CANCER IMMUNOLOGY cancer immunotherapy for organ specific tumors. [2 ed.] 9783030579494, 3030579492

Table of contents :
Preface
Acknowledgment
Contents
Abbreviations
Contributors
1: Cancer Immunotherapy Confers a Global Benefit
1.1 Introduction
1.2 Incidence, Morbidity, and Mortality of Cancers: Why Is a New Therapeutic Avenue Indicated?
1.2.1 Cancer Incidence
1.2.2 Cancer Mortality Rate
1.3 History of Immunotherapy of Cancers
1.4 Immunotherapy Is Going Upstream to Combat Cancers
1.4.1 Prophylactic Implication of Immunotherapy
1.4.2 Therapeutic Implication of Immunotherapy
1.5 Strategies of Cancer Immunotherapy
1.5.1 Immunotherapy Acts to Eliminate Immunosuppression
1.5.2 Immunotherapy Boosts the Antitumor Immune Responses and Enhances Killing of the Tumor Cell
1.5.2.1 Activated DCs and T Cells Are Pivotal in Cancer Immunotherapy
1.5.2.2 Materials of Activating DCs and T Cells
1.6 At Which Line of Treatment?
1.7 Monotherapy or Combined Therapy?
1.8 Monitoring the Immunological and Clinical Responses to Immunotherapy
1.9 Limitations of Cancer Immunotherapy
1.10 Supportive Therapy
1.11 Effect of Immunotherapy on Health-Related Quality of Life of Cancer Patients
1.12 Cost-Effectiveness of Cancer Immunotherapy
1.13 Concluding Remarks
References
2: Immunotherapy for Pediatric Solid Tumors
2.1 Introduction
2.2 Solid Tumors
2.2.1 Sarcomas
2.2.1.1 Osteosarcoma
2.2.1.2 Ewing Sarcoma
2.2.1.3 Soft-Tissue Sarcomas
Rhabdomyosarcoma
Non-rhabdomyosarcoma Soft-Tissue Sarcomas
2.2.2 Neuroblastoma
2.2.3 Nephroblastoma
2.2.4 Hepatoblastoma
2.2.5 Systemic Germ Cell Tumors
2.2.6 Central Nervous System Tumors
2.2.6.1 Embryonal Tumors
Medulloblastoma
CNS Primitive Neuroectodermal Tumors
Atypical Teratoid/Rhabdoid Tumors
2.2.6.2 Gliomas
Low-Grade Gliomas
High-Grade Gliomas
Brainstem Gliomas
Ependymomas
2.2.6.3 Pineal Region Tumors
2.2.7 Retinoblastoma
2.3 Immune Therapy and Pediatric Solid Tumors
2.3.1 Tumor-Targeting Monoclonal Antibodies (mAbs)
2.3.1.1 Cell Surface Immune Targets
Gangliosides
B7-H3
RANK-L
2.3.1.2 Growth Factor Receptors and Oncogenes
HER2
Insulin-Like Growth Factor-1 Receptors
Epidermal Growth Factor Receptor Family
Platelet-Derived Growth Factor
Vascular Endothelial Growth Factor
2.3.1.3 Immunomodulatory Monoclonal Antibodies
Cytotoxic T Lymphocyte Antigen 4 and Programmed Death Receptor 1
2.3.2 Adoptive Cell Transfer
2.3.3 Anticancer Vaccines
2.3.3.1 Peptide-Based Vaccines
2.3.3.2 Dendritic Cell-Based Vaccines
2.3.3.3 Genetically Modified Tumor Vaccines
2.3.3.4 Other Adoptive Cell Therapies
2.4 Concluding Remarks
References
3: Immunotherapeutic Strategies for Multiple Myeloma
3.1 Introduction
3.2 Immune Therapy for Myeloma: Overcoming Tumor-Associated Immune Suppression
3.3 Antibody-Mediated Strategies
3.3.1 CS1
3.3.2 CD38
3.3.3 PD-1/PD-L1
3.3.4 Antibody Conjugates and Bispecific Antibodies
3.4 Cellular Immunotherapy for Multiple Myeloma
3.4.1 Allogeneic Transplantation
3.4.2 Myeloma Vaccines
3.4.2.1 Peptide-Based Myeloma Vaccines
3.4.2.2 Cell-Based Myeloma Vaccines
3.4.3 Adoptive Cell Therapy
3.4.3.1 Marrow-Infiltrating T Cells
3.4.3.2 NK Cell Therapy
3.4.4 Engineered T Cells
3.4.4.1 TCR T Cells
3.4.4.2 CAR T Cells
3.5 Concluding Remarks
References
4: Immunopathology and Immunotherapy of Myeloid Leukemia
4.1 Introduction
4.2 Immunopathology of Acute Myeloid Leukemia
4.2.1 Causes of Genetic Alterations
4.2.1.1 Primary AML
4.2.1.2 Secondary AML
Acute Myeloid Leukemia with Myelodysplasia-Related Changes (AML-MDS)
Therapy-Related Myeloid Neoplasms (t-AML)
4.2.2 Genes Affected in AML
4.2.3 Models for Leukemogenesis Through Gene Alterations
4.2.4 The Leukemic Stem Cell
4.2.4.1 Phenotype of the LSC
4.2.4.2 Clinical Relevance of the LSC
4.2.5 How Do Gene Alterations in the LSC Lead to the Clinical Presentation of AML?
4.3 Immunotherapy for AML
4.3.1 Antigens to Target in AML
4.3.1.1 Antigens Presented by MHC After Internal Processing
4.3.1.2 Surface Antigens
4.3.2 Current Immunotherapeutic Strategies for AML
4.3.2.1 Active Immunotherapeutic Strategies
Peptide Vaccination
Dendritic Cell Vaccination
4.3.2.2 Passive Immunotherapeutic Strategies
Monoclonal Antibodies
Adoptive T-Cell Transfer
Adoptive NK Cell Transfer
4.4 Concluding Remarks
References
5: Immunopathology and Immunotherapy of Acute Lymphoblastic Leukemia
5.1 Immunopathology of Lymphoblastic Leukemia
5.1.1 General Considerations
5.1.1.1 Lymphocyte Development as Biological Basis of Disease
5.1.1.2 Genetics in Acute Lymphatic Leukemia
Numerical Chromosome Changes
Hyperdiploid
Hypodiploid
Structural Changes
MLL Rearrangements
BCR-ABL
ETV6-RUNX1
Molecular Mutations
5.1.2 Immune Phenotype and Targets in Acute Lymphatic Leukemia
5.1.2.1 Cell Surface Marker
5.1.2.2 Challenges for Immunophenotyping as MRD Marker
5.2 Immunotherapy for Acute Lymphatic Leukemia
5.2.1 Cellular Approaches
5.2.1.1 T Cells and Modified T Cells
5.2.1.2 NK Cell Approaches
5.2.2 Antibodies
5.2.2.1 CD20 Antibodies
5.2.2.2 CD22 Antibody
5.2.2.3 CD52 Antibody
5.2.2.4 CD19 Antibody
Blinatumomab
SGN-CD19A
5.2.3 Stem Cell Transplantation
5.2.3.1 Allogeneic Stem Cell Transplantation (Allo SCT)
References
6: Immunopathology and Immunotherapy of Hodgkin Lymphoma
6.1 Introduction
6.2 Immunopathology of Hodgkin Lymphoma
6.3 General Concepts of Monoclonal Antibodies
6.3.1 The Structure of Monoclonal Antibodies
6.3.2 Choosing the Optimal Antibody
6.4 CD30
6.4.1 CD30 Monoclonal Antibodies
6.4.1.1 MDX-060 (5F11)
6.4.1.2 MDX-1401
6.4.1.3 Chimeric-AC10
6.4.1.4 SGN-30
6.4.2 CD30 mAb-Drug Conjugates
6.4.2.1 Brentuximab Vedotin
6.5 CD20
6.5.1 Rituximab
6.6 CD40
6.6.1 Lucatumumab (HCD122)
6.7 CD80
6.7.1 Galiximab (IDEC-114)
6.8 Immune Checkpoint Inhibitors
6.8.1 CTLA-4
6.8.1.1 Ipilimumab
6.8.2 PD-1
6.8.2.1 Nivolumab
6.8.2.2 Pembrolizumab
6.8.2.3 Sintilimab
6.8.2.4 Tislelizumab
6.9 Therapeutic Efficacy of Cytokines
6.9.1 Interleukin-2 (IL-2)
6.9.2 An IL-2-IL-12 Fusion Protein Targeting Hodgkin Lymphoma
6.10 Bispecific Monoclonal Antibodies
6.11 Novel Immunotherapeutic Treatment Strategies in HL
6.11.1 Immunotoxins
6.12 Chimeric Antigen Receptor-Modified T Cells
6.12.1 Anti-CD30 CAR T Cell
6.13 Concluding Remarks
References
7: Immunopathology and Immunotherapy of Non-Hodgkin Lymphoma
7.1 Introduction
7.2 Immunopathology of NHL
7.3 CD30
7.3.1 M67
7.3.2 SGN-30
7.4 CD20
7.4.1 Effector Mechanisms of CD20 mAbs
7.4.2 Rituximab
7.4.2.1 Mechanisms of B-Cell Depletion by Rituximab
7.4.2.2 Rituximab in Diffuse Large B-Cell Lymphoma (DLBCL)
7.4.2.3 Rituximab in Mantle Cell Lymphoma
7.4.2.4 Rituximab in Follicular Lymphoma
7.4.2.5 Rituximab Incorporated with Carboplatin/Cisplatin-Based Chemotherapy
7.4.3 Targeting CD20 with New Anti-CD20 mAbs
7.4.4 First-Generation Anti-CD20 mAbs
7.4.4.1 Reengineered Rituximab
7.4.4.2 Tositumomab (B1)
7.4.4.3 Veltuzumab (hA20, IMMU-106)
7.4.4.4 Ocrelizumab (PRO70769, rhuH27)
7.4.5 Second-Generation CD20 mAb
7.4.5.1 Ofatumumab (Arzerra, HuMax-2F2)
7.4.6 Third-Generation CD20 mAb
7.4.6.1 PRO131921 (RhumAb v114)
7.4.6.2 AME-133v (LY2469298)
7.4.6.3 GA-101 (RO5072759, Obinutuzumab)
7.4.7 Small Modular Immunopharmaceutical Anti-CD20 Protein
7.4.7.1 TRU-015
7.5 CD22
7.5.1 Epratuzumab
7.5.2 Inotuzumab Ozogamicin (CMC-544)
7.6 CD40
7.6.1 Dacetuzumab (SGN-40)
7.6.2 Lucatumumab (HCD122, Formerly CHIR-12.12)
7.7 CD19
7.7.1 XmAb5574
7.7.2 Blinatumomab (MT102/MEDI-538)
7.7.3 hu-DM4/SAR3419
7.8 CD37
7.8.1 Tetulomab (HH1)
7.9 CD52
7.9.1 Alemtuzumab (CAMPATH-1H)
7.10 CD80
7.10.1 Galiximab (IDEC-114)
7.11 CD74 and HLA-DR
7.11.1 Milatuzumab (IMMU-115, hLL1), Naked and Conjugated
7.11.2 Apolizumab (Hu1D10, Remitogen)
7.11.3 IMMU-114 (hL243g4P)
7.11.4 LYM-1
7.11.5 Selective High-Affinity Ligands (SHALs)
7.12 CD1d and NK Cells
7.12.1 CD1d
7.12.2 Function of NK Cells in NHL
7.12.3 Adoptive Transfer of Highly Cytotoxic NK Cells
7.13 Therapeutic Efficacy of Antibody-Targeted Cytokines
7.13.1 Interferon-α (IFN-α)
7.13.2 Interleukin-2 (IL-2)
7.13.3 Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)
7.13.3.1 Mapatumumab (HGS-ETR1, TRM-1)
7.13.3.2 Lexatumumab (HGS-ETR2)
7.13.3.3 Conatumumab (AMG 655)
7.14 Novel Immunotherapeutic Treatment Strategies
7.14.1 Molecular Engineered Antibodies
7.14.2 Radioimmunoconjugates
7.14.2.1 Radioimmunotherapy for Follicular Lymphoma
7.14.2.2 CD20-Directed Radioimmunotherapy
131I-Tositumomab
90Y-Ibritumomab Tiuxetan Monotherapy
7.14.2.3 CD37-Directed RIT
7.14.3 Adoptive Cell Transfer of Genetically Modified T Cells
7.14.3.1 Redirecting T-Cell Specificity with Transgenic TCRs
7.14.3.2 Redirecting T-Cell Specificity with CARs
7.14.3.3 Other Instances of Genetic Engineering of Adoptively Transferred T Cells
7.14.4 Immune Checkpoint Blockade Therapy
7.14.5 NK Cell-Mediated Immunotherapy
7.14.6 Bispecific Antibodies
7.15 Vaccines
7.15.1 Salmonella Vaccine
7.15.2 DNA Vaccines
7.15.3 Epitope-Driven Vaccine Design
7.15.4 Preclinical Efficacy of Epitope-Driven DNA Vaccines Against B-Cell Lymphoma
7.16 Concluding Remarks
References
8: Immunotherapy of Gastric and Esophageal Cancers
8.1 Introduction
8.2 Current Immunotherapeutic Modalities for Esophageal and Gastric Cancers
8.2.1 Monoclonal Antibodies
8.2.1.1 Anti-HER2 mAbs
8.2.1.2 Anti-EGFR mAbs
8.2.1.3 Anti-VEGF mAbs
8.2.1.4 Anti-MET mAbs
8.3 Adoptive Cell Therapy
8.3.1 Immune Checkpoint Inhibitors
8.3.2 Dendritic Cell-Based Vaccination in Gastric and Esophageal Cancers
8.3.3 Protein- or Peptide-Based Vaccines in Gastric and Esophageal Cancers
8.3.4 Personalized Peptide Vaccination (PPV) Immunotherapy in Gastric and Esophageal Cancer
References
9: Hepatobiliary Tumors: Immunopathology and Immunotherapy
9.1 Introduction
9.2 Epidemiology
9.3 Current Treatment
9.4 Immunopathology
9.4.1 Hepatocellular Carcinoma (HCC)
9.4.2 Cholangiocarcinoma
9.4.3 Biomarker and Current Molecular Targeted Therapies
9.5 Inflammatory and Oxidative Stress Pathway
9.5.1 NF-κB Signaling Pathway
9.5.2 STAT3 Pathways
9.5.3 Cross Talk Between NF-κB and STAT3 Pathways
9.5.4 Mechanism Underlying Immunosuppression in HCC
9.6 Progress in Immunotherapy
9.6.1 Cancer Vaccines
9.6.1.1 HCC Cell Vaccines
9.6.1.2 Antigen Peptide Vaccines
9.6.1.3 DC Vaccines
9.6.2 Adoptive Cell Therapy (ACT)
9.6.3 CIK Cells
9.6.4 TILs
9.6.5 NK Cells
9.6.6 Chimeric Antigen Receptor (CAR) T Cells
9.6.7 Immune Checkpoint Inhibitors
9.6.8 CTLA-4 Inhibitors
9.6.9 PD-1 Inhibitors
9.6.10 PD-L1 Inhibitors
9.6.11 Oncolytic Viro-Therapy
9.7 Conclusion
References
10: Immunology and Immunotherapy of Colorectal Cancer
10.1 Introduction: Immunity, Infection, and Inflammation
10.2 Gut Microbiota, Inflammation, and Colorectal Cancer
10.3 Obesity, Metabolic Syndrome, Cancer Cachexia, Stress, and Inflammation
10.4 CRC Prevention by Nonsteroidal Anti-inflammatory Drugs
10.5 Colorectal Cancer Microenvironment: TILs, DCs, and Tregs
10.6 Mechanisms of Immunosuppression
10.7 Immunotherapy for Colorectal Cancer
10.7.1 Consensus Molecular Subtypes of CRC
10.7.2 Key Immunotherapeutic Trials in CRC
10.7.3 Other Approaches Tested in Humans
10.7.3.1 Other Monoclonal Antibodies
10.7.3.2 Adoptive Cell Transfer
10.7.3.3 Lymphodepletion
10.7.3.4 Vaccines
References
11: Immunotherapy in Nonmelanoma Skin Cancers
11.1 Introduction
11.2 Immunotherapy for Keratinocyte Cancers
11.2.1 Nonspecific Immunotherapy
11.2.1.1 Employing Delayed-Type Hypersensitivity
11.2.1.2 Interferons
11.2.1.3 Interleukin-2
11.2.1.4 Toll-Like Receptor Agonists
11.2.2 Specific Immunotherapy
11.2.2.1 Immunotherapy via Antibodies
11.2.2.2 Adoptive T-Cell Immunotherapy
11.2.2.3 Cancer Vaccines
11.2.3 Oncolytic Viruses
11.3 Conclusion
References
12: Immunopathology and Immunotherapy of Melanoma
12.1 Global Statistics on Melanoma
12.2 Immunology of Melanoma
12.2.1 Inflammatory Mediators
12.2.1.1 Histopathology
12.2.1.2 Mast Cells (MCs)
12.2.1.3 Macrophages
12.2.1.4 Neutrophils
12.2.1.5 Natural Killer (NK) Cells
12.2.2 Inflammatory Pathways
12.2.2.1 Mitogen-Activated Protein Kinase (MAPK)
12.2.2.2 Nuclear Factor-kappaB (NF-κB)
12.2.3 Immune Responses
12.2.3.1 T-Cells
12.2.3.2 B-Cells
12.2.3.3 Dendritic Cells (DCs)
12.3 Immunotherapy for Melanoma
12.3.1 Immune Checkpoint Inhibitors
12.3.1.1 Efficacy
12.3.1.2 Predictive Biomarkers
12.3.1.3 Adverse Events
12.3.2 Anti-CTLA-4 Monoclonal Antibodies
12.3.2.1 Efficacy
12.3.2.2 Adverse Events
Immune-Related Adverse Effects (irAEs)
Death Rate
Rash
12.3.3 Anti-PD-1 Monoclonal Antibodies
12.3.3.1 Efficacy
12.3.3.2 Adverse Events
Vitiligo
Pneumonitis
Atypical Responses
12.3.4 Cytokine-Based Immunotherapies
12.3.4.1 IFN
Efficacy
Adverse Events
12.3.4.2 IL-2
Efficacy
Adverse Events
12.3.5 Vaccines
12.3.5.1 Efficacy
12.3.5.2 Adverse Events
12.3.6 GM-CSF
12.3.7 Biochemotherapy or Chemoimmunotherapy
12.3.7.1 Efficacy
12.3.7.2 Adverse Events
References
13: Immunopathology as a Basis for Immunotherapy of Head and Neck Squamous Cell Carcinoma
13.1 The Immune Profile of HNSCC
13.1.1 Immune Responses in HNSCC
13.1.2 Wt p53-Specific T-cells
13.1.3 Virus-Derived Antigen-Specific T-cells
13.1.4 Suppression of T-Cells in the Cancer-Bearing Host
13.1.5 Role of Regulatory T-Cells
13.1.6 Tumor Immune Escape
13.2 Immune Features of Tumor-Derived Exosomes
13.3 Reversing Immune Escape
13.4 Immunotherapeutic Approaches Targeting Cancer Stem Cells
13.5 Current Vaccination Strategies
13.6 Immune Checkpoint Inhibitors
13.7 Concluding Remarks
References
14: Immunotherapy and Immunosurveillance of Oral Cancers: Perspectives of Plasma Medicine and Mistletoe
14.1 Introduction
14.2 Trapping an Advanced Squamous Cell Carcinoma of the Tongue by Continuous Repeated Peritumoral Injection of Mistletoe Preparation
14.3 Concluding Remarks
References
15: Immunopathology of Bone and Connective Tissue Cancers and Immunotherapy of Sarcomas
15.1 Introduction
15.2 Coley’s Toxin and Toll-Like Receptors
15.3 Sarcoma Antigens as Targets for Immunotherapy
15.3.1 NY-ESO-1
15.3.2 SSX
15.3.3 ALK
15.3.4 HHV8
15.4 Preclinical Models of Immunotherapy for Sarcoma
15.4.1 Methylcholanthrene (MCA)
15.4.2 p53 and Nf1
15.5 Undifferentiated Pleomorphic Sarcoma
15.6 Clinical Applications of Immunotherapy for Sarcoma
15.6.1 Adoptive Cell Therapy
15.6.1.1 Lymphokine-Activated Killers (LAKs)
15.6.1.2 Cytokine-Induced Killers (CIKs)
15.6.1.3 Natural Killers (NKs)
15.6.1.4 Engineered T-Cells
15.6.1.5 Chimeric Antigen Receptors (CARs)
15.6.2 Sarcoma Immunotherapy of the Future: CTLA-4 and PD-1 Manipulation
15.6.2.1 CTLA-4
15.6.2.2 PD-1
15.6.2.3 Other Checkpoint Inhibitors
15.7 Concluding Remarks
References
16: Immunopathology and Immunotherapy of Central Nervous System Cancer
16.1 Introduction
16.2 Antitumor Mechanisms of the Immune System
16.3 Immune Compartment of the CNS
16.4 CNS Tumor-Derived Immunosuppression
16.4.1 Tumor Cells
16.4.2 Glioma Cancer Stem Cells
16.4.3 Tumor-Associated Macrophages/Microglia
16.4.4 Myeloid-Derived Suppressor Cells
16.4.5 Lymphocytes and Regulatory T Cells
16.5 STAT3 Pathway
16.6 Cytomegalovirus in Glioma
16.7 Immunoediting in CNS Cancer
16.8 Immunotherapy
16.8.1 Adoptive Therapy
16.8.2 Vaccination Strategies
16.8.2.1 Autologous Tumor Material
16.8.2.2 Dendritic Cell-Based Vaccination Strategies
16.8.2.3 Antigen-Specific Peptide Strategies
16.8.2.4 Heat Shock Protein Peptide Complex 96
16.8.3 Immunotherapy Targeting CNS Cancer-Induced Immunosuppression
16.8.4 Monoclonal Antibodies
16.9 Concluding Remarks
References
17: Immunotherapy of Lung Tumors
17.1 Introduction
17.2 Cancer Staging and Histology
17.3 The Vaccines and Cellular Therapies
17.3.1 GVAX
17.3.2 IDM-2101
17.3.3 Belagenpumatucel-L
17.3.4 MAGE-3
17.3.5 MUC1
17.3.6 EGF Vaccine
17.3.7 TG4010
17.3.8 FANG
17.3.9 Talactoferrin
17.3.10 TAG Plasmid Vaccine
17.4 Dendritic Cells
17.5 The Monoclonal Antibodies
17.5.1 Ziv-Aflibercept (Zaltrap®)
17.5.2 Bevacizumab
17.5.3 Cetuximab
17.5.4 Necitumumab
17.5.5 The EGFR Inhibitor Rash
17.5.6 Durvalumab
17.5.7 Atezolizumab
17.5.8 Pembrolizumab
17.5.9 Nivolumab
17.5.10 Ipilimumab
17.6 Adverse Effects Related to Immunotherapy
17.6.1 Measurement of Immune Response to Monoclonals
17.7 The Mutations
17.8 Chemoprevention
17.9 Discussion
References
18: Immunotherapy in Bladder and Renal Cancers
18.1 Bladder cancer
18.1.1 Introduction
18.1.2 Histological Subtypes and Staging
18.1.2.1 Non-muscle Invasive Bladder Cancer
18.1.2.2 Muscle Invasive Bladder Cancer
18.1.3 Immunotherapy in Bladder Cancer
18.1.3.1 Immunotherapy in NMIBC
Intravesical Bacillus Calmette–Guérin (BCG) Immunotherapy
History
Efficacy
Side Effects
Mechanism of the Antitumor Effect
The Role of Bladder Cancer Cells
The Role of the Immune System
Optimal BCG Dose and Schedule
Clinicopathologic Prognostic Factors of Non-Muscle Invasive Bladder Cancer
Markers Predicting Response to BCG
Cell Cycle Regulators
Apoptosis Inhibitors
Angiogenesis and Proliferation Markers
Inflammatory Markers
Cell Adhesion Molecules
Combination of BCG and INF-α
Checkpoint inhibitors
Vaccine Therapy
Gene Therapy
18.1.3.2 Immunotherapy in MIBC
Checkpoint Inhibitors
Atezolizumab
Pembrolizumab
Durvalumab
Avelumab
Nivolumab
Ipilimumab
Nivolumab and Ipilimumab Combination
Pembrolizumab and Radiation
Novel Tumor-Targeted Immunotherapeutics
18.2 Renal Cancer
18.2.1 Introduction
18.2.2 Immunotherapy in Renal Cancer
18.2.2.1 Localized RCC
Immunotherapy
Traditional Immunotherapy
Vaccine-Based Therapy
Antibody-Dependent Cytotoxic Agents
Immune Checkpoint Inhibitors
Targeted Therapy
VEGF-Targeted Tyrosine Kinase Inhibitors (VEGFR-TKI)
Mammalian Target of Rapamycin (mTOR) Inhibitors
18.2.2.2 Metastatic RCC
Immunotherapy
Historical Cytokines
Vaccine-Based Therapy
Adoptive T-Cell Therapy
Immune Checkpoint Inhibitors
Ongoing Combination Strategies
Targeted Therapy
VEGF Inhibitors
mTOR Inhibitors
18.3 Future Directions
References
19: Immunopathology of Specific Cancers in Males and Females and Immunotherapy of Prostate and Cervical Cancer
19.1 Introduction
19.2 Prostate Cancer: Past, Present, and Future
19.3 Immunotherapy of Prostate Cancer
19.4 Cervical Cancer: What We Know and What We Need to Know
19.5 The Immunotherapy of Cervical Cancer
19.6 Concluding Remarks
References
20: Immunology and Immunotherapy of Ovarian Cancer
20.1 Introduction
20.2 The Role of Cytokines in Neovascularization of Epithelial Ovarian Cancer (EOC)
20.2.1 Characterization of VEGF Function
20.2.2 VEGF in Ovarian Cancer Patients
20.2.3 Role of VEGF for Ovarian Cancer Growth, Dissemination, and Metastases
20.3 The Role of Pro-Inflammatory Cytokines in Ovarian Cancer
20.3.1 Inflammation and Cancer: General Remarks
20.3.2 Inflammatory Reaction and the Risk of Ovarian Cancer
20.3.3 Inflammation and Ovarian Cancer Growth and Dissemination
20.3.3.1 Tumor Necrosis Factor-α
20.3.3.2 Interleukin-10
20.3.3.3 COX and PGE2
20.3.3.4 Interleukin-23 and Th17 Cells
20.3.3.5 Macrophage Migration Inhibitory Factor
20.3.3.6 Macrophage Colony-Stimulating Factor
20.3.3.7 Chemokines
20.4 Regulatory and Inflammatory Cells in Ovarian Cancer
20.5 Immune Checkpoint Proteins and Their Inhibitors
20.5.1 The CTLA-4 Checkpoint Molecule
20.5.2 The PD-1 Checkpoint Molecule
20.5.2.1 Anti-PD-1/PD-L1 Monoclonal Antibodies in Animal and Human Trials
20.6 Cytokines in Diagnosis and Prognosis of Ovarian Cancer
20.6.1 Diagnosis
20.6.2 Prognosis
20.7 Immunotherapy of Ovarian Cancer
20.7.1 Monoclonal Antibodies
20.7.1.1 Bevacizumab
20.7.1.2 Catumaxomab
20.7.1.3 Oregovomab and Abagovomab
20.7.1.4 Trastuzumab and Pertuzumab
20.7.1.5 Farletuzumab
20.7.2 Cytokines
20.7.3 Cancer Vaccines
20.7.3.1 Dendritic Cell-Based Vaccines
20.7.3.2 Peptide-Based, Genetic and Epigenetic Vaccines
20.7.4 Adoptive Immunotherapy Using Autologous T-Cells
20.7.5 Cytokine-Induced Killer Cells
20.7.6 Chimeric Antigen-Receptor T-Cells (CAR-T Cells)
20.7.7 Targeting Tumor-Associated Macrophages (TAMs)
20.8 Conclusion
References
21: Immunopathology and Immunotherapy for Breast Cancer
21.1 Introduction
21.2 Breast Cancer and the Immune System
21.3 Gene Expression and Molecular Classification
21.4 Immunotherapy for Breast Cancer
21.4.1 Targeted Therapy by Using Monoclonal Antibodies (mAb)
21.4.2 Vaccines
21.4.3 Immune Checkpoint Inhibitors
21.4.3.1 Anti-CTLA-4 Antibodies
21.4.3.2 Anti-PD-1 Antibodies
21.4.3.3 Anti-PD-L1 Antibodies
21.4.4 Adoptive T-cell therapy
21.5 Conclusion
References
22: Immunology and Immunotherapy of Graft-Versus-Host Disease
22.1 Introduction
22.2 GVHD
22.3 Pathogenesis of Acute GVHD
22.3.1 Phase I: Conditioning
22.3.2 Phase II: Activation
22.3.3 Phase III: Effector Phase
22.4 Natural Control of GVHD
22.5 Graft-Versus-Tumor Effect
22.6 Prevention of GVHD
22.7 Treatment of Acute GVHD
22.8 Targeted Approaches
22.8.1 Targeting Cytokines
22.8.2 Targeting Co-stimulation
22.8.3 Targeting Cell Subsets
22.8.3.1 B Cells
22.8.3.2 NK Cells
22.8.3.3 Mesenchymal Stem Cells
22.8.3.4 Treg Cells
22.9 Concluding Remarks
References
Index

Citation preview

Cancer Immunology Cancer Immunotherapy for Organ-Specific Tumors Nima Rezaei Editor Second Edition

123

Cancer Immunology

Nima Rezaei Editor

Cancer Immunology Cancer Immunotherapy for Organ-­Specific Tumors Second Edition

Editor Nima Rezaei Research Center for Immunodeficiencies Children’s Medical Center Tehran University of Medical Sciences Tehran Iran Department of Immunology School of Medicine Tehran University of Medical Sciences Tehran Iran Network of Immunity in Infection Malignancy and Autoimmunity (NIIMA) Universal Scientific Education and Research Network (USERN) Tehran Iran

ISBN 978-3-030-57948-7    ISBN 978-3-030-57949-4 (eBook) https://doi.org/10.1007/978-3-030-57949-4 © Springer Nature Switzerland AG 2015, 2020 This work is subject to copyright. All rights are reserved 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 Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

This book would not have been possible without the continuous encouragement by my parents and my wife, Maryam. I wish to dedicate it to my daughters, Ariana and Arnika, with the hope that progress in diagnosis and treatment of these diseases may result in improved survival and quality of life for the next generations, and at the same time that international collaboration in research will happen without barriers. Whatever I have learnt comes from my mentors. This book is therefore dedicated also to all of them, but most importantly to the patients and their families whose continuous support has guided me during the years.

Preface

The rapid flow of studies in the field of cancer immunology during the last decade has increased our understanding of the interactions between the immune system and cancerous cells. In particular, it is now well known that such interactions result in the induction of epigenetic changes in cancerous cells and the selection of less immunogenic clones as well as alterations in immune responses. Understanding the cross talk between nascent transformed cells and cells of the immune system has led to the development of combinatorial immunotherapeutic strategies to combat cancer. Cancer Immunology Series, a three-volume book series, is intended as an up-to-date, clinically relevant review of cancer immunology and immunotherapy. The first edition of book was published 4 years ago, which was very welcomed by the readers, made us to work on second edition of the book in such a short period of time. Volume I, Cancer Immunology: A Translational Medicine Context, is focused on the immunopathology of cancers. Volume II, Cancer Immunology: Bench to Bedside Immunotherapy of Cancers, is a translation text explaining novel approaches in the immunotherapy of cancers; and finally, Volume III, vii

Preface

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Cancer Immunology: Cancer Immunotherapy for Organ-Specific Tumors, thoroughly addresses the immunopathology and immunotherapy of organ-­ specific cancers. In Volume III, immunopathology and immunotherapy of various cancers categorized on an organ-specific basis either are discussed in detail. Notably, the principal focus is to put the basic knowledge gained on tumor immunology and immunotherapy in Volumes I and II into clinical perspective, with the aim to educate clinicians on the most recent approaches used in the immunotherapy of various tumors. Twenty-two chapters are allocated to meet this purpose. At the very beginning, an overview of the beneficial effects of immunotherapy is outlined in Chap. 1; then in Chap. 2, various aspects of immunotherapy for pediatric solid tumors are discussed. Thereafter, five chapters are devoted to hematological malignancies, specifically their immune microenvironment as well as the immunotherapeutic approaches; multiple myeloma, myeloid and lymphoid leukemias as well as Hodgkin and non-Hodgkin lymphomas are discussed in Chaps. 3–7. Due to the global prevalence of gastrointestinal tumors, precise discussions are brought up in Chaps. 8–10; esophageal and gastric, hepatobiliary, and colon cancers are tackled down one by one, respectively. Skin cancers, including melanoma and nonmelanoma as well as head, neck, and oral tumors, are illustrated in Chaps. 11–14. A chapter is allocated to the immunopathology and immunotherapy of bone and connective tissue tumors, followed by descriptions on progresses made on immunotherapy of central nervous system, in Chaps. 15 and 16, respectively. Chapter 17 focuses on immunotherapy of lung cancer, while Chaps. 18–20 aim to educate the reader on the immunopathology and immunotherapy of genitourinary tract tumors. Chapter 21 provides the readers with the most important details on the application of immunotherapy in breast cancers. To put an end to this volume and actually to the whole book series, immunology and immunotherapy of graft versus host disease as a common complication of organ transplantation would be highlighted. The Cancer Immunology Series is the result of valuable contribution of more than 300 scientists from more than 100 well-known universities/institutes worldwide. I would like to hereby acknowledge the expertise of all contributors, for generously devoting their time and considerable effort in preparing their respective chapters. I would also like to express my gratitude to the Springer Nature publication for providing me the opportunity to publish the book. Finally, I hope that this translational book will be comprehensible, cogent, and of special value for researchers and clinicians who wish to extend their knowledge on cancer immunology. Tehran, Iran

Nima Rezaei

Acknowledgment

I would like to express my gratitude to the Editorial Assistant of this book, Dr. Mahsa Keshavarz-Fathi. With no doubt, the book would not have been completed without her contribution. Nima Rezaei, MD, PhD

ix

Contents

1 Cancer Immunotherapy Confers a Global Benefit����������������������   1 Zahra Aryan, Mahsa Keshavarz-Fathi, Håkan Mellstedt, and Nima Rezaei 2 Immunotherapy for Pediatric Solid Tumors ��������������������������������  49 Lauren Nicholls and Lisa M. Kopp 3 Immunotherapeutic Strategies for Multiple Myeloma����������������  75 Jessica J. Liegel and David E. Avigan 4 Immunopathology and Immunotherapy of Myeloid Leukemia������������������������������������������������������������������������������������������ 103 Sylvia Snauwaert, Farzaneh Rahmani, Bart Vandekerckhove, and Tessa Kerre 5 Immunopathology and Immunotherapy of Acute Lymphoblastic Leukemia���������������������������������������������������������������� 119 Thomas Stübig and Nicolaus Kröger 6 Immunopathology and Immunotherapy of Hodgkin Lymphoma���������������������������������������������������������������������������������������� 135 Maryam Ebadi, Mahsa Keshavarz-Fathi, Yi Zeng, Maria Gkotzamanidou, and Nima Rezaei 7 Immunopathology and Immunotherapy of Non-­Hodgkin Lymphoma���������������������������������������������������������������������������������������� 159 Maryam Ebadi, Mohammad Amin Sadeghi, Nishitha M. Reddy, and Nima Rezaei 8 Immunotherapy of Gastric and Esophageal Cancers������������������ 213 Ali Sanjari-Moghaddam, Fatemeh Sadeghi, and Saeed Soleyman-Jahi 9 Hepatobiliary Tumors: Immunopathology and Immunotherapy�������������������������������������������������������������������������������� 241 Nazanin Momeni Roudsari, Naser-Aldin Lashgari, Saeideh Momtaz, and Amir Hossein Abdolghaffari 10 Immunology and Immunotherapy of Colorectal Cancer������������ 261 Oscar J. Cordero, Rubén Varela-Calviño, and Begoña Graña-Suárez xi

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11 Immunotherapy in Nonmelanoma Skin Cancers������������������������� 291 Fateme Rajabi 12 Immunopathology and Immunotherapy of Melanoma���������������� 305 Amene Saghazadeh and Nima Rezaei 13 Immunopathology as a Basis for Immunotherapy of Head and Neck Squamous Cell Carcinoma�������������������������������������������� 333 Xu Qian, Thomas K. Hoffmann, Andreas M. Kaufmann, and Andreas E. Albers 14 Immunotherapy and Immunosurveillance of Oral Cancers: Perspectives of Plasma Medicine and Mistletoe���������������������������� 355 Hans-Robert Metelmann, Thomas von Woedtke, Kai Masur, Peter Hyckel, Sander Bekeschus, Fred Podmelle, and Christian Seebauer 15 Immunopathology of Bone and Connective Tissue Cancers and Immunotherapy of Sarcomas�������������������������������������������������� 363 Sumana Narayanan and Joseph J. Skitzki 16 Immunopathology and Immunotherapy of Central Nervous System Cancer������������������������������������������������������������������ 379 Sara Hanaei, Víctor Andrés Arrieta, and Adam M. Sonabend 17 Immunotherapy of Lung Tumors �������������������������������������������������� 427 Shahe Boghossian 18 Immunotherapy in Bladder and Renal Cancers �������������������������� 451 Neda Khalili, Nastaran Khalili, and Nima Rezaei 19 Immunopathology of Specific Cancers in Males and Females and Immunotherapy of Prostate and Cervical Cancer���������������� 475 Maurizio Chiriva-Internati, Fabio Grizzi, Leonardo Mirandola, and Jose A. Figueroa 20 Immunology and Immunotherapy of Ovarian Cancer���������������� 487 Jacek R. Wilczyński, Marek Nowak, and Miłosz Wilczyński 21 Immunopathology and Immunotherapy for Breast Cancer�������� 541 Negar Ghaffari, Sepideh Razi, Mahsa Keshavarz-­Fathi, and Nima Rezaei 22 Immunology and Immunotherapy of Graft-Versus-Host Disease���������������������������������������������������������������������������������������������� 557 Doreen Haase and Farzaneh Afshari Index���������������������������������������������������������������������������������������������������������� 575

Contents

Abbreviations

5-ASA 5-Aminosalicylic acid 5-FU 5-Fluorouracil AA Anaplastic astrocytoma AA Arachidonic acid ACT Adoptive cell therapy ADCC Antibody-dependent cell-mediated cytotoxicity ADCP Ag-dependent cellular phagocytosis ADCs Antibody-drug conjugates AFP Alpha-fetoprotein Ag Antigens AIDS Acquired immunodeficiency syndrome AIM Antigen isolated from immunoselected melanoma AJCC The American Joint Committee on Cancer AKAP4 A-kinase anchor protein 4 ALCL Anaplastic large cell lymphoma ALDH1 Aldehyde dehydrogenase-1 ALK Anaplastic lymphoma kinase ALL Acute lymphatic leukaemia Allo SCT Allogeneic stem cell transplantation AML Acute myeloid leukemia AMP Adenosine monophosphate AO Anaplastic oligodendroglioma AOA Anaplastic oligoastrocytoma AOM Azoxymethane AP-1 Activating protein-1 APC Adenomatosis polyposis coli APC Antigen-presenting cells APLs Aspirin-triggered lipoxins APM Antigen-processing machinery AS04 Adjuvant system 04 ASCT Autologous stem cell transplantation ATCs Autologous tumor cells ATF Activating transcription factor ATG Anti-thymocyte globulin ATR Antitumor responses ATRTs Atypical teratoid-rhabdoid tumors BAFF B-cell-activating factor xiii

xiv

BBB Blood-brain barrier BCC Basal cell carcinoma BCG Bacillus Calmette-Guerin BCMA B-cell maturation antigen bFGF Basic fibroblast growth factor BID Bowel inflammatory disease BM Bone marrow BMI Body mass index BMSCs BM stromal cells BMT Bone marrow transplantation B-NHLs B-cell non-Hodgkin’s lymphomas BTLA B- and T-lymphocyte attenuator C Chemotherapy CAC Colitis-associated cancer CAFs Cancer-associated fibroblasts CAK Cytokine activated cells CAR Chimeric antigen receptor CD Cytosine deaminase CDC Complement-dependent cytotoxicity CDR Complementary-determining region CEA Carcinoembryogenic antigen cHL Classical HL CHP Cholesterol-bearing hydrophobized pullulan CI Confidence interval CIK Cytokine-induced killer cILCs Colonic innate lymphoid cells CIN Cervical intraepithelial neoplasia CIS Carcinoma in situ CLL Chronic lymphocytic leukemia CLP Common lymphoid progenitor CMC Complement-mediated cytotoxicity CML Chronic myeloid leukaemia CMP Common myeloid progenitor cells CMV Cytomegalovirus CNS Central nervous system COG Children’s Oncology Group COX Cyclooxygenase COX-2 Cyclooxygenase-2 CPG ODN CpG oligodeoxynucleotides CR Complete remission CR Complete response CRC Colorectal cancer CRI Cancer-related inflammation CRP C-reactive protein CRPC Castration-resistant prostatic carcinoma CSCs Cancer stem cells CSF-1 Colony-stimulating factor CTAs Cancer/testis antigens

Abbreviations

Abbreviations

xv

CTL Cytotoxic T lymphocyte CTL4 Cytotoxic T lymphocyte antigen-4 CTLA Cytotoxic T-lymphocyte-associated CTLA-4 Cytotoxic T lymphocyte-associated antigen 4 CTLs Cytotoxic T lymphocytes CTTNB1 Beta-catenin gene DALY Disability-adjusted life year DAMPs Damage-associated molecular patterns DAPK Death-associated protein kinase DC Dendritic cell DFI Disease-free interval DFS Disease-free survival DHA Docosahexaenoic acid DHFR Dihydrofolate reductase DKK1 Dickkopf-related protein 1 DLBCL Diffuse large B-cell lymphoma DLI Donor lymphocyte infusion DMFI Distant-metastasis-free interval DMH Dimethylhydrazine DR5 Death receptor 5 DSS Dextran sulfate sodium DTH Delayed-type hypersensitivity EAU European Association of Urology EBV Epstein-Barr virus ECAD E-cadherin ECM Extracellular matrix ECP Extracorporeal photochemotherapy EFS Event-free survival EGCs Esophageal and gastric cancers EGF Epidermal growth factor EGFR Epidermal growth factor receptor EMEA European Medicines Agency EMT Epithelial-mesenchymal transition EOC Epithelial ovarian cancer EORTC European Organisation for Research and Treatment of Cancer EP1 Prostaglandin E receptor-1 EPA Eicosapentaenoic acid Eph Ephrin ER Estrogen receptor ERK1/2 Extracellular signal-regulated kinase 1/2 ESCC Esophageal squamous cell carcinoma ESHAP  Etoposide, doxorubicin, methylprednisolone, cytarabine, and cisplatin ET-1 Endothelin-1 ET A R Endothelin A receptor EWSR1 Ewing’s sarcoma breakpoint region 1 FAP Familial adenomatous polyposis FasL Fas ligand

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FcR Fc receptor FDA Federal Drug Administration FFS Failure-free survival FGF Fibroblast growth factor FGF2 Fibroblast growth factor 2 FGFR 4 Fibroblastic growth factor receptor 4 FIGO International Federation of Gynecology and Obstetrics FL Follicular lymphoma FLI1 Friend leukemia virus integration 1 FOLFIRI 5-Fluorouracil, leucovorin, irinotecan FOLFOX 5-Fluorouracil, leucovorin, oxaliplatin FOXp3 Forkhead box P3 FRα Folate receptor α GAA Glioblastoma-associated antigen GBM Glioblastoma multiforme GC Gemcitabine and carboplatin G-CSF Granulocyte-CSF GCT Germ cell tumors GI Gastrointestinal GISTs Gastrointestinal stromal tumors GITR Glucocorticoid-induced tumor necrosis factor receptor GLSG German low-grade lymphoma study group Gly Glycine GM CSF Granulocyte-macrophage colony-stimulating factor GMP Good manufacturing practice GPC3 Glypican-3 GPI Glycosylphosphatidylinositol GPR9 G protein-coupled receptor 9 GSC Glioma stem cells GSTP1 Glutathione S-transferase P1 GSTP1 Glutathione S-transferase p1 gene GVH Graft-versus-host GVHD Graft versus host disease GVL Graft-versus-leukaemia GVT Graft-versus-tumor HAART Highly active antiretroviral treatment HAMA Human anti-mouse antibody HBV Hepatitis B virus HCC Hepatocellular carcinoma HDI Human development index HDL High-density lipoprotein HER2 Human epidermal growth factor receptor 2 HETE Hydroperoxyeicosatetraenoic acid HGG High-grade glioma HHV8 Human herpesvirus 8 HIF Hypoxia-inducible factor HIV Human immunodeficiency virus HL Hodgkin’s lymphoma

Abbreviations

Abbreviations

xvii

HLA Human leukocyte antigen HLA-G Human leukocyte antigen G HMG High-mobility group HMGB1 High-mobility group box 1 HNSCC Squamous cell carcinoma of the head and neck HOX Homeobox HPCs Hematopoietic progenitor cells HPV Human papillomavirus HRS Hodgkin and Reed-Sternberg HRT Hormone replacement therapy HSC Hematopoietic stem cells HSCT Hematopoietic stem cell transplantation HSP Heat shock protein HSPPCs Heat shock protein peptide complexes HSPs Heat shock proteins HTLV-I Human T-lymphotropic virus-I HVG Host-versus-graft IAP Inhibitor of apoptosis protein IBD Inflammatory bowel disease ICE Ifosfamide, carboplatin, and etoposide IDH Isocitrate dehydrogenase IDO Indoleamine 2,3-dioxygenase IEDB Immune Epitope Database and Analysis Resources IFN Interferon IFN-α Interferon-α IFNγ Interferon gamma IGF-1 Insulin-like growth factor-1 IGF-1R Insulin-like growth factor 1 receptor IGF-BPs Insulin-like growth factors binding proteins IGFs Insulin-like growth factors IGKC Immunoglobulin κ C IHC Immunohistochemistry IL Interleukin IL-10 Interleukin-10 IL-18 Interleukin-18 IL-18R IL-18 receptor IL-2 Interleukin-2 IL-4 Interleukin-4 IL-6 Interleukin-6 IL-8 Interleukin-8 IMSCs Immature myeloid suppressor cells IMTs Inflammatory myofibroblastic tumors INF-γ Gamma interferon INGR International Neuroblastoma Risk Group iNOS Inducible nitric oxide synthase INSS International Neuroblastoma Staging System Ipb Ipilimumab IPI International prognostic index

xviii

IRC Immune-related criteria irPFS Immune-related progression-free survival irRC Immune-related response criteria IRS Intergroup Rhabdomyosarcoma Study I-TAC Interferon-inducible T-cell α-chemoattractant ITK Inducible T cell kinase IV Intravenous IVIG Intravenous immunoglobulin kg Kilogram KHL Keyhole limpet hemocyanin KIF Kinesin superfamily protein KIR Killer-cell immunoglobulin-like receptor KLH Keyhole limpet hemocyanin KO Knockout KRAS Kristin rat sarcoma KS Kaposi’s sarcoma KSHV Kaposi’s sarcoma herpesvirus LAA Leukemia-associated antigen LAK Lymphokine-activated killer LCMC Lung Cancer Mutation Consortium LDH Lactate dehydrogenase LDL Low-density lipoprotein LMP1 Latent membrane protein 1 LOH Loss of heterozygosity LOX Lipoxygenase LPA Lysophosphatidic acid LPS Lipopolysaccharide LSA Leukemia-specific antigen LSC Leukemic stem cell LT Lymphotoxins LTs Leukotrienes M Months mAb Monoclonal antibody MALP-2 Macrophage-activating lipopeptide MAPKs Mitogen-activated protein kinases MCA Methylcholanthrene MCL Mantle cell lymphoma MCP-1 Monocyte chemotactic protein 1 MCP-3 Monocyte chemoattractant protein-3 MCPs Macrophage chemotactic proteins M-CSF Monocyte colony-stimulating factor MDS Myelodysplasia MDSCs Myeloid-derived suppressor cells MFH Malignant fibrous histiocytoma mg Milligram MGMT Methylguanine-DNA-methyltransferase MGMT O(6)-methylguanine-DNA methyltransferase MGUS Monoclonal gammopathy of undetermined significance

Abbreviations

Abbreviations

xix

MHC Major histocompatibility complex MHC I Major histocompatibility complex I MHC II Major histocompatibility complex II MIATA Minimal information about T cell assays MIF Migration inhibitory factor MiHA Minor histocompatibility antigens MIP-3α Macrophage inflammatory protein-3 MLL Mixed lineage leukemia MM Multiple myeloma MMAE Monomethyl auristatin E MMP Matrix metalloproteinases MP Myeloid progenitors MPIF-1 Myeloid progenitor inhibitory factor-1 MPL Monophosphoryl lipid A MR Minor response MRD Minimal residual disease MSC Mesenchymal stem cells MSI-H High-level microsatellite-unstable MTD Maximum tolerated dose mTOR Mammalian target of rapamycin MTP-PE Muramyl tripeptide phosphatidylethanolamine MTX Methotrexate MUC Mucin MVD Microvessel density N Nodes NA Not available NCI The National Cancer Institute NF Nuclear factor NF-κB Nuclear factor-κB NHL Non-Hodgkin’s lymphoma NK Natural killer NKT Natural killer T NKTCs Natural killer T cells NLPHL Nodular lymphocyte predominant HL NMIBC Nonmuscle, invasive bladder cancer NMSCs Non-melanocytic skin cancers NO Nitric oxide NRAS Neuroblastoma RAS oncogene NRSTS Non-rhabdomyosarcoma soft tissue sarcomas NSAIDs Nonsteroid anti-infl ammatory drugs NSCLC Non-small cell lung carcinoma NTS Nuclear targeting sequence OFA Ofatumumab ORR Overall response rate OS Osteosarcoma OS Overall survival OT 18α-Olean-12-ene-3β-23,28-triol P Placebo

xx

PAI-1 Plasminogen activator inhibitor type 1 PAMPs Pathogen-associated molecular patterns PanINs Pancreatic intraepithelial neoplasias PAR-1 Protease-activated receptor-1 PBLs Peripheral blood lymphocytes PBMC Peripheral blood mononuclear cells PC Pancreatic cancer PD Progressive disease PD1 Programmed death-1 PD-1 Programmed cell death-1 PDAC Pancreatic ductal adenocarcinoma PDCs Plasmacytoid dendritic cells PDEGF Platelet-derived endothelial cell growth factor PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor PD-L1 Programmed cell death-1 ligand 1 PFS Progression-free survival PGE2 Prostaglandin E2 PGs Prostaglandins Phe Phenylalanine PI3K Phosphatidylinositol 3-kinase PIKC Phosphoinositol kinase C PK Pharmacokinetics PL Placebo PMNs Polymorphonuclear neutrophils PNETs Primitive neuroectodermal tumors Poly-A:U Polyadenylic-polyuridylic acid Poly-I:C Polyinosinic-polycytidylic acid PPARγ Peroxisome proliferator-activated receptor-γ PPARδ Peroxisome proliferator-activated receptor-δ PPV Personalized peptide vaccination PR Partial regression PR Partial response PR Progesterone receptor pRB Retinoblastoma protein PRRs Pattern recognition receptors PUFAs Polyunsaturated fatty acids PXA Pleomorphic xanthoastrocytoma R Rituximab RANK Receptor activator of nuclear factor-kappa B RARB Retinoic acid receptor beta RASSF1 RAS association domain family protein 1 Rb Retinoblastoma RCC Renal cell cancer RCTs Randomized-controlled trials RECIST Response Evaluation Criteria In Solid Tumors RFS Relapse-free survival RHAMM Receptor for hyaluronan-mediated motility

Abbreviations

Abbreviations

xxi

RIC Reduced-intensity conditioning rIL-2 Recombinant IL-2 RMS Rhabdomyosarcoma RNS Reactive nitrogen species ROS Reactive oxygen species RR Response rate RRMM Relapsed or refractory MM RT Radiation therapy RTK Receptor tyrosine kinase RXRs Retinoid X receptors SART Squamous cell carcinoma antigen recognized by T cells SASP Senescence-associated secretory phenotype SC Subcutaneous SCC Squamous cell carcinoma SCCHN Squamous cell carcinoma of the head and neck sCD30 Soluble CD30 scFv Single-chain variable-fragment SCLCs Small cell lung carcinomas SCP-1 Stromal cell-derived protein SD Stable disease SDF-1 Stromal cell-derived factor-1 SEREX Serological analysis of antigens by recombinant expression cloning SERMs Selective estrogen response modulators sIL-2R Soluble interleukin-2 SIR Standardized incidence ratio SL Salmonella SLN Sentinel lymph node SLP Synthetic long peptides SPTs Second primary lung tumors SRE Skeletal-related events SSX Synovial sarcoma X chromosome breakpoint STAT Signal transducer and activator of transcription STAT-3 Signal transducer and activator of transcription-3 STS Soft tissue sarcomas SWOG South West Oncology Group T Thickness TA Tumor antigen TAA Tumor-associated antigen TADCs Tumor-associated dendritic cells TAM Tumor-related macrophage TAMs Tumor-associated macrophages TAMs Tumor-associated macrophages/microglia TAMs Tumor-associated monocytes/macrophages TANs Tumor-associated neutrophils TAP-1 Abnormal transport proteins TApDCs Tumor-associated plasmacytoid dendritic cells TCC Transitional cell carcinoma

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TCR T cell receptor TDSFs Tumor-derived soluble factors TERT Telomerase reverse transcriptase TF Tissue factor TGF Transforming growth factor TGFBI Transforming growth factor-β-inducible gene-h3 TGFβ Transforming growth factor-β Th T helper Th1 T helper 1 Th17 T helper 17 Th2 T helper 2 TIC Tubal intraepithelial carcinoma TIL Tumor-infiltrating lymphocytes TIM-3 Immunoglobulin- and mucin domain-containing protein 3 TIMP1 Tissue inhibitor of metalloproteinase 1 TKI Tyrosine kinase inhibitor TLR Toll-like receptor TLS Tertiary lymphoid structures TMZ Temozolomide TNBC Triple-negative breast cancer TNF Tumor necrosis factor TNFR Tumor necrosis factor receptor TNFR1 TNF receptor 1 TNF-α Tumor necrosis factor-α TP53 Tumor protein 53 TRAIL Tumor necrosis factor-related apoptosis-inducing ligand TRAILR2 TNF-related apoptosis-inducing ligand receptor 2 Treg Regulatory T cell TRM Treatment-related mortality TRUC Tbet−/− and Rag2−/− ulcerative colitis TSA Tumor-specific antigens TSP-1 Thrombospondin-1 TTP Time to tumor progression TTS Time to tumor survival TUM-CAM Tumor chorioallantoic membrane TUR Transurethral resection TX Thromboxane U Unit UCB Umbilical cord blood uPA Urokinase plasminogen activator uPAR Urokinase plasminogen activator receptor UPS Undifferentiated pleomorphic sarcoma URI Upper respiratory tract infection UTI Urinary tract infection VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptors VEGFR1 Vascular endothelial growth factor receptor 1 VH Variable heavy

Abbreviations

Abbreviations

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VIN Vulvar intraepithelial neoplasia VISTA V-domain immunoglobulin suppressor of T cell activation VL Variable light VNTR Variable number of tandem repeats VPF Vascular permeability factor WHIM Warts, hypogammaglobulinemia, infections, myelokathexis WHO World Health Organization WT1 Wilms tumor antigen WT1 Wilms tumor gene 1 XA Xanthoastrocytoma YLD Years lived with disability YLL Years of life lost βHCG Beta subunit of human chorionic gonadotropin

Contributors

Amir Hossein Abdolghaffari  Department of Toxicology & Pharmacology, Faculty of Pharmacy, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran Gastrointestinal Pharmacology Interest Group (GPIG), Universal Scientific Education and Research Network (USERN), Tehran, Iran Farzaneh Afshari  Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran Andreas  E.  Albers Department of Otorhinolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany Berlin Institute of Health, Berlin, Germany Víctor Andrés Arrieta  Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, USA PECEM, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico Zahra  Aryan Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran David  E.  Avigan Department of Medicine, Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Sander Bekeschus  ZIKplasmatis, Leibniz Institute for Plasma Science and Technology, INP Greifswald e.V, Greifswald, Germany

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Shahe  Boghossian National Health Service-NHS UK, Royal Glamorgan Hospital, Wales, UK Maurizio  Chiriva-Internati Departments of Lymphoma & Myeloma, Gastroenterology, Hepatology& Nutrition, MDANDERSON Cancer Center, Kiromic BioPharma, The University of Texas, Houston, TX, USA Oscar  J.  Cordero Department of Biochemistry and Molecular Biology, University of Santiago de Compostela, CIBUS Building, Campus Vida, Santiago de Compostela (Galicia), Spain Maryam  Ebadi Division of Hematology and Oncology, Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA Jose A. Figueroa  Kiromic LLC, Lubbock, TX, USA Negar  Ghaffari  Faculty of Medicine, Mazandaran University of Medical Sciences, Mazandaran, Iran Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran Maria  Gkotzamanidou Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Begoña  Graña-Suárez Service of Oncology, Galician Health System (SERGAS), Universitary Hospital Complex of A Coruña (CHUAC), A Coruña (Galicia), Spain Fabio  Grizzi Department of Immunology and Inflammation, Humanitas Research and Clinical Center, Rozzano, Milan, Italy Doreen Haase  Atrial Fibrillation NETwork, Münster, Germany Sara Hanaei  Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Universal Scientific Education and Research Network (USERN), Tehran, Iran Thomas  K.  Hoffmann Department of Otolaryngology, Head and Neck Surgery, University of Ulm, Ulm, Germany Peter  Hyckel Department of Oral and Maxillo-Facial Surgery/Plastic Surgery, Jena University, Jena, Germany Andreas M. Kaufmann  Clinic for Gynecology, Charité-Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany Berlin Institute of Health, Berlin, Germany Tessa Kerre  Department of Hematology, Ghent University Hospital, Ghent, Belgium Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium

Contributors

Contributors

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Mahsa  Keshavarz-Fathi Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Breast Cancer Association (BrCA), Universal Scientific Education and Research Network (USERN), Tehran, Iran Nastaran  Khalili  Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran Neda  Khalili Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran Lisa M. Kopp  Department of Epidemiology and Biostatistics, Mel and Enid Zuckerman College of Public Health, The University of Arizona, Arizona, USA Nicolaus  Kröger Department of Stem Cell Transplantation, Center for Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Naser-Aldin Lashgari  Department of Toxicology & Pharmacology, Faculty of Pharmacy, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Jessica  J.  Liegel Department of Medicine, Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Kai  Masur ZIKplasmatis, Leibniz Institute for Plasma Science and Technology, INP Greifswald e.V, Greifswald, Germany Håkan  Mellstedt Department of Oncology, Cancer Centre Karolinska, Karolinska University Hospital Solna, Stockholm, Sweden Hans-Robert Metelmann  Department of Oral and Maxillo-Facial Surgery/ Plastic Surgery, Greifswald University, Ferdinand-­Sauerbruch-­Strasse DZ 7, Greifswald, Germany Leonardo Mirandola  Kiromic LLC, Lubbock, TX, USA Saeideh Momtaz  Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), and Faculty of Pharmacy, Tehran, Iran Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran Gastrointestinal Pharmacology Interest Group (GPIG), Universal Scientific Education and Research Network (USERN), Tehran, Iran Sumana  Narayanan Department of Surgical Oncology, Mount Sinai, Miami, FL, USA

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Lauren  Nicholls Department of Pediatrics, Section of HematologyOncology, Baylor College of Medicine, Texas, USA Marek  Nowak Department of Gynecological Surgery and Gynecologic Oncology, Polish Mother’s Health Center Research Institute, Lodz, Poland Fred  Podmelle Department of Oral and Maxillo-Facial Surgery/Plastic Surgery, Greifswald University, Ferdinand-­ Sauerbruch-­ Strasse DZ 7, Greifswald, Germany Xu  Qian Department of Clinical Laboratory, Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, China Department of Clinical Laboratory, Zhejiang Cancer Hospital, Hangzhou, China Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, China Department of Otorhinolaryngology, Head and Neck Surgery, CharitéUniversitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany Farzaneh Rahmani  Student’s Scientific Research Center, Tehran University of Medical Sciences (TUMS), Tehran, Iran NeuroImaging Network (NIN), Universal Scientific Education and Research Network (USERN), Tehran, Iran Fateme  Rajabi Network of Dermatology Research (NDR), Universal Scientific Educational and Research Network (USERN), Tehran, Iran Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran Sepideh  Razi Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran Student Research Committee, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Nishitha  M.  Reddy  Hematology and Stem Cell Transplantation Section, Division of Hematology/Oncology, Department of Medicine, VanderbiltIngram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA Nima Rezaei  Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran

Contributors

Contributors

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Nazanin  Momeni Roudsari  Department of Toxicology & Pharmacology, Faculty of Pharmacy, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Fatemeh  Sadeghi Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran Mohammad  Amin  Sadeghi Department of Immunology, Molecular Immunology Research Center, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Department of Medicine, Tehran University of Medical Sciences, Tehran, Iran Amene  Saghazadeh Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Ali Sanjari-Moghaddam  School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Christian Seebauer  Department of Oral and Maxillo-Facial Surgery/Plastic Surgery, Greifswald University, Ferdinand-­ Sauerbruch-­ Strasse DZ 7, Greifswald, Germany Department of Plasma Medicine, Leibniz Institute for Plasma Science and Technology, INP Greifswald e.V, Greifswald, Germany Joseph J. Skitzki  Department of Surgical Oncology, Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA Sylvia  Snauwaert Department of Hematology, AZ Sint-Jan BruggeOostende AV, Bruges, Belgium Saeed  Soleyman-Jahi Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), St. Louis, MO, USA Division of Gastroenterology, School of Medicine, Washington University in Saint Louis, St. Louis, MO, USA Cancer Research Center, Cancer Institute of Iran, Tehran, Iran Adam  M.  Sonabend Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Thomas  Stübig Department of Internal Medicine II, Haematology and Oncology, University Medical Center Schleswig Holstein, Kiel, Germany Bart  Vandekerckhove Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium Rubén  Varela-Calviño Department of Biochemistry and Molecular Biology, University of Santiago de Compostela, CIBUS Building, Campus Vida, Santiago de Compostela (Galicia), Spain

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Jacek R. Wilczyński  Department of Gynecological Surgery and Gynecologic Oncology, Medical University of Łódź, Lodz, Poland Cancer Immunology Project (CIP), Universal Scientific Education of Research Network (USERN), Lodz, Poland Miłosz Wilczyński  Department of Gynecological and Endoscopic Surgery and Gynecologic Oncology, Polish Mother’s Health Center Research Institute, Lodz, Poland Thomas  von Woedtke  Department of Plasma Medicine, Leibniz Institute for Plasma Science and Technology, INP Greifswald e.V, Greifswald, Germany Yi  Zeng Department of Pediatrics, Steele Children’s Research Center, University of Arizona, Tucson, AZ, USA

Contributors

1

Cancer Immunotherapy Confers a Global Benefit Zahra Aryan, Mahsa Keshavarz-Fathi, Håkan Mellstedt, and Nima Rezaei

Contents 1.1

Introduction 

 2

I ncidence, Morbidity, and Mortality of Cancers: Why Is a New Therapeutic Avenue Indicated?  1.2.1    Cancer Incidence  1.2.2    Cancer Mortality Rate  1.2

1.3

 2  3  5

History of Immunotherapy of Cancers 

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1.4 Immunotherapy Is Going Upstream to Combat Cancers  1.4.1    Prophylactic Implication of Immunotherapy  1.4.2    Therapeutic Implication of Immunotherapy 

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1.5 Strategies of Cancer Immunotherapy  1.5.1    Immunotherapy Acts to Eliminate Immunosuppression  1.5.2    Immunotherapy Boosts the Antitumor Immune Responses and Enhances Killing of the Tumor Cell  1.5.2.1  Activated DCs and T Cells Are Pivotal in Cancer Immunotherapy  1.5.2.2  Materials of Activating DCs and T Cells 

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Z. Aryan Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran M. Keshavarz-Fathi Cancer Immunology Project (CIP), Universal Scientific Education and Research Network (USERN), Tehran, Iran

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N. Rezaei (*) Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran e-mail: [email protected]

School of Medicine, Tehran University of Medical Sciences, Tehran, Iran H. Mellstedt Department of Oncology, Cancer Centre Karolinska, Karolinska University Hospital Solna, Stockholm, Sweden © Springer Nature Switzerland AG 2020 N. Rezaei (ed.), Cancer Immunology, https://doi.org/10.1007/978-3-030-57949-4_1

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At Which Line of Treatment? 

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1.7

Monotherapy or Combined Therapy? 

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1.8

 onitoring the Immunological and Clinical Responses M to Immunotherapy 

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1.9

Limitations of Cancer Immunotherapy 

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Supportive Therapy 

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1.11

 ffect of Immunotherapy on Health-Related Quality E of Life of Cancer Patients 

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1.12

Cost-Effectiveness of Cancer Immunotherapy 

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1.13

Concluding Remarks 

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References 

1.1

Introduction

Cancer is a major public health issue which can affect every individual. Worldwide, cancer is one of the leading causes of mortality, morbidity, and decreased quality of life. Additionally, incidence of cancers is growing, and it would be the main source of burden on both patients and societies, particularly in low- to medium-resource countries. A total of one-fifth of overall cancers can be prevented by immunization against oncogenic infections. Thus, national vaccination programs against viruses such as HPV help prevent cancers and are regarded as the primary level of prevention using immunotherapy. On the other hand, current standards of care have failed to do much for many cancer patients; hence, a new therapeutic avenue like immunotherapy is needed to improve the care of cancer patients. With regard to current status of cancers worldwide including considerable incidence, morbidity, mortality rate, and insufficiency of current mainstays of cancer management including surgical approaches, chemotherapy, and radiotherapy, immunotherapy holds great promise in combating cancers. In this chapter, a glance at the overall status of morbidity, mortality, and burden of cancers worldwide has been made. Then the utility of immunotherapy at primary, secondary, and tertiary levels of prevention from cancers is discussed. At the end immunerelated response criteria for cancer immunotherapy as well as cost-effectiveness of cancer immunotherapy have been discussed.

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1.2

Incidence, Morbidity, and Mortality of Cancers: Why Is a New Therapeutic Avenue Indicated?

Nowadays, cancer has become a global health issue with respect to its worldwide increase in incidence and burden. New cancer cases were estimated to be 18.1 million in 2018, whereas it is expected to rise to 22.2 million in 2030. This is alarming since increase in incidence of cancers outnumbers the proportional increase in population worldwide [1, 2]. Another unpleasant fact is the high mortality of this growing issue. Of 18.1 million new cancer cases in 2018, 48.4% were diagnosed in Asia, 23.4% in Europe, 21% in Americas, 5.8% in Africa, and 1.4% in Oceania. On the other hand, of 9.6 million deaths, 57.3% occurred in Asia, 20.3% in Europe, 14.4% in Americas, 7.3% in Africa, and 0.7% in Oceania [2]. Considering these absolute numbers of new cases and deaths owing to cancers, low- to middle-­income countries are at emergent need for appropriate heath policies to fight cancers. In the following years, it is also estimated that new cases will mostly occur in low- to medium-­ resource countries due to two major reasons: (1) increase in the incidence of cancers associated with westernized lifestyle including colorectal, breast, and prostate cancers and (2) increase in the incidence of infection-related cancers (stomach, liver, and cervical cancers and less importantly lymphomas and Kaposi’s sarcoma) owing

1  Cancer Immunotherapy Confers a Global Benefit

to the dramatic increase in the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and human papillomavirus (HPV) infections, particularly in sub-Saharan Africa and East Asia [1, 3]. These data shed light on “global cancer transition” that should be considered when establishing priorities to control cancers. One of the best strategies to control cancer pandemic, particularly in a low-resource setting, is to provide vaccination against oncogenic viruses considered to have prophylactic use in immunotherapy to fight cancers [4–7].

1.2.1 Cancer Incidence Worldwide incidence of overall cancers was estimated to be 197.9 per 100,000 for all cancers, 218.6 per 100,000 for men, and 182.6 per 100,000 for women in 2018, and it is predicted to grow in the future. In 2018, the highest age-standardized incidence of overall cancers per 100,000 was estimated for men in Australia/New Zealand with 571.2 per 100,000. By contrast, the least incidence was estimated to be 95.6 per 100,000 men in

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Western Africa [2] (Fig. 1.1). Based on the countries, the highest age-standardized incidence of overall cancers per 100,000 was estimated for Australia with 468.0, followed by New Zealand with 438.1, Ireland with 373.7, Hungary with 368.1, and the United States with 352.2 (Fig. 1.2) [8]. With respect to these data, incidence of cancer is the highest in high-resource countries, possibly due to westernized lifestyle and exposure to a wide range of pollutants and carcinogens. However, it does not mean that cancers are of less importance in restricted-resource regions as explained by several reasoning: (1) An absolute number of new cancer cases are higher in less developed countries with respect to overall population and (2) underestimation of cancers in restricted-resource setting is a possibility considering the worldwide inequality in health system facilities. More than the environment, host-related characteristics are also major determinants of cancer incidence. Since cancer development is the result of immune system defeat in the war against tumor cells, immune-deficient states are believed to predispose subjects to several cancers. The most common form of immune deficiency is sec-

Estimated age-standardized incidence and mortality rates (World) in 2018, all ages Incidence Mortality

Females

Males

Australia and New Zealand

North America

Western Europe

Populations

Northern Europe

Southern Europe

Central and Eastern Europe

Polynesia

Eastern Asia

Southern Africa

South America 600 Data source: Globocan 2018 Graph production: Global Cancer Observatory (http://gco.iarc.fr)

500

400

300

200

100

0

100

200

300

400

500

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ASR (World) per 100 000

Fig. 1.1  Estimated age-standardized incidence and mortality rates of overall cancers per 100,000 in different countries. (Reproduced by permission from International Agency for Research on Cancer (IARC) [8])

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Fig. 1.2  Estimated age-standardized incidence rate of overall cancers per 100,000 in different countries. (Reproduced by permission from IARC [8])

ondary types, though both primary and secondary forms are associated with cancer development [9–12]. Infection-related cancers, but not other cancers, have increased incidence in these subjects [10, 12]. Acquired immunodeficiency syndrome (AIDS), as one of the most important causes of secondary immunodeficiency, is associated with increased malignancies [10]. AIDS-­ defining cancers encompass Kaposi’s sarcoma, cervical cancer, and non-Hodgkin lymphoma (NHL) with a standardized incidence ratio (SIR) of 3640.0, 5.8, and 76.7, respectively. Interestingly, Kaposi’s sarcoma risk of incidence is up to 3640 times higher in patients with AIDS compared to the normal population [10]. More interestingly, subjects who received transplantation and are immunosuppressed with drugs have SIR of 208.0 for Kaposi’s sarcoma and are also at increased risk for other infection-related cancers [10]. Hence, immunodeficiency is an important risk factor for cancer development. All the interventions targeting immune system directly or indirectly to improve immunity will reduce infections in immune-deficient patients and thereby prevent infection-related cancers [11, 12]. In addition to immunodeficiency, chronic inflam-

matory states and autoimmunities predispose individuals to cancer development [13–15]. Incidence of cancers also varies in different age groups. It is suggestive of up to 20 times increase in incidence of cancers by aging. Accumulating genetic alterations in a long period of time and gradual deterioration of immune system by aging, regarded as immunosenescence, are all attributed to increased incidence of cancers with increase in age [16–18]. Interestingly, types and course of cancers are also different between children and adults. Leukemia is the most common cancer in childhood while lung, breast, colorectal, and prostate cancers are the most common cancers in adults. With respect to rapid expansion of elder population all around the world, there is an emerging need for novel strategies to prevent and treat cancers [18]. In addition to environmental factors and host characteristics, incidence of cancers varies with respect to cancer sites. Some cancers are so common, while some are relatively uncommon The highest incidence of cancer is attributed to breast, prostate, lung, colorectal, and cervix uteri cancers with incidence of 46.3, 29.3, 22.5, 19.7, and 13.1 per 100,000, respectively. Beyond the range,

1  Cancer Immunotherapy Confers a Global Benefit

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Estimated age-standardized incidence and mortality rates (World) in 2018, worldwide, both sexes, all ages Breast Prostate Lung Colorectum Cervix uteri Stomach Liver Corpus uteri Thyroid Ovary Oesophagus Bladder Non-Hodgkin lymphoma Leukaemia Pancreas Kidney Lip, oral cavity Brain, central nervous system Melanoma of skin Gallbladder Larynx Testis Multiple myeloma Nasopharynx Oropharynx Hodgkin lymphoma Hypopharynx Vulva Penis Salivary glands Kaposi sarcoma Vagina Mesothelioma

0

Incidence Mortality

10

20

Data source: Globocan 2018 Graph production: Global Cancer Observatory (http://gco.iarc.fr)

30

40

ASR (World) per 100 00

Fig. 1.3  Worldwide age-standardized incidence and mortality rates of cancers in 2018. (Reproduced by permission from IARC [8])

cancer of penis, cancer of salivary glands, Kaposi’s sarcoma, cancer of vagina, and mesothelioma are the least common cancers worldwide with incidence of 0.80, 0.59, 0.50, 0.37, and 0.31 per 100,000, respectively (Fig. 1.3) [8]. As described in the following, fatality of cancers with high incidence is not low; notably, some like lung cancers also have the highest mortality rates among cancers. As current surgical, radiotherapeutic, and chemotherapeutic approaches failed to improve the outcome of cancers, novel approaches like immunotherapy may be the solution.

170.2, followed by Hungary with 155.8, Serbia with 150.7, Zimbabwe with 146.7, and Slovakia with 141.4 [8]. As evident, area of residence affects cancer status. Population composition particularly mean age of people, lifestyle, environmental factors including pollutants and status of infectious diseases in that area, and vaccination and screening programs are determinants of cancer incidence in each geographic area. However, mortality rate of cancers is affected by access to health facilities together with natural course of disease and its fatality. Proportion of cancer death to new cases is the highest in Africa; conversely the least proportional death has been recorded in North America. Disparities in receiv1.2.2 Cancer Mortality Rate ing immunotherapy also exist in nationwide perspective in which patients with lower Worldwide mortality rate of overall cancers was socioeconomic status are less likely to benefit estimated to be 101.1 per 100,000 in 2018, and it from novel efficacious therapeutic modalities is also predicted to grow in the future if health [19]. Access to current standards of care as well systems do not improve worldwide. In contrast to as novel treatments affects the outcome of canhigher incidence of cancers in developed high-­ cers. It implies that our interventions are efficaresource countries, the mortality rate of cancers cious in changing the outcome of cancers but is higher in low-resource countries (Fig. 1.4). The should be spread worldwide equally. To reach highest age-standardized mortality rates of can- this goal, spread of knowledge about new theracers per 100,000 are recorded in Mongolia with peutic modalities as well as investment of

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Fig. 1.4  Estimated age-standardized mortality rate of overall cancers per 100,000 in different countries. (Reproduced by permission from IARC [8])

i­nternational and national organizations on cancer is needed. Similar to incidence, patient’s characteristics affect the outcome of cancer. Immunodeficient patients are at increased risk of cancers and also have higher risk for cancer-related mortality. However, investigations indicate that cancer-­ related mortality will dramatically decrease by interventions aimed to preserve immune system functions [20]. As expected, mortality rate of cancers also increases with aging (Fig.  1.2), which may be explained by higher fatality of cancers occurring in elderly as well as deterioration of immune functions in this group of patients [21]. The greatest determinant of cancer mortality is the site of the tumor and its stage at diagnosis. A 5-year survival of less than 10% in patients with pancreatic, liver, esophageal, and lung cancers warrants the need for novel, more efficacious therapeutic modalities. In other words, more than 90% of patients with these fatal cancers will be deceased in only 5 years. Regardless of sex, the highest mortality rates are seen in patients with lung, breast, colorectal, liver, and stomach cancers with age-standardized mortality rates of 18.6, 13, 8.9, 8.5, and 8.2 deaths per

100,000 [8]. Age-standardized mortality rate of different cancers in both sexes is depicted in Fig. 1.3.

1.3

History of Immunotherapy of Cancers

First experience of cancer immunotherapy dates back to 1898 when William B. Coley succeeded to treat inoperable sarcomas by intratumoral injections of Streptococcus pyogenes and Serratia marcescens toxins [22]. This challenging observation of administration of bacterial products to already cancer patients with weakened immune system constructed the cornerstones of today’s cancer immunotherapy. For ensuing 50 years, the progress of cancer immunotherapy was slow with only sporadic documents of successful treatments that were mostly irreproducible. However, further studies paved the road to immunotherapy of cancers. In this way, another important step was the attempt of Maurice Hilleman to invent hepatitis B vaccine. Hepatitis B vaccine prevents the spread of hepatitis B virus (HBV) infection and its consequences such as development of hepatocellular carcinoma (HCC) by induction of

1  Cancer Immunotherapy Confers a Global Benefit

active immunity against HBV.  Concurrent with these investigations, in 1976, post-resection intravesical instillation of bacillus Calmette-­ Guérin (BCG) was shown to prolong the survival of patients with bladder cancer. Indeed, cancer immunotherapy was evolving in both prophylactic and therapeutic approaches. Since the 1980s, emerging field of cancer immunotherapy was revolutionized by introduction of cytokines, monoclonal antibodies (mAbs), and adoptive cell therapy in treatment of cancers. Since then, cytokines and mAbs were tested not only as stand-­ alone therapeutic modalities but also in combinational schedules with chemotherapy [23–25] and radiotherapy [26]. Interferon (IFN)-α was approved for hairy cell leukemia in 1986, and it was the first immunotherapeutic drug approved for use in melanoma patients in 1995, owing to comprehensive studies by Kirkwood and his colleagues [27–29]. Rituximab is the first mAb which received Food and Drug Administration (FDA) approval for NHL in 1997 [30]. Immunotherapy also showed efficacy in postsurgical management of patients with cancers. One of the first reports dates back to 1988 when Grohn et al. employed levamisole adjuvant immunotherapy in patients with breast cancer with equivocal results [31]. Nearby these events, in 1986, recombinant HBV vaccine was developed, and efforts to constructing human papillomavirus (HPV) vaccine were initiated. The first HPV vaccine was approved by the FDA in 2007. Similar to HBV vaccine, HPV vaccine prevents viral induced cancers, in particular cervical cancers of the genital area and anus and oropharyngeal cancers. Despite common concept about vaccines, cancer vaccination can be applied to treat neoplastic lesions as secondary line of prevention. HPV vaccination was used to treat vulvar intraepithelial neoplasia (VIN) with promising results in 2009 [32]. In addition, dendritic cell (DC)-based, peptide-based, and combined vaccines were introduced to treat cancer patients in the recent decade [33]. Gene therapy, by providing the opportunity to manipulate the immune system, holds a great promise for cancer immunotherapy [33]. Gene

7

transfer with novel biologic or nonbiologic delivery vehicles enabled scientists to genetically modulate T cells to combat tumors [33]. Combination of adoptive T-cell therapy with gene transfer was one of the most important steps in the field of cancer immunotherapy. In this way, T-cell receptor (TCR) gene transfer was one of the greatest achievements in treating cancers, reached in 2001 [34]. Interestingly, further investigations suggest hopes for a combination of cancer vaccines with current mainstays of cancer treatments. One of the most interesting studies was conducted by Antonia et al. on patients with extensive-stage small-cell lung cancer in 2007 [35]. The patients received dendritic cells transduced with the full-length wild-type p53 gene delivered via an adenoviral vector as cancer vaccine prior to chemotherapy. Significant clinical response was observed in more than half of the patients owing to pre-chemotherapeutic stimulation of the immune system by cancer vaccine [35]. Another progress was the combination of adoptive T-cell therapy with radiotherapy made in 2005. In this experience, combined radiotherapy with intratumoral injection of the cancer vaccine was promising in patients with refractory hepatoma [36]. Vaccine-based therapeutics aim to enhance endogenous immune response against cancers, while adoptive T-cell therapy is based on infusion of primed tumor-specific T cells. Finally, sipuleucel-T (Provenge), an active autologous dendritic cell-based vaccine, received FDA approval for patients with castration-resistant prostate cancer in 2010 [37]. Sipuleucel-T is the first and the sole FDA-approved therapeutic cancer vaccine. Unfortunately, no adoptive T-cell therapy has yet obtained FDA approval maybe due to its obstacles in providing sufficient amounts of primed specific T cells [38]. However, cancer-testis antigens of MAGE family with restricted expression in tumor cells hold promise for the future of not only cancer vaccines but also adoptive T-cell therapies [39, 40]. Almost all the progresses in immunotherapy are owed to progresses in understanding the immunopathology of cancers. This is well reflected in the development of novel monoclonal antibodies and immune adjuvants through the

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past decade. Ipilimumab (also known as MDX-­ ­ 010-­ 20  in clinical trials) received FDA approval for the treatment of patients with advanced metastatic melanoma in 2011. Ipilimumab blocks the inhibitory effects of cytotoxic T lymphocyte-­associated antigen 4 (CTLA4) on presenting the tumor antigens and improves cytotoxic T lymphocyte function [41, 42]. Pegylated IFN-α has been approved for treating patients with advanced melanoma in March 2011 [33]. Another progress was the discovery of pattern recognition receptors and targeting them in cancer immunotherapy; imiquimod (Aldara), a Toll-like receptor 7 agonist, was employed in the treatment of VIN since 2008 [43]. Imiquimod has been approved by the FDA for external genital warts, papilloma, superficial basal cell carcinoma, and actinic keratosis [44]. Imiquimod administration results in endogenous induction of IFN production [44]. Through more than a century of experience with immunotherapy, scientists and health-care providers aimed to reinstate immune surveillance against tumor cells in either primary lesions or metastases by immunotherapy. Immunotherapy provides a dynamic and specific formation of adaptive immune response that fights tumor-­ mediated immunoediting. This effect of immunotherapy promises long-term protection against relapse of cancers, while it may not be efficacious as other drugs, radiotherapy, and surgery in immediate debulking of tumor masses. Hence, combinational therapies may be the key to improve both tumor progression and overall survival of the patients. Despite brilliant progresses in the field of cancer immunotherapy, it is still in its infancy and may provide definite treatment for all cancers in its maturity.

1.4

I mmunotherapy Is Going Upstream to Combat Cancers

Immunotherapy helps health-care providers prevent not only cancer development but also its further progression and cancer-related complications offering prevention from cancers at all levels. Modulation of immune responses in favor of

enhancing tumor cell detection and immune clearance of these cells is what immunotherapy does. In addition, immunotherapy helps recover an injured or completely destroyed immune system after intensive cancer therapies as occurred in intensive chemotherapy schedules. Chronic inflammation owing to infectious etiologies or continuous sterile inflammation appears to cause cancers of variable origins [45]. Targeting the immune system to control infections known as causes of variable cancer as well as conditions associated with chronic inflammation (i.e., autoimmunities) results in a dramatic decrease in the incidence of cancers [46]. This application of immunotherapy prevents cancers at the primary level. Interestingly, not all subjects with predisposing chronic inflammation state develop cancers. For instance, the human herpesvirus 8 (HHV8) causes Kaposi’s sarcoma in the context of HIV caused by immunodeficiency or drug-induced immunodeficiency. Hence, immunotherapeutic approaches prevent the spread of HIV in the community and can be regarded as a primordial level of prevention from cancers. A considerable numbers of vaccines and immune adjuvants as well as monoclonal antibodies are developed to combat cancers at primary and primordial stages. On the other hand, a broad spectrum of immunotherapeutic medications have been developed to treat patients with cancers. At this stage, immunotherapy acts as the second level of prevention from cancers. Adoptive cell therapy, therapeutic cancer vaccines, immune adjuvants, cytokines, monoclonal antibodies, and gene therapies are already established to treat different cancers. Treatment of an already diagnosed patient with any type of cancer is designated as the secondary level of prevention from cancer. This is the most known kind of use of immunotherapy to combat cancers; however, it acts lately after establishing the cancer and usually can eliminate cancer in a limited number of patients. Today, immunotherapy is considered as a first line of treatment of a wide range of cancers. Immunotherapy also offers hope for patients failed with other available therapeutics and patients with end-stage cancers. In addition,

1  Cancer Immunotherapy Confers a Global Benefit

combination of immunotherapy with almost all available therapeutic approaches has been tested and holds promises at least to increase progression-­free survival of patients. Many new immunotherapeutic drugs are now under development, and many of them are in clinical trials. Finally, immunotherapy can ameliorate the toxic effects of other available therapeutic modalities, known as supportive immunotherapy. At this stage, immunotherapy prevents further disabilities due to cancer progression or therapies and improves quality of life of patients. Accordingly, immunotherapy also provides a tertiary level of prevention from different cancers.

1.4.1 Prophylactic Implication of Immunotherapy It is best to prevent cancer development than to prevent cancer progression and its related complications. Immunotherapy is defined by the treatment of disease using enhancement or suppression of immune responses. Accordingly, it can be used to treat cancers as well as treat underlying diseases that cause cancers. The latter is prophylactic use of immunotherapy for cancers. Eliminating infections known to cause cancers and improving the immune system by eliminating chronic inflammation states and immunodeficiency are the bases of prophylactic cancer immunotherapy. Despite complex expensive immunotherapeutic approaches employed at other levels of prevention from cancers, immunotherapy at primary level includes simple inexpensive methods. To follow a healthy lifestyle, daily intake of anti-inflammatory drugs, vaccination against oncogenic viruses, and finally immunotherapies aimed to control spread of HIV are all acting to prevent cancers at primary or primordial level. Increased body mass index (BMI) is believed to increase the risk of several cancers [3]. Correction of lifestyle by doing regular exercise, eating fresh foods full of antioxidants, restricting calorie intake, and eating low-fat foods lead to prevention of cancers [47–49]. The main mechanism is inhibiting tumorigenesis and providing

9

inappropriate microenvironment for tumor growth; however, a healthy lifestyle improves immune function [47, 48, 50]. Indeed, the main antitumor mechanism of having a healthy lifestyle is to prevent tumorigenesis, but combating chronic inflammation state as well as enhancing the function of effector innate cells to eliminate tumor cells should also be acknowledged [50– 54]. From this perspective, a healthy lifestyle acts as an old, inexpensive, simple immunomodulatory way of preventing cancers at primary level [47, 48]. Thus, healthy lifestyle can be regarded as the first ancient immunotherapy employed against cancers. Infection-related cancers are caused by a variety of viral and bacterial agents. Chronic inflammation, mutagenesis by integration of pathogen genes with host genome, and induction of immunodeficiency are all believed to be mechanisms by which infectious agents can cause cancers. HBV is one of the most important agents known to cause HCC. The HBV vaccine is able to induce immunity against HBV infection in more than 95% of vaccinated subjects. Thereby, it can prevent HBV-caused HCC [55]. In endemic areas of HBV infection like Taiwan, it has been shown that HBV vaccination has resulted in decrease of HCC incidence from 1.08 per 100,000 to 0.49 per 100,000, suggestive of a 50% decrease in HCC incidence [56]. Interestingly, in some areas like Alaska, known as an endemic area of HBV infection in the United States, HBV vaccination has eliminated HCC in children [57]. In addition, in coinfected patients with HBV and hepatitis C virus (HCV), immunotherapy with IFN-α reduces the risk of HCC development [58]. Hence, active immunotherapy against HBV infection leads to significant reduction in HCC occurrence, and it is logical to assume HBV vaccine as a prophylactic immunotherapy against cancers. HPV vaccine is another golden step in the history of cancer immunotherapy. HPV-16 and HPV-18 are responsible for more than 70% of cervical cancers worldwide. However, HPV-6 and HPV-11 are also targeted in newer generations of HPV vaccine. The best target group of vaccine administration is young girls who are not still sexually active. Indeed, the vaccine is benefi-

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cial for someone who is not infected with the virus. Quadrivalent vaccination against HPV (16, 18, 6, and 11) results in 98% (95% confidence interval of 86–100%) protection against HPV-16 or HPV-18 cancer-related lesions. It also confers weak protection against other cervical neoplastic lesions [59]. As the cervical cancer holds the second place in incidence and DALYs of cancers of women all around the world, its prevention is of utmost importance. HPV vaccine is a cost-­ effective approach to reduce cervical cancer incidence worldwide [60, 61]. In addition to cervical cancer, HPV-16 and HPV-18 are implicated in the development of other perineal and perianal neoplastic lesions as well as squamous cell carcinoma of the head and neck and oropharyngeal cancers. HPV vaccination can reduce the risk of these cancers but with a lesser extent [62]. Vaccination of women also offers protection against HPV-related cancers in men owing to herd immunity; however, covering boys in vaccination programs is not without clinical benefits and needs further investigations [62]. Interestingly, vaccination of women with high-­ grade VIN with a mix of oncoproteins E6 and E7 from HPV-16 resulted in relief of VIN-related symptoms in 60% of patients, highlighting the important role of HPV vaccine for cervical cancers [32]. By contrast to HBV and HPV vaccine, other immunotherapeutic approaches to control HIV and Epstein-Barr virus (EBV) are on the way. In the development of anti-HIV agents, targeting the adaptive immune system failed due to progressive involvement of the adaptive system. However, recent studies herald promises in targeting the innate immunity by targeting DCs, pattern recognition receptors, and alarmins [63]. Prevention from spread of HIV and progression of HIV infection to AIDS averts AIDS-associated syndromes and AIDS-defining cancers (Kaposi’s sarcoma, cervical cancer, and NHL). Immunotherapeutic approaches combat HIV spread, and others used to prevent infections in immunodeficient patients whether primary or secondary are regarded as prophylactic implica-

Z. Aryan et al.

tion of cancer immunotherapy [11, 63]. Autosomal recessive hyper-IgE syndrome, X-linked agammaglobulinemia, common variable immunodeficiency, X-linked lymphoproliferative disease, IL-2-inducible T-cell kinase (ITK) deficiency, epidermodysplasia verruciformis, and warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome are primary immunodeficiency diseases with increased risk of infection-related cancers. Granulocyte-macrophage colony-stimulating factor (GM-CSF), intravenous immunoglobulin (IVIG) administration, and allogeneic hematopoietic stem cell transplantation (HSCT) provide benefits for these patients [11]. EBV is one of the most common viral infections with more than 90% seropositivity worldwide [64]. It is implicated in the development of several cancers including Burkitt’s lymphoma, NK or T-cell lymphoma, Hodgkin lymphoma, and nasopharyngeal carcinomas particularly in subjects with incompetent immune system [64]. EBV-associated lymphoproliferative disorders are of utmost clinical importance in patients who have undergone transplantation. Immunotherapy with adoptive T-cell transfer specific for EBV antigens promises hope to prevent EBV-associated lymphoproliferative disorders in vulnerable subjects with EBV viremia [65–67]. Sterile chronic inflammation and immune dysregulation states like autoimmunities also predispose individuals to cancer development. Sterile chronic inflammation is strongly associated with colorectal cancer development. Patients with ulcerative colitis are at increased risk of colorectal cancers that might be prevented by anti-inflammatory drugs like 5-aminosalicylic acid (5-ASA) [68]. Another group of patients suffering from familial adenomatous polyposis (FAP) have chronic inflammation state and 100% increased risk of colorectal cancers. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, is approved to be used for prevention of colorectal cancer in this group of patients [68]. In subjects with no underlying inflammatory disease of the gastrointestinal tract, nonsteroidal anti-inflammatory

1  Cancer Immunotherapy Confers a Global Benefit

drugs (NSAIDs) reduce the risk of colorectal cancer by 18–39%; however, to date no NSAIDs have been approved to prevent sporadic tumor prevention [69–71]. Similarly, monoclonal antibodies used in the treatment of patients with autoimmunities that predispose patients to malignancies can be regarded as another prophylactic immunotherapeutic approach [72]. Altogether, immunotherapy can be used to prevent the development of cancers. Prophylactic use of immunotherapy, also regarded as immunoprevention, offers benefits for a wide range of cancers particularly infection-related cancers. Both active immunizations with vaccines and passive immunizations with monoclonal antibodies and cytokines are employed in prophylactic immunotherapy. Many other prophylactic immunotherapeutic modalities may be introduced in the future.

1.4.2 Therapeutic Implication of Immunotherapy Immunotherapy currently has been set as a key component of therapeutic regimens of many cancers [33]. Bone marrow transplantation (BMT) following ablative/non-myeloablative bone marrow therapies is now the standard of care of many hematological malignancies. Similarly donor lymphocyte infusion following failed BMT is an accepted immunotherapy for the treatment of relapsed hematological malignancies [73–75]. Once cancer develops, immunotherapy helps the patient’s immune system fight with tumor cells to prevent cancer progression and finally elimination of cancer [76]. Benefits of immunotherapy are not restricted to patients with advanced stages of cancers, and by contrast, patients with early stages of cancers are good candidates for immunotherapy. Bacillus Calmette-Guérin (BCG) for early-stage bladder carcinoma [77–79] and sipuleucel-­T immunotherapy for castration-resistant prostate cancer are all examples of approved immunotherapies employed at different stages of urological can-

11

cers [37, 80, 81]. In addition, immunotherapy offers hope for approximately all types of cancers. FDA-approved immunotherapeutic drugs are now available for chronic lymphocytic leukemia (CLL) [82, 83], NHL [30, 84, 85], Hodgkin lymphoma (HL) [86, 87], acute leukemia [88], breast cancer [89], lung cancer [90], colorectal cancers [69, 91–93], bladder cancer [78, 79], prostate cancer [80, 81], renal cell carcinoma [29, 94], basal cell carcinoma [44, 95], melanoma [96–98], cervical cancer [5, 32], hepatocellular carcinoma [55, 56, 58], and soft-­tissue tumors [99, 100]. Promisingly, a large number of immunotherapies are also under investigation. Table  1.1 shows current FDA-approved immunotherapies to treat different cancers. Immunotherapeutic weapons are of wide categories: immunomodulator monoclonal antibodies whether agonistic or blocking, cytokines [interleukin (IL)-2, IFN-α, IL-12, GM-CSF, and tumor necrosis factor-α (TNF)-α], therapeutic cancer vaccines particularly DC vaccines, adoptive T-cell transfer, gene therapy, and novel immune adjuvant and delivery vehicles are all available to help cancer patients. Elimination of immunosuppression and boosting of immune responses against tumor cells are what immunotherapy does. These effects of immunotherapy offer long-term antitumor immune response that fights with already established cancer, prevents its progression, and prevents new metastases. Accordingly, immunotherapy should not logically become restricted to patients with advanced and metastatic cancers. Despite initial experiences with immunotherapy on patients who failed with other therapeutics, today, immunotherapy is set to become the first-line treatment either in combination with other therapeutic modalities or as stand-alone therapy. In addition, to accurately measure the immunotherapy-induced tumor destruction, immune-related response criteria have been developed and should be used in clinical practice and future researches. In the following, different aspects of cancer immunotherapy as therapeutic (second level of cancer prevention) have been described.

mAb

Immune checkpoint inhibitor

Antibody–drug conjugate

Category Antibody–drug conjugate

Targeting CD38

Mechanism of action HER2-directed antibody conjugated to cytotoxic chemotherapy Nectin-4-directed antibody conjugated to microtubule inhibitor Blockade of PD-L1

Daratumumab (Darzalex)

Atezolizumab (Tecentriq)

Enfortumab vedotin-ejfv (Padcev)

•  2019: Adult patients with locally advanced or metastatic urothelial cancer, previously treated with PD-1/PD-L1 blockade or chemotherapy in the neoadjuvant/adjuvant, locally advanced, or metastatic setting [103] •  2019: Combination therapy as first line for adults with metastatic NSCLC without EGFR/ALK aberrations [104], combination therapy as first line for ES-SCLC [105], PD-L1-positive unresectable advanced or metastatic triple-negative BC [106, 107], combination therapy as first line for metastatic NSqNSCLC without EGFR/ALK aberrations [108, 109] • 2017: Locally advanced or metastatic urothelial carcinoma not eligible for chemotherapy [110] • 2016: Metastatic NSCLC without EGFR/ALK aberrations that progressed despite chemotherapy [111], locally advanced or metastatic urothelial carcinoma that progressed despite chemotherapy [112] •  2019: Combination therapy for adults with newly diagnosed MM who are eligible for ASCT [113], combination therapy for MM ineligible for transplant [114] • 2016: Combination therapy for progressive MM [115, 116] • 2015: MM with ≥3 prior treatments [117]

Immunotherapeutic modality Year and indication(s) of approval Fam-trastuzumab • 2019: Unresectable or metastatic HER2-positive BC with deruxtecan-­nxki (Enhertu) ≥2 prior anti-HER2 treatments in the metastatic setting [102]

Table 1.1  FDA-approved immunotherapeutic agents to treat cancers [101]

Neutropenia, thrombocytopenia, infusion reactions

Hyperglycemia, peripheral neuropathy, ocular disorders, skin reactions, infusion-site extravasation Immune-mediated pneumonitis, immunemediated hepatitis, immune-­mediated colitis, immune-mediated endocrinopathies, infections, infusion-­related reactions

Adverse effects Interstitial lung disease/ pneumonitis, neutropenia, left ventricular dysfunction

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Immune checkpoint inhibitor

Blockade of PD-1

Pembrolizumab (Keytruda)

• 2019: Combination therapy for advanced endometrial carcinoma without MSI-H/dMMR that progressed despite systemic therapy [118], advanced PD-L1-positive esophageal SCC that progressed despite systemic therapy [119–121], metastatic SCLC that progressed despite prior therapy [122, 123], first-line treatment for metastatic or unresectable recurrent SCCHN [124], combination therapy as first line for RCC [125], first line for stage III NSCLC not candidates for surgery/chemoradiation or metastatic PD-L1-positive NSCLC without EGFR/ALK aberrations [126], adjuvant therapy for melanoma with involvement of lymph node(s) following complete resection [127] • 2018: Adult and pediatric recurrent or metastatic MCC [128], HCC previously treated with sorafenib [129], combination therapy as first line for metastatic NSCLC [130], combination therapy as first line for metastatic NSqNSCLC without EGFR/ALK aberrations [131, 132], adult and pediatric patients with r/r PMBCL [133], recurrent or metastatic PD-L1-positive cervical cancer that progressed despite chemotherapy [134] • 2017: Advanced gastric or GEJ cancers that progressed after ≥2 prior lines of treatment [135], solid tumors in adult and pediatric patients with MSI-H and/or dMMR that progressed despite prior treatments [136, 137], combination therapy as first line for NSCLC regardless of PD-L1 expression [138], advanced or metastatic bladder cancer that progressed despite chemotherapy [139, 140], r/r cHL [141] • 2016: First line for metastatic PD-L1-positive NSCLC without EGFR/ALK aberrations [142], recurrent or metastatic SCCHN that progressed despite chemotherapy (NCT01848834) • 2015: Unresectable or metastatic melanoma [76, 77], metastatic PD-L1-expressing NSCLC that progressed despite chemotherapy [143] • 2014: Unresectable or metastatic melanoma following ipilimumab and a BRAF inhibitor (in case of BRAF V600 mutation) [144]

(continued)

Immune-mediated pneumonitis, immunemediated hepatitis, immune-mediated colitis, immune-mediated endocrinopathies, infusion-related reactions, immune-mediated nephritis and renal dysfunction, immunemediated skin adverse reactions

1  Cancer Immunotherapy Confers a Global Benefit 13

Mechanism of action CD79b-directed antibody conjugated to antimitotic cytotoxic

Blockade of PD-L1

Targeting VEGFR2 (KDR)

Category Antibody–drug conjugate

Immune checkpoint inhibitor

mAb

Table 1.1 (continued)

Ramucirumab (Cyramza)

Avelumab (Bavencio)

Immunotherapeutic modality Polatuzumab vedotin-piiq (Polivy) Adverse effects Peripheral neuropathy, infusion-related reactions, myelosuppression, serious and opportunistic infections, progressive multifocal leukoencephalopathy, tumor lysis syndrome, hepatotoxicity Immune-mediated •  2019: Combination therapy as first line for RCC [146] • 2017: Locally advanced or metastatic bladder cancer [147], pneumonitis, immunemediated hepatitis, metastatic MCC in patients ≥3 years old [148] immune-mediated colitis, immune-mediated endocrinopathies, infusion-related reactions, immune-mediated nephritis and renal dysfunction, major adverse cardiovascular events Hemorrhage, • 2019: A single agent for HCC with AFP ≥400 ng/mL and gastrointestinal previously treated with sorafenib [149, 150] •  2015: mCRC that progressed after first-line treatment [151] perforations, impaired wound healing, arterial • 2014: Combination therapy for metastatic NSCLC that progressed despite chemotherapy and NSCLC with EGFR/ thromboembolic events, hypertension, infusionALK aberrations that progressed despite approved treatment [152], combination therapy for advanced gastric related reactions, or GEJ adenocarcinoma [153], single agent for advanced or worsening of preexisting hepatic impairment, metastatic gastric or GEJ adenocarcinoma that progressed reversible posterior despite chemotherapy [154] leukoencephalopathy syndrome, proteinuria including nephrotic syndrome, thyroid dysfunction

Year and indication(s) of approval • 2019: Combination therapy for adult patients with r/r DLBCL after ≥2 prior therapies [145]

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IL-3 fused to truncated diphtheria toxin, targeting CD123 (alpha chain of the IL-3 receptor) CD30-directed cytotoxin

Cytokine

Blockade of PD-1

CD22-directed cytotoxin

Immune checkpoint inhibitor

Antibody–drug conjugate

Antibody–drug conjugate

Her2-directed cytotoxin

Antibody–drug conjugate

• 2018: Combination therapy for previously untreated systemic anaplastic large-cell lymphoma or other CD30expressing peripheral T-cell lymphomas [158], combination therapy for adult patients with previously untreated stage III or IV (cHL) in combination with chemotherapy [159] • 2011: Refractory HL, refractory ALCL [87] • 2018: Metastatic or locally advanced cutaneous SCC who are not candidates for curative surgery or curative radiation [160]

Brentuximab vedotin (Adcetris)

Moxetumomab • 2018: Adult r/r HCL received ≥2 prior systemic therapies pasudotox-tdfk (Lumoxiti) [161]

(continued)

Severe and fatal immunemediated adverse reactions, infusion-related reactions Capillary leak syndrome, hemolytic uremic syndrome, renal toxicity, infusion-related reactions, electrolyte abnormalities

Cytopenia (all lineages), peripheral sensory neuropathy, nausea and vomiting, fatigue, URI, pyrexia

• 2018: BPDCN in patients ≥2 years old [157]

Tagraxofusp-erzs (Elzonris)

Cemiplimab-rwlc (Libtayo)

Hepatotoxicity, left ventricular dysfunction, embryo-fetal toxicity, pulmonary toxicity, infusion-related reactions, hypersensitivity reactions, hemorrhage, thrombocytopenia, neurotoxicity Capillary leak syndrome, hypersensitivity reactions, hepatotoxicity

• 2019: Adjuvant treatment for HER2-positive early BC with residual invasive disease after neoadjuvant treatment by trastuzumab [155] • 2013: Single agent for HER2-positive, metastatic BC that progressed despite treatment by trastuzumab and/or chemotherapy [156]

Ado-trastuzumab emtansine (Kadcyla)

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Mechanism of action Blockade of PD-1

Blockade of CTLA-4

Category Immune checkpoint inhibitor

Immune checkpoint inhibitor

Table 1.1 (continued)

Ipilimumab (Yervoy)

Immunotherapeutic modality Nivolumab (Opdivo) Year and indication(s) of approval • 2018: Metastatic SCLC that progressed despite chemotherapy and at least one other line of therapy [162] • 2017: Advanced HCC previously treated by sorafenib [163, 164], MSI-H, and/or dMMR-positive mCRC that progressed despite chemotherapy [165], locally advanced or metastatic bladder cancer that progressed despite adjuvant or neoadjuvant chemotherapy [166] • 2016: SCCHN that progressed despite chemotherapy [167], r/r cHL that progressed despite autologous transplant and brentuximab vedotin [168, 169] • 2015: Advanced RCC treated with prior anti-angiogenic agents [170], combined with ipilimumab for unresectable or metastatic melanoma without BRAF V600 mutation [171], advanced non-squamous NSCLC that progressed despite chemotherapy [172], advanced squamous NSCLC that progressed despite chemotherapy [173, 174] • 2014: Unresectable or metastatic melanoma following ipilimumab and a BRAF inhibitor [175] • 2018: Combined with nivolumab for the treatment of patients ≥12 years old with MSI-H or dMMR mCRC that progressed despite prior treatment [176] •  2015: Adjuvant treatment of cutaneous melanoma with positive regional lymph nodes of more than 1 mm after complete resection [177] • 2011: Unresectable or metastatic melanoma [41]

Immune-mediated enterocolitis/colitis, immune-mediated hepatitis, immune-mediated dermatitis/skin adverse reactions, immune-mediated neuropathies, immunemediated endocrinopathies, immune-mediated pneumonitis, immunemediated nephritis and renal dysfunction, immunemediated encephalitis, infusion reactions, other immune-mediated adverse reactions, including ocular manifestations, embryofetal toxicity

Adverse effects Immune-mediated pneumonitis, immunemediated hepatitis, immune-mediated colitis, immune-mediated endocrinopathies, infusion-related reactions, immune-mediated nephritis and renal dysfunction, immunemediated skin adverse reactions, immunemediated encephalitis, complications of allogeneic HSCT after nivolumab

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Inhibitor of VEGF-A

Targeting CD19 and providing first and co-stimulatory signaling through CD3ζ and 4-1BB

Targeting CD19 on cancer cells and CD3 on T cells

mAb

CAR T-cell therapy

Bispecific mAb

Blinatumomab (Blincyto)

Tisagenlecleucel (Kymriah™)

Bevacizumab (Avastin)

• 2018: Combination therapy followed by single-agent bevacizumab for stage III or IV disease after initial surgical resection epithelial ovarian, fallopian tube, or primary peritoneal cancer [178] • 2014: Combination therapy for recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer resistant to chemotherapy [179], combination therapy for persistent, recurrent, or metastatic cervical cancer [180] • 2013: Combination therapy for mCRC that progressed during a first-line bevacizumab-based treatment [181] • 2009: Combined with IFN-α for metastatic renal cell carcinoma [182], single agent for glioblastoma that progressed despite prior treatment [183, 184] • 2006: Combination therapy as first line for locally advanced, metastatic, or recurrent NSCLC [185], secondline treatment for mCRC [186] • 2004: Combination therapy as first line for mCRC [187] • 2018: Adult patients with r/r large B-cell lymphoma after two or more lines of systemic therapy including DLBCL not otherwise specified, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma [188] • 2017: B-cell ALL up to age of 25 not responding to treatment or relapsed ≥2 times [189] • 2018: Adult and pediatric patients with B-cell precursor ALL in first or second complete remission with minimal residual disease (MRD) greater than or equal to 0.1% [190] • 2017: r/r B-cell ALL [191] • 2014: r/r B-cell ALL with negative Philadelphia chromosome [192]

(continued)

Cytokine release syndrome, neurological toxicities, infections and febrile neutropenia, prolonged cytopenias, hypogammaglobulinemia Cytokine release syndrome, neurological toxicities, infections, tumor lysis syndrome, neutropenia and febrile neutropenia, effects on the ability to drive and use machines, elevated liver enzymes, pancreatitis, leukoencephalopathy

Gastrointestinal perforations and fistulae, surgery and wound healing complications, hemorrhage, arterial and venous thromboembolic events, hypertension, posterior reversible encephalopathy syndrome, renal injury and proteinuria, infusion reactions, ovarian failure, congestive heart failure

1  Cancer Immunotherapy Confers a Global Benefit 17

Mechanism of action Blockade of PD-L1

Targeting CD19 and providing first and co-stimulatory signaling through CD3ζ and CD28

Targeting CD33 to deliver a toxic agent

Targeting CD22 to deliver a toxic agent

Inhibitor of PDGFRα

Targeting CD20

Category Immune checkpoint inhibitor

CAR T-cell therapy

Antibody–drug conjugate

Antibody–drug conjugate

mAb

mAb

Table 1.1 (continued)

Obinutuzumab (Gazyva)

Olaratumab (Lartruvo®)

Inotuzumab ozogamicin (Besponsa®)

Gemtuzumab ozogamicin (Mylotarg™)

Axicabtagene ciloleucel (Yescarta™)

Immunotherapeutic modality Durvalumab (Imfinzi)

• 2016: Combination therapy for soft-tissue sarcoma not curable by surgery or radiation therapy, and usually responding to anthracycline-based chemotherapy [200] • 2016: Combination therapy followed by obinutuzumab monotherapy for r/r FL despite rituximab-containing regimen [201] • 2013: Combination therapy for untreated CLL [202]

• 2017: B-cell ALL, for adults with Philadelphia chromosome that progressed despite targeted therapy [199]

• 2017: CD33-positive AML in patients above the age of 2 with relapse or not responding to first therapy [196–198]

• 2017: DLBCL in patients who had follicular lymphoma, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma [195]

Year and indication(s) of approval • 2018: Unresectable stage III NSCLC that not progressed following concurrent chemotherapy and radiation [193] • 2017: Locally advanced or metastatic bladder cancer that progressed despite chemotherapy [194]

Hepatitis B reactivation, progressive multifocal leukoencephalopathy, infusion reactions, tumor lysis syndrome, infections, neutropenia, thrombocytopenia

Adverse effects Immune-mediated pneumonitis, immunemediated hepatitis, immune-mediated colitis, immune-mediated endocrinopathies, infusion-related reactions, infection Cytokine release syndrome, neurologic toxicities, hypersensitivity reactions, serious infections, prolonged cytopenias, hypogammaglobulinemia Hepatotoxicity, including VOD, infusion-related reactions, hemorrhage Hepatotoxicity, including hepatic VOD, increased risk of posttransplant non-relapse mortality, myelosuppression, infusion-related reactions, QT interval prolongation Infusion-related reactions

18 Z. Aryan et al.

Selectively replicate in Talimogene laherparepvec • 2015: Recurrent melanoma lesions metastasized to the skin (T-VEC, or Imlygic®) tumor cells and and lymph nodes [208] secrete granulocyte macrophage colonystimulating factor (GM-CSF) Targeting GD2 Dinutuximab (Unituxin™) • 2015: Combined with granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid (RA), for high-risk neuroblastoma [209]

Oncolytic virus therapy

mAb

mAb

• 2015: Combination therapy for initial treatment of metastatic squamous NSCLC [207]

• 2015: Combination therapy for MM received 1–3 prior treatments [206]

Elotuzumab (Empliciti®) Targeting signaling lymphocytic activation molecule F7 (SLAMF7) Necitumumab Targeting the (Portrazza™) epidermal growth factor receptor (EGFR)

• 2016: Recurrent or progressive CLL, responded to therapy after ≥2 lines of treatment [203] • 2014: Combination therapy for previously untreated cases with CLL inappropriate for fludarabine-based chemotherapy [204] • 2009: Chronic lymphocytic lymphoma refractory to fludarabine and alemtuzumab [205]

mAb

Ofatumumab (Arzerra)

Targeting CD20

mAb

(continued)

Serious infusion reactions, pain and peripheral neuropathy, capillary leak syndrome, hypotension, infection, neurological disorders of the eye, bone marrow suppression, electrolyte abnormalities, atypical hemolytic uremic syndrome, embryo-fetal toxicity

Infusion reactions, hepatitis B virus reactivation, hepatitis B virus infection, progressive multifocal leukoencephalopathy, tumor lysis syndrome, cytopenias Infusion reaction, infections, second primary malignancies, hepatotoxicity Cardiopulmonary arrest, hypomagnesemia, venous and arterial thromboembolic events, dermatologic toxicities, infusion-related reactions, in non-squamous NSCLC—increased toxicity and increased mortality Herpetic infection, injection-site complications

1  Cancer Immunotherapy Confers a Global Benefit 19

Mechanism of action Targeting Her2

Targeting RANK ligand

Targeting EGFR

Targeting CD20

Targeting Her2

Category mAb

mAb

mAb

mAb

mAb

Table 1.1 (continued)

Trastuzumab (Herceptin)

Rituximab (Rituxan)

Cetuximab (Erbitux)

Denosumab (Xgeva)

Immunotherapeutic modality Pertuzumab injection (Perjeta)

• 2010: Combination therapy for metastatic gastric or GEJ adenocarcinoma overexpressing HER2 [226] • 2006: Early-stage BC after primary therapy [227] • 1998: HER2-positive BC [228]

Year and indication(s) of approval • 2013: Combination therapy as a neoadjuvant for HER2positive, locally advanced, inflammatory, or early-stage BC [210] • 2012: Combination therapy for HER2-expressing metastatic BC not received prior anti-HER2 or chemotherapy [211] • 2013: Bone giant-cell tumor unresectable or with severe morbidity causing resection, in adults and adolescents matured skeletally [212] • 2010: For prevention of skeletal related events (SREs) in solid tumor metastases to bone, except for multiple myeloma [213–215] • 2012: Combination therapy as first line for EGFR-positive Kras-negative mCRC [216] • 2011: Combination therapy as first line for recurrent locoregional and/or metastatic squamous SCCHN [217] • 2006: Combined with radiation for advanced SCCHN and as a single agent for recurrent or metastatic SCCHN that progressed despite chemotherapy [218] • 2004: Combination therapy for EGFR-positive metastatic CRC [219] • 2011: Maintenance therapy for CD20-positive follicular B-cell NHL [220] • 2010: combination therapy for CLL [221] • 2006: First-line treatment for low-grade or follicular, CD20-positive B-cell NHL [222], combination therapy as first-line treatment of CD20-positive DLBCL [223–225]

Infusion reactions, tumor lysis syndrome, mucocutaneous reactions, progressive multifocal leukoencephalopathy, hepatitis B reactivation with fulminant hepatitis, infections, cardiac arrhythmias, renal toxicity, bowel obstruction, and perforation Gastrointestinal side effects, hepatotoxicity, metabolic effects

Skin toxicities most commonly acne

Adverse effects Left ventricular dysfunction, infusionrelated reactions, hypersensitivity reactions/ anaphylaxis URI, UTI, cataracts, rash, gastrointestinal manifestations

20 Z. Aryan et al.

Boosting antitumor immunity

TLR2 and TLR4 agonists

Infections, pancytopenia Vascular leak syndrome

Neutropenia, similar to rituximab

Depression, pancytopenia, bleeding, fatigue, flulike symptoms BCG-osis in case of immunosuppression

• 2001, 2007: CLL • 1998: Advanced-stage melanoma [234] • 1992: Metastatic RCC [235] • 1997: r/r CD20-positive, B-cell, low-grade, or follicular NHL [236] • 1996: Advanced melanoma [237] • 1986: HCL [238]

BCG

IFN-α2B

Rituximab (Rituxan, Mabthera) or 131 I-rituximab

Alemtuzumab (Campath) [233] Recombinant IL-2 (aldesleukin)

• 2002: r/r Low-grade, follicular, or transformed B-cell NHL [231, 232]

Ibritumomab tiuxetan (Zevalin), Y-90, In-111 ibritumomab

• 1990: Superficial bladder cancer [239]

FDA Food and Drug Administration, CAR chimeric antigen receptor, DLBCL diffuse large B-cell lymphoma, ALL acute lymphoblastic leukemia, NSCLC non-small cell lung cancer, EGFR epidermal growth factor receptor, ALK anaplastic lymphoma kinase, SLAMF7 signaling lymphocytic activation molecule F7, GD2 disialoganglioside GD2, GM-CSF granulocyte-macrophage colony-stimulating factor, IL-2 interleukin-2, MSI-H high microsatellite instability, dMMR DNA mismatch repair deficiency, SCLC small-cell lung cancer, SCC squamous cell carcinoma, SCCHN squamous cell cancer of the head and neck, HSCT hematopoietic stem cell transplantation, PD-L1 programmed death ligand 1, CTLA-4 cytotoxic T-lymphocyte-associated antigen 4, VEGFR2 vascular endothelial growth factor receptor-2, CLL chronic lymphocytic leukemia, Her2 human epidermal growth factor receptor 2, ASCT autologous stem cell transplant, RANK receptor activator of nuclear factor kappa-Β, TLR toll-like receptor, VEGF-A vascular endothelial growth factor-A, mAb monoclonal antibody, AML acute myeloid leukemia, CHOP cyclophosphamide, doxorubicin, vincristine, and prednisone, PDGF platelet-derived growth factor, IFN interferon, BCG bacillus Calmette–Guérin, CPS combined positive score, PMBCL primary mediastinal large B-cell lymphoma, mCRC metastatic colorectal cancer, MCC Merkel cell carcinoma, HCC hepatocellular carcinoma, NSqNSCLC non-squamous non-small cell lung cancer, r/r refractory or relapsed, cHL classical Hodgkin lymphoma, MM multiple myeloma, AFP alpha fetoprotein, GEJ gastroesophageal junction, ES-SCLC extensive-stage small-cell lung cancer, BC breast cancer, BPDCN blastic plasmacytoid dendritic cell neoplasm, ALCL systemic anaplastic large-cell lymphoma, HL Hodgkin lymphoma, HCL hairy-cell leukemia, NHL non-Hodgkin lymphoma, FL follicular lymphoma, ALK anaplastic lymphoma kinase, VOD veno-occlusive liver disease

Cytokine

mAb

Boosting antitumor immunity, growth factor for T cells Targeting CD20, delivering radiotherapeutic agents Boosting antitumor immunity

Cytokine

mAb

Radiolabeled mAb

• 2004: Basal cell carcinoma, actinic keratosis [230]

Boosting antitumor immunity Targeting CD20 to deliver radiotherapeutic agents Targeting CD52

TLR7 agonist

• 2006: mCRC [229]

Imiquimod (Aldara)

Targeting epidermal Panitumumab (vectibix) growth factor receptor

mAb

• 2010: Hormone-refractory prostate cancer [37] Cardiovascular events, chills, fever, fatigue, nausea, and headache Skin toxicities most commonly dry skin, rash, acne Hypopigmentation, redness, scarring Hypersensitivity reactions, pancytopenia

Sipuleucel-T (Provenge)

Increasing tumor antigen presentation

DC vaccine

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1.5

Strategies of Cancer Immunotherapy

Two main strategies of cancer immunotherapy to treat cancer patients are (1) reduction of immunosuppressive milieu and (2) boosting of antitumor responses.

1.5.1 Immunotherapy Acts to Eliminate Immunosuppression Immunotherapy reduces immunosuppression by blocking the negative regulatory receptors and inhibitory checkpoints [CTLA-4 and programmed death-1 (PD-1)], blocking immunosuppressive cytokines [transforming growth factor-β (TGF-β), IL-10, and TNF], blocking immunosuppressive enzymes [indoleamine 2,3-­dioxygenase (IDO)], and targeting the T regulatory cells. Blockade or inhibition of negative regulators of the immune system enhances endogenous antitumor responses as well as antitumor activity of the immune system following other therapeutic approaches. CTLA-4 (CD152) is expressed on T cells in combination with a wide variety of other immune cells [240]. It acts as a negative immune regulatory receptor that switches off T-cell attacks to tumor cells. Indeed, CTLA-4 is one of the main players in establishing peripheral tolerance [240]. It competes with CD28 to bind to B7-1/B7-2 with higher affinity and avidity [240]. Two mAbs have been developed to block CTLA-4: ipilimumab (MDX-010) and tremelimumab (CP-675,206). Both of these antibodies are under investigation for a wide range of cancers. Tremelimumab has been tested in the treatment regimen of patients with metastatic or refractory melanoma, colorectal cancer, and prostate cancer. Unfortunately, favorable response in terms of tumor regression and improvement of survival was only detected in less than 10% of patients [97, 241–243]. Results of trials with ipilimumab were more positive, and ipilimumab has received FDA approval for patients with metastatic melanoma [41, 98]. In addition, ipilimumab combined with

nivolumab has been approved for the treatment of patients above the age of 12 years old who had metastatic colorectal cancer with MSI-H or dMMR who showed progression despite prior treatments [76, 244–246]. PD-1 is an inhibitory receptor present on activated T cells and B cells and has two main ligands PD-L1 and PD-L2. PD-L1 has a broad expression on not only immune cells but also nonimmune and tumor cells, whereas PD-L2 expression is restricted to antigen-presenting cells (APCs). PD-L1 is one of the most important actors in the maintenance of immunosuppressive microenvironment around tumor cells. PD-1 blocking antibody, CT-011, is under investigation for patients with advanced hematological malignancies and multiple myeloma [247, 248]. Anti-inflammatory cytokines IL-10 and TGF-β are produced by tumor cells and suppress antitumor responses. Anti-TGF-β antibodies are now in development for cancer immunotherapy. Initial experiences on animal models of osteosarcoma reduced T regulatory cell numbers and increased cytotoxic T lymphocyte numbers, thereby preventing the growth of new metastases [249]. Fresolimumab, a fully human anti-TGF-β, is now produced and may be tested in the treatment of cancer patients [250]. Anti-IL-10 antibodies and anti-IL-10 receptor antibodies have potential antitumor activity, but they have not currently entered into clinical trials for cancer patients [251, 252]. In addition, infliximab, an anti-TNF-α antibody, was tested in patients with RCC and resulted in 16% partial response and 16% stable disease among recipients [253]. TNF-α is an inflammatory cytokine, and its increased levels have been associated with poor prognosis in cancer patients [253]. IDO catalyzes degradation of essential l-­ tryptophan amino acid and keeps it away from activated T-cells needing it for clonal expansion. Tumor cells as well as plasmacytoid DCs present in tumor-draining lymph nodes express high amounts of IDO leading to indirect suppression of antitumor responses. 1-Methyl tryptophan (1MT) inhibits IDO and prevents tumor cell growth of variable origins, but it is still under laboratory investigations [254–256].

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Fig. 1.6 Immunotherapy boosts antitumor immune responses and kills tumor cells. Dendritic cell (DC) vaccines and adoptive T-cell transfer are two main ways of enhancing antitumor responses. Tumor antigens, immune adjuvants, and stimulatory cytokines help improve DC functions and provide cytotoxic T lymphocyte. Tumor cells themselves are the best source of antigens to produce cancer-reactive T cells. Monoclonal antibodies act as immunostimulatory agents to direct stimulation of T cells or bind tumor cells and activate complement system and

antibody-dependent cell-mediated cytotoxicity (ADCC). TLR Toll-like receptor, MPL monophosphoryl lipid A, MHC II major histocompatibility complex II, TNF tumor necrosis factor, IFN interferon, IL interleukin, GM-CSF granulocyte-macrophage colony-stimulating factor, VEGF vascular endothelial growth factor, EGFR epidermal growth factor receptor, HER2/NEU human epidermal growth factor receptor 2, GITR glucocorticoid-induced tumor necrosis factor receptor

T regulatory cells are a strong source of inhibitory signals, preventing the effect of endogenous antitumor responses and inhibiting sufficient response to immunotherapeutic agents boosting immune system. Denileukin diftitox, a conjugate of diphtheria toxin and IL-2, has efficacy in the treatment of patients with T-cell lymphoma, B-cell NHL, and melanoma. Further studies demonstrated its efficacy in enhancing cancer vaccine responses by depletion of T regulatory cells. Indeed, denileukin diftitox is a targeted therapy to kill T regulatory cells [84, 257–259]. Other potential targets to weaken T regulatory responses include IL-35 and MFGE8 that block T regulatory functions [260, 261].

Figure  1.5 summarizes the immunotherapies aimed at inhibiting immunosuppression to treat cancers.

1.5.2 Immunotherapy Boosts the Antitumor Immune Responses and Enhances Killing of the Tumor Cell 1.5.2.1 Activated DCs and T Cells Are Pivotal in Cancer Immunotherapy Immunotherapy boosts the immune responses against cancers by providing primed T cells

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either in vivo or ex vivo. Therapeutic cancer vaccines provide the opportunity to prime the T cells in vivo, while adoptive T-cell transfer gifts ex vivo primed T cells to the immune system of cancer patients. DC vaccines constitute the most popular therapeutic cancer vaccines developed to treat a wide variety of cancers. First, DCs should be cultured from patients’ peripheral blood mononuclear cells (PBMCs). Then they should be matured (most commonly with inflammatory cytokine cocktails) and loaded with tumor antigens. Finally, they are reintroduced to the patient’s body to activate T cells and enhance antitumor responses. Some researchers prefer in vivo maturation of DCs by injection of these cells into an inflamed tissue as a simple, inexpensive, and physiologic way of maturation that enhances migration of DCs to draining lymph nodes [262]. Furthermore, antigens can also be loaded in vivo using antibodies that bind DC surface like DEC205 [263, 264]. DC vaccines are under investigation (phase I/II clinical trial) for high-­ grade glioma [265, 266], glioblastoma [267], hepatocellular carcinoma [268], pancreatic cancer [269], colorectal cancer [270], metastatic melanoma [271, 272], multiple myeloma [273– 275], acute leukemia [276, 277], breast cancer [278], ovarian cancer [279, 280], RCC [281], and non-small cell lung cancer [282]. On the other hand, adoptive T-cell transfer relies on in vitro expansion of T cells harvested from cancer patients and reintroduction of these manipulated and primed T cells into the patient’s circulation. These T cells can be harvested from four major sites: (1) PBMC, (2) resections from draining lymph nodes, (3) malignant effusions, and (4) directly from tumor biopsies. However, the quantity and quality of harvested T cells from each site differ significantly; PBMCs are an easy site to obtain T cells, while biopsy-derived T cells are more reactive against tumor antigens [76, 283]. Thereafter, T cells will be engineered to express T-cell receptors (TCR) necessary for tumor recognition or to express T bodies (a chimeric antigen receptor that directly binds tumor antigens) [284]. Finally, T cells can be expanded with exposure to relevant tumor antigens, activating mAbs and T-cell growth factors like IL-15 [285].

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CD8+ cytotoxic T lymphocytes constitute the main cells produced and transferred in adoptive T-cell therapy [286]. Adoptive T-cell therapy is now underway for neuroblastoma [287], hepatocellular cancers [288], gastric cancer [289], metastatic melanoma [290–292], hematological malignancies [293–295], colorectal cancer [296, 297], posttransplant lymphoproliferative diseases [298, 299], breast cancer [300], ovarian cancer [162, 163], advanced lung cancer [164, 165], RCC [166], and nasopharyngeal carcinoma [167]. Recently, an HLA-independent targeting of cancer cells, which is directed by nonconventional T cells, has been reported. It has been observed that some ligands by cancer cells present on the monomorphic MHC class 1-related protein (MR1) and are recognized by nonconventional T cells. MR1, with limited polymorphism, is an ideal candidate for designing immunotherapeutics for almost all the patients [301]. Moreover, the TCR responsible for this recognition could be adopted by T cells from patients with various types of cancer, which leads to enhanced cytotoxicity against the established tumor. This method showed promising results in the destruction of autologous and allogeneic melanoma [301].

1.5.2.2 Materials of Activating DCs and T Cells Tumor-specific and tumor-associated antigens as well as immunostimulatory cytokines and immune adjuvants help in activating DCs and priming the T or natural killer (NK) cells in vivo or ex vivo. Cancer-testis antigens are mainly expressed in germ cells and also appear on tumors of variable origin. However, these antigens are rarely expressed by other human cell types under physiologic conditions. Of all cancer-testis antigens, MAGE-1 family has obtained growing interest as the potential target for different cancers at variable stages [302, 303]. MAGE-A3 and NY-ESO-1, two cancer-testis antigens, have been used to develop cancer vaccines against melanoma [304, 305], lung cancer [306], ovarian carcinoma [307], and prostate cancer [308]. In addition to tumor-specific antigens, tumor-­associated antigens with narrow dis-

1  Cancer Immunotherapy Confers a Global Benefit

tribution in tumor cells are widely used in cancer immunotherapy like tumor lysate antigens in DC-loaded vaccines [309]. Interestingly, antigens of oncogenic viruses also exert immunostimulatory effects able to induce strong antitumor responses [310]. Tumor cells of EBV-associated nasopharyngeal carcinoma express EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2) which are EBV antigens [310]. Intradermal administration of MVA-EL vaccinations, which encode an EBNA1/LMP2 fusion protein, results in boosting T-cell response against tumors [310]. Indeed, EBV-targeted immunotherapy promises hopes for patients with refractory or metastatic EBV-associated cancers [311]. Antigens can be delivered to DCs via different ways including fusion with tumor cells, loading of tumor lysates, long overlapping peptide mixtures or specific antigenic peptides, exposure with recombinant proteins, and transfection with genes encoding tumor antigens [269, 278, 312, 313]. Recombinant antigens offer specific targeting of tumor cells with high safety and efficacy. Sipuleucel-T is a DC-approved vaccine for prostate cancer which contains ex vivo-primed DCs with recombinant PA2024 protein fused with GM-CSF [37]. Interestingly, specific tumor antigens can be selectively delivered to patients to enhance in vivo DC uptake and presentation of tumor antigens. This direct delivery of tumor antigens is known as peptide vaccination. Subcutaneous injection of modified 9-mer WT1 peptides to patients with Wilms’ tumor results in increased frequency of CD14+, CD16+, CD33+, and CD85+ DCs [314]. However, for improving the efficacy of peptide vaccines, addition of cytokines particularly GM-CSF is of significant benefit. GM-CSF acts mainly via enhancement of antigen presentation by promotion of recruitment and maturation of DCs. Autologous or allogeneic irradiated tumor cells engineered to produce GM-CSF (GVAX) have been tested in some cancers like metastatic melanoma, colorectal cancers, non-small cell lung cancer, pancreatic cancer, and castration-resistant prostate cancer with promising results [315–322]. This approach is expensive and technically difficult; thus, it did

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not gain popularity among researchers and clinicians. In addition to therapeutic cancer vaccines, cytokine monotherapy is used for the promotion of tumor death and boosting T-cell responses. Isolated limb perfusion with TNF-α is approved for the treatment of patients with locally advanced soft-tissue tumors [99, 100]. TNF-α is a proinflammatory cytokine involved in systemic inflammation, acute-phase reaction, and constitutional symptoms of cancer patients like cachexia, whereas it also inhibits tumor growth and promotes apoptotic cell death. To overcome systemic unpleasant effects of TNF-α together with selective use of its antitumor effects, it is approved for local delivery in tumor repertoire [99, 100]. Other immunostimulatory cytokines acting as T-cell growth factors, increasing survival of T cells, and enhancing T-cell responses to tumor antigens are used in cancer immunotherapy. IL-2 and IFN-α have received FDA approval for patients with unresectable metastatic melanoma and renal cell carcinoma (RCC) [94, 292, 323–325]. With the discovery of pattern recognition receptors, novel immune adjuvants gained considerable popularity among researchers [326]. Toll-like receptor (TLR) agonists have received FDA approval for use as an immune adjuvant for cancer immunotherapy. Monophosphoryl lipid A (MPL), a TLR4 agonist, has been used in Cervarix®. Cervarix® is a vaccine against HPV16 and HPV-18 and prevents HPV-related cancers [95]. Imiquimod, a TLR7 agonist, received FDA approval for basal cell carcinoma, external genital warts, and actinic keratosis [95]. In addition, the development of new immunotherapeutic agents using other TLR agonists like CpG oligonucleotides continues to be an area of active research [326]. PF-3512676, a TLR9 agonist, is now in phase II clinical trial for patients with metastatic melanoma. Intravenous or intradermal administration of PF-3512676  in melanoma patients results in activation of DCs in sentinel lymph nodes and expansion of cancerreactive cytotoxic CD8+ T cells [327, 328]. These immunological changes were in association with partial clinical response in 10% and stable disease in 15% of treated melanoma

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Fig. 1.5  Cancer immunotherapy eliminates immunosuppressive milieu of cancer patients. Cytotoxic T lymphocyte-­ associated antigen 4 (CTLA-4) and programmed death-1 (PD-1) are expressed by activated T cells and act as T-cell checkpoint blockers that inhibit T-cell functions. These are blocked by anti-CTLA-4 and anti-PD-1 monoclonal antibodies. Interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) are inhibitory cytokines produced by T regulatory cells and tumor cells.

These inhibitory cytokines are blocked by specific monoclonal antibodies that are under investigations in laboratory. Denileukin diftitox, a conjugate of IL-2 and diphtheria toxin, kills T regulatory cells and enhances endogenous or induced antitumor responses. l-Methyl tryptophan inhibits indoleamine 2,3-dioxygenase (IDO). IDO inhibits T-cell expansion by degradation of essential amino acid of tryptophan

patients with PF-3512676 monotherapy [329]. Similarly, PF-3512676 is in phase I clinical trial for patients with basal cell carcinoma and NHL [330, 331]. PF-3512676 was also evaluated for cutaneous T-cell lymphoma, chronic lymphocytic leukemia, metastatic esophageal squamous cell carcinoma, and non-small cell lung cancer [332–337]. Finally, immunomodulator mAbs can act as agonists of stimulatory receptors on immune cells. Stimulatory mAbs have been developed for glucocorticoid-induced tumor necrosis factor receptor (GITR), OX40 (CD134), CD40, and CD137. These mAbs are now underway for a wide range of cancers from hematological malignancies to solid tumors. They have recently entered into clinical trials and showed promising results [338–341]. Of note, mAbs act as immunomodulators, trigger complement activation, induce antibody-dependent cell-mediated cytotoxicity (ADCC), and also are able to provide opportunity for targeted delivery of cytotoxic materials to malignant cells (for instance

131 I-tositumomab, known as radioimmunotherapy) [342]. Figure 1.6 summarizes immunotherapies aimed at stimulating antitumor immune responses.

1.6

At Which Line of Treatment?

Since the introduction of cancer immunotherapy, it has been used mainly as the last line of treatment of patients with advanced disease. It does not mean that cancer immunotherapy is restricted to patients who relapsed with other standards of care. As this emerging field is still in its infancy and its safety and efficacy are not well evaluated, patients who accept to enter into trials are usually at advanced stages, have several metastases, and are inoperable. Indeed, immunotherapy provides hope for who is disappointed from other therapeutic approaches. Nonetheless, the question of why it cannot be considered as a first-line therapy of cancer patients at different stages remains to be answered.

1  Cancer Immunotherapy Confers a Global Benefit

For different cancers, it has been shown that considering immunotherapy as the first-line treatment did not compromise patients’ prognosis and their quality of life, but inversely improved progression-­free survival and sometimes overall survival of patients. For metastatic colorectal cancer, addition of panitumumab to standard chemotherapy as first-line therapy resulted in significant improvement in progression-free survival of patients without deterioration of patients’ quality of life [343]. Panitumumab is approved for patients with metastatic refractory colorectal cancers, but it has several benefits (at least improvement of progression-free survival) for patients who have not received previous chemotherapy [91]. Similarly, use of ipilimumab in combination with paclitaxel and carboplatin improved progression-free survival of patients with non-­ small cell lung cancer who had not previously taken any medication [244]. On the other hand, immunotherapy is associated with better results in patients with early stages of cancers. BCG for superficial bladder cancer is a historical example of this claim. In addition, vaccination with HPV-­ 16 oncoproteins in women with high-grade VIN resulted in relief of VIN-related symptoms in 60% of patients [32]. Accordingly, immunotherapy is of benefit as the first-line treatment of patients with advanced or early stages of cancer. However, this hypothesis should be assessed in future studies.

1.7

 onotherapy or Combined M Therapy?

For many years there was a dogma that chemotherapy and radiotherapy have deleterious effects on immunity and thereby effects of combined immunotherapy may be subsided. Initial experiences opposed to this dogma dating back to animal studies in the 1970s when intratumoral injection of cytotoxic drugs enhanced systemic immune response against tumors, cleared distant metastases, and promoted protective immunity with rechallenge with tumor cells [344]. In addition, systemic delivery of chemotherapy enhanced antitumor responses without induction of T regulatory

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cell depletion [345]. These observations suggested that cytotoxic chemotherapy may not be always immunosuppressive. Further studies unveiled that immunostimulatory or immunosuppressive effects of chemotherapeutic drugs depend on drug/dosage and schedule of treatment [38]. In addition, radiotherapy breaks immunosuppressive tumor microenvironment and enhances tumor antigen presentation. Accordingly radiotherapy with nonfatal doses for the immune system may enhance the efficacy of cancer treatment to be combined with immunotherapy [346]. Under schedule that does not suppress effector cytotoxic T lymphocyte, induction of apoptotic death of tumor cells by chemotherapy/radiotherapy results in enhanced tumor antigen presentation and subsequent T-cell activation. This is known as immunogenic cell death and constructs the basis of combined immunotherapy with chemotherapy/radiotherapy [347]. In addition, this combined therapy reduces the chance of tumor escape and resistance similar to multidrug therapy. Combined therapies are now on the way for wide varieties of cancers. Immunotherapy can be combined with radiotherapy, chemotherapy, targeted therapy (like tyrosine kinase inhibitors), and surgery [348–352]. Interestingly, radioimmunotherapy is an emerging field with introduction of mAbs bearing radioactive agents. Yttrium-90-ibritumomab tiuxetan is a mAb against CD20 conjugated to yttrium-90 and is used to treat relapsed B-cell malignancies [353]. 131 I-rituximab and 131I-tositumomab are other radiolabeled mAbs against CD20 [352, 354]. One of the most famous combined chemotherapies/immunotherapies is used in the treatment of hematological malignancies. Combined CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy with rituximab as first-line treatment in non-Hodgkin lymphoma [355], CHOP plus 131I-tositumomab in non-­ Hodgkin lymphoma [352], and CHOP plus rituximab in untreated mantle cell lymphoma [353, 356] are examples of combined chemotherapy with immunotherapy in hematological malignancies. Other promising results have been obtained in pancreatic cancer, one of the most lethal cancers worldwide. Algenpantucel-L immunotherapy combined with gemcitabine and 5-fluorouracil-based

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chemoradiotherapy improves progression-free survival of patients with resected pancreatic cancer [357]. Furthermore, yttrium-90-labeled humanized clivatuzumab tetraxetan with gemcitabine resulted in partial response in 16% and stable disease in 42% of patients with advanced pancreatic cancer [358]. However, combined immunotherapy with chemotherapy does not always improve clinical outcome. In esophageal cancer patients, intratumoral administration of 111 In-labeled dendritic cells (DC) in combination with preoperative c­ hemotherapy did not improve immune nor clinical response [359]. Targeted therapy with tyrosine kinase inhibitors particularly those inhibiting vascular endothelial growth factor receptors (VEGFR) promises hope for the treatment of several cancers. Both small molecules inhibiting this receptor like axitinib [360] (received FDA approval for refractory RCC in 2012) and mAbs targeting VEGFR like bevacizumab (received FDA approval for metastatic colorectal cancer, RCC, and glioblastoma multiform in 2004) are now available. Despite axitinib which is a chemotherapeutic, bevacizumab belongs to immunotherapeutic agents due to activation of complement system and ADCC when binding VEGFR [361, 362]. Interestingly, combination of targeted therapy with immunotherapy has also been evaluated in patients with RCC. Combination of SU5416, VEGFR inhibitor, and IFN-α2B (received FDA approval for hairy-cell leukemia and advanced-­ stage melanoma in the 1990s) was tested in patients with RCC with no beneficial effects. By contrast, this combination leads to fatal events in 6.5% of treated patients and thereby is discouraged [363]. Finally, immunotherapy confers benefits to patients who have undergone surgery for complete resection or debulking of cancer. IFN-α2B after resection of melanoma in patients with high risk of relapse improves survival and decreases risk of relapse but jeopardizes quality of life of patients owing to IFN toxicities [364]. In addition to postsurgical benefits, immunotherapy can be employed prior to surgery and resection of tumor masses. Neoadjuvant chemotherapy like induction of resistance cells, difficulty in resec-

tion, and false shrinkage of tumor on imaging results in fast growth of residual tumor cells after resection; thus, neoadjuvant immunotherapy is more advantageous compared to neoadjuvant or induction chemotherapy [365]. Neoadjuvant immunotherapy has been tested for several cancers; carcinoembryonic antigen (CEA)-derived MHCII-loaded DC vaccination prior to resection of colorectal metastases was promising [270]. In addition, adoptive T-cell transfer prior or after surgery/radiotherapy of glioblastoma multiforme patients is an emerging solution for this lethal cancer [366, 367].

1.8

Monitoring the Immunological and Clinical Responses to Immunotherapy

It should be borne in mind that immunotherapy-­ induced tumor destruction may appear with delay after a period of tumor progression and metastasis. By contrast, response to other standards of care of cancer patients including surgery, radiotherapy, and chemotherapy appears early with obvious reduction of tumor size and metastasis [368]. It highlights the significance of the development of immune-related response criteria nearby classic World Health Organization (WHO) criteria and Response Evaluation Criteria in Solid Tumors (RECIST). WHO criteria and RECIST measure response to cancer therapy mostly with respect to tumor shrinkage and appearance of new metastases [369–372]. However, immune-related response criteria should be applied to measure response to cancer immunotherapy considering the distinct biology of tumor cell killing using each cancer therapy approach [371]. One of the evidences of this distinction is the immune infiltrate of tumor mass caused by immunotherapy that is responsible for delayed but effective antitumor responses [368]. Major determinants of safety and efficacy of cancer immunotherapy encompass specificity of therapy or quality of primed T cells, quantity of primed T cells against cancer, and half-life of induced response against cancer [33]. This also

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underscores the significance of a reliable unique assay to measure immunological response to immunotherapy. Such universal standard assays let us compare immune responses in different trials. The minimal information about T-cell assays (MIATA) project aims at establishing universal criteria to assess immunological response to cancer immunotherapy [373, 374]. Of note is that the best site of assessment for immune response is tumor microenvironment rather than peripheral blood or distant sites from tumor origin due to immunosuppressive effect of tumor microenvironment [373, 374].

tions from pancytopenia to isolated neutropenia; and respiratory or urinary tract infections [39, 96, 376–378]. On the other hand, some drugs are associated with specific toxicities: peripheral sensory neuropathy with brentuximab vedotin [379], skin toxicities with panitumumab and cetuximab [380], and hypertension and hemorrhage with bevacizumab [381]. These side effects restrict the use of immunotherapy as FDA revoked bevacizumab approval for breast cancer due to its fatal side effects.

1.9

Despite inevitable side effects of immunotherapy to treat cancers, immunotherapy also offers hope for rehabilitation and reconstruction of destroyed nonmalignant tissues during cancer treatment. This use of immunotherapy is the tertiary level of prevention from cancers which completes the treatise of cancer immunotherapy. The most famous one is GM-CSF following myelosuppressive chemotherapy. Not only GM-CSF but also granulocyte-CSF (G-CSF) increase the maturation and release of myeloid linage including DCs and neutrophils. These functions of G(M)-CSF on DCs are used to construct more efficient therapeutic cancer vaccines (known as GVAX); however, increase of neutrophil count is pivotal in the supportive therapy of cancer patients. Neutropenia predisposes cancer patients to a wide range of bacterial infections and increases mortality of patients. G(M)-CSF efficiently reduces the risk of infection-related morbidity and mortality [382–384]. GM-CSF has FDA approval for recovery following HSCT of several hematological malignancies and is part of the guidelines of supportive therapy of many other countries other than the United States [385]. However, it has supportive implication in several malignancies like breast cancer [384]. In breast cancer patients that received chemotherapy, use of GM-CSF reduces asthenia, anorexia, stomatitis, myalgia, dysgeusia, and nail disorders [386]. Vitamins can also act as supportive immunotherapeutic agents. High-dose methotrexate (MTX) which inhibits dihydrofolate reductase

Limitations of Cancer Immunotherapy

Several obstacles limit the implication of cancer immunotherapy including technical obstacles, and side effects of immunotherapeutic drugs. Harvesting sufficient amounts of T cells or DCs from cancer patients and activating them are not always easy and inversely are associated with technical difficulties, high costs, and different interindividual efficacies. Similarly, provision of autologous whole-tumor cell vaccines expressing GM-CSF is also difficult and expensive [76, 368]. In addition, novel adjuvants are needed to optimize therapy with cancer vaccines. Despite considerable investment on cancer vaccine development, less is paid on the development of vaccine adjuvant components [375]. On the other hand, bypass of tumor tolerance is inevitably associated with the break of peripheral tolerance to self-antigens. Accordingly, autoimmune manifestations are the most common adverse events of cancer immunotherapy. In addition some drugs such as IFN induce fatal toxicities leading to drug discontinuation. As immunotherapy is a systemic treatment, adverse events may appear all over the body. Various side effects are observed with the administration of various immunotherapeutic drugs including gastrointestinal involvement with colitis, nausea, vomiting, and hepatotoxicity; skin involvement with rash and pruritus; endocrine involvement like adrenalitis and hypophysitis; hematological manifesta-

1.10 Supportive Therapy

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(DHFR) is used in the chemotherapy of a wide range of lymphoproliferative diseases as well as breast cancer [387]. DHFR is a pivotal enzyme in folic acid metabolism and is required for thymidine synthesis and cell replication [387]. Accordingly, folic acid derivatives can be used to rescue bone marrow as well as gut epithelial cells. The beneficial effects of folic acid supplementation are more chargeable to nonmalignant cells justifying the use of folic acid even concomitant with chemotherapy [387].

1.11 Effect of Immunotherapy on Health-Related Quality of Life of Cancer Patients Symptomatic treatment is one essential component of care of cancer patients. Cancer patients have constitutional symptoms like decreased appetite and fever owing to systemic inflammation. Psychiatric complaints like depression and insomnia also are common among the patients. In addition, chemotherapy and radiotherapy as well as surgical resection of tumor masses exacerbate general condition of patients at least for a short period of time surrounding the therapy. In this manner mastectomy is one of the overwhelming events in the care of breast cancer patients with undeniable psychiatric effects like depression. Interestingly, depression reduces the overall survival of breast cancer patients controlling for other variables [388]. Similarly, immunotherapy is not free of side effects and also may cause toxicities for several organs. However, it is shown that certain immunotherapeutic drugs improve health-related quality of life of patients. Cytokine-­induced killer (CIK) cell transfer for patients with several cancers including hepatocellular carcinoma and gastric cancer homological malignancies improves patients’ quality of life. CIK improves appetite and sleep, in addition to relieving pain [389]. Sometimes immunotherapy has equivocal effects on quality of life. Addition of cetuximab in chemotherapeutic regimen of patients with metastatic colorectal cancer does not impact patients’ quality of life but improves overall survival [390]. Similarly, use of

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ipilimumab for advanced melanoma or panitumumab for metastatic colorectal cancer does not impact the quality of life of treated patients [343, 391]. On the other hand, immunotherapy may negatively impact the quality of life of treated patients. IL-2 or IFN-α2B therapy for RCC and melanoma patients may cause depressive but not anxiety symptoms in the first week of treatment [324].

1.12 Cost-Effectiveness of Cancer Immunotherapy Not all cancer immunotherapeutic drugs are expensive or technically difficult to be developed. Vaccination against oncogenic viruses like HPV offers a cost-effective solution to prevent cancers more prominently in limited-resource settings [392]. Several studies from African low-income countries confirmed the cost-effectiveness of girl vaccination against HPV [392]. Prevention from cancer development is of utmost importance in countries without organized programs of cervical cancer screening [392]. In addition to low-income countries, developed countries which provide access to both screening and therapeutic programs for cervical cancer benefit from HPV vaccination [61]. Cost-effectiveness of cancer prophylaxis in such a setting underscores the importance of primary prevention. In this way, both the health system and people will benefit as people experience better status of health while spending less. On the other hand, immunotherapy aiming at cancer treatment is usually expensive considering either therapeutic cancer vaccines or adoptive T-cell therapy. However, it should be borne in mind that other therapies for cancers are not inexpensive and predispose patients to life-­threatening side effects. Hence, cost-effectiveness of other therapies for cancer patients is also under question, but these are all that can be done to save patients. Immunotherapy provides hopes for patients disappointed from other drugs or have metastatic advanced stages of disease. In addition, immunotherapy is now available for pancreatic cancer, esophageal cancer, liver cancer, and

1  Cancer Immunotherapy Confers a Global Benefit

lung cancer which have a 5-year mortality of more than 90% [17, 57, 90, 244, 268, 320, 321, 336, 337, 359, 393, 394]. It suggests that immunotherapy for treatment of cancer patients is unlikely to be cost effective but is the only hope of several patients to live 1 day more. Considering supportive immunotherapy, administration of GM-CSF is shown to be cost effective in the treatment of neutropenia and prevention of cancer/chemotherapy-related sequelae on bone marrow. Increase of neutrophil count is associated with reduction in infection-related morbidity and mortality of cancer patients concurrent with improvement in their health-related quality of life [382, 395]. Indeed, cancer immunotherapy has approved economic benefit at primary and tertiary levels of care, and at second level, it provides hope for patients who have recurrent/relapsed end-stage disease.

1.13 Concluding Remarks Immunotherapy can be active or passive, rapid in onset of effects, or associated with delayed response, specific or nonspecific. Active immunization against cancers includes different vaccines, cell-based therapies, peptide-based therapies, cytokines, and gene therapies, while the passive immunization against cancers is also provided by developing variable mAbs. These antibodies exert their effects via complement activation and ADCC that are rapid in onset. Of note, monoclonal antibodies also exert immunomodulator effects by targeting the immune system rather than tumor-associated antigens. Innate immune actors are more important in the induction of passive immunization against cancers, while adaptive immunity plays the central role in active immunization. In recent years, a large number of immunotherapeutic drugs obtained FDA approval, and novel combinations for cancer prevention and treatments are underway. Moreover, investigation of novel unconventional T cells targeting cancer cells through an HLA-­ independent mechanism lightens the way toward the design of “one-size-fits-all” immunotherapeutics for many cancer types.

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Today, cancer is an important global issue with high incidence, mortality, and considerable burden on the health system. Considering failure of chemotherapy, radiotherapy, or surgery in treatment of many cancer patients, a new therapeutic avenue is indicated. Immunotherapy could be the solution providing protection against cancer at all levels of care. Prophylactic use of immunotherapy with immunomodulation to treat diseases predisposes individuals to cancer, and vaccination against oncogenic viruses is beneficial for both health-care providers and people. Therapeutic use of immunotherapy not only might offer hope for those who are disappointed from other therapies, but also can come into first line of treatment and be applied in the earlier phases of cancer development. Moreover, supportive immunotherapy helps rescue patients following intensive therapies. Accordingly, cancer immunotherapy confers a global benefit, and everybody all around the world has the right to benefit from this novel therapeutic avenue.

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Z. Aryan et al. Oncology Group Study E1499. J Clin Oncol. 2012;30(25):3119–26. 354. Kruger PC, Cooney JP, Turner JH. Iodine-131 rituximab radioimmunotherapy with BEAM conditioning and autologous stem cell transplant salvage therapy for relapsed/refractory aggressive non-­ Hodgkin lymphoma. Cancer Biother Radiopharm. 2012;27(9):552–60. 355. Cunningham D, Hawkes EA, Jack A, Qian W, Smith P, Mouncey P, et al. Rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone in patients with newly diagnosed diffuse large B-cell non-Hodgkin lymphoma: a phase 3 comparison of dose intensification with 14-day versus 21-day cycles. Lancet. 2013;381(9880):1817–26. 356. Delarue R, Haioun C, Ribrag V, Brice P, Delmer A, Tilly H, et al. CHOP and DHAP plus rituximab followed by autologous stem cell transplantation in mantle cell lymphoma: a phase 2 study from the Groupe d’Etude des Lymphomes de l’Adulte. Blood. 2013;121(1):48–53. 357. Hardacre JM, Mulcahy M, Small W, Talamonti M, Obel J, Krishnamurthi S, et al. Addition of algenpantucel-­L immunotherapy to standard adjuvant therapy for pancreatic cancer: a phase 2 study. J Gastrointest Surg. 2013;17(1):94–100; discussion 100-1. 358. Ocean AJ, Pennington KL, Guarino MJ, Sheikh A, Bekaii-Saab T, Serafini AN, et  al. Fractionated radioimmunotherapy with (90) Y-clivatuzumab tetraxetan and low-dose gemcitabine is active in advanced pancreatic cancer: a phase 1 trial. Cancer. 2012;118(22):5497–506. 359. Fujiwara S, Wada H, Miyata H, Kawada J, Kawabata R, Nishikawa H, et  al. Clinical trial of the intratumoral administration of labeled DC combined with systemic chemotherapy for esophageal cancer. J Immunother. 2012;35(6):513–21. 360. Motzer RJ, Escudier B, Tomczak P, Hutson TE, Michaelson MD, Negrier S, et  al. Axitinib versus sorafenib as second-line treatment for advanced renal cell carcinoma: overall survival analysis and updated results from a randomised phase 3 trial. Lancet Oncol. 2013;14(6):552–62. 361. Atmaca A, Al-Batran SE, Werner D, Pauligk C, Guner T, Koepke A, et al. A randomised multicentre phase II study with cisplatin/docetaxel vs oxaliplatin/docetaxel as first-line therapy in patients with advanced or metastatic non-small cell lung cancer. Br J Cancer. 2013;108(2):265–70. 362. Heng DY, Mackenzie MJ, Vaishampayan UN, Bjarnason GA, Knox JJ, Tan MH, et  al. Primary anti-vascular endothelial growth factor (VEGF)refractory metastatic renal cell carcinoma: clinical characteristics, risk factors, and subsequent therapy. Ann Oncol. 2012;23(6):1549–55. 363. Lara PN Jr, Quinn DI, Margolin K, Meyers FJ, Longmate J, Frankel P, et al. SU5416 plus interferon alpha in advanced renal cell carcinoma: a phase II California Cancer Consortium Study with biological

1  Cancer Immunotherapy Confers a Global Benefit and imaging correlates of angiogenesis inhibition. Clin Cancer Res. 2003;9(13):4772–81. 364. Cole BF, Gelber RD, Kirkwood JM, Goldhirsch A, Barylak E, Borden E.  Quality-of-life-adjusted survival analysis of interferon alfa-2b adjuvant treatment of high-risk resected cutaneous melanoma: an Eastern Cooperative Oncology Group study. J Clin Oncol. 1996;14(10):2666–73. 365. Glynne-Jones R, Grainger J, Harrison M, Ostler P, Makris A.  Neoadjuvant chemotherapy prior to preoperative chemoradiation or radiation in rectal cancer: should we be more cautious? Br J Cancer. 2006;94(3):363–71. 366. Lillehei KO, Mitchell DH, Johnson SD, McCleary EL, Kruse CA.  Long-term follow-up of patients with recurrent malignant gliomas treated with adjuvant adoptive immunotherapy. Neurosurgery. 1991;28(1):16–23. 367. Dillman RO, Duma CM, Ellis RA, Cornforth AN, Schiltz PM, Sharp SL, et  al. Intralesional lymphokine-activated killer cells as adjuvant therapy for primary glioblastoma. J Immunother. 2009;32(9):914–9. 368. Mellman I, Coukos G, Dranoff G.  Cancer immunotherapy comes of age. Nature. 2011;480(7378):480–9. 369. Hunter R.  WHO handbook for reporting results of cancer treatment. Int J Radiat Biol. 1980;38(4):481. 370. Nishino M, Jackman DM, Hatabu H, Yeap BY, Cioffredi LA, Yap JT, et al. New Response Evaluation Criteria in Solid Tumors (RECIST) guidelines for advanced non-small cell lung cancer: comparison with original RECIST and impact on assessment of tumor response to targeted therapy. AJR Am J Roentgenol. 2010;195(3):W221–8. 371. Wolchok JD, Hoos A, O’Day S, Weber JS, Hamid O, Lebbe C, et  al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin Cancer Res. 2009;15(23):7412–20. 372. Shim JH, Lee HC, Kim SO, Shin YM, Kim KM, Lim YS, et  al. Which response criteria best help predict survival of patients with hepatocellular carcinoma following chemoembolization? A validation study of old and new models. Radiology. 2012;262(2):708–18. 373. Britten CM, Janetzki S, van der Burg SH, Huber C, Kalos M, Levitsky HI, et  al. Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond. Cancer Immunol Immunother. 2011;60(1):15–22. 374. Janetzki S, Britten CM, Kalos M, Levitsky HI, Maecker HT, Melief CJ, et  al. “MIATA”-minimal information about T cell assays. Immunity. 2009;31(4):527–8. 375. Cheever MA.  Twelve immunotherapy drugs that could cure cancers. Immunol Rev. 2008;222:357–68.

47 376. Fecher LA, Agarwala SS, Hodi FS, Weber JS. Ipilimumab and its toxicities: a multidisciplinary approach. Oncologist. 2013;18(6):733–43. 377. Palumbo MO, Kavan P, Miller WH Jr, Panasci L, Assouline S, Johnson N, et  al. Systemic cancer therapy: achievements and challenges that lie ahead. Front Pharmacol. 2013;4:57. 378. Alatrash G, Jakher H, Stafford PD, Mittendorf EA. Cancer immunotherapies, their safety and toxicity. Expert Opin Drug Saf. 2013;12:631–45. 379. de Claro RA, McGinn K, Kwitkowski V, Bullock J, Khandelwal A, Habtemariam B, et al. U.S. Food and Drug Administration approval summary: brentuximab vedotin for the treatment of relapsed Hodgkin lymphoma or relapsed systemic anaplastic large-cell lymphoma. Clin Cancer Res. 2012;18(21):5845–9. 380. Ocvirk J, Heeger S, McCloud P, Hofheinz RD.  A review of the treatment options for skin rash induced by EGFR-targeted therapies: evidence from randomized clinical trials and a meta-analysis. Radiol Oncol. 2013;47(2):166–75. 381. Letarte N, Bressler LR, Villano JL.  Bevacizumab and central nervous system (CNS) hemorrhage. Cancer Chemother Pharmacol. 2013;71(6):1561–5. 382. Lee S, Knox A, Zeng IS, Coomarasamy C, Blacklock H, Issa S. Primary prophylaxis with granulocyte colony-­stimulating factor (GCSF) reduces the incidence of febrile neutropenia in patients with non-Hodgkin lymphoma (NHL) receiving CHOP chemotherapy treatment without adversely affecting their quality of life: cost-benefit and quality of life analysis. Support Care Cancer. 2013;21(3):841–6. 383. Younis T, Rayson D, Thompson K. Primary G-CSF prophylaxis for adjuvant TC or FEC-D chemotherapy outside of clinical trial settings: a systematic review and meta-analysis. Support Care Cancer. 2012;20(10):2523–30. 384. Chan A, Fu WH, Shih V, Coyuco JC, Tan SH, Ng R.  Impact of colony-stimulating factors to reduce febrile neutropenic events in breast cancer patients receiving docetaxel plus cyclophosphamide chemotherapy. Support Care Cancer. 2011;19(4):497–504. 385. Aapro MS, Bohlius J, Cameron DA, Dal Lago L, Donnelly JP, Kearney N, et  al. 2010 update of EORTC guidelines for the use of granulocyte-­ colony stimulating factor to reduce the incidence of chemotherapy-induced febrile neutropenia in adult patients with lymphoproliferative disorders and solid tumours. Eur J Cancer. 2011;47(1):8–32. 386. Martin M, Lluch A, Segui MA, Ruiz A, Ramos M, Adrover E, et al. Toxicity and health-related quality of life in breast cancer patients receiving adjuvant docetaxel, doxorubicin, cyclophosphamide (TAC) or 5-fluorouracil, doxorubicin and cyclophosphamide (FAC): impact of adding primary prophylactic granulocyte-colony stimulating factor to the TAC regimen. Ann Oncol. 2006;17(8):1205–12. 387. Robien K, Schubert MM, Yasui Y, Martin P, Storb R, Potter JD, et  al. Folic acid supplementation during methotrexate immunosuppression is not associated

48 with early toxicity, risk of acute graft-versus-host disease or relapse following hematopoietic transplantation. Bone Marrow Transplant. 2006;37(7):687–92. 388. Giese-Davis J, Collie K, Rancourt KM, Neri E, Kraemer HC, Spiegel D.  Decrease in depression symptoms is associated with longer survival in patients with metastatic breast cancer: a secondary analysis. J Clin Oncol. 2011;29(4):413–20. 389. Hontscha C, Borck Y, Zhou H, Messmer D, Schmidt-­ Wolf IG. Clinical trials on CIK cells: first report of the international registry on CIK cells (IRCC). J Cancer Res Clin Oncol. 2011;137(2):305–10. 390. Lang I, Kohne CH, Folprecht G, Rougier P, Curran D, Hitre E, et al. Quality of life analysis in patients with KRAS wild-type metastatic colorectal cancer treated first-line with cetuximab plus irinotecan, fluorouracil and leucovorin. Eur J Cancer. 2013;49(2):439–48. 391. Revicki DA, van den Eertwegh AJ, Lorigan P, Lebbe C, Linette G, Ottensmeier CH, et al. Health related

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2

Immunotherapy for Pediatric Solid Tumors Lauren Nicholls and Lisa M. Kopp

Contents 2.1

Introduction 

 50

2.2 Solid Tumors  2.2.1    Sarcomas  2.2.1.1  Osteosarcoma  2.2.1.2  Ewing Sarcoma  2.2.1.3  Soft-Tissue Sarcomas  2.2.2    Neuroblastoma  2.2.3    Nephroblastoma  2.2.4    Hepatoblastoma  2.2.5    Systemic Germ Cell Tumors  2.2.6    Central Nervous System Tumors  2.2.6.1  Embryonal Tumors  2.2.6.2  Gliomas  2.2.6.3  Pineal Region Tumors  2.2.7    Retinoblastoma 

 50  50  50  50  51  51  52  52  52  53  53  54  55  55

2.3 Immune Therapy and Pediatric Solid Tumors  2.3.1    Tumor-Targeting Monoclonal Antibodies (mAbs)  2.3.1.1  Cell Surface Immune Targets  2.3.1.2  Growth Factor Receptors and Oncogenes  2.3.1.3  Immunomodulatory Monoclonal Antibodies  2.3.2    Adoptive Cell Transfer  2.3.3    Anticancer Vaccines  2.3.3.1  Peptide-Based Vaccines  2.3.3.2  Dendritic Cell-Based Vaccines  2.3.3.3  Genetically Modified Tumor Vaccines  2.3.3.4  Other Adoptive Cell Therapies 

 56  56  56  58  60  61  63  63  64  66  66

2.4

Concluding Remarks 

References 

L. Nicholls Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine, Texas, USA

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L. M. Kopp (*) Department of Epidemiology and Biostatistics, Mel and Enid Zuckerman College of Public Health, The University of Arizona, Arizona, USA e-mail: [email protected]

© Springer Nature Switzerland AG 2020 N. Rezaei (ed.), Cancer Immunology, https://doi.org/10.1007/978-3-030-57949-4_2

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L. Nicholls and L. M. Kopp

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2.1

Introduction

Over 12,000 children and adolescents are diagnosed each year with cancer in the United States [1]. Cancer remains the leading cause of ­non-­accidental death in the pediatric age group. While the incidence of childhood cancer has increased over the last 40 years, the overall cure rates have significantly improved [1, 2]. There have been improvements in the outcome of children with solid tumors; however these have lagged behind those seen in hematologic malignancies.

2.2

Solid Tumors

Pediatric solid tumors include those located within the central nervous system (CNS), neural tumors, and those outside the CNS. Multi-agent chemotherapy, radiation, and surgery have greatly improved survival in children and adolescents with localized solid tumors. Unfortunately the same is not true for those with metastatic or recurrent disease, which may benefit from novel alternate therapies including immunotherapy [3].

2.2.1 Sarcomas The most common pediatric and adolescent sarcomas include osteosarcoma, Ewing sarcoma, and the soft-tissue sarcoma, rhabdomyosarcoma.

2.2.1.1 Osteosarcoma Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. The cell of origin is thought to be mesenchymal stem cells with an osteoid component [4]. The incidence of OS is approximately 4.5 cases per million per year in the population and it occurs primarily in adolescents with more than half of cases found in those less than 25 years of age [4, 5]. The most common sites for OS are the long bones of the distal femur, proximal tibia, and proximal humerus. Approximately 20% of children and adolescents will have metastatic disease

at diagnosis usually involving bones and lungs [6]. Multimodal therapy consists of 10 weeks of neoadjuvant chemotherapy followed by local control with tumor resection and 20  weeks of adjuvant chemotherapy [4, 6]. Radiation therapy has not been found to be as effective and is only recommended for palliation [6]. In children and young adults with localized disease, the overall survival is around 60–70% and for those with metastatic disease the prognosis is poor ranging from 25% to 40%.

2.2.1.2 Ewing Sarcoma Ewing sarcoma is the second most common bone tumor in children and adolescents [7]. The source of the Ewing sarcoma cell is still unknown; however, it is thought to derive from neuroectodermal cells with either neuronal or epithelial origins [8]. The most common cytogenetic translocation found in Ewing sarcoma is the balanced translocation of t(11;22), which at the molecular level involves the fusion of the Ewing sarcoma breakpoint region 1 (EWSR1) in 22q12 with the Friend leukemia virus integration 1 (FLI1) gene in 11q24. This fusion is found in 85% of Ewing sarcoma, yet multiple alternative gene fusions have been found between these two genes [9]. The overall incidence of Ewing sarcoma is almost three cases per million in the population per year and as with osteosarcoma, Ewing sarcoma occurs primarily in adolescents [7]. The most common sites of disease are the long bones of the lower extremity followed by the pelvis and chest wall. Metastatic disease is found in 25% of children and adolescents at diagnosis; the most common locations for metastasis are the lungs and other bones. Multimodal therapy includes chemotherapy and local control with either radiation or surgery. There have been no prospective studies comparing radiation versus surgery; however no advantage has been shown utilizing one modality versus the other [4, 6, 10]. Children and adolescents with localized disease have survival rates between 60% and 70% and those with metastatic disease have a much poorer outcome with 20–30% survival [4, 7, 10].

2  Immunotherapy for Pediatric Solid Tumors

2.2.1.3 Soft-Tissue Sarcomas The soft-tissue sarcomas (STS) are the most common extracranial solid tumors found in children and young adults accounting for more than 7% of cancer cases [11]. They are comprised of a diverse group of malignant connective tissue tumors including rhabdomyosarcoma (RMS) and non-rhabdomyosarcoma soft-tissue sarcomas (NRSTS). Rhabdomyosarcoma Rhabdomyosarcoma comprises almost half of the STS and the overall incidence of RMS is 4.5 cases per million in the population per year [11, 12]. RMS is derived from immature skeletal muscle and the two largest subgroups are embryonal (ERMS) and alveolar (ARMS) rhabdomyosarcoma. These groups are classified according to their histologic and biologic features. ERMS is associated with allelic loss of chromosome 11, found more often in younger children, and is associated with a better prognosis. ARMS has two commonly seen gene translocations, t(2;13) and t(1;13), and it is distributed equally throughout childhood and adolescence [12]. Forty percent of RMS cases are found in the head and neck, 20% arise in genitourinary sites, 20% from the extremities, and 20% from other sites [13]. The most common site for metastasis is the lungs, followed by bone and rarely the bone marrow. The prognosis for RMS is determined by multiple factors including primary site, stage and group, pathology, and age. Treatment includes multi-agent chemotherapy and local control with surgery and radiation. Chemotherapy is minimal for low-risk disease and is intensified for intermediate- and high-risk disease. Children and adolescents with low-risk disease have the most favorable outcome with failure-free survival (FFS) of 90%, those with intermediate-risk disease have a 70% FFS, and those with high-risk disease have a poor FFS of 20% [14]. Non-rhabdomyosarcoma Soft-Tissue Sarcomas NRSTS are a heterogeneous group of tumors derived from similar cells to mesenchymal cells. Due to the individual rarity of these tumors, they are commonly grouped together with the highest

51

incidence of NRSTS occurring in infants and young adults [11]. Tumors 5  cm and those that are unresectable have an overall survival of 50% and children and adolescents with metastatic disease have an overall survival of 10%. Chemotherapy and radiation therapy have been used with some of the NRSTS; however their efficacy is poor as the majority of these tumors are not sensitive to these modalities [15].

2.2.2 Neuroblastoma Neuroblastoma is the second most common extracranial solid tumor and the most common tumor found in infancy. The incidence is 4.9 cases per million per year [16]. It is an embryonal tumor of the autonomic nervous system and can arise anywhere in tissues of the sympathetic nervous system. The most common location of primary tumors is the abdomen. Approximately 50% of patients will present with metastatic disease. Children with low-risk disease only require surgery for treatment and have a survival of >98%. Those with intermediate-risk disease require surgery and moderate chemotherapy, and the survival rate remains excellent, 90–95%. In children with high-risk disease survival is very poor, 40–50%, despite intense treatment with surgery, chemotherapy, radiation, and immunotherapy [17]. Neuroblastoma is the only pediatric solid tumor in which immunotherapy has become the standard of care. Disialoganglioside GD2 is a protein that is uniformly expressed on neuroblastoma cells. Anti-GD2 antibodies have shown an improvement in the overall survival in clinical trials [18, 19] and the Children’s Oncology Group (COG) completed a randomized phase III study with an improvement in overall survival from 46% to 66% with the anti-GD2 antibody, dinutuximab, in combination with IL-2 and GM-CSF [17, 19]. This led to the FDA approval of dinutuximab in early 2015 and will be discussed in more detail in the upcoming sections on immunotherapy treatment.

52

2.2.3 Nephroblastoma Nephroblastoma, also known as Wilms’ tumor, is the most common malignant renal tumor of childhood. The incidence rate is eight cases per million per year in children younger than 15  years of age in North America. Nephroblastoma tumor is composed of blastemal, stromal, and epithelial cells. Tumors that contain anaplastic cells are classified as unfavorable as these are more aggressive tumors with a worse prognosis [20]. There have been specific genetic alterations found to be associated with the tumorigenesis of nephroblastoma. The tumor-suppressor gene WT1 was the first gene identified in the development of nephroblastoma. Loss of heterozygosity of chromosomes 16q and 1p has also been found in more aggressive nephroblastoma [21, 22]. Treatment includes surgery, chemotherapy, and radiation therapy. Patients with favorable histology tumors have an excellent prognosis, even with metastatic disease. Patients with stage I and II disease receive chemotherapy, whereas patients with stage III and IV disease as well as anaplastic tumors receive intensified chemotherapy and radiation therapy [23, 24]. Children with favorable histology and stage I, II, and III disease have a 4-year survival of >90%, whereas those with stage IV and V disease had a survival of >79% in the most recently completed clinical trials. Children with focal anaplasia have a better prognosis than those with diffuse anaplasia; however even with diffuse anaplasia survival is >70% for stage I, II, and III disease. Children with stage IV disease with focal or diffuse anaplasia have a very poor prognosis of 90% for children with stage I and II low-risk disease. Intermediate-risk disease prognosis is around 70% and survival is less than 30% in children with metastatic high-risk disease [28].

2.2.5 Systemic Germ Cell Tumors Germ cell tumors (GCT) arise from primordial cells involved in gametogenesis and can occur at multiple sites in the body with a variety of histological subtypes including embryonal carcinoma, yolk sac tumors, choriocarcinoma, and teratomas (mature and immature) [29]. Malignant germ cell tumors arising outside of the central nervous system account for 2–4% of all pediatric and adolescent cancers. The annual incidence is eight cases per million people in those less than 20 years of age [30]. In pubertal and postpubertal adolescents and adults, isochromosome p12 is present in the majority of tumors tested. However, in prepubertal children this is rarely present indicating a clear cytogenetic difference between these groups of patients [29, 31]. The most common sites of metastasis include the lungs, liver, and local lymph nodes. Chemotherapy treatment is the standard of care for tumors classified as intermediate or high risk after initial biopsy or complete resection [29, 32–35]. Radiation is not utilized in the initial treatment of malignant GCTs, as the majority of these tumors are very sensitive to chemotherapy. The prognosis is excellent for children with testicular, ovarian, and sacrococcygeal GCTs: stages I–III >85% and stage IV >80% [34, 35]. Children with refractory disease have a much worse prognosis of 30–50% [34, 36].

2  Immunotherapy for Pediatric Solid Tumors

2.2.6 C  entral Nervous System Tumors Collectively, tumors of the central nervous system (CNS) are the most common solid tumor in children. Primary CNS tumors are the leading cause of both cancer-related morbidity and mortality in pediatrics [37]. Overall incidence of childhood CNS tumors from birth to age 19 in the United States is 5.26 cases per 100,000 persons [38]. Childhood brain tumors are biologically diverse, and have widely variable presenting features, therapeutic approaches, and prognoses. Presenting features are generally related to tumor location, and approximately half of pediatric brain tumors arise in the posterior fossa [39]. The 5-year overall survival for CNS tumors in children is 73.6% [40]. Pediatric CNS tumors can be grouped into four major categories: embryonal tumors, gliomas, pineal region tumors, and craniopharyngiomas. We will review the most common categories including embryonal and glioma tumors.

2.2.6.1 Embryonal Tumors

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[43]. The 5-year overall survival for average-risk patients is 80% and for high-risk patients it is 60–65% [42]. CNS Primitive Neuroectodermal Tumors Central nervous system primitive neuroectodermal tumors (CNS PNET) describe a diverse group of rare and highly malignant childhood embryonal brain tumors [44, 45]. CNS PNETs can occur in the cerebral hemispheres, pineal gland, brain stem, and spinal cord. They account for less than 5% of childhood brain tumors [44, 46]. CNS PNET is now known to be genetically distinct from medulloblastoma [45]. However, because CNS PNETs are rare, they are generally treated on the same protocols as high-risk medulloblastoma, with best safe resection followed by craniospinal irradiation and chemotherapy [44, 46]. Metastatic spread is only found in 3% of patients at diagnosis [47]. The overall survival for CNS-PNET is 50–60% and factors associated with poor prognosis include young age, disseminated disease, non-pineal location, and subtotal tumor resection [45, 46].

Atypical Teratoid/Rhabdoid Tumors Atypical teratoid/rhabdoid tumors (ATRT) are Medulloblastoma The most common brain tumor of childhood is rare and aggressive malignant brain tumors most medulloblastoma, representing approximately often diagnosed in infants and very young chil20% of malignant brain tumors in patients dren. The incidence is estimated to be 0.1–0.5 per ≤19 years [41]. There are four histologic variants million children per year [48]. ATRTs account for of medulloblastoma: classic, desmoplastic-­ less than 5% of childhood brain tumors and are nodular, large-cell-anaplastic, and medulloblas- characterized by a loss of function in SMARCB1 toma with extensive nodularity. Additionally, (also known as hSNF5/INI-1), a tumor-­suppressor medulloblastomas are classified by molecular gene [49]. Pathologic diagnosis is made based on subtype which are thought to represent distinct loss of INI-1 staining on immunohistochemistry. cells of origin: WNT, SHH, group 3, and group 4 Primary tumors can occur in the supratentorial [42]. Approximately 14% of patients have meta- region, posterior fossa, or spinal cord [48]. static disease at diagnosis, which is almost always Metastatic disease can be seen in the brain and leptomeningeal dissemination [43]. Treatment spinal cord, although in some cases these are consists of surgical resection followed by cranio- additional primary tumors in patients with germspinal irradiation with boost to the primary tumor line INI-1 mutations. Treatment approaches and any sites of metastatic disease, followed by include surgical resection, radiation therapy, systemic chemotherapy Patients are considered high-dose chemotherapy often requiring autoloaverage risk if they are ≥3 years old at diagnosis, gous stem cell rescue, as well as intrathecal chehave 90% 10-year overall survival [51]. High-Grade Gliomas High-grade gliomas (HGG) account for 8–12% of brain tumors in pediatric oncology. Anaplastic astrocytomas (grade III) and glioblastoma multiforme (grade IV) are considered HGG [54]. These HGG are characterized histologically by nuclear atypia, high cellularity, many mitotic figures, and cellular pleomorphism; the presence of

L. Nicholls and L. M. Kopp

necrosis or microvascular proliferation is specific for glioblastoma multiforme [55]. Molecular profiling has identified marked differences between pediatric and adult HGG.  Common pediatric molecular aberrations include p53 mutation, PDGFR-alpha overexpression, and over-­ activation of the Akt pathway [55, 56]. Disseminated disease in HGG is extremely rare [54]. Treatment for pediatric HGGs typically consists of surgical resection followed by radiation therapy and temozolomide. Several cooperative group trials have failed to demonstrate further benefit from additional chemotherapy [56]. Children 85% [71] and the 5-year event-free survival for NGGCT is 72% for localized disease and 68% for metastatic disease [72].

2.2.7 Retinoblastoma Retinoblastoma is the most common malignant ocular tumor in childhood, affecting approximately 300 children in the United States per year, almost always before 5  years. Mutation of the RBI gene is implicated in retinoblastoma pathogenesis, with somatic mutations occurring in 85% of patients with unilateral disease [73]. Patients with bilateral retinoblastoma are presumed to have a germline mutation, although the majority of cases occur in the absence of family history due to new mutations arising [73]. Retinoblastoma patients are classified by group and stage. Group is based on the degree of tumor involvement within the eye, while stage describes the degree of tumor involvement outside of the eye [74]. Trilateral retinoblastoma occurs in 5–10% of patients with a germline RB1 mutation, and refers to bilateral ocular retinoblastoma along with a primary CNS lesion which most

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commonly occurs in the pineal region [74]. Distant metastatic disease is rare in developed nations, but can occur throughout the brain and spinal cord, and lymphatic spread to regional nodes and hematogenous metastases to bone or bone marrow have been reported [73–75]. The primary goal of therapy is to eradicate disease, with secondary goal to preserve the eye, and finally to conserve as much vision as possible. Systemic chemotherapy is effective but must be consolidated with a local intervention such as cryotherapy or laser treatment [76]. Intra-arterial chemotherapy injected directly into the ophthalmic artery is a newer technique which has become more widely used in recent years [77]. Enucleation with orbital implant is associated with high disease control rate, but has significant impact on the quality of life [76]. The 5-year overall survival rate is 96.5%, with the majority of deaths occurring in patients with metastatic disease [77].

2.3

Immune Therapy and Pediatric Solid Tumors

2.3.1 Tumor-Targeting Monoclonal Antibodies (mAbs) Monoclonal antibodies (mAbs) are a type of immune therapy in which the drug binds to the cell surface of the tumor cell allowing the immune system to recognize the tumor cell as foreign and attack. These mAbs can mediate response through antibody-dependent cell-mediated toxicity (ADCC) and complement-mediated toxicity (CMC) or these mAbs bind to the surface and then deliver a toxin into the cell [78]. There are a multitude of mAbs in clinical trials and many more are in development; we will cover the mAbs currently being utilized. Table  2.1 contains the current open clinical trials utilizing mAbs.

2.3.1.1 Cell Surface Immune Targets Gangliosides Disialoganglioside (GD2) is a carbohydrate antigen expressed normally on the tissues of the central nervous system, peripheral nerves, and skin

melanocytes whose true function is not known. GD2 is expressed uniformly on neuroblastoma, melanoma, and osteosarcoma while some sarcomas, lung tumors, and brain tumors may also express GD2 [79–81]. Murine, mouse-human chimeric, and humanized versions of anti-GD2 antibodies have been studied in neuroblastoma clinical trials for over 20 years [82]. The first generation of anti-GD2 mAbs included 3F8, 14G2a, and ch14.18. Reversible pain, fever, tachycardia, and urticaria were the most common toxicities reported in initial trials of murine 3F8 and 14G2a antibodies. Phase I and II studies revealed moderate responses in patients with relapsed/recurrent neuroblastoma. In a later phase II study 3F8 was combined with granulocyte colony macrophage-­ stimulating factor (GM-CSF) to augment granulocyte/monocyte antibody-dependent cellular cytotoxicity (ADCC). Patients tolerated this combination without significant toxicity and those with bone marrow involvement benefited most [79, 83]. A Children’s Oncology Group (COG) phase III clinical trial in children with newly diagnosed high-risk neuroblastoma randomized children to receive immunotherapy with the humanized anti-GD2 antibody ch14.18, dinutuximab, combined with IL-2 alternating with GM-CSF versus standard-of-care therapy. Children randomized to receive dinutuximab had significantly improved rates of event-free survival (66% vs. 46% at 2  years, P  =  0.01) and overall survival compared to standard therapy alone [19]. Subsequently, dinutuximab was FDA approved as part of first-line therapy for high-risk neuroblastoma patients in early 2015. GD2 is also uniformly present in osteosarcoma human cell lines with several having a higher expression than neuroblastoma cell lines [80, 81]. There are two open phase II clinical trials (NCT02484443 and NCT02502786) using GD2 mAbs (dinutuximab and Hu3F8, respectively) in combination with GM-CSF open to adolescents and young adults with relapsed/ refractory osteosarcoma. Second-generation GD2 mAbs have been developed and are in early clinical trials. Hu14.18-IL-2 is a fusion protein of a humanized second-generation anti-GD2 antibody and IL-2.

2  Immunotherapy for Pediatric Solid Tumors

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Table 2.1  Current targeted immunotherapy clinical trials for pediatric solid tumors: monoclonal antibodies Study title Dinutuximab in combination with sargramostim in treating patients with recurrent osteosarcoma Humanized monoclonal antibody 3F8 (Hu3F8) with granulocyte-macrophage colony-stimulating factor (GM-CSF) in the treatment of recurrent osteosarcoma Therapy for children with advanced-stage neuroblastoma Racotumomab in patients with high-risk neuroblastoma Enoblituzumab (MGA271) in children with B7-H3-­expressing solid tumors Denosumab in treating patients with recurrent or refractory osteosarcoma Combination chemotherapy with or without ganitumab in treating patients with newly diagnosed metastatic Ewing sarcoma External beam radiation therapy and cetuximab followed by irinotecan and cetuximab for children and young adults with newly diagnosed diffuse pontine tumors and high-grade astrocytomas Intra-arterial infusion of erbitux and bevacizumab for relapsed/refractory intracranial glioma in patients under 22 Nimotuzumab in combination with radio-chemotherapy for the treatment of brainstem tumor in children A study of olaratumab alone and in combination with standard chemotherapies in children with cancer Study of the IDO pathway inhibitor, indoximod, and temozolomide for pediatric patients with progressive primary malignant brain tumors Bevacizumab, temsirolimus alone and in combination with valproic acid or cetuximab in patients with advanced malignancy and other indications Metronomic and targeted anti-angiogenesis therapy for children with recurrent/ progressive medulloblastoma (MEMMAT) Metronomic therapy for pediatric patients with solid tumors at high risk of recurrence (metronomic) A study of ramucirumab (LY3009806) in children with refractory solid tumors Pediatric MATCH: Targeted therapy directed by genetic testing in treating pediatric patients with relapsed or refractory advanced solid tumors, non-Hodgkin lymphomas, or histiocytic disorders Nivolumab with or without ipilimumab in treating younger patients with recurrent or refractory solid tumors or sarcomas Durvalumab and tremelimumab in patients with advanced rare tumors A study to evaluate the safety, tolerability, pharmacokinetics, immunogenicity, and preliminary efficacy of atezolizumab (anti-­programmed death-ligand 1 [PD-L1] antibody) in pediatric and young adult participants with solid tumors A phase II trial of avelumab in patients with recurrent or progressive osteosarcoma Nivolumab with or without stereotactic radiosurgery in treating patients with recurrent, advanced, or metastatic chordoma Pembrolizumab in treating younger patients with recurrent, progressive, or refractory high-grade gliomas, diffuse intrinsic pontine gliomas, or hypermutated brain tumors A study of pembrolizumab (MK-3475) in pediatric participants with an advanced solid tumor or lymphoma (MK-3475-051/KEYNOTE-051) SARC028: a phase II study of the anti-PD1 antibody pembrolizumab (MK-3475) in patients with advanced sarcomas Durvalumab in pediatric and adolescent patients Expanded access for nivolumab (Opdivo)

Phase II

NCT no. NCT02484443

II

NCT02502786

II II I II III

NCT01857934 NCT02998983 NCT02982941 NCT02470091 NCT02306161

II

NCT01012609

I/II

NCT01884740

II

NCT02672241

I

NCT02677116

I

NCT02052648

I

NCT01552434

II

NCT01356290

I

NCT02446431

I II

NCT02564198 NCT03155620

I/II

NCT02304458

II I/II

NCT02879162 NCT02541604

II I

NCT03006848 NCT02989636

I

NCT02359565

I/II

NCT02332668

II

NCT02301039

I n/a

NCT02793466 NCT03126643

58

Phase I and II studies of patients with r­ efractory/ relapsed neuroblastoma have been completed and toxicities were similar to ch14.18. The phase II study revealed a response rate of around 21% for patients with non-bulky disease [84, 85]. Another second-generation GD2 mAb, Hu14.18K332A, is a humanized ch14.18 mAb with a mutation to alanine at lysine 322 in order to limit complement fixing and thus pain associated with anti-GD2 [79]. A phase I clinical trial in patients with recurrent/refractory neuroblastoma, melanoma, osteosarcoma, and Ewing sarcoma has been completed [86]. Hu14.18K332A is currently being tested in children with newly diagnosed high-risk neuroblastoma in a phase II clinical trial in combination with cyclophosphamide and topotecan (NCT01857934). Unlike GD2, GD3 is a disialoganglioside that is not expressed on normal tissues. GD3 expression is found in melanomas, soft-tissue sarcomas, and tumors of neuroectodermal origin including Wilms’ tumor, neuroblastoma, and retinoblastoma [65, 87]. Similar to GD3, N-glycosylated ganglioside NeuGc-GM3, GM3, is expressed primarily in neoplasms and not in normal tissues. Racotumomab is a murine mAb, an IgG1, anti-­ idiotype to NeuGcGM3, which is delivered as an intradermal injection. It has been tested in phase 1 and II clinical trials in melanoma, breast, and non-small cell lung cancer (NSCLC). There have been prolonged responses and minimal toxicity and currently a phase III clinical trial in NSCLC is ongoing [88]. Racotumomab was tested in a phase I study in refractory/recurrent pediatric tumors; it was well tolerated with no maximum tolerated dose reached [89]. There are no clinical trials open in the United States, but there is a phase II study of racotumomab currently recruiting in Argentina (NCT02998983). B7-H3 The surface immunomodulatory glycoprotein B7 homolog 3 protein (B7-H3) is overexpressed in some human tumors and inhibits natural killer cells and T cells leading to promotion of tumor growth in multiple cancers including neuroblastoma [90]. The murine antibody 8H9 is specific for the B7-H3 protein, 4Ig-B7H3, and has been

L. Nicholls and L. M. Kopp

radiolabeled with 131I (131I-8H9) to target solid tumors in xenografts. 131I-8H9 has been utilized in a phase I study along with radiolabeled anti­GD2 antibody 3F8 (131I-3F8) to target patients with central nervous system metastases of neuroblastoma. Either 131I-8H9 or 131I-3F8 was given intrathecally to 21 patients (1 patient received both mAbs, 17 patients 131I-8H9 and 3 patients 131 I-3F8) along with systemic chemotherapy of irinotecan and temozolomide. There was minimal toxicity and 17 patients remained free of CNS neuroblastoma with a median of 33 months [91]. B7-H3 is also being targeted with the mAb, enoblituzumab (MGA271), that is in phase I clinical trials for children with B7-H3-expressing solid tumors (NCT02982941). RANK-L The cytokine RANK-L is a TNF family member expressed on the surface of osteoblasts and is released by activated T cells. RANK-L has been found to be critical to osteoclast formation, function, and survival. Dysregulation in bone remodeling has been found to be key in the pathophysiology of bone metastasis, and RANK-L plays an essential role in this process [92]. Denosumab is a humanized mAb that binds RANK-L ligand and has been used in phase II and III clinical trials in multiple myeloma (MM), metastatic breast, and prostate cancer [92–95]. Denosumab is thought to have utility in osteosarcoma because of its direct effects on bone tumor pathophysiology and expression in 75% of osteosarcomas correlating with poor response to neoadjuvant chemotherapy [96]. Denosumab is currently being studied in a COG phase II clinical trial (NCT02470091) in patients with recurrent/ refractory osteosarcoma.

2.3.1.2 Growth Factor Receptors and Oncogenes HER2 HER2 overexpression has been found in pediatric medulloblastoma, nephroblastoma, and osteosarcoma [87, 97]. However, its use in pediatric solid tumors has been limited. The HER2-targeting mAb, trastuzumab, was found to be safe when

2  Immunotherapy for Pediatric Solid Tumors

combined with chemotherapy in a phase II trial of newly diagnosed patients with metastatic osteosarcoma. Forty-one children and adolescents with HER2+ tumors received trastuzumab, yet survival was not significantly prolonged [98]. As this study did not discriminate based on the overexpression of HER2 in tumors, future directions would include using trastuzumab in children and adolescents with only overexpression of HER2. Pediatric tumors that express HER2, similar to GD2, have recently been incorporated into chimeric antigen receptor T cells as discussed further under adoptive cell transfer [99, 100].

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[106]. An ongoing COG randomized phase II/III study opened in December 2014 comparing standard multi-agent chemotherapy with or without the IGF-1R antibody, ganitumab, in patients with newly diagnosed metastatic Ewing sarcoma (NCT02306161).

Epidermal Growth Factor Receptor Family Epidermal growth factor receptor (EGFR) family of tyrosine kinase receptors are found to be dysregulated in many cancers including brain tumors [107]. High-grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG) have been found to have overexpression of EGFR [56, 108]. Insulin-Like Growth Factor-1 Receptors Two anti-EGFR mAbs are being tested in pediatInsulin-like growth factor-1 receptor (IGF-1R) ric brain tumors, cetuximab and nimotuzumab. pathway has been found to be important in the Cetuximab is being tested in children and young growth of solid tumors, specifically sarcomas. adults with newly diagnosed DIPG and HGG in Previously, it was difficult to target IGF-1R the phase II setting in combination with radiation because of its similarity to the insulin receptor followed by cetuximab plus chemotherapy leading to toxicities occurring without specific (NCT01012609). There is also an ongoing phase inhibition. In the past decade there has been I/II trial of intra-arterial infusion of cetuximab development of humanized mAbs that target and bevacizumab (anti-VEGF mAb) for children IGR-1R without major toxicities [101]. The and adolescents with relapsed/refractory intraPediatric Preclinical Testing Program evaluated cranial gliomas (NCT01884740). A phase II the human antibody, SCH 717454, in solid tumor study of nimotuzumab as a single agent in pediatxenograft models and found broad antitumor ric patients with progressive DIPG was comactivity in Ewing sarcoma, osteosarcoma, RMS, pleted and found modest activity, but only 2 of 44 and neuroblastoma [102]. Cixutumumab (IMC-­ patients had a partial response with no complete A12), a fully human IgG1 mAb against IGF-1R, responses [109]. Another pilot/phase II study in was used in a phase I/II trial in pediatric patients 25 children with DIPG in which nimotuzumab with refractory solid tumors. The drug was well was combined with vinorelbine and radiation tolerated; however there was limited single-agent showed an increased progression-free survival of activity [103]. 8.5  months and overall survival of 15  months It is thought that IGF-1R mAbs will work best [110]. There is currently an ongoing phase II in combination with other targeting agents such study of nimotuzumab in combination with temoas mTOR inhibitors, which have been shown to zolomide and radiation for children with newly increase the IGF-1R serine/threonine kinase diagnosed or progressive brainstem tumors AKT. The combination of mTOR inhibition and (NCT02672241). IGF-1R AKT inhibition leads to more effective killing of RMS, osteosarcoma, and Ewing sar- Platelet-Derived Growth Factor coma cell lines [101, 104, 105]. In a recent clini- Platelet-derived growth factors (PDGF) and their cal trial, which included 20 patients with receptors (PDGFR) have been found to be refractory Ewing sarcoma and desmoplastic expressed on a variety of tumors and its expressmall-round cell tumor, cixutumumab was com- sion coincides with increased tumor growth, bined with the mTOR inhibitor temsirolimus. invasiveness, and poor outcomes. PDGF are This combination was well tolerated with one thought to change the microenvironment of complete response and five partial responses tumors and contribute to tumor growth and

60

metastasis [111]. Olaratumab is a fully human recombinant IgG1 mAb blocking PDGF-AA and PDGF-BB from binding with PDGFRα. It was granted accelerated FDA approval in combination with doxorubicin for soft-tissue sarcomas in adult patients in 2016 [112]. Olaratumab is currently open as a phase I study in combination with either doxorubicin, vincristine and irinotecan, or ifosfamide (physician choice) in refractory/recurrent solid tumors including CNS tumors (NCT02677116). Vascular Endothelial Growth Factor It is well known that the growth and metastasis of solid tumors are dependent on angiogenesis. Many pro-angiogenic factors and receptors have been found to contribute to regulating tumor growth including vascular endothelial growth factor (VEGF). As VEGF is overexpressed in many cancers, this is a useful target for tumor vascular inhibition [113]. Bevacizumab is a humanized antibody targeting VEGF-A and VEGFR-2 complex [114]. It has been approved for use in a multitude of adult cancers including renal cell carcinoma, glioblastoma, colorectal cancer, and breast and ovarian cancer [115]. Bevacizumab has been tested in a variety of clinical trials in pediatric solid tumors; however the results have been mixed. Bevacizumab did show some prolonged responses in retrospective reviews and case series of pediatric and adolescent patients with sarcomas, glioblastoma, medulloblastoma, and low-grade glioma [116– 119]. However, four recently published phase II clinical trials each showed no increase in event-­ free survival when bevacizumab was added to conventional chemotherapy for child and adolescent patients with metastatic soft-tissue sarcomas [120], newly diagnosed osteosarcoma [121], recurrent/refractory gliomas [122], and recurrent/ refractory neuroblastoma [123]. There are studies open evaluating bevacizumab in combination with chemotherapy and small-molecule inhibitors in pediatric and adolescent patients with relapsed/refractory gliomas (NCT01884740 and NCT02052648) and solid tumors (NCT01552434). Bevacizumab is also combined with metronomic chemotherapy, which is being

L. Nicholls and L. M. Kopp

tested in children with recurrent/refractory medulloblastoma (NCT01356290) and recurrent/ refractory solid tumors (NCT02446431). Ramucirumab is another human mAb which targets VEGFR2 by blocking VEGF ligands. It is approved for adenocarcinoma and NSCLC [115]. Ramucirumab is currently being tested in a phase I study through COG in children and adolescents with refractory or recurrent solid tumors (NCT02564198) and in a phase II clinical trial in the pediatric MATCH study. This study is testing a plethora of targeted drugs for relapsed/refractory solid tumors (NCT03155620).

2.3.1.3 Immunomodulatory Monoclonal Antibodies Cytotoxic T Lymphocyte Antigen 4 and Programmed Death Receptor 1 Cytotoxic T lymphocyte antigen 4 (CTLA4) is a member of the immunoglobulin superfamily, expressed on the surface of T cells, and transmits an inhibitory signal. T-cell activation through the T-cell receptor engagement and CD28 leads to increased expression of CTLA4, an inhibitory receptor for B7 molecules. CTLA4 is also found in regulatory T cells in which CTLA-4 blockade leads to their decreased immunosuppression. Programmed death receptor 1 (PD1) and its ligands (PD-L1 and PD-L2) are also part of the immune checkpoint pathway. In inflammatory environments, PD 1 can decrease T-cell activity, while in the tumor microenvironment PD1 ligand inhibits antitumor lymphocytes [124]. PD1 is also highly expressed on T regulatory cells and when engaged by its ligand it is thought to enhance T regulatory proliferation [125]. Ipilimumab, an anti-CTLA-4 antibody, in a phase III clinical trial of metastatic melanoma patients increased survival by 20% [126]. The anti-PD1 antibody nivolumab has shown tumor responses in adult solid tumors including melanoma, lung cancer, and renal cancers [127, 128]. The results of combination nivolumab and ipilimumab in stage III and IV melanoma had an impressive increase in median progression-free survival to 11.5  months compared to 2.9  months with ipilimumab alone (P  16  years of age with advanced rare tumors (NCT02879162). Recently 451 pediatric tumors were evaluated for PD-L1 expression and tumorassociated immune cells. Thirty-nine pediatric tumors expressed at least 1% PD-L1 with the highest frequency occurring in Burkitt lymphoma, glioblastoma multiforme, and neuroblastoma. Currently there are multiple phase I and phase II clinical trials testing PD-1 and PD-L1 inhibitors in pediatric solid tumors: atezolizumab (anti-PD-L1) in nonneural solid tumors (NCT02541604); avelumab (anti-PD-L1) in recurrent osteosarcoma (NCT03006848); nivolumab with or without radiation in recurrent/ metastatic chordoma (NCT02989636); pembrolizumab (anti-PD-1) in recurrent/refractory high-­ grade gliomas, diffuse intrinsic pontine gliomas, and hypermutated brain tumors (NCT02359565); pembrolizumab in relapsed/refractory non-CNS solid tumors (NCT02332668); pembrolizumab in advanced sarcomas (NCT02301039); and durvalumab (anti-PDL-1) in recurrent/refractory solid tumors and CNS tumors (NCT02793466). Bristol-Myers Squibb has begun an expanded access for its anti-PD-1 drug, nivolumab, and it is available to patients up to 17  years of age with refractory pediatric tumors (NCT03126643). There are many more upcoming clinical trials to evaluate anti-PD-1 and anti-PDL1 tumors.

2.3.2 Adoptive Cell Transfer The use of autologous chimeric antigen receptor (CAR) T cells targeted against a cancer cell-­ specific antigen has demonstrated benefit in

61

B-cell leukemia and lymphomas. Efforts to apply this strategy toward the treatment of refractory pediatric solid tumors have presented several challenges. CAR T-cell therapy requires identification of a specific tumor-associated antigen against which to direct the CAR T cell. Targetable epitopes have not been identified for many pediatric tumors. Tumor heterogeneity and unique microenvironmental features in pediatric solid tumors may affect response [132]. There is emerging evidence that negative selection for tumor cells that do not express the target antigen following CAR T-cell therapy contributes to treatment failure [133]. In addition, persistence of tumor-directed CAR T cells is required for durable disease control, and this has proven difficult to achieve. Table  2.2 contains the current open clinical trials utilizing CAR T cells. Largely due to known expression of GD-2 in neuroblastoma, this is the first pediatric solid tumor in which CAR T cells have been tested in clinical trials. In a phase I trial, patients with EBV seropositivity and recurrent/refractory neuroblastoma received both autologous activated T Table 2.2  Current targeted immunotherapy clinical trials for pediatric solid tumors: chimeric antigen receptor (CAR) T cells Study title Third-generation GD-2 chimeric antigen receptor and iCaspase suicide safety switch, neuroblastoma, GRAIN (GRAIN) Engineered neuroblastoma cellular immunotherapy (ENCIT)-01 A pilot study of genetically engineered NY-ESO-1-specific NY-ESO-1c259T in HLA-A2+ patients with synovial sarcoma (NY-ESO-1) CMV-specific cytotoxic T lymphocytes expressing CAR targeting HER2 in patients with GBM (HERT-GBM) Genetically modified T cells in treating patients with recurrent or refractory malignant glioma Her2 chimeric antigen receptor expressing T cells in advanced sarcoma

Phase NCT no. I NCT01822652

I

NCT02311621

I/II

NCT01343043

I

NCT01109095

I

NCT02208362

I

NCT00902044

62

cells and Epstein-Barr Virus (EBV)-specific CTLs that were genetically modified to recognize GD2. Three of the eleven patients with active disease had a complete response and no significant toxicity was observed. A median follow-up of 96  weeks revealed low-level persistence of both types of GD2-CAR T cells and association with longer survival [134, 135]. Interestingly, patients who received the GD2CAR T cells did not experience significant pain as has been observed in anti-GD2 mAb therapy [135]. Enthusiasm for GD2-CAR T cells continues; there is a recently completed phase I trial in GD2+ solid tumors using escalating doses of anti-GD2-CAR T-cell infusions (NCT02107963). A phase I study of third-generation anti-GD2 CAR T cells for refractory neuroblastoma patients (NCT01822652) uses GD2-CAR T cells which integrates the CD28 and OX40 costimulatory endodomains with the hope of increasing persistence and antitumor effects [136]. This CAR also contains an iCaspase suicide safety switch that can be activated leading to programmed cell death to prevent unanticipated toxicities such as cytokine storm [137, 138]. Unfortunately, no objective responses or persistence of GD2.CD28. OX40 CAR T cells were seen at 6  weeks posttreatment in 11 neuroblastoma patients. Further investigation has raised concern that the presence of CD28 costimulatory domain instead enhances CAR T-cell exhaustion [139]. A completed phase I study used donor-derived CAR T cells directed against GD2 following allogeneic stem cell transplant to enhance the graft-versus-tumor effect posttransplant (NCT01460901). CD-171 is a new candidate epitope, which is homogeneously and abundantly expressed on neuroblastoma cells, and it plays a role in oncogenesis by regulating tumor cell differentiation, proliferation, migration, and invasion. The majority of heavily pretreated patients have demonstrated the ability to generate adequate CE7 CAR T cells to have antitumor effect on mice [132]. There is an active phase I trial of CE7-derived CAR T cells targeting CD-171 (NCT02311621). Synovial cell sarcoma commonly expresses the cancer testis antigen NY-ESO-1. A recent study (NCT0067048) treated 18 NY-ESO-1-­

L. Nicholls and L. M. Kopp

positive synovial sarcoma patients with cytoreductive chemotherapy followed by infusion of NY-ESO-1-transduced autologous T cells. Ten patients achieved partial response, four of which were durable for >10  months. One patient achieved a complete response, which at the time of publication was sustained for 20  months. Treatment was generally well tolerated; there was one toxic death from septic shock in a neutropenic patient [140]. There is an ongoing phase I/II clinical trial to further evaluate the efficacy and tolerability of this approach (NCT01343043). Pediatric rhabdomyosarcoma and non-­ rhabdomyosarcoma soft-tissue sarcomas have very poor prognosis if they are refractory to up-­ front therapy or relapse following initial response. New preclinical data has identified ErbB2 as a promising target antigen that is highly expressed in pediatric rhabdomyosarcomas as well as other sarcomas. In vitro ErbB2-transduced CAR T cells demonstrated superior recognition, infiltrating, proliferating, and killing capacities of ErbB2 CAR T cells in pediatric rhabdomyosarcoma compared to previously utilized cytokine-induced killer (CIK) cells [141]. This promising work will likely be moved forward into clinical trials in the near future. Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in up to 80% of glioblastoma multiforme (GBM) brain tumors. CD133+ tumor stem cells are believed to mediate chemoresistance. HER2-specific CAR T cells were successfully generated from GBM patients and demonstrated potent antitumor activity in  vitro, including targeting of CD133+ cells [142]. There is an active phase I trial using dose escalation of HER-2 CAR T cells in GBM patients (NCT01109095). A recent case report describes a 50-year-old man with multiply recurrent, multifocal GBM who achieved disease control for 7.5 months with intraventricular infusions of IL13R-alpha 2-directed CAR T cells. IL13R-alpha 2 is an antigen associated with poor survival in glioma. Co-stimulation via 4-1BB (CD137) was incorporated to enhance T-cell persistence [143]. There is an ongoing phase I trial exploring this therapeutic

2  Immunotherapy for Pediatric Solid Tumors

approach further; children aged 12  years and older are eligible (NCT02208362). CAR T-cell therapy directed against HER2 has been used to target other pediatric solid tumors. The results of an ongoing phase I clinical trial in 19 patients with advanced pediatric sarcomas utilizing HER2-CAR T cells were recently published [100]. Patients with HER2-positive refractory/recurrent sarcomas received escalating doses of HER2-CD28 T cells (NCT00902044). The cells persisted for 6 weeks without toxicities and responses were seen in some of the patients including four patients with stable disease [100]. Further potential targets for CAR-based therapy are currently under investigation for pediatric solid tumors. Specific areas of interest going forward include optimization of co-stimulatory domains to enhance CAR T-cell function and persistence, and investigation of combination immunotherapeutic approaches [139].

2.3.3 Anticancer Vaccines While there is agreement that cancer vaccines are more effective in patients with minimal residual disease, devoid of major immunosuppressive effects from T regulatory or myeloid suppressive cells, there is still no consensus for the “optimal” tumor vaccine. Vaccines derived from total tumor cells such as lysates, irradiated, genetically modified, apoptotic, or necrotic tumor cells or tumor-­ derived chaperone proteins, allow for a wider array of antigens reducing the emergence of tumor escape variants, seen with single peptides, and do not require identification of specific antigens [82, 144]. In addition to the antigen component of the vaccine, an adjuvant is also essential. Adjuvants have ranged from attenuated bacterial products; emulsions such as Montanide or liposomal adjuvants; tensoactive agents such as saponins, alum, and other minerals; and cytokines such as GM-CSF [82, 145]. DNA or RNA vaccines use the patient’s own cells to transcribe and translate nucleic acids into proteins for processing, which avoids HLA haplotype restriction [146]. As in adult cancers, the number of vaccine trials continues to grow with much anticipation

63 Table 2.3  Current targeted immunotherapy clinical trials for pediatric solid tumors: anticancer vaccines Study title Bivalent vaccine with escalating doses of the immunological adjuvant OPT-821, in combination with oral β-glucan for high-risk neuroblastoma Vaccine immunotherapy for recurrent medulloblastoma and primitive neuroectodermal tumor (re-MATCH) Dendritic cell vaccine with or without gemcitabine pretreatment for adults and children with sarcoma Gene-modified T cells, vaccine therapy, and nivolumab in treating patients with stage IV or locally advanced solid tumors expressing NY-ESO-1 (NYM) Allogeneic tumor cell vaccination with oral metronomic cytoxan in patients with high-risk neuroblastoma (ATOMIC) A two-part phase IIb trial of vigil in Ewing sarcoma

Phase NCT no. I/II NCT00911560

I/II

NCT01326104

I

NCT01803152

I

NCT02775292

I/II

NCT01192555

II

NCT02511132

of its role in solid tumor therapy. Table 2.3 contains the current open clinical trials utilizing anticancer vaccines.

2.3.3.1 Peptide-Based Vaccines An ongoing phase I/II trial of GD2 and GD3 antigens with OPT-821 adjuvant in combination with oral β-glucan recently enrolled 15 neuroblastoma patients in complete or very good partial remission. In the phase I component (NCT00911560) of 13 who completed the series of vaccinations, 12 remain relapse free at the time of publication (median of 32 months) and 1 with a single-node relapse [147]. The Wilms’ tumor antigen (WT1) is expressed on many pediatric solid malignancies including nephroblastoma (Wilms’ tumor), neuroblastoma, and RMS particularly the alveolar subtype [148]. WT1 was ranked as the most promising tumor antigen by the NCI in 2009 [149]. It has been targeted in multiple trials in patients with leukemia. A phase I/II trial was completed in patients with

64

relapsed tumors with overexpression of WT1 protein and HLA-A*2402 positive. The HLA-­ A*2402-restricted, 9mer-modified WT1 peptide was emulsified with Montanide ISA51 adjuvant and given intradermally to patients. The trial included four pediatric and young adult patients with sarcomas. The only adverse effect was injection-site erythema. WT1-specific CTLs were found in three of the four patients with one having a complete response and one stable ­disease [150]. WT1 may prove to be a useful target for pediatric solid tumors [148]. A recent clinical trial identified three broadly expressed glioma-associated antigens (GAA) in high-grade pediatric glioma: interleukin-13 receptor alpha 2, EphA2, and surviving. Peptide vaccines were created using Montanide as the delivery vehicle and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethocellulose (poly-ICLC) as an immunoadjuvant. Toxicities were grades 1–2 only. Thirteen of 21 evaluable patients developed an immune response to at least one GAA.  Twenty-six patients were evaluated for response. Best response was stable disease in 19 patients, partial response in 2 patients, minor response in 1 patient, and sustained disease-free state for 2 patients with grossly resected disease prior to vaccine. Two patients had early progression. Five patients developed pseudoprogression during vaccine therapy. Pseudoprogression refers to intra-­tumoral edema and/or contrast enhancement, which can be seen in the setting of immunotherapy disease response. Median survival on this trial was 13.2  months, with one patient surviving greater than 38 months at the time of publication [146]. Carcinoembryonic antigen glypican-3 (GPC3) is over-expressed in many pediatric solid tumors, particularly yolk sac tumors and hepatoblastoma. GPC3 is a cell-surface proteoglycan, which promotes cell growth, development, and response to several growth factors. In a completed phase I trial, 18 refractory pediatric solid tumor patients received GPC3 peptide vaccine intradermally every 2  weeks until they developed progressive disease. GPC3 expression was confirmed in 17/18 patients; the remaining patient’s tumor was not evaluable. Hepatoblastoma tumors were

L. Nicholls and L. M. Kopp

noted to have particularly robust GPC3 expression. The vaccine was well tolerated, with only grade 1–2 toxicities. Patients were classified according to disease status prior to vaccine therapy: tumor progression (6), partial remission (4), and remission (8). One patient in the partial remission group and six patients in the remission group had complete response after 2  months of vaccine therapy. Seven of the 18 patients mounted a GPC3 cytotoxic lymphocyte (CTL) response, which correlated with improved progression-free and overall survival. Five patients with hepatoblastoma in remission and who generated a CTL response to the vaccine had overall survival of 27 months or more, indicating that this therapy is most efficacious in the minimal residual disease setting [151].

2.3.3.2 Dendritic Cell-Based Vaccines The largest dendritic cell vaccine trial in pediatric patients published to date included 30 patients with recurrent or metastatic Ewing sarcoma or alveolar rhabdomyosarcoma. Patients with confirmed t(11;22) or t(2;13) translocations had an initial cell harvest to collect autologous T cells and then received cytoreductive chemotherapy, radiation therapy, and/or surgery. This was followed by infusion of autologous T cells and DCs pulsed with tumor-specific peptides derived from tumor-specific breakpoints and E7 as a model antigen. Immune responses were seen in 39% of patients toward the tumor translocation breakpoint peptides; however responses were limited becoming undetectable 6  weeks later. The survival in the tumor vaccine group was 43% but it was difficult to ascertain the vaccine effect versus historical control rates in this high-risk population. There were no significant toxicities reported with the vaccine [152]. Multiple smaller phase I and pilot studies have been completed utilizing a variety of DC vaccines. A pilot study used a cancer vaccine in five patients with relapsed/refractory neuroblastoma or sarcoma post-chemotherapy, radiation, and autologous peripheral blood stem cell transplant. Patients received autologous DCs pulsed with tumor-specific synthetic peptides or tumor lysates. Delayed-type hypersensitivity (DTH) to

2  Immunotherapy for Pediatric Solid Tumors

the tumor was detected in all five patients. There was no significant toxicity and one of the neuroblastoma patients had stable disease for 27 months and even more impressive an Ewing sarcoma patient had a complete response for 77 months [153]. Active IL-12-secreting type 1 DC vaccine was completed in pediatric and adolescent patients with a variety of refractory or metastatic solid tumors in a phase I study. Fourteen patients received subcutaneous and eight intranodal vaccine injections. The majority of patients did have a positive DTH test. No serious toxicities occurred and all patients given the vaccine intranodally were alive at the end of the trial as opposed to about half of the subcutaneously treated patients. However, the follow-up period was short (2–13 months). The majority of patients did not have measurable tumor responses except for one patient with adrenocortical carcinoma who did achieve a partial response of lung metastases. The remaining patients demonstrated stabilization of disease [154]. Another DC vaccine trial in refractory pediatric solid tumors once again showed no serious adverse events and DTH response was found in seven of the ten patients that completed the immunization series. Regression of multiple metastatic sites was found in one patient and five patients had stable disease with a 16–30-month follow-up [155]. A phase I vaccine study in 11 neuroblastoma patients was conducted using DCs pulsed with tumor RNA after standard chemotherapy, surgery, radiation, and high-dose chemotherapy with stem cell rescue. Of the three patients evaluated for tumor-specific response, two demonstrated a response. One of these patients remained alive with stable disease 14 months after diagnosis [156]. Cancer testis antigens (CTAs) are a group of antigens expressed on many tumor types including pediatric sarcomas and neuroblastoma. CTAs comprise 70 families with over 140 antigens [87, 157]. Their biologic function is not fully understood, but because of their immunogenicity, they are being studied as T-cell targets for vaccine and adoptive cellular therapy. However not all antigens are immunogenic in all patients and expression of

65

the antigens may vary between patients. A recently completed phase I study evaluated the use of decitabine with an autologous dendritic cell CTAspecific (MAGE-A1, MAGE-A3, NY-ESO-1) vaccine for patients with refractory/recurrent sarcomas and neuroblastoma (NCT01241162). Decitabine is given initially as it is a hypomethylating agent that has been shown to upregulate CTA.  The group has published the case of their first vaccine recipient, a boy with refractory bone marrow neuroblastoma. He went into remission after three cycles of the vaccine and had no evidence of disease at the time of publication, 1 year postvaccination [151, 158]. Ten total patients received a median of 2.5 cycles. Therapy was generally well tolerated; five of ten patients had myelosuppression from decitabine. One patient had a possible hypersensitivity reaction to the vaccine requiring discontinuation. Four patients had objective responses, two of which were durable. Six of nine evaluable patients mounted a T-cell response to the vaccine. The two patients with durable responses were the only patients in which both CD4 and CD8 antibody responses were demonstrated. The authors note that while decitabine chemotherapy upregulates target antigen expression, it also causes myelosuppression; co-administration of GM-CSF to enhance immune response may improve the efficacy in future studies [159]. DNA or RNA vaccines use the patient’s own cells to transcribe and translate nucleic acids into proteins for processing, which avoids HLA haplotype restriction [160]. An active phase I/II tumor vaccine clinical trial uses autologous dendritic cells loaded ex  vivo with individualized tumor-specific mRNA in patients with recurrent medulloblastoma or primitive neuroectodermal tumor (NCT01326104). The presence of myeloid-derived suppressor cells in tumor tissue is negatively associated with immunotherapy response, likely due to their inhibitory effect on T cells. Suppression of MDSC is therefore expected to enhance the efficacy of immunotherapeutic agents [161]. An active clinical trial uses gemcitabine for MDSC suppression in combination with autologous dendritic cell vaccine in refractory sarcoma patients (NCT01803152).

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Combination immunotherapy may lead to synergistic antitumor effects. The use of anti­PD1/PD-L1 therapy is expected to enhance and prolong T-cell response to tumor vaccines through inhibition of regulatory T cells [162]. An ongoing clinical trial for patients with high-risk, NY-ESO-expressing tumors utilizes conditioning chemotherapy followed by infusion of NY-ESO-­ 1-transduced T cells, then anti-PD-1 immunotherapy, and finally peptide-pulsed dendritic cell vaccine (NCT02775292).

2.3.3.3 Genetically Modified Tumor Vaccines Rousseau et al. initially conducted a phase I study in relapsed advanced neuroblastoma patients using an allogeneic neuroblastoma tumor cell vaccine combining lymphotactin with IL-2. Lymphotactin encourages lymphocyte chemotaxis and works synergistically with IL-2. The only adverse event was reversible panniculitis and bone pain. Measurable responses were detected in the majority of patients as well as complete remission in two and partial response in one [163]. They followed this with another phase I trial in seven patients with recurrent neuroblastoma utilizing a tumor vaccine consisting of autologous instead of allogeneic neuroblastoma cells that were genetically modified to secrete IL-2 and lymphotactin with minimal toxicity. Tumor-specific immune responses were measurable in five of six patients, and two of the seven patients had stable disease throughout the study [164]. This group of investigators then went on to complete a phase I/II study in high-risk neuroblastoma utilizing autologous neuroblastoma cells genetically modified to secrete IL-2. Thirteen patients with limited tumor burden were enrolled consisting of those who had achieved a complete response, very good partial response, or partial response to their initial therapy. There were no serious toxicities and median event-free survival was 22 months for patients in first remission with four patients alive and three without disease recurrence [165]. This study is being built upon with combining metronomic cyclophosphamide and adding additional tumor-associated antigens (NCT01192555) [138].

L. Nicholls and L. M. Kopp

Vigil (formerly known as FANG) is a tumor peptide vaccine created from autologous tumor cells transfected with the rhGMCSF transgene and the RNAi bi-shRNAfurin. Thirty patients with relapsed or progressive Ewing sarcoma underwent surgical harvest of tumor cells and vaccine was created. For 16 patients, vaccine construction was successful and they received Vigil; 14 patients did not receive Vigil and were used as the comparison group (6 due to vaccine contamination, 3 had insufficient viable tumor cells, 5 chose other treatment). The vaccine was well tolerated; all toxicity was grade 1 and consisted mostly of injection-site reactions. Patients that received Vigil had a 17.2-month improvement in overall survival compared to those that did not. One-year overall survival was 73% in patients that received Vigil and 23% in the comparison group. One patient had a partial response, but ultimately progressed. Six patients had stable disease for at least 3 months [166]. There is an ongoing phase 2 study of Vigil compared to systemic chemotherapy in patients with metastatic Ewing sarcoma that have failed at least two prior regimens (NCT02511132).

2.3.3.4 Other Adoptive Cell Therapies Autologous or allogeneic cytotoxic T cells and natural killer (NK) cells can be utilized against tumors. An ongoing phase I clinical trial infuses autologous ex  vivo-expanded NK cells directly into the fourth ventricle via Ommaya reservoir in patients with recurrent posterior fossa tumors (NCT02271711) [160]. NK cells are an attractive tool to both optimize disease control and minimize toxicity following allogenic bone marrow transplant. NK cells engraft relatively early, and they both stimulate and regulate the immune system. Future studies aim to capitalize on NK cells’ ability to enhance immune reconstitution and graft-versus-tumor effect, and also potentially mediate graft-versus-­ host disease via their immunoregulatory capabilities [167]. An ongoing phase 2 study of haploidentical stem cell transplant for patients of any age with high-risk Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, osteosarcoma, or high-risk CNS tumors adds infusion of donor NK

2  Immunotherapy for Pediatric Solid Tumors Table 2.4  Current targeted immunotherapy clinical trials for pediatric solid tumors: natural killer cell-based therapy Study title Fourth ventricle infusions of autologous ex vivo-expanded NK cells in children with recurrent posterior fossa tumors Phase II STIR trial: haploidentical transplant and donor natural killer cells for solid tumors (STIR)

Phase NCT no. I NCT02271711

incorporate targeted immunotherapeutic strategies with more conventional regimens for a greater therapeutic impact in pediatric solid tumors.

References II

NCT02100891

cells on day 7 to enhance graft-versus-tumor effect (NCT02100891). Table  2.4 contains the current open clinical trials utilizing NK cell therapies.

2.4

67

Concluding Remarks

Pediatric cancer comprises only 1% of newly diagnosed cancers in the United States and approximately 40% of those cancers comprise the solid tumors [1]. Pediatric solid tumors that are metastatic or recurrent have a very poor survival, which has not significantly changed over the past 50 years. Despite the poor prognosis of these pediatric solid tumors, the numerous pediatric clinical translation research reviewed herein is evidence of the optimism that immunotherapy will have a distinct role in the treatment of pediatric solid tumors. One of the primary reasons for the plethora of pediatric phase I and II trials for this small population of patients is the acknowledgment of the value of these clinical trials by pediatric oncologists. The collaborative efforts of the Children’s Oncology Group as well as European Pediatric Groups have been the driving force behind many of these trials. The undeniable success of the anti-GD2 antibody dinutuximab in metastatic neuroblastoma is due to the ability of COG to organize meaningful large-scale immunotherapy clinical trials [19]. This confirmed that immunotherapy not only is feasible but can also improve the overall survival and quality of life of pediatric patients with solid tumors that carry a poor prognosis. Future clinical collaborative clinical trials in other solid tumors will continue to

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73 164. Russell HV, Strother D, Mei Z, Rill D, Popek E, Biagi E, et al. Phase I trial of vaccination with autologous neuroblastoma tumor cells genetically modified to secrete IL-2 and lymphotactin. J Immunother. 2007;30(2):227–33. 165. Russell HV, Strother D, Mei Z, Rill D, Popek E, Biagi E, et  al. A phase 1/2 study of autologous neuroblastoma tumor cells genetically modified to secrete IL-2  in patients with high-risk neuroblastoma. J Immunother. 2008;31(9):812–9. 166. Ghisoli M, Barve M, Mennel R, Lenarsky C, Horvath S, Wallraven G, et  al. Three-year follow up of GMCSF/bi-shRNA(furin) DNA-transfected autologous tumor immunotherapy (vigil) in metastatic advanced Ewing’s sarcoma. Mol Ther. 2016;24(8):1478–83. 167. Palmer JM, Rajasekaran K, Thakar MS, Malarkannan S. Clinical relevance of natural killer cells following hematopoietic stem cell transplantation. J Cancer. 2013;4(1):25–35.

3

Immunotherapeutic Strategies for Multiple Myeloma Jessica J. Liegel and David E. Avigan

Contents 3.1

Introduction

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3.2

I mmune Therapy for Myeloma: Overcoming TumorAssociated Immune Suppression

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3.3 Antibody-Mediated Strategies 3.3.1    CS1 3.3.2    CD38 3.3.3    PD-1/PD-L1 3.3.4    Antibody Conjugates and Bispecific Antibodies

 77  78  79  81  81

3.4 Cellular Immunotherapy for Multiple Myeloma 3.4.1    Allogeneic Transplantation 3.4.2    Myeloma Vaccines 3.4.2.1  Peptide-Based Myeloma Vaccines 3.4.2.2  Cell-Based Myeloma Vaccines 3.4.3    Adoptive Cell Therapy 3.4.3.1  Marrow-Infiltrating T Cells 3.4.3.2  NK Cell Therapy 3.4.4    Engineered T Cells 3.4.4.1  TCR T Cells 3.4.4.2  CAR T Cells

 83  83  84  85  86  88  89  89  90  90  90

3.5

Concluding Remarks

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References

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3.1

J. J. Liegel (*) · D. E. Avigan Department of Medicine, Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA e-mail: [email protected]

Introduction

Multiple myeloma (MM) is the second most common hematologic malignancy with greater than 30,000 cases estimated in 2018 [1]. It represents a progressive disorder mediated by malignant plasma cells that may clinically present with elevated monoclonal immunoglobulins or skewing of free light chains in the serum or urine, renal

© Springer Nature Switzerland AG 2020 N. Rezaei (ed.), Cancer Immunology, https://doi.org/10.1007/978-3-030-57949-4_3

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disease, osteolytic bone lesions, cytopenias due to marrow infiltration, and suppression of the normal B-cell repertoire leading to humoral immunodeficiency. Despite significant progress in the treatment of myeloma since alkylator-­based therapies, currently approved biologic agents such as immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), monoclonal antibodies, and histone deacetylase inhibitors are not curative. Immunotherapy is becoming increasingly incorporated into the armamentarium for cancer treatment and is being actively explored in myeloma. Current immunotherapy strategies in myeloma include the use of monoclonal antibodies, vaccines, and adoptive cell transfer which seek to immunologically target myeloma cells and effectively interact and amplify host tumor-specific immunity. One unique potential of cancer immunotherapy is the triggering of diverse immune mechanisms to overcome tumor heterogeneity and create antitumor memory that is crucial to prevent disease recurrence.

J. J. Liegel and D. E. Avigan

The microenvironment is characterized by increased presence of inhibitory cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) that directly down-­modulate T-cell activation, polarization of macrophages and dendritic cells towards a tolerizing phenotype, and presence of stromal cells that release inhibitory cytokines. Interactions between myeloma cells and stromal elements in the microenvironment lead to release of soluble factors such as adhesion molecules, growth factors, cytokines, and chemokines that collectively promote tumor survival and immune escape [2]. MDSCs are a subset of immature myeloid cells that fail to differentiate normally. Marrow samples of patients with myeloma have been shown to have increased MDSCs which can be directly induced by myeloma cells and serve to promote disease escape by inhibiting antigen-specific CD8(+) T-cell responses [3, 4]. MDSCs can also induce other tolerizing cells such as regulatory T cells. Increasing Tregs and associated anti-­inflammatory cytokines, a decreased 3.2 Immune Therapy Th1/Th2 ratio, and elevated peripheral blood Treg/Th17 ratio all have been correlated with for Myeloma: Overcoming myeloma progression and/or survival [5, 6]. Tumor-Associated Immune The tumor environment is also impacted by Suppression metabolic stress such as hypoxia that impairs Immune escape is a critical aspect of oncogene- T-cell function and also may provide steric barsis. Malignant transformation and disease evolu- riers for T-cell penetration and effector-to-tartion are associated with gradual loss of get interface. Hypoxia can also encourage immunogenicity of the tumor cell population via metastasis due to decreased E-cadherin and immune editing, presentation of antigen in the increased CXCR4 leading to migration [7]. absence of positive costimulation, and upregulaAntigen processing and presentation are tion of inhibitory ligands that promote immune altered in the tumor microenvironment. tolerance and T-cell exhaustion. Another critical Dendritic cells (DCs) express an immature phefactor in this process is the development of a notype with reduced maturation and expression tumor microenvironment that exerts an immuno- of costimulatory molecules such as CD80 as suppressive effect on T-cell activation and myeloma advances due to the presence of TGF-β immune recognition. The evolution of clinically and IL-10 [8]. Increase in the immunosuppresactive multiple myeloma is associated with pro- sive enzyme indoleamine 2,3-dioxygenase gressive immunologic dysfunction resulting in (IDO) in the DC population has also been shown increased risk of infection and loss of myeloma-­ to be associated with a tolerizing effect on specific immunity. The development of clinically potentially reactive T-cell populations [9]. efficacious immunotherapy requires a nuanced Plasmacytoid DCs can also directly foster understanding of these disease-specific effects on myeloma growth via cytokines such as vascular the immunologic milieu. endothelial growth factor (VEGF) and IL-3

3  Immunotherapeutic Strategies for Multiple Myeloma

which are secreted upon their interaction with myeloma cells [10]. T-cell derangements further blunt the myeloma-specific response, due to downregulation of T-cell activation and eventual T-cell exhaustion and senescence. The negative costimulatory molecule programmed death 1 (PD-1) normally supports prevention of autoreactivity to maintain an equilibrium of host immunity. Tumor cells can take advantage of this pathway as a means of preventing T-cell activation that blocks the killing of malignant cells by effector cells. The ligand PD-L1 has been shown to be aberrantly expressed in primary myeloma cells [11] and PD-1 expression on T cells and natural killer (NK) cells is upregulated [12, 13]. Other inhibitory factors such as CTLA-4, LAG-3, and TIM-3 are also expressed [14] while the positive costimulatory molecule CD28 is conversely reduced in T cells of myeloma patients [15]. Furthermore, phenotypes of T-cell exhaustion, CD8+CD28negPD-1+, and senescence, CD8+CD28negCD57+, are present in patients with relapsed disease and more pronounced at the site of disease in the marrow than in peripheral blood [16]. NK cells constitute a key cellular subset of the innate immune system with the potential to lyse target cells upon engagement of tumor ligands for NK-activating receptors or “missing self” recognition whereby lack of MHC class I incites cytotoxicity [17]. Myeloma cells elude surveillance as the disease evolves, and studies show NK cell alterations as MGUS progresses to MM [18] including shedding of the ligand for the activating receptor NK group 2 member D (NKG2D) [19] and upregulation of MHC class I [20]. Compared to healthy controls, patients with MM also have reduced expression of other activating receptors including DNAM-1 and NKp30 on NK cells [21]. Furthermore, the production of TGF-­ β1 and IL-10, expansion of tolerizing cells such as MDSCs in the myeloma microenvironment, and increased PD-1 expression as noted above can additionally suppress NK cell function.

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Overall, the microenvironment and immune milieu generated by myeloma promote tolerance and lose capacity to check the growth of the malignant plasma cell clone.

3.3

Antibody-Mediated Strategies

The efficacy of antibody therapy in cancer treatment is potentially dependent on several mechanisms including direct induction of cytotoxicity with signaling induced by binding to the target as well as activation of effector cell populations. In myeloma, use of monoclonal antibodies (mAbs) can trigger antibody-dependent cellmediated cytotoxicity (ADCC) by facilitating binding of tumor antigens to Fcγ receptors on NK cells, neutrophils, and macrophages. After activation of Fc receptors, cytotoxicity is mediated through at least two different mechanisms: one involving the release of perforin and granzyme from effector cells, and the other involving death ligands Fas ligand and TRAIL [22]. Alternatively, cell lysis may be accomplished by the antibody-­mediated activation of the classical complement cascade at the tumor site (CDC). Further tumor killing may occur as DCs can engulf the resultant apoptotic cells, and subsequently present tumor antigens on MHC class I and II molecules, to activate secondary CD8+ cytotoxic T lymphocytes (CTLs). In addition, cross-presentation can be mediated by phagocytosis of dying antibody-­ coated tumor cells through Fcγ receptors [23]. As such, efficacy of antibody therapy may be impacted by the underlying immune competence of the patient. Antibody-based therapy has been pursued in an effort to selectively target myeloma cells while minimizing toxicity to normal tissues. Antibody therapies have focused on cell surface markers expressed by plasma cells such as CD38, CD138, B-cell maturation antigen (BCMA), and tumor adhesion molecule CS1. Summary of published outcomes for monoclonal antibody therapies is shown in Table 3.1.

J. J. Liegel and D. E. Avigan

78 Table 3.1  Monoclonal antibodies in myeloma Monoclonal antibodies Elotuzumab

Isatuximab

CD38

Phase I/II Phase Ib Phase Ib

MOR202

CD38

Phase I/IIa Phase I/IIa Phase I/IIa

Clinical outcomes Monotherapy: No objective responses With Rd: ORR: 79%, PFS 18.5 months With Vd: ORR 66%, PFS 9.7 months With Pd: ORR 53%, PFS 10.3 months Monotherapy: ORR 35%, median PFS 3.7 months With Rd: ORR 93%, 12 months PFS 83% With Pd: ORR 60%, median PFS 8.8 months With Vd: ORR 83%, 12 months PFS 61% With Rd: ORR 93%, 30 months PFS 71% With VMP: ORR 91%, median PFS 36 months, 3-year OS 78% With VTd/ASCT: ORR 93%, 18 months PFS 93% With RVd/ASCT: ORR 99%, 24 months PFS 96% Monotherapy: ORR 24% (>10 mg/kg), median PFS 3.7 months With Rd: ORR 56%, median PFS 8.5 months With Pd: ORR 65% Monotherapy: ORR 29% With Rd: 5 of 7 patients with PR With Pd: 2 of 5 patients with CR

SLAMF7 CD352

Phase 1 Phase I Phase I/IIa Phase I/IIa Phase I/IIa Phase I Phase 1

Monotherapy: ORR 60%, PFS 7.9 months NCT02561962 Monotherapy: ORR 15%, median PFS 3 months With Rd: ORR 77%, median PFS 16.4 months With Pd: ORR 79% NCT02462525 NCT02954796

CD19-CD3 BCMA-CD3 FCRH5-CD3 BCMA-CD3 BCMA-CD3 BCMA-CD3 BCMA-CD3

Phase I Phase I Phase I Phase I Phase I Phase I Phase I

NCT03173430 Monotherapy: ORR 70%, CR 50% NCT03275103 NCT03287908 NCT03269136 NCT03145181 Monotherapy: ORR 83%, 33% sCR

Daratumumab

Target SLAMF7

Trial Phase I Phase III Phase II

CD38 RRMM

Phase II Phase III Phase Ib Phase III Phase III Phase III Phase III Phase II

CD38 Up front

Antibody-drug conjugates GSK2857916 BCMA AMG 224 BCMA BT062 CD138 (indatuximab) ABBV-838 SGN-CD352A Bispecific antibodies Blinatumomab AMG420 BFCR4350A AMG 701 PF-06863135 JNJ-64007957 CC-93269

ORR = CR + VGPR + PR SD stable disease, PR partial response, MR minimal response, CR complete response, sCR stringent complete response, PFS progression-free survival

3.3.1 CS1 CS1 is a cell surface glycoprotein (CD2 subset 1, CRACC, SLAMF7, CD319, or 19A24) member of the immunoglobulin gene superfamily that supports bone marrow stroma adhesion. It is highly expressed in CD138-purified primary tumor cells from the majority of MM patients. Elotuzumab is a humanized anti-CS1 mAb which binds with high affinity to MM cells, inhibiting

their adhesion to bone marrow stromal cells, and triggering ADCC especially after pretreatment with lenalidomide in preclinical studies [24]. CS1 is also expressed at a low level in NK cells in which it serves an activating role. Thus elotuzumab facilitates a CS1-CS1 interaction between effector NK cells and CS1(+) target cells to enhance NK cell ADCC against MM [25]. Elotuzumab demonstrated minimal single-­ agent activity in phase I clinical trials of patients

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with relapsed/refractory myeloma (RRMM) [26] but combinations of elotuzumab with PIs or IMiDs have achieved responses. The phase III ELOQUENT-2 trial of elotuzumab, lenalidomide, and dexamethasone (ELd) vs. Ld reported ORR of 79% vs. 66% and median PFS of 18 vs. 14 months after 3-year extended follow-up [27]. Most common adverse events were infusion reactions in up to 10%; the majority were grade 1 or 2. This study formed the basis for FDA approval in 2015 of ELd in MM after 1–3 prior therapies. A randomized phase II study of elotuzumab with bortezomib and dexamethasone (EBd) achieved a marginal response over Bd alone (ORR 66% vs. 63%) and median progression-free survival (PFS) benefit was 10 versus 7 months [28]. While PFS duration appears longer with ELd compared to EBd, the absolute median PFS benefit of adding elotuzumab is short with either. A subsequent trial combining elotuzumab with pomalidomide (EPd) yielded a slightly higher benefit with median PFS 10 months compared to 5 months for pomalidomide alone [29].

3.3.2 CD38 Malignant plasma cells strongly express CD38, a glycoprotein that functions in cell adhesion, signal transduction, and calcium signaling. In addition to FcγR-mediated ADCC or phagocytosis, cross-linking of tumor-bound CD38 mAb can induce direct programmed cell death. Furthermore, CD38 mAbs can assist in depleting CD38+ Tregs, regulatory B cells, and MDSCs potentially improving the host immune response [30]. Daratumumab is a fully human anti-CD38 mAb that demonstrated both ADCC and CDC against myeloma in preclinical studies [31] and significant enhancement in combination with lenalidomide [32]. Single agent daratumumab in patients with heavily pretreated RRMM yielded moderate ORR of 35% but was not durable [33]. Subsequent trials of daratumumab were undertaken in combination with PIs or IMiDs that demonstrated markedly enhanced responses with a subset of patients showing no evidence of minimal residual disease (MRD) leading to FDA

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approvals for RRMM.  The phase III CASTOR trial of daratumumab with bortezomib and dexamethasone (DVd) yielded ORR 83% vs. 63%, with MRD negativity of 10% vs. 2% in patients treated with DVd as compared to Vd, respectively. 12-month PFS was 61% vs. 27% amongst patients for whom 66% were refractory to bortezomib [34]. The phase III POLLUX trial of daratumumab with lenalidomide and dexamethasone (DRd) reported an ORR of 93% vs. 76% and 12-month PFS 83% vs. 61%, [35] for DRd and Rd, respectively, although in comparison to the CASTOR trial only 18% of patients had previously received lenalidomide. Notably 22.4% of patients receiving DRd achieved the threshold below MRD. Use of daratumumab with pomalidomide and dexamethasone (DPd) in the APOLLO trial achieved ORR of 60% and median PFS of 8.8 months in patients among whom 71% were double refractory to PI and IMiDs [36]. Use of carfilzomib with daratumumab and dexamethasone (DKd) in RRMM is being evaluated in the CANDOR trial in which the majority of patients were lenalidomide refractory. A phase I study reported ORR of 84% with 12-month PFS of 74% and lower rates of neutropenia than combinations with IMiDs [37] and a phase III trial of DKd versus Kd is underway. Based on these results several options are now available for use of daratumumab and incorporation into quadruplet therapies in RRMM is also being explored. Several trials in the up-front setting have sought to define the role of daratumumab for newly diagnosed MM. In patients not eligible for autologous stem cell transplantation (ASCT) the phase III MAIA study led to FDA approval of DRd as frontline therapy in 2019 [38]. DRd achieved superior CR and MRD negativity as compared to Rd, with 30-month PFS of 71% versus 56%; however in subgroup analysis the PFS benefit was less in those with high-risk cytogenetics. Notably, patients in the daratu­ mumab group had more lenalidomide discontinuations due to neutropenia and infections but were treated longer while grade 3 or 4 infections were similar in both groups. Frontline use of daratumumab along with bortezomib, melphalan, and prednisone (D-VMP) was also evaluated in the

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phase III ALCYONE trial [39] in patients ineligible for ASCT. ORR (91%) and MRD negativity (22%) for D-VMP was superior to VMP and appears comparable to DRd. The most recent analysis suggests that addition of daratumumab translates into improved duration of response with a median PFS of 36 months for D-VMP versus 19 months for VMP as well as OS benefit [40]. Another phase III trial (CEPHEUS) is evaluating the efficacy of up-front daratumumab with RVd in non-­transplant candidates and results are eagerly awaited. Incorporation of up-front daratumumab is also underway in patients eligible for ASCT.  The phase III CASSIOPEIA trial of bortezomib, thalidomide, and dexamethasone (VTd, standard induction in Europe) evaluated daratumumab for four pre-ASCT induction and two post-ASCT consolidation cycles. At day 100 post-ASCT, higher sCR (29% versus 20%) and higher MRD negativity (64% versus 44%) were achieved with Dara-VTd than VTd [41]. PFS at 18 months was significantly higher in the daratumumab group (93% versus 85%) but unlike the MAIA trial, the benefit was observed in patients with high-risk cytogenetics. In the USA the randomized phase II GRIFFIN trial evaluated standard RVd induction and lenalidomide maintenance with or without daratumumab given before and after transplant. Improved sCR at the end of consolidation was observed in the daratumumab group (42% versus 32%), but not within the high-risk cytogenetic subset. Of those patients who achieved CR or better after consolidation, MRD-­ negative rates were 59% for Dara-RVd versus 24% for RVd [42]. In both CASSIOPEIA and GRIFFIN trials the addition of daratumumab lowered median stem cell yield but did not affect the percent of patients able to proceed to transplant and grade 3 or 4 infections were similar in both groups. Longer term data from these randomized studies will be needed to determine whether MRD negativity is linked to improved duration of response as seen in the ALCYONE trial and a phase III trial of frontline Dara-RVD with ASCT is underway (PERSEUS). Of note, MRD status has been linked to duration of response in

J. J. Liegel and D. E. Avigan

many prior myeloma trials. Very high rates of MRD negativity more than 80% were recently reported from an initial trial (MASTER) incorporating daratumumab with carfilzomib, lenalidomide, and dexamethasone (KRd) both as induction before ASCT and in some patients as consolidation afterwards as determined by MRD status [43]. A similar phase II trial of first-line daratumumab with KRd using weekly carfilzomib dosing that included a substantial number of high-risk patients also reported high rates with 77% MRD negativity after eight cycles but prior to ASCT [44]. This has led to a multicenter randomized trial (ADVANCE) comparing up-front daratumumab plus KRd with standard of care. Other anti-CD38 mAbs tested in humans include the fully human MOR202 and chimeric isatuximab (SAR650984). As a single agent, MOR202 achieved partial response (PR) or better in 29% of patients treated with a median of four prior lines of therapy. Responses were heightened in combination with lenalidomide and pomalidomide in early trials [45]. Isatuximab monotherapy in heavily pretreated RRMM patients with a median of five prior therapies yielded ORR of 24% with median PFS of 3.7 months, quite similar to daratumumab [46]. Isatuximab in combination with lenalidomide and dexamethasone in preliminary phase Ib results reported ORR of 62% [47]. Phase III ICARIA study is ongoing (NCT02990338) to evaluate this combination in a randomized trial. For a summary of outcomes of mAbs in MM, see Table 3.1. Durable responses to CD38 antibody therapy may be due in part to the impact of therapy on secondary activation and expansion of tumor-­ specific adaptive immune responses as manifested by clonal expansion of T cells observed in some patients treated with CD38 antibody therapy. A larger increase in T-cell receptor (TCR)β clonality was observed in patients treated with DRd compared to Rd suggesting clonal expansion [48]. And amongst responders to isatuximab, polyclonal T-cell and IgG responses against CD38 and other myeloma antigens were noted suggesting antigen spreading [49].

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Adverse effects of CD38 antibodies are most commonly infusion-related reactions that can occur in nearly half of patients and can be reduced with the leukotriene antagonist montelukast and acutely managed with temporary cessation of the infusion and/or additional antihistamines or corticosteroids. Neutropenia is the most common grade 3 or 4 adverse effect likely due to effects on normal CD38+ myeloid cells. CD38 antibodies interfere with red blood cell antibody screen causing a pan-agglutination. If transfusion is required, blood products can be obtained by more extensive erythrocyte antigen phenotyping if done prior to CD38 therapy or via genotyping. Myeloma monitoring is also affected as CD38 antibodies are detected as a monoclonal IgG-­ kappa on serum protein electrophoresis that can co-migrate with the patient’s M-protein and mask response. In this case daratumumab-specific IFE reflex assay can be used to distinguish the two.

inferred senescence rather than exhaustion [54]. As an avenue to restore T-cell function, use of IMiDs which have a directly stimulating effect on CTLs has been explored in combination with PD-1 inhibition. In vitro, addition of lenalidomide further enhanced checkpoint blockademediated myeloma cytotoxicity and IFNγ production by effector cells [55]. Phase I and II trials of this therapeutic combination showed promise. In a phase I study of pembrolizumab, lenalidomide, and dexamethasone (KEYNOTE-023) in which 51% of patients were double refractory to an IMiD and PI, ORR was 50% and immune-related adverse events occurred in 10% [56]. A phase II study of pomalidomide and pembrolizumab in RR MM patients, of whom 73% were refractory to both an IMiD and PI, achieved ORR of 60% with a median duration of response of 14.7 months and median PFS of 17.4 months [57]. Autoimmune events included pneumonitis in 13%, hypothyroidism in 10%, hepatitis in 2%, and adrenal insufficiency in 2%. Only one autoimmune event was grade 3 and all resolved with interruption of therapy and methylprednisolone. However additional safety concerns arose in phase III trials of pembrolizumab (KEYNOTE-185 with lenalidomide and KEYNOTE-183 with pomalidomide) [58, 59]. Both trials were halted after interim results showed an increased risk of death for the pembrolizumab arms compared to control arms. Use of checkpoint inhibitors remains an area of interest in myeloma, with clinical trials evaluating PD1 blockade combined with other modalities that evoke a potent T-cell response, such as vaccines and adoptive cell therapies. Furthermore, other checkpoints are being explored in RRMM such as anti-LAG3, anti-OX40, and anti-TIGIT.

3.3.3 PD-1/PD-L1 The PD-1/PD-L1 pathway provides a critical inhibitory signal that disrupts immune activation and promotes tolerance. Rationale for blockade targeted against PD-1 or its ligands in myeloma was supported by observations of increased expression of PD-1 and its ligands in the tumor microenvironment thought to contribute to immune evasion by myeloma cells [50]. In preclinical studies, myeloma cell growth was suppressed with the use of anti-PDL2 Ab and completely inhibited in a PD-1-deficient mouse model [51]. Higher serum PD-L1 levels have also been associated with reduced ORR and shorter PFS during up-front treatment [52]. However an initial trial of the anti-PD-1 mAb nivolumab in hematologic malignancies did not achieve objective responses in the myeloma cohort [53]. More recent studies evaluating cytotoxic T-cell clones as opposed to global T cells provide evidence to explain a suboptimal response to checkpoint blockade alone. T-cell clones from MM patients exhibited low expression of PD-1 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and a phenotype that

3.3.4 Antibody Conjugates and Bispecific Antibodies Antibody-drug conjugates (ADC) link mAbs to cytotoxic agents, typically antitubulin, to be released upon target antigen binding. They thus rely less on host effector cells for tumor killing. In myeloma, such agents have been developed

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using CD138 or BCMA targets. CD138 is a proteoglycan involved in cell adhesion expressed on plasma cells and commonly overexpressed on MM cells. Indatuximab ravtansine is an ADC which links CD138 (syndecan 1) to the maytansinoid drug DM4. Monotherapy with indatuximab yielded poor objective response [60]. In combination with lenalidomide, ORR was 77% with a median duration of 12 months, and median PFS of 16.4 months. Amongst patients previously exposed to both lenalidomide and bortezomib, ORR was 54% and increased to 79% when indatuximab was combined with pomalidomide although duration of response in prior IMiD-­ exposed patients was not reported [61]. BCMA is a cell surface protein in the tumor necrosis factor receptor superfamily involved in B-cell maturation. BCMA is upregulated during the late stages of normal B-cell differentiation, and thus expressed on both normal and malignant plasma cells but not on naïve or memory B cells making it an enticing antigen target in MM [62]. Belantamab mafadotin (GSK2857916) is an ADC which employs a humanized IgG1 antibody targeting BCMA conjugated to the microtubule-­ disrupting agent monomethyl auristatin-F. ADCC is also enhanced due to afucosylation of the Fc domain that increases affinity to FCγRIIIa expressed on effector cells. Recently updated data from a phase I trial of GSK2857916 has demonstrated ORR of 60% with a median PFS of 12 months and for patients refractory to IMiDs, PIs, and daratumumab median PFS was 6 months [63]. These are impressive results for monotherapy in heavily pretreated patients leading to FDA breakthrough therapy designation in late 2017. Adverse events were notable for infusion reactions and corneal events such as blurred vision, dry eye, or photophobia most commonly grade 1 or 2. Two other BCMA-targeting ADCs have demonstrated preclinical efficacy in MM. HDP-1 has an amanitin-based payload that binds to RNA pol II to inhibit transcription and thus may be more effective in lower proliferation diseases [64]. MEDI2228 is armed with a DNA-cross-­ linking pyrrolobenzodiazepine dimer tesirine and has demonstrated superior in vitro activity than monomethyl auristatin-F [65].

J. J. Liegel and D. E. Avigan

Bispecific antibodies (bsAbs) are recombinant antibodies with dual specificity for two epitopes in the form of single-chain variable fragments (scFv) binding to both a tumor surface antigen and CD3. In cancer immunotherapy, employment of such bispecific T-cell engagers (BiTE antibodies) approximates malignant cells to effector cells whose activation triggers T-cell-dependent cellular cytotoxicity [66]. This process is independent of TCR specificity or MHC restriction. Blinatumomab is a CD3-CD19 bsAb that has proven efficacious in B-cell acute lymphoblastic leukemia. CD19 expression has been reported on MM stem cells [67] and this has fueled efforts to investigate CD19 as a target in myeloma. A phase I trial of blinatumomab after salvage autologous stem cell transplant (ASCT) is underway (NCT03173430). As with ADCs, more promising BiTE antibodies in myeloma are targeting BCMA. AMG420 is a very-short-half-life CD3BCMA bsAb given as a continuous infusion. In a phase I trial in RRMM with median four prior lines, AMG420 demonstrated ORR of 70% at the recommended dose, including 50% MRDnegative CR with some responses lasting more than 1 year [68]. Of note, two patients died from infection-related complications, aspergillus, and hepatitis, but were not deemed to be related to therapy. CC-93269 is a T-cell engager with an asymmetric two-arm humanized IgG that binds bivalently to BCMA and monovalently to CD3ℇ in a 2+1 format [69]. In a phase I trial in RRMM with majority of patients triple refractory to IMiD, PI, and anti-CD38 agents, high response rates were shown with ORR 83%, including 90% MRD negativity and 33% sCR; however durability is yet unknown [70]. BFCR4350A is an antiFcRH5-­CD3 bsAb antibody directed against the tumor-­associated antigen Fc receptor-like protein 5. FcRH5 is an immune receptor translocation-­associated protein in the Fc receptor homolog family and a B-cell lineage marker overexpressed on myeloma cells. In contrast to BCMA, it is also expressed on naïve and memory B cells. Preclinical studies of BFCR4350A further elucidated the mechanism of T-cell engagers. Binding of tumor antigen led to its

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clustering in the cell-­cell interaction site, excluding the inhibitory molecule CD45 from the synapse fostering activation of TCR signaling [71]. BFCR4350A potently killed primary myeloma cells and led to tumor regression in animal models. The inhibitory tumor microenvironment remains a challenge for CD3 bsAbs as optimal T-cell activation and proliferation require not only antigen-specific CD3 ligation but also proper costimulation and cytokine production. Activity can be inhibited by the suppressive accessory cells in the tumor microenvironment such as Tregs. Cell surface antigen-loss variants can also occur, all leading to immune evasion. AMG701 is in development as an extended half-­ life designed for once-weekly dosing which has shown enhanced cytotoxicity when combined with IMiDs in the presence of immunosuppressive osteoclasts or bone marrow stromal cells in preclinical studies [72]. The most notable side effects of CD3-­ activating bsAbs include cytokine release syndrome (CRS) and neurotoxicity, especially in patients with high tumor burden [73]. CRS arises in the setting of rapid immune cell activation associated with high levels of cytokine production including high IL-6 and IFN-γ. Clinical manifestations can range from flulike symptoms such as persistent high fever, rigors, or myalgia in mild cases to more severe sepsis-like picture including hypotension, hypoxia, and multi-organ dysfunction and can be fatal. Neurotoxicity can also occur including tremors, seizures, and/or encephalopathy. Prophylactic dexamethasone and incremental dosing of bsAbs can prevent CRS or neurotoxicity. The IL-6 inhibitor tocilizumab has been used successfully if CRS develops and additional dexamethasone has been effective for neurological symptoms. Severe CRS has occurred in studies with BiTE antibodies in myeloma despite dexamethasone prophylaxis at which time treatment is administered with tocilizumab and/or additional dexamethasone once grade 2 or above. Of 42 patients treated with AMG420, grade 2 to 3 CRS developed in three patients. Of 19 patients treated with CD-93269, grade 2 CRS developed in 5

patients and 1 patient had a grade 5 event with suspected concomitant infection. Table 3.1 lists additional novel CD3 bsAbs currently in phase I trials.

3.4

Cellular Immunotherapy for Multiple Myeloma

3.4.1 Allogeneic Transplantation The unique potential efficacy of cellular immunotherapy for myeloma is highlighted by the observation that allogeneic transplantation induces durable remissions in a subset of patients due to the graft-versus-myeloma effect mediated by alloreactive lymphocytes [74, 75]. A retrospective comparison of autologous (ASCT) versus myeloablative allogeneic transplantation (allo HSCT) revealed improved event-free and overall survival at 10 years for allo HSCT [76]. However, allogeneic transplantation is associated with higher treatment-related morbidity and mortality due to regimen-related toxicity, infection during the period of immune reconstitution, and graft-versus-host disease due to the lack of specificity of the alloreactive lymphocytes. Efforts to limit toxicity through the use of reduced intensity-­ conditioning regimens have resulted in a decrease in treatment-related mortality but a concomitant increase in the risk of relapse. Subsequently the use of ASCT for cytoreduction prior to a reduced-­ intensity allo HSCT was evaluated. A total of six prospective trials have compared sequential ASCT with ASCT followed by reduced-intensity allo HSCT and have shown conflicting results [77]. A meta-analysis of these trials showed an expected increase in treatment-associated mortality in patients undergoing allo HSCT which was offset by higher CR rate but only a nonsignificant trend towards improved overall survival (OS) at 3 years compared to tandem ASCT [78]. Thus, although potentially curative, allo HSCT is considered only for patients with early relapse less than 24 months after primary therapy that included ASCT and/or high-risk features (cytogenetics, extramedullary disease, plasma cell leukemia) [79].

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The ability of donor lymphocyte infusions (DLI) to induce remission in the setting of relapse after allo HSCT provides clear evidence of an effective graft-versus-myeloma effect [80]. Prophylactic DLI following allo HSCT has been recently shown to improve molecular remission and PFS [81]. The anti-myeloma effect is presumably mediated primarily by donor T cells and DLI has been shown to reconstitute the TCR repertoire [82]. DLI is associated with a higher risk of GVHD which has previously been one of the only predictive factors linking allogeneic T cells to anti-myeloma effect [83]. However studies have now demonstrated the presence of myeloma-­ specific CTL responses and antibodies specific for myeloma-associated antigens. High-titer antibodies against BCMA [84] as well as CTLs specific for WT1 antigen [85] were associated with response after DLI, with the latter in the absence of GVHD.  Such observations have led to an ongoing phase 1 trial of WT1-primed donor T cells following T-cell-depleted allogeneic HSCT in RRMM or plasma cell leukemia (NCT01758328). Early results reported attainment of CR lasting more than 2 years post-WT1 infusions in two of four patients with persistent disease after transplant [86]. However, even in the highest risk patients, there is now hope that the risk of allo HSCT can be avoided and supplanted by an autologous graft-versus-myeloma effect of cellular based immune therapies which have the advantage of selectively targeting malignant cells with significantly less toxicity.

3.4.2 Myeloma Vaccines Investigators have explored the development of cancer vaccine therapy in an effort to induce the expansion of cytotoxic T cells that selectively target tumor-specific antigens while minimizing toxicity due to off-target effects. Vaccine efficacy is dependent on the identification of antigens that demonstrate relative selectivity as unique to the malignant clone, aberrantly expressed on the malignant cells or restricted to early development and not found in normal adult tissue. In myeloma, several antigens have been

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explored. The idiotype protein is unique to the malignant plasma cell, the M protein reflective of genetic rearrangement of the immunoglobulin gene during plasma cell differentiation [87]. Cancer-testis antigens are normally expressed in germ cells during early development, involved in epigenetic regulation of transcription, and are expressed in several malignancies including myeloma, especially in advanced disease [88]. MAGE-1/A1/A2/A3/C1, LAGE-1, PASD1, and NY-ESO amongst others have induced in  vivo humoral and cellular myeloma immunity leading to therapeutic use [89–91]. Aberrantly expressed proteins associated with increased oncogene expression include WT1, MUC1, survivin, RHAMM, and DKK1. Antigens selective to malignant or normal plasma cell differentiation include CS1, CD138, XBP1, and BCMA. There has been an increasing interest in the identification of neoantigens that arise from mutational events associated with malignant transformation. These antigenic targets are potentially more immunogenic as they are foreign to the host and are recognized by high-­affinity T cells that are not deleted as a part of thymic mediated tolerance towards self-antigens. Investigators are exploring the use of neoantigens as peptide vaccines with ongoing work in neoantigen identification evaluation for recognition by the host repertoire. Vaccine efficacy is also dependent on the effective presentation of antigen in the context of immunostimulatory signals that favor immune activation rather than tolerance. APCs can present antigen upon direct infection by pathogens such as viruses; however the primary mode of generating effector CD8+ T-cell response to cancer is via cross-presentation [92]. Exogenous antigen is detected, internally processed, and presented on MHC class I by APCs to naive T cells. For a robust cytotoxic response, both binding of the antigen-MHC complex by the TCR/CD3 complex and costimulation by APCs are ­necessary [93] DCs are the most potent and efficient APCs, capable of presenting tumor-derived antigen and inducing cancer-specific immunity [94]. Cross-priming by DCs is essential for antitumor immune response in  vivo, as shown by

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Table 3.2  Vaccines in myeloma Vaccine APC8020, DC pulsed with idiotype protein PVX-410 Ongoing phase II

Clinical setting After autologous HSCT

Myeloma GVAX Ongoing phase II

nCR after lenalidomide-­ combination therapy

DC/myeloma cell fusion Ongoing phase II

Active disease with median 4 prior regimens After autologous HSCT

Smoldering myeloma

LitVacc, DC pulsed with autologous lysate Ongoing phase II

Clinical outcomes Vaccine cohort, N = 27, median PFS 1.5 years Database control, N = 124, median PFS 1.5 years Vaccine cohort, N = 12, SD in 7 at 12 months Lenalidomide cohort, N = 9, PR in 1, MR in 4, SD in 4 NCT01718899, with lenalidomide and citarinostat Observation group, N = 16, median PFS 17.9 months Vaccine with lenalidomide, N = 15, median PFS not reached NCT03376477, with lenalidomide Vaccine alone, N = 16, SD for at least 2 months in 11 Vaccine alone, N = 36, 17% conversion from VGPR/PR to nCR/CR, median 2-year PFS 57% NCT02728102, with lenalidomide maintenance ACTRN12613000344796

SD stable disease, PR partial response, MR minimal response, CR complete response, nCR near-complete response, PFS progression-free survival

severely impaired response to syngeneic tumors in mice genetically modified to lack the two main cross-­ presenting DC subsets, lymphoid organresident CD8+ DCs, and migratory CD103+ DCs [95]. Mouse CD8+ DCs are resident DCs derived from a marrow precursor that continuously seeds the lymphoid organs. They are efficient at cross-­ presentation of exogenous antigen on MHC class I and are major producers of IL-12 on activation. Although CD8+ DCs do not exist in humans, there is evidence for a human counterpart BDCA3+Necl2+Clec9A+ that may correspond to the mouse CD8+Necl2+Clec9A+ DC subset [96] and subsequent work has identified a functional equivalence [97]. Lastly, vaccine efficacy depends on the presence of functionally competent immune effector cells and the kinetics of response is affected by the bulk of disease and proliferative rate. Smoldering myeloma may provide an effective platform for vaccination given that host immunity is more intact than with clinically advanced disease. However, presence of antigen may be more limited and defining endpoints predictive for outcome may provide logistical challenges. Of note, the period following autologous transplantation may offer an advantageous platform for vaccination due to a lower burden of disease and reduced tumor-mediated immune suppression. Lymphoid reconstitution after high-dose

chemotherapy and stem cell rescue is characterized by the transient reversal of tumor tolerance due to the relative depletion of accessory cells such as Tregs. Regulatory T cells are decreased in number and function with skewed CD8:CD4 providing a window for increased vaccine response to eliminate residual myeloma cells, generate long-term memory, and prevent recurrence [98]. Summary of published outcomes for myeloma vaccines is shown in Table 3.2.

3.4.2.1 Peptide-Based Myeloma Vaccines Peptide vaccines have been developed targeting myeloma antigens recognized as epitopes presented to T cells by MHC molecules. Peptide-­ based vaccines characteristically involve the use of peptide antigen(s) administered with immune adjuvants such as granulocyte macrophage colony-­stimulating factor (GM-CSF), IL-12, or toll-like receptor ligand poly-ICLC to recruit native antigen-presenting cells to the vaccine bed for subsequent internalization and antigen presentation. Peptide-based vaccination is highly feasible and potentially tumor selective but is limited by the need for HLA restriction, immune deficiencies of the native DC populations, and a potentially narrow CD8-mediated response that limits the opportunity for epitope spreading.

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One of the first clinical trials incorporating peptide vaccination for multiple myeloma used the idiotype protein, the product of the unique VDJ rearrangement of monoclonal plasma cells, in conjunction with GM-CSF and IL-12 [99, 100]. In subsequent trials antigen-specific T-cell responses were evoked and associated with prolonged time to progression [101]. However, idiotype protein is a weak immunogen and lack of immunologic response was associated with increased Tregs and higher burden disease. RHAMM-R3 plays a role in signaling via mitotic spindles and is associated with tumor progression in myeloma as centrosome abnormalities promote genomic instability. This peptide has been investigated in phase I/II clinical trials and demonstrated immune response with increased RHAMM-specific T cells and reduction of free light chain in patients with myeloma in partial or near-complete remission (nCR) after autologous HSCT [102, 103]. Wilms’ tumor-1 (WT1), a transcription factor for differentiation in kidneys and gonads, is immunogenic in myeloma [104]. WT1 vaccine given in RRMM setting evoked an immune response, but minimal clinical response [105]. Mucin1 (MUC1) is an epithelial mucin glycoprotein that also has cell signaling capacity. It is overexpressed in malignancies including myeloma, and binds multiple MHC class I and II alleles. A phase I/II study of biweekly vaccines using the complete MUC1 signal domain with GM-CSF in MUC1-positive MM patients with minimal residual or biochemical disease following ASCT achieved immune response with increase in anti-MUC1 antibodies, IFN-γ, and T-cell-mediated cytotoxicity. Stable disease or improvement for 17–41 months was achieved in 11 of 15 patients [106]. Although disease remission is not striking, these examples highlight the capacity to generate myeloma-­ specific immune response. Multiple-antigen peptide (MAP) vaccines have been developed to increase responsiveness to tumor antigens. A phase I trial vaccinated patients with DCs pulsed with keyhole limpet hemocyanin, (KLH), a hapten carrier, and electroporated with MAGE3, survivin, an anti-­ apoptotic protein, and BCMA after auto

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HSCT. Immune response to at least one antigen was demonstrated in only 2 of 12 patients [107]. DKK1 is a protein associated with lytic bone lesions in myeloma, shown to inhibit differentiation of osteoblasts and enhance osteoclasts [108]. Preclinical studies of DKK1 vaccine protected mice from developing myeloma and eradicated active disease. The vaccine was enhanced by use of anti-B7H1 or OX40 agonist through reduced IL-10 and Foxp3+ Tregs [109]. A vaccine combining DKK1 with membrane protein MMSA-1 may be a novel target for lytic bone disease. Compared to single epitope, there were increased cytotoxicity in vitro, increased CD4+ and CD8+ T cells in  vivo, and improved bony destruction and survival in myeloma-bearing severe combined immunodeficient mice [110]. PVX-410 multipeptide vaccine consisting of HLA-A2 peptides derived from X-box-binding protein 1 (XBP1), CD138, and CS1 antigen is being explored in smoldering myeloma. XBP1 is a transcription factor involved in endoplasmic reticulum stress response to unfolded protein and also regulates plasma cell differentiation through control of IL-6 and has been implicated in disease progression [111]. Preclinical studies with the peptide cocktail can induce HLA-restricted expansion and activation of myeloma-specific CTLs, upregulation of activation marker CD69, costimulatory receptor 4-1BB, and increased IFN-γ [112]. In phase I/IIa studies, 5 of 12 patients treated with PVX-410 alone had progression to active disease within 9 months, and the remaining 7 had stable disease at 12 months. With PVX-410 plus lenalidomide fivepatients had partial or minimal responses, three had stable disease through 12 months, and one had progression to active myeloma [113]. PVX-410 and lenalidomide are also being evaluated with anti-­ PD-­ 1 agent durvalumab (NCT02886065). Overall, peptide vaccines are highly feasible but evidence of clinical efficacy remains limited.

3.4.2.2 Cell-Based Myeloma Vaccines Further manipulation of cellular vaccine manufacturing can incorporate cellular components from both tumor and APCs. Investigators have demonstrated that functionally competent DCs

3  Immunotherapeutic Strategies for Multiple Myeloma

may be generated ex vivo from monocyte precursor populations in the peripheral blood via cytokine-­ mediated differentiation [114]. Individual or whole-cell-derived tumor antigens can be pulsed or loaded onto ex vivo DCs which then traffic to local lymph nodes to prime CD8+ T cells in vivo thru costimulation and secretion of cytokines such as IL-12 [115]. Circulating DC populations have been identified as myeloid and plasmacytoid in origin with the capacity to elicit Th1 and Th2 responses, respectively. As noted above, plasmacytoid DCs have been shown to contribute to the stromal environment in myeloma to promote tolerance; thus functional deficiencies in DCs derived from MM patients may impact their ability to elicit immunologic responses. Alternatively, ex vivo generation yields immunostimulatory myeloid DCs [116]. Vaccinations consisting of idiotype-pulsed DCs with idiotype/KLH boosters co-injected with GM-CSF were capable of inducing idiotype-­ specific CTL responses in only a few patients while all patients mounted KLH-specific T-cell responses. When vaccinated after ASCT, two patients who had idiotype-specific T-cell response elicited by vaccination remained in clinical partial response at 25 and 29 months while all other patients progressed. In addition to demonstrating some efficacy for DC-based vaccines in myeloma these studies also established feasibility of vaccine administration after ASCT [117, 118]. Similar trials evaluating idiotype-pulsed DC vaccines in early-stage MM elicited immune responses in a subset of patients but clinical effect was minimal [119]. In a single-arm phase II, DCs pulsed with idiotype protein following autologous transplant demonstrated an improvement in the overall survival of 5.3 vs. 3.4 years compared to historical controls, suggesting induction of long-term memory [120]. Administration of intranodal injection of CD40 ligand-matured DCs pulsed with idiotype in patients with smoldering or stable myeloma led to immune response in nine of nine patients and six of nine had stable disease at 1 year which persisted in four patients at 5 years [121]. Other efforts have used protein transduction to introduce more immunogenic antigens such as NY-ESO into the cytosol of DCs

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where they are processed by the proteasome, yielding NY-ESO-1 peptides presented by HLA class I that elicit NY-ESO-1-specific T lymphocytes [122]. However, vaccines using single-tumor antigens remain at risk of tumor evasion by the immune response through downregulated expression of the targeted antigen. Vaccines using whole-tumor antigens have been developed to optimize priming of effector cells by presenting the entire antigenic repertoire. For instance, a murine myeloma model demonstrated the emergence of idiotype-negative variants following idiotype-based vaccinations but not with irradiated myeloma cell-based vaccines [123]. The use of allogeneic myeloma cell lines as a source of whole-cell tumor antigen has been tested in the setting of minimal residual disease. Myeloma GVAX is a GM-CSF-based vaccine consisting of two allogeneic myeloma cell lines, coupled to a GM-CSF-secreting cell line to recruit DCs to the site of vaccination and present myeloma-derived antigens. Patients in nCR (absence of an M-spike but persistence of detectable immunofixation for 6 months) were vaccinated and continued on lenalidomide maintenance alone. At a median follow-up of at least 36 months, the vaccine group had not reached a median PFS in comparison to 17.9 months in the group that continued on lenalidomide combination therapy [124]. A larger randomized phase II study is planned. Vaccination using primary myeloma cells as a source of antigen would potentially drive a polyclonal immune attack against multiple tumor-­ associated antigens that is better able to target the tumor heterogeneity. DCs are capable of presenting peptides from engulfed apoptotic cells on MHC class I which can provide a source of whole-tumor antigen to be incorporated into myeloma vaccines. DCs pulsed with both apoptotic bodies and cell lysate can generate ­ myeloma-­ specific CTL responses [125, 126]. DCs cultured with apoptotic bodies from irradiation stimulated significantly greater T-cell proliferation than primary myeloma cells [127]. Comparison of irradiated cells versus transfection with myeloma total RNA by electroporation demonstrated that both methods of DC pulsing

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effectively primed autologous myeloma cytotoxicity and Th1 response [128]. In vivo, release of tumor antigens via cell lysis may amplify this antitumor response via cross-presentation of tumor-derived peptides on native MHC class I molecules, resulting in further activation of CD8+ cytotoxic T lymphocyte [129]. The authors’ group has developed a vaccine platform in which patient-derived myeloma cells are fused with autologous DCs, creating a hybridoma which expresses a broad array of both shared myeloma antigens and potentially neoantigens arising in the context of enhanced DC costimulation. Preclinical models demonstrated that DC-myeloma cell fusions cultured with GM-CSF induced DC maturation with expression of IL-12 and CCR7, a chemokine receptor necessary for DC migration to draining lymph nodes, and potently stimulated tumor killing via cytotoxic T cells [130]. In a murine model, fusions generated from a plasmacytoma cell line and autologous DCs were protective against challenge with syngeneic myeloma cells, and addition of IL-12 resulted in eradication of established disease [131]. A phase I trial administered serial vaccinations of DC fusion vaccine and GM-CSF in patients with RRMM with a median of four prior treatments. Biopsy of the vaccine bed demonstrated a dense infiltrate of CD8+ T cells consistent with T-cell expansion occurring at the site. Expansion of myeloma-specific T cells was detected in the majority of patients as manifested by the percent of CD4+ and/or CD8+ T cells expressing IFN-γ following ex vivo exposure to autologous tumor lysate. On SEREX analysis, humoral responses against novel proteins were noted after vaccination. These findings were consistent with the induction of myeloma-specific immunity in patients with advanced disease. Nearly 70% of patients demonstrated evidence of disease stabilization for at least 2 months or longer, with two patients having stable disease for more than 2 years [132]. A subsequent phase II clinical study of myeloma-DC fusion vaccination was conducted following ASCT as it was postulated that response would be augmented during lymphopoietic

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reconstitution with associated depletion of Tregs. 24% of patients who achieved a partial response following transplant converted to CR/nCR greater than 100 days posttransplant during the period after vaccination, consistent with a vaccine-­mediated elimination of residual disease in a subset of patients [133]. Vaccines were generally well tolerated with most common toxicity erythema and induration at the injection site. Patients in this study did not receive maintenance therapy with lenalidomide which enhances T-cell activation and has improved progression-free survival after auto HSCT [134, 135]. In vitro studies with lenalidomide reduced Tregs and T-cell PD-1 expression and augmented immune response to DC/fusion vaccine [136]. A first-of-­ its-kind national trial of the personalized DC/ MM fusion vaccine is being conducted under the auspices of the CTN oncology cooperative group (CTN 1401) in which myeloma patients undergoing autologous transplantation are randomized in the posttransplant period to receive DC fusion vaccine and lenalidomide maintenance versus lenalidomide maintenance. Each of the participating sites was trained at vaccine production with centralized characterization to assess release criteria and monitoring of immune correlates. Alternative strategies for DC-based vaccines include the use of pDCs augmented by exposure to toll-like receptor agonists to restore immunogenicity [137] or checkpoint inhibition as PD-L1 is expressed on APCs [138].

3.4.3 Adoptive Cell Therapy A fundamental limitation to the development of effective immunotherapy for myeloma is the availability of immunocompetent effector cells to stimulate and target myeloma cells. Observation of the association of more rapid recovery of the absolute lymphocyte count postASCT with improved outcomes in myeloma has led investigators to study the effect of adoptive cell transfer (ACT). Autologous lymphocytes can be obtained via leukapheresis from peripheral blood or from marrow and activated ex vivo by CD3/CD28 bead ligation. In this setting,

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improved pneumococcal immunity after ASCT was demonstrated with the use of ACT of in vivo vaccine-primed T cells combined with posttransplant pneumococcal vaccine [139]. Subsequent approaches used ACT in conjunction with cancer vaccines to boost myeloma immunity. In one study of patients treated with vaccine derived from hTERT and survivin followed by ACT, a median PFS of 20 months and projected 2-year OS of 83% were observed. Patient outcomes correlated with higher post-HSCT CD4+ and lower FOXP3+ T cells [140]. Other phase II trials that incorporated vaccine-primed autologous T cells after auto HSCT have included lenalidomide maintenance. A MAGE-A3 peptide vaccine plus agonist toll-­ like receptor-3 and GM-CSF demonstrated 88% MAGE-A3-specific CD8 T cells with 2-year PFS of 56% and OS of 74% [141]. An idiotype antigen conjugated to KLH was compared to KLH-­only vaccine with similar 2-year PFS of 81% and 83% at median follow-up of 26 months. PFS may have been affected by higher use of maintenance therapy in the KLH-only group but a trend was noted towards improved CR in the idiotype group [142]. However overall, ACT using polyclonal T cells has not achieved PFS consistently different than historical controls after ASCT. Subsequent efforts in ACT have focused on better selection of myeloma-specific cells or genetic manipulation of effector cells.

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of patients achieved a very good partial response (VGPR) or better and amongst this group, median OS at 7 years has not yet been reached [144, 145]. The independent impact of the MIL infusion following autologous transplant is currently being assessed. Two ongoing randomized phase II trials after auto HSCT in myeloma are comparing MILs versus GM-CSF expressing myeloma vaccine-primed MILs (NCT10145460), and in high-risk disease comparing tadalafil and lenalidomide with and without MILs (NCT01858558).

3.4.3.2 NK Cell Therapy NK cell-mediated immunity may play a crucial aspect as part of standard biologic therapy for MM.  IMiDs can induce proliferation of CD4+ and CD8+ T cells via the TCR and enhance production of Th1 cytokines IL-2 and IFNγ and inhibit IL-2-mediated induction of Tregs [146]. Increased transcription of IL-2 also stimulates NK cell proliferation and cytotoxicity. Lenalidomide has been shown to modulate NK cell activity via upregulation of activating ligand ULBP-1 (binds to NKG2D receptor) and decreased expression of inhibitory PD-L1 on MM cells, overall resulting in improved NK cell recognition and lysis of MM tumor targets [12, 147]. A monoclonal antibody against inhibitory KIR receptors has been developed, IPH2101. Phase I trials using this agent in patients with RRMM achieved objective responses in combination with lenalidomide but not monotherapy, 3.4.3.1 Marrow-Infiltrating T Cells albeit serious adverse effects were noted includBone marrow-infiltrating cells (MILs) are ing cytopenias and cytokine release syndrome enriched for myeloma reactive memory T cells [148, 149]. and also express CXCR4 which improves honing Killer-cell immunoglobulin-like receptor to the marrow once reinfused. Following CD3/ (KIR)-ligand mismatch predicting for NK activaCD28 activation, MILs have demonstrated tion was protective against relapse in patients enhanced ex  vivo proliferation and myeloma-­ with MM undergoing allogeneic SCT [150]. A specific cytotoxicity as well as a long-term sur- phase I/II study of lenalidomide given early after vival advantage after ACT as compared to allogeneic SCT for MM resulted in an increase of peripheral blood lymphocytes in a murine activating receptors NKp30 and NKp44 on NK myeloma model [143]. A clinical trial was per- cells, as well as an increase in NK cell-mediated formed using ACT with MILs plus IL-2 3 days cytotoxicity directed against myeloma associated after ASCT in 25 patients. Clinical outcomes with CR rates improving from 24% to 42% [151]. directly correlated with Th1 phenotype in the Ex vivo expansion and adoptive transfer have ex vivo product and tumor specificity of T cells been evaluated with NK cells generated from obtained from the BM after transplant. Over 30% autologous or haplo-identical family donors. In a

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phase I trial including myeloma, NK cell infusion was well tolerated, but only one of seven patients had a partial response [152]. An allogeneic off-the-shelf umbilical cord blood-expanded natural killer cell (PNK-007) is being investigated in combination with recombinant IL-2 after ASCT.  Preliminary results of a phase II trial in high-risk patients with active disease demonstrated 83% VGPR or better and 65% CR posttransplant with median PFS not reached at 22-month follow-up [153]. An alternative strategy is the development of NK CARs, in which human NK cells are genetically engineered to express a CAR that is specific to a myeloma-­ associated target. NK CARs are potentially safer than CAR T cells because they are less likely to cause a CRS and to induce GVHD in the allogeneic setting. In preclinical studies, NK CARs targeting CCND3 subset 1 (CS1) potently lysed human myeloma cells but have not yet been clinically investigated in myeloma [154].

3.4.4 Engineered T Cells Infusion of unmodified cells such as those used in ACT relies on the endogenous T-cell repertoire. Conversely, genetic engineering of T cells to express synthetic TCRs can redirect a large clonal population to a specific antigen via ex vivo transfection of autologous lymphocytes with viral vectors, typically gamma-retrovirus or lentivirus. CRISPR-Cas9 gene editing technology is also being used to disrupt the expression of PD1 or to encode safety switches. Typically, genetically engineered cells undergo expansion with CD3/ CD28 beads. Expansion and survival of the engineered T cells are enhanced through the prior administration of lymphodepleting chemotherapy, characteristically with a fludarabine and/or alkylator-based regimen.

3.4.4.1 TCR T Cells Affinity-enhanced TCR T cells involve TCR modification to generate a receptor with high specificity and sensitivity to tumor antigen peptide-­MHC complex. T cells have been developed that recognize NY-ESO-1 and LAGE-1

J. J. Liegel and D. E. Avigan

[155] which are expressed in up to 60% of patients with advanced MM. In a phase I/II trial of 20 patients, administration of NY-ESO-1/ LAGE-1-directed T cells after autologous HSCT [156] resulted in 70% of patients achieving nCR/ CR, 20% PR, and 10% stable disease. In responders, TCR T cells were detected up to 2 years while progressive disease correlated with their loss. Two patients relapsed with NY-ESO-1/ LAGE-1 antigen-negative subclones suggesting antigen escape. Infusions were well tolerated without apparent CRS.  At median follow-up of 30 months, the median PFS was 19.1 months, and median OS was 32.1 months which was promising in a heavily pretreated population. In a lung cancer model, NY-ESO TCR T cells were shown to become profoundly hypofunctional accompanied by upregulation of PD1, Tim3, and Lag3 that were co-expressed in a high percentage of cells [157]. Gene editing of NY-ESO TCR T cells has been employed to disrupt endogenous TCR and PD-1 genes in an effort to improve functional potency and persistence. This will be evaluated in a first-in-human phase I trial of CRISPR gene editing in melanoma, sarcoma, and myeloma [158]. Trials are also underway combining NY-ESO TCR T cells and checkpoint inhibitors. Antigen specificity is a major limitation with TCR T cells as use requires extensive in  vitro testing to evaluate cross-­ reactivity to normal tissue epitopes that can cause off-target toxicity. Initial use with a MAGE-A3 TCR led to fatal cardiogenic shock with T-cell infiltration in myocardial tissue on autopsy due to recognition of an unrelated peptide from myocyte protein titin [159].

3.4.4.2 CAR T Cells Chimeric antigen receptor T cells (CARs or CARTs) are engineered to express antigen-­ binding regions of scFvs of both heavy and light chains of a tumor-specific murine or humanized monoclonal antibody. The surface scFv is fused with a linker to the transmembrane domain CD3ζ (zeta) chain which transmits activation signals upon target binding. An important advance was the co-transduction of costimulatory molecules to facilitate constitutive T-cell

3  Immunotherapeutic Strategies for Multiple Myeloma

expansion and survival. Second- and third-generation CARs encode one or two intracellular domains, respectively, such as CD28, 4-1BB, CD278 (ICOS), or OX40 [160]. CAR targets are limited to extracellular antigens but possess a very high affinity, often several orders of magnitude higher than TCR T cells, and tumor killing is independent of MHC. Summary of published outcomes for CAR T-cell therapies is shown in Table 3.3.

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The primary toxicities associated with CAR T-cell therapy have been cytokine release syndrome (CRS), neurotoxicity, termed CAR T-cell-­ related encephalopathy (CRES), and related inflammatory events such as macrophage-­ activating syndrome. CRS arises from the cytokine storm associated with peak T-cell expansion and characteristically presents with fever and may progress to hypoxia and hypotension. Earlier onset is typically associated with increased sever-

Table 3.3  T-cell therapies in myeloma T cell therapy NY-ESOc259 TCR

Target/co-stim NY-ESO-1 LAGE-1

N Dose 20 1–10 × 109 after salvage ASCT

Clinical outcomes ORR 80%, CR/nCR 70%, median PFS 19.1 months NCT03399448 5 × 107 after salvage ORR 80%, 0 CR ASCT Median PFS 185 days 10-fold higher dose NCT02794246 0.3–9 × 106 after 3 doses ORR 25%, CR 17% Cy + flu ORR 48%, 10% CR 1–5 × 108 (cohort 1) 1 × 107–5 × 108 after Cy Cohort 3 median PFS 125 days (cohorts 2 and 3) 1.5–8 × 108 after 3 days ORR 85%, CR 45% Cy + flu Median PFS 11.8 months

Toxicity None

NYCE T cells CTL019

NY-ESO CD19/4-­1BB

10

No severe CRS

CTL019 CAR-BCMA

BCMA/CD28

12

CART-BCMA

BCMA/4-­1BB

25

bb2121

BCMA/4-­1BB

33

bb21217

BCMA/4-­1BB

ORR 83%, CR 25%

LCAR-B38M

BCMA/4-­1BB

JNJ-4528

BCMA/4-­1BB

22 1.5–4.5 × 108 after 3 days Cy + flu 57 0.5–7 × 106/kg in 3 split doses after Cy 25 0.5–1 × 106/kg after 3 days Cy + flu

JCARH125

BCMA/4-­1BB

ORR 82%, CR 27%

FCARH143

BMCA/4-­1BB

P-BCMA-101

BMCA/4-­1BB

CART-138 κ.CAR KITE-585 EGFRt/BCMA ± lenalidomide Anti-LeY CM-CS1

CD138/4-­1BB κ light chain BCMA/CD28 BCMA/4-1BB

14 0.5–4.5 × 108 after 3 days Cy + flu 11 0.5–1.5 × 108 after 3 days Cy + flu 21 0.5–8.5 × 108 after 3 days Cy + flu 5 2–9 × 108 7 9 × 106–1.9 × 108/m2

Lewis Y antigen/CD28 NKG2D

ORR 88%, CR 68% ORR 91%, CR 29%

ORR 100%, CR 36%

16% treated with tocilizumab 30% Gr 3 CRS 21% tocilizumab 15% ICANS 6% Gr 3 CRS 20% tocilizumab 39% Gr 1–2 3% ICANS Gr 4 5% ≥Gr 3 CRS 9% ≥Gr 3 ICANS 7% ≥Gr 3 CRS 11% tocilizumab 8% ≥Gr 3 CRS 80% tocilizumab 4% ICANS Gr 3 9% ≥Gr 3 CRS 7% ≥Gr 3 ICANS No severe CRS

ORR 100% at maximum No severe CRS dose 80% stable disease No severe CRS 57% stable disease No severe CRS NCT03318861 NCT03070327 NCT01716364 NCT02203825

ORR = CR + VGPR + PR SD stable disease, CR complete response, nCR near-complete response, PFS progression-free survival, CRS cytokine release syndrome, ICANS immune effector cell-associated neurotoxicity syndrome

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ity. Therapeutic options include targeting IL-6 such as with the tocilizumab and steroids. Neurotoxicity characteristically may follow CRS or occur independently and patients may manifest somnolence, altered mental status, seizures, and obtundation. Intensive monitoring and prompt recognition and management of these latter toxicities with the use of steroids or the anti-­IL-­6 agent tocilizumab are essential to mitigate potentially life-threatening complications. The CAR T-cell therapy-associated toxicity (CARTOX) working group has now published algorithms and guidelines for the use of CAR T-cell products [161]. To date, the incidence of severe CRS and neurotoxicity may be somewhat lower in myeloma as compared to lymphoma and acute leukemia although the precise impact of the different CAR constructs and targets, underlying disease, and disease stage and bulk has not been fully elucidated. Of note, there is increasing appreciation of the presence of prolonged cytopenias and risk of infection that has been reported and the relationship with CAR therapy as compared to the underlying disease and prior therapy is being studied. Dramatic results of CD19-directed CARs for the treatment of relapsed refractory acute lymphocytic leukemia and relapsed or refractory non-Hodgkin’s lymphoma have led to FDA approval of this personalized cell therapy in these settings. CD19-specific CAR T cells have been studied in myeloma due to the potential of targeting the CD19+ plasma cell precursor. In a clinical trial in which CD19 CARTs (CTL019) were infused following salvage auto HSCT, a single patient with multiply relapsed disease experienced a complete remission lasting more than 12 months [162]. In updated results of ten patients, six had achieved VGPR to the combination of high-dose melphalan and CAR T cells; however all patients ultimately experienced disease progression at a median PFS of 185 days. Grade 3 autologous GVHD occurred in one patient and resolved with steroids; no other major adverse events were reported [163] A study with tenfold higher dose after first-line auto HSCT is ongoing. Off-target effects of CD19-directed CARs include B-cell aplasia and hypogammaglobulinemia requiring immune globulin repletion.

J. J. Liegel and D. E. Avigan

An alternative target is BCMA which is highly expressed on benign and malignant plasma cells with limited expression on normal B cells or other tissues [164]. Efficacy was initially demonstrated at the National Cancer Institute in RRMM patients with an ORR of 81%, 63% VGPR, or CR, including eradication of soft-tissue plasmacytomas and marrow MRD negativity. Median event-free survival was 31 weeks, with responses associated with CAR T-cell expansion and reduction in serum BCMA as well as with toxicity, including severe CRS which was reversible in all cases [165]. A phase I trial at the University of Pennsylvania (CART-BCMA) infused split doses of T cells over 3 days and observed best expansion and response (ORR of 64%) with the use of cyclophosphamide for lymphodepletion and higher cell dose of 1–5 × 108 cells [166]. However only a few responses were durable (>11 months) with BCMA expression noted to increase in most patients at the time of progression. A larger multicenter phase I trial (bb2121) in heavily pretreated RRMM has reported even higher response rates with longer durability [167]. Infusion of CAR T cells was given after cyclophosphamide and fludarabine with ORR of 85% and median duration of response of 10.9 months. VGPR or better was observed only with doses of 1.5  ×  108 or higher; 45% had CR or sCR. Of note almost half the patients had high-­ risk cytogenetics. The most common grade 3 or higher toxicities were cytopenias expected from lymphodepletion. CRS was common but mostly grades 1–2 and only one episode of neurologic toxicity required treatment with glucocorticoids. Some patients achieved a deepening of response over time with conversions from VGPR to CR as late as 15 months [168]. Response was associated with higher circulating CAR T-cell levels but not with tumor BCMA expression levels. At a median follow-up of 11 months, 50% of responses are ongoing with several beyond 12 months, while relapses have occurred even after achievement of CR and MRD negativity. Longer follow-up is required to determine whether bb2121 will achieve long-term durable remissions. A large non-randomized phase II trial is ongoing (KarMMa-1) while the use of bb2121 is also

3  Immunotherapeutic Strategies for Multiple Myeloma

being investigated in earlier lines of treatment (KarMMa-2, KarMMa-3). Based on the encouraging phase I data, bb2121 was granted breakthrough therapy designation by the US and European regulatory agencies and is anticipated to receive first-in-class approval for BCMA-­ targeted CAR T-cell therapy. A modified manufacturing process (bb21217) uses the bb2121 construct cultures with a PI3 kinase inhibitor to enrich for memory-like T cells for enhanced persistence [169]. LCAR-B38M consists of two different heavy-­ chain variable domains which target different epitopes of the same BCMA antigen. Results were reported from a trial in China using three split doses over 1 week following cyclophosphamide. Response rates reported to date (ORR 88%, sCR 68%) are slightly superior to bb2121 but not directly comparable as the patients were less heavily pretreated [170]. CRS occurred in 90% but was grade 3 or higher in only 7% of patients. Median PFS was 15 months but for those who achieved MRD negativity PFS was 24 months. The CARTITUDE-1 trial is ongoing in the USA with an identical product (JNJ-4528) given as a single dose in RRMM patients with a median of five prior lines of therapy [171]. Similar ORR to LCAR-B38M has been shown thus far in preliminary results but CR rate was lower at 29%. Grade 3 or higher CRS was also similar at 8% but one was grade 5. Other BCMA-targeting CAR products are also undergoing testing. JCARH125 is a BCMA CAR construct with a fully human scFv with nanomolar binding affinity to allow for activity on target cells with low BCMA density but minimize tonic signaling to reduce exhaustion. Manufacturing was designed to enrich central memory CD4 and CD8 T cells [172]. Notably activity was seen in patients with high levels of soluble BMCA which can inhibit CAR T-cell recognition of myeloma cells [173]. FCARH143 also has a fully humanized BCMA scFv, manufactured with mixed CD4 and CD8 cells yielding a 1:1 ratio in the final product. 100% ORR was reported in a small group of patients, all of whom were at high risk, but relapses developed with reduction in BCMA binding capacity or BCMA

93

loss on surviving plasma cells [174]. Lastly, P-BCMA-101 is manufactured with an anti-­ BCMA centyrin in a nonviral transposon system designed to yield higher stem cell memory T cells. Preliminary results were promising with 100% ORR at the maximum dose but durability is unknown [175]. Other targets including CD138 and kappa light chain have yielded durable partial response or stable disease in a small number of patients [176, 177]. Preclinical models for CD38 CAR T cells have shown cytotoxicity against T-cell lines and primary malignant T cells from multidrug-­ resistant myeloma patients [178]. A novel CAR approach fused the full-length human NK receptor NKG2D rather than an scFv to potentially target multiple ligands on myeloma cells. However a phase 1 trial of CM-CS1 T cells in hematologic malignancies including myeloma failed to demonstrate any objective responses or CAR T-cell persistence although no lymphodepleting conditioning was used [179]. CAR targets in MM are summarized in Table 3.3. Off-the-shelf allogeneic products are being investigated that may allow for the development of a universal source of cells markedly easing the logistics of T-cell generation and administration. CRISPR or TALEN gene editing to inactivate the TCRα constant gene of T cells is an approach to prevent GVHD. Allogeneic products are in preclinical development in myeloma, P-BMCA-­ ALLO1 targets BCMA, and UCARTCS1 targets SLAMF7 [180, 181]. NK CARs may have some advantages as compared to CAR T cells as it is hypothesized because that they are less likely to cause CRS or induce GVHD [182]. Genetic engineering of NK cells to express myeloma-specific targets such as CS1 has shown activity with potent lysis of human myeloma cells and prolonged survival in an aggressive orthotopic MM xenograft mouse model [154]. In general, dose of infused CAR cells and peak expansion as detected in peripheral blood have been correlated with response as well as toxicity in MM trials to date. Reduction in serum BCMA levels is also associated with response. It is unclear if the difference in response rates for different products could be due to variations of

J. J. Liegel and D. E. Avigan

94

BCMA epitopes used. It also does not appear that myeloma BCMA expression is needed for response. Lymphodepletion appears to be important as cohorts that did not receive this had reduced CAR expansion and response. It is also unclear what effect the costimulatory domain has, as for instance CAR-BCMA which is the only product to use CD28 had similar response rates to BCMA CART which like all other BCMA CARs uses 4-1BB.  The product composition at the time of apheresis can have an effect, with higher CD4:CD8 T-cell ratios and CD45RO−CD27+CD8+ (more naïve or stem cell memory-like phenotype) associated with response [166]. CAR T-cell therapy has shown remarkable promise in myeloma with overall less treatment-­ related toxicity relative to the experience in lymphoma or ALL.  However durability remains a concern and immune escape has been noted due to the emergence of antigen-negative variants, lack of persistence of the CAR T cells, and downregulation of CAR T-cell function due to the tumor microenvironment. While antigen loss and lack of T-cell persistence appear to be general mechanisms of relapse, further studies are needed. In an effort to improve long-term response, earlier use of CAR T-cell therapies is being investigated. Collecting T cells prior to heavy therapy exposure may enhance T-cell fitness for improved functionality and effectiveness [183]. BMT CTN 1901 will evaluate CAR T cells as consolidation after ASCT in patients with high-risk cytogenetics and BMT CTN 1902 will evaluate CAR T cells as consolidation in patients who achieved less than VGPR after ASCT. Minimizing of antigen escape combination therapy of multiple CAR T-cell targets such as BCMA with CD19 or CD38 is being explored with infusion of two products or one dual-­ targeting product. The addition of IMiDs following CAR T-cell infusion is also being investigated. An interesting observation of possible antigen spreading was observed following the initiation of lenalidomide during the use of engineered T cells. A patient who relapsed with NY-ESO-1/ LAGE-1+ disease received a second T-cell infusion which led to marrow response, concomitant increase in CD8+ T cells, and emergence of two

dominant non-NY-ESO TCR clonotypes [156]. This strategy could be beneficial in reducing relapses after either TCR or CAR T cells. A phase I trial is evaluating BCMA CARs that encode a truncated epithelial growth factor elimination gene as a safety switch with and without lenalidomide [172]. Of note, we are studying the potential synergy between vaccine and CAR T-cell therapy, hypothesizing that vaccination will result in the broadening of the T-cell repertoire while maintaining T-cell activation and expansion. Recently, the use of agents to increase surface BCMA in order to avoid antigen loss has been reported. BCMA is a membrane-bound receptor serving as a survival signal on plasma cells that can be cleaved by gamma-secretase [184]. Cleavage leads to soluble BCMA which is known to correlate with worse clinical outcomes [185]. The use of gamma-secretase inhibitors in vitro has been shown to increase surface BCMA on myeloma cell lines and patient samples and improve tumor recognition by CAR T cells [186]. This has led to the first clinical trial combining gamma-secretase inhibitors with BCMA CAR T-cell therapy (NCT03502577).

3.5

Concluding Remarks

Disease evolution of multiple myeloma is associated with progressive immune dysregulation and an immunosuppressive tumor microenvironment that facilitates disease expansion. Reversal of this phenomenon using cancer immunotherapy can generate enhanced myeloma-specific effector cell responses. Antibody therapy in myeloma exhibits activity via improved targeting of myeloma-associated antigens to enhance innate immunity via ADCC, CDC, more precise d­ elivery of cytotoxic agents, or approximation of tumor and effector cells. Based on promising results, addition of anti-CD38 therapy to frontline myeloma therapy is likely to become a new standard of care. Checkpoint inhibitors targeting PD-1/PD-L1 pathway in combination with IMID therapy held promise but later phase III trials have observed a higher treatment-associated

3  Immunotherapeutic Strategies for Multiple Myeloma

mortality. Use of myeloma vaccines has created unique platforms to reeducate host effector cells to target early or minimal residual disease. And in the most heavily pretreated patients, use of CAR T cells has led to impressive successes, particularly with the use of BCMA targets but risk of CRS and CRES limits this therapy to the multiply relapsed setting where it is outweighed by the need for rapid reduction and limited other options for disease control. Engineered T-cell therapies remain susceptible to tumor escape by antigen downregulation under immune pressure and development of T-cell anergy. Therapies that target multiple antigens or combination use with IMiDs and gamma-secretase inhibitors will attempt to overcome this. ADCs have also demonstrated impressive results in heavily pretreated RRMM with minimal toxicity and are likely to become a clear choice in patients who cannot tolerate lymphodepletion or prefer to avoid the risks of CRS and neurotoxicity. BiTE antibodies are also promising, but similar to CAR T cells need to be given at a tertiary center that can manage the side effects. The use of the optimal combination strategies at the ideal time points to address the complex nature of myeloma progression and immune dysregulation will likely need to be developed for long-term disease eradication.

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3  Immunotherapeutic Strategies for Multiple Myeloma 137. Ray A, Tian Z, Das DS, Coffman RL, Richardson P, Chauhan D, Anderson KC. A novel TLR-9 agonist C792 inhibits plasmacytoid dendritic cell-induced myeloma cell growth and enhance cytotoxicity of bortezomib. Leukemia. 2014;28:1716–24. 138. Ray A, Das DS, Song Y, Richardson P, Munshi NC, Chauhan D, Anderson KC.  Targeting PD1-PDL1 immune checkpoint in plasmacytoid dendritic cell interactions with T cells, natural killer cells and multiple myeloma cells. Leukemia. 2015;29:1441–4. 139. Rapoport AP, Stadtmauer EA, Aqui N, et  al. Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer. Nat Med. 2005;11:1230–7. 140. Rapoport AP, Aqui NA, Stadtmauer EA, et  al. Combination immunotherapy using adoptive T-cell transfer and tumor antigen vaccination on the basis of hTERT and survivin after ASCT for myeloma. Blood. 2011;117:788–97. 141. Rapoport AP, Aqui NA, Stadtmauer EA, et  al. Combination immunotherapy after ASCT for multiple myeloma using MAGE-A3/Poly-ICLC immunizations followed by adoptive transfer of vaccine-primed and costimulated autologous T cells. Clin Cancer Res. 2014;20:1355–65. 142. Qazilbash MH, Stadtmauer EA, Baladandayuthapani V, et  al. Randomized phase II trial of combination idiotype vaccine and anti-CD3/Anti-CD28 costimulated autologous T cells in patients with multiple myeloma post-autotransplantation. Blood. 2016;128:4548. 143. Noonan K, Matsui W, Serafini P, Carbley R, Tan G, Khalili J, Bonyhadi M, Levitsky H, Whartenby K, Borrello I.  Activated marrow-infiltrating lymphocytes effectively target plasma cells and their clonogenic precursors. Cancer Res. 2005;65:2026–34. 144. Noonan KA, Huff CA, Davis J, et  al. Adoptive transfer of activated marrow-infiltrating lymphocytes induces measurable antitumor immunity in the bone marrow in multiple myeloma. Sci Transl Med. 2015;7:288ra78. 145. Noonan KA, Borrello IM. Marrow infiltrating lymphocytes: their role in adoptive immunotherapy. Cancer J. 2015;21:501–5. 146. Quach H, Ritchie D, Stewart A, Neeson P, Harrison S, Smyth M, Prince H.  Mechanism of action of immunomodulatory drugs (IMiDS) in multiple myeloma. Leukemia. 2010;24:22–32. 147. Benson DM, Bakan CE, Zhang S, et al. IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect. Blood. 2011;118:6387–91. 148. Benson DM, Hofmeister CC, Padmanabhan S, et al. A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed/refractory multiple myeloma. Blood. 2012;120:4324–33. 149. Benson DM, Cohen AD, Jagannath S, Munshi NC, Spitzer G, Hofmeister CC, Efebera YA, Andre P, Zerbib R, Caligiuri MA. A phase I trial of the anti-­ KIR antibody IPH2101 and lenalidomide in patients

101 with relapsed/refractory multiple myeloma. Clin Cancer Res. 2015;21:4055–61. 150. Kröger N, Shaw B, Iacobelli S, et  al. Comparison between antithymocyte globulin and alemtuzumab and the possible impact of KIR-ligand mismatch after dose-reduced conditioning and unrelated stem cell transplantation in patients with multiple myeloma. Br J Haematol. 2005;129:631–43. 151. Wolschke C, Stübig T, Hegenbart U, et  al. Postallograft lenalidomide induces strong NK cell-mediated antimyeloma activity and risk for T cell-mediated GvHD: results from a phase I/II dose-­ finding study. Exp Hematol. 2013;41:134– 142.e3. 152. Szmania S, Lapteva N, Garg T, et  al. Ex vivo-­ expanded natural killer cells demonstrate robust proliferation in  vivo in high-risk relapsed multiple myeloma patients. J Immunother. 2015;38:24–36. 153. Shah N, Mehta R, Li L, et  al. Phase II study of ex vivo expanded cord blood natural killer cells for multiple myeloma. JCO. 2018;36:8006. 154. Chu J, Deng Y, Benson DM, et  al. CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in  vitro and in  vivo antitumor activity against human multiple myeloma. Leukemia. 2014;28:917–27. 155. Li Y, Moysey R, Molloy PE, et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display. Nat Biotechnol. 2005;23:349–54. 156. Rapoport AP, Stadtmauer EA, Binder-Scholl GK, et  al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med. 2015;21:914–21. 157. Moon EK, Ranganathan R, Eruslanov E, Kim S, Newick K, O’Brien S, Lo A, Liu X, Zhao Y, Albelda SM.  Blockade of programmed death 1 augments the ability of human T cells engineered to target NY-ESO-1 to control tumor growth after adoptive transfer. Clin Cancer Res. 2016;22:436–47. 158. Baylis F, McLeod M.  First-in-human phase 1 CRISPR gene editing cancer trials: are we ready? Curr Gene Ther. 2017;17:309–19. 159. Linette GP, Stadtmauer EA, Maus MV, et  al. Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood. 2013;122:863–71. 160. Lim WA, June CH.  The principles of engineering immune cells to treat cancer. Cell. 2017;168:724–40. 161. Neelapu SS, Tummala S, Kebriaei P, et al. Chimeric antigen receptor T-cell therapy—assessment and management of toxicities. Nat Rev Clin Oncol. 2018;15:47–62. 162. Garfall AL, Maus MV, Hwang W-T, et al. Chimeric antigen receptor T cells against CD19 for multiple myeloma. N Engl J Med. 2015;373:1040–7. 163. Garfall AL, Stadtmauer EA, Maus MV, et  al. Pilot study of anti-CD19 chimeric antigen receptor T cells (CTL019) in conjunction with salvage autologous stem cell transplantation for advanced multiple myeloma. Blood. 2016;128:974.

102 164. Carpenter RO, Evbuomwan MO, Pittaluga S, Rose JJ, Raffeld M, Yang S, Gress RE, Hakim FT, Kochenderfer JN.  B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma. Clin Cancer Res. 2013;19:2048–60. 165. Brudno JN, Maric I, Hartman SD, et al. T cells genetically modified to express an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of poor-prognosis relapsed multiple myeloma. J Clin Oncol. 2018;36:2267–80. 166. Cohen AD, Garfall AL, Stadtmauer EA, et  al. B cell maturation antigen–specific CAR T cells are clinically active in multiple myeloma. J Clin Invest. 2019;129:2210–21. 167. Raje N, Berdeja J, Lin Y, et  al. Anti-BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma. N Engl J Med. 2019;380:1726–37. 168. Berdeja JG, Lin Y, Raje N, et  al. Durable clinical responses in heavily pretreated patients with relapsed/refractory multiple myeloma: updated results from a multicenter study of bb2121 anti-­ Bcma CAR T cell therapy. Blood. 2017;130:740. 169. Berdeja JG, Alsina M, Shah ND, et  al. Updated results from an ongoing phase 1 clinical study of bb21217 anti-Bcma CAR T cell therapy. Blood. 2019;134:927. 170. Zhao W-H, Liu J, Wang B-Y, et al. A phase 1, open-­ label study of LCAR-B38M, a chimeric antigen receptor T cell therapy directed against B cell maturation antigen, in patients with relapsed or refractory multiple myeloma. J Hematol Oncol. 2018;11:141. 171. Madduri D, Usmani SZ, Jagannath S, et al. Results from CARTITUDE-1: a phase 1b/2 study of JNJ-­ 4528, a CAR-T cell therapy directed against B-cell maturation antigen (BCMA), in patients with relapsed and/or refractory multiple myeloma (R/R MM). Blood. 2019;134:577. 172. Smith EL, Mailankody S, Ghosh A, et  al. Development and evaluation of a human single chain variable fragment (scFv) derived Bcma targeted CAR T cell vector leads to a high objective response rate in patients with advanced MM. Blood. 2017;130:742. 173. Mailankody S, Htut M, Lee KP, Bensinger W, Devries T, Piasecki J, Ziyad S, Blake M, Byon J, Jakubowiak A. JCARH125, anti-BCMA CAR T-cell therapy for relapsed/refractory multiple myeloma: initial proof of concept results from a phase 1/2 multicenter study (EVOLVE). Blood. 2018;132:957. 174. Green DJ, Pont M, Sather BD, et  al. Fully human Bcma targeted chimeric antigen receptor T cells

J. J. Liegel and D. E. Avigan administered in a defined composition demonstrate potency at low doses in advanced stage high risk multiple myeloma. Blood. 2018;132:1011. 175. Gregory T, Cohen AD, Costello CL, et al. Efficacy and safety of P-Bcma-101 CAR-T cells in patients with relapsed/refractory (r/r) multiple myeloma (MM). Blood. 2018;132:1012. 176. Guo B, Chen M, Han Q, Hui F, Dai H, Zhang W, Zhang Y, Wang Y, Zhu H, Han W. CD138-directed adoptive immunotherapy of chimeric antigen receptor (CAR)-modified T cells for multiple myeloma. J Cell Immunother. 2016;2:28–35. 177. Ramos CA, Savoldo B, Torrano V, et  al. Clinical responses with T lymphocytes targeting malignancy-­associated κ light chains. J Clin Invest. 2016;126:2588–96. 178. Drent E, Groen RWJ, Noort WA, et al. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma. Haematologica. 2016;101:616–25. 179. Nikiforow S, Werner L, Murad J, et al. Safety data from a first-in-human phase 1 trial of NKG2D chimeric antigen receptor-T cells in AML/MDS and multiple myeloma. Blood. 2016;128:4052. 180. Wang X, Barnett BE, Martin C, et al. Production of universal anti-Bcma CAR-T cells with reduced alloreactivity, but potent effector function for the treatment of multiple myeloma. Blood. 2017;130:503. 181. Mathur R, Zhang Z, He J, et al. Universal SLAMF7-­ specific CAR T-cells as treatment for multiple myeloma. Blood. 2017;130:502. 182. Rezvani K, Rouce R, Liu E, Shpall E. Engineering natural killer cells for cancer immunotherapy. Mol Ther. 2017;25:1769–81. 183. Garfall AL, Dancy EK, Cohen AD, et al. T-cell phenotypes associated with effective CAR T-cell therapy in postinduction vs. relapsed multiple myeloma. Blood Adv. 2019;3:2812–5. 184. Laurent SA, Hoffmann FS, Kuhn P-H, et  al. γ-Secretase directly sheds the survival receptor BCMA from plasma cells. Nat Commun. 2015;6:7333. 185. Sanchez E, Li M, Kitto A, et al. Serum B-cell maturation antigen is elevated in multiple myeloma and correlates with disease status and survival. Br J Haematol. 2012;158:727–38. 186. Pont MJ, Hill T, Cole GO, et al. γ-Secretase inhibition increases efficacy of BCMA-specific chimeric antigen receptor T cells in multiple myeloma. Blood. 2019;134:1585–97.

4

Immunopathology and Immunotherapy of Myeloid Leukemia Sylvia Snauwaert, Farzaneh Rahmani, Bart Vandekerckhove, and Tessa Kerre

Contents 4.1

Introduction 

 104

4.2 Immunopathology of Acute Myeloid Leukemia  4.2.1    Causes of Genetic Alterations  4.2.1.1  Primary AML  4.2.1.2  Secondary AML  4.2.2    Genes Affected in AML  4.2.3    Models for Leukemogenesis Through Gene Alterations  4.2.4    The Leukemic Stem Cell  4.2.4.1  Phenotype of the LSC  4.2.4.2  Clinical Relevance of the LSC  4.2.5    How Do Gene Alterations in the LSC Lead to the Clinical Presentation of AML? 

 104  104  104  104  105  105  106  106  107

4.3 Immunotherapy for AML  4.3.1    Antigens to Target in AML  4.3.1.1  Antigens Presented by MHC After Internal Processing  4.3.1.2  Surface Antigens  4.3.2    Current Immunotherapeutic Strategies for AML  4.3.2.1  Active Immunotherapeutic Strategies  4.3.2.2  Passive Immunotherapeutic Strategies 

 108  109  109  109  109  110  110

4.4

Concluding Remarks 

References 

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 111  112

S. Snauwaert Department of Hematology, AZ Sint-Jan Brugge-­ Oostende AV, Bruges, Belgium

B. Vandekerckhove Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium

F. Rahmani Student’s Scientific Research Center, Tehran University of Medical Sciences (TUMS), Tehran, Iran

T. Kerre (*) Department of Hematology, Ghent University Hospital, Ghent, Belgium

NeuroImaging Network (NIN), Universal Scientific Education and Research Network (USERN), Tehran, Iran

Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium email: [email protected]

© Springer Nature Switzerland AG 2020 N. Rezaei (ed.), Cancer Immunology, https://doi.org/10.1007/978-3-030-57949-4_4

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4.1

Introduction

Acute myeloid leukemia (AML) is the most common myeloid leukemia, with a median prevalence of 3–8 cases per 100,000. The median age at presentation is about 70  years, and men are more commonly affected than women (ratio 3:2). Risk factors for acquiring AML include exposure to ionizing radiation, benzene, and cytotoxic chemotherapy [1]. AML is a heterogeneous clonal disorder, consisting of hematopoietic progenitor cells and  “malignant blasts,” characterized by maturation arrest, uncontrolled proliferation, and resistance to apoptosis. Untreated, the disease is fatal within weeks to months, most commonly due to fatal infection, bleeding diathesis particularly in the acute promyelocytic leukemia (APL) subtype, or organ failure due to malignant infiltration [1]. Especially APL patients are at risk for  early death, less than 30  days after the diagnosis, this  risk increasing with delayed first dose of standard all-trans retinoic acid (ATRA) therapy and increased age of the patient [2].

4.2

Immunopathology of Acute Myeloid Leukemia

It is now generally accepted that AML originates from genetic alterations in normal hematopoietic stem cells (HSC) or common myeloid progenitor cells (CMP), giving rise to the leukemic stem cell (LSC), from which the bulk of leukemic blasts rise, ultimately leading to the clinical presentation of AML.

4.2.1 Causes of Genetic Alterations 4.2.1.1 Primary AML Primary and secondary AML are, respectively, defined based on the absence or presence of a preexisting condition or therapy. According to the most recent, revised World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia, published in 2016

[3], primary AML is classified into three major subcategories: (1) AML with recurrent genetic abnormalities, (2) AML with myelodysplasia-­ related changes, and (3) AML with the absence of known genetic alterations, i.e., not otherwise specified (NOS). This highlights the importance of genetic abnormalities in AML that bear prognostic importance and therapeutic implications and above all are a major pathogenetic mechanism for AML. In primary AML, the causes for the alterations are largely unknown, and one can say that the mutations are simply a result of “bad luck” during mitosis. Nonetheless, rare cases of familial hematological malignancies have been described [4, 5], in which a genetic predisposition also exists for other primary cancers and possible immunodeficiency. Mutations in genes as RUNX1 and CEBPA, as well as in GATA2, have been identified in these families [6, 7]. This same mechanism is also applicable to the higher incidence of AML and other cancers in patients with specific genetic disorders such as Down syndrome [8] and Fanconi anemia [9].

4.2.1.2 Secondary AML AML is considered secondary, if it evolves from a preexisting myelodysplastic syndrome (MDS) or if the patient received chemotherapy and/or radiotherapy for an unrelated disease. Acute Myeloid Leukemia with Myelodysplasia-Related Changes (AML-MDS) MDS is seen as a premalignant condition in AML occurring with myelodysplasia-related changes. Exome and whole-genome sequencing has revealed that AML and MDS share only few common mutated genes, but still higher than expected to occur by chance, suggesting that a fraction of recurrent mutations are involved in both AML and MDS [10]. Using a microarray and meta-analysis of genetic and biological signatures, firm ground of shared genetic dysregulation is identified in fibroblast growth factor receptor (FGFR), IL-6/interferon, and B-cell receptor signaling pathway in both entities [3].

4  Immunopathology and Immunotherapy of Myeloid Leukemia

Therapy-Related Myeloid Neoplasms (t-AML) Predisposition to therapy-related AML is determined by the chemotherapy regimen, e.g., anthracyclines such as daunorubicin and epipodophyllotoxins such as etoposide, as well as host factors, e.g., specific polymorphisms of detoxification enzymes or DNA repair genes (reviewed in [11]). Therapy-related AML with epipodophyllotoxins is associated with a favorable prognosis and signature mutations such as KMT2A/MLL-MLLT3, while alkylating agents herald a t-AML associated with an initial MDS phase, latency in diagnosis, and poor outcome [12]. Other mechanisms have been suggested for t-AML generation including dysfunctions in cellular metabolic pathways associated with post-­ HSCT t-AML [13]. Fortunately, up to 50% of t-AMLs present with favorable risk fusion genes such as PML-RARA [12].

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Alternatively, more committed progenitor cells can undergo transforming events, partially reprogramming these cells, resulting in the reacquisition of stem cell characteristics such as self-renewal [22]. This phenomenon is supported by evidence on heterogeneity within the LSC phenotype [26], and the fact that transduction of strictly defined populations of long-term repopulating HSC, short-term repopulating HSC, and lineage-committed common myeloid precursors (CMP) with the leukemia-associated mixed-­ lineage leukemia (MLL) protein can reproduce AML phenotype in these populations [27]. The classical two-hit leukemogenesis model is still a popular model to describe AML pathogenesis (Fig.  4.1). This model describes how AML blasts develop from normal blasts by two types of genetic damage. The first (class 1) hit results in constitutive activation of cell-surface receptors or receptor-associated tyrosine kinases and signaling adaptors, which leads to survival or proliferative advantage of the lineage. Typical 4.2.2 Genes Affected in AML class 1 mutations affect RAS, FLT3, and KIT genes. These genetic aberrations from the first hit With regard to which genes are inducing AML, are not sufficient to produce a typical AML, but numerous studies have been done in mice with rather result in a myeloproliferative disorder [28, induced expression of oncogenes in normal bone 29]. The second hit (class 2) blocks myeloid difmarrow, resulting in the development of AML ferentiation, preparing grounds for accumulation [14]. Human genetic studies are typically more of prosurvival-related mutations in the lineage scarce [15]. The authors refer the reader to review already hit by an oncogenic mutation. This is articles which have nicely summarized the cur- exemplified in the formation of fusion genes rent knowledge of the translocations and/or resulting from the translocation t(8;21) or invermutations involved in AML and their interrela- sion inv(16), respectively. Both the AML1-ETO tionship [16–18] and the importance of next-­ and CBFβ-MYH11 fusion genes created by generation sequencing in the future management t(8;21) and inv(16) respectively, result in alteraof AML [19]. tions of the core-binding factor β (AML1-CBFβ), a transcription factor which regulates a number of hematopoiesis-specific genes and is essential 4.2.3 Models for Leukemogenesis for normal development of the hematopoietic Through Gene Alterations system [30–32]. Similarly to class 1 hits, these abnormalities alone do not cause leukemia in AML can be the result of normal HSC acquiring mouse models [28, 29, 33, 34]. Importantly, studa sequence of mutations, as evidenced by the ies on human AML lineages suggest that class 1 shared CD34+CD38− phenotype of both HSC and and class 2 lesions occur sequentially more often LSC [20–25], cytogenetic abnormalities detected than do two class 1 or two class 2 hits, further in in the CD34+CD38− cells of AML patients [24, support of the two-hit model. 25], and heterogeneity self-renewal potential and The “minimal two-hit” model was later challongevity of different LSC linages [23]. lenged by cumulative evidence on new mutations

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106 Pre-LSC

HSC

Leukemic blasts

HIt II

HIt I

MP

LSC

HIt I

HIt I Pre-LMP

HIt I HIt III (E pigenetic factors)

Normal myelold cells

Fig. 4.1  Models of pathogenesis of AML. Three possible scenarios for the development of the LSC are shown. Hematopoietic stem cells (HSC) or myeloid progenitors (MP) or both populations are potential targets for primary (Hit I) and secondary (Hit II) hits. Usually, one single mutation leads to a preleukemic stem cell (pre-LSC) of myeloid progenitor (pre-LMP), and a second mutation

(Hit II) results in the formation of a leukemic stem cell (LSC) that finally gives rise to the bulk of the leukemic blasts, although the LSC can also—albeit to a lower extent—lead to normal differentiated cells. The role of epigenetic changes (Hit III) has more  recently become clear, but the exact mechanism has not been fully unraveled

and especially the role of epigenetics in AML.  Epigenetic regulation of gene expression is mediated chiefly by either DNA methylation or posttranslational modifications such as histone modifications that regulate chromatin structure. It is now known that malignant LSC harbors not only several class 1 and class 2 mutations but also epigenetic abnormalities such as DNA hypomethylation and hypoacetylation of chromatin, as well as gene-specific hypo- or hypermethylation [35, 36], changes designated as class 3 hits. Indeed, recent evidence suggests that class 3 hits can occur very early during AML clonal progression and remain stable during disease progression, making them suitable for therapeutic targeting [37]. Furthermore, several germ-line mutations with previously undermined significance are now found to predispose to epigenetic aberrancies and global dysregulation of gene expression [37] necessary to maintain leukemic transformation. Genome-wide epigenetic studies focusing on DNA methylation in AML are being

published [38, 39] suggest a multiple/complex hit model to generate AML. To understand the entire picture of molecular pathogenesis in AML, gene rearrangement, gene copy number, DNA methylation, and expression profiles are needed to be analyzed together with gene mutations [40]. Indeed one would be right saying that every AML has its own story to tell. Finally, the role of miRNAs in the development of AML, as in other cancers, has been shown by several studies comparing miRNA signature between AML and ALL, between AML and normal CD34+ cells, and between  different AML samples (reviewed in [41]).

4.2.4 The Leukemic Stem Cell 4.2.4.1 Phenotype of the LSC Most AML cells are unable to proliferate extensively, and undergo senescence like any other cell in the body. Only a subset of these cells preserve

4  Immunopathology and Immunotherapy of Myeloid Leukemia

clonogenic properties, suggesting that, similar to normal hematopoiesis, leukemia may be maintained by a small population of stem cells [20, 21, 42, 43]. Based on transplantation experiments in SCID and later NOD/SCID mice, Dick and his colleagues concluded that AML-initiating cells or LSCs are CD34+CD38−, a phenotype that is similar to normal HSC [20, 21]. In addition, serial transplantation experiments performed by Shultz et al. in xenotransplant-permissive NOD/ SCID/IL2Rγ−/− (NSG) mice demonstrated that long-term engraftment and self-renewal capacity of human AML cells resided exclusively in the CD34+CD38− population [44], but not other subpopulations. LSCs were shown to be mainly in the G0 phase of the cell cycle, confirming their quiescent nature [44, 45]. Despite these studies, controversy arose about the immunophenotype of the LSC (Table  4.1) [46]. It became clear that although the LSC is contained within the CD34+CD38− population in most patients, some exceptions exist where LSC can be found in the CD34+CD38+ cells as well [20, 21, 26] or the CD34− fraction (K, I) [26]. Moreover, identifying a more refined immunophenotype discriminating LSC from normal HSC would enable clinicians to better evaluate the minimal residual disease (MRD) after therapy and design LSC-targeted therapies. Some of the identified surface markers associated with LSC

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include the C-type lectin-like 1 (CLL-1/MICL/ CLEC12A), c-MPL, CD123, CD44, CD47, CD96, CD26, and CD25 [47–55], but a unique phenotype has not been established thus far.

4.2.4.2 Clinical Relevance of the LSC If LSC, as defined in mouse models, are also relevant in the clinic, they constitute the ideal targets for consolidation therapy against MRD [56]. In 2005, van Rhenen et  al. demonstrated that a high frequency of CD34+CD38− LSCs at the time of diagnosis predicts high-rate MRD after chemotherapy and poor overall, diseasefree, and relapse-free survival, both in an in vivo model and in correlation studies in patients [57]. Presence of CD34+CD38− blasts also correlates with older age of diagnosis and adverse cytogenetic profile in AML [58]. Interestingly, the relative ability of AML cells to successfully engraft in immunodeficient mice, a property directly correlated with LSC population, correlates with adverse clinical features [59]. In 2010, two groups independently demonstrated that HSCand LSC-enriched populations share very similar transcriptional “stem cell-like” or “self-­renewal” gene expression signatures that reflect stem cell function in  vivo [60] and that they are predictive of adverse clinical outcome in AML [60, 61]. The predictive value of this LSC score appeared to be independent of other

Table 4.1  Advantages and disadvantages of immunotherapy for acute myeloid leukemia, depending on the antigen targeted Antigen Examples LSA >Mutations: Flt3, NPM1 >Translocations: AML1-ETO, DEK-CAN, PML-RARα LAA WT1, AurAkinase, Bcl2, Muc1, SSX21P MIHA

HA-1, HA-2 (hematopoietic specific)

Advantages Specificity Oncogenicity LSC expression LSC expression Broad applicability (AML and other types of cancer) High-avidity T cells available AG recognized by both CD4+ and CD8+ T cells Potential multivalent response

Disadvantage(s) Expression restricted to defined AML subgroups → use limited to small patient populations Low avidity of T cells for AG Potential toxicity to normal tissues Use limited to allo-HSCT Limited number of AG defined Potential cause of GVHD

LSA leukemia-specific antigen, LAA leukemia-associated antigen, MIHA minor histocompatibility antigen [57, 58] Adapted by permission from Nature Reviews Cancer (Copyright 2004. Bleakley and Riddell [62] and Immunotherapy. Copyright 2013)

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risk factors in multivariate Cox regression analysis, which further supports the clinical relevance of LSC [60, 61].

4.2.5 H  ow Do Gene Alterations in the LSC Lead to the Clinical Presentation of AML? Genetic alterations lead to hyperproliferation (class 1) and differentiation block (class 2), which further enhance the risk of genetic damage. This leads to a vicious feed-forward loop that ultimately gives rise to this life-threatening disease. Critical events in this  loop include the following: • Clonal outgrowth and uncontrolled, limitless expansion, which is achieved by mutations that lead to constitutive activation of pathways involved in cell cycle. These mutations might activate or upregulate proto-oncogenes or abrogate the normal function of proteins involved in the restriction point of the cell cycle [62]. • Not only inefficient maturation from the malignant blasts is seen, but  also from the residual normal stem cells. The latter cannot be attributed to  a maturation arrest due to mutation (as with malignant blasts), but contributing factors are  altered marrow cytokine milieu produced by the malignant blasts, inhibition of the normal differentiation, and—to a lesser extent—crowding effect of aberrantly proliferating cells. • Constitutive release of chemokines by the malignant blasts that also express several chemokine receptors maintains the feedback loop. Other cytokines especially the hematopoietic growth factors, angioregulatory factors, and matrix metalloproteinase (MMP) system are also released by the AML cells. These factors improve their propagation and survival [63]. • Expression of P-glycoprotein (Pgp, MDR1 or ABCB1) and plasma membrane transporters enables AML cells to efflux a variety of substrates from the cytoplasm, including chemotherapeutic agents. This leads to the

development of resistance to chemotherapy [64]. • Resistance to apoptosis and defective or proficient DNA damage response [56] enables accumulation of disease-promoting mutations.

4.3

Immunotherapy for AML

Despite the progress that has been made in the past decades, AML still remains a therapeutic challenge. Primary induction failure is especially  common in elderly patients, and even if chemotherapy is successful at inducing remission, the probability of relapse is high [65]. Immunotherapy for AML was put forward almost 40  years ago when it was first speculated that AML blasts were phenotypically  distinct from normal blasts. This led to preliminary attempts to improve immune responsiveness to AML by the administration of inactivated autologous AML blasts with BCG [66]. Next, an important insight into the role of the immune system in controlling AML came from allogeneic HSCT and the observed graft-versus-leukemia (GVL) effect. Increasing evidence exists that the success of allo-HSCT in curing AML is largely attributable to this GVL effect, especially in the context of non-myeloablative HSCT [67–71]. Both donor NK cells and donor T cells contribute to the suppression and elimination of leukemic cells [69]. Although allo-HSCT remains to be the most successful post-remission therapy in AML, it has its price of important morbidity and mortality due to infections, toxicity due to conditioning regimen, and possibly detrimental acute and chronic graft-­ versus-­ host disease (GVHD) [67, 68]. These important complications limit the applicability of HSCT in patients of older age and those with comorbidities. Targeted forms of immunotherapy were thereafter a main focus of interest, due to their specificity and the fact that they do not require conditioning and have less side effects. The biggest challenge here lies in identifying the ideal tumor antigen, present on LSCs, but low to absent on normal hematopoietic cells and other vital tissues.

4  Immunopathology and Immunotherapy of Myeloid Leukemia

4.3.1 Antigens to Target in AML 4.3.1.1 Antigens Presented by MHC After Internal Processing Major histocompatibility complex (MHC)presented antigens are the main targets for T-cell-­ mediated immunotherapy, as in tumor vaccination and adaptive T-cell therapy. Three types of MHC-­ presented antigens, which are primarily peptides and proteins, are described in AML: leukemia-­ specific antigens (LSA), leukemia-associated antigens (LAA), and minor histocompatibility antigens (MIHA). LSA arise when mutations or translocations lead to formation of new, leukemia-­ specific antigens, making them ideal for targeted immunotherapy. LAA are expressed both on normal and leukemic cells, including LSC. LAA can serve as immunotherapy targets if they are either significantly overexpressed on leukemic cells and are not expressed on vital tissues and organs, or if  their physiological expression is restricted to certain developmental stages, i.e., they are not expressed by mature non-leukemic hematopoietic stem cells [69, 72–74]. Finally, hematopoietic-­ specific MIHA that differ between donor and recipient could be efficient targets for AML immunotherapy in the context of previous allo-­ HSCT [69, 71, 75–79]. Correct identification of these MIHA antigens is of paramount importance as both GVHD and GVL are driven by T-cell response to MIHA [69]. A ranking of the most promising cancer antigens is reviewed by Cheever et  al. [80], and by other groups [80, 81]. Some examples of the advantages and disadvantages of these antigen types are summarized in Table 4.1.

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CD44(v6), CD47, and IL-3R (CD123) [84]. Indeed, bi- and tri-specific monoclonal antibodies are developed that engage both NK cells and cytotoxic T cells to direct cytotoxicity against residual AML disease. Toxin and radio-­ conjugated monoclonal  antibodies can further increase the efficacy of mAbs [85]. These antibodies and their applications are reviewed in [85, 86]. These antigens however, have the disadvantage of being also expressed on normal cells and  tissues, resulting in important side effects, as seen with the anti-CD33 antibodies [85, 87] (see section “Monoclonal Antibodies”). One way to overcome self-toxicity of these agents is through generation of leukemia-specific antigens, through knockout of the native leukocyte antigens from autologous HSCs before they are infused in the context of  HSCT [88]. These antigens would then be recognized as leukemia specific by the progeny of engrafted HSCs. Unfortunately, a defective engraftment and differentiation are common in genetically engineered autologous HSCs, hindering application of this method in patients [89].

4.3.2 Current Immunotherapeutic Strategies for AML

Active immunotherapy (e.g., modified leukemic cells, peptide, DNA, or dendritic cell-based vaccinations) requires a patient with an well functioning immune system and can only exploit the available T-cell receptor (TCR) repertoire of the patient. Meanwhile, high-affinity TCR-bearing T cells specific for self-antigens (TAA) are expected to be deleted after negative selection in the thy4.3.1.2 Surface Antigens Surface-expressed leukemia-specific antigens mus. In addition, the question is whether active are ideal targets for monoclonal antibodies immunotherapy will be able to combat the immu(mAbs) and chimeric antigen receptor-modified nosuppressive milieu produced by the tumor T cells (CAR T cells). Surface antigens on AML microenvironment in the host immune system. that are already targeted by mAbs or chimeric Passive immunotherapeutic strategies like adopT-cell receptors include CD33 [82], Flt3L-ITD tive transfer of AML-specific T cells or NK cells [83], PR1/HLA-A2 [84], as well as a few tumor-­ are expected to be more potent therapies to target associated antigens and LSC markers including LAA and MIHA. Immunotherapy with mAbs has CLL1, CD33, CD13, CLL-1 and CD38 proven efficacy in AML as well.

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4.3.2.1 Active Immunotherapeutic Strategies

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dritic cells (DCs) as professional APCs to prime naïve T cells [100]. Furthermore, the synthetic peptide approach which may miss the immunoPeptide Vaccination dominant epitopes is replaced by the addition of Among peptide-based vaccines, the HLA-A2-­ whole protein or mRNA transfection of DCs with binding nonamer peptides PR-1, PRTN3, prefer- the genetic codes of dominant epitopes. Most of entially expressed antigen in melanoma the strategies use DCs which are derived from (PRAME), peptide epitopes of Wilms’ tumor 1 monocytes, and only those strategies used in clin(WT1), receptor for hyaluronan-mediated motil- ical trials are mentioned here: ity (RHAMM), as well as hTERT are among the • DCs pulsed with leukemic cell lysates [101], most widely researched and developed peptide apoptotic leukemic cells, or modified WT1 vaccines for AML [90–94]. peptides [102] have been successfully Leukemia vaccines are combined with adjuvants explored in small clinical trials. such as montanide or keyhole limpet hemocyanin • Even more promising are mRNA-­ (KLH), with or without concurrently administered electroporated DCs. In 2010, a phase I/II clinigranulocyte-macrophage colony-­stimulating factor cal trial (clinicaltrials.gov ID: NCT00834002) (GM-CSF), to maximize their efficacy. The potency investigated the effect of vaccination with of peptide vaccines may potentially be increased by full-length WT1 mRNA-electroporated autolmodifying peptides to enhance TCR affinity or by ogous dendritic cells in ten patients with using synthetic long peptides (SLP)(instead of exact AML, and in five of them, a molecular remisnatural MHC-­binding nonamer peptides) delivering sion was reached, although not always persistantigens in a more efficient and stable manner and ing. Priming DCs to produce IL-15 or IL-15 optimize their presentation by antigen-presenting receptor alpha has also proved efficacy to cells (APCs). Toll-like receptor (TLR) ligand-pepeliminate human AML blasts via potentiation tide conjugates constitute an attractive vaccination of antitumoral NK cell activity [103]. modality, sharing the peptide antigen and a defined • In an attempt to generate WT1-presenting adjuvant in one single molecule. Antibodies directed DCs with a longer in  vivo persistence, against surface antigens of T cells and NK cells Stripecke and colleagues recently developed a such as autoantibodies against 4-1BB and protricistronic lentiviral vector co-expressing a grammed cell death-1 (PD-1) ligand on T cells, truncated form of WT1, granulocyte-­ APCs, and NK cells enhance the diversity of T-cell macrophage colony-stimulating factor (GM-­ response and potentiate the resulting antitumor CSF), and IL-4, which was used for the activity [95, 96] while antagonizing/decreasing transduction of human monocytes. This led to T-cell exhaustion within an immunosuppressing a rapid self-differentiation of these cells into tumor milieu [97, 98]. “SmartDC/tWT1” that showed very promisUnfortunately, these studies comprise small ing potential for use as immunotherapy against and diverse groups of patients treated with differWT1-expressing tumors [104]. ent vaccines according to different administration schedules, making it difficult to draw meaningful 4.3.2.2 Passive Immunotherapeutic Strategies conclusions about the true efficacy of peptide-­ based vaccinations in AML. Nonetheless, clinical responses to vaccines range from reduction in Monoclonal Antibodies marrow blasts to complete remissions in a small Due to their antigen specificity and minimal toxnumber of patients. icity, monoclonal antibodies have proven to be an excellent targeted anti-AML and even anti-LSC Dendritic Cell Vaccination therapy. Antibody-dependent T-cell-mediated In order to circumvent the limitations inherent to cytotoxicity (ADCC), complement activation, peptide vaccines [99], researchers have intensely direct pro-apoptotic effect, and inhibition of sigstudied the possibility to use antigen-loaded den- nal transduction cascades that are essential for

4  Immunopathology and Immunotherapy of Myeloid Leukemia

homeostasis, proliferation, or interaction with the microenvironment [105] are among possible therapeutic mechanisms of monoclonal antibodies. mAbs are often conjugated with radioisotopes or toxins leadint to enhanced killing of the recognized target. Radioisotope-­ coupled anti-CD45 antibodies have successfully been used as part of the conditioning before allo-HSCT. Anti-CD33 mAbs are as mentioned widely studied and have proven clinical efficacy while posing important toxicity in several trials [87, 102, 106]. High level of expression of CD33 on LSC has led to continuation of these trials. Promising trials have used anti-CD33 mAbs in more fractionated, less toxic administration in conjugation with chemotherapy [106, 107]. Alternatively, mAbs are designed to block the immune-regulatory effect of molecules, such as cytotoxic T lymphocyte antigen-4 (CTL4) or PD-1, and thereby unleash cytotoxic T lymphocyte function [90, 95, 97].

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responses after HSCT, or even more ideally, in an allogeneic setting [111–113]. A new promising immunotherapeutic strategy, using PBMC transduced with the genetic construct of a chimeric antigen receptor (CAR), is developed through in vitro studies. A CAR usually consists of the VH and VL domain of a tumor antigen-directed antibody coupled to the CD3ζ chain, alone or combined with the signaling motifs of CD28 or CD137 to enhance the signaling. Also here, the largest challenge remains the identification of a CAR-target on AML that is not present (or only at low level) on normal hematopietic cells.

Adoptive NK Cell Transfer NK cells have an important antileukemic effect, mediated through killer-cell immunoglobulin-­ like receptor (KIR). In the context of haploidentical HSCT, NK cell adoptive therapy for AML is being explored in clinical trials (NCT01904136). Similar to dendritic cells, NK cells activated by priming signal CNDO-109 and/or IL-2 or IL-15 Adoptive T-Cell Transfer have been successfully implemented in AML Various research has been done to direct T cells patients with promising results in relapse or more specifically toward the AML cells. In AML refractory acute leukemia and considerable patients, autologous- or donor-derived antigen-­ increase in relapse-free survival [114, 115]. specific T cells can be isolated from peripheral blood by pMHC-multimer staining, CD137- or CD154-based assays, IFNγ-secretion techniques, 4.4 Concluding Remarks or repeated ex vivo stimulation with antigen and subsequent expansion. Importantly, all of these This chapter reviewed both the immunopatholtechniques still require the availability of preex- ogy of AML and currently explored immunotheristing high-affinity antigen-specific T cells in the apeutic strategies, in  vitro and in clinical trials. patient or the donor in the context of allo-HSCT The genetic alterations leading to the differentia[108–110]. As for LAA, these are usually absent tion block and hyperproliferation of leukemic due to negative selection in the thymus as part of blasts were also discussed. The potential clinical the normal induction of central tolerance. In relevance of the LSC in the pathogenesis of AML order to circumvent the issue of tolerance and and its therapeutic relevance were also emphainduce LAA specificity to T cells, peripheral sized. LSC might be one of the reasons why blood mononuclear cells (PBMC) can be trans- AML still remains a tremendous therapeutic duced with a high-affinity TCR recognizing challenge nowadays. New immunotherapeutic LAAs present on AML blasts and LSCs. Such a strategies are awaiting further validation and high-affinity TCR can be isolated from LAA-­ clinical trials. Various immunotherapeutic stratespecific T cells generated in vivo or in vitro in an gies using T cells, NK cells, dendritic cells, and autologous setting such as from tissue-infiltrating monoclonal antibodies, and the possible target lymphocytes of a patient with complete clinical antigens, were discussed.

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116 logic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS. Blood. 2009;113(26):6541–8. https://doi. org/10.1182/blood-2009-02-202598. 94. Greiner J, Schmitt A, Giannopoulos K, Rojewski MT, Götz M, Funk I, et al. High-dose RHAMM-R3 peptide vaccination for patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma. Haematologica. 2010;95(7):1191–7. https://doi.org/10.3324/haematol.2009.014704. 95. Kerage D, Soon MSF, Doff BL, Kobayashi T, Nissen MD, Lam PY, et  al. Therapeutic vaccination with 4-1BB co-stimulation eradicates mouse acute myeloid leukemia. Onco Targets Ther. 2018;7(10):e1486952. https://doi.org/10.1080/2162 402x.2018.1486952. 96. Melief CJM, van der Burg SH.  Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines. Nat Rev Cancer. 2008;8(5):351– 60. https://doi.org/10.1038/nrc2373. 97. Tan J, Chen S, Lu Y, Yao D, Xu L, Zhang Y, et  al. Higher PD-1 expression concurrent with exhausted CD8+ T cells in patients with de novo acute myeloid leukemia. Chin J Cancer Res. 2017;29(5):463–70. https://doi.org/10.21147/j. issn.1000-9604.2017.05.11. 98. Khan S, Bijker MS, Weterings JJ, Tanke HJ, Adema GJ, van Hall T, et al. Distinct uptake mechanisms but similar intracellular processing of two different toll-­ like receptor ligand-peptide conjugates in dendritic cells. J Biol Chem. 2007;282(29):21145–59. https:// doi.org/10.1074/jbc.M701705200. 99. Van Driessche A, Gao L, Stauss HJ, Ponsaerts P, Van Bockstaele DR, Berneman ZN, et al. Antigen-­ specific cellular immunotherapy of leukemia. Leukemia. 2005;19(11):1863–71. https://doi. org/10.1038/sj.leu.2403930. 100. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392(6673):245– 52. https://doi.org/10.1038/32588. 101. Lee J-J, Kook H, Park M-S, Nam J-H, Choi B-H, Song W-H, et al. Immunotherapy using autologous monocyte-derived dendritic cells pulsed with leukemic cell lysates for acute myeloid leukemia relapse after autologous peripheral blood stem cell transplantation. J Clin Apher. 2004;19(2):66–70. https:// doi.org/10.1002/jca.10080. 102. Anguille S, Van de Velde AL, Smits EL, Van Tendeloo VF, Juliusson G, Cools N, et al. Dendritic cell vaccination as postremission treatment to prevent or delay relapse in acute myeloid leukemia. Blood. 2017;130(15):1713–21. https://doi. org/10.1182/blood-2017-04-780155. 103. Van den Bergh J, Willemen Y, Lion E, Van Acker H, De Reu H, Anguille S, et  al. Transpresentation of interleukin-15 by IL-15/IL-15Ralpha mRNA-­ engineered human dendritic cells boosts antitumoral natural killer cell activity. Oncotarget. 2015;6(42):44123–33. https://doi.org/10.18632/ oncotarget.6536.

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5

Immunopathology and Immunotherapy of Acute Lymphoblastic Leukemia Thomas Stübig and Nicolaus Kröger

Contents 5.1 Immunopathology of Lymphoblastic Leukemia  5.1.1    General Considerations  5.1.1.1  Lymphocyte Development as Biological Basis of Disease  5.1.1.2  Genetics in Acute Lymphatic Leukemia  5.1.2    Immune Phenotype and Targets in Acute Lymphatic Leukemia  5.1.2.1  Cell Surface Marker  5.1.2.2  Challenges for Immunophenotyping as MRD Marker 

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5.2 Immunotherapy for Acute Lymphatic Leukemia  5.2.1    Cellular Approaches  5.2.1.1  T Cells and Modified T Cells  5.2.1.2  NK Cell Approaches  5.2.2    Antibodies  5.2.2.1  CD20 Antibodies  5.2.2.2  CD22 Antibody  5.2.2.3  CD52 Antibody  5.2.2.4  CD19 Antibody  5.2.3    Stem Cell Transplantation  5.2.3.1  Allogeneic Stem Cell Transplantation (Allo SCT) 

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References 

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T. Stübig Department of Internal Medicine II, Haematology and Oncology, University Medical Center Schleswig Holstein, Kiel, Germany e-mail: [email protected] N. Kröger (*) Department of Stem Cell Transplantation, Center for Oncology, University Medical Center Hamburg-­ Eppendorf, Hamburg, Germany e-mail: [email protected]; [email protected]­hamburg.de

5.1

Immunopathology of Lymphoblastic Leukemia

5.1.1 General Considerations The incidence of acute lymphoblastic leukemia (ALL) is about 1–4/100,000 persons per year. Most of the cases will occur in children below 6 years of age and approximately 85% of all ALL cases will be of a B-cell phenotype.

© Springer Nature Switzerland AG 2020 N. Rezaei (ed.), Cancer Immunology, https://doi.org/10.1007/978-3-030-57949-4_5

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5.1.1.1 Lymphocyte Development as Biological Basis of Disease Acute lymphatic leukemia rises from lymphoid progenitors. In humans LIN−/CD34+/CD38− cells mark a stem and progenitor population, in which three different subcompartments can be found: CD90+/CD45RA−, CD90−/CD45RA−, and CD90−/CD45RA+. The LIN−CD34+/ CD38−/CD90+/CD45RA− fraction is highly enriched in HST [1]. The common lymphoid progenitor (CLP) can be defined as LIN−/CD10+/ CD34+ [2]. From this CLP, B cells continue to differentiate into pre-proB-cells, which then become pro-B-cells, large pre-B-cells, and small pre-Bcells and thereafter turn into immature B cells. This differentiation is highly regulated by a vast number of transcription factors, which are specifically expressed over a specific time period to ensure the correct development of a B cell (reviewed in [3]). B-ALLs are heterogenic diseases, with an accumulation of abnormal cells. Traditionally, B-ALL cells have been compared to their normal counterparts in B-cell development. This was mostly done because of the morphology and immune phenotypical similarities. However, this head-to-head comparison misses some ALL features, for example up to 30% of ALL cases express myeloid markers [4], and makes the direct comparison with normal B cells difficult. Further nowadays research on chromosome changes in ALL has marked that some of the initiating change occurs very early (e.g., being of parental origin), while the cell that turns to be malignant reaches a later stage in development [3]. The genetic changes that occur, and in some cases lead to the development of B-ALL, are discussed below. The increasing role of the signaling of the pre-B-cell receptor and the signal transduction by this receptor should be mentioned. During the B-cell development the pre-B-­ cell receptor has a dual function: it promotes survival and proliferation and subsequently it induces differentiation in the B-cell compartment [5]. Two downstream targets, which are mainly important for the tumor-suppressive function of the pre-B-cell receptor, are IKAROS and AIOLOS [6]. Interestingly, in 80% of the BCR-­ ABL+ ALL the gene responsible for IKAROS (IKZF1) has been deleted [7], underlining the

importance of those events in the signal transduction for the development of ALL. In parallel to AML there is a two-hit model for ALL.  The first “hit” is posed to be a chromosomal abnormality (the majors are listed below). However, this first “hit” is not sufficient for the induction of an ALL.  Therefore, a second “hit” like deletion of tumor-suppressor genes is needed to fully generate an ALL.

5.1.1.2 Genetics in Acute Lymphatic Leukemia Numerical Chromosome Changes Hyperdiploid

High hyperploidy (>50 chromosomes) can be found in up to 30% of all cases in children [8]. In contrast, the number of hyperploid cases in adults is significantly lower—around 10%. Hyperploidy is associated with a good prognosis in children. This may be explained by the higher sensitivity against chemotherapy [9]. The impact of hyperploidy in adults is less clear. While some reports found a benefit, others contradict these findings [10, 11]. Hypodiploid

Hypoploidy is defined as less than 46 chromosomes in a cell. Approximately 10% of children and nearly 10% of the adult cases will show a hypoploid chromosome content [12]. Patients with hypoploidy have an inferior outcome compared to patients with a hyperploid leukemia [13]. This is even more of importance since the event-free survival depends on the number of chromosomes and patients having less than 44 chromosomes showed an 8-year EFS of 30% [14]. Structural Changes MLL Rearrangements

In ALL cells there are several rearrangements involving the MLL gene at chromosome 11q23. The most common are t(4;11)(q21;q23), t(11;19) (q23;p13.3), and t(9;11)(p22;q23), which lead to the fusion of the 5′ portion of the MLL with the 3′ portion of AFF1, MLLT1, and MLLT3 [15, 16]. Besides these frequent translocation partners there are over 50 known other partners that fuse

5  Immunopathology and Immunotherapy of Acute Lymphoblastic Leukemia

to MLL. Interestingly, there are two major breaking clustering regions in the MLL gene between exon 5 and exon 11 regardless of the fusion partner [17]. Fusion proteins will keep the transcription repressing domain of MLL and gain the 3′ portion of the partner, which is mostly a transcription factor. MLL rearrangements are usually associated with poor outcome [18–20]. BCR-ABL

The translocation of 9q to chromosome 22q leads to the formation of the Philadelphia (Ph) chromosome. This fusion protein is the hallmark of the chronic myeloid leukemia (CML), but is also found in ALL. Around 5% of the children and up to nearly 30% of adults will show the t(9;22) (q34;q11.2), which can be detected by conventional cytogenetics, FISH, or PCR [12, 21, 22]. The latter is often used for quantification, which makes this method extremely interesting for detection of minimal residual disease (MRD) [23]. Genetically, the Ph chromosome is a fusion of the 5′ portion of the BCR gene to the 3′ portion of the Abelson leukemia virus proto-oncogene (ABL1). The breaking points of BCR cluster into two regions: the major clustering region (M-bcr), where mostly the breaking point in CML occurs, and the minor (m-bcr), where mostly the breaking point in ALL is found. Therefore, two different translocation products can occur, the p210  kDa and the smaller p180–190  kDa, depending on the involved breaking point. Patients will show only one of the two possible fusion samples [23, 24]. Detection of the t(9;22) in patients with ALL leads to an adverse prognosis for the disease [25, 26]. Tyrosine kinase inhibitors are active in Ph+  ALL, but after the first response to treatment, a large portion of patients relapse, even during treatment [27, 28]. ETV6-RUNX1

The translocation t(12;21)(p13:q22) leads to the fusion protein ETV6-RUNX1. This is the most frequent structural chromosome change in pediatric ALL, which occurs in nearly 30% of the cases [29, 30]. While being quite frequent in childhood lymphatic leukemia, ETV6-RUNX1 transcripts are rare in adults with a frequency

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below 5% [31, 32]. RUNX1 is a transcription factor that regulates a vast number of genes that are important for human hematopoiesis [33]. Occurrence of the ETV6-RUNX1 translocation is associated with a good prognosis in childhood ALL. This is especially seen in the group of younger children (1–9 years of age) rather than in the group of children above the age of 10 years [14, 32, 34, 35]. A hypothesized mechanism is that the occurrence of the translocation sensitizes the malignant cell towards classical chemotherapy drugs used in ALL protocols [36, 37]. Molecular Mutations With the availability of high-throughput sequencing new molecular mutations and their impact on the outcome of ALL have occurred. Most of them are involved in B-cell development (e.g., PAX5, IKFZ1, EBF1), which can be found in two-thirds of all B-ALLs and CDKN2A/CDKN2B mutations, which occur in over 80% of T-ALLs. Recently, a new subtype of B-ALL has been described, the BCR-ABL1-like ALL. This subentity has already been introduced as a provisional entity in the WHO2016 classification. This entity is based on a gene expression profile, which is very similar to BCR-ABL1-positive ALL. Retrospective analyses have shown an inferior outcome; however, the impact of the BCR-­ABL1-­like ALL in the context of risk-adapted therapy regimens is not fully clarified. Furthermore, a standardized diagnostic approach to define BCR-ABL1-like ALL is also missing. However, more relevant than the exact diagnosis of BCR-ABL1-like ALL itself is the identification of molecular mutations associated with this gene expression profile. Studies have shown that molecular aberrations in CRLF2, JAK mutations, or ABL-rearrangements may be a therapeutic target in BCR-ABL1-like ALLs.

5.1.2 Immune Phenotype and Targets in Acute Lymphatic Leukemia 5.1.2.1 Cell Surface Marker The expression of proteins at the cell surface is of extreme importance for the immune system to recognize antigens.

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ALL cells show a vast number of antigens that are linked to normal B-cell development. In a more simplified way, one can define three major subgroups which can be classified by their immune phenotype. The early precursor or pro-­ ALL is marked by the expression of CD19, cytoplasmic CD79a, cytoplasmic CD22, and nuclear TdT expression. The intermediate stage or common ALL is marked by CD10 and the late precursor or pre-B-ALL stage is marked by the cytoplasmic expression of the μ chain. Typical phenotypes are listed in Table 5.1. Lymphoblasts are positive for CD10, surface CD22, CD24, Pax5, and TdT in most cases. The expression of CD34 and CD20 varies. CD45 may be absent. Myeloid markers may also be expressed like CD13, CD15, CD33, and CD68 [38] (Table 5.2).

5.1.2.2 Challenges for Immunophenotyping as MRD Marker The immune phenotype of ALL blasts is frequently used as a marker for the detection of minimal residual disease (MRD). In the modern treatment protocols, the intensification of treatment according to the amount of MRD levels has improved the overall survival and outcome in nearly every study on ALL [40–50]. Besides the molecular MRD detection of the B-cell receptor/ T-cell receptor rearrangement, flow cytometry is widely used as MRD detection method. As MRD levels are critical for therapy intensification the requirements for MRD analysis by flow have increased. Today’s approaches follow recommendations like the EUROFLOW guidelines. Those guidelines offer the chance to reach a sen-

Table 5.1  Different phenotypes of ALL Stage Early precursor

Immune phenotype HLA-DR, TdT, cyCD22, cyCD79a, CD19, CD22 (variable)

Intermediate precursor (common)

HLA-DR, TdT, cyCD22, cyCD79a, CD19, CD10, CD20 (variable), CD22 (variable)

Late precursor

HLA-DR, TdT (variable), cyCD22, cyCD79a, CD19, CD10, CD20, CD22 (variable), cytoplasmic μ

Target CD19 Blinatumomab CD22 Inotuzumab CD19 Blinatumomab CD20 Rituximab, ofatumumab CD22 Inotuzumab CD19 Blinatumomab CD20 Rituximab, ofatumumab CD22 Inotuzumab

Table 5.2  Function of different surface marker CD Other name CD19 CD20

Function Forms complex with CD21 and CD81; co-receptor for B-cells, binds cytoplasmic tyrosine kinases and PI3K Oligomer of CD20 is involved in Ca2+ transport and B-cell activation

CD22 BL-CAM

Binds sialoconjugates

CD52 CAMPATH-1, HE5

Unknown

Antibody Blinatumomab SGN-CD19A Rituximab Ofatumumab Obinutuzumab Inotuzumab Epratuzumab Alemtuzumab

5  Immunopathology and Immunotherapy of Acute Lymphoblastic Leukemia

sitivity of 10−5 to ensure valid results [39]. However, the problem with modern therapy regimens is that most of them especially the immunebased treatment influences the immune phenotype of ALL.  The use of anti-CD19 CAR-T or antibodies against CD19, CD20, or CD22 (see below) alters the expression level or total expression on ALL blasts. Therefore, a carful design of flow panels with robust backbone markers to detect ALL blasts is needed.

5.2

Immunotherapy for Acute Lymphatic Leukemia

5.2.1 Cellular Approaches 5.2.1.1 T Cells and Modified T Cells T cells as part of the adoptive immune system have the ability to recognize and kill tumor cells. This quality is part of the concept of donor lymphocyte infusions (DLI, discussed below) as cellular therapy against various types of hematological cancers [51–53]. However, the response rates of ALL to DLI are inferior compared to other hematological cancer types and mostly below 15% [54, 55]. Possible explanations may be that ALL is an aggressive disease, where time for priming the naïve T cells is missing and ALL cells lack costimulatory molecules [56]. Even ex  vivo prestimulation of T cells with CD3/CD28 antibodies does not enhance the benefit of DLI for ALL patients [57]. Another approach to enhance the T-cell toxicity towards ALL is an ex vivo priming against known tumor antigens. WT-1 and BCRABL are the best studied antigens so far and first reports are promising, showing that the priming may lead to a better control of the tumor cells [58]. An alternative to conventional T cells for adoptive immunotherapy is to use genetically modified T cells. Here the α and β subunit of the T-cell receptor (TCR) of a tumor-specific T-cell clone is used. First results in solid tumor like melanoma were promising [59], but due to the antigen restriction of the T-cell clone the application in hematology was limited [60, 61]. Besides, many tumors downregulate the HLA

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molecule, thereby lowering the T-cell ability of recognition [62]. A chance to avoid the limitations of TCR gene transfer is to use chimeric antigen receptors (CARs). CARs are composed of a single-chain variable-fragment (scFv) antibody specific to a tumor antigen, fused to a transmembrane domain and a T-cell signaling motif. DNA constructs were stably incorporated by either lentiviral, γ-retroviral transduction, electroporation, or “Sleeping Beauty” transposon. While first CAR-T cells were only incorporated with T-cell receptor CD3ζ signaling domain and showed only minor proliferation and persistence capacity, newer generations of CAR-T incorporate a number of co-stimulatory domains like CD137, CD28, CD27, or CD134. These co-stimulatory molecules keep CAR-T more potent and longer persistent and help to expand them in vivo. The most used combination of co-stimulatory domains is CD137 and CD28. CD137 has shown to prolong the persistence, while CD28 increases the killing capacity of CAR-T [63]. Especially the expansion is a critical point in CAR-T treatment. First, the modified T cells were ex  vivo expanded to 3 × 106 cells/kg. After the administration of the cells, they expanded 1000-fold within 2 weeks. As powerful as this expansion is, the question of how stable these cells are is still under debate. Reports showed that CAR-T could be detectable for 3 months, but others showed a persistence up to 39 months [64, 65]. Several studies have shown very promising results for CAR-T cells in the relapsed/refractory ALL setting. Most of the trials have shown CR rates between 69 and 90%, which is impressive considering that the included patients were heavily pre-treated. Even more impressive is that using the CAR-T cells MRD negativity was achieved and those patients showed an OS of 76% at 6 months [66] and a LFS of 49% at 18  months. However, all trials showed a variance in the applied cell dose and differed in the used CAR construct. The difference of the applied CAR-T-­ cell approaches makes the results difficult to compare, but clearly, those data are a robust basis for the current CAR-T-cell trials.

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Additionally to autologous anti-CD19 CAR-T cells, some studies investigated allogeneic CAR-T cells for relapse after allogeneic stem cell transplantation. While induction of graft vs. host disease (GvHD) was the major drawback in the published data, only one report showed an induction of GvHD [67]. However, those who did not observe a GvHD induction used lower cell dosages and showed promising 40% remission rates [68, 69]. Further studies have to confirm whether allogeneic CAR-T cells without prior allo SCT are also a therapeutic option, as reported by Cai et al. [70, 71]. As powerful as CAR-T cells are, their therapeutical use has side effects; most of them are mild and reversible; however, neurotoxicity, cytokine release syndrome (CRS), and on-tumor off-target effects are more of concern. Generally reported CNS side effects included delirium, dysphasia, akinetic mutism, and seizures. But neurotoxicity was the reason why the phase II ROCKET study was stopped in Summer 2016 after five patients died, two of them due to CNS edema. If the used CAR-T construct and/or the preparative conditioning regimen causes the observed effects is still under debate. Hyper-­cytokine production of the activated CAR-T cells causes the cytokine release syndrome. While nearly all patients will experience a mild form of CRS, one-third will show some severe symptoms including hypotension by vascular leaking, which could lead to multi-organ failure [72, 73]. The on-tumor offtarget effect describes the phenomenon that CAR-T cells cannot discriminate between normal CD19+ B cells and malignant CD19+ blasts. Therefore, a complete B-cell depletion occurs, which can lead to agammaglobulinemia, which increases the risk of infections. However, this can be managed by substitution of IV immunoglobulin and antibiotic prophylaxis. Further challenges in the treatment of CAR-T cells represent the relapses after CAR-T-cell therapy. Reasons for a relapse are a loss of functional CAR-T cells and CD19-negative ALL relapses. To increase the persistence of CAR-T cells an adequate lympho-depletion is essential. If further addition of BTK inhibitors or PDL1 inhibitors is helpful is under investigation. In the case of

CD19-negative relapses, first promising CAR-T-­ cell approaches using an anti-CD22 CAR-T construct are reported [74, 75].

5.2.1.2 NK Cell Approaches NK cells are part of the innate immune system. In contrast to B or T cells, NK cells do not have receptors that were rearranged during their maturation, making them less specific for one special antigen. Instead, the receptors expressed on the NK cell surface have more the function of tightly controlling NK cell activation. One of those receptors is the killer-immunoglobulin-like receptor (KIR, CD158) family. This family consists of different members that have activating, as well as inhibitory, functions on NK cells. NK cell cytotoxicity is triggered by tumor cells, which lack the expression of some of the MHC class I molecules, which is referred to as “missing self” hypothesis [76]. Inhibitory KIRs recognize groups of HLA-A, HLA-B, and HLA-C alleles. When KIR-inhibitory NK cells target cells lacking the corresponding HLA class I ligand, the target cell will be lysed (KIR-ligand model) [77]. Up to now NK cell alloreactivity seems not to be beneficial in the treatment of ALL [78], but some reports with genetically modified NK cells provide some encouraging data. Retroviral or electroporation of NK cells to induce a CD19-­ targeting CAR led to increased NK cell-mediated killing of ALL cell lines as well as primary patients’ ALL blasts [79–81].

5.2.2 Antibodies 5.2.2.1 CD20 Antibodies The CD20 molecule is an integral membrane protein that is specific to B cells and seems to be important for calcium transport across the cell membrane [82]. The expression of CD20 is linked to a poor prognosis [83]. CD20 is expressed on leukemic blast cells of about 50% of the patients with B-lineage ALL. Rituximab is a chimeric mouse/human antibody that has dramatically changed the therapy of non-Hodgkin lymphoma. Since CD20 is also expressed in B-ALL cells, the antibodies have

5  Immunopathology and Immunotherapy of Acute Lymphoblastic Leukemia

been used also in the ALL setting. Reports have shown that the addition of CD20 antibodies to conventional chemotherapy leads to a higher rate of complete response, as well as a better overall survival [84, 85]. In a larger study, the French ALL study group showed that the addition of rituximab leads to a longer event-free survival in Ph patients without increasing side effects [86]. Ofatumumab is a second-generation antiCD20 antibody. Ofatumumab shows more ­ potency in direct antibody-dependent c­ ytotoxicity (ADCC), as well as in complement-dependent cytotoxicity (CDC) [87]. First clinical reports have shown that ofatumumab in combination with conventional chemotherapy offers a benefit [88, 89]. Obinutuzumab is a type II humanized glycol-­ engineered anti-CD20 antibody. Obinutuzumab has been reported to be highly active in CLL; however, besides interesting preclinical data there are no clinical trials in ALL with obinutuzumab.

5.2.2.2 CD22 Antibody CD22 molecule expression is found in more than 90% of B-lineage ALL. Functionally, CD22 leads to the downregulation of CD19 after its phosphorylation. Further, CD22 is rapidly internalized after activation and therefore is highly interesting for toxin-linked antibodies [90]. Inotuzumab ozogamicin (INO) is an anti-CD22 antibody that is linked to calicheamicin. Calicheamicin is a toxic antibiotic that causes double-strand breaks in the DNA. In a first phase II trial of nearly 49 patients, 57% responded to the immunotoxin and showed an OS of 5.1 months. Of note is that a MRD negativity of 63% was achieved when IDO was used as a single agent. Based on those promising results a phase III study showed a longer PFS and OS with INO (median PFS, 5.0 vs. 1.8 months, P day 28), and high leukocyte count at diagnosis that have been identified as high-risk patients. In these, allo SCT seems to be favorable as consolidation therapy [106–108]. However, it has to be emphasized that this data is mainly based on myeloablative treatment protocols and a related donor. Of note is that some studies failed to support the beneficial use of allo SCT in high-risk ALL patients [109, 110]. With growing evidence that detectable MRD is also a high-risk feature of ALL, more patients who are MRD positive were treated with allo SCT.  In fact, detection of MRD by molecular techniques has been shown to be an even better tool for discrimination of which patients should be transplanted and not [111]. Patients who achieve molecular MRD negativity showed a significant lower relapse rate after SCT (126 vs. 18 months, respectively; P = 0.007) [112]. This holds also true for children [113]. With incorporation of MRD positivity as an indication for allo SCT in ALL, the pool of potential candidates for a stem cell transplantation expanded. One way to lower TRM is to reduce the intensity of the preparative conditioning (RIC). Patients in the advanced stage of ALL, due to older age or heavy pretreatment, can be transplanted after RIC [114, 115]. Registry data from the EBMT showed that use of RIC protocols reduced TRM in the context of ALL, but also in this data set there was an increase in the relapse rate (RIC: 47% vs. MAC 31%, P 90% depletion was reached within minutes, followed by slower depletion in the lymph nodes and spleen (approximately 60–70% depletion in 24 h) and the slowest depletion in the peritoneal cavity (significant depletion only after a week) [62, 63]. With respect to the lymphoid organs, the position of B cells in the organ itself also determines the rate of depletion by rituximab, which could explain the relative resistance of mantle-zone B cells towards this therapy [64]. Another key feature of rituximab is its favorable toxicity profile. No dose-limiting toxicity at a single dose of 500  mg/m2 was observed in a

7  Immunopathology and Immunotherapy of Non-Hodgkin Lymphoma

phase I clinical study. Moreover, a weekly four-­ time infusion schedule of 375  mg/m2 was well tolerated [59, 65]. Despite successes obtained with rituximab therapy, resistance still remains a challenging issue in FL, as disease relapse occurs in almost 60% of patients during the first 5 years and eventually occurs in all patients with FL in the long term. Even though the precise mechanism of resistance remains unknown, it is thought that the characteristics of both patient and lymphoma contribute to it. Low expression of CD20 on the cell surface [66], high expression of complement regulatory proteins [67], expression of anti-apoptotic genes, and blockade of effector NK cells due to the deposition of C3 fragments of the complement system [68] are possible mechanisms for the resistance of lymphoma cells to rituximab [13]. Furthermore, loss of the CD20 epitope after rituximab infusion [69] or consumption of the extracellular portion of CD20-mAb complexes by phagocytic cells may also be involved [45]. In addition, survival signals secreted by the microenvironment may prevent mAB-induced death of lymphoma cells [70]. As mentioned before, a considerable difference in B-cell depletion by rituximab between peripheral blood, lymph nodes, and spleen has been observed, with B cells in the lymph nodes being the least susceptible [71]. Patient characteristics also affect the therapeutic efficacy of rituximab. Genetic analysis of FcγR polymorphism in cancer patients treated with mAb-based rituximab and trastuzumab revealed that ADCC is one of the critical factors responsible for the clinical efficacy of therapeutic Abs [72–74]. Considerable progress has been made in the treatment of B-NHL by the use of rituximab either as a single agent or in combination with chemotherapy. However, treatment-refractory relapse is an inevitable fate in a considerable proportion of patients, highlighting the need for new therapeutic modalities [75].

7.4.2.2 Rituximab in Diffuse Large B-Cell Lymphoma (DLBCL) Initial clinical trials on relapsed or refractory DLBCL patients revealed that rituximab was well tolerated and resulted in complete (CR) or partial response (PR) in one-third of the patients [76].

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The addition of rituximab (R) to CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy was regarded as a significant breakthrough in the treatment of DLBCL.  The R-CHOP regimen has become widely accepted in this regard. The final outcome is influenced by various pathways. The number of patients who reach high-dose therapy and ASCT is increased with prior R-CHOP salvage therapy; moreover, the outcome is improved when the regimen is used as posttransplantation maintenance therapy. Overall, rituximab salvage therapy for DLBCL is effective and well tolerated [77]. Coiffier et al. [60] compared the efficacy of CHOP and R-CHOP in elderly DLBCL patients and concluded that the addition of 375 mg/m2 rituximab on day 1 of each cycle significantly improves CR/Cru (73% vs. 63%, P  =  0.005), leading to a significantly greater event-free survival (EFS) (47% vs. 29%, P  =  0.00002), progression-free survival (PFS) (54% vs. 30%, P