Background Malignant melanoma is a common oral cancer in dogs with a 3-month median survival for advanced stage disease. Inflammatory cytokines exert potent anticancer activities, but are limited by their systemic toxicity. Intratumoral JEN-101 is a protein-engineered, locally-retained interleukin-12 (IL-12) cytokine that binds to aluminum hydroxide. Here, a preclinical study was conducted to determine safety profile, tolerability, cytoreductive capacity, and immune responses of intratumoral JEN-101 in dogs with melanoma.

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 of the trial was to determine the safety and tolerability of JEN-101 in dogs with advanced melanoma. A 3+3 dose-escalation design with four dose levels (1, 3, 10, and 20 µg/kg) of JEN-101 injected intratumorally every three weeks for 4 cycles was evaluated in pet dogs. In the absence of toxicity or clinical progression, dogs could receive a second course of 4 cycles. Dogs were monitored for adverse events and clinical responses. Peripheral blood, serum, and tumor biopsies were collected at baseline and at pre-specified timepoints for pharmacokinetic and immune analyses, which included serum cytokines, immune cell phenotype, and gene expression assessments. Descriptive statistics were used for analyses.

Results Eighteen dogs were enrolled and JEN-101 was well tolerated. Documented adverse events included fever, lethargy, and isolated elevated liver enzymes. Five dogs experienced grade 3 events and no grade 4 events were observed. Thirteen dogs completed at least 4 doses of JEN-101 and seven dogs completed the full 8 doses. Pharmacokinetic analysis revealed a trend towards C_max within 8 hours of injection and maximum median serum IL-12 concentration was 1.1 ng/mL at the highest dose. An increase in serum interferon-g and IL-10 within 8 hours of injection was seen, and responding dogs demonstrated decreased serum IL-10, recruitment of local CD3⁺ T cells, and increased pro-inflammatory and antigen processing gene expressions were identified in responding lesions.

Conclusions JEN-101 was well tolerated in dogs with advanced melanoma with evidence of biological and therapeutic activity. The potential of anchored IL-12 immunotherapy in dogs with melanoma merits further investigation in dogs with melanoma and our approach represents an immune competent model to inform an ongoing human clinical trial (NCT06171750).

Acknowledgements We are grateful to the patients, dog owners, and referring veterinarians who participated in the clinical trial. The authors wish to thank Dane Wittrup, PhD at MIT for scientific guidance and design input. The authors also wish to thank Cheryl Kent and Gail Iodice for clinical operations support and Heather Kelley 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) 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.