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Ep 2655624 B1 (19) TZZ _T (11) EP 2 655 624 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/62 (2006.01) C07K 16/46 (2006.01) 29.11.2017 Bulletin 2017/48 (86) International application number: (21) Application number: 11820905.5 PCT/US2011/066947 (22) Date of filing: 22.12.2011 (87) International publication number: WO 2012/088461 (28.06.2012 Gazette 2012/26) (54) LINKER PEPTIDES AND POLYPEPTIDES COMPRISING SAME LINKERPEPTIDE UND DIESE ENTHALTENDE POLYPEPTIDE PEPTIDES COUPLEURS ET POLYPEPTIDES LES COMPORTANT (84) Designated Contracting States: US-A1- 2002 103 353 AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • SHERIDAN DOUGLAS L ET AL: "A new way to PL PT RO RS SE SI SK SM TR rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro (30) Priority: 23.12.2010 US 201061426993 P transposition reaction", BMC NEUROSCIENCE, BIOMED CENTRAL, LONDON, GB, vol. 3, no. 1, (43) Date of publication of application: 19 June 2002 (2002-06-19), page 7, XP021015026, 30.10.2013 Bulletin 2013/44 ISSN: 1471-2202, DOI: 10.1186/1471-2202-3-7 • DATABASE UniProt [Online] 23 October 2007 (73) Proprietor: Biogen MA Inc. (2007-10-23), "SubName: Cambridge, MA 02142 (US) Full=AGAP006375-PA;", XP002676589, retrieved from EBI accession no. UNIPROT:A7UU88 (72) Inventors: Database accession no. A7UU88 -& HOLT R A ET • MILLER, Brian, Robert AL: "The Genome Sequence of the Malaria San Diego, CA 92122 (US) Mosquito Anopheles gambiae", SCIENCE, • GLASER, Scott AMERICAN ASSOCIATION FOR THE San Diego, CA 92107 (US) ADVANCEMENT OF SCIENCE, WASHINGTON, • CARAVELLA, Justin DC; US, vol. 298, 4 October 2003 (2003-10-04), Cambridge, MA 02141 (US) pages 129-149, XP002254396, ISSN: 0036-8075, • FOLEY, Susan DOI: 10.1126/SCIENCE.1076181 Milford, MA 01757 (US) • WANG R ET AL: "Enhancement of engineered • HRONOWSKI, Xiaoping trifunctional enzyme by optimizing linker Bedford, MA 01730 (US) peptides for degradation of agricultural • AIVAZIAN, Tigran, Dikran by-products", ENZYME AND MICROBIAL San Diego, CA 92130 (US) TECHNOLOGY, STONEHAM, MA, US, vol. 47, no. 5, 6 October 2010 (2010-10-06), pages 194-199, (74) Representative: Miller, David James XP027234468, ISSN: 0141-0229 [retrieved on Mathys & Squire LLP 2010-08-22] The Shard • VOELKEL T ET AL: "Optimized linker sequences 32 London Bridge Street for the expression of monomeric and dimeric London SE1 9SG (GB) bispecific single-chain antibodies", PROTEIN ENGINEERING, OXFORD UNIVERSITY PRESS, (56) References cited: SURREY, GB, vol. 14, no. 10, 1 October 2001 WO-A1-2010/010051 WO-A2-2005/017121 (2001-10-01), pages815-823, XP002185481, ISSN: WO-A2-2007/137279 WO-A2-2010/138803 0269-2139, DOI: 10.1093/PROTEIN/14.10.815 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 2 655 624 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 655 624 B1 • PING LU ET AL: "Bifunctional enhancement of a • PIHKALA P ET AL: "An antigen-mediated Î-glucanase-xylanase fusion enzyme by selection system for mammalian cells that optimization of peptide linkers", APPLIED produce glycosylated single-chain Fv", MICROBIOLOGY AND BIOTECHNOLOGY, BIOCHEMICAL AND BIOPHYSICAL RESEARCH SPRINGER, BERLIN, DE, vol. 79, no. 4, 16 April COMMUNICATIONS, ACADEMIC PRESS INC. 2008 (2008-04-16) , pages 579-587, XP019623621, ORLANDO, FL, US, vol. 324, no. 4, 26 November ISSN: 1432-0614 2004 (2004-11-26), pages 1165-1172, • HOEDEMAEKER F J ET AL: "A single chain Fv XP004609138, ISSN: 0006-291X, DOI: fragment of P-glycoprotein-specific monoclonal 10.1016/J.BBRC.2004.09.178 antibody C219. Design, expression, and crystal • ROCH C ET AL: "Differences in gene expression structure at 2.4 A resolution", JOURNAL OF of human xylosyltransferases and determination BIOLOGICAL CHEMISTRY, THE AMERICAN of acceptor specificities for various SOCIETY OF BIOLOGICAL CHEMISTS, INC, US, proteoglycans", BIOCHEMICAL AND vol. 272, no. 47, 21 November 1997 (1997-11-21), BIOPHYSICAL RESEARCH COMMUNICATIONS, pages 29784-29789, XP002225635, ISSN: ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 0021-9258, DOI: 10.1074/JBC.272.47.29784 391, no. 1, 1 January 2010 (2010-01-01), pages • S-H WANG ET AL: "Construction of single chain 685-691, XP026907965, ISSN: 0006-291X, DOI: variable fragment (scFv) and BiscFv-alkaline 10.1016/J.BBRC.2009.11.121 [retrieved on phosphatase fusion protein for detection of 2009-11-26] Bacillus anthracis", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 78, no. 4,15 February 2006 (2006-02-15), pages997-1004, XP002464471, ISSN: 0003-2700, DOI: 10.1021/AC0512352 • SCHOUTEN A ET AL: ’Improving scFv antibody expression levels in the plant cytosol’ FEBS LETTERS, ELSEVIER, AMSTERDAM, NL vol. 415, no. 2, 29 September 1997, pages 235 - 241, XP002298966 ISSN: 0014-5793 2 EP 2 655 624 B1 Description Background of the Invention 5 [0001] The application of protein engineering techniques to fusion protein design has produced a number of formats that have been shown to have altered, and in some cases, improved pharmacodynamic, biodistribution, and activity profiles. Linker peptides are frequently an important part of these constructs. [0002] Linker peptides are synthetic sequences of amino acids that are commonly used to physically connect polypep- tide domains. Most linker peptides are composed of repetitive modules of one or more of the amino acids glycine and 10 serine. The standard 15 amino acid (GGGGS)3 linker peptide has been well-characterized (e.g., within the context of an antibody single-chain Fv (scFv) domain) and has been shown to adopt an unstructured, flexible conformation. In addition, this linker peptide does not interfere with assembly and binding activity of the domains it connects. (Freund, C. et al., 1993. FEBS 320:97). [0003] Unfortunately, when polypeptide constructs comprising linker peptides are expressed in a host cell, the linker 15 peptides can serve as substrates for post-translational modification. Such post-translational modification can create heterogeneity in the protein product produced. Thus, new linker peptides that reduce or eliminate the addition of post- translational modifications would enable the production of more homogenous polypeptide preparations; such homoge- neous preparations are critical for clinically relevant polypeptides. Holt et al. (2002) Science 298(5591):129-49 describes a study to determine the genome sequence of the malaria mosquitoAnopheles gambiae. WO 2010/138803 and WO 20 2007/137279 provide methods and compositions for detecting, preventing and treating HERV-K + cancers. Schouten et al. (1997) FEBS Lett. 415(2):235-41 reports an improvement in scFv antibody expression levels in the plant cytosol. Pihkala et al. (2004) Biochem Biophys Res Commun. 324(4):1165-72 describes an antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv. WO 2010/010051 relates to single-chain fusion proteins comprising three soluble TNF superfamily (TNFSF) cytokine domains and nucleic acid molecules encoding the fusion 25 proteins. Roch et al. (2010) Biochem Biophys Res Commun. 391(1): 685-91 investigates differences in gene expression of human xylosyltransferases and determination of acceptor specificities for various proteoglycans. Summary of the Invention 30 [0004] The invention is based, at least in part, on the finding that linker peptides which lack the amino acid sequence GSG reduce or eliminate the addition of post-translational modifications to the polypeptides which comprise them. More specifically, the novel linker peptides disclosed herein reduce the ability of enzymes to link carbohydrate adducts to polypeptides comprising these linker peptides, e.g., reduce the ability of xylosyltransferase to link xylose to polypeptides. Surprisingly, this is true even though the amino acid sequence GSG alone was not previously thought to be sufficient 35 for xylosyltransferase-mediated addition of xylose to proteins. Moreover, polypeptides comprising the linker peptides disclosed herein have been found to exhibit reduced aggregation and increased pH stability. Thus, the inclusion of the linker peptides disclosed herein in polypeptide molecules leads to increased homogeneity, increased pH stability and reduced aggregation. Based on the disclosure that is contained herein, the present invention provides a binding molecule comprising an scFv 40 moiety and an Fc moiety, wherein the scFv moiety and the Fc moiety are genetically linked by a linker peptide that comprises an amino acid sequence selected from the group consisting of: (GGGGA)nGGGGS; (GGGGQ)2GGGGS; 45 (GGGPS)2GGGGS; GGGGS(PGGGS)2; and (GGGGA)2GGGGS; wherein n is greater than 1, and wherein the linker peptide lacks the sequence GSG and reduces or eliminates the 50 addition of a post-translational modification to the binding molecule upon expression in a host cell, wherein the post- translational modification is the addition of a xylose adduct. [0005] In a further aspect, the invention provides an scFv molecule comprising a VH and a VL region, wherein the VH and VL region are genetically linked by a linker peptide, wherein the linker peptide comprises an amino acid sequence selected from the group consisting of: 55 (GGGGA)nGGGGS; (GGGGQ)2GGGGS; (GGGPS)2GGGGS; 3 EP 2 655 624 B1 GGGGS(PGGGS)2; (GGGGA)2GGGGS; (GGGGA)3; and (GGGGA)4, 5 wherein n is greater than 1, and wherein the linker peptide lacks the sequence GSG and reduces or eliminates the addition of a post-translational modification to the binding molecule upon expression in a host cell, wherein the post- translational modification is the addition of a xylose adduct.
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