A Bead-Based Screening Platform for Identifying Monoclonal Antibodies That Disrupt PD-1/PD-L1 Interactions.

Monoclonal antibodies (mAbs) targeting immune checkpoint pathways such as programmed cell death protein 1 (PD-1)/PD-L1 are central to modern immunotherapy, yet scalable methods to assess their functional blockade remain limited. We present a bead-based flow cytometry assay for quantifying the inhibition of PD-1/PD-L1 interaction by antibodies. Recombinant human PD-1 protein was conjugated to polystyrene beads, and its interaction with recombinant human PD-L1 protein labeled with a fluorochrome was measured. The inhibitory activity of an anti-PD-L1 mAb was quantified based on their ability to disrupt this interaction. The assay was validated for intra- and inter-assay precision, in addition, functionality was confirmed using a T cell coculture assay. The assay demonstrated dose-dependent inhibition by the αPD-L1 mAb, with a calculated mean IC of 3.122 µg/mL. The method proved to be reproducible for the determination of antibody blocking activity, with relative standard deviation (RSD) < 20% between three independent runs. At the concentration approximating the IC detected on the bead assay, the antibody significantly restored CD69 expression on the T cell surface (p = 0.0001) in a coculture in vitro system. In addition, the methodology could successfully distinguish the blocking capacity of two anti-PD-L1 antibodies with different affinities. This high-throughput compatible platform offers a reliable tool for screening PD-1/PD-L1 blocking antibodies, supporting immunotherapy discovery and development.