118. Efficient Infection of Head and Neck Cancer Cells by Ad5/35 Hybrid Virus

Abstract

Top of pageAbstract Most adenoviral vectors that are currently being employed as gene delivery vehicles for cancer gene therapy are derivatives of adenovirus serotype 5 (Ad5). These vectors enter target cells via two specific interactions: between the viral fiber knob and the coxsackievirus and adenovirus receptor (CAR) and the penton base and integrins αVβ3 and αVβ5. However, adenovirus-mediated gene transfer to many important gene therapy targets is limited due to the low expression levels of CAR. Redirecting Ad5-based vectors to other, preferentially target specific cellular receptors and simultaneously abrogating the interaction between the viral capsid proteins and their cellular counterparts, has thus become a major goal in gene therapy. Group B adenovirus-based vectors have recently gained interest as attractive gene delivery vehicles. Subgroup B2 adenoviruses, including Ad35, infect cells in a CAR-independent manner and may thus transduce cells that are resistant to Ad5 infection. Notably, human CD46 was recently identified as the cellular attachment receptor for these viruses. Squamous cell carcinoma of the head and neck (HNSCC) has several characteristics that make it a suitable target for gene therapy. Most HNSCC are localized and thus easily accessible by direct intratumoral injection. In addition, metastases of the tumor are a late-stage occurrence and therefore early loco-regional control of the disease is paramount. On the basis of these favorable characteristics, gene delivery strategies that utilize adenoviruses have been developed for HNSCC. However, effective in vivo transduction has been a limiting factor in the initial clinical trials using standard Ad5-based vectors for this disease. In order to determine the feasibility of adenoviral retargeting to head and neck cancer, primary HNSCC cells collected from cancer patients were used. Full clinical as well as histological data is available on these cells. Our retargeting scheme was based on the Ad5/35 hybrid system, where the serotype 5 fiber has been replaced with the fiber of serotype 35. Flow cytometric analyses demonstrated that the level of CD46 expression was uniformly high in all primary cells studied (85–99%, average positive ratio 96%). In contrast, CAR expression was clearly lower and also highly variable (1.6–62%, average positive ratio 30%). αV integrin expression was between 39–98% with an average expression of 82%. In situ stainings for b-galactosidase gene expression demonstrated that Ad5/35.lacZ was clearly more effective than Ad5.lacZ in transducing primary HNSCC cells, and quantification of b-galactosidase expression revealed 3 to 58-fold differences in transduction efficacy between the two viruses. As expected, the infection efficacies of the viruses correlated with the receptor expression levels. When the viral transduction and receptor expression data was compared against the clinical data, the difference between the efficacies of the two viruses was found to be clearest in tumors with a high degree of malignancy, although the trend was not statistically significant. These data suggest that the group B2 adenovirus cell entry pathway is highly efficient in transducing HNSCC cells. These results warrant further development of the Ad5/35 hybrid system for head and neck cancer.