[摘 要] 目的：探讨构建靶向CD7抗原的嵌合共刺激受体（CCR）并制备该受体修饰的健康供者来源γδ T细胞，以评估其对急 性T淋巴细胞白血病（T-ALL）的体内与体外杀伤作用。方法：构建携带CD7-DAP10-CCR的慢病毒载体，并转导健康人外周血 γδ T细胞，制备靶向CD7抗原的CCR γδ T细胞（CD7-DAP10-CCR-γδ T），所获细胞借助表达CD64、CD86和CD137L的人工抗原 提呈细胞（aAPC）进行体外扩增。采用Annexin Ⅴ/7-AAD方法检测CD7-DAP10-CCR-γδ T对T-ALL细胞（Jurkat）、CD7缺陷型 Jurkat细胞（CD7? Jurkat）及异体健康人原代αβ T细胞的体外杀伤作用，共设置了3组效靶比（E∶T = 1∶1、1∶3和1∶10），孵育时间 为18~24 h。其中，Jurkat细胞作为CD7阳性靶细胞，CD7? Jurkat作为CD7阴性靶细胞以验证杀伤特异性，而异体健康人原代αβ T细胞则作为CD7阳性正常细胞对照，用于评估CD7-DAP10-CCR-γδ T细胞的脱靶效应。此外，在T-ALL荷瘤免疫缺陷小鼠体 内验证药效，通过定期对荷瘤免疫缺陷小鼠进行活体成像、体质量检测及生存期观察，评估CD7-DAP10-CCR-γδ T细胞在荷瘤免 疫缺陷小鼠体内的药效。结果：成功利用aAPC体外制备出CD7-DAP10-CCR-γδ T细胞，其平均扩增倍数超过10 000倍。体外 杀伤实验表明，该细胞对T-ALL细胞具有较高的杀伤能力（P < 0.01），对Jurkat 细胞具有较高的毒性（P < 0.01），对CD7? Jurkat细 胞杀伤作用有限，而对高表达CD7的正常原代αβ T细胞基本无杀伤作用；荷瘤免疫缺陷小鼠体内药效试验结果显示，相对于对照 PBS组，经CD7-DAP10-CCR-γδ T细胞治疗后，荷瘤免疫缺陷小鼠的生存期显著延长（P < 0.01）。结论：体外借助aAPC能成功制 备CD7-DAP10-CCR-γδ T细胞，并且CD7-DAP10-CCR-γδ T细胞在体外、小鼠体内均对T-ALL细胞具有较强的杀伤作用，表明 CD7-DAP10-CCR-γδ T细胞具备对T-ALL的治疗潜力。

[Abstract] Objective: To develop a chimeric costimulatory receptor (CCR) targeting the CD7 antigen and prepare CCR-modified γδ T cells from healthy donors for the evaluation of its in vitro and in vivo cytotoxic effects against T-cell acute lymphoblastic leukemia (T-ALL) cells. Methods: Lentiviral vectors carrying CD7-DAP10-CCR were constructed and γδ T cells in the peripheral blood of healthy individuals were transduced to prepare CCR γδ T cells targeting the CD7 antigen (CD7-DAP10-CCR-γδ T). The obtained cells were expanded in vitro using artificial antigen-presenting cells (aAPC) expressing CD64, CD86, and CD137L. The in vitro cytotoxic effects of CD7-DAP10-CCR-γδ T cells against T-ALL cells (Jurkat), CD7-deficient Jurkat cells (CD7? Jurkat), and healthy donor primary αβ T cells were detected using the Annexin Ⅴ/7-AAD assay. The experiment was performed at three effector-to-target (E∶T) ratios (1∶1, 1∶3, and 1∶10), with Jurkat cells as CD7 positive target cells, CD7? Jurkat cells as CD7 negative target cells to verify the killing specificity, and healthy donor primary αβ T cells as CD7 positive normal control cells to evaluate the off-target effects of CD7 DAP10-CCR-γδ T cells. The incubation time was 18-24 h. Furthermore, the in vivo efficacy was evaluated in an immunodeficient mouse model bearing T-ALL xenografts. In vivo imaging of tumor-bearing immunodeficient mice was regularly conducted, their body weight and length of survival monitored to evaluate in vivo efficacy of CD7-DAP10-CCR-γδ T cells in tumor-bearing immunodeficient mice. Results: CD7-DAP10-CCR-γδ T cells were successfully prepared in vitro using aAPC, achieving an average expansion fold exceeding 10 000. In vitro cytotoxicity assays demonstrated that these cells exhibited significantly high killing activity against T-ALL cells and significantly high toxicity against Jurkat cells (P < 0.01), while showing limited cytotoxicity against CD7? Jurkat cells and negligible effects on normal primary CD7-high αβ T cells. In vivo efficacy experiment on tumor-bearing immunodeficient mice indicated that treatment with CD7-DAP10-CCR-γδ T cells resulted in a significant prolongation of survival compared with the PBS control group. Conclusion: CD7-DAP10-CCR-γδ T cells can be successfully generated in vitro using aAPC. CD7-DAP10-CCR-γδ T cells demonstrate strong cytotoxicity against T-ALL cells both in vitro and in vivo, which suggests therapeutic potential of CD7-DAP10-CCR-γδ T cells against T-ALL cells.

[基金项目] 国家生物药技术创新中心“揭榜挂帅”技术攻关项目（No.NCTIB2023XB01003）；安徽省科技重大专项项目 （No.202203a07020030）；安徽省重点研究与开发计划项目（No.2023s07020010）；苏州市科技计划项目（No.SYG2024066）