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The acidic tumour microenvironment (TME) and the abundance of tumour associated macrophages (TAMs) are key features of solid tumours that drive immune suppression, support tumour growth and limit the efficacy of approved therapies. We identified the pH sensing GPCR, GPR65, as a key determinant of low pH induced immune suppression in human cancers, particularly in TAMs, based on the following observations: 1) cancer patients who are homozygous for a hypomorphic coding variant in GPR65 (I231L) show a statistically significant increase in survival and altered expression of key immune system genes compared to other genotypes; 2) single cell RNA sequencing (scRNAseq) data from multiple solid tumors show that GPR65 and downstream pathway genes are ubiquitously expressed in myeloid and other innate immune cells in human cancers; and 3) low pH acting via GPR65 profoundly alters gene expression in human macrophages in vitro, bringing about a pronounced suppression of proinflammatory cytokines and a marked upregulation of tissue repair genes. These findings identify GPR65 as a novel innate immune check point, and suggest that GPR65 inhibition could be highly beneficial in cancer. Indeed, previous work has shown that genetic ablation of the GPR65 signaling pathway in B16.F10 tumour bearing mice upregulates immunostimulatory genes in TAMs and significantly reduces tumor growth1. Pathios has identified potent and selective antagonists of human GPR65 with excellent oral bioavailability and pharmacokinetics. In line with their potencies in recombinant cell systems, lead molecules are able to inhibit low pH induced cAMP elevations in primary human macrophages with IC50 values in the single digit nM range. In macrophages subjected to acidic conditions, the inhibitory effects on cAMP are accompanied by a reduction in the expression of anti inflammatory and tissue repair genes, and the enhancement of immunostimulatory genes. In particular, GPR65 inhibition counteracts the pronounced low pH induced suppression of key Type I/II interferon (IFN) response genes and chemokines such as CXCL9, CXCL10 and CXCL11. Following oral administration in tumour bearing mice, lead compounds up-regulate the expression of anti-tumor immune response genes at systemic exposures that significantly suppress GPR65 signalling in vitro. Results from tumour growth inhibition studies in mice with our GPR65 inhibitors will be also be presented. In summary, ‘Macrophage Conditioning’ via GPR65 inhibition holds substantial promise as a novel immunoncology strategy to counteract the immunosuppressive effects of the acidic TME on TAMs, and could be deployed either as monotherapy or in combination with T cell checkpoint inhibitors or other standard of care treatments. 1Nat. Immunol. 19:1319 Citation Format: Barbara Cipriani, David Miller, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Suorav Sarkar, Zoe Smith, Rhoanne McPherson, Anastasia Nika, Preeti Singh, Toszka Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. Inhibition of GPR65 counteracts low pH induced immunosuppressive polarization of macrophages: In vitro and in vivo characterization of potent, selective and orally bioavailable small molecule GPR65 antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2162.

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Whilst the advent of Immune Checkpoint Blockade has revolutionized the management of cancer, a significant proportion of patients have limited or absent response to these therapies. A key cause of this immune insensitivity is the hostile solid tumor microenvironment (TME) dominated by immunosuppressive myeloid cells. We previously identified the acid sensing G protein coupled receptor (GPCR), GPR65, as a primary determinant of these suppressive cells. In mice, genetic deletion of Gpr65 or oral administration of small molecule GPR65 inhibitors in vivo causes a profound repolarization of immunosuppressive tumor associated macrophages, an increase in infiltrating effector cells and potent anti tumor effects in syngeneic models. In TCGA data, across all tumors, patients homozygous for a hypomorphic coding variant in GPR65 (I231L) show increased overall survival, providing compelling genetic evidence of the clinical potential of GPR65 inhibition. To further explore the translational potential of GPR65 we employed a range of techniques to define the human biology of this receptor in different contexts. At the mechanistic level, single cell RNA sequencing (scRNAseq) of human PBMCs obtained from healthy donors demonstrated a pronounced effect of low pH on the myeloid compartment, with a clear polarization of these cells toward an immunosuppressive character and modulation of GPR65 expression. In parallel, pharmacological inhibition of GPR65 in human monocyte derived macrophages exposed to low pH demonstrated that equivalent gene expression changes are primarily due to GPR65 activation. To examine the relevance of these findings to the intact acidic human TME, we performed studies in fresh primary human tumor histocultures from clear cell renal cell carcinoma (ccRCC) patients with immunohistochemically confirmed high macrophage infiltration and carbonic anhydrase 9 (CA9) expression. In these cultures, GPR65 inhibition caused a dose dependent suppression of a geneset closely overlapping with that modulated by GPR65 in primary macrophages. Furthermore, we observed a marked decrease in immune suppressive IL10 secretion with coincident elevation of specific proinflammatory chemokines. Consistent with these findings, in vivo administration of a small molecule GPR65 inhibitor elicited similar changes in human CA9 expressing RCC PDX tumors implanted in myeloid boosted CD34+ stem cell engrafted NCG mice. In summary, inhibition of GPR65 provides a unique and genetically validated approach to favorably modify the immunosupressive TME with features highly conserved between mouse and human contexts. We propose that GPR65 inhibition holds significant clinical promise, with specific evidence around ccRCC as a potential standout indication. Citation Format: Barbara Cipriani, Alastair Corbin, David Miller, Alan Naylor, Faraz Khan, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Mussa Quareshy, Anastasia Nika, Preeti Singh, Gavin Knox, Darryl Turner, Satish Sankaran, Nandini Pal Basak, Toszka Bohn, Tobia Bopp, Surya Koturan, Bo Sun, Benjamin Fairfax, Tom McCarthy, Stuart Hughes. The translational biology of small molecule GPR65 inhibitors: shared effects between mouse models and human primary tumors highlight the unique transformative potential of targeting a genetically validated innate immune checkpoint [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 668.