Novel immuno and stem cell based therapies. Sendi Montanič, Uroš Rajčevič and Vladka Čurin Šerbec

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1 Passamonti S. (Editor) The Partners and the Objectives of Trans2Care, an Italy-Slovenia cross-border network of science and healthcare institutions pp / ISBN Transregional Network for Innovation and Technology Transfer to Improve Health Care Novel immuno and stem cell based therapies Sendi Montanič, Uroš Rajčevič and Vladka Čurin Šerbec PP10-Blood Transfusion Centre of Slovenia, Ljubljana Abstract The main areas of the research at the Blood Transfusion Center of Slovenia are cell therapies, stem cells and new diagnostic reagents, based on monoclonal antibodies (mabs). Our laboratories are equipped for cell-culture work (certified GLP; GMP in reconstruction), molecular biology, immunological techniques and biochemistry. In the course of our research we conducted studies and applied projects (the last mainly industrial) beside production of reagents, with common topic - the antibodies. They were prepared and used to optimise and validate ELISA tests for quality control assessment of the end-product (polyclonal antibodies, in production of drugs), to study protein structure, for immunodiagnostics and as potential immunotherapeutics. Among different mabs, which we produced in the last decade, a panel of mabs against prion protein (PrP) and mabs against bilitranslocase (prepared in collaboration with the University of Trieste) will be studied with different project partners in the scope of Trans2Care. We are fully dedicated to translate the results of the basic research onto applied, possibly clinical level and to contribute to improved healthcare in a sense of advanced modes of immunotherapeutics development and cell based therapies. Index Terms human prion protein, prion, bilitranslocase, monoclonal antibodies, immunodiagnostics, immunotherapy, stem cells, cell therapy 1 Introduction Blood Transfusion Centre of Slovenia (BTCS) is a national blood bank, responsible for the supply of safe blood and blood components in our country. Beside that, we are providing diagnostic and therapeutic services and blood-derived drugs to our hospitals. We are a strong partner of the University of Ljubljana, giving lectures and providing experimental work to students of graduate studies at Faculty of Medicine, 93

2 Faculty of Pharmacy, Faculty of Chemistry and Chemical Technology and Faculty of Health Sciences. We are also involved in postgraduate programs of Biomedicine and Biotechnology at the University of Ljubljana. The same personnel is engaged in research and development at BTCS, being members of three research groups: Tissue Typing Center, Transfusion Medicine and Biomedicine. As of 2004 BTCS has an ISO 9001 standard and is a WHO Collaborating Center and an EFI (European Federation of Immunogenetics) member. 2 Research at the BTCS Research activities at the BTCS started as early as 1953, when the institution was registered for the research and development in medical biotechnology and medical sciences. In the course of next four decades, activities in this field intensified, especially in the research. During last 15 years the three forementioned research groups, conducted 33 national research projects, 3 national programmes, 6 industrial projects and 6 international projects, financed by Slovene Research Agency, by Ministry for Higher Education, Science and Technology, by industrial partners as well as the European Comission. 18 young researchers/phd students were trained at the BTCS in the scope of these projects, covering medicine, life sciences and biotechnology. Our development is closely related to our routine operations and services (blood collection and processing, blood grouping, cell therapeutical services) and assures constant education and follow-up in transfusion medicine. The main areas of our research are cell therapies, stem cells and new diagnostic reagents, based on monoclonal antibodies (mabs). Our laboratories are equipped for cell-culture work (certified GLP; GMP in reconstruction), molecular biology, immunological techniques and biochemistry. On the basis of our research, we were invited to take part of the educational programmes at the University of Ljubljana (see introduction). 2.1 Biomedicine research group Our research group evolved from the department for the producion of diagnostic reagents, in charge of production of diagnostic reagents for blood grouping under GMP (Good Manufacturing Practice) conditions. Our first research project included the production of potent mouse IgM monoclonal antibodies (mabs) against AB0 blood group system in early ninetees of the last century, which were introduced into routine work in the form of diagnostic reagents, registered in In consequent years, we conducted research and applied projects (the last mainly for the industry) beside production of reagents, with common topic - the antibodies. They were prepared and used to optimise and validate ELISA tests for quality control assessment of the endproduct (polyclonal antibodies, in production of drugs), to study protein structure, for diagnostics and as potential therapeutics (mabs). Among different mabs, which we produced in the last decade, a panel of mabs against prion protein (PrP) [1, 2, 4, 6, 9, 10] and mabs against bilitranslocase (prepared in collaboration with the University of Trieste, started in 2006) [11] will be studied with different project partners in the scope of Trans2Care. 94

3 3 Contribution to Trans2Care 3.1 Bilitranslocase Our current, ongoing collaboration with the T2C partners includes the research of mabs against bilitranslocase in collaboration with University of Trieste (prof. S. Passamonti). This collaboration will further develop as we anticipate our first joint publications [11]. Figure 1: ICC staining of HepG2 cells with anti-bilitranslocase mab 6E4/1F2 (40x, 2.4s). 3.2 Prion diseases In our research on prion diseases we constructed and tested a panel of mabs against prion protein (PrP) enabling us to distinguish and differentiate between the wild type PrPc and its pathogenic form PrPSc, which is of a great diagnostic value in Creutzfeld Jacob s disease (CJD). This research yielded EU and US patents for these antibodies as well as our common publications with T2C partners SISSA prof. G. Legname and University of Trieste - prof. R. Gennaro [1, 6]. In the frame of T2C we are aiming at upgrading this collaboration in two possible directions: the use of proteomics for the identification of novel biomarkers of prion diseases using animal models of CJD as well as clinical samples of CJD. These biomarkers could help us elicit the molecular background of prion diseases as well as serve as potential diagnostic or therapeutic tools. Alternatively we aim at using the expertise and specialized infrastructure (GMP laboratory standards) of the BTCS in the field of cell based therapeutics to reaserch a potential of neural stem cell based therapies for prion diseases using animal models of CJD. 95

4 Figure 2: Immunohistochemistry of mab V5B2 binding to the pathogenic form of PrPSc in CJD brain section, compared to normal brain section. (a) Diffuse PrPSc plaque depositions are visible in a synaptic section of cerebellum (40x). (b) PrPSc negative staining of normal brain hypocampus (20x). 4 Conclusions In the frame of T2C, we offer our above mentioned expertise to project partners, within the fields of development of immunotherapeutics, immunodiagnostics, cell based therapies and proteomics as well as whereever sinergies are anticipated. Thus far, we have proven our capacity of inter-regional collaborations and we are looking forward to expanding the ongoing and new collaborations with Slovene and Italian partners. We are fully dedicated to translate the results of the basic research onto applied, possibly clinical level and to contribute to improved healthcare in a sense of advanced modes of immunotherapeutics development and cell based therapies. Acknowledgement The financial support of the Fondo europeo di sviluppo regionale (Evropski sklad za regionalni razvoj) for the Trans2Care project is greatly appreciated. The financial support by the Slovene Research Agency through the research grant P is acknowledged. We also acknowledge the contribution of the collaborators working on the projects in the past (please see the references) from within the BTCS and outside. 96

5 References [1] V. Čurin Šerbec, Antibodies capable to selectively detect prion PrPSc isoforms. (2000) patent no. EP (2005, granted European patent) and US 7,098,317 B1 (2006, granted USA patent). [2] V. Čurin Šerbec, M. Bresjanac, M. Popović, K. Pretnar Hartman, V. Galvani, R. Rupreht, M. Černilec, T. Vranac, I. Hafner, R. Jerala, Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt-Jakob s disease-affected and normal brain tissue, J. Biol. Chem., vol. 279, no. 5, p , [3] T. Vranac, K. Pretnar Hartman, M. Popović, A. Venturini, E. Žerovnik, V. Čurin Šerbec. A single prion potein peptide can elicit a panel of isoform specific monoclonal antibodies, Peptides vol. 27, no. 11, p , [4] M. Černilec, T. Vranac, I. Hafner-Bratkovič, S. Koren, A. Colja Venturini, M. Popović, P. Juntes, V. Čurin Šerbec, Identification of an epitope on the recombinant bovine PrP that is able to elicit prominent immune response in wild-type mice, Immunology Letters 113, p , [5] M. Ghielmetti, T. Vranac, V. Čurin Šerbec. Prion protein peptides as vaccines, Mini Rev. Med. Chem. vol 9, no. 4, p , [6] M. Kosmač, S. Koren, G. Giachin, T. Stoilova, R. Gennaro, G. Legname, V. Čurin Šerbec, Epitope mapping of a PrP(Sc)-specific monoclonal antibody: Identification of a novel C-terminally truncated prion fragment, Mol. Immunol., vol. 48, no. 5, p , [7] K. Trontelj, M. Reberšek, M. Kandušer, V. Čurin Šerbec, M. Šprohar, D. Miklavčič, Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells, Bioelectrochemistry, vol. 74, no. 1, p , [8] K. Trontelj, M. Usaj, V. Čurin Šerbec, D. Miklavcic. Fusion of cells with electrofusion, Med. Razgl, no. 49, p , [9] N. Škrlj, V. Čurin Šerbec, M. Dolinar. Single-chain Fv antibody fragments retain binding properties of the monoclonal antibodies raised against peptide P1 of the human prion protein, Applied Biochemistry and Biotechnology, vol.160, no. 6, p , [10] N. Škrlj, T. Vranac, M. Popović, V. Čurin Šerbec, M. Dolinar M., Specific isoform: Developing and characterization of a humanized single-chain variable antibody fragment binding of the pathogenic prion, PLoSOne, vol. 6, no. 1, p 1-9, [11] S. Montanič, M. Terdoslavich, V. Čurin-Šerbec, S. Passamonti. An anti sequence monoclonal antibody for the investigation of bilitranslocase in the biosphere. Publication pending. Contact info Sendi Montanič, Uroš Rajčevič and Vladka Čurin Šerbec are with Blood Transfusion Centre of Slovenia, Centre for the production of diagnostic reagents and for research & research group Biomedicine. Šlajmerjeva 6, 1000 Ljubljana, Slovenia. 97