Construction And Anti-HCC Activity Of GPC3 Specific Chimeric Antigen Receptor T-Cell

Hepatocellular carcinoma(HCC),which is a serious worldwide health problem and the fourth mortality of cancer-related death all around the world.The incidence of liver cancer in China ranked ninth in the world in 2018,with more than 13.4 people per 100,000 having liver cancer.The human and material resources invested in the treatment of hepatocellular carcinoma every year are countless,which is a huge public health burden.However,there is not any superior method for the diagnosis and treatment of liver cancer.Fortunately,in recent years,immunotherapy for liver cancer has gradually shown some possibilities.There are many signal pathways and potential molecular targets in hepatocellular carcinoma,so many molecular targeted drugs are under clinical study.Phosphatidylinositol 3(Glypican 3,GPC3)is one of the potential targets,which is hardly expressed in non-maligna nt tissues but highly expressed in liver cancer tissues,making it a possible target to diagnosis or treat liver cancer with high specificityChimeric Antigen Receptor(CAR)T cell therapy is a very popular personalized therapy for tumor in recent years.Similar to other immunotherapies,the main idea of CAR-T therapy is to break through the tumor microenvironment and enable the body's own T lymphocytes to kill the tumor tissue in order to get the treatment done.It performs well in the treatment of leukemia,and recently breakthroughs have been made against the type of solid tumors.In this study,CAR gene components and lentiviral infection conditions were explored to obtain the best CAR construction and lentivirus infection conditions,and human primary T lymphocytes were engineered by lentivirus infection system to stabilize the expression of GPC3-specific CAR.The CAR-T construction platform was preliminarily established,and their effective secretion of cytokines IL-2,TNF-a and IFN-y was significantly different from the control group.Effective T cell activation and cytokine release are effective preconditions for T cells to break through tumor microenvironment and kill tumors.Indeed,GPC3-specific CAR-T cells showed good in vitro cytotoxicity against Huh7,HepG2,Hep3B and other six liver cancer cell lines.When E:T(Effector:Target)was 10:1,the maximum killing efficiency of all liver cancer cell lines was over 95%on the fourth day.The antitumor activity in vitro was excellent,and the tumor inhibition ability in vivo was also excellent Huh7 cells were pre-tumorigenic in SCID mice subcutaneously,and the therapeutic dose of CAR-T was determined to be 2.5×106 CAR-T cells in the exploration experiment of five inj ections and three groups of different doses.In formal in vivo experiment,we also pre-induced subcutaneous tumorigenesis in SCID mice,and selected mice with tumor size of 75-100 mm3 24 days after tumor grafting,and divided them into the 2.5x106 Anti-GPC3-CAR-T treatment group and the PBS control group.One injection every two days for a total of four injections.By observing the growth trend of tumors,we can see that GPC3-specific CAR-T cells show significant tumor inhibition effect in vivo.On the last day,the average tumor size of the control group was 1.8 times that of the CAR-T treatment group.In the end,we preliminarily evaluated the specificity of Anti-GPC3-CAR-T by dissecting mice in the CAR-T group and the control group to obtain liver,lung,spleen and tumor for immunohistochemical detection of T-cell target CD8 and liver cancer target GPC3.From the experimental results,T cell signals could not be detected in other GPC3 negative tissues,indicating that GPC3 specific CAR-T cells can specifically target GPC3-positive tumor tissues,and have certain specificity and safety.To sum up,this study successfully completed the preliminary exploration of CAR-T construction platform,obtained GPC3 specific CAR-T with good activation and cytokine secretion in vitro,verified its in vitro cytotoxicity and tumor inhibition ability in vivo,and preliminarily evaluated its specificity and safety.It has not only identified the basis for immunotherapy research of liver cancer,but also established a new platform for other types of tumor treatment research.