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Engineering human antibody derivatives, which specifically recognise and ablate new blood vessels, for the therapy of angiogenesis-related pathologies

   
Project

QLK3-2001-01495

Cell factory area

3.1.3

EU Contribution

1 677 851 Euro

Duration

36 months

Type

Research project

Starting date

01-12-2001

Keywords
angiogenesis
phage Ab display libraries
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ABSTRACT
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Angiogenesis is a typical process that underlies cancer, rheumatoid arthritis and the majority of blinding ocular disorders. The ability to selectively target and/or ablate new blood vessels will open diagnostic and therapeutic opportunities. Recently, our network has collaborated in the isolation of antibodies (Ab) which selectively recognise new-forming blood vessels. Two good-quality markers of angiogenesis have been identified. The corresponding high-affinity phage Ab, efficiently targets tumoural neovasculature invivo. We now propose to chemically modify anti-angiogenesis Ab and to test the biomedical potential of their conjugates. In particular, we will use radionuclides and fluorophores to investigate the potential of our labelled Ab for image diagnostics. We will also couple them to drugs, cytokines etc. and evaluate their therapeutic potential in suitable animal models. We will continue to search for novel markers of angiogenesis.

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OBJECTIVES
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Our network has already been able to produce antibodies (Ab) that target new blood vessels in vivo, while sparing the pre-existing ones. We plan to use our Ab against the ED-B domain of fibronectin and the C-domain of tenascin C, two markers of angiogenesis, to pursue the following objectives:

  • Use radionuclides and fluorophores to investigate the potential of anti-angiogenesis Ab for image diagnostics. Our goal is to establish (in animal models, and eventually by immunoscintigraphy in cancer patients) whether anti-angiogenesis Ab can improve the early detection of angiogenesis-related diseases.
  • Couple Ab to drugs, cytokines etc. and evaluate their therapeutic potential in animal models. Preliminary experiments performed by us in animal models of both tumoural and non-tumoural angiogenesis indicate that therapeutic benefits are to be expected.
  • Continue to search for novel angiogenesis markers, and to produce specific Ab using human Ab phage display libraries.

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DESCRIPTION OF THE WORK
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Two human antibody fragments (AF), "L19" and "TN11", with sub-nanomolar affinity for markers of angiogenesis have been produced and characterised by us (A. Pini et al., J.B.C. 273, 21769-76 (1998); B. Carnemolla et al., Am. J. Pathol, 154, 1345-52 (1999); F. Viti et al., Cancer Res., 59, 347-52 (1999); Tarli et al., Blood 94, 1, 192-8 (1999). For both AF, the recombinant antigens are available, which facilitate purification by affinity chromatography and immunoreactivity determination.

These AF will be used to perform the following experiments:

In vivo imaging:

  • a) High-specific radio-labelling of AF. Labelling experiments at activities >5 mCi/mg AF with I-123 and Tc-99m are essential to warrant future clinical investigations.
  • b) Biodistribution of tumour models in animals, using radio-labelled AF.
  • c) Imaging of ocular angiogenesis, using fluorescently labelled AF and immunophotodetection (D. Neri et al., Nature Biotech., 13, 1271-5).
  • Therapy experiments:
  • d) Conjugation of AF to I.R. photosensitizers. Immunophotodynamic therapy in animal models of tumoural and non-tumoural angiogenesis.
  • e) Conjugation of AF to doxorubicin (a standard chemotherapeutic agent toxic for endothelial cells), using linkers that mediate the slow drug release. Evaluation of the anti-tumour activity of the conjugates.
  • f) Cloning/expression in eukaryotic cells of antibody-cytokine fusions (IL-2, IL-12, TNF-a, IFN-g). Evaluation of therapeutic performances in syngeneic murine tumour models, by monitoring tumour size and by histochemistry.
  • g) Cloning/expression and in vivo testing of antibody-(truncated tissue factor) fusion proteins, which are expected to mediate the selective blood coagulation in tumoural neovasculature. Novel markers of angiogenesis: In a previous Biotech-2 Project, we aimed at discovering novel markers of angiogenesis using a combination of 2D-gel electrophoresis and antibody phage technology. We plan to continue the activity of this project to ensure that the best markers are used.

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DELIVERABLES
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  • Methods for the high-activity radioactive and fluorescent labelling of antibodies of antibody fragments or smaller binding molecules.
  • In vivo evaluation of labelled antibodies for imaging of angiogenesis-related diseases.
  • Antibody derivatives for therapy experiments.
  • In vivo evaluation of the therapeutic performance of antibody derivatives.
  • Samples of endothelial cells for the discovery of novel markers of angiogenesis.
  • 2D-gel electrophoretic analysis of angiogenesis samples.
  • Panning of antibody phage display libraries on angiogenesis samples.

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CONSORTIUM
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COORDINATOR
  Prof. Paolo Neri
Biologia Molecolare
Università degli Studi di Siena
53100 Siena, Italy
Tel: +39 0577234904/913
Fax: +39 0577234903
Nerip@unisi.it

PARTNERS
  Dr. Ludger Dinkelborg
DG R Molecular Imaging
New research fields
Schering Aktiengesellschaft
13342 Berlin, Germany
Tel: +49 3046817404
Fax: +49 3046816609
ludger.dinkelborg@schering.de

Prof. Hartwig Kosmehl
Institute of Pathology
Klinikum der Friedrich-Schiller
Universitaet Jena
07740 Jena, Germany
Tel: +49 3641933195
Fax: +49 3641933411
kosmehl@bach.med.uni-jena.de

Prof. Luciano Zardi
Biologia Cellulare
Istituto Nazionale per la
Ricerca sul Cancro
16132 Genova, Italy
Tel: +39 0105600413
Fax: +39 010258032
lzardi@cisi.unige.it

Duccio Neri
Philogen S.r.L.
53100 Siena, Italy
Tel: +39 057742038
Fax: +39 057742151
philogen@tin.it

Prof. Dario Neri
Institut für Pharmazeutische
Wissenschaften
Swiss Federal Institute of
Technology Zürich
8057 Zürich, Switzerland
Tel: +41 16356063
Fax: +41 16356886
neri@pharma.anbi.ethz.ch
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