Construction And Characterization Of CAR-T Cells Targeting GPC3-Positive Hepatocellular Carcinoma

Background and objective:Hepatocellular carcinoma(HCC)is one of the most common and most vicious malignancies.Its incidence is the 3rd highest in China,and is still on the rise,and its mortality is also among the highest.Progression of HCC is rather rapid,many can hardly be resected upon diagnosis,and it’s sensitive to neither chemotherapy nor radiotherapy,leading to extremely poor prognosis and survival of the patients.Treatment of liver cancer is still a worldwide problem,and a more effective treatment is urgently needed.In recent years,with the rapid development of cell biology and tumor immunology,the importance of tumor immunity in development,progression,and treatmen t of tumors has been well appreciated,and breakthrough progresses have been made in the field of tumor immunotherapy,such as chimeric antigen receptor-modified T cell(CAR-T)and immunological checkpoint suppression by antibody.These technologies showed very good efficacy in the treatment of tumor,and brings hope for a cure of malignancies.CAR molecule combines single-chain antibody fragment(scFV)that target specific tumor antigen with intracellular domain of T-cell receptor that activate T cells.CAR-modified T-lymphocytes can effectively recognize antigen-positive tumor cells and get activated in a non-MHC context,thus effectively kill the tumor cells.Glypican-3(GPC3)is a heparan sulfate proteoglycan(HSPG)expressed on cell surface.This protein is highly expressed in the mesodermal tissues and in the placenta during embryogenesis,but in adults,it is expressed only in kidney,lung,and ovaries at low levels,and not in normal heart,liver,and brain tissues.Studies have shown that GPC3 expression is highly correlated with liver cancer,the patients showed higher rate of GPC3 expression,and the rate increases as the disease progresses.So,GPC3 is considered an ideal target for HCC therapy.This study intends to construct a CAR that integrates anti-GPC3 scFv(GC33),and the intracellular domain of CD28,CD137,CD3ζ,and package this anti-GPC3-CD28-CD137-CD3ζ cassette into lentivirus to get CAR-T cells.Then we shall determine expression of this CAR molecule on the surface of the T cells,and tes t the killing efficiency of such CAR-T against GPC3-positive cells,in an expectation to find a new approach for the treatment of GPC3-positive HCC.Method:CAR motif against GPC3 was designed and ligated into the lentiviral expression vector pCDH-GPC3-CAR,then lentivirus was packaged to infect T cells.The expression of anti-GPC3 CAR in T cell was determined by western blot,and the capacity and specificity of the CAR-T cells to kill GPC3-positve HCC cells were determined by flow cytometry,real-time cell analysis(RTCA),and ELISA.Result:1.The recombinant plasmid pCDH-CMV-GPC3-CAR-EF1α-copGFP was successfully constructed as determined by restriction endonuclease digestion,DNA gel electrophoresis and DNA sequencing.And corresponding lentiviral particles were successfully packaged.2.Peripheral blood mononuclear cells(PBMC)were successfully isolated and gene modification of activated T cells were accomplished by lentivirus infection.Flow cytometry and Western Blot showed that CAR molecules were successfully expressed on T cells.3.Real-time cell analysis showed that the CAR-T cells significantly inhibited the proliferation of GPC3-positive Huh-7 hepatoma cells,but had no obvious inhibitory effect on GPC3-negative SK-HEP-1 hepatoma cells.LDH release assay demonstrated that the CAR-T cells have a direct killing effect on GPC3-positive Huh-7 cells.ELISA assay determined high level of IFN-γ in the supernatant,suggesting that the CAR-T effectively secreted IFN-γ when co-cultured with Huh-7 cells.Conclusion:Our results showed that the GPC3-CAR-T cells can secret very high level of IFN-γ,and effectively and specifically kill GPC3-positve HCC cells,thus laying basis for future preclinical and clinical studies on GPC3-CAR-T cells.