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Archive > Volume 29 Issue 7 > 2022,29(7):623-630. DOI:10.3872/j.issn.1007-385X.2022.07.003 Prev Next
Basic Research
Tumor-killing effects of CD19 and CD22 bi-specific CAR-T cells on tumor cells
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Abstract:
Objective: To design and prepare a bi-specific chimeric antigen receptor (CAR)-T cell targeting both CD19 and CD22 antigens on the surface of B lymphocytes and to detect its tumor-killing effects in vitro and in vivo. Methods: Second-generation CAR molecules containing CD19 ScFv (human originated) and CD22 ScFv (CD3ε chain as costimulatory domain) were connected with P2A self-cleaving peptide. The synthesized fragment was packaged into lentivival vector pLTR-CMV-MCS. The lentiviruses were packaged with HEK-293T cells and infected with healthy human T cells to prepare CAR-19-22-T cells. Single targeted second-generation CAR-T cells were constructed with the same CD19 ScFv CAR molecules and CD22 ScFv CAR molecules respectively. Prostate cancer 3M cells expressing luciferase,CD19 and/or CD22 were constructed as target cells. Various CAR-T cells were co-cultured with target cells. Luciferase and ELISA methods were used to detect the target-cell killing ability of each group of CAR-T cells and their cytokine secretion. Mouse leukemia models were constructed by injecting Raji-Luc cells through the tail veins of the mice. Different groups of CAR-T cells were then injected respectively for treatment and the treatment efficacy was evaluated. Results: For CAR-19-22-T cells cultured for 7 days, the expression rates of CAR-19 and CAR-22 were 13.7% and 14.3% respectively.The killing rates of 3M-CD19-Luc, 3M-CD22-Luc and 3M-CD19-CD22-Luc cells by CAR-19-22-T cells at 10∶1 efficiency target ratio were significantly higher than those of T cells [(78.1±14.4)% vs (11.1±4.3)%, (46.7± 10.7)% vs (12.4±2.7)%, (90.5±4.3)% vs (14.3±3.7)%, all P<0.01]. CAR-19-22-T cells co-cultured with 3M-CD19-Luc, 3M-CD22-Luc or 3M-CD19-CD22-Luc target cells secreted significantly lower amounts of IFN-γ, TNF-α and IL-2 than those for CAR-19-T or CAR-22-T (P<0.05 or P<0.01). CAR-19-22-T cells had significantly better treatment efficacy on mice infected with Raji-Luc cells. The survival time of these mice was significantly longer than that of the mice in the T-cell group (P<0.01) and compared with those of the tumor-bearing mice in the CAR-19-T group and CAR-22-T group, was not statistically significant (all P>0.05). Conclusion: Bi-specific CAR-19-22-T cells were successfully designed and prepared, which could effectively kill tumor cells expressing CD19 and/or CD22 antigens and have a significant treatment effect on mouse leukemia model of Raji-Luc cells.
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