## 5 Clinical Studies
The expression of HGF after the procedure reached the appropriate level, and pulmonary fibrosis was reduced, confirming that EP could also be used to repair alveolar wounds and stop the process of pulmonary fibrosis [79]. Regarding immunogen electrotransfer, the most extensively used cytokine gene in GET is IL-12 [80]. Interleukin 12 is a proinflammatory agent, which mainly enhances cytotoxicity of various immune cells, such as NK cells and T lymphocytes. It evokes an IFN\(\gamma\) production and increases the expression of MHC molecules [62]. IL-12 boosts the proliferation of CD8+ cells and reduces PD1 expression [81, 82]. Furthermore, the IL-12 enhances its secretion by dendritic cells, which creates a positive feedback loop [83]. Preclinical studies on IL-12 GET using rodents have been very successful, as 47% of mice with the complete regression of murine malignant melanoma B16.F10 was achieved after IL-12-plasmid intertumoral injection followed by EP. Additionally, five of seven mice remained healthy for 100 days after being challenged with tumor cells [60]. Another research showed that 80% of mice were cured for over 100 days. Furthermore, only 37.5% of mice developed lung nodules, whereas in a control group, these nodules appeared in 87.5%, which shows that the following treatment may prevent metastases [84]. The high effectiveness of IL-12 GET treatment on murine SA-1 fibrosarcoma was also proven, while 90% complete response rate was obtained after using an intertumoral injection and 60% of cured animals remained resistant to tumor cells [85]. IL-12 GET may be combined with other methods, such as an electrotransfer of IL-12 plasmid with IL-2 plasmid. Interleukin 2 is responsible for the proliferation and differentiation of T lymphocytes, such as CD8+ cells. It also constitutes a strong mitogen and may prevent the activation of Treg cells. IL-2 is used in the treatment of renal carcinoma and metastatic melanoma, but in GET therapy, the dosage of this cytokine is much lower, which allows us to avoid the toxic systemic effect. The usage of the described combination in research resulted in an increased concentration of dendric cells and macrophages in the tumor tissue and the development of anti-tumor immune memory. A total of 71% of treated mice obtained the complete regression of the B16.F10 melanoma and 80% rejected tumor cells after secondary challenge [68]. IL-12 can also function as an adjuvant when it is combined with electrochemotherapy because it enhances the antitumor effect of cytotoxic drugs [54, 61, 86]. Electrochemotherapy can induce only a local effect, whereas immunotherapy can contribute to the cure of metastases. IL-12 GET used as an adjuvant is very efficient. It intensifies the response rate and may induce a systemic effect [86]. Interleukin 12 was also used in conjunction with tumor necrosis factor-\(\alpha\) (TNF\(\alpha\)), a cytokine with direct cytotoxic activity. IL-12 and TNF\(\alpha\) GET resulted in 79% of the complete responses of the tumor, which was confirmed by the vast accumulation of immune cells in the tumor [87]. The IL-12 GET was also tested in treating the canine transmissible venereal tumor (CTVT) in beagle dogs. This treatment resulted in complete tumor regression and elicited a systemic response, which led to a cure of distant tumors and prevented the appearance of a new tumor [88]. In conjunction with electrochemotherapy, it resulted in high efficacy in the treatment of mast cell tumors in dogs. The complete response rate was 72% and no side effects were noticed [89]. The phase I trial of IL-12 plasmid in vivo EP was performed on patients with metastatic melanoma. This study resulted in tumor necrosis in 76% of lesions. Treated tumors showed the infiltration of lymphocytes. Notably, 10% of patients obtained the complete regression of all lesions and another 42% demonstrated a partial response. The major side effect was temporary pain after applying electric pulses [90]. Interleukin 12 is not the only mediator used in GET.