Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering

Michael J Lowden; Eric K Lei; Greg Hussack; Kevin A Henry

doi:10.1007/978-1-0716-3381-6_26

Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering

Methods Mol Biol. 2023:2702:489-540. doi: 10.1007/978-1-0716-3381-6_26.

Authors

Michael J Lowden¹, Eric K Lei¹, Greg Hussack¹, Kevin A Henry^{2

3}

Affiliations

¹ Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada.
² Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada. kevin.henry@nrc-cnrc.gc.ca.
³ Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada. kevin.henry@nrc-cnrc.gc.ca.

PMID: 37679637
DOI: 10.1007/978-1-0716-3381-6_26

Abstract

Next-generation DNA sequencing (NGS) technologies have made it possible to interrogate antibody repertoires to unprecedented depths, typically via sequencing of cDNAs encoding immunoglobulin variable domains. In the absence of heavy-light chain pairing, the variable domains of heavy chain-only antibodies (HCAbs), referred to as single-domain antibodies (sdAbs), are uniquely amenable to NGS analyses. In this chapter, we provide simple and rapid protocols for producing and sequencing multiplexed immunoglobulin variable domain (V_HH, V_H, or V_L) amplicons derived from a variety of sources using the Illumina MiSeq platform. Generation of such amplicon libraries is relatively inexpensive, requiring no specialized equipment and only a limited set of PCR primers. We also present several applications of NGS to sdAb discovery and engineering, including: (1) evaluation of phage-displayed sdAb library sequence diversity and monitoring of panning experiments; (2) identification of sdAbs of predetermined epitope specificity following competitive elution of phage-displayed sdAb libraries; (3) direct selection of B cells expressing antigen-specific, membrane-bound HCAb using antigen-coupled magnetic beads and identification of antigen-specific sdAbs, and (4) affinity maturation of lead sdAbs using tandem phage display selection and NGS. These methods can easily be adapted to other types of proteins and libraries and expand the utility of in vitro display technology.

Keywords: Antibody; Next-generation DNA sequencing; Phage display; Protein engineering; Single-domain antibody; VHH.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

B-Lymphocytes
Cell Surface Display Techniques
High-Throughput Nucleotide Sequencing
Immunoglobulin Heavy Chains / genetics
Single-Domain Antibodies* / genetics
Technology

Substances

Single-Domain Antibodies
Immunoglobulin Heavy Chains