637. Specific Oncolytic Adenovirus Driven by Human A33 Promoter Region for Colorectal Cancer Therapy
Authors: Eduardo G. Cafferata, Daniela R. Maccio, Maria V. Lopez, Diego L. Viale, Osvaldo L. Podhajcer
Journal: Molecular Therapy (2006)

A33 antigen is a membrane-bound protein that is expressed only in intestinal ephitelium and is over-expressed in most colon cancers. A33 is found in 95% of primary and metastatic colon cancer cells and in 38% of diffuse gastric cancers with uniform antigen expression but is absent in most other normal tissues and tumor types. Based on these characteristics, we hypothesized that A33 promoter might be useful in the design of a conditional replicative adenovirus. For this purpose we cloned an A33 promoter fragment (A33Pr) that includes the 5´UTR and extends from −105 bp to +307 bp upstream of the luciferase gene. Using luciferase activity as a gene reporter we demonstrated that A33Pr was in average 10-fold more active in colon carcinoma cells (LoVo, T84 and HT29) than in melanoma (A375) or breast (T47D) cancer cell lines. We next constructed a conditionally replicating adenovirus (AV22EL) where E1A was placed under the control of A33Pr. We examined by crystal violet staining the adenovirus lytic capacity of AV22EL on different cell lines. AV22EL induced specific oncolysis of human colorectal cancer cell lines that expressed A33 such as LoVo, T84 and HT29 and have negligible lytic capacity on cells that lacked or had minimal A33 expression such as A375 (melanoma), WI-38 and HFL-1 (normal human fibroblasts), endothelial cells and CCD841 (normal colon cells). AV22EL was active in colon cancer cells even at a MOI of 1. This specific lytic capacity was also observed in multicellular spheroids. AV22EL inhibited homotypic spheroids made of LoVo cells but had no effect on spheroids made of A375 cells. Viral yield of AV22EL in colon carcinoma cells was increased in average up to 10,000 fold compared to melanoma and normal cells.The antitumor activity of AV22EL was examined in BALB/c nu/ nu mice carrying s.c. human colon cancer xenografts. When tumors reached an average volume of 100 mm3, mice were injected intratumorally for three times with 1x1010 vp/mouse of AV22EL or Ad-betagal. Treatment with AV22EL resulted in 60% inhibition of LoVo colon cancer xenografts growth. These data suggest that AV22EL is a potential oncolytic agent for the treatment of human colon cancers. A33 antigen is a membrane-bound protein that is expressed only in intestinal ephitelium and is over-expressed in most colon cancers. A33 is found in 95% of primary and metastatic colon cancer cells and in 38% of diffuse gastric cancers with uniform antigen expression but is absent in most other normal tissues and tumor types. Based on these characteristics, we hypothesized that A33 promoter might be useful in the design of a conditional replicative adenovirus. For this purpose we cloned an A33 promoter fragment (A33Pr) that includes the 5´UTR and extends from −105 bp to +307 bp upstream of the luciferase gene. Using luciferase activity as a gene reporter we demonstrated that A33Pr was in average 10-fold more active in colon carcinoma cells (LoVo, T84 and HT29) than in melanoma (A375) or breast (T47D) cancer cell lines. We next constructed a conditionally replicating adenovirus (AV22EL) where E1A was placed under the control of A33Pr. We examined by crystal violet staining the adenovirus lytic capacity of AV22EL on different cell lines. AV22EL induced specific oncolysis of human colorectal cancer cell lines that expressed A33 such as LoVo, T84 and HT29 and have negligible lytic capacity on cells that lacked or had minimal A33 expression such as A375 (melanoma), WI-38 and HFL-1 (normal human fibroblasts), endothelial cells and CCD841 (normal colon cells). AV22EL was active in colon cancer cells even at a MOI of 1. This specific lytic capacity was also observed in multicellular spheroids. AV22EL inhibited homotypic spheroids made of LoVo cells but had no effect on spheroids made of A375 cells. Viral yield of AV22EL in colon carcinoma cells was increased in average up to 10,000 fold compared to melanoma and normal cells. The antitumor activity of AV22EL was examined in BALB/c nu/ nu mice carrying s.c. human colon cancer xenografts. When tumors reached an average volume of 100 mm3, mice were injected intratumorally for three times with 1x1010 vp/mouse of AV22EL or Ad-betagal. Treatment with AV22EL resulted in 60% inhibition of LoVo colon cancer xenografts growth. These data suggest that AV22EL is a potential oncolytic agent for the treatment of human colon cancers.