Incorporation of tumor antigen epitopes into cytokine signal peptides enhance the efficacy of immuno-gene therapy

Immuno-gene therapy is a promising approach for the control of cancer. Because of the essential role in activating and expanding T cell responses to antigens, cytokines are comprehensively studied for cancer therapy. The efficacy of cancer immunotherapy is diminished by the fact that spontaneous tumors often downregulate antigen expression and/or presentation. The density of tumor antigen in conjunction with major histocompatibility complex (MHC) class I molecules on the cell surface affects cytotoxic T cell (CTL) function. In this study, we have developed a novel approach to augment the effect of cytokine-based cancer immuno-gene therapy through the coupling of antigen expression/presentation with cytokine production by expression of antigen epitopes within cytokine signal peptides. We first investigated the possibility of modifying cytokine signal peptides with antigen epitopes without aborting function. We inserted the genes encoding the MHC class I restricted antigenic epitopes of chicken ovalbumin (OVA), tyrosinase related protein 2 (TRP-2), and oncoprotein HER2 into the signal sequence of the interleukin-2 (IL-2) gene, replacing part of the signal sequence at different positions. Our results showed that these modified signal peptides still functioned, as indicated by cytokine secretion. We then demonstrated that an antigen epitope contained within the modified signal peptide could be processed properly and presented on the tumor cell surface. Furthermore, using a murine melanoma model, we studied the effect of antigen epitope modified IL-2 and IFN-gamma on the immuno-gene therapy of malignancy. Tumor cells showed enhanced immunogenicity as indicated by increased susceptibility to CTL lysis in vitro and decreased tumor growth in vivo after gene modification with the antigen epitope-containing cytokine expression vectors. Vaccination with TRP-2 epitope-containing IFN-gamma gene-modified B16 cells resulted in protection against wild type tumor challenge. In addit