Discovery Of Novel Prostaglandin E2 Receptor 4 Antagonist And Its Application In Cancer Immunotherapy

Immune checkpoint blockade(ICB)therapy is revolutionizing the treatment paradigm in oncology.The approvals of anti-CTLA-4,anti-PD-1 and anti-PD-L1 antibodies in a broad range of human cancer types have established immunotherapy as a feasible therapeutic strategy for patients with advanced cancers.However,these ICB therapies fail to control malignancies progression among a large population of patients.Increasing evidence indicated that the tumor immunosuppressive microenvironment consisting of tumor-associated myeloid cells(TAMCs)and immunosuppressive factors play critical roles in primary and adaptive resistance to immunotherapy.Therefore,blocking the activations of immunosuppressive TAMCs in tumor microenvironment is a new direction of cancer immunotherapy.Plenty of studies have demonstrated that COX2/Prostaglandin E2(PGE₂)signaling is a constitutive activated key pathway in advanced cancer patients.PGE₂ as a crucial tumor-sustaining bioactive lipid mediator of inflammation,is involved in inducing immunosuppression in tumor microenvironment through binding to its four downstream receptors(EP1,EP2,EP3,EP4).In this study,we demonstrated that PGE₂ mediates the differentiation and immunosuppression function of TAMCs,including tumor-associated macrophages(TAM)and myeloid-derived suppressor cells(MDSCs),mainly through EP4receptors.In order to investigate the feasibility of EP4 as a novel target for cancer immunotherapy,we constructed the EP4 calcium flux screening platform to evaluate the EP4 antagonistic activities of a series of compounds.Finally,YJ-3-164 was identified as a novel,effective and selective EP4 antagonist,IC₅₀=3.0 n M.We also found YJ-3-164 could inhibit the EP4downstream pathways,which were mediated by c AMP and?-arrestin.Next,we found oral administration of 75 mg/kg YJ-3-164 daily inhibited tumor growth in multiple syngeneic mouse tumor models,and the growth inhibition rates of Pan02,4T1,MC38 and CT26 transplanted tumors were 44.0%,23.7%,50.6%and 51.5%,respectively.Notably,YJ-3-164 showed no effect on CT26 tumor-bearing nude mice and tumor cells in vitro,indicating that the antitumor effect of YJ-3-164 is depended on immune system.Next,our data showed that YJ-3-164 not only effectively inhibited PGE₂-induced differentiation of macrophages and MDSCs and polarization of M2 type macrophage in vitro,but also significantly up-regulated the proportion of anti-tumor M1 type macrophages,as well as down-regulated the proportion of M2 type macrophages and MDSCs in vivo.Meanwhile,YJ-3-164 treatment enhanced the anti-tumor effect of M1 type macrophages by raising their expression of TNF-?,CCL2 and CCL5,and impaired the pro-tumor effect of M2 type macrophages and MDSCs by reducing their expression of immunosuppressive factors.Furthermore,YJ-3-164 recruited more T cells into the tumor microenvironment,raised the expression of anti-tumor immune factors such as Gzm B,IFN-?and TNF-?,enhanced T cell mediated anti-tumor immune response.To further explore whether YJ-3-164 can sensitize ICB therapy,we combined YJ-3-164and anti-PD-1 antibody in vivo.Our results showed that YJ-3-164 and anti-PD-1 antibody combination significantly impeded tumor progression in CT26 and MC38 colorectal syngeneic mouse tumor models.Microarray results showed that inflammatory and immune-related pathways,such as cytokine-cytokine receptor interaction,were key signaling pathways taken part in anti-tumor activities of the combination therapy.Meanwhile,the combination treatment also repressed pro-tumor cytokines IL-6 and CXCL-1 secretion.Furthermore,the synergic effect of YJ-3-164 and PD-1 blockade was also observed in AOM/DSS induced colorectal cancer model.More CD8⁺cytotoxic T cells infiltrated into mouse intestine under the combination treatment.In addition,the crucial inflammatory cytokines,including IL-6,CXCL1 and ARG-1 were remarkably suppressed by the combination treatment rather than either agent used alone.All these data suggested that YJ-3-164 improved the efficacy of immune checkpoint blockade therapy and induced a durable antitumor immune response.Taken together,our study showed that EP4 is an important regulator of TAMCs immunosuppressive function,and identified a new selective EP4 antagonist YJ-3-164 through multiple EP4 signaling pathways relatad functional assays.We also confirmed YJ-3-164treatment alone in vivo inhibited the immune suppression effect of TAMCs,and enhanced the T cell mediated anti-tumor immunity.Additionally,YJ-3-164 could sensitize anti-PD-1antibody in treatment of colorectal cancer.Therefore,we proposed that shifting TAMCs'immunosuppressive function and improving the immuno-inhibitory tumor microenvironment by YJ-3-164 could be a promising novel strategy for cancer therapy.