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Background Canine oral malignant melanoma is an aggressive neoplasm with limited therapeutic options and a median survival of approximately three months in advanced-stage disease. JEN-101 is a protein-engineered interleukin-12 (IL-12) designed for intratumoral delivery and local retention through binding to deposited aluminum hydroxide. Here, we evaluated the safety, tolerability, immunologic, and cytoreductive activity of intratumoral JEN-101 in dogs with spontaneous melanoma. We report findings from the initial weight-based dose-escalation cohort and an expanded cohort where JEN-101 was administered using a tumor volume dosing strategy.Methods The clinical study was approved by the University of Illinois IACUC, conducted at the College of Veterinary Medicine, and all pet owners provided written informed consent. The primary objective was to evaluate safety and tolerability of intratumoral JEN-101 in dogs with advanced-stage malignant melanoma. An initial 3+3 dose-escalation design was employed, testing four dose levels (1, 3, 10, and 20 µg/kg) administered intratumorally every three weeks for up to four cycles. In the absence of dose-limiting toxicity or progressive disease, a second treatment course of four additional cycles was permitted. Following dose-escalation, an expansion cohort using a tumor volume-normalized dosing strategy was implemented to further investigate safety, cytokine exposure, and local immune responses. Clinical monitoring included adverse event reporting and tumor response evaluation. Blood, serum, and tumor biopsies were collected at baseline and predefined timepoints for pharmacokinetics, multiplex cytokine analysis, immune cell profiling, and transcriptomics. Descriptive statistics were used for data analysis.Results Twenty-four dogs were enrolled, and JEN-101 was well tolerated. Documented adverse events included fever, lethargy, and isolated elevated liver enzymes. Treatment-related events were limited to grade 3; no grade 4 events occurred. Nineteen dogs received ≥4 doses, and nine completed all 8 doses. Pharmacokinetic analysis showed dose-related serum cytokine peaks within 8 hours. Weight-based dosing was associated with more consistent systemic exposure to IL-12, IFN-γ, and IL-10 based on AUC measurements. Responding dogs demonstrated increased systemic IFN-γ and IL-10 AUC levels and local recruitment of CD3 + T cells. Increased proinflammatory and antigen-processing gene expression was identified in responding lesions. JEN-101 was well tolerated with evidence of biological and therapeutic activities.Conclusions JEN-101 was safe and biologically active in dogs with advanced melanoma. Weight-based dosing induced a more robust therapeutic response and greater systemic immune activation compared to tumor volume-based delivery. These findings support continued development of anchored IL-12 immunotherapy in canine melanoma and underscore the value of immunocompetent dog models in guiding human clinical trials ( NCT06171750).Acknowledgements We are grateful to the patients, dog owners, and referring veterinarians who participated in the clinical trial. The authors wish to thank Karl Dane Wittrup, Professor at MIT for scientific guidance and design input. The authors also wish to thank Cheryl Kent and Gail Iodice for clinical operations support and Saran Vardhanabhuti for biostatistical support. The authors would like to acknowledge Hui Xu, PhD, Huimin Zhang, and Renee Walker at the Tumor Engineering and Phenotyping Shared Resource (TEP-SR) at the Cancer Center at Illinois for assistance with histology and NanoString experiments. The authors would like to thank Gary McNeil from McNeil Scientific Consulting for protein purification and analytical support, teams at ATUM Bio, Beantown Biotech, Charles River Labs, Ichor Life Sciences, VitroVivo Biotech and Certara for their technical and analytical contributions.Ethics Approval The study protocol for the treatment of advanced melanoma in pet dogs was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign. All pet dog owners provided written informed consent before enrollment in the trial.