General Data

Project Phases

The objectives of the project are implemented in work packages (DS), which are divided into individual tasks.

Project description

Immunotherapies, based on the encouraging results of preclinical and initial clinical trials, have greatly influenced cancer treatment guidelines in recent years, especially for immunogenic tumours. In addition to immune-based antibodies, the development of basic cancer research is focused on DNA vaccines and therapies. Significant progress has also been made in the development of plasmid DNA. The most advanced plasmid DNA and electroporation immunotherapy is interleukin-12 (IL -12) immunotherapy, which is already on the priority list to become an orphan drug in the US. Plasmid DNA alone cannot cross the cell membrane, so a physically mediated transfer system, electroporation, is used to transfer the plasmid to target cells. Electroporation is currently used in electrochemotherapy (ECT) (combination of cytotoxic drugs, such as bleomycin or cisplatin, with electroporation). The application of electrical pulses increases the permeability of the cell membrane, allowing increased uptake of the drug. Determination of bleomycin concentration in blood serum or plasma and target tumour tissue in a human clinical trial suggested the possibility of reducing bleomycin dose in elderly patients.

Electroporation can also be used for gene transfer (GET). GET with IL -12 is one of the most advanced immune gene therapies. It has already been shown to be safe and effective in preclinical and clinical studies in dogs with spontaneous tumours. Preclinical studies in mice have demonstrated the induction of long-term antitumor immunity, one of the main effects of GET with IL -12. The antitumor effect of GET IL-12 is multifaceted and includes antiangiogenic effect, activation of cytotoxic T lymphocyte activity, and activation of M1 macrophages, with systemic cytokine concentration lower than recombinant cytokines or adenoviral administration, which means safer treatment. An important advantage of this type of treatment is the low immunogenicity of plasmids compared to viral vectors, which allows repeated DNA application. A clinical trial in the treatment of malignant melanoma metastases in humans has shown that in some cases intratumoral GET with IL -12 can have a systemic "abscopal" effect on distant, untreated tumours. GET IL-12 may also increase the efficacy of standard therapies such as radiotherapy and ECT that cause immunogenic cell death.

The combination of ECT and GET with IL -12 is already used to treat dogs with cancers of various histologies, but many questions remain regarding bleomycin dosing, the site of administration of the plasmid encoding IL -12, and the reasons for individual differences in response to therapy. With the aim of safer and more efficient use of the plasmid encoding canine IL -12, we have already developed a suitable antibiotic resistance gene-free plasmid that complies with the EMA regulations for gene therapy. Although peritumoral administration of the plasmid encoding IL -12 has already been shown to be effective, preclinical studies suggest that intratumoral administration may have an even better antitumor effect, which we would like to evaluate in the proposed project.

The main objective of the proposed project is to optimize the components of the combination therapy in order to improve the efficacy of the treatment and reduce the side effects. To this end, we will establish a protocol for conducting a clinical trial, including a risk assessment in dogs with spontaneous peripheral tumours, in which we expect to determine the pharmacokinetic profile of bleomycin in dogs of different ages and body weights and to determine an appropriate time window for electroporation after cytostatic administration. We also expect GET to be as successful or more successful with intratumoral administration of IL -12 than with peritumoral administration. By determining the expression of selected tumour infiltrating lymphocytes (TLIs) (CD8+, regulatory T lymphocytes (Treg)) and programmed death receptors and ligands (PD-1/ PD-L1) in tissue samples prior to treatment, we will be able to determine the frequency and number of treatments in correlation with clinical data. A better response to treatment is expected in animals in which there are fewer Treg and PD-1 positive cells in the tumour microenvironment, and in animals in which IL -12 GET treatment will result in a decrease in circulating Treg and CD8+ lymphocytes. Prior to treatment, we will also analyze the composition of the intestinal microbiota and, in correlation with the success of the therapy and other biomarkers, we will prepare recommendations for regulating the intestinal microbiota.

Structure of the project group

SICRIS