An immunoproteomic approach to characterize the CAR interactome and signalosome

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 prostate cancer-specific 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 anti-tumor 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 co-stimulatory 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 novel optimized immune receptors.

Proteomic-based evaluation of nuclear transport of NLS-tagged trastuzumab-emtansine with enhanced cytotoxic potency

“…In contrast, studies on proteomic profiling of biopharmaceuticals with respect to their dynamics inside cells is scarce. Only recently has there been important advances that have utilized proteomic profiling to determine resistance mechanisms to biopharmaceuticals such as Tmab, T-DM1 and other emerging mAb-based drugs (41)(42)(43)(44). However, these approaches have not specifically addressed intracellular transport.…”

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies

“…These results are of tremendous benefit for the design of new, safer, and more potent CAR molecules. This work represents an example where LC-MS based proteomics uncovered important changes upon CAR expression and identified co-stimulatory factors [72].…”

“…In a more recent study, Romello and co-workers combined transcriptomics, immunoprecipitation (IP) MS to identify pathways that are differently regulated upon expression of second or third generation anti-prostate stem cell antigen (PSCA) CARs and then performed untargeted phosphoproteomics to identified signaling pathway differences upon CAR activation [72]. Second and third CAR designs essentially differ by the structure/composition of their intracellular signaling endodomains.…”

Clinical translation of gene medicine

“…Chimeric antigen receptors (CARs) are transduced into T cells ex vivo , creating expandable antigen‐specific T cells with sustained CAR expression. The CAR‐T cells have resulted in substantial clinical benefits, predominantly in the treatment of hematological malignancies, which has lead to the approval of YESCARTA™ and KYMRIAH™ by the FDA in 2017 . Current research aims to expand CAR therapy to solid tumors, including melanoma, breast cancer and sarcoma …”

“…cell anemia, 26 Wiskott-Aldrich syndrome, 27 ADA-SCID, 12 adrenoleukodystrophy 28 and metachromatic leukodystrophy. 29 30 Current research aims to expand CAR therapy to solid tumors, including melanoma, breast cancer and sarcoma. 31,32 In vivo gene delivery strategies have been attracting much attention in the field of gene therapy.…”