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BackgroundAlthough NSAIDs and COX2 inhibitors show clinical promise, their toxicities limit their cancer therapeutic use. PGE2 is a metabolite from the COX2 pathway, well known to mediate immunosuppressive functions through EP2 and EP4 receptors and downstream cAMP signaling in immune cells, which suppresses their anti-tumoral activities. Similarly, PGD2, a metabolite of HPGDS, known to bind DP1, has recently been proposed to play pro-tumoral functions. OKN4395 is a novel small molecule, highly selective and potent against EP2, EP4, and DP1, intended to block the pro-tumor activities of PGE2 and PGD2, while avoiding other prostanoid receptors to reduce side effects.MethodsOKN4395’s efficacy and selectivity were rigorously characterized in a series of vitro and vivo preclinical assays. Given that the biology of DP1 is not fully understood, we confirmed using primary immune cell that its signaling was similar to the one of EP2/EP4 and further evaluated the redundancy of PGD2 with PGE2 in vitro. In addition, combination potential with anti-PD1 was evaluated.ResultsA comprehensive selectivity screen confirmed that OKN4395 selectively inhibits EP2, EP4, and DP1 receptors, with no off-target effects on other prostanoid receptors. In vitro, OKN4395 exhibited robust potency in counteracting the immunosuppressive effects of PGE2 and PGD2 in human T cells and NK cells. Our data demonstrated that PGE2 and PGD2 can fully compensate for each other’s effects; notably, the blockade of EP2, EP4, and DP1 receptors was able to effectively restore NK and CD8+ T cell anti-tumor functions when both PGE2 and PGD2 were present at high concentrations. In a mixed lymphocyte reaction assay, we showed that the well-characterized activity of anti-PD1 on IFN-γ secretion was fully blocked by prostaglandins and could only be rescued by the activity of OKN4395. Furthermore, the potent anti-tumor effect of OKN4395 in combination with anti-PD1 was demonstrated using an in vitro killing assay. In vivo, OKN4395 significantly reduced tumor growth as a monotherapy and demonstrated instances of complete tumor regression when combined with anti-PD1. Finally, pharmacokinetic and tolerability profiles from non-clinical studies supported further clinical development.ConclusionsOur studies demonstrate that triple inhibition of EP2, EP4, and DP1 with OKN4395 is more effective than dual EP2/EP4 inhibition in restoring anti-tumor immune responses in presence of both PGE2 and PGD2. These compelling results strongly endorse the clinical development of OKN4395, currently in a Phase 1 trial (NCT06789172), as a novel immunotherapy for solid tumors, both as a standalone treatment and in combination with anti-PD1.Ethics ApprovalAll human derived material used in the study were obtained under ethics and regulatory approvals.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their functional role in gliomagenesis and their potential as treatment targets. To identify therapeutically targetable opportunities among aGPCR family members in unbiased fashion, we analyzed expression levels of all aGPCRs in GBM and non-neoplastic brain tissue. Using bulk and single cell transcriptomic and proteomic data, we show that CD97 (ADGRE5), an aGPCR previously implicated in GBM pathogenesis, is the most promising aGPCR target in GBM, by virtue of its abundance in all GBM tumors and its de novo expression profile in GBM compared to normal brain tissue and neural progenitors. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGC) in vitro and in vivo. Transcriptomic and metabolomic data from PDGCs suggest that CD97 promotes glycolytic metabolism. The oncogenic and metabolic effects of CD97 are mediated by the MAPK pathway. Activation of MAPK signaling depends on phosphorylation of the cytosolic C-terminus of CD97 and recruitment of β-arrestin. Using single-cell RNA-sequencing and biochemical assays, we demonstrate that THY1/CD90 is the most likely CD97 ligand in GBM. Lastly, we show that targeting of PDGCs with an anti-CD97 antibody-drug conjugate in vitro selectively kills tumor cells but not human astrocytes or neural stem cells. Our studies identify CD97 as an important regulator of tumor metabolism in GBM, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it for therapeutic purposes.