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Introduction: Cancer cells can escape tumor-specific T cell responses via engagement of inhibitory immune checkpoints. PD-L1/PD-1 interaction is one of the major checkpoints that limit effector T cell function against cancer cells, and monoclonal antibodies that block this interaction have been approved as therapies in multiple tumor indications. As a next generation therapy, small molecule inhibitors of PD-L1 potentially have advantageous properties compared to approved monoclonal antibodies, such as better penetration into solid tumors, reduced immunogenicity, lack of Fc-mediated side effects, convenience of oral administration, and lower cost of goods. We therefore embarked on an effort to identify and develop an orally available small molecule capable of targeting PD-L1/PD-1 interactions. Methods: Inhibition of the PD-L1/PD-1 interaction was measured using a binding assay, followed by a cell culture system assessing PD-1 inhibition of T cell receptor (TCR) activation. Human T cell responses were assessed in vitro using the mixed lymphocyte reaction (MLR) assay, and a human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. For in vivo studies CCX559 was dosed orally in a syngeneic tumor model and in a human tumor cell/PBMC co-implantation model in immune deficient mice. Results: Using structural information and focused medicinal chemistry, we identified CCX559 as a potent inhibitor of PD-L1 interaction with PD-1. CCX559 prevented PD-L1/PD-1 inhibition of TCR signaling in a cell-based reporter assay, increased IFNγ secretion in allogeneic MLR assays, and increased tumor cell killing by human PBMCs. We demonstrated that CCX559 potentially employs multiple mechanisms to inhibit PD-L1, which are distinct from those published for human anti-PD-L1 antibodies. In murine tumor models, orally administered CCX559 reduced tumor growth similarly to a clinically-approved anti-human PD-L1 antibody. Summary: CCX559 is a highly potent, small molecule PD-L1 inhibitor that can be orally administered. CCX559 enhanced primary human T cell activity in vitro and demonstrated anti-tumor efficacy in two murine tumor models. Based on its unique mechanism of PD-L1 inhibition, strong anti-tumor activity, desirable drug properties, and good safety profile, we plan to advance CCX559 into clinical development in the first half of 2021. Citation Format: Chris Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Scamp, Vicky Chhina, Alice Kumamoto, Ryan Ong, Ton Dang, Ashton Easterday, Niky Zhao, Shirley Liu, Rajinder Singh, Israel Charo, Kathleen Sullivan, Thomas J. Schall, Penglie Zhang. CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1274.

Introduction: Antibody-based therapies targeting the Programmed cell Death-1/ Programmed Death-Ligand 1 (PD-1/PD-L1) immune checkpoint axis have achieved great success in cancer immunotherapy in recent years. As a next generation therapy, small molecule inhibitors of PD-1/PD-L1 offer the potential for increased tumor penetration, shorter half-life (to better manage immune related adverse events), and lower cost. We therefore embarked on an effort to identify and develop orally available small molecules capable of targeting PD-L1. Methods: We designed and optimized a number of small molecule PD-L1 inhibitors which potently disrupted the interaction of PD-1 with PD-L1. Active compounds were first profiled by an ELISA assay measuring inhibition of the PD-1/PD-L1 interaction, followed by a functional cell-based reporter assay, mixed lymphocyte reaction (MLR) assay, and human peripheral blood mononuclear cell (PBMC)-mediated tumor cell killing assay. Targeted medicinal chemistry efforts were employed to improve potency and oral bioavailability, and candidate compounds were then evaluated in murine tumor models. Due to the specific reactivity of these compounds to human PD-L1 (but not murine PD-L1), a syngeneic tumor model with murine MC-38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 tumor model) was used. Results: The optimized PD-L1 inhibitors were highly potent in cell-based reporter assay, the MLR assay and PBMC-mediated tumor cell killing assays. These compounds also possess high oral bioavailability and desirable safety profiles. In the MC38-hPD-L1 tumor model, Lead compounds potently reduced tumor growth similarly to an anti-human PD-L1 antibody, which was used as a positive control for the experiments. The tumor microenvironment analysis by flow cytometry demonstrated that these compounds almost completely occupied human PD-L1 on the tumor cells in vivo, and thus could potently block the interaction of PD-1/PD-L1 and enhance the immune responses against tumor. Summary: We have identified and advanced unique small molecule inhibitors of human PD-L1 by rational design and optimization. Molecules resulting from these efforts exhibited marked inhibition of the PD-1/PD-L1 interaction and signaling in vitro, and potent anti-tumor effects in an animal model. Citation Format: Shijie "Chris" Li, Marta Vilalta, Linda S. Ertl, Yu Wang, Yibin Zeng, Pingchen Fan, Christopher Lange, Darren McMurtrie, Ju Yang, Rebecca Lui, Ryan Ong, Vicky Chhina, Alice Kumamoto, Simon Yau, Ton Dang, Ashton Easterday, Shirley Liu, Rajinder Singh, Israel Charo, Thomas J. Schall, Penglie Zhang. Anti-tumor effect of orally available small molecule PD-L1 inhibitors in a murine model of colon adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5693.