靶向前列腺干细胞抗原的嵌合抗原受体T细胞构建及其抗肿瘤作用

洪娟; 陈运凡; 沈俊杰; 徐艳敏; 钱程

doi:10.16016/j.1000-5404.201712225

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靶向前列腺干细胞抗原的嵌合抗原受体T细胞构建及其抗肿瘤作用

基础医学 | 更新时间：2026-01-09

- 靶向前列腺干细胞抗原的嵌合抗原受体T细胞构建及其抗肿瘤作用
- Construction of T cells expressing chimeric antigen receptor targeting prostate stem cell antigen and its anti-tumor effect in tumor-bearing mice
- 第三军医大学学报 2018年40卷第12期页码：1053-1059
- 作者机构：
  
  400038 重庆，陆军军医大学(第三军医大学)第一附属医院生物治疗中心
- 作者简介：
  
  [ 钱程，E-mail:cqian8634@gmail.com QIAN Cheng, E-mail:cqian8634@gmail.com
- 基金信息：
  
  国家重点研发计划(2016YFC1303405);陆军军医大学(第三军医大学)第一附属医院重大技术创新计划重大项目(SWH2016ZDCX1004)
- DOI：10.16016/j.1000-5404.201712225
  中图分类号： R322.64; R392-33; R73-362
- 收稿：2017-12-24，
  
  修回：2018-02-02，
  
  纸质出版：2018-01
- 稿件说明：
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洪娟, 陈运凡, 沈俊杰, 等. 靶向前列腺干细胞抗原的嵌合抗原受体T细胞构建及其抗肿瘤作用[J]. 第三军医大学学报, 2018,40(12):1053-1059.

Juan HONG, Yunfan CHEN, Junjie SHEN, et al. Construction of T cells expressing chimeric antigen receptor targeting prostate stem cell antigen and its anti-tumor effect in tumor-bearing mice[J]. Journal of Third Military Medical University, 2018, 40(12): 1053-1059.
洪娟, 陈运凡, 沈俊杰, 等. 靶向前列腺干细胞抗原的嵌合抗原受体T细胞构建及其抗肿瘤作用[J]. 第三军医大学学报, 2018,40(12):1053-1059. DOI： 10.16016/j.1000-5404.201712225.

Juan HONG, Yunfan CHEN, Junjie SHEN, et al. Construction of T cells expressing chimeric antigen receptor targeting prostate stem cell antigen and its anti-tumor effect in tumor-bearing mice[J]. Journal of Third Military Medical University, 2018, 40(12): 1053-1059. DOI： 10.16016/j.1000-5404.201712225.

摘要

目的

制备表达靶向前列腺干细胞抗原(prostate stem cell antigen，PSCA)的嵌合抗原受体修饰的T细胞(chimeric antigen receptor T-cell，CAR-T)

研究其嵌合抗原受体(chimeric antigen receptor，CAR)的表达及其对肿瘤细胞的体内外杀伤作用。

方法

基因合成靶向PSCA的单克隆抗体的轻链和重链可变区(PSCA ScFv)，利用酶切将其插入慢病毒载体中。病毒感染T淋巴细胞后，通过流式细胞术检测PSCA-CAR在T淋巴细胞中表达的阳性率。利用体外杀伤实验及ELISA实验分别检测PSCA-CAR-T细胞对PSCA阳性的HeLa细胞及PSCA阴性的T24细胞的杀伤效果、特异性及其细胞因子分泌情况。利用NOG小鼠背部皮下注射HeLa细胞建立移植瘤模型。

结果

酶切结果表明慢病毒载体pL-PSCA-G4H-28TM-28BBζ构建成功，DNA测序结果表明PSCA-CAR的载体序列正确；病毒感染T淋巴细胞后CAR表达阳性率为53.9%；获得的PSCA-CAR-T细胞不管是在高效靶比短时作用条件下还是低效靶比长时作用条件下，其对PSCA阳性肿瘤细胞的杀伤作用均明显强于PSCA阴性肿瘤细胞(

0.01)；同时获得的PSCA-CAR-T在受到PSCA阳性肿瘤细胞刺激时，分泌的细胞因子量显著高于对照的T细胞(

0.01)，而在受到PSCA阴性肿瘤细胞刺激时几乎不分泌细胞因子。体内实验证实PSCA-CAR-T细胞在体内有显著的肿瘤抑制效果(

0.01)。

结论

成功制备了靶向PSCA的CAR-T细胞，该CAR-T细胞能特异杀伤PSCA阳性的肿瘤细胞。

Abstract

Objective

To construct genetically modified T cells expressing chimeric anti

gen receptor (CAR) targeting prostate stem cell antigen (PSCA) and test the cytotoxicity of these cells against tumor cells both

in vitro

and in a tumor-bearing mouse model.

Methods

The sequences encoding the light and heavy chain variable regions of a monoclonal antibody targeting PSCA were synthesized and integrated into lentiviral vectors using restriction enzymes. Human T lymphocytes isolated from peripheral blood mononuclear cells were infected with the recombinant lentivirus and the positivity rate of PSCA-CAR expression in the T lymphocytes was detected using flow cytometry. Cytotoxic assay was performed to evaluate the specific cytotoxicity of the genetically modified T cells against the PSCA-positive HeLa cells and PSCA-negotive T24 cells

and ELISA was used to detect cytokine secretion by the cells. The antitumor effect of PSCA-CAR-T cells was also tested in a NOG mouse model bearing HeLa cell xenograft.

Results

DNA sequencing analysis suggested correct synthesis of PSCA-CAR sequences

and enzyme digestion verified successful construction of the lentiviral vector PL-PSCA-G4H-28TM-28BBζ. The positivity rate of CAR expression was 53.9% in the T lymphocytes after infection with the lentiviral vector. The PSCA-CAR-T cells showed a significantly stronger cytotoxicity against PSCA-positive tumor cells than against PSCA-negative tumor cells (

0.01) both in short-term co-incubation at a high effective-target ratio and in long-term co-incubation at a low effective-target ratio. The PSCA-CAR-T cells secreted significantly higher levels of cytokines than the control T cells upon stimulation by PSCA-positive tumor cells (

0.01)

but the secretions were almost undetectable upon stimulation by PSCA-negative tumor cells. In the

in vivo

experiment

PSCA-CAR-T cells exhibited a significant tumor-suppressive effect in the tumor-bearing mice (

0.01).

Conclusion

PSCA-CAR-T cells capable of specific killing of PSCA-positive tumor cells are successfully constructed both

in vitro

and

in vivo

关键词

Keywords

references

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