Laboratory of Molecular Biology & Immunobiotechnology

Head of the lab: Dr. Avgi Mamalaki
Τel: 210 6478838, Email: amamalaki@pasteur.gr

About

The laboratory of Molecular Biology and Immunobiotechnology was founded in January 2004, having as its primary goal the exploitation of experimental findings that will be used for the development of novel diagnostic and therapeutic approaches in important fields of the biomedical science.

The laboratory of Molecular Biology and Immunobiotechnology focuses on:

  • The development of new biotechnological drugs (production of recombinant biomolecules such as antigens, antibodies and peptides) of known and novel molecular targets, and
  • Functional studies of molecular targets and biomolecules for the diagnosis and the therapeutic applications of several diseases

The main achievements of the laboratory include the:

  • Production of rodent recombinant antibody fragments of monoclonal antibodies
  • Production of human recombinant antibody fragments
  • In vitro affinity maturation of human antibody fragments, and
  • Production of recombinant proteins or peptides in heterologous expression systems (bacteria, yeast, protozoa)

The production of recombinant antibody fragments is a technology based on the construction of combinatorial cDNA libraries of Fab or scFv antibody fragments that are expressed on the surface of filamentous bacteriophages (phage display antibody libraries). These libraries allow the isolation of highly specific antibodies since they include the complete repertoire of antibodies expressed in B lymphocytes deriving from specific tissue. The laboratory of Molecular Biology and Immunobiotechnology is the pioneer lab in Greece where this technology has been applied and evolved with great success..

The current research activity of our lab focuses on:

a) New research activity concerning the:

1. Cancer Immunotherapy and HER-2/neu. A combined approach of genes, proteins and drugs

2. Immunodiagnosis and Immunotherapy of Metabolic Disease, concerning Hepcidin.

b) On-going research activity that was previously developed in the Laboratory of Molecular Neurobiology and Immunology, during the last decade and concerns the:

  1. Immunotherapy of Autoimmune Diseases – Myasthenia Gravis
Members

Head of the lab: Mamalaki Avgi, Senior Researcher, amamalaki@pasteur.gr

Postdoctoral fellows: M. Marinou, mmm@pasteur.gr, K. Vourvouhaki, kvourvouhaki@pasteur.gr

Graduate Students:

M. Belimezi, mariabelimezi@pasteur.gr
B. Filippi, bfilippi@pasteur.gr
V. Koliaraki, vkoliaraki@pasteur.gr
E. Protopapadaki, protopapadaki@pasteur.gr

Research programs

Research programs

1. Cancer Immunotherapy and HER-2/neu. A combined approach of genes, proteins and drugs.
(M.BELIMEZI, B. FILIPPI, K. VOURVOUHAKI, A. MAMALAKI)

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:

  • 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
  • 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
  • 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
  • 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

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

  • 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.
  • 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.
Funding

Research funds

2002 – 2005

  1. ROJECT PENED 2001:
    Title: Genetically modified lymphocytes in cancer immunotherapy
  2. PROJECT PENED 2001:
    Title: Interactions of Protein molecules – Bioreactive molecules
  3. PROJECT EPΑΝ 2003:
    Title: Hepcidin, a novel iron-regulatory hormone implicated in the anemia of chronic disease and in hemochromatosis: Development of diagnostic tools, study of the mechanism of action and design of potential therapeutic interventions
  4. PROJECT EPΑΝ 2003:
    Title: Profiling changes in gene and protein expression in HER-2/neu overexpressing human tumor cell lines:identification of novel immunogenic proteins acting as diagnostic markers and specific targets for cancer immunotherapy
  5. SCIENTIFIC AND TECHNOLOGICAL COOPERATION BETWEEN RTD ORGANISATIONS
    IN GREECE AND RTD ORGANISATIONS IN KANADA (2004):
    Τitle: Hepcidin, a novel iron-regulatory hormone implicated in the anemia of chronic disease and in hemochromatosis.
Publications

Selected publications

  1. Tzartos, S., Cung, M., Demange, P., Loutrari, H., Mamalaki, A., Marraud, M., Papadouli, I., Sakarellos, C., Tsikaris, V. The main immunogenic region (MIR) of the acetylcholine receptor and the anti-MIR antibodies. Molecular Neurobiology, 5:1-29, 1991.
  2. Mamalaki, A., Trakas, N., Tzartos, S. Bacterial expression of a single-chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies. European Journal of Immunology, 23:1831845, 1993.
  3. Mamalaki, A. and Tzartos, S.  Nicotinic Acetylcholine Receptor: Structure, Function and Main Immunogenic Region. Advances in Neuroimmunology, 4:339-354, 1994.
  4. Kioussi, C., Mamalaki, A., Mirski, R., Jessen, K., Matsas, R. Expression of endopeptidase-24.11/ (common acute lymhoblastic leukemia CD10) in the sciatic nerve in the adult rat after lesion and during regeneration. European Journal of Neuroscience, 7:951-961, 1995.
  5. Mamalaki, A., Boutou, E., Patsavoudi, E., Hurel, C., Tzartos, S. and Matsas, R. BM88: cDNA cloning of a novel neuron-specific molecule from pig nervous system that accelarates the differentiation of transfected mouse neuroblastoma cells. Journal of Biological Chemistry, 270:14201-14208, 1995.
  6. Tzartos, S., Cung, M., Mamalaki, Α., Marraud, M., Papanastasiou, D., Sakarellos, C., Sakarellos-Daitsiotis, M. and Tsantili, P. Antigenic Structure of the Muscle Nicotinic Acetylcholine Receptor. Annales de  Medecine Interne, 147:442-445, 1996.
  7. Gaitanou, M., Mamalaki, Α., Merkouri, E. and Matsas, R. Purification and cDNA cloning of mouse BM89 antigen shows that it is identical with the synaptic vesicle protein synaptophysin. Journal of Neuroscience Research, 48:507-514, 1997.
  8. Tzartos, S., Barkas, T., Cung, M., Mamalaki, A., Marraud, M., Orlewski, P., Papanastasiou, D., Sakarellos, C., Sakarellos-Daitsiotis, M., Tsantili, P. and Tsikaris, V. Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor. Immunological Reviews, 163: 89-120, 1998.
  9. Tzartos, SJ., Tsantili, P., Papanastasiou, D., Mamalaki, A. Construction of single-chain Fv fragments of anti-MIR monoclonal antibodies. Annals of the New York Academy of Science, 841:475-7, 1998.
  10. Gomez, J., Boutou, E., Hurel, C., Mamalaki, A., Kentroti, S., Vernadakis A. and Matsas, R. Overexpression of the neuron specific molecule, BM88, in mouse neuroblastoma cells: altered responsiveness to growth factors. Journal of Neuroscience Research, 51:119-128, 1998.
  11. Tsantili, P., Tzartos S. and Mamalaki, A. High affinity scFv antibody fragments protecting the human nicotinic acetylcholine receptor. Journal of Neuroimmunology, 94:15-27, 1999.
  12. Papanastasiou, D., Mamalaki, A., Eliopoulos, E., Poulas, K., Liolitsas, C. and Tzartos, S. Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor. Journal of Neuroimmunology, 94:182-195, 1999.
  13. Martin-Ruiz, C. M., Court, J.A., Molnar, E., Lee, M., Gotti, C., Mamalaki, A., Tsouloufis, T., Tzartos, S., Ballard, C., Perry, R.H. and Perry, E.K. Alpha 4 but not alpha 3 and 7 nicotinic acetylcholine receptor subunits are lost from the temporal cortex in Alzheimer’s disease. Journal of  Neurochemistry, 73(4):1635-40, 1999.
  14. Baxevanis, C.N., Spanakos, G., Voutsas, I.F., Gritzapis, A.D., Tsitsilonis, O.E., Mamalaki, Α. and Papamichail, M. Increased generation of autologous tumor-reactive lymphocytes by anti-CD3 monoclonal antibody and prothymosin alpha. Cancer Immunology Immunotherapy, 48(2-3):71-84, 1999.
  15. Kleinjung, J., Petit, M.C., Orlewski, P., Mamalaki, A., Tzartos, S., Tsikaris, V., Daitsiotis-Sakarellos, M., Sakarellos, C., Marraud, M. and Cung, M.T. The Third-Dimensional Structure of the Complex between an Fv Antibody Fragment and an Analog of the Main Immunogenic Region of the Acetylcholine Receptor: A Combined Two-Dimensional NMR, Homology and Molecular Modeling Approach. Biopolymers, 53(2):113-28, 2000.
  16. Kontou, Μ., Leonidas, D.D., Vatzaki, E.H., Acharya, K. R., Mamalaki, A., Oikonomakos, N.G. and Tzartos, S.J. The crystal structure of a Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor. European Journal of Biochemistry, 267(8):2389-2397, 2000.
  17. Tsouloufis, T., Mamalaki, A., Remoundos, M., Tzartos, S.J. Reconstitution of conformationally dependent epitopes of the N-terminal extracellular domain of human muscle AChR α subunit expressed in E.coli: implications for myasthenia gravis therapeutic approaches. International Immunology, 12(9):1255-1265, 2000.
  18. Boutou, E., Matsas, R. and Mamalaki, A. Ιsolation of a mouse brain cDNA expressed in developing neuroblasts and mature neurons.  Molecular Brain Research, 86:153-167, 2001.
  19. Gaitanou, M., Buanne, P., Pappa, C., Mamalaki, A., Tirone, F. and Matsas, R. Cloning, expression and localization of human BM88 shows that it maps to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis, Biochemistry Journal 355:715-724, 2001.
  20. Perez, S., Sotiropoulou, P., Sotiriadou, N., Mamalaki, A., Gritzapis, A., Echner, H., Voelter, W., Pawelec, G., Papamichai,l, M., Baxevanis, CN. HER-2/neu-derived peptide 884-899 is expressed by human breast, colorectal and pancreatic adenocarcinomas and is recognized by in-vitro-induced specific CD4(+) T cell clones. Cancer Immunol Immunotherapy 50(11):615-24, 2002.
  21. Sotιriadou, N., Perez, S., Gritzapis, A., Sotiropoulou PA, Echner, H., Heinzel, S., Mamalaki, A., Pawelec, W., Voelter, W., Baxevanis, CN., and Papamichail, M. Peptide HER-2/neu (776-788) represents a, naturally processed broad MCH class II restricted T cell epitope. British Journal of Cancer 85(10), 1527-1534, 2001
  22. Psaridi, L., Mamalaki, A., Remoundos M., and Tzartos, S. Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha -bungarotoxin binding. J Biol Chem. 277, 30, 26980-26986, 2002.
  23. Gritzapis A., Mamalaki A., Kretsovali Α., Papamatheajis J., Belimezi Μ., Perez S., Baxevanis C. and Papamichail M. Redirecting mouse T hybridoma against human breast and ovarian carcinomas: in vivo activity against HER-2/neu expressing cancer cells. Br J Cancer. 2003 Apr 22; 88(8):1292-300.
  24. Mamalaki A., Gritzapis A., Kretsovali Α., Belimezi Μ., Papamatheajis J., Perez S., Papamichail M and Baxevanis C. In vitro and in vivo antitumor activity of a mouse CTL hybridoma expressing chimeric receptors bearing the single chain Fv from HER-2/neu- specific antibody and the γ-chain from Fc(ε) RI. Cancer Immunol Immunother. 52(8): 513-22, 2003.
  25. Psaridi-Linardaki, L., Mamalaki, A., and Tzartos, S. Future therapeutic strategies in autoimmune Myasthenia Gravis. Annals of the New York Academy of Science, 998: 539-548, 2003.
  26. V. Avramopoulou, A. Mamalaki and S. Tzartos. Soluble oligomeric and ligand-binding extracellular domain of human neuronal alpha7 acetylcholine receptor expressed in the yeast Pichia pastoris: Replacement of the hydrophobic Cys-loop by the hydrophilic loop of ACh-binding protein enhances protein solubility. J. Biol.Chem., 279(37):38287-93.  2004.
  27. Loukia Psaridi-Linardaki, Nikos Trakas, Avgi Mamalaki and Socrates J. Tzartos Specific immunoadsorption of the autoantibodies from myasthenic patients using the extracellular domain of the human muscle acetylcholine receptor α-subunit. Development of an antigen-specific therapeutic strategy.  Journal of Neuroimmunology, 159:183-191, 2005.
  28. Fostieri E, Tzartos SJ, Berrih-Aknin S, Beeson D, Mamalaki A. Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies. Eur J Immunol. 35: 632–643, 2005.

BOOK EDITED:

29. S. Tzartos and A. Mamalaki. “Basic Immunology – Functions and Disorders of the Immune System” by
A.  Abbas and A. Lichtman. Ed. Paschalides, 2004.   PATENT:    Production of recombinant fragments of muscle acetylcholine receptor and their use for ex vivo immunoadsorption of anti-ACh receptor antibodies from myasthenic patients. Tzartos, S.J., Psaridi-Linardaki, L., Kostelidou, K. and Mamalaki, A. Greek patent: (No. 1004240, 2002).