Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4+ and CD8+ T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies.

中文翻译：

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CRISPR 介导的 TCR 替代产生优越的抗癌转基因 T 细胞

过继转移经过基因修饰以表达癌症特异性 T 细胞受体 (TCR) 的 T 细胞已显示出对血液肿瘤和实体瘤的显着治疗潜力。然而，用转基因 TCR 转导 T 细胞的一个主要问题是 TCR 在受体细胞中的预先存在的表达。这些内源性 TCR 与转基因 TCR 竞争表面表达并允许形成混合二聚体。由内源性和转基因 TCR 之间的错配形成的混合二聚体可能具有自身反应性特异性。为了规避这些问题，我们设计了一个系统，其中使用成簇的规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 技术从受体细胞中敲除内源性 TCR-β，同时转导癌症-选择的反应性受体。这种 TCR 替代策略导致转基因 αβ 和 γδ TCR 的表面表达显着增加，进而转化为工程化 T 细胞对其靶癌细胞系的更强、更多功能的反应。此外，与标准 TCR 转导的 T 细胞或用于研究 TCR 活性的常规模型代理系统相比，TCR 加 CRISPR 修饰的 T 细胞对抗原的敏感性高达一千倍。最后，使用泛癌反应性 γδ TCR 转导与 CRISPR/Cas9 敲除内源性 αβ TCR 一起使用，可以更有效地将 CD4+ 和 CD8+ T 细胞重新定向到一组已确定的血癌和原发性、患者来源的 B细胞急性淋巴细胞白血病原始细胞与标准 TCR 转移相比。