Engineering of pH-regenerable anti-insulin single chain variable fragments (scFvs) for continuous insulin monitoring

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ID: 332166
2026
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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.
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Authors Qianming Xu, Ellie D. Wilson, Mai Hamasaki, Hirobumi Suzuki, A TANAKA, Junko Okuda‐Shimazaki, Miho Oda, Daimei Miura, Koji Sode, Ryutaro Asano
Journal The Journal of Biochemistry
Year 2026
DOI
10.1093/jb/mvag071
URL
https://doi.org/10.1093/jb/mvag071
Keywords Keywords not found

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