Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Exp Cell Res. 2011 Nov 1;317(18):2630-41. doi: 10.1016/j.yexcr.2011.08.015. Epub 2011 Aug 28.
The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.
T 淋巴细胞在抗肿瘤免疫中的关键作用促使人们开发出了针对肿瘤细胞的新型靶向和激活 T 细胞的策略。重组 DNA 技术已被用于生成非 MHC 限制的嵌合抗原受体（CAR）。在这里，我们构建了一组含有抗 MUC1 纳米抗体和信号及共信号结构域（CD3ζ/CD28）的重组 CAR，其间隔区来源于人 IgG3，具有一个或两个铰链序列重复或 FcγRII 铰链区。PhiC31 整合酶系统被用于研究重组效率是否可以招募高表达和稳定表达 T 细胞嵌合受体基因。核定位信号（NLS）和两种不同启动子（CMV 和 CAG）对 PhiC31 整合酶在人 T 细胞系中的效力的影响进行了评估。整合酶与 NLS 共同存在时，分别介导 ZCHN-attB 和 ZCHHN-attB 盒整合 T 细胞中 CAR 表达增加 7.6 倍和 8.5 倍。我们的结果表明，PhiC31 整合酶对 Jurkat 细胞系的高效且稳定转导是用于癌症免疫治疗的嵌合抗肿瘤 T 细胞生成的一种可行方式。
