632. Oncolytic Adenoviruses Targeted to the HPV E6 and E7 Oncoproteins as a Novel Treatment for Head & Neck Squamous Cell Carcinomas

Abstract

Introduction:Recent reports have shown that the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has been steadily increasing. HNSCCs are known to have a high recurrence rate and compared to their HPV-negative counterparts, these tumors have a unique biology which necessitates the development of novel treatment modalities. The HPV E6 and E7 oncoproteins are attractive therapeutic targets as they interact with key cell cycle regulatory components, namely p53 and pRb. Our research group has applied conditionally replicating oncolytic adenoviruses (CRAd) with modifications in the E1a region of the genome allowing for selective replication in E6 and E7 expressing HNSCCs.Methods:The in vitro transduction efficiency of E1-deleted luciferase expressing vectors with fiber modifications was assessed in multiple HPV-positive and negative cell lines. The CRAds were designed with a distinct deletion in the E1a region of the adenoviral genome (D24 and CB016) intended to allow for selective replication in HPV-positive HNSCC cells. Additionally, the CRAds have a luciferase transgene in the E3 region that is expressed in a replication dependent manner. By using a luciferase assay, the degree of viral replication was analyzed in numerous HNSCC cell lines. In vitro cell viability following viral infection was analyzed with crystal violet and MTS assays. A HPV-positive cell line (UPCI SCC 090) was used to establish subcutaneous tumors in female nude mice. They were subsequently treated with either one intratumoral viral injection or four injections (given every fourth day).Results:The 5/3 fiber modification significantly increased viral infectivity in all tested HNSCC cell lines. The 5/3 CB016 vector replicated selectively in HPV-positive cell lines, while the 5/3 D24 virus replicated in all cell lines regardless of HPV status. Both of the vectors demonstrated profound cytocidal effects in the crystal violet and MTS assays. For the in vivo experiments, a single intratumoral injection of virus demonstrated an anti-tumor effect for only one week following injection. Given this limited effect, an additional in vivo experiment was performed to analyze the efficacy of multiple intratumoral injections (3.5 x1011 vp/injection). This setup resulted in statistically significant tumor growth suppression at day 26 when compared to the saline control group (p<0.05 for 5/3 CB016, p<0.01 for 5/3 D24).Conclusion:CRAds designed to target HPV-positive HNSCCs demonstrated tremendous, yet selective in vitro cytocidal effects. The profound tumor growth suppression in a mouse model (particularly by multiple injections) shows the excellent potential of our viruses to be clinically translated as a new therapeutic entity for this emerging disease process.