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Inducible IL-7 expression enhances proliferation and in vitro antitumor activity of GPC3-specific CAR-T cells
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Abstract:
Objective: To explore the effects of induced IL-7 expression on proliferation and in vitro antitumor activities of glypican-3 (GPC3)- specific chimeric antigen receptor gene modified-T (CAR-T) cells. Methods: The GPC3 CAR sequence fragment was inserted into the BamHⅠ/EcoRⅠs ite of the GV400 vector using seamless cloning, constructing second-generation CAR lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7. The lentiviruses were packaged with 293T cells and transfected into healthy human T cells to prepare CAR-T cells， which were divided into non-transduced T cell (NT) group, GPC3-BBZ CAR-T cell group and GPC3-BBZ-NFAT-IL-7 CAR-T cell group. The expression of CAR in CAR-T cells of each group was determined by flow cytometry. IL-7 mRNA expression level in CAR-T cells activated by GPC3 protein was determined by qPCR. The proliferation ability of CAR-T cells under GPC3 antigen stimulation was evaluated by cell counting. The secretion levels of IL-7, IFN-γ, and TNF-α by CAR-T cells after stimulation with tumor cells was determined using ELISA. The cytotoxicity of CAR-T cells against human hepatocellular carcinoma Huh-7 cells was tested by real-time cell analyzer (RTCA). Results: Lentiviral vectors GPC3-BBZ and GPC3-BBZ-NFAT-IL-7 were successfully constructed, and GPC3-specific CAR-T cells were prepared. After activation with GPC3 antigen, GPC3-BBZ-NFAT-IL-7 CAR-T cells effectively expressed IL-7 mRNA (P < 0.01) and exhibited stronger proliferation capacity (P < 0.05). Compared with GPC3-BBZ CAR-T cells, GPC3-BBZ-NFAT-IL-7 CAR-T cells co-cultured with GPC3-positive Huh-7 cells secreted higher levels of IL-7, IFN-γ, and TNF-α (P < 0.01 or P < 0.001). The RTCA results showed that the cytotoxicity of GPC3-BBZ-NFAT-IL-7 CAR-T cells against GPC3-positive Huh-7 cells was significantly higher than that of GPC3-BBZ CAR-T cells (P < 0.05). Conclusion: GPC3-specific CAR-T cells with inducible IL-7 expression were successfully prepared, which exhibited immune activity and tumor cell killing capacity in vitro.
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