Site-specific Effector Protein Functionalization to Create Bead-based Avidity Model Systems

Abstract The cooperative effect of multiple affinity binding interactions creating a stable bond, known as avidity, is particularly important in assessing the potency of potential drugs such as monoclonal antibodies, CAR T, or NK cells to treat cancer. However, predicting avidity based on in vitro single affinity interactions has limitations and often fails to describe the avidity effects observed in vivo . Acoustic force-based assays have recently emerged as a reliable method for direct avidity measurements, expressed as adhesion forces, which positively correlate with drug efficacy. However, to better understand avidity, in particular for cell-cell interactions and correlate it with affinity, a cell model system with controlled avidity-related properties is needed. This study presents a method for producing a cell model system using “effector beads” that can be used in acoustic force spectroscopy-based avidity assays or any other bead-based avidity assay. The protein of interest is biotinylated in vivo in E.coli , purified and subsequently mixed with streptavidin coated beads to create effector beads. The results demonstrate the dependency of rupture force on the receptor surface density and force loading rate, thus providing valuable information for designing future effector bead assays as well as cell avidity measurements for screening and characterization purposes.