中国生物工程杂志

Objective: To construct T cells encoding chimeric antigen receptor (CAR) targeting glypican-3 (GPC3) and chimeric antigen costimulatory receptor (CCR) targeting epithelial cell adhesion molecule (EPCAM), and analyze their activities in vitro. Methods: Gene fragments encoding EPCAM-CCR and GPC3-CAR was cloned into lentivirus vector plasmid. The recombinant CCR+CAR pCDH vector was identified by enzyme digestion, PCR and sequencing. T lymphocytes were isolated, activated and expanded. A stable combinatorial CAR modified cell line was generated using the lentivirus. Western blot, RT-PCR and flow cytometry (FCM) were used to verify the expression of CCR+CAR in CCR+CAR T cells. The secretion of cytokines IL-2, IL-4 and INF-γ was detected by ELISA. Results: The CCR+CAR pCDH lentiviral recombinant plasmid was successfully constructed. Human T lymphocytes were successfully isolated, activated and expanded. Human T lymphocytes were successfully infected with CCR+CAR pCDH lentivirus. RT-PCR and Western blot showed that the target protein was successfully expressed, and FCM analysis showed that the expression rate of CCR+CAR on CCR+CAR T cells was about 42%. ELISA was used to detect CCR+CAR co-culture with HepG2 and Lu-7 expressing GPC3 and EPCAM, the secretion levels of IL-2, INF-γ and IL-4 were higher, and the co-culture of human normal liver cells L-02 did not find effective activation of CCR+CAR T cells. Conclusions: The combined CCR+CAR T cells were successfully obtained, and the immune effect of CAR T combined with the two antigen recognition signals was attempted in hepatocellular carcinoma cells. The result laid a foundation for the immune effects in the subsequent in vitro experiments, and provided a potentially novel approach to augment the off-target effect of CAR-T immunotherapy for solid tumors.