Antibody Engineering: Methods and ProtocolsBenny K. C. Lo Springer Science & Business Media, 03.02.2008 - 562 Seiten The exquisite binding specificity of antibodies has made them valuable tools from the laboratory to the clinic. Since the description of the murine hybridoma technology by Köhler and Milstein in 1975, a phenomenal number of mo- clonal antibodies have been generated against a diverse array of targets. Some of these have become indispensable reagents in biomedical research, while others were developed for novel therapeutic applications. The attractiveness of an- bodies in this regard is obvious—high target specificity, adaptability to a wide range of disease states, and the potential ability to direct the host’s immune s- tem for a therapeutic response. The initial excitement in finding Paul Ehrlich’s “magic bullet,” however, was met with widespread disappointment when it was demonstrated that murine antibodies frequently elicit the human anti-murine an- body (HAMA) response, thus rendering them ineffective and potentially unsafe in humans. Despite this setback, advances in recombinant DNA techniques over the last 15–20 years have empowered the engineering of recombinant antibodies with desired characteristics, including properties to avoid HAMA. The ability to p- duce bulk quantities of recombinant proteins from bacterial fermentation also fueled the design of numerous creative antibody constructs. To date, the United States Food and Drug Administration has approved more than 10 recombinant antibodies for human use, and hundreds more are in the development pipeline. The recent explosion in genomic and proteomic information appears ready to deliver many more disease targets amenable to antibody-based therapy. |
Inhalt
3 | |
11 | |
Toward a Unified Modeling Method | 51 |
PCR Cloning of Human Immunoglobulin Genes | 117 |
Antibody Humanization by CDR Grafting | 135 |
Selection of Human Antibodies from Phage Display Libraries | 161 |
Production of Human SingleChain Antibodies | 177 |
Selection of Internalizing Antibodies for Drug Delivery | 201 |
Kevin Brady and Benny K C Lo 19 Antibody Affinity Maturation by Chain Shuffling | 319 |
Marks | 327 |
Antibody Affinity Maturation by Random Mutagenesis | 345 |
Developing a Minimally Immunogenic Humanized Antibody | 361 |
Antibody Purification by Column Chromatography | 379 |
Robert Karlsson and Anita Larsson | 389 |
Kinetic Exclusion Assays to Study HighAffinity Binding | 417 |
by Fluorescence Spectroscopy | 431 |
Expression and Isolation of Recombinant Antibody Fragments | 245 |
Expression of Recombinant Antibodies in Mammalian Cell Lines | 255 |
Human Antibody Production Using InsectCell Expression Systems | 269 |
Rainer Fischer and Paul Christou 18 Directed Mutagenesis of Antibody Variable Domains | 301 |
Alexandra Huhalov Daniel I R Spencer and Kerry A Chester | 465 |
Paloma de Prada and Donald W Landry 30 Recombinant Immunotoxins in the Treatment of Cancer | 495 |
557 | |
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affinity Amersham Biosciences amino acid analysis anti antibody fragments antibody libraries antigen antigen-binding assay bacterial baculovirus beads BIAcore binding Biol bispecific buffer canonical classes Centrifuge chromatography cloning coli column concentration conformation containing culture database diabodies digested diluted domains ELISA elution enzyme epitope expression framework heavy-chain human antibodies hybridoma IMGT immobilized immunoglobulin Immunol immunotoxin incubate injection insect cells interactions Kabat kinked ligand light chains linker loop medium membrane Methods Molecular molecules monoclonal antibody murine NaCl Natl Note nucleotide overnight PCR product peptides phage antibody phage display plasmid plate polymerase primer protein Protocols purification reaction reagents recombinant repertoire residues restriction enzyme Resuspend ribosome display room temperature scFv selection sequence single-chain sodium solution step structure supernatant surface TAE buffer target tion transgenic tube variable regions vector VL gene Wash µg/mL