Liu Y, Liu Y, Tang K J, Chen Z Q, Mou J L, Xu Y X, Xing H Y, Tian Z, Rao Q, Wang M, Wang J X
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Zhonghua Xue Ye Xue Za Zhi. 2022 Apr 14;43(4):279-286. doi: 10.3760/cma.j.issn.0253-2727.2022.04.003.
To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, Vector-T (56.58±19.8) %, =0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % (13.06± 4.23) %, <0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, <0.001; TNF-α: (1732±560) pg/ml, <0.01]. We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.
构建靶向CD52的嵌合抗原受体（CAR）T细胞（CD52 CAR-T），并验证CD52 CAR-T细胞对CD52阳性白血病的作用。通过分子克隆将携带4-1BB共刺激结构域的第二代靶向CD52的CAR连接到慢病毒载体中。采用四质粒系统，由293T细胞制备并包装慢病毒。用荧光素标记细胞表面抗原，以评估感染后CD52 CAR-T细胞的表型。采用流式细胞术和酶联免疫吸附测定法在体外评估CD52 CAR-T细胞对CD52阳性细胞系的特异性细胞毒性。①成功构建了表达质粒pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP，并用于转导表达新型靶向CD52的CAR的T细胞。②第6天，慢病毒转导后，CD52靶向的CAR-T几乎杀死了CD52阳性T细胞[CD52 CAR-T（4.48±4.99）%，载体T（56.58±19.8）%，P = 0.011]。③用靶向CD52的CAR转导的T细胞凋亡水平较低，并且可以在体外长期扩增。④CD52 CAR可促进T细胞分化为中枢记忆T细胞和效应记忆T细胞，而具有CD45RA(+)效应记忆表型的T细胞比例降低。⑤CD52 CAR-T细胞可特异性杀死CD52阳性的HuT78-19t细胞，但对CD52阴性的MOLT4-19t细胞无杀伤作用。对于CD52 CAR-T细胞，在效靶比为1∶1共培养24小时时，HuT78-19t细胞的残留百分比为（2.66±1.60）%，而MOLT4-19t细胞的存活百分比为（56.66±5.74）%（P<0.001）。⑥脱颗粒实验结果证实，HuT78-19t细胞可显著激活CD52 CAR-T细胞，但不能激活MOLT4-19t细胞[（57.34±11.25）%比（13.06±4.23）%，P<0.001]。⑦与载体T细胞相比，CD52 CAR-T细胞与HuT78-19t细胞共培养时释放更多细胞因子[干扰素-γ：（3706±226）pg/ml，P<0.001；肿瘤坏死因子-α：（1732±560）pg/ml，P<0.01]。我们成功制备了具有抗白血病作用的CD52 CAR-T细胞，这可能为进一步的免疫治疗提供基础。
