Recombinant antibodies / Frank Breitling, Stefan Dübel.

Format:

Book

Author/Creator:

Breitling, Frank.

Contributor:

Dübel, Stefan.

Language:

English

German

Subjects (All):

Recombinant antibodies.

Physical Description:

viii, 161 pages : illustrations ; 26 cm

Edition:

English edition.

Place of Publication:

New York : John Wiley ; Heidelberg : Spektrum, [1999]

Summary:

Recombinant Antibodies is the first general overview of the emerging field of antibody engineering and will serve as the definitive source for researchers and engineers in biology and medicine, biochemistry, immunology, and molecular biology, as well as anyone who is interested in biotechnology and antibody engineering.

Contents:

1.1 How the Body Manufactures Antibodies 1

1.1.1 The Chance Combination of Peptide Building Blocks Leads to a Vast Variety of Antibodies 1

1.1.2 The Specificity of Antigen Binding is Determined by the Hypervariable Domains 2

1.1.3 The Constant Regions Stabilize the Antibody Molecule 4

1.1.4 The Constant Regions Mediate the Effector Functions 4

1.1.5 Antibody Binding Improves During an Immune Response 4

1.1.6 B-Lymphocytes are Expanded through Clonal Selection 5

1.1.7 Only those Memory Cells Survive that Code for Higher Affinity Antibodies 7

1.1.8 Evolution Invented Antibody Engineering Millions of Years Ago 7

1.2 Antibody Production: Established Methods and New Approaches 8

1.2.1 Antibodies are Widely Used in Research and Diagnosis 8

1.2.2 Antisera Contain Polyclonal Antibodies 9

1.2.3 Immortal B-Lymphocytes Produce Monoclonal Antibodies 9

1.2.4 Recombinant Antibodies are Genetically Engineered Antibody Fragments 11

1.2.5 Fv Fragments are the Smallest Antigen-Binding units 11

1.2.6 Why Recombinant Antibodies? 12

Chapter 2 Building Recombinant Antibody Fragments 15

2.1.1 Bacteria can Produce Recombinant Antibody Fragments 15

2.1.2 The Humoral Immune System can be Imitated in Bacteria 15

2.2 Sources of Antibody Genes 20

2.2.1 Combinatorial Assembly of Antibody Genes 20

2.2.2 Complexity of Antibody Gene Libraries 20

2.2.3 What is the real Complexity of Antibody Gene Libraries? 22

2.2.4 Cloning Antibody Genes from Hybridoma Cell Lines 23

2.2.5 Gene Libraries from Immunized Donors 28

2.2.6 Universal Antibody Gene Libraries from Nonimmunized Donors 30

2.2.7 Genomic Antibody Gene Libraries 31

2.2.8 Hybrid and Semisynthetic Antibody Gene Libraries 31

2.2.9 Synthetic Antibody Gene Libraries with Random Sequences in the CDRs 32

2.2.10 In Vivo Recombination Systems enable "Superlibraries" 33

2.3 From Diversity to Specifity: The Selection of Recombinant Antibodies from Gene Libraries 34

2.3.1 Selection of Recombinant Antibodies with Classical Expression Systems 35

2.3.2 Genes Coupled to their Gene Products 35

2.3.3 Peptides can be Presented on the Surface of Bacteriophages 36

2.3.4 Recombinant Antibodies Anchored on the Surface of Filamentous Phages 36

2.3.5 Billions of Different Antibody Clones can be Tested for Binding with the Help of Display Vectors 39

2.3.6 Display Vectors Simplify the Selection of Human Recombinant Antibody Fragments 43

2.3.7 Antigen-Specific Infection of Bacteria 46

2.3.8 Other Prokaryotic Display Vectors 46

2.3.9 Antibodies can also be Anchored on the Surface of Eukaryotic Viruses 48

2.4 Antibody Engineering 48

2.4.1 Why Antibody Engineering? 48

2.4.2 Mouse-Human Chimeras 49

2.4.3 Framework Regions of the Variable Domain from Mouse Antibodies can be Humanized 51

2.4.4 Three-Dimensional Structure of Antibodies can be Modeled on a Computer 53

2.4.5 Efficient Screening Systems help in the Humanization of Antibodies through Chain Shuffling 54

2.4.6 Affinity of Recombinant Antibodies can be Increased by Repeated Mutation and Selection 56

2.4.7 Antibody Genes can be Mutated with Gene Synthesis or with the Help of the Polymerase Chain Reaction 57

2.4.8 "Sexual" PCR Combines Several Mutations 59

2.4.9 Fv Fragments are Stabilized through a Peptide Bond Linking the Variable Domains 61

2.4.10 Fv Fragments can be Stabilized through Internal Disulfide Bridges 63

2.4.11 Camel Antibodies Contain only One Variable Domain 65

2.4.12 Antibodies can take over the Function of Enzymes 66

Chapter 3 Antibodies With New Functions 79

3.1 Why Bispecific and Bifunctional Antibodies? 79

3.2 New Functions through Homologous Fusion Partners: Bispecific Antibodies 82

3.2.1 Bispecific Antibodies Unite the Binding Properties of Two Different Monoclonal Antibodies into One Molecule 82

3.2.2 Recombinant Production of Bispecific Antibodies 85

3.2.3 Creating Bispecific Antibodies with Help from Heterologous Binding Domains 88

3.2.4 "Universal" Bispecific Antibodies 90

3.2.5 Recombinant Bispecific Antibodies can be Distinctly Smaller than an IgG 90

3.3 New Functions through Heterologous Fusion Partners: Bifunctional Antibodies 91

3.3.1 What are Bifunctional Antibodies? 91

3.3.2 Bifunctional Antibodies can be Used as Immunotoxins 92

3.3.3 Radioimmunotoxins 97

3.3.4 Intracellular Antibodies 98

3.3.5 Recombinant Antibodies can be Anchored on the Surface of Cells 100

Chapter 4 Production and Purification of Recombinant Antibody Fragments 109

4.1 Properties of Recombinant Antibodies and Choice of Expression System 109

4.1.1 Structural Characterization of an Antibody: The Definition of the Hypervariable Regions (CDRs) 109

4.1.2 Biochemical Characterization of an Antibody: Specificity and Affinity 113

4.1.3 Different Applications of Antibodies Require Different Expression Systems 119

4.2 Prokaryotic Expression Systems 119

4.2.1 Production in E. coli 119

4.2.2 Production in Gram-Positive Bacteria 126

4.3 Eukaryotic Expression Systems for Recombinant Antibodies 126

4.3.1 Expression in the Cytoplasm 127

4.3.2 The Cells of the Immune System are the Natural Production Sites For Antibodies 127

4.3.3 Production in Other Mammalian Cells 128

4.3.4 Production of Recombinant Antibody Fragments in Insect Cells (Baculovirus System) 129

4.3.5 Production of Recombinant Antibody Fragments in Plants 131

4.3.6 Production of Recombinant Antibody Fragments in Fungi 131

4.3.7 Production in Cell-Free Systems 132

4.4 Purification of Recombinant Antibodies and Antibody Fragments 133

4.4.1 Physical Separation Methods are the First Steps in Every Antibody Purification 133

4.4.2 Affinity Chromatography: the Key to Efficient Purification of Recombinant Proteins 133

4.4.3 Affinity Chromatographic Purification with the Help of the Binding Properties of the Antibody Portion 134

4.4.4 Affinity Chromatographic Purification with the Help of a Heterologous Fusion Component 138

4.4.5 Special Case Human Therapeutic Agents: Removal of Bacterial Endotoxins 145

4.4.6 Storage of Purified Recombinant Antibodies 145.

Notes:

Includes bibliographical references and index.

ISBN:

0471178470

OCLC:

40964704