[摘 要] 目的：制备低亲和力的CD117 CAR-T细胞，探讨其对急性髓系白血病（AML）细胞Kasumi-1的体外杀伤效应。方 法：调取CD117低亲和力抗体巴佐利单抗（barzolvolimab）和Fab-79D VH和VL序列，设计VH-(G4S)3-VL结构的单链抗体，分别 构建带4-1BB共刺激分子的经典二代CAR分子，经基因合成后分别亚克隆至pMFG逆转录病毒载体，获得CD117-79D CAR和 CD117-0159 CAR质粒。将两种CAR质粒分别包装制备逆转录病毒，检测其滴度合格后转导活化后的T细胞，构建CD117-79D CAR-T和CD117-0159 CAR-T细胞，采用流式细胞术检测两种CAR-T细胞的阳性率。将未转导T细胞与两种CAR-T细胞分别与 CD117+ Kasumi-1细胞共培养，通过流式细胞术检测Kasumi-1细胞凋亡率，以评估两种CAR-T细胞的抗肿瘤活性。结果：成功构 建 CD117-79D CAR-T 和 CD117-0159 CAR-T 细胞，其阳性率分别为（59.4 ± 2.6）%、（62.5 ± 1.2）%。未转导 T 细胞、CD117-79D CAR-T和CD117-0159 CAR-T细胞体外培养均能稳定增殖，且三者的增殖能力均无显著差异（均P > 0.05）。体外杀伤Kasumi-1 细胞结果显示，不同效靶比条件下，CD117-79D CAR-T和 CD117-0159 CAR-T细胞较未转导T细胞展现出显著增强的杀伤能力 （P < 0.05或P < 0.01），但两种CAR-T细胞的杀伤效率无显著差异（P > 0.05）。结论：成功构建低亲和力的CD117-79 CAR-T和 CD117-0159 CAR-T细胞，体外实验证实其可有效杀伤CD117+ Kasumi-1细胞，为AML的靶向治疗提供了实验依据。

[Abstract] Objective: To generate low-affinity CD117-targeting CAR-T cells and investigate their in vitro cytotoxic effects on acute myeloid leukemia (AML) Kasumi-1 cells. Methods: Low-affinity CD117-targeting monoclonal antibody (barzolvolimab) and Fab-79D VH and VL sequences were retrieved for the design of single-chain variable fragments (scFvs) with a VH-(G4S)?-VL configuration. These scFvs were then integrated individually into canonical second-generation chimeric antigen receptor (CAR) constructs harboring the 4-1BB costimulatory domain. Subsequent to gene synthesis, the resultant CAR sequences were subcloned into the pMFG retroviral vectors, yielding the CD117-79D CAR and CD117-0159 CAR plasmids, respectively. The two CAR plasmids were subjected to separate retroviral packaging and production procedures. After validation of viral titers, the recombinant retroviruses were transduced into activated T cells to generate CD117-79D CAR-T and CD117-0159 CAR-T cell products. The CAR-positive fractions of the two CAR-T cell populations were quantified utilizing flow cytometry. Untransduced T cells and the two engineered CAR-T cells were co-cultured independently with CD117 ? Kasumi-1 cells. Thereafter, the apoptotic rates of Kasumi-1 cells were detected via flow cytometry for the comparative evaluation of the anti-tumor activity of the two CAR-T cell products. Results: CD117-79D CAR-T and CD117-0159 CAR-T cells were successfully constructed, with positive rates of (59.4 ± 2.6)% and (62.5 ± 1.2)% , respectively. Untransduced T cells, CD117-79D CAR-T cells, and CD117-0159 CAR-T cells all exhibited stable proliferation in in vitro cultivation, and no statistically significant difference was observed in their proliferative abilities (all P > 0.05). Results of the in vitro cytotoxicity assay against Kasumi-1 cells demonstrated that, in varying effector-to-target ratios, both CD117-79D CAR-T and CD117-0159 CAR-T cells exerted significantly enhanced cytotoxicity compared with untransduced T cells (P < 0.05 or P < 0.01). However, no statistically significant difference was detected in the killing efficiency between the two CAR-T cell products (P > 0.05). Conclusion: CD117-79D CAR-T and CD117-0159 CAR-T cells with low-affinity were successfully generated. In vitro experiments verified that these CAR-T cells could effectively eliminate CD117? Kasumi-1 cells, thereby providing experimental evidence for the targeted therapy of AML.

[基金项目] 王建勋高层次人才科研启动经费（90011451310032）