A disulfide-stabilized human VL single-domain antibody library is a source of soluble and highly thermostable binders

From National Research Council Canada

DOI	Resolve DOI: https://doi.org/10.1016/j.molimm.2017.07.006
Author	Search for: Henry, Kevin A.¹; Search for: Kandalaft, Hiba¹; Search for: Lowden, Michael J.¹; Search for: Rossotti, Martin A.¹; Search for: van Faassen, Henk¹; Search for: Hussack, Greg¹; Search for: Durocher, Yves¹; Search for: Kim, Dae Young¹; Search for: Tanha, Jamshid¹
Name affiliation	National Research Council Canada. Human Health Therapeutics
Format	Text
Type	Article
Journal title	Molecular Immunology
ISSN	0161-5890 1872-9142
Volume	90
Pages	190–196
Subject	single-domain antibody; protein engineering; human VL; phage display; disulfide bond; thermostability
Abstract	We have previously shown that incorporation of a second intradomain disulfide linkage into camelid VHH and human VH/VL single-domain antibodies confers increased thermostability. Here, we explored the effects of introducing an additional disulfide linkage, formed between Cys48 and Cys64 (Kabat numbering), into a phage-displayed synthetic human VL library. In comparison to an identical library bearing only the highly conserved Cys23-Cys88 disulfide linkage, the disulfide-stabilized VL library tolerated a similar degree of randomization but retained a higher level of functional diversity after selection with protein L. Both libraries yielded soluble, antigen-specific VLs that recognized a model antigen (maltose-binding protein) with similar affinities, in the micromolar range; however, the disulfide-stabilized antigen-specific VLs were much more thermostable (average ΔTm ∼10 °C) than non-disulfide-stabilized VLs. This work provides proof-of-concept for building synthetic antibody libraries using disulfide-constrained immunoglobulin domains, thus avoiding pitfalls of post-hoc disulfide linkage engineering such as impaired antigen binding and reduced expression yield.
Publication date	2017-08-15
Publisher	Elsevier
Language	English
Peer reviewed	Yes
NPARC number	23002130
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Record identifier	34a122ba-3c4d-416d-b65e-6032d9b5129a
Record created	2017-08-24
Record modified	2017-08-24

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Date modified:: 2020-02-28