Humanized monoclonal antibody TNT-3-mediated truncated tissue factor for the treatment of H22 hepatoma-bearing mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effects of humanized monoclonal antibody-3 (huTNT-3) mediated truncated tissue factor (tTF) on the H(22) hepatoma-bearing mice, and to explore its mechanisms.</p><p><b>METHODS</b>The coagulation activity of the huTNT-3/tTF fusion protein was detected by clotting assay and clotting factor X (FX) activation test in vitro. Mouse hepatoma cell line H(22) cells were inoculated subcutaneously into mice to establish the mouse models of hepatoma. The mice were randomly divided into two groups to be injected once with huTNT-3/tTF fusion protein or tTF protein labeled with rhodamine B isothiocyanate (RBITC), respectively. The localization of huTNT-3/tTF fusion protein in the mouse hepatoma tissue was analyzed by confocal laser scanning microscopy 24 hour after the injection. Fifteen mice were randomly divided into three groups to be injected with the huTNT-3/tTF fusion protein, tTF protein or phosphate buffered saline (PBS) once, respectively. The tumor size was measured every two days to calculate the tumor volume. Ten days after the injection the mice were sacrificed. Samples of the tumor, heart, livers, spleen, lung, kidney and brains of the mice were taken for histopathological examination.</p><p><b>RESULTS</b>Both the huTNT-3/tTF fusion protein and tTF protein effectively promoted blood coagulation. Under the conditions of Ca(2+), the coagulation time in the 1.5, 3, 6 µmol/L huTNT-3/tTF groups was (12.90 ± 0.60) min, (10.39 ± 0.40) min and(8.15 ± 0.24) min, respectively, and the coagulation time of the 1.5, 3, 6 µmol/L tTF groups was (14.23 ± 0.46) min, (12.10 ± 0.49) min and (9.83 ± 0.52) min, respectively, the difference between the two groups was not significant (F = 0.145, P = 0.705). The huTNT-3/tTF fusion protein was similar to the tTF protein in the ability of activating FX (t = 0.101, P > 0.05). The confocal laser scanning microscopic analysis showed that RBITC-fluorescence labeled huTNT-3/tTF fusion protein was enriched in the hepatoma tissue. The tumor volume of the huTNT-3/tTF fusion protein group was significantly lower than that of the tTF and PBS groups (both P < 0.001), however, there was not significant difference between the tTF and PBS groups (t = -0.616, P > 0.05). The survival time of the huTNT-3/tTF group was (25.5 ± 2.5) d, significantly longer than that of the PBS group (17.3 ± 1.9) d and the tTF group (18.6 ± 1.9) d, (both P < 0.05).</p><p><b>CONCLUSION</b>The huTNT-3/tTF fusion protein retains the coagulation ability and has the capability of targeting to tumor vasculature, and induces thrombosis in the tumor vessels, thus to suppress the growth of hepatoma in the mice.</p>

Arginine-glycine-aspartic polypeptide mediated truncated tissue factor therapy for colorectal carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the therapy effects of (arginine-glycine-aspartic, RGD)(3)-truncated tissue factor (tTF) fusion protein on colorectal carcinoma in mice.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>