centro biotecnologie avanzate

LABORATORY OF: Therapeutic Recombinant Proteins
CONTACT PERSON: Prof.Luciano Zardi
Phone +39 0105737437 E-mail: luciano.zardi@poste.it

Description of Laboratory and Expertise:

The activity of the Laboratory of therapeutic recombinant proteins is focused on the generation by molecular biology techniques of recombinant proteins having therapeutic activity, with special regards to neoplastic and degenerative chronic diseases.
The laboratory is organized in the following units:
Molecular Biology: for the generation of cDNA constructs, transformation and/or transfection of cells and selection of the positive clones. For the conjugation of recombinant proteins to different markers and substances for photodynamic therapy.
Cell culture: for the maintenance of the necessary cell lines and for massive culture of transfected mammalian cells.
Protein Biochemistry: for the purification of recombinant proteins and their validation (quality control).
Histology and Immuno-histochemistry: for testing new antibody on animal and human tissues and for discovering new antigens
Preclinical studies: for therapeutic preclinical studies on animal models.

Abstract of Activities:

The laboratory has long tradition in comparative studies on the extra-cellular matrix (ECM) of normal and pathologic tissues as well as in the generation of antibodies (both murine monoclonal and human recombinant) specific for components of the ECM from pathologic tissues. The laboratory has also an agreement with the biotech company Philogen that gives to the laboratory the permission to use a library of recombinant antibody.
At present the laboratory is involved in the following studies:
Generation of antibodies conjugated to photosensitizing molecules for the photodynamic therapy of tumours.
Generation of human recombinant polyvalent and polyspecific antibodies.
Validation of a new linker for generation of polyvalent fusion protein.
Validation of new tumour associated antigens and their validation in vitro and in vivo.
Validation of a new therapeutic approach to chronic inflammatory and neoplastic diseases using complex recombinant proteins.

Detailed Research Activities:

The vast majority of existing drugs localize preferentially in normal organs, rather than at the tumour or pathologic sites, often leading to unacceptable toxicities and to poor cure rates. The targeted delivery of therapeutics to pathologic tissues is one of the most promising avenues towards a more efficacious and better-tolerated drugs. In the Laboratory of Therapeutic Recombinant Proteins we use disease-associated extracellular matrix (ECM) components as targets and high affinity human recombinant antibodies as ligands for the delivery of therapeutics . As a therapeutic molecule we preferentially use cytokines such as IL2, IL12 TNF-alpha, IL10, without excluding other types of agents.
In the lesion, both neoplastic or due to degenerative inflammatory diseases, the ECM of the normal tissue is remodelled through proteolytic degradation of existing ECM components and the neo-synthesis of new ones. This generates a tumour ECM which differs from that of normal tissues. Furthermore, it is now accepted that this provisional ECM generates a more suitable environment for cellular migrations in which angiogenesis is a crucial step. Thus, many of these ECM antigens are common to almost all tumours and represent pan-tumour antigens. Our groups have clearly demonstrated that the ”remodelling environment” (pH, Borsi et al 1995; presence of cytokines, Balza et al., 1988) leads to a deregulation of the pattern of alternative splicing of the pre-mRNA of a number of ECM components (Borsi et al., 1992; Carnemolla et al., 1989), thereby generating novel ECM components. These ECM antigens represent an appealing target for the selective delivery of therapeutics, since they are more abundant and genetically more stable than cell surface antigens.
The use of phage display libraries for the generation of human monoclonal antibodies is simple and not particularly expensive. This technique is also extremely flexible, since it is possible, for instance, to generate pre-absorbed libraries, thereby removing antibodies directed to unwanted antigens (for instance, antigens present in normal tissues) and making it possible to generate antibodies to specific groups of antigens or to particular epitopes of a molecule. For example, it is extremely simple to generate anti-idiotype antibodies to a particular IgG by pre-absorbing the library with a crude preparation of IgG, since all the antibodies reacting with common epitopes on IgG molecules will be removed and the selection of antibodies will be directed on epitopes of the idiotype. After generation, scFvs can then easily be manipulated to increase their affinity or used as building blocks for generating more complex molecules, such as, for instance, fusion proteins consisting of the antibody and a cytokine. Thus, in general, we consider extremely useful - both from a scientific standpoint and from a possible industrial point of view - the power and flexibility of this technique, which, in our opinion, has yet to be sufficiently disseminated and, for many reasons, to be appropriately exploited.
We have already exploited the advantages of phage display libraries for the generation of antibodies to tumour antigens and have pioneered the development of human monoclonal antibodies and their derivatives, capable of selective in vivo localization at sites of angiogenesis (Neri & Bicknell, 2005). For example, after extensive preclinical testing, three derivatives (Carnemolla et al 2002; Halin et al 2002; Borsi et al 2003) of the human L19 antibody (Pini et al., 1998) specific to the EDB domain of fibronectin (a marker of angiogenesis; Zardi et al., 1987; Castellani et al., 1994; Castellani et al., 2002) are currently in industrial and clinical development (Santimaria et al., 2003). We now seek to test the use of phage display libraries for the generation of new molecules useful in the diagnosis and the therapy.

Applications and Developments:

The recombinant proteins generated in the laboratory may find application the therapy of neoplastic and degenerative diseases.

Ongoing collaborations:

Prof. Dr. Dario Neri, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETHZ, Zurich, Switzerland
Dr. Laura Borsi, Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
Prof. Dr. Giuliano Mariani, Department of Oncology, University of Pisa, Pisa, Italy
Prof. Dr. Hartwig Kosmehl, HELIOS Klinikum Erfurt, Institute of Pathology, Erfurt, Germany
Prof. Silvio Parodi, Cattedra di Oncologia, University of Genova, Italy

Most recent and significant publications:

Castellani P., Borsi L., Carnemolla B., Birò A., Dorcaratto A., Viale G.L., Neri D. and Zardi L. Differentiation between high- and low-grade astrocytoma using a human recombinant antibody to the extra domain-B of fibronectin. Am J Pathol. 2002 Nov;161(5):1695-700.

Borsi L, Balza E, Carnemolla B, Sassi F, Castellani P, Berndt A, Kosmehl H, Biro A, Siri A, Orecchia P, Grassi J, Neri D, and Zardi L. Selective targeted delivery of TNFalpha to tumor blood vessels. Blood. 2003 Dec 15;102(13):4384-92. Epub 2003 Aug 21.

Balza E, Mortara L, Sassi F, Monteghirfo S, Carnemolla B, Castellani P, Neri D, Accolla RS, Zardi L and Borsi L. Targeted delivery of tumor necrosis factor-alpha to tumor vessels induces a therapeutic T cell-mediated immune response that protects the host against syngeneic tumors of different histologic origin. Clin Cancer Res. 2006 Apr 15;12(8):2575-82.

ijink BM, Neri D, Leemans CR, Budde M, Dinkelborg LM, Stigter-van Walsum M, , and van Dongen GA. Radioimmunotherapy of head and neck cancer xenografts using 131I-labeled antibody L19-SIP for selective targeting of tumor vasculature
J Nucl Med. 2006 Jul;47(7):1127-35.

Avignolo C, Bagnasco L, Biasotti B, Melchiori A, Tomati V, Bauer I, Salis A, Chiossone L, Mingari MC, Orecchia P, Carnemolla B, Neri D, Zardi L and Parodi S. Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.FASEB J. 2007 Nov 29

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