Single Domain Antibodies Greg

Hussack Kevin A Henry

Visit to download the full and correct content document:
https://ebookmeta.com/product/single-domain-antibodies-greg-hussack-kevin-a-henry
/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Therapeutic Antibodies Methods and Protocols 1st

Edition Gunnar Houen

https://ebookmeta.com/product/therapeutic-antibodies-methods-and-
protocols-1st-edition-gunnar-houen/

Domain Storytelling A Collaborative Visual and Agile

Way to Build Domain Driven Software 1st Edition Stefan
Hofer Henning Schwentner

https://ebookmeta.com/product/domain-storytelling-a-
collaborative-visual-and-agile-way-to-build-domain-driven-
software-1st-edition-stefan-hofer-henning-schwentner/

Single Dad Billionaire s Nanny A Single Dad Age Gap

Romance 1st Edition Lynne Sweet

https://ebookmeta.com/product/single-dad-billionaire-s-nanny-a-
single-dad-age-gap-romance-1st-edition-lynne-sweet/

Semantic Control for the Cybersecurity Domain

Investigation on the Representativeness of a Domain
Specific Terminology Referring to Lexical Variation 1st
Edition Claudia Lanza
https://ebookmeta.com/product/semantic-control-for-the-
cybersecurity-domain-investigation-on-the-representativeness-of-
a-domain-specific-terminology-referring-to-lexical-variation-1st-
Henry Dresser and Victorian ornithology: Birds, books
and business 1st Edition Henry A. Mcghie

https://ebookmeta.com/product/henry-dresser-and-victorian-
ornithology-birds-books-and-business-1st-edition-henry-a-mcghie/

Dichronauts Greg Egan

https://ebookmeta.com/product/dichronauts-greg-egan/

Fractures in Sport Greg A. J. Robertson & Nicola

Maffuli

https://ebookmeta.com/product/fractures-in-sport-greg-a-j-
robertson-nicola-maffuli/

Scientific Reasoning and Argumentation The Roles of

Domain Specific and Domain General Knowledge 1st
Edition Frank Fischer (Editor)

https://ebookmeta.com/product/scientific-reasoning-and-
argumentation-the-roles-of-domain-specific-and-domain-general-
knowledge-1st-edition-frank-fischer-editor/

Financial Statements Greg Shields

https://ebookmeta.com/product/financial-statements-greg-shields/
Methods in
Molecular Biology 2446

Greg Hussack
Kevin A. Henry Editors

Single-Domain
Antibodies
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Single-Domain Antibodies

Methods and Protocols

Edited by

Greg Hussack and Kevin A. Henry

Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada
Editors
Greg Hussack Kevin A. Henry
Human Health Therapeutics Research Centre Human Health Therapeutics Research Centre
National Research Council Canada National Research Council Canada
Ottawa, ON, Canada Ottawa, ON, Canada

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2074-8 ISBN 978-1-0716-2075-5 (eBook)
https://doi.org/10.1007/978-1-0716-2075-5
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2022, Corrected Publication 2022
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

Nearly 50 years after their first isolation, monoclonal antibodies continue to play vital roles
in research and medicine. Expansion of antibody therapeutic formats to include multi-
specific antibodies, chimeric antigen receptors (CARs), and various types of “armed”
antibody conjugates has placed antibody fragments in the spotlight. For applications requir-
ing fragments, the naturally occurring autonomous variable domains of camelid heavy
chain-only antibodies (VHHs, nanobodies) and shark immunoglobulin new antigen recep-
tors (VNARs) have significant advantages over fragments derived from conventional tetra-
meric antibodies such as Fabs or scFvs. Collectively known as single-domain antibodies
(sdAbs), these molecules are the minimal antigen-binding competent representations of
vertebrate adaptive immune receptors.
The many attractive properties of sdAbs (high monovalent affinity; stability and revers-
ible unfolding; solubility and monomericity; ease of recombinant production in microbial
and eukaryotic cells; modularity in a variety of fusion formats; small size enabling tissue
penetration; recognition of “cryptic” epitopes inaccessible to conventional antibodies) are
now well established. These properties form the basis for a variety of applications of sdAbs as
affinity adsorbents, crystallization chaperones, tracers for in vivo imaging, intrabodies for
studying cellular processes, diagnostic agents, and multi-functional therapeutics for a range
of disease indications. As research tools, sdAbs have been invaluable in advancing several
fields of biological science.
This edition of Single-Domain Antibodies: Methods and Protocols, an update on the 2012
book of the same name, is divided into five sections. Part I provides a general introduction to
sdAbs. Part II covers techniques used for isolation of sdAbs from several platforms. Part III
includes techniques for production of sdAbs and related molecules in various organisms.
Part IV addresses strategies for sdAb engineering, humanization, multimerization, labeling,
and characterization. Part V comprises chapters dedicated to specific sdAb applications.
Since the publication of the last edition, significant progress has been made in the site-
specific labeling of sdAbs which can be useful in several applications.
We hope the contents of the book are useful and accessible to scientists embarking on
investigations in this area for many years to come.

Ottawa, Canada Greg Hussack

Kevin A. Henry

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I INTRODUCTION TO SINGLE-DOMAIN ANTIBODIES

1 Nanobodies: From Serendipitous Discovery of Heavy Chain-Only
Antibodies in Camelids to a Wide Range of Useful Applications . . . . . . . . . . . . . . 3
Fangling Ji, Jun Ren, Cécile Vincke, Lingyun Jia, and Serge Muyldermans
2 Overview, Generation, and Significance of Variable New Antigen Receptors
(VNARs) as a Platform for Drug and Diagnostic Development . . . . . . . . . . . . . . . 19
Samata S. Pandey, Marina Kovaleva, Caroline J. Barelle,
and Obinna C. Ubah

PART II ISOLATION OF SINGLE-DOMAIN ANTIBODIES

3 Llama DNA Immunization and Isolation of Functional Single-Domain

Antibody Binders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Frédéric Trempe, Martin A. Rossotti, Tahir Maqbool, C. Roger MacKenzie,
and Mehdi Arbabi-Ghahroudi
4 Preparation of Immune and Synthetic VNAR Libraries as Sources
of High-Affinity Binders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Jahaziel Gasperin-Bulbarela, Olivia Cabanillas-Bernal, Salvador Dueñas,
and Alexei F. Licea-Navarro
5 Isolation of Single-Domain Antibodies to Transmembrane Proteins
Using Magnetized Yeast Cell Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Kaitlyn Bacon, Stefano Menegatti, and Balaji M. Rao
6 A Transgenic Heavy Chain IgG Mouse Platform as a Source
of High Affinity Fully Human Single-Domain Antibodies for Therapeutic
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Dubravka Drabek, Rick Janssens, Rien van Haperen, and Frank Grosveld

PART III EXPRESSION OF SINGLE-DOMAIN ANTIBODIES

7 Cytoplasmic Production of Nanobodies and Nanobody-Based Reagents

by Co-Expression of Sulfhydryl Oxidase and DsbC Isomerase . . . . . . . . . . . . . . . . 145
Ario de Marco
8 Small-Scale Secretory VHH Expression in Saccharomyces cerevisiae . . . . . . . . . . . . 159
Michiel M. Harmsen, Marga van Hagen-van Setten,
and Peter T. J. Willemsen
9 Production of Single-Domain Antibodies in Pichia pastoris . . . . . . . . . . . . . . . . . . 181
Yusei Matsuzaki, Kaho Kajiwara, Wataru Aoki, and Mitsuyoshi Ueda
10 Production of Designer VHH-Based Antibodies in Plants . . . . . . . . . . . . . . . . . . . 205
Henri De Greve
vii
viii Contents

PART IV SINGLE-DOMAIN ANTIBODY ENGINEERING AND CHARACTERIZATION

11 Assessing the Aggregation Propensity of Single-Domain Antibodies

upon Heat-Denaturation Employing the ΔTm Shift . . . . . . . . . . . . . . . . . . . . . . . . . 233
Patrick Kunz
12 Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation
DNA Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Michael J. Lowden, Henk van Faassen, Shalini Raphael, Shannon Ryan,
Greg Hussack, and Kevin A. Henry
13 Engineering pH-Sensitive Single-Domain Antibodies . . . . . . . . . . . . . . . . . . . . . . . 269
Tosha M. Laughlin and James R. Horn
14 Humanization of Camelid Single-Domain Antibodies . . . . . . . . . . . . . . . . . . . . . . . 299
Traian Sulea
15 Creation of Multimeric Single-Domain Antibodies
Using Bacterial Superglues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Paul J. Wichgers Schreur, Sandra van de Water, and Jeroen Kortekaas
16 Introducing Cysteines into Nanobodies for Site-Specific Labeling . . . . . . . . . . . . 327
Simon Boje Hansen and Kasper Røjkjær Andersen
17 Affinity-Guided Site-Selective Labeling of Nanobodies
with Aldehyde Handles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Anders M€ a rcher and Kurt V. Gothelf
18 Cytoplasmic Expression of Nanobodies with Formylglycine Generating
Enzyme Tag and Conversion to a Bio-Orthogonal Aldehyde Group . . . . . . . . . . 357
Da Li, Qiang Peng, Chungdong Huang, Berlin Zang, Jun Ren,
Fangling Ji, Serge Muyldermans, and Lingyun Jia
19 Site-Specific Fluorescent Labeling, Single-Step Immunocytochemistry,
and Delivery of Nanobodies into Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Jan Gettemans
20 Design and Validation of Site-Specifically Labeled Single-Domain
Antibody-Based Tracers for in Vivo Fluorescence Imaging and Image-Guided
Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Noemi B. Declerck, Lukasz Mateusiak, and Sophie Hernot
21 Design and Preparation of Photobodies: Light-Activated Single-Domain
Antibody Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Zahide Yilmaz, Benedikt Jedlitzke, and Henning D. Mootz

PART V APPLICATIONS OF SINGLE-DOMAIN ANTIBODIES

22 Visualizing Filoviral Nucleoproteins Using Nanobodies Fused to the Ascorbate

Peroxidase Derivatives APEX2 and dEAPX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
Laura Jo Sherwood and Andrew Hayhurst
23 Development of Glypican-2 Targeting Single-Domain Antibody CAR T Cells
for Neuroblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Nan Li and Mitchell Ho
24 Single-Domain Antibodies for Intracellular Toxin Neutralization . . . . . . . . . . . . . 469
Timothy F. Czajka and Nicholas J. Mantis
 Contents ix

25 Generation of Single-Domain Antibody-Based Recombinant

Immunotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Bryan D. Fleming and Mitchell Ho
26 X-ray Crystal Structure Analysis of VHH–Protein Antigen Complexes . . . . . . . . 513
Angham M. Ahmed and Cory L. Brooks
27 Functionalization of Magnetic Beads with Biotinylated Nanobodies
for MALDI-TOF/MS-Based Quantitation of Small Analytes. . . . . . . . . . . . . . . . . 531
Gabriel Lassabe, Macarena Pı́rez-Schirmer,
and Gualberto González-Sapienza
28 Nanobody-Based Assays for the Detection of Environmental
and Agricultural Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Feng Wang and Hong Wang
29 Peptide-Tag Specific Nanobodies for Studying Proteins in Live Cells . . . . . . . . . . 555
Funmilayo O. Fagbadebo and Ulrich Rothbauer
30 Nanobody-Based GFP Traps to Study Protein Localization and Function
in Developmental Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Shinya Matsuda, Gustavo Aguilar, M. Alessandra Vigano,
and Markus Affolter
31 Optogenetic Activation of Intracellular Nanobodies. . . . . . . . . . . . . . . . . . . . . . . . . 595
Daseuli Yu and Heo Won Do
Correction to: Optogenetic Activation of Intracellular Nanobodies. . . . . . . . . . . . . . . . C1

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
Contributors

MARKUS AFFOLTER • Biozentrum, University of Basel, Basel, Switzerland

GUSTAVO AGUILAR • Biozentrum, University of Basel, Basel, Switzerland
ANGHAM M. AHMED • Department of Chemistry and Biochemistry, California State
University Fresno, Fresno, CA, USA
KASPER RØJKJÆR ANDERSEN • Department of Molecular Biology and Genetics, Aarhus
University, Aarhus C, Denmark
WATARU AOKI • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
University, Kyoto, Japan; Core Research for Evolutionary Science and Technology
(CREST), Japan Science and Technology Agency (JST), Tokyo, Japan
MEHDI ARBABI-GHAHROUDI • Human Health Therapeutics Research Centre, National
Research Council Canada, Ottawa, ON, Canada; Department of Biochemistry,
Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada; Department
of Biology, Carleton University, Ottawa, ON, Canada
KAITLYN BACON • Department of Chemical and Biomolecular Engineering, North Carolina
State University, Raleigh, NC, USA
CAROLINE J. BARELLE • Elasmogen Limited, Aberdeen, UK
CORY L. BROOKS • Department of Chemistry and Biochemistry, California State University
Fresno, Fresno, CA, USA
OLIVIA CABANILLAS-BERNAL • Biomedical Innovation Department, CICESE, Zona Playitas,
Ensenada, Mexico
TIMOTHY F. CZAJKA • Department of Biomedical Sciences, University at Albany School of
Public Health, Albany, NY, USA
NOEMI B. DECLERCK • Laboratory for In Vivo Cellular and Molecular Imaging, ICMI-
BEFY/MIMA, Vrije Universiteit Brussel, Brussels, Belgium
HENRI DE GREVE • VIB-VUB Center for Structural Biology, Brussels, Belgium; Structural
Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium
ARIO DE MARCO • University of Nova Gorica (UNG), Nova Gorica, Slovenia
HEO WON DO • Life Science Research Institute, Korea Advanced Institute of Science and
Technology, Daejeon, Republic of Korea; KAIST Institute for the BioCentury, Korea
Advanced Institute of Science and Technology, Daejeon, Republic of Korea
DUBRAVKA DRABEK • Cell Biology Department, Erasmus Medical Center, Rotterdam, The
Netherlands; Harbour BioMed, Rotterdam, The Netherlands
SALVADOR DUEÑAS • Biomedical Innovation Department, CICESE, Zona Playitas,
Ensenada, Mexico
FUNMILAYO O. FAGBADEBO • Pharmaceutical Biotechnology, Eberhard Karls University
Tuebingen, Tuebingen, Germany
BRYAN D. FLEMING • Laboratory of Molecular Biology, National Cancer Institute, National
Institutes of Health, Bethesda, MD, USA
JAHAZIEL GASPERIN-BULBARELA • Biomedical Innovation Department, CICESE, Zona
Playitas, Ensenada, Mexico
JAN GETTEMANS • Department of Biomolecular Medicine, Faculty of Medicine and Health
Sciences, Ghent University, Ghent, Belgium

xi
xii Contributors

GUALBERTO GONZÁLEZ-SAPIENZA • Cátedra de Inmunologı́a, Facultad de Quı́mica,

Instituto de Higiene, UdelaR, Montevideo, Uruguay
KURT V. GOTHELF • Department of Chemistry and Interdisciplinary Nanoscience Centre
(iNANO), Aarhus University, Aarhus, Denmark
FRANK GROSVELD • Cell Biology Department, Erasmus Medical Center, Rotterdam, The
Netherlands; Harbour BioMed, Rotterdam, The Netherlands
SIMON BOJE HANSEN • Department of Molecular Biology and Genetics, Aarhus University,
Aarhus C, Denmark
MICHIEL M. HARMSEN • Wageningen Bioveterinary Research, Lelystad, The Netherlands
ANDREW HAYHURST • Disease Intervention and Prevention, Texas Biomedical Research
Institute, San Antonio, TX, USA
KEVIN A. HENRY • Human Health Therapeutics Research Centre, Life Sciences Division,
National Research Council Canada, Ottawa, ON, Canada; Department of Biochemistry,
Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON,
Canada
SOPHIE HERNOT • Laboratory for In Vivo Cellular and Molecular Imaging, ICMI-BEFY/
MIMA, Vrije Universiteit Brussel, Brussels, Belgium
MITCHELL HO • Laboratory of Molecular Biology, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, Bethesda, MD, USA
JAMES R. HORN • Department of Chemistry and Biochemistry, Northern Illinois University,
DeKalb, IL, USA
CHUNGDONG HUANG • Liaoning Key Laboratory of Molecular Recognition and Imaging,
School of Bioengineering, Dalian University of Technology, Dalian, Liaoning, China
GREG HUSSACK • Human Health Therapeutics Research Centre, Life Sciences Division,
National Research Council Canada, Ottawa, ON, Canada
RICK JANSSENS • Cell Biology Department, Erasmus Medical Center, Rotterdam, The
Netherlands; Harbour BioMed, Rotterdam, The Netherlands
BENEDIKT JEDLITZKE • Department of Chemistry and Pharmacy, Institute of Biochemistry,
University of Muenster, Münster, Germany
FANGLING JI • Liaoning Key Laboratory of Molecular Recognition and Imaging, School of
Bioengineering, Dalian University of Technology, Dalian, Liaoning, China
LINGYUN JIA • Liaoning Key Laboratory of Molecular Recognition and Imaging, School of
Bioengineering, Dalian University of Technology, Dalian, Liaoning, China
KAHO KAJIWARA • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
University, Kyoto, Japan
JEROEN KORTEKAAS • Department of Virology and Molecular Biology, Wageningen
Bioveterinary Research, Lelystad, The Netherlands; Laboratory of Virology, Wageningen
University, Lelystad, The Netherlands
MARINA KOVALEVA • Elasmogen Limited, Aberdeen, UK
PATRICK KUNZ • Coriolis Pharma Research GmbH, Martinsried, Germany
GABRIEL LASSABE • Cátedra de Inmunologı́a, Facultad de Quı́mica, Instituto de Higiene,
UdelaR, Montevideo, Uruguay
TOSHA M. LAUGHLIN • Department of Chemistry and Biochemistry, Northern Illinois
University, DeKalb, IL, USA
DA LI • Liaoning Key Laboratory of Molecular Recognition and Imaging, School of
Bioengineering, Dalian University of Technology, Dalian, Liaoning, China
NAN LI • Laboratory of Molecular Biology, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, MD, USA
 Contributors xiii

ALEXEI F. LICEA-NAVARRO • Biomedical Innovation Department, CICESE, Zona Playitas,

Ensenada, Mexico
MICHAEL J. LOWDEN • Human Health Therapeutics Research Centre, Life Sciences Division,
National Research Council Canada, Ottawa, ON, Canada
C. ROGER MACKENZIE • Human Health Therapeutics Research Centre, National Research
Council Canada, Ottawa, ON, Canada
NICHOLAS J. MANTIS • Department of Biomedical Sciences, University at Albany School of
Public Health, Albany, NY, USA; Division of Infectious Diseases, New York State
Department of Health, Wadsworth Center, Albany, NY, USA
TAHIR MAQBOOL • Cedarlane Labs, Burlington, ON, Canada
ANDERS MA€ RCHER • Department of Chemistry and Interdisciplinary Nanoscience Centre
(iNANO), Aarhus University, Aarhus, Denmark
LUKASZ MATEUSIAK • Laboratory for In Vivo Cellular and Molecular Imaging, ICMI-
BEFY/MIMA, Vrije Universiteit Brussel, Brussels, Belgium
SHINYA MATSUDA • Biozentrum, University of Basel, Basel, Switzerland
YUSEI MATSUZAKI • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
University, Kyoto, Japan
STEFANO MENEGATTI • Department of Chemical and Biomolecular Engineering, North
Carolina State University, Raleigh, NC, USA; Biomanufacturing Training and
Education Center (BTEC), North Carolina State University, Raleigh, NC, USA
HENNING D. MOOTZ • Department of Chemistry and Pharmacy, Institute of Biochemistry,
University of Muenster, Münster, Germany
SERGE MUYLDERMANS • Liaoning Key Laboratory of Molecular Recognition and Imaging,
School of Bioengineering, Dalian University of Technology, Dalian, Liaoning, China;
Cellular and Molecular Immunology Laboratory, Vrije Universiteit Brussel, Brussels,
Belgium
SAMATA S. PANDEY • Elasmogen Limited, Aberdeen, UK
QIANG PENG • Liaoning Key Laboratory of Molecular Recognition and Imaging, School of
Bioengineering, Dalian University of Technology, Dalian, Liaoning, China
MACARENA PÍREZ-SCHIRMER • Cátedra de Inmunologı́a, Facultad de Quı́mica, Instituto de
Higiene, UdelaR, Montevideo, Uruguay
BALAJI M. RAO • Department of Chemical and Biomolecular Engineering, North Carolina
State University, Raleigh, NC, USA; Biomanufacturing Training and Education Center
(BTEC), North Carolina State University, Raleigh, NC, USA
SHALINI RAPHAEL • Human Health Therapeutics Research Centre, Life Sciences Division,
National Research Council Canada, Ottawa, ON, Canada
JUN REN • Liaoning Key Laboratory of Molecular Recognition and Imaging, School of
Bioengineering, Dalian University of Technology, Dalian, Liaoning, China
MARTIN A. ROSSOTTI • Human Health Therapeutics Research Centre, National Research
Council Canada, Ottawa, ON, Canada
ULRICH ROTHBAUER • Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen,
Tuebingen, Germany; Natural and Medical Sciences Institute at the University of
Tuebingen, Reutlingen, Germany
SHANNON RYAN • Human Health Therapeutics Research Centre, Life Sciences Division,
National Research Council Canada, Ottawa, ON, Canada
LAURA JO SHERWOOD • Disease Intervention and Prevention, Texas Biomedical Research
Institute, San Antonio, TX, USA
xiv Contributors

TRAIAN SULEA • Human Health Therapeutics Research Centre, National Research Council
Canada, Montreal, QC, Canada; Institute of Parasitology, McGill University, Sainte-
Anne-de-Bellevue, QC, Canada
FRÉDÉRIC TREMPE • Human Health Therapeutics Research Centre, National Research
Council Canada, Ottawa, ON, Canada
OBINNA C. UBAH • Elasmogen Limited, Aberdeen, UK
MITSUYOSHI UEDA • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto
University, Kyoto, Japan; Core Research for Evolutionary Science and Technology
(CREST), Japan Science and Technology Agency (JST), Tokyo, Japan
SANDRA VAN DE WATER • Department of Virology and Molecular Biology, Wageningen
Bioveterinary Research, Lelystad, The Netherlands
HENK VAN FAASSEN • Human Health Therapeutics Research Centre, Life Sciences Division,
National Research Council Canada, Ottawa, ON, Canada
MARGA VAN HAGEN-VAN SETTEN • Wageningen Bioveterinary Research, Lelystad, The
Netherlands
RIEN VAN HAPEREN • Cell Biology Department, Erasmus Medical Center, Rotterdam, The
Netherlands; Harbour BioMed, Rotterdam, The Netherlands
M. ALESSANDRA VIGANO • Biozentrum, University of Basel, Basel, Switzerland
CÉCILE VINCKE • Cellular and Molecular Immunology Laboratory, Vrije Universiteit
Brussel, Brussels, Belgium; Myeloid Cell Immunology Laboratory, VIB Center for
Inflammation Research, Brussels, Belgium
FENG WANG • College of Food Science, South China Agricultural University, Guangzhou,
China
HONG WANG • College of Food Science, South China Agricultural University, Guangzhou,
China
PAUL J. WICHGERS SCHREUR • Department of Virology and Molecular Biology, Wageningen
Bioveterinary Research, Lelystad, The Netherlands
PETER T. J. WILLEMSEN • Wageningen Bioveterinary Research, Lelystad, The Netherlands
ZAHIDE YILMAZ • Department of Chemistry and Pharmacy, Institute of Biochemistry,
University of Muenster, Münster, Germany
DASEULI YU • Life Science Research Institute, Korea Advanced Institute of Science and
Technology, Daejeon, Republic of Korea
BERLIN ZANG • Liaoning Key Laboratory of Molecular Recognition and Imaging, School of
Bioengineering, Dalian University of Technology, Dalian, Liaoning, China
 Part I

Introduction to Single-Domain Antibodies

 Chapter 1

Nanobodies: From Serendipitous Discovery of Heavy

Chain-Only Antibodies in Camelids to a Wide Range
of Useful Applications
Fangling Ji, Jun Ren, Cécile Vincke, Lingyun Jia, and Serge Muyldermans

Abstract
The presence of unique heavy chain-only antibodies (HCAbs) in camelids was discovered at Vrije Uni-
versiteit Brussel (VUB, Brussels, Belgium) at a time when many researchers were exploring the cloning and
expression of smaller antigen-binding fragments (Fv and Fab) from hybridoma-derived antibodies. The
potential importance of this discovery was anticipated, and efforts were immediately undertaken to
understand the emergence and ontogeny of these HCAbs as well as to investigate the applications of the
single-domain antigen-binding variable domains of HCAbs (nanobodies). Nanobodies were demonstrated
to possess multiple biochemical and biophysical advantages over other antigen-binding antibody fragments
and alternative scaffolds. Today, nanobodies have a significant and growing impact on research, biotech-
nology, and medicine.

Key words Camels, Llamas, Heavy chain antibodies, Single domain antibodies, Nanobodies, VHHs

1 Introduction

A serendipitous discovery in the laboratory of Professor R. Hamers

(Vrije Universiteit Brussel) revealed that camelids (camels, dromed-
aries, llamas, and alpacas) produced, in addition to conventional
antibodies, unique antibodies lacking light chains in their blood
[1]. These heavy chain-only antibodies (HCAbs) consisted of a
dimer of identical heavy chains, in which the CH1 domain is
missing and the variable heavy chain domains harbor remarkable
amino acid substitutions in an otherwise conserved region impli-
cated in VL domain pairing in conventional antibodies [2]. The
HCAbs from a dromedary infected with trypanosomes were able to
immuno-capture trypanosome antigens, proving that HCAbs were
active in antigen recognition [1]. From the architecture of the
HCAb it was anticipated that each variable domain could bind to
a cognate antigen. Hence, the variable domain of a HCAb is the

Greg Hussack and Kevin A. Henry (eds.), Single-Domain Antibodies: Methods and Protocols,
Methods in Molecular Biology, vol. 2446, https://doi.org/10.1007/978-1-0716-2075-5_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

3
4 Fangling Ji et al.

Fig. 1 Top: schematic genome organization of the igh locus on chromosome 6 in the Bactrian camel. The IGHV
genes (grey) are used to generate the VH domains of classical heterotetrameric antibodies and the IGHVH
genes (amber) are used for recombination with IGHD (red) and IGHJ (green) genes to form the VHH domains.
The VHH domains are then recombined with polypeptides encoded by dedicated IGHGH genes (cyan) devoid of
CH1 domains. These truncated H-chains dimerize in the absence of a light chain and form the homodimeric
HCAb. Cloning and expressing the IGHVH-IGHD-IGHJ recombination product in microorganisms yields soluble
nanobodies (Nbs). Bottom: for reference, the structure of a classical heterotetrameric antibody is shown. The
antigen binding fragment (Fab) of classical antibodies is the equivalent of the Nb of HCAbs

equivalent of the Fab region of a conventional antibody (Fig. 1).

The unconventional variable domains of HCAbs were originally
referred to as VHHs (variable domains of the heavy chains of
HCAbs). VHHs can be cloned using recombinant DNA
 Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in. . . 5

technologies, selected for antigen-binding, and expressed in bacte-

ria or yeast [3, 4]. The recombinant VHH domain has a diameter of
about 2.5 nm and a length of about 4.5 nm. Because of these
dimensions in the nanometer scale, the name “nanobody”
(Nb) was registered by Ablynx. By definition, a Nb is the recombi-
nant, antigen-binding variable domain derived from a functional
heavy chain-only antibody naturally occurring in camelids.
At the time of the serendipitous discovery of functional HCAbs
in camelids, many laboratories were exploring the expression of
Fabs (antigen-binding fragments) or scFvs (single chain variable
fragments) derived from classical antibodies [5, 6] in microorgan-
isms. However, production yields were not consistently high. In
addition, the first antibody humanization protocol was reported
around this time, whereby the antigen-binding loops of murine VH
and VL domains were grafted onto human VH and VL scaffolds to
generate a reshaped Fv [7]. This success coincided with the cloning
of VH and VL (or Fab) repertoires, soon followed by the selection
of antigen-binding scFvs by phage display [8]. However, early
attempts to isolate functional Fabs and scFvs were not always
successful because: (1) the cloning efficiency of VH and VL reper-
toires was suboptimal, (2) the original VH-VL pair that was affinity
matured in vivo during immunization or vaccination was scrambled
during scFv assembly, (3) the best scFv binders were often difficult
to express in high yields, and (4) the selected scFvs were fragile and
sometimes unstable under physiological conditions.
In view of the difficulties encountered with scFvs and Fabs, it
was logical to attempt to substitute these with VHHs [9], while
concurrently investigating the structure [10] as well as the bio-
chemical and biophysical properties of Nbs [11]. Fortunately,
Nbs were well expressed in bacteria and folded into stable mono-
meric entities; these unique properties offer several advantages over
other antigen-binding fragments. As a result, many researchers
throughout the world have adopted Nb technology and developed
multiple applications for these molecules far beyond the wildest
dreams and original expectations of the discoverers of camelid
HCAbs.

2 Emergence and Ontogeny of VHHs and HCAbs

At the time of their discovery, it was clear that HCAbs circulating in

blood of camelids were of the IgG isotype. They could be purified
on Protein A columns and a subset could also be purified on
Protein G columns [1]. The widespread occurrence of HCAbs in
both old-world Camelidae (Bactrian camel and dromedary) and
new-world Camelidae (llama, guanaco, alpaca, and vicuña), and
their absence in Ruminantia (cattle, antelopes, and giraffes) and
Suidae (pigs and hippopotami) was a first indication that HCAbs
6 Fangling Ji et al.

emerged after the Tylopoda split from other Artiodactyla and

before Laminae and Camelinae became geographically
separated [12].
All camelids possess dedicated genes to assemble HCAbs
(Fig. 1). The igh locus contains dedicated IGHGH genes bearing
point mutations at the 50 splicing site of the intron between the
CH1 and hinge exons [13, 14]. Consequently, the splicing site is
no longer recognized such that the CH1 exon is eliminated during
splicing, explaining the lack of a CH1 domain in HCAbs. The llama
and alpaca has two IGHGH genes bearing this particular point
mutation (IGHGH2a and IGHGH2b genes), whereas in Bactrian
camels and dromedaries there appears to be three IGHGH genes
(IGHGH2a, IGHGH2b and IGHGH3) encoding the constant
regions of HCAbs [15, 16]. The IGHV gene cluster contains
IGHVH genes dedicated to the generation of HCAbs.
An initial study suggested the presence of 33 IGHVH genes in
camels, nearly as many as the IGHV genes of conventional hetero-
tetrameric antibodies (39 unique genes) [17]. This study was
mainly based on sequencing of cloned amplicons obtained after
PCR from dromedary genomic DNA, which may have been biased.
In contrast, deep sequencing of Bactrian camel DNA suggested
that only 13 IGHV genes and four IGHVH genes are present in this
species [16]. The IGHVH genes are distinguishable from IGHV
genes since they encode Tyr or Phe at position 42, Arg or Cys at
position 50, and Gly at position 52 instead of conserved Val, Leu,
and Trp, respectively. The IGHV and IGHVH genes are inter-
spersed in the cluster and can recombine with any of a set of
IGHD and IGHJ genes located upstream of the IGHM gene
[17, 18]. Therefore somatic hypermutation, introduced after the
initial IGHVH-IGHD-IGHJ recombination event, expands the
diversity of the primary repertoire, probably in an antigen-
independent fashion. Of note, the IGHV genes can also be used
to generate HCAbs. Camels and llamas even have an IGHV gene
closely related to human IGHV4 genes that appears equally well
suited for the generation of classical antibodies and HCAbs [19].

3 Identification of Antigen-Specific Nbs

Several techniques are available to isolate Nbs against virtually any

target starting from an immune, naı̈ve, or synthetic Nb library
[20]. Antigen-binding Nbs are retrieved successfully and rapidly
form these libraries by phage display, bacterial display, yeast display,
ribosome display, or a combination of these technologies. Other
strategies have been developed as well, such as mass spectrometry-
based techniques [21] or CIS display [22].
The key step in generation of immune Nb libraries is the
immunization of a llama, alpaca, dromedary, or Bactrian camel
Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in. . . 7

with the antigen. These animals are available throughout the world
and their immunization requires authorization from an animal
ethics committee. The immunization step might take 6–12 weeks
and requires about 0.5 mg of antigen in total although the exact
amount of antigen will largely depend on its immunogenicity. The
quality of the antigen is very important since properly folded, stable
proteins are most likely to elicit a vigorous immune response.
Aggregated, degraded, proteolyzed, or unstable proteins, as well
as flexible oligopeptides or small haptens, are all weaker immuno-
gens. Weaker immunogenicity does not guarantee that it will be
impossible to raise an immune response, as several Nbs have been
generated against small or flexible targets [23]. Of note, multiple
antigens can be mixed and used to immunize a single animal. After
the immunization, a small aliquot of anti-coagulated blood
(50–100 mL) contains sufficient HCAb-producing B cells to
clone the Nb repertoire and to construct the immune library. An
adequate immune Nb repertoire comprises approximately 106–108
clones.
While immune Nb libraries can be cloned from blood or lymph
nodes of immunized camelids, several groups invested in the gen-
eration of transgenic mice in which IGHM and/or IGHG genes
were truncated to have their CH1 domain deleted [24, 25]. Subse-
quently, transgenic mice were generated in which endogenous
murine IGHV genes were inactivated and either camelid IGHVH
genes or autonomous human IGHV genes were knocked in. The
immunization of such transgenic mice has advantages over llama or
camel immunization since lower amounts of antigen will be
required and animal husbandry costs are typically less. In addition,
the use of human IGHV genes in transgenic mice results in genera-
tion of HCAbs equipped with an autonomous yet fully human VH
domain. This may avoid immunogenicity issues when the resulting
Nbs are employed for human therapy.
At least in Europe, there is a strong drive to forbid the immu-
nization of animals for antibody production since alternative tech-
nologies are available whereby a naı̈ve or synthetic VHH library is
used as the primary source for selecting antigen-specific Nbs
[26]. The use of naı̈ve or synthetic Nb libraries bypasses the
requirement to immunize. However, for naı̈ve libraries one should
clone a large repertoire obtained from a large volume of blood from
several camelids. Even then, in view of the absence of VH-VL
combinatorial diversity and the presence of only a handful
IGHVH genes in the genome of camels [16], there remain doubts
regarding the diversity that can be obtained in a naı̈ve library and
the antigen-binding affinity of isolated Nbs. This does not exclude
the possibility to retrieve practical binders from naı̈ve libraries
[27]. However, naı̈ve libraries should contain at least 1010 individ-
ual clones, which is the maximum that can be handled in phage
8 Fangling Ji et al.

display selections, and larger libraries can be employed using ribo-

some display.
To construct synthetic libraries, one should first select a few
VHHs that are well expressed and stable. These VHHs will serve as
scaffolds in which codons for amino acids of the antigen-binding
loops are randomized with mutagenic oligonucleotides. Here
again, the larger the library size (i.e., the larger the sequence
diversity) the higher the potential antigen-binding affinity of the
retrieved Nbs. Library size limitations will be dictated by the selec-
tion approach: phage display can handle up to 1010 clones, while
ribosome display can handle approximately 1014 clones [28, 29].
Potent Nb selection procedures (based on bacteriophage, bac-
terial, yeast, or ribosome display) have been developed to retrieve
the best possible Nbs from immune, naı̈ve, or synthetic Nb reper-
toires. Currently, the combination of an immune library with phage
display is still the most widely used approach. This is partially for
historical reasons as it was the first selection technology that yielded
satisfactory results [9] and secondly because phage display is an
accessible and very robust technology used in many laboratories.
All these display technologies employ an enrichment step for poten-
tial antigen-binding candidates, with final identification of antigen-
binding Nbs achieved by choosing a limited number of individual
clones after the last round of enrichment. These clones are cultured
and tested individually in ELISA or in functional screens. Alterna-
tively deep sequencing techniques can be implemented, while the
NestLink technology might also be beneficial in specific cases [30].
Finally, using an immune library that is by definition already
enriched in target-specific Nbs, it is possible to envisage a pheno-
typic selection rather than selection for antigen binding alone.
Although this strategy has been attempted using a large
non-immune single pot scFv library, it would be predicted to be
more successful using an immune Nb library, for example, in iden-
tifying neutralizing binders against viruses, intracellular pathogens,
or even toxins. Cloning and expressing the immune Nb library
directly in host cells of the virus, pathogen, or toxin, and selecting
transformed cells that resist challenge with the lethal component
provides immediate access to neutralizing Nbs [31].

4 Favorable Characteristics of Nanobodies

For in-depth characterization of Nbs, selected candidates should be

expressed in large quantities in microorganisms and purified. Pro-
duction in yeast or bacteria and purification of monoclonal Nbs is
many times cheaper and faster than the production of hybridoma
antibodies [3, 4]. The expression of Nbs in the periplasm of Gram-
negative bacteria (such as Escherichia coli) is often preferred and
Nbs are easily extracted using osmotic shock. Already highly
 Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in. . . 9

enriched in the periplasmic extract, recombinant Nbs bearing

C-terminal His6 tags are further purified by a single immobilized
metal affinity chromatography (IMAC) step yielding Nbs that are
sufficiently pure for more detailed characterization.
In addition to periplasmic expression, recloning the Nbs for
expression in Gram-positive bacteria [32], in the cytoplasm of
Gram-negative bacteria engineered to have a less reducing cytosol
(e.g., Rosetta-gami B, Shuffle T7 Express, or Origami 2) [33], or in
yeast might result in higher yields. Unfortunately, these microor-
ganisms grow more slowly than E. coli. Therefore, it is usually
preferred to postpone this recloning to a later stage when a smaller
number of clones (a single clone or a few lead candidates at most)
need to be characterized and when large amounts of purified pro-
tein are required. In any case, the production cost of Nbs is low, due
to the high expression levels achieved in microorganisms.
Due to the stringent selection conditions applied during pan-
ning, Nbs typically recognize their cognate antigen with high spec-
ificity and affinity. The single domain nature of Nbs and their
prolate shape results in a paratope with a preference to recognize
concave epitopes [10]. This contrasts with the flat or concave
paratopes of classical antibodies (scFvs) that prefer planar or convex
epitopes [34]. Nbs are highly soluble and tolerate exposure to
non-physiological conditions. Hence, Nbs resist lyophilization
and nebulization, and they can be sprayed and inhaled [35]. The
resilience of Nbs to organic solvents and extremes of pH means
they can be used to target antigens, such as pesticides and herbi-
cides, that are extracted in nonaqueous solutions [36]. The amino
acid sequences of Nbs share a high degree of sequence identity with
the variable domains of human immunoglobulin heavy chains [37]
and as a result Nbs have a low immunogenicity risk profile in
humans [38].
The small size of Nbs and the absence of the Fc region means
that intravenously administered Nbs are rapidly extravasated and
diffuse well into tissues. There are even indications that Nbs might
cross the blood–brain barrier [39]. However, their small size, far
below the renal retention cut off, ensures that Nbs will be rapidly
eliminated from the body via the kidneys. It is estimated that the
serum half-life for Nbs would be approximately 20 min [40]. This is
a disadvantage in that only a small fraction of the administered Nbs
will reach their target. However, an advantage of short half-life is
that Nbs conjugated to a toxic substance are rapidly removed from
the body, reducing off-target damage to healthy cells.

5 Tailoring Nanobody Candidates for Particular Applications

The high expression yields and robust behavior of Nbs makes them
amenable to further engineering. Tailoring the activity of Nbs is
10 Fangling Ji et al.

occasionally associated with decreased expression yields and

decreased binding activity, but these reductions remain
manageable.
Humanization of a selected Nb is an engineering task that is
regularly proposed for Nbs intended to be administered in humans
[41]. Since the wild type Nb sequence already shares high sequence
identity with human VH domains, Nb humanization is rather
straightforward [42]. Moreover, codon optimization will most
likely be performed during the development of therapeutic Nbs.
In vitro Nb gene synthesis is the most appropriate time to perform
Nb humanization; however, the necessity of Nb humanization is
questionable in view of the low immunogenicity risk of these
molecules [38].
Cloning two Nb genes in a tandem will generate bivalent,
biparatopic, or bispecific constructs, depending on whether:
(1) the genes encoding two identical Nbs, (2) the genes encoding
two different Nbs targeting different epitopes on the same antigen,
or (3) the genes encoding two Nbs against different antigens are
cloned in tandem, respectively. Bivalent, biparatopic, and bispecific
Nbs are coming to the forefront of therapeutic and diagnostic
applications as they might increase the functional affinity for an
antigen, or bring two different antigens in proximity. The latter is
important to generate bispecific T-cell engagers whereby, for exam-
ple, an anti-CD19 moiety targeting B cells is fused with an anti-
CD3 moiety targeting T cells [43], as well as when a Nb against
serum albumin is included to increase the blood circulation half-life
of the molecule [44, 45].
The sequence and length of the linker between the two Nb
genes should be optimized. Options include flexible linkers com-
prising one or several repeats of Gly4Ser [46] as well as sequences
based on human IgA sequences that might be more rigid and
protease resistant [47]. However, the linkage of the C-terminus
of one Nb to the N-terminus of a second Nb might compromise
the paratope accessibility of the C-terminally located Nb
[48]. Therefore, the order of the Nbs within a single polypeptide
chain is also a critical parameter and should be tested empirically.
While pentavalent Nb constructs have been generated, their
expression yields became problematically low. Therefore, to achieve
very high valency, another strategy is to fuse the Nb with a penta-
merization domain such as the B domain of verotoxin or Shiga
toxin [49]. Using this pentamerizing scaffold, a decavalent/bispe-
cific construct was produced by cloning one Nb at the 50 end of the
verotoxin B subunit and another Nb at its 30 end [50]. Moreover,
the insertion of a Nb in a solvent exposed helix of ferritin allowed
production of a chimeric protein assembling spontaneously in a
24-mer, leading to an unforeseen avidity increase [51].
In addition to the genetic fusion of Nb genes in a head-to-tail
bivalent, biparatopic, or bispecific construct, Nbs can be joined via
Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in. . . 11

their C-terminal ends using various other methods. This strategy

retains maximal antigen-binding activity of the component Nb
units and can be achieved by inserting a Cys at the C-terminal
end of the Nb, leading to spontaneous dimerization after disulfide
bond formation [52]. Alternatively, protein ligation methods
might be considered. Sortase A, a staphylococcal transpeptidase
recognizing the Sortag sequence LPXTG cloned at the
C-terminal end of a Nb, will cut between Thr and Gly residues
[53]. The acyl-enzyme intermediate is then susceptible to nucleo-
philic attack from peptides or proteins with an N-terminal Gly
residue. Reactive peptides (GGG-P, where P denotes any probe
such as biotin or an unnatural amino acid) can be obtained by
chemical peptide synthesis. Alternatively, a recombinant Nb with
an upstream MHHHHHHENLYFQGGG sequence can be
expressed and purified by IMAC before being cleaved with TEV
protease (recognizing the ENLYFQ sequence and cleaving
between Q-G). The resulting cleaved Nb with three Gly residues
at its N-terminal end can then serve as the nucleophile to react with
the Sortase-acyl intermediate and form the Nb dimer construct
[54]. The problem with this strategy is that the Sortag sequence
will be present in the final dimerized construct serving as a substrate
for Sortase cleavage. The substitution of Sortase A and its tag by the
Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, and its
recognition sequence, might provide an elegant solution for an
incoming nucleophile to produce a ligated protein resistant to
further reactions [55].
In another strategy, formylglycine-generating enzyme (FGE)
has emerged as a robust tool for site-specific protein modification
[56]. FGE is an aerobic oxidase that specifically recognizes the five-
amino acid motif CXPXR (termed “aldehyde tag”) and catalyzes
the conversion of Cys to a non-canonical, aldehyde-containing
formylglycine residue. This aldehyde group, absent in native pro-
teins, is a versatile bio-orthogonal reactive chemical handle for
bioconjugation. A stabilized FGE, immobilized on Sepharose,
served as a continuous-flow bio-catalysis system to improve the
aldehyde formation in the absence of additional exogenous Cu
(II) ions [57].
The use of the Avi tag sequence, recognized by the BirA
enzyme, at the C-terminal end of Nbs is a versatile strategy for
in vivo biotinylation of Nbs [58]. Subsequently, the biotin moiety
can be recognized by streptavidin-conjugated particles or enzymes.
Furthermore, other tags such as SpyTag and SpyCatcher have been
used to form covalently-linked dimeric, trimeric, and even hepta-
meric Nb conjugates [59].
The intracellular expression of Nbs with their stress-resistant
folds allows the use of Nbs as intrabodies and generation of split Nb
formats. The chromobody, produced by intracellular expression of
a genetic fusion between a Nb and a fluorescent protein (e.g.,
12 Fangling Ji et al.

monomeric red fluorescent protein) was a first and highly successful

engineered Nb-based tool to trace antigens in living cells [60]. This
example of intracellular expression of Nb genetic fusions was rap-
idly followed and expanded to Nbs linked to the F-box, the von
Hippel-Lindau protein, and transcription activation or
DNA-binding domains. These innovative tools were employed to
specifically degrade particular cytoplasmic proteins or to activate
reporter or killing genes at particular times during cell development
[61–64].
In all of these fusion constructs, the Nb was intact and preceded
the fusion partner. However, it is also possible to split the Nb into
two parts or to integrate an entire exogenous protein within the Nb
structure. In the first case, the two halves of the Nb were linked to
light inducible heterodimerization domains to form “optobodies.”
The half Nb fragments fail to assemble and recognize their target,
but exposure to blue light induces the heterodimerization process
and restores the antigen-binding capacity of the Nb [65]. In an
alternative approach, a bacterial circularly permutated dihydrofo-
late reductase (cpDHFR) was inserted within the third antigen-
binding loop of the Nb [66]. This did not prevent the Nb moiety
from associating with its cognate antigen. However, the presence of
cofactors or inhibitors of the cpDHFR, such as nicotinamide ade-
nine dinucleotide phosphate and/or trimethoprim, provoked a
conformational reorganization that disrupted antigen recognition
by the Nb. Such chemogenetic control of antigen-recognition by
modified Nbs is a useful approach to investigate various biological
processes.
Finally, inserting a large protein within the β hairpin loop in
between the A and B strands of a Nb generates a “Megabody,” that
retains the full antigen-binding capacity of the Nb. Such Megabo-
dies are instrumental for structure determination of membrane
proteins by single particle cryo-electron microscopy [67]. These
unique activities demonstrate the flexibility exhibited by Nbs in
producing novel protein functionalities for innovative research
and discovery.

6 Perspectives

The most critical question in the field is: “what are the applications
for which Nbs are the best choice?” Nbs have been proposed as
research tools as well as for diagnostic and therapeutic applications
[20]. However, there are many alternative formats of affinity bin-
ders available, including DARPins, affibodies, monobodies, antic-
alins, and non-proteinaceous aptamers. These alternatives share
many beneficial properties with Nbs, except for one: they rely on
the availability of an extremely large and highly diverse synthetic
repertoire, while Nbs can be retrieved from (smaller) immune
Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in. . . 13

libraries enriched for antigen binders. The immunization of a cam-

elid and the construction of a relatively small Nb library is straight-
forward, even for non-experts. In addition, many commercial
entities offer this service. Therefore, obtaining Nbs from immune
libraries is probably one of the most accessible and affordable
techniques to obtain high affinity, target-specific binding reagents.
Multiple Nb-based tools have been developed to trace, to
purify, and to investigate the structure and biochemical properties
of target proteins. The possibility to express Nbs intracellularly and
function as intrabodies illustrates that they work equally well in
living organisms as well as in in vitro experiments. As such, Nbs
have been used to investigate the status of protein–protein interac-
tions in living cells in real time [68] as well as transcription factor
dynamics in live embryos [69]. Furthermore, the small size of
fluorescently labelled Nbs make them ideal probes in super-
resolution microscopy [70] as well as useful tools for the crystalli-
zation and study of dynamic or membrane proteins [71].
Monoclonal antibodies are highly valued in diagnostic tests
where these antibodies are used to capture and detect the antigen
from a complex mixture. The antigen specificity of Nbs also permits
their use as antigen capturing and detection reagents, with an
added benefit of robust stability in extreme temperature, chemical,
and pH environments. We expect that Nbs will replace monoclonal
antibodies for the development of low-cost diagnostic tools, such
as lateral flow assays in field tests or in cases where sensitivity is not
the highest priority and where the presence of a nearby laboratory is
lacking. When sensitivity and specificity are more of a concern, then
Nbs might be preferred over monoclonals to detect active infec-
tions, as their association with the antigen might be less obstructed
by endogenous host antibodies [72]. Nb engineering in biotiny-
lated bivalent constructs might increase the avidity and thus
enhance the diagnostic sensitivity. Directional immobilization of
these Nbs at high density on magnetic particles improves antigen
capture and concentration from diluted solutions. Furthermore,
the resilience to elevated concentrations of organic solvents might
facilitate the detection of herbicides and pesticides extracted in
nonaqueous solutions from food substances.
Their small size, low immunogenicity, and fast blood clearance
makes Nbs labelled with radionuclides practical for noninvasive
in vivo imaging [73] as well as for use in targeted radionuclide
therapy [74, 75]. In these applications, it is critical to employ
probes of minimal size such as monomeric Nb derivatives [76].
The fast extravasation and tissue biodistribution forms the basis
for use of Nbs in therapeutic applications to neutralize toxins or
infectious pathogens (bacteria, viruses, or even plant viruses). As
such, several Nbs were identified aiming at the neutralization of
toxins (Shiga toxin, anthrax, scorpion, or snake venom) [77] or to
neutralize viruses (severe acute respiratory syndrome coronavirus-2,
14 Fangling Ji et al.

respiratory syncytial virus, rotavirus, or influenza) [78]. Toxins circu-

lating in blood might also be removed ex vivo by apheresis. For this
type of application, Nbs are covalently linked to resin for the produc-
tion of affinity adsorbents [79].
Nbs will also be useful in multidomain constructs. The gene
encoding a tumor-specific Nb can be cloned in expression vectors
upstream from genes encoding transmembrane regions and signal-
ing cytoplasmic domains. Transduction of natural killer (NK) or
primary T cells with such constructs results in chimeric antigen
receptors (CAR)-NK or CAR-T cells that induce anti-tumor effects
[80]. Several of these constructs are currently involved in clinical
trials.
Finally, the autonomous behavior of Nbs and their feasibility to
be assembled as independent affinity reagents in multidomain con-
structs formed the basis of several additional therapeutic applica-
tions [81]. Cablivi® is the first clinically approved Nb comprised of
a dimerized and humanized Nb against von Willebrand factor that
is administered to treat thrombotic thrombocytopenic purpura.
However, several other therapeutic Nb constructs are in the pipe-
line and have reached advanced clinical stages, underscoring the
growing excitement for this unique antibody format.

Acknowledgments

The work was funded by the National Natural Science Foundation

of China (Grant No. U20A20263), the National Key R&D Pro-
gram of China (2016YFC1103002), and the Fundamental
Research Funds for the Central Universities (DUT20LAB121,
DUT20YG107, DUT21ZD207, and DUT21LK10).

References
1. Hamers-Casterman C, Atarhouch T, Muylder-
mans S et al (1993) Naturally-occurring anti-
bodies devoid of light-chains. Nature 363:
446–448
2. Muyldermans S, Atarhouch T, Saldanha J et al
(1994) Sequence and structure of VH domain
from naturally occurring camel heavy chain
immunoglobulins lacking light chains. Protein
Eng Des Sel 7:1129–1135
3. Arbabi-Ghahroudi M, Tanha J, MacKenzie R
(2005) Prokaryotic expression of antibodies.
Cancer Metastasis Rev 24:501–519
4. van der Linden RHJ, de Geus B, Frenken LGJ
et al (2000) Improved production and func-
tion of llama heavy chain antibody fragments
by molecular evolution. J Biotechnol 80:
261–270
5. Skerra A, Plückthun A (1988) Assembly of a
functional immunoglobulin Fv fragment in
Escherichia coli. Science 240:1038–1041
6. Better M, Chang CP, Robinson RR et al
(1988) Escherichia coli secretion of an active
chimeric antibody fragment. Science 240:
1041–1043
7. Riechmann L, Clark M, Waldmann H et al
(1988) Reshaping human antibodies for ther-
apy. Nature 332:323–327
8. McCafferty J, Griffith AD, Winter G et al
(1990) Phage antibodies: filamentous phage
displaying antibody variable domains. Nature
348:552–554
9. Arbabi Ghahroudi M, Desmyter A, Wyns L
et al (1997) Selection and identification of sin-
gle domain antibody fragments from camel
 Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in. . .
heavy-chain antibodies. FEBS Lett 414:
521–526
10. Desmyter A, Transue TR, Arbabi Ghahroudi M
et al (1996) Crystal structure of a camel single-
domain VH antibody fragment in complex
with lysozyme. Nat Struct Biol 3:803–811
11. Dumoulin M, Conrath K, Van Meirhaeghe A
et al (2002) Single-domain antibody fragments
with high conformational stability. Protein Sci
11:500–515
12. Nguyen VK, Su C, Muyldermans S et al (2002)
Heavy-chain antibodies in Camelidae; a case of
evolutionary innovation. Immunogenetics 54:
39–47
13. Nguyen VK, Hamers R, Wyns L et al (1999)
Loss of splice consensus signal is responsible for
the removal of the entire CH1 domain of the
functional camel IGG2A heavy-chain antibo-
dies. Mol Immunol 36:515–524
14. Woolven BP, Frenken LGJ, van der Logt P et al
(1999) The structure of the Ilama heavy chain
constant genes reveals a mechanism for heavy-
chain antibody formation. Immunogenetics
50:98–101
15. Henry KA, van Faassen H, Harcus D et al
(2019) Llama peripheral B-cell populations
producing conventional and heavy chain-only
IgG subtypes are phenotypically indistinguish-
able but immunogenetically distinct. Immuno-
genetics 71:307–320
16. Ming L, Wang Z, Yi L et al (2020)
Chromosome-level assembly of wild Bactrian
camel genome reveals organization of immune
gene loci. Mol Ecol Resour 20:770–780
17. Nguyen VK, Hamers R, Wyns L et al (2000)
Camel heavy-chain antibodies: diverse germ-
line VHH and specific mechanisms enlarge the
antigen-binding repertoire. EMBO J 19:
921–930
18. Achour I, Cavelier P, Tichit M et al (2008)
Tetrameric and homodimeric camelid IgGs
originate from the same IgH locus. J Immunol
181:2001–2009
19. Deschacht N, De Groeve K, Vincke C et al
(2010) A novel promiscuous class of camelid
single-domain antibodycontributes to the
antigen-binding repertoire. J Immunol 184:
5696–5704
20. Muyldermans S (2021) A guide to: generation
and design of nanobodies. FEBS J 288:
2084–2102
21. Fridy PC, Li Y, Keegan S et al (2014) A robust
pipeline for rapid production of versatile nano-
body repertoires. Nat Methods 11:1253–1260
22. Odegrip R, Coomber D, Eldridge B et al
(2004) CIS display: in vitro selection of
15
peptides from libraries of protein-DNA com-
plexes. Proc Natl Acad Sci U S A 101:
2806–2810
23. Kim HJ, McCoy MR, Majkova Z et al (2012)
Isolation of alpaca anti-hapten heavy chain sin-
gle domain antibodies for development of sen-
sitive immunoassay. Anal Chem 84:1165–1171
24. Janssens R, Dekker S, Hendriks RW et al
(2006) Generation of heavy-chain-only antibo-
dies in mice. Proc Natl Acad Sci U S A 103:
15130–15135
25. Teng Y, Young JL, Edwards B et al (2020)
Diverse human VH antibody fragments with
bio-therapeutic properties from the Crescendo
mouse. New Biotechnol 55:65–76
26. Laustsen AH, Greiff V, Karatt-Vellatt A et al
(2021) Animal immunization, in vitro display
technologies, and machine learning for anti-
body discovery. Trends Biotechnol, in press.
https://doi.org/10.1016/j.tibtech.2021.
03.003
27. Monegal A, Ami D, Martinelli C et al (2009)
Immunological applications of single-domain
llama recombinant antibodies isolated from a
naı̈ve library. Protein Eng Des Sel 22:273–280
28. Zimmermann I, Egloff P, Hutter CAJ et al
(2018) Synthetic single domain antibodies for
the conformational trapping of membrane pro-
teins. eLife 7:1–32
29. Zimmermann I, Egloff P, Hutter CAJ et al
(2020) Generation of synthetic nanobodies
against delicate proteins. Nat Protoc 15:
1707–1741
30. Egloff P, Zimmermann I, Arnold FM et al
(2019) Engineered peptide barcodes for
in-depth analyses of binding protein libraries.
Nat Methods 16:421–428
31. Schmidt FI, Hanke L, Morin B et al (2016)
Phenotypic lentivirus screens to identify func-
tional single domain antibodies. Nat Microbiol
1:1–21
32. Mizukami M, Tokunaga H, Onishi H et al
(2014) Highly efficient production of VHH
antibody fragments in Brevibacillus choshinensis
expression system. Protein Expr Purif 105:
23–32
33. Li D, Ji F, Huang C et al (2019) High expres-
sion achievement of active and robust anti-β
2 microglobulin nanobodies via E. coli hosts
selection. Molecules 24:2860
34. Mitchell LS, Colwell LJ (2018) Analysis of
nanobody paratopes reveals greater diversity
than classical antibodies. Protein Eng Des Sel
31:267–275
35. Van Heeke G, Allosery K, de Brabandere V et al
(2017) Nanobodies® as inhaled
16 Fangling Ji et al.
biotherapeutics for lung diseases. Pharmacol
Ther 169:47–56
36. Zhang JR, Wang Y, Dong JX et al (2019)
Development of a simple pretreatment immu-
noassay based on an organic solvent-tolerant
nanobody for the detection of carbofuran in
vegetable and fruit samples. Biomol Ther 9:
576
37. Klarenbeek A, El Mazouari K, Desmyter A et al
(2015) Camelid Ig V genes reveal significant
human homology not seen in therapeutic tar-
get genes, providing for a powerful therapeutic
antibody platform. MAbs 7:693–706
38. Ackaert C, Smiejkowska N, Xavier C et al
(2021) Immunogenicity risk profile of nanobo-
dies. Front Immunol 12:e632687
39. Li T, Bourgeois JP, Celli S et al (2012) Cell-
penetrating anti-GFAP VHH and
corresponding fluorescent fusion protein
VHH-GFP spontaneously cross the blood-
brain barrier and specifically recognize astro-
cytes: application to brain imaging. FASEB J
26:3969–3979
40. De Vos J, Devoogdt N, Lahoutte T et al (2013)
Camelid single-domain antibody-fragment
engineering for (pre)clinical in vivo molecular
imaging applications: adjusting the bullet to its
target. Expert Opin Biol Ther 13:1149–1160
41. Rossotti MA, Bélanger K, Henry KA et al
(2021) Immunogenicity and humanization of
single-domain antibodies. FEBS J, in press.
https://doi.org/10.1111/febs.15809
42. Vincke C, Loris R, Saerens D et al (2009)
General strategy to humanize a camelid
single-domain antibody and identification of a
universal humanized nanobody scaffold. J Biol
Chem 284:3273–3284
43. Chanier T, Chames P (2019) Nanobody engi-
neering: toward next generation immu-
notherapies and immunoimaging of cancer.
Antibodies 8:13
44. Roovers RC, Laeremans T, Huang L et al
(2007) Efficient inhibition of EGFR signalling
and of tumour growth by antagonistic anti-
EGFR nanobodies. Cancer Immunol Immun-
other 56:303–317
45. Coppieters K, Dreier T, Silence K et al (2006)
Formatted anti-tumor necrosis factor alpha
VHH proteins derived from camelids show
superior potency and targeting to inflamed
joints in a murine model of collagen-induced
arthritis. Arthritis Rheum 54:1856–1866
46. Huston JS, Levinson D, Mudgett-Hunter M
et al (1988) Protein engineering of antibody
binding sites: recovery of specific activity in an
anti-digoxin single-chain Fv analogue
produced in Escherichia coli. Proc Natl Acad
Sci U S A 85:5879–5883
47. Li D, Ren J, Ji F et al (2020) Peptide linker
affecting the activity retention rate of VHH in
immunosorbents. Biomol Ther 10:1–12
48. Conrath KE, Lauwereys M, Wyns L et al
(2001) Camel single-domain antibodies as
modular building units in bispecific and biva-
lent antibody constructs. J Biol Chem 276:
7346–7350
49. Zhang J, Tanha J, Hirama T et al (2004) Pen-
tamerization of single-domain antibodies from
phage libraries: a novel strategy for the rapid
generation of high-avidity antibody reagents. J
Mol Biol 335:49–56
50. Stone E, Hirama T, Tanha J et al (2007) The
assembly of single domain antibodies into bis-
pecific decavalent molecules. J Immunol Meth-
ods 318:88–94
51. Fan K, Jiang B, Guan Z et al (2018) Fenobody:
a ferritin-displayed nanobody with high appar-
ent affinity and half-life extension. Anal Chem
90:5671–5677
52. Massa S, Xavier C, De Vos J et al (2014) Site-
specific labeling of cysteine-tagged camelid
single-domain antibody-fragments for use in
molecular imaging. Bioconjug Chem 25:
979–988
53. Schmohl L, Schwarzer D (2014) Sortase-
mediated ligations for the site-specific modifi-
cation of proteins. Curr Opin Chem Biol 22:
122–128
54. Sarpong K, Bose R (2017) Efficient sortase-
mediated N-terminal labeling of TEV protease
cleaved recombinant proteins. Anal Biochem
521:55–58
55. Rehm FBH, Harmand TJ, Yap K et al (2019)
Site-specific sequential protein labeling cata-
lyzed by a single recombinant ligase. J Am
Chem Soc 141:17388–17393
56. Rabuka D, Rush J, DeHart G et al (2012) Site-
specific chemical protein conjugation using
genetically encoded aldehyde tags. Nat Protoc
7:1052–1067
57. Peng Q, Zang B, Zhao W et al (2020) Efficient
continuous-flow aldehyde tag conversion using
immobilized formylglycine generating enzyme.
Cat Sci Technol 10:484–492
58. Saerens D, Frederix F, Reekmans G et al
(2005) Engineering camel single-domain anti-
bodies and immobilization chemistry for
human prostate-specific antigen sensing. Anal
Chem 77:7547–7555
59. Khairil Anuar INA, Banerjee A, Keeble AH et al
(2019) Spy&Go purification of SpyTag-
proteins using pseudo-SpyCatcher to access
 Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in. . .
an oligomerization toolbox. Nat Commun 10:
1–13
60. Rothbauer U, Zolghadr K, Tillib S et al (2006)
Targeting and tracing antigens in live cells with
fluorescent nanobodies. Nat Methods 3:
887–889
61. Caussinus E, Kanca O, Affolter M (2012)
Fluorescent fusion protein knockout mediated
by anti-GFP nanobody. Nat Struct Mol Biol
19:117–121
62. Fulcher LJ, Macartney T, Bozatzi P et al
(2016) An affinity-directed protein missile sys-
tem for targeted proteolysis. Open Biol 6:
160255
63. Fulcher LJ, Hutchinson LD, Macartney TJ
et al (2017) Targeting endogenous proteins
for degradation through the affinity-directed
protein missile system. Open Biol 7:170066
64. Tang JCY, Szikra T, Kozorovitskiy Y et al
(2013) A nanobody-based system using fluo-
rescent proteins as scaffolds for cell-specific
gene manipulation. Cell 154:928–939
65. Yu D, Lee H, Hong J et al (2019) Optogenetic
activation of intracellular antibodies for direct
modulation of endogenous proteins. Nat
Methods 16:1095–1100
66. Farrants H, Tarnawski M, Müller TG et al
(2020) Chemogenetic control of nanobodies.
Nat Methods 17:279–282
67. Uchański T, Masiulis S, Fischer B et al (2021)
Megabodies expand the nanobody toolkit for
protein structure determination by single-
particle cryo-EM. Nat Methods 18:60–68
68. Herce HD, Deng W, Helma J et al (2013)
Visualization and targeted disruption of pro-
tein interactions in living cells. Nat Commun 4:
2660
69. Bothma JP, Norstad MR, Alamos S et al (2018)
LlamaTags: a versatile tool to image transcrip-
tion factor dynamics in live embryos. Cell 173:
1810–1822.e16
70. Pleiner T, Bates M, Trakhanov S et al (2015)
Nanobodies: site-specific labeling for super-
resolution imaging, rapid epitope-mapping
and native protein complex isolation. eLife 4:
1–21
17
71. Uchański T, Pardon E, Steyaert J (2020)
Nanobodies to study protein conformational
states. Curr Opin Struct Biol 60:117–123
72. Morales-Yánez F, Trashin S, Hermy M et al
(2019) Fast one-step ultrasensitive detection
of Toxocara canis antigens by a nanobody-
based electrochemical magnetosensor. Anal
Chem 91:11582–11588
73. Keyaerts M, Xavier C, Heemskerk J et al
(2016) Phase I study of 68Ga-HER2-nano-
body for PET/CT assessment of HER2-
expression in breast carcinoma. J Nucl Med
57:27–33
74. D’Huyvetter M, Vincke C, Xavier C et al
(2014) Targeted radionuclide therapy with a
177
Lu-labeled anti-HER2 nanobody. Thera-
nostics 4:708–720
75. Dekempeneer Y, Keyaerts M, Krasniqi A et al
(2016) Targeted alpha therapy using short-
lived alpha-particles and the promise of nano-
bodies as targeting vehicle. Expert Opin Biol
Ther 16:1035–1047
76. Debie P, Lafont C, Defrise M et al (2020) Size
and affinity kinetics of nanobodies influence
targeting and penetration of solid tumours. J
Control Release 317:34–42
77. Alirahimi E, Kazemi-Lomedasht F, Shahbazza-
deh D et al (2018) Nanobodies as novel thera-
peutic agents in envenomation. Biochim
Biophys Acta Gen Subj 1862:2955–2965
78. Koenig PA, Das H, Liu H et al (2021)
Structure-guided multivalent nanobodies
block SARS-CoV-2 infection and suppress
mutational escape. Science 371:eabe6230
79. Zhang L, Zang B, Huang C et al (2019)
One-step preparation of a VHH-based immu-
noadsorbent for the extracorporeal removal of
β2-microglobulin. Molecules 24:2–13
80. Bao C, Gao Q, Li L et al (2021) The applica-
tion of nanobody in CAR-T therapy. Biomol
Ther 11:1–18
81. Jovčevska I, Muyldermans S (2019) The thera-
peutic potential of nanobodies. BioDrugs 34:
11–26
 Chapter 2

Overview, Generation, and Significance of Variable New

Antigen Receptors (VNARs) as a Platform for Drug
and Diagnostic Development
Samata S. Pandey, Marina Kovaleva, Caroline J. Barelle,
and Obinna C. Ubah

Abstract
The approval of the first VHH-based drug caplacizumab (anti-von Willebrand factor) has validated a
two-decade long commitment in time and research effort to realize the clinical potential of single-domain
antibodies. The variable domain (VNAR) of the immunoglobulin new antigen receptor (IgNAR) found in
sharks provides an alternative small binding domain to conventional monoclonal antibodies and their
fragments and heavy-chain antibody-derived VHHs. Evolutionarily distinct from mammalian antibody
variable domains, VNARs have enhanced thermostability and unusual convex paratopes. This predisposi-
tion to bind cryptic and recessed epitopes has facilitated both the targeting of new antigens and new
(neutralizing) epitopes on existing antigens. Together these unique properties position the VNAR platform
as an alternative non-antibody binding domain for therapeutic drug, diagnostic and reagent development.
In this introductory chapter, we highlight recent VNAR advancements that further underline the exciting
potential of this discovery platform.

Key words Sharks, Single-domain antibodies, Variable new antigen receptors (VNARs), Antibody
reformatting, Immunization, Phage display, Protein expression

1 Introduction

One could postulate that nature’s goal for single binding domain-
based moieties is to enhance host protection through the evolution
of new binding specificities and characteristics that make them
capable of recognizing targets (e.g., G protein-coupled receptors,
enzyme active sites, viral surface proteins) considered inaccessible
to conventional antibodies [1]. Despite recent advances in protein
engineering that have successfully reduced the size of classical anti-
bodies to smaller fragments (Fab, single chain-Fv, VHH, VH, VL),
even smaller binding entities have been developed such as Affibo-
dies, DARPins, Fynomers, and Adnectins. Almost overlooked,

Greg Hussack and Kevin A. Henry (eds.), Single-Domain Antibodies: Methods and Protocols,
Methods in Molecular Biology, vol. 2446, https://doi.org/10.1007/978-1-0716-2075-5_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

19
20 Samata S. Pandey et al.

nature also produced another reductionist solution some 450 mil-

lion years ago in cartilaginous fish. It was not until the early 1990s
that sharks were discovered to possess unconventional “antibodies”
(antibody-like immunoglobulins) as part of the complex make-up
of their immune system, known now as immunoglobulin new
antigen receptors (IgNARs) [2]. Heavy-chain only IgNARs have
now been identified in several different species of sharks including
nurse shark (Ginglymostoma cirratum) [2–4], spotted wobbegong
shark (Orectolobus maculatus) [5], spiny dogfish (Squalus
acanthias) [6, 7], banded houndshark (Triakis scyllium) [8], bam-
boo shark (Chiloscyllium plagiosum) [9], small-spotted catshark
(Scyliorhinus canicula) [10], smooth dogfish (Mustelus canis)
[6, 7], and horn shark (Heterodontus francisci) [11]. In 2003, the
immunization of nurse sharks [12] demonstrated that the variable
domain of IgNAR, known as the variable new antigen receptor
(VNAR), was indeed autonomous in its antigen binding and per-
forms the same functions as the variable VH/VL domains of classi-
cal antibodies.

2 Structures and Properties of IgNAR and VNAR

IgNAR is a homodimer of heavy chains. The antigen-binding site is

composed of two identical, independent variable domains
(VNARs). Interestingly, these VNARs bear only two
complementarity-determining regions (CDRs), CDR1 and
CDR3. The loss of variability caused by the lack of a CDR2 is
compensated by the evolution of a long and variable CDR3, rang-
ing from 5 to 27 residues [3, 13, 14]. Additional diversity in the
VNAR domain is provided by two hypervariable regions (HV),
HV1 and HV2. The absence of a CDR2 loop contributes signifi-
cantly to making VNARs the smallest (~11 kDa) known naturally
occurring immunoglobulin-like proteins with a full antigen-
binding site [15].
VNARs display a wide variety of CDR loop lengths and struc-
tures. To date, three major isotypes of VNARs have been defined
based on the position and the number of cysteine residues located
within both the framework regions (FRs) and CDRs of the domain
(Fig. 1). Canonical cysteine residues are present in all three isotypes
in FR1 and FR3b, with the key difference between isotypes result-
ing from the number and location of non-canonical cysteines. Type
I VNARs possess two cysteine residues in CDR3 that are paired
with cysteine residues in FR2 and FR4. To date, this isotype has
only been identified in nurse sharks [2, 3, 16, 17].
Type II VNARs possess two paired cysteines in CDR1 and
CDR3, which form a stabilizing disulfide bond between the pro-
truding CDR loops [18]. The CDR3 of this isotype lacks the
second cysteine residues found in Type I VNARs. This allows
 Overview, Generation, and Significance of Variable New Antigen Receptors... 21

Fig. 1 VNAR classification by isotype families. VNARs can be differentiated into three major families based on
their non-canonical disulfide bonds and sequence motifs. C cysteine residue, W tryptophan residue,
F phenylalanine residue, FW framework region

Type II VNARs to adopt a protrusive structure and bind cryptic or

catalytic epitopes [15, 18–20]. Type IIb VNARs, also known as
Type IV VNARs, are similar to Type II VNARs but lack the
non-canonical cysteine residues, allowing the CDRs to move away
from one another in a more flexible and less physically constrained
manner [14, 21].
Type III VNARs are predominantly expressed in young sharks
and are hypothesized to form part of their “innate” immune sys-
tem. This isotype has shorter and less diverse CDR3s due to germ-
line fusion of two of the three diversity gene segments [3]. Type III
VNARs are similar to Type II, with the addition of a conserved
tryptophan residue at position 31 in CDR1, stabilizing the core of
the protein and a phenylalanine residue at position 96 in CDR3.
This invariant phenylalanine can participate in polar and non-polar
interactions, significantly increasing the loop conformations
beyond those simply afforded by sequence diversity [18]. As the
shark matures, levels of Type I and Type II IgNAR predominate
with Type III levels subsiding. Type IIIb VNARs lack the
non-canonical cysteine residues of Type III VNARs while main-
taining the invariant tryptophan in CDR1 and phenylalanine in
CDR3. Type IIb and Type IIIb VNARs are similar in that they
contain identical canonical cysteine residues allowing them to form
disulfide linkage between FR1 and FR3b [14, 22].
Recently, a Type V VNAR has been proposed, originating from
the horn shark (H. francisci) and bearing a pair of non-canonical
cysteine residues in CDR3 and a pair of cysteine residues in CDR1
22 Samata S. Pandey et al.

[23]. This observation has yet to be confirmed by additional studies

in horn sharks or different shark species.
VNARs retain the classic immunoglobulin fold except for the
absence of the C0 and C00 strands [15, 18]. Despite their similar
folding patterns, VNARs share only 25–30% sequence identity with
the heavy-chain variable domains of mammalian antibodies.
Greater sequence identity is shared with mammalian antibody
light chains and T-cell receptors. Furthermore, using a bioinfor-
matic approach and the crystal structure of 5A7, a hen egg
lysozyme-binding VNAR, 24 hallmark VNAR residues have been
identified; 21 of 24 residues showed sequence similarity with VL
and TCR Vα domains, while the remaining three residues, L18,
T/L34, and E/Q57, were identified as VNAR-specific residues.
This conservation of sequence has led to homology modeling of
VNARs using Ig VL domains as templates, as they also possess
short CDR2s [3, 17].
The structures of VNAR paratopes are markedly different from
those of conventional antibodies. These structural properties
enable binding to recessed clefts, enhanced thermal stability, and,
through multiple contact residues in a single-domain, high affinity
binding. Despite the absence of CDR2, VNARs achieve large
sequence diversity from their extended CDR3 loops. The long
length of this loop is stabilized by inter- and intra-loop disulfide
bridges which hold the loops in a flexible finger-like projection. The
VNAR domain of all isotypes typically forms a convex antigen-
binding site that is ideal for binding poorly accessible protein clefts
and cavities. Exploitation of this predisposition has generated a
biologics platform capable of generating binders to intractable
antigenic epitopes such as enzyme active sites [19, 20] and parasite
ectodomains [24]. The interface between the paired variable
domains of a classical antibody requires conservation of hydropho-
bic residues. These hydrophobic patches are absent in VNARs,
replaced by charged residues that help to increase their solubility
[2, 19, 25]. In summary, VNARs demonstrate high thermal stabil-
ity and refolding ability [26], as well as high binding affinity to a
range of antigens (picomolar to low nanomolar range), without the
requirement for affinity maturation [4, 21, 27, 28].

3 Generation of Antigen-Specific VNARs

Several techniques exist for the isolation of antigen-specific VNARs

from naı̈ve, immune, or synthetic libraries. In all cases, VNAR
library construction and generation requires the amplification of a
diverse VNAR gene repertoire using variable domain-specific pri-
mers and subsequent cloning into suitable display and recombinant
protein expression vectors [4, 6, 11, 13, 27–29].
Overview, Generation, and Significance of Variable New Antigen Receptors... 23

Naturally derived naı̈ve libraries are usually small in size and

have limited diversity but can still deliver antigen-specific VNARs
[7]. These libraries can be cloned quickly and are cost-effective, as
they avoid a lengthy immunization process. To increase the size of a
naı̈ve library, it can be built by combining the repertoires from
several animals. Feng et al. combined the VNAR repertoires of six
nurse sharks delivering a library size of 1.2  1010 unique clones
[30]. However, relying on the existing natural repertoire has typi-
cally yielded lower binding affinity VNARs (in the laboratory) when
compared with those derived from an immunized VNAR library
[7, 29]. Many groups favor immunization as the preferred route to
high affinity, antigen-specific VNAR generation.
Immunized libraries can afford to be smaller in size than syn-
thetic libraries, as natural in vivo affinity maturation to a specific
antigen frequently leads to the isolation of target-specific VNARs
with high affinity without extensive rounds of enrichment and
selection [4, 29]. Another sometimes overlooked advantage of
shark immunization is the likelihood of eliciting an immunogenic
IgNAR response to antigens that are highly conserved proteins in
humans and other mammals. As a result of 450 million years of
evolutionary divergence between elasmobranchs and humans, most
mammalian proteins are seen as “foreign” by the shark immune
system. However, a shark immunization campaign with the goal of
isolating antigen-specific VNARs can be a time-consuming process,
taking ~4–6 months and requiring custom reagents for quantifying
IgNAR titers in shark sera. The IgNAR response in sharks to an
antigen occurs more slowly than responses of mammalian IgM or
IgG. However, the capacity to generate a specific humoral response
and the subsequent in vivo maturation of this response clearly
existed very early in vertebrate evolution [31], with IgNARs play-
ing a role as affinity matured binders, tailor-made to recognize a
particular class of antigens that contained protein cavities or can-
yons. Since the first detection of an antigen-driven IgNAR response
and isolation of antigen-specific VNAR [12], VNARs have been
raised successfully against diverse targets for both therapeutic and
diagnostic purposes and from multiple shark species (Table 1).
Synthetic and semi-synthetic libraries can be designed and built
to contain theoretically 1 trillion (1012) unique clones. Typically,
this is achieved by utilizing native shark FRs as a scaffold on to
which diversity can be engineered through randomization of one or
more antigen-binding loops. Diversity can be introduced by direct
mutagenesis of individual amino acids or synthesis/amplification of
fully randomized loops. Most synthetic VNAR repertoires employ
hypervariable CDR3 loops as the primary paratope for antigen
interaction. The characteristics of a diverse synthetic/semi-
synthetic VNAR library can also be influenced by the VNAR isotype
chosen as the scaffold for library construction, affecting the stability
and paratope shapes of individual clones [9, 23, 28, 40].
24 Samata S. Pandey et al.

Table 1
Summary of immune, naı̈ve, and synthetic library-derived VNARs and their applications

Therapeutic
Library type Shark species Common name application Antigen References
Naı̈ve Mustelus canis Smooth dogfish Cholera Cholera toxin [7]
Naı̈ve Orectolobus Spotted Various cancers Aurora-A [32]
maculatus wobbegong kinase
shark
Immunized Ginglymostoma Nurse shark Uveitis, ICOSL, [4, 16]
cirratum inflammatory TNF-α
disease
Immunized Ginglymostoma Nurse shark NA HEL [12]
cirratum
Immunized Ginglymostoma Nurse shark Ebola virus VP40 [33]
cirratum
Immunized Heterodontus Horn shark Inflammatory TNF-α [11]
francisci disease
Immunized Squalus Spiny dogfish Half-life extension HSA [29]
acanthias
Immunized Triakis scyllium Banded hound Malaria PfHRP2 [34]
shark
Synthetic/semi- Squalus Spiny dogfish Rheumatoid ICOSL [35]
synthetic acanthias arthritis
Synthetic/semi- Squalus Spiny dogfish Anti-toxins SEB, BoNT/ [6]
synthetic acanthias A
Synthetic/semi- Ginglymostoma Nurse shark NA Leptin [36]
synthetic cirratum
Synthetic/semi- Chiloscyllium Bamboo shark Cancer VEGF, [23, 37]
synthetic plagiosum EpCAM
Synthetic/semi- Triakis scyllium Banded hound Antiviral VHSV [8]
synthetic shark
Synthetic/semi- Orectolobus Wobbegong NA Tom70 [13]
synthetic maculatus shark
Synthetic/semi- Orectolobus Wobbegong Antiviral HBeAg of [38]
synthetic maculatus shark HBV
Synthetic/semi- Orectolobus Wobbegong Malaria AMA1 [39]
synthetic maculatus shark
Synthetic/semi- Orectolobus Wobbegong Periodontitis Gingipain K [5]
synthetic maculatus shark protease
AMA1 apical membrane antigen 1, BoNT/A botulinum neurotoxin A, EpCAM epithelial cell adhesion molecule, HBeAg
hepatitis B e-antigen, HBV hepatitis B virus, HEL hen egg lysozyme, HSA human serum albumin, ICOSL induced
costimulatory ligand, NA not applicable, PfHRP2 histidine-rich protein 2 of Plasmodium falciparum, SEB staphylococ-
cal enterotoxin B, TNF-α tumor necrosis factor alpha, Tom70 70 kDa outer membrane translocase receptor from human
mitochondria, VEGF vascular endothelial growth factor, VHSV viral hemorrhagic septicemia virus, VP40 40 kDa viral
protein of Ebola virus
 Overview, Generation, and Significance of Variable New Antigen Receptors... 25

Antigen-specific binders can be isolated from VNAR libraries

using different display technologies such as phage display [4, 6, 12,
29], yeast display [28, 41, 42], or ribosome display
[43]. Subsequent analyses of the selection outputs and screening
for target specificity of individual VNARs is achieved using enzyme-
linked immunosorbent assay, fluorescence-activated cell sorting, or
next-generation DNA sequencing [4, 29, 30, 37]. In vitro affinity
maturation can be adopted to improve the affinity of low affinity
VNARs; this approach can increase affinity by 10- to 20-fold using
error-prone PCR or low fidelity RNA polymerase approaches
[9, 17, 43, 44]. Fennell et al. showed that a single point mutation
at position 61 (SerΔArg) improved the affinity of several different
VNARs against different antigens [17]. The biophysical properties
of isolated antigen-specific VNARs can be enhanced in parallel with
in vitro affinity maturation [44]. While VNARs are clearly respon-
sive to affinity maturation, it is also evident that when synthetic
VNAR libraries are well designed, low nanomolar and picomolar
binders can sometimes be obtained to different antigens without
the need for affinity maturation. This may be a consequence of the
multiple points of antigen contact afforded by the long CDR3s and
their ability through paratope-shaping to extend into the pockets
and grooves found in many protein classes [13].

4 VNAR Reformatting and Humanization

While the relatively small size of VNAR domains confers advantages

in both therapeutic and diagnostic applications, there are potential
limitations to their use. Their smaller size enables better tissue
uptake, biological membrane permeation for site-specific drug
delivery, and intracellular targeting of antigens [16, 45]. Their
short serum half-life and rapid clearance from systemic circulation
makes them suitable, at ~11 kDa, as diagnostic imaging reagents.
However, for many therapeutic applications, VNAR domains with
an extended serum half-life may be required. In vivo half-life exten-
sion of VNAR domains has been achieved through multimerization
(dimer, trimer) of therapeutic VNARs with half-life extending
VNAR domains that recognize serum albumin from multiple spe-
cies [21, 44]. Alternatively, an IgG Fc fragment has been fused to a
therapeutic VNAR domain to improve its serum half-life and result-
ing efficacy [4, 16, 46]. VNAR-Fc fusions are also capable of
Fc-mediated effector functions, such as antibody-dependent cellu-
lar cytotoxicity and complement-dependent cytotoxicity. However,
at ~75 to 80 kDa, VNAR-Fc fusions remain smaller than a classical
monoclonal antibody (mAb; ~150 kDa), while possibly providing
better tissue penetration, and are less complex to manufacture at
scale. More recently, the super-potency of 103 kDa Quad-X™ (and
Quad-Y™) VNAR-Fc formats was demonstrated [4, 46]. These are
26 Samata S. Pandey et al.

essentially quadrivalent VNAR domains fused to an IgG1 Fc frag-

ment at both N- and C-terminal positions (Fig. 2). The ease of
reformatting the VNAR domains is attributed to the absence of
variable domain pairing.
While there is no definitive evidence that VNAR domains are
inherently immunogenic to humans in vivo, and the limited data
available suggests they are not [44, 47], efforts to humanize or
de-immunize VNARs are often performed regardless. The first
reported humanized VNAR was an anti-serum albumin VNAR
clone called E06. Humanization was achieved using a human germ-
line V kappa light chain (DPK9) as a template [48]. Both the
humanized VNARs, now known as soloMERs™, and their native
parental VNARs, were shown to have a very low response index in a
classical dendritic cell-T-cell proliferation assay suggesting that nei-
ther domain class contained detrimental T-cell epitopes [44]. Strelt-
sov and colleagues reported a level of structural similarity between
the I-set family of immunoglobulins and VNARs [18], and this
observation was used to design, build, and screen a library using
I-set proteins as an alternative scaffold. From this work a fully
human I-protein/VNAR against the chemokine receptor CXCR4
was isolated that displayed binding to its target in the low nano-
molar affinity range [49]. This domain has been reformatted as a
VNAR-Fc (AD-214) for half-life extension and is currently in Phase
1 clinical trials in Australia, with orphan drug designation by the US
Food and Drug Administration, for idiopathic pulmonary fibrosis
(ClinicalTrials.gov identifier: NCT04415671).

5 Expression and Purification of VNAR Domains

VNARs and their reformatted constructs including soloMERs™

are readily produced at scale using existing bioprocessing systems.
Their small size and minimal post-translation modification means
that they are easily expressed in Escherichia coli and Pichia pastoris
[4, 21, 46, 50]. IgG Fc fused VNAR constructs (VNAR-Fc, Quad-
X™, and Quad-Y™, Fig. 2) require additional post-translational
modifications, including glycosylation in their Fc region, and there-
fore heterologous expression of these constructs has been success-
fully conducted in mammalian cells such as HEK293 and CHO
[4, 46, 51].
VNARs and their reformatted versions generally express well in
both prokaryotic and eukaryotic systems with scalable yields rang-
ing from 100 to 400 mg/L for monomeric VNAR domains
[52, 53], 200 mg/L for dimeric constructs, 100 mg/L for trimeric
constructs, and 150 mg/L for IgG Fc fused VNARs [46, 53]. Yields
 Overview, Generation, and Significance of Variable New Antigen Receptors... 27

Fig. 2 Schematic illustration of multivalent VNAR formats. These multivalent formats represent both multi-
paratopic and multi-specific constructs and are achieved through molecular biology and protein engineering
techniques. The linear multivalent VNAR constructs (dimers and trimers) can be created with an anti-human
serum albumin half-life extending VNAR as part of the multimer

of VNAR-Fc constructs of up to 460 mg/L have recently been

achieved in non-optimized, transient CHO cells.
Purification of non-Fc-fused domains is achieved via the addi-
tion of genetically encoded purification/detection tags (poly-
histidine, hemagglutinin, or c-Myc) to either the N- or
C-terminal position of the protein [4, 9, 15, 29]. Through design,
such tags can be omitted from humanized soloMER™ constructs
by incorporating a protein L-binding site in FR1 (as part of the
humanization protocol). This facilitates both detection (quantifica-
tion) and purification of the final product and can be used in parallel
with more traditional protein-A affinity purification that has been
used successfully for VNAR-Fc fusions [48]. VNAR domains typi-
cally possess robust biophysical properties which means that they
can tolerate harsh purification conditions such as low pH used for
protein A and L elution [44, 54].
28 Samata S. Pandey et al.

6 Current Trends in VNAR Development

The first published VNAR was isolated against the apical membrane
antigen 1 of Plasmodium falciparum [5] and since then VNARs
have been generated against several therapeutic targets, as well as
for other non-therapeutic applications. An anti-tumor necrosis
factor (TNF)-α super-potent Quad-X™ VNAR demonstrated
superior in vivo efficacy when compared head-to-head with
Humira® in a transgenic mouse model of spontaneous polyarthritis.
The linear non-human Fc-fused multivalent anti-TNF-α constructs
all demonstrated picomolar neutralizing potency against TNF-α
[4, 46]. The anti-TNF-α Quad-X™, with an ND50 of ~2 pM in
the gold standard L929 fibrosarcoma cell line neutralization assay
and a binding affinity to TNF-α of 17 pM, is the most potent and
high affinity anti-TNF-α biologic isolated from any platform. Using
a surrogate anti-mouse TNF-α VNAR generated from a proprietary
synthetic library platform, equivalent potency of the VNAR and the
“standard of care” steroid therapy was demonstrated in an in vivo
rat experimental model of uveitis [55]. In addition to TNF-α
antagonism, as part of a growing armory targeting chronic inflam-
matory diseases, VNARs have been successfully generated and are
undergoing preclinical development against: B cell-activating fac-
tor, transferrin receptor 1, induced costimulatory ligand (ICOSL),
and vascular endothelial growth factor (VEGF165) [16, 45, 51, 56,
57].
VNARs have been generated against multiple notable oncology
targets, including the onco-embryonic tyrosine kinase ROR1 [58],
aurora-A kinase [32], delta-like ligand 4 [59], epithelial cell adhe-
sion molecule, ephrin type-A receptor 2, and human serine
protease [9].
The VNAR platform is being considered as an alternative drug
administration/delivery approach because of the relatively small
size, biophysical properties, and biological stability of VNARs. As
well as co-developing a first-in-class VNAR-drug conjugate (also
known as a soloMER™ drug conjugate), innovative modalities are
being explored for target-specific drug delivery. Nanotechnology is
increasingly being considered as a safe way to deliver toxic payloads
compartmentalized inside nanoparticles, especially when delivery is
achieved using target-specific antibodies/binding domains, mini-
mizing off-target toxicities [60]. Recently, site-specific conjugation
of an anti-delta-like ligand 4 (DLL4) VNAR (anti-angiogenesis)
was demonstrated to poly(lactic-co-glycolic) acid PEGylated nano-
particles through surface maleimide functional groups. These
nanoconjugates were shown to specifically bind DLL4 with high
affinity and were preferentially internalized by DLL4-expressing
pancreatic cancer cell lines and endothelial cells [59].
Overview, Generation, and Significance of Variable New Antigen Receptors... 29

An increasing number of VNAR studies have shown the flexi-

bility of these robust domains in accessing challenging and hard-to-
reach biological compartments. An anti-VEGF165 VNAR gener-
ated via an immunized horn shark phage display library was used to
provide proof-of-concept data for intraocular penetration. The
VNAR could be detected in aqueous humor 3 h after topical
administration in New Zealand rabbits with healthy eyes
[56]. Another anti-ICOSL VNAR generated following nurse
shark immunization showed efficient corneal penetration as a
monomeric domain (11 kDa) in wild-type BALB/C mice 20 min
after application of the VNAR onto the scratched cornea (used to
mimic inflammation). In this study, the impact of molecular size on
biological membrane penetration was clearly demonstrated, with a
VNAR-Fc construct (80 kDa) showing almost four-fold lower
penetration across the cornea, while a commercial anti-ICOSL
mAb (150 kDa) was just above the level of detection in the anterior
fluid [16].
VNARs are known to have exceptional tolerance to extreme
environmental conditions such as high temperature. This thermo-
tolerance appears to make VNARs ideal candidates for certain rapid
diagnostic tests (RDTs). Current RDTs developed using mAbs can
have reduced shelf-lives due to their temperature sensitivities and
storage requirements, resulting in limited use in hot but
low-resource regions where maintaining refrigerated supply chains
can be challenging. A malaria biomarker-specific VNAR has been
published and its utility as an RDT in malaria diagnosis is currently
being explored [34]. The small size of VNARs potentially makes
them ideal as in vivo diagnostic imaging tools too. Their small
hydrodynamic radius is below the molecular weight cut-off for
glomerular filtration, and therefore VNARs are cleared rapidly
following initial systemic exposure. This rapid clearance may help
to improve in vivo signal to noise ratios in the imaged tissues.
Several groups have proposed this hypothesis but to date no data
in either non-human primates or humans have been forthcoming.
As VNARs progress further into the mainstream of biologics
discovery, several applications for their use have been published. In
the field of biodefence, VNARs have been developed against staph-
ylococcal enterotoxin B, ricin, and botulinum toxin with affinities
in the nanomolar range [6]. VNARs have also been isolated and
characterized as bioprocessing tools for downstream purification or
depletion of a specific isoform of strand-exchange engineered
domain homodimers [61]. This study was particularly interesting
as it demonstrated the ability of VNARs to distinguish two closely
related proteins (Fc regions of IgG molecules), a difference that
had proved impossible to separate using traditional antibody
approaches.
30 Samata S. Pandey et al.

7 Summary

With 400 million years of evolution behind their refinement as

binding domains, it is unsurprising that VNARs are showing enor-
mous potential for disruptive success in the fields of drug discovery,
diagnostic tool development, and bioprocessing. For certain anti-
gen classes, especially proteins with pockets, grooves, or canyons, it
is now clear that VNAR binders can outperform their conventional
mAb counterparts. VNAR isolation is technically easy to achieve,
either via immunization of several shark species, or by utilizing a
growing number of large and diverse synthetic VNAR libraries.
Because of their simple molecular architecture, they are readily
amenable to extensive reformatting to increase their valency,
improve their affinity, enhance their biophysical properties, or for
humanization for therapy. Their distinct evolutionary ancestry dis-
tinguishes them from classical antibodies and affords any VNAR
platform a clear intellectual property position and freedom to oper-
ate, making their route to commercialization easier in the complex
landscape of biologics drug discovery.

References
1. Holliger P, Hudson PJ (2005) Engineered
antibody fragments and the rise of single
domains. Nat Biotechnol 23:1126–1136
2. Greenberg AS, Avila D, Hughes M et al (1995)
A new antigen receptor gene family that under-
goes rearrangement and extensive somatic
diversification in sharks. Nature 1995:168–173
3. Diaz M, Stanfield RL, Greenberg AS et al
(2002) Structural analysis, selection, and
ontogeny of the shark new antigen receptor
(IgNAR): identification of a new locus prefer-
entially expressed in early development. Immu-
nogenetics 2002:501–512
4. Ubah OC, Steven J, Kovaleva M et al (2017)
Novel, anti-hTNF-α variable new antigen
receptor formats with enhanced neutralizing
potency and multifunctionality, generated for
therapeutic development. Front Immunol 8:
1780
5. Nuttall SD, Krishnan UV, Hattarki M et al
(2001) Isolation of the new antigen receptor
from wobbegong sharks, and use as a scaffold
for the display of protein loop libraries. Mol
Immunol 38:313–326
6. Liu JL, Anderson GP, Goldman ER (2007)
Isolation of anti-toxin single domain antibo-
dies from a semi-synthetic spiny dogfish shark
display library. BMC Biotechnol 7:1–10
7. Liu JL, Anderson GP, Delehanty JB et al
(2007) Selection of cholera toxin specific
IgNAR single-domain antibodies from a naive
shark library. Mol Immunol 44:1775–1783
8. Ohtani M, Hikima J, Jung T et al (2013) Vari-
able domain antibodies specific for viral hem-
orrhagic septicemia virus (VHSV) selected
from a randomized IgNAR phage display
library. Fish Shellfish Immunol 34:724–728
9. Zielonka S, Weber N, Becker S et al (2014)
Shark attack: high affinity binding proteins
derived from shark vNAR domains by stepwise
in vitro affinity maturation. J Biotechnol 191:
236–245
10. Crouch K, Smith LE, Williams R et al (2013)
Humoral immune response of the small-
spotted catshark, Scyliorhinus canicula. Fish
Shellfish Immunol 34:1158–1169
11. Camacho-Villegas T, Mata-Gonzalez T,
Paniagua-Solis J et al (2013) Human TNF
cytokine neutralization with a vNAR from Het-
erodontus francisci shark: a potential therapeu-
tic use. MAbs 5:80–85
12. Dooley H, Flajnik MF, Porter AJ (2003) Selec-
tion and characterization of naturally occurring
single-domain (IgNAR) antibody fragments
from immunized sharks by phage display. Mol
Immunol 40:25–33
13. Nuttall SD, Krishnan UV, Doughty L et al
(2003) Isolation and characterization of an
IgNAR variable domain specific for the
 Overview, Generation, and Significance of Variable New Antigen Receptors...
human mitochondrial translocase receptor
Tom70. Eur J Biochem 270:3543–3554
14. Streltsov VA, Varghese JN, Carmichael JA et al
(2004) Structural evidence for evolution of
shark Ig new antigen receptor variable domain
antibodies from a cell-surface receptor. Proc
Natl Acad Sci U S A 101:12444–12449
15. Zielonka S, Empting M, Grzeschik J et al
(2015) Structural insights and biomedical
potential of IgNAR scaffolds from sharks.
MAbs 7:15–25
16. Kovaleva M, Johnson K, Steven J et al (2017)
Therapeutic potential of shark anti-ICOSL
VNAR domains is exemplified in a murine
model of autoimmune non-infectious uveitis.
Front Immunol 8:1121
17. Fennell B, Darmanin-Sheehan A, Hufton S
et al (2010) Dissection of the IgNAR V
domain: molecular scanning and orthologue
database mining define novel IgNAR hallmarks
and affinity maturation mechanisms. J Mol Biol
400:155–170
18. Streltsov VA, Carmichael JA, Nuttall SD
(2015) Structure of a shark IgNAR antibody
variable domain and modeling of an early-
developmental isotype. Protein Sci 14:
2901–2909
19. Stanfield RL, Dooley H, Flajnik MF et al
(2004) Crystal structure of a shark single-
domain antibody V region in complex with
lysozyme. Science 305:1770–1773
20. Stanfield RL, Dooley H, Verdino P et al (2007)
Maturation of shark single-domain (IgNAR)
antibodies: evidence for induced-fit binding. J
Mol Biol 367:358–372
21. Müller MR, Saunders K, Grace C et al (2012)
Improving the pharmacokinetic properties of
biologics by fusion to an anti-HSA shark
VNAR domain. MAbs 4:673–685
22. Kovaleva M, Ferguson L, Steven J et al (2014)
Shark variable new antigen receptor biolo-
gics—a novel technology platform for thera-
peutic drug development. Expert Opin Biol
Ther 14:1527–1539
23. Cabanillas-Bernal O, Dueñas S, Ayala-Avila M
et al (2019) Synthetic libraries of shark vNAR
domains with different cysteine numbers
within the CDR3. PLoS One 14:e0213394
24. Henderson KA, Streltsov VA, Coley AM et al
(2007) Structure of an IgNAR-AMA1 com-
plex: targeting a conserved hydrophobic cleft
broadens malarial strain recognition. Structure
15:1452–1466
31
25. Roux KH, Greenberg AS, Greene L et al
(1998) Structural analysis of the nurse shark
(new) antigen receptor (NAR): molecular con-
vergence of NAR and unusual mammalian
immunoglobulins. Proc Natl Acad Sci U S A
95:11804–11809
26. Liu JL, Zabetakis D, Brown JC et al (2014)
Thermal stability and refolding capability of
shark derived single domain antibodies. Mol
Immunol 59:194–199
27. Nuttall SD, Krishnan UV, Doughty L et al
(2002) A naturally occurring NAR variable
domain binds the Kgp protease from Porphyr-
omonas gingivalis. FEBS Lett 516:80–86
28. Konning D, Rhiel L, Empting M et al (2017)
Semi-synthetic vNAR libraries screened against
therapeutic antibodies primarily deliver anti-
idiotypic binders. Sci Rep 7:9676
29. Müller MR, O’Dwyer R, Kovaleva M et al
(2012) Generation and isolation of target-
specific single-domain antibodies from shark
immune repertoires. Methods Mol Biol 907:
177–194
30. Feng M, Bian H, Wu X et al (2019) Construc-
tion and next-generation sequencing analysis
of a large phage-displayed VNAR single-
domain antibody library from six naive nurse
sharks. Antib Ther 2:1–11
31. Dooley H, Flajnik MF (2005) Shark immunity
bites back: affinity maturation and memory
response in the nurse shark, Ginglymostoma
cirratum. Eur J Immunol 35:936–945
32. Burgess SG, Oleksy A, Cavazza T et al (2016)
Allosteric inhibition of Aurora-A kinase by a
synthetic vNAR domain. Open Biol 6:160089
33. Goodchild SA, Dooley H, Schoepp RJ et al
(2011) Isolation and characterisation of
Ebolavirus-specific recombinant antibody frag-
ments from murine and shark immune
libraries. Mol Immunol 48:2027–2037
34. Leow CH, Fischer K, Leow CY et al (2018)
Isolation and characterization of malaria
PfHRP2 specific VNAR antibody fragments
from immunized shark phage display library.
Malar J 17:383
35. O’Dwyer R, Kovaleva M, Zhang J et al (2018)
Anti-ICOSL new antigen receptor domains
inhibit T cell proliferation and reduce the
development of inflammation in the collagen-
induced mouse model of rheumatoid arthritis.
J Immunol Res 2018:4089459
36. Shao C, Secombes CJ, Porter AJ (2007) Rapid
isolation of IgNAR variable single-domain
32 Samata S. Pandey et al.
antibody fragments from a shark synthetic
library. Mol Immunol 44:656–665
37. Zielonka S (2015) The shark strikes twice: gen-
eration of mono-and bispecific high-affinity
vNAR antibody domains via step-wise affinity
maturation. PhD thesis, Technische Uni-
versit€at Darmstadt. http://tuprints.ulb.tu-
darmstadt.de/4481/
38. Walsh R, Nuttall S, Revill P et al (2011) Tar-
geting the hepatitis B virus precore antigen
with a novel IgNAR single variable domain
intrabody. Virology 411:132–141
39. Nuttall SD, Humberstone KS, Krishnan UV
et al (2004) Selection and affinity maturation
of IgNAR variable domains targeting Plasmo-
dium falciparum AMA1. Proteins 55:187–197
40. Grzeschik J, Könning D, Hinz SC et al (2018)
Generation of semi-synthetic shark IgNAR
single-domain antibody libraries. Methods
Mol Biol 1701:147–167
41. Könning D, Zielonka S, Sellmann C et al
(2016) Isolation of a pH-sensitive IgNAR vari-
able domain from a yeast-displayed, histidine-
doped master library. Mar Biotechnol (NY) 18:
161–167
42. Könning D, Kolmar H (2018) Beyond anti-
body engineering: directed evolution of alter-
native binding scaffolds and enzymes using
yeast surface display. Microb Cell Fact 17:32
43. Kopsidas G, Roberts AS, Coia G et al (2006) In
vitro improvement of a shark IgNAR antibody
by Qβ replicase mutation and ribosome display
mimics in vivo affinity maturation. Immunol
Lett 107:163–168
44. Steven J, Müller MR, Carvalho MF et al (2017)
In vitro maturation of a humanized shark
VNAR domain to improve its biophysical prop-
erties to facilitate clinical development. Front
Immunol 8:1361
45. Stocki P, Szary J, Rasmussen CL et al (2021)
Blood-brain barrier transport using a high
affinity brain-selective VNAR antibody target-
ing transferring receptor 1. FASEB J 35:
e21172
46. Ubah OC, Steven J, Porter AJ et al (2019) An
anti-hTNF-α variable new antigen receptor for-
mat demonstrates superior in vivo preclinical
efficacy to Humira® in a transgenic mouse
autoimmune polyarthritis disease model.
Front Immunol 10:526
47. Ubah OC, Porter AJ, Barelle CJ (2020) In
vitro ELISA and cell-based assays confirm the
low immunogenicity of VNAR therapeutic
constructs in a mouse model of human RA:
an encouraging milestone to further clinical
drug development. J Immunol Res 2020:
7283239
48. Kovalenko OV, Olland A, Piché-Nicholas N
et al (2013) Atypical antigen recognition
mode of a shark immunoglobulin new antigen
receptor (IgNAR) variable domain character-
ized by humanization and structural analysis. J
Biol Chem 288:17408–17419
49. Griffiths K, Dolezal O, Cao B et al (2016)
i-bodies, human single domain antibodies that
antagonize chemokine receptor CXCR4. J Biol
Chem 291:12641–12657
50. Bojalil R, Mata-González MT, Sánchez-
Muñoz F et al (2013) Anti-tumor necrosis fac-
tor VNAR single domains reduce lethality and
regulate underlying inflammatory response in a
murine model of endotoxic shock. BMC
Immunol 14:1–7
51. H€asler J, Flajnik MF, Williams G et al (2016)
VNAR single-domain antibodies specific for
BAFF inhibit B cell development by molecular
mimicry. Mol Immunol 75:28–37
52. Leow HC, Fischer K, Leow YC et al (2019)
Cytoplasmic and periplasmic expression of
recombinant shark VNAR antibody in Escher-
ichia coli. Prep Biochem Biotechnol 49:
315–327
53. Ubah OC, Buschhaus MJ, Ferguson L et al
(2018) Next-generation flexible formats of
VNAR domains expand the drug platform’s
utility and developability. Biochem Soc Trans
46:1559–1565
54. Griffiths K, Dolezal O, Parisi K et al (2013)
Shark variable new antigen receptor (VNAR)
single domain antibody fragments: stability and
diagnostic applications. Antibodies 2:66–81
55. Pepple KL, Wilson L, Van Gelder RN et al
(2019) Uveitis therapy with shark variable
novel antigen receptor domains targeting
tumor necrosis factor alpha or inducible T-cell
costimulatory ligand. Transl Vis Sci Technol 8:
11
56. Camacho-Villegas TA, Mata-González MT,
Garcı́a-Ubbelohd W et al (2018) Intraocular
penetration of a vNAR: in vivo and in vitro
VEGF165 neutralization. Mar Drugs 16:113
57. Matz H, Dooley H (2019) Shark IgNAR-
derived binding domains as potential diagnos-
tic and therapeutic agents. Dev Comp Immu-
nol 90:100–107
 Overview, Generation, and Significance of Variable New Antigen Receptors...
58. Cotton G, Thom J, Trumper P et al (2020)
Exploiting the properties of VNAR domains
for the development of novel efficacious pro-
tein drug conjugates targeting the oncofetal
protein ROR1. In: Proceedings of the annual
meeting of the American Association for Can-
cer Research 2020; 2020 Apr 27–28 and Jun
22–24. Philadelphia (PA): AACR; Cancer Res
2020;80(16 Suppl): Abstract nr 538
59. Leach A, Smyth P, Ferguson L et al (2020)
Anti-DLL4 VNAR targeted nanoparticles for
targeting of both tumour and tumour
33
associated vasculature. Nanoscale 12:
14751–14763
60. Jain S, Doshi AS, Iyer AK et al (2013) Multi-
functional nanoparticles for targeting cancer
and inflammatory diseases. J Drug Target 21:
888–903
61. Buschhaus MJ, Becker S, Porter AJ et al (2019)
Isolation of highly selective IgNAR variable
single-domains against a human therapeutic
Fc scaffold and their application as tailor-
made bioprocessing reagents. Protein Eng
Des Sel 32:385–399
Another random document with
no related content on Scribd:
aside; and what was a greater Misfortune, the Prince and his
Retinue beheld with winking Eyes, all their Hovels on fire ashore:
The Occasion this; we happening to have all our Colours flying, and
some Guns let off in honour of the 28th of May, another Leader on
shore misinterpreting it as a particular Respect to Jacobus, grew
jealous, seized his House during the Revel, his Wives, and his
Dashees, drank up all his Brandy, eat all his Victuals, cudgelled his
People, and set both his Houses on fire.
Next Morning, on unravelling the Mystery, the Surprize was over,
and all became good Friends again. They have very little Knowledge
or Use of Fire-Arms, because no Trade scarce; their Weapons being
Spears, Arrows, and Clubs, and it is a bloody Battle among them,
when half a dozen of a side are knocked down.
 A
V O YA G E
TO
Brasil, and the West-Indies.
From Cape Lopez (parting with the Coast) we came in sight of the
Island Annabona, the Breezes small at South, and Calms
alternatively; hereabout we cruised three or four days, for our
Consort the Swallow, that some how or other was separated; and
missing her, bore away for Brasil.
In the Passage it may be first observed, that when we had sailed
ninety Leagues to the Westward, and got into 3°° S. the Winds that
were at South veered, so as to become a true S E. Trade, that
carried us four or five Knots.——And still as we advanced farther
Westward, it blew fresher at E S E. 7, 8, and 9 Knots constantly, with
neither Thunder nor Lightning. Quære, whether this distance is not a
proper Medium to allow for the Attraction of all Exhalations by the
Land; at least that they considerably abate after that length from all
Shores, allowing for Latitude, and as they are Montainous or Plain.
We see within this Dimension, (plainly) that it takes off the Influence
of the Sun, and varys the Trade Wind towards itself: Nor is it so
astonishing, since Animals themselves obey; several sorts of Fish
and Fowl have a periodical return to such and such places, and not
so of those inhabiting the more stable Element of Land: Wherefore it
is highly rational to think, that as the fluid Elements they live in yield
to the attractive Power of the Earth and Planets, so also their
Inhabitants have their Instinct more sensibly fated by them.
 Secondly, in this Trade-Wind sailing, we are every day diverted
with flying Fish, Bonetos, and Sea-Fowl; the Sails require little labour
in trimming, the Ship goes steady, and the Bowl unslung; so that at
leaving such a Country we might cheerfully sing,

How happy were we, when the Wind blew abaft!

One of these cheerful Evenings, eleven at night, the full Moon
became totally eclipsed, a Darkness surprizing, as it was
unexpected; when she had recovered her Light, we repeated our
Sacrifice in Bowls, and fell into Reflections and Admiration of that
Power which supports the Regularity of the planetary Motions, and
the Sublimity of that Art which can so exactly calculate them: They
demonstrate the Sphericity of the Earth, because Countries, as they
are farther East or West, observe them hours sooner or later,
according to their Longitude, which could not be on a Plain, but
visible to all at once.
This Longitude therefore in a general View, is the same thing as
Time, the difference of it being the distance East and West of any
two places, allowing 15 Degrees, or 300 Leagues to an Hour; the
whole 24 being lost or gained in a Circum-navigation of the Globe,
West or Eastward.
A natural, tho’ hitherto incorrect way of estimating the Parts of
Longitude in those Runs, till Instruments and Rules are discovered,
is, I think, First, to make exact Tables of the Sun’s Risings and
Settings, at Places commonly departed from, and those we go to for
every day in the year: and then, Secondly, to carry two proved
Watches of equal Goodness, kept in equal Warmth, and freest from
Motion and Weather, to measure the difference of time where you
are, by the same edge of the Sun the Tables were made from; the
Minutes sooner or later, according as you go East or West, is so
many Leagues of Longitude for that day. I would insinuate by this
only my Opinion, that those literal Improvers of Time, the Watch
Makers, bid as fair for the Discovery of Longitude as the Astronomer;
for if Watches can be made not to err above two or three Minutes in
the time a Ship is running 1000 Leagues, or if they do err more, a
Rule could be found how much, (like as an Azimuth corrects the
common Compass;) or if any Movement could be depended on only
from Observation to Observation, then the Error would be no greater
than what is met in different Quadrants, observing Latitude. As it is, it
seems a proper Method to correct or assist the present Rules of
calculating meridional Distance.
We made this Passage of 8 or 900 Leagues to Brasil in three
Weeks; but having elsewhere given my Observations on the Country,
I shall only take notice that the Trade blowing very fresh, and
bringing in a great Swell, we hastened from the Coast to our
intended Ports in the West-Indies.
In our Progress thither, a Remark or two: First, that in the
Navigation from Brasil, we crossed the Æquinoctial, two or three
Degrees W. of Cape Roque, keeping on with a pleasant S E. Trade
that gradually lessened, and in 4°° North Latitude, left us in Calms,
Rains, and uncertain Squalls, (varying round the Compass;) That
this continued for several days, till we drew in or near the Parallel of
Barbados, and then we as gradually had obtained the Trade to
Northward of the East, running 150 Leagues with it, that is, to
Barbados.
Secondly, The reason of Calms, and Rains met in Latitudes
between 4°° and 11°° N. (with a little Variation, as the Sun is of this
or that side the Equinoctial) is probably from a Contest between the
N E. and S E. Trade; but whether the passing them be more
favourable far to the Eastward or Westward, I am uncertain.
 BARBADOS.
Barbados was discovered by Sir William Carter, in King James I’s
time, planted to little purpose until 1627; since which, the Crops have
been so advantageous, as to have raised the Price of Ground thirty
fold.
We anchored here in Carlisle-Bay the beginning of August, the
resort of most Shipping who load at this Island. The Bay is made by
Needham and Pelican Points; the Anchorage 20 Fathom, so clear
Water that you may see to the bottom; but so foul and rocky, that the
Cables are always buoy’d up with Cask. In the bottom of the Bay
stands Bridgetown, the principal of the Island, and is the Residence
of the Governor, Factors, and Merchants, who transact their
Business here and at their Plantations alternately. There is only one
large Church, with an Organ, and about twenty Chappels at different
parts of the Island, all Episcopal, there having been no Dissenters
these many years. The People are for the most part polite and well-
bred, promoting Trade by a magnificent way of living; the chief of
them are Colonels, or Captains of Militia, and in the Assembly are
divided into a Party-Interest, on the civil Affairs of the Island, their
chief Distinction; murmuring, or elate, just as they are in or out of the
Governor’s Favour, who can abate in the Customs, or imploy in the
Application: (tho’ by the way the fewer Officers, and those Menials,
the better advantage to him.)
The whole is a sweet Spot of Earth, not a Span hardly uncultivated
with Sugar-Canes; all sides bend with an easy Declivity to the Sea,
and is ever green: This delight to the Planter has its Inconveniencies,
that there is no Recreation out of Business, but in Drinking or
Gaming.
 The Propriety was given by King James I. to the Earl of Carlisle;
and Anno 1661, King Charles II. purchased it back of Lord Kinoul,
that Earl’s Heir, allowing him 1000l. per Ann. Acknowledgment.
Anno 1663, an Act passed by the President (who acts as
Governor in their absence) the Council and Assembly, for 4½ in
Specie Duty of all Commodities, the Produce of the Island, which it’s
computed will amount to 10000l. per Annum.—Madeira Wines
imported, at 4l. 10s. per Pipe, raises 7000l. and this, with one Pound
of Gunpowder per Tun on each Ship, is appropriated for Stores, and
Repairs to Forts.
The Governor is appointed by the King, his Salary formerly used to
arise at an uncertain Sum of 4 or 5000l. per Ann. from Presents and
Perquisites, since fixed at 1200l., 2000l. and now is 6000l. And as
the Council, a part of their Constitution, is in a manner of his own
Nomination, being appointed by Letters of Mandamus, as they have
Power to make Demands on ancient Perquisites, and sway in the
Application of the publick Money; there are various ways of obliging,
and his Party will always be uppermost in the Legislature, which
consists of him, the Council of twelve, and an Assembly of twenty
two, chose at the several Parishes by a Majority of Votes.
One Law is, That no Inhabitant shall be carried off the Island
without Leave; whoever engages in the Project, is liable to the
Debts; so that when a Family sees Ruin approaching, (a frequent
Case of late years) their Remedy is stealing away in Boats to some
other Place of Subsistence; and if they cannot this way escape a
hard Creditor, they comfort themselves in dying, that it may be their
Lot next. Those who depart fairly, are obliged to give publick notice
at the Secretary’s Office; and no body objecting in twenty one days,
are at liberty.
Another Act in 1676, passed against the Industry of the Quakers,
whose Conversion of the Negroes, it was pretended, hazarded the
Safety of the Island. They are computed at 80 or 90000, and are
countenanced in Polygamy; yet not dangerous, because no
Mountains to fly to, Detections and Executions would soon follow
their Rebellions. The English are reckoned 20000, the Women
among them most Scotch and Irish, very homely, and great
Swearers. The Men, contrarily, are very gay, clean, and handsome,
from mean Originals, often succeeding with rich Widows; it being but
Justice to link a fat Plantation to the truely nauseous Draught of
Matrimony.
The way of feeding such a Multitude, and providing Necessaries in
an Island yielding little besides Sugar, is principally by their Fisheries
and Importations.
The Sea gives them great plenty of flying Fish, Dolphins,
Barricuda and King Fish, particularly the first; they bait with their own
Specie, which thrown about, the Fish fly in such numbers to the
Boats, that they take them up with Dip-nets, and sometimes the
Dolphins with them; the Season goes off at the Autumnal Equinox.
Their Importations by Ships from England, Ireland, New-England,
Pensylvania, Carolina, or New-York, constantly supplying any Defect
of Food or Necessaries, every Vessel bringing them something or
other of this kind, which the Merchants keep in store and sell the
Planters occasionally, who give their Sugars, Rum, and Molosses in
return. The Price in what I was acquainted is, viz.
Bought,
Rum at 1s. 2d. per Gallon.
Citron Water 40 0
Pickled Pepper 10 0
Preserved Ginger 5 0 per lb.
Sugar, twenty Shillings a hundred; and before
our Improvements (says Gee) the
Portuguese sold for 7 and 8l. a hundred.
Cocoa, 3 or 4l.
Aloes 4d. per lb.

Sold,
Salt Beef and Pork, 40 Shillings for a
Cask of 2 Cwt.
Bisket, 17s. per hundred
 Candles, 6½ per lb. &c.
Exchange 30 per Cent. or more.

I have heard that the Custom-house Books had one year 35000
Hogsheads of Sugar entred, which at 10l. per Hogshead, amounts to
350000l. Every Acre was supposed 10s. a year Profit to the national
Stock of England, besides what the Planter got, and Mouths fed by
it; but I must observe, the Crops of late years have very much failed,
and put many of them under great Necessities. The Soil fertile in the
Age past, seems now growing old, and past its teeming-time; they
endeavour to mend this by a few Cattle kept for the sake of Manure;
few, I say, because Land imploy’d this way, gives not 1/10 its Value.
Wherefore when a thoughtless Man has joined to unlucky Events
and Seasons an inadvertent way of living, he falls a Prey to the more
astronomical Heads of Factors, who supply him with Food and
Necessaries. The Hardships of many Planters at this time, through
such Inclemencies, cannot be better laid open to the Reader, than in
transcribing part of a Sermon, that I am informed was preached by
Command of his Excellency the Governor, May 1734.

A Charity S e r m o n at Bridgetown,
for the two Parishes, St. Philip, and Christ-Church.

“Here I should have left off, but I am commanded by his Excellency

the Governor, to exhort you to that Charity, the Necessity of which
has been laid before ye.
“Remember therefore, that one of the ways observed of seeking
God is, by obeying the Dictates of his Holy Spirit, that Humanity and
Charity undepraved Nature feels towards all that are poor and
distressed.
“What an excellent Grace of Christianity this is, St. Paul from the
Spirit of God teacheth, (1 Cor. xiii.) saying, that when the Gift of
Tongues, of Prophecies, of Miracles shall cease, a greater, even that
of Charity, shall never cease in the Church militant, never in the
Church triumphant; nor can there be any greater Inducements to
provoke us to Charity, than first, it covers a Multitude of Sins, and
next, bringeth God himself (as he is pleased to esteem it) in debt to
us; for he who giveth to the Poor lendeth to the Lord, and look, what
he layeth out shall be paid him again; paid in Blessings here, and
hereafter eternal Life, if no mortal Sin continue in the Giver, to hinder
these blessed Effects.—I need say no more to ye who read the
Bible, how dear to God those Christians are, who according to their
Ability are liberal to poor Persons and Families; so that what remains
for me to say, is to expatiate a little upon the miserable State of the
Poor of these two Parishes, and leave the whole to your pious
Consideration.
“In one of these, St. Philip’s, mine Eyes beheld all the Signs of an
approaching Famine; the Face of the Earth appeared as it were a
dry Crust, burnt up and gaping for its watry Nutriment; hardly any
thing green appeared, and I am told, the Face of the Country is
much the same in Christ-Church Parish. Now how miserable must it
be with the single Poor, and with Families! I assure you, several are
come into ours, and others are gone farther Leeward to seek for
Work and Food. You who are tender Parents, consider how terrible it
must be for Families with nothing in their House, nothing growing on
their Land, not a grain of any thing to support themselves and dear
helpless Children: No Money, and no Credit, no Relief from without,
and no Bread, nor Water either, hardly within or without. I have heard
of poor Men going about for Work, to sustain their own Bodies,
forced to leave Wife and Children at home to starve; sure your
Hearts must relent, and every one of you give according as you are
able, with a free Mind, and willing Heart. But here some may object,
Why should I give to those two Parishes, when our own Poor may be
in as great Want? I answer, some may be so; but the Calamity
(blessed be God) is not so general here; it is not so bad with us in
that one necessary Article of Water. Thirst is terrible, let us then pity
our poor Brethren, their Wives and Children, who go so far for Water
that they have not due time to get their Bread, were there Work for
them to earn it by.
“I believe, you know we have here poor Families in great want,
and I could wish our Vestry would meet, particularly to consider it;
but in the mean time, let us not forget the poorer People of these two
Parishes, as now perishing for want of Food; yea, his L——p and the
Council’s Belief is, (you hear) that some have already died for want
of Bread.
“What Christian Man or Woman then in Affluence and Plenty, can
have an Heart so hard as not to bestow liberally on so great, so sad,
so calamitous a Necessity and Misery? and what poorer Christian,
who has somewhat, tho’ little above his daily Wants, but will fling his
Mite to stop so dread an Evil?
“What Christian Woman, who has young and helpless Children of
her own, and Bread to give them, but whose Bowels must yearn and
Heart ake to hear, that in these two Parishes are many Infants crying
at the empty Breasts of their Mothers, and their Mothers weeping
and languishing at the same time for Bread to sustain themselves.
“What compassionate Fathers or Brothers but must grieve to
understand, that grown Children too young to work, are now starving
in these Parishes, and their Parents and Brothers nothing to relieve
them.
“What good Children but must bleed at heart to see their Parents
starving? yet such is the Fate of some in these Parishes.
“Christians consider, that one way of keeping Famine from us of
this Parish, is to bestow our Charity in a Proportion to their Wants,
and our Ability: That is the likeliest Method to move God to give us
fruitful Seasons, to renew our Springs, and bring a cheerful Green
over the Face of our Plants and Seeds.
“May the blessed Spirit, &c.”

The Consequence of this Distress now among the Barbadians, is

shifting their old Habitations; several impelled by Necessity, and
Wants, (stronger Motives than Religion;) are stealing away to mend it
where they can.
The Sufferings of these Islanders, I think, will carry some
Similitude to larger Countries; where the remarkable Decay, or Loss
of one single Branch of Trade, it’s observed, will sensibly affect
Multitudes, not only those immediately concerned in the retailing,
who must change Trades, infringing on others, or seek other
Countries, but also those not concerned; because as an
extraordinary Trade stamps an extraordinary Value on Land, and that
on Provisions, when the one fails, or changes hands, as it has and
will do, (Venice, the Hans Towns, Antwerp, Holland, and which by
the way, shews all Countries bordering on the Sea, within 50°° of
Latitude, equally advantageous for Trade) the other ought to give
way for the lowering of Provisions, and Charge of Subsistence to the
Poor, (some ways of it being supposed now to be cut off or
curtailed:) and if Landlords do it slowly, the Law should oblige;
because, as publick Virtue is no private Man’s Profession, he will
take his Lands into his own hands, tho’ with Loss, rather than submit
to the Reduction of his Rents; and because he can afford it, will
hoard, and suffer Grain to decay and spoil, before he will fall the
Price.
In our Plantations, the inferior sort of Merchants are not unlike
Sharpers in Gaming; they by a better Skill, know how to prey on the
Wants, the Weakness, and Passions of their Customers (the
Planters and Artificers) chaining them down by degrees to their
Service; many of the Inconsiderate being ruined without knowing it,
till the very Day they want Victuals.
 SUGAR-CANES.
In the Wars between Holland and Portugal in Brasil, a Dutch-Man
arrived here from thence, who taught them the way of Planting and
making Sugars. They are set out between August and December, six
Inches deep, and do not come to Maturity until one year and a
quarter: when ripe, which is known by their Colour, they cut them up
with a Bill, and send them to the Wind-mills, which presses out the
Juice so clean, the Canes by being an hour or two in the Sun,
become fit for Fuel.
The Liquor must not remain in the Cistern above a day, for fear of
souring; it is therefore by a Gutter conveyed to the Copper or Boyler,
and in the boiling, the Filth scummed off; thence it’s conveyed into
the second and third, and in the last, called the Tack, is boiled to a
Consistency, and turned into a Grain by throwing in of Temper, which
is only the Infusion of Lime and Water made strong according to the
Goodness of the Cane. Nine Pounds of Juice makes one of
Muscovado, and one of Molossus.
From hence it is carried to the cooling Cistern, till fit to put in Pots,
which have Holes at Bottom to drain off the Molossus.
Of these Molossus again, they sometimes make another worse
Sugar, called Paneels. Of the Scum, coarse Molossus, Washings of
the Boilers and Pots, fermented together, is made Rum.
To refine Sugar, is to boil it over again, and clarify with the same
Lime-Water and Eggs, reckoned better than the clayed Sugars of
this Region, made by putting a clayey Earth mixed with Water to the
thickness of a Batter upon them, and repeated three or four times
according to the degree of Whiteness design’d; both ways carry the
Treacle and Molossus downwards, but the former most esteemed,
as mixing less, and purging to better purpose. Lime refines from
Impurities, and imparts a softer Taste, experienced in throwing it into
Wells of hard Water; the best refin’d in Loaves comes back to the
Sugar-Colonies from England, sell at 50 or 100 per Cent. Advance,
and are of common Use; they must be kept dry, a hot and moist Air
dissolving them.
From Molossus, Distillers make a clean Brandy, and it gives a
pretty tasted Spirit to Malt Liquors, boiled and worked in the Tun.
Besides Rum and Sugars, they have Quantities of Ginger, Aloes,
Tamarinds, Citron, Cassia, Coloquintida, Cassava, Limes, Oranges,
Guavas, Pine-Apples, Mastick, Cedar, Cotton and Palmeto Trees,
prickled Pear; but our Apples and Pears, nor any of our Shrub-Fruits,
Goose-berry or Currant, will thrive. Of the Potato they make a brisk
Small-beer, called Mobby.
About two or three years ago, the low Price of Sugars, that had
reduced and beggar’d the Planters, brought on a Complaint, and Bill
in Parliament in their favour. They urged, according to the best of my
Remembrance, that the northern Colonies, especially New-England,
being suffered to trade with the French Islands, was in a great part
the Occasion of this, and a Loss to the Nation; for they took off all
the French Molossus, which before they had no use for, but sold it
our Islands at very low Prices.
The French therefore were helped by this Sale, to afford their
Sugars cheaper, and still more enabled by a nearer Way of Living;
by the Customs being taken off, allowing them to go thence to any
Market, and other Encouragements to undersell, and take the foreign
Markets from us, who were clogged with all those Inconveniencies.
The New-England People alledged, their Trade seemed the least
essential Article in the Injury complained of; for unless our Islands
found means to take off the other Impediments, and bring their
Sugars to as cheap, or cheaper Price than the French and Dutch,
they would be the same in respect to foreign Markets; and if new
Grounds are better, or more wanted in Plantations, there are enough
at Jamaica, St. Christopher’s, &c. to redress the Evil. But this is not
in their View, say they; the more Lands are employed, the less will
be the Value of the present Estates, an impolitick Reduction of all
prodigal Expences; for every Island singly, reckon their Happiness in
part, not from the flourishing Condition of another, but from
Casualties, and bad Seasons; the less quantity there is to answer
the Demand, the higher the Price.
Barbados formerly used to buy the French and Dutch Sugars,
making all that Trade go through their own hands, till in 1715, laying
a Duty turned the Channel, and they would now make up that
oversight by imposing their own Price on us.
The Northern Colonies deserve Favour, they think, as vastly
superior in Number and Trade, take off more of the Manufactures of
England for themselves, and their Trade with the Indians, who
exchange Furrs and Pelfry to make Hats; for the same Reason, they
want more Molossus to manufacture among themselves, than our
Islands can sell, or if they could, cannot take off one quarter of the
Lumber, Horses, and refuse Fish, with which we trade with the
French, not only for Rum and Molossus (which may as well come to
us this way, as through their hands) but sometimes also Money; and
without which we have no means of purchasing, nor could get rid of
our Produce and Industry, which is very unreasonable.
To lay a Tax of six-pence a Gallon on French Molossus, is the
same as a Prohibition, which their Country cannot so easily bear.
They take 20000 Hogsheads a year (each 100 Gallons) from the
Dutch and French, which is 50000l. whereas they have no Specie to
pay it, their Currency being all Paper, and that but 30000l. Besides, it
would be the first Tax on a charter’d Colony from England, where
they have no Representatives.
Lastly, it was said, the French buy their Negroes, and Sugar-
Materials (Mills, Coppers, &c.) 40 per Cent. dearer than us; therefore
for our Islands to say they cannot afford as cheap, is to say, they will
not abate of their Pride and Luxury, but help to maintain it by a Tax
on our more humble Industry.
 T h e W E S T- I N D I E S .
For a general Idea of the West-Indies, we may understand by that
Term, all the Continent, Sea, and Islands, from Terra Firma to
Florida, or from near the Equinoctial to 28°° of N. Latitude; and if you
include Bermudas, to 32°°. The main Land in this Circuit divided into
Spanish Provinces, is more peculiarly called the Spanish West-
Indies, they possessing all, unless to the Southward in Guiana and
Paria, where there are a few English, Dutch, and French,
interspersed on the Rivers and Coast of Oronoko, Surinam, and
Amazons.
They import hence to Europe, besides Rum and Sugars, great
quantities of Cocoa, Indigo, Cotton, Logwood, Ginger, Lignum-vitæ,
Cochineel, Snuff, Cassia, Aloes, Pimento, Tortoise-shell, Dyers, and
other Wood, a Variety of Drugs, and above all, prodigious Quantities
of Plate, and some Gold.
The Islands in this Sea are the Charibbees, Sotovento, Antilles,
and Bahama.
Charibbees were the lesser Antilles, about 30 in number, whereof
the French have Martinico, St. Lucia, Bartholomew, Deseada,
Granada, Marigalant, Guadalupe, and Santa Cruz. To the Dutch
belong in whole or part, Saba, Eustatia, St. Vincent, and Tobago, or
Tobacco Island; so called, from the Plenty of that Weed there, or the
Weed so called, as first transplanted thence. The rest are English,
and of them Barbados is chief. Others next of Note are Antegoa,
Nevis, St. Christopher’s, and Montserrat; which have a separate
Governor, stiled General of the Leeward Islands, their principal
Produce with us, is Rum and Sugars; but the French, besides these,
cultivate Cocoa, and Indigo: and as the managing of more Lands
naturally gives Plenty, and makes room for an Increase of People,
the French Policy of late years has considerably increased their
Colonies at Martinico and Hispaniola; some say 40000 settled there
at the French King’s Expence, with the Addition of a year’s
Maintenance, to countenance their Mississipi Settlements, and these
further Views of drawing over Men’s Affections, by affording
Europeans the West-India Commodities, at the cheapest rate, and
strengthning themselves against the Resentment of any who dislike
it.
In some are found large Caves that run half a Mile under ground,
supposed the Dwelling-places of the old Natives, who quickly
forsook them to the new Inmates; tho’ Dampier says, he met some of
these Charibbees at St. Lucia, and St. Vincent, and others say the
like of Curasao: The Name imports Cannibals, an Inhumanity
charged on them at the Discovery, as a proper Accusation for
Dispossessors.
Sotovento Isles lie E S E. and W N W. along the Terra Firma,
called so because the Spaniards in their Voyages to Mexico, make
them one after another sub vento (to Leeward.) Of these, the Dutch
have Curasao, Oruba, and Berraire. The Spaniard the others, (La
Trinidad, and Margarita, chief;) from whence, and the Antilles, they
have of late years very much infested this Navigation, with their
Guard le Costas, confiscating the English Effects in Reprisal, it is
supposed, for the Loss of their Fleet near Messina, 1718.
The greater Antilles are, Cuba, Hispaniola, Portorico, and
Jamaica; the three former, Spanish.
Cuba is principal; a very pleasant and flourishing Island, the
Spaniard building and improving for Posterity, without dreaming, as
the English Planters do, of any other Home. They make the best
Sugars in the West-Indies. It was from this Island, (Velasquez
Governor,) that Cortez in 1518, made his Expedition and Conquest
of Mexico.
The Havana, its chief Port and Town, is esteemed the richest in
America; for besides its own valuable Produce, the Spanish Fleets
from all parts on the Main, make up here in their return to Europe.
 The Islands on the South Side of it, and the Camaines, are
resorted to for the largest and best Turtle.
Porto-Rico, and Hispaniola (the diminutive of Hispania) are Islands
we make, in our Passage to Jamaica, famous of late for their Guard
le Costas. These Privateering Fellows, when they are not acting by
lawful Commission, they know the Governor’s Mind, and bring in
Ships on a pretence they are trading with the King of Spain’s
Subjects in a clandestine and prohibited manner; if they find any
Pieces of Eight, it is a Condemnation; an Encouragement in
searching a Ship, to deposite some there themselves: Or if this Trick
fails, they are yet detained, and on various Pretences lengthned out
with Law-suits, till ruined. We called, after weighing from Barbados,
at Sancto Domingo, the chief Town of Hispaniola, where we found
three English Masters of Ships under these Hardships. They had got
the better in Law, but with such Charge and Delay, that it had spoiled
their Ships and Voyages; and lest that should not do it effectually,
their Damages are against the Captains of the Privateers, who are
perhaps the Governor’s servile Dependants, and not worth a Groat.
Sancto Domingo Harbour has 15 Fathom Water at the Bar, and
the Entrance defended by several Batteries. The Town is the
Residence of an Arch-Bishop, and a President from Spain, who lives
in a House that is said to have been built and occupied by
Christopher Columbus himself. To this Officer (on account of its prior
Settlement) Appeals come from all the Spanish West-India Islands,
whose Sentence is definitive, unless called by a particular
Commission to Old Spain. They buy their Places, it seems, and
consequently execute them oppressively.
The Island is diminished of its Inhabitants, for this, or a securer
and better Settlement on the Continent; so that the French now,
about Petit Guavas, equal, if not outnumber them, tho’ both together
are vastly short of what its Extent and Fertility deserves. A Soil that
produces any thing; their Sea and Rivers full of Fish, and the
Country spread with Forests of Cabbage and Palm-Trees, in which
are prodigious Numbers of wild Hog and Beef, which the Hunters of
different Nations at certain Seasons shoot, the latter for their Hides;
and the Pork, they jerk (as they call it) that is, strip it from the Bones,
and then salting the Flesh a little, dry it in the Sun.
Bahamas, so called from the Principal, or Lucayes from Lucayone
(new Providence, the largest of them) where the English have a
Governor: They are noted for a dangerous and rapid Chanel,
commonly called the Gulph of Florida, through which the Spanish
Fleets always take their Passage to Europe, and are frequently
shipwrecked.
The Pyrates often take their rise here, or if not, seldom fail in the
Course of their Adventures to visit these Seas. There are Multitudes
of little Islands and Kays, besides this Division above, that afford
Refreshments of wild Hog, Cow, Goat, Sheep, Parrots, Guanas,
Turtle, and Fish; many of them uninhabited, and seldom visited but
on that account, whereby they are a natural and good Security. The
Sailor, when he would express the Intricacy of any Path-way, stiling it
the Caribbees.
They commonly make their Beginning here after this manner;
when any Spanish Ship is wrecked in Florida, the Jamaicans fit out
Vessels to fish upon her, (the best I believe, being always pleas’d
with going shares in such Voyages, which may be judged of by their
Treatment of the Galleon cast away on Jamaica, a very few Years
ago) and dispute a Right of Plunder with the Spaniard himself, who
is also fitted from the Havana on these Accidents, to recover what
they can; the Contest therefore is with various Fortune, and
sometimes turns to a bad account.
Our Logwood-Cutters from Campechy and Honduras, who have
been unfortunate by the frequent Visits of the Spaniards to destroy
that Trade, remove hither, or those to them, to consult of Reparations
to their broken Fortunes. Saunterers also, who are turtling from
different Parts, do all together make a considerable Resort
sometimes, and being prompted to Revenge for the Injuries
sustained, they combine and furnish out a little Sloop perhaps
against them at first, who finding little come by confining their Ways
and Means to the Spaniards only, who sail in Fleets, they fall at last
on any Nation; the Transition being easy from a Buccanier to a
Pyrate; from plundering for others, to do it for themselves.
These Logwood-Cutters, (since mentioned) I must observe, were
originally settled at the Bay of Campechy, but with a contested Right
that made it hazardous, the Spaniard opposing the Legality, and
when uppermost, treating them as Pyrates, which our People have
frequently returned again with Interest. It was taken 1659, by Sir
Christopher Mins. In 1678 again, by the English and French
Privateers; and what Licence the Peace of Utrecht gave, I am
uncertain, but they are since drove out, and now support themselves
with their Arms at the Bay of Honduras.
They are about 500 (Merchants and Slaves,) and have taken up
their Residence at a Place called Barcaderas, about 40 Miles up a
narrow River full of Alligators; and what is a greater Inconvenience
against transporting their Effects, is a strong Current in it from the
Freshes up Land, and the Banks being covered with Shrubs, that
makes it difficult to walk and tow the Boats; covered also with infinite
Numbers of Sand-Flies, and Muskitos. They live in Pavilions; a
Servant at their time of lying down to rest, shaking them till cleared of
these Vermin, that are an unsufferable Plague and Impediment to
Sleep.
At the Season (once a year) they move their Pavilions from the
pleasurable Spots, the better to attend the Logwood cutting, which
carries them sometimes many Miles from this principal Residence, to
follow the Wood, which runs in a Line or Vein (like Minerals in the
Earth) of some Miles perhaps, and then as many, without a Stick of
it. They cut it into large Pieces, and leave it on the Ground till the
Land-Flood favours their bringing it into the River, and then Canoos
are laden away with it, to lay in store at Barcaderas, where the Chief
are still left residing.
They have all good Arms, and knowing the Spanish Clemency,
defend themselves desperately, if attacked; which has happened
seldomer than at Campechy, and always by Sea.
A Servant, which is the first Step with Seamen into the Trade, is
hired at a Tun of Logwood per Month, and has one Day in seven for
himself, making together about 10l. a Month to him; hence, if
thoughtful and sober, they in time become Masters, join Stock, and
trade independently. They have a King, chose from among their
Body, and his Consort is stiled Queen, agreeing to some Laws by
common Consent, as a Guide to them.
The Ships that come into the Bay, are on their Guard also, fetch it
down in flat-bottomed Boats, each Crew being allowed on the
Voyage, a Bottle of Rum and some Sugar, and row generally in the
Night, as freest from those stinging Flies, and rest in the Day.
The Exchange with Ships is for Money, Beer, Flower, or any sort of
Provisions and Necessaries; these, the cunningest reserve in Store
against the Wants and Demands of the Inconsiderate, and so make
extraordinary Returns.
It may not be improper to conclude this Head with an Observation
or two on the Channel and Current of Florida, which I submit to the
more Skilful.
This Gulph is as dangerous a Navigation as any known; the
Spaniards often experience it, because it’s an Addition to the
Danger, that they have unwieldy Ships, and lubberly Seamen. We
commit Errors, I imagine, by our common Charts, which lay down the
Channel double the Breadth it is; the most intelligent in the Passage
having assured me, it is not above 16 or 18 Leagues over; and
therefore when a Storm happens, build on a false Supposition.
The Spaniard is likewise over-careful to be safe; the nicer
Observations made on Shoals, Currents, or Winds, either here or in
the Bay, when and how to make them advantageous, are from an
imagined Security against any maritime Power, committed only to
their Admiral (according to common Report) whose Light the Fleet
are to follow; and for their better Recovery of any shipwrecked Cargo
in the Gulph, (frequent in losing the Admiral,) they have a Garrison at
St. Augustine, on the Florida Shore, a barren Spot where they are
almost starved, and which would not be worth keeping but for this.
Ships and Vessels may, and often have sailed through this Channel
from the N End to Cuba, or the Bay of Mexico, notwithstanding the
common Opinion, on account of the Current, that is against it. They