Activity comparison of humanized CD19 CAR-T cells with murine CD19 CAR-T on Nalm-6 cells and xenograft tumor model

Abstract

Objective: To compare the activity difference of the high affinity humanized CD19 chimeric antigen receptor (CAR)-T cells and murine CD19 CAR-T cells. Methods: Peripheral venous blood T cells from 8 healthy volunteers were collected and infected with humanized and murine CD19 CAR lentivirus. Human and murine CD19 CAR-T cells were prepared and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The cytotoxicity of CD3(+) T cells, humanized and murine CD19 CAR-T cells to NALM-6 cells was detected by lactate dehydrogenase assay. Thirty BAL B/c nude mice transplanted with NALM-6 cells were randomly divided into 3 groups with 10 mice in each group and injected humanized CD19 CAR-T cells, mouse CD19 CAR-T cells and control CD3(+) T cell via tail vein, respectively. The proportion of NALM-6 cells in peripheral blood and the proportion of CD19 CAR-T cells in T cells from the vein of the inner canthus were detected by flow cytometry. The overall survival of BAL B/c nude mice was observed. Results: The proliferation of mouse and humanized CD19 CAR-T cells were (68.50±0.93)% and (80.63±1.41)%, respectively (t=20.353, P<0.001) after cultured in vitro for 24 hours, and were (91.38±1.41)% and (148.13±1.25)%, respectively (t=85.364, P<0.001) after cultured for 48 hours. When the effect to target ratio was 1∶1, there was no difference between the humanized and murine CD19 CAR-T cell group after co-culture for 24 hours (P=0.169), while the killing activity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells (P<0.01) after 48 hours of co-culture. When the effect to target ratio was 4∶1, the cytotoxicity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells in co-culture for 24 and 48 hours (P<0.01). On the seventh day of CD19 CAR-T cell therapy, the proportion of NALM-6 cells in the peripheral blood of BAL B/c nude mice decreased to the lowest level in the humanized CD19 CAR-T cell group and the murine CD19 CAR-T cell group. After 21 days, the proportion of NALM-6 cells in the murine CD19 CAR-T cell group was higher than that in the humanized CD19 CAR-T cell group (P(21 d)=0.001, P(28 d)<0.001, P(35 d)<0.001). The proportion of humanized and murine CD19 CAR-T cells in the peripheral blood reached the peaks after 7 days of therapy, and the proportion of humanized CD19 CAR-T cells was higher than that of murine CAR-T cells (P(7 d)=0.002). The CD19 CAR-T cells disappeared in the peripheral blood in the murine CD19 CAR-T cell group after 14 days of therapy, while in the humanized CD19 CAR-T cell group it disappeared after 21 days of therapy. The median survival of BAL B/c nude mice in the murine CD19 CAR-T cell group and the humanized CD19 CAR-T cell group was 42 days and 63 days, respectively (χ(2)=15.382, P<0.001). Conclusions: High affinity humanized CD19 CAR-T cells have stronger proliferation, higher cytotoxicity and longer survival time compared with those of murine CD19 CAR-T cells. The results indicate that the clinical efficacy of humanized CD19 CAR-T cells would be better than that of murine CD19 CAR-T cells.