Regulation of immune responses is tightly controlled through a balance of co-stimulatory and inhibitory checkpoint receptors, often exploited by many cancers. Therefore, therapeutics that block inhibitory receptors have proved to be powerful agents to restore anti-tumor immune responses. One key inhibitory checkpoint receptor that is the target of several therapeutic agents in the clinic is programmed cell death 1 (PD-1). PD-1 is expressed on T-cells, while it’s ligands, PD-L1 or PD-L2, are expressed on the surface of tumor cells or antigen presenting cells. Like many other immunoglobulin receptors, PD-1 harbors immunoreceptor tyrosine inhibitory motifs (ITIMs) in it’s cytoplasmic tail that are important signaling motifs. When its ligand, e.g. PD-L1, binds to PD-1, Src family kinases phosphorylate the ITIM motif, resulting in the recruitment of SH2-domain containing phosphatases, SHP-1 and SHP-2, which are involved in inhibiting the T-cell response.
Here, we present a robust assay for quantifying SHP recruitment to PD-1 using our proprietary enzyme fragment complementation (EFC) technology. Jurkat cells expressing the PD-1 and SHP-1 proteins, each fused to a fragment of our EFC system, are co-incubated with ligand-presenting cells. This results in PD-1 activation and SHP-1 recruitment to the PD-1 receptor, bringing together the two EFC fragments and generating a light signal. We demonstrate the suitability of the assay for quantifying pathway activation as well as inhibition of PD-1 signaling by both anti-ligand (anti-PD-L1) and anti- receptor (anti-PD-1) antibodies. The assay is rapid (<4 hours), extremely robust, and has an excellent assay window (>20-fold) with unparalleled sensitivity. In summary, the EFC-based PD-1 assay provides a valuable tool for both drug screening & characterization assays, with a possible role in lot release testing and stability studies during drug manufacture. This also provides a proof of concept for developing assays for other therapeutically relevant checkpoint receptors, such as TIGIT and CD47.