Kavli Affiliate: Michael W. Young
| Authors: Yi Sun, Tyler J. Florio, Sagar Gupta, Michael C. Young, Quinlen F. Marshall, Samuel E. Garfinkle, Georgia F. Papadaki, Hau V. Truong, Emily Mycek, Peiyao Li, Alvin Farrel, Nicole L. Church, Shereen Jabar, Matthew D. Beasley, Ben R. Kiefel, Mark Yarmarkovich, Leena Mallik, John M. Maris and Nikolaos G. Sgourakis
| Summary:
Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy1. We have previously developed a PC-CAR targeting a neuroblastoma- associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes2. Here, we determine the 2.1 Å structure of the PC-CAR:PHOX2B/HLA-A*24:02/β2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.