Engineered single chain variable fragments (scFvs) with improved pH-dependent kinetics for use in continuous...

One challenge in the continuous monitoring of insulin lies with the unavailability of a high-affinity biological recognition element (BRE) with kinetic parameters sufficient to track fluctuating concentrations of insulin in vivo. An approach to overcome this limitation is to engineer a high-affinity BRE to selectively modulate its binding kinetics in response to an external signal. Herein, we design and evaluate changes in the pH-dependent binding kinetics of an anti-insulin single chain variable fragment (scFv) we previously employed in a point-of-care insulin sensor. We predicted the scFv structure in complex with human insulin and selected scFv residues directly involved in insulin binding for histidine substitution. We identify one mutation, T32H, that improves the pH-sensitivity of the wild-type (WT); the K _D of the T32H mutant is calculated to be 145.5 ± 83.1 nM at pH 7.4 and 17.4 ± 5.1 nM at pH 6.0 - an average of an 8.4× difference between the two conditions and a 3.8× increase in pH-sensitivity from the WT. We design a bio-layer interferometry (BLI) assay to interrogate the improved pH-sensitivity of the T32H mutant in tracking fluctuating insulin concentrations in dynamic pH conditions and find that improved pH-sensitivity can be leveraged to improve biosensor regeneration. These results suggest the potential for pH-sensitive antibodies to improve the development of in vivo continuous monitoring systems.