Internal Medicine III, University Cancer Center (UCT), Research Center for Immunotherapy (FZI), University Medical Center (UMC) of the Johannes Gutenberg University and German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany.
Division of Molecular Oncology of Gastrointestinal Tumors, German Cancer Research Center (DKFZ), Heidelberg, Germany.
PLoS One. 2020 Sep 9;15(9):e0238875. doi: 10.1371/journal.pone.0238875. eCollection 2020.
To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRβ V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αβTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order β-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.
为了便于对肿瘤反应性 T 细胞克隆衍生的 T 细胞受体（TCR）进行临床前测试，有必要开发方便、快速的克隆策略来生成 TCR 表达构建体。在此，我们描述了一个 pDONR™221 载体骨架，该骨架允许生成编码优化的双顺反子αβTCR 构建体的 Gateway™ 兼容入口克隆。它携带 P2A 连接的 TCR 恒定区和 Type IIS 限制酶 BsmBI 和 BsaI 的头对头定向识别位点，用于分别无缝克隆 TCRα 和 TCRβ V(D)J 区。还纳入了其他成熟的 TCR 优化，以增强 TCR 的功能。这包括用其密码子优化的鼠类对应物替换人αβTCR 恒定区以进行嵌合，添加第二个链间二硫键，并按β-P2A-α的顺序排列 TCR 链。我们通过在原代人 T 细胞中克隆和功能测试三种黑色素瘤反应性 TCR 来举例说明我们的载体骨架的实用性。
