Osteosarcoma is a common primary bone tumour. Although fairly manageable when localised, high-grade forms carry a risk of metastasis, recurrence and face limited effective and often toxic treatments. Human leukocyte antigen (HLA)-based immunotherapy, such as TCR-engineered T cells or peptide vaccines, can offer new alternatives by training the immune system to survey and target tumour-specific peptides presented by HLA molecules. Leveraging in-depth immunopeptidomics, peptide antigens can be detected and exploited in HLA-based immunotherapy. Cancer-testis antigens (CTAs), normally restricted to germ cells but aberrantly expressed in tumours, are promising targets for immunotherapy. Discovering HLA-presented CTA-derived peptides, presented on primary, relapsed, and metastatic sites, can grant the possibility for treating and preventing the progression of osteosarcoma.
In this study, patient-derived osteosarcoma cell lines, U2OS, B143, and SJSA, as well as tissue specimens, including a middle-lobe lung metastasis biopsy and a paired primary–metastatic tumour from an orthotopic B143 CDX mouse model, were used. Immunopeptidomics was performed according to our SAPrIm protocol with a pan HLA-I antibody (W6/32). The eluted peptides were analysed by LC-MS/MS on the Orbitrap Exploris 480 or Astral mass spectrometers. The identified peptides were matched against the reviewed human proteome database (Uniprot) and subsequently cross-referenced against the CTdatabase and the HLA Ligand Atlas to select CTA-derived peptides that are absent in healthy tissues.
Across all three cell lines and three tissues, 19 distinct HLA-I alleles were expressed, which overall cover nearly 82% of the global population according to the IEDB database. In total, >19000 cell-line- and >21000 tissue-derived unique peptide-HLAs were identified in this study. Overall, 49 distinct CTA proteins with 296 CTA-derived peptides were identified. Among them, ATAD2, MAGE-A6/8, MAGE-B3, and PRAME were shared across at least two cell lines and one metastatic specimen, with a total of 32 unique peptide sequences with no evidence of expression in healthy tissues.
In our study, we profiled tumour-restricted HLA-presented peptides in osteosarcoma, conserved across primary and metastatic lesions, with minimal expression in normal cells. These peptides offer significant translational value by potentially eliminating residual malignant cells or micrometastases, which are often missed by conventional methods, chemotherapy and surgery. Several identified CTAs in this study, including MAGE-A6/8 and PRAME, are currently being investigated in clinical trials for other solid malignancies. Conclusively, our data nominates targets for a durable therapeutic response to treat osteosarcoma and prevent relapse or metastatic progression.