979. The Gene Expression and Function of Adenoviral E3-Genes in Murine Tumour Cell Lines

Abstract

We recently developed a number of immunocompetent murine tumour models that enabled the evaluation of adenoviruses with classical E3 deletions, dl309 (E3B deleted (RIDa/RIDb complex and 14.7kD)) and dl704 (E3 gp19K deleted). The E3B genes protect infected cells from tumour necrosis factor (TNF) and Fas mediated apoptosis, and internalizes epidermal growth factor receptor (EGFR). In addition, E3 gp19K prevents newly synthesised major histocompatibility complex (MHC) class 1 molecules from reaching the cell surface, thus avoiding immune recognition of infected cells. Three murine tumour cell lines (CMT64, JC and CMT93) and two human control tumour cell lines (A549 and H460) were characterised in vitro after infection with Ad5, (wild type E3), dl309, dl704 and dl312 (E1A deleted, wild type E3). The dl309 mutant was more potent compared to Ad5 in each cell line tested while dl704 was less potent than Ad5. Compared with Ad5, dl309 viral replication was higher in every cell line at each time point tested while dl704 replication was lower than Ad5. In one murine cell line (CMT64), viral burst was similar to the observed levels in the human H460 cells while two cell lines (JC and CMT93) had lower or no replication. Adenoviral E3 gene expression (E3B and gp19K) was supported in each murine tumour line. In addition, the E3 proteins had similar function in the murine cell lines as previously reported for human cell lines. We demonstrated EGFR, Fas and TNFR1 internalisation/degradation following Ad5 and dl704 infection while Ad5 and dl309 infection resulted in loss of extracellular MHC class 1 receptor expression/detection. The decrease in MHC class 1 cell surface expression was not observed following dl704 infection. These results strengthen our previous findings using murine immunocompetent xenograft models in the evaluation of interactions between the host immune system and tumour cells infected with oncolytic viral mutants. The further development of these murine tumour models will aid in the improved design of more efficacious E3-deletion mutants.