Ewing Sarcoma (ES) is the second most common primary bone tumour in children, adolescents, and young adults. Patients with localised disease have ~70% five-year survival, but those with metastatic, recurrent, or relapsed disease face <30% survival under current therapies. ES is driven by a fusion protein, most commonly EWS-FLI1, which merges FET and ETS family genes to create a potent oncogenic transcription factor. Human leucocyte antigens (HLA)-dependent T cell-based immunotherapy offers a promising avenue by targeting peptide-antigens presented on HLA recognised and lyse tumour cells. To explore this, we performed a comprehensive immunopeptidomics analysis to define the HLA class I-presented antigen repertoire in Ewing Sarcoma.
Seven ES cell lines were cultured with or without 100IU/ml interferon-gamma for 72 hours, and 100 million cells per condition (n=1) were harvested. HLA class-I peptides were isolated using SAPrIm workflow, and analysed using LC-MS/MS on an Orbitrap Exploris 480. Thirteen patient-derived tumour tissues (10–50mg), were similarly processed and analysed on an Orbitrap Astral. The data were searched against the human proteome, including the fusion sequences using PEAKS software, and downstream analysis included referencing against TANTIGEN, IEDB and HLA-ligand Atlas database to identify potential targets.
Across ES cell lines, we identified 52,373 HLA class-I peptides from 10,438 source proteins, predicted to bind across 23 distinct alleles. Of these, 1381 peptides corresponded to 150 known tumour-associated antigens, highlighting potential therapeutic targets. Strikingly, we discovered a fusion-derived peptide spanning the junction presented on HLA, validated via spectral matching with a synthetic reference peptide. This discovery confirms that the fusion protein can be processed and presented on HLA, providing a highly specific immunotherapy target for ES. Single-cell RNA sequencing using stimulated T cells with the fusion-peptide, uncovered four distinct T-cell receptors capable of recognising the identified fusion peptide, showcasing the feasibility of TCR-T cell therapy. In parallel, we mapped 98,450 HLA class-I peptides to 12,258 source proteins in patient-derived tumours. Almost half of HLA class-I peptides found in cell lines was observed in tissues, highlighting the translational relevance of our models to uncover clinically actionable targets.
This study presents a highly comprehensive immunopeptidomics analysis of Ewing Sarcoma, integrating cell lines and patient-derived tumours data. Our findings provide a rich and diverse repertoire of HLA class-I peptides, enabling us to search for potential targets for developing HLA-dependent T cell-based therapies against ES, like the fusion-derived peptide.