PROJECT SUMMARY Invasive bladder cancer is a lethal disease that often requires life-altering surgical removal of the bladder to prevent or limit metastasis. Recent approvals of five checkpoint inhibitors for advanced or refractory bladder cancers demonstrate its responsiveness to immunotherapy. However, less than half of advanced bladder cancer patients benefit from checkpoint immunotherapy and antitumor responses are often transient. Our previous preclinical studies in mice demonstrated that intravesical immunotherapy with interleukin- 12 (IL-12) formulated with the mucoadhesive biopolymer chitosan (CS), i.e., CS/IL-12, can eliminate nearly all established orthotopic bladder tumors in a T cell-dependent manner. These studies also demonstrated that intravesical CS/IL-12 can induce robust abscopal responses with the elimination of distant, untreated bladder tumors in most mice. Although these data are promising, several limitations of implanted murine bladder tumor models have hindered clinical translation. Thus, this application proposes to evaluate the safety and activity of intravesical CS/canine IL-12 (caIL-12) immunotherapy in pet dogs with spontaneous invasive urothelial carcinoma (UC) of the bladder. There are numerous similarities between canine and human bladder cancers including mechanisms of tumorigenesis, rates and sites of metastasis, and distinguishable molecular subtypes. Given these similarities, treatments found to be successful in dogs are more likely to be successful in people. The objectives of this project are: 1) to demonstrate that intravesical CS/caIL-12 immunotherapy can safely induce antitumor immunity against canine invasive UC; and 2) to determine if canine bladder cancer is a useful model for the evaluation of this and other novel immunotherapies. The first objective will help prepare intravesical CS/IL-12 for translation into human clinical trials, while the second objective will help bladder cancer researchers overcome the limitations of rodent models and provide a more faithful representation of human bladder cancer. To accomplish these objectives, 2 specific aims have been designed. Aim 1 is focused on safety, as CS/caIL-12 has never been evaluated in dogs. After synthesizing and validating recombinant caIL-12 in vitro, we will perform a dose-escalation study of intravesical CS/caIL-12 in pet dogs with spontaneous invasive UC of the bladder. Aim 1 will establish a recommended dose (RD) based on safety readouts that utilize a combination of clinical examinations and laboratory tests. Proposed pharmacokinetic and immunophenotyping studies will investigate the possible systemic uptake and dissemination of intravesical immunotherapy and the resulting immune impacts. Aim 2 will assess antitumor and immunological responses to the RD of intravesical CS/caIL-12 in an expanded cohort of dogs with bladder cancer. Correlative studies will determine if intravesical CS/caIL-12 influences T cell infiltration, the tumor- imm...