Authors
Qianming Xu, Ellie D Wilson, Mai Hamasaki, Hirobumi Suzuki, Ayumi Tanaka, Junko Okuda-Shimazaki, Miho Oda, Daimei Miura, Koji Sode, Ryutaro Asano
Published in
Journal of biochemistry. Oct 12, 2026. Epub Oct 12, 2026.
Abstract
To realize the continuous monitoring of insulin, the development of a regenerable engineered antibody is expected, whose binding constant is modulated by the mild in situ stimulation for future in vivo application. In this study, we aimed to develop pH-responsive anti-insulin antibodies based on a clinically established analytical antibody, the HUI-018 anti-insulin antibody. Its high-resolution co-crystal structure with human insulin (PDB ID: 6Z7W) provides a uniquely precise blueprint for molecular engineering by preparing engineered HUI-018 single-chain variable fragment (scFv). Among the engineered variants, the N93H/E101K mutant exhibited the biggest pH-responsiveness, showing a 5.2-folds increased dissociation rate at pH 6.0 while maintaining binding affinity and specificity comparable to wild-type (WT) at pH 7.4. Repeated cycles of insulin binding at pH 7.4 and regeneration at pH 6.0 demonstrated that the N93H/E101K mutant achieved substantially higher regeneration and rebinding efficiencies than WT. These results demonstrate that engineered pH-responsive antibody enables efficient binding site regeneration through mild pH change. To our knowledge, this is the first report of an anti-insulin antibody capable of binding site regeneration at pH 6.0, representing an important step toward antibody-based continuous insulin monitoring and continuous sensing of protein biomarkers.
PMID:
42861879
Bibliographic data and abstract were imported from PubMed on 12 Oct 2026.
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