[CANCER RESEARCH 61, 711–716, January 15, 2001] Targeted Delivery of Tissue Factor to the ED-B Domain of Fibronectin, a Marker of Angiogenesis, Mediates the Infarction of Solid Tumors in Mice 1 Fredrik Nilsson, Hartwig Kosmehl, Luciano Zardi, and Dario Neri 1 Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, CH-8057 Zurich, Switzerland [F. N., D. N.]; Institute of Pathology, Friedrich Schiller University, D-07740 Jena, Germany [H. K.]; and Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, I-16132 Genoa, Italy [L. Z.] ABSTRACT The selective thrombosis of tumor blood vessels, leading to the starva- tion and subsequent death of tumor cells, is an attractive anticancer strategy. Here we report that a fusion protein, consisting of an antibody fragment specific for the oncofoetal ED-B domain of fibronectin fused to the extracellular domain of tissue factor, selectively targets tumor blood vessels in vivo. Furthermore, this fusion protein mediates the complete and selective infarction of three different types of solid tumors in mice. At the highest doses administered, complete tumor eradication was observed in 30% of the mice treated without apparent side effects. These results are of therapeutic relevance because the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is ex- pressed in the majority of aggressive solid tumors but is undetectable in normal vessels and tissues. INTRODUCTION Angiogenesis, i.e., the proliferation of new blood vessels from preexisting ones, is a characteristic feature of aggressive solid tumors and relevant disorders such as age-related macular degeneration, diabetic retinopathy, and rheumatoid arthritis (1, 2). Molecules capa- ble of inhibiting angiogenesis or of selectively targeting and destroy- ing new blood vessels, would be promising agents for the treatment of angiogenesis-related diseases (3–9). A novel approach to cancer therapy based on the antibody-directed targeting of the human coagulation-inducing protein TF 2 to tumor vasculature has recently been proposed (10, 11). The approach is based on the concept that thrombosis of tumor vessels may stop the supply of nutrients and oxygen to tumor cells, thereby causing their death. TF is a cell-surface glycoprotein and a major initiator of blood coagulation (12). At sites of injury, blood comes in contact with the membrane-bound TF, which forms a complex with the serine protease FVIIa present in blood. The resulting complex activates factors IX and X, which leads to thrombin activation and ultimately to blood clotting. tTF consisting of only the extracellular soluble domain (residues 1–219), exhibits an ability to activate the clotting cascade in solution that is five orders of magnitude lower than normal but fully active when retargeted to a membrane surface (10). In a first article, Huang et al. (10) used a bispecific antibody to target tTF to an artificial marker of angiogenesis (MHC-II) experi- mentally induced on tumor vascular endothelium by grafting in mice neuroblastoma cells that had been transfected with the IFN- gene. In that study, the investigators observed extensive intravascular throm- bosis of the tumor and complete regressions in 38% of the mice treated (10). In a second study, immunoconjugates were used to target tTF to a naturally occurring marker of tumor vascular endothelium, VCAM-1 (11). VCAM-1 is expressed by vascular endothelial cells in Hodgkin’s lymphoma and in various solid tumors in mice and humans. It is also expressed in some vessels of thyroid, kidney, and thymus in humans and in heart and lung in mice (11). In this study, the authors observed selective localization of tTF to VCAM-1-expressing vessels in the tumor, causing thrombosis of those vessels, a 50% reduction of tumor growth rate, but no complete remission. The immunoconjugate also localized to VCAM-1-expressing vessels in the lungs and heart, but did not induce thrombosis at these sites. An immunohistochemical evaluation of a monoclonal anti-PS antibody in the mice showed that the VCAM-1-expressing vessels in the tumor also expressed PS, whereas VCAM-1-expressing vessels in the lungs and heart lacked PS. The authors concluded that PS expression on the luminal aspect of blood vessels is needed to provide the procoagulant surface upon which coagulation complexes can assemble. The targeted delivery of tTF would be of significant therapeutic relevance if it were directed against a naturally occurring marker of angiogenesis that is expressed in the majority of aggressive solid tumors but undetectable in normal vessels and tissues, and if it mediated the selective thrombosis of tumor blood vessels. A good quality marker for both tumoral and nontumoral neovas- culature is the ED-B domain of fibronectin (a sequence of 91 amino acids identical in mouse, rat, rabbit, dog, and man) that can be inserted into the fibronectin molecule by a mechanism of alternative splicing (13–17). B-FN accumulates around neovascular structures in aggres- sive tumors and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions, but is otherwise undetectable in normal adult tissues. To date, the production of monoclonal antibodies directly recog- nizing the ED-B domain in B-FN has not been possible using hybri- doma technology because of tolerance. However, this problem has been overcome using antibody phage technology (18) with large synthetic antibody repertoires (19, 20). Several antibody fragments specific for the ED-B domain of fibronectin have been generated recently. These antibody fragments stain vascular structures in tumor sections and selectively target tumor neovasculature, as shown in tumor-bearing mice using IR fluorescence and radioactive techniques (21, 22). Increased binding affinity leads to improved targeting of tumoral angiogenesis, as demonstrated by biodistribution studies per- formed using the L19 antibody fragment with affinity for the ED-B domain in the pM range and L19 mutants with reduced affinity (23). In this study we investigated whether the selective antibody-medi- ated targeting of tTF to B-FN would result in thrombosis of tumor blood vessels. B-FN is a component of the modified extracellular matrix that surrounds tumor blood vessels, and it was therefore not known whether the delivery of a procoagulant agent at this abluminal site would be capable of mediating the complete and selective intralu- minal coagulation of tumor blood vessels. In this article we show that a fusion protein consisting of the L19 Received 7/14/00; accepted 11/13/00. 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 Krebforschung Schweiz and the Bundesamt fu ¨r Bilding und Wissenschaft are 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.ethz.ch. 3 The abbreviations used are: TF, tissue factor; tTF, truncated form of TF; VCAM-1, vascular cell adhesion molecule-1; PS, phosphatidylserine; B-FN, fibronectin containing ED-B. 711 Research. on February 17, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from