Antibody Methods and Protocols

326 Pages·2012·3.85 MB·English

by  Hilmar Ebersbach

M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Antibody Methods and Protocols
Edited by
Gabriele Proetzel
The Jackson Laboratory, Bar Harbor, ME, USA
Hilmar Ebersbach
NIBR Biologics Center, Novartis Institutes for BioMedical Research, Basel, Switzerland
Editors
Gabriele Proetzel Hilmar Ebersbach
The Jackson Laboratory NIBR Biologics Center
Bar Harbor, ME, USA Novartis Institutes for BioMedical Research
Basel, Switzerland
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-61779-930-3 ISBN 978-1-61779-931-0 (eBook)
DOI 10.1007/978-1-61779-931-0
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2012938221
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Preface
Antibodies play such a central role in research and development due to their high versatility
and universal applicability that research without them would be inconceivable. Their use
ranges from protein localization, cell separation, and screening to functional assays being
applied in many formats including high-throughput assays. With the breakthrough in the
generation of monoclonal antibodies, the practical potential of antibodies due to their
almost-designer specifi cities was immediately recognized, especially regarding their applica-
tions in diagnostics and therapeutics. However, surprisingly it still took until the 1990s for
antibodies to begin to make a substantial impact in drug development and to be applied as
effective therapeutics. T he industry has since grown into a multibillion-dollar market, and
34 therapeutic antibodies have been FDA approved, most of them still being on the market
in 2012. Additionally, literally hundreds of antibodies are now in the development pipeline.
These are being generated by a variety of platforms incorporating many technical enhance-
ments, such as improved half-life and effector functions. This rapidly growing fi eld is the
result of many advancing technologies allowing current developments to take advantage of
molecular engineering to create tailor-made antibodies. New antibody formats and scaf-
folds are being explored, exemplifi ed by bispecifi cs and antibody drug conjugates.
This volume, Antibody Methods and Protocols , attempts to provide insight into the gen-
eration of antibodies using in vitro and in vivo approaches, as well as technical aspects for
screening, analysis, and modifi cation of antibodies and antibody fragments. Even though
this volume covers subjects as diverse as classical methods, such as hybridoma technology
and phage display, to the more recent developments including Fc engineering, it is still
beyond the scope of any single volume to present the multitude of techniques now available
for antibody isolation, screening, and modifi cation. Instead, we have focused on basic pro-
tocols for isolating antibodies and, at the same time, selected a range of specifi c areas with
the aim of providing guides for the overall process of antibody isolation and characteriza-
tion as well as protocols for enhancing classical antibodies and antibody fragments. The
antibody process begins with antigen generation and presentation; this is discussed in the
fi rst chapter. An overview of in vitro approaches is presented in the chapters by Ron Geyer,
Dev Sidhu, Konstantin Petropoulos, Christoph Rader, Georg Thom, and Elisabetta
Traggiai. These cover phage display, ribosome display, as well the use of human B cells for
antibody isolation. Chapters by E-Chiang Lee, Michel Cogné, and Chonghui Zhang dis-
cuss the usefulness of mice in the development of antibodies, in particular genetically engi-
neered mice to develop human and humanized antibodies directly in the mouse. We touch
upon biophysical and biochemical characterization, affi nity measurements by surface plas-
mon resonance, and glycosylation analysis with chapters by Michael Schräml and Christiane
Jäger. Further, we have included a description of antibody fragments, cloning approaches,
and modi fi cation by pegylation presented in chapters by Christoph Rader and Simona
Jevševar. More recent developments in the fi eld of antibody engineering addressing half-life
extension, effector function modulation, and the rising fi eld of bispecifi c antibodies, as well
as approaches for antibody decoration including antibody drug conjugates, are covered in
v
vi Preface
chapters by Ulrich Brinkmann, Gloria Meng, and Michel Cogné. While we cannot address
all the new and exciting developments in this fast-developing fi eld, we believe that this volume
provides a broad and useful background to support ongoing efforts and encourages the
development of new imaginative approaches.
The assembly of this volume would not have happened without the commitment of all
contributors, their discussions and rapid responses, making this a valuable and relevant
contribution to antibody methods and protocols. We are also grateful to Dr. Michael V. Wiles
for his help in editing of this volume and providing many dinners.
We like to thank Dr. John Walker for the opportunity to assemble this work and his
encouragement and help throughout the process. We wish to thank also the team from
Humana Press, especially David Casey, for continuous support.
We hope that this volume will provide useful insights for both experts and novices and
that it will stimulate further development of antibody approaches and encourage the com-
munity to continuously share ideas and protocols.
Bar Harbor, ME, USA Gabriele Proetzel
Basel, Switzerland Hilmar Ebersbach
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Antigen Presentation for the Generation of Binding Molecules . . . . . . . . . . . . 1
Hilmar Ebersbach, Gabriele Proetzel, and Chonghui Zhang
2 Recombinant Antibodies and In Vitro Selection Technologies. . . . . . . . . . . . . 11
C. Ronald Geyer, John McCafferty, Stefan Dübel,
Andrew R.M. Bradbury, and Sachdev S. Sidhu
3 Phage Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Konstantin Petropoulos
4 Generation of Human Fab Libraries for Phage Display . . . . . . . . . . . . . . . . . . 53
Christoph Rader
5 Selection of Human Fab Libraries by Phage Display . . . . . . . . . . . . . . . . . . . . 81
Christoph Rader
6 Ribosome Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
George Thom and Maria Groves
7 Hybridoma Technology for the Generation of Monoclonal Antibodies . . . . . . 117
Chonghui Zhang
8 The Application of Transgenic Mice for Therapeutic
Antibody Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
E-Chiang Lee and Michael Owen
9 Production of Human or Humanized Antibodies in Mice . . . . . . . . . . . . . . . . 149
Brice Laffleur, Virginie Pascal, Christophe Sirac, and Michel Cogné
10 Immortalization of Human B Cells: Analysis of B Cell Repertoire
and Production of Human Monoclonal Antibodies. . . . . . . . . . . . . . . . . . . . . 161
Elisabetta Traggiai
11 Kinetic Screening in the Antibody Development Process. . . . . . . . . . . . . . . . . 171
Michael Schräml and Matthias Biehl
12 Temperature-Dependent Antibody Kinetics as a Tool
in Antibody Lead Selection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Michael Schräml and Leopold von Proff
13 Determination of Antibody Glycosylation by Mass Spectrometry. . . . . . . . . . . 195
Christiane Jäger, Claudia Ferrara, Pablo Umaña, Anne Zeck,
Jörg Thomas Regula, and Hans Koll
14 Cloning, Expression, and Purification of Monoclonal
Antibodies in scFv-Fc Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Jiahui Yang and Christoph Rader
vii
viii Contents
15 PEGylation of Antibody Fragments for Half-Life Extension . . . . . . . . . . . . . . 233
Simona Jevševar, Mateja Kusterle, and Maja Kenig
16 Bispecific Antibody Derivatives Based on Full-Length IgG Formats. . . . . . . . . 247
Michael Grote, Alexander K. Haas, Christian Klein,
Wolfgang Schaefer, and Ulrich Brinkmann
17 Generation of Fluorescent IgG Fusion Proteins in Mammalian Cells . . . . . . . . 265
Alexander K. Haas, Klaus Mayer, and Ulrich Brinkmann
18 Methods to Engineer and Identify IgG Variants with Improved
1
FcRn Binding or Effector Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Robert F. Kelley and Y. Gloria Meng
19 Class-Specific Effector Functions of Therapeutic Antibodies . . . . . . . . . . . . . . 295
Virginie Pascal, Brice Laffleur, and Michel Cogné
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Contributors
MATTHIAS BIEHL (cid:129) Roche Diagnostics GmbH, Penzberg, Germany
ANDREW R.M. BRADBURY (cid:129) Los Alamos National Laboratory , Los Alamos , NM , USA
ULRICH BRINKMANN (cid:129) Large Molecule Research, Roche Pharma Research
and Early Development, Penzberg, Germany
MICHEL COGNÉ (cid:129) CNRS UMR6101, Contrôle des Réponses Immunes
B et Lymphoproliférations, Université de Limoges, Limoges , France
STEFAN DÜBEL (cid:129) Technische Universität Braunschweig, Braunschweig, Germany
HILMAR EBERSBACH (cid:129) NIBR Biologics Center, Novartis Institutes for BioMedical
Research , Basel , Switzerland
CLAUDIA FERRARA (cid:129) pRED, Pharma Research and Early Development,
Roche Glycart AG, Schlieren, Switzerland
C. RONALD GEYER (cid:129) University of Saskatchewan, Saskatoon , SK , Canada
MICHAEL GROTE (cid:129) Large Molecule Research, Roche Pharma Research and Early
Development, Penzberg, Germany
MARIA GROVES (cid:129) MedImmune Ltd, Cambridge, UK
ALEXANDER K. HAAS (cid:129) Large Molecule Research, Roche Pharma Research
and Early Development, Penzberg, Germany
CHRISTIANE JÄGER (cid:129) pRED, Pharma Research and Early Development,
Roche Glycart AG, Schlieren, Switzerland
SIMONA JEVŠEVAR (cid:129) Sandoz Biopharmaceuticals, Mengeš, Lek Pharmaceuticals d.d,
Mengeš , Slovenia
ROBERT F. KELLEY (cid:129) Antibody Engineering, Genentech Inc, South San Francisco,
CA , USA
MAJA KENIG (cid:129) Sandoz Biopharmaceuticals, Mengeš, Lek Pharmaceuticals d.d,
Mengeš , Slovenia
CHRISTIAN KLEIN (cid:129) Large Molecule Research, Roche Pharma Research
and Early Development, Schlieren, Switzerland
HANS KOLL (cid:129) pRED, Pharma Research and Early Development,
Roche Diagnostics GmbH, Penzberg, Germany
MATEJA KUSTERLE (cid:129) Sandoz Biopharmaceuticals, Mengeš, Lek Pharmaceuticals d.d,
Mengeš , Slovenia
BRICE LAF FL EUR (cid:129) CNRS UMR6101, Contrôle des Réponses Immunes
B et Lymphoproliférations, Université de Limoges, Limoges , France
E-CHIANG LEE (cid:129) Kymab Ltd, Meditrina, Cambridge, UK
KLAUS MAYER (cid:129) Large Molecule Research, Roche Pharma Research and Early
Development, Penzberg, Germany
JOHN MCCAFFERTY (cid:129) University of Cambridge, Cambridge, UK
ix
x Contributors
Y. GLORIA MENG (cid:129) Biochemical and Cellular Pharmacology , Genentech Inc,
South San Francisco, CA , USA
MICHAEL OWEN (cid:129) Kymab Ltd, Meditrina, Cambridge, UK
VIRGINIE PASCAL (cid:129) CNRS UMR6101, Contrôle des Réponses Immunes
B et Lymphoproliférations, Université de Limoges, Limoges , France
KONSTANTIN PETROPOULOS (cid:129) MorphoSys AG, Martinsried/Planegg, Germany
GABRIELE PROETZEL (cid:129) The Jackson Laboratory , Bar Harbor , ME , USA
LEOPOLD VON PROFF (cid:129) Roche Diagnostics GmbH, Penzberg, Germany
CHRISTOPH RADER (cid:129) Experimental Transplantation and Immunology Branch,
Center for Cancer Research, National Cancer Institute, National Institutes
of Health, Bethesda , MD, USA; Department of Cancer Biology
and Department of Molecular Therapeutics, The Scripps Research Institute,
Scripps Florida Jupiter, FL, USA
JÖRG THOMAS REGULA (cid:129) pRED, Pharma Research and Early Development,
Roche Diagnostics GmbH, Penzberg, Germany
WOLFGANG SCHAEFER (cid:129) Large Molecule Research, Roche Pharma Research
and Early Development, Penzberg, Germany
MICHAEL SCHRÄML (cid:129) Roche Diagnostics GmbH, Penzberg, Germany
SACHDEV S. SIDHU (cid:129) University of Toronto , Toronto , ON , Canada
CHRISTOPHE SIRAC (cid:129) CNRS UMR6101, Contrôle des Réponses Immunes
B et Lymphoproliférations, Université de Limoges, Limoges , France
GEORGE THOM (cid:129) MedImmune Ltd, Cambridge, UK
ELISABETTA TRAGGIAI (cid:129) Translational Science, Novartis Institute
for Biomedical Research, Basel , Switzerland
PABLO UMAÑA (cid:129) pRED, Pharma Research and Early Development,
Roche Glycart AG, Schlieren, Switzerland
JIAHUI YANG (cid:129) Experimental Transplantation and Immunology Branch,
Center for Cancer Research, National Cancer Institute, National Institutes
of Health, Bethesda , MD, USA
ANNE ZECK (cid:129) NMI, Naturwissenschaftliches und Medizinisches Institut,
Universität Tübingen, Reutlingen, Germany
CHONGHUI ZHANG (cid:129) NIBR Biologics Center, Novartis Institutes for BioMedical
Research , Cambridge, MA , Switzerland