Comparative analysis of anti-MICA scFv affinities: Insights from three label-free biophysical methods and biological...

Antibody-antigen affinity is a key factor in the development of therapeutic antibodies, influencing candidate selection and their clinical efficacy. We used a combination of three label-free biophysical techniques: SPR, ITC, and fluorescence quenching, to rank anti-MICA single-chain variable fragments (scFvs), complemented by ELISA. We evaluated a wild-type (WT) scFv and its Beta mutant across the platforms, complementing this with molecular dynamics simulations and functional assays. The affinity constants (K_D) varied depending on the method used, but SPR provided the most precise discrimination. Flow cytometry confirmed stronger binding of the WT scFv to MICA-expressing gastric cell lines, which was consistent with the results obtained using SPR. Simulations indicated greater conformational stability and more favourable antigen contacts for the WT scFv. Together, these results highlight the value of orthogonal measurements for robust affinity assessment and support WT as a promising lead for further development. This benchmark provides practical guidance for selecting affinity assays in discovery pipelines.