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

We have previously shown that incorporation of a second intradomain disulfide linkage into camelid VH and human V/V 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 V library. In comparison to an identical library bearing only the highly conserved Cys23-Cys88 disulfide linkage, the disulfide-stabilized V 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 Vs that recognized a model antigen (maltose-binding protein) with similar affinities, in the micromolar range; however, the disulfide-stabilized antigen-specific Vs were much more thermostable (average ΔT ∼10°C) than non-disulfide-stabilized Vs. 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.