Adoptive transfer of T cells that express a chimeric antigen receptor (CAR) is an approved immunotherapy that may be curative for some hematological cancers. To better understand the therapeutic mechanism of action, we systematically analyzed CAR signaling in human primary T cells by mass spectrometry. When we compared the interactomes and the signaling pathways activated by distinct CAR-T cells that shared the same antigen-binding domain but differed in their intracellular domains and their in vivo antitumor efficacy, we found that only second-generation CARs induced the expression of a constitutively phosphorylated form of CD3ζ that resembled the endogenous species. This phenomenon was independent of the choice of costimulatory domains, or the hinge/transmembrane region. Rather, it was dependent on the size of the intracellular domains. Moreover, the second-generation design was also associated with stronger phosphorylation of downstream secondary messengers, as evidenced by global phosphoproteome analysis. These results suggest that second-generation CARs can activate additional sources of CD3ζ signaling, and this may contribute to more intense signaling and superior antitumor efficacy that they display compared to third-generation CARs. Moreover, our results provide a deeper understanding of how CARs interact physically and/or functionally with endogenous T cell molecules, which will inform the development of novel optimized immune receptors.

表达嵌合抗原受体（CAR）的T细胞的过继转移是一种经过认可的免疫疗法，对于某些血液系统癌症可能是治愈的。为了更好地了解作用的治疗机制，我们通过质谱系统分析了人类原代T细胞中的CAR信号传导。当我们比较相互作用的基因组和由共享相同的抗原结合结构域但细胞内结构域和体内抗肿瘤功效不同的不同CAR-T细胞激活的信号通路时，我们发现只有第二代CARs诱导了a的表达。 CD3ζ的组成型磷酸化形式，类似于内源性物种。这种现象与共刺激域或铰链/跨膜区域的选择无关。相反，这取决于细胞内结构域的大小。而且，第二代设计还与下游二级信使的更强磷酸化有关，这已通过全球磷酸化蛋白质组分析得到了证明。这些结果表明，第二代CAR可以激活CD3ζ信号传导的其他来源，与第三代CAR相比，它们可能显示出更强的信号传导和更强的抗肿瘤功效。此外，我们的结果提供了对CAR如何与内源性T细胞分子进行物理和/或功能相互作用的更深入的了解，这将为新型优化免疫受体的发展提供信息。这些结果表明，第二代CAR可以激活CD3ζ信号传导的其他来源，与第三代CAR相比，它们可能会显示出更强的信号传导和更强的抗肿瘤功效。此外，我们的结果提供了对CAR如何与内源性T细胞分子进行物理和/或功能相互作用的更深入的了解，这将为新型优化免疫受体的发展提供信息。这些结果表明，第二代CAR可以激活CD3ζ信号传导的其他来源，与第三代CAR相比，它们可能会显示出更强的信号传导和更强的抗肿瘤功效。此外，我们的结果提供了对CAR如何与内源性T细胞分子进行物理和/或功能相互作用的更深入的了解，这将为新型优化免疫受体的发展提供信息。