Construction and Functional Verification of CAR-T Cells Targeting CD70 Using the Extracellular Domain of CD27 as the Chimeric Antigen Receptor
WANG Bing, GUO Ruiting, ZHAO Mingfeng*
This study constructed CAR-T (chimeric antigen receptor T) cells targeting CD70 using partial peptides of the extracellular segment of CD27 molecule as chimeric antigen receptor and verified its function in vitro. The expression of CD70 targets antigen in AML (acute myeloid leukemia ) cell lines was detected by flow cytometry. The second generation anti-CD70 Lentivirus expression vector containing 4-1BB costimulatory factor was constructed by genetic engineering method, and the corresponding Lentivirus was prepared to infect activated human CD3+ T cells to obtain the second generation CAR-T cells targeting CD70. Flow cytometry was used to detect the killing function of CD70 CAR-T cells targeting AML cell lines in vitro; the secretion level of cytokines was measured by CBA kit (including IL-2, IL-4, IL-6, IL-10, TNF-α、 IFN-γ). The research results indicated that CD70 was expressed in AML cell lines. And the CD70 CAR-T significantly and specifically killed AML cells expressing CD70 when the effector target ratio was 1׃1, 2׃1 and 5׃1. Compared with the untransduced T cells, CD70 CAR-T cells killed target cells and secreted higher levels of inflammatory cytokines, such as interleukin-6, interleukin-10 and interferon-γ (P<0.05). In summary, this study the CD70 targeting CAR-T cells have been successfully constructed and have excellent killing functions, which provided the foundation for further improvement and optimization of CD70 CAR-T in the future.
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