Antibody Engineering Protocols

Methods in Molecular Biology ' Series John M. WalkeVy SERIES EDITOR

35. Plant Cell Electroporation and Eledrofusion Protocols, edited by Jac A. Nkkoloff. 1995

54. YAC Protocols, edited by David Markie, 1995 53. Yeast Protocols: Methods in Cell and Molecular Biology,

edited by Ivor H. Evans, 1995 52. Capillary Electrophoresis: Principles. Inslrumenlalion,

and Applications, edited by Kevin D. Altria, 1995 51. Antibody Engineering Protocols, edited by SudhirPaul,

1995 50. Species Diagnostics Protocols: PCR and Other Nucleic

Acid Methods, edited by Justin P. Clapp, 1995 49. Plant Gene Transfer and Expression Protocols,

edited by Heddwyn Jones, 1995 48. Animal Cell Electroporation and Electrofusion Proto­

cols, edited by Jac A. Nickoloff, 1995 47. Electroporation Protocols for Microorganisms, edited by

Jac A. Nickoloff, 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny

Hoviard and David M. Whitcombe, 1995

45. Monoclonal Antibody Protocols, edited by William C.

Davis, 1995 44. Agrobacterium Protocols, edited by Kevan U. A. Gartland

and Michael R. Davey, 1995 43. In Vitro Toxicity Testing Protocols, edited by Sheila

O'Hare and Chris K. Atterwill, 1995 42. ELISA: Theory and Practice, by John R. Crowther, 1995 41. Signal Transduction Protocols, edited by OaviW.4. ATcniia//

and Stephen J Hill, 1995 40. Protein Stability and Folding: Theory and Practice,

edited by Bret A. Shirley, 1995 39. Baculovirus Expression Protocols, edited by Christopher

D. Richardson, 1995 38. Cryopreservation and Freeze-Drying Protocols,

edited by John G. Day and Mark R. McLellan, 1995 37. In Vitro Transcription and Translation Protocols,

edited by Martin J. Tymms, 1995 36. Peptide Analysis Protocols, edited by Ben M. Dunn and

Michael W. Pennington, 1994

35. Peptide Synthesis Protocols, edited by Michael W. Pennington and Ben M. Dunn, 1994

34. Immunocytochemicai Methods and Protocols, edited by lorette C. Javois, 1994

33. In Situ Hybridization Protocols, edited by K. H. Andy Choo, 1994

32. Basic Protein and Peptide Protocols, edited by John M.

Walker, 1994

31. Protocols for Gene Analysis, edited by Adrian J. Harwood,

1994

30. DNA-ProteIn Interactions, edited by G. Geoff Kneale, 1994

29. Chromosome Analysis Protocols, edited by John R. Gosden, 1994

28. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, edited by Peter G. Isaac, 1994

27. Biomembrane Protocols: //. Architecture and Function, edited by John M. Graham and Joan A. Higgins, 1994

26. Protocols for Oligonucleotide Conjugates: Synthesis and Analytical Techniques, edited by Sudhir Agrawal, 1994

25. Computer Analysis of Sequence Data: Part II, edited by Annette M. Griffin and Hugh G. Griffin, 1994

24. Computer Analysis of Sequence Data: Part 1, edited by Annette M. Griffin and Hugh G. Griffin, 1994

23. DNA Sequencing Protocols, edited by Hugh G. Griffin and Annette M. Griffin, 199S

22, Microscopy, Optical Spectroscopy, and Macroscopic Techniques, edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1993

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993

20. Protocols for Oligonucleotides and Analogs: Synthesis and Properties, edited by Sudhir Agraval, 1993

19. Biomembrane Protocols: /. Isolation and Analysis, edited by John M. Graham and Joan A. Higgins, 1993

18. Transgenesis Techniques: Principles and Protocols, edited by David Murphy and David A. Carter, 1993

17. Spectroscopic Methods and Analyses: NMR, Mass Spec­trometry, and Metalloprotein Techniques, edited by Chris­topher Jones, Barbara Mulloy, and Adrian H. Thomas. 1993

16. Enzymes of Molecular Biology, edited by Michael M. Burrell. 1993

15. PCR Protocols: Current Methods and Applications, edited by Bruce A. White, 1993

14. Glycoprotein Analysis In Biomedicine, edited by Eliza­beth F. Hounsell, 1993

13. Protocols in Molecular Neurobiology, edited by Alan Longstaff and Patricia Revest, 1992

12. Pulsed-Field Gel Electrophoresis: Protocols, Methods, and Theories, edited by Margit Burmeister and Levy Ulanovsky, 1992

II. Practical Protein Chromatography, edited by Andrew Kenney and Susan Fov/ell, 1992

10. Immunochemical Protocols, edited by Margaret M. Manson, 1992

9. Protocols in Human Molecular Genetics, edited by Chris­topher G. Mathew, 1991

8. Practical Molecular Virology: Viral Vectors for Gene Expression, edited by Mary K. I. Collins, 1991

7. Gene Transfer and Expression Protocols, edited by Edward J. Murray, 1991

6. Plant Cell and Tissue Culture, edited by Jeffrey W. Pollard and John M. Walker, 1990

5. Animal Cell Culture, edited by ye/f re)' W. Pollard and John M. Walker. 1990

Methods in Molecular Biology™ • S I

Antibody Engineering

Protocols

Edited by

Sudhir Paul University of Nebraska Medical Center, Omaha, NE

H u m a n a P r e s s ^ f e Totowa, New Jersey

© 1995 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

All rights reserved.

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Main entry under title:

Methods in molecular biology'".

Antibody engineering protocols / edited by Sudhir Paul, p. cm. ~ (Methods in molecular biology'" ; 51)

Includes index. ISBN 0-89603-275-2 (alk. paper) 1. Immunoglobulins-Biotechnology. 2. Protein engineering.

I. Paul, Sudhir. II. Series: Methods in molecular biology'" (Totowa, NJ); 51. TP248.65.149A585 1995 616.07'93-dc20 95-423

CIP

Preface

Antibodies are the instraments of immune defense and attack. They can bind small atomic arrays as well as large epitopes with high affinity. Antibody Engineering Protocols presents advanced protocols in the field of antibody engineering, reviews of basic principles and methodology, and a historical perspective on the development of cur­rently held beliefs about antibody structure-function relationships. The topics cover analysis of antibody sequences, three-dimensional structure, delineation of antibody characteristics in polyclonal mixtures, phage display of natural and synthetic antibodies, and antibody catalysis.

Ligand recognition by antibodies occurs primarily at a subset of amino acid residues located in the complementarity determining regions (CDRs) found in the light (L) and heavy (H) chain subunits. Specific antibodies are developed by immunization with molecules identical to or related in structure to the target ligand. The immune repertoire from nonimmunized individuals also contains pre-existing specificities that can be selected by screening libraries composed of hybridoma cells or phage particles displaying F domains or indi­vidual variable domains of the light (VJ and heavy (V ) chains. Ran­dom or site-directed mutagenesis in vitro can be used to refine the pre-existing specificities or produce new specificities de novo. Another level at which new specificities may be generated in vitro is V^ and V^ domain-swapping and CDR-swapping. The former procedure embodies a variation of natural mechanisms of generating antibody diversity. The latter procedure produces new intramolecular CDR combinations not found in nature. Application of anti-idiotypic imagery can also be utilized to replicate and modify the active site of antibodies and enzymes.

In the biological sciences, many conceptual advances have been driven by new methodology. In the case of antibody research, recent

vi Preface

methodological developments make possible mechanistic study of the role of antibodies in immune defense and autoimmune disease, and encourage the belief that antibody fragments will find important uses in industry and medicine. In particular, the discovery of cataly­sis by natural antibodies offers simple means to derive new catalysts from the immune response. Inmiunization with transition-state and charged-substrate analogs has previously been observed to generate esterolytic and amidolytic antibodies. Combined with the natural pro­pensity of the immune system to produce catalytic antibodies, the "nudge" toward catalysis by substrate and transition state analog immunizations may permit development of efficient catalysts.

I thank each of the authors for painstaking descriptions of the nuts and bolts of their techniques. Donna Dutch and Angela Plendl typed many of the manuscripts, scanned them into the computer, and generally helped in generating a comprehensible text, for which I am in their debt. I thank John Walker for editorial guidance and Humana Press for producing this volume.

Sudhir Paul

Contents

Preface v

Contributors ix

Ch. 1. SEQHUNT: A Program to Screen Aligned Nucleotide and Amino Acid Sequences,

George Johnson, Tai Te Wu, and Elvin A. Rabat / Ch. 2. Molecular Modeling of Antibody-Combining Sites,

David M. Webster and Anthony R. Rees 17 Ch. 3. Structure and Properties of Human Immunoglobulin Light-Chain Dimers,

Fred J. Stevens and Marianne Schiffer 51 Ch. 4. Crystallographic and Chromatographic Methods for Study

of Antibody Light Chains and Other Proteins, Marianne Schiffer and Fred J. Stevens 83

Ch. 5. Detection of Human Variable Gene Family Expression at the Single-Cell Level,

MoncefZouali 99 Ch. 6. Purification of Reduced and Alkylated Antibody Subunits,

Mei Sun and Sudhir Paul / / / Ch. 7. Murine Monoclonal Antibody Development,

Donald R. Johnson 123 Ch. 8. ComparativePropertiesof Polyclonal and Monoclonal Antibodies,

L. Scott Rodkey 139 Ch. 9. Evaluation of Antibody Clonality,

L. Scott Rodkey 151 Ch. 10. Affinity Immunoblotting,

Keith A. KnisleyandL. Scott Rodkey 165 Ch. 11. Epitope and Idiotope Mapping Using Monoclonal Antibodies,

Srinivas Kaveri 171 Ch. 12. Anti-Idiotypic Antibodies That Mimic Opioids,

Jay A. Glasel and Dianne Agarwal 183 Ch. 13. CaX&Xyiic Aniiho&its: Structure and Possible Applications,

Howard Amital, Ilan Tur-Kaspa, Zeev Tashma, Israel Hendler, and Yehuda Shoenfeld 203

Ch. 14. Preparation and Assay of Acetylcholinesterase Antibody, Alain Friboulet and Ladan Iz/adyar-Demichele 211

vii

via Contents

Ch. 15. DNA Hydrolysis by Antibodies, Alexander G. Gabibov and Oxana Makarevitch 223

Ch. 16. Screening Strategies for Catalytic Antitransition-State Analog Antibodies, Alfonso Tramontano 237

Ch. 17. Expression of Chimeric Immunoglobulin Genes in Mammalian Cells, Sergey M. Deyev and Oleg L. Polanovsky 251

Ch. 18. Single-Chain Anti-DNA F^, Michael Polymenis and B. David Stollar 265

Ch. 19. Molecular Cloning of Antiground-State Proteolytic Antibody Fragments, Qing-Sheng Gao and Sudhir Paul 281

Ch. 20. Cloning and Bacterial Expression of an Esterolytic sF^, Rodger G. Smith, Mark T. Martin, Rosa Sanchez,

and John H. Kenten 297 Ch. 21. Site-Directed Mutagenesis of Antibody-Variable Regions,

Qing-Sheng Gao and Sudhir Paul 319 Ch. 22. Synthetic Antibody Gene Libraries for In Vitro Affinity Maturation,

Su-jun Deng, C. Roger MacKenzie, and SaranA. Narang 329 Ch. 23. Chaperonins in Phage Display of Antibody Fragments,

Eskil Soderlind, Marta Duenas, and Carl A. K. Borrebaeck... 343 Ch. 24. Phage-Display Libraries of Murine and Human Antibody Fab Fragments,

Jan Engberg, Peter Sejer Andersen, Leif Kofoed Nielsen, Morten Dziegiel, Lene K. Johansen, and Bjarne Albrechtsen 355

Ch. 25. Selection of Human Immunoglobulin Light Chains from a Phage-Display Library,

Sonia Tyutyulkova, Qing-Sheng Gao, and Sudhir Paul 377 Ch. 26. Purification of Antibody Light Chains by Metal Affinity

and Protein L Chromatography, Sonia Tyutyulkova and Sudhir Paul 395

Ch. 27 Rapid Purification of Recombinant Antibody Fragments for Catalysis Screening,

Han Huang, Brian Fichter, Robert Dannenbring, and Sudhir Paul 403

Ch. 28. Assay of Radiolabeled VIP Binding and Hydrolysis by Antibodies, Han Huang and Sudhir Paul 409

Ch. 29 Methods of Measuring Thyroglobulin and Peptide-Methylcoumarinamide Hydrolysis by Autoantibodies,

Lan Li, Ravishankar Kalaga, Srinivas Kaveri, and Sudhir Paul 417

Ch. 30. Radiolabeling of Antibodies for Therapy and Diagnosis, Janina Baranowska-Kortylewicz, Glenn V. Dalrymple,

Syed M. Quadri, and Katherine A. Harrison 423

Index 441

Contributors

DiANNE AGARWAL • Department of Biochemistry, University of Connecticut Health Center, Farmington, CT

BJARNE ALBRECHTSEN • Department of Biology, Danmarks Farmaceutiske H0jskole, Copenhagen, Denmark

HOWARD AMITAL • Department of Medicine 'B' and Research Unit of Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University

PETER SEJER ANDERSEN • Department of Biology, Royal Danish School of Pharmacy, Copenhagen, Denmark

JANINA BARANOWSKA-KORTYLEWICZ • Department of Radiology, University of Nebraska Medical Center, Omaha, NE

CARL A. K. BORREBAECK • Department of Immunotechnology, Lund University, Lund, Sweden

GLENN V. DALRYMPLE • Department of Radiology, University of Nebraska Medical Center, Omaha, NE

ROBERT DANNENBRING • Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE

SU-JUN DENG • Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada

SERGEY M . DEYEV • Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

MARTA DUENAS • Department of Immunotechnology, Lund University, Lund, Sweden

MORTEN DZIEGIEL • Department of Biology, Royal Danish School of Pharmacy, Copenhagen, Denmark

JAN ENGBERG • Department of Biology, Royal Danish School of Pharmacy, Copenhagen, Denmark

BRIAN FIGHTER • Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE

ix

jc Contributors

ALAIN FRIBOULET • Laboratoire de Technologie Enzymatique, Universite de Technologie de Compiegne, France

ALEXANDER G . GABIBOV • Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

QING-SHENG GAO • Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE

JAY A . GLASEL • Department of Biochemistry, University of Connecticut Health Center, Farmington, CT

KATHERINE A . HARRISON • Department of Radiology, University

of Nebraska Medical Center, Omaha, NE ISRAEL HENDLER • Israeli Defense Forces-Medical Corps, Tel-Aviv, Israel HAN HUANG • Department of Anesthesiology, University

of Nebraska Medical Center, Omaha, NE LADAN IZADYAR-DEMICH6LE • Laboratoire de Technologie

Enzymatique, Universite de Technologie de Compiegne, France

LENE K . JOHANSEN • Department of Biology, Royal Danish School of Pharmacy, Copenhagen, Denmark

DONALD R . JOHNSON • Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE

GEORGE JOHNSON • Department of Biochemistry and Molecular Cell Biology, Northwestern University, Evanston, IL

ELVIN A . KABAT • Department of Microbiology, Columbia University, New York, NY

RAVISHANKAR KALAGA • Department of Anesthesiology, University

of Nebraska Medical Center, Omaha, NE SRINIVAS KAVERI • INSERM U28, Hopital Broussais, Paris, France JOHN H . KENTEN • Proneuron, Inc., Rockville, MD KEITH A. KNISLEY • Department of Cell Biology and Biochemistry,

Texas Tech University Health Science Center, Lubbock, TX LAN L I • Department of Anesthesiology, University of Nebraska

Medical Center, Omaha, NE C. ROGER MACKENZIE • Institute for Biological Sciences, National

Research Council of Canada, Ottawa, Ontario, Canada OxANA MAKAREVITCH • Engelhardt Institute of Molecular Biology,

Russian Academy of Sciences, Moscow, Russia

Contributors xi

MARK T. MARTIN • IGEN Inc., Rockville, MD SARAN A. NARANG • Institute for Biological Sciences, National

Research Council of Canada, Ottawa, Ontario, Canada LEIF KOFOED NIELSEN • Department of Biology, Royal Danish

School of Pharmacy, Copenhagen, Denmark SuDHiR PAUL • Department of Anesthesiology, University

of Nebraska Medical Center, Omaha, NE OLEG L. POLANOVSKY • Engelhardt Institute of Molecular Biology,

Russian Academy of Sciences, Moscow, Russia MICHAEL POLYMENIS • Department of Biochemistry, Tufts University

School of Medicine, Boston, MA SYED M . QUADRI • Department of Radiology, University

of Nebraska Medical Center, Omaha, NE ANTHONY R. REES • School of Biology and Biochemistry, University

of Bath, UK L. SCOTT RODKEY • Department of Pathology, University of Texas-

Houston Medical School, Houston, TX ROSA SANCHEZ • IGEN Inc., Rockville, MD MARIANNE SCHIFFER • Center for Mechanistic Biology

and Biotechnology, Argonne National Laboratory, Argonne, IL YEHUDA SHOENFELD • Department of Medicine 'B' and Research

Unit of Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University

RODGER G. SMITH • IGEN Inc., Rockville, MD ESKIL SODERLIND • Department of Immunotechnology, Lund University,

Lund, Sweden FRED J. STEVENS • Center for Mechanistic Biology and

Biotechnology, Argonne National Laboratory, Argonne, IL B. DAVID STOLLAR • Department of Biochemistry, Tufts University

School of Medicine, Boston, MA MEI SUN • Department of Anesthesiology, University of Nebraska

Medical Center, Omaha, NE ZEEV TASHMA • Israeli Defense Forces-Medical Corps, Tel-Aviv,

Israel ALFONSO TRAMONTANO • IGEN Research Institute, Rockville, MD ILAN TUR-KASPA • Israeli Defense Forces-Medical Corps, Tel-Aviv, Israel

xii Contributors

SoNiA TYUTYULKOVA • Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE

DAVID M . WEBSTER • School of Biology and Biochemistry, University of Bath, UK

TAI TE WU • Department of Biochemistry and Molecular Cell Biology, Northwestern University, Evanston, IL

MoNCEF ZouALi • Dcpartemcnt d'Immunologie, Institut Pasteur, Paris, France