Discovery of tumor-specific antigens in melanoma tissue biopsies via integrated proteogenomic analysis

Background Despite significant progress in therapeutic outcomes, melanoma remains frequently resistant to immunotherapy. Efforts to improve response rates include the development of vaccines targeting tumor neoantigens; however, the optimal tumor-associated antigens (TAAs) to target remain uncertain. Recent evidence suggests that the most relevant TAAs may originate from non-mutated protein sequences, including those derived from the so-called dark proteome (1). In this study, we conducted an immunopeptidomic analysis of tumor samples obtained from patients with metastatic melanoma before initiation of systemic therapy Methods A total of 35 tumor biopsy samples from melanoma patients were subjected to native tissue lysis, followed by immunoaffinity purification of HLA class I complexes using a pan HLA class I antibody (W6/32) and subsequent elution of the bound immunopeptides. The isolated immunopeptides were analyzed using data-independent acquisition (DIA) mass spectrometry on an Exploris 480 mass spectrometer (Thermo Scientific) equipped with FAIMS Pro. Data analysis was performed using Spectronaut 20 (Biognosys) using directDIA. For peptide identification, a customized human proteome database was constructed to enable the detection of tumor-associated antigens (TAs). This database included the canonical and isoforms human proteome, a previously reported TA reference dataset (1), and an in-house generated “dark genome” database derived from tumor RNA-sequencing data. Results Analysis of these 35 melanoma tissue samples yielded in total over 120,000 unique immunopeptides with an average of ~15,000 per sample, with notable inter-sample variability (ranging from ~6,000 to >28,000 identifications). All samples displayed the expected HLA class I length distribution, with a predominant population of 9-mer peptides. Across the cohort, approx. 70 previously reported tumor-associated antigens (TAs) were identified, spanning multiple categories including tumor-associated antigens (TAA), lineage-specific antigens (LSA), and aberrantly expressed tumor-specific antigens (aeTSA). These TAs originated from both annotated open reading frames (ORFs) and noncanonical translation events such as frameshifts, noncoding RNA (ncRNA), and 5′UTR-derived peptides. From the RNA-seq-derived database, approximately 22 immunopeptides were mapped to sequences originating from both annotated genes (18 peptides) and previously uncharacterized genomic regions (4 peptides; dark genome). Together, these results demonstrate that the applied proteogenomic immunopeptidomics workflow enables the detection of tumor-specific and neoantigenic peptides directly from clinical melanoma biopsy samples, underscoring its potential utility in translational immuno-oncology research. Reference: (1) Apavaloaei et al. Nature Cancer, 2025