GPR65 Is A Critical Innate Immune Checkpoint In Tumour Associated Macrophages: Human Genetic Validation And Discovery Of Potent and Selective GPR65 Antagonists

By Tia Byer |
01 November 2021
The activation of GPR65 on immune cells by the acidic tumour microenvironment and the resultant immunosuppression may be one of the principal reasons many cancers do not respond to current T Cell checkpoint therapies. Chief Executive Officer Stuart Hughes delves into some of Pathios Therapeutics recent developments in macrophage conditioning and its promise to enable a successful anti-tumour immune response.

Presented by Stuart Hughes, Chief Executive Officer at Pathios Therapeutics

Transcribed by Tia Byer

Pathios Therapeutics is a cutting-edge biotech company that does not use the term ‘Innate Immune Checkpoint’ lightly. Instead, it is a qualification that they are incredibly compelled by and with recent research into the validation of GPR65, Pathios is excited to propose GPR65 as a critical innate immune checkpoint in human cancers. Findings show that GPR65 activation by an acidic microenvironment is a key causative factor in creating a ‘cold’ tumour which therefore may explain why many cancers do not currently respond adequately to T cell checkpoint inhibitors.

Based in Oxford, UK, Pathios Therapeutics has a sole interest in pursuing small molecule inhibitors of the pH sensing GPR65 for applications in cancer immunotherapy. In 2021, Pathios announced the discovery of potent, selective, and orally bioavailable small molecule inhibitors of this immune checkpoint. Proprietary understanding of GPR65 derives from human genetics, single-cell transcriptomics, and computational modelling. Pathios’ ‘Macrophage Conditioning’ approach holds significant promise as a strategy for targeting the innate immune systems in cancers and, as such, holds substantial clinical promise.

GPR65 – What is it?

GPR65 is a pH-activated G-protein-coupled receptor (GPCR) on immune cells. It belongs to the pH-sensing GPCR family and is highly enriched on cells of the immune system and especially on macrophages. GPR65 is somewhat unusual because its natural ligand is a proton, so an acidic environment activates it. In macrophages, GPR65 non-redundantly transduces an extracellular low pH signal into an intracellular elevation in cyclic adenosine monophosphate (cAMP). While this mechanism is also found in other immune cells, particularly T cells and NK cells, Pathios believe the pathway is particularly important and prominent in macrophages. GPR65 and its downstream signalling partner, ICER (CREM gene), are highly expressed in tumour-associated macrophages in a range of human tumours. Examples include renal cell carcinoma (RCC), ovarian cancer, melanoma, glioblastoma and breast cancer. 

Impact on the Tumour Microenvironment:

The presence of the GPR65 receptor in macrophages is highly relevant when contemplating the tumour microenvironment. Most immune cells that are present in solid tumours are macrophages. These are primarily immunosuppressive pro-tumourigenic cells that are essentially locked in a wound repair state, suppressing inflammation, supporting vascularisation, and encouraging tissue growth. Unsurprisingly, a higher content of these cells is typically associated with a worse prognosis or a worse response to a range of treatments. Many tumours also exhibit a low pH microenvironment which negatively impacts response to immunotherapy. Tumour pH is typically in the range of 6.5 to 7.2 due to the high expression of glycolytic and acidifying enzymes. Acidic tumours tend to have a worse response to immunotherapy as highly glycolytic acidic tumours present an additional barrier to achieving a successful immune response.

Low pH causes a pronounced and widespread alteration in gene expression including, the upregulation of growth factors, neutrophil chemoattractants and angiogenic factors, and the downregulation of key cytokines. Other alterations include a suppression of both chemokines that attract T cells and NK cells, and type I/II inteferon (IFN) response genes. The capacity of GPR65 signalling to suppress the expression of pro-inflammatory genes involves the transcriptional repressor, ICER. The activation of GPR65 by low pH leads to an elevation in ICER, a shortened isoform of the CREM gene that lacks a transactivation domain, but which binds with high affinity to the promoters of a host of pro-inflammatory genes to inhibit their expression.

The Solution: Small molecule GPR65 inhibitors

By blocking GPR65, Pathios propose that pH-induced immunosuppressive pro-tumourigenic polarisation of macrophages in the tumour microenvironment can be reversed, and in doing so, support a successful immune response to be used either as a monotherapy or in combination with currently approved checkpoints. One of the key molecules to emerge from their active drug discovery programme is PTT-3196 (see Figure 1). PTT-3196 is a selective small-molecule inhibitor that can inhibit GPR65 in multiple contexts such as in human macrophages and peripheral blood mononuclear cells (PBMCs).

Most significantly, this molecule can dose-dependently counteract low pH-induced suppression of anti-tumour immunostimulatory pathways and reduce expression of pro-tumourigenic immunosuppressive and tissue repair pathways. It also robustly activates the innate immune system in vivo as evidenced by an increase in key chemokines and Type I/II IFN genes following oral dosing to mice that have been reconstituted with stem cell-derived human immune cells and that have been implanted with RCC patient-derived xenograft (PDX) tumours.

Figure 1. Characterisation of the Selective GPR65 Inhibitor: Effects of PTT-3196 in HEK cells stably overexpressing either GPR65, GPR4 or GPR68 (left) and in human PBMCs (right).

Forward Outlook:

Human genetics supports the assertion of GPR65 as an innate immune checkpoint since homozygosity for a reduced function coding variant of GPR65 (I231L) provides a significant survival benefit across multiple cancers. A host of evidence and data shows that suppressing the signalling of this receptor will be clinically important, enabling a successful immune response to the disease. Pathios’ macrophage condition strategy holds significant promise for targeting the innate immune system, and here at Oxford Global, we look forward to seeing its impact on the field. 

Speaker Biographies

Stuart Hughes, Chief Executive Officer at Pathios Therapeutics – Hughes is Chief Executive Officer of Pathios and brings over 20 years experience in scientific leadership and drug discovery expertise. Hughes has worked across several therapeutic areas including CNS disorders, immunology and oncology, and has deep expertise in guiding early stage drug discovery programs from inception toward clinical development. He has gained wide-ranging experience from positions in both academic and industrial settings. Previous positions include Research Fellow and Senior Lecturer at Cardiff University, Principal Research Scientist at Eli Lilly and most recently, Senior Director at Vertex Pharmaceuticals. He holds a PhD in Neuroscience from Cardiff University.

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