221. The Utilization of Capsid-Optimized Adeno-Associated Virus (AAV) Vectors for Cancer Immunotherapy

Abstract

Cancer, a leading cause of death in the human population today, is generally being treated with chemotherapeutic methods, which have adverse toxic side effects. Immunotherapy represents an attractive alternative to common treatment since it is based on activation of host immune system.In current studies we assessed the ability the of AAV vectors expressing antigen to activate a specific T-cell response in vivo. The ovarian albumin (OVA), commonly used in mouse models as an antigen for immunization studies was used to evaluate immune response. AAV6-WT-OVA and capsid-optimized AAV6-S662V+T492V-OVA vectors were injected intramuscularly in C57BL/6 mice with a dose of 5×10e11 vgs/mouse. Enchased green fluorescent protein (AAV6-EGFP) was used as negative control. In two weeks after injection, blood was collected and a number of OVA-specific T-cells were analyzed by stain with iTAg MHC Class I Murine Tetramer. The data shown suggest that the administration of AAV vectors expressing OVA led to a robust activation (approximately 9%) of specific T-cells compared to the mock and AAV-WT treated animals (less than 1%).Next, we evaluated the killing ability of these OVA specific T-cell. Splenocytes from C57BL/6 mice i.m. injected with AAV6-WT and AAV6-mutant vectors were isolated in 14 days after injection. OVA-CD8 cells were expanded in vitro in the presence of a predominant for C57BL/6 mice, SIINFEKL peptide. Stimulated T-cells were used for killing assay against mouse prostate cell line RM1 stably expressing OVA. Two-color fluorescence assay of cell-mediated cytotoxicity was used as described above to estimate percentage of dead/alive target cells and generate a killing curve with different effectors to target cell ratio. Results of these experiments suggest that OVA-CD8 cells isolated from mice injected with AAV-S662V+T492V-OVA vectors have higher killing activity compared with OVA-CD8 cells from AAV-WT-OVA injected mice. Control T-cell do not show significant cytotoxicity which eliminates the possibility of autoreactive response.Finally, we evaluated the ability of capsid-optimized AAV6 vectors to initiate a protective anti-cancer immune response. Prostatic acid phosphatase (PAP), a gene up regulated in both human and mouse prostate cancer, was used as a specific target. C57BL/6 mouse subcutaneously injected prostate cancer cell line, RM1, was used as an animal model. We genetically modified this cell line for a stable expression of firefly luciferase (FLuc) to monitor progression of tumor growth in live animals. First, animals were immunized with AAV6-S663V+T492-PAP or AAV6-WT-PAP vectors. Two weeks later mice were challenged with RM1-FLuc cells by subcutaneous injection. The effect of treatment on tumor growth was evaluated by whole body life imaging. Results indicate suppression of tumor growth by AAV6-S663V+T492-PAP for approximately four weeks in comparison to one week and two weeks for negative control AAV6-GFP and AAV6-WT-PAP treated mice, respectively.In conclusion, successful inhibition of tumor growth in this artificial animal model would set the stage for potential clinical application.