The goal of this project is to develop effective molecularly-defined immune adjuvants for active immunization against cancer. Although tolerance to cancer is reversible, immunization against cancer antigens faces substantial hurdles related to tolerance and immune regulation. Potent immune adjuvants will be necessary for successful active immunization against cancer antigens, which are inherently poorly immunogenic. We apply recent knowledge about molecules that regulate growth, activation and survival of innate and acquired immune cells to study adjuvant strategies and underlying mechanisms. We use genetic adjuvants (plasmid DNA incorporating Fc domains of IgG) combined with DNA vaccines against cancer antigens. DNA encoding cytokines have been carefully selected for analysis based on results of extensive preliminary screening and on our understanding about how these molecules regulate activation and survival of innate and acquired immune cells. Specific aim 1 is built on experimental results from our screens showing that DNA encoding an IL-12/Fc fusion molecule is most effective at enhancing T cell responses and tumor immunity. A phase I clinical trial is proposed to assess safety of IL-12/Fc DNA and compare the relative immunologic efficacy of vaccination with tyrosinase DNA plus IL-12/Fc DNA adjuvant to tyrosinase DNA alone in patients with stage III melanoma. Specific aim 2 examines why ligation of Fc domains to cytokines markedly increases adjuvanticity while unexpectedly decreasing their bioavailability. Genetic approaches are used to address the hypothesis that adjuvant effects are amplified by interactions with Fc receptors on innate immune cells. Specific aim 3 studies DNA adjuvants combined with vaccination in treatment models of melanoma, including treatment in a transgenic model that develops de novo melanoma. Subaims investigate a) the role of IFNgamma produced by NK cells for adjuvanticity by cytokine and cytokine/Fc cDNAs, and b) adjuvant effects of IL-23 DNA, which is strongly implicated in the pathogenesis of inflammatory autoimmune diseases. Specific aim 4 investigates the role of cytokine DNA adjuvants for potentiating antigen-specific responses elicited by vaccination during homeostatic recovery of the immune system after non-myeloablative treatments. The overall goal is to develop the most potent DNA adjuvants that can be applied to vaccines delivered during homeostatic recovery.