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Methods in
Molecular Biology 1827
Damien Nevoltris
Patrick Chames Editors
Antibody
Engineering
Methods and Protocols
Third Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Antibody Engineering
Methods and Protocols
Third Edition
Edited by
Damien Nevoltris
Garvan Institute of Medical Research, Immunology Division, Darlinghurst, NSW, Australia
Patrick Chames
Institut Paoli-Calmettes, CRCM, Aix Marseille University, CNRS, INSERM, Marseille, France
Editors
Damien Nevoltris Patrick Chames
Garvan Institute of Medical Research Institut Paoli-Calmettes
Immunology Division CRCM, Aix Marseille University, CNRS, INSERM
Darlinghurst, NSW, Australia Marseille, France
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8647-7 ISBN 978-1-4939-8648-4 (eBook)
https://doi.org/10.1007/978-1-4939-8648-4
Library of Congress Control Number: 2018950851
© Springer Science+Business Media, LLC, part of Springer Nature 2018

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Preface
The use of antibody-based therapeutics has grown exponentially in the past few decades,
now representing a large component of therapeutic drugs that was dominated by small
organic molecules up until the late 1990s. Antibodies have proven versatile in treating a
variety of diseases including cancer, autoimmunity, infectious diseases, or even neurodegen-
erative disorders. As of 2017, 70 therapeutic antibodies have been approved by the FDA,
and more than 550 promising candidates are in different phases of clinical trials. They
currently represent 20% of the top 100 selling drugs, up from just 1% in 2007. However,
major improvements and breakthroughs have been necessary to achieve these impressive
results.
The 1970s were the start of great revolutions in the field: Gerald Edelman and Rodney
Porter were awarded the Nobel Prize for their work on the molecular structure of
antibodies, the first atomic resolution structure of an antibody fragment was published,
followed by the groundbreaking development of hybridoma technology by Georges
J. F. Köhler and César Milstein. This technology allowed antibodies to be produced and
characterized as monoclonals, starting the modern era of antibody engineering.
Despite this revolution, the success of antibodies as therapeutic molecules was not
immediate, and most clinical studies led to disappointments. First murine antibodies used
as treatment had many limitations, such as a short in vivo half-life, limited tumor penetra-
tion, inefficient recruitment of host effector functions, and most of all, immune response
from the patient against the injected antibody, also called “HAMA” response, referring to
the production of neutralizing human anti-mouse antibodies.
For many years, researchers developed strategies to abrogate this problem; the journey
toward antibody humanization began. Fully murine antibodies first progressed to chimeras,
where variable regions from murine origin were assembled onto human constant domains,
then to humanized antibodies by insertion of only the relevant CDRs onto human antibody
scaffolds. Finally, fully human antibodies were generated, directly in genetically modified
mice, selected from human synthetic antibody libraries or by sequencing of human plasma
cells.
However, immunogenicity was not the only factor holding up the development of
antibodies. Indeed, the classical architecture of immunoglobulin molecules bears some
inherent limitations. Many innovative formats have been explored to overcome these
major hurdles, such as reducing the antibody to its minimal functional size, modulating
the valency, the (multi) specificity, increasing the half-life, and enhancing the recruitment of
immune effector cells.
As the demand for monoclonal antibodies in research and clinical applications continues
to increase, the necessity to develop even more efficient molecules is crucial. Antibody
engineering has become a key discipline for generation of innovative antibodies-based
molecules used in research, diagnostics, and therapy.
v
vi Preface
This third edition of Antibody Engineering: Methods and Protocols remains in the lineage
of its predecessors and gives the readers complete and easy access to a variety of antibody
engineering techniques. From the generation of native, synthetic, or immune antibody
libraries, the selection of lead candidates thanks to different powerful and innovating
display technologies, to their production, characterization, and optimization, this handbook
provides the reader with an extensive toolbox to create the powerful molecules of tomorrow.
Darlinghurst, NSW, Australia Damien Nevoltris

Marseille, France Patrick Chames
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I IN SILICO METHODS

1 Antibody Design and Humanization via In Silico Modeling . . . . . . . . . . . . . . . . . . 3
Vinodh B. Kurella and Reddy Gali
2 Antibody Affinity Maturation by Computational Design . . . . . . . . . . . . . . . . . . . . . 15
Daisuke Kuroda and Kouhei Tsumoto
3 Use of IMGT® Databases and Tools for Antibody Engineering
and Humanization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Marie-Paule Lefranc, François Ehrenmann, Sofia Kossida,
Véronique Giudicelli, and Patrice Duroux
PART II GENERATION OF DIVERSITY
4 Construction of Human Naı̈ve Antibody Gene Libraries . . . . . . . . . . . . . . . . . . . . . 73

Michela Pasello, Alessandra Mallano, Michela Flego, Silvia Zamboni,
Anna Maria Giudice, and Katia Scotlandi
5 Construction of Synthetic Antibody Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Déborah Caucheteur, Gautier Robin, Vincent Parez, and
Pierre Martineau
6 Construction of Histidine-Enriched Shark IgNAR Variable Domain
Antibody Libraries for the Isolation of pH-Sensitive vNAR Fragments . . . . . . . . 109
Doreen Könning, Steffen Hinz, Julius Grzeschik, Christian Schröter,
Simon Krah, Stefan Zielonka, and Harald Kolmar
7 Display Technologies for Generation of Ig Single Variable Domains. . . . . . . . . . . 129
Vladimir Bobkov, Bas van der Woning, and Hans de Haard
8 A Streamlined Approach for the Construction of Large Yeast
Surface Display Fab Antibody Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Simon Krah, Julius Grzeschik, Simon Rosowski, Ramona Gaa,
Iris Willenbuecher, Deniz Demir, Lars Toleikis, Harald Kolmar,
Stefan Becker, and Stefan Zielonka
PART III SELECTIONS OF LEAD CANDIDATE

9 Phage Display and Selections on Purified Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Magali Colazet and Patrick Chames
10 Selection of Antibodies to Transiently Expressed Membrane
Proteins Using Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Martina L. Jones, Stephen M. Mahler, and Sumukh Kumble
vii
viii Contents
11 Selection of Antibody Fragments Against Structured DNA by

Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Mahdi Zeraati, Marcel E. Dinger, and Daniel Christ
12 Selection of Antibody Fragments by Yeast Display . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Nathalie Scholler
13 Rapid Selection of High-Affinity Antibody scFv Fragments
Using Ribosome Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Birgit Dreier and Andreas Plückthun
14 In Vitro Selection of Single-Domain Antibody (VHH) Using
cDNA Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Naoto Nemoto, Shigefumi Kumachi, and Hidenao Arai
15 Sequencing and Affinity Determination of Antigen-Specific B
Lymphocytes from Peripheral Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Peter Schofield, Rodrigo Vazquez-Lombardi, Mahmoud Abdelatti,
Damien Nevoltris, Christopher C. Goodnow, Daniel Christ,
and Joanne H. Reed
PART IV PRODUCTION OF RECOMBINANT ANTIBODIES
16 Expression of IgG Monoclonals with Engineered Immune

Effector Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Rodrigo Vazquez-Lombardi, Damien Nevoltris,
Romain Rouet, and Daniel Christ
17 An IRES-Mediated Tricistronic Vector for Efficient Generation of
Stable, High-Level Monoclonal Antibody Producing CHO
DG44 Cell Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Jessna H. M. Yeo, Mariati, and Yuansheng Yang
18 Production, Purification, and Characterization of Antibody-TNF
Superfamily Ligand Fusion Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Martin Siegemund, Nadine Beha, and Dafne Müller
PART V FC ENGINEERING
19 Chemoenzymatic Defucosylation of Therapeutic Antibodies for
Enhanced Effector Functions Using Bacterial α-Fucosidases . . . . . . . . . . . . . . . . . 367
Chao Li, Tiezheng Li, and Lai-Xi Wang
20 Fc Glyco- and Fc Protein-Engineering: Design of Antibody
Variants with Improved ADCC and CDC Activity . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Christian Kellner, Stefanie Derer, Katja Klausz, Sophia Rosskopf,
Tim Wirt, Thies Rösner, Anna Otte, Elisa Cappuzzello,
and Matthias Peipp
21 Fc Engineering: Tailored Synthetic Human IgG1-Fc Repertoire
for High-Affinity Interaction with FcRn at pH 6.0 . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Abhishek Saxena, Bingxin Bai, Shin-Chen Hou, Lianlian Jiang,
Tianlei Ying, Shane Miersch, Sachdev S. Sidhu, and Donghui Wu
Contents ix
PART VI CHARACTERIZATION, OPTIMIZATION, AND INNOVATIVE FORMATS
22 Measuring Antibody-Antigen Binding Kinetics Using Surface

Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
Stephen Hearty, Paul Leonard, Hui Ma, and Richard O’Kennedy
23 Parallel Evolution of Antibody Affinity and Thermal Stability
for Optimal Biotherapeutic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Edward Franklin, Orla Cunningham, and Brian Fennell
24 The Use of Somatic Hypermutation for the Affinity Maturation
of Therapeutic Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Peter M. Bowers, William J. Boyle, and Robert Damoiseaux
25 Selection and Use of Intracellular Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
Sandrine Moutel, Clément Nizak, and Franck Perez
26 Site-Specific Radioactive Labeling of Nanobodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
Maxine Crauwels, Sam Massa, Charlotte Martin, Cecilia Betti,
Steven Ballet, Nick Devoogdt, Catarina Xavier, and Serge Muyldermans
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Contributors
MAHMOUD ABDELATTI Garvan Institute of Medical Research, Sydney, NSW, Australia;

Faculty of Medicine, St. Vincent’s Clinical School, University of New South Wales, Sydney,
NSW, Australia
HIDENAO ARAI Epsilon Molecular Engineering, Inc., Saitama, Japan
BINGXIN BAI Laboratory of Antibody Engineering, Shanghai Institute for Advanced
Immunochemical Studies, ShanghaiTech University, Shanghai, China
STEVEN BALLET Research Group of Organic Chemistry, Departments of Chemistry and
Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium
STEFAN BECKER Protein Engineering and Antibody Technologies, Merck KGaA, Darmstadt,
Germany
NADINE BEHA Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart,
Germany
CECILIA BETTI Research Group of Organic Chemistry, Departments of Chemistry and
VLADIMIR BOBKOV argenx BVBA, Zwijnaarde, Belgium
PETER M. BOWERS Clinical and Translational Science Institute, UCLA David Geffen
School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
WILLIAM J. BOYLE Clinical and Translational Science Institute, UCLA David Geffen
School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
ELISA CAPPUZZELLO Division of Stem Cell Transplantation and Immunotherapy,
Department of Medicine II, University Hospital Schleswig-Holstein and Christian-
Albrechts, University of Kiel, Kiel, Germany; Department of Surgery, Oncology and
Gastroenterology, Oncology and Immunology Section, University of Padua, Padua, Italy
DÉBORAH CAUCHETEUR Institut de Recherche en Cancérologie de Montpellier (IRCM),
Montpellier, France; INSERM, U1194, Montpellier, France; Université de Montpellier,
Montpellier, France; Institut Régional du Cancer de Montpellier, Montpellier, France
PATRICK CHAMES Institut Paoli-Calmettes, CRCM, Aix Marseille University, CNRS,
INSERM, Marseille, France
DANIEL CHRIST Garvan Institute of Medical Research, Sydney, NSW, Australia; Faculty of
Medicine, St. Vincent’s Clinical School, University of New South Wales, Sydney, NSW,
Australia
MAGALI COLAZET Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS,
INSERM, Institut Paoli-Calmettes, Aix Marseille University, Marseille, France
MAXINE CRAUWELS Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussel,
Belgium; In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel,
Brussels, Belgium
ORLA CUNNINGHAM Biomedicine Design, Pfizer, Dublin, Ireland
ROBERT DAMOISEAUX Department of Molecular and Medical Pharmacology, California
NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA
HANS DE HAARD argenx BVBA, Zwijnaarde, Belgium
DENIZ DEMIR Protein Engineering and Antibody Technologies, Merck KGaA, Darmstadt,
Germany
xi
xii Contributors
STEFANIE DERER Institute of Nutritional Medicine, Molecular Gastroenterology, University

Hospital Schleswig-Holstein, Campus Lübeck, Lübeck, Germany
NICK DEVOOGDT In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit
Brussel, Brussels, Belgium
MARCEL E. DINGER Garvan Institute of Medical Research, Sydney, NSW, Australia;
Faculty of Medicine, St. Vincents Clinical School, University of New South Wales, Sydney,
NSW, Australia
BIRGIT DREIER Department of Biochemistry, University of Zurich, Zurich, Switzerland
PATRICE DUROUX IMGT®, The International ImMunoGeneTics Information System®,
Laboratoire d’ImmunoGénétique Moléculaire (LIGM), Institut de Génétique Humaine
(IGH), UMR 9002 CNRS-UM, Université de Montpellier, Montpellier Cedex, France
FRANÇOIS EHRENMANN IMGT®, The International ImMunoGeneTics Information
System®, Laboratoire d’ImmunoGénétique Moléculaire (LIGM), Institut de Génétique
Humaine (IGH), UMR 9002 CNRS-UM, Université de Montpellier, Montpellier Cedex,
France; UMR 1202 BIOGECO, INRA, Université Bordeaux, Site de recherches Forêt Bois
de Pierroton, Cestas Cedex, France
BRIAN FENNELL Biomedicine Design, Pfizer, Dublin, Ireland
MICHELA FLEGO National Center for Global Health, The National Institute of Health,
Rome, Italy
EDWARD FRANKLIN Biomedicine Design, Pfizer, Dublin, Ireland
RAMONA GAA Protein Engineering and Antibody Technologies, Merck KGaA, Darmstadt,
Germany
REDDY GALI The Harvard Clinical and Translational Science Center and Countway
Library of Medicine, Harvard Medical School, Boston, MA, USA; Department of
Biomedical Informatics, Harvard Medical School, Boston, MA, USA
ANNA MARIA GIUDICE CRS Development of Biomolecular Therapies, Experimental
Oncology Lab, Rizzoli Orthopedic Institute, Bologna, Italy
VÉRONIQUE GIUDICELLI IMGT®, The International ImMunoGeneTics Information
France
CHRISTOPHER C. GOODNOW Garvan Institute of Medical Research, Sydney, NSW,
Australia; Faculty of Medicine, St. Vincent’s Clinical School, University of New South
Wales, Sydney, NSW, Australia
JULIUS GRZESCHIK Institute for Organic Chemistry and Biochemistry, Technische
Universit€at Darmstadt, Darmstadt, Germany
STEPHEN HEARTY School of Biotechnology, Dublin City University, Dublin, Ireland;
National Centre for Sensor Research, Dublin City University, Dublin, Ireland
STEFFEN HINZ Institute for Organic Chemistry and Biochemistry, Technische Universit€ at
Darmstadt, Darmstadt, Germany
SHIN-CHEN HOU Laboratory of Antibody Engineering, Shanghai Institute for Advanced
LIANLIAN JIANG Laboratory of Antibody Engineering, Shanghai Institute for Advanced
MARTINA L. JONES ARC Training Centre for Biopharmaceutical Innovation, Australian
Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland,
St. Lucia, QLD, Australia
Contributors xiii
DOREEN KÖNNING Institute for Organic Chemistry and Biochemistry, Technische

Universit€at Darmstadt, Darmstadt, Germany; Antibody-Drug Conjugates and Targeted
NBE Therapeutics, Merck KGaA, Darmstadt, Germany
CHRISTIAN KELLNER Division of Stem Cell Transplantation and Immunotherapy,
Department of Medicine II, University Hospital Schleswig-Holstein and Christian-
Albrechts, University of Kiel, Kiel, Germany
KATJA KLAUSZ Division of Stem Cell Transplantation and Immunotherapy, Department of
Medicine II, University Hospital Schleswig-Holstein and Christian-Albrechts, University of
Kiel, Kiel, Germany
HARALD KOLMAR Institute for Organic Chemistry and Biochemistry, Technische Universit€ at
SOFIA KOSSIDA IMGT®, The International ImMunoGeneTics Information System®,
Laboratoire d’ImmunoGénétique Moléculaire (LIGM), Institut de Génétique Humaine
(IGH), UMR 9002 CNRS-UM, Université de Montpellier, Montpellier Cedex, France
SIMON KRAH Institute for Organic Chemistry and Biochemistry, Technische Universit€ at
Darmstadt, Darmstadt, Germany; Protein Engineering and Antibody Technologies, Merck
KGaA, Darmstadt, Germany
SHIGEFUMI KUMACHI Epsilon Molecular Engineering, Inc., Saitama, Japan
SUMUKH KUMBLE ARC Training Centre for Biopharmaceutical Innovation, Australian
Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland,
St. Lucia, QLD, Australia
VINODH B. KURELLA Protein Engineering, Immuno-Oncology Division, Intrexon
Corporation, Germantown, MD, USA; Merrimack Pharmaceuticals, Protein Engineering,
Cambridge, MA, USA
DAISUKE KURODA Department of Bioengineering, School of Engineering, The University of
Tokyo, Tokyo, Japan
MARIE-PAULE LEFRANC IMGT®, The International ImMunoGeneTics Information
France
PAUL LEONARD School of Biotechnology, Dublin City University, Dublin, Ireland; National
Centre for Sensor Research, Dublin City University, Dublin, Ireland
CHAO LI Department of Chemistry and Biochemistry, University of Maryland, College
Park, MD, USA
TIEZHENG LI Department of Chemistry and Biochemistry, University of Maryland, College
Park, MD, USA
DAFNE MÜLLER Institute of Cell Biology and Immunology, University of Stuttgart,
Stuttgart, Germany
HUI MA School of Biotechnology, Dublin City University, Dublin, Ireland
STEPHEN M. MAHLER ARC Training Centre for Biopharmaceutical Innovation,
Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of
Queensland, St. Lucia, QLD, Australia
ALESSANDRA MALLANO National Center for Global Health, The National Institute of
Health, Rome, Italy
MARIATI Bioprocessing Technology Institute, Agency for Science, Technology and Research
(A*STAR), Singapore, Singapore
CHARLOTTE MARTIN Research Group of Organic Chemistry, Departments of Chemistry and
xiv Contributors
PIERRE MARTINEAU Institut de Recherche en Cancérologie de Montpellier (IRCM),

Montpellier, France; INSERM, U1194, Montpellier, France; Université de Montpellier,
Montpellier, France; Institut Régional du Cancer de Montpellier, Montpellier, France
SAM MASSA Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussel,
Belgium; In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel,
Brussels, Belgium
SHANE MIERSCH Banting and Best Department of Medical Research, Terrence Donnelly
Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON,
Canada
SANDRINE MOUTEL CNRS, UMR144, Paris, France; Institut Curie, PSL Research
University, Paris, France
SERGE MUYLDERMANS Cellular and Molecular Immunology, Vrije Universiteit Brussel,
Brussel, Belgium
NAOTO NEMOTO Graduate School of Science and Engineering, Saitama University,
Saitama, Japan; Epsilon Molecular Engineering, Inc., Saitama, Japan
DAMIEN NEVOLTRIS Garvan Institute of Medical Research, Immunology Division,
Darlinghurst, NSW, Australia
CLÉMENT NIZAK Laboratoire de Biochimie, ESPCI Paris, PSL Research University, CNRS
UMR8231 Chimie Biologie Innovation, Paris, France
RICHARD O’KENNEDY School of Biotechnology, Dublin City University, Dublin, Ireland;
National Centre for Sensor Research, Dublin City University, Dublin, Ireland; Qatar
Foundation and Research Complex, Hamad Bin Khalifa University, Education City,
Doha, Qatar
ANNA OTTE Division of Stem Cell Transplantation and Immunotherapy, Department of
Kiel, Kiel, Germany
VINCENT PAREZ Institut de Recherche en Cancérologie de Montpellier (IRCM), Montpellier,
France; INSERM, U1194, Montpellier, France; Université de Montpellier, Montpellier,
France; Institut Régional du Cancer de Montpellier, Montpellier, France
MICHELA PASELLO CRS Development of Biomolecular Therapies, Experimental Oncology
Lab, Rizzoli Orthopedic Institute, Bologna, Italy
MATTHIAS PEIPP Division of Stem Cell Transplantation and Immunotherapy, Department
of Medicine II, University Hospital Schleswig-Holstein and Christian-Albrechts, University
of Kiel, Kiel, Germany
FRANCK PEREZ CNRS, UMR144, Paris, France; Institut Curie, PSL Research University,
Paris, France
ANDREAS PLÜCKTHUN Department of Biochemistry, University of Zurich, Zurich,
Switzerland
THIES RÖSNER Division of Stem Cell Transplantation and Immunotherapy, Department of
Kiel, Kiel, Germany
JOANNE H. REED Garvan Institute of Medical Research, Sydney, NSW, Australia; Faculty
of Medicine, St. Vincent’s Clinical School, University of New South Wales, Sydney, NSW,
Australia
GAUTIER ROBIN Institut de Recherche en Cancérologie de Montpellier (IRCM), Montpellier,
France; INSERM, U1194, Montpellier, France; Université de Montpellier, Montpellier,
France; Institut Régional du Cancer de Montpellier, Montpellier, France
Contributors xv
SIMON ROSOWSKI Institute for Organic Chemistry and Biochemistry, Technische Universit€ at
SOPHIA ROSSKOPF Division of Stem Cell Transplantation and Immunotherapy, Department
of Medicine II, University Hospital Schleswig-Holstein and Christian-Albrechts, University
of Kiel, Kiel, Germany
ROMAIN ROUET Garvan Institute of Medical Research, Sydney, NSW, Australia
ABHISHEK SAXENA Laboratory of Antibody Engineering, Shanghai Institute for Advanced
PETER SCHOFIELD Garvan Institute of Medical Research, Sydney, NSW, Australia; Faculty
of Medicine, St Vincent’s Clinical School, University of New South Wales, Sydney, NSW,
Australia
NATHALIE SCHOLLER Kite, a Gilead Company, Santa Monica, CA, USA
CHRISTIAN SCHRÖTER Antibody-Drug Conjugates and Targeted NBE Therapeutics, Merck
KGaA, Darmstadt, Germany
KATIA SCOTLANDI CRS Development of Biomolecular Therapies, Experimental Oncology
Lab, Rizzoli Orthopedic Institute, Bologna, Italy
SACHDEV S. SIDHU Laboratory of Antibody Engineering, Shanghai Institute for Advanced
Immunochemical Studies, ShanghaiTech University, Shanghai, China; Banting and Best
Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular
Research, University of Toronto, Toronto, ON, Canada; Department of Molecular Genetics,
University of Toronto, Toronto, ON, Canada
MARTIN SIEGEMUND Institute of Cell Biology and Immunology, University of Stuttgart,
Stuttgart, Germany
LARS TOLEIKIS Protein Engineering and Antibody Technologies, Merck KGaA, Darmstadt,
Germany
KOUHEI TSUMOTO Department of Bioengineering, School of Engineering, The University of
Tokyo, Tokyo, Japan; Medical Proteomics Laboratory, The Institute of Medical Science, The
University of Tokyo, Tokyo, Japan
BAS VAN DER WONING argenx BVBA, Zwijnaarde, Belgium
RODRIGO VAZQUEZ-LOMBARDI Garvan Institute of Medical Research, Darlinghurst/
Sydney, NSW, Australia
LAI-XI WANG Department of Chemistry and Biochemistry, University of Maryland, College
Park, MD, USA
IRIS WILLENBUECHER Protein Engineering and Antibody Technologies, Merck KGaA,
Darmstadt, Germany
TIM WIRT Division of Stem Cell Transplantation and Immunotherapy, Department of
Kiel, Kiel, Germany
DONGHUI WU Laboratory of Antibody Engineering, Shanghai Institute for Advanced
CATARINA XAVIER In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit
Brussel, Brussels, Belgium
YUANSHENG YANG Bioprocessing Technology Institute, Agency for Science, Technology and
Research (A*STAR), Singapore, Singapore
JESSNA H. M. YEO Bioprocessing Technology Institute, Agency for Science, Technology and
Research (A*STAR), Singapore, Singapore
TIANLEI YING Key Laboratory of Medical Molecular Virology of MOE/MOH, Shanghai
Medical College, Fudan University, Shanghai, China
xvi Contributors
SILVIA ZAMBONI Department of Neuroscience, The National Institute of Health, Rome,

Italy
MAHDI ZERAATI Garvan Institute of Medical Research, Sydney, NSW, Australia; Faculty of
Medicine, St. Vincent’s Clinical School, University of New South Wales, Sydney, NSW,
Australia
STEFAN ZIELONKA Institute for Organic Chemistry and Biochemistry, Technische
Universit€
at Darmstadt, Darmstadt, Germany; Protein Engineering and Antibody
Technologies, Merck KGaA, Darmstadt, Germany
Part I
In Silico Methods
Chapter 1
Antibody Design and Humanization via In Silico Modeling

Vinodh B. Kurella and Reddy Gali
Abstract
Antibody humanization process converts any nonhuman antibody sequence into humanized antibodies.
This can be achieved using different methods of antibody design and engineering. This chapter will
primarily focus on antibody design using a homology model followed by framework shuffling of murine
to human germline sequence for humanization. Historically, mouse antibodies have been humanized using
sequence-based approaches, in which all the murine frameworks are replaced with most homologous
human germline sequence or related scaffold. Most often this humanized antibody design, when tested,
has a significantly reduced binding or no binding to the cognate antigen. This is due to noncompatibility of
mouse CDRs being supported by non-native human framework scaffold. This mismatch between mouse,
human structural fold, and elimination of key conformational residues often leads to antibody humaniza-
tion failures. Recently, there has been advent of homology modelor structure-guided antibody humaniza-
tion. Instead of humanization based on linear sequence, this approach takes into account the tertiary
structure and fold of the mouse antibody. A mouse homology model of the fragment variable is created, and
based on sequence alignment with human germline, residues that are different in mouse are replaced with
humanized sequence in the model. Energy minimization is applied to this humanized model that also
delineates residues which might have steric clashes due to change in the overall tertiary conformation of the
humanized antibody. Therefore, a homology model-guided with rational mutations, and reintroduction of
key conformational residues from mouse antibody not only eliminates steric clashes but might also restore
function in relation to binding affinity to its antigen.
Key words Antibody design, Humanization, De-immunization, Antibody homology model, PIGS,
Rosetta, Antibody model, Prediction of Immunoglobulin Structures, Mouse antibody humanization,
Homology model-guided humanization, Structure-based antibody engineering
1 Introduction
Historically, antibodies have been generated mainly using mouse

models. These antibodies worked best for research and diagnostic
applications, but did not fare well in human therapeutic use. In the
early days of therapeutic antibody development and use, mouse
monoclonal antibody against a specific cancer target was adminis-
tered directly to human patients. This led to generation of human
anti-mouse antibody response (HAMA), which not only neutra-
lized this therapeutic antibody but also led to severe allergic
Damien Nevoltris and Patrick Chames (eds.), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology,
vol. 1827, https://doi.org/10.1007/978-1-4939-8648-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2018
3
4 Vinodh B. Kurella and Reddy Gali
reaction in humans. Researchers then replaced mouse Fc (fragment

crystalline) and part of Fab (fragment antigen binding) with human
antibody sequences, a chimeric antibody. This chimera, based on
the sequence, did reduce overall immunogenicity of the mouse
mAb but still did contain substantial mouse residues. Greg Winter
at Medical Research Council (MRC, UK) came up with a novel idea
of taking the mouse CDRs of both heavy and light chains and
transplanting them directly onto a fully human antibody scaffold.
In this method, CDRs are the only sequences that are mouse, and
the rest of the antibody contains human sequence. This technique
is popularly known as CDR replacement [1]. It was a groundbreak-
ing technique at that time and is still considered a gold standard in
antibody humanization.
Over the years, there have been numerous methods developed
for undertaking antibody humanization or antibody de-immuniza-
tion. Although CDR replacement dramatically reduces the mouse
residues in the humanized antibody, however, in most cases it
results in a significant drop in affinity toward the antigen. This is
due to sequence and structural differences between mouse CDRs
being supported by non-native human frameworks. Therefore,
humanization becomes an iterative process in which numerous
designs have to be made, tested, and ranked based on functional
readout (e.g., affinity measurement). Other humanization tech-
nique adopts sequence-based conversion of a mouse mAb to a
humanized antibody. It involves replacement of only those residues
to human germline sequences, which are not conserved between
mouse and human. Another technique involves a more conserva-
tive approach, in which replacement is done only for one framework
at a time instead of all the four frameworks [2]. In this approach,
designs are tested, and sequential framework replacement of mouse
to human is accomplished, driven by affinity measurements and
functional data. Framework shuffling is a method to create huma-
nized antibody variants based on sequence conservation and ran-
domization of heavy and light chains from different variable chain
germline sequences [3]. Super humanization is another technique
in which mouse CDRs are also humanized along with the frame-
works [4]. Given HAMA responses against a mouse mAb are
antibody mediated, researchers have mapped out the surface-
exposed residues in the mouse mAb and replaced only those with
corresponding human sequences, also known as antibody resurfa-
cing [5]. This is achieved by delineating surface-exposed residues
using either X-ray/NMR structures or homology model of the
mouse mAb fragment variable region (Fv). Recently, this approach
is further optimized, in which the mouse mAb Fv model is first
generated, and then non-conserved residues in mouse are mutated
to reflect human germline sequences. This humanized homology
model has mouse CDRs and human frameworks, which is energy
minimized using one of these force fields (GROMOS,
Antibody Design and Humanization 5
CHARMM). If there are steric clashes between frameworks and

CDRs/frameworks regions, these can be visualized in the chimeric
homology model. Sequence or structure-based back or novel muta-
tions can be introduced to avoid these steric clashes. This approach
is well adopted by the scientific community as a homology model-
guided antibody humanization [6–9].
1.1 Antibody Antibodies can be classified into canonical and noncanonical clas-
Structures ses. Antibody heavy chain can be further divided into four frame-
works, which encompass CDRs (H1, H2, H3) and light chain
containing CDRs (L1, L2, L3). All these frameworks have a very
high degree of similar structural fold. CDRs L1–L3 and H1 and H2
can be assigned to canonical classes. Based on the length and
sequence, these loops can be modeled based on the structural
templates available from these canonical classes in protein data
bank. Given the diversity and uniqueness of CDR H3, it does not
belong to any canonical family. In the recent past, there has been
plethora of antibody structures determined via NMR, X-ray crys-
tallography, as well as cryo-EM techniques. Most of these antibo-
dies have sequence and structural similarity between them that has
led to creation and development of different algorithms for anti-
body homology model prediction, as opposed to resource-intensive
experimental structure determination.
These algorithms primarily use antibody sequence information
to extract structural templates from protein data bank (PDB),
which results in creation of homology model of an antibody.
Given tremendous growth in biologics in the recent past over
small molecules, different researchers, as well as commercial com-
panies, have developed antibody model prediction algorithms.
Publically available antibody modeling algorithms are Rosetta anti-
body modeling, Web antibody modeling (WAM), structure-based
antibody prediction server (SAbPred), Prediction of ImmunoGlob-
ulin Structures (PIGS), and Kotai (antibody builder). Commer-
cially, there are a number of antibody modeling products, such as
Biovia’s Discovery Studio, Schrodinger’s BioLuminate, and Chem-
ical Computing Group’s MOE and Macromoltek. A brief descrip-
tion of some of these prediction algorithms is described.
1.2 Web Antibody Antibody modeling algorithm follows a series of steps to obtain a
Modeling (WAM) homology model from the sequences of variable heavy and light
chains. It consists of sequence-based search for framework and
canonical loop regions to find the most homologous structure
from protein data bank. Noncanonical antibody regions are built
either using knowledge-based search from databases or ab initio
model building, using CONGEN conformational search
[10]. Final conformational homology model is selected from the
five lowest energy models, ranked based on torsion angles closest to
the original PDB template.
1.3 Rosetta Antibody In this algorithm, antibody variable heavy and light chain is split
Modeling into frameworks and CDR sequences. These sequences then
become input for template search in the curated protein data
bank for the most similar sequences, and use the most homologous
structure as a template to build a homology model. As framework
regions are mostly conserved across different antibodies, a frame-
work 3D model is built. CDR-H3 structure is the most variable and
challenging to predict; hence its structural fold is created by de
novo model building. Once VH–VL orientation is chosen via
sequence blast of the whole Fv (fragment variable—VH þ VL)
against the PDB, the modeled CDR loops for VH and VL are
grafted onto this framework template for further refinement and
model building. Engrafted model is energy minimized, and an
ensemble of high-resolution models <3000 are ranked based
on Rosetta score that reflects probable entropies in each model,
and the lowest energy is ranked as the top model [11].
1.4 Prediction In general, this antibody variable homology modeling server algo-
of Immunoglobulin rithm is similar to those mentioned above. However, there are
Structures (PIGS) some differences in the workflow and strategies implemented in
PIGS. A sequence-based analysis is performed to obtain homolo-
gous framework sequences of known antibody structures from
protein data bank. Antibody CDRs with canonical loops are mod-
eled based on known antibody structures and grafted upon the
framework model. Both heavy and light chain models (if the paren-
tal templates are different) then are packed together based on
conserved interface residues at the heavy and light chains from
known antibody structures. Finally, energy minimization of the
side chains is carried out via SCWRL4.0 (side chain with
backbone-dependent rotamer library), results in the final antibody
homology model generation [12].
2 Materials
Antibody humanization can be undertaken either using commer-

cially available tools (Schrodinger’s BioLuminate, Biovia’s Discovery
Studio, Chemical Computing Group’s MOE and Macromoltek) or
publically available web servers (Prediction of Immunoglobulin
Structures- PIGS; Rosetta antibody modeling; structure-based anti-
body prediction server- SAbPred; and Kotai -antibody builder). To
describe and delineate differences and limitations between these
algorithms will be beyond the scope of this chapter. Given, publically
available resources and servers can be accessed by everyone with a
computer and internet access. This chapter will primarily focus on
utilizing public servers for antibody homology model building and
humanization.
1. To delineate boundaries of mouse heavy and light chain, user

can input the whole heavy chain and light chain from the
mouse IgG sequence separately into IMGT (the international
ImMunoGeneTics information system®) DomainGapAlign
alignment tool using default settings (http://imgt.org/
3Dstructure-DB/cgi/DomainGapAlign.cgi) [13]. As an
example, a mouse antibody from protein data bank code
(3MBX) with the variable heavy (VH) and light
(VL) sequence (see Notes 1 and 2).
VH—
EVTLKESGPGILQPSQTLSLTCSFSGFSLSTYGMGVGWIRQPSGKGLEWLA
HIWWDDVKRYNPALKSRLTISKDTSGSQV
FLKIASVDTSDTATYYCARMGSDYDVWFDYWGQGTLVTVSA
VL—
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYNNNQKNYLAWYQQKPGQS
PKLLIYWASTRESGVPDRFTGSGSGTDFTLT
ISSVKAEDLAVYYCQQYYSYPFTFGSGTKLEIK
2. Once the variable heavy (VH) and variable light (VL) chains are
defined as above, these VH and VL sequences will become the
starting material to be used as input for antibody homology
model building. Model can be built by accessing any of the
antibody modeling servers:
PIGS—http://circe.med.uniroma1.it/pigs/index.php
Rosetta—http://rosie.rosettacommons.org/antibody
SAbPred—http://opig.stats.ox.ac.uk/webapps/sabdab-
sabpred/WelcomeSAbPred.php
Kotai—https://sysimm.ifrec.osaka-u.ac.jp/kotaiab/
3. Mouse antibody homology model is built by one of the above
servers; the model can be visualized via molecular visualization
software programs, such as PyMOL, DeepView-Swiss-
PDBViewer, or UCSF Chimera. These programs can be down-
loaded as given below:
PyMOL—https://www.pymol.org/
DeepView-Swiss-PDBViewer—http://spdbv.vital-it.ch/dis
claim.html
UCSF Chimera—http://www.cgl.ucsf.edu/chimera/
4. Energy minimization of the humanized antibody model can be
undertaken by DeepView-Swiss-PDBViewer. This software can
be downloaded from this website.
http://spdbv.vital-it.ch/disclaim.html
3 Methods
1. The first step is to obtain the mouse antibody variable and

heavy and light sequences and create a homology model via
any of the antibody homology modeling software. Input
mouse antibody sequences (VH and VL) into either antibody
homology modeling software (BioLuminate, Discovery studio,
or MOE) or any of these public web servers (e.g., PIGS,
Rosetta, SAbPred, or Kotai). (For an example, refer to Sub-
heading 2, step 1, for a mouse antibody sequence.) Use PIGS
web server as a starting tool for this exercise (Fig. 1a, b).
PIGS—http://circe.med.uniroma1.it/pigs/index.php
Rosetta—http://rosie.rosettacommons.org/antibody
SAbPred—http://opig.stats.ox.ac.uk/webapps/sabdab-
sabpred/WelcomeSAbPred.php
Kotai—https://sysimm.ifrec.osaka-u.ac.jp/kotaiab/
2. Human framework selection can be primarily accomplished
from two sources:
(a) IMGT database can be used to obtain most identical
(percentage identity) human germline repertoire for
both VH chain and VL chains. IMGT’s DomainGapAlign
tool can be used to obtain the most similar corresponding
human germline sequences for heavy and light chain sep-
arately (http://imgt.org/3Dstructure-DB/cgi/Dom
ainGapAlign.cgi) (see Fig. 2). Use default settings; under
species drop-down menu, choose Homo sapiens
(Humans), and then click “Align and IMGT-gap my
sequence for VH and VL separately.
(b) Blast antibody sequences VH and VL separately to find
the most identical human framework in PDB. Igblast tool
can be utilized for this analysis (https://www.ncbi.nlm.
nih.gov/igblast/).
Human framework selection criteria can be made based
on percentage identity to individual VH and VL chains or
human frameworks with highest overall identity for both
chains (VH and VL).
3. Mutate and replace mouse residues in the homology model
created in step 1 to human residues, either found in the align-
ment using IMGT’s DomainGapAlign alignment (step 2a) or
human antibody structure obtained from PDB (step 2b).
Replacement of mouse residues to human residues can be
performed using PyMOL>wizard> mutagenesis tool https://
www.pymol.org/ (see Note 3). In addition, user can subject
the resulting chimeric model to Ramachandran plot validation
using PDBsum Generate option (this step is optional) (https://
www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.
html).
Fig. 1 (a) Antibody humanization steps are described in this workflow. As

described in this flow chart, there are numerous steps in this process that
need usage of different antibody modeling servers, sequence alignment tools,
and protein visualization software. (b) Visual representation of antibody humani-
zation design process. Different steps involved in antibody humanization are
annotated based on each stage of the design development. A mouse antibody
4. This humanized homology model has human frameworks and

mouse CDRs (chimeric), which is subjected to energy mini-
mizations using Swiss-PDBViewer (spdbv) software. Upload
this humanized model in the spdbv software, and select all, and
then under tools, select “compute energy (Force Field).” The
output “energy report” can be copied into a word document
for review. Residues with high total energies need to be exam-
ined further. This can be carried out using PyMOL software
(see Note 4).
5. Energy-minimized humanized model is then examined both
visually and energetically for steric clashes. Based on the degree
of entropies score, as well as extent of steric clashes, some residues
may be replaced either to novel mutations or back mutation
(parental mouse) residues. For example (Figs. 3 and 4), if there
is a steric clash between two residues in the framework regions,
then the residue that is not conserved across different germlines is
mutated to fix the steric clash (see Note 5).
6. Once residue replacement is completed, humanized homology
model is again subjected to energy minimization (step 4) to
examine amelioration of these steric clashes (Fig. 3b). If no
further steric clashes are found, then this humanized model is
designated as design one.
7. To design additional humanized variants, one can choose the
second highest identical human framework from step 2 and
carry out the engineering until step 6 to obtain design two.
Repeat steps from 2 to 6 to obtain a minimal set of 20 different
humanized variants for experimental testing (see Fig. 1a, b).
4 Notes
1. To explore some additional examples of mouse antibodies for

humanization, refer to [14], as it lists 17 different mouse
antibodies for humanization using the same methods as men-
tioned in this chapter.
ä
Fig. 1 (continued) sequence was utilized to create a 3D homology model, CDRs

in orange, light chain in blue, and heavy chain in purple, and sequences that are
different in mouse are highlighted in yellow. Once human germline sequence is
obtained from either IMGT or Abysis database and aligned with mouse antibody
sequence, those residues that were different are changed to reflect human
sequences (green—step 3). This chimeric humanized model can also be vali-
dated via Ramachandran plot using PDBsum Generate option (step 4—optional).
The humanized model (human framework and mouse CDRs) was then subjected
to energy minimization using Swiss-PDBViewer (spdbv) software (step 5). If no
steric clashes are found, this will result in creation of the first humanized
antibody design (step 7)
Fig. 2 Mouse antibody and corresponding human germline sequence alignment using IMGT DomainGapAlign
tool. Mouse antibody sequence (PDB ID—3MBX) is aligned with the most identical human germline sequence.
Mouse residues in both heavy and light chain (yellow) were mutated to human germline residues (green),
whereas the mouse CDRs (orange) were unchanged. Mouse variable heavy chain is 71.4% (identical) to human
germline IGHV2-5*09; a total of 24 amino acids needed to be replaced. Whereas mouse variable light chain is
82.3% (identical) to human germline IGKV4-1*01, as such, only 12 amino acids needed to be replaced
2. If using PIGS for antibody model building with VH and VL as

input sequence from PDB ID—3MBX antibody, there will be
two warnings after the submission for template selection. One
being “No IG satisfying search criteria among first 20, and
other is H3 canonical structure not defined.” To circumvent
first warning, under results, select threshold 40 results, instead
of 20 (default).
3. Mutagenesis tool in PyMOL is recommended for mutating and
replacing mouse antibody residues in the heavy chain and light
chain to corresponding residues in the human germline gene
(Fig. 2). In PyMOL, select wizard and mutagenesis, it will
prompt to “pick a residue” of the mouse amino acid, and
mutate to corresponding human amino acid from Fig. 2.
(If using 3MBX as an example, in the heavy chain
(VH) position 1, mouse glutamate (E) needs to be mutated
to glutamine (Q) for humanization.) (see Fig. 2).
4. Energy minimization of the humanized chimeric model using
Swiss-PDBViewer (spdbv). Once the hybrid-humanized model
is opened using Swiss-PDBViewer, it might give an error of
“unrealistic B factor.” Please ignore and close this generic error.
Compute energy (force field), under tools, is first selected.
Make sure the pop-up window has all the boxes checked,
including “show energy report.” The energy report is usually
saved in “temp” folder, which can be found inside the original
folder, where Swiss-PDBViewer is installed
(SPDBV_4.1.0_OSX “folder name.” It is recommended to
use desktop folder as a destination for software installation).
In 3MBX, there are many residues with high total energies. As
an example, only a couple of them are described here. Residues
in the heavy chain position 24 (PHE) and in the light chain
position 46 (LEU) have very high electrostatic or total score.
Fig. 3 Energy minimization of the humanized 3MBX mouse antibody in Swiss-PDBViewer (in vacuo) via
GROMOS96 force field. First 30 residues of the heavy chain are only shown, as an example, in this. (a) Heavy
chain residue phenylalanine (Phe) 24 has high total energy score, due to a steric clash with asparagine (Asn) at
position 76. (b) Phenylalanine is conserved; as such asparagine was mutated to serine (mouse residue not
shown in this figure) that had a significant impact on lowering the overall total energy of the Phe 24 residue
Fig. 4 Steric clashes in heavy and light chains of the humanized mouse antibody
3MBX. (a) Heavy chain framework 1, residue phenylalanine (Phe) 24 has a steric
clash with asparagine (Asn) of framework 3 at position 76 (Phe is number 24 in
the model but is 25 in IMGT alignment; Asn number is 76 in the homology model
but 85 in the IMGT alignment). Phe is conserved between mouse and human
germline; Asn was replaced with serine (Ser) to fix this steric clash. (b) In the
light chain framework 2, leucine 46 (position 46 in the model, which corresponds
to position 52 in IMGT numbering scheme) had a steric clash with tryptophan
(W) in the CDR H3 loop. To fix this clash, leucine (Leu) 46 was mutated to
isoleucine (Ile)
5. To fix these steric clashes, if using 3MBX as an example, in the

heavy chain (VH) framework 1, phenylalanine (Phe) at position
24 has a steric clash with framework 3, asparagine (Asn)
76 (homology model numbering is different than IMGT align-
ment numbering; see Fig. 4 for details). As Phe is conserved
between mouse and human, Asn was back mutated to Ser
(mouse) residue to relieve this steric clash. In the VL, leucine
(Leu) 46 has a steric clash with tryptophan (W) in the CDR H3
loop. To fix this clash, leucine (Leu) 46 can be mutated to
isoleucine (Ile) (see Fig. 4).
References
1. Riechmann L, Clark M, Waldmann H, Winter 2. Harding FA, Stickler MM, Razo J, DuBridge
G (1988) Reshaping human antibodies for RB (2010) The immunogenicity of humanized
therapy. Nature 332(6162):323–327. https:// and fully human antibodies: residual
doi.org/10.1038/332323a0
immunogenicity resides in the CDR regions. design. MAbs 7(6):1045–1057. https://doi.

MAbs 2(3):256–265 org/10.1080/19420862.2015.1076600
3. Dall’Acqua WF, Damschroder MM, Zhang J, 9. Apgar JR, Mader M, Agostinelli R, Benard S,
Woods RM, Widjaja L, Yu J, Wu H (2005) Bialek P, Johnson M, Gao Y, Krebs M,
Antibody humanization by framework shuf- Owens J, Parris K, St Andre M, Svenson K,
fling. Methods 36(1):43–60. https://doi. Morris C, Tchistiakova L (2016) Beyond
org/10.1016/j.ymeth.2005.01.005 CDR-grafting: structure-guided humanization
4. Tan P, Mitchell DA, Buss TN, Holmes MA, of framework and CDR regions of an anti-
Anasetti C, Foote J (2002) “Superhumanized” myostatin antibody. MAbs 8(7):1302–1318.
antibodies: reduction of immunogenic poten- https://doi.org/10.1080/19420862.2016.
tial by complementarity-determining region 1215786
grafting with human germline sequences: 10. Whitelegg NR, Rees AR (2000) WAM: an
application to an anti-CD28. J Immunol 169 improved algorithm for modelling antibodies
(2):1119–1125 on the WEB. Protein Eng 13(12):819–824
5. Roguska MA, Pedersen JT, Keddy CA, Henry 11. Weitzner BD, Jeliazkov JR, Lyskov S, Marze N,
AH, Searle SJ, Lambert JM, Goldmacher VS, Kuroda D, Frick R, Adolf-Bryfogle J, Biswas N,
Blattler WA, Rees AR, Guild BC (1994) Dunbrack RL Jr, Gray JJ (2017) Modeling and
Humanization of murine monoclonal antibo- docking of antibody structures with Rosetta.
dies through variable domain resurfacing. Proc Nat Protoc 12(2):401–416. https://doi.org/
Natl Acad Sci U S A 91(3):969–973 10.1038/nprot.2016.180
6. Mader A, Kunert R (2010) Humanization stra- 12. Marcatili P, Olimpieri PP, Chailyan A, Tramon-
tegies for an anti-idiotypic antibody mimicking tano A (2014) Antibody modeling using the
HIV-1 gp41. Protein Eng Des Sel 23 prediction of immunoglobulin structure
(12):947–954. https://doi.org/10.1093/pro (PIGS) web server [corrected]. Nat Protoc 9
tein/gzq092 (12):2771–2783. https://doi.org/10.1038/
7. Chang DK, Kurella VB, Biswas S, Avnir Y, nprot.2014.189
Sui J, Wang X, Sun J, Wang Y, Panditrao M, 13. Ehrenmann F, Kaas Q, Lefranc MP (2010)
Peterson E, Tallarico A, Fernandes S, IMGT/3Dstructure-DB and IMGT/Domain-
Goodall M, Zhu Q, Brown JR, Jefferis R, Mar- GapAlign: a database and a tool for immuno-
asco WA (2016) Humanized mouse G6 anti- globulins or antibodies, T cell receptors, MHC,
idiotypic monoclonal antibody has therapeutic IgSF and MhcSF. Nucleic Acids Res 38(Data-
potential against IGHV1-69 germline gene- base issue):D301–D307
based B-CLL. MAbs 8(4):787–798. https:// 14. Kurella VB, Gali R (2014) Structure guided
doi.org/10.1080/19420862.2016.1159365 homology model based design and engineering
8. Choi Y, Hua C, Sentman CL, Ackerman ME, of mouse antibodies for humanization. Bioin-
Bailey-Kellogg C (2015) Antibody humaniza- formation 10(4):180–186. https://doi.org/
tion by structure-based computational protein 10.6026/97320630010180
Chapter 2
Antibody Affinity Maturation by Computational Design

Daisuke Kuroda and Kouhei Tsumoto
Abstract
The immune systems protect our bodies from foreign molecules or antigens, where antibodies play
important roles. Antibodies evolve over time upon antigen encounter by somatically mutating their
genome sequences. The end result is a series of antibodies that display higher affinities and specificities to
specific antigens. This process is called affinity maturation. Recent improvements in computer hardware and
modeling algorithms now enable the rational design of protein structures and functions, and several works
on computer-aided antibody design have been published. In this chapter, we briefly describe computational
methods for antibody affinity maturation, focusing on methods for sampling antibody conformations and
for scoring designed antibody variants. We also discuss lessons learned from the successful computer-aided
design of antibodies.
Key words Antibody engineering, Affinity maturation, Computer-aided design, Molecular simula-
tions, Sampling, Scoring
1 Introduction
Computational methods are widely accepted as essential tools for

drug discovery of small molecules, helping with tasks such as
screening candidate compounds, evaluating drug-likeness, and
optimizing physicochemical and pharmacokinetic properties
[1]. In these contexts, computational methods are generally not
considered substitutes for “wet lab” experiments but rather are a
way to generate testable hypotheses, thus helping to guide experi-
ments and interpret the results. In recent years, computational
methods have garnered much attention in regard to antibody
research and drug discovery [2, 3]. The applications of those
tools in this field include affinity improvement [4–7], specificity
control [8], engineering for thermostability [9, 10], aggregation
propensity [11, 12], and humanization [13–19]. In this chapter, we
briefly review successes regarding improving the affinity of antibo-
dy–antigen interactions and discuss how potential affinity-
enhancing mutations might be identified. Before describing
Damien Nevoltris and Patrick Chames (eds.), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology,
vol. 1827, https://doi.org/10.1007/978-1-4939-8648-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2018
15
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dame tú la fuerça y yo porne la
voluntad. Las cosas de onrra que
pones delante conozcolas con la
razon y niegolas con ella misma.
Digo que las conozco y aprueuo
si las ha de vsar onbre libre de mi
pensamiento, y digo que las niego
para comigo pues pienso avnque
busque graue pena que escogí
onrrada muerte. El trabaio que
por mi as recebido y el deseo que
te he visto me obligauan á ofrecer
por tí la vida todas las vezes que
fuere menester, mas pues lo
menos della me queda de beuir
seate satisfacion lo que quisiera y
no lo que puedo. Mucho te ruego
pues esta será la final buena obra
que tú me podras hazer y yo
recebir que quieras leuar á
Laureola en vna carta mia nueuas
con que se alegre, porque della
sepa como me despido de la vida
y de mas dalle enoio, la qual en
esfuerço que la leuarás quiero
començar en tu presencia y las
razones della sean estas.
CARTA DE LERIANO Á
LAUREOLA
Pues el galardon de mis afanes
auie de ser mi sepoltura ya soy a
tiempo de recebirlo. Morir no
creas que me desplaze, que
aquel es de poco iuyzio que
aborrece lo que da libertad. ¿Mas
que haré que acabará comigo el
esperança de verte graue cosa
para sentir? Dirás que cómo tan
presto en vn año ha o poco mas
que ha que soy tuyo desfallescio
mi sofrimiento; no te deues
marauillar que tu poca esperança
y mi mucha pasion podian bastar
para más de quitar la fuerça al
sofrir, no pudiera pensar que á tal
cosa dieras lugar si tus obras no
me lo certificaran.
Siempre crey que forçara tu
condicion piadosa á tu voluntad
porfiada, como quiera que en esto
si mi vida recibe el daño mi dicha
tiene la culpa, espantado estoy
cómo de tí misma no te dueles.
Dite la libertad, ofrecite el
coraçon, no quise ser nada mio
por serlo del todo tuyo, pues,
¿cómo te querrá seruir ni tener
amor quien sopiere que tus
propias cosas destruyes? Por
cierto tú eres tu enemiga. Si no
me querias remediar porque me
saluara yo, deuieraslo hazer
porque no te condenaras tú.
Porque en mi perdicion ouiese
algund bien deseo que te pese
della, mas si el pesar te avie de
dar pena no lo quiero, que pues
nunca biuiendo te hize seruicio no
seria iusto que moriendo te
causase enoio. Los que ponen los
oios en el sol quanto mas lo miran
mas se ciegan, y assi quanto yo
más contenplo tu hermosura mas
ciego tengo el sentido. Esto digo
porque de los desconciertos
escritos no te marauilles: verdad
es que á tal tienpo escusado era
tal descargo, porque segund
quedo mas estó en disposicion de
acabar la vida que de desculpar
las razones.
Pero quisiera que lo que tú auias
de ver fuera ordenado, porque no
ocuparas tu saber en cosa tan
fuera de tu condicion. Si
consientes que muera porque se
publique que podiste matar, mal
te aconseiaste, que sin
esperiencia mia lo certificava la
hermosura tuya; si lo tienes por
bien porque no era merecedor de
tus mercedes, pensaua alcançar
por fé lo que por desmerecer
perdiese, y, con este
pensamiento, osé tomar tal
cuydado. Si por ventura te plaze
por parecerte que no se podria
remediar sin tu ofensa mi cuyta,
nunca pense pedirte merced que
te causase culpa. ¿Cómo auia de
aprouecharme el bien que á ti te
viniese mal? Solamente pedí tu
respuesta por primero y
postrimero galardon. Dexadas
mas largas te suplico, pues
acabas la vida que onrres la
muerte, porque si en lugar donde
van las almas desesperadas ay
algun bien, no pediré otro si no
sentido para sentir que onrraste
mis huesos por gozar aquel poco
espacio de gloria tan grande.
EL AUCTOR
Acabada la habla y carta de
Leriano, satisfaziendo los oios por
las palabras con muchas
lagrimas, sin poderle hablar
despedime dél, auiendo aquella,
segund le vi, por la postrimera vez
que lo esperaua ver; y puesto en
el camino puse su sobrescrito á
su carta porque Laureola en
seguridad de aquel la quisiese
recebir. Y llegado donde estaua,
acordé de gela dar, la qual
creiendo que era de otra calidad
recebio, y començo y acabó leer;
y como en todo aquel tiempo que
la leya nunca partiese de su
rostro mi vista, vi que quando
acabó de leerla quedó tan
enmudecida y turbada como si
gran mal touiera, y como su
turbacion de mirar la mia no le
escusase, por asegurarme hizo
me preguntas y hablas fuera de
todo proposito, y para librarse de
la conpañia que en semeiantes
tienpos es peligrosa, porque las
mudanças públicas no
descubriessen los pensamientos,
retraxose. Y assí estuuo aquella
noche sin hablarme nada en el
propósito, y otro dia de mañana
mandome llamar y despues que
me dixo quantas razones
bastauan para descargarse del
consentimiento que daua en la
pena de Leriano, dixome que le
tenia escrito pareciéndole
inumanidad perder por tan poco
precio un onbre tal; y porque con
el plazer de lo que le oya estaua
desatinado en lo que hablaua, no
escriuo la dulceza y onestad que
ouo en su razonamiento. Quien
quiera que la oyera pudiera
conocer que aquel estudio auie
vsado poco: ya de enpachada
estaua encendida, ya de turbada
se tornaua amarilla. Tenia tal
alteracion y tan sin aliento la
habla como si esperara sentencia
de muerte; en tal manera le
tenblaua la boz que no podía
forçar con la discrecion al miedo.
Mi respuesta fué breve porque el
tienpo para alargarme no me
daua lugar, y despues de besalle
las manos recebi su carta, las
razones de la qual eran tales.
CARTA DE LAUREOLA Á
LERIANO
La muerte que esperauas tú de
penado merecia yo por culpada si
en esto que hago pecase mi
voluntad, lo que cierto no es assí,
que más te escriuo por redemir tu
vida que por satisfazer tu deseo.
Mas, triste de mi, que este
descargo solamente aprouecha
para conplir comigo, porque si
deste pecado fuese acusada no
tengo otro testigo para saluarme
sino mi intencion, y por ser parte
tan principal no se tomaria en
cuenta su dicho, y con este
miedo, la mano en el papel, puse
el coraçon en el cielo, haziendo
iuez de mi fin aquel á quien la
verdad de las cosas es
manifiesta.
Todas las vezes que dudé en
responderte fue porque sin mi
condenacion no podias tú ser
asuelto. Como agora parece que
puesto que tú solo y el levador de
mi carta sepays que escreui, qué
sé yo los iuycios que dareys
sobre mi; y digo que sean sanos
sola mi sospecha me amanzilla.
Ruegote mucho quando con mi
respuesta en medio de tus
plazeres estés mas vfano, que te
acuerdes de la fama de quien los
causó, y auiso te desto, porque
semeiantes fauores desean
publicarse teniendo mas
acatamiento á la vitoria dellos que
á la fama de quien los da. Quanto
meior me estouiera ser afeada
por cruel que amanzillada por
piadosa, tú lo conosces, y por
remediarte vsé lo contrario. Ya tú
tienes lo que deseauas y yo lo
que temia. Por Dios te pido que
enbueluas mi carta en tu fe,
porque si es tan cierta como
confiesas no se te pierda ni de
nadie pueda ser vista, que quien
viese lo que te escriuo pensaria
que te amo, y creeria que mis
razones antes eran dichas por
disimulacion de la verdad que por
la verdad. Lo qual es al reues,
que por cierto mas las digo, como
ya he dicho, con intencion
piadosa que con voluntad
enamorada. Por hazerte creer
esto querria estenderme y por no
ponerte otra sospecha acabo, y
para que mis obras recibiesen
galardon iusto auia de hazer la
vida otro tanto.
EL AUCTOR
Recibida la carta de Laureola
acordé de partirme para Leriano,
el qual camino quise hazer
acompañado, por leuar comigo
quien á él y á mí ayudase en la
gloria de mi enbaxada, y por
animarlos para adelante llamé los
mayores enemigos de nuestro
negocio que eran
Contentamiento, y Esperança, y
Descanso, y Plazer, y Alegría, y
Holgança. Y porque si las
guardas de la prision de Leriano
quisiesen por leuar conpañía
defenderme la entrada, pense de
yr en orden de guerra, y con tal
pensamiento, hecha vna batalla
de toda mi conpañía, seguí mi
camino, y allegado á vn alto
donde se parecia la prision,
viendo los guardadores della mi
seña que era verde y colorada, en
lugar de defenderse pusieronse
en huyda tan grande que quien
mas huya mas cerca pensaua
que yua del peligro. Y como
Leriano vido sobre á ora tal
rebato, no sabiendo qué cosa
fuese, pusose á vna ventana de la
torre, hablando verdad, mas con
flaqueza de espíritu que con
esperança de socorro. Y como
me vio venir en batalla de tan
hermosa gente, conocio lo que
era, y lo vno de la poca fuerça y lo
otro de supito, bien perdido el
sentido, cayó en el suelo de
dentro de la casa. Pues yo que no
leuaua espacio, como llegué al
escalera por donde solia sobir
eché á descanso delante, el qual
dió estraña claridad á su tinibra, y
subido á donde estaua el ya
bienauenturado, quando le ví en
manera mortal pense que yua á
buen tienpo para llorarlo y tarde
para darle remedio, pero socorrio
luego Esperança que andaua allí
la mas diligente y echandole vn
poco de agua en el rostro tornó
en su acuerdo, y por más
esforçarle dile la carta de
Laureola, y entre tanto que la leya
todos los que leuaua comigo
procurauan su salud. Alegria le
alegraua el coraçon, Descanso le
consolaua el alma, Esperança le
bolvia el sentido, Contentamiento
le aclaraua la vista, Holgança le
restituya la fuerça, Plazer le
abiuaua el entendimiento, y en tal
manera lo trataron que quando lo
que Laureola le escrebió acabó
de leer estaua tan sano como si
ninguna pasion vuiera tenido. Y
como vido que mi diligencia le dio
libertad echabame muchas vezes
los brazos encima, ofreciendome
á él y á todo lo suyo, y pareciale
poco precio segund lo que
merecia mi seruicio. De tal
manera eran sus ofrecimientos
que no sabía responderle como
yo deuia y quien él era. Pues
despues que entre él y mí
grandes cosas pasaron, acordó
de yrse á la corte, y antes que
fuesse estuuo algunos dias en
vna villa suya por rehazerse de
fuerças y atauios para su partida,
y como se vido en disposicion de
poderse partir pusolo en obra, y
sabido en la corte como yua,
todos los grandes señores y
mancebos cortesanos salieron á
recebirle. Mas como aquellas
cerimonias vieias touiesse
sabidas, mas vfana le daua la
gloria secreta que la onrra
pública, y así fue acompañado
hasta palacio. Quando besó las
manos á Laureola pasaron cosas
mucho de notar, en especial para
mí que sabia lo que entre ellos
estaua: al vno le sobraua
turbacion, al otro le faltaua color;
ni él sabie qué dezir, ni ella qué
responder, que tanta fuerça tienen
las pasiones enamoradas que
sienpre traen el seso y discrecion
debaxo de su vandera; lo que allí
vi por clara esperiencia.
Y puesto que de las mudanças
dellos ninguno touiese noticia por
la poca sospecha que de su
pendencia auia, Persio, hijo del
señor de Gavia miró en ellas,
trayendo el mismo pensamiento
que Leriano traya; y como las
sospechas celosas escudriñan las
cosas secretas, tanto miró de allí
adelante las hablas y señales dél,
que dió crédito á lo que
sospechaua: y no solamente dió
fé á lo que veya, que no era nada,
mas á lo que ymaginaua él que
era todo. Y con este maluado
pensamiento, sin más
deliberacion ni conseio, apartó al
rey en vn secreto lugar y dixole
afirmadamente que Laureola y
Leriano se amauan y que se
veyan todas las noches despues
que él dormia, y que gelo hazia
saber por lo que deuie á la onrra y
á su seruicio. Turbado el rey de
cosa tal, estouo dubdoso y
pensatiuo sin luego determinarse
á responder, y despues que
mucho dormio sobre ello, tovolo
por verdad, creyendo segund la
virtud y auctoridad de Persio que
no le diria otra cosa. Pero con
todo esso primero que deliberase
quiso acordar lo que deuie hazer,
y puesta Laureola en vna carcel
mandó llamar á Persio y dixole
que acusase de traydor á Leriano,
segun sus leyes, de cuyo
mandamiento fue mucho
afrontado. Mas como la calidad
del negocio le forçaua á otorgarlo,
respondió al rey que aceutaua su
mando y que daua gracias á Dios
que le ofrecia caso para que
fuesen sus manos testimonio de
su bondad; y como semeiantes
autos se acustumbran en
Macedonia hazer por carteles y
no en presencia del rey, enbió en
vno Persio á Leriano las razones
siguientes:
CARTEL DE PERSIO PARA

LERIANO
Pues procede de las virtuosas
obras la loable fama, iusto es que
la maldad se castigue porque la
virtud se sostenga, y con tanta
diligencia deue ser la bondad
anparada que los enemigos della
si por voluntad no la obraren, por
miedo la vsen. Digo esto, Leriano,
porque la pena que recebirás de
la culpa que cometiste sera
castigo para que tú pagues y
otros teman, que si á tales cosas
se diese lugar no sería menos
fauorecida la desvirtud en los
malos, que la nobleza en los
buenos.
Por cierto mal te as aprovechado
de la limpieza que eredaste; tus
mayores te mostraron hazer
bondad y tú aprendiste obrar
trayzion; sus huessos se
leuantarian contra tí si supiesen
como ensuziaste por tal error sus
nobles obras. Pero venido eres á
tienpo que recibieras por lo
hecho, fin en la vida y manzilla en
la fama. Malauenturados aquellos
como tú que no saben escoger
muerte onesta; sin mirar el
seruicio de tu rey y la obligacion
de tu sangre touiste osada
desuerguença para enamorarte
de Laureola, con la qual en su
camara, despues de acostado el
rey, diuersas vezes as hablado,
escureciendo por seguir tu
condicion tu claro linage, de cuya
razon te rebto por traydor, y
sobrello te entiendo matar ó echar
del canpo; ó lo que digo hazer
confesar por tu boca, donde
quanto el mundo durare sere en
exenplo de lealtad; y atreuome á
tanto confiando en tu falsía y mi
verdad. Las armas escoge de la
manera que querras y el canpo.
Yo de parte del rey lo hago
seguro.
RESPUESTA DE LERIANO
Persio, mayor seria mi fortuna
que tu malicia si la culpa que me
cargas con maldad no te diese la
pena que mereces por iusticia. Si
fueras tan discreto como malo,
por quitarte de tal peligro antes
deuieras saber mi intencion que
sentenciar mis obras. Á lo que
agora conozco de tí, mas curauas
de parecer bueno que de serlo.
Teniendote por cierto amigo todas
mis cosas comunicaua contigo y
segund parece yo confiaua de tu
virtud y tú vsauas de tu condicion.
Como la bondad que mostrauas
concertó el amistad, assi la
falsedad que encobrías causó la
enemiga. ¡Ó enemigo de tí
mismo! que con razon lo puedo
dezir, pues por tu testimonio
dexarás la memoria con cargo y
acabarás la vida con mengua.
¿Por que pusiste la lengua en
Laureola que sola su bondad
basta a si toda la del mundo se
perdiese para tornarla á cobrar?
Pues tú afirmas mentira clara y yo
defiendo causa iusta, ella quedará
libre de culpa y tu onrra no de
verguença. No quiero responder á
tus desmesuras porque hallo por
mas onesto camino vencerte con
la persona que satisfazerte con
las palabras. Solamente quiero
venir a lo que haze al caso, pues
allí está la fuerça de nuestro
debate. Acusasme de traydor y
afirmas que entré muchas vezes
en su camara de Laureola
despues del rey retraydo. Á lo vno
y á lo otro te digo que mientes,
como quiera que no niego que
con voluntad enamorada la miré.
Pero si fuerça de amor ordenó el
pensamiento, lealtad virtuosa
causó la lynpieza dél; assi que
por ser della fauorecido y no por
ál lo pensé. Y para mas afearte te
defendere no solo que no entré
en su camara, mas que palabras
de amores iamás le hablé, pues
quando la intencion no peca saluo
está el que se iuzga, y porque la
determinacion desto ha de ser
con la muerte del vno y no con las
lenguas dentramos, quede para el
dia del hecho la sentencia, la qual
fio en Dios se dara por mí, porque
tú reutas con malicia y yo
defiendo con razon y la verdad
determina con iusticia. Las armas
que á mí son de señalar sean a la
bryda segund nuestra costumbre,
nosotros armados de todas
pieças, los cauallos con cubiertas
y cuello y testera, lanças yguales
y sendas espadas sin ninguna
otra arma de las vsadas, con las
quales defendiendo lo dicho, ó
(te) haré desdezir ó echaré del
campo sobrello.
EL AUCTOR
Como la mala fortuna enbidiosa
de los bienes de Leriano vsase
con él de su natural condicion,
diole tal reues quando le vido
mayor en prosperidad. Sus
desdichas causauan pasion á
quien las vio y conbidan á pena á
quien las oye. Pues desando su
cuyta para hablar en su reuto,
despues que respondio al cartel
de Persio como es escrito,
sabiendo el rey que estauan
concertados en la batalla,
aseguró el canpo, y señalando el
lugar donde hiziesen, y
ordenadas todas las cosas que en
tal auto se requerian segund las
ordenanças de Macedonia,
puesto el rey en vn cadahalso,
vinieron los caualleros cada vno
acompañado y fauorecido como
merecía y guardadas en ygualdad
las onrras dentrambos entraron
en el canpo: y como los fieles los
dexaron solos, fueronse el vno
para el otro donde en la fuerça de
los golpes mostraron la virtud de
los animos, y quebradas las
lanças en los primeros encuentros
pusieron mano á las espadas, y
assi se conbatian que quien
quiera ouiera enbidia de lo que
obrauan y compasion de lo que
padecian.
Finalmente, por no detenerme en
esto que parece cuento de
ystorias vieias, Leriano le cortó á
Persio la mano derecha, y como
la meior parte de su persona le
viese perdida dixole: Persio,
porque no pague tu vida por la
falsedad de tu lengua deues te
desdezir. El qual respondio: haz lo
que as de hazer, que aunque me
falta el braço para defender no
me fallece coraçon para morir. Y
oyendo Leriano tal respuesta
diole tanta priesa que lo puso en
la postrimera necesidad; y como
ciertos caualleros sus parientes le
viesen en estrecho de muerte
suplicaron al rey mandase echar
el baston, que ellos le fiauan para
que dél hiziese iusticia si
claramente se hallase culpado; lo
qual el rey assi les otorgó. Y
como fuesen despartidos, Leriano
de tan grande agrauio con mucha
razon se sentio, no podiendo
pensar porqué el rey tal cosa
mandase. Pues como fueron
despartidos, sacaronlos del canpo
yguales en cerimonia avnque
desyguales en fama, y assi los
leuaron á sus posadas donde
estuvieron aquella noche; y otro
dia de mañana avido Leriano su
conseio, acordó de yr á palacio á
suplicar y requerir al rey en
presencia de toda su corte, le
mandase restituir en su onrra
haziendo iusticia de Persio. El
qual como era malino de
condicion y agudo de iuyzio, en
tanto que Leriano lo que es
contado acordaua, hizo llamar
tres onbres muy conformes de
sus costumbres que tenia por
muy suyos, y iuramentandolos
que le guardasen secreto dió á
cada uno infinito dinero porque
dixesen y iurasen al rey que
vieron hablar á Leriano con
Laureola en lugares sospechosos
y en tienpos desonestos, los
quales se profirieron á afirmarlo y
iurarlo hasta perder la vida
sobrello. No quiero dezir lo que
Laureola en todo esto sentia
porque la pasion no turbe el
sentido para acabar lo
començado, porque no tengo
agora menos nueuo su dolor que
quando estaua presente. Pues
tornando á Leriano que mas de su
prision della se dolia que de la
Vitoria dél se gloriaua, como supo
que el rey era leuantado fuese á
palacio y presentes los caualleros
de su corte hizole una habla en
esta manera.
LERIANO AL REY
Por cierto, señor, con mayor
voluntad sufriera el castigo de tu
iusticia que la verguença de tu
presencia, si ayer no leuara lo
meior de la batalla, donde si tú lo
ouieras por bien, de la falsa
acusacion de Persio quedara del
todo libre: que puesto que á vista
de todos yo le diera el galardon
que merecia, gran ventaia va de
hizieralo á hizolo. La razon por
que despartir nos mandaste no la
puedo pensar, en especial
tocando á mi mismo el debate,
que aunque de Laureola
deseases vengança, como
generoso no te faltaria piedad de
padre, comoquiera que en este
caso, bien creo quedaste
satisfecho de tu descargo. Si lo
heziste por conpasion que auias
de Persio, tan iusto fuera que la
vuieras de mi onrra como de su
vida, siendo tu natural. Si por
ventura lo consentiste por verte
aquexado de la suplicacion de
sus parientes, quando les
otorgaste la merced deuieras
acordarte de los seruicios que los
mios te hizieron, pues sabes con
quanta costança de coraçon,
quantos dellos en muchas
batallas y combates perdieron por
tu seruicio las vidas. Nunca
hueste iuntaste que la tercia parte
dellos no fuese. Suplicote que por
iuyzio me satisfagas la onrra que
por mis manos me quitaste: cata
que guardando las leyes se
conseruan los naturales. No
consientas que biua onbre que
tan mal guarda las preeminencias
de sus pasados, porque no
corronpan su benino los que con
él participaren. Por cierto no
tengo otra culpa sino ser amigo
del culpado, y si por este indicio
merezco pena, damela avn que
mi inocencia della me asuelua,
pues conserué su amistad
creyendole bueno y no iuzgandole
malo. Si le das la vida por seruirte
del, digote que te sera el mas leal
cizañador que puedas hallar en el
mundo. Requierote contigo
mismo, pues eres obligado á ser
ygual en derecho, que en esto
determines con la prudencia que
tienes y sentencies con la iusticia
que vsas. Señor, las cosas de
onrra deuen ser claras, y si á este
perdonas por ruegos, ó por ser
principal en tu reyno, ó por lo que
te plazera, no quedará en los
iuyzios de las gentes por
desculpado del todo; que si vnos
creyeren la verdad por razon,
otros la turbarán con malicia: y
digote que en tu reyno lo cierto se
sepa. Nunca la fama leua lexos lo
cierto; como sonará en los otros
lo que es pasado, si queda sin
castigo publico; por Dios, señor,
dexa mi onrra sin disputa, y de mi
vida y lo mio ordena lo que
quisieres.
EL AUCTOR
Atento estuuo el rey á todo lo que
Leriano quiso dezir, y acabada su
habla respondiole que el auria su
conseio sobre lo que deuiese
hazer, que en cosa tal con
deliberacion se auie de dar la
sentencia. Verdad es que la
respuesta del rey no fue tan dulce
como deuiera, lo qual fue porque
si á Laureola daua por libre
segund lo que vido, él no lo
estaua de enoio; porque Leriano
penso de seruilla auiendo por
culpado su pensamiento avnque
no lo fuese su entencion: y asi por
esto como por quitar el escandalo
que andaua entre su parentela y
la de Persio mandóle yr á vna villa
suya que estaua dos leguas de la
corte, llamada Susa, entre tanto
que acordaua en el caso. Lo que
luego hizo con alegre coraçon
teniendo ya á Laureola por
desculpada, cosa que él tanto
deseaua.
Pues como del rey fue despedido,
Persio que siempre se trabaiaua
en ofender su onrra por condicion
y en defenderla por malicia, llamó
los coniurados antes que

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Ebffiledoc - 259download Textbook Antibody Engineering Methods and Protocols Damien Nevoltris Ebook All Chapter PDF

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Antibody Engineering Methods and

Protocols Damien Nevoltris

Antibody Arrays: Methods and Protocols Kelly C.

Computer-Aided Tissue Engineering: Methods and

Systems Metabolic Engineering Methods and Protocols 1st

Yeast Metabolic Engineering Methods and Protocols 1st

Zebrafish Methods and Protocols Koichi Kawakami

SNAREs: Methods and Protocols Rutilio Fratti

Epitranscriptomics: Methods and Protocols Narendra

Phytoplasmas: Methods and Protocols Rita Musetti

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Printed on acid-free paper

Darlinghurst, NSW, Australia Damien Nevoltris

PART I IN SILICO METHODS

PART II GENERATION OF DIVERSITY

4 Construction of Human Naı̈ve Antibody Gene Libraries . . . . . . . . . . . . . . . . . . . . . 73

PART III SELECTIONS OF LEAD CANDIDATE

11 Selection of Antibody Fragments Against Structured DNA by

PART IV PRODUCTION OF RECOMBINANT ANTIBODIES

16 Expression of IgG Monoclonals with Engineered Immune

PART VI CHARACTERIZATION, OPTIMIZATION, AND INNOVATIVE FORMATS

22 Measuring Antibody-Antigen Binding Kinetics Using Surface

MAHMOUD ABDELATTI  Garvan Institute of Medical Research, Sydney, NSW, Australia;

STEFANIE DERER  Institute of Nutritional Medicine, Molecular Gastroenterology, University

DOREEN KÖNNING  Institute for Organic Chemistry and Biochemistry, Technische

PIERRE MARTINEAU  Institut de Recherche en Cancérologie de Montpellier (IRCM),

SILVIA ZAMBONI  Department of Neuroscience, The National Institute of Health, Rome,

Antibody Design and Humanization via In Silico Modeling

Historically, antibodies have been generated mainly using mouse

reaction in humans. Researchers then replaced mouse Fc (fragment

CHARMM). If there are steric clashes between frameworks and

Antibody humanization can be undertaken either using commer-

1. To delineate boundaries of mouse heavy and light chain, user

1. The first step is to obtain the mouse antibody variable and

Fig. 1 (a) Antibody humanization steps are described in this workflow. As

4. This humanized homology model has human frameworks and

1. To explore some additional examples of mouse antibodies for

Fig. 1 (continued) sequence was utilized to create a 3D homology model, CDRs

2. If using PIGS for antibody model building with VH and VL as

5. To fix these steric clashes, if using 3MBX as an example, in the

immunogenicity resides in the CDR regions. design. MAbs 7(6):1045–1057. https://doi.

Antibody Affinity Maturation by Computational Design

Computational methods are widely accepted as essential tools for

CARTEL DE PERSIO PARA

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MAHMOUD ABDELATTI Garvan Institute of Medical Research, Sydney, NSW, Australia;

STEFANIE DERER Institute of Nutritional Medicine, Molecular Gastroenterology, University

DOREEN KÖNNING Institute for Organic Chemistry and Biochemistry, Technische

PIERRE MARTINEAU Institut de Recherche en Cancérologie de Montpellier (IRCM),

SILVIA ZAMBONI Department of Neuroscience, The National Institute of Health, Rome,