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1 Preparation, structure and function of monoclonal antibodies 1 1.1 Introduction 1 1.2 The role of antibodies in the immune response 2 1.3 Structure and function of antibodies 2 1.4 The organisation of antibody genes 11 1.5 Antigen-binding affinity and avidity 13 1.5.1 Affinity 13 1.5.2 Avidity 14 1.6 Generation of monoclonal antibodies 14 1.6.1 Hybridoma technology 14 1.6.2 Human monoclonal antibodies 18 1.6.3 Human MAbs from transgenic mice 20 1.6.4 Isolation of antibodies by phage display 21 2 Antibody engineering: design for specific applications 27 2.1 Introduction 27 2.2 Isolation of variable region genes 28 2.3 Overcoming immunogenicity 29 2.3.1 Chimeric and humanised antibodies 29 2.3.2 Antibody fragments to reduce immunogenicity 37 2.3.3 Chemical modification to reduce immunogenicity 38 2.3.4 Immunosuppressive therapy 40 2.4 Antibody fragments 40 2.4.1 Antibody fragments from proteolysis of IgG 40 2.4.2 Recombinant antibody fragments 42 Fab-based fragments 43 Fv-based fragments 45 Multivalent antibody fragments 48 2.5 Antibodies with multiple specificities 50 2.6 Engineering effector functions, 54 2.6.1 Engineering natural effector functions 54 2.6.2 Attachment of diagnostic or therapeutic agents 58 Chemical conjugates 58 Site-specific attachment 63 Fusion proteins 65 2.7 Engineering pharmacokinetics and biodistribution 67 2.7.1 Pharmacokinetics of IgG 68 2.7.2 Pharmacokinetics of antibody fragments 71 2.7.3 Clearance 74 2.7.4 Chemical modification 75 2.7.5 Fc region to extend half-life 75 3 Monoclonal antibodies in research and diagnostic applications 77 3.1 Introduction 77 3.2 Immunoassays in diagnostics and research 77 3.2.1 Radioimmunoassay 78 3.2.2 Immunoradiometric assay 79 3.2.3 Non-isotopic immunoassays 83 3.2.4 Improving sensitivity 85 3.2.5 Assay formats 89 3.2.6 Advantages of monoclonal antibodies in immunoassay 91 3.3 Immunosensors 92 3.3.1 Mass-detecting immunosensors 93 3.3.2 Electrochemical immunosensors 94 3.3.3 Optical immunosensors 95 3.4 Immunocytochemistry 97 3.5 Flow cytometry and cell sorting (FACS) 99 3.6 Western blotting (immunoblotting) 100 3.7 Immunopurification 102 3.8 Antibodies in structural biology 106 3.9 In vivo diagnostics 106 3.9.1 Radioimmunodetection of human tumours 107 Tumour-associated antigens 107 Form of antibody 109 Radioisotopes 110 Two- and three-step targeting approaches 114 3.9.2 Radioimmunoguided surgery 116 3.9.3 Non-tumour radioimmunodetection 116 4 Monoclonal antibodies in therapeutic applications 119 4.1 Introduction 119 4.2 Cancer 120 4.2.1 Cancer therapy with unmodified (naked) antibodies 121 4.2.2 Anti-idiotype antibodies 123 4.2.3 Bispecific antibody-mediated effector cell targeting 125 4.2.4 Other approaches to recruit the immune system using MAbs 127 4.2.5 Radioimmunotherapy 128 Form of antibody 129 Radioisotopes 132 Clearance mechanisms and two-step targeting 136 Clinical results with RIT - 137 4.2.6 Immunotoxins 139 4.2.7 Drug conjugates 142 4.2.8 Antibody-directed enzyme prodrug therapy (ADEPT) 144 4.2.9 Vascular targeting 146 4.3 Infectious disease 147 4.3.1 Antiviral antibodies 147 4.3.2 Bacterial sepsis 148 4.4 Cardiovascular disease 149 4.4.1 Inhibition of platelet aggregation 149 4.4.2 Thrombolysis 150 4.5 Disorders of the immune system/inflammatory diseases 151 4.5.1 The inflammatory response 151 4.5.2 Blocking inflammatory mediators 153 Anti-TNF antibodies in rheumatoid arthritis and inflammatory bowel disease 153 Anti-C5 154 4.5.3 Blocking adhesive interactions 154 4.5.4 Antibodies which directly inhibit T cell activation and proliferation 155 4.5.5 Antibody treatment of allergy 158 5 Production of monoclonal antibodies 161 5.1 Introduction 161 5.2 Expression of antibodies in mammalian cells 161 5.2.1 Transient expression systems 162 5.2.2 Stable expression systems 163 5.2.3 Expression of antibody fragments in mammalian cells 167 5.3 Expression in Escherichia coli 167 5.3.1 Intracellular expression of antibody fragments in E. coli 168 5.3.2 Secretion of antibody fragments from E. coli 170 5.4 Expression in other microbial systems 172 5.5 Expression in plants 173 5.6 Production in transgenic animals 174 5.7 Expression in insect cells 175 5.8 Production of monoclonal antibodies - cell culture 175 5.9 Purification of monoclonal antibodies 176 5.9.1 Purification of IgG 176 5.9.2 Purification of IgM 181 5.9.3 Purification of monoclonal antibody fragments 181 5.9.4 Purification for therapeutic use 185 6 Prospects for engineered antibodies in biotechnology 187 6.1 Gene therapy 187 6.1.1 Intracellular antibodies 187 6.1.2 Other applications of MAbs in gene therapy 188 6.2 Applications of antibodies in plants 189 6.3 Catalytic antibodies 189 6.4 Towards drug design 190 6.5 Improving affinity 191 6.6 Summary and prospects 192 7 References 195