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Institute of Biosciences and Bioresources

National Research Council of Italy

Maddalena De Virgilio

Role: Researcher
Section: Researchers and Technologists
Division: Bari
Tel: (39) 0805583400-214
E-mail: maddalena.devirgilio@ibbr.cnr.it


Project: (TOXIC CHIMERAS): Pichia pastoris as a host for secretion of toxic saporin chimeras for the targeted therapy of human cancer.

Brief project description

Plant and bacterial toxins have been widely used for the production of tumor-directed chimeras in which a targeting moiety, generally an antibody fragment or a receptor ligand, is linked to a catalytic, toxic domain. Compared to classical inhibitors that diffuse into the whole organism and potentially affect most if not all types of cells, chimeric toxins have the major advantage of providing a targeted treatment of tumor cells without affecting healthy ones.

Among plant toxins, Ribosome Inactivating Proteins (RIPs), such as saporin, are potent inhibitors of protein synthesis and induce apoptosis in intoxicated mammalian cells. Compared to other plant RIPs, such as ricin, saporin has received less attention for clinical application. Clinical trials with saporin-containing immunotoxins showed the efficacy of these molecules and their potential for application in the therapy of haematological malignancies. Notably no drug-related toxicity were reported in this studies.

A major bottleneck in the development of recombinant toxins is the choice of an appropriate expression system. In this regard both prokaryotic and eukaryotic hosts have been used. Pichia pastoris is a microbial host that recapitulates many of the co- and postranslational events that occur in the ER of mammalian cells, allowing correctly folded polypeptides to reach the extracellular medium. Recently it has been shown that the P. pastoris (strain GS115) can tolerate the expression and secretion of high amount of saporin.The secreted recombinant proteins were purified from yeast culture medium and found to be endowed with the expected biological activity.

Therefore we have at our disposal the biotechnological tools to produce sufficient amount of recombinant chimeric toxins for pre-clinical and clinical investigation on the targeted therapy of human cancer.

Project aim

The chimeras ATF-saporin and OPG-saporin have been already expressed and purified from Pichia pastoris medium by ion exchange chromatography.

ATF-saporin consisting of the aminoterminal fragment (ATF) of human urokinase was fused to the plant toxin saporin, in order to target the human urokinase receptor (uPAR) over expressed at the surface of many different tumoral cells.

We have also observed that this chimera efficiently induces apoptosis in human monocytic leukaemia and human myeloma cell lines.

OPG-saporin consisting af saporin and osteoprotegerin, a soluble ligand of CD138 receptor overexpressed at the surface of human myeloma plasmacells ,as expected, efficiently induces apoptosis exclusively in myeloma cell lines expressing the CD138 receptor, as deduced from preliminary observation. In addition, the OPG-saporin induces apotosis to higher extent on myeloma cell lines, as compared to ATF-saporin.

We intend to provide sufficient amount of purified recombinant toxins for testing on animal models.

Pubblications :

de Virgilio M., Degryse B .Harnessing the destructive power of ricin to fight human cancer. In: Ricin Toxin. Cherwonogrodzky J., Betham Science Publishers, (accepted).

de Virgilio M, Lombardi A, Caliandro R, Fabbrini MS. Ribosome Inactivating Proteins: from plant defense to tumor attak. Toxins, 2010, 2: 2699-737.

de Virgilio M, Silvestris F. Urokinase receptor (uPAR) ligand based recombinant toxins for human cancer therapy. Curr Pharm Des, 2011, 17: 1979-83.

Avesani L., Vitale A., Pedrazzini L., de Virgilio M., Pompa A., Barbante A., Gecchele E., Dominaci P., Morandini F., Brozzetti A., Falorni A., Pezzetti M. Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant.

Plant Biotechnol J., 2010 8: 862-72

Foresti O., De Marchis F., de Virgilio M., Klein E.M., Arcioni S., Bellucci M., Vitale A.Protein domains involved in assembly in the endoplasmic reticulum promote vacuolar delivery when fused to secretory GFP, indicating a protein quality control pathway for degradation in the plant vacuole.

Molecular Plant 2008 1: 1067-1076.

de Virgilio M., Francesca De Marchis, Michele Bellucci, Davide Mainieri,Marika Rossi, Eugenio Benvenuto, Sergio Arcioni and Alessandro VitaleThe Human Immunodeficiency Virus Antigen Nef Forms Protein Bodies in Leaves of Transgenic Tobacco When Fused to Zeolin. J. Exp. Bot. 2008 59: 2815-29

 

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