Selective targeted delivery of TNFα to tumor blood vessels . Short title: Delivery of TNFα to tumor vessels. Scientific Section Heading: Hemostasis, Thrombosis & Vascular Biology

We sought to enhance the selective toxicity of tumor necrosis factor alpha (TNFα) in order to permit its systemic use in cancer therapy. Since ligand-targeted therapeutics has proven successful in improving the selective toxicity of drugs, we prepared a fusion protein (L19mTNFα) composed of mouse TNFα and a high affinity antibody fragment (L19 scFv) to the ED-B domain of fibronectin, a marker of angiogenesis. L19mTNFα was expressed in mammalian cells, purified and characterized. L19mTNFα was an immunoreactive and biologically active homotrimer. Radiolabeled L19mTNFα selectively targeted tumor neo-vasculature in tumor-bearing mice, where it accumulated selectively and persistently (tumor-blood ratio of the %ID/g of 700, 48h from injection). L19mTNFα showed a greater anticancer therapeutic activity than both mTNFα and TN11mTNFα, a control fusion protein in which an antibody fragment, irrelevant in the tumor model used, substituted L19, and this activity was further dramatically enhanced by its combination with Melphalan or the recently reported fusion protein L19-IL2. In conclusion, L19mTNFα allows concentrating therapeutically active doses of TNFα at the tumor level, thus opening new possibilities for the systemic use of TNFα in cancer therapy. Corresponding author E-mail: [email protected]