No abstract

We have developed an approach to cancer gene therapy in which the oncolytic effects of an adenoviral vector have been combined with selective expression of a soluble form of transforming growth factor (TGF)-beta receptor II fused with Fc (sTGFbetaRIIFc). We chose to use adenoviral dl01/07 mutant because it can replicate in all cancer cells regardless of their genetic defects. An oncolytic adenovirus expressing sTGFbetaRIIFc (Ad.sT- betaRFc) was constructed by homologous recombination. Infection of MDA-MB-231 and MCF-7 human breast cancer cells with Ad.sTbetaRFc produced sTGFbetaRIIFc, which was released into the media. The conditioned media containing sTGFbetaRIIFc could bind with TGF-beta 1 and inhibited TGF-beta-dependent transcription in target cells. Infection of MDA-MB-231, MCF-7, and 76NE human breast cancer cells with Ad.sTbetaRFc resulted in high levels of viral replication, comparable to that of a wild-type dl309 virus. Although some viral replication was observed in actively dividing normal human lung fibroblasts, there was no replication in nonproliferating normal cells. Direct injection of Ad.sTbetaRFc into MDA-MB-231 human breast xenograft tumors grown in nude mice resulted in a significant inhibition of tumor growth, causing tumor regression in more than 85% of the animals. These results indicate that it is possible to construct an oncolytic virus expressing sTGFbetaRIIFc in which both viral replication and transgene expression remain intact, and the recombinant adenovirus is oncolytic in a human tumor xenograft model. On the basis of these results we believe that it may be feasible to develop a cancer gene therapy approach using Ad.sTbetaRFc as an antitumor agent.

Our laboratory is interested to develop oncolytic adenoviral vectors that can be administered systemically for the treatment of breast cancer. To restrict viral replication in breast tumor cells, we have constructed mhTERTAd.sTβRFc, a 01/07 based adenoviral vector expressing the soluble form of TGFβ receptor II fused with human Fc IgG1 (sTGFβRIIFc) gene, in which viral replication is under the control of modified human telomerase reverse transcriptase (mhTERT) promoter. In addition, mhTERTAd.sTβRFc-mediated sTGFβRIIFc production would target growth factor-β (TGFβ) pathway known to contribute to the tumor progression breast cancer metastasis. We chose to use mhTERT promoter because it was found to be relatively more active (approximately 20-times) in breast cancer cells compared to normal human cells. We showed that infection of MDA-MB-231 and MCF-7 breast cancer cells for 48 hrs with mhTERTAd.sTβRFc produced high levels of sTGFβRIIFc (greater than 1 μg/ml) in the medium. Breast cancer cells produced nearly 6,000-fold increase in the viral titers during 48 hrs infection period. However, mhTERTAd.sTβRFc replication was attenuated in normal cells. Infection of breast cancer cells with a replication deficient virus Ad(E1-).sTβRFc also produced high levels of sTGFβRIIFc, but under these conditions no detectable viral replication was observed. Adenoviral-mediated production of sTGFβRIIFc was shown to bind with TGFβ-1, and abolished the effects of TGFβ-1 on downstream SMAD-3 phosphorylation. The administration of mhTERTAd.sTβRFc intravenously into MDA-MB-231 human xenograft bearing mice resulted in significant inhibition of tumor growth, and production of sTGFβRIIFc in the blood. On the other hand, intravenous injection of Ad(E1-).sTβRFc did not exhibit significant inhibition of the tumor growth, but resulted in the sTGFβRIIFc in the blood, suggesting that viral replication along with sTGFβRIIFc protein production play a critical role in inducing inhibition of tumor growth. These results warrant future investigation of mhTERTAd.sTβRFc as an anti-tumor agent in vivo.