一个新的CD19嵌合抗原受体的构建以及其对抗白血病的功能研究
技术资料下载地址: http://www.ncbi.nlm.nih.gov/pubmed/26840021文献来源: Oncotarget
Construction of a new anti-CD19 chimeric antigen receptor and the anti-leukemia function study of the transduced T cells.
Abstract
Chimeric antigen receptor (CAR) transduced T cells have been used to efficiently kill the target tumor cells depending on the single chain variable fragment (scFv) against the specific tumor associated antigen. Here we show the high specific cytotoxicity of the CAR-T cells with very low effector to target cell (E:T) ratio owing to the CD19-scFv, which was constructed in our laboratory and proved to be highly effective in our previous study. Four plasmids containing three generation of CAR were constructed by cloning the CD19-CAR fragment into the lentiviral vector pCDH. CD3 positive T cells were successfully transduced and the CAR protein expression was confirmed by flow cytometry and Western blot. When cocultured with CD19 positive leukemia cell line Nalm-6 cells, CAR-T cells showed specific cytotoxicity: the percentage of target cells decreased to 0 in 24 hours; IL-2, IFN-γ and TNF-α produced in cocultured supernatants increased obviously; and the cytotoxicity reached more than 80%, still remarkable even when the E:T ratio was as low as 1:4. Dynamic change of cell interaction between CAR-T and leukemia cells was visually tracked by using living cells workstation for the first time. A NOD/SCID B-ALL murine model was established using Nalm-6 cells inoculation with a morbidity rate of 100%, and the survival time was prolonged statistically with CAR-T cell treatment. These data demonstrate that the CAR-T cells we prepared could be a promising treatment strategy for CD19 positive tumor diseases.
KEYWORDS:
CD19; T cell; cellular immunotherapy; chimeric antigen receptor; lentivirus
一个新的CD19嵌合抗原受体的构建以及其对抗白血病的功能研究
摘要：
嵌合抗原受体(CAR)转移的T细胞通过单链可变片段（scFv）对抗肿瘤相关抗原，已经能够有效的用于杀灭靶向肿瘤细胞。在这篇文章中，我们显示了CAR-T细胞具有较高的特异性细胞毒性，这在我们实验室构建并且在之前的研究中也证明了其实非常有效的。通过克隆CD19-CAR片段到慢病毒载体pCDH 中，构建了4个含有3代CAR的质粒。然后成功转导了CD33阳性T细胞，通过流式细胞仪和Western杂交能够证实CAR蛋白在T细胞表面成功表达。当与CD19阳性白血病细胞Nalm-6细胞共培养时，CAR-T细胞显示了特异性的细胞毒性：在24小时之内，靶细胞的数量下降；IL-2、IFN-γ和TNF-ααγ共培养上清液中产生的量明显增加；甚至当E:T比例下降 到1:4时，细胞毒性仍然增加了80%。CAR-T细胞和白血病细胞之间的动态相互作用第一时间被实时细胞工作站可视化的追踪下来，使用Nalm-6 细胞构建了一个NOD/SCID B-ALL小鼠模型，通过使用CAR-T细胞治疗，模型的存活时间得到了明显的延长。这些数据说明了，我们制备的CAR-T细胞是一种对抗CD19阳性肿瘤疾病的一种有前景的治疗策略。
关键词：
免疫治疗；CD19细胞；T细胞；慢病毒；嵌合抗原受体；
技术资料分享自爱康得生物技术
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