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Background Bispecific T cell engagers (TCEs) have demonstrated significant clinical benefit in solid tumors as evidenced by the approvals of tebentafusp and tarlatamab. Despite these initial clinical successes, there remains a paucity of solid tumor targets with expression profiles suitable for TCE therapies. A key benefit of peptide-HLA (pHLA) targets is access to the intracellular proteome, which harbors most cancer-specific antigens. To date, little progress has been made in diversifying pHLA targets by both identifying novel antigens and broadening patient populations beyond HLA-A2. Enara developed its EDAPT ® platform to discover and validate novel antigens derived from genomic sequences historically thought to be ‘dark’ or non-coding. These Dark Antigens® exhibit properties of optimal cancer targets, including minimal expression in normal tissues, homogeneous expression within tumors, robust presentation on the surface of tumor cells, and shared expression across patients with diverse solid tumor types.Methods The EDAPT pipeline was used to identify an HLA-A3-presented peptide in multiple, primary tumor samples including colon, lung, breast and ovarian carcinomas, that mapped to an alternative open reading frame (ORF) in the FOXM1 gene, termed DARKFOX TM. Mice from a humanized transgenic platform were immunized with the pHLA complex and expanded B cell clonotypes were characterized for binding to the DARKFOX peptide (DARKFOX-A3) but not off-target peptides. Binders were engineered into a bispecific TCE format and characterized in functional assays. In vivo pharmacology studies in mice with human immune cells were performed.Results DARKFOX-A3-specific binders discovered from mouse immunizations exhibited low nM binding affinity for the target pHLA. Binders were reformatted as scFv’s and engineered into bispecific TCEs, which redirected and activated T cells to specifically and robustly kill cancer cell lines expressing a range of DARKFOX levels. In vitro co-culture assays with normal cell models and binding motif analyses indicate a specific profile of the pHLA binding moiety. In vivo studies demonstrate anti-tumor activity of a DARKFOX-A3-targeting TCE in a xenograft mouse model. Expression analyses of DARKFOX show robust and homogeneous expression of the antigen across a range of solid tumor types.Conclusions Targeting DARKFOX-A3 with bispecific TCEs demonstrates a highly promising in vitro and in vivo preclinical profile for the treatment of solid tumors. These TCEs exhibit robust and selective redirection of T cell activity toward DARKFOX-expressing cells across multiple tumor types. Affinity-enhanced binders derived from these sequences will be selected to advance into IND-enabling studies with Enara’s EnTiCE® half-life-extended TCE platform.