目的：针对CD7阳性的急性髓系白血病（acute myeloid leukemia，AML）细胞开发一种新型的CD7嵌合抗原受体修饰 的T细胞（CD7-CAR-T），观察其对CD7阳性AML细胞的杀伤作用。方法：基于CD7纳米抗体序列、CD28和4-1BB共刺激域序 列构建CD7-CAR慢病毒载体，包装病毒颗粒并与CD7蛋白阻断剂（protein expression blocker，PEBL）慢病毒共感染人T细胞， 制 备CD7-CAR-T；应用实时无标记动态细胞分析技术（real time cellular analysis，RTCA）验证CD7-CAR-T对CD7过表达的293T细 胞的特异性杀伤能力，流式细胞术检测CD7-CAR-T对CD7高、中、低表达的AML细胞（KG-1、HEL、Kasumi-1细胞）的增殖和细 胞因子分泌的影响。结果：成功构建CD7-CAR-T 细胞并阻断其表面 CD7 的表达，与 T 细胞相比，CD7-CAR-T 细胞可以抑 制 CD7-293T 细胞的增殖并促进其分泌 TNF、Granzyme B 和 INF-γ，可显著促进 CD7 阳性的 KG-1 细胞和 HEL细胞的凋亡 （t=147.1、 P＜0.01； t=23.57、 P＜0.01）和细胞 因 子 分 泌（P＜0.05 或 P＜0.01），而对低表达 CD7 的 Kasumi-1 细胞无显著影响 （t=0.7058、 P＞0.05）。结论： CD7-CAR-T细胞能够特异性杀伤CD7阳性的AML细胞。

Objective: To develop a new type of CD7 chimeric antigen receptor modified T cell (CD7-CAR-T) for the treatment of CD7 positive acute myeloid leukemia (AML), and to observe its killing effect on CD7 positive AML cells. Methods: The CD7-CAR lentiviral vector was constructed based on the CD7 Nanobody sequence and costimulatory domain sequence of CD28 and 4-1BB. The lentiviral particles were packaged and used to co-transfect human T cells with protein expression blocker (PEBL), so as to prepare CD7CAR-T cells. Real time cellular analysis (RTCA) was used to monitor the cytotoxicity of CD7-CAR-T cells on CD7 overexpressed 293T cells. Flow cytometry assay was used to detect the effect of CD7-CAR-T cells on proliferation and cytokine secretion of AML cells with high, medium and low CD7 expressions (KG-1, HEL and Kasumi-1 cells, respectively). Results: CD7-CAR-T cell was successfully constructed and its surface expression of CD7 was successfully blocked. Compared with T cells, CD7-CAR-T cells could significantly inhibit the proliferation of CD7-293T cells and promote the release of TNF, Granzyme B and INF-γ; in addition, CD7CAR-T cells also significantly promoted the apoptosis (t=147.1, P<0.01; t=23.57, P<0.01) and cytokine release (P<0.05 or P<0.01) in CD7 positive KG-1 and HEL cells, but had little effect on Kasumi-1 cells that only expressed minimal CD7 antigen (t=0.7058, P>0.05). Conclusion: CD7-CAR-T cells can specifically kill CD7-positiveAMLcells in vitro.

国家重点研发计划资助项目（No. 2016YFC1303403）；国家自然科学基金资助项目（No. 81872431；31471283）；江苏省高等学校优势 学科建设工程资助项目；协同创新重大项目资助（No. XYXT-2015304）；江苏省“六大人才高峰工程”资助项目（No. SWYY-CXTD-010）；江苏省高 等学校自然科学基金资助项目（No. 19KJD320003）