AbstractAntibodies that target the tumor microenvironment can be used to deliver pro‐inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody–cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi‐specific fusion protein that combines interleukin‐2 and a single amino acid mutant of tumor necrosis factor. The two pro‐inflammatory payloads were fused to the L19 antibody, a clinical‐grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor‐bearing mice at three doses of 30 μg in a 2‐day interval and promoted rapid tumor eradication in murine models, more efficiently than single‐agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin‐2 and tumor necrosis factor to neoplastic sites.
