HER family plays a significant role in cell growth, survival and differentiation and their mechanism is mediated through signal transduction pathways.In addition ,they are also involved in pathogenesis of cancers such as breast, ovarian, renal, colon and lung carcinomas[23–25].This family consists of four main members naming HER-1, HER-2, HER-3 and HER-4 which are also known as ErbB1, ErbB2, ErbB3 and ErbB4[26].Among all these four members,HER2 is the most important one and its overexpression is the main cause of aggressive forms of cancer and as a consequence poor clinical outcome.HER2 belongs to a tyrosine kinase receptor family consisting of three domains. Other receptors of EGFR family prefer to conform a heterodimerized complex with HER2. [1, 2] This receptor is found to be over expressed in different cancers such as ovarian, breast and gastrointestinal tumors. Breast cancer HER2 positive patients has shown poor prognosis. DNA vaccination procedure is based on designing a special plasmid encoding a specific Antigen [3–8]. These Antigens should be safe and Highly immunogenic[9].These cancer vaccines belongs to the field of cancer immunotherapy and has progressed a lot in recent years. [10] Despite of promising achievements in animal studies such as mice, DNA vaccines were not successful in humans and large animals [11]. Therefore, using chemokines as an important regulator factor lead to immigration of both innate and adaptive cells to the injection site and therfore will increase immunr response to intended antigens [12–14]. The foundation of this study was based on designing a novel recombinant construction with high immunogenicity feature that can be expressed in in-vitro transfection.The Bioinformatic Analysis Tools were used for designing an appropriate immunogenic structure with the ability of expression in host cells.According to the results of previous PhaseII trials of different cancers studies, including non-small-cell lung, ovarian and prostate cancers,combination therapy has indicated more promising results [27–29].In our study different domain of HER family were used. we designed a unique recombinat structure of combined HER2 and HER3.Despite of promising results of DNA vaccination,some molecular approaches have been taken in order to improve the efficacy of vaccination.Using Immunostimulatory propeties of cytokines will affect the function of effector T cells.We used CCL20 according to a study conducted by Weilong Chen in 2018.Several similar studies the same as our plasmid have been also conducted. In order to get the highest amount of gene expression, we used “Gen Script” and tried to balance the GC content of the gene. By Using Bioinformatics Tools, we put HER 2 and HER3 and GFP in designed plasmid.In order to improve expression we used CMV promoter .In some DNA-vaccine based studies also CMV promoter has been applied[30].In a pioneer study conducted by Megan Jo Miller in 2014 the combination immunotherapy targeting different pathways leads to long term effective antitumor immunity in HER expressing cancers. In Megan study different HER combination with monoclonal antibady therapy were evaluated according to previous conducted studies. Fundamental basis of these studies were based on the lack of intrinsic kinase activity of HER3 along with the inability to conform Homodimer. Several previous studies have provided enough evidence for the active HER3 Heterodimers complex with other members of HER family. The efficacy of the constructed plasmid was evaluated through the delivery of Her2-nue plasmid through gene gun and jet injector to BALB-c mice.This protocle was successful and 70% of mice showed protective immunity after 50 days [31].Norell H et al. conducted a pilot clinical trial.In theire studies The recombinant constructions were plasmids encoding a full length signaling deficient version of the oncogene Her2 with the co-administration of GM-CSF and IL-2 and the selected groups were metastatic Her2-expressing breast carcinoma patients treated with trastuzumab.During the long term follow-up, a significant increase of MHC class II restricted T-cell responses were detected[32].
Many methods have been established to improve transfection efficacy[33, 34],but we used Lipofectamine method for transfection.
Two previous conducted studies compare the transfection efficacy through different reagents including jetPEI,lipofectamine 2000 and activated dendrimer(Superfact) and all these methods efficacy were evaluted in order to select the best transfection process.(6)According to experiment conducted by Tao Wang ,transfection carried out by lipofectamin indicated more efficacy.