BCG therapy consists of weekly installations of a suspension of viable bacteria into the bladder lumen. The BCG suspension is retained for 2 hrs after which the bacteria are eliminated through micturition. The applicant's studies demonstrate that the antitumor response induced by such a treatment regimen requires attachment and retention of the bacteria followed by the induction of antitumor immunity. Attachment is mediated by receptor/ligand interaction with the mycobacterial fibronectin attachment protein (FAP) functioning as the receptor and bladder fibronectin functioning as the ligand. Histochemical studies show that BCG are retained in areas associated with tumor cells and/or sites of epithelial cell disruption. Thus, BCG attachment mechanisms are responsible for focusing the immune response to sites of retention which include tumor foci. In-vivo studies demonstrate a requirement for both CD4+ and CD8+ T cell subsets for the expression of antitumor activity. No tumor specific immune response was observed suggesting that the immune response to BCG is of prime importance. In this regard, bladder tumor cells have been observed to act as antigen presenting cells and to function as target cells for fas-mediated lysis by CD4+ T cells. The implication of these data are that the response to BCG is either directly or indirectly responsible for antitumor activity. It is the applicant's hypothesis that fibronectin-mediated bacterial attachment is required for the delivery of BCG to the tumor site and that T cell immunity directed toward BCG is responsible for the expression of antitumor activity. He proposes to further characterize FAP-mediated fibronectin binding and to characterize the role of CD4+ and CD8+ T cell responses in bladder tumor immunity. To this end, he will clone, sequence, and characterize fibronectin proteins. Characterization will include determining the active binding sites and relative affinities in order to optimize attachment of bacteria to fibronectin. The intent of the characterization is to develop recombinant BCG that exhibit high binding properties in order to enhance immunological activity to BCG by introducing higher antigen loads. Immunological characterization of the BCG-induced immune response will be pursued by developing recombinant BCG expressing known antigens for helper and cytotoxic T cells. These antigens will provide target antigens for characterizing the response in both naive and immune animals using T cell receptor transgenic mice specific for the recombinant antigens. Moreover, the in vitro models will allow analysis of the influence of tumor cells on induction and maintenance of the immune response.
