Research ArticleCAR-T CELLS

An immunoproteomic approach to characterize the CAR interactome and signalosome

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Science Signaling  12 Feb 2019:
Vol. 12, Issue 568, eaap9777
DOI: 10.1126/scisignal.aap9777

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The long and short of CAR activation

B cell malignancies can be therapeutically targeted by the adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR). In T cells, CARs that encode specific signaling domains have unique functional responses to antigen recognition. Ramello et al. used immunoproteomics to understand how CARs with distinct intracellular domains activated signaling in human T cells. Independently of specific signaling domains, the overall length of CAR intracellular activation domains determined whether a CAR interacted with the signaling protein CD3ζ and promoted strong T cell signaling. These data may explain why some CARs can stimulate antigen-independent tonic signaling, which leads to progressive CAR-T cell inactivation.

Abstract

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.

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