[CANCER RESEARCH 63, 3202–3210, June 15, 2003] Synergistic Therapeutic Effects of a Tumor Targeting Antibody Fragment, Fused to Interleukin 12 and to Tumor Necrosis Factor  1 Cornelia Halin, Verena Gafner, Maria Elena Villani, Laura Borsi, Alexander Berndt, Hartwig Kosmehl, Luciano Zardi, and Dario Neri 2 Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, CH-8057 Zurich, Switzerland [C. H., V. G., M. E. V., D. N.]; Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, I-16132 Genoa, Italy [L. B., L. Z.]; Institute of Pathology, Friedrich Schiller University, D-07740 Jena, Germany [A. B.]; and Institute of Pathology, HELIOS-Klinikum, Erfurt, Germany [H. K.] ABSTRACT The potent antitumor activity of certain cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the thera- peutic index of cytokines in cancer therapy consists of fusing them to monoclonal antibodies capable of a selective localization at the tumor site. We have constructed fusion proteins of interleukin-12 (IL-12) and tumor necrosis factor (TNF-) with L19, an antibody fragment specific to the extradomain B of fibronectin which has been shown to target tumors in animal models and in patients with cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of cancer but do not lead to complete remissions of established aggressive tumors. In this article, we have evaluated the tumor-targeting properties and the anticancer activities of combinations of the two antibody-cytokine fusion proteins, as well as of a triple fusion protein between IL-12, L19, and TNF-. Although all fusion proteins were active in vitro, the triple fusion protein failed to localize to tumors in vivo and to show significant thera- peutic effects. By contrast, the combination of IL-12-L19 and L19-TNF- displayed potent synergistic anticancer activity and led to the eradication of F9 teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with tumor cells, a delayed onset of tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. Potent anticancer effects were achieved at doses of IL-12-L19 and L19-TNF- (2 g  2 g/mouse), which were at least 5-fold lower than the maximal-tolerated dose. The combined administration of the two fusion proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion proteins. These results show that the targeted delivery of cytokines to the tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12-L19 and L19-TNF- appears to be synergistic in vivo. INTRODUCTION Systemic administration of cytokines, such as IL 3 -2, TNF-, gran- ulocyte macrophage colony-stimulating factor, or IL-12 can render some nonimmunogenic tumors immunogenic, activating a protective immunity (1–3). However, systemic administration of cytokines is often associated with severe toxic side effects, which prevent the administration of a curative dose. One possible way of increasing the therapeutic index of certain cytokines consists of using them for locoregional treatments, in the case of localized tumors. Indeed, the perfusion of an isolated limb with TNF- (often in combination with IFN- and melphalan; Ref. 4) allows reaching therapeutic concentrations of TNF-, with a thera- peutic benefit for patients with in transit melanoma metastases and limb salvage in soft tissue sarcoma patients (5). Consequently, this application of TNF- was approved for use by the European Agency for the Evaluation of Medicinal Products (6). Another possible way of increasing the therapeutic index of cyto- kines consists of fusing them to antibodies, which mediate a prefer- ential accumulation of the cytokine at the tumor site. Indeed, in the past decade, several groups have reported different antibody-cytokine fusions for different tumor-associated antigens (7–10). These novel proteins were shown to retain both antibody and cytokine functions and to show superior anticancer activities as compared with equiva- lent amounts of free cytokine (and antibody). We have previously described the targeted delivery of IL-12 and TNF- to the subendothelial extracellular matrix by fusion of the cytokine to scFv(L19), a high-affinity human antibody fragment spe- cific to the EDB domain of fibronectin, a marker of angiogenesis (11–14). 4 IL-12-L19 dramatically enhanced the therapeutic index of IL-12 in both s.c. and metastatic murine tumor models (15). However, com- plete tumor regression could only be observed occasionally. Further- more, when complete regression was observed, tumors could be reestablished in cured animals upon reinjection of tumor cells, indi- cating the absence of a lasting vaccination effect. Similarly, L19-TNF- exhibited an excellent tumor uptake in mu- rine cancer models because of the vasoactive properties of TNF- (16, 17) 4 and to the homotrimeric nature of the fusion protein, resulting in a high binding avidity (18). Administration of L19-TNF- led to a substantial inhibition of tumor growth, which could further be delayed by melphalan. However, complete cures were rarely observed. 4 The combination of different cytokine-based immunotherapies, showing synergistic rather than only additive antitumor effects, may represent an avenue for additional improvement of therapeutic effi- cacy. For instance, Reisfeld et al. (19) have shown that a partially protective memory T-cell immune response induced by a cellular tumor vaccine (tumor cells genetically engineered to secrete IL-12) could be additionally boosted by administration of a tumor-specific antibody-IL-2 fusion protein. Other groups have reported similar synergistic effects of IL-2 and IL-12 when administering combina- tions of recombinant IL-12 and IL-2 or when combining gene therapy with administration of recombinant cytokines (20, 21). Comparable enhancement of therapeutic activity has been demonstrated in murine tumor models with combinations of recombinant IL-12 and TNF- (22) or when combining TNF- gene-transduced cancer vaccines with administration of recombinant IL-12 (23). In this study, we have combined the therapeutic activity of the Received 12/18/02; accepted 4/10/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Financial support from the Bundesamt fu ¨r Bildung und Wissenschaft (EC Project; to D. N.), the Swiss National Science Foundation (SNF and TANDEM Project; to D. N.), the European Community (to L. Z. and H. K.), and Associazione Italiana Ricerca sul Cancro (to L. Z.) is gratefully acknowledged. 2 To whom requests for reprints should be addressed, at Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Building 36 M14, Winterthur- erstrasse 190, CH-8057 Zurich, Switzerland. Phone: 41-1-635-60-63; Fax: 41-1-635-68- 86; E-mail: neri@pharma.anbi.ethz.ch. 3 The abbreviations used are: IL, interleukin; TNF-, tumor necrosis factor ; EDB, extradomain B of fibronectin; L19, human antibody specific for the EDB domain of fibronectin; IL12-L19, fusion protein of IL-12 and the human antibody fragment L19; L19-TNF-, fusion protein of the human antibody fragment L19 with TNF-; ILT, triple fusion protein of IL-12 with the antibody fragment L19 and TNF-; PHA, phytohemag- glutinin. 4 L. Borsi, E. Balza, B. Carnemolla, F. Sassi, P. Castellani, A. Berndt, H. Kosmehl, A. Siri, P. Orecchia, J. Grass, D. Neri, and L. Zardi. Selective targeted delivery of TNF- to tumor blood vessels, submitted for publication. 3202 Research. on June 21, 2017. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from