Abstract
In the last twenty years, interferon-α (IFN-α) has gained success as an immunotherapy for treating such cancers as hairy cell leukemia, malignant melanoma, and renal cell cancer. Our goal was to improve the effectiveness of IFN-α therapy by genetically modified the IFN-α gene to encode a tumor-targeting peptide fused to a functional IFN-α protein. To ensure the targeting peptide worked, a genetically modified reporter gene encoding a secreted alkaline phosphatase (SEAP) gene and different mini-peptides were used to determine distribution and targeting ability. The DNA fragment encoding the most effective peptide was selected to modify the IFN-α gene construct for therapeutic trials. This fusion gene encoded the peptide with the amino acid sequence of C-D-G-R-C, and demonstrated a higher localization of the genetically modified gene product in the tumor local area. Tumor volume and animal survival was measured over several weeks to compare the anti-tumor effects of the IFN-α to CDGRC-IFN-α treatments. Results indicate an increase in therapeutic efficacy due to treatment with the CDGRC-IFN-α gene over the wild-type IFN-α gene. Flow cytometry was performed and it was determined that both of the tumor targeted gene products, CDGRC-SEAP and CNGRC-SEAP share a high affinity for the receptor, Aminopeptidase N (CD13). In order to determine the mechanism responsible for the enhanced anti-tumor effect by CDGRC-IFN-α gene therapy, the T cell infiltration, subsequent CTL activity, and tumor vessel density were confirmed through immunostaining. An increase in number of CD8+ T cells was seen, as well as an increase in activity of cytotoxic T cells. Decreased vessel density in CDGRC-IFN-α treated animals suggest that this therapy enhanced anti-angiogenisis. A high level of non-specific activity was detected in the CTL assay, suggesting involvement of other immune cells, such as NK cells. Overall, this study describes the first example of using a genetically modified immunostimulatory gene encoding tumor-targeted IFN-α for treating tumors. This novel concept may have the potential for increasing therapeutic efficacy of several current cancer treatments.
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