Tissue-factor fusion proteins induce occlusion of tumor vessels

Abstract

A variety of fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or derivates of these peptides, have been synthesized. These binding motif peptides target av-integrins or aminopeptidase N (CD13), respectively, on tumor endothelial cells. After expression and deposition as inclusion bodies in Escherichia coli BL21 (DE3), the tTF-fusion proteins were refolded and purified in a multi-step chromatography process. The upscaling process of fusion protein synthesis in order to produce amounts needed for clinical studies is presented. The proteins retained their specific proteolytic ability to activate FX by FVIIa and were able to bind to endothelial cells in vitro. Western blot analysis, analytic chromatography, FX coagulation assay and in vivo experiments have been performed to test for the in vitro stability of the tTF-NGR protein after long-term incubation at 5°C or 25°C, respectively. In vivo xenograft studies in nude mice bearing different malignant human tumors (mammary carcinoma SKBR3, adenocarcinoma of the lung A549) revealed that intravenous or subcutaneous administration of tTF-NGR or -RGD fusion proteins, but not the tTF protein without binding motif, induced thrombosis of tumor vessels which led to significant tumor growth retardation or regression. The anti-vascular mechanism of the tTF fusion proteins was verified by the molecular imaging methods such as magnetic resonance imaging (MRI) and fluorescence reflectance imaging (FRI); MRI showed a reduction of the relative tumor blood volume (BV) and FRI the formation of fibrin in the tTF-fusion protein treated tumors.