In vitro and in vivo cytotoxic activity of humanized CD19 CAR-T cells against Raji cell line / 中华微生物学和免疫学杂志

ABSTRACT

Objective To investigate the different functions of humanized and murinized CD19 chimeric antigen receptor ( CAR)-T cells against Raji cell line in vitro and in vivo. Methods Peripheral blood samples were collected from eight patients with lymphoma who were going to receive CD19 CAR-T cell therapy and used for the preparation of peripheral blood mononuclear cells ( PBMC) as well as humanized and murinized CAR-T cells. Cell proliferation and cytotoxicity were detected with CCK-8 and LDH assays, respectively. A tumor-bearing mouse model was established by injecting BALB/c female nude mice with flu-orescent Raji cells. Changes in tumor volume in these mice were observed by in vivo imaging technology. The transfection efficiency and amount of CAR-T cells in the mice were detected with flow cytometry. Re-sults No statistical difference in transfection efficiency was found between humanized and murinized CAR-T cells, nor in cell proliferation at 24 h of culture in vitro(P=0. 104). The proliferation of humanized CAR-T cells showed a significant increase compared with that of murinized CAR-T cells at 48 h of culture ( P=0. 009). Similarly, the cytotoxicity of the two types of CAR-T cells against Raji cells showed no significant difference at 24 h at any effector/target (E/T) ratio (1 1 or 4 1), and that of humanized CAR-T cells was higher than that of murinized CAR-T cells at both E/T ratios at 48 h (E/T ratio=1 1, P=0. 005;E/T ratio=4 1, P=0. 008). Moreover, the cytotoxicity of CAR-T cells was higher than that of PBMC in any case. Tumor volumes in mice were reduced 14 d after humanized or murinized CAR-T cell therapy, while the mice in the PBMC control group suffered tumor progression. Tumor volume began to increase in mice 21 d after murinized CAR-T cell therapy, while no significant change was observed in the mice treated with hu-manized CAR-T cells. All of the mice died 25 d after murinized CAR-T cell therapy, while the deaths among those under humanized CAR-T cell therapy occurred on 31 d. The proportion of CAR-T cells in mice reached the peak 7 d after receiving humanized or murinized CAR-T cell therapy, while that in the humanized group was significantly higher than that in the murinized group at any time point (P4 d=0. 001, P7 d=0. 000, P14 d=0. 003). Murinized CAR-T cells became undetectable on 21 d, while humanized CAR-T cells on 35 d. The maximum survival time for mice in the PBMC and murinized and humanized CAR-T cell groups was 20 d, 25 d and 53 d, respectively. Conclusions Compared with murinized CD19 CAR-T cells, humanized CD19 CAR-T cells showed stronger proliferation potential and cytotoxicity and remained in vivo detectable for a longer period of time. This study indicated that humanized CD19 CAR-T cells were superior to murinized CD19 CAR-T cells for the treatment of B cell lymphoma.