Characterization and humanization of VNARs targeting human serum albumin from the whitespotted bamboo shark (Chiloscyllium plagiosum)

Abstract

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6's ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions.