While vaccine strategies for the generation of tumor specific immunity in patients continue to have great promise, to date they have been less than overwhelming in their antitumor efficacy. During the last period of funding supported by this award, our studies utilizing tetramer analysis, functional assessment of tumor associated, lymph node, and systemic populations and the generation of a family of epitope specific vaccines, have resulted in our defining an active immune escape mechanism in which the tumor microenvironment and draining LN manifest a balance between effector and regulatory cells resulting in functional anergy in the periphery. Our underlying hypothesis/strategy is that by modulating the tumor microenvironment we will be able to induce an effective tumor specific systemic response. We will focus on the use of in-situ gene transfer using poxvirus recombinants, as we believe this will provide us an opportunity to immunize to the optimal combination of tumor-associated antigens. Our findings that local immune modulation can lead to peripheral responsiveness has led us to hypothesize that local intervention has the potential to enhance the response to tumor antigen encoding vaccines given in the periphery thus enhancing systemic effector function. Thus, while there is clear agreement that the development of systemic responsiveness is crucial, if what goes on in the tumor microenvironment, as our studies have shown, blocks the development of effective systemic immunity or even worse, sets up an environment where immunization actually hinders the desired response via the expansion of a negative regulatory component as we describe, modulating the tumor-host environment may be critical in inducing effective antitumor immunity. Findings from our preclinical studies supported by this award have been translated into 5 clinical trials establishing intratumoral gene transfer using poxvirus as safe, allowing prolonged expression of encoded transgenes, and as having the ability to change the immune milieu by altering the infiltrate makeup. More specifically, we will:1. Characterize the tumor microenvironment / draining lymph node / and systemic immunity axis as a means of further identifying targets for manipulation using vaccinia virus recombinants: The tumor microenvironment as a factory for systemic unresponsiveness; 2. Evaluate localized modulation of antigen presentation in the tumor-DLN compartment for enhanced antitumor responses; 3. Evaluate localized modulation of Treg and effector functions using receptor-based and antibody-based genetic fusion molecules as adjuncts to vaccines; and 4. Evaluate systemic modulation of tumor-induced regulatory mechanisms using antibodies and small molecules as adjuncts to vaccines. If successful, the studies outlined in this proposal will: 1) Significantly enhance our understanding of the regulation of antitumor immunity in tumor bearing murine models with direct relevance to human host-tumor interactions; and 2) provide a rationally designed vaccine strategy for translation to clinical trial. While vaccine strategies for the generation of tumor specific immunity in patients continue to have great promise, to date they have been less than overwhelming in their antitumor efficacy. While there is clear agreement that the development of systemic responsiveness is crucial, if what goes on in the tumor microenvironment, as our studies have shown, blocks the development of effective systemic immunity or even worse, sets up an environment where immunization actually hinders the desired response via the expansion of a negative regulatory component as we describe, modulating the tumor-host environment may be critical in inducing effective antitumor immunity. If successful, the studies outlined in this proposal will: 1) Significantly enhance our understanding of the regulation of antitumor immunity in tumor bearing murine models with direct relevance to human host-tumor interactions; and 2) provide a rationally designed vaccine strategy, focused on the combined local and systemic immune modulation, for translation to clinical trial.