Fig. 2: Engineering pMHC-specific CAR receptors.
From: RETRACTED ARTICLE: Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs
a, Ranked binding affinity of 10LH scFv to PHOX2B (blue) and a panel of 95 peptides presented on HLA-A*24:02 peptides (orange) demonstrate high target binding and negligible binding to HLA-A*24:02 pMHCs. b, Cross-reactivity algorithm identifies CAR constructs with significant off-target binding and informs prioritization of highly selective receptors (selective receptors marked with arrow). Peptide score represents the predicted cross-reactivity based on the amino acid sequences of normal-tissue peptides; overall score is calculated on the basis of peptide score, binding affinity and normal tissue expression; T, peptides reported in the normal tissue immunopeptidome; F, peptides absent in normal immunopeptidome. ND, not determined. c, Example counterstaining of top CAR clones with target (x-axis) and off-target (y-axis) peptides on HLA-A*24:02 reveals selective target binding in 10LH and 302LH constructs. d, Left, flow cytometry plot of predicted cross-reactive peptides compared with PHOX2B shows cross-reactive binders ABCA8 and MYO7B. Right, flow mean fluorescence intensity (MFI) used to calculate degree of binding relative to PHOX2B in table. e, Functional screening of ABCA8 and MYO7B shows CAR killing through ABCA8 only at a supraphysiological concentration of 50 µM versus through PHOX2B at 0.1 µM. ABCA8 and MYO7B were not detected in the normal tissue immunopeptidome, and none of the peptides predicted by sCRAP that were detected in the normal immunopeptidome (FDFTI, SLC23A2 and TNS4) show binding to 10LH. The experiment was performed once on the entire panel of CAR constructs and repeated for 10LH and 302LH on an expanded panel of peptides. f, Representative BLItz plot at 200 nM PHOX2B pMHC shows fast on rate for 10LH and 302LH and exceptionally slow off-rate for 10LH (k d = 7.6 × 10−4 s−1). g, Alanine scan of QYNPIRTTF reveals that mutations in five residues (N3A, I5A, R6A, T7A and T8A) result in significant abrogation of binding to PC-CAR 10LH (n = 2; data are mean ± s.d.). h, PHOX2B–HLA-A*24:02 crystal structure paired with alanine scan of 10LH enables mapping of peptide–receptor interface, revealing spatial conformation of five receptor contact residues (left, top view; right, side view of pMHC complex). Created with BioRender.com.
