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Chimeric antigen receptor (CAR) transduced T cells have been used to efficiently kill the target tumor cells depending on the single chain variable fragment (scFv) against the specific tumor associated antigen. Here we show the high specific cytotoxicity of the CAR-T cells with very low effector to target cell (E:T) ratio owing to the CD19-scFv, which was constructed in our laboratory and proved to be highly effective in our previous study. Four plasmids containing three generation of CAR were constructed by cloning the CD19-CAR fragment into the lentiviral vector pCDH. CD3 positive T cells were successfully transduced and the CAR protein expression was confirmed by flow cytometry and Western blot. When cocultured with CD19 positive leukemia cell line Nalm-6 cells, CAR-T cells showed specific cytotoxicity: the percentage of target cells decreased to 0 in 24 hours; IL-2, IFN-γ and TNF-α produced in cocultured supernatants increased obviously; and the cytotoxicity reached more than 80%, still remarkable even when the E:T ratio was as low as 1:4. Dynamic change of cell interaction between CAR-T and leukemia cells was visually tracked by using living cells workstation for the first time. A NOD/SCID B-ALL murine model was established using Nalm-6 cells inoculation with a morbidity rate of 100%, and the survival time was prolonged statistically with CAR-T cell treatment. These data demonstrate that the CAR-T cells we prepared could be a promising treatment strategy for CD19 positive tumor diseases.

Construction of a new anti-CD19 chimeric antigen receptor and the anti-leukemia function study of the transduced T cells