Antibody Engineering (2^nd Ed., 2nd ed. 2012)
Methods and Protocols, Second Edition
Methods in Molecular Biology Series, Vol. 907

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? 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.

Part I. In silico Methods

1. Use of IMGT® Databases and Tools for Antibody Engineering and Humanization

Marie-Paule Lefranc, François Ehrenmann, Chantal Ginestoux, Véronique Giudicelli, and Patrice Duroux

2. Computer-Assisted Modeling of Antibody Variable Domains

Oscar H. P. Ramos

Part II. Generation of Diversity

3. Cloning Single-Chain Antibody Fragments (scFv) from Hybridoma Cells

Lars Toleikis and André Frenzel

4. Human in Cell scFv Library from Infiltrating B Cell

Sylvie Peraldi-Roux                                   

5. Construction of Human Naive Antibody Gene Libraries

Michael Hust, André Frenzel, Torsten Meyer, Thomas Schirrmann, and Stefan Dübel  

6. Synthetic Customized scFv Libraries  

Gautier Robin and Pierre Martineau        

7. Selection of Stable scFv Antibodies by Phage Display       

Eeva-Christine Brockmann       

8. Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

Cécile Vincke, Carlos Gutiérez, Ulrich Wernery, Nick Devoogdt, Gholamreza Hassanzadeh-Ghassabeh, and Serge Muyldermans

9. Generation and Isolation of Target Specific Single Domain Antibodies from Shark Immune Repertoires

Mischa Roland Müller, Ronan O’Dwyer, Marina Kovaleva, Fiona Rudkin, Helen Dooley, and Caroline Jane Barelle

10. Generation of Human Single Domain Antibody Repertoires by Kunkel Mutagenesis

Kip Dudgeon, Romain Rouet, Kristoffer Famm, and Daniel Christ

Part IV. Selections of Lead Candidate

11. Phage Display and Selections on Purified Antigens

Julie Matz and Patrick Chames    

12. Phage Display and Selections on Cells

Klervi Even-Desrumeaux and Patrick Chames

13. Humanization by CDR Grafting and Specificity-Determining Residue Grafting  

Jin Hong Kim and Hyo Jeong Hong

14. Humanization by Guided Selections

Sang Jick Kim and Hyo Jeong Hong            

15. Selection of Antibody Fragments by Yeast Display   

Nathalie Scholler      

16. Evolution of Antibodies in vitro by Ribosome Display

Bryan M. Edwards and Mingyue He              

17. Mammalian Cell Display of Full Length IgG       

Chen Zhou and Wenyan (David) Shen

Part V. Production of Recombinant Antibodies

18. Production of Antibody Fragments in Escherichia coli

Tomohisa Katsuda, Hiroyuki Sonoda, and Hideki Yamaji        

19. Production of Antibody Derivatives in the Methylotrophic Yeast Pichia pastoris

Steve Schoonooghe, Jannick Leoen, and Jurgen Haustraete                

20. Monoclonal Antibody Expression in Mammalian Cells

Richard (Yi) Zhang and Wenyan (David) Shen        

21. Production of Recombinant Antibodies in Drosophila melanogaster S2 Cells

Daniel X. Johansson, Thomas Krey, and Oskar Andersson

22. Production of Antibody Fragments Using the Baculovirus-Insect Cell System

Takanori Furuta, Takafumi Ogawa, and Hideki Yamaji                

23. Transient and Stable Expression of Antibodies in Nicotiana Species

J. Christopher Hall, Michael D. McLean and Freydoun Garabagi    

Part VI. Variable Domain Optimization

24. Measuring Antibody-Antigen Binding Kinetics Using Surface Plasmon Resonance

Stephen Hearty, Paul Leonard, and Richard O’Kennedy               

25. Affinity Determination of Biotinylated Antibodies by Flow Cytometry

Klervi Even-Desrumeaux and Patrick Chames       

26. Affinity Maturation of Antibodies: Optimized Methods to Generate High Quality scFv Libraries and Isolate IgG Candidates by High Throughput Screening

Laurence Renaut, Céline Monnet, Olivier Dubreuil, Ouafa Zaki, Fabien Crozet, Khalil Bouayadi, Hakim Kharrat and Philippe Mondon   

27. Affinity Maturation by Semi Rational Approaches  

Rodrigo Barderas, Johan Desmet, Philippe Alard, and J. Ignacio Casal       

28. Molecular Scanning Mutagenesis: Combining Ribosome Display, Random Mutagenesis and Bioinformatic Analysis for Protein Engineering

Alfredo Darmanin-Sheehan, William James Jonathan Finlay Orla Cunningham, and Brian Joseph Fennell    

Part VII. Fc Engineering

29. Fucose-Targeted Glyco-Engineering of Pharmaceutical Cell Lines

Christiane Ogorek, Ingo Jordan, Volker Sandig, and Hans Henning von Horsten

30. Fc engineering: Design, expression and functional characterization of antibody variants with improved effector function

Stefanie Derer, Christian Kellner, Sven Berger, Thomas Valerius and Matthias Peipp  

31. Fc engineering: Serum half life modulation through FcRn binding

Tove Olafsen                          

32. Monoclonal Antibody Lead Characterization - In vitro and in vivo methods

Axel Hernandez, Julie Parmentier, Youzhen Wang, Jane Cheng and Gadi Gazit Bornstein 

Part VIII. Innovative Formats

33. Production, generation and characterization of scFv TNF ligand fusion proteins

Andrea Fick, Agnes Wyzgol and Harald Wajant     

34. Antibody-IL-2 fusion proteins for tumor targeting

Andreas A. Hombach and Hinrich Abken   

35. Recombinant immunotoxins containing low endotoxins for clinical and animal studies

Masanori Onda

36. Chimeric antigen receptors for T-cell based therapy

Eleanor J. Cheadle, Vicky Sheard, Andreas A. Hombach, Markus Chmielewski, Tobias Riet, Cor Berrevoets, Erik Schooten, Cor Lamers, Hinrich Abken, Reno Debets, David E. Gilham              

37. Selection and Use of Intracellular Antibodies (Intrabodies)

Sandrine Moutel, Clément Nizak, and Franck Perez     

38. Radiolabeled Antibodies for Imaging and Therapy 

Jacques Barbet, Manuel Bardiès, Mickael Bourgeois, Jean-François Chatal, Michel Chérel, François Davodeau, Alain Faivre-Chauvet, Jean-François Gestin, and Françoise Kraeber-Bodéré 

39. Design and Production of Multimeric Antibody Fragments, Focused on Diabodies with Enhanced Clinical Efficacy

Glenn A. Powers, Peter J. Hudson, and Michael P. Wheatcroft                                

40. Production of Bispecific Antibodies: Diabodies and Tandem scFv

Nora Hornig and Aline Färber-Schwarz   

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts

Includes supplementary material: sn.pub/extras