一种新的CD123嵌合抗原受体T细胞的构建及其抗白血病作用探究

OBJECTIVE: To construct a new CD123-specific chimeric antigen receptor in order to provide a foundation for immunotherapy of CD123 positive leukemia. METHODS: A hybridoma strain（6E11）capable of stably secreting CD123 antibody was obtained by a monoclonal screening technique, and the hybridoma cells were expanded and injected intraperitoneally to the pretreated Balb/c mice. Ascites was collected and purified to obtain the monoclonal antibody（mAb）. The affinity and specificity of 6E11 mAb were measured. The variable regions of the heavy and light chains of the 6E11 mAb were cloned by RT-PCR from the 6E11 mouse hybridoma. We generated a new CD123 specific chimeric antigen receptor with a scFv fragment derived from 6E11 antibody, designated as 6E11 CAR. T cells were transduced with lentiviral supernatant from 293T cells transfected with 6E11 CAR plasmid to generate 6E11 CAR-T cells. The specific cytotoxicity of 6E11 CAR-T against CD123(+) acute myeloid leukemia（AML）cell lines and primary AML cells in vitro were evaluated by co-culture experiments, degranulation experiments and cytokine releasing assay. RESULTS: ① A hybridoma cell line 6E11 stably secreting anti-human CD123 antibody was developed and its variable region sequences were obtained. ② The 6E11 mAb has high affinity for CD123 protein (Kd value: 2.1 nmol/L). The 6E11 mAb specifically recognizes CD123(+) cell line THP-1 cells and does not respond to CD123(−) cell line Jurkat cells. ③ 6E11 CAR-T cells were successfully generated with a CAR expression rate higher than 60％. ④ 6E11 CAR-T cells could specifically kill CD123(+) MV4-11 cell line but had no killing effect on the CD123(−) K562 cell line. Compared with vector-T cells, 6E11 CAR-T cells have higher killing rate to MV4-11 cells [(98.60 ± 1.20)％ vs (20.28±6.74)％, P<0.001]. ⑤ MV4-11 cells activated 6E11 CAR-T cells significantly but not Vector-T cells [(26.33±3.30)％ vs (1.17±0.06)％, P<0.001]. ⑥ 6E11 CAR-T cells released more cytokines than vector-T cells when co-cultured with MV4-11 [IL-2:(92.90±1.51) pg/ml vs (6.05±3.41) pg/ml, P<0.001; TNF-α: (1 407.20±91.95) pg/ml vs (7.86±0.85) pg/ml, P<0.001; IFN-γ: (5 614.60±170.17) pg/ml vs (8.42±2.70) pg/ml, P<0.001]. The IFN-γ, IL-2 and TNF-α in the 6E11 CAR-T group were similar to those in the Vector-T group when co-cultured with K562. ⑦ 6E11 CAR-T cells could be activated by bone marrow mononuclear cells (BMMNC) derived from CD123 (+) AML patients and effectively kill these BMMNC cells from CD123(+) AML patients. CONCLUSION: 6E11 hybridoma cell line can stably secrete highly specific monoclonal antibodies against human CD123, which can be used to detect the expression of human CD123. It can also be used to target human CD123 protein in tumor immunotherapy. CD123 CAR-T cells with 6E11 Ig variable region sequence have specific anti-leukemic activity in vitro, which may provide a new option for further clinical research of AML.