List of Contributors xvii
Preface xxi
Historical Perspective: What Makes Antibody-Drug Conjugates Revolutionary? xxiii
Part I What is an Antibody-Drug Conjugate 1
1 Typical Antibody-Drug Conjugates 3
John M. Lambert
1.1 Introduction 3
1.1.1 A Simple Concept 3
1.1.2 Turning Antibodies into Potent Anticancer Compounds 4
1.1.3 What is a Typical ADC and How Does it Act? 4
1.1.4 Simple Concept, but Not So Simple to Execute 5
1.2 The Building Blocks of a Typical ADC 6
1.2.1 The Antibody 6
1.2.1.1 Antibody Isotype in ADCs 7
1.2.1.2 Functional Activity of the Antibody Moiety in ADCs 8
1.2.2 The Payload 9
1.2.2.1 DNA-Targeting Payloads 11
1.2.2.2 Payloads Targeting Tubulin 11
1.2.3 Linker Chemistries 12
1.3 Building an ADC Molecule 13
1.3.1 Conjugation of Payloads to Antibodies at Lysine Residues 13
1.3.2 Conjugation of Payloads to Antibodies at Cysteine Residues 17
1.4 Attributes of a Typical ADC 19
1.4.1 Structural Attributes of a Typical ADC 19
1.4.2 Functional Characteristics of a Typical ADC 20
1.4.2.1 In Vitro Properties 20
1.4.2.2 In Vivo Efficacy 20
1.4.2.3 Pharmacokinetics of ADCs 23
1.5 Summary 24
Acknowledgment 24
Abbreviations 25
References 25
Part II Engineering, Manufacturing, and Optimizing Antibody-Drug Conjugates 33
2 Selecting Optimal Antibody-Drug Conjugate Targets Using Indication-Dependent or Indication-Independent Approaches 35
Jay Harper and Robert Hollingsworth
2.1 Characteristics of an Optimal ADC Target 35
2.2 Indication-Dependent ADC Target Selection 40
2.3 Indication-Independent ADC Target Selection 48
2.4 Concluding Remarks and Future Directions 50
Acknowledgments 52
References 52
3 Antibody-Drug Conjugates: An Overview of the CMC and Characterization Process 59
Philip L. Ross and Janet Wolfe
3.1 Introduction 59
3.2 ADC Manufacturing Process 60
3.2.1 Conjugation 62
3.2.2 Conjugation - Next¿Generation Chemistry 64
3.2.2.1 Conjugation - Novel Payloads 64
3.2.2.2 Conjugation - Linker Design 65
3.2.3 mAb Engineering 66
3.2.4 Purification 68
3.2.5 Formulation 68
3.3 Characterization 70
3.3.1 Quality and Stability Testing 70
3.3.2 Biochemical and Microbiological Testing 74
3.3.3 Extended Characterization 74
3.4 Comparability 76
3.5 Concluding Remarks 76
Abbreviations 77
References 78
4 Linker and Conjugation Technology; and Improvements 85
Riley Ennis and Sourav Sinha
4.1 Overview 85
4.2 Noncleavable 86
4.3 Cleavable Linkers and Self¿Immolative Groups 86
4.4 Differences in Therapeutic Window of Cleavable and Noncleavable Linkers 88
4.5 Improving Therapeutic Window with Next¿Generation Linker Technologies 89
4.6 Site¿Specific Conjugation, Homogeneous Drug Species, and Therapeutic Window 91
4.7 Influence of Linkers on Pharmacokinetics and ADME 93
4.8 PEG Linkers to Optimize Clearance, Solubility, and Potency 93
4.9 Linkers to Optimize for Drug Resistance 94
4.10 Improving Solid Tumor Penetration with Linkers 96
4.11 Analytical Methods for Characterizing Linker Pharmacodynamics 96
4.12 Conclusion 98
References 99
5 Formulation and Stability 105
Kouhei Tsumoto, Anthony Young, and Satoshi Ohtake
5.1 Introduction 105
5.2 Stability Considerations for ADCs 106
5.2.1 Physical Stability 106
5.2.2 Chemical Stability 111
5.3 Formulation Approaches 115
5.4 Logistical Considerations 123
5.5 Summary and Close 125
References 126
6 QC Assay Development 131
Xiao Hong. Chen and Mate Tolnay
6.1 Introduction 131
6.2 Drug¿töAntibody Ratio 132
6.3 Drug Loading Distribution 133
6.3.1 Lysine¿Linked ADCs 134
6.3.2 Cysteine¿Linked ADCs 134
6.4 Positional Isomers 136
6.5 ADC Concentration 136
6.6 Drug¿Related Substances 137
6.7 Antigen Binding Assays and Potential Impact of Drug Conjugation 137
6.8 Cell¿Based Cytotoxicity Assays 139
6.9 Assays to Monitor Fc¿Dependent Effector Functions to Characterize Additional Possible Mechanisms of Action 140
6.10 Immunogenicity Assays to Monitor the Immune Response to ADC 142
6.11 Conclusions 144
6.12 Key Guidance Documents 145
Acknowledgments 145
References 145
7 Occupational Health and Safety Aspects of ADCs and Their Toxic Payloads 151
Robert Sussman and John Farris
7.1 Introduction 151
7.2 Background on ADCs 152
7.2.1 Payloads 153
7.2.2 Linker Technologies 154
7.2.3 Antibodies 156
7.2.4 Partial Conjugates 156
7.3 Occupational Hazard Assessment of ADCs and Their Components 157
7.4 Occupational Implications and Uncertainties 159
7.4.1 Routes of Occupational Exposure 159
7.4.2 Binding Efficiency (Payload to Antibody) 159
7.4.3 Unintended Targets 160
7.4.4 Free Payload in Conjugation Formulation 160
7.4.5 Local Effects in the Lung 160
7.5 General Guidance for Material Handling 160
7.5.1 Handling of Powders 162
7.5.2 Handling of Solutions 162
7.6 Facility Features and Engineering Controls 163
7.6.1 HVAC and Air Pressure Relationships 164
7.6.2 Air Changes and Airflow 164
7.6.3 Recirculation and Filtration of Room Air 164
7.6.4 Changing Areas 164
7.6.5 Designated Areas 165
7.7 Specific Operational Guidance 165
7.7.1 Payload Synthesis 165
7.7.2 Conjugation 166
7.7.3 Lyophilization 166
7.7.4 Cleaning 167
7.8 Personal Protective Equipment 167
7.8.1 Chemical Protective Clothing 167
7.8.1.1 Protective Clothing 167
7.8.1.2 Gloves 167
7.8.1.3 Eye and Face Protection 168
7.8.2 Respiratory Protection 168
7.9 Training 168
7.9.1 Potent Compound Awareness Training 169
7.9.2 Standard Operating Procedures for Synthesizing and Handling ADCs 169
7.10 Industrial Hygiene Monitoring 169
7.10.1 Air Monitoring 170
7.10.2 Surface Monitoring 170
7.11 Medical Surveillance Program 171
7.12 Summary and Future Direction 172
References 172
Part III Nonclinical Approaches 177
8 Bioanalytical Strategies Enabling Successful ADC Translation 179
Xiaogang Han, Steven Hansel, and Lindsay King
8.1 Introduction 179
8.2 ADC LC/MS Bioanalytical Strategies 182
8.2.1 Nonregulated Unconjugated Payload Bioanalysis 183
8.2.2 Intact Protein Bioanalysis by LC/MS: Measurement of Drug¿töAntibody Ratio 184
8.2.3 ADC Pharmacokinetic Bioanalysis by LC/MS 186
8.2.4 Calculated Conjugated Payload Determination 187
8.2.5 Conjugated Payload Quantitation of Cleavable Linker ADCs 188
8.2.6 Conjugated Payload Quantitation by Peptide¿Based Analysis 189
8.3 Non¿Regulated ADC Pharmacokinetic and Immunogenicity Support Using Ligand Binding Assays 190
8.3.1 ADC Ligand Binding Assays 190
8.3.2 Reagents 191
8.3.3 ADC Reference Standards 192
8.3.4 Total Antibody Assays 192
8.3.5 ADC Assays 193
8.3.6 Target Interference in ADC Measurement 194
8.3.7 ADC Immunogenicity Assays 194
8.4 Biodistribution Assessment 195
8.5 Regulated ADC Pharmacokinetics and Immunogenicity Evaluation 196
8.5.1 ADC Assays in Regulated Studies 196
8.5.2 Regulated Ligand Binding Assays 197
8.5.3 Regulated LC/MS/MS Quantitation of Unconjugated Payload 198
8.5.4 Regulated Conjugated Payload LC/MS Assays 199
8.5.5 Regulated Anti¿therapeutic Assays 199
8.6 ADC Biomeasures and Biomarkers 199
8.7 Summary 200
References 201
9 Nonclinical Pharmacology and Mechanistic Modeling of Antibody- Drug Conjugates in Support of Human Clinical Trials 207
Brian J. Schmidt, Chin Pan, Heather E. Vezina, Huadong Sun, Douglas D. Leipold, and Manish Gupta
9.1 Introduction 207
9.2 Cell Line Testing 210
9.2.1 Antigen Density 211
9.2.2 Antigen and Antibody-Drug Conjugate Internalization 211
9.2.3 Payload Processing and Binding 213
9.3 Xenograft Models 214
9.3.1 Payload Bystander Effects 215
9.3.2 Biomarker Assays 216
9.4 Nonclinical Testing to Support Investigational New Drug Applications 216
9.4.1 Antibody-Drug Conjugate Efficacious Dose Range 218
9.5 Mechanistic Modeling of Antibody-Drug Conjugates 220
9.5.1 Tumor Tissue Transport Considerations 221
9.5.2 Subcellular Trafficking 225
9.5.3 Shed Antigen and Endosomal Processing 225
9.5.4 Enhanced Pharmacokinetic Modeling to Enable Antibody-Drug Conjugate Pharmacology Predictions 226
9.5.5 Mechanistic Modeling of Antibody-Drug Conjugate Pharmacology: Accounting for Uncertainties 227
9.6 Target¿Mediated Toxicity of Antibody-Drug Conjugates 228
9.7 Considerations for Nonclinical Testing Beyond Antibody-Drug Conjugate Monotherapies 229
9.8 Summary 230
Acknowledgments 231
References 231
10 Pharmacokinetics of Antibody-Drug Conjugates 245
Amrita V. Kamath
10.1 Introduction 245
10.2 Pharmacokinetic Characteristics of an ADC 246
10.2.1 ADC Biodistribution 248
10.2.2 ADC Clearance 249
10.3 Unique Considerations for ADC Pharmacokinetics 250
10.3.1 Linker Stability 250
10.3.2 Site of Conjugation and Drug Load 252
10.3.3 Cytotoxic Drug 253
10.4 Tools to Characterize ADC PK/ADME 254
10.4.1 Bioanalytical Methods 254
10.4.2 In Vitro Assays 255
10.4.3 In Vivo Studies 256
10.4.4 Pharmacokinetic/Pharmacodynamic (PK/PD) Models 256
10.5 Utilization of ADC Pharmacokinetics to Optimize Design 257
10.6 Pharmacokinetics of Selected ADCs 259
10.6.1 AdöTrastuzumab Emtansine (Kadcyla®) 259
10.6.2 Brentuximab Vedotin (Adcetris®) 261
10.7 Summary 261
References 262
11 Path to Market Approval: Regulatory Perspective of ADC Nonclinical Safety Assessments 267
M. Stacey Ricci, R. Angelo De Claro, and Natalie E. Simpson
11.1 Introduction 267
11.2 FDA Experience with ADCs 268
11.3 Regulatory Perspective of the Nonclinical Safety Assessment of ADCs 269
11.3.1 Regulatory Guidance Available for Nonclinical Studies 270
11.3.1.1 Species Selection 272
11.3.1.2 Study Duration and Dose Regimen 275
11.3.1.3 Study Test Article 276
11.3.1.4 Pharmacology Studies 278
11.3.1.5 Pharmacokinetics/Toxicokinetics 279
11.3.1.6 Genotoxicity 280
11.3.1.7 Developmental and Reproductive Toxicology 280
11.3.1.8 First-in-Human Dose Selection 280
11.4 Concluding Remarks 282
References 283
Part IV Clinical Development and Current Status of Antibody-Drug Conjugates 285
12 Antibody-Drug Conjugates: Clinical Strategies and Applications 287
Heather E. Vezina, Lucy Lee, Brian J. Schmidt, and Manish Gupta
12.1 Antibody-Drug Conjugates in Clinical Development 287
12.2 Therapeutic Indications 291
12.3 Transitioning from Discovery to Early Clinical Development 292
12.4 Challenges and Considerations in the Design of Phase 1
Studies 293
12.5 First-in-Human Starting Dose Estimation 293
12.6 Dosing Strategy Considerations 294
12.7 Dosing Regimen Optimization 295
12.8 Phase 1 Study Design 297
12.9 Supportive Strategies for Phase 1 and Beyond 299
12.10 Clinical Pharmacology Considerations 301
12.11 Organ Impairment Assessments 301
12.12 Drug-Drug Interaction Assessments 302
12.13 Immunogenicity 303
12.14 QT/QTc Assessments 303
12.15 Pharmacometric Strategies 307
12.16 Using Physiologically Based Pharmacokinetic and Quantitative Systems Pharmacology Models with Clinical Data 308
12.17 Summary and Conclusions 311
Acknowledgments 311
References 311
13 Antibody-Drug Conjugates (ADCs) in Clinical Development 321
Joseph McLaughlin and Patricia LoRusso
13.1 Introduction and Rationale 321
13.2 Components of ADCs in Development 321
13.2.1 Antibody 321
13.2.2 Linker 327
13.2.3 Payload 328
13.3 Landscape of ADCs 329
13.3.1 History of ADCs 329
13.3.2 FDA Approved ADCs 329
13.4 Clinical Use of ADCs 330
13.5 Future of ADCs 330
13.6 ADCs in Development 330
13.6.1 Hematological Malignancies and Renal Cell Carcinoma 330
13.6.1.1 Auristatins (MMAE and MMAF) 330
13.6.1.2 Maytansinoids (DM1 and DM4) 332
13.6.1.3 Pyrrolobenzodiazepines (PBDs) 334
13.6.1.4 Calicheamicins 335
13.6.1.5 Others 335
13.6.2 Solid Malignancies 335
13.6.2.1 Auristatins (MMAE and MMAF) 335
13.6.2.2 Maytansinoids (DM1 and DM4) 338
13.6.2.3 Others 339
13.7 Future Directions 340
References 340
14 ADCs Approved for Use: Trastuzumab Emtansine (Kadcyla ® , T-DM1) in Patients with Previously Treated HER2-Positive Metastatic Breast Cancer 345
Gail D. Lewis Phillips, Sanne de Haas, Sandhya Girish, and Ellie Guardino
14.1 Introduction 345
14.2 Preclinical Development of T-DM 1 348
14.3 Early Clinical Studies of T-DM 1 357
14.3.1 Phase I Adverse Events (AEs) 357
14.3.2 Phase I Efficacy 358
14.3.3 Dosing Schedule 359
14.3.4 Phase II Trials 359
14.4 Clinical Pharmacology and Pharmacokinetics 361
14.5 Phase III Studies of T-DM1 in Patients with HER2-Positive MBC 362
14.5.1 EMILIA Trial 363
14.5.2 TH3RESA Trial 367
14.5.3 Treatment Exposure 369
14.5.4 Biomarkers as Predictors of Efficacy 369
14.6 Future Directions 371
14.7 Summary 373
References 374
15 ADCs Approved for Use: Brentuximab Vedotin 381
Monica Mead and Sven de Vos
15.1 Introduction 381
15.2 Early Efforts to Target CD30 with Monoclonal Antibodies 383
15.3 BV: Preclinical Data 386
15.3.1 Clinical Data: Safety/Tolerability 388
15.3.2 Clinical Data: Efficacy 391
15.3.3 CD30 Expression Level and Response to BV 393
15.4 Clinical Context 394
15.5 Mechanisms of Resistance 395
15.6 Current Research 397
15.7 Discussion 400
References 401
16 Radioimmunotherapy 409
Savita V. Dandapani and Jeffrey Wong
16.1 History of Radioimmunotherapy 409
16.2 Radioisotopes 410
16.3 Chemistry of RIT 411
16.4 Radioimmunotherapy Antibody Targets in Use Today (Table 16.2) 412
16.4.1 Hematologic Malignancies 412
16.4.1.1 CD20 412
16.5. Other Hematologic Targets 415
16.5.1 Lymphomas 415
16.5.1.1 Lym-1, CD22, CD 25 415
16.5.2 Leukemias 417
16.5.2.1 CD33 417
16.6 Solid Tumors 417
16.6.1 CEA (Carcinoembryonic Antigen) 418
16.6.2 Other RIT in Solid Tumors 419
16.7 Combination Therapy with RIT: Chemotherapy and/or Radiation 420
16.7.1 RIT and Chemotherapy 420
16.8 RIT and External Beam Radiation Treatment (EBRT) 421
16.9 RIT and EBRT and Chemotherapy 421
16.10 RIT Administration 422
16.11 Future of RIT 422
References 423
Part V Future Perspectives in Antibody-Drug Conjugate Development 431
17 Radiolabeled Antibody¿Based Imaging in Clinical Oncology 433
Bart S. Hendriks and Daniel F. Gaddy
17.1 Introduction 433
17.2 Applications for Clinical Antibody Imaging 434
17.3 Antibodies as Imaging Agents 435
17.4 Nuclear Imaging - Gamma Camera (Planar) Scintigraphy and SPECT 439
17.4.1 Tumor Detection and Staging 440
17.4.1.1 CEA 441
17.4.1.2 PSMA 441
17.4.1.3 TAG¿72 443
17.4.1.4 Pancarcinoma Antigen 443
17.4.2 Diagnostic Assessment 444
17.4.2.1 HER2 444
17.4.2.2 EGFR 445
17.4.3 Dosimetry for Radioimmunotherapy 445
17.4.4 Early Assessment of Response 447
17.5 Nuclear Imaging ¿ PET 448
17.5.1 68Ga 448
17.5.2 64Cu 449
17.5.3 89Zr 451
17.5.4 124I 454
17.6 Commercialization Considerations 456
17.7 Summary 461
References 462
18 Next-Generation Antibody-Drug Conjugate Technologies 473
Amy Q. Han and William C. Olson
18.1 Introduction 473
18.2 Novel Cytotoxic Payloads and Linkers 474
18.2.1 Microtubule Inhibitors 474
18.2.2 Benzodiazepine Dimers 474
18.2.3 Anthracyclines 477
18.2.4 Amatoxins 478
18.2.5 Disulfide Rebridging 479
18.2.6 FleximerTM Polymeric Linkers 481
18.3 Tailoring Antibodies for Use as ADCs 482
18.3.1 Engineered Cysteines 483
18.3.2 Enzyme-Assisted Conjugation 484
18.3.2.1 Microbial Transglutaminase 484
18.3.2.2 Formylglycine-Generating Enzyme (FGE) 485
18.3.2.3 Glucosyltransferases and Other Glycan Engineering 486
18.3.3 Non-Native Amino Acids and Selenocysteine 487
18.3.4 Alternative Formats and Masked Antibodies 488
18.3.5 ADCs Beyond Oncology 489
18.4 Conclusions 491
References 491
Index 505
