Background An important facet of the hostile tumor microenvironment (TME) and a recognized driver of cancer immunosuppression is extracellular acidification which results from the glycolytic nature of rapidly proliferating cancer cells. Pathios identified GPR65, a pH-sensing G protein-coupled receptor (GPCR), as the primary sensor of this acidity in human immune cells. Activation of GPR65 is particularly pronounced in macrophages where it leads to the downregulation of key chemokines and anti-tumorigenic cytokines, and the upregulation of pro-tumorigenic wound repair and remodeling factors. In dendritic cells GPR65 signaling suppresses antigen presentation pathways, whereas in T cells and natural killer cells it leads to the suppression of chemokine receptors required for effective tumor homing. Consequently, acid sensing by GPR65 can explain tumor resistance to current immunotherapies. Validating the relevance of this target in immuno-oncology, cancer patients homozygous for the hypomorphic GPR65 variant (I231L) exhibit profoundly improved survival compared to patients with other genotypes.

Methods Pathios has developed PTT-4256, a first-in-class small molecule inhibitor of GPR65. The in vitro efficacy of PTT-4256 under low pH conditions was determined by cAMP screening, gene expression and cytokine release profiling of primary immune populations. Anti-tumor activity was assessed using the MC38 and B16.F10 syngeneic mouse models, whilst immunomodulatory effects in MC38 tumor-infiltrating leukocytes were profiled by targeted RNAseq. Notably, PTT-4256 is markedly more potent at human GPR65 versus the mouse receptor. To determine its full potential in patients and to develop an accurate prediction of efficacious human exposure, we employed a range of approaches including molecular pharmacology, site-directed mutagenesis, PK/PD modelling, and genetically engineered mice.

Results PTT-4256 fully counteracts the low pH-driven immunosuppressive transcriptional program in human and murine immune cells and restores the secretion of key pro-inflammatory cytokines and chemokines in macrophages. PTT-4256 displays an excellent oral PK profile across species and elicits significant monotherapy efficacy in MC38 and B16.F10 mouse models. Through molecular modelling and site-directed mutagenesis we identified three key residues that explained the difference in potency of PTT-4256 between mouse and human GPR65. Incorporating these residues in a transgenic knock-in mouse fully restored human pharmacology in vitro and provided an important translational platform.

Conclusions GPR65 is a key checkpoint on tumor-infiltrating immune cells that links the chronically acidic TME to tumor-promoting immunosuppression. The GPR65 inhibitor PTT-4256 restores anti-tumor immunity and demonstrates pronounced single-agent efficacy in mice, and is therefore being advanced towards clinical development.

Ethics Approval Protocols or procedures involving the care and use of animals in studies in China were reviewed and approved by the Institutional Animal Care and Use Committee of Crown Bioscience. During studies, the care and use of animals was conducted in accordance with the regulation of the Association for Assessment and Accreditation of Laboratory Animal Care.

Studies involving the welfare and use of animals within the UK complied with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and Council of 22/September/2010 on the protection of animals used for scientific purposes and UK Home Office guidance on the implementation of the Act and applicable codes of practice for the care and housing of laboratory animals.