Antibody-Drug Conjugates and Cellular Metabolic Dynamics

Preface
Contents
Chapter 1: Antibody-Drug Conjugates: Basic Concepts and Structures
1.1 Basic Concepts
1.2 Monoclonal Antibodies Used for ADC Development
1.3 Linkers
1.4 Effector Molecules
1.4.1 Tubulin Inhibitors
1.4.2 DNA-Damaging Antibiotics
1.5 Challenges and Prospective
References
Chapter 2: Relationship Between Target and Specific Action of Antibody-Drug Conjugates
2.1 Introduction
2.2 Tumor Associated Antigens
2.2.1 Membrane Proteins Associated with Cell Proliferation
2.2.2 Carcinoembryonic Antigen
2.2.3 Leukocyte Differentiation Antigen
2.3 Tumor Specific Antigen
2.3.1 Tumor Specific pMHC
2.3.1.1 Tumor-Specific pMHC Under Investigation as Targets of Antibody Drugs
2.3.1.2 Tumor-Specific pMHC as Potential Targets of Antibody Drugs
2.3.2 Tumor Specific Mutant Extracellular Domain of Membrane Protein
2.4 Conclusion
References
Chapter 3: The Internalization and Therapeutic Activity of Antibody Drug Conjugates
3.1 Introduction
3.2 Experimental Steps for Evaluating Internalization
3.2.1 Materials
3.2.2 Internalization Assay by Flow Cytometry
3.2.3 Intracellular Localization Assay by Confocal Microscope
3.3 Strategies to Improve Internalization Efficiency
3.3.1 Identifying Antibody Binding Epitopes
3.3.2 Antibody Drug Conjugate Toxins
3.3.3 Application of Bispecific Antibody
3.3.4 Application of Biparatopic Antibody
3.4 Conclusion
References
Chapter 4: The Internalization and Intracellular Trafficking of ADCs
4.1 Introduction
4.2 The Endocytosis Pathways of ADC
4.2.1 Clathrin Mediated Endocytosis (CME)
4.2.2 Caveolae Mediated Endocytosis (CavME)
4.2.3 Macropinocytosis
4.3 Intracellular Trafficking of ADC
4.3.1 Early Endosome (EE)
4.3.2 Late Endosome (LE)
4.3.3 Lysosome
4.4 Drug Release
4.5 Methods in Tracing the Intracellular Transport of ADC
4.5.1 Flow Cytometry Analysis to Analyze Endocytosis
4.5.2 Confocal Microscopy to Monitor Internalization and Intracellular Trafficking of ADC
4.5.3 The Colocalization of Internalized ADC and Lysosome
4.6 Conclusions
References
Chapter 5: Distribution and Metabolism of Antibody-Drug Conjugates
5.1 Preface
5.2 The Mechanism of Action of ADC in Cells
5.2.1 The Release of Cytotoxic Payloads
5.2.2 Mechanism of Action and Metabolism of Cytotoxic Payloads
5.2.3 Metabolism of Antibody Parts in ADCs
5.3 Methods and Procedures in Studying Distribution and Metabolism of ADC
5.3.1 Ligand Binding Assay
5.3.2 Liquid Chromatography-Tandem Mass Spectrometry Assay
5.3.3 Radiolabeled Antibody Assay
5.4 Summary
References
Chapter 6: Application of Antibody Fragments in ADCs
6.1 Introduction
6.2 Antibody Fragment Drug Conjugates (AFDCs)
6.2.1 Materials
6.2.2 Methods
6.2.2.1 Expression and Purification of Fab and Fab-CH3mut Miniaturized Antibodies
6.2.2.2 Preparation of Fab-vcMMAE and Fab-CH3mut-vcMMAE
6.2.2.3 Affinity of Fab, Fab-CH3mut, OFA, Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMAME
6.2.2.4 In Vitro Anti-Tumor Activity of Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE
6.2.2.5 Apoptosis-Inducing Activity of Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMMAE on Tumor Cell
6.2.2.6 In Vivo Biodistribution of Fab, Fab-CH3mut and OFA
6.2.2.7 Antitumor Activity and Toxicity of Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMMAE in Vivo
6.3 Overview of Antibody Fragments
6.4 Summary
References
Chapter 7: Novel Targeting Carriers in Antibody-Drug Conjugates
7.1 Introduction
7.2 The Feasibility of TRAIL as a Conjugate Carrier
7.2.1 TRAIL and its Receptor
7.2.2 Cell Apoptosis Inducing and Anti-Tumor Activity of TRAIL
7.2.3 Study on TRAIL as a Cytotoxin Carrier
7.2.3.1 Materials
7.2.3.2 Experiments [28]
7.3 Review Study on Other Novel Carriers
7.4 Summary
References
Chapter 8: Site-Specified Conjugating Technology and Application
8.1 Introduction
8.1.1 Engineered Cysteine
8.1.2 Insertion of Unnatural Amino Acids
8.1.3 Enzyme-Chemical Conjugation
8.2 Site-Specified Conjugation of Mutated Cysteine
8.2.1 Principle of Site-Specified Conjugation of Mutated Cysteine
8.2.2 Main Experimental Materials
8.2.3 Main Experimental Procedures [20] (DAR = 2 or DAR = 4)
8.2.3.1 Acquisition of Cysteine Mutant Antibodies
8.2.3.2 Synthesis and Analysis of Ab-vcMMAE
8.3 Site-Specific Conjugation Through Sortase A
8.3.1 Direct Enzymatic Approach
8.3.1.1 Materials
8.3.1.2 Methods
8.3.2 Chemo-Enzymatic (Srt A) Strategy
8.3.2.1 Materials
8.3.2.2 Methods[24]
8.4 Identification of Conjugation Sites by Mass Spectrometry
8.4.1 Materials
8.4.2 Methods[25]
8.5 Conclusion
References
Chapter 9: Determination of Drug-to-Antibody Ratio of ADCs
9.1 Introduction
9.2 Experimental Procedures for RP-HPLC and HIC to Determine DAR
9.2.1 Determination of DAR by RP-HPLC
9.2.1.1 Experimental Conditions and Methods
9.2.1.2 Data Processing
9.2.2 Determination of DAR by HIC
9.2.2.1 Experimental Conditions and Methods
9.2.2.2 Data Processing
9.2.3 Case Analysis [18]
9.3 The Experimental Procedure for the Determination of DAR by LC-MS
9.3.1 Instruments and Reagents
9.3.1.1 Instruments
9.3.1.2 Reagents
9.3.2 Method
9.3.2.1 Sample Preparation
9.3.2.2 LC-MS Analysis
9.3.2.3 Data Processing
9.3.3 Case Analysis [18]
9.3.3.1 Relative Molecular Mass of Light and Heavy Chains
9.3.3.2 Peptide Map Detection
9.3.3.3 Results
9.4 Conclusion
References
Chapter 10: Pharmacokinetic Study of Antibody-Drug Conjugates
10.1 Introduction
10.2 The Application of ELISA-Based Methods in Studying Pharmacokinetic of ADC
10.2.1 ELISA-Sandwich Technique
10.2.1.1 Total Antibody Detection
10.2.1.2 Total ADC Detection
10.2.1.3 Active ADC Detection
10.2.2 Ligand Binding Assay Based on ELISA
10.2.2.1 Detection of Active Antibody
10.2.2.2 Detection of Active ADC
10.2.3 Semihomogeneous Assay
10.2.3.1 Specific SHA
10.2.3.2 Non-specific SHA
10.3 The Application of Flow Cytometry in Studying Pharmacokinetics of Antibody-Drug Conjugates
10.3.1 Making Biotinylated Anti-MMAE Antibody
10.3.2 Quantitative OFA-HL and OFA-MMAE with Flow Cytometry Method
10.3.3 The Lower Limit of Quantification Is Affected by Selection of Fluorescein
10.3.4 Quantification of OFA-HL and OFA-HL-MMAE in Blood Samples
10.4 Summary
References