777. A Novel Pan-Oncolytic Adenovirus Therapeutically Effective in Different Cancer Cell Types

SPARC is a matricellular protein that is overexpressed in malignant and stromal components of human melanomas. We have previously shown that a 1.3 Kb SPARC promoter region was effective in driving suicide gene expression both in melanoma and endothelial cells leading to the elimination of melanoma tumors in vivo in nude mice suggesting that SPARC promoter could be a good candidate for generating a conditional replicating oncolytic adenovirus. By using luciferase expression as a reporter gene, we performed a detailed analysis of the activity and specificity of different fragments of SPARC promoter. We selected a promoter sequence extending from -512 bp to +35 bp, named F512, that showed the best ratio of activity vs. specificity in human melanoma cells compared to human cervix, breast and colon carcinoma cell lines. We constructed two adenoviral vectors in which the E1A gene was driven by F512. Ad-F512 lacks the E3 region while Ad-I-F512-E3 has an intact E3 region and an insulator sequence between the ITR and F512. The lytic capacity of both viruses was initially tested in vitro on a panel of malignant cells, melanoma (n=3), colon (n=2), breast (n=3) and cervix (n=1); and normal cells (bone marrow derived cells, WI-38 fibroblasts and BAEC endothelial cells).Unexpectedly, both adenoviruses were equally efficient in inducing lysis of SPARC-expressing and non-expressing malignant cells. In general, malignant cells that were sensitive or resistant to wild type Ad5 lytic effect were also sensitive or resistant to both recombinant viruses lytic effect. Interestingly, colon cancer cells became more sensitive than melanoma cells to the recombinant viruses. In addition, both viruses showed 3 to 5 logs lower lytic capacity in normal cells than wild type Ad5. Thus, CRAds based on SPARC promoter seem to replicate specifically in cancer cells while preserving normal cells. Ad-F512 was able to complement cell spreading of an E1-E3- replication defective vector expressing b-galactosidase indicating that this virus could be combined with non replicative therapeutic adenovirus. Finally, nude mice harboring SB2 melanoma tumors (mean 120 mm 3 ) were treated intratumoraly with three administrations of 1010 vp/mouse of Ad-F512 or Ad-I-F512-E3. The treatment resulted in a potent antitumor effect. In 3 out of 5 mice, the tumor was completely eliminated, and one mouse showed a slight tumor growth, while in mice treated with control Ad-b-gal virus we observed no therapeutic benefit