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Transferrin Fusion Protein Libraries Transferrin-Fusionsproteinbibliotheken Banques De Proteines De Fusion De La Transferine

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Transferrin Fusion Protein Libraries Transferrin-Fusionsproteinbibliotheken Banques De Proteines De Fusion De La Transferine (19) & (11) EP 1 539 221 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 38/40 (2006.01) A61K 38/16 (2006.01) 23.12.2009 Bulletin 2009/52 A61K 38/41 (2006.01) C07K 14/79 (2006.01) (21) Application number: 03751903.0 (86) International application number: PCT/US2003/026779 (22) Date of filing: 28.08.2003 (87) International publication number: WO 2004/020588 (11.03.2004 Gazette 2004/11) (54) TRANSFERRIN FUSION PROTEIN LIBRARIES TRANSFERRIN-FUSIONSPROTEINBIBLIOTHEKEN BANQUES DE PROTEINES DE FUSION DE LA TRANSFERINE (84) Designated Contracting States: US-A1- 2003 221 201 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR • PARK E ET AL: "Production and characterization of fusion proteins containing transferrin and (30) Priority: 30.08.2002 US 406977 P nerve growth factor" JOURNAL OF DRUG 10.03.2003 US 384060 TARGETING, HARWOOD ACADEMIC 09.07.2003 US 485404 P PUBLISHERS GMBH, DE, vol. 6, no. 1, 1998, pages 53-64, XP002960815 ISSN: 1061-186X (43) Date of publication of application: • PARISE F ET AL: "Construction and in vitro 15.06.2005 Bulletin 2005/24 functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a (73) Proprietor: Biorexis Pharmaceutical Corporation therapeutic tool for familial New York, NY 10017-5755 (US) hypercholesterolemia" HUMAN GENE THERAPY, MARY ANN LIEBERT, NEW YORK ,NY, US, vol. (72) Inventors: 10, no. 7, 1 May 1999 (1999-05-01), pages • PRIOR, Christopher, P. 1219-1228, XP009057500 ISSN: 1043-0342 New York, NY 10017-5755 (US) • SHIN ET AL.: ’Transferin-antibody fusion • TURNER, Andrew, J. proteins are effective in brain targeting’ PROC. Durham NG 27713 (US) NATL. ACAD. SCI. USA vol. 92, March 1995, pages • SADEGHI, Homayoun 2820 - 2824, XP001126925 Hillsborough, NC 27278 (US) • ALI ET AL.: ’Trasnferin trojan horses as a rational approach for the biological delivery of (74) Representative: Cripps, Joanna Elizabeth et al therapeutic peptide domains’ J. BIOL. CHEM. vol. Mewburn Ellis LLP 274, no. 24, 20 August 1999, pages 24066 - 24073, 33 Gutter Lane XP002985527 London • GALLOP ET AL.: ’Applications of combinatorial EC2V 8AS (GB) technologies to drug discovery. 1. Background and peptide combinatorial libraries’ J. MED. (56) References cited: CHEM. vol. 37, no. 9, 29 April 1994, pages 1234 - WO-A-03/020746 US-A- 5 026 651 1251, XP002911078 US-A- 5 672 683 US-A- 5 986 067 • MACGILLIVRAY ET AL.: ’The primary structure of human serum transferin’ J. BIOL. CHEM. vol. 258, no. 6, 25 March 1983, pages 3543 - 3553, XP002985528 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 539 221 B1 Printed by Jouve, 75001 PARIS (FR) EP 1 539 221 B1 Description RELATED APPLICATIONS 5 [0001] This application claims priority to U.S. Provisional Application 60/485,404, filed July 9, 2003, U.S. Patent Ap- plication 10/384,060, filed March 10, 2003, and U.S. Provisional Application 60/406,997, filed August 30, 2002. FIELD OF THE INVENTION 10 [0002] The present invention relates to phage libraries containing proteins or peptides with extended serum stability and/or in vivo circulatory half-life, particularly to proteins or peptides fused to or inserted in a transferrin molecule modified to reduce or inhibit transferrin receptor binding. BACKGROUND OF THE INVENTION 15 [0003] Therapeutic proteins or peptides in their native state or when recombinantly produced are typically labile mol- ecules exhibiting short periods of serum stability or short in vivo circulatory half-lives. In addition, these molecules are often extremely labile when formulated, particularly when formulated in aqueous solutions for diagnostic and therapeutic purposes. 20 [0004] Few practical solutions exist to extend or promote the stability in vivo or in vitro of proteinaceous therapeutic molecules. Polyethylene glycol (PEG) is a substance that can be attached to a protein, resulting in longer-acting, sustained activity of the protein. If the activity of a protein is prolonged by the attachment to PEG, the frequency that the protein needs to be administered may be decreased. PEG attachment, however, often decreases or destroys the protein’s therapeutic activity. While in some instance PEG attachment can reduce immunogenicity of the protein, in other instances 25 it may increase immunogenicity. [0005] Therapeutic proteins or peptides have also been stabilized by fusion to a protein capable of extending the in vivo circulatory half-life of the therapeutic protein. For instance, therapeutic proteins fused to albumin or to antibody fragments may exhibit extended in vivo circulatory half-life when compared to the therapeutic protein in the unfused state. See U.S. Patents 5,876,969 and 5,766,883. 30 [0006] Another serum protein, glycosylated human transferrin (Tf) has also been used to make fusions with therapeutic proteins to target delivery to the interior of cells or to carry agents across the blood-brain barrier. These fusion proteins comprising glycosylated human Tf have been used to target nerve growth factor (NGF) or ciliary neurotrophic factor (CNTF) across the blood-brain barrier by fusing full-length Tf to the agent. See U.S. Patents 5,672,683 and 5977,307. In these fusion proteins, the Tf portion of the molecule is glycosylated and binds to two atoms of iron, which is required 35 for Tf binding to its receptor on a cell and, according to the inventors of these patents, to target delivery of the NGF or CNTF moiety across the blood-brain barrier. Park et al (Journal of Drug Targeting Vol 6, No.1 pp 53- 64) explores the ability to use genetic fusions of transferrin as carrier for brain targeting and delivery. Transferrin fusion proteins have also been produced by inserting an HIV-1 protease target sequence into surface exposed loops of glycosylated transferrin to investigate the ability to produce another form of Tf fusion for targeted delivery to the inside of a cell via the Tf receptor 40 (Ali et al. (1999) J. Biol. Chem. 274(34):24066-24073). See also Shin et al (Proc. Natl. Acad. Sci. USA Vol. 92, pp2820-2824). [0007] Serum transferrin (Tf) is a monomeric glycoprotein with a molecular weight of 80,000 daltons that binds iron in the circulation and transports it to various tissues via the transferrin receptor (TfR) (Aisen et al. (1980) Ann. Rev. Biochem. 49: 357-393; MacGillivray et al. (1981) J. Biol. Chem. 258: 3543-3553, U.S. Patent 5,026,651). Tf is one of 45 the most common serum molecules, comprising up to about 5-10% of total serum proteins. Carbohydrate deficient transferrin occurs in elevated levels in the blood of alcoholic individuals and exhibits a longer half life (approximately 14-17 days) than that of glycosylated transferrin (approximately 7-10 days). See van Eijk et al. (1983) Clin. Chim. Acta 132:167-171, Stibler (1991) Clin. Chem. 37:2029-2037 (1991), Arndt (2001) Clin. Chem. 47(1):13-27 and Stibler et al. in "Carbohydrate-deficient consumption", Advances in the Biosciences, (Ed Nordmann et al.), Pergamon, 1988, Vol. 71, 50 pages 353-357). [0008] The structure of Tf has been well characterized and the mechanism of receptor binding, iron binding and release and carbonate ion binding have been elucidated (U.S. Patents 5,026,651, 5,986,067 and MacGillivray et al. (1983) J. Biol. Chem. 258(6):3543-3546). [0009] Transferrin and antibodies that bind the transferrin receptor have also been used to deliver or carry toxic agents 55 to tumor cells as cancer therapy (Baselga and Mendelsohn, 1994), and transferrin has been used as a non-viral gene therapy vector to deliver DNA to cells (Frank et al., 1994; Wagner et al., 1992). The ability to deliver proteins to the central nervous system (CNS) using the transferrin receptor as the entry point has been demonstrated with several proteins and peptides including CD4 (Walus et al., 1996), brain derived neurotrophic factor (Pardridge et al., 1994), glial 2 EP 1 539 221 B1 derived neurotrophic factor (Albeck et al.), a vasointestinal peptide analogue (Bickel et al., 1993), a beta-amyloid peptide (Saito et al., 1995), and an antisense oligonucleotide (Pardridge et al., 1995). [0010] Transferrin fusion proteins have not, however, been modified or engineered to extend the in vivo circulatory half-life of a therapeutic protein nor peptide or to increase bioavailability by reducing or inhibiting glycosylation of the Tf 5 moiety nor to reduce or prevent iron and/or Tf receptor binding. SUMMARY OF THE INVENTION [0011] As described in more detail below, the present invention includes a phage library containing a plurality of fusion 10 proteins, each comprising a first transferrin (Tf) polypeptide fused to at least one second polypeptide, wherein the Tf peptide has reduced affinity for a transferrin receptor (TfR). [0012] In a preferred embodiment, the modified Tf fusion proteins comprise a human transferrin Tf moiety that has been modified to reduce or prevent iron and receptor binding and optionally glycosylation. 15 BRIEF DESCRIPTION OF THE DRAWINGS [0013] Figure 1 shows an alignment of the N and C Domains of Human (Hu) transferrin (Tf) (amino acids I-331 and 332-679 20 of SEQ ID NO: 3, respectively) with similarities and identities highlighted. Figure 2A-2B show an alignment of transferrin sequences from different animal species.
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