Single-Domain Antibodies: Methods and Protocols (Methods in Molecular Biology, 2446) 1071620746, 9781071620748

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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-bystep 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 Human Health Therapeutics Research Centre National Research Council Canada Ottawa, ON, Canada

Kevin A. Henry Human Health Therapeutics Research Centre National Research Council 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 multispecific antibodies, chimeric antigen receptors (CARs), and various types of “armed” antibody conjugates has placed antibody fragments in the spotlight. For applications requiring fragments, the naturally occurring autonomous variable domains of camelid heavy chain-only antibodies (VHHs, nanobodies) and shark immunoglobulin new antigen receptors (VNARs) have significant advantages over fragments derived from conventional tetrameric 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 reversible 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 sitespecific 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

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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 . . . . . . . . . . . . . . Fangling Ji, Jun Ren, Ce´cile 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 . . . . . . . . . . . . . . . Samata S. Pandey, Marina Kovaleva, Caroline J. Barelle, and Obinna C. Ubah

PART II

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3

19

ISOLATION OF SINGLE-DOMAIN ANTIBODIES

3 Llama DNA Immunization and Isolation of Functional Single-Domain Antibody Binders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Fre´de´ric 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 ˜ as, Jahaziel Gasperin-Bulbarela, Olivia Cabanillas-Bernal, Salvador Duen 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 . . . . . . . . . . . . . . . . Ario de Marco 8 Small-Scale Secretory VHH Expression in Saccharomyces cerevisiae . . . . . . . . . . . . Michiel M. Harmsen, Marga van Hagen-van Setten, and Peter T. J. Willemsen 9 Production of Single-Domain Antibodies in Pichia pastoris . . . . . . . . . . . . . . . . . . Yusei Matsuzaki, Kaho Kajiwara, Wataru Aoki, and Mitsuyoshi Ueda 10 Production of Designer VHH-Based Antibodies in Plants . . . . . . . . . . . . . . . . . . . Henri De Greve vii

145 159

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Contents

PART IV 11

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13 14 15

16 17

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21

SINGLE-DOMAIN ANTIBODY ENGINEERING AND CHARACTERIZATION

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

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PART V APPLICATIONS OF SINGLE-DOMAIN ANTIBODIES 22

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Visualizing Filoviral Nucleoproteins Using Nanobodies Fused to the Ascorbate Peroxidase Derivatives APEX2 and dEAPX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 Laura Jo Sherwood and Andrew Hayhurst Development of Glypican-2 Targeting Single-Domain Antibody CAR T Cells for Neuroblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Nan Li and Mitchell Ho Single-Domain Antibodies for Intracellular Toxin Neutralization . . . . . . . . . . . . . 469 Timothy F. Czajka and Nicholas J. Mantis

Contents

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

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

489 513

531

547 555

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595 C1 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, ICMIBEFY/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 DUEN˜AS • 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 GONZA´LEZ-SAPIENZA • Ca´tedra de Inmunologı´a, Facultad de Quı´mica, 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, Mu¨nster, 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 • Ca´tedra de Inmunologı´a, Facultad de Quı´mica, 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, ICMIBEFY/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, Mu¨nster, 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 PI´REZ-SCHIRMER • Ca´tedra de Inmunologı´a, Facultad de Quı´mica, 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, SainteAnne-de-Bellevue, QC, Canada FRE´DE´RIC 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 CE´CILE 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, Mu¨nster, 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, Ce´cile Vincke, Lingyun Jia, and Serge Muyldermans Abstract The presence of unique heavy chain-only antibodies (HCAbs) in camelids was discovered at Vrije Universiteit 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, biotechnology, 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, dromedaries, 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 implicated 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

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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. . .

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technologies, selected for antigen-binding, and expressed in bacteria 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 recombinant, 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 microorganisms. 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 repertoires 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 biochemical and biophysical properties of Nbs [11]. Fortunately, Nbs were well expressed in bacteria and folded into stable monomeric 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.

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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 ˜ a), and new-world Camelidae (llama, guanaco, alpaca, and vicun their absence in Ruminantia (cattle, antelopes, and giraffes) and Suidae (pigs and hippopotami) was a first indication that HCAbs

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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 heterotetrameric 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 interspersed 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 antigenindependent 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].

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Identification of Antigen-Specific Nbs Several techniques are available to isolate Nbs against virtually any target starting from an immune, naı¨ve, 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 spectrometrybased 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

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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 immunogens. 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 generation of transgenic mice in which IGHM and/or IGHG genes were truncated to have their CH1 domain deleted [24, 25]. Subsequently, 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 generation 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 immunization of animals for antibody production since alternative technologies are available whereby a naı¨ve or synthetic VHH library is used as the primary source for selecting antigen-specific Nbs [26]. The use of naı¨ve or synthetic Nb libraries bypasses the requirement to immunize. However, for naı¨ve 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 naı¨ve library and the antigen-binding affinity of isolated Nbs. This does not exclude the possibility to retrieve practical binders from naı¨ve libraries [27]. However, naı¨ve libraries should contain at least 1010 individual clones, which is the maximum that can be handled in phage

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display selections, and larger libraries can be employed using ribosome 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 selection 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, bacterial, yeast, or ribosome display) have been developed to retrieve the best possible Nbs from immune, naı¨ve, or synthetic Nb repertoires. 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 potential antigen-binding candidates, with final identification of antigenbinding 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. Alternatively 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 phenotypic 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 identifying 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].

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Favorable Characteristics of Nanobodies For in-depth characterization of Nbs, selected candidates should be expressed in large quantities in microorganisms and purified. Production 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 Gramnegative bacteria (such as Escherichia coli) is often preferred and Nbs are easily extracted using osmotic shock. Already highly

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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 microorganisms 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 protein 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 panning, Nbs typically recognize their cognate antigen with high specificity 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 herbicides, 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.

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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

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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 example, an anti-CD19 moiety targeting B cells is fused with an antiCD3 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 comprising 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 pentamerization domain such as the B domain of verotoxin or Shiga toxin [49]. Using this pentamerizing scaffold, a decavalent/bispecific 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

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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 nucleophilic 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 fiveamino 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 proteins, 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 heptameric 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.,

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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 rapidly 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 dihydrofolate reductase (cpDHFR) was inserted within the third antigenbinding 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 adenine 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 Megabodies 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.

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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 binders available, including DARPins, affibodies, monobodies, anticalins, 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

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libraries enriched for antigen binders. The immunization of a camelid and the construction of a relatively small Nb library is straightforward, 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 interactions 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 superresolution microscopy [70] as well as useful tools for the crystallization 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 infections, as their association with the antigen might be less obstructed by endogenous host antibodies [72]. Nb engineering in biotinylated 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,

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respiratory syncytial virus, rotavirus, or influenza) [78]. Toxins circulating in blood might also be removed ex vivo by apheresis. For this type of application, Nbs are covalently linked to resin for the production 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 signaling 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 constructs formed the basis of several additional therapeutic applications [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 pipeline 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 Program of China (2016YFC1103002), and the Fundamental Research Funds for the Central Universities (DUT20LAB121, DUT20YG107, DUT21ZD207, and DUT21LK10). References 1. Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally-occurring antibodies 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 function of llama heavy chain antibody fragments by molecular evolution. J Biotechnol 80: 261–270

5. Skerra A, Plu¨ckthun 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 therapy. 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 single 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 singledomain 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 antibodies. 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 heavychain 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 indistinguishable but immunogenetically distinct. Immunogenetics 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 germline 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 nanobody repertoires. Nat Methods 11:1253–1260 22. Odegrip R, Coomber D, Eldridge B et al (2004) CIS display: in vitro selection of

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peptides from libraries of protein-DNA complexes. 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 single domain antibodies for development of sensitive immunoassay. Anal Chem 84:1165–1171 24. Janssens R, Dekker S, Hendriks RW et al (2006) Generation of heavy-chain-only antibodies 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 antibody 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 naı¨ve 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 proteins. 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 functional 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 expression 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

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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 immunoassay 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 target 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 nanobodies. Front Immunol 12:e632687 39. Li T, Bourgeois JP, Celli S et al (2012) Cellpenetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the bloodbrain barrier and specifically recognize astrocytes: 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, Be´langer 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 engineering: toward next generation immunotherapies 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 antiEGFR nanobodies. Cancer Immunol Immunother 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 bivalent antibody constructs. J Biol Chem 276: 7346–7350 49. Zhang J, Tanha J, Hirama T et al (2004) Pentamerization 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 bispecific decavalent molecules. J Immunol Methods 318:88–94 51. Fan K, Jiang B, Guan Z et al (2018) Fenobody: a ferritin-displayed nanobody with high apparent affinity and half-life extension. Anal Chem 90:5671–5677 52. Massa S, Xavier C, De Vos J et al (2014) Sitespecific 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) Sortasemediated ligations for the site-specific modification of proteins. Curr Opin Chem Biol 22: 122–128 54. Sarpong K, Bose R (2017) Efficient sortasemediated 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 catalyzed by a single recombinant ligase. J Am Chem Soc 141:17388–17393 56. Rabuka D, Rush J, DeHart G et al (2012) Sitespecific 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 antibodies 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 SpyTagproteins 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 system 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 fluorescent 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, Mu¨ller TG et al (2020) Chemogenetic control of nanobodies. Nat Methods 17:279–282 67. Uchan´ski T, Masiulis S, Fischer B et al (2021) Megabodies expand the nanobody toolkit for protein structure determination by singleparticle cryo-EM. Nat Methods 18:60–68 68. Herce HD, Deng W, Helma J et al (2013) Visualization and targeted disruption of protein interactions in living cells. Nat Commun 4: 2660 69. Bothma JP, Norstad MR, Alamos S et al (2018) LlamaTags: a versatile tool to image transcription 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 superresolution imaging, rapid epitope-mapping and native protein complex isolation. eLife 4: 1–21

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71. Uchan´ski T, Pardon E, Steyaert J (2020) Nanobodies to study protein conformational states. Curr Opin Struct Biol 60:117–123 72. Morales-Ya´nez F, Trashin S, Hermy M et al (2019) Fast one-step ultrasensitive detection of Toxocara canis antigens by a nanobodybased electrochemical magnetosensor. Anal Chem 91:11582–11588 73. Keyaerts M, Xavier C, Heemskerk J et al (2016) Phase I study of 68Ga-HER2-nanobody for PET/CT assessment of HER2expression 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. Theranostics 4:708–720 75. Dekempeneer Y, Keyaerts M, Krasniqi A et al (2016) Targeted alpha therapy using shortlived alpha-particles and the promise of nanobodies 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, Shahbazzadeh D et al (2018) Nanobodies as novel therapeutic 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 immunoadsorbent for the extracorporeal removal of β2-microglobulin. Molecules 24:2–13 80. Bao C, Gao Q, Li L et al (2021) The application of nanobody in CAR-T therapy. Biomol Ther 11:1–18 81. Jovcˇevska I, Muyldermans S (2019) The therapeutic 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 predisposition 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

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Introduction One could postulate that nature’s goal for single binding domainbased 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 antibodies to smaller fragments (Fab, single chain-Fv, VHH, VH, VL), even smaller binding entities have been developed such as Affibodies, 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

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nature also produced another reductionist solution some 450 million 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], bamboo 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 performs the same functions as the variable VH/VL domains of classical antibodies.

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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, ranging 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 significantly to making VNARs the smallest (~11 kDa) known naturally occurring immunoglobulin-like proteins with a full antigenbinding site [15]. VNARs display a wide variety of CDR loop lengths and structures. 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 resulting 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 protruding CDR loops [18]. The CDR3 of this isotype lacks the second cysteine residues found in Type I VNARs. This allows

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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 system. This isotype has shorter and less diverse CDR3s due to germline 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 maintaining 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

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[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 bioinformatic 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 antigenbinding 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 hydrophobic 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 stability 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].

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Generation of Antigen-Specific VNARs Several techniques exist for the isolation of antigen-specific VNARs from naı¨ve, 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 primers and subsequent cloning into suitable display and recombinant protein expression vectors [4, 6, 11, 13, 27–29].

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Naturally derived naı¨ve 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 naı¨ve 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 typically 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 synthetic 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 playing a role as affinity matured binders, tailor-made to recognize a particular class of antigens that contained protein cavities or canyons. 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/semisynthetic 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].

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Table 1 Summary of immune, naı¨ve, and synthetic library-derived VNARs and their applications

Library type

Shark species

Common name

Therapeutic application

Antigen

Naı¨ve

Mustelus canis

Smooth dogfish

Cholera

Cholera toxin [7]

Naı¨ve

Orectolobus maculatus

Spotted wobbegong shark

Various cancers

Aurora-A kinase

[32]

Immunized

Ginglymostoma cirratum

Nurse shark

Uveitis, inflammatory disease

ICOSL, TNF-α

[4, 16]

Immunized

Ginglymostoma cirratum

Nurse shark

NA

HEL

[12]

Immunized

Ginglymostoma cirratum

Nurse shark

Ebola virus

VP40

[33]

Immunized

Heterodontus francisci

Horn shark

Inflammatory disease

TNF-α

[11]

Immunized

Squalus acanthias

Spiny dogfish

Half-life extension

HSA

[29]

Immunized

Triakis scyllium

Banded hound shark

Malaria

PfHRP2

[34]

Synthetic/semi- Squalus synthetic acanthias

Spiny dogfish

Rheumatoid arthritis

ICOSL

[35]

Synthetic/semi- Squalus synthetic acanthias

Spiny dogfish

Anti-toxins

SEB, BoNT/ [6] A

Synthetic/semi- Ginglymostoma synthetic cirratum

Nurse shark

NA

Leptin

[36]

Synthetic/semi- Chiloscyllium synthetic plagiosum

Bamboo shark

Cancer

VEGF, EpCAM

[23, 37]

Synthetic/semi- Triakis scyllium synthetic

Banded hound shark

Antiviral

VHSV

[8]

Synthetic/semi- Orectolobus synthetic maculatus

Wobbegong shark

NA

Tom70

[13]

Synthetic/semi- Orectolobus synthetic maculatus

Wobbegong shark

Antiviral

HBeAg of HBV

[38]

Synthetic/semi- Orectolobus synthetic maculatus

Wobbegong shark

Malaria

AMA1

[39]

Synthetic/semi- Orectolobus synthetic maculatus

Wobbegong shark

Periodontitis

Gingipain K protease

[5]

References

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 staphylococcal 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

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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 enzymelinked 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 responsive 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].

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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 extension 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 species [21, 44]. Alternatively, an IgG Fc fragment has been fused to a therapeutic VNAR domain to improve its serum half-life and resulting efficacy [4, 16, 46]. VNAR-Fc fusions are also capable of Fc-mediated effector functions, such as antibody-dependent cellular 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

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essentially quadrivalent VNAR domains fused to an IgG1 Fc fragment 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 germline 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 neither domain class contained detrimental T-cell epitopes [44]. Streltsov 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 nanomolar 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).

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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, QuadX™, and Quad-Y™, Fig. 2) require additional post-translational modifications, including glycosylation in their Fc region, and therefore heterologous expression of these constructs has been successfully 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 ranging 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

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Fig. 2 Schematic illustration of multivalent VNAR formats. These multivalent formats represent both multiparatopic 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 addition of genetically encoded purification/detection tags (polyhistidine, 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 (quantification) 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 typically 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].

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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 inflammatory diseases, VNARs have been successfully generated and are undergoing preclinical development against: B cell-activating factor, 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 adhesion 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, minimizing 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 nanoparticles 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...

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An increasing number of VNAR studies have shown the flexibility of these robust domains in accessing challenging and hard-toreach biological compartments. An anti-VEGF165 VNAR generated 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 thermotolerance 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 staphylococcal 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.

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Summary With 400 million years of evolution behind their refinement as binding domains, it is unsurprising that VNARs are showing enormous potential for disruptive success in the fields of drug discovery, diagnostic tool development, and bioprocessing. For certain antigen 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 distinguishes them from classical antibodies and affords any VNAR platform a clear intellectual property position and freedom to operate, 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 undergoes 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 preferentially expressed in early development. Immunogenetics 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 antibodies 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) Variable domain antibodies specific for viral hemorrhagic 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 smallspotted 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 Heterodontus francisci shark: a potential therapeutic use. MAbs 5:80–85 12. Dooley H, Flajnik MF, Porter AJ (2003) Selection 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 earlydevelopmental isotype. Protein Sci 14: 2901–2909 19. Stanfield RL, Dooley H, Flajnik MF et al (2004) Crystal structure of a shark singledomain 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. Mu¨ller 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 biologics—a novel technology platform for therapeutic drug development. Expert Opin Biol Ther 14:1527–1539 ˜ as S, Ayala-Avila M 23. Cabanillas-Bernal O, Duen 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 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure 15:1452–1466

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25. Roux KH, Greenberg AS, Greene L et al (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence 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 Porphyromonas 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 antiidiotypic binders. Sci Rep 7:9676 29. Mu¨ller MR, O’Dwyer R, Kovaleva M et al (2012) Generation and isolation of targetspecific single-domain antibodies from shark immune repertoires. Methods Mol Biol 907: 177–194 30. Feng M, Bian H, Wu X et al (2019) Construction and next-generation sequencing analysis of a large phage-displayed VNAR singledomain 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 fragments 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 collageninduced 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

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antibody fragments from a shark synthetic library. Mol Immunol 44:656–665 37. Zielonka S (2015) The shark strikes twice: generation of mono-and bispecific high-affinity vNAR antibody domains via step-wise affinity maturation. PhD thesis, Technische Universit€at Darmstadt. http://tuprints.ulb.tudarmstadt.de/4481/ 38. Walsh R, Nuttall S, Revill P et al (2011) Targeting 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 Plasmodium falciparum AMA1. Proteins 55:187–197 40. Grzeschik J, Ko¨nning D, Hinz SC et al (2018) Generation of semi-synthetic shark IgNAR single-domain antibody libraries. Methods Mol Biol 1701:147–167 41. Ko¨nning D, Zielonka S, Sellmann C et al (2016) Isolation of a pH-sensitive IgNAR variable domain from a yeast-displayed, histidinedoped master library. Mar Biotechnol (NY) 18: 161–167 42. Ko¨nning D, Kolmar H (2018) Beyond antibody engineering: directed evolution of alternative 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, Mu¨ller MR, Carvalho MF et al (2017) In vitro maturation of a humanized shark VNAR domain to improve its biophysical properties 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 targeting 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 format 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, Piche´-Nicholas N et al (2013) Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized 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-Gonza´lez MT, Sa´nchez˜ oz F et al (2013) Anti-tumor necrosis facMun 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 Escherichia 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-Gonza´lez MT, Garcı´a-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 IgNARderived binding domains as potential diagnostic and therapeutic agents. Dev Comp Immunol 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 protein drug conjugates targeting the oncofetal protein ROR1. In: Proceedings of the annual meeting of the American Association for Cancer 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

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associated vasculature. Nanoscale 12: 14751–14763 60. Jain S, Doshi AS, Iyer AK et al (2013) Multifunctional 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 tailormade bioprocessing reagents. Protein Eng Des Sel 32:385–399

Part II Isolation of Single-Domain Antibodies

Chapter 3 Llama DNA Immunization and Isolation of Functional Single-Domain Antibody Binders Fre´de´ric Trempe, Martin A. Rossotti, Tahir Maqbool, C. Roger MacKenzie, and Mehdi Arbabi-Ghahroudi Abstract Genetic immunization is a simple, cost-effective, and powerful tool for inducing innate and adaptive immune responses to combat infectious diseases and difficult-to-treat illnesses. DNA immunization is increasingly used in the generation of monoclonal antibodies against targets for which pure proteins are unavailable or are difficult to express and purify (e.g., ion channels and receptors, transmembrane proteins, and emerging infectious pathogens). Genetic immunization has been successfully utilized in small inbred laboratory animals (mostly rodents); however, low immunogenicity of DNA/RNA injected into large mammals, including humans, is still a major challenge. Here, we provide a method for the genetic immunization of llamas, using a combination of biolistic transfection with a gene gun and intradermal injection with a DERMOJET® device, to elicit heavy-chain IgG responses against epidermal growth factor receptor (EGFR). We show the technique can be used to generate single-domain antibodies (VHHs) with nanomolar affinities to EGFR. We provide methods for gene gun bullet preparation, llama immunization, serology, phage-display library construction and panning, and VHH characterization. Key words Single-domain antibody, VHH, Camelid, DNA immunization, Gene gun, DERMOJET®, Phage-display

1

Introduction Protein immunization of laboratory animals has been the most effective way to generate antibodies against therapeutically important targets ranging from cancer biomarkers to infectious agents. This technique has resulted in dozens of antibodies (monoclonals, polyclonals, and fragments) that have been approved by the US Food and Drug Administration and the European Medicines Agency [1]. However, protein immunization is time and labor intensive and may not be applicable for emerging infectious pathogens, or for proteins with complex structures, such as multi-pass membrane proteins, where sufficient quantities are difficult to express and purify in active conformations [2–4].

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_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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DNA immunization was initially introduced as a vaccination platform in the 1990s following several studies which showed that genetic material (DNA) could be directly transferred into mouse muscle tissue in vivo, and that expressed gene products could subsequently be detected [5]. The presence of anti-human growth hormone antibodies was demonstrated in another study in which DNA-coated gold microprojectiles were propelled into the cells of a living animal using a hand-held biolistic system [6]. These studies established a new class of “third generation” vaccines that are now considered critical tools in fighting emerging pathogens [7]. Advantages of DNA immunization include its relative simplicity, absence of impurities commonly associated with protein immunization, speed and flexibility in immunogen design, and efficiency in generating antibodies against conformationally sensitive epitopes [4, 8]. However, genetic immunization also has many challenges, including inconsistency of the immune response in large, outbred animals and low immunogenicity of injected DNA in clinical studies. Comparison of immunization data between mice and large animals showed that transfection efficiency and subsequent expression of the target protein are key factors in the success of DNA immunization. Numerous strategies have been developed to increase transfection efficiency including improved DNA delivery systems, use of adjuvants, and use of heterologous prime-boost schemes. In some instances, DNA immunization can elicit strong humoral immune responses and responding B cells can be used to produce monoclonal antibodies [3, 8, 9]. For example, biolistic transfection via gene gun, intramuscular injection, and intradermal injection of DNA have been used to generate monoclonal antibodies against membrane proteins (e.g., G-protein coupled receptors), intracellular proteins (e.g., BCL-6 and MAL T1), bacterial toxins (e.g., Clostridium difficile toxin A), and viral envelope proteins (e.g., influenza H5N1 and hepatitis B virus) [3]. DNA immunization in large animals has largely been studied for the purpose of vaccination. However, the discovery of unique heavy-chain antibodies in the Camelidae family by Hamers-Casterman et al. in 1993 [10], as well as the single-domain antibodies (sdAbs or VHHs) derived therefrom, created new opportunities for genetic immunization and antibody production against complex targets in large animals. VHHs have many desirable therapeutic properties including small size, stability, and modularity [11–13]. Like fragments derived from conventional tetrameric antibodies, sdAbs can be generated against proteins and other biomolecules using in vitro display technologies to mine the repertoires of naı¨ve and/or immunized animals [14]. Immunization of llamas with DNA encoding difficult-toaccess targets such as membrane proteins has resulted in elicitation of sdAbs with functional activity against several targets including the chemokine receptors CXCR7 [15] and ChemR23 [16], the

Llama DNA Immunization and Isolation of Functional Single-Domain Antibody. . .

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ribosyltransferase ART2.2 [17], the G-protein coupled receptor VPAC1 [18], and the epidermal growth factor receptor (EGFR) [19]. While several of these reports have implied that DNA immunization in the Camelidae is routine and generally successful in yielding high-affinity VHHs, our experience has been variable; DNA immunization has resulted in poor immunogenicity for some targets, eliciting inconsistent or undetectable immune responses. Here, we provide detailed protocols for DNA immunization of llamas and isolation and functional characterization of the resulting VHHs. The methods described include: (1) preparation of cDNA constructs encoding a gene of interest; (2) plasmid preparation for DERMOJET® injections and preparation of gold nanoparticles for gene gun delivery; (3) llama immunization and analysis of polyclonal immune responses; (4) phage-displayed VHH library construction and panning; (5) identification of enriched VHH-phage clones and phage ELISA; (6) VHH binding studies by ELISA and flow cytometry, and (7) reformatting the resulting VHHs into VHH-Fc fusions. These protocols are presented as a case study of the single-pass membrane protein EGFR [19]. We caution that variability may be observed at the level of the llama immune response or in the isolation of functional VHH binders against different immunogens. Thus, proceeding with the library construction and panning campaigns, in the absence of a clear positive polyclonal immune response against the immunogen, is expected to have variable success in the isolation of sdAbs.

2

Materials Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 M Ω cm at 25
C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. All oligonucleotide primers listed in this protocol were purchased commercially, desalted but with no additional purification unless otherwise indicated.

2.1 Large-Scale EGFRvIII-pTT5 Plasmid Preparation

1. Two pTT5 expression vectors [20] encoding: (1) 6  Histagged human EGFRvIII ectodomain (UniProt P00533: residues 1–29/Gly/297–645), and (2) membrane-tethered human EGFRvIII-ECD-TM (UniProt P00533: residues 1–29/Gly/297–668). 2. Electrocompetent Escherichia coli DH5α cells (NEB, Ipswich, MA, USA) or equivalent. 3. Sterile double-distilled water (ddH2O).

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4. SOC medium: 20 g of tryptone, 5 g of yeast extract, 0.58 g of NaCl, and 0.19 g of KCl in 1 L of ddH2O. Autoclave, cool and add 0.4% (w/v) filter-sterilized glucose and 10 mM filtersterilized MgCl2. Store at 20
C in 1 mL aliquots. 5. LB broth (per L): 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl dissolved in ultrapure H2O. Sterilize by autoclaving for 20 min at 121
C. 6. 100 mg/mL ampicillin disodium salt (Amp) stock solution prepared in ultrapure water. Sterilize by 0.22 μm filtration and store at 20
C. 7. LB agar (per L): prepare LB broth and add 15 g of agar. Sterilize by autoclaving. Cool to ~50
C and add filtersterilized Amp to a final concentration of 100 μg/mL. 8. Sterile 70% (v/v) glycerol solution. Autoclave to sterilize. 9. 0.22 μm Millex-GV Syringe filter unit. 10. Disposable electroporation cuvettes, 0.1 cm gap width. 11. MicroPulser™ electroporator or equivalent. 12. NanoDrop™ One Microvolume UV-Vis spectrophotometer or similar instrument. 13. Qiagen® EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA, USA). 14. 0.5 M ethylenediaminetetraacetic acid (EDTA) stock solution (per L): 186.1 g EDTA-Na2·2H2O in ultrapure H2O. Adjust pH to 8.0 with NaOH and store at room temperature. 15. 50 TAE buffer (per L): 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA solution in ultrapure H2O. Store at room temperature. Dilute 1:50 in H2O to prepare 1 TAE buffer. 16. 1% (w/v) agarose gel, prepared in 1 TAE buffer. 17. Agarose gel electrophoresis materials and equipment, including molecular size standards, DNA loading dye, and gel stain. 2.2 Preparation of Gene Gun Bullets for Llama Immunization

1. 1.0 μm diameter gold microcarriers (Bio-Rad, Hercules, CA, USA). 2. 0.05 M spermidine solution. 3. 1 M CaCl2. 4. Ethyl alcohol, anhydrous. 5. Cartridge Kit (Bio-Rad): includes 0.5 g polyvinylpyrolidone (PVP), five desiccant pellets, five cartridge collection/storage vials, and 15 m of Tefzel™ tubing. 6. 10 mL plastic syringe. 7. 1.5 mL microcentrifuge tubes.

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8. 15 mL Falcon tubes. 9. Microcentrifuge. 10. Ultrasonic cleaner (e.g., Fisher FS3 or Branson 1210). 11. Vortex mixer. 12. Analytical balance. 13. Tubing prep station (Bio-Rad). 14. Micropipettors (20 μL, 200 μL, and 1000 μL) and tips. 15. Serological pipettes (5 mL and 10 mL) and pipette gun. 16. Nitrogen tank (grade 4.8 or higher). 17. Nitrogen tank regulator. 2.3 Llama DNA Immunization and Peripheral Blood Mononuclear Cell Isolation

1. Helios gene gun (Bio-Rad). 2. Helium tank (grade 4.5 or higher). 3. Adjustable wrench. 4. Helium pressure regulator with a CGA 580 female fitting (Bio-Rad). 5. Ear protection. 6. Gene gun gold bullets pre-adsorbed with plasmid DNA. Prepare sufficient bullets to deliver 10 μg of DNA per bombardment for 12 bombardments per gene gun immunization. 7. Plasmid DNA solution (1 mg/mL). Prepare sufficient plasmid to administer 1 mg per immunization by intradermal injection. 8. DERMOJET® device (AKRA Dermojet, Pau, France). 9. Male or female llama. 10. 1 and 2 mL syringes fitted with 21-gauge, 1- to 1.5-inch long needles, and 10–15 mL vacutainers for blood collection. 11. Heparin-coated tubes. 12. RPMI 1640 medium. 13. LymphoPrep® tubes (Progen, Heidelberg, Germany) or equivalent. 14. RNAlater® (Thermo Fisher, Waltham, MA, USA).

2.4 Monitoring of Immune Responses

1. Recombinant 6  His-tagged EGFR ectodomain (GenScript, Piscataway, NJ, USA). 2. Recombinant in vivo biotinylated 6  His-tagged human EGFRvIII ectodomain (ACROBiosystems, Cambridge, MA, USA). 3. Pre-immune and immune llama sera. 4. Phosphate-buffered saline (PBS, per L): 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 dissolved in ultrapure

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H2O. Adjust pH to 7.4 with HCl, sterilize by autoclaving and store at room temperature. 5. PBS-T: PBS containing 0.05% (v/v) Tween-20. 6. PBS containing 5% (w/v) skim milk. 7. PBS-T containing 2% (w/v) bovine serum albumin (BSA). 8. Goat anti-llama IgG antibody, horseradish peroxidase (HRP) conjugated (Cedarlane, Burlington, Canada). 9. 3,30 ,5,50 -Tetramethylbenzidine (TMB) liquid substrate. 10. 1 M H3PO4. 11. 96-well microtiter plates. 12. Multiskan™ FC Microplate photometer (Thermo Fisher) or similar instrument. 13. 96-well deep well plates. 2.5 Phage-Displayed VHH Library Construction

1. Llama peripheral blood mononuclear cells (PBMCs). 2. PureLink™ RNA Mini Kit (Thermo Fisher). 3. qScript® cDNA supermix (Quanta Biosciences, Gaithersburg, MD, USA). 4. dNTP Mix, 10 mM each. 5. RNase/DNase-free water. 6. Platinum™ Taq DNA polymerase and 10 buffer (Thermo Fisher). 7. Primers (10 pmol/μL) for library construction, colony PCR, and sequencing (Table 1). 8. FastDigest SfiI, XhoI, and PstI restriction endonucleases and buffers (Thermo Fisher). 9. pMED1 phagemid vector [21]. 10. T4 DNA ligase (5 U/μL) (Thermo Fisher). 11. E. coli TG1 electrocompetent cells (Lucigen, Middleton, WI, USA). 12. 100 mg/mL Amp stock solution: see Subheading 2.1. 13. 50 mg/mL kanamycin (Kan) stock solution prepared in ultrapure water. Sterilize by 0.22 μm filtration and store at 20
C. 14. 2 M glucose: 360 g glucose per L dissolved in ultrapure ddH2O. Sterilize by 0.22 μm filtration and store at room temperature. 15. SOC medium: see Subheading 2.1. 16. LB broth and agar: see Subheading 2.1. 17. 2  YT medium: 16 g tryptone, 10 g yeast extract, 5 g NaCl in 1 L of ddH2O. Autoclave to sterilize. Supplement with Amp (100 μg/mL), Kan (50 μg/mL), and/or glucose as needed.

Llama DNA Immunization and Isolation of Functional Single-Domain Antibody. . .

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Table 1 Oligonucleotide primers used for phage-displayed VHH library construction, colony PCR, and DNA sequencing Name

Sequence (50 –30 )

Purpose

MJ1

GCC CAG CCG GCC ATG GCC SMK GTG CAG CTG GTG GAK TCT GGG GGA

Library construction

MJ2

GCC CAG CCG GCC ATG GCC CAG GTA AAG CTG GAG GAG TCT GGG GGA

Library construction

MJ3

GCC CAG CCG GCC ATG GCC CAG GCT CAG GTA CAG CTG GTG GAG TCT

Library construction

CH2FORTA4 CGC CAT CAA GGT ACC AGT TGA

Library construction

CH2B3-F

GGG GTA CCT GTC ATC CAC GGA CCA GCT GA

Library construction

MJ7

CAT GTG TAG ACT CGC GGC CCA GCC GGC CAT GGC C

Library construction

MJ8

CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA Library CGG TGA CCT GG construction

PN2

CCC TCA TAG TTA GCG TAA CGA TCT

Colony PCR, sequencing

M13RP

CAG GAA ACA GCT ATG AC

Colony PCR, sequencing

18. 2  YT-Amp plates: 2  YT medium, 15 g agar in 1 L of ddH2O. Autoclave, cool to ~55
C. Supplement with Amp (100 μg/mL) and/or Kan (50 μg/mL) as needed. 19. PBS: see Subheading 2.4. 20. 80% glycerol solution. Autoclave to sterilize. 21. M13KO7 helper phage (NEB). 22. Polyethylene glycol (PEG)/NaCl: 20% (v/v) PEG 8000, 146.1 g NaCl in 1 L of ddH2O. Autoclave and store at room temperature. 23. QIAquick® gel extraction and QIAquick® PCR purification kits (Qiagen). 24. Agarose gel electrophoresis materials and equipment, including molecular size standards, DNA loading dye and gel stain. 25. 0.5 M EDTA stock solution: see Subheading 2.1. 26. 1 TAE buffer: see Subheading 2.1. 27. 1% agarose gel, prepared in 1 TAE buffer. 28. Disposable electroporation cuvettes, 0.1 cm gap width. 29. MicroPulser™ electroporator or similar instrument.

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30. Heating block. 31. NanoDrop™ One Microvolume UV-Vis spectrophotometer or similar instrument. 32. E. coli TG1 cells streaked on a minimal media plate. 33. 37
C shaker incubator. 34. Thermal cycler. 35. 0.22 μm Steriflip-GP sterile centrifuge tube top filter unit and 0.22 μm Stericup-GP sterile vacuum filtration system. 36. 70% (v/v) ethanol. 37. 15 mL and 50 mL Falcon tubes. 38. Pasteur pipettes. 39. RNase AWAY™. 40. Taq DNA polymerase and 10 PCR buffer (GenScript). 2.6 VHH-PhageDisplay Library Panning and Screening

1. Recombinant in vivo biotinylated 6  His-tagged human EGFRvIII ectodomain. 2. Recombinant streptavidin (Jackson ImmunoResearch, West Grove, PA, USA). 3. StartingBlock™ (Thermo Fisher). 4. PBS: see Subheading 2.4. 5. PBS-T: see Subheading 2.4. 6. PBS-T containing 5% BSA. 7. E. coli TG1 cells streaked on a minimal media plate. 8. M13KO7 helper phage. 9. 20% PEG/2.5 M NaCl: see Subheading 2.5. 10. 2  YT medium, selective antibiotics (Amp, Kan), and 2 M glucose (see Subheading 2.5). 11. NanoDrop™ One Microvolume UV-Vis spectrophotometer or similar instrument. 12. Primer PN2 (10 pmol/μL) (Table 1). 13. Primer M13RP (10 pmol/μL) (Table 1). 14. dNTP Mix, 10 mM each. 15. RNase/DNase-free water. 16. Platinum™ Taq DNA polymerase and 10 buffer. 17. Agarose gel electrophoresis materials and equipment, including molecular size standards, DNA loading dye and gel stain. 18. 0.1 M triethylamine, prepared fresh daily. 19. 1 M Tris-HCl: 121.4 g Tris in 1 L of ddH2O. Adjust to pH 7.4 with 3 M HCl, filter-sterilize, and store at 4
C.

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20. 96-well, flat bottom Costar® tissue culture plates. 21. Sorvall high-speed swinging-bucket refrigerated bench-top centrifuge or equivalent. 22. 0.22 μm Steriflip-GP sterile centrifuge tube top filter unit and 0.22 μm Stericup-GP sterile vacuum filtration system. 23. Mouse anti-M13 antibody, HRP conjugated (Cedarlane). 24. TMB substrate. 25. 1 M H3PO4. 26. 96-well microtiter plates. 27. Multiskan™ FC Microplate photometer or similar instrument. 28. Sterile 80% glycerol. 29. Taq DNA polymerase and 10 PCR buffer. 30. 8-well microwell strips. 2.7 VHH Subcloning, Bacterial Expression, and Purification

1. Sequence of VHH of interest cloned into pMRO-BAP-H6 expression vector [22]. 2. E. coli BL21 (DE3) electrocompetent cells (NEB). 3. Sterile ddH2O. 4. SOC medium: see Subheading 2.1. 5. LB broth and agar: see Subheading 2.1. Supplement with Amp and/or Kan as needed. 6. Disposable electroporation cuvettes, 0.1 cm gap width. 7. MicroPulser™ electroporator or similar instrument. 8. E. coli AVB101 cells harboring the pACYC184 plasmid (Avidity, Aurora, CO, USA). 9. Sterilized 500 mL baffled flasks. 10. Protease inhibitor tablets. 11. Emulsiflex-C5 homogenizer or similar instrument. 12. 15 mL and 50 mL falcon tubes. 13. 35 mg/mL chloramphenicol (Cam) stock solution prepared in anhydrous ethyl alcohol, filter sterilized. Store at 20
C in the dark. 14. 100 mM D-(+)-biotin solution: dissolve 1.52 g of D-biotin in 0.1 M NaOH, filter-sterilize (0.22 μM), and store at 20
C. 15. 100 mM ATP solution: prepare a 1000 stock solution by dissolving 2.75 g ATP in 50 mL of water, filter-sterilize (0.22 μM), aliquot, and store at 20
C. 16. 2 M glucose: see Subheading 2.5. 17. 37
C shaker incubator.

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18. Sorvall RT6000B refrigerated high-speed centrifuge with swinging bucket rotor or similar instrument. 19. 1 M isopropyl-β-D-thio-galactopyranoside (IPTG): dissolve 2.4 g of IPTG in 10 mL of ultrapure H2O. Sterilize by 0.22 μm filtration. Store at 20
C. 20. 0.22 μm Steriflip-GP sterile centrifuge tube top filter unit and 0.22 μm Stericup-GP sterile vacuum filters. 21. 3.5 kDa MWCO dialysis tubing. 22. NanoDrop™ One Microvolume UV-Vis spectrophotometer or similar instrument. 23. PBS: see Subheading 2.4. 24. 500 mL polypropylene centrifuge bottles. ¨ KTA fast protein liquid chromatography (FPLC) system or 25. A similar instrument. 26. HisTrap™ HP immobilized metal affinity chromatography (IMAC) column (GE Healthcare, Piscataway, NJ, USA). 27. IMAC buffer A: 10 mM HEPES, pH 7.0, containing 500 mM NaCl. 28. IMAC buffer B: 10 mM HEPES, pH 7.0, containing 500 mM NaCl and 500 mM imidazole. 29. 500 mM imidazole. 30. 5 M NaCl. 2.8 Transient Expression of VHH-Fc Fusions in HEK2936E Cells

1. Sequence of VHH of interest cloned into a pTT5 vector upstream of human IgG1 Fc. 2. HEK293-6E cells. 3. Gibco™ FreeStyle™ F17 expression medium (Thermo Fisher). 4. Gibco™ GlutaMAX™ supplement (Thermo Fisher). 5. Gibco™ antibiotic-antimycotic (100) (Thermo Fisher). 6. Gibco™ Pluronic™ F-68 non-ionic surfactant (100) (Thermo Fisher). 7. Gibco™ Geneticin™ selective antibiotic (50 mg/mL G418 sulfate) (Thermo Fisher). 8. PEIpro® DNA transfection reagent (Polyplus Transfection, New York, NY, USA). 9. Corning disposable baffled polycarbonate Erlenmeyer flasks with vented screw caps, sterile, 125 mL and 500 mL. 10. TC20™ Automated Cell Counter or similar instrument. 11. Cell counting slides. 12. 0.4% (w/v) trypan blue.

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13. Humidified shaking incubator at 37
C with 5% CO2, 60% humidity. 14. Orbital shaker set at 100 rpm. 15. Feed solution: 20% (w/v) tryptone N1 (TN1) prepared in FreeStyle™ F17 expression medium. Sterilize through a 0.22 μm filter. 16. Plasmid DNA of interest purified using an EndoFree Plasmid Maxi Kit (Qiagen) or equivalent. 17. SDS-PAGE gels and apparatus, including 2 Laemmli buffer, molecular size standards and stain (e.g., Coomassie blue). 18. Serological pipettes (5 mL and 10 mL) and pipette gun. ¨ KTA FPLC. 19. A 20. MabSelect™ SuRe™ column (GE Healthcare). 21. 100 mM citric acid, pH 3.0. 22. 1 M Tris, pH 8.0. 2.9 VHH Soluble ELISA

1. Human EGFR-Fc fusion protein (GenScript). 2. Rhesus and mouse EGFR-Fc fusion proteins (Sino Biological, Beijing, China). 3. PBS: see Subheading 2.4. 4. PBS-T: see Subheading 2.4. 5. PBS containing 2% skim milk. 6. PBS-T containing 5% BSA. 7. Goat anti-6  His antibody conjugated to HRP (Cedarlane). 8. TMB substrate. 9. 1 M H3PO4. 10. 96-well microtiter plates. 11. Multiskan™ FC Microplate photometer or similar instrument.

2.10 Assessment of VHH and VHH-Fc Fusion Cell Binding

1. MDA-MB-468 (EGFR-positive) and MCF7 (EGFR-negative) cells (ATCC, Manassas, VA, USA). 2. RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B. 3. PBS: see Subheading 2.4. 4. TC20™ Automated Cell Counter or similar instrument. 5. Cell counting slides. 6. Humidified incubator at 37
C with 5% CO2, 60% humidity. 7. PBS containing 1% BSA. 8. Tissue culture treated T75 flasks.

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9. Streptavidin R-phycoerythrin conjugate (SA-PE) (Thermo Fisher). 10. Mirrorball® microplate cytometer (SPT Labtech, Melbourn, UK). 11. Accutase™ cell dissociation reagent (Thermo Fisher). 12. Hank’s Balanced Salt Solution (HBSS). 13. Nunc™ MicroWell 96-well optical bottom plates. 14. Live cell imaging buffer (LCIB) (Thermo Fisher) containing 1% BSA. 15. Alexa Fluor® 488 (AF488)-conjugated donkey anti-human IgG (Jackson ImmunoResearch). 16. 5 mM DRAQ5™ fluorescent probe solution (Thermo Fisher).

3

Methods In this protocol, we initially immunized a llama with cDNA coding for EGFRvIII with the goal of obtaining EGFRvIII-specific VHHs; however, all isolated antibodies bound equivalently to both EGFRvIII and wild-type EGFR.

3.1 Large-Scale EGFRvIII-pTT5 Plasmid Preparation

1. Transform 50 μL of electrocompetent DH5α cells (see Note 1) with 1–2 μL (50–100 ng) of the purified pTT5-EGFRvIII ECD TM and pTT5-EGFRvIII ECD H6 plasmids (see Note 2) using a MicroPulser™ electroporator [23]. Immediately add 1 mL of pre-warmed SOC medium and transfer the mixture to a 15 mL conical tube. Incubate for 1 h at 37
C with 180 rpm shaking. 2. Spread 100 μL of the transformed cells on LB-Amp plates and incubate overnight at 32
C. 3. Pick an isolated colony and grow in 100 mL of LB-Amp overnight at 37
C with 250 rpm shaking. 4. The next day, harvest the bacterial cells by centrifugation at 5000  g for 10 min at 4
C. Purify DNA using the EndoFree Plasmid Maxi Kit following the manufacturer’s instructions. Repeat this step if the yield is lower than 1 mg of purified plasmid DNA. 5. Pass the purified plasmid DNA through a 0.22 μm filter unit and measure the DNA concentration with a spectrophotometer [24] (see Note 3). Store the DNA at 4
C if transfection is planned within several days or at 20
C for long-term storage. 6. Run ~200 ng of plasmid DNA on a 1% agarose gel to assess the quality and purity of the plasmid [24].

Llama DNA Immunization and Isolation of Functional Single-Domain Antibody. . .

3.2 Preparation of Gene Gun Bullets for Llama Immunization

49

The method presented here essentially follows the Helios Gene Gun System Instruction Manual. However, several steps have been modified/optimized. 1. Weigh 25 mg of gold nanoparticles in a 1.5 mL microfuge tube and add 100 μL of 0.05 M spermidine. Vortex for 5 s, then sonicate in a water bath for 3–5 s to break up gold clumps (see Note 4). 2. Add 50 μg of plasmid DNA (1 μg/μL) obtained as described above (see Subheading 3.1) encoding EGFRvIII ectodomain to the spermidine-coated gold particles. Use a 50:50 mixture of the plasmids encoding 6  His-tagged human EGFRvIII ectodomain and membrane-tethered human EGFRvIII ectodomain. Mix by vortexing for 5 s (see Notes 5 and 6). 3. Continue vortexing at medium speed while adding exactly 100 μL of 1 M CaCl2 dropwise to the mixture (see Note 7). 4. Incubate the mixture at room temperature for 10 min and then centrifuge at maximum speed for 15 s. Remove the supernatant and vortex briefly to resuspend the pellet in the remaining volume (see Note 8). 5. Prepare a stock solution of 20 mg/mL PVP in anhydrous ethyl alcohol and then make a working solution of 0.05 mg/mL PVP in 3.5 mL of anhydrous ethyl alcohol. 6. Wash the gold pellet three times with 1 mL of anhydrous ethyl alcohol. At each step, microcentrifuge at maximum speed for 5 s and resuspend the pellet in 1 mL of anhydrous ethyl alcohol. After the last centrifugation step, discard the supernatant and resuspend the gold pellet in 200 μL of 0.05 mg/mL PVP. 7. Transfer the gold solution into a 15 mL conical tube. Rinse the microcentrifuge tube with an additional 200 μL of 0.05 mg/ mL PVP in ethanol and transfer to the 15 mL conical tube. Bring the volume to 3 mL with 0.05 mg/mL PVP (see Note 9). 8. Set up the tubing prep station and connect it to a nitrogen tank. Ensure all syringe adaptor tubing is connected tightly. 9. Cut a 3000 piece of gold-coat tubing and ensure it is dry by purging with nitrogen. 10. Vortex the gold-DNA suspension and ensure the gold particles are dispersed evenly in the suspension. Quickly draw the solution into the 3000 (76.2 cm) gold-coat tubing connected to a 10 mL syringe by adaptor tubing. Make sure no bubbles are generated and that there is a 2–300 (5–7.6 cm) of empty space at each end of the tubing.

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11. Keep the coated tubing in a horizontal position and slide it into the tubing support cylinder of the tubing prep station until the tubing passes the O-ring. 12. Allow the DNA-gold suspension to settle for 3–5 min and then remove the ethanol solution using a syringe attached to the end of the tubing at a rate of no more than 0.5–100 per s (1.2–2.5 cm per s). The entire process will take about 30–45 s. 13. Detach the syringe from the tubing. Immediately turn the tubing 180
and allow the gold to coat the inside surface of the tubing for 3–4 s. 14. Turn the switch to ON and allow the gold to coat the tubing for 20–30 s. Then open the valve to allow nitrogen to flow at 0.35–0.4 L per min to dry the tubing for 15 min, while it continues to rotate. 15. Turn off the tubing prep station and remove the tubing from the tubing support cylinder. 16. Cut the tubing into 0.500 (1.3 cm) pieces with the tubing cutter equipped with a cartridge storage vial in the tube collector base. 17. Cap the vial tightly, label, wrap with parafilm and store at 4
C until further use. 3.3 Llama DNA Immunization and Peripheral Blood Mononuclear Cell Isolation

All immunizations are performed at the Cedarlane facility in Burlington, ON, Canada (http://www.cedarlane.ca) and are based on the protocol provided here and following the guidelines set by the Canadian Council on Animal Care. If feasible, use two llamas for immunization as the quality of the immune response varies between individual animals. 1. Set up the gene gun and load the gene gun cartridge with the 0.500 (1.3 cm) gold-coated tubing (see Subheading 3.2). 2. On day 1, conduct a pre-immune bleed (10–15 mL) and then immunize the llama by biolistic gene gun transfection six times (weeks 0, 2, 4, 6, 9, and 12) (Fig. 1a). Each immunization consists of 12 bombardments administered at 600 psi to shaved sites on the neck and hind limb (10 μg of total DNA per immunization) (see Note 10). 3. Four additional immunizations (weeks 16, 20, 24, and 28) are administered by intradermal injection of 1 mg (1 mg/mL) of DNA using a DERMOJET® device. 4. As test bleeds, collect blood (10– 15 mL) into 50 mL Falcon tubes at weeks 4 and 12 (during the gene gun immunization) and weeks 24 and 28 after the DERMOJET® immunization. Store the collected test bleeds overnight at 4
C. Prepare the sera the next day by centrifuging for 10 min at 2700  g at

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Fig. 1 Llama genetic immunization schedule and serum response monitoring. (a) A llama was immunized six times with DNA using a gene gun (weeks 0, 2, 4, 6, 9, and 12) and four times with a DERMOJET® device (weeks 16, 20, 24, and 28). Sera and PBMCs were collected. (b) Serum titration ELISA against recombinant human EGFR ectodomain. The immune response to EGFR was specific with no binding to blocker. Gene gun bombardment alone did not elicit a significant immune response; however, boosting by DERMOJET® injection led to a significant increase in immune response. The gene gun immunization may also have contributed to the overall success of DNA immunization. (Figure re-used with permission from [19])

4
C. Store the sera at 4
C for short-term use and at 20
C long term. 5. For the production bleed, collect 50 mL of blood 5 days after both the week 24 and 28 immunizations into heparin-coated tubes (or tubes containing other anti-coagulants such as EDTA). Place the blood on ice immediately. 6. Dilute 50 mL of blood at a 1:1 ratio in RPMI 1640 medium. 7. Centrifuge five 10 mL LymphoPrep™ tubes at 400  g for 1 min at 18–22
C to displace the liquid to the bottom of the tube before use.

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8. Slowly add 20 mL of diluted blood to each of LymphoPrep™ tube and centrifuge the tubes at 800  g for 20 min at 18–22
C. 9. Collect the PBMCs that have formed an interface between the two layers using a Pasteur pipette. 10. Dilute the harvested fraction with 20 mL of RPMI 1640 medium and pellet the cells by centrifugation at 250  g for 10 min at 18–22
C. 11. Count the cells using trypan blue staining and dispense in aliquots of 5  106 to 1  107 cells per mL. Freeze on dry ice and then store at 80
C till further use. For extended storage at 80
C prior to library construction, add 0.5 mL of RNAlater® to prevent or minimize RNA degradation prior to freezing. 3.4 Llama Immune Response Analysis

1. Coat a 96-well microtiter plate with 100 μL of EGFRvIII or wild-type EGFR (1–5 μg/mL) in PBS overnight at 4
C. 2. The next day, rinse the wells twice with PBS (300 μL/well), then block the wells with 200 μL/well of PBS containing 5% skim milk for 2 h at room temperature. Coat additional wells with skim milk only or irrelevant proteins to evaluate the specificity of the immune response against the antigen. After blocking, wash all plates with PBS (300 μL/well). 3. Prepare a dilution master plate using a 96 deep-well plate. Prepare a dilution range of sera from 1/100 to 1/100,000 in PBS-T containing 2% BSA. Transfer, using a multichannel pipette, 100 μL of each dilution to the plates prepared in step 2. Incubate for 1.5 h at room temperature. 4. Wash the wells five times with 300 μL/well of PBS-T. After each wash empty the plate and remove excess moisture by inverting the plates and firmly tapping on tissue paper. Next add 100 μL/well of goat anti-llama IgG-HRP (diluted 1: 10,000 in PBS-T containing 2% BSA). Incubate for 1 h at 37
C (see Note 11). 5. Wash the wells again as described in step 4. Add TMB substrate (100 μL/well) and incubate at room temperature for 3–10 min. Stop the reaction with 1 M H3PO4 (100 μL/ well). Read the absorbance at 450 nm with a microtiter plate reader. In the example presented here, serum titers against wild-type EGFR are shown but sera showed equivalent reactivity with EGFRvIII (Fig. 1b).

3.5 Phage-Displayed VHH Library Construction

The PBMCs collected 5 days after week 24 and 28 immunization (see step 11 in Subheading 3.3) are used as a source of mRNA for phage-display library construction (see Note 12). Complementary DNA (cDNA) is synthesized from total RNA and amplified by a

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53

Fig. 2 Maps of pMED1 phagemid vector and pMRO-BAP-H6 expression vector. pMRO-BAP-H6 is a derivative of pET28a (Novagen). In both vectors, the VHHs are cloned between the two SfiI restriction enzyme sites which generate different ends upon digestion, thereby avoiding self-ligation. In pMRO-BAP-H6, the biotinylation signal preceding the 6  His tag mediates the conjugation of a biotin moiety specifically at the VHH C-terminus via the biotin ligase BirA. The T7 promoter allows for high yield expression of VHHs

two-step PCR. Amplified VHH are then cloned into the phagemid vector pMED1 (Fig. 2) and used to transform E. coli TG1 cells, creating the VHH library. The phagemid vector allows the VHH to be expressed as a fusion to the minor coat protein (pIII) on the surface of filamentous phages when the bacterial cells in the library are superinfected with a helper phage (Fig. 3a, b). 1. Take 5  107 cells (4–5 tubes of cells in total) and isolate total RNA using the PureLink™ RNA Mini Kit according to the manufacturer’s instructions. Measure the RNA concentration and purity at A260 nm and A280 nm with a spectrophotometer [24]. Typical RNA yields range from 20 to 50 μg per 5  107 cells with an A260/280 ratio > 1.8. 2. Use a total of 2.5 μg of RNA to synthesize cDNA in a total reaction volume of 20 μL, using qScript® cDNA supermix containing oligo(dT) and random hexamer primers according to the manufacturer’s instructions (see Note 13). Perform 4–5 reactions independently and then pool them. 3. For the amplification of the VHH encoding regions, perform PCRs using various amounts of the cDNA from step 2 as template (0.5–5 μL). Prepare two master mixes: each contains equimolar mixes of the three framework region (FR) 1-specific primers MJ1, MJ2, and MJ3 and one or the other of the

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Fig. 3 Overview of library construction, phage panning and isolation of EGFR-specific VHHs. (a) Schematic representation of RNA extraction, cDNA synthesis, and PCR amplification of VHH-CH2 fragments. The rearranged VHH repertoire is gel-purified and used as a template for a second PCR using SfiI-MJ7 and MJ8-SfiI primers. (b) A phagemid library is constructed and phage rescued using M13KO7 helper phage. (c) Phage displaying VHHs panned against recombinant EGFRvIII to isolate antigen-specific VHHs. (d) After four rounds of panning, VHHs are screened by phage ELISA. (e) Positive, unique binders are subcloned into the high-yielding pMRO-BAP-H6 expression vector in frame with a biotinylation acceptor peptide (BAP) and a 6  His tag. The BAP sequence is bolded and Lys (K) residue for biotin attachment shown in red

CH2-specific primers CH2FORTA4 and CH2B3-F (Table 1; see Note 14). Set up the PCR reactions as follows in a total volume of 50 μL: 5 μL of 10 Platinum™ Taq DNA polymerase buffer, 0.5 μL of MJ1–3 primer mix (10 pmol/μL each), 0.5 μL of CH2FORTA4 or CH2B3-F primer (10 pmol/μL), 1 μL of 10 mM dNTPs, 0.5–5 μL of cDNA, 0.5 μL of Platinum™ Taq DNA polymerase, and sterile ddH2O to 50 μL. Perform the PCR reaction using the following thermal cycling parameters: 94
C for 3 min; 30 cycles of 94
C for 1 min, 55
C for 30 s, and 72
C for 30 s; 72
C for 7 min. 4. Analyze 5 μL of the PCR reactions on a 1% agarose gel [24]. Identify the cDNA volume that gives the best yield in terms of amplifying the VHH-CH2 fragments and repeat the PCR experiment under the same conditions. Perform at least eight PCR reactions each for each of the two master mixes. Separate the PCR reactions on 1% agarose gels, visualize the products (see Note 15), excise the band corresponding to the

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55

VHH harboring fragment, and extract the DNA using the QIAquick® Gel Extraction kit following the manufacturer’s instructions. Measure the DNA concentration by spectrophotometry. 5. Re-amplify the purified products from step 4 (20–30 ng of first round PCR per reaction) in a second nested PCR under the same conditions as above but using different primers. Prepare only a single master mix this time containing the FR1 and FR4-specific primers MJ7 and MJ8 (Table 1). Primers MJ7 and MJ8 will introduce the SfiI sites for subsequent cloning. Perform a total of 40 PCR reactions and pool the reactions. Analyze 5 μL of the PCR reaction on a 1% agarose gel, expecting to see relatively wide bands ranging from 400 to 450 bp, which correspond to the VHH fragments. Purify the PCR products with the QIAquick® PCR purification kit and determine the concentration (see Note 16). 6. Digest 10 μg of the PCR products with FastDigest SfiI (5 U/μ g DNA) overnight at 50
C in a final volume of 200 μL and subsequently analyze 5 μL on a 1% agarose gel. The digested DNA should appear the same as the undigested DNA from step 5. Repurify the digested DNA with the QIAquick® PCR purification kit and measure the concentration. 7. Digest 30 μg of pMED1 phagemid vector with FastDigest SfiI (5 U/μg DNA) overnight at 50
C in a final volume of 200 μL. Then, add 1 μL (10 U) of FastDigest XhoI and PstI restriction enzymes and incubate for an additional 2 h at 37
C to prevent self-ligation of pMED1. Examine 5 μL of the digested pMED1 (~750 ng) on a 1% agarose gel. Include a sample of the undigested vector (750 ng) to ensure that the vector has been completely linearized. Purify the digested vector with a QIAquick® PCR purification kit and measure the concentration. 8. Ligate the SfiI-digested VHH DNA with SfiI-digested pMED1 vector in a total volume of 100 μL as follows: 20 μg of SfiI-linearized pMED1, 3.5 μg of SfiI-digested VHH insert, 10 μL of 10 T4 DNA ligase buffer, 5 μL of T4 DNA ligase, and sterile ddH2O to 100 μL. Incubate at room temperature for 60 min (see Note 17). 9. Purify the ligation reaction using a QIAquick® PCR purification kit. Divide the ligation between two spin columns and elute the DNA in a final volume of 35 μL of sterile ddH2O per column. Pool the eluted material and measure the concentration. 10. Transform 50 μL of electrocompetent TG1 E. coli cells with 3 μL of the purified ligated material using 0.1 cm cuvettes and a MicroPulser™ electroporator. Add 1 mL of pre-warmed SOC medium and transfer the electroporated cells into a 50 mL

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conical tube. Incubate for 1 h at 37
C with 180 rpm shaking. Repeat the transformation for the remaining DNA for a total of 20 transformations. 11. Next, pool all the transformed cells in SOC in a 50 mL tube. From this tube, prepare 103, 104, and 105 fold dilutions in LB medium. Spread 100 μL of the diluted cells on pre-warmed LB-Amp plates and incubate overnight at 32
C. 12. Amplify the library and generate a bank of cells by transferring the pooled transformed bacteria into 500 mL of 2  YT-Amp containing 2% glucose and incubate 3-4 h with 220 rpm shaking at 37
C. 13. Centrifuge the cells at 5000  g for 20 min at 4
C. Discard the supernatant and resuspend the cells in 200 mL of 2  YT-Amp containing 2% glucose. Make dilutions of the cells in 2  YT and measure the A600 nm. Use this value to calculate the density (in cells/mL) of the library cells (A600 nm ¼ 1 corresponds to approximately 109 E. coli TG1 cells per mL). Add 20 mL of sterile 70% glycerol solution to the cell stock, make several aliquots of approximately 1010 bacterial cells/vial and freeze the library cells at 80
C. 14. Count the colonies on the titer plates from step 11 and determine the total library size. 15. Perform colony PCR on the colonies from the titer plates in a total volume of 15 μL to determine the insert ratio for the library. Prepare a master mix for 50 PCR reactions as follows: 80 μL of 10 PCR buffer, 16 μL of 10 mM dNTPs, 8 μL each of PN2 and M13RP primers (10 pmol/μL; Table 1), 8 μL of Taq DNA polymerase, and 680 μL of ddH2O. Touch single colonies from the titer plates with a sterile P10 pipette tip and swirl in the PCR tubes. Place the reaction tubes in a thermal cycler and perform the PCR as follows: 94
C for 5 min; 30 cycles of 94
C for 30 s, 55
C for 30 s, and 72
C for 1 min; 72
C for 7 min. 16. Analyze 5 μL of each PCR reaction on a 1% agarose gel to identify the clones with full inserts (400–450 bp VHH + 180 bp flanking sequence ¼ approximately 600 bp band). Sequence these PCR amplicons using the M13RP primer (Table 1) using Sanger sequencing. M13RP is upstream of the VHH sequences [24] (see Notes 18 and 19). 17. To produce the library phage, thaw 1–2 mL of frozen library cells from step 13. Use the thawed cells to inoculate 200 mL of 2  YT-Amp containing 2% glucose at 37
C with 220 rpm until the cell culture density reaches an A600 nm of 0.5 (2–3 h). Infect the cells with a 20 excess of M13KO7 helper phage (2  1012 plaque-forming units, pfu) for 30 min at 37
C without agitation followed by 30 min at 37
C with shaking

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at 250 rpm. Pellet the infected cells by centrifugation at 5000  g for 10 min at 4
C. Resuspend the pellets in 200 mL of 2  YT-Amp-Kan and grow overnight at 37
C with 250 rpm shaking. This step is necessary to remove any traces of glucose which act as a repressor of VHH expression during the first step of library generation. 18. To purify the phage, pellet the overnight culture (10,000  g, 15 min, 4
C), filter the 200 mL of supernatant through a 0.22 μm filter unit, then add 1/5 the volume (40 mL) of 20% PEG/2.5 M NaCl to the filtered supernatant and mix thoroughly. Incubate for 1 h on ice to precipitate the phage particles, centrifuge as above and discard the supernatant. Resuspend the phage pellet in 1.5 mL of sterile PBS. Determine the titer of the phage by serial dilution and infecting TG1 E. coli cells (see Notes 20 and 21) and store phage at 80
C. Use the purified phage as the input phage for the first round of panning. 3.6 VHH-PhageDisplay Library Panning and Screening

3.6.1 Panning

The VHH library displayed on the surface of phage is panned against the target antigen EGFRvIII directly immobilized on a solid surface (microtiter plates). A second library selection is carried out in the same manner except that biotinylated EGFRvIII is immobilized on streptavidin-coated wells. After four rounds of panning individual colonies are screened for their specificity against EGFRvIII by monoclonal phage ELISA, in which the binding of VHH-displaying phage to EGFRvIII is detected using an anti-M13 antibody-HRP conjugate (Fig. 3c, d). 1. In the first panning campaign, coat an 8-well microwell strip or a 96-well microtiter plate with 20 μg/well (two wells) of recombinant EGFRvIII (100 μL/well diluted in PBS) overnight at 4
C. 2. The next day, discard the well contents and wash with 250 μL/ well of PBS. Block for 2 h at 37
C with 200 μL/well of StartingBlock™. Start growing a 10 mL culture of E. coli TG1 cells in 2  YT medium in a sterile 50 mL conical tube (see Note 22). 3. Remove the blocking buffer and add ~1012 input phage (see Subheading 3.5) in 100 μL of PBS-T to the blocked wells for 2 h at 37
C. Remove unbound phage, wash five times with PBS-T and five times with PBS (300 μL per wash), and elute the bound phage by incubating with freshly prepared 0.1 M triethylamine (100 μL/well) for exactly 10 min at room temperature. Pipette the elution solution up and down several times in the well, transfer the contents to a 1.5 mL tube, and neutralize with 50 μL of 1 M Tris-HCl, pH 7.4. Keep the tube on ice.

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4. Titrate the output phage by infection of E. coli TG1 cells. Keep 100 μL of the exponentially growing E. coli TG1 cells from step 2 as the titer negative control and infect the remaining cells (~9.9 mL) with 100 μL of the eluted phage by incubating the mixture at 37
C for 15 min without shaking, followed by incubation with shaking at 220 rpm at 37
C for 1 h. Store the remaining eluted phage at 80
C. Titrate the infected cells (see Note 23). 5. Superinfect the infected bacterial cells (~10 mL) with 1011 pfu of M13KO7 helper phage as described (see Subheading 3.5). Subsequently, add Kan to a final concentration of 50 μg/mL and incubate overnight at 37
C with 250 rpm shaking. 6. The next day, purify the phage by precipitation with one fifth the volume of PEG/NaCl, resuspend in 200 μL of PBS and determine the phage titer as described above (see Subheading 3.5). Use the purified phages as the input phage for the next round of panning. 7. To assess the progress of the panning, perform colony PCR and DNA sequencing on titer plate colonies of the eluted phages as described above (see Subheading 3.5). We routinely sequence 15 to 20 clones in each of the first two rounds, 25 clones in the third round, and 50 to 100 clones in the fourth round. 8. Repeat the panning process (steps 1–7) for three additional rounds using the amplified phage from the previous round as the input phage for the next round. We recommend reducing the amount of coated antigen by 5 μg for each subsequent round (e.g., 15 μg of EGFRvIII in round two) (see Note 24). Additionally, increase the number of washes before elution of the phages by five additional washes in each subsequent round of selection (e.g., 10 washes round two, 15 washes round three). 9. In the second panning campaign, site-specific biotinylated EGFRvIII is captured on a streptavidin-coated well. Coat an 8-well microwell strip with 2 μg/mL of streptavidin diluted in PBS (100 μL). Incubate overnight at 4
C and the next day block the wells as described in step 2. 10. Add 100 ng of biotinylated EGFRvIII/well diluted in PBS and incubate for 1 h at room temperature to capture the biotinylated protein. Wash the wells five times with PBS-T (300 μL/ well), then add the library phage and proceed with panning as described in steps 2–8 (see Note 25). 3.6.2 Phage ELISA

1. For monoclonal phage amplification, the starting material is the titer plates from selected rounds of panning (see Subheading 3.6.1). In the morning, prepare a 96-well round-bottom culture plate containing 200 μL/well of 2  YT-Amp and inoculate 94 colonies from the titration plate from the fourth

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round of panning. To do this use P10 pipette tips, touching the media in the 96-well plate with individual colonies. Leave wells H11 and H12 empty as controls. Place the plate in a humidified chamber (e.g., an empty tip box with a lid containing wet tissue paper) and incubate at 37
C with 110 rpm shaking until the A600 of the cultures reaches 0.4 (4–5 h). Next, superinfect the cells with 5  109 pfu of M13KO7 for 30 min without shaking. Then add 20 μL of 500 μg/mL Kan prepared in 2  YT, place the plate in the humid chamber box, and continue growing the cultures at 37
C overnight with 110 rpm shaking. 2. Coat a 96-well microtiter plate with recombinant EGFRvIII at 1 μg/mL in 100 μL of PBS at 4
C. 3. The next day, spin down the overnight culture plate at 3600  g for 15 min at 4
C, using a bench-top centrifuge with a swinging plate bucket rotor. Transfer the supernatant to a new plate and keep it on ice or at 4
C (master plate) to be used in step 5 below. 4. Prepare a glycerol stock by adding 25 μL/well of sterile 70% glycerol to the bacterial culture pellets, seal the lid with parafilm, label, and store at 80
C. This plate can be used for colony PCR and plasmid sequencing (step 8). 5. Block the microtiter plate from step 2 using 200 μL/well of PBS-T containing 5% BSA and incubate at room temperature for 2 h. Prepare a duplicate plate containing only blocker to evaluate monoclonal VHH-phage binding in the absence of antigen. 6. Discard the blocking buffer and wash the plates with PBS-T (three 250 μL washes/well). Add 50 μL of PBS-T containing 5% BSA to all wells and then add 50 μL/well of phage supernatants (clones 1–94) from the master plate (step 3) to the corresponding wells. Include a negative control in duplicate (109 pfu M13KO7 helper phage in PBS-T containing 5% BSA added to the wells H11 and H12). Incubate the plate for 1.5 h at room temperature. 7. Discard unbound phage particles and wash the wells with PBS-T (five 250 μL washes/well). Add 100 μL/well of antiM13 IgG-HRP conjugate (previously diluted 1:5000 in PBS-T containing 5% BSA) and incubate for 1 h at room temperature. 8. Remove the unbound anti-M13 IgG-HRP, wash the plate five times with PBS-T and add 100 μL/well of TMB substrate. Incubate for 5–10 min, stop the reaction with 100 μL of 1 M H2SO4 and measure absorbance at 450 nm using a microplate reader. 9. Perform colony PCR and Sanger sequencing of positive clones as described above (see Subheading 3.5).

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3.7 VHH Subcloning, Bacterial Expression and Purification

3.7.1 VHH Expression

Gene synthesis and subcloning of EGFR-specific VHHs are performed at Twist Bioscience or through a similar provider (for monomeric VHHs in pMRO-BAP-H6) (see Note 26 and Fig. 2). The DNA sequences are optimized for expression in E. coli and individual plasmid constructs transformed as described above (see Subheading 3.1). Note that pMRO-BAP-H6 is Kan and not Amp resistant. 1. Transform electrocompetent E. coli BL21 (DE3) cells as described above (see Subheading 3.1). 2. Prepare starter E. coli cultures for each VHH clone by inoculating 10 mL of LB-Kan containing 1% glucose with a single colony harboring pMRO-BAP-H6 with inserts encoding VHHs of interest. Grow overnight at 37
C with 220 rpm shaking. 3. The next day, transfer each overnight culture into 200 mL of LB-Kan in a 500 mL baffled flask and grow at 37
C with continuous shaking at 220 rpm until an A600 of 0.6–0.8 is reached (1.5–2 h). 4. Induce VHH expression by adding 1 μL of 1 M IPTG (final concentration 5 μM) and grow overnight at 28
C with shaking at 220 rpm (see Note 27). 5. The next day, transfer the bacterial culture to a 500 mL centrifuge bottle and harvest cells by centrifuging at 4000  g 15 min at 4
C. 6. Resuspend the pellet in 30 mL of sterile PBS. Add an appropriate volume of a 10 protease inhibitor cocktail (see Note 28). 7. Transfer each sample to a 50 mL conical tube and keep the samples on ice. 8. Use an Emulsiflex-C5 or similar instrument to lyse the bacteria at a pressure of 20,000 psi. Alternative, sonication can be used to lyse the bacteria. 9. Centrifuge the lysate at 17,000  g for 30 min at 4
C. 10. To perform the BirA-AviTag site-specific biotinylation of the VHHs (Fig. 3e), add 5 mL of BirA cleared bacterial extract (see Subheading 3.7.2), 350 μL of 100 mM D-biotin, and 350 μL of 100 mM ATP. Incubate for 2 h at 37
C with slow agitation or in a water bath with periodic manual agitation. 11. Centrifuge the mixture at 17,000  g for 30 min at 4
C. 12. Repeat step 11 once to remove any impurities. 13. Remove the supernatant into a new 50 mL tube, add PBS to a final volume of 45 mL, then add 1.8 mL of 500 mM imidazole and 3.25 mL of 5 M NaCl.

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14. Purify VHHs using a 5 mL HiTrap™ chelating HP column on ¨ KTA FPLC system [25–27]. an A 15. Pool the fractions corresponding to the VHH peak and buffer exchange into PBS (see Note 29) by dialysis or using an Amicon® Ultra-15 Centrifugal Filter Unit. Analyze the quality of the proteins by SDS-PAGE and size exclusion chromatography as described previously [27]. Store the samples at 20
C. 3.7.2 Preparation of the BirA Extract

1. Prepare 10 mL of LB supplemented with 10 μg/mL Cam (see Note 30). Inoculate the medium with one colony of E. coli AVB101 (see Note 31) and grow overnight at 37
C with 250 rpm shaking. 2. The next day, transfer 5 mL of the starter culture to a 2 L flask containing 500 mL of LB/Cam and continue growing under the same conditions until the A600 reaches 0.6–0.8. 3. Induce expression of the biotin ligase by adding 1 mM IPTG and incubate for 4 h at 37
C. 4. Transfer the bacterial culture to a 500 mL centrifuge bottle and harvest cells by centrifuging at 4000  g for 15 min at 4
C. 5. Resuspend the pellet in 50 mL of PBS and lyse the bacteria using the Emulsiflex-C5 or by sonication as described above (see Subheading 3.7.1). 6. Centrifuge the mixture at 17,000  g for 30 min at 4
C. 7. Repeat step 6 once to remove any impurities. 8. Prepare 5 mL aliquots and store at 80
C.

3.8 Transient Expression of VHH-Fc Fusions in HEK2936E Cells

Gene synthesis and subcloning of EGFR-specific VHH-Fc fusions in pTT5 are performed by GeneArt (see Note 32) or a similar provider. The DNA sequences are optimized for expression in HEK293-6E cells (see Note 33), individual plasmid constructs transformed into E. coli and plasmid DNA is purified as described above (see Subheading 3.1). 1. Thaw one vial of HEK293-6E cells (5  106 cells/tube) for 2 min in a water bath at 37
C and transfer the cell suspension to a 125 mL Erlenmeyer flask containing 25 mL of pre-warmed complete FreeStyle™ F17 expression medium to obtain a cell density of 0.25  106 cells/mL (see Note 34). Check the cell density and viability, which should be more than 90%, using Trypan blue staining and a TC20™ automated cell counter. Incubate the flask on an orbital shaker at 110–130 rpm in a 37
C humidified incubator supplemented with 5% CO2. 2. Continue to grow the cells until the cell density reaches approximately 1.5–1.8  106 cells/mL. This should take around 72 h. Check the cell density and viability every 24 h.

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3. Prepare 500 mL flasks containing 90 mL of pre-warmed complete FreeStyle™ F17 expression medium and inoculate with 10 mL of the culture from step 2. Grow the cells as described in step 1 until the density reaches approximately 1.5  106 cells/ mL. This should take 60–72 h (see Note 35). 4. For each transfection, add 5 mL of FreeStyle™ F17 expression medium (without supplements) to a 50 mL Falcon tube. Add 100 μg of pTT5-VHH-Fc fusion plasmid and vortex briefly. Then add 100 μL of PEIpro® DNA transfection reagent and vortex for three 1 s pulses. 5. Incubate the mixture for 5 min at room temperature and then add to each flask containing cells from step 3. Return the flasks to the orbital shaking incubator. 6. After 24 h, feed the culture with 10 mL of 20% TN1 pre-warmed to 37
C and continue growing for 4–5 days. It is recommended that the cell number and viability be monitored using Trypan blue staining and a TC20™ automated cell counter. Viability should be above 90% for up to 4 days (see Note 36). 7. Harvest the cell supernatant between 5 and 7 days posttransfection by centrifugation at 200  g for 5 min at 4
C. Filter the supernatant through a 0.22 μm filter to remove any cell debris. Save 100 μL for analysis by SDS-PAGE. 8. Evaluate the expression of the VHH-Fc by SDS-PAGE to confirm the presence of the protein. 9. Load the remaining volume directly on a 5 mL MabSelect™ ¨ KTA FPLC. Wash the colSuRe™ column connected to an A umn with PBS and elute in 100 mM citric acid, pH 3.0. Neutralize immediately using 1 M Tris, pH 8.0. 10. Buffer exchange the VHH-Fc fusion protein into PBS (see Note 29) by dialysis or using an Amicon® Ultra-15 Centrifugal Filter Unit. Analyze the quality of the proteins by SDS-PAGE and size exclusion chromatography as described previously [27]. Store the samples at 4
C. 3.9 VHH Soluble ELISA

Binding specificity of the isolated VHHs is determined by ELISA using EGFR ectodomains fused to human IgG1 Fc domain as the coating antigen. Cross-reactivity studies against rhesus and mouse EGFR are performed against the same antigens (Fig. 4a). Surface plasmon resonance (SPR) binding studies can also be performed as described previously [19, 23, 25, 26] to determine affinities for EGFR of multiple species and for epitope binning (Fig. 4b, c). 1. Dilute human, rhesus, and mouse EGFR-Fc in PBS to 1 μg/ mL and coat microtiter plates with 100 μL/well overnight at 4
C.

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Fig. 4 Characterization of EGFR-binding VHHs raised by DNA immunization. (a) Binding of VHHs elicited by DNA immunization to human, rhesus, and mouse EGFR-Fc in ELISA. (b) Epitope binning of EGFR VHHs elicited by DNA immunization by surface plasmon resonance (SPR). (c) SPR-derived monovalent affinities (pH 7.4, 25
C) of EGFR VHHs elicited by DNA immunization for EGFR of various species. n.b., no binding. KD values are expressed as the means  standard deviations of three independent experiments. EG2 was included for reference as a VHH raised by protein antigen immunization. (d) Binding of EGFR-specific VHH-Fcs to EGFRpositive cells by Mirrorball® microplate cytometry. VHH-Fc fusions were serially diluted and incubated with MDA-MB-431 cells and binding was detected using an AF488-labeled anti-human IgG1 Fc secondary antibody. (Figure re-used with permission from [19])

2. The next day, block plates with 200 μL/well of PBS containing 2% skim milk for 1 h at 37
C. 3. Prepare three-fold serial dilutions of VHHs in PBS-T containing 5% BSA and add these to the wells (100 μL) for 2 h at room temperature. 4. Wash the plates five times with PBS-T and add 100 μL/well of HRP-conjugated rabbit anti-6  His antibody diluted 1: 10,000 in PBS-T containing 5% BSA. 5. After 1 h of incubation, wash the plates five times with PBS-T then add 100 μL/well of TMB substrate. Stop the reaction with 100 μL/well of 1 M H3PO4 and read the absorbance at 450 nm using a microplate reader. 3.10 Assessment of VHH and VHH-Fc Fusion Cell Binding

Cell binding assays using Mirrorball® fluorescence plate cytometry are used to analyze the cell binding of the isolated VHHs and VHH-Fc fusions to cell lines positive and negative for EGFR.

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1. Thaw one vial each of MDA-MB-468 (EGFR+) and MCF7 (EGFR) cells in a 37
C water bath. Transfer the contents to a T75 flask containing 10 mL of complete medium (RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B). 2. Grow cells at 37
C in a humidified 5% CO2 atmosphere until they reach 70–80% confluency. 3. For the Mirrorball® cytometry assay, detach each cell line from the T75 flask by aspirating the complete media, washing with HBSS, and detach cells with Accutase™ solution. Transfer each cell suspension to two 15 mL conical tubes and centrifuge for 5 min at 300  g at room temperature. 4. Discard the supernatant and resuspend each cell pellet in 10 mL of HBSS. Centrifuge for 5 min at 300  g at room temperature, discard the supernatant, and resuspend the cells in complete medium to achieve a cell density of approximately 1  105 cells/mL. 5. Prepare one plate for each cell line. Dispense 50 μL/well (~5000 cells) in a Nunc MicroWell 96-well optical bottom plate and place it back in the incubator (37
C, 5% CO2) for 24 h. 6. The next day, prepare three-fold serial dilutions of biotinylated VHHs or VHH-Fc fusions in LCIB containing 1% BSA in a 96-well dilution plate, starting at a concentration of 5 μM. Prepare a sufficient volume for both the EGFR+ and EGFR plates. Keep the dilutions on ice until use. 7. Take the plates containing the cells out of the incubator, remove the supernatants, and add 100 μL of each antibody dilution to each plate. Incubate for 2 h at 4
C. 8. With a multichannel pipette remove the antibody solutions carefully without disturbing the cells. 9. Wash once with LCIB without disturbing the cells and discard the supernatant. 10. Add 100 μL/well of SA-PE at 40 μg/mL (for VHH detection) or AF488-conjugated donkey anti-human IgG at 30 μg/mL (for VHH-Fc detection) diluted in LCIB containing 1% BSA. Incubate the plate at 4
C for 1 h. 11. Repeat steps 8 and 9 and proceed to stain live cells by addition 100 μL of 1 μM DRAQ5™ fluorescent probe for 10 min at 4
C. After a final wash with LCIB, data are acquired on a Mirrorball® microplate cytometer and analyzed using Cellista software.

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12. Plot the data using GraphPad Prism and EC50s are determined by curve fitting using the equation for one-site specific binding with Hill slope plots (Fig. 4d).

4

Notes 1. The E. coli DH5α strain is ideal for large-scale plasmid production and has not observed any unusual recombination events in this strain. 2. These constructs encode the native human leader sequence of EGFR. Replacing this sequence with llama leader sequences may improve expression following DNA immunization depending on the antigen. We have not tested this. 3. In contrast to a conventional spectrophotometer, the NanoDrop™ One Microvolume UV-Vis Spectrophotometer (or instruments with similar technology) can measure absorbance at very low volumes (1 μL) without the use of cuvettes. 4. It is important to determine the optimal mixing ratio between plasmid DNA and the gold particles. This is referred to as the DNA Loading Ratio (DLR). The amount of microcarrier delivered per target is referred to as the Microcarrier Loading Quantity (MLQ). A DRL of 2 and MLQ of 0.5 worked best in our hands [19]. 5. The amounts of spermidine, gold nanoparticles, and plasmid DNA need to be determined in advance based on Table 2 of the Bio-Rad gene gun manual. The ratio selected here worked best for our plasmid. The volume of plasmid DNA must remain at 100 μL if this protocol is followed. 6. If multiple plasmid DNA coatings are desired, then use an appropriate amount of each DNA preparation so that the total amount does not exceed 100 μg. 7. The volume of CaCl2 added should be equal to the volume of spermidine. 8. To test the efficiency of DNA adsorption, measure the absorbance of the DNA remaining in the supernatant and compare it with the input amount. 9. The DNA-coated gold solution can be stored for up to 2 months at 20
C. To freeze, tighten the cap of the tube and secure it with parafilm. To use it again, allow the solution to thaw at room temperature prior to removing the parafilm. 10. Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set

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out in the Ontario Ministry of Agriculture, Food and Rural Affairs Animals for Research Act, R.S.O. 1990, c. A.22. 11. Alternatively, use monoclonal antibodies specific for camelid heavy chain only antibody isotypes [27]. 12. It is strongly recommended that before starting the RNA extraction all surfaces and instruments are cleaned to minimize the presence of RNases. For this propose, use commercially available RNase AWAY™ or similar reagents. 13. Occasionally, it may be necessary to optimize the amount of input RNA, but generally 3–5 μg of total RNA per cDNA synthesis reaction results in a good yield of cDNA. 14. Prepare the primer solutions in autoclaved ddH2O and always store at 20
C to prevent degradation. We typically prepare a 10 working solution (10 μM) in 100 μL aliquots. 15. Three bands are obtained following RT-PCR: one with a size of ffi850 bp, which corresponds to conventional antibodies, and two close bands with sizes in the range of 550–650 bp, which correspond to heavy-chain antibodies and contain the VHH genes. We frequently observe that the CH2B3-F primer gives a banding pattern which consists of an intense VHH band and a faint conventional antibody band. The aim of optimizing the PCR reaction is to increase the intensity of the VHH-CH2/ VHH-CH2b bands relative to the conventional antibody band. However, differential intensities of the two VHH bands with respect to each other are routinely observed. 16. In all steps, elute the bound DNA with sterile ddH2O as opposed to the manufacturer’s recommended elution buffer. 17. A small-scale ligation can be performed with different molar ratios of digested insert (VHH) to digested vector (pMED1). Typically, a 1:3 ratio works best. We also recommend performing colony PCR to test for in-frame insertion before proceeding with the large-scale ligation and electroporation. A library size of close to the number of input lymphocytes (5  107) gives a good representation of the VHH repertoire; in llamas less than half of the immunoglobulin repertoire is heavy-chain antibody. 18. Generally, a 0.5–1 μL aliquot of the PCR product can be used directly for DNA sequencing and will give clean sequencing reads. If the sequencing quality is poor, purify the PCR product with a QIAquick® PCR purification kit before DNA sequencing. Either upstream (M13RP) or downstream primer (PN2) gives sufficient read coverage for VHH fragments. 19. When analyzing the library sequences, VHHs can be distinguished from VHs by examining the four amino acids at positions 37, 44, 45, and 47 using Kabat definitions. VHHs

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characteristically have Phe or Tyr at position 37, Glu at position 44, Gln, Arg, or Cys at position 45, and Gly, Ser, Leu, or Phe at 47. By contrast, VHs have Val, Gly, Leu, and Trp at these four positions, respectively. 20. To prepare a stock plate of E. coli TG1 cells, streak out a frozen glycerol stock of TG1 on a minimal medium (M9) agar plate [24] supplemented with 1 μg/mL thiamine (vitamin B1). Incubate at 37
C for at least 24 h. Seal the plate with parafilm and store at 4
C for up to 1 month. It is recommended to grow the TG1 cells on minimal media to ensure that the F pilus, which mediates phage infection, is maintained on the cells. Thiamine is added to the media since TG1 cells are auxotrophic for thiamine. 21. To determine the titer of the phage, prepare 106, 108, 1010, and 1012 serial dilutions of phage in PBS, then mix 10 μL of each dilution with 100 μL of the exponentially growing E. coli TG1 cells. Incubate the cells at room temperature for 15 min and subsequently plate them on LB-Amp plates. In the morning, count the colonies and determine the titer. Phage titers are typically 5  1012 to 1  1013 colony-forming units/mL from a 100 mL culture. 22. From an E. coli TG1 minimal medium plate (see Note 20), pick a single colony and inoculate 10 mL of 2  YT medium (no antibiotics) in a sterile 50 mL Falcon tube and incubate at 37
C with shaking at 220 rpm. Increase the volume as necessary if conducting multiple panning campaigns simultaneously. Measure the A600 in a spectrophotometer with disposable cuvettes using 2  YT as the blank. Stop the incubation when A600 ¼ 0.3 to 0.4 (this usually takes 2–3 h, but it could take longer). 23. Prepare serial dilutions (102 to 106) of the infected cells in 2  YT medium. Spread 100 μL of each dilution on LB-Amp plates and also plate 100 μL of the uninfected cells as a negative control. Incubate at 32
C overnight. Keep the plates parafilmsealed and stored at 4
C for clonal analysis (colony PCR, sequencing, and phage ELISA). 24. To minimize the chance of obtaining VHH-phage binders against the blocking proteins, it is recommended to alternate the blocking buffer used in each round of panning (e.g., use PBS containing 1% casein in the first and third rounds, and PBS containing 1% BSA in the second and fourth rounds). This step is essential when panning naı¨ve or synthetic libraries but less important for immune VHH libraries. 25. To avoid the enrichment of antibodies specific for streptavidin, there is an option to perform a pre-incubation step of the library with a blocked well containing streptavidin only. This

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step is essential when panning naı¨ve or synthetic libraries but less important for immune VHH libraries. 26. Genes encoding unique VHH sequences obtained after panning are synthesized commercially and cloned into the pMROBAP-H6 vector between the SfiI restriction enzyme sites. 27. A test experiment should be performed when working with a new VHH construct for protein expression using a small volume of culture (10 mL) and applying variable conditions: different IPTG concentrations (5–800 μM), growth temperatures (16
C, 28
C, and 37
C), and expression times (3 h or overnight). 28. Ten mL of a 10 protease inhibitor cocktail is sufficient to inhibit proteases in 20 g of bacterial pellet. Therefore, it is advised to determine the mass of the pellet to ensure addition of sufficient protease inhibitor. 29. PBS may be supplemented with 3 mM EDTA to chelate any residual Ni2+ and to prevent protein oxidation and degradation. Ensure that small amounts of EDTA will not adversely affect downstream assays. 30. Dilute the Cam stock solution to 1 mg/mL in ultrapure H2O before use and store the working stock at 4
C for up to 30 days. Protect from light to avoid degradation. 31. Strain AVB101 is an E. coli B strain (hsdR, lon11, sulA1) harboring a plasmid encoding the biotin ligase BirA, whose expression is induced by the addition of IPTG. The enzyme recognizes the AviTag encoded at the C-terminus of the VHHs. For simplicity, we prefer to perform the enzymatic biotinylation post expression as needed. 32. Genes encoding VHH-Fc fusions were ordered, synthesized, and subcloned into pTT5 by GeneArt (Thermo Fisher Scientific). Approximately 5 μg plasmid DNA (pTT5-VHH-Fc) is received and a sample is transformed for large-scale plasmid preparation. 33. We use HEK 293-6E cells, a HEK293 derivative that constitutively expresses a truncated version of EBNA-1 and that was adapted for growth in suspension and serum free medium. The cell line was developed at NRC (Montreal, Canada) and is available under license. Conventional HEK293 cells (e.g., 293T) could also be used. 34. The complete medium is generated by supplementing each L with 20 mL of GlutaMAX™, 10 mL of 100 Pluronic™ F-68 non-ionic surfactant, 10 mL of antibiotic-antimycotic, and 1 mL of geneticin (G-418). 35. The cell density at transfection should range from 1  106 to 2  106 cells/mL, with cell viability greater than 95%.

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Transfection at 1.5  106 cells/mL is usually optimal, yielding the highest amount of recombinant protein. 36. Increasing the length of expression until cell viabilities decrease to 65% or lower may increase yield but could also complicate downstream processing and promote proteolytic degradation of the expressed protein.

Acknowledgments This work was supported by the National Research Council Canada. We gratefully acknowledge the excellent assistance of Henk van Faassen, Shalini Raphael, Mary Foss, Hong Tong-Sevinc, Debbie Callaghan, and Sonia Leclerc. References 1. Lu RM, Hwang YC, Liu IJ et al (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27:1 2. Eden T, Menzel S, Wesolowski J et al (2018) A cDNA immunization strategy to generate nanobodies against membrane proteins in native conformation. Front Immunol 8:1989 3. Hobernik D, Bros M (2018) DNA vaccines— how far from clinical use? Int J Mol Sci 19: 3605 4. Liu S, Wang S, Lu S (2018) Using DNA immunization to elicit monoclonal antibodies in mice, rabbits, and humans. Hum Gene Ther 29:997–1003 5. Wolff JA, Malone RW, Williams P et al (1990) Direct gene transfer into mouse muscle in vivo. Science 247:1465–1468 6. Tang DC, DeVit M, Johnston SA (1992) Genetic immunization is a simple method for eliciting an immune response. Nature 356: 152–154 7. Babiuk LA, Pontarollo R, Babiuk S et al (2003) Induction of immune responses by DNA vaccines in large animals. Vaccine 21:649–658 8. Kutzler MA, Weiner DB (2008) DNA vaccines: ready for prime time? Nat Rev Genet 9: 776–788 9. Leitner WW, Ying H, Restifo NP (1999) DNA and RNA-based vaccines: principles, progress and prospects. Vaccine 18:765–777 10. Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363: 446–448

11. Muyldermans S (2001) Single domain camel antibodies: current status. J Biotechnol 74: 277–302 12. Harmsen MM, De Haard HJ (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77:13–22 13. de Marco A (2011) Biotechnological applications of recombinant single-domain antibody fragments. Microb Cell Fact 10:44 14. Bradbury AR, Sidhu S, Dubel S et al (2011) Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol 29:245–254 15. Maussang D, Mujic´-Delic´ A, Deschamps FJ et al (2013) Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo. J Biol Chem 288: 29562–29572 16. Peyrassol X, Laeremans T, Gouwy M et al (2016) Development by genetic immunization of monovalent antibodies (nanobodies) behaving as antagonists of the human chemR23 receptor. J Immunol 196:2893–2901 17. Koch-Nolte F, Reyelt J, Scho¨ssow B et al (2007) Single domain antibodies from llama effectively and specifically block T cell ectoADP-ribosyltransferase ART2.2 in vivo. FASEB J 21:3490–3498 18. Peyrassol X, Laeremans T, Lahura V et al (2018) Development by genetic immunization of monovalent antibodies against human vasoactive intestinal peptide receptor 1 (VPAC1), new innovative, and versatile tools to study

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VPAC1 receptor function. Front Endocrinol 9: 153 19. Rossotti MA, Henry KA, van Faassen H et al (2019) Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling. Biochem J 476: 39–50 20. Durocher Y, Perret S, Kamen A (2002) Highlevel and high throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30:E9 21. Arbabi-Ghahroudi M, MacKenzie R, Tanha J (2009a) Selection of non-aggregating VH binders from synthetic VH phage-display libraries. Methods Mol Biol 525:187–216 22. Rossotti MA, Pirez M, Gonzalez-Techera A et al (2015) Method for sorting and pairwise selection of nanobodies for the development of highly sensitive sandwich immunoassays. Anal Chem 87:11970–11914 23. Arbabi-Ghahroudi M, Tanha J, MacKenzie R (2009b) Isolation of monoclonal antibody

fragments from phage display libraries. Methods Mol Biol 502:341–364 24. Arbabi Ghahroudi M, Desmyter A, Wyns L et al (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414: 521–526 25. Hussack G, Arbabi-Ghahroudi M, Mackenzie CR et al (2012) Isolation and characterization of Clostridium difficile toxin-specific singledomain antibodies. Methods Mol Biol 911: 211–239 26. Baral TN, MacKenzie R, Arbabi Ghahroudi M (2013) Single-domain antibodies and their utility. Curr Protoc Immunol 103:Unit 2.17 27. 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 indistinguishable but immunogenetically distinct. Immunogenetics 71:307–320

Chapter 4 Preparation of Immune and Synthetic VNAR Libraries as Sources of High-Affinity Binders Jahaziel Gasperin-Bulbarela, Olivia Cabanillas-Bernal, Salvador Duen˜as, and Alexei F. Licea-Navarro Abstract The shark-derived autonomous variable antibody domains known as VNARs are attractive tools for therapeutic and diagnostic applications due to their favorable properties like small size (approximately 12 kDa), high thermal and chemical stability, and good tissue penetration. Currently, different techniques have been reported to generate VNAR domains against targets of therapeutic interest. Here, we describe methods for the preparation of an immune VNAR library based on bacteriophage display, and for the preparation of a synthetic library of VNAR domains using a modified protocol based on Kunkel mutagenesis. Finally, we describe procedures for in silico maturation of a VNAR using a bioinformatic approach to obtain higher affinity binders. Key words Shark, Antibody, VNAR, Immune library, Synthetic library, In silico affinity maturation

1

Introduction Due to their molecular specificity, antibodies are biochemical tools of great interest as therapeutic and diagnostic agents. Shark-derived autonomous variable domain antibodies, known as VNARs, are highly soluble single-domain antibodies with a molecular weight of approximately 12 kDa [1]. These small domains are of significant interest for scientific research and therapeutic applications due to their high affinity for antigens, high thermal and chemical stability, and enhanced tissue penetration [2–5]. Over the past 20 years, VNAR domains have been produced against many target antigens of pharmaceutical and therapeutic interest, including Ebolavirus nucleoprotein [6], tumor necrosis factor-α [7, 8], vascular endothelial growth factor [7, 9], Plasmodium falciparum histidine-rich protein 2 [10], and Aurora A kinase [11], amongst numerous others. VNAR domains can be isolated from naı¨ve and immune libraries. Naı¨ve libraries are generated by amplifying the genes

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_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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encoding VNAR domains from naı¨ve animals (not previously immunized with an antigen of interest) [12], while immune libraries are prepared by amplifying the VNAR-encoding repertoire of animals immunized with a target antigen [13]. The construction of immune libraries of VNAR domains requires immunization of a shark, RNA extraction, and subsequent steps to amplify the immune repertoire. VNAR libraries can be generated from the repertoires of all cartilaginous fish, including all types of sharks and rays. While these libraries routinely offer high-affinity binders to specific targets [4], their diversity may be limited; moreover, library preparation is costly, labor-intensive, and must be repeated for each immunization campaign. In recent years, advances in protein engineering have allowed for the construction of synthetic libraries of antibodies, including VNAR domains [9, 14–16]. These synthetic libraries avoid animal immunization, allowing for the rapid generation of moderate- to high-affinity binders with specificity for practically any antigen. Furthermore, preparation of synthetic libraries allows the use of VNAR frameworks that are pre-selected for desirable characteristics, including high expression yield, solubility, and low immunogenicity. Both immune and synthetic libraries offer distinct advantages. Selecting one or the other will depend on the tools available and the characteristics of the target antigen. For example, a small antigen that does not elicit an immune response in an organism requires coupling to a carrier protein to induce an antibody response. However, binders to this target could be directly selected from a synthetic library. Synthetic libraries can also be beneficial when facilities for maintaining sharks are not available. However, if the necessary conditions and organisms are in place, use of an immune VNAR library generated for an antigen of interest is the preferred option for obtaining high-affinity binders. While high-affinity binders can be obtained from both immune and synthetic VNAR libraries, affinity maturation of VNARs is occasionally required to increase target binding affinity [15, 17]. Strategies for affinity maturation of VNAR domains in silico have been developed [18], allowing for the virtual screening of hundreds of variants that would be impractical to synthesize, express, purify, and test in vitro. This chapter describes methods for the preparation of an immune VNAR library based on bacteriophage display and for the preparation of a synthetic library of VNAR domains using a protocol based on Kunkel mutagenesis [19] (Fig. 1). In addition, we describe a proposed procedure for in silico affinity maturation of a VNAR using bioinformatic tools to obtain higher affinity binders.

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Fig. 1 Graphical representation of the procedures used for preparation of an immune VNAR library based on bacteriophage display and preparation of a synthetic library of VNAR domains using a modified protocol derived from Kunkel mutagenesis

2

Materials

2.1 Preparation of Immune VNAR Libraries 2.1.1 Horn Shark (Heterodontus francisci) Immunization

1. Complete and incomplete Freund’s adjuvant. 2. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4. Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.245 g of KH2PO4 in 800 mL of distilled water. Adjust the pH of the solution to 7.4 and then add distilled water to a final volume of 1 L. Sterilize by autoclaving. 3. 3 mL syringe with 23-gauge needle. 4. Antigen solution: prepare stocks of the antigen at a concentration of 5 μg/mL in PBS. For each horn shark immunization, use 200 μL of antigen solution for a total of 1 μg. Note that many protocols for shark immunization in the literature use higher amounts of antigen (~30–250 μg per immunization) although this is typically for larger shark species. For complete cell immunization, prepare a suspension of 1
106 cells in a maximum of 1 mL of PBS.

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2.1.2 RNA Isolation

1. Diethyl pyrocarbonate (DEPC)-treated water. Dissolve 0.1% (v/v) DEPC in distilled water and incubate overnight with agitation. Autoclave for 15 min at 121  C and 15 psi to inactivate any residual DEPC. 2. TRI reagent® (also sold as TRIzol™). Take appropriate safety precautions as TRI reagent® is a mixture of guanidine thiocyanate and phenol. 3. 50 mL centrifuge tubes. 4. Tissue homogenizer. 5. 1-bromo-3-chloropropane (BCP). 6. Isopropanol (molecular biology grade). 7. Absolute ethanol (molecular biology grade). 8. 75% (v/v) ethanol: mix 37.5 mL of absolute ethanol with 12.5 mL of DEPC-treated water. 9. NanoDrop Lite. 10. DNA/RNA electrophoresis equipment and buffers.

2.1.3 cDNA Synthesis

1. SuperScript™ III First-Strand Synthesis SuperMix, including Annealing Buffer, First-Strand Reaction Mix and SuperScript™ III/RNaseOUT™ Enzyme Mix (Invitrogen, Carlsbad, CA, USA). 2. PCR tubes. 3. Thermal cycler. 4. DEPC-treated water. 5. Primer R564 (SfiI restriction endonuclease site underlined): 50 -GTG GAG CAG GCC GGC CTG GCC GTT CAC AGT CAG CAC GGT GCC AGC TC-30 .

2.1.4 Amplification of VNAR Coding Regions

1. GoTaq® Flexi DNA polymerase (Promega, Madison, WI, USA), including 5
buffer and 25 mM MgCl2, or another suitable PCR amplification system. 2. Primer F557 (SfiI restriction endonuclease site underlined): 50 -AGG CGG GGC CCA GGC GGC CGC ACG GCT TGA ACA AAC ACC-30 . 3. Primer F558 (SfiI restriction endonuclease site underlined): 50 -AGG CGG GGC CCA GGC GGC CCA ACG GGT TGA ACA AAC ACC-30 . 4. Primer F559 (SfiI restriction endonuclease site underlined): 50 -AGG CGG GGC CCA GGC GGC CAC AAG GGT AGA CCA AAC ACC-30 . 5. Primer F560 (SfiI restriction endonuclease site underlined): 50 -AGG CGG GGC CCA GGC GGC CGC AAG GGT GGA CCA AAC ACC-30 .

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6. Primer F561 (SfiI restriction endonuclease site underlined): 50 -AGG CGG GGC CCA GGC GGC CGC ATG GGT AGA CCA AAC ACC-30 . 7. Primer F562 (SfiI restriction endonuclease site underlined): 50 -AGG CGG GGC CCA GGC GGC CGC AAG CCT GGA CCA AAC ACC-30 . 8. Primer F563 (SfiI restriction endonuclease site underlined): 50 -AGG CGG GGC CCA GGC GGC CGC ATT GAC GGA CCA AAC ACC-30 . 9. Primer R564: see Subheading 2.1.3. 10. Sterile distilled water (autoclaved). 11. 10 mM dNTP mix. 12. DNA electrophoresis equipment and buffers. 13. QIAquick DNA Gel Extraction Kit (Qiagen, Hilden, Germany) or similar kit. 14. NanoDrop Lite. 15. Thermal cycler. 2.1.5 Construction of Immune VNAR Libraries

1. Purified pComb3x vector [20] (at least 20 μg). This vector is Addgene plasmid #63890. 2. Purified VNAR amplicons. 3. SfiI restriction endonuclease (20 U/μL) and buffer. 4. T4 DNA ligase (400 U/μL) and buffer. 5. Electrocompetent (1
1010 colony-forming units [cfu]/μg) F0 amber suppressor strain of Escherichia coli, such as TG1 (Lucigen, Madison, WI, USA), XL1-Blue (Stratagene, La Jolla, CA, USA), or ER2738 (Lucigen). 6. Electroporation cuvettes with 0.2 cm gap. 7. Electroporator. 8. 2
YT medium: dissolve 10 g of yeast extract, 16 g of tryptone, and 5 g of NaCl in 950 mL of deionized water. Adjust pH to 7.0, bring to 1 L with water and autoclave. 9. 2
YT-glucose and carbenicillin (2
YT-GC) medium: 2
YT medium containing 2% (w/v) glucose and 100 μg/ mL carbenicillin. Prepare 1 L of 2
YT medium and after autoclaving, allow to cool to 45–60  C, add 50 mL of filtersterilized 40% glucose (400 g/L in water), and 1 mL of filtersterilized carbenicillin (100 mg/mL). 10. 2
YT-GC agar plates: prepare 2
YT-GC medium, adding 11 g/L of bacteriological agar before autoclaving. Pour into 100- and 150-mm diameter Petri dishes. Unless otherwise stated 100 mm plates are used.

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11. 3 M sodium acetate, pH 5.2. 12. Ethanol (100% and 70%). 13. Sterile toothpicks (autoclaved). 14. pComb3x-specific primer Ompseq: 50 -AAG ACA GCT ATC GCG ATT GCAG-30 . 15. pComb3x-specific primer gback: 50 -GCC CCC TTA TTA GCG TTT GCC ATC-30 . 16. QIAprep Spin Miniprep Kit (Qiagen). 2.2 Preparation of Synthetic VNAR Libraries 2.2.1 Production and Purification of UracilContaining ssDNA

1. pComb3x vector. 2. VNAR scaffold (Fig. 2). 3. E. coli CJ236 strain (New England Biolabs, Ipswich, MA, USA). 4. 2
YT-GC medium (see Subheading 2.1.5). 5. 2
YT-GC agar plates (see Subheading 2.1.5). 6. Chloramphenicol. 7. M13KO7 helper phage (New England Biolabs). 8. Uridine. 9. PEG/NaCl solution: 20% (v/v) PEG 8000 and 2.5 M NaCl. Dissolve 40 g of PEG 8000 and 36.53 g of NaCl in 180 mL of water. Bring to 250 mL and autoclave. After autoclaving two phases are formed, allow to cool down and mix vigorously to homogenize. 10. PBS (see Subheading 2.1.1). 11. 0.22 μm syringe filter. 12. E.Z.N.A.® M13 DNA Mini Kit (Omega Bio-tek, Norcross, GA, USA) or similar M13 phage ssDNA purification kit.

2.2.2 Mutagenic dsDNA Production

1. 10
TM buffer: 0.5 M Tris, 0.1 M MgCl2, pH 7.5. 2. 10 mM ATP. 3. 100 mM dithiothreitol (DTT). 4. T4 polynucleotide kinase (10 U/μL) and buffer. 5. 10 mM dNTPs. 6. T4 DNA ligase (400 U/μL) and buffer. 7. T7 DNA polymerase (10 U/μL) and buffer. 8. Thermal cycler. 9. QIAquick PCR Purification Kit (Qiagen) or similar. 10. E. coli TG1 strain (Lucigen). 11. 2
YT medium (see Subheading 2.1.5). 12. 2
YT-GC medium (see Subheading 2.1.5).

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Fig. 2 Protein sequence of the T1 VNAR domain used as a scaffold for synthetic library construction. The different regions of the VNAR are labeled. The gene encoding the T1 VNAR was designed to include three stop codons (not amber) immediately before the CDR3 which are intended to be eliminated by mutagenic oligonucleotides. FW framework region, CDR complementarity-determining region, HVR hypervariable region

13. 2
YT-GC agar plates (see Subheading 2.1.5). 14. Electroporation cuvettes with 0.2 cm gap and electroporation device. 15. Glycerol. 16. Primer Ompseq (see Subheading 2.1.5). 17. Primer gback (see Subheading 2.1.5). 18. Mutagenic complementarity-determining region 3 (CDR3) oligonucleotide (see Note 1). 2.3 Propagation of Phage Particles from VNAR Libraries

1. 2
YT-GC medium (see Subheading 2.1.5) 2. M13KO7 helper phage. 3. Antibiotics: carbenicillin (100 mg/mL) and kanamycin (50 mg/mL), sterile filtered. 4. PEG/NaCl solution (see Subheading 2.2.1). 5. PBS (see Subheading 2.1.1). 6. 0.22 μm syringe filter. 7. 60% (v/v) glycerol solution. Dilute 60 mL of glycerol with 40 mL of water and autoclave. 8. 50 mL centrifuge tubes. 9. 1.5 mL microcentrifuge tubes.

2.4 In Silico Maturation of VNARs

1. VNAR protein sequence (Fig. 2). 2. Computer with internet access. A computer with at least a fourth-generation Intel I5 processor and 8 GB of RAM can be used.

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3. PyMOL Molecular Graphics System, Version 1.8 (Schro¨dinger, New York, NY, USA). 4. MODELLER, NAMD, and VMD programs. These programs are multiplatform. 5. For molecular dynamics, a server is recommended. For example, a server tower with 24 12-core 2.7 GHz processors and 125 GB of RAM. A 50 ns run can last up to 5 days.

3

Methods

3.1 Preparation of Immune VNAR Libraries

3.1.1 Horn Shark Immunization

This section describes the construction of an immune VNAR library following immunization of a horn shark (Heterodontus francisci) and the isolation of RNA from the spleen. The library construction and amplification were based on the work of Barbas et al. [20]. Using this methodology, a library size of at least 109 individual clones can be expected. All procedures described in this section were performed in accordance with the Mexican guidelines NOM-062-ZOO-1999 and adhered to the requirements defined by the Institutional Animal Care and Use Committee of the Center for Scientific Research and Higher Education at Ensenada. 1. Place the shark on a preparation table and hold the shark firmly from the head and the precaudal tail (see Note 2). Avoid keeping the animal out of the water for more than 5 min. 2. Harvest a blood sample (~2 mL) from the caudal vein before the first immunization. Allow the blood to clot by incubating for 15 min at room temperature (RT), then centrifuge at 1,000
g for 10 min at 4  C. Collect the serum, aliquot, and store at 80  C. 3. Mix the antigen solution (1 μg in 200 μL; note that most immunization protocols in the literature use larger shark species and inject a larger amount of antigen, typically 30–250 μg per immunization) with an equal volume of complete Freund’s adjuvant and inject subcutaneously between the first and second dorsal fin (see Note 3). Harvest a blood sample 1 week after each immunization and process as in step 2. 4. Two weeks after the first immunization, perform a second immunization but mixing the antigen solution (1–250 μg) in an equal volume of incomplete Freund’s adjuvant. Inject subcutaneously as described in step 2. 5. Boosts are performed at 2-week intervals by intravenous injection of the antigen solution (1–250 μg) in PBS into the caudal vein. Repeat for a total of eight immunizations. 6. For immunizations with whole cells, inject 1
106 cells in PBS intravenously. A total of four immunizations are suggested in

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this case. Adjuvant the priming immunization with complete Freund’s adjuvant and subsequent boosts with incomplete Freund’s adjuvant. 7. One week after the last boost, euthanize the shark and extract the spleen by mid-ventral dissection. 3.1.2 RNA Isolation

1. All materials need to be autoclaved and rinsed with DEPCtreated water before use. 2. Place 1–2 g of spleen in a 50 mL centrifuge tube. Add 20 mL of TRI reagent® (50–100 μL per 1 mg of tissue, see Note 4). Homogenize the spleen using a tissue homogenizer. Allow the mixture stand for 5 min at RT. 3. Add 2 mL of BCP (0.1 mL per 1 mL of TRI reagent®). Mix for 15 s by vortexing and incubate for 15 min at RT. 4. Centrifuge at 12,000
g for 20 min at 4  C. 5. Carefully transfer the upper aqueous phase to a 50 mL centrifuge tube (see Note 5) and add 10 mL of isopropanol (0.5 mL per 1 mL of TRI reagent® used). Mix and incubate for 10 min at RT. 6. Centrifuge at 12,000
g for 20 min at 4  C. 7. Discard the supernatant and wash with 20 mL (equivalent to the volume of TRI reagent® used) of 75% ethanol diluted with DEPC-treated water. Vortex the sample for 15 s. 8. Centrifuge at 12,000
g for 10 min at 4  C. 9. Aspirate the supernatant and air-dry the RNA pellet for 5–10 min. Avoid over drying the pellet since this will reduce its solubility. 10. Resuspend the pellet in DEPC-treated water (0.5–1 mL) and incubate for 10 min at 50  C to enhance solubility. Let the mixture cool to RT and quantify the RNA by spectrophotometry at 260 nm (1 OD260 ¼ 40 μg/mL) and at 280 nm to assess purity (OD 260/280 should be ~2.0). Prepare multiple aliquots and store at 80  C or on ice if using immediately. 11. Evaluate the integrity of the RNA (0.5–1 μg) by electrophoresis in a 1% agarose gel. Two distinct bands should be observable, corresponding to 28S and 18S ribosomal RNA.

3.1.3 cDNA Synthesis

1. Maintain the RNA sample, SuperScript™ III First-Strand Synthesis reagents, and all other reagents on ice. 2. In a PCR tube on ice, mix 1 μg of RNA (volume not to exceed 6 μL) with 1 μL of annealing buffer and 1 μL of 10 μM primer R564 oligonucleotide. Bring to a final volume of 8 μL with DEPC-treated water. Mix gently by pipetting up and down, then centrifuge briefly (2,000
g for 5 s at 4  C).

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3. Incubate the reaction in a pre-heated thermal cycler at 65  C for 5 min (see Note 6), then immediately place on ice for at least 1 min to cool down. 4. Add 10 μL of First-Strand Reaction Mix and 2 μL of SuperScript™ III/RNaseOUT™ Enzyme Mix to the reaction. Mix gently by pipetting up and down and centrifuge at 2,000
g for 5 s at RT. 5. Incubate for 50 min at 55  C in a thermal cycler and terminate the reaction at 85  C for 5 min. Immediately place the cDNA on ice and proceed to the amplification of VNAR regions, or at 20  C for long-term storage. 3.1.4 Amplification of VNAR Coding Regions

Perform separate PCR reactions for each of the sense oligonucleotides (F557, F558, F559, F560, F561, F562, and F563) with the common antisense oligonucleotide (R564) to retain the diversity of the immune repertoire. Sense and antisense oligonucleotides contain an SfiI restriction endonuclease site for cloning into the pComb3x phagemid vector (see Note 7). All steps should be carried out on ice. 1. Prepare a PCR master mix by combining: 40 μL of 5
Green GoTaq® Flexi Buffer, 24 μL of 25 mM MgCl2, 4 μL of 10 mM dNTP mix, 1 μL of GoTaq® DNA polymerase (5 U/μL), and 0.4 to 1 μg of cDNA. Bring to a final volume of 184 μL with sterile water (see Note 8). Mix gently by pipetting up and down, then centrifuge at 2,000
g for 5 s at RT. 2. In seven separate tubes, mix 1 μL of each 10 μM sense oligonucleotide into the corresponding tube and 1 μL of 10 μM common antisense R564 oligonucleotide into all tubes. 3. Add 23 μL of the PCR master mix to each of the seven oligonucleotide tubes. Mix by pipetting up and down and centrifuge briefly (2,000
g for 5 s at RT) to collect. 4. Perform PCR with the following thermal cycling parameters: 95  C for 2 min, 30 cycles of 30 s at 95  C, 30 s at 56  C, and 60 s at 72  C, and a final extension of 10 min at 72  C. Once the reaction is complete, store at 4  C indefinitely. 5. Evaluate 3 μL of each reaction by electrophoresis in a 2% agarose gel. An efficient amplification results in a single amplicon of approximately 400 bp. 6. If a successful amplification is obtained, pool the seven PCR reactions for preparative gel electrophoresis. Excise the ~400 bp band and gel purify using the QIAquick Gel Extraction Kit, according to the manufacturer’s instructions. 7. Quantify the concentration of the PCR amplicon by spectrophotometry at 260 nm (1 OD260 ¼ 50 μg/mL) and at 280 nm to assess purity (ratio OD 260/280 should be ~1.8). Evaluate

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the purity and integrity of the DNA (100 ng) by electrophoresis in a 2% agarose gel (see Note 9). 3.1.5 Construction of Immune VNAR Libraries

1. Digest the pComb3x vector and PCR amplicon (insert) with SfiI restriction endonuclease. Prepare a separate reaction for the vector and insert as follows: mix 10 μg of DNA, 2.5 μL of 20 U/μL SfiI, an appropriate volume of buffer to attain 1
final concentration, and sterile water to a final volume of 50 μL. Incubate at 50  C for 5 h. 2. Purify the digested DNA by preparative gel electrophoresis in a 2% agarose gel using the QIAquick Gel Extraction Kit or similar, according to the manufacturer’s instructions. 3. Perform a small-scale ligation to assess digestion and ligation efficiency. In PCR tubes, mix 100 ng of digested pComb3x vector with digested insert, using vector to insert ratios of 1:1, 1:3, and 1:5. Add 1 μL of 10
ligase buffer and 1 μL of T4 DNA ligase (400 U/μL). Bring to 10 μL with sterile water. As a control, prepare a reaction without insert. 4. Incubate for 16 h at RT. Heat inactivate at 65  C for 10 min. 5. Transform 50 μL of an electrocompetent E. coli strain containing the F-factor and amber suppressor genes such as XL1-Blue, TG1, or ER2738 (see Note 10) with 3 μL of each ligation by electroporation. Use pre-chilled 0.2 cm electroporation cuvettes and pulse at 2.5 kV, 200 Ω, and 4 ms. Recover the electroporated cells immediately by adding 1 mL of pre-warmed 2
YT medium supplemented with 2% glucose to the cuvette, then rinse each cuvette twice with 1 mL of medium. Collect the electroporated bacterial suspension in a single 50 mL centrifuge tube and incubate for 1 h at 37  C with 250 rpm shaking. 6. Plate 100 μL of bacterial suspension serially diluted (101 to 104 in 2
YT-GC) on 2
YT-GC agar plates (see Note 11). Incubate overnight at 37  C. 7. Calculate the number of transformants as follows (see Note 12): No: of cf u
Volume recovery media ðμLÞ
Dilution Factor Volume plated ðμLÞ 8. Perform a large-scale ligation using the vector to insert ratio that gave the highest number of transformants. Mix 1 to 1.5 μg of digested vector with the corresponding amount of insert, add 20 μL of 10
ligase buffer and 20 μL of T4 DNA ligase (400 U/μL), and bring to 200 μL with sterile water. Incubate for 16 h at RT. Heat inactivate at 65  C for 10 min.

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9. Ethanol-precipitate the ligation product by mixing with 20 μL of 3 M sodium acetate, pH 5.2 (1/10 volume). Vortex for 10 s, add 500 μL of 100% ethanol (2.5 volumes), vortex again, and incubate for 30 min at 80  C. 10. Centrifuge at >16,000
g for 20 min at 4  C to pellet DNA. Discard the supernatant and wash with 1 mL of ice-cold 70% ethanol. Centrifuge again for 5 min and discard the supernatant. Repeat the wash step twice. Remove all of the ethanol and air-dry the pellet. Resuspend in 30 μL of sterile water. Store at 20  C until use. 11. Mix the 30 μL of precipitated ligation product with 400 μL of electrocompetent E. coli cells. Divide between four electroporation cuvettes and electroporate as described in step 5. 12. To estimate the library size and diversity, plate 100 μL of serially diluted bacterial suspension (101 to 1012 in 2
YT-GC medium) onto 2
YT-GC agar plates. Incubate overnight at 37  C. 13. Centrifuge the remaining bacterial suspension at 1,250
g for 10 min, discard supernatant, resuspend the pellet gently by pipetting and plate the cells by dividing between four 150-mm diameter 2
YT-GC agar plates. Incubate at 37  C overnight. A bacterial lawn will grow. 14. To recover bacteria, rinse each plate three times with 1 mL of 2
YT-GC medium. Keep cells on ice for later use in phage library propagation (see Subheading 3.3). 15. To estimate the diversity of the library, randomly select at least 50 colonies on plates from the serial dilutions (step 12) and evaluate them by colony PCR with the pComb3x-specific oligonucleotides Ompseq and gback. To prepare 50 reactions (20 μL each), mix: 200 μL of 5
Green GoTaq® Flexi Buffer, 120 μL of 25 mM MgCl2, 20 μL of 10 mM dNTP mix, 20 μL of each oligonucleotide (10 μM), 5 μL of GoTaq® DNA polymerase (5 U/μL), and 615 μL of sterile water. Pick a colony with a sterile toothpick and submerge in the PCR reaction. Perform the thermal cycling as described above (see step 4 in Subheading 3.1.4 and Note 13). 16. For clones that show an amplification product of the expected size (~620 bp), inoculate 2 mL cultures of 2
YT-GC medium and grow overnight at 37  C with 250 rpm shaking. The next day, extract plasmid DNA using a QIAprep Spin Miniprep Kit according to the manufacturer’s instructions. Sequence the plasmids to evaluate library diversity using both pComb3x oligonucleotides (Ompseq and gback) for bidirectional sequencing.

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3.2 Preparation of Synthetic VNAR Libraries

This section describes the construction of a synthetic VNAR library using a modified protocol derived from the Kunkel mutagenesis technique [19]. Using this protocol, it is possible to construct synthetic VNAR libraries containing up to 109 individual clones. We present this methodology in detail using as a case study a synthetic library prepared from the previously reported VNAR scaffold “T1” and the mutagenic oligonucleotide T1-MUT-F [9] to introduce library diversity.

3.2.1 Production and Purification of UracilContaining ssDNA

1. Select a VNAR scaffold (see Note 14) (Fig. 2) for the construction of the synthetic library and clone it into the phagemid vector pComb3x between the SfiI restriction endonuclease sites as described above (see Subheading 3.1.5). 2. Transform chemically competent CJ236 E. coli cells (or another dut/ung strain) with the phagemid vector carrying the template VNAR sequence by mixing 50 μL of cells with 1 ng of vector. Incubate at 42  C for 1 min. Resuspend the cells in 1 mL of 2
YT-GC medium and plate 50 μL of the transformed cells on 2
YT-GC agar plates. Incubate overnight at 37  C. 3. Randomly select 10 transformed colonies and inoculate each one in 10 mL of 2
YT-GC medium containing 5 μg/mL chloramphenicol. Incubate the cultures at 37  C with 250 rpm shaking until an optical density at 600 nm (OD600) of 0.4–0.8 is reached. 4. Infect the 10 mL cultures with a 20-fold excess of M13KO7 helper phage particles per cell (see Note 15). Incubate the cultures at 37  C for 30 min with no agitation, then centrifuge the cells at 1,250
g for 15 min. Discard the supernatant and resuspend the pellet in 40 mL of 2
YT-GC medium containing 50 μg/mL kanamycin and 0.25 μg/mL uridine. Incubate the cultures at 37  C with shaking at 250 rpm for 20 h. 5. Centrifuge the cultures at 1,750
g for 15 min at 4  C. Recover the supernatant and add 1/5 the volume of PEG/NaCl (8 mL). Mix gently and incubate on ice for 1 h to precipitate the phage. 6. Centrifuge at 1,750
g for 15 min at 4  C and discard the supernatant. Resuspend each phage pellet in 0.5 mL of sterile PBS. Centrifuge the solution containing the phage at 5,000
g for 15 min at 4  C to remove any remaining bacteria. Collect the supernatant and filter through a 0.22 μm filter. 7. Extract uracil-containing ssDNA from the phage solution using an E.Z.N.A.® M13 DNA Mini Kit and elute in 100 μL of ultrapure water.

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8. Quantify the DNA by measuring absorbance at 260 nm. Evaluate 1 μL of purified DNA by electrophoresis on a 1% agarose gel (see Note 16). 3.2.2 Mutagenic dsDNA Production

1. Phosphorylate the mutagenic oligonucleotides (see Note 1) by adding the following components to a PCR tube: 2 μL of 10
TM buffer, 2 μL of 10 mM ATP, 1 μL of 100 mM DTT, 11 μL of H2O, 2 μL of T4 polynucleotide kinase (10 U/μL), and 2 μL of 330 ng/L mutagenic oligonucleotide. Incubate the reaction for 1 h at 37  C. 2. Immediately prepare the annealing reaction by combining: 20 μg of purified uracil-containing ssDNA (see Subheading 3.2.1), 20 μL of phosphorylated mutagenic oligonucleotide, 25 μL of 10
TM buffer, and H2O to a final volume of 250 μL. Perform the annealing in a thermal cycler using the following program: 3 min at 90  C, 3 min at 50  C, and 5 min at 20  C. Place the reaction on ice. 3. Prepare the extension reaction by combining: 10 μL of 10 mM ATP, 15 μL of 100 mM DTT, 25 μL of 10 mM dNTPs, 6 μL of T4 DNA ligase (400 U/μL), and 4 μL of T7 DNA polymerase (10 U/μL). Add 60 μL of this reaction to the 250 μL annealing reaction (step 2) and incubate for 20 h at 20  C. Purify the mutagenic dsDNA using a QIAquick PCR Purification Kit and elute the dsDNA in 35 μL of ultrapure water. Quantify the dsDNA by measuring absorbance at 260 nm and evaluate 1 μL of the purified dsDNA by electrophoresis in a 1% agarose gel. 4. Gently mix 80 μg of dsDNA (maximum volume of 200 μL) with 700 μL of electrocompetent TG1 E. coli cells. Divide between four 0.2 cm electroporation cuvettes and electroporate using the conditions described above (see step 5 in Subheading 3.1.5). Immediately recover the electroporated cells by adding 1 mL of 2
YT medium containing 2% glucose to each cuvette, then rinse each cuvette twice with 1 mL of medium. Collect the electroporated cell suspension in a single 50 mL centrifuge tube and incubate for 1 h at 37  C with 250 rpm shaking. 5. To estimate the library size and diversity, prepare serial dilutions of the culture (101 to 1012 in 2
YT-GC medium) and plate 100 μL of the dilutions on 2
YT-GC agar plates. 6. Centrifuge the culture at 1,250
g for 10 min, discard the supernatant, resuspend the pellet gently by pipetting, and plate the cells by dividing equally between four 2
YT-GC agar plates (150 mm). Incubate at 37  C overnight. A bacterial lawn is expected to grow.

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7. To recover the bacteria, rinse each plate three times with 1 mL of 2
YT-GC medium. Keep the cells on ice for later use in the phage library preparation (see Subheading 3.3). 8. To estimate the diversity of the library, randomly select 50–100 colonies from the serial dilutions plates and evaluate them by colony PCR with Ompseq and gback oligonucleotides as described above (see step 15 in Subheading 3.1.5). 9. For clones that show an amplification product of the expected size (~620 bp), inoculate 2 mL cultures of 2
YT-GC medium and grow overnight at 37  C with 250 rpm shaking. The next day, extract plasmid DNA using a QIAprep Spin Miniprep Kit. Sequence the plasmids to evaluate library diversity using both pComb3x oligonucleotides (Ompseq and gback) for bidirectional sequencing (see Note 17). 3.3 Propagation of Phage Particles from VNAR Libraries

1. To propagate phage particles from the immune or synthetic VNAR libraries, use cells recovered from the 150 mm plates (see step 14 in Subheading 3.1.5 for immune libraries and step 7 in Subheading 3.2.2 for synthetic libraries). In a 250 mL flask containing 50 mL of 2
YT-GC medium, inoculate several drops of the cell suspension recovered from the 150 mm plates until an OD600 between 0.05 and 0.1 is reached (usually 50–100 μL of cell suspension). Shake at 250 rpm and 37  C until an OD600 between 0.4 and 0.8 is obtained. Infect the cultures with a 20-fold excess of M13KO7 helper phage per cell (add ~50 μL of phage, 1011 plaque-forming units [pfu]). Incubate at 37  C for 30 min with no agitation. Transfer the infected cultures to a 50 mL centrifuge tube and centrifuge the cells at 1,750
g for 15 min. Discard the supernatant and resuspend the pellet in 300 mL of 2
YT medium supplemented with 100 μg/mL carbenicillin and 50 μg/mL kanamycin. Incubate the cultures at 28  C for 20 h with 250 rpm shaking. 2. Centrifuge at 5,000
g for 20 min at 4  C. Collect the supernatant and add 1/5 the volume of PEG/NaCl (60 mL). Mix gently and incubate on ice for 1 h to precipitate the phage. 3. Centrifuge at 5,000
g for 15 min at 4  C and discard the supernatant. Gently resuspend the phage pellet in a final volume of 20 mL of PBS. Precipitate the phage again by adding 1/5 the volume of PEG/NaCl (4 mL) and incubate on ice for 20 min. Centrifuge at 5,000
g for 15 min at 4  C and discard the supernatant. Resuspend the phage pellet by gently adding 20 mL of PBS. Centrifuge the solution containing the phage at 14,000
g for 15 min at 4  C to remove any remaining bacteria. Collect the supernatant and filter through a 0.22 μM filter.

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4. Add 10 mL of 60% glycerol solution, mix by inversion, and prepare 1 mL aliquots in a 1.5 mL microcentrifuge tube. The phage library can be used immediately for VNAR panning experiments or stored at 80  C for later use. 3.4 In Silico Maturation of VNARs

3.4.1 Homology Modeling

This section describes the in silico affinity maturation of a VNAR via homology modeling using MODELLER software [21] followed by refinement with molecular dynamics simulations using VMD [22] and NAMD [23]. The reader should be aware of the following points before proceeding. First, homology modeling of VNARs is challenging and in many cases approximate due to the limited number of VNAR structures available in public databases and their generally long CDR3 loops whose structures cannot be accurately predicted. Second, antibody-antigen docking, even when high-resolution structures of both proteins are available independently, is only accurate enough for hypothesis generation and is not conclusive. Third, even for high-quality antibody-antigen co-crystal structures, in silico mutagenesis has limited power to predict the impact of substitutions on binding affinity and other parameters. Thus, we recommend that the following procedure be used in exploratory fashion and not as a substitute for validated methods such as epitope mapping and in vitro mutagenesis to enhance affinity. 1. Perform a BLASTP search on the website http://blast.ncbi. nlm.nih.gov/Blast.cgi (see Note 18) to identify potential VNAR templates (solution structures obtained by nuclear magnetic resonance or X-ray diffraction) to be used in homology modeling with MODELLER software. 2. Select three complete sequences with more than 70% identity to the VNAR of interest. 3. Search for structures on the Protein Data Bank (PDB) website (http://www.rcsb.org) using the PDB IDs corresponding to the sequences in the previous step. 4. Install MODELLER software version 9.25 downloaded from http://salilab.org/modeller/download_installation.html (see Note 19). 5. Download the advanced modeling protocol documents provided by MODELLER from http://salilab.org/modeller/ tutorial/advanced.html and place them in the modeler/bin/ qseq1 directory created earlier. 6. Write the query protein sequence to a document with “.ali” extension (see Note 20). 7. Use the shell to run the scripts in order. First, align the templates using the script “salign.py” (see Note 21).

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8. Perform an alignment of the templates with the query sequence using the script “align2d_mulot.py” (see Note 21). 9. For constructing the new model of the query VNAR sequence based on alignment with the multiple templates, use the script “model_mult.py” (see Note 21). 10. Choose the generated 3D model in the modeler/bin/qseq1 folder. 3.4.2 Molecular Dynamics

1. Download and install the NAMD and VMD programs from https://www.ks.uiuc.edu/Research/namd. 2. Create a folder containing all of the topology documents and executables necessary to perform the molecular dynamics simulations (toppar_water_ions_namd.str, par_all36_prot.prm, par_all36m_prot.prm, to_all36_prot.rtf, namd.conf, and the protein structure in PDB format) (see Note 22). 3. Use the shell to prepare the 3D model. Add the CHARM36 force field to the protein structure in the VMD program using the “automatic PSF builder” tool (see Note 23). 4. Add the cubic water box TIP3P in the molecule using the TK console of the VMD program. First use the command “package require solvate,” then add the water box with “Solvate protein. psf portein.pdb - t 15 -o protein_wb.pdb”. 5. The protein coordinates in the water box are obtained using the next set of commands: “set everyone [atomselect top all]”, “measure mixmax $everyone”, and finally “measure center $everyone”. 6. Modify the executable “namd.conf” in the section “Periodic Boundary Conditions” with the previous step’s coordinates. 7. Add ions to the water box with the “Add Ions” tool in the VMD program. This will produce a structure called “protein_wb_ion.psf” and “protein_wb_ion.pdb”. 8. In the shell, use the script “namd.conf” to calculate molecular dynamics. 9. Once the dynamics simulations have been performed, you will obtain various files containing the molecular dynamics results. 10. Load the structures “protein_wb_ion.psf” and “protein_wb_ion.pdb” and the file with a “.dcd” extension in the VMD program. 11. Perform the root mean square deviation (RMSD) calculation for the molecular dynamics simulations using the “RMSD Trajectory Tool.” Perform the alignment and then graph the RMSD of the structure’s backbone. 12. Select the most thermodynamically stable structure using the WMC PhysBio tool and perform clustering. Select the

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Backbone box, select the number of structures (five) and a cutoff of 2.0, and press “calculate.” 13. Select the structure with the longest existence time in the molecular dynamics simulation. 14. Save the structure’s coordinates with the longest existence time in PDB format in file> Save coordinates. 15. Perform structural analysis of the refined model using the online tool PROCHECK (https://servicesn.mbi.ucla.edu/ PROCHECK/) (see Note 24). 3.4.3 Protein–Protein Docking

To evaluate the query VNAR’s potential binding site on its antigen, a protein–protein coupling protocol can be applied using the ClusPro web tool. The global coupling algorithm considers different putative complexes with favorable surface complementarities. The complexes obtained are filtered, selecting those that have good electrostatic energy and are free from solvation for subsequent grouping. The PyMOL Molecular Graphics System is used to visualize and select the best results. 1. For global docking, log into the ClusPro online server at https://cluspro.bu.edu/login.php with your name and password (see Note 25). 2. In the “Dock” tool, load the Receptor (antigen) and Ligand (VNAR) protein structures in PDB format. Also perform docking of the natural ligand if one exists for the antigen of interest. 3. Run the analysis on the server with the default parameters. 4. Select the four most probable interaction models from the results. To identify local protein–protein interaction regions, the “Peptiderive” tool is used in the ROSIE server with the default configuration by the developers. For the coupling calculation, Peptiderive provides a graph with potential protein– protein interaction residues involved in binding, classified according to the Rosetta scoring function in Rosetta Energy Units (REU). 5. For local docking, log onto the ROSIE online server at https://rosie.rosettacommons.org. 6. Use the “Peptiderive” tool to perform local docking. Load the four complexes obtained in step 4 in the ClusPro platform. 7. Perform the molecular dynamics of the four interaction as described above (see Subheading 3.4.2). 8. Select the complex with the lowest interaction energy and the most stable RMSD value (see Note 26).

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Once the results of the natural ligand/antigen complex and the VNAR/antigen complex have been analyzed and compared, attempts can be made to mature the VNAR to improve affinity for antigen. 1. Analyze the binding site between the VNAR and the antigen using PyMOL. Look for the amino acids with the lowest interaction force. 2. Analyze the binding site between the natural ligand and the antigen to identify amino acids present in both the natural ligand and the VNAR CDR3 but that play larger roles in the natural ligand/antigen interaction. 3. Perform mutagenesis of the VNAR using the Wizard ! Mutagenesis tool in PyMOL. Exchange one or two amino acids with a lower interaction score in the VNAR/antigen complex for amino acids with better interaction scores in the ligand/antigen complex. 4. Execute the molecular dynamics procedure (see Subheading 3.4.2) to refine the structure of the modified VNAR. 5. Carry out the procedures for global docking (see steps 1–4 in Subheading 3.4.3) and local docking (see steps 5–8 in Subheading 3.4.3). 6. Once the complex with the lowest interaction energy has been selected, compare the matured VNAR/antigen complex and the original VNAR/antigen complex. 7. Using the amino acid sequences of the matured and original VNARs, infer the DNA-coding sequence of the mature VNAR and synthesize the corresponding gene. Be sure to add a His tag for protein purification. Following cloning into an expression vector and transformation of an appropriate E. coli strain, the affinity-matured VNAR can be purified by immobilized metal affinity chromatography and the affinity measured by surface plasmon resonance.

4

Notes 1. A crucial step in the construction of synthetic libraries is the design of the mutagenic oligonucleotides. These can be designed considering the length of the CDR3 of the VNAR scaffold. However, some studies have shown that it is possible to design more than one mutagenic oligonucleotide in the construction of a synthetic library [24], with different lengths of the CDR3 in a single library. All codons within the CDR3 must encode NNK, where N is an equimolar mixture of A, G, C, and T, and K is an equimolar mixture of G and T

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(the 20 amino acids and the amber stop codon are encoded in the degenerate codon NNK). Leave six to seven triplets before and after the CDR3 for overlap during the alignment reaction. The mutagenic oligonucleotides must not include the three stop codons (not amber) that are included in the framework sequence. The stop codons will be eliminated by the mutagenic oligonucleotides. A successful annealing and extension of the mutagenic oligonucleotide results in the correct display of the protein on the surface of the phage. The mutagenic oligonucleotide, T1-MUT-F, that was utilized with the framework T1 (Fig. 2) in our previous work [9], has the following sequence: 50 GGCACATACTATTGCAAGG CANNKNNKNNKNNKNNKNNKNNKNNKNNKNNKN NKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKN NKTATTATGGGGCCGGCACG-30 . 2. Horn sharks are easy to control when taken out of the water, and they will generally remain immobile during the process. However, some force is necessary in case of sudden movements. This protocol is designed for use with adult specimens (>0.6 m in length). 3. Immediately after removing the syringe from the skin of the shark, use the thumb and press firmly at the injection site for at least 30 s to prevent the expulsion of the antigen-adjuvant solution. Gently spread the injected solution through the muscle massaging with the thumb. 4. Usually, the spleen from an adult horn shark weighs ~5 g. Multiple extractions can be performed. 5. Three phases are formed after centrifugation of BCP-TRI reagent® homogenates: a red phase at the bottom of the tube containing protein and insoluble material (organic phase), a white band in the interphase (DNA), and a colorless phase containing RNA. Avoid disrupting the interphase. 6. Place the samples in the thermal cycler when it reaches 65  C. A complete program consists of 65  C for 5 min, 55  C for 50 min, 85  C for 5 min, and 4  C indefinitely. Including a hold/pause step between the first and second steps of at least 15 min for the addition of the SuperScript™ enzyme mix. 7. Sense oligonucleotides include the SfiI restriction endonuclease site 50 - GGCCCAGGCGGCC -30 and antisense oligonucleotides includes the SfiI restriction endonuclease site 50 - GGCCAGGCCGGCC -30 . Note that these sites differ to avoid self-ligation. If another vector is used, modify the restriction sites as required and ensure the sequences remain in frame. 8. A mix without cDNA can be prepared to evaluate the purity of the reagents and/or specificity of oligonucleotides. No amplification signal should be observed.

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9. Densitometric analyses can be used to corroborate DNA concentrations by comparing band intensities with a DNA ladder (such as Promega 100 bp DNA Ladder) with established masses for each band. 10. An amber suppressor strain is necessary to produce VNARs in fusion with protein pIII of bacteriophage M13. Common names for amber suppression gene are glnV, glnX, SupE, or Su2. 11. Use carbenicillin at a constant concentration of 100 μg/mL. If a different phagemid vector from pComb3x is used, make sure to use the appropriate antibiotic. 12. The following is an example for calculating transformation efficiency. The total volume of recovery media used was 3 mL (3000 μL) and 100 cfu were obtained after plating 100 μL of the 103 dilution. Thus, the library size would be [(100 cfu
3000 μL)/100 μL]
103 ¼ 3
106. 13. When using Ompseq and gback oligonucleotides to amplify a cloned VNAR fragment in pComb3x, a fragment of approximately 620 bp is expected. If a phagemid vector other than pComb3x is used, exchange the Ompseq/gback oligonucleotides for the appropriate oligonucleotides. 14. Factors to consider in the selection of VNAR frameworks include: the presence or absence of cysteines within CDR3, expression yield, stability and solubility, as well as other properties of particular interest of a known VNAR fragment. To improve efficiency during the selection, the VNAR gene used as a framework can be designed to include three stop codons (not amber) just before the CDR3 (Fig. 2). The stop codons are intended to be eliminated with the mutagenic oligonucleotides to avoid display of the original domain. 15. Add 1 μL of helper phage M13KO7 (1011 pfu) per each 1 mL of culture. 16. A predominant single band with a higher electrophoretic mobility represents the desired dU-ssDNA. However, faint bands of a higher molecular weight may be visible since dU-ssDNA can adopt higher order structures. 17. Mutagenesis using NNK degenerate codons does not prevent the appearance of hydrophobic amino acid residues, as well as Cys residues, in positions that do not naturally harbor such residues in CDR3. This may result in the incorrect folding of some domains. However, Kunkel oligonucleotide-directed mutagenesis offers efficiencies that range from 50 to 90% [25], and it is possible to build synthetic libraries of VNARs in which at least 70% are mutagenized using this method [9].

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18. The reference database to perform the BLAST search must be the PDB. 19. Follow all the developer’s instructions for creating a MODELLER account (https://salilab.org/modeller/registration. html). Once the program is installed, create a folder within the modeller/bin directory (e.g., qseq1). 20. Example of the query sequence document: P1; qseq sequence: qseq::::::: 0.00: 0.00. GDCPPWCGARCRLQVGVSVGGLAKWNGLYYC * 21. To perform modeling in MODELLER, you must use the command mod9.25 to run the scripts (example: mod9.25 salign.py). 22. The topology documents to perform the molecular dynamics can be found at: https://www.ks.uiuc.edu. 23. It is recommended to place the VMD and NAMD programs in the directory where all the topology documents are located. 24. The main elements to consider in the results of the model in PROCHECK are the Ramachandran plot and the summary. A good quality model would be expected have over 90% of residues in the most favored regions in the Ramachandran plot. 25. Register for the ClusPro online service. 26. The lower the variation in the RMSD of the interaction complex, the more stable the binding between the antigen and the ligand.

Acknowledgments This chapter is dedicated to the memory of Dr. Jorge Gavilondo. References 1. Roux KH, Greenberg AS, Greene L et al (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins. Proc Natl Acad Sci U S A 95:11804–11809 2. 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 3. 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 4. 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 5. Camacho-Villegas T, Mata-Gonza´lez M, Garcı´a-Ubbelohd W et al (2018) Intraocular penetration of a vNAR: in vivo and in vitro VEGF165 neutralization. Mar Drugs 16:113 6. Goodchild SA, Dooley H, Schoepp RJ et al (2011) Isolation and characterisation of ebolavirus-specific recombinant antibody

Preparation of Immune and Synthetic VNAR Libraries as Sources of High. . . fragments from murine and shark immune libraries. Mol Immunol 48:2027–2037 7. Camacho-Villegas T, Mata-Gonzalez T, Paniagua-Solis J et al (2013) Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark. MAbs 5:80–85 8. 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 ˜ as S, Ayala-Avila M 9. Cabanillas-Bernal O, Duen et al (2019) Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3. PLoS One 14:e0213394 10. 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 17:1–15 11. Burgess SG, Oleksy A, Cavazza T et al (2016) Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain. Open Biol 6:160089 12. 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 13. Dooley H (2003) Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Mol Immunol 40:25–33 14. Shao CY, Secombes CJ, Porter AJ (2007) Rapid isolation of IgNAR variable singledomain antibody fragments from a shark synthetic library. Mol Immunol 44:656–665 15. Zielonka S, Weber N, Becker S et al (2014) Shark attack: high affinity binding proteins

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derived from shark vNAR domains by stepwise in vitro affinity maturation. J Biotechnol 191: 236–245 16. Ko¨nning D, Rhiel L, Empting M et al (2017) Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver antiidiotypic binders. Sci Rep 7:9676 17. Nuttall SD, Humberstone KS, Krishnan UV et al (2004) Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1. Proteins 55:187–197 ˜ as S, Mun ˜ oz PLA et al 18. Milla´n-Go´mez D, Duen (2018) In silico-designed mutations increase variable new-antigen receptor single-domain antibodies for VEGF165 neutralization. Oncotarget 9:28016–28029 19. Kunkel TA (1985) Rapid and efficient sitespecific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492 20. Barbas C, Burton D, Scott J et al (eds) (2001) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press, New York 21. Webb B, Sali A (2014) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinform 47:5.6.1–5.6.32 22. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14:33–38 23. Phillips JC, Braun R, Wang W et al (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26:1781–1802 24. Liu JL, Anderson GP, Delehanty JB et al (2007) Selection of cholera toxin specific IgNAR single-domain antibodies from a naı¨ve shark library. Mol Immunol 44:1775–1783 25. Firnberg E, Ostermeier M (2012) PFunkel: efficient, expansive, user-defined mutagenesis. PLoS One 7:e52031

Chapter 5 Isolation of Single-Domain Antibodies to Transmembrane Proteins Using Magnetized Yeast Cell Targets Kaitlyn Bacon, Stefano Menegatti, and Balaji M. Rao Abstract The isolation of binding ligands from yeast-displayed combinatorial libraries has typically relied on the use of a soluble, recombinantly expressed form of the target protein when performing magnetic selections or fluorescence-activated cell sorting. When identifying binding ligands, appropriate target protein expression and subsequent purification represents a significant bottleneck. As an alternative, we describe the use of target proteins expressed on the surface of magnetized yeast cells in the selection of yeast-displayed nanobody libraries. In this approach, yeast cells displaying the target protein also co-express an iron oxide-binding protein; incubation with iron oxide nanopowder results in magnetization of targetdisplaying cells. Alternatively, target-displaying cells are magnetized by nonspecific adsorption of iron oxide nanopowder. Subsequently, any library cells that interact with the magnetized target cells can be isolated using a magnet. Here, we detail protocols for the isolation of binders to membrane protein targets from a yeast display nanobody library using magnetized yeast cell targets. We provide guidance on how to generate magnetic yeast cell targets as well as library selection conditions to bias the isolation of high affinity binders. We also discuss how to assess the affinity and specificity of the isolated nanobodies using flow cytometry. Key words Yeast magnetization, Yeast surface display, Protein engineering, Library screening, Ligand discovery, Membrane proteins, Nanobody

1

Introduction Recent advances in clinical biomarker discovery have led to an increased demand for engineered ligands to target proteins, specifically overexpressed membrane proteins, for both therapeutic and diagnostic applications [1–4]. To meet this demand, large combinatorial libraries, generated using display platforms like yeast display [5] and phage display [6], are commonly screened using a combination of a magnetic [7] and fluorescence [8]-based selections to identify engineered proteins with novel-binding properties and affinities [9]. Screening strategies have typically utilized a soluble form of the target of interest to identify putative binding

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_5, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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proteins. Consequently, each target must be recombinantly expressed and purified, which is time-consuming, tedious, and not feasible for all proteins. Yeast display has emerged as an alternative strategy for expressing target proteins when screening combinatorial libraries [10]. In the most commonly used yeast surface display platform, a protein of interest is expressed as an N- or C-terminal fusion to the Aga2 subunit of the yeast mating protein a-agglutinin [5, 11]. The Aga2 subunit is connected to the cell-wall-associated Aga1 subunit by disulfide bonds, which tethers the protein of interest to the yeast cell wall. The surface-displayed protein can thus interact with any molecules in solution. This method is particularly powerful for proteins that require a eukaryotic host for appropriate expression [12]. However, yeast surface display may not be an appropriate choice to express all proteins, especially those that require complex post-translational modifications for function, as yeast glycosylation patterns may vary from those of higher order eukaryotes [13, 14]. Despite this limitation, yeast surface display has been used to successfully express the extracellular domains of multiple mammalian membrane proteins and other clinically relevant biomarkers [12, 15–18]. Here, we describe an established method for isolating binding proteins from a yeast display library using yeastdisplayed protein targets. The principal challenge in screening yeast display libraries using yeast-displayed targets is the separation of target-bound library cells from non-binding cells. Unlike the screening of phage display libraries against yeast-displayed targets, centrifugation cannot be used to separate target-bound yeast from non-binding yeast. To efficiently separate binders, we have developed two different routes to magnetize yeast cells expressing the target protein. One route employs the surface display of an engineered protein, SsoFe2, with affinity for iron oxide [10, 19]. Yeast cells that express SsoFe2 are magnetized upon incubation with iron oxide nanopowder. The other route relies on the nonspecific adsorption of iron oxide particles to the yeast surface [20]. After magnetization using either route, the target cells are incubated with the yeast display library of scaffold proteins, such as nanobodies. Any library cell complexed with the target-displaying cells can be isolated using a magnet and expanded for subsequent screening rounds (Fig. 1). The library cells and target cells utilize yeast surface display plasmids with distinct nutritional selection markers allowing for the selective expansion of only the bound library cells. Selection stringency may be increased between screening rounds to bias the isolation of high affinity binders without the need to perform fluorescenceactivated cell sorting. Here, we describe a method to screen a yeast display library of nanobodies using a yeast-displayed target [21]. Binding proteins derived from the nanobody scaffold are attractive for their high

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Fig. 1 General strategy for the isolation of binding proteins from a yeast display nanobody library when performing selections against magnetic yeast cells expressing the target protein. Yeast cells expressing the target protein can be magnetized in two ways: (1) affinity-based interaction between iron oxide nanoparticles and surface displayed SsoFe2, a protein with affinity for iron oxide, or (2) nonspecific adsorption of iron oxide to the yeast cell surface. After magnetization, the target cells are incubated with a yeast display nanobody library. Any library cells that bind the magnetized target cells can be separated from the non-binding library cells using a magnet. The target-bound library cells can be expanded in appropriate media for additional screening rounds

specificity and ease of recombinant expression. Specific recommendations for generating plasmids for the co-expression of a target protein and SsoFe2 as yeast surface fusions are discussed (Fig. 2). Next, we describe detailed procedures for magnetizing target cells utilizing affinity based or nonspecific iron oxide adsorption followed by suggestions for library selections using the magnetized target cells. Lastly, we provide detailed discussion on how to analyze the affinity of a putative binder isolated from the screens by performing yeast surface titrations of the target cells with a recombinantly expressed, soluble form of the putative binder. While the methods presented here consider the screening of a nanobody library using a yeast display platform, magnetic yeast targets can be utilized when screening libraries generated using other display platforms, as well as for the selection of binders derived from other scaffolds. For example, we have utilized magnetic yeast targets to identify cyclic peptide binders from mRNA display libraries [20]. Additionally, we have isolated functional binders from yeast display libraries of Sso7d [10] and cyclic peptide mutants [22] after performing selections against magnetic yeast targets. When considering the isolation of binders to membrane protein targets, the yeast display platform limits display to only the extracellular domain of the membrane protein rather than the full-length protein. Despite this limitation, we have used this

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Fig. 2 Simultaneous display of two proteins on the yeast surface as fusions to Aga2. Here, the target protein and SsoFe2 are both expressed under the control of a GAL1 promoter from a single mRNA transcript using a T2A ribosomal skipping peptide. Expression of each protein can be quantified via immunofluorescent detection of the fused epitope tags

method to isolate binders to a membrane protein target (mitochondrial membrane protein TOM22) that were functional in the context of the natively expressed membrane protein [10]. We expect the use of magnetic yeast targets will become a powerful tool to increase the efficiency of isolating binding proteins to membrane protein targets.

2

Materials Prepare all media and buffers utilizing deionized water at room temperature. Store yeast growth and induction media at 4
C for up to 2 months. Always examine media for contamination prior to use. Other buffers, unless otherwise noted, can be stored at room temperature. Yeast cultures can be saved at 4
C for a few days. However, they should be kept at 80
C for long-term storage.

2.1 Yeast Strains, Plasmids, and Libraries

1. Saccharomyces cerevisiae strain EBY100 [5]. This strain is leucine (Leu) and tryptophan (Trp) deficient and can be transformed with plasmids with appropriate selectable markers. 2. Plasmid pCT302-T2A-EFE2 [10]. This plasmid was constructed from the pCT302 plasmid containing a Leu selectable

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marker for yeast display [23]. Protein expression is under the control of a galactose inducible promoter, GAL1/GAL10. This plasmid affords the co-expression of a protein target of interest and SsoFe2 (iron oxide-binding protein) via a T2A ribosomal skipping peptide. 3. Plasmid pCT302, containing a Leu selectable marker for yeast display [23]. This plasmid affords the expression of a single protein as a yeast surface fusion. 4. A yeast display library of nanobody variants from the Kruse lab [21]. The nanobody variants are encoded within a plasmid harboring a Trp selectable marker. 2.2 Yeast Growth Media and Plates

1. Yeast Peptone Dextrose (YPD) medium (see Note 1): 10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose in an appropriate amount of water is autoclaved. When making plates, add 182 g/L sorbitol and 12 g/L agar prior to autoclaving. 2. SDSCAA medium (see Note 2): 20 g/L dextrose, 6.7 g/L Difco™ yeast nitrogen base without amino acids (Becton Dickinson, Franklin Lakes, NJ, USA), 1.62 g/L synthetic drop-out mix minus leucine without yeast nitrogen base (US Biological, Salem, MA, USA), 5.4 g/L Na2HPO4, and 8.6 g/L NaH2PO4·H2O in an appropriate amount of water. Use a 0.22-μm bottle top filter to sterilize. 3. SDSCAA plates: Autoclave 5.4 g of Na2HPO4, 8.6 g of NaH2PO4·H2O, 182 g of sorbitol, and 12 g of agar dissolved in 900 mL of water. Separately, add 20 g of dextrose, 6.7 g of Difco™ yeast nitrogen base without amino acids, and 1.62 g of synthetic drop-out mix minus leucine without yeast nitrogen base in 100 mL of water. Sterilize with a 0.22-μm filter. After the autoclaved solution is below a temperature of 50
C, add the non-autoclaved, filtered solution (see Note 3). 4. SGSCAA medium (see Note 4): 20 g/L galactose, 2 g/L dextrose, 6.7 g/L Difco™ yeast nitrogen base without amino acids, 1.62 g/L synthetic drop-out mix minus leucine without yeast nitrogen base, 5.4 g/L Na2HPO4, and 8.6 g/L NaH2PO4·H2O in an appropriate amount of water. Use a 0.22-μm bottle top filter to sterilize. 5. Yglc4.5 medium (see Note 5): Dissolve 3.8 g/L synthetic drop-out mix minus tryptophan without yeast nitrogen base (US Biological), 6.7 g/L yeast nitrogen base without amino acids (catalog #M878, HiMedia, Mumbai, India), 10.4 g/L sodium citrate, 7.4 g/L citric acid monohydrate, 20 g/L dextrose, and 100,000 units/L each of penicillin and streptomycin in an appropriate amount of water. Adjust the pH to 4.5 and sterile filter using a 0.22-μm bottle top filter.

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6. SDCAA.NB medium (see Note 6): Dissolve 3.8 g/L synthetic drop-out mix minus tryptophan without yeast nitrogen base, 6.7 g/L yeast nitrogen base without amino acids, 20 g/L dextrose, and 100,000 units/L each of penicillin and streptomycin in an appropriate amount of water. Adjust the pH to 6 and sterile filter using a 0.22-μm bottle top filter. 7. SGCAA.NB medium (see Note 7): Dissolve 3.8 g/L synthetic drop-out mix minus tryptophan without yeast nitrogen base, 6.7 g/L yeast nitrogen base without amino acids, 20 g/L galactose, and 100,000 units/L each of penicillin and streptomycin in an appropriate amount of water. Adjust the pH to 6 and sterile filter using a 0.22-μm bottle top filter. 8. pCT302.FS (see Note 8): 6.7 g/L Difco™ yeast nitrogen base without amino acids and 10% glycerol. Filter-sterilize with a 0.22-μm filter. 9. NB.FS (see Note 9): Yglc4.5 medium containing 10% dimethyl sulfoxide. Sterilize with a 0.22-μm filter. 2.3 Molecular Cloning for Yeast Surface Display

1. Primers for amplification of DNA encoding a protein to be expressed as a yeast surface fusion (see Note 10). 2. Phusion™ High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) or other high-fidelity polymerase. 3. PCR thermocycler. 4. Gel electrophoresis equipment. 5. Restriction enzymes NheI and BamHI-HF (New England Biolabs, Ipswich, MA, USA). 6. Antarctic phosphatase (New England Biolabs). 7. T4 DNA ligase (Promega, Madison, WI, USA). 8. 9K Series Gel and PCR purification kit (BioBasic, Markham, Canada). 9. Electrocompetent Escherichia coli NovaBlue™ cells (SigmaAldrich, St. Louis, MO, USA). 10. Electroporator. 11. Electroporation cuvettes. 12. LB medium: 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. Autoclave. 13. LB plates with carbenicillin: Autoclave 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 12 g/L agar in water. After the autoclaved solution has cooled below 50
C, add sterile-filtered carbenicillin to a final concentration of 100 μg/mL. 14. GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific).

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1. Frozen-EZ Yeast Transformation Kit™ (Zymo Research, Irvine, CA, USA). This kit allows for easy and fast transformation of plasmids into yeast. Other yeast transformation protocols can be utilized, such as the lithium acetate method [24]. 2. Spectrophotometer and cuvettes. 3. Cryo-tubes.

2.5 Surface Expression and Protein-Binding Quantification by Flow Cytometry

1. Spectrophotometer and cuvettes. 2. Chicken anti-c-Myc antibody (catalog #A-21281, Thermo Fisher Scientific). 3. Rabbit-anti-HA antibody (catalog #PA1-985, Thermo Fisher Scientific). 4. Goat-anti-chicken DyLight® 488 (GAC488) secondary antibody (catalog #GtxCk-003-D488NHSX, ImmunoReagents, Raleigh, NC, USA). 5. Donkey-anti-rabbit DyLight® 488 (DAR488) secondary antibody (catalog #DkxRb-003-D488NHSX, ImmunoReagents). 6. Streptavidin R-phycoerythrin conjugate (SA-PE) (catalog #S866, Thermo Fisher Scientific). 7. Bench top flow cytometer. 8. Phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) (0.1% PBSA): Dissolve 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4, and 1 g/L BSA in water. Adjust pH to 7.4. Filter-sterilize with a 0.22-μm bottle top filter. Store at 4
C. 9. Rotator.

2.6 Library Selections Using Magnetic Yeast Targets

1. Spectrophotometer and cuvettes. 2. Iron oxide (catalog# AA4714114, Thermo Fisher Scientific). 3. PBS containing 1% BSA (1% PBSA): Dissolve 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4, and 10 g/L BSA in water. Adjust pH to 7.4. Filter-sterilize with a 0.22-μm bottle top filter. Store at 4
C. 4. Magnets with holders for 1.7/2 mL centrifuge tubes and 15 mL tubes. (e.g., DynaMag™-2 and DynaMag™-15, Thermo Fisher Scientific). 5. PBS containing 1% BSA and 0.05% Tween-20 (1% PBSA + 0.05% Tween): Dissolve 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4, and 10 g/L BSA in water. Add 0.5 mL Tween-20 per L. Adjust pH to 7.4. Filtersterilize with a 0.22-μm bottle top filter. Store at 4
C. 6. Vortex mixer.

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2.7 Plasmid Extraction for Clone Identification

1. Zymoprep™ Yeast Plasmid Miniprep Kit II (Zymo Research). 2. Electrocompetent E. coli NovaBlue™ cells (Sigma-Aldrich). 3. Electroporator. 4. Electroporation cuvettes. 5. LB medium: see Subheading 2.3 for preparation. 6. LB plates with carbenicillin: see Subheading 2.3 for preparation. 7. GeneJET Plasmid Miniprep Kit.

2.8 Molecular Cloning for Recombinant Nanobody Expression

1. Plasmid or synthetic gene block encoding a nanobody of interest. 2. Primers for amplification of nanobody DNA (see Note 11). 3. pET-22b(+) vector (Sigma-Aldrich). 4. NdeI and XhoI restriction enzymes (New England Biolabs). 5. Molecular cloning supplies (see Subheading 2.3).

2.9 Recombinant Nanobody Expression

1. E. coli Rosetta™ cells (Sigma-Aldrich). 2. Mix and Go!™ E.coli Transformation Kit (Zymo Research). 3. Carbenicillin stock solution. 4. Chloramphenicol stock solution. 5. Ampicillin stock solution. 6. LB medium: see Subheading 2.3 for preparation. 7. LB plates with carbenicillin and chloramphenicol: Autoclave 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 12 g/L agar in water. After the autoclaved solution has cooled below 50
C, add sterile-filtered carbenicillin and chloramphenicol to concentrations of 100 μg/mL and 25 μg/mL, respectively. 8. 2  YT medium: Autoclave 16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl in water. 9. Spectrophotometer and cuvettes. 10. Isopropyl β-D-1-thiogalactopyranoside (IPTG). 11. Sonic dismembrator (sonicator). 12. 0.45-μm syringe filter. 13. Fast protein liquid chromatography system. 14. Immobilized metal affinity chromatography (IMAC) column. (e.g., 5 mL Bio-Scale™ Mini-Profinity™ IMAC cartridge, Bio-Rad, Hercules, CA, USA). 15. IMAC buffer A: Dissolve 50 mM Tris and 300 mM NaCl in water. Adjust pH to 8 and sterile filter with a 0.22-μm bottle top filter.

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16. IMAC buffer B: Dissolve 50 mM Tris, 300 mM NaCl, and 500 mM imidazole in water. Adjust pH to 8 and sterile filter with a 0.22-μm bottle top filter. 17. SDS-PAGE equipment such as the XCell SureLock™ MiniCell Electrophoresis System, Nu-PAGE™ 4–12% Bis-Tris Protein Gels, Nu-PAGE™ MES SDS Running Buffer (20), Novex™ Sharp Pre-stained Protein Standard, and Imperial™ Stain (Thermo Fisher Scientific). 18. SnakeSkin™ dialysis tubing (Thermo Fisher Scientific).

with

appropriate

MWCO

19. PBS: Dissolve 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, and 0.24 g/L KH2PO4 in water. Adjust pH to 7.4. Filtersterilize with a 0.22-μm bottle top filter. 20. Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 21. EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific). 22. Vivaspin® sample concentrator with appropriate MWCO (Cytiva, Marlborough, MA, USA).

3

Methods All procedures should be carried out at room temperature, unless otherwise noted. Antibody solutions should always be kept cold.

3.1 Cloning Target Genes of Interest into pCT302-T2A-EFE2 for Affinity-Based Magnetization

The following protocol can be used to introduce DNA encoding the target protein of interest into a plasmid that affords concurrent expression of the target protein and SsoFe2 as yeast surface fusions. This plasmid construct is utilized for the affinity-based magnetization approach. If you wish to isolate binders to a membrane protein target, we recommend using the extracellular domain of the membrane protein as your target for selection. This cloning strategy can also be used to co-express nonspecific proteins on the yeast surface along with SsoFe2 for use in negative selections. 1. Amplify the gene of interest by PCR using Phusion™ HighFidelity DNA polymerase to introduce NheI and BamHI restriction sites at the 50 and 30 ends of the gene, respectively (see Note 10). 2. Double digest 1–4 μg of pCT302-T2A-EFE2 with 20 U of NheI and BamHI for 2 h at 37
C in a 50 μL reaction. Add 1 μL of Antarctic phosphatase and 5 μL of Antarctic phosphatase reaction buffer. Incubate for 1 h at 37
C, followed by heatinactivation at 80
C for 2 min. In parallel, digest 1 μg of the amplified insert with 20 U of NheI and BamHI for 2 h at 37
C in a 50 μL reaction.

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3. Purify the digested vector and insert using a 9K Series Gel and PCR purification kit. 4. Ligate digested insert with digested pCT302-T2A-EFE2 backbone in a 10 μL reaction that contains 1 μL of T4 DNA ligase, 1 μL of ligase buffer, 50 ng of digested backbone, a 5 molar excess of digested insert, and water. Perform the ligation overnight (16 h) at 16
C followed by heat-inactivation at 70
C for 10 min. 5. Transform electrocompetent E. coli NovaBlue™ cells with 10 μL of the ligation reaction using the following electroporation conditions: 1.6 kV, 25 μF, 200 Ω, and a 2 mm electroporation cuvette. Recover transformed cells in 1 mL of LB medium for 1 h in a shaking incubator at 37
C. Plate recovered cells on LB-carbenicillin plates. Grow overnight at 37
C. 6. Harvest DNA of individual colonies from overnight E. coli cultures using a GeneJET Plasmid Miniprep Kit. Send for sequencing to confirm correct insertion. 3.2 Yeast Transformation and Preparation of Yeast Stock Solutions

1. Utilizing the Frozen-EZ Yeast Transformation Kit™, prepare 50 μL aliquots of chemically competent EBY100 yeast cells as detailed in the manufacturer’s instructions. After a slow freeze, aliquots can be stored at 80
C for future use. 2. To transform a plasmid into EBY100 yeast, mix 500 ng of plasmid with 50 μL of competent EBY100 yeast cells. Follow the transformation protocol outlined by the manufacturer of the Frozen-EZ Yeast Transformation Kit™. 3. If transforming a plasmid with a Leu selectable marker, plate the transformed cells on SDSCAA (-Leu) plates. Incubate plates at 30
C until single colonies appear (~2 days). 4. To begin a liquid culture using the transformed cells, inoculate 5 mL of SDSCAA medium with a single colony from the SDSCAA plate. Grow the culture at 30
C with 250 rpm shaking until the yeast reach an optical density (OD) between 5 and 8 (~2 days). 5. To freeze cells grown in SDSCAA, transfer 1 mL of yeast culture into a cryo-tube and centrifuge at 2,500  g for 5 min. Discard the supernatant and resuspend the cells in 1.5 mL of pCT302.FS. 6. To freeze cells grown in SDCAA.NB, transfer the appropriate amount of yeast culture into a cryo-tube and centrifuge at 2,500  g for 5 min. Discard the supernatant and resuspend the cells in NB.FS. When freezing heterogeneous cell populations, like naı¨ve and enriched nanobody library populations, freeze aliquots representing at least a ten-fold excess of the population’s estimated diversity.

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7. Slow freeze the cells for storage at 80
C. Yeast cells can be stored at 80
C for years. 8. When thawing a homogeneous cell population, first streak the cells onto a plate containing appropriate drop-out mix and start a fresh culture using a single colony. Thawed heterogeneous, nanobody library cell populations (naı¨ve and enriched) should not be plated, but rather expanded in fresh SDCAA.NB medium at an OD of 1. 3.3 Flow Cytometry Analysis of Target Proteins Displayed on the Yeast Cell Surface

The following protocol can be used to confirm display of a target protein on the yeast surface through immunofluorescent detection of epitope tags expressed as fusions to the target protein. 1. Grow a 5 mL culture of yeast harboring the pCT302-T2AEFE2 plasmid affording dual display of the target protein and SsoFe2 at 30
C overnight in SDSCAA medium. 2. Measure the OD600 of the culture. Harvest 5  107 cells by centrifugation at 12,000  g for 1 min. Resuspend the cells in 5 mL of SGSCAA medium. Incubate for 16–24 h at 20
C with 250 rpm shaking. 3. Measure the OD600 of the induced culture. Aliquot 2  106 cells for each sample. Prepare an experimental sample, a secondary-only labeling control sample, and an unlabeled control sample. Expression can be quantified by detecting the HA or c-Myc tags expressed as fusions to the displayed target protein. However, c-Myc detection is preferred (see Note 12). 4. Centrifuge for 1 min at 12,000  g. Remove the supernatant. Wash once with 1 mL of 0.1% PBSA. 5. Resuspend the experimental sample cells in 50 μL of a 1:100 dilution of chicken-anti-c-Myc (or rabbit-anti-HA) antibody in 0.1% PBSA. Incubate for 20 min at room temperature on a rotator. 6. Add 1 mL of cold 0.1% PBSA to the cells, and centrifuge for 1 min at 12,000  g. Remove the supernatant. Keep cells on ice after this step. 7. Resuspend the primary labeled experimental cells and the secondary-only control in 50 μL of a 1:250 dilution of GAC488 (or DAR488) in 0.1% PBSA. Place these cells on ice in the dark for 10 min. 8. Add 1 mL of cold 0.1% PBSA to the cells. Centrifuge for 1 min at 12,000  g. Remove the supernatant. Keep samples on ice in the dark until analysis. 9. Prior to analysis, resuspend the cells in 500 μL of 0.1% PBSA. Quantify the fluorescence of at least 50,000 events using the unlabeled cell control to distinguish appropriate gates (see Notes 13 and 14).

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3.4 Recovery of Frozen LibraryYeast Cells

1. Thaw frozen aliquots of the yeast nanobody library at room temperature, such that the number of thawed cells exceeds the expected diversity by at least ten-fold. For example, the naı¨ve nanobody yeast library has a diversity of 5  108 [21], so at least 5  109 cells should be thawed when recovering the naı¨ve library. 2. Prepare cells in Yglc4.5 medium at an OD600 of 1 and recover overnight at 30
C with 250 rpm shaking. 3. After overnight expansion, passage the cells into fresh medium at an OD600 of 1, maintaining the same volume used previously. Expand overnight. Repeat for an additional third expansion.

3.5 Affinity-Based Magnetization of Yeast Cells Displaying Target Protein

The following protocol details how yeast cells harboring SsoFe2 and a target protein can be magnetized for use in the selection of nanobody libraries. The protocol can be scaled linearly to magnetize different cell concentrations as needed. We recommend use of this affinity-based magnetization approach over the subsequently detailed nonspecific adsorption approach as the affinity interaction between SsoFe2 and the iron oxide particles increases the probability that the iron oxide remains bound to the target cells over the course of the selection. This strategy can be used to magnetize cells displaying specific proteins for both negative and positive selections. 1. Grow three 5 mL cultures of yeast harboring the pCT302T2A-EFE2 plasmid encoding the target protein gene overnight at 30
C in SDSCAA medium. Assume the OD after overnight growth will be between 4 and 8. 2. Measure the OD of the cultures. Harvest 5  108 cells by centrifugation at 12,000  g for 1 min. Resuspend the cells in 50 mL of SGSCAA medium. Incubate overnight at 20
C with 250 rpm shaking. 3. Measure the OD of the culture. Aliquot 5  108 cells into a 50 mL sterile conical tube and centrifuge at 12,000  g for 1 min. Discard the supernatant. 4. Resuspend the cells in 10 mL of 1% PBSA. Transfer to a 15 mL sterile conical tube. Centrifuge at 12,000  g for 1 min. Discard the supernatant. 5. Resuspend the cells in 10.5 mL of 1% PBSA. Use 100 μL of this solution to measure the OD600 of the resuspended cells. This is the initial OD. 6. Combine 40 mg of iron oxide with 10 mL of sterilized deionized water.

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7. Shake the iron oxide solution vigorously. Add 2.6 mL of the iron oxide solution to the aliquoted cells. Incubate on a rotator for 20 min at room temperature (see Note 15). 8. Afterwards, place the conical tube on a magnet. Wait 1 min for the iron oxide to move towards the sides of the tube prior to removing the unbound cell fraction. 9. Use 100 μL of the unbound cell fraction to take an OD600. This is the final OD. 10. Calculate the number of cells magnetized (CellsMag) using the following formula: CellsMag ¼ ðVolInitial Þ  ðODInitial Þ  ðVolFinal Þ  ðODFinal Þ

ð1Þ

where VolInitial is the volume of the cells prior to the addition of the iron oxide solution (10.4 mL), ODInitial is the OD600 of the cells prior to the addition of the iron oxide solution, VolFinal is the volume after the addition of the iron oxide solution (13 mL), and ODFinal is the OD600 of the unbound cell fraction. 11. Remove the conical tube from the magnet and add 10 mL of 1% PBSA to the magnetized cell solution. Invert the tube five times to wash the magnetized cells. 12. Place the conical tube back on the magnet. Wait 1 min for the iron oxide to move towards the sides of the tube. Remove the supernatant. Repeat the wash step two more times. Finally, resuspend the washed magnetized cells in 1 mL of 1% PBSA. 13. Calculate the volume of magnetized cell solution to be incubated for the screening round (Screen) using the following formula: Screen ¼

Desired  ð1 mLÞ Magnetized

ð2Þ

where Desired is the number of magnetized cells needed for the screening round and Magnetized is the number of cells actually magnetized in the solution. In the first screening round, aliquot 1  108 magnetized cells for use. 14. Aliquot the appropriate volume of magnetized cell solution for the screening round into a new 10 mL conical tube. Place the tube onto the magnet. Remove the supernatant. Resuspend in 10 mL of fresh 1% PBSA. Incubate for 1 h on a rotator to block any unbound iron oxide. 15. Afterwards, place the tube on the magnet. Remove the supernatant. Add 10 mL of 1% PBSA to the blocked magnetized cell fraction. Invert the tube five times to wash the blocked magnetized cells.

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16. Place the conical tube back on the magnet. Wait 1 min for the iron oxide to move towards the sides of the tube. Remove the supernatant. Repeat wash step two more times. The cells are now prepared for use in screening a nanobody library. 3.6 Magnetization of Target-Displaying Yeast Cells by Nonspecific Surface Adsorption of Iron Oxide

Yeast cells displaying the target protein of interest can also be magnetized via nonspecific adsorption of iron oxide nanoparticles. When utilizing this strategy, the target-displaying yeast does not have to co-express SsoFe2, which can simplify cloning efforts. However, we have observed that a greater number of target cells dissociate from the iron oxide particles when using the nonspecific adsorption method over the course of a selection compared to the affinity-based approach utilizing SsoFe2 display [10]. In addition, iron oxide can adsorb to any yeast surface protein, including the displayed target. If iron oxide complexes with the displayed target protein, then a portion of the target protein may be occluded from interaction with the library binders, thereby influencing the efficiency of the library selection. The suggested magnetization protocol can be scaled linearly to magnetize other cell concentrations. This strategy can be used to generate magnetized target cells for both negative and positive selections. 1. Generate a plasmid that affords the expression of the target protein alone as a yeast surface fusion. Specifically, amplify (see Note 10) and clone the target gene of interest into the pCT302 plasmid harboring a Leu selectable marker between the NheI and BamHI sites. Follow the cloning protocol described above (see Subheading 3.1). 2. Transform competent EBY100 yeast cells with the constructed plasmid as described above (see Subheading 3.2). 3. If desired, expression of the target protein on the surface of yeast cells harboring the pCT302-target protein plasmid can be analyzed utilizing immunofluorescent staining and flow cytometry as described above (see Subheading 3.3). 4. Grow three 5 mL cultures of yeast containing the pCT302 plasmid encoding the target protein gene overnight at 30
C in SDSCAA medium. After overnight growth, assume the OD600 will be between 4 and 8. 5. Measure the culture’s OD600. Harvest 5  108 cells by centrifugation at 12,000  g for 1 min. Resuspend the cells in 50 mL of SGSCAA medium. Incubate overnight at 20
C with 250 rpm shaking. 6. Measure the culture’s OD600. Aliquot 5  108 cells into a 50 mL sterile conical tube followed by centrifugation at 12,000  g for 1 min. Remove the supernatant.

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7. Resuspend the cells in 10 mL of PBS and transfer to a 15 mL sterile conical. Centrifuge at 12,000  g for 1 min. Discard the supernatant (see Note 16). 8. Resuspend the cells in 10.5 mL of PBS. Use 100 μL of this solution to measure the OD600 of the resuspended cells. This is the initial OD. 9. Add 40 mg of iron oxide to 10 mL of sterile deionized water. 10. Mix the iron oxide solution vigorously. Incubate 2.6 mL of the iron oxide solution with the aliquoted cells. Incubate on a rotator for 20 min at room temperature. 11. Place the conical tube on a magnet. Wait 1 min before removing the unbound cell fraction. 12. Use 100 μL of the unbound cell fraction to measure an OD600. This is the final OD. 13. Calculate the number of cells magnetized (CellsMag) using Eq. 1 (see Subheading 3.5). 14. Remove the conical tube from the magnet and add 10 mL of PBS to the magnetized cell solution. Invert the tube five times to wash the magnetized cells. 15. Place the conical tube on the magnet. Wait 1 min before removing the supernatant. Repeat the wash step twice. Resuspend the washed magnetized cells in 1 mL of PBS. 16. Calculate the volume of magnetized cell solution to be incubated for the screening round using Eq. 2 (see Subheading 3.5). In the first screening round, aliquot 1  108 magnetized cells for use. 17. Aliquot the appropriate volume of magnetized cell solution for the screening round into a new 10 mL conical tube. Place the tube onto the magnet. Discard the supernatant. Resuspend the magnetized cells in 10 mL of 1% PBSA. Incubate for 1 h on a rotator to block any unbound iron oxide. 18. Place the tube on a magnet and remove the supernatant. Add 10 mL of 1% PBSA to the blocked magnetized cell fraction. Invert the tube five times to wash the blocked magnetized cells. 19. Place the conical tube on the magnet. Wait 1 min before removing the supernatant. Repeat the wash step twice. The cells are now prepared for use in screening a nanobody library. 3.7 Initial Screening of a Nanobody Yeast Display Library Against a Magnetic Yeast Cell Target

The following details the steps to perform when screening a yeast display library using magnetic yeast cell targets. Cells magnetized utilizing the affinity or nonspecific approach described above can be used interchangeably in the following protocol.

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1. Aliquot 5  109 freshly recovered naı¨ve nanobody library cells. Centrifuge at 3,000  g for 2 min. Remove the supernatant. Resuspend cells in 500 mL of SDCAA.NB medium. Grow overnight at 30
C with 250 rpm shaking. 2. Measure the OD600 of the overnight library culture. Aliquot 5  109 cells and centrifuge at 3,000  g for 2 min. Remove the supernatant and resuspend the cells in 500 mL of SGCAA. NB medium. Incubate the cells overnight at 20
C with 250 rpm shaking. 3. Inoculate a 200 mL EBY100 culture at an OD600 of 1. Grow overnight at 30
C with 250 rpm shaking. 4. Measure the OD600 of the induced culture and the EBY100 cells the following morning. Aliquot 5  109 library cells and 5  109 EBY100 cells into the same tube. Centrifuge at 3,000  g for 2 min. Remove the supernatant (see Note 17). 5. Wash the aliquoted cells with 10 mL of 1% PBSA. Centrifuge at 3,000  g for 2 min and remove the supernatant. Repeat this wash an additional time. 6. Resuspend the washed cells in 10 mL of 1% PBSA. 7. If performing a negative selection, add the washed library/ EBY100 cell population to the non-target magnetized cells (see Note 18). If not, skip to step 11. 8. Incubate for 1 h at room temperature with rotation. 9. After 1 h, place the selection tube onto a magnet. After 1 min, remove the unbound cell fraction into a clean tube. To ensure effective removal of any negative selection magnetic cells, place the separated unbound cell fraction onto the magnet and remove the supernatant after 1 min. Repeat two more times. 10. If performing additional negative selections, add the unbound cell fraction to the next set of non-target magnetized cells. Repeat steps 7–9. If no more negative selections are to be performed, proceed to step 11. 11. Add the unbound cell fraction to the magnetized target cells for positive selection. 12. Incubate at room temperature for 1 h with rotation. 13. After the incubation, place the selection tube onto a magnet. After 1 min, remove the unbound cell fraction to a waste tube. Keep the tube containing the magnetized target cells and any bound library cells. 14. Add 10 mL of 1% PBSA to the isolated magnetic target cells. Invert the tube five times to wash the bound library cells. Place the tube on a magnet. After 1 min, remove the supernatant as waste. Repeat this wash step four times.

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15. Mix the isolated magnetic target cells with 20 mL of SDCAA. NB medium. Expand for 1–2 days at 30
C with 250 rpm shaking. 16. After the cells reach an OD600 of 4–5, remove any iron oxide present in the expanded population using a magnet. Centrifuge the expanded cells separated from the iron oxide at 3,000  g for 2 min. Remove the supernatant, resuspend the cells in 100 mL of fresh SDCAA.NB medium, and grow overnight at 30
C with 250 rpm shaking. These expanded cells can be used for additional screening rounds. 17. Additional rounds of screening are encouraged with greater selection stringency to bias the isolation of higher affinity binders. For example, in subsequent rounds, all selections can take place in 1% PBSA + 0.05% Tween. We suggest incubating 5  108 library cells + 5  109 EBY100 cells with 5  107 magnetized target cells in the second round and 5  107 library cells + 5  109 EBY100 cells with 1  107 magnetized target cells in the third round. The stringency of the wash steps can also be increased by washing with 1% PBSA + 0.05% Tween and vortexing after each step (15 and 30 s, respectively, for rounds 2 and 3) (see Notes 19 and 20). 3.8 Identification of Individual Clones

1. Passage the cells isolated from the final screening round overnight at a low OD600 (~0.2). 2. Isolate plasmid from the passaged population using the Zymoprep™ Yeast Plasmid Miniprep Kit II following the manufacturer’s instructions. 3. Transform the isolated plasmid into electrocompetent E. coli NovaBlue™ cells using the following electroporation conditions: 1.6 kV, 25 μF, 200 Ω, and a 2 mm electroporation cuvette. Recover transformed cells in 1 mL of LB for 1 h at 37
C with 250 rpm shaking. Plate recovered cells on an LB-carbenicillin plate. Grow overnight at 37
C. 4. Harvest DNA of individual colonies from overnight E. coli cultures using a GeneJET plasmid miniprep kit. Send for sequencing to confirm nanobody sequences (see Notes 21 and 22).

3.9 Cloning Individual Nanobodies into pET-22b(+) Vector for Recombinant Expression

1. Amplify the nanobody DNA by PCR using Phusion™ HighFidelity DNA polymerase and an isolated plasmid or a synthetic gene block as template to introduce NdeI and XhoI restriction sites at the 50 and 30 ends of the DNA, respectively (see Note 11). 2. Digest 1–4 μg of pET-22b(+) with 20 U of NdeI and XhoI for 2 h at 37
C in a 50 μL reaction followed by Antarctic phosphatase treatment as described above (see Subheading 3.1). In

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parallel, digest 1 μg of insert with 20 U of NdeI and XhoI for 2 h at 37
C in a 50 μL reaction. 3. Clean up the digested backbone and insert using a 9K Series Gel and PCR purification kit. 4. Ligate the digested insert with digested pET-22b(+) backbone in a 10 μL reaction as described above (see Subheading 3.1). 5. Transform electrocompetent E. coli NovaBlue™ cells with 10 μL of the ligation reaction described above (see Subheading 3.1). 6. Harvest DNA of individual colonies from overnight E. coli cultures using a GeneJET Plasmid Miniprep Kit. Send for sequencing to confirm correct insertion. 3.10 Recombinant Expression and Purification of Individual Nanobodies

1. Transform chemically competent E. coli Rosetta cells prepared using the Mix and Go!™ E.coli Transformation Kit with pET-22b(+) plasmids encoding individual nanobodies. Follow the manufacturer’s recommendations. Plate the transformed cells on LB plates containing carbenicillin and chloramphenicol. Grow overnight at 37
C. 2. Inoculate 5 mL of LB medium with a single colony from the plate. Add ampicillin and chloramphenicol at a concentration of 100 μg/mL and 25 μg/mL, respectively. Grow overnight at 37
C with 250 rpm shaking. 3. Inoculate 1 L of 2  YT medium with the overnight 5 mL culture. Add ampicillin and chloramphenicol at a concentration of 100 μg/mL and 25 μg/mL, respectively. Grow at 37
C with 250 rpm shaking until the OD600 reaches between 0.6 and 0.8 (~5 h). 4. Induce protein expression using 0.5 mM IPTG. Incubate overnight at 20
C with 250 rpm shaking (~16 h). 5. Centrifuge the induced cells for 12 min at 3,000  g. Remove the supernatant. 6. Resuspend the cells in 35 mL of IMAC buffer A. 7. Sonicate the cells on ice. Pulse for 10 s followed by a 10 s pause for 6 min in total. 8. Centrifuge the lysed cells at 15,000  g for 22 min. 9. Filter-sterilize the supernatant with a 0.45-μm syringe filter. 10. Load the filtered supernatant onto a pre-equilibrated IMAC column. Wash with 40 mL of IMAC buffer A followed by elution with a 40 mL linear gradient of IMAC buffer B. Perform all steps at a flow rate of 2 mL/min. 11. Analyze fractions via SDS-PAGE. Pool fractions containing the protein of interest.

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12. Dialyze pure fractions into PBS or another appropriate buffer to remove imidazole using SnakeSkin™ Dialysis Tubing. 13. Determine protein concentration using a Pierce™ BCA Protein Assay Kit following the manufacturer’s instructions. 14. Biotinylate protein using a 5:1 molar excess of EZ-Link™ Sulfo-NHS-LC-Biotin overnight at 4
C following the manufacturer’s recommendations (see Note 23). 15. Dialyze biotinylated protein against 50 mM Tris-HCl, pH 7.5, containing 300 mM NaCl to remove unreacted biotin. 16. Optional: concentrate the protein using a Vivaspin® sample concentrator following the manufacturer’s recommendations. 17. Determine protein concentration using a Pierce™ BCA Protein Assay Kit following the manufacturer’s instructions. 3.11 NanobodyBinding Affinity Estimation by Yeast Surface Titration

The following protocol can be used to estimate the binding affinity of an individual nanobody for a target protein via yeast surface titration using a previously outlined method [11]. The binding protein is expressed in a soluble form while the target protein is displayed as a yeast surface fusion. 1. If selections were performed against cells expressing the target protein and SsoFe2, then clone the target gene of interest into the pCT302 plasmid harboring a Leu selectable marker between the NheI and BamHI sites (see Subheading 3.1). Transform competent EBY100 yeast cells with the constructed plasmid following the methods described above (see Subheading 3.2). 2. Grow a 5 mL culture of yeast harboring the pCT302 plasmid affording the display of the target protein of interest alone. 3. Measure the OD600 of the culture. Harvest 5  107 cells by centrifugation at 12,000  g for 1 min. Resuspend the cells in 5 mL of SGSCAA medium. Incubate for 16–24 h at 20
C with 250 rpm shaking. 4. Measure the OD600 of the induced culture. Pellet 2  106 cells per sample at 12,000  g for 1 min. Prepare at least 10–12 experimental samples that will be labeled with varying concentrations of the binding nanobody, spanning at least two orders of magnitude below and above the expected KD. Also, prepare a secondary-only control sample and an unlabeled control sample (see Note 24). 5. Remove the supernatant from each sample. Wash the cell pellet once with 1 mL of 0.1% PBSA. 6. Prepare different dilutions of the soluble nanobody in 0.1% PBSA (50 μL volume). Resuspend each experimental sample in

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a different dilution. Incubate for 30 min at room temperature on a rotator (see Note 25). 7. Add 1 mL of cold 0.1% PBSA and centrifuge for 1 min at 12,000  g. Remove the supernatant. Keep cells on ice after this step. 8. Resuspend the experimental samples and the secondary reagent control sample in 50 μL of a 1:250 dilution of SA-PE in 0.1% PBSA. Place these cells on ice in the dark for 10 min. 9. Add 1 mL of cold 0.1% PBSA to the cells. Centrifuge for 1 min at 12,000  g. Remove the supernatant. Keep samples on ice in the dark until analysis. 10. Prior to analysis, resuspend the cells in 500 μL of 0.1% PBSA. Quantify the fluorescence of at least 150,000 events (see Note 26). 11. Fit an appropriate gate, using the unlabeled cell population, to the raw flow cytometry data. Process the data to obtain the mean fluorescence value corresponding to the interaction of the nanobody (SA-PE) for all experimental samples and SA-PE alone for the secondary-only reagent control. Subtract the mean fluorescence of the secondary-only control sample from each experimental sample’s mean fluorescence value. Steps 2–11 should be repeated two additional times to obtain three independent sets of binding data in total. Using a global nonlinear least squares regression, fit the data across the three repeats to the equation: F ¼

F Max ½L 0 , K D þ ½L 0

where F is background subtracted mean fluorescence value of the nanobody interaction, FMax is the background subtracted mean fluorescence value when nanobody binding reaches saturation, [L]0 is the concentration of nanobody incubated, and KD is the equilibrium dissociation constant describing the interaction between the nanobody and the target protein. The fitted parameters are a single KD across all replicates and a different FMax value for each repeat.

4

Notes 1. YPD medium is non-selective and is utilized for culturing EBY100 cells. 2. SDSCAA medium lacks leucine and is used for the selection of yeast cells carrying the pCT302 plasmid containing a Leu selectable marker.

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3. Yeast nitrogen base and the drop-out mix are heat sensitive. They should only be added to the autoclaved media once it is cooler than 50
C. 4. SGSCAA medium lacks leucine and is used to induce protein expression. 5. Yglc4.5 medium is used for recovering the nanobody library cells from freezer stocks. 6. SDCAA.NB medium lacks tryptophan and is used for passaging the nanobody library cells. 7. SGCAA.NB medium lacks tryptophan and is used for inducing protein expression of the nanobody library cells. 8. pCT302.FS is a freezing solution utilized for the long-term storage of yeast cells harboring the pCT302 plasmid at 80
C. 9. NB.FS is a freezing solution utilized for the long-term storage of the nanobody library cells at 80
C. 10. An example set of primers for PCR amplification of a target protein of interest is provided here. Forward Primer: 50 -AAAAAA GCTAGC NNN NNN NNN NNN NNN NNN-30 ; Reverse Primer: 50 -TTTTTT GGATCC NNN NNN NNN NNN NNN NNN-30 . In this example, NNN anneals to the coding sequence of the target protein (without leader peptide) to be expressed as a yeast surface fusion. When considering membrane proteins, we recommend expressing the extracellular domains only. Design primers to amplify from the first to the last codon of the protein of interest. Do not amplify leader peptides. Design primers to have an annealing temperature between 58 and 62
C. Do not include a start or stop codon within the primer sequences. These primers can be used when introducing the gene of interest between the NheI and BamHI sites of both pCT302-T2A-EFE2 and pCT302. In both plasmids, a start codon precedes the N-terminal prepro sequence while a stop codon is encoded downstream of the BamHI site. 11. An example set of primers for PCR amplification of a nanobody of interest is provided here with restriction enzyme sites in underline bold and nanobody coding region in italics. Forward Primer: 50 -GTCTCG CATATG GCACAAGTTCAGCTTGTAGAGT-30 ; Reverse Primer: 50 -GTCACT CTCGAG CGATGATACAGTTACTTGGGTAC -30 . The nanobody-specific sequences can be modified depending on your application. Note that in the forward primer, the GCA codon encoding alanine immediately following the NdeI site was an artifact of the template nanobody used in this example; most nanobodies will not contain this residue. Design primers to have an annealing temperature between 58 and 62
C. Do not include a start or stop codon within the primer sequences. The NdeI

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restriction site contains a start codon while a stop codon is encoded downstream of the C-terminal His tag present in the pET-22b(+) backbone. 12. We recommend quantifying target protein expression via the c-Myc tag. Positive detection of the c-Myc tag assesses complete translation of the expressed target protein. 13. The yeast can also be labeled with an antibody against the expressed target protein in a similar manner using appropriate controls and gates. 14. Expression of the SsoFe2 protein used for magnetization can also be quantified in a similar manner using an anti-Flag or anti-V5 antibody. The SsoFe2 protein is fused to a Flag tag at its N-terminus and a V5 tag at its C-terminus. We recommend using anti-V5 antibody (Thermo Fisher Scientific, catalog# R960-25) and donkey-anti-mouse DyLight® 633 secondary antibody (ImmunoReagents, catalog# DkxMu-003D633NHSX). 15. The iron oxide will settle over time. It is important to shake the tube vigorously prior to transferring the iron oxide solution to the cells to ensure the iron oxide is suspended throughout. 16. We have observed that yeast cells displaying certain proteins may magnetize better in different pH buffers when utilizing nonspecific iron oxide adsorption. If the target cells do not magnetize in PBS, pH 7.4, then try to utilize a different buffer with varying pH. For example, if the isoelectric point of the displayed target protein is ~4, then utilize a buffer with pH ~5 for magnetization as this will allow the iron oxide particles and the displayed target protein to have opposite charges. The isoelectric point of iron oxide is ~7 [25]. 17. Excess non-displaying EBY100 cells are added to reduce the isolation of nonspecific binders due to interactions between yeast surface proteins. 18. When performing a negative selection utilizing affinity-based magnetization, cells displaying only SsoFe2 or cells displaying SsoFe2 and a nonspecific protein can be used. When utilizing nonspecific magnetization, we suggest performing negative selections against cells displaying a nonspecific protein target. It is important to include a negative selection step to remove any library members that interact with yeast surface proteins or tags included in the yeast surface display construct. Negative selections can also be used to ensure the isolation of nanobodies with selectivity for the target over other proteins. 19. Beyond the first few rounds, negative selections can continue to be performed using non-target magnetized cells. As an alternative, negative selections can also be performed by

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incubating cells displaying non-target proteins (no SsoFe2, Leu nutritional marker) with the library cells and magnetized target cells. If this approach is taken, we suggest replacing the EBY100 non-expressing yeast with the yeast displaying the non-target protein. 20. After the third round of screening, we suggest testing individual nanobodies for their binding characteristics. If superior affinities are desired after this initial analysis, additional rounds of screening can be performed with greater selection stringency (e.g., include a soluble competitor protein, increase the concentration of non-target-displaying yeast cells, perform selections in the presence of fetal bovine serum, or increase incubation times for wash/vortex steps). If the Aga1-Aga2 yeast surface display platform is used, then affinity discrimination can be enhanced by incubating the library cells with dithiothreitol to reduce the surface display level of each library member [26]. A lower surface density may favor the isolation of higher affinity nanobodies by reducing the avidity between library-yeast and target-yeast. Lastly, a mutagenized library can be generated via error prone PCR and screened in a similar manner to isolate higher affinity binders. 21. The isolated plasmid population can also be amplified via PCR and interrogated using next-generation DNA-sequencing platforms. This method allows identification of the entire recovered population. However, when utilizing this method, you will not isolate plasmid encoding each individual nanobody and may need to purchase synthetic gene blocks if you wish to perform additional analysis of individual clones. 22. We typically sequence 10–20 individual colonies if not performing next-generation DNA sequencing. 23. While the expressed protein contains a His tag, we recommend biotinylating the protein for subsequent analysis as we have had difficulties analyzing protein binding using immunofluorescent His tag detection. 24. Titration experiments to determine binding affinity are typically performed in triplicate. After the first titration, the concentrations used for labeling can be adjusted to span an appropriate range around the expected KD. 25. For low concentrations of nanobodies, ensure that the nanobody concentration exceeds the effective concentration of the target protein in solution by ten-fold. For example, in a volume of 50 μL, 2  106 cells displaying approximately 50,000 copies of target protein will have an effective concentration of (2  106 cells/0.00005 L)  (5  104 target protein copies/ cell)  (1 mol/6.022  1023 copies) ¼ 3.3 nM. Accordingly,

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labeling using nanobody concentrations lower than 33 nM should be performed in larger volumes. 26. A similar immunofluorescence-based procedure can be used to determine the selectivity of the isolated nanobodies for the target protein over other proteins of interest. Complete titration curves do not need to be developed when analyzing specificity. Binding at a few selected concentrations can be compared. To perform selectivity analysis, nonspecific proteins can be displayed as yeast surface fusions. Interactions of the nanobody with yeast cells displaying nonspecific proteins can be quantified via immunofluorescence, as described, for selected nanobody concentrations.

Acknowledgments This work was funded by a National Science Foundation Grant (CBET 1511227). KB kindly acknowledges support from an NSF Graduate Research Fellowship and a National Institute of Health Molecular Biotechnology Training Fellowship (NIH T32 GM008776). References 1. Tainsky MA (2009) Genomic and proteomic biomarkers for cancer: a multitude of opportunities. Biochim Biophys Acta 1796:176–193 2. Boschetti E, D’Amato A, Candiano G et al (2018) Protein biomarkers for early detection of diseases: the decisive contribution of combinatorial peptide ligand libraries. J Proteome 188:1–14 3. Crutchfield CA, Thomas SN, Sokoll LJ et al (2016) Advances in mass spectrometry-based clinical biomarker discovery. Clin Proteomics 13:1 4. Josic D, Clifton JG, Kovac S et al (2008) Membrane proteins as diagnostic biomarkers and targets for new therapies. Curr Opin Mol Ther 10:116–123 5. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557 6. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317 7. Ackerman M, Levary D, Tobon G et al (2009) Highly avid magnetic bead capture: an efficient selection method for de novo protein engineering utilizing yeast surface display. Biotechnol Prog 25:774–783

8. VanAntwerp JJ, Wittrup KD (2000) Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol Prog 16:31–37 9. Banta S, Dooley K, Shur O (2013) Replacing antibodies: engineering new binding proteins. Annu Rev Biomed Eng 15:93–113 10. Bacon K, Burroughs M, Blain A et al (2019) Screening yeast display libraries against magnetized yeast cell targets enables efficient isolation of membrane protein binders. ACS Comb Sci 21:817–832 11. Gera N, Hussain M, Rao BM (2013) Protein selection using yeast surface display. Methods 60:15–26 12. Wadle A, Mischo A, Imig J et al (2005) Serological identification of breast cancer-related antigens from a Saccharomyces cerevisiae surface display library. Int J Cancer 117:104–113 13. Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3:119–128 14. Gupta SK, Shukla P (2018) Glycosylation control technologies for recombinant therapeutic proteins. Appl Microbiol Biotechnol 102: 10457–10468 15. Zhou Y, Zou H, Zhang S et al (2010) Internalizing cancer antibodies from phage libraries

Isolation of Single-Domain Antibodies to Transmembrane Proteins Using. . . selected on tumor cells and yeast-displayed tumor antigens. J Mol Biol 404:88–99 16. Zhao L, Qu L, Zhou J et al (2014) High throughput identification of monoclonal antibodies to membrane bound and secreted proteins using yeast and phage display. PLoS One 9:e111339 17. Cochran JR, Kim YS, Olsen MJ et al (2004) Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments. J Immunol Methods 287:147–158 18. Johns TG, Adams TE, Cochran JR et al (2004) Identification of the epitope for the epidermal growth factor receptor-specific monoclonal antibody 806 reveals that it preferentially recognizes an untethered form of the receptor. J Biol Chem 279:30375–30384 19. Cruz-Teran CA, Bacon K, McArthur N et al (2018) An engineered Sso7d variant enables efficient magnetization of yeast cells. ACS Comb Sci 20:579–584 20. Bacon K, Bowen J, Reese H et al (2020) Use of target-displaying magnetized yeast in screening mRNA-display peptide libraries to identify ligands. ACS Comb Sci 22:738–744

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21. McMahon C, Baier AS, Pascolutti R et al (2018) Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol 25:289–296 22. Bacon K, Blain A, Burroughs M et al (2020) Isolation of chemically cyclized peptide binders using yeast surface display. ACS Comb Sci 22: 519–532 23. Midelfort KS, Hernandez HH, Lippow SM et al (2004) Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody. J Mol Biol 343: 685–701 24. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34 25. Schwegmann H, Feitz AJ, Frimmel FH (2010) Influence of the zeta potential on the sorption and toxicity of iron oxide nanoparticles on S. cerevisiae and E. coli. J Colloid Interface Sci 347:43–48 26. Stern LA, Csizmar CM, Woldring DR et al (2017) Titratable avidity reduction enhances affinity discrimination in mammalian cellular selections of yeast-displayed ligands. ACS Comb Sci 19:315–323

Chapter 6 A Transgenic Heavy Chain IgG Mouse Platform as a Source of High Affinity Fully Human Single-Domain Antibodies for Therapeutic Applications Dubravka Drabek, Rick Janssens, Rien van Haperen, and Frank Grosveld Abstract The antibody repertoires of transgenic mice expressing human heavy chain only antibodies (HCAbs) can be retrieved from immune cells after antigen challenge. Compared with genetically modified rodents expressing conventional human antibodies (tetramers consisting of two heavy chains paired with two light chains), there is no chain pairing problem, since each antibody consists of a heavy chain dimer which is solely responsible for antigen binding. HCAbs can be obtained by classical hybridoma fusion, or the generation of phage libraries or eukaryotic cell libraries displaying or secreting HCAbs. Combined transcriptomic/serum proteomic approaches can also be used to determine the repertoire of antibodies, as well as single cell technologies such as the Beacon system that enable capture of immune cells of interest, analysis, and sequencing of antibodies in a short period of time. Here, we describe a protocol for obtaining monoclonal HCAbs from immunized Harbour transgenic mice through the generation and screening of HEK cell libraries of secreted antibodies. The method can be used routinely and is fast and affordable for everyone. Selected VH regions (single domains) are sequenced and individual HCAbs can be produced and purified from the same expression vector that is used for library generation (hIgG1 Fc). They can also be cloned into other expression plasmids and reformatted to equip them with a particular effector function, modify lifespan in serum, or optimize valency and avidity depending on the specific aim. Key words Transgenic mouse, Immunization, Heavy chain only antibody (HCAb), HCAb libraries, VH, HEK cells

1

Introduction In addition to conventional tetrameric antibodies, camelids produce functional heavy chain only antibodies (HCAbs). These are encoded by a distinct set of IGHVsegments (IGHVH segments). HCAbs lack the entire CH1 domain of the heavy chain constant region and possess conserved hydrophilic amino acid substitutions in the region that normally participates in the hydrophobic interaction with the light chain in conventional antibodies [1]. Since mice are smaller, easier, and more economical to keep and handle than

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_6, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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camelids, we initially generated transgenic mice containing a hybrid llama/human heavy chain immunoglobulin locus (containing two unrearranged llama VHH regions, human D and J regions, and human constant regions) as a proof of principle [2, 3] to demonstrate that transgenic mice can go through the entire B cell development process and generate functional HCAbs. This was followed by the generation of a series of transgenic mice containing different numbers of human VH regions that produce functional, soluble, high affinity, fully human HCAbs [4, 5]. All mouse immunoglobulin loci were deleted to prevent any expression of endogenous mouse immunoglobulins. While camelid VHH regions might cause an undesirable immune reaction when administered in humans, human VHs present a safer therapeutic option. Their small size and short half-life are attractive for diagnostic purposes while the ease of engineering them into multi-specific agents makes them extremely appealing for the development of therapeutics. In this chapter, we describe the procedures involved in obtaining antigen-specific HCAbs from the 9VH3 Harbour transgenic mouse containing nine human VH regions (Fig. 1). The process starts with the immunization. It is followed by monitoring antigenspecific titers during the immunization process and the selection of animals for library generation. Selected animals are sacrificed and their lymphoid organs collected. Antigen-specific B cells and plasma cells are used for purification of total RNA, followed by reverse transcription/cDNA synthesis and the amplification of human VH regions. The VHs are ligated into a mammalian expression vector followed by transformation of Escherichia coli competent cells. Single colonies are picked and grown in 96-well plates. Individual DNA plasmids are extracted and used to transiently transfect HEK cells in the same format. Supernatants are tested for antigen binding using ELISA. The DNA corresponding to selected positive clones is sequenced and medium-scale production is performed by transient transfection of FreeStyle 293-F cells with the same DNA plasmid. HCAbs are purified using protein A affinity columns. Individual HCAbs are used for further functional characterization, affinity measurements, and epitope binning experiments that are outside the scope of this chapter. If desired, one or multiple VHs can be engineered into multivalent, multi-specific molecules.

2

Materials

2.1 Immunization and Testing of Antigen-Specific Titers

1. Insulin syringe with needle. 2. Antigen (see Note 1). 3. Adjuvants: incomplete Freund’s adjuvant (IFA) and Ribi (see Note 2). 4. Ethylenediaminetetraacetic acid (EDTA)-coated microfuge tubes.

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Fig. 1 Schematic representation of the workflow and expression vector for HCAb recovery from Harbour transgenic 9VH3 mice. The pCAG hygro hIgG1 plasmid contains the leader sequence from human VH3–23 as a secretion signal, PvuII and BstEII cloning sites for insertion of VHs, human IgG1 hinge, CH2, and CH3 domains, and a stop codon. The vector contains a hygromycin (HygR) resistance marker for eukaryotic cell expression/selection and an ampicillin (AmpR) resistance gene for bacterial selection. We have produced pCAG hygro variants encoding human IgG2, IgG3, and IgG4 isotypes

5. Sterile lancet. 6. Microcentrifuge. 7. PBS. 8. Nunc™ MaxiSorp® 96-well ELISA plates.

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9. PBS containing 1% BSA, 1% fat-free milk powder, and 0.1% Tween-20. 10. PBS containing 0.1% Tween-20. 11. Horseradish peroxidase (HRP)-conjugated rabbit polyclonal anti-mouse IgG (Dako/Agilent, Santa Clara, CA, USA). 12. BM Blue POD tetramethylbenzidine substrate. 13. 1 M H2SO4. 14. ELISA plate reader. 2.2 Dissection, Collection of Lymphoid Organs, and Isolation of Immune Cells

1. Isoflurane, Evans blue solution (500 mg in 10 mL PBS), and insulin syringe with needle. 2. Scissors and fine forceps (Dumont 5). 3. Light microscope. 4. PBS containing 0.5% BSA and 2 mM EDTA. 5. 23-gauge needle attached to 5 mL syringe. 6. Cell strainer (40 μm nylon filter). 7. 2 mL syringe with plunger. 8. Plastics (3 cm Petri dishes or 6-well plates; 50 mL conical tubes; 15 mL conical tubes; microfuge tubes). 9. CD138+ Plasma Cell Isolation Kit, Mouse (Miltenyi Biotec, Bergisch Gladbach, Germany). 10. MACS magnet stand, columns (LD and MS), and separators. 11. Biotinylated antigen (see Note 3). 12. EZ-Link™ NHS-PEG12-Biotin (Thermo Fisher Scientific, Waltham, MA, USA). 13. Dimethyl sulfoxide. 14. Dynabeads™ M-280 streptavidin (Invitrogen, Carlsbad, CA, USA). 15. NanoDrop spectrophotometer. 16. Magnetic stand for microfuge tubes. 17. Automated cell counter. 18. Amicon centrifugal filtration device with appropriate MW cutoff for antigen of interest. 19. PBS containing 0.5% BSA. 20. Rotating wheel.

2.3

RNA Isolation

1. TRI® Reagent (Sigma-Aldrich, St. Louis, MO, USA). 2. Chloroform. 3. Isopropanol. 4. 75% (v/v) ethanol.

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5. Distilled water (dH2O). 6. NanoDrop spectrophotometer. 2.4 cDNA Synthesis and Amplification of VHs

1. Oligo(dT)12–18 primer (500 μg/mL). 2. dNTP Mix (10 mM each). 3. dH2O. 4. 0.1 M dithiothreitol. 5. RNaseOUT™ (40 units/μL). 6. SuperScript™ II reverse transcriptase with 5
first-strand buffer (Invitrogen). 7. Phusion® High Fidelity DNA Polymerase (NEB, Ipswich, MA, USA). 8. Primer mIgG1 rev GACGGTGACC-30 ).

(50 - AATCCCTGGGCACTGAAGA

9. Primer Lib 3–23 for (50 - GTGTCCAGTGTGAGGTG CAGCTG-30 ). 10. Primer Lib 3–11 for (50 - GTGTCCAGTGTCAGGTG CAGCTG-30 ). 2.5 Generation and Screening of HCAb Libraries

1. NanoDrop spectrophotometer. 2. Agarose. 3. Ethidium bromide (5 mg/mL). 4. 10
TBE buffer: dissolve 1090 g Tris, 556 g borate, and 93 g EDTA in a final volume of 10 L dH2O. 5. Gel tray, combs, electrophoresis tank, and power supply. 6. Long wavelength UV box. 7. Sharp sterile razor blade. 8. NucleoSpin® Extract II PCR Purification and Gel Extraction kit (Macherey-Nagel, Du¨ren, Germany). 9. Phosphatase-treated, PvuII/BstEII-digested pCAG hygro hIgG1 vector (Fig. 1). 10. PvuII HF restriction enzyme and 10
CutSmart buffer (NEB). 11. BstEII HF restriction enzyme and 10
CutSmart buffer (NEB). 12. T4 DNA ligase and buffer (Promega, Madison, WI, USA). 13. Analytical balance.

2.6 Transformation of Bacteria

1. MegaX DH10B T1R Electrocomp™ cells (Invitrogen). 2. Lysogeny broth (LB).

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3. Ampicillin stock: 0.1 mg/mL in dH2O (2000
), filter sterilized. 4. 10 cm LB agar plates containing ampicillin: autoclave 500 mL of LB containing 8 g agar. When cooled but still liquid, add 250 μL of ampicillin stock and pour into 10 cm Petri dishes. 5. Terrific broth (TB): add 20 g tryptone, 24 g yeast extract, and 4 mL glycerol to 900 mL of dH2O. Stir until dissolved and sterilize by autoclaving. Allow the solution to cool to ~60  C and add 100 mL of sterile phosphate buffer (0.17 M KH2PO4, 0.72 M K2HPO4). 6. Nunc™ 96-well polypropylene storage microplates (for growing bacteria and DNA storage). 7. TE buffer: 15 mM Tris, 10 mM EDTA. 8. DNase-free RNase stock (10 mg/mL). 9. TE/RNase buffer: add 2 mL of RNAse stock to 148 mL of TE buffer. This amount should be sufficient for 2,000 wells. 10. 1% (w/v) SDS, 0.2 M NaOH. 11. 3 M potassium acetate (CH3CO2K), pH 5.5. 12. Isopropanol. 13. 70% ethanol. 2.7 HEK Cell Libraries

1. Centrifuge with adaptor to accommodate 96-well plates. 2. Multichannel pipettes and sterile tips. 3. 100
non-essential amino acids. 4. Penicillin/streptomycin solution. 5. DMEM supplemented with 10% fetal calf serum, non-essential amino acids, penicillin, and streptomycin. 6. Flat-bottom 96-well plates. 7. Round-bottom 96-well plates. 8. Lipofectamine™ 2000 (Invitrogen). 9. OptiMEM™ medium (Thermo Fisher).

with

GlutaMAX™

supplement

10. 384-well ELISA plates. 11. Items for ELISA (see Subheading 2.1 items 7, 9, 10, 12, 13, and 14). 12. HRP-conjugated anti-human IgG1 antibody (clone KT46; Absea, Beijing, China). 2.8 Sequencing of VHs

1. Sequencing primer for VH3–23 leader (50 - TACACCGGTC CACCATGGAGTT-30 ).

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1. DNA plasmid-encoding HCAb (~200 μg). 2. Disposable plastic 250 mL Erlenmeyer flasks with filter top. 3. FreeStyle 293-F cells (Thermo Fisher). 4. FreeStyle F17 medium supplemented with GlutaMAX™ and Pluronic F-68 (Thermo Fisher; both supplements 1:100 dilution). 5. Polyethylenimine (PEI; Polysciences, Warrington, PA, USA). Dissolve in water to make a 1 mg/mL stock solution followed by 0.22 μM filtration. 6. Tryptone N1 (TN1). Dissolve in water to make a 20% (w/v) stock solution followed by 0.22 μM filtration. 7. Centrifuge. 8. Plastic 50 mL tubes. 9. Protein A agarose fast flow resin. 10. 0.1 M glycine containing 150 mM NaCl (pH 3.5). 11. 1 M Tris-HCl (pH 8.0). 12. Amicon Ultra-4 Centrifugal Filter Unit, 30 kDa cutoff. 13. PBS. 14. Automated cell counter.

3

Methods

3.1 Mouse Immunization and Serology 3.1.1 Immunization

1. Select a group of 6–10 9VH3 Harbour transgenic mice, 6–8 weeks old, of both genders (see Note 4). 2. Freshly prepare the antigen/adjuvant mixture immediately prior to use. 3. Immunize in 2-week intervals using 20–50 μg of antigen per mouse. The optimal number of immunizations is six. 4. For the priming immunization, use a mixture of IFA and antigen (1:1 volumetric ratio). Add aqueous antigen solution and vortex until an emulsion is formed. Prepare 20–50 μg of antigen per mouse in 100 μL volume and inject subcutaneously (50 μL in the right and left groin/inguinal area of each animal). 5. For the following five boosts, use a mixture of Ribi and antigen. Dissolve contents of a standard Ribi vial in 2 mL of PBS. Prepare 20–50 μg of antigen in 200 μL volume and inject 50 μL subcutaneously in the right and left groin each and 100 μL intraperitoneally. For the last boost, inject all 200 μL intraperitoneally. 6. Wait 3–5 days after the last injection, then sacrifice animals and isolate immune cells as described below.

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3.1.2 Measurement of Antigen-Specific HCAb Titers in Blood by ELISA

After the fourth immunization, perform the first ELISA, and after the sixth immunization perform the second ELISA (see Note 5). 1. Restrain the mouse. Keeping the head fixed, take the lancet in the other hand and aim it at the facial vein (see Note 6). Collect blood in an EDTA-coated tube (maximum 100 μL volume). 2. Spin blood for 10 min at 1,500
g. Leave the pelleted cells behind and transfer plasma to a fresh tube. Store at 20  C if not using immediately. 3. Coat wells of a 96-well ELISA plate with 5 μg/mL antigen in PBS for 2 h at room temperature (RT). 4. Block wells with 300 μL of blocking buffer (PBS containing 1% BSA, 1% fat-free milk powder and 0.1% Tween-20) per well for 30 min at RT. Wash three times with PBS containing 0.1% Tween-20. 5. Make plasma dilutions: to 3 μL of mouse plasma sample add blocking buffer to a final volume of 600 μL (200-fold dilution). Prepare 1,000-, 3,000-, 7,500-, 15,000-, 30,000-, 60,000-, and 120,000-fold dilutions. 6. Add 50 μL of each plasma dilution to the wells and incubate for at least 2 h at RT (overnight incubations should be conducted at 4  C). 7. Wash plate five times with 300 μL of PBS containing 0.1% Tween-20. 8. Add 50 μL of HRP-conjugated anti-mouse IgG (1/1,500 diluted in blocking buffer). 9. Incubate for 2 h at RT. 10. Wash plate five times with 300 μL of PBS containing 0.1% Tween-20. 11. Add 50 μL of BM Blue POD substrate. Incubate for 3–10 min, then stop the reaction with 50 μL of 1 M H2SO4. 12. Read absorbance at 450 nm.

3.2 Preparation of Immune Cells

If not experienced with finding and dissecting lymph nodes, see Note 7 before starting the experiment and follow instructions accordingly. 1. Euthanize immunized animals showing satisfactory antibody titers in accordance with institutional guidelines. 2. Dissect lymph nodes first (see Note 7 and Fig. 2) followed by hind limb long bones (tibias and femurs) and spleens. At all times keep dissected organs in ice cold PBS containing 0.5% BSA and 2 mM EDTA.

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Fig. 2 Schematic drawing of lymphoid organs of the mouse as a guide to dissection. Lymph nodes are depicted in dark blue; the spleen is shown in red; and the femurs and tibias are drawn in a red dotted pattern

3. Pull the entire leg bones, including femur, knee, and tibia, up and away from the body, carefully cutting away the connective tissue and muscle connecting the leg to the skin using scissors. 4. Overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia. 5. Trim the bones from the remaining muscle, connective tissue, and cartilage at the epiphysis to enable access to the bone marrow (BM).

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6. Flush bones with PBS containing 0.5% BSA and 2 mM EDTA using a 23-gauge needle attached to a 5 mL syringe to obtain BM cells. Repeat if necessary until the BM is completely removed. Bones should be white after removing the BM. Collect the BM cells in a Petri dish, pipette up and down to make a single cell suspension, and pass through a 40 μm cell strainer attached to a 50 mL tube. 7. Dissect spleen, remove connective and fat tissue, and make a single cell suspension by gently pressing the spleen tissue between a 2 mL syringe rubber plunger and a cell strainer attached to a 50 mL conical tube. Make sure to add enough buffer (PBS containing 0.5% BSA and 2 mM EDTA) to prepare the cell suspension. At the end of the procedure, only connective tissue should remain on the cell strainer. 8. Pool BM and spleen cells for isolation of plasma cells (see Subheading 3.2.1). 9. Use a microscope to remove surrounding fat and connective tissue from lymph nodes and prepare single cell suspension by passing through the cell strainer as for spleen cells. Proceed with affinity purification of antigen-specific B cells (see Subheading 3.2.2). 3.2.1 Isolation of Plasma Cells from BM and Spleen by Magnetic Sorting

For magnetic sorting of plasma cells from mice, we use a CD138+ plasma cell isolation kit following the manufacturer’s instructions. 1. Determine the number of leukocytes using a cell counter (see Note 8). 2. Centrifuge cells at 300
g for 10 min at 4  C. 3. Remove the supernatant and resuspend the cells in 400 μL of fresh PBS containing 0.5% BSA and 2 mM EDTA per 108 cells. 4. Add 100 μL of non-plasma cell depletion cocktail per 108 cells. 5. Mix well and incubate for 10 min at 4–8  C. 6. Wash cells by adding 5–10
the volume of PBS containing 0.5% BSA and 2 mM EDTA used in step 3. Centrifuge at 300
g for 10 min and remove the supernatant completely. 7. Resuspend the cell pellet in 900 μL of PBS containing 0.5% BSA and 2 mM EDTA per 108 cells. 8. Add 100 μL of Anti-Biotin MicroBeads (supplied with the CD138+ plasma cell isolation kit) per 108 cells. 9. Mix well and incubate for 15 min at 4  C. 10. Wash the cells by adding 5–10
volume of PBS containing 0.5% BSA and 2 mM EDTA and centrifuge at 300
g for 10 min. Remove the supernatant completely.

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11. Resuspend the cell pellet in PBS containing 0.5% BSA and 2 mM EDTA for depletion with the LD column (500 μL for up to 1.25
108 cells). 12. Place the LD Column in the magnetic field of an MACS separator (see Note 9). 13. Prepare column by rinsing with 2 mL of PBS containing 0.5% BSA and 2 mM EDTA. 14. Apply the cell suspension to the column. 15. Collect unlabeled cells which pass through and wash the column twice with 1 mL of PBS containing 0.5% BSA and 2 mM EDTA. Perform washing steps by adding buffer successively once the column reservoir is empty. Collect total effluent which contains the unlabeled pre-enriched plasma cell fraction. 16. Centrifuge the cells at 300
g for 10 min. Pipette off supernatant completely. 17. Resuspend the cell pellet in 400 μL of PBS containing 0.5% BSA and 2 mM EDTA per 108 cells (initial starting cell number). 18. Add 100 μL of CD138 MicroBeads (supplied with the CD138+ plasma cell isolation kit) per 108 cells. 19. Mix well and incubate for 15 min at 4–8  C. Wash the cells by adding 5–10 volumes of PBS containing 0.5% BSA and 2 mM EDTA and centrifuge at 300
g for 10 min. Pipette off the supernatant completely. 20. Resuspend up to 108 cells in 500 μL of PBS containing 0.5% BSA and 2 mM EDTA and proceed with the positive selection using MS columns. 21. Place MS column in the magnetic field of an MACS separator (see Note 10). 22. Prepare column by rinsing with 500 μL of PBS containing 0.5% BSA and 2 mM EDTA. 23. Apply the cell suspension to the column. 24. Collect unlabeled cells which pass through and wash the column three times with 500 μL of PBS containing 0.5% BSA and 2 mM EDTA, adding buffer once the column reservoir is empty. 25. Remove the column from the separator and place it on a suitable collection tube (15 mL). 26. Pipette 1 mL of PBS containing 0.5% BSA and 2 mM EDTA onto the column. Immediately flush out the fraction with magnetically labeled cells (plasma cells) by firmly applying the plunger supplied with the column.

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27. Repeat the magnetic separation procedure as described in steps 21–26 using a new MS column. 28. Spin down the purified plasma cells in a microfuge at 300
g for 5 min. Remove the supernatant and proceed with RNA isolation (see Subheading 3.3 and Note 11). 3.2.2 Isolation of Antigen-Specific B Cells from Lymph Nodes

1. Biotinylate the protein antigen using a 20-fold molar excess of EZ-Link™ NHS-PEG12-Biotin according to the manufacturer’s instructions (see Note 12). Prepare ~100 μg of protein to be biotinylated in PBS according to calculations (see Note 13), then add the appropriate volume of 250 mM EZ-Link™ NHS-PEG12-Biotin stock solution. Mix and incubate for 30 min at RT. 2. Remove unreacted biotin reagent using an Amicon centrifugal filtration device with appropriate molecular weight cut off size for the antigen used. Spin down and buffer exchange with PBS (up to ten times). 3. Measure the concentration of the biotinylated protein using a NanoDrop spectrophotometer (absorbance at 280 nm) (see Note 14). 4. Wash lymph node cells in a microfuge tube twice with PBS containing 0.5% BSA. Each wash step includes adding buffer, resuspending cells, centrifugation at 4  C for 5 min at 300
g, and discarding the supernatant. 5. For labeling of cells with biotinylated antigen, use ~2 μg of biotinylated protein per 1
107 cells (a typical yield per lymph node). Adjust the amount of biotinylated protein based on the number of mice and lymph nodes collected. Use 25 μL of Dynabeads™ M-280 streptavidin for up to 1
108 cells. 6. Incubate the biotinylated antigen with Dynabeads™ M-280 streptavidin in a microfuge tube containing 1 mL of PBS containing 0.5% BSA for 30 min at 4  C on a rotating wheel. 7. Place the microfuge tube on a magnetic stand, discard the supernatant, and wash the beads (pellet) three times with PBS/0.5% BSA by exposing the tube to the magnetic field. 8. Add the lymph node single cell suspension in 1 mL of PBS containing 0.5% BSA to the antigen-labeled Dynabeads and incubate for 30 min at 4  C with rotation. 9. Wash three times in the same buffer by exposure to the magnetic field.

3.3 Total RNA Isolation

1. Lyse the pellet in TRI® Reagent. One mL of the reagent is sufficient to lyse 5–10
106 cells (see Note 15). 2. To ensure complete dissociation of nucleoprotein complexes, allow the samples to stand for 5 min at RT.

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3. Add 0.2 mL of chloroform per mL of TRI® Reagent (see Note 16). 4. Cover the sample tightly, shake vigorously for 15 s, and allow it to stand for 2–15 min at RT. 5. Centrifuge the resulting mixture at 12,000
g for 15 min, 2–8  C. Centrifugation separates the mixture into three phases: a red organic phase (containing protein), an interphase (containing DNA), and a colorless upper aqueous phase (containing RNA). 6. Transfer the aqueous phase to a fresh tube and add 0.5 mL of isopropanol per mL of TRI® Reagent. 7. Mix the sample. Allow the sample to stand for 5–10 min at RT. 8. Centrifuge at 12,000
g for 10 min, 2–8  C. The RNA precipitate will form a pellet on the side and bottom of the tube. 9. Remove the supernatant and wash the RNA pellet by adding a minimum of 1 mL of 75% ethanol per 1 mL of TRI® Reagent. 10. Centrifuge at 7,500
g for 5 min, 2–8  C. 11. Briefly air-dry the RNA pellet for 5–10 min. Do not let the RNA pellet dry completely, as this will greatly decrease its solubility. 12. Dissolve the RNA pellet in 20–40 μL of dH2O and measure the concentration using a NanoDrop spectrophotometer (see Note 17). 3.4 cDNA Synthesis and Amplification of VHs

Use 3 μg of RNA in a 20 μL reaction volume for first strand cDNA synthesis using SuperScript™ II reverse transcriptase and oligo (dT) primers. Prepare separate reactions for plasma cell cDNA and lymph node antigen-specific B cell cDNA. This will yield two VH libraries (see Note 18). 1. Add the following components to a nuclease-free microfuge tube: 1 μL of oligo(dT) primer, ~3 μg of total RNA, and 1 μL of dNTP mix. 2. Add dH2O to a 12 μL total volume. 3. Heat the mixture to 65  C for 5 min and quickly chill on ice. Collect the contents of the tube by brief centrifugation and add 4 μL of 5
first-strand buffer, 2 μL of 0.1 M dithiothreitol and 1 μL of RNase OUT™. 4. Mix the contents of the tube gently. 5. Incubate at 42  C for 2 min. Add 1 μL (200 U) of SuperScript™ II RT and mix by pipetting gently up and down. 6. Incubate at 42  C for 50 min. 7. Inactivate the reaction by heating at 70  C for 15 min.

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8. Use 2 μL of the resulting cDNA as a template in a 50 μL total volume of PCR reaction. Add 10 μL of 5
HF Phusion® buffer, 1 μL of 10 mM dNTPs, 0.5 μM primer mIgG1 rev, 0.5 μM primers Lib 3–23 for/Lib3–11 for, 1.5 μL of 100% dimethyl sulfoxide, 0.5 μL of Phusion® DNA polymerase, and dH2O to 50 μL. Cycle the temperature as follows: 98  C for 30 s; 28 cycles of 98  C for 10 s, 68  C for 30 s, and 72  C for 30 s; 72  C for 10 min. 9. Purify the PCR product using Nucleospin® Extract II PCR purification kit according to the protocol provided by the manufacturer. Use 15–50 μL dH2O to elute the DNA. 10. Measure the concentration of the DNA using a NanoDrop spectrophotometer. 11. Perform a double PvuII/BstEII digest for 1 h at 37  C using PvuII HF and BstEII HF (see Note 19). Perform the digest in 100 μL total volume containing 10 μL of CutSmart 10
buffer, 1 μL of each enzyme, 1–5 μg of VH DNA, and H2O to 100 μL. Stop the reaction with the loading buffer provided with the restriction enzymes. 3.5 Cloning of VHs into Mammalian Expression Vector

1. Prepare a 1.8–2% agarose gel in 0.5
TBE buffer containing ethidium bromide (50 μL stock solution/L gel). 2. Load the digested sample in a well of a preparative gel. 3. Load the DNA molecular weight marker (see Note 20). 4. Run the gel in electrophoresis buffer (~100–150 V). 5. Move the gel to an open UV box (see Note 21) and remove it from the gel tray as plastic will block much of the UV light. With a clean, sterile razor blade, excise the VH fragment band (~350 bp), placing the gel slice in a microfuge tube. 6. Using a scale, weigh the tube containing the gel fragment after taring the balance with an empty tube. 7. Purify the VH fragments from the gel using the Nucleospin® Extract II gel extraction kit according to the manufacturer’s protocol, using dH2O for elution (~20 μL). 8. Measure the concentrations of VH fragments using a NanoDrop spectrophotometer. 9. Prepare ligation reactions in a volume of 10 μL containing 1 μL of T4 DNA ligase, 1 μL of 10
T4 DNA ligase buffer, 20 ng of insert (purified PvuII/BstEII digested VH fragments), 150 ng of phosphatase-treated PvuII/BstEII-digested pCAG hygro hIgG1 vector, and dH2O to 10 μL. 10. Incubate the reactions at 4  C overnight.

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1. Use 1 μL of ligation to transform MegaX DH10B T1R Electrocomp™ cells according to the manufacturer’s protocol (20 μL of competent cells per electroporation at 2 kV, 200 Ω, 25 μF). 2. Add 980 μL of recovery medium to the cuvette (this is provided with the competent cells). 3. Transfer the contents to a 15 mL Falcon tube. 4. Incubate for 1 h at 37  C with 100 rpm shaking. 5. Plate the bacteria on LB agar plates containing ampicillin at dilutions suitable for picking individual colonies. Pick ~1,000 colonies per library for optimal results.

3.6.2 Generation of the Bacterial Library and Isolation of Plasmid DNA

1. Grow picked colonies in 380 μL of TB medium containing ampicillin in Nunc™ 96-well polypropylene storage microplates overnight at 37  C. 2. Centrifuge plates at 200
g for 5 min. 3. Discard growth medium by vigorously turning the plate upside down. 4. Resuspend pellet in 75 μL of TE/RNase buffer (per sample: 74 μL of TE buffer and 1 μL of RNase stock). 5. Add 75 μL of lysis buffer (1% SDS, 0.2 M NaOH) and incubate at RT for 10 min. 6. Add 75 μL of neutralization buffer (3 M potassium acetate, pH 5.5). 7. Slowly shake plates on a vortex to mix. 8. Centrifuge the plates for 10 min at 3,200
g. 9. Move the supernatant to a new 96-well plate using a multichannel pipette. 10. Add 120 μL of isopropanol and leave at RT for 20 min. 11. Centrifuge at 3,200
g for 30 min. 12. Discard supernatant by decanting. 13. Wash pellet by adding 300 μL of 70% ethanol. 14. Centrifuge at 3,200
g for 10 min. 15. Discard supernatant and leave plates to dry. 16. Dissolve DNA pellet in 30 μL of H2O. 17. Measure the concentration of the plasmid DNA from a few wells (randomly) using a NanoDrop spectrophotometer to estimate the average yield.

3.7 Generation of HEK Cell Library

1. Grow HEK 293 T adherent cells in flat-bottom 96-well plates containing 200 μL of expansion medium (DMEM supplemented with 10% fetal calf serum, non-essential amino acids,

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penicillin, and streptomycin). At the time of transfection, cells should be 80% to 90% confluent. 2. In a new round-bottom 96-well plate, add 50 μL per well of OptiMEM™ medium, 0.5 μL of lipofectamine™ 2000, and ~100 ng (2–4 μL) of plasmid DNA. Use a multichannel pipette to move DNA from a 96-well plate containing plasmid DNA (see Note 22). 3. Leave at RT for 15 min. 4. Add the plasmid/lipofectamine™ 2000 mixtures from the round bottom plates to the flat bottom plates containing HEK 293T cells. 5. Incubate transfected cells at 37  C under a humidified atmosphere containing 5% CO2 for 48 h. 6. Coat 384-well ELISA plates with antigen (~20 μL, 5 μg/mL in PBS) for 2 h at RT. 7. Empty the wells by shaking plates upside down and block with 100 μL of blocking buffer (PBS containing 1% BSA, 1% fat-free milk powder and 0.1% Tween-20) for 1 h at RT. 8. Empty wells by shaking upside down and wash with 200 μL PBS containing 0.1% Tween-20 five times. To avoid pipetting, submerge the plates in wash buffer each time. 9. Take 80 μL of supernatant from each well containing transiently transfected HEK 293T cells and place it into the 384 ELISA plates. Use a multichannel pipette to ensure that transfer from one plate format (96 well) to the other (384 well) does not cause problems with identification of clones later. 10. Incubate for 2 h at RT. 11. Empty ELISA plate wells and wash as previously. 12. Add ~20 μL of secondary HRP-conjugated anti-human IgG antibody (1:2,000 dilution) and incubate for 1 h at RT. 13. Empty ELISA plates and wash five times with PBS containing 0.1% Tween-20. 14. Add 20 μL of BM Blue POD substrate and incubate for 5–10 min (see Note 23). 15. Stop the reaction with 10 μL of 1 M H2SO4. 16. Measure the absorbance at 450 nm using an ELISA plate reader. 3.8 Sequencing of VHs

1. Sequence the VH regions of HCAb expression vector DNA using the VH3–23 leader primer. 2. Align each of the sequences with germline genes contained in the IMGT database (http://www.imgt.org/IMGT_vquest/ vquest). This will provide information about which VH

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germline is used, which D and J segments are used, and show the complementarity-determining regions and somatic hypermutations. For large numbers of sequences, use IMGT/ HighV-QUEST. 3. Align all obtained sequences and discard identical sequences. If there are no potential posttranscriptional modifications that could cause liabilities for further development, proceed with production. 3.9 Medium-Scale (100 mL) Production of Full-Length HCAbs

1. Split FreeStyle 293-F cells at an appropriate density (around 1
106 cells/mL) 1 day before transfection. Count the cells using a cell counter. The next day, the cell density should be around 2
106 cells/mL and the cell viability should be 97%. 2. Add the HCAb expression plasmid DNA (200 μg) into a 15 mL tube and add 1 mL of fresh medium (see Note 24). 3. Add PEI to the same tube (ratio 1 μg DNA:4 μL PEI stock). Mix well and incubate at RT for 15 min. 4. Add the mixture (DNA/PEI in medium) dropwise to a flask containing FreeStyle 293-F cells (100 mL). 5. Incubate the transfected cells at 37  C under a humidified atmosphere containing 5% CO2 with 120 rpm shaking. 6. Supplement the cells with 2.5 mL of freshly prepared 20% TN1 (0.5% w/v final concentration) around 24 h post transfection. 7. Harvest cells 5–6 days later by transferring the cell suspension to two 50 mL tubes. 8. Centrifuge for 30 min at 2,900
g, 4  C. 9. Pack around 0.4 mL of protein A fast flow resin in an empty column, and rinse/equilibrate with 10 column volumes (CV) of PBS. Collect the supernatant and load it on the protein A column. Apply the supernatant to the column by gravity, discard the flow through. 10. Wash the column with 10 CV of PBS. 11. Elute the HCAb with 1 mL of 0.1 mM glycine, 150 mM NaCl (pH 3.5). Elute into a 15 mL tube containing 1 mL of 1 M Tris-HCl, pH 8.0. Mix immediately after elution. 12. Buffer exchange into PBS and concentrate the sample using an Amicon Ultra 4 filter centrifugation device (30 kDa) by centrifugation at 2,900
g, 4  C. Repeatedly centrifuge (~10 times), bringing the volume down to less than 1 mL. Discard the flow through at each step and add new PBS (3–4 mL) to the filter device.

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13. Transfer the concentrated samples to clean 1.5 mL or 2 mL tubes. Measure the protein concentration using a NanoDrop spectrophotometer (absorbance at 280 nm). 14. Store the final products at 4  C for further characterization.

4

Notes 1. The immunization method described in this chapter is applicable to most antigens including soluble proteins, peptide conjugates, and cell membrane preparations. DNA/RNA immunizations are not covered in this protocol. 2. The authors of this chapter are obliged by Dutch animal welfare regulations and law to use IFA and Ribi adjuvant. Other countries and institutions might have different regulations. Harbour mice have been successfully immunized using other adjuvants outside of the Netherlands. 3. Prepare biotinylated antigen 1 day in advance. Store at 4  C before use. 4. If using male mice, place them in separate cages to prevent fighting and potential injury. 5. Mice whose sera yield saturated ELISA signal at 1/1,000 dilution or higher are candidates for continued immunization after the first ELISA. Animals whose sera fall below this threshold are not moved forward to subsequent experiments. Antigenspecific HCAb titers should improve after further boosts yielding a saturated signal at higher dilutions in the second ELISA. 6. Alternative blood draw techniques can be used in accordance with approved animal procedures in other institutions. A single drop of blood is sufficient for testing. 7. Lymph nodes can be difficult to locate. They are generally “pearly white” and/or translucent in color and can blend in with surrounding fat. The easiest nodes to find are the mesenteric, brachial, and inguinal nodes (Fig. 2). The other nodes are generally much smaller and more difficult to identify. The most commonly used and easily visible are inguinal lymph nodes, which are also the closest to the sites of both subcutaneous injections in inguinal areas and intraperitoneal injections. Lymph nodes can be visualized by injecting a solution of Evans blue. For this purpose, anesthetize the mouse with isoflurane (place the animal in a beaker containing cotton wool soaked in isoflurane). Inject ~10 μL of dye subcutaneously into a foot pad. Return the mouse to its cage and wait for ~10 min. Euthanize the animal and start dissection. Lymph nodes will stain blue. The dye does not interfere with any subsequent procedure.

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8. As a guideline, one can expect 1–2
108 lymphocytes per spleen, 1–2
107 cells from the BM of one femur/tibia and 1–2
107 cells from all lymph nodes, except the mesenteric lymph nodes that could yield 2–3
107 lymphocytes. Volumes for magnetic sorting are for up to 108 cells. When working with fewer than 108 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2
108 leukocytes use twice the volume of all indicated reagents). 9. Separators for the LD column are: MidiMACS, QuadroMACS, VarioMACS, and SuperMACS. The maximum number of total leukocytes per LD column is 5
108. 10. Separators for the MS column are: MiniMACS, OctoMACS, VarioMACS, and SuperMACs. The maximum number of total leukocytes per MS column is 2
108. 11. The pellet might be difficult to see. It is not necessary to count the cells. Use 1 mL of TRI® Reagent for both plasma cells and antigen-specific B cells. This amount is more than sufficient. 12. Proteins in Tris or other amine-containing buffers must be exchanged into a suitable buffer (i.e., amine-free PBS) prior to biotinylation. 13. Calculate the quantity in millimoles of EZ-Link™ NHS-PEG12-Biotin to add to the reaction to achieve a 20-fold molar excess as follows: 1 mL protein



mg protein mmol protein
mL protein mg protein

20 mmol Biotin Reagent mmol protein

¼ mmol Biotin Reagent: The value of 20 in this equation corresponds to the suggested 20-fold molar excess of biotin reagent for a 2 mg/mL protein sample. Calculate microliters of 250 mM EZ-Link™ NHS-PEG12-Biotin stock solution to add to the reaction as follows: 1, 000, 000 μL L ¼ L 250 mmol ¼ μL Biotin Reagent stock solution:

mmol Biotin Reagent


14. Purified protein samples can be accurately measured using direct absorbance at 280 nm. Absorbance at 280 nm is mostly due to the aromatic chains on the amino acids tryptophan and tyrosine. Protein A280 is the most popular quantification

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method because it is fast and simple, requires no reagents or standard curves, and consumes very little sample. Sample purity can also be assessed via the A260/A280 value. The ideal ratio is ~0.6. 15. After the cells have been homogenized or lysed in TRI® Reagent, samples can be stored at 70  C for up to 1 month. If clumps are visible, the samples can be optionally sonicated. 16. The chloroform used for phase separation should not contain isoamyl alcohol or other additives. 17. The final preparation of RNA is free of DNA and proteins. It should have an A260/A280 ratio of 1.7. 18. A 20 μL reaction volume can be used for 1 ng–5 μg of total RNA. We separately prepare a plasma cell library (from BM and spleen cells) and an antigen-specific B cell library (from lymph nodes). The number of positive clones from antigen-specific B cells is generally higher than from plasma cells. Unique antibodies are isolated from each library. 19. If using a different BstEII enzyme active at 60  C, cut the DNA with PvuII at 37  C for 1–2 h in the appropriate buffer, purify, and then and cut with BstEII at 60  C for 1–2 h. Do not digest for longer because of potential star activity. 20. Use any appropriate size marker that will show bands between 100 and 500 base pairs. 21. Wear proper UV protection, especially for the eyes. Try to isolate the gel fragment in the shortest period of time possible using a long wave (365 nm) UV light source to prevent DNA damage. 22. Prepare a mixture of OptiMEM™ and lipofectamine™ 2000 (sufficient for all plates) and aliquot 50.5 μL per well. 23. The incubation time with substrate could be longer (up to 30 min) and could be shortened by incubation at 37  C. 24. Midipreps or maxipreps of the selected HCAb vector DNA will be required. To accomplish this, transform E. coli cells with the plasmid, plate on LB agar plates containing ampicillin, pick a single colony, culture in 500 mL of growth medium containing ampicillin, and follow the instructions of any kit for DNA purification. References 1. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797 2. Janssens R, Dekker S, Hendriks RW et al (2006) Generation of heavy-chain-only antibodies in

mice. Proc Natl Acad Sci U S A 103: 15130–15135 3. Laventie BJ, Rademaker HJ, Saleh M et al (2011) Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing

A Transgenic Heavy Chain IgG Mouse Platform as a Source of High Affinity. . . Staphylococcus aureus leukotoxins. Proc Natl Acad Sci U S A 108:16404–16409 4. Drabek D, Janssens R, de Boer E et al (2016) Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells. Front Immunol 7:619

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5. Yu-Shi S, Lu YW, Liu Z et al (2018) A biparatopic agonistic antibody that mimics fibroblast growth factor 21 ligand activity. J Biol Chem 293:5909–5919

Part III Expression of Single-Domain Antibodies

Chapter 7 Cytoplasmic Production of Nanobodies and Nanobody-Based Reagents by Co-Expression of Sulfhydryl Oxidase and DsbC Isomerase Ario de Marco Abstract Nanobodies are stable molecules that can often fold correctly even in the absence of the disulfide bond (s) that stabilize their three-dimensional conformation. Nevertheless, some nanobodies require the formation of disulfide bonds, and therefore they are commonly expressed from vectors that promote their secretion into the oxidizing environment of the Escherichia coli periplasm. As an alternative, the bacterial cytoplasm can be an effective compartment for producing correctly folded nanobodies when sulfhydryl oxidase and disulfide-bond isomerase activities are co-expressed from a recombinant vector. The larger volume and wider chaperone/foldase availability of the cytoplasm enable the achievement of high yields of both nanobodies and nanobody-tag fusions, independently of their redox requirements. Among other examples, the protocol described here was used to successfully produce nanobody fusions with fluorescent proteins that do not fold correctly in the periplasm, nanobodies with Fc domains, and nanobodies containing free cysteine tags. Key words Single-domain antibodies, Redox conditions, Fusion immunoreagents, Nanobody functionality, Chromobodies

1

Introduction Recombinant antibody fragments cannot replace conventional antibodies for therapeutic applications that require effector function in vivo, but otherwise possess several advantages in many research and diagnostic applications. Their sequence is defined and therefore their clonality is assured [1], they can be produced inexpensively in Escherichia coli, their engineering and 1:1 site-specific modification is straightforward, and they can be expressed as fusion proteins with tags that provide further functionalities [2–4]. Nanobodies are the smallest available antibody fragment that still conserves the binding specificity of the original IgG. Their tiny dimensions and high stability contributes to their large use in super resolution

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_7, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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microscopy, in the preparation of polymeric therapeutic reagents, and as tools for in vivo imaging [5–10]. Furthermore, their short sequences are suitable for rational design, in silico modeling, and selection methods which exploit deep sequencing [11–13]. Approximately 10 years ago our group explored the possibility of developing a general method for the cytoplasmic production of proteins that require the formation of disulfide bonds for correct folding and applied it to nanobodies and nanobody fusion proteins [14, 15]. This approach is not based on inhibition of the cell’s reductive metabolism [16], a strategy that can succeed but usually provides low protein yields. In contrast, wild type bacteria harboring the nanobody-encoding plasmid are simply co-transformed to overexpress a sulfhydryl oxidase (SOX), for instance Erv1p from the inter-membrane mitochondrial space of Saccharomyces cerevisiae, which promotes efficient formation of disulfide bonds even in a reducing environment [15]. This system also enables co-expression of the disulfide bond isomerase DsbC, the availability of which becomes useful when nanobody-fusion proteins have more than two cysteines. The system is very robust: the cells grow normally and the intact cytoplasmic folding machinery assures high yields of correctly folded nanobodies. Furthermore, this approach has the advantage of enabling the folding of fusions between nanobodies and proteins that do not fold efficiently in the oxidizing environment of the periplasm, as is the case for most fluorescent proteins [17]. Applying this method, we also produced complex proteins with multiple disulfide bonds in the bacterial cytoplasm, such as nanobody-Fc fusions [18] and nanobody-alkaline phosphatase fusions. Furthermore, this method is suitable for expressing nanobodies with a diverse array of tags including SNAP-Tag, CLIP-Tag, HaloTag, free cysteine, Sortase-Tag, AviTag, SpyTag and SpyCatcher, mVirD2, ALFA-tag, and fluorescent proteins (EGFP, mCherry, mRuby3, and mClover3) [3, 15, 18–21]. Proteins expressed in the cytoplasm of SOX-overexpressing cells were monodisperse and at least as functional as those expressed in the periplasm, but their yields were significantly higher [18]. Since the plasmid expressing SOX and DsbC confers chloramphenicol resistance, both ampicillin and kanamycin expression vectors can be used for the nanobody construct (Fig. 1). We have also demonstrated that the system can be applied to scFv fragments and expect that it could be suitable for increasingly complex proteins that are disulfide bond-dependent for correct folding.

2 2.1

Materials Vectors

1. A vector (see Note 1) for the arabinose-inducible cytoplasmic expression of SOX and DsbC (SOX plasmid, see Note 2).

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Fig. 1 Schematic representation of the procedure for the cytoplasmic expression of nanobodies and nanobody fusion constructs. Bacterial cells are first co-transformed with two plasmids (a), one enabling the expression of SOX and DsbC and the other the target nanobody construct. All three proteins will accumulate in the cytoplasm but the independent induction systems present in the two plasmids allow for their temporally asynchronous expression. More precisely, the protocol will trigger an initial accumulation of the foldase/ isomerase enzymes (b), followed by the expression of the nanobody (c). Under these conditions, nanobodies and nanobody fusion constructs can form their disulfide bonds and attain their native conformations even in the reducing environment of the bacterial cytoplasm

2. A vector for the isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible cytoplasmic expression of nanobodies or nanobody-fusion constructs (see Note 3).

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2.2 Preparation of SOX-Overexpressing Bacteria

1. SOB++ medium: 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCl, and 0.186 g of KCl in 1 L of H2O. Autoclave to sterilize and supplement with sterile 10 mM MgSO4 and 10 mM MgCl2. 2. E. coli BL21 (DE3) cells. 3. Dimethyl sulfoxide (DMSO). 4. 10 mM HEPES, 15 mM CaCl2, 250 mM KCl. Adjust the pH to 6.7 with KOH and then add 55 mM MnCl2. 5. Luria Bertani (LB) broth: 10 g of tryptone, 10 g of NaCl, and 5 g of yeast extract in 1 L of H2O. Sterilize by autoclaving. 6. LB agar plates: prepare LB medium then add 15 g of agar prior to autoclaving. Allow to cool to ~50  C before adding selective antibiotics. 7. Refrigerated preparative centrifuge with corresponding rotor and tubes. 8. Erlenmeyer flasks. 9. Liquid nitrogen. 10. SOX plasmid. 11. Microcentrifuge tubes (1.5 mL). 12. Petri dishes. 13. Chloramphenicol stock solution (10 mg/mL). 14. Spectrophotometer. 15. Sterile 80% (v/v) glycerol.

2.3 Small-Scale Protein Production

1. LB broth (see Subheading 2.2) or Terrific Broth (TB): 20 g of tryptone, 24 g of yeast extract, and 4 mL of glycerol in 900 mL of H2O. Sterilize by autoclaving, then add 100 mL of sterile phosphate buffer (0.17 M KH2PO4, 0.72 M K2HPO4). 2. 1 M IPTG dissolved in H2O. 3. 20% (w/v) arabinose. 4. 40% (w/v) glucose. 5. E. coli BL21 (DE3) SOX-overexpressing bacteria. 6. Nanobody expression vector (see Subheading 2.1). 7. 1 mg/mL DNase I. 8. 100 mg/mL lysozyme. 9. Ni-NTA magnetic beads and magnetic rack. 10. 50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl and 5 mM MgCl2. 11. 50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl, 15 mM imidazole, and 0.02% Triton X-100.

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12. Phosphate-buffered saline containing 0.1% Triton X-100 (PBST): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 0.1% (v/v) Triton X-100. 13. Water bath sonicator. 14. Tubes for microbiology (12–15 mL). 15. 10 mg/mL chloramphenicol, 100 mg/mL ampicillin, and/or 50 mg/mL kanamycin stock solutions, dissolved in H2O, and filter sterilized. 16. SDS sample buffer 2: 100 mM Tris pH 6.8, 4% (w/v) SDS, 20% glycerol, bromophenol blue (4.8 mg/100 mL). 17. SDS-PAGE apparatus and gels. 18. Colloidal blue. 2.4 Large-Scale Cytoplasmic Protein Production

1. Culture medium (LB or TB): see Subheadings 2.2 and 2.3 for preparation. 2. 1 M IPTG dissolved in H2O. 3. Arabinose (powder). 4. 40% glucose. 5. E. coli BL21 (DE3) SOX-overexpressing co-transformed with nanobody constructs.

bacteria

6. 1 mg/mL DNase I. 7. 100 mg/mL lysozyme. 8. Ni-NTA chromatographic column. 9. 50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl and 5 mM MgCl2. 10. 50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl and 15 mM imidazole. 11. 50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl and 200 mM imidazole. 12. Centrifuge with a rotor suitable for 50 mL tubes. 13. Tubes for microbiology (12–15 mL). 14. Fast protein liquid chromatography (FPLC) instrument. 15. 10 mg/mL chloramphenicol, 100 mg/mL ampicillin, and/or 50 mg/mL kanamycin stock solutions, dissolved in H2O and sterile filtered. 16. Gel filtration column with adaptors for FPLC equipment. 17. 25 mM Tris-HCl, pH 7.8, containing 150 mM NaCl. PBS is an acceptable alternative.

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2.5 Nanobody Concentration Determination

3

1. Spectrophotometer. 2. Cuvette.

Methods

3.1 Preparation of Competent SOXExpressing BL21 (DE3) Cells

1. Grow E. coli BL21 (DE3) cells in 250 mL of SOB++ medium using a 2 L flask at 30  C with 270 rpm shaking until the OD600 reaches 0.4. 2. Pellet the bacteria in a refrigerated centrifuge (10 min at 10,000  g). 3. Gently resuspend the pellets on ice using 100 mL of ice-cold transformation buffer (10 mM HEPES, 15 mM CaCl2, 250 mM KCl, 55 mM MnCl2, pH 6.7). 4. Incubate for 20 min on ice and harvest the cells by centrifugation (10 min at 10,000  g). 5. Resuspend cells in 18.6 mL of transformation buffer supplemented with 1.4 mL of DMSO and incubate for 20 min on ice. 6. Aliquot the competent cells in microcentrifuge tubes, snap freeze in liquid nitrogen and store at 80  C. Cells will remain competent for at least 6 months (see Note 4). 7. Transform an aliquot of 30 μL of the E. coli BL21 (DE3)competent cells with 1.5 μL (50 ng) of SOX plasmid by incubating for 30 min on ice, heat shocking for 40 s at 42  C, and placing again on ice for 2 min. 8. Add 120 μL of LB, incubate for 1 h at 37  C with 270 rpm shaking, and plate on LB agar plates containing 25 μg/mL chloramphenicol. 9. Collect colonies corresponding to E. coli BL21 (DE3) SOX cells after overnight growth of the plates at 37  C. 10. Start a pre-culture by inoculating 2 mL of LB containing 25 μg/mL chloramphenicol with a single colony. Take an aliquot of the pre-culture, add glycerol to 20% of the final volume, and store at 80  C. Use the remaining pre-culture to inoculate SOB++ containing 25 μg/mL chloramphenicol as in step 1. Repeat the above procedure to prepare competent SOX-overexpressing cells. These cells will be used later for co-transformation with the vector harboring the nanobody construct.

3.2 Small-Scale Protein Production

1. Transform an aliquot of 30 μL of the E. coli BL21 (DE3) SOX-competent cells with 1.5 μL (50 ng) of a nanobody plasmid by incubating 30 min on ice, heat shocking for 40 s at 42  C, and placing again on ice for 2 min.

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2. Add 120 μL of LB, incubate for 1 h at 37  C with 270 rpm shaking, and plate on LB agar plates containing 25 μg/mL chloramphenicol and either 100 μg/mL ampicillin or 50 μg/ mL kanamycin (depending on nanobody vector resistance marker). Grow overnight at 37  C. 3. Inoculate a colony of the SOX and nanobody plasmid co-transformed cells in a microbiology (12–15 mL) tube containing 2 mL of LB medium, 1% glucose, 25 μg/mL chloramphenicol and either 100 μg/mL ampicillin or 50 μg/mL kanamycin (depending on nanobody vector resistance marker). 4. Grow overnight at 30  C in an inclined rack (30 ) inside a shaking incubator (180 rpm). 5. The next day, add 100 μL of the pre-cultures to a 50 mL Erlenmeyer flask containing 15 mL of LB (or TB), 25 μg/mL chloramphenicol and either 100 μg/mL ampicillin or 50 μg/ mL kanamycin (depending on nanobody vector resistance marker). 6. Let the bacteria grow at 37  C in an orbital shaker (210 rpm) until the OD600 reaches 0.4, then switch the temperature to 20  C. 7. Induce the expression of SOX (and DsbC) by adding 0.5% arabinose (see Note 5). 8. After 30 min (the OD600 of the culture will have reached approximately 0.6), add 0.2 mM IPTG to induce nanobody expression. 9. Let the culture grow for 18 h at 20  C and then harvest the pellet by centrifuging (15 min at 11,000  g, 4  C). 10. Remove the medium and store the pellet at

20  C.

11. Add 35 μL of Ni-NTA magnetic bead slurry to a 2 mL microcentrifuge tube. 12. Set the tube into a magnetic rack and carefully remove the solution. 13. Transfer the tube to a standard rack and resuspend the beads in 400 μL of PBST. 14. Set the tube into the magnetic rack and remove the buffer. 15. Transfer the tube to a standard rack and resuspend the beads in 50 μL of lysis buffer (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl and 5 mM MgCl2). 16. Resuspend the bacterial pellet in 500 μL of lysis buffer. 17. Sonicate the bacterial suspension for 5 min in a water bath at room temperature. 18. Add lysozyme to a final concentration of 1 mg/mL and DNase I to a final concentration of 50 μg/mL. Incubate for 30 min at room temperature with continuous rocking. No viscous

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material, indicating the presence of undigested nucleic acids, should be detectable at the end of this step (see Note 6). 19. Separate the supernatant fraction by centrifugation (5 min at 16,100  g at room temperature). Take a sample from both pellet and supernatant and add an equal volume of 2 SDS sample buffer. 20. Add the supernatant from step 19 to the tubes containing pretreated beads from step 15. Incubate the tubes for 30 min at room temperature with constant rotation. 21. Separate the beads from the supernatant using the magnetic rack and discard the supernatant. 22. Remove the magnet, resuspend the beads in 400 μL of washing buffer (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl, 15 mM imidazole, and 0.02% Triton X-100) and incubate for 30 min at room temperature with constant rotation. 23. Repeat steps 21 and 22. 24. Separate the beads from the supernatant using the magnetic rack and carefully discard the buffer. 25. Remove the magnet, resuspend the beads in 25 μL of 2 SDS sample buffer, and boil the sample. 26. Perform SDS-PAGE with fractions from the cell pellet, supernatant and beads, and stain the gel with colloidal blue (see Note 7). 3.3 Large-Scale Cytoplasmic Protein Production

1. Inoculate a single colony of E. coli BL21 (DE3) cells co-transformed with the SOX and nanobody plasmids (see step 2 in Subheading 3.2) in a 200 mL Erlenmeyer flask containing 25 mL of LB medium, 1% glucose, 25 μg/mL chloramphenicol and either 100 μg/mL ampicillin or 50 μg/mL kanamycin (depending on nanobody vector resistance marker). 2. Grow overnight at 30  C inside a shaking incubator (180 rpm). 3. The next day, add 3 mL of the pre-cultures to a 2 L Erlenmeyer flask containing 500 mL of LB (or TB), 25 μg/mL chloramphenicol and either 100 μg/mL ampicillin or 50 μg/mL kanamycin (depending on nanobody vector resistance marker). 4. Grow bacteria at 37  C in an orbital shaker (210 rpm) until the OD600 reaches 0.4, then switch the temperature to 20  C. 5. Induce the expression of SOX (and DsbC) by adding the optimized arabinose concentration determined above (see step 7 in Subheading 3.2). 6. After 30 min (the OD600 of the culture should have reached approximately 0.6), add 0.2 mM IPTG to induce nanobody expression.

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7. Grow the culture for 18 h at 20  C and then harvest the pellet by centrifuging (15 min at 11,000  g, 4  C). 8. Remove the medium and store the pellet at

20  C.

9. Resuspend the bacterial pellet in 20 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl and 5 mM MgCl2). 10. Sonicate for 5 min in a water bath at room temperature. 11. Add lysozyme to a final concentration of 1 mg/mL and DNase I to a final concentration of 50 μg/mL. Incubate for 30 min at room temperature with continuous rocking. 12. Separate the supernatant fraction by centrifugation (15 min, 10,000  g at room temperature). No viscous material, indicating the presence of undigested nucleic acids, should be detectable at the end of this step. 13. Load the supernatant onto an Ni-NTA chromatographic column pre-equilibrated in washing buffer (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl and 15 mM imidazole) and connected to an FPLC system. 14. Wash the column with washing buffer until the OD280 reaches baseline and then elute the protein complex with elution buffer (50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl and 200 mM imidazole). 15. Buffer exchange the fractions corresponding to the elution peak into size exclusion chromatography buffer (25 mM TrisHCl, pH 7.8, containing 150 mM NaCl) (see Note 8). 16. Pre-equilibrate the size exclusion chromatography column with five volumes of size exclusion chromatography buffer. 17. Load the sample. 18. Run the gel filtration column at a suitable flow rate (1 mL/ min), monitor the absorbance signal at 280 nm, and collect fractions. 19. Analyze the peak distribution (see Note 9). 3.4 Nanobody Concentration Determination

1. Start with buffer-exchanged nanobody protein following Ni-NTA purification (see step 15 in Subheading 3.3) or nanobody protein post-size exclusion chromatography (see step 19 in Subheading 3.3) (see Note 10). 2. Prepare a UV spectrophotometer and cuvettes (see Note 11). 3. Fill the cuvette with sufficient material to ensure an accurate measurement (see Note 12). 4. Perform a scan between 240 and 340 nm and record the absorbance. 5. Record the absorbance values at 260, 280, and 340 nm.

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6. Use the value at 280 nm in combination with the protein extinction coefficient to calculate the nanobody concentration (see Note 13). 7. Calculate the ratio of Abs 260 nm/Abs 280 nm. The value should be 0.6  0.1, above which nucleic contamination becomes problematic. 8. Calculate the value 100  Abs 340 nm/[Abs 280 nm Abs 340 nm]. The value should be 100) in the first round. The enrichment may even decrease with each round, as

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selection stringency also increases (e.g., enrichment values 10–90). If the enrichment ratio is close to a value of 1 after the second round or beyond, it is likely that the selection stringency may be too high and selection criteria can be made less stringent (e.g., smaller drop in antigen concentration or keeping low pH selection at pH 4.0). 14. The remaining rounds of phage selection (second round and beyond) can be performed entirely automated using the Kingfisher. For round two, in two row A wells, add 100 μL of amplified phage to either 100 μL of 0.2 μM biotinylated antigen (sample) or 0.2 μM BSA (control). In row B wells add 100 μL of washed beads in a total of 200 μL TBS-T/BSA. In row C through G wells add 200 μL of TBS-T/BSA. In row H add 200 μL of sodium acetate, pH 4.5. Program the Kingfisher to incubate Row A wells with mixing for 15 min, before transferring SAV Beads. Capture SAV Beads with mixing for 15 min. Then, transfer beads successively to each wash well C through G, incubating with mixing for 20 s before transferring to next wash well. The final transfer to the pH-sensitive selection (wells H) should include incubation with mixing for 10 min before removing SAV Beads. 15. Amplify the sample output for a third round of selection by following steps 8–12. Perform a phage titer for the amplified phage input, as well as both sample and control output wells using 10 μL of each sample. 16. In the third round of selection, repeat steps 13–15. However, the biotinylated antigen and BSA control concentrations can be reduced ten-fold to 20 nM and the pH-sensitive selection buffer can be raised to pH 5.0 (i.e., if the enrichment is not too close to 1). 17. A fourth round of selection may be pursued further increasing the stringency of binding at pH 7.4 (by reducing antigen concentration) or pH sensitivity (by increasing pH or decreasing contact time). 3.10 Assessment of pH-Sensitive Binding Using Phage ELISA

1. Using up to 23 individual colonies from the third and/or fourth round output clones, inoculate individual 300 μL cultures of 2
YT-Carb medium containing 1010 M13KO7 helper phage/mL within a 96-well deep well plate. Repeat this process for a colony that was transformed/infected with the Wt sdAb phagemid to serve as a control. Mount the deepwell plate at a slight angle and incubate at 37  C overnight with shaking at 235 rpm (see Note 27). 2. The same day use carbonate buffer to dilute concentrated stocks of antigen and BSA to final working concentrations of 2 μg/mL. Each individual clone will require four separate wells

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to test binding at two pH values (each pH includes a control well). For each clone include a Wt sdAb control (up to 24), add 100 μL/well of the antigen or BSA solution into two wells of a 96-well microtiter plate. Store plate overnight at 4  C. 3. The next morning, centrifuge the culture plate for 10 min at 3,700 rpm (1,400
g) using a swinging bucket rotor and collect the phage-containing supernatant for each sample. Store at 4  C until ready to use. 4. Continue microtiter plate preparation by removing the overnight coating solutions by inverting and then gently stamping on a paper towel to remove excess liquid. 5. Next, block all wells by adding 200 μL of PBS-T/BSA to all sample and control wells. Incubate at room temperature with gentle shaking for 2 h. Invert plate to remove liquid. 6. Wash the plate five times with PBS-T/BSA. Invert to remove liquid and gently stamp on paper towels between each wash to remove remaining liquid. 7. To prepare phage samples, using a 96-well deep well plate, add 100 μL of each phage-containing supernatant collected in step 3 to 200 μL of PBS-T/BSA to dilute the phage into pH 7.4 buffer. Repeat this process with acetate-T/BSA to dilute the phage into pH 4.0 buffer. 8. Add 100 μL of the diluted phage (whether pH 7.4 or pH 4.0) to the relevant sample and control wells. Incubate at room temperature for 1 h with shaking. 9. After incubation, wash the plate three times with PBS-T/BSA (for pH 7.4 wells) or acetate-T/BSA (for pH 4.0 wells). Then, wash all wells two times with PBS-T/BSA. 10. Generate a dilution of an anti-M13 antibody HRP conjugate in the range of 1:2,500 to 1:4,000 using PBS-T/BSA. Add 100 μL of the diluted antibody to each well and incubate at room temperature with shaking for 30 min (see Note 28). 11. Wash all wells four times with PBS-T/BSA and twice with PBS-B. 12. Add 100 μL of TMB substrate to each well and incubate at room temperature for 10 min. Quench the reaction with 100 μL 1 M sulfuric acid and immediately measure the absorbance at 450 nm (see Note 29). 13. Relative to background signals and the wild-type clone, evaluate the binding response for the two pH conditions to assess pH-dependent sdAb clones. 14. Positive clones can then be sub-cloned, expressed, and purified as soluble sdAb monomers, and characterized in binding and functional assays.

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Notes 1. QIAprep 2.0 spin columns may be purchased separately from the kit. 2. The total number of unique clones should be calculated by counting codons for the wild-type and His residues, as well as any additional codons that are introduced in the degenerate combination to include His. 3. Most amino acid residues can be exchanged for His with only one or two nucleotide changes, resulting in 2 or 4 codons. Some residues, such as tryptophan, require additional degeneracy and may even introduce stop codons. Introduction of stop codons is allowed as long as the total diversity is below the maximum number of phage produced. In addition, it is ideal to avoid using codons that are not frequently used by E. coli. 4. Ultimately, the 1010 library limit stems from limitations in electroporation of E. coli SS320 cells with the degenerate phagemid vector (see step 4 in Subheading 3.6). It is important not to exceed the 1010 limit, otherwise 100% coverage of the library is not achieved. It is recommended that prior to electroporation of the library, the user performs a test electroporation including serial dilutions to determine electroporation efficiency and verify that 1010 transformants can be reached. The library diversity can always be reduced to be below the experimental electroporation number. In situations where the designed library exceeds the upper limit of 1010, the user may choose to probe only one or two regions (e.g., CDR1 and CDR3). This approach has the advantage of selecting potential intraloop cooperative effects, which tend to stem from nearby residues. 5. Since Kunkel mutagenesis is not 100% efficient, the use of a stop-codon template ensures only newly mutated (i.e., combinatorial mutant VHH variants) will produce full VHH polypeptides, thus eliminating Wt bias. 6. By leaving large stretches of complementary base pairs between stop codons, the problem can arise of a mixture of a primer properly annealing and primer partially annealing to the template strand. 7. When starting from a single colony, culture growth can be less predictable; therefore, it is often convenient to start more than one 2 mL culture. 8. The phage pellet is a faint white color. It is advised to carefully note the expected location where the pellet should form to avoid inadvertent disruption. If a phage pellet is accidently

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disrupted or difficult to observe, collect the supernatant and repeat centrifugation. 9. Make sure ethanol has been added to the PE buffer before use. 10. On a molar scale, the oligonucleotide:template ratio should be 3:1. Values provided assume an oligonucleotide:template length ratio of approximately 1:100. 11. The reaction can be stored in a 20  C freezer when complete. 12. A QIAquick DNA purification kit (Qiagen) may be used instead. 13. Work by the Kay lab suggested that growing at 25  C can enhance single-strand DNA yield by two- to seven-fold [23]. 14. Multiple tubes are used to purify the ssDNA as the ssDNA yield is often higher than the capacity of a single column (sometimes even more than two or three columns). The resulting uracilcontaining stop codon ssDNA template may be used for additional libraries, if stop codon coverage is suitable. For a single combinatorial library generation, a yield of 20 μg of ssDNA is suggested. 15. Two spin columns are used due to the high amount of DNA present. For some high yield phagemids, three or four spin columns may be used for maximum recovery. 16. Kunkel mutagenesis is approximately 50–80% efficient; therefore, it is expected to observe the original base at some frequency. 17. Increasing the number of clones sent for sequencing will increase the reliability of the estimates of successful mutagenesis efficiency. Modern high-throughput sequencing may also be used to greatly enhance sampling/reliability. For troubleshooting, it may also be of interest to determine the success rate of each primer. 18. The total library size of 1
1010 represents the “typical” upper limit for a routine phage display preparation and is dependent on the efficiency of the electroporation of E. coli SS320 cells with the dsDNA library and subsequent packaging into phage particles. It is important for the user to establish that they can reach this threshold (or what the “actual” threshold may be running through the protocol). The user can alter the size of the library, if needed. For those less familiar with phage display, one may want to choose to sample fewer residues and produce a library with a lower theoretical diversity. For example, if the final library theoretical diversity was 1
107, there would be plenty of room (103) to compensate for suboptimal electroporation.

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19. To avoid introducing bubbles into the system, avoid using the pipette’s second stop “blow out.” 20. If using a phagemid with an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter, IPTG should be included in the 500 mL culture. 21. It is important to stress that the number of experimental transformants sets the limitation on library coverage. For example, if a library had a total theoretical diversity of 1
1010, yet only 1
108 transformants were achieved, only 1% of the designed library would be sampled moving forward. This fractional coverage would not yield suitable results for the combinatorial His scanning approach. 22. Be mindful of potential phage contamination and proper decontamination. 23. For antigen concentrations of ~1 mg/mL, a greater than 20-fold molar excess of EZ-Link™ Sulfo-NHS-SS-Biotin reagent to antigen is recommended. For a 15 kDa hypothetical antigen under these conditions, this ratio is 75:1. Add this volume to the antigen solution and incubate on ice for 2 h. 24. Alternatively, size exclusion chromatography or desalting columns may be used to remove unreacted biotin. 25. If a Kingfisher instrument is not available, selection washes and incubations may be carried out in a microcentrifuge tube. Brief 10 s centrifugation spins followed by placing in a magnetic stand allows efficient condition changes. 26. The use of disposable 50 mL conical tubes helps reduce the chance of phage contamination; however, it is important to verify the maximum g force to avoid potential tube collapse/ shattering. Not all conical tubes can tolerate higher g forces. Alternatively, Oakridge tubes and a JA-20 rotor can be used. 27. While we have had success growing small-scale cultures in deep well plates, sometimes larger phage yields may be necessary, which can be generated by increasing the overnight culture volume to 5 mL. 28. While our early work used a GE Healthcare anti-M13-HRP antibody conjugate, it is no longer manufactured. In our hands, the anti-M13-HRP antibody conjugate from Sino Biological worked, but produced lower signal/noise. More recently, we have used the anti-M13-g8p monoclonal antibody from Antibody Design Laboratories, which may be an even better substitute with a higher signal/noise. Other antibody systems, such as anti-FLAG antibodies can also be explored for detection. 29. The absorbance can be measured at 652 nm before quenching.

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Acknowledgments This work is supported by NIH grant 1R15GM124607 to J.R.H. References 1. Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363: 446–448 2. Rahbarizadeh F, Rasaee MJ, ForouzandehMoghadam M et al (2005) High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli. Protein Expr Purif 44:32–38 3. Henry KA, MacKenzie CR (2018) Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs 10: 815–826 4. Laursen NS, Friesen RHE, Zhu X et al (2018) Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin. Science 362:598–602 5. De Munter S, Ingels J, Goetgeluk G et al (2018) Nanobody based dual specific CARs. Int J Mol Sci 19:403 6. Dong JX, Lee Y, Kirmiz M et al (2019) A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons. eLife 8: e48750 7. Hussack G, Arbabi-Ghahroudi M, van Faassen H et al (2011) Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. J Biol Chem 286:8961–8976 8. Hussack G, Hirama T, Ding W et al (2011) Engineered single-domain antibodies with high protease resistance and thermal stability. PLoS One 6:e28218 9. Hoey RJ, Eom H, Horn JR (2019) Structure and development of single domain antibodies as modules for therapeutics and diagnostics. Exp Biol Med 244:1568–1576 10. Schonichen A, Webb BA, Jacobson MP et al (2013) Considering protonation as a posttranslational modification regulating protein structure and function. Annu Rev Biophys 42: 289–314

11. Chaparro-Riggers J, Liang H, DeVay RM et al (2012) Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9. J Biol Chem 287:11090–11097 12. Igawa T, Ishii S, Tachibana T et al (2010) Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization. Nat Biotechnol 28:1203–1207 13. Tawfik DS, Chap R, Eshhar Z et al (1994) pH on-off switching of antibody hapten binding by site-specific chemical modification of tyrosine. Protein Eng 7:431–434 14. Davenport KR, Smith CA, Hofstetter H et al (2016) Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns. J Chromatogr B Analyt Technol Biomed Life Sci 1021: 114–121 15. Ito W, Sakato N, Fujio H et al (1992) The His-probe method: effects of histidine residues introduced into the complementaritydetermining regions of antibodies on antigenantibody interactions at different pH values. FEBS Lett 309:85–88 16. Sidhu SS, Lowman HB, Cunningham BC et al (2000) Phage display for selection of novel binding peptides. Methods Enzymol 328: 333–363 17. Sidhu SS, Weiss GA (2004) Constructing phage display libraries by oligonucleotide-directed mutagenesis. In: Clackson T, Lowman HB (eds) Phage display: a practical approach. Oxford University Press, Oxford, pp 27–41 18. Murtaugh ML, Fanning SW, Sharma TM et al (2011) A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches. Protein Sci 20:1619–1631 19. Pershad K, Sullivan MA, Kay BK (2011) Dropout phagemid vector for switching from phage

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displayed affinity reagents to expression formats. Anal Biochem 412:210–216 20. Kunkel TA (1985) Rapid and efficient sitespecific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492 21. DeLano WL (2002) The PyMOL molecular graphics system. https://pymol.org/2/. Accessed 5 Jan 2021

22. Tonikian R, Sidhu SS (2012) Selecting and purifying autonomous human variable heavy (VH) domains. Methods Mol Biol 911: 327–353 23. Huang R, Fang P, Kay BK (2012) Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phagedisplay libraries. Methods 58:10–17

Chapter 14 Humanization of Camelid Single-Domain Antibodies Traian Sulea Abstract Humanization of therapeutic antibodies derived from animal immunizations is often required to minimize immunogenicity risks in humans, which can cause potentially harmful and serious side effects and reduce antibody efficacy. Humanization is typically applied to conventional monoclonal antibodies derived in rodents as well as single-domain antibodies isolated from camelids and sharks (VHHs and VNARs). A streamlined protocol is described here for sequence humanization of camelid VHHs, which represent a promising biotherapeutic format with many desirable attributes. From human framework selection and complementarity-determining region grafting strategies to empirical scoring for prioritization of backmutations, step-by-step instructions, and templates are provided along with bioinformatics resources to assist each step of the humanization process. Alternative approaches, warnings, and caveats are also presented. Key words Human framework, Complementarity-determining region grafting, Structural model, Back-mutations, Empirical scoring, Humanized variants, VHH, Single-domain antibody

1

Introduction Monoclonal antibodies (mAbs) represent the largest segment of approved biotherapeutics, which has grown exponentially in number, market value, and range of disease indications during the past decade [1–4]. A significant proportion of mAbs is currently derived from animals by immunization with human antigens. Animalderived antibodies can lead to immunogenicity in humans, which in turn can cause reduced antibody efficacy and even adverse or lethal side effects. For example, antibodies derived via mouse hybridoma technology [5, 6] can generate human anti-mouse antibodies [7]. Therefore, therapeutic antibodies derived from non-human sources have to be “humanized” for use in humans.

Supplementary Information The online version of this chapter (https://doi.org/10.1007/978-1-0716-20755_14) contains supplementary material, which is available to authorized users. 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_14, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Through humanization, most of the antibody structure is replaced with the human counterpart [8, 9]. A first approach is called chimerization, in which a chimeric antibody is generated by replacing the constant regions of the heavy and light chains of a murine IgG antibody with the constant regions of a functionally equivalent human IgG isotype. Chimerization reduces the non-human content of a mAb to ~33%. The term humanization typically refers to also replacing most of the variable domains of the heavy and light chains of the source antibody with human sequences, to reach a non-human content of only ~5%. During the humanization of variable domains, the complementaritydetermining regions (CDRs) of the source antibody are retained in order to preserve antigen binding, whereas the framework regions (FRs) are replaced with human FRs in a procedure known as CDR grafting [10]. Clinical data indicates that antibody humanization significantly reduces the incidence of a marked antiantibody response in humans [11]. Currently, about 41% of approved therapeutic antibodies and antibody-drug conjugates are humanized, 14% are chimeric, and only 4% are fully murine [4]. Development of alternate antibody discovery technologies such as transgenic mice, single B-cell soring, and phage, yeast, or mammalian display led to ~38% of approved antibodies having entirely human sequences [3, 4]. Single-domain antibodies (sdAbs), also called nanobodies, are very attractive biologics that present several advantages over conventional mAbs in terms of ability to access cryptic and concave epitopes, increased stability, high modularity, and smaller size, thus potentially leading to excellent efficacy, pharmacokinetics, and developability profiles [12–16]. Animal sources of sdAbs are mainly the camelids (alpaca, llama, and dromedary camel) and cartilaginous fishes (sharks) which produce heavy-chain-only isotypes devoid of light chains and with antigen binding mediated by only one variable domain [12]. These variable domains, called VHH (camelids) and VNAR (sharks) require humanization for use in humans. The protocols described here focus on the humanization of camelid VHHs, which are very popular due to their versatility for engineering and are currently being tested in late-stage clinical trials with one marketing approval [17], whereas the development of shark-derived VNARs is still at a preclinical stage [12, 18]. While similar to the humanized universal scaffold method [19], the VHH humanization protocol described here further streamlines the technique. Methods are presented for human framework selection, CDR-grafting strategy, and most importantly, a simple scheme for empirical scoring of mutated FR position for prioritization of backmutations. Bioinformatics resources to assist each step of the humanization process are recommended based on prior experience.

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Materials Most tools exemplified in this section can be accessed online via webservers and downloaded for local installation. They may be substituted with similar tools appropriate for each task. 1. Human template sequence: It is recommended to use the VH3-23/DP47 (IGHV3-23*01) and IGHJ4*04 human germline sequence as templates for most VHH humanization campaigns. The sequences of these templates are available from the IMGT database [20] (http://www.imgt.org/) (Fig. 1a). A portion of the CDR3 can be left unspecified as the entire CDR3 will be grafted from the VHH to be humanized. The VH3-23/DP47 human sequence provides the most similar framework region for the majority of camelid-derived VHH sequences (see Notes 1 and 2). 2. Protein sequence alignment software: Use the MAFFT [21] webserver (https://mafft.cbrc.jp/alignment/server/) with default settings or download a local copy (https://mafft.cbrc. jp/alignment/software/). 3. Homology modeling software: Use the ABodyBuilder [22] webserver (http://opig.stats.ox.ac.uk/webapps/newsabdab/ sabpred/abodybuilder/) to build three-dimensional models of the VHH. Chose MODELLER as the method to use for ab initio modeling and Kabat as the numbering scheme of the final models. This tool can also be used indirectly for sequence numbering and CDR/FR delineation according to widely accepted rules/definitions (Kabat [23, 24], Chothia [25, 26], and IMGT [27]) as an alternative to the tools listed in steps 5d and 5e below. 4. Molecular graphics software: Download a local copy of PyMOL (Schro¨dinger, LLC) (https://pymol.org/) and visit PyMOL Wiki (https://pymolwiki.org/), a community-run support site of the PyMOL molecular viewer. 5. Antibody sequence annotation tools (not required in the standard protocol described here, but discussed in the Notes section). (a) Identification of the closest human germline VH family can be done with the SUBIM program [28] implemented in the Human Subgroup Assignment server (http://www. bioinf.org.uk/abs/hsubgroup.html) for the three main human VH branches.

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Fig. 1 Generation of a camelid VHH sequence with a 100% humanized framework. (a) Assembly of the human VH template from the human variable and junction germline regions. Intervening amino-acid residues in the hypervariable CDR3 are arbitrarily shown as “X”. (b) Delineation of CDR and FR segments on the sequence alignment between the source camelid VHH M79 and the human VH template. CDR loops are defined according to Kabat for CDR2 and CDR3, and the union of Kabat and Chothia definitions for CDR1. Mutated positions between camelid and human FRs are identified below the sequence by “x”. Kabat numbering is shown above the sequence alignment. (c) Assembly of the humanized VHH sequence is achieved by combining CDR segments of the source camelid VHH and FR segments of the human VH template

(b) Identification of the closest human germline VH sequence can be done with the IMGT/V-QUEST [29, 30] server (http://www.imgt.org/IMGT_vquest/input). This requires the nucleotide sequence of the antibody, which can be obtained by back-translation of the protein sequence using a tool such as the Reverse Translate server (https://www.bioinformatics.org/sms2/rev_trans.html). (c) Consensus sequences corresponding to all seven human VH subgroups [31] can be accessed from AHo’s Amazing Atlas of Antibody Anatomy resources (https://www.bioc. uzh.ch/plueckthun/antibody/Sequences/ and https:// www.bioc.uzh.ch/plueckthun/antibody/Modelling/ index.html); antigen contact propensities along the VH sequence can also be found at this online resource. (d) Rules for CDR definitions according to Kabat [23, 24] and Chothia [25, 26] can be found among the resources of Andrew C. R. Martin’s group (University College London, UK) (http://www.bioinf.org.uk/abs/info. html#cdrdef and http://www.bioinf.org.uk/abs/info. html#cdrid) and according to IMGT by using the IMGT/V-QUEST tool [29, 30] (see step 5b above). (e) Antibody sequence and structure numbering according to Kabat [24] and Chothia [25, 26] can be done with the

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AbNum [32] server (http://www.bioinf.org.uk/abs/ abnum/), whereas IMGT sequence numbering [27] and CDR correspondence between various rules using the IMGT scientific char t (http://www.imgt.org/ I M G T S c i e n t i fi c C h a r t / N u m b e r i n g / IMGTcorrespondence.html). (f) Repositories of human germline sequences can be accessed from IMGT [20] (http://www.imgt.org/) and VBASE2 [33] (http://www.vbase2.org/) databases. 6. Immunogenicity prediction software (not required in the standard protocol described here, but discussed in the Notes section): Use the IEDB T-cell epitope prediction and de-immunization [34] webserver (http://tools.iedb.org/deimmunization/) with default settings. 7. Relative folding stability calculations (not required in the standard protocol described here, but discussed in the Notes section): Use the machine learning-based mCSM-Stability [35] webserver (http://biosig.unimelb.edu.au/mcsm/stability/) with default settings by uploading the homology model (in PDB format) generated with the software suggested at step 3 above, and a list of single-point mutations, e.g., as suggested by de-immunization predictions with the tool recommended at step 6 above.

3

Methods This section describes a standard protocol for humanization of a sdAb, starting from a camelid VHH domain sequence. The sequence of the camelid VHH called M79 that binds serum albumin [36] is used as an example.

3.1 Delineation of CDRs and FRs

The CDR and FR sequences of the camelid VHH to be humanized and the human VH template are first determined by applying the rules and tools provided in the Materials section. This humanization protocol uses the Kabat [23, 24] definition of CDR2 and CDR3 and the union of Kabat [23, 24] and Chothia [25, 26] definitions for CDR1. This is shown in Fig. 1b on a sequence alignment between the VHH M79 and the human VH template. This also serves as a model to delineate the CDR and FR segments for other camelid VHHs subjected to humanization. See Note 3 regarding the choice of CDR/FR definition recommended here for humanization, as well as alternate options. At this step, residues that have to be mutated in the four FR segments between the source VHH sequence and the human VH template can be flagged (denoted by an “x” in Fig. 1b).

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3.2 CDR Grafting into Human FRs

The next step is to “stitch” the three CDR segments of the source camelid VHH sequence to the four FR segments of the human VH template, in the appropriate order. This is illustrated in Fig. 1c, and it follows naturally from the sequence alignment previously constructed (Fig. 1b). At the end of this step, the amino-acid sequence of a fully (100%) humanized VHH is generated, whereby the entire FR is of human origin and the CDR is of camelid origin.

3.3 Design of BackMutation Variants

In our experience, the 100% humanized VHH variant generated in the previous step will likely possess weakened antigen-binding affinity relative to the parental VHH, to varying degrees depending on the particular antibody. To address this possible problem, a straightforward procedure for the design of alternative humanized sequences is presented with an aim to mitigate antigen-binding affinity loss. This procedure typically consists of introducing “back-mutations,” which are amino-acid residues in the source antibody framework that may directly or indirectly impact antigen binding. A general standardized template is provided here to assist mapping and ranking of those potentially problematic positions and guide the introduction of back-mutations in the 100% humanized variant. The goal of this step is to find an optimal balance between the degree of humanness and retaining antigen binding, by proposing only a small number of humanized variants for recombinant production and testing. The back-mutation step is critical to the overall success of the humanization process.

3.3.1 Mapping and Ranking Liabilities at Mutated Positions

Figure 2a provides a template that can be used to score and rank the liabilities of any humanized VHH sequence by: (a) substituting the illustrated humanized sequence with the user’s humanized sequence, (b) ensuring the FR and CDR boundaries are correctly matched, and (c) updating the mutated positions relative to the source antibody sequence. This template includes a simple scoring system based on a set of attributes and features annotated for each position along the VHH FR sequence. These attributes and features are detailed in Table 1 along with their associated empirical scores. Some of these attributes relate to a potential direct involvement in antigen binding (positions labeled with C, c, A and a codes), others to a predicted role in maintaining the conformation of the CDRs (positions labeled with S, V, and v codes), or other important properties like solubility that can indirectly affect antigen binding (positions labeled with the s code) [19, 37–41]. The degree of amino-acid residue burial or surface exposure, which can be calculated based on a modeled antibody structure (vide infra), is not considered in this scoring system (see Note 4). The larger the empirical score, the more important that particular attribute is with respect to its predicted effect on binding affinity. One can simply sum up the scores at a given position to calculate its total back-mutation score (BM-Score), as annotated above the

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Fig. 2 Representative template used to determine the liabilities at various FR positions of the 100% humanized VHH sequence and to assist in the selection and prioritization of back-mutations. (a) The back-mutation score (BM-Score) calculated along the humanized sequence. Attributes described in Table 1 used to calculate the BM-Score are indicated above the humanized sequence. Mutated positions between camelid and human FRs are identified below the humanized sequence by “x”. (b) Mapping of positions with a BM-Score > 0 on the 3D homology model of the source camelid VHH, which is shown as a ribbon diagram with CDR loops colored in brown and FR segments in grey. Positions identified in panel (a) as having a BM-Score > 0 are rendered as spheres centered on Cα atoms, color-coded in tones of blue from the highest value shown in dark blue to the lowest value shown in light blue. Positions with a BM-Score > 1 are labeled by Kabat numbering and those with a BM-Score ¼ 1 are unlabeled for clarity. Two orthogonal views are shown Table 1 Scoring system of sequence positions for back-mutations Code

Attribute

Score

C

Kabat position 94 (part of CDR3 according to IMGT definition)

5

c

Kabat position 93 (part of CDR3 according to IMGT definition)

4

S

FR2 position required for stability and CDR3 conformation (Kabat 37 and 47)

4

s

FR2 position required for VHH solubility (Kabat 44 and 45)

3

A

Residue belongs to CDR4 (also called CDR2a or D-E loop)

1

a V v a

FR position with propensity for antigen contact Position in the Vernier zone

a

1

b

FR residue adjacent to CDR that is not part of the Vernier zone

1 c

Retrieved from AHo’s Amazing Atlas of Antibody Anatomy online resource (see step 5c in Subheading 2) According to [9, 37] c This annotation requires a 3D homology model (see Subheading 3.3.2) b

1

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humanized sequence shown in Fig. 2a. By focusing on the positions with the largest BM-Scores at the humanized positions (marked by an “x”), one arrives at a prioritization among all FR positions for back-mutation to the respective amino acid in the source VHH sequence. Only the v code annotation needs to be updated for any particular VHH, which is done using a 3D structural model as described in the next section. 3.3.2 Using Homology Models for Structural Information

Running the ABodyBuilder webserver with the camelid VHH sequence input for the heavy chain will produce a 3D homology model of the VHH structure that can be download for visual inspection and geometric measurements on a local computer. After loading and rendering of the homology model in the PyMOL molecular graphics software, a sequence editor is typically used to highlight the three CDR loops both on the sequence and structure according to delineation shown in Figs. 1b and 2a. To determine the residues corresponding to the v annotation in Fig. 2a, a selection of the three CDR loops can be made and then modified to include only residues within 5 Å around the CDR selection. This can be done in PyMOL by executing the following sequence of actions: “A > modify > around > residues within 5 A” on the “(sele)” tab located in the PyMOL side panel. The resulting new selection of FR residues will then be used to update the v annotation and the BM-Scores in Fig. 2a for the particular VHH subjected to humanization. The BM-Scores can also be optionally mapped on the modeled 3D structure of the VHH, with an example mapping illustrated in Fig. 2b (also see Supplemental Material). In addition, the structural model is useful to closely examine the interactions made by the amino-acid residues substituted during humanization, with the protein environment and particularly with the CDR. This may affect the prioritization of certain residues especially those in the low-to-medium range of the BM-Score. See Note 5 for additional considerations about the use of 3D structural models in VHH humanization.

3.3.3 Incremental Generation of BackMutated Sequences

Ideally, the effects of various single-point back-mutations would be probed independently. The protocol described here provides a more cost-efficient alternative, whereby various back-mutations are added progressively in the order of importance according to BM-Score values. The recommended order in which back-mutations are progressively added is shown in Table 2, with examples of resulting variants presented in Fig. 3 for the M79 VHH. Humanized variants generated using this method will have a decreased degree of humanness with a correspondingly decreased risk of affinity loss as more back-mutations are amassed. Experimental testing on the panel of humanized variants will determine variants that achieve an optimal balance between these two important

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Table 2 Suggested order for incrementing back-mutations into humanized variants Varianta

Kabat position (region)

1

94 (FR3-Kabat; CDR3-IMGT)

2

37 and 47 (FR2)

3

93 (FR3-Kabat; CDR3-IMGT)

4

44 and 45 (FR2)

5

Next highest scored;b 1–2 positions

6

Next highest scored;b 1–2 positions

a

Each variant includes the back-mutations of all previous variants Based on Fig. 2 and Table 1

b

Fig. 3 Example of a set of humanized variants with positions back-mutated to the source camelid VHH. The Humanized v0 variant with a 100% human FR has the same sequence as shown in Fig. 2. Humanized variants v1 through v5 progressively introduce back-mutations following the calculated BM-Score values and the guidelines provided in Table 2. Back-mutated positions are indicated by blue arrows coded by color intensity in the order of priority (dark blue—first priority; light blue—last priority)

parameters. Based on our experience with this humanization method, VHH antigen-binding affinity may evolve across the set of back-mutated humanized variants designed in this manner (see Note 6 examples). For additional properties of the humanized variants in terms of their expected immunogenicity, suitability for Protein A affinity purification, and multi-factorial experimental characterization, see Notes 7–9, respectively.

4

Notes 1. A complete picture of camelid germline V gene repertoires and the classification of VH (IGHV) and VHH (IGHVH) genes is still missing [18]. At present, the vast majority of camelid VHH genes appear to be homologous to the human VH3 family and the humanization protocol presented here is directed to those VHHs. Another promiscuous class of V genes in camelids was identified that is closely related to the human VH4 family; however, this class largely contributes to the conventional heterodimeric antibody

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repertoire and lacks the hallmark solubilizing VHH residues in FR2. Nevertheless, antigen-specific VH4-family fragments with VHH-like stability and solubility have been isolated from an immune llama library [42]. For humanization of camelid VH4-like VHHs and VNARs isolated from sharks, identification of appropriate human FRs by mining human germline databases (see tools suggested in steps 5a, 5b, and 5f in Subheading 2) should be performed and then followed by the same humanization principles described here for camelid VH3-like VHH humanization. 2. Aside from using specific human germline sequences as FR templates, it is possible to use human consensus sequences determined for various human VH families [31]. To locate the appropriate human VH family consensus sequence, use the resources given at Subheading 2, step 5c. Note, however, that consensus sequences are artificial and although idiosyncratic somatic hypermutation have been evened out, they may contain unnatural sequence motifs which are immunogenic. 3. The delineation of CDR/FR proposed here can be replaced by other widely accepted definitions (see step 5d in Subheading 2). Alternative humanization approaches are based on grafting abbreviated CDR segments (in which the anchors of the CDR loops are also humanized) or specificity-determining regions (SDR, further reducing the CDR to only residues directly engaged in antigen contacts) [8, 9, 43]. The CDR/FR delineation employed in this protocol (i.e., Kabat/Chothia for CDR1 and Kabat for CDR2 and CDR3) adopts the more structurally conservative philosophy of grafting a larger CDR into a smaller human FR, which lowers the risk of reduced antigen-binding affinity. 4. While the so-called veneering or resurfacing humanization method only targets surface-exposed FR positions [8, 9, 44], the CDR grafting and back-mutation scoring protocol presented here is not influenced by the degree of surface exposure or burial of humanized positions. The decision not to include this attribute into back-mutation decisions was based on the molecular mechanisms of T-cell responses against foreign antigens. The antigen is proteolytically cleaved in antigenpresenting cells and the resulting short peptides are loaded into MHC-II and presented to the T-cell receptor, regardless if they are buried in the core of the folded protein antigen or located at the surface. 5. Generating a homology model for the source VHH is not required when an experimentally derived 3D structure is available from X-ray crystallography or NMR spectroscopy. Moreover, if the experimental 3D structure of the VHH–antigen

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complex is also available, some annotations in the back-mutation scoring scheme in Fig. 2a can be replaced with the precise knowledge of the antigen contacts provided by the structure of the complex. A homology model of the humanized VHH could also be built and compared with the structure of the source VHH in terms of interactions made with CDR residues at mutated FR positions, or in terms of predicted changes in interactions with the antigen (if the source VHH–antigen complex structure is known). Aside from examining the degree of conformational changes and displacements, molecular graphics software can be used to examine polar contacts, hydrophobic packing, and charge reversals around mutated position adjacent to the CDR. 6. VHH humanization using this protocol has been recently published [36]. This and other unpublished data indicate that the so-called 100% humanized variant with a Lys residue at position 94 generally loses significant antigen-binding affinity and can be eliminated altogether from the panel of humanized variants. Thus, the variant back-mutated at position 94 (variant 1 in Table 2) can be considered as the most humanized variant, which unfortunately almost always suffers from significant reduction in antigen binding. In some cases, restoration of antigen binding occurs after back-mutations at positions 37/47 (variant 2 in Table 2). However, in other cases, especially if the source antibody has a non-alanine residue at position 93, recovery of antigen binding only occurs after backmutation at position 93. Finally, in some cases, antigen binding only gradually recovers as additional back-mutations are introduced. Such a gradual restoration of antigen binding was observed during humanization of a VHH with a very short CDR3 [36], which may explain why back-mutations at many positions supporting the entire CDR are required to recover the original binding affinity. 7. It is important to appreciate that, somewhat paradoxically, increasing the degree of human sequence via CDR grafting does not necessarily guarantee decreased immunogenicity in humans. This is due to the junctions between source CDR and human FR segments. T-cell immunogenicity elicited via MHC class-II antigen-presenting molecules is triggered by 9- to 22-mer peptides generated by proteolytic digestion of foreign antigens in antigen-presenting cells. While T-cell epitopes located inside the FR are eliminated through humanization, T-cell epitopes that overlap with the CDR/FR junctions are not. The presence of MHC-II-binding peptides in the parent VHH and in its humanized variants can be predicted with in silico tools. While the majority of potential T-cell epitopes are eliminated by humanization, in some cases, FR humanization

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leads to increased predicted binding affinity and promiscuity to human MHC-II alleles for certain T-cell epitopes at CDR/FR junctions. Humanized variants displaying these epitopes should be deprioritized or subjected to de-immunization by selecting alternate back-mutations. De-immunization predictions with automated single-residue scanning mutagenesis tools can greatly assist the selection of appropriate mutations in the FR in problematic VHHs (see tool recommendation at step 6 in Subheading 2). The panel of suggested de-immunizing mutations can be further analyzed by stability predictions to ensure correct folding of the mutant VHH (see suggested tool in step 7 of Subheading 2). 8. Humanized VHH variants generated with this protocol have a high likelihood of being amenable to tagless purification by protein A affinity chromatography. Required FR residues for binding to protein A are G15, D17, R19, R66, T68, S70, Q81, N82A, and S82B, which are provided by the FR template recommended here [45, 46]. However, other required positions are in the CDR2 as defined according to the Kabat rule, including K/T57, Y59, K64, and G65 [45]. The latter three residues are inside CDR2 according to the Kabat definition but in the FR according to the IMGT definition and therefore could be subjected to humanization relatively safely if protein A-based purification is required. The only outlier is position 57, located inside CDR2 by all definitions, and its mutation to an amino acid tolerated for protein A binding [45] should be verified for retention of antigen binding. 9. The focused set of humanized variants can be produced recombinantly in various expression systems (e.g., bacteria, yeast, or mammalian cells) and tested in a variety of assays. A dataset consisting of expression yield, heterogeneity, aggregation, folding stability, binding affinity, and specificity, along with the calculated degree of humanness and the number, promiscuity, and affinity of predicted T-cell epitopes, is typically required for informed lead selection. References 1. DeFrancesco L (2019) Drug pipeline 1Q19. Nat Biotechnol 37:579–580 2. Kaplon H, Muralidharan M, Schneider Z et al (2020) Antibodies to watch in 2020. MAbs 12: 1703531 3. Lu RM, Hwang YC, Liu IJ et al (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27:1 4. Kang TH, Jung ST (2020) Reprogramming the constant region of immunoglobulin G

subclasses for enhanced therapeutic potency against cancer. Biomolecules 10:382 5. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497 6. Little M, Kipriyanov SM, Le Gall F et al (2000) Of mice and men: hybridoma and recombinant antibodies. Immunol Today 21:364–370 7. Klee GG (2000) Human anti-mouse antibodies. Arch Pathol Lab Med 124:921–923

Humanization of Camelid Single-Domain Antibodies 8. Gonzales NR, De Pascalis R, Schlom J et al (2005) Minimizing the immunogenicity of antibodies for clinical application. Tumour Biol 26:31–43 9. Safdari Y, Farajnia S, Asgharzadeh M et al (2013) Antibody humanization methods—a review and update. Biotechnol Genet Eng Rev 29:175–186 10. Riechmann L, Clark M, Waldmann H et al (1988) Reshaping human antibodies for therapy. Nature 332:323–327 11. Hwang WYK, Foote J (2005) Immunogenicity of engineered antibodies. Methods 36:3–10 12. Konning D, Zielonka S, Grzeschik J et al (2016) Camelid and shark single domain antibodies: structural features and therapeutic potential. Curr Opin Struct Biol 45:10–16 13. Desmyter A, Spinelli S, Roussel A et al (2015) Camelid nanobodies: killing two birds with one stone. Curr Opin Struct Biol 32:1–8 14. Hussack G, Hirama T, Ding W et al (2011) Engineered single-domain antibodies with high protease resistance and thermal stability. PLoS One 6:e28218 15. Kijanka M, Dorresteijn B, Oliveira S et al (2015) Nanobody-based cancer therapy of solid tumors. Nanomedicine (Lond) 10: 161–174 16. Van Audenhove I, Gettemans J (2016) Nanobodies as versatile tools to understand, diagnose, visualize and treat cancer. EBioMedicine 8:40–48 17. Morrison C (2019) Nanobody approval gives domain antibodies a boost. Nat Rev Drug Discov 18:485–487 18. Arbabi-Ghahroudi M (2017) Camelid singledomain antibodies: historical perspective and future outlook. Front Immunol 8:1589 19. 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 20. Lefranc MP, Giudicelli V, Duroux P et al (2015) IMGT®, the international ImMunoGeneTics information system® 25 years on. Nucleic Acids Res 43(Database issue): D413–D422 21. Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20:1160–1166 22. Leem J, Dunbar J, Georges G et al (2016) ABodyBuilder: Sutomated antibody structure prediction with data-driven accuracy estimation. MAbs 8:1259–1268

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23. Wu TT, Kabat EA (1970) An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J Exp Med 132:211–250 24. Kabat EA, Wu TT (1991) Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementaritydetermining regions to binding of antibodycombining sites. J Immunol 147:1709–1719 25. Chothia C, Lesk AM (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol 196:901–917 26. Al-Lazikani B, Lesk AM, Chothia C (1997) Standard conformations for the canonical structures of immunoglobulins. J Mol Biol 273:927–948 27. Lefranc MP, Pommie C, Ruiz M et al (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 27:55–77 28. Deret S, Maissiat C, Aucouturier P et al (1995) SUBIM: a program for analysing the Kabat database and determining the variability subgroup of a new immunoglobulin sequence. Comput Appl Biosci 11:435–439 29. Brochet X, Lefranc MP, Giudicelli V (2008) IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res 36(Web Server issue):W503–W508 30. Giudicelli V, Brochet X, Lefranc MP (2011) IMGT/V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences. Cold Spring Harb Protoc 6:695–715 31. Knappik A, Ge L, Honegger A et al (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57–86 32. Abhinandan KR, Martin AC (2008) Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol 45:3832–3839 33. Retter I, Althaus HH, Munch R et al (2005) VBASE2, an integrative V gene database. Nucleic Acids Res 33(Database issue): D671–D674 34. Dhanda SK, Grifoni A, Pham J et al (2018) Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity. Immunology 153:118–132

312

Traian Sulea

35. Pires DE, Ascher DB, Blundell TL (2014) mCSM: predicting the effects of mutations in proteins using graph-based signatures. Bioinformatics 30:335–342 36. van Faassen H, Ryan S, Henry KA et al (2020) Serum albumin-binding VHHs with variable pH sensitivities enable tailored half-life extension of biologics. FASEB J 34:8155–8171 37. Foote J, Winter G (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol 224:487–499 38. Conrath K, Vincke C, Stijlemans B et al (2005) Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH. J Mol Biol 350:112–125 39. Bond CJ, Wiesmann C, Marsters JC et al (2005) A structure-based database of antibody variable domain diversity. J Mol Biol 348: 699–709 40. Sircar A, Sanni KA, Shi J et al (2011) Analysis and modeling of the variable region of camelid single-domain antibodies. J Immunol 186: 6357–6367 41. Makabe K, Nakanishi T, Tsumoto K et al (2008) Thermodynamic consequences of mutations in vernier zone residues of a

humanized anti-human epidermal growth factor receptor murine antibody, 528. J Biol Chem 283:1156–1166 42. Deschacht N, De Groeve K, Vincke C et al (2010) A novel promiscuous class of camelid single-domain antibody contributes to the antigen-binding repertoire. J Immunol 184: 5696–5704 43. Kashmiri SV, De Pascalis R, Gonzales NR et al (2005) SDR grafting—a new approach to antibody humanization. Methods 36:25–34 44. Padlan EA (1991) A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligandbinding properties. Mol Immunol 28:489–498 45. Henry KA, Sulea T, van Faassen H et al (2016) A rational engineering strategy for designing protein A-binding camelid single-domain antibodies. PLoS One 11:e0163113 46. Graille M, Stura EA, Corper AL et al (2000) Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci U S A 97:5399–5404

Chapter 15 Creation of Multimeric Single-Domain Antibodies Using Bacterial Superglues Paul J. Wichgers Schreur, Sandra van de Water, and Jeroen Kortekaas Abstract Multimerization of single-domain antibodies (sdAbs) is instrumental for construction of antibody molecules with high avidity, extended in vivo half-life, and tailor-made biological activity. Two-component superglues, based on bacterium-derived peptides (Tags) and small protein domains (Catchers) that form isopeptide bonds when in close proximity, enable the creation of multimers by simply mixing of the individual components. Here, we provide detailed methods for the construction of sdAbs and scaffolds bearing genetically fused superglue components and their assembly into multimeric complexes. Key words Single-domain antibody, VHH, Multimers, Superglue, SpyTag:SpyCatcher, SnoopTag: SnoopCatcher

1

Introduction Multimers of sdAbs, such as camelid-derived variable domains of heavy-chain-only antibodies (VHHs), offer several advantages over their monomeric counterparts. Multivalent and multispecific antibodies show improved avidity/affinity, specificity, functionality, and in vivo pharmacokinetics compared to monomeric sdAbs [1, 2]. Conventionally, genetic fusion is used to create sdAb multimers. However, selection of the best sdAb combinations and formats including optimal linker length can be very challenging and time consuming, as all combinations should be constructed and expressed individually [3]. Furthermore, head-to-tail genetic fusions of sdAbs may reduce the affinity of the second or subsequent sdAbs in the complex, due to steric interference with their antigen-binding complementarity-determining regions (CDRs) [4]. The discovery of bacterial superglues enables the creation of unique and novel protein architectures. Molecular superglues are based on short peptides and small protein domains derived from

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_15, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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bacterial proteins that form an irreversible covalent isopeptide bond upon close contact [5]. One of the first and most frequently used two-component bacterial superglues, referred to as SpyTag:SpyCatcher, was developed by splitting the immunoglobulin-like collagen adhesion domain (CnaB2) of the fibronectin-binding protein (FbaB) of Streptococcus pyogenes into a peptide (SpyTag) and a protein fragment (SpyCatcher) [6]. The irreversible covalent amide bond is formed within minutes after mixing the two components and occurs without the need for specific buffers or enzymes. Furthermore, bond formation is highly specific and robust under various conditions [5]. The SpyTag:SpyCatcher superglue has already been applied in several disciplines and triggered the search for similar peptide-protein partners such as SnoopTag and SnoopCatcher [7, 8]. Here, we describe the design and production of Tag-conjugated sdAbs and scaffolds containing Catchers and provide guidance in the use of these individual building blocks to generate higher order sdAb-based multimers (Fig. 1).

2

Materials All materials should be stored as indicated, or according to the manufacturer’s instructions. Furthermore, all buffers should be prepared using double distilled water (ddH2O).

2.1 Construction of sdAbs and Scaffolds with Genetically Fused Tags and Catchers

1. DNA cloning and sequence analysis software (e.g., SnapGene, Clone Manager, DNAStar). 2. DNA-encoding sdAb(s) of interest. Genes encoding sdAbs previously cloned into a phagemid vector, expression vector, or other vector are well suited. 3. Elastin-like protein (ELP) scaffold amino acid sequence and DNA sequence (Table 1) (see Note 1). 4. SpyTag, SnoopTag, SpyCatcher, and SnoopCatcher amino acid or DNA sequences (Table 1) (see Note 1). 5. Yeast episomal expression plasmids pRL480-stuffer and pRL481-stuffer (available from Addgene; see Note 2). These plasmids encode the following: yeast signal sequence, QVQ-stuffer (1 kb)-VSS, (GGGGS)3 linker, SpyTag (pRL480) or SnoopTag (pRL481), GAA linker, and the C-terminal His6 Tag. The stuffer can be replaced by a VHH of interest by cloning using PstI and BstEII restriction sites. 6. Escherichia coli expression plasmid pQE-80L in ddH2O (Qiagen, Hilden, Germany) (see Note 3). 7. Temperature controlled shaking incubator. 8. Plasmid mini- or midiprep kits.

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Fig. 1 Schematic representation of superglue-based sdAb multimerization. Following characterization of monomeric sdAbs for antigen specificity, Spy and Snoop-Tagged sdAb variants are produced which are covalently coupled to scaffolds of interest containing SpyCatchers and/or SnoopCatchers. The sdAb multimers can be used with or without purification in assays of choice. (Figure produced using BioRender.com)

9. PstI, BstEII, EcoRI, and HindIII restriction endonucleases and buffers (preferably high fidelity [HF] versions). 10. Rapid DNA Switzerland).

Dephosphorylation

Kit

(Roche,

Basel,

11. DNA Clean & Concentrator Kit and Gel DNA Recovery kit (Zymo Research, Irvine, CA, USA). 12. T4 DNA ligase (400 U/μL) and buffer (New England Biolabs, Ipswich, MA, USA). 13. Chemically competent JM109 E. coli cells for amplification of plasmids (Promega, Madison, WI, USA). Store at
70  C. 14. Super Optimal broth with Catabolite repression (SOC) medium: 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCl, 10 mL of 250 mM KCl, final volume 1 L of ddH2O. Autoclave, then supplement with 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose. 15. 2 Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). 16. PCR primer VHH-For: 50 - CTAGTGCGGCCGCTGGA GACGGTGACCTGGGT-30 .

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Table 1 Amino acid sequences of protein domains for construction of multimeric sdAbs using bacterial superglues Domain/tag

Amino acid sequence

SpyTag

AHIVMVDAYKPTK

SpyCatcher

DIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEED SATHIKFSKRDEDGKELAGATMELRDSSGKTIS TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFT VNEQGQVTVNGKATKGDAHIDGPQGIWGQLEWKK

SnoopTag

KLGDIEFIKVNK

SnoopCatcher

SSGLVPRGSHMKPLRGAVFSLQKQHPDYPDIYGAI DQNGTYQNVRTGEDGKLTFKNLSDGKYR LFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVP QDIPATYEFTNGKHYITNEPIPPKGPQGIWGQLDGHGVG

ELP (1–3)a

NL(GVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPG EGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPG)1-3GLL

N-terminal his Tag MKGSSHHHHHH C-terminal His tag HHHHHHStopb Twin Strep-tag

GSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

Yeast signal peptide

MMLLQAFLFLLAGFAAKISA

(GGGGC)3 linker GGGGSGGGGSGGGGS

a

Example SpyTagged VHH

MMLLQAFLFLLAGFAAKISAQVQ{-VHH sequence-} VSSGGGGSGGGGSGGGGSAHIVMVDAYKPTKGAAHHHHHHStopb

SnoopCatcherELP2SpyCatcherTwinStrepTagc

MKGSSHHHHHHSSGLVPRGSHMKPLRGAVFSLQKQHPDYPDIYGAID QNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIV AFQIVNGEVRDVTSIVPQDIPATYEFTNGKHYITNEPIPPKGPQGIWGQ LDGHGVGNLGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVG VPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGTSVPG VGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPG VGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLDIPTTENLYFQGA MVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELR DSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQG QVTVNGKATKGDAHIDGPQGIWGQLEWKKGSAWSHPQFEKGGGSGGG SGGSAWSHPQFEKStopb

Dependent on the application multiple ELP domains can be encoded within one scaffold. The sequence within brackets (..) should be copied b Stop codon c Add the following DNA sequences immediately flanking this amino acid sequence when ordering synthetic genes (restriction sites underlined): N-terminal, 50 -GAATTCATTAAAGAGGAGAAATTAACT-30 ; C-terminal, 50 -AAGCTT-30

Creation of Multimeric Single-Domain Antibodies Using Bacterial Superglues

17. PCR primer VHH-Rev: GAGTCTGGRGGAGG-30 .

317

50 - GATGTGCAGCTGCAG

18. Thermal cycler. 19. Agarose gel electrophoresis reagents and equipment. 20. Luria–Bertani (LB) broth: 10 g of tryptone, 10 g of NaCl, 5 g of yeast extract in 1 L of ddH2O. Autoclave and store at 4–12  C. 21. 100 mg/mL ampicillin stock in ddH2O (1,000). Filter sterilize with a syringe and 0.22 μm filter. Store at
20  C. 22. 25 mg/mL kanamycin stock in ddH2O (1,000). Filter sterilize with a syringe and 0.22 μm filter. Store at
20  C. 23. LB agar plates containing 100 μg/mL ampicillin (LBamp100) and LB agar plates containing 25 μg/mL kanamycin (LBkana25). Prepare LB as in step 20 but add 10 g/L agar prior to autoclaving. Wait until medium has cooled (~50  C) prior to adding antibiotics. Store at 4–12  C for up to 4 weeks. 24. NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific) or similar instrument. 25. Access to Sanger sequencing service. 26. Sequencing primer CAAGCCTTC-30 .

BOLI166:

50 - ATGATGCTTTTG

27. Sequencing primer Revseq-pUC19: 50 - TCACACAGGAAA CAGCTATGAC-30 . 2.2 Expression of ELP Scaffolds in E. coli

1. LBkana25 plates: see Subheading 2.1. 2. Chemically competent E. coli BL21 cells (New England Biolabs). 3. LB broth: see Subheading 2.1. 4. Sterile (vented) 50 mL Erlenmeyer flasks (e.g., polyethylene terephthalate G copolyester [PETG] or glass). 5. Sterile (vented) 2 L Erlenmeyer flasks (e.g., PETG or glass). 6. 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) in ddH2O. Filter sterilize with a syringe and 0.22 μm filter. Store at
20  C. 7. Temperature controlled shaking incubator suitable for Erlenmeyer flasks. 8. Sterile 50 mL polypropylene tubes. 9. pH meter. 10. Qiagen lysis buffer A: 100 mM NaH2PO4, 10 mM Tris-HCl, 6 M guanidine hydrochloride. Adjust pH to 8.0 using NaOH. 11. Slow speed centrifuge for 50 mL tubes. 12. Microfuge for 2 mL microcentrifuge tubes.

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13. Sonicator (e.g., Branson Sonifier 250) or similar instrument. 14. Polypropylene columns (5 mL) for gravity flow chromatography with Ni-NTA Agarose (Qiagen). 15. Ni-NTA agarose (Qiagen). 16. Qiagen wash buffer C: 100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea. Adjust pH to 6.3 using HCl. 17. Qiagen elution buffers D (100 mM NaH2PO4, 10 mM TrisHCl, 8 M urea; adjust pH to 5.9 using HCl) and E (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea; adjust pH to 4.5 using HCl). 18. Native elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole. Adjust pH to 8.0 using NaOH. 19. Amicon Ultra-2 Centrifugal Filter Unit (molecular weight cut-off 30 kDa). 20. Tris-buffered saline (TBS): 50 mM Tris-HCl, 350 mM NaCl, pH 7.6. 2.3 Expression of Spy/Snoop-Tagged SdAbs in Yeast

2.4 Coupling of Tagged SdAbs to ELP Scaffolds and Purification of Multimers

Materials required for generalized sdAb expression in yeast are described in Harmsen et al. (see Chapter 8, Small-Scale Secretory VHH Expression in Saccharomyces cerevisiae). Readers may also refer to similar published protocols [9, 10]. A purified yeast expression plasmid encoding the Tagged versions of the sdAbs (e.g., pRL480_sdAbinterest, pRL481_sdAbinterest) are required (see Subheading 3.1). Alternatively, expression of Spy/Snoop-Tagged sdAbs could be performed in other systems such as E. coli [7]. 1. Total protein quantification kit/equipment (e.g., UV-Vis spectrophotometer). 2. Purified Tagged-sdAb from Subheading 2.3/3.3, preferably at a concentration > 0.5 mg/mL. 3. Purified Catcher-containing ELP scaffold from Subheading 2.2/3.2, preferably at a concentration > 0.5 mg/mL. 4. Thermomixer for 1.5 mL and 2 mL tubes. 5. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4. 6. TBS: see Subheading 2.2. 7. Gravity Flow Strep-Tactin® XT purification system (IBA Lifesciences, Go¨ttingen, Germany): (a) Gravity Flow Strep-Tactin® XT Sepharose 1 mL prepacked columns. (b) Wash buffer W: 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA).

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(c) Elution buffer BXT: 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 50 mM biotin, pH 8.0. (d) 10 mM NaOH. (e) Regeneration buffer R: 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM 2-[40 -hydroxy-benzeneazo] benzoic acid, pH 8.0. (f) Amicon Ultra-2 Centrifugal Filter Unit (molecular weight cut-off 30 kDa). 8. SDS-PAGE reagents (see Note 4): (a) NuPAGE™ Novex gel system (Thermo Fisher Scientific). (b) NuPAGE™ 4–12% (w/v), Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well. (c) NuPAGE™ 3-(N-morpholino)propanesulfonic (MOPS) SDS Running Buffer (20).

acid

(d) NuPAGE™ Sample Reducing Agent (10). (e) NuPAGE™ LDS Sample Buffer (4). (f) Novex™ Sharp Pre-stained Protein Standard. (g) GelCode™ Blue Stain Reagent.

3

Methods

3.1 Construction of Tagged sdAb Expression Vectors

1. Determine the DNA and amino acid sequence of an sdAb of interest. For a VHH, the sequence should start with the amino acids QVQ and end at VSS covering IMGT positions 1–128 (Kabat positions 1–113). DNA encoding the sdAb can be in any vector of choice. 2. Amplify the VHH of interest in a 50 μL PCR reaction containing: 25 μL of 2 Phusion Flash High-Fidelity PCR Master Mix, 1 μL of primer VHH-For (final concentration 1 μM), 1 μL of primer VHH-Back (final concentration 1 μM), template DNA (1–10 ng), and ddH2O to 50 μL final volume. Cycle the reaction as follows: 15 s at 98  C; 35 cycles of 15 s at 98  C, 15 s at 55  C, and 15 s at 72  C; and 5 min at 72  C. Alternatively order DNA encoding the VHH of interest as a gene block from a company offering gene synthesis, making sure to include PstI and BstEII sites in the VHH sequence (see Notes 5 and 6). 3. Purify the PCR product using a DNA Clean & Concentrator Kit according to the manufacturer’s instructions. Synthetic gene blocks do not need additional purification. 4. Digest 100–500 ng of the purified PCR product or gene block with PstI-HF and BstEII-HF restriction endonucleases in a 10–20 μL reaction mixture containing 0.5 μL of each enzyme

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for 3 h at 37  C (see Note 6). Purify the digested product with the DNA Clean & Concentrator Kit according to the manufacturer’s instructions. Quantitate DNA by spectrophotometry. 5. Transform 30 μL of chemically competent E. coli JM109 cells with 10 ng of the pRL480-stuffer or pRL481-stuffer plasmids. Heat shock the mixture for 80 s at 42  C, incubate on ice for 2 min, and add 900 μL of SOC medium. Place in a shaking incubator at 37  C for 1 h, then plate 50 μL of transformed cells neat on an LBamp100 plate. Grow overnight at 37  C. 6. Pick single colonies of E. coli JM109 cells harboring the pRL480-stuffer or pRL481-stuffer plasmid and inoculate 10 mL of LB containing 100 μg/mL ampicillin. Grow the culture overnight at 37  C and extract plasmid DNA using a miniprep kit according to the manufacturer’s instructions the following morning. 7. Digest 10 μg of pRL480-stuffer and/or pRL481-stuffer in a 50 μL reaction mixture containing 1–2 μL of the PstI-HF and BstEII-HF endonucleases for 4 h at 37  C. Dephosphorylate the digested vector by adding 6 μL of alkaline phosphatase (from the Rapid DNA Dephosphorylation Kit), 10 μL of phosphatase buffer, and 34 μL of water and incubate for 2 h at 37  C. Subsequently purify the linearized plasmid from an agarose gel (the largest fragment running at ~6,500 bp) using a Gel DNA Recovery Kit according to the manufacturer’s instructions. The smaller fragment (1 kb) represents the stuffer. Quantitate DNA by spectrophotometry. 8. Ligate the PstI-HF and BstEII-HF-digested VHH genes of interest into the digested pRL480 and/or pRL481 vectors as follows. Combine 50 ng of vector, 20–80 ng of VHH insert, 1 μL of T4 DNA ligase buffer, 1 μL (400 U) of T4 DNA ligase, and water to a final volume of 10 μL. Incubate for 1 h at room temperature. After cloning, the resulting plasmid will encode: yeast signal sequence—VHH—(GGGGS)3 linker—Spy/SnoopTag—GAA linker—C-terminal His6 tag (Table 1) (see Note 7). 9. Transform 30 μL of chemically competent JM109 cells with 10 μL of the ligation mixture on ice in thin-walled microcentrifuge tubes. Heat shock the mixture for 80 s at 42  C, incubate on ice for 2 min, and add 900 μL of SOC medium. Place in a shaking incubator at 37  C for 1 h, then plate 50 μL of transformed cells neat on an LBamp100 plate. Pellet the remaining cells by centrifugation (4,300  g, 10 min), resuspend in 50–100 μL of SOC, and plate the entire volume on a second LBamp100 plate. 10. Following overnight incubation at 37  C, grow three single colonies from the LBamp100 plates for miniprep purification

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using standard procedures. Inoculate 10 mL of LBamp100 with a single colony and incubate overnight in a shaking incubator at 37  C with 250 rpm shaking. The next day, isolate plasmid DNA for analytical restriction digestion. Digest 500 ng of purified plasmid with PstI-HF and BstEII-HF restriction endonucleases in 10–20 μL reaction mixtures containing 0.5 μL of each enzyme for 3 h at 37  C. 11. Assess the digests by agarose gel electrophoresis. Confirm plasmid insert sequences of clones with the correct restriction enzyme digestion pattern (~320 bp for the VHH insert and ~6,500 bp for the vector), by bidirectional Sanger sequencing using primers BOLI166 and Revseq-pUC19. 12. Store the purified plasmids at
20  C. 3.2 Construction of Catcher-Containing Scaffold Expression Vector

1. Design your scaffold of interest encoding SpyCatchers and/or SnoopCatchers. In Table 1, a design strategy is presented for a scaffold comprising two ELP domains (ELP-2) with an N-terminal SpyCatcher and a C-terminal SnoopCatcher. The organization is as follows: N-terminal His-tag, SpyCatcher, ELP 1 and ELP 2, SnoopCatcher, TwinStrepTag (see Notes 8 and 9). 2. Add an appropriate 50 (e.g., EcoRI) and 30 (e.g., HindIII) restriction enzyme site and order the synthetic DNA construct encoding the Catcher-containing ELP scaffold from a service provider (see Note 5). Make sure to include a 50 ribosomal binding site in the synthetic construct immediately following the EcoRI site (Table 1). We recommend gene synthesis and most companies can directly clone the fragment into a plasmid of interest. If gene synthesis and subcloning is done by a provider, steps 3 through 7 can be excluded. If the gene is synthesized but not subcloned into the expression vector, or if construction of the scaffolds is accomplished using methods other than gene synthesis (see Note 10), proceed with steps 3 through 7. 3. Digest the synthetic gene construct (200–500 ng) and the pQE-80L vector (2 μg) with EcoRI and HindIII, in 10–20 μL reaction mixture containing 0.5–1 μL of each enzyme and appropriate enzyme buffer for 3 h at 37  C. Purify the digested insert and linearized vector using a DNA Clean & Concentrator Kit according to the manufacturer’s instructions. 4. Ligate the EcoRI and HindIII-digested scaffold of interest into pQE-80L digested with the same enzymes. Mix vector (50 ng) and scaffold insert (40–120 ng) with 1 μL of T4 DNA ligase buffer and 1 μL of T4 DNA ligase, then add water up to 10 μL total volume. Incubate for 1 h at room temperature. 5. Transform 30 μL of chemically competent E. coli JM109 cells with 10 μL of the ligation mixture on ice in thin-walled

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microcentrifuge tubes. Heat shock the mixture for 80 s at 42  C, incubate on ice for 2 min, then add 900 μL SOC medium and place in shaking incubator at 37  C for 1 h. Plate 50 μL of transformed cells neat on a LBKana25 plate. Pellet the remaining cells by centrifugation (4,300  g, 10 min), resuspend in 50–100 μL of SOC, and plate the entire volume on a second LBKana25 plate. 6. Following overnight incubation at 37  C, grow single colonies for miniprep purification using standard procedures (e.g., 10 mL LBKana25, overnight growth at 37  C in a shaking incubator, 250 rpm). Check for correct insertion of the purified plasmid by restriction enzyme digestion with EcoRI and HindIII followed by analytical agarose gel electrophoresis (~1,500 bp for His-Spy-ELP2-Snoop-TwinStrep and ~4,600 bp for vector). Confirm the sequence by Sanger sequencing of clones with the correct restriction enzyme digestion pattern. 7. Store the purified plasmids at
20  C. 3.3 Expression of ELP Scaffolds in E. coli

1. Introduce the expression plasmid encoding the ELPs with Catchers (see Subheading 3.2) in E. coli BL21 cells by transformation. Combine 50 μL of chemically competent E. coli BL21 cells with 1 μL of purified plasmid (0.1 ng) for 30 min on ice. Heat shock the bacteria for 10 s at 42  C, add 1 mL of SOC medium, and recover for 1 h at 37  C with 250 rpm shaking. Plate the transformed bacteria on an LBkana25 plate. 2. Incubate overnight at 37  C. 3. Inoculate 25 mL of pre-warmed LBkana25 medium with a single colony and incubate overnight at 37  C with 225 rpm shaking (see Note 11). 4. Transfer the overnight culture to a 2 L Erlenmeyer flask containing 500 mL of pre-warmed LBkana25 and incubate at 37 C with 225 rpm shaking. Measure the optical density every 30 min until OD600 reaches 0.6, starting from 1.5 h post sub-culture. 5. When the OD600 reaches 0.6, add 500 μL of 1 M IPTG (final concentration 1 mM) and continue the incubation for 4 h at 37  C with 250 rpm shaking (see Note 12). 6. Collect the induced bacteria by centrifugation in 50 mL tubes (10 min, 4,300  g) and discard the supernatant. 7. Freeze the bacterial cell pellets in the 50 mL tubes at
20  C (short or long term). 8. Thaw the tubes with bacterial pellet for 15 min on ice. 9. Resuspend the pellet in 3 mL of lysis buffer A (100 mM NaH2PO4, 10 mM Tris-Cl, 6 M guanidine hydrochloride, pH 8.0) per 50 mL tube. Combine the contents of two

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50 mL tubes (3 mL suspension each) in a single 15 mL tube. Alternatively, combine the contents of all ten 50 mL tubes (3 mL suspension each) in one 50 mL tube. 10. Incubate for 30 min on ice. 11. Sonicate six times for 10 s on ice (rest for 10 s between each sonication). 12. Centrifuge the lysate for 30 min at 4  C (15,000  g) and pour the supernatant into a clean 50 mL tube. 13. Add 3 mL of 50% Ni-NTA agarose resin, divide the mixture into three 50 mL tubes, and incubate for 30 min on ice on a shaking platform. 14. Apply the mixture to a polypropylene column still containing the lower stopper, the Ni-NTA agarose resin and sample mixture. Remove the lower stopper and collect the flow-through. Take a sample of the flow-through for SDS-PAGE analysis. 15. Wash the column twice with 12 mL of buffer C. 16. Elute the column with 4  0.5 mL of buffer D, 4  0.5 mL of buffer E, and 4  0.5 mL of native elution buffer (collect each fraction individually, 12 in total). 17. Assess each individual elution fractions, as well as a sample of the flow-through from step 14, by SDS-PAGE and determine the protein concentration by NanoDrop (OD280). 18. Pool the fractions containing protein of the expected size range (Catcher-ELP1-Catcher ~40 kDa, Catcher-ELP2-Catcher ~50 kDa, Catcher-ELP3-Catcher ~60 kDa) and buffer exchange into TBS using 30 kDa MWCO Amicon Ultra15 mL centrifugal filters. Following the pooling of fractions, add the pooled fractions to the centrifugal filter and fill with the maximal volume of TBS. Centrifuge for 20 min at 4,300  g, discard flow-through and fill with the maximum volume of TBS. Repeat this process two additional times. 19. Determine the protein concentration by NanoDrop (OD280). 20. Store the purified protein at
70  C. 3.4 Expression of Spy/Snoop-Tagged sdAbs in Yeast

Using the pRL480 (SpyTag) or pRL481 (SnoopTag) plasmid encoding your Spy- or SnoopTagged sdAb of interest (see Subheading 3.1), transform Saccharomyces cerevisiae (baker’s yeast) for expression and purification. Follow the protocol for transformation, production, and purification of VHHs as described in Harmsen et al. (see chapter 8, Small-Scale Secretory VHH Expression in Saccharomyces cerevisiae). Readers may also refer to similar published protocols [9, 10]. Alternatively, expression of Spy/ Snoop-Tagged sdAbs can be accomplished in E. coli [7].

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3.5 Coupling of Tagged sdAbs to ELP Scaffolds and Purification of Multimers

1. Determine the optimal coupling conditions by mixing the purified Tagged-sdAbs (obtained in Subheading 3.4) with the purified ELP scaffolds of interest (obtained in Subheading 3.3) at 0.5:1, 1:1, 2:1, and 3:1 molar ratios. Incubate 25 pmol of purified ELP scaffold with various amounts of the tagged sdAbs (12.5, 25, 50, and 75 pmol) in a final volume of 20–50 μM of PBS or TBS (see Notes 13 and 14). Place the tubes in a thermomixer set to 20  C and 300 rpm for 3 h. Following coupling, the optimal ratio can be found by subjecting the different mixtures to SDS-PAGE. An optimal ratio is considered the ratio in which nearly all ELP scaffold molecules have shifted to the size range resembling coupled molecules, but using the least amount of sdAb molecules possible. 2. Couple a larger amount of sdAb to the scaffold (2–4 mg) of interest using the optimal molar ratio determined in step 1. 3. Remove uncoupled tagged-sdAbs by StrepTactin affinity chromatography (see Note 15) by first washing the column with two column volumes (CV) of buffer W. 4. Load the complexes onto the column and wash 5 with 1 CV of buffer W. 5. Elute the complexes 6 with 0.5 CV of BXT. 6. Buffer exchange into PBS using 30 kDa MWCO Amicon centrifugal devices. 7. To regenerate the column after use, add 2 mL of 10 mM NaOH. Wash the column with 1 mL of buffer R. The column should turn orange. Wash the column with 8 mL of buffer W. 8. Use the multimeric complexes in biological assays of interest such as virus neutralization tests [9].

4

Notes 1. Various ELP-, Tag-, and Catcher-encoding plasmid constructs have been deposited to Addgene (www.addgene.org) by the group of Prof. Dr. Mark Howarth and are available for non-commercial use. As an alternative to an ELP scaffold, multimeric scaffold proteins that assemble into a nanoparticle or any other type of linker/scaffold could be used (e.g., lumazine synthase, aldolase, IMX313, ferritin), depending on your specific application. 2. pRL480 and pRL481 plasmids are available through Addgene (www.addgene.org). pRL480 and pRL481 are both derivatives of pRL188 [10], and pRL188 is derived from plasmid YEplac181 (Genbank accession number X75460.1; Creative

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Biogene, Shirley, NY, USA) [11]. Instead of using pRL188based vectors, other yeast expression vectors can also be used. 3. Instead of using pQE-80 L other E. coli expression vectors can also be used (e.g., pDEST14, pET28a). 4. We use the NuPAGETM Novex gel system and reagents but alternative SDS-PAGE systems can also be used. 5. To improve expression yields the VHH of interest could be codon optimized for expression in yeast prior to gene synthesis. The ELP scaffold could be codon optimized for expression in E. coli. Most gene synthesis companies offer online codon optimization tools. 6. The 50 PstI restriction endonuclease site is naturally present in nearly all VHH genes at IMGT/Kabat position 4–5 (CTGCAG; QVQLQES). The 30 BstEII restriction endonuclease site near IMGT position 122 /Kabat position 107 (GGTCACC; QVTVSS) is also naturally present in nearly all VHH genes. 7. The placement of the superglue tag is flexible; however, the preferred location for a VHH is at the C-terminus to reduce the chances of interference with antigen binding by the CDRs. 8. In principle, there are no limits for the type of scaffold and number of catchers/tags although increasing the overall length will reduce production yields and may complicate cloning. 9. To remove the TwinStrepTag, an enterokinase site (DDDDK) can be included immediately before the Tag. For longer in vivo serum half-life, an albumin-binding domain can be included following the C-terminal Catcher domain and linked with a (GGGGS)3 or similar linker. 10. If a gene encoding your scaffold of interest (consisting of SpyCatchers and/or SnoopCatchers) is not obtained by commercial gene synthesis, the components of the construct can be generated and assembled using other methods. For instance, amplify your scaffolds using standard PCR methods, overlap PCR, or Gibson ligation, and include the EcoRI and HindIII restriction sites by 50 primer overhangs to either the 50 or 30 ends of the fragment, respectively. Purify the final assembled fragment with the DNA Clean & Concentrator kit. 11. If the colonies are >7 days old, viability of the bacteria might drop and you could try to inoculate with a group of colonies instead although it is preferable to streak a new plate. 12. Depending on the specific gene product (scaffold), a shorter or longer IPTG induction time might result in higher protein yields.

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13. Although the Tag:Catcher reactions are robust under a wide variety of conditions, optimization of complex formation is recommended. Optimizing the overall protein concentrations and time of complex formation are the main parameters. 14. If a Tagged sdAb can bind to multiple Catchers within one ELP scaffold molecule, the molar concentration of the sdAb should be adapted accordingly. Thus, if a bivalent construct is made using an ELP scaffold with two SpyCatchers, twice as much SpyTagged sdAb is needed for a 1:1 molar ratio. 15. By using a slight excess of sdAbs compared to scaffold molecules, there is no need to remove unsaturated scaffold molecules. Removing unbound sdAbs is sufficient.

Acknowledgments We thank Dr. Michiel Harmsen for his careful reading of the protocol. References 1. Laursen NS, Friesen RH, Xhu X et al (2018) Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin. Science 362:598–602 2. Hultberg A, Temperton NJ, Rosseels V et al (2011) Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PLoS One 6:e17665 3. Iezzi ME, Policastro L, Werbajh S et al (2018) Single-domain antibodies and the promise of modular targeting in cancer imaging and treatment. Front Immunol 9:273 4. Els Conrath K, Lauwereys M, Wyns L et al (2001) Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem 276: 7346–7350 5. Veggiani G, Zakeri B, Howarth M (2014) Superglue from bacteria: unbreakable bridges for protein nanotechnology. Trends Biotechnol 32:506–512 6. Zakeri B, Fierer JO, Celik E et al (2012) Peptide tag forming a rapid covalent bond to a

protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109:E690–E697 7. Veggiani G, Nakamura T, Brenner MD et al (2016) Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A 113:1202–1207 8. Tan LL, Hoon SS, Wong FT (2016) Kinetic controlled Tag-catcher interactions for directed covalent protein assembly. PLoS One 11:e0165074 9. Wichgers Schreur PJ, van de Water S, Harmsen M et al (2020) Multimeric single-domain antibody complexes protect against bunyavirus infections. eLife 9:e52716 10. Harmsen MM, van Solt CB, Fijten HPD et al (2007) Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease. Vet Microbiol 120:193–206 11. Gietz RD, Sugino A (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74: 527–534

Chapter 16 Introducing Cysteines into Nanobodies for Site-Specific Labeling Simon Boje Hansen and Kasper Røjkjær Andersen Abstract We have developed a generally applicable methodology for cysteine mutagenesis of nanobody (Nb) framework region serine residues. This strategy allows for subsequent labeling with thiol-reactive compounds without disrupting Nb antigen binding. We provide a protocol for production, labeling, and affinity determination of cysteine-engineered Nbs (cys-Nbs) with Alexa Fluor 488-maleimide and the mercury compound para-chloromercuribenzoic acid (PCMB). Alexa Fluor 488- and PCMB-labeled cys-Nbs can be used for immunofluorescence microscopy and experimental phasing in crystallography, respectively. Key words Nanobody, Site-specific labeling, Cysteine, Mercury, Maleimide, Experimental phasing, Imaging

1

Introduction Nanobodies (Nbs) are versatile molecular tools, with applications in many fields of biological sciences including immunofluorescence microscopy and structural biology [1, 2]. As a miniaturized antibody fragment, they excel in many areas where conventional antibodies have traditionally been used. The ease and low cost of Nb production in Escherichia coli, high stability, solubility, and versatility in subsequent applications makes Nbs superior to conventional antibodies in many regards [3]. One example is in immunofluorescence microscopy, where use of conventional antibodies can have disadvantages through the inherent larger size of antibodies (~150 kDa compared to ~15 kDa for Nbs), causing inefficient tissue penetration and often requiring using fixed tissues [1, 4]. In structural biology, Nbs have proven successful as crystallization chaperones, enabling the generation of well-diffracting crystals, while heavy-atom derivatization facilitates solving of 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_16, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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phase problem and subsequent model building of macromolecular structures [5–8]. Here, we present an easy and accessible method to produce site-specific labeled Nbs, utilizing an introduced free thiol from an engineered cysteine residue. Introduction of a free solvent exposed cysteine is achieved by mutagenesis of conserved framework serine residues. We show how the free thiol can be covalently conjugated using maleimide compounds, allowing for labeling of Nbs with molecules such as fluorophores to be used in immunofluorescence microscopy. Furthermore, we show how to utilize the thiol for heavy-atom derivatization of Nbs with mercury compounds. The resulting labeled Nbs can be used not only as crystallization chaperones, but also for de novo phasing in macromolecular crystallography [2, 9]. Importantly, we additionally show how labeling of the Nb scaffold does not interfere with Nb antigen-binding properties. We assume the selection, cloning and initial characterization of a Nb candidate has already been performed, protocols for which can be found in a previous edition of Methods in Molecular Biology [10]. As an example here, we use a Nb from a previous study (called Nb_36), which is an anti-complement component 5 Nb [9]. The protocol is divided into the following sections: (1) bioinformatic analysis of conserved serines in Nb scaffolds to identify sites for introduction of cysteines, (2) PCR mutagenesis, (3) expression of cysteine-nanobodies (cys-Nbs) in E. coli and cys-Nb purification, (4) labeling of cys-Nbs with Alexa Fluor 488 maleimide, (5) labeling of cys-Nbs with mercury, (6) purification of monomeric cys-Nbs, and (7) affinity determination of mercury labeled cys-Nbs by biolayer interferometry (BLI).

2

Materials

2.1 Buffers, Media, and Solutions

Use ultrapure water (18.2 MΩ cm at 25
C) to prepare all media, solutions, and buffers. 1. Lysogeny broth (LB): 10 g of tryptone, 10 g of NaCl, and 5 g of yeast extract in 1 L of ultrapure water. Autoclave. 2. LB containing 1.5% (w/v) agar (LA). 3. Chemically competent E. coli DH5α cells (Thermo Fisher Scientific, Waltham, MA, USA). 4. Chemically competent E. coli LOBSTR cells carrying the pRIL plasmid (Kerafast, Boston, MA, USA). 5. Ampicillin (100 mg/mL in water, filter-sterilized), chloramphenicol (34 mg/mL in 100% ethanol), or other appropriate selective antibiotics. 6. dNTP mix, 2 mM each.

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Table 1 Primers used for inverse PCR mutagenesis to generate serine to cysteine mutations at Kabat positions S7, S70, S82b, and S112 of nanobodies Tm (
C)

Inverse PCR primers C7 C7f: TGT GGG GGA GGC TTG GTG CAG

61.7

C7r: CTC CAC GAG CTG CAC CTG

58.6

C70 C70f: TGT AAG GAC AAC GCC AAG AAG ACA G

57.5

C70r: G ATG GTG AAT CGG CCC TTC

56.0

C82b C82bf: TGT CTG AAA CCT GAG GAC ACG G

55.4

C82br: G TTC ATT TGC AGA TAC ACT GTC TTC TTG

56.3

C112 C112f: TGT TCA CAC CAC CAC CAC CAC CAC TGA G

64.6

C112r: GAC GGT GAC CTG GGT CCC CTG

64.3

Cysteine TGT codons highlighted in bold in all forward primers indicate mutagenized codon to generate a solvent exposed cysteine

7. Phusion® High-Fidelity DNA polymerase and buffer (NEB, Ipswich, MA, USA). 8. Polynucleotide kinase (PNK) (Thermo Fisher Scientific). 9. T4 DNA ligase and buffer (Thermo Fisher Scientific). 10. DpnI endonuclease (Thermo Fisher Scientific). 11. Primers for inverse PCR as shown in Table 1. 12. Isopropyl β-D-1-thiogalactopyranoside (IPTG). 13. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 in 1 L deionized water. Adjust pH to 7.4. 14. PBS, pH 7.4, containing: 400 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol, and 1 mM benzamidine (see Note 1). 15. PBS, pH 7.4, containing 400 mM NaCl, 400 mM imidazole, and 5 mM β-mercaptoethanol (see Note 1). 16. 10 mM HEPES, pH 7.6, containing 150 mM NaCl and 2 mM dithiothreitol (see Note 1). 17. 10 mM HEPES, pH 7.6, containing 150 mM NaCl (see Note 1). 18. 20 mM sodium acetate, pH 5.5 (see Note 1).

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19. 20 mM sodium acetate, pH 5.5, containing 500 mM NaCl (see Note 1). 20. PBS, pH 7.4, containing 0.01% (v/v) Tween-20 (see Note 1). 21. Para-chloromercuribenzoic acid (PCMB) (see Note 2). 22. Alexa Fluor 488 maleimide (see Note 2). 23. Monomethoxy polyethylene glycol (MPEG)-maleimide (see Note 2). 24. Iodoacetamide (see Note 2). 25. Vivaspin® centrifugal concentrator, 5 kDa MWCO. 26. 1 mL Protino™ Ni-NTA agarose column. 27. Superdex™ 75 Increase 10/300 GL gel filtration column. 28. Mono S® 5/50 GL cation exchange column. 29. PD-10 desalting column. 30. E.Z.N.A. Plasmid Mini Kit (Omega Bio-Tek, Norcross, GA, USA). 31. E.Z.N.A. Cycle Pure Kit (Omega Bio-Tek). 32. Octet® Anti-Penta-HIS biosensors. 33. 96-well microplates. 34. Coomassie brilliant blue. 2.2

Equipment

1. PCR thermocycler. 2. Labsonic M® sonicator. 3. Agarose gel electrophoresis equipment, stain, and imager. 4. SDS-PAGE equipment, stain, and imager. 5. Fast protein liquid chromatography (FPLC) system. 6. NanoDrop spectrophotometer. 7. Typhoon Trio Variable Mode Scanner. 8. Octet® BLI system.

2.3

Software

1. CLC Genomics Workbench (Qiagen, Valencia, CA, USA). 2. Abnum web tool (http://www.bioinf.org.uk/abs/abnum/). 3. ImageJ (https://imagej.nih.gov/ij/). 4. GraphPad Prism (GraphPad Software, San Diego, CA, USA; http://www.graphpad.com).

3

Methods We analyzed 41 Nb-antigen structures from the Protein Data Bank (PDB) to identify ideal sites for labeling Nbs (see Note 3). Superpositioning the Nbs revealed a primary antigen contact surface

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Fig. 1 (a) Structure of Nb_36 (PDB: 5NLU) in cartoon representation. The light-brown surface (“binding face”) indicates the antigen-interacting buried surface, while the purple surface (“free face”) is solvent exposed and the ideal area for labeling. The six serine positions on the free face are shown as yellow spheres and labeled in red text. Complementarity-determining regions (CDRs) 1, 2, and 3 are colored green, red, and blue, respectively. Framework β-strands are labeled according to [3]. (b) Nb consensus sequence from Fig. 2 alignment in Kabat numbering with the six serine positions highlighted in bold red. Part (a) was prepared in PyMOL Molecular Graphics System, version 2.4 (Schro¨dinger, LLC) and reproduced from [9], with permission of the International Union of Crystallography 3.1 Structural Bioinformatics Analysis to Identify Nb Framework Region Serines for Cysteine Mutagenesis

comprising framework β-strands C, C0 , C00 , and F [3], while framework β-strands A, B, D, E, and G did not engage in protein–protein interactions and remained solvent exposed (Fig. 1a). Based on this structural analysis, the exposed face is optimal for labeling. One labeling strategy is to use nucleophilic thiols of solvent exposed cysteines to conjugate compounds [11]. The 41 Nb amino acid sequences were aligned using CLC Genomics Workbench software and six conserved serines were identified on the exposed face as candidate residues for cysteine mutagenesis (Figs. 1a and 2). For convenience, the alignment consensus sequence is Kabat numbered

Fig. 2 Alignment of 41 Nb amino acid sequences generated and visualized with CLC Genomics Workbench software. Sequences were retrieved from 40 Nb-antigen structures deposited in the PDB, with the addition of the Nb_36 sequence (PDB: 5NLU). The six positions (colored red in the consensus) were identified based on framework localization, conservation, and inspection of Nb-antigen structures to ensure that the approach was generally applicable. PDB codes of structures analyzed: 3g9a, 3ogo, 4bel, 4c57, 4y8d, 4wgv, 4x7e, 4x7f, 4lgp, 4lgs, 4lhq, 4i13, 4eig, 3k74, 4fhb, 4eiz, 4mqt, 4weu, 4wem, 4wen, 3sn6, 3p0g, 4ldl, 4ocm, 4w6x, 4w6y, 3cfi, 4cdg, 4kml, 3ezj, 4xt1, 4p2c, 4s10, 4grw, 3stb, 4i0c, 4aq1, 4krm, 3j69, 3j6a, and 5nlu

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using the Abnum webtool [12] and shown in Fig. 1b along with the six candidate positions for cysteine introduction: S7, S17, S21, S70, S82b, and S112 (see Note 4). 3.2 Introducing a Cysteine Residue into Nbs by Inverse PCR Mutagenesis

Below we outline protocols to generate cysteine mutagenized Nbs at positions S7, S70, S82b, and S112. The subsequent mutant constructs are referred to as C7, C70, C82b, and C112, respectively. Our template Nb is the anti-complement component 5 Nb_36 (PDB: 5NLU) cloned into the bacterial expression vector pET-22b(+) as a C-terminal 6  His fusion [9] (see Note 5). 1. Mutagenize serine positions by inverse PCR [13]. Each of the four reactions should contain 10 μL of 5 Phusion® HF buffer, 5 μL of 2 mM each dNTP, 5 μL of 5 μM each forward and reverse primers (Table 1), 100 ng of DNA template, 0.5 μL of Phusion® High-Fidelity DNA polymerase (2 U/μL), and ultrapure water to 50 μL. 2. Cycle the temperature of the reactions as follows: 98
C for 30 s; 24 cycles of 98
C for 10 s, 57
C for 20 s, and 72
C for 180 s. Altering the annealing temperature by 5
C may enhance PCR yield (see Note 6). Analyze PCR products using agarose gel electrophoresis. 3. Purify the PCR products using the E.Z.N.A. Cycle Pure Kit following the manufacturer’s instructions. 4. Perform blunt end ligation by mixing 16.5 μL of the purified PCR product (1–2 μg), 2 μL of T4 DNA ligase buffer (10), and 0.5 μL of PNK (10 U/μL). Incubate for 30 min at 37
C. 5. Add 0.5 μL of T4 DNA ligase (30 U/μL) and incubate for 1 h at room temperature. 6. Add 0.5 μL of DpnI (10 U/μL) and incubate for 30 min at 37
C. 7. Mix the ligated plasmid with 25 μL of chemically competent E. coli DH5α cells and incubate on ice for 5 min. 8. Heat-shock E. coli DH5α cell-DNA mixtures at 42
C for 30 s and place back on ice for 2 min. 9. Add 200 μL of LB and incubate for 1 h at 37
C. 10. Spread transformed E. coli cells onto LA plates containing appropriate antibiotics for selection (100 μg/mL ampicillin for the pET-22b(+) vector). Incubate plates at 37
C overnight. 11. Pick single colonies (see Note 7) and inoculate 5 mL of LB supplemented with appropriate selection antibiotics (100 μg/ mL ampicillin for the pET-22b(+) vector). Incubate in a shaking incubator (100 rpm) at 37
C overnight (see Note 8).

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Fig. 3 (a) Gel filtration chromatogram of Nb_36 C82b purification on a Superdex™ 75 Increase 10/300 GL column. Two peaks were observed and labeled 1 and 2. Dashed lines indicate fractions analyzed by SDSPAGE in (b). Two bands are observed corresponding to a dimeric ~24 kDa form (peak 1 in a) and a monomeric ~14 kDa form (peak 2 in a). Fractions from peak 2 in (a) were pooled for subsequent labeling steps. (c) Cation exchange chromatogram of Nb_36 C82b purification on a Mono® S 5/50 column. Peak 1 mainly contained free iodoacetamide from quenching after PCMB labeling. Dashed lines surrounding peak 2 indicate fractions analyzed by SDS-PAGE in (d). Peak 2 contained the ~14 kDa monomeric labeled Nb_36 C82b. Fractions of peak 2 were pooled for subsequent BLI analysis. *Dimeric Nb band in (b), ** monomeric Nb bands in (b) and (d)

12. Pellet E. coli cultures by centrifugation at 3,000  g for 10 min at room temperature and discard the supernatant. 13. Purify plasmids from the E. coli pellets using the E.Z.N.A. Plasmid Mini Kit following the manufacturer’s instructions. 14. Sequence the Nb insert (see Note 9) and identify a plasmid preparation containing the desired cysteine residue. 3.3 Expression and Purification of Cys-Nbs

1. Mix 50–100 ng of purified plasmid encoding the cys-Nb construct with 25 μL of chemically competent E. coli LOBSTR cells [14] (see Note 10) and incubate on ice for 5 min. 2. Heat-shock E. coli LOBSTR cell-DNA mixtures at 42
C for 30 s and place back on ice for 2 min.

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3. Add 200 μL of LB and incubate for 1 h at 37
C. 4. Spread transformed E. coli cells onto LA plates containing appropriate selection antibiotics (100 μg/mL ampicillin for the pET-22b(+) vector and 35 μg/mL chloramphenicol for LOBSTR pRIL). Incubate plates at 37
C overnight. 5. Pick or scrape colonies and inoculate 100 mL of LB supplemented with appropriate selection antibiotics (100 μg/mL ampicillin for the pET-22b(+) vector and 35 μg/mL chloramphenicol for LOBSTR pRIL). Incubate in a shaking incubator (100 rpm) at 37
C overnight (see Note 8). 6. Use 20 mL of overnight culture to inoculate 2 L of LB supplemented with appropriate selection antibiotics (100 μg/mL ampicillin for the pET-22b(+) vector and 35 μg/mL chloramphenicol for LOBSTR pRIL). Incubate in a shaking incubator (150 rpm) at 37
C until log growth phase is reached (OD600 of 0.5–0.8, typically after 2–4 h). 7. Induce cys-Nb expression by addition of IPTG to a final concentration of 0.2 mM and incubate in a shaking incubator (150 rpm) at 18
C overnight (see Note 11). 8. Centrifuge cells at 5,000  g for 15 min and resuspend the E. coli cell pellet in 50 mL of PBS, pH 7.4, containing 400 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol, and 1 mM benzamidine (see Note 12). 9. Lyse E. coli cells by sonication. Follow the manufacturers’ guidelines for your sonicator (see Notes 13 and 14). 10. Centrifuge the lysate at 16,000  g for 30 min and collect the cleared supernatant. 11. Initiate immobilized metal affinity chromatography (IMAC) purification by equilibrating the Protino™ Ni-NTA agarose column in PBS, pH 7.4, containing 400 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol, and 1 mM benzamidine, and apply cleared supernatant to the column. 12. Wash the column with 10 column volumes (CV) of PBS, pH 7.4, containing 400 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol, and 1 mM benzamidine, or until baseline is reached when monitoring 280 nm absorbance on an FPLC system. 13. Elute cys-Nbs using two subsequent isocratic steps at 50% and 100% PBS, pH 7.4, containing 400 mM NaCl, 400 mM imidazole, and 5 mM β-mercaptoethanol (5 CV each). Analyze IMAC fractions using 15% SDS-PAGE. 14. Pool elution fractions and concentrate sample using a centrifugal concentrator (5 kDa MWCO) to an appropriate volume for subsequent gel filtration (see Note 15).

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Fig. 4 (a) Nb_36 C7, C70, C82b, and C112, unmodified () or labeled with Alexa Fluor 488-maleimide (+). The Nbs were analyzed by SDS-PAGE and the gel was subsequently imaged on a Typhoon Trio Variable Mode Scanner. (b) SDS-PAGE analysis of Nb_36 C7, C70, C82b, and C112 PCMB labeling. Labeling was quantified using an MPEG-maleimide reagent to induce a size shift on SDS-PAGE (**). Free thiol reactivity was visualized with Hg(OAc)2, inducing a smeary band appearance. (c) Labeling was quantified in ImageJ and reported as an approximate percentage (ratio of MPEG-maleimide reacted Nb to monomeric PCMB labeled Nb). * in (a) and (b) shows dimeric Nb fractions. C7 and C112 Nbs dimerize more readily than C70 and C82b Nbs (see Note 5). Figure 4 was reproduced from [9] with permission of the International Union of Crystallography

15. Initiate size exclusion chromatography (SEC) purification by equilibrating the Superdex™ 75 Increase 10/300 gel filtration column in 10 mM HEPES, pH 7.6, containing 150 mM NaCl and 2 mM dithiothreitol and apply the sample through a capillary loop at a flow rate of 0.8 mL/min. 16. Elute protein with 1.5 CV isocratic elution in 10 mM HEPES, pH 7.6, containing 150 mM NaCl and 2 mM dithiothreitol. Analyze SEC fractions using 15% SDS-PAGE (Fig. 3a, b). 17. Pool elution fractions and optionally concentrate the sample using a centrifugal concentrator (5 kDa MWCO) to the desired concentration (see Note 16). 3.4 Labeling of CysNbs with Alexa Fluor 488-Maleimide

1. Equilibrate the PD-10 column in 10 mM HEPES, pH 7.6, containing 150 mM NaCl (see Notes 17 and 18). 2. Apply 500 μL of SEC-purified cys-Nb sample to the equilibrated PD-10 column and let the sample fully absorb into the column bed.

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3. Elute the cys-Nb from the PD-10 column using 5 CV of 10 mM HEPES, pH 7.6, containing 150 mM NaCl. Collect 0.5–1 mL fractions in microcentrifuge tubes (see Note 19). 4. Immediately identify protein-containing fractions by measuring absorbance at 280 nm on a NanoDrop spectrophotometer (see Note 20). Calculate or estimate beforehand the molarity of the cys-Nb in the fractions. Pool all of the protein-containing fractions. 5. Mix the cys-Nb with a 1.5-fold molar excess of Alexa Fluor 488-maleimide and incubate the labeling reaction for 1 h on ice (see Note 21). 6. To quantify labeling efficiency, retrieve an aliquot of the reaction (~50–100 μg) for SDS-PAGE analysis and then react with a 10-fold molar excess of MPEG-maleimide for 1 h on ice. 7. Quench the labeling by addition of a 100-fold molar excess of dithiothreitol. 8. Analyze Alexa Fluor 488 labeling by reducing 15% SDS-PAGE. Scan the gel at 490 nm excitation and 525 nm emission with a Typhoon Trio Variable Mode Scanner. Stain the gel with Coomassie brilliant blue after the fluorescence scan (Fig. 4a). 9. Using ImageJ software [15], compare band intensities of Alexa Fluor 488-maleimide cys-Nb reactions with band intensity of MPEG-maleimide cys-Nbs to quantify the degree of Alexa Fluor 488 labeling. 3.5 PCMB Labeling of Cys-Nbs

1. Follow steps 1–4 in Subheading 3.4. 2. Mix the cys-Nb with a 5-fold molar excess of PCMB and incubate the labeling reaction for 1 h on ice. 3. To quantify labeling efficiency, retrieve an aliquot of the reaction (~50–100 μg) for SDS-PAGE analysis and then react with a 10-fold molar excess of MPEG-maleimide for 1 h on ice. 4. Quench the labeling by addition of a 100-fold molar excess of iodoacetamide (see Note 22). 5. Analyze labeling solutions by non-reducing 15% SDS-PAGE and Coomassie brilliant blue staining (Fig. 4b). 6. Using ImageJ software [15], compare band intensities of PCMB-cys Nb reactions with band intensities of MPEGmaleimide cys-Nbs to quantify the degree of PCMB labeling (Fig. 4c).

3.6 Purification of Monomeric Labeled Cys-Nbs

1. Initiate ion exchange chromatography (IEX) purification by equilibrating the Mono S® 5/50 cation exchange column in 20 mM sodium acetate, pH 5.5, at a flow rate of 0.5 mL/min.

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Fig. 5 (a) Schematic outline of 96-well plate setup for Octet® BLI experiments shown in (b) and (c). Blue, orange, and red indicate wells containing buffer (PBS, pH 7.4, containing 0.01% Tween-20), Nb sample, and antigen dilution series samples, respectively. The concentration of the antigen dilution series is indicated in nM. (b) and (c) show BLI binding curves of unmodified Nb_36 and PCMB-labeled Nb_36 C82b to a dilution series of complement component 5. The equilibrium dissociation constant (KD) was similar for both Nb preparations, showing that labeling of the C82b position with PCMB does not disturb binding. (b) and (c) were reproduced from [9] with permission of the International Union of Crystallography

2. Dilute the labeled cys-Nb 1:1 v/v in 20 mM sodium acetate, pH 5.5, and apply the sample to the Mono S® 5/50 cation exchange column through a capillary loop.

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3. Wash the Mono S® 5/50 cation exchange column in 20 mM sodium acetate, pH 5.5, until baseline absorbance at 280 nm is reached, using an FPLC system. Elute the labeled cys-Nb over a 20 CV 50–100% gradient of 20 mM sodium acetate, pH 5.5, containing 500 mM NaCl. Analyze IEX fractions by 15% SDSPAGE (Fig. 3c, d). 4. Pool elution fractions for downstream applications. 3.7 Analysis of Binding Capability of Labeled Cys-Nbs by BLI

Prepare a 96-well plate following the scheme outlined in Fig. 5a and the protocol below. Fill each well with 250 μL of buffer or sample. Optionally, run a similar control experiment in parallel with the unmodified Nb (see Note 23). 1. Buffer exchange labeled cys-Nbs into PBS, pH 7.4, using the SEC or PD-10 protocols described above (see Note 24). 2. Pool elution fractions and concentrate sample using a centrifugal concentrator to 2.5 mg/mL. Supplement PBS with 0.01% Tween-20 for BLI experiments. 3. Mix a 1:1 volume dilution series of the Nb target antigen in PBS, pH 7.4, containing 0.01% Tween-20. We recommend an upper starting concentration of 1,000–2,000 nM. Depending on the affinity and kinetics of the Nb assayed, the concentrations of the dilutions may need to be adjusted. 4. Equilibrate an Octet® Anti-Penta-HIS biosensors in PBS, pH 7.4, containing 0.01% Tween-20 for 15 min. 5. Initiate the BLI experiment on an Octet® system using the steps below (Fig. 5a). 6. Obtain a baseline reading for the biosensors in PBS, pH 7.4, containing 0.01% Tween-20 for 180 s (column 1 in Fig. 5a). 7. Load labeled cys-Nbs on the biosensors for 300–600 s, until 0.2 nm saturation is reached (column 2 in Fig. 5a). 8. Wash the labeled cys-Nb-loaded biosensors in PBS, pH 7.4, containing 0.01% Tween-20 for 300 s (column 3 in Fig. 5a). 9. Associate the labeled cys-Nb-loaded biosensors with an antigen dilution series for 600–1,800 s or until equilibrium is reached (column 4 in Fig. 5a). 10. Dissociate the labeled cys-Nb:antigen complex in PBS, pH 7.4, containing 0.01% Tween-20 for 600–3,600 s or until baseline is reached (column 5 in Fig. 5a). 11. Analyse the BLI data in GraphPad Prism or similar, and fit the data using an appropriate (1:1 in most cases) association/dissociation model (Fig. 5b, c).

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Notes 1. Buffers used for column purifications should be filtered and degassed to prolong column longevity. 2. Para-chloromercuribenzoic acid, maleimide, and iodoacetamide compounds are toxic. Read the safety instructions carefully. Use caution and appropriate protective equipment for your own and your colleagues’ safety. Ensure proper handling, treatment, and disposal of contaminated materials and equipment. 3. PDB codes for analyzed Nb-antigen structures: 3g9a, 3ogo, 4bel, 4c57, 4y8d, 4wgv, 4x7e, 4x7f, 4lgp, 4lgs, 4lhq, 4i13, 4eig, 3 k74, 4fhb, 4eiz, 4mqt, 4weu, 4wem, 4wen, 3sn6, 3p0g, 4ldl, 4ocm, 4w6x, 4w6y, 3cfi, 4cdg, 4kml, 3ezj, 4xt1, 4p2c, 4 s10, 4grw, 3stb, 4i0c, 4aq1, 4krm, 3j69, 3j6a, and 5nlu. 4. We have experimentally tested positions S7, S70, S82b, and S112 as outlined throughout this protocol and as reported in [9]. We identified S17 and S21 as two additional positions potentially viable as alternatives for cysteine mutagenesis and labeling, but have not yet tested these experimentally. 5. Introduction of free cysteines promotes Nb dimer formation through disulfide bonds. In our hands, Nbs containing the C70 and C82b cysteines were less prone to dimerization than Nbs containing C7 and C112 (Fig. 4b). 6. If available, use a PCR thermocycler able to generate a temperature gradient and empirically determine the optimal annealing temperature for each Nb. 7. Some clones will not be mutagenized, therefore make sure to pick multiple single colonies for culturing and subsequent sequencing. Screening 10 or more individual colonies is recommended to obtain the desired mutation. 8. To reduce time spent transforming E. coli in the future, prepare 20% glycerol stocks of the E. coli strain used for DNA amplification and protein expression. Use sterile glycerol to reduce the risk of contamination. Store E. coli glycerol stocks at 80
C. 9. For pET-22b(+) and vectors with the T7 promoter, use T7 forward sequencing primer (50 -TTAATACGACTCACTAT AGGG-30 ). 10. The BL21-derived E. coli strain LOBSTR (LOw Background STRain) is genetically modified to reduce expression of common IMAC contaminant proteins [14]. Purification of Nbs from the LOBSTR strain by Ni-NTA IMAC and SEC typically yields >95% pure protein preparations in our hands (Fig. 3a,

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b). BL21 or other BL21-derived E. coli strains are acceptable alternatives for Nb expression. 11. Alternatively, express proteins for 3 h at 37
C and harvest cells on the same day. Be aware that rapid growth and overexpression of proteins may promote inclusion body formation. In case of inclusion body formation, it is advisable to reduce E. coli growth and translation speeds by lowering the temperature as outlined in this protocol. 12. The E. coli cell pellet can optionally be stored for months at 20
C before subsequent lysis and purification steps. 13. Cell lysis by other means such as French press or chemical lysis are also viable. For sonication, we use a Labsonic M® sonicator with the following settings: 60% amplitude and 0.6 cycle. Sonication of E. coli cells suspended in PBS, pH 7.4, containing 400 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol, and 1 mM benzamidine is performed in a 50 mL tube placed in an ice bath for three times 5 min. Submerge the sonotrode 1 cm into the E. coli cell suspension for optimal wave propagation throughout the liquid. 14. Because of the introduced free thiol, cys-Nbs readily form disulfide-linked dimers (Figs. 3b and 4b), which in our hands are not easily reduced unless high concentrations of reducing agents are used. This may in turn jeopardize the canonical stabilizing disulfide linkage of the Nb. Therefore, purify cys-Nbs in the presence of a reducing agent up until labeling when a buffer exchange is implemented using a PD-10 column (see Subheadings 3.4 and 3.5). Furthermore, cys-Nbs starting from cell lysis throughout the labeling reactions should be kept at 4
C to minimize dimer formation through disulfide linkages. 15. Consult manufacturer guidelines for optimal volume and amounts for the gel filtration column you intend to use. For the Superdex™ 75 Increase 10/300 column used in this method, a sample volume of 300 μL loaded onto a 500 μL capillary loop is optimal. Ensure not to exceed the solubility limit of the cys-Nb. This will be clone dependent but the concentration in the 300 μL of injected cys-Nb should be 200) are highly soluble in this compartment since they naturally have at least one intra-domain disulfide bond and are usually derived from phage display selection platforms that traffic through the periplasm. The periplasm also provides the opportunity to load APEX2 with heme during recombinant expression since it can be supplemented in the growth medium and readily diffuses through the outer membrane. The model sdAbs described in this protocol were generated by selections on live virus preparations of the genera Marburgvirus and Ebolavirus and were all found to be specific for nucleoprotein (NP) [4, 5]. Subsequent X-ray crystallography studies revealed the breadth of sdAb engagement mechanisms and helped to explain the cross-reactivity profiles among different filoviral species [6, 7]. Mammalian cells expressing recombinant NP or cells infected with virus could be visualized after single-step probing with sdAb-APEX2 fusion proteins and development with either the colorimetric substrate DAB or the fluorescent reporter

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Amplex™ UltraRed [3]. No background binding to untransfected or uninfected cells was apparent and the ability to distinguish between the filoviral genera was retained, indicating that APEX2 was an ideal reporter to faithfully relay the binding characteristics of the sdAb. The sdAb-APEX2 fusions also had utility as probes for western blots, being able to specifically recognize antigens of interest within the complex milieu of cell lysates transferred to synthetic membranes. During these studies, we also showed that tandem dimers of sdAbs yielded superior signals compared with monomeric sdAb constructs, though their yields were 1/3 to 1/4 those of the monomers. More recently, we applied the APEX2 methodology to sdAbs specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen [8]. We showed that APEX2 dependably relayed the sensitivity and specificity of sdAbs for SARS-CoV-2 within a panel of other human and zoonotic coronaviruses, further pointing to its usefulness as a molecular probe. Here, we describe the methods employed to generate and use anti-filoviral monomeric sdAb-APEX2 fusion proteins and introduce an advancement on the approach that capitalizes on a devolved and dimeric version of APEX2. We previously noticed that while sdAb-APEX2 fusion proteins expressed well with good purity, they expressed less well than sdAbs alone. This suggested that the monomeric APEX2 enzyme might not be an optimal partner for overexpression and purification. We speculated that reverting to a dimeric enzyme with native protein surfaces may improve product quantity and quality and potentially enhance performance through avidity of the attached sdAb. We therefore removed the resurfacing mutations K14D and E112K, while retaining the catalysis-enhancing mutations W41F and A134P and the periplasmic compatibility mutation C31S. The resultant “retro” APEX2 derivative was named dEAPX for dimeric enhanced ascorbate peroxidase. Since dEAPX and APEX2 fusions are expressed, purified, and utilized in equivalent manners, we showcase the performance differences between these two systems. dEAPX fusions have improved expression in E. coli and greater purity, for at least one of the sdAb fusion partners. The dEAPX format also offers additional signal strength over the original monomeric APEX2 format when used in the whole range of probe scenarios. The sdAb-dEAPX fusions even exhibited superior performance over tandem dimers of sdAbs fused to monomeric APEX2 for detection virus-infected cells, demonstrating the combined effects of avidity and an enzyme:sdAb ratio of 1:1 rather than 0.5:1. The assembly of tandem sdAbs can be quite complex for some clones and repetitive sequences may not be amenable to rapid whole gene synthesis. Furthermore, some sdAbs engage their antigens using approaches that benefit from free N-termini and can lose affinity when placed in tandem. Consequently, the much simpler to

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make, yet higher performing sdAb-dEAPX is our recommended probe for applications where maximum sensitivity is required and smallest size or monomeric character is not a concern. The sdAbdEAPX constructs were used as probes of foci of all five species of the genus Ebolavirus and Marburg marburgvirus to offer a convenient and cost-effective means to assay samples for infectious virus particles or virions. The methods described below should be applicable to the study of any given antigen the user is interested in by simply fusing the APEX2 or dEAPX reporter modules to antigen-specific recombinant affinity scaffolds of choice. We focus on the production and purification of the probes and their final uses as we assume the reader will have their own platforms for generating and analyzing their antigen-specific sdAbs of choice and can simply exchange the existing probe(s) for APEX2- or dEAPX-based constructs.

2

Materials Aqueous solutions should be prepared with type I ultrapure water with a resistivity of 18.2 MΩ cm. Local and federal guidelines should be followed for chemical and biological hazard handling, particularly when it involves pathogens or select agents (see Note 1).

2.1 Recombinant Protein Expression and Purification 2.1.1 Equipment and General Supplies

1. Wide test tubes with fluted caps (see Note 2). 2. Bellco 250 mL flasks with 4 bottom baffles (available off-theshelf in cases of 12). The 2.5 L flasks with 4 bottom baffles are special order from Bellco though other baffled configurations may work. 3. Foam bungs and foil for pre-autoclaving empty 250 mL flasks and for capping and autoclaving large flasks with media (see Note 3). 4. Refrigerated orbital incubator for shaker flasks. 5. Swing-out centrifuge rotor, mid-capacity: Beckman Allegra 6R or equivalent (see Note 4). 6. Poly-Prep® chromatography columns (Bio-Rad, Hercules, CA, USA). 7. Amicon Ultra-4 10 kDa ultrafiltration devices. 8. Fast performance liquid chromatography (FPLC) instrument set up for size exclusion chromatography (SEC) with a SuperdexTM 200 Increase 10/300 GL column (GE Healthcare, Pittsburgh, PA, USA) equilibrated in phosphate-buffered saline (PBS) (see Note 5).

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Fig. 1 sdAb-dEAPX fusion protein expression vector pecan319 which enables the user to clone out the resident sdAb with another sdAb or protein-based affinity reagents via SfiI/NcoI and NotI restriction sites. Amp, ampicillin resistance gene; lacI, lac repressor gene; tHP, terminator; 35 trp and 10 lac, tac promoter; pelB, pectate lyase signal sequence; sdAb, single-domain antibody; dEAPX, dimeric enhanced ascorbate peroxidase; Gly3His6, polyhistidine tag; tlpp, terminator. The nucleotide sequence of the gBlocks™ (IDT, Coralville, IA, USA) encoding dEAPX-Gly3His6 between NotI and HindIII (underlined) is also displayed if a user prefers to synthesize the reporter gene for insertion into their own construct

9. E. coli TunerTM or RosettaTM-competent cells (Sigma-Aldrich, St. Louis, MO, USA). 10. 750 mL centrifuge bottles. 11. Electric pipettor. 12. 10 mL serological pipettes. 13. Large curved spatula. 14. Expression vector encoding sdAb-APEX2 (pecan278) [3] or sdAb-dEAPX fusion proteins (pecan319) (Fig. 1).

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2.1.2 Stock Solutions

1. Glycerol. 2. Antifoam (Sigma-Aldrich). 3. 20% (w/v) glucose: dissolve 200 g of anhydrous dextrose in 1 L of water and then filter through a 0.22 μm bottle top filter into a sterile 1 L bottle (store at 4  C) (see Note 6). 4. DifcoTM Terrific broth (TB, BD Biosciences, San Jose, CA, USA). To prepare ready-made medium for small volumes and starter cultures, weigh out 47.6 g of powdered medium, pour 4.8 g of glycerol on top, and dissolve all in 900 mL of water. Add one drop of antifoam. Dispense 90 mL aliquots into 100 mL bottles and autoclave. Once autoclaved, add 10 mL of 20% sterile glucose to each aliquot in a laminar flow hood or biological safety cabinet to ensure sterility. Once opened, store at 4  C. For the mid-scale volumes (450 mL), weigh out 21.4 g of broth powder and 2.2 g of glycerol onto 450 mL of water in a 2.5 L baffled flask, wait until dissolved, add one drop of antifoam, and then autoclave. 5. 20 mg/mL ampicillin stock solution: dissolve 1 g ampicillin in 50 mL of water. Add 10 μL to every 1 mL of medium to achieve a final concentration of 200 μg/mL. Store at 4  C. Add to sterile medium after autoclaving and cooling (see Note 7). 6. 3 mg/mL chloramphenicol stock solution: dissolve 0.15 g of chloramphenicol in 50 mL of ethanol. Add 10 μL to every 1 mL of medium to achieve a final concentration of 30 μg/ mL. Store at 4  C. Add to sterile medium after autoclaving and cooling (see Note 7). 7. 2 YT agar plates (per L): 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, 15 g of agar. Autoclave, then supplement with 2% glucose, 200 μg/mL ampicillin, and 30 μg/mL chloramphenicol. 8. 1 M Tris–HCl, pH 7.5: dissolve 121.14 g of Tris base in 1 L of water. Adjust pH to 7.5 with concentrated HCl. 9. 0.75 M sucrose in 100 mM Tris–HCl, pH 7.5: Dissolve 256.7 g of sucrose in 900 mL of water and add 100 mL of 1 M Tris–HCl. Sterile filter and store at 4  C. 10. 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8.0: add 186.1 g of disodium EDTA·2H2O to 1 L of water. Adjust pH to 8.0 with NaOH pellets while stirring. Autoclave. 11. 1 mM EDTA, pH 7.5: Add 2 mL of 0.5 M EDTA, pH 8.0, stock solution to 998 mL of water. Sterile filter and store at 4  C. 12. 0.5 M MgCl2: dissolve 11.9 g of MgCl2 in 250 mL water, sterile filter, and store at 4  C.

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13. 10 immobilized metal affinity chromatography (IMAC) buffer: 0.2 M Na2HPO4, 5 M NaCl, 0.2 M imidazole, 1% (v/v) Tween-20, pH 7.5. Dissolve 53.61 g of Na2HPO4·7H2O, 292.2 g of NaCl, 13.6 g of imidazole, and 10 mL of Tween-20 to 1 L of water. Adjust pH to 7.5 with HCl once all components have dissolved. Sterile filter and store at room temperature. Prepare 1 IMAC buffer from the 10 stock and store at 4  C until ready for use. 14. 10 IMAC buffer containing 500 mM imidazole: add 17 g of imidazole (check the grade to decrease UV interference) to 450 mL of 1  IMAC buffer and adjust pH to 7.5 with HCl. 15. 25 PBS: to around 600 mL of stirring water, gradually add: 54.4 g of Na2HPO4·7H2O, 160 g of NaCl, 4 g of KCl, and 4.8 g of KH2PO4. Increase volume to 800 mL and sterile filter once all components have dissolved. Store at 4  C until required. Warm to dissolve the crystalline precipitate and mix with 19.2 L of water in a 20 L carboy to make 1  PBS and store at room temperature. Filter sterilize and de-gas if buffer is used for FPLC. 16. Protein quantification reagents and/or UV spectrophotometer with small volume cuvettes. 17. SDS-PAGE gels and apparatus, including molecular size markers, 5 Laemmli buffer, and Coomassie Blue stain. 2.1.3 Solutions Made Fresh at Time of Expression

We generally work with an even number of protein expressions to maintain balance and often perform four at a time. The volumes below are for 4  500 mL cultures. 1. 1.4 M ammonium acetate: add 107.8 g of ammonium acetate to 1 L of H2O. Autoclave. 2. 10 mM hemin: add 136.5 mg of hemin to 20 mL of 1.4 M ammonium acetate and dissolve by vortexing. Add 5 mL of the hemin solution to each 450 mL of medium (previously autoclaved and cooled) while swirling the flask. 3. 100 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), dioxane-free: dissolve 0.5 g of IPTG in 21 mL of water just before use. Add 5 mL per 500 mL culture to achieve 1 mM final concentration. 4. 1 mg/mL hen egg lysozyme (6 mL total) in Tris-sucrose solution (0.75 M sucrose in 100 mM Tris–HCl, pH 7.5). 5. Ni SepharoseTM High Performance resin (GE Healthcare).

2.2 Probing Transfected Cells with sdAb-APEX2 or sdAbdEAPX

1. HEK293T cells. 2. Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 4.5 g/L glucose, L-glutamine, sodium pyruvate, 5% (v/v) fetal bovine serum, and penicillin/streptomycin.

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3. T75 flasks. 4. Trypsin/EDTA. 5. Mammalian expression vector driving production of antigen of interest. We use the puma2 vector to express most of the NPs shown here and puma4 to express Sudan ebolavirus (SUDV) NP. These are from a set of vectors with various posttranscriptional regulatory sequences to test which one is optimal [8]. 6. Pre-warmed serum free medium: for HEK293T this is DMEM. 7. Polyethylenimine (PEI; Sigma-Aldrich). 8. 10% (v/v) phosphate-buffered formalin. 9. PBS: see Subheading 2.1. 10. 0.1% (v/v) Triton X-100 in PBS: dissolve 250 μL of Triton X-100 in 250 mL of PBS and store at room temperature (see Note 8). 11. 2% (w/v) bovine serum albumin (BSA), 0.05% Tween-20 in PBS: dissolve 10 g of fraction V BSA in 400 mL of PBS, then add 250 μL of Tween-20. Adjust volume to 500 mL with PBS and sterile filter. Store at 4  C. 12. 0.1% Tween-20 in PBS (PBST) 13. The colorimetric substrate metal-enhanced SigmaFastTM DAB (Sigma-Aldrich), available in pre-weighed tablet form and prepared according to the manufacturer’s instructions by dissolving one of each tablets in 5 mL of water immediately before use. 14. The fluorescent substrate Amplex™ UltraRed (Thermo Fisher, Waltham, MA, USA; supplied as 1 mg per tube). Resuspend in 340 μL of dimethyl sulfoxide to produce a 10 mM stock and vortex well (any unused stock can be stored at 20  C). Immediately before developing, make a solution of 50 μM Amplex™ UtraRed plus 10 mM hydrogen peroxide in PBS and use 200 μL per well of an ibidi slide. 15. Hoechst counterstain. 16. ibidi μ-Slide 8-well polymer coverslips (ibidi, Fitchburg, WI, USA). We find these coverslips to yield the best imaging and fluorescence microscopy data. If using DAB, one can use standard transparent plasticware for bright-field microscopy. 17. Microscopes: fluorescence.

bright

field

for

DAB,

fluorescent

for

18. sdAb-APEX2 or sdAb-dEAPX fusion proteins produced in Subheading 3.1.

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1. SDS-PAGE gels and apparatus (e.g., Bio-Rad Mini-PROTEAN®), including molecular size markers, 5 Laemmli buffer, and Coomassie Blue stain. 2. Semi-dry electrotransfer system (e.g., Ancos or Owl™). 3. Immobilon® P polyvinylidene difluoride membrane (SigmaAldrich). 4. 2% (w/v) non-fat dried milk in PBS (MPBS), freshly made each day. Stir to homogenize. 5. Colorimetric DAB substrate: see Subheading 2.2. 6. Chemiluminescent (Thermo Fisher).

substrate,

SuperSignal™

West

Pico

7. Image capture: flatbed scanner or digital camera for colorimetric development, digital imaging system for colorimetric and chemiluminescent imaging. 8. sdAb-APEX2 or sdAb-dEAPX fusion proteins produced in Subheading 3.1. 2.4 Employing sdAbAPEX2 or sdAb-dEAPX as a Tracer in ELISA

1. 96-well microtiter plates. 2. Antigen of interest and capture reagent if desired (see Subheading 3.4). 3. sdAb-APEX2 or sdAb-dEAPX fusion proteins produced in Subheading 3.1 (see Note 9). 4. MPBS: see Subheading 2.3. 5. PBST: see Subheading 2.2. 6. PBS: see Subheading 2.1. 7. SuperSignal™ ELISA Pico substrate (Thermo Fisher) (see Note 10). 8. Luminometer. 9. Multichannel pipette.

2.5 Probing VirusInfected Cells with sdAb-APEX2 or sdAbdEAPX

1. Items 6–15 from Subheading 2.2. 2. Vero E6 cells or other cells of interest. 3. DMEM supplemented with 5% fetal bovine serum and penicillin/streptomycin. 4. T75 flasks. 5. 24-well tissue culture plates (for DAB probing and bright-field microscopy). 6. 96-well ibidi plates (for Amplex™ UltraRed staining and fluorescent microscopy). 7. Trypsin/EDTA.

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8. Viruses of interest. The examples described herein were Marburg marburgvirus (MARV) Angola or Musoke, Zaire ebolavirus (EBOV) Kikwit, SUDV Boniface, Reston ebolavirus (RESTV) Reston, Taı¨ Forest ebolavirus (TAFV) and Bundibugyo ebolavirus (BDBV). 9. For staining of foci, a semi-solid overlay is required. For the pilot experiment shown here, we employed an agarose-based overlay using 0.6% (w/v) low melt agarose (SeaPlaque™ GTG™ agarose) prepared in minimal essential medium (MEM) without pH indicator supplemented with 5% fetal calf serum, penicillin, and streptomycin (see Note 11). 10. sdAb-APEX2 or sdAb-dEAPX fusion proteins produced in Subheading 3.1.

3

Methods

3.1 Recombinant Protein Expression and Purification

1. Transform E. coli Tuner™ competent cells with vector pecan278 encoding sdAb-APEX2 [3] or vector pecan319 encoding sdAb-dEAPX by heat shock following the manufacturer’s instructions (Fig. 1). Plate on 2 YT agar plates containing 2% glucose, 200 μg/mL ampicillin, and 30 μg/mL chloramphenicol. Grow at 30  C overnight. 2. Inoculate a 3.5 mL starter culture of TB (containing 2% glucose, 200 μg/mL ampicillin, and 30 μg/mL chloramphenicol) in a test tube with a fresh colony using a blue (1 mL) pipette tip. Alternatively, transfer an equivalent sized scrape from a glycerol stock. Grow this culture at a slant at 30  C with vigorous, though not manic, shaking for 7 h (~200 rpm). We usually perform four different expressions at one time. 3. Once satisfied that all cultures are growing, subculture into 50 mL of TB (containing 2% glucose, 200 μg/mL ampicillin, and 30 μg/mL chloramphenicol) in a 250 mL baffled flask and continue to grow overnight with ~200 rpm shaking at 30  C. By 9:00 the next morning, these cultures should be very dense and will serve as the starters for the mid-scale expression. 4. Inoculate each 50 mL starter culture using good sterile technique into 450 mL of TB containing 0.1 mM hemin without glucose and antibiotics in a 2.5 L baffled flask. Grow for 3 h at 25  C with ~200 rpm shaking (see Notes 3 and 12). Induce expression by addition of 5 mL of 100 mM IPTG and grow for a further 3 h at 25  C with ~200 rpm shaking. 5. Transfer each culture to a pre-weighed 750 mL Beckman centrifuge bottle and centrifuge at 3,500 rpm for 20 min at 4  C in a Beckman R6 benchtop centrifuge or equivalent (2,800  g at rmax). Decant the supernatant back into the flask and bleach.

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Drain the pellet briefly over some paper towels and then weigh the bottle to calculate the approximate wet weight of cells (see Note 13). 6. Place the bottles in a large tray of ice and add 14 mL of ice-cold 0.75 M sucrose in 100 mM Tris–HCl, pH 7.5 to each bottle. One by one, use a large curved spatula to resuspend the pellets. If noticeable small clumps remain, use an electric pipettor with a 10 mL pipette to finish resuspending the small clumps. Once all pellets are resuspended place the ice tray on a benchtop orbital platform and rotate gently. Add 1.4 mL of the 1 mg/mL hen egg lysozyme solution to each suspension. Slowly, dribble in 28 mL of ice-cold 1 mM EDTA solution and leave for 15 min. Add 2 mL of 0.5 M MgCl2 and continue swirling for 15 min (see Note 14). 7. Prior to using the Ni Sepharose™ resin, swirl the suspension of beads provided in 20% ethanol and retrieve about 750 μL into a 2 mL Eppendorf tube. Pellet in a microfuge briefly (5,000  g, 30 s, room temperature), pour off the supernatant and resuspend in 1 mL of 1 IMAC buffer. Centrifuge again (5,000  g, 30 s, room temperature), drain pellet, and resuspend in 1 mL of 1 IMAC buffer. The final volume of the resin should be 500 μL. 8. Centrifuge the bottles at 2,800  g for 20 min at 4  C. Pour the supernatant into a 50 mL Falcon tube on ice (see Note 15). Add one tenth the volume of 10 IMAC buffer (~4.5 mL) and swirl to mix. Add the 500 μL prewashed Ni Sepharose™ resin in 1 IMAC buffer and leave the tube horizontal on ice, gently shaking on the orbital platform shaker for 1 h. 9. Centrifuge the tubes for 5 min at 2,800  g at 4  C and pour off the supernatant leaving the resin in the bottom of the tube. Add 45 mL of ice-cold 1 IMAC buffer and gently invert several times. Repeat the centrifugation, decanting, and washing step. Before pouring off the supernatant for the second time, take 3 mL using a 5 mL pipette and use this to resuspend the Ni Sepharose™ resin pellet that remains. Transfer the 3 mL of resin the Poly-Prep® column. Cap and leave in the fridge overnight or allow the resin to settle and continue. 10. Remove the buffer above the settled resin. Add 400 μL of elution buffer (1 IMAC buffer containing 500 mM imidazole) to void the 0.5–0.6 mL of Ni Sepharose™ resin. Place the Poly-Prep® column over an Amicon Ultra-4 10 kDa ultrafiltration device and add 2 mL more elution buffer to elute the His-tagged proteins. 11. Concentrate the proteins to 200 μL by centrifugation for several min at 2,000  g. The required time to achieve this volume will vary between preparations depending on the

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Fig. 2 Size exclusion chromatography of the monomeric (APEX2) and dimeric (dEAPX) fusion proteins of antiMARV NP sdAb A (MA) and anti-EBOV NP sdAb ZE (ZE) with predicted molecular weights indicated. It is interesting to note that, at these high concentrations, the APEX2 fusion proteins appear to have a small proportion of multimers present while the dEAPX fusion proteins appear strictly dimeric, with no higher molecular weight or monomeric forms apparent

quantity of protein present so start with 10 min then extend time as appropriate. Once done, transfer to a round bottom 2 mL microcentrifuge tube on ice. 12. Using a 1 mL glass syringe apply concentrated protein to the gel-filtration column (SuperdexTM 200 Increase 10/300 GL) equilibrated in PBS. Collect the peak fractions and pool. Examples of the chromatograms for sdAb-APEX2 and sdAb-dEAPX are shown in Fig. 2 (see Note 16). Add glycerol to 50% (v/v), aliquot, and store at 80  C. An aliquot can be thawed for quantification by UV adsorption and then 1 and 5 μg analyzed by SDS-PAGE to gauge purity (Fig. 3). 3.2 Probing Transfected Cells with sdAb-APEX2 or sdAbdEAPX

1. Grow HEK293T cells in DMEM supplemented with 4.5 g/L glucose, L-glutamine, sodium pyruvate, 5% fetal bovine serum, and penicillin/ streptomycin in a T75 flask (see Note 17). 2. Approximately 18 h prior to transfection (see Note 18), seed 7.5  104 HEK293T cells in 300 μL of medium per ibidi chamber. Add 0.5 μL of PEI (1 mg/mL, pH 7) to 14.5 μL of serum-free DMEM for each well to be transfected. Make an amount appropriate for the total number of wells. Add mammalian expression vector DNA-encoding antigen of interest (125 ng in 1.25 μL made to 15 μL with serum-free DMEM). Add an additional 15 μL of PEI+DMEM to make a final volume of 30 μL. Allow to equilibrate for 20 min at room temperature and then gently add to cells (see Note 19). 3. After 24 h (or user-defined time depending on antigen), remove medium by gently dumping into a waste container, dab on a paper towel and wash cells gently by dipping one

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Fig. 3 Coomassie Blue-stained SDS-PAGE of 5 μg and 1 μg of the purified monomeric (APEX2) and dimeric (dEAPX) sdAb fusions to gauge purity. Molecular weights of markers in kDa are shown. MA, anti-MARV NP sdAb A; ZE, anti-EBOV NP sdAb ZE

side and then the other into warm serum-free DMEM in a multichannel reservoir. Repeat wash step and dab on paper towel again (see Note 20). 4. Gently add 10% phosphate-buffered formalin to fix cells for 1 h at 4  C. 5. Gently wash slides three times with 300 μL of PBS. 6. Add sufficient 0.1% Triton X-100 in PBS to cover and permeabilize cells for 10 min. 7. Wash slides three times with 300 μL of PBS. 8. Block with 300 μL of 2% BSA, 0.05% Tween-20 in PBS for 1 h. 9. Replace blocking solution with 200 μL of various concentrations of the sdAb-APEX or sdAb-dEAPX probes diluted in PBS containing 2% BSA and 0.05% Tween-20 to determine the optimal concentration (we suggest 100, 10, 1, and 0.1 nM as a starting range). 10. Wash three times with 300 μL of PBST, then twice with 300 μL of PBS. 11. For DAB staining add 200 μL of dissolved DAB tablet and incubate for 1 min. Monitor color development and develop according to preference by eye. Stop by dumping and washing twice with water. 12. For fluorescent staining, add 200 μL of the Amplex™ UltraRed reagent for 10 min. Monitor the color development in the supernatant. Stop by dumping and washing twice in 300 μL of PBS. 13. Apply 200 μL of Hoechst for 10 min. Wash twice with 300 μL of PBS.

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Fig. 4 Comparison of the ability of monomeric sdAb-APEX2 and dimeric sdAb-dEAPX probes to detect a range of recombinant filovirus NPs expressed in HEK293T cells. At 24 h post-transfection cells were fixed, permeabilized, and probed with 10 nM of each of the fusions. Fluorescent signals were developed using Amplex™ UltraRed and images captured microscopically at 10 magnification. (a) Probing with anti-EBOV sdAb ZE fusions; (b) probing with anti-MARV sdAb MA fusions. Recombinant human codon optimized NP genes expressed in HEK293T cells: EBOV, Zaire ebolavirus Kikwit; SUDV, Sudan ebolavirus Boniface; RESTV, Reston ebolavirus Reston; TAFV, Taı¨ Forest ebolavirus; BDBV, Bundibugyo ebolavirus; MARV, Marburg marburgvirus Musoke

14. Store slides at 4  C in a sealed container with a moist paper towel to keep the slides from drying and protect from light. For long-term storage, wrap slides with parafilm or cling-film. 15. Image DAB stained slides by light microscopy. Image fluorescence slides on an Eclipse Ti confocal microscope (Nikon) with NIS Elements Imaging Software and use ImageJ within Fiji to process the images. Figure 4 provides an example of the data that can be generated using the process. The differentially reactive anti-EBOV sdAb ZE (ZE) was more prone to bind a variety of different NPs as a dimeric dEAPX construct as opposed to the monomeric APEX2 construct. The antiMARV sdAb A (MA) showed enhanced MARV NP signal as a dimeric dEAPX construct compared with the monomeric APEX2 format.

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1. Laboratories will have their preferred methods of resolving proteins by electrophoresis and transferring to membranes that may suit the specific requirements of particular samples and antigens. We typically use the Bio-Rad Mini-PROTEAN® SDS-PAGE apparatus with home-made Laemmli gels and the Ancos or Owl™ semi-dry blot approach for transfer to Immobilon® P membranes. For the subsequent procedure, we assume the user has already accomplished these steps. 2. Immediately after the transfer submerge the membrane in MPBS and leave for at least 1 h with gentle rocking (for us this step is at the end of a day and we leave overnight at 4  C). 3. Pour off the MPBS and replace with a minimal volume of MPBS (usually 20 mL to cover two gel transfers on the same membrane) containing the desired concentration of probe (10 nM in this example; dependent on binding affinity, see Note 21). Rock gently at room temperature for 1 h. 4. Pour off the probe solution, wash with 20 mL of PBST by pouring and then add 20 mL more PBST and rock for 5 min. Pour off and repeat twice. 5. Pour off PBST and perform two washes with PBS. 6. When ready to develop signal, drain the membrane. Add either: (i) colorimetric substrate DAB and develop according to preference by eye and stop with a water wash, or (ii) chemiluminescent substrate and capture image on X-ray film in a dark room and develop or using an imager if available (see Note 22). Figure 5 provides an example of the data that can be generated using this process where the sdAb constructs have a challenging task of detecting the NP C-terminal domain as monomeric APEX2 formats (with ZE sdAb just visible on cognate EBOV) yet are much improved when used in the dimeric dEAPX format.

3.4 Employing sdAbAPEX2 or sdAb-dEAPX as a Tracer in ELISA

Each laboratory will have its own ELISA protocol depending on the application. Typically, this would involve either coating antigens directly in microtiter plates and blocking the wells or coating capture antibodies, blocking the wells, and then capturing antigens. The idea is to exchange the final detection step using a secondary enzyme conjugate, with an sdAb-APEX2 or sdAb-dEAPX fusion (see Note 9). For the subsequent procedure, we assume the user has already accomplished these steps. It is essential that the wells have been fully blocked at some point during the procedure and that the antigen is in a format that will be recognized by the tracing sdAb. 1. Tap the plate on a stack of paper towels or carefully aspirate to remove all of the previous solution into disinfectant if it contained an infectious agent.

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Fig. 5 Comparison of the ability of monomeric sdAb-APEX2 and dimeric sdAbdEAPX probes to detect a range of recombinant filovirus NP C-termini using western blotting. Approximately 100 ng of His6-nluc-NP C-terminal fusion proteins were transferred from SDS-PAGE to Immobilon® P membranes and probed with: (a) anti-His6 horseradish peroxidase conjugate (1/10,000 dilution); (b) anti-EBOV sdAb ZE-APEX2 or anti-MARV sdAb MA-APEX2 (both at 10 nM); or (c) anti-EBOV sdAb ZE-dEAPX or anti-MARV sdAb MA-dEAPX (both at 10 nM). Each pair of APEX2 and dEAPX fusions of a particular sdAb were processed side by side to ensure the data could be compared. The images were captured on a Bio-Rad imager as bright field, then chemiluminescent (47 s exposure) and then merged within the software. E, EBOV; S, SUDV; R, RESTV; T, TAFV; B, BDBV; M, MARV. Molecular weight markers are indicated in kDa

2. Use a multichannel pipette to add 100 μL of sdAb-APEX2 or sdAb-dEAPX probe in MPBS to each well and cover the plate with a plate sealer. The concentration will need to be determined empirically or guided by the EC50 value.

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Fig. 6 Using sdAb-APEX2 and sdAb-dEAPX as tracers for the final step in a sandwich ELISA. Here, we used the monoclonal affinity reagent sandwich assay (MARSA) format where neutravidin-coated wells capture sitespecifically biotinylated sdAbs which then serve to capture titrated purified recombinant NP polymer previously generated in HEK293T cells. Each probe (100 nM) was then used as the final tracer followed by chemiluminescent development. A single high concentration of the non-cognate NP served as a negative control. MA, anti-MARV sdAb A; ZE, anti-EBOV sdAb, EBOV, Zaire ebolavirus Kikwit; MARV, Marburg marburgvirus Musoke; NP, nucleoprotein

3. Incubate the plate for 1 h in a moist sandwich box static, or for 6–10 min on a plate shaker. 4. Wash wells three times with 300 μL/well of PBST and two times with 300 μL/well of PBS (see Note 23). 5. Develop plate with 50–100 μL per well of SuperSignalTM ELISA Pico substrate warmed to room temperature using the injector on a Turner Luminometer (or equivalent) with a 2 s integration. 6. Read signals in a luminometer and plot relative light units (RLU) against the variable of interest. Figure 6 provides an example of data that can be generated using this process. 3.5 Probing VirusInfected Cells with sdAb-APEX2 or sdAbdEAPX

Probing virus-infected cells is essentially the same as the process for probing transfected cells (see Subheading 3.2) after a suitable time has elapsed for sufficient viral replication. Ensure the inactivation procedure has been validated on-site as this will be mandated for select agents. Figure 7 provides an example of the data that can be generated using this process and reveals that the dEAPX format is superior to the more complex tandem sdAb-APEX2 format, for both the anti-EBOV sdAb ZE and the anti-MARV sdAb MA. 1. For the focus forming assay, Vero E6 cells are seeded 2 days prior to infection in T75 flasks in DMEM supplemented with 5% fetal bovine serum and penicillin/streptomycin and grown in a humidified CO2 incubator at 37  C.

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Fig. 7 Superior detection of viral infection of Vero E6 cells using sdAb-dEAPX over monomeric sdAb-APEX2 or even tandem dimeric sdAb fused to monomeric APEX2. 8  103 pfu of live EBOV Kikwit or MARV Angola were used to infect cells. After 24 h, following fixation and permeabilization, cells were probed with (a) 10 nM of the anti-EBOV sdAb ZE or (b) 10 nM of the anti-MARV sdAb A MA probes. Development employed the fluorescent substrate Amplex™ UltraRed with images captured microscopically at 10 magnification. (ZE)2 and (MA) 2 represent tandem dimers of the respective sdAb. EBOV, Zaire ebolavirus Kikwit; MARV, Marburg marburgvirus Musoke

2. One confluent T75 flask is trypsinized in 5 mL trypsin EDTA, then added to 21 mL DMEM/5% fetal bovine serum. Seed two 24-well plates at 0.5 mL per well. 3. Prior to infection, remove culture medium and replace with 250 μL of DMEM. Plates are taken into the BSL-4 laboratory, viral dilutions are added, and the plates are rocked at 37  C in a humidified CO2 incubator for 1 h. 4. Virus is removed and 0.5 mL of 0.6% SeaPlaqueTM GTGTM agarose MEM overlay is added to each plate and allowed to solidify before returning to the incubator (plates are static). Leave one plate for 48 h and the other for 96 h. 5. One plate is removed after 48 h, placed in formalin in a sealed box and left at 4  C for 24 h. The plate is removed, the overlays are removed, and the plate is again placed in formalin for 24 h before being removed from the BSL-4 laboratory via the chemical dunk tank. The process is repeated at 96 h post-infection for the second plate. 6. The plates are permeabilized, blocked, probed, and developed with DAB in the same manner as the transfected cells in Subheading 3.2. All washes can be done with a squirt bottle as, after fixation, the monolayer is stable. Images were captured on an EVOS microscope with the 40 objective. Figure 8 provides an example of the foci of virus-infected cells detected using the sdAb-dEAPX constructs 96 h post-infection.

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Fig. 8 Employing sdAb-dEAPX fusions to detect foci of virus-infected Vero E6 cells 96 h post-infection. Cells were either probed with (a) 10 nM anti-EBOV sdAb ZE-dEAPX or (b) 10 nM anti-MARV sdAb MA-dEAPX. Color development employed DAB and images were captured at 40 magnification. EBOV, Zaire ebolavirus Kikwit; SUDV, Sudan ebolavirus Boniface; RESTV, Reston ebolavirus Reston; TAFV, Taı¨ Forest ebolavirus; BDBV, Bundibugyo ebolavirus; MARV, Marburg marburgvirus Musoke

4

Notes 1. Be sure to consult with your Biological Safety Officer, Environmental Health and Safety staff, Institutional Biosafety Committee and Alternate/Responsible Official to ensure compliance as appropriate. 2. We try and recycle as much as possible and soak these tubes and caps in bleach, rinse well with water, dry, autoclave, and reuse. The tubes should last for years. 3. Baffles significantly increase the aeration imparted to shake flask cultures in orbital incubators and are vital in achieving high cell wet weights. Ensure you set the orbital speed to achieve maximal aeration of the culture, as observed by eye, and not necessarily the maximum speed.

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4. A versatile swing-out rotor with a variety of inserts is extremely helpful here as initially pelleting the cells as a thin layer on the bottom of a 750 mL pot is key to resuspending the cells evenly and quickly as opposed to struggling with a large compacted pellet in a fixed angle rotor. The subsequent clarification of the periplasm and IMAC purification can all be accomplished in such an instrument and do not require particularly high speeds. 5. We have an AKTA FPLC (GE Healthcare) and prefer to try and generate as pure a reagent as possible though, it should be possible to use IMAC pure material if FPLC is not available. If this is the case, we would suggest dialyzing into PBS, checking purity on a gel, freezing in glycerol in small aliquots, and empirically titrating for each application. 6. We try to use reusable glass filtration devices where possible fitted with disposable 0.22 μm filters to minimize single-use plastic ware. Wash, dry, and autoclave before each use. 7. Our expression system relies on ampicillin selection of a very high copy number replicon, and we routinely use E. coli Tuner™ or Rosetta cells™ as the hosts. This latter strain contains the pRARE plasmid to constitutively supply rare tRNA codons and relies on chloramphenicol selection. 8. Our antigens of interest are within the cell and so we require a membrane permeabilization step for probing. If your antigen is cell surface displayed, you should not require this step depending on what your goals are (simple detection or more complex processing studies). 9. Our test system herein is a monoclonal antigen reagent sandwich assay or MARSA [4, 9] where a single antibody is used as captor and tracer which works well for polyvalent antigens. We first employ neutravidin-coated ELISA wells to capture a singly biotinylated version of the sdAb within Bio-Plex® buffer. After washing, a titration of recombinant polyvalent NP within MPBS is then applied, washed again, and the sdAb-APEX2 or sdAb-dEAPX tracers added in MPBS. 10. We have used colorimetric measurement at 570 nm for Amplex™ UltraRed development previously [3], but the dynamic range is more limited. In our hands, by comparison with our standard Gaussia luciferase (Gluc)-based sdAb fusions and using this exact same MARSA system and NP preparations the dynamic range and chemiluminescent signal strengths of Gluc (which is naturally a monomer) to APEX2 are equivalent. 11. For us this was a pilot experiment to mimic how we do plaque assays and used an agarose overlay. Even more convenient would be methylcellulose or Avicell which both allow easier removal of the matrix for fixation.

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12. While sdAbs are renowned for high productivity and solubility, other recombinant antibody fragments such as scFvs are not, and we typically grow cultures prior to induction at 30  C and then induce at 25  C. Here, since dextrose is absent in the mid-scale volumes we use 25  C before and after to ameliorate the negative consequences of leaky expression. A temperature of 25  C can be difficult to maintain in an ambient lab depending on the location and a refrigerated orbital incubator is a great help and worth the expense. 13. Here, we usually obtain 6–8 g, anything less indicates an issue with the construct interfering with growth. While we have not seen this with sdAb genes, this can happen with scFvs and we would suggest decreasing IPTG to 0.1 mM and/or inducing in the presence of glucose to balance productivity of recombinant protein with cell biomass. 14. In the original description of the optimized osmotic shock protocol [10], it is vital not to have locally high concentrations of EDTA, and so we gently shake on a tray of ice and add the EDTA over the course of 30 to 60 s dropwise. 15. If you notice thick, viscous pellets (we have never seen this with >200 sdAbs), this may indicate that your affinity handle has a problem. This often occurs with scFvs. 16. Between chromatography runs flush the injection loop and also run a PBS sample to ensure no residual protein remains on the column which may confound interpretation of the next protein being purified. The hemin-loaded recombinant proteins should appear as brown bands migrating down the column, and the collected fractions should have a brown tinge to them. 17. Laboratories will have their own transient expression systems of choice, or antigen positive and negative null lines to test the performance of the sdAb probes and ensure no background binding occurs. We routinely use PEI-mediated transfection of HEK293T cells for a first look at performance of the probes since these cells generate large amounts of protein within 24 h owing to the high gene expression obtainable with SV40 origin containing vectors. Although we routinely achieve transfection efficiencies of 80–90% in HEK293T cells, they are fairly fragile and can be labile to washing procedures so we advise coating culture surfaces with 0.01% (w/v) poly-L-lysine (0.1% solution is available from Millipore Sigma) in sterile water for a few min and rinsing 3 with PBS before plating cells. If the probes perform satisfactorily, we then switch to a cell line permissive for virus infection which, in our hands for these viruses, are Vero E6 cells which produce modest amounts of proteins and are more difficult to transfect efficiently with PEI (5–10%).

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18. It is always important to include a control transfection set up along-side using, e.g., β-galactosidase, to ensure each set of transfections is performing as expected before spending time processing further. 19. Prior to using PEI, it is recommended to determine the optimal ratio of PEI:DNA since excess PEI is detrimental to the cells. The complexing reaction should not be mixed in polypropylene plastic; we use polystyrene. A sterile 96-well plate works well for small volumes and can be saved for repeated use until most or all of the wells have been used. For large-scale transfections in 10 cm plates, we use Falcon round bottom polystyrene tubes. 20. All washes and liquid additions that do not require a specific volume (essentially everything except the probe and substrate) can be added with the multichannel reservoirs. This avoids disrupting the monolayer by pipetting. 21. Here, we used 10 nM for these particular constructs. The user may need to titrate based on the affinity and specificity of the sdAb being used, and also the amount of antigen thought to be present. 22. Be sure to have a control or use an anti-tag antibody to ensure that the protein behaves as expected during SDSPAGE and transfers to membranes. Some very large targets can be reluctant to enter SDS-PAGE gel matrices and may be poorly transferred, while smaller targets can actually migrate through a membrane. Bear in mind that if the epitope is absolutely conformational and discontinuous, the denaturing sample approach (boiling, SDS, reducing agent) is unlikely to work. However, we have often found that sufficient linear information exists as part of many conformational epitopes that can be leveraged by probe avidity. 23. For most antigens, we do this with squirt bottles over a sink and fill each well to brimming before dumping the wash buffer out. For pathogens or toxins, ensure you use a more controlled method within a biosafety cabinet at the appropriate containment level, e.g., using a multichannel for washing and a self-contained vacuum line aspirating to disinfectant (or house vacuum bubbling through two bleach traps), or plate washer within a biosafety cabinet.

Acknowledgments This work was supported by NIH NIAID grant award R01AI112851.

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References 1. Martell JD, Deerinck TJ, Sancak Y et al (2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 30:1143–1148 2. Lam SS, Martell JD, Kamer KJ et al (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods 12:51–54 3. Sherwood LJ, Hayhurst A (2019) Periplasmic nanobody-APEX2 fusions enable facile visualization of Ebola, Marburg, and Mĕngla` virus nucleoproteins, alluding to similar antigenic landscapes among Marburgvirus and Dianlovirus. Viruses 11:364 4. Sherwood LJ, Hayhurst A (2013) Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA) formulation. PLoS One 8:e61232 5. Sherwood LJ, Osborn LE, Carrion R Jr et al (2007) Rapid assembly of sensitive antigencapture assays for Marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level 4. J Infect Dis 196 (Suppl. 2):S213–S219

6. Garza JA, Taylor AB, Sherwood LJ et al (2017) Unveiling a drift resistant cryptotope within Marburgvirus nucleoprotein recognized by llama single-domain antibodies. Front Immunol 8:1234 7. Sherwood LJ, Taylor AB, Hart PJ et al (2019) Paratope duality and gullying are among the atypical recognition mechanisms used by a trio of nanobodies to differentiate Ebolavirus nucleoproteins. J Mol Biol 431:4848–4867 8. Sherwood LJ, Hayhurst A (2021) Toolkit for quickly generating and characterizing molecular probes specific for SARS-CoV-2 nucleocapsid as a primer for future coronavirus pandemic preparedness. ACS Synth Biol 10:379–390 9. Sherwood LJ, Hayhurst A (2012) Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures. Sci Rep 2:807 10. Neu HC, Heppel LA (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 240:3685–3692

Chapter 23 Development of Glypican-2 Targeting Single-Domain Antibody CAR T Cells for Neuroblastoma Nan Li and Mitchell Ho Abstract Chimeric antigen receptors (CARs) are engineered fusion proteins constructed from antigen-recognition, signaling, and costimulatory domains. CARs can be expressed in T cells with the purpose of reprogramming the T cells to specifically target tumor cells. This strategy thereby avoids the requirement for antigen processing and presentation by the target cell. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan with highly tumor-specific expression in neuroblastoma compared with nonmalignant cells. Therefore, GPC2 is an attractive target candidate for CAR T-cell therapy. Single-domain antibodies (sdAbs) can access epitopes different from those targeted by single-chain variable fragments and, because of their stability and modularity, could serve as ideal antigen-recognition domains in CAR T cells. Here, we describe a protocol for generating GPC2-targeted sdAb CAR T cells. We also present a methodology for assessing the efficiency of CAR expression on human T cells and their ability to kill GPC2-positive neuroblastoma cells in vitro and in vivo. The method described here is applicable to the production of CAR T cells derived from all types of sdAbs including VHHs and VNARs. Key words Adoptive T-cell therapy, Glypican, Neuroblastoma, Nanobody, Lentivirus, Single-domain antibody

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Introduction The concept of using an antibody-based chimeric antigen receptor (CAR) to redirect the effector functions of T cells was first described almost 30 years ago [1]. CARs are composed of an extracellular antigen-recognition domain, a transmembrane spacer, and intracellular signaling domains. The standard antigenrecognition domain typically consists of a single-chain variable fragment (scFv), which serves as the targeting moiety. The intracellular portion consists of signaling domains necessary for activation of T cells. CD19-targeted CAR T cells have been investigated clinically for the treatment of B-cell malignancies. Adoptive transfer of CD19-directed CAR T cells has generated complete and durable

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_23, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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remissions in patients with refractory/relapsed B-cell malignancies, which has led to the approval of four CD19 CAR T-cell products by the US Food and Drug Administration [2–5]. Following this success, much attention has been devoted to the development of CAR T cells for the treatment of other hematological malignancies and solid tumors. However, selecting appropriate solid tumor targets is challenging. The expression of most target candidates is not tumor specific. They are also expressed on healthy tissues, which increase the risk of on-target, off-tumor adverse effects. Glypicans (GPCs) are a group of heparan sulfate proteoglycans that are attached to the external surface of the plasma membrane via a glycosyl-phosphatidylinositol anchor [6]. There are six GPC family members (GPC1–GPC6) in mammals [7, 8]. GPC3 is expressed in over 70% of hepatocellular carcinoma (HCCs) [7, 9], but is not detected in non-malignant tissues [10]. GPC3-targeted CAR T cells were shown to inhibit the growth of HCC xenografts in mice [11]. We previously developed a GPC3-targeted CAR based on the hYP7 scFv and demonstrated that CAR (hYP7) T cells could eliminate tumors in mice bearing orthotopic HCC xenografts [12]. The clinical development of this GPC3 CAR T cell is underway in China (NCT04121273) and the United States (NCT05003895). Recently, we and others demonstrated that another GPC, GPC2, was highly expressed in neuroblastoma, one of the deadliest childhood cancers, and minimally expressed in normal tissues [13–15]. Moreover, integration of clinical and genomic information from neuroblastoma patients revealed a correlation between high GPC2 expression and poorer neuroblastoma prognosis and overall survival [13]. These findings suggest that GPC2 is an attractive target for CAR T-cell therapy. Conventional antibodies and/or their fragments cannot always bind certain antigenic epitopes because of their large size and the challenges in generating optimal scFvs. By contrast, single-domain antibodies (sdAbs) can access cavities and buried epitopes that are unreachable by conventional antibodies due to their small size (12–15 kDa). SdAbs also do not require the additional engineering steps associated with generation of scFvs from conventional antibodies. Moreover, sdAbs have been shown to be effective for generation of functional CAR T cells [16]. Therefore, sdAbs could serve as suitable antigen-recognition domains in CAR T cells. Several gene transfer platforms have been used to introduce the CAR transgene into primary T cells. The transgene insertion can be performed by virus-mediated transduction, transfection of messenger RNA, Sleeping Beauty transposons, and piggyBac transposons [17]. Lentiviruses have been widely used in the clinic since they can transduce terminally differentiated cells such as T cells and have been designed to include a variety of safety features [18]. In general terms, production of lentiviruses is accomplished by transiently transfecting HEK-293T cells with a transfer vector, packaging plasmid(s), and an enveloping plasmid [19].

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We isolated a human heavy chain variable domain antibody (LH7) targeting GPC2 by phage display technology [13]. In a proof-of-concept study, we showed that LH7 CAR T cells were able to kill GPC2-positive neuroblastoma cells in culture and mouse models. The GPC2-targeted CAR construct contains an upstream green fluorescent protein (GFP) reporter for cell tracking. As shown in Fig. 1, the GPC2 CAR lentiviral construct (named pMH260) contains: (1) the GFP reporter sequence; (2) the selfcleaving T2A sequence; and (3) the CAR sequence consisting of the

Fig. 1 Schematic of the CAR (LH7) lentiviral vector pMH260. (a) The construct consists of GFP, self-cleavable T2A, and the CAR cassette including the LH7 single-domain antibody, CD8α hinge, CD8α transmembrane domain (TM), 4-1BB costimulatory, and CD3ζ domains. (b) Plasmid map of pMH260

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Fig. 2 Schematic of CAR (LH7) T-cell production and evaluation in mouse models. Flow chart of the production of CAR (LH7) T cells and determination of their efficacy in NSG mice bearing human neuroblastoma metastatic xenografts. The analysis of CAR T cells includes measurement of cytokine/chemokine secretion, determination of the CAR vector-positive cells by droplet digital PCR (ddPCR), sequencing of the CAR vector integration site, and evaluation the effect on cancer cell signaling pathways

LH7 heavy chain variable domain antibody, the hinge and transmembrane regions of CD8α, the signaling domain of costimulatory molecule 4-1BB, and the cytoplasmic domain of CD3ζ chain. The entire sequence was synthesized and then cloned into the pLenti6.3 backbone lentiviral vector. This chapter focuses on the generation of GPC2-targeted CAR T cells using a lentiviral-based gene transfer approach. The antitumor activity of CAR T cells is determined using neuroblastoma cells expressing GPC2 both in vitro and in vivo. A brief overview of this process is shown in Fig. 2.

2

Materials Prepare all reagents in a sterile environment at room temperature. Follow all institutional safety requirements when disposing of waste materials.

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1. Lentiviral plasmid pMH260 [pLenti6.3 expressing GFP-T2AGPC2 CAR (LH7)] (see Note 1). 2. Second-generation lentiviral packaging plasmid psPAX2 (Addgene #12260). 3. Vesicular stomatitis virus (VSV)-G envelope expressing plasmid pMD2.G (Addgene #12259). 4. Chemically competent Escherichia coli Stbl3™ cells (Thermo Fisher Scientific, Waltham, MA, USA). 5. LB broth and agar plates: 10 g of tryptone, 10 g of NaCl, and 5 g of yeast extract in 1 L of ultrapure water. For plates, add 15 g of agar. Autoclave, then supplement with 100 μg/mL ampicillin when cool. 6. EndoFree® Plasmid Maxi Kit (Qiagen, Valencia, CA, USA). 7. 0.45 μm filters. 8. Incubator.

2.2 Cell Culture and Maintenance

1. HEK-293T cells cryopreserved in liquid nitrogen (see Note 2). 2. Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin–streptomycin. 3. RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin. 4. Luciferase-expressing IMR5 (GPC2-positive) neuroblastoma cell line cryopreserved in liquid nitrogen. 5. Phosphate-buffered saline (PBS), pH 7.4: dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 1 L of ddH2O. Autoclave. 6. 0.25% (w/v) trypsin-ethylenediaminetetraacetic acid (EDTA). 7. 0.4% (w/v) trypan blue solution. 8. Automated cell counter. 9. RPMI-1640 medium supplemented with 20% heat-inactivated FBS, 1% L-glutamine, and 10% (v/v) dimethyl sulfoxide. 10. Cryogenic vials. 11. CO2 incubator.

2.3 Lentivirus Production

1. 0.01% (w/v) poly-L-lysine (optional; see Note 3). 2. Cell culture grade water. 3. HEK-293T cells. 4. DMEM supplemented with 10% heat-inactivated FBS, 1% Lglutamine, and 1% penicillin–streptomycin. 5. 100 mm cell culture dishes.

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6. Serum-free DMEM. 7. Lentiviral plasmids: see Subheading 2.1. 8. CalFectin™ mammalian DNA transfection reagent (SignaGen, Rockville, MD, USA) (see Note 4). 9. Sterile 0.45 μm filter/storage bottle systems (low protein binding). 10. Lenti-X™ concentrator (Takara Bio, Mountain View, CA, USA) (see Note 5). 11. RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin. 12. 70% (v/v) ethanol and 10% (v/v) bleach. 2.4 Lentivirus Titration

1. HEK-293T cells. 2. DMEM supplemented with 10% heat-inactivated FBS, 1% Lglutamine, and 1% penicillin–streptomycin. 3. Sterile 12-well cell culture plates. 4. 5% (w/v) bovine serum albumin (BSA) in PBS containing 0.1% (w/v) sodium azide. 5. PBS: see Subheading 2.2. 6. 1% (w/v) formaldehyde in PBS (see Note 6). 7. 5 mL round-bottom polystyrene tubes. 8. Flow cytometer.

2.5 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

1. 50 mL buffy coat from approximately 300 mL fresh whole human blood. 2. Sterile conical 15 mL and 50 mL tubes. 3. Sterile PBS: see Subheading 2.1. 4. Ficoll-Paque. 5. Leucosep™ 50 mL tube (Greiner Bio-One, Kremsmu¨nster, Austria). 6. ACK (ammonium-chloride-potassium) lysing buffer. 7. Complete human PBMC culture medium: RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% Lglutamine, and 1% penicillin–streptomycin. 8. Freezing medium: RPMI-1640 medium supplemented with 20% heat-inactivated FBS, 1% L-glutamine, and 10% dimethyl sulfoxide.

2.6 Activation of T Cells Within PBMCs

1. RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin. 2. Sterile 24-well cell culture plate.

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3. Dynabeads® Human T-Activator CD3/CD28 for T-Cell Expansion and Activation (Thermo Fisher Scientific, Waltham, MA, USA) (see Note 7). 4. Recombinant human interleukin (IL)-2 (Chiron, Emeryville, CA, USA). Store aliquots at
20  C. 2.7 Lentiviral Transduction of Human T Cells Within PBMCs

1. Lentiviruses encoding CAR (LH7 antibody).

2.8 CAR T-Cell Expansion

1. RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin.

2. 10 mg/mL protamine sulfate stock in PBS, filter sterilized (0.22 μm). Store aliquots at
20  C (see Note 8).

2. Freezing medium: RPMI-1640 medium supplemented with 20% heat-inactivated FBS, 1% L-glutamine, and 10% dimethyl sulfoxide. 3. Recombinant human IL-2. 4. DynaMag™-15 magnet (Thermo Fisher Scientific). 2.9 In Vitro Cytotoxicity Assay

1. Luciferase-expressing IMR5 cells. 2. RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin. 3. Sterile 96-well cell culture plates. 4. RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin. 5. Steady-Glo® luciferase assay system (Promega, Madison, WI, USA). 6. Luminescence plate reader.

2.10 In Vivo Testing of CAR T Cells

1. Luciferase-expressing IMR5 cells. 2. RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin. 3. T75 flasks. 4. Sterile PBS. 5. Sterile 1 mL syringe. 6. Sterile 27-gauge needle. 7. 5- to 6-week-old NOD-scid IL2rγnull (NSG) mice. 8. D-luciferin. 9. IVIS Imaging System (PerkinElmer, Waltham, MA, USA).

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Methods Carry out all procedures at room temperature in a Class II biosafety cabinet unless otherwise stated.

3.1 Lentivirus Production

Prior to attempting this protocol, be aware of the serious biosafety concerns involved. Lentivirus is a modified form of human immunodeficiency virus (HIV). Although unable to replicate in a host, it must be handled with caution. All work with lentiviruses must be performed in BSL2 designated hoods or viral vector rooms. All handling, storage, and disposal of biohazard waste must be in accordance with institutional rules and regulations. 1. Transform three aliquots of chemically competent E. coli Stbl3™ cells with 10 ng of pMH260, psPAX2, and pMD2.G, respectively, following the manufacturer’s instructions. Plate each transformation on LB agar plates containing 100 μg/ mL ampicillin and grow overnight at 37  C. 2. Pick single colonies and inoculate 100 mL cultures of LB containing 100 μg/mL ampicillin. After overnight growth at 37  C with 220 rpm shaking, purify plasmid DNA using the EndoFree® Plasmid Maxi Kit following the manufacturer’s instructions. 3. (Optional) Before seeding HEK-293T cells, coat a 100 mm cell culture dish with 5 mL of 0.01% poly-L-lysine. Rock gently to ensure even coating of the culture surface. After 5 min, remove solution by aspiration and thoroughly rinse surface with sterile cell culture grade water. Dry for at least 2 h before introducing cells and medium. 4. Seed ~2  106 HEK-293T cells from liquid nitrogen stock in 100 mm cell culture dishes. Rapidly thaw cells in a 37  C water bath and resuspend in 10 mL of HEK-293T complete growth medium (DMEM supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin). Centrifuge at 300  g for 5 min to pellet cells, then resuspend in 10 mL of fresh HEK-293T complete growth medium and add to the cell culture dish. Grow for 2–3 days at 37  C, 5% CO2. 5. One day prior to transfection (Day 0), trypsinize and count cells. Seed 7  106 HEK-293T cells per 100 mm dish (see Note 9). 6. On the day of transfection (Day 1), remove supernatant and replace with 5 mL of fresh HEK-293T complete growth medium 30–60 min before transfection. 7. For each dish, dilute 16 μg of total DNA (8 μg of GPC2 CAR lentiviral plasmid pMH260, 2 μg of VSV-G envelope

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expressing plasmid pMD2.G, and 6 μg of packaging plasmid psPAX2) prepared in step 2 into 500 μL of serum-free DMEM (see Note 10). Vortex gently to mix. 8. For each dish, dilute 48 μL of CalFectin™ (DNA: CalFectin™ ratio 1:3) into 500 μL of serum-free DMEM. Vortex gently to mix. 9. Apply diluted CalFectin™ reagent immediately to the diluted DNA solution all at once (see Note 11). Vortex immediately to allow CalFectin™-DNA complexes to form. 10. Incubate for 10 min at room temperature (see Note 12). 11. Apply the 1 mL of CalFectin™-DNA complexes dropwise onto the medium in each dish and distribute the mixture by gently swirling the dish (see Note 13). 12. One day after transfection (Day 2), aspirate medium from the transfected cells and disinfect the aspirated medium with 10% bleach (see Note 14). Then, add 10 mL of fresh complete HEK-293T growth medium to the transfected cells (see Note 15). 13. Harvest the lentivirus-containing supernatants 48–72 h posttransfection. Centrifuge at 500  g for 5 min and then filter through a sterile 0.45 μm filter (see Note 16). 14. If concentrated lentivirus is needed, transfer clarified supernatant to a sterile container and combine one volume of LentiX™ Concentrator with three volumes of clarified supernatant. 15. Incubate mixture at 4  C for 30 min or overnight (see Note 17). 16. Centrifuge sample at 1,500  g for 45 min at 4  C. After centrifugation, an off-white pellet will be visible. 17. Carefully remove supernatant, taking care not to disturb the pellet. 10. Gently resuspend the pellet in 1/10 to 1/100 of the original volume using complete PBMC growth medium (RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% Lglutamine, and 1% penicillin–streptomycin) (see Note 18). 18. Immediately titrate lentivirus or store at
80  C in single-use aliquots (see Note 19). 3.2 Lentivirus Titration

1. One day before adding lentivirus (Day 0), trypsinize and count HEK-293T cells. Seed 1  105 cells in 1 mL of complete HEK-293T growth medium (DMEM supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin– streptomycin) in each well of a 12-well cell culture plate. 2. Use one well to trypsinize and count cells the next day (Day 1). Remove 500 μL of medium from each well.

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3. For non-concentrated lentivirus, transduce cells with 500 μL, 100 μL, 50 μL, 20 μL, and 10 μL of crude lentivirus-containing supernatant. Complete the volume to 500 μL with complete HEK-293T growth medium. Keep one well as an uninfected control. 4. For concentrated lentivirus, transduce the cells with 10 μL, 1 μL, 10
1 μL, 10
2 μL, and 10
3 μL of lentivirus in 500 μL of complete HEK-293T growth medium. 5. 48–72 h after transduction, prepare samples for flow cytometry. First, trypsinize and collect cells in microcentrifuge tubes. 6. Centrifuge samples at 500  g for 5 min at 4  C. Remove lentiviral supernatant and disinfect the collected supernatant with 10% bleach. 7. Resuspend the pellet in 500 μL of 1% formaldehyde and incubate for 5 min at room temperature (see Note 20). 8. Centrifuge samples at 500  g for 5 min at 4  C. Wash cells with PBS once. 9. Resuspend the pellet in 1 mL of FACS buffer (5% BSA in PBS containing 0.1% sodium azide) and analyze cells for transduction (GFP expression) using a flow cytometer. Untransduced HEK-293T cells serve as a negative gating control. 10. Calculate the lentivirus titer using the following equation (see Note 21): Titer (transduction units [TU]/mL) ¼ (F  Cn/ V)  FD, where F ¼ % of GFP-positive cells/100 (see Note 22), Cn ¼ number of target cells counted on Day 1, V ¼ volume of supernatant (in mL), and FD ¼ fold dilution. 3.3 Isolation of PBMCs from Buffy Coats

1. Dilute 50 mL buffy coat (see Note 23) with 2–4 volumes of PBS (see Note 24). 2. Pipette 15 mL of Ficoll-Paque into a Leucosep tube. Close the tube and centrifuge at 1,000  g for 30 s at 20  C. The FicollPaque is now located below the porous barrier. 3. Transfer 35 mL of diluted cell suspension into the prepared Leucosep tube. 4. Centrifuge at 140  g for 10 min at room temperature in a swinging-bucket centrifuge without brake, then at 670  g for 15 min. 5. Three layers occur above the barrier: a plasma layer, the interphase consisting of PBMCs, and a small layer of Ficoll-Paque. Discard the plasma layer. 6. Transfer the interphase above the barrier, which contains the PBMCs, to a new 50 mL conical tube. 7. Fill the conical tube with PBS, mix, and centrifuge at 300  g for 5 min at room temperature. Carefully remove supernatant completely.

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8. Add 40 mL of ACK lysing buffer into the conical tube and incubate at room temperature for 3–5 min. Lysis of red cells should be evident during the incubation. 9. Collect the PBMCs by centrifugation at 300  g for 5 min at room temperature. 10. Aspirate the supernatant without disturbing the pellet. 11. Wash the cells with 10 mL of PBS. 12. Collect the PBMCs by centrifugation at 300  g for 5 min at room temperature. 13. Resuspend cell pellet in complete PBMC growth medium (RPMI-1640 medium supplemented with 10% heatinactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin) and count cells. A typical yield is approximately 150 to 250 million PMBCs from 50 mL buffy coat and only eight million are required for the procedures described below. Freeze down excess cells at eight million cells per mL in PBMC and human T-cell freezing medium (RPMI-1640 medium supplemented with 20% heat-inactivated FBS, 1% L-glutamine, and 10% dimethyl sulfoxide) in a cryogenic vial (see Note 25). 3.4 Activation of Human T Cells Within PBMCs

1. If necessary, thaw frozen PBMCs (one vial, 8  106 cells) rapidly in a 37  C water bath. Pellet (300  g, 5 min) and resuspend cells in complete PBMC growth medium (RPMI1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin). Count the cells using trypan blue solution. 2. Seed 1  106 cells in 1 mL of complete PBMC growth medium per well in a 24-well cell culture plate. One or two wells are typically sufficient for in vitro cytotoxicity assays, while five to six wells are necessary for in vivo mouse testing. Include one well to serve as the mock transduced control. 3. Add 50 international units (IU)/mL of human IL-2. 4. Wash the Dynabeads® Human T-Activator CD3/CD28 before use as follows. First, resuspend the Dynabeads® in the vial (i.e., vortex for 30 s, or tilt and rotate for 5 min). 5. Transfer the desired volume of Dynabeads® to a tube. The product contains 4  107 beads/mL. To maintain a bead-tocell ratio of 2:1, 50 μL of Dynabeads® is needed for 1  106 PBMCs. 6. Add an equal volume of PBS or at least 1 mL and mix (vortex for 5 s or keep on a roller for at least 5 min). 7. Centrifuge at 300  g for 5 min and discard the supernatant. 8. Resuspend the washed Dynabeads® in the same volume of culture medium as the initial volume of Dynabeads® taken from the vial.

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9. Add Dynabeads® Human T-Activator CD3/CD28 to PBMCs at a bead-to-cell ratio of 2:1. 10. Activate T cells within PBMCs by incubation at 37  C, 5% CO2 for 24 h before transduction. 3.5 Lentiviral Transduction of Human T Cells Within PBMCs

1. Take a sample of 2  106 PBMCs containing activated T cells to serve as the mock transduced control. Process the mock transduced cells in the same manner as the CAR lentivirus transduced cells, except adding growth medium instead of lentivirus in step 5. 2. For the remaining PBMCs, calculate the volume of high-titer lentivirus required for transduction of PBMCs after activation with anti-CD3/CD28 Dynabeads® using the following equation and a multiplicity of infection (MOI) of 5: MOI ¼ Titer ðpfu=mLÞ  Volume ðmLÞ=PBMC number: 3. Add 1 μL of 10 mg/mL protamine sulfate per mL to cells to achieve a final concentration of 10 μg/mL. 4. Centrifuge cells at 1,000  g for 60–90 min. 5. Resuspend cells in the volume of high-titer lentivirus (or growth medium for mock transfected cells) calculated in step 2. Add 50 IU/mL of human IL-2 to the culture. Incubate overnight at 37  C, 5% CO2. 6. (Optional) After overnight culture, the transduction procedure can be repeated to enhance transduction efficiency.

3.6 CAR T-Cell Expansion

1. Collect transduced cells (see Subheading 3.5) 24 h after transduction in a 15 mL conical tube. 2. Centrifuge at 300  g for 5 min and discard supernatant. 3. Resuspend cells in complete T-cell growth medium (RPMI1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin) at a density of 0.5  106 cells/mL. 4. Add 100 IU/mL of human IL-2. 5. The percentage of CD3+ T cells is typically >95% of total cells 1-week post-activation of PBMCs. CAR (LH7) T-cell growth should be assessed by trypan blue counting every other day with fresh medium/human IL-2 added as required (see Note 26). 6. Assessment of transduction efficiency based on GFP expression (see Subheading 3.7) and functional activity of the CAR (LH7) T cells (see Subheading 3.8) can be performed shortly after the transduction process. However, the requirements of the individual experiment will determine the number of CAR T cells required and this will determine the duration of cell expansion required.

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7. At the end of T-cell expansion, remove beads from cells using the DynaMag™ magnet. Freeze down excess CAR (LH7) T cells in PBMC and human T-cell freezing medium (RPMI1640 medium supplemented with 20% heat-inactivated FBS, 1% L-glutamine, and 10% dimethyl sulfoxide) and place in liquid nitrogen. 3.7 Determination of the Efficiency of CAR Expression in T Cells

1. Count mock (untransduced) and CAR (LH7) T cells and transfer 1  106 cells from each sample to microcentrifuge tubes. 2. Centrifuge at 500  g for 5 min and discard supernatant. 3. Resuspend the pellet in FACS buffer (5% BSA in PBS containing 0.1% sodium azide) and analyze cells for CAR transduction efficiency based on GFP expression using a flow cytometer. Mock T cells serve as a negative gating control (see Note 27).

3.8 In Vitro Cytotoxicity Assay

1. Thaw cryopreserved IMR5 (target) cells rapidly in a 37  C water bath. Pellet and resuspend cells in complete IMR5 cell culture medium (RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin– streptomycin). Count the cells using trypan blue solution. 2. Seed 2  103 luciferase-expressing IMR5 cells in 50 μL of complete IMR5 cell culture medium in each well of a 96-well plate. Allow cells to attach by incubating overnight at 37  C, 5% CO2. 3. Prepare dilutions of CAR (LH7) T cells (effector) in T-cell growth medium at different Effector (E)/Target (T) ratios. The highest ratio is 50:1 (see Note 28). 4. Add 50 μL of CAR (LH7) T cells to each well. Each E:T ratio is assessed in triplicate. 5. Incubate the co-cultures at 37  C for 20–24 h. 6. Before measuring luciferase activity, save the supernatants for cytokine analysis and store at
20  C. Cytokine analysis falls outside the scope of this chapter but several published methods and assays (e.g., ELISA, Luminex, MSD) are available. 7. Add 60 μL of Steady-Glo® reagent to each well to lyse IMR5 cells. Incubate the plate protected from light at room temperature for at least 5 min. 8. Read luminescence on a luminescence plate reader. 9. Results are analyzed as percent killing based on luciferase activity in wells with target cells alone: % killing ¼ 100
[(RLU from well with effector and target cells)/(RLU from well with target cells)  100].

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3.9 In Vivo Testing of CAR T Cells

Work in aseptic conditions at all times. 1. Thaw cryopreserved luciferase-expressing IMR5 cells as above (see step 1 in Subheading 3.8). Seed cells in a T75 flask and grow at 37  C, 5% CO2. 2. Trypsinize and count luciferase-expressing IMR5 cells. Then, wash cells with ice-cold PBS twice. Prepare 2  106 cells in 200 μL of PBS (1  107 cells/mL). Place cells on ice. 3. Fully resuspend cells. Intravenously inoculate 2  106 luciferase-expressing IMR5 cells in 200 μL PBS into each NSG mouse (Day 0). 4. Two weeks after tumor transplantation, intraperitoneally inject 3 mg of luciferin into each mouse and image animals 10 min later (see Note 29) using the IVIS Imaging System. Perform imaging once a week to assess tumor growth. Living Image software is used to analyze the bioluminescence signal flux for each mouse as photons per second per square centimeter per steradian (photons/s/cm2/sr). 5. Randomly group the mice that show steady increase of bioluminescence into three groups [PBS, mock CAR-T (untransduced T cells), and CAR (LH7)] during the fourth week after tumor inoculation. Image the mice in each group using the IVIS Imaging System. The bioluminescence signals will represent the tumor burdens of IMR5 metastases before treatment. 6. Count mock and CAR (LH7) T cells (see Note 30) and wash cells with ice-cold PBS twice. Prepare single cell suspension of desired cell density (see Note 31), and intravenously inject mock or CAR (LH7) T cells into mice from each group. Inject equivalent volumes of PBS into mice from the PBS group. 7. Image the mice thereafter once every week to monitor tumor growth. Cytokine secretion in the serum of mice treated with CAR T cells can be measured by ELISA or other assay. Euthanize mice when there are signs of severe sickness like graftversus-host disease, high tumor burden (see Note 32), or body weight loss. Snap freeze mouse spleen and human neuroblastoma xenograft tumors after euthanasia. Extract genomic DNA from the collected tissues and perform droplet digital PCR (ddPCR) to quantify the CAR vector-positive cells as reported previously [12]. CAR lentivector integration in the T-cell genome can be analyzed using linker-mediated PCR based on a protocol described previously for measuring viral infection in HIV patients [20]. Monitor the overall survival from the date of transplantation until death.

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Notes 1. There is a wide diversity of lentiviral vectors available from commercial and noncommercial sources that are suitable for CAR expression in primary T cells. The lentiviral titer is affected by a combination of factors including, but not limited to, size and structure of transgene RNA and efficiency of lentiviral elements to support virus assembly. Consequently, small-scale testing of the transduction efficiency of CAR-containing lentiviral supernatants on primary T cells is strongly encouraged to provide an estimate of the likely quantities of lentiviral plasmid that will be required to generate sufficient CAR T cells for in vitro and in vivo studies. 2. HEK-293T cells or derivatives (i.e., 293FT cells) can all be used to package lentivirus. The packaging cell line needs to contain the SV40 large T antigen. The main function of the large T antigen is to help plasmids with an SV40 origin of replication to replicate and be retained within the cell after transfection. This will in turn allow the cell to produce more viruses during packaging. 3. HEK-293T cells loosely attach to culture dishes and easily detach. Poly-L-lysine solutions are typically used to coat plasticware for optimal attachment and growth of cells. 4. Many transient transfection systems can be used to produce lentivirus, such as Fugene, polyethylenimine (PEI), CaCl2, and Lipofectamine 2000. Empirical testing of transfection reagents in pilot studies is recommended for generating high-titer lentivirus (see Note 1). 5. The Lenti-X™ Concentrator provides a fast and simple method for concentrating lentiviral stock. Lentivirus can also be concentrated via ultracentrifugation if appropriate equipment is available. 6. 1% formaldehyde is sufficient to fix cells and inactivate lentiviral vectors. Increasing formaldehyde concentration (up to 4%) will increase the autofluorescence of cells. Cells fixed with formaldehyde can be stored in the dark at 4  C for several hours. 7. There are various commercial human CD3/CD28 T-cell activators. Pilot studies are necessary to test other sources of T-cell activators (i.e., bead-to-cell ratios). 8. In addition to protamine sulfate, polybrene at 8 μg/mL can be used to improve lentiviral transduction efficiency. 9. Cells should be 90% confluent. Wait until cells reach this density before transfection.

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10. Opti-MEM is not suitable to dilute DNA and CalFectin™ reagent as it disrupts transfection complexes. 11. Do not mix the solutions in the opposite order. 12. Never keep the CalFectin™-DNA complexes for longer than 20 min prior to transfection. 13. HEK-293T cells detach very easily from the dish. Gently add the CalFectin™-DNA complex and mix. 14. Sterilize all materials in a 10% bleach solution starting from this point. Never move open viral preparations out of the hood. Anything that touches virus must be decontaminated with 10% bleach for at least 30 min. 15. HEK-293T cells can detach easily from the dish. Add the fresh medium slowly and gently to avoid cell detachment. 16. If filtering, use only cellulose acetate or polyethersulfone (low protein binding) filters. Do not use nitrocellulose filters. Nitrocellulose binds surface proteins on the lentiviral envelope. 17. Incubation times can be as short as 15 min and up to 1 week at 4  C with minimal loss in viral titer. Thorough cooling of the sample is essential, so larger volumes (>100 mL) may require longer incubation times. 18. Try to avoid creating bubbles when resuspending lentivirus. It is also critical that the pellet is never left without medium and allowed to dry out. 19. Lentiviruses stored at
80  C are suitable for use for up to 1 year. After long-term storage, we recommend retitering the viral stock before transducing your mammalian cell line of interest. 20. This step will fix the cells and inactivate the viral particles. Samples can thus be removed from the BSL2 laboratory at this point. 21. Titers of concentrated lentivirus are usually more than 2  107 TU/mL. 22. Choose dilutions yielding 1–10% GFP-positive cells for titer calculations. 23. The buffy coat should be processed within 8 h of whole blood sampling and should be supplemented with anticoagulants (e.g., heparin, EDTA, or citrate). 24. The purity of mononuclear cells is improved when blood samples are more diluted. 25. PBMCs can be used either freshly or after thawing cryopreserved aliquots of cells. Thaw cryopreserved cells quickly in a 37  C water bath with gentle agitation, then add to 10 mL of complete PBMC growth medium (RPMI-1640 medium

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supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin–streptomycin) and centrifuge for 5 min at 300  g to remove dimethylsulfoxide. 26. Human T cells continue to expand at a rapid rate for the first 10–12 days after activation and then begin to plateau. During this time, the culture should be readjusted to a cell density of 0.5  106 cells/mL by the addition of fresh complete T-cell growth medium (RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin– streptomycin). Fresh human IL-2 should be added to achieve the desired final concentration (100 IU/mL). 27. Typical transduction efficiencies are 30–50% based on GFP expression. 28. The 50:1 is the ratio before normalization. If the transduction efficiency is 50%, the normalized E:T ratio would be 25:1. 29. The kinetic curve for each cell type could be different. It is recommended to take images every 5–10 min up to approximately 40 min after injection of luciferin. This will indicate the kinetic curve for luciferin uptake in your model. Once you have established your curve, you can follow the imaging procedure and use the optimal peak time point to image thereafter (10–20 min after luciferin injection is ideal for most models). 30. Either fresh or frozen T cells can be used for animal studies. Fresh cells tend to have better viability than thawed frozen cells. It is recommended to perform in vitro cytotoxicity assay before injection into animals. 31. In our studies, 5–20 million mock or CAR (LH7) T cells per mouse are generally used. 32. For the metastatic IMR5 xenograft model, we consider bioluminescence signals reaching 1  1010 photons/s/cm2/sr as high tumor burden and euthanize the mice. The radiance unit of photons/s/cm2/sr is the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian (sr). However, the end point should be determined based on the luciferase expression level in tumor cells and the imaging system.

Acknowledgments This work was supported by the Intramural Research Program of NIH, Center for Cancer Research (CCR), National Cancer Institute (NCI) (Z01 BC010891 and ZIA BC010891 to M.H.). The authors thank Alan Hoofring (NIH) for graphic illustration of the GPC2-targeted CAR T-cell production and evaluation. The content of this publication does not necessarily reflect the views or

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policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. M.H. and N.L. are inventors on patent applications describing antibodies to glypican 2 that are assigned to the National Institutes of Health. The antibodies targeting GPC2 are available for licensing, in a wide range of fields of use, from the National Cancer Institute, NIH. If you are interested in obtaining a license, please contact the principal investigator Dr. Mitchell Ho at [email protected]. References 1. Gross G, Waks T, Eshhar Z (1989) Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A 86:10024–10028 2. Brentjens RJ, Davila ML, Riviere I et al (2013) CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapyrefractory acute lymphoblastic leukemia. Sci Transl Med 5:177ra38 3. Grupp SA, Kalos M, Barrett D et al (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368: 1509–1518 4. Maude SL, Frey N, Shaw PA et al (2014) Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371: 1507–1517 5. Kochenderfer JN, Yu Z, Frasheri D et al (2010) Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood 116: 3875–3886 6. Hacker U, Nybakken K, Perrimon N (2005) Heparan sulphate proteoglycans: the sweet side of development. Nat Rev Mol Cell Biol 6: 530–541 7. Ho M, Kim H (2011) Glypican-3: a new target for cancer immunotherapy. Eur J Cancer 47: 333–338 8. Li N, Gao W, Zhang YF et al (2018) Glypicans as cancer therapeutic targets. Trends Cancer 4: 741–754 9. Baumhoer D, Tornillo L, Stadlmann S et al (2008) Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4387 tissue samples. Am J Clin Pathol 129:899–906 10. Yamauchi N, Watanabe A, Hishinuma M et al (2005) The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol 18:1591–1598

11. Gao H, Li K, Tu H et al (2014) Development of T cells redirected to glypican-3 for the treatment of hepatocellular carcinoma. Clin Cancer Res 20:6418–6428 12. Li D, Li N, Zhang YF et al (2020) Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology 158:2250–2265. e20 13. Li N, Fu H, Hewitt SM et al (2017) Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma. Proc Natl Acad Sci U S A 114:E6623–E6631 14. Bosse KR, Raman P, Zhu Z et al (2017) Identification of GPC2 as an oncoprotein and candidate immunotherapeutic target in high-risk neuroblastoma. Cancer Cell 32:295–309.e12 15. Li N, Spetz MR, Ho M (2020) The role of glypicans in cancer progression and therapy. J Histochem Cytochem 68:841–862 16. Xu J, Chen LJ, Yang SS et al (2019) Exploratory trial of a biepitopic CAR T-targeting B cell maturation antigen in relapsed/refractory multiple myeloma. Proc Natl Acad Sci U S A 116:9543–9551 17. Harris E, Elmer JJ (2020) Optimization of electroporation and other non-viral gene delivery strategies for T cells. Biotechnol Prog: e3066 18. Zufferey R, Dull T, Mandel RJ et al (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72: 9873–9880 19. van der Loo JC, Wright JF (2016) Progress and challenges in viral vector manufacturing. Hum Mol Genet 25:R42–R52 20. Maldarelli F, Wu X, Su L et al (2014) Specific HIV integration sites are linked to clonal expansion and persistence of infected cells. Science 345:179–183

Chapter 24 Single-Domain Antibodies for Intracellular Toxin Neutralization Timothy F. Czajka and Nicholas J. Mantis Abstract Ricin is a plant-derived toxin with a history as a biothreat agent. The toxin’s enzymatic subunit, ricin toxin A chain (RTA), is a ribosome-inactivating protein that, when delivered into the cytoplasm of mammalian cells, arrests protein synthesis with extraordinary efficiency. Once within the cytoplasm, RTA is shielded from circulating toxin-neutralizing antibodies. Here, we describe methods we developed to neutralize RTA within the cytoplasm of Vero cells using DNA-based delivery of alpaca-derived single-domain antibodies (VHHs) targeting RTA’s active site. We describe the design of the VHH expression vectors, assessment of transient expression of VHHs in Vero cells by enzyme-linked immunosorbent assay and western blotting, and cytotoxicity studies. While the protocols here are specific to ricin, they are easily modified for other toxins or even intracellular pathogens such as viruses. Key words Toxin, Antibody, Nanobody, Biodefense, Therapeutics, Single-domain antibody

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Introduction Camelid-derived single domain antibodies (sdAbs or VHHs) are increasingly being exploited for their unique biological properties in the development of antidotes for fast-acting and highly lethal biothreat toxins such as botulinum neurotoxin (BoNT), anthrax toxin, and ricin toxin [1–3]. For prophylactic development, the most desirable antibodies are those that block toxin entry into target cells, and therefore prevent toxin-induced cell death entirely. Indeed, VHHs that target receptor-binding domains of anthrax toxin and BoNT are highly effective at toxin neutralization [4– 7]. However, therapeutic applications of these VHHs are complicated by the fact that these three toxins are rapidly internalized into host cells and thus shielded from circulating antibodies [8, 9]. Moreover, once the catalytic subunits of BoNT, anthrax, and ricin are delivered into the cytoplasm they can wreak havoc on cellular processes. As a case in point, ricin’s catalytic subunit, ricin toxin A

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_24, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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chain (RTA), is a ribosome-inactivating protein that depurinates a universally conserved adenosine within the sarcin/ricin loop (SRL) of 28S rRNA [10]. With a Kcat of 1500 ribosomes per minute, RTA can readily interfere with protein translation and trigger the onset of the ribotoxic-stress response and programmed cell death [11, 12]. Efforts to identify small molecule inhibitors of RTA have proven difficult, in part because RTA’s active site is an open pocket and not easily “druggable” [13]. The handful of small molecule inhibitors that have been identified against RTA have had limited success in vivo due to issues of solubility, toxicity, and/or biodistribution [14]. We are interested in employing VHHs as intracellular antibodies, or “intrabodies,” to interfere with RTA within the cytoplasm of a host cell. The current model for RTA-mediated ribosome inactivation involves two steps. In step one, RTA interacts with the C-terminus of the acidic P stalk proteins of the 60S ribosome [15–18]. These largely unstructured ribosomal proteins have weak affinity for the so-called ribosomal recruitment site located on the underside of RTA that is normally occluded by ricin’s binding subunit [17, 18]. In step two, RTA is guided to the SRL, where depurination of the single conserved adenine residue within the SRL is catalyzed by the coordinated effort of five active site residues (Fig. 1; pink) [19]. The depurination event renders the ribosome irreversibly inactive [10]. Our lab has described a collection of alpaca-derived VHHs that target RTA’s active site [20, 21]. Co-crystal structures of seven VHHs in complex with RTA demonstrated binding modes in which the active site cleft was either physically occluded or capped [21]. The binding affinities of the VHHs for RTA ranged from 0.3 nM to 10 nM, as determined by surface plasmon resonance. The VHHs were tested for their ability to inhibit RTA’s capacity to inactivate ribosomes in a cell-free, in vitro translation (IVT) assay. Three VHHs, including V2A11, had high RTA inhibitory activity (>70%) in the IVT assay. The same VHHs also interfered with RTA’s RNA N-glycosidase activity in an rRNA depurination reaction with yeast ribosomes as substrate. The structure of V2A11 bound to RTA revealed that the VHH complementaritydetermining region 3 (CDR3) penetrated RTA’s active site and engaged with two catalytic residues involved in stacking the adenine substrate within the SRL. Thus, by all measures, V2A11 bind RTAs with high affinity and physically block RTA’s enzymatic activity. With this structural information in hand, we sought to determine whether VHHs like V2A11 were capable of neutralizing RTA within the context of the cytoplasm as “intrabodies.” To do this we transiently transfected Vero cells with expression vectors encoding RTA-specific VHHs and then challenged the cells with increasing doses of ricin toxin. Using this approach, we have demonstrated potent intracellular neutralization of RTA [21]. In the future, the

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Fig. 1 Ricin toxin A chain (RTA) and VHH-binding sites. (a) Structure of RTA (gray) with active site cleft highlighted in pink. PDB ID: 1RTC. (b) VHH epitopes (black) on RTA, as determined by X-ray crystallography. Image created using PyMOL and BioRender

same platform will be used to probe other potentially critical cytosolic events in RTA’s journey to the SRL, including P-stalk binding. Ultimately, intrabodies are powerful tools not only for potential therapeutic purposes but also for tracking the toxin after it has been secreted from the endoplasmic reticulum. In this chapter, we describe methods for the design of VHH intrabody expression plasmids, Vero cell transfection, intrabody expression profiling, and assessment of intrabody toxin neutralizing activity. We use a combination of enzyme-linked immunosorbent assay (ELISA) and western blotting approaches to measure functional intracellular expression levels. Altogether, these assays should provide a translatable approach to develop and test antibodies against a wide variety of intracellular targets.

2

Materials

2.1 Common Equipment and Reagents

1. Biosafety Cabinet (BSC). 2. Vero cells (American Type Culture Collection, Manassas, VA, USA). 3. Complete Dulbecco’s Minimal Essential Medium (DMEM): DMEM supplemented with fetal bovine serum (10% v/v) and penicillin/streptomycin. 4. T75 flasks. 5. Hemocytometer and trypan blue.

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6. Sterile phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4. Prepare as 10
concentrate. 7. Trypsin/ethylenediaminetetraacetic acid (EDTA): 0.05% (w/v) trypsin, 0.53 mM EDTA in Hank’s balanced salt solution with sodium bicarbonate. 8. Multichannel pipettors (10–100 μL range and 30–300 μL range). 9. Gibco Opti-MEM™-Reduced Serum Medium (Thermo Fisher, Waltham, MA, USA). 10. Lipofectamine® 3000 transfection reagent with P3000™ enhancer reagent (Thermo Fisher). 11. gBlock™ containing VHH-coding sequences with C-terminal E-tag and flanked by 50 and 30 UTRs (see Subheading 3.1). 12. pcDNA3.1 vector (Thermo Fisher). 13. Primer 50 UTR-F (50 -CAATATGCAGCGGATCCGGGTCC-30 ) (see Note 1). 14. Primer 30 UTR-R (50 -CTGATGGCCCAGGGCCCGATCTAG -30 ) (see Note 1). 15. Primer VHH-F (50 -CTTATTCGCGGCCGCACCATG-30 ) (see Note 1). 16. Primer VHH-R (50 - GTGATGCTCTAGATTTTAACGCGG30 ) (see Note 1). 17. One ShotTM TOP10 chemically competent Escherichia coli cells (Thermo Fisher). 18. LB broth and agar: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl. For plates, add 15 g/L agar. Autoclave and let cool prior to adding antibiotics. 19. 100 μg/mL carbenicillin. 20. 10 mL bacterial culture tubes. 21. Glycerol. 22. Cryovials. 23. Plasmid miniprep kit. 24. NanoDrop spectrophotometer. 25. Agarose gel electrophoresis equipment and supplies, including molecular weight marker. 26. QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). 27. DNA Clean & Concentrator™-5 Kit (Zymo Research, Irvine, CA, USA). 28. BamHI and ApaI restriction enzymes (REs). 29. T4 DNA ligase.

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30. Super Optimal broth with Catabolite repression (SOC) medium: 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCl, 10 mL of 250 mM KCl, final volume 1 L of ddH2O. Autoclave, then supplement with 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose. 2.2 Cell Lysis for Intrabody Detection Assays

1. 6-well tissue culture plates. 2. Vacuum aspirator. 3. Disposable glass pipettes. 4. Bead mill homogenizer. 5. Refrigerated microcentrifuge capable of holding 2 mL tubes and achieving 15,700
g. 6. 1 mm glass beads. 7. 2 mL screw cap tubes. 8. Disposable cell scrapers (1.8 cm
25 cm). 9. PBS: see Subheading 2.1. 10. 2
RIPA buffer: 100 mM Tris–HCl, pH 7.5, containing 300 mM NaCl, 2% (w/v) NP-40, 0.2% (w/v) SDS, and 2% (w/v) sodium deoxycholate. Store at 4  C in the dark.

2.3 Detection of Functional Intracellular VHHs by ELISA

1. Spectrophotometer (450 nm). 2. Immulon® 4 HBX flat bottom 96 well ELISA plates. 3. 96-well “U” bottom dilution plates. 4. PBS containing 0.1% (v/v) Tween-20 (PBS-T): for a 2 L solution, combine 1.8 L H2O, 0.2 L 10
PBS (see Subheading 2.1), and 2 mL Tween-20. 5. PBS-T containing 2% (v/v) goat serum: combine 490 mL of PBS-T with 10 mL of goat serum. 6. KPL SureBlue™ 3,30 ,5,50 -tetramethylbenzidine (TMB) Microwell Peroxidase Substrate (SeraCare, Milford, MA, USA). 7. 1 M H3PO4. 8. Anti-RTA monoclonal antibody (mAb) PH12 [22] (see Note 2). 9. Goat anti-E tag polyclonal IgG conjugated to horseradish peroxidase (HRP). 10. RTA.

2.4 Western Blot Detection of Intracellular VHH Expression

1. Imager capable of fluorescent and ECL channels (e.g., iBright Imager; see Note 3). 2. Mini-PROTEIN® TGX™ precast gels (4–15%, 10-well comb, 30 μL/well) (Bio-Rad, Hercules, CA, USA). 3. Protein electrophoresis tank.

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4. Transfer insert. 5. Blot stack frames. 6. Plastic sponge pads (two per frame). 7. Filter/blotting paper (two per frame). 8. Nitrocellulose membrane. 9. 10
SDS-PAGE running buffer: 30.2 g/L Tris base, 188 g/L glycine, 15% SDS. 10. Protein molecular size ladder. 11. 4
sample loading buffer: 200 mM Tris–HCl, pH 6.8, containing 8% SDS, 0.4% (w/v) bromophenol blue, and 40% (v/v) glycerol. Supplement with 10% (w/v) dithiothreitol immediately prior to use. 12. Towbin transfer buffer: 25 mM Tris, 192 mM glycine, pH 8.3. Prepare as 10
concentrate and store at 4  C. Immediately prior to use, dilute with ddH2O and add 20% (v/v) methanol. 13. Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T): 50 mM Tris–HCl, pH 7.5, containing 150 mM NaCl and 0.1% Tween-20. 14. TBS-T containing 5% (w/v) bovine serum albumin (BSA). 15. Goat anti-E tag polyclonal IgG conjugated to HRP. 16. Pierce ECL Plus substrate. 17. Transparency film. 2.5 Ricin Cytotoxicity Assay

1. Luminometer. 2. White 96-well treated cell culture plates. 3. Serocluster® 96 well “U” bottom dilution plates. 4. Vacuum aspirator. 5. Ricin toxin (RCA-I; dialyzed to remove sodium azide if necessary). 6. CellTiter-Glo® (Promega, Madison, WI, USA).

3

Methods Carry out all cell culture procedures in a BSC unless otherwise specified.

3.1 Intrabody Expression Vectors

DNA sequences of intrabody construct components can be found in Table 1. A linear depiction of an intrabody construct is shown in Fig. 2.

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Table 1 Hinge, E-tag, and untranslated region DNA sequences Component Sequence (50 –30 ) Hinge

GAA CCC AAG ACA CCA AAA CCA CAA

E-tag

GGT GCG CCG GTG CCG TAT CCG GAC CCG CTG GAA CCG CGT

50 UTR

GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCG TTGCAGGCCTTATTC

30 UTR

GCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAA TGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTG TAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTC TTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTAGATCG

Fig. 2 Intrabody pcDNA expression vector. (a) VHH DNA (orange) is fused in frame with a short C-terminal hinge region (blue) and an E-tag (pink). The E-tagged VHH ORF is controlled by either CMV or T7 promoters (light grey). Restriction enzyme (RE) sites for generation of new VHH intrabodies and/or alternative fusion domains are denoted above. Primers used for DNA sequence confirmation are shown below. (b) DNA sequence of a representative VHH gBlock™ (V1D3). Colors are consistent with the linear depiction in (a) and all RE sites shown in (a) are boxed. Regions 50 of the 50 UTR (grey) and 30 of the 30 UTR (grey) are irrelevant DNA randomly generated to contain 50% GC content which will be removed during digestion. Image created in BioRender

1. Design intrabody construct to be inserted downstream of CMV and T7 promoters and upstream of a poly(A) region in the pcDNA3.1 plasmid. We routinely have these constructs synthesized commercially as dsDNA fragments (e.g., gBlocks™ from IDT, Coralville, IA, USA). Each intrabody vector contains the following components (Fig. 2a). (a) The 50 UTR from hydroxysteroid 17-beta dehydrogenase 4 to increase mRNA stability. (b) A BamHI RE sequence at the 50 end. (c) The 30 UTR from albumin.

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(d) An ApaI RE sequence at the 30 end. (e) A VHH sequence, optimized for human codons using the codon optimization tool by IDT or a similar provider. (f) A sequence encoding an E-tag peptide linked to the 30 end of VHH via a hinge region. (g) NotI and AscI RE sequences at the 50 and 30 ends of the VHH-hinge sequence, respectively, to allow for exchange of VHHs. (h) An XbaI RE sequence located at the 30 end of the E-tag to allow for insertion of alternative tags and/or fusion partners (e.g., designing fluorescent fusion intrabodies or multimeric constructs). 2. Generate intrabody-containing pcDNA3.1 plasmids using RE digestion and ligation. Digest pcDNA3.1 plasmid and the dsDNA fragment (gBlock™; Fig. 2b) consisting of the DNA sequences described in step 1 with BamHI and ApaI. Ligate digested product into pcDNA3.1 using T4 DNA ligase (see Note 4). (a) Transform chemically competent E. coli TOP10 cells with 10 ng of pcDNA3.1 and plate on LB agar containing 100 μg/mL carbenicillin. Grow overnight at 37  C. (b) Pick single colonies containing the pcDNA3.1 vector and inoculate 5 mL of LB-carbenicillin (100 μg/mL) overnight at 37  C with 250 rpm shaking. Perform a miniprep on 3–4 mL of the culture using a miniprep kit following the manufacturer’s instructions. (c) Prepare two 30 μL RE digestions at 37  C for 2 h (see Note 5). For the pcDNA3.1 vector: 1 μg of DNA, 3 μL 10
CutSmart® Buffer, 1 μL of BamHI (20 U), 1 μL of ApaI (50 U), and H2O to 20 μL. For the synthetic dsDNA insert: 20 μL of DNA (10 ng/μL), 3 μL of 10
CutSmart® Buffer, 1 μL of BamHI (20 U), 1 μL of ApaI (50 U), and 5 μL of H2O. (d) Separate digested DNA by loading into a 1% (w/v) agarose gel alongside a 1 kb DNA ladder and run at 120 V for approximately 1 h. Excise the band at the appropriate size (about 6 kb for digested vector and 750 bp for the insert). Purify DNA and elute in 30 μL using a QIAquick Gel Extraction kit. (e) Further concentrate the digested insert to 6 μL using the DNA Clean & Concentrator™-5 kit. (f) Prepare a 20 μL ligation reaction at room temperature for 4 h or at 16  C overnight as follows (see Note 5): 8 μL of H2O, 3 μL of digested vector, 6 μL of digested insert,

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2 μL of 10
T4 DNA ligase buffer, and 1 μL (5 U) of T4 DNA ligase. 3. Transform chemically competent E. coli TOP10 cells with the ligation product and plate on LB agar with carbenicillin (100 μg/mL). (a) Add 2 μL of the ligation reaction to 10 μL of thawed E. coli TOP10 cells. Incubate on ice for 30 min. (b) Heat shock E. coli at 42  C for 30 s and place on ice for 2 min. (c) Add 200 μL of SOC medium and incubate in a shaking incubator (250 rpm) at 37  C for 30 min. (d) Spread 10 μL or 100 μL of bacteria on LB agar plates containing 100 μg/mL carbenicillin (two plates per ligation). (e) Incubate plate overnight at 37  C. 4. Pick three or more colonies from plates and inoculate each in 5 mL of LB-carbenicillin (100 μg/mL) broth. Grow overnight at 37  C with 250 rpm shaking (see Note 6). 5. Prepare glycerol stocks of overnight cultures by adding 500 μL of bacterial cells to 170 μL of glycerol (25% final concentration) in a cryogenic vial and store at 80  C. Isolate plasmid DNA using a miniprep kit from the remaining culture. 6. Measure the concentration of isolated plasmids from transformed E. coli using a spectrophotometer and store at 20  C. 7. Confirm successful insertion in pcDNA3.1 plasmids via bidirectional Sanger sequencing using forward and reverse primers (50 UTR-F and 30 UTR-R for initial the VHH insertion described in steps 2–6; VHH-F and VHH-R for VHH exchange, see Note 4). 8. After sequence confirmation, plasmids are ready to use for subsequent cell-based assays. 3.2 Transfection of Vero Cells and Confirmation of Intrabody Expression 3.2.1 Growth and Passaging of Vero Cells

1. Grow Vero cells in 10 mL of complete DMEM in a T75 flask in a 37  C incubator (5% CO2). 2. Passage cells by dilution (1:10) twice weekly as follows. Remove medium from cells with a vacuum aspirator. Rinse cells by adding 10 mL sterile PBS and aspirating. Add 2 mL of trypsin-EDTA and incubate at 37  C for 10–15 min to detach cells from the flask. Gently shake or tap flask to help detachment. Add 8 mL of DMEM to cells. Remove 9 mL of cells for seeding larger culture volumes (if desired) or discard. Add 9 mL fresh DMEM to remaining 1 mL of Vero cells in a new T75 flask and return to incubator.

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3. Experiment Day 1. For seeding Vero cells, determine the cell density of 9 mL of cells from step 2 using a hemocytometer and adjust to a concentration of 1
105 cells/mL by diluting in DMEM. 4. Seed 2.5 mL of Vero cells per well in 6-well tissue culture plates. 5. Incubate overnight at 37  C (5% CO2). 3.2.2 Transfection of Vero Cells

Volumes described below are for a single well of a 6-well plate with 50 μL overage. Reagent preparation is performed in a BSC. 1. Experiment Day 2. Combine 4.5 μL Lipofectamine® 3000 reagent with 150 μL Opti-MEM™ medium. Mix by briefly vortexing. 2. Combine 1.46 μg of pcDNA3.1 encoding VHH intrabody, 4.37 μL of P3000™ enhancer reagent, and Opti-MEM™ (to 150 μL final volume) and mix by vortexing. 3. Add 150 μL of Lipofectamine® mixture (step 1) to DNA/ P3000™ mixture (step 2). Mix by repeated pipetting with a P200 pipette. 4. Incubate at room temperature for 10–15 min. 5. Add 250 μL of transfection mixture per well to Vero cells in 6-well plates (see step 4 in Subheading 3.2.1). 6. Mix by gently rocking plate for several seconds to ensure mixture is dispersed throughout medium. 7. Return to 37  C incubator.

3.2.3 ELISA

A schematic of the intrabody ELISA experiment is shown in Fig. 3a. 1. Experiment Day 3. Dilute capture antibody (anti-RTA mAb PH12) to 1 μg/mL in PBS. 2. Apply 100 μL/well of capture antibody to wells of a 96-well microtiter plate. 3. Cover to prevent evaporation and incubate overnight at 4  C. 4. Experiment Day 4. Invert ELISA microtiter plate to remove unbound antibody and wash plate four times by submerging in PBS-T. Invert and pat plate dry on paper towels. 5. Block by adding 200 μL/well of PBS-T containing 2% goat serum and incubate at room temperature for 2 h. 6. Remove blocking buffer by inversion and pat dry. 7. Dilute RTA to 1 μg/mL in PBS-T containing 2% goat serum. Add 100 μL/well to all wells. Incubate at room temperature for 1 h.

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Fig. 3 Intrabody ELISA workflow. (a) On day 1, seed Vero cells in 6-well tissue culture plates. On day 2, transfect with VHH expression vectors. On day 3, coat an ELISA plate with capture antibody. On day 4, serially dilute lysates in blocking buffer. Detect captured and functional intracellular VHH expression via their ability to bind RTA captured on ELISA plates. VHHs are detected via C-terminal E-tags. (b) Up to six intrabody-transfected wells may be tested in duplicate on a single ELISA plate when lysates are diluted down columns. Columns 11 and 12 are reserved for mock-transfected cells (Lipofectamine® alone, LNP). The final row (row H) is treated with blocking buffer only to establish a baseline absorbance value. Image created in BioRender

8. During the waiting periods for steps 5–7, lyse transfected Vero cells (from Subheading 3.2.2) and dilute as follows (see Note 7): (a) Aspirate medium from Vero cells and replace with 1 mL/ well of ice-cold PBS. Perform this step twice. (b) Aspirate PBS and add 150 μL/well of RIPA buffer (diluted 1:1 from 2
stock). Incubate plates on ice for 5 min with gentle rocking to ensure buffer coverage. (c) Tilt plate and scrape cells until pooled in buffer to one side. Pipette 150 μL of suspended cells from each well into 2 mL screw cap homogenization tube pre-loaded with 2 mm glass beads and kept on ice (see Note 8). (d) Incubate for 30 min on ice with intermittent homogenization at 5 m/s for 5 s (see Notes 8 and 9). (e) Centrifuge lysate for 10 min at 15,700
g at 4  C. (f) Following centrifugation, dilute lysate in 150 μL of PBS-T containing 2% goat serum (1:1) and transfer 150 μL to the top row of a “U” bottom dilution plate in duplicate. This is not the ELISA plate.

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(g) Add 100 μL of PBS-T containing 2% goat serum to the remaining wells of the dilution plate and serially dilute cell lysate three-fold down the plate columns, leaving the final row with this blocking buffer only (Fig. 3b for plate map; see Notes 10–12). 9. After the RTA capture step is complete (step 7), wash the plate four times as described in step 4 and transfer the contents of the dilution plate (step 8) to the ELISA plate (80 μL/well). Incubate at room temperature for 1 h. 10. Wash plate four times as described in step 4. Pat dry and then add 100 μL/well HRP-conjugated anti-E-tag antibody diluted 1:10,000 in PBS-T containing 2% goat serum. Incubate at room temperature for 30 min. 11. Wash plate four times as described in step 4. Add 100 μL/well TMB (equilibrated to room temperature) and develop for 8–10 min until blue chromagen hue is evident (see Note 13). 12. Add 100 μL/well of 1 M H3SO4. 13. Measure absorbance (450 nm) using a spectrophotometer. 3.2.4 Western Blotting

1. Experiment Day 4. Lyse Vero cells as described in Subheading 3.2.3 (step 8) with 100 μL of RIPA buffer. 2. Add 12 μL of lysed sample to 8 μL of loading buffer and boil at 95  C for 10 min. For blank samples, use 12 μL of RIPA buffer and 8 μL of loading buffer. 3. Assemble gel box and TGX precast gel (4–15% gradient) and place gel inside electrophoresis insert. Place insert in gel box. 4. Fill inner reservoir between gel(s) and/or buffer dam with Tris-glycine SDS-PAGE running buffer until it runs over the top of the wells. 5. Fill gel box with running buffer up to the “two gels” mark. 6. Load 5–10 μL of blank sample into all wells and pre-run gel at 70 V for 20 min. This step pre-loads wells with SDS, not already incorporated in the gel and ensures a more even subsequent run for samples. 7. Load 5 μL of ladder and 15 μL of samples (12 μL lysate +8 μL 4
loading buffer) as needed. Add 15 μL of blank sample to all remaining unloaded wells. 8. Run gel at 130 V until dye front reaches white plastic at the bottom of the insert, roughly 60 min. 9. Cut nitrocellulose membrane to be the same size as the gel, with a notch to mark orientation (see Note 14). 10. Retrieve gel(s) from gel box after electrophoresis. Immediately immerse gels in transfer buffer and incubate for 10 min with gentle rocking.

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11. Prepare blot stacks as follows: (a) Place the stack frame in a clean Pyrex dish with clear side down. Place on plastic sponge on clear side and add transfer buffer until sponge barely floats. (b) Wet one piece of filter paper in buffer and place on sponge. (c) Add nitrocellulose membrane on top of filter paper in preferred orientation (protein transfer will occur directly down onto the upper surface of membrane). (d) Position gel over membrane in preferred orientation (see Notes 14 and 15). (e) Wet and add second filter paper on gel. Roll pipette gently over stack with slight pressure to push out air bubbles. (f) Wet and add second sponge pad, close stack, and carefully secure latch. 12. Place transfer modules without block stack inserted and ice pack in gel box and add cold, clean transfer buffer until box is three quarters full (see Note 15). 13. Transfer blot stack(s) into transfer module, hinge side up. Make sure clear side faces red and black side faces black. 14. Fill gel box with buffer to “4 Gels/Blotting” line. 15. Run transfer at 350 mA for 60 min. 16. After transfer, place membrane(s) in square petri dish in TBS-T containing 5% BSA at 4  C overnight (see Note 16). 17. Experiment Day 5. Remove blocking buffer from membrane and add 4–5 mL of secondary antibody (goat anti-E tag polyclonal IgG conjugated to HRP) diluted 1:10,000 in TBS-T containing 5% BSA. Incubate at room temperature for 60 min with rocking. 18. Remove antibody and wash three times for 10 min in TBS-T, rocking at room temperature. 19. Prepare 1 mL of Pierce ECL Plus substrate per blot (mix reagents 40:1, Solution A:B). Note that larger volumes may be required for bigger membranes. 20. Place membrane on a sheet of transparency film, apply ECL substrate, and place a second sheet of transparency film on the membrane so it is fully saturated and sandwiched between films. 21. Image immediately using an iBright Imager. 22. Use “Universal Mode” to allow visualization of Alexa Fluor 647 and ECL channels for fluorescent ladders and samples, respectively.

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Fig. 4 Determining the neutralizing potential of RTA intrabodies. (a and b) Vero cells are seeded in a 96-well tissue culture plate overnight and subsequently transfected with lipid nanoparticles (Lipofectamine®) containing plasmids encoding the intrabody of interest. Empty lipid nanoparticles (LNP) are used as vehicle controls. One day following transfection, cells are exposed to serial dilutions of ricin toxin for 2 h. Cells in column 11 are treated with medium alone and serve as “live” cell controls. Approximately 48 h post ricin exposure, cell viability is assessed using CellTiter-Glo® to detect intracellular ATP. The percent viable cells are calculated as follows: [(experimental well/“live” control average)
100]. Image created in BioRender

23. Prolonged ECL incubation before imaging will result in “burned membrane.” 3.3 Ricin Challenge and Cell Viability Assessment 3.3.1 Growth of Vero Cells

Please see Fig. 4a for a schematic of the cytotoxicity experiment.

1. Experiment Day 1. Culture, count, and dilute Vero cells to 1
105 cells/mL in DMEM as described above (see Subheading 3.2.1). Pipette 100 μL/well in white 96-well cell culture plates. 2. Incubate overnight at 37  C with 5% CO2.

3.3.2 Transfection of Vero Cells

Volumes described allow for 30 transfected wells at 10 μL/well with 100 μL overage. Reagent ratios and DNA concentrations are identical to those used in Subheading 3.2.2. 1. Experiment Day 2. Combine 6 μL of Lipofectamine® 3000 Reagent with 200 μL Opti-MEM™ and mix by vortexing. 2. Combine 1.94 μg DNA, 5.83 μL P3000™ enhancer reagent, and Opti-MEM™ (to 200 μL final volume) and mix by vortexing.

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3. Add 200 μL Lipofectamine® 3000 mixture (step 1) to DNA mixture (step 2) and mix by pipetting up and down with a P200 pipette. 4. Incubate at room temperature for 10–15 min. 5. Add 10 μL of transfection mixture per well (see Notes 17 and 18 and Fig. 4b). 6. Return plates to 37  C incubator overnight. 3.3.3 Ricin Treatment

Volumes described are for treating one plate of cells with ricin. 1. Experiment Day 3. Prepare ricin (50 μg/mL) in DMEM from stock (5 mg/mL; previously dialyzed to exchange buffer with sterile PBS) by diluting 9 μL into 891 μL of DMEM. Add 140 μL/well to six wells of a sterile 96-well dilution plate (column 2, rows B–G; Fig. 4b). 2. Add 120 μL/well DMEM to remaining wells of dilution plate. Serially dilute ricin from step 1 fivefold along rows, stopping at column 10 so that column 11 will remain DMEM only. 3. Remove Vero cells from incubator (from Subheading 3.3.2) and aspirate media. Transfer 100 μL/well from dilution plate to cell culture plate. 4. Return cells to the 37  C incubator for 2 h. 5. After 2 h incubation, aspirate ricin media from cells and add 100 μL/well of DMEM (see Note 19). 6. Return cells to the 37  C incubator overnight.

3.3.4 Assessment of Vero Cell Viability

1. Experiment Day 5. Approximately 48 h after treating cells with ricin, apply 100 μL/well of thawed CellTiter-Glo™ reagent (diluted 1:5 in PBS) directly to cells in medium (see Note 20). 2. Incubate at room temperature for 15 min. 3. Measure luminescence using a luminometer (see Note 21). 4. Determine cell viability as a percentage of “live” control cells (transfected but not treated with ricin; Fig. 4b column 10). The formula used for viability calculations is: [(experimental well/“live” control average)
100] (Fig. 5; see Note 22).

4

Notes 1. The forward primer, VHH F, is designed to bind the 50 UTR sequence. The reverse primer, VHH R, is designed to overlap the 3’ UTR and E-tag sequences. 2. For ELISAs, any mAb can be used for capturing your antigen of interest. However, it is imperative that the mAb be of sufficiently high affinity and that it does not compete with the VHH

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Fig. 5 Neutralization of ricin toxin by intrabody V1D3. Cells were transfected with pcDNA vectors encoding the anti-ricin toxin VHH V1D3 (left; open circles), a control anti-botulinum neurotoxin VHH ciA-H7 (right; open circles), or Lipofectamine (LNP) alone (both graphs; closed circles) and then challenged with serial dilutions of ricin toxin. Cell viability was measured using CellTiter-Glo® 2 days after ricin challenge. (Left) Representative ricin dose–response curves showing protection by a high affinity anti-RTA neutralizing intrabody (V1D3) that binds near the toxin’s active site compared to mock-transfected cells (LNP). Ricin neutralization is demonstrated by a shift in cell viability to the right, showing increased viability at higher concentrations of ricin. (Right) Ricin dose–response curves of cells transfected with an anti-botulinum neurotoxin VHH (ciA-H7) compared to mock-transfected cells (closed circles), demonstrating no protection from ricin by a non-binding intrabody

of interest. Perform preliminary competition ELISAs to select an appropriate capture mAb. Similarly, use purified VHHs to generate standard curves before subjecting cell lysates to testing. 3. The iBright Imager’s Universal Setting is compatible with both channels; however, other imagers may also be appropriate. 4. For subsequent intrabody preparations, we recommend synthesizing a gBlock™ containing 50 UTR, 30 UTR, and E-tag sequences but no VHH (make sure to include NotI and AscI RE sites). Clone this “empty” gBlock™ into pcDNA3.1 as described in Subheading 3.1. Then, use NotI and AscI enzymes to digest and ligate a VHH-hinge sequence of interest (produced either as a gBlock™ or by PCR) into the vector already containing 50 UTR, 30 UTR, and E-tag sequences. 5. For RE digestions and ligations, the enzyme(s) should be the final component added to the reaction mixture. 6. We advise picking at least three colonies from each plate to be grown separately in LB broth to ensure at least one successful VHH insertion is retrieved.

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7. Be sure to visually inspect cell growth prior to lysis to confirm similar levels of confluency among all wells to be tested. When pipetting Vero cells following RIPA buffer treatment, use a 1 mL pipette tip, since the lysate is viscous. 8. Homogenization tubes do not need to be filled to capacity with beads. Add just enough to cover the bottom of the 2 mL tube. 9. Homogenize cells/lysates twice: once at the start of incubation and then again ~15 min later. 10. For the lysate ELISA, be aware that expression levels will vary among different intrabodies. Therefore, the recommended dilution series used may need to be adjusted. 11. As positive controls for the intrabody ELISA, we recommend using purified VHHs to establish a standard curve. The experiment is set up the same; however, purified VHH with a C-terminal E-tag is diluted down the columns of the ELISA plate instead of cell lysate. This will establish appropriate experimental conditions (i.e., RTA is captured on the plate surface, there is no VHH competition with the coating mAb, and the secondary anti-E tag antibody works for detection) and reveal any binding differences between VHHs that may account for differences in lysate binding curves (e.g., high affinity will result in more robust binding curves for purified VHHs, but these proteins may not express well as intrabodies and thus result in low detection levels, see Note 10). 12. As noted above, intrabody expression levels will vary depending on the specific VHH. For example, many VHHs harbor a non-canonical disulfide bond that tethers the CDR3 to the framework regions. While this linkage results in higher thermal resistance and pH-stability, the formation of the disulfide bond is unfavorable in the cytosol. As such, we have found that VHHs with these this secondary disulfide bond express poorly as intrabodies. 13. Applying TMB to the ELISA plate(s) may result in bubbles. After applying stop solution, use a pipette tip to pop any air bubbles that remain. These may affect absorbance. 14. For orientation marking on nitrocellulose membrane, a small diagonal cut out of one upper corner is sufficient. Handle nitrocellulose membrane with broad, flat, untextured forceps to minimize direct handling and prevent creasing/tearing. 15. When setting up transfer cassette, handle the gel from the bottom as this is denser and less likely to tear. For gel transfer, transfer buffer used in blot stack assembly may be reused multiple times. However, only clean buffer (1 or 2 uses) should be added to gel box for electrophoresis.

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16. Any appropriately sized container to allow the membrane to remain flat and submersed in blocking buffer is sufficient. Cover container overnight to avoid evaporation. 17. For cytotoxicity assays all 96 wells of each plate are seeded. However, only the inner 60 wells (rows B–G, columns 2–11) will be subjected to transfection since we have observed that wells along the outside perimeter of the plate demonstrate high degrees of variability, possibly due to evaporation issues. 18. For cytotoxicity assays, Vero cells are transfected either with the intrabody/intrabodies of interest or Lipofectamine® vehicle control in triplicate (Figs. 2b and 3). This set up ensures that each plate has its own internal control. 19. While we recommend removing ricin from cells after 2 h, we have extended this up to 1 additional hour without results being affected for Vero cells. However, at least 2 h is recommended to allow toxin internalization. 20. CellTiter-Glo® should be stored at 20  C in approximately 45 mL aliquots and kept away from light, either using opaque conical tubes or wrapping tubes in foil. Make sure it is completely thawed when preparing to add to cells. 21. Any plate reader capable of luminescence detection, such as the Spectramax L Microplate Reader (Molecular Devices) should be appropriate. 22. When analyzing cell viability as a function of ricin concentration, the cytotoxicity curves of cells transfected with neutralizing VHHs often do not fit well as sigmoidal functions. This may be due to transfection and/or expression efficiency limitations. Therefore, the concentration of ricin necessary to achieve 50% cell viability (EC50) may not be the best method for analysis. We recommend a two-way ANOVA to compare cell viabilities across all concentrations between transfected and mock-transfected cells.

Acknowledgments This work was supported by research award AI125190 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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References 1. Vrentas CE, Moayeri M, Keefer AB et al (2016) A diverse set of single-domain antibodies (VHHs) against the anthrax toxin lethal and edema factors provides a basis for construction of a bispecific agent that protects against anthrax infection. J Biol Chem 291: 21596–21606 2. Vance DJ, Tremblay JM, Rong Y et al (2017) High-resolution epitope positioning of a large collection of neutralizing and nonneutralizing single-domain antibodies on the enzymatic and binding subunits of ricin toxin. Clin Vaccine Immunol 24:e00236–e00217 3. Miyashita SI, Zhang J, Zhang S et al (2021) Delivery of single-domain antibodies into neurons using a chimeric toxin-based platform is therapeutic in mouse models of botulism. Sci Transl Med 13:eaaz4197 4. Lam KH, Tremblay JM, Vazquez-Cintron E et al (2020) Structural insights into rational design of single-domain antibody-based antitoxins against botulinum neurotoxins. Cell Rep 30:2526–2539.e6 5. Yao G, Lam KH, Weisemann J et al (2017) A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding. Sci Rep 7:7438 6. Mukherjee J, Tremblay JM, Leysath CE et al (2012) A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model. PLoS One 7:e29941 7. Moayeri M, Leysath CE, Tremblay JM et al (2015) A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model. J Biol Chem 290:6584–6595 8. Connan C, Popoff MR (2017) Uptake of clostridial neurotoxins into cells and dissemination. Curr Top Microbiol Immunol 406:39–78 9. Moayeri M, Leppla SH, Vrentas C et al (2015) Anthrax pathogenesis. Annu Rev Microbiol 69: 185–208 10. Endo Y, Mitsui K, Motizuki M et al (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem 262:5908–5912 11. Endo Y, Tsurugi K (1988) The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain

with ribosomes and with rRNA. J Biol Chem 263:8735–8739 12. Iordanov MS, Pribnow D, Magun JL et al (1997) Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA. Mol Cell Biol 17:3373–2281 13. Wahome PG, Robertus JD, Mantis NJ (2012) Small-molecule inhibitors of ricin and Shiga toxins. Curr Top Microbiol Immunol 357: 179–207 14. Jasheway K, Pruet J, Anslyn EV et al (2011) Structure-based design of ricin inhibitors. Toxins (Basel) 3:1233–1248 15. Chiou JC, Li XP, Remacha M et al (2008) The ribosomal stalk is required for ribosome binding, depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae. Mol Microbiol 70:1441–1452 16. May KL, Li XP, Martı´nez-Azorı´n F et al (2012) The P1/P2 proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells. FEBS J 279:3925–3936 17. Shi WW, Tang YS, Sze SY et al (2016) Crystal structure of ribosome-inactivating protein ricin A ahain in complex with the C-terminal peptide of the ribosomal stalk protein P2. Toxins (Basel) 8:296 18. Fan X, Zhu Y, Wang X et al (2016) Structural insights into the interaction of the ribosomal P stalk protein P2 with a type II ribosomeinactivating protein ricin. Sci Rep 6:37803 19. Ready MP, Kim Y, Robertus JD (1991) Sitedirected mutagenesis of ricin A-chain and implications for the mechanism of action. Proteins 10:270–278 20. Angalakurthi SK, Vance DJ, Rong Y et al (2018) A collection of single-domain antibodies that crowd ricin toxin’s active site. Antibodies (Basel) 7:45 21. Rudolph MJ, Czajka TF, Davis SA et al (2020) Intracellular neutralization of ricin toxin by single-domain antibodies targeting the active site. J Mol Biol 432:1109–1125 22. O’Hara JM, Kasten-Jolly JC, Reynolds CE et al (2014) Localization of non-linear neutralizing B cell epitopes on ricin toxin’s enzymatic subunit (RTA). Immunol Lett 158:7–13

Chapter 25 Generation of Single-Domain Antibody-Based Recombinant Immunotoxins Bryan D. Fleming

and Mitchell Ho

Abstract The discovery of single-domain antibodies has opened new avenues for drug development. Single-domain antibodies, also known as nanobodies, can access buried epitopes that are inaccessible to conventional antibodies. These antigen-binding domains have a high level of solubility and stability, which makes them well suited for therapeutic development. This chapter will discuss the design, production, and testing of single-domain antibody-based recombinant immunotoxins. Recombinant immunotoxins are chimeric proteins that combine the specificity of an antibody with the ribosomal-inhibitory domain of a bacterial toxin. Immunotoxins using the Pseudomonas exotoxin domain have been well studied in clinical trials. Recently, an anti-CD22 immunotoxin was granted marketing approval for use in patients with relapsed or refractory hairy cell leukemia. This supports the idea that treatment with recombinant immunotoxins can be explored for cancers that have not responded to standard therapies. Key words Single-domain antibody, Nanobody, Recombinant immunotoxin, Pseudomonas exotoxin A, Tandem-binding domain

1

Introduction The discovery of single-domain antibodies (sdAbs) has opened new avenues for drug discovery. These molecules represent the smallest variable antigen-binding domains found in nature [1, 2]. Despite their small size, sdAbs display a level of specificity and affinity comparable to conventional tetrameric IgGs [3]. An elongated complementarity-determining region (CDR) 3 region allows for binding to recessed epitopes deep inside of protein clefts [2– 4]. This unique binding characteristic provides new opportunities for treating diseases that have not been targetable by conventional antibodies. SdAbs have been described in both shark and camelid species [1, 2, 5]. These binding domains are resistant to extremes in thermal and osmotic conditions, due to their high solubility and inherent ability to refold [6]. The robust nature and relative

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_25, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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simplicity of these antibodies support their future development as therapeutic drugs. The last few decades have seen a surge in antibody-based therapeutics entering the drug discovery pipeline. This includes antibody-based recombinant immunotoxins being explored as anti-cancer drugs. These chimeric proteins combine the antigen specificity of an antibody with a ribosomal inhibitory domain isolated from plant or bacterial species [7, 8]. Pseudomonas exotoxin-based recombinant immunotoxins targeting CD22positive leukemias have recently been granted marketing approval due to the high level of effectiveness and patient safety observed in clinical trials [9]. Immunotoxins based on the anti-glypican 3 (GPC3) sdAb HN3 currently in preclinical development have shown potent inhibition of hepatocellular carcinoma-derived cell lines [10–12]. HN3 is a sdAb that has been shown to inhibit the Wnt/Frizzled-signaling pathway by blocking binding of Wnt to GPC3 [10, 13]. Recombinant immunotoxins are difficult to produce in eukaryotic systems due to the toxic nature of the ribosomal inhibitory domain. To circumvent this, a prokaryotic production system has been developed for immunotoxin production. This protocol will describe the production, purification, and subsequent testing of sdAb-based recombinant immunotoxins. HN3-based immunotoxins used for treating liver cancer are the focus of this protocol. We describe the production of HN3-PE38, which combines the HN3 sdAb with domain II and domain III of the Pseudomonas exotoxin. We also describe HN3-ABD-T20 which contains a tandem-binding domain (anti-GPC3 and serum albumin binding domain [ABD]) combined with a deimmunized domain III-only version of the Pseudomonas exotoxin known as T20. T20 uses a furin-cleavable linker to replace domain II, which was found to be highly antigenic and made the immunotoxin susceptible to lysosomal degradation [14]. Removal of domain II, while retaining the furin-cleavage site, was found to increase lysosomal resistance and had no negative effect on immunotoxin processing [14]. Additionally, six point mutations were introduced into domain III to reduce antigenicity. The belief was that by mutating amino acids predicted to be important for MHC class II peptide binding, it would be possible to dampen the T helper response associated with immunotoxin treatment [15]. It should be mentioned that while this protocol describes the use of a human VH sdAb for immunotoxin construction, it is also possible to construct immunotoxins containing camelid VHHs and shark VNARs. For antigen-binding domains derived from a conventional IgG, a single-chain Fv [16] or disulfide-stabilized Fv [17] format is required. For production of immunotoxins in these formats, please refer to our previously published protocol from which the current method was adapted [18]. An overview of different antigenbinding domain formats and exotoxin versions is shown in Fig. 1. Discovery of sdAbs is generally accomplished by panning phage display libraries [5, 19]. Once the sdAb has been cloned into an

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Fig. 1 Overview of different antigen-binding domain formats and Pseudomonas exotoxin A versions used for construction of immunotoxins

expression vector, immunotoxin production can be induced in bacteria using isopropyl-β-D-1-thiogalactopyranoside (IPTG). This triggers the formation of inclusion bodies that are later isolated, purified, and dissolved in a guanidine buffer. The solubilized inclusion bodies are fully reduced before refolding and column purification. This method yields proteins with high purity (>95%) and generally has acceptable protein recovery (10–20%). The cytotoxic activity of immunotoxins is generally tested in cellbased assays before experiments in animal models. Production and testing of recombinant immunotoxins require less than 2 weeks and the steps are summarized in Table 1.

2

Materials

2.1 Generation of Recombinant Immunotoxin Expression Vectors 2.1.1 Isolation of Phagemid DNA

1. Phagemid clone (e.g., pComb3X) encoding sdAb of interest. 2. 100 mg/mL ampicillin in water. Filter-sterilize. 3. LB broth: 10 g of tryptone, 10 g of NaCl, and 5 g of yeast extract per 1 L H2O. Autoclave. Supplement with 100 μg/mL ampicillin for selection of pComb3X. 4. Falcon round-bottom polypropylene test tubes. 5. QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA).

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Table 1 Recombinant immunotoxin production and testing steps Step

Day

Procedure

Section

1

1

Transformation of E. coli

3.2.1

2

2

Fermentation, IPTG induction, and cell harvesting

3.2.2

3

3

Inclusion body isolation

3.2.3

4

3

Inclusion body solubilization and denaturing

3.2.4

5

4

Protein refolding

3.2.5

6

6

Dialysis

3.2.5

7

7

Q-Sepharose chromatography

3.2.6

8

7

MonoQ chromatography

3.2.6

9

8

TSK chromatography

3.2.6

10

9–12

Cytotoxicity testing

3.3.1

2.1.2 Identification of sdAb Sequence

1. pComb3X sequencing primer: 50 -CGT ATG TTG TGT GGA ATT GTG AGC G-30 . 2. Molecular biology-grade water. 3. PCR tubes (0.2 mL).

2.1.3 PCR Amplification of sdAb

1. Sense primer: 50 -CAT ATG CAG GTG CAG CTG GTG CAG-30 (see Subheading 3.1.3 for more information on primer design). 2. Anti-sense primer HN3-PE38 cloning: 50 -AAG CTT TGG CCG CAC TTG AGG AGA CGG TGA CCA GG-30 (HindIII site, linker, sdAb) 3. Anti-sense primer HN3-T20 cloning: 50 - AAG CTT TGA GGA GAC GGT GAC CAG GC-30 (HindIII site, sdAb). 4. ExpandTM High Fidelity PCR system (Roche, Basel, Switzerland). 5. dNTPs. 6. Thermocycler. 7. UltraPure agarose. 8. 50 Tris-acetate ethylenediaminetetraacetic acid (EDTA) (TAE) buffer: 2 M Tris, 1 M glacial acetic acid, 50 mM EDTA. 9. Ethidium bromide or other DNA stain. 10. DNA gel loading buffer. 11. DNA ladder. 12. QIAquick Gel Extraction Kit (Qiagen). 13. HN3-PE38 (pMH236) plasmid to serve as the template for amplification of sdAb HN3. Plasmids can be obtained by contacting the corresponding author.

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1. Restriction enzymes (NdeI and HindIII). 2. NanoDrop or other small volume spectrophotometer. 3. Rapid DNA Ligation Kit (Roche). 4. One Shot™ TOP10 chemically competent Escherichia coli cells (Invitrogen, Carlsbad, CA, USA). 5. SOC medium: 20 g of tryptone (2% w/v), 5 g of yeast extract (0.5% w/v), 0.584 g of NaCl (10 mM), 0.186 g of KCl (2.5 mM), 2.033 g of MgCl2·6H2O (10 mM), 2.465 g of MgSO4·7H2O (10 mM), and deionized water to 1 L. Adjust pH to 7.0 with NaOH and autoclave the solution. Prepare a 1 M glucose solution (180.15 g/L) and filter sterilize. Add 20 mL of the glucose solution after the medium has cooled. 6. 2 YT agar (per L): 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, and 15 g of agar dissolved in ultrapure H2O. Sterilize by autoclaving, cool to ~50  C and add 25 μg/mL chloramphenicol. 7. LB broth: see Subheading 2.1.1. 8. Falcon round-bottom polypropylene test tubes. 9. QIAprep Spin Miniprep Kit. 10. HN3-PE38 (pMH236) or HN3-ABD-T20 (pMH294) plasmid to provide the desired immunotoxin fragment and the required pRB98 backbone. Plasmids can be obtained by contacting the corresponding author.

2.1.5 Construction of Tandem-Binding Domains

1. sdAb1 sense primer: 50 -CAT ATG CAG GTG CAG CTG GTG-30 (NdeI site, sdAb1) (see Subheading 3.1.5 for more information on primer and Ultramer® design). 2. sdAb1—Linker anti-sense Ultramer®: 50 -TCC AGA CCC ACT ACC CGA CCC GCT CCC TGA GCC ACT CCC AGA GCC GGA CCC TGA GGA GAC GGT GAC CAG G-30 ([GS]8G linker sequence, sdAb1) (see Subheading 3.1.5 for more information on primer and Ultramer® design). 3. Linker—ABD sense Ultramer®: 50 -GGG TCC GGC TCT GGG AGT GGC TCA GGG AGC GGG TCG GGT AGT GGG TCT GGA CCT GGA TCT TCT CTG CAA GTA GAT TTG G-30 ([GS]8G linker sequence, ABD) (see Subheading 3.1.5 for more information on primer and Ultramer® design). 4. ABD—Linker anti-sense Ultramer®: 50 -AAG CTT ACC ACT TCC ACT CCC CGA TCC TGA GCC GGA CCC CGA GCC AGA TCC ACT TCC AGG CAA GGC TGC AAG AAT CTC G-30 (HindIII site, [GS]8G linker sequence, ABD) (see Subheading 3.1.5 for more information on primer and Ultramer® design).

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5. (GS)8G linker anti-sense primer: 50 -AAG CTT ACC ACT TCC ACT CCC C -30 (HindIII site, [GS]8G linker sequence) (see Subheading 3.1.5 for more information on primer and Ultramer® design). 6. pMH236 and pMH294 to serve as templates, Plasmids can be obtained by contacting the corresponding author. 7. Reagents in Subheadings 2.1.3 and 2.1.4. 2.2 Production of Recombinant Immunotoxins

1. One Shot™ BL21 Star™ (DE3) chemically competent E. coli cells (Invitrogen).

2.2.1 Transformation of E. coli

3. 2 YT agar plates: see Subheading 2.1.4.

2.2.2 Fermentation

1. TB medium (per L): 24 g of yeast extract, 20 g of tryptone, 4 mL of glycerol, and 100 mL of phosphate buffer (0.17 M KH2PO4, 0.72 M K2HPO4).

2. SOC medium: see Subheading 2.1.4.

2. 20% (v/v) glucose solution: prepare in deionized water and sterilize using a 0.22 μm filter top flask. 3. 1 M MgSO4: prepare in deionized water and sterilize using a 0.22 μm filter top flask. 4. 25 mg/mL chloramphenicol: prepare in 100% ethanol. 5. Pyrex shaker flask with extra-deep baffles (2 L). 6. L-shaped spreader. 7. IPTG. 8. Nalgene™ 1,000 mL polycarbonate centrifuge bottles. 9. TES buffer: 50 mM Tris–HCl (pH 8.0), 20 mM EDTA, 100 mM NaCl. 10. SDS-PAGE gels, molecular weight standards, and electrophoresis equipment. 11. Desktop refrigerated centrifuge. 2.2.3 Inclusion Body Isolation

1. TES buffer: see Subheading 2.2.2. 2. Nalgene™ 250 mL polycarbonate centrifuge bottles. 3. Lysozyme. 4. Triton X-100. 5. IKA T25 digital ULTRA-TURRAX homogenizer with 18G dispersion tool. 6. Sorvall RC5B centrifuge.

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1. GTE buffer: 6 M guanidine-HCl, 100 mM Tris–HCl (pH 8.0), 2 mM EDTA. 2. Nalgene™ 50 mL polycarbonate centrifuge bottles. 3. Dithiothreitol. 4. IKA T25 digital ULTRA-TURRAX homogenizer with 10G dispersion tool. 5. Pierce™ Coomassie Plus reagent (Thermo Fisher, Waltham, MA, USA).

2.2.5 Refolding

1. 100 mM Tris–HCl (pH 8.0), 1 mM EDTA, and 0.5 M arginine. Prepare in a 2 L flask with a stir bar. Chill to 4  C. Adjust the pH to 9.5 with 10 N NaOH. 2. Oxidized L-glutathione. 3. 20 mM Tris–HCl (pH 7.4) containing 100 mM urea. Prepare 50 L and chill to 4  C. Add urea at a concentration of 100 mM immediately prior to use. 4. Dialysis tubing with 14 kDa MWCO. 5. 50 L dialysis tank. 6. 0.45 μM filter top flask.

2.2.6 Chromatography

1. Chromatography buffer A: 20 mM Tris–HCl, 1 mM EDTA, pH 7.4. 2. Chromatography buffer B: 1 M NaCl in chromatography buffer A. 3. 10 phosphate-buffered saline (PBS) (per L): 25.6 g Na2HPO4·7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4. 4. Tricorn 10/100 column (Cytiva, Marlborough, MA, USA). 5. Q-Sepharose fast flow resin (Cytiva). 6. MonoQ 10/100 GL column (Cytiva). 7. TSKgel G3000SW column (Tosoh Bioscience, King of Prussia, PA, USA). 8. AKTA Pure or equivalent fast protein liquid chromatography (FPLC) instrument.

2.3 Cytotoxicity and Functional Assays 2.3.1 Cell Proliferation Inhibition

1. 96-well cell culture-treated plates. 2. A431 cell line stably expressing human GPC3 (G1 cells) to serve as a positive control and parental A431 cells to serve as the negative control. Cell lines are available upon request from the corresponding author. 3. Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine.

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4. Cell culture-grade PBS. 5. Automated cell counter or other method to determine cell concentration and viability 6. Cell Counting Kit-8 (WST reagent) (Dojindo, Rockville, MD, USA). 7. ELISA plate reader. 8. Opti-MEM™ reduced serum medium, no phenol red. 9. Trypsin-EDTA (0.25% w/v), phenol red. 2.3.2 ADP-Ribosylation Assay

1. 20 mM Tris–HCl (pH 7.4), containing 1 mM EDTA. 2. RIPA buffer (Thermo Fisher). 3. cOmplete™ mini protease inhibitor (Roche). 4. 15 mL conical tubes. 5. 2 mL microcentrifuge tubes. 6. 1 M dithiothreitol. 7. Biotinylated NAD+ (Trevigen, Gaithersburg, MD, USA). 8. Molecular biology-grade water. 9. 4 Laemmli buffer: 10 mL 1 M Tris (pH 6.8), 4 g of SDS, 20 mL of glycerol, 10 mL of β-mercaptoethanol, 0.1 g of bromophenol blue, and H2O to 50 mL. 10. SDS-PAGE gels, molecular weight standards, and electrophoresis equipment. 11. Western blot transfer equipment and materials. 12. Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST): in 1 L H2O, dissolve 6.05 g of Tris and 8.76 g of NaCl. Bring pH to 7.5 and add 1 mL Tween-20. 13. Horseradish peroxidase (Invitrogen).

(HRP)-conjugated

streptavidin

14. Pierce ECL substrate (Thermo Fisher). 15. Bio-Rad ChemiDoc instrument and Image Lab software.

3

Methods

3.1 Subcloning of sdAb into Expression Vector 3.1.1 Isolation of Phagemid DNA

1. Identify a sdAb of interest by panning of a phage-displayed library using a well-established protocol [20]. For the purposes of this protocol, we assume that a binding sdAb-phagemid clone (pComb3X) and matched single E. coli TG1 colony are available. 2. Inoculate 3 mL of LB broth containing 100 μg/mL ampicillin with a single E. coli TG1 colony containing the phagemid clone of interest.

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3. Incubate culture overnight at 37  C in a shaking incubator at 250 rpm. 4. Isolate DNA for the phagemid of interest using a QIAprep Spin Miniprep Kit according to the manufacturer’s instructions. 3.1.2 Identification of sdAb Sequence

1. Retrieve the phagemid Subheading 3.1.1.

DNA

that

was

isolated

in

2. Perform Sanger sequencing to identify the sdAb nucleic acid sequence. If the phagemid has a pComb3X backbone, then the sequencing primer 50 CGT ATG TTG TGT GGA ATT GTG AGC G 30 can be used. 3. Identify the open reading frame containing the sdAb sequence (see Note 1). 3.1.3 PCR Amplification of sdAb

1. Design PCR primers to amplify the sdAb sequence identified in Subheading 3.1.2. If pRB98 will serve as the backbone for the immunotoxin, then the sense primer should start with an NdeI restriction site (CAT ATG) followed by the first 15–20 nucleotides in the sdAb sequence. The sense primer used for HN3 cloning is 50 -CAT ATG CAG GTG CAG CTG GTG CAG-30 (NdeI site). The anti-sense primer design will vary depending on whether the immunotoxin will contain domain II of the Pseudomonas exotoxin. If the immunotoxin will contain PE38 (domain II + III), then the anti-sense primer will start with (AAG CTT TGG CCG CAC T) followed by the first 15–20 nucleotides of the sdAb’s anti-sense sequence. The anti-sense primer used for HN3-PE38 cloning is 50 -AAG CTT TGG CCG CAC TTG AGG AGA CGG TGA CCA GG-30 (HindIII site, linker, sdAb). Immunotoxins containing T20 (deimmunized domain III-only versions) require an anti-sense primer starting with the HindIII restriction site (AAG CTT) followed by the first 15–20 nucleotides of the sdAb’s anti-sense sequence. The anti-sense primer used for HN3-T20 cloning is 50 -AAG CTT TGA GGA GAC GGT GAC CAG GC-30 (HindIII site, sdAb). Vector maps for HN3-PE38 (pMH236) and HN3-ABD-T20 (pMH294) can be found in Fig. 2a, b, respectively. The nucleotide sequence highlighting the HindIII restriction site used to clone into PE38 immunotoxins (Fig. 2c) and domain III only immunotoxins like T20 (Fig. 2d) are shown. 2. Set up a PCR reaction containing: 200 ng of phagemid DNA template, 1 μL each of the sense and anti-sense primers (5 μM), 2.5 μL of 10 PCR buffer with 15 mM MgCl2, 0.5 μL of 10 mM dNTPs, 0.5 μL of Expand™ High Fidelity DNA polymerase and molecular biology-grade water to 25 μL.

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Fig. 2 Vector maps and partial sequences of immunotoxin expression vectors. (a) Vector map for pRB98-HN3PE38 (pMH236). (b) Vector map for pRB98-HN3-ABD-T20 (pMH294). (c, d) Nucleotide sequence of the linker peptides located between the sdAb and toxin domains. The short flexible linker used in immunotoxins containing domain II and III (c) and the furin-cleavable linker used in domain III-only versions (d) are shown. Abbreviations: ABD, albumin-binding domain; CAT, chloramphenicol acetyltransferase; ori, origin of replication

3. Perform the PCR reaction as follows: 95  C for 5 min; 32 cycles of 95  C for 40 s, 56  C for 40 s, and 72  C for 60 s; 72  C for 7 min. The PCR reaction can be frozen at 20  C until gel purification can be performed. 4. Prepare a 1.5% (w/v) agarose gel in 1 TAE buffer containing 1 μg/mL ethidium bromide. 5. Mix the PCR reaction from step 3 with gel loading dye, then load the entire sample into the agarose gel. Another well should be loaded with an appropriate DNA ladder. 6. Excise the DNA band that corresponds to the predicted size of the PCR product. Typically, the amplified DNA product for an sdAb-coding region will be around 400 bp in length.

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7. Extract the DNA from the agarose gel using a QIAquick Gel Extraction Kit following the manufacturer’s instructions. Quantitate the DNA using a NanoDrop spectrophotometer. 3.1.4 Construction of Immunotoxin Plasmid

1. Obtain either the pMH236 vector if producing PE38containing immunotoxins or pMH294 if producing T20-containing immunotoxins by contacting the corresponding author. 2. Add 0.5 μL (50 ng) of the desired immunotoxin plasmid DNA to 25 μL of ice cold One ShotTM TOP10 chemically competent E. coli cells. Incubate tube on ice for 5–30 min, heat shock for 60 s at 42  C, then rest the cells on ice for 2 min. Add 250 μL of SOC medium and incubate the transformed E. coli cells in a shaking 37  C incubator for 60 min at 250 rpm. 3. Spread 25, 50, and 100 μL of the transformed bacteria on three LB plates containing 25 μg/mL chloramphenicol to assure single colony isolation. 4. Incubate plates overnight at 37 formation.



C to allow for colony

5. Select 4–8 colonies for culture in 3 mL of LB broth with 25 μg/mL chloramphenicol. Grow the cultures overnight at 37  C with 250 rpm shaking. 6. The next day, isolate plasmids using a QIAprep Spin Miniprep Kit following the manufacturer’s instructions. Quantitate the DNA using a NanoDrop spectrophotometer. 7. To prepare the desired pRB98-immunotoxin backbone (pMH236 or pMH294) and the purified sdAb insert from step 7 in Subheading 3.1.3 for the ligation reaction, they will need to be double digested with NdeI/HindIII to prepare sticky ends. In separate tubes, combine 1 μL of NdeI, 1 μL of HindIII, 2.5 μL of 10 NEBuffer 2.1, sdAb insert (1 μg) or vector (3–5 μg) DNA, and molecular biology-grade water to 25 μL. 8. Incubate the reactions at 37  C for 1–2 h to allow for restriction enzyme digestion (see Note 2). 9. Purify the digested pRB98-immunotoxin backbone (pMH236 or pMH294) and the sdAb insert using agarose gel electrophoresis and a QIAquick Gel Extraction Kit following the manufacturer’s instructions (see steps 4–7 in Subheading 3.1.3). The digested sdAb insert will be around 400 bp in size and the pRB98-immunotoxin backbone will be around 4.5 kb for PE38-containing vectors and 4.1 kb for T20-containing vectors. 10. Use a NanoDrop or other small volume spectrophotometer to determine the concentration of the purified DNA.

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11. Set up a ligation reaction containing 50 ng of the desired digested pRB98-immunotoxin backbone (pMH236 or pMH294) with a 5-molar excess of insert (see Note 3). Using the Rapid DNA Ligation Kit, this reaction can be performed in 5 min. The DNA should be combined and diluted to 8 μL in molecular biology-grade water. Add 2 μL of 5 DNA dilution buffer and mix thoroughly. Add 10 μL of 2 T4 DNA ligase buffer and mix thoroughly. Finally, add 1 μL of T4 DNA ligase and mix thoroughly. Incubate the tube at room temperature for 5–30 min (see Note 4). 12. Add 2 μL of the ligation product to 50 μL of ice cold One Shot™ TOP10 chemically competent E. coli cells. Continue with the procedure described earlier in steps 2–6 of this Subheading to transform E. coli, grow and select colonies, isolated clonal plasmids by miniprep, and quantitate by NanoDrop. 13. The resulting plasmids can be digested with NdeI and HindIII, then visualized by agarose gel electrophoresis to confirm the presence of the sdAb insert. Briefly, combine 0.5 μL of NdeI, 0.5 μL of HindIII, 1 μL 10 NEBuffer 2.1, and 8 μL of purified plasmids from step 12. Incubate at 37  C for 1–2 h to allow for restriction enzyme digestion (see Note 2). Visualization of the digested bands is possible by performing agarose gel electrophoresis. 14. If desired, Sanger sequencing can be used to confirm proper in-frame insertion of the sdAb sequence. 3.1.5 Construction of Tandem-Binding Domains

The construction of tandem-binding domains can be used to generate immunotoxins that are bivalent (containing two copies of the same antibody), biparatopic (targeting different epitopes on the same antigen), and bispecific (targeting different antigens). Using a tandem-binding domain can improve the binding avidity of the immunotoxin leading to better cytotoxic activity. We have constructed bispecific HN3-immunotoxins targeting GPC3 and mouse serum albumin by incorporating both a llama nanobody and a bacterial-derived ABD into the T20 immunotoxin backbone [12]. These immunotoxins were shown to have improved serum half-life due to their high affinity for mouse serum albumin. This resulted in immunotoxins with 5–10-fold higher potency when tested in mouse xenograft models [12]. The HN3-ABD-T20 immunotoxin construct described in Fig. 2b contains a bacterialderived ABD flanked by 17 amino acid (GS)8G flexible spacers. Standard (G4S)3 flexible spacers can be substituted if desired. This protocol will describe how to prepare the HN3-ABD tandembinding domain by overlap extension PCR using Ultramers® (primers longer than 60 bp). However, any combination of linkers and binding domains can be cloned into the pRB98-T20 immunotoxin backbone as long as they start and end with in frame NdeI and

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HindIII sites, respectively. It is worth mentioning that commercial synthesis of this fragment should be considered due to the relatively low cost and time commitment. 1. Obtain the pMH236 vector to serve as the template for amplification of sdAb1(HN3) and the pMH294 vector to serve as the ABD template by contacting the corresponding author. 2. Follow steps 2–6 in Subheading 3.1.4 to generate pMH236 and pMH294 plasmid stocks. 3. Set up the sdAb1—Linker PCR reaction containing: 200 ng of pMH236 template, 1 μL of sdAb1 sense primer: (50 -CAT ATG CAG GTG CAG CTG GTG-30 , NdeI site, sdAb1) (5 μM), 1 μL of sdAb1—Linker anti-sense primer: (50 -TCC AGA CCC ACT ACC CGA CCC GCT CCC TGA GCC ACT CCC AGA GCC GGA CCC TGA GGA GAC GGT GAC CAG G-30 , [GS]8G linker sequence, sdAb1) (5 μM), 2.5 μL of 10 PCR buffer with 15 mM MgCl2, 0.5 μL of 10 mM dNTPs, 0.5 μL of Expand™ High Fidelity DNA polymerase, and molecular biology-grade water to 25 μL. 4. Set up the ABD flanked by (GS)8G linkers PCR reaction containing: 200 ng of pMH294 template, 1 μL of Linker—ABD sense primer: (50 -GGG TCC GGC TCT GGG AGT GGC TCA GGG AGC GGG TCG GGT AGT GGG TCT GGA CCT GGA TCT TCT CTG CAA GTA GAT TTG G-30 , [GS]8G linker sequence, ABD) (5 μM), 1 μL of ABD—Linker antisense primer: (50 -AAG CTT ACC ACT TCC ACT CCC CGA TCC TGA GCC GGA CCC CGA GCC AGA TCC ACT TCC AGG CAA GGC TGC AAG AAT CTC G-30 , [GS]8G linker sequence, ABD) (5 μM), 2.5 μL of 10 PCR buffer with 15 mM MgCl2, 0.5 μL of 10 mM dNTPs, 0.5 μL of Expand™ High Fidelity DNA polymerase, and molecular biology-grade water to 25 μL. 5. Perform the PCR reaction on tubes from steps 3 and 4 as follows: 95  C for 5 min; 32 cycles of 95  C for 40 s, 56  C for 40 s, and 72  C for 60 s; 72  C for 7 min. The PCR reaction can be frozen at 20  C until gel purification can be performed. 6. Perform agarose gel electrophoresis to purify PCR products (see steps 4–7 in Subheading 3.1.3 for details on gel extraction). The sdAb1 (HN3) product will be around 400 bp and the ABD—Linkers product will be 264 bp in size. 7. Set up the overlap extension PCR reaction to combine the containing: 50 ng of sdAb1—Linker PCR product (amplified in step 3, purified in step 6), 50 ng of ABD flanked by (GS)8G linkers PCR product (amplified in step 4, purified in step 6), 1 μL of sdAb1 sense primer: (50 -CAT ATG CAG GTG CAG

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CTG GTG-30 , NdeI site, sdAb1) (5 μM), 1 μL of (GS)8G linker anti-sense primer: (50 -AAG CTT ACC ACT TCC ACT CCC C-30 , HindIII, [GS]8G linker sequence) (5 μM), 2.5 μL of 10 PCR buffer with 15 mM MgCl2, 0.5 μL of 10 mM dNTPs, 0.5 μL of Expand™ High Fidelity DNA polymerase and molecular biology-grade water to 25 μL. 8. Perform the PCR reaction on the tube from step 7 as follows: 95  C for 5 min; 32 cycles of 95  C for 40 s, 56  C for 40 s, and 72  C for 60 s; 72  C for 7 min. The PCR reaction can be frozen at 20  C until gel purification can be performed. 9. Perform agarose gel electrophoresis to purify PCR products (see steps 4–7 in Subheading 3.1.3 for details on gel extraction). The full-length PCR products should be 624 bp in size. If low PCR efficiency is observed, then return to the overlap extension PCR in step 7 with the following modification. Omit the primer pair (sdAb1 sense and [GS]8G linker anti-sense) from the PCR tube and perform the following reaction: 95  C for 5 min, then 10 cycles of 95 C for 40 s, 56  C for 40 s, and 72  C for 60 s. At this point, the primer pair is added to the reaction tube and an additional 20–25 cycles are performed as follows: 95  C for 40 s, 56  C for 40 s, and 72  C for 60 s, followed by a final elongation step of 72  C for 7 min. 10. Clone the tandem-binding domain into the pMH294 expression vector using the NdeI and HindIII double digestion method described in steps 7–14 in Subheading 3.1.4. 3.2 Preparation of Recombinant Immunotoxins 3.2.1 Transformation of E. coli

1. Thaw one vial of BL21 Star (DE3) chemically competent E. coli cells on ice. 2. Add 200–600 ng of pRB98-immunotoxin plasmid produced in step 12 of Subheading 3.1.4 or step 10 of Subheading 3.1.5. 3. Incubate cells on ice for 30 min. 4. Heat shock cells in a water bath set to 42  C for 60 s. 5. Rest the cells on ice for 2 min. 6. Add 1 mL of SOC medium. 7. Incubate tubes at 37  C for 1 h with 225 rpm shaking. 8. Plate 100 μL of transformed E. coli cells onto each of 10 2 YT/chloramphenicol plates. 9. Incubate plates overnight at 37  C overnight. Additional incubation time may be required for plasmids with low transformation efficiency. 10. Warm 1 L of TB to 37  C overnight in the incubator. 11. Autoclave two 2 L baffled flask for each immunotoxin being produced.

Generation of Single-Domain Antibody-Based Recombinant Immunotoxins 3.2.2 Fermentation

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1. Add 20 mL of 20% glucose, 1.68 mL of 1 M MgSO4, and 25 μg/mL chloramphenicol to each L of pre-warmed TB medium. 2. Save 1 mL of complete TB to serve as a spectrophotometry blank. 3. Pipet 5 mL of TB onto each of the 10 culture plates from Subheading 3.2.1 and dislodge the bacteria with an L-shaped spreader or equivalent. Combine the scraped bacteria into a single tube and homogenize by pipetting. 4. Add 5–20 mL bacterial inoculum to each 1 L of TB to obtain a starting OD600 of between 0.15 and 0.20. 5. Split the inoculated 1 L TB evenly between two 2 L baffled flasks (500 mL each). 6. Incubate the flasks in a 37  C shaking incubator (250 rpm) for 2–4 h. 7. The OD600 should be checked every 30 min after the first 2 h of culture. 8. The bacterial culture will be ready for IPTG induction when the OD600 reaches between 2 and 3 (see Note 5). 9. Save a 500 μL pre-induction sample on ice for later analysis. 10. Prepare 0.1 M IPTG (25 mg IPTG/mL) in TB and add 5 mL to each 500 mL culture. 11. Incubate the bacterial cultures for an additional 90 min in the 37  C shaking incubator (250 rpm). 12. Save a 250 μL aliquot of the post-induction bacteria on ice for later analysis. 13. Transfer the bacterial culture into a Nalgene™ 1,000 mL polycarbonate centrifuge bottle and pellet the bacteria by centrifuging at 4,500  g for 30 min at 4  C in a Sorvall RC3B centrifuge. 14. Decant the bacterial supernatant and store the bacterial pellet at 80  C. 15. Centrifuge the pre- and post-induction culture samples from steps 9 and 12 in a benchtop-refrigerated centrifuge at maximum speed for 2 min. 16. Remove the supernatant and resuspend the bacterial pellet in 0.1 mL of TES buffer. Sonicate the bacterial pellet for 10–20 s. 17. Determine the protein concentration of the TES extract from pre- and post-induction culture samples after pelleting cellular debris from step 16 using a NanoDrop spectrophotometer. Load equal amounts (10–15 μg) of pre- and post-induction protein on an SDS-PAGE gel to confirm protein induction.

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3.2.3 Inclusion Body Isolation

1. Remove the bacterial pellets from the 80  C freezer (from step 14 of Subheading 3.2.2) and allow the centrifuge bottles to warm to room temperature. The storage of frozen bacterial pellets serves as an experimental break point. The bacteria can be frozen for many months at 80  C with little effect on inclusion body quality. 2. Resuspend the pellets in 160 mL of TES buffer and transfer to a Nalgene™ 250 mL polycarbonate centrifuge bottle. 3. Add 6.5 mL of lysozyme (5 mg/mL) and homogenize the bacteria using the homogenizer with the 18G dispersion tool. 4. Incubate the bacteria for 30 min at room temperature. Mix by hand every 10 min to ensure full lysis. 5. Add 20 mL of 25% (v/v) Triton X-100 and mix with the homogenizer. 6. Incubate the bacteria for 30 min at room temperature. Mix by hand every 10 min to ensure full lysis. 7. Centrifuge the containers at 12,000  g at 4  C in a Sorvall 5B centrifuge for 50 min. Discard the supernatant. 8. Add 160 mL of TES and 20 mL of 25% Triton X-100 to each container and resuspend the pellet using the homogenizer. Incubate for 5–10 min at room temperature. 9. Centrifuge the containers at 12,000  g at 4  C in a Sorvall 5B centrifuge for 30 min. Discard the supernatant. 10. Repeat steps 8 and 9 twice more, for a total of three wash steps. 11. Add 180 mL of TES to each container and resuspend the pellet using the homogenizer. Incubate for 5–10 min at room temperature. 12. Centrifuge the containers at 12,000  g at 4  C g in a Sorvall 5B centrifuge for 30 min. Discard the supernatant. 13. Repeat steps 11 and 12 twice more, for a total of three rinse steps. 14. Inclusion bodies can be frozen at 80  C indefinitely.

3.2.4 Solubilization and Denaturing

1. Thaw the inclusion bodies and transfer them to a Nalgene™ 50 mL polycarbonate centrifuge bottle. 2. Add 10 mL of GTE buffer and homogenize the inclusion bodies using the homogenizer with the 10G dispersion tool. 3. Determine the protein concentration with the Pierce™ Coomassie Plus reagent following the manufacturer’s instructions. 4. Dilute the inclusion bodies with GTE to achieve a 10 mg/mL solution. 5. Add dithiothreitol at a concentration of 10 mg/mL. Mix well by gentle inversion.

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6. Incubate at room temperature overnight. 7. Centrifuge the denatured protein solution at 12,000  g at 4  C in a Sorvall 5B centrifuge for 10 min. 8. Transfer the clarified supernatant to a fresh Nalgene™ 50 mL polycarbonate centrifuge bottle. 9. Determine the concentration of the clarified supernatant using the Pierce™ Coomassie Plus reagent following the manufacturer’s instructions. 10. Adjust the final concentration of inclusion bodies to 10 mg/mL using GTE buffer containing 10 mg/mL dithiothreitol. 11. Solubilized inclusion bodies can be aliquoted and frozen at 80  C until a later time. 3.2.5 Refolding

It is important to perform the refolding reaction in a 4  C cold room. Keeping the refolding buffer cold and constantly stirring with a magnetic bar will help to ensure proper protein refolding. 1. Prepare 1 L of refolding buffer (100 mM Tris–HCl, pH 8.0, 1 mM EDTA, 0.5 M arginine) for every 100 mg of inclusion bodies to be refolded. Chill buffer to 4  C and adjust the pH to 9.5. 2. Add 551 mg/L oxidized L-glutathione to the refolding buffer just before proceeding to the next step. 3. Using a 10 mL pipette, add the solubilized inclusion bodies in a dropwise fashion to the refolding buffer while stirring with a magnetic stir bar. The inclusion bodies should be added quickly over a period of 15–20 s. 4. Continue mixing the refolding buffer with the magnetic stir bar for an additional 2–3 min. 5. Stop stirring the refolding buffer and store at 4  C for 36–48 h. 6. Transfer the refolded protein to fresh dialysis tubing. The protein should be dialyzed against 50 L of cold dialysis buffer (20 mM Tris–HCl, pH 7.4, containing 100 mM urea) at 4  C for 16–36 h. 7. Filter the dialyzed protein though a 0.45 μm filter top flask to remove any protein aggregates. If the protein is turbid after dialysis, it may be necessary to centrifuge before passing through the filter top flask.

3.2.6 Chromatography

The chromatography protocol consists of three separate column purification steps. The first step uses an anion exchange Q-Sepharose resin to concentrate the 1 L of refolded protein down to about 20 mL. The second step is another anion exchange step using a MonoQ column. These anionic exchange steps are

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possible due to the low isoelectric point of the Pseudomonas exotoxin domain (pI ¼ ~5). The buffers used to refold and dialyze the protein have pHs of 9.5 and 7.5, respectively. This means that most Pseudomonas exotoxin-based immunotoxins will carry a negative change throughout the purification process. The final purification step serves as both a buffer exchange step (Tris–HCl/EDTA/NaCl to PBS) and as a preparative size exclusion chromatography step. 1. Pack an empty Tricorn 10/100 column with 10 mL of fresh Q-Sepharose fast flow resin (see Note 6). 2. Connect the Tricorn column to the FPLC and wash the resin at 10 mL/min with 50 mL of buffer A, 50 mL of buffer B, and another 50 mL of buffer A. This will help to pack the resin and will remove any contaminates from the resin. 3. Load the refolded protein at 10 mL/min until entire sample has been passed through the resin. It is important to collect and retain the column flow-through until protein elution has been confirmed. 4. Wash the loaded column at 10 mL/min with 50 mL of buffer A to dislodge any protein that has nonspecifically bound. 5. Elution of the protein should be performed at 2 mL/min using a 35% buffer B gradient over a 15 min period. The eluted protein should be collected in 2 mL fractions. If low protein recovery is observed, then the conductivity of the dialyzed protein may be too high for efficient Q-Sepharose binding. It may be necessary to return the column flow-through collected in step 3 to the dialysis step in step 6 of Subheading 3.2.5 for additional time. This will lower the conductivity of flowthrough and may result in better protein binding. 6. Fractions corresponding to the protein peak (measured via absorbance at 280 nm) should be pooled together. 7. Save a 50 μL sample of the pooled protein to determine the protein concentration and for SDS-PAGE analysis. 8. Dilute the pooled protein with a 5 volume of buffer A to reduce high salt concentration due to the Q-Sepharose elution with buffer B. 9. Flush the FPLC lines with buffer A before attaching the MonoQ column. 10. Connect the MonoQ column to the FPLC and wash the resin at 4 mL/min with 80 mL of buffer A, 80 mL of buffer B, and another 80 mL of buffer A. This will help to condition the resin and will remove any contaminates. 11. Load the diluted protein from step 8 at 2 mL/min until entire sample has been passed through the resin.

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12. Wash the loaded column at 1 mL/min with 30 mL of buffer A to dislodge any protein that has nonspecifically bound. 13. Elution of the protein should be performed at 1 mL/min using a 50% buffer B gradient over a 90 min period. The eluted protein should be collected in 2 mL fractions. 14. Fractions corresponding to the protein peak (measured via absorbance at 280 nm) should be pooled together. 15. Save a 50 μL sample of the pooled protein to determine the protein concentration and for SDS-PAGE analysis. 16. Cleaning of the MonoQ column should be conducted according to the manufacturer’s instructions. Briefly, wash the column with 30 mL of 1 M NaOH (1 mL/min), followed by 50 mL of buffer A, 50 mL of buffer B, and 50 mL of buffer A at 2–5 mL/min. If long-term storage is required, then the column should be filled with 20% (v/v) ethanol and stored at 4  C. 17. Flush the FPLC system with PBS for at least 20 mL to remove any traces of NaOH. Traces of NaOH can seriously damage a TSKgel column. 18. Connect and equilibrate the TSKgel G3000SW column on the FPLC at 0.5–1 mL/min with 300 mL of PBS (10 column volumes) (see Note 7). 19. The injection loop will be utilized for the protein loading in the next step. Prepare your protein from step 14 at 2 mg/mL and use the syringe port to load 0.5 mL per TSK run (see Note 8). 20. Collect the flow-through in 1 mL fractions. 21. Repeat steps 19 and 20 until all the protein has been passed through the column. 22. Clean the TSK column at 1 mL/min with water for 100 mL, followed by 300 mL of 20% ethanol at 0.5 mL/min. 23. Use SDS-PAGE to visualize the final purity of the purified immunotoxins. 24. Immunotoxins should be stored at 80  C for long-term storage. 3.3 Cytotoxicity/ Functional Assays 3.3.1 Cell Proliferation Inhibition

Functional testing of the immunotoxins is important to confirm proper refolding of the antibody domain and the enzymatic ribosomal inhibitory domain. Determining the concentration of immunotoxins that inhibit 50% of the cellular proliferation (IC50) can be used to establish the sensitivity of various cell lines to immunotoxin treatment or to compare the activity of different immunotoxin constructs. Cell proliferation assays are best performed in parallel with an antigen-positive cell line and an antigen-negative cell line. This helps to differentiate between antigen-specific cell killing and nonspecific cell killing.

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1. In a T75 flask, grow the antigen-positive and antigen-negative cells required to test the specificity of your immunotoxin. For testing GPC3-specific immunotoxins, we use the G1 (A431 cells stably transfected with GPC3) and A431 (epidermoid carcinoma) cell lines. Cells should be grown to 80–90% confluency to ensure cells are healthy and actively dividing. 2. Aspirate to remove used growth medium. Wash cells with PBS, then aspirate to remove dead cells. Add 3–5 mL of pre-warmed 0.25% trypsin-EDTA solution to cover the cell surface. Incubating the flask at 37  C for 5–10 min can help speed cell dissociation by trypsin digestion. 3. Resuspend the trypsinized cells with DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine. Determine the concentration of resuspended cells by using an automated cell counter or other method capable of determining cell concentration and viability. 4. Centrifuge at 300  g for 5 min or until the cells have been pelleted. 5. Aspirate the medium and resuspend cells in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine. The cell density should be 8  104 cells/mL. This will result in 5,000 cells/well when plated in the next step (see Note 9). 6. Plate 180 μL of cells per well in the middle 60 wells of a 96-well plate. 7. Incubate the cells overnight at 37  C in a 5% CO2 incubator to allow for cell attachment. 8. Prepare the immunotoxins to be tested at 10 μg/mL in PBS, then serially dilute the immunotoxins 1:3 in PBS for a total of 8 dilutions. 9. Transfer 20 μL of the diluted immunotoxins to the appropriate wells on the 96-well plate. The last set of wells should have 20 μL of PBS added to serve as the untreated control. 10. Grow the cells for 2–3 days at 37  C incubator to allow for the inhibition of cell proliferation. 11. Prepare the WST reagent by diluting it 1:10 in Opti-MEM™ reduced serum medium with no phenol red. Prepare enough reagent to plate 100 μL/well plus 2–3 extra wells (200–300 μL) to serve as background controls. 12. Incubate the plates in the 37  C incubator until a dark orange color has formed. This will generally take around 30 min; however, it can take as long as 3 h. It will depend directly on the cell density in the well and the metabolic activity of the cell lines used.

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13. Measure the absorbance of the plate at 450 nm with the reference wavelength set to 650 nm. 14. The absorbance measured in the untreated wells should be considered 100% cell proliferation and will be used to determine the level of inhibition observed in the treated wells. The percent cell proliferation should be graphed vs concentration of the immunotoxin (in ng/mL) to calculate the IC50. 3.3.2 ADP-Ribosylation Assay

Pseudomonas exotoxin inhibits ribosomal activity by modifying elongation factor 2 (EF2) through an ADP-ribosylation step. If biotinylated NAD+ is present, then EF2 will become biotinylated during the modification step. By visualizing the amount of biotinylated EF2, it is possible to gain a better understanding of the level of exotoxin activity. This assay is particularly helpful when comparing different versions of mutated exotoxins or to confirm a cell line’s sensitivity to immunotoxin treatment. 1. Add a cOmplete™ mini protease inhibitor tablet to 10 mL of ice-cold RIPA buffer. 2. Using steps 1–3 from Subheading 3.3.1 as a guide, resuspend 1  107 A431 cells and transfer to a 15 mL centrifuge tube. 3. Pellet the cells at 300  g for 5 min, then aspirate the growth medium. 4. Resuspend the cells in 100–200 μL of cold RIPA buffer and transfer to a 2 mL microcentrifuge tube. 5. Incubate the tube for 30 min on ice. Pipetting up and down several times halfway through the incubation will help to increase cell lysis efficiency. 6. Clarify the cell lysis by centrifuging at 13,000  g for 5 min. 7. Transfer clarified supernatant to a fresh tube. 8. Determine the protein concentration of the clarified supernatant and the RIPA buffer containing protease inhibitor using a NanoDrop (absorbance at 280 nm). To calculate μg of protein in cell lysates, determine the concentration of the clarified supernatant and subtract the concentration in the RIPA buffer containing protease inhibitor. 9. Prepare the reaction by combining: 4 μL of ADP-ribosylation buffer (20 mM Tris–HCl, pH 7.4, containing 1 mM EDTA), 1 μL of 1 M dithiothreitol, 1 μL of biotinylated NAD+, 5 μg of cell lysate, 20 ng of immunotoxin and water to 20 μL. The immunotoxins should be added last to assure that all the reactions run for the same amount of time. 10. Incubate the reaction at room temperature for 60 min. 11. Add 4 Laemmli buffer to stop the reaction. 12. Run an SDS-PAGE with 10–20 μL of reaction per well.

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13. Transfer the protein to a nitrocellulose membrane using a western blot sandwich system (100 W for 60 min). 14. Wash the blot with TBST for 10 min. Repeat two times. There is no need for a blocking step since the HRP-streptavidin used in the next step has a high specificity. 15. Add 1:100,000 diluted HRP-streptavidin prepared in TBST. Incubate for 30 min at room temperature with rocking. 16. Wash the blot for 15 min in TBST. Repeat two times. 17. Develop the blot with ECL reagent and image on a ChemiDoc imager. 18. Use the densitometry feature in the Image Lab software to quantify the band intensity of the modified EF2. This will be the band that corresponds to 95 kDa.

4

Notes 1. The pComb3x plasmid uses an OmpA leader sequence (MKK TAIAIAVALAGFATVAQAA). The sdAb sequence starts imme diately after the leader sequence. 2. The digestion reaction can be conducted overnight for increased digestion efficiency. 3. The ratio of insert to backbone can be adjusted between 3:1 and 8:1 depending on ligation efficiency. 4. The ligation reaction can be performed overnight to increase ligation efficiency. 5. Bacterial cultures with an OD600 over 0.6 should be diluted 1/10 in TB before reading. 6. Q-Sepharose fast flow resin has a protein-binding capacity of 20 mg/mL. 7. If this is the first time the TSK column has been used, then run 2 mg of bovine serum albumin through the column to prevent nonspecific binding of immunotoxins. 8. Up to 8 mg of protein can be run per TSK run, but this will have a negative impact on the column lifespan and may reduce protein recovery. 9. The number of cells seeded in the well will depend on how fast the cells divide. It is important that the cells not reach confluency during the 3-day incubation period. Anywhere from 2,500 to 10,000 cells can be plated per well.

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Acknowledgments This work was supported by the Intramural Research Program of NIH, Center for Cancer Research (CCR), National Cancer Institute (NCI) (Z01 BC010891 and ZIA BC010891 to M.H.). The NCI holds patent rights to anti-GPC3 antibodies including HN3 in many jurisdictions, including the United States (e.g., US Patent 9409994, US Patent 9206257, US Patent 9394364, US Patent 9932406, US Patent Application 62/716169, US Patent Application 62/369861), China, Japan, South Korea, Singapore, and Europe. Claims cover the antibodies themselves as well as conjugates that use the antibodies, such as recombinant immunotoxins, ADCs, bispecific antibodies, and modified T-cell receptors/CARs, and vectors expressing these constructs. Anyone interested in licensing these antibodies can contact the principal investigator Dr. Mitchell Ho ([email protected]) for additional information. References 1. Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363: 446–448 2. Greenberg AS, Avila D, Hughes M et al (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173 3. Desmyter A, Decanniere K, Muyldermans S et al (2001) Antigen specificity and high affinity binding provided by one single loop of a camel single-domain antibody. J Biol Chem 276:26285–26290 4. De Genst E, Silence K, Decanniere K et al (2006) Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 103: 4586–4591 5. Feng M, Bian H, Wu X et al (2019) Construction and next-generation sequencing analysis of a large phage-displayed VNAR singledomain antibody library from six naive nurse sharks. Antibd Ther 2:1–11 6. Kunz P, Zinner K, Mu¨cke N et al (2018) The structural basis of nanobody unfolding reversibility and thermoresistance. Sci Rep 8:7934 7. Vitetta ES, Uhr JW (1985) Immunotoxins: Redirecting nature’s poisons. Cell 41:653–654 8. Pastan I, Willingham MC, FitzGerald DJ (1986) Immunotoxins. Cell 47:641–648 9. Kreitman RJ, Pastan I (2020) Development of recombinant immunotoxins for hairy cell leukemia. Biomolecules 10:1140

10. Gao W, Tang Z, Zhang YF et al (2015) Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis. Nat Commun 6:6536 11. Wang C, Gao W, Feng M et al (2016) Construction of an immunotoxin, HN3-mPE24, targeting glypican-3 for liver cancer therapy. Oncotarget 8:32450–32460 12. Fleming BD, Urban DJ, Hall MD et al (2020) Engineered anti-GPC3 immunotoxin, HN3-ABD-T20, produces regression in mouse liver cancer xenografts through prolonged serum retention. Hepatology 71: 1696–1711 13. Li N, Wei L, Liu X et al (2019) A frizzled-Like cysteine-rich domain in glypican-3 mediates Wnt binding and regulates hepatocellular carcinoma tumor growth in mice. Hepatology 70: 1231–1245 14. Weldon JE, Xiang L, Chertov O et al (2009) A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity. Blood 113: 3792–3800 15. Mazor R, Zhang J, Xiang L et al (2015) Recombinant immunotoxin with T-cell epitope mutations that greatly reduce immunogenicity for treatment of mesothelin-expressing tumors. Mol Cancer Ther 14:2789–2796 16. Pastan I, FitzGerald D (1991) Recombinant toxins for cancer treatment. Science 254: 1173–1177 17. Brinkmann U, Reiter Y, Jung SH et al (1993) A recombinant immunotoxin containing a

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disulfide-stabilized Fv fragment. Proc Natl Acad Sci U S A 90:7538–7542 18. Pastan I, Ho M (2010) Recombinant immunotoxins for treating cancer. In: Kontermann R, Du¨bel S (eds) Antibody Engineering, 2nd edn. Springer, Heidelberg 19. Feng M, Gao W, Wang R, Chen W, Man Y-G, Figg WD, Wang XW, Dimitrov DS, Ho M (2013) Therapeutically targeting glypican-3 via a conformation-specific single-domain

antibody in hepatocellular carcinoma. Proc Natl Acad Sci U S A 110(12):E1083–E1091. Epub 2013 Mar 5. PMID: 23471984. PMC3607002. https://doi.org/10.1073/ pnas.1217868110 20. Flajnik MF, Dooley H (2009) The generation and selection of single-domain, V region libraries from nurse sharks. Methods Mol Biol 562:71–82

Chapter 26 X-ray Crystal Structure Analysis of VHH–Protein Antigen Complexes Angham M. Ahmed and Cory L. Brooks Abstract VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies. A detailed molecular analysis of VHH interactions with their cognate protein antigens is valuable for understanding structure–function relationships and for protein engineering. The majority of VHHs bind to folded proteins and thus recognize discontinuous three-dimensional epitopes. While multiple approaches exist for dissecting the interaction between a protein antigen and a VHH, X-ray crystallography remains the highest resolution method available. Here, we provide an updated procedure for determining and analyzing the X-ray structure of a VHH in complex with a protein antigen. We describe the recombinant expression and purification of VHHs and protein antigens, purification and analysis of protein complexes, crystallization, and optimization, X-ray structure determination by molecular replacement, and analysis of the complex. Key words Single-domain antibody, Nanobody, VHH complementarity-determining region, Crystallography, Crystal structure, VHH–protein complex

1

Introduction Heavy-chain only antibodies (HCAbs) are naturally produced by the Camelidae family (camel, llama, alpaca) and some cartilaginous sharks [1]. These antibodies were serendipitously discovered in 1989 by a student during analysis of antibodies in the serum of a dromedary camel at the Vrije Universiteit Brussel [2]. HCAbs represent 30–50% of the overall circulating antibody in the sera of these animals [2]. HCAbs are distinct from conventional IgG antibodies as they lack a light chain. A series of solubility enhancing mutations combined with defective splicing of the CH1 domain prevents the heavy-chain from pairing with the light chain [3]. The single variable domain (VHH) can be cloned and expressed independently, resulting in one of the smallest antibody fragments

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_26, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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which retains antigen binding [4]. VHHs possess a variety of advantageous structural and functional properties including: small size, stability under a range of pH and temperatures, solubility, high binding affinity, tissue penetration, and low cost of production in microbial systems [5]. The combination of these properties has made VHHs attractive tools for basic research and biomedical applications including diagnostics and therapeutics [6]. For example, a bivalent VHH (caplacizumab) for treating thrombosis has recently been approved and entered the pharmaceutical market in Europe [7] and the USA. As with other antibodies, the antigen-binding region (paratope) of a VHH is formed by the complementarity-determining region (CDR) loops. Since VHHs do not have cognate paired VL domains, the paratope is formed by three CDR loops, as opposed to the six CDR loops found in conventional antibodies. This difference in paratope arrangement results in an architecture that is either flat or convex, a geometry rarely observed in conventional antibodies [8]. Amongst the three CDR loops, CDR3 frequently dominates the VHH–antigen interaction and is capable of reaching epitopes inaccessible to conventional IgGs [6]. Determining the molecular basis of VHH–antigen-binding interactions is important for understanding structure–function relationships during the development of VHHs as biomedical tools and therapeutics [7, 9]. The majority of VHHs isolated to date bind folded protein antigens and often target discontinuous epitopes [10], which makes dissection of the VHH–antigen interaction challenging using non-structural approaches. While a variety of approaches can provide structural information on the nature of the VHH–antigen interaction, X-ray crystallography remains the primary technique for producing data at atomic-level resolution. Modern synthetic biology combined with advances in X-ray crystallography technology have made determining VHH–antigen interactions affordable and accessible. In this chapter, we provide an update on protocols used to purify VHHs and associated protein antigens, crystallize the complexes, and analyze the resulting structures [11]. The protocol assumes the researcher has already obtained a VHH from an immunized animal or an alternative source such as naı¨ve or synthetic libraries [12]. The genes encoding VHHs and protein antigens are codon optimized and produced using gene synthesis. The synthetic genes are cloned into expression vectors and both the VHH and antigen are purified using immobilized metal affinity chromatography (IMAC). VHH–antigen complexes are co-purified using size exclusion chromatography (SEC). The VHH–antigen complex is crystallized and complex formation in the crystal verified using SDS-PAGE. The crystals are harvested, X-ray diffraction data are collected, and the VHH– antigen three-dimensional complex structure is determined and analyzed.

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Materials VHH Purification

1. A synthetic VHH gene, codon optimized for Escherichia coli expression and sub-cloned into a periplasm-targeted expression vector, such as pET22b(+) (Novagen, Millipore-Sigma, Burlington, MA, USA), containing N-terminal pelB secretion signal and C-terminal His6 tag. 2. Chemically competent E. coli BL21 (DE3) cells (Novagen, Millipore-Sigma). 3. Luria–Bertani (LB) medium and plates: 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl dissolved in 1 L of dH2O. For plates, add 15 g of agar per L. Autoclave and store at room temperature or 4
C for longer periods. Prepare 6 L at a time. 4. 100 mg/mL ampicillin dissolved in dH2O. Filter sterilize, aliquot into 15 mL conical tubes, and store at 20
C. 5. 50% (v/v) glycerol. Autoclave. 6. 0.4 M isopropyl β-D-1 thiogalactopyranoside (IPTG): 1 g of IPTG dissolved in 10.5 mL of dH2O. Aliquot 1 mL of stock solution into 1.5 mL microcentrifuge tubes and store at 20
C. 7. Sorvall™ high performance centrifuge polypropylene copolymer (PPCO) centrifuge bottles (1 L), with aluminum cap. 8. 1 M Tris–HCl, pH 6.8 or 8.0: dissolve 121.1 g of Tris base in 800 mL of dH2O. Adjust the pH with HCl. Autoclave and store at room temperature. 9. 0.5 M ethylenediaminetetraacetic acid (EDTA): add 186.1 g of disodium EDTA·2H2O to 800 mL of dH2O. Adjust the pH to 8.0 with NaOH pellets and stir vigorously. Autoclave and store at room temperature. 10. Tris-EDTA-sucrose (TES) buffer: 0.2 M Tris–HCl, pH 8.0, containing 0.5 mM EDTA and 0.5 M sucrose. For a 1 L solution, dissolve 171.2 g of sucrose into 700 mL dH2O, 200 mL of 1 M Tris–HCl, pH 8.0, and 1 mL of 0.5 M EDTA. Bring to a final volume of 1 L with dH2O. Store at 4
C. 11. 0.1 M phenylmethanesulfonyl fluoride (PMSF): dissolve 871 mg of PMSF in 50 mL of isopropyl alcohol and store at 20
C. 12. Isotemp™ Hot Plate Stirrer. 13. Nalgene™ Oak Ridge high-speed PPCO centrifuge tubes (50 mL) with sealing cap. 14. Sorvall LYNX 4000 centrifuge or similar instrument. 15. Regenerated cellulose dialysis tubing, MWCO 3 kDa.

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16. HisPur™ Ni-NTA resin (Thermo Scientific, Waltham, MA, USA). 17. Conical polypropylene tubes (15 mL and 50 mL). 18. IMAC buffer A: 50 mM Tris, pH 8.0, containing 500 mM NaCl. For 1 L, dissolve 29.22 g of NaCl in 800 mL of dH2O. Add 50 mL of 1 M Tri–HCl, pH 8.0, then bring the final volume to 1 L with dH2O and store at 4
C. 19. IMAC buffer B: 50 mM Tris, pH 8.0, containing 500 mM NaCl and 1 M imidazole. For 500 mL, dissolve 14.61 g of NaCl and 34.04 g of imidazole in 300 mL dH2O, add 25 mL of 1 M Tris–HCl, pH 8.0, and bring the final volume to 500 mL with dH2O. Store at 4
C. 20. Wash buffer 1: combine 49.5 mL of IMAC buffer A with 0.5 mL of IMAC buffer B. The concentration of imidazole in this buffer is 10 mM. 21. Wash buffer 2: combine 49 mL of IMAC buffer A with 1 mL of IMAC buffer B. The concentration of imidazole in this buffer is 20 mM. 22. Elution buffer 1: combine 3.6 mL of IMAC buffer A with 0.4 mL of IMAC buffer B. The concentration of imidazole in this buffer is 100 mM. 23. Elution buffer 2: combine 3 mL of IMAC buffer A with 1 mL of IMAC buffer B. The concentration of imidazole in this buffer is 250 mM. 24. Elution buffer 3: combine 32 mL of IMAC buffer A with 2 mL of IMAC buffer B. The concentration of imidazole in this buffer is 500 mM. 25. Econo-column® chromatography column (Bio-Rad, Hercules, CA, USA) fitted with a one-way Luer Lok™ stopcock (Promega, Madison, WI, USA). 26. Rotator. 27. Dialysis buffer A: 50 mM Tris, pH 8.0, containing 300 mM NaCl. For 3.8 L, dissolve 66.62 g of NaCl in 3 L of dH2O and 190 mL of 1 M Tris–HCl, pH 8.0. Bring the final volume to 3.8 L with dH2O and store at 4
C. 28. Dialysis buffer B: 25 mM Tris, pH 8.0, containing 150 mM NaCl. For 3.8 L, dissolve 33.31 g NaCl in 3 L of dH2O and 95 mL of 1 M Tris–HCl, pH 8.0. Bring the final volume to 3.8 L with dH2O and store at 4
C. 29. NanoDrop Lite Spectrophotometer. 30. 14% (w/v) SDS-PAGE gels.

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31. 4 SDS-PAGE sample loading buffer: for 30 mL, dissolve 2.4 g of SDS and 0.12 g bromophenol blue in 6.0 mL of 1 M Tris–HCl, pH 6.8, 9.6 mL of 100% glycerol, and 2.4 mL of 14.3 M 2-mercaptaethanol. Add dH2O to 30 mL, aliquot in 1.5 mL microcentrifuge tubes and store at 20
C. 32. CostarR Mini Centrifuge or similar benchtop centrifuge. 33. Isotemp™ Digital Dry Baths/Block Heater. 34. SDS-PAGE electrophoresis chamber and PowerPac™ HC High-Current Power Supply. 35. 10 Laemmli SDS-PAGE buffer: for 2 L, dissolve 60.6 g of Tris base, 288.2 g of glycine, and 20.0 g of SDS in 1.5 L of dH2O. Bring the final volume of 2 L with dH2O. Store at room temperature. 36. PageRuler™ prestained protein ladder, 10–180 kDa. 37. Coomassie Blue staining solution: for 1 L, combine 2.5 g of Coomassie Brilliant Blue R-250 dye, 400 mL of methanol, 70 mL of glacial acetic acid, and 530 mL of dH2O. Store at room temperature. 38. Coomassie Blue destaining solution: for 3 L, combine 1590 mL of dH2O, 210 mL of glacial acetic acid, and 1200 mL methanol. Store at room temperature. 39. ChemiDoc™ MP Imaging System. 2.2 Cytoplasmic Purification of Protein Antigen

1. Synthetic gene-encoding protein antigen of interest, codon optimized for E. coli expression and sub-cloned into pET28a (+) (Novagen) or similar vector. 2. Chemically competent E. coli SHuffle® T7 Express cells (New England Biolabs, Ipswich, MA, USA). 3. LB medium: see Subheading 2.1. 4. 50 mg/mL kanamycin stock: dissolve in dH2O, aliquot into 15 mL conical tubes and store at 20
C. 5. 0.4 M IPTG: see Subheading 2.1. 6. Tris-buffered saline (TBS): 25 mM Tris, pH 8.0, containing 150 mM NaCl. For 1 L, dissolve 8.76 g of NaCl in 900 mL of dH2O and 25 mL of 1 M Tris–HCl, pH 8.0. Bring to 1 L with dH2O, autoclave, and store at 4
C. 7. Sorvall™ high performance centrifuge PPCO centrifuge bottles (1 L), with aluminum cap. 8. 0.1 M PMSF: see Subheading 2.1. 9. Model 505 Sonic Dismembrantor. 10. Nalgene™ Oak Ridge High-Speed PPCO Centrifuge Tubes 50 mL. 11. SORVALL LYNX 400 superspeed centrifuge.

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12. HisPur™ Ni-NTA resin. 13. Conical polypropylene tubes, 15 mL and 50 mL. 14. IMAC buffer A and buffer B, wash buffers 1 and 2, and elution buffers 1–3: see Subheading 2.1. 15. Econo-column® chromatography column equipped with one-way Luer Lok™ stopcocks. 16. NanoDrop Lite Spectrophotometer. 17. Dialysis buffers: see Subheading 2.1. 18. 14% SDS-PAGE gels, electrophoresis equipment, and buffers: see Subheading 2.1. 2.3 Co-purification of VHH and Protein Antigen Using SEC

1. Eppendorf 6810R swinging bucket centrifuge. 2. Amicon Ultra-4 centrifugal filter 10,000 NMWL. 3. Air-Tite™ all-plastic Henke-Ject™ syringes. 4. BD disposable syringes with Luer Lok™ tips 5. AccuSpin Microcentrifuge 17. 6. 0.45 μm Target2™ PVDF syringe filters. 7. 100 μL syringes for HPLC instruments. 8. NGC Quest™ 10 chromatography system equipped with an ENrich™ SEC 70 10  300 column (Bio-Rad). 9. Disposable culture tubes 12  75 mm borosilicate glass heavy wall. 10. NanoDrop Lite Spectrophotometer. 11. 12–14% SDS-PAGE gels, equipment, and reagents: see Subheading 2.1.

2.4 Crystal Screening and Optimization

1. Eppendorf 6810R swinging bucket centrifuge. 2. Amicon Ultra-4 centrifugal filter 10,000 NMWL. 3. AccuSpin Microcentrifuge 17. 4. Crystal Gryphon liquid handling instrument (Art Robinson Instruments, Sunnyvale, CA, USA). 5. Crystal screens: PACT premier™ (Molecular Dimensions, Maumee, OH, USA), JCSG-plus™ (Molecular Dimensions), Index HT (Hampton Research, Aliso Viejo, CA, USA), and PEGRx HT™ (Hampton Research). 6. INTELLI-PLATE® Instruments).

96-well

plates

(Art

7. AlumaSeal II sealing films (Hampton Research). 8. Adhesive PCR plate seals. 9. Fisherbrand™18 L low temperature incubator. 10. Leica M165 stereomicroscope.

Robinson

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11. 24-well plate for hanging drop vapor diffusion technique (Hampton Research). 12. Unbreakable plastic microscope cover slips. 13. High-vacuum grease. 14. Liquid nitrogen. 15. Liquid nitrogen transfer vessels. 16. 50% glycerol solution as a cryoprotectant. 17. CrystalWand™ magnetic (Hampton Research). 18. CrystalCapTM Copper HT CryoLoop (Hampton Research). 19. Universal V1-Puck (Uni-Puck) (MiTeGen, LLC, Ithaca, NY, USA). 20. Puck separator tools set (MiTeGen, LLC). 21. Puck dewar loading tool, 1 tool (MiTeGen, LLC). 22. Bent cryo tongs for puck manipulation (MiTeGen, LLC). 23. Double puck loading dewar with lid, 1 dewar (MiTeGen, LLC). 24. Shelved puck shipping cane set: shelved shipping cane, locking rod, and hooked handle (MiTeGen, LLC). 2.5 SDS-PAGE Screening for Complex Formation

1. Optimization plate containing the putative VHH–antigen complex crystals. 2. 1.5 mL microcentrifuge tubes. 3. 12% SDS-PAGE gels, gel electrophoresis equipment, and buffers: see Subheading 2.1.

2.6 X-ray Data Collection, Structure Determination, and Analysis

1. Access to a synchrotron. 2. Linux or Macbook with the following software installed: (a) No-Machine (https://www.nomachine.com) (b) XDS (http://xds.mpimf-heidelberg.mpg.de) (c) Xia2 (https://xia2.github.io/). (d) Python-based Hierarchical ENvironment for Integrated Xtallography (Phenix) software suite (https://www.phe nix-online.org/). (e) Crystallographic Object-Oriented Toolkit (COOT) software (https://www2.mrc-lmb.cam.ac.uk/personal/ pemsley/coot/). (f) PyMOL Molecular Graphics System (Schro¨dinger, LLC; https://www.pymol.org/).

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Methods

3.1 VHH Purification Using IMAC

1. Transform chemically competent E. coli BL21 (DE3) cells with 10 ng of VHH-pET22b vector by heat shock following the manufacturer’s instructions. Plate 50 μL of transformed cells on LB plates containing 100 μg/mL ampicillin and grow overnight at 37
C. 2. The next day, pick single colonies and inoculate 5 mL of LB medium containing 100 μg/mL ampicillin. After overnight growth at 37
C with 225 rpm shaking, create glycerol stocks by mixing 0.5 mL of culture with 0.5 mL of 50% sterile glycerol. Place at 80
C for long-term storage. 3. Prepare and autoclave six 4 L Erlenmeyer flasks each containing 1 L of LB medium. Also prepare and autoclave 500 mL of LB medium in a glass bottle. 4. Add 150 mL of LB medium supplemented with 100 μg/mL ampicillin to a sterile 500 mL Erlenmeyer flask. Using sterile technique, inoculate the medium with a colony from the LB plate containing 100 μg/mL ampicillin or the glycerol stock of E. coli BL21 (DE3) cells harboring the VHH-pET22b plasmid from step 2. Grow the culture overnight at 37
C with 225 rpm shaking. 5. The following day, inoculate six 4 L Erlenmeyer flasks, each containing 1 L of LB medium supplemented with 100 μg/mL ampicillin by adding 20 mL of the overnight culture from step 4 into each flask. Grow the cultures at 37
C with 225 rpm shaking until the optical density at 600 nm (OD600) reaches 0.3. Lower the temperature to 30
C and continue growing until the OD600 reaches 0.6–0.8. Induce protein expression by adding 1 mL of 0.4 M IPTG (0.4 mM final concentration) to each culture and continue growing (30
C, 225 rpm) overnight. 6. Harvest the cells by centrifugation at 4
C, 5,000  g, for 10 min. Discard the supernatant and place the tubes containing the cell pellet on ice or freeze them at 20
C for later use. 7. Extract periplasmic proteins using an adaptation of a standard osmotic shock procedure [13]. Briefly, resuspend the cell pellets from step 6 in 60 mL of ice-cold TES buffer (0.2 M Tris– HCl, pH 8.0, containing 0.5 mM EDTA, and 0.5 M sucrose) containing 0.1 mM PMSF, then transfer into a 400 mL beaker and place on ice. 8. Incubate on ice and mix continuously with a magnetic stir bar for 30 min on a stir plate. 9. Add 60 mL of ice-cold dH2O and continue mixing as in step 7 for an additional 30 min.

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10. Centrifuge the periplasmic extract at 4
C, 19,000  g, 30 min. Gently transfer the supernatant into regenerated cellulose dialysis tubing and discard the pellet. 11. Dialyze the supernatant against 3.8 L of dialysis buffer A (50 mM Tris, pH 8.0, containing 500 mM NaCl) at 4
C for 1.5 h. Transfer the dialyzed supernatant into four 50 mL conical tubes, each containing 1 mL of 50% HisPur™ Ni-NTA resin slurry. Allow the His-tagged VHH to bind the Ni-NTA resin by rotating at 4
C for 45 min. 12. Pour the Ni-NTA-protein mixture into an empty Econo-column® chromatography column (1 column volume, CV, is 2 mL) fitted with a stopcock and collect the flow-through. 13. Wash the Ni-NTA resin to remove nonspecifically bound protein by sequentially adding 30 CV (60 mL) each of IMAC wash buffers 1 and 2. Allow the wash buffer to drain by gravity flow and discard the flow-through. 14. Elute the VHH by sequentially adding 2 CV (4 mL) of IMAC elution buffers 1, 2, and 3. Collect each elution fraction separately. 15. Measure the A280 of each elution using the NanoDrop spectrophotometer and determine the concentration of the purified VHH. 16. Assess the purity of the VHH by running the post-IMAC purification collected fractions (flow-through, wash 1 and 2, and elutions 1–3) on a 14% SDS-PAGE gel. Add 10 μL of 4 SDS-PAGE reducing dye and 30 μL of each sample fraction to 1.5 mL microcentrifuge tubes. Heat the samples at 80
C for 10 min, then load 15 μL into the SDS-PAGE gel and run the gel at 200 V for 40 min. Coomassie stain and destain the gel. Image the gel using a Bio-Rad ChemiDocTM MP Imaging System or similar imaging system. The dense bands seen at ~12–15 kDa are the VHH. 17. Combine the elution fractions containing VHH and dialyze against 3.8 L of dialysis buffer B overnight at 4
C. 3.2 Antigen Purification Using IMAC

Many antigens can be expressed in the less reducing cytoplasm of E. coli SHuffle® T7 Express cells. However, some antigens may need to be produced in the periplasm or a different host (i.e., yeast or mammalian cells). 1. Transform chemically competent E. coli SHuffle® T7 Express cells with 10 ng of pET28a(+) vector encoding the protein antigen of interest following the manufacturer’s instructions. Plate 50 μL of transformed cells on LB agar plates containing 50 μg/mL kanamycin and grow overnight at 37
C.

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2. The next day, pick single colonies and inoculate 5 mL of LB medium containing 50 μg/mL kanamycin. After overnight growth at 37
C with 225 rpm shaking, create glycerol stocks by mixing 0.5 mL of culture with 0.5 mL of 50% sterile glycerol. Place at 80
C for long-term storage. 3. Prepare and autoclave six 4 L Erlenmeyer flasks each containing 1 L of LB medium. Also prepare and autoclave 500 mL of LB medium in a glass bottle. 4. Add 150 mL of LB medium supplemented with 50 μg/mL into a sterile 500 mL Erlenmeyer flask. Inoculate the medium with a single colony from the LB/Kan plate or glycerol stock of E. coli SHuffle® T7 Express cells harboring the protein antigenpET28a(+) plasmid from step 2. Grow the culture overnight at 30
C with 225 rpm shaking. 5. The following day, inoculate the six 4 L Erlenmeyer flasks each containing 1 L of LB medium supplemented with 50 μg/mL kanamycin by adding 20 mL of overnight culture from step 4 into each flask. Grow the cultures at 30
C with 225 rpm shaking until OD600 reaches 0.6–0.8. Lower the temperature to 16
C and induce protein expression by adding 250 μL of 0.4 M IPTG stock (0.1 mM final concentration) and continue growing overnight at 16
C with 225 rpm shaking. 6. Harvest the cells by centrifugation at 5,000  g, 4
C, for 10 min. Discard the supernatant and place the tubes containing the cell pellet on ice or freeze them at 20
C for later use. 7. Resuspend the cell pellets in 60 mL of ice-cold TBS buffer supplemented with 1 mM PMSF. 8. Sonicate the cell homogenate with a model 505 Sonic dismembranator using 40% amplitude with 5 s burst on and 10 s off for a total time of 10 min. Avoid foaming as it is an indication that the probe is not properly submerged into the cell mixture. 9. Centrifuge the cell lysate at 19,000  g, 4
C, 30 min. Gently transfer the supernatant into regenerated cellulose dialysis tubing while taking care to avoid disturbing the cell pellet. 10. For IMAC purification of the protein antigen, see steps 11–17 in Subheading 3.1. 3.3 Co-purification of VHH–Antigen Complex Using SEC 3.3.1 Small-Scale Co-Purification

The purpose of this experiment is to confirm that the VHH binds a protein antigen by monitoring shifts in peak retention volumes on a SEC column. The retention volumes of the following three protein samples are monitored: VHH, the protein antigen, and the VHH– antigen complex (Fig. 1a). 1. For the VHH and antigen complex formation, combine both proteins (VHH and antigen) using a 1.5 molar excess of VHH (1 antigen:1.5 VHH). For example, combine ~100 μg of VHH

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Fig. 1 Co-purification of the VHH–antigen complex by SEC. (a) Chromatogram of a trial SEC run to confirm the co-purification of a VHH–antigen complex (protein antigen: blue line, VHH: green dashed line, and VHH in complex with antigen: red line). The shift in retention volume on the chromatogram of the protein complex indicates that the VHH is bound to the antigen, forming a stable protein complex. (b) Large-scale co-purification of the VHH bound to the antigen. The large peak corresponds to the VHH–protein complex as demonstrated by SDS-PAGE (inset) where the protein antigen appears at ~30 kDa and the VHH at ~15 kDa

with 90 μg of a 40 kDa antigen. Allow the protein complex to form on ice for 3 to 4 h or overnight at 4
C. 2. Remove protein precipitate/aggregates by centrifuging and filtering the protein complex using an AccuSpin Microcentrifuge 17 (17,000  g, 1 min, room temperature) and a 0.45 μm PVDF syringe filter, respectively.

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3. Using a Bio-Rad NGC Quest 10 chromatography system, equilibrate the ENrich™ SEC 70 10  300 column with 2 CV of TBS. Separately inject 100 μL of VHH (0.2 mg/mL), antigen (0.25 mg/mL), and VHH–antigen complex (0.45 mg/mL). Elute the protein over 1 CV with the same buffer at a flow rate of 0.5 mL/min. No fractions are collected. 4. Determine if the VHH binds to its target antigen based on the peak shift and retention volume of the VHH–antigen complex compared to the peak and retention volume of antigen alone (Fig. 1a). The VHH-antigen complex is expected to elute earlier at a lower retention volume. 3.3.2 Large-Scale CoPurification

1. Once confirmed by small-scale SEC that the VHH–protein antigen complex can be separated, the complex is purified by large-scale preparative SEC (Fig. 1b). Combine both proteins (VHH and antigen) using 1.5 molar excess of VHH (1 antigen:1.5 VHH). For example, 6 mg of VHH is combined with 5.4 mg of a 40 kDa antigen. The protein complex is then incubated on ice for 3–4 h or overnight at 4
C. 2. Concentrate the VHH–antigen complex to 1 mL using an Amicon Ultra-4 centrifugal filter (NMWL ¼ 10 kDa) in a swinging bucket Eppendorf 6810R centrifuge (4,000  g, 4
C, 15 min). The final concentration should be 5–20 mg/mL. 3. Transfer the concentrated VHH–antigen protein complex into a 1.5 mL microfuge tube. Remove protein precipitate/aggregates by centrifugation and filtration using an AccuSpin Microcentrifuge 17 (17,000  g, 1 min, room temperature) and a 0.45 μm PVDF syringe filter, respectively. 4. Using a Bio-Rad NGC Quest 10 chromatography system, equilibrate the ENrich™ SEC 70 10  300 column with 2 CV of TBS buffer. Inject 1 mL of the concentrated VHH– antigen complex onto the column. The protein complex is eluted over 1 CV using a flow rate of 0.5 mL/min. Collect 0.5 mL fractions. 5. Run samples from the fractions corresponding to the major peaks on the SEC chromatogram on 12–14% SDS-PAGE (see step 16 in Subheading 3.1). Pool the fractions containing the complex and then determine the concentration of the VHH– antigen complex (Fig. 1b).

3.4 Crystal Screening and Optimization

1. Concentrate the SEC-purified VHH–antigen complex (obtained in Subheading 3.3.2) to 5–20 mg/mL using an Amicon Ultra-4 centrifugal filter (NMWL ¼ 10 kDa) in a swinging bucket centrifuge 6810R (4,000  g, 4
C, 15 min).

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2. Transfer the VHH–antigen complex to a new 1.5 mL microfuge tube. Centrifuge and filter to remove any protein aggregates using an AccuSpin Microcentrifuge 17 (17,000  g, 1 min, room temperature) and a 0.45 μm Target2™ PVDF syringe filter, respectively. 3. Crystal screening is performed using drop vapor diffusion with sparse matrix crystallization screens (Index HT and PEGRx HTTM and/or PACT premierTM and JCSG-plusTM). A Crystal Gryphon liquid handling robot is used to dispense 0.3 μL of crystal screen reagent and 0.3 μL of VHH–antigen complex (at 5–20 mg/mL) into the wells of an INTELLI-PLATE® 96 well plate. Seal the plate with Thermo adhesive PCR plate seals. Place the plate in a FisherbrandTM 18L low temperature incubator. 4. Examine the plates for crystal formation using an M165 stereomicroscope daily for 7 days, and then weekly until crystals form. 5. Once crystals form in the initial screen, an optimization step is typically required to obtain diffraction-quality crystals. Crystal optimization is carried out using the hanging drop vapor diffusion method in 24-well plates. Starting with the condition from the initial screen which yielded crystals, create an optimization screen by varying the concentration (e.g., 21–27%) of precipitant along the Y-axis of the 24 well plate, and vary the pH (e.g., 8.3–8.8) along the X-axis of the plate. Use a total volume of 0.5–1 mL for the reservoir. 6. Place 1 μL of VHH–antigen complex (at 5–20 mg/mL) and 1 μL of optimized crystal screen reagent on FisherbrandTM unbreakable plastic microscope cover slips, seal the cover slips on Hampton Research 24-well plate on Dow Corning™ Highvacuum grease and place the plate in a FisherbrandTM 18L low temperature incubator. 7. Examine the plates for crystal formation using a Leica M165 stereomicroscope daily for 7 days and then weekly until crystals form. 8. Once suitable crystals have been identified, the crystals are collected, frozen in liquid nitrogen, and stored in a crystal dewar filled with liquid nitrogen several days prior to crystal collection. 9. Place a MiTeGen puck Universal V1-Puck (Uni-Puck) into a MiTeGen double puck loading dewar with lid and add liquid nitrogen. For detailed instructions on the use of the Uni-Puck system, contact beamline staff or see the user manual created by the Stanford Synchrotron Radiation Light Source: (https:// smb.slac.stanford.edu/facilities/hardware/cryotools/Unipuck/Uni-puck_Directions.pdf)

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10. Remove the cover slip containing the VHH–antigen crystals. Under a Leica M165 stereomicroscope, add 1 μL of 50% glycerol (25% final concentration) and 1 μL of the remaining precipitant-buffer mixture solution from the optimization next to the crystal drop on the cover slip. Using the appropriate size of the Hampton Research CrystalCapTM Copper HT CryoLoop, collect a single VHH–antigen crystal, directly place it in the glycerol mixture, flash freeze the crystal in liquid nitrogen, and then place it in the crystal puck while keeping record of the crystal position in puck and crystal growth conditions. 11. Once all the VHH–antigen crystals have been frozen, use the MiTeGen bent cryo tongs for puck manipulation to cap the crystal puck and lock the cap using the locking rod. 12. Remove the dewar cap and pull out the puck from liquid nitrogen using the MiTeGen hooked handle. Quickly place the puck containing the collected crystals in the shelved shipping cane and place it back in the dewar. 3.5 VHH–Antigen Complex Screening Using SDS-PAGE

1. The purpose of this experiment is to confirm that formed crystals contain VHH–antigen complexes. Collect crystals of the VHH–antigen complex from an optimized 24-well plate and gently place them into a 2 μL drop composed of 50% mother liquor (from step 10 in Subheading 3.4) and 50% dH2O. This step will “wash” the crystals of excess protein. 2. Gently transfer the crystals to a new 10 μL drop as in step 1 and aspirate the entire mixture with a pipette into a 1.5 mL microfuge tube while viewing under the microscope. 3. Resuspend the mixture in 10 μL of 4 SDS-PAGE reducing sample loading buffer in the tube to dissolve the crystals and using an AccuSpin Microcentrifuge 17 centrifuge, centrifuge the sample at 17,000  g for 1 min at room temperature. 4. Heat the sample in Isotemp™ Hot Plate Stirrer for 10 min at 85
C and centrifuge the sample at 17,000  g for 1 min at room temperature. 5. Run the dissolved crystals on a 12% SDS-PAGE gel (see step 16 in Subheading 3.1). If the complex is present in the crystals, the gel should show two bands corresponding to the antigen (~30 kDa) and VHH (~1215 kDa) (Fig. 2).

3.6 VHH-Antigen Structure Determination and Analysis

1. X-ray diffraction data are collected remotely at synchrotron facilities. We typically use the Canadian Macromolecular Crystallography Facility (CMCF) or Stanford Synchrotron Radiation Lightsource (SSRL) for data collection. 2. Details of X-ray data collection are beyond the scope of this chapter and are presented elsewhere [14]. The non-expert is encouraged to consult with synchrotron beamline staff for

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Fig. 2 SDS-PAGE screening of crystals for the presence of VHH–antigen complex. Crystals are dissolved and run on SDS-PAGE to verify the presence of VHH-antigen complex. Lane 1: molecular weight marker; lane 2: dissolved crystal demonstrating presence of protein antigen (~30 kDa) and VHH (~15 kDa). The crystals were collected, washed of excess protein, dissolved, heated, and ran on 12% SDS-PAGE. The image of the gel shows the presence of two bands in the same lane (lane 2) at the molecular weight that corresponds to the antigen (~30 kDa) and VHH (~15 kDa) and confirms the presence of the VHH–antigen complex in the crystal

assistance with data collection. With modern beamlines equipped with pixel detectors, X-ray data can be collected at a full 360-degree in a few minutes. 3. Once the X-ray diffraction data has been collected, the data can be processed using XDS (http://xds.mpimf-heidelberg. mpg.de), xia2 (https://xia2.github.io/), or HKL2000 (https://www.hkl-xray.com). 4. X-ray structure determination of the VHH–antigen complex can frequently be accomplished using molecular replacement [15]. The method requires high quality models of the VHH and protein antigen structure. 5. To select appropriate models for molecular replacement, search the Protein Data Bank using Protein BLAST and select models for the VHH and antigen with the highest degree of sequence identity. There will be many suitable VHH models. If only low sequence homology models of the protein antigen are available, in some cases molecular replacement can be achieved using predicted models generated in Rosetta [16].

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Fig. 3 Representative sample output of PDBsum analysis of VHH–antigen interactions. The output displays a summary table of interacting surface areas and interaction types, a graphical representation of the sum of the interactions with antigen (red circle) and VHH (light blue circle) shown, the total number and type of contacts and a detailed list of each interacting residues

6. To prepare the models for molecular replacement, download the structure files of both the VHH and the protein antigen. Using PyMOL, remove extra copies of the protein molecules, ligands, ions, and water molecules. We have found it useful to remove the CDR3 sequence from the VHH as it can introduce packing clashes during the molecular replacement search. 7. X-ray data quality is assessed using Xtriage as implemented in Phenix [17]. The program checks for issues associated with

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Fig. 4 Structural analysis of VHH CDR loop interactions with antigen. Examination of polar contacts between the CDR loops found on the VHH (yellow) and the protein antigen (pink) using PyMOL

data quality such as outliers, presence of translational non-crystallographic symmetry, twining, and other pathologies. 8. Molecular replacement is carried out using Phaser by inputting the following files: experimental diffraction data (.mtz file), the molecular replacement search models (.pdb files), and the sequence files of both the VHH and antigen. If the VHH– antigen complex structure is solved, the TFZ-score will be 8 or higher. The structure is then refined in Phenix using 5–10 cycles. 9. After the initial refinement, automated model building can be carried out using Phenix and the VHH–antigen complex structure is manually fitted to the electron density maps using the modeling software Coot [18]. Further refinement cycles are carried out in Phenix to obtain a structure that accurately fits the electron density map. Model quality is assessed by a variety of metrics including R factors, clash score, Ramachandran outliers, sidechain outliers, and RSRZ outliers [19]. 10. Once the structure of the VHH–antigen complex has been modeled, automated analysis of the interactions between the VHH and the antigen can be performed using PDBsum (http://www.ebi.ac.uk/pdbsum) [20]. The analysis provides a variety of information, including a graphical representation

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summarizing the interacting interface between the VHH and antigen, a detailed summary of all interfacing residues and their contacts, and a summary table of the total interaction surface area and type of polar contacts involved in the interaction (Fig. 3). 11. A detailed structural examination of the interface is then carried out using PyMOL. Taking the summary information from PDBsum, the crystallographer can examine the interactions of the CDR loops and determine which interactions are involved in protein–protein interaction and which may simply be due to crystal packing. PyMOL can also be used to create publication quality representations of the VHH–antigen interface (Fig. 4).

References 1. Muyldermans S (2020) Applications of nanobodies. Annu Rev Anim Biosci 9:401–421 2. Arbabi-Ghahroudi M (2017) Camelid singledomain antibodies: historical perspective and future outlook. Front Immunol 8:1589 3. Vincke C, Muyldermans S (2012) Introduction to heavy chain antibodies and derived nanobodies. Methods Mol Biol 911:15–26 4. Fang T, Duarte JN, Ling J et al (2016) Structurally defined αMHC-II nanobody-drug conjugates: a therapeutic and imaging system for B-cell lymphoma. Angew Chem Int Ed Engl 55:2416–2420 5. Henry KA, MacKenzie CR (2018) Editorial: single-domain antibodies – biology, engineering and emerging applications. Front Immunol 9:41 6. Yang EY, Shah K (2020) Nanobodies: next generation of cancer diagnostics and therapeutics. Front Oncol 10:1182 7. Akiba H, Tamura H, Kiyoshi M et al (2019) Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody. Sci Rep 9:15481 8. Henry KA, MacKenzie CR (2018) Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs 10: 815–826 9. Gershoni JM, Roitburd-Berman A, DimanTov DD et al (2007) Epitope mapping: the first step in developing epitope-based vaccines. BioDrugs 21:145–156 10. Forsstro¨m B, Axn€as BB, Rockberg J et al (2015) Dissecting antibodies with regards to linear and conformational epitopes. PLoS One 10:e0121673

11. Toride King M, Brooks CL (2018) Epitope mapping of antibody-antigen interactions with X-ray crystallography. Methods Mol Biol 1785:13–27 12. White B, Huh I, Brooks CL (2019) Structure of a VHH isolated from a naive phage display library. BMC Res Notes 12:154 13. Quan S, Hiniker A, Collet J-F et al (2013) Isolation of bacteria envelope proteins. Methods Mol Biol 966:359–366 14. Dauter Z (2017) Collection of X-ray diffraction data from macromolecular crystals. Methods Mol Biol 1607:165–184 15. Evans P, McCoy A (2008) An introduction to molecular replacement. Acta Crystallogr D Biol Crystallogr 64:1–10 16. DiMaio F (2017) Rosetta structure prediction as a tool for solving difficult molecular replacement problems. Methods Mol Biol 1607: 455–466 17. Liebschner D, Afonine PV, Baker ML et al (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75:861–877 ˜ al A, Lohkamp B, Emsley P (2020) Cur18. Casan rent developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci 29: 1069–1078 19. Read RJ, Adams PD, Arendall WB 3rd et al (2011) A new generation of crystallographic validation tools for the protein data bank. Structure 19:1395–1412 20. Laskowski RA, Jabłon´ska J, Pravda L et al (2018) PDBsum: structural summaries of PDB entries. Protein Sci 27:129–134

Chapter 27 Functionalization of Magnetic Beads with Biotinylated Nanobodies for MALDI-TOF/MS-Based Quantitation of Small Analytes Gabriel Lassabe, Macarena Pı´rez-Schirmer, and Gualberto Gonza´lez-Sapienza Abstract Over the last two decades, the variable domains from heavy chain-only antibodies in camelids (nanobodies) have emerged as valuable immunoreagents for analytical and diagnostic applications. One prominent use of nanobodies is for the detection of small molecules due to their ease of production, resistance to solvents used in sample extraction, facile genetic manipulation, and small size. These last two properties make it possible to produce biotinylated nanobodies in vivo, which can be loaded in an orientated manner on magnetic beads covered with avidin, creating high-density immunoadsorbenpi twbch ""ts. The method described here details the use of nanobody-based adsorbents to concentrate small molecular weight analytes for subsequent quantitative analysis by MALDI-TOF mass spectrometry. Quantitation requires the inclusion of an internal standard (IS), a compound with properties similar to those of the analyte, enabling compensation for uneven distribution during crystallization of the MALDI-TOF matrix. Since nanobody generation against small compounds requires conjugation to carrier proteins, the same conjugation chemistry can be used to synthesize the IS. By design the IS cross reacts with the capture nanobody and can be preloaded in the immunoadsorbent, facilitating quantitative detection of the target compound. Key words VHH, Nanobody, Immunoconcentration, Immunoadsorbent, Streptavidin, Quantitative MALDI-TOF, Hapten, Internal standard

1

Introduction Mass spectrometry (MS) has been broadly applied for the identification and characterization of small molecular weight compounds. Although a powerful technique, the ability of MS to identify target compounds can be limited by the complexity of the sample and matrix. Hence, the combination of MS with chromatographic purification systems, such as high performance liquid chromatography (HPLC) has become the main instrumental analytical method for quantitative analysis of trace compounds [1, 2]. These methods

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_27, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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have high sensitivity, but in the case of very low abundance compounds, often require the concentration of large sample volumes and cumbersome clean-up steps. This can be costly and disadvantageous when a large number of samples requires analyses [3, 4]. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a simple to use highthroughput technique, which requires minimal sample preparation and has a high tolerance to matrix interference. The method is highly sensitive, with sub-femtomole detection limits, and in reflectron mode achieves exquisite ion resolution allowing precise mass identification of a compound. Due to irregular crystallization of the matrix during sample preparation, the analyte tends to be unevenly distributed along the target spot, making quantitative applications of MALDI-TOF MS difficult [5]. This can be overcome by the use of an internal standard (IS), preferably an isotope-labeled variant of the analyte, or more practically, molecules with similar physicochemical properties to the target analyte [6–8]. Depending on the matrix, the method produces ions in the low m/z range that can make the interpretation of the spectra of low molecular weight compounds difficult. This problem can be circumvented by changes in the matrix or by replacing the matrix with nanoparticles [9] or surface-assisted laser desorption MS [10]. Previously, we reported a new method for highly sensitive quantitation of small molecules by combining the analytical power of MALDI-TOF MS with a simple and fast purification/ concentration step driven by magnetic beads (MBs) coated with high affinity nanobodies (Fig. 1) [11]. The small size of nanobodies (~15 kDa) and the possibility of manipulating their genetic sequence to allow for in vivo biotinylation permits tightly packed and orientated immobilization of nanobodies on streptavidincoated beads, resulting in highly efficient immunoadsorbents. After immunoconcentration, the beads are directly mixed with the matrix and transferred to the MALDI-TOF MS target plate where the laser energy disrupts the nanobody–analyte interaction without the need for an elution step. Using these reagents, we selectively purified and concentrated trace amounts of microcystins (the main toxic metabolites produced by cyanobacteria) from water and other complex untreated samples such as serum, thereby enhancing the sensitivity of MALDI-TOF MS and simplifying the workflow by avoiding clean-up steps. Moreover, the beads can be loaded with an optimized amount of IS as a ready-to-use reagent for quantitative measurements. This is facilitated by using an IS that is typically an isotope-labeled or analyte analog, which will also be bound by the anti-analyte nanobody. The method is particularly useful for analytes that belong to a family of congeners sharing a common structure that can be recognized by the same nanobody. This is the case for microcystins: a nanobody loaded on beads reacts with the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-

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Fig. 1 Schematic overview of the method for functionalization of magnetic beads with biotinylated nanobodies for MALDI-TOF MS-based quantitation of small molecular weight analytes. Initially, the gene encoding an antianalyte nanobody is cloned into the pINQ-BtH6 expression vector and transformed into E. coli cells harboring a second vector pCY216 to produce the in vivo biotinylated nanobody (Nb). The purified nanobody is then bound to streptavidin-coated magnetic beads and preloaded with an internal standard (IS) to generate the ready-touse immunoadsorbent. For the analysis, the analyte in the sample is captured on the beads, thoroughly washed, resuspended, and directly loaded in the MALDI-TOF MS for analysis

4,6-dienoic acid group that is present in different variants of these toxins. Therefore, all toxin variants present in a sample will be immunoconcentrated and detected. Here, we present a detailed protocol for the generation of nanobody-based immunoadsorbent beads and their use in combination with MALDI-TOF MS for the quantification and identification of toxins, drugs, or other small compounds, preferably in the 800–4,000 Da range. This is the optimal window for MALDI-TOF MS using the alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix, but this range can be extended to smaller analytes by modifying the conditions as mentioned above. This protocol involves a workflow that was originally designed for the detection of microcystin congeners, but it can be applied to other analytes providing that the corresponding nanobody is available or can be

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developed. The protocol presented here assumes a target-specific nanobody is available and includes the following steps: (1) cloning, expression, and purification of the nanobody with a biotin moiety at the C-terminus, (2) generation of the immunoadsorbent by immobilization of the nanobody on streptavidin MBs and loading of the IS, (3) capture and immunoconcentration of the analyte, and (4) analysis by MALDI-TOF MS.

2

Materials The pINQ-BtH6 plasmid, derived from pET-28a(+) (Novagen, Merck, Darmstadt, Germany) and containing the expression cassette shown in Fig. 2 downstream of the lac operator and ribosomebinding site, is used for nanobody expression and in vivo biotinylation.

2.1 Nanobody Cloning into the pINQBtH6 Expression Vector

1. pCY216, BirA (Escherichia coli biotin ligase) expression vector (GenBank: AAD22470.1). This plasmid construct is freely available to researchers by request to the corresponding authors [12]. 2. The pET-28a(+)-derived expression vector pINQ-BtH6. This plasmid is freely available to researchers by request to the corresponding authors (Fig. 2). 3. QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). 4. QIAquick Gel Extraction Kit (Qiagen). 5. QIAquick PCR Purification Kit (Qiagen). 6. Forward primer FwSfiI: 50 - GGCCCAGGCGGCC N20-30 , where N20 corresponds to the first 20 nucleotides at the 50 end of the VHH. 7. Reverse primer RvSfiI: 50 - GGCCGGCCTGGCC N20-30 , where N20 corresponds to the reverse complement of the last 20 nucleotides at the 30 end of the VHH. 8. OmpA seq primer: 50 -GGCCGGCCTGGCC-30 . 9. Sterile MilliQ H2O. 10. Taq DNA polymerase, 10 Taq Buffer, 25 mM MgCl2, and 2.5 mM dNTP mix (Thermo Fisher Scientific, Waltham, MA, USA). 11. Thermal cycler. 12. Vacuum concentrator. 13. SfiI restriction endonuclease and 10 buffer G (Thermo Fisher). 14. T4 DNA ligase and 10 ligase buffer (Thermo Fisher). 15. Agarose.

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Fig. 2 Schematic illustration of the expression cassette used for in vivo biotinylation of nanobodies. The two SfiI restriction endonuclease sites are non-complementary allowing directional cloning of the nanobody (VHH) gene and are identical to those found in the pComb3x vector. RBS, ribosome-binding site; OmpA leader sequence, MKKTAIAIAVALAGFATVAQA; Biotin tag, GLNDIFEAQKIEWHE (E. coli biotin ligase peptide substrate sequence); His6 tag, 6 histidine. The pINQ-BtH6 vector can be requested from our laboratory or it can be assembled by inserting the synthetic gene for the OmpA-nanobody-biotin-tag into the XbaI and XhoI sites of the pET-28a(+) vector (Novagen)

16. 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8.0: add 186.1 g of disodium EDTA·2H2O to 800 mL of H2O. While stirring, adjust the pH to 8.0 with NaOH pellets. Autoclave. 17. 50 TAE: 2 M Tris-acetate, 50 mM EDTA. Dissolve 242 g of Tris base in 800 mL of deionized water, add 57.1 mL of glacial acetic acid and 100 mL of 0.5 M EDTA, pH 8.0, and adjust the volume to 1 L. 18. 10,000 GelRed Nucleic Acid Stain. 19. 1% (w/v) and 2% agarose gels: Add 1 or 2 g of agarose to 100 mL of 1 TAE buffer. Heat in a microwave until the agarose is completely dissolved. Let the solution cool to 60  C, add 10 μL of 10,000 GelRed Nucleic Acid Stain, and pour into the electrophoresis gel cast. 20. Electrocompetent E. coli DH5α cells (Thermo Scientific) for plasmid amplification. 21. Electrocompetent E. coli BL21(DE3) cells (Novagen). 22. SOB medium: dissolve 20 g of tryptone, 5 g of yeast extract, and 0.5 g of NaCl in 1 L of deionized H2O. Adjust pH to 7.0 if required and autoclave for 20 min at 121  C. 23. Super Optimal broth with Catabolite repression (SOC): To 1 L of SOB medium, add 10 mL of 1 M MgCl2 (autoclaved) and 20 mL of 20% (w/v) glucose (filter sterilized). For long-term

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storage, dispense into 50 mL aliquots and freeze at 80  C. Prepare single use aliquots of 1 mL and store at 20  C. 24. Electroporation instrument. 25. Electroporation cuvettes, 0.2 cm gap. 26. 50 mg/mL kanamycin, filter sterilized. 27. 35 mg/mL chloramphenicol, filter sterilized. 28. Luria Broth (LB) plates: Dissolve 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 15 g of agar in 1 L of deionized H2O. Autoclave for 20 min at 121  C, let the solution to cool to 55  C, then supplement with 100 μg/mL ampicillin and 35 μg/mL chloramphenicol. 2.2 Expression of Biotinylated Nanobodies

1. LB medium: Dissolve 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl in 1 L of deionized H2O. Autoclave for 20 min at 121  C. 2. 40% (v/v) glucose stock solution: dissolve 40 g of D-glucose in 50 mL of deionized H2O while heating and stirring. Bring volume to 100 mL with H2O, filter sterilize and store at 4  C. 3. 50 mg/mL kanamycin, filter sterilized. 4. 35 mg/mL chloramphenicol, filter sterilized. 5. 20% (w/v) L-arabinose stock solution: dissolve 20 g of L-arabinose in 50 mL of deionized H2O while stirring. Bring volume to 100 mL with H2O, filter sterilize, and store in aliquots of 1 mL at 20  C. 6. 100 mM biotin stock solution: Equilibrate biotin to room temperature before opening. Dissolve 244 mg of D-biotin in 10 mL of deionized H2O. Filter sterilize and store in 1 mL aliquots at 20  C. 7. 0.5 M isopropyl β-D-1-thiogalactopyranoside (IPTG) stock solution: Equilibrate IPTG to room temperature before opening. Dissolve 1.19 g of IPTG in 10 mL of deionized H2O. Filter sterilize and store in 1 mL aliquots at 20  C for up to 1 year. 8. Centrifuge. 9. Sonicator. 10. 1 phosphate-buffered saline (PBS): dissolve 8 g of NaCl, 0.2 g of KCl, 0.24 g of KH2PO4, and 1.44 g of Na2HPO4 in 900 mL of deionized H2O while stirring. Adjust the pH to 7.4 with NaOH and bring volume to 1 L with H2O. Autoclave. 11. HisTrap™ HP 1 mL Ni-NTA column. ¨ KTA fast protein liquid chromatography (FPLC) purification 12. A system or similar. 13. Buffer A: dissolve 6 g of NaH2PO4, 17.53 g of NaCl, and 1.36 g of imidazole in 900 mL of deionized H2O while

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stirring. Adjust the pH to 8.0 and bring volume to 1 L with H2O. Filter through 0.22 μm filters. 14. Buffer B: dissolve 6 g of NaH2PO4, 17.53 g of NaCl, and 34 g of imidazole in 900 mL of deionized H2O while stirring. Adjust the pH to 8.0 and bring volume to 1 L with H2O. Filter through 0.22 μm filters. 15. Cellulose dialysis membrane, cut off 100) which express the Pep-tagged antigen as well as control cells. Export individual CB and background fluorescence values from each image to a data analysis software. Calculate the mean CB fluorescence intensities and standard deviation after subtraction of the background fluorescence. Test the resulting data set for statistical significance. 3.8 Characterizing PepCB Interactions in Live Cells: FRAP

The following protocol outlines a previously documented FRAP experiment [31]. 1. Seed 8
103 U2OS cells (from step 6 of Subheading 3.4) in 100 μL of DMEM supplemented with 10% FCS and 1% penicillin/streptomycin in each well of a 96-well SCREENSTAR™

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plate. Transfect with plasmid DNAs encoding both GFP-actin and actin-CB [35] or PepCB and PepActin using the method described in steps 8–13 of Subheading 3.4. 2. Perform the FRAP recordings on a Zeiss Confocal Laser Scanning Microscope at 63
magnification using a 488 nm argon laser. The software can be configured to bleach a region of interest for the desired time interval. In our hands, 20% laser output and 100% transmission is sufficient to bleach a 2.2
2.2 μm region of interest for 2.4 s. Set the microscope control to acquire prebleach and postbleach images at 1% laser transmission and the pinhole opened to 1.5 Airy units (see Note 12). 3. Use ImageJ to extract the fluorescence intensity values and export into a data analysis software. Calculate mean fluorescence values after correcting for background signal and loss of fluorescence over time (see Note 12). 4. To determine the half-time of recovery, first export the normalized mean fluorescence values into Origin 7.5 and plot the fluorescent recovery curves. Execute a curve fit using an exponential function. The exponential function is given as:
I ðt Þ ¼ A 1  e kt , where I(t) is the signal intensity dependent on time, A is the value of intensity at the endpoint, and k is the time constant. The half time of recovery t1/2 is then determined by: ln 0:5 k 3.9 Live-Cell Imaging of PepActin using PepCB to Monitor the Effects of Compound Treatment on the Cytoskeleton

Previously, we have documented the potential of the PepCB to trace endogenous antigens comprising a PepTag. As shown in Fig. 5, we demonstrated over time the effects of treatment with cytochalasin D, an actin polymerization inhibitor, on the reorganization of actin fibers using time-lapse imaging of U2OS cells stably expressing the PepCB in combination with transiently transfected PepActin [31]. The following protocol outlines the steps necessary for such experiments. 1. Seed 8
103 cells (U2OS_E02, the stable monoclonal cell lines generated in Subheading 3.5 and validated in Subheading 3.6) in each well of a 96-well black μClear® plate. 2. Transfect U2OS_E02 cells with the PepActin expression construct from Subheading 3.2 using the method described in Subheading 3.4. 3. Follow the post-transfection guidelines described in steps 11– 13 of Subheading 3.4.

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Fig. 5 PepCB can be used to visualize the distribution and reorganization of Pep-tagged proteins in living cells upon treatment with exogenous compounds. Shown are representative fluorescence images of U2OS_E02 cells transiently expressing PepActin displaying the PepCB signal at indicated time points. Immediately after starting the time series cells were treated with 2 μM cytochalasin D (actin polymerization inhibitor) for 10 min. Subsequently, cytochalasin D was removed and cells were continuously imaged for additional 30 min. Scale bar 50 μm. Images were taken from [31] with permission

4. Adjust the environmental settings of the ImageXpress Micro XL system (see step 3 in Subheading 3.7) about 1 h before imaging. Program the time-lapse imaging settings. 5. Identify wells with 50–75% transfection efficiency of PepActin by microscopy (see Note 13). Use this time to set the exposure time, focus, and imaging coordinates of the microscope. 6. Using the 40
magnification, acquire pre-treatment images of the designated wells (see Note 13). 7. Prepare a 2 μM dilution of cytochalasin D or dimethyl sulfoxide (control) in DMEMgfp2 anti-bleaching live-cell visualization medium. 8. Exchange the medium in the designated wells to either the treatment or control medium. 9. Start the time-lapse imaging immediately. Acquire every site at 5 min intervals (see Note 14). 10. After 10 min, change the treatment or control medium in the designated wells to DMEMgfp2 anti-bleaching live-cell visualization medium. 11. Continue the time-lapse imaging immediately. Acquire every site at 5 min intervals for as long as needed.

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Notes 1. Since the PepNb cDNA sequence was produced by gene synthesis, the sequence was modified to contain a BglII restriction site a few nucleotides upstream of the start codon to facilitate easy cloning into our array of bacterial and mammalian expression vectors. The BstEII recognition site is in the conserved FR4 (30 end). The cDNA sequence of PepNb is shown, with BglII and BstEII restriction sites bolded and underlined:

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agatctccggccatggctgacgtgcagctgcaggagagcggcggcggcctgg tgcagcccggcggcagcctgaggctgagctgcgccgccagcggcaacatcgtgag catcgacgccgccggctggttcaggcaggcccccggcaagcagagggagcccgtg gccaccatcctgaccggcggcgccaccaactacgccgacagcgtgaagggcaggtt caccatcagcagggacaacgccaagaacaccgtgtacctgcagatgaacagcctgaa gcccgaggacaccgccgtgtactactgctacgcccccatgatctactacggcggca ggtacagcgactactggggccagggcacccaggtcacc. This cDNA is available from our lab. Regarding the backbone fragment, pure DNA preparations of chromobody expression vectors [8, 35] are available on request from our lab. Depending on the desired application, other fluorescent protein vectors may also be considered. 2. The control plate (vector-only ligation) should have significantly fewer colonies than the vector + insert plates. Contrary results are indicative of inefficient restriction digestion of the vector. In this case, the digestion should be repeated with confirmation by agarose gel electrophoresis. 3. Plasmid preparations for mammalian cell transfections need to be endotoxin-free. 4. The annealing temperature is dependent on the primer pair used. Loading 5 μL of the PCR product on a 1% agarose gel is advised to ascertain if the amplification was effective before proceeding to the next step. The expected amplicon sizes are 5,136 bp (PepActin) and 6,082 bp (GFP-PCNAPep). For the KLD treatment, we have found that using 2 μL of the PCR product increases the chances of obtaining positive clones. We also recommend incubation times for the KLD treatment of a minimum of 30 min. In our hands, longer incubation times correlate with higher blunt-end ligation success rates. After transformation, successful mutation can be initially verified by colony PCR using one primer complementary to the inserted PepTag and the other primer annealing on the vector backbone. However, this is not necessary as DpnI destroys the “mother” vector and only the amplicons are ligated. 5. Digestion with XbaI may be affected by Dam methylation. Thus, we recommend that the plasmid DNA should be prepared from cultures of dam - E. coli strains (e.g., dam / dcm  competent E. coli cells from NEB). 6. The PstI recognition site exists in the FR1 region of most Nbs. The BspEI restriction enzyme cuts in the vector backbone just before the (Gly4Ser)3 linker connecting the Nb to the fluorescent protein. We routinely use these restriction sites when shuttling Nbs in our mammalian expression vectors. A different choice of restriction enzymes may be necessary depending on the target Nb and vector sequence. The similar size of the

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ActinNb and PepNb-coding sequence makes it difficult to identify positive clones by colony PCR. Clones can be randomly selected and verified by Sanger sequencing. 7. Prolonged incubation may lead to degradation of DNA-Lipofectamine™ complexes and thereby reduce overall transfection efficiency. We recommend including a positive transfection control such as a mammalian expression plasmid-encoding GFP. A control for the PepCB intracellular binding, such as the expression construct for a protein of interest lacking the PepTag, should also be included. 8. Prepare a killing curve to determine the minimum antibiotic concentration needed to deplete all non-transfected cells first before proceeding with stable cell line generation. 9. Cells should be split into new plates if they become fully confluent. Some aliquots can also be frozen as polyclonal back-up stocks at this point. Most cells lacking the transfected plasmid should be dead by up to 9 days after transfection. 10. Ensure that you acquire a good quality image. Over- and under-exposed images can introduce bias when acquiring images for quantification. 11. If the protein is nuclear, a reference image from the DAPI channel is sufficient to create the nuclear filter mask. For other cell structures, staining with an appropriate organelle marker is advised. When creating the custom module, selecting an optimal width range and minimum fluorescence above local background are important to limit bias. 12. Five pre-bleach and 145 post-bleach images have been found to be sufficient in our hands. For statistical significance, more than 10 replicate acquisitions are done for each construct. 13. We noticed that selecting wells with more cells showing clear structural features of the exogenous protein (e.g., PepActin) improves the quality of visual data obtained during compound treatment. Though the imaging media prevents bleaching, take care not to over-expose images before starting the time-lapse series. 14. Consider acquisition time and delays when programming the time-lapse series. Selecting too many imaging sites may prolong the time interval between images. References 1. Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448 2. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797

3. Kaiser PD, Maier J, Traenkle B et al (2014) Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells. Biochim Biophys Acta 1844:1933–1942

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4. Helma J, Cardoso MC, Muyldermans S et al (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209:633–644 5. Rothbauer U, Zolghadr K, Tillib S et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3: 887–889 6. Van Audenhove I, Van Impe K, Ruano-Gallego D et al (2013) Mapping cytoskeletal protein function in cells by means of nanobodies. Cytoskeleton 70:604–622 7. Zolghadr K, Mortusewicz O, Rothbauer U et al (2008) A fluorescent two-hybrid assay for direct visualization of protein interactions in living cells. Mol Cell Proteomics 7: 2279–2287 8. Zolghadr K, Gregor J, Leonhardt H et al (2012) Case study on live cell apoptosis-assay using lamin-chromobody cell-lines for highcontent analysis. Methods Mol Biol 911: 569–575 9. Pellis M, Pardon E, Zolghadr K et al (2012) A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional nanobodies. Arch Biochem Biophys 526: 114–123 10. Moutel S, Bery N, Bernard V et al (2016) NaLi-H1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. eLife 5:e16228 11. Wagner TR, Rothbauer U (2020) Nanobodies right in the middle: intrabodies as toolbox to visualize and modulate antigens in the living cell. Biomolecules 10:1701 12. Rothbauer U, Zolghadr K, Muyldermans S et al (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7: 282–289 13. Kirchhofer A, Helma J, Schmidthals K et al (2010) Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol 17:133–138 14. Ries J, Kaplan C, Platonova E et al (2012) A simple, versatile method for GFP-based superresolution microscopy via nanobodies. Nat Methods 9:582–584 15. Schornack S, Fuchs R, Huitema E et al (2009) Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J 60: 744–754 16. Caussinus E, Kanca O, Affolter M (2011) Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat Struct Mol Biol 19:117–121 17. Traenkle B, Rothbauer U (2017) Under the microscope: single-domain antibodies for live-

cell imaging and super-resolution mcroscopy. Front Immunol 8:1030 18. Meyer T, Begitt A, Vinkemeier U (2007) Green fluorescent protein-tagging reduces the nucleocytoplasmic shuttling specifically of unphosphorylated STAT1. FEBS J 274: 815–826 19. Huang Z, Zhang C, Chen S et al (2013) Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility. Microb Cell Fact 12:25 20. Zhang F, Moniz HA, Walcott B et al (2014) Probing the impact of GFP tagging on Robo1heparin interaction. Glycoconj J 31:299–307 21. Montecinos-Franjola F, Bauer BL, Mears JA et al (2020) GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission. Sci Rep 10:14,777 22. Hosein RE, Williams SA, Haye K et al (2003) Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila. J Eukaryot Microbiol 50: 403–408 23. Snapp EL (2009) Fluorescent proteins: a cell biologist’s user guide. Trends Cell Biol 19: 649–655 24. Jarvik JW, Telmer CA (1998) Epitope tagging. Annu Rev Genet 32:601–618 25. Brizzard B (2008) Epitope tagging. Biotechniques 44:693–695 26. Braun MB, Traenkle B, Koch PA et al (2016) Peptides in headlock—a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Sci Rep 6:19,211 27. Go¨tzke H, Kilisch M, Martı´nez-Carranza M et al (2019) The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat Commun 10:4403 28. De Genst EJ, Guilliams T, Wellens J et al (2010) Structure and properties of a complex of alpha-synuclein and a single-domain camelid antibody. J Mol Biol 402:326–343 29. De Genst E, Silence K, Decanniere K et al (2006) Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 103: 4586–4591 30. Pardon E, Laeremans T, Triest S et al (2014) A general protocol for the generation of nanobodies for structural biology. Nat Protoc 9: 674–693 31. Traenkle B, Segan S, Fagbadebo FO et al (2020) A novel epitope tagging system to

Peptide-Tag Specific Nanobodies for Studying Proteins in Live Cells visualize and monitor antigens in live cells with chromobodies. Sci Rep 10:1–13 32. Strokappe NM, Hock M, Rutten L et al (2019) Super potent bispecific llama VHH antibodies neutralize HIV via a combination of gp41 and gp120 epitopes. Antibodies (Basel) 8:38 33. Keller BM, Maier J, Secker KA et al (2018) Chromobodies to quantify changes of endogenous protein concentration in living cells. Mol Cell Proteomics 17:2518–2533 34. Keller BM, Maier J, Weldle M et al (2019) A strategy to optimize the generation of stable chromobody cell lines for visualization and

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quantification of endogenous proteins in living cells. Antibodies (Basel) 8:10 35. Panza P, Maier J, Schmees C et al (2015) Live imaging of endogenous protein dynamics in zebrafish using chromobodies. Development 142:1879–1884 36. Virant D, Traenkle B, Maier J et al (2018) A peptide tag-specific nanobody enables highquality labeling for dSTORM imaging. Nat Commun 9:930 37. Leonhardt H, Rahn HP, Weinzierl P et al (2000) Dynamics of DNA replication factories in living cells. J Cell Biol 149:271–280

Chapter 30 Nanobody-Based GFP Traps to Study Protein Localization and Function in Developmental Biology Shinya Matsuda, Gustavo Aguilar, M. Alessandra Vigano, and Markus Affolter Abstract Synthetic protein-binding tools based on anti-green fluorescent protein (GFP) nanobodies have recently emerged as useful resources to study developmental biology. By fusing GFP-targeting nanobodies to wellcharacterized protein domains residing in discrete sub-cellular locations, it is possible to directly and acutely manipulate the localization of GFP-tagged proteins-of-interest in a predictable manner. Here, we describe a detailed protocol for the application of nanobody-based GFP-binding tools, namely Morphotrap and GrabFP, to study the localization and function of extracellular and intracellular proteins in the Drosophila wing imaginal disc. Given the generality of these methods, they are easily applicable for use in other tissues and model organisms. Key words Nanobody, GFP, Morphotrap, GrabFP, Protein binders, Drosophila

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Introduction A fundamental question in developmental biology is how proteins function in large networks to control a variety of cellular processes. However, it remains challenging to dissect the precise molecular role of proteins due to the lack of appropriate tools to directly manipulate proteins. In recent years, protein binders have emerged as versatile tools to study protein localization and function during development. Single chain variable fragments (scFvs), nanobodies (VHHs), Designed Ankyrin Repeat Proteins (DARPins), and other similar classes of molecules are relatively small, modular protein binders that can recognize their targets with high affinity and can be expressed in vivo. By fusing a protein binder to a wellcharacterized functional domain, a variety of tools have recently been generated to directly manipulate proteins of interest (POIs) in vivo in a predictable manner [1–6].

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_30, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Among protein binders directed against commonly used tags, nanobodies against green fluorescent protein (GFP) have been intensively utilized to manipulate GFP-tagged POIs [7]. vhhGFP4 is one of the best characterized nanobodies that binds to GFP and some of its close derivatives (Venus, YFP, and EYFP), but does not bind to Cerulean, dsRed, or mCherry [8, 9]. GFP traps (Morphotrap and the GrabFP system) are based on tissue-specific expression of an anti-GFP nanobody (vhhGFP4) fused to different transmembrane domains targeted to distinct cell compartments. Morphotrap is a fusion protein consisting of vhhGFP4, the mouse CD8 protein as a transmembrane scaffold, and mCherry as a marker, in which vhhGFP4 is facing the extracellular milieu along the entire cell surface (Fig. 1a) [2]. Morphotrap was originally used to trap GFP-tagged Dpp, a critical secreted morphogen in Drosophila [10, 11], in order to test the requirement of GFP-Dpp dispersal [2]. Analogous to Morphotrap, the GrabFPExt toolbox was developed by fusing vhhGFP4 to transmembrane domains with discrete localization along the apicobasal axis and to mCherry or TagBFP for visualization purposes. Different transmembrane domains were used to localize vhhGFP4 to the extracellular space in the apical compartment (GrabFP-AExt), or in the basolateral compartment (GrabFP-BExt) of a tissue of interest (Fig. 1a) [12]. For each of these GrabFPExt constructs, a second version was designed in which the vhhGFP4 domain faces the intracellular milieu (GrabFPInt), thus allowing to manipulate intracellularly tagged POIs (Fig. 1a). An additional construct was produced in which vhhGFP4 is deposited in the basement membrane (GrabFP-ECM) (Fig. 1a) [12]. These GFP trap systems can be used to re-localize or trap GFP-tagged POIs in the extracellular or intracellular region of different compartments of interest (Fig. 1b). All constructs of the GrabFP system were implemented as Gal4 and LexA-inducible transgenes [12]. In this chapter, we describe a protocol for applying GFP traps in the Drosophila wing disc as a model system. The Drosophila wing imaginal disc is a sac-like structure consisting of two cell layers: the pseudo-stratified disc proper epithelium (DPE) and the squamous peripodial epithelium (Fig. 2a). The DPE represents the future adult wing, hinge, as well as notum, and has been used as a leading paradigm to study wing growth, patterning, and morphogenesis. We assume that readers are familiar with Drosophila melanogaster husbandry and genetics. A useful introduction to the nomenclature and practice of Drosophila genetics can be found in [13]. Given the availability of many rescue GFP fusion transgenes and functional GFP fusion knock-ins in different species [14–19], as well as recent progress in genome engineering allowing the insertion of GFP tags in genes of interest, GFP traps can be easily applied to various target proteins in different species.

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Fig. 1 Overview of GFP traps. (a) Schematic illustration of the localization of GrabFPExt and GrabFPInt. In the GrabFPExt tool box, the GFP-binding nanobody vhhGFP4 is orientated facing the extracellular space to trap POI::GFP along different apico-basal axes on the cell surface of GrabFPExt producing cells. In the GrabFPInt tool box, vhhGFP4 is orientated facing the intracellular space to trap POI::GFP along different apico-basal axes inside of GrabFPExt producing cells. All GFP traps are fused to mCherry, with the exception of GrabFP-BExt which is fused to TagBFP for visualization purpose. (b) Schematic illustration showing the relationship of different types of POI::GFP and vhhGFP4 of GrabFPExt or GrabFPInt. GrabFPExt can target extracellular proteins and extracellularly tagged transmembrane proteins, while GrabFPInt can target intracellular proteins and intracellularly tagged transmembrane proteins. GFP green fluorescent protein, POI protein of interest

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Materials 1. D. melanogaster stocks. Available GFP trap stocks in the Bloomington Drosophila Stock Center include: (a) 68176: M{lexAop-UAS-GrabFP.A.Ext.mCh}ZH-35B (b) 68177: M{lexAop-UAS-GrabFP.A.Ext.mCh}ZH-86Fb (c) 68178: M{lexAop-UAS-GrabFP.A.Int.mCh}ZH-86Fb (d) 68173: M{lexAop-UAS-GrabFP.B.Ext.TagBFP}ZH-35B

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Fig. 2 Schematic overview of experimental setups. (a) Schematic illustration of the wing imaginal disc. The wing disc is subdivided into four compartments during development (anterior/posterior and dorsal/ventral compartments) and consists of the future adult wing blade (wing pouch), hinge, as well as the notum. (b) Schematic illustration of the wing disc for testing trapping POI::GFP by Morphotrap. In the control wing disc (left), GFP-POI is expressed in the source cells (region A) (here in the anterior stripe of cells along the A-P compartment boundary as an example). POI::GFP distribution outside of region A could be visualized in the surrounding cells (target cells), if POI::GFP can bind to these cells. In the wing disc where Morphotrap is concomitantly expressed in region A (right), POI::GFP is expected to be trapped in region A and therefore, the distribution of POI::GFP in the target cells is expected to be eliminated. (c) Schematic illustration of the wing disc for testing the efficiency of trapping POI::GFP using tissue-specific expression of Morphotrap. In the control wing disc (left), POI::GFP is expressed in the source cells (region A) (here in the anterior stripe of cells along the A-P compartment boundary as an example). Upon Morphotrap expression in the target cells (region B) (here in the entire P compartment as an example), POI::GFP is expected to accumulate there. In the wing disc where Morphotrap is concomitantly expressed in region A (right), POI::GFP is expected to be trapped in the region A and therefore, the accumulation of POI::GFP in region B is expected to be eliminated. (d) Schematic illustration of the wing disc for testing the efficiency of trapping POI::GFP using clonal expression of Morphotrap. In the control wing disc (left), POI::GFP is expressed in the source cells (region A) (here in the anterior stripe of cells along the A-P compartment boundary as an example). Upon clonal Morphotrap expression in the target cells, POI::GFP is expected to accumulate there. In the wing disc where Morphotrap is concomitantly expressed in region A (right), POI::GFP is expected to be trapped in region A and therefore, the clonal accumulation of POI::GFP is expected to be eliminated. GFP green fluorescent protein, POI protein of interest, DPE pseudostratified disc proper epithelium, PPE squamous peripodial epithelium

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(e) 68174: M{lexAop-UAS-GrabFP.B.Ext.TagBFP}ZH-86Fb (f) 68175: M{lexAop-UAS-GrabFP.B.Int.mCh}ZH-86Fb (g) 68179: M{lexAop-UAS-GrabFP.ECM.mCh}ZH-35B (h) 68170: M{lexAop-UAS-morphotrap.ext.mCh}ZH-35B (i) 68171: M{lexAop-UAS-morphotrap.ext.mCh}ZH-86Fb (j) 68172: M{lexAop-UAS-morphotrap.int.mCh}ZH-86Fb 2. Basic fly husbandry materials (fly vials, fly food, binocular microscope, CO2, fly station, and baker’s yeast). 3. Incubators (29  C, 25  C, and 18  C). 4. Rotator. 5. Dissection forceps. 6. 1.5 mL microcentrifuge tubes. 7. Pipettes (P200 and P1000) and tips. 8. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4. 9. 30% (v/v) glycerol. 10. 4% (w/v) paraformaldehyde (PFA), diluted from 32% PFA with PBS. 11. Vectashield® mounting medium (Vector Labs, Burlingame, CA, USA). 12. Coverslips (22-mm square no. 1 coverslip). 13. Nail polish. 14. Slide glasses. 15. Depression slide for dissection. 16. Binocular microscope. 17. Fluorescence stereomicroscope. 18. Confocal microscope. 19. Ice.

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Methods We first describe a protocol to apply GFP traps to trap extracellular GFP-tagged POIs in order to test the requirement of their dispersal using Morphotrap as an example. In a second protocol, we describe the application of GFP traps for intracellular, GFP-tagged POIs. The most time-consuming steps in these protocols is the design and generation of the appropriate fly stocks for each experiment. Detailed protocols have been previously published [20, 21] and can also be found online (https://flycrispr.org; http://www. crisprflydesign.org).

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3.1 Trapping Secreted GFP-Tagged POIs

This protocol describes GFP trapping of extracellular GFP-tagged POIs, but trapping using other tags and specific VHHs is also possible (see Note 1). 1. Generate or obtain the following fly stocks (locations of transgenes are arbitrary): (a) Stock 1: A-Gal4 (a tissue-specific Gal4 line expressed in the source cells) (b) Stock 2: A-Gal4, tubGal80ts (see Note 2) (c) Stock 3: UAS-POI::GFP (see Note 3) (d) Stock 4: UAS-POI::GFP; UAS-Morphotrap (e) Stock 5: POI::GFP knock-in allele (see Note 4) or POI:: GFP under endogenous regulation (see Note 5). (f) Stock 6: POI::GFP; UAS-Morphotrap 2. Set up control and experimental crosses. For the control cross of UAS-POI::GFP (Fig. 2b, left), cross Stock 1 (or Stock 2) with Stock 3 to express POI::GFP in region A. For the control cross of POI::GFP knock-in allele, cross Stock 1 (or Stock 2) with Stock 5. For the experimental cross of UAS-POI::GFP (Fig. 2b, right), cross Stock 1 (or Stock 2) with Stock 4 to express POI::GFP and Morphotrap in region A in order to trap POI::GFP in region A. For the experimental cross of POI::GFP knock-in allele, cross Stock 1 (or Stock 2) with Stock 6 to express Morphotrap in region A in order to trap POI::GFP in region A. 3. Keep each cross at 25  C in a fly tube supplemented with baker’s yeast for 2 days. Change the tubes daily and keep them at a stable temperature (see Note 6). It takes around 5 days at 25  C for fertilized eggs to become late third instar larvae. If Stock 2 is used, keep the crosses at 18  C, until a temperature shift to 29  C. If the temperature is shifted after 6, 7, or 8 days at 18  C (during the third instar stages), it takes about 36 h, 27 h, or 18 h, respectively, until larvae reach the late third instar stages. This timing should be decided based on the expression level and effects of overexpression of POI::GFP. To collect the late third instar larvae, flood the fly tube with 30% glycerol. The larvae will float to the surface making them easy to harvest. 4. Collect the late third instar larvae in cold PBS. Select larvae expressing GFP and GFP trap (visible via mCherry) with a fluorescence stereomicroscope (see Note 7). Five to six larvae for each condition are sufficient for the first trial to check how the distribution of the GFP-tagged POI changes. 5. Dissect the larvae under a binocular microscope to expose the wing discs in cold PBS in a depression slide and transfer to

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1 mL of cold PBS in a 1.5 mL microcentrifuge tube on ice. Dissection and mounting protocols have been previously published [22, 23]. 6. Carefully remove the PBS and add 4% PFA in PBS for 20 min at room temperature on a rotator to fix the wing discs. 7. Carefully remove the fixative and wash the fixed wing discs by exchanging the fixative with 1 mL of PBS. Repeat this step three times at 10 min intervals (see Note 8). 8. Remove as much PBS as possible and add a drop of Vectashield® to the fixed wing discs. Leave overnight at 4  C. 9. Mount the wing discs on a regular microscope slide. Use the larval brain as a spacer and apply a 22-mm square no. 1 coverslip and seal with nail polish. 10. Image the wing discs using a confocal microscope. Compare the GFP signal from both conditions (control and experimental). In the control, the POI::GFP signal is expected in the producing cells as well as in the surrounding cells. Upon GFP trap expression, the GFP signal is expected to accumulate on the cell surface of producing cells and to be reduced from the receiving cells (Fig. 2b) (see Note 9). For a specific example, please see our previously published work [2, 24]. 3.2 Testing the Trapping Efficiency of Secreted GFP-Tagged POIs

If POI::GFP trapping is efficient in the producing cells, POI::GFP levels in the receiving cells should be reduced. However, the level of POI::GFP in the target tissue may be too low to confirm if the trapping of POI::GFP is efficient. To circumvent this, we suggest trapping GFP-tagged POI in the surrounding cells to artificially stabilize GFP-tagged POI on the surface of surrounding cells, thus revealing POIs otherwise diffusing away or being degraded. This “sensitized” setup can be then used to test the efficiency of blocking dispersal of a POI by Morphotrap. If the trapping is efficient, Morphotrap expression in the source cells should eliminate the accumulation of GFP-tagged POI by Morphotrap in the surrounding cells (see Note 10). 1. Generate or obtain the following fly stocks: (a) Stock 7: UAS-POI::GFP; LexAop-Morphotrap (see Note 11) (b) Stock 8: UAS-POI::GFP; UAS/LexAop-Morphotrap (see Note 12) (c) Stock 9: A-Gal4, B-LexA (B-LexA; a tissue-specific LexA expressed in the target cells (tentatively named “BLexA”)) (see Note 13) (d) Stock 10: hsFLP; act>stop>LexA ; A-Gal4 (e) Stock 11: POI::GFP; UAS-Morphotrap

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(f) Stock 12: B-Gal4 (a tissue-specific Gal4 expressed in the target cells) (g) Stock 13: A-Gal4, B-Gal4 (h) Stock 14: hsFLP ; act>stop>Gal4 (i) Stock 15: hsFLP ; act>stop>Gal4; A-Gal4 2. Set up control and experimental crosses for tissue-specific expression of Morphotrap. For the control cross of UASPOI::GFP (Fig. 2c, left), cross Stock 7 with Stock 9 to express POI::GFP in region A and Morphotrap in region B in order to trap POI::GFP spreading from region A into region B. For the control cross of POI::GFP knock-in allele, cross Stock 11 with Stock 12 to express Morphotrap in region B in order to trap POI::GFP spreading from region A into region B. For the experimental cross of UAS-POI::GFP (Fig. 2c, right), cross Stock 8 with Stock 9 to express POI::GFP in region A and Morphotrap in region A and B in order to block POI::GFP spreading from region A into region B. For the experimental cross of POI::GFP knock-in allele, cross Stock 11 with Stock 13 to express Morphotrap in region A and B in order to block POI::GFP spreading from region A into region B. 3. Set up control and experimental crosses for clonal expression of Morphotrap (see Note 14). For the control cross of UAS-POI:: GFP (Fig. 2d, left), cross Stock 7 with Stock 10 to express POI::GFP in region A and Morphotrap in clones of cells in order to trap POI::GFP spreading from region A into clones of cells. For the control cross of POI::GFP knock-in allele, cross Stock 11 with Stock 14 to express Morphotrap in clones of cells in order to trap POI::GFP spreading from region A into clones of cells. For the experimental cross of UAS-POI::GFP (Fig. 2d, right), cross Stock 8 with Stock 10 to express POI::GFP in region A and Morphotrap in region A and in clones of cells in order to block POI::GFP spreading from region A into clones of cells. For the experimental cross of POI::GFP knock-in allele, cross Stock 11 with Stock 15 to express Morphotrap in region A and in clones of cells to block POI::GFP spreading from region A into clones of cells. 4. Fix and mount the wing discs as described in Subheading 3.1. 5. Image the wing discs using a confocal microscope. Compare the GFP signal from both conditions (control and experimental). In the control sample, the secreted POI::GFP signal is expected to be trapped in region B or in the clones. If POI:: GFP is efficiently trapped in region A, a GFP signal is expected to be strongly reduced in region B or in the clones (Fig. 2c, 2d)

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(see Note 15). For a specific example, please see our previously published work [2, 24]. 3.3 Requirement of Dispersal of a GFPTagged POI

Efficient trapping of POI::GFP by Morphotrap allows testing the requirement for POI::GFP dispersal. In this case, it is important to replace all the POI with POI::GFP, either by rescuing mutants by expressing POI::GFP or by using endogenous knock-in alleles. 1. Generate or obtain the following fly stocks: (a) Stock 16: POI*; A-Gal4 (POI*; a mutant allele for POI) (b) Stock 17: POI*, UAS-POI::GFP (c) Stock 18: POI*, UAS-POI::GFP ; UAS-Morphotrap (d) Stock 19: POI::GFP ; A-Gal4 (e) Stock 20: POI::GFP (f) Stock 21: POI::GFP ; UAS-Morphotrap 2. Set up control and experimental crosses. For the control cross of UAS-POI::GFP, cross Stock 16 with Stock 17 to express POI::GFP in region A in order to rescue the POI mutant by POI::GFP. For the control cross of POI::GFP knock-in allele, cross Stock 19 with Stock 20. For the experimental cross of UAS-POI::GFP, cross Stock 16 with Stock 18 to express POI::GFP and Morphotrap in region A in order to rescue the POI mutant by GFP::POI and concomitantly trap GFP::POI in region A. For the experimental cross of POI::GFP knock-in allele, cross Stock 19 with Stock 21 to express Morphotrap in region A to trap POI::GFP in region A. 3. Fix and mount the wing discs as described in Subheading 3.1. 4. Image the wing discs using a confocal microscope. Compare the GFP signal from both conditions (control and experimental). Analyze the effects of blocking dispersal of POI on signaling, target genes, patterning, and growth with immunostainings if necessary (see Note 16). For a specific example, please see our previously published work [2, 24].

3.4 Re-Localization of Transmembrane Proteins or Intracellular POIs

1. Generate or obtain the following fly stocks: (a) Stock 22: A-Gal4 (b) Stock 23: UAS-POI::GFP (c) Stock 24: UAS-POI::GFP ; UAS-GrabFPInt (d) Stock 25: POI::GFP ; A-Gal4 (e) Stock 26: POI::GFP (f) Stock 27: POI::GFP ; UAS-GrabFPInt 2. Set up control and experimental crosses. For the control cross of UAS-POI::GFP, cross Stock 22 with Stock 23 to express POI::GFP (see Note 17). For the control cross

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of POI::GFP knock-in allele, cross Stock 25 with Stock 26. For the experimental cross of UAS-POI::GFP, cross Stock 22 with Stock 24 to express POI::GFP and GrabFPInt in region A in order to re-localize POI::GFP to the compartment of interest. For the experimental cross of POI::GFP knock-in allele, cross Stock 25 with Stock 27 to express GrabFPInt in order to re-localize all the POI::GFP. 3. Fix and mount the wing discs as described in Subheading 3.1. 4. Image the wing discs using a confocal microscope. Compare the GFP signal from both conditions (control and experimental). POI::GFP is expected to be re-localized to a distinct sub-cellular compartment, depending on the GrabFPInt used (see Note 18). For a specific example, please see our previously published work [12].

4

Notes 1. In case the POI is not functional when fused to GFP, the POI can alternatively be tagged with HA and subsequently be trapped with an HA trap based on an anti-HA scFv [24]. Alternatively, other short peptide tags and the corresponding binders might be used [25]. 2. If overexpression of a POI throughout development adversely impacts developmental processes, the effect can be minimized by applying tub-Gal80ts to temporally control the expression of the POI. At the permissive temperature of 18  C, Gal80ts represses Gal4 activity. At the restrictive temperature of 29  C, Gal80ts can no longer block Gal4 activity. Thus, POI expression can be temporally controlled upon temperature shift. Given the temperature-dependency of the UAS/Gal4 system, control of the POI expression levels can also be achieved by incubating the stocks at different temperatures. When used in combination with other transcriptional systems, it should be noted that LexA-controlled stocks do not show temperature sensitivity [26]. 3. The function of a POI can be affected by the insertion of GFP. Rescue of a POI mutant by the GFP-tagged POI is important to validate appropriate protein function. POI mutants are available from stock centers such as Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/Home/Search) or can be generated by CRISPR approaches. 4. GFP knock-in alleles can be generated by CRISPR approaches, or by protein traps (e.g., MiMIC transposons if available for genes of interest). Regardless of how a GFP knock-in allele is generated, it is important to assure that the entire population of

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the POI is tagged to investigate the outcome of a given manipulation. 5. Numerous Drosophila stocks containing a GFP-tagged gene under its native regulatory region are available [19]. When using such transgenes, the experiments should ideally be performed in rescue setups (see Subheading 3.3). 6. If a detailed phenotypic analysis is required (e.g., assaying tissue growth or tissue size), 2–4 h egg collection is recommended to minimize variation among samples. Too many larvae in one tube also affect developmental speed. Normally around 30 larvae in one tube appears to more or less synchronize their development. Keeping the tubes well hydrated throughout the 5 days is fundamental to ensure that larvae can access food properly. Selecting larvae with identical sex may also reduce variation among samples. 7. GFP and mCherry signals in the wing disc can be detected through the cuticle using a standard fluorescent microscope. Using balancers expressing fluorescent proteins (color balancer) or a balancer with a dominant marker (such as Tb) is helpful to identify the genotype, when the transgenes are not homozygous viable. 8. If necessary, immunostaining can be performed after fixation [22]. Fixed samples can be stored at 4  C for several days. 9. Note that the GFP signal is enhanced upon binding to vhhGFP4 [12]. Immunostaining for GFP can reveal the POI level independently of this enhancement of GFP signal. 10. The potential leakage may be problematic when the resulting phenotypes, upon manipulation, are milder than expected. To reduce the problem of potential leakage, it may be better to use a GFP knock-in allele rather than overexpression of the POI. 11. The transgene should be inserted into the same position as pUASTLOTattB for direct comparison. Ideally, the LexAopGFP trap is generated from the UAS/LexAop-GFP trap upon Cre expression. 12. The pUASTLOTattB vector contains both UAS and LexAop and can thus respond to both Gal4 and LexA. Upon Cre expression, one or the other of the two “enhancers” can be removed in order to create flies responding only to Gal4 or to LexA [27]. Plasmids containing pUASTLOTattB as a backbone can be found in Addgene (https://www.addgene.org) (such as ID163702, ID163917, ID163925, ID163926, and ID163927). 13. If both LexA and Gal4 stocks are available to trap the POI, compare the expression level and choose a line giving stronger

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expression to trap the POI in the source cells to minimize potential leakage. 14. Cross virgins from the stock containing hsFLP with males from the corresponding stocks so that all the progenies have hsFLP. 15. Even if the trapping is efficient, it may be impossible to demonstrate that there is no leakage. Generation of membranetethered versions of POIs may bypass the problem. 16. If the phenotypes upon manipulation are milder than expected, the minor phenotypes could be due to leakage of the POI or independent of the dispersal of the POI. Comparison of the phenotypes caused by the GFP trap with that caused by the membrane-tethered version of the POI, if available, may distinguish these two possibilities. Alternatively, blocking downstream signaling of POI in the target tissue upon GFP trap expression can also show if the minor phenotypes are still dependent on signaling mediated by POI. 17. Overexpression of POI::GFP should not affect the producing cells. To avoid potential problems by overexpression of a POI, the use of endogenously tagged POI::GFP is recommended for intracellular manipulation. 18. Re-localization of GFP-tagged POIs using a GFP trap removes the POI from the original location and re-localizes it to a new location. Therefore, the effect of re-localization in homozygous conditions could be due to a combination of loss of localization from the original location and gain of localization upon re-localization. To analyze the effect of re-localization without affecting protein function in the original location, manipulation in heterozygous condition may be better since non-tagged POIs can remain functional in the original location (see [7] for more details). References 1. Helma J, Cardoso MC, Muyldermans S et al (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209:633–644 2. Harmansa S, Hamaratoglu F, Affolter M et al (2015) Dpp spreading is required for medial but not for lateral wing disc growth. Nature 527:317–322 3. Bieli D, Alborelli I, Harmansa S et al (2016) Development and application of functionalized protein binders in multicellular organisms. Int Rev Cell Mol Biol 325:181–213 4. Kaiser PD, Maier J, Traenkle B et al (2014) Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells. Biochim Biophys Acta 1844:1933–1942

5. Plu¨ckthun A (2015) Designed ankyrin repeat proteins (DARPins): Binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol 55:489–511 6. Aguilar G, Matsuda S, Vigano MA et al (2019) Using nanobodies to study protein function in developing organisms. Antibodies (Basel) 8:16 7. Aguilar G, Vigano MA, Affolter M et al (2019) Reflections on the use of protein binders to study protein function in developmental biology. Wiley Interdiscip Rev Dev Biol 8:e356 8. Saerens D, Pellis M, Loris R et al (2005) Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol 352:597–607

Nanobody-Based GFP Traps to Study Protein Localization and Function in. . . 9. Rothbauer U, Zolghadr K, Muyldermans S et al (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7: 282–289 10. Matsuda S, Harmansa S, Affolter M (2016) BMP morphogen gradients in flies. Cytokine Growth Factor Rev 27:119–127 11. Matsuda S, Affolter M (2017) Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc. eLife 6:e22319 12. Harmansa S, Alborelli I, Bieli D et al (2017) A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila. eLife 6:e22549 13. Greenspan RJ (2004) Fly pushing: the theory and practice of Drosophila genetics, 2nd edn. CSHL Press, Cold Spring Harbor 14. Morin X, Daneman R, Zavortink M et al (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci U S A 98:15,050–15,055 ˜ ones AT et al 15. Kelso RJ, Buszczak M, Quin (2004) Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster. Nucleic Acids Res 32: D418–D420 16. Buszczak M, Paterno S, Lighthouse D et al (2007) The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175:1505–1531 17. Asakawa K, Kawakami K (2009) The Tol2mediated Gal4-UAS method for gene and enhancer trapping in zebrafish. Methods 49: 275–281

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18. Kawakami K, Abe G, Asada T et al (2010) zTrap: Zebrafish gene trap and enhancer trap database. BMC Dev Biol 10:105 19. Sarov M, Barz C, Jambor H et al (2016) A genome-wide resource for the analysis of protein localisation in Drosophila. eLife 5:e12068 20. Ringrose L (2009) Transgenesis in Drosophila melanogaster. Methods Mol Biol 21:3–19 21. Venken KJT, Bellen HJ (2007) Transgenesis upgrades for Drosophila melanogaster. Development 134:3571–3584 22. Spratford CM, Kumar JP (2014) Dissection and immunostaining of imaginal discs from Drosophila melanogaster. J Vis Exp 91:51792 23. Blair SS (2007) Dissection of imaginal discs in Drosophila. Cold Spring Harb Protoc 2007: pdb.prot4794 24. Matsuda, S., Schaefer, J.V., Mii, Y. et al. Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc. Nat Commun 12, 6435 (2021). 25. Vigano MA, Ell C-M, Kustermann MMM et al (2021) Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster. Development 148: dev191700 26. Yagi R, Mayer F, Basler K (2010) Refined LexA transactivators and their use in combination with the Drosophila Gal4 system. Proc Natl Acad Sci U S A 107:16,166–16,171 27. Kanca O, Caussinus E, Denes AS et al (2014) Raeppli: a whole-tissue labeling tool for live imaging of Drosophila development. Development 141:472–480

Chapter 31 Optogenetic Activation of Intracellular Nanobodies Daseuli Yu and Heo Won Do Abstract Intracellular antibody fragments such as nanobodies and scFvs are powerful tools for imaging and for modulating and neutralizing endogenous target proteins. Optogenetically activated intracellular antibodies (optobodies) constitute a light-inducible system to directly control intrabody activities in cells, with greater spatial and temporal resolution than intracellular antibodies alone. Here, we describe optogenetic and microscopic methods to activate optobodies in cells using a confocal microscope and an automated fluorescence microscope. In the protocol, we use the examples of an optobody targeting green fluorescent protein and an optobody that inhibits the endogenous gelsolin protein. Key words Optobody, Intracellular antibody fragment, Nanobody, scFv, Optogenetics, Split-antibody fragment

1

Introduction Precise targeting of endogenous proteins and manipulation of their functions is essential to understand the roles of individual proteins in specific cellular events. Small molecules have been widely used to mediate protein inhibition in cells, but these molecules are prone to induce off-target effects. Genetic knockdown is hard to distinguish from selective alterations in the conformational state of endogenous proteins. Limitations of these conventional methods may be overcome by the introduction of intracellular antibodies (intrabodies) into cells to manipulate the function of endogenous proteins [1–3]. Due to their high target-specificity and stability in cells [4], single-chain variable fragments (scFvs) and smaller antibody fragments such as single-domain antibodies (VHHs, nanobodies) have been widely used to efficiently assess the functions of endogenous proteins and their roles in signaling pathways [5, 6]. Despite the

The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/ 10.1007/978-1-0716-2075-5_32 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_31, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022, Corrected Publication 2022

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widespread use of intrabodies in cell biology, general methods for inducing intrabody activity in cells rely on transcriptional regulation, similar to chemical-induced intrabody expression [7, 8]. Finetuning the regulation of intrabody activity is more difficult at the transcript level than at the protein level, limiting the ability of intrabodies to dissect complex signaling networks. The lack of methods to control intrabody activity inside cells can be overcome by using an optogenetically activatable intrabody, or optobody [9]. Direct modulation of protein activity at the posttranslational level provides advantages in speed and precision [10]. Moreover, an optogenetic approach using light allows more precise spatial and temporal regulation of protein levels [11]. Optogenetics has enabled the control of light-responsive protein modules, leading to hetero- or homo-dimerization [12, 13], oligomerization [14], dissociation [15], and conformational changes [16]. Using a number of methods providing precise control of protein function [12–17], expanded optogenetic strategies have enabled the observation and perturbation of the dynamics of protein signals in cells and organisms [17]. Based on the advantages of optogenetics, optobodies have been generated to spatio-temporally induce the activity of an intrabody in a cell. Optobody systems use light-responsive hetero-dimerization proteins such as nMag-pMag proteins [12] or iLID-SspB proteins [13], fused to split intrabody fragments. The dimerization of lightresponsive proteins results in the reassembly of split intrabody fragments, activating the intrabody function to bind its target protein. Here, we describe two different protocols to activate optobodies: (1) a green-fluorescent protein (GFP)-specific optobody targeting mitochondria-localized GFP using a confocal microscope, and (2) a gelsolin (GSN)-specific optobody directly targeting and inhibiting endogenous GSN function using an automated fluorescence microscope.

2 2.1

Materials Plasmids

1. GFP optobody plasmids. The intracellular antibody fragment vhhGFP4 (see Note 1) was the template for the GFP optobody. The N-terminal fragment of vhhGFP4 (amino acids 1–65) was fused to nMagHigh1 and inserted into the EcoRI site of piRFP682-C1 (resulting in N-terminal fusion with red fluorescent protein, RFP). The C-terminal fragment of vhhGFP4 (amino acids 66–117) was fused to pMagHigh1 and inserted into the EcoRI site of pFusionRed-C1 (resulting in N-terminal fusion with FusionRed). In both vectors, the nanobody subsequence was separated from the fluorescent moiety by (GGGGS)3 linkers (see Notes 2–5) (Figs. 1, 2, and 3). Direct requests for the GFP optobody plasmids to the corresponding author. 2. GSN optobody plasmids. The GSN nanobody (GSN Nb11), which inhibits GSN activity [18] (see Notes 1 and 5), was the

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Fig. 1 Split site on GFP nanobody. (a, b) Schematic representation of the secondary structure (a) and sequence (b) of a GFP nanobody and its split site (green arrow). Figure reproduced with permission from [9]

Fig. 2 Alignment of protein sequence of intrabodies. The amino acid sequences of GFP nanobody, mCherry nanobody, and scFvGCN4 VH were aligned using T-Coffee. The green arrow indicates the split site. Figure reproduced with permission from [9]

template for the GSN optobody. The GSN optobody plasmids were generated in the same manner as the GFP optobody plasmids. Direct requests for the GSN optobody plasmids to the corresponding author. 3. pTOMM20-GFP-N1. To achieve mitochondrial localization of the target of the GFP optobody, this plasmid was

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Fig. 3 Design of an optobody. (a) Schematic design of an optobody DNA construct. (b) Schematic depiction of blue light-induced activation of an optobody. Figure reproduced with permission from [9]

constructed by fusing the outer mitochondrial membrane signal peptide from human TOMM20 (amino acids 1–33, NM_014765) and GFP (see Note 6). The DNA sequenceencoding TOMM20 signal peptide was inserted between the EcoRI and BamHI sites of pEGFP-N1 (Clontech, Takara Bio, Shiga, Japan). 4. piRFP682-C1 and pFusionRed-C1. The piRFP682-C1 and pFusionRed-C1 vectors were generated by replacing EGFP in pEGFP-C1 (Clontech) with iRFP682 and FusionRed, respectively. The DNA sequences encoding fluorescent proteins were inserted between the AgeI and BsrGI sites of pEGFP-C1. 2.2

Cell Culture

1. HeLa cells (ATCC CCL-2, Manassas, VA, USA). 2. Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 3. NIH/3T3 cells (ATCC CRL-1658). 4. DMEM supplemented with 10% bovine calf serum and penicillin/streptomycin. 5. T75 flasks. 6. 96-well plates.

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7. Incubator (37
C and 10% CO2). 8. jetPRIME reagent and jetPRIME buffer (Polyplus, New York, NY, USA). 9. Neon™ Transfection System including neon transfection device, neon pipette, and neon pipette station (Invitrogen, Carlsbad, CA, USA). 10. Neon™ Kits containing neon tips, neon tubes, resuspension buffer R, and electrolytic buffer E (Invitrogen). 11. 5 μg/mL fibronectin. 12. Dulbecco’s phosphate-buffered saline (DPBS). 13. 0.25% (w/v) trypsin-ethylenediaminetetraacetic acid. 14. 1.5 mL microcentrifuge tubes. 15. Hemocytometer. 16. Microcentrifuge. 2.3

Microscopes

1. Nikon A1R confocal microscope (Nikon, Tokyo, Japan). 2. ImageXpress Micro XL high-throughput automated microscope with a transmitted light option and an environment control hardware option (Molecular Devices, Sunnyvale, CA, USA). 3. 60 Plan Apochromat VC and 20 Plan Apochromat microscope objectives (Nikon). 4. Multi-line Argon Laser (488 nm/40 mW) and Coherent Sapphire Solid Laser (561 nM/20 mW) (Nikon) (see Note 7). 5. Fluorescein isothiocyanate (FITC), Texas Red, and Cy5 HQ fluorescence filter cubes (Nikon). 6. Chamlid TC live cell instrument system (Live Cell Instrument, Namyangju, Republic of Korea) consisting of a stage-top incubator, a lens warmer, a temperature controller, a gas flow rate controller for CO2, a humidifier, and a CO2 gas cylinder. 7. Image analysis software: NIS-Elements (Nikon ver 4.1), ImageJ, MATLAB (R2015a), and Microsoft Excel.

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Methods

3.1 Photo-Activation of GFP Optobody 3.1.1 Transfection of Cells with jetPRIME

1. Thaw and culture HeLa EG cells in DMEM supplemented with 10% bovine calf serum and penicillin/streptomycin in T75 flasks in a 10% CO2 incubator at 37
C. When cells become confluent, trypsinize and harvest them. 2. Plate cells at 1.2  104 cells per well in a 96-well plate in 200 μL of DMEM supplemented with 10% bovine calf serum and

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penicillin/streptomycin. Incubate the cells for at least 12 h at 37
C in an atmosphere containing 10% CO2 (see Note 8). 3. For each well to be transfected, dilute 150 ng of GFP optobody (75 ng of each plasmid encoding the two fragments) and 50 ng of pTOMM20-GFP-N1 in 5 μL of jetPRIME buffer in a 1.5 mL microcentrifuge tube. 4. In a second 1.5 mL microcentrifuge tube, add 0.4 μL of jetPRIME to 4.6 μL of jetPRIME buffer (see Note 9). Add the 5 μL of jetPRIME reagent mixture to the DNA solution prepared in step 3. Following thorough mixing by pipetting, incubate at room temperature for 10 min. 5. Add the 10 μL of DNA-jetPRIME mixture dropwise into each well containing cells (from step 2). Incubate the plate for 24 h at 37
C in an atmosphere containing 10% CO2 (see Note 10). 3.1.2 Imaging and PhotoActivation of GFP Optobody Using an A1R Confocal Microscope

1. To maintain environmental conditions (37
C and 10% CO2), turn on the Chamlide TC system at least 10 min before imaging of live cells. Turn off the lights in the microscope room and place the 96-well plate from step 5 of Subheading 3.1.1 onto the warmed plate chamber. 2. Select appropriate fluorescence channels for imaging GFP, FusionRed, and iRFP682. Identify cells with FusionRed and iRFP682 signals using a 60 oil-immersion objective. 3. To simultaneously stimulate cells with blue light and image GFP fluorescence, adjust the laser power, scan speed, and scan number of the GFP channel for photo-stimulation (see Note 11). 4. Run neutral density (ND) sequential acquisition in NIS-Elements for sequential imaging of GFP optobody without a GFP signal followed by TOMM20-GFP (see Note 12) (Fig. 4).

3.1.3 Imaging Analysis

1. Perform Pearson’s correlation analysis of GFP optobody fragments and TOMM20-GFP by downloading the custom MATLAB scripts (see Note 13). 2. Prepare two fluorescent channels as separate stacked “.tif” files in the same directory. 3. Open the script in the MATLAB program. Record the file name, time interval and minimum cell. Run the script and follow the directions on the screen (see Note 14). 4. After calculating Pearson’s correlation coefficient, perform statistical analysis using Microsoft Excel (Fig. 5).

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Fig. 4 Optogenetic activation of a GFP optobody. Fluorescence images of HeLa cells expressing both fragments of a GFP optobody are shown. Signals from the GFP optobody N-terminal fragment (GFP nanobody N65; RFP), the GFP optobody C-terminal fragment (GFP nanobody C66; FusionRed), and TOMM20-EGFP (MitoGFP) are shown before and 1 h after illumination with 488-nm light every 2 min. Scale bar, 10 μm. Figure reproduced with permission from [9]

Fig. 5 Pearson’s correlation for signals from each GFP optobody fragment (RFP or FusionRed) and TOMM20GFP (Mito-GFP). HeLa cells transfected with GFP optobody and TOMM20-GFP were monitored immediately after illumination with 488-nm light every 2 min. Correlations were calculated for single cells (n ¼ 27). Values represent means  standard errors. Figure reproduced with permission from [9]

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3.2 Inhibition of Cell Migration by GSN Optobody 3.2.1 Transfection of Cells with a Neon Electroporation Transfection System

1. Add 70 μL of 5 μg/mL fibronectin to wells of a 96-well plate. Incubate the plate for 30 min at 37
C. The fibronectin solution was discarded and the wells were washed three times with DPBS. 2. Thaw and grow NIH/3T3 cells in DMEM supplemented with 10% bovine calf serum and penicillin/streptomycin in T75 flasks until 80% confluency is reached. Pre-warm electrolytic buffer E and resuspension buffer R from the Neon™ kits. 3. Fill a Neon tube with 3.5 mL of electrolytic buffer E and insert it into a Neon pipette station. Set the optimal voltage (1450 V), width (10 ms), and pulse (3) for NIH/3T3 cells (see Note 15). 4. Prepare two 1.5 mL microcentrifuge tubes as in steps 3 and 4 of Subheading 3.1.1, one for diluted DNA and the other for complete culture medium. Place DNA-encoding GSN optobody (600 ng, 300 ng of each fragment) in the first tube in a final volume of 9 μL of water. In the second tube, place 1 mL of DMEM supplemented with 10% bovine calf serum and penicillin/streptomycin. 5. Trypsinize and harvest NIH/3T3 cells. Transfer 1.8  105 cells to a new 1.5 mL microcentrifuge tube. Centrifuge at 200  g for 30 s at room temperature. Discard the supernatant and wash the cell pellet with 1 mL of DPBS. 6. Centrifuge the cells at 200  g for 30 s at room temperature. Discard the supernatant and resuspend the cell pellet in 11 μL of pre-warmed resuspension buffer R, gently pipetting the cells to avoid generating air bubbles. Add the 11 μL of resuspended cells to the DNA-containing tube from step 4 and mix well by pipetting. 7. Tightly attach a 10 μL Neon tip to a Neon pipette. Fill the tip with the DNA-cell mixture (see Note 16). Insert the Neon pipette into the Neon tube placed in the Neon pipette station. Press the “Start” button on the screen. 8. After the “Completed” signal is displayed on the screen, transfer the DNA-cell mixture into the 1 mL of DMEM supplemented with 10% bovine calf serum from step 4 and mix well by pipetting. 9. For each well of the fibronectin-coated plate from step 1, add 50 μL of the electroporated cells from step 8 to 200 μL of DMEM supplemented with 10% bovine calf serum and penicillin/streptomycin. Transfer 250 μL of this suspension into a well of a fibronectin-coated 96-well plate. Incubate the plate for 24 h at 37
C and 10% CO2 (see Note 17).

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1. To maintain environmental conditions (37
C and 10% CO2), turn on the Environment Control Hardware at least 10 min before imaging of live cells. Place the 96-well plate from step 9 in Subheading 3.2.1 on the warmed plate chamber. 2. Set up the “Plate Acquisition Setup” and select a 20 objective. For multipoint and time-lapse imaging, designate sites on the well and set intervals at 3 min. Determine the time required for one loop to image the multisite position (see Note 18). 3. Adjust the exposure time and autofocus options. Save two “Plate Acquisition Setup” files, one for imaging Texas Red and Cy5 fluorescence channels for tracking the GSN optobody, and the other for imaging the FITC channel for photostimulation (see Note 19). 4. Open “Edit Journal” to run both types of “Plate Acquisition Setup” and to load the files, followed by running of the journal.

3.2.3 Imaging Analysis

1. To analyze distance and instantaneous speed, open the “MTrackJ” plugin in ImageJ software and click the “Add” button to determine cell movement. Select cells expressing Texas Red and Cy5 signals and follow the migration track by sequential clicking (Fig. 6a). 2. Display migration tracks using the “Plot_At_Origin” program (Fig. 6b). Calculate the migration distance and instantaneous speed using the “Speed” program [19] (see Note 20) (Fig. 7a, b).

Fig. 6 Disruption of cell migration by a GSN optobody. (a) Migration path of two NIH3T3 cells using the “MTrackJ” plugin on ImageJ. (b) Tracking the migration of a group of NIH3T3 cells expressing a GSN optobody, with and without light stimulation. Each line represents the migration path of one cell (n ¼ 50), monitored every 3 min for 5 h with simultaneous blue light illumination. Figure reproduced with permission from [9]

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Fig. 7 Analysis of cell migration using a GSN optobody. (a, b) Quantitation of migration distance (a) and instantaneous speed (b) of NIH/3T3 cells expressing FusionRed, GSN nanobody, and GSN optobody with and without stimulation with light. Instantaneous speed at 0 h (Light) and 2 h (+Light) was measured (n 132 for each group). Figure reproduced with permission from [9]

4

Notes 1. The coding sequence of the anti-GFP intrabody vhhGFP4 was derived from the expression plasmid pcDNA3_NSlmbvhhGFP4 (Addgene; plasmid 35579). The coding sequence of the GSN nanobody (GSN Nb11) in the pCMV/myc/ER vector (Life Technologies, Carlsbad, CA, USA) was kindly provided by Jan Gettemans (Ghent University, Belgium). 2. For split site selection of intracellular antibody fragments, the sequences of variable heavy chain domains (e.g., nanobody and scFv VH domain) were aligned with vhhGFP4 using T-Coffee multiple sequence alignment. The split-antibody fragment was generated by splitting between residues S65 and V66 of vhhGFP4 (Fig. 2). 3. Codon optimization was performed using the GenScript website (http://www.genscript.com/cgi-bin/tools/rare_codon_ analysis) followed by gene synthesis of nMagHigh1 and pMagHig1. nMagHigh1 and pMagHigh1 are mutants of nMag and pMag, respectively. This pair of mutants has the highest binding affinity upon blue light stimulation and the slowest dissociation rate in the dark [13]. 4. These vectors were constructed by PCR amplification of DNA encoding the vhhGFP4 N-terminal fragment (residues 1–65), (GGGGS)3 linker, and nMagHigh1 and insertion into the EcoRI site of piRFP682-C1 by Gibson assembly. PCR products encoding the vhhGFP4 C-terminal fragment (residues 66–117), (GGGGS)3 linker, and pMagHigh1 were cloned into the EcoRI site of pFusionRed-C1 by Gibson assembly.

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5. The GSN nanobody (GSN Nb11) specifically recognizes the GSN G2 domain, inhibiting GSN function [18]. 6. The outer mitochondrial membrane signal peptide should be positioned at the N-terminal of the protein. A PCR product encoding TOMM20 (amino acids 1–33) was digested with EcoRI and BamHI and inserted into pEGFP-N1 (Clontech). 7. Wavelengths longer than 561 nm have no effects on the photoactivation of Magnet proteins. 8. To prevent the cells from congregating on the edge of wells, the plates should not be agitated horizontally; rather, they should be mixed immediately and thoroughly with a pipette. 9. A 1:2 ratio of DNA to jetPRIME reagent is recommended to achieve high transfection efficiency. 10. After transfection, the plate should be wrapped in aluminum foil to protect the cells from exposure to light. 11. To confirm activation of the GFP optobody, the target protein GFP should be monitored. However, the 488 nm laser for GFP activates magnet protein dimerization, thereby activating GFP optobody. A co-imaging system that captures GFP signals was utilized for photo-activation and imaging of GFP. A 488 nm laser with a power density of 490 μW mm2 (measured with an ADCMT optical power meter) was used for photo-excitation. 12. The coefficient of correlation between each GFP optobody fragment and mitochondria-localized GFP represents the efficiency of GFP optobody activation. The iRFP682 and FusionRed signals represent the N-terminal and C-terminal fragment of GFP optobody, respectively. Thus, cells co-imaged with TOMM20-EGFP are in a state of photostimulation by the 488 nm laser. 13. The custom MATLAB scripts are available in the Supplementary Code file of a previous publication [9]. 14. For precise segmentation, cells in images should be separated into single cells. 15. The conditions for electroporation of NIH/3T3 cells were optimized to increase the transfection efficiency. 16. Care is required during resuspension and electroporation to prevent bubble formation in the electroporation tips. These bubbles can cause a spark, reducing transfection efficiency and cell viability. 17. If cells are plated at high density, their migration paths can converge and interfere with each other. Cells should therefore be plated as single cells to prevent clustering. 18. Cell migration is usually monitored by acquiring images for at least 4 h. Illumination with blue light (for excitation of GFP) at 3 min intervals was sufficient to activate GSN optobody.

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19. Filter sets used included Texas Red for red fluorescent proteins like FusionRed, Cy5 for near-infrared proteins like iRFP682, and FITC for green fluorescent proteins. Photo-activation was performed at a light intensity of 40 μW mm2, achieved by an exposure time of 500 ms in our system. 20. The supplementary data files “Plot_at_Origin” and “Speed” were downloaded and the protocol described in our previous publication was followed [19].

Acknowledgments Figures 1, 2, 3, 4, 5, 6b, and 7 were reproduced with permission from [9]. References 1. Marschall ALJ, Du¨bel S, Bo¨ldicke T (2015) Specific in vivo knockdown of protein function by intrabodies. MAbs 7:1010–1035 2. Miller TW, Messer A (2005) Intrabody applications in neurological disorders: progress and future prospects. Mol Ther 12:394–401 3. Mujic´-Delic´ A, de Wit RH, Verkaar F et al (2014) GPCR-targeting nanobodies: attractive research tools, diagnostics, and therapeutics. Trends Pharmacol Sci 35:247–255 4. Muyldermans S, Cambillau C, Wyns L (2001) Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 26: 230–235 5. Helma J, Cardoso MC, Muyldermans S et al (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209:633–644 6. Staus DP, Strachan RT, Manglik A et al (2016) Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature 535:448–452 7. Bethuyne J, De Gieter S, Zwaenepoel O et al (2014) A nanobody modulates the p53 transcriptional program without perturbing its functional architecture. Nucleic Acids Res 42: 12,928–12,938 8. Daniel K, Icha J, Horenburg C et al (2018) Conditional control of fluorescent protein degradation by an auxin-dependent nanobody. Nat Commun 9:3297 9. 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 10. Rakhit R, Navarro R, Wandless TJ (2014) Chemical biology strategies for

posttranslational control of protein function. Chem Biol 21:1238–1252 11. Tischer D, Weiner OD (2014) Illuminating cell signalling with optogenetic tools. Nat Rev Mol Cell Bio 15:551–558 12. Kawano F, Suzuki H, Furuya A et al (2015) Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat Commun 6:6256 13. Guntas G, Hallett RA, Zimmerman SP et al (2015) Engineering an improved lightinduced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc Natl Acad Sci U S A 112:112–117 14. Park H, Kim NY, Lee S et al (2017) Optogenetic protein clustering through fluorescent protein tagging and extension of CRY2. Nat Commun 8:30 15. Wang H, Vilela M, Winkler A et al (2016) LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat Methods 13: 755–758 16. Wu YI, Frey D, Lungu OI et al (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461: 104–108 17. Lee S, Park H, Kyung T et al (2014) Reversible protein inactivation by optogenetic trapping in cells. Nat Methods 11:633–666 18. Van den Abbeele A, De Clercq S, De Ganck A et al (2010) A llama-derived gelsolin singledomain antibody blocks gelsolin-G-actin interaction. Cell Mol Life Sci 67:1519–1535 19. Gorelik R, Gautreau A (2014) Quantitative and unbiased analysis of directional persistence in cell migration. Nat Protoc 9:1931–1943

Correction to: Optogenetic Activation of Intracellular Nanobodies Daseuli Yu and Heo Won Do

Correction to: Chapter 31 in: 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_31 The original version of this chapter was inadvertently published with wrong format of the author’s name “Won Do Heo”. Now, the correct format of the author’s name has been updated in this chapter.

The updated version of this chapter can be found at https://doi.org/10.1007/978-1-0716-2075-5_31 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_32, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

INDEX A Affinities.......................... 5, 7–10, 12–14, 22, 23, 25–30, 46, 62, 63, 71, 72, 86, 89, 95–97, 102, 106, 109, 111, 113, 117, 121–140, 154–156, 161, 166, 182, 183, 187, 192, 194, 195, 199–201, 245–267, 270, 275, 304, 305, 307–310, 313, 324, 328, 335, 338, 357, 358, 365, 383, 390, 397, 410, 412, 417, 419, 421, 422, 429–431, 433, 441, 447, 448, 470, 483–485, 489, 500, 514, 532, 538, 552, 557, 581, 604 Affinity maturation.............. 22, 23, 25, 72, 86, 245–267 Agarose .....................40, 43, 48, 54–56, 79–81, 84, 125, 127, 134, 164–166, 169, 173, 175, 184, 185, 187, 188, 193, 194, 199, 200, 208–210, 216, 222, 247, 253, 255–257, 261, 267, 317, 318, 320–323, 329–331, 335, 377, 381, 413, 436, 444, 446, 472, 476, 492, 498–502, 534, 535, 539, 540, 545, 559, 562, 563, 566, 567, 571, 576 Aggregation propensity ....................................... 233–243 Agrobacterium-mediated transformation .................... 206 Albumin binding domain (ABD) .............................................. 493, 498, 501 Aldehyde handles ................................................. 345–355 Aldehyde tags ........................................11, 347, 352, 358 Alexa Fluor 488........................................... 328, 330, 337 Alpaca........................................................... 5, 6, 300, 513 Antibiotics .................................................. 43, 46, 67, 77, 91, 148, 207–208, 216–218, 220, 221, 287, 317, 328, 331, 335, 362, 367, 385, 387, 411, 416, 421, 436, 472, 559, 560, 570, 577 Antigens............................................................. 3, 4, 7–10, 12–14, 19–30, 51, 53, 58, 59, 62–64, 71–73, 78, 88, 89, 92, 122, 123, 127, 128, 131, 136, 138, 160, 161, 166, 173, 174, 178, 181, 182, 188, 195, 201, 210, 224, 225, 228, 246, 260, 261, 263, 265, 269, 271, 273–275, 289–293, 296, 299, 300, 302, 304, 305, 308–310, 315, 325, 329, 338, 342, 374, 390, 391, 410, 413–415, 419, 427, 429, 430, 434, 435, 437, 441, 446–448, 451, 465, 483, 490, 500, 513–530, 548–553, 555–557, 566, 571, 573, 574 Antigen-specific B cells ................................................. 140 Arabidopsis thaliana ..................................................... 206

Ascorbate peroxidase (APX) ....................... 428, 429, 431 Ascorbate peroxidase derivative (APEX2) .......... 427–448 Avidity............ 10, 13, 45, 117, 161, 313, 429, 448, 500

B Back mutations.............................................300, 304–310 Baker’s yeast ...............................160, 162, 323, 585, 586 Bicine ..........................................160, 163, 168, 170, 175 Bioinformatics ................................ 22, 72, 300, 328, 331 Biolayer interferometry (BLI) ...............................328, 329, 334, 338, 342 Biolistic transfection........................................................ 38 Bioluminescent enzyme immunoassay (BLEIA) ...................................547, 548, 552, 553 Bio-orthogonal...............................................11, 357–370 Biotin-acceptor peptide ................................................ 538 Biotin ligase ............ 53, 61, 68, 534, 535, 538, 540, 545 Biotinylation ......................................................11, 53, 54, 60, 68, 139, 154, 165, 166, 173, 174, 177, 178, 273, 289, 532, 534, 535, 541, 545 Biparatopic.............................................................. 10, 500 BirA ............................. 11, 53, 60, 61, 68, 154, 534, 541 Bivalent ..........................10, 13, 182, 212, 326, 500, 514 Bone marrow................................................................. 129 Buffy coats ...........................................456, 460, 461, 466

C Camel...................................................4–7, 154, 300, 513 Camelidae ............................................5, 38, 39, 396, 513 Cancers ..............................................................24, 28, 37, 182, 345, 452, 454, 467, 468, 490, 511 Catchers ...............................................314, 322, 325, 326 Cell migration ...................................................... 602–605 Chimeric antigen receptor T-cell (CAR T) ..............................................14, 451–467 Chinese hamster ovary (CHO) cells ..................... 26, 183 Chromatography ................................................. 9, 46, 89, 102, 149, 153, 155, 165, 173, 192, 269, 270, 310, 318, 324, 329, 335, 337, 346, 350, 358, 359, 383, 397–399, 401, 402, 417, 419, 422, 430, 433, 447, 492, 495, 505, 506, 514, 516, 518, 521, 523, 524, 531, 535, 552 Chromobodies ......................................11, 555, 573, 576

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, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

607

SINGLE-DOMAIN ANTIBODIES: METHODS AND PROTOCOLS

608 Index

Click chemistry..................................................... 374, 397 Clones .................................................7–9, 23, 26, 39, 56, 58, 59, 78, 82, 83, 85, 102, 107, 110, 113, 117, 122, 126, 136, 140, 163, 185, 191, 192, 216, 246–248, 255–257, 260, 263, 264, 270, 274, 275, 285, 290, 291, 293–295, 314, 320–322, 340, 341, 429, 431, 484, 491, 496, 497, 502, 563, 565, 567, 568, 570, 576, 577, 588 Co-crystal structure ...................................................... 246 Colonies................................................42, 43, 48, 53, 56, 58–61, 66, 67, 82, 83, 85, 104, 105, 110, 112, 117, 122, 135, 140, 150, 151, 168, 169, 171, 172, 176, 183, 188, 191, 192, 199, 215–218, 220, 226, 227, 257, 260, 265, 279, 282, 284, 285, 287, 289, 290, 294, 320–322, 325, 331, 335, 340, 363, 367, 369, 382, 388, 417, 436, 476, 477, 484, 496, 498, 500, 520, 522, 538–540, 563, 567, 576, 577 Colony PCR ........................... 42, 43, 56, 58, 59, 66, 67, 82, 85, 215, 216, 227, 257, 260, 563, 567, 577 Combinatorial His scanning library ................... 276, 278, 279, 286, 290 Competent cells................................. 122, 135, 150, 155, 161, 168, 169, 189, 217, 265, 272–274, 282, 436 Complementarity determining regions (CDRs).............................................. 77, 137, 181, 245, 300, 313, 331, 374, 397, 410, 470, 514, 556 Complementary DNAs (cDNAs)........................... 39, 42, 48, 51, 53, 54, 66, 74, 79, 80, 90, 122, 125, 133, 134, 374, 376, 380, 381, 386, 387, 561, 575, 576 Conjugations ................28, 53, 345–347, 349, 350, 352, 353, 358, 368, 397, 398, 400, 402, 404, 405, 544 CRISPR/Cas9............................ 557, 563–566, 569–570 Cryptic epitopes ..................................................v, 21, 300 Crystallography ................................................... 308, 328, 331, 336, 338, 428, 471, 514, 525 Crystal structures ................ 22, 271, 413, 416, 513–530 Cysteine-maleimide chemistry ............................ 397, 398 Cysteines ................ 20, 21, 91, 146, 154, 161, 163, 167, 178, 327–342, 345, 359, 397, 398, 400, 402, 428 Cytoplasm........................................................9, 146, 147, 358, 391, 421, 469, 470, 521, 545 Cytotoxicity .................................................. 25, 457, 461, 463, 467, 474, 481, 486, 492, 495–496, 506–510

D Degenerate oligonucleotides .......................275–277, 279 DERMOJET ............................................... 39, 41, 50, 51 Developability......................................233–235, 239, 300 Developmental biology........................................ 581–592 Diagnostics ........................................... 10, 12, 13, 19–30, 71, 95, 122, 145, 182, 183, 269, 357, 374, 514 Differential scanning fluorimetry ................................. 233 Diol probe ...........................................346, 347, 349, 352

Dissection .............................................................. 79, 123, 129, 514, 585, 587 Disulfide...................................................... 11, 20–22, 96, 146, 147, 154, 167, 182, 212, 340–342, 358, 370, 397, 400, 421, 428, 485 Disulfide bond isomerase (DsbC) .....................................146, 147, 151, 154 DNA ............................................ 4, 6, 25, 37–69, 74–76, 80–82, 84, 85, 90, 91, 100, 102–104, 110, 112, 122, 125–127, 133–138, 140, 159, 161, 164, 165, 167–170, 175, 176, 184, 185, 188–190, 196, 199, 205, 208, 209, 212, 215–217, 226, 227, 247, 248, 253, 255–257, 259–261, 263, 267, 271, 272, 276, 277, 279, 282, 284–286, 295, 314–316, 319–321, 325, 329, 331, 340, 369, 375, 376, 380–382, 386, 395, 416, 448, 456, 458, 459, 464, 466, 472, 474–478, 481, 483, 491–493, 496–500, 534, 538–540, 559–569, 571, 572, 576, 598, 600, 602, 604, 605 DNA immunization ..................................................37–69 DNA sequencing........................................ 25, 43, 58, 60, 61, 66, 117, 164, 205, 245–267, 314, 316, 369, 375, 382, 395, 474–476, 540, 561, 598 Double antibody sandwich (DAS) ELISA...........................160, 166–167, 174 Drosophila ...........................................582, 583, 590, 591 DsbC isomerase.................................................... 145–156

E Electrophoresis ........................................... 40, 43, 74, 75, 79–81, 84, 100, 103, 125, 134, 164, 165, 184, 188, 196, 198, 199, 209, 211, 225, 247, 249, 253, 255, 256, 317, 321, 322, 329, 331, 377, 381, 400, 402, 404, 441, 472, 479, 485, 494, 496, 498, 500–502, 517–519, 535, 560, 576 Electroporation .................................................40, 43, 45, 66, 75, 77, 81, 82, 84, 100, 102, 104, 110, 135, 160, 165, 170–172, 175, 189, 206, 209, 217–218, 248, 249, 256, 259, 271, 273, 277, 279, 285, 286, 294, 295, 416, 536, 539, 602, 605 Empirical scoring .......................................................... 300 Enzyme linked immunosorbent assay (ELISA)............................................. 8, 39, 47, 51, 53, 54, 58, 62, 63, 67, 122–124, 126, 128, 136, 138, 160, 166, 167, 174, 176, 178, 194, 210, 223, 224, 274, 275, 290, 435, 441, 443, 446, 471, 472, 478–480, 485, 496, 547, 548, 550, 551 Episomal vector ............................................................. 160 Epitope tags....................................................98, 105, 412 Error-prone PCR ...........................................25, 117, 255 Escherichia coli ..................................................... 8, 26, 39, 75, 100, 102, 122, 145, 159, 182, 185, 207, 271, 327, 358, 377, 397, 409, 428, 455, 472, 493, 515, 534, 559

SINGLE-DOMAIN ANTIBODIES: METHODS F Fast protein liquid chromatography (FPLC)............................................ 46, 47, 61, 62, 149, 153, 155, 329, 335, 339, 359, 361, 363, 413, 422, 430, 433, 446, 495, 506, 507, 535, 541 Fc fusion ..................................................... 25, 27, 39, 47, 48, 61–64, 68, 206, 212, 214, 217, 218, 223, 224 Filovirus ................................................................ 427–448 Flow cytometry .......... 39, 101, 105, 107, 114, 395, 460 Fluorescence .................................. 63, 95, 105, 114, 233, 235, 238, 241, 242, 337, 347, 349, 370, 380, 395, 396, 402, 404, 428, 434, 440, 551, 561, 569, 573–575, 577, 585, 586, 596, 599–601, 603 Fluorescence enzyme immunoassay (FEIA)......................................547, 548, 551, 553 Fluorescence recovery after photobleaching (FRAP)............................................. 561, 573, 574 Fluorescent labeling ............................................. 373–391 Focus forming assay ...................................................... 442 Foot and mouth disease virus (FMDV) ........................................... 160, 166, 174 Formulations .......................................234, 235, 238–240 Formylglycine-generating enzymes (FGE) ..................................................11, 357–370 Fully human antibody.......................................... 121–140 Fusion proteins................................................47, 62, 145, 146, 163, 205, 212, 223, 224, 374, 428, 429, 431, 434–436, 438, 442, 548, 556, 562, 582

G Gal4.............................................582, 586, 588, 590, 591 Gateway cloning...........................................214–217, 227 Gelsolin (GSN) ...................................596, 597, 602–605 Gene gun ............................................... 38–41, 49–51, 64 Genetic code expansion ................................................ 410 Glycosylation ......................... 26, 96, 160, 184, 200, 212 Glypicans .............................................................. 452, 468 GrabFP .......................................................................... 582 Green fluorescent protein (GFP) .............. 184, 195, 201, 219, 227, 374, 413, 415, 416, 421, 453, 460, 462, 463, 467, 556, 577, 581–592, 596–601, 605

H Half-life................................ 9, 10, 24–27, 122, 325, 500 Haptens................................................544, 548, 550–553 Heat-denaturation................................................ 233–243 Heavy chain antibodies ................ 38, 161, 181, 245, 396 Helper phage .............................................. 43, 53, 54, 56, 58, 59, 76, 77, 83, 85, 91, 260, 262, 271, 273, 274, 280, 286, 290, 291 High performance liquid chromatography (HPLC).................................................... 249, 253, 346–352, 354, 364, 518, 531

AND

PROTOCOLS Index 609

His-tag ............................53, 54, 89, 116, 117, 192, 193, 197, 199, 200, 228, 316, 320, 346, 347, 355, 369 Histidines........................... 176, 177, 270, 278, 422, 535 Homology modelling ..................................................... 86 Horn sharks ..............................20–22, 24, 29, 73, 78, 90 Human embryonic kidney (HEK293) cells .......................................... 68, 183 Human framework........................................................ 300 Humanization ...............................................5, 10, 25–27, 30, 300, 301, 303–305, 307–310 Humanized variants ...................304, 305, 307, 309, 310 Hydroxylamine compounds ...............347, 350, 352–354

I Igh locus ........................................................................ 4, 6 Illumina..................... 246, 250, 253, 258, 261, 263, 264 Imaging..................................................... 13, 25, 29, 146, 182, 345, 369, 374, 380, 386, 395, 396, 398, 410, 434, 435, 440, 457, 464, 467, 482, 517, 521, 556, 561, 571, 573–575, 577, 600, 603, 605 Immobilize metal affinity chromatography (IMAC) ................... 46, 102, 103, 112, 335, 340, 383, 387, 433, 437, 446, 514, 516, 518, 520–522 Immune library ................................ 7, 8, 71, 72, 85, 357 Immune responses ............ 7, 38, 39, 41, 50, 51, 72, 183 Immunizations ................................................ 5–7, 12–13, 20, 23, 29, 30, 37–69, 72, 73, 78, 79, 122, 124, 127–128, 138, 206, 299, 397, 548 Immunoadsorbents ......................................532–534, 541 Immunoconcentration...............532, 534, 538, 543, 544 Immunocytochemistry......................................... 373–391 Immunodetection ......................................................... 428 Immunofluorescence microscopy ....................... 327, 328 Immunogenicity .................................................... 7, 9, 10, 13, 38, 39, 72, 182, 299, 303, 307, 309 Immunoglobulins ............................................... 9, 20, 22, 26, 66, 122, 166, 173, 177, 181, 374, 410 Immunotoxins..................................................... 490, 491, 493, 497, 498, 500, 501, 506–510 In silico maturation............................................ 77, 86–89 Internal standard ......................................... 532, 533, 544 Intrabodies .......................................................... 470–472, 474–482, 484–486, 595–597, 604 Inverse PCR ......................................................... 329, 331 In vivo fluorescence imaging ............................... 395–405 Iron oxide ......................96, 97, 101, 106–109, 111, 116

K Kunkel mutagenesis ........72, 73, 83, 272, 279, 294, 295

L Lentiviral transduction................................ 457, 462, 465 Lentivirus .................. 455, 456, 458–460, 462, 465, 466

SINGLE-DOMAIN ANTIBODIES: METHODS AND PROTOCOLS

610 Index

LexA...................................................................... 587, 591 Libraries ...........................................................6–8, 12, 13, 22–26, 29, 30, 39, 42, 43, 51–59, 66–68, 71–92, 95–99, 101, 104–111, 115–117, 122, 125, 126, 133, 135, 136, 140, 246–250, 255–261, 263–265, 267, 270, 271, 273–279, 284–287, 289–291, 294–296, 308, 490, 496, 545 Library selections ...........................................97, 101, 107 Ligand discovery ............................................................. 95 Ligations ............................................................11, 55, 66, 81, 82, 104, 112, 134, 135, 163, 168, 169, 256, 320, 325, 331, 382, 387, 476, 477, 484, 493, 498, 500, 510, 540, 562, 563, 566–568, 576 Linked protonation ....................................................... 270 Liquid chromatography-high resolution mass spectrometry ...................................................... 360 Live cell imaging .......................... 48, 555–557, 571, 574 Llama ............................ 5–7, 38–42, 48–51, 64, 66, 122, 154, 161, 163, 167, 300, 308, 365, 370, 500, 513 Lymphocytes .......................................................... 66, 139

M Magnetic beads ................................................... 151, 274, 291, 531–545 Maleimides............................................................. 28, 328, 330, 340, 342, 345, 399, 401 Mass spectrometry ...................................... 422, 531, 532 Megabodies ..................................................................... 12 Melting temperature (Tm).................................................. 233, 241, 421 Membrane proteins.................................... 12, 13, 37–39, 95–98, 103, 115, 182 Mercury ................................................................ 328, 342 Metal complexation ...................................................... 347 Methanol-inducible promoter...................................... 183 Microscopy ...................12, 13, 145, 374, 376, 383, 395, 410, 412, 428, 434, 435, 440, 556, 558, 561, 575 Mirrorball ........................................................... 48, 63, 64 MiSeq.......................................................... 246, 250, 253, 254, 258, 261, 263, 264 Molecular dynamics .....................................78, 86–89, 92 Molecular replacement ........................................ 527–529 Monoclonal antibodies ........................................... 25, 38, 66, 228, 299, 390, 427, 547 Morphotrap ..................................................582, 584–588 Multi-copy integration.................................................. 184 Multi-cycle kinetics .............................................. 201, 266 Multimeric ........................................................... 160, 166, 313–326, 476 Multimers ....................................................................... 27, 313–315, 318, 324, 438 Mutagenesis.......................................................23, 86, 89, 91, 246–249, 255, 257–261, 265, 275, 276, 285, 295, 310, 328, 329, 331, 340, 564

N Naı¨ve library ....................................................... 7, 23, 106 Nanobodies .............................................. 5, 8–12, 96, 97, 99, 102, 104–106, 108–110, 112–118, 145–156, 205, 300, 327–343, 345–355, 357, 373–391, 396, 409, 411, 416, 427–448, 500, 531–547, 555–577, 581–583, 595–606 Neuroblastoma..................................................... 451–467 Neutralization .................................................13, 28, 135, 160, 177, 324, 469–486 Next-generation DNA sequencing (NGS)......................................... 25, 117, 245–267 N-hydroxysuccinimide (NHS) ester .............................................. 345, 374 Nicotiana benthamiana ...............................................206, 209, 211, 212, 214, 217–220, 225 Ni-NTA magnetic beads ............................................... 148

O Octet ....................................................194, 329, 330, 338 Optobodies........................................................... 596–605 Optogenetic activation ........................................ 595–606 Optogenetics ........................................................ 595–606

P Panning...................................... 9, 39, 43, 53–59, 67, 68, 86, 246, 249, 260–265, 267, 274, 289, 490, 496 Para-chloromercuribenzoic acid......................... 330, 340 Paratopes ...................................................... 9, 10, 22, 23, 386, 410, 412, 415, 416, 514 Peptide tags .......................................................... 556, 590 Peripheral blood mononuclear cells (PBMCs) ................................................42, 51, 52, 456, 457, 460–462, 466 Phage display ...................................................5–8, 25, 29, 51, 95, 96, 161, 167, 246, 270, 273–275, 277, 285, 286, 289, 290, 295, 428, 453, 490, 538 Pharmacokinetics ........................................ 300, 313, 396 Photobodies ......................................................... 409–411 Photocaging ......................................................... 411, 412 Photoporation ................... 375, 376, 380, 385, 386, 391 pH sensitivity........................................................ 291, 292 Pichia pastoris ........................................ 26, 159, 181–201 Plasma cells ........................ 122, 123, 129–133, 139, 140 Plasmid extraction ......................................................... 102 Plasmids ................................................ 39–41, 45, 47–49, 59–62, 64, 68, 75, 82, 85, 96–107, 110, 112–115, 117, 122, 123, 135–137, 140, 146–148, 150, 151, 154, 160–164, 167–171, 175–178, 184, 185, 188–190, 197, 207, 209, 216, 217, 226, 255, 282, 284, 285, 314, 315, 318, 320–324, 328, 330, 331, 334, 363, 367, 369, 375–378, 380–382, 386, 389, 397, 410,

SINGLE-DOMAIN ANTIBODIES: METHODS 411, 416, 417, 421, 446, 452, 453, 455, 456, 458, 459, 465, 471, 472, 475–477, 482, 492–494, 498, 500, 501, 510, 520, 522, 534, 538–540, 545, 558, 560, 562–570, 574, 576, 577, 596–598, 600, 604 Polyclonal antibody...............................37, 390, 427, 547 Polymerase.................................................. 25, 42–45, 54, 56, 74, 76, 80, 82, 84, 100, 103, 110, 125, 134, 167, 208, 215, 247, 253, 255–257, 259, 261, 272, 276, 279, 284, 329, 331, 376, 380, 386, 497, 501, 502, 559, 566, 572 Polymerase chain reaction (PCR) ...................... 6, 42–45, 53–56, 58, 59, 66, 67, 74, 76, 79–82, 84, 85, 100, 103, 104, 110, 112, 115, 117, 125, 134, 161, 163, 167–169, 175, 184, 185, 188, 189, 199, 208, 215, 216, 227, 236, 237, 240, 247, 249, 253–257, 260, 261, 265–267, 272, 279, 284, 315, 317, 319, 325, 328, 329, 331, 340, 375–377, 380, 381, 386, 387, 414, 419, 421, 422, 464, 484, 492, 497, 498, 500–502, 518, 525, 534, 538–540, 545, 560, 562, 563, 565–568, 571, 572, 576, 577, 604, 605 Primary T cells................................................14, 452, 465 Primers........................................................ 22, 39, 42, 43, 53–56, 66, 74–77, 79, 100, 102, 115, 125, 126, 133, 134, 136, 161, 164, 167, 168, 184, 188, 190, 199, 208, 215, 216, 227, 247–250, 253–257, 259–261, 265–267, 276, 279, 282, 284, 285, 294, 295, 315, 317, 319, 321, 325, 329, 331, 340, 376, 380, 381, 386, 421, 472, 475, 477, 483, 492–494, 497, 500–502, 534, 538, 540, 558, 559, 563–568, 571, 572, 576 Propargyl glycine.................................................. 374, 375 Protein A .........................5, 27, 122, 127, 137, 307, 310 Protein aggregation .................................... 238, 239, 387 Protein engineering ......................... 19, 27, 72, 235, 239 Protein expressions .................................. 22, 68, 99, 112, 115, 116, 340, 412, 419, 430–433, 436, 520, 522 Protein–protein docking................................................. 88 Protein purifications.............................................. 89, 383, 387, 417, 419–422 Protein stability .................................................... 234, 270 Pseudomonas exotoxin A ............................................... 491 Purification tag .............................................................. 365

Q Quantitative MALDI-TOF .........................532, 543–544

R Random mutagenesis..........................247, 256, 263–265 Recombinant antibodies ............145, 155, 181, 428, 447 Recombinant immunotoxins............................... 489–511 Reverse transcription..................................................... 122

AND

PROTOCOLS Index 611

Ricin ............................. 29, 469–471, 474, 481–484, 486 RNA ..................... 25, 42, 51–54, 66, 72, 74, 78, 79, 90, 122, 123, 125, 131–133, 138, 140, 452, 465, 470

S Saccharomyces cerevisiae......................................... 98, 146, 159–178, 183, 318, 323 Scaffolds................................................................. 5, 8, 10, 23, 26, 76, 77, 83, 96, 97, 199, 300, 314–316, 318, 320, 322, 324–326, 328, 396, 430, 582 SDS-PAGE ............................................... 47, 61, 62, 103, 112, 149, 152, 155, 161, 186, 187, 191–194, 198, 200, 211, 224–226, 228, 289, 319, 323–325, 329, 334–337, 339, 352, 370, 379, 383, 387, 388, 402, 404, 413, 417–419, 422, 433, 435, 438, 439, 441, 442, 448, 474, 479, 494, 496, 503, 507, 508, 514, 516–519, 521, 523, 525, 527, 537, 541 Sepharose ............ 11, 318, 359–361, 364–366, 368, 369 Shake-flask ..................................................................... 444 Sharks............................................. 20–24, 29, 30, 72, 73, 78, 79, 90, 181, 300, 308, 374, 489, 490, 513 Single-cycle kinetics .................................... 195, 201, 266 Single-domain antibody...........................................37–69, 235, 243, 409–423, 431, 453, 555 Site-directed mutagenesis ................................... 249, 270, 421, 559, 562, 564 Site-saturating mutagenesis ................................. 250, 265 Site-specific labeling .........................................v, 327–342, 373–391, 395–405 Size exclusion chromatography (SEC) ......................... 61, 62, 153, 183, 192, 200, 296, 336, 338, 340, 358, 367, 385, 388, 397, 399, 402, 417, 419, 420, 430, 438, 514, 518, 522–524 SnoopTag SnoopCatcher................................................. 314, 316 Sortase ...........................................................11, 374–377, 379–381, 383, 384, 386, 388, 389 Spectrophotometry .........55, 79, 80, 256, 320, 503, 539 Spleens .................... 78, 79, 90, 128–130, 139, 140, 464 Split antibody fragment ................................................ 604 SpyTag SpyCatcher.......................................11, 146, 314, 316 Stable cell lines ..................................................... 557, 577 Standard curves ........ 140, 174, 484, 485, 544, 551, 553 Stop codons.............................................. 77, 90, 91, 115, 116, 123, 263, 271–273, 275, 277, 279, 282–285, 294, 295, 316, 368–370, 411, 416, 421 Streptavidin.......................43, 48, 58, 67, 101, 123, 131, 166, 174, 252, 261, 274, 496, 534, 537, 542, 545 Structural models ................................................. 305, 306 Sulfhydryl oxidase (SOX) .................................... 145–156 Superglues ............................................................ 313–326

SINGLE-DOMAIN ANTIBODIES: METHODS AND PROTOCOLS

612 Index

Surface plasmon resonance (SPR).......................... 62, 63, 89, 184, 187, 194, 200, 263, 266, 470 Synchrotrons ........................................................ 519, 525 Synthetic libraries ..............................................22, 23, 28, 67, 68, 72, 73, 77, 83, 85, 89, 91, 357, 514

T Tag ................................................. 11, 43, 116, 163, 228, 247, 248, 254, 255, 314, 316, 320, 325, 326, 357–370, 380, 381, 398, 400, 421, 431, 472, 474, 481, 485, 515, 535, 549, 550, 552, 556 Tandem binding domain ....................490, 493, 500, 502 T-DNA........................................................ 206, 208, 212, 214, 216, 218, 220–222, 227 Therapeutics ............................ 10, 12–14, 23–25, 28, 38, 71, 95, 121–140, 145, 146, 182, 183, 233, 234, 269, 270, 299, 300, 357, 469, 471, 490, 514 Toxins8, 10, 13, 14, 24, 29, 38, 160, 226, 246, 252, 255, 259, 448, 469–486, 498, 533 Transfections ........................................38, 46, 48, 50, 62, 68, 122, 136, 137, 437, 447, 448, 452, 456, 458, 459, 465, 466, 471, 472, 475, 478–483, 486, 561, 569, 570, 573, 575–577, 599, 602, 605 Transformations ..................... 56, 89, 91, 101, 102, 104, 112, 122, 125, 126, 135, 150, 155, 160, 161, 163, 165, 168, 170–172, 175, 176, 183, 185, 188–191, 196, 197, 206, 215, 217, 220, 227, 256, 257, 259, 284, 286, 289, 322, 323, 364, 421, 458, 492, 494, 501, 545, 562, 563, 565, 576 Transgenic mice.....................................28, 122, 127, 300 Transgenic plants..........................................206, 223–226 Transient expression................................................ 46, 61, 206, 209, 214, 217–220, 223, 225, 447, 567 Transmembrane proteins ............. 95–118, 583, 588, 590

U Unnatural amino acids..........................11, 368, 410, 411 Uracil ............................................................................. 176

V Variable heavy (VH)............... 4, 5, 10, 19, 20, 122, 133, 134, 136, 245, 301–304, 307, 308, 490, 597, 604 Variable heavy (VH) domain .........................3–5, 10, 604 Variable new antigen receptor (VNAR).................. 19–30, 71–92, 300, 308, 490

Vectors .............................. 22, 39, 42, 45, 46, 53–55, 66, 68, 75, 76, 80, 81, 83, 89–91, 102, 104, 110, 122, 123, 125, 134, 136, 140, 146–148, 150, 151, 154, 155, 159–162, 167, 184, 188, 189, 192, 193, 196–198, 200, 206, 208, 209, 212, 214–218, 221, 226, 227, 247, 255–257, 259, 266, 271, 277, 282, 294, 314, 319–322, 325, 331, 335, 340, 358, 359, 361, 362, 365, 369, 375–377, 381–383, 387, 411, 416, 419, 431, 434, 436, 437, 447, 452–454, 458, 465, 470, 472, 474–477, 479, 484, 491–494, 496–502, 511, 514–516, 520, 521, 533–536, 538–540, 545, 552, 561–568, 576, 585, 596, 598, 604 VHH ............................... 4, 7, 19, 38, 39, 42–48, 53–55, 57, 60–64, 66–68, 122, 159–178, 205–228, 245, 247, 248, 255, 259, 269, 270, 294, 300–310, 313–316, 318–321, 323, 325, 381, 409, 428, 469–471, 473–479, 483–485, 490, 513–530, 534, 535, 538–540, 552, 556, 581, 595 VHH-Fc.............................................................39, 46, 47, 61–64, 68, 206, 208–218, 223–228 VHH–protein complex ........................................ 523, 524 Virus................................8, 24, 160, 199, 206, 324, 427, 428, 430, 444, 447, 455, 458, 465, 466, 557, 571 Virus-infected cells ............ 427, 429, 435–436, 442–444 VNAR-Fc............................................................ 25–27, 29 VNAR reformatting ..................................................25, 26

W Western blots....................................................... 225, 228, 428, 429, 435, 441, 472, 474, 496, 510

X X-ray crystal structure .......................................... 513–530 X-ray diffraction ....................................86, 514, 525, 527

Y Yeast magnetization .....................................106, 108–109 Yeasts...........................5, 6, 8, 9, 25, 40, 42, 75, 95–118, 126, 148, 159–165, 167–172, 175–177, 183, 185, 186, 189, 199, 207, 248, 271–273, 300, 310, 314–318, 320, 323, 325, 328, 359, 360, 362, 377, 378, 397, 413, 432, 455, 470, 472, 473, 491, 493, 494, 515, 521, 535, 536, 559, 560 Yeast surface display ............................................... 96, 100