P70 Construction and studying expression in eukaryotic cells of allel-specific HLA-A∗0201 DNA-vaccine against breast cancer

BackgroundBreast cancer ranks the first among female oncological diseases and ranks the second after lung cancer among causes of cancer related deaths. Use of contemporary anticancer drugs significantly enhanced possibilities of cancer therapy. However, most of medicines against cancer have such disadvantages as toxicity, narrowness of therapeutic action, and emergence of drug resistance. Recently, an approach based on potential of dendritic cells (DCs) to induce formation of cytotoxic T-cell immune response against cancer cells is widely used. Priming of dendritic cells by DNA-constructs encoding immunogenic epitopes of tumor antigens is regarded as a rather promising approach to design cancer vaccines. The aim of this study is to construct, obtain and study expression in eukaryotic cells of recombinant plasmid – candidate DNA-vaccines inducing T-cell immune response against breast cancer.Materials and methodsUsing the PolyCTLDesigner software, DNA-vaccine construct was made encoding polyepitope T-cell immunogen BC-A∗0201. Gene encoding target immunogen was synthesized and cloned in the composition of plasmid pcDNA3.1 that is eukaryotic expression vector. Accuracy of gene insertion encoding polyepitopeimmunogen was verified using restriction analysis. Obtained target plasmid pBC-A∗0201 – candidate DNA-vaccine was used for evaluation of gene expression in transfected eukaryotic cells 293T and dendritic cells by means of flow cytofluorometry and immunohistochemistry using MAT to p24 epitope-marker inserted into the structure of target plasmid. Dendritic cells were obtained from peripheral blood precursors by 2-h plastic adhesion. The nonadherent cells were removed 2h later and the adherent monolayer was cultivated in the medium containing growth factors (GM-CSF and IL-4) according to (Naik, 2010; Ganul and Khranovskaya, 2012). Transfection of immature DCs (iDC) was performed by using the reagents purchased from Promokine. Further cultivation of iDCs was carried out in the medium containing TNF- α to obtain fully mature DCs (mDC). Analysis of mDCsurface markers expression was made using MAO (HLA-DR, CD83, CD86) and flow cytofluorometer (BD FACSCalibur).ResultsPlasmid pBC-A∗0201 encoding epitopes of breast cancer antigens was constructed and produced in preparative amount. Using the method of intracellular staining of product of expression by specific MAO 29F2 to epitope p24, we showed expression of gene BC-A∗0201 in eukaryotic cells 293T and dendritic cells transfected by plasmid pBC-A∗0201. Maturity of dendritic cells was also proved.ConclusionThus, obtained candidate DNA-vaccine encoding target polyepitopeimmunogen provides synthesis of relevant protein in the eukaryotic cells culture. In future, we plan to study biological activity of DNA-vaccine to evaluate level of induction of cytotoxic response against SKBR3 breast cancer cells