In current studies we assessed the ability the of capsid-optimized adeno-associated virus (AAV) vectors expressing antigen to activate a specific T-cell response in vivo. We have showed that the efficacy of wild-type (WT) AAV vectors can be significantly enhanced by substituting critical serine (S) and threonine (T) residues on their capsids to valine (V). These residues were identified by analysis of the AAV capsid crystal structure and they can be recognized and phosphorylated by common serine/threonine cellular kinases such as JNK and p38 MAPK. Several different amino acids were tested and (V) was chosen because of the similarity of its structure with both (S) and (T), and lack of recognition by kinases. Thus, these modifications can prevent kinase-mediated phosphorylation of the AAV capsid, subsequent ubiquitination and proteasome-mediated degradation of the vectors. Next, the ovarian albumin (OVA), commonly used in mouse models as an antigen for immunization studies was used to evaluate ability of these vectors to initiate immune response. AAV6-WT-OVA and capsid-optimized AAV6-S662V+T492V-OVA vectors were injected intramuscularly in C57BL/6 mice with a dose of 5x10e11 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 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 isolated from splenocytes of C57BL/6 mice i.m. injected with AAV6-WT and AAV6-mutant vectors. Two-color fluorescence assay of cell-mediated cytotoxicity was used 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 approximately 2-fold higher killing activity compared with OVA-CD8 cells from AAV-WT-OVA injected mice.
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 reduction or 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. 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.
Citation Format: Munjal Pandy, Kellee Britt, George Aslanidi. Reprogramming immune response with capsid-optimized AAV vectors for immunotherapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A072.
- ©2016 American Association for Cancer Research.