Antibody Engineering

More than ever, antibodies are being recognized as a major drug modality in a variety of diseases, including cancer, autoimmune diseases, infectious diseases, or even neurodegenerative disorders. Over 30 therapeutic antibodies have been approved and novel molecules are entering clinical trials at an average rate of 50 per year and that is predicted to continue well into the future. Notwithstanding the many achievements already made in the field, there is still a lot of room for improvements for these molecules in terms of activity, and a plethora of approaches have been attempted to optimize these molecules. Antibody Engineering: Methods and Protocols, Second Edition was compiled to give complete and easy access to a variety of antibody engineering techniques, starting from the creation of antibody repertoires and efficient ways to select binders from these repertoires, to their production in various hosts, their detailed characterization using various well established techniques, and to the modification and optimization of these lead molecules in terms of binding activity, specificity, size, shape, and more. Written in the successful Methods in Molecular Biology(TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and easily accessible, Antibody Engineering: Methods and Protocols, Second Edition serves as an invaluable resource for both experts and those new to the field, and most of all as a source of inspiration for the creation of the antibodies of tomorrow.

Use of IMGT® Databases and Tools for Antibody Engineering and Humanization.- Computer-Assisted Modeling of Antibody Variable Domains.- Cloning Single-Chain Antibody Fragments (scFv) from Hybridoma Cells.- Human in Cell scFv Library from Infiltrating B Cell.- Construction of Human Naive Antibody Gene Libraries.- Synthetic Customized scFv Libraries.- Selection of Stable scFv Antibodies by Phage Display.- Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs.- Generation and Isolation of Target Specific Single Domain Antibodies from Shark Immune Repertoires.-Generation of Human Single Domain Antibody Repertoires by Kunkel Mutagenesis.- Phage Display and Selections on Purified Antigens.- Phage Display and Selections on Cells.- Humanization by CDR Grafting and Specificity-Determining Residue Grafting.-Humanization by Guided Selections.- Selection of Antibody Fragments by Yeast Display.- Evolution of Antibodies in vitro by Ribosome Display.- Mammalian Cell Display of Full Length IgG.- Production of Antibody Fragments in Escherichia coli.-Production of Antibody Derivatives in the Methylotrophic Yeast Pichia pastoris.- Monoclonal Antibody Expression in Mammalian Cells.- Production of Recombinant Antibodies in Drosophila melanogaster S2 Cells.- Production of Antibody Fragments Using the Baculovirus-Insect Cell System.- Transient and Stable Expression of Antibodies in Nicotiana Species.- Measuring Antibody-Antigen Binding Kinetics Using Surface Plasmon Resonance.- Affinity Determination of Biotinylated Antibodies by Flow Cytometry.- Affinity Maturation of Antibodies: Optimized Methods to Generate High Quality scFv Libraries and Isolate IgG Candidates by High Throughput Screening.- Affinity Maturation by Semi Rational Approaches.- Molecular Scanning Mutagenesis: Combining Ribosome Display, Random Mutagenesis and Bioinformatic Analysis for Protein Engineering.- Fucose-Targeted Glyco-Engineering of PharmaceuticalCell Lines.- Fc engineering: Design, expression and functional characterization of antibody variants with improved effector function.- Fc engineering: Serum half life modulation through FcRn binding.- Monoclonal Antibody Lead Characterization - In vitro and in vivo methods.- Production, generation and characterization of scFv TNF ligand fusion proteins.- Antibody-IL-2 fusion proteins for tumor targeting.- Recombinant immunotoxins containing low endotoxins for clinical and animal studies.- Chimeric antigen receptors for T-cell based therapy.- Selection and Use of Intracellular Antibodies (Intrabodies).- Radiolabeled Antibodies for Imaging and Therapy.- Design and Production of Multimeric Antibody Fragments, Focused on Diabodies with Enhanced Clinical Efficacy.- Production of Bispecific Antibodies: Diabodies and Tandem scFv.