Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects.
10.3760/cma.j.issn.0253-2727.2022.04.003
- VernacularTitle:靶向CD52嵌合抗原受体T细胞的制备及其抗白血病作用研究
- Author:
Yan LIU
1
;
Yu LIU
1
;
Ke Jing TANG
1
;
Zhao Qi CHEN
1
;
Jun Li MOU
1
;
Ying Xi XU
1
;
Hai Yan XING
1
;
Zheng TIAN
1
;
Qing RAO
1
;
Min WANG
1
;
Jian Xiang WANG
1
Author Information
1. 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.
- Publication Type:Journal Article
- Keywords:
CD52;
Chimeric antigen receptor T cells;
Immunotherapy
- MeSH:
CD52 Antigen;
Cell Line, Tumor;
Humans;
Immunotherapy, Adoptive/methods*;
Lentivirus/genetics*;
Leukemia;
Receptors, Antigen, T-Cell;
Receptors, Chimeric Antigen/genetics*
- From:
Chinese Journal of Hematology
2022;43(4):279-286
- CountryChina
- Language:Chinese
-
Abstract:
Objective: 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. Methods: 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. Results: 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) %, vs Vector-T (56.58±19.8) %, P=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 (P<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) % vs (13.06± 4.23) %, P<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, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.
