Targeted TNF Potentiates the Activity of Bispecific T-cell Engagers in Solid Tumors by Turning Cold Tumors Hot

Abstract Colorectal cancer remains a major global health burden and an area of urgent unmet medical need. Immunotherapy has shown limited success in colorectal cancer as most patients present with an immune-excluded, “cold” tumor microenvironment (TME). In this study, we report a dual-modality approach to treating colorectal cancer by combining the tumor necrosis factor (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 carcinoembryonic antigen (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 colorectal cancer. Immunohistochemistry confirmed coexpression of CEA and EDB across microsatellite-stable and -instable tumors. In vitro, L19-TNF in combination with a CEAxCD3 TCE significantly 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 intercellular adhesion molecule 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.