A Selective Tumor Microvasculature Thrombogen that Targets a Novel Receptor Complex in the Tumor Angiogenic Microenvironment Amr El-Sheikh, 1 Per Borgstrom, 2 Gourab Bhattacharjee, 1 Mattias Belting, 1,3 and Thomas S. Edgington 1 1 Department of Immunology, The Scripps Research Institute; 2 Vascular Biology and Angiogenesis Program, The Sidney Kimmel Cancer Center, La Jolla, California and 3 Department of Cell and Molecular Biology, Lund University, Lund, Sweden Abstract We have previously shown that part of the heparin-binding domain of the vascular endothelial growth factor (VEGF), designated HBDt, localizes very selectively to surfaces of the endothelial cells of i.t blood vessels. Here, we have coupled the HBDt to the extracellular domain of tissue factor (TFt), to locally initiate the thrombogenic cascade. In tumor- bearing mice, infusion of this HBDt.TFt results in rapid occlusive thrombosis selective only for tumor microvascula- ture with resultant infarctive destruction of tumors. We now show that infusion of an optimal combination of this HBDt.TFt and its requisite cofactor ( factor VIIa) in tumor models results in significant tumor eradication. Binding studies and confocal microscopy indicate that the target for the HBDt.TFt seems to be a trimolecular complex of chondroitin C sulfate proteoglycan, neuropilin-1, and VEGF receptor-2, overexpressed together only in highly angiogenic sites of the tumor microenvironment. The HBDt.TFt was also colocalized with the trimolecular receptor complex in endothelial sprouts from tumor tissues, and its binding inhibited the growth of such sprouts. In vitro , we show that the HBDt structure has its highest affinity for chondroitin 6 sulfate. We show the potential of this HBDt.TFt as a candidate therapeutic and elucidate its target in vivo . (Cancer Res 2005; 65(23): 11109-17) Introduction To search and discover novel targets specific for tumor vascular endothelium is a challenging task but a critical aspect of developing more effective tumor therapeutics. The existence of such targets expands on our perception of the uniqueness of the tumor microenvironment. To be an optimally effective target, it should not only be expressed selectively by tumor microvasculature but should also be a common feature found in a broad spectrum of solid tumors. We previously showed that the rather novel vascular endothelial growth factor (VEGF) heparin-binding domain substructure (HBDt), when expressed on the surface of a modified M13 phage, selectively enabled accumulation in vivo on the vascular surface of the CD31-positive endothelium of tumor vasculature (1). The VEGF 165 isoform is known to bind to VEGF receptor-2 (VEGFR-2, KDR), neuropilin-1 (Npn-1), and an unknown heparan sulfate (HS) proteoglycan (2). In addition to VEGFR-2, Npn-1 is also known to be highly expressed by endothelial and tumor cells (3) and is known to play important roles in endothelial mitogenesis, tumor cell migration, and invasion (4–7). The role of Npn-1 expression in tumor angiogenesis has been observed in several types of tumors (4, 8). Furthermore, it was reported that expression of Npn-1 in tumors seems to correlate with a shift from benign stromal tissue to that associated with malignancies (9). The VEGF 165 isoform, which contains HBDt, is known to participate in the formation of molecular complexes, containing VEGFR-2 and Npn-1. The HBDt-encoded VEGF/exon 7 is thought to interact with the Npn-1 b1b2 domain (10–12). The b1b2 region of Npn-1 was also analyzed and shown to be a heparin- binding domain, which enhanced the binding of VEGF 165 to Npn- 1 (13). The size of the oligosaccharide fragment needed for enhancement of binding was at least 20-monosaccharide unit in length, suggesting the potential involvement of a large polysac- charide chain, possibly a chondroitin sulfate proteoglycan (CSPG). We fused the HBDt structure to the extracellular domain of human tissue factor (TFt) through a linker to explore the feasibility of targeting the truncated extracellular domain of TFt to the tumor microvessel plasmalemma in a mode to locally initiate the thrombogenic cascade. Local infarctive necrosis of tissue follows loss of local vascular perfusion; a concept conserved in evolution from marine invertebrates to mammals. We have now analyzed the ability of the HBDt.TFt fusion protein to bind heparin and other carbohydrate moieties in vitro and to endothelial cells in culture. We also investigated the effect of HBDt.TFt addition on angiogenic sprouting ex vivo . We also analyzed the complex dynamics of targeting of the HBDt.TFt protein to tumor intravascular microenvironment when infused in vivo in various murine tumor models, monitoring thrombus formation by real-time intravital microscopy. We investigated the effect of HBDt.TFt administration and the resultant throm- bosis on the destruction of tumors. Finally, we confirmed docking of HBDt.TFt in the i.t. vasculature and colocalization with its target receptor molecules by confocal microscopy in vivo and ex vivo . Our data show that HBDt.TFt accumulates in i.t. vasculature preferentially, attributable to recognition of a unique target, and docks on tumor endothelium plasmalemma, in a manner to permit functional initiation of the coagulation cascade. This results in vascular occlusion of tumors, resulting in local infarctive necrosis and significantly reducing the mass of solid tumors. We support the potential of this strategy for therapeutic purposes and illustrate the unique biology of the tumor microenvironment. Requests for reprints: Thomas S. Edgington, Department of Immunology (SP258), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-784-8225; Fax: 858-784-8480; E-mail: tse@scripps.edu. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2733 www.aacrjournals.org 11109 Cancer Res 2005; 65: (23). December 1, 2005 Research Article Research. on July 1, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from