Evaluation of small molecule PD-1/PD-L1 inhibitors in a T cell reporter system

Antibodies that block PD-1 signaling, known as immune checkpoint inhibitors (ICIs), have revolutionized cancer treatment. Small molecule inhibitors of the PD-1/PD-L1 interaction could serve as promising alternatives to antibody-based ICIs, offering advantages including reduced cost, the option of oral administration, and their small size allows for deeper tissue penetration. Numerous such compounds have been described, but many have not been evaluated in well-defined cellular systems, and comparative studies are scarce. We tested eleven small molecule inhibitors using Jurkat-PD-1-reporter cells activated by stimulator cells expressing PD-L1. Additionally, their effects on T cell reporter activation and viability were evaluated. We found that ARB-272572, INCB086550, Evixapodlin, and PD-1/PD-L1 Inhibitor 3 completely reversed the inhibitory effects of PD-1. Except for PD-1/PD-L1 Inhibitor 3, these compounds blocked PD-1 inhibition with EC50 values in the low nanomolar range. Interestingly, seven PD-1 inhibitors failed to fully block PD-1 inhibition in our cellular assay. BMS-1166 and PD-L1-IN3 showed some blocking capacity but could not fully restore the activation of PD-1 reporter cells. AUNP-12, BMS1, BMS202, CA170, and PD-1/PD-L1-IN-9 were ineffective at reducing PD-1-mediated reporter inhibition. Moreover, our data indicate that adverse effects on T cell reporter activation compromise the activity of several of the tested compounds. In summary, our results revealed that the majority of small molecule PD-1/PD-L1 blockers possess a limited capacity to reverse PD-1 inhibition in a T cell reporter platform and highlight the importance of cellular assays to assess the therapeutic potential of drugs targeting the PD-1/PD-L1 axis.