To model T cell antigen engagement, Gudipati and Rydzek et al. developed a glass-supported lipid bilayer displaying antigens (ROR1 and cytomegalovirus (CMV)-pMHC) capable of activating ROR1-CAR, CMV-specific CD8+ T cells. CAR signaling (intracellular calcium flux and IFNγ secretion) was less efficient than TCR signaling, requiring ~1000x higher target antigen density. Compared to intact TCR, synapses formed by CAR engagement demonstrated reduced CD3ζ ITAM phosphorylation and ZAP-70 activity, and recruited more antigen to initiate a response. Varying the CAR antibody, target, or costimulatory domain minimally affected sensitivity.
Contributed by Alex Najibi
ABSTRACT: Rational design of chimeric antigen receptors (CARs) with optimized anticancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative, single-molecule, live-cell imaging and found the sensitivity of CAR T cells toward antigen approximately 1,000-times reduced as compared to T cell antigen-receptor-mediated recognition of nominal peptide-major histocompatibility complexes. While CARs outperformed T cell antigen receptors with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-protein kinase ZAP-70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR designs, which presently limit the efficacy of CAR T cell therapies to target tumors with diminished antigen expression.