Study On Anti-tumor Activity And Mechanism Of EP4 Receptor Antagonist BY001 Combined With Anti-PD-1 Antibody For Prostate Cancer

Prostate cancer is the most prevalent urological malignancies in middle-aged and elderly men and the second leading cause of cancer-related death in men worldwide.The incidence and mortality of prostate cancer in China are increasing daily in recent years,which greatly threats human health.Because the pathogenesis of prostate cancer is concealed,at the time of initial diagnosis,most of prostate cancer patients in China have been in the middle and late stage and miss the opportunity for radical treatment.Endocrine therapy is the main treatment for the patients with advanced prostate cancer.However,the vast majority of patients after treatment progress to refractory castration-resistant prostate cancer?CRPC?.At present,the drugs approved by the FDA for the treatment of CRPC patients are cabazitaxel,abiraterone,apalutamide,enzalutamide and radium-223.These drugs extend the median overall survival of CRPC patients for no more than 2 years.Immunotherapy is a new strategy for tumor therapy in recent years.Sipuleucel-T is the first tumor therapy vaccine approved by the FDA for the treatment of asymptomatic or mild metastatic CRPC.However,the survival benefits of patients are relatively limited.Although immune checkpoint inhibitors show better curative effect in the treatment of tumor,such as melanoma,renal cell carcinoma and non-small cell lung cancer,the treatment is not effective in prostate cancer.Combination therapy is not only a hot spot in immunotherapy,but also an important strategy to improve the therapeutic effect for prostate cancer.Therefore,this paper aims to improve the therapeutic effect of immune checkpoint inhibitors in prostate cancer through combination therapy.Studies have shown that COX-2/PGE₂/EP4?PTGER4?signaling pathway plays an important role in the tumor immune microenvironment.EP4 receptor antagonists have immunotherapeutic effect in colon and breast cancer.The signaling pathway is highly activated in prostate cancer,but there is no research on the immunotherapy of prostate cancer.In order to explore the distribution and fuction of EP4 receptor in the tumor microenvironment,we performed the single-cell transcriptome sequencing of tumor tissue samples from prostate cancer patients and analyzed the results.Combined with subsequent differential expression gene enrichment analysis and bioinformatics analysis of existing databases,we identified EP4 receptor as a specific target for cancer immunotherapy in prostate cancer.We selected BY001,a novel small molecule antagonist of EP4 receptor,which was synthesized and screened in our laboratory.Then we researched the function and mechanism of monotherapy and combined therapy with PD-1 antibody in prostate cancer.In vitro,on the one hand,BY001 could directly target EP4 receptor to inhibit the formation of myeloid-derived suppressor cells?MDSC?and the secretion of immunosuppressive factor ARG1 and i NOS in MDSC cells.BY001 also could enhance the proliferation,survival and effector function of T cells.On the other hand,BY001 indirectly reversed the proportion of MDSC and T cells in tumor microenvironment by targeting chemokines secreted by tumor cells to exert the immunomodulatory function.Next,we evaluated the antitumor activity of BY001 and PD-1 antibody in prostate cancer syngeneic tumor model.The inhibition rate of tumor growth was only about 40%by PD-1 antibody or BY001 alone.Though the therapeutic effect was not significant,BY001 can promote the infiltration of CD8⁺T cells and inhibit the infiltration of MDSC cells in tumor tissues,which implied that BY001 could alter the immunosuppressive microenvironment of prostate cancer.Since CD8⁺T cells were still in a state of exhaustion after BY001 treatment and the clinical combination treatment strategies were exploring,we attempted to conduct the combination therapy of BY001 and anti-PD-1 antibody.The combination therapy significantly inhibited the growth of prostate tumor,promoted the increase of CD8⁺T cells and the enrichment of NK cells,and inhibited the infiltration of MDSC cells in tumor tissues.The combination therapy enhanced the expression of CXCL9,CXCL10 and CXCL11 chemokines associated with T cell recruitment,while inhibitd the expression of CCL2,CCL4,CCL5,CXCL5,CXCL12 and CXCL16 chemokines associated with MDSC cell recruitment.Moreover,the combination therapy also promoted the expression of IFN-?and TNF-?and inhibited the expression of ARG1.The combination therapy induced a more effective tumor immune response,inhibited the growth,metastasis or recurrence of tumor,and prolonged the survival of mice in an in situ and re-challenged animal model of prostate cancer.To explore the mechanism of combination therapy,we found there were more colocalization of CD8⁺TCF1⁺T cells and MHC?⁺antigen presenting cells after treatment,which indicated that CD8⁺TCF1⁺T cells combined with MHC-?⁺antigen presenting cells formed an independent anti-tumor base to maintain the immune response.Furthermore,the combination therapy mainly exerted anti-tumor effects through CD8⁺T cells.Collectively,the combination therapy of BY001 and anti-PD-1 antibody played a synergistic therapeutic effect in prostate cancer,it can transform prostate cancer with poor response into sensitive tumor for immunotherapy.The combination therapy inhibited the growth of tumor,prolonged the survival and maintained immune response.This study provides a new strategy for the clinical treatment of CRPC.