The Research Of Treatment For HCC With Mesothelin Targeting Chimeric Antigen Receptor Modified T Cells

Hepatocellular carcinoma(HCC) is the sixth most common cancer and the third most common cause of cancer mortality worldwide with 7,500,000 new cases each year. Because of the fast pace of life, great work pressure, unhealthy eating habits and poor environment, the high incidence of cancer and younger trend has become increasingly evident.The potentially curative therapies can only induced limited effect include surgical resection, liver transplantation, chemotherapy, chemoembolization, radiofrequency ablation and proton beam therapy. And due to the limit of early diagnosis technology, the overall five-year survival of liver cancer is only 10%, while five-year survival can reach 50%-70% with early effective treatment. Thus, it remains an urgent need for effective, life-prolonging strategies in the management of patients with liver cancer. In recent years, the rise of immunotherapy, which uses its own immune system against cancer, brought new hope to cancer patients. However, cunning tumor cells can escape the attack of immune cells by none or low expression of MHC molecules, which brought more difficulties for treatment coupled with severely damaged immune system of patients.CAR-T cells, chimeric antigen receptor modified T cells, can overcome MHC mediated immune escape mechanism for adoptive cell therapy, and establish tumor immunity and eliminate small or large tumor burden rapidly. It is considered to be a promising therapeutic strategy due to the great success of CD19 CAR-T cells in the malignant hematologic disease. It has opened up a new era in tumor biological therapy.Chimeric antigen receptor modified T(CAR-T) cells based immuno-therapy has achieved outstanding success in treating B-cell hematologic malignancies,but there are facing many obstacles when treating solid tumors. The possible reasons include: immuno-suppressive microenvironment within solid tumors, lack of specific target like CD19, low transfection efficiency, the small number and low affinity of CAR. In this study, we developed the second-generation mesoCAR-T cells based on piggy Bac transposon system, and investigated their potential in treating HCC.(1) We developed the second-generation mesothelin targeting CAR-T(mesoCAR-T) cells with 4-1BB co-stimulatory module by piggy Bac transposon system. Meso CAR was expressed by 66.0% of mesoCAR-T cells post stimulation with k562-meso cells, the expressions of activation markers were tested by FCM, and showed greater activation of mesoCAR-T cells than control T cells.(2) We detect the expression levels of mesothelin among a variety of tumor cell lines by RT-PCR and Western Blot. Then, mesoCAR-T cells exerted cytotoxicity toward SMMC-7721, BEL-7404 cells(high expression of mesothelin). The cytotoxic efficiency of mesoCAR-T cells towards mesothelin expression cell lines was stronger than low or no expression cell lines.The results showed that the mesoCAR-T cells have a certain specificity.(3) By detecting the mesoCAR-T cell cytokine,we found that IFN- γ, TNF-α, and IL2 secretion was significantly higher than that of the control T cells. The IFN- γ secretion of mesoCAR-T cells were also directly proportional to the mesothelin expression of the tumor cells when co-cultured with six different tumor cells.(4) Additionally, mesoCAR-T cells showed greater inhibitory and proliferative capability than control T cells within SMMC-7721 cell xenografts.The mesoCAR-T cells showed good transfection efficiency, the secretion of cytokines, and the cytotoxic in vitro and in vivo. This study provides basis for the application of mesoCAR-T cells to treat HCC.