Scientific background: Spontaneous tumors in dogs represent an excellent model for testing new therapies and for their translation into human oncology. One such (new/novel) therapy is electrochemotherapy, which has proved to be effective in treating tumors in dogs and humans. Electrochemotherapy is a treatment with great success locally, but not systemically. Interleukin 12 (IL-12) electrogenetherapy has proved highly effective in inducing systemic effect in preclinical and clinical studies. IL-12 is an inflammatory cytokine that increases the activity of antitumor immune response through interferon γ (IFNɣ). In clinical studies of a combined electrochemotherapy and IL-12 electrogenetherapy treatment of spontaneous tumors in dogs, good antitumor efficacy has been demonstrated. In these studies plasmids encoding human or feline IL-12, which do not have a complete homology to canine IL-12, were used. In addition, these plasmids carried an ampicillin resistance gene which is disapproved by regulatory agencies. Therefore, the preparation of plasmids encoding canine IL-12 with or without an antibiotic resistance gene is essential for use in further clinical studies on dogs.
Methods: Firstly, plasmids encoding canine IL-12 under a constitutive or a tissue specific promotor with or without the gene for antibiotic resistance were prepared and evaluated. The experiments were carried out in in vitro conditions on human and canine normal and tumor cell lines. The IL-12 expression on mRNA and protein levels was monitored following gene electrotransfer. In in vivo conditions, the antitumor effect of IL-12 gene electrotransfer of canine melanoma cell line xenografts on immunocompromised mice was evaluated. Also we observed the induced immune response after IL-12 gene electrotransfer. Lastly, we registered a clinical study to the competent authorities and began to enroll dogs with spontaneous tumors. Dogs were treated with a combination of electrochemotherapy and IL-12 electrogenetherapy. During the course of the therapy we measured the size of the tumors and took blood, swabs, urine and stool samples to determine the safety and efficacy of the therapy.
Results: The new plasmid encoding canine IL-12 has a similar or a higher level IL-12 expression on mRNA and protein levels in in vitro conditions. In in vivo conditions on immunocompromised mice the therapeutic efficacy after IL-12 gene electrotransfer was found to be comparable between plasmids encoding canine and human IL-12. In the case of plasmids encoding canine IL-12 under the constitutive and the tissue-specific promoter without the antibiotic resistance gene, it has been found that the removal of the antibiotic resistance gene does not reduce the cytotoxicity or the expression level of the transgene. In in vivo conditions, it was found that the plasmid with the constitutive promoter had a higher expression of the transgene than a plasmid with a tissue-specific promoter. In addition, gene electrotransfer of the plasmid with the constitutive promoter caused an increase of serum IL-12, while in the case of the tissue specific promotor, IL-12 expression was limited to the tumor. In the case of the constitutive promoter, an increased infiltration of granzyme B positive cells was observed in the tumor and spleen, indicating a systemic effect of the treatment. Also, an increased infiltration of M1 macrophages and dendritic cells in the tumor was observed after the IL-12 gene electrotransfer. In our clinical study 55 dogs were enrolled, 42 dogs with skin tumors and 13 with oral tumors. In the case of skin tumors 84 % complete responses were achieved, whereas in oral tumors only 15 % complete responses were achieved. When screening for the presence of plasmid DNA, no plasmid DNA was detected in the stool, urine and serum samples. Plasmid DNA was present in skin swabs in some patients. The highest concentration of plasmid DNA (100 ng/mL) was detected immediately after the therapy, but after 4 weeks no plasmid DNA was detected. Lastly, the percentage of immune cells was measured after the therapy. We observed a statistically significant decline in the percentage of regulatory T cells at the end of the observation period compared to the time of the treatment.
Conclusion: The new plasmid encoding canine IL-12 with kanamycin resistance gene has the same or a higher expression level of IL-12 and comparable antitumor effect than the plasmid encoding human IL-12. The use of a new plasmid in combination with electrochemotherapy for the treatment of spontaneous skin and oral tumors in dogs has proved to be safe and efficient. Additionally, newly prepared plasmids encoding canine IL-12 under the constitutive or the tissue-specific promoter without antibiotic resistance genes serves as an effective tool for further clinical studies, which are indispensable for translating this therapy into human medicine.
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