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| Research Programs |
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1. Cancer Immunotherapy and HER-2/neu. A combined approach of genes, proteins and drugs.
(M.BELIMEZI, B. FILIPPI, K. VOURVOUHAKI, A. MAMALAKI) |
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The oncoprotein HER2/neu (c-erbB-2) is a member of the erbB receptor family with tyrosine kinase activity. It is the principal component of the heterodimers that are formed among members of the erbB family (EGFR, erbB-3, erbB-4) in the presence of growth factors, which result in the activation of those signaling pathways that lead to cell proliferation and differentiation. Any alteration in the tightly regulated signaling cascade of HER-2/neu cause severe abnormalities and carcinogenesis. HER2/neu is overexpressed in approximately 30% of human breast and ovarian cancer, and has been associated with aggressiveness and poor prognosis. In addition, HER2/neu has been shown to be overexpressed, in a large number of other human malignancies including ovarian, colon, lung, prostate and cervical cancers. HER2/neu overexpression in neoplasmatic cells activates the immune system, as it is detected from the increased title of antibodies against HER2/neu as well as from the presence of increased numbers of T lymphocytes with immunological memory for HER-2/neu protein in the peripheral blood. Current therapeutic approaches aim to the inhibition of HER2/neu signaling and to reduction of its expression in the cells..
Our aim is to:
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Identify the pathways that are involved in the immunogenicity and the malignancy of cancer cells that they overexpress the HER-2/neu antigen. By using cDNA microarrays we will identify genes overexpressed in HER-2/neu positive cancer cell lines that will serve as potential therapeutic targets in the future
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Isolate antibody fragments able to inhibit the activity of cancer antigens. For this reason, we have developed combinatorial phage display antibody libraries from the mRNA of B lymphocytes deriving from invasive lymph nodes obtained from patients with breast cancer overexpressing HER-2/neu. We have isolated human recombinant Fab fragments that they bind to the extracellular domain of the HER-2/neu oncoprotein
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Investigate whether HER-2/neu oncoprotein can activate the immune system leading to tumour rejection. The extracellular domain of HER-2/neu, which contains many antigenic epitopes, was produced in heterologous protein expression systems and is tested as vaccine in experimental cancer models simultaneously with cancer cells
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In collaboration with the Immunology and Immunotherapy Cancer Center of “Agios Savas” hospital and the Institute of Molecular Biology and Biotechnology (I.M.B.M.) of Crete, we have established the production of prototype constructs that they encode tumour-specific chimeric receptors. The innovation of this approach is based on the construction of cytotoxic cells with anti-cancer activity. Their function is determined by the expression of chimeric receptors and involves: 1) the recognition of the cancer cell through the variable region of a specific antibody, and 2) the destruction of the cancer cell through the activation mechanisms that are induced by the signaling components (ζ-chain) of the TCR/CD3 complex or the γ-chain of the Fc receptors for IgG. |
2. Immunodiagnosis and Immunotherapy of Metabolic Disease, concerning Hepcidin.
(V. KOLIARAKI, M. MARINOU, A. MAMALAKI) |
Hepcidin is a cysteine-rich peptide with antimicrobial activity, generated in the liver and secreted in the plasma. Iron levels, inflammatory stimuli and hypoxia regulate its expression. Hepcidin exerts a very important function in controlling body iron homeostasis in the body. It functions as an iron-regulatory hormone that controls the absorption of dietary iron and its release from cells. The expression of hepcidin appears to be very important in the context of anemia of chronic disease, the most frequent anemia among hospitalized patients, which is associated with impaired iron utilization for erythropoiesis. In addition, hepcidin may also have an important role in disorders of primary and secondary iron overload, such as hereditary hemochromatosis, thalassemias etc.
In this project, we are in process to express in heterologous expression systems the two peptide forms of hepcidin identified in the plasma (peptides of 25 and 20 amino acids). The expression of hepcidin is important in order to study its function in iron metabolism and to produce new drugs for the treatment of anemias and iron overload. Moreover, recombinant antibody fragments against hepcidin can be used as therapeutic tools in diagnosis and therapy. |
3. Immunotherapy of Autoimmune Diseases - Myasthenia Gravis
(E. FOSTIERI, E. PROTOPAPADAKI, S. TZARTOS, A. MAMALAKI |
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Myasthenia gravis (MG) is an autoimmune disease characterized by fatigability and weakness of the skeletal muscles. The interaction of autoantibodies with acetylcholine receptors (AChR) leads to the destruction of functional AChRs at the neuromuscular junction. The 2/3 of the anti-AChR antibodies in rats and many myasthenic antibodies are directed against an extracellular region of the AChR α-subunit, named main immunogenic region (MIR). Anti-MIR mAbs lead to the destruction of AChR in cell cultures and experimental MG in animals. Fab fragments of anti-MIR mAbs, being univalent, do not bind the complement and do not cause antigenic modulation, whereas they are capable of protecting the AChR from the modulating activity of human MG sera in cell cultures and they do not induce in vivo experimental myasthenia. Moreover, we have shown that scFv fragments of anti-MIR mAbs and their humanized version can protect the AChR in cell cultures against the destructive activity of autoantibodies from myasthenic sera.
The aim of the project is to isolate a number of human origin Fab antibody fragments that are capable to compete with the majority of autoantibodies, in order to study and use them in therapeutic application in Myasthenia gravis
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By introducing the “phage-antibody” technology we have isolated human Fabs for AChR protection. Anti-AChR human antibody fragments have been cloned from thymic B lymphocytes of a myasthenic patient. Further optimization of their affinity has been achieved by “chain shuffling” technology and new Fabs with dramatically improved binding ability for human AChR have been isolated that bind to a new strong antigenic epitope. These Fabs have high binding affinity for the human receptor and compete for AChR binding with autoantibodies. Fabs are also capable of protecting surface AChR in cell culture against antigenic modulation due to MG sera.
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Another approach to produce fully human antibodies is to use “humanized” mice that are transgenic for the human Ig locus and have disrupted the endogenous expression of immunoglobulins. These mice were immunized with the extracellular domain of the AChR α-subunit expressed in E.coli or in Pichia pastoris. One of the isolated mAbs was able to bind well in the intact human AChR, whereas its Fab fragment was able to protect the AChR from the binding of anti-MIR mAbs and the pathogenic action of MG sera with high anti-MIR autoantibodies. |
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