Presentation Information
[P01-097]Development of pH-responsive antibodies for continuous insulin monitoring
○Qianming XU1, Ellie Dewese Wilson2, Hirobumi Suzuki1, Junko Okuda-Shimazaki1, Koji Sode2, Ryutaro Asano1 (1. Tokyo University of Agriculture and Technology (Japan), 2. University of North Carolina at Chapel Hill (USA))
Keywords:
Antibody,Biosensor,Biosensing,Protein engineering,Biomedical engineering,Healthcare material,Immonosensor,Biotechnology
We strategically engineered pH-responsive anti-insulin single chain variable fragments (scFvs), which are promising biological recognition elements (BREs) for continuous insulin monitoring systems.
Antibodies are widely used as BREs in various clinical analyses for the diagnosis of cancer, neurodegenerative diseases, and viral infections, and in the development of biosensors. However, there lies a tradeoff with utilizing highly sensitive and selective BREs in continuous monitoring systems as target binding is typically favored thermodynamically. Therefore, developing technologies/methods/devices to regenerate antibodies both in situ and in vivo is crucial to expand their utility for in vivo continuous sensing devices. To realize this, we have focused on engineering antibodies that are capable of binding site regeneration through a mild pH change inspired by the well-documented approach to confer pH-dependent binding through histidine (His) mutagenesis [1]. As the pKa of histidine is near physiological pH, we can alter the electrostatic interactions between antibodies and their targets by introducing His residues at specific regions within the binding interface and mildly changing pH.
Our group is developing an in vivo, real-time, and continuous monitoring system of insulin by developing pH-responsive anti-insulin scFvs. We recently reported an anti-insulin scFv with histidine mutations that exhibited faster dissociation at physiological pH [2]. Here, we utilized the co-crystal structure of a different anti-insulin antibody in complex with insulin to design mutants to achieve faster dissociation at lower pH, ideal for continuous monitoring applications. We strategically designed mutations in the antigen binding site of this antibody and expressed the scFv in Escherichia coli. Following affinity chromatography and gel filtration chromatography, the affinity and the specificity of the mutants were evaluated by biolayer interferometry (BLI) and enzyme-linked immunosorbent assay (ELISA), respectively.
We obtained promising mutant scFvs that showed decreased affinity at pH 6.0, while maintaining high affinity and specificity to insulin at pH 7.4. We further performed repeated monitoring of insulin with BLI by incubating the mutant scFvs with various concentrations of insulin in the association step and regenerating with pH 6.0 buffer. The results demonstrated concentration-dependent binding signals, even during decreasing insulin concentrations. Our results revealed that we engineered an scFv whose binding site was successfully regenerated by a mild pH change. We are now working on electrochemical devices for continuous monitoring.
[1] Igawa T. et al., Nat. Biotechnol., 28, 1203–1207 (2010)
[2] Wilson E. et al., RSC Adv., 15, 25337–25348 (2025)
Antibodies are widely used as BREs in various clinical analyses for the diagnosis of cancer, neurodegenerative diseases, and viral infections, and in the development of biosensors. However, there lies a tradeoff with utilizing highly sensitive and selective BREs in continuous monitoring systems as target binding is typically favored thermodynamically. Therefore, developing technologies/methods/devices to regenerate antibodies both in situ and in vivo is crucial to expand their utility for in vivo continuous sensing devices. To realize this, we have focused on engineering antibodies that are capable of binding site regeneration through a mild pH change inspired by the well-documented approach to confer pH-dependent binding through histidine (His) mutagenesis [1]. As the pKa of histidine is near physiological pH, we can alter the electrostatic interactions between antibodies and their targets by introducing His residues at specific regions within the binding interface and mildly changing pH.
Our group is developing an in vivo, real-time, and continuous monitoring system of insulin by developing pH-responsive anti-insulin scFvs. We recently reported an anti-insulin scFv with histidine mutations that exhibited faster dissociation at physiological pH [2]. Here, we utilized the co-crystal structure of a different anti-insulin antibody in complex with insulin to design mutants to achieve faster dissociation at lower pH, ideal for continuous monitoring applications. We strategically designed mutations in the antigen binding site of this antibody and expressed the scFv in Escherichia coli. Following affinity chromatography and gel filtration chromatography, the affinity and the specificity of the mutants were evaluated by biolayer interferometry (BLI) and enzyme-linked immunosorbent assay (ELISA), respectively.
We obtained promising mutant scFvs that showed decreased affinity at pH 6.0, while maintaining high affinity and specificity to insulin at pH 7.4. We further performed repeated monitoring of insulin with BLI by incubating the mutant scFvs with various concentrations of insulin in the association step and regenerating with pH 6.0 buffer. The results demonstrated concentration-dependent binding signals, even during decreasing insulin concentrations. Our results revealed that we engineered an scFv whose binding site was successfully regenerated by a mild pH change. We are now working on electrochemical devices for continuous monitoring.
[1] Igawa T. et al., Nat. Biotechnol., 28, 1203–1207 (2010)
[2] Wilson E. et al., RSC Adv., 15, 25337–25348 (2025)
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