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Construction of anti-CD38 CAR-T cells with targeted inhibition of CD38 by shRNA and preliminary identification of its functions
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Abstract:
Objective:To investigate whether targeted inhibition of CD38 by shRNA can enhance the anticancer function of anti-CD38 CAR T cells. Methods: The anti-CD38 CAR molecules with targeted inhibition of CD38 by shRNA were constructed. After being successfully packaged with retroviral vector, the CAR molecules were transferred into human primary T cells to prepare CAR-T cells,which were then divided into shRNA1-CD38 CAR-T group, shRNA2-CD38 CAR-T group and control group (shR-NC-CD38 CAR-T cells). The relative expression of CD38 mRNA in CAR-T cells was detected by qPCR method, and the proliferation index of CAR-T cells cultured ex vivo for 0-14 days was calculated. The proliferation of CAR-T cells co-cultured with human Burkitt lymphoma Raji-luc cells or human multiple myeloma peripheral blood B lymphocytes RPMI-8226-luc was detected by CFSE method. The killing efficiency of CAR-T cells against Raji-luc and RPMI-8226-luc cells at different effector-target ratios (1∶1, 1∶2, 1∶4, 1∶8) was detected by luciferase chemiluminescence. The level of IFN- γ in the supernatant of CAR-T cells co-cultured with Raji-luc cells or RPMI-8226-luc cells was detected by ELISA. The expression of PD-1, one of the T cell exhaustion biomarkers, on the surface of CAR-T cells was detected by flow cytometry. Results: The titers of shR-NC-CD38 CAR, shRNA1-CD38 CAR and shRNA2-CD38 CAR retroviral vectors were all about 1×107 copies/mL. The transduction efficiency (CAR positive rate) in cells of shR-NC-CD38 CAR-T group, shRNA1-CD38 CAR-T group and shRNA2-CD38 CAR-T group was 60.3%, 67.0% and 57.4%, respectively. Compared with the control group, the expression level of CD38 mRNA in shRNA2-CD38 CAR-T group was significantly lower (P<0.01),indicating the successful construction of shRNA-CD38 CAR-T cells. For the cells in shRNA2-CD38 CAR-T group, their ex vivo proliferation ability was stronger, the killing efficiency against two CD38 positive tumor cells was higher, the release level of IFN-γ was higher, and the surface expression level of PD-1 was lower, as compared with the other two groups (P<0.05). Conclusion: A novel anti[1]CD38 CAR-T cells with targeted inhibition of CD38 by shRNA was successfully constructed, which exhibited obvious advantages in antitumor function.
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