Antibody-Drug Conjugates

Kenneth J. Olivier, Jr., PhD, is Head of Toxicology, Discovery Regulatory, Bioanalytical Assay Development, Pharmacokinetics and Discovery Project Management at Merrimack Pharmaceuticals and has over 13 years’ experience in the biotechnology and pharmaceutical industries.Sara A. Hurvitz, MD, is an Associate Professor of Medicine at the University of California, Los Angeles (UCLA); Co-Director of the Santa Monica-UCLA Outpatient Oncology Practice; Medical Director of the Clinical Research Unit of the Jonsson Comprehensive Cancer Center of UCLA; and Director of the Breast Oncology Program, Division of Hematology-Oncology, at UCLA.

• List of Contributors xviiPreface xxiHistorical Perspective: What Makes Antibody–Drug Conjugates Revolutionary? xxiiiPart I What is an Antibody–Drug Conjugate 11 Typical Antibody–Drug Conjugates 3John M. Lambert1.1 Introduction 31.1.1 A Simple Concept 31.1.2 Turning Antibodies into Potent Anticancer Compounds 41.1.3 What is a Typical ADC and How Does it Act? 41.1.4 Simple Concept, but Not So Simple to Execute 51.2 The Building Blocks of a Typical ADC 61.2.1 The Antibody 61.2.1.1 Antibody Isotype in ADCs 71.2.1.2 Functional Activity of the Antibody Moiety in ADCs 81.2.2 The Payload 91.2.2.1 DNA-Targeting Payloads 111.2.2.2 Payloads Targeting Tubulin 111.2.3 Linker Chemistries 121.3 Building an ADC Molecule 131.3.1 Conjugation of Payloads to Antibodies at Lysine Residues 131.3.2 Conjugation of Payloads to Antibodies at Cysteine Residues 171.4 Attributes of a Typical ADC 191.4.1 Structural Attributes of a Typical ADC 191.4.2 Functional Characteristics of a Typical ADC 201.4.2.1 In Vitro Properties 201.4.2.2 In Vivo Efficacy 201.4.2.3 Pharmacokinetics of ADCs 231.5 Summary 24Acknowledgment 24Abbreviations 25References 25Part II Engineering, Manufacturing, and Optimizing Antibody–Drug Conjugates 332 Selecting Optimal Antibody–Drug Conjugate Targets Using Indication-Dependent or Indication-Independent Approaches 35Jay Harper and Robert Hollingsworth2.1 Characteristics of an Optimal ADC Target 352.2 Indication-Dependent ADC Target Selection 402.3 Indication-Independent ADC Target Selection 482.4 Concluding Remarks and Future Directions 50Acknowledgments 52References 523 Antibody–Drug Conjugates: An Overview of the CMC and Characterization Process 59Philip L. Ross and Janet Wolfe3.1 Introduction 593.2 ADC Manufacturing Process 603.2.1 Conjugation 623.2.2 Conjugation – Next‐Generation Chemistry 643.2.2.1 Conjugation – Novel Payloads 643.2.2.2 Conjugation – Linker Design 653.2.3 mAb Engineering 663.2.4 Purification 683.2.5 Formulation 683.3 Characterization 703.3.1 Quality and Stability Testing 703.3.2 Biochemical and Microbiological Testing 743.3.3 Extended Characterization 743.4 Comparability 763.5 Concluding Remarks 76Abbreviations 77References 784 Linker and Conjugation Technology; and Improvements 85Riley Ennis and Sourav Sinha4.1 Overview 854.2 Noncleavable 864.3 Cleavable Linkers and Self‐Immolative Groups 864.4 Differences in Therapeutic Window of Cleavable and Noncleavable Linkers 884.5 Improving Therapeutic Window with Next‐Generation Linker Technologies 894.6 Site‐Specific Conjugation, Homogeneous Drug Species, and Therapeutic Window 914.7 Influence of Linkers on Pharmacokinetics and ADME 934.8 PEG Linkers to Optimize Clearance, Solubility, and Potency 934.9 Linkers to Optimize for Drug Resistance 944.10 Improving Solid Tumor Penetration with Linkers 964.11 Analytical Methods for Characterizing Linker Pharmacodynamics 964.12 Conclusion 98References 995 Formulation and Stability 105Kouhei Tsumoto, Anthony Young, and Satoshi Ohtake5.1 Introduction 1055.2 Stability Considerations for ADCs 1065.2.1 Physical Stability 1065.2.2 Chemical Stability 1115.3 Formulation Approaches 1155.4 Logistical Considerations 1235.5 Summary and Close 125References 1266 QC Assay Development 131Xiao Hong. Chen and Mate Tolnay6.1 Introduction 1316.2 Drug‐to‐Antibody Ratio 1326.3 Drug Loading Distribution 1336.3.1 Lysine‐Linked ADCs 1346.3.2 Cysteine‐Linked ADCs 1346.4 Positional Isomers 1366.5 ADC Concentration 1366.6 Drug‐Related Substances 1376.7 Antigen Binding Assays and Potential Impact of Drug Conjugation 1376.8 Cell‐Based Cytotoxicity Assays 1396.9 Assays to Monitor Fc‐Dependent Effector Functions to Characterize Additional Possible Mechanisms of Action 1406.10 Immunogenicity Assays to Monitor the Immune Response to ADC 1426.11 Conclusions 1446.12 Key Guidance Documents 145Acknowledgments 145References 1457 Occupational Health and Safety Aspects of ADCs and Their Toxic Payloads 151Robert Sussman and John Farris7.1 Introduction 1517.2 Background on ADCs 1527.2.1 Payloads 1537.2.2 Linker Technologies 1547.2.3 Antibodies 1567.2.4 Partial Conjugates 1567.3 Occupational Hazard Assessment of ADCs and Their Components 1577.4 Occupational Implications and Uncertainties 1597.4.1 Routes of Occupational Exposure 1597.4.2 Binding Efficiency (Payload to Antibody) 1597.4.3 Unintended Targets 1607.4.4 Free Payload in Conjugation Formulation 1607.4.5 Local Effects in the Lung 1607.5 General Guidance for Material Handling 1607.5.1 Handling of Powders 1627.5.2 Handling of Solutions 1627.6 Facility Features and Engineering Controls 1637.6.1 HVAC and Air Pressure Relationships 1647.6.2 Air Changes and Airflow 1647.6.3 Recirculation and Filtration of Room Air 1647.6.4 Changing Areas 1647.6.5 Designated Areas 1657.7 Specific Operational Guidance 1657.7.1 Payload Synthesis 1657.7.2 Conjugation 1667.7.3 Lyophilization 1667.7.4 Cleaning 1677.8 Personal Protective Equipment 1677.8.1 Chemical Protective Clothing 1677.8.1.1 Protective Clothing 1677.8.1.2 Gloves 1677.8.1.3 Eye and Face Protection 1687.8.2 Respiratory Protection 1687.9 Training 1687.9.1 Potent Compound Awareness Training 1697.9.2 Standard Operating Procedures for Synthesizing and Handling ADCs 1697.10 Industrial Hygiene Monitoring 1697.10.1 Air Monitoring 1707.10.2 Surface Monitoring 1707.11 Medical Surveillance Program 1717.12 Summary and Future Direction 172References 172Part III Nonclinical Approaches 1778 Bioanalytical Strategies Enabling Successful ADC Translation 179Xiaogang Han, Steven Hansel, and Lindsay King8.1 Introduction 1798.2 ADC LC/MS Bioanalytical Strategies 1828.2.1 Nonregulated Unconjugated Payload Bioanalysis 1838.2.2 Intact Protein Bioanalysis by LC/MS: Measurement of Drug‐to‐Antibody Ratio 1848.2.3 ADC Pharmacokinetic Bioanalysis by LC/MS 1868.2.4 Calculated Conjugated Payload Determination 1878.2.5 Conjugated Payload Quantitation of Cleavable Linker ADCs 1888.2.6 Conjugated Payload Quantitation by Peptide‐Based Analysis 1898.3 Non‐Regulated ADC Pharmacokinetic and Immunogenicity Support Using Ligand Binding Assays 1908.3.1 ADC Ligand Binding Assays 1908.3.2 Reagents 1918.3.3 ADC Reference Standards 1928.3.4 Total Antibody Assays 1928.3.5 ADC Assays 1938.3.6 Target Interference in ADC Measurement 1948.3.7 ADC Immunogenicity Assays 1948.4 Biodistribution Assessment 1958.5 Regulated ADC Pharmacokinetics and Immunogenicity Evaluation 1968.5.1 ADC Assays in Regulated Studies 1968.5.2 Regulated Ligand Binding Assays 1978.5.3 Regulated LC/MS/MS Quantitation of Unconjugated Payload 1988.5.4 Regulated Conjugated Payload LC/MS Assays 1998.5.5 Regulated Anti‐therapeutic Assays 1998.6 ADC Biomeasures and Biomarkers 1998.7 Summary 200References 2019 Nonclinical Pharmacology and Mechanistic Modeling of Antibody– Drug Conjugates in Support of Human Clinical Trials 207Brian J. Schmidt, Chin Pan, Heather E. Vezina, Huadong Sun, Douglas D. Leipold, and Manish Gupta9.1 Introduction 2079.2 Cell Line Testing 2109.2.1 Antigen Density 2119.2.2 Antigen and Antibody–Drug Conjugate Internalization 2119.2.3 Payload Processing and Binding 2139.3 Xenograft Models 2149.3.1 Payload Bystander Effects 2159.3.2 Biomarker Assays 2169.4 Nonclinical Testing to Support Investigational New Drug Applications 2169.4.1 Antibody–Drug Conjugate Efficacious Dose Range 2189.5 Mechanistic Modeling of Antibody–Drug Conjugates 2209.5.1 Tumor Tissue Transport Considerations 2219.5.2 Subcellular Trafficking 2259.5.3 Shed Antigen and Endosomal Processing 2259.5.4 Enhanced Pharmacokinetic Modeling to Enable Antibody–Drug Conjugate Pharmacology Predictions 2269.5.5 Mechanistic Modeling of Antibody–Drug Conjugate Pharmacology: Accounting for Uncertainties 2279.6 Target‐Mediated Toxicity of Antibody–Drug Conjugates 2289.7 Considerations for Nonclinical Testing Beyond Antibody–Drug Conjugate Monotherapies 2299.8 Summary 230Acknowledgments 231References 23110 Pharmacokinetics of Antibody–Drug Conjugates 245Amrita V. Kamath10.1 Introduction 24510.2 Pharmacokinetic Characteristics of an ADC 24610.2.1 ADC Biodistribution 24810.2.2 ADC Clearance 24910.3 Unique Considerations for ADC Pharmacokinetics 25010.3.1 Linker Stability 25010.3.2 Site of Conjugation and Drug Load 25210.3.3 Cytotoxic Drug 25310.4 Tools to Characterize ADC PK/ADME 25410.4.1 Bioanalytical Methods 25410.4.2 In Vitro Assays 25510.4.3 In Vivo Studies 25610.4.4 Pharmacokinetic/Pharmacodynamic (PK/PD) Models 25610.5 Utilization of ADC Pharmacokinetics to Optimize Design 25710.6 Pharmacokinetics of Selected ADCs 25910.6.1 Ado‐Trastuzumab Emtansine (Kadcyla®) 25910.6.2 Brentuximab Vedotin (Adcetris®) 26110.7 Summary 261References 26211 Path to Market Approval: Regulatory Perspective of ADC Nonclinical Safety Assessments 267M. Stacey Ricci, R. Angelo De Claro, and Natalie E. Simpson11.1 Introduction 26711.2 FDA Experience with ADCs 26811.3 Regulatory Perspective of the Nonclinical Safety Assessment of ADCs 26911.3.1 Regulatory Guidance Available for Nonclinical Studies 27011.3.1.1 Species Selection 27211.3.1.2 Study Duration and Dose Regimen 27511.3.1.3 Study Test Article 27611.3.1.4 Pharmacology Studies 27811.3.1.5 Pharmacokinetics/Toxicokinetics 27911.3.1.6 Genotoxicity 28011.3.1.7 Developmental and Reproductive Toxicology 28011.3.1.8 First-in-Human Dose Selection 28011.4 Concluding Remarks 282References 283Part IV Clinical Development and Current Status of Antibody–Drug Conjugates 28512 Antibody–Drug Conjugates: Clinical Strategies and Applications 287Heather E. Vezina, Lucy Lee, Brian J. Schmidt, and Manish Gupta12.1 Antibody–Drug Conjugates in Clinical Development 28712.2 Therapeutic Indications 29112.3 Transitioning from Discovery to Early Clinical Development 29212.4 Challenges and Considerations in the Design of Phase 1Studies 29312.5 First-in-Human Starting Dose Estimation 29312.6 Dosing Strategy Considerations 29412.7 Dosing Regimen Optimization 29512.8 Phase 1 Study Design 29712.9 Supportive Strategies for Phase 1 and Beyond 29912.10 Clinical Pharmacology Considerations 30112.11 Organ Impairment Assessments 30112.12 Drug–Drug Interaction Assessments 30212.13 Immunogenicity 30312.14 QT/QTc Assessments 30312.15 Pharmacometric Strategies 30712.16 Using Physiologically Based Pharmacokinetic and Quantitative Systems Pharmacology Models with Clinical Data 30812.17 Summary and Conclusions 311Acknowledgments 311References 31113 Antibody–Drug Conjugates (ADCs) in Clinical Development 321Joseph McLaughlin and Patricia LoRusso13.1 Introduction and Rationale 32113.2 Components of ADCs in Development 32113.2.1 Antibody 32113.2.2 Linker 32713.2.3 Payload 32813.3 Landscape of ADCs 32913.3.1 History of ADCs 32913.3.2 FDA Approved ADCs 32913.4 Clinical Use of ADCs 33013.5 Future of ADCs 33013.6 ADCs in Development 33013.6.1 Hematological Malignancies and Renal Cell Carcinoma 33013.6.1.1 Auristatins (MMAE and MMAF) 33013.6.1.2 Maytansinoids (DM1 and DM4) 33213.6.1.3 Pyrrolobenzodiazepines (PBDs) 33413.6.1.4 Calicheamicins 33513.6.1.5 Others 33513.6.2 Solid Malignancies 33513.6.2.1 Auristatins (MMAE and MMAF) 33513.6.2.2 Maytansinoids (DM1 and DM4) 33813.6.2.3 Others 33913.7 Future Directions 340References 34014 ADCs Approved for Use: Trastuzumab Emtansine (Kadcyla ® , T-DM1) in Patients with Previously Treated HER2-Positive Metastatic Breast Cancer 345Gail D. Lewis Phillips, Sanne de Haas, Sandhya Girish, and Ellie Guardino14.1 Introduction 34514.2 Preclinical Development of T-DM 1 34814.3 Early Clinical Studies of T-DM 1 35714.3.1 Phase I Adverse Events (AEs) 35714.3.2 Phase I Efficacy 35814.3.3 Dosing Schedule 35914.3.4 Phase II Trials 35914.4 Clinical Pharmacology and Pharmacokinetics 36114.5 Phase III Studies of T-DM1 in Patients with HER2-Positive MBC 36214.5.1 EMILIA Trial 36314.5.2 TH3RESA Trial 36714.5.3 Treatment Exposure 36914.5.4 Biomarkers as Predictors of Efficacy 36914.6 Future Directions 37114.7 Summary 373References 37415 ADCs Approved for Use: Brentuximab Vedotin 381Monica Mead and Sven de Vos15.1 Introduction 38115.2 Early Efforts to Target CD30 with Monoclonal Antibodies 38315.3 BV: Preclinical Data 38615.3.1 Clinical Data: Safety/Tolerability 38815.3.2 Clinical Data: Efficacy 39115.3.3 CD30 Expression Level and Response to BV 39315.4 Clinical Context 39415.5 Mechanisms of Resistance 39515.6 Current Research 39715.7 Discussion 400References 40116 Radioimmunotherapy 409Savita V. Dandapani and Jeffrey Wong16.1 History of Radioimmunotherapy 40916.2 Radioisotopes 41016.3 Chemistry of RIT 41116.4 Radioimmunotherapy Antibody Targets in Use Today (Table 16.2) 41216.4.1 Hematologic Malignancies 41216.4.1.1 CD20 41216.5. Other Hematologic Targets 41516.5.1 Lymphomas 41516.5.1.1 Lym-1, CD22, CD 25 41516.5.2 Leukemias 41716.5.2.1 CD33 41716.6 Solid Tumors 41716.6.1 CEA (Carcinoembryonic Antigen) 41816.6.2 Other RIT in Solid Tumors 41916.7 Combination Therapy with RIT: Chemotherapy and/or Radiation 42016.7.1 RIT and Chemotherapy 42016.8 RIT and External Beam Radiation Treatment (EBRT) 42116.9 RIT and EBRT and Chemotherapy 42116.10 RIT Administration 42216.11 Future of RIT 422References 423Part V Future Perspectives in Antibody–Drug Conjugate Development 43117 Radiolabeled Antibody‐Based Imaging in Clinical Oncology 433Bart S. Hendriks and Daniel F. Gaddy17.1 Introduction 43317.2 Applications for Clinical Antibody Imaging 43417.3 Antibodies as Imaging Agents 43517.4 Nuclear Imaging – Gamma Camera (Planar) Scintigraphy and SPECT 43917.4.1 Tumor Detection and Staging 44017.4.1.1 CEA 44117.4.1.2 PSMA 44117.4.1.3 TAG‐72 44317.4.1.4 Pancarcinoma Antigen 44317.4.2 Diagnostic Assessment 44417.4.2.1 HER2 44417.4.2.2 EGFR 44517.4.3 Dosimetry for Radioimmunotherapy 44517.4.4 Early Assessment of Response 44717.5 Nuclear Imaging ‐ PET 44817.5.1 68Ga 44817.5.2 64Cu 44917.5.3 89Zr 45117.5.4 124I 45417.6 Commercialization Considerations 45617.7 Summary 461References 46218 Next-Generation Antibody–Drug Conjugate Technologies 473Amy Q. Han and William C. Olson18.1 Introduction 47318.2 Novel Cytotoxic Payloads and Linkers 47418.2.1 Microtubule Inhibitors 47418.2.2 Benzodiazepine Dimers 47418.2.3 Anthracyclines 47718.2.4 Amatoxins 47818.2.5 Disulfide Rebridging 47918.2.6 FleximerTM Polymeric Linkers 48118.3 Tailoring Antibodies for Use as ADCs 48218.3.1 Engineered Cysteines 48318.3.2 Enzyme-Assisted Conjugation 48418.3.2.1 Microbial Transglutaminase 48418.3.2.2 Formylglycine-Generating Enzyme (FGE) 48518.3.2.3 Glucosyltransferases and Other Glycan Engineering 48618.3.3 Non-Native Amino Acids and Selenocysteine 48718.3.4 Alternative Formats and Masked Antibodies 48818.3.5 ADCs Beyond Oncology 48918.4 Conclusions 491References 491Index 505