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

From National Research Council Canada

DOIResolve DOI: https://doi.org/10.1016/j.molimm.2017.07.006
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Name affiliation
  1. National Research Council of Canada. Human Health Therapeutics
FormatText, Article
Journal titleMolecular Immunology
ISSN0161-5890
1872-9142
Volume90
Pages190–196
Subjectsingle-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
PublisherElsevier
LanguageEnglish
Peer reviewedYes
NPARC number23002130
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Record created2017-08-24
Record modified2020-03-16
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