTempTM 56 benchtop shaker/incubator (Woodbridge, NJ, USA). 8. Eppendorf 5810R, Rotor A-4-81 with MTP adapter. 9. 2xYT media pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl. 10. 2xYT-T: 2xYT, containing 50 μg/mL tetracycline. 11. 10xGA: 1 M glucose, 1 mg/mL ampicillin. 12. 2xYT-GA: 2xYT, 100 mM glucose, 100 μg/mL ampicillin. 13. 2xYT-AK: 2xYT, containing 100 μg/mL ampicillin, 50 μg/mL kanamycin. 14. Glycerol (99.5%). 2.3

Phage Titration

1. E. coli XL1-Blue MRF’ (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]). 2. 2xYT-GA agar plates (2xYT-GA + 1.5% (w/v) agar-agar).

2.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. 96-well U-bottom polypropylene (PP) microtiter plates (Greiner Bio-One, Frickenhausen, Germany). 2. AeraSeal breathable Victorville, USA).

sealing

film

(Excel

Scientific,

3. Potassium phosphate buffer pH 7.2–7.4: 2.31% (w/v) (0.17 M) KH2PO4 + 12.54% (w/v) (0.72 M) K2HPO4. 4. Buffered 2xYT pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl, 10% (v/v) potassium phosphate buffer. 5. Buffered 2xYT-SAI: buffered 2xYT containing 50 mM saccharose + 100 μg/mL ampicillin + 50 μM isopropyl-beta-D-thiogalactopyranoside (IPTG). 2.5 ELISA of Soluble Monoclonal Antibody Fragments

1. Mouse α-His-tag monoclonal antibody (α-Penta His, Qiagen, Hilden, Germany). 2. Mouse α-myc-tag monoclonal antibody (9E10) (Sigma, Mu¨nchen, Germany). 3. Mouse α-pIII monoclonal antibody PSKAN3 (Mobitec, Go¨ttingen, Germany). 4. Goat α-Mouse IgG serum, (Fab specific) HRP conjugated (Sigma). 5. Oligonucleotide primers MHLacZ-Pro_f (5′ GGCTCGTATG TTGTGTGG 3′) and MHgIII:r (5′ CTAAAGTTTTGTCGTC TTTCC 3′).

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Methods

3.1 Coating of Microtiter Plate Wells

1. (A) Protein antigen: For the first panning round, use 1–5 μg protein/well per panning, for the following rounds use 0.1–1 μg protein/well for more stringent conditions. Dissolve the antigen in 150 μL PBS (see Note 1) and incubate it in a polystyrene (PS) microtiter plate well (MTP) overnight at 4 °C. (B) Oligopeptide antigen: Coat 200 ng streptavidin/well per panning (see Note 2) then block the streptavidin coated well with 300 μL 2% BSA-PBST (w/v). Wash the coated microtiter plate wells 3× with PBST using an ELISA washer (see Note 3). Use 500–1000 ng oligopeptide for each panning round. Dissolve the oligopeptide in 150 μL PBS, transfer into a streptavidin-coated MTP well and incubate overnight at 4 °C. 2. Wash the coated microtiter plate wells again 3× with MilliQ-T using an ELISA washer (see Note 3).

3.2

Panning

1. (A) Block the antigen-coated MTP wells with MPBST for 1 h at RT, or overnight at 4 °C. The wells have to be completely filled. Afterward, wash the blocked antigen-coated wells 3× with MiliQ-T (see Note 3). (B) In parallel, block an additional MTP well (without antigen!) per panning with panning block solution for 1 h at RT, or overnight at 4 °C, for preincubation of the antibody gene library. The MTP wells have to be completely filled. When using biotinylated antigens, use a streptavidin MTP well (see Note 2). Wash 3× times with MilliQ-T (see Note 3). Incubate 1011–1012 antibody phage (you should use 50–100× excess of phage particles compared to the library size) from the library in 150 μL panning block for 1 h at RT. This step removes unspecific binders which often occur from the antibody gene libraries due to incorrect folding of individual antibodies. 2. Transfer the preincubated antibody phage library to the blocked MTP wells or fill 1011–1012 amplified phage solved in panning block solution (final volume 150 μL) from the previous panning round in the blocked MTP wells. Incubate at RT for 2 h for binding of the antibody phage. When using biotinylated antigens add 5 μg streptavidin for competition per MTP well. 3. Remove the unspecific bound antibody phage by stringent washing. Therefore, wash the wells 10× with an ELISA washer in the first panning round. In the following panning rounds increase the number of washing steps (20× in the second panning round, 30× in the third panning round, etc.) (see Note 3).

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4. Elute bound antibody phage with 150 μL Trypsin (10 μg/mL) solution for 30 min at 37 °C (see Note 4). 5. After the last panning round, use 10 μL of the eluted phage for titration and determine cfu/mL (see Subheading 3.3 Phage titration). 6. Inoculate 50 mL 2xYT with an overnight culture of E. coli TG1 (see Note 5) in 100 mL Erlenmeyer flasks and grow at 250 rpm and 37 °C to O.D.600 0.4–0.5 (see Note 6). 7. Fill 150 μL exponentially growing E. coli TG1 (O.D. ~0.5) in a polypropylene (PP) Deepwell MTP well and mix with 150 μL of the eluted phage. Incubate the bacteria for 30 min at 37 °C without shaking and 30 min at 37 °C and 650 rpm (see Note 7). 8. Add 1000 μL of 2xYT and 150 μL 10xGA (see Note 8) and incubate for 1 h at 37 °C and 650 rpm. O.D. should be around ~0.5 (~5 × 108 cells/mL). 9. Infect the bacteria with 50 μL M13K07 helperphage (2 × 1011 phage particles/mL = 1 × 1010 phage particles, MOI 1:20 ). Incubate for 30 min at 37 °C without shaking, followed by 30 min at 37 °C and 650 rpm. 10. Centrifuge the MTP plate at 3220 × g (e.g., use Eppendorf 5810R, Rotor A-4-81 with MTP carriers). Remove the complete supernatant with a pipette. Do not destroy the pellet (see Note 9). 11. Add 950 μL 2xYT-AK and incubation overnight at 30 °C and 650 rpm to produce new antibody phage. 12. Centrifuge the MTP plate at 3220 × g. Transfer the supernatant (~1 × 1012 scFv-phage/mL) into a new PP MTP. The supernatant can be directly used for the next panning round. Therefore, mix 50 μL supernatant with 100 μL panning block. 3.3

Phage Titration

1. Inoculate 30 mL 2xYT-T in a 100 mL Erlenmeyer flask with E. coli XL1-Blue MRF’ (see Note 10) and grow overnight at 37 °C and 250 rpm. 2. Inoculate 50 mL 2xYT-T with 500 μL overnight culture and grow at 250 rpm and 37 °C up to O.D.600 ~ 0.5 (see Note 6). 3. Make serial dilutions of the phage suspension in PBS. The number of eluted phage depends on several parameters (e.g., antigen, library, panning round, washing stringency, etc.). In case of a successful enrichment, the titer of eluted phage usually is around 103–105 cfu/mL after the first panning round and increases two to three orders in magnitude per additional panning round (see Note 11). The phage titer after reamplification should be 1012–1013 cfu/mL.

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4. Infect 50 μL bacteria with 10 μL phage dilution and incubate 30 min at 37 °C. 5. You can perform titrations in two different ways: (A) plate the 60 μL infected bacteria on 2xYT-GA agar plates (9 cm petri dishes). (B) pipette 10 μL (in triplicates) on 2xYT-GA agar plates. Here, about 20 titer spots can be placed on one 9 cm petri dish. Dry drops on work bench. 6. Incubate the plates overnight at 37 °C. 7. Count the colonies and calculate the cfu or cfu/mL titer according to the dilution. 3.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. Fill each well of a 96-well U-bottom PP MTP with 150 μL 2xYT-GA. 2. Pick 92 clones with sterile tips from the last panning round and inoculate each well (see Note 12). Seal the plate with a breathable sealing film. 3. Incubate overnight in a microtiter plate shaker at 37 °C and 800 rpm. 4. (A) Fill a new 96-well polypropylene microtiter plate with 180 μL 2xYT-GA and add 10 μL of the overnight cultures. Incubate for 2 h at 37 °C and 800 rpm. (B) Add 30 μL glycerol solution to the remaining 140 μL overnight cultures. Mix by pipetting and store this masterplate at -80 °C. 5. Pellet the bacteria in the microtiter plates by centrifugation for 10 min at 3220 × g. Remove the glucose containing media by carefully pipetting (see Note 9). 6. Add 180 μL buffered 2xYT-SAI (containing saccharose, ampicillin and 50 μM IPTG) and incubate overnight at 30 °C and 800 rpm (see Notes 13 and 14). 7. Pellet the bacteria by centrifugation for 10 min at 3220 × g in the microtiter plates. Transfer the antibody fragmentcontaining supernatant to a new polypropylene microtiter plate and store at 4 °C for few days or directly proceed with testing the antibody binding.

3.5 ELISA of Soluble Monoclonal Antibody Fragments

1. To analyze the specific monoclonal soluble antibody fragment binding to the antigen, coat 100–200 ng antigen/well overnight at 4 °C. As control coat 100–200 ng BSA or streptavidin/well (for coating see Subheading 3.2, step 1). 2. Wash the coated microtiter plate wells 3× with MilliQ-T (washing procedure see Subheading 3.2, step 1 and Note 3).

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3. Block the antigen-coated wells with MPBST (BSA-PBST for biotinylated targets) for 2 h at RT. The wells have to be completely filled. 4. Fill 50 μL MPBST in each well and add 50 μL of antibody solution (see Subheading 3.2, step 4). Incubate for 1.5 h at RT (or overnight at 4 °C). 5. Wash the microtiter plate wells 3× with MilliQ-T (washing procedure see Subheading 3.2, step 1 and Note 3). 6. Incubate 100 μL/well mouse α-myc tag antibody (clone 9E10) solution for 1 h (appropriate dilution in MPBST (BSA-PBST for biotinylated targets)). 7. Wash the microtiter plate wells 3× with MilliQ-T (washing procedure see Subheading 3.2, step 1 and Note 3). 8. Incubate 100 μL/well goat α-mouse HRP conjugate for 1 h (appropriate dilution in MPBST (BSA-PBST for biotinylated targets)). 9. Wash the microtiter plate wells 3× with MilliQ-T (washing procedure see Subheading 3.2, step 1 and Note 13). 10. Shortly before use, mix 19-parts TMB substrate solution A and 1-part TMB substrate solution B. Add 100 μL of this TMB solution mix into each well and incubate for 30 min. 11. Stop the color reaction by adding 100 μL 1 N sulfuric acid solution per well. The color turns from blue to yellow. 12. Measure the extinction at 450 nm using an ELISA reader (reference wavelength 620 nm). 13. Identify positive candidates with a signal (on antigen) 10× over noise (on control protein, e.g., BSA) (see Note 15). 14. Sequence the DNA of the selected scFv for identification of unique clones using the oligonucleotide primers MHLacZPro_f and MHgIII_r. We suggest analyzing the antibody sequences using VBASE2 (www.vbase2.org) (Tool: Fab/scFab/scAb/scFv Analysis). 3.6 Fast On-Rate Panning

For the selection of monoclonal antibodies with fast on-rates, the previous described panning protocol needs to be adjusted slightly. Instead of the 2 h incubation on the antigen, the time can be shortened to 10 min. All other steps are performed exactly as described above (see Subheading 3.2). But, be aware that titers can be lower than usual (up to 100-fold), which can but not necessarily has to influence the number of unique antibodies selected from the panning. To have a first estimation of the on-rate the screening ELISA can be adjusted as well. All steps except for the scFv incubation are performed as described before (see Subheading 3.5). The scFv incubation time can be shortened

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from 1.5 h to 2 min, 5 min or 10 min (depending on the needed properties). It is recommended to incubate scFvs from the same batch in an additional MTP for the standard 1.5 h incubation as a control. Therefore, the on-rate antibodies can be compared directly to the standard 1.5 h incubation control. With the fast on-rate panning the antibody selection time can be drastically reduced.

4

Notes 1. If the protein is not binding properly to the microtiter plate surface, try bicarbonate buffer (50 mM NaHCO3, pH 9.6) (this buffer is recommended by Nunc for MaxiSorp plates). 2. More hydrophobic oligopeptides may need to be dissolved in PBS containing 5%–100% DMSO. If biotinylated oligopeptides are used as antigen for panning, dissolve 200 ng streptavidin in 150 μL PBS and coat overnight at 4 °C. Coat two wells for each panning, one well for the panning and the second one for the preincubation of the library to remove streptavidin or unspecific binders! Pour out the wells and wash thrice with PBST. Dissolve 100–500 ng biotinylated oligopeptide in PBS and incubate for 1 h at RT. Alternatively, oligopeptides with a terminal cysteine residue can be coupled to BSA and coated overnight at 4 °C. When working with biotinylated oligopeptides, it is important to use 2% BSA in PBST solution instead of 2% MPBST. Soluble streptavidin (1–5 μg) should be added into the library well for competition at least in the first panning round to further reduce streptavidin binders. 3. The washing should be performed with an ELISA washer (e.g., Agilent BioTek Elx50) to increase the stringency and reproducibility. To remove antigen or blocking solutions, wash thrice with Milli-QT (“standard washing” protocol for BioTek washer). If no ELISA washer is available, wash manually thrice with Milli-QT. After binding of antibody phage, wash ten times with PBST (“stringent bottom washing” protocol in case of BioTek washer). If no ELISA washer is available, wash manually ten times with PBST and ten times with PBS. For stringent off-rate selection, increase the number of washing steps or additionally incubate the microtiter plate in 1 L PBS for several days. 4. Phagemids like pHAL14 [12, 29], pHAL30 [21], or pHAL52 [19] have coding sequences for a trypsin-specific cleavage site between the antibody fragment gene and the gIII. Trypsin also cleaves within antibody fragments but does not degrade the phage particles including the pIII that mediates the binding of the phage to the F-pili of E. coli required for the infection. We observed that proteolytic cleavage of the antibody fragments

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from the antibody::pIII fusion by trypsin not only increases the elution but also enhances the infection rate of eluted phage particles, especially when using Hyperphage for packaging. 5. E. coli TG1 is growing much faster compared to XL1-Blue MRF’. This strain allows performing one full panning round per day. 6. If the bacteria have reached O.D.600 ~ 0.5 before they are needed, store the culture immediately on ice to maintain the F-pili on the E. coli cells; however, be careful, store no longer than 20 min! M13K07 helper phage (kan+) or other scFvphage (amp+) can be used as positive control to check the infectibility of the E. coli cells. 7. After 1 h of incubation, an O.D.600 0.4–0.5 is reached, corresponding to ~5 × 108 bacteria. 8. The high concentration of glucose is necessary to efficiently repress the Lac promoter controlling the antibody::pIII fusion gene on the phagemid. Low glucose concentrations lead to an inefficient repression of the lac promoter and background expression of the antibody::pIII fusion protein. Background antibody expression is a strong selection pressure frequently causing mutations in the phagemid, especially in the promoter region and the antibody::pIII fusion gene. Bacteria with this kind of mutations in the phagemids proliferate faster than bacteria with nonmutated phagemids. Therefore, the 100 mM glucose has to be included in every step of E. coli cultivation except during the phage production, here compliment instead of glucose with IPTG (see Note 13). 9. To not destroy the pellet, remove the supernatant carefully by touching the side of the well with the pipette tip and aspirate slowly. An alternative is to manually shake out the supernatant (do it with a fast movement of your wrist). 10. Use E. coli XL1-Blue MRF’ for titer determination and production of soluble antibodies. The plasmid quality and yield using this strain are better compared to TG1. Furthermore, the XL1-Blue MRF’ slower growth rate and the more regular colony shape compared to the TG1 allow for a more accurate picking of single colonies for the screening. 11. When the antibody gene library was packaged using Hyperphage, the titer of the eluted phage after the second panning may not increase as strongly or even decreases slightly due to the change from polyvalent to monovalent display. 12. We recommend picking 92 clones when using a 96-well microtiter plate. Use the wells H3, H6, H9, and H12 for controls. H3 and H6 are negative controls (these wells will not be inoculated, but used as negative control for the following

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ELISA with soluble antibodies). We inoculate the wells H9 and H12 with a clone containing a phagemid encoding a known antibody fragment. In ELISA, the wells H9 and H12 are coated with the antigen corresponding to the control antibody fragment in order to check scFv production and the ELISA setup. 13. The appropriate IPTG concentration for induction of antibody or antibody::pIII expression depends on the vector design. A concentration of 50 μM was well suited for vectors with a Lac promoter like pIT2 [30], pHENIX [31], pHAL14 [12, 29], or pHAL30 [21]. 14. Buffered culture media and the addition of saccharose enhance the production of many but not all scFvs [32]. We observed that antibody::pIII fusion proteins and antibody phage sometimes show differences in antigen binding in comparison to soluble antibody fragments, because some antibodies can bind the corresponding antigen only as pIII fusion [33, 34]. Therefore, we recommend performing the screening procedure only by using soluble antibody fragment, to avoid false-positive binders. 15. The background (noise) signals should be about O.D.450/ 620 ~ 0.02 after 30 min TMB incubation time.

Acknowledgments This protocol is an updated and revised version of [16, 28]. References 1. Parmley SF, Smith GP (1988) Antibodyselectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305 – 318 2. Breitling F, Du¨bel S, Seehaus T, Klewinghaus I, Little M (1991) A surface expression vector for antibody screening. Gene 104:147 –153 3. Hawlisch H, Mu¨ller M, Frank R, Bautsch W, Klos A, Ko¨hl J (2001) Site-specific anti-C3a receptor single-chain antibodies selected by differential panning on cellulose sheets. Anal Biochem 293:142 –145 4. Moghaddam A, Borgen T, Stacy J, Kausmally L, Simonsen B, Marvik OJ, Brekke OH, Braunagel M (2003) Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J Immunol Methods 280:139 –155

5. Hust M, Maiss E, Jacobsen H-J, Reinard T (2002) The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease. J Virol Methods 106: 225 –233 6. Schu¨tte M, Thullier P, Pelat T, Wezler X, Rosenstock P, Hinz D, Kirsch MI, Hasenberg M, Frank R, Schirrmann T, Gunzer M, Hust M, Du¨bel S (2009) Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus. PLoS ONE 4:e6625 7. Keller T, Kalt R, Raab I, Schachner H, Mayrhofer C, Kerjaschki D, Hantusch B (2015) Selection of scFv antibody fragments binding to human blood versus lymphatic endothelial surface antigens by direct cell phage display. PLoS ONE 10:e0127169

Antibody Selection via Phage Display in Microtiter Plates 8. Rezaei J, RajabiBazl M, Ebrahimizadeh W, Dehbidi GR, Hosseini H (2016) Selection of single chain antibody fragments for targeting prostate specific membrane antigen: a comparison between cell-based and antigen-based approach. Protein Pept Lett 23:336 –342 9. Frenzel A, Ku¨gler J, Wilke S, Schirrmann T, Hust M (2014) Construction of human antibody gene libraries and selection of antibodies by phage display. Methods Mol Biol 1060: 215 –243 10. Ayriss J, Woods T, Bradbury A, Pavlik P (2007) High-throughput screening of single-chain antibodies using multiplexed flow cytometry. J Proteome Res 6:1072 –1082 11. Hoet RM, Cohen EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem L, Frans N, Daukandt M, Pieters H, van Hegelsom R, Neer NC, Nastri HG, Rondon IJ, Leeds JA, Hufton SE, Huang L, Kashin I, Devlin M, Kuang G, Steukers M, Viswanathan M, Nixon AE, Sexton DJ, Hoogenboom HR, Ladner RC (2005) Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat Biotechnol 23:344 –348 12. Hust M, Meyer T, Voedisch B, Ru¨lker T, Thie H, El-Ghezal A, Kirsch MI, Schu¨tte M, Helmsing S, Meier D, Schirrmann T, Du¨bel S (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152:159 –170 13. J€ager V, Bu¨ssow K, Wagner A, Weber S, Hust M, Frenzel A, Schirrmann T (2013) High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 13:52 14. Frenzel A, Schirrmann T, Hust M (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8: 1177 –1194 15. Kuhn P, Fu¨hner V, Unkauf T, Moreira GMSG, Frenzel A, Miethe S, Hust M (2016) Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display. Proteomics Clin Appl 10:922 – 948 16. Russo G, Meier D, Helmsing S, Wenzel E, Oberle F, Frenzel A, Hust M (2018) Parallelized antibody selection in microtiter plates. Methods Mol Biol 1701:273 –284 17. Trott M, Weiβ S, Antoni S, Koch J, von Briesen H, Hust M, Dietrich U (2014) Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody. PLoS ONE 9: e97478

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18. Chan SW, Bye JM, Jackson P, Allain JP (1996) Human recombinant antibodies specific for hepatitis C virus core and envelope E2 peptides from an immune phage display library. J Gen Virol 77(Pt 10):2531–2539 19. Bertoglio F, Fu¨hner V, Ruschig M et al (2021) A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2RBD interface and is tolerant to most known RBD mutations. Cell Rep 36(4):109433 20. Bertoglio F, Meier D, Langreder N et al (2021) SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface. Nat Commun 12:1577 21. Ku¨gler J, Wilke S, Meier D, Tomszak F, Frenzel A, Schirrmann T, Du¨bel S, Garritsen H, Hock B, Toleikis L, Schu¨tte M, Hust M (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol 15:10 22. Schofield DJ, Pope AR, Clementel V, Buckell J, Chapple SD, Clarke KF, Conquer JS, Crofts AM, Crowther SRE, Dyson MR, Flack G, Griffin GJ, Hooks Y, Howat WJ, Kolb-KokocinskiA, Kunze S, Martin CD, Maslen GL, Mitchell JN, O’Sullivan M, Perera RL, Roake W, Shadbolt SP, Vincent KJ, Warford A, Wilson WE, Xie J, Young JL, McCafferty J (2007) Application of phage display to high throughput antibody generation and characterization. Genome Biol 8:R254 23. Glanville J, Zhai W, Berka J, Telman D, Huerta G, Mehta GR, Ni I, Mei L, Sundar PD, Day GMR, Cox D, Rajpal A, Pons J (2009) Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire. Proc Natl Acad Sci USA 106:20216 – 20221 24. de Wildt RM, Mundy CR, Gorick BD, Tomlinson IM (2000) Antibody arrays for highthroughput screening of antibody-antigen interactions. Nat Biotechnol 18:989 –994 25. Rothe C, Urlinger S, Lo¨hning C et al (2008) The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J Mol Biol 376(4):1182–1200. Epub 2007 Dec 15 26. Prassler J, Thiel S, Pracht C et al (2011) HuCAL PLATINUM, a synthetic Fab library optimized for sequence diversity and superior performance in mammalian expression systems. J Mol Biol 413(1):261–278. Epub 2011 Aug 12

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27. Tiller T, Schuster I, Deppe D et al (2013) A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. MAbs 5(3):445–470. Epub 2013 Apr 9 28. Schirrmann T, Hust M (2010) Construction of human antibody gene libraries and selection of antibodies by phage display. Methods Mol Biol 651:177 –209 29. Kirsch M, Hu¨lseweh B, Nacke C, Ru¨lker T, Schirrmann T, Marschall H-J, Hust M, Du¨bel S (2008) Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV). BMC Biotechnol 8:66 30. Goletz S, Christensen PA, Kristensen P, Blohm D, Tomlinson I, Winter G, Karsten U (2002) Selection of large diversities of antiidiotypic antibody fragments by phage display. J Mol Biol 315:1087 –1097

31. Finnern R, Pedrollo E, Fisch I, Wieslander J, Marks JD, Lockwood CM, Ouwehand WH (1997) Human autoimmune anti-proteinase 3 scFv from a phage display library. Clin Exp Immunol 107:269 –281 32. Hust M, Steinwand M, Al-Halabi L, Helmsing S, Schirrmann T, Du¨bel S (2009) Improved microtitre plate production of single chain Fv fragments in Escherichia coli. New Biotechnol 25:424 –428 33. Goffinet M, Chinestra P, Lajoie-Mazenc I, Medale-Giamarchi C, Favre G, Faye J-C (2008) Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection. BMC Biotechnol 8:34 34. Lillo AM, Ayriss JE, Shou Y, Graves SW, Bradbury ARM (2011) Development of phagebased single chain Fv antibody reagents for detection of Yersinia pestis. PLoS ONE 6: e27756

Chapter 13 Antibody Selection in Solution Using Magnetic Beads Philip Alexander Heine, Maximilian Ruschig, Nora Langreder, Esther Veronika Wenzel, Maren Schubert, Federico Bertoglio, and Michael Hust Abstract Antibody phage display is a valuable in vitro technology to generate recombinant, sequence-defined antibodies for research, diagnostics, and therapy. Up to now (autumn 2022), 14 FDA/EMA-approved therapeutic antibodies were developed using phage display, including the world best-selling antibody adalimumab. Additionally, recombinant, sequence-defined antibodies have significant advantages over their polyclonal counterparts. For a successful in vitro antibody generation by phage display, a suitable panning strategy is highly important. We present in this book chapter the panning in solution and its advantages over panning with immobilized antigens and give detailed protocols for the panning and screening procedure. Key words Antibody phage display, Panning, Antibody selection, Panning in solution, Streptavidin, Biotinylated antigens

1

Introduction Antibody phage display is an important technology to select recombinant antibodies. This in vitro method overcomes restrictions of animal-based selection methods in respect of toxicity and immunogenicity of certain antigens [1]. Phage display is based on the work of G.P. Smith, who received together with Sir G.P. Winter the half of the Nobel Prize 2018 in Chemistry for this technology [2–4]. For antibody phage display, F. Breitling and S. Du¨bel invented the most common phagemid system, which links the genotype encoded on the phagemid with the phenotype displayed on the phage surface [5]. Most commonly, the single-chain Fragment variable (scFv) is displayed on the minor coat protein (pIII) of the phage, but there are also other possibilities, like the Fragment antigen binding (Fab) [6, 7]. The selection procedure of antibody fragments from antibody gene libraries is called “panning”

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_13, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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according to the gold washer procedure [2]. In this panning process, the antigen can be immobilized on different surfaces like column matrixes [5], nitrocellulose [8], magnetic beads [9], or polystyrene [10]. Beyond that there is a panning strategy to select antibodies in solution [11]. This approach has the advantage of a higher reaction surface during the panning and an antibodyantigen interaction without sterical hindrance due to immobilization. Furthermore, a selection for antibodies against cell surface markers, e.g., cancer targets, can be performed directly on cells [12, 13]. For panning in solution, the antigen is incubated with the antibody library, e.g., HAL9/10 [14], in solution, and afterward, a “pull-down” of the antigen using magnetic beads is performed. For the pull-down a high affine tag on the antigen like biotin is needed to bind all antigens to the magnetic beads coated with streptavidin [11]. To avoid selection of streptavidin binders, a preincubation of the library on the beads is essential. After the preincubation, panning, and pull-down processes, a stringent washing is performed to remove not- or weak-binding antibodies. The stringency of the washing is increased by each panning round to select the most affine binders. All remaining binders can be eluted enzymatically, because of a trypsin cleavage site between the pIII and the scFv fragment. Afterward, E. coli bacteria will be infected with the eluted phage first and subsequently with the helper phage for phage amplification for the next panning round. When using E. coli TG1 strains, one panning round can be performed per day. Usually, three to four panning rounds are necessary to select antigen-specific antibody fragments with a high affinity. The number of antigen-specific antibody phage clones increases during the panning rounds, whereas the library diversity decreases. Therefore, more than three panning rounds are only recommended, if there are no or less antigen-specific antibody fragments after third panning round to avoid losing binders. For screening, the infected E. coli are plated out. Thereby, single clones can be picked, resulting in a monoclonal assay. These clones produce the soluble scFv they encode on the plasmid (link of pheno- and genotype) in a well of a multi-titer plate. Afterward, the binding scFv can be identified in ELISA [15], immunoblot [10], flow cytometer [16], or functional assays [17, 18]. The gene fragment of binding antibody fragments can be subcloned in any other antibody format, like scFv-Fc or IgG [6, 15, 19, 20]. The selection process is shown in Fig. 1. Antibody selection with phage display can be performed with naı¨ve libraries [14, 18, 23] or immune libraries [24–26]. Naı¨ve libraries contain a large repertory of different antibody clones, whereas immune libraries [14] derived from patients [24] or immunized animals [27–30] contain in vivo affinity maturated antibody clones. These libraries are valuable sources for antibodies against all kinds of targets, like proteins, sugars, or DNA.

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Fig. 1 Schema of antibody (scFv) phage display selection and screening. (Modified figure from former publications [21, 22])

The following protocols describe the panning and screening processes to select monoclonal antibodies in solution. The antibody selection can be performed in 3 days, and the screening will take additional 3 days afterward.

2 Materials 2.1 Preparation of the Magnetic Beads

1. Protein LoBind Tubes (Eppendorf, Hamburg, Germany). 2. Magnetic streptavidin beads Dynabeads™ M-280 Streptavidin (Thermo Fisher Scientific, Waltham, USA). 3. Magnetic Rack 16-Tube (Bio-Rad, Hercules, USA).

SureBeads™

Magnetic

Rack

4. Phosphate-buffered saline (PBS): 140 mM NaCl + 2.7 mM KCl + 1.8 mM KH2PO4 + 10 mM Na2HPO4 • 2 H2O. 5. BioLock – biotin blocking solution (IBA GmbH, Go¨ttingen, Germany) (optional). 2.2

Panning

1. Antibody phage library. 2. BSA-PBST: 2% BSA in PBS with 0.05% Tween 20, prepare fresh. 3. Panningblock: 1% milk powder and 1% BSA in PBS with 0.05% Tween 20, prepare fresh.

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4. M-PBST: 2% milk powder in PBS with 0.05% Tween 20, prepare fresh. 5. 96-well polystyrene Germany).

MTP

(Sarstedt

AG,

Nu¨mbrecht,

6. Overhead shaker Grant Bio PTR-30 (Grant Instruments, Cambridge, GB). 7. 10 μg/mL trypsin in PBS. 8. E. coli TG1 (supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rK-mK-) [F′ traD36 proAB lacIqZΔM15]). 9. Filter tips Biosphere® (Sarstedt, Nu¨mbrecht, Germany). 10. M13K07 helper phage (Thermo Fisher Scientific, Waltham, USA). 11. Round bottom polypropylene (PP) DeepWell 96 MTPs (Greiner, Frickenhausen, Germany). 12. Thermoshaker VorTemp 56 (Labnet, Edison, USA). 13. Eppendorf 5810R, Rotor A-4-81 with MTP adapter. 14. 2xTY media pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl. 15. 2xTY-T: 2xTY, containing 50 μg/mL tetracycline. 16. 2xTY-GA: 2xTY, 100 mM glucose, 100 μg/mL ampicillin. 17. 2xTY-AK: 2xTY, containing 100 μg/mL ampicillin, 50 μg/ mL kanamycin. 18. Glycerol (80%). 2.3

Phage Titration

1. E. coli XL1-Blue MRF’ (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]). 2. 2xTY-GA agar plates (2xTY-GA + 1.5% (w/v) agar-agar).

2.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. 96 well U-bottom polypropylene (PP) microtiter plates (Greiner Bio-One, Frickenhausen, Germany). 2. AeraSeal breathable sealing film (Excel Scientific, Victorville, USA). 3. Potassium phosphate buffer: 2.31% (w/v) KH2PO4 + 12.54% (w/v) (0.72 M) K2HPO4.

(0.17

M)

4. Buffered 2xTY pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl, 10% (v/v) potassium phosphate buffer. 5. Buffered 2xTY-SAI: buffered 2xTY containing 50 mM saccharose +100 μg/mL ampicillin +50 μM isopropyl-beta-D-thiogalactopyranoside (IPTG).

Antibody Selection in Solution Using Magnetic Beads

2.5 ELISA of Soluble Monoclonal Antibody Fragments

1. 96-well polystyrene Germany).

MTP

(Sarstedt

AG,

265

Nu¨mbrecht,

2. M-PBST: 2% milk powder in PBS with 0.05% Tween 20, prepare fresh. 3. Mouse α-His-tag monoclonal antibody (α-Penta His, Qiagen, Hilden, Germany). 4. Mouse α-myc-tag monoclonal antibody (9E10) (Sigma, Mu¨nchen, Germany). – Hyper -Myc-M (Abcalis GmbH, Braunschweig, Germany) (optional). 5. Mouse α-pIII monoclonal antibody (PSKAN3) (Mobitec, Go¨ttingen, Germany). 6. Goat α-Mouse IgG polyclonal (Fc specific) HRP conjugated (A0168) (Sigma, Mu¨nchen, Germany). 7. TMB solution: mix 20 parts of TMB-A with 1 part of TMB-B directly prior use TMB-A: 50 mM citric acid, 30 mM potassium citrate, pH 4.1 TMB-B: 90% (v/v) ethanol, 10% (v/v) acetone, 10 mM tetramethylbenzidine, 80 mM H2O2. 8. Stop Solution: 1 N H2SO4. 9. Oligonucleotide primers MHLacZ-Pro_f (5′ GGCTCGTATG TTGTGTGG 3′) and MHgIII:r (5′ CTAAAGTTTTGTCG TCTTTCC 3′).

3

Methods The time schedule for the complete procedure from antibody selection to identification on monoclonal antibodies is given in Table 1.

3.1 Preparation of the Magnetic Beads

1. Calculate needed amount (see Note 1). 2. Transfer the desired volume of magnetic beads in a Protein LoBind Tube. 3. Add an equal volume of PBS (or at least 1 mL). 4. Mix the tube (vortex for 5 s, or keep on an overhead shaker for at least 5 min). 5. Place the tube on a magnetic rack for 1 min and discard the supernatant. 6. Resuspend in same volume PBS as the initial volume beads taken from the vial.

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Table 1 Time schedule for antibody selection (panning) and screening of monoclonal antibodies Day Procedure steps

Preparation steps

0

–

Overnight culture of E. coli TG1

1

First panning round Infection of E. coli TG1 with eluted phage Infection with helper phage Antibody phage production overnight

Overnight culture of E. coli TG1

2

Second panning round Infection of E. coli TG1 with eluted phage Infection with helper phage Antibody phage production overnight

Overnight culture of E. coli XL1-blue MRF’

3

Third panning round – Infection of E. coli XL1-blue MRF’ with eluted phage Titration on agar plates

4

Picking clones for screening Culture overnight

–

5

Production of soluble scFv overnight

Coating of MTP wells for screening ELISA

6

Screening ELISA

–

3.2

Panning

1. For library preincubation dilute 1011–1012 antibody phage (you should use ~100× more phage particles compared to the library size) from the library in 150 μL BSA-PBST. 2. Incubate the library in a MTP well coated and blocked with 330 μL panning block for 1 h at RT (optional). 3. Transfer the library in a 1.5 mL LoBind tube and add 450 μL BSA-PBST. Add the calculated amount of streptavidin beads from Subheading 3.1, step 6 (10× more binding capacity than needed, see Note 1) and incubate 60 min at RT with overhead shaking. This step removes unspecific binders which can be present in the antibody gene libraries due to incorrect folding of individual antibodies. Preincubation with uncoupled beads further allows getting rid of unwanted streptavidin specific binders. 4. Place the tube on a magnetic rack, wait 1 min to let the beads attach on the tube wall. 5. Carry over the preincubated antibody phage library to a new protein LoBind tube and add 100–500 ng biotinylated antigen. Incubate at RT for 2 h with overhead shaking for binding of the antibody phage (see Note 2). 6. Add the calculated amount of streptavidin beads and incubate 30 min with overhead shaking at RT to pull down the phageantibody::biotinylated antigen complex with the beads.

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7. Place the tube on a magnetic rack, wait 5 min. 8. Remove the unspecifically bound antibody phage by stringent washing. Therefore, wash the tubes 10× with PBST in the first panning round. In the following panning rounds, increase the number of washing steps (20× in the second panning round, 30× in the third panning round, etc.) (see Note 3). 9. Elute bound antibody phage with 150 μL trypsin solution for 30 min at 37 °C (see Note 4). 10. Place the tube on a magnetic rack, wait for 1 min, and collect supernatant containing eluted phages. 11. After the third panning round, use 10 μL of the eluted phage for titration (see titering Subheading 3.3). 12. Inoculate 50 mL 2xTY with an overnight culture of E. coli TG1 (see Note 5) in 100 mL Erlenmeyer flasks and incubate at 250 rpm and 37 °C till exponential growth phase is reached O.D.600 0.4–0.5 (see Note 6). 13. Fill 150 μL exponentially growing E. coli TG1 in a polypropylene (PP) DeepWell MTP well and mix with 150 μL of the eluted phage. Incubate the bacteria for 30 min at 37 °C without shaking and 30 min at 37 °C and 650 rpm (see Note 7). 14. Add 1150 μL of 2xTY-GA (see Note 8) and incubate for 1 h at 37 °C and 650 rpm. 15. Infect the bacteria with 2 × 1011 phage particles/mL (=1 × 1010 phage particles, MOI 1:20 ) M13K07 helper phage (use filter tips to avoid potential cross-contamination). Incubate for 30 min at 37 °C without shaking, followed by 30 min at 37 °C at 650 rpm. 16. Centrifuge the MTP plate at 3220 × g (e.g., use Eppendorf 5810R, Rotor A-4-81 with MTP carriers). Remove the complete supernatant with a pipette. Do not destroy the pellet (see Note 9). 17. Add 950 μL 2xTY-AK and incubate overnight at 30 °C and 650 rpm to produce new antibody phage. 18. Centrifuge the MTP plate at 3220 × g. Transfer the supernatant (~1 × 1012 scFv-phage/mL) into a new cryovial. The produced antibody phages can directly be used for the next panning round. 3.3

Phage Titration

1. Inoculate 30 mL 2xTY-T in a 100 mL Erlenmeyer flask with E. coli XL1-Blue MRF’ (see Note 10) and grow overnight at 37 °C and 250 rpm. 2. Inoculate 50 mL 2xTY-T with 500 μL overnight culture and grow at 250 rpm at 37 °C up to O.D.600 ~ 0.5 (see Note 6).

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3. Make serial dilutions of the phage suspension in PBS (use filter tips). The number of eluted phage depends on several parameters (e.g., antigen, library, panning round, washing stringency etc.). In case of a successful enrichment, the titer of eluted phage usually is 103–105 phages per well after the first panning round and increases two to three orders in magnitude per additional panning round (see Note 11). The phage preparation after reamplification of the eluted phage has a titer of about 1012–1013 phage/mL. 4. Infect 50 μL bacteria with 10 μL phage dilution (use filter tips) and incubate 30 min at 37 °C. 5. You can perform titrations in two different ways: (A) Plate the 60 μL infected bacteria on 2xTY-GA agar plates (9 cm Petri dishes). (B) Pipet 10 μL (in triplicate) on 2xTY-GA agar plates. Here, about 20 titering spots can be placed on one 9 cm Petri dish. Dry drops on work bench. 6. Incubate the plates overnight at 37 °C. 7. Count the colonies and calculate the cfu or cfu/mL titer according to the dilution. 3.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. Fill each well of a 96-well U-bottom PP MTP with 150 μL 2xTY-GA. 2. Pick 92 clones with sterile tips from the third panning round and inoculate each well (see Note 12). Seal the plate with a breathable sealing film. 3. Incubate overnight in a microtiter plate shaker at 37 °C and 850 rpm. 4. (A) Fill a new 96-well polypropylene microtiter plate with 180 μL 2xTY-GA and add 10 μL of the overnight cultures. Incubate for 2 h at 37 °C and 850 rpm. (B) Add 30 μL glycerol solution to the remaining 140 μL overnight cultures. Mix by pipetting and store this masterplate at -80 °C. 5. Pellet the bacteria in the microtiter plates by centrifugation for 10 min at 3200 × g and 4 °C. Remove 190 μL glucosecontaining media by carefully pipetting (do not disturb the pellet) (see Note 9). 6. Add 180 μL buffered 2xTY-SAI and incubate overnight at 30 ° C and 850 rpm (see Notes 13 and 14). 7. Pellet the bacteria by centrifugation for 10 min at 3200 × g in the microtiter plates. Transfer the antibody fragment containing supernatant to a new polypropylene microtiter plate, and use it directly or store at 4 °C but not longer than 3 days.

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3.5 ELISA of Soluble Monoclonal Antibody Fragments

269

1. Immobilization of Antigens. To analyze the antigen specificity of the monoclonal soluble antibody fragments, coat 100–200 ng antigen per well of an MTP plate overnight at 4 °C. As control, coat 100–200 ng BSA or streptavidin per well (see Note 15). 2. Wash the coated microtiter plate wells thrice with PBST (washing procedure; see Note 16). 3. Block the antigen coated wells with M-PBST for 1 h at RT. The wells must be completely filled. Completely empty the wells after blocking. 4. Fill 50 μL M-PBST in each well and add 50 μL of antibody solution (coming out of Subheading 3.4). Incubate for 1 h at RT (or overnight at 4 °C). 5. Wash the microtiter plate wells thrice with PBST (see Note 16). 6. Incubate 100 μL α-myc tag antibody solution for 1 h at RT (appropriate dilution in M-PBST). 7. Wash the microtiter plate wells thrice with PBST (see Note 16). 8. Incubate 100 μL goat α-mouse HRP conjugate secondary antibody ( 1:10 ,000 in M-PBST) 1 h at RT. 9. Wash the microtiter plate wells thrice with PBST (see Note 16). 10. Shortly before use, mix 20 parts of TMB substrate solution A and 1 part TMB substrate solution B. Add 100 μL of this TMB solution into each well and incubate for 1–15 min at RT. 11. Stop the color reaction by adding 100 μL 1 N sulfuric acid. The color turns from blue to yellow. 12. Measure the extinction at 450 nm using an ELISA reader (see Note 17). 13. Identify positive candidates with a signal (on antigen) five to ten times over noise (on control protein, e.g., streptavidin or BSA) (see Note 18). 14. Sequence the DNA of the selected scFv with the oligonucleotide primers MHLacZ-Pro_f and MHgIII_r (if HAL9/HAL10 Library is used). We suggest analyzing the antibody sequences using VBASE2 (www.vbase2.org) (Tool: Fab/scFab/scAb/ scFv Analysis).

4

Notes 1. Calculate the amount of bead per biotinylated antigen accordingly to the manufacturer protocol, and then use ten times the calculated amount. Double this amount of beads so to have one half for library preincubation and the other half for the panning selection process.

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2. Instead of using a LoBind tube, a normal 1.5 mL Eppendorf tube can be used when preincubated 1 h with BSA-PBST and overhead shake. 3. The washing is performed manually. Vortex tube for 5 s, place the tube on a magnetic rack, wait for 1 min, discard supernatant, and finally add fresh PBST to repeat the washing step. 4. Phagemids like pHAL14 [19, 31] or pHAL30 [14] have coding sequences for a trypsin-specific cleavage site between the antibody fragment gene and the gIII. Trypsin also cleaves within antibody fragments but does not degrade the phage particles including the pIII that mediates the binding of the phage to the F pili of E. coli required for the infection. We observed that proteolytic cleavage of the antibody fragments from the antibody::pIII fusion by trypsin increases not only the elution but also enhances the infection rate of eluted phage particles, especially when using Hyperphage as helper phage. 5. E. coli TG1 is growing much faster compared to XL1-Blue MRF’. This strain allows to perform one panning round per day. 6. If the bacteria have reached O.D.600 ~ 0.5 before they are needed, store the culture immediately on ice to maintain the F pili on the E. coli cells for up to 1 h. M13K07 helper phage (kan+), or other scFv-phage (amp+) can be used as positive control to check the infectibility of the E. coli cells. 7. After 1 h, a concentration of OD600 0.4 to 0.5 is reached, corresponding to ~4 × 108 bacteria/mL. 8. Higher concentration of glucose is necessary to efficiently repress the lac promoter controlling the antibody::pIII fusion gene on the phagemid. Low glucose concentrations lead to an inefficient repression of the lac promoter and background expression of the antibody::pIII fusion protein. Background antibody expression is a strong selection pressure frequently causing mutations in the phagemid, especially in the promoter region and the antibody::pIII fusion gene. Bacteria with this kind of mutations in the phagemids proliferate faster than bacteria with nonmutated phagemids. Therefore, 100 mM glucose must be included in every step of E. coli cultivation except during the phage production! 9. To not destroy the pellet, remove the supernatant carefully by having the pipette tip on the side of the well. An alternative is to manually shake out the supernatant (do it with a fast movement of your wrist). 10. Use E. coli XL1-Blue MRF’ for titering and production of soluble antibodies. The plasmid quality and yield in this strain are higher compared to TG1.

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11. When the antibody gene library was packaged using Hyperphage, the titer of the eluted phage after the second panning may not increase as strongly or even decreases slightly due to the change from oligovalent to monovalent display. 12. We recommend picking 92 clones when using a 96-well microtiter plate. Use the wells H3, H6, H9, and H12 for controls. H3 and H6 are negative controls, these wells will not be inoculated and just contain the bacteria medium. We inoculate the wells H9 and H12 with a clone containing a phagemid encoding a known antibody fragment. In ELISA, the wells H9 and H12 are coated with the antigen corresponding to the control antibody fragment in order to check scFv production and detection by ELISA. 13. The appropriate IPTG concentration for induction of antibody or antibody::pIII expression depends on the vector design. A concentration of 50 μM is well suited for vectors with a Lac promoter like pIT2 [32] , pHENIX [33] , pHAL14 [19], or pHAL30 [14]. 14. Buffered culture media and the addition of saccharose enhance the production of many but not all scFvs [34]. We observed that antibody::pIII fusion proteins and antibody phage sometimes show differences in antigen binding in comparison to soluble antibody fragments, because some antibodies can bind the corresponding antigen only as pIII fusion [35, 36]. Therefore, we recommend performing the screening procedure only by using soluble antibody fragments, to avoid false-positive binders. On the other hand, some scFv binding as antibody phage, but not also soluble scFv, binds as scFv-Fc after recloning. 15. While using biotinylated antigens for panningPanning in solution, we recommend to use streptavidinStreptavidin as control for the screening ELISA to identify possible biotin binders. 16. Microtiter plate wells washing should be performed with an ELISA washer (e.g., Tecan Columbus Plus). To remove antigen or blocking solutions, wash thrice with PBST (“standard washing protocol” for Tecan washer). If no ELISA washer is available, wash manually thrice with PBST. 17. A measurement at 450 nm and 620–650 nm as reference wavelength can improve the readout pattern in ELISA. 18. The background (noise) signals should be about O. D.450 ~ 0.02 after 5–30 min TMB incubation time.

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Acknowledgments This review contains updated and revised parts of former protocols [37]. References 1. Winter G, Milstein C (1991) Man-made antibodies. Nature 349:293 –299 2. Parmley SF, Smith GP (1988) Antibodyselectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305 – 318. https://doi.org/10.1016/0378-1119 (88)90495-7 3. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552 –554 ˜ a E (2019) The 2018 4. Barderas R, Benito-Pen Nobel Prize in Chemistry: phage display of peptides and antibodies. Anal Bioanal Chem 411:2475 –2479. https://doi.org/10.1007/ s00216-019-01714-4 5. Breitling F, Du¨bel S, Seehaus T, Klewinghaus I, Little M (1991) A surface expression vector for antibody screening. Gene 104:147 –153 6. Hoet RM, Cohen EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem L, Frans N, Daukandt M, Pieters H (2005) Generation of high-affinity human antibodies by combining donor-derived and synthetic complementaritydetermining-region diversity. Nat Biotechnol 23:344 –348 7. Omar N, Lim TS (2018) Construction of naive and immune human Fab phage-display library. In: Phage display. Springer, New York, USA, pp 25–44 8. Hawlisch H, Mu¨ller M, Frank R, Bautsch W, Klos A, Ko¨hl J (2001) Site-specific anti-C3a receptor single-chain antibodies selected by differential panning on cellulose sheets. Anal Biochem 293:142 –145 9. Moghaddam A, Borgen T, Stacy J, Kausmally L, Simonsen B, Marvik OJ, Brekke OH, Braunagel M (2003) Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J Immunol Methods 280:139 –155 10. Hust M, Maiss E, Jacobsen H-J, Reinard T (2002) The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease. J Virol Methods 106: 225 –233

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Antibody Selection in Solution Using Magnetic Beads Grasshoff M, Wenzel EV, Russo G, Kro¨ger A, Brunotte L, Ludwig S, Fu¨hner V, Kr€amer SD, ˇ icˇin-Sˇain L, Du¨bel S, Varani L, Roth G, C Schubert M, Hust M (2021) SARS-CoV2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface. Nat Commun 12:1577 . https://doi.org/10.1038/ s41467-021-21609-2 19. Hust M, Meyer T, Voedisch B, Ru¨lker T, Thie H, El-Ghezal A, Kirsch MI, Schu¨tte M, Helmsing S, Meier D (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152:159 –170 20. J€ager V, Bu¨ssow K, Wagner A, Weber S, Hust M, Frenzel A, Schirrmann T (2013) High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 13:1 –20 21. Kuhn P, Fu¨hner V, Unkauf T, Moreira GMSG, Frenzel A, Miethe S, Hust M (2016) Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display. Proteomics Clin Appl 10:922 – 9 4 8 . h t t p s : // d o i . o r g / 1 0 . 1 0 0 2 / p r c a . 201600002 22. Frenzel A, Schirrmann T, Hust M (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8: 1177 –1194. https://doi.org/10.1080/ 19420862.2016.1212149 23. Schofield DJ, Pope AR, Clementel V, Buckell J, Chapple SD, Clarke KF, Conquer JS, Crofts AM, Crowther SR, Dyson MR (2007) Application of phage display to high throughput antibody generation and characterization. Genome Biol 8:1 –18 24. Bertoglio F, Fu¨hner V, Ruschig M, Heine PA, Abasi L, Klu¨nemann T, Rand U, Meier D, Langreder N, Steinke S, Ballmann R, Schneider K-T, Roth KDR, Kuhn P, Riese P, Sch€ackermann D, Korn J, Koch A, Chaudhry MZ, Eschke K, Kim Y, Zock-Emmenthal S, Becker M, Scholz M, Moreira GMSG, Wenzel EV, Russo G, Garritsen HSP, Casu S, Gerstner A, Roth G, Adler J, Trimpert J, Hermann A, Schirrmann T, Du¨bel S, ˇ icˇin-Sˇain L, Frenzel A, Heuvel JV den, C Schubert M, Hust M (2021) A SARS-CoV2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to most known recently emerging RBD mutations. 2020.12.03.409318 25. Trott M, Weiß S, Antoni S, Koch J, Von Briesen H, Hust M, Dietrich U (2014) Functional characterization of two scFv-Fc

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Improved microtitre plate production of single chain Fv fragments in Escherichia coli. New Biotechnol 25:424 –428. https://doi.org/ 10.1016/j.nbt.2009.03.004 35. Goffinet M, Chinestra P, Lajoie-Mazenc I, Medale-Giamarchi C, Favre G, Faye J-C (2008) Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection. BMC Biotechnol 8:34 . https://doi. org/10.1186/1472-6750-8-34 36. Lillo AM, Ayriss JE, Shou Y, Graves SW, Bradbury ARM (2011) Development of phage-

based single chain Fv antibody reagents for detection of Yersinia pestis. PLoS One 6: e27756. https://doi.org/10.1371/journal. pone.0027756 37. Wenzel EV, Roth KDR, Russo G, Fu¨hner V, Helmsing S, Frenzel A, Hust M (2020) Antibody phage display: antibody selection in solution using biotinylated antigens. In: Zielonka S, Krah S (eds) Genotype phenotype coupling: methods and protocols. Springer, New York, pp 143–155

Chapter 14 Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning Theam Soon Lim, Angela Chiew Wen Ch’ng, Brenda Pei Chui Song, and Jing Yi Lai Abstract Phage display is a technique that allows the presentation of unique proteins on the surface of bacteriophages. The phage particles are usually screened via repetitive rounds of antigen-guided selection and phage amplification. The main advantage of this approach lies in the physical linkage between phenotype and genotype. This feature allows the isolation of single unique clones from a panning campaign consisting of a highly diverse population of clones. Due to the high-throughput nature of this technique, different approaches have been developed to assist phage display selections. One of which involves utilizing a streptavidin-coated solid-phase extraction (SPE) tip that is mounted to an electronically controlled motorized multichannel pipette. In this chapter, we will entail the procedures involved in the adaptation of a commercial SPE tip (MSIA™ streptavidin D.A.R.T’s®) as the solid phase. This protocol is an updated version of a previous protocol with some minor refinements. Key words Solid-phase extraction (SPE), Biopanning, Disposable Automation Research Tips (D.A.R. T’s®), Mass spectrometry immunoassay (MSIA™), Monoclonal antibodies, Phage display

1

Introduction There has been an increase in bacteriophage application for the evaluation of protein-protein interaction, antibody generation, and ligand identification since its inception by George P. Smith [1]. However, phage particles have also been implemented for the development of nanostructures like nanotubes, nanobatteries, and nanorods [2]. Phage display technology leverages on the ability of filamentous phage to display the resulting phenotype on its surface fused to the minor coat protein creating a physical link between the phenotype and genotype. Every phage particle is designed to house one unique antibody sequence for presentation, thus allowing the selection of target-specific antibody presenting phage particles [3, 4].

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_14, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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The antibody selection process via affinity enrichment or commonly known as biopanning requires the use of an antibody phage library [3]. The biopanning process takes advantage of the binding strength (affinity) of the antibody and target molecules to achieve clonal enrichment. During this process, the target-specific antibodies are then concentrated from repeating cycles of target binding of phage displaying antibody, washing steps, and rescue steps [3, 5, 6]. Generally, the biopanning process starts with the immobilization of the target antigen on a solid surface such as immunotubes or high protein binding microplates, or in some cases on streptavidincoated surfaces when using biotinylated antigens [3]. Apart from traditional direct antigen immobilization onto polystyrene surfaces, the use of nanoparticles like magnetic beads contributed to enhance the binding efficiency by increasing the surface area for binding [7, 8]. Solid-phase extraction (SPE) is a common sample preparation method utilized in mass spectrometry (MS)-based proteomics as a replacement for liquid-liquid extraction protocols to achieve sample isolation, cleanup, and concentration from a complex matrix [9, 10]. The concept of SPE allows the differences in affinity between the target antigen, and interferents to the bound receptor on the solid phase allow for sample separation. SPE allows the attachment of targets in liquid phase to active sites located on the solid phase permitting the adsorption of the analytes selectively onto the surface of the solid phase. Then, a wash step is performed to remove the unwanted matrix components from the solid phase. Finally, an elution step using appropriate solvents will allow the dissociation of the target from the solid phase in the resulting liquid fraction [10]. The solid-phase packing materials can be found in various types of single-use containers such as micropipette tips, magnetic beads, 96-well plates, microcolumn, and cartridges [11]. The use of SPE does not only allow for a highly consistent high-throughput approach but also includes other advantages such as low reagent consumption, ease of manipulation, high enrichment factor, high concentration rate due to a smaller size, high sensitivity due to a large binding surface area, absence of emulsion, and flexibility [9, 10]. By applying the principles of affinity separation by antibody and antigen interaction in immunoaffinity methods, we adopted the similar concept on the Mass Spectrometric Immunoassay (MSIA™) system. The MSIA™ system was originally developed for the purification and concentration of proteins for mass spectrophotometry (MS) analysis [12]. The Mass Spectrometric Immunoassay (MSIA™) Streptavidin Disposable Automation Research Tips (D.A.R.T’s®) (MSD) was designed as a miniaturized version of a conventional SPE to extract biotinylated proteins via affinity selection utilizing the packed streptavidin in the form of a pipette tip [13]. The immobilization process of the biotinylated target protein

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Fig. 1 (a) Schematic representation of MSIA™ streptavidin D.A.R.T’s. (b) Immobilization of biotinylated antigen-containing buffer to streptavidin within the porous matrix of D.A.R.T’s® through repeated aspirating and dispensing

to the conjugated streptavidin in the pipette tip via streptavidinbiotin interaction allows for a convenient and rapid target immobilization process [14]. The process involves a set of repeated aspirating and dispensing of the sample in liquid through the MSD until saturation of the porous matrix of the tip with the biotinylated antigen is achieved. This is a rapid and selective process taking into account the affinity of the streptavidin-biotin interaction [15]. The MSD panning approach starts with the immobilization of biotinylated antigens onto the streptavidin surface to allow presentation of the target antigen on the solid phase [16, 17]. The main mechanical action manipulating the liquid flowing through the MSD tips is the rate of aspiration and dispensing. The process involved for the capture of the biotinylated antigen on the streptavidin packed MSD tips is highlighted in Fig. 1. Considering the number of aspiration and dispensing cycles required to achieve saturation, it is advisable to have an automated pipette to generate consistent aspiration and dispensing rates to ensure consistency. Antigen immobilization is done by saturating the streptavidin on the porous matrix with biotinylated antigen followed by several washing and blocking steps. This step is critical to remove unbound proteins and eliminate nonspecific binding [3, 18]. To achieve this, the use of an electronically controlled motorized multichannel pipette with predefined parameters such as number of cycles, setting of speed, and cycle volume for aspirating and dispensing is critical. The flexibility to adjust these parameters is also important to optimize the biopanning process as optimization of the conditions may be required for different target molecules. This is ensued

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Fig. 2 Process of MSIA™ Streptavidin D.A.R.T’s® antibody biopanning approach

by the introduction of the antibody phage library to the antigencoupled tips. The antibody library phage preparation is then circulated through the target immobilized MSD tips by repeated aspiration and dispensing. The constant flow of the liquid through the MSD tips will help to promote affinity binding of specific antibodies to the target antigen. The tips are then washed to detach any remaining unbound, nonspecific, or low-affinity antibodies. Finally, the target bound antibody phage particles are eluted by using an appropriate elution buffer and rescued by re-amplification with a suitable E.coli strain for use in subsequent rounds of biopanning like any ordinary panning protocol [3, 16]. The entire MSD biopanning process is shown in Fig. 2. To discover and obtain target-specific antibodies, incorporation of the MSD system in the biopanning process has the main advantage of being an economical alternative for automated panning protocol. This is due to the possibility of adapting any liquid handling device already at your disposal or the purchase of an electronic pipette. Although the proof of concept was carried out using a 12-channel electronic multichannel pipette, we envisage that the approach can also be adapted for a higher throughput if

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the MSDs are mounted on 96 tip liquid handling systems. Taken together, the adaptation of MSD coupled with an electronic programmable pipette or liquid handling equipment would give rise for an alternative semi-automated biopanning system for antibody development programs.

2

Materials

2.1 Preparation of Phage Display Antibody Library 2.1.1 Phage Display Antibody Libraries and E. Coli Host Strains

2.1.2 Preparation of Antibody Library

1. An in-house naı¨ve scFv antibody phagemid library [19] was used (see Note 1). The HAYLY (Human AntibodY LibrarY) ver 1.0 was cloned in the pLABEL phagemid which employs pIII minor coat protein for antibody display. The library size is estimated at 2 × 109 CFU/mL. 2. E. coli XL1Blue: tetracycline resistant; endonuclease (endA) deficient which greatly improves the quality of miniprep DNA; recombination (recA) deficient which improves insert stability; hsdR mutation prevents the cleavage of cloned DNA by the EcoK endonuclease system; lacIqZΔM15 gene on the F´ episome which allows for blue-white color screening. 1. Sterile conical centrifuge tubes: 15 and 50 mL. 2. 1.5 mL sterile microcentrifuge tubes. 3. Erlenmeyer flasks (1 L and 2 L). 4. Sterile Petri dishes 94 mm × 16 mm. 5. 2 YT: 16 g tryptone, 10 g yeast extract, and 5 g NaCl in 1 L dH2O, autoclave and store at room temperature. 6. 50 mg/mL ampicillin stock solution: 0.5 g ampicillin in 10 mL of 50% (v/v) ethanol, filter sterilize, and store at -20 °C. 7. 30 mg/mL kanamycin stock solution: 0.3 g kanamycin in 10 mL of dH2O, filter sterilize, aliquot, and store at -20 °C. 8. 40% glucose stock solution: 40 g glucose in 100 mL of dH2O, autoclave, and store at room temperature. 9. 80% glycerol: 80 mL glycerol in 20 mL of dH2O, autoclave, and store at room temperature. 10. 2 YT agar: 31 g premixed 2 YT and 15 g agar in 1 L dH2O, autoclave, cool to 55 °C, add 2% glucose and appropriate antibiotics. 11. 20% polyethylene glycol 6000/2.5 M NaCl (PEG/NaCl) solution: Prepare 200 g PEG and 146 g NaCl in 1 L of dH2O, autoclave, and store at room temperature. 12. PBS buffer: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 1 L dH2O, adjust to pH 7.4, autoclave, and store at room temperature.

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13. M13KO7 helper phage (NEB). 14. 2 mL cryogenic vials. 2.2 Phage Display MSD Biopanning

1. Target antigen: biotinylated recombinant antigen (see Note 2) in PBS/bicarbonate buffer (0.1 M NaHCO3, pH 8.6 see Note 3). 2. TG1 E. coli cell: contains the lacIqZΔM15 gene on the F´ episome which allows blue-white screening for recombinant plasmids. 3. 0.1 M NaHCO3/bicarbonate buffer: 0.84 g NaHCO3 in 100 mL of dH2O, adjust to pH 8.6. 4. 0.5% PBS-T: 5 mL Tween 20 into 1 L PBS. 5. 3% PTM: 3 g skimmed milk in 100 mL 0.1% PBS-T. 6. 0.2 M glycine, pH 2.2: 1.5 g glycine in 100 mL dH2O, adjust to pH 2.2, autoclave, and store at room temperature. 7. 1 M Tris–HCl: 3.0275 g Tris–HCl in 25 mL dH2O, adjust to pH 9.1. 8. MSIA™ Streptavidin D.A.R.T’s® (Thermo Scientific) (see Note 4).

2.3

Phage ELISA

1. High protein binding 96-well microtiter plate and 96-well strip microtiter plate. 2. Anti-M13 horseradish peroxidase (HRP)-conjugated monoclonal antibody. 3. 2% BSA: 2 g BSA in 100 mL of 0.1% PBS-T. 4. ABTS developing solution: add one 10 mg tablet ABTS in 5 mL of 50 mM citric acid, 5 mL of 50 mM trisodium citrate, and 10 μL H2O2. Store in dark.

2.4

DNA Sequencing

1. Plasmid miniprep kit. 2. Primers: LMB3_Fw -5′ CAGGAAACAGCTATGAC 3′ and PIII_Rv -5′ GTTAGCGTAACGATCTAA 3′.

2.5 Soluble Antibody Fragment Detection

1. 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) stock solution: 2.38 g IPTG in 10 mL of dH2O, aliquot and store at -20 °C. 2. 1 × TES buffer: 30 mM Tris–HCl (pH 8), 1 mM EDTA, and 20% sucrose. 1.5 mL 1 M Tris–HCl (pH 8.0), 0.05 mL 1 M EDTA and 10 g sucrose into dH2O in a total volume of 50 mL. Store at 4 °C. 3.

1:5 dilution 1 × TES buffer: 10 mL of 1 × TES buffer into 40 mL dH2O. Store at 4 °C.

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4. PBS-T with 3% milk (3% PTM): 3 g skimmed milk in 100 mL of 0.1% PBS-T. 5. Horseradish peroxidase-anti-c-Myc antibody. 6. PBS buffer: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 1 L dH2O, adjust to pH 7.4, autoclave, and store at room temperature.

3

Methods

3.1 Preparation of Antibody Library Phage

In order to prepare sufficient starting material for the biopanning process to be conducted, the naı¨ve HAYLY antibody library was amplified from the library stock. Since the phagemid system is employed, an additional co-infection with M13KO7 helper phage is required during the packaging process of the antibody library phage. 1. Thaw the glycerol stock of the antibody library and start culturing in 500 mL of 2 YT containing 2% glucose and ampicillin (100 μg/mL) whereby the starting inoculation is at OD600 ~ 0.1. 2. Grow the culture at 37 °C with 200 rpm shaking until OD600 ~ 0.5. 3. Divide the culture equally into two flasks: one is for phage packaging, whereas the other is stored as the first-generation stock.

3.2 Library Phage Packaging

1. For phage packaging purpose, co-infect the culture with M13KO7 helper phage (1011 CFU/mL) by incubation at 37 °C static for 30 min (see Note 5). 2. Centrifuge the culture at 1726 × g for 30 min and discard the supernatant. 3. Reconstitute the pellet with 250 mL of 2 YT medium containing 0.1% glucose, ampicillin (100 μg/mL), and kanamycin (60 μg/mL) with gentle mixing (see Note 6). 4. Grow the bacteria in 2 YT medium o/n at 30 °C with 180 rpm agitation. This step is to amplify/package phagemid-bearing phage particles. 5. The next day, centrifuge the culture at 1726 × g for 30 min to collect phage-containing supernatant. 6. To the supernatant, add an additional 1/6 of the total supernatant volume with PEG/NaCl and chill on ice for 1 h. to precipitate the phage. 7. Centrifuge the mixture at 1726 × g for 30 min.

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8. Discard the supernatant and air-dry the white color phage pellet. 9. Resuspend the pellet with 1 mL of PBS buffer. 10. Centrifuge the mixture at maximum speed (21,130 × g) for 20 min. Additional centrifugation may be required to ensure total removal of bacterial culture in the supernatant. 11. Store the supernatant containing the library phage at 4 °C until ready for use. 12. Perform phage titration to estimate the amount of phage particles present. Prepare a series of 1:10 phage dilution by adding 10 μL of phage with 90 μL PBS. Then, add 200 μL of the TG1 culture (OD600 ~ 0.5) to the 100 μL final volume of phage dilution prepared earlier and incubate static at 37 °C for 30 min. Spot 10 μL of each dilution, TG1 and PBS (as negative controls) on 2 YT agar plates containing ampicillin (100 μg/ mL) and kanamycin (60 μg/mL) respectively. The phage particles with the desired antibody phagemid genome would survive on ampicillin agar plate after infection with TG1 pilusbearing bacteria, whereas phage with M13KO7 genome would only survive on kanamycin plates. Incubate the agar plates o/n at 37 °C and calculate the number of colonies on the ampicillin agar plate using the equation as shown below: Amount of phage ðCFU=mLÞ =

3.3 Preparation of First-Generation Stock

No of colonies × dilution factor × 100 ðin 1 mLÞ

1. For the first-generation library stock, leave the culture to grow o/n at 37 °C with 200 rpm agitation. 2. The next day, centrifuge the culture at 1726 × g for 30 min to collect the cell pellet that is later resuspended with fresh 5 mL of 2 YT containing 2% glucose and ampicillin (100 μg/mL). 3. Add 20% glycerol to the mixture, aliquot into cryogenic vials for storage at -80 °C.

4

Phage Display Biopanning This updated MSD antibody phage display biopanning is based on the earlier version of the protocol by Chin et al. (2016) [16]. Nonetheless, in order to choose the best biopanning approach that is suitable for your laboratory, there are several aspects that you may need to take into consideration. First is to select the biopanning approach based on equipment/materials available in your laboratory. In this context, we utilized the MSIA™ streptavidin D.A.R.

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T’s® solid-phase extraction tips and a Finnpipette™ Novus i Electronic 12-channel Pipette (Thermo Fisher Scientific). Alternatively, it is possible to use any other electronic pipette with programming capabilities, streptavidin-coated SPE tips, and an adjustable pipette stand. Secondly, the target antigen used has to be biotinylated. The biotinylated target antigen is critical in this particular situation as MSD allows for easy capture of biotinylated antigen to be used as the biopanning solid phase. The biopanning conditions proposed in this protocol may require some variations depending on the targets. If there is a high background formed during biopanning, a repetition of selection rounds is needed to produce suitable enrichment of target antibodies. Modifications to the flow rate or cycle number may be required to optimize the panning process for different targets. 4.1 MSIA™ Streptavidin D.a.R.T’s® Loading of Biotinylated Antigen

1. Mount the MSD to a multichannel electronic for loading of biotinylated target antigens to take place. 2. Load biotinylated recombinant antigen at 100 μg (see Note 7) in bicarbonate buffer to MSD by continuous aspiration and dispensing. Set the electronic pipette program for 999 cycles at a speed setting of 5 with a fixed volume of 150 μL. Continuous aspiration and dispensing at a moderate speed could help in binding the biotinylated targets to the streptavidin in the MSD. 3. Wash the MSD two times (20 cycles, speed setting 8, and volume 200 μL) with 0.5% PBS-T followed by one time (20 cycles, speed setting 8, and volume 200 μL) with PBS. The antigen captured MSD is now ready for use in biopanning.

4.2 MSIA™ Streptavidin D.a.R.T’s® Antibody Biopanning

1. Block the antigen-coupled tip with 3% PTM with continuous aspiration and dispensing at 500 cycles with a speed setting of 5 and volume of 200 μL. 2. At the same time, pre-incubate ~1012 phage particles (see Note 8) of the antibody library with 3% PTM to minimize background from the system. 3. Subsequently, wash the MSD two times (20 cycles, speed setting 8, and volume 200 μL) with 0.5% PBS-T followed by one time (20 cycles, speed setting 8, and volume 200 μL) with PBS. 4. Capture the antibody phage in PTM by performing repetitive pipetting with a fixed volume of 150 μL, 999 cycles repeat, and a speed setting of 5. 5. Rinse the MSD for five rounds with 0.5% PBS-T and another five rounds with PBS (see Note 9). Each wash cycle constitutes 20 cycles of aspirating and dispensing with a speed setting of 8 and volume of 200 μL.

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6. Meanwhile, inoculate 20 mL of 2YT with 200 μL of TG1 overnight culture at 37 °C with shaking (200 rpm) until OD600 ~ 0.5 is reached. 7. Elute the bound phages by using 100 μL of 0.2 M glycine, pH 2.2 (see Note 10) with 300 cycles of repetitive pipetting (speed setting 3) (see Note 11). 8. Immediately neutralize the eluted fraction with 1 M Tris–HCl, pH 9.1 to achieve pH 7. This step has to be done immediately to prevent the decrease of phage infectivity. 9. Infect the eluted phage particles with an exponentially growing 2–4 mL TG1 culture (OD600 ~ 0.5) from step 6 by incubating at 37 °C for 30 min and static for phage rescue. 10. At the same time, perform phage titration as described in Subheading 3.2, step 12 (see Note 12). 11. Centrifuge the infected culture at 9000 × g for 10 min. 12. Discard the supernatant and mix the cell pellet with 20 mL 2 YT medium containing 2% glucose and ampicillin (100 μg/ mL). 13. Grow the culture at 37 °C with 200 rpm agitation for approximately 3–4 h. (see Note 13). 14. Equally divide the culture into two where one is kept as glycerol stock as described earlier (Subheading 3.3) in 1 mL 2YT medium instead of 5 mL and the other half is co-infected with helper phage (~1010 CFU/mL) and incubated at 37 °C and static for 30 min. 15. Centrifuge the co-infected culture at 9000 × g for 10 min. 16. Reconstitute the pellet with the same volume of 2 YT medium containing 0.1% glucose, ampicillin (100 μg/mL), and kanamycin (60 μg/mL). Grow the culture o/n at 30 °C with 180 rpm agitation. 17. The next day, centrifuge the culture at 9000 × g for 30 min to collect phage-containing supernatant. 18. Perform phage precipitation (Subheading 3.2, steps 6–11) and titration (Subheading 3.2, step 12) as previously described. The final volume for the antibody phage in PBS is 300 μL. Repeat this process for two to five rounds in order to obtain target-enriched phages (see Note 14).

5

Phage ELISA To determine the enrichment pattern of each biopanning round of the selection process, polyclonal phage ELISA is performed. Following positive enrichment, screening of individual clones from the

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panning round exhibiting the highest enrichment will be conducted. The phage from the positive round will be rescued and plated out to isolate individual phage clones. Propagate the monoclonal antibody phage and screen with monoclonal ELISA. 5.1 Polyclonal Phage ELISA

1. For three rounds of biopanning, coat three wells of high protein binding microtiter plate with 100 μL of target antigen (10 μg) in bicarbonate buffer/PBS buffer o/n at 4 °C with 700 rpm agitation. Coat another three wells with 300 μL of 2% BSA concurrently to be used as background control. 2. The next day, wash the plate three times with 0.5% PBS-T. 3. Block the wells with 300 μL of 2% BSA for 1–2 h. with 700 rpm agitation to reduce nonspecific binding. 4. Add 100 μL of (109) enriched phage particles in the 2% BSA from the biopanning process to the wells coated with target as well as preblocked coated wells. Incubate the plate for 1–2 h. with 700 rpm agitation. 5. Wash the plate thrice with 0.5% PBS-T. 6. Add 150 μL of anti-M13 HRP ( 1:5000 ) in 2% BSA to the wells and incubate for 1–2 h. with 700 rpm agitation. 7. Wash the plate three times with 0.5% PBS-T. 8. Add 150 μL of the ABTS developing solution to detect bound phage particles. After 30 min incubation in the dark, the absorbance reading at 405 nm (OD405) is recorded with a microplate reader. The incubation is done in dark as ABTS solution is light-sensitive.

5.2 Monoclonal Phage Propagation

1. Plate out the phage particles infected with bacteria from the biopanning with the highest enrichment. Dilute the polyclonal phage in 1:10 serial dilution until 10-10 and further infect with 200 μL of TG1 culture (OD600 ~ 0.5) at 37 °C, static for 30 min. Then, 100 μL of the infected culture is plated on 2 YT agar plate containing ampicillin (100 μg/mL). The plates are incubated at 37 °C for o/n. 2. Pick a total of 93 single colonies and grow in 2 YT containing 2% glucose and ampicillin (100 μg/mL) at 37 °C, 900 rpm for o/n in a round bottom microtiter plate. On the plate, wells in position A2 were left empty as negative control, while position A1 was cultured with a known clone as positive control (see Note 15). 3. The next day, inoculate the 10 μL of the o/n culture in 200 μL of 2 YT containing 2% glucose and ampicillin (100 μg/mL) and further grown at 37 °C, 900 rpm, for 2.5 h. The o/n culture is added with glycerol to a final concentration of 20% and kept at -80 °C as stock.

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4. After the incubation, add 20 μL of M13KO7 helper phage (~109) for co-infection by static incubating at 37 °C for 30 min. 5. Centrifuge the culture at 563 × g for 10 min. 6. After discarding the supernatant, resuspend the cell pellet with 220 μL of 2 YT containing 0.1% glucose, ampicillin (100 μg/ mL), and kanamycin (60 μg/mL). 7. Incubate the culture at 30 °C with 900 rpm agitation for o/n. 8. The next day, centrifuge the culture at 563 × g for 10 min and phage-containing supernatant is collected as well as kept at 4 ° C until ready for use. 5.3 Monoclonal ELISA

6

Perform the monoclonal phage ELISA in a similar way as the polyclonal phage ELISA (Subheading 5.1) described earlier. A total of 50 μL sample monoclonal antibody phage is used to perform monoclonal ELISA. In addition, positive and negative controls are included in the ELISA to ensure the validity of the ELISA (see Note 15).

DNA Sequencing 1. Miniprep of clones that showed positive binding activities using a plasmid miniprep kit. 2. The purified plasmid-DNA is sent for sequencing with LMB3 forward and pIII reverse primers.

7

Generation of Soluble Antibody Fragments After verification of positive binding clones with high specificity toward the antigen of interest, the expression of the selected antibody fragment in soluble form is done. However, the expression of soluble antibody fragment using an amber suppressor E.coli strains will result in the attachment of the pIII minor coat protein to the antibody fragment. Alternatively, by infecting the target clones into non-suppressor strains of E.coli such as Top10 F′, the amber stop codon will be read, and the soluble antibody fragment will be expressed devoid of the pIII.

7.1 Expression and Extraction of Soluble Antibody

1. Pick single colonies from the target-specific monoclonal antibody bacteria colonies growing on the agar plate. Culture the colonies o/n at 37 °C in 5 mL 2 YT medium containing 2% glucose and 100 μg/mL ampicillin.

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2. The next day, inoculate the o/n culture in 100 mL 2 YT medium containing ampicillin (100 μg/mL) and 0.1% glucose at 1:100 ratio and further grow it at 37 °C to OD600nm = 0.6. 3. Induce the lac promoter from pLABEL phagemid with 1 mM IPTG, and further express the clones o/n at 25 °C with 160 rpm agitation for 16 h. 4. At the following day, centrifuge the culture at 1726 × g for 30 min to collect the cell pellet in 100 mL fraction. 5. Resuspend the cell pellet fraction of 100 mL expressed antibody in 1 mL of cold 1 × TES buffer. 6. Add 1.5 mL of 1:5 dilution cold 1 × TES buffer and mix gently. Incubate the mixture on ice for 1 h. The protein extraction method used is by hypotonic shock to release soluble antibodies especially in the periplasmic region of the bacteria. 7. Centrifuge at 9000 × g for 10 min and collect the antibody containing supernatant. Keep the soluble antibody at -20 °C until ready for use. 7.2

Soluble ELISA

1. Coat 10 μg/well of target antigen on the microtiter plate o/n at 4 °C in PBS buffer and 3% PTM as background control (see Note 15). 2. Wash the wells three times with 0.5% PBS-T. 3. Block the wells with 3% PTM for 1 h. with 700 rpm agitation to reduce nonspecific binding. 4. Wash the wells thrice with 0.5% PBS-T. 5. Incubate the monoclonal soluble antibody with target-coated wells and preblocked wells for 1 h. with 700 rpm agitation. 6. Add anti-c-Myc-HRP antibody ( 1:2500 in PTM) into the wells and incubate for 1 h. with 700 rpm agitation. 7. Add ABTS developing solution in dark to detect bound antibody carrying the peroxidase enzyme that converts the substrate into color product. Record the absorbance reading (OD405nm) using a microplate reader.

8

Analysis After performing MSD biopanning, a typical enrichment pattern for polyclonal phage ELISA will be observed for a successful biopanning campaign. The isolation of target-specific antibodies from the antibody library will be conducted through monoclonal phage propagation and monoclonal antibody ELISA. Phage enrichment ratios may be used to gauge the enrichment process of the panning experiment. A similar polyclonal antibody ELISA pattern to

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Fig. 3 Phage ELISA results showing the enrichment pattern after biopanning process of the scFv antibody library on the target antigen. Bound phage particles were detected via incubation with anti-M13 conjugated with HRP

conventional methods can be expected using the MSD biopanning protocol. Figure 3 shows the typical polyclonal ELISA readout from the biopanning rounds in previous experiments using the approach. Soluble monoclonal antibody ELISA must be performed to validate the functionality of isolated monoclonal antibodies in soluble form. To determine the identity and diversity of the positive clones, the isolated positive clones are sequenced. This will allow the identification of functional and soluble antibody clones that can be applied for multiple downstream applications.

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Notes 1. Apart from naı¨ve scFv libraries, any phage display antibody library with different antibody formats including domain and Fab libraries could also be utilized with this approach. 2. Due to the strong binding of streptavidin tetramer to biotin, this interaction allows for a highly efficient coupling of biotinylated targets to the streptavidin on the porous matrix of the tip. Biotinylated antigens can either be produced via chemical conjugation with biotin or using in vivo biotinylation systems (i.e., avi-tag and biotin ligase systems). 3. To allow continuous aspiration and dispensing with adjustable program settings, an electronically controlled motorized multichannel pipette is needed for MSD biopanning. With the exception of Finnpipette™ Novus i Electronic 12-channel that we applied, other programable electronic multichannel pipettes can be used. Other liquid handling systems that can conduct programmed aspiration and dispensing would also be adaptable.

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4. Static incubation of the culture is required for efficient infection of bacterial cell with the phage by avoiding pilus destruction. 5. Different antibiotic resistance genes carried by the antibody phagemid (ampicillin resistant) and M13K07 helper phage (kanamycin resistant) enable selection of antibody of interestbearing bacteria. 6. In case the coupling of biotinylated targets to streptavidin D.A. R.T’s® is inefficient, scale up the optimum amount of targets. 7. Since the ratio of output phage and input phage used may affect the efficient enrichment during biopanning, make sure identical amount of input phage is utilized for each round of selection round. 8. To remove weak binders, imposing an increasingly stringent washing process for each increased round of biopanning may increase the enrichment ratio of specific to nonspecific binders and attenuates the nonspecific binding of background phage. 9. Elution buffer used for MSD biopanning can be varied. Acidic elution buffer with low pH does not disrupt the binding of streptavidin to biotin because streptavidin-biotin interaction is generally stable over wide range of pH. Other elution buffers like triethylamine, trypsin, or competitive elution can also be used. 10. To retrieve and recover the high-affinity phage antibodies more effectively, the cycle volume and number of cycles for the elution step may be modified by increasing the cycles of repetitive pipetting (300–500 cycles). Nevertheless, extended cycles of pipetting should not exceed 10 min during elution step because prolonged exposure to low pH may adversely alter the infectivity and causes phage degradation. 11. Even though the amount of eluted phage obtained from each round of selection process will vary, ensure the amount of the empty phage is fewer than the target-specific phage. 12. A single round of biopanning step can be completed in 1 day. It is also possible to perform overnight growth by plating out the rescued phage. 13. Generally, three to five rounds of biopanning are necessary for obtaining good enrichment of phage binders. 14. Optimization of biopanning conditions could depend on the titers after each round of biopanning process. A rise in the titers after each round of biopanning would be expected implying that the selection of binders. If the titer of non-binders on the kanamycin agar plate is higher than the titer of binders on the ampicillin agar plate, it may be necessary to repeat the biopanning process.

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Acknowledgments The authors would like to acknowledge the support of the Malaysian Ministry of Higher Education Fundamental Research Grant Scheme [FRGS/1/2018/STG05/USM/02/2 – (203/CIPPM/ 6711658)]. References 1. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315 –1317 ˜ a E (2019) The 2018 2. Barderas R, Benito-Pen Nobel Prize in Chemistry: phage display of peptides and antibodies. Anal Bioanal Chem 411:2475 –2479 3. Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM et al (2020) Phage display derived monoclonal antibodies: from bench to bedside. Front Immunol 11:1986 –2023 4. Wen J, Yuan K (2021) Phage display technology, phage display system, antibody library, prospects and challenges. Adv Appl Microbiol 11:181 –189 5. Davydova E (2022) Protein engineering: advances in phage display for basic science and medical research. Biochemist 87:146 –167 6. Xu P, Ghosh S, Gul A, Bhamore J, Park JP, Park TJ (2021) Screening of specific binding peptides using phage-display techniques and their biosensing applications. TrAC Trends Anal Chem 137:116229 –116247 7. McConnell S, Dinh T, Le M-H, Spinella D (1999) Biopanning phage display libraries using magnetic beads vs. polystyrene plates. BioTechniques 26:208 –214 8. Takakusagi Y, Takakusagi K, Sakaguchi K, Sugawara F (2020) Phage display technology for target determination of small-molecule therapeutics: an update. Expert Opin Drug Discov 15:1199 –1211 9. Hamidi S (2023) Recent advances in solidphase extraction as a platform for sample preparation in biomarker assay. Crit Rev Anal Chem 53:199–210 10. S´cigalski P, Kosobucki P (2020) Recent materials developed for dispersive solid phase extraction. Molecules 25:4869 –4895 11. Dugheri S, Marrubini G, Mucci N, Cappelli G, Bonari A, Pompilio I et al (2020) A review of

micro-solid-phase extraction techniques and devices applied in sample pretreatment coupled with chromatographic analysis. Acta Chromatogr 33:99 –111 12. Stevens KG, Pukala TL (2020) Conjugating immunoassays to mass spectrometry: solutions to contemporary challenges in clinical diagnostics. Trends Anal Chem 132:116064 –116095 13. Seidi S, Tajik M, Baharfar M, Rezazadeh M (2019) Micro solid-phase extraction (pipette tip and spin column) and thin film solid-phase microextraction: miniaturized concepts for chromatographic analysis. TrAC Trends Anal Chem 118:810 –827 14. Chisvert A, Ca´rdenas S, Lucena R (2019) Dispersive micro-solid phase extraction. TrAC Trends Anal Chem 112:226 –233 15. Trenchevska O, Nelson R, Nedelkov D (2016) Mass spectrometric immunoassays in characterization of clinically significant proteoforms. Proteomes 4:13 –33 16. Chin CF, Ler LW, Choong YS, Ong E, Ismail A, Tye G et al (2015) Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning. J Microbiol Methods 120:6 – 14 17. Wilchek M, Bayer EA (1988) The avidin-biotin complex in bioanalytical applications. Anal Biochem 171:1 –32 18. Solemani Zadeh A, Gr€asser A, Dinter H, Hermes M, Schindowski K (2019) Efficient construction and effective screening of synthetic domain antibody libraries. Methods Protoc 2:17 –36 19. Mohd Ali MR, Sum JS, Aminuddin Baki NN, Choong YS, Nor Amdan NA, Amran F et al (2021) Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology. Int J Biol Macromol 168:289 – 300

Chapter 15 Magnetic Nanoparticle-Based Semi-automated Panning for High-Throughput Antibody Selection Angela Chiew Wen Ch’ng, Zolta´n Konthur, and Theam Soon Lim Abstract Bio-panning is a common process involved in recombinant antibody selection against defined targets. The biopanning process aims to isolate specific antibodies against an antigen via affinity selection from a phage display library. In general, antigens are immobilized on solid surfaces such as polystyrene plastic, magnetic beads, and nitrocellulose. For high-throughput selection, semi-automated panning selection allows simultaneous panning against multiple target antigens adapting automated particle processing systems such as the KingFisher Flex. The system setup allows for minimal human intervention for pre- and post-panning steps such as antigen immobilization, phage rescue, and amplification. In addition, the platform is also adaptable to perform polyclonal and monoclonal ELISA for the evaluation process. This chapter will detail the protocols involved from the selection stage until the monoclonal ELISA evaluation with important notes attached at the end of this chapter for optimization and troubleshooting purposes. Key words Panning, Antibody library, Monoclonal antibodies, Phage display, Semi-automated, Magnetic nanoparticle

1

Introduction Directed evolution of antibodies by display technologies like phage display has helped to drive the rapid growth of recombinant human antibodies in biomedical applications such as laboratory research, medical diagnostics, and therapeutics [1]. A total of 570 antibody therapeutics were reported to be at various stages of clinical trials [2]. However, only 79 monoclonal antibodies (mAbs) were approved in the European Union (EU) and the United States by December 2019 [1, 2]. The numbers are based on the various novel display technologies such as yeast display [3], ribosome display [4], mammalian cell display [5–7], bacterial display [8–10], covalent DNA display [11, 12], and mRNA display [13] for human antibody generation. Even with a plethora of display methods, phage display remains a popular method for human mAbs generation due to its robustness and efficiency in antibody development [14, 15].

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_15, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 The antibody phage library construction and classification into naı¨ve, immune, synthetic, and semisynthetic antibody library based on the source of antibody genes

The phage display selection process begins with the establishment of an antibody library with a diverse repertoire. In general, there are several different types of antibody libraries such as naı¨ve, immune, and synthetic antibody libraries [16, 17]. Each of these libraries has different characteristics based on the source of the cDNA genes, which are either derived from healthy or infected donors or generated by chemical synthesis [18], as shown in Fig. 1. The diversity and size of the library would generally influence the quality of a library and subsequent mAbs isolated from it [19, 20]. It is anticipated that the larger the library, the more diverse the library repertoire and as a direct consequence the higher

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Fig. 2 The general phage display panning procedure, including antigen immobilization, antigen-phage antibody binding, binder-specific antibody phage elution, E. coli infection, and re-amplification process for several panning rounds to obtain a good enrichment of target specific binders

the probability of enriching high-affinity antigen-specific binders [21, 22]. The choice of library for selection is greatly dependent on the antigen specificity [23, 24]. In general, phage display selection is a repetitive process that allows continuous enrichment of antigen-specific clones by continuous isolation and multiplication of a pool of binding clones [17, 25]. The process involves several key stages, namely, affinity-induced target binding, isolation, and amplification before analysis as shown in Fig. 2. To facilitate target capture, antigens are anchored to solid supports, such as high binding plastic surfaces [26], magnetic particles [27], or nitrocellulose membranes [28]. However, plastic surfaces such as polystyrene immune tubes or microtiter wells are the most commonly used surface. These surfaces serve as a physical surface to capture and present the antigen for affinity binding [29]. Then, a collection of unique phage particles derived by the library is allowed to bind with the immobilized antigen. This is followed by a wash step that is needed to remove nonspecific, unbound, or even weak binders from the solid surface. The remaining phage particles are then rescued by elution using salts, pH dissociation, or even enzymatic cleavage [30–33]. However, a

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direct rescue without the need of phage elution is conducted with magnetic bead selection. The re-amplified phage can then be used for subsequent panning rounds for clone enrichment or analysis. The panning process will continue until a satisfactory enrichment pattern is obtained which normally requires two to five rounds of biopanning selection. A fully automated system refers to a pipeline of predefined processes that are programmed and conducted without human intervention [34, 35]. In some laboratories, a fully automated process is difficult to be accomplished due to the high cost involved. Even so, laboratory automation can help ensure a more reproducible, efficient, and faster processes to be carried out Therefore, semi-automated processes are more commonly found in research labs where automation is only applied in particular stages in a pipeline and human involvement is still required in some steps within certain process stages. The automation on several stages in the biopanning process can help to reduce human errors and increase consistency in the entire process [36, 37]. Highthroughput biopanning is also possible with the use of such a system because it can handle 96-well microtiter plates. The application of magnetic nanoparticles and a magnetic particle processor for semi-automated biopanning is an ideal compromise [37]. This chapter is an updated version of the previous chapter published in 2020 [37]. A pin-based magnetic particle processor (MPP) (KingFisher Flex, Thermo Scientific) is used for semiautomated panning process. The MPP design comprises an array of magnetic pins matching the standard 96-well microtiter plate layout on a moving arm to physically relocate the magnetic particles from one well to another as shown in Fig. 3. The moving arm is aligned according to the positions of a standard 96-well microtiter plate. The MPP magnetic pins are layered with a plastic cover to function as a border between magnet particles from the magnetic pins. The instrument comes with an intuitive software to control the movements of the magnetic pins for specific incubation times, temperature, capture release frequency, plate position movement, frequency, and speed of pin movement. The adaptation of the MPP system for biopanning is preferred over the conventional microtiter plate panning when dealing with a large sample size with advantages of higher reproducibility in sample handling with minimal errors. Additionally, MPP was used to control the transfer of magnetic particles from one well to another for a reduced background caused by nonspecific binder selections. The automated transfer ensures minimal volume transfer which aids to avoid any carryover of extra washing solutions which might contain nonspecific binders. The panning protocol is generally conducted using a MPP for four rounds of consecutive selection as shown in Fig. 2. A general MPP protocol was standardized as parameters for biopanning when

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Fig. 3 (a) Consumables used in KingFisher Flex instrument including KingFisher 96 microplates and a KingFisher 96 tip comb for KingFisher magnets; (b) the rotating table of KingFisher Flex magnetic particle processor of Thermo Fisher Scientific; and (c) the capturing, releasing, and transferring procedure of MPP by the magnetic head covered with plastic comb. (1) The rod-shaped magnet is covered by plastic comb and moves into the solution containing preloaded magnetic beads. (2) Incubation process is done by mixing and moving the covered magnet up and down slowly. (3) Moving the covered magnet to the subsequent plate to transfer the magnetic beads to new solution. (4) The magnet is removed from the plastic cover, the beads slowly suspended into the solution again. (5) The magnet head and plastic cover were removed up to the starting position and the next stage process was continued

dealing with protein targets for enrichment of antibodies binders as shown in Tables 1 and 2. Besides, parameters can be personalized when dealing with different targets. As the semi-automated protocol requires some human intervention in particular stages in the process, the protocol is designed to keep human intervention at a minimum. Multichannel pipettors are used in 96-well format MPP when

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Table 1 Overview on the plate position for automated magnetic particles panning procedure with Kingfisher Flex Plate no.

Panning round 1

Panning round 2

Panning round 3

Panning round 4

1

Bead plate

Bead plate

Bead plate

Bead plate

2

Wash plate 1

Wash plate 1

Wash plate 1

Wash plate 1

3

Phage plate

Phage plate

Phage plate

Phage plate

4

Wash plate 2

Wash plate 2

Wash plate 2

Wash plate 2

5

Release plate

Wash plate 3

Wash plate 3

Wash plate 3

6

E. coli culture plate

Release plate

Wash plate 4

Wash plate 4

7

–

E. coli culture plate

Release plate

Wash plate 5

8

–

–

E. coli culture plate

Release plate

–

–

–

E. coli culture plate (next transfer)

~140 min

~150 min

~160 min

~170 min

Total time

Table 2 The automated magnetic particles in operation mode for Round 4 panning process Plate no. Plate name

Process

Volume (μL) Time (min)

1

Bead plate

Blocking the antigen loaded on magnetic beads with PTM

200

60

2

Wash plate 1 Wash 1 of the magnetic beads in PBST

200

10

3

Phage plate

Selection of antibody phage with magnetic beads 200

60

4

Wash plate 2 Wash 2 of the magnetic beads in PBST

200

10

5

Wash plate 3 Wash 3 of the magnetic beads in PBST

200

10

6

Wash plate 4 Wash 4 of the magnetic beads in PBST

200

10

7

Wash plate 5 Wash 5 of the magnetic beads in PBST

200

10

8

Release plate Released the magnetic beads with specific binders after wash with PBST

200

5-10

Total time

180

human participation is required for multi-well selection handling. Human intervention is required in the stages such as antigen loading to magnetic beads, phage selection via affinity selection, phage rescue amplification, and confirmation on panning result by ELISA. The evaluation of the panning round consists of two stages of ELISA, with the first focusing on polyclonal level analysis and second stage

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Fig. 4 Top panel: Plate setup for magnetic bead ELISA on MPP. Bottom panel: a typical ELISA result highlighting the enrichment pattern of the antibody phage panning rounds for one target. Pooled phage obtained from panning rounds against semi-synthetic scFv library were added to ELISA well coated with the target antigen

involving monoclonal evaluation. Generally, polyclonal analysis will indicate a rising enrichment pattern as shown in Fig. 4. If good enrichment is attained in polyclonal level analysis, the enriched binders in a specific selection round will be subsequently involved in the selection of individual clones in a 96-well format. The individual clones can be either packaged or expressed independently as antibody presenting phage particles or antibody protein for monoclonal ELISA analysis as shown Fig. 5. The application of MPP and biopanning can help solve the bottlenecks associated with conventional panning strategies especially for high-throughput panning. Therefore, the MPP protocol could be an ideal alternative to the conventional bio-panning methods considering the reasonable cost involved for a semi-automated process as compared to a fully automated process for antibody panning campaigns.

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Fig. 5 Top panel: plate setup for magnetic bead ELISA on MPP. Bottom panel: a typical monoclonal ELISA result of selected clones. Individual antibody expression clones were chosen from the second round of phage display selection procedure. Several soluble antibody clones from the pooled phage indicate the binding of binders to the respective antigen in the form of soluble antibody

2

Materials

2.1 Loading of Magnetic Beads

1. Dynabeads™ M-280 Streptavidin (Invitrogen Dynal AS, Oslo, Norway). 2. Phosphate-buffered saline (PBS): 8 g/L NaCl, 0.2 g/L KCL, 1.44 g/L Na2HPO4•2 H2O and 0.24 g/L KH2PO4, pH 7.4. 3. Phosphate-buffered saline Tween (PBST): PBS and 0.1% Tween-20. 4. Mini-PROTEAN Tri-Glycine gel for SDS PAGE. 5. Protein CBB-Assay Solution.

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2.2 Semi-automated Panning Using a Magnetic Particle Processor

299

1. E. coli suppressor strain TG1 genotype: supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rK– mK–) [F´ traD36 proAB lacIq ZΔM15] (Stratagene, Santa Clara, CA, USA). 2. KingFisher 96 microplate (200 μL) (Thermo Fisher Scientific, Massachusetts, USA). 3. KingFisher 96 tip comb for KingFisher magnets (Thermo Fisher Scientific, Massachusetts, USA). 4. 5 mL polystyrene maxi binding immunotube. 5. 96-well U-bottom polypropylene (PP) microtiter plates. 6. AeraSeal breathable sealing film (Sigma-Aldrich, Taufkirchen, Germany). 7. Phosphate-buffered saline (PBS): 8 g/L NaCl, 0.2 g/L KCL, 1.44 g/L Na2HPO4•2 H2O and 0.24 g/L KH2PO4, pH 7.4. 8. Phosphate-buffered saline Tween (PBST): PBS and 0.1% Tween-20. 9. Phosphate-buffered saline Tween Milk powder (PTM): PBS, 1% Tween-20, 2% non-fat dry milk powder, prepare fresh. 10. 2YT medium: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, and 0.5% NaCl, pH 7.0. 11. 10×Amp/Glu solution: 1 mg/mL ampicillin, and 20% (w/v) glucose in 2YT medium.

2.3 Packaging of Phagemids

1. M13K07 helper phage (Invitrogen, Massachusetts, US). 2. 96-well filtration plate: MultiScreenHTS plates with hydrophilic Durapore PVDF membrane with 0.65 mm pore size (Millipore, Germany). 3. 2YT-AG-2: 2YT medium containing 100 μg/mL ampicillin, 2% (w/v) glucose. 4. 2YT-AKG: 2YT medium containing 100 μg/mL ampicillin, 60 μg/mL kanamycin, 0.1% (w/v) glucose. 5. Glycerol solution: 80% (w/v) glycerol in distilled water, and then autoclave.

2.4 Titration of Phage Particles

1. 2YT-AG agar plates: 2YT medium containing 100 μg/mL ampicillin, 2% (w/v) glucose and 1.5% (w/v) agar-agar. 2. 2YT-KG agar plates: 2YT medium containing 60 μg/mL kanamycin and 1.5% (w/v) agar-agar.

2.5 Magnetic Particle ELISA of Polyclonal Antibody Phage

1. 96-well polystyrene microtiter plates. 2. Anti-M13 horseradish peroxidase (HRP)-conjugated monoclonal antibody (GE Healthcare, USA). 3. ABTS developing solution: Dissolve one ABTS tablet (50 mg) in 50 mL of the ABTS buffer solution. Store in dark (Roche, Basel, Switzerland).

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2.6 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. E. coli nonsuppressor strain HB2151 genotype: K12 ara D (lac-proAB) thi/F0 proA+B lacIq lacZDM15 (Stratagene, Santa Clara, CA, USA). 2. 2YT-AG-1: 2YT medium containing 100 μg/mL ampicillin, 1% (w/v) glucose. 3. 2YT-AG-0.1: 2YT medium containing 100 μg/mL ampicillin, 0.1% (w/v) glucose. 4. 2YT-AI: 2YT medium containing 100 μg/mL ampicillin, 9 mM isopropyl-b-D-thiogalactopyranoside (IPTG).

2.7 ELISA of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. Phosphate-buffered saline (PBS): 8 g/L NaCl, 0.2 g/L KCL, 1.44 g/L Na2HPO4•2 H2O and 0.24 g/L KH2PO4, pH 7.4. 2. Phosphate-buffered saline milk powder (PM): PBS, 2% non-fat dry milk powder, prepare fresh. 3. Phosphate-buffered saline Tween milk powder (PTM): PBS, 1% Tween-20, 2% nonfat dry milk powder, prepare fresh. 4. Anti-c-myc-peroxidase from mouse IgG1κ (Roche, Basel, Switzerland). 5. Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Invitrogen, Massachusetts, USA). 6. ABTS developing solution: Dissolve one ABTS tablet (50 mg) in 50 mL of the ABTS buffer solution. This solution has a lightgreen color. Store in dark (Roche, Basel, Switzerland).

3

Methods All the designed protocols in this section are applied for highthroughput antibody selection of more than one target antigen in parallel. The semi-automated phage display panning procedure requires minimal human intervention. The antibody library used can be either naı¨ve, immune, or synthetic regardless of the format. With the standard protocol outlined in the chapter, the sample handling via this MPP semi-automated selection will become more straightforward to produce high-throughput results with minimal human intervention.

3.1 Loading of Magnetic Beads

1. Pipette out 1 mg of Dynabeads™ M-280 Streptavidin magnetic beads (100 μL of the purchased stock). 2. Wash the magnetic beads 5 minutes (min) for three times with either 1 mL PBS or PBST at room temperature (RT). At the same time, dissolve 100–200 μg of biotinylated protein antigen or 1–2 μg of biotinylated peptide antigen in at least 300 μL PBS. Then, resuspend the antigen solution with washed magnetic beads and incubate the mixture overnight (o/n) at 4 °C

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or 1 hour (h) at RT on a Multi RS-60 Programmable rotator (Biosan, Latvia) (see Subheading 4, Notes 1 and 2). 3. After conjugation, wash the conjugated beads three times in 1 mL of PBS containing 0.1% BSA or 0.1% (v/v) PBS Tween20 (PBST). The washing process is conducted either on a rotator for 5 min or by pipette aspiration at RT. Discard the leftover washing solution with the help of a DynaMag™-2 magnet (Invitrogen, USA) for 2–3 min. 4. Discard the washing solution before resuspending the magnetic beads with the same volume of PBS. Lastly, store the antigen-loaded bead stock at 4 °C until use. 5. The conjugation process should be evaluated with SDS PAGE and Bradford assay. The conjugated beads should be electrophoresed on SDS PAGE to confirm the protein antigen loaded on magnetic beads. At the same time, quantify the concentration of the antigen protein before and after conjugation by using Bradford assay to estimate the binding capacity of the magnetic beads. The peptide conjugation can be done using the same magnetic beads, and the peptide conjugation will be different due to its size. Normally an ELISA will be used to determine the leftover binding capacity with the use of Biotin HRP for detection. 3.2 Semi-automated Panning on Magnetic Particle Processor

1. Culture a single colony of TG1 in 5 mL of 2YT at 37 °C, 200 rpm o/n (see Subheading 4, Note 3). 2. Inoculate 20 mL 2YT in a 100 mL Erlenmeyer flask with 0.2 mL of a fresh overnight TG1 at 37 °C and 200 rpm until OD600 ~0.5 (see Subheading 4, Notes 3 and 4). 3. Organizing bead plate (Plate no. 1). Fill the positions A1–A12 of a KingFisher 96 microplate with 190 μL PTM. Aspirate 10 μL of antigen-loaded magnetic beads for each antigen to the specified position, namely, magnetic beads of antigen 1 to position A1, magnetic beads of antigen 2 to position A2, and so on (total of 12 antigens in this design). 4. Pre-incubate the unselected phage library with unloaded magnetic beads with PTM to deplete selection matrix binders as well as the blocking agent binders before subjected to affinity binding toward antigen. In a 5 mL polystyrene maxi-binding immunotube, add 0.1 mg empty Dynabead M-280 Streptavidin to 1 × 1011 – 1 × 1012 phage particles, and then top up with PTM to a final volume of 200 μL and incubate for 1–2 h at RT on a Multi RS-60 Programmable rotator (Biosan, Latvia) (see Subheading 4, Note 5). 5. Then, transfer the phage library into the specific well on phage plate after separation using DynaMag™-2 magnet (Invitrogen, USA) for 2–3 min. The magnetic beads are then discarded.

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6. Organize the phage plate (Plate no. 3 as shown in Fig. 3 and Table 1) for round 1 magnetic bead panning. Fill positions A1– A12 of a KingFisher 96 microplate with 200 μL of the pre-incubated phage library solution. The following rounds of panning will continue with step 9. 7. Organize phage-plate for subsequent rounds and fill positions A1–A12 of a KingFisher 96 microplate (200 μL) with 100 μL PTM. Pipette 100 μL of the amplified phage solutions from the previous panning round according to the same antigen order in positions A1–A12 (see Subheading 4, Note 6). 8. Organize wash plate (Plate no. 2, 4, 5, 6, 7 as shown in Fig. 3 and Table 1) and fill positions A1–A12 of a KingFisher 96 microplate with 200 μL PBST (see Subheading 4, Note 7). 9. Organize release plate (Plate no. 5, 6, 7, 8 as shown in Fig. 3 and Table 1) and fill positions A1–A12 of a KingFisher 96 microplate with 100 μL PBS. 10. Place the plates in the KingFisher Flex plate holder table according to the plate numbering in Table 1 before initiating the magnetic bead-based panning program. The magnetic beads should then be relocated from plate to plate according to the set program. 11. Incubate the beads in each plate. The beads should be kept in suspension by moving the KingFisher 96 tip comb up and down in the wells at medium speed (30–50 mm/s) during incubation as shown in Fig. 3c. Immediately prepare the E. coli culture plate and pipette into positions A1–A12 of a 96-well U-bottom PP microtiter plates with 200 μL of E. coli TG1 (OD600 ~0.5 as mentioned in step 2) once the panning program has finished. Place E. coli culture plate in Kingfisher 96 instrument and start the transfer program. This program simply transfers the beads from the release plate to the E. coli culture plate (see Subheading 4, Note 8). 12. Remove the selection stock plate from the KingFisher Flex instrument, cover with plastic lid, and incubate static for 30 min at 37 °C. 13. Remove the beads. The culture is mixed with 20 μL 10×Amp/ Glu solution before sealing with a breathable sealing film and incubate in IEMS Incubator/Shaker (Thermo Fisher Scientific, Dreieich, Germany) for 2–2.5 h at 37 °C and 1140 rpm (see Subheading 4, Note 9). 14. Then, proceed to packaging of phage particle protocol (Subheading 3.4). 15. Refer to Fig. 3 for the actual positions and operating mode.

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3.3 Packaging of Phage Particles

303

1. Take out the selection stock plate. Then, add 200 μL of pre-warmed 2YT-AG medium to culture and mix thoroughly by pipetting up and down and transfer 200 μL into 96-well filtration plate. 2. Seal the selection stock plate again with breathable sealing film and continue incubation in a microplate shaker o/n at 37 °C at 1140 rpm (see Subheading 4, Note 9). 3. Add 20 μL M13K07 helper phage ~1011 phage particles into culture in 96-well filtration plate and cover it before incubating stationary for 30 min at 37 °C (see Subheading 4, Notes 10 and 11). 4. Filter the bacterial culture by centrifuging the microtiter plate for 5 min at 2000 rpm. 5. Discard the supernatant with remaining M13K07 helper phage. 6. Resuspend bacteria in 220 μL prewarmed 2YT-AKG and transfer to a fresh 96-well U-bottom PP microtiter plate. Seal phage production plate with breathable sealing film, and incubate in a microplate shaker o/n at 30 °C at 1140 rpm (see Subheading 4, Note 12). 7. The next day, add 160 μL glycerol solution to selection stock plate. Then mix and store as glycerol stock at -80 °C. 8. Pellet down the bacteria in phage production plate by centrifugation 10 min at 2000 rpm. Transfer supernatant carefully without disturbing the pellet to a 96-well filtration plate. 9. Place filtration plate on top of a new 96-well U-bottom PP microtiter plate and fix with sticky tape. 10. Filter antibody presenting phage particles to remove possible contaminating E. coli cells by centrifugation for 2–5 min at 2000 rpm. 11. Store the filtrate. Discard bacteria pellets (see Subheading 4, Note 13). 12. Add 50 μL PBS to each well of the phage stock plate and mix thoroughly. Use 100 μL for the next round of selection and 10 μL for phage titration (see Subheading 4, Notes 14– 16).

3.4 Titration of Phage Particles

1. Culture 5 mL of 2YT in a 50 mL falcon tube with a single clone of TG1 from a M9 minimal agar plate and incubate o/n at 37 ° C and 200 rpm. 2. Culture 20 mL 2YT in a 100 mL Erlenmeyer flask with 200 μL of overnight culture and grow at 37 °C and 200 rpm until OD600~0.5. 3. Make 1:10 serial dilutions of phage suspension in PBS.

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Table 3 Automated magnetic based polyclonal antibody ELISA protocol for Kingfisher Flex Plate no.

Plate name

Procedure

Volume (μL)

Time (min)

1

Bead plate

Blocking of antigen-conjugated and control magnetic beads with PTM

200

60

2

Wash plate 1

Wash of magnetic beads in PBST

200

10

3

Phage plate

Incubation of magnetic beads in antibody phage 200 library/antibody phage stock of the selection rounds

60

4

Wash plate 2

Wash 1 of magnetic beads in PBST

200

20

5

M13 Antibody Plate

Incubation of antigen-conjugated and control beads with anti-M13 HRP (1:5000) in PTM

200

60

6

Wash plate 3

Wash 3 of magnetic beads in PBST

200

10

7

Substrate plate

Incubation of magnetic beads in ABTS

200

30a

8

–

–

–

–

Total time

170

a

At this stage, the ABTS incubation takes place at plate shaker at RT for maximum 30 min before measuring its absorbance. The magnetic beads must be removed before measuring the absorbance in plate reader at 405 nm

4. Infect 100 μL of E. coli TG1 to phage dilutions and incubate for 30 min at 37 °C without shaking. 5. Spot 10 μL of each dilution on 2YT-AG and 2YT-K agar plates for enriched library. Incubate plates o/n at 37 °C after the droplets are dried (see Subheading 4, Notes 16 and 17). 3.5 ELISA of Polyclonal Antibody Phage

1. Preparing bead plate (Plate no. 1 as shown in Fig. 3 and Table 3). Fill each well of a KingFisher 96 microplate with 190 mL 2% PTM, and add 10 μL of antigen-loaded bead stock according to plate layout. Then add magnetic beads of antigen 1 to positions A1–D1, beads of antigen 2 to positions A2–D2, and so on. 2. Prepare bead plate (Plate no. 1 as shown in Fig. 3 and Table 3). Empty magnetic beads are used as a negative control. Take 5 mg (500 μL) Dynabeads™ M-280 Streptavidin magnetic beads and wash three times with 1.5 mL PBS at RT. Discard the last wash solution and resuspend in 500 μL. Add 10 μL to positions E1-H12 (note: the wash can be done on rotator). 3. Prepare phage plate (Plate no. 3 shown in Fig. 3 and Table 3). Fill each position of a KingFisher 96 microplate with 50 μL PTM. Add 50 μL of phage solution from the phage stock plates of the individual rounds to plate according to the layout shown in

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Fig. 5. Add phage stocks of selection rounds 1 to 4 on antigen 1 to position A1-D1 and E1-H1, respectively. Add phage stocks of selection rounds 1–4 on antigen 2 to position A2-D2 and E2-H2, respectively, and so on. 4. Prepare wash plates (Plate no. 2, 4, 6 as shown in Fig. 3 and Table 3). Fill each well of KingFisher 96 microplate with 200 μL PBST. 5. Prepare M13 antibody plates (Plate no. 7 as shown in Fig. 3 and Table 3). Adding 4 μL mouse monoclonal anti-M13 HRP-conjugated to 20 mL 2% PTM (1:5000). Fill each well of KingFisher 96 microplate with 200 μL antibody solution. 6. Place the plates in the Kingfisher Flex plate holder table and start magnetic bead-based ELISA program. The program should be set to move magnetic beads from plate to plate and incubate the beads in each plate (Fig. 3c). During all incubations, the beads should be kept in suspension by moving plastic tips up and down in the wells at medium speed (30–50 mm/s). 7. While ELISA program is running, prepare the substrate plate (Plate no. 7 as shown in Fig. 3 and Table 3). Dissolve one ABTS tablet (50 mg) in 50 mL substrate buffer. 8. Once beads are incubated in the substrate and color developed for 30 min, remove beads from the substrate by transferring them back to wash plate 4. 9. Take out substrate plate from the Kingfisher Flex plate holder table, and measure substrate specific extinction at 405 nm in an ELISA reader. 10. For each individual selection target, evaluate enrichment by plotting the obtained values for antigen-loaded and control magnetic beads of each phage selection rounds next to each other (Fig. 4). 3.6 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. Inoculate 5 mL of 2YT in a 50 mL PP tube with a single colony of HB2151 from a 2YT agar plate (without antibiotic) and grow shaking o/n at 37 °C and 250 rpm. 2. Inoculate 20 mL 2YT in a 100 mL Erlenmeyer flask with 0.2 mL of o/n HB2151 culture and incubate shaking at 37 ° C and 200 rpm until OD600 ~ 0.4–0.5. 3. Meanwhile, prepare a 1:10 dilution series of the desired panning round from the corresponding phage stock plate by adding 10 μL phage to 90 μL PBS. 4. Add 100 μL of TG1 E. coli cell (OD600 ~ 0.4–0.5) to phage dilutions and incubate for 30 min at 37 °C.

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5. Mix infected E. coli cultures and plate out 10 μL of each dilution series on a 2YT-AG agar plate. Once dried, incubate plates top-down at 37 °C o/n. 6. Pick 92 clones into 96-well U-bottom PP microtiter plate filled with 100 μL 2YT-AG-1: 2YT medium containing 100 μg/mL ampicillin, 1% (w/v) glucose (see Subheading 4, Note 18). 7. Leave positions H3, H6, H9, and H12 empty for controls. Seal mother plate with breathable sealing film and incubate in a microplate shaker o/n at 37 °C and 1140 rpm. 8. Next day, inoculate fresh 96-well U-bottom PP microtiter plate containing 200 μL 2YT- AG-0.1 with 2 μL of the o/n culture and incubate daughter plate for 150 min at 37 °C and 1140 rpm. 9. Add 100 μL glycerol solution to each well of the mother plate and store as glycerol stock -80 °C. 10. Induce soluble antibody fragment production in daughter plate by adding 25 μL of 2YT containing 100 μg/mL ampicillin and 9 mM IPTG to each well and continue incubating o/n at 30 °C, 1140 rpm. 11. Pellet down the bacteria by centrifuge the microtiter plates for 10 min at 3000 rpm. 12. Transfer soluble monoclonal antibody fragment containing culture supernatant into fresh 96-well U-bottom PP microtiter plate and store until further use at 4 °C. Discard pelletcontaining plate. 3.7 ELISA of Soluble Monoclonal Antibody Fragments in Microtiter Plates

1. To analyze the antigen specificity of the soluble antibody fragment, coat half of a Matrix 96-well microtiter plate (positions A1-H6) by transferring (a) 10 μg protein antigen in 100 μL PBS or (b) 10–20 μg peptide antigen in 100 μL PBS to each well. At the same time, coat the other half of the plate (positions A7-H12) with 100 μL/well of an appropriate negative control, such as bovine serum albumin (100 μg/mL in PBS) and incubate microtiter plate o/n at 4°C (see Subheading 4, Note 14). 2. Discard coating solution and wash all wells three times with PBS by using Wellwash™ Microplate Washer (Thermo Fisher Scientific, Dreieich, Germany). 3. Block all wells by completely filling them with 2% PM and incubate for 1 h at RT. 4. Discard blocking solution and wash all wells three times with PBS by using Wellwash™ Microplate Washer (Thermo Fisher Scientific, Dreieich, Germany).

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5. Fill each well with 20 μL PTM and 80 μL soluble antibody fragment solution of the respective 46 clones to each half of the plate (containing target antigen and a negative control, respectively) and incubate for 1 h at RT. 6. Discard soluble antibody fragment solution and wash wells three times with PBST. 7. Add 100 μL of Anti-c-myc-Peroxidase from mouse IgG1κ (1: 2500 in PM) to each well and incubate for 1 h at RT. 8. Discard Anti-c-myc-Peroxidase from mouse IgG1κ solution. Then, wash the wells three times with PBST. 9. Meanwhile, prepare substrate by dissolving one ABTS tablet (50 mg) in 50 mL substrate buffer. 10. Finally, add 100 μL of substrate to each well and allow to develop for 30 min at RT in the dark (see Subheading 4, Note 19). 11. Read substrate-specific extinction at 405 nm in an Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific™, Dreieich, Germany). 12. Plot the ELISA results for antigen and background protein for each soluble monoclonal antibody fragment next to each other to identify positive clones with an acceptable signal to background ratio (Fig. 5).

4

Notes 1. The large surface area of magnetic nanoparticles increases protein accessibility toward the library phage particles for affinity binding with less steric hindrance [38]. Alternatively, surfaceactivated magnetic beads such as Tosyl-activated, carboxylic acid, amine, or epoxy beads can be used for protein attachment [39–42]. 2. Biotinylated antigens produced either by chemical conjugation or enzymatic bioconjugation methods [43] can be used. 3. The single colonies of TG1 are obtained by plating the TG1 stock on M9 minimal agar plate supplement with 0.2% glucose, 1 mM magnesium sulfate, 0.1 μM calcium chloride, and 1 μg/ mL thiamine hydrochloride for selection against TG1 with F-pili expression. Besides TG1, E. coli strains such as XL1 Blue, SS320, and ER2738 can be used for phagemid re-amplification with the help of a helper phage as the E. coli strains contain F´ episome [44, 45]. The F-episome is needed for the production F-pili which are necessary for the infection process by M13 phage [46].

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4. Phage or phagemid infect F+-E-coli through the sex pili. Therefore, E. coli must be grown at 37 °C to reach the log phase of growth (OD at 600 nm = 0.4–0.5) for sex pili production which is efficient for infection [47]. The growing temperature of E. coli is crucial for phage display as a low growing temperature (200 rpm) for 1 h After this incubation, add 1 × 1011 pfu M13K07 helper phage (see Note 6), mix well, and incubate for another 60 min at 37 °C in a shaking incubator. After this incubation, pellet the cells by spinning them at 3000 g for 10 min, and resuspend the cells in 20 mL of LB-GAT and add 20 μL of kanamycin. Incubate the cells O/N at 30 °C in a shaking incubator. 3. The following day, pellet the bacteria by spinning at 3000 g for 15 min, and transfer the supernatant (~20 mL) to a new tube and add in 5 mL of 5× PEG/NaCl. Keep the tube on ice or at 4 °C for 1 h to precipitate the amplified phage. During this

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incubation, periodically shake the tube. At this time, begin a culture of XL1-Blue (~5 mL per sample). After 1 h, pellet the precipitated phages by spinning at 6000 g for 30 min. Carefully decant the supernatant, taking care not to disturb the phage pellet (see Note 7). 4. Carefully resuspend the phages in 1 mL of PBS (see Note 8). Be sure to wash the sides of the tube to remove the phage smear. Spin the 1 mL of phage at 14,000 rpm at 4 °C for 5 min in a microcentrifuge to pellet any insoluble particles or remnants of E. coli. Titer the phages by inoculating 50 μL of OD600 0.5–1.0 XL1-Blue with 1 μL of 10-8–10-12 dilutions of this phage stock. Wait 15 min after inoculation before plating the cells on 10 cm LB-GAT plates. Incubate the plates at 37 °C O/N. 5. The next day, calculate the phage titer by multiplying the number of colonies on each plate by the dilution factor. This is the concentration of phages per microliter (see Note 9). Freeze down several aliquots of the amplified phages in 15% glycerol and store at -80 °C, and use ~1 × 1012 pfu phages to continue panning. 6. Repeat the above panning protocol three to four times, each time decreasing the amount of biotinylated target pMHC used. This will improve the specificity of the selected phages. 3.4 Clone Selection for Dynabeads Panning

1. After the final round of selection and amplification, titer the selected phage pool as before. After quantifying, freeze down several aliquots of the phage (as explained in Subheading 3.3, step 5) and inoculate 1 mL of an OD600 0.3–0.6 culture of HB2151 E. coli. Inoculate using a concentration of phages that gives between 100 and 200 colonies per 50 μL of culture. Plate 50 μL per plate on 3–5 LB-Amp plates and incubate O/N at 30 °C. 2. The next day, pick colonies from the plates and inoculate them into a 96-well DeepWell™ plate with each well containing 400 μL LB-Amp and seal it with AxygenTM microplate sealing film. Be sure to include at least one negative control well per experiment. This well should include the uninfected HB2151 in LB alone (see Note 10). Incubate the 96-well plate on a shaker at 37 °C for 3–6 h. After the incubation, add 200 μL of 50% glycerol-LB per well. These now constitute the monoclonal glycerol stocks. Take 50 μL of each stock and inoculate a new 96-well plate with 400 μL of LB-Amp per well as before. Incubate until the OD600 reaches about 0.4 on over half of the plate (~3–6 h). Afterward, freeze down the first 96-well plate at -80 °C for long-term storage.

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3. Once the cells have reached an OD600 of ~0.4, add 200 μL of LB-Amp + 0.5 mM IPTG to induce soluble scFv production, and incubate O/N with shaking at 28 °C. The next day, centrifuge the plates at 3000 g for 15 min and transfer supernatant to a new plate for screening. This supernatant now contains soluble scFv from the monoclonal stocks. The next step will be to screen the clones for proper binding activity and specificity. 3.5 Clone Selection for Cell Panning

1. After the final round of selection and amplification, titer the selected phage pool as before. After quantifying, freeze down several aliquots of the phage (as explained in Subheading 3.3, step 5), and inoculate 1 mL of XL1-Blue culture with OD600 of 0.3–0.6. Inoculate using a concentration of phages that gives between 100 and 200 colonies per 50 μL of culture. Plate 50 μL per plate on LB-GAT plates and incubate O/N at 30 °C. 2. Pick colonies and inoculate them to 400 μL of 2xYT-GAT media in the 2 mL 96-deep well. Incubate at 30 °C, with shaking at 250 rpm O/N with a microplate sealing film. 3. Transfer 15 μL of the O/N bacterial culture to 400 μL of fresh 2YT-GAT media in a new 2 mL 96-deep well. Incubate at 37 ° C, with shaking at 250 rpm with a microplate sealing film until OD600 reaches around 0.2 (~3 h). 4. Before infection, dilute the M13KO7d3 helper phage (1 × 1013 virions/mL) 1:50 into D-PBS, and then add 25 μL of the diluted M13KO7d3 helper phage to the bacterial culture. Mix gently and incubate at 37 °C for 1 h (without shaking). Centrifuge the infected bacterial culture at 4500 rpm for 25 min, and remove the supernatant as much as possible. Add 400 μL of 2xYT-Amp-Tet-Kan-Glu-IPTG medium to resuspend the bacteria pellet. Seal the plate with a microplate sealing film and incubate O/N at 30 °C with 250 rpm shaking. 5. The next day, centrifuge culture at 4500 rpm for 25 min. The supernatant containing single clone phage is ready to be used for FACS screening.

3.6 ELISA Screening for pMHC Binding Phage Clones

1. For each 96-well plate, coat two ELISA plates with 50 μL/well BSA-biotin at 10 μg/mL in PBS. Cover or seal the plate and incubate O/N at 4 °C (see Note 11). 2. The next morning, wash the plates three to five times with PBS. After washing, add 50 μL/well of streptavidin at 10 μg/mL in PBS and incubate for 1 h at RT. 3. Wash the plates five times with PBS and coat the plates with 50 μL/well of either biotinylated target pMHC or control pMHC at 5 μg/mL in PBS. Incubate the plates for 1 h at RT (see Note 11).

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4. Wash the plates three to five times with PBS and then add 150 μL/well of blocking buffer (2% BSA) to block the binding of proteins to the plates. Incubate the plates at RT for 60 min (see Note 11). 5. Wash the plate three to five times and add 100 μL of each monoclonal stock supernatant, or purified scFv/scFv-Fc diluted in the dilution buffer (0.5% BSA), to the plate. Incubate the plates at RT for 1 h. 6. Wash the plates five times and add 100 μL/well of mouse antiV5 antibody at 0.5 μg/mL in dilution buffer if detecting soluble scFv. If detecting scFv-Fc (human Fc) fusion proteins, add 100 μL/well of goat-anti human HRP at 0.5 μg/mL in dilution buffer. Incubate the plates at RT for 1 h. 7. If V5 tag is used for detection, wash the plates five times and add 100 μL/well of goat anti-mouse HRP at 0.5 μg/mL. Incubate the plates at 4 °C for 1 h. If Fc is used for detection, skip this step and begin development of the plates. 8. During incubation prepare the development buffer by adding two OPD tablets to 40 mL of OPD buffer. Keep at 4 °C until ready to use. 9. Before developing the plates, wash them thoroughly (at least five times) with PBS. Right before use, add 40 μL of 30% H2O2 to the development buffer and mix well. Immediately add 150 μL/well of the development buffer and incubate the plates at RT in the dark. Check the reaction every 5 min and stop the reaction after 30 min, when positive control wells turn dark yellow, or when negative control wells begin to turn light yellow, whichever comes first. Stop the reaction by adding 30 μL of the stopping solution (5 N H2SO4). Add the acid quickly and carefully, making sure to not let too much time pass between the first wells and the final wells being stopped. Once all wells have been stopped, tap the sides of the plate to mix well. The acid is denser than the development solution and should mix on its own quite readily. 10. Read the plate on a spectrophotometer set to wavelength 490 nm. Wells with target pMHC-binding scFvs should have an OD490 above background (i.e., negative control wells) by at least three times the standard deviation of the background. Compare the binding against the target pMHC to the control pMHC to determine specificity (see Note 12). 3.7 FACS Screening for pMHC Binding Phage Clones

1. One day before the FACS staining experiment, load the T2 cells with the same control and target peptides used in the panning step (see Note 4 for peptide loading). 2. On the day of FACS screening, collect cells in a 15 mL or 50 mL tube. Spin down the cells at 500 g for 5 min. Discard

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the supernatant. Resuspend the cells in cold D-PBS and spin down again at 500 g for 5 min. Discard the supernatant and resuspend the cells in FACS buffer at four million cells/mL. Add 50 uL of suspension cells (0.2 million cells) into each well of a 96-well plate. 3. Add 50 uL of phage supernatant from Subheading 3.5, step 5 to each well. Incubate the cell/phage mixture at 4 °C for 1 h. Spin down the mixture at 500 g for 5 min. Wash the cells twice with 200 μL of cold FACS buffer with 0.05% Tween-20 per well and once with 200 uL of cold FACS buffer per well. 4. Add 100 μL of primary antibody (1:200 dilution, anti-M13 mouse Ab) in cold FACS buffer for each well. Incubate the mixture at 4 °C for 1 h. After incubation, wash the cells once with cold FACS buffer. 5. Add 100 μL of secondary antibody (1:200 dilution, PE conjugated anti-mouse IgG (H + L)) in FACS buffer into each well. Incubate the mixture at 4 °C for 0.5 h in the dark. Wash the cells twice with cold FACS buffer. 6. Cell fixation (optional): Resuspend the cells in each well with 150 μL of fixation buffer (1:3 dilution in FACS buffer). Save the plate with a cover at 4 °C in a dark place to avoid evaporation and light exposure. The samples can be stored up to 3 days if the sample cannot be run on the same day. This step can be avoided if the sample is run on the same day; resuspend the cells in each well with 150 μL of cold FACS buffer. 7. Perform FACS measurement for each sample according to the manufacturer’s instruction. 3.8 Large-Scale Expression

1. Identify the positions of the positive clones in the monoclonal glycerol stocks from Subheading 3.4, step 2 and use them to inoculate 3 mL of HB2151 E. coli in LB with an OD600 of 0.6 and incubate at 30 °C O/N in a shaking incubator (>200 rpm). Subculture the 1 mL culture to 0.2 L of LB-Amp, and incubate at 37 °C in a shaking incubator until OD600 reaches 0.6. 2. Add IPTG to a final concentration of 0.5 mM, and incubate the culture at 30 °C O/N in a shaking incubator. The following day, pellet the cells at 2500 g for 15 min at 4 °C. Resuspend the pellet in 40 mL PBS and add polymyxin B to a final concentration of 1 μM. Shake at 37 °C for 30 min. Polymyxin is an antibiotic that lyses the outer cell wall of bacteria and releases soluble scFv from the periplasmic space into solution. 3. Spin down and collect the supernatant and add imidazole to a final concentration of 40 mM. 4. While incubating, prepare GraviTrap column by washing it with 10 mL of washing buffer (see Note 13).

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5. Apply the supernatant with 40 mM imidazole onto the column. 6. Wash with 10 mL of washing buffer. 7. Elute with 3 mL of elution buffer with 250 mM imidazole. If the starting volume of culture is small or if there is evidence that the yield is not good, step-wise elution with 500 uL elution buffer can be used. 8. A 10 K MWCO Slide-A-Lyzer cassette was used to dialyze the collected fractions to PBS O/N at 4 °C. RT is acceptable if necessary (see Note 14). Measure the OD280 after dialysis to determine concentration and run the samples on SDS-PAGE or HPLC to determine purity. 9. Finally, aliquot the dialyzed protein and freeze it at -80 °C. Minimize freeze/thaw cycles to prevent precipitation or degradation of the scFv. 3.9 Generating Fc Fusion Proteins

1. Starting from the monoclonal stocks, miniprep the clones of interest and sequence the scFv regions using sequencing primers for the vector used in the library. After determining the sequences, design primers with relevant 5′ and 3′ restriction sites (i.e., 5′ EcoRI and 3′ BglII) to PCR amplify the scFv sequences. Be sure to check the scFv sequence for these restriction sites; and if necessary use alternative restriction sites found on the pFUSE-hIgG1-Fc vector, or perform Gibson cloning [27]. Digest the PCR fragment and vector for 1 h at 37 °C, and gel purify it using a 1% agarose gel. Ligate for 30 min at RT using a 3:1 insert to vector molar ratio and transform the plasmid into competent E. coli. Plate cells on LB-Amp plates and incubate O/N at 37 °C. Pick five to ten colonies and miniprep them. Screen by restriction digest to validate that the insert and vector bands match the approximate sizes of the pFUSE vector (4 kb) and scFv (~800 bp). Sequence the screened plasmids and select one with the correct sequence. If necessary, amplify the selected plasmid via midiprep or maxiprep (minimum 30 μg). 2. For transient transfection, begin culturing Expi293F cells according to manufacturer’s instructions. Prepare cells 1 day before transfection with cell density of 2 × 106 cells/mL. On the day of transfection, count the number of cells and ensure viability is above 95%. 3. Dilute 30 μg plasmid DNA into 1.5 mL of OptiMEM® I reduced-serum medium. Simultaneously mix 80 μL of ExpiFectamine™ with 1.5 mL of OptiMEM® I reduced-serum. Incubate for no more than 5 min at RT and add the diluted DNA into the diluted transfection reagent. Incubate the mixture for another 20 min at RT.

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4. While waiting, prepare 75 × 106 Expi293F cells, and top up the volume to 25.5 mL with warmed up fresh medium and place in shaking incubator until use. 5. After 20 min incubation, slowly add the 3 mL transfection mixture to the prepared cells while swirling the flask. Incubate the cells for 4–6 days and harvest when viability drops below ~70%. 6. On the day of harvest, spin down supernatant at >3000 g for 1 h at 4 °C. Store at 4 °C until ready to begin purification. If one will be storing the supernatant for more than 2 days, it is best to add sodium azide (0.05% final) to prevent bacterial growth. Right before purification, pass the supernatant through a 0.22 μm filter to remove any precipitate or bacterial/fungal growth. 7. To purify the Fc-Fusion proteins, it is best to use protein-A beads (e.g., MabSelect). For larger volumes use an FPLC machine if possible. For smaller volumes, briefly, mix proteinA resin with the filtered 293F supernatant and incubate the mixture O/N at 4 °C with rotation. The next day, load the resin onto a gravity column. Wash with 10 column volume (CV) of PBS before eluting with a pH gradient (i.e., from pH 7 to pH 2.5). Elute into 1.0 CV fractions, 5 fractions per condition, and check OD280 for each fraction to identify the optimal elution conditions. Neutralize each fraction with 0.1 CV of 1 M Tris–HCl (pH 9.0); combine and dialyze the fractions of interest in PBS or another suitable buffer (see Note 15). Aliquot and store samples at -80 °C for longterm storage.

4

Notes 1. It is important to use the same MHC protein for control and target pMHCs to eliminate as much unwanted binding to MHC as possible. The selection of control peptides is critical in eliminating cross-reactivity to normal tissues and can be difficult in certain applications for at least two reasons. First, there is very limited information on the repertoire of “presentable” peptides in normal cells from different tissues. Second, recently it was found that around 25% of cell surface pMHC-I are derived from proteasome-catalyzed peptide splicing [28], adding further to the complexity of normal peptide repertoire. Under circumstances where a control peptide is not obvious, we generally recommend a bioinformatics approach to include a pool of homologous peptides as controls. Additionally, clones can be screened against cell lines that are known to express or not express the target peptide of interest. This is a very important step to confirm immunoreactivity.

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2. Depending on the library used, it may be necessary to generate the phages from E. coli stocks. In this case, follow the manufacturer’s instructions, which should be similar to the steps from Subheading 3.3 (Amplification). 3. While PBS and PBS-T are the most common buffers for washing during the panning and screening steps, any buffer that is compatible with downstream in vitro or in vivo studies can be used, as long as it does not disturb antigen-antibody interactions. The method of washing can affect the stringency of selection. Longer incubations with wash buffer or using more wash steps is thought to select for higher affinity binders. Feel free to adjust these steps as necessary. 4. To load peptides on the MHC of T2 cells, wash T2 cells twice with IMDM medium (without FBS). Dilute the cells with IMDM medium to 1 × 106 cells/mL, and add proper volume of cells to the 6-well plate or 10 cm dish. Add control or target peptides on to the cells (50 ug peptide per 1 × 106 cells). Culture the cells at 28 °C, 5% CO2 O/N. Loading peptides at 28 °C instead of normal culture temperature (37 °C) increases the loading efficiency. 5. During this step, be careful not to break up the agar when scraping the plate. It is easiest to use a wide scraper to prevent this. The bacteria come off quite easily, and it is not necessary to remove all traces of bacteria. If some agar does get into the mix, simply spin down the solution at a low speed (i.e., 100 g) briefly and transfer the supernatant to a fresh tube. 6. Helper phage provides the machinery necessary to package phagemid into mature virion for secretion. 7. Often the phage pellet forms only a faint smear on the side of the tube and is hard to identify. To make it easier to find, mark the bottom of the tube at the location where a pellet is expected to form before spinning. On an angled rotor, this is typically on the side of the tube facing away from the rotor. The smear is also apparent if the tube is held to the light and rotated. If no smear or pellet can be seen, simply wash the tube carefully, and test for phage by using the tittering scheme explained in Subheading 3.3, step 4 but start at 101 and go up until 1010. 8. When using a library generated by animal immunization, it is important to know if the target protein was conjugated to a carrier protein or used with an adjuvant. To improve specificity, the phage pellet can be dissolved in PBS mixed with the adjuvant or protein conjugate. For instance, if the immunization used BSA as a carrier protein, resuspending the phage pellet in 1% BSA in PBS will negatively select the BSA-binding phages before panning, thus reducing the chance that these phages are selected and amplified. However, be sure not to use this same

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protein in the blocking or dilution buffers during the screening. 9. If at any stage it is clear that the selected phages are not amplifying or binding with enough specificity, simply go back to a frozen aliquot and continue panning/screening from there with less stringency. With each subsequent screening, the concentration of binding phages should increase steadily. To accurately quantify the concentration, it is often necessary to dilute the phage even further than 10-12. 10. It is recommended to pick as many colonies as possible to increase the chances of finding a good clone. Be aware, however; picking more than 95 clones per experiment means doing more than two ELISAs per experiment. Always include a negative control well but feel free to scale up screening as much as necessary. A positive control is only necessary if no binding is detectable after a primary screen. 11. The protein used during the panning steps (in this case BSA) should match the protein used in the blocking and dilution buffers in the screening ELISA; however, it does not need to be BSA. Be sure to use a protein that was not used in generating the phage library. BSA-biotin can be easily replaced with another biotinylated protein, if necessary. 12. For screening it is best to test the monoclonal stocks against both the target pMHC as well as the control pMHC to validate the specificity of the selected phage. However, for convenience one can screen a larger selection against the target pMHC first and then perform a secondary screen with high binders against the control pMHC. 13. Note that at this stage it is impossible to definitively separate higher affinity binders from more stable sequences. Higher OD490 at this stage only means more scFvs were left bound by the end of the ELISA, but it does not accurately distinguish between more efficient expression of the scFvs and more efficient binding to the targets. An internal control that detects the expression level of scFv can be implemented. 14. While this protocol uses a nickel purification method, this can easily be replaced with other methods such as protein-A (antiFc), protein-L (anti-kappa chain), or anti-V5 affinity chromatography. Each method has its benefits, but nickel purification is only used in this case for its convenience. Protein-L will sometimes have improved purity over nickel, but it doesn’t bind all scFv sequences equally well. Protein-A can only bind to Fc-fusion or full IgG proteins, and anti-V5 requires that the construct has a V5 tag. Similarly, all steps can be performed by FPLC, although small-scale purifications should be limited to gravity columns.

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15. To fill the dialysis cassette, it is easiest to use a 22-gauge hypodermic needle fitted onto a small 1 mL syringe. Be sure not to overfill the cassette or let it sink into the dialysis buffer. When adding the sample to the cassette, keep the pointed end of the needle angled slightly downward with the cassette held parallel to the floor. This will help prevent any accidental puncturing of the membrane. The final volume after dialysis can sometimes change quite dramatically from the starting volume, so do not be alarmed if the volume appears to have dropped by up to 50%. 16. It is difficult to determine the optimal buffer formula for a given protein before enough of it can be successfully purified, but buffer optimization can substantially improve the stability of a given protein, during both short-term and long-term storage. Similarly, the buffers used during affinity chromatography can have enormous impact on the overall yield and purity of the final product. The buffers listed above should be considered as a starting point but can and should be optimized for each construct produced. References 1. Smith GP (1985) Filamentous fusion phage – novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317. https://doi.org/10. 1126/Science.4001944 2. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G et al (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides1. J Mol Biol 296(1):57–86. https://doi.org/10.1006/jmbi.1999.3444 3. Griffiths AD, Williams SC, Hartley O, Tomlinson IM, Waterhouse P, Crosby WL et al (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J 13(14):3245–3260 4. Balint RF, Larrick JW (1993) Antibody engineering by parsimonious mutagenesis. Gene 137(1):109–118. https://doi.org/10.1016/ 0378-1119(93)90258-5 5. Devlin JJ, Panganiban LC, Devlin PE (1990) Random peptide libraries: a source of specific protein binding molecules. Science 249(4967): 404–406 6. Luzzago A, Felici F, Tramontano A, Pessi A, Cortese R (1993) Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides. Gene

128(1):51–57. https://doi.org/10.1016/ 0378-1119(93)90152-S 7. McLafferty MA, Kent RB, Ladner RC, Markland W (1993) M13 bacteriophage displaying disulfide-constrained microproteins. Gene 128(1):29–36. https://doi.org/10.1016/ 0378-1119(93)90149-W 8. Cwirla SE, Peters EA, Barrett RW, Dower WJ (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci U S A 87(16):6378–6382 9. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554 10. Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19(15):4133–4137. https://doi. org/10.1093/nar/19.15.4133 11. Gram H, Marconi LA, Barbas CF, Collet TA, Lerner RA, Kang AS (1992) In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc Natl Acad Sci U S A 89(8):3576–3580 12. ørum H, Andersen PS, øster A, Johansen LK, Riise E, Bjørnvad M et al (1993) Efficient

Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage. . . method for construction comprehensive murine Fab antibody libraries displayed on phage. Nucleic Acids Res 21(19):4491–4498. https://doi.org/10.1093/nar/21.19.4491 13. Hoogenboom HR, Winter G (1992) By-passing immunisation. J Mol Biol 227(2): 381–388. https://doi.org/10.1016/00222836(92)90894-P 14. Barbas CF 3rd (1995) Synthetic human antibodies. Nat Med 1(8):837–839 15. Dao T, Yan S, Veomett N, Pankov D, Zhou L, Korontsvit T et al (2013) Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med 5(176):176ra33. https://doi.org/10.1126/ scitranslmed.3005661 16. Kurosawa N, Midorikawa A, Ida K, Fudaba YW, Isobe M (2020) Development of a T-cell receptor mimic antibody targeting a novel Wilms tumor 1-derived peptide and analysis of its specificity. Cancer Sci 111(10): 3516–3526. https://doi.org/10.1111/cas. 14602 17. Chames P, Hufton SE, Coulie PG, UchanskaZiegler B, Hoogenboom HR (2000) Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library. Proc Natl Acad Sci U S A 97(14):7969–7974 18. Liu H, Xu Y, Xiang J, Long L, Green S, Yang Z et al (2016) Targeting alpha-fetoprotein (AFP)-MHC complex with CAR T cell therapy for liver cancer. Clin Cancer Res. https://doi. org/10.1158/1078-0432.ccr-16-1203 19. Ahmed M, Lopez-Albaitero A, Pankov D, Santich BH, Liu H, Yan S et al (2018) TCR-mimic bispecific antibodies targeting LMP2A show potent activity against EBV malignancies. JCI Insight 3(4). https://doi.org/10.1172/jci. insight.97805 20. Chang AY, Dao T, Gejman RS, Jarvis CA, Scott A, Dubrovsky L et al (2017) A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I

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antigens. J Clin Invest 127(7):2705–2718. https://doi.org/10.1172/JCI92335 21. Klatt MG, Dao T, Yang Z, Liu J, Mun SS, Dacek MM et al (2022) A TCR mimic CAR T cell specific for NDC80 is broadly reactive with solid tumors and hematological malignancies. Blood. https://doi.org/10.1182/blood. 2021012882 22. Douglass J, Hsiue EH-C, Mog BJ, Hwang MS, DiNapoli SR, Pearlman AH et al (2021) Bispecific antibodies targeting mutant RAS neoantigens. Sci Immunol 6(57):eabd5515. https:// doi.org/10.1126/sciimmunol.abd5515 23. Hsiue EH-C, Wright KM, Douglass J, Hwang MS, Mog BJ, Pearlman AH et al (2021) Targeting a neoantigen derived from a common TP53 mutation. Science 371(6533):eabc8697. https://doi.org/10.1126/science.abc8697 24. Barbas CF 3rd, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88(18):7978–7982 25. Schoonbroodt S, Steukers M, Viswanathan M, Frans N, Timmermans M, Wehnert A et al (2008) Engineering antibody heavy chain CDR3 to create a phage display Fab library rich in antibodies that bind charged carbohydrates. J Immunol 181(9):6213–6221 26. Rauchenberger R, Borges E, ThomassenWolf E, Rom E, Adar R, Yaniv Y et al (2003) Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J Biol Chem 278(40):38194–38205. https:// doi.org/10.1074/jbc.M303164200 27. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5): 343–345. https://doi.org/10.1038/nmeth. 1318 28. Liepe J, Marino F, Sidney J, Jeko A, Bunting DE, Sette A et al (2016) A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 354(6310):354–358. https://doi.org/10.1126/science.aaf4384

Chapter 18 Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS Adi Amir, David Taussig, Almog Bitton, Limor Nahary, Anna Vaisman-Mentesh, Itai Benhar, and Yariv Wine Abstract Antibody libraries came into existence 30 years ago when the accumulating sequence data of immunoglobulin genes and the advent of PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. As other antibody discovery tools, phage display is not an offthe-shelf technology and not offered as a kit but rather requires experience and expertise for making it indeed very useful. Next-generation sequencing (NGS) coupled with bioinformatics is a powerful tool for analyzing large amount of DNA sequence output of the panning. Here, we demonstrate how NGS analysis of phage biopanning (phage-Seq) of complex antibody libraries can facilitate the antibody discovery process and provide insights regarding the biopanning process (see Fig. 1). Key words Synthetic library, Phage display, Single-chain antibodies, scFv (single-chain variable fragment), VH (variable region of antibody heavy chain), VL (variable region of antibody light chain), FR (variable framework region), CDRs (complementarity determining regions), NGS (next-generation sequencing, HTS (high-throughput sequencing), Phage-Seq

1

Introduction The antibody phage display was the first and remains the most widely used method of gaining access to antibody libraries today (reviewed in [1, 2]). This method, in its most common format, is based on the expression of functional antibody fragments fused with the minor coat protein (g3p) of the filamentous M13 bacteriophage (phage) [3]. First demonstrated in 1990, it provided the basis for rapidly isolating recombinant antibodies from antibody libraries based on antigen-binding by individual library clones

Authors Adi Amir and David Taussig have equally contributed to this chapter. Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_18, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Overview of phage panning and analysis using phage-Seq. Phage panning process starts from naı¨ve “Ronit-1” library with expected scFv diversity of 2
1011. Cycles 1 and 3 are executed on antigen immobilized on plate, while in cycles 2 and 4, the antigen is coupled to magnetic beads. Phage panning is followed by either phage-ELISA or phage-Seq for analysis. (Created with BioRender.com)

[4]. In such systems, the genetic information encoding for the displayed molecule is physically linked to its product via the displaying phage particle that carries the antibody-coding gene as part of the encapsulated DNA. The most popular antibody formats present in libraries were the single-chain variable fragment (scFv), developed by the laboratories of Sir Gregory Winter at the Medical Research Council, Cambridge, UK [5] and Melvyn Little at Heidelberg, Germany [6]. Burton and Lerner’s research group at Scripps Research Institute, La Jolla, CA, USA, pioneered the fragment antigen-binding (Fab’) as the primary antibody format present in libraries [3]. The first libraries that were built from natural

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sources of sequence diversity, namely, animal or human B cells, were soon followed by libraries into which sequence diversity was inserted artificially [7]. Antibody libraries are classified as “immune libraries” when the source for antibody genes is an immunized donor [3, 7], or as “nonimmune” or “naı¨ve” libraries when the source for antibody genes is a donor (animal or human) that was not intentionally immunized for the purpose of the library construction [8]. Diversity can be artificially inserted into antibodies (by inserting random sequences into the antigen-binding site), resulting in a “semi-synthetic antibody library” or a “synthetic antibody library” [2, 9, 10]. An advantage of synthetic libraries is that scFv/Fab antibodies can be isolated against any desired target from a sufficiently large library [8, 10–14]. Phage libraries are enriched for specific binding clones by subjecting the phage to repetitive rounds of affinity selection followed by screening and sequencing of antigen-specific clones (a process also known as “panning” or “biopanning”). The screening step following the enrichment can be technically challenging and time-consuming; it is arduous to identify binders with lower representation within the pool and to evade dominant clones within the enriched population [15] (Fig. 1). Despite the fact that next-generation sequencing (NGS) technologies have provided a tremendous advance in sequence applications [16], they still have limitations concerning sequence read length [17]. Phage-Seq (see Fig. 2) is the utilization of NGS in the antibody phage display panning process. By using this methodology, one can gain insight into the phage panning process, including the enrichment of antibody libraries during the phage panning process. This can mainly be achieved by focusing the phage-Seq analysis on the antibody complementary determining regions (CDRs) as these are the key elements for antigen binding. The current NGS technology applied by Illumina is limited to 2
300bp for NGS instruments with high accuracy (Illumina, MiSeq). In order to meet this constraint, we designed primers that would amplify the variable region of the antibody heavy (VH) and light (VL) chains on two DNA amplicons, each of which overlapped the complementary determining region of the heavy chain (CDRH3), which is considered the most diverse CDR. Our phage-Seq approach is composed of three consecutive PCR reactions that will allow the reconstruction of the full scFv (i.e., both VH and VL parts of the scFv) (see Fig. 1). The first PCR is used to amplify the scFv encoding region from the phagemid backbone. The second PCR amplifies separately the VH and the VL parts of the scFv, with an overlap intended to allow assembly of the full-size scFv later on. Finally, the sequencing adaptors are attached during the third PCR.

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Fig. 2 Overview of phage-Seq library preparation steps. We use three PCR steps, the first to extract the full scFv region (PCR1), the second to separate VH and VL regions (PCR2), and the third to add the NGS sequencing adaptors with indexes creating the VH and VL phage-Seq amplicons (PCR3)

In 2009, we published within the MMB series a protocol for designing a human synthetic combinatorial library (“Ronit 1 library”) of scFvs [18]. In 2017, we published an additional, revised chapter within the MMB series, again describing the panning prosses of that human synthetic scFv antibody phage display library, using the n-CoDeR principle [12, 14, 19]. Here, we describe an updated version of the phage display and NGS library preparation protocols and demonstration of data from different cycles of the panning phage-Seq analysis. We create a phage-Seq library representing the antibody genes carried by the phage population after each cycle and demonstrate how bioinformatics tools that may facilitate the prediction of scFv clone binders. The actual construction of the original library [20] will not be described in this chapter; however, we describe it schematically in Fig. 3, and for additional details we refer the reader to the original chapter [18], or to the article where the construction of the “Ronit 1 library” was initially described [20]. One can also request a copy from the corresponding author.

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Fig. 3 An outline of the “Ronit 1” human-synthetic library construction. A: Human cDNA libraries from spleens, lymph nodes, and peripheral blood leukocytes are used as templates for PCR amplification of each CDR

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Materials Panning

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25  C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. Specific vendors are referred to in the following list of materials. We do not endorse any of these commercial entities, as similar products offered by other vendors may perform equally well. 1. Bacterial glycerol stock of a phage display library (in house production). 2. Glycerol. 3. Bacterial growth media: (a) YTAG: 2
YT medium: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, A: 100 μg/L ampicillin, G: 1% D-glucose. (b) YTAK: 2
YT medium, A: 100 μg/L ampicillin; K: 50 μg/L kanamycin. (c) Difco™ LB Broth, Lennox (BD, USA). (d) LBA: LB + 100 μg/mL ampicillin. (e) LBGA: LB + 0.4% D-glucose +100 μg/mL ampicillin. 4. Helper phage: A variety of helper phages are available for the rescue of phagemid libraries, such as VCS-M13 (Stratagene, La Jolla, CA, USA) and M13KO7 (Bio-Rad Laboratories, Hercules, CA, USA). According to the literature, there are helper phages that improve display scFv in 3 + 3 system [21, 22]. 5. Filtrap – Filter System 0.45μm CA (Corning, NY, USA). 6. PEG/NaCl (PEG6000-8000 200 g/L (Sigma, Israel); NaCl 146.1 g/L). 7. Phosphate buffers: (a) 10
phosphate-buffered saline (PBS) was purchased from Sigma, Israel.

ä Fig. 3 (continued) individually into CDR pools. B: The amplified CDR pools are mixed with oligonucleotides encoding framework regions, and intact cassettes encoding variable domains (VH and VL) are assembled using a two-step overlap-extension PCR. C: The amplified variable domain pools are mixed with oligonucleotides containing restriction sites (NcoI and the 50 of the VH; NotI at the 30 of the VL), and intact cassettes encoding scFv are assembled using overlap-extension PCR. D: The newly assembled scFv cassettes are cloned into pCC phagemid vector in frame by the NcoI and NotI restriction sites

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(b) PBST: PBS supplemented with 0.05% Tween-20 detergent (Sigma, Israel). (c) 3% MPBS: 3% skim milk powder in PBS. 8. For capture of his-tagged proteins – magnetic nickel beads: Dynabeads® His-Tag Isolation and Pulldown (Novex by Life Technologies Ltd., UK/Thermo Fisher Scientific cat.no. 10103D). For capture of biotinylated proteins and peptides: Dynabeads® M-280 Streptavidin (Invitrogen by Life Technologies/Thermo Fisher Scientific cat.no. 11206D). 9. 24-well cell culture plates (Merck cat.no. CLS3526). 10. Bovine serum albumin (BSA) (Merck cat.no. A3294). 11. Triethylamine (Merck cat.no. 90340). 12. 1.5 M Tris (HCl) solution pH 7.0. 13. E. coli strains: XL-1 Blue and TG-1 (see Note 1). 14. HRP-conjugated anti-M13 antibody (Sino Biological cat.no. 11973-MM05T-H). 15. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Bio-Lab cat. no. 16242352). 16. Triton-X100 (a nonionic surfactant) (Merck cat.no. 9036-19-5). 2.2

Phage-Seq

1. PCR. (a) Thermocycler. (b) Phusion DNA polymerase (NEB cat.no. M0530L). (c) Deoxynucleotide (dNTP) mix (NEB cat.no. N0447L). (d) Ultrapure water (upW). 2. Gel electrophoresis. (a) Agarose (Hylabs cat.no. K18100-500). (b) Tris acetate EDTA (TAE) buffer (Merck cat.co. T9650). (c) SYBR Safe DNA gel dye (Invitrogen cat.no. S33102). 3. DNA recovery from gel. (d) Zymoclean Gel DNA Recovery Kit (uncapped) (Zymo research cat.no. D4001). (e) Thermal block. 4. NGS sequencing. (f) Illumina MiSeq SY-410-1003).

instrument

(Illumina

cat.no.

(g) Illumina MiSeq Reagent Kit v3 2
300 bp (Illumina cat. no. MS-102-3003).

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3.1 Affinity Selection of scFv-Displaying Phages on Immobilized Antigen 3.1.1 Growth and Helper Phage Rescue of the Library (See Note 2)

1. To generate a full representation of the library, inoculate an aliquot of the bacterial library glycerol stock, 5–10 copies of each clone (~ 1
1010 clones) into 100 mL YTAG (~OD600nm ¼ 0.1). 2. Grow with 250 RPM OD600nm ¼ 0.4–0.6.

shaking

at

37



C

until

3. Infect the cells with helper phage at the multiplicity of infection (MOI) of 20 (number of helper phage particles/number of target bacteria, considering that 1 OD600nm ~ 2
108 bacteria/ mL) (see Note 1) and shake for a few seconds. 4. Incubate at 37  C for 30 min without shaking and then for additional 30 min with 250 RPM shaking. 5. Spin the infected cells at 3300 g for 10 min and resuspend the pellet in 200 mL of YTAK medium. Incubate overnight at 30  C with 250 RPM shaking. 6. Spin the culture at 8000 g for 10 min at 4  C and filter the supernatant with a 0.45 mm Filtrap. 7. Add 1/5 volume PEG/NaCl to the supernatant (50 mL PEG/NaCl to 200 mL YTAK). Mix well and keep on ice for 1 h or overnight in 4  C. 8. Spin at 10,800 g for 30 min at 4  C. Discard the supernatant and spin at 10,800 g for 5 min at 4  C. Discard the supernatant and resuspend the pellet in 0.5–5 mL of sterile PBS. 9. Store the phage supernatant at 4  C for short-term storage (and skip to step 11), or add sterile glycerol (to a final 15% v/v) for long-term storage at 80  C. “Rescued” phages that have been stored for no longer that 1 week at 4  C should be used as input for panning. 10. Before the panning procedure, precipitate phages using PEG/NaCl as described above, to remove the glycerol, and resuspend in sterile PBS. 11. To titer the phage stock, make serial tenfold dilutions of the phages in sterile PBS. Transfer logarithmic E. coli cells (see Note 1) into a sterile 96-well plate (90 μL/well) and infect with 10 μL of diluted phages (infect with the 107–1013 dilutions; take into consideration that 10 μL are already 102/mL dilution. For instance, 10 μL of phages into 990 μL PBS is 103/mL dilution. Taking 10 μL of 103/mL dilution to infect 90 μL of E. coli is already 105/mL dilution if seeding all 100 μL of infected bacteria. If seeding only 10 μL of infected bacteria, it is a 106/mL dilution. Mix by pipetting up and down and incubate at 37  C for 1 h. Plate the 100 μL infected cells on

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YTAG plates and grow overnight at 37  C. On the next day, count the colonies and multiply that number by the dilution factor to calculate the population size (AKA phage titer). Phage stock titer should be 1012–1013/mL. 3.1.2 Affinity Selection (Panning) on Immobilized Antigen

There are various approaches for antigen immobilization (e.g., plastic plates, polystyrene beads, immunotubes, and magnetic pull-down beads) that enable the enrichment of binders from a phage display library by applying sequential affinity-selection (panning) cycles. We found that using alternating phage capture approaches (i.e., a different antigen immobilization method in every other cycle) helps depleting phage clones that bind the surfaces of the solid phases used for protein immobilization (see Note 3). As a routine, we advise using two alternating complexes: (i) magnetic beads in the first and third cycles and (ii) a 24-well plate in the second and fourth cycles (for more efficient antigen binding, use high-binding plates). In addition, to deplete phages that may bind the blocking protein, we recommend using two alternating blocking solutions: 3% BSA in PBS and 3% skim milk powder in PBS. Due to space limitations, we describe here a selection method using magnetic nickel beads (IMAC) for immobilizing a 6
His-tagged antigen. For biotinylated antigens (proteins and peptides), we use streptavidin magnetic beads (Dynabeads M-280) in the bead-capture cycles. Please refer to our previously published protocol [18] for the description of using a 24-well plate for antigen immobilization (see Note 4). A scheme of library construction and affinity selection is shown in Fig. 4. The selection efficiency depends on many factors, such as the selection condition (an immobilized antigen on a plate or beads, or a cell-displayed antigen); the antigen concentration in a solution or its density on the surface of a solid phase; and antigen purity and the number of washes and the duration of each wash. To preserve rare binders, we recommend performing the initial panning cycles using relatively high antigen concentrations and short washes and employing more stringent washing conditions in later selection cycles (see Note 5). All incubations described below are performed at RT (room temperature, about 25  C) unless mentioned otherwise. Blocking 1. Using the suitable magnet, capture 1 mg of magnetic nickel beads (ensure excess of beads over antigen in terms of binding capacity) for 1 min, remove the supernatant (supe), wash with 1 mL PBS and capture again. Remove the supe and resuspend the beads in 1 mL of blocking solution (3% BSA in PBS), and incubate 1 h in a rotating platform (see Notes 6 and 7).

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Fig. 4 Scheme of library construction and affinity selection. Steps 1–3 are the library construction and phage preparation steps. Steps 4–7 describe an affinity selection cycle which should be repeated about four times to obtain sufficient enrichment of antigen binders allowing characterization of monoclonal phage clones (step 8)

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2. Suspend 1012 phages in 1 mL of blocking solution (this is the first panning input), and incubate for 1 h in a rotating platform. This blocking step decreases nonspecific binding of phages to the beads. Binding 3. If you wish to deplete phages that bind specific regions in your antigen, refer to Note 8. Otherwise, proceed to step 4. 4. Transfer the blocked phages to an Eppendorf tube containing 10 μg of a 6
His-tagged protein antigen. Incubate for 1 h in a rotating platform. 5. Capture the blocked beads (from step 1) for 1 min on the magnet and remove the supe. Transfer the antigen-phage mix to the beads. Incubate 30–60 min in a rotating platform. In this step, phage-antigen-bead complexes are formed. Washing 6. Capture the beads for 1 min on the magnet, remove the supe, add 1 mL of PBST, mix by relatively gentle vortexing, and return to the magnet. Repeat four more times with PBST and five times with PBS (see Note 5). Elution 7. Remove the excess PBS from the beads and elute phages by adding 1 mL of 100 mM triethylamine pH 13.0 (14 μL trimethylamine (7.18 M) in 1 mL ultrapure water, freshly prepared on the day of use), and incubate for 20 min on a rotating platform. 8. Capture the beads for 2 min on the magnet and transfer the eluted 1 mL phages into a 13 mL polypropylene culture tube containing 1 mL of 1.5 M Tris (HCl) pH 7.0. Mix by vortexing. Neutralized phages can be stored for several days at 4  C or (better) used to immediately infect E. coli cells (see Notes 1 and 9) as in step 9. The neutralized phage solution is the first panning output. Infection 9. Add 1 mL of the neutralized output phages (store the other half at 4  C) to 5 mL of an exponentially growing culture (OD600nm ¼ 0.4–0.6) of E. coli XL-1 Blue cells in 2
YT medium (see Note 1). Mix well and incubate at 37  C for 30 min without shaking and then for additional 30 min with shaking at 250 RPM. You may perform step 11 during this incubation time.

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10. Centrifuge the infected E. coli XL-1 Blue culture at 3300 g for 10 min. Resuspend the pellet in 1 mL YTAG and spread on two 15 cm YTAG plates. Grow overnight at 30  C. Titration 11. Transfer exponential E. coli XL-1 Blue cells into a 96-well plate (90 μL/well). Infect with serial tenfold dilutions of the input (1010–1012) and output phages (103–109). Incubate at 37  C for 1 h without shaking. Plate on YTAG plates. Grow overnight at 37  C to determine the panning input and output titer (see Subheading 3.1.1, step 11 for determining phage titer). Output phage titer should be 104 and  107 / mL. Amplification of Output Phages and Further Selection Cycles 12. The first selection cycle is the most important one. Any errors made at this point will only be amplified in the following selection cycles. You should recover at least 104 phages as cycle 1 panning output. If you obtain fewer phages, it is probable that a mistake had occurred. Repeat the infection of the remaining 1 mL of eluted, neutralized phages (see Notes 10 and 11); otherwise, continue to further selection cycles. 13. Using a cell scraper, scrape the output cells (from step 10) into 10 mL of YTAG medium. Plate serial tenfold dilutions onto YTAG plates to determine how much the library was amplified during the overnight growth. Prepare glycerol stocks (by adding glycerol to a final concentration of 15% v/v) and store 1 mL aliquots at 80  C. 14. Once you know the titer of the scraped bacteria, inoculate (in 100 mL YTAG medium) with a number of cells that yield at least 20 copies of phage output, i.e., (scraped bacteria titer)/(phage output titer)  20. For instance, having a bacterial titer of 109/mL and phage titer of 106/mL, inoculate 20 μL of scraped bacteria (2
107 cells). 15. Continue with phage rescue as in Subheading 3.1.1, steps 2–11. 16. Use 1–5
1011 phages as input for the next panning cycle. Store the remaining phages at 4  C (see Note 12). 17. Repeat the selection for a total of three to four cycles. In each cycle, decrease the size of phage input and the antigen’s concentration (a factor of 2–10 is reasonable) (see Notes 5, 12, and 13). 18. Monitor the ratio between panning input and panning output in each cycle. With successful enrichment of antigen binders, you should observe a descending input/output ratio.

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Phage ELISA serves as the primary method for screening scFvdisplaying phage clones that specifically bind the antigen. Therefore, for reliable results, high-phage titers are critical as well as the number of scFv molecules displayed on an average phage. We addressed those issues by comparing phages that were produced in various strains of E. coli and found that phages rescued from E. coli TG-1 cells yield the strongest signals in phage ELISA. Consequently, TG-1 cells should be infected with output phages of the last panning cycle (XL-1 Blue cells should also be infected for preparing glycerol stocks and for phage rescue of further panning cycles, if required). Preparation of Single-Phage Clones 1. Use 50–100 μL of phages from the last output cycle (either those eluted and neutralized at the end of the last panning cycle or rescued phages from XL-1 Blue cells) to infect a 5 mL exponential E. coli TG-1 culture growing in 2
YT medium. Mix well and incubate at 37  C for 30 min without shaking and then for additional 30 min shaking at 250 RPM. 2. Spread dilutions of infected cells on YTAG plates to obtain single, isolated colonies and grow overnight at 37  C. 3. On the following day, use sterile inoculation loops or pipette tips to pick single colonies into single wells in a 96-well plate containing 100 μL/well of YTAG. Keep one well sterile for blank control. Grow overnight at 30  C with gentle shaking (100–150 RPM, to avoid contamination between wells; we use a plastic box with sponges or styrofoam to stabilize the plate). This is the master plate. Phage Rescue 4. On the next day, dilute the cells 1/10 by transferring 10 μL from each well of the master plate into a new 96-well plate containing 90 μL/well of YTAG. Grow to mid-log at 37  C shaking at 100–150 RPM (approx. 2 h for TG-1 cells). This is the rescue plate. 5. Initiate rescue by adding 11 μL of 1010/mL helper phage per well. Incubate at 37  C for 30 min without shaking and then for additional 30 min shaking at 100–150 RPM. 6. Spin the rescue plate at 3200 g for 10 min at 14  C. Discard the supernatant quickly and add 150 μL/well of YTAK. Grow overnight at 30  C shaking at 100–150 RPM. Proceed to step 7 on this day.

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Phage ELISA 7. Coat two ELISA plates (50 μL per well) at 1–5 μg/mL of antigen (1) or control protein (2) diluted in PBS, overnight at 4  C. 8. On the following day, wash the ELISA plates with PBST and block with 300 μL/well of 3% MPBS for 1 h at 37  C. 9. Wash three times with PBST and add 50 μL/well of PBST to the control protein-coated wells. 10. Spin the rescue plate at 3200 g for 10 min at 14  C. Transfer 50 μL/well of supernatant into the control protein-coated wells (already containing 50 μL PBST). Mix well by pipetting up and down and transfer 50 μL into the antigen-coated wells (see Note 14). 11. Complete phage ELISA by adding 50 μL/well of anti-phage secondary antibodies (e.g., HRP-conjugated anti-M13) and developing with an appropriate substrate. 12. Repeat the procedure at least once (including rescue from the same master plate) to discriminate false positives and to confirm specificity of initial binders (use a different control protein each time). Use the master plate to inoculate validated clones on YTAG plates (to obtain well-isolated, single colonies). 13. (Optional) Proceed to step 14 to perform another screening phase by expressing soluble scFv antibodies (see Note 15). Otherwise, skip to step 21. Soluble ELISA 14. Prepare 5 mL LBA starters by inoculating single colonies of phage ELISA-verified binders and grow overnight at 37  C shaking at 250 RPM. 15. Per clone, keep 1 mL for glycerol stock (15% v/v) and 3 mL for plasmid DNA preparation. Add the remaining 1–9 mL LBGA in a 50 mL tube and grow at 37  C shaking at 250 RPM. 16. At OD600nm ¼ 0.8, cool cells to 30  C and induce scFv expression by adding IPTG to a 0.5 mM final concentration. Incubate for 3–4 h at 30  C shaking at 250 RPM. 17. Collect the cells by centrifugation at 3300 g for 10 min. Resuspend the pellet(s) in 1 mL of PBS + 0.1% TritonX100, and lyse the cells preferably by sonication. 18. Spin the cell extracts at 12,000 g for 15 min at 4  C. Collect the soluble fractions (supernatants); these fractions contain the soluble scFv molecules (see Note 16).

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19. Dilute the soluble fractions 1:1 in PBST and perform ELISA (plates coated with 1–5 μg/mL of antigen and control proteins). Use anti-tag antibodies for detection (see Note 17). 20. If an assessment of scFv expression levels is required, conduct a western blot analysis with 10 μL of soluble fraction alongside a series of dilutions from a reference scFv protein of known concentration. Evaluation of Antibody Diversity 21. Prepare plasmid preparations for all positive, antigen-specific clones. The diversity can be assessed by sequencing the scFv domain. 3.3

Phage-Seq

Phage-Seq enables the capture of scFv clones that persist over consecutive phage display biopanning cycles. The methodology described herein is based on the generation of two separated phage-Seq libraries derived from amplification of all CDRH1-3 of the scFv-VH (designated as VH amplicon) and all CDRL1-3 of the scFv-VL fused with the CDRH3 (designated the VL amplicon). This protocol in an amendment of a protocol that was published in 2018 [23]. Prepare PCR reaction according to Table 2 for each sample. The sequences of the required PCR primers are given in Table 1. 1. Perform PCR under the following conditions. No. of cycles

PCR step

Temperature [ C]

Time [mm:ss]

1

Initial denaturation

98

03:00

15

Denaturation Annealing Elongation

98 55 72

00:30 00:30 00:45

1

Final elongation

72

07:00

–

Hold

4

1

2. Apply PCR products on 1.5% agarose gel in TAE. Use SYBR Safe as gel dye and visualize bands under blue light. 3. Extract DNA-Gel bands corresponding to 900 bp (those are the extracted scFv sequences). 4. Recover DNA using Zymoclean Gel DNA Recovery kit. Elute DNA in 10 μL of upW. 5. Prepare PCR step 2 reaction with the following primers (Table 3) for amplification of VH and VL amplicon according to Table 4. Use 5 μL of recovered DNA in step 5 as template for each reaction.

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Table 1 Nucleotide sequence of primers PCR1 Primer

Nucleotide sequence (50 –30 )

TAB-RI primer forward PCR1

CCATGATTACGCCAAGCTTTGGAGCC

CBD-AS primer reverse PCR1

GAATTCAACCTTCAAATTGCC

Table 2 Composition of PCR1 reactions Component

Volume [μL]

NTC [μL]

upW

44.5

9.3

dNTPs [10 μM]

1

0.2

Primer FW TAB-RI

1

0.2

Primer REV CBD-AS

1

0.2

Phusion polymerase

0.5

0.1

a

2

X

50

10

Template

Total a

Templates are phagemids recovered from each biopanning cycle

6. Perform PCR under the following conditions. No. of cycles

PCR step

Temperature [ C]

Time [mm:ss]

1

Initial denaturation

98

03:00

20

Denaturation Annealing Elongation

98 58 72

00:30 00:30 00:30

1

Final elongation

72

07:00

–

Hold

4

1

7. Load PCR products onto 1.5% agarose gel in TAE. Use SYBR Safe as gel dye and visualize bands under blue light. 8. Extract DNA-Gel bands corresponding to 460 bp and 510 bp for VH and VL amplicons, respectively. 9. Recover DNA using Zymoclean Gel DNA Recovery kit. Elute DNA in 8 μL of upW. 10. Prepare PCR step 3 reactions with Illumina TruSeq primers (Table 5), according to Table 6. The reverse primer comprises the sample identifying index for NGS. Use all the recovered DNA in step 10 as template for each reaction.

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Table 3 Nucleotide sequence of primer PCR2 Primer

Nucleotide sequence (5’-3’)

VH part extraction Primer

CCCTCCTTTAATTCCCGAGGTGCAGCTGTTGGAGTCT

forward PCR2 *Kappa

GAGGAGAGAGAGAGAGGGAGACTGCGTCAACACGATATC

primer reverse PCR2 *Lambda

GAGGAGAGAGAGAGAGTGGCTGAGTCAGCACGATATC

primer reverse PCR2 VL part extraction Primer

CCCTCCTTTAATTCCCGGCCGTGTATTACTGTGCAAG

forward PCR2 Primer

GAGGAGAGAGAGAGAGTATTTGCGCCACCTGCGG

reverse PCR2 a

VH reverse primers are mixed 1:1 before PCR reaction preparation (red, sequence used for the addition of NGS adaptors in PCR3 step; blue, master framework (FR) annealing region)

Table 4 Composition of PCR2 reactions Component

Volume [μL]

NTC [μL]

upW

42.5

9.3

dNTPs [10 μM]

1

0.2

Primer FW

1

0.2

Primer REV

1

0.2

Phusion polymerase

0.5

0.1

Template

4

X

Total

50

10

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Table 5 Nucleotide sequence of primer PCR3 Primer

Nucleotide sequence (5’-3’)

Primer forward PCR3

AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT[N NNN]CCCTCCTTTAATTCCC

Primer reverse PCR3

CAAGCAGAAGACGGCATACGAGAT[XXXXXX] GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCT[NNNN]GAGGAGAGAGAGAGAG

(red, parts identify the overlap region with primers used in PCR2) [XXXXXX] ¼ 6 bp index [NNNN] ¼ 4 bp required diversity region

Table 6 Composition of PCR3 reactions Component

Volume [μL]

NTC [μL]

upW

42.5

9.3

dNTPs [10 μM]

1

0.2

Primer FW

1

0.2

Primer REV

1

0.2

Phusion polymerase

0.5

0.1

Template

4

X

Total

50

10

11. Perform PCR under the following conditions. No. of cycles

PCR step

Temperature [ C]

Time [mm:ss]

1

Initial denaturation

98

03:00

8

Denaturation Annealing Elongation

98 56 72

00:30 00:30 00:30

1

Final elongation

72

07:00

–

Hold

4

1

12. Apply PCR products (VH and VL amplicons) on 1.5% agarose gel in TAE. Use SYBR Safe as gel dye and visualize bands under blue light.

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Fig. 5 scFv VH CDR3 length distribution across phage-Seq cycles. Histogram of unique VH CDR3 length as identified using phage-Seq of VH amplicon. Y-axis represents the number of phage-Seq reads associated with each CDR3 length. X-axis represents the CDR3 length in amino acids. CDR3 lengths of scFv clones identified by phage-ELISA are marked by vertical gray lines

13. Extract DNA-Gel bands corresponding to 590 bp and 640 bp for VH and VL amplicon, respectively. 14. Recover DNA using Zymoclean Gel DNA Recovery kit. Elute DNA in 8 μL of upW. 15. Phage-Seq library pooling and sequencing: (a) Pool phage-Seq libraries derived from step 15 to achieve final concentration of 1 nM and add 15% Phix library. (b) Sequence using MiSeq 2
300 bp reagent kit v3. We used this protocol to generate the VH and VL amplicons derived from cycles 2–4 of phage display biopanning products. As can be seen in Fig. 5, the CDR3 length profile as identified in the VH amplicon changes along with the progression of the biopanning cycles. Although the difference in counts of scFv clones is explained by the greater depth of sequencing obtained in cycle 4 (C4), a clear selection toward VH CDR3 of the length of 10 amino acids can be observed. Interestingly, none of the phage-ELISA identified clones exhibited the VH CDR3 length of 10 amino acids, suggesting that affinity selection (clone abundance) is not mainly driven by specific affinity. Furthermore, the scFv clone with a VH CDR3 length of 23 amino acids indicated that high affinity binders can be found

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Fig. 6 scFv VL CDR3 length distribution across phage-Seq cycles. Histogram of unique VL CDR3 length as identified using phage-Seq of VL amplicon. Y-axis represents the number of phage-Seq reads associated with each CDR3 length. X-axis represents the CDR3 length in amino acids. CDR3 lengths of scFv clones identified by phage-ELISA are marked by vertical gray lines

among the super rare clones, which further underlines the difficulty of identifying high affinity binders by phage-ELISA. We were not able to observe this same phenomenon with the VL CDR3 lengths (see Fig. 6). When looking at the most abundant scFv clones (defined by CDRH3 and CDRL3 pair), the only clone identified by phageELISA is found within the low abundant clones in the phage-Seq data (see Fig. 7). The top represented clones do not share CDRs with the clones identified using phage-ELISA. This coupled with their extreme expansion compared to other clones in cycles 3 and 4 indicates that their biological properties, such as bacterial expression, are the root of their competitive advantage (see Fig. 8).

4

Notes 1. Filamentous phages infect F+ E. coli via the sex pili. For sex pili production and efficient infection by phage, E. coli must be grown at 37  C and be in the exponential (logarithmic) growth phase (OD600 nm of 0.4–0.6).

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Fig. 7 scFv VH and respective VL CDR3 length distribution across phage-Seq cycles. Histogram of unique VH and respective VL CDR3 length as identified using phage-Seq of VH amplicon. Y-axis represents the number of phage-Seq reads associated with each CDR3 length. X-axis represents the CDR3 length in amino acids. CDR3 lengths of scFv clones identified by phage-ELISA are marked by vertical gray lines

2. All glassware that had been used for phage work should be immersed in a diluted solution (5%) hypochlorite (chlorine bleach) before being sterilized by autoclaving. 3. Some phages in the library stick to the surfaces that are used to immobilize the antigen. These phages will be eluted with the antigen-bound phages and therefore they will be amplified. Although sticky phages can be discriminated in the screening phase, it is recommended to deplete them sooner by not repeating an immobilization method in the following cycle. 4. The detailed affinity selection process relates to immobilized antigens or to panning using soluble antigen followed by capture using magnetic beads. This approach is suitable for protein and peptide antigens. In addition to protein and peptides [24– 26], we successfully isolated from the “Ronit 1 library” and from immune scFv phage libraries antibodies that bind different antigens such as hapten-carrier conjugates [16], antigen-expressing mammalian cells (with counter selection on antigen-negative cells) [27], crystalline facets of semiconducting materials [28], and whole fungal cells (unpublished data).

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Fig. 8 Relative frequency of paired VH and VL CDR3 across phage-Seq cycles. Marked in red is the one clone identified by phage-ELISA within the phage-Seq data. The top 20 clones according to frequency are listed in black. Y-axis represents the number of phage-Seq reads (in log scale) associated with each VH CDR3 and its respective VL CDR3. X-axis represents the cycle number

5. Decrease antigen concentration on progressing cycles to enrich the high-affinity binders’ population. Standard antigen concentrations for the first four cycles: 10, 5, 1, and 0.1 μg/mL. Perform short washes (1–5 min each) in the first cycle and increase the durations in the following cycles (10 min or longer). The number of the washes can be increased in the following cycle up to 20 washes. 6. When using polystyrene surfaces (such as 24-well plates) for immobilization, the antigen must be carrier-free (CF) to prevent amplifying phages that bind the carrier protein. The antigen does not have to be CF when using affinity-based immobilizing methods. 7. To deplete sticky phages that could bind BSA, use a different blocking solution in the second cycle (for instance, 3% MPBS).

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You may use BSA again in the third cycle and so on. Using the same blocking reagent for all cycles usually leads to the isolation of blocker-specific antibodies at the expense of antigenspecific ones. 8. Some antigens contain regions or domains that are common in other proteins, such as immunoglobulin domains or conserved regions between homologous proteins from different organisms. This might result in amplification of clones that bind those regions and fewer clones that bind antigen-specific regions. To isolate antigen-specific clones, perform a depletion step in which a control protein is immobilized. Allow phage-control protein complexes to form during 1 h incubation with rotation, and collect the unbound phages before exposing them to the desired antigen. By doing this we were able to isolate anti-idiotype antibodies that specifically bind the CDRs of Remicade (anti human TNF-α), when Avastin (antihuman VEGF-A) had served as the control protein (unpublished data). If there is no control antigen, it is recommended to perform depletion on he blocked matrix (e.g., beads, plate). 9. E. coli strains such as XL-1 Blue, but not TG1, which possess the recA1 genotype, are less likely to insert DNA mutations that result from recombination. Therefore, use XL-1 Blue cells for amplification of output phages and storage. 10. Few or no colonies on plates after the first panning cycle may indicate that the cells lost the F pilus and were not infected by output phages or that antigen coating was not efficient – start a cell culture from a single colony on a minimal plate. Grow the cells at no lower than 37  C. Optimize coating and blocking buffers and conditions of the wells. 11. Too many colonies (>107) after the first panning cycle: This may be due to inadequate blocking of wells (optimize coating and blocking conditions of the wells); inadequate blocking of phages (block the phages with the same blocking solution used to block the wells); insufficient washing (increase the number of washes). 12. When your library is sufficiently large (>109 clones), you should be able to isolate high-affinity binders against most antigens (affinity of 108–109 M). To preferably isolate the high-affinity binders, apply “off-rate selection” by prolonging the washing time: after the 20 washes (Subheading 3.1.2, step 6), fill the well again with PBS supplemented with 1% BSA and drain it after 15 min; repeat several times so that accumulated washing time is from 1 h to overnight (and even longer). It may be advantageous to run several panning wells in parallel, each with a different total duration of washing to determine the

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optimal conditions for your library and particular antigen. It is also suggested to decrease the input by a factor of 10 for each progressive panning cycle. 13. If no positive binders are identified after three to four panning cycles, the enrichment is insufficient. Perform additional panning cycles or start from scratch using a different panning approach. 14. When performing a phage ELISA (as described in [29]), we usually coat half a plate (columns 1–6) with antigen and the other half with a control protein such as BSA. After coating and blocking, the control half of the plate is filled with 100 μL/well of PBST. To these wells, 100 μL of rescued phages from the picked clones are added and mixed, and then 100 μL are transferred to the antigen-coated wells. The plate is further developed by incubation with anti-phage and secondary antibodies and the appropriate substrate. This gives an important specificity control and helps avoid carrying nonspecific (“sticky”) phage clones to further validation and characterization steps. 15. Some phage-displayed antibodies lose their ability to bind the antigen when expressed in soluble formats (scFv, IgG, etc.), and some of them bind it nonspecifically. Therefore, a soluble antibody format screening step is recommended to eliminate such misleading phage clone hits. 16. To prevent degradation of the soluble scFv, it is strongly recommended to add a protease inhibitor cocktail to the soluble fractions and to store them at 4  C (short term) or at 20  C (long term). 17. Select the detection antibody according to the tag of your library. While in our pCC phagemid there is a CBD tag, more common phagemid vectors have a hexa-histidine tag, a myc tag, or both.

Acknowledgments In 2009, we published in “Methods in Molecular Biology” a chapter describing the construction of a large human synthetic singlechain Fv antibody library displayed on phage, where in vivo formed complementarity determining regions (CDRs) were shuffled combinatorially onto germline-derived human variable region frameworks [18]. The present chapter is a revision and update of that chapter, not including the part of library construction. We are grateful to past and present members of the Benhar and Wine Labs for their contributions in optimizing the antibody phage display protocols described herein.

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References 1. Benhar I (2007) Design of synthetic antibody libraries. Expert Opin Biol Ther 7(5):763–779 2. Hoogenboom HR (2005) Selecting and screening recombinant antibody libraries. Nat Biotechnol 23(9):1105–1116 3. Burton DR, Barbas CF 3rd, Persson MA, Koenig S, Chanock RM, Lerner RA (1991) A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci U S A 88(22):10134–10137 4. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554 5. Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G, By-passing immunization. (1991) Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222(3):581–597 6. Breitling F, Dubel S, Seehaus T, Klewinghaus I, Little M (1991) A surface expression vector for antibody screening. Gene 104(2):147–153 7. Clackson T, Hoogenboom HR, Griffiths AD, Winter G (1991) Making antibody fragments using phage display libraries. Nature 352(6336):624–628 8. Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14(3):309–314 9. Barbas CF 3rd, Bain JD, Hoekstra DM, Lerner RA (1992) Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc Natl Acad Sci U S A 89: 4457–4461 10. Griffiths AD (1993) Production of human antibodies using bacteriophage. Curr Opin Immunol 5(2):263–267 11. de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC, Henderikx P et al (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274(26):18218–18230 12. Holt LJ, Bussow K, Walter G, Tomlinson IM (2000) By-passing selection: direct screening for antibody-antigen interactions using protein arrays. Nucleic Acids Res 28(15):E72 13. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G et al (2000) Fully

synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296(1):57–86 14. Soderlind E, Strandberg L, Jirholt P, Kobayashi N, Alexeiva V, Aberg AM et al (2000) Recombining germline-derived CDR sequences for creating diverse singleframework antibody libraries. Nat Biotechnol 18(8):852–856 15. Nannini F, Senicar L, Parekh F, Kong KJ, Kinna A, Bughda R et al (2021) Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments. MAbs 13(1):1864084. https://doi.org/10.1080/19420862.2020. 1864084 16. Saggy I, Wine Y, Shefet-Carasso L, Nahary L, Georgiou G, Benhar I (2012) Antibody isolation from immunized animals: comparison of phage display and antibody discovery via V gene repertoire mining. Protein Eng Des Sel 25(10):539–549. https://doi.org/10.1093/ protein/gzs060 17. Lovgren J, Pursiheimo JP, Pyykko M, Salmi J, Lamminmaki U (2016) Next generation sequencing of all variable loops of synthetic single framework scFv-Application in antiHDL antibody selections. New Biotechnol 33(6):790–796. https://doi.org/10.1016/j. nbt.2016.07.009 18. Nahary L, Benhar I (2009) Design of a human synthetic combinatorial library of single-chain antibodies. Methods Mol Biol 525:61–80, xiv. https://doi.org/10.1007/978-1-59745554-1_3 19. Jirholt P, Ohlin M, Borrebaeck CA, Soderlind E (1998) Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework. Gene 215(2):471–476 20. Azriel-Rosenfeld R, Valensi M, Benhar I (2004) A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions. J Mol Biol 335: 177–192 21. Oh MY, Joo HY, Hur BU, Jeong YH, Cha SH (2007) Enhancing phage display of antibody fragments using gIII-amber suppression. Gene 386(1-2):81–89. https://doi.org/10. 1016/j.gene.2006.08.009 22. Baek H, Suk KH, Kim YH, Cha S (2002) An improved helper phage system for efficient isolation of specific antibody molecules in phage

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display. Nucleic Acids Res 30(5):e18. https:// doi.org/10.1093/nar/30.5.e18 23. Vaisman-Mentesh A, Wine Y (2018) Monitoring phage biopanning by next-generation sequencing. Methods Mol Biol 1701:463– 473. https://doi.org/10.1007/978-1-49397447-4_26 24. Nahary L, Trahtenherts A, Benhar I (2009) Isolation of scFvs that inhibit the NS3 protease of hepatitis C virus by a combination of phage display and a bacterial genetic screen. Methods Mol Biol 562:115–132. https://doi.org/10. 1007/978-1-60327-302-2_9 25. Trahtenherts A, Benhar I (2009) An internalizing antibody specific for the human asialoglycoprotein receptor. Hybridoma (Larchmt) 28(4):225–233. https://doi.org/10.1089/ hyb.2009.0019 26. Ofir K, Berdichevsky Y, Benhar I, AzrielRosenfeld R, Lamed R, Barak Y et al (2005) Versatile protein microarray based on

carbohydrate-binding modules. Proteomics 5(7):1806–1814. https://doi.org/10.1002/ pmic.200401078 27. Shimoni M, Herschhorn A, Britan-Rosich Y, Kotler M, Benhar I, Hizi A (2013) The isolation of novel phage display-derived human recombinant antibodies against CCR5, the major co-receptor of HIV. Viral Immunol 26(4):277–290. https://doi.org/10.1089/ vim.2012.0029 28. Artzy Schnirman A, Zahavi E, Yeger H, Rosenfeld R, Benhar I, Reiter Y et al (2006) Antibody molecules discriminate between crystalline facets of a gallium arsenide semiconductor. Nano Lett 6(9):1870–1874. https://doi. org/10.1021/nl0607636 29. Benhar I, Reiter Y (2001) Phage display of single-chain antibodies (scFvs). In: Colligan J (ed) Current protocols in immunology. Wiley, pp 10.9B.1–10.9B.39

Chapter 19 Selection of Affibody Affinity Proteins from Phagemid Libraries Kim Anh Giang, Per-A˚ke Nygren, and Johan Nilvebrant Abstract Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories. Key words Phage display, Affibody, Antigen preparation, Phage-ELISA, Signal normalization

1

Introduction Phage display is a well-established procedure to enrich binders from affinity protein libraries utilizing the genotype-phenotype linkage provided by the phage particle architecture. The rapid development of recombinant DNA technology now allows for a fully in vitro process where target binding affinity protein clones can be isolated from synthetically generated libraries and the sequence-identified binders later produced via genes recovered from individual bacterial clones or produced via gene synthesis. Since this approach does not require animal immunization for the generation of a binding protein repertoire, it allows binding proteins to be generated based on essentially any stable protein scaffold that can accommodate a diversified binding surface (e.g., a non-antibody scaffold protein). This protocol describes a step-by-step procedure to select affibody affinity proteins from combinatorial libraries displayed on filamentous phage, but the protocol can in principle be applied to any class

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_19, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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of binding protein library displayed on phage particles using a similar phagemid vector. Affibody molecules are small (ca. 6.5 kDa, 58 amino acids) binding proteins based on a three-helix bundle framework derived from one of the domains of Staphylococcal Protein A [1, 2]. Combinatorial libraries are generated by simultaneous randomization of 13–14 surface-exposed positions in helices one and two localized on a flat surface of the domain (Fig. 1a), which in the native protein forms a high affinity interface for binding to immunoglobulin Fc. Diversification of positions in this binding interface to generate diverse libraries from which affibodies to completely different targets than IgG Fc can be selected is typically achieved via utilization of oligonucleotide building blocks containing specified mixes of trinucleotide codons at the randomized positions. Compared to the use of so-called degenerate codons (e.g., NNG/T), this allows for a high degree of control over the introduced diversity while avoiding unwanted codons, e.g., cysteine and proline, that may become involved in undesired disulfide bonds or destabilize the expected helical secondary structure, respectively. Our protocol utilizes a phagemid-encoded affinity protein (here affibody) library fused in-frame to an albumin binding domain tag (denoted ABD) and a truncated phage coat protein 3 (p3) in conjunction with M13K07 helper phage (Fig. 1b). Of note, upon phage assembly in the bacterial host, this system will result in the generation of four different types of phage particles that may display an affibody-tagp3 fusion protein or only wild-type full-length p3 and harbor either phagemid- or helper phage ssDNA, respectively (Fig. 1c). To isolate antigen-binding clones, a phage library stock is prepared via helper phage superinfection of bacterial cells harboring the phagemid library, and this phage library is then cycled through affinity selections (involving library re-amplifications between selection rounds) on labeled – typically, chemically, or enzymatically biotinylated – antigen in four to five rounds, followed by clonal binding analyses including phage-ELISA as described in more detail below (Fig. 1d). In general, affibody molecules and other non-antibody scaffold proteins are based on small and stable protein domains that can be conveniently expressed in bacteria [3–5]. The small size and relatively simple composition compared to larger antibody fragments can offer advantages over antibody-based binders for certain applications, e.g., in in vivo tumor imaging applications where a small probe size is linked to a rapid tissue distribution and blood clearance (e.g., low background) [6]. Furthermore, small modular binding units as such are ideal as gene fusion appendages to other proteins to provide a targeting function [7], e.g., in immunotoxins [8] or as building blocks to create multi-specific constructs for various applications, for example, receptor blockage [9], simultaneous inhibition of different cytokines [10], or synergistic binding

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Fig. 1 Overview of the affibody phage display system. (a) Schematic representation of the 58 aa affibody affinity protein three-helix bundle scaffold. Positions in helices one and two that are combinatorially randomized to generate libraries and shown in red and are numbered. (b) The protocol is based on the use of a combined phagemid + helper phage system for phage display of library members. The phagemid harbors an IPTG-inducible (and glucose-suppressible) lac promoter-driven expression cassette encoding for a signal peptide (OmpA) in fusion with a library member (the affinity protein), a suitable detection tag (e.g., an albumin binding domain or a FLAG-tag), an amber stop codon (TAG), and a truncated version of the Fd phage coat protein 3 (p3). In glnV/SupE suppressor E. coli strains (e.g., ER2738, XL1-Blue, TG-1), the amber stop codon can be read-through via incorporation of a glutamine amino acid. If the phagemid vector is transformed into a non-suppressor strain (e.g., BL21, RV308), the amber codon is read as a stop codon, resulting in the production of soluble (not p3-fused) library member-tag fusion protein that can be purified and analyzed. The phagemid also contains an antibiotic marker (Amp/Carb) and signals for replication in E. coli and phage particle packing. For phage particles to be produced, a phagemid-containing cell needs to be infected with helper phage particles, providing the helper phage genome encoding all necessary phage proteins. M13K07 helper phage (kan resistance marker) is used as helper phage in the present protocol. (c) The use of a phagemid + helper phage system results in the production of four different types (i–iv) of phage particles, of which only one type (i) is of the desired kind. This particle type displays the phagemid-encoded library member as fused to the truncated p3 proteins, typically in a monovalent fashion, alongside copies of wild-type p3 encoded from the helper phage genome present in the same cell. (d) Schematic representation of the cyclic selection/amplification process, involving incubation of the phage library with biotinylated target protein, capture of phage/target complexes onto paramagnetic beads, washing, elution of captured phage, infection of E. coli, and production of a new phage stock (enriched for target binding library members) for the next round. After ca. 4–5 cycles, infection of E. coli with the eluted phage gives individual colonies/clones that can be analyzed further (e.g., using phage-ELISA, DNA sequencing)

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to several epitopes of virulence factors [11]. The lack of native cysteine residues in the affibody sequence enables facile site-specific modification with, e.g., maleimide fluorophores or other thiolreactive probes to an added unique cysteine residue, as exemplified by affibody-drug conjugates [12]. The full affibody sequence may also be produced via solid-phase peptide synthesis [13, 14] and thereby enable incorporation of non-natural amino acid derivatives with unique properties, e.g., for click chemistry. An alternative approach based on recombinant incorporation of non-canonical amino acids via engineered orthogonal tRNA-aminoacyl tRNAsynthetase pairs is also possible [15, 16]. Next-generation DNA sequencing has become an increasingly popular means to screen and analyze the output from library selection campaigns, particularly using Illumina HiSeq or MiSeq sequencing-by-synthesis technology [17, 18]. However, short read lengths (typically 3–400 bp without using paired-end reads), technical limitations in retaining cognate pairs of two-chain antibody heavy- and light-chain variable gene fragments, or covering the full reading frame of larger affinity protein classes and challenges to translate observed sequence convergences into true functional hits represent current challenges that often require creative experimental and bioinformatic workarounds [19–22]. The small size of the single polypeptide chain affibody scaffold (58 amino acids = 174 base pairs) circumvents many of these common pitfalls since the full sequence can be contained within a single read. Moreover, the small size also facilitates the conversion of in silicoidentified sequences of interest into physical genes via affordable manufacture of the corresponding short synthetic DNA with low inherent error rates. Nevertheless, even if such endeavors also are performed in our laboratory in parallel, the current protocol describes manual handling of clones, phage-ELISA screening for function, Sanger-DNA sequencing, and a final retrieval of individual affibody genes from phagemid inserts in corresponding bacterial colonies. Thus, although the approach is amenable for both automation of some steps and deep sequencing of selection outputs, the described manual strategy works well for most situations in our hands, can be applied to several antigens or selection strategies in parallel, and does not require any specialized laboratory equipment.

2

Materials 1. α-M13-HRP: anti-M13 monoclonal antibody, horse radish peroxidase conjugate. 2. 0.5 M HAc: adjust pH with NaOH to 2.8 and filter sterilize (0.2 μm).

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3. 1 M Tris–HCl: adjust pH to 8 (with HCl) and filter sterilize (0.2 μm). 4. 1.5 mL microcentrifuge tubes. 5. 2 M H2SO4. 6. 25 mM Tris–HCl, 2 mM EDTA. 7. 5% (w/v) BSA: 5% (w/v) bovine serum albumin in PBS. Mix and filter sterilize (0.2 μm). 8. 96-well deep-well plates (1 mL). 9. Baffled E-flasks (5 L). 10. Blood agar/carb plates: 40 g/L blood agar, 100 μg/mL carbenicillin. 11. Blood agar/kan plates: 40 g/L blood agar, 25 μg/mL kanamycin. 12. Blood agar/tet plates: 40 g/L blood agar, 10 μg/mL tetracycline. 13. Bovine serum albumin (BSA). 14. Carbonate buffer: 0.03 M Na2CO3, 0.07 M NaHCO3. Mix, adjust pH to 9.6 with HCl/NaOH and filter sterilize (0.2 μm). 15. Cooled centrifuge and centrifuge bottles (variable size). 16. Clear Flat-Bottom Immuno Nonsterile 384-Well Plates (Thermo Fisher Scientific, Rockford, IL, USA). 17. D-glucose. 18. DMSO: dimethyl sulfoxide. 19. Dynabeads M-280 Streptavidin (Thermo Fisher Scientific). 20. E. coli ER2738 (Lucigen, Middleton, WI, USA). 21. E. coli XL1-Blue (Agilent, Santa Clara, CA, USA). 22. E-flasks (regular; 5 L and 1 L). 23. EZ-Link Sulfo-NHS-LC-LC-Biotin Scientific).

(Thermo

Fisher

24. Freeze dryer. 25. Heating block. 26. M13K07 helper phage (New England Biolabs, Ipswich, MA, USA). 27. Magnetic rack. 28. Microplate reader (for A450 nm). 29. PBS: phosphate-buffered saline, 150 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4·H2O. Adjust pH to 7.4 with HCl, filter sterilize (0.2 μm), and autoclave. 30. PBST: PBS, 0.05% (v/v) Tween 20. Adjust pH to 7.4 with HCl, filter sterilize (0.2 μm) and autoclave.

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31. PEG/NaCl: 20% (w/v) PEG8000, 15% (w/v) NaCl. Mix and filter sterilize (0.2 μm). 32. Petri dishes. 33. Rotamixer. 34. SDS-PAGE gels and reducing SDS-PAGE sample loading buffer. 35. Shaking table at 37 °C. 36. Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific). 37. Spectrophotometer (for A600 nm). 38. TMB substrate (TMB Substrate Kit, Thermo Fisher Scientific). 39. TSB + Y medium: 30 g tryptic soy broth, 5 g yeast extract. Add water to 1 L and autoclave. 40. TSB + Y/carb/IPTG: TSB + Y, 100 μg/mL carbenicillin, 1 mM isopropyl β-D-1 thiogalactopyranoside. 41. TSB + Y/carb/kan medium: TSB + Y, 100 μg/mL carbenicillin, 25 μg/mL kanamycin. 42. TSB + Y/carb/tet medium: TSB + Y, 100 μg/mL carbenicillin, 10 μg/mL tetracycline. 43. TSB + Y/tet medium: TSB + Y, 10 μg/mL tetracycline.

3

Methods

3.1 Phage Stock Preparation

This protocol describes the production of an affibody displaying phage particle stock based on a preexisting affibody library, previously electroporated to an appropriate strain of E. coli (here strain ER2738, containing a pili-encoding F’episome and capable of amber suppression; see Fig. 1b) and prepared as glycerol stocks. In principle, glycerol stocks of phage particles, rather than phagemid containing bacteria, can also be stored and used for library reamplification or directly used for the first round of biopanning. 1. Inoculate an appropriate number (ca. 1–5× the library size) of freshly thawed E. coli cells containing the affibody library to 750 mL TSB+Y medium supplemented with appropriate antibiotics and 20% (w/v) D-glucose in a baffled 5-l E-flask (see Note 1). For instance, TSB+Y/carb/tet medium contains carbenicillin to select for cells containing a phagemid carrying a β-lactamase gene and tetracycline to select for the pili-encoding F’episome of E. coli ER2738. 2. Incubate at 37 °C for 30 min with shaking at 200 rpm to mid-log phase (OD600 = 0.5) (see Note 2). 3. Superinfect the cultures with helper phage M13K07 at an MOI of 5 (see Note 3).

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4. Swirl gently and incubate at 37 °C without shaking for 15 min, followed by shaking at 70 rpm for 15 min. 5. Pellet superinfected bacteria by centrifuging at 6000× g for 20 min at 4 °C. 6. Resuspend the pellet in a small volume of TSB+Y and inoculate 750 mL TSB+Y/carb/IPTG, preheated to 37 °C, in baffled 5-l E-flasks. 7. Incubate at 37 °C with shaking at 90 rpm for 2 h before adding kanamycin (25 μg/mL final concentration) to select for bacteria co-infected with helper phage M13K07. 8. Incubate at 37 °C and 90 rpm overnight. 9. Inoculate 5 mL TSB+Y/tet medium with a single colony of E. coli XL1-Blue from a fresh blood agar/tet plate. Grow at 37 °C and 150 rpm overnight (to be used in step 12). 10. After overnight incubation (ca. 16–18 h), transfer phage producing bacterial cultures to centrifuge bottles, and pellet bacteria by centrifugation for 20 min at 6000× g at 4 °C. 11. Transfer the supernatants to fresh centrifuge bottles containing 1/5 total volume of cold PEG/NaCl solution to precipitate phage. Incubate for 45 min on ice. 12. Re-inoculate the E. coli XL1-Blue overnight culture 1:100 to 50 mL TSB + Y/tet medium. Grow at 37 °C and 150 rpm to mid-log phase (OD600 = 0.6–0.8) and use for phage titration (see step 19). 13. Spin for 45 min at 20,000× g at 4 °C to pellet precipitated phage. Decant the supernatant. 14. Resuspend phage pellets in total volume 320 mL of prechilled PBS buffer by gentle pipetting. Always use filter tips when handling phage to avoid pipette contamination. 15. Pellet residual bacteria by centrifuging at 6000× g for 20 min at 4 °C. 16. Transfer the phage-containing supernatants to new centrifuge bottles containing 1/5 final volume of PEG/NaCl, and incubate for 30 min on ice to precipitate phage. 17. Spin for 30 min at 15,000× g at 4 °C to pellet precipitated phage. Carefully decant the supernatants. 18. Use a pipette to resuspend the phage pellets in the smallest volume possible of cold PBST, and transfer to a 15 mL tube. Rinse the centrifuge bottles with an additional small volume of cold PBST and transfer to the same 15 mL tube. 19. Determine carb-resistant phage concentration by infecting log-phase E. coli XL1-Blue cells with serial dilutions of phage. Dilute 10 μL in 90 μL PBS and prepare 12 tenfold dilutions.

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Transfer 10 μL of each dilution to a 96-well round bottom plate, and add 100 μL of log-phase E. coli XL1-Blue cells. Incubate w/o shaking for 45 min at 37 °C, and plate 5 μL of each dilution on blood agar/carb plates using a multi-pipette. Optional: plate samples also on blood agar/kan plates to determine kan-resistant helper phage concentration (see Note 4). 20. Plate larger volumes (50–100 μL) of selected dilutions on blood agar/carb plates to obtain individual colonies to determine the percentage of phage harboring a correctly sized affibody gene, using standard colony PCR procedure, using PCR primers annealing at sites flanking the affibody insert. Optional: sequence phagemids harboring the affibody gene to assess the quality and diversity of the prepared affibody stock (we generally sequence 96 clones). 21. Use aliquots of the affibody phage stock directly for selection experiments. Alternatively, it can be stored short term at 4 °C or frozen as glycerol stocks. 3.2 Target Antigen Preparation

In this protocol, the use of an appropriately biotinylated target antigen is critical for a successful selection, since enrichment of antigen binders depends on the immobilization of binder-target antigen complexes using streptavidin-coated paramagnetic beads. Alternative selection schemes, which are not covered here, rely on the use of antigen-coated microtiter wells or immunotubes for selections (where target antigens do not need to be biotinylated). Biotinylation can be achieved via chemical labeling of primary amines available in the antigen sequence (e.g., N-terminus and side chains of lysine residues). The following protocol describes biotin labeling using EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific), the removal of excess nonreacted biotin using a dialysis cassette (see Note 5), and estimation of degree of labelling by mass spectrometry (see Note 6). 1. Dissolve EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) in 50% (v/v) DMSO. Make this solution immediately before use as the NHS moiety will begin to hydrolyze after preparation. 2. Incubate the target antigen (typically ca. 100–200 μg depending on the molecular weight and number of planned selection strategies) with a 15× molar excess of biotin for 1.5 h at room temperature [11] with end-over-end (eoe) rotation. 3. Stop the reaction by adding 1/7 total volume of 1 M Tris–HCl (pH 8). 4. Prepare 2 l PBS for use as dialysis buffer and transfer 1 L to a beaker. Store the remaining 1 L at 4 °C.

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5. Hydrate a dialysis cassette of appropriate size, with an appropriate molecular weight cutoff (not allowing the antigen pass through the pores), in the dialysis buffer. 6. Inject the sample into the dialysis cassette using a needle and syringe. 7. Dialyze the sample using a buoy and slow magnetic stirring for 1–2 h at 4 °C. 8. Exchange the dialysis buffer to fresh prechilled 1 L PBS. Incubate overnight with slow magnetic stirring at 4 °C. 9. Remove the sample from the dialysis cassette using a needle and syringe, and transfer to a new tube. 10. Measure the sample volume and the absorbance at 280 nm to determine the protein concentration and yield. 11. To determine the degree of labeling, prepare streptavidin beads for a binding test by washing two aliquots of 40 μL (10 mg/ mL) Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) 10× with 500 μL PBS in 1.5 mL microcentrifuge tubes using a magnetic rack. 12. Add 2 μg of the biotinylated target antigen, based on absorbance, to one of the bead tubes to a final volume of 50 μL with PBST. Add 50 μL PBST to the second tube as a negative control. 13. Incubate for 1 h at RT with eoe rotation. Mix by pipetting every 15 min. 14. Spin down and collect the supernatants using a magnetic rack. Lyophilize the supernatants. 15. Resuspend the dried supernatant samples in reducing SDS-PAGE sample loading buffer and boil. 16. Prepare bead samples by washing the beads once with 500 μL PBST followed by heating at 95 °C for 10 min in reducing SDS-PAGE sample loading buffer. 17. Analyze the supernatant samples and bead-derived samples (do not load the beads, only the sample loading buffer) in parallel to a reference sample of 2 μg of biotinylated target antigen on an SDS-PAGE gel. Successful biotin labeling will show most of the labeled protein in the bead sample and a lower amount of non-labeled protein in the supernatant sample (see Note 7). 3.3 Phage Display Selection

The following protocol describes the general procedure for an affibody phage display selection cycle. Selections are usually performed for a total of four to five cycles, with increased stringency with each cycle (e.g., decreased target antigen concentration, increased total time of wash, etc.). An overview of the experimental steps is shown in Fig. 2.

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Fig. 2 Schematic overview of the experimental procedures involved in one round of phage display selection. An overnight culture of E. coli is started on day 0 and used to inoculate a larger culture that is infected with phage particles that are eluted from antigen-coated magnetic beads (day 1). A phage stock is incubated with biotinylated antigen, and bound phage particles are captured on streptavidin-coated magnetic beads, while unbound phage particles are washed away. Bacteria infected with target-binding phage particles are grown on agar plates supplemented with carbenicillin (to select for bacteria carrying phagemid) and glucose (to suppress expression of affibody-tag-p3 fusion). Following overnight incubation, bacterial cells are scraped from the agar plate on day 2 and used to inoculate liquid growth medium. Phagemid-carrying bacterial cells are superinfected with M13K07 helper phage, collected by centrifugation, and used to inoculate an overnight culture supplemented with carbenicillin, kanamycin, and IPTG to produce a new phage stock. On day 3 phage particles are concentrated from the supernatant of the bacterial culture via two successive precipitation steps using PEG/NaCl. In addition to serving as input for the following round of selection, the phage stock may be assayed by, e.g., colony PCR of individual bacterial colonies infected with phage particles or for target/tag binding via phage-ELISA

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1. To prepare for a culture to be used later for infection with the phage eluate: Inoculate 5 mL TSB+Y/tet medium with a single colony of E. coli XL1-Blue from a fresh blood agar/tet plate. Grow at 37 °C and 150 rpm overnight (see Subheading 3.1, step 1). 2. Prepare blocked tubes by adding 500 μL 1% (w/v) BSA in PBST to 1.5 mL microcentrifuge tubes and store at 4 °C. Remove the blocking solution before use. 3. Prepare beads (Dynabeads M-280 Streptavidin, Thermo Fisher Scientific) by washing twice with 500 μL PBS using a magnetic rack. Use an amount of beads in excess of what is required to immobilize the desired amount of biotin-labeled target antigen, depending on the capacity of the beads. 4. Immobilize target antigen onto the beads by incubating biotinlabeled target antigen with the beads in PBST to a final volume of 50 μL for 1 h at RT with eoe rotation. Use an amount of target antigen that in the final selection volume corresponds to the desired antigen concentration for the particular selection round (see Note 8). 5. Remove the supernatant and add 500 μL 1% (w/v) BSA in PBST to the beads to post-block for 30 min at RT with eoe rotation. 6. Remove the blocking solution and wash the blocked beads once with PBST to remove excess BSA. 7. Add phage stock, 5% (w/v) BSA and PBST to the beads in a final selection volume of 1 mL, with a final concentration of 1% (w/v) BSA (see Note 9). Incubate for 3 h at RT with eoe rotation. 8. Inoculate the E. coli XL1-Blue overnight culture (from Subheading 3.1, step 1) 1:100 to 50 mL TSB+Y/tet medium. Grow at 37 °C and 150 rpm to mid-log phase (OD600 = 0.6–0.8) and use for phage output titration (see Subheading 3.3, step 13) and phage output amplification (see Subheading 3.3, step 14). 9. Place the selection tube in a magnetic rack and remove the supernatant after 10 min. 10. Wash the beads with 1 mL PBST for a total wash time of 5 min (in later cycles, the washing time may be extended significantly to increase the selection stringency; see Note 10). 11. Transfer the solution (round one) or last wash (later rounds) containing both beads and wash solution to a new blocked tube in a magnetic rack. Remove and discard the supernatant. 12. To elute phage, incubate the beads with 500 μL 0.5 M HAc, pH 2.8 for 15 min with eoe rotation after which the

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supernatant (containing the enriched and eluted phages) is transferred to a new blocked tube containing 500 μL 1 M Tris–HCl (pH 8) for neutralization. 13. Evaluate the selection eluate using titration on E. coli cells and colony PCR (N.B. should not be performed after the first cycle, to avoid losing low copy binders): determine the phage concentration by infecting log-phase E. coli XL1-Blue cells with serial dilutions of the phage eluate. Dilute 10 μL of eluate in 90 μL PBS and prepare 12 such tenfold dilutions. Transfer 10 μL of each dilution to a 96-well round bottom plate, and add 100 μL of log-phase E. coli XL1-Blue cells. Incubate still for 45 min at 37 °C and plate 5 μL of each dilution on blood agar/carb plates. Optional: plate samples also on blood agar/ kan plates to determine the helper phage concentration (see Note 11). Plate larger volumes of selected dilutions on blood agar/carb plates to determine the percentage of clones harboring a phagemid with correct affibody gene insert, using standard colony PCR procedure. 14. Infect 100 mL log-phase E. coli XL1-Blue cells with the whole (neutralized) phage eluate volume (rinse the tube carefully). In later cycles (when binding clones have been significantly amplified in numbers), the volume of bacteria may be decreased, and only half of the eluate volume can be used (see Note 12). 15. Swirl gently and incubate at 37 °C without shaking for 25 min, followed by shaking at 70 rpm for 15 min. 16. Pellet infected bacteria by centrifuging at 3500× g for 10 min at 4 °C. 17. Resuspend the pellet in 2 mL TSB + Y and plate on large blood agar/carb plates supplemented with 1% (w/v) D-glucose, preheated to 37 °C. Incubate overnight at 37 °C (see Note 13). 18. Add a few mL of TSB + Y to the agar plate and collect the bacterial cells by scraping. Transfer the collected bacteria to a new 15 mL tube, mix well, and measure OD600 on a 1:100 dilution. 19. To start the preparation of a new (binder-enriched) phage stock for the next selection round, inoculate 200 mL TSB + Y/carb medium with an appropriate portion of scraped cells such that the starting OD600 = 0.1. Grow at 37 °C and 150 rpm until OD600 = 0.5–0.8. Residual cells can be stored as frozen glycerol stocks. 20. Superinfect 30 mL of the culture with helper phage M13K07 at an MOI of 5. 21. Swirl gently and incubate at 37 °C without shaking for 25 min, followed by shaking at 70 rpm for 15 min.

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22. Pellet superinfected bacteria by centrifuging at 3500× g for 10 min at 4 °C. 23. Resuspend the pellet in 1 mL TSB+Y and transfer to a new E-flask containing 150 mL TSB+Y/carb/IPTG medium. 24. Incubate at 37 °C with 150 rpm for 2 h before addition of kanamycin (25 μg/mL final concentration) to select for bacteria co-infected with helper phage M13K07. 25. Incubate at 37 °C and 150 rpm overnight. 26. Inoculate 5 mL TSB+Y/tet medium with a single colony of E. coli XL1-Blue from a fresh blood agar/tet plate. Grow at 37 °C and 150 rpm overnight (to be used in Subheading 3.3, step 29). 27. After overnight incubation (ca. 16–18 h), transfer the phage producing bacterial culture to a centrifuge bottle, and pellet bacteria by centrifugation for 20 min at 4000× g at 4 °C. 28. Transfer the supernatant to a fresh centrifuge bottle containing 1/5 total volume of cold PEG/NaCl solution to precipitate phage. Incubate 45 min on ice. 29. Inoculate the E. coli XL1-Blue overnight culture 1:100 to 50 mL TSB+Y/tet medium. Grow at 37 °C and 150 rpm to mid-log phase (OD600 = 0.6–0.8), and use for phage titration (see Subheading 3.3, step 37). 30. Spin for 45 min at 15000× g at 4 °C to pellet precipitated phage. Decant the supernatant. 31. Resuspend the phage pellet in 4 mL cold 25 mM Tris–HCl, 2 mM EDTA. 32. Pellet residual bacteria by centrifuging at 4000× g for 15 min at 4 °C. 33. Transfer the phage-containing supernatants to new centrifuge tubes containing 1/5 final volume of PEG/NaCl, and incubate for 45 min on ice to precipitate phage. 34. Spin for 30 min at 15000× g at 4 °C to pellet precipitated phage. Carefully decant the supernatants. 35. Use a pipette to resuspend the phage pellets in 1 mL of cold PBST, and transfer to a new 1.5 mL microcentrifuge tube. 36. Determine phage concentration by infecting log-phase E. coli XL1-Blue cells with serial dilutions of phage as described in Subheading 3.1, step 19. 37. Plate larger volumes of selected dilutions on blood agar/carb plates to get colonies to use to determine the percentage of clones harboring a phagemid with a correct affibody gene insert, using a standard colony PCR procedure and insertflanking primers specific for the phagemid vector.

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38. Subheading 3.1, steps 1–37 describe the procedure for one selection cycle and ensuing phage stock amplification, which we recommend repeating four to five times with increasing stringency in each cycle. Use the output affibody phage stock from each cycle as the input affibody phage stock of the next cycle (see Subheading 3.1, step 7). 39. Following the final cycle, the procedure is completed after Subheading 3.1, step 12. Log-phase E. coli XL1-Blue cells are subsequently infected with a portion of the eluate from the final selection round by incubating for 45 min at 37 °C, without shaking, and plated on blood agar/carb plates. Clones harboring a phagemid containing a correctly sized affibody gene (usually close to 100% in a successful selection campaign) are identified using standard colony PCR procedure, and such clones proceed to functional screening in monoclonal phageELISA (see Subheading 3.4). 3.4 Phage-ELISA Screening

An initial binding screen to identify candidate affibody binders is done by performing a monoclonal phage-ELISA, where phage particles are produced from individual colonies generated from the eluate obtained after the final selection cycle (Fig. 3). Each monoclonal phage sample produced is assayed for binding to a set of proteins, including the target antigen, a protein which binds to the tag that is present between the affibody and truncated p3 and relevant controls. A similar procedure can be applied also on polyclonal phage samples to monitor the enrichment of target binding phage populations throughout the biopanning experiments. 1. Inoculate individually grown E. coli colonies containing the affibody phagemid, based on colony PCR, to 500 μL TSB + Y/carb medium in a 96-well deep-well plate. Bookkeep which colonies are picked. Cover with a breathable sealing film and incubate overnight at 250 rpm at 37 °C. 2. Inoculate 30 μL of each overnight culture to 720 μL TSB + Y/ carb medium in a new 96-well deep-well plate for 2 h at 250 rpm at 37 °C. 3. Superinfect the grown bacteria cultures with M13K07 helper phage at an MOI of 5 by adding M13K07 diluted in 100 μL TSB + Y/carb medium. Incubate without shaking for 30 min at 37 °C. 4. Add 150 μL TSB + Y/carb/kan/IPTG and incubate overnight at 250 rpm at 37 °C. The final concentrations should be 100 μg/mL carbenicillin, 25 μg/mL kanamycin, and 1 mM IPTG. 5. Coat ELISA plates (Clear Flat-Bottom Immuno Nonsterile 384-Well Plates, Thermo Fisher Scientific) with 30 μL target antigen at 1–10 μg/mL or control protein(s) at 1–20 μg/mL,

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Fig. 3 Monoclonal phage-ELISA. Individual colonies obtained after infection of E. coli XL1-Blue (or ER2738 or TG-1) with a neutralized phage eluate from a selection can be used to inoculate individual cultures in, e.g., deep-well culture plates for production of small-scale monoclonal phage stocks through superinfection of the cultures with helper phage. Such individual (i.e., clonal) phage particle preparations, each corresponding to a particular library member clone, can be subjected to binding analyses in ELISA to investigate the ability of the selected, and here phage-displayed, library member to bind the target. Parallel tests of the same phage stocks to control proteins (incl. streptavidin) and the cognate ligand of any included tag can give useful information about the characteristics of the library member. An interesting comparison is the ratio of signals obtained toward the target and tag ligand, respectively, where a high value may reflect a display of a high affinity binder. Detection of phage particles in the ELISA assay is easily performed using an HRP-conjugated anti-M13 antibody. Transfer of the chosen colonies to also a master replica plate makes it easy to later go back and pick the most interesting clones for further analyses (e.g., DNA sequencing, phagemid purification, subcloning, expression, etc.)

per well in 0.1 M carbonate buffer (see Note 14). Cover the plates, spin down briefly, and incubate still or with gentle shaking at 4 °C overnight. 6. Wash the ELISA plate twice with PBST and block with 60 μL 1% (w/v) BSA in PBS per well for 1 h at RT with gentle shaking. 7. Collect the phage from the overnight cultures by centrifuging for 15 min at 3000× g at 4 °C and transferring the phage supernatants to a new 96-well deep-well plate. Do not disturb the pellets. 8. Remove the blocking solution from the ELISA plate, and add 10 μL phage supernatant in 20 μL PBST per well, so that each phage sample is incubated with a set of wells (target and control). Incubate for 1 h at RT with gentle shaking. 9. Wash the ELISA plate thrice with PBST.

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10. Add 30 μL α-M13-HRP diluted in PBST according to the manufacturer’s instructions (typically 1:5000) to each well, and incubate for 30 min at RT with gentle shaking. 11. Wash the ELISA plate twice with PBST and once with PBS. 12. Add 30 μL TMB substrate (TMB Substrate Kit, Thermo Fisher Scientific). 13. Stop the reaction after 5–15 min by adding 30 μL 2 M H2SO4. 14. Measure absorbance at 450 nm using a microplate reader. A positive ELISA result should show a high absorbance signal against positive control protein(s) and a relatively high absorbance signal against the target antigen compared to negative control protein(s). Affibody-expressing phage eliciting a positive ELISA result should be retraced to their original bacterial colonies to send for sequencing. Subclone unique and ELISA positive affibody binders to an expression vector of choice for further screening and characterization of affinity proteins on the protein level.

4

Notes 1. The volume of bacterial glycerol stock and total culture volume will depend on the library size as well as the viable cell concentration in the glycerol stock. If possible, it is recommended to use a volume of bacterial cells that oversample the library size by a factor of 10. However, for practical reasons this is not always feasible since it would result in very large culture volumes for the initial phage stock preparation. 2. OD600 will not be measured directly after inoculation, to avoid removal of bacteria carrying unique copies of library members. 3. To obtain highly concentrated M13K07, helper phage can be reamplified from individual phage plaques formed upon E. coli infection and visible in top agar using standard procedures. 4. The expected phage concentration is 1012–1013 cfu/mL and the titers from blood agar/carb should be tenfold higher than those from blood agar/kan. 5. Excess nonreacted biotin can also be removed through dialysis using dialysis tubing or desalting columns. We recommend dialysis cassettes since our experience is that sample loss is minimized when using this procedure. 6. Mass spectrometry can be used to determine the number of biotin labels per antigen. The number of biotins per antigen should be 1–2, to avoid discommoding the dissociation of biotin from streptavidin coated paramagnetic beads. Overmodification may also negatively affect available binding epitopes

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in the antigen structure. We recommend adjusting the molar excess of biotin in the biotin labeling reaction depending on the number of primary amine containing amino acid residues in the antigen. If available, recombinant target antigen produced with an Avi tag that is enzymatically labeled with biotin only at the lysine residue of the Avi tag is a recommended option as the number of biotins per antigen would not exceed 1. 7. The combined band intensities of the supernatant sample and the bead sample should correspond to the intensity of the 2 μg of reference antigen loaded, or otherwise could indicate a higher degree of labeling such that antigen dissociating from the beads during washing and boiling steps is prevented. Expect to see bands corresponding to streptavidin (ca. 16 kDa as a denatured monomer) and bovine serum albumin (ca. 66 kDa, which is present in the bead storage solution) present in the bead product. 8. Biotinylated antigen may either be immobilized on beads before or after incubation with the phage-displayed library. We commonly use pre-immobilized target in early cycles and solution-based selection followed by capture of antigen-phage complexes on streptavidin-coated magnetic beads in later cycles. However, to remove nonbiotinylated antigen that may interfere with enrichment of antigen-binding clones, it is critical that antigen is immobilized on beads before exposure to the phage-displayed library in the first round of selection when target-binding clones may not be present in redundant numbers. Based on previous observations, we recommend using 100–200 nM antigen concentration and rather work with longer washing time than decreasing antigen concentration below ca. 15 nM [23]. 9. The volume of phage stock added will depend on the phage stock titer and should preferably cover the library diversity 10–100 times since not all phages will display a library member (see Fig. 1c). Typically, for the first round using a library of a 1010 diversity, aim for the use of 1012–1013 cfu of phage stock. Since target availability may also be a limiting factor, a too large volume of phage stock may result in an undesirably low target concentration. Furthermore, before this selection step, the phage stock can be subjected to preselection, in which the phage stock volume of interest is preincubated in conditions like the selection step, but excluding the biotinylated target, to remove nonspecific binders to the beads and the reaction tube material. 10. The total washing time can be increased with each subsequent cycle, for increased selection stringency, and is the product of the duration of each wash step (during which the beads are

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incubated with PBS with end-over-end rotation) and the total number of washes. Preferably the number of washes in the first cycles should be few, as to avoid losing potential binders due to physical handling of the beads when removing the wash solution. We commonly use washing times 5 min in cycle 1, 10 min in cycle 2, 15 min in cycle 3, and 20 min in cycle 4. The elution of phage can also be done using trypsin cleavage: the washed beads are incubated with 0.5 mL 0.25 mg/mL trypsin (Gibco Life Technologies) in 1xTST (25 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.05% (v/v) Tween 20, pH 8) supplemented with 1 mM CaCl2 for 30 min at room temperature with endover-end rotation. 11. The expected phage concentration in the eluate is 106– 108 cfu/mL, and the titers from blood agar/carb should be at least tenfold higher than those from blood agar/kan. 12. Phage eluate that is not used for infection and preparation of a new phage stock can be kept as a glycerol stock for retrospective analyses (e.g., sequencing), or enable a re-initiation of the selection procedure at this stage. We commonly use 100 mL of bacterial culture for infection with eluate in rounds 1–2, 50 mL in round 3, and 25 mL in rounds 4–5. 13. The petri dish area can be decreased for each cycle; we generally use large 245 × 245 mm dishes in rounds 1–2 and round 140 mm diameter dishes in later rounds. In later cycles phage eluate may be used directly to infect a liquid culture of log phase XL1-Blue cells followed by helper phage superinfection to generate phage stocks for the following round. 14. Affibody co-expression with albumin binding domain [11] on the surface of the phage facilitates the use of human serum albumin (HSA) as a positive control for in-frame expression of the affibody. Absorbance signal measured from immobilized HSA could also give an estimate of the amount of affibodydisplaying phage that should be available to bind target antigen and/or negative control(s). Negative controls could be BSA or SA, which are present during the selection cycles, or an unrelated antigen which may have been present in a parallel selection track.

Acknowledgments We would like to acknowledge former and present members of the KTH lab who have helped improving this protocol over the years.

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References 1. Nilsson B, Moks T, Jansson B, Abrahmsen L, Elmblad A, Holmgren E, Henrichson C, Jones TA, Uhlen M (1987) A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng 1(2):107–113 2. Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA (1997) Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nat Biotechnol 15(8):772–777 3. Gebauer M, Skerra A (2020) Engineered protein scaffolds as next-generation therapeutics. Annu Rev Pharmacol Toxicol 60:391–415 4. Stahl S, Graslund T, Eriksson Karlstrom A, Frejd FY, Nygren PA, Lofblom J (2017) Affibody molecules in biotechnological and medical applications. Trends Biotechnol 35(8): 691–712 5. Vazquez-Lombardi R, Phan TG, Zimmermann C, Lowe D, Jermutus L, Christ D (2015) Challenges and opportunities for non-antibody scaffold drugs. Drug Discov Today 20(10):1271–1283 6. Tolmachev V, Orlova A (2020) Affibody molecules as targeting vectors for PET imaging. Cancers (Basel) 12(3):651 7. Hober S, Lindbo S, Nilvebrant J (2019) Bispecific applications of non-immunoglobulin scaffold binders. Methods 154:143–152 8. Ding H, Altai M, Yin W, Lindbo S, Liu H, Garousi J, Xu T, Orlova A, Tolmachev V, Hober S, Graslund T (2020) HER2-specific pseudomonas exotoxin a PE25 based fusions: influence of targeting domain on target binding, toxicity, and in vivo biodistribution. Pharmaceutics 12(4):391 9. Malm M, Bass T, Gudmundsdotter L, Lord M, Frejd FY, Stahl S, Lofblom J (2014) Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albuminbinding domain allows for affinity purification and in vivo half-life extension. Biotechnol J 9(9):1215–1222 10. Yu F, Gudmundsdotter L, Akal A, Gunneriusson E, Frejd F, Nygren PA (2014) An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo. MAbs 6(6):1598–1607 11. Johan N, Sophia H (2013) The albumin-binding domain as a scaffold for protein engineering. Comput Struct Biotechnol J 6: e201303009

12. Sochaj-Gregorczyk AM, Serwotka-Suszczak AM, Otlewski J (2016) A novel AffibodyAuristatin E conjugate with a potent and selective activity against HER2+ cell lines. J Immunother 39(6):223–232 13. Engfeldt T, Renberg B, Brumer H, Nygren PA, Karlstrom AE (2005) Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein. Chembiochem 6(6): 1043–1050 14. Lindgren J, Ekblad C, Abrahmsen L, Eriksson Karlstrom A (2012) A native chemical ligation approach for combinatorial assembly of affibody molecules. Chembiochem 13(7): 1024–1031. https://pubmed.ncbi.nlm.nih. gov/24688717/ 15. Galindo Casas M, Stargardt P, Mairhofer J, Wiltschi B (2020) Decoupling protein production from cell growth enhances the site-specific incorporation of noncanonical amino acids in E. coli. ACS Synth Biol 9(11):3052–3066 16. Kanje S, Hober S (2015) In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies. Biotechnol J 10(4):564–574 17. Rouet R, Jackson KJL, Langley DB, Christ D (2018) Next-generation sequencing of antibody display repertoires. Front Immunol 9: 118 18. Zambrano N, Froechlich G, Lazarevic D, Passariello M, Nicosia A, De Lorenzo C, Morelli MJ, Sasso E (2022) High-throughput monoclonal antibody discovery from phage libraries: challenging the current preclinical pipeline to keep the pace with the increasing mAb demand. Cancers (Basel) 14(5):1325 19. Ferrara F, Teixeira AA, Naranjo L, Erasmus MF, D’Angelo S, Bradbury ARM (2020) Exploiting next-generation sequencing in antibody selections – a simple PCR method to recover binders. MAbs 12(1):1701792 20. Nannini F, Senicar L, Parekh F, Kong KJ, Kinna A, Bughda R, Sillibourne J, Hu X, Ma B, Bai Y, Ferrari M, Pule MA, Onuoha SC (2021) Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments. MAbs 13(1):1864084 21. Noh J, Kim O, Jung Y, Han H, Kim JE, Kim S, Lee S, Park J, Jung RH, Kim SI, Park J, Han J,

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Lee H, Yoo DK, Lee AC, Kwon E, Ryu T, Chung J, Kwon S (2019) High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library. MAbs 11(3): 532–545 22. Spiliotopoulos A, Owen JP, Maddison BC, Dreveny I, Rees HC, Gough KC (2015) Sensitive recovery of recombinant antibody clones

after their in silico identification within NGS datasets. J Immunol Methods 420:50–55 23. Astrand M, Nilvebrant J, Bjornmalm M, Lindbo S, Hober S, Lofblom J (2016) Investigating affinity-maturation strategies and reproducibility of fluorescence-activated cell sorting using a recombinant ADAPT library displayed on staphylococci. Protein Eng Des Sel 29(5): 187–195

Part IV Complementary Approaches for Antibody Phage Display Selections

Chapter 20 Antibody Affinity and Stability Maturation by Error-Prone PCR Nora Langreder, Dorina Scha¨ckermann, Tobias Unkauf, Maren Schubert, Andre´ Frenzel, Federico Bertoglio, and Michael Hust Abstract Human antibodies are the most important class of biologicals, and antibodies – human and nonhuman – are indispensable as research agents and for diagnostic assays. When generating antibodies, they sometimes show the desired specificity profile but lack sufficient affinity for the desired application. In this article, a phage display-based method and protocol to increase the affinity of recombinant antibody fragments is given. The given protocol starts with the construction of a mutated antibody gene library by error-prone PCR. Subsequently, the selection of high-affinity variants is performed by panning on immobilized antigen with washing conditions optimized for off-rate-dependent selection. A screening ELISA protocol to identify antibodies with improved affinity and an additional protocol to select antibodies with improved thermal stability is described. Key words Antibody phage display, Panning, Off-rate panning, Antibody affinity maturation, Antibody thermal stability maturation, Antibody selection

1

Introduction In the past years, monoclonal antibodies have been the fastestgrowing class of pharmaceutical proteins with a global sales revenue of nearly $163 billion in 2019, representing approximately 70% of the total sales of all biopharmaceutical products [1]. The combined worldwide sales are predicted to be nearly $300 billion in 2025 [2]. Furthermore, antibodies are indispensable reagents in many fundamental biochemical methods such as affinity chromatography, enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, Western blotting, or flow cytometry. The rapid expansion of genomics, proteomics, and other biotechnology fields has

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_20, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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led to a growing demand for antibodies as high-affinity reagents to specifically recognize, e.g., peptides and proteins but also carbohydrates and haptens [3]. Despite the availability of other in vitro approaches like yeast and ribosome display, phage display is still the most robust technology. It allows the screening of large antibody libraries with theoretical complexities of up to 1011 different clones [4], hence almost entirely covering the structural diversity of naturally occurring antibodies (2
1012) [5]. When compared to animal-based generation of antibodies, many advantages are offered by in vitro technologies such as phage display, ribosome display, or yeast display: enhanced throughput by parallelization and miniaturization; the stringent control of biochemical and physical selection conditions, e.g., pH, temperature, or provision of a ligand; and the possibility to use proteins which cannot be used for immunization, e.g., toxic proteins, evolutionary conserved proteins, or different conformational variants [6]. However, even complex naι__ve antibody libraries sometimes show a proportion of just 1–10 binders to a given target per 107 clones [7]. This can result in the selection of low affinity clones with KD values above 100 nM. Although exhibiting low affinities, these antibodies can still provide useful starting molecules for the construction of mutagenized libraries [8–10] or light chain shuffling [11, 12] for in vitro affinity maturation. Light chain shuffling combines selected heavy chain variable (VH) genes with a variety of light chain variable (VL) genes. By shuffling the VL genes, it may be possible to select antibodies with increased affinity or other desired improved properties. But the drastic randomization could also lead to an epitope drift and thus a loss of biological activity [13]. A more complex but promising approach is the introduction of diversity by a defined collection of natural CDRs. Here, the parental epitope is retained and the number of sequence liabilities is reduced [14]. For the creation of mutagenized libraries from a parental antibody gene, many methods have been developed. Nearly all approaches generate diversity by introducing mutations at the nucleotide level. Nonstochastic techniques often use alanine-scanning or sitedirected mutagenesis to generate limited collections of specific variants but require prior knowledge of the respective antibodyantigen interaction [15]. Stochastic methods like error-prone PCR [16], mutator bacterial strains [17], and site-specific saturation mutagenesis of complementary determining regions (CDR) [18, 19] are random mutagenesis methods. The use of mutator bacterial strains like

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E. coli mutD5 or E. coli XL1-Red results in the mutation of the antibody gene but also the vector backbone, making a subsequent recloning of the antibody gene necessary. Directed mutagenesis of the CDR is only limited to the immediate antigen binding sites of the antibody; hence, a limitation of the affinity maturation potential of an antibody might occur. CDRs are directly involved in target recognition, but framework regions are the foundations of the VH and VL structures and thus are of high importance for the CDRs’ presentation. Mutations within the framework region of a given antibody can therefore stabilize and improve the antibody-antigen interactions [20]. The most common technique to introduce random mutations is error-prone PCR [16] as it bypasses both limitations by targeting only the antibody gene which than can be directly used for the construction of a mutagenized library. Selectivity for high-affinity binders during panning can be relatively low even when binders do differ by a factor of 10 in affinity [7]. Successful selection of high-affinity mutants is only achieved by phage display panning approaches [21] using many harsh and long washing steps. The use of multiple forms of the target antigen in sequential selection rounds, and the inclusion of competitor proteins can drive the selected pool toward a highly specific set of epitopes. In this chapter, the affinity maturation is described with the classical panning approach on immobilized antigen with extended washing and incubation steps. This is the easiest and most straightforward approach with great potential and therefore a good protocol to start with. Alternatively, the panning process can be performed in solution. Here, the selection process can be either performed by systematically reducing the antigen concentration or by competition with free antigen or soluble antibody fragments [22, 23]. A protocol for these approaches has been presented in the previous edition of this book [24]. Monoclonal binders can be tested and affinity ranked by using crude cell supernatants from 96-well production in a competitive ELISA approach [25] or solution equilibrium titration (SET) using highly sensitive electrochemiluminescence [26]. Specificity can be validated, for example, by flow cytometry [27] or peptide arrays such as PEPperCHIP [28]. Affinities are usually determined by surface plasmon resonance (SPR) [29, 30], microscale thermophoresis (MST) [31], or biolayer interferometry (BLI) [32]. A rapid and easy screening procedure to rank lead candidates for thermal stability from crude E. coli production supernatant is described within these protocols.

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Materials Error-Prone PCR

1. Template DNA fragment gene).

(e.g.,

phagemid

containing

antibody

2. Site-specific DNA-oligo primer sets. 3. GeneMorph II Random Mutagenesis Kit (Agilent Technologies, Santa Clara, USA). 4. PCR thermocycler. 5. PCR cleanup kit. 6. Agarose gel and agarose gel electrophoresis device. 2.2 Library Construction

1. Phage display compatible phagemid (e.g., pHAL14 [33], pHAL30 [34], pCOMB3x [35]). 2. Restriction enzymes NcoI, NotI. 3. Dephosphorylation phosphatase, CIP)

enzyme

(e.g.,

30K

centrifugal

calf

intestinal

4. T4 DNA-ligase. 5. PCR cleanup kit. 6. Amicon® Ultra-0.5 (Merck KGaA).

filter

devices

7. Electrocompetent E. coli ER2738 cells (Lucigen Corporation, Middleton, USA); Genotype: [F0 proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10 (tetr)] fhuA2 glnVΔ(lac-proAB) thi-1Δ(hsdSmcrB)5. 8. SOC medium: 0.5% (w/v) yeast extract, 2.0% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM MgSO4, 20 mM glucose. 9. Electroporation unit. 10. Tabletop thermomixer. 11. 9 cm sterile petri dishes. 12. 25 cm sterile plastic dishes. 13. 2YT medium pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl. 14. 2YT-GA medium: 2YT, containing 100 μg/mL ampicillin, 100 mM glucose. 15. 2YT-GA agar: 2YT, containing 100 μg/mL ampicillin, 100 mM glucose, 1.5% (w/v) agar-agar. 16. 80% (v/v) glycerol solution. 17. 1.8 mL Cryotubes.

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1. Phagemid-specific DNA-oligo primer set. 2. GoTaq DNA polymerase. 3. Agarose gel and agarose gel electrophoresis device. 4. PCR cleanup kit.

2.4 Library Packaging

1. 2YT-GA: 2YT, containing 100 μg/mL ampicillin, 100 mM glucose. 2. 2YT-AK: 2YT, containing 100 μg/mL ampicillin, 50 μg/mL kanamycin. 3. Helper phage M13K07 (Stratagene). 4. Incubation shaker (Infors HT Multitron incubator). 5. Sorvall Centrifuge RC 6+. 6. Polyethyleneglycol (PEG) solution: 20% (w/v) PEG 6000, 2.5 M NaCl in water. 7. Phage dilution buffer: 10 mM Tris–HCl pH 7.5, 20 mM NaCl, 2 mM EDTA. 8. 0.45 μm syringe filter (Whatman).

2.5

Titration

1. 2YT-T: 2YT, containing 50 μg/mL tetracycline. 2. 2YT-GA agar: 2YT, containing 100 μg/mL ampicillin, 100 mM glucose, 1.5% (w/v) agar-agar. 3. Incubation shaker (Infors HT Multitron incubator). 4. Incubator. 5. E. coli XL1 Blue MRF0 (Stratagene); Genotype: Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F0 proAB lac IqZΔM15 Tn10 (Tetr)]. 6. PBS pH 7.4: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4*2H2O, 0.24 g KH2PO4 in 1 L water solution. 7. 9 cm sterile Petri dishes.

2.6 Selection by Panning

1. Maxisorp stripe (Nunc). 2. 96-well ELISA Costar plate (Corning). 3. 24-deep-well plate. 4. PBS pH 7.4: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4*2H2O, 0.24 g KH2PO4 in 1 L water solution. 5. PBS-T: PBS + 0.05% Tween 20. 6. MPBS-T: 2% skim milk in PBS-T; prepare fresh. 7. Panning block solution: 1% (w/v) skim milk + 1% (w/v) BSA in PBS-T; prepare fresh. 8. 10 μg/mL trypsin in PBS. 9. 2YT-T: 2YT, containing 50 μg/mL tetracycline.

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10. 2YT-GA agar: 2YT, containing 100 μg/mL ampicillin, 100 mM glucose, 1.5% (w/v) agar-agar. 11. 15 cm sterile Petri dishes. 12. E. coli XL1-Blue MRF0 (Stratagene); Genotype: Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F0 proAB lac IqZΔM15 Tn10 (Tetr)]. 13. Breathable sealing film (VWR International, Radnor, USA). 14. Incubator. 15. Incubation shaker (Infors HT Multitron incubator). 16. Microtiter plate thermo shaker. 17. ELISA washer. 2.7 Production of Soluble, Monoclonal Antibody Fragments

1. 96-well U-bottom polypropylene (PP) microtiter plates (Greiner Bio–One, Frickenhausen, Germany). 2. Breathable sealing film (VWR International, Radnor, USA). 3. 2YT-GA: 2YT, containing 100 μg/mL ampicillin, 100 mM glucose. 4. 2YT-A containing 50 μM isopropyl-beta-D-thiogalactopyranoside (IPTG). 5. 80% (v/v) glycerol solution. 6. Microtiter plate thermo shaker.

2.8 Monoclonal ELISA and Thermal Stability Screening

1. 96-well ELISA Costar plate (Corning). 2. PBS pH 7.4: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4*2H2O, 0.24 g KH2PO4 in 1 L water solution. 3. MPBS-T: 2% skim milk in PBS-T; prepare fresh. 4. dH2O-T: dH2O + 0,05% Tween 20. 5. α-tag antibody (e.g., for pHAL vectors: Mouse α-myc-tag monoclonal antibody (9E10, Sigma-Aldrich, Munich, Germany) or Mouse α-Penta His-tag monoclonal antibody (Qiagen, Hilden, Germany)). 6. Goat α-Mouse IgG serum (Fab-specific), HRP conjugated (Sigma-Aldrich, Munich, Germany). 7. Alternatively to 5. + 6.: α-tag antibody directly coupled to HRP, e.g., for myc-tag Hyper-myc-HRP [36]. 8. TMB solution A, pH 4.1: 10 g citric acid solved in 100 mL water, add 9.73 g potassium citrate, add H2O to make 1 L. 9. TMB solution B: 240 mg tetramethylbenzidine, 10 mL acetone, 90 mL ethanol, 907 mL 30% H2O2. 10. 0.5 M H2SO4.

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11. Microtiter plate thermo shaker. 12. Incubator. 13. ELISA washer. 14. ELISA reader.

3 3.1

Methods Error-Prone PCR

1. Design specific primers for your phagemid flanking your gene of interest approximately 50–100 bp up and downstream respectively. To increase DNA quality for cloning and number of error-prone PCR rounds, a nested approach with a second pair of primers is feasible. Consider that the restriction sites that are used for library cloning should be included into these primers. 2. Perform PCR by using GeneMorph II Random Mutagenesis Kit (Agilent Technologies) according to manufacturer’s instructions. As described there, the mutation frequency depends on the initial template DNA amount and the number of amplification cycles. Recommended: Use 1–15 ng purified template DNA and 30 cycles for error-prone PCR in order to achieve a high mutation frequency. The indicated amount of DNA refers to the amplified sequence only, not the total vector. For instance, 10–150 ng DNA of a 1000 bp plasmid will be used if the amplified sequence is 100 bp (see Note 1). 3. Validate successful amplification on an agarose gel. In case the PCR product is not clean, cut out the respective band. If the PCR product is clean, avoiding a gel extraction can improve the following cloning steps. 4. Clean up DNA with a PCR purification kit. 5. 1–15 ng of this DNA can be used in an additional error-prone PCR. PCR and cleanup steps can be repeated three to four times until no distinct amplification band can be obtained anymore (see Note 2).

3.2 Library Construction

1. Digest the PCR product and phage display vector with suitable restriction enzymes (e.g., NcoI and NotI can be used for pHAL vectors pHAL14 [33], pHAL30 [34], and pSEX81 [37]). For increased ligation efficiency, any suitable dephosphorylation enzyme can be added to the vector digest. Incubate digestion according to manufacturer’s instructions. Optional: Heat inactivate all enzymes according to manufacturer’s instructions. 2. Clean up digested DNA and determine the respective DNA concentrations of vector and PCR product (see Note 3).

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3. Use approximately 1 μg vector DNA for ligation. Adjust the amount of PCR product accordingly. A molar ratio of vectorinsert of 1:3 is recommended. Perform ligation overnight at 16  C. Optional: Heat inactivation of ligation according to ligase manufacturer’s instructions. 4. Clean up ligation by washing four times with H2O using Amicon® Ultra-0.5 30K centrifugal filter devices (Merck KGaA) according to manufacturer’s instructions. Final volume should be around 25 μL. 5. Mix half of the pre-chilled ligation mix with 25 μL electrocompetent ER2738 E. coli cells (Lucigen) and perform electroporation at 1.7 kV, 4-5 ms pulse (Bio-Rad, electroporation unit). The remaining ligation can be used for a second transformation that gets pooled with the first one in Subheading 3.2, step 8. 6. Resuspend transformed cells immediately in 950 μL prewarmed SOC medium, and incubate for 1 h at 37  C and 650 rpm in a tabletop thermomixer. 7. Take 10 μL of the cell suspension and make a dilution series in 2xYT or LB medium. For successful transformations, 1
107– 5
108 clones can be expected. Plate an aliquot of each dilution and calculate the transformation efficiency after overnight incubation at 37  C. The average of all dilution plates with countable colonies is the maximum theoretical diversity (size) of the library. 8. Plate the rest of the cell suspension to 25 cm
25 cm 2xYT-GA agar plates. Incubate overnight at 37  C. 9. Add 30 mL 2xYT medium to each plate and incubate on a rocker for 15 min. Carefully scrape cells from the plate using a spatula. 10. If possible, start directly with the library packaging and use fresh cell suspension for inoculation (Subheading 3.4, step 1). As backup and for later library packaging, prepare glycerol stocks of your library by mixing 250 μL 80% (v/v) glycerol and 750 μL cell suspension in 1.8 mL cryotubes. Shock freeze aliquots in liquid nitrogen and store at 80  C. 3.3 Library Validation

1. Pick up to 30 clones from the plates used for determination of transformation efficiency. 2. Perform colony-PCR (10 μL scale) using a primer set which amplifies the gene of interest. 3. Analyze a 5 μL aliquot by agarose gel electrophoresis to check upon rate of successful insert integration. The percentage of these “positive” clones indicates the quality of the library.

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4. Send 96 clones in a 96-well plate for sequencing with appropriate primers. 5. Determine the average mutation rate and percentage of functional scFv by aligning the obtained sequences with the parental template sequence. 3.4 Library Packaging

1. Gently thaw a library glycerol stock on ice or use fresh cell suspension from the library construction (Subheading 3.2, step 10). 2. Inoculate 100 mL 2xYT-GA medium in a 500 mL flask with cell suspension directly from the glycerol stock or fresh cell suspension to an initial OD600 of about 0.1. 3. Grow the cells at 37  C and 250 rpm to an OD600 of ~0.5 (logarithmic growth phase). 4. Transfer 25 mL culture into a sterile 50 mL polypropylene tube and add 2.5
1011 cfu M13K07. Mix gently (see Note 4). 5. Incubate at 37  C for 30 min without and 30 min with shaking at 250 rpm. 6. Centrifuge the suspension for 10 min at 3200
g to pellet the cells. Discard the supernatant to remove remaining glucose and resuspend the pellet in 20 mL 2xYT-AK and transfer the cells in a 1000 mL shake flask containing 230 mL 2xYT-AK media. Due to the selection with kanamycin, only M13K07 (Kanr) infected cells will survive and produce antibody phage. 7. Incubate the cells overnight at 30  C and 210 rpm (minimum of 20 h production time is recommended). 8. Centrifuge the culture for 30 min at 12,000
g and 4  C (Sorvall Centrifuge RC 6+; F9S 4x1000Y rotor) to pellet the cells. If the supernatant is not clear, repeat the centrifugation step. 9. Separate the cleared supernatant from the cells, and precipitate the phage by adding 1/5 of the final volume ice-cold PEG/NaCl solution. Mix well (vortex) and incubate for 2 h or overnight on ice at 4  C with gentle shaking. 10. Pellet the phage by centrifugation for 1 h at 20,450
g and 4  C (Sorvall Centrifuge RC 6+; SLA3000 rotor), and discard the supernatant. 11. Put the tubes upside down on tissue paper to remove the PEG solution completely. 12. Resuspend the phage pellet in 10 mL phage dilution buffer, and filter the solution through a 0.45 μm syringe filter (Whatman).

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13. Precipitate phage a second time by adding 1/5 of the final volume ice-cold PEG/NaCl and incubate for 2 h or overnight on ice at 4  C with gentle shaking. 14. Pellet phage particles by centrifugation for 30 min at 47,810
g and 4  C (Sorvall centrifuge RC 6+; SS34 rotor). 15. Completely remove remaining PEG/NaCl and resuspend pellet in 1 mL phage dilution buffer. Transfer the suspension into a 1.5 mL tube. 16. Optional: Remaining cell debris might be removed by an additional centrifugation step (2 min, 4  C, 16,000
g). 17. Titer phage solution. Phage can be stored at 4  C. 3.5

Titration

1. Prepare a 5 mL overnight culture of E. coli XL1-Blue MRF0 in 2xYT-T medium by shaking at 250 rpm and 37  C. 2. Inoculate 50 mL fresh 2xYT-T medium with the overnight culture to an OD600 of about 0.05. Grow culture at 250 rpm and 37  C up to OD600 ~0.5 (see Note 5). 3. Make a serial dilution of the phage solution in PBS. The library titer after packaging (Subheading 3.4) is expected in the range of 1011–1013 cfu/mL. The number of eluted phage during panning (Subheading 3.6) depends on several parameters such as antigen, library, panning round, washing stringency, etc. The phage titer can vary from 103 to 107 cfu/mL. The phage preparation after re-amplification of the eluted phage has a titer of about 1012– 1013 cfu/mL. 4. Infect 50 μL bacteria with 10 μL of each phage dilution, and incubate 30 min at 37  C without shaking. 5. Plate the 60 μL infected bacteria on 2xYT-GA agar plates, and incubate plates overnight at 37  C. 6. Count the colonies and calculate the colony-forming units (cfu) titer according to the respective dilution.

3.6 Selection by Panning

1. Antigen well: Coat antigen overnight at 4  C in different amounts of 1–50 ng per well in a Maxisorp stripe (Nunc) using PBS Buffer (total volume 150 μL). Pre-incubation well: Fill one well in a 96-well ELISA Costar plate (Corning) with 330 μL Panning block solution, and incubate overnight at 4  C. (see Note 6). 2. Antigen well: Block microtiter plate stripe with 330 μl MPBS-T for 1 h at room temperature. Pre-incubation well: Incubate the appropriate cfu of antibody phage from the library in Panning block solution (total volume 150 μL) for 1 h at room temperature. The amount of phage depends on the total size of the library as determined in Subheading 3.2, step 7. The amount of phage should exceed

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the library size by factor 100. If your library size is, e.g., 108, use 1010 cfu phage particles. Antigen well: Discard blocking solution by washing three times with PBS-T (see Note 7). 3. Transfer the pre-incubated library from the pre-incubation well to the antigen well, and incubate for 1 h at room temperature. 4. Wash harshly with PBS-T, e.g., 20 times. To remove unbound or weakly bound phage, stringent washing is necessary (see Note 7). 5. Put the stripe in 2 L sterile PBS, incubate under soft stirring at 4  C for 1 week. 6. It is recommended to repeat steps 5 and 6 one time weekly (2–4 weeks total). To speed up the process, shorter incubation times are possible. The optimal amount of washing steps and incubation time is individual for each target and needs to be tested. For this, it is recommended to elute phage at different time points for further production of antibody fragments (Subheading 3.7) and screening ELISA (Subheading 3.8). 7. Elute with 200 μL trypsin solution for 30 min at 37  C (see Note 8). 8. Use 10 μL of phage solution for titration as described in Subheading 3.5. 9. Inoculate 50 mL 2YT-T with an overnight culture of E. coli XL1-Blue MRF0 in a 100 mL flask and grow at 250 rpm and 37  C until the culture reaches OD600 ~0.5. 10. Infect 4.5 mL of the culture in a 24-deep well plate with the remaining 190 μL trypsin-phage solution. Seal the plate with breathable sealing film and incubate for 30 min without and 30 min with shaking at 37  C and 450 rpm in a microtiter plate shaker. 11. Harvest the infected bacteria by centrifugation of the 24-well plate for 10 min at 3200
g. Resolve the pellet in 250 μL 2xYT-GA and plate the bacteria on 2xYT-GA agar plates (15 cm sterile petri dishes). Grow overnight at 37  C. 12. Pick single colonies from these plates to produce soluble monoclonal antibody fragments (Subheading 3.7). 3.7 Production of Soluble Monoclonal Antibody Fragments

1. Fill each well of a 96-well U-bottom polypropylene plate with 150 μL 2xYT-GA medium. 2. Pick 92 clones with sterile tips from the desired panning round, and inoculate each well. Use the wells H3, H6, H9, and H12 for controls. H3 and H6 are negative controls – these wells will not be inoculated. Inoculate the wells H9 and H12 with the clone containing the phagemid encoding for the parental antibody fragment. Seal the plate with breathable sealing film.

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3. Incubate overnight in a microtiter plate shaker at 37  C and 800 rpm. 4. (A) Fill a new 96-well polypropylene microtiter plate with 150 μL 2xYT-GA per well, and add 10 μL of the overnight cultures to each well. Incubate at 37  C and 800 rpm for 2 h. (B) Add 45 mL 80% (v/v) glycerol solution to the remaining 140 mL overnight cultures. Mix by pipetting and store this master plate at 80  C. 5. Pellet the bacteria in the microtiter plates by centrifugation at 3200
g and 4  C for 10 min. Carefully remove the glucosecontaining medium above the pellets without disturbing the pellet. 6. Add 160 μL 2xYT-A supplemented with 50 μM IPTG, seal the plate with a breathable sealing film. Incubate overnight at 30  C and 800 rpm (see Notes 9 and 10). 7. Pellet the bacteria by centrifugation at 3200
g for 10 min. Supernatant, containing the soluble antibody fragments, can be used directly in ELISA (Subheading 3.8). Alternatively, transfer the supernatant carefully into a new 96-well plate without disturbing the bacteria pellet, and store at 4  C for a short time. 3.8 ELISA of Soluble Monoclonal Antibody Fragments

1. To analyze the binding affinity of the soluble monoclonal antibody fragments to its antigen, coat 100 ng antigen per well in PBS (total volume 100 μL) for 1 h at room temperature or overnight at 4  C in a 96-well ELISA Costar plate (Corning). 2. Block the antigen coated wells with 330 μL MPBS-T for 1 h at room temperature. 3. Wash three times with H2O-T (see Note 7). 4. Fill each well with 60 μL MPBS-T, and transfer 40 μL antibody solution from the production plate (Subheading 3.7, step 7). If high signals are expected for the parental antibody fragment, it is reasonable to use 80–90 μL MPBS-T and just 10–20 μL antibody solution in order to obtain greater signal differences. Incubate for 1 h at room temperature. 5. Wash three times with H2O-T (see Note 7). 6. Incubate wells with 100 μL α-tag antibody solution for 1 h at room temperature. Dilute the antibody according to manufacturer’s instruction in MPBS-T. (Alternatively, it is possible to use an α-tag antibody directly conjugated with HRP. In this case, skip steps 7 and 8. The antibody should be wellcharacterized because due to the loss of signal amplification by the secondary antibody the resulting signals could be lower.) 7. Wash three times with H2O-T (see Note 7).

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8. Incubate wells with 100 μL of appropriate HRP conjugate (e.g., goat α-mouse HRP conjugate if α-tag antibody is of murine origin). Dilute according to manufacturer’s instruction in MPBS-T. 9. Wash three times with H2O-T (see Note 7). 10. Shortly before use, mix 10 parts TMB solution A with 0.5 parts TMB solution B. Add 100 μL of the prepared TMB solution into each well, and incubate for 1–15 min until a bright blue color is developed. Don’t over incubate so that signal differences are possible to be detected. 11. Stop the reaction by adding 100 μL 0.5 M sulfuric acid to each well. The color turns from blue to yellow. 12. Measure the A450nm – A620nm in an ELISA reader to identify candidates with a higher signal compared to the parental antibody fragment (see Note 11). 13. Sequence candidates to eliminate identical clones and to identify the number and position of the inserted mutations. 3.9 Stability Screening of Soluble Monoclonal Antibody Fragments

1. To screen soluble monoclonal antibody fragments for increased thermal stability, coat 100 ng antigen per well in PBS (total volume 100 μL) for 1 h at room temperature or overnight at 4  C in a 96-well ELISA Costar plate (Corning). 2. In a new polypropylene 96-well plate, mix 10–40 μL of each antibody solution with 60–90 μL PBS. Incubate the plate at elevated temperature for 1 h. The exact temperature depends on the stability of the parental antibody. If no prior knowledge is available, prepare multiple plates and incubate at 48  C, 50  C, 52  C, and 54  C, respectively. 3. Block the antigen coated wells with 330 μL MPBS-T for 1 h at room temperature. 4. Wash three times with H2O-T (see Note 7). 5. Add the heat-treated antibody solutions to the antigen-coated wells, and incubate for 1 h at room temperature. 6. Follow the standard screening procedure as described in Subheading 3.8, starting with step 5.

4

Notes 1. A total of 2–3 rounds of error-prone PCR can be performed by using the same primer set. After more rounds, the PCR product yield and quality will decrease rapidly. Switch to the next inner primer set if more rounds of error-prone PCR are required.

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2. By inserting random mutations, there is the chance of creating stop codons or frame shifts. Therefore, a higher number of mutations reduce the rate of full-length and in-frame sequences on amino acid level. Thus, it is important to find a balance between a sufficient number of inserted mutations and resulting complete sequences. 3. Vectors containing only small inserts 90%, select random clones for inoculation and incubate in 1–2 mL of 2× YT-GA medium overnight in a 48- or 96-deep well plate. When using 2 mL cultures in a 48-deep well plate, pool two plates together before pelleting by centrifugation (see Note 2). 3.5

Plasmid Isolation

1. Perform plasmid isolation with the NucleoSpin 96 Plasmid Transfection Grade Kit (Macherey-Nagel) according to the manufacturer’s instructions. 2. Determine DNA yield. 3. Use the isolated plasmids for the transfection of mammalian cells in 48- or 96-well format. The complete workflow is given in Table 4.

Antibody Batch Cloning

4

417

Notes 1. Mini-XL Spin columns have a higher DNA binding capacity than regular Mini Prep Kits and are therefore preferably used 2. Instead of using 96-deep-well plates for bacterial cultivation, we recommend using 48-well plates

References 1. Mullard A (2021) FDA approves 100th monoclonal antibody product. Nat Rev Drug Discov 20:491–495 2. Lu R-M, Hwang Y-C, Liu I-J et al (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27:1 3. Taylor PC, Adams AC, Hufford MM et al (2021) Neutralizing monoclonal antibodies for treatment of COVID-19. Nat Rev Immunol 21:382–393 4. Blum B, Neum€arker BKJ (2021) Lessons from Globalization and the COVID-19 Pandemic for Economic, Environmental and Social Policy. World 2:308–333 5. Valldorf B, Hinz SC, Russo G et al (2022) Antibody display technologies: selecting the cream of the crop. Biol Chem 403:455–477 6. Schirrmann T, Hust M (2010) Construction of Human Antibody Gene Libraries and Selection of Antibodies by Phage Display. In: Immunotherapy of Cancer. Humana Press, Totowa, NJ, pp 177–209 7. Breitling F, Du¨bel S, Seehaus T et al (1991) A surface expression vector for antibody screening. Gene 104:147–153 8. Frenzel A, Hust M, Schirrmann T et al (2013) Expression of Recombinant Antibodies. Front, Immun, p 4 9. Wenzel EV, Bosnak M, Tierney R et al (2020) Human antibodies neutralizing diphtheria toxin in vitro and in vivo. Scientific Reports 10:571 10. Fuchs M, K€ampfer S, Helmsing S et al (2014) Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B. BMC Biotechnol 14:68 11. Ossipow V, Fischer N (2014) Monoclonal Antibodies: Methods and Protocols, vol 1131. Humana Press Incorporated, Totowa, NJ 12. Kaplon H, Chenoweth A, Crescioli S et al (2022) Antibodies to watch in 2022. MAbs 14:2014296

13. Li Z, Krippendorff B-F, Sharma S et al (2016) Influence of molecular size on tissue distribution of antibody fragments. MAbs 8:113–119 14. Yokota T, Milenic DE, Whitlow M et al (1992) Rapid Tumor Penetration of a Single-Chain Fv and Comparison with Other Immunoglobulin Forms. Cancer Res 52:3402–3408 15. Hudson PJ, Souriau C (2003) Engineered antibodies. Nat Med 9:129–134 16. Brinkmann U, Kontermann RE (2017) The making of bispecific antibodies. MAbs 9:182– 212 17. Sterner RC, Sterner RM (2021) CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J 11:69 18. Bertoglio F, Meier D, Langreder N et al (2021) SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface. Nat Commun 12:1577 19. Liu JL, Zabetakis D, Acevedo-Ve´lez G et al (2013) Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6trinitrotoluene. Analytica Chimica Acta 759: 100–104 20. J€ager V, Bu¨ssow K, Wagner A et al (2013) High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 13:52 21. Schneider K-T, Kirmann T, Wenzel EV et al (2021) Shelf-Life Extension of Fc-Fused Single Chain Fragment Variable Antibodies by Lyophilization. Front Cell Infect Microbiol 11:717689 22. Schirrmann T, Bu¨ssow K (2010) Transient Production of scFv-Fc Fusion Proteins in Mammalian Cells. In: Kontermann R, Du¨bel S (eds) Antibody Engineering. Scholars Portal, Berlin, Heidelberg, pp 387–398 23. Bertoglio F, Fu¨hner V, Ruschig M et al (2021) A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2RBD interface and is tolerant to most known RBD mutations. Cell Rep 36:109433

Chapter 22 Deep Mining of Complex Antibody Phage Pools Tulika Tulika and Anne Ljungars Abstract An important, and rapidly growing class of drugs are antibodies which can be discovered through phage display technology. In this technique, antibodies are typically first enriched through consecutive rounds of selection on a target antigen with amplification in bacteria between each selection round. Thereafter, a subset of random individual clones is analyzed for binding in a screening procedure. This results in discovery of the most abundant antibodies in the pool. However, there are multiple factors affecting the enrichment of antibodies during the selection resulting in a very complex output pool of antibodies. A few antibodies are present in many copies and others only in a few copies, where the most abundant antibodies are not necessarily the functionally best ones. In order to utilize the full potential of the output from a phage display selection, and enable discovery of low abundant, potentially functionally important clones, deep mining technologies are needed. In this chapter, two methods for deep mining of an antibody pool are described, protein depletion and antibody blocking. The methods can be applied both when the target is a single antigen and on complex antigen mixtures such as whole cells and tissues. Key words Phage display, Deep mining, Antibody discovery, Target discovery, Phenotypic screening, Complex antigen mixtures, Cell panning

1

Introduction Antibodies are today the fastest growing class of drugs [1], with more than 100 antibodies approved by FDA [2], where the top-selling antibody, Humira, has sold for US$ 20 billion per year during the last years [3]. For discovery of therapeutic antibodies, the most common strategy is a target-based approach. In this strategy, a target is selected based on a hypothesis that it has an important disease-modulating effect, thereafter antibodies against the target are developed. An alternative is phenotypic discovery, in this strategy, functionally active antibodies are discovered without knowing the specificity beforehand. Thereafter, the specificity, the target, of the functionally active antibodies is determined. For therapeutic antibodies, there is today a limited target space where multiple antibodies are developed in parallel against a few targets

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_22, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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[1, 4]. For small molecule drugs, it has been shown that a targetbased discovery is most successful for discovery of best in class drug, whereas phenotypic discovery is more successful for discovery of first-in-class drugs [5, 6]. Inspired by this, in order to increase the target space, phenotypic discovery has gained increased attention for discovery of antibodies [7, 8]. Although successful, often only a handful of targets have been discovered when phenotypic discovery has been applied on whole cells [9–14], indicating that there is a need for refined antibody discovery methods to enable discovery of additional antibodies and targets. Furthermore, in the case of target-based discovery, studies have shown that discovered antibodies often bind to larger, well-exposed loops and epitopes on antigens [15]. These epitopes are often prone to changes over time due to mutations, which might make previously discovered antibodies less effective or even useless [16, 17]. For discovery of more broadly neutralizing antibodies, that bind conserved epitopes which can be buried in the antigen, advanced antibody discovery methods might be needed. A commonly used method for discovery of antibodies, is phage display technology [18–20], which can be applied in principle to any type of antigen and even complex antigen mixtures such as whole cells [21, 22], cell lysates [23], viruses [24, 25], bacteria, etc. Traditionally, first multiple sequential selections on the target antigen with amplification in between each round is performed to enrich a pool of phages displaying antibodies that are specific to the antigen of interest. Thereafter, a small number of random clones from the output pool is screened for binding to the target, and finally, the genes encoding a few binding antibodies are Sanger sequenced to determine the unique ones. However, during the selection process, multiple factors affect the enrichment of antibodies, including, antigen concentration, antigen accessibility, antibody affinity, antibody display level on phages, and phage amplification in bacteria, etc. [26, 27] As a result, the output from a selection, even on a single target, is very complex and contains a few high-abundant clones but many more clones of low frequency as illustrated in Fig. 1. If a complex target is included during the selection, like a whole cell, there will be hundreds of different cell surface receptors present at different density on the cell. Antibodies binding to these different receptors will be enriched in parallel resulting in an even more complex output pool. The traditionally discovered antibodies, found through direct screening of the output from a phage display selection, might therefore not be the clones binding the most relevant cell surface receptor, or the right epitope of an antigen. In order to fully utilize the output from a phage display selection, and allow discovery of not only the most abundant antibodies, deep mining technologies are needed [28]. Potentially, by applying these methods, more targets can be discovered in

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Fig. 1 Schematic picture of the output pool after several consecutive rounds of antibody phage display selections and strategies for discovery. The most abundant antibodies are easily found through direct screening whereas deep mining strategies are needed for discovery of more rare antibodies. (Picture adopted from Ljungars et al. [28])

phenotypic discoveries, and also when needed more broadly binding antibodies can be discovered. In this chapter, methods to shift the outcome from phage display selections and discover rare, low-abundant clones are described. The methods are described as applied on selections on whole cells, however, they can also be adopted and applied on other targets in a similar way.

2

Materials 1. Phage pool: Pool of antibody displaying phages (e.g., naı¨ve phage display antibody library, immune phage display antibody library, or an enriched antibody phage pool). 2. Coating buffer: 0.1 M Sodium Carbonate pH 9.5. For 1 L solution dissolve 2.75 g of Na2CO3 and 6.22 g of NaHCO3 in 1 L of deionized H2O. Adjust pH with NaOH or HCl if necessary. 3. 1/8″ Polystyrene beads (Polyscience #17175). 4. Streptavidin beads: Dynabeads M-280 Streptavidin (Thermo Fisher Scientific #11205D). 5. Tosylactivated beads: Dynabeads M-280 (Thermo Fisher Scientific # 14203).

Tosylactivated

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6. PBS: PBS w/o Ca/Mg (Gibco #14190230). 7. 20% BSA stock solution: Dissolve 100 g Protease Free BSA (Albumin, Bovine Fraction V) (Sigma-Aldrich # A-3059) in sterile PBS to final volume of 500 mL. Sterilize by filtration through 0.2 μm filter. 8. 10% Tween stock solution: Add 1 mL Tween 20 to 9 mL sterile PBS. Mix the two solutions completely by vortexing. Sterilize by filtration through 0.2 μm filter. 9. PBST: PBS + 0.05% Tween 20. Add 250 μL 10% Tween stock solution to 50 mL PBS. 10. PBST+5% BSA: Add 12.5 mL 20% BSA stock solution to 37.5 mL PBST. 11. Heat inactivated FBS (HI FBS): Heat inactivate the FBS for 30 min at 56 °C. 12. 0.5 M EDTA (Invitrogen #15575020). 13. 10% NaN3: Dissolve 1 g of Sodium Azide in 10 mL of water. Sterilize by filtration through 0.2 μm filter. 14. 25X Complete (Roche #11873580001): Dissolve 1 tablet in 2 mL PBS. 15. 10 mg/mL Trypsin: Dissolve 500 mg Trypsin (Sigma-Adrich #T4799) in sterile PBS to final volume of 50 mL. 16. 2 mg/mL Aprotenin: Dissolve 10 mg Aprotinin (SigmaAldrich 10236624001) in sterile PBS to final volume of 5 mL. Sterilize by filtration through 0.2 μm filter.

3

Methods

3.1 Protein Depletion Followed by Panning on Whole Cells

When panning on whole cells is performed, antibodies are typically enriched against a few cell surface receptors. If these receptors are known, and they can be produced recombinantly, discovery of antibodies binding to these receptors can be avoided through protein depletion (Fig. 2a). Depending on the nature of the receptors, competition with proteins (Subheading 3.2) can be included either after the protein depletion or as an alternative to protein depletion (Fig. 2b). Multiple types of beads can be used for coating of proteins. A few examples of coating beads, including passive absorption (coating on polystyrene beads), specific binding (binding to streptavidin beads), and chemical coupling (tosyl-activated beads), are listed below.

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Fig. 2 To reduce the number of antibodies binding to targets of low interest the corresponding protein can be included during the selection for protein depletion (a) or competition (b). (Picture adopted from Ljungars et al. [28])

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3.1.1 Coating of Polystyrene Beads

1. Dilute the protein to 0.1 μM in coating buffer. Add 4 polystyrene beads per depletion tube (see Note 1). 2. Incubate the beads with end over end rotation overnight at +4 °C. 3. Wash the beads 3 times with 10 mL PBST. For washing add buffer, let beads sediment, aspirate buffer (see Note 2). 4. Block the beads in 1 mL PBST+5% BSA for 1 h at room temperature with end over end rotation. 5. Store the coated and blocked beads at +4 °C until use (see Note 3).

3.1.2 Binding to Streptavidin Beads

1. If biotinylated antigens are available, streptavidin beads can be used to capture the antigen. Couple the beads according to the manufacturer’s instructions. Use approximately 200 pmole of protein to 4 mg beads for one depletion. 2. After the last step in the manufacturer’s instructions, concentrate the beads on the magnetic rack and discard the supernatant. 3. Block the beads in 1 mL PBST+5% BSA for 1 h at room temperature with end over end rotation. 4. Store the coated and blocked beads at +4 °C until use (see Note 3).

3.1.3 Coupling of Tosylactivated M-280 Beads

1. Couple the beads according to the manufacturer’s instructions. Use approximately 200 pmole of protein to 1.8 mg beads for one depletion. 2. After the last step in the manufacturer’s instructions, concentrate the beads on the magnet and discard the supernatant. 3. Block the beads in 1 mL PBST+5% BSA for 1 h at room temperature with end over end rotation. 4. Store the coated and blocked beads at +4 °C until use (see Note 3).

3.1.4 Pool

Depletion of Phage

1. In a 2 mL Eppendorf tube, mix all beads that will be used for depletion of the phage pool. Remove the buffer. 2. Add the phage pool in PBST + 5% BSA. Try to keep a relatively low volume (100–500 μL) (see Note 4). 3. Incubate the phage pool with the beads for approximately 2 h at room temperature with end over end rotation (see Note 5). 4. Collect the unbound phages and use them for selection.

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Table 1 Concentrations and volumes of reagents to be mixed for a cell selection Reagent

Stock conc.

Final conc.

300 μL

Phages HI FBS

100%

10%

80 μL

EDTA

0.5 M

2 mM

3.2 μL

Complete

25X

1X

32 μL

NaN3

10%

0.02%

1.6 μL

PBS

–

–

383.2 μL

Total 3.1.5

Panning on Cells

Volume stock added

800 μL

1. Add between 1·107 and 1·108 cells to a 2 mL tube and spin down the cells to remove any buffer or media (see Note 6). 2. Add the depleted phages from step 4 in Subheading 3.1.4 above and additional reagents to the cells according to Table 1. Incubate for 2–4 h on ice on a rocking table (see Note 7). 3. Transfer cells and phages to a larger tube (10 mL or 50 mL falcon tube) for washing. 4. Add 10 mL PBS+10% FBS and incubate for 5 min on ice. 5. Spin down the cells, 300 g for 10 min at +4 °C. Discard the supernatant with unbound phages. 6. Repeat steps 4–5, 2 times resulting in a total of 3 washes. Perform the last wash with PBS only (see Note 8).

3.1.6 Elution of Phages Using Trypsin

1. Add 400 μL trypsin at 1 mg/mL and incubate for 30 min at room temperature with end over end rotation (see Note 9). 2. Stop the trypsin digestion by addition of aprotinin to a final concentration of 0.2 mg/mL. 3. Spin down the cells/lysed cells and recover the eluted phages. 4. Continue and amplify the eluted phages according to standard procedures.

3.2 Panning on Whole Cells with Protein Competition 3.2.1 Calculations of Protein Concentrations

Before starting panning, decisions should be made on which proteins to include and at what concentrations. Typically avoid proteins that can bind to the cells, e.g., a soluble ligand binding to a cell surface receptor. (Such a ligand can then bind to the receptor on the cell and when panning is performed on cells with the ligand present it can possibly enable discovery of binders to the ligand.) Concentration of the different proteins is key during the selection. Typically, 100–1000 times excess of competing proteins in solution

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Table 2 Total number of receptor molecules in a mixture of different number of cells depending on the number of receptors per cell # Receptors/cell # Cells

1000

10,000 8

9

100,000

1,000,000

1.0·10

1.0·10

10

1.0·1011

1.0E5

1.0·10

1.0E6

1.0·109

1.0·1010

1.0·1011

1.0·1012

1.0E7

1.0·1010

1.0·1011

1.0·1012

1.0·1013

1.0E8

1.0·1011

1.0·1012

1.0·1013

1.0·1014

Table 3 Total number of molecules depending on antigen concentration in a 1 mL solution Antigen concentration (nM)

Total number of molecules

1

6.0·1011

10

6.0·1012

100

6.0·1013

1000

6.0·1014

10000

6.0·1015

compared to the expected total number of cell surface expressed receptors is added. See Tables 2 and 3, e.g., calculations on the total number of molecules in solution and on the cell surface. Based on the calculations using Tables 2 and 3, prepare a mixture of the proteins that will be used for competition. 3.2.2

Panning on Cells

1. Add between 1·107 and 1·108 cells to a 2 mL tube and remove any buffer (see Note 6). 2. Add the mixed proteins (in this example, 200 μL) that will be used for competition. 3. Add phages (depleted from step 4 in Subheading 3.1.4 above or undepleted) and additional reagents according to Table 4. Incubate for 2–4 h on ice on a rocking table (see Note 7). 4. Transfer cells and phages to a larger tube (4 or 50 mL falcon tube) for washing. 5. Add 10 mL PBS+10% FBS and incubate for 5 min on ice. 6. Spin down the cells, 300 g for 10 min at +4 °C. Discard the supernatant with unbound phages. 7. Repeat steps 5–6, 2 times resulting in totally 3 washes. Perform the last wash with PBS only (see Note 8).

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Table 4 Concentrations and volumes of reagents to be mixed for a cell selection with protein depletion Reagent

Stock conc.

Final conc.

300 μL

Phages Protein mix

4 μM

1 μM

200 μL

HI FBS

100%

10%

80 μL

EDTA

0.5 M

2 mM

3.2 μL

Complete

25X

1X

32 μL

NaN3

10%

0.02%

1.6 μL

PBS

–

–

183.2 μL

Total 3.2.3 Elution of Phages Using Trypsin

Volume stock added

800 μL

1. Transfer cells to 2 mL Eppendorf tube. 2. Add 400 μL trypsin at 1 mg/mL and incubate for 30 min at room temperature with end over end rotation (see Note 9). 3. Stop the trypsin digestion by addition of Aprotinin to a final concentration of 0.2 mg/mL. 4. Spin down the cells/lysed cells and recover the eluted phages. 5. Continue and amplify the eluted phages according to standard procedures.

3.3 Panning on Whole Cells with Antibody Blocking

During panning, either on complex antigen mixtures such as whole cells or even on a single antigen the output pool of antibodies typically contains a few antibodies present in high abundance. Using these antibodies to block their respective epitopes/targets during new selections can facilitate discovery of new antibodies targeting different epitopes and/or targets as depicted in Fig. 3.

3.3.1 Calculations of Antibody Concentrations

In order to avoid finding the already discovered antibodies again, and antibodies binding the same or overlapping epitopes to discovered ones, a 10–100 times excess of the blocking antibody compared to cell surface receptors and antibodies displayed on phages should be used. For example of calculations of antibody concentrations see Table 5. Examples of total number of cell surface receptors are found in Table 2.

3.3.2

Panning on Cells

1. Add between 1·107 and 1·108 cells to a 2 mL tube and remove any buffer or media by spinning down the cells (see Note 6). 2. Add the antibodies at a concentration calculated to be in excess compared to the number of cell surface receptors (see Note 10). Incubate the cell antibody mixture for 1 h on ice on a rocking table.

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Fig. 3 Antibodies can be included during a phage display selection to block binding to epitopes and targets to which antibodies have already been discovered. Thereby, additional, new antibodies can be discovered. (Picture adopted from Ljungars et al. [28]) Table 5 Total number of molecules depending on antibody concentration in a 1 mL solution calculated with an antibody molecular weight of 150 kDa Antibody concentration (μg/mL)

Total number of molecules

0.15

6.0·1011

1.5

6.0·1012

15

6.0·1013

150

6.0·1014

1500

6.0·1015

3. Add phages (depleted or undepleted) and additional reagents according to Table 6. Incubate for 2–4 h on ice on a rocking table (see Note 7). 4. Transfer cells and phages to a larger tube (10 or 50 mL falcon tube) for washing. 5. Add 10 mL PBS+10% FBS and incubate for 5 min on ice. 6. Spin down the cells, 300× g for 10 min at +4 °C. Discard the supernatant with unbound phages. 7. Repeat steps 5–6, 2 times resulting in a total of 3 washes. Perform the last wash with PBS only (see Note 8). 3.3.3 Elution of Phages Using Trypsin

1. Transfer cells to a 2 mL tube. 2. Add 400 μL trypsin at 1 mg/mL and incubate for 30 min at room temperature with end over end rotation (see Note 9). 3. Stop the trypsin digestion by addition of Aprotinin to a final concentration of 0.2 mg/mL.

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Table 6 Concentrations and volumes of reagents to be mixed for a cell selection with antibody blocking Reagent

Stock conc.

Final conc.

Volume stock added 300 μL

Phages IgG pool

2.7 mg/mL of each IgG 1.0 mg/mL 300 μL

HI FBS

100%

10%

80 μL

EDTA

0.5 M

2 mM

3.2 μL

Complete 25X

1X

32 μL

NaN3

10%

0.02%

1.6 μL

PBS

–

–

83.2 μL

Total

800 μL

4. Spin down the cells/lysed cells and recover the eluted phages. 5. Continue and amplify the eluted phages according to standard procedures.

4

Notes 1. Use 4 beads per protein included for depletion and coat with approximately 25 pmole/bead. To get a good mixing of the beads use 2 mL Eppendorf tubes with 1 mL buffer for coating 2. For easy washing of many beads in parallel, beads + buffer can be poured out into a tea strainer where the beads are collected and transferred to a 50 mL Falcon tube 3. Beads are typically used the same day or the day after coating/ coupling. Antigen stability on the coated beads is dependent on the antigen and longer storage may affect the antigen quality 4. The whole volume will be used later for selection on cells, and it is, therefore, important to keep a low volume and to avoid tween which might disrupt the cells later. If many different depletions are to be performed resulting in an increased volume of the beads, depletion may be run sequentially, first incubating with one batch of proteins and then transfer the unbound phages to a second batch of proteins 5. Incubation can be prolonged and run overnight. For longer incubation times incubate at +4 °C instead of room temperature 6. Viability of the cells is crucial, handle the cells gently and keep on ice or at +4 °C at all times

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7. Volume is a compromise in this step, it should be kept low to receive a high concentration of both phages and antigens. However, a sufficiently large volume should be added to allow a good mixture between phages and cells. Typically between 0.2 mL and 1 mL is used 8. To avoid any residues of FBS after washing, which will inhibit the trypsin digestion, the last wash is performed in PBS only 9. The concentration of trypsin may vary depending on the activity of the trypsin. Typically, a higher concentration than used on protein selections on beads is needed due to the high number of proteins present on the cells 10. Different antibody formats can be used. IgGs have higher apparent affinity due to the bivalent binding resulting in avidity compared to scFv, Fabs, and Nanobodies, and can be more successful. However, due to the larger size of the IgG molecule, more steric hindrance of phage binding might occur compared to when using a smaller antibody format for blocking. Depending on the number of antibodies, and their concentration, the total volume of the antibody mixture might need to be reduced before usage to minimize the volume during the selection References 1. Carter PJ, Lazar GA (2018) Next generation antibody drugs: pursuit of the “high-hanging fruit”. Nat Rev Drug Discov 17:197–223. https://doi.org/10.1038/nrd.2017.227 2. Mullard A (2021) FDA approves 100th monoclonal antibody product. Nat Rev Drug Discov 20:491–495. https://doi.org/10.1038/ d41573-021-00079-7 3. Urquhart L (2022) Top companies and drugs by sales in 2021. Nat Rev Drug Discov 21: 251–251. https://doi.org/10.1038/d41573022-00047-9 4. Martineau P, Watier H, Pe`legrin A, Turtoi A (2019) Targets for MAbs: innovative approaches for their discovery & validation, LabEx MAbImprove 6th antibody industrial symposium, June 25–26, 2018, Montpellier, France. MAbs 11:812–825. https://doi.org/ 10.1080/19420862.2019.1612691 5. Swinney DC, Anthony J (2011) How were new medicines discovered? Nat Rev Drug Discov 10:507–519. https://doi.org/10.1038/ nrd3480 6. Eder J, Sedrani R, Wiesmann C (2014) The discovery of first-in-class drugs: origins and evolution. Nat Rev Drug Discov 13:577–587. https://doi.org/10.1038/nrd4336

7. Minter RR, Sandercock AM, Rust SJ (2017) Phenotypic screening—the fast track to novel antibody discovery. Drug Discov Today Technol 23:83–90. https://doi.org/10.1016/j. ddtec.2017.03.004 8. Gonzalez-Munoz AL, Minter RR, Rust SJ (2016) Phenotypic screening: the future of antibody discovery. Drug Discov Today 21: 150–156. https://doi.org/10.1016/j.drudis. 2015.09.014 9. Fransson J, Tornberg U-C, Borrebaeck CAK, Carlsson R, Frende´us B (2006) Rapid induction of apoptosis in B-cell lymphoma by functionally isolated human antibodies. Int J Cancer 119:349–358. https://doi.org/10. 1002/ijc.21829 10. Veitonm€aki N, Hansson M, Zhan F, Sundberg A, Lo¨fstedt T, Ljungars A et al (2013) A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo. Cancer Cell 23:502–515. https://doi. org/10.1016/j.ccr.2013.02.026 11. Rust S, Guillard S, Sachsenmeier K, Hay C, Davidson M, Karlsson A et al (2013) Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’

Deep Mining of Complex Antibody Phage Pools breast cancer cell type. Mol Cancer 12:11. https://doi.org/10.1186/1476-4598-12-11 12. Sandercock AM, Rust S, Guillard S, Sachsenmeier KF, Holoweckyj N, Hay C et al (2015) Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling. Mol Cancer 14:147. https://doi. org/10.1186/s12943-015-0415-0 13. Williams GS, Mistry B, Guillard S, Ulrichsen JC, Sandercock AM, Wang J et al (2016) Phenotypic screening reveals TNFR2 as a promising target for cancer immunotherapy. Oncotarget 7:68278–68291. https://doi. org/10.18632/oncotarget.11943 14. Ljungars A, Ma˚rtensson L, Mattsson J, Kovacek M, Sundberg A, Tornberg U-C et al (2018) A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia. Npj Precision Onc 2:1–4. https://doi.org/10.1038/ s41698-018-0061-2 15. Fong RH, Banik SSR, Mattia K, Barnes T, Tucker D, Liss N et al (2014) Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy. J Virol 88: 14364–14379. https://doi.org/10.1128/ JVI.01943-14 16. Krammer F (2019) The human antibody response to influenza A virus infection and vaccination. Nat Rev Immunol 19:383–397. https://doi.org/10.1038/s41577-0190143-6 17. Sun W, Kang DS, Zheng A, Liu STH, Broecker F, Simon V et al (2019) Antibody responses toward the major antigenic sites of influenza B virus hemagglutinin in mice, ferrets, and humans. J Virol 93:e01673–e01618. https://doi.org/10.1128/JVI.01673-18 18. Ledsgaard L, Ljungars A, Rimbault C, Sørensen CV, Tulika T, Wade J et al (2022) Advances in antibody phage display technology. Drug Discov Today 27:2151–2169. https://doi. org/10.1016/j.drudis.2022.05.002 19. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making Antibodies by Phage Display Technology. Ann Rev Immunol 12:433–455. https://doi.org/10.1146/ annurev.iy.12.040194.002245 20. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains.

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Nature 348:552–554. https://doi.org/10. 1038/348552a0 21. Lipes BD, Chen Y-H, Ma H, Staats HF, Kenan DJ, Gunn MD (2008) An entirely cell-based system to generate single-chain antibodies against cell surface receptors. J Mol Biol 379: 261–272. https://doi.org/10.1016/j.jmb. 2008.03.072 22. Jones ML, Alfaleh MA, Kumble S, Zhang S, Osborne GW, Yeh M et al (2016) Targeting membrane proteins for antibody discovery using phage display. Sci Rep 6:26240. https://doi.org/10.1038/srep26240 23. Even-Desrumeaux K, Nevoltris D, Lavaut MN, Alim K, Borg J-P, Audebert S et al (2014) Masked selection: a straightforward and flexible approach for the selection of binders against specific epitopes and differentially expressed proteins by phage display. Mol Cell Proteomics 13:653–665. https://doi.org/10. 1074/mcp.O112.025486 24. Kirsch MI, Hu¨lseweh B, Nacke C, Ru¨lker T, Schirrmann T, Marschall H-J et al (2008) Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV). BMC Biotechnol 8:66. https://doi.org/10.1186/1472-6750-8-66 25. Bertoglio F, Meier D, Langreder N, Steinke S, Rand U, Simonelli L et al (2021) SARS-CoV2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface. Nat Commun 12:1577. https://doi.org/10.1038/ s41467-021-21609-2 26. Derda R, Tang SKY, Whitesides GM (2010) Uniform amplification of phage with different growth characteristics in individual compartments consisting of monodisperse droplets. Angew Chem Int Ed Engl 49:5301–5304. https://doi.org/10.1002/anie.201001143 27. Bradbury ARM, Marks JD (2004) Antibodies from phage antibody libraries. J Immunol Methods 290:29–49. https://doi.org/10. 1016/j.jim.2004.04.007 28. Ljungars A, Svensson C, Carlsson A, Birgersson E, Tornberg U-C, Frende´us B et al (2019) Deep mining of complex antibody phage pools generated by cell panning enables discovery of rare antibodies binding new targets and epitopes. Front Pharmacol 10:847. https://doi.org/10.3389/fphar.2019.00847

Chapter 23 High-Throughput IgG Reformatting and Expression Using Hybrid Secretion Signals and InTag Positive Selection Technology Georgina Sansome, Veronika Rayzman, Irene Kiess, Michael J. Wilson, Con Panousis, and Chao-Guang Chen Abstract We have previously published protocols for high-throughput IgG reformatting and expression, that enable rapid reformatting of phage-displayed antibody Fab fragments into a single dual expression vector for full IgG expression in Expi293F cells (Chen et al. Nucleic Acids Res 42:e26, 2014; Chen et al. Methods in Molecular Biology, vol 1701, 2018). However, when working with phage clones from a naı¨ve library containing highly diverse N-terminal sequences, where the 5′ PCR primers bind, the PCR step can become cumbersome. To overcome this limitation, we have investigated and found that the C-terminal 7 amino acid residues of the human antibody VH1 secretion signal can be replaced with those from ompA or pelB bacterial signals to form hybrid signal sequences that can drive strong IgG expression in Expi293F cells. The use of such hybrid signals allows any Fab fragment in the library to be amplified and cloned into the IgG expression vector using only a single 5′ PCR primer targeting the bacterial secretion signal of the light or heavy chain, thus dramatically simplifying the IgG reformatting workflow. Key words Phage display, IgG reformatting, InTag positive selection, Secretion signal, Hybrid secretion signal, In-Fusion cloning, Ligation-independent cloning, Mammalian cell expression, Transient transfection

1

Introduction IgG reformatting and expression are key steps in antibody discovery using phage display technology when the ultimate clinical or diagnostic application format is an intact IgG. In a typical antibody discovery workflow, positive phage clones are isolated by screening a phage display antibody library against the antigen of interest. These clones are then reformatted and expressed in E. coli as soluble Fabs or scFvs for biophysical and functional analysis to narrow

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_23, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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down the number of lead candidates. The selected few clones are finally reformatted into intact IgGs and expressed in mammalian cells for detailed characterization before a lead antibody is selected for further development. The E. coli expression step is a lengthy and laborious procedure that is often hindered by problems including low and variable expression of antibody fragments and complex purification protocols. In addition, antibody fragments produced in E. coli are often not suitable for screening in biological assays requiring Fc-dependent effector functions and antibody avidity. Furthermore, lipopolysaccharide (LPS) contamination can compromise in vitro, and in vivo functional assays employed during the candidate screening process. Ideally, biophysical and functional screening of all unique antigen-binding phage clones should be undertaken in IgG format, however, the complexity and challenges associated with IgG reformatting necessitate the use of an E. coli expression/screening step to narrow the number of candidates for IgG reformatting and mammalian expression. Reformatting antibody fragments from a phage display vector to an intact IgG in a mammalian expression vector presents several significant challenges due to the fundamental differences in the architecture of bacterial and mammalian expression vectors. First, while the light and heavy chains can be expressed from a single promoter in bacteria, the bacterial promoter needs to be replaced by two mammalian promoters (or one promoter and one IRES) for IgG expression in mammalian cells. Second, bacterial secretion signals need to be replaced with their mammalian counterparts. Third, a polyadenylation domain needs to be added to both light and heavy chains for mammalian expression. Fourth, an additional heavy chain constant domain sequence needs to be added to convert a Fab to a full IgG. Finally, the addition of a constant region and the swap of secretion signals require perfect in-frame fusions between coding regions, which demands the incorporation of appropriate cut sites at the exact positions if a cut-paste cloning strategy is used. Several IgG reformatting methods have been reported where the light and heavy chains are generated in separate vectors and the IgG expressed by co-transfection in mammalian cells [1, 2] or cloned sequentially into a single mammalian expression vector [3, 4]. However, these methods suffer from several key limitations such as multiple and laborious cloning steps; the use of restriction digestion for the preparation of antibody inserts from the phage display vectors, which can result in the loss of clones containing internal restriction sites; and most importantly, the requirement for single colony screening to discriminate the correct recombinant clone from cloning background that results from uncut and re-ligated vector.

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To address these problems, we have previously developed a one-step zero-background IgG reformatting method with inserttagged (InTag) positive selection [5, 6]. This high-throughput (HTP) method allows the light chain (LC) and the variable domain of the heavy chain (VH) to be amplified by a single duplex PCR from a phage clone and subsequently cloned, in conjunction with an InTag adaptor, into an IgG mammalian expression vector (Fig. 1). The InTag adaptor contains a SV40pA for the light chain, as well as a second CMV promoter and a secretion signal for the heavy chain. In addition, the InTag adaptor contains an antibiotic resistance marker such as kanamycin resistance gene (KanR) to enable direct selection of recombinant clones in liquid culture following transformation, thus avoiding the laborious and time-consuming step of single colony screening. Furthermore, we have incorporated In-Fusion cloning, a type of ligationindependent cloning, into the workflow to streamline the process by eliminating PCR purification and restriction enzyme digestion steps. Compared with the traditional cut-paste cloning methods, the In-Fusion cloning method not only requires fewer manipulations but also eliminates the potential for clone loss due to the presence of internal cut sites. One limitation of the original IgG reformatting protocol is, however, the use of 5′ PCR primers targeting the N-terminal sequences of the LC and VH regions for PCR amplification due to the requirement of replacing the bacterial signals with mammalian secretion signals for IgG expression. While the protocol can work well for a library with a small number of germline sequences, it can become cumbersome for a naı¨ve library, where the N-terminal sequences are very diverse. Reformatting of phage clones isolated from such a diverse library without modifying their N-terminal sequences would therefore require: (1) a large number of specific 5′ primers to be synthesized for both LC and VH regions; (2) the N-terminal sequences of both LC and VH regions of each phage clone to be clearly determined by DNA sequencing; and (3) individual PCRs to be set up for each clone using the correct specific primers. To overcome this limitation, we have investigated and found that the C-terminal 7 amino acid residues (long enough for a PCR primer) from bacterial signals ompA or pelB can be fused with the N-terminal 12 amino acid sequence of human antibody VH1 secretion signal to form hybrid signals (hVH1_ompA and hVH1_pelB, Table 1) that still retain the capacity to drive strong IgG expression in Expi293F cells [unpublished data]. This is consistent with the previous findings, where a hybrid signal between the pelB bacterial signal and a mammalian signal was found to be functional in driving scFv-Fc fusion proteins in mammalian cells [7, 8]. We also

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A)

P2

P1 P S

P3

rbs

LC

P4 S

VH

G1 CH1 Gene III

ampR LC

Duplex PCR

VH NgoMIV

B)

AfeI

pA1 KanR

pCMV S

InTag adaptor pA1 KanR

pCMV S

C) LC

VH In-Fusion cloning pA1

ApaLI pCMV

KanR

pCMV S

NheI IgG4 CH

S Intron

pA2

ampR Transformation, KanR selection

D)

ApaLI pCMV

S

Intron

AfeI

NgoMIV LC

pA1 KanR

pCMV S

NheI VH

IgG4 CH

pA2

ampR

Fig. 1 Schematic of zero-background IgG reformatting using InTag positive selection and hybrid secretion signals. (a) Amplification of antibody fragments from phage vector. The light chain (LC) and VH region are amplified by a duplex PCR using P1, P2, P3, and P4 primers. (b) Isolation of the InTag adaptor. The adaptor containing the SV40pA (pA1), KanR gene, CMV promoter, and the hVH1-pelB hybrid signal is isolated by NgoMIV and AfeI digestion. (c) In-Fusion cloning of LC and VH regions using InTag positive selection. PCR fragments were cloned along with the InTag adaptor into the expression vector using In-Fusion cloning. The resulting recombinant plasmids were selected in liquid media containing kanamycin. (d) Final IgG reformatted mammalian expression vector. This dual expression vector contains two CMV promoters to drive both the light chain (LC) and heavy chain (VH/IgG4 CH) expression from their respective hybrid signals. The LC contains an intron (marked by a narrow black box) at the 5′ untranslated region to enhance IgG expression and a SV40pA signal (pA1), whereas the heavy chain contains a BGHpA signal (pA2). Bacterial signal sequences (S) are shown in red and the human VH1 signal sequence (S) are shown in purple. P lac promoter, S ompA or pelB signal peptides, rbs ribosome binding site, pA1 SV40pA, pA2 BGHpA, pCMV CMV promoter, KanR kanamycin resistance marker, AmpR ampicillin resistance marker

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Table 1 Amino acid sequences of secretion signals Signal Name

Amino acid sequence

Note

Light chain ompA

MKKTAIAIAVALAGFATAVQA

hVH1

MDWTWRILFLVAAATGAHS

hVH1_ompA hybrid

MDWTWRILFLVAAFATAVHA

Used in the phage library Wild Type Used in IgG expression vector

Heavy chain pelB

MKYLLPTAAAGLLLLAAQPAMA

Used in phage library

hVH1

MDWTWRILFLVAAATGAHS

Wild Type

hVH1_pelB hybrid

MDWTWRILFLVAAAQPALA

Used in IgG expression vector

The amino acid sequences of the bacterial secretion signals ompA and pelB used for light and heavy chain expression in CSL’s naı¨ve Fab phage display libraries are shown in red, while that of human VH1 secretion signal is shown in black. The hybrid signals hVH1_ompA and hVH1_pelB used for IgG mammalian expression, contain residues from human VH1 (in black) and bacterial signals (in red). Residues in blue are introduced mutations for the purpose of cloning

discovered that the C-terminal 7 amino acid region can tolerate mutations, thus allowing the incorporation of restriction sites for cloning [unpublished data]. Therefore, since ompA and pelB bacterial signals are used for the light and heavy chain expression in our naı¨ve phage display libraries, only a single 5′ PCR primer targeting to the C-terminal 7 amino acid region of ompA and another targeting that of pelB are required to amplify all the Fab clones from the naı¨ve libraries, even without the pre-determination of their nucleotide sequences (Figs. 1 and 2), thus dramatically simplifying the PCR step of the IgG reformatting workflow. The updated and simplified HTP IgG reformatting strategy is illustrated in Fig. 1 and the detailed primer designs are shown in Figs. 2 and 3. Briefly, the LC and VH regions are amplified in a duplex PCR using P1, P2, P3, and P4 primers. P1 and P3 are 5′ primers binding to ompA and pelB signal regions respectively, whereas P2 and P4 are 3′ primers binding to the vector sequence downstream of the light chains (kappa and lambda) or to the start of the heavy chain constant region. The LC and VH fragments are then cloned along with an InTag adaptor into the mammalian expression vector using In-Fusion cloning. The KanR selection marker contained within the InTag adaptor will allow recombinant clones to be selected in liquid culture without single colony screening, thus dramatically shortening the cloning workflow and

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I. Light chain hybrid signal (hVH1_ompA) design A)

M K K T A I A I A V A L A G F A T A V Q A 5’ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACGGCCGTGCAGGCA 3’TACTTTTTCTGTCGATAGCGCTAACGTCACCGTGACCGTCCAAAGCGATGCCGGCTCGTCCGT | ||||| ||||||||||| ||| P1: 5’GCCTTCGCCACGGCCGTGCACGCA 3’ B) |||||||||||||||||||| C) 5’ ATGGACTGGACCTGGCGCATCCTGTTTCTGGTGGCCTTCGCCACGGCCGTGCACGCA 3’ TACCTGACCTGGACCGCGTAGGACAAAGACCACCGGAAGCGGTGCCGGCACGTGCGT M D W T W R I L F L V A F A T A V H A D)

M

D

W

T

W

R

I

L

F

L

V

A

A

A

T

G

A

H

S

II. Heavy chain hybrid signal (hVH1_pelB) design A)

M K Y L L P T A A A G L L L L A A Q P A M A 5’ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCC 3’TACTTTATGGATAACGGATGCCGTCGGCGACCTAACAATAATGAGCGACGGGTTGGTCGGTACGCC ||||||||||||||| ||||| B) P3: 5’GTGGCCGCTGCCCAACCAGCGCTGGCC 3’ |||||||||||||||||||| C) 5’ ATGGACTGGACCTGGCGCATCCTGTTTCTGGTGGCCGCTGCCCAACCAGCGCTGGCC 3’ TACCTGACCTGGACCGCGTAGGACAAAGACCACCGGCGACGGGTTGGTCGCGACCGG M D W T W R I L F L V A A A Q P A L A D)

M

D

W

T

W

R

I

L

F

L

V

A

A

A

T

G

A

H

S

Fig. 2 Hybrid signal design and sequences. (a) Phage display vector. The nucleotide and amino acid sequences of the bacterial secretion signals ompA (slightly modified from wildtype) and pelB used for the light chain (I) and heavy chain (II) respectively in the phage display library are shown. (b) PCR primers. The sequences of 5′ PCR primers (P1, P3), which bind to the bacterial secretion signals are shown in bold (I and II) with the homologies to the phage clone and the mammalian expression vector (I) or the InTag adaptor (II) indicated respectively. (c) IgG expression vector and InTag adaptor. The nucleotide and amino acid sequences of the hybrid secretion signals in the IgG mammalian expression vector or InTag adaptor are shown (I and II). The amino acid residues from the human antibody VH1 secretion signal are shown in black, with those from the bacterial signals highlighted in red. Restriction sites ApaLI and AfeI were introduced and are underlined with resulting amino acid changes represented in blue. (d) Human antibody VH1 secretion signal sequence

improving efficiency. The in-frame fusion between the C-terminal region of ompA or pelB bacterial signals with the N-terminal region of human antibody VH1 secretion signal forms hVH1_ompA or hVH1_pelB hybrid signals to drive IgG expression in Expi293F cells. The detailed protocols for HTP IgG reformatting and mammalian expression using hybrid secretion signals are described in this Chapter.

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I. Light chain Kappa or Lambda – end of constant region A)

5’ 3’ B) C)

5’ 3’

K S F N R G E C * (Kappa) T V A P T E C S * (Lambda) nnnnnnnnnnnnnnnnnnnnnnnnTAGGCCGGCCCGGCCTAATCTATT nnnnnnnnnnnnnnnnnnnnnnnnATCCGGCCGGGCCGGATTAGATAA |||||||||||||||||||||||| P2: 3’< ATCCGGCCGGGCCGGATTAGATAA 5’ |||||||||||||||||||| nnnnnnnnnnnnnnnnnnnnnnnnTAGGCCGGCCCGGCCTAATCTATT nnnnnnnnnnnnnnnnnnnnnnnnATCCGGCCGGGCCGGATTAGATAA K S F N R G E C * (Kappa) T V A P T E C S * (Lambda)

II. Heavy chain – start of constant region A)

B) C)

AST K G P S V F P L A P S S K S T S 5’ AAGGGCCCATCGGTCTTCCCGCTAGCACCCTCCTCCAAGAGCACCTCT 3’ TTCCCGGGTAGCCAGAAGGGCGATCGTGGGAGGAGGTTCTCGTGGAGA ||||||||||| |||| ||||| ||| P4: 3’< AAGGGCGATCGCGGGACGAGGTCCTC 5’ |||||||||||||||||||| 5’ AAGGGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCC 3’ TTCCCGGGTAGCCAGAAGGGCGATCGTGGGAGGAGGTTCTCGTGGAGA AST K G P S V F P L A P C S R S T S

Fig. 3 Reverse primers for the amplification of light and heavy chains. (a) Phage display vector. The nucleotide and amino acid sequences at the end of kappa and lambda light chains (I) or the start of heavy chain constant region (II) are shown. (b) Reverse PCR Primers. The sequences of reverse PCR primers are shown in bold, with the homologies to the phage clone and the mammalian expression vector or the InTag adaptor indicated respectively. Primer P2 binds to the end of both kappa and lambda light chains (I). P4 binds to near the start of human IgG1 CH1 constant region and contains mismatches for conversion from human G1 to human G4 (II). The amino acid residues of IgG1, which are different to IgG4 are shown in red. (c) InTag adaptor or IgG expression vector. The nucleotide and amino acid sequences of the relevant regions in the InTag adaptor (I) or the IgG expression vector (II) are shown. The restriction sites used for the digestion of the InTag adaptor and the IgG expression vector are underlined

2

Materials

2.1 General Reagents

1. 1% Agarose gel: Dissolve 1 g of agarose in 100 mL of 1× TAE buffer by microwaving. 2. 2YT media: To prepare 1 L measure ~900 mL of ddH2O and dissolve 16 g Bacto Tryptone, 10 g Bacto Yeast Extract and 5 g NaCl. Then adjust pH to 7.0 with 5 M NaOH. Adjust to 1 L with ddH2O and sterilize by autoclaving. 3. 50% Glucose in ddH2O.

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4. 96-well PCR plate. 5. 96-well flat bottom plate (Costar). 6. AccuPrime Pfx DNA polymerase (Thermo Fisher Scientific, Waltham, United States). 7. AirPore plate seal (QIAGEN, Hilden, Germany). 8. Ampicillin (Amp) filter-sterilized stock solution (100 mg/mL in sterile ddH2O). Working concentration is 100 μg/mL. 9. CutSmart Buffer (New England Biolabs, Ipswich, United States). 10. Ethidium Bromide. Working concentration is 10 μg/mL. 11. Kanamycin (Kan) stock solution (50 mg/mL in sterile ddH2O). Working concentration is 50 μg/mL. 12. Luria-Bertani (LB) Broth: To prepare 1 L, dissolve 10 g Bacto Tryptone, 5 g Bacto Yeast Extract and 10 g NaCl, adjust pH to 7.5, and bring to 1 L with ddH2O. Sterilize by autoclaving. 13. PCR plate seals. 14. QIAquick Gel Extraction Kit (QIAGEN). 15. QIAquick PCR Purification Kit (QIAGEN). 16. QIAprep Spin Miniprep Kit (QIAGEN). 17. Restriction enzymes: ApaLI, NheI, NgoMIV, and AfeI (New England Biolabs, Ipswich, Massachusetts, United States). 18. Super optimal broth (SOC) recovery media: 2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose. Sterilize by filtration through a 0.2 μm filter. 19. Sterile ddH2O. 20. T4 DNA Ligase and Buffer (Promega, Madison, United States). 21. Tris-acetate-EDTA (TAE) buffer: To prepare 1 L of 50× TAE dissolve the following components in 600 mL of ddH2O: 242 g Tris base (FW = 121), 57.1 mL glacial acetic acid and 100 mL 0.5 M EDTA (pH 8.0). Adjust the final volume to 1 L with ddH2O. Prior to use dilute to a 1× working solution (40 mM Tris (pH 7.6), 20 mM acetic acid, 1 mM EDTA). 2.2 Preparation of Linearized Vector and Adaptor for Cloning

1. IgG expression vector (Fig. 1) (see Note 1). 2. InTag adaptor vector containing SV40pA_KanR_CMV_pelB hybrid signal (Fig. 1) (see Note 2). 3. Restriction enzymes: ApaLI, NheI, NgoMIV, and AfeI.

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Table 2 Primers for LC and VH amplification and reformatting Primer Name

Sequence 5′ – 3′

P1

GCCTTCGCCACGGCCGTGCACGCA

P2

AATAGATTAGGCCGGGCCGGCCTA

P3

GTGGCCGCTGCCCAACCAGCGCTGGC

P4

CTCCTGGAGCAGGGCGCTAGCGGGAA

The primers were designed as illustrated in Figs. 2 and 3. All primers contain a 20 bp homologous region (underlined) required for In-Fusion cloning. P1 and P2 primers are used to amplify both the kappa and lambda light chains. Primers P3 and P4 are used to amplify the VH regions from the Fab-on-phage clones isolated from CSL naı¨ve antibody libraries

2.3 PCR Amplification of LC and VH Regions

1. Primers used for LC and VH amplification from CSL naı¨ve Fab-on-phage antibody library are listed in Table 2 and Figs. 2 and 3 (see Notes 3 and 4). 2. Overnight phagemid-containing bacterial culture.

2.4 In-Fusion Cloning

1. In-Fusion HD Cloning System CE (Clontech, Mountain View, United States) (see Note 5). 2. Purified vectors and adaptors (from Subheading 2.2). 3. Cloning Enhancer (Clontech). 4. Stellar chemically competent cells (Clontech). 5. 24-well round bottom deep well plates (Whatman, Little Chalfont, United States).

2.5 Isolation of Plasmid DNA and Sequencing Analysis

1. QIAcube (optional) (QIAGEN). 2. Light chain sequencing primer: SqCMVpro1F: 5′ TAATACGACTCACTATAGGG 3. Heavy chain sequencing primer: SqCMVpro2F: 5′ ATTAACCCTCACTAAAGGGA

2.6 Transient Transfection

1. Miniprep plasmid DNA. 2. Expi293F™ Cells (Thermo Fisher Scientific). 3. ExpiFectamine™ 293 Transfection Kit. Contents include ExpiFectamine 293 reagent, ExpiFectamine 293 Transfection Enhancer 1, and ExpiFectamine 293 Transfection Enhancer 2 (Thermo Fisher Scientific). 4. Expi293™ Expression Medium (Thermo Fisher Scientific). 5. Antibiotic-antimycotic (100×) (Thermo Fisher Scientific). 6. Opti-MEM® I Reduced-Serum Medium (Thermo Fisher Scientific).

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7. Filter-sterilized LucraTone Lupin – 20% in ddH2O (Merck Millipore, Billerica, United States). 8. Hemocytometer with trypan blue or cell counter. 9. 50 mL TubeSpin Bioreactor tubes (TPP, Trasadingen, Switzerland). 10. 0.22 μm Steriflip-GP sterile centrifuge tube top filter unit (Merck Millipore, Billerica, United States).

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Methods

3.1 Preparation of Linearized Vector and Adaptor

1. Set up a restriction enzyme digestion of IgG expression vector by combining 5 μg of DNA with 50 units ApaLI and 50 units NheI, 20 μL of CutSmart buffer and ddH2O to a final reaction volume of 200 μL. Incubate at 37 °C for 1–3 h. 2. Separate the digested vector on a 1% agarose/TAE gel along with a DNA molecular weight marker. Excise the vector fragment and purify with QIAquick gel extraction kit. Elute in 30 μL of Buffer EB and quantitate DNA using a Trinean Xpose or similar spectrophotometer. 3. To prepare the InTag adaptor, digest 5 μg of the adaptor plasmid with 50 units AfeI and 50 units NgoMIV, 20 μL of CutSmart buffer, and ddH2O to a final reaction volume of 200 μL. Incubate at 37 °C for 1–3 h (see Note 6). 4. Separate the digested adaptor vector on a 1% agarose/TAE gel along with a DNA molecular weight marker. Excise the 2 kb fragment of the InTag adaptor and purify with QIAquick gel extraction kit. Elute in 30 μL of Buffer EB and quantitate DNA using a Trinean Xpose or similar spectrophotometer.

3.2 PCR Amplification of Antibody Light Chain and Variable Heavy Chain

1. Inoculate 2 μL of bacterial glycerol stock containing phagemid to 120 μL of 2YT media supplemented with 2% glucose and 100 μg/mL ampicillin in a flat bottom 96-well plate. 2. Seal with an AirPore Tape sheet and incubate overnight at 37 ° C with shaking at 255 rpm in an Infors Microtron Shaking Incubator (3 mm pitch). 3. Prepare PCR template by diluting 2 μL of overnight culture with 198 μL of ddH2O in a 96-well plate. 4. Prepare a PCR master mix in multiples of 10 μL containing 2 μL of 10× AccuPrime pfx reaction mix, 0.2 μL of AccuPrime pfx DNA polymerase, 0.2 μL of each of the LC (P1, P2) and VH primers (P3, P4) (Note: primer stocks are 20 μM). 5. Aliquot 10 μL of the master mix into appropriate wells of a 96-well PCR plate.

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6. Add 10 μL of diluted bacterial culture containing phagemid to corresponding wells (reaction final volume is 20 μL). 7. Seal the plate with a PCR plate seal. 8. Pulse-spin the plate to ensure all contents are in the bottom of the wells. 9. Transfer the plate to a PCR machine and start the following PCR program (see Note 7): 1 cycle of 94 °C for 3 min; 5 cycles of 94 °C for 30 s, 50 °C for 30 s, and 68 °C for 1 min; 25 cycles of 94 °C for 20 s, 60 °C for 30 s and 68 °C for 1 min; and 1 cycle of 68 °C for 10 min. 10. Load 5 μL of PCR product on a 1% agarose gel containing 10 μg/mL ethidium bromide alongside a DNA molecular weight marker. Each PCR reaction should generate two products: (i) a higher band on the gel corresponding to the amplified LC (~700 bp); and (ii) a lower band corresponding to the amplified VH (~450 bp) (see Note 8). 11. Proceed with cloning or store PCR products at -20 °C. 3.3 In-Fusion Cloning (Also see Note 5)

1. Take a fresh aliquot of Cloning Enhancer (see Note 9) from 20 °C and place immediately on ice. 2. Add 2 μL of Cloning Enhancer into the appropriate number of wells of a 96-well PCR plate. This is best done in a 96-well cooling block. 3. Transfer 5 μL of PCR products to the plate containing the 2 μL of Cloning Enhancer. 4. Cover with a suitable PCR plate seal. 5. Incubate in a PCR thermal cycler at 37 °C for 15 min, followed by 15 min incubation at 80 °C. 6. Transfer the plate to ice or 96-well cooling block or if not proceeding, store Cloning Enhancer-treated PCR products at -20 °C. 7. For the In-Fusion cloning prepare a master mix in multiples of 9 μL containing 60 ng linearised vector, 50 ng InTag adaptor, and 2 μL 5× In-Fusion HD enzyme Premix (see Note 10). 8. Dispense 9 μL per well of In-Fusion cloning mix into a 96-well PCR plate, then transfer 1 μL of Cloning Enhancer-treated PCR product to each well. 9. Incubate in a PCR thermal cycler at 50 °C for 15 min. 10. Transfer the plate immediately to ice or store at -20 °C if not proceeding with transformation.

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Transformation

1. Thaw Stellar competent cells on ice just prior to use. 2. Aliquot 20 μL cells into 96-well PCR plate on ice or a cooling block. 3. Add 2 μL In-Fusion reaction mix to 20 μL cells (see Note 11). 4. Heat-shock the cells for 30 s at 42 °C (if using a thermal cycler, set block to 42 °C, and do not close the thermal cycler lid). 5. Return the plate to ice or cooling block for 2 min. 6. Add 80 μL pre-warmed SOC to each well. 7. Cover with a plate seal. 8. Incubate at 37 °C for 1 h without shaking. 9. Transfer 100 μL transformation mix to 4 mL of LB containing 50 μg/mL Kan in a 24-well round bottom deep well plate (see Note 12). 10. Cover with an AirPore Tape sheet and incubate at 37 °C with shaking at 220 rpm for up to 2 days.

3.5 IgG Reformatting Using the Cut-Paste Method

1. Scale up the PCR reaction described in Subheading 3.2 to 50 μL scale. 2. Analyze 5 μL of the PCR product on 1% agarose/TAE gel as described in Subheading 3.2. 3. Purify the PCR products using QIAquick PCR Purification Kit and elute the DNA in 50 μL of Buffer EB. 4. Digest the PCR products with 20 units each ApaLI, NgoMIV, AfeI, and NheI enzymes in a final volume of 50 μL in 1 × CutSmart buffer, at 37 °C for 2 h. 5. Purify the DNA again using QIAquick PCR Purification Kit and elute the DNA in 30 μL of Buffer EB. 6. Set up a ligation reaction in a final volume of 10 μL containing 60 ng linearized vector, 50 ng InTag adaptor, 50–100 ng of purified PCR products, 1 μL 10× Ligase Buffer, and 1 μL T4 DNA Ligase (1 unit). 7. Incubate the reaction at room temperature for 1–3 h or at 15 °C for 4–18 h. 8. For transformation, add 3–5 μL ligation mix to 50 μL chemically competent Stellar cells and incubate on ice for 15 min. 9. Heat-shock the cells for 60 s at 42 °C and return the tube to ice for 2 min. 10. Add 500 μL pre-warmed SOC to each transformation and incubate at 37 °C with shaking at 220 rpm for 1 h. 11. Transfer 500 μL to 4 mL of LB containing 50 μg/mL kanamycin in 24-well deep well plates. 12. Seal with an AirPore Tape sheet and incubate at 37 °C with shaking at 220 rpm for up to 2 days.

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1. Isolate plasmid miniprep DNA by transferring 2 mL of 2-day culture into 2 mL Eppendorf tube and pelleting bacterial cells by centrifugation at 6800× g for 3 min at room temperature. 2. Remove the supernatant and proceed with isolation of DNA using QIAprep Spin Miniprep Kit either manually or using QIAGEN QIAcube according to the manufacturer’s instructions. Elute the DNA in 100 μL Buffer EB. 3. DNA minipreps are sequenced with SqCMVpro1F and SqCMVpro2F primers using the ABI BigDye Terminator Cycle Sequencing Kit. 4. Reformatted IgG sequences are aligned and compared to the original phage sequences (see Note 13).

3.7 Transient Transfection

1. Culture Expi293F cells in Expi293F Expression media supplemented with 10 mL/L antibiotic-antimycotic solution by seeding at 0.3–0.5 × 106 cells/mL. Cell density should not exceed 5 × 106 cells/mL. 2. On the day of transfection use a hemocytometer and trypan blue exclusion (or another method of cell counting) to determine the cell counts and viability. Only transfect if viability is greater than 95% and the cell count is between 3–5 × 106 cells/ mL. 3. Pellet the required number of cells (1.25 × 107 per 5 mL transfection) and resuspend with Expi293 Expression Medium to a final concentration of ~3 × 106 cells/mL. 4. For each 5 mL transfection transfer 4.1 mL of the cells to each TubeSpin Bioreactor tube and shake at 250 rpm, 37 °C, 8% CO2. 5. Aliquot 5 μg of plasmid DNA into a sterile 24-well plate and dilute with 0.25 mL of Opti-MEM I Reduced Serum Medium. For multiple transfections use a multidispenser pipette to add reagents. 6. In a separate tube dilute 13.5 μL ExpiFectamine in 0.25 mL of Opti-MEM per 5 mL transfection and incubate at room temperature for 5 min. 7. Add the diluted ExpiFectamine to the DNA to give a final volume of 0.5 mL per transfection and incubate at room temperature for 20 min to allow the DNA and ExpiFectamine Reagent complexes to form. 8. Add 0.5 mL of the complex to each tube containing 1.25 × 107 cells to give a final volume of 4.6 mL. Incubate the cells at 37 °C, 8% CO2, 250 rpm for 16–20 h.

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9. Add 400 μL master mix containing 25 μL of Enhancer 1, 250 μL Enhancer 2, and 125 μL LucraTone Lupin to each TubeSpin Bioreactor tube. 10. Harvest the supernatants 5 days after transfection by centrifugation at 3000× g for 20 min. 11. Filter the supernatant using a 0.22 μm Steriflip-GP sterile centrifuge tube top filter unit prior to analysis and purification as described by Schmidt et al. [9].

4

Notes 1. The IgG expression vector described in this Chapter is a CSL proprietary vector generated from fragments synthesized by GeneArt (Thermo Fisher Scientific) using standard molecular biology techniques. Other mammalian expression vectors such as pcDNA3.1 can be readily adapted for use with IgG reformatting using InTag positive selection and hybrid signals via the insertion of a heavy chain constant region (species and isotype of choice) and the hVH1_ompA hybrid signal described in this Chapter. We found that incorporation of an intron in the light chain expression cassette (e.g., an intron before the secretion signal – see Fig. 1) is important to ensure overall high IgG expression level [unpublished data]. 2. The InTag adaptor was generated from fragments synthesized by GeneArt and cloned into pUC57 plasmid vector using standard molecular biology techniques. The InTag adaptor contains the SV40 pA, KanR gene, CMV promoter, and the hVH1-pelB hybrid signal and serves two key roles in this one-step zero-background IgG reformatting strategy. Firstly, it provides the necessary regulatory elements (a polyadenylation site for the light chain, the CMV promoter and a hybrid signal peptide for the heavy chain) for IgG expression in mammalian cells. Secondly, it provides a distinct antibiotic resistance marker (KanR) to facilitate the positive selection of recombinant clones. The final IgG expression constructs will contain two CMV promoters. However, we find no instability issues with these constructs despite the presence of these duplications. 3. The primers described here are tailored for the CSL naı¨ve Fabon-phage antibody libraries and therefore might need to be modified accordingly when different phage display libraries and/or expression vectors are used. A single 5′ primer (P1) targeting the ompA secretion signal and a single 3′ primer (P2) were designed to amplify all the light chain sequences in the naı¨ve libraries. Likewise, a single forward primer (P3) which

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binds to the pelB signal sequence is designed to amplify all the heavy chain sequences in the naı¨ve libraries. This primer is used in conjunction with a single reverse primer (P4), which anneals to the start of the heavy chain constant region for the amplification of all the VH sequences. In our system, we convert IgG1 isotype of Fab-on-phage to IgG4 in the mammalian expression vector, hence, the P4 primer sequence is identical to IgG4 but not to IgG1. All the primers contain a 20 bp sequence (underlined, Table 2) at the 5′end homologous to their corresponding vector or InTag adaptor region for In-Fusion cloning. We have found that increasing the overlap sequence from 15 bp to 20 bp results in higher efficiency cloning when using multiple inserts. Refer to the In-Fusion cloning manual for the appropriate design of this 20 bp extension region. 4. We recommend ordering PAGE-purified primers for the best quality possible as current oligonucleotide synthesis technologies often produce by-products that are either prematurely terminated or contain internal deletions in the sequence. These by-products, especially the ones with internal deletions, could contaminate the final IgG construct DNA as our HTP IgG reformatting strategy relies on bulk cloning without single colony screening. As we see batch-to-batch variations in primer quality, re-ordering the primer from the same or different manufacturers is sometimes necessary. We also recommend the primers to be dissolved and stored in Tris-EDTA (TE) buffer, pH 8.0 at -20 °C for long-term usage. 5. Clontech has updated the In-Fusion Cloning System to the In-Fusion Snap Assembly System. The new system has not been tested in our hands yet, but as reported by Clontech, In-Fusion Snap Assembly outperforms In-Fusion HD in terms of efficiency and consistency while maintaining exceptionally high accuracy. 6. Since the InTag adaptor contains the kanamycin resistance marker used for the final recombinant clone selection, there is a potential for the adaptor plasmid to contaminate the ligation mixture. Therefore, extra care needs to be taken to ensure that the plasmid digestion and gel extraction procedures are performed well so that there is no carryover of uncut plasmid prior to cloning. Quality control can be performed by transforming the isolated InTag adaptor DNA fragment and culturing the transformants in LB supplemented with kanamycin to ensure there is no growth. Although we rarely encounter a contamination problem with the InTag adaptor, this can be overcome by double selection with ampicillin in addition to kanamycin, as our InTag adaptor plasmid lacks the AmpR gene.

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7. The duplex PCR conditions used here were optimized for our primers and will need to be modified accordingly if different primers are used. 8. Agarose gel analysis of PCR products as a quality control step is optional but recommended, especially when the number of samples is manageable. On the agarose gel, apart from the intended LC and VH bands, we can sometimes observe a larger PCR product representing the full-length Fab fragment amplified by Primers P1 and P4 (Fig. 1). Although this Fab fragment can be cloned into the expression vector by itself, the recombinant clone containing this Fab fragment will be eliminated by InTag positive selection with kanamycin due to the absence of the InTag adaptor in the clone. This protocol can be adapted for automation using a liquid handler such as Eppendorf epMotion. For accuracy, we recommend diluting the Cloning Enhancer-treated PCR product with ddH2O to a final volume of 24 μL and adding 4 μL to a 6 μL In-Fusion cloning master mix. 9. Extra care needs to be taken for the handling and storage of the In-Fusion HD Cloning Kit to avoid a decrease in cloning efficiency. It is recommended to be stored in small aliquots to minimize freezing and thawing. 10. We have tried various ratios of inserts, InTag adaptor, and vector and found that the cloning method can tolerate wide variations in ratios. Therefore, quantitation and normalization of the PCR products is not required prior to cloning, thus facilitating the HTP operation. 11. It is important that the volume of In-Fusion reaction mix added to the competent cells does not exceed 10% of the volume of competent cells used to minimize transformation inhibition. We normally use the Stellar chemically competent cells provided with the In-Fusion HD Cloning Kit. We have also successfully used a range of other chemically competent cells such as TOP10 (Thermo Fisher Scientific) and AlphaSelect Gold Efficiency (BIOLINE). We strongly recommend the use of competent cells with a transformation efficiency ≥1 × 108 cfu/μg, and naturally, the competent cells need to be sensitive to ampicillin and kanamycin, which are used for clone selection. If low cloning efficiency is observed with certain bacterial strains, better results may be obtained by diluting the reaction mix with TE buffer 5–10 times prior to transformation. 12. For liquid culture following transformation, we use a 24-well deep well plate with a final volume of 4 mL/well. Cultures can just as easily be grown in tubes; however, the use of a 24-well

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deep well plate enables the application of either automation or adjustable spacing pipettes for sample transfer. We typically grow the cultures for approximately 40 h to ensure consistent DNA yields as overnight growth is often inadequate. 13. Due to the nature of bulk cloning without single colony isolation, we occasionally observe double or multiple sequencing signals from the same DNA sample. One cause of this is that the starting phage material is not clonal. Another cause is that the primer is not 100% pure and contains a certain percentage of by-products with internal deletion(s) (also see Note 4). One solution to these problems is to plate the culture out and pick single colonies for subsequent sequencing analysis to isolate the correct clone. References 1. Sarantopoulos S, Kao CY, Den W, Sharon J (1994) A method for linking VL and VH region genes that allows bulk transfer between vectors for use in generating polyclonal IgG libraries. J Immunol 152:5344–5351 2. Jones ML, Seldon T, Smede M, Linville A, Chin DY, Barnard R, Mahler SM, Munster D, Hart D, Gray PP, Munro TP (2010) A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods 354:85–90 3. Sanna PP, Samson ME, Moon JS, Rozenshteyn R, De Loqu A, Williamson RA, Burton DR (1999) pFab-CMV, a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies. Immunotechnology 4:185–188 4. Jostock T, Vanhove M, Brepoels E, Gool RV, Daukandt M, Wehnert A, Hegelsom RV, Dransfield D, Sexton D, Devlin M, Ley A, Hoogenboom H, Mu¨llberg J (2004) Rapid generation of functional IgG antibodies derived from Fab-on-phage display libraries. J Immunol Methods 289:65–80 5. Chen CG, Fabri LJ, Wilson MJ, Panousis C (2014) One-step zero-background IgG reformatting of phage-displayed antibody fragments

enabling rapid and high-throughput lead identification. Nucleic Acids Res 42(4):e26. https:// doi.org/10.1093/nar/gkt1142 6. Chen CG, Sansome G, Wilson MJ, Panousis C (2018) High-throughput IgG reformatting and expression. In: Hust M, Lim T (eds) Phage display: methods in molecular biology, Humana Press, vol 1701, New York 7. Valadon P, Garnett JD, Testa JE, Bauerle M, Oh P, Schnitzer JE (2006) Screening phage display libraries for organ-specific vascular immunotargeting in vivo. Proc Natl Acad Sci U S A. 103(2):407–412. https://doi.org/10. 1073/pnas.0506938103 8. Yoon H, Song JM, Ryu CJ, Kim YG, Lee EK, Kang S, Kim SJ (2012) An efficient strategy for cell-based antibody library selection using an integrated vector system. BMC Biotechnol 12: 62. https://doi.org/10.1186/1472-675012-62 9. Schmidt PM, Abdo M, Butcher RE, Yap MY, Scotney PD, Ramunno ML, Martin-Roussety G, Owczarek C, Hardy MP, Chen CG, Fabri LJ (2016) A robust robotic high-throughput antibody purification platform. J Chromatogr A 455:9–19. https://doi.org/10.1016/j.chroma. 2016.05.076

Chapter 24 Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols Mary R. Ferguson, Qiana M. Mendez, Felicity E. Acca, Cassandra D. Chapados, Holland A. Driscoll, Kezzia S. Jones, Gregory Mirando, Michael P. Weiner, and Xiaofeng Li Abstract To develop reproducible results, it is critical that all reagents used in an experiment be validated in an alternative or independent method. We present two such independent methods for determining the specificity of antibodies: (1) “MILKSHAKE,” which can be used to validate the liability and specificity of antibodies directed against post-translationally-modified epitopes, and (2) “Sundae,” which is a more complete alanine-like scanning method that can be used to better understand the binding and bioactivity of specific residues of a protein. We apply both of these methods to the interaction between an antibody and its antigen. Key words Alanine scanning, Antibody specificity, Antibody validation, ELISA, Western

1

Introduction A current cause for concern in the scientific literature is the poor reproducibility of published experimental results because of a lack of rigor in reagent validation and inter-experimental consistency. This is especially true for antibodies (Abs) used in experiments and a topic of several excellent reviews and conferences [1–13]. As a result of these concerns, many detailed recommendations for Ab validation before beginning experimental work have been proposed [3–6, 13]. Even beyond affinity reagent-antigen binding, the reproducibility issue drastically increases in complexity when validating Abs against post-translationally modified (PTM) targets. Often Abs are sold as being post-translational modification specific, however, when tested under the recommended protocol appear to have some affinity to the non-modified state of the epitope (Fig. 1). Thus a real difficulty in validating any anti-PTM Abs is access to a

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_24, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Commercially available phospho-specific Abs show varying degrees of specificity to phospho epitopes in MILKSHAKE. Simulated MILKSHAKE Western blots tested with catalog Abs from three different vendors. Abs A and C show binding specific to the phosphorylated epitope. Catalog Ab B, however, binds to the phosphorylated as well as the non-phosphorylated MILKSHAKE residue (arrow). For data and additional Ab validation examples see Ref. 14

validated antigen or cell lysate. It is especially difficult to generate cell lysates and purified antigen wherein the modified amino acid in the protein is present at either 100% (fully modified) or 0% (fully non-modified) in the sample [14]. Even when a particularly specific and targeted treatment is known for a particular PTM, other variables such as batch to batch variation, cell health, modifyingenzyme activity, storage conditions, and researcher technique can still persist to allow for errors in an appropriate validation. Alanine scanning is a technique that is often used to determine the contribution of a specific amino acid residue to the stability or function of a given protein [15, 16]. Alanine is used because it is non-bulky and chemically inert. The alanine methyl group can be used to mimic the secondary structure preferences that the other amino acids possess. Sometimes bulky amino acids such as the branched-chain amino acids valine and leucine are used where the conservation of the size of mutated residues is needed.

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Alanine scanning is further used to determine whether a specific amino acid residue plays a significant role in bioactivity by replacing the naturally occurring amino acid with alanine. This can often be performed without the requirement for highly purified protein and often uses the method of site-directed mutagenesis to change the residues in question to alanine [17, 18]. Once the change is made, the protein can be retested for bioactivity with the alanine replaced. Often these tests can yield a quantitative measurement. However, one limitation of alanine scanning is that it only reveals whether alanine as a replacement at a specific site retains bioactivity. It reveals nothing about the effect of the other 18 amino acids (i.e., the set of 20 amino acids minus the native and alanine residues). So a true picture of the importance of a specific residue for interaction or bioactivity cannot be painted if only alanine is used for scanning (Fig. 2). To summarize, the most reliable method for validation of antiPTM-specific Abs is one in which each antigen sample contains the target sequence with either 100% modification or 100% non-modification at the residue or epitope of interest. The MILKSHAKE protocol provides that certainty for a Western blot assay. This protocol uses a modified maltose binding protein (MBP) and the sortase enzyme to produce surrogate proteins containing the modified or non-modified epitope sequence (Fig. 3). These MILKSHAKE proteins can then be used in Western blot to evaluate the PTM specificity of catalog Abs (Fig. 4). To determine more completely the binding contribution of a specific amino acid at a specific site, we have developed a protocol we term “Sundae” (because we are using multiple ‘flavors’ of amino acids at the site of interest, not just alanine). Using Sundae, we replace the targeted site with up to 20 different amino acids (Fig. 5) and retest for bioactivity. The Sundae method is able to distinguish varying reactivity to each amino acid in ELISA (Fig. 6).

2

Materials and Reagents Materials Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ-cm at 25
C). Prepare and store all reagents at room temperature unless indicated otherwise. Follow all waste disposal regulations when disposing of waste materials. Media and Solutions 1. Rich Broth: deionized water, to 950 mL, Tryptone, 10 g, NaCl, 5 g, Yeast extract, 5 g. Combine the above reagents and mix until the solutes have dissolved. Adjust the volume of the solution to 1 L with deionized water and autoclave. Before use, add glucose, 2 g.

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Fig. 2 MILKSHAKE and Sundae workflow. (a) MILKSHAKE proteins used in Western blot result from a conjugation reaction between a target peptide (with or without a PTM) and a modified MBP using the sortase enzyme. These proteins can then be tested to determine the specificity of PTM Abs. (b) Sundae proteins contain genetically encoded target sequences in which one residue has been replaced by all 20 amino acids. These proteins can be used in ELISA to determine binding affinity of Abs when this single residue is replaced. (c) Trays containing multiple small compartments (sold as bead organizers at hobby stores) facilitate testing a variety of different primary Abs in MILKSHAKE Western blot

2. Column buffer: 20 mM Tris–HCL, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM DTT. 3. Elution buffer: column buffer with maltose to 10 mM (final). 4. 10 Tris-NaCl buffered saline (TBS): 1.5 M Sodium Chloride, and 0.25 M Tris, pH 7.2-7.5. 5. 1 TBST: 1 TBS with final concentration of 0.1% Tween 20. 6. 3% MTBST: 1 TBST with 3% nonfat dry milk powder. 7. 5% BSA-TBST: 1 TBST with 5% bovine serum albumin. 8. 10 Sortase Buffer: 200 mM Tris–HCl, 1.5 M NaCl, 50 mM CaCl2, 2 mM beta-mercaptoethanol prepared in sterile water.

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Fig. 3 Plasmid map of pMAL-c6T. This vector [New England Biolabs] was modified to construct both the sortase acceptor plasmid for MILKSHAKE and the Sundae proteins

9. 2 Laemmeli + DTT: 2 Laemmli sample buffer [BioRad], 200 mM DTT 10. 1 PBST: 1 PBS with final concentration of 0.1% Tween 20.

3

Methods Carry out all procedures at room temperature unless otherwise specified.

3.1 Construction of Sortase-Acceptor Plasmid

To begin construction of a sortase-acceptor plasmid (pAT27 or equivalent), the commercially available plasmid pMAL-c6t can be purchased from New England BioLabs (NEB catalog #: N0378S, see Note 1). A sortase conjugation site (LPETG, encoded in the

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Fig. 4 MILKSHAKE Validation of Vendor catalog Abs. (a) Blot loaded with unconjugated MILKSHAKE and four versions of MILKSHAKE protein containing the target sequence for JNK1 with or without phosphorylation at Thr183 and Tyr185. Blot probed with anti-HA Ab. Each lane containing conjugated MILKSHAKE protein shows reactivity with anti-HA and the unconjugated sample shows no reactivity. (b) Blot loaded with unconjugated MILKSHAKE and four versions of MILKSHAKE protein containing the target sequence for JNK1 with or without phosphorylation at Thr183 and Tyr185. Blots probed with vendor Abs labeled for sale as specific binders to the JNK1 protein, only when phosphorylated at both Thr183 and Tyr185. Vendor Ab 1 binds non-specifically to phospho as well as the non-phospho_JNK MILKSHAKE proteins. Vendor Abs 2 and 3 recognize MILKSHAKE when both sites are phosphorylated and phospho_Tyr185 but are not able to bind when Thr183 alone is phosphorylated. Vendor Ab 4 is able to bind all three phosphorylated versions of MILKSHAKE. (c) Loading as in (b) for Abs against ERK1 phospho-positions 202 (phospho-threonine) and 204 (phospho-tyrosine). Vendor Ab 5 is able to bind when either residue is phosphorylated. Vendor Abs 6 and 7 are able to bind only when position 202 is phosphorylated. None of the three vendor Abs bind unphosphorylated residues. In all blots, M ¼ size marker

plasmid DNA as: TTACCGGAAACTGGT) with a preceding Gly4Ser peptide linker (GGGGS encoded in the DNA as: GGTGGCGGTGGCTCG) are cloned into the parent vector in-frame with the MBP into the multiple cloning site using EcoRI and NotI. The resulting plasmid pAT27 expresses a modified MBP (MILKSHAKE) containing the sortase conjugation site in its C-terminus (Fig. 3). The recombinant vector can be transformed

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Fig. 5 Sundae proteins shed light on which residues are important for Ab binding. 2F5 is a broadly neutralizing human monoclonal Ab known to bind the sequence 657-EQELLELDKWASLW-670 found in the human immunodeficiency virus (HIV). Sundae proteins were constructed with an insert of this HIV sequence in which the underlined residue (EQELLELXKWASLW) was replaced with each of the naturally occurring amino acids (Sundae-A for alanine etc.). These Sundae proteins were then tested in ELISA for binding to 2F5. ELISA plates were coated with 1 μg mL1 of Sundae antigens and primary Ab 2F5 [Polymun Scientific] was tested using four-fold dilutions in 2% BSA. The 2F5 Ab binds strongly to a full-length gp41 HIV protein [Abcam], a native HIV sequence peptide as well as the Sundae-D protein containing the sequence EQELLELDKWASLW. Binding is reduced when tested against the Sundae proteins containing each of the other 19 amino acids as well the negative control, Sundae-Empty (MBP with no HIV sequence present). These data suggest that the D at position 664 is critical for 2F5 binding

into NEB Express cells and frozen in 15% glycerol stock solution and stored at 80
C. Purified plasmid DNA can be stored at 20
C in Tris-EDTA solution. 3.2 Preparation of MILKSHAKE Protein Inoculant

1. Inoculate 5 mL of LB media supplemented with ampicillin (final concentration of 100 μg mL1) in a 14 mL snapcap culture tube with a scraping of a glycerol stock of the sortase acceptor plasmid in NEB Express cells. (see Note 2). 2. Incubate tube with shaking at 37
C overnight.

3.3 Expression of MILKSHAKE Protein

1. Inoculate 75 mL of Rich Broth media supplemented with ampicillin (final concentration of 100 μg mL1) in a 250 mL baffled glass flask with 750 μL of the overnight culture. 2. Incubate 250 mL flask shaking at 37
C until an OD600 ¼ 0.5 is reached (approximately 1–3 h). Determine OD600 using a spectrophotometer. 3. Add 15 μL of 1 M IPTG stock to each flask (final concentration 0.3 mM).

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Fig. 6 Ab reactivity varies depending on which residue is present in Sundae proteins. Different rabbit IgG clones (measured at 5 μg mL1) show different reactivity when a single residue in the Ab’s epitope (an aspartic acid in the native sequence) is replaced with other amino acids (Sundae-A, Sundae-E, etc.). IGT-0037 is able to bind the original amino acid, aspartic acid as well as a peptide version containing aspartic acid (D). However, IGT-0034 is able to bind both those antigens as well as Sundae proteins with glutamic acid and proline substitutions (Sundae-E and Sundae-P). IGT-0035 recognizes tryptophan and cysteine (Sundae-W and Sundae-C) as well at that same position in the epitope sequence but not glutamic acid. IGT-0042 is only able to recognize the peptide version of the antigen sequence and not any of the Sundae proteins. None of these Abs were able to bind alanine at this position. Unlike alanine scanning, the Sundae method can fully interrogate the contribution of a single residue to Ab binding

4. Incubate flasks with shaking at 37
C for an additional 2 h. 5. Pour culture from one 250 mL flask into one 250 mL centrifuge tube. (see Note 3). 6. Centrifuge tubes in a high-speed floor centrifuge at 5000 g for 20 min at 4
C. 7. Discard supernatant in biohazardous liquid waste container. 8. Freeze pellet overnight at 80
C.

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1. Thaw pellets at room temperature on a benchtop. Add 2 mL of B-PER II Reagent [Thermo] or up to 4 mL per gram of pellet. Incubate for 15 min at room temperature on a nutator or equivalent piece of equipment. 2. Transfer sample to a 45 mL centrifuge tube and place on ice. 3. Centrifuge sample at 15,000 g for 15 min at 4
C in a highspeed floor centrifuge. 4. While the sample is spinning, prepare one 2 mL purification column in a cold room or deli case refrigerator for each sample. Snap off the bottom of the column and set the column up in a rack above a waste container to catch the flow through. 5. Add 2 mL of amylose resin [New England BioLabs] to each column and allow to drain via gravity. Be sure to shake resin stock bottle to resuspend resin before pipetting into column. 6. Wash each column once with 2 mL of ultra-pure water followed by 2 mL of column buffer. 7. Load the clarified sample onto the washed column by pouring, being careful not to disturb the cell pellet. Discard pellet. (see Note 4). 8. Allow sample to flow by gravity. Collect the flow through in a clean tube and pour back over column a second time without collecting the flow through. 9. Wash each column with 12 mL column buffer. 10. Place a new sterile tube under the column. 11. Add 1 mL of elution buffer 3 times. Make sure to allow buffer to flow through completely before adding the next 1 mL. Collect each elution fraction in a separate tube. 12. Place eluted protein on ice. Add glycerol to a final concentration of 15% (333 μL of 45% glycerol stock per 1 mL eluate). 13. Quantitate the amount of protein in the sample using a Biotek plate reader or equivalent. Use elution buffer with 15% glycerol as your blank. (see Note 5). 14. Label each tube with the concentration of MILKSHAKE protein in mg mL1. 15. Store MILKSHAKE protein at 4
C for short term. For longterm storage, flash freeze, the sample in liquid nitrogen and store at 80
C. Be sure to wear appropriate personal protective equipment when working with liquid nitrogen.

3.5 Modified Maltose Binding Protein Conjugation to Peptide 3.5.1 Conjugation Controls

1. Positive control: Use anti-HA as a secondary Ab [Abcam] in a separate blot to confirm conjugation reaction was successful. 2. Negative control: Load unconjugated MILKSHAKE protein in one lane per blot to confirm primary Ab binding is specific to conjugated peptide and not to the MILKSHAKE protein itself (see Note 6).

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3.5.3 Modified Maltose Binding Protein Conjugation to Peptides

1. Design and order two peptides per target being tested: (i) one peptide should be the modified peptide containing the N-terminal sortase compatible site and a C-terminal HA tag and (ii) the other peptide will be the non-modified version containing both an N-terminal sortase compatible site and a C-terminal HA tag (see Note 7). 1. Reactions will be arranged in sets of three in a 96-well deepwell plate: (i) unconjugated MILKSHAKE protein negative control, (ii) MILKSHAKE protein conjugated to the modified peptide, and (iii) MILKSHAKE protein conjugated to the non-modified peptide. 2. For wells containing unconjugated MILKSHAKE protein, mix the following per well: 10 Sortase buffer (11 μL), MILKSHAKE protein (0.145 mg), 1 PBS to a final volume of 113 μL. 3. For wells containing “MILKSHAKE protein conjugated to the modified peptide” mix the following per well: modified peptide, 4 μL of a 10 mg mL1 stock (in DMSO), 10 Sortase Buffer (11 μL), MILKSHAKE protein (0.145 mg), Sortase Enzyme (1.5 μL) [Moradec LLC] and 1 PBS to a final well volume of 113 μL. 4. For wells containing “MILKSHAKE protein conjugated to the non-modified peptide” mix the following per well: non-modified peptide, 4 μL of a 10 mg mL1 stock (in DMSO), 10 Sortase Buffer (11 μL), MILKSHAKE protein (0.145 mg), Sortase Enzyme (1.5 μL) and 1 PBS to a final well volume of 113 μL (see Note 8). 5. Seal the reactions with plate tape and incubate with shaking at 250 RPM at 37
C for 2 h. 6. After incubation, centrifuge plate for 5 min in tabletop centrifuge at 2100 g to make sure reaction volume is at the bottom of the well. 7. Add 113 μL of 2 Laemmli + DTT buffer to each well. Transfer each mixture to separate and labeled 1.7  g Eppendorf tubes. 8. Incubate tubes containing conjugation reactions at 95
C for 10 min. Reaction is now ready to load in a gel for Western blot (see Note 9).

3.6 Ab Validation Using Western Blot 3.6.1 Protein Electrophoresis and Membrane Transfer

1. Use the lanes described below for each Ab to be tested. Load up to 15 μL (or 5 μg) of sample (unconjugated MILKSHAKE protein or conjugated MILKSHAKE protein) per lane. Load 5 μl of a protein standard [Precision Plus from BioRad or equivalent] per lane (see Note 10): (Lane 1) Protein standard, (Lane 2) unconjugated MILKSHAKE protein, (Lane 3)

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modified peptide conjugated to MILKSHAKE protein, and (Lane 4) non-modified peptide conjugated to MILKSHAKE protein. 2. Run the gel(s) until the dye front reaches the bottom of the gel. If using a Criterion apparatus [BioRad], we typically run the gel (s) at 180 V for 40 min. 3. Transfer the proteins in the gel to a nitrocellulose membrane according to the Western blot supplier’s protocol (BioRad Protocol 10019593). 4. When transfer is complete, cut the membranes between the molecular weight standard lanes to generate separate blots. 5. Place membranes in separate compartments in a tray and wash with 1 TBS for 5 min on a platform shaker. We use a compartmentalized plastic tray such as one purchased from a hobby shop (Fig. 2). 6. Discard the 1 TBS and add 3% MTBST (enough volume to cover the membrane) to each blot. Incubate at room temperature for 1 h on a platform shaker with gentle shaking to block the membranes. 7. Discard blocking solution and wash membranes with 1 TBST 3 times for 5 min each on a platform shaker at room temperature. 8. During washes, dilute Ab(s) to be tested in 5% BSA-TBST at a concentration of 1 μg mL1 (or according to vendor recommendations). Remove final 1 TBST wash from blots and add primary Ab to the corresponding blot(s). For secondary only or anti-HA blots, add 5% BSA-TBST only. 9. Incubate blots overnight at 4
C on a platform shaker with gentle shaking. 3.6.2 Developing and Exposing the Membrane

1. Discard primary Abs and wash membranes with 1 TBST 3 times for 5 min each on a platform shaker at room temperature. 2. Discard TBST and add secondary Ab diluted in 3% MTBST. For starting conditions, try a 1:5000 dilution. Incubate at room temperature for 1 h on a platform shaker with gentle shaking. 3. Remove secondary Ab and wash membranes with 1 TBST 3 times for 5 min each at room temperature on a platform shaker with gentle shaking. 4. Develop the membranes with ECL Reagent [BioRad] or equivalent for at least 10 s. Mix together equal volumes of the Clarity Western ECL Substrates [BioRad] and then add a small volume

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of the mixture to each membrane (just enough to cover the surface of the membrane). It is important to note not to mix the substrates together until immediately before use. 5. Image the membranes using an appropriate available imaging system. 3.6.3 Interpretation of Data

1. Expected band size of MILKSHAKE protein is 42 kDa (Fig. 4). (a) In the anti-HA blot, verify the presence of the HA tag on all of the conjugation reactions but NOT on the unconjugated reactions. (b) Verify PTM-specific binding for your positive control Abs (if available). Most general Abs will not bind the peptide. Check the vendor’s catalog page to determine whether you should expect binding to your conjugated MILKSHAKE protein. (c) As an example, a phospho-specific Ab should only bind the phospho-conjugated MILKSHAKE protein. Phospho-independent Abs should bind to both phosphoand to the non-phospho-conjugated MILKSHAKE proteins. Non-phospho specific Abs should bind only to the nonphospho-conjugated MILKSHAKE proteins. No Abs should bind to the unconjugated MILKSHAKE proteins (Fig. 4).

3.7 Construction of Genetically Encoded Target Sequence Plasmid (Sundae)

To begin construction of a Sundae expression plasmid (pAT28 or equivalent), the commercially available plasmid pMAL-c6T was purchased from NEB (catalog #: N8108S, see Note 1). A sequence from the target of interest (10–14 amino acids) preceded by a GSGS linker was cloned into the parent vector in-frame with the MBP into the multiple cloning site using NotI and EcoRI (see Note 11). The resulting plasmid pAT28 expresses a modified MBP containing a short sequence of the target of interest in its C-terminus (termed “Sundae”). In order to further probe Ab binding to a specific residue in a target sequence, a set of 20 Sundae plasmids should be constructed, each with a different amino acid at the site of interest (Fig. 2). The recombinant vector(s) can be transformed into NEB Express cells and frozen in 15% glycerol stock solution and stored at 80
C. Purified plasmid DNA can be stored at 20
C in Tris-EDTA solution.

3.8 Sundae Protocol—Preparation of Inoculant

1. Follow the same steps as Subheading 3.2 for each Sundae plasmid.

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3.9 Expression of Sundae Protein

1. Follow the same procedure as Subheading 3.3 for each Sundae plasmid.

3.10 Purification of Sundae Protein

1. Follow the same procedure as Subheading 3.4 for each Sundae plasmid.

3.11 Ab Validation Using Sundae ELISA

1. Coat wells of a 96-well microtiter plate with 50 μL of the Sundae protein(s) (antigen) diluted to 1 μg mL1 in 1 PBS and incubate, sealed, overnight at 4
C. 2. Following day, wash plates 3 times with PBS and block the wells with 50 μL of 2% BSA diluted in 1 PBS. Incubate for 1 h. Wash 3 times with 1 PBS. 3. Add 50 μL per well of primary Ab using four-fold dilutions in 2% BSA beginning at 30 μg mL1. Incubate for 1 h. Wash 3 times with 1 PBST. 4. Add 50 μL of anti-Protein A HRP conjugated Ab using 1:5000 dilution (or other appropriate secondary Ab) to each well and incubate for 1 h. Wash 3 times with 1 PBST. 5. Add 50 μL of room temperature TMB to each well and allow color to develop for 5–10 min. 6. Add 50 μL of 0.16 M H2SO4 to each well to quench the reaction. Be sure to wear PPE and dispose of acidic solutions in a hazardous waste stream. 7. Read absorbance at 450 nm in an appropriate available plate reader.

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Notes 1. The more recent pMAL-c6T vector is a replacement for NEB #N8108, pMAL™-c5X Vector. Like the pMAL-c5x vector, the pMAL-c6T vector is designed to produce maltose-binding protein (MBP) fusions in the cytoplasm. The MBP has been engineered for tighter binding to amylose resin. The vector expresses an N-terminal his-tagged malE gene followed by a multiple cloning site containing a TEV protease recognition sequence, allowing MBP to be cleaved from the protein of interest after purification. 2. This plasmid contains a modified maltose binding protein (MILKSHAKE protein) with a C-terminal sortase site. The protein is expressed in NEB Express Cells (New England BioLabs Catalog # C2523H). Genotype: fhuA2 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210:: Tn10--TetS) endA1 delta(mcrC-mrr)114::IS10. See Fig. 3 for details on this plasmid.

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3. Do not overfill centrifuge bottles or tubes to avoid spillage and damage to the centrifuge. Make sure tubes are weighed and balanced to prevent faulting. 4. If the sample volume is large and it will require multiple additions. 5. When using the Biotek plate reader, use a protein quantification setting for a percent extinction coefficient of 1.4. 6. The anti-HA Ab recognizes the C-terminal HA tag on the peptide. Confirm the presence of the HA tag in a conjugated reaction and the absence of HA tag in the unconjugated sample. 7. When ordering peptides, request 2 mg of peptide with 90% purity. When peptides arrive (depending on the vendor this takes approximately 4–6 weeks), resuspend them in DMSO to a concentration of 10 mg mL1. An example of one peptide set for the target akt1_473 (a PTM phospho-site) would consist of the following two peptides (sortase compatible site, target sequence, HA tag); Phospho_akt1_473_HA: GGGSGSS and ERRPHFPQF{pSer}YSASGTA YPYDVPDYA Non-Phospo_akt1_473_HA GGGSGSS ERRPHFPQFSYSASGTA YPYDVPDYA

8. Determine how many reactions you will need to test all of your Abs for each target. Each conjugation reaction will allow you to load approximately 11 lanes using 15 μL per lane in Western blot. If you have additional Abs to test, set up additional reactions in the plate. 9. The denatured reactions may be stored at 4
C for up to 1 month before use in a Western blot. Settling may occur during 4
C storage, so be sure to vortex these samples before use. 10. As an added control, include one additional lane per blot containing 15 μL MILKSHAKE protein conjugated to an irrelevant modified-HA peptide. This control can confirm that binding of the primary Ab is specific to the target peptide. 11. An example of two Sundae plasmids for the MPER target (found in the human immunodeficiency virus HIV) would consist of the following (pMAL-c6T sequence, GS linker, target sequence): MPER-Sundae-D: MLMGGR GSGS EQELLELDKWASLW MPER-Sundae-E: MLMGGR GSGS EQELLELEKWASLW

Acknowledgments The authors would like to thank Emily P. Fuller and Brian K. Kay for insightful discussions.

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This research was funded by NIH Appl. ID 1R44GM148998-01 “Platform for the High Throughput Generation and Validation of Affinity Reagents” and NIH Appl. ID 1R43GM146473-01 “Method for the validation by Western analysis of affinity reagents against post-translationally modified proteins. I. survey of existing Abs, and II. development of method improvements.” References 1. Adhikari S, Nice EC, Deutsch EW et al (2020) A high-stringency blueprint of the human proteome. Nat Commun 11(1):5301. https:// doi.org/10.1038/s41467-020-19045-9 2. Aebersold R, Agar JN, Amster IJ et al (2018) How many human proteoforms are there? Nat Chem Biol 14(3):206–214. https://doi.org/ 10.1038/nchembio.2576 3. Bordeaux J, Welsh A, Agarwal S et al (2010) Antibody validation. BioTechniques 48(3): 1 9 7 – 2 0 9 . h t t p s : // d o i . o r g / 1 0 . 2 1 4 4 / 000113382 4. Bourbeillon J, Orchard S, Benhar I et al (2010) Minimum information about a protein affinity reagent (MIAPAR). Nat Biotechnol 28(7): 6 5 0 – 6 5 3 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nbt0710-650 5. Bradbury A, Plu¨ckthun A (2015) Reproducibility: standardize antibodies used in research. Nature 518(7537):27–29. https://doi.org/ 10.1038/518027a 6. Bradbury AM, Plu¨ckthun A (2015) Antibodies: validate recombinants once. Nature 520(7547):295. https://doi.org/10.1038/ 520295b 7. Bradbury AR, Plu¨ckthun A (2015) Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents. Protein Eng Des Sel 28(10): 303–305. https://doi.org/10.1093/protein/ gzv051 8. Jonasson K, Berglund L, Uhlen M (2010) The 6th HUPO Ab Initiative (HAI) workshop: sharing data about affinity reagents and other recent developments September 2009, Toronto, Canada. Proteomics 10(11): 2066–2068. https://doi.org/10.1002/pmic. 201090040 9. Shankar G, Devanarayan V, Amaravadi L et al (2008) Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal 48(5):1267–1281. https://doi.org/10.1016/j.jpba.2008.09.020

10. Taussig MJ, Fonseca C, Trimmer JS (2018) Antibody validation: a view from the mountains. New Biotechnol 45:1–8. https://doi. org/10.1016/j.nbt.2018.08.002 11. Taussig MJ, Stoevesandt O, Borrebaeck CA et al (2007) ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome. Nat Methods 4(1):13–17. https://doi.org/10.1038/ nmeth0107-13 12. Uhlen M, Bandrowski A, Carr S et al (2016) A proposal for validation of antibodies. Nat Methods 13(10):823–827. https://doi.org/ 10.1038/nmeth.3995 13. Voskuil JLA, Bandrowski A, Begley CG et al (2020) The antibody society’s antibody validation webinar series. MAbs 12(1):1794421. https://doi.org/10.1080/19420862.2020. 1794421 14. Jones KS, Chapman AE, Driscoll HA et al (2022) MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot. BioTechniques 72(1):11–20. https://doi. org/10.2144/btn-2021-0078 15. Morrison KL, Weiss GA (2001) Combinatorial alanine-scanning. Curr Opin Chem Biol 5(3): 302–307. https://doi.org/10.1016/s13675931(00)00206-4 16. Weiss GA, Watanabe CK, Zhong A et al (2000) Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc Natl Acad Sci U S A 97(16):8950–8954. https:// doi.org/10.1073/pnas.160252097 17. Costa GL, Bauer JC, McGowan B et al (1996) Site-directed mutagenesis using a rapid PCR-based method. Methods Mol Biol 57: 239–248. https://doi.org/10.1385/089603-332-5:239 18. Costa GL, Weiner MP (2006) Rapid PCR sitedirected mutagenesis. CSH Protoc 2006:pdb. prot4144. https://doi.org/10.1101/pdb. prot4144

Chapter 25 Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display Maria T. Tsoumpeli, Anitha Varghese, Jonathan P. Owen, Ben C. Maddison, Janet M. Daly, and Kevin C. Gough Abstract Peptide phage display has historically been used to epitope map monoclonal antibodies. More recently, by coupling this method with next-generation sequencing (so-called next-generation phage display, NGPD) to mass screen peptide binding events, the methodology has been successfully applied to map polyclonal antibody responses to infection. This leads to the identification of panels of mimotopes that represent the pathogen’s epitopes. One potential advantage of using such an approach is that the mimotopes can represent not just linear epitopes but also conformational epitopes or those produced from posttranslational modifications of proteins or from other non-protein macromolecules. The mapping of such complex immunological recognition of a pathogen can inform novel serological assay development and vaccine design. Here, we provide detailed methods for the application of NGPD to identify panels of mimotopes that are recognized specifically by antibodies from individuals with a particular infection. Key words Phage display, Next generation sequencing, Biopanning, Bioinformatic analysis, Epitope mapping, Antibody, Peptide ELISA

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Introduction The mapping of polyclonal antibody responses to infectious diseases to identify individual epitopes is vital to facilitate the development of novel serological assays and multi-peptide vaccines. Mapping antibody responses at the epitope level can underpin the development of assays for the epidemiological investigation of infections that are closely related or that emerge into new geographical regions, for instance, due to climate change or due to the more frequent interactions of human and/or production animal populations with wildlife species. Understanding antibodymediated recognition of a pathogen can reveal immuno-protective

Joint first authors: Maria T. Tsoumpeli and Anitha Varghese. Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_25, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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responses in the host after infection or vaccination and facilitate study of protective immunity. However, the mapping of antibody responses to infection is challenging in many respects. Antipathogen responses are complicated with polyclonal antibodies recognizing a wide range of epitopes, only a subset of which correlate with protection; and responses show high idiotypic variation. Until recently, conventional screening for infection-specific epitopes involved the resolution of pathogen proteins on acrylamide gels, western blotting with polyclonal sera, and the identification of recognized proteins, e.g., by mass spectrometry methods or microsequencing [1–3]. However, the resolving power of such methods is limited, and the sensitivity is relatively low. More recently, nextgeneration phage display (NGPD) has been applied to map polyclonal antibody responses to infections [4, 5]. This method combines the very high diversity of phage display with the screening power of next-generation sequencing platforms (NGS). In this procedure, billions of random peptide variants are displayed on the outside of phage particles, with each phage particle encoding and displaying a single peptide variant. This physically links phenotype (peptide binding property) to genotype (gene fragment) and allows the potential screening of many millions of ligand binding events by NGS analysis of the peptide genes isolated after binding of the phage-peptides. The method identifies peptide mimotopes of the pathogen’s epitopes. These are peptides that mimic the shape of an epitope but do not necessarily share its linear sequence. Pathogen epitopes are often conformational and cannot be designed from the pathogen proteome (for instance by screening overlapping peptides to reveal immunogenic linear epitopes [6]). This ‘unbiased’ approach to discover peptide mimotopes can potentially reveal a far more comprehensive set of immunogenic peptides representing not only linear epitopes but also conformational protein epitopes, post-translational modifications of proteins and other non-protein macromolecules, all of which can contribute to antibody recognition of a pathogen. Here, we describe a strategy for mapping polyclonal antibody responses to an infection using NGPD. Fundamentally, this involves biopanning against the target (infected) IgG samples (Figs. 1 and 2). In a first round of biopanning, the phage library is bound to irrelevant antigen (usually beads coated in irrelevant immunoglobulin, IgG) in a subtraction step to remove non-specific binders. The non-bound phage is then interacted with immobilized IgG isolated from serum samples taken from multiple infected individuals. Following extensive washing and elution with a pH shift, this round of biopanning produces a sub-library that is enriched for phage-peptides that include those that bind to IgGs that are infection-specific. This sub-library is propagated in bacteria and the purified phage is again subtracted for non-specific binders as before. The subtracted phage are then bound to a wide range of samples from infected individuals and in parallel, samples from the control cohort (which could be, e.g.,

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Fig. 1 Schematic of the next-generation phage display process. (1) The phage library is grown from bacterial glycerol stock. (2) Phage library is centrifuged to remove bacterial remnant, and the supernatant PEG is precipitated to further purify phage. (3) A subtractive biopanning step is carried out to remove phage that bind non-specifically to the agarose beads and the control antibodies (red). (4) The phage that did not bind in the subtraction step are then mixed with target antibody (blue). This step is carried out against serum samples from multiple infected individuals. (5) Following extensive washing, the bound phage are then eluted with a pH shift. This represents the enriched sub-library from the biopanning (output phage from round 1 of biopanning). (6) The rescued sub-library is infected into TG1 bacteria and amplified to make an enriched phage stock. Steps 2–5 are then repeated with the enriched sub-library from round 1 and binding is carried out against panels of both target and control IgG samples. (7–8) After the second round of biopanning, the individual sub-libraries eluted from each IgG sample are used as template to PCR amplify the peptide gene fragment and this is then analyzed by NGS. (9) Boinformatic analysis of binding events allows the identification of peptide sequences that were bound at relatively high levels by multiple target IgG samples compared to binding to the control IgG. (Figure made using Biorender)

samples from uninfected, infected with alternate pathogen, or vaccinated individuals). Following extensive washing and elution of bound phage, the peptide gene fragment in phage sub-libraries isolated against each IgG sample are amplified by PCR and analyzed by NGS. Bioinformatic analysis of the frequency of peptide gene sequences in each sub-library allows comparison of the binding events between infected vs. control groups, facilitating the identification of candidate infection-specific peptides. As mentioned above, subtraction of non-specific phage is carried out in each round of panning to help drive the selection of infection-specific peptide mimotopes. The phage library is

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Fig. 2 Schematic of next-generation phage display biopanning strategy. Here, the phage peptide library is subtracted against beads that are coated with a pool of 10 control serum samples. Phage that did not bind in this subtraction step are then introduced to beads coated with 10 target serum samples, and after extensive washing, the eluted phage represent an enriched sub-library (Round 1). The output phage from round 1 are amplified in TG1 bacteria and then again subtracted against beads bound with IgG from the same 10 control serum samples. This sub-library is then bound to IgG from 20 target samples and 20 control samples (Round 2). The NGS analysis of the enriched phage-peptides for each individual sample after round 2 of selection are processed to compare the relative binding in each of the 20 target samples to that in the pool of control samples 1-10 (red box). This is carried out by generating Z scores for each sample. Peptides that have high Z scores above an arbitrary cut-off value (e.g., 2.5 or 5) for the highest number of the target samples are selected for further characterization as synthetic peptides. As a further assessment of specificity, further control samples (11-20) can be treated in the same way as the target samples to generate Z scores by comparison of binding to the pool of control samples 1–10. The Z scores for control samples can allow the deselection of any peptides where enrichment was seen above the cut-off value used

subtracted against bead-immobilized IgG from a control cohort, before each panning round. This is carried out to remove binders to the blocked solid support, and to remove binders against the control IgG that bind non-paratope regions or that bind the paratope of the control antibodies. Furthermore, during the NGS analysis, the selection of specific peptide mimotopes which are recognized only by infection-specific IgG is further driven by bioinformatic analysis that selects peptides enriched against the infection-specific IgG over the control IgG. This bioinformatic sorting also selects peptides that are being specifically enriched across a wide range of the samples in the infected cohort and so are more likely to be of diagnostic value. Combinations of synthetic peptides are then assessed by serological ELISA to select the best-performing assay for detecting infection, this will use serum samples from both target (infected) cohorts and control cohorts that are independent from those used in the phage biopanning. The presented methods

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describe the panning approach, the bioinformatic sorting for infection-specific peptides, and the validation of peptide recognition in ELISAs. The sensitivity of the NGPD approach for identifying mimotopes could transform our ability to dissect the antibody response to infectious diseases for the development of diagnostic tests and vaccines. Most B-cell epitopes are thought to be conformational [7], therefore, phage libraries constructed using overlapping portions of a pathogen gene sequence, e.g., will only represent a limited portion of the epitope repertoire recognized by antibodies generated in response to infection. The NGPD approach was previously reported to identify mimotopes that could diagnose bacterial infection (including distinguishing between infected and vaccinated animals, DIVA), demonstrated using serum samples from pigs or poultry experimentally infected with Salmonella enterica [4, 5]. These data suggest that the approach has the capacity to detect subtle differences in antibody repertoires and has potential application in developing DIVA assays to existing vaccines. Such an application is based on the chemical or genetic modification of the vaccine pathogen resulting in changes in epitopes [8] that can be resolved using the NGPD methods. The DIVA strategy is diagnostically challenging for killed or attenuated vaccines but is important for identifying individuals that become infected despite vaccination when attempting to control infectious disease outbreaks.

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Materials General

Reagents should be Analytical Reagent grade. All solutions, buffers, and media should be autoclaved or filter sterilized as appropriate. Equipment required: thermal cycler, agarose gel electrophoresis tank and power supply, shaking incubator, Qubit 2.0 Fluorometer 2.0 (Invitrogen), Ion Proton sequencing machine or service provider. Serum samples will be from two cohorts: target (infected) and control (uninfected, infected with alternate pathogen or vaccinated individuals). A selection of each cohort will be used to carry out the biopanning. Independent serum samples from each cohort will be required to assess immunological assay accuracy.

2.2 Biopanning Against Polyclonal Immunoglobulin G

1. TG1 Escherichia coli: F′ traD36 proAB lacIqZ ΔM15] supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK – mK –), (Lucigen).

2.1

2. Minimal media (M9) plates: (i) Make sterile 5x M9 salts, mix 15 g Disodium hydrogen phosphate (Anhydrous)], 7.5 g Potassium dihydrogen phosphate (Monobasic), 2.5 g Ammonium Chloride, 1.25 g Sodium Chloride, and distilled water to 500 mL. (ii) Prepare sterile 20% (w/v) Magnesium chloride.

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(iii) Prepare sterile 100 mg/mL thiamine-hydrochloride, filter sterilize using a 0.45 μm micron filter. (iv) Mix 1.5 g agar powder in 75 mL distilled water, autoclave and allow to cool to 50 °C then add 25 mL 5x M9 salts, 1.6 mL 25% glucose, 100 μl Magnesium chloride, and 50 μl thiamine hydrochloride. Pour out 20 mL per 9 cm diameter plate and allow to set. Make fresh before use and store plates for up to 1 week at 4 °C. 3. Phage-peptide library, for example [9, 10]. 4. Protein G agarose beads (Pierce) (see Note 1). 5. Ultrapure™ DNase/RNase-Free Distilled Water. 6. Agarose. 7. 6x DNA loading dye (NEB). 8. Q5® High-Fidelity DNA Polymerase (NEB). 9. Deoxynucleotide (dNTP) Solution Mix. 10. Nancy 520 (Sigma-Aldrich). 11. TAE buffer (agarose gel running buffer): 2 M Tris–HCl, 1 M acetic acid, 0.05 M EDTA pH 8. (50x). 12. Macherey-Nagel™ NucleoSpin™ Gel and PCR Clean-up Kit. 13. 1 kb DNA molecular weight marker. 14. DNA isolation kit (QIAprep Spin Miniprep Kit, Qiagen). 15. 2YT media (500 mL): add 8 g of tryptone, 5 g yeast, and 2.5 g NaCl to 500 mL water. 16. 2YT agar: add 7.5 g agar to 2YT media prior to autoclaving. 17. 2YTA/2YTA agar: As above, with addition of ampicillin stock kept at 150 mg/mL to final concentration of 150 μg/mL. 18. 2YTAG and 2YTAG agar: Add filter sterilized 25% (w/v) D-(+)-Glucose stock solution in distilled water to the cooled molten media to a final concentration of 1% (w/v) glucose. 19. 1xPBS (phosphate buffered saline) made by dissolving 1 PBS tablet (VWR chemicals) in 500 mL water. Alternatively, make up a solution of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4. 20. 6xPBS (phosphate buffered saline) made by dissolving 3 PBS tablets in 250 mL water. Alternatively, make up a solution of 822 mM NaCl, 16.2 mM KCl, 60 mM Na2HPO4, 10.8 mM KH2PO4. 21. Qubit™ dsDNA BR Assay Kit (ThermoFisher Scientific). 22. Bioassay dish (L × W × H: 245 mm × 245 mm × 25 mm, Merck).

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23. 18% PBSM: add 1.8 mg instant dried skimmed milk to 10 mL of 6× PBS solution and mix thoroughly, use on the day. 24. 3% PBSM: 1 mL 18% PBSM +5 mL 1x PBS, use on the day. 25. 0.1% PBS-Tween wash buffer: 100 mL PBS + 100 μl Tween 20. 26. Freezing media: 2YT/Amp/Glucose media +20% glycerol made fresh and used on the day. 27. Phage elution buffer (100 mM triethylamine solution). 28. 1 M Tris, pH 7.4 buffer: dissolve 6.05 g of Tris base in 30 mL of distilled water, adjust pH to 7.5 with 5 M HCl, and adjust final volume to 50 mL with addition of distilled water. 29. Helper phage: MK13KO7 (Stratagene) and used at 2 × 1012 pfu/mL. 30. Kanamycin stock: 50 mg/mL used in media at 50 μg/mL. 31. PEG-8000 20% (w/v) /2.5 M NaCl: dissolve 200 g of PEG-8000 and 146.1 g of NaCl in 1 L distilled water. 32. QIAprep Spin M13 kit (Qiagen). 33. Agencourt AMPure XP kit (Beckman Coulter). 34. Forward1NGS primer: 5′- GTAATCCTTGTGGTATCG GATGCTGTCTTTCGCTGC-3′. 35. Reverse1NGS primer: 5′CTAGAACATTTCACT TACGGTTTTCCCAGTCACG-3′. 36. Akey-BC-linker1 (Forward): 5′CCATCT CATCCCTGCGTGTCTCCGACTCAG xxxxxxxxxxGTAATCCTTGTGGTATCG-3′. N.B. Underlined is the 10 bp barcoded region, with many variants available to use. This region varies for every primer. Codons in bold are the homologous linker regions, identical for both first and second step PCRs. 37. P1prim-linker2 (Reverse): CCTCTCTATGGGCAGTCGGTGATCTAGAACATTTCACTTAC-3′. N.B. Codons in bold are the homologous linker regions, identical for both first and second step PCRs. 2.3 Bioinformatic Analysis to Identify Enriched Peptides Specific for Infection 2.4 Confirmation of Peptide Specificity for Infection

None

1. Peptides (crude or up to 90% purity) (see Note 2). 2. Nunc Immunoplate F96 MaxiSorp (ThermoFisher Scientific). 3. 1x PBS (Phosphate buffered saline): 1x PBS is made by dissolving 1 tablet of pre-made PBS (VWR chemicals) in 500 mL distilled water. This is the ELISA coating buffer.

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4. 3% PBSM is prepared by adding 3 g instant dried skimmed milk to 100 mL of 1× PBS solution. 5. 3% PBS-T: is prepared by adding 1 mL of Tween-20 (MP Biomedicals) to 1 L of 1 × PBS solution. 6. 1.5 mL tubes. 7. 50 mL Falcon tubes. 8. Rabbit anti-human IgG-Alkaline phosphatase conjugated (Sigma-Aldrich). 9. SIGMA-FAST™ p-Nitrophenyl phosphate buffer (SigmaAldrich).

3

Methods

3.1 Biopanning Against Polyclonal Immunoglobulin G

The panning strategy aims to isolate peptide mimotopes that are recognized by antibodies specific for a particular infection (Figs. 1 and 2). This involves subtraction of phage-peptides against control sera and then enrichment of peptides against the target sera (see Note 3). After this, the enriched sub-library is screened against a panel of serum samples from infected individuals and a panel of serum samples from control individuals. PCR amplification of the peptide gene region within the bound phage and subsequent NGS allows the bioinformatic analysis of peptide binding events. This identifies those peptides that bind consistently to antibody from numerous infected individuals but do not bind to antibody from control samples. The physical subtraction of non-specific mimotopes as well as in silico deselection of non-specific mimotopes enhances identification of peptides that are only recognized by infection-specific IgG. The method described here uses a 16mer peptide library displayed on gene VIII within the pc89 phagemid (in TG1) produced as previously described [10]. 1. Production of phage for the phage-peptide library (Fig. 1, steps 1 and 2). (1) Streak minimal media plates with TG1. Incubate at 37 °C overnight (see Note 4). (2) Grow the phagemid library in TG1 until mid-log: to a 1 L flask, add 250 μl of glycerol library stock to 250 mL of 2YTAG and grow to OD600nm 0.4 to 0.6 in a shaking incubator set to at least 200 rpm 37 °C. (3) Add M13KO7 helper phage to a multiplicity of infection (MOI) of 10 and incubate for 60 min at 37 °C static. This assumes the bacteria is at 4 x108/mL at mid log and M13KO7 is used at 2 × 1012 pfu/mL.

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(4) Pellet bacteria by centrifugation at 3000× g for 10 min and discard supernatant. Remove all traces of supernatant with a further spin (30 s). Discard any supernatant. (5) Resuspend cell pellet in 500 mL of 2YTA containing 50 μg/mL kanamycin, grow shaking at 200 rpm overnight at 30 °C. (6) The next day, pick a single TG1 colony from a minimal plate and inoculate into 10 mL 2YT in a 50 mL centrifuge tube. Incubate at 37 °C shaking to OD600nm 0.4 to 0.6. (7) Centrifuge the phage library culture for 20 min at 8000× g. Collect the supernatant. Precipitate phage by adding 125 mL 20% (w/v) PEG-8000/2.5 M NaCl. Place on ice for 1 h, centrifuge 8000× g for 30 min at 4 °C. Discard the supernatant, centrifuge a further two times at 8000× g for 30 s, and remove any residual supernatant. (8) Resuspend the phage pellet. Add 25 mL of PBS and incubate for 10 min on ice. Pipette gently to resuspend the phage pellet, then mix by rotation for 30 min at RT. Centrifuge at 13000× g for 10 min and collect the supernatant. (9) Dilute 10 μl of phage through five 100-fold serial dilutions into PBS. Add 20 μl of the dilutions to 180 μl of the mid-log TG1 culture. Alongside a TG1 only control. Leave at 37 °C static for 30 min. Spread 100 μl of the infected TG1 onto 2YTAG agar plates. Incubate plates at 37 °C overnight. Count colonies for each dilution and calculate the original number of phage in the stock. N.B. expected to be ~1 × 1013 /mL. No colonies should grow for the TG1-only control. (10) Phage can be stored for 7–14 days at +4 °C or stored long term at -80 °C as a glycerol stock (following the addition of glycerol to 20% v/v). 2. Preparing agarose beads for biopanning. (1) The starting agarose bead slurry volume is dependent on the number of samples in each individual biopanning experiment and the number of pre-panning subtraction steps (see Note 5). Pipette 100 μl bead slurry into a 1.5 mL microcentrifuge tube, allow the beads to settle, and remove the supernatant. Wash the beads twice in 1 mL 1x PBS and centrifuge at 2500× g between washes to remove the supernatant. (2) Resuspend beads in 600 μl 1x PBS after the final wash and use 50 μl aliquot for each biopanning experimental serum sample. 3. Subtraction of peptide binders to control serum IgG (Fig. 1, step 3).

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(1) Block the PEG- precipitated phage library (0.5 mL for each panning sample) in a 50 mL centrifuge tube by addition of 18% (w/v) MPBS to a final concentration of 3% MBPS. Incubate for 1 hour at room temperature on a roller mixer. (2) Pool control serum samples by pipetting 2 μl of each sample (e.g., 10 samples) into a single microcentrifuge tube and mix by pipetting. (3) Add 50 μl of the prepared agarose beads to the pooled serum, gently mix, and incubate for 30 min at room temperature on a rotator mixer. Wash the beads twice in 1 mL 1x PBS and centrifuge at 2500× g between washes to remove the supernatant. Then add to the blocked phage library and incubate for 60 min. (4) At the end of the incubation step, centrifuge at 2500× g and transfer the phage supernatant to a clean 50 mL tube taking care to avoid disturbing the bead pellet. 4. Selection of phage peptide binders to the target serum IgG (Fig. 1, steps 4 and 5). (1) Into 1.5 mL microcentrifuge tubes, pipette 5 μl of each target serum samples (e.g., 10 samples) and 50 μl of prepared agarose beads. Gently mix the beads and serum and incubate for 45 min on a rotator mixer at room temperature. (2) Following the incubation, pipette 1 mL of 3% milk-PBS (3% M-PBS) into each tube, gently mix, and incubate for a further 30 min. (3) Centrifuge the microcentrifuge tubes at 2500× g for 1 min to pellet the beads. Discard the supernatant without disturbing the bead pellet. (4) Wash the beads twice in 1 mL 1x PBS and centrifuge at 2500× g between washes to discard the supernatant. The serum antibody coupled agarose beads are now ready for use in biopanning. (5) Add 0.5 mL of subtracted phage to all the microcentrifuge tubes containing the agarose bead-antibody complex. The phage-peptide library is then left to interact with the antibody-beads complex, overnight at 4 °C on a rocking platform. (6) Following overnight binding, the tubes are centrifuged at 2500× g for 2 min to pellet the beads. The supernatant is discarded, and the beads are washed three times with 1 mL PBS-0.1% Tween 20 buffer, and then a further three times with 1 mL PBS buffer (see Note 6). (7) The bound phage is eluted from the beads by changing the pH, add 250 μl of 100 mM Triethylamine solution to each

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tube, agitate to mix the beads in the solution, and incubate for 10 min at room temperature. Centrifuge the tubes at 2500× g for a minute to pellet the beads (see Note 7). (8) Carefully transfer the supernatants (~250 μl each) to fresh microcentrifuge tubes containing 250 μl 1 M Tris–HCl buffer which will neutralise the pH. The eluted phage sub-libraries can be stored 4 °C for up to 1 week. 5. Titrating enriched phage and making glycerol stocks. (1) Use half of each eluted phage sub-library to infect 10 mL of E. coli TG1 bacterial culture (OD600 0.4–0.8). Mix and incubate at 37 °C static for 30 min. The remaining phage are stored at 4 °C as a backup. (2) Pellet bacteria by spinning at 3000× g for 10 min. Resuspend pellet in 1 mL 2YT. Remove 10 μl of cells to sterile Eppendorf, and plate the remaining cells onto 2YTAGagar bioassay dishes. Incubate overnight at 37 °C. (3) Carry out a ten-fold serial dilution of the 10 μl of bacteria in 1% (v/v) glucose in 2YT media. Plate 100 μl of this dilution series out onto 9 cm 2YTAG-agar plates and incubate overnight at 37 °C. The expected titer should be ~104 to 107 phage /mL eluted phage. (4) Prepare glycerol stocks of each enriched phage sub-library by scraping the bacteria from the bioassay dishes using a cell spreader into 5 mL of 2YTAG media. Add glycerol to 20% (v/v) and mix on a rotating mixer for 15 min before storage at -80 °C as 1 mL aliquots. 6. Screening the enriched phage-peptide sub-library for specific binders. (1) Pool an aliquot of all enriched phage sub-libraries from round 1 of biopanning. Produce and purify phage particles as described above for the original library (Fig. 1 step 6). (2) To screen the enriched library for specific IgG binders in the ‘infected serum samples’ the process is carried out exactly as described for the first round of biopanning. The round 1 enriched phage-peptides are subtracted against the pool of control samples (e.g., n = 10), then they are bound, washed, and eluted against a panel of IgG samples (see Note 3) from the infected cohort (e.g., n = 20) and against a cohort of control sample (e.g., n = 20) in parallel (see Fig. 2). The eluted, neutralized phage from each sample are then again used to produce glycerol stocks. N.B. when more than 10 samples are produced, selected examples can be used to demonstrate the titer of eluted phage is in the expected range of ~104 to 107 phage /mL.

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Fig. 3 Scheme for the amplification of the peptide gene region for NGS analysis. The first PCR step amplifies around a 250 bp fragment of the phagemid DNA containing the gene fragment encoding the displayed peptide. After purification this amplicon is reamplified with primers that are homologous to linker regions in the step 1 primers and also code for a distinct barcode (Akey-BC-linker1) or a universal P1 adapter (P1prim-linker2), both primers contain regions compatible with Ion Torrent sequencing. Unique barcoded primers are used for each phage sample

7. Preparation of peptide gene fragments for NGS analysis (Fig. 1, Step 7). (1) After the screening step, phagemid DNA is purified from the bacterial glycerol stocks stored after the round 2 screening using QIAprep Spin Miniprep Kit and following the manufacturer’s instructions. (2) DNA extracted from each glycerol stock is used as template for a 2 step PCR amplification with primers designed to give a final product containing the required tags for the Ion Torrent NGS analysis (Fig. 3). In the first step, ForwardNGS and ReverseNGS primers specific for phagemid vector pc89 are used to amplify the 16-mer peptide sequence flanked by two small linker regions resulting in a fragment size of ~250 bp. Linker sequences homologous to the primers used in the second step of PCR are present

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in these step 1 primers. PCR reactions are comprised of 10 ng of the DNA template, distilled water (to 50 μl), 10 μl Q5 buffer, 10 μl GC enhancer, 1 μl dNTPs, 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM) and 0.5 μl of Q5 enzyme. The PCR conditions are 95 °C for 3 min, [95 °C for 30 s, 60 °C for 30 s, 68 °C for 30 s] x30 and last 72 °C for 5 min. (3) Amplicons are resolved on a 3% (w/v) agarose gel and the product gel purified (e.g., using the Macherey-Nagel™ NucleoSpin™ Gel and PCR Clean-up Kit). (4) For the second step PCR, purified amplicon (1 μl) from the first step PCR is the template. Primers contain a distinct barcode (Akey-BC-linker1) or a universal P1 primer compatible with Ion Torrent sequencing (P1prim-linker2). Unique barcoded primers are used for each phage sample. PCR set up is as previously, PCR conditions are 95 °C for 3 min, [95 °C for 30 s, 63 °C for 30 s, 68 °C for 30 s] x12, and 72 °C for 5 min. (5) Resolve PCR products on a 3% (w/v) agarose/1x TEA gel to confirm the presence of amplicon of ~310 bp. Amplicon concentration is estimated using Qubit and all samples are pooled to the same concentration. (6) Pooled PCR products are resolved through a 3% (w/v) agarose/1x TAE gel and purified using the MachereyNagel™ NucleoSpin™ Gel and PCR Clean-up Kit. (7) DNA is then further purified using the Agencourt AMPure XP kit and resolved through a 3% (w/v) agarose gel to confirm a single product of the expected size. (8) The concentration of the pooled DNA is again measured by Qubit and diluted to that required by the commercial Ion Torrent service provider (200 ng in a 30–40 μl volume). 3.2 Bioinformatic Analysis to Identify Enriched Peptides Specific for Infection

Ion Torrent S5 amplicon sequence data from a 540 chip run is processed using a custom Perl pipeline running under Linux Ubuntu (Fig. 1, step 9). A single script capable of performing the majority of the processing steps was developed to extract FASTQ data from a “.gz” compressed file and demultiplex DNA sequences using Ion Torrent barcode adapters introduced during the PCR amplification stage. Translation of DNA to protein in all 3 reading frames using a custom codon table is then performed by the script, and the frame containing the randomized library peptides flanked by two fixed vector amino acid sequences is identified. The vector flanking sequences themselves are then removed after

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identification, leaving a FASTA file for each barcode ID containing peptide sequences representing those sequences that had been selected during panning and recovered by PCR amplification. 1. The script, along with associated text files identifying the amino acid sequences flanking the NNK encoded randomized 16mer peptide insert, are available at https://github.com/UoNADAS-KGough/NGS_Scripts01.git and is run from the Linux terminal with a set of example command line options as follows (see Note 8): 2. perl pipeline.pl --infile input.fastq.gz --outfile example_output --barcodes barcodes.txt --left AEGEF.txt --right DPAKA.txt -fulloutput. 3. A second Perl script is then used to compare pairs of FASTA files corresponding to a “target panning” against IgG from infected individuals and a “control panning” against control (e.g., non-infected) IgG samples. Comparisons are performed on individual target-infected panning enrichments against a pool of control samples generated in silico (in a single FASTA file) from a number of individual control samples (see Note 9). 4. For each identified peptide within a target infected sample and its corresponding control (from a pool of samples), a Z-score value is determined using a two proportion Z test and is used to rank peptide sequences based on relative statistical importance, with those at the top of the list being peptides that were enriched the most during the biopanning [11]. 5. The Z score equation is given by:

z=

p1 - p2 p1ð1 - p1Þ n1

þ p2ð1n2- p2Þ

Where n1 = sample 1 size, n2 = sample 2 size, p1 = proportion 1, p2 = proportion 2. 6. The Perl script “compareZ_2.1.pl” is used to generate Z-score value tables from a pair of FASTA files of panning-enriched peptide sequences stripped of N-terminal and C-terminal flanking sequences. As well as generating Z score values, an optional switch “--percentage” can add columns containing each peptide’s percentage as determined from both the target infected and control FASTA files. 7. The script itself needs to be supplied with two FASTA files (here labeled file1.fasta and file2.fasta corresponding to a single target sample and 10 in silico pooled control samples, respectively) containing the randomized peptide sequences generated by pipeline.pl and is again run from the Linux terminal:

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8. perl compareZ_2.1.pl file1.fasta file2.fasta >Z_output.txt, 9. The text file “Z_output.txt” generated by the script contains tab-separated columns consisting of every peptide sequence from both input files in the first column, along with their respective frequencies in columns 2 and 3 and their corresponding Z-score value in column 4. 10. Peptides are automatically ranked in descending numerical order of the Z score value, with those peptide sequences at the top of the list representing the highest Z score values, i.e., the most enriched peptides in the target sample compared to the controls and therefore the most likely candidates for further investigation as diagnostic peptides. 11. Peptides are selected for further analysis if they return a Z-score value exceeding an arbitrary cut off, e.g., 2.5. Those peptides that matched this criterion are subsequently filtered such that, e.g., only those seen at least 3 times out of the 10 individual target (infected) samples are retained, allowing for narrowing the list of peptides to those which are consistently enriched across multiple target samples (see Note 10). It should be noted that a range of Z score cut-off values are typically tested. For example, 2, 2.5, 5, and 10 have been examined in our analysis, with the final value that is applied dependent on the relative enrichment of peptides in the target sample panning and control sample panning. 12. The above is carried out for the “training cohort” comprised of the 10 target samples used in both biopanning rounds and compared to the pool of the 10 control samples used for subtraction in both biopanning rounds. The analysis is also then carried out for a “test cohort,” consisting of an additional 10 infected IgG samples and 10 control IgG samples, all compared to the same pool of 10 control samples to generate Z scores (see Fig. 2). Peptides that occur the greatest number of times in infected samples in both the training and test cohorts (but not seen above the cut off in the control test cohort) are selected for peptide synthesis and ELISA screening. 3.3 Confirmation of Infection-Specific Diagnostic Peptides

1. Candidate peptides for screening should be synthesized using a commercial service to crude purity. The peptides were synthesized with the N-terminal residues AEGEF and C-terminal amino acids DPAKA flanking the peptide sequence, and with an amidated C-terminus. This more closely represents display on the phage. (see Note 2). 2. Wash Nunc Immunoplate F96 MaxiSorp plates 3 times with 1x PBS.

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3. Coat 100 μl of peptide resuspended in PBS (10 μg/mL) onto Nunc Immunoplate F96 MaxiSorp in duplicated wells (see Note 11); wrap in cling film and store plates at 4 °C overnight. 4. Wash plates 3 times with 1x PBS. Add 400 μl of 3% PBSM onto each well; incubate static at RT for 1 h to block the plate. In the meantime, dilute sera (e.g., 1:100, but this will depend on the infection-specific antibody titer) using 3% PBSM as diluent. 5. Wash plates 3 times with 1x PBS. Add 100 μl of diluted sera per well (duplicates); incubate static at RT for 1 h. Screening against a minimum of 20 control and target serum samples is recommended for this preliminary characterization. These should be distinct samples from those used in biopanning (see Note 2). 6. Wash plates 3 times with 1x PBS-T and then 3 times with 1x PBS. Add 100 μl of species-specific anti-IgG AP conjugate (e.g., rabbit anti-human IgG AP conjugate at the recommended manufacturers dilution using 3% PBSM as diluent) to each well; incubate static at RT for 1 h. 7. Prepare SIGMA-FAST™ p-Nitrophenyl phosphate buffer as per manufacturer’s instructions (add each tablet to 20 mL of distilled water, leave it rotating in dark for 30 min to 1 h before use). 8. Wash plates 3 times with 1x PBS-T and then 3 times with 1x PBS. Add 100 μl of SIGMA-FAST™ p-Nitrophenyl phosphate buffer to each well and incubate in dark; plates can be read at 405 nm once yellow colour has developed, typically after 1 h to 16 h incubation. 9. Select peptides that give ELISA signals above the background (an irrelevant peptide control, can be used Note 11) against the target serum samples that are at least 2x that produced with control serum samples (see Note 3). Assays can be developed using multiple peptides that together show the highest assay performance to differentiate the target serum samples from the controls.

4

Notes 1. Coupling to solid support. We describe using Protein G immobilization, depending on species IgG may bind better to Protein A, in this case, protein A agarose beads can be substituted. We have also had success binding antibody to the surface of maxisorp plates for the panning steps. 2. Peptides for screening should be synthesized (a crude purity level is sufficient for this screening) and with a blocked C terminus. It is advisable to include a few amino acids

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(we routinely use 5 at the N and C terminus) that derive from the display system so that the peptide is presented in a similar context to that which it was displayed on the phage particle. Peptides can also be synthesized biotinylated and bound to streptavidin-coated microplates. This can increase the amount of oriented peptide that is available in the ELISA assay and may be useful when infection-specific antibody is at low titer. 3. The example given here and depicted in Fig. 2 uses 10 control serum samples to subtract non-specific mimotopes and then 10 serum samples from infected individuals to select candidate diagnostic peptides. The strategy then uses a further 10 samples from each cohort in round 2 of biopanning to further inform the selection of peptides only recognized by infection-specific IgG. The control serum samples can be varied depending on the assay specificity that is required. For example, using controls from uninfected ‘healthy’ individuals can be used to search for infection-specific markers; alternatively, using controls from individuals with infection with a closely related pathogen can yield peptides capable of differentiating the infecting pathogens; and using controls from vaccinates can yield peptides that can differentiate infected from vaccinated individuals. Also of note, the number of samples in each cohort (target and control) will influence the success of the approach, the more challenging the distinction to be made (e.g., between infection- and vaccination-induced antibodies), the more samples are likely to be required. It is recommended to use a minimum of 20 samples in each cohort. Verification of the diagnostic potential of peptides by ELISA can initially use the same serum sample cohorts as in the biopanning to confirm the bioinformatics analysis, but also using independent samples from both cohorts is strongly recommended to confirm assay accuracy. 4. As a control, also streak TG1 onto a 2YTAG agar plate and grow overnight at 37 °C. There should be no growth; any growth indicates contamination of the TG1 stock. 5. A larger starting volume is better as that takes into account any loss of bead during the preparatory PBS washes and is easier to visualize, especially during the phage rescue stage. For 10 target screening samples, a minimum starting bead slurry volume of 50 μl is required. This can be resuspended in 300 μl PBS and 30 μl of the resulting bead suspension can be used for each sample. 6. The beads should appear translucent by the final wash. The wash solution should not have any trace of the milk block. If there are traces of milk block, repeat wash step until the beads look translucent.

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7. Elution can also utilize a 0.2 M glycine-HCl pH 2.2 buffer, with a similar neutralization step. 8. The Perl script described here can read either a compressed FASTQ file in “.gz” (GNU zip) format or alternatively the compressed file can be uncompressed manually prior to reading by the script as a FASTQ file. The “--outfile” option sets the prefix which will be applied to the output data files. The option “--fulloutput” is provided to create text files of all output, namely it generates demultiplexed FASTA files, translations of every demultiplexed DNA sequence in each of three forward translational frames using a custom codon table, and a tab-separated text file containing each sequence identified between the flanking regions in descending numerical frequency order for each barcode. It is also possible to reverse complement each DNA sequence before translation using the option “—revcomp.” A barcode is identified as a match and all nucleotides up to and including the barcode sequence are removed from the read if it begins within the first 5 nucleotides of the query DNA sequence and is an exact match to the barcode sequence. A list of Ion Torrent barcodes must be provided in a plain text file (described here as barcodes.txt) that is in the following tab-or space-separated format: BC01 CTAAGGTAAC BC02 TAAGGAGAAC ... BC96 TTAAGCGGTCA Identified sequences are “binned” according to their respective barcode sequence ID, and translated in all three frames in the forward direction. Optionally, DNA sequences can be reverse complemented at this stage prior to translation if PCR primers used for template amplification were designed such that Ion Torrent sequencing was performed on the reverse strand. Translation of the DNA sequences is performed using a custom codon table. Essentially, stop codons are translated to “o” for ochre stop codons, “p” for opal stop codons, and “q” for amber stop codons, with amber stop codons being replaced with the amino acid glutamine (Q) if a peptide containing such a stop codon is subsequently selected for chemical synthesis. N.B. TG1 is an amber suppressor strain and so peptides containing an amber stop should be displayed on the phage. Identification of amino acid sequences flanking the randomized peptide insert ensures that the N-terminal flanking region, the peptide insert, and the C-terminal flanking region are all maintained in frame. Typically 5 or more amino acids N-

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and C-terminal to the insert are used for identification. To allow for PCR/sequencing errors, any amino acid can be negated if required using the ^ character within square brackets; e.g., AE[^G]EF will identify a flanking region where glycine does not occur at the third position of this pentameric amino acid sequence. Two text files each containing either the N-terminal or C-terminal flanking regions must be provided. Where flanking region sequences have included negated residues, each file may contain multiple flanking peptide sequences, in which case all possible flanking region combinations between the two files will be tested against the target peptide sequences to see if matches occur. If a match is found, only the peptide sequence between the flanking regions is retained; all amino acid sequences before and after the insert are discarded. If only one of the N-terminal or C-terminal flanking sequences is identified, the corresponding randomized peptide will not be retained for further analysis. For a translated sequence with flanking regions AEGEF and DPAKA from a pC89 pVIII derived 16mer peptide library described previously [4, 5, 9, 10] and used in this work, an example sequence before and after flanking sequence identification is shown, with the target flanking regions underlined: Before: >KESPI:07775:02799 LSFAAEGEFLTYNARKADPRLSAMLDPAKAAFDSLQA After: >KESPI:07775:02799 LTYNARKADPRLSAML 9. Where library-derived peptides have been identified correctly as occurring between the N-terminal and C-terminal flanking sequences but do not match the expected insert size (16 amino acids in this case), a Perl script run from the Linux terminal prompt can be used to remove incorrect length sequences prior to calculating Z scores: perl Nmerfastafilter.pl --infile input.fasta --outfile output.fasta --length 16 This will remove all peptide sequences in a FASTA file that are not exactly 16 amino acid sequences long, generating a file called output.fasta containing the filtered FASTA sequences. This step may not be necessary dependent on library construction method and quality 10. Standard Linux tools can be used to extract peptides from the tabulated output where a particular Z-score value is observed. For example, given that the peptide sequence is in column

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1 and its corresponding Z-score value is in column 4, the program “awk” can be used to extract peptides matching a particular criteria (in this case having a Z-score value of equal to or greater than 2.5) whilst skipping the header on line 1: awk ‘NR>1 & & $4>=2.5 {print $1;}’ < Z_output.txt > peptides_Z2.5_list.txt These peptide lists from multiple comparisons (here labeled file1.txt to file5.txt) can be concatenated using the standard Linux “cat” command and replicate occurrences counted and ranked using “sort” and “uniq” by piping the data from one command to the next using the Linux pipe symbol “|”: cat file1.txt file2.txt file3.txt file4.txt file5.txt > peptide_list.txt sort peptide_list.txt | uniq -c | sort -nr > ranked_peptide_list.txt The output file “ranked_peptide_list.txt” should now contain the number of times each peptide was observed to be enriched above the chosen cut-off value across all the files joined by “cat” and, depending on the Z score cut-off chosen, is indicative of statistically significant enrichment across multiple panning samples or replicates. Given that 5 text files of peptides were joined together using the “cat” command above, a value of 3 in the resulting “ranked_peptide_list.txt” file would indicate that that peptide was seen in 3 out of 5 possible occasions with a Z score of 2.5 or higher. Selective filtering of sequencing data, to identify patterns of enrichment of particular peptides across multiple replicate pannings or panning with multiple infected samples where common antibodies would perhaps be expected to occur because of similar immune responses can provide the basis for screening potential binders prior to time-consuming functional assays 11. Always test serum samples in duplicate or triplicate and use the mean signal. Also, include wells coated with an irrelevant peptide to determine the background of the assay and therefore the signal-to-noise ratio for each sample. Include wells coated with PBS only to determine any issues with secondary antibody background signal. References 1. Capello M, Cappello P, Linty FC et al (2013) Autoantibodies to ezrin are an early sign of pancreatic cancer in humans and in genetically engineered mouse models. J Haematol Oncol 6:67 2. Xiong X, Wang X, Wen B et al (2012) Potential serodiagnostic markers for Q fever identified in

Coiella burnetii by immunoproteomic and protein microaraay approaches. BMC Microbiol 12:35 3. Tjalsma H, Schaeps RMJ, Swinkels D (2008) Immunoproteomics: from biomarker discovery to diagnostic applications. Proteomics Clin Appl 2:167–180

Polyclonal Antibody Mapping 4. Naqid IA, Owen JP, Maddison BC et al (2016) Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals. Sci Rep 6:31186 5. Naqid IA, Owen JP, Maddison BC et al (2016) Mapping polyclonal antibody responses to bacterial infection using next generation phage display. Sci Rep 6:24232 6. Xu GJ, Kula T, Xu Q et al (2016) Comprehensive serological profiling of human populations using a synthetic human virome. Science 348: aaa0698 7. Ferdous S, Kelm S, Baker TS et al (2019) B-cell epitopes: discontinuity and conformational analysis. Mol Immunol 114:643–650 8. Fan Y-C, Chiu H-C, Chen L-K et al (2015) Formalin inactivation of Japanese encephalitis virus vaccine alters the antigenicity and

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immunogenicity of a neutralization epitope in envelope protein domain III. PLoS Negl Trop Dis 9(10):e0004167 9. Felici F, Castagnoli L, Musacchio A et al (1991) Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J Mol Biol 222(2):301–310 10. Tsoumpeli MT, Gray A, Parsons AL et al (2022) A simple whole-plasmid PCR method to construct high-diversity synthetic phage display libraries. Mol Biotechnol 64(7):791–803 11. Zhang H, Torkamani T, Jones TM et al (2011) Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems. Proc Natl Acad Sci U S A 108(33): 13456–13461

Chapter 26 Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering Michael J. Lowden, Eric K. Lei, Greg Hussack, and Kevin A. Henry Abstract Next-generation DNA sequencing (NGS) technologies have made it possible to interrogate antibody repertoires to unprecedented depths, typically via sequencing of cDNAs encoding immunoglobulin variable domains. In the absence of heavy-light chain pairing, the variable domains of heavy chain-only antibodies (HCAbs), referred to as single-domain antibodies (sdAbs), are uniquely amenable to NGS analyses. In this chapter, we provide simple and rapid protocols for producing and sequencing multiplexed immunoglobulin variable domain (VHH, VH, or VL) amplicons derived from a variety of sources using the Illumina MiSeq platform. Generation of such amplicon libraries is relatively inexpensive, requiring no specialized equipment and only a limited set of PCR primers. We also present several applications of NGS to sdAb discovery and engineering, including: (1) evaluation of phage-displayed sdAb library sequence diversity and monitoring of panning experiments; (2) identification of sdAbs of predetermined epitope specificity following competitive elution of phage-displayed sdAb libraries; (3) direct selection of B cells expressing antigen-specific, membrane-bound HCAb using antigen-coupled magnetic beads and identification of antigen-specific sdAbs, and (4) affinity maturation of lead sdAbs using tandem phage display selection and NGS. These methods can easily be adapted to other types of proteins and libraries and expand the utility of in vitro display technology. Key words Antibody, Single-domain antibody, VHH, Phage display, Next-generation DNA sequencing, Protein engineering

1

Introduction Single-domain antibodies (sdAbs), also called VHHs, are the monomeric antigen-binding variable domains of camelid heavy chainonly antibodies and have many desirable therapeutic properties, including small size, stability, and modularity [1]. Rare autonomous VH and VL domains have also been isolated or engineered from species that do not produce such molecules naturally (e.g., human [2], mouse [3]). Despite bearing only three complementarity-determining region (CDR) loops in the absence of a paired VL domain, sdAbs achieve antigen binding affinities

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_26, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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comparable with those of conventional VH:VL antibodies [4]. Due to their compact footprints and efficient concentration of contact residues in three-dimensional space, sdAbs may have preferred access to recessed antigenic clefts on proteins [5] and their evolutionary emergence subsequent to the origins of heterotetrameric conventional antibodies has been speculated to relate to potential roles in antiviral defense [6]. In vitro display libraries, including phage display libraries, are now routinely constructed from large repertoires of sdAbs (VHH, VNAR, VH, or VL domains) as well as from paired VH:VL repertoires in Fab and scFv libraries [7]. The applications of sdAbs in biology, biotechnology, and medicine are diverse. The development of massively parallel next-generation DNA sequencing (NGS) technologies at the beginning of the twenty-first century has fueled rapid progress in many areas of research. Highthroughput sequencing of antibody repertoires has been applied to detect B-cell malignancies, to discover novel antibodies, to guide vaccine development, and to understand autoimmunity [8]. The genetic characteristics of immunoglobulin variable domain repertoires (i.e., sequence diversity among homologous germline gene segments; high clonal diversity of B-cell populations; potential for somatic mutation) make them challenging templates for NGS. The major challenges include: (1) sequence variation between germline genes at the 5′ end [leader sequences and framework region (FR) 1], necessitating either multiplex PCR or 5′ RACE approaches [9], both of which may bias repertoire diversity; (2) ontogeny of immunoglobulin variable domains through somatic recombination of gene segments, making regions of each B-cell receptor unique at the nucleotide level with no genomic comparison possible; and (3) difficulties in distinguishing rare somatic variants from sequencing errors, which can be mitigated by barcoding of individual cDNA molecules [10]. Nevertheless, several approaches have been developed for interrogating immunoglobulin VH and VL repertoires, most of which rely on the availability of large amounts of input material (e.g., whole peripheral blood lymphocytes [9]; high-frequency B-cell subsets obtained from large blood samples [11, 12]; bulk antigen-specific B cells [13]; phage-displayed libraries [14]) or are limited in throughput to thousands of sequences (e.g., linked VH and VL amplicons generated from single B cells [15, 16]). In this chapter, we describe a basic set of techniques for NGS of immunoglobulin variable domains (VHH, VH, or VL) using the Illumina MiSeq platform. The methods outlined here are simple and inexpensive, requiring only standard molecular biology skills and equipment available in most labs, and are meant to serve as a starting point for investigators wishing to undertake NGS analyses of antibody repertoires and/or antibody libraries. In particular, we illustrate a number of examples in which NGS is used: (1) to probe

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Fig. 1 Overview of applications of high-throughput DNA sequencing to sdAb discovery and engineering described in this chapter

the diversity and functionality of sdAb libraries, as well as to observe the behavior of individual library members under selection [17]; (2) to identify sdAbs of predetermined epitope specificity following competitive elution [18, 19], (3) to directly identify antigenspecific sdAbs following selection of B cells on antigen-coupled magnetic beads, and (4) to affinity mature lead sdAbs [20] (Fig. 1).

2

Materials Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 M Ω cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature unless indicated otherwise. Carefully follow all waste disposal regulations when disposing of hazardous waste materials. Institutional and other relevant research ethics approvals must be obtained prior to starting work with animal and/or human samples. All oligonucleotides listed in this protocol were purchased commercially, desalted, but with no additional purification unless otherwise indicated.

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2.1 Core NGS Workflow

1. Micropipettes (P10, P20, P200, P1000) and sterile filter tips.

2.1.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

3. Freshly obtained whole blood sample from human, mouse, or llama (see Note 1).

2. 1.5 mL microcentrifuge tubes.

4. BD VacutainerTM blood collection tubes with sodium citrate (BD Biosciences, San Jose, CA, USA; see Note 2). 5. Sterile hypodermic needles of appropriate length and gauge (see Note 3). 6. 0.5 M ethylenediaminetetraacetic acid (EDTA) stock solution (per L): 186.1 g EDTA-Na2∙2H2O in ultrapure H2O. Adjust pH to 8.0 with NaOH, and store at room temperature. 7. Phosphate-buffered saline (PBS): 8 g (137 mM) NaCl, 0.2 g (2.37 mM) KCl, 1.44 g (10 mM) Na2HPO4, 0.24 g (1.8 mM) KH2PO4 dissolved in ultrapure H2O. Adjust pH to 7.4 with HCl, sterilize by autoclaving, and store at room temperature. 8. PBS supplemented with 2 mM EDTA (per L): prepare as above but add 4 mL 0.5 M EDTA stock solution prior to adjusting pH. 9. Histopaque®-1077 (Sigma-Aldrich, St. Louis, USA) or FicollPacque® PLUS (GE Healthcare, Chicago, IL, USA) density separation media, stored at 4 °C. 10. LympholyteⓇ-M (Cedarlane Labs, Burlington, Canada) density separation medium. 11. 50 mL Falcon tubes. 12. SorvallTM LegendTM refrigerated tabletop centrifuge with swinging bucket rotor (Thermo-Fisher Scientific, Waltham, MA, USA), or similar instrument. 13. Pasteur pipettes. 14. TC20TM automated cell counter (Bio-Rad Laboratories, Hercules, CA, USA), or similar instrument, with counting slides and 0.4% (w/v) Trypan blue solution. 15. Mr. FrostyTM freezing container (Thermo-Fisher Scientific), or similar instrument. 16. Isopropanol. 17. -80 °C freezer. 18. Fetal bovine serum. 19. Tissue-culture grade dimethyl sulfoxide (DMSO). 20. NuncTM cryobank vials (Sigma-Aldrich). 21. Cryogenic storage dewar containing an appropriate volume of liquid nitrogen.

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22. 37 °C water bath. 23. RPMI medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. 2.1.2 RNA Extraction and cDNA Synthesis

1. Micropipettes (P10, P20, P200, P1000) and sterile filter tips. 2. RNase AWAY™ surface decontaminant (Thermo-Fisher Scientific). 3. RNeasyⓇ Mini Kit (Qiagen, Hilden, Germany), or similar product. 4. RNase-free TE buffer (Thermo-Fisher Scientific). 5. NanoDropⓇ ND-1000 spectrophotometer (Thermo-Fisher Scientific), or similar instrument. 6. -80 °C freezer. 7. qScriptTM cDNA supermix (Quanta Biosciences, Beverley, MA, USA), or similar product. 8. 0.2 mL PCR tubes, strips, or plates. 9. GeneAmp® PCR System 9700 thermal cycler (Thermo-Fisher Scientific) or similar instrument. 10. -20 °C freezer.

2.1.3 Construction and Panning of PhageDisplayed sdAb Libraries 2.1.4 Illumina MiSeq Sequencing

See Subheading 3.1.3 and references therein.

1. Micropipettes (P10, P20, P200, P1000) and sterile filter tips. 2. 1.5 mL microcentrifuge tubes. 3. Gene-specific first round PCR primers as in Table 1 bearing universal tags. The gene-specific regions of these primers are adapted from those found in the following sources: human lymphocytes [21, 22]; mouse lymphocytes [23]; llama lymphocytes [24]. 4. Barcoded second round tagging PCR primers as in Table 2. All primers in this table should be purified either by HPLC or polyacrylamide gel electrophoresis. 5. AmpliTaq Gold® DNA polymerase with PCR Buffer II and MgCl2 (Thermo-Fisher Scientific). 6. dNTP mix, 10 mM each. 7. GeneAmp® PCR System 9700 thermal cycler, or similar instrument. 8. 0.2 mL PCR tubes, strips, or plates. 9. Agarose gel electrophoresis equipment and power supply. 10. 0.5 M EDTA stock solution (see Subheading 2.1.1 for preparation).

Mouse lymphocytes

Heavy chain (igh)

Human lymphocytes

Heavy chain (igh)

Lambda light chain (igl)

Kappa light chain (igk)

Locus

Template

seqF-VH1 seqF-VH2 seqF-VH3 seqF-VH4 seqF-VH5 seqF-VH6 seqF-VH7

seqF-VH1 seqF-VH2 seqF-VH3 seqF-VH4 seqF-VH5 seqF-VH6 seqR-JH seqR-IgM seqR-IgG seqR-IgA seqF-VK1 seqF-VK2 seqF-VK3 seqR-CK seqF-VL1 seqF-VL2 seqF-VL3 seqF-VL4 seqF-VL5 seqF-VL6 seqF-VL7 seqF-VL8 seqF-VL9 seqF-VL10 seqR-CL

Primer name

CGCTCTTCCGATCTCTG(N4-6)AGRTYCAGCTGCARCAGTCT CGCTCTTCCGATCTCTG(N4-6)AGGTCCAACTGCAGCAGCC CGCTCTTCCGATCTCTG(N4-6)TCTGCCTGGTGACWTTCCCA CGCTCTTCCGATCTCTG(N4-6)GTGCAGCTTCAGGAGTCAG CGCTCTTCCGATCTCTG(N4-6)GAGGTGAAGCTTCTCGAGTC CGCTCTTCCGATCTCTG(N4-6)GAAGTGAAGCTGGTGGAGTC CGCTCTTCCGATCTCTG(N4-6)ATGKACTTGGGACTGARCTGT

CGCTCTTCCGATCTCTG(N4-6)GGCCTCAGTGAAGGTCTCCTGCAAG CGCTCTTCCGATCTCTG(N4-6)GTCTGGTCCTACGCTGGTGAAACCC CGCTCTTCCGATCTCTG(N4-6)CTGGGGGGTCCCTGAGACTCTCCTG CGCTCTTCCGATCTCTG(N4-6)CTTCGGAGACCCTGTCCCTCACCTG CGCTCTTCCGATCTCTG(N4-6)CGGGGAGTCTCTGAAGATCTCCTGT CGCTCTTCCGATCTCTG(N4-6)TCGCAGACCCTCTCACTCACCTGTG TGCTCTTCCGATCTGAC(N4-6)CTTACCTGAGGAGACGGTGACC TGCTCTTCCGATCTGAC(N4-6)GGTTGGGGCGGATGCACTCC TGCTCTTCCGATCTGAC(N4-6)SGATGGGCCCTTGGTGGARGC TGCTCTTCCGATCTGAC(N4-6)CTTGGGGCTGGTCGGGGATG CGCTCTTCCGATCTCTG(N4-6)ATGAGGSTCCCYGCTCAGCTCCTGGG CGCTCTTCCGATCTCTG(N4-6)CTCTTCCTCCTGCTACTCTGGCTCCCAG CGCTCTTCCGATCTCTG(N4-6)ATTTCTCTGTTGCTCTGGATCTCTG TGCTCTTCCGATCTGAC(N4-6)CAGCAGGCACACAACAGAGGCAGTTCC CGCTCTTCCGATCTCTG(N4-6)GCACAGGGTCCTGGGCCCAGTCTG CGCTCTTCCGATCTCTG(N4-6)GCTCTGTGACCTCCTATGAGCTG CGCTCTTCCGATCTCTG(N4-6)GGTCTCTCTCSCAGCYTGTGCTG CGCTCTTCCGATCTCTG(N4-6)GTTCTTGGGCCAATTTTATGCTG CGCTCTTCCGATCTCTG(N4-6)GAGTGGATTCTCAGACTGTGGTG CGCTCTTCCGATCTCTG(N4-6)GCTCACTGCACAGGGTCCTGGGCC CGCTCTTCCGATCTCTG(N4-6)GCTTACTGCACAGGATCCGTGGCC CGCTCTTCCGATCTCTG(N4-6)ACTCTTTGCATAGGTTCTGTGGTT CGCTCTTCCGATCTCTG(N4-6)TCTCACTGCACAGGCTCTGTGACC CGCTCTTCCGATCTCTG(N4-6)ACTTGCTGCCCAGGGTCCAATTC TGCTCTTCCGATCTGAC(N4-6)CACCAGTGTGGCCTTGTTGGCTTG

Sequence (5′–3′)a,b

Table 1 First round gene-specific PCR primers used to amplify genes encoding rearranged VH, VL, or VHH domains from lymphocytes or phage-displayed libraries for Illumina MiSeq Sequencing

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Lambda light chain (igl)

Kappa light chain (igk)

seqF-VH8 seqF-VH9 seqF-VH10 seqF-VH11 seqF-VH12 seqF-VH13 seqF-VH14 seqF-VH15 seqF-VH16 seqF-VH17 seqR-JH seqR-IgM seqR-IgG1 seqR-IgG2 seqR-IgA seqF-VK1 seqF-VK2 seqF-VK3 seqF-VK4 seqF-VK5 seqF-VK6 seqF-VK7 seqF-VK8 seqF-VK9 seqF-VK10 seqF-VK11 seqF-VK12 seqF-VK13 seqF-VK14 seqF-VK15 seqF-VK16 seqF-VK17 seqF-VK18 seqF-VK19 seqR-CK seqF-VL1 seqF-VL2 seqF-VL3 seqR-CL

CGCTCTTCCGATCTCTG(N4-6)CAGTGTGAGGTGAAGCTGGT CGCTCTTCCGATCTCTG(N4-6)CCAGGTTACTCTGAAAGAGTC CGCTCTTCCGATCTCTG(N4-6)TGTGGACCTTGCTATTCCTGA CGCTCTTCCGATCTCTG(N4-6)TGTTGGGGCTGAAGTGGGTTT CGCTCTTCCGATCTCTG(N4-6)ATGGAGTGGGAACTGAGCTTA CGCTCTTCCGATCTCTG(N4-6)AGCTTCAGGAGTCAGGACC CGCTCTTCCGATCTCTG(N4-6)CAGGTGCAGCTTGTAGAGAC CGCTCTTCCGATCTCTG(N4-6)ATGCAGCTGGGTCATCTTCTT CGCTCTTCCGATCTCTG(N4-6)GACTGGATTTGGATCACKCTC CGCTCTTCCGATCTCTG(N4-6)TGGAGTTTGGACTTAGTTGGG TGCTCTTCCGATCTGAC(N4-6)CTYACCTGAGGAGACDGTGA TGCTCTTCCGATCTGAC(N4-6)CATGGCCACCAGATTCTTATC TGCTCTTCCGATCTGAC(N4-6)AGGGAAATARCCCTTGACCAG TGCTCTTCCGATCTGAC(N4-6)AGGGAAGTAGCCTTTGACAAG TGCTCTTCCGATCTGAC(N4-6)GAATCAGGCAGCCGATTATCAC CGCTCTTCCGATCTCTG(N4-6)TGATGACCCARACTCCACT CGCTCTTCCGATCTCTG(N4-6)GCTTGTGCTCTGGATCCC CGCTCTTCCGATCTCTG(N4-6)CTGCTGCTCTGGGTTCC CGCTCTTCCGATCTCTG(N4-6)CAGCTTCCTGCTAATCAGTG CGCTCTTCCGATCTCTG(N4-6)CTCAGATCCTTGGACTTHTG CGCTCTTCCGATCTCTG(N4-6)TGGAGTCACAGACYCAGG CGCTCTTCCGATCTCTG(N4-6)TGGAGTTTCAGACCCAGG CGCTCTTCCGATCTCTG(N4-6)CTGCTMTGGGTATCTGGT CGCTCTTCCGATCTCTG(N4-6)CWTCTTGTTGCTCTGGTTTC CGCTCTTCCGATCTCTG(N4-6)GATGTCCTCTGCTCAGTTC CGCTCTTCCGATCTCTG(N4-6)CCTGCTGAGTTCCTTGGG CGCTCTTCCGATCTCTG(N4-6)CTGCTGCTGTGGCTTACA CGCTCTTCCGATCTCTG(N4-6)CCTTCTCAACTTCTGCTCT CGCTCTTCCGATCTCTG(N4-6)AGGGCCCYTGCTCAGTTT CGCTCTTCCGATCTCTG(N4-6)ATGAGGGTCCTTGCTGAG CGCTCTTCCGATCTCTG(N4-6)GAGGTTCCAGGTTCAGGT CGCTCTTCCGATCTCTG(N4-6)CCATGACCATGYTCTCACT CGCTCTTCCGATCTCTG(N4-6)ATGGAAACTCCAGCTTCATTT CGCTCTTCCGATCTCTG(N4-6)ATGAGACCGTCTATTCAGTT TGCTCTTCCGATCTGAC(N4-6)GCACCTCCAGATGTTAACTG CGCTCTTCCGATCTCTG(N4-6)GCCTGGAYTTCACTTATACTC CGCTCTTCCGATCTCTG(N4-6)TGGCCTGGACTCCTCTCTT CGCTCTTCCGATCTCTG(N4-6)ACTCAGCCAAGCTCTGTG TGCTCTTCCGATCTGAC(N4-6)AGCTCCTCAGRGGAAGGTG

(continued)

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CGCTCTTCCGATCTCTG(N4-6)GCAATTCCTTTAGTTGTTCCTTTCTATTCTCAC TGCTCTTCCGATCTGAC(N4-6)GAGGTTTTGCTAAACAACTTTCAACAGTTTC CGCTCTTCCGATCTCTG(N4-6)GCCCAGCCGGCCATGGCC TGCTCTTCCGATCTGAC(N4-6)TGAGGAGACGGTGACCTGG

seqF-FdTc seqR-FdTc seqF-MJ7d seqR-MJ8d

Various

Phage-displayed VHH/VH/VL library

b

Universal tag sequences are indicated in underlined italics, random nucleotides in bold and gene-specific sequences annealing to target template in ordinary type N4-6 indicates a stretch of 4–6 N-nucleotides. Data quality is marginally improved by substituting an equimolar mixture of three primers in the place of primers listed in this table, each bearing a different number of N-nucleotides (4, 5, or 6) c Primers seqF-FdT and seqR-FdT are designed to amplify sdAb (VHH, VH, or VL) libraries in fd-tet vectors. For other vectors, substitute the gene-specific sequences of these primers with vector-specific primers immediately flanking genes encoding antibody variable regions d Primers seqF-MJ7 and seqR-MJ8 are designed to amplify camelid VHH libraries in phagemid vector pMED1 and anneal in the pelB leader sequence and VHH FR4, respectively. The gene-specific regions of these primers correspond to the primers used for library cloning. For other libraries, substitute the gene-specific sequences of these primers with those used for cloning of the library

a

CGCTCTTCCGATCTCTG(N4-6)SMKGTGCAGCTGGTGGAKTCTGGGGGA CGCTCTTCCGATCTCTG(N4-6)CAGGTAAAGCGGAGGAGTCTGGGGGA CGCTCTTCCGATCTCTG(N4-6)CAGGCTCAGGTACAGCTGGTGGAGTCT TGCTCTTCCGATCTGAC(N4-6)CGCCATCAAGGTACCAGTTGGA TGCTCTTCCGATCTGAC(N4-6)GGGGTACCTGTCATCCACGGACCAGCTGA

seqF-MJ1 seqF-MJ2 seqF-MJ3 seqR-CH2 seqR-CH2b3

Heavy-chain only (igh)

Llama lymphocytes

Sequence (5′–3′)a,b

Primer name

Locus

Template

Table 1 (continued)

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AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG

CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCTCTACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

P5-seqF

P7-index1-seqR

P7-index2-seqR

P7-index3-seqR

P7-index4-seqR

P7-index5-seqR

P7-index6-seqR

P7-index7-seqR

P7-index8-seqR

P7-index9-seqR

P7-index10-seqR

P7-index11-seqR

P7-index12-seqR

P7-index13-seqR

P7-index14-seqR

P7-index15-seqR

P7-index16-seqR

P7-index17-seqR

P7-index18-seqR

P7-index19-seqR

(continued)

Sequence (5′–3′)a

Primer name

Table 2 Universal second-round barcoded PCR primers used to tag first-round amplicons (regardless of template type) with MiSeq indexes and adapter sequences

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Sequence (5′–3′)a

CAAGCAGAAGACGGCATACGAGATGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCGAAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCCACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGCTACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGCTCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATAGGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCTTTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTAGTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCCGGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATATCGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTGAGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCGCCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGCCATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATAAAATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTGTTGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATATTCCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATAGCTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGTATAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

Primer name

P7-index20-seqR

P7-index21-seqR

P7-index22-seqR

P7-index23-seqR

P7-index24-seqR

P7-index25-seqR

P7-index26-seqR

P7-index27-seqR

P7-index28-seqR

P7-index29-seqR

P7-index30-seqR

P7-index31-seqR

P7-index32-seqR

P7-index33-seqR

P7-index34-seqR

P7-index35-seqR

P7-index36-seqR

P7-index37-seqR

P7-index38-seqR

P7-index39-seqR

Table 2 (continued)

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CAAGCAGAAGACGGCATACGAGATGTCGTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCGATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGCTGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATATTATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATGAATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTCGGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATCTTCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

CAAGCAGAAGACGGCATACGAGATTGCCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

P7-index41-seqR

P7-index42-seqR

P7-index43-seqR

P7-index44-seqR

P7-index45-seqR

P7-index46-seqR

P7-index47-seqR

P7-index48-seqR

MiSeq P5/P7 adapters are indicated in italics, index sequences in bold underline and universal tag sequences annealing to first-round PCR products in regular type

a

CAAGCAGAAGACGGCATACGAGATTCTGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

P7-index40-seqR

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11. 50× TAE buffer (per L): 242 g (2 M) Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA solution in ultrapure H2O. Store at room temperature. Dilute 1:50 in H2O to prepare 1× TAE buffer. 12. 1% (w/v) agarose gel, prepared in 1× TAE buffer. 13. PureLink® PCR purification kit (Life Technologies, Carlsbad, CA, USA). 14. Phusion® High-Fidelity DNA polymerase with Phusion HF buffer and MgCl2 (Thermo-Fisher Scientific). 15. QIAquick® gel extraction kit (Qiagen). 16. Dark Reader® transilluminator, or similar instrument. 17. GelGreen™ nucleic acid gel stain (Biotium, Fremont, CA, USA). 18. Agencourt® AMPure® XP beads (Beckman Coulter, Brea, CA, USA). 19. 70% (v/v) ethanol. 20. Sterile ultrapure H2O. 21. NanoDropⓇ instrument.

ND-1000

spectrophotometer,

or

similar

22. Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), or similar instrument. 23. MiSeq Sequencing System (Illumina, San Diego, CA, USA). 24. 500-cycle Reagent Kit V2 or 600-cycle Reagent Kit V3 (Illumina). 2.1.5

Data Analysis

1. FastQC version quality control tool [25], version 0.11.5. Available online at http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/. 2. FLASH paired-end read overlapping tool [26], version 1.2.11. Available online at https://ccb.jhu.edu/software/FLASH/. 3. FASTX toolkit, including FASTQ quality filtering tool [27], version 0.0.14. Available online at http://hannonlab.cshl.edu/ fastx_toolkit/. 4. R version 3.3.2, including packages “seqinr” and “stringdist.” Available online at https://cran.r-project.org/.

2.2 Identification of sdAbs Against Prespecified Epitopes 2.2.1

Isolation of PBMCs

All materials and reagents are listed under Subheading 2.1.1 (except for LympholyteⓇ-M density separation medium, which is used for mouse PBMC isolation).

NGS for sdAb Discovery & Engineering 2.2.2 RNA Extraction and cDNA Synthesis

All materials and reagents are listed in Subheading 2.1.2.

2.2.3 Construction of Phage-Displayed sdAb Libraries

See Subheading 3.2.3 and references therein.

2.2.4 Panning of PhageDisplayed sdAb Libraries with Competitive Elution

501

1. Micropipettes (P10, P20, P200, P1000) and sterile filter tips. 2. 1.5 mL microcentrifuge tubes. 3. Cryopreserved TGF-β3 immune sdAb library cells prepared as described under Subheading 3.2.3. 4. 2×YT broth and agar (per L): 16 g tryptone, 10 g yeast extract, 5 g NaCl dissolved in ultrapure H2O. For agar plates, add 15 g agar. Sterilize by autoclaving, cool to ~50 °C, and add filtersterilized ampicillin to a final concentration of 100 μg/mL if desired. 5. 100 mg/mL kanamycin B sulfate salt stock solution prepared in ultrapure H2O. Sterilize by passing through a 0.22 μm filter and store at -20 °C. 6. 50 mg/mL ampicillin disodium salt stock solution prepared in ultrapure H2O. Sterilize by passing through a 0.22 μm filter and store at -20 °C. 7. 2 M/36% (w/v) glucose (per L): 360 g glucose dissolved in ultrapure H2O. Sterilize by passing through a 0.22 μm filter and store at room temperature. 8. M13KO7 helper phage. 9. GENESYSTM 20 visible spectrophotometer (Thermo-Fisher Scientific), or similar instrument. 10. 1.5 mL 10 mm path length disposable cuvettes. 11. 37 °C incubator with shaker. 12. J2-21M/E high-speed centrifuge, or similar instrument. 13. Stericup-GP Express® PLUS membrane filters. 14. 20% (w/v) polyethylene glycol (PEG)/2.5 M NaCl solution (per L): 200 g PEG (average molecular weight 6000 or 8000 Da), 146.1 g (2.5 M) NaCl dissolved in warm ultrapure H2O. Sterilize by autoclaving, and store at room temperature. 15. PBS (per L): 8 g (137 mM) NaCl, 0.2 g (2.37 mM) KCl, 1.44 g (10 mM) Na2HPO4, 0.24 g (1.8 mM) KH2PO4 dissolved in ultrapure H2O. Adjust pH to 7.4 with HCl, sterilize by autoclaving, and store at room temperature. 16. Escherichia coli strain TG1 glycerol stock. 17. 5× M9 salts (per L): 64 g Na2HPO4·7H2O, 15 g KH2PO4, 2.5 g NaCl, 5.0 g NH4Cl dissolved in ultrapure H2O. Adjust to pH 7.4 with NaOH pellets, sterilize by autoclaving, and store at room temperature.

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18. 1 M MgSO4 solution (per L): 120.4 g MgSO4 dissolved in ultrapure H2O. Sterilize by autoclaving, and store at room temperature. 19. 1 M CaCl2 solution (per L): 219.1 g CaCl2·6H2O dissolved in ultrapure H2O. Sterilize by autoclaving, and store at room temperature. 20. M9 minimal media (per L): 200 mL 5× M9 salts, 5.5 mL 2 M/ 36% glucose, 2 mL 1 M MgSO4, 0.1 mL 1 M CaCl2. For agar plates, add 15 g agar first to 800 mL ultrapure H2O, autoclave, and cool to 50 °C. Aseptically add the components above, sterilize by passing through a 0.22 μm filter, and store at room temperature. 21. NUNC MaxiSorpTM 96-well plates. 22. Recombinant TGF-β3 (a gift from Andrew Hinck, University of Texas Health Science Center, San Antonio, TX; also available commercially from R & D Systems, Minneapolis, MN, USA). 23. PBS containing 5% (w/v) skim milk. 24. PBS containing 1% (w/v) bovine serum albumin (BSA) and 0.1% (v/v) Tween-20. 25. PBS containing 0.05% Tween-20. 26. Soluble dimeric type II TGF-β receptor ectodomain (produced as described in [28]; alternatively, a soluble dimeric type II TGF-β receptor ectodomain fused to human IgG1 Fc is commercially available from R & D Systems). 27. Anti-RSV glycoprotein F Ab (Synagis; Creative Biolabs, Shirley, NY, USA). 28. 15 mL and 50 mL Falcon tubes. 29. 50 mL high-speed centrifuge tubes. 30. 250 mL and 500 mL Ultra Yield™ flasks. 31. NanoDrop® ND-1000 spectrophotometer, or similar instrument. 2.2.5 Illumina MiSeq Sequencing

All materials and reagents are listed under Subheading 2.1.4. Note that of the 1st round primers listed in Table 1, only seqF-MJ7 and seqR-MJ8 are needed. Note that of the 2nd round primers listed in Table 2, the forward primer P5-seqF is needed, while the number of barcoded reverse primers depends on the number of samples in the experiment.

2.2.6

All materials are listed under Subheading 2.1.5.

Data Analysis

NGS for sdAb Discovery & Engineering

2.3 Direct Selection of Antigen-Specific B Cells from PBMCs and Identification of Antigen-Specific sdAbs 2.3.1

503

All materials and reagents are listed under Subheading 2.1.1. Note that in the example presented here, PBMCs are derived from a llama immunized with recombinant MBP-Int277 protein.

Isolation of PBMCs

2.3.2 Preparation of MBP-Int277-Coupled Magnetic Beads

1. MBP-Int277 protein [29] (GenScript, Piscataway, NY, USA). 2. Ovalbumin (Sigma-Aldrich). 3. Dynabeads® M-270 Epoxy (Life Technologies). 4. 0.1 M sodium phosphate buffer, pH 8.0 (per L): 2.62 g NaH2PO4•H2O,14.42 g Na2HPO4•2H2O dissolved in ultrapure H2O. Adjust pH to 8.0 with NaOH and store at room temperature. 5. 3 M ammonium sulfate in 0.1 M sodium phosphate buffer (per L): dissolve 396 g [NH4]2SO4 in 0.1 M sodium phosphate buffer, pH 8.0. Store at room temperature. 6. PBS containing 0.5% BSA. 7. Magnetic separation rack. 8. Tube rotator. 9. 1.5 mL microcentrifuge tubes. 10. Vortex mixer. 11. Analytical balance.

2.3.3 Positive Selection of MBP-Int277-Reactive B cells

1. PBS containing 0.1% BSA. 2. 1.5 mL microcentrifuge tubes. 3. 4 °C refrigerator. 4. Magnetic separation rack. 5. Refrigerated microcentrifuge. 6. MBP-Int277-coupled and ovalbumin-coupled magnetic beads prepared under Subheading 3.3.2. using materials under Subheading 2.3.2.

2.3.4 RNA Extraction and cDNA Synthesis

All materials and reagents are listed under Subheading 2.1.2.

2.3.5 Illumina MiSeq Sequencing

All materials and reagents are listed under Subheading 2.1.4. In addition, the following materials are required: 1. Primer MJ1 (5’-GCC CAG CCG GCC ATG GCC SMK GTG CAG CTG GTG GAK TCT GGG GGA-3’). 2. Primer MJ2 (5’-GCC CAG CCG GCC ATG GCC CAG GTA AAG CTG GAG GAG TCT GGG GGA-3’).

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3. Primer MJ3 (5’-GCC CAG CCG GCC ATG GCC CAG GCT CAG GTA CAG CTG GTG GAG TCT-3’). 4. Primer CH2 (5’-CGCCATCAAGGTACCAGTTGGA-3’). 5. Primer CH2b3 (5’-GGGGTACCTGTCATCCACGGACCAG CTGA-3’). Note that of the first round primers listed in Table 1, only seqFMJ1, SeqF-MJ2, SeqF-MJ3, seqR-CH2, and seqR-CH2b3 are needed. Note that of the second round primers listed in Table 2, the forward primer P5-seqF is needed, while the number of barcoded reverse primers depends on the number of samples in the experiment. 2.3.6

Data Analysis

2.4 Affinity Maturation of sdAbs Using NGS 2.4.1 Construction of Random sdAb Mutagenesis Libraries

All materials are listed under Subheading 2.1.5. 1. Micropipettes (P10, P20, P200, P1000) and sterile filter tips. 2. 1.5 mL microcentrifuge tubes. 3. DNA encoding sdAb of interest (e.g., a PCR amplicon, sdAbdisplaying phage or phagemid clone, or expression vector containing sequences encoding a sdAb of interest). In this chapter, the sdAb is the Clostridioides difficile-specific camelid VHH A26.8. 4. QIAprep® spin miniprep kit (Qiagen). 5. NanoDrop® instrument.

ND-1000

spectrophotometer,

or

similar

6. Platinum® Taq DNA polymerase with 10× PCR buffer and 50 mM MgCl2 (Thermo Fisher). 7. dNTP mix, 10 mM each. 8. Diversify™ PCR Random Mutagenesis Kit (Takara Bio, Mountain View, CA, USA). 9. Primer MJ1 (5′-GCC CAG CCG GCC ATG GCC SMK GTG CAG CTG GTG GAK TCT GGG GGA-3′). 10. Primer MJ2 (5′-GCC CAG CCG GCC ATG GCC CAG GTA AAG CTG GAG GAG TCT GGG GGA-3′). 11. Primer MJ3 (5′-GCC CAG CCG GCC ATG GCC CAG GCT CAG GTA CAG CTG GTG GAG TCT-3′). 12. Primer MJ7 (5′-CAT GTG TAG ACT CGC GGC CCA GCC GGC CAT GGC C-3′). 13. Primer MJ8 (5′-CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCT GG-3′). 14. Sterile ultrapure or molecular biology-grade H2O. 15. 0.2 mL PCR tubes, strips, or plates. 16. GeneAmp® PCR System 9700 thermal cycler, or similar instrument.

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17. Agarose gel electrophoresis equipment and power supply. 18. 0.5 M EDTA stock solution (see Subheading 2.1.1 for preparation). 19. 50× TAE buffer (see Subheading 2.1.4 for preparation). 20. 1% agarose gel, prepared in 1× TAE buffer. 21. PureLink® PCR purification kit. 22. pMED1 phagemid vector [30], other phagemid vector, or phage vector. 23. FastDigest® SfiI, PstI, and XhoI restriction enzymes (Thermo Fisher). 24. T4 DNA ligase (Life Technologies). 25. E. coli TG1 electrocompetent cells (Stratagene, La Jolla, CA, USA). 26. Disposable electroporation cuvettes, 0.2 cm gap width. 27. MicroPulserTM electroporator or similar instrument. 28. 2 M/36% glucose (see Subheading 2.2.4 for preparation). 29. SOC broth (per L): 20 g tryptone, 5 g yeast extract, 0.58 g NaCl, 0.19 g KCl, 0.95 g MgCl2, 1.2 g MgSO4 dissolved in ultrapure H2O. Sterilize by autoclaving, cool to ~50 °C, then add 10 mL 2 M glucose solution using sterile technique. 30. 100 mg/mL ampicillin stock solution (see Subheading 2.2.4 for preparation). 31. 2×YT broth and agar (see Subheading 2.2.4 for preparation). 32. 37 °C incubator with shaker. 33. 15 mL and 50 mL FalconTM tubes. 34. 500 mL Ultra YieldTM flasks. 35. 2.0 mL cryovials. 36. Primer M13RP (5′-CAG GAA ACA GCT ATG AC-3′). 37. Primer -96gIII (5′-CCC TCA TAG TTA GCG TAA CGA TCT-3′). 38. 80% (v/v) glycerol: 80 mL glycerol, 20 mL ultrapure H2O. Sterilize by autoclaving, and store at room temperature. 39. -80 °C freezer. 40. Eppendorf 5415R microcentrifuge, or similar instrument. 41. SorvallTM LegendTM refrigerated tabletop centrifuge with swinging bucket rotor, or similar instrument. 2.4.2 Construction of Site-Saturating sdAb Mutagenesis Libraries

1. Micropipettes (P10, P20, P200, P1000) and sterile filter tips. 2. 1.5 mL microcentrifuge tubes.

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3. A phage or phagemid clone encoding a sdAb of interest. In this example, the sdAb is the C. difficile-specific camelid VHH A26.8. 4. QIAprep® spin miniprep kit. 5. NanoDrop® instrument.

ND-1000

spectrophotometer,

or

similar

6. QuikChange II site-directed mutagenesis kit (Agilent). 7. Mutagenic primers (as shown for the model sdAb A26.8 in Table 3) designed to alter the codon encoding each targeted CDR residue to NNK, purified by polyacrylamide gel electrophoresis or HPLC. 8. Sterile ultrapure or molecular biology-grade H2O. 9. 0.2 mL PCR tubes, strips, or plates. 10. GeneAmp® PCR System 9700 thermal cycler or similar instrument. 11. E. coli TG1 electrocompetent cells. 12. Disposable electroporation cuvettes, 0.2 cm gap width. 13. MicroPulserTM electroporator, or similar instrument. 14. 2 M/36% glucose (see Subheading 2.2.4 for preparation). 15. SOC broth (see Subheading 2.4.1 for preparation). 16. 2×YT broth and agar (see Subheading 2.2.4 for preparation). 17. 100 mg/mL ampicillin stock solution (see Subheading 2.2.4 for preparation). 18. 37 °C incubator with shaker. 19. 15 mL and 50 mL FalconTM tubes. 20. 500 mL Ultra YieldTM flasks. 21. 2.0 mL cryovials. 22. 80% glycerol: 80 mL glycerol, 20 mL ultrapure H2O. Sterilize by autoclaving, and store at room temperature. 23. -80 °C freezer. 24. Eppendorf 5415R microcentrifuge, or similar instrument. 25. SorvallTM LegendTM refrigerated tabletop centrifuge with swinging bucket rotor, or similar instrument. 2.4.3 Panning of sdAb Mutagenesis Libraries

1. Micropipettes (P10, P20, P200, P1000) and sterile filter tips. 2. 1.5 mL microcentrifuge tubes. 3. Cryopreserved (-80 °C) random and site-saturating sdAb library cells prepared as described under Subheadings 3.4.1 and 3.4.2 using materials listed under Subheadings 2.4.1 and 2.4.2.

CDR2

a

Region

G74

K72

V71

S70

D69

A68

Y67

Y66

T65

S64

T63

G62

T59

S58

S57

I56

V55

Position

(continued)

CTCGTACCAGTCGAGCTAATMNNTGCTACAAACTCACGCTCCG CGGAGCGTGAGTTTGTAGCANNKATTAGCTCGACTGGTACGAG CGTACCAGTCGAGCTMNNAACTGCTACAAACTCACGCTCCGC GCGGAGCGTGAGTTTGTAGCAGTTNNKAGCTCGACTGGTACG CTCGTACCAGTCGAMNNAATAACTGCTACAAACTCACGCTCCGC GCGGAGCGTGAGTTTGTAGCAGTTATTNNKTCGACTGGTACGAG GCATAGTATGTGCTCGTACCAGTMNNGCTAATAACTGCTACAAACTCAC GTGAGTTTGTAGCAGTTATTAGCNNKACTGGTACGAGCACATACTATGC CTGCATAGTATGTGCTCGTACCMNNCGAGCTAATAACTGCTACAAAC GTTTGTAGCAGTTATTAGCTCGNNKGGTACGAGCACATACTATGCAG CATAGTATGTGCTCGTMNNAGTCGAGCTAATAACTGCTACAAACTCACG CGTGAGTTTGTAGCAGTTATTAGCTCGACTNNKACGAGCACATACTATG GAGTCTGCATAGTATGTGCTMNNACCAGTCGAGCTAATAACTGCTA TAGCAGTTATTAGCTCGACTGGTNNKAGCACATACTATGCAGACTC CACCGAGTCTGCATAGTATGTMNNCGTACCAGTCGAGCTAATAAC GTTATTAGCTCGACTGGTACGNNKACATACTATGCAGACTCGGTG CTTCACCGAGTCTGCATAGTAMNNGCTCGTACCAGTCGAGCTAAT ATTAGCTCGACTGGTACGAGCNNKTACTATGCAGACTCGGTGAAG TTCACCGAGTCTGCATAMNNTGTGCTCGTACCAGTCG CGACTGGTACGAGCACANNKTATGCAGACTCGGTGAA CCCTTCACCGAGTCTGCMNNGTATGTGCTCGTACCAG CTGGTACGAGCACATACNNKGCAGACTCGGTGAAGGG GCCCTTCACCGAGTCMNNATAGTATGTGCTCGTACCAGTCGAG CTCGACTGGTACGAGCACATACTATNNKGACTCGGTGAAGGGC GCCCTTCACCGAMNNTGCATAGTATGTGCTCGTACCAG CTGGTACGAGCACATACTATGCANNKTCGGTGAAGGGC TGGTGAACCGGCCCTTCACMNNGTCTGCATAGTATGTGCTC GAGCACATACTATGCAGACNNKGTGAAGGGCCGGTTCACCA ATGGTGAACCGGCCCTTMNNCGAGTCTGCATAGTATG CATACTATGCAGACTCGNNKAAGGGCCGGTTCACCAT AGATGGTGAACCGGCCMNNCACCGAGTCTGCATAG CTATGCAGACTCGGTGNNKGGCCGGTTCACCATCT CTCTGGAGATGGTGAACCGMNNCTTCACCGAGTCTGCATAG CTATGCAGACTCGGTGAAGNNKCGGTTCACCATCTCCAGAG

Sequence (5′–3′)

Table 3 Oligonucleotide primers for creating site-saturating mutagenesis libraries of sdAb A26.8

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Y117

D116

Y115

E114

N113

P112

D112A

Q112B

L111B

R111A

T111

R110

Q109

S108

N107

Note that primers for mutagenesis of CDR2 using Kabat definition are provided

a

A105

CDR3

V106

Position

Region

Table 3 (continued)

TCTCGTACGCTGCGAATTTACMNNACAGAAATAAACGGCCGTGTC GACACGGCCGTTTATTTCTGTNNKGTAAATTCGCAGCGTACGAGA CAGTCTCGTACGCTGCGAATTMNNTGCACAGAAATAAACGGCCGT ACGGCCGTTTATTTCTGTGCANNKAATTCGCAGCGTACGAGACTG TCGTACGCTGCGAMNNTACTGCACAGAAATAAACGGCCGT ACGGCCGTTTATTTCTGTGCAGTANNKTCGCAGCGTACGA CAGTCTCGTACGCTGMNNATTTACTGCACAGAAATAAACGGCCGTG CACGGCCGTTTATTTCTGTGCAGTAAATNNKCAGCGTACGAGACTG GGGTCCTGCAGTCTCGTACGMNNCGAATTTACTGCACAGAAAT ATTTCTGTGCAGTAAATTCGNNKCGTACGAGACTGCAGGACCC TGGGGTCCTGCAGTCTCGTMNNCTGCGAATTTACTGCACAG CTGTGCAGTAAATTCGCAGNNKACGAGACTGCAGGACCCCA TTGGGGTCCTGCAGTCTMNNACGCTGCGAATTTACTGCACAGA TCTGTGCAGTAAATTCGCAGCGTNNKAGACTGCAGGACCCCAA TACTCATTGGGGTCCTGCAGMNNCGTACGCTGCGAATTTACTG CAGTAAATTCGCAGCGTACGNNKCTGCAGGACCCCAATGAGTA CTCATTGGGGTCCTGMNNTCTCGTACGCTGCGA TCGCAGCGTACGAGANNKCAGGACCCCAATGAG GTAGTCATACTCATTGGGGTCMNNCAGTCTCGTACGCTGCGAATT AATTCGCAGCGTACGAGACTGNNKGACCCCAATGAGTATGACTAC GTAGTCATACTCATTGGGMNNCTGCAGTCTCGTACGCTG CAGCGTACGAGACTGCAGNNKCCCAATGAGTATGACTAC CCCAGTAGTCATACTCATTMNNGTCCTGCAGTCTCGTACGC GCGTACGAGACTGCAGGACNNKAATGAGTATGACTACTGGG CCCAGTAGTCATACTCMNNGGGGTCCTGCAGTCTC GAGACTGCAGGACCCCNNKGAGTATGACTACTGGG CTGGCCCCAGTAGTCATAMNNATTGGGGTCCTGCAGTCTC GAGACTGCAGGACCCCAATNNKTATGACTACTGGGGCCAG CCTGGCCCCAGTAGTCMNNCTCATTGGGGTCCTGC GCAGGACCCCAATGAGNNKGACTACTGGGGCCAGG CCTGGCCCCAGTAMNNATACTCATTGGGGTCCTGCA TGCAGGACCCCAATGAGTATNNKTACTGGGGCCAGG GTCCCCTGGCCCCAMNNGTCATACTCATTGGGGTCC GGACCCCAATGAGTATGACNNKTGGGGCCAGGGGAC

Sequence (5′–3′)

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4. 2×YT broth and agar (see Subheading 2.2.4 for preparation). 5. 50 mg/mL kanamycin B stock solution (see Subheading 2.2.4 for preparation). 6. 100 mg/mL ampicillin stock solution (see Subheading 2.2.4 for preparation). 7. 2 M/36% glucose (see Subheading 2.2.4 for preparation). 8. M13KO7 helper phage. 9. GENESYSTM instrument.

20

visible

spectrophotometer

or

similar

10. 1.5 mL 10 mm path length disposable cuvettes. 11. 37 °C incubator with shaker. 12. J2-21M/E high-speed centrifuge, or similar instrument. 13. Stericup-GP Express® PLUS membrane filters. 14. 20% PEG/2.5 M NaCl solution (see Subheading 2.2.4 for preparation). 15. PBS (see Subheading 2.1.1 for preparation). 16. E. coli strain TG1 glycerol stock. 17. 5× M9 salts (see Subheading 2.2.4 for preparation). 18. 1 M MgSO4 solution (see Subheading 2.2.4 for preparation). 19. 1 M CaCl2 solution (see Subheading 2.2.4 for preparation). 20. M9 minimal media (see Subheading 2.2.4 for preparation). 21. Nunc MaxiSorpTM 96-well plates. 22. Recombinant streptavidin. 23. EZ-Link™ N-hydroxysulfosuccinimide (Thermo Fisher).

(NHS)-biotin

24. 1 M Tris–HCl (per L): 121.1 g Tris base dissolved in ultrapure H2O. Adjust pH to 8.0 with HCl, sterilize by autoclaving, and store at room temperature. 25. 3.5 kDa MWCO dialysis tubing. 26. Casein powder, biotin-free. 27. C. difficile toxin A (TcdA; List Biological Laboratories, Campbell, CA, USA). 28. PBS containing 0.1% Tween-20. 29. 100 mM triethylamine (per mL): 14 μL ≥99% triethylamine in ultrapure H2O. The pH of this solution will be approximately 11. 30. 100 mM glycine (per L): 7.5 g glycine dissolved in ultrapure H2O. Adjust pH to 2.0 using concentrated HCl. 31. 15 mL and 50 mL FalconTM tubes. 32. 250 mL Ultra

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2.4.4 Illumina MiSeq Sequencing

All materials and reagents are listed under Subheading 2.1.4. Note that of the 1st round primers listed in Table 1, only seqF-MJ7 and seqR-MJ8 are needed. Note that of the 2nd round primers listed in Table 2, the forward primer P5-seqF is needed, while the number of barcoded reverse primers depends on the experiment.

2.4.5

All materials are listed under Subheading 2.1.5.

3

Data Analysis

Methods

3.1 Core NGS Workflow

3.1.1

Isolation of PBMCs

In the protocols that follow, we describe workflows for interrogating repertoires of single immunoglobulin variable domains (VH, VL, or VHH) using Illumina MiSeq sequencing, both directly from lymphocytes as well as from phage-displayed libraries. While many of the applications we highlight pertain to phage-displayed sdAb libraries, we also include methods for sequencing directly from lymphocytes; these are valuable both for investigations of in vivo immune responses as well as for evaluating the quality of phagedisplayed libraries constructed from natural sources of diversity (Fig. 2). 1. Collect whole blood samples directly into citrated tubes using a sterile hypodermic needle of appropriate length and gauge (see Note 4). 2. Dilute blood samples with two volumes of ice-cold PBS containing 2 mM EDTA. 3. Gently layer 35 mL of diluted blood sample onto 15 mL HistopaqueⓇ-1077 or Ficoll-PacqueⓇ PLUS (human and llama blood samples) or LympholyteⓇ-M (mouse blood samples) in a 50 mL Falcon tube. Centrifuge at 400× g for 30 min at room temperature with no brake. 4. Using a Pasteur pipette, carefully aspirate and discard the topmost plasma layer. 5. Using a Pasteur pipette, carefully aspirate the PBMC layer (interphase) and transfer to a new 50 mL Falcon tube containing 30 mL PBS supplemented with 2 mM EDTA. Centrifuge at 200× g for 10 min at room temperature, then carefully remove supernatant. Repeat this step twice. 6. Prior to final wash, count cells to estimate density and viability (should be ≥95%; see Note 5). 7. Resuspend cells in sterile freeze medium (90% (v/v) fetal bovine serum, 10% (v/v) DMSO) equilibrated to room temperature at a density of 5–10 × 106 cells/mL. Aliquot into cryovials.

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Fig. 2 Evaluation of the potential effects of bias on VH domains sequenced directly from human B cells. (a) A rearranged IGHV3-encoded VH domain-containing plasmid was doped into cDNA derived from 104 CD19+ human B cells at the indicated ratios (assuming 300 copies of immunoglobulin heavy-chain mRNA per cell), then VHs were amplified and sequenced as described in this chapter. Recovery rates of the plasmid-encoded VH show no major deviation from the expected frequency. (b) IGHV gene family usage from three different subpopulations of CD19+ B cells from a single individual, sequenced as described in this chapter. Data are representative of approximately 50,000–200,000 sequences per B-cell pool. Each subpopulation has widely different gene usage, indicating that all of the multiplexed PCR primers are able to amplify their targets

8. Place cryovials inside a Mr. FrostyTM freezing container containing fresh isopropanol, and place in -80 °C freezer overnight. 9. The next day, move cells on dry ice to liquid nitrogen storage. 10. To thaw cells, move cryovials as quickly as possible from liquid nitrogen storage into a 37 °C water bath. Gently agitate until no visible ice crystals remain, then add to 15 mL warm RPMI media supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a 50 mL Falcon tube. Pellet cells at 200× g for 5 min at room temperature, then resuspend in PBS or other isotonic buffer (see Note 6). 3.1.2 RNA Extraction and cDNA Synthesis

1. Prior to commencing work, spray work area, micropipettes and gloves with RNase AWAYTM. Extract total cellular RNA from ≥104–106 PBMCs or ≥102–105 purified B-cells and elute in

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30 μL RNase-free TE buffer. Store RNA at -80 °C if not using immediately for reverse transcription. 2. Measure RNA concentration and A260/A280 ratio using a spectrophotometer (see Note 7). 3. In a 0.2 mL PCR tube, combine 16 μL RNA with 4 μL qScriptTM cDNA supermix. In a thermal cycler, incubate at 25 °C for 5 min, 42 °C for 1 h, then 85 °C for 5 min. Store cDNA at -20 °C. 3.1.3 Construction and Panning of PhageDisplayed Single-Domain Antibody Libraries

Detailed protocols for constructing, rescuing, and panning phagedisplayed camelid VHH libraries [24, 30] and human synthetic VH/ VL libraries [31, 32] have been previously published.

3.1.4 Illumina MiSeq Sequencing

High-throughput sequencing of phage-displayed sdAb libraries during all stages of their construction (at the DNA level, prior to transformation of E. coli; from phage or phagemid DNA in E. coli cells; from phage or phagemid DNA encapsulated in the phage virion) as well as during their selection can yield highly valuable information. Both phage particles themselves, phage/phagemid single-stranded DNA and phage/phagemid replicative form DNA isolated from E. coli cells are appropriate templates for NGS. We caution that phage particles can inhibit PCR, and can be successfully amplified only within a relatively narrow window of ~105–107 particles per PCR reaction. We also caution that replicative form DNA isolated from infected E. coli cells (e.g., overnight cultures for the purpose of phage amplification) will reflect growth advantages conferred by library variants, which can significantly bias the composition of synthetic sdAb libraries. 1. Amplify genes encoding rearranged VH/VL/VHH domains in 25 μL PCR reactions containing 1× ABI Buffer II, 1.5 mM MgCl2, 200 μM each dNTP, 5 pmol each primer or primer mixture from Table 1, 1 U of AmpliTaq Gold DNA polymerase and 1 μL of template. For amplification directly from lymphocytes, equimolar mixtures of all forward primers annealing in FR1 for a given locus (e.g., human igh) should be used in a single reaction. Depending on the application, either a single reverse primer or a mixture of isotype-specific primers annealing in constant regions may be used. Template may be cDNA derived from lymphocytes, single-stranded or replicative form DNA (1–100 ng/μL) or phage particles (~106 particles/μL). Cycle the reactions as follows: 95 °C for 7 min; 35 cycles of (94 °C for 30 s, 55 °C for 45 s, and 72 °C for 2 min); 72 °C for 10 min. 2. Electrophorese 5 μL aliquots of the PCRs in 1% agarose gels in TAE buffer and confirm amplification of a ~400 bp band.

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3. Purify the amplicons using a PureLink® PCR purification kit (300 bp cutoff; see Note 8). 4. Conduct second round “tagging” PCRs in 50 μL reaction volumes containing 1× Phusion HF Buffer, 1.5 mM MgCl2, 200 μM each dNTP, 10 pmol of each primer pair from Table 2 (e.g., P5-seqF and P7-index1-seqR; see Note 9), 0.25 U Phusion High-Fidelity DNA polymerase, and 5 μL of first-round PCR as template. Cycle the reactions as follows: 98 °C for 30 s; 20 cycles of (98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s); 72 °C for 5 min. 5. Electrophorese 5 μL aliquots of the PCRs in 1% agarose gels in TAE buffer and confirm amplification of a ~450–500 bp band. 6. Pool equal volumes of all second-round amplicons (see Notes 10 and 11) and purify using a PureLink® PCR purification kit (300 bp cutoff). Subsequently, gel purify the pooled library from a 1% agarose gel in TAE buffer using a QIAquick® gel extraction kit and elute in 50 μL EB. Perform final cleanup of the pooled NGS library using 90 μL of AMPure XP beads and elute in 20 μL ultrapure H2O. 7. Measure pooled amplicon purity and concentration using a High Sensitivity DNA Analysis kit on a BioAnalyzer 2100 instrument (Fig. 3a). 8. Sequence the pooled amplicons on an Illumina MiSeq Sequencing System using a 500-cycle Reagent Kit V2 or 600-cycle Reagent Kit V3 and a ≥5% PhiX genomic DNA spike. Diluting the amplicons to ~7–8 pM should yield a cluster density between 800–1000 K/mm2. 3.1.5

Data Analysis

Construction and selection of phage-displayed antibody libraries has historically been a “black box,” with limited insight into the processes governing the generation of a library and the behavior of its members. NGS provides a window into these processes by allowing us to sample a significant proportion of library diversity over the course of various types of manipulations. Template R scripts enabling the following analyses can be obtained from the authors by request. 1. Visualize per-base quality scores for forward and reverse reads using FastQC (Fig. 3b; see Note 12). In many cases, poor data quality cannot be compensated for in the analysis and will artificially inflate diversity estimates. 2. Merge forward and reverse paired-end reads using FLASH with default parameters. High-quality data with an appropriate degree of overlap (≥20 bp) should yield ≥90% merged sequences (see Note 13).

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Fig. 3 Quality control metrics for VH/VL/VHH amplicon sequencing using the Illumina MiSeq platform. (a) Representative Bioanalyzer 2100 trace of a VHH amplicon library produced from a phage-displayed library and purified as described in this chapter. The bulk of the DNA should be distributed around the expected library size (in this case, ~500–600 bp); the presence of smaller molecular weight bands suggests primer multimer contamination and means samples should be repurified. (b) Representative results of FastQC for amplicon sequencing of VH/VL/VHH domains on the MiSeq as described in this chapter. Results for forward paired-end reads are shown but reverse reads will be similar; quality scores decline with read length but should not dip appreciably below Q25. If quality scores fall below Q20 at the 3′ end of most reads, merging of forward and reverse reads will be inefficient and resequencing is recommended

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3. Quality filter the merged sequences using the FASTQ quality filtering tool within the FASTX toolkit. Accept only sequences with Q30 scores over ≥95% of bases in the read. Generally, this will result in discarding of ~30% of the lowest-quality reads. 4. Convert data from .fastq to .fasta format and read into R using the read.fasta function of package “seqinr.” Strip all primerencoded non-antibody sequences. 5. Translate nucleotide sequences to protein using the translate function of package “seqinr.” 6. Assess library functionality by determining the proportion of nucleotide sequences that are in-frame (Fig. 4). All in vitro display libraries, regardless of their method of construction, will contain genetic imperfections at some frequency (e.g., single nucleotide indels introduced by PCR primers). The frequency of out-of-frame library variants should remain static when phagemid library DNA is transformed into E. coli cells, assuming sufficient glucose is present to inhibit protein production, but can increase dramatically when cells are grown to high density and the library is rescued with helper phage. This phenomenon can be counteracted somewhat by rescue with M13KO7ΔpIII hyper phage, which forces pIII-sdAb expression from phagemid DNA. Large increases in the proportion of out-of-frame sequences after rescue with helper phage is indicative of strong growth advantages for cells that do not produce a potentially toxic pIII-sdAb protein; loss of diversity through this mechanism is a major challenge for synthetic libraries. 7. Using protein sequences, determine the frequency of stop codons amongst library members, and as above (for phagemid libraries), assess whether the frequency of stop codons increases when rescued with helper phage. This can readily be accomplished by pattern matching the ‘*’ character. Library members bearing stop codons are expected to be present to some degree, depending on the randomization strategy, but should represent a minor proportion of the library. 8. Using protein sequences, determine the frequency of each amino acid at each randomization position and assess the degree of deviation from planned randomization (Fig. 5a). Strong regression toward the parental residue suggests intolerance to substitution, and library redesign should be considered. 9. Determine the frequency of the most commonly observed library sequences. Immune VHH libraries are expected to have major variations in frequency, reflecting immunodominance of particular B-cell clones, but synthetic libraries should have a relatively flat landscape, and the presence of highfrequency variants (especially the parental scaffold) signifies lost library “space” and lower overall diversity.

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Fig. 4 Evaluation of phagemid sdAb library functionality using Illumina MiSeq sequencing. (a) A human VL domain was randomized synthetically in vitro and the resulting library transformed into E. coli TG1 cells, then library phage particles were rescued using M13KO7 helper phage. The randomization design called for three CDR3 lengths (DNA size shown with black arrows). Out-of-frame variants are rare in the library but very

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10. Determine the proportion of library members with a given number of randomized residues using the stringdist function of package “stringdist” (Fig. 5b). For synthetic antibody libraries with high theoretical diversity that are interrogated to limited depth, this analysis is more robust to the effects of sequencing error and undersampling than more complex measures of diversity. 11. Reduce the analysis to only regions of highest sequence diversity (for instance, CDR3 for VHH libraries, or randomization positions for synthetic libraries) by parsing these regions to new data objects. This is done to reduce the impact of sequencing error for longer sequences on subsequent analyses. 12. Measure enrichment of library variants over the course of one or more types of selection (Fig. 6). The observed foldenrichments will depend both on the library and the selection, but minimally should be ≥10. Carefully defined analyses of such data are strongly predictive of antibody phenotype. 3.2 Identification of sdAbs Against Prespecified Epitopes

This workflow describes the identification of sdAbs from an immune VHH library targeting a prespecified epitope (the site of interaction between TGF-β3 and the type II TGF-β receptor). The strategy requires the availability of an affinity reagent targeting the desired epitope.

3.2.1

See Subheading 3.1.1 for PBMC isolation protocol.

Isolation of PBMCs

3.2.2 RNA Extraction and cDNA Synthesis

See Subheading 3.1.2 for RNA extraction and cDNA synthesis protocol.

3.2.3 Construction of Phage-Displayed sdAb Libraries

Detailed protocols for constructing, rescuing, and panning phagedisplayed camelid VHH libraries [24, 30] and human synthetic VH/ VL libraries [31, 32] have been previously published.

3.2.4 Panning of PhageDisplayed sdAb Libraries with Competitive Elution

VHH-displaying phage are rescued from the libraries following superinfection with M13KO7 helper phage. The resulting library phage are allowed to bind to immobilized antigen (TGF-β3) and then competitively eluted with either a known affinity reagent

ä Fig. 4 (continued) common in the rescued phage, suggesting that E. coli cells producing in-frame library protein are at a significant growth disadvantage. (b) A dromedary VHH domain was randomized synthetically in vitro and the resulting library transformed into E. coli TG1 cells, then library phage particles were rescued using either M13KO7 helper phage or M13KO7ΔpIII hyper phage. The randomization design called for a single CDR3 length (DNA size shown with black arrow). Out-of-frame VHHs make up the large majority of the helper phage-rescued library, but a minor proportion of the hyper phage-rescued library. (c) An immune VHH phagemid library was constructed from the lymphocytes of an immunized llama, transformed into E. coli TG1 cells and then rescued with M13KO7 helper phage. Through all steps, changes in the relative frequencies of individual VHHs were measured. Frequencies are stable for most VHHs in the library (red parentheses: 95% of data; blue parentheses: 75% of all data), with slightly larger bias introduced at the library construction step compared to the phage rescue step

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Fig. 5 Evaluation of phage-displayed sdAb library diversity using Illumina MiSeq sequencing. (a) A dromedary VHH domain was randomized synthetically in vitro and the resulting library transformed into E. coli TG1 cells. The randomization design called for full randomization (20 amino acids) at six positions. A significant bias toward the parental residue is observed at four positions (P2, P3, P4, P5). (b) A human VL domain was randomized synthetically in vitro and the resulting library transformed into E. coli TG1 cells. The design of the library called for full randomization of up to 16 residues and partial randomization of two additional residues. The proportion of library members bearing the indicated number of randomized positions (‘NGS data’) is shown, along with the expected distribution for a library reaching the theoretical maximum diversity under this design (‘Theoretical’, 2 × 2 × 2016 members)

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Fig. 6 Enrichment of antigen-specific VHHs over the course of a selection experiment. An immune VHH phagemid library was constructed from the lymphocytes of a llama immunized with three antigens, transformed into E. coli TG1 cells, and then rescued with M13KO7 helper phage. Individual VHHs making up the library are shown as rectangles, with size proportional to their frequency in the library. Colored squares represent antigen-specific VHHs that were isolated and characterized after several rounds of selection. Many of these VHHs can be identified by their enrichment after a single round of selection, instead of the conventional strategy of waiting for them to rise to high frequency after multiple rounds of panning

targeting a desired epitope (in this case study, soluble dimeric type II TGF-β receptor ectodomain) or an irrelevant competitor.

1. Inoculate 200 mL of 2×YT containing 100 μg/mL ampicillin and 2% glucose in a 500 mL flask with 0.5 mL of cryopreserved library cells (~5 × 1010 cells; see Subheadings 2.1.3 and 3.1.3 and references therein for library construction protocols). Grow at 37 °C with 250 rpm shaking until OD600 reaches ~0.5. 2. Using the OD600 measurement, calculate the number of cells (1 OD600 = ~8 × 108 cells/mL) and add a 20-fold excess of M13KO7 helper phage. Incubate at 37 °C without shaking for 30 min, then at 37 °C with 250 rpm shaking for 30 min. 3. Pellet cells in 50 mL Falcon tubes by centrifugation at 5000× g for 10 min. Resuspend cells in 200 mL of 2×YT containing 100 μg/mL ampicillin and 50 μg/mL kanamycin in a 500 mL flask. Grow overnight at 37 °C with 250 rpm shaking. 4. The next morning, pellet cells by centrifugation at 5000× g for 10 min. Filter the supernatant through a 0.22 μm Stericup-GP Express® PLUS filter. In 50 mL centrifuge tubes, add 1/5 the volume of 20% PEG/2.5 M NaCl, and invert several times to thoroughly mix. Incubate on ice for 1 h.

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5. Centrifuge the tubes at 10,000× g for 30 min. Resuspend pellets in a total volume of 1 mL PBS. Transfer to a microcentrifuge tube, centrifuge at maximum speed for 2 min, and collect the supernatant. 6. Determine library phage concentration spectrophotometrically using the formula: virions/mL = (A269 - A320)·6 × 1016/ number of bp in phage or phagemid genome. 7. Coat wells of Nunc MaxiSorpTM plates overnight at 4 °C with 10 μg of TGF-β3 in 35 μL of PBS. Ensure to coat at least 1 well for the active competitor and 1 well for the irrelevant competitor (see Note 14). 8. Aspirate wells and block with 200 μL of PBS containing 5% skim milk for 1 h at 37 °C. 9. Add ~1012 library phage particles to each well, diluted in PBS containing 1% BSA and 0.1% Tween-20, and incubate at room temperature for 2 h. 10. Wash wells five times with PBS containing 0.05% Tween-20. 11. Wash wells five times with PBS. 12. To elute, add 50 μL PBS containing 100 μg/mL soluble dimeric type II TGF-β receptor ectodomain or 50 μL PBS containing 100 μg/mL anti-RSV glycoprotein F Ab (Synagis). Incubate at RT for 1 h. 13. Add half (50 μL) of the eluted phage from step 12 to 2 mL of log-phase E. coli TG1 cells (OD600 = 0.3–0.4) in a 15 mL Falcon tube. Incubate at 37 °C for 30 min without shaking, then for 30 min with 250 rpm shaking. 14. Add the 2 mL infected E. coli TG1 cells to 10 mL 2×YT containing 100 μg/mL ampicillin in a 50 mL Falcon tube. Incubate at 37 °C for 1 h with 250 rpm shaking. 15. Add 1011 M13KO7 helper phage and incubate at 37 °C for 30 min without shaking, then for 30 min with 250 rpm shaking. 16. Transfer the superinfected cells to 90 mL of 2×YT containing 100 μg/mL ampiciillin in a 250 mL flask, then add 50 μg/mL kanamycin. Grow overnight at 37 °C with 250 rpm shaking. 17. Repeat steps 4 through 16 to perform rounds 2 and 3 of panning, ensuring to save half of the eluted phage from each round. Store the eluted phage at -20 °C. 3.2.5 Illumina MiSeq Sequencing

See Subheading 3.1.4 for Illumina MiSeq sequencing protocol. Use primers seqF-MJ7 and seqR-MJ8 for first round PCR amplifications of unselected library phage, phage eluted from immobilized TGF-β3 using active competitor (soluble dimeric type II TGF-β receptor ectodomain), and phage eluted from immobilized

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TGF-β3 using irrelevant competitor. While a single round of panning is typically sufficient to detect enrichment of binders targeting epitopes overlapping that of the affinity reagent used for competitive elution, performing additional rounds of panning and sequencing the eluted phage can increase confidence in the results. 3.2.6

Data Analysis

Refer to Subheading 3.1.5 for general considerations for data analysis. 1. Visualize per-base quality scores for forward and reverse reads using FastQC. In many cases, poor data quality cannot be compensated for in the analysis and will artificially inflate diversity estimates. 2. Merge forward and reverse paired-end reads using FLASH with default parameters. 3. Quality filter the merged sequences using the FASTQ quality filtering tool within the FASTX toolkit. Accept only sequences with Q30 scores over ≥95% of bases in the read. 4. Convert data from .fastq to .fasta format and read into R using the read.fasta function of package “seqinr.” Strip all primerencoded non-antibody sequences. 5. Translate nucleotide sequences to protein using the translate function of package “seqinr.” 6. Reduce the analysis to CDR3 sequences only by parsing the conserved N-terminal amino acid consensus sequences (YYC); the C-terminus of CDR3 is a constant 10 amino acid residues away from FR4 in the PCR amplification protocol, and somatic insertions or deletions in FR4 are rare. 7. Measure enrichment of library variants over the course of competitive elution and irrelevant elution by dividing the frequency of each CDR3 sequence in the post-selection sdAbphage by its frequency in the unselected library (Fig. 7). For immune VHH libraries, a signal of enrichment driven by dissociation of high-frequency binders (especially those with fast off-rates) may be apparent in the irrelevant competitor selection. Therefore, identify sdAb CDR3 sequences showing the highest differences in enrichment comparing the active and irrelevant competitors.

3.3 Direct Selection of Antigen-Specific B Cells from PBMCs and Identification of Antigen-Specific sdAbs

This workflow describes the identification of antigen-specific sdAb sequences by comparative NGS analysis of B cells within unselected PBMCs and following positive selection on antigen-coupled magnetic beads, as well as simultaneous mock selection on irrelevant antigen-coupled magnetic beads. The source of PBMCs should be an animal immunized with the target antigen of interest and in which a vigorous serum antibody response is evident. In this case, study triplicate positive and triplicate mock selections are performed.

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Fig. 7 Enrichment of sdAb sequences in single-round panning against TGF-β3 following competitive elution with soluble dimeric type II TGF-β receptor ectodomain or an irrelevant competitor (Synagis). (a) Fold enrichment following competitive and irrelevant elution. (b) Differences in fold enrichment calculated as: fold enrichment (soluble dimeric type II TGF-β receptor ectodomain elution) minus fold enrichment (Synagis elution) 3.3.1

Isolation of PBMCs

3.3.2 Preparation of MBP-Int277-Coupled Magnetic Beads

See Subheading 3.1.1 for PBMC isolation protocol. Note that antigen-coupled magnetic beads should be prepared and ready for selection prior to PBMC isolation (if using fresh PBMCs) or prior to PBMC thawing (if using cryopreserved PBMCs). 1. For each target selection to be performed, weigh 3 mg of Dynabeads® M-270 Epoxy from the tube (approximately 2 × 108 beads) and place in a 1.5 mL microcentrifuge tube. In this case study two different target selections are performed, 6 mg of beads are used. 2. Resuspend the 6 mg beads in 1 mL of 0.1 M sodium phosphate buffer, pH 8.0. Vortex to mix and place on tube rotator for 10 min at room temperature. 3. Using the magnetic rack, remove the buffer from the beads. Resuspend in 120 μL of 0.1 M sodium phosphate buffer, pH 8.0. At this stage, split the beads into two 1.5 mL microcentrifuge tubes (Tube A and Tube B). 4. To Tube A, add 60 μg of MBP-Int277 protein in 60 μL of PBS (see Note 15). To Tube B, add 60 μg of ovalbumin in 60 μL of PBS. 5. To both Tubes A and B, add 60 μL of 3 M ammonium sulfate in 0.1 M sodium phosphate buffer. Vortex to mix and place on tube rotator for 20 h at room temperature. 6. The next day, wash the beads in both Tube A and Tube B four times with 1 mL of PBS containing 0.5% BSA using the magnetic rack. 7. Resuspend the beads in both Tube A and Tube B in 100 μL of PBS containing 0.5% BSA. The beads are now ready to use.

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1. Isolate or thaw cryopreserved PBMCs as described under Subheading 3.1.1. Count the cells and assess viability (see Note 16). In this protocol, 5 × 106 cells are used per selection (6 selections, 3 × 107 PBMCs). 2. Wash PBMCs three times in 1 mL of PBS containing 0.1% BSA in a refrigerated microcentrifuge. Use gentle centrifugation to pellet cells (200× g, 5 min, 4 °C). 3. Resuspend PBMCs at 5 × 106 cells/mL in PBS containing 0.1% BSA. Set aside approximately 5 × 106 PBMCs on ice; these are the unselected PBMCs. 4. Prepare three 1.5 mL microcentrifuge tubes for positive selection with MBP-Int277 coupled magnetic beads. To each tube add 5 × 106 PBMCs (1 mL). Then, to each tube add 33 μL (1 mg) of MBP-Int277 coupled magnetic beads prepared under Subheading 3.3.2 (Tube A). 5. Prepare three 1.5 mL microcentrifuge tubes for mock selection with ovalbumin-coupled magnetic beads. To each tube add 5 × 106 PBMCs (1 mL). Then, to each tube add 33 μL (1 mg) of ovalbumin-coupled magnetic beads prepared under Subheading 3.3.2 (Tube B). 6. Rotate tubes for 30 min at 4 °C. 7. Wash the magnetic beads in each tube three times with 1 mL of PBS containing 0.1% BSA using the magnetic rack.

3.3.4 RNA Extraction and cDNA Synthesis

See Subheading 3.1.2 for RNA extraction and cDNA synthesis protocol. Note that lysis buffer is added directly to magnetic beads, which are then removed using the magnetic rack prior to subsequent steps.

3.3.5 Illumina MiSeq Sequencing

See Subheading 3.1.4 for Illumina MiSeq sequencing protocol. However, note that three additional steps are required prior to beginning step 1 of Subheading 3.1.4. 1. Amplify genes encoding rearranged VH/VHH domains in two 25-μL PCR reactions. The first PCR reaction contains 1× ABI Buffer II, 1.5 mM MgCl2, 200 μM each dNTP, 1.7 pmol primer MJ1, 1.7 pmol primer MJ2, 1.8 pmol primer MJ3, 5 pmol primer CH2, 1 U of AmpliTaq Gold DNA polymerase, and 1 μL of cDNA from Subheading 3.3.4. Cycle the reaction as follows: 95 °C for 7 min; 35 cycles of (94 °C for 30 s, 55 °C for 45 s, and 72 °C for 2 min); 72°C for 10 min. The second PCR reaction is identical but replace primer CH2 with primer CH2b3.

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2. Electrophorese 5 μL aliquots of the PCRs in 1% agarose gels in TAE buffer. The first PCR reaction (MJ1, MJ2, MJ3 + CH2) should produce two bands at ~600 bp and ~850 bp. The second PCR reaction (MJ1, MJ2, MJ3 + CH2b3) should produce one predominant band at ~650 bp. 3. If positive, gel purify the 600 bp band of the first PCR (CH2 primer) and the 650 bp band of the second PCR (CH2b3 primer) from a 1% agarose gel in TAE buffer using a QIAquick® gel extraction kit and elute in 50 μL EB buffer. Use this purified amplicon as template for NGS. For preparation of amplicon libraries for Illumina MiSeq sequencing under Subheading 3.1.4, use an equimolar mixture of forward primers seqF-MJ1, seqF-MJ2, and seqF-MJ3 in combination with the reverse primer seqR-MJ8 for 1st round PCR amplification. 3.3.6

Data Analysis

Refer to Subheading 3.1.5 for general considerations for data analysis. 1. Visualize per-base quality scores for forward and reverse reads using FastQC. In many cases, poor data quality cannot be compensated for in the analysis and will artificially inflate diversity estimates. 2. Merge forward and reverse paired-end reads using FLASH with default parameters. 3. Quality filter the merged sequences using the FASTQ quality filtering tool within the FASTX toolkit. Accept only sequences with Q30 scores over ≥95% of bases in the read. 4. Convert data from .fastq to .fasta format and read into R using the read.fasta function of package “seqinr.” Strip all primerencoded non-antibody sequences. 5. Translate nucleotide sequences to protein using the translate function of package “seqinr.” 6. Reduce the analysis to CDR3 sequences only by parsing the conserved N-terminal amino acid consensus sequences (YYC); the C-terminus of CDR3 is a constant 10 amino acid residues away from FR4 in the PCR amplification protocol, and somatic insertions or deletions in FR4 are rare. 7. For each of the triplicate positive selections and mock selections, measure enrichment of library variants by dividing the frequency of each CDR3 sequence in the post-magnetic bead selection B cells by its frequency in B cells among unselected PBMCs (Table 4). Antigen-specific sdAbs should demonstrate enrichment on antigen-coupled beads but not on irrelevant antigen-coupled beads (see Note 17).

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Table 4 Frequencies of sdAbs in the unselected B-cell repertoire and post-selection on antigen-coupled magnetic beads Post-selection MBP-Int277

Ovalbumin

sdAb

Unselected PBMCs

VHH1

2.8 × 10-6

3.1 × 10-2 1.4 × 10-5 9.5 × 10-5 5.6 × 10-6 ND

VHH4

8.0 × 10-4

2.9 × 10-2 1.7 × 10-5 4.7 × 10-3 1.3 × 10-3 3.0 × 10-3 3.9 × 10-6 0.5

VHH9

ND

1.7 × 10-5 1.2 × 10-2 4.6 × 10-3 2.5 × 10-6 ND

2.0 × 10-6 1.1

6.2 × 10-3 4.2 × 10-3 ND

2.0 × 10-6 0.2

VHH10 1.4 × 10-6

Exp1

ND

Exp2

Exp3

Exp1

Exp2

ND

Exp3

KD (nM)a

5.9 × 10-6 7.1

ND not detected a Monovalent binding affinities determined by surface plasmon resonance [29]

3.4 Affinity Maturation of sdAbs Using NGS 3.4.1 Construction of Random sdAb Mutagenesis Libraries by Error-Prone PCR

To produce libraries of randomly mutagenized sdAbs (in this case, the C. difficile toxin A-specific camelid VHH A26.8 [30]), template DNA is PCR-amplified in the presence of mutagenic dNTP analogs, dGTP and Mn2+ [33]. The resulting amplicon is rapidly cloned in a single step between the SfiI sites of the pMED1 phagemid vector. 1. If required, prepare template sdAb DNA. This can be accomplished either by PCR amplification or plasmid (phagemid) purification from a single sdAb clone. Any monoclonal source of DNA is suitable, including single B cells. Purify the template sdAb DNA using the QIAprep® spin miniprep kit, eluting in 50 μL of buffer EB or H2O, and quantitate spectrophotometrically. 2. For sdAb templates that are not already cloned into pMED1 or related phagemid vector, amplify the sdAb coding region in a 50 μL PCR reaction containing 1–10 ng of template sdAb DNA, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 μM primer MJ1, MJ2 and/or MJ3 (see Note 18), 0.2 μM primer MJ8, and 0.2 μL of Platinum® Taq DNA polymerase. Cycle the reaction as follows: 94 °C for 2 min; 30 cycles of (94 °C for 30 s, 56 °C for 45 s, and 72 °C for 1 min); 72 °C for 10 min. Confirm amplification of a ~400 bp band by analytical 1% agarose gel electrophoresis in 1× TAE buffer, then purify and quantitate the amplicon as described above. This step is not required for sdAb clones in pMED1, which can be amplified directly using primers MJ7 and MJ8. 3. Amplify the template sdAb DNA from step 1 or 2 in a 50 μL mutagenic PCR reaction containing 1–10 ng of template sdAb DNA, 1× TITANIUMTM Taq buffer, 160 μM MnSO4, 80 μM dGTP, 1× Diversify® dNTP mix, 0.2 μM primer MJ7, 0.2 μM

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primer MJ8 and 1 μL TITANIUMTM Taq DNA polymerase. This corresponds to buffer condition 2 in the Diversify™ PCR Random Mutagenesis Kit. Cycle the reaction as follows: 94 °C for 30 s; 25 cycles of (94 °C for 30 s, 68 °C for 1 min); 68 °C for 1 min. Confirm amplification of a ~400 bp band by analytical agarose gel electrophoresis (see Note 19). Purify the amplicon using a PureLink® PCR purification kit, eluting in 30 μL of buffer E1, and quantitate spectrophotometrically. 4. Digest 5–10 μg of pMED1 and 5–10 μg of mutagenized sdAb insert in 50–μL reactions, each containing 1 μg of DNA, 1× FastDigest® buffer, and 3 μL of FastDigest® SfiI. Incubate the reactions at 50°C for several hours or, preferably, overnight. 5. The next day, add 1 μL each of FastDigest® PstI and XhoI to the pMED1 restriction digestions. These enzymes cut in between the two SfiI sites, preventing vector religation. Incubate for another 1 h at 37 °C. 6. Pool the pMED1 vector and mutagenized sdAb insert digests (separately) and purify them using the PureLink® PCR purification kit, eluting in 50 μL of sterile ultrapure H2O. Quantitate DNA by spectrophotometry. 7. Prepare 15 μL ligation reactions consisting of 500 ng of SfiIdigested mutagenized sdAb insert, 200 ng of SfiI-digested pMED1, 1× T4 DNA ligase buffer, and 1 U of T4 DNA ligase in ultrapure H2O. Sufficient vector and insert should be available for at least 5–10 ligation reactions. Incubate at 16 °C for several hours or preferably overnight. 8. Pool and purify the ligation reactions using the PureLink® PCR purification kit, eluting in the same volume of sterile ultrapure H2O as the total ligation reaction volume. Quantitate DNA by spectrophotometry. 9. Thaw electrocompetent E. coli TG1 cells on ice for 15 min. Pre-chill electroporation cuvettes on ice or at -20 °C at the same time. Mix ~300 ng of ligation reaction per 50 μL electrocompetent cells and incubate on ice for several minutes. Prepare 5–10 transformations in this manner to ensure an adequate library size. 10. Transfer the E. coli TG1:ligation mixtures to cuvettes. Ensure no air bubbles are present and electroporate at 1800 V (25 μF and 200 Ω) using a MicroPulserTM or similar instrument. The pulse time should be ~5 ms. Immediately transfer the electroporated cells to 1 mL of pre-warmed (37 °C) SOC media. Pool the transformations and incubate at 37 °C with shaking for 1 h. 11. Titer the transformed E. coli TG1 cells by preparing serial dilutions in 2×YT media. We recommend at minimum plating 100 μL of the 10-2 and 10-4 dilutions on 2×YT/ampicillin

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plates. Incubate the plates overnight at 37 °C. Plating additional dilutions (e.g., 10-3, 10-5) may yield a more accurate titer. 12. Transfer the remaining transformed E. coli TG1 cells to a 500 mL flask containing 200 mL of 2×YT/ampicillin media supplemented with 2% glucose. Incubate at 37 °C overnight with 250 rpm shaking. 13. The next day, pellet cells by centrifugation in 50 mL Falcon tubes at 5000× g for 10 min. Resuspend cells in 20 mL of 2×YT/ampicillin containing 2% glucose and 25% glycerol. Aliquot in cryovials and store at -80 °C. These are cryopreserved library cells. 14. Count colonies on titer plates from step 11 and calculate the overall library size in colony-forming units (CFU). 15. Perform colony PCR on 20–40 single colonies on plates from step 11. Each 15 μL colony PCR reaction should contain 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 μM primer M13RP, 0.2 μM primer -96GIII, and 0.05 μL of Platinum® Taq DNA polymerase. Touch a sterile pipette tip to each colony, then briefly touch the tip to the reaction master mix aliquoted in PCR tubes. Cycle the reactions as follows: 94 °C for 2 min; 35 cycles of (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s); 72 °C for 10 min. Electrophorese the amplicons in a 1% TAE agarose gel. Colonies bearing phagemids with sdAb inserts will show a band between 600 and 700 bp while empty phagemid vectors will show a band between 300 and 400 bp. 16. Multiply the % insert calculated in step 15 by the overall library size calculated in step 14 to obtain the functional library size in CFU. 17. Sequence 20–40 clones from the library to confirm mutagenesis (Fig. 8a). Either colony PCR products or phagemid DNA (purified from overnight cultures of single colonies) can be used as template for bidirectional Sanger sequencing with M13RP or -96gIII primers. The library can also be interrogated using high-throughput sequencing to confirm mutagenesis. 3.4.2 Construction of Site-Saturating sdAb Mutagenesis Libraries

To produce libraries of sdAbs (in this case, the C. difficile toxin A-specific camelid VHH A26.8) bearing all possible single-residue substitutions in CDRs, template DNA is PCR-amplified using pairs of mutagenic primers. The resulting amplicons are pooled, residual methylated template DNA is digested with Dpn1 [34], and mutagenized DNA mixtures are used to transform electrocompetent E. coli TG1 cells.

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Fig. 8 Sequence analysis of random (a) and pooled CDR2/CDR3 site-saturating (b) A26.8 sdAb mutagenesis libraries. The libraries were generated as described under Subheadings 2.4.1/3.4.1 and 2.4.2/3.4.2,

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1. Prepare template sdAb DNA as described under Subheading 3.4.1. For this step, the template must be a double-stranded phagemid or phage replicative form DNA harboring a sdAbcoding insert. In the example here the template was DNA encoding the A26.8 sdAb cloned between the SfiI sites of the pMED1 phagemid vector. 2. Amplify template sdAb DNA in 20 μL reactions consisting of 1× reaction buffer, 2 ng dsDNA template, 50 ng each of a mutagenic primer pair, 0.4 μL dNTP mix, and 1 U of PfuUltra DNA polymerase (supplied with the QuikChange II sitedirected mutagenesis kit). Perform 1 reaction for each CDR residue targeted for mutation. Cycle the reactions as follows: 95 °C for 30 s; 16 cycles of (95 °C for 30 s, 55 °C for 1 min, 68 °C for 5 min) (see Note 20). 3. Add 0.4 μL (4 U) DpnI (supplied with the QuikChange II sitedirected mutagenesis kit) to each mutagenesis reaction, mix, and incubate at 37 °C for 1 h. 4. Pool all mutagenesis reactions corresponding to each of the sdAb CDRs in a single tube (3 tubes total; see Note 21). 5. Thaw electrocompetent E. coli TG1 cells on ice for 15 min. Pre-chill electroporation cuvettes on ice or at -20 °C at the same time. Mix ~3 μL of each of the three pooled mutagenesis reactions per 50 μL electrocompetent cells and incubate on ice for several minutes. Prepare 5–10 transformations in this manner to ensure an adequate library size. 6. Transfer the E. coli TG1:mutagenesis reaction mixtures to cuvettes. Ensure no air bubbles are present and electroporate at 1800 V (25 μF and 200 Ω) using a MicroPulserTM or similar instrument. The pulse time should be ~5 ms. Immediately transfer the electroporated cells to 1 mL of pre-warmed (37 °C) SOC media. Pool the transformations and incubate at 37 °C with shaking for 1 h. 7. Titer the transformed E. coli TG1 cells by preparing serial dilutions in 2×YT media. We recommend at minimum plating 100 μL of the 10-2 and 10-4 dilutions on 2×YT/ampicillin plates. Incubate the plates overnight at 37 °C. Plating additional dilutions (e.g., 10-3, 10-5) may yield a more accurate titer. ä Fig. 8 (continued) respectively, then interrogated using Illumina MiSeq sequencing (2 × 105 reads analyzed per library). Amino acid representation and the Shannon conservation metric are shown for each sdAb position. The proportion of library variants bearing the indicated numbers of amino acid mutations compared with A26.8 is indicated. Positions are shown using IMGT numbering

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8. Transfer the remaining transformed E. coli TG1 cells to a 500 mL flask containing 200 mL of 2×YT/ampicillin media supplemented with 2% glucose. Incubate at 37 °C overnight with 250 rpm shaking. 9. The next day, pellet cells by centrifugation in 50 mL Falcon tubes at 5000× g for 10 min. Resuspend cells in 20 mL of 2×YT/ampicillin containing 2% glucose and 25% glycerol. Aliquot in cryovials and store at -80 °C. These are cryopreserved library cells. 10. Count colonies on titer plates from step 7 and calculate the overall library size in CFU. 11. Sequence 20–40 clones from each library to confirm mutagenesis (Fig. 8b). Either colony PCR products (see step 15 under Subheading 3.4.1) or phagemid DNA (purified from overnight cultures of single colonies) can be used as template for bidirectional Sanger sequencing with M13RP or -96gIII primers. The libraries can also be interrogated using high-throughput sequencing technologies to confirm mutagenesis. 3.4.3 Panning of Random and Site-Saturating sdAb Mutagenesis Libraries

VHH-displaying phage are rescued from the libraries following superinfection with M13KO7 helper phage. The resulting phage libraries are selected against biotinylated antigen (TcdA) using thermodynamic (affinity) or kinetic (off-rate) panning to enrich for sdAb variants with improved binding affinity. 1. Inoculate 200 mL of 2×YT containing 100 μg/mL ampicillin and 2% glucose with 0.5 mL of cryopreserved library cells (~5 × 1010 cells; see Subheadings 3.4.1 and 3.4.2 for library construction protocols). Grow at 37 °C with 250 rpm shaking until OD600 reaches ~0.5. 2. Using the OD600 measurement, calculate the number of cells (1 OD600 = ~8 × 108 cells/mL) and add a 20-fold excess of M13KO7 helper phage. Incubate at 37 °C without shaking for 30 min, then at 37 °C with 250 rpm shaking for 30 min. 3. Pellet cells by centrifugation at 5000× g for 10 min. Resuspend cells in 200 mL of 2×YT containing 100 μg/mL ampicillin and 50 μg/mL kanamycin. Grow overnight at 37 °C with 250 rpm shaking. 4. The next morning, pellet cells by centrifugation at 5000× g for 10 min. Filter the supernatant through a 0.22 μm Stericup-GP Express® PLUS filter. In 50 mL centrifuge tubes, add 1/5 the volume of 20% PEG/2.5 M NaCl and invert several times to thoroughly mix. Incubate on ice for 1 h. 5. Centrifuge the tubes at 10,000× g for 30 min. Resuspend pellets in a total volume of 1 mL PBS. Transfer to a microcentrifuge tube, centrifuge at maximum speed for 2 min, and collect the supernatant.

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6. Determine library phage concentration spectrophotometrically using the formula: virions/mL = (A269 - A320)·6 × 1016/ number of bp in phage or phagemid genome. 7. Prepare biotinylated TcdA antigen: combine 0.5 mg TcdA with 25 μL of 10 mM EZ-Link™ NHS-biotin in a total volume of 0.5 mL PBS. Incubate at room temperature for 1 h then quench the reaction with 50 μL of 1 M Tris–HCl, pH 8.0. Remove unreacted and hydrolyzed biotin reagent by overnight dialysis against 0.5–1 L of PBS at 4 °C. 8. On day 1, coat wells of Nunc MaxiSorpTM plates overnight at 4 °C with either 10 μg of unbiotinylated TcdA in 100 μL of PBS (standard panning) or 10 μg of streptavidin in 100 μL of PBS (thermodynamic and kinetic/off-rate panning). 9. For kinetic/off-rate panning: on day 1, mix 1011 library phage with 25 ng biotinylated TcdA in 100 μL PBS containing 1% (w/v) biotin-free casein and 0.1% Tween-20. Incubate at room temperature for 2 h, then add 10 μg unbiotinylated TcdA and incubate overnight at room temperature (phage A). 10. The next day (day 2), aspirate wells and block with 200 μL of PBS containing 2% biotin-free casein at 37 °C for 1 h (see Note 22). 11. For thermodynamic panning: mix 1011 library phage with 2 nM (0.6 μg/mL) biotinylated TcdA in 100 μL PBS containing 1% biotin-free casein and 0.1% Tween-20 for 2 h at room temperature (phage B; see Note 23). 12. To unbiotinylated TcdA-coated wells, add 1011 library phage in 100 μL PBS containing 1% biotin-free casein and 0.1% Tween-20 (standard panning). To streptavidin-coated wells, add either phage A (kinetic/off-rate panning) or phage B (thermodynamic panning). Incubate for 2 h at room temperature. 13. Wash wells five times quickly with 300 μL of PBS containing 0.05% Tween-20. Wash five more times with 300 μL of PBS containing 0.05% Tween-20 for 5 min each wash. 14. Elute bound phage with 50 μL of 100 mM triethylamine for 10 min followed by 50 μL of 100 mM glycine. Neutralize both elutions immediately with 50 μL of 1 M Tris–HCl, pH 8.0, and pool the elutions. 15. Add half (50 μL) of the eluted phage from step 14 to 2 mL of log-phase E. coli TG1 cells (OD600 = 0.3–0.4). Incubate at 37°C for 30 min without shaking then for 30 min with 250 rpm shaking. 16. Add the 2 mL infected E. coli TG1 cells to 10 mL 2×YT containing 100 μg/mL ampicillin. Incubate at 37 °C for 1 h with 250 rpm shaking.

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17. Add 1011 M13KO7 helper phage and incubate at 37 °C for 30 min without shaking then for 30 min with 250 rpm shaking. 18. Transfer the superinfected cells to 90 mL of 2×YT containing 100 μg/mL ampicillin in a 250 mL flask, then add 50 μg/mL kanamycin. Grow overnight at 37 °C with 250 rpm shaking. 19. Repeat steps 4 through 18 to perform rounds 2 and 3 of panning, ensuring to save half of the eluted phage from each round. Store the eluted phage at -20 °C. 3.4.4 Illumina MiSeq Sequencing

The eluted phage from panning of random and site-saturating sdAb mutagenesis libraries can now be interrogated using Illumina MiSeq sequencing, and through comparison with the unselected libraries, enrichment of affinity-enhancing substitutions can be detected. 1. Amplify genes encoding rearranged VHHs in 25-μL PCR reactions containing 1× ABI Buffer II, 1.5 mM MgCl2, 200 μM each dNTP, 5 pmol each of primers NGS-MJ7 and NGS-MJ8 (see Note 24), 1 U of AmpliTaq Gold® DNA polymerase, and 1 μL of library or eluted phage (~106 particles/μL; see Note 25). Cycle the reactions as follows: 95 °C for 7 min; 35 cycles of (94 °C for 30 s, 55 °C for 45 s, and 72 °C for 2 min); 72 °C for 10 min. 2. Electrophorese 5 μL aliquots of the PCRs in 1% agarose gels in TAE buffer and confirm amplification of a ~400 bp band. 3. Purify the amplicons using a PureLink® PCR purification kit (300 bp cutoff). 4. Conduct second round “tagging” PCRs in 50 μL reaction volumes containing 1× Phusion HF Buffer, 1.5 mM MgCl2, 200 μM each dNTP, 10 pmol of each primer pair (e.g., P5-seqF and P7-index1-seqR; see Note 26), 0.25 U of Phusion® HighFidelity DNA polymerase, and 5 μL of first-round PCR as template. Cycle as follows: 98 °C for 30 s; 20 cycles of (98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s); 72 °C for 5 min. 5. Electrophorese 5-μL aliquots of the PCRs in 1% agarose gels in TAE buffer and confirm amplification of a ~450–500 bp band. 6. Pool equal volumes of all second-round amplicons (see Note 27) and purify using a PureLink® PCR purification kit (300 bp cutoff). Subsequently, gel purify the pooled library from a 1% agarose gel in TAE buffer using a QIAquick® gel extraction kit and elute in 50 μL of EB. Perform final cleanup of the pooled NGS library using 90 μL of AMPure XP beads and elute in 20 μL of ultrapure H2O. 7. Measure pooled amplicon purity and concentration using a High Sensitivity DNA Analysis kit on a BioAnalyzer 2100 instrument.

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8. Sequence the pooled amplicons on an Illumina MiSeq Sequencing System using a 500-cycle Reagent Kit V2 or 600-cycle Reagent Kit V3 and a ≥5% PhiX genomic DNA spike. Diluting the amplicons to ~7–8 pM should yield a cluster density between 800 and 1000 K/mm2. 9. Visualize per-base quality scores for forward and reverse reads using FastQC. In many cases, poor data quality cannot be compensated for in the analysis and will artificially inflate diversity estimates. 10. Merge forward and reverse paired-end reads using FLASH with default parameters. High-quality data with an appropriate degree of overlap (≥20 bp) should yield ≥90% merged sequences. 11. Quality filter the merged sequences using the FASTQ quality filtering tool within the FASTX toolkit. Accept only sequences with Q30 scores over ≥95% of bases in the read. Generally, this will result in discarding of ~30% of the lowest-quality reads. 12. Convert data from .fastq to .fasta format and read into R using the read.fasta function of package “seqinr.” Strip all primerencoded non-antibody sequences. 13. Translate nucleotide sequences to protein using the translate function of package “seqinr.” 14. For each sdAb position, calculate the frequency of each amino acid. In the example here (sdAb A26.8), the result is a matrix with 124 columns (the total number of sdAb residues) and 21 rows (20 residues plus stop codon), with each cell containing a percentage frequency. 15. Measure enrichment at each sdAb position over the course of a panning experiment (Fig. 9). The absolute values of foldenrichments will depend on the library, antigen, and panning strategy (see Note 28). 3.4.5 Expression of sdAbs and Affinity Determination by Surface Plasmon Resonance

Detailed protocols for sdAb expression, purification, and affinity determination by surface plasmon resonance have been published elsewhere [24, 30]. Because affinity-enhancing substitutions are identified from high-throughput sequencing data and not at the level of individual clones, constructs encoding sdAb variants must be synthesized rather than subcloned. Although sdAb variants bearing multiple substitutions may be enriched during panning of random mutagenesis libraries, we recommend testing single residue substitutions prior to combining multiple substitutions. Our heuristic for combining substitutions is generally as follows: (1) identify and determine the affinities of single-residue substitutions; (2) produce all pairwise combinations of substitutions showing ≥2-fold decreased KD, or to reduce the number of variants tested, pairwise combinations of the 3–5 substitutions showing the largest

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Fig. 9 Enrichment of sdAb clones bearing affinity-enhancing amino acid substitutions as shown by Illumina MiSeq sequencing. (a) Proportion of sdAbs bearing the T111S substitution (see Fig. 10) in a random

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Fig. 10 Surface plasmon resonance sensorgrams showing interactions of sdAb A26.8 and its affinity matured variants with C. difficile TcdA. TcdA was immobilized by amine coupling on sensor chips CM5 and sdAbs were flowed over the surfaces at the indicated concentrations. (a) Binding of wild-type A26.8 was analyzed using multi-cycle kinetics (left) as well as single-cycle kinetics analysis (right). (b) Binding of single mutants (T111S, Q109R, T63K) was analyzed using either multi-cycle kinetics or single-cycle kinetics analysis. (c) Binding of a double mutant (T111S/Q109R) and a triple mutant (T111S/Q109R/T63K) was analyzed using multi-cycle kinetics analysis. Black lines show data and red lines show fits to a 1:1 interaction model. Kinetic and affinity constants are shown for each variant, as well as the KD fold change with reference to wild-type A26.8

ä Fig. 9 (continued) mutagenesis library and three rounds of standard, thermodynamic/affinity or kinetic/offrate panning. (b) Enrichment scores at sdAb A26.8 CDR positions after three rounds of thermodynamic/affinity panning of a random mutagenesis library. (c) Enrichment scores at sdAb A26.8 CDR positions after three rounds of kinetic/off-rate panning of a pooled CDR2/CDR3 site-saturating mutagenesis library. Enrichment scores of 1 (blue) imply increasing frequency following selection. Positions are shown using IMGT numbering and the parental residue at each position is marked with an X

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decreases in KD; (3) starting from the 3–5 double mutants showing the largest decreases in KD, produce triple mutants by adding additional single-site substitutions one a time (Fig. 10). Continue this process for testing of variants bearing four or more substitutions.

4

Notes 1. While numbers of PBMCs and circulating peripheral B cells vary by species and by individual, a typical yield might be ~106 PBMCs/mL whole blood, of which ~10% is made up by B lymphocytes. 2. Other anticoagulants (e.g., EDTA, heparin) have been used successfully, although the potential for PCR inhibition makes these reagents less suitable. 3. 1-inch, 22- or 22-gauge needles are appropriate for collection of human and llama blood samples, while 25-gauge or smaller needles can be used for mice. 4. Fresh blood samples with anticoagulant can be kept on ice or at 4 °C for several hours but should be processed to the point of cryopreservation of PBMCs as quickly as possible. It is possible to store fresh blood samples overnight at the same temperature, but significant loss of B cells should be expected. 5. Prior to cryopreservation, purified PBMCs should be either used immediately for nucleic acid extraction or stored at 4 °C in RNAlater solution or an equivalent product. 6. B cells and/or B-cell subsets can be further purified using magnetic bead selection and/or fluorescence-activated cell sorting, the parameters for which will vary depending on the application. 7. Expect yields of approximately 5 μg RNA per 106 cells. In a volume of 30 μL, this is equivalent to a concentration of 167 ng/μL; thus, below a threshold of ≤105 cells, spectrophotometric measurements may be unreliable. 8. For amplification of VHH genes directly from llama lymphocytes, PCRs using the CH2b3 primer will produce two bands around ~600 bp and ~800 bp. The smaller band must be purified by gel extraction rather than PCR purification to exclude conventional VH/VL antibodies. 9. The forward primer (P5-seqF) is the same for all second-round PCRs. Ensure each sample is barcoded using a single reverse primer from Table 2 bearing a unique index sequence. 10. If very even coverage across samples is important, secondround PCRs can be purified individually and then pooled in

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equimolar amounts prior to final cleanup. As a general rule, we find this to be unnecessary, as sample volumes can be adjusted based on band intensity on analytical agarose gels. 11. While we include primers for multiplexing up to 48 different samples in Table 2, the degree of multiplexing depends on sequence depth required. Multiplexing 12 samples using a 500-cycle Reagent Kit V2 will yield about 106 reads per sample prior to merging and quality filtering. 12. In many circumstances, poor data quality can be improved simply by increasing the amount of PhiX DNA spiked into the run. Reducing clustering density may also improve data quality. 13. Merging of synthetically randomized phage-displayed VH/VL libraries of defined CDR length will generally be more successful than merging of natural repertoires and libraries constructed from natural sources of diversity, from which variable domains with extremely long CDR3s may be lost at this step. 14. An additional well can be coated with antigen (TGF-β3) and eluted with PBS alone as a blank control. Data for this experiment will be similar to the irrelevant competitor well. 15. It is critical that the antigens being coupled to the beads are in a non-Tris containing buffer. 16. PBMC viability should be at least 95%. 17. An additional useful control is to select PBMCs from a different animal (not immunized with the target antigen) on target antigen-coupled and irrelevant antigen-coupled magnetic beads and to analyze these samples by NGS as well. For these PBMCs, enrichment should be similar in all selections. 18. Assuming the sdAb sequence is known, choose one of primers MJ1, MJ2, or MJ3 to maximize identity with the sdAb FR1 N-terminus. If the sequence is not known, an equimolar mixture of all three forward primers can be used. 19. If weak amplification occurs, perform multiple 50 μL reactions at this step to improve yield. Standard PCR troubleshooting rules apply, so increasing the number of cycles or decreasing annealing temperature may also improve yield. 20. Adjust the extension time based on the size of the dsDNA template (1 min per kb). 21. While in theory all mutagenesis reactions can be pooled in a single tube at this step, we recommend preparing sitesaturation libraries for each CDR separately. There are two reasons for this. First, if mutagenesis is unsuccessful for some positions, it will simplify diagnosis of the problem and allow only part of the procedure to be repeated. Second, it permits panning of each CDR library separately.

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22. It is suggested to start the E. coli TG1 culture to be used for amplification of the eluted phage (step 15) at this stage. If a single colony on a M9 minimal media plate is unavailable, commercial competent cells can be used as a source of inoculum. 23. The concentration of biotinylated antigen should be below the KD of the sdAb of interest. Ideally, multiple concentrations of antigen should be tested here (for instance, 0.5, 0.1, 0.05, and 0.01-fold the KD concentration). Too high of a concentration will result in inefficient selection of higher-affinity binders, while very low concentrations may prevent selection of variants with modestly improved affinity and yield very few output phage. 24. The gene-specific sequences of primers NGS-MJ7 and NGS-MJ8 can be modified to yield primers that will anneal to other vector sequences flanking sdAb inserts. 25. Eluted phage can often successfully be used directly as templates for PCR amplification. However, concentrations significantly higher than 106 phage particles per reaction can inhibit PCR. If PCR is not successful, phagemid DNA can instead be extracted from overnight E. coli TG1 cultures for phage amplification and used as the template for this PCR. 26. Ensure that the second round tagging PCR for each library and eluted phage sample uses the same forward primer (P5-seqF) and a different reverse primer bearing a unique index sequence (e.g., P7-index1-seqR). 27. If very even coverage across samples is important, secondround PCRs can be purified individually and then pooled in equimolar amounts prior to final cleanup. As a general rule, we find this to be unnecessary, as sample volumes can be adjusted based on band intensity on analytical agarose gels. 28. We recommend testing substitutions showing the highest enrichment scores in each sdAb CDR. Consistency of enrichment across multiple rounds of panning is also a strong indicator of robustness of the analysis.

Acknowledgments This work was supported by funding from the National Research Council Canada. This chapter is an amalgamation and extension of two previous Methods in Molecular Biology chapters [17, 20]. Conflict of Interest Statement The authors have no conflicts of interest to declare.

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Part V Epitope Mapping and Biomarker Discovery by Phage Display

Chapter 27 Biomarker Discovery by ORFeome Phage Display Philip Alexander Heine, Rico Ballmann, Praveen Thevarajah, Giulio Russo, Gustavo Marc¸al Schmidt Garcia Moreira, and Michael Hust Abstract Phage display is an efficient and robust method for protein-protein interaction studies. Although it is mostly used for antibody generation, it can be also utilized for the discovery of immunogenic proteins that could be used as biomarkers. Through this technique, a genome or metagenome is fragmented and cloned into a phagemid vector. The resulting protein fragments from this genetic material are displayed on M13 phage surface, while the corresponding gene fragments are packaged. This packaging process uses the pIII deficient helperphage, called Hyperphage (M13KO7 ΔpIII), so open reading frames (ORFs) are enriched in these libraries, giving the name to this method: ORFeome phage display. After conducting a selection procedure, called “bio-panning,” relevant immunogenic peptides or protein fragments are selected using purified antibodies or serum samples, and can be used as potential biomarkers. As ORFeome phage display is an in vitro method, only the DNA or cDNA of the species of interest is needed. Therefore, this approach is also suitable for organisms that are hard to cultivate, or metagenomic samples, for example. An additional advantage is that the biomarker discovery is not limited to surface proteins due to the presentation of virtually every kind of peptide or protein fragment encoded by the ORFeome on the phage surface. At last, the selected biomarkers can be the start for the development of diagnostic assays, vaccines, or protein interaction studies. Key words ORFeome phage display, Oligopeptide phage display, ORF selection, Peptide phage display, Biomarker, Panning, Identification immunogenic proteins, Protein interaction

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Introduction Phage display is a powerful tool for protein-protein interaction studies. Currently, it is mainly used for antibody discovery [1–4], but it is also suitable for epitope mapping [5], target identification or validation [6], and identification of immunogenic proteins or biomarkers from genomic [7, 8], metagenomic [9] or cDNA libraries [10]. Such biomarkers can be, e.g.,, allergens [11–13], or immunogenic proteins from bacterial pathogens like Mycobacterium tuberculosis [4], Mycoplasma mycoides [14], the rickettsia

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_27, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Cowdria ruminantium [15], eukaryotic pathogens like Taenia solium [16] or viruses like SARS-CoV-2 [17]. To perform ORFeome phage display, a DNA- or cDNAderived genomic or metagenomic library needs to be constructed. For this first step, the genetic material of interest is fragmented and inserted into a phagemid vector, e.g., pHORF3 [18]. Since the insertion procedure is random, this process results in many wrongly-oriented and out-of-frame molecules [19, 20]. The functional molecules, in their turn, encode peptides or protein fragments that are displayed by fusion to an M13 phage coat protein, typically minor coat protein 3 (pIII), which is fused downstream of the inserted peptide sequence on the phagemid, e.g., on the vector pHORF3 [18]. To package this fusion-proteins into phage particles, a helperphage is needed to provide the genetic information of all other structural phage proteins. This helperphage, however, is not able to eliminate the “junk” sequences comprehended by the inverted and out-of-frame ORF fragments and, therefore, their number must be decreased to increase library quality [18, 19]. To do this for genomic or metagenomic libraries, a special variant of this helperphage, named Hyperphage, is used to enrich ORFs in the library [21, 22]. This Hyperphage has a deletion in the pIII gene (pIII
genotype), but carries functional pIII proteins (pIII+ phenotype) provided by the phagemid for infection of E. coli. It also has a kanamycin resistance, which allows selection for double-infected clones. During the packaging process with Hyperphage, the (pHORF3) phagemid is the only source of pIII, which leads to an incorporation of up to five peptide-pIII fusion proteins in each produced phage particle only when the gene fragment encoding the peptide or protein fragment is in the same reading frame with gIII. This way, if the inserts are not in frame or in wrong orientation, several stop codons are formed, leading to no pIII fusions proteins and no infective phage particles produced [19]. Thus, ORFeome phage display combines the positive selection of ORFs from whole genomes or metagenomes with the functional display of protein fragments encoded by these sequences (Fig. 1). The selection procedure from phage display libraries is called “panning”, named after the gold washer procedure [2]. To avoid the enrichment of false-positive peptides or protein fragments in this method, it is highly recommended to deplete anti phage binders from the antibody source by preincubation with M13 phage particles. Furthermore, it is possible that the library contains proteins or fragments that can bind antibodies in general, e.g., Protein A (Staphylococcus aureus) [23]. Therefore, the library should be preincubated on unrelated antibodies to deplete phage particles that display such molecules. Such steps for the elimination of binders and antibodies are called “negative selection.” For the “positive selection,” precleared library and antibody source are incubated together, and the non-bound phage can be washed

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Fig. 1 [7]: ORFeome phage display combines the enrichment of open reading frames (ORF) and the functional display of proteins on the surface of phage particles. Randomly fragmented genomic DNA from pathogens or microbial communities is cloned into a phage display vector (phagemid; M13 ori: intergenic region for packaging [26]) upstream of the minor coat protein III (pIII) gene. When using a special helperphage (“Hyperphage”, deleted pIII gene), the phagemid encoded pIII fusion protein is the only pIII source. Infectious phage particles are only assembled if the cloned DNA sequence is in frame with the pIII gene and does not contain any stop codon leading to an enrichment of ORFs whereas the encoded protein is displayed at the

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away while the bound ones can be eluted, e.g., with trypsin. According to the genome size and the library diversity, a second panning round could be helpful, to enrich antibody-specific phage particles as it is recommended for metagenomic libraries. After positive selection via panning, their recognition must be confirmed, e.g., in a screening-ELISA. Here, the setup includes capturing the pentavalent phage particles or immobilizing them directly on microtiter plates (MTP), and then incubated with the same source of antibodies used in the positive selection. In this ELISA, similar negative selection procedure may be considered to select specific binders. Further, positive hits shall be sequenced and aligned to the original genome or blasted to identify the corresponding biomarker, using public databases. Recombinant production of the identified protein, followed by a titration ELISA with the corresponding antibody is also recommended as a validation step. ORFeome phage display for biomarker identification using pHORF system was used first to identify biomarkers from two different Mycoplasma species, pathogens causing Bovine Respiratory Diseases [18]. Furthermore, novel and already described immunogenic proteins from Salmonella Typhimurium are identified as well as biomarkers from Neisseria gonorrhoeae [7, 8]. This technique has also been shown to be as effective as mass spectrometry for target identification after monoclonal antibody generation [6]. ORFeome phage display with a eucaryotic cDNA library resulted in the discovery of new immunogenic saliva proteins from Ixodes scapularis (tick) [10]. Further, a major immunogenic epitope of the SARS-CoV-2 spike protein was identified with ORFeome phage display [17]. Besides its vast flexibility for different applications, ORFeome phage display is independent of limitations like cell surface accessibility, transcriptome, or proteome and can therefore be used for biomarker discovery regardless of cultivation-, conditional difficulties, or cell barriers. The following protocol describes the library construction, panning, and screening process to select immunogenic proteins from procaryotic [7, 8, 18] or eucaryotic [10, 17] pathogens as well as whole metagenomes [9]. Novel biomarkers, discovered by ORFeome phage display can play a valuable role in the development of diagnostics [24] and vaccines.

ä Fig. 1 (continued) same time on the phage particle. In a panning procedure using immobilized immune sera, these libraries can be enriched for immunogenic peptides in multiple panning rounds. Afterward, the monoclonal binders must be validated in a screening ELISA. DNA of positive clones can be sequenced to identify the biomarker

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Materials

2.1 Isolation of Genomic DNA

1. DNA isolation kit.

2.2 Amplification of Genomic DNA

1. illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit (GE Healthcare, Freiburg, Germany). 2. DNA-free water. 3. PCR reaction tubes. 4. Thermocycler. 5. Agarose (Peqlab, Erlangen, Germany). 6. TAE-buffer 50x: 2 M Tris–HCl, 1 M acetic acid, 0.05 M EDTA pH 8. 7. Electrophoresis chamber.

2.3 Fragmentation of DNA

1. Biorupter Plus (Diagenode, Seraing, Belgium). 2. Amicon Ultra Centrifugal Filters (30 k) (Merck Millipore, Tullagree, Ireland). 3. Gel and PCR purification kit. 4. Agarose. 5. TAE-buffer 50x. 6. Electrophoresis chamber.

2.4

DNA End Repair

1. Fast End Repair Waltham, USA).

Kit

(Thermo

Fisher

Scientific,

2. Gel and PCR purification kit. 2.5 Library Construction

1. Phagemid (in this protocol pHORF3). 2. PmeI-HF (NEB, Frankfurt am Main, Germany). 3. CIP (NEB, Frankfurt am Main, Germany). 4. CutSmart Buffer (NEB, Frankfurt am Main, Germany). 5. NucleoSpin Gel and PCR clean-up (Macherey-Nagel, Du¨ren, Germany). 6. T4 DNA ligase (Promega, Mannheim, Germany). 7. Amicon Ultra Centrifugal Filters (30 k) (Merck Millipore, Tullagree, Ireland). 8. Glycerol. 9. 0.1-cm electroporation cuvette. 10. Electroporator.

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11. SOC medium pH 7.0: 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05% (w/v) NaCl, 20 mM Mg solution, 20 mM glucose (sterilize magnesium and glucose separately, add solutions after autoclaving). 12. PolysYTrene dish with lid (245 mm x 245 mm x 25 mm). 13. 2xYT medium pH 7.0: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl. 14. 2xYT-GA agar: 2xYT, 100 mM glucose, 100 μg/mL ampicillin, 1.2% (w/v) agar-agar. 15. Electrocompentent E. coli TOP10F’ (Invitrogen, Carlsbad, USA) (F0 [lacIq Tn10(tetR)] mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR nupG recA1 araD139 Δ(ara-leu)7697 galU galK rpsL(StrR) endA1 λ-). 16. Liquid Nitrogen. 17. Single-use Drigalsky spatulas. 18. 2-ml cryo vials. 19. 10-cm Petri dishes. 20. Agarose. 21. TAE-buffer 50x. 22. Electrophoresis chamber. 23. Optional: QIAxcel Advanced System (QIAGEN, Hilden, Germany). 2.6 Antigen Library Packaging

1. 2xYT medium pH 7.0. 2. 2xYT-T: 2xYT + 20 μg/mL tetracycline. 3. 2xYT-GA: 2xYT + 100 mM glucose +100 μg/mL ampicillin. 4. 2xYT-GA agar plates. 5. 2xYT-AK: 2xYT + 100 μg/mL ampicillin +50 μg/mL kanamycin. 6. 10-cm Petri dishes. 7. Hyperphage (Progen, Heidelberg, Germany). 8. 1-mL cuvettes and spectrophotometer 600-nm wavelength 9. 100 and 1000-mL glass shake flasks. 10. 50-mL tubes. 11. 0.45-μM syringe filters. 12. Syringe. 13. Incubator for shake flasks. 14. Eppendorf centrifuge (Eppendorf, Hamburg, Germany). 15. Sorvall Centrifuge RC5B Plus, rotor F9S and SS34 (Thermo Fisher Scientific, Waltham, USA) and respective tubes.

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Table 1 Oligonucleotide primers Oligonucleotide primer

Sequence 50 -30

MHLacZ-Pro_f

GGCTCGTATGTTGTGTGG

MHgIII_r

CTAAAGTTTTGTCGTCTTTCC

16. Polyethylenglycol-Sodium Chloride (PEG-NaCl) solution: 20% (w/v) PEG 6000, 2.5 M NaCl. 17. Phosphate buffered saline (PBS) pH 7.4: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4x2H2O, 0.24 g KH2PO4 in 1 L. 2.7

Colony PCR

1. Oligonucleotide primer (Table 1). 2. GoTaq DNA Polymerase and buffer (Promega, Frankfurt am Main, Germany). 3. dNTP Mix. 4. DNA-free H2O. 5. Thermocycler.

2.8 Antigen Panning and Screening-ELISA

1. 96-well ELISA Costar plate (Corning, Corning, USA). 2. Phosphate buffered saline (PBS) pH 7.4. 3. PBST: PBS + Tween 20 0.05% (v/v). 4. 2% M-PBST: skimmed milk powder 2% (w/v) diluted in PBST. 5. ELISA washer, Vermont, USA).

e.g.,

ELx50

(BioTek;

Winooski,

6. E. coli TOP10F’ (Invitrogen, Carlsbad, USA) (F0 [lacIq Tn10 (tetR)] mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR nupG recA1 araD139 Δ(ara-leu)7697 galU galK rpsL(StrR) endA1 λ-). 7. 1-mL cuvettes and spectrophotometer 600-nm wavelength. 8. Trypsin (1 mg/mL stock). 9. Eppendorf centrifuge (e.g., 5415 D). 10. 2xYT medium pH 7.0. 11. 2xYT-T. 12. 2xYT-GA. 13. 2xYT-GA agar plates. 14. 2xYT-AK. 15. 96-well U-shaped polypropylene plate. 16. Hyperphage (Progen, Heidelberg, Germany). 17. PEG-NaCl.

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18. Anti-IgG Fc-specific antibody HRP-conjugated. 19. Anti-M13 phage (pVIII) HRP-conjugated (GE Healthcare, Freiburg, Germany). 20. TMB solution. 21. 1 N H2SO4.

3

Methods

3.1 Isolation of Genomic DNA

Prepare genomic DNA from a bacterial culture (or other source of bacteria) using any commercial kit suitable for the preparation of genomic DNA. It is also possible to use metagenomic DNA (e.g., from bacterial communities like fecal samples) or cDNA from mRNA isolation (e.g., for eukaryotic cells).

3.2 Amplification of Genomic DNA (Optional)

Approximately 20 μg template DNA is needed for library construction. If DNA amounts are insufficient for library construction, they can isothermally be amplified by multiple displacement amplification (MDA) using commercial kits like the illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit: 1. Dilute 10-100 ng of template DNA in 10 μL DNA-free H2O Milli-Q and mix with 10 μL 2 X Denaturation Buffer provided with the kit. 2. Denature DNA for 3 min at 95  C and subsequently cool down on ice. 3. Transfer the denatured DNA to the reaction cake, ensure complete reconstitution of the cake, and incubate for 2 h at 30  C using a thermocycler. 4. Inactivate the polymerase for 10 min at 65  C. 5. Analyze the amplified DNA on a 1% (w/v) TAE agarose gel (see Note 1).

3.3 DNA Fragmentation

1. Dissolve three times 1.25 μg of the input DNA in 100 μL water in one 1,5-mL Eppendorf tube for each library. You can load 3-6 Eppendorf tubes in the BiorupterPlus. 2. Fragment the DNA with the BiorupterPlus with 70 cycles; 30 s on/ 30 s off. 3. Pool 3 samples (3x 100 μL) of the same library in a Amicon® Ultra-0.5 mL Centrifugal Filters Ultracel®-30 K, add 200 μL MilliQ and centrifuge 10 min at 14,000 g. Afterward, elute the filtrate by centrifugation at 1000 g for 2 min in a fresh tube. You should now have ~20 μL sample volume from each centrifugal filter.

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4. Measure the DNA concentration with the nanodrop. The DNA concentration should be between 100 ng/μL and 200 ng/μL. 5. Dilute 1 μL of each sample with 4 μL MilliQ and 1 μL 6x loading dye and analyze the fragment size distribution with agarose gel electrophoresis (1.5% agarose gel, 120 V, 30 min). The fragment size should be between 75 bp and 600 bp. 3.4 Removal of Cohesive Ends

Sonication of DNA results in fragments with 50 or 30 overhangs. The cohesive ends need to be repaired and fragments must be phosphorylation to allow blunt-end cloning in the linearized phage display vector. Removal of cohesive ends and phosphorylation can be performed using the Fast End Repair Kit or any other commercial kit (Table 2): 1. Incubate the reaction for 15 min at 20  C (do not exceed incubation time) and purify using a PCR purification kit. Elute in 30 μL of the provided elution buffer or H2O Milli-Q. 2. Measure the DNA concentration with the nanodrop. The DNA concentration should be between 100 ng/μL and 200 ng/μL.

3.5 PhagemidFragment Ligation and Library Construction

The preparation of the phagemid varies display method used. In this protocol, phagemid that allows the cloning in pHORF3, which has a PmeI as cloning digestion as described in Table 3.

with the kind of phage it is necessary to use a a blunt end, such as site. Thus, perform the

Table 2 Reagents to be added on reaction for DNA-ends repair Fragmented DNA (final amount 5 μg)

X μL

10 X end repair reaction mix

5 μL

End repair enzyme mix

2.5 μL

H2O Milli-Q

Up to 50 μL

Table 3 Reagents to be added on the linearization of the phagemid Phagemid (total 5 μg)

X μL

Buffer CutSmart 10 X (NEB, Frankfurt am Main, Germany) 2 μL PmeI (10 U/μL, NEB, Frankfurt am Main, Germany)

1 μL

H2O Milli-Q

Up to 20 μL

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Table 4 Reagents to be added on the ligation of gene fragments with phagemid Digested ~4-kb phagemid (total 1 μg)

X μL

DNA fragments up to 1.5 kb (total 1.4 μg)

Y μL

T4 DNA ligase buffer 10 X (Promega)

10 μL

T4 DNA ligase (3 U/μL, Promega)

3.33 μL

H2O Milli-Q

Up to 100 μL

1. Incubate the reaction for 2 h at 37  C and add 1 μL of calfintestinal alkaline phosphatase (10 U/μL, NEB, Frankfurt am Main, Germany). 2. Incubate for 1 h at 37  C and purify the reaction using the NucleoSpin Gel and PCR clean-up kit. (see Note 2) Elute in 30 μL Milli-Q water. 3. Perform ligation reaction for 16 h at 16  C (Table 4). 4. Inactivate the ligase for 10 min at 65  C ( see Note 3) and perform a buffer exchange using Amicon® Ultra-0.5 mL Centrifugal Filters Ultracel®-30 K. For this, add 400 μL of Milli-Q water in the reaction and centrifuge (10 min, 10,000 g). Repeat this buffer exchange with 500 μL of Milli-Q water for 2-4 more times before collecting the final volume as instructed by the manufacturer (place the centrifugal filter inverted in a fresh collection tube and centrifuge 2 min at 1000 g.) 5. Mix 10 μL purified ligation mix with 25 μL electrocompetent E. coli TOP10F’ or E. coli SS320, in a 1.5 mL Eppendorf tube, transfer the volume to a pre-chilled 0.1-mm cuvette, and keep it on ice for 1 min. 6. Perform electroporation for bacteria (Ω; 1.8 kV; pulse ~4.8 ms) and immediately add 1 mL of the appropriate pre-warmed recovery medium. 7. Transfer the cells to a 1.5 mL tube and incubate at 37  C for 1 h and 650 rpm. 8. Take 10 μL of the tube and dilute it in 10 mL of 2xYT (first dilution). From this tube, transfer 100 μL to 1 mL of 2xYT (second dilution) and from the second dilution another 100 μL to 1 mL of 2xYT (third dilution). Finally, plate 100 μL of each dilution (final dilution factor 10
4, 10
5, 10
6) onto a 10 cm 2xYT-GA agar plate and incubate overnight at 37  C (see Note 4). 9. Plate the remaining volume of the transformation mix onto a 245  245  25 mm plate supplemented with 2xYT-GA agar and incubate at 37  C overnight.

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Table 5 Composition of a colony PCR Solution or component

Volume

Final concentration

dH2O

7.5 μL

GoTaq buffer (5x)

2 μL

1

dNTPs (10 mM each)

0,2 μL

200 μM each

MHLacZPro_f 10 μM

0,1 μL

0,1 μM

MHgIII_r10 μM

0,1 μL

0,1 μM

GoTag (5 U/ μl)

0,1 μL

0.5 U

Template

Picked colonie from dilution plate

10. Perform the colony counting on the 10 cm plates. 11. On the 245  245  25 (“pizza plate”) mm plate, add 20 mL of 2xYT and incubate on a shaker for 20 min. 12. With a Drigalsky spatula, carefully scrape the cells from the plate’s surface. (see Note 5) Then, collect the liquidcontaining cells with a serological pipette in a 50 ml tube and supplement with 20% (v/v) glycerol and distribute 1 mL in each of 6 cryovials. 13. Flash freeze the cells in liquid nitrogen and wait for 5 min. Then, carefully take the tubes with proper protective gloves and store the tubes at
80  C promptly. 3.6 Library Quality Control

1. From the 10 cm plates used for counting, take at least 20 colonies to perform a colony PCR. For this PCR, use one tube containing the empty phagemid used for the library construction as a negative control (Table 5). 2. Check the size of each fragment by agarose gel electrophoresis. (see Note 6). 3. Count the number of positive clones (those showing a larger amplicon compared to the negative control) expecting to have at least 80% (16/20) of the clones positive (this quality measurement is called “insert rate”). If the number is much below 80%, consider repeating previous steps, mainly the phagemid preparation or ligation (see Note 7).

3.7 Library Packaging and ORF Enrichment

1. Gently thaw the library previously stored at
80  C on. Inoculate 200 mL of 2xYT-GA in a 500-mL shake flask with the library (OD600 ¼ 0.1). 2. Incubate the shake flask at 37  C, 250 rpm until logarithmic growth is reached (OD600  0.5). Then, transfer 25 mL (1.25  1010 cells) of the culture to a 50-mL tube and add 2.5  1011 cfu (MOI 1:20 ) of Hyperphage.

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3. Incubate the tube for 30 min at 37  C without shaking, and another 30 min at 37  C and 250 rpm. 4. Pellet the cells at 3220xg for 10 min, RT. Discard the supernatant, suspend the cells in 10 mL of 2xYT-AK, and transfer them to a 1000-mL shake flask containing 390 mL of the same medium. Incubate the flask at 30  C, 250 rpm for 24 h. 5. Transfer the culture to a 1000-mL centrifuge tube and centrifuge for 10 min at 10,000 g, 4  C. The supernatant contains the phage. Collect the supernatant into another 1000-mL centrifuge tube (see Note 8), add 1/5 volume (100 mL) of PEG-NaCl solution, mix thoroughly, and incubate the tube at 4  C overnight. In parallel, inoculate a 100-mL shake flask containing 25 mL of 2xYT-T with E. coli TOP10F’ and incubate at 37  C, 250 rpm overnight. 6. Centrifuge the tube containing the supernatant with PEG-NaCl 10,000 g, 1 h, 4  C and discard the supernatant. 7. Suspend the pellet containing phage in 10 mL of pre-chilled PBS, filter the suspension with a 0.45 μm filter, and transfer to another 50-mL centrifuge tube. 8. Add 1/5 volume (2,5 mL) of PEG-NaCl solution and incubate 1 h on ice on a rocker. 9. Centrifuge the suspension 20,000 g, 30 min, 4  C and discard the supernatant. 10. Suspend the pellet in 1 mL of Phage dilution buffer, transfer to a 1.5-mL tube, and centrifuge at 16,000 g, 30 min, 4  C to remove remaining bacteria. 11. Transfer the supernatant to a cryovial and store it at 4  C until further use. 12. Take the E. coli TOP10F’ overnight culture, make another 30-mL 2xYT-T culture in a 100-mL shake flask with initial OD600  0.1, and incubate at 37  C, 250 rpm until OD600  0.5. 13. Prepare six 1.5-mL tubes for phage dilution, three with 990 μL and three with 900 μL of PBS. First, use 10 μL of the phage prepared in step 11 to make the three 100-fold dilutions on the tubes with 990 μL. Then, make three ten-fold dilutions by adding 100 μL of the last tube on the remaining three tubes with 900 μL (these will be dilutions 10
2, 10
4, 10
6, 10
7, 10
8, and 10
9). 14. Prepare four 1.5-mL tubes with 50 μL of E. coli TOP10F’ cells in each and transfer 10 μL of the last four phage dilutions to each tube (these will be dilutions 10
8, 10
9, 10
10, 10
11 on the plate). (see Note 9). 15. Incubate the tubes at 37  C for 30 min without shaking.

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16. Divide one 2xYT-GA agar plate into 4 parts and make three 10-μL droplets of each of the four dilutions on each part. Let the droplets dry under the biological cabinet for 5 min and incubate the plate at 37  C for 16 h. 17. Spread the remaining volume (30 μL) of the two intermediary dilutions (10
9, and 10
10) on 2xYT-GA agar plates. 18. Count the colonies on countable droplets and calculate the titer as the arithmetic mean of the 3 droplets and multiply per 6, so the result will be in cfu/mL. This quality measurement is called “library titer”. 19. From the other plate, pick at least 20 colonies, analyze insert rate and size by colony PCR and sequence the DNA expecting to have at least 50% (10/20) of the clones with in-frame and correct sequence. This quality measurement is called in-frame rate. The insert rate should be higher after packaging. 20. Analyze the colony PCR by electrophoresis (see Note 10). 3.8

Colony PCR

3.9

Antigen Panning

Choose 10–20 single colonies per transformation. Set up the 10 μL PCR reaction per colony as follows (see Table 1 for primer sequences). Suggested PCR program: 95  C, 120 s + 95  C, 15 s; 54  C, 20 s; 72  C, 120 s (25 cycles) + 72  C 10 min + 4  C forever. 1. Inoculate an overnight culture of E. coli TOP10F’ in 30 mL 2xYT-T in a 100 mL flask. 2. Coat 1 well of a 96-well high-binding ELISA plate with 1 μg of a purified antibody suitable to capture the desired antibody isotype and species (diluted in 200 μL of PBS) (following referred to as “selection wells”). In parallel, coat 2 wells with 5  1010 cfu Hyperphage (in 150 μl PBS) (following referred to as “pre-clearance wells”). It is also recommended, to coat one wells with an antibody isotype control. Coating can be performed at 4  C overnight. (see Note 11). 3. On the next day, remove the solutions and add 350 μL 2% M-PBST in each of the wells to saturate the protein binding capacity (1 h at room temperature). 4. Wash the pre-clearance wells three times with PBST. 5. Dilute the serum 1:100 – 1:1000 with PBST (2  150 μL), transfer to two of the pre-clearance wells, and incubate for 1 h at RT. 6. Transfer the sera to the second pre-clearance wells and incubate for 1 h at RT. 7. Dilute 1  1011 cfu (or at least 100-fold excess of library size) of the library in 150 μL MPBST and apply the libraries to preincubation well with the antibody isotype control.

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8. Remove excess serum in the selection wells by 3 washing steps with PBST. Transfer the library to the captured serum antibodies. Incubate for 2 h at RT. 9. After approximately one hour, inoculate 30 mL 2xYT-T with the E. coli TOP10F’ overnight culture (OD600 ¼ 0.1) and cultivate at 37  C and 250 rpm until logarithmic growth is reached (OD600 ¼ 0.5, approximately 1.5 h). 10. Remove non-bound antigen-phage in 10 washing cycles using PBST. (see Note 12). 11. Elute the bound phage using 200 μL Trypsin solution (10 μg/ mL in PBS) for 30 min at 37  C and pool the elutions (The phagemid encoded pIII fusion protein harbors a trypsin site). 12. Prepare three 1.5-mL tubes with 90 μL PBS or 2x YT medium for phage dilution. First, use 10 μL of the phage prepared on step 11 to make the three ten-fold dilutions on the tubes with 90 μL. (these will be dilutions 10
2, 10
3, 10
4). The dilutions have to be adjusted each panning round according to the expected elution titers. (see Note 13). 13. Prepare four 1.5-mL tubes with 50 μL of E. coli TOP10F’ cells in each and transfer 10 μL of the non-diluted phage and each of the respective dilutions (these will be dilutions 10
2, 10
3, 10
4, 10
5 on the plate). 14. Incubate the tubes at 37  C for 30 min without shaking. 15. Divide one 2xYT-GA agar plate into 4 parts and make three 10-μL droplets of each of the four dilutions on each part. Let the droplets dry under the biological cabinet for 5 min and incubate the plate at 37  C for 16 h. 16. Use the remaining 380 μL of eluted phage to infect 5 mL of the E. coli TOP10F’ culture and incubate for 30 min at 37  C without shaking. 17. Pellet the cells for 10 min at 3220xg and discard the supernatant. Suspend the pellet in up to 500 μL and plate on a 15 cm 2xYT-GA agar plate. Incubate at 37  C overnight. 18. On the 15 cm plate, add 5 mL of 2xYT and incubate on a shaker for 20 min. 19. With a Drigalsky spatula, carefully scrape the cells from the medium surface (see Note 14). Collect the liquid containing the cells and inoculate 30 mL 2xYT-GA (OD600 ¼ 0.1) and incubate at 37  C and 250 rpm until logarithmic growth is reached (OD600 ¼ 0.5). 20. Collect 5 mL of the culture (~2.5  109 cells) and infect with 5  1010 cfu Hyperphage (MOI 1:20 ). Incubate for 30 min at 37  C without shaking and for another 30 min at 37  C and 250 rpm.

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21. Pellet the cells for 10 min at 3220 g and discard the supernatant. Suspend the cells in 1 mL 2xYT-AK and transfer to a 100 mL shaking flask with 29 mL 2xYT-AK. Incubate at 30  C and 250 rpm overnight. 22. Pellet the cells for 10 min at 3220 g and transfer the supernatant to another tube (the supernatant contains the phage). 23. Add 1/5 volume (ca. 6 mL) PEG-NaCl, mix thoroughly, and incubate for 1 h on ice to precipitate phage. 24. Pellet the phage for 1 h at 3220 g and 4  C. Discard and completely remove the supernatant. 25. Suspend the pellet in 1 mL of phage dilution buffer, transfer to a 1.5-mL tube, and centrifuge at 16,000 g, 30 min, 4  C to remove remaining cells. Transfer the supernatant to a cryovial and store at 4  C until next use. 26. Repeat the steps above for another 1 two 2 rounds of panning. Stop at step 14 in the last panning round. 27. Instead of applying 10 μL droplets to the agar plates, plate the whole 60 μL for each dilution on an individual 2xYT-GA agar plate in order to allow screening of individual clones. 3.10 Monoclonal Phage Production and Screening ELISA

1. In a 96-well U-bottom propylene plate, add 180 μL/well of 2xYT-GA. 2. Pick 92 colonies from the plates described in the last step of the previous part. In this same plate, include 2 wells (H3 and H9) with medium only, 1 well (H6) with a colony to produce a non-related phage, and 1 well (H12) with the same colony added on H11. 3. Add a breathable sealing tape over the plate and incubate at 37  C, 800 rpm, for 6 h (this will be called “Master plate”). (see Note 15). 4. In a new 96-well U-shaped propylene plate, add 180 μL/well of 2xYT-GA, and transfer 10 μL of the previously grown plate to this new one. Store the Master plate, supplemented with 20% (v/v) glycerol at
80  C or without glycerol at 4  C, and incubate the new one at 37  C, 800 rpm, for 2 h. 5. Dilute purified Hyperphage in 2xYT to the concentration of 1  1011 cfu/mL, and add 50 μL/well (5  109 cfu/well). 6. Incubate 30 min at 37  C without shaking, followed by 30 min at 37  C with 800 rpm. 7. Centrifuge the plate 3220 g for 10 min at RT, remove the supernatant, and add 190 μL/well of 2xYT-AK. 8. Incubate the plate overnight at 30  C, 800 rpm.

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9. Centrifuge the plate at 3220 g for 10 min, RT, transfer 150 μL of each supernatant to a new plate, and add 40 μL/ well of PEG-NaCl solution. 10. Incubate the plate 1 h at 4  C, and centrifuge 3220 g for 1 h at 4  C. 11. Completely remove the supernatant ensuring not to touch the pellet and suspend each pellet in 150 μL of PBS. 12. Shake the plate for 5 min under 500 rpm, and centrifuge 3220 g for 10 min at 4  C to pellet remaining bacteria. 13. Coat each well of a high-binding ELISA plate with an antiM13 (pVIII specific) antibody of a species different to the used serum at 4  C overnight (see Note 16). 14. Discard the content on the ELISA plate and add 350 μL/well of 2% M-PBST. 15. Add 50 μL/well of 2% M-PBST (except well H9), and then add 50 μL of the supernatant from the phage production plate (step 12), diluting the phage 1:2 . On well H9, add 3  108 cfu of Hyperphage as a negative control and incubate for 1.5 h at RT to capture the monoclonal oligopeptide phage. 16. Wash the plate 3 times with PBST. 17. Dilute the serum according to a previously determined dilution (e.g., titration ELISA on cell lysate) in 2% M-PBST, and add 100 μL/well on each well, except H12. On H12, add 100 μL of 2% M-PBST only. (see Note 16). 18. Incubate 1.5 h at room temperature, and wash the plate 3 times with PBST. 19. Dilute an appropriate detection antibody-HRP conjugate in 2% M-PBST, and add 100 μL/well on each well, except H12. On H12, add 100 μL of an anti-M13 (pVIII) HRP-conjugated antibody in 2% M-PBST. 20. Incubate 1 h at room temperature, and wash the plate 3 times with PBST. 21. Add 100 μL/well of TMB ELISA developing solution and incubate at room temperature until single wells exhibit a significant blue color (5-30 min). Stop the reaction by adding 100 μL/well of 1 N H2SO4 (the blue color will turn yellow). Acquire the data with an ELISA plate reader at 450 nm, using 620 nm as a reference wavelength. 22. Sequence the positive clones and align them to the target genome. For metagenome libraries, the hit sequences can be blasted. Both could be done on DNA or protein level. 23. A validation of the hits is recommended. Produce the corresponding protein recombinantly and validate the binding on protein level in an ELISA.

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Notes 1. The amplified DNA appears as smear between about 15 kb and 1 kb. The genomic template DNA appears as rather distinct band >20 kb. 2. Check the manufacturer’s FAQ’s for the use of CIP on blunt ends. Some manufacturers recommend adjusted protocols for the dephosphorylation of blunt ends (NEB recommends 50  C for dephosphorylation of blunt ends). 3. Ligase inactivation is crucial. Skipping this step will negatively influence the transformation rates. 4. Transformation rates between >107 clones per transformation are expected. 5. It is important to scrape all colonies as well as possible. 6. Colony PCR can be analyzed by agarose gel electrophoresis or capillary electrophoresis like on Qiaxcel Advance system. There will be a certain distribution of insert size depending on cut-offs used for library construction. The better the resolution of the used technique the more precise is the estimation of mean insert size. We usually prefer to use capillary electrophoresis. 7. There is one rule of thumb: The higher the insert rate the better the performance of ORF enrichment. However, we found the ORF filtering to be quite efficient even with libraries of 50% insert rate and less. 8. If the supernatant still contains bacteria consider another centrifugation step as this will alleviate the filtration step after the first precipitation. 9. Depending on the library you should expect phage titers >1010. 10. Mean insert size often decreases after ORF enrichment. 11. Optional: Pre-clearance of the library with non-relevant serum antibodies to remove non-specific binders from the library. Therefore, immobilize the serum capture antibody in another 2 wells and perform antibody capturing in parallel to the capturing in the selection wells. Perform pre-clearance of the library in parallel to the pre-clearance step of the serum used for selection (1 h immobilization and 2 h pre-clearance). 12. Washing cycles can be increased with the panning rounds (1st round: 10 cycles, 2nd round: 20 cycles, 3rd round: 30 cycles). 13. Typical dilutions are 103–104 total cfu in panning round 1 and 106–107 total cfu in panning round 2.

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14. Consider storing 2  1 mL of the scraped at
80  C (supplemented with 20% (v/v) glycerol). 15. Alternatively, this step can be performed at 34  C overnight. 16. This can be prepared in parallel with the phage production. 17. If you experience high background, consider further dilution of the serum and competitive addition of E. coli cell lysate.

Acknowledgments This review contains updated and revised parts of former protocols [25]. References 1. Winter G, Milstein C (1991) Man-made antibodies. Nature 349(6307):293–299 2. Parmley SF, Smith GP (1988) Antibodyselectable filamentous fd phage vectors: affinity purification of target genes. Gene 73(2): 305–318 3. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554 4. Breitling F, Du¨bel S, Seehaus T, Klewinghaus I, Little M (1991) A surface expression vector for antibody screening. Gene 104(2):147–153 5. Fu¨hner V, Heine PA, Helmsing S, Goy S, Heidepriem J, Loeffler FF, Du¨bel S, Gerhard R, Hust M (2018) Development of neutralizing and non-neutralizing antibodies targeting known and novel epitopes of TcdB of Clostridioides difficile. Front Microbiol 9 6. Moreira GMSG, Ko¨llner SMS, Helmsing S, Ja¨nsch L, Meier A, Gronow S, Boedeker C, ˆ N, ConceiDu¨bel S, Mendonc¸a M, Moreira A c¸˜ao FR, Hust M (2020) Pyruvate dehydrogenase complex-enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies. Sci Rep 10(1):15267 7. Meyer T, Schirrmann T, Frenzel A, Miethe S, Stratmann-Selke J, Gerlach GF, Strutzberg-Minder K, Du¨bel S, Hust M (2012) Identification of immunogenic proteins and generation of antibodies against SalmonellaTyphimurium using phage display. BMC Biotechnol 12(1):29 8. Connor DO, Zantow J, Hust M, Bier FF, von Nickisch-Rosenegk M (2016) Identification of novel immunogenic proteins of Neisseria

gonorrhoeae by phage display. PLoS One 11(2):e0148986 9. Zantow J, Just S, Lagkouvardos I, Kisling S, Du¨bel S, Lepage P, Clavel T, Hust M (2016) Mining gut microbiome oligopeptides by functional metaproteome display. Sci Rep 6(1): 34337 10. Becker M, Felsberger A, Frenzel A, Shattuck WMC, Dyer M, Ku¨gler J, Zantow J, Mather TN, Hust M (2015) Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva. BMC Biotechnol 15(1):43 11. Rhyner C, Weichel M, Flu¨ckiger S, Hemmann S, Kleber-Janke T, Crameri R (2004) Cloning allergens via phage display. Methods San Diego Calif 32(3):212–218 12. Crameri R, Walter G (1999) Selective enrichment and high-throughput screening of phage surface-displayed cDNA libraries from complex allergenic systems. Comb Chem High Throughput Screen 2(2):63–72 13. Kodzius R, Rhyner C, Konthur Z, Buczek D, Lehrach H, Walter G, Crameri R (2003) Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays. Comb Chem High Throughput Screen 6(2):147–154 14. Miltiadou DR, Mather A, Vilei EM, Du Plessis DH (2009) Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) by using phage display. BMC Microbiol 9:215 15. Fehrsen J, du Plessis DH (1999) Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium. Immunotechnology Int J Immunol Eng 4(3–4):175–184

Biomarker Discovery by ORFeome Phage Display 16. González E, Robles Y, Govezensky T, Bobes RJ, Gevorkian G, Manoutcharian K (2010) Isolation of neurocysticercosis-related antigens from a genomic phage display library of Taenia solium. J Biomol Screen 15(10):1268–1273 17. Ballmann R, Hotop S-K, Bertoglio F, Steinke S, Heine PA, Chaudhry MZ, Jahn D, Pucker B, Baldanti F, Piralla A, Schubert M, ˇ icˇin-Sˇain L, Bro¨nstrup M, Hust M, Du¨bel S C (2022) ORFeome phage display reveals a major immunogenic epitope on the S2 subdomain of SARS-CoV-2 spike protein. Viruses 14(6): 1326 18. Ku¨gler J, Nieswandt S, Gerlach GF, Meens J, Schirrmann T, Hust M (2008) Identification of immunogenic polypeptides from a mycoplasma hyopneumoniae genome library by phage display. Appl Microbiol Biotechnol 80(3): 447–458 19. Stratmann T, Kang AS (2005) Cognate peptide-receptor ligand mapping by directed phage display. Proteome Sci 3(1):7 20. Hust M, Meysing M, Schirrmann T, Selke M, Meens J, Gerlach G-F, Du¨bel S (2006) Enrichment of open reading frames presented on bacteriophage M13 using Hyperphage. BioTechniques 41(3):335–342

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21. Rondot S, Koch J, Breitling F, Du¨bel S (2001) A helper phage to improve single-chain antibody presentation in phage display. Nat Biotechnol 19(1):75–78 22. Soltes G, Hust M, Ng KKY, Bansal A, Field J, Stewart DIH, Du¨bel S, Cha S, Wiersma EJ (2007) On the influence of vector design on antibody phage display. J Biotechnol 127(4): 626–637 23. Forsgren A, Sjo¨quist J (1966) ‘Protein a’ from S. Aureus: I. pseudo-immune reaction with human γ-globulin. J Immunol 97(6):822–827 24. Ramli SR, Moreira GMSG, Zantow J, Goris MGA, Nguyen VK, Novoselova N, Pessler F, Hust M (2019) Discovery of Leptospira spp. seroreactive peptides using ORFeome phage display. PLoS Negl Trop Dis 13(1):e0007131 25. Zantow J, Moreira GMSG, Du¨bel S, Hust M (2018) ORFeome phage display. In: Hust M, Lim TS (eds) Phage display: methods and protocols. Springer, New York, pp 477–495 26. Johnston S, Ray DS (1984) Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin. J Mol Biol 177(4):685–700

Chapter 28 Mapping Epitopes by Phage Display Stephan Steinke, Kristian Daniel Ralph Roth, Ruben Englick, Nora Langreder, Rico Ballmann, Viola Fu¨hner, Kilian Johannes Karl Zilkens, Gustavo Marc¸al Schmidt Garcia Moreira, Allan Koch, Filippo Azzali, Giulio Russo, Maren Schubert, Federico Bertoglio, Philip Alexander Heine, and Michael Hust Abstract Monoclonal antibodies (mAbs) are valuable biological molecules, serving for many applications. Therefore, it is advantageous to know the interaction pattern between antibodies and their antigens. Regions on the antigen which are recognized by the antibodies are called epitopes, and the respective molecular counterpart of the epitope on the mAbs is called paratope. These epitopes can have many different compositions and/or structures. Knowing the epitope is a valuable information for the development or improvement of biological products, e.g., diagnostic assays, therapeutic mAbs, and vaccines, as well as for the elucidation of immune responses. Most of the techniques for epitope mapping rely on the presentation of the target, or parts of it, in a way that it can interact with a certain mAb. Among the techniques used for epitope mapping, phage display is a versatile technology that allows the display of a library of oligopeptides or fragments from a single gene product on the phage surface, which then can interact with several antibodies to define epitopes. In this chapter, a protocol for the construction of a single-target oligopeptide phage library, as well as for the panning procedure for epitope mapping using phage display is given. Key words Epitope mapping, Phage display, Monoclonal antibodies, Panning, Paratope, Epitope, Biomarkers

1

Introduction Antibodies or B-cell receptors, as well as T-cell receptors, are key molecules of the adaptive immune system since they specifically recognize non-self or altered self-structures, called antigens [1, 2]. These antigens can have different biochemical compositions (e.g., lipids, carbohydrates, nucleic acids, etc.), but the most common ones are proteins, peptides, or glycans, usually derived from pathogens. Under certain pathological circumstances, self-antigens can also be targeted by the immune system, leading to autoimmune

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6_28, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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diseases, or as part of responses against certain types of cancer [3– 5]. Among the molecules of the immune system, the antibodies play an important role in the host organism, since they specifically recognize antigens and either act directly or trigger further immune responses against them [6]. The binding of an antibody to its target is usually highly specific, meaning that normally one antibody can only recognize a certain part of the antigen; in rare cases, however, antibodies can bind to different epitopes, in a phenomenon called multi- or polyspecificity [7, 8]. Such binding specificity is driven by a defined part of their structure, called “paratope”, which consists of amino acids contained in the complementarity-determining regions (CDRs) that go through affinity maturation in order to become highly selective. The recognized target, in turn, also has a small part of its structure, called “epitope,” which is the counterpart of the paratope [9, 10]. Therefore, an epitope is an amino acid sequence that allows the interaction with the paratope via non-covalent interactions (i.e., ionic interactions, hydrogen bonds, hydrophobic interactions, etc.) [11]. In principle, there are two possible kinds of epitopes: continuous, and discontinuous. The former type is characterized by amino acids that are very close to each other in the protein sequence (usually among a sequence of 4–30 amino acids), while the latter contains amino acids that are far from each other on the primary structure, but very close on the tertiary or quaternary structure. Although this epitope nomenclature has been used extensively in the literature, it is sometimes very difficult to distinguish between these types, as can be illustrated by the definition of a “hybrid epitope,” which can be formed in specific stretches that contain secondary structures [12–14]. Hence, since secondary structures bring together amino acid residues that are not directly neighbored in the amino acid sequence, the distinction into different epitope types in the literature remains an open topic for research. Due to their high specificity and stability, antibodies, especially monoclonal antibodies (mAbs), are valuable molecules for therapy against cancer, autoimmune diseases, or infections [15–17]. For infectious diseases, knowing the epitopes recognized by antibodies from the host against a pathogen allows their use in vaccine development [18, 19], as well as permits the use of a mAb against certain epitope as a therapeutic molecule [20]. In the diagnostic field, mAbs are used for the direct detection of pathogens or other biomarkers with diagnostic value. In most mAbs applications, it is advantageous to define the epitope, since it may be crucial not only to enhance the performance of diagnostics, therapy, or vaccines [21, 22], but also to understand the nature of immune responses [23]. The principle of all techniques available for epitope mapping is to provide a target protein or oligopeptide that can be tested against a certain mAb. From the interaction of this antigen-antibody pair, the different methods differ in their practicality and, most importantly,

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resolution to define a minimum number of amino acids that are essential for the interaction. The most used techniques for this purpose are site-directed mutagenesis, high throughput mutagenesis, array-based oligopeptide scanning, mass spectrometry, and X-ray co-crystallography. The site-directed mutagenesis consists of adding mutations on a gene in a way that some amino acids will be changed [24]. This way, the role of this amino acid can be verified by testing the modified protein against the studied antibody and checked for changes in reactivity. Although it allows the study of both continuous and discontinuous epitopes, only few mutations can be added at a time in order to allow proper conclusions. Thus, it is essential to have previous information of the binding region, making this method very laborious, timeconsuming, and not standalone. The high throughput mutagenesis tries to overcome these problems since a library is generated containing mutations on every position of a certain target [25]. This way, each variant is expressed and tested against the studied mAb. In an array-based oligopeptide scanning, overlapping and non-overlapping peptides are synthesized and immobilized on a surface (e.g., on chips, plates, or nitrocellulose membranes) that allow antibody testing [26]. There is also the possibility to combine different peptides or modify them in a way that discontinuous epitopes can also be mapped [27, 28]. Although the immobilized peptides can be used to characterize different antibodies, this method is less practical due to its relatively high cost and need of a set of complex techniques to be performed. Mass spectrometrybased epitope mapping is usually performed in-solution, where antibody and antigen are mixed together to form an antibodyantigen complex. Afterward, proteolytic degradation of the antigen is performed, followed by various washing steps to remove unbound parts. Specifically bound antigen peptides are then isolated from the antibody and subsequently prepared for the mass spectrometric analysis [14]. Among the above-mentioned techniques, X-ray co-crystallography is considered the gold standard for epitope mapping, since it can give interaction information not only on amino acid level, but also on atomic level [29, 30]. The method consists of mixing both the antigen and the studied antibody in optimal concentrations with a suitable buffer for the development of a two-protein (antigen-antibody) crystal, which is then diffracted with an X-ray source to determine the tridimensional structure of the protein complex. Even though crystallography is the approach that gives the most refined and reliable data, the development and diffraction of crystals is still a limitation, mainly when the target has special characteristics, e.g., when it is a lipophilic or a highly flexible protein. So far, although these techniques have been shown to be effective, none is considered an easy-to-do method that can be performed in most of the situations. This often leads to the search for new alternatives, such as bioinformatic

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analysis [31] or adaptation of other procedures, e.g., H/Deexchange mass spectrometry [32, 33]. Since most of these techniques need a lot of resources and time, a fast and cheap epitope mapping alternative is still required, which can help solve and/or complement the information for epitope mapping. Currently, phage display technology is being extensively used to generate useful antibodies for diagnostics, therapy, or basic research [34–38]. However, it was primarily used to present small proteins and peptides on the phage surface, which led to the Nobel Prize in Chemistry in 2018 [39, 40]. Due to the high-throughput nature of this method, libraries of peptides or parts of proteins could be used for pathogen research, mainly for the identification of novel biomarkers for diagnostics, therapy, or even vaccine applications [41]. The same approach allows protein-protein interaction studies, such as antigen-antibody binding. Moreover, the main advantage of this technique is that both the phenotype (oligopeptide on phage surface) and genotype (coding sequence inside the phage) are present in the same system while keeping relatively high library size and diversity when compared to other display methods [42, 43]. This way, it was possible to adapt the technology for the display of oligopeptides encoded by DNA fragments from different organisms and sources [44, 45]. Because phage display technology allows the presentation of many different oligopeptides in a library scale with virtually any source of DNA, it is attractive for epitope mapping applications, since different parts of a certain target can be displayed [46]. In addition, by having coupled phenotype and genotype, further techniques, such as the site-directed mutagenesis, can be easily used in combination with phage display to refine the results [47]. The use of phage display for epitope mapping is based on two strategies regarding the used library: (1) the use of a random peptide library, (2) or a library containing parts of only one defined target [48]. In the former, small oligopeptides (approximately 20 amino acids) with random sequences are displayed and used to perform panning against the studied mAb. However, since these oligopeptides are random and not directly related to the actual target, the resulting sequences show conserved properties, but have to be analyzed carefully to determine the corresponding parts on the target [49]. On the other hand, the latter strategy, which uses libraries containing sequences of a single target (called “single-gene library”), can provide more reliable information regarding the recognized epitope, since parts of the antigen can be directly defined as the epitope without further complex analysis. Besides this single-target approach, it has been shown that an epitope mapping with a genome library, built with the ORFeome phage display is also possible [21]. The protocol describing ORFeome phage display is shown in the previous chapter of this book. However, mapping epitopes with a single-gene library is more precise, because it shows only epitopes for one particular

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antigen, and therefore the collection and data analyses are simpler. Although the literature describes the epitope mapping by phage display as being more reliable for linear epitopes, there are also data describing its effectiveness for conformational ones [50, 51]. Ultimately, the high versatility of phage display allows using this method to perform a complete research pipeline over antibody and antigen interactions, making it a standalone technique for the discovery of biomarkers, antibody generation, and epitope mapping [52]. This protocol is an improved and updated version of the epitope mapping protocol described by Moreira et al. [53]. It shows a step-by-step guide, starting with single-gene library construction until the interpretation of epitope data.

2

Materials

2.1 Antigen Library Construction

1. Primers for gene amplification (individually designed for each target by the researcher). 2. Phusion DNA polymerase + buffer 5
(NEB, Frankfurt, Germany). 3. dNTP mix (10 mM each). 4. Agarose. 5. TAE-buffer 50
: 2 M Tris–HCl, 1 M acetic acid, 0.05 M EDTA, pH 8.0. 6. NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA). 7. Gel and PCR purification kit (Macherey-Nagel, Du¨ren, Germany). 8. Sonicator Bioruptor® Plus Sonication System (Diagenode, Seraing, Belgium). 9. Amicon® Ultra-0.5 mL Centrifugal Filters Ultracel®-30 K (Millipore). 10. Fast DNA End Repair kit (Thermo Fisher Scientific). 11. Phagemid (in this protocol the pHORF3 [41] is used). 12. PmeI endonuclease + buffer (NEB). 13. Calf intestine phosphatase (CIP; NEB). 14. T4 ligase + buffer (Promega). 15. E. coli TOP10 F0 (Thermofisher), genotype: F0 {lacIq, Tn10(TetR)} mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG. 16. MicroPulser Electroporator (Bio-Rad).

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17. SOC medium pH 7.0: 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05% (w/v) NaCl, 20 mM Mg solution, 20 mM glucose (sterilize magnesium and glucose separately, add solutions after autoclavation). 18. Ampicillin (100 mg/mL stock). 19. Kanamycin (50 mg/mL stock). 20. Tetracyclin (20 mg/mL stock). 21. 2 M Glucose (autoclaved). 22. 2xTY medium: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl. 23. 2xTY-glycerol: 1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl, 16% (v/v) glycerin. 24. 2xTY-T: 2xTY, 20 μg/mL tetracycline. 25. 2xTY-GA: 2xTY, 100 mM glucose, 100 μg/mL ampicillin 26. 2xTY-GA agar plates: 2xTY-GA, 1.5% (w/v) agar-agar. 27. 2xTY-AK: 2xTY, 100 μg/mL ampicillin, 50 μg/mL kanamycin. 28. Single-use Drigalsky spatulas. 29. 10-cm Petri dishes. 30. 24.5
24.5
2.5 cm plates. 31. 2 mL cryovials. 32. Liquid Nitrogen. 33. 80  C freezer. 34. Hyperphage for oligovalent display (Progen, Heidelberg, Germany). 35. 1 mL cuvettes and spectrophotometer with 600 nm wavelength. 36. Taq DNA polymerase + buffer 5
(Promega, Heidelberg, Germany). 37. 100 and 500-mL glass shake flasks. 38. 50 mL tubes. 39. Incubator for shake flasks. 40. Refrigerated centrifuge with holders for 15 and 50 mL tubes and plates. 41. Sorvall Centrifuge RC5B Plus, rotor GS3 and SS34 (Thermo Fisher Scientific), and respective tubes. 42. Polyethylenglycol-Sodium Chloride (PEG-NaCl) solution: 20% (w/v) PEG 6000, 2.5 M NaCl. 43. Phosphate buffered saline (PBS) pH 7.4: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4.2H2O, 0.24 g KH2PO4 in 1 L.

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44. Phage dilution buffer (PDB) pH 7.5: 10 mM Tris–HCl, 20 mM NaCl, 2 mM EDTA. 45. E. coli XL1-Blue MRF‘(Agilent, Santa Clara, CA, USA), genotype: Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F0 proAB lac IqZΔM15 Tn10 (Tetr)]. 2.2 Antigen Panning and Screening

1. 96-well ELISA Costar plate (Corning). 2. PBS. 3. PBS-T (PBS, Tween-20 0.05% (v/v)). 4. Panning block solution (skimmed milk powder 1% (w/v), bovine serum albumin (BSA) 1% (w/v) diluted in PBS-T). 5. BioTek ELx50 plate washer (Agilent, Waldbronn, Germany). 6. E. coli TG1 (Lucigen, Middleton, WI, USA), genotype: [F0 traD36 proAB lacIqZ ΔM15] supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK- mK-). 7. E. coli XL1-Blue MRF’. 8. 1 mL cuvettes and spectrophotometer 600 nm wavelength. 9. 24-deep well plate. 10. VorTemp 56 incubator (Labnet, Edison, USA). 11. Trypsin for phage elution (10 μg/mL). 12. M13K07 helperphage for monovalent display (Agilent). 13. Refrigerated centrifuge for 15 and 50 mL tubes and plates (Eppendorf, Hamburg, Germany). 14. 2xTY-T. 15. 2xTY-GA. 16. 2xTY-GA agar. 17. 2xTY-AK. 18. 96-well U-shaped polypropylene plate. 19. hyperphage for oligovalent display (Progen). 20. Non-related phage (as negative control). 21. PEG-NaCl. 22. 2% MPBS-T (skimmed milk powder 2% (w/v), diluted in PBS-T). 23. 96-well flat-bottom polystyrene ELISA plate. 24. Anti-Fc specific HRP-conjugated (Sigma Aldrich, Mu¨nchen, Germany). 25. Anti-M13 phage (pVIII) HRP-conjugated (GE Healthcare, Mu¨nchen, Germany).

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26. TMB solutions: TMB-A: 50 mM citric acid, 30 mM potassium citrate, pH 4.1; TMB-B: 90% (v/v) ethanol, 10% (v/v) acetone; 10 mM tetramethylbenzidine; 1 mL 30% H2O2; mix 19-parts of TMB-A with 1-part of TMB-B. 27. 1 N H2SO4. 28. ELISA plate reader with 450 nm filter (Tecan).

3

Methods

3.1 Gene Amplification, Fragmentation, and End-Repair

1. Design primers for the gene of interest depending on the DNA source used (see Note 1). 2. Amplify the gene using polymerase chain reaction in duplicate (Table 1). 3. Run an agarose gel to check the amplification (band size, specificity, etc.). 4. Mix the two duplicate reactions and purify using NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel), eluting the DNA with Milli-Q water twice in different tubes, first with 30 μL and then with 20 μL. 5. Quantify the eluted DNA using NanoDrop and mix at least 1 μg in a total volume of 100 μL of Milli-Q water. 6. Fragment the DNA using the Bioruptor® Plus Sonication System (Diagenode) following the manufacturer’s instructions to obtain fragments between 100 and 500 bp. Standard program is set as: 70 times 30 s sonication with 30 s interval at low power, all at 4  C in water bath. It may be necessary to consider using gene fragments of different sizes and thus, sonication settings should change accordingly (see Note 2).

Table 1 Reagents to be added on the gene amplification PCR DNA (50 ng/μL plasmid, or 200 ng/μL genome)

1 μL

dNTP mix (10 mM each)

1 μL

5
HF buffer

10 μL

Primer forward + reverse (10 μM each)

2.5 μL + 2.5 μL

Phusion® DNA polymerase (2 U/μL)

0.5 μL

H2O Milli-Q

32.5 μL 50 μL

Total volume 



Suggested PCR program: 98 C, 30 s + 98 C, 10 s + Tm, 15 s + 72  C, 30 s/1 kb (30 cycles) + 72  C, 5 min + 4  C, forever

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Table 2 Reagents to be added on reaction for DNA-ends repair Fragmented DNA (final amount 0.8–1 μg)

X μL

10
end repair reaction mix

5 μL

End repair enzyme mix

2.5 μL

H2O Milli-Q

Up to 50 μL

Table 3 Reagents to be added on the linearization of the phagemid Phagemid (total 5 μg)

X μL

10
CutSmart buffer (NEB)

2 μL

PmeI (10 U/μL, NEB)

1 μL

H2O Milli-Q

Up to 20 μL

7. Run an 1.5% agarose gel loading 5 μL of the sample to check the actual size of the fragments (see Note 3). 8. Concentrate the fragments using Amicon Ultra-0.5 mL Centrifugal Filters Ultracel-30 K (Millipore) following the manufacturer’s instructions. 9. Quantify the DNA using NanoDrop. 10. Repair the ends of the fragment using Fast DNA End Repair kit (Thermo Scientific) according to the manufacturer’s instructions (Table 2). 11. Incubate the reaction at 20  C for 15 min (do not let it stand longer) and purify reaction using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel). Elute in 20 μL Milli-Q water. 3.2 PhagemidFragment Ligation and Library Construction

1. The preparation of the phagemid varies with the kind of phage display method used. In this protocol, it is necessary to use a phagemid that allows the cloning in a blunt end, such as pHORF3 (see Note 4), which has PmeI as cloning site. Thus, perform the digestion as described in Table 3. 2. Incubate the reaction for 1.5 h at 37  C and add 1 μL of calfintestinal alkaline phosphatase (CIP, 10 U/μL, NEB). 3. Incubate 1 h at 50  C, stop the reaction for 2 min at 80  C, and purify the reaction using the NucleoSpin Gel and PCR cleanup kit (Macherey-Nagel). Elute in 20 μL Milli-Q water. 4. Perform ligation reaction for 16 h (overnight) at 16 (Table 4).



C

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Table 4 Reagents to be added on the ligation of gene fragments with phagemid Digested 4 kb phagemid (total 1 μg) Gene fragments 150–500 bp (total 0.75 μg)

X μL a

Y μL

10
T4 DNA ligase buffer (Promega)

10 μL

T4 DNA ligase (3 U/μL, Promega)

3.5 μL

H2O Milli-Q

Up to 100 μL

a

The range 100–500 is considered because the described sonication procedure usually results in a smear of different sizes. In this case, the considered average size of the fragments is 300 bp, and thus 0.75 μg should be added. However, if obtained average size is different, it is important to maintain the molar ratio of 1:10 (vector:insert)

5. Inactivate the ligation for 10 min at 65  C and clean the reaction using Amicon® Ultra-0.5 mL Centrifugal Filters Ultracel®-30 K (Millipore). For this, add 300 μL of Milli-Q water in the reaction and centrifuge (10 min, 14,000
g). Repeat this washing with 400 μL of Milli-Q water 2 more times before collecting the final volume as instructed by the manufacturer. 6. Mix  10 μL (500 ng) of the purified ligation with 25 μL electrocompetent E. coli TOP10F’ (Thermo Scientific) in a 0.2 mL tube, transfer the volume to a 0.1 mm cuvette and keep it on ice for 1 min. 7. Perform electroporation for bacteria (Ω; 1.8 kV; pulse 5.3 ms long) and immediately add 1 mL of SOC medium pre-warmed at 37  C. 8. Transfer the cells to a 1.5 mL tube and incubate at 37  C for 1 h at 650 RPM. 9. Take 10 μL of the tube and make ten-fold dilutions until 105 in 2xYT. 10. Plate the 103 and 105 dilutions onto 2xYT-GA agar (10 cm plates) and grow it overnight at 37  C. 11. Plate the remaining  990 μL of the transformation onto a 24.5
24.5
2.5 cm plate (“pizza plate”) with 2xYT-GA agar and incubate at 37  C for 16 h. 12. Count the colonies on the 10-cm agar plates (see Note 5). 13. On the 24.5
24.5
2.5-cm plate, add 30 mL of 2xYT and incubate on a shaker for 30 min. 14. Carefully remove the cells from the medium surface with a Drigalsky spatula. Then, take the liquid-containing cells with a serological pipette and mix it gently in a 50-mL falcon tube. Distribute 900 μL in each of 6 cryovials and mix it with 300 μL 80% (v/v) glycerol.

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Table 5 Reagents to be added on the PCR for insert rate calculation dNTP mix

0.2 μL

MgCl2 25 mM

0.8 μL

5
GoTaq® flexi buffer

2 μL

Primer forward + reverse (10 mM each)

0.5 μL + 0.5 μL

GoTaq® DNA polymerase (5 U/μL)

0.05 μL

H2O Milli-Q

5.95 μL 10 μL

Total volume 



Suggested PCR program: 95 C, 5 min + 95 C, 30 s + Tm, 30 s + 72  C, 1 min/1 kb (30 cycles) + 72  C, 5 min + 4  C, forever

15. Immerse the cryovials containing the cells into liquid nitrogen and wait for 5 min. Then, carefully take the tubes and promptly store them at 80  C. 3.3 Library Quality Control and Packaging

1. From the 10-cm plates used for counting (Subheading 3.2, step 10), take at least 20 colonies to perform a colony PCR. Make one tube containing the empty phagemid used for the library construction as a negative control (Table 5, see Note 6). 2. To check the size of each fragment, prepare a 1.5% agarose gel and run the samples at 120 V for 25 min (see Note 7). 3. Count the number of positives (those above the band of the negative control) expecting to have at least 50% (10/20) of the clones positive (this quality measurement is called “insert rate”). Due to ORF-enrichment, the insert rate will be higher after phage packaging. If the number is much below 50%, consider repeating the previous steps, mainly the phagemid preparation or ligation. 4. Add 200 mL of 2xYT-GA in a 1-L shake flask. Then, take the library stored at 80  C (Subheading 3.2, step 15) and inoculate the culture to an OD600  0.1. 5. Incubate the shake flask at 37  C, 250 RPM until OD600  0.5. Then, transfer 25 mL (1.25
1010 cells) of the culture to a 50-mL tube and add 2.5
1011 CFU (MOI 1:20 ) of hyperphage. 6. Incubate the tube for 30 min at 37  C without shaking and then further 30 min at 37  C, 250 rpm. 7. Centrifuge the tube at 3220
g, 10 min, at RT. Then, discard the supernatant, suspend the cells in 25 mL of 2xYT-AK medium and transfer them to a 2000 mL shake flask containing 400 mL of the same medium. Incubate the flask at 30  C, 250 rpm for 20–24 h.

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8. Transfer the culture to a 500-mL centrifuge tube and centrifuge at 12,000
g, 30 min, 4  C. If the supernatant is clear, transfer it into fresh 500-mL centrifuge tubes, if not repeat the centrifugation step until supernatant is clear. Add 1/5 volume (80 mL) of PEG-NaCl solution. Incubate the tubes at 4  C on ice overnight. In parallel, inoculate a 100-mL shake flask containing 20 mL of 2xYT-T with E. coli XL1-Blue MRF’ and incubate at 37  C, 250 RPM overnight. 9. Centrifuge the tube containing the supernatant with PEG-NaCl at 21,000
g, 1 h, 4  C, and discard the supernatant. 10. Suspend the pellet containing phage in 10 mL of pre-chilled PDB (phage dilution buffer) and transfer the volume to a 50-mL centrifuge tube. 11. Filter the suspension with a 0.45 μm filter and transfer to another 50 mL centrifuge tube. 12. Add 1/5 volume (2 mL) of PEG-NaCl solution and incubate overnight on ice at 4  C. 13. Centrifuge the suspension 48,000
g, 30 min, 4  C and discard the supernatant. 14. Suspend the pellet in 1 mL of PDB, transfer to a 1.5 mL tube and centrifuge at 16,000
g, 1 min. 15. Transfer the supernatant to a cryovial and store it at 4  C for further use or mix it with 20% (v/v) glycerol and store the library at 80  C. 16. Take the E. coli XL1-Blue MRF’ culture, make another 20 mL 2xYT-T culture in a 100-mL shake flask with initial OD600  0.1 and incubate at 37  C, 250 RPM until OD600  0.5. 17. Take 10 μL of the phage suspension and make 100-fold dilutions until 1010 in PBS. 18. Prepare four 1.5 mL tubes with 50 μL of E. coli XL1-Blue MRF’ cells and transfer 10 μL of the last three phage dilutions to each tube (these will be dilutions 108, 1010, 1012 on the plate). 19. Incubate the tubes at 37  C for 30 min without shaking. 20. Plate all 60 μL of each dilution on 2xYT-GA agar plates. Let the plates dry under the biological cabinet and incubate the plate at 37  C overnight. 21. Count the colonies and calculate the titer as the arithmetic mean of all dilutions, so the final result will be in CFU/mL. This quality measurement is called “library titer” (see Note 8).

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22. Perform again a quality control for the insert rates as described above (Subheading 3.3, steps 1–3). The number of positive clones should increase. 23. Sequence at least 20 positive colonies and check if they have the right sequence and are in-frame. It is good to have at least 60% (12/20) of the clones in-frame. This quality measurement is called “in-frame rate” (see Note 9). 3.4

Antigen Panning

1. Coat one well of a 96-well ELISA plate with 1.5 μg of a purified monoclonal antibody diluted in 150 μL of PBS (recommended well A1, called “mAb wells”) (see Note 10). 2. Add 300 μL of Panning Block solution in another well (recommended well A3, called “block well”) and incubate the plate at 4  C overnight. 3. On the next day, mix the single-target library with Panning Block solution to a final volume of 150 μL in a 1.5 mL tube (the final amount of phage should be around1
108 – 1
1010). 4. Clear the block and mAb wells. In the block well, add 150 μL of the library (or 5
109/well). In the mAb well, add 300 μL of Panning Block solution and incubate the plate 30 min at RT. It may be necessary to perform additional negative selections to reduce background phage enrichment (see Note 11). 5. Take the blocking solution out of the antibody well and wash three times with 300 μL of PBS-T (using the BioTek ELX50/ 8 plate washer from Agilent is recommended). 6. Transfer the phage library pre-incubated in the block well to the mAb well and incubate 1.5 h at RT. 7. Remove the library from the mAb well and wash the well, i.e., 10 times with 300 μL of PBS-T (using the BioTek ELX50/ 8 plate washer from Tecan). 8. Elute the binding phage by adding 200 μL of 10 μg/mL trypsin diluted in PBS for 30 min at 37  C. 9. Store the eluted phage at 4  C in 0.2 mL tubes. Before presiding to step 11 read Note 12. 10. Inoculate 25 mL of 2xYT in a 100-mL shake flask with E. coli TG1 and incubate overnight at 37  C, 250 rpm. 11. On the next day inoculate 300 μL of the E. coli TG1 overnight culture in fresh 25 mL 2xYT medium (initial OD600 ¼ 0.08–0.1). Incubate at 37  C, 250 rpm for 1.5 h until OD600  0.5 and use for step 12. 12. In a 24-deep well plate, add 1 mL of E. coli TG1. Then add 100 μL of eluted phage from the previous panning round into

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the well and incubate 30 min at 37  C without shaking, followed by 30 min at 37  C and 450 rpm. 13. Centrifuge the plate at 2500
g for 10 min at RT. Discard the supernatant, add 5 mL of pre-warmed 2xYT-GA and incubate at 37  C and 450 rpm for 30 min until OD600  0.5 is reached. Then, add the helperphage M13K07 (5
1010 total, MOI 1:20 ) for 30 min at 37  C without shaking. Then, incubate 30 min at 37  C and 450 rpm. 14. Centrifuge the plate at 2500
g for 10 min at RT. Remove supernatant completely (be careful with the pellet). Add 5 mL 2xYT-AK, suspend the pellet and incubate at 30  C and 450 rpm overnight. 15. In addition, prepare the panning plates as described above in steps 1–2. 16. On the next day, centrifuge the 24-well plate (3220
g, 10 min, RT), collect and mix the supernatants in a 15 mL tube. 17. Mix 50 μL of the mixed supernatant from the first panning round with 100 μL of Panning block solution. 18. Take the contents out of the block and mAb wells. In the block wells, add 150 μL of the supernatant mixed with Panning block solution. In the mAb wells, add 300 μL of Panning Block solution and incubate the plate 30 min at RT. 19. Take the blocking solution out of the antibody well and wash three times with 300 μL of PBS-T (using the BioTek ELX50/ 8 plate washer from Tecan). 20. Transfer the supernatant from the block well to the mAb well and incubate 1.5 h at RT. 21. Remove the library from the mAb well and wash the well 10 times with 300 μL of PBS-T (using the BioTek ELX50/ 8 plate washer from Tecan). 22. Elute the binding phage by adding 200 μL of 10 μg/mL trypsin diluted in PBS for 30 min at 37  C. 23. Store all volume of eluted phage at 4  C in 0.2 mL tubes. 3.5 Monoclonal Phage Production and Screening

1. Inoculate 25 mL of 2xYT-T medium with a culture of E. coli XL1-Blue MRF’ in a 100-mL flask and incubate overnight. 2. On the next day, inoculate 300 μL of E. coli XL1-Blue MRF’ overnight culture in 25 mL 2xYT (OD600 ¼ 0.08–0.1). Incubate at 37  C, 250 rpm for 1.5 h until OD6000.5 and use for step 3. 3. Use the eluted phage from the previous panning rounds and make two ten-fold dilutions by adding 20 μL of the infected E. coli XL1-Blue MRF’ into 180 μL of 2xYT.

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4. Spread 25 μL from the first dilution and 50 μL from the second dilution onto 2xYT-GA agar in 10-cm plates. Store the dilutions at 4  C until the next day (see Note 13). 5. Centrifuge the 24-well plate at 2500
g for 10 min at RT, discard 1 mL of the supernatant, suspend the pellet in the remaining medium volume, and spread it onto one 10-cm plate with 2xYT-GA agar as a backup (see Note 14). 6. Incubate all the plates at 37  C overnight and then store them at 4  C or directly start working on the next day (step 8). 7. In a 96-well U-bottom propylene plate, add 150 μL/well of 2xYT-GA. 8. Use 200 μL pipette tips to pick 92 colonies from the agar plates described in step 6. In this same 96-well plate, include 2 wells (H3 and H9) with medium only, 1 well (H6) with a colony to produce a non-related phage (hyperphage), and 1 well (H12) with the same colony added in H11. A suggested design for this plate is shown below. 9. Add a breathable membrane over the plate and incubate at 37  C, 800 RPM, for 6 h (this will be called “Master plate”) (see Note 15). 10. In another 96-well U-shaped propylene plate, add 160 μL/ well of 2xYT-GA and transfer 20 μL from the previously grown plate to this new one. Store the master plate at 4  C and incubate the new one at 37  C, 800 rpm, for 2 h. 11. Dilute purified hyperphage in 2xYT to the concentration of 2
1011 CFU/mL and add 50 μL/well (2
109 CFU/well). 12. Incubate 30 min at 37  C without shaking, followed by 30 min at 37  C and 800 rpm. 13. Centrifuge the plate at 3200
g for 10 min at RT, remove the supernatant by inverting the plate very quickly over a discard and add 180 μL/well of 2xYT-AK. 14. Incubate the plate at overnight 30  C, 800 rpm. 15. Centrifuge the plate at 3200
g for 10 min at RT. 16. Add 50 μL blocking solution and 50 μL of the supernatant from the plates centrifuged in the previous step (step 15) on the ELISA plates diluting the phage 1:2 (Note 16). 17. Incubate the ELISA plates for 1.5 h at RT and wash the plates 3 times with 300 μL/well of dH2O-Tween. 18. Add 100 μL HRP-conjugated goat anti-M13 (pVIII) antibody (1,40,000) (see Note 17). and incubate at RT for 45 min. 19. Wash the plates 3 times with 300 μL/well of dH2O-Tween. 20. Add 100 μL/well of TMB ELISA developing solution and let it stand for 15 min at room temperature. Then, stop the reaction by adding 100 μL/well of 1 N H2SO4. Acquire the

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data with an ELISA plate reader at 450 nm, using 620 nm as a reference wave length (see Note 18). 3.6 Selection and Sequencing of Positive Hits and Epitope Determination

1. Based on the signal obtained in the ELISA, set the maximum signal as 100%. Then, classify the clones according to their signal as low (10–30%), medium (30–70%), and high (70–100%) reactions (see Note 19). 2. For each panning round, select colonies from all three groups (low to high signal). 3. Take the Master plate stored in Subheading 3.5, step 4 and use it as source of the selected colonies to send them for sequencing. 4. Analyze the DNA sequences checking for their quality, i.e., if there are out-of-frame sequences or premature stop codons discard these sequences (see Note 20). 5. With the remaining sequences, perform their translation with, e.g., TranSeq tool (EBI, https://www.ebi.ac.uk/Tools/st/ emboss_transeq) to obtain the corresponding amino acid sequence (see Note 21). 6. Align all the amino acid sequences with ClustalOmega tool (EBI, http://www.ebi.ac.uk/Tools/msa/clustalo) and define the regions with high identity and similarity. 7. Select a region with no more than 25 amino acids as the minimal epitope region (see Note 22).

4

Notes 1. If using a plasmid as source of the gene, it is recommended to design primers annealing to the plasmid, but near the gene. This way, it is possible to amplify different genes if the same plasmid is used. If using genomic DNA as source of the gene, it is recommended to design primers annealing to the gene. 2. This way, by setting the sonicator to 150 bp, it is expected to have most of the displayed peptides with 50 amino acid, but also some smaller (20 amino acids) and some bigger (100 amino acids). However, it is worth considering that certain structures of the epitope can only be formed by augmenting the size of the displayed peptide. If it occurs, an epitope can only be mapped by building libraries with bigger DNA fragments. In this case, it is recommended to build multiple libraries in parallel, e.g., one with 150 bp fragments, another with 400 bp fragments and another with 1250 bp fragments. Although this alternative can allow the detection of fragments containing epitopes, it is essential to know which kind of

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epitope is being searched (continuous, discontinuous, etc.) and determine if this technique will be useful. 3. Usually, the DNA is seen as a smear with a concentrated band on the expected size. However, it can happen that the smear is broader with no concentrated band or the concentrated band is slightly above the expected size. In these cases, it is accepted to work in a range of up to 500 bp (not more than this). If the smear or a band has >500 bp, the sonication conditions should be optimized. 4. This protocol is written based on the pHORF3 system for ORFeome display [54, 55]. Briefly, this phagemid allows cloning fragments in a blunt PmeI restriction site. This way, cloned fragments that are in frame will be expressed in fusion with pIII, allowing the peptide corresponding to the cloned fragment to be displayed on the surface of the phage particles. 5. The expected number of independent clones is 106–108. This amount is actually above the number of clones necessary to cover the gene length. For example, a gene with 1200 bp would need 2100 cloned fragments of 150 bp to completely cover its sequence when walking one nucleotide upstream at a time. It means that a titer of 2.1
103 is enough to cover the gene. This number is obtained by the following formula: N ¼ 2
ða  b þ 1Þ N is the number of cloned fragments (independent clones); a is the size of the gene; and b is the average size of the fragmented DNA. The calculation considers that the fragments can be cloned in 2 possible orientations in the blunt-end ligation. This is why the number of cloned fragments needed to cover the gene (a – b + 1) is multiplied by 2. This formula is just an approximation for the researcher in a simple manner. If specific applications are needed, the formula is to be modified accordingly 6. The pair of primers used for colony PCR (including negative control) depends on the phagemid system. However, regardless of the phagemid used, it is recommended to have primers annealing on the phagemid backbone and have enough bp distance from the restriction site used for cloning. This way, the negative control will be well distinguishable from positive clones, which should be above this size. 7. This step can sometimes require better resolution. Initially, it is possible to optimize the running and imaging conditions to detect small size differences between the fragmnts, i.e. increase agarose gel concentration or running with lower voltage. Nevertheless, the optimal evaluation of the band size is done with

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capillary electrophoresis (e.g. using QIAxcel Advanced System, QIAGEN, Hilden, Germany). 8. The expected titer is 103–109 CFU per mL. If the titer is much below this value, consider repeating the phage production (steps 4–16). 9. The forward primer of the colony PCR can be used for sequencing. Anyway, the sequences should be analyzed regarding the reading frame of the cloned fragments, which should be in-frame with the gIII contained in the vector (e.g., frame “+2” in pHORF3 vector). Sequences containing only parts of the vector, if present, should be counted as negative. After the analysis, the number of positives (i.e., in-frame sequences) should be about 60%. If this number is much lower, e.g., 30% consider repeating the sequencing with 20 additional clones. If the low in-frame rate persists, try repackaging your library or consider to reset the library construction. 10. Normally, coating mAbs directly onto the ELISA plate surface is not a problem. Nevertheless, some antibodies can have their activity considerably reduced or even eliminated after attaching to the plastic surface. If this is the case, coat the plate for 1 h at RT with 100 μL/well of an antibody against the Fc part of the studied mAb diluted in PBS to 2–4 μg/mL, and block by adding 300 μL/well of 2% MPBS-T for 30 min prior to adding the mAb. 11. To improve the ratio of specific and reduce the effect of unspecific fragments. Perform a competition with an unrelated control antibody. Add 5 μg of the control antibody to the pre-incubated library and perform the preincubation of the library as usual. 12. Normally, one panning round is sufficient to find specific epitopes for the tested antibodies. However, it may be necessary to perform an additional panning round in case of low hit rates. It must be considered that a second panning round can decrease the diversity of the detected epitopes. Nevertheless, it can be used as partial confirmation of the results obtained in the screening of the first panning round. For a second panning round, proceed to step 11. 13. Considering that the number of phage containing the epitope, or epitopes, of the studied antibody can be highly variable (especially in the panning round 1), there is no rule for the number of phage eluted in the first and second round. This way, the number of colonies obtained after diluting and spreading the infected bacteria on agar plates is variable. The protocol suggests a volume that is more likely to give the researcher enough colonies to start a screening which is at least 46 isolated colonies for each panning round. However, if the plates have too few or too many colonies, the spreading step should be

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repeated using the dilutions stored at 4  C in this same step. In this case, the spread volume should be adjusted accordingly. 14. The backup plates can be stored at 4  C until the end of the screening. If the epitope mapping procedure is considered successful, the backup plates contain most of the selected clones. Thus, it is recommended to store these clones longterm, considering that further studies can become interesting as the researcher’s work goes on. For this, add 5 mL of 2xYT + 16% glycerol over the plate and use a Drigalski spatula to scrape the cells from the plate. Then, take 1 mL of the scraped cells, add into cryovials (make at least 2 cryovials) and store them promptly at 80  C. 15. The growth of colonies in the 96-well U-shaped polypropylene plate can also be done overnight. The step described in the protocol uses 6 h of incubation to reduce the time of the procedure by 1 day, but it is optional. 16. Although during the phage production most of the E. coli cell contents (e.g., cell debris, proteins, etc.) are removed by centrifugation, it is possible that they are still present in the final preparation. Usually, the high specificity of the studied mAb allows ignoring these “undesired” protein contents. However, if high levels of background are noticed after the screening (i.e. high reactions with the non-related phage used as negative control), it is recommended to immobilize the phage using anti-phage antibody. In this case, coat the plate for 1 h at RT with 100 μL/well of goat anti-M13 (pVIII) antibody (Sigma Aldrich) diluted in PBS to 2–4 μg/mL. Then, block by adding 300 μL/well of 2% MPBS-T for 30 min prior to adding the phage. It is also possible to test the produced monoclonal phage against a negative control antibody (usually the one used for negative selection) in order to reduce false positive hits. 17. The specifications of the used anti-Fc specific antibody depends on the properties of the mAb used in the procedure. Usually, the studied mAbs are IgG and have human or mouse Fc parts, allowing the use of goat anti-mouse or human IgG Fc HRP-conjugated antibodies. If the studied mAb has another isotype (IgA, IgE, IgM, etc.) or if the Fc part is from another species (rat, rabbit, etc.), the researcher should choose the best HRP-conjugated option for the work. 18. As mentioned in the introduction of this chapter, the complete applications of this technique are not completely defined. At the moment, it is known that continuous epitopes have higher chances to be mapped compared to discontinuous ones. Therefore, if the result of the screening ELISA is negative (even in the repetition), it is more likely that the studied mAb recognizes a conformational epitope that cannot be mapped by this

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procedure. However, it is worth trying the alternative described on Note 2 before discarding the use of this method. 19. This classification has the aim to individualize the analysis to every antibody-antigen pair, considering that each pair may show different interaction strengths that affect the maximum signal. It is also possible to perform the analysis as signal-tonoise ratio in case a plate with a negative antibody control is used. In this case, it is necessary to carefully determine a cut-off for the ratio, since different antibody-antigen pairs may respect different patterns, hence not allowing the same cut-off to be used for every pair. A third option is to combine both interpretation approaches. In any case, a possible production bias must be considered because some clones may be better producible than others and therefore, have lower or higher signals in the ELISA. This means that the signal intensity does not represent better or worse binders, but simply antigen molecules that are produced in more or less amount and, therefore, affect the signal intensity. Thus, the ranking of binders at this stage should be as broad as possible. 20. Usually, it is expected that every sequence is useful for the analysis. But beware that in some cases not all sequences are suited for analysis (e.g., out-of-frame, stop codons, bad sequencing profiles, etc.) and therefore should be excluded to obtain a good analysis of the epitope map. If the majority of sequences are unreliable, consider to re- sequencing the samples or picking new colonies from the master plate for a new sequencing. 21. In this step, it is important to observe the size of the obtained amino acid sequences. The smaller the fragments are, the more refined the epitope mapping will be. Thus, having at least one sequence with not more than 50 amino acids (one with 100 amino acids) are obtained, it is suggested to select new colonies for sequencing from the Master plate. 22. For the definition of the final epitope, it is better if the analyzed sequences contain at least one with 1 × 1012, diluted 1:10 in 2% MPBS for each Ag being screened. 100 μL of diluted library is added to each well of the plate and incubated for 1 h at room temperature with shaking at ~600 rpm on a microplate shaker. If the phagemid contains a trypsin cleavage site between the scFv and phage coat III protein gpIII, then after rigorous washing (10 times) with PBST, bound phage can be eluted by an addition of 100 μL per well of 1× trypsin in 1× PBS buffer and incubating with shaking at ~600 rpm on a microplate shaker at room temperature for 10 min. (see Note 2) 4. Transfer the eluate in a new 96-well deep-well plate containing 200 μL per well exponentially growing E. coli TG1 (OD600 of 0.4–0.6). Transduce for 30 min at 37 °C without shaking. (see Note 3) 5. Pellet the transduced cells by spinning the 96-well deep-well plate at 3000 rpm in a Sorvall RT-7 with RTH-750 rotor for 10 min. Pour off excess supernatant gently so as to not disturb the pellet. 6. Resuspend the pellet in each well with 600 μL 2YT medium supplemented with ampicillin (100 g μL-1) and 1% glucose. Cover the 96-well deep-well block with a plastic plate seal and grow with shaking at ~600 rpm on a microplate shaker at 30 oC overnight. 7. The next day: for each well, dilute 5 μL of the overnight cultures into 200 μL fresh 2YT medium containing 100 μg μL-1 ampicillin and 1% glucose in a new 96-well deep-well plate. Cover the plate with a plastic plate seal and incubated with shaking at 37 oC until the culture OD600 reaches 0.4. (see Note 4) 8. When OD600 reaches 0.4, 16 μL KM13 helper phage (Multiplicity of Infection (MOI) of 20) or hyperphage (Progen, PRHYPE, MOI of 10) is added to each well and then incubated at 37 oC for 30 min without shaking. 9. Transduced cells are then pelleted by spinning at 3000 rpm for 10 min.

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10. Pour off excess supernatant gently so as to not disturb the pellet. 11. Resuspend the pellets using 600 μL per well of 2YT media supplemented with ampicillin (100 ug μL-1), kanamycin (50 ug μL-1), and 0.1% glucose. Cover the plate with a plastic plate seal. 12. Incubate the plate with shaking at ~600 rpm on a microplate shaker at 30 °C overnight. 13. Apply the resulting phage supernatant to another Ag-coated Maxisorp microplate. 14. This is the beginning of 2nd round bio-screening process. This whole process from step 1 to step 14 will be repeated for total of 3–5 rounds. 15. At the end of final biopanning round, we plate bio-screened phages on agar plates to pick single clones: At the end of the final round, we elute the bound phage from the biopanning plate by adding 100 μL per well with 1× trypsin in 1× PBS. 16. Incubate the plate with shaking at ~600 rpm on a microplate shaker at room temperature for 10 min. 17. Transfer the eluents from the biopanning plate to a 96-well deep-well plate. 18. Transduce TG1 cells with eluted phage by adding 200 μL of mid-log phase (OD600 of 0.4–0.6) TG1 cells to the 100 μL of eluate already in the deep-well plate. Incubate without shaking at 37 °C for 30 min. 19. Pool the transduced cells from each well in a column on the plate (corresponding to each Ag being screened on the plate). For example, for the first Ag being screened, pool column 1 (wells A1-H1). 20. Take 1 mL of pooled cells to make a serial dilution of by diluting 100-fold down to 1:1,000,000 (10-6). Plate 250 μL of each dilution (10-2 , 10-4 , 10-6 ) onto large TYE plates (150 mm diameter) supplemented with ampicillin (final concentration of 100 μg μL-1) and glucose (final concentration of 1%) individually. (see Note 5). Store the remaining pooled transduced eluates at 4 °C overnight in case this step needs to be repeated. 21. Incubate overnight at 37 °C. 22. The next day, prepare 2YT media supplemented with 100 μg mL-1 ampicillin and 1% glucose and transfer 200 μL per well into 96-well deep-well plates. One plate will be required for each Ag being screened. 23. For each Ag target, from the agar plates of transduced eluates, pick 88 single colonies into columns 1–11 of a deep-well plate

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containing the prepared media. Leave the last column (12) free of single colonies for ELISA controls. Pick a single colony from positive control plate (separately prepared) into wells A12 through D12, leaving wells E12 through H12 as mediumonly negative control wells. Cover the plate with a plastic plate seal. 24. Incubate the deep-well plate with shaking at ~250 rpm at 37 °C overnight. 25. These single clones will be analyzed by scFv expression and single-point ELISA testing to identify the best hits (See Note 6). 26. Once the mod1-specific hits are identified, scaled-up soluble scFv proteins from those hits are then expressed by inducing with IPTG and purified with standard HisPur Cobalt Resin column purification. 27. Perform standard titration ELISA to analyze purified scFv proteins (Subheading 3.4). Typical positive clones from phase I will be mod1-peptide specific antibodies as shown in Fig. 3a. 28. Best mod1-specific antibodies will be moved forward to DisMat or AffMat to in vitro envolve to NAT-specific antibodies. Alternatively, to generate a final model1-specific antibody, the positive scFv can be subcloned into IgG vectors and retested using titration ELISA following standard protocols (Subheading 3.4) and validated by applications.

Fig. 3 Simulated examples of scFv protein or full IgG protein titration ELISA results of site-specific antibodies discovered by Epivolve (a) Mod1-specific scFv or IgG protein titration ELISA results from anti-mod1 discovery phage display biopanning in phase I. A typical anti-Mod1 Ab will demonstrate specific binding to the mod1 peptide, and absence of binding to the NAT-biotin-peptide, or NAT-full-length protein, or scrambled mod1 peptide. (b) and (c) are scFv or IgG protein titration ELISA results of two different clonotypes of final antiNAT Abs: (b) a mod1-independent Ab which demonstrates binding to mod1-peptide, NAT-peptide, NAT-fulllength protein, and absence of binding to scrambled Mod1 peptide; (c) a NAT-specific Ab which demonstrates binding to NAT-peptide, NAT-full-length protein, and absence of binding to mod1-peptide and scrambled Mod1 peptide

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3.4 Site-Specific scFv Protein Titration ELISA

1. Coat the Maxisorp 96-well plates with 100 μL per well of NeutrAvidin at a final concentration of 10 μL mL-1. Incubate at 4 °C overnight. 2. Wash NeutrAvidin-coated plates three times with 250 μL per well of 1× PBS using a handheld electronic multichannel pipette or plate washer. (see Note 7) 3. Discard the last wash and gently tap the plates on a dry paper towel to remove residual PBS. Add 250 μL per well of 3% BSA in 1× PBS to block each plate using a handheld multichannel electronic pipette. Incubate the plates at room temperature for 1 h. 4. Prepare the Ag dilutions in an untreated 96-well plate. (a) Dilute each target Ag to 10 μg mL-1 in 1× PBS and add 300 μL to Row A of each corresponding column of the 96-well plate. (b) Add 150 μL of 1× PBS to Rows B-H of the 96-well plate. (c) Move 150 μL from Row A to Row B (1:2 dilution) using a multichannel pipette. Be sure to mix each well significantly by pipetting up and down several times. (d) Continue the 1:2 serial dilutions until Row G. Final Ag concentrations will be per mL: 10 μg, 5 μg, 2.5 μg, 1.25 μg, 0.62 μg, 0.31 μg, 0.16 μg. It is important to be sure to use new pipette tips for each serial dilution. (e) DO NOT add Ag to Row H. Leave row H as 150 μL 1× PBS. It is the negative control. 5. Wash the NeutrAvidin-coated plates three times with 250 μL per well of 1× PBS using BioTek plate washer. 6. Using a hand-held multichannel pipette, transfer 100 μL of the serially diluted Ags from the untreated 96-well plate to the NeutrAvidin-coated 96-well plate. In the last column of the plate, add 100 μL of each Ag at starting concentration of 10 μg mL-1 to one well for the secondary only negative control. Incubate the plates at room temperature for 1 h. 7. Wash the plates three times with 250 μL per well of 1× PBS on the BioTek plate washer. 8. Using a hand-held multichannel electronic pipette, add 250 μL per well of 3% BSA/ in 1× PBS block solution to the washed wells of the plates. Incubate the plates at room temperature for 1 h. 9. While the plates are in block, prepare the scFv dilutions in 1.7 mL microcentrifuge tubes: add 1 μg of scFv protein to 1 mL of 3% BSA in 1× PBS block solution for a final concentration of 1 μg mL-1 and mix well.

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10. Wash the Ag-coated plates three times with 250 μL per well of 1× PBS on the BioTek plate washer. 11. Using an electronic multichannel pipette, add 100 μL of scFv (at 1 μg mL-1) to Rows A-H of the corresponding column on the plate. Add 3% BSA in 1× PBS block only (no scFv) to the last column for secondary only control. Incubate the plates at room temperature for 1 h. 12. Wash the plates four times with 250 μL per well of 1× PBST on plate washer. Discard the last wash and gently tap the plates on a dry paper towel to remove residual 1× PBST. 13. Add 100 μL per well of the appropriate secondary Ab diluted 1: 5000 in 3% BSA in 1× PBS per well to all ELISA plates, including the secondary only column. Incubate the plates at room temperature for 1 h. 14. Wash the plates three times with 250 μL per well of 1× PBST on the BioTek plate washer. 15. Add 100 μL per well of Ultra TMB developing reagent (ThermoFisher, Catalog number 34028) that has come to room temperature. 16. To stop the developing reaction, add 50 μL per well of 2 M H2SO4 stop solution. 17. Place plates on Perkin Elmer Envision plate reader (or equivalent) and measure the absorbance at 450 nm. A typical result of mod1-specific Abs discovered from phase I biopanning is shown in Fig. 3a. Typical results of final NAT-specific Abs discovered from phase II and phase III biopanning are shown in Fig. 3b, c. 3.5

Error-Prone PCR

DisMat (Discovery Maturation) and AffMat (Affinity Maturation) phage libraries are used for directed evolution from mod1-specificic antibodies to either mod1-independent, or NAT-specific site-specific antibodies. Several methods for the manufacturing of directed evolution mutagenesis libraries are known. Two of the methods are described below. The first is AXM mutagenesis method which is we have previously developed [2]. This “AXM mutagenesis” method relies on the ability of T7 exonuclease to sequentially hydrolyze DNA in the 5′ → 3′ direction and its inability to hydrolyze DNA that contains several phosphorothioate groups at its 5′ terminus [20]. A large (i.e., 800 nt long), mutated DNA fragment is produced using polymerase chain reaction (PCR) conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains phosphorothioate linkages at its 5′ end. Treatment of the error-prone generated PCR product with T7 exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-

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stranded DNA segment, or “megaprimer.” The use of this megaprimer in a Kunkel-like mutagenesis reaction takes advantage of the E. coli DNA base excision repair pathway to bias nucleotide basechanges between the megaprimer and a complementary uracilated DNA sequence in favor of the in vitro synthesized megaprimer [21– 23]. A second method is the Error-Prone PCR developed by Cadwell and Joyce [24]. The protocol is as follows. 1. 10 μL of 10× mutagenic PCR buffer combined with 10 μL of 10× dNTP mix (2 mM dGTP, 2 mM dATP, 10 mM dCTP, 10 mM dTTP), 20 fmole of input DNA, 30 pmole each of forward and reverse primers, and H2O for a final volume of 88 μL. 2. 10 μL of 5 mM MnCl2 is added to the reaction, mixed well, and the absence of any precipitate need to be visually verified. 2 μL of Taq DNA polymerase is added to bring the final volume to 100 μL. Cycling conditions consist of 30 cycles of: 94 °C 1 min, 55 °C 1 min, and 72 °C 1 min. It is important to use standard Taq polymerase. It is important NOT to use a proofreading polymerase. 3. After the PCR is complete, cleanup is performed using the Qiagen QIAquick PCR Purification Kit (Qiagen, catalog number 28104) according to the manufacturer’s protocol. The purified DNA is eluted in 40 μL of Buffer EB. For scFvs template DNA, we expect a PCR amplification product of approximately 800 base pairs. 4. The EP-PCR product is then cloned into the appropriate expression vector using standard conditions. (see Note 8) 3.6 Discovery Maturation (DisMat) and Affinity Maturation (AffMat) Biopanning

Both the DisMat and AffMat biopanning can be performed using the protocol previously described (Subheading 3.3) with the following modifications.

3.6.1 DisMat Modifications

DisMat is used to convert mod1-specific Abs to anti-NAT Abs. Typically, we choose 6–10 positive mod1-specific hits to move forward to Discovery Maturation (DisMat). Three rounds of positive selecting bio-screenings are used against the NAT-biotin-peptide. And in the 2nd and 3rd round, we add a 10× concentration of the non-biotinylated NAT-peptide during the bio-screening as competitors.

3.6.2 AffMat Modifications

AffMat is used to evolve the binding affinities of DisMatted Abs to NAT peptides and NAT full-length proteins. Typically, we will pick 2–3 best NAT-specific hits to move forward to AffMat. Three rounds of positive selecting bio-screenings are used against the NAT full-length proteins (if not available, then use NAT-biotin-

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peptides). And in the 2nd round and 3rd round, we successively reduce 10-fold screening Ag concentration while coating the plates. Additionally, we use a range of 10-fold to 100-fold excess of the non-biotinylated NAT-peptides as competitors. 3.7 Converting scFv Hits into Full-Length IgG Proteins and Final Antibody Validation.

4

Final validated positive scFv hits should be sequenced and assembled into suitable plasmid expression vectors for IgG expression and purification. Final IgGs can be validated by our innovative Western-Blot based MILKSHAKE and Sundae methods published recently [25] and also described in a separate chapter in this book [26]. Final validation of the Epivolve-derived Abs will depend on the desired specific applications.

Notes 1. Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ-cm at 25 ° C). Prepare and store all reagents at room temperature unless indicated otherwise. Follow all waste disposal regulations when disposing of waste materials 2. It is important to note that trypsin elution method will only work with phage libraries constructed with trypsin-cleavage phagemid vectors, which contains a trypsin cleavage site between the scFv and phage coat III protein gpIII, such as pIT-2 based vectors. If another type of elution condition is needed, use the recommended elution condition for that specific library in use 3. The eluted selected phage can be stored at 4 °C for no longer than 6 h if needed. 4. Make a duplicate 96-well deep-well plate which grows the same cells at the same timeline as the experimental plate. Use this plate to monitor the cell growth by measuring OD600 to avoid any contamination concerns to the experimental plate 5. If autoclaved glass beads are available, use autoclaved glass beads to spread the culture thoroughly over the agar plate. Allow the plate to dry with beads on the agar at 37 °C for around 20 min before inverting the plates to incubate overnight. Alternatively, cell spreaders can be used for plating cells on agar plates 6. Selection criteria of the best hits to move forward from phageinduced cell supernatant ELISA: we export the ELISA data at OD450 absorbance into Excel file. We calculate the ratio of absorbance of each well over the absorbance from background well (medium-only control wells of E12 through H12). We consider a positive hit only when all the following 3 criteria will

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be met: (1) the Ab can detect the Ag coated on plate at 2.5 μg mL-1 concentration; (2) the absorbance OD450 must be ≥0.2; (3) The binding absorbance signal must be ≥2-fold over absorbance on background wells 7. For consistency between rounds and different experiments, it is recommended that electronic washing conditions such as Biotek plate washer will be used. Manual pipetting may deliver variable results 8. We typically use heteroduplex formation cloning (Kunkel mutagenesis) after T7 exonuclease treatment to make the mutagenesis libraries [2, 3]. But standard enzymatic digestion followed by standard ligase ligation to a vector cloning will also work for making the mutagenesis libraries

Acknowledgments The authors would like to thank Dr. Brian K. Kay for insightful comments and discussion. This research was funded by NIH Appl. ID 1R43GM146473-01 and NIH Appl. ID 1R44AI177126-01. Patent protection for the Epivolve technology has been submitted for Abbratech Inc. References 1. Fuller EP, O’Neill RJ, Weiner MP (2022) Derivation of splice junction-specific antibodies using a unique hapten targeting strategy and directed evolution. New Biotechnol 71:1–10. https://doi.org/10.1016/j.nbt.2022.06.003 2. Holland EG, Buhr DL, Acca FE et al (2013) AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins. J Immunol Methods 394(1-2): 55–61. https://doi.org/10.1016/j.jim.2013. 05.003 3. Batonick M, Holland EG, Busygina V et al (2016) Platform for high-throughput antibody selection using synthetically-designed antibody libraries. New Biotechnol 33(5 Pt A):565–573. https://doi.org/10.1016/j.nbt.2015.11.005 4. Van Deventer JA, Wittrup KD (2014) Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation. Methods Mol Biol (Clifton, N.J.) 1131:151–181. https://doi.org/10.1007/ 978-1-62703-992-5_10 5. Zahnd C, Amstutz P, Plu¨ckthun A (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods 4(3):269–279. https://doi.org/ 10.1038/nmeth1003

6. Novotny CP, Lavin K (1971) Some effects of temperature on the growth of F pili. J Bacteriol 107(3):671–682. https://doi.org/10.1128/ jb.107.3.671-682.1971 7. Marks JD, Hoogenboom HR, Bonnert TP et al (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222(3):581–597. https:// doi.org/10.1016/0022-2836(91)90498-u 8. Hoogenboom HR, Griffiths AD, Johnson KS et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19(15):4133–4137. https://doi.org/10.1093/nar/19.15.4133 9. Sheets MD, Amersdorfer P, Finnern R et al (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci U S A 95(11):6157–6162. https://doi.org/10. 1073/pnas.95.11.6157 10. Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat

Develop Site-Specific Antibodies Using Epivolve Technology Biotechnol 14(3):309–314. https://doi.org/ 10.1038/nbt0396-309 11. de Haard HJ, van Neer N, Reurs A et al (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274(26):18218–18230. https:// doi.org/10.1074/jbc.274.26.18218 12. Haidaris CG, Malone J, Sherrill LA et al (2001) Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J Immunol Methods 257(1–2):185–202. https://doi.org/10. 1016/s0022-1759(01)00463-x 13. Knappik A, Ge L, Honegger A et al (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296(1):57–86. https://doi.org/10.1006/jmbi.1999.3444 14. Sidhu SS, Li B, Chen Y et al (2004) Phagedisplayed antibody libraries of synthetic heavy chain complementarity determining regions. J Mol Biol 338(2):299–310. https://doi.org/ 10.1016/j.jmb.2004.02.050 15. Rauchenberger R, Borges E, Thomassen-Wolf E et al (2003) Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J Biol Chem 278(40): 38194–38205. https://doi.org/10.1074/jbc. M303164200 16. Nelson B, Sidhu SS (2012) Synthetic antibody libraries. Methods Mol Biol (Clifton, N.J.) 899:27–41. https://doi.org/10.1007/978-161779-921-1_2 17. Strachan G, McElhiney J, Drever MR et al (2002) Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli. FEMS Microbiol Lett 210(2):257–261. https://doi.org/10.1111/j. 1574-6968.2002.tb11190.x 18. Zhao Q, Buhr D, Gunter C et al (2018) Rational library design by functional CDR

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resampling. New Biotechnol 45:89–97. https://doi.org/10.1016/j.nbt.2017.12.005 19. Kiss MM, Babineau EG, Bonatsakis M et al (2011) Phage ESCape: an emulsion-based approach for the selection of recombinant phage display antibodies. J Immunol Methods 367(1–2):17–26. https://doi.org/10.1016/j. jim.2010.09.034 20. Nikiforov TT, Rendle RB, Kotewicz ML et al (1994) The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization. PCR Methods Appl 3(5):285–291. https://doi. org/10.1101/gr.3.5.285 21. Kunkel TA (1985) Rapid and efficient sitespecific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82(2):488–492. https://doi.org/10.1073/pnas.82.2.488 22. Scholle MD, Kehoe JW, Kay BK (2005) Efficient construction of a large collection of phage-displayed combinatorial peptide libraries. Comb Chem High Throughput Screen 8(6):545–551. https://doi.org/10. 2174/1386207054867337 23. Huang R, Fang P, Kay BK (2012) Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phagedisplay libraries. Methods (San Diego, Calif.) 58(1):10–17. https://doi.org/10.1016/j. ymeth.2012.08.008 24. Cadwell RC, Joyce GF (1994) Mutagenic PCR. PCR Methods Appl 3(6):S136–S140. https://doi.org/10.1101/gr.3.6.s136 25. Jones KS, Chapman AE, Driscoll HA et al (2022) MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot. BioTechniques 72(1):11–20. https://doi. org/10.2144/btn-2021-0078 26. Ferguson FM, Mendez MQ, Acca EF et al (this volume) Validation and the determination of antibody bioactivity using MILKSHAKE and sundae protocols. In: Phage display: methods and protocols. Springer, New York

INDEX A Affibody ................................................................ 373–390 Affinity maturation...................................... v, 7, 8, 34, 40, 207, 229, 231, 238–241, 248, 396, 397, 504–510, 525–536, 564, 588, 597–599 Alpaca antibody ............................................................. 142 Antibody engineering ............................................ 108, 207, 328 fragments ...............................................4–7, 9, 16, 17, 61, 94, 108, 142, 143, 149, 163, 207, 228, 247, 248, 251, 254–256, 258, 261, 262, 264, 265, 268–271, 286–287, 300, 305–307, 310, 328, 347, 374, 397, 398, 405–408, 412, 434, 436, 437, 468, 544, 566, 580 gene libraries................................... 17–19, 25, 31, 33, 252, 257, 261, 266, 271, 324

B Batch cloning ....................................................... 411–417 Biomarker ................................................v, 109, 543–560, 564, 566, 567 Biopanning ................................................... 94, 103, 111, 126, 275–289, 294, 297, 334, 349, 361, 362, 365, 378, 382, 468–483, 588, 589, 592, 594, 595, 597, 598

C Camel antibody .................................................6, 61, 108, 142, 227–228, 489 Cell panning ........................................................ 248, 316, 331, 334–335, 337 Chicken libraries........................................................77–90 Combinatorial libraries ............................... 350, 373, 374 Complementarity determining region (CDR) ............... 9, 17, 18, 61, 66, 73, 349, 351, 352, 489, 506, 529, 535, 537, 538, 592 Cytokines ......................................................149–187, 374

D

Diagnostics .................................................... v, 10, 15, 77, 79, 229, 291, 412, 433, 470, 471, 481, 483, 546, 564, 566

E Epitope mapping............................................. v, 150, 543, 564–567, 581, 582 Epivolve ................................................................ 587–600 Error-prone PCR ............................... 395–409, 525–527, 588, 589, 597–598

F Fibronectin type III (FN3)........................................... 206 Filamentous phage ..........................................64, 65, 108, 120, 149–151, 158–160, 275, 366, 373 Fragment antigen binding (Fab).............. 16, 61, 62, 261 Fragment crystallizable/constant (Fc) ............5, 62, 110, 114, 116 Framework................................................ 4, 9, 17, 18, 60, 61, 64, 65, 122, 231, 232, 234, 352, 363, 374, 397, 490, 537, 590

G Golden gate ............................17, 40, 191–194, 197, 200

H Heavy chain .....................................................4–6, 16, 17, 40, 41, 47, 55, 60, 62, 80, 101, 108, 109, 122, 123, 143, 227, 349, 396, 434–439, 441, 442, 446, 447, 489, 494, 496, 511 High-throughput DNA sequencing..................................................... 490 panning ................................. 291–310, 412, 533, 566 reformatting ................................................... 433–449 Human libraries............................................................... 18 Hybrid secretion signals ...................................... 433–449

I IgG reformatting.................................................. 433–449

Deep mining......................................................... 420, 421

Michael Hust and Theam Soon Lim (eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology, vol. 2702, https://doi.org/10.1007/978-1-0716-3381-6, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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604 Index

Immune libraries .............................................7, 8, 17, 18, 33, 34, 40, 41, 46, 47, 54, 55, 79, 89, 90, 94, 145, 248, 262, 328, 349 Immunoglobulin ........................................ 60, 77, 82, 94, 107, 108, 123, 143, 369, 374, 468, 490, 510, 592 Immunoglobulin G (IgG) .............................60, 108, 196 Immunoglobulin new antigen receptor (IgNAR).......................................... 227–229, 231, 234, 238, 239 InTag positive selection ....................................... 433–449 In vitro selection ..............................................60, 94, 412

K Kunkel mutagenesis ............................................ 150, 155, 168–170, 205–224, 600

L Light chain (LC) .............................................4, 5, 17, 24, 33, 34, 40–43, 47–49, 55, 56, 65, 80, 99, 101, 104, 108, 110, 396, 435–438, 441–443, 446, 448, 592 Llama libraries ....................................109, 110, 119–121, 140–143, 492, 493, 503, 510, 517, 519, 536

M M13 phage ................................... 16, 144, 307, 544, 590 Magnetic particles ............................................... 293–296, 299, 301–302, 308 Membrane proteins.............................109, 142, 315–325 Microtiter plates ........................................... 54, 110, 121, 142, 211, 219, 220, 223, 247–258, 264, 268, 269, 271, 280, 285, 287, 294, 299, 300, 302, 303, 305, 306, 308, 310, 315, 321–322, 331, 400, 401, 404–406, 463, 546 MILKSHAKE protocol ................................................ 453 Mutagenesis.................................................. 9, 62, 65, 66, 73, 155, 156, 170–173, 181–185, 192, 396–398, 401, 453, 504–507, 525–531, 533–535, 537, 565, 566, 588, 590, 597, 598, 600

N Naı¨ve libraries ...........................8, 40, 248, 262, 437, 446 Nanoparticles............................................... 276, 294, 307 NeutrAvidin (NA)............................................... 195, 202, 211, 220, 223, 593, 596 Next generation sequencing (NGS) .................. 206, 217, 224, 347–370, 468–470, 474, 478–479, 490–495, 504–515, 518, 521, 524–537

O Open reading frame (ORF) selection .......................... 544 ORFeome ............................................543–560, 566, 579 Ostrich libraries ............................................................... 89

P Panning......................................................v, 4, 33, 34, 41, 45–46, 52–54, 56, 87, 88, 108, 109, 114, 123, 126–129, 140–142, 144, 145, 156, 174–175, 247–250, 252–257, 261–263, 266–271, 277, 278, 283, 284, 287, 293, 294, 296, 297, 299, 301, 302, 305, 308–310, 316, 319, 321, 324, 329, 330, 333–338, 342, 343, 348–350, 352–359, 367, 369, 370, 397, 399, 404, 405, 412, 413, 416, 422–429, 469–471, 474, 476, 480–482, 486, 493, 501, 506, 512, 515, 519–522, 529, 531–533, 535, 537, 538, 544, 546, 549–550, 554–557, 559, 568–570, 574, 576, 577, 580 Phage ............................................................ 4, 15, 39, 78, 94, 108, 191, 205, 247, 261, 275, 315, 328, 347, 373, 396, 412, 420, 468, 490, 566, 588 Phage display ............................................ v, vi, 4, 6–8, 10, 15–56, 59–73, 81, 94, 108, 140, 144, 149, 150, 159, 183, 191, 201, 205–224, 247, 248, 261–263, 275–289, 291–293, 298, 300, 308, 315–317, 319–321, 327, 328, 347, 349, 350, 352, 355, 361, 365, 373, 375, 381–386, 396–398, 401, 412, 413, 416, 420, 421, 428, 433, 434, 437–439, 446, 467–486, 490, 543–560, 563–583, 588–590, 592, 595 Phagekines ..................................................................... 150 p-MHC binding antibodies................................. 327–344 Polyclonal antibody......................................... v, 120, 140, 196, 287, 299, 304–305, 467, 468 Polymerase chain reaction (PCR) ...........................17–22, 24–31, 33, 40, 42, 47–51, 54, 77, 79–81, 83–85, 87, 94, 95, 99–102, 111, 115, 120, 122, 123, 125, 127, 129, 131, 132, 143, 144, 156, 171, 192, 194, 197, 198, 201, 208, 212, 214–217, 222, 230–232, 234–241, 340, 349–353, 361–364, 380, 382, 384–386, 398, 399, 401, 402, 407, 408, 413, 415, 416, 435–444, 448, 469, 472, 474, 475, 478–480, 484, 485, 490, 493, 494, 497, 499, 500, 504–506, 511–513, 515, 518, 521, 523–527, 530, 531, 536–538, 547, 549, 551–555, 559, 567, 570–573, 579, 580, 591, 597, 598

PHAGE DISPLAY: METHODS R Rabbit libraries ........................................................93–105 Rolling circle amplification (RCA)................40, 205–224

S ScFv-Fc ....................................................... 5, 6, 248, 249, 262, 271, 328, 338, 412, 415, 435 Shark libraries ....................................................... 229–241 Single-chain fragment variable (scFv) .................. 5–7, 10, 15–35, 40, 61, 77–79, 84–87, 89, 90, 94, 108, 109, 163, 248, 253, 255, 258, 261–263, 266, 269, 271, 279, 288, 297, 319–324, 328, 329, 337–340, 343, 348–350, 352, 359–361, 365–367, 370, 403, 412, 430, 490, 588–590, 592, 593, 595–597, 599 Single domain antibody (sdAbs) .......................... 5, 6, 40, 107–145, 207, 489, 490, 512 Solid-phase extraction (SPE)............................... 275–289 Streptavidin................................................. 111, 114, 116, 118, 121, 123, 126, 128, 129, 131, 133, 135–142, 202, 210, 217, 223, 247, 252, 254, 256, 262, 263, 266, 269, 276–278, 280, 282–284, 288, 289, 298, 300, 301, 304, 329, 331, 333, 337, 353, 355, 377, 381, 383, 387, 389, 421, 422, 424, 509, 531 Sundae protocols.................................................. 451–464

AND

PROTOCOLS Index 605

Synthetic libraries ................................................ 9, 17, 18, 60, 61, 90, 145, 248, 347–370, 515, 517

T Therapeutic antibodies .................... 15, 16, 65, 248, 419 Therapy ........................................................ v, 10, 16, 150, 229, 412, 564, 566

V Variable fragment of the heavy chain (VH)............4–6, 8, 17–20, 22, 24–27, 29–31, 33, 34, 40, 47, 55, 61, 62, 65, 66, 77–79, 82–85, 89, 99, 100, 108, 111, 349, 350, 396, 412, 435–437, 441–443, 447, 448, 489, 490, 494, 496, 510–512, 514, 515, 523, 536, 537 Variable fragment of the light chain (VL) ..............4–6, 8, 17, 18, 21–22, 27–29, 31, 33, 34, 40, 47, 62, 77–79, 82–85, 89, 99, 108, 350, 396, 412, 489, 490, 494, 496, 510, 512, 514–516, 518, 536, 537 VHH .................................................. 6, 16, 61, 108, 109, 111, 120, 122–126, 128, 129, 131–144, 227–229, 490, 494, 496, 504, 506, 510, 512, 514, 515, 517–519, 521, 523, 525, 526, 536

Z Z domain .............................................................. 193, 196