Abstract Background Immune therapies are currently under intensive investigation providing in many cases excellent responses in different tumors. Other possible approach for immunotherapy is a targeted intratumoral delivery of interleukin 12 (IL-12), a cytokine with anti-tumor effectiveness. Due to its immunomodulatory action, it can be used as an imunostimulating component to in situ vaccinating effect of local ablative therapies. We have developed a phIL12 plasmid devoid of antibiotic resistance marker with a transgene for human IL-12 p70 protein. The plasmid can be delivered intratumorally by gene electrotransfer (GET). Patients and methods Here we present a first-in-human clinical trial protocol for phIL12 GET (ISRCTN15479959, ClinicalTrials NCT05077033). The study is aimed at evaluating the safety and tolerability of phIL12 GET in treatment of basal cell carcinomas in patients with operable tumors in the head and neck region. The study is designed as an exploratory, dose escalating study with the aim to determine the safety and tolerability of the treatment and to identify the dose of plasmid phIL12 that is safe and elicits its biological activity. Conclusions The results of this trail protocol will therefore provide the basis for the use of phIL12 GET as an adjuvant treatment to local ablative therapies, to potentially increase their local and elicit a systemic response.

/pdf/treatment-of-skin-tumors-with-intratumoral-interleukin-12-3jdaeco7.pdf

Treatment of Skin Tumors with Intratumoral Interleukin 12 Gene Electrotransfer in the Head and Neck Region: A First-in-human Clinical Trial Protocol

Electroporation (EP) allows for the transport of molecules into the cytoplasm with significant effectiveness by forming transient pores in the cell membrane using electric pulses. This can be used for cellular transport (RE—reversible electroporation) or ablation (IRE—irreversible electroporation). The first of described options fortifies medicine with novel possibilities: electrochemotherapy (ECT), which creates promising perspectives for cancer treatment, and gene electrotransfer (GET), a powerful method of DNA delivery as well as immunogen electrotransfer. The review constitutes a comprehensive explanation of the mechanism of EP in the case of GET, its present and prospective employment in medicine, including gene delivery, vaccinations, therapy, and transfection, are also presented.

/pdf/electroporation-in-clinical-applications-the-potential-of-18fhr688.pdf

Electroporation in Clinical Applications—The Potential of Gene Electrotransfer and Electrochemotherapy

http://www.cell.com/article/S2405844022001670/pdf

Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues

Expression of GFP and DsRed fluorescent proteins after gene electrotransfer of tumour cells in vitro.

Introduction In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca2+, causing the induction of cell death. The effectiveness of CaEP has already been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT. Methods The effect of CaEP was tested in vitro as well as in vivo in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells. Results In vitro, CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed in vivo; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature. Conclusions Mice bearing 4T1 tumors responded better to CaEP in vivo than mice bearing B16-F10 tumors, even though a similar response was observed in vitro. Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.

https://www.frontiersin.org/articles/10.3389/fimmu.2023.1189960/pdf

The effectiveness of calcium electroporation combined with gene electrotransfer of a plasmid encoding IL-12 is tumor type-dependent

Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans.

Matjaz Peterka

Papers

Non-Clinical In Vitro Evaluation of Antibiotic Resistance Gene-Free Plasmids Encoding Human or Murine IL-12 Intended for First-in-Human Clinical Study