163 Unveiling the non-canonical dark immunopeptidome of head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) using mass spectrometry and watchmaker total RNA-Seq

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Background Recent studies have shown that the vast majority of a cancer’s neoantigens are not presented by MHC molecules, 1 2 offering a possible explanation for why clinical trials that successfully generated neoantigen-reactive T cells have not translated into improved clinical outcomes. These same studies have revealed that non-canonical peptides (NCPs)—referred to as cancer’s ‘Dark Matter’—represent the dominant cancer-specific epitopes actually presented by MHC. Some of these NCPs are shared across different cancer types, contribute to tumor progression, and correlate with poor patient prognosis, underscoring their potential as promising targets for cancer immunotherapy. Advances in mass spectrometry and next-generation sequencing technologies have significantly enhanced the ability to detect these peptides. Here we characterized the immunopeptidome of head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines to look for dark matter elements shared with a dark matter cancer vaccine, DPV-001, administered as immunotherapy to patients with HNSCC and NSCLC.3 Methods HNSCC cell lines were subject to treatment with detergent in the presence of a protease inhibitor. HLA complexes were purified by anti-HLA-I antibody (w6/32) and HLA-bound peptides eluted and analyzed by the Orbitrap mass spectrometer (ORBITRAP FUSION LUMOS with FAIMS-Pro interface (Thermofisher Scientific). Simultaneously, total RNA-Seq libraries were generated using RNA library prep kits with Polaris depletion (WatchMaker). RNA-Seq libraries were converted to searchable peptide databases and spectrum matching was performed using the PEAKS-X software. Peptides identified in RNA-Seq and mass spectrometry data underwent database searching using standard and custom databases to identify canonical versus NCP. NCP identified in both the mass spectrometry and RNA-Seq data were output and annotated with genomic information utilizing Peptide-PRISM. 4 Results Our current assessment characterizing the immunopeptidome of 10 HNSCC and 3 NSCLC cell lines derived from 9 different patients, revealed 74,578 unique canonical and 2506 unique NCP present in both mass spectrometry and RNA-Seq data. At least 119 NCP were found shared across 2 or more patients with common HLA super families. We are now evaluating the overlap between these 119 NCP and different NCP that trace to the same transcripts with dark matter proteins found in DPV-001.Conclusions Ongoing analysis has identified dark antigens presented by HNSCC and NSCLC that represent immunotherapy targets. Some of these dark antigens are contained in the DPV-001 vaccine administered to patients with this cancer. Current efforts are evaluating which antigens are immunogenic and whether they have oncogenic properties.References Ely ZA, Kulstad ZJ, Gunaydin G, Addepalli S, Verzani EK, Casarrubios M, Clauser KR, Wang X, Lippincott IE, Louvet C, Schmitt T. Pancreatic cancer-restricted cryptic antigens are targets for T cell recognition. Science. 2025 May 8;388(6747):eadk3487.Apavaloaei A, Zhao Q, Hesnard L, Cahuzac M, Durette C, Larouche JD, Hardy MP, Vincent K, Brochu S, Laverdure JP, Lanoix J. Tumor antigens preferentially derive from unmutated genomic sequences in melanoma and non-small cell lung cancer. Nature Cancer. 2025 May 22:1–9.Fox BA, Urba WJ, Jensen SM, Page DB, Curti BD, Sanborn RE, Leidner RS. Cancer’s dark matter: lighting the abyss unveils universe of new therapies. Clinical Cancer Research. 2023 Jun 13;29(12):2173–5.Erhard F, Dölken L, Schilling B, Schlosser A. Identification of the cryptic HLA-I immunopeptidome. Cancer Immunology Research. 2020 Aug 1;8(8):1018-26.

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