Ewing Sarcoma (ES) is the second most prevalent primary bone tumour in children, adolescents, and young adults. Despite intensive multimodal therapies, treatment options remain limited in efficacy, with less than 20% chance to survive after five years for patients with recurrent or metastatic disease. Personalised and precision medicine such as HLA-dependent T cell-based immunotherapy is a potential therapy for ES patients. It uses the body’s immune system to target peptide antigens displayed on human leucocyte antigens (HLA) and destroy the tumour cells. In this study, we present an in-depth immunopeptidomics analysis, identifying peptide antigens present on HLA class I in a cohort of ES cell lines.
We analysed the immunopeptidome of eight ES cell lines under basal condition and 100IU/ml interferon-gamma (IFNγ) treatment for 72hr. Our optimised immunopeptidomics pipeline allowed us to identify 59,129 HLA class I peptides, matched against the human proteome database, across the cohort by processing 100 million cells per cell line and condition (n=1). We found that IFNγ treatment increased HLA class I peptide antigens by 2-fold, resulting in more than 60% of unique peptides detected in each cell line post-treatment. Identified peptides were found to be sourced from 10,250 proteins. Similarly, IFNγ also increased the diversity of source proteins, where over 65% were found after treatment in each cell line. Moreover, 26 tumour-associated antigens were shared across our cohort. Our findings suggest that an antigen discovery approach using in-depth immunopeptidomics analysis and IFNγ stimulation can help identify potential peptide antigen targets for ES immunotherapy.