Differential protein-protein interactions underlie signaling mediated by the TCR and a 4-1BB domain–containing CAR

Abstract

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)–mediated signaling, but the number and type of protein interaction–mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.

Regulation of temporal cytokine production by co-stimulation receptors in TCR-T cells is lost in CAR-T cells

“…We hypothesise that this regulation by the 4-1BB co-stimulation receptor will be lost in 2nd generation CARs that rely on the 4-1BB rather than the CD28 co-stimulation domain. Moreover, we predict that 3rd generation CARs that contain both CD28 and 4-1BB co-stimulation domains would not exhibit higher cytokine production by engagement of CD28 and 4-1BB receptors, as their signalling pathways would be fully activated by the CAR [ 36 , 37 ].…”

CAR-NK Cell Therapy: A Transformative Approach to Overcoming Oncological Challenges

“…Year Cell Type Generation Intracellular Signaling Domains P Kvacskay [14] 2024 CAR-T First CD3ζ Y Wang [15] 2024 CAR-T Second CD28, 4-1BB with CD3ζ Ritmeester-Loy [16] 2024 CAR-T Third CD28, 4-1BB, and CD3ζ R Basar [13] 2020 CAR-NK First CD3ζ or NK cell activating domains (e.g., 2B4)…”