Gpr65 Is A Critical Mediator Of Low Ph Induced Immunosuppressive Signalling In Tumor Associated Macrophages: Human Target Validation Of Gpr65 As A Novel Innate Immune Checkpoint And Discovery Of Potent, Selective Gpr65 Antagonists.

Abstract Tumor-associated macrophages (TAMs) are the major innate immune component in the microenvironment of solid tumors. These cells are highly heterogeneous and plastic but often display a pronounced immunosuppressive phenotype that supports primary tumor growth and metastasis. A recently identified determinant of the immunosuppressive properties of TAMs is the activation of the pH-sensing G protein-coupled receptor, GPR65, on these cells by the acidic microenvironment that is inherent to many advanced solid tumours1. Previous work in mouse macrophages has shown that GPR65 activation leads to an elevation of inducible cAMP early repressor (ICER), an isoform of the CREM gene, which in turn suppresses the expression of a host of proinflammatory mediators1. Consistent with a high expression of GPR65 and CREM in human myeloid cells, and particularly in human TAMs, we now show that equivalent immunosuppressive signaling is also present in human macrophages in response to low pH. Further substantiating GPR65 as an innate immune checkpoint in human cancers, we also show that subjects that are homozygous for a hypomorphic coding variant in GPR65 (I231L) have a significantly improved survival across a range of cancers compared to other genotypes. This survival advantage is maintained in patients with highly glycolytic tumors that would otherwise be predicted to respond poorly to immunotherapy, and is consistent with studies showing that genetic deletion of ICER attenuates the growth of anti-PD-1-resistant B16.F10 melanoma tumors in mice1. With a view to developing a therapeutic agent able to reverse low pH-dependent immunosuppression in TAMs, we identified potent and selective small molecule inhibitors of GPR65 from a screening campaign. These molecules were able to fully ablate the acidic pH-induced expression of ICER in human macrophages with downstream implications for pro-inflammatory cytokines known to support T cell anti-tumor responses. Additional medicinal chemistry optimization led to molecules exhibiting excellent oral bioavailability in preclinical species and systemic exposure that completely suppressed GPR65 signaling following oral dosing in mice. Profiling of these inhibitors in relevant mouse tumor models is currently ongoing. In conclusion, we have (i) demonstrated that GPR65 is a key determinant of low pH-induced immunosuppression in human macrophages and thus an important innate immune checkpoint in cancer, and (ii) identified selective small molecule inhibitors of GPR65 with potent in vivo activity. This work provides the basis for developing novel “macrophage conditioning” therapeutic agents that may have utility across a range of cancers, either as single agents or in combination with other approved immunoncology drugs. 1Nat Immunol 19:1319. Citation Format: Barbara Cipriani, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Zoe Smith, Louise Healy, John Unitt, Jessica Holien, Rhoanne McPherson, Anastasia Nika, Jessy Cartier, Tozska Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. GPR65 is a critical mediator of low pH induced immunosuppressive signalling in tumor associated macrophages: Human target validation of GPR65 as a novel innate immune checkpoint and discovery of potent, selective GPR65 antagonists [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 1631.