Abstract Background: Bispecific T cell engagers are an emerging immunotherapy for solid tumors, capable of potently redirecting polyclonal cytotoxic T cells to lyse tumor cells independent of their native specificity. This mechanism enables potent immune activation and has delivered landmark clinical benefit in solid tumors, exemplified by tarlatamab and tebentafusp, which achieved unprecedented efficacy even in immunologically ‘cold’ settings such as small-cell lung cancer and uveal melanoma. Progress in solid tumors is constrained by the limited availability of antigens with sufficient tumor specificity to ensure efficacy with a robust therapeutic window. To address this, we discovered and validated DARKFOX™, a novel cancer-specific Dark Antigen® encoded by an alternative open reading frame (altORF) of the canonical FOXM1 gene. DARKFOX is highly prevalent, tumor-specific, and homogeneously expressed within tumors, with a robustly presented HLA-A3 peptide (DARKFOX-A3). We have developed TCR-mimic antibodies with picomolar affinity for DARKFOX-A3 and incorporated them into our EnTiCE® T cell engager platform, engineered for best-in-class functional and manufacturability properties. Methods: Antibodies specific for DARKFOX-A3 were generated from mice immunized with the antigen and subsequently affinity-matured in vitro to achieve high-affinity binding to the peptide-HLA complex. The optimized sequences were reformatted as single-chain variable fragments (scFvs) and engineered into Enara’s EnTiCE® T cell engager platform. Proteins were expressed in mammalian systems and purified for use in functional assays, analytical characterization, and in vivo efficacy studies. Expression analyses were performed using RNA in situ hybridization. Results: DARKFOX-A3-specific binders incorporated into Enara’s optimized EnTiCE® half-life-extended T cell engager format achieved robust yields and high purity using a scalable mammalian expression system. ENA101 demonstrated pM binding affinity for DARKFOX-A3 and potently redirected and activated T cells, inducing cancer cell lysis with low-pM EC values across multiple DARKFOX-A3+ targets. In vitro co-culture assays with normal cell models, together with binding motif analyses, confirmed a highly specific pHLA binding profile. In vivo, ENA101 exhibited strong anti-tumor efficacy in xenograft models. Planned first-in-human studies are supported by expression data indicating high prevalence of DARKFOX across major tumor types, such as squamous NSCLC, enabling patient enrolment without antigen pre-screening. Conclusions: ENA101 exhibits the hallmarks of a best-in-class bispecific T cell engager for solid tumors, including potent and highly specific redirection of T cell activity against antigen-positive cells across multiple tumor types. CMC activities and IND-enabling studies are currently in progress. Citation Format: Terri Cornforth, Colette Johnston, Duncan Howie, Hope Adamson, Rachel Bergin, Alexandra Black, Laura Blackholly, Ellen Border, Carl Cox, Eleanor Denham, Magdalenda von Essen, Vicki Jefferson, Luke Johnson, Emily Lam, Jack Lewis, Mengqiu Li, Tatiana Lobry, Heather McIntyre, Isabel Mount, Joana Senra, Martha Simpson, Andrea Stacey, Emily Tye, Maria Vologianni, Joseph Dukes, . ENA101: A first-in-class bispecific T cell engager targeting a DARKFOX peptide presented by solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4052.
Cornforth et al. (Fri,) studied this question.