Patrice Yeh

AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice

TIMP-2 is a natural matrix metalloproteinase (MMP) inhibitor that prevents the degradation of extracellular matrix proteins. It abolishes the hydrolytic activity of all activated members of the metalloproteinase family and in particular that of MT1-MMP, MMP-2, and MMP-9, which are selective for type IV collagenolysis. Since MMPs have been implicated in both cancer progression and angiogenesis, we generated a recombinant adenovirus to deliver human TIMP-2 (AdTIMP-2) and evaluated its anticancer efficiency in three murine models. Our results demonstrated that overexpression in vitro of TIMP-2 inhibited the invasion of both tumor and endothelial cells without affecting cell proliferation. Its in vivo efficiency has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer MDA-MB231 in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three types significantly reduced tumor growth rates by 60--80% and tumor-associated angiogenesis index by 25--75%. Lung metastasis of LLC tumor was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.

Blockage of urokinase receptor reduces in vitro the motility and the deformability of endothelial cells

The binding of urokinase (u‐PA) to its cell surface receptor (u‐PAR) is critical for tumor cell invasion. Here, we report that the disruption of this binding by an u‐PAR antagonist ATF‐HSA inhibits in vitro the motility of endothelial cells in a dose‐dependent manner. This inhibition was also observed when the cells were first stimulated with potent angiogenic factors, including bFGF or VEGF. [3H]thymidine incorporation assay demonstrated that ATF‐HSA did not affect the cell proliferation. ATF‐HSA was more potent than plasmin inhibitors, suggesting that it exerts its effects not solely by inhibiting the remodeling of the extracellular matrix. In fact, analysis of the cell shape change during migration revealed for the first time that its effect is related to a decrease in cell deformability. These results suggest that u‐PAR antagonist may be a new approach to control angiogenesis.

Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice.

Systemic administration of Ad5-based recombinant adenovirus leads to preferential transduction of the liver. Using this property, we have assessed the potential of venous viral injection to deliver a recombinant antiangiogenic adenovirus to treat cancer dissemination and improve survival. The results demonstrate that venous injection of adenovirus AdmATF, which encodes a secretable mouse ATF (amino-terminal fragment of urokinase) known to inhibit angiogenesis, suppressed angiogenesis induced by colon cancer metastasis growth in mice liver and improved survival. Nude mice were injected intravenously with 5 X 10(9) PFU of AdmATF and subsequently challenged after a 3-day interval by intrasplenically injected human colon carcinoma cells (LS174T, 3 x 10(6)) that home to liver. Microscopic inspection revealed that, within the AdmATF-pretreated mice (n = 8), the size and number of liver-metastasized nodules on day 30 were remarkably reduced (80% in number, p < 0.05) compared with control mice (n = 7) pretreated in parallel with a control adenovirus. Metastatic growth-related liver weight gain was also inhibited up to 90%. AdmATF-specific capability that offers liver resistance to the apparition and growth of liver metastasis was shown to correlate with the inhibition of peritumoral and intratumoral angiogenesis (reduced by 79%, p < 0.01 as shown by anti-vWF immunostaining of liver sections) and a twofold increase in tumor necrotic area and an eightfold increase in apoptotic tumor cell number. This protective effect was still observed when the mice were challenged 10 days after venous AdmATF injection (visible metastasis nodules: 6.3+/-3.1, n = 7 for control mice versus 2.7+/-2.9, n = 10 for treated mice, p < 0.05). More importantly, the mean survival has been prolonged from 45.1 days (n = 9) to 83.3 days (n = 10, p < 0.05). Altogether, the high efficacy, although transient, in this experimental mice model strongly advocates the plausibility of transforming the liver into a dissemination resistant organ by antiangiogenic gene therapy through systemic delivery approach.

Blockage of the urokinase receptor on the cell surface: Construction and characterization of a hybrid protein consisting of the N‐terminal fragment of human urokinase and human albumin

Receptor‐bound urokinase is likely to be a crucial determinant in both tumor invasion and angiogenesis. We report here that a yeast‐derived genetic conjugate between human serum albumin and the 1–135 N‐terminal residues of urokinase (u‐PA) competitively inhibits the binding of exogenous and endogenous u‐PA to its cell‐anchored receptor (u‐PAR). This hybrid molecule (ATF‐HSA) also inhibits in vitro pro‐urokinase‐dependent plasminogen activation in the presence of u‐PAR bearing cells. These effects are probably responsible for the observed in vitro inhibition of tumor cell invasion in a reconstituted basement membrane extract (Matrigel).

Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses.

Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1α (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro, as well as transgene amplification and persistence in vivo. Retrovirus titers of >105 infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10- to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin.

Adenoviruses (Part II): Improvement of Adenoviral Vectors for Human Gene Therapy: E1 and E4 Deleted Recombinant Adenoviruses

We thank Dr. Gary Ketner (Baltimore, Maryland, U.S.A.) for providing us with many adenoviral E4 deletants, and the W162 cell line. We also thank Dr. P. Boulanger (Montpellier, France) for providing us with antibodies raised against the adenoviral fiber protein, Dr. T. Shenk (Princeton, New Jersey, U.S.A.) and Dr. P. Hearing (Stony Brook, New York, U.S.A.) for their kind gift of antibodies raised against the E4 gene products. We thank A. Gillardeaux, M. Lepeut, I. Mahfouz and C. Jullien for excellent technical assistance, Dr. J.M. Guillaume for the production of viral stocks, and Dr. M.Lakich for critical reading of the manuscript. This work was supported by Bioavenir (Rhone-Poulenc and French Ministry of Research and Industry).