Introduction to Antibody Engineering

Preface
Contents
1: Introduction by the Editors
2: Antibodies: A History of Their Discovery and Properties
	What You Will Learn in This Chapter
	2.1 Introduction
	2.2 Early Beginnings
	2.3 Serum as the Protection Matrix
	2.4 Specificity and the Nature of Proteins
	2.5 Theories of Protein Structure
	2.6 Antibody Sequences: Homogeneous or Heterogeneous?
	2.7 γ-Globulin: Isotypes and Allotypes
	2.8 The Structural Basis of Antibody Recognition Revealed
	2.9 The Origins of Antibody Diversity
	2.10 The Fc Region and Effector Functions
	2.11 The Modern Era
	2.12 The Antibody Engineering Revolution
	References
3: Monoclonal AntibodiesMonoclonal antibodies (mAbs) and HybridomasHybridomas
	What You Will Learn in This Chapter
	3.1 The First Monoclonal Antibodies
	3.2 Sequence, Structure and Function of Antibodies
		3.2.1 Overall Structure
		3.2.2 Variable Domains
		3.2.3 Constant Domains
	3.3 The Hybridoma Method
		3.3.1 The Starting Point: Antibody-Producing B-Cells
		3.3.2 ImmunizationImmunizations
		3.3.3 Myeloma Cell Lines Suitable for Fusion
		3.3.4 Cell FusionCell fusion
		3.3.5 Identification of Individual Hybridoma Clones
		3.3.6 Monoclonality
	3.4 From Mouse to Human Monoclonal Antibodies
		3.4.1 Chimeric Antibodies
		3.4.2 Nomenclature of Monoclonal Antibodies
		3.4.3 Humanized Antibodies
		3.4.4 Fully Human Monoclonal Antibodies
	3.5 Antibody Fragments
		Questions
		Take Home Message
	References
4: Antibody Display Systems
	What You Will Learn in This Chapter
	4.1 Introduction
	4.2 Phage DisplayPhage display
	4.3 YeastYeast Surface Display
	4.4 Mammalian DisplayMammalian display
	4.5 E. coli DisplayE. coli display
	4.6 Ribosome Display
	4.7 cDNA DisplaycDNA display
	4.8 B-Cell Cloning
		Take Home Message
	References
5: Transgenic AnimalsTransgenic animals for the Generation of Human Antibodies
	What You Will Learn in This Chapter
	5.1 The Concept of Using Transgenic Animals to Generate Human Therapeutic Antibodies
	5.2 Strategies in Developing Transgenic Platforms for Human Antibody Discovery
		5.2.1 Transgenic Strategies
		5.2.2 Animals with Knockout of Endogenous Ig Production
		5.2.3 Human IgH Gene Loci in Transgenic Constructs
		5.2.4 Human Igĸ and Igλ Gene Loci in Transgenic Constructs
	5.3 Examples of Currently Available Transgenic Platforms for Human Antibody Discovery
		5.3.1 HuMAb-MouseHuMab-Mouse and XenoMouse
		5.3.2 Tc Mouse, Tc Bovine and Tc Goat (Caprine)
		5.3.3 OmniRat, OmniMouseOmniMouse and OmniChicken
		5.3.4 VelociMouseVelociMouse, Kymouse and Trianni Mouse
		5.3.5 Transgenic Platforms Expressing Different Formats of Antibodies
			5.3.5.1 OmniFlicOmniFlicand OmniClicOmniClic
			5.3.5.2 HCAb Transgenic Mice (Harbour Biomed), Humabody VH Platform and UniRat
	5.4 Strategies in Antibody Discovery from Transgenic Platforms
		Take Home Messages
	Further Reading
	References
6: Applications of Antibodies in Therapy, Diagnosis, and Science
	What You Will Learn in This Chapter
	6.1 Application of Antibodies in Therapy
		6.1.1 Cetuximab
		6.1.2 Trastuzumab
		6.1.3 Rituximab
		6.1.4 Ipilimumab
		6.1.5 Nivolumab
		6.1.6 PembrolizumabPembrolizumab (Keytruda)
		6.1.7 Avelumab
		6.1.8 Adalimumab
		6.1.9 BlinatumomabBlinatumomab (Blincyto)
		6.1.10 Emicizumab
	6.2 Applications of Antibodies in Diagnosis
	6.3 Application of Antibodies in Science
		6.3.1 Immunoprecipitation
		6.3.2 Chromatin ImmunoprecipitationChromatin immunoprecipitation (ChIP)
		6.3.3 Western Blotting
		6.3.4 Enzyme-Linked Immunosorbent Assay (ELISAEnzyme-linked immunosorbent assay (ELISA))
		6.3.5 ImmunohistochemistryImmunohistochemistry (IHC)/ImmunocytochemistryImmunocytochemistry (ICC)
		6.3.6 Immuno-PCR and Proximity Ligation Assay
		6.3.7 Flow CytometryFlow cytometry
		6.3.8 Mass SpectrometryMass spectrometry (MS)
			Take Home Message
	References
		Further Reading
7: Bispecific Antibodies
	What You Will Learn in This Chapter
	7.1 Introduction
		Questions
	7.2 History of Bispecific Antibodies
		Questions
	7.3 Mode of Action of Bispecific Antibodies Today
		7.3.1 BsAbs for Bridging Different Cell Types
		7.3.2 Signaling Inhibition by Bridging Receptor Molecules
		7.3.3 Overcoming Ligand Redundancy
		7.3.4 Targeting Two Epitopes of the Same Antigen: Biparatopic bsAbs
		7.3.5 Enzyme Mimetics
		7.3.6 BsAbs Enabling Transport: Piggyback Molecules
			Questions
	7.4 Formats of Bispecific Antibodies
		7.4.1 Fragment-Based Bispecific Antibodies
		7.4.2 Symmetric Ab Formats
		7.4.3 Asymmetric bsAb Formats
			Questions
	7.5 Outlook for Bispecific Antibodies
	7.6 Conclusion
		Take Home Messages
	References
8: Antibody-Drug Conjugates
	What You Will Learn in This Chapter
	8.1 Introduction to ADCs
	8.2 Milestones of ADC Development
	8.3 Targets and Antibodies Utilized for ADCsAntibody-drug conjugates (ADCs)
	8.4 Cytotoxic ADC Payloads
	8.5 ADC Linkers
	8.6 Conjugation Technologies for ADC Generation
		Take Home Messages
	References
9: Alternative Binding Scaffolds: Multipurpose Binders for Applications in Basic Research and Therapy
	What You Will Learn in This Chapter
	9.1 What Are Alternative Binding Scaffolds, What Are Their Features, and What Is Their Potential?
	9.2 Details on the Most Advanced Alternative Binding Scaffolds
		9.2.1 Tenth Type III Domain of Fibronectin (Monobodies, Adnectins)
		9.2.2 Affibodies
		9.2.3 Affitins/Nanofitins
		9.2.4 Avimers
		9.2.5 Cysteine-Knot Miniproteins (Knottins)
		9.2.6 Designed Ankyrin Repeat Proteins
		9.2.7 Fynomers
		9.2.8 Kunitz Domain Inhibitors
		9.2.9 Lipocalins (Anticalins)
	9.3 Alternative Binding Scaffolds in the Clinic
	9.4 Alternative Binding Scaffolds as Tools in Basic Research and Beyond
	9.5 Future Directions
		Take Home Messages
	References
10: Chimeric Antigen Receptor (CAR) Redirected T Cells
	What You Will Learn in This Chapter
	10.1 The Prototype Chimeric Antigen Receptor (CAR, T-Body, Immunoreceptor)
	10.2 Modular Composition of a CAR: The Extracellular CAR Domains
		10.2.1 The Binding Domain
		10.2.2 The Spacer Domain
		10.2.3 The Transmembrane Region
		10.2.4 Intracellular Signaling Domains
	10.3 TRUCK: The ``Fourth Generation´´ CAR T Cell
	10.4 CAR T Cells with Dual Specificities, Combinatorial Antigen Recognition, and Conditional CARs
	10.5 Universal CARs with Engrafted Multiple Antigen Specificities
	10.6 Switch CARs Convert a Suppressor into an Activation Signal
	10.7 Inhibitory CARs: iCARs
	10.8 Allogeneic CAR T Cells and CAR NK Cells
	10.9 Manufacturing CAR T Cells for Clinical Applications
	10.10 Adoptive Therapy with CAR T Cells Induces Lasting Remissions in Hematologic Malignancies
	10.11 The Challenges of CAR T Cells in Treating Solid Cancer
	10.12 CAR T Cell Therapy Is Associated with Toxicities
		10.12.1 Systemic Toxicities
		10.12.2 ``On-Target Off-Tumor´´ Toxicities
		10.12.3 ``Off-target Off-tumor´´ Toxicities
		10.12.4 Strategies to Ameliorate CRS
	10.13 Challenges in CAR T Cell Therapy
		10.13.1 Which Is the Best Tumor-Selective Antigen Suitable for Targeting?
		10.13.2 How to Optimize the CAR Architecture?
		10.13.3 How to Establish Long-Term Cancer Control After CAR T Cell Therapy?
		10.13.4 Which T Cell Subset Provides Long-Term Tumor Control?
		10.13.5 How Can CAR T Cell Manufacturing Be Adapted to an Increasing Number of Patients?
		10.13.6 How to Balance CAR T Cell Efficacy and Side Effects?
		10.13.7 Do We Need Specific Pre-conditioning for Each Cancer Type?
		10.13.8 How to Activate the Immune Network to Induce a Broad Inflammatory Antitumor Response?
	10.14 CAR T Cells for the Treatment of Other Diseases
		Take Home Messages
	References
11: Improvement of Key Characteristics of Antibodies
	What You Will Learn in This Chapter
	11.1 Improving the Natural Functions of Antibodies to Create Therapeutics
	11.2 Antigen Binding
		11.2.1 Binding Epitope and Affinity
		11.2.2 Creating a Therapeutic Antibody with pH Sensitivity or `pH Switch´
	11.3 Improving and Optimising the Interactions with Fc Receptors
	11.4 Complement Activation and Inactivation
	11.5 Improving Serum Half-Life to Decrease Dosing Frequency
	11.6 Immunogenicity of Therapeutic Antibodies
	11.7 Antibody Fragments
	11.8 Improving Biophysical Properties of Therapeutic Antibodies
		11.8.1 Aggregation
		11.8.2 Thermal Denaturation
		11.8.3 Fragmentation, Deamidation and Oxidation
		11.8.4 Computational Design Tools
			Take Home Messages
	References
12: Engineering Therapeutic Antibodies for Development
	What You Will Learn from This Chapter
	12.1 Introduction
	12.2 Safety Considerations
	12.3 Examples of Target-Dependent Side Effects of mAbs
	12.4 Immunogenicity of Biotherapeutics
	12.5 What Causes Immunogenicity?
	12.6 Mechanisms Leading to an ADA Response
		12.6.1 T Cell-Dependent Immune Response
	12.7 T Cell-Independent Immune Response
	12.8 Clinical Consequences of Immunogenicity
	12.9 Engineering mAbs for Safety and Minimal Immunogenicity
	12.10 Humanization
	12.11 Selection of Isotypes
	12.12 Heterogeneity of Therapeutic mAbs
	12.13 Quality Attributes of mAbs Can Impact Safety and Efficacy
	12.14 Protein Engineering to Control mAb Variants
	12.15 Improved Manufacturability to Control mAb Variants
	12.16 mAb Industrial Manufacturing
	12.17 Considerations for Intended Use and mAb Formulation into Drug Product
	12.18 Formulation Development
	12.19 Closing Remarks
		Take Home Messages
	References
13: Eukaryotic Expression Systems for Upstream Processing of Monoclonal Antibodies
	What You Will Learn in This Chapter
	13.1 Introduction
		13.1.1 Antibodies and Different Antibody Formats
		13.1.2 Product Quality Attributes
			13.1.2.1 Glycosylation
			13.1.2.2 Charge Distribution
			13.1.2.3 Molecular Size
			13.1.2.4 Oxidation
			13.1.2.5 Structure
	13.2 Antibody Production in Mammalian Cells
		13.2.1 Host Cell Lines
		13.2.2 Cell Culture Medium
		13.2.3 Environmental Cultivation Parameters
		13.2.4 Mode of Bioreactor Operation
	13.3 Antibody Production in Yeast and Filamentous Fungi
		13.3.1 General Characteristics of the Expression Systems
		13.3.2 Glycosylation and Glycoengineered Production Strains
		13.3.3 Antibody Production in Yeast
		13.3.4 Antibody Production in Filamentous Fungi
	13.4 Conclusion
		Take Home Messages
	References
14: Antibody Validation
	What You Will Learn in This Chapter
	14.1 Introduction
		14.1.1 State of the Art and Problem Setting
			Questions
		14.1.2 Advantages of Polyclonal Antibodies
			Questions
	14.2 Cornerstones for Antibody Validation for Different Scientific Applications
		14.2.1 Standards for Reporting the Use of Antibodies in Scientific Publications
			Questions
		14.2.2 Publicly Available Antibody Databases
			Questions
		14.2.3 Minimum Information About a Protein Affinity Reagent Proposal
			Questions
	14.3 Scientific Incentives for Antibody Validation
		Questions
	14.4 The Human Protein Atlas
		14.4.1 Introduction to the Human Protein Atlas
		14.4.2 History of Human Protein Atlas
		14.4.3 Standard Antibody Validation
		14.4.4 Enhanced Antibody Validation
			Questions
	14.5 Start-off Simple: 10 Validation Rules for Diagnostic and Analytical Antibodies
		Questions
	14.6 Conclusion
		Take Home Messages
	References
Index