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Background 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.Interleukin-12 (IL-12), has shown substantial promise in preclinical studies but underperforms in humans, largely due to dose-limiting toxicities (DLTs). To address this, we developed a strategy to localize IL-12 delivery to target bladder tissues through coformulation with chitosan (CS) and intravesical administration. This approach significantly enhances antitumor efficacy while reducing systemic exposure. Success in preclinical research has enabled progression into comparative oncology studies in pet dogs with spontaneous invasive urothelial carcinoma (UC).Methods Client-owned dogs with histologically confirmed invasive UC were enrolled in 3+3 dose escalation study. Canine IL-12 (caIL-12) was produced, purified and validated in house. Enrolled subjects received a co-formulation of increasing doses of caIL-12 in a 1% chitosan acetate solution (CS/caIL-12) via a urinary catheter with a 1 hour dwell. Dogs were monitored for adverse events using modified VCOG-CTCAE criteria. Dissemination of caIL-12 was assessed via serum cytokine measurements.Results As of June 2025, 17 treatments across 6 dose levels have been completed. One DLT, grade 3 diarrhea, was observed at the highest dose level – 4.155mg/m 2 caIL-12 (table 1). Markers of potential toxicity have largely remained within normal ranges (figure 1). Out-of-range values are mostly isolated to individual patients within their respective cohorts and/or individual endpoints among those measured. Similarly, serum cytokine analyses reveal no substantial systemic responses to intravesical CS/IL-12.Conclusions Intravesical CS/caIL-12 treatments have been generally well-tolerated. Five dose levels have been completed and the sixth is in progress. One grade 3 toxicity was observed at the highest dose level, indicating that identification of a maximum tolerated dose may be imminent. Serum cytokine analyses reveal minimal systemic immune responses. Additional dogs will be enrolled to finalize a recommended dose to be used in a planned Phase II activity study.Acknowledgements This research is supported by a grant from the National Cancer Institute (U01CA272258).Ethics Approval Studies involving animals were approved by the IACUC at NC State University (22-229, 25-190).Abstract 855 Table 1Adverse events following intravesical chitosan/interleukin-12 immunotherapy in client-owned dogs with invasive urothelial carcinomaAbstract 855 Figure 1Hematologic and biochemical safety/toxicity marker in dogs following treatment with intravesical CS/caIL-12