Generation of Human Interferon Gamma and Tumor Necrosis Factor Alpha Chimeric TNT-3 Fusion Proteins

Abstract

Studies have shown that cytokines can effectively treat solid tumors by a direct cytotoxic effect as well as by immunomodulation. Both human interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) have been used to treat a variety of colon carcinoma cell lines and tumors in patients. These cytokines, however, are dose limited by their toxicity and fast clearance rates when given intravenously. To improve their therapeutic value, we now report on the generation of two new fusion proteins consisting of human IFNγ and TNFα genetically linked to the C-terminal portion of chTNT-3, a monoclonal antibody (MAb), which targets human solid tumors by binding to intracellular antigens exposed in degenerating cells associated with tumor necrosis. In vitro characterization studies demonstrate that both the IFNγ and TNFα fusion proteins are able to maintain their binding affinity to antigen as well as their direct cytotoxic effect and immunomodulatory functions. When both fusion proteins are combined at optimal doses, they demonstrate a 30% direct cellular cytotoxicity of human colon carcinoma cells of which approximately 14% can be attributed to apoptosis. In vivo, these agents were studied for their pharmakocinetic clearance rates and their ability to target human colon carcinomas heterotransplanted in nude mice. The results of these studies show that, compared with chTNT-3 parental antibody, both fusion proteins have a substantially shorter whole body half-life, yet are able to target tumor in a similar manner. As each of these fusion proteins are cleared from the circulation and normal tissues, tumor-to-normal-tissues ratios rise demonstrating the retention of these reagents in tumor. The generation of long-acting and targeted human IFNγ and TNFα antibody fusion proteins will enable investigators to study the role of these potent immunostimulatory cytokines in the treatment of human solid tumors.