Prostaglandin E2 (PGE2) generated by the cyclooxygenase (COX) pathways regulates both inflammation and tumor microenvironment (TME). Among the four E-type prostanoid receptors EP1-4, it has been demonstrated that PGE2 exhibits its immune suppression and tumorigenic function through engagement with EP2 and EP4. Mechanistically, tumor-derived PGE2 acts on EP2 and EP4 in immune cells (e.g., NK cells and dendritic cells) and elicits immunosuppression in TME1,2. Importantly, the deletion of both EP2 and EP4, but not deletion of either receptor, is required to reverse PGE2-mediated immune evasion. Furthermore, high levels of PGE2-EP2/EP4 associated inflammatory signature have been shown in multiple human cancer types to correlate with poor prognosis and worse response to immune checkpoint blockade in the clinic1. Therefore, PGE2-EP2/EP4 signaling likely acts as a key regulatory node linking inflammation and immunosuppression in TME, and its inhibition may lead to a less immunosuppressive TME and enhance the response to immune checkpoint inhibitors. COX inhibitors have been used to block PGE2 synthesis but cause cardiovascular and gastrointestinal risks due to the inhibition of all prostanoids synthesis3. Thus, therapeutically targeting both EP2 and EP4 is an effective and more selective approach to limit the immunosuppressive functions of PGE2 while avoiding potential side effects associated with COX inhibition. Here we describe the discovery of a potent and selective EP2/EP4 dual antagonist DX-002. At 10 nM concentration, it displayed >90% inhibition of PGE2 binding to EP2 or EP4, whereas no significant inhibition was seen towards PGE2 binding to EP1 or EP3. In human NK cells DX-002 reversed PGE2-mediated inhibition of IFN-γ production more potently than selective EP2 or EP4 antagonist. DX-002 has excellent pharmacokinetics profiles across all animal species. In the mouse tumor model, treatment of DX-002 produced superior tumor regression as a monotherapy over selective EP2 or EP4 antagonists. In addition, DX-002 also provided significant synergistical effect for tumor regression in combination with anti-PD1 in both immune checkpoint inhibitor insensitive and sensitive models, in association with increased tumor infiltration of CD8+ T cells. Based on its in vitro and in vivo profiles along with other properties, DX-002 is being profiled as a development candidate as a potential cancer immunotherapeutic agent. Citation Format: Lintong Li, Guorui Yao, Wei Zheng, Yongshuai Chai, Zhongying Huang, Guojian Ma, Linlin Nie, Dengfeng Xu, Jiuquan Yang, Nanxin Li. DX-002, a dual EP2 and EP4 antagonist, alters the tumor microenvironment to enhance tumor immunogenicity and responses to immune checkpoint inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5175.