Turning cold tumors hot: Targeted TNF potentiates the activity of bispecific T cell engagers in solid tumors

Colorectal cancer (CRC) remains a major global health burden and an area of urgent unmet medical need. Immunotherapy has shown limited success in CRC, since most patients present with an immune-excluded, “cold” tumor microenvironment (TME). Here, we introduce a novel dual-modality approach combining the TNF-based fusion protein directed to the extradomain B (EDB) of fibronectin, L19-TNF, which induces localized intratumoral inflammation and facilitates T cell infiltration, with a CD3-based bispecific T cell engager (TCE) targeting CEA, which mediates antigen-specific cytotoxicity. Together, these agents aim to remodel the TME, convert “cold” tumors into inflamed, “hot” lesions, and broaden the therapeutic reach of immunotherapy in CRC. Immunohistochemistry confirmed co-expression of CEA and EDB across microsatellite-stable and -instable tumors. In vitro , L19-TNF in combination with CEAxCD3 TCE synergistically enhanced tumor cell killing and CD8⁺ T-cell proliferation. In vivo , the combination induced complete tumor regression in most animals, prolonged survival, and conferred durable protection against tumor rechallenge. Furthermore, mechanistic analyses revealed enhanced TCE extravasation, upregulated ICAM-1 expression, and increased CD8⁺ T-cell infiltration, indicating vascular modulation and remodeling of the TME toward an inflamed, “hot” phenotype. These findings confirm that targeted delivery of TNF to the TME can effectively enhance the activity of immunotherapeutic agents, such as T cell redirecting therapies, in challenging tumor settings.