Second-generation And Third-generation GPC3-CAR Modified T Lymphocytes Targeting Hepatocellular Carcinoma In Vitro

Backgroud and ObjectiveCAR(Chimeric Antigen Receptor)is a chimeric receptor that can specifically recognize and bind tumor antigens,which is fused with the ITAMs of T cells by genetic engineering techniques,that is,by combining the high affinity of antibody to the tumor antigen with T Lymphocyte killing mechanism that makes T cells express specific CAR,which gives T-cells tumor-targeted cytotoxic activity.Specific CAR modified T lymphocyte targeted therapy for cancer has become the focus of immunotherapy in recent years.CAR structure,continuous improvement,has developed to the fourth and fifth generation since 1989,but the current study is still more for the second and third generation CAR.The third generation of CAR differs from the second generation in that two co-stimulatory molecules are fused to increase the cytotoxicity and proliferative activity of T cells,but may also enhance the destruction of normal tissue.Currently,the majority of the CARs used in clinical trials is the second generation CAR.whether the third generation CAR is better than the second generation CAR remains to be verified.Glypylan-3(GPC3)is up-regulated in primary liver cancer and is not expressed in normal liver tissue,benign liver lesions and adjacent tissues because of its specific expression in hepatocellular carcinoma,which makes it a popular target for hepatocellular carcinoma immunotherapy in recent years.In this study,the second generation lentiviral expression vector of GPC3-CAR(hereinafter referred to as LV-GPC3-G2)was further designed on the basis of the third generation lentiviral expression vector of GPC3-CAR(hereinafter referred to as LV-GPC3-G3)by infecting human T lymphocytes,to observe their killing hepatocarcinoma cells effect in vitro,and compare the anti-liver cancer effectiveness and targeting of the second generation and third generation,which provides the experimental basis for the GPC3-CAR modified T lymphocyte application in the treatment of liver cancer.Methods1.The GPC3 single chain antibody sc Fv was fused with 4-1BB and CD3? fragments to construct the second-generation chimeric antigen receptor lentivirus expression vector LV-GPC3-G2.2.After sequencing and double enzyme digestion,293 FT cells were transfected into lentivirus with lentiviral packaging plasmid REV,RRE and VSVG,and the virus titer was determined by ELISA.3.T lymphocytes were extracted from human peripheral blood using the Pan T Cell Isolation Kit.The cultivation of T lymphocytes were optimized by using cytokines.LV-GPC3-G2,LV-GPC3-G3,and empty vector control group LV-MOCK were infected with T lymphocytes(hereinafter referred to as G2,G3,MOCK).The virus infection efficiency and T cell survival rate were detected by flow cytometry.The expression of target protein GPC3 was detected by Western-blot.4.The expression of GPC3 in Hep G2,Huh7,HCC-LM3,Hep3 B,SK-HEP-1,97 H and97L were detected by PCR.The hepatocarcinoma cell lines were screened for different positive and non-expressed GPC3 cells for the cytotoxicity assays.5.Killing experiments were divided into four groups:LV-GPC3-G2,LV-GPC3-G3 modified T lymphocytes(G2,G3),empty virus vector LV-MOCK infected T lymphocytes,and uninfected T lymphocytes toward various HCC cells at different effector:target ratios was measured after coculturing by CCK8 kit and Annexin V/7-AAD apoptosis detection kit using flow cytometry(FCM)to determine the cytotoxicity of GPC3CAR-T cells.6.ELISA was used to detect the cytokine secretion: the supernatant was taken after 24 hours at the effector:target ratio of 10:1.To further evaluate the cytotoxicity of GPC3CAR-T cells to hepatocarcinoma cells,cytokines IFN-? and IL-2 in the supernatant were detected by Quantikine ELISA.Results1.The second generation lentiviral expression vector LV-GPC3 CAR was constructed correctly by double enzyme digestion and the nucleotide sequencing analysis.2.The lentivirus vector was packing with Lip3000 kit and the titer of lentivirus was2.0×108 TU/m L determined by ELISA.3.The cultivation of T lymphocytes were optimized by using IL-2(300U/m L)and Human T-Activator CD3/CD28 beads(25?l/1×106 cells),and T lymphocytes could be expanded for 42 days continuously.Human T lymphocytes were infected with20 MOI of LV-GPC3-G2,LV-GPC3-G3 respectively.The expression of LV-GPC3-G2 and LV-GPC3-G3 were detected by flow cytometry(G2 infection rate(%)69.70 ± 0.3345,cell survival rate(%)85.53 ± 0.1392;G3 infection rate(%)69.40 ± 0.2573,cell survival rate(%)87.31 ± 0.1090).Western-blot demonstrated T cells expression of the second generation and third generation GPC3 CAR correctly after infection.4.The expression of GPC3 in HCC cell lines was detected by PCR from high to low:Hep G2> Hep3B> Huh-7> Hcc-LM3> 97H> 97 L and SK-HEP-1 did not express GPC3.5.The second generation and third generation GPC3CAR-T were incubated with Hep G2,Huh7,SK-HEP-1 cells respectively,and the empty vector vector LV-MOCK and T cells without LV-GPC3 CAR were used as the negative control groups.Compared with the control groups,the second and third generation GPC3CAR-T cells could efficiently kill GPC3-positive HCC cells(each groups as follows: Hep G2 group P*(G2 vs.MOCK)=0.016/P*(G3vs.MOCK)=0.018<0.05;Huh7 group P*(G2 vs.MOCK)=0.023/P*(G3 vs.MOCK)=0.027<0.05),but not the GPC3-negative cells in vitro.These cytotoxic activities seemed to be positively correlated with GPC3 expression levels in the target cells.The killing efficiency between the second generation and third generation GPC3-CAR T showed no significant differences.6.The levels of cytokines IFN-? and IL-2 in the supernatant were detected.After incubated with the second generation and third generation GPC3-CAR T cells,the cytokines IFN-? and IL-2 of Hep G2 and Huh7 groups were significantly higher than that of SK-HEP-1(P <0.05).Conclusions The second generation of GPC3 chimeric antigen receptor lentiviral is constructed successfully.The cultivation of human peripheral blood T lymphocytes is optimized.The second and third generation GPC3 CAR were successfully infected with T lymphocytes and were stably cultured and expanded in vitro.T cells modified by the second generation and third generation GPC3 CAR could effectively kill GPC3-positive HCC cells but not GPC3-negative cells in vitro.These cytotoxic activities seemed to be positively correlated with GPC3 expression levels in the target cells.The killing efficiency between the second generation and third generation GPC3-CAR T showed no significant differences.