嵌合抗原受体 (CAR) T 细胞已成功用于治疗各种血癌，但副作用限制了其潜力。在这里，我们开发了嵌合接头蛋白 (CAP) 和 CAR 酪氨酸激酶 (CAR-TK)，其中细胞内 z T 细胞受体 (TCR z) 链被细胞内蛋白结构域取代，以刺激 TCR z 链下游的信号传导。 CAP 包含适配器结构域和 ZAP70 的激酶结构域，而 CAR-TK 仅包含 ZAP70 结构域。我们假设 CAP 和 CAR-TK 比 CAR 更有效，因为它们会绕过定义 TCR 信号阈值的步骤和上游分子的抑制调节。 CAP 的作用太强，并且在体外表现出强效信号传导。相比之下，与传统的 CD19-28 z-CAR-T 细胞相比，CAR-TK 在患有白血病的 NSG 小鼠中表现出较高的抗肿瘤功效，并显着增强了长期肿瘤清除率。 CAR-TK 以独立于激酶 Lck 的方式被激活，与 28 z-CAR 相比，显示出更慢的磷酸化动力学和更长的信号传导。 Lck 抑制减轻了 CAR-TK 细胞的耗竭并改善了长期功能。因此，可以利用 CAR-TK 的独特信号传导特性来提高经工程改造以表达抗肿瘤嵌合受体的 T 细胞的体内功效。

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor.