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Frontiers in Anti-Cancer Drug Discovery
 9781681087016, 1681087014

Table of contents :
Content: Cover
Title
Biblography
End User License Agreement
Contents
Preface
List of Contributors
Recent Advances and New Insights for the Therapeutic Intervention of Cancer
Nhi Nguyen1, Stina George Fernandes2, Ekta Khattar2 and Yinghui Li1,*
TARGETING DNA DAMAGE RESPONSE (DDR) IN CANCER
DNA Damage Response Pathways
Base Excision Repair (BER)
DNA Mismatch Repair (MMR)
Nucleotide Excision Repair (NER)
DNA Double Strand Break Repair
DDR Associated Cell Signalling Pathways
DDR as an Anticancer Drug Target
DDR Inhibitors in Clinical Development Combining DDR Inhibitors with DNA Damaging Radiotherapy and ChemotherapyFuture Prospects of DDR Targeting Agents in Cancer Treatment
Synthetic Lethality
DDR with Epigenetic Compounds
IMMUNOTHERAPY
Common Immunotherapeutic Approaches
Cytokine Therapy
Antibody Immunotherapy
Therapeutic Cancer Vaccination Therapy
Oncolytic Viruses Therapy
Adoptive Cell Transfer Therapy
The Basis Behind CAR-T and CD19 CAR-T Therapy
Advantages of CD19 CAR-T Cell Therapy
Common Side Effects and Considerations of CD19 CAR-T Cell Therapy Clinical Application and Drug Development of CD19 CAR-T Cell TherapyTisagenlecleucel
Axicabtagene Ciloleucel
Future Perspective of CAR and CD19 CAR-T Cell Therapy
CAR Construct Delivery System
Novel CAR-T Models
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCE
Dabrafenib in Melanoma
Meredith McKean and Rodabe N. Amaria*
INTRODUCTION
Mechanism of Action
Pharmacokinetics and Pharmacodynamics
Clinical Evaluations
Phase I and Phase II Studies
Phase III Studies
Dabrafenib for CNS Metastases
Dabrafenib/Trametinib Combination Therapy Adjuvant Combination Targeted TherapyNeoadjuvant Combination Targeted Therapy
Dabrafenib/Trametinib for CNS Metastases
Safety
Immunotherapy and Targeted Therapy
BRAF V600 Mutation in Other Malignancies
CONCLUSIONS
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Targeting Autophagy in Cancer Therapy
Maria Ines Vaccaro1,* and Claudio Daniel Gonzalez2
AUTOPHAGY IS AN EVOLUTIONARY HIGHLY PRESERVED PHYSIOLOGICAL PROCESS
Signaling Pathway for Autophagy
Autophagic Process
Functions of Autophagy ALTERATIONS IN AUTOPHAGY ARE ASSOCIATED WITH TRANSITION FROM NORMAL TO NEOPLASTIC CELLS, AS WELL AS WITH CANCER CELL SURVIVAL AND RESISTANCE TO CHEMOTHERAPYAutophagy in Normal to Neoplastic Cells
Autophagy in Cancer Cell Survival
Autophagy in Resistance to Chemotherapy
ACTIVATION AND INHIBITION OF AUTOPHAGY IN CHEMOTHERAPY OF CANCER
Cancer Cell Response After Autophagy Enhancement
Cancer Cell Response After Inhibition of Autophagy
AUTOPHAGY IN IMMUNOTHERAPY FOR CANCER
CONCLUSIONS AND PERSPECTIVES
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES

Citation preview

Frontiers in Anti-Cancer Drug Discovery (Volume 9) Edited by Atta-ur-Rahman, FRS

Honorary Life Fellow, Kings College,University of Cambridge,Cambridge, UK

& M. Iqbal Choudhary H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

 

)URQWLHUVLQ$QWL&DQFHU'UXJ'LVFRYHU\ Volume # 9 Editors: Atta-ur-Rahman and M. Iqbal Choudhary ISSN (Online): 1879-6656 ISSN (Print): 2451-8395 ISBN (Online): 978-1-68108-701-6 ISBN (Print): 978-1-68108-702-3 ©2018, Bentham eBooks imprint. Published by Bentham Science Publishers – Sharjah, UAE. All Rights Reserved.

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CONTENTS PREFACE ................................................................................................................................................ i LIST OF CONTRIBUTORS .................................................................................................................. iv CHAPTER 1 RECENT ADVANCES AND NEW INSIGHTS FOR THE THERAPEUTIC INTERVENTION OF CANCER ........................................................................................................... Nhi Nguyen, Stina George Fernandes, Ekta Khattar and Yinghui Li TARGETING DNA DAMAGE RESPONSE (DDR) IN CANCER ........................................... DNA Damage Response Pathways ......................................................................................... Base Excision Repair (BER) ......................................................................................... DNA Mismatch Repair (MMR) ..................................................................................... Nucleotide Excision Repair (NER) ................................................................................ DNA Double Strand Break Repair ................................................................................ DDR Associated Cell Signalling Pathways ............................................................................ DDR as an Anticancer Drug Target ........................................................................................ DDR Inhibitors in Clinical Development ...................................................................... Combining DDR Inhibitors with DNA Damaging Radiotherapy and Chemotherapy Future Prospects of DDR Targeting Agents in Cancer Treatment ......................................... Synthetic Lethality ......................................................................................................... DDR with Epigenetic Compounds ................................................................................ IMMUNOTHERAPY ..................................................................................................................... Common Immunotherapeutic Approaches ............................................................................. Cytokine Therapy .......................................................................................................... Antibody Immunotherapy .............................................................................................. Therapeutic Cancer Vaccination Therapy .................................................................... Oncolytic Viruses Therapy ............................................................................................ Adoptive Cell Transfer Therapy ............................................................................................. The Basis Behind CAR-T and CD19 CAR-T Therapy ................................................... Advantages of CD19 CAR-T Cell Therapy ................................................................... Common Side Effects and Considerations of CD19 CAR-T Cell Therapy ................... Clinical Application and Drug Development of CD19 CAR-T Cell Therapy ............... Tisagenlecleucel ............................................................................................................ Axicabtagene Ciloleucel ................................................................................................ Future Perspective of CAR and CD19 CAR-T Cell Therapy ........................................ CAR Construct Delivery System .................................................................................... Novel CAR-T Models ..................................................................................................... CONCLUSION ............................................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCE .................................................................................................................................. CHAPTER 2 DABRAFENIB IN MELANOMA ................................................................................ Meredith McKean and Rodabe N. Amaria INTRODUCTION .......................................................................................................................... Mechanism of Action .............................................................................................................. Pharmacokinetics and Pharmacodynamics ............................................................................. Clinical Evaluations ................................................................................................................ Phase I and Phase II Studies ......................................................................................... Phase III Studies ........................................................................................................... Dabrafenib for CNS Metastases ....................................................................................

1 3 3 3 4 4 5 6 6 6 12 14 14 14 15 18 18 19 27 28 28 29 30 30 34 35 36 38 38 38 40 42 42 42 42 64 65 67 67 68 68 69 70

Dabrafenib/Trametinib Combination Therapy ............................................................. Adjuvant Combination Targeted Therapy ..................................................................... Neoadjuvant Combination Targeted Therapy ............................................................... Dabrafenib/Trametinib for CNS Metastases ................................................................. Safety ...................................................................................................................................... Immunotherapy and Targeted Therapy ................................................................................... BRAF V600 Mutation in Other Malignancies ........................................................................ CONCLUSIONS ............................................................................................................................. CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ...............................................................................................................................

71 76 76 78 79 82 84 85 85 85 85 86

CHAPTER 3 TARGETING AUTOPHAGY IN CANCER THERAPY .......................................... Maria Ines Vaccaro and Claudio Daniel Gonzalez AUTOPHAGY IS AN EVOLUTIONARY HIGHLY PRESERVED PHYSIOLOGICAL PROCESS ........................................................................................................................................ Signaling Pathway for Autophagy .......................................................................................... Autophagic Process ................................................................................................................. Functions of Autophagy .......................................................................................................... ALTERATIONS IN AUTOPHAGY ARE ASSOCIATED WITH TRANSITION FROM NORMAL TO NEOPLASTIC CELLS, AS WELL AS WITH CANCER CELL SURVIVAL AND RESISTANCE TO CHEMOTHERAPY ............................................................................ Autophagy in Normal to Neoplastic Cells .............................................................................. Autophagy in Cancer Cell Survival ........................................................................................ Autophagy in Resistance to Chemotherapy ............................................................................ ACTIVATION AND INHIBITION OF AUTOPHAGY IN CHEMOTHERAPY OF CANCER ......................................................................................................................................... Cancer Cell Response After Autophagy Enhancement .......................................................... Cancer Cell Response After Inhibition of Autophagy ............................................................ AUTOPHAGY IN IMMUNOTHERAPY FOR CANCER ......................................................... CONCLUSIONS AND PERSPECTIVES .................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ...............................................................................................................................

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CHAPTER 4 SELECTIVE ANTI-CANCER DRUGS AGAINST MULTI-DRUG RESISTANCE Tijana Stanković, Ana Podolski-Renić, Jelena Dinić and Milica Pešić INTRODUCTION .......................................................................................................................... TARGETING ABC TRANSPORTERS’ ACTIVITY ................................................................. Verapamil Related Compounds .............................................................................................. Niguldipine Analogs ............................................................................................................... Quinine Isomers ...................................................................................................................... Cyclosporine A Derivatives .................................................................................................... Synthetic Taxanes ................................................................................................................... Tetrahydroisoquinolin-ethyl-phenylamine Based Compounds .............................................. Quinoline Derivatives ............................................................................................................. Other Second-Generation Inhibitors ....................................................................................... More Selective ABC Transporters’ Inhibitors – Third-Generation ........................................ Multifunctional Drugs as Fourth-Generation Inhibitors ......................................................... Peptidomimetics ............................................................................................................

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93 93 94 95 96 96 97 99 102 102 103 104 106 109 109 109 109

114 116 116 117 117 118 118 119 119 120 120 122 122

Surfactants and Lipids ................................................................................................... Dual Ligands ................................................................................................................. ROS PRODUCTION AND VULNERABILITY OF MDR CANCERS .................................... ROS Production via Facilitation of ABC Transporters’ ATPase ........................................... ROS Production via Iron Chelation ........................................................................................ GLUTATHIONE DEPLETION .................................................................................................... ENERGETIC ALTERATIONS .................................................................................................... CHANGES IN PLASMA MEMBRANE ...................................................................................... AUTOPHAGY MODULATION ................................................................................................... MICROTUBULE COMPOSITION ............................................................................................. OTHER EXAMPLES OF CS ........................................................................................................ NOVEL STRATEGIES FOR MDR TREATMENT ................................................................... CONCLUDING REMARKS ......................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENT ............................................................................................................. REFERENCES ............................................................................................................................... CHAPTER 5 PRO-APOPTOTIC AND ANTI-TELOMERASE ACTIVITY OF NATURALLY OCCURRING COMPOUNDS ............................................................................................................... Mahendar Porika, Radhika Tippani and Nazneen Firdous INTRODUCTION .......................................................................................................................... Cancer ..................................................................................................................................... Telomerase .............................................................................................................................. TELOMERASE COMPLEX AND ITS FORMATION ............................................................. TELOMERASE AND CANCER .................................................................................................. NON TELOMERIC ACTIVITIES OF TELOMERASE ........................................................... TELOMERASE AND OXIDATIVE STRESS ............................................................................ TELOMERASE AND DNA DAMAGE/APOPTOSIS/DRUG RESISTANCE ........................ NATURAL SOURCES AS ANTICANCER AGENTS ............................................................... THE NEED FOR NATURISTIC ANTICANCER THERAPY .................................................. Plant Sources ........................................................................................................................... Abnormal Savda Munzig ........................................................................................................ Apigenin .................................................................................................................................. Berberine ................................................................................................................................. Boldine .................................................................................................................................... Butylidenephthalide ................................................................................................................ Crocin ...................................................................................................................................... Curcumin ................................................................................................................................. (-) Epigallocatechin-3-Gallate ................................................................................................ Gambogic Acid ....................................................................................................................... Genistein ................................................................................................................................. Helenalin ................................................................................................................................. Indole- 3-carbinol .................................................................................................................... Papaverine ............................................................................................................................... Pterostilbene ............................................................................................................................ Quercetin ................................................................................................................................. Resveratrol .............................................................................................................................. Silymarin ................................................................................................................................. Sulforaphane ........................................................................................................................... Triptolide .................................................................................................................................

123 124 125 126 127 128 130 131 132 135 137 140 143 161 161 161 161 193 193 193 194 195 196 197 198 200 202 203 203 204 204 205 205 206 206 207 207 208 209 209 210 210 211 211 212 212 213 214

Wogonin .................................................................................................................................. CONCLUSION ............................................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 6 THE PROSPECTS OF CDK TARGETING AND CLINICAL APPLICATION OF CDK INHIBITORS IN CANCER ......................................................................................................... Aditi Gangopadhyay and Aparna Gangopadhyay INTRODUCTION .......................................................................................................................... REGULATION OF CDK ACTIVITY IS CRUCIAL FOR CELL CYCLE CONTROL ........ The Four Modes of CDK Regulation ...................................................................................... Molecular Biology of Cyclin-Dependent Cell Cycle Regulation ........................................... Transition from G1 to S ................................................................................................. Transition from S to G2 ................................................................................................. G2 to M Transition ........................................................................................................ DEREGULATION OF CDKS IN CANCERS ............................................................................. CDK TARGETING IN CANCER ................................................................................................ Mechanism of CDK Inhibition ............................................................................................... The Development of CDK Inhibitors ..................................................................................... Inspired by Nature ................................................................................................................... Computer-Aided Drug Discovery (CADD) ............................................................................ THE CLINICAL DEVELOPMENT OF CDK INHIBITORS ................................................... The Evolution of Early Clinical Non-Selective CDK Inhibitors ............................................ First Generation CDK Inhibitors .................................................................................. Attempting to Improve Clinical Outcomes - Advent of Second Generation CDK Inhibitors ....................................................................................................................... Selectivity is the Key ...................................................................................................... A Beacon in CDK Research: Selective Dual CDK 4/6 Inhibitors ................................ Ushering in a New Age-Selective Dual CDK4/6 Inhibitors in the Clinic ..................... Clinical Trials of Selective Dual CDK 4/6 Inhibitors in Breast Cancer ....................... Palbociclib .................................................................................................................... Selective Dual CDK 4/6 Inhibitors in the Pipeline ................................................................. Selective Dual CDK 4/6 Inhibitors in Other Cancers ............................................................. Palbociclib .................................................................................................................... Ribociclib ...................................................................................................................... Abemaciclib ................................................................................................................... Challenges in CDK Inhibitor Therapy .................................................................................... FUTURE SCOPE ............................................................................................................................ CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 7 ADVANCEMENTS IN ANTICANCER DRUG DELIVERY ................................... Hemant K.S. Yadav, Abrar E. Srouji, Alyazya Mohammed and Manar Dibi INTRODUCTION .......................................................................................................................... HISTORY ........................................................................................................................................ NEW DRUGS FOR ANTICANCER THERAPY ........................................................................ Bavencio (Avelumab) ............................................................................................................. Nerlynx (Neratinib) .................................................................................................................

214 215 216 216 216 216 234 235 236 236 237 238 239 240 240 241 242 242 243 244 245 245 245 247 248 249 250 251 251 263 264 264 265 266 267 269 270 270 270 270 287 287 288 289 290 291

Zejula (Niraparib) ................................................................................................................... Xermelo (Telotristat Ethyl) ..................................................................................................... Imfinzi (Durvalumab) ............................................................................................................. Opdivo (Nivolumab) ............................................................................................................... dTCApFs ................................................................................................................................. Apalutamide (Erleada) ............................................................................................................ Lutathera ................................................................................................................................. Blinatumomab ......................................................................................................................... DRUG-ANTIBODY CONJUGATES ........................................................................................... CANCER DRUG DISCOVERY BY REPURPOSING ............................................................... EXAMPLES OF REPURPOSED NON-CANCER DRUGS ...................................................... Thalidomide ............................................................................................................................ Aspirin ..................................................................................................................................... Metformin ............................................................................................................................... COX-2 Inhibitors .................................................................................................................... Psychiatric Drugs .................................................................................................................... Statins ...................................................................................................................................... COMBINATION THERAPY IN COMBATING CANCER ...................................................... Nivolumab and Ipilimumab Combination Therapy ................................................................ Combination of Pharmaceutical Agents Targeting Antioxidant Response Pathways ............ Carbonic Anhydrase Inhibitors in Combination Therapy ....................................................... Autocrine Growth Factors in Cancer Survival ........................................................................ Anti-Angiogenesis Therapeutic Agents in Combination Therapy .......................................... Histone Deacetylase Inhibitors in Combination Therapy ....................................................... CONCLUSION ............................................................................................................................... CONSENT FOR PUBLICATION ................................................................................................ CONFLICT OF INTERESTS ....................................................................................................... ACKNOWLEDGEMENT ............................................................................................................. REFERENCES ...............................................................................................................................

291 293 293 294 295 296 297 297 298 300 300 300 301 301 302 303 304 304 305 306 307 307 307 308 309 310 310 310 310

SUBJECT INDEX ................................................................................................................................... 315

i

PREFACE No other area of drug development has received as much attention as the field of anti-cancer drug discovery and development. Despite major advancements in the understanding of the molecular basis of cancers, and the advent of several new classes of chemotherapeutics, prognosis in certain forms of cancers has not been improved substantially. This has led to a re-thinking of current drug discovery paradigm, and several out of the box ideas have emerged. The literature in this field is now enriched with several new approaches and extraordinary targets, which deserve to be highlighted for the benefit of the scientific community. The 9th volume of the book series entitled, “Frontiers in Anti-Cancer Drug Discovery” comprises seven comprehensive review articles in this field. They cover therapeutic advancements against various cancers, study of the mechanisms of action of various anticancer drugs, and drug candidates, including natural products, and recent advancements in the new and novel target identification related to various types of cancers. These articles represent the state-of-the-art in the field of anti-cancer drug discovery and development and provide an in-depth understanding of the subject. The first article in the book series, contributed by Li et al., is a general review of the key issues and developments in cancer treatment, and emergence of targeted therapies. Compounds which induce DNA damaging response (DDR) in tumour cells have been developed as effective drugs for cancer treatment. The article provides examples and advantages of several drugs which target DDR through a variety of mechanisms. Li et al. have also critically reviewed notable findings in cancer immunotherapies. These include development of cytokines, oncolytic viruses, cancer vaccination, and adaptive cell transfer. The most exciting of them is the anti-CD19 CAR cell immunotherapy. These recent developments have brought new hopes in cancer chemotherapy. Cutaneous melanoma is the most aggressive form of skin cancer with poor prognosis. Over half the cases of cutaneous melanoma are reported to have activating mutation in BRAF kinase leading to the activation of kinase pathway and resulting in unregulated cell growth. BRAF kinase inhibition was therefore identified as a treatment option for cutaneous melanoma. Clinical outcome, however, has been only marginal. McKean and Amaria have critically reviewed the successes and limitations of various BRAF kinase inhibitors such as Dabrafenib and Vemurafenib, approved in recent years for the treatment of malignant cutaneous melanoma. Resistance against BRAF kinase inhibitors has been the key issue. The authors have also described the use of various combinational therapies including targeted therapy, immunotherapy, and dual inhibitors of BRAF and MEK. They concluded that BRAF inhibition is an attractive target for the development of anti-cancer drugs for other forms of cancers. Macroautophagy is a process that sequesters senescent or damaged organelles and proteins in autophagosomes for recycling of their useful components. This process is also involved in the removal of cells death due to classical type 1 apoptosis. Therefore, macroautophagy was primarily considered as a protection mechanism for cells against various stressors and wear and tear. Autophagy may also lead to a form of non-apoptotic type 2 programmed cell death. Cell protective (anti-apoptotic) or cell damaging role of autophagy seems to depend on many factors, including microenvironment of the cells. Autophagy and its role in diverse cells and conditions has been the focus of research for quite some time, with contradictory results. Recent studies have identified autophagy as the mechanism of survival of cancerous cells,

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under adverse environmental conditions. A review by Vaccaro and Gonzalez covers this interesting topic, with special emphasis on the role of autophagy in cancerous cell cycle, as a new target for anti-cancer drug development. Several classes of drugs which modulate autophagy and their mechanisms of action, have been discussed in this interesting article. Drug resistance is a cross cutting issue in various classes of drugs, including anti-bacterial, anti-fungal, and anti-viral drugs. In the case of anti-cancer drugs, drug resistance limits the choice of treatment, thus creating havoc both in health care professionals and patients. Ironically, drug resistance in target cells also generates a collateral sensitivity (CS) towards other drugs, compared to the parent drug against which resistance has been developed. CS networks are therefore now targeted for drug discovery against infections and cancer. This novel approach has opened a window of opportunity for new drug development. Pesic et al. have contributed a review of recent attempts to exploit collateral sensitivity in MDR phenotype cancer cells as the target for the development of new anti-cancer agents. The recent literature on the mechanism of action of MDR selective drugs and their potential use in combination therapy for cancer treatment has been professionally reviewed. Telomerase enzymes are among the most studied targets for a variety of purposes, including anti-ageing drugs. Telomers serve as protective caps at the end of DNA strands, and they are responsible for maintaining the length and integrity of chromosomes, thus playing a key role in cell survival. The enzyme telomerase is responsible for maintaining the length of telomers. This has been correlated in the uncontrolled growth of cancer cells, and its activity has been detected in over 80% of cancers. Porika et al. have focused their contribution on the potential of telomerase as a novel target for anti-cancer drug development. Telomerase inhibition is presented as an important approach to specifically target cancer cells. They have presented various classes of natural products which inhibit telomerase activity, and can thus serve as specific anti-cancer leads. The mechanism of inhibition of cancerous cell proliferation, and the results of clinical studies in some telomerase inhibitors are presented at great length. Gangopadhhyay and Gangopadhyay have reviewed the prospects of targeting cyclindependent kinases (CDKs) as drug discovery targets against various cancers. CDKs regulate cell cycle progression. CDK inhibitors can be used for the treatment of cancers as they can prevent the proliferation of cancer cells. In certain cancers, CDKs are overexpressed and thus lead to unregulated growth and proliferation. Inhibition of CDKs has been vigorously pursued in recent years for anti-cancer drug discovery, and US-FDA has approved several drugs of this class. The results of clinical studies and experiences on CDK inhibitors as anti-cancer drugs, as well as associated complications including resistance, are presented in this detailed review. Key issues with the CDK inhibitors include resistance and low selectivity. The authors have critically reviewed the advantages and merits of the use of dual CDK inhibitors and combination therapies, along with present and future prospects of selective CDK inhibitors for the treatment of various cancer types. Last but not least, the review by Yadav et al. describes several recent developments in the field of cancer treatment. Initially, several new classes of anti-cancer drugs capable of effectively targeting cancer cells have been described. Interestingly, these drugs include both well-established anti-cancer drugs and non-cancer agents such as statins, NSAIDs, and antidiabetics. The authors have also presented results of recent studies on combination anti-cancer therapies versus single drug treatment. The above articles by prominent researchers in selected fields have made this volume another important treatise for scientists and research scholars. We are grateful to all the authors for their excellent and scholarly contributions for the 9th volume of this internationally recognized

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eBook series. The editorial team of Bentham Science Publishers deserves appreciation for efficient processing and timely management of this publication. The coordination and liaison by Ms. Fariya Zulfiqar (Manager Publications), under the leadership of Mr. Shehzad Naqvi (Editorial Manger Publication) & Mr. Mahmood Alam (Director Publications), are duly acknowledged. We also hope that like the previous volumes of this internationally reputed book series, the current compilation will also receive wide readership and appreciation.

Atta-ur-Rahman, FRS Kings College University of Cambridge, Cambridge UK & M. Iqbal Choudhary H.E.J. Research Institute of Chemistry International Center for Chemical and Biological Sciences University of Karachi, Karachi Pakistan

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List of Contributors Abrar E. Srouji

Department of Pharmaceutics, RAK College of Pharmaceutical Sciences, RAK Medical & Health Sciences University, UAE

Aditi Gangopadhyay

University of Calcutta, Kolkata, West Bengal, India Jhargram Raj College, Paschim Medinipur, West Bengal, India

Alyazya Mohammed

Department of Pharmaceutics, RAK College of Pharmaceutical Sciences, RAK Medical & Health Sciences University, UAE

Ana Podolski-Renić

Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia

Aparna Gangopadhyay

University of Calcutta, Kolkata, West Bengal, India Chittaranjan National Cancer Institute, Kolkata, West Bengal, India

Claudio Daniel Gonzalez University Institute CEMIC (Center for Medical Education and Clinical Research), Buenos Aires, Argentina Ekta Khattar

Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS (Deemed-to-be University), Vile Parle (West), Mumbai, India

Hemant K.S Yadav

Department of Pharmaceutics, RAK College of Pharmaceutical Sciences, RAK Medical & Health Sciences University, UAE

Jelena Dinić

Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia

Mahendar Porika

Department of Biotechnology, Kakatiya University, Warangal, Telangana, India

Manar Dibi

Department of Pharmaceutics, RAK College of Pharmaceutical Sciences, RAK Medical & Health Sciences University, UAE

Maria Ines Vaccaro

Institute of Biochemistry and Molecular Medicine (CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

Meredith McKean

Department of Melanoma Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA

Milica Pešić

Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia

Nazneen Firdous

Department of Obstetrics and Gynaecology, Uzhgorod National University, Uzhgorod, Ukraine

Nhi Nguyen

School of Biological Sciences (SBS), Nanyang Technological University (NTU), 60 Nanyang Drive, Singapore

Radhika Tippani

Department of Biotechnology, Kakatiya University, Warangal, Telangana, India

Rodabe N. Amaria

Department of Melanoma Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA

v Stina George Fernandes

Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS (Deemed-to-be University), Vile Parle (West), Mumbai, India

Tijana Stanković

Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia

Yinghui Li

School of Biological Sciences (SBS), Nanyang Technological University (NTU), 60 Nanyang Drive, Singapore

Frontiers in Anti-Cancer Drug Discovery, 2018, Vol. 9, 1-63

1

CHAPTER 1

Recent Advances and New Insights for the Therapeutic Intervention of Cancer Nhi Nguyen1, Stina George Fernandes2, Ekta Khattar2 and Yinghui Li1,* School of Biological Sciences (SBS), Nanyang Technological University (NTU), 60 Nanyang Drive 637551 Singapore 2 Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS (Deemed-to-be University), Vile Parle (West), Mumbai 400056 India 1

Abstract: Traditionally, anti-cancer treatments mainly focus on chemotherapies, radiation therapy and surgery. However, these treatments are limited in terms of their specificity for targeting only cancer cells. DNA damaging chemotherapy is one of the most common treatment modalities of cancer. Current progress of targeted therapy that relies on DNA damage response (DDR) in cancer offers a vast therapeutic window by specifically targeting DDR functions in patient specific tumours. Recent developments in immunotherapy – therapy that boosts the body’s immune system to fight against cancer cells, have shown promising results. Currently, different approaches of immunotherapies such as cytokine, antibody, oncolytic virus, adoptive cell transfer therapies or cancer vaccination have made significant progress in treating different cancer types. This paper seeks to provide an overview of the recent developments of drugs targeting DDR and various immunotherapeutic approaches, with specific focus on the anti-CD19 CAR-T cell therapy. Finally, the paper will give a perspective on future directions of DDR therapy, CAR-T cell therapy as well as the combination of different cancer therapies for effective cancer treatment regime. Over the past few years, the decipher of different DNA damage response mechanisms enables the development of novel inhibitors for cancer treatment. Furthermore, recent advances in genomic editing technologies and scientific discoveries reveal important roles of the immune system in the context of cancer development. This also prompts the plethora of drug developments that have vast potential in the treatment of different types of cancer diseases. This paper will give an overview of recent drug developments in chemotherapeutic agents targeting DNA damage response as well as recent immunotherapies that have been successful in the treatment of human cancers. The contents discussed in the paper are summarized in (Fig. 1).

* Correspondence author Yinghui Li: Nanyang Technological University (NTU), School of Biological Sciences (SBS), 60 Nanyang Drive, 637551 Singapore; Tel: 6563162871; Email: [email protected]

Atta-ur-Rahman and M. Iqbal Choudhary (Eds.) All rights reserved-© 2018 Bentham Science Publishers

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Keywords: Adoptive Cell Transfer Therapies, Antibody Therapy, Anti-CD19 CAR-T Cell Therapy, Combinational Approaches, Cytokine Therapy, DNA Damage Response, DNA Damage Response Inhibitors, Immunotherapy, Oncolytic Virus Approach. Base exicion repair

DNA mismatch repair

DDR pathways Nucleotide excision repair

DDR associated cell signaling pathways

Targeting DNA damage response (DDR)

DDR as an anticancer drug target Future prospects of DDR targeting agents in cancer treatment

THERAPEUTIC INTERVENTION OF CANCER

DNA double strand break repair DDR inhibitiors in clinical development Combining DDR inhibitors with DNA damaging radiotherapy and chemotherapy Synthetic lethality DDR with epigenetic compounds

Cytokine therapy

Common immunotherap eutic approaches

Antibody immunotherapy

Therapeutic cancer vaccination therapy

Oncolytic viruses therapy

The basis behind CAR-T and CD19 CAR-T therapy

Immunotherapy

Advantages of CD19 CAR-T cell therapy

Adoptive cell transfer therapy

Common side effects and considerations of CD19 CAR-T cell therapy

Conclusion

Clinical application and drug development of CD19 CAR-T cell therapy Future perspective of CAR and CD19 CAR-T cell therapy

Fig. (1). Summary diagram of the chapter.

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TARGETING DNA DAMAGE RESPONSE (DDR) IN CANCER Cells encounter thousands of DNA damaging events every day and various mechanisms have evolved to combat the damage [1]. Induction, detection and resolution of the DNA damage involves various intra and inter-cellular signalling events mediated by enzymes, adaptor proteins as well as other macromolecules. These events are collectively termed as DNA damage response (DDR). The consequences of DDR can be cell cycle arrest, transcription and replication regulation leading to repair or bypass of the DNA damage. Depending on the type of DNA damage, different repair and response mechanisms are activated. The type of DDR signalling also depends on the cell cycle stage during which it is activated. DDR is important for normal human development as it contributes to genome maintenance which is essential for cellular function and health. Loss of efficient DDR may lead to mutations and thus promote cancer initiation and development. While loss of DDR is deleterious to normal cell functioning, it is extremely useful for cancer cells. Cancer cells have to maintain endless replication capacity and overcome high rate of apoptosis to survive, thus they rely heavily on some of the DDR pathways and components making them attractive therapeutic targets [2]. This section discusses the type of DDR and current therapeutic approaches targeting DNA repair proteins and pathways in cancer. DNA Damage Response Pathways Different types of DNA damage initiate different signalling pathways and repair mechanisms. However, there is no redundancy and different pathways might compensate and cross talk amongst each other to determine the final outcome. The major repair pathways in human cells depending on type of damage are described below. Base Excision Repair (BER) DNA bases encounter damage due to various reasons including oxidation, deamination and alkylation. These modifications result in incorrect base pairing and thus lead to mutations in the DNA. Such base lesions which are small and non-helix distorting are repaired by base excision repair pathway. BER is active during all stages of the cell cycle. The major steps of BER comprise the removal of damaged DNA base by enzymatic action of DNA glycosylase, creating an apyrimidic/apurinic (AP) site. Examples of DNA glycosylases include 3-methyladenine DNA glycosylase (AAG/MAG1), 8-oxoguanine DNA glycosylate (OGG), uracil DNA glycosylase (UDG/UNG) and endonuclease III like DNA glycosylases. AP site is processed by AP endonuclease (APE1) by cleaving the phosphodiester backbone located at

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5’ position to the AP site. This generates a 3’ hydroxyl group and a 5’ deoxyribose phosphate moiety (5’dRP). The resulting single-strand break can then be repaired by either short-patch (where a single nucleotide is replaced, predominant mode of repair) or long-patch BER (where 2–10 new nucleotides are synthesized) [3]. The selection also depends on relative concentration of ATP or the nature of 5’dRP intermediates, cell cycle stage, and whether the cell is terminally differentiated or dividing [4]. Short patch BER is catalyzed by polymerase β (pol β) and by pol λ in the absence of pol β. These polymerases insert only one nucleotide and in addition possess a lyase domain that removes the 5’dRP that is generated during AP endonuclease cleavage. DNA ligase III together with its cofactor XRCC1 catalyzes the nicksealing step and completes the short patch BER. Long patch BER is catalyzed by pol δ and pol ε along with PCNA that acts as a processivity factor. They carry out displacement synthesis in which 5’ end of DNA is displaced to form a flap. This 5’ flap is removed by FEN1 flap endonuclease followed by ligation of the break by DNA ligase I [5]. DNA Mismatch Repair (MMR) MMR repairs base-base mismatches or insertion/deletion loops that are generated by erroneous replication or recombination. In eukaryotes, the mismatch is recognized by MutS family of proteins. MutSα (Msh2/Msh6) pathway is involved primarily in base substitution and small-loop mismatch repair while MutSβ (Msh2/Msh3) pathway is involved in small-loop repair, in addition to large-loop (around 10 nucleotide loops) repair but does not perform base substitution [6 - 8]. This is followed by incision through MutLα which is composed of MLH1 and PMS2 proteins [7]. It functions as a DNA endonuclease upon activation by mismatch and other proteins like MutS and PCNA. MutLβ (MLH1 and PMS1) and MutLγ (MLH1 and MLH3) are also known to function in mismatch repair but their roles are not well known. This incision recruits exonuclease which incises the DNA strand 5’ or 3’ depending on the incision. Single strand binding protein RPA (Replication Protein A) coats the region and DNA polymerase III is recruited to fill in the gap which is sealed by DNA ligase. PCNA is also known to play important roles in MMR pathway [9]. Nucleotide Excision Repair (NER) NER mainly removes DNA damage induced by Ultra Violet light (UV), which results in bulky adducts of DNA typically thymine dimers and 6,4-photoproducts. NER in eukaryotes can be divided into two sub-pathways in the initial damage recognition step: global genomic NER (GG-NER or GGR) and transcription coupled NER (TC-NER or TCR) [10]. Later steps involving dual incision, repair

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and ligation are common in both sub pathways. In TCR, when RNA polymerase encounters damaged DNA, it stalls and the damaged site recruits XPG and CSB proteins while in GGR, the damaged site is recognized by XPE/DDB2 and XPC/hHR23B. These complexes have distortion recognition properties. Following damage recognition, XPA, RPA, XPG and TFIIH are recruited. TFIIH then unwinds the DNA helix, following which, XPG (uses its endonuclease activity to cut 3’side of lesion) and XPF/ERCC1 (uses its endonuclease activity to cut 5’side of lesion) cut and excise the lesion. Subsequently, DNA polymerase fills the gap and DNA ligase seals the nick to restore normal nucleotide sequence. DNA Double Strand Break Repair Double strand breaks (DSB) in DNA are extremely harmful if left unrepaired or incorrectly repaired. Normal cells generate DSB through endogenous processes such as V(D)J recombination, DNA replication and exogenous agents like ionizing radiation and chemotherapeutics [11]. Cells can repair DSB through two pathways: the first is homologous recombination (HR) where either a homologous chromosome or sister chromatid is used as a template to repair the DNA break and the second is non-homologous end joining (NHEJ) where DNA breaks are directly joined without or with very little sequence homology. In HR pathway, following DNA damage, there is an initial resection step by the core complex consisting of Mre11, Nbs1 and Rad50 (MRN) along with nucleases Exo1 and CTIP. This resection creates single stranded DNA which is then bound and stabilized by RPA which activates ATR kinase and also recruits Rad51, BRCA1, BRCA2 and other factors. This complex then searches for the homologous repair template. Once template is found, the DNA polymerases, resolvases and DNA ligase I reseal the repaired break for this high fidelity repair [12]. In mammals, NHEJ is the preferred pathway for DSB repair. NHEJ can be further divided into two biochemically and genetically distinct pathways: classic – NHEJ (C-NHEJ), and alternative NHEJ (A-NHEJ). The mechanism for C-NHEJ is known in great depth. Seven proteins have been shown to be essential for this pathway namely: Ku70, Ku-86, the DNA dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, X-ray cross complementing 4 (XRCC4), XRCC4-like factor (XLF) and DNA ligase IV [13]. Heterodimer of Ku70 and 86 recognizes and binds to the broken ends of DNA as a ring encircling DNA duplex [14]. This bridge of Ku dimer structurally supports the DNA and also aligns the ends of DNA thus protecting it from degradation. Following this DNA-PKcs is recruited which is then activated when it binds and phosphorylates artemis protein, and the resulting artemis/DNA-PKcs complex is

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believed to possess nuclease activity that cleaves the 5’ and 3’ DNA overhangs [15]. NHEJ requires two DNA blunt ends in order to perform ligation. Once the blunt ends are placed, the XRCC4/DNA ligase IV ligation complex is recruited to ligate the broken DNA ends. Ligation step is performed by DNA ligase IV, but it requires the binding of XRCC4. XRCC4 protein plays a regulatory role by stabilizing DNA ligase IV, stimulating its ligase activity, and directing the ligase to the site of DNA breaks via its recognition helix and DNA-binding capacity. A-NHEJ functions when the key proteins in C-NHEJ are less functional or absent. The mechanism and steps of A-NHEJ are not very well known. Major proteins involved are PARP1, DNA Ligase III/XRCC1, DNA Ligase I, MRN, Polynucleotide kinase and FEN1. A-NHEJ pathway utilizes microhomology at the DNA breakpoint regions, consisting of shared sequence (ranging from 5 to 22 bp) between the two DNA ends, hence it is also known as microhomology-mediated end-joining (MMEJ). Recent studies suggest that MMEJ is functional in both normal and cancer cells [16, 17]. DDR Associated Cell Signalling Pathways DDR simultaneously activates signalling response pathways in the cell. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia Rad3 related (ATR) proteins form the key regulators of DDR and maintain genome homeostasis. Double strand breaks primarily activate ATM kinase while any damage to replicating chromosomes activates ATR kinase although DSB can also activate ATR but that is dependent on ATM and MRN complex [18, 19]. In response to DNA damage, several hundred proteins are phosphorylated majorly by ATM kinase or ATR kinase while DNA-PKcs also regulates a smaller number of target proteins but it is mainly restricted to NHEJ pathway [20 - 24]. Chk1 and Chk2 represent major downstream kinases of ATR and ATM respectively. Activation of DDR kinase signalling further activates cell cycle checkpoint pathways, mainly cell cycle arrest and DNA repair pathway components. DDR as an Anticancer Drug Target DDR in cancer cells is different as compared to that in normal cells and that provides a rationale for drug targeting. The major differences include: loss of one or more DDR pathways resulting in dependency of cancer on remaining pathways, elevated levels of replication stress and also higher levels of endogenous DNA damage. DDR Inhibitors in Clinical Development A DDR deficiency in cancer cells that results in dependency on a particular DDR

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pathway or target presents an opportunity to use inhibitors of the pathway or target so as to block repair in such cancer cells. This concept is known as synthetic lethality. In the context of cancer DDR therapeutics, synthetic lethality exploits the possibility that, in cancers, one loss-of-function event is genetic and distinct to the tumor whereas the second loss-of-function event can be triggered pharmacologically using a DDR inhibitor. An example of such synthetic lethality is observed in the case of PARP inhibition. PARP enzyme activity is important for chromosome relaxation following which PARP dissociates from the DNA for SSB repair to proceed. PARP inhibitors are designed with a NAD+ mimetic core to compete with NAD+ such that they stall PARP1 onto SSBs and these PARP1DNA complexes block replication. Such trapped PARP1-DNA complexes will further result in toxic DSBs by virtue of stalled and collapsed replication forks. In normal cells, HR would be activated to repair these DBS, however HR deficiency in cancer cells would direct the cells to initiate less efficient NHEJ, resulting in increased genomic instability and thus cell death. This has been investigated in the cancers with defective forms of the tumor suppressor genes BRCA1 and BRCA2, whose normal function involves repair of DSBs by HR. It has been demonstrated that PARP inhibitor activity in BRCA homozygous mutant (BRCA-/-) is almost 1000-fold as compared to BRCA heterozygous (BRCA-/+) and WT BRCA (+/+) because PARP inhibition in BRCA-deficient tumor cells will exhibit greater effects than in normal cells which are WT or heterozygous for BRCA1 or BRCA2 [25]. In cases of germline BRCA mutations, the tumors will lack both copies of functional BRCA and normal cells will harbour at least one functional copy. In non-BRCA, HR deficient cancers, numerous approaches like DNA sequence analysis of HR genes, analysis of DNA rearrangements or mutational patterns due to loss of HR pathway, an immunohistochemistry approach, etc., are being exploited for selection of a single non-BRCA, HR-associated deficiency. Further examples of inhibitors targeting HR by exploiting synthetic lethality phenotype include Rad52 inhibitors in BRCA1/2 cancers, ATR inhibition combined with PARP inhibition and CDK inhibition with PARP inhibition [26 28]. The first PARP inhibitor to be clinically tested was olaparib (AZD2281). Currently, there are several PARP inhibitors in clinical trials (Table 1). One important challenge is the identification of agents that are truly synthetically lethal, because if the agent only functions to increase sensitivity of the lesion to another agent, monotherapy may not be an option.

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Table 1. DDR Inhibitors in Clinical Development [328]. Pathway Base Excision Repair

Target

Compound

Latest Stage of Development and Trial Details

Clinical Trial Identifier(s)

APE1

Methoxyamine Phase II in combination with TMZ NCT02395692, in glioblastoma NCT00892385, NCT01658319, NCT00692159

PARP

E7016

Phase I in combination with TMZ NCT01127178 in advanced solid tumors

PARP

Niraparib

Phase III as monotherapy in breast NCT01847274, cancer and as maintenance NCT01905592 monotherapy in ovarian cancer NCT02657889, NCT02354131, NCT02476552, NCT02655016, NCT02354586, NCT00749502, NCT01294735

PARP

Olaparib

Olaparib licensed for use. Olaparib NCT02476968 Phase IV as maintenance monotherapy in ovarian cancer.

PARP

Olaparib

Phase III as monotherapy, maintenance monotherapy and in combination with chemotherapy or cediranib in multiple tumor types (ovarian, breast, gastric, pancreatic)

NCT01844986, NCT01874353, NCT01924533, NCT02000622

PARP

Olaparib

Phase II as monotherapy and as combination therapy in various tumor types

NCT01972217, NCT01078662, NCT01063517, NCT02345265, NCT01583543, NCT00912743, NCT02340611, NCT01081951, NCT01650376, NCT02208375, NCT02446704, NCT01513174, NCT01116648, NCT00753545, NCT00679783, NCT00494234, NCT00494442, NCT00628251

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(Table 1) cont.....

Pathway Base Excision Repair

Target

Compound

Latest Stage of Development and Trial Details

Clinical Trial Identifier(s)

PARP

Olaparib

Phase I as monotherapy and as combination therapy in various tumor types

NCT01851265, NCT01758731, NCT01929603, NCT01921140, NCT02398058, NCT01296763, NCT01894256, NCT01894243, NCT01390571, NCT01237067, NCT02511795

PARP

Rucaparib

Phase III as maintenance monotherapy in ovarian, primary peritoneal and fallopian tube cancers

NCT01968213, NCT02042378, NCT02740712, NCT03533946 NCT02986100, NCT01891344, NCT01482715, NCT00664781, NCT01009190

PARP

Talazoparib

Phase III as monotherapy in advanced and/or metastatic breast cancer

NCT01945775, NCT02282345, NCT03070548, NCT03042910, NCT01286987, NCT03077607, NCT02034916, NCT01776437, NCT02049593, NCT01399840, NCT02358200, NCT02316834

PARP

Veliparib

Phase III in combination with chemotherapy in multiple tumor types

NCT02032277, NCT02106546, NCT02152982, NCT02163694, NCT02264990, NCT02470585

PARP

Veliparib

Phase I and II as monotherapy and NCT01472783, as combination therapy in various NCT01690598, tumor types NCT01386385, NCT01711541, NCT02483104, NCT01199224, NCT01642251, NCT02210663, NCT01193140, NCT02305758

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(Table 1) cont.....

Pathway

Target

Compound

Latest Stage of Development and Trial Details Phase I as monotherapy in ASTs

Clinical Trial Identifier(s)

Non homologous DNA-PKcs End joining DNA-PKcs

CC-115

NCT01353625

MSC2490484A Phase I as monotherapy or in combination with RTx in ASTs and CLL

NCT02316197

Homologous Recombination Repair

ATR

VX-970

Phase I in combination with RTx and cisplatin in HNSCC

NCT02157792

ATR

AZD6738

Phase l drug efficacy

NCT01955668

Checkpoint Inhibitors

ATM

AZD0156

Phase I as monotherapy or in combination with cytotoxic chemotherapy, olaparib or novel anti-cancer therapies in advanced tumors

NCT02588105

CHK1

GDC-0575

Phase I as monotherapy and in combination with cytotoxic chemotherapy in ASTs or lymphoma

NCT01564251

CHK1

MK-8776

Phase II in combination with cytarabine in myeloid leukemia

NCT01870596, NCT00779584

CHK1 and CHK2

LY2606368

Phase I in combination With Ralimetinib in Advanced or Metastatic Cancer

NCT02778126, NCT02514603, NCT02860780, NCT01115790

WEE1

AZD1775

Phase II as monotherapy and in NCT02272790, combination with chemotherapy in NCT02593019, multiple tumor types NCT02101775, NCT02194829, NCT01357161

WEE1

AZD1775

Phase I as monotherapy and as combination therapy in ASTs

ATR (listed above)

NCT02610075, NCT02482311, NCT02207010, NCT03315091, NCT02341456, NCT02508246, NCT02194829, NCT00648648

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(Table 1) cont.....

Pathway Topoisomerase inhibitors

Target

Compound

Latest Stage of Development and Trial Details

Clinical Trial Identifier(s)

Topo I

Belotecan

Licensed for use

Topo I

CRLX101

Phase II as monotherapy and in combination with RT, cytotoxic chemotherapy or bevacizumab in various tumor types

Topo I

Irinotecan

Licensed for use

Topo I

LMP 400

Phase I as monotherapy in ASTs and lymphomas

NCT01051635, NCT01794104

Topo I

LMP 776

Phase I as monotherapy in ASTs and lymphomas

NCT01051635

Topo I

NKTR-102

Phase III as monotherapy in locally recurrent or metastatic breast cancer

NCT01492101, NCT01663012, NCT01773109, NCT01457118, NCT00856375, NCT01991678, NCT00806156, NCT00802945, NCT01876446, NCT01976143, NCT00598975

Topo I

Topotecan

Licensed for use

Topo II

Doxorubicin

Licensed for use

Topo II

Epirubicin

Licensed for use

Topo II

Etoposide

Licensed for use

Topo II

Idarubicin

Licensed for use

Topo II

Mitoxantrone

Licensed for use

Topo II

Teniposide

Licensed for use

NCT00333502, NCT01380769, NCT01652079, NCT02010567, NCT02187302, NCT02648711, NCT02389985, NCT01612546

Replication stress represents one of the several hallmarks of cancer wherein dissociation of DNA polymerase from the replisome complex creates stretches of single-stranded DNA at the replication fork and this results in recruitment of replication protein A (RPA) which triggers DDR, and predominantly activates ATR kinase pathway [29]. Insufficient concentration of nucleotides or other replication factors can also result in replication stress [30]. For example, G1/S checkpoint defect in cancer cells due to pRB deficiencies coupled with CDKN2A deletion or Cyclin D1/Cyclin E amplification can cause premature entry into

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S-phase and then stalling of DNA replication due to insufficient replication resources [31]. Cyclin E and MYC amplification result in rapid replication origin firing causing collision of replication and transcription machinery [32, 33]. MYC overexpression also generates high levels of ROS that causes oxidative damage to DNA [34]. Since cancers are associated with deprivation of cell-cycle checkpoints, increased expression of oncogenes, elevated levels of intrinsic ROS, etc., cancer cells will experience greater replication stress as compared to normal cells upon chemotherapy. This elevated replication stress coupled with the loss of DDR mechanisms can help design cancer-specific and DDR target-specific strategies for a number of proteins functioning in replication stress. RPA recruitment to the ssDNA at the stalled replication fork shields the ssDNA from cleavage and engages ATR and SMARCAL1 (helicase). By activating Chk1 and ribonucleotide reductase M2 (RRM2), ATR inhibits new replication firing and accumulation of ssDNA respectively. Accumulation of ssDNA and subsequent consumption of RPA needs to be prevented because exhaustion of all RPA will result in the remainder ssDNA being converted to lethal DSBs. Apart from the ATR-CHK1 pathway, WEE1-CDK pathway also needs investigation for designing replication stress-driven therapies in cancer [35]. WEE1 and CDKs are involved in replication origin firing and inhibition of WEE1 prevents inhibition of CDK1 and CDK2 via phosphorylation by WEE1, thereby allowing increased replication origin firing, reduction in nucleotide levels and an increase in Mus81Eme1 endonuclease-mediated DSBs [36]. As CDK1 functions as a G2/M checkpoint protein, its upregulation will force early entry of cells into mitosis eventually leading to cell death in the absence of completely replicated DNA. Studies reveal that Chk1 inhibition causes much higher levels of γH2AX induction as compared to ATR inhibition, suggesting that Chk1 inhibition kills cells at much lower levels of replication stress whereas ATR inhibition selectively destroys cells that exhibit stress levels beyond a certain threshold [37]. It has been demonstrated that ATR-Chk1 may not always follow a linear pathway with respect to replication origin firing, which can be prevented even in the presence of ATR inhibitor wherein alternative pathway may be utilized involving DNA-PK and ATM [31]. Table 1 enlists several targets and compounds with which clinical trials are being conducted. Combining DDR Inhibitors with DNA Damaging Radiotherapy and Chemotherapy Over the course of many years, ionizing radiation and systemic chemotherapy have been used for cancer treatment apart from surgery. 1 Gy of ionizing radiation can generate about 1000 SSBs and 35 DSBs due to the oxygen free radicals

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produced, radiation contributes to around 40% of cures [38, 39]. However, one major disadvantage is the collateral damage to normal healthy cells. One approach to overcome this problem is to identify and utilize a tumor’s intrinsic sensitivity to radiation and generate a patient-specific treatment plan. In this way, more sensitive tumors can be given lower doses of radiation whereas resistant tumors can be approached more aggressively. Another approach is combining DDR inhibitor treatment with radiotherapy to sensitize the tumor and to increase therapeutic index of the treatment. A preclinical trial evaluated radiosensitisation effects of PARP inhibitor olaparib in BRCA2-deficient and BRCA2-complemented isogenic cell lines and several human head and neck squamous cell carcinoma cell lines [40]. This study demonstrated that radiation followed by 7-hour exposure to olaparib, at concentrations much lower than those required to induce cell death unassisted, were adequate to radiosensitise the cells. Also, olaparib could radiosensitise the BRCA2-deficient cells at lower doses than in BRCA2-complemented samples. However, it still needs to be ascertained if this kind of combination therapy clinically helps to enhance therapeutic index. One major challenge faced by research of this nature is the requirement of a syngenic or orthotopic immunecompetent host model in which antitumour activity and normal tissue toxicity in response to radiation can be studied simultaneously. Another challenge is the duration of the clinical study as dose escalation studies (to gauge chronic radiation-induced toxicities) require months of follow up in the cohort drug design. Also localized disease control is not a true measure of overall survival of a patient. Combining DDR inhibitors with DNA-damaging chemotherapy also bears its own challenges as they involve systemic delivery of the drug and many of the drugs show overlapping toxicities with DDR inhibitors. Frequently used DNAdamaging chemotherapies are: a. Platinum salts for induction of covalent crosslinks of DNA bases e.g. carboplatin, cisplatin, oxaliplatin, etc. b. Alkylating agents for modification of DNA bases e.g. temozolomide c. Topoisomerase inhibitors e.g. Topoisomerase 1-camptothecin, topotecan and irinotecan and Topoisomerase 2- etoposide, doxorubicin. Topoisomerases function to relax the DNA supercoil by nicking the DNA, rotating the strands and then relegating them. Topoisomerase inhibitors produce non-productive Topoisomerase-DNA cleavage complexes (Topcc) post DNA cleavage but before relegation. Topoisomerase 2 nicks both strands of DNA and its inhibitors produce DSBs while Topoisomerase 1 nicks one strand and its

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inhibitors produce SSBs which get converted to DSBs due to stalled replication forks. Topoisomerase inhibitors differ from PARP inhibitors as their target enzymes normally have error-free activity and are not part of any DDR machinery and also that their therapeutic index is so much smaller than PARP inhibitors. The choice of chemotherapy to be used in conjunction with a DDR agent depends on the type of DNA damage. For instance, Top1 inhibitors can be used in combination with ATM, ATR or PARP inhibitors as all three operate in DDR resulting from Top1 inhibition [41]. Also, a combination of PARP inhibitor and Temozolomide can be used as PARP is required for repair of TMZ-generated SSBs [42]. Identifying an effective combination of drug dosage and schedule is still a major challenge in multiple studies that are being conducted. However, generating such data holds clinical significance as it has been shown that combination therapy is able to re-sensitize chemo-resistant tumors. One approach to alleviate the toxicity of chemotherapy plus DDR agent in healthy cells is to allow a gap of two to three days between chemotherapy and treatment with DDR agent. This gapped schedule approach exhibited differential effects of platinum-induced DNA damage in tumor against bone marrow. In the bone marrow, resolution of DNA damage took place within 48 hours following therapy whereas in tumor cells, gamma-H2AX foci could be detected even after 72 hours. An alternative strategy is to use targeted delivery agents like nanoparticles which is currently being explored. Future Prospects of DDR Targeting Agents in Cancer Treatment Synthetic Lethality Initially, combination therapy of DDR inhibitors began with chemotherapy, with MGMT inhibitor O6-benzylguanin being used in conjunction with alkylating agent BCNU [43]. However, this combination was poorly tolerated. A major development came in 2005, when PARP inhibitor olaparib treatment in BRCAdeficient cancers helped to signify the concept of synthetic lethality. In the last decade, DDR inhibitors targeting essential replication proteins, inhibitors that enhance differential S-phase replication stress between cancers and normal cells, G2/M checkpoint inhibitors, inhibitors that exploit genetic deficiencies in cancers, etc., have been developed. Thus, identifying novel genetic combinations with synthetic lethality holds therapeutic potential. DDR with Epigenetic Compounds Epigenetic modulators are also being tested with DDR inhibitors. For example, histone lysine methyltransferase (HKMT) inhibitors have been shown to

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cooperate with PARP inhibitor activity, thereby preventing retention of BRCA1/BARD1 complex at DSBs and inhibiting cancer cell growth [44]. IMMUNOTHERAPY The essential role of the immune system as the natural defence against cancers has been studied for years. It has also been observed in clinical researches and epidemiological studies that immunosuppressed patients have higher risks of developing different types of cancers. Amongst them, the incidence of Kaposi’s sarcoma, a type of cancer developed from the opportunistic infection of human herpes virus type 8 (HHV-8) in lymph node and skin, has been shown to be 400 to 500 higher in HIV-positive or organ transplant patients compared to the control population [45]. The termination of immunosuppressive treatments reduces the risk for Kaposi’s sarcoma, suggesting a possible association between immunosuppression and tumour development in certain individuals [45, 46]. Furthermore, studies among patients showing spontaneous tumour regression after episodes of bacterial infections present convincing evidence that the immune system plays a significant role in combating cancer development [47 - 49]. Among them, the injection of attenuated Baccillus Calmette-Guerin bacterium for the treatment of superficial bladder cancer has been in practice since 1976 [49]. This suggests that the enhancement of one’s immune system has the potential to counter tumorous growth. These observations build the basis for continuous efforts in harnessing the immune system for effective cancer treatments. In recent years, immunotherapy has shown promises as a powerful and specific treatment for cancers. Immunotherapy is considered as a biological therapy, which makes use of substances produced by living organisms to counter cancerous growth. There are several advantages of immunotherapies in cancer treatments. Unlike chemotherapy and radiotherapy, immunotherapy is more specific and does not target normal rapidly dividing cells such as hair follicles, bone marrow or intestinal epithelial cells [50 - 52]. Due to the specificity of immunotherapy, damages to adjacent normal tissue can be reduced as compared to the standard chemotherapy and radiation therapy. Furthermore, immunotherapy can be modified to target non-dividing cancerous cells such as rare cancer stem cells, which are associated with cancer recurrence. Location-wise, modified immune cells can target areas that are unreachable by surgery. Given the heterogeneous nature of tumour, there are variations in responses to general treatments among different patients having the same type of tumour. Therefore, the flexibility of immunotherapy allows greater specific and effective targets of cancers. Personalized treatments of immunotherapy designed to target neoantigens unique to a certain population have the potential to reduce the problem of heterogeneity among patients [53]. Considering these advantages,

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immunotherapy presents a paradigm shift in cancer treatment routines in addition to the traditional methods. The regular use of immunotherapy in combination with standard radiation therapy, chemotherapies and surgery is foreseeable in standard cancer treatment of the future. Approaches to immunotherapy are categorized into five broad branches: adoptive cell transfer approach, vaccination approach, antibody therapy, cytokine approach and oncolytic virus approach [54, 55]. In the past few years, there have been numerous clinical trials and FDA-approved drugs in response to positive findings highlighting the effectiveness of immunotherapy in the treatments of different cancer types. This review aims to: (i) give a brief overview of these immunotherapeutic approaches by discussing their mechanisms of action, their advantages and disadvantages as well as highlighting currently approved drugs in each area (Table 2), (ii) discuss in detail the newly approved anti - CD19 chimeric antigen receptor (CAR) T cells therapy and its vast potential. Table 2. Current FDA-approved immunotherapeutic drugs. Treatment Strategy

Modes of Immunotherapeutic Approaches

Target

Cytokine therapy

Drug Name (Year Approved) IFNα-2b (1986) Cetuximab (2004)

mAbs targeting proliferation pathways

EGFR-targeting mAbs

Panitumumab (2006) Necitumumab (2015)

HER-2 targeting mAbs

Monoclonal antibody Antibody immunotherapy immunotherapy (mAbs)

EGFR

mAbs targeting blood vessel formation mechanism

HER-2

Trastuzumab (1998) Pertuzumab (2012)

VEGF-A

Bevacizumab (2004)

VEGF-R2

Ramucimumab (2014) Rituximab (1997)

mAbs targeting cancer-specific antigens

Lineage-specific antigen (LSA)targeting mAbs

CD20

Non lineagespecific antigen CD52 (NLSA)-targeting mAbs

Obinutuzumab (2013) Ofatumumab (2009) Alemtuzumab (2001)

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(Table 2) cont.....

Treatment Strategy

Modes of Immunotherapeutic Approaches

Target

Drug Name (Year Approved)

CD38

Daratumumab (2015)

SLAMF7 Elotuzumab (2015) mAbs targeting CTLA-4 mAbs immune checkpoints

CTLA-4

Iplimumab (2011) Nivolumab (2014)

PD-1/PDL-1 axis PD-1 mAbs

Monoclonal antibody Antibody immunotherapy immunotherapy (mAbs)

Pembrolizumab (2014) Durvalumab (2017)

PDL-1

Atezolizumab (2016) Avelumab (2017)

mAbs targeting other factors Bispecific mAbs Antibody-drug and Antibody-drug antibody-radioisotopes conjugate conjugates

PDGFR-α Olaratumab (2016) RANKL

Denosumab (2013)

CD19 and Blinatumab (2014) CD3 HER2

Ado-trastuzumab emtansine (2013)

CD22

Inotuzumab ozogamicin (2017)

CD33

Gemtuzumab ozogamicin (2017)

CD30

Brentuximab vedotin (2011)

CD20

(90)Y-ibritumab tiuxetan (2002)

CD20

(131)I-tositumomab (2003) (withdrawn in 2014)

Therapeutic cancer vaccination therapy

NA

Sipuleucel-T (2010)

Oncolytic viruses therapy

NA

Talimogene laherparepvec (2015)

CD19

Tisagenlecleucel (2017)

CD19

Axicabtagene ciloleucel (2017)

Antibody immunotherapy Antibody-radioactive conjugates

Tumour-infiltrating Adoptive cell lymphocyte therapy transfer therapy

CAR-T cell therapy

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Common Immunotherapeutic Approaches Cytokine Therapy Cytokines are signalling molecules that cells use to communicate with one another. They play a major role in the development of both innate and adaptive immune systems. For most immune cells, cytokines are essential in their proliferation, differentiation and prolonged survival necessary for effective functioning of the immune system. Harnessing the role of cytokines in immunotherapy enhances or inhibits certain immunologic responses in leukocytes. Various clinical trials with interferon (IFN)-α, interleukin – 2 (IL-2), IL-12, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GMCSF) and tumour necrosis factors (TNFs) suggest their roles in effective treatment of cancer [56]. Among them, IFN-α and IL-2 cytokines have been approved by the FDA for the treatment of different cancer types [56]. IFN-α was the first cytokine therapy to be approved by the FDA for the treatment of hairy cell leukemia (HCL) in 1986 [57]. Subsequently, IFN-α was also permitted as a treatment for several cancer types such as Kaposi’s sarcoma, metastatic melanomas [58] and chronic myelogenous leukemia for instance [59], [60 - 63]. IFN-α belongs to type I IFN - cytokines that are produced upon viral invasion by various immune cells [64, 65]. Through tyrosine kinase (Tyk) and Jak/Stat signalling, IFN-α activates antigen specific B and T cells, which aids in the maturation of dendritic cells (DCs) as well as promote cancer cell apoptosis [56]. Among HCL patients treated with IFN-α, 77% showed significant improvements in red blood cell count and granulocyte level [66]. After the success of IFN-α, IL-2 was also approved by the FDA in 1992 to treat melanoma and renal cell cancer [67, 68]. IL-2 is a globular glycoprotein cytokine that acts through Janus kinase signal transducers and activators of transcription (JAK/STAT) signalling to promote natural killer (NK) cells, antigen-specific CD8+ and CD4+ T cell expansion [69 - 72]. IL-2 also mediates antibodies secretion from IgM-stimulated B cells. High doses of IL-2 therapy against metastatic renal cancer used over different durations have shown moderate responses of 14% with 5% of the patients having complete remissions (CR) [73 75]. In addition, low dose of IL-2 treatment has been shown to increase the amount of CD56+ CD3- NK population from 450% to 900% [76]. Even though some positive results have been observed in many clinical trials, the use of cytokine therapy as monotherapy is limited in its potential in cancer treatment. In terms of effectiveness, IL-2 and IFN-α administrations have induced many side effects in patients [77]. This is because cytokines act within a short distance, which results in high concentrations of cytokines being administered

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peritoneally or subcutaneously to achieve desired effects [78 - 83]. This large dosage of cytokines causes flu-like symptoms like nausea, fever or neuropsychological effects like mania or depression. In fact, FDA immediately terminated all IL-12 trials after a phase II clinical trials of IL-12 therapy caused severe side effects to 15 over 17 patients, resulting in the death of two patients [84]. Flu-like symptoms caused by severe toxicities of IFN-α are also well documented [85, 86]. Furthermore, some cytokines used in immunotherapy have been implicated in immunosuppression. In fact, IL-2 plays a role in the maintenance of peripheral regulatory T cells (Tregs) – T cell subpopulation that mediate immunosuppression and immune escape of cancerous cells [87]. This compromises on the effectiveness of IL-2 as a cytokine therapy. Antibody Immunotherapy Monoclonal Antibody Immunotherapy Monoclonal antibody (mAb) therapy has been used a long time ago as a useful tool to target specific cancer-related antigens. These cancer-specific antigens can be expressed on cancer cells or as active members in cancer proliferation pathways, blood vessel formation or immune checkpoint mechanism [88, 89]. Furthermore, conjugation of radioactive substances or other anticancer drugs like cytokines and toxins with mAbs has been shown to enhance the effectiveness of tumour targeting in mAb therapy [90]. The most commonly used mAb is IgG mAb, which has two regions: a variable region and a constant fragment. The variable region is antigen-specific while the constant fragment helps bring together antigen-specific cells and leukocytes for elimination. Furthermore, the constant fragment of mAb facilitates the immune role of mAb against antigenspecific cells like antibody-dependent cell cytotoxicity (ADCC), complementdependent cytotoxicity (CDC) or antibody-dependent phagocytosis (ADCP) [91, 92]. Hence, mAb functions as a useful tool in cancer immunotherapy by facilitating the killing of cancer cells using ADCC, CDC and ADCP, inhibiting specific ligand-receptor interaction on cancer cells or regulating checkpoint molecules on immune cells [91, 92]. MAbs Targeting Cell Proliferation Pathways Spontaneous mutations of genes involved in proliferation pathways lead to uncontrolled divisions of cancer cells. Among them, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor HER2, members of ErbB family of receptor cytokine kinases [93, 94] are cell surface receptors that have been widely implicated in the autonomic growth of various cancer types [95, 96]. They have extracellular ligand binding domains, through which specific ligands bind and activate growth-related pathways [97]. Therefore, several mAbs

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have been approved by the FDA to specifically target these EGFR and HER2 extracellular domains involved in the downstream proliferation pathways. EGFR-targeting mAbs EGFR is a transmembrane tyrosine kinase receptor protein, which is the most extensively researched member of the ErbB family [94, 98]. Overexpression of EGFR leads to the dysregulation of downstream pathways implicated in several types of tumour formation [98, 99]. Cetuximab (approved in 2004), a human recombinant mAb used in colorectal cancer (CRC) and head and neck cancer treatments, binds specifically to EGFR extracellular domain and competitively inhibits the binding of EGFR ligand [100]. This prevents the activation of EGFRassociated growth pathways, proinflammatory cytokines secretion, matrix metalloproteinases production, angiogenesis as well as apoptotic induction [101]. Using similar mechanisms of targeting EGFR extracellular domain, panitumumab (approved in 2006) and necitumumab (approved in 2015) are also used in the treatment of metastatic colon cancer [102 - 104] and squamous non-small cell lung cancer (NSCLC) [105], respectively. HER2-targeting mAbs Besides EGFR, HER2 overexpression is also implicated in several cancer types such as breast, gastric, ovarian, endometrial and oesophageal cancers [106]. Unlike EGFR, HER2 has no known ligand and its activation is dependent on receptor dimerization with other ligand-activated receptors from EGFR family. Several drugs targeting HER2 take advantage of these mechanisms. Trastuzumab (approved in 1998), a humanized mAb, binds to HER2 receptor and inhibits its dimerization, preventing the activation of downstream proliferation pathways [107]. Trastuzumab is also known to target HER2-positive tumour cells by ADCC and to block HER2 activation by proteolytic cleavage. In combination with chemotherapeutic agents, trastuzumab has been approved for the treatment of breast, metastatic gastrointestinal cancers and gastric or gastrooesophageal junction adenocarcinoma [108 - 113]. In addition, Pertuzumab (first approved in 2012) is a recombinant humanized mAb that binds to HER2 extracellular dimerization domain and inhibits HER2 heterodimerization [114]. Pertuzumab was approved for the treatment of breast cancer in combination with other chemotherapies [115, 116]. MAbs Targeting Blood Vessel Formation Mechanism For successful delivery of nutrients and metastasis of fast-growing cancerous cells, angiogenesis - the formation of new blood vessels is essential. Hence, genes encoding regulators of angiogenesis are often upregulated in various types of

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tumours. Among them, vascular endothelial growth factor (VEGF) is one of the most important activators of angiogenesis, presenting an ideal target for anticancer therapies [117 - 119]. By binding to its VEGF receptors (VEGF-R), VEGF stimulates endothelial cells to form new blood vessels. Among different subtypes of VEGF, VEGF-A plays the most crucial role in angiogenesis by binding to its target receptor VEGF-R1 and 2 [119]. Therefore, VEGF-A and VEGF-R1 or 2 are reasonable targets for mAbs in immunotherapy. Bevacizumab (first approved in 2004) is a recombinant humanized mAb that targets VEGF-A and blocks its interaction with VEGF-R [120]. As a result, VEGF downstream pathways are inhibited, preventing the formation of new blood vessels. Currently, bevacizumab was approved for the treatment of solid tumours such as metastatic CRC (mCRC) (by European Medicines Agency and not FDA), glioblastoma, NSCLC, renal cell carcinoma , cervical cancer and ovarian cancer [121 - 124]. Besides bevacizumab, ramucirumab (first approved in 2014), a recombinant mAb, is the newest approved drug being included in the family of VEGF-targeting mAbs immunotherapy [125]. Ramucirumab binds VEGF-R2 and inhibits VEGFmediated metastatic pathway by changing the receptor conformation [126, 127]. Ramucirumab was approved for the treatment of metastatic gastric and gastroesophageal cancers, NSCLC and mCRC [128]. MAbs Targeting Cancer-Specific Antigens In the haematological field, there have been extensive researches on antigens expressed by different subtypes of leukocytes through different stages of development. These distinct antigens in the form of lineage-specific antigens (LSAs) and non-lineage specific antigens (NLSAs) are often abnormally expressed on tumorous leukocytes [129]. Therefore, many mAbs have been designed and approved to target these LSAs and NLSAs as haematological cancer immunotherapies. LSA-targeting mAbs Under normal circumstances, different LSAs are present at different specific developmental stages of leukocytes, which provide effective targets for mAbs in cancer treatment. In B cell development, cluster of difference (CD) 19, 20, 22 are associated with different B cell developmental stages prior to final development of B cell into plasma cells [130]. Once bound, these mAbs mediate ADCC, ADCP or CDC, leading to the eradication of tumour cells as discussed above. Therefore, many mAbs are developed to target CD20 in malignant B cells [131]. Among them, rituximab (first approved in 1997) was the first approved CD20-targeting mAb for the treatment of B cell, nHL, chronic lymphocytic leukemia (CLL), follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) [132 -

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136]. Rituximab is a chimeric human-mouse mAbs that binds to CD20 on both normal and malignant B cells [137, 138]. Because CD20 is not expressed in hematopoietic stem cells, pro-B cells or mature plasma cells, normal B cell population is regenerated from stem cells after several months or years while malignant cells are eliminated [139]. Using the same CD20-targeting mechanism, ibritumumab (first approved 2002), tositumomab (first approved 2003), ofatumumab (first approved 2009) and obinutuzumab (first approved 2013) [140 143] are also mAbs approved for the treatment of different types of haematological cancer [144 - 146]. The effectiveness of CD20 targeting mAbs prompts the investigation of other LSA targets such as CD19, CD22 or CD79b used in conjugation with other therapies in clinical trials [147 - 149]. Despite successes in the treatments of B cell malignancies, the efficacy of LSA-targeting mAbs is limited in patients who do not express LSAs on cancerous B cells. Therefore, there is a need for the identification and design of NLSA-targeting mAbs. NLSA-targeting mAbs In contrast to LSAs, NLSAs are expressed on cancerous leukocytes but are not present exclusively on a certain subset of leukocytes [129]. NLSAs like chemokine, proliferation pathway receptors, microenvironmental mediators or overexpressed antigens are involved in different tumorigenic pathways such as abnormal proliferation, differentiation and immune escape mechanisms of malignant haematological cells [129]. Examples of NLSAs include CD52 - a glycoprotein on monocytes and lymphocytes [150], CD38 – a transmembrane receptor involved in signal transduction in myeloid and lymphoid cells [151, 152] and SLAMF7 – a glycoprotein expressed on plasma cells, natural killer cells and CD8+ T cells [153, 154]. These NLSAs are overexpressed in cancerous states, making them ideal targets for mAbs for effective cancer treatments. CD52 is overexpressed in different haematological cancers such as acute lymphoblastic leukemia (ALL), CLL or T cell prolympholitic leukemia [150 - 152, 154 - 157]. Therefore, anti-CD52 mAb alemtuzumab (initially approved in 2001) [158] is developed and approved as a treatment for CLL [159 - 161]. Using the same rationale, other approved mAbs targeting different NLSAs include anti-CD38 daratumumab (first approved in 2015) for the treatment of multiple myeloma (MM) [162, 163] and anti-SLAMF7 elotuzumab (approved in 2015) for the treatment of MM [164, 165]. In addition, CCR4, a chemokine transmembrane receptor, which facilitates the pathological development of different T lymphocyte subtypes, is also a target for therapeutic mAbs. Currently, mogamulizumab is the only approved anti- CCR4 mAb (first approved in 2012 by Japan, validated by European Medicines Agency and pending status by FDA) for

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the treatment of cutaneous T-cell lymphoma [166 - 168]. MAbs Targeting Immune Checkpoints The immune system functions effectively by keeping the delicate balance between immune reactivity and immune suppression. This is important for the body to mount effective responses against harmful agents while avoiding autoimmune reactions against self-antigens using immune checkpoints mechanism. Immune checkpoints are inhibitory signalling pathways that contribute to self-tolerance. It has been established that several types of cancer cells can escape immune surveillance by various methods, leading to regression of different cancer types [169]. Among them, modifications of immunoregulatory checkpoint pathways in innate and acquired immune systems have been widely employed by cancer cells to evade detection and elimination by different leukocytes [170]. Therefore, many mAbs used in immunotherapies have been designed to block these immune checkpoint pathways such as programmed death-1/programmed death ligand-1 (PD-1/PDL-1) axis and cytotoxic T lymphocyte antigen 4 (CTLA-4) on T lymphocytes. This restores the effectiveness of the immune system in targeting tumorous cells. Hence, second-generation mAbs have the ability to target receptors expressed on either tumour cells or immune cells. Therefore, these different types of mAb work together in a complementary fashion to kill tumour cells and at the same time, restore the host immune reactivity against cancerous growth. CTLA-4 mAb CTLA-4 is an inhibitory receptor expressed on activated cytotoxic CD8+ T cells. This receptor is a structural homolog of CD28 receptor – a co-stimulatory molecule expressed on T cells. Even though both CTLA-4 and CD28 bind to B7 family ligands, CTLA-4 has greater affinity for these ligands than CD28 [171]. The binding of CTLA-4 receptors with its B7 family ligands expressed on activated DCs dampens cytotoxic T cell functions by reducing T cell proliferation and IL-2 secretion [172]. Therefore, mAbs blocking CTLA-4 ligand-receptor interaction have been effective in the treatment of several cancer types. Iplimumab (first approved in 2011) is the first anti-CTLA-4 mAb approved for the treatment of metastatic and cutaneous melanoma [173, 174]. Several other anti-CTLA-4 mAbs such as tremelimumab have also been showing positive results in clinical trials. PD-1/PDL-1 Axis mAbs Like CTLA-4, PD-1 receptor is a member of CD28 family, which is expressed on T and B cells [175]. Its ligands, PDL-1 and PDL-2 express differentially on

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leukocytes. PDL-1 is expressed constitutively on T cells and antigen-presenting cells (APCs) as well as other non-haematological cells such as neurons, vascular endothelial cells as well as cancerous cells in renal, ovarian, urothelial cancers, just to name a few. In contrast, PDL-2 is mostly expressed in an inducible manner on macrophages and DCs [176]. Targeting PD-1/PDL-1 is more effective than PD-1/PDL-2 interaction as PD-1/PDL-1 interaction is more potent in mediating immune suppression in peripheral tissues [177]. Therefore, many mAbs targeting PD-1/PDL-1 have been approved for the treatment of various cancer types. In fact, treatments with PD-1/PDL-1 targeting mAbs have yielded huge success with limited side effects in a wide spectrum of cancer types. For instance, nivolumab (first approved in 2014) is a mAb that targets PD-1 and thus, inhibits its interaction with PDL-1 and PDL-2 [178, 179]. This enhances T cell activation in anti-tumour immunity against metastatic melanoma, metastatic squamous NSCLC, classical Hodgkin lymphoma (cHL), metastatic head and neck squamous cell carcinoma (HNSCC), metastatic or locally advanced urothelial carcinoma (UC), mCRC as well as hepatocellular carcinoma (HCC) from 2014 to 2017 [178]. Similar to nivolumab, pembrolizumab (first approved in 2014) is a humanized mAb that also targets PD-1 and inhibits its interaction with PDL-1 or PDL-2 [180, 181]. Pembrolizumab is used in the treatment of metastatic melanoma, metastatic NSCLC, HNSCC, cHL, mUC, CRC as well as gastric and gastroesophageal junction adenocarcinoma from 2014 to 2017 [180]. Durvalumab is the latest PD-1 targeting mAb to be approved in 2017 for the treatment of UC. In addition to the target of PD-1, several mAbs are also designed and approved to target PDL-1 [182]. Atezolizumab (approved in 2016) is a humanized mAb that targets PDL-1 and blocks its interaction with PD-1. Atezolizumab is used for the treatment of UC and mNSCLC [183]. Avelumab, the latest anti-PDL-1 mAb is approved in 2017 for the treatment of Merkel cell carcinoma and UC [184]. MAbs Targeting Other Factors Due to the effectiveness of mAbs in immunotherapy, the potential of mAbs that target other cancer-related factors involved in tumour microenvironement maintenance or aberrant osteoclast activation pathway are also investigated. Tumour microenvironment is necessary for cancer cells to evade immune surveillance and elimination [185]. Hence, olaratumab (approved in 2016) a recombinant human mAb that targets platelet-derived growth factor receptor receptor alpha (PDGFR-α) has been designed to target microenvironment-related pathways [186]. This is because pathologic PDGFR-α activation has been shown to be involved in the maintenance of tumour microenvironment, facilitating the immune escape ability of cancer cells [187]. Olaratumab is used in the treatment of soft tissue sarcoma [186].

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Besides regulating tumour microenvironment, metastatic cancer cells invade bones and aberrantly activate osteoclasts, leading to bone destruction. This activation is due to the interaction between receptor activator of NF-kappa B ligand (RANKL) and its RANK receptor expressed on osteoclast. Therefore, denosumab (approved in 2013), a human mAb, binds RANKL and inhibits RANK/RANKL interaction, blocking aberrant osteoclast activation. Denosumab has been used to reduce bone adsorption in solid tumours. Bispecific mAb Bispecific mAbs are engineered antibodies, which comprise of two arms with dual specificities for two different antigens on effector and tumour cells [188]. The most commonly investigated bispecific mAb is the bispecific T cell engager (BITE), which binds cytotoxic T cell with tumour cells. This brings cytotoxic T cells close to cancer cells, facilitating the elimination of cancer cells. Blinatumab (first approved in 2014) is a BITE targeting CD19 expressed on tumour B cells and CD3 expressed on T cells. Blinatumab is currently used in the treatment of Bcell ALL (B-ALL) [188, 189]. Since the first approval in 1997, the usage of mAbs as the first or second line therapy for different cancer types emphasises the important role of mAb in cancer treatment. In fact, the approval of many mAbs in 2017 and at least 4 other mAbs waiting to be approved by the end of 2018 demonstrate the huge potential of mAbs in the treatment of various cancer types [190]. However, different types of mAbs still focus on a limited pool of biomarkers such as HER2, EGFR or some common checkpoint inhibitors. This is a pressing issue especially since cancer cells spontaneously mutate, leading to the loss of target antigens for mAbs. Hence, identification of more tumour-specific antigens allows a more complete elimination of cancer cells by mAbs. Furthermore, the success story of mAbs targeting checkpoint inhibitors is due to the identification of correct pathways contributing to immunosuppressive mechanism. Therefore, continued research on tumour immune microenvironment proves a necessary step in designing effective therapeutic mAbs. Antibody-drug and Antibody-Radioisotopes Conjugates Antibody-drug conjugates (ADCs) and antibody-radioisotope conjugates (ARCs) comprise of a mAb covalently conjugated to a cytotoxic agent or radioisotope via a linker respectively. ADCs and ARCs take advantage of the specificity of mAb in binding to the targets as well as the antineoplastic feature of cytotoxic agents in the elimination of cancerous growth. In addition, ARCs are also used in imaging to accurately identify regions of tumourous growth. It has also been shown that ADCs increase cytotoxic agents’ stability in circulation and specificity to tumour

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environments and thus, reducing the side effects of cytotoxic agents when taken alone. The developments of ADCs and ARCs have been promising with several drugs approved in 2017 and 2018 for the treatment of both solid and haematological tumours. Antibody-Drug Conjugates Among the ADCs, ado-trastuzumab emtansine (approved in 2013) is a humanized anti-HER2 mAb conjugated to a microtubule inhibitor DM1 for the treatment of HER2 positive metastatic breast cancer. As mentioned before, HER2 is implicated in the proliferation pathway of cancer cells. Thus, ado-trastuzumab emtansine provides a specific delivery of DM1 to the tumour clusters. Inotuzumab ozogamicin (approved in 2017) is a humanized anti-CD22 mAb conjugated to calicheamicin for the treatment of precursor B-ALL. As mentioned before, CD22 is a B cell-specific surface antigen. It has been observed that CD22 is expressed in more than 90% of ALL cases, making it an ideal candidate for B-ALL immunotherapy [191]. Calicheamicin is a cytotoxic agent that induces DNA strand break and thus, reduces the proliferation of cancerous cells [192, 193]. Hence, the combination of anti-CD22 mAb with calicheamicin in inotuzumab ozogamicin has proven effective in the treatment of relapsed B-ALL patients [194]. In addition, gemtuzumab ozogamicin is another approved drug in 2017 for the treatment of acute myeloid leukemia (AML). Gemtuzumab ozogamicin is an anti-CD33 mAb conjugated to cytotoxic calicheamicin derivative [195]. Normally, CD33 is present on different types of myeloid cells as well as some subsets of B, T and NK cells [196, 197]. Even though there are variable expressions of CD33 among AML patients, almost all patients have CD33 expression on leukemia blasts [198 - 200]. Furthermore, CD33 is overexpressed in acute promyelocytic leukemia (APL), making CD33 a potential candidate for ADC target [201, 202]. Brentuximab vedotin is another ADC (first approved in 2011) for the treatment of cHL and primary cutaneous anaplastic large cell lymphoma. This ADC comprises of anti-CD30 human chimeric mAb conjugated to microtubule disrupting agent monomethyl auristatin E – a cytotoxic drug preventing the rapid proliferation of cancer cells [203 - 205]. CD30 is a surface receptor overexpressed on some tumour cells but minimally present on normal cells [206]. In fact, the presence of Reed-Sternberg cells marked by CD30 expression is a typical characteristic of cHL, making CD30 a good target for ADC treatment in cHL [206, 207]. Antibody-Radioactive Conjugates Currently, there are two FDA-approved ARCs that are used as immunotherapy: (90)Y-ibritumomab tiuxetan (approved in 2002) and (131)I-tositumomab

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(approved in 2003) for the treatment of non-Hodgkin Lymphoma (nHL) [51]. Several other ARCs are approved for imaging and detection purposes, which shall not be discussed in this review. These ARCs are anti-CD20 murine mAb conjugated with radioisotopes yttrium-90 or iodine-131 respectively. This conjugation in ARC provides a more specific delivery of radioisotopes into CD20-specific tumour clusters, killing cancer cells by inducing DNA damage [208]. Therapeutic Cancer Vaccination Therapy The field of therapeutic cancer vaccination has been evolving rapidly in recent years. The idea of vaccination, even though not entirely novel, has been investigated for its potential in anti-cancer immunotherapy. Cancer vaccinations attempt to trigger immune responses against cancer cells by presenting the whole tumour cells or DCs incorporated with tumour-associated antigen (TAA) to the patient’s immune system. This whole cancer cells or TAA-specific DCs can be derived from the patient’s peripheral-blood mononuclear cells (PBMCs) (autologous) or from a different source (allogenic). Once vaccines containing these factors are injected into the patient, cancer-specific T cells will be activated through antigen presentation by DCs. These T cells differentiate and proliferate with the help of suitable activation signals, which can be provided by vaccine adjuvants. Finally, activated, antigen-educated T cells will seek and destroy cancer cells [209, 210]. Vaccines against oncogenic viruses are also investigated for their anti-cancer potential. The discussion of this type of vaccination will not be included in this review. There are several types of cancer vaccinations including, (i) genetic vaccines (ii) peptide and protein vaccines, (iii) whole tumour cell vaccines as well as (iv) immune cell vaccines currently under active clinical trials. However, the only vaccine approved for immunotherapy is sipuleucel-T (approved in 2010) for the treatment of metastatic castrate-resistant prostate carcinoma [211]. Sipuleucel-T is an immune cell vaccine, which consists of autologous PBMCs including PA2024-activated APCs. PA2024 is a recombinant protein containing activator, granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with a prostate cancer antigen, prostatic acid phosphatase [212]. Different from other conventional cancer treatments like chemotherapy, the efficacy of cancer vaccination can only be observed over a period of time, in order for the immune system to be fully activated. Hence, the usage of cancer vaccination is more beneficial in the earlier stages of cancer. In patients with cancers in later stages, cancer vaccinations should be used in combination with other conventional and immunotherapeutic treatments. As the designs of cancer vaccinations are based on specific TAAs, similar to mAbs, novel TAAs should be identified. No single TAA will be sufficient and a range of different TAAs should be used in vaccination for a more complete activation of the immune system

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[209]. In a recent clinical trial, six patients treated for melanoma with a vaccine targeting up to 20 peptide TAAs in combination with PD-1 mAbs either experience no recurrence or CR [213]. This favourable result demonstrates the potential of cancer vaccination, especially in combination with other treatments. Oncolytic Viruses Therapy Oncolytic viruses therapy is classified as an emerging class of anti-cancer therapy, which has characteristics of both biologic therapy and immunotherapy [214]. Taking advantage of viral abilities to invade cells, attenuated viruses can be used to eliminate specific cancer cells by lysing. These modified viruses are harmless against normal cells but have the ability to lyse cancerous cells. This is because most cancerous cells have lost the ability to defend against viral infection, making them vulnerable targets for oncolytic viruses [214, 215]. Currently, talimogene laherparepvec (T-VEC) (approved in 2015), a herpes-simplex-1 virus (HSV-1) is the only oncolytic virus therapy approved for the treatment of advanced melanoma. T-VEC is engineered to express GM-CSF, a cytokine used to promote DC maturation and proliferation, which helps boost tumour-antigen presentation and T cell reactivity [216 - 218]. Unlike other treatments, the usage of live oncolytic viruses for the treatment of various cancer types present unique safety considerations. So far, oncolytic viruses are tolerable in various clinical treatments. However, as a “live” treatment, oncolytic viruses can replicate, which requires special attention in handling and management of this therapy, especially in immunocompromised patients and related healthcare workers [219]. Dosing of oncolytic viral treatment should be optimized as well, as a small amount of viral injection at the tumour site can actually replicate [220]. Furthermore, the usage of oncolytic virus therapy in combination with PD-1 mAb has shown favourable results. In fact, an overall response rate of 62% has been documented with oncolytic viruses/PD-1 combination for the treatment of advanced melanoma [221]. This result, again, emphasizes the importance of immunotherapeutic combinations in the treatment of various cancer types. Adoptive Cell Transfer Therapy Adoptive cell therapy (ACT) is one of the most promising fields in immunotherapy that is currently under many investigations and expansions. This therapy involves the isolation followed by in-vitro expansion and activation of patient immune cells before infusing them back to the patient. There are many types of ACT, including cytokine or lymphokine-activated killer cell therapy and DC therapy. However, the most widely investigated ACT type is the tumourinfiltrating lymphocyte (TIL) therapy, which uses mostly T cells. Tumourinfiltrating T lymphocytes can be extracted directly from the patients [222] or

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taken from in-vivo engineered, tumour-specific T lymphocytes using the technique called chimeric antigen receptor T-cell (CAR-T cell) therapy. Since its first conceptualization in 1989 [223], the approvals of CD19 CAR-T cell therapy tisagenlecleucel and axicabtagene ciloleucel in August and November, 2017 for the treatment of B-ALL and large B-cell lymphoma respectively mark important turning points in the history of ACT development as an immunotherapy, especially as therapies for B cell malignancies [224]. This review will focus on the effort of CD19 CAR-T therapy thus far as well as critically discuss the potential of CD19 CAR-T therapy in the future. The Basis Behind CAR-T and CD19 CAR-T Therapy CAR is a recombinant receptor that contains antigen-specific segment linked to a T-cell signalling domain [225 - 227]. The antigen-specific segment is an extracellular single-chain variable fragment (scFv) that targets tumour antigens such as CD19, CD20 or CD22. In contrast, T-cell signaling domain contains an intracellular T cell receptor (TCR) signaling domain CD3 (CD3 zeta) (in the case of first-gen CAR-T) that activates and stimulates T cell proliferation, differentiation and cytotoxic activities for tumour cell elimination. Due to the inefficiency [228 - 230] of this first-gen CAR-T, second and third-gen CAR-Ts are developed with additional T-cell signaling domains like costimulatory domains from CD28, CD134 or CD137. These additional domains better stimulate T cell activity [231 - 235]. Complete CAR construct is transferred into a delivery vector like retrovirus [236 - 241] or lentivirus [224, 242, 243] in order to incorporate this construct into the T cell genome. These T cells are isolated from the patient’s own pool or from allogeneic donor. After being genetically modified, CAR-T cells are infused into the same patient. Like CD20, CD19 is a surface antigen homogenously expressed on normal B as well as cancer cells that is needed for normal B cell development [231, 244]. In fact, CD19 is present in more than 95% of nHL cases [245], making CD19 an ideal target for nHL treatments, especially in patients with CD20 downregulation. Because CD19 expression is limited to B cells and follicular DC, targeting CD19 does not affect other haematological cells, including haematopoietic stem cells. Therefore, normal B cells can be generated from haematopoietic stem cells after the treatment [246]. Ig replacement therapy (IVIg) can also be employed to counter hypogammaglobulinemia resulted from B cell aplasia – low or absent of B cell count when CD19 CAR-T cell therapy targets normal B cells [247, 248]. CD137 is a member of TNF receptor and is usually expressed on immune cells including activated T cells [249, 250]. Costimulatory signal mediated by CD137 promotes T cell proliferation and survival [251]. Both tisagenlecleucel and

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axicabtagene cilolucel are third-gen CD19 CAR-T whose intracellular fragment includes both CD28 and CD137 costimulatory fragments besides CD3 fragment. The production of CD19 CAR-T cells uses Good Manufacturing Practice (GMP) system before infusion back to the patients. Currently, closed automated GMP process instead of the conventional manual one is under investigation [252, 253]. Advantages of CD19 CAR-T Cell Therapy In terms of efficacy, it is undeniable that CD19 CAR-T cells are powerful against relapsed and refractory B lymphomas and leukemias, especially when other treatments have failed. CD19 CAR-T cell therapy is also a durable treatment option with CAR-T cells persisting for more than 10 years after infusion [254]. Furthermore, it is shown that CD19 CAR-T cells containing CD137 domain is more persistent than CD28 domain [233, 255, 256]. In terms of safety, the use of autologous T cells obtained from the patients lower the risk of graft-versus-host diseases (GVHD). In fact, GVHD associated with the use of allosteric haematopoietic stem cell transplant (AHST) in the treatment of B cell malignancies account for 15% to 40% mortality rate [226]. Furthermore, CD19 CAR-T cells are more specific, reducing the risk of off-tumour targeting of normal cells in conventional therapies. Chemotherapy and radiotherapy are effective in targeting rapidly replicating cells. However, they are unable to differentiate between normal and cancerous proliferating cells, leading to many adverse effects [226, 257]. Common Side Effects and Considerations of CD19 CAR-T Cell Therapy Even though CAR-T cell therapy generally and CD19 CAR-T cell therapy specifically are promising approaches for B cell malignancies, there are many factors that affect the efficient and safe use of CD19 CAR-T cells. Immunogenicity against CAR-T Cell There is a risk of immune reactivity against murine scFv domain on CAR-T cells, which reduces the long-term persistence of CAR-T cells. In fact, this effect has been noticed in many clinical trials [228, 258, 259]. In terms of safety, this rejection of CAR-T cells causes acute anaphylaxis for patients after a few infusions with CAR-T cells [260]. One way to reduce this reactivity is to reduce host B cell population prior to the treatment of CD19 CAR-T cells in order to impede the formation of CD19specific antibodies against murine antigen on CAR-T cells. In fact, lymphodepletion has been shown to increase in vivo expansion and persistence of CAR-T cells [243, 261]. However, this approach does not address T cell-mediated

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targeting of CAR-T cells [262]. Another way is to use humanized scFv for CAR designs in the future. As of now, there is no evidence that the presence of antiCD19 mAb prior or induced by the treatment would affect the efficacy and safety of axicabtagene ciloleucel and tisagenlecleucel [263, 264]. In fact, the hostdirected immune response is observed to be severe for CAR-T cells targeting solid tumours compared to B cell malignancies [265]. In a clinical trial using CAR-T cells targeting carbonic anhydrase IX – a transmembrane protein expressed on clear-cell renal cell carcinoma but not on normal cells [266] in renal carcinoma, severe liver toxicities were observed, with no objective anti-tumour effect measured. There were evidences of host reactivity against CAR-T cells in these trials, making solid tumours more prone to host-directed immunogenicity [229]. Loss of Target Antigens Loss of target antigens CD19 on cancerous B cells due to downregulation or mutation [267, 268] contributes to the resistance of CD19+ tumour from CD19 CAR-T therapy. In fact, 10%-20% relapsed cases in B-ALL is due to CD19 negative B cells [269, 270]. The ability of cancerous B cells to escape CD19 CAR T cell activity contributes to refraction from treatments and relapse. Thus, there is a need for combinations of CD19 CAR-T cells with other antigen-specific CAR-T cells, in the case of pooled CAR-T cells. In fact, the combination of CD19 and CD123 treatments have shown favorable results against antigen-negative relapses after CD19 CAR-T treatment in clinical study [271]. Solid Tumour Targeting The successes of CD19 and other antigen-specific CAR-T cells in haematological diseases are not transferable to solid tumours due to numerous reasons. One of the explanations for this inefficiency is due to antigen heterogenicity in solid tumours [272]. Unlike CD19, which is relatively specific and homogenously expressed on B cells, solid tumours contain a diverse range of different antigens that may not be as specific. Hence, there is a need to identify novel specific markers for CAR construct designs. Furthermore, precise delivery of CAR-T cells to solid tumour sites presents a challenging issue compared to haematologic cancers. The fibrotic nature of solid tumour makes it difficult for CAR-T cells to reach their targets [265]. Cytokine Release Syndrome Cytokine release syndrome (CRS) is the most commonly observed and serious side effect in CAR-T and CD19 CAR-T therapies. In CRS, inflammatory cytokines are released by activated CD19 CAR-T cells which causes immune

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reactions [273]. CRS toxicity is often linked to peak blood CAR-T cells, peak serum C-reactive protein level as well as cytokine levels such as IL-6, IFN [62, 80, 85], and TNF [80]. Severe CRS is associated with the IL-6 cytokine [262]. This cytokine release may cause related symptoms such as fevers, hypotension, cytopenia and reversible neurological events [238, 248, 274]. Most CRS are resolved using IL-6 specific mAb such as tocilizumab, vasopressors, corticosteroids as well as ventillatory support in combination with CD19 CAR-T cell [238, 275]. Furthermore, additional testing like cytokine profiling can be helpful in predicting and managing CRS before or after CD19 CAR-T cell treatment. Biomarkers predicting the possibility of CRS are also under investigation [276]. Neurological Toxicities Neurological toxicities such as confusion, ataxia, aphasia or seizures are especially prevalent in CD19 CAR-T therapy, with the frequency of severe toxicities ranging from 25% to 57% of patients in most clinical trials (62,67,80,85,90). Neurological side effects are also observed in the treatment using CD19/CD3 bispecific antibody blinatumab, which supports the specific role of CD19 in neurological toxicities. Current managing options for neurological toxicities include steroids and anti-epileptic drugs. Severe neurotoxic effects can be life-threatening. In fact, three patients in a single trial in phase 2 sponsored by Juno therapeutics treated with JCAR015 (CD18/CD3signaling domain combination for B-ALL) passed away. These deaths were due to cerebral edemas, a sign of neurotoxicity [277]. Given the severity with CD19 CAR-T cells but not in other antigen-specific CAR T cells, CD19 specific scFv on this CAR construct may target the same antigen expressed on unknown nervous tissues [278 - 280]. Another possibility is that these neurological events are linked to CRS caused by the release of cytokines such as IL-6 or IFN [276]. As CD19 CAR-T cells have been found in the cerebrospinal fluid in some patients [281, 282], neurotoxicity can also be due to the translocation of active CD19 CAR-T cells across the bloodbrain barrier [283]. However, the exact aetiologies of CD19 or any other CAR--related neurological toxicity remain to be determined. Off-Tumour Effects Off-target toxicity is also the main issue that needs to be addressed in the usage of CAR-T cells. In off-target toxicity, CAR-T cells recognize non-cancerous antigens on normal cells and thus cause damages to other normal tissues [284]. Recognition of similar antigens on other tissues by CAR-T cells seems to be a more serious issue in solid tumour treatments as compared to B cell malignancies. In fact, the usage of T cells targeting MAGE - an antigen specific to testes cancer

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has shown life-threatening consequences in clinical trials. A patient died after the treatment with these T cells due to cardiac toxicity, which was due to the offtarget recognition of similar protein titin in cardiac muscles [285]. Compared to off-target toxicity, on-target off-tumour toxicity is more usually observed in CD19 CAR T cell therapy. CD19 CAR-T cells recognize the correct antigen, in this case CD19, but the antigen is expressed on normal cells like normal B cells [286]. All these toxicities cause autoimmune reactivity against CD19-expressed normal tissues, leading to disease like B cell aplasia [226, 243, 287]. As explained before, B cell aplasia is manageable by the usage of IVIg after CD19 CAR-T cell treatment [224, 274, 288]. The most serious on-target offtumour effect observed on CAR-T cell therapy is the use of HER-2-specific CAR T cells. As explained before, HER-2 is overexpressed on various types of tumours, prompting the clinical trial of third-gen HER-2 specific CAR-T cells as treatments for metastatic cancers. However, one patient suffered from respiratory failure and passed away due to the on-target off tumour recognition of HER-2 expressed on normal lung cells by CAR-T cells [289]. Therefore, suitable targets for CAR-T cells should be identified to reduce the incidences of on-target off tumour effect of CAR-T cell therapy. In fact, choosing the right target requires the consideration for the level of target antigens on normal tissues and affinity of CAR scFv for antigens. Modification of doses and CAR design with lower scFv affinity may also help [284]. Economic Issues with CAR-T Cell Therapy Beyond the technical consideration of CAR-T cell therapy, it is important to take into account the economic aspects of CAR-T cell therapy. The requirements for specialized manufacturing facilities, including GMP facilities limit the accessibility of patients to CAR-T cell therapy in terms of the high cost involved. In fact, tisagenlecleucel, priced at $475,000 [290] for a single infusion, is likely to be a barrier to most patients. This is due to the highly personalized nature of CAR-T cell manufacturing which requires manual, labour-intensive, failure-prone processes. In fact, as the number of T cells obtained is highly dependent on each patient, large variations of expansion rate, ranging from 23.6 to 385-fold of T cell expansion within similar culture durations in a single trial have been reported [282]. This unpredictability and variability may require several trials and errors before developing the final product, contributing to the hefty price tag of CAR-T cell therapy. Currently, there are many variations in technologies employed to produce CAR-T cells, which causes variations in the final product between different CAR-T producing centers. Therefore, there is a need for more streamlined, centralized production of CAR-T cells before being cryo-shipped to the centers nearer to the patients for further manipulation [291]. This means that

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one central GMP facility will be in charge of manufacturing CAR-T cells after receiving apheresis products from patients, ensuring the uniformity in production [292]. Clinical Application and Drug Development of CD19 CAR-T Cell Therapy Among CAR-T cells that target different antigens, CD19 CAR-T cells have gained positive results, with tisagenlecleucel having an impressive 82.5% overall remission rate in the treatment of relapsed and/or refractory B-ALL in 63 patients based on pivotal and supportive clinical trials by Novartis [293]. Similarly, axicabtagene ciloleucel was also approved after the objective response rate of 72% for the treatment of relapsed and refractory large B-cell lymphoma based on the ZUMA-1 clinical trial by Kite Pharma. Relapsed cancer refers to the recurrence of the disease in patients who have undergone previous treatments for this cancer. Refractory CD19 CAR-T cell therapy has been shown to be effective against B cell leukemia such as B-ALL and CLL as well as several B cell nHL subtypes such as DLBCL, FL, mantle-cell lymphoma and marginal zone lymphoma in clinical trials. Since the first pre-clinical trial in 2003 [247, 294], there are about 157 completed or active clinical studies of CD19 CAR-T cell therapy registered on clinicaltrials.gov. Among the clinical trials done for second-gen CAR-T cell therapy, trials done by Memorial Sloan Kettering Cancer Center, National Cancer Institute, The University of Pennsylvania and Fred Hutchinson Cancer Research Center published their results. These trials also demonstrated the potential of CD19 CAR-T cells in B cell leukemia treatment [262]. All of these clinical trials used second-gen CD19 CAR- T cell therapy with variations in the design of signaling domains using either CD28/CD3 or CD137/CD3 combination. The number of patients recruited in these trials varied in the range of 20 to 32 patients. The results obtained from these studies are impressive, with more than 80% rate of CR seen among the recruits, even with different criteria for recruitment and CD19 CAR-T designs among the trials. However, the side effects observed which include cytokine release syndrome (CRS), B cell aplasia, neuropathic diseases were common among the trials [224, 238, 262, 281, 295]. These favorable preliminary results prompted further testing of CD19 CAR-T cell potential in larger cohort studies, including landmark clinical trials that lead to the approval of second-gen CD19 CAR-T tisagenlecleucel and axicabtagene ciloleucel at the end of 2017 by the FDA.

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Tisagenlecleucel Tisagenlecleucel (experimental name CTL019) is an autologous CD19 CAR-T cell drug that is genetically modified using lentivirus .The CAR design consists of an extracellular murine CD19-specific scFv next to a CD8 hinge and an intracellular signaling domain with CD137/CD3 combination. These fragments are fused together using a transmembrane region. T cells in CTL019 are extracted and enriched from PBMCs of the patients by leukapheresis. These T cells are then transduced with lentiviruses carrying CAR to make CD19 CAR-T cells [263] The approval of tisagenlecleucel was dependent on the results from three key clinical trials including two supportive studies and one pivotal study [296]. The results in these clinical studies are analyzed using overall remission rate (ORR), which includes complete remission (CR) and complete remission with incomplete blood count (CRi) [296]. The criteria for CR is first defined for the treatment of acute myeloid leukemia [297], which is adapted for relapsed and refractory BALL treatment. CR indicates a normalization blood count for for at least 4 weeks after treatments including