Liu M J, Mu J, Yuan T, Cui R, Meng J X, Jiang Y Y, Li Y M, Deng Q
The First Central Clinical College of Tianjin Medical University, Department of Hematology, Tianjin First Central Hospital, Tianjin 300192, China.
Zhonghua Xue Ye Xue Za Zhi. 2021 Feb 14;42(2):140-145. doi: 10.3760/cma.j.issn.0253-2727.2021.02.009.
To investigate the characteristics and cytotoxicity in vitro of the residual leukemia cells in the culture system that caused the accidental transfer of CD19 chimeric antigen receptor (CAR) into leukemia cells during the preparation of autologous CD19 CAR-T cells of relapsed/refractory B-cell acute lymphoblastic leukemia. ①Peripheral blood mononuclear cells (PBMC) of 30 patients with relapsed/refractory B-cell acute lymphoblastic anemia (R/R B-ALL) who accepted CD19 CAR-T cell therapy and six healthy volunteers were collected. ②The residual leukemia cells were analyzed by flow cytometry in the system after the PBMCs of R/R B-ALL patients were sorted by CD3 magnetic beads. ③ CD3(+) T cells from patients and healthy volunteers were transfected with CD19 CAR and CD22 CAR lentivirus to prepare CD19 CAR-T and CD22 CAR-T cells. ④The Nalm-6 cell line was resuscitated and the Nalm-6 cells with CD19 CAR lentivirus were transfected to prepare CD19 CAR-Nalm-6 cells. The patient's primary ALL cells were transfected with CD19 CAR lentivirus at the same time. ⑤The transfection rates were analyzed by flow cytometer, the cell proliferation was analyzed by the CCK-8 method, and the cell-killing activities were detected by the lactate dehydrogenase method. ① Among the 30 R/R B-ALL patients who received CD19 CAR-T cell therapy, two patients had 2.04% and 3.32% residual leukemia cells in CD3(+) T cells. After 4 days in culture, the residual leukemia cells disappeared and could not be detected by a flow cytometer with prolonged cultivation in vitro. ② The proliferation of CD19 CAR-Nalm-6 cells was higher than that of the Nalm-6 cells. ③ The killing activity of the CD19 CAR-T cells on Nalm-6 cells was higher than that of the CD19 CAR-Nalm6 cells at a target ratio of 1∶1 on 24, 48, 72 h, respectively. The cytotoxicity of CD22 CAR-T cells on CD19 CAR-Nalm-6 cells was significantly higher than that of CD19 CAR-T cells. ④ The cytotoxicity of CD22 CAR-T alone on CD19 CAR-Nalm-6 cells was higher than that of CD19 CAR-T combined with CD22 CAR-T at the same target ratio. The residual leukemia cells in the culture system in the preparation of CD19 CAR-T cells may lead to the introduction of CD19 CAR into leukemia cells and results in the failure of the CD19 CAR-T cell therapy. Detecting the residual leukemia cells in the culture system via flow cytometry before transfection with CD19 CAR lentivirus is needed. Thus, CD22 CAR-T cell therapy could be used as one of the salvage treatments.
为研究在复发/难治性B细胞急性淋巴细胞白血病自体CD19嵌合抗原受体（CAR）-T细胞制备过程中导致CD19 CAR意外转入白血病细胞的培养体系中残留白血病细胞的体外特性及细胞毒性。①收集30例接受CD19 CAR-T细胞治疗的复发/难治性B细胞急性淋巴细胞贫血（R/R B-ALL）患者及6名健康志愿者的外周血单个核细胞（PBMC）。②用CD3磁珠分选R/R B-ALL患者的PBMC后，采用流式细胞术分析体系中的残留白血病细胞。③将患者和健康志愿者的CD3(+) T细胞用CD19 CAR和CD22 CAR慢病毒转染，制备CD19 CAR-T和CD22 CAR-T细胞。④复苏Nalm-6细胞系，用CD19 CAR慢病毒转染Nalm-6细胞制备CD19 CAR-Nalm-6细胞。同时用CD19 CAR慢病毒转染患者的原发性ALL细胞。⑤用流式细胞仪分析转染率，采用CCK-8法分析细胞增殖情况，用乳酸脱氢酶法检测细胞杀伤活性。①在30例接受CD19 CAR-T细胞治疗的R/R B-ALL患者中，2例患者的CD3(+) T细胞中有2.04%和3.32%的残留白血病细胞。培养4天后，残留白血病细胞消失，体外长时间培养后流式细胞仪检测不到。②CD19 CAR-Nalm-6细胞的增殖能力高于Nalm-6细胞。③在靶效比为1∶1时，CD19 CAR-T细胞在24、48、72 h对Nalm-6细胞的杀伤活性高于CD19 CAR-Nalm6细胞。CD22 CAR-T细胞对CD19 CAR-Nalm-6细胞的细胞毒性显著高于CD19 CAR-T细胞。④在相同靶效比下，单独的CD22 CAR-T细胞对CD19 CAR-Nalm-6细胞的细胞毒性高于CD19 CAR-T细胞与CD22 CAR-T细胞联合使用时。在制备CD19 CAR-T细胞的培养体系中残留白血病细胞可能导致CD19 CAR转入白血病细胞，导致CD19 CAR-T细胞治疗失败。在用CD19 CAR慢病毒转染前，需要通过流式细胞术检测培养体系中的残留白血病细胞。因此，CD22 CAR-T细胞治疗可作为挽救治疗方法之一。
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