Glycosylphosphatidylinositol (GPI) anchored interleukin 2 (IL-2) and interleukin 12 (IL-12) cloning, expression and application in melanoma immunotherapy

Cancer immunotherapy using cytokines such as interleukin 2 (IL-2) and{09}interleukin 12 (IL-12) has shown encouraging anti-tumor effectiveness. However, the severe adverse side effects associated with the systemic high dose administration of these cytokines have hampered their clinical applications. Much research has been undertaken to overcome this problem. In this study, we developed a novel approach to target cytokines directly to tumors using glycosylphosphatidylinositol (GPI) anchoring technology, with which a GPI anchoring signal sequence is genetically added to the C-terminus of the cytokine protein. As a result, the expressed proteins are anchored onto the cell surface so that a local high dose of the cytokines can be achieved without severe side effects.; The first cytokine we chose to test this approach was IL-2. By using DNA cloning technique, a fusion gene consisting of human IL-2 cDNA and a GPI anchoring signal sequence from decay accelerating factor (DAF) was constructed. When transfected into B16F0 murine melanoma tumor cells, the fusion protein was expressed on cell surface with GPI anchors and functions as indicated by ELISA assay and T cell infiltration analysis. In vivo tumor studies demonstrated that GPI anchored IL-2 significantly inhibits tumor growth when compared with free IL-2, but without the severe side effects.; We then extended this technology to IL-12. As expected, GPI anchored IL-12 showed its biological activity. More importantly, when GPI anchored IL-2 and GPI anchored IL-12 co-existed, a significant synergistic anti-tumor effect was observed. Indeed, 190% more T cells were infiltrated into B16F0 tumors expressing both GPI anchored IL-2 and GPI anchored IL-12 than in regular B16F0 tumors, while B16F0 tumors expressing GPI anchored IL-2 alone or GPI anchored IL-12 alone infiltrated only 33% and 28% more T cells, respectively, than regular B16FO tumors. The synergistic anti-tumor effect was also observed in both tumor growth and animal survival studies.; GPI anchored cytokines represent a promising new development in cytokine mediated immunotherapy. GPI anchored cytokines, in combination with gene therapy, have the potential of being a useful clinical practice in fighting cancer without the severe adverse side effects.