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 obvioushigh 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 1520 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.

Includes supplementary material: sn.pub/extras

Klappentext
With 10 recent FDA approvals of therapeutic antibody drugs and hundreds more in the biotechnology pipeline, it is clear that engineered antibodies will play a major role in treating human diseases in the 21st century. In Antibody Engineering: Methods and Protocols, leading researchers and clinicians present a core collection of cutting-edge techniques for the generation, expression, optimization, and characterization of recombinant antibodies. Readily reproducible protocols for lead generation range from the cloning of human immunoglobulin genes to the generation of human recombinant antibodies by humanization approaches, molecular display technologies, and transgenic animals. The techniques for antibody expression and optimization utilize not only bacterial and mammalian cell cultures, but also insect cell cultures and transgenic plants. State-of-the-art technologies are described for the characterization of antigen-binding affinity and specificity with novel applications in radioimmunotargeting, cancer immunotherapy, drug abuse, and proteomics. Each fully tested protocol is described in step-by-step detail and includes background material, equipment and reagent lists, and tips on troubleshooting and avoiding pitfalls.
Comprehensive and up-to-date, Antibody Engineering: Methods and Protocols provides both laboratory and clinical investigators with a diverse array of the fundamental techniques upon which new and imaginative drug technologies can be successfully developed.

Inhalt
Antibody Sequence And Structure.- Internet Resources for the Antibody Engineer.- The Kabat Database and a Bioinformatics Example.- IMGT, The International ImMunoGeneTics Information System®, http://imgt.cines.fr.- Antibody Variable Regions.- Studying Antibody Conformations by Ultracentrifugation and Hydrodynamic Modeling.- Antibody-Lead Generation.- PCR Cloning of Human Immunoglobulin Genes.- Antibody Humanization by CDR Grafting.- Selection of Human Antibodies from Phage Display Libraries.- Production of Human Single-Chain Antibodies by Ribosome Display.- Production of Human Antibodies from Transgenic Mice.- Selection of Internalizing Antibodies for Drug Delivery.- Engineering Multivalent Antibody Fragments for In Vivo Targeting.- Production of Recombinant Bispecific Antibodies.- Antibody Expression And Optimization.- Expression and Isolation of Recombinant Antibody Fragments in E. coli.- Expression of Recombinant Antibodies in Mammalian Cell Lines.- Human Antibody Production Using Insect-Cell Expression Systems.- Antibody Production in Transgenic Plants.- Directed Mutagenesis of Antibody Variable Domains.- Antibody Affinity Maturation by Chain Shuffling.- Antibody Affinity Maturation by Random Mutagenesis.- Developing a Minimally Immunogenic Humanized Antibody by SDR Grafting.- Antibody Characterization and Novel Applications.- Antibody Purification by Column Chromatography.- Affinity Measurement Using Surface Plasmon Resonance.- Kinetic Exclusion Assays to Study High-Affinity Binding Interactions in Homogeneous Solutions.- Characterization of Antibody-Antigen Interactions by Fluorescence Spectroscopy.- Antibody Epitope Mapping Using Arrays of Synthetic Peptides.- Mapping Antibody:Antigen Interactions by Mass Spectrometry and Bioinformatics.- Radiometal Labeling ofAntibodies and Antibody Fragments for Imaging and Therapy.- Production and Characterization of Anti-Cocaine Catalytic Antibodies.- Recombinant Immunotoxins in the Treatment of Cancer.- Antibodies in Proteomics.- Targeting of Antibodies Using Aptamers.