Spontaneous canine malignant melanoma provides an excellent pre‐clinical model to study DNA vaccines for melanoma immunotherapy. A USDA‐approved xenogeneic human tyrosinase (huTYR) plasmid DNA vaccine delivered intramuscularly induces detectable immune responses and has clinical activity in some dogs with melanoma. The objective of this pilot study was to evaluate the feasibility, safety and immunogenicity of huTYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma. DNA microseeding utilizes a modified tattooing device as an alternate and potentially more potent delivery method for DNA immunization. DNA was delivered to shaved inner thigh skin of six companion dogs with melanoma approximately every 14 days for a planned total of four vaccination time points. An anti‐huTYR ELISA was used to test pre‐ and post‐treatment sera. Biopsies of treated skin were obtained for detection of huTYR transgene expression. DNA microseeding was well tolerated with no significant toxicity detected beyond local site irritation, and there were no signs of autoimmunity. huTYR‐expressing cells were observed in biopsies of huTYR DNA microseeding sites. Increased humoral anti‐huTYR antibodies were seen in two of five evaluable dogs following microseeding compared to baseline. DNA microseeding is well tolerated in companion dogs with melanoma. Further investigation is needed to determine if combining DNA microseeding with other immunotherapy regimens potentiates this delivery platform for cancer immunotherapy.

Background: Canine malignant melanoma provides a model to study DNA vaccine delivery systems. A xenogeneic human tyrosinase (huTYR) DNA vaccine delivered by Biojector2000 received United States Department of Agriculture licensure when it appeared to prolong survival of dogs with melanoma compared to historical, stage-matched controls and to stimulate immune responses in some dogs. The current study evaluates toxicity, transgene expression, and antibody responses to huTYR in companion dogs with spontaneously developing melanoma following delivery of huTYR DNA to the skin via a modified tattoo device, a method termed DNA microseeding. Methods: Five companion dogs with melanoma were scheduled to receive huTYR DNA at two sites (Site A and Site B) on the inner thigh by DNA microseeding every 2 weeks for 4 administrations at a range of huTYR DNA doses; 2 dogs (50 μg [Site A] and 100 μg [Site B]); 2 dogs (200 μg [Site A] and 400 μg [Site B]); and 1 dog (83 μg [Site A] and 83 μg commercial huTYR plasmid [Site B]). Vaccine site biopsies were obtained to determine transgene expression 24 hours after the 1st and 3rd vaccination time-points, and 48 hours after the 2nd and 4th vaccination time-points. Blood samples were obtained at baseline and 2 weeks after the 2nd, 3rd, and 4th vaccinations to quantify TYR-specific antibodies via indirect ELISA. Results: No toxicity, beyond local site irritation, related to the vaccine administration was observed. The 3 dogs with known disease at study entry received 3, 1 and 4 treatments before discontinuation of treatment due to progressive disease. The 2 dogs without evidence of disease at study entry received all 4 planned treatments and remained without evidence for recurrence after treatment for the duration of the study (6 weeks). Only rare huTYR+ cells with macrophage-like morphology were observed in some vaccine site biopsies. A significant increase in anti-huTYR IgG was detected at Day 57 compared to pre-treatment in 2 of the 4 evaluable dogs (p = 0.03 Wilcoxon Mann Whitney), and these were the 2 dogs without evidence of disease at study entry. Baseline anti-huTYR IgG levels were also greater compared to IgG levels against an irrelevant control antigen. Conclusions: While microseeding of huTYR plasmid DNA resulted in only rare transgene expression at DNA doses up to 400 μg, humoral responses against huTYR were substantially boosted in 2 of 4 evaluable dogs. Additional testing is needed to determine if DNA microseeding enhances huTYR DNA vaccine immunogenicity compared to Biojector delivery. Citation Format: Cindy L. Zuleger, Chulhi Kang, Erik A. Ranheim, Ilene Kurzman, Michael D. Macklin, Michael A. Newton, David M. Vail, Jedd D. Wolchok, Elof Eriksson, Mark R. Albertini. Pilot study of DNA microseeding to activate immune rejection of canine melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2366.