Fig 1: Detection of tumor self-antigen specific CD8 T cells in normal donors.a. Gating used to select CD8 T cells. Similar gating strategy was used to select live singlets for Jurkat and primary cells transduced with CAR constructs (without selection for CD3 and exclusion of CD14, CD19, and CD4). b. Four normal donors stained with IGFBPL1 dextramer on x-axis and GFRA2 on y-axis shows rare population of antigen-specific cells varying by donor and antigen (left panel showing unstained and right panel showing tetramer stained). c. Frequencies and MFIs of antigen specific cells across donors. d. Top constructs generated from normal donor-derived antigen-specific TCRs found by single-cell sequencing show range of antigen binding PHOX2B, TH, and IGFBPL1 as compared to DMF5 receptor targeting MART-1. Screening for antigen specific T cells in 3 donors reveals that PHOX2B TCRs have minimal target binding, suggesting that PHOX2B self-antigen is immunogenically silent and warrants targeting using synthetic scFv receptors.
Fig 2: Development of antigen-specific CARs for neuroblastoma antigens.a. ELISA of PHOX2B scFv A7 using PHOX2B p/MHC and decoy peptide on HLA-A*24:02. b. Schematic of second-generation CAR constructs. c. A7 CAR transduced into primary CD8 cells binds PHOX2B dextramer but not HLA-A*02:01 dextramer. d. A7 CAR preferentially binds PHOX2B dextramer but cross-reacts with mismatched peptides PBK and CHRNA3 on HLA-A*24:02 at high affinity. e. A7 CAR potently kills HLA-A*24:02 neuroblastoma lines, but also kills HLA+/antigen- tumor cells. f. Tetramer and dextramer gating strategy for pMHC staining.
Fig 3: Neuroblastoma CTCs expressing characteristic markers are isolated using cell size-based separation. (A) Schema of experimental design and 3 serial timepoints where blood and/or bone marrow samples were obtained from patients for CTC enrichment. (B) Representative photomicrographs of cytospots of CTC-enriched fractions demonstrating PHOX2B- and TH-positivity of isolated large, atypical cells on immunohistochemical staining (arrows; NBL20 at initial diagnosis and NBL10 following induction chemotherapy, respectively; scale bar: 50 µm), and corresponding bone marrow aspirate with neuroblastoma tumor cell infiltration for size comparison (NBL20 at initial diagnosis; H&E, scale bar: 50 µm). (C) Relative gene expression of PHOX2B and TH in CTC-enriched and waste fractions from blood samples taken at diagnosis.
Fig 4: An antigen discovery and prioritization process identifies PHOX2B as a target for immunotherapy.a, Summary of tumour-antigen discovery and CAR-engineering workflow: (1) integrated genomics and immunopeptidomics process; (2) target validation; (3) CAR engineering; and (4) cross-HLA tumour killing. b, Computational filtering of 9,117 peptide instances identified by immunopeptidomics in primary tumours (1% FDR) resulted in 56 neuroblastoma-specific peptides (33 unique peptides) derived from 29 distinct proteins. c, Primary neuroblastoma tumour immunopeptidome compared with 190 normal tissues. Each point on the x-axis represents one of 5,832 unique peptides identified in primary tumours, with the proportion of neuroblastoma tumours presenting a given peptide annotated above axis in dark blue and the proportion of normal tissue expressing the identical peptide below the axis in light blue. The green horizontal bar indicates 1,492 peptides not previously observed in the normal tissue immunopeptidome. Parent genes from neuroblastoma-specific peptides resulted in the top two GO enrichment terms: noradrenergic neuron differentiation and sympathetic nervous system development. The arrow denotes 351 recurring peptides presented in neuroblastoma that were not previously detected in normal tissues. d, Five antigens differentially expressed in PDX and primary tumours and further prioritized for analysis, HLA allele frequency, relative peptide abundance (percentile rank annotated below pMHC), predicted pMHC-binding affinity, and relevance to neuroblastoma tumourigenesis. e, PHOX2B expression in RNA-seq data from 153 neuroblastoma tumours versus 1,641 normal tissues in GTEx. PHOX2B expression is restricted to tumours, compared with the immunotherapy target HER2 and the neuroblastoma chemotherapy target TOP1 (note differences in the y-axis scales). FPKM, fragments per kilobase of transcript per million mapped reads. f, Crystal structure of PHOX2B 9mer QYNPIRTTF (red) refolded with HLA-A*24:02 (grey). g, Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) of neuroblastoma tissue shows binding of all CRC proteins at the PHOX2B locus and association with a H3K27Ac super-enhancer mark. h, RNA-seq of fetal tissue shows that PHOX2B is expressed in early development and downregulated before birth across seven tissues. Created with BioRender.com.
Fig 5: Structural basis of CARs binding to PHOX2B peptide presented on multiple HLAs.a, PHOX2B–HLA-A*24:02 crystal structure and models of PHOX2B in complex with HLA-A*23:01, HLA-B*14:02 and HLA-C*07:02. b, The charged and polar R151, Q155 and R69 residues of HLA-C*07:02 align with key 10LH interaction residues I5, R6 and I7 (MHC residues in blue and PHOX2B–10LH interaction residues in red). R151, Q155 and R69 create steric and charged hindrance of key peptide-binding residues. c, Staining of the PHOX2B PC-CAR 10LH reveals strong binding to HLA-A*24:02, HLA-A*23:01 and HLA-B*14:02, but not to HLA-C*07:02. CD19, CD19-directed CAR; 10LH, PHOX2B PC-CAR; UT, untransduced T cells. Created with BioRender.com.
Supplier Page from Abcam for Anti-PHOX2B antibody [EPR14423] - C-terminal