Adenoviral-encoded GMCSF-CAIX antigen fusion gene transfer into human peripheral blood monocyte-derived dendritic cells to induce antigen-specific cellular immune responses: preclinical studies

2871 Metastatic renal cell carcinoma (mRCC) is resistant to conventional modes of therapy. Carbonic anhydrase IX (CAIX), a cell surface tumor-associated antigen, is expressed by most clear cell renal cell carcinomas (RCC) and has implications in RCC diagnosis, prognosis, and response to immunotherapy, and represents a novel approach to tumor-specific targeted therapy. Adenoviral-mediated, tumor-associated antigen transfer is a promising strategy to manipulate the immunostimulatory properties of DCs to create tumor vaccines.
 In the present study, we constructed a recombinant adenovirus encoding GMCSF-CAIX fusion gene (Ad-GMCAIX) using the AdEasy system. Human immature dendritic cells (DCs) were generated from CD14+ monocycles derived from peripheral blood mononuclear cells (PBMC) of healthy donors. DCs were infected with Ad-GMCAIX to raise specific CTLs responses from autologous peripheral blood lymphocytes. The expression level of CAIX in the DCs was confirmed by RT-PCR, flow cytometric assay and western blot analysis. Lymphocytes from the same donors were repeatedly stimulated by genetically modified DCs to generate CAIX-specific cytotoxic T lymphocytes (CTLs). ELISA was used to measure the secretion of IFN-gamma in the supernatants containing effectors cells stimulated with modified DCs. The ability of CTLs to lyse target cells expressing CAIX was performed in vitro by quantitative measurements of release of lactate dehydrogenase (LDH). Our results demonstrated that DCs were efficiently transduced with Ad-GMCAIX and the expression of CAIX was significantly increased. The Ad-GMCAIX significantly increased the number of mature DCs via up-regulation of CD83, CD86 and HLA-DR as compared to unstimulated DCs. Ad-GMCAIX transduced DCs were able to induce CAIX -specific CTLs against RCC cancer cells expressing CAIX in vitro . Moreover, the DCs modified by Ad-GMCAIX produced higher levels of interferon gamma when compared with unstitumated DCs.
 Our results suggest that Ad-GMCAIX critically alters DC immune function and leads to the generation of CAIX specific CTLs, critical to the development of CAIX-based immunotherapy strategies. This finding demonstrates the exciting potential of CAIX to serve as an effective treatment modality for advanced RCC patients. These studies form the foundation for a planned clinical trial using dendritic cells pulsed with GMCAIX in patients with advanced kidney cancer.