The Effects And Mechanism Of PD-1 Monoclonal Antibody Combined With CPI-203 And Immunomodulator NP19 On Liver Cancer

BACKGROUD:The highly malignant degree of primary hepatocellular carcinoma(HCC)accelerated patients’ case fatality rate(CFR),the 5-year survival rate of HCC patients is only about 14%.Due to the limited clinical treatments,immune checkpoint inhibitors for the treatment of liver cancer are being launched around the world.Monoclonal antibody against PD-1(PD-1 mAb)plays an anti-tumor role,but its effective rate is low.It has been reported that the expression of PD-L1,which was induced by IFN-γ in the tumor microenvironment may play a key role for the efficacy of immune checkpoint inhibitors.Therefore,the efficacy of immunotherapy can be improved by blocking the PD-L1 in the tumor microenvironment and preventing immune escape.TCGA database analysis showed that:BRD4 was highly expressed in liver tissues of liver cancer patients.Therefore,we designed the study about the effects and mechanism of PD-1 mAb combined with CPI-203 and immunomodulator NP19 on liver cancer.METHODS:1.Firstly,we established a liver cancer model with BALB/c mice.Then treat it with PD-1 mAb(αPD-1).The PD-L1 expression in the tumor tissues was detected by immunohistochemistry.2.In vitro,lentivirus transfection technique was used to verify the effect of PD-L1 on tumor cells.WB and qPCR experiments were used to detect the effect of BRD4 inhibitor CPI-203 on PD-L1 expression at RNA and protein levels.3.The mechanism of CPI-203 regulating the expression of PD-L1 was studied by qPCR,cell transfection technology,Chromatin immunoprecipitation assay(ChIP)and dual luciferase reporter gene experiment.4.The efficacy of CPI-203 combined with PD-1 mAb(αPD-1)in the treatment of liver cancer in vivo,and the adverse reactions of the combined treatment were monitored.5.To evaluate the antitumor efficacy of NP19,a small molecule immunomodulator targeting PD-L1,and its combination with PD-1 mAb in BALB/c mouse hepatocellular carcinoma model.RESULTS:1.In BALB/c mouse hepatocellular carcinoma model,we observed that the increase of PD-L1 protein during the treatment of PD-1 mAb(αPD-1),which is not conducive to the anti-tumor effects of PD-1 mAb(αPD-1).2.In vitro experiments showed that IFN-y induced high expression of PD-L1 protein in HepG2 cells,which can be blocked in the presence of the BRD4 inhibitor CPI-203.At the same time,the malignant migration characteristics of HCC cells were enhanced with high expression of PD-L1.3.Cell transfection experiments showed that knockdown of BRD4 expression in HepG2 and HepG2.2.15 cells with siRNA could inhibit the expression of PD-L1,overexpression of BRD4 with DNA plasmid promoted the expression of PD-L1.ChIP assay confirmed that CPI-203 can inhibit the binding of BRD4 to the PD-L1 promoter,region;The results of double luciferase reporter gene assay suggested that CPI-203 inhibited the transcriptional activity of PD-L1 gene promoter.4.Compared with PD-1 mAb monotherapy group,the tumor volume of mice in CPI-203 and PD-1 mAb combination group decreased significantly and the antitumor effect appeared at the beginning of the treatment.Further pathological and biochemical tests showed that lung tissue,intestinal tissue,liver and kidney function and bone marrow function were normal in the combined treatment group.5.NP19 single or combined with PD-1 mAb can synergistically exert better anti-liver cancer therapeutic effect,and didn’t induce immune-related adverse events in mice.CONCLUSSION:BRD4 inhibitor CPI-203 can increase the response rate of PD-1 mAb in liver cancer immunotherapy by inhibiting the increase of PD-L1 in the tumor microenvironment,and the combination of PD-1 mAb showd low toxic and side effect on mice and good tolerance.NP19,which plays an anti-liver cancer effect by acting directly on PD-L1,can synergistically exert better anti-liver cancer therapeutic effect when combined with PD-1 mAb,primsing a potential clinical immunotherapy method to reduce the incidence of clinical resistance to immunetherapy.