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T细胞急性淋巴细胞白血病细胞（T-ALL）的体内与体外杀伤作用。 方法：构建了靶向CD7 抗原的CCR γδ T细胞（CD7-DAP10-CCR-γδ T），利用慢病毒转染技术将其导入健康人外周血来源的γδ T细胞，并通过表达CD64、CD86、CD137L的人工抗原呈递细胞（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）及正常原代αβ T细胞的体外杀伤作用。此外，在T-ALL荷瘤免疫缺陷小鼠体内药效试验验证，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细胞对荷瘤免疫缺陷小鼠的体内药效。 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10000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 细胞具有非常高的毒性（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得到了显著延长。 结论：aAPC体外能成功制备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。

Objective: To develop a chimeric costimulatory receptor targeting CD7 and prepare 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: γδ T cells expressing the CD7-targeting chimeric costimulatory receptor (CD7-DAP10-CCR-γδ T) were constructed. Using lentiviral transduction, the receptor was introduced into human peripheral blood-derived γδ T cells from healthy donors, followed by ex vivo expansion with artificial antigen-presenting cell (aAPC) expressing CD64, CD86, and CD137L. The cytotoxic activity of CD7-DAP10-CCR-γδ T cells against T-ALL cells (Jurkat), CD7-deficient Jurkat cells (CD7? Jurkat), and normal primary αβ T cells was assessed using the Annexin V/7-AAD assay. Furthermore, the in vivo efficacy was evaluated in an immunodeficient mouse model bearing T-ALL xenografts. Tumor burden was monitored regularly via in vivo imaging, and body weight changes and survival rates were recorded. Results: CD7-DAP10-CCR-γδ T cells were successfully expanded using aAPC, achieving an average expansion fold exceeding 10000. In vitro cytotoxicity assays demonstrated that these cells exhibited significantly high killing activity against T-ALL cells and 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 the T-ALL xenograft model, treatment with CD7-DAP10-CCR-γδ T cells resulted in a significant prolongation of survival compared to the PBS control group. Conclusion: This study establishes a robust aAPC-driven protocol for generating CD7-DAP10-CCR-γδ T cells with scalable expansion ( > 10 000-fold) and CD7-specific anti-tumor efficacy. The engineered cells exhibited selective cytotoxicity against T-ALL while sparing normal T cells, highlighting their therapeutic promise for relapsed/refractory T-ALL with mitigated on-target/off-tumor risks.

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)