Kinetic measurements of membrane target level and on-cell binding of biotherapeutic antibodies using flow-cytometry

Abstract Characterization of cell surface target expression is critical for the development of therapeutics that bind membrane proteins or use changes in target abundance as efficacy biomarkers. Target level is often analyzed with flow-cytometry by converting signal intensity of fluorophore-conjugated target-specific monoclonal antibodies (mAbs) into an Ab binding capacity (ABC) number, as determined at binding equilibrium using a calibrator. Here, we examined the feasibility of simultaneous estimation of ABC as well as mAb binding and dissociation rates in cell populations of interest, by mathematical modeling of experimentally observed time-dependent changes in the mAb-target complex formation. First, using three different target-specific mAbs, conjugated to a fluorophore in an equimolar ratio, we demonstrated that by modeling mAb binding kinetics, simultaneous quantification of ABC and apparent affinity (Kd) by flow cytometry was feasible, and the estimates were similar to calibration-based benchmark measurements with these mAbs. Next, by comparing different fluorophore formats of the same mAbs, we found that while kinetic data fitting with employed model can estimate the ABC and apparent affinity for mAbs, regardless of fluorophore type, the results can be sensitive to the model’s assumptions of fluorophore to Ab conjugation ratio. Collectively, our findings indicated that the described experimental and modeling approaches can be applied for the concomitant analysis of membrane target level and Ab on-cell affinity, circumventing the need for complex multiple experiments, expediting data delivery for drug discovery. Graphical Abstract