A disulfide-stabilized human VL single-domain antibody library is a source of soluble and highly thermostable binders. (October 2017)

• Record Type: Journal Article
• Authors: Henry, Kevin A.; Kandalaft, Hiba; Lowden, Michael J.; Rossotti, Martin A.; van Faassen, Henk; Hussack, Greg; Durocher, Yves; Kim, Dae Young; Tanha, Jamshid
• Is Part Of: Molecular immunology. Volume 90(2017:Oct.)
• Page Start: 190
• Page End: 196
• Publication Date: 2017-10
• Subjects: CDR complementarity-determining region -- FR framework region -- sdAb single-domain antibody -- VL variable light chain immunoglobulin domain; Single-domain antibody -- Protein engineering -- Human VL -- Phage display -- Disulfide bond -- Thermostability; Immunochemistry -- Periodicals; Molecular biology -- Periodicals; Immunochemistry -- Periodicals; Allergy and Immunology -- Periodicals; Molecular Biology -- Periodicals; Immunochimie -- Périodiques; Biologie moléculaire -- Périodiques; Immunochemistry; Molecular biology; Periodicals; Electronic journals; 571.96
• DOI: 10.1016/j.molimm.2017.07.006
• Languages: English
• ISSNs: 0161-5890

Abstract

Abstract: We have previously shown that incorporation of a second intradomain disulfide linkage into camelid VH H 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 VL s that recognized a model antigen (maltose-binding protein) with similar affinities, in the micromolar range; however, the disulfide-stabilized antigen-specific VL s were much more thermostable (average Δ T m ∼10 °C) than non-disulfide-stabilized VL s. 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.