A Novel CDC25B Promoter Based Oncolytic Adenovirus Suppresses Tumor Growth in an Orthotopic Human Pancreatic Cancer Model

HELGA L. WEBER; GIDEKEL MANUEL; DAVID T CURIEL; EDUARDO A. CAFFERATA; OSVALDO L PODHAJCER

Congreso; 14th Annual Meeting of the American Society of Gene & Cell Therapy; 2011

Pancreatic Cancer is one of the most aggressive types of cancer, with an overall 5 year survival rate of approximately 3-5%. In the present study we constructed a novel conditionally replicative oncolytic adenovirus (CRAd) with the aim to target pancreatic cancer. Based on global gene expression studies from the literature we decided to clone the human version of the cdc25B promoter to regulate adenovirus E1A expression, since cdc25B gene is overexpressed in more than 70 % of pancreatic tumors (in addition to gastric and colon cancer) while faint or no expression was observed in chronic pancreatitis and pancreatic normal cells. After in vitro analysis of different promoter variants, we selected a 468 bp fragment extending from -426/+42 that includes two SP1 sites, a TATA box and an INR motif. Using luciferase activity as a reporter gene we observed that the 0.5 Kb Cdc25-Pr was in average 10-fold more active in pancreatic cancer cells (SW1990, BxPC3, HS766T, MIA Paca2 and Panc1) than in fibroblasts (WI-38, HFL-1 and HACAT) and human microendothelial cells (hMEC-1). These in vitro lytic effects correlated with cdc25B expression in the different cell types. Parallel studies demonstrated that pancreatic cancer cells exhibited 2-fold increased infection with adenovirus pseudotyped with a chimeric 5/3 (shaft/knob) fiber, compared with the parental adenovirus type 5 or pseudotyped with RGD residues. The novel AV-5/3-Cdc25(468) exhibited a strong in vitro lytic effect on pancreatic, gastric and colon cancer cells, even at MOIs of 0.1, while it showed a highly attenuated effect on different normal fibroblasts. Moreover, it showed a synergistic effect with gemcitabine, in highly resistant, SW1990 pancreatic cancer cells. In vivo administration of AV-5/3-Cdc25(468) combined with 15 mg/kg gemcitabine, on mice harboring ectopic SW1990 pancreatic tumors, almost abrogated tumor growth with 100% survival of treated mice after 100 days follow up. Moreover, mice harboring 15-days old SW1990 orthotopic tumors, injected intratumorally once or twice with the virus, in combination or not with gemcitabine, exhibited in average 70%-80% reduction in tumor size at day 45 compared to control PBS-treated mice. The persistence of the CRAd at the end of the experiment was confirmed by E4 DNA analysis. Interestingly, in mice treated with the CRAd+gemcitabine a return to normal levels of alanine transaminase, aspartate aminotransferase and pancreatic amylase was observed. These mice also exhibited more than 90% reduction in CA19.9 serum levels compared to control PBS-treated mice. In order to evaluate their safety, we analyzed CRAd replication on ex-vivo hamster tissue explants. With the exception of liver, Ad5/3-WT replicated in every Syrian hamster tissue tested, while AV-5/3-Cdc25(468) exhibited no replication nor in liver, neither in stomach, lung or pancreas. In conclusion, these data demonstrate that AV-5/3-Cdc25 (468) is an effective oncolytic agent for pancreatic cancer treatment.