P61

Melanoma is the most aggressive and dangerous skin cancer. In 2013 about 23.8% of patients developed III–IV stages of a disease in Russia (http://clinical-pharmacy.ru). The most common approaches to control melanoma are surgical resection, chemotherapy, radiotherapy, and immunotherapy (Wildemore et al., 2001). In the case of immunotherapy interferon preparations are the most commonly used, however, it is impossible to increase overall survival rate. Vaccine therapy is more promising method including engineered DNA-vaccines. The goal of this work is to design and study candidate DNA-vaccines against melanoma pMEL-TCI-A0201 and pMEL-TCI in vitro and ex vivo. Using original software we designed two T-cell immunogens MEL-TCI-A0201 and MEL-TCI which are candidate DNA-vaccines against melanoma. The first allele-specific immunogen MEL-TCI-A0201 comprises CTL-epitopes restricted by only one allelic variant HLA-A∗0201 that is prevailing in global population. The second universal immunogen MEL-TCI comprises CTL-epitopes restricted by 12 major allelic variants of HLA class I molecules. Furthermore, DNA-construct pcDNA-mart1 carrying full-length variant of antigen MART-1 was designed. This construct was used as a positive control. Genes encoding target immunogens have been synthesized and cloned into vector plasmid DNA3.1 (Antonets et al., 2010, Bazhan et al., 2010). Evidence of the target genes expression in vitro after transfection of 293T-cells with obtained DNA-vaccine constructs p MEL-TCI and pMEL-TCI-A0201 were evaluated using three methods: reverse transcription-PCR to determine specific polyepitope immunogen mRNA; western immunoblotting assay with 29F2 monoclonal antibodies to “marker” epitope at the C-terminus of the polyepitope constructs; flow cytometry assay of stained 293T cells transfected with obtained plasmids using FITC-labeled 29F2 monoclonal antibodies. It was shown that all obtained plasmids were provided expression of target genes in eukaryotic cells. Plasmids pMEL-TCI and pMEL-TCI-A0201 were used for transfection of human monocyte derived dendritic cells to estimate capacity of polyepitope constructs to induce specific T-cell immune response by IFNγ ELISpot analysis ex vivo. The study was carried out in collaboration with researchers from laboratory of molecular immunology at Scientific Research Institute of Clinical immunology, Siberian Branch, Russian Academy of Medical Sciences. License for conducting the study was approved by the local ethic committee at this institute. Cell culture of human melanoma Mel Is (HLA-A∗0201+, MART-1+) was kindly provided by N. N. Blokhin Russian Cancer Research Center (RAMS) and was used as target cells. Monocyte suspension and suspension of co-incubated monocytes with dendritic cells transfected with vector plasmid DNA3.1 were used as negative controls. Co-incubated monocytes with dendritic cells transfected with plasmid pcDNA-mart1, encoding full-length melanoma antigen MART-1, were used as positive control. It was shown that in the presence of melanoma cell lysates co-incubated with monocytes and dendritic cells transfected with plasmids pMEL-TCI-A0201 and pcDNA-mart1 (positive control) reliably more IFNγ-producing cells are detected compared with negative control samples in the presence of lysate of melanoma cells. Thus, it was shown that obtained plasmids pMEL-TCI-A0201, pMEL-TCI, and pcDNA-mart1 are able to provide expression of target proteins in eukaryotic cells. Moreover using IFNγ ELISpot assay we demonstrated that DNA vaccine pMEL-TCI-A0201 induced T-cell immune response against human melanoma antigens and its efficiency is equal to full-length melanoma antigen MART-1. This study was supported by Federal Program “Research and development on priority directions of scientific-technological complex of Russia for 2007–2012”, State contrast No. 16.512.11.2186