Simultaneous Measurement of Total and Occupied/Free PD-1 on a Single Cell by Mass Cytometry: Development of a Novel Method for Receptor Occupancy Assessment

Receptor occupancy (RO) assays are often used to quantify and characterize the binding profiles of antibody-based therapeutic drugs to target receptors, providing valuable pharmacodynamic insights. Programmed cell death protein 1 (PD-1) is a key immune checkpoint that suppresses T cell inflammatory activities, and monoclonal antibodies targeting PD-1 have been developed to stimulate T activity against a wide range of malignancies. Although flow cytometry has enabled PD-1 RO assessment, current applications have largely focused on individual RO measurements. The development of antibody-based strategies that allow quantitative RO assessment at the single-cell level, together with simultaneous characterization of broader immune phenotypes, remains a challenge. Here, we described two mass cytometry-based methods for PD-1 RO measurement. First, we utilized two non-competing anti-PD-1 antibodies (clones EH12.2H7 and J105) and an anti-human IgG4 antibody (clone G17-4), which has high specificity to pembrolizumab and nivolumab, to simultaneously measure detectable/accessible PD-1 and occupied PD-1, respectively. Second, we used metal-conjugated PD-1 inhibitors to directly characterize RO. Using these methods, we demonstrated that neither anti-PD-1 antibody clone directly competed with pembrolizumab or nivolumab, and that PD-1 receptor binding sites were down-regulated after PD-1 inhibitor treatments. Finally, either approach can be used to approximate PD-1 RO in conjunction with high parameter assessment of other important surface and intracellular markers by mass cytometry. Our findings showed that mass cytometry RO assays can be incorporated into high parameter analyses of immune cell surface and intracellular phenotypes, facilitating high complexity analysis of immunotherapy responses in preclinical and clinical stages of drug development.