Study On Production Of HBsAg Specific CAR-T Cells And Its Killing Effect On Hepatocellular Carcinoma

OBJECTIVEChimeric antigen receptors(CAR)recognize specific antigens.In this study,HBsAg-CAR containing a 4-1BB costimulatory signal domain was designed and transduced into T cells using a lentiviral vector to manufacture HBsAg-CAR-T cells.The ability to kill hepatocellular carcinoma cells expressing HBsAg protein was evaluated in in vitro and in vivo experiments.METHEDS(1)HBsAg-CAR was constructed and transduced into T cells by a lentiviral vector to manufacture HBsAg-CAR-T cells.Transduction efficiency,phenotypic changes and viability of CAR-T cells were detected by flow cytometry.(2)Hepatocellular carcinoma cells were cultured and the expression level of HBsAg in hepatocellular carcinoma cells was detected by Western-blot.(3)HBsAg-CAR-T cells were co-cultured with hepatocellular carcinoma cells in vitro.The specific killing ability of HBsAg-CAR-T cells against HBsAg-expressing hepatocellular carcinoma cells were detected.(4)Subcutaneous xenotransplanted PLC/PRF/5 tumor model using NPG mice was established and HBsAg-CAR-T cells were injected through tail vein.To observe the tumor growth by measuring the tumor volume,to detect the number of T cells in peripheral blood by flow cytometry,to detect T cell infiltration by immunohistochemistry,to detect cytokine levels in peripheral blood and to observe tumor necrosis by HE staining.(5)CD8 CD62L T cell and CD8 CD62L-T cell subsets were sorted by magnetic beads and CD62L CAR-T and CD62L-CAR-T cells were manufactured by lentiviral transduction.The activation phenotype and terminal differentiation phenotype on CAR-T cells were detected by flow cytometry.The ability to kill hepatocellular carcinoma cells were evaluated in vitro.(6)In the subcutaneous xenotransplanted PLC/PRF/5 tumor model,the ability of CD62L CAR-T and CD62L-CAR-T cells to inhibit tumor and the viability of T cells in vivo were observed,and the expression of tumor-related genes in tumor tissues were detected.RESULTS(1)We constructed a lentiviral vector containing HBsAg-CAR(anti-HBsAg single-chain antibody-4-1BB-CD3 intracellular region).HBsAg-CAR was transduced into T cells with a transduction efficiency between 40 and 80%.During in vitro culture,HBsAg-CAR-T cells expressed higher amplification capacity and viability,and the expression rate of CAR remained stable.(2)HBsAg-CAR-T cells were co-cultured with HBsAg-expressing hepatocellular carcinoma cells in vitro and released high level of IL-2,IFN-y,TNF-a and GM-CSF.The killing effect of hepatocellular carcinoma cells was significantly higher than that of untransduced T cells.HBV DNA content in the supernatant was also significantly reduced.(3)The tumor growth of mice treated with HBsAg-CAR-T was significantly inhibited.The tumor volume of HBsAg-CAR-T group was significantly smaller than that of untransduced T cell treatment group on the 21st day after T cell injection.The level of IFN-y and TNF-a in peripheral blood was higher in HBsAg-CAR-T group,and there were more T cells infiltrating in tumor tissues.The pathological results showed that HBsAg-CAR-T cells led to obvious necrosis in tumor tissues.(4)We successfully sorted CD8 CD62L T and CD8 CD62L-T cell subsets with high cell purity.CD62L CAR-T cells expressed lower terminal differentiation phenotype during in vitro culture.(5)CD62L CAR-T cells showed stronger anti-tumor ability in vivo.Compared with CD62L CAR-T cells,CD62L CAR-T cells inhibited tumor growth in vivo for a long time,and reduced the expression of tumor-associated genes CCND1 and CTNNB1 in tumor tissues.CONCLUSIONS(1)We designed HBsAg-CAR containing 4-1BB co-stimulatory signal domain,and manufactured HBsAg-CAR-T cells,which showed strong cell expansion ability and stable CAR expression rate in vitro.(2)In vitro,HBsAg-CAR-T cells could specifically recognize and kill HBsAg-positive hepatocellular carcinoma cells,and released high level of anti-tumor cytokines.(3)HBsAg-CAR-T cells significantly inhibited the growth of HBsAg-positive hepatocellular carcinoma in vivo,which showed strong anti-tumor ability.(4)HBsAg-specific CAR-T cells derived from CD8+CD62L+T cell subsets were less differentiated.CD62L+CAR-T had a high proliferative capacity,which could inhibit tumor growth persistently in vivo.