Preparation Of PD-1 MAb And Mechanism Study Of Its Reversal Effect On Immunosuppressive Jurkat Cell

Background:Tumor cells can escape from the clearance of immune system via a variety of ways,among which the PD-1/PD-L1 checkpoint has been playing a crucial role.Tumor cells recognize the PD-1 proteins on the effector T cells by overexpressing PD-1 ligand(PD-L1),thus activate the PD-1/PD-L1 checkpoint to transmit immunosuppressive signals sequentially reduce the activity and cytokine secretion of effector T cells,and finally resulting in immune escape of tumor cells from host immune clearance.Therefore,reversing immunologic tolerance of tumor cells by blocking PD-1/PD-L1 checkpoint has become a new direction for anti-tumor immunotherapy.Monoclonal antibodies(mAbs)targeting PD-1/PD-L1 checkpoint have demonstrated dominant therapeutic ability in a variety of malignant neoplasms,yet reports concerning their clinical trials in patients with liver cancer are still limited.Liver cancer has long been recognized as a life-threatening malignant tumor with high mortality rate,moreover,the number of deaths from liver cancer in China each year accounts for about half of that across the world,which renders PD-1/PD-L1 checkpoint inhibitors,to have a broad application prospect in treating liver cancer as a promising immunotherapy.Currently,it remains to be proved whether PD-1 can act as a biomarker for immunotherapy and prognosis,or whether mAbs blocking PD-1/PD-L1 checkpoint can be applied to the treatment of liver cancer,therefore,further studies on the effect and mechanism of anti-PD-1 m Abs are of great significance to the development of immunotherapy drugs,as well as the diagnosis and treatment of liver cancer patients.At present,a number of monoclonal antibodies targeting PD-1/PD-L1 checkpoint are under clinical trials,and exhibit encouraging curative effects.But the low response rate still limits its utilization in only a small number of patients.Moreover,unfavorable toxic effects and adverse events were often observed in treatment,which counteracts the effectiveness and restricts the clinical application of these antibodies.Therefore,it is significant,both theoretically and practically,to develop less toxic monoclonal antibodies for immunotherapy of liver cancer,and to figure out how to maximize their efficacy in tumor immunotherapy.Objective:This study is aimed at preparing and screening out a new mouse anti-human PD-1 monoclonal antibody with high specificity and high blocking bioactivity,and sequentially studying the clinical value of the expression of PD-1 in HCC tissues with the high specific antibody,while investigating the reversal effect and mechanism of high blocking antibody to immunosuppressive Jurkat cell.Methods:1.Preparation and identification of mouse anti-human PD-1 m AbThe mouse anti-human PD-1 monoclonal antibody was prepared from immunizing BALB/c mice with the recombinant human PD-1 protein as the immunogen.The sub-isotypes,specificity and cytotoxicity of the anti-PD-1 m Abs were identified.2.Research on the clinical significance of the expression of PD-1 in HCC tissue with self-developed anti-PD-1 m AbHE staining and immunohistochemistry were used to detect the expression of PD-1 in tumor tissues of patients with hepatocellular carcinoma by the high specific antibody,to analyze the correlation between the expression of PD-1 on the tumor tissues and the clinicopathological features of patients,and to evaluate the feasibility of utilizing anti-PD-1 mAb in treating liver cancer.3.Study on the effect of anti-PD-1 m Ab reversing the immunosuppression of Jurkat cellsHepG2 cells were co-cultured with Jurkat cells to screen out mouse anti-human PD-1 mAb with highest blocking bioactivity;ELISA assay was used to detec t the effect of antiPD-1 mAb on the secretion of anti-tumor cytokines in Jurkat cells;while MTT assay was used to measure the effect of anti-PD-1 m Ab on cytotoxic activity of Jurkat cells to Hep G2 cells,to study the reversal effect of anti-PD-1 m Ab on the immunosuppression of Jurkat cells in the co-culture system.4.Preliminary study on the mechanism of anti-PD-1 mAb reversing the immunosuppression of Jurkat cellsOn the basis of previous experiments,HepG2 cells and Jurkat cells were co-cultured,flow cytometry and Western Blot assay were used to detect the changes of cell cycle and apoptosis of Jurkat cells in the co-culture system.PD-1/PD-L1 pathway was blocked in coculture system by the anti-PD-1 m Ab with high blocking bioactivity,to observe the changes of apoptosis,the expression of apoptosis related proteins and upstream or downstream regulatory molecules in Jurkat cells,thus investigating the mechanism of anti-PD-1 m Ab reversing the immunosuppression of Jurkat cells.Results:1.Fourteen mouse anti-human PD-1 monoclonal antibodies were obtained,named 1C4,1H8,2C4,2H7,3E5,5B2,5H7,7C9,7E5,7G12,9A5,9B4,9E11 and B1C4.2.The results of ELISA and Western Blot showed that,anti-PD-1 mAb 2C4,5B2,5H7,7E5,7G12,9A5,9B4,9E11 and B1C4 could recognize the immunogenic PD-1 protein.In immunohistochemistry assay,anti-PD-1 m Ab 9E11 exhibited highest specificity in recognizing the PD-1 protein expressed in T cells in hepatocellular carcinoma.3.The study of the expression of PD-1 in HCC tissues and its clinical value by using the m Ab 9E11 showed that,PD-1+T lymphocytes were mainly distributed in the peritumoral tissue adjacent to tumor margin area.The expression of PD-1 in HCC tissues was highly related to the size and differentiation degree of the tumor,less related to the presence or absence of thrombosis in patients,and not related to other clinicopathological features such as gender,age,liver cirrhosis,hepatitis B infection,TNM stage and tumor location.4.Anti-PD-1 m Ab 2H7 and B1C4 had the strongest blocking bioactivity,and could significantly enhance Jurkat cells function to kill target Hep G2 cells by upregulating its cytokine secretion.While In high concentration,the anti-PD-1 mAb 2H7 and B1C4 were toxic to Jurkat cells with prolonged reaction time.5.Anti-PD-1 m Ab B1C4 could decrease the proportion of Jurkat cells in the S phase and increase the proportion of cells in G2/M phase;anti-PD-1 m Ab B1C4 could also inhibit the apoptosis of Jurkat cells induced by co-culture system and affect the expression of PI3K/AKT related proteins of Jurkat cells in co-culture system,such as downregulating the expression of pro-apoptotic protein Bax and PTEN protein that inhibits the phosphorylation of AKT,upregulating the phosphorylation level of AKT and anti-apoptotic protein m TOR.Conclusions:1.Fourteen mouse anti-human PD-1 monoclonal antibodies were successfully prepared and obtained,they can be used as antibodies in ELISA,Western Blot and IHC according to their differences in antigen recognition.2.The expression of PD-1 in HCC tissues is closely related to the size and differentiation degree of the tumor;the monoclonal antibody targeting PD-1 has potential application in the diagnosis and treatment of poorly differentiated HCC patients.3.The prepared anti-PD-1 mAbs 2H7 and B1C4 with lower cytotoxicity can reverse the immunosuppression of Jurkat cells,and significantly improve the ability of Jurkat cells to kill tumor cells.4.Anti-PD-1 m Ab B1C4 can activate the PI3K/AKT signaling pathway to block the immune negative regulation of PD-1/PD-L1 checkpoint,and to weaken the apoptosis of Jurkat cells induced by PD-L1 derived from tumor cells.In the end,the immunosuppression of Jurkat cells induced by Hep G cells was reversed.