RICERCA ITALIANA     

Laboratory of immunobiotechnology and functional proteomics

Università degli Studi di Genova

Abstract

The general objective of this application is the construction of an advanced Laboratory located at the Centro per le Biotecnologie Avanzate (CBA) in Genova, devoted to Immunobiotechnology and Functional Proteomics. The Laboratory is based on the vision of a site to integrate sophisticated technological platforms, which require large infrastructures and equipment, with innovative research in the perspective of technological transfer and communication between basic and industrial research. The activity of the Laboratory will be focused on receptors in membrane-bound or soluble form, with emphasis on immunobiotechnology and intercellular communications. The Laboratory is viewed as a site for integration of advanced, complementary know-hows, both in terms of technology and of scientific innovation. As a basis of the Laboratory, the applicants intend to build-up technological platforms, such as production of murine and human monoclonal antibodies, expression of receptors and muteins thereof, signal transduction, transcriptional profiling, functional proteomics, molecular modeling for drug design. On this orizontal basis, represented by technological platforms, innovative, interrelated research lines will be activated, aimed at identifying new molecules and paradigms in the field of immunobiotechnology and intercellular communication. The molecules and functions characterized as well as reagents generated in the context of their analysis (e.g. monoclonal antibodies, muteins, etc.) will be candidate for industrial transfer. Transfer to industry will be based on new molecules as targets for drug development, validated using diverse approaches including generation of genetically modified mice. To provide a site for integration between technological platforms and innovative research aimed at industrial transfer, the Laboratory will provide the context for bidirectional interactions between innovative research groups and innovation-based industry. Indeed, the applicant groups include academic, university-based research laboratories (e.g. two MIUR Centers of Excellence), non-profit academic institutions and industry. Technological platforms, innovative research and integration between academic and industrial research will be developed with a focus on topics of great interest in the perspective of transfer, such as the identification of inhibitory and activatory receptors of the immune system, membrane-bound and soluble innate immunity receptors, inflammatory cytokine receptors, chemokine receptors, and intercellular communication systems based on signal metabolites and contact.
In the context of this general vision based on integration and complementarity between different levels and approaches, the Laboratory will represent a core facility open to external access, in particular to industrial groups, both conventional pharmaceutical industry and small biotech companies, having innovation as common denominator . The credibility of the application is based on the track record of the applicants from the point of view of both innovation and industrial transfer of molecules and methodologies. The activation of the Laboratory will also serve the function of higher education to scientific research of young students and postdoctoral fellows at the National and International level. The Laboratory will be located at CBA, in the perspective to become an essential core facility of this center. The CBA is specially suited to host a Laboratory devoted to Immunobiotechnology and Functional Proteomics, inasmuch as it is a functioning Center of the Liguria Region, already working as company incubator with special emphasis on providing advanced diagnostic services. Therefore the Laboratory will fit into the institutional missions of CBA. For these reasons, this application is in line with the Guidelines of the Italian Government in the field of Science and Technology (13/03/2002, Axis 2) , as well as with Axis 4 of the same Guidelines. <<<

Principal Investigator

Lorenzo MORETTA, Universita' degli Studi di GENOVA

Research Goal

Immune system cells express a wide spectrum of surface molecules that, after interaction with specific ligands, transport inside the cell the information regulating their function. It is increasingly evident that the different components of both innate and adaptive immunity do not act independently but communicate thanks to receptor/ligand interactions and binding/release of soluble factors. The present view is that the immune response (e.g. against pathogens or tumor cells) is not the result of the action of a single cell type but of a coordinated action of multiple components of the immune system. These observations can be extended to other systems (such as the neuroendocrine system) that are also regulated by multifaceted interactions among different cell types. Thus the principal task of this proposal is to build-up a new Laboratory dedicated to the analysis of cell-to-cell communications (with particular interest to those involving the immune system) using strategies and methods of functional proteomics. This main objective will be accomplished by setting up different principal technological platforms. These will be devoted to the identification of membrane molecules with biological functions (e.g. receptors, ectoenzymes and transporters); the analysis and characterization of their cellular or soluble ligands (receptors), of their substrates and products (ectoenzymes) and of the translocated molecules (transporters); the identification of the novel molecules and of the co-ordinated systems participating in the transduction of signals generated upstream (receptor/ligand interaction, ectoenzyme-dependent production of signaling metabolites) .
One the objective is the set-up of a platform based on the generation of monoclonal antibodies (mAbs) specific for different proteins with biological function, with particular regard to the receptors and their ligands. The approach was very successful in identifying new molecules of considerable biological interest. mAbs are an inexhaustible source of specific reagents that can be easily used for phenotypic, functional, and biochemical characterization and identification of proteins specifically recognized through purification and analysis of amino acid sequence by mass spectrometry or cDNA library screening. In addition, mAbs are used more and more frequently in the clinical practice for diagnostic purposes and innovative therapeutic approaches. Some of the proponents have a recognized experience in this field. Actually, thanks to the generation of new mAbs, they identified a large number of receptor or co-receptor molecules essential to the function of different components of the immune system. These reagents contributed to the understanding of mechanisms underlying the immune response and are now routinely used worldwide thanks to their marketing. In this context, it is worth mentioning that the company (Dompé SpA) involved in this proposal has a well documented expertise in the field of cloning and expression of fully human monoclonal antibodies. This expertise, unique in Italy, is also based on a pilot plant facility for the production clinical grade of fully human monoclonal antibodies with a 300 liters fermentator.
Another objective will be the development of platforms for the purification of membrane proteins. Thanks to the platform illustrated above, it will be possible to generate and develop new technologies for the purification of membrane molecules such as receptors and their cellular ligands, enzymes and transporters. One of the experimental systems used by the proponents is based on purification with affinity chromatography, protein separation on polyacrylamide gel, protein identification through colorimetric methods and analysis of tryptic fragments through mass spectrometry. Though successful, it is a long and complex experimental procedure associating technologically advanced systems with largely obsolete methods. These reasons make the whole process not very effective and reproducible. In addition, the phases preceding the analysis of tryptic fragments require the use of large quantities of initial cell material and considerable loss of this latter during the different phases of purification/separation. Our objective is to join the specific skills of proponents to set up technological platforms aimed at isolating and identifying complex structures as membrane proteins. On the one hand, we will use advanced technologies as liquid phase mass spectrometry, BIAcore sensor chips and plasmon resonance, and molecular modeling of receptor-ligand interactions, while on the other we will use biological tools including not only mAbs but also soluble chimeric receptors or ligands, stable transfectants expressing one or the other molecule and enzyme-substrate or transporter-solute complexes. Moreover, the application of the Multidimensional Protein Identification Technology (MudPIT, available with the RU 004 of ITB) will allow the characterisation and quantitative analysis of protein complexes, in particular those on the membrane.
The expertise and technologies described above will be used to build up platforms for the study of protein/ligand interactions and for the analysis of structure-function-regulation relationships of membrane receptors. Experimental and clinical data clearly show that membrane proteins and glycoproteins are not isolated and independent entities but belong to molecular complexes in continuous development (e.g. receptors and co-receptors), moving rapidly along the membrane and localizing in restricted areas. Moreover, these complexes are associated with clusters of transmembrane and cytoplasmic molecules inducing a cascade of transduction signals reaching the cell nucleus. It is therefore essential to analyze and understand the molecular bases that, on the one hand, regulate the interaction of surface molecules and, on the other, allow surface molecules to transmit correctly the information derived from the interaction with specific ligands.
The different technological platforms described in this proposal will allow the integration of data on the structure of a particular receptor, the nature of its ligand, the interaction with other membrane molecules or with signal transduction mechanisms. This will allow in-depth understanding of the molecular mechanisms underlying the different cell functions. <<<

Timescale

60 months