Bioengineering / Biyomühendislik
Permanent URI for this collectionhttps://hdl.handle.net/11147/4529
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Article Citation - WoS: 12Citation - Scopus: 12Immunogenicity of a Xenogeneic Multi-Epitope Her2+ Breast Cancer Dna Vaccine Targeting the Dendritic Cell Restricted Antigen-Uptake Receptor Dec205(Elsevier, 2022) Gül, Ayten; Döşkaya, Mert; Can, Hüseyin; Karakavuk, Muhammet; Anıl İnevi, Müge; Sağlam Metiner, Pelin; Atalay Sahar, EsraBreast cancer was ranked first in global cancer incidence in 2020, and HER2 overexpression in breast cancer accounts for 20–30% of breast cancer patients. Current therapeutic strategies increase the survival rate, but resistance to them occurs frequently, and there is an urgent need to develop novel treatments such as DNA vaccines which can induce a specific and long-lasting immune response against HER2 antigens. To enhance the immunogenicity of DNA vaccines, dendritic cells (DCs) can be targeted using multi-epitope proteins that provide accurate immune focusing. For this purpose, we generated a DNA vaccine encoding a fusion protein composed of 1) in silico discovered antigenic epitopes of human and rat HER2 proteins (MeHer2) and 2) a single-chain antibody fragment (ScFv) specific for the DC-restricted antigen-uptake receptor DEC205 (ScFvDEC). The xenogeneic multi-epitope DNA vaccine (pMeHer2) encodes three only T-cell epitopes, two only B-cell epitopes, and two T and B cell epitopes, and pScFvDEC-MeHer2 vaccine additionally encodes ScFvDEC introduced at the N terminus of the MeHer2. Then, mouse groups were immunized with pScFvDEC-MeHer2, pMeHer2, pScFvDEC, pEmpty, and PBS to determine the elicited immune response. pScFvDEC-MeHer2 vaccinated mice showed a strong IgG response (P < 0.0001) and pScFvDEC-MeHer2 induced a significant IgG2a increase (P < 0.01). The percentages of both IFN-γ secreting CD4 and CD8 T cells were higher in mice immunized with pScFvDEC-MeHer2 compared with the pMeHer2. pScFvDEC-MeHer2 and pMeHer2 secreted significantly higher levels of extracellular IFN-γ compared with to control groups (P < 0.0001). In addition, the IFN-γ level of the pScFvDEC-MeHer2 vaccine group was approximately two times higher than the pMeHer2 group (P < 0.0001). Overall, this study identified the pScFvDECMeHer2 construct as a potential DNA vaccine candidate, supporting further studies to be conducted on HER2+ animal models.Article Citation - WoS: 14Citation - Scopus: 15Development and Verification of a Three-Dimensional (3d) Breast Cancer Tumor Model Composed of Circulating Tumor Cell (ctc) Subsets(Springer, 2020) Anıl İnevi, Müge; Sağlam Metiner, Pelin; Kabak, Evrim Ceren; Gülce İz, SultanBreast cancer is one of the most common cancer types among women in which early tumor invasion leads to metastases and death. EpCAM (epithelial cellular adhesion molecule) and HER2 (human epidermal growth factor receptor 2) are two main circulating tumor cell (CTC) subsets in HER2+ breast cancer patients. In this regard, the main aim of this study is to develop and characterize a three-dimensional (3D) breast cancer tumor model composed of CTC subsets to evaluate new therapeutic strategies and drugs. For this reason, EpCAM(+) and HER2(+) sub-populations were isolated from different cell lines to establish 3D tumor model that mimics in situ (in vivo) more closely than two-dimensional (2D) models. EpCAM(+)/HER2(+) cells had a high proliferation rate and low tendency to attach to the surface in comparison with parental MDA-MB-453 cells as CTC subsets. Aggressive breast cancer subpopulations cultured in 3D porous chitosan scaffold had enhanced cell-cell and cell-matrix interactions compared to 2D cultured cells and these 3D models showed more aggressive morphology and behavior, expressed higher levels of pluripotency marker genes, Nanog, Sox2 and Oct4. For the verification of the 3D model, the effects of doxorubicin which is a chemotherapeutic agent used in breast cancer treatment were examined and increased drug resistance was determined in 3D cultures. The 3D tumor model comprising EpCAM(+)/HER2(+) CTC subsets developed in this study has a promising potential to be used for investigation of an aggressive CTC microenvironment in vitro that mimics in vivo characteristics to test new drug candidates against CTCs.Article Citation - WoS: 6Citation - Scopus: 7A Biodesign Approach To Obtain High Yields of Biosimilars by Anti-Apoptotic Cell Engineering: A Case Study To Increase the Production Yield of Anti-Tnf Alpha Producing Recombinant Cho Cells(Humana Press, 2018) Gülce İz, Sultan; Anıl İnevi, Müge; Sağlam Metiner, Pelin; Ayyıldız Tamiş, Duygu; Kisbet, NazlıRecent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-α MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.