Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T-cell receptor (TCR) signaling, while its role in CAR signaling is unknown. By electroporation of clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.

中文翻译：

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IL-2 诱导型 T 细胞激酶缺陷维持针对肿瘤细胞的嵌合抗原受体 T 细胞治疗。


尽管嵌合抗原受体 （CAR） T 细胞疗法在治疗癌症（尤其是白血病）方面取得了革命性的成就，但一些关键挑战仍然限制了其治疗效果。特别相关的是癌症的复发，这在很大程度上是由于 CAR-T 细胞在体内的耗竭和短暂持续存在。IL-2 诱导型 T 细胞激酶 （ITK） 是 T 细胞受体 （TCR） 信号传导强度的关键调节剂，而其在 CAR 信号传导中的作用尚不清楚。通过将成簇的规则间隔短回文重复序列 （CRISPR） 相关蛋白 9 （Cas9） 核糖核蛋白 （RNP） 复合物电穿孔到 CAR-T 细胞中，我们成功地高效地删除了 CD19-CAR-T 细胞中的 ITK。大量和单细胞 RNA 测序 （scRNA-seq） 分析揭示了 ITK 缺陷型 CD19-CAR-T 细胞中耗竭的下调和记忆基因特征的上调。我们的结果进一步表明，T 细胞耗竭显着减少，T 细胞记忆增强，CAR-T 细胞在体外和体内的扩增和持久性显着改善。此外，ITK 缺陷的 CD19-CAR-T 细胞显示出更好的肿瘤复发控制。我们的工作提供了一种有前途的策略，可以靶向 ITK 开发用于临床的可持续 CAR-T 产品。