Coelectrotransfer to skeletal muscle of three plasmids coding for antiangiogenic factors and regulatory factors of the tetracycline-inducible system: tightly regulated expression, inhibition of transplanted tumor growth, and antimetastatic effect.

We describe an approach employing intramuscular plasmid electrotransfer to deliver secretable forms of K1-5 and K1-3-HSA (a fusion of K1-3 with human serum albumin), which span, respectively, five and three of the five kringle domains of plasminogen. A tetracycline-inducible system (Tet-On) composed of three plasmids coding, respectively, for the transgene, the tetracycline transcriptional activator rtTA, and the silencer tTS was employed. K1-3-HSA and K1-5, produced from C2C12 muscle cells, were found to inhibit endothelial cell (HMEC-1) proliferation by 30 and 51%, respectively. In vivo, the expression of the transgene upon doxycycline stimulation was rapid, stable, and tightly regulated (no background expression) and could be maintained for at least 3 months. Blood half-lives of 2.1 and 3.7 days were found for K1-5 and K1-3-HSA, respectively. The K1-5 protein was secreted from muscle into blood at a level of 45 ng/ml, which was sufficient to inhibit MDA-MB-231 tumor growth by 81% in nude mice and B16-F10 melanoma cell lung invasion in C57BL/6 mice by 73%. PECAM-1 immunostaining studies revealed modest tumor vasculature in mice expressing K1-5. In contrast, K1-3-HSA, although secreted into blood at much higher level (250 ng/ml) than K1-5, had no effect on tumor growth.

Combined effects of docetaxel and angiostatin gene therapy in prostate tumor model.

The anti-tumor effects of adenovirus-delivered angiostatin (AdK3) in association with docetaxel (Taxotere) have been evaluated in a human-origin prostate tumor model. In vitro, human endothelial cells were 50- to 100-fold more sensitive to docetaxel than prostate cell lines (PC3, LNCaP, and DU145), and the combination regimen of docetaxel and AdK3 was significantly more cytotoxic for endothelial cells than either treatment alone. PC3 cells, which display the highest sensitivity to docetaxel, were then grafted onto athymic mice for an evaluation of the combined regimen as a therapy. The combination of a single intratumoral injection of AdK3 (2 x 10(9) pfu) and a single intravenous injection of docetaxel (15 mg/kg) was compared with the injection of AdK3 alone on preestablished mice bearing PC3-derived tumors with a mean tumor volume of 60 +/- 11 or 205 +/- 46 mm3. Significant antitumoral effects were observed only in mice receiving the combined treatment. We showed that all PC3 tumors regressed in the AdK3-docetaxel combination group and that 40 to 83% totally regressed. In all cases, this regimen was tightly correlated with a marked decrease in intratumoral vascularization. Our experimental data show that attacking both endothelial and tumoral compartments is an efficient and logical strategy, making this bitherapy approach clinically promising.