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Background Targeted immunotherapies are a highly promising treatment strategy for solid tumors, exemplified by the recent FDA approvals of tebentafusp and afamitresgene autoleucel, which target Class I peptide-HLA (pHLA) antigens. A major hurdle in developing such therapies is the scarcity of truly cancer-specific antigens that are shared across broad patient populations. Recent studies revealed that unannotated regions of the genome are a rich source of tumor-specific targets called Dark Antigens ®. Using our EDAPT® pipeline, we identified a novel target, DARKFOXTM, which encodes multiple peptides including a dominant one presented by HLA-A*03 (DARKFOX-A3). DARKFOX is encoded by an alternative open reading frame (altORF) within the FOXM1 gene, which is highly expressed across various solid tumors and implicated in cancer progression.Methods RNAseq-based de novo transcript assemblies were used to discover unannotated ORFs with cancer-specific expression. Discovery-based immunopeptidomics (DDA-MS) on primary tumors confirmed ORF translation and Class I HLA presentation. Validation techniques included targeted immunopeptidomics (PRM-MS), Ribo-seq, and RNA in situ hybridization (RNA-ISH). Immunogenicity was assessed using healthy donor-derived T cells.Results DARKFOX was identified from esophageal adenocarcinoma RNAseq data as an altORF within the canonical FOXM1 transcript. DDA-MS identified peptides from DARKFOX in multiple primary tumor tissues (including colon, lung, breast and ovarian carcinomas) but not in normal tissues. The predominant peptide was presented by HLA-A*03. Ribo-seq analysis supported altORF translation, and PRM-MS on DARKFOX knockout cells confirms the DARKFOX-A3 peptide’s origin. Validation across tumor and normal tissues using RNA-ISH and PRM-MS confirmed consistent cancer-enrichment and homogeneous intra-tumoral expression. Outside of tumors, DARKFOX-A3 appeared only in B-cell rich tissues, at low levels, suggesting a wide therapeutic window.The DARKFOX-A3 peptide was immunogenic, as evidenced by T cell responses in multiple healthy donors. TCRs from responding donors were isolated and confirmed to be specific for the DARKFOX-A3 peptide. Jurkat-T cells expressing one of these TCRs showed correlation of T cell activation level with the quantified peptide when co-cultured with cancer cells, as determined by PRM-MS.Conclusions DARKFOX represents a compelling target for cancer immunotherapy that is highly prevalent in multiple solid tumors. It is characterised by minimal presence in normal tissues, homogeneous intratumoral expression, and robust presentation by HLA on the surface of tumor cells. ENA101, an EnTiCE ® bispecific T-cell engager targeting DARKFOX-A3, is currently in IND-enabling studies.