Background Currently approved PD-L1/PD-1 targeted immuno-oncology therapy involves intravenous administered monoclonal antibodies against PD-1 or PD-L1 that can effectively block the interaction between PD-1/PD-L1 proteins located on cell membrane surfaces. Small-molecule PD-L1 inhibitors have been demonstrated to have distinct mechanisms from and potential advantages over well-known mAbs by targeting both the surface and intracellular PD-L1. This study focuses on the discovery and identification of two novel orally active small-molecule inhibitors, CU-B103 and CU-B206.

Methods The ALPHA assay was used for PD-L1/PD-1 interaction evaluation. T cell activation was assessed through NFAT-PD-1 Jurkat T cell reporter assay and IFN-γ/IL-2 ELISA. Mechanisms of PD-L1 signaling blockade were studied using flow cytometry, confocal imaging, and western blotting with enzyme digestion. The anti-tumor effects were evaluated in 2D and 3D tumor models using co-cultures of PBMCs and A375 cells. Immune cell infiltration in the 3D model was examined by confocal microscopy. In vivo efficacy and tissue penetration were investigated in humanized PD-L1 mice bearing MC38-hPD-L1 tumors. MDCKII-WT, MDCKII-BCRP and Caco-2 monolayers were used in in vitro transporter studies.

Results CU-B103 and CU-B206 potently inhibited hPD-L1/hPD-1 protein-protein interaction (IC50 = 0.2 nM), and effectively induced Jurkat T cell activation and rescued IFN-γ/IL-2 secretion from human T cells when co-cultured with hPD-L1 overexpressing cells. Distinct from the anti-PD-L1 antibody atezolizumab, the compounds promoted PD-L1 internalization, intracellular retention and degradation, leading to a long-lasting loss of PD-L1 signal from the cell surface. They also altered the glycosylation pattern of PD-L1. In the 2D tumor-killing assay format, they demonstrated T cell cytolytic activity comparable to anti-PD-L1 antibody. In the 3D tumor spheroid model, both compounds exerted a greater cytotoxic effect vs the antibody, with greater increased immune cell infiltration and decreased tumor size. Consistent with the in vitro mechanistic studies, the orally dosed CU-B206 demonstrated in vivo target occupancy in mice bearing MC38 hPD-L1 tumor, at a level much greater than anticipated based on the corresponding in vitro assay. Additionally, CU-B206, at Ctrough/EC50 ratio of <0.2, exhibited comparable efficacy as approved mAbs. CU-B206 also showed excellent tumor and brain exposure in mice, consistent with its high passive permeability and insusceptibility to P-gp and BRCP efflux transporters.

Conclusions CU-B103 and CU-B206 are small-molecules with distinct mechanisms in inhibiting PD-L1, and with demonstrated corresponding in vitro and in vivo efficacy profiles. They exhibited favorable ADME/PK properties in preclinical studies, with acceptable safety profile, supporting further development as potential preclinical candidates.

Acknowledgements This work is mainly supported by Rachadapisek Sompoch Endowment Fund (Chulalongkorn University, Bangkok, Thailand), and partially supported by Thai Government Budget Grant (Thailand).