Molecularly engineered truncated tissue factor with therapeutic aptamers for tumor-targeted delivery and vascular infarction

Selective occlusion of tumor vasculature has proven to be an effective strategy for cancer therapy. Among vascular coagulation agents, the extracellular domain of coagulation-inducing protein tissue factor, truncated tissue factor (tTF), is the most widely used. Since the truncated protein exhibits no coagulation activity and is rapidly cleared in the circulation, free tTF cannot be used for cancer treatment on its own but must be combined with other moieties. We here developed a novel, tumor-specific tTF delivery system through coupling tTF with the DNA aptamer, AS1411, which selectively binds to nucleolin receptors overexpressing on the surface of tumor vascular endothelial cells and is specifically cytotoxic to target cells. Systemic administration of the tTF-AS1411 conjugates into tumor-bearing animals induced intravascular thrombosis solely in tumors, thus reducing tumor blood supply and inducing tumor necrosis without apparent side effects. This conjugate represents a uniquely attractive candidate for the clinical translation of vessel occlusion agent for cancer therapy.

Establishment and characterization of a targeting tumor vesseles system for inducing thrombosis / 中国药学杂志

METHODS: Chemical cross-linking technique was used to prepare a cross-linking agent of the A6-Streptavidin (A6-SA), MF-A6-SA and Biotein-tTF (B-tTF). FX coagulation assay was used to test MF-A6-SA:B-tTF system's FX activity. Fluorescence microscopy and prussian blue staining were used to simultaneously observe the targeting activity of MF-A6-SA:B-tTF with an external magnetic field. Hemagglutination was directly used to study the system's biological amplification by SA/B. Biodistribution experiment was used to observe the toxicity of MF-A6-SA:B-tTF.

Experimental study of (RGD)_3-tTF fusion proteins binding specifically to tumor vasculature in the colonic carcinoma nude mice model / 中国癌症杂志

Background and purpose: Tumor vasculature is increasingly recognized as a target for cancer therapy. In recent years, a fusion protein consisting of the extra cellular domain of tissue factor (truncated tissue factor, tTF) was fused to the antibody selectively binding to tumor vasculature. Antibody-truncated tissue factor(Ab-tTF) fusion protein specifically induced thrombotic occlusion of tumor vessels resulting in tumor growth retardation or regression in some types of solid tumors. However, there were still some disadvantages in the above approach. We constructed and expressed that the (RGD)_3-tTF fusion protein with peptides arginine-glycine-aspartic acid (GRGDSP, abbr. RGD)as the carrier of tTF to explore whether it bad the capability of targeting to tumor vasculature in the colonic carcinoma model. Methods: The (RGD)_3-tTF fusion gene consisting of the tTF was fused to three series-wound peptides RGD. The (RGD)_3-tTF construct was expressed in Escherichia coil BL21(DE_3). The fusion protein was purified through Nickel affinity chromatography column. The activity of inducing blood coagulation was detected by clotting assay and coagulation factor X (FX) activation assay. The specific binding to integrins α_vβ_3 was analyzed by indirect enzyme linked immunosorbent assay (ELISA). All these were compared with the fusion protein RGD-tTE Colonic nude mice models were randomly divided into 3 groups (1 nude mice per group).Tumors were stained by the (RGD)_3-tTE RGD-tTF fusion protein and tTF which were labeled with Fluorescein Isothiocyanate(FITC). The location of the (RGD)_3-tTF fusion protein in the colonic carcinoma bearing nude mice tissue was analyzed by immunofluorescence assay. Results: The (RGD)_3-tTF fusion protein retained tissue factor thrombogenic activities. With increasing concentration, the clotting time was shortened correspondingly. Under the conditions of Ca~(2+), the clotting time was 9.96±0.56 min when the concentration was 6 μmol/L(P<0.01). The (RGD)_3-tTF fusion protein could activise F X above 6 μmol/L concentration, which was similar to RGD-tTF fusion (F=0.147, P>0.05). The ability of the (RGD)_3-tTF fusion protein binding specifically to integrins α_vβ_3 was stronger than that of the RGD-tTF fusion protein in the same concentration (F=164.81, P<0.01), which was apparently indicated by the A_(405nm) 1.25 and 0.95 when the concentration was 0.24 μmol/L. Immunofluorescence assay showed that the (RGD)_3-tTF fusion protein was assembling in the tumor vasculature of the colonic carcinoma bearing nude mice. Conclusion: The (RGD)_3-tTF fusion protein which retained tissue factor thrombogenic activities could bind specifically and efficiently to tumor vasculature in the colonic carcinoma bearing mice through binding to the tumor marker integrins α_vβ_3. It might be a promising foundation for further studies on the colon cancer molecular targeted therapy with tTF as an effective factor.