Abstract 3156: A surface plasmon resonance-based screening platform to identify potential PD-1/PD-L1 inhibitors from a FDA-approved drugs library for cancer immunotherapy

Abstract The PD-1/PD-L1 axis plays a pivotal role in immune checkpoint regulation in cancer, making its inhibition a key strategy in cancer immunotherapy. However, the development of small molecule inhibitors targeting PD-1/PD-L1 interaction has been hindered by the lack of robust high-throughput screening platforms. In this study, we developed a high-throughput screening platform based on surface plasmon resonance (SPR) technology, utilizing a 384-well plate format. This platform integrates a dual-step coupling method, employing both amine and bio-streptavidin coupling to optimize PD-1 orientation on the sensor chip.We screened a library of 2, 764 FDA-approved drugs using this SPR platform with a 1:1 ratio of drug to PD-L1, alongside two positive control PD-L1 inhibitors. Three compounds, RB, MB, and VP, were identified as hits, showing PD-1/PD-L1 blockade rates of 13.1%, 9.6%, and 6.7%, respectively, compared to the positive controls BMS-1166 (33%) and BMS-202 (8.4%). Structural validation through saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy confirmed the binding of RB to PD-L1, with a unique STD peak at 7.6 ppm, further supported by a WaterLOGSY control experiment indicating a negative nuclear overhauser effect to water. The dissociation constants KD of RB were determined using SPR, revealing moderate binding affinities to PD-L1.Cytotoxicity assays conducted on Jurkat T cells and A375 melanoma cells demonstrated that RB exhibited no toxicity across a concentration range from 100 μM to 0.01 μM. Functional assays using a co-culture model of T cells and cancer cells, designed to replicate the tumor microenvironment, showed that RB treatment modulated IL-2 secretion and induced apoptosis in A375 cancer cells. Proteomic analysis of sorted Jurkat T cells from the co-culture model via bottom-up proteomics revealed key proteins and signaling pathways regulated by RB.Overall, these findings suggest that RB exerts significant PD-1/PD-L1 inhibitory activity and may hold promise as a repurposed drug candidate for cancer immunotherapy. Citation Format: Huifang Li, Ang Cai, Navindra P. Seeram, Chang Liu. A surface plasmon resonance-based screening platform to identify potential PD-1/PD-L1 inhibitors from a FDA-approved drugs library for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3156.