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Tables of Contents for Recombinant Antibodies
Chapter/Section Title
Page #
Page Count
Preface
IX
Chapter 1 Background
1
14
1.1 How the Body Manufactures Antibodies
1
7
1.1.1 The Chance Combination of Peptide Building Blocks Leads to a Vast Variety of Antibodies
1
1
1.1.2 The Specificity of Antigen Binding is Determined by the Hypervariable Domains
2
2
1.1.3 The Constant Regions Stabilize the Antibody Molecule
4
1
1.1.4 The Constant Regions Mediate the Effector Functions
4
1
1.1.5 Antibody Binding Improves During an Immune Response
4
1
1.1.6 B-Lymphocytes are Expanded through Clonal Selection
5
2
1.1.7 Only those Memory Cells Survive that Code for Higher Affinity Antibodies
7
1
1.1.8 Evolution Invented Antibody Engineering Millions of Years Ago
7
1
1.2 Antibody Production: Established Methods and New Approaches
8
7
1.2.1 Antibodies are Widely Used in Research and Diagnosis
8
1
1.2.2 Antisera Contain Polyclonal Antibodies
9
1
1.2.3 Immortal B-Lymphocytes Produce Monoclonal Antibodies
9
2
1.2.4 Recombinant Antibodies are Genetically Engineered Antibody Fragments
11
1
1.2.5 Fv Fragments are the Smallest Antigen-Binding units
11
1
1.2.6 Why Recombinant Antibodies?
12
3
Chapter 2 Building Recombinant Antibody Fragments
15
64
2.1 Introduction
15
5
2.1.1 Bacteria can Produce Recombinant Antibody Fragments
15
1
2.1.2 The Humoral Immune System can be Imitated in Bacteria
15
5
2.2 Sources of Antibody Genes
20
14
2.2.1 Combinatorial Assembly of Antibody Genes
20
1
2.2.2 Complexity of Antibody Gene Libraries
20
2
2.2.3 What is the real Complexity of Antibody Gene Libraries?
22
1
2.2.4 Cloning Antibody Genes from Hybridoma Cell Lines
23
5
2.2.5 Gene Libraries from Immunized Donors
28
2
2.2.6 Universal Antibody Gene Libraries from Nonimmunized Donors
30
1
2.2.7 Genomic Antibody Gene Libraries
31
1
2.2.8 Hybrid and Semisynthetic Antibody Gene Libraries
31
1
2.2.9 Synthetic Antibody Gene Libraries with Random Sequences in the CDRs
32
1
2.2.10 In Vivo Recombination Systems enable "Superlibraries"
33
1
2.3 From Diversity to Specifity: The Selection of Recombinant Antibodies from Gene Libraries
34
14
2.3.1 Selection of Recombinant Antibodies with Classical Expression Systems
35
1
2.3.2 Genes Coupled to their Gene Products
35
1
2.3.3 Peptides can be Presented on the Surface of Bacteriophages
36
1
2.3.4 Recombinant Antibodies Anchored on the Surface of Filamentous Phages
36
3
2.3.5 Billions of Different Antibody Clones can be Tested for Binding with the Help of Display Vectors
39
4
2.3.6 Display Vectors Simplify the Selection of Human Recombinant Antibody Fragments
43
3
2.3.7 Antigen-Specific Infection of Bacteria
46
1
2.3.8 Other Prokaryotic Display Vectors
46
2
2.3.9 Antibodies can also be Anchored on the Surface of Eukaryotic Viruses
48
1
2.4 Antibody Engineering
48
21
2.4.1 Why Antibody Engineering?
48
1
2.4.2 Mouse-Human Chimeras
49
2
2.4.3 Framework Regions of the Variable Domain from Mouse Antibodies can be Humanized
51
2
2.4.4 Three-Dimensional Structure of Antibodies can be Modeled on a Computer
53
1
2.4.5 Efficient Screening Systems help in the Humanization of Antibodies through Chain Shuffling
54
2
2.4.6 Affinity of Recombinant Antibodies can be Increased by Repeated Mutation and Selection
56
1
2.4.7 Antibody Genes can be Mutated with Gene Synthesis or with the Help of the Polymerase Chain Reaction
57
2
2.4.8 "Sexual" PCR Combines Several Mutations
59
2
2.4.9 Fv Fragments are Stabilized through a Peptide Bond Linking the Variable Domains
61
2
2.4.10 Fv Fragments can be Stabilized through Internal Disulfide Bridges
63
2
2.4.11 Camel Antibodies Contain only One Variable Domain
65
1
2.4.12 Antibodies can take over the Function of Enzymes
66
3
2.5 Summary
69
10
Chapter 3 Antibodies With New Functions
79
30
3.1 Why Bispecific and Bifunctional Antibodies?
79
3
3.2 New Functions through Homologous Fusion Partners: Bispecific Antibodies
82
9
3.2.1 Bispecific Antibodies Unite the Binding Properties of Two Different Monoclonal Antibodies into One Molecule
82
3
3.2.2 Recombinant Production of Bispecific Antibodies
85
3
3.2.3 Creating Bispecific Antibodies with Help from Heterologous Binding Domains
88
2
3.2.4 "Universal" Bispecific Antibodies
90
1
3.2.5 Recombinant Bispecific Antibodies can be Distinctly Smaller than an IgG
90
1
3.3 New Functions through Heterologous Fusion Partners: Bifunctional Antibodies
91
11
3.3.1 What are Bifunctional Antibodies?
91
1
3.3.2 Bifunctional Antibodies can be Used as Immunotoxins
92
5
3.3.3 Radioimmunotoxins
97
1
3.3.4 Intracellular Antibodies
98
2
3.3.5 Recombinant Antibodies can be Anchored on the Surface of Cells
100
2
3.4 Summary
102
7
Chapter 4 Production and Purification of Recombinant Antibody Fragments
109
46
4.1 Properties of Recombinant Antibodies and Choice of Expression System
109
10
4.1.1 Structural Characterization of an Antibody: The Definition of the Hypervariable Regions (CDRs)
109
4
4.1.2 Biochemical Characterization of an Antibody: Specificity and Affinity
113
6
4.1.3 Different Applications of Antibodies Require Different Expression Systems
119
1
4.2 Prokaryotic Expression Systems
119
7
4.2.1 Production in E. coli
119
7
4.2.2 Production in Gram-Positive Bacteria
126
1
4.3 Eukaryotic Expression Systems for Recombinant Antibodies
126
7
4.3.1 Expression in the Cytoplasm
127
1
4.3.2 The Cells of the Immune System are the Natural Production Sites For Antibodies
127
1
4.3.3 Production in Other Mammalian Cells
128
1
4.3.4 Production of Recombinant Antibody Fragments in Insect Cells (Baculovirus System)
129
2
4.3.5 Production of Recombinant Antibody Fragments in Plants
131
1
4.3.6 Production of Recombinant Antibody Fragments in Fungi
131
1
4.3.7 Production in Cell-Free Systems
132
1
4.4 Purification of Recombinant Antibodies and Antibody Fragments
133
22
4.4.1 Physical Separation Methods are the First Steps in Every Antibody Purification
133
1
4.4.2 Affinity Chromatography: the Key to Efficient Purification of Recombinant Proteins
133
1
4.4.3 Affinity Chromatographic Purification with the Help of the Binding Properties of the Antibody Portion
134
4
4.4.4 Affinity Chromatographic Purification with the Help of a Heterologous Fusion Component
138
7
4.4.5 Special Case Human Therapeutic Agents: Removal of Bacterial Endotoxins
145
1
4.4.6 Storage of Purified Recombinant Antibodies
145
10
Index
155