In vitro construction and amplification and primary functional analysis of antiCD19 chimeric antigen receptor(CD19-CAR) modifiedTcells

LI Jian; Tian Fang; Jiang Pengjun; Kong Xiangtu; WU Jian; Yin Tingting; Xing Yun; Jin Liang; Hao Ruidong; Liu Gentao; Zhu Xuejun

Return

In vitro construction and amplification and primary functional analysis of antiCD19 chimeric antigen receptor(CD19-CAR) modifiedTcells

VernacularTitle:嵌合抗原受体修饰的CD19-CAR-T的体外构建、扩增及初步功能鉴定
Author: LI Jian ^{1
,

2} ; TIAN Fang ³ ; JIANG Pengjun ⁴ ; KONG Xiangtu ⁴ ; WU Jian ³ ; YIN Tingting ⁴ ; XING Yun ⁵ ; JIN Liang ⁵ ; HAO Ruidong ⁶ ; LIU Gentao ⁶ ; ZHU Xuejun ⁴
Author Information

1. 1. a. Department of Hematology, Jiangsu Provincial Hospital of TCM Affiliated to Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu, China
2. 2. College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, Jiangsu, China
3. b. Central Laboratory, Jiangsu Provincial Hospital of TCM Affiliated to Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu, China
4. 1. a. Department of Hematology
5. 2. College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, Jiangsu, China
6. 3. Shanghai Biomed-Union Biotechnology Co. Ltd., Shanghai 201321, China
Publication Type:Journal Article
Keywords: CD19 chimeric antigen receptor; leukemia; lymphoma; cytotoxicity
From: Chinese Journal of Cancer Biotherapy 2018;25(4):389-393
CountryChina
Language:Chinese
Abstract: [Abstract] Objective: To establish a chimeric antigen receptor（CAR）modified T cells specifically targeting CD19 molecule (CD19CAR-T cells) and to testify their in vitro killing effect on target cells. Methods: CD19-CAR fragments yielded by PCR were constructed into pCDH-GFP lentiviral vectors by molecular cloning technology. The packaged lentiviral particles were transducted into CD3+ T cells of donors. Transduction efficiency was measured by flow cytometry and PCR. The in vitro cytotoxicity of obtained CD19CAR-T cells against CD19+ Ramos cells was tested by 7-AAD staining. Results: The amplification folds of CD3+ T cells increased to (78.8± 23.2) folds after in vitro culture for 10 days, and about (58.3±5.4)% cells expressing GFP.About (57.4±9.3)% CD19+Ramos cells were specifically killed by the CD19-CAR-T cells in vitro at the E∶T ratio of 5∶1. Conclusion: This study successfully established an effective method for constructing and amplifying CD19-CAR-T cells in vitro, which showed profound efficiency and specific cytotoxity against CD19+ Ramos cells.And this report might provide an experimental evidence for clinical treatment of CD19+ B cell neoplasmas.
Full text:20180412.pdf