A number of cytokines are either approved drugs or are in advanced clinical trials, yet these biopharmaceuticals do not typically localize efficiently in solid tumors and manifest their therapeutic potential at the expense of severe side effects. The targeted delivery of cytokines to solid tumors is a promising avenue for increasing the therapeutic index of these biopharmaceuticals. We engineered a fusion protein between scFv(L19), a human antibody fragment specific to the EDB domain of fibronectin, and a cysteine-free mutant of murine interferon-g. The resulting fusion protein was capable of targeting new blood vessels in solid tumors, and the targeting efficiency was strikingly increased in tumorbearing knockout mice lacking the interferon-g receptor. ScFv(L19)-interferon-g displayed a strong antitumor effect in both subcutaneous and metastatic murine F9 teratocarcinomas, but was not efficacious as single agent when used to treat C51 and CT26 tumors. The potency of this fusion protein could be substantially enhanced by combination with doxorubicin and other immunocytokines. These findings are of clinical relevance, as the EDB domain is a marker of angiogenesis, with identical sequence in mouse and man, which is abundantly expressed in a variety of aggressive solid tumors but is undetectable in most normal tissues. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience. wiley.com/jpages/0020-7136/suppmat/index.html. ' 2005 Wiley-Liss, Inc.Key words: tumor targeting; tumor therapy; scFv antibody fragment; immunocytokine; interferon-g; mice A quarter of a century after their discovery, 1 monoclonal antibodies have become the most rapidly expanding class of pharmaceuticals for the treatment of many human diseases, including cancer. Since 1997, with the FDA approval of the first anticancer therapeutic antibody, a chimeric anti-CD20 antibody for the treatment of non-Hodgkin's lymphoma 2 (Rituxan), several antibodies have been approved for the treatment of cancer. The last FDA-approved antibodies in February 2004 are Erbitux and Avastin, the latter being the first approved therapy designed to inhibit angiogenesis.Despite these promising developments, the efficacy of the approved clinical anticancer antibodies needs to be enhanced since cure is rare. 3,4 One avenue for the improvement of the therapeutic performance of anticancer antibodies relies on the engineering of IgG binding affinity to Fc receptors, either by mutation or by glycosylation engineering. 5-7 Another avenue, which represents the focus of our laboratory, consists in the development of antibody derivatives, which can deliver a bioactive agent to the tumor environment (e.g., drugs, toxins, radionuclides, photosensitizers, procoagulant factors, cytokines).The targeted delivery of anticancer cytokines to the tumor environment appears to be a particularly promising strategy. A number of cytokines are either approved drugs or are in advanced clinical trials, yet these biopharmaceutical...