Extended Data Fig. 14: Presentation of the PHOX2B peptide by multiple HLA allotypes.
From: Targeting of intracellular oncoproteins with peptide-centric CARs
a. Schematic of hypothesis proposing that PHOX2B peptide QYNPIRTTF detected by immunopeptidomics can be presented by additional HLA alleles after undergoing a common antigen processing pathway. b. Population-scale presentation across the length of the PHOX2B protein (all potential 9mers on x-axis) generated by ShinyNAP8 predicts PHOX2B peptide QYNPIRTTF to be presented by an additional 8 HLA alleles in addition to HLA-A*24:02. Additionally, QYNPIRTTF was found to bind additional common HLA alleles HLA-C*07:01, HLA-C*06:02, HLA-A*29:02, and HLA-A*32:01 using NetMHCpan 4.165 and also predicted by HLAthena44. c. Size exclusion chromatography of PHOX2B peptide QYNPIRTTF refolded with HLA-A*23:01, HLA-B*14:02, and HLA-C*07:02 shows formation of stable pMHC complex with HLA-A*23:01 and HLA-C*07:02, and minimal complex with HLA-B*14:02. d. PC-CAR 10LH binds PHOX2B on HLA-A*23:01 demonstrates higher binding than 302LH, in concordance with observed in vivo activity (Fig. 4g). e. 10LH CAR kills HLA-A*23:01/PHOX2B− WM873 cells when pulsed with PHOX2B peptide but not with CHRNA3 peptide n = 2 technical replicates; reported as mean +/− SD. f. Matched peptide search identifies unfragmented peaks in additional neuroblastoma tumors (NBSD shown). Unfragmented NBSD peaks are within 0.006 Da m/z of peaks in which PHOX2B peptide QYNPIRTTF was identified by MS/MS in other samples and eluted within one minute of fragmented peaks. While validated MS/MS peaks were found in 2/8 PDX tumors and 2/8 primary tumors, peaks with m/z and retention times matched to validated QYNPIRTTF peaks were identified in 6/8 PDX tumors and 7/8 primary tumors. Created with BioRender.com.
