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.
Author Info: (1) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (2) Medizinische Klinik un
Author Info: (1) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (2) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, Wurzburg, Germany. (3) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (4) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, Wurzburg, Germany. (5) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. (6) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. Roche Diagnostics GmbH, Penzberg, Germany. (7) Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria. (8) Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria. (9) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, Wurzburg, Germany. (10) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. (11) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, Wurzburg, Germany. Hudecek_M@ukw.de. (12) Center for Pathophysiology, Infectiology and Immunology, Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria. johannes.huppa@meduniwien.ac.at.
