PROJECT SUMMARY/ABSTRACT The standard of care for most patients with non-muscle-invasive bladder cancer (NMIBC) is cystoscopic resection followed by intravesical therapy with bacillus Calmette-Guérin (BCG). Outcomes for patients treated with BCG are comparable to those of patients treated with radical cystectomy, and are superior to intravesical chemotherapy; however, there is still a substantial risk of recurrence of bladder cancer among BCG-treated patients. Currently, there are no reliable methods to predict an individual patient's outcome. The mechanism of action of BCG therapy for bladder cancer remains an area of active investigation. Using the MB49 orthotopic mouse model of BCG therapy, we have compiled data demonstrating that BCG elicits an immune response to tumor-specific antigens. Mice cured of MB49 bladder cancer by BCG therapy were specifically resistant to a subsequent challenge with subcutaneous MB49 tumors. This resistance was not seen in mice that received intravesical BCG therapy in the absence of a bladder tumor. Furthermore, adoptive transfer of T-cells from mice surviving MB49 bladder cancer after BCG therapy conferred a survival advantage to naïve mice instilled with intravesical MB49, but there was no advantage using T-cells from mice who received intravesical BCG therapy in the absence of a bladder tumor. These results strongly suggest that an initial inflammatory reaction invoked by BCG results in T-cell-dependent tumor immunity and put forward the possibility that interventions to enhance tumor-specific immunity should enhance BCG efficacy. Based on these data, we propose a model of BCG efficacy with the following components: A) The first phase of BCG therapy involves engulfment of BCG by tumor cells, leading to local inflammation and immune-mediated tumor cell lysis. B) Tumor cell lysis in the context of BCG-induced inflammation primes a T-cell-dependent, neoantigen-directed immune response that results in elimination of tumor cells in the bladder and subsequent tumor immunity. We will test the hypotheses generated by this model to identify strategies to enhance the efficacy of BCG therapy by: 1) testing BCG strains with enhanced capacity to infect bladder tumor cells, enhanced or impaired survival within bladder cancer cells, or enhanced ability to induce an inflammatory response in a mouse model of bladder cancer; 2) determining the magnitude and diversity of the BCG-induced tumor neoepitope-specific T-cell response and testing whether neoepitope vaccination can enhance BCG-induced tumor elimination in a mouse model of bladder cancer. We will also attempt to develop a clinically useful tool to predict an individual patient's likelihood of therapeutic response to BCG therapy by determining whether mutational or neoantigen load predict the therapeutic efficacy of BCG in human patients with NMIBC.