IL-2-inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells. IL-2-inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several significant 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 TCR signaling, while its role in CAR signaling is unknown. By electroporation of 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. Overall design: To investigate the transcriptomic regulation by ITK in CD19-CAR-T cells, we generated ITK-KO CD19-CAR-T cells in which ITK gene has been deleted by Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing technology.

尽管嵌合抗原受体（chimeric antigen receptor, CAR）T细胞疗法在癌症尤其是白血病治疗中取得了革命性进展，但仍存在多项显著限制其治疗效能的挑战。其中尤为关键的是癌症复发问题，这在很大程度上源于CAR-T细胞在体内的耗竭及存续时间短暂。白细胞介素-2诱导性T细胞激酶（IL-2-inducible T cell kinase, ITK）是T细胞受体（T cell receptor, TCR）信号强度的关键调节因子，但其在CAR信号通路中的作用尚不清楚。我们通过将Cas9核糖核蛋白（ribonucleoprotein, RNP）复合物电转染至CAR-T细胞中，以高效率在CD19-CAR-T细胞中成功敲除了ITK基因。批量RNA测序与单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析显示，ITK缺陷型CD19-CAR-T细胞中耗竭相关基因特征表达下调，而记忆相关基因特征表达上调。本研究结果进一步证实，ITK缺陷可显著减轻T细胞耗竭、增强T细胞记忆功能，并在体外及体内均有效促进CAR-T细胞的扩增与存续。此外，ITK缺陷型CD19-CAR-T细胞可更好地抑制肿瘤复发。本研究提出了一种靶向ITK的极具前景的策略，用于开发可临床应用的长效CAR-T产品。
总体实验设计：为探究ITK对CD19-CAR-T细胞的转录调控作用，我们通过成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats, CRISPR）介导的基因编辑技术构建了ITK敲除的CD19-CAR-T细胞。