Characterization of Clinically Evaluated Small-Molecule Inhibitors of PD-L1 for Immunotherapy

Cancer immunotherapy aims to employ the immune system to target cancer cells. The PD-1/PD-L1 axis is a critical immune checkpoint that tumors exploit to evade immune surveillance. In this study, we characterized three small-molecule PD-L1 inhibitors, Evixapodlin, MAX-10181, and INCB086550, currently undergoing clinical trials for cancers such as non-small cell lung cancer, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, and melanoma. Using the homogeneous time resolved fluorescence assay, we confirmed that each compound potently disrupts human PD-1/PD-L1 binding with IC₅₀ values in the nanomolar range. PD-L1 oligomerization upon inhibitor binding was demonstrated through NMR analysis and confirmed by X-ray crystallography, which finally elucidated the binding interactions that stabilize these inhibitors at the PD-L1 interface. Cellular assays revealed dose-dependent T-cell activation, demonstrating the immunomodulatory potential of each compound and its cytotoxicity profiles. These findings underscore the promise of small-molecule PD-L1 inhibitors as viable alternatives to antibody-based therapies in cancer immunotherapy.