thromboplastin and arginyl-glycyl-aspartic-acid

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 degrees C or 25 degrees 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.

Topics: Animals; Antineoplastic Agents; Blood Vessels; Escherichia coli; Gene Expression; Humans; Male; Mice; Mice, Nude; Neoplasms; Oligopeptides; Recombinant Fusion Proteins; Thromboplastin

To explore the therapy effects of (arginine-glycine-aspartic, RGD)(3)-truncated tissue factor (tTF) fusion protein on colorectal carcinoma in mice.. The (RGD)(3)-tTF fusion gene, constructed with tTF and three series-wound peptides RGD, was expressed in Escherichia coli BL21 (DE(3)). The fusion protein was purified through Nickel affinity chromatography column. The coagulation activity of the (RGD)(3)-tTF fusion protein was detected by clotting assay in vitro. Mice colorectal cancer cells line CT26 were inoculated subcutaneously into mice to establish colorectal cancer model. Four mice were randomly divided into two groups to be injected with the (RGD)(3)-tTF or tTF fusion protein labeled with rhodamine B isothiocyanate (RBITC) at a single dose of 50 microg respectively. The location of the (RGD)(3)-tTF fusion protein in the colorectal carcinoma bearing mice tissue was analyzed by using in vivo optical imaging one hour after the injection and confocal microscopy twenty-four hours after the injection. Fifteen mice bearing colorectal carcinoma were randomly divided into three groups for injection with the (RGD)(3)-tTF, tTF fusion protein or phosphate buffered saline (PBS) at a single dose of 50 microg respectively. The tumor size was measured daily to calculate the tumor volume. Five days after the injection, the mice were killed to harvest tumor tissues, hearts, livers, spleens, lung, kidneys and brains to observe valid thrombogenesis and tumor necrosis.. With the concentration of the (RGD)(3)-tTF fusion protein increased, the clotting time was shorten correspondingly under the conditions of Ca(2+), and the clotting time was (8.6 +/- 0.2) min when the concentration was 6 micromol/L, and it was >30 min in the group of 0 micromol/L (P < 0.05). The coagulation activity of (RGD)(3)-tTF and tTF fusion protein was alike (F = 0.09, P > 0.05). The in vivo optical imaging and confocal microscopy analyses showed that RBITC fluorescence labeling (RGD)(3)-tTF fusion protein was assembled in the tumor vasculature. On the first, third, fifth day after injection, the tumor volume of (RGD)(3)-tTF fusion protein group was (120.8 +/- 4.8) mm(3), (93.8 +/- 3.4) mm(3), (132.2 +/- 7.7) mm(3) respectively, which was significantly smaller than that of the tTF group [(181.4 +/- 13.8) mm(3), (333.0 +/- 32.0) mm(3), (514.0 +/- 11.5) mm(3)] and PBS group [(182.6 +/- 11.5) mm(3), (332.8 +/- 21.0) mm(3), (524.2 +/- 16.7) mm(3)] (both P < 0.05). However, there was no significant difference in the tumor volume between the latter two groups (P > 0.05).. The (RGD)(3)-tTF fusion protein is capable of targeting to tumor vasculature and inducing thrombogenesis for suppressing the tumor growth in the colorectal carcinoma mice model, and it's expected to be a new therapy for colorectal cancer.

Topics: Animals; Colorectal Neoplasms; Genetic Vectors; Male; Mice; Mice, Nude; Neoplasm Transplantation; Oligopeptides; Plasmids; Recombinant Fusion Proteins; Thromboplastin

To develop a new fusion protein (RGD)3/tTF for the therapy of the selective thrombosis of tumor blood vessels. The fused gene (RGD) 3/tTF was reconstructed by PCR, was cloned into vector pET22 b(+), and expressed in E. coli BL21 (DE3). The fusion protein was purified through Nickel-affinity chromatography column. The tTF activity of the fusion protein was detected by clotting assay and F X activation assay. The specific binding of (RGD) 3/tTF to alphavbeta3 was analyzed by indirect ELISA. The recombinant plasmid pET22 b(+)/(RGD)3/tTF was obtained and expressed in E. coli BL21 (DE3). The purified fusion protein could induce blood coagulation, activiate F X. The ability of (RGD) 3/tTF binding specifically to alphavbeta3 was increased by 32%, compared with RGD/tTF. A new fusion protein (RGD) 3/tTF was successfully expressed in E. coli BL21 (DE3). The expressed proteins retained tTF activity and showed a higher binding to alphavbeta3 than that of RGD/tTF.

Topics: Animals; Blood Coagulation; Chromatography, Affinity; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Factor Xa; Gene Expression; Humans; Integrin alphaVbeta3; Mice; Oligopeptides; Polymerase Chain Reaction; Protein Binding; Recombinant Fusion Proteins; Thromboplastin

Tools for monitoring modern target-specific antiangiogenic and antivascular therapies are highly desirable because treatment strategies are time consuming, expensive, and yet sometimes ineffective. Therefore, the aim of this experimental study was to evaluate the predictive value of steady-state ultrasmall particles of iron oxide (USPIO; SH U 555 C)-enhanced magnetic resonance imaging (MRI) for early assessment of antivascular tumor-treatment effectiveness.. Mice were inoculated with an HT-1080 fibrosarcoma xenograft and subjected to target-specific antivascular therapy using a selective thrombogenic vascular-targeting agent (truncated tissue factor fused to RGD peptide) or saline as control. Four to 8 hours after treatment, the USPIO-induced change in the transverse relaxation rate DeltaR2* was measured by MRI, and the vascular volume fraction (VVF) was calculated by calibrating DeltaR2* of the tumor by DeltaR2* of muscle tissue. Treatment response was defined by histologic grading of vascular thrombosis and tumor necrosis.. After thrombogenic treatment, half of the HT-1080 xenograft-bearing animals showed only minor (=nonresponder) whereas the other half showed extensive tumor thrombosis (=responders). For responders, a significant decrease of DeltaR2* and VVF was observed compared with the control group (DeltaR2*: controls: 16 +/- 1 s-1 vs. responder: 4 +/- 2 s-1; P < 0.001) whereas DeltaR2* and VVF remained nearly unchanged for nonresponders (DeltaR2*: nonresponder 14 +/- 2 s-1). VVF and DeltaR2* values correlated inversely with the histologic grading of vascular thrombosis and tumor necrosis (VVF: r = -0.8; DeltaR2*: r = -0.71; P < 0.01).. USPIO-enhanced MRI allows a noninvasive, early assessment of treatment efficacy of thrombogenic vascular-targeting agents.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Contrast Media; Female; Ferric Compounds; Fibrosarcoma; Humans; Magnetic Resonance Imaging; Mice; Mice, Nude; Oligopeptides; Predictive Value of Tests; Recombinant Fusion Proteins; Thromboplastin; Time Factors; Treatment Outcome; Xenograft Model Antitumor Assays

Tissue Factor (TF) is a cell membrane receptor protein that is the initiator of the extrinsic pathway of the blood coagulation cascade and normally released from damaged tissues. By substituting the attachment site with a tumor delivery agent, this potent thrombogenic protein in its truncated form (tTF) can be targeted to the tumor where it can initiate clotting, thereby occluding the tumor's blood supply and causing rapid tumor destruction. To test the therapeutic potential of this vascular targeting approach, three fusion proteins, chTNT-3/tTF, chTV-1/tTF, and RGD/tTF, which target DNA exposed in degenerative areas of tumors, fibronectin on the tumor vascular basement membrane, and alpha nu beta 3 on the luminal side of tumor vessels, respectively, were developed and tested for their antitumor effects. Antigen binding and clotting assays demonstrated that each of the fusion proteins retained their antigen binding and thrombogenic activities. In vivo studies in mice bearing established MAD109 lung and Colon 26 carcinomas revealed that all three reagents induced histological evidence of microregional thrombosis and massive cell necrosis. Of interest, the chTV-1/tTF and RGD/tTF fusion proteins induced thrombosis in small and medium sized tumor vessels, whereas the chTNT-3/tTF induced clotting in relatively larger vessels. Treatment studies showed that chTNT-3/tTF and chTV-1/tTF but not RGD/tTF had a significant inhibition of tumor growth. These studies demonstrate that multiple targets exist which can be used to localize tTF to occlude tumor vessels in two diversely different murine tumor models. To attain a significant antitumor effect, however, these thrombogenic agents had to occlude medium and large vessels within the tumor. Additional studies are warranted to identify maximal conditions for inducing therapeutic vascular coagulation as a new and potent method of cancer therapy.

Topics: Animals; Female; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Neoplasms, Experimental; Oligopeptides; Recombinant Fusion Proteins; Thromboplastin; Thrombosis; Tumor Cells, Cultured