105. Enhanced Oncolytic Activity Mediated by a Novel Human Adenovirus Type 6 Based Vector

Abstract

Most existing oncolytic adenoviruses (AdV) are based on human AdV type 5 (hAdV-5). Clinical efficacy of hAdV-5 based oncolytic viruses is limited by variable expression levels of coxsackie- and adenovirus receptor (CAR) in different tumor cells and insufficient replication rates. Additionally, high prevalence of neutralizing antibodies against hAdV-5 resulting in lower efficiency makes hAdV-5 a less suitable candidate for systemic application. Recent studies have highlighted human adenovirus type 6 (hAdV-6) as a promising candidate for oncolytic and vaccine vectors. Thus, development of novel oncolytic AdV based on hAdV-6 may help overcoming these limitations. We further hypothesised that oncolytic efficacy of the candidate virus can be augmented by expression of RNAi suppressor protein P19 (Lecellier et al., Nature 2005) as has been shown previously for hAdV-5 (Rauschhuber et al, Sci. Rep. 2013). Here we aim at evaluating a novel hAdV-6-based, p19-containing oncolytic AdV as novel candidate for oncolytic applications in different tumour cell lines. We cloned a P19-containing hAdV-6 based virus (hAdV-6FP19) by a novel seamless recombineering technique (Zhang et al, unpublished). In order to allow P19 expression from the adenoviral vector genome, the P19 cDNA was fused via an internal ribosome entry site (IRES) to the late fiber gene. After release of the respective recombinant adenoviral genomes from plasmids containing the complete DNA molecule, linearized viral DNA was transfected into HEK293 cells for virus reconstitution. After initial amplification steps which were monitored by virus specific PCRs upscaling and virus purification using cesium-chloride density-gradient ultracentrifugation was performed. Rescue and amplification efficiencies were comparable to commonly used hAdV-5 based vectors. Various cancer cell lines from different origin were used to perform oncolysis assays. This included A549 (lung carcinoma), HCT 116 (colon carcinoma), Hela (cervical carcinoma) and Huh7 (hepatocellular carcinoma) cells which were infected with hAdV-6FP19, hAdV-6 and hAdV-5 at various multiplicities of infection (MOI). 2-3 days after infection cells were fixed and stained with methylene blue. We observed significantly higher cell lysis (up to 100-fold) for hAdV-6FP19 infected cells as compared to hAdV-5 and 6 at identical MOIs. Higher cell lysis rates for hAdV-6FP19 compared to wildtype virus were present in all evaluated cell lines, suggesting significantly enhanced oncolytic potential for hAdV-6FP19. In total we believe that hAdV6-based vectors hold great promise for oncolytic applications and that their oncolytic effectiveness can be further improved by RNAi-suppression.