CD3ζ ITAM diversity determines chimeric antigen receptor force and function.

The Chimeric antigen receptor (CAR) T cell therapy is a powerful treatment for hematologic malignancies; however, clinical responses vary depending on CAR T cell function and persistence. The optimization of CAR design has gained considerable interest with a new focus on calibrating CAR intracellular signals via the immunoreceptor tyrosine-based activation motifs (ITAMs) to increase the fitness of CAR T cells. Evidence suggests that unique ITAM motifs at positions 1, 2, or 3 (ITAMs-A, B, and C respectively) of the CD3ζ signaling domain may have non-redundant roles in signal strength, T cell functionality, persistence, and memory formation. However, the individual contributions of each CD3ζ ITAM in transmitting force and activating mechanosensitive signals are yet unexplored. We have found that human CARs expressing specific ITAM sequences (ITAM-A, ITAM-B, or ITAM-C only) generate differing amounts of force and bond lifetimes after interacting with their target antigen, CD19. Therefore, a CAR may act as a mechanosensor similar to a T cell receptor, discriminating the quality of the interaction with antigen as cellular-derived forces are applied to the bond. The higher transmission of force directly correlates with better anti-tumor efficacy in vitro and in vivo. Hence, understanding the mechanotransduction in CARs has important implications in “tuning” the activation of a CAR T cell without changing specificity.