The long-term goal of this product development plan is to develop a recombinant bacille Calmette-Gu[unreadable]rin (rBCG) strain that displays reduced toxicity and improved cancer immunotherapeutic properties. We believe that we can achieve this goal by altering both the tissue-binding and proinflammatory properties of BCG. The central hypothesis of this project is that genetic manipulation of BCG to enhance factors associated with antitumor activity will improve the safety and potency of this biologic. To address this hypothesis we propose to complete the studies in the following specific aims: Specific Aim 1 :- Construction of rBCG that constitutively expresses a tissue adherence factor - The objective of this aim is to produce an rBCG that constitutively over-expresses fibronectin attachment protein, FapB. This protein and the attachment to fibronectin were shown to be necessary for BCG-mediated protection against bladder cancer in animal models. Strains that carry a recombinant constitutive FapB expression cassette will be verified genotypically and phenotypically. Specific Aim 2 :- Construction of rBCG with altered immunostimulatory properties - The objective of this aim is to produce rBCG strains that express the RD1 region, which has been shown to increase the immunostimulatory properties of BCG, and a pro-apoptosis factor, which we predict will increase antitumor activity and ameliorate the reactogenicity of BCG. BCG strains expressing RD1, a pro- apoptosis factor, or both will be validated genotypically and phenotypically. This project will be milestone-driven and at the conclusion of each milestone the Senior Executive Team (SET) will review the results and recommend one of the following decisions: Go: proceed to the next stage; Redirect: based on incomplete deliverables, changed objectives or strategy; No-go: terminate the project. In addition to the milestone review, the SET will also review plans for continuation activities. Overall, we believe that these studies will result in the development of an improved bladder cancer therapeutic and contribute to our understanding of the BCG-host interaction in the development of protection against bladder cancer. [unreadable] [unreadable]