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Protein Scaffolds Proteingerüste Structures Protéiques (19) TZZ _ _T (11) EP 2 215 246 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/10 (2006.01) C12N 15/62 (2006.01) 07.01.2015 Bulletin 2015/02 C07K 14/78 (2006.01) C40B 40/10 (2006.01) (21) Application number: 08845766.8 (86) International application number: PCT/US2008/012398 (22) Date of filing: 31.10.2008 (87) International publication number: WO 2009/058379 (07.05.2009 Gazette 2009/19) (54) PROTEIN SCAFFOLDS PROTEINGERÜSTE STRUCTURES PROTÉIQUES (84) Designated Contracting States: US-B1- 6 482 410 US-B1- 6 818 418 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • KOIDE A ET AL: "The fibronectin type III domain RO SE SI SK TR as a scaffold for novel binding proteins", JOURNAL OF MOLECULAR BIOLOGY, LONDON, (30) Priority: 31.10.2007 US 984209 P GB, vol. 284, no. 4, 11 December 1998 (1998-12-11), pages 1141-1151, XP004455886, (43) Date of publication of application: ISSN: 0022-2836, DOI: DOI:10.1006/JMBI. 11.08.2010 Bulletin 2010/32 1998.2238 • BATORI V ET AL: "Exploring the potential of the (73) Proprietor: MedImmune, LLC monobody scaffold:effects of loop elongation on Gaithersburg, MD 20878 (US) the stability of a fibronectin type III domain", PROTEIN ENGINEERING, OXFORD UNIVERSITY (72) Inventors: PRESS, SURREY, GB, vol. 15, no. 12, 1 January • WU, Herren 2002 (2002-01-01), pages 1015-1020, Boyds,MD 20841 (US) XP002996047, ISSN: 0269-2139, DOI: DOI: • BACA, Manuel 10.1093/PROTEIN/15.12.1015 Gaithersburg,MD 20878 (US) • KOIDE AKIKO ET AL: "Monobodies: antibody •SWERS,Jeffrey mimics based on the scaffold of the fibronectin Rockville,MD 20852 (US) type III domain", METHODS IN MOLECULAR • CHACKO, Benoy BIOLOGY, HUMANA PRESS INC, NJ, US, vol. 352, Gaithersburg,MD 20878 (US) 1 January 2007 (2007-01-01), pages 95-109, XP009102789, ISSN: 1064-3745 (74) Representative: Ttofi, Evangelia • KARATAN E ET AL: "Molecular Recognition MedImmune Limited Properties of FN3 Monobodies that Bind the Src Milstein Building SH3 Domain", CHEMISTRY AND BIOLOGY, Granta Park CURRENT BIOLOGY, LONDON, GB, vol. 11, no. Cambridge 6, 1 June 2004 (2004-06-01), pages 835-844, Cambridgeshire CB21 6GH (GB) XP025940205, ISSN: 1074-5521, DOI: DOI: 10.1016/J.CHEMBIOL.2004.04.009 [retrieved on (56) References cited: 2004-06-25] WO-A2-2005/056764 US-A- 6 160 089 US-A1- 2006 073 559 US-A1- 2007 098 681 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 2 215 246 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 215 246 B1 • GILL D S ET AL: "Biopharmaceutical drug discovery using novel protein scaffolds", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 17, no. 6, 1 December 2006 (2006-12-01), pages653-658, XP024962817, ISSN: 0958-1669, DOI: DOI:10.1016/J.COPBIO. 2006.10.003 [retrieved on 2006-12-01] 2 EP 2 215 246 B1 Description 1. FIELD OF THE INVENTION 5 [0001] This invention relates to protein scaffolds that specifically bind a target and methods of making, screening and using such scaffolds. 2. BACKGROUND OF THE INVENTION 10 [0002] This invention relates to protein scaffolds useful, for example, for the generation of products having novel binding characteristics. [0003] Proteins having relatively defined three-dimensional structures, commonly referred to as protein scaffolds, may be used as reagents for the design of engineered products. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce 15 libraries of proteins from which desired products may be selected. One particular area in which such scaffolds are useful is the field of antibody mimetic design. [0004] While therapeutic antibodies are known with some successful examples on the market (HERCEPTIN®, AVAS- TIN®, SYNAGIS®), there is a growing interest in generating antibody fragments as therapeutic proteins. The advantages are the ease of manipulation by molecular biology techniques in order to obtain desired binding characteristics, the ability 20 to express such fragments in microbial systems, and the expectation that antibody fragments will have better tissue penetration than full-length antibodies. One example is REOPRO®. [0005] In addition, there have been efforts to develop small, non-antibody therapeutics,i.e., antibody mimetics, in order to capitalize on the advantages of antibodies and antibody fragments, such as high affinity binding of targets and low immunogenicity and toxicity, while avoiding some of the shortfalls, such as the requirement for intradomain disulfide 25 bonds which require proper refolding, and the tendency for antibody fragments to aggregate and be less stable than full-length IgGs. One example is a "minibody" scaffold, which is related to the immunoglobulin fold, which is designed by deleting three beta strands from a heavy chain variable domain of a monoclonal antibody (Tramontano et al., J. Mol. Recognit. 7:9, 1994). This protein includes 61 residues and can be used to present two hypervariable loops, much like complementarity determining regions (CDRs) in antibodies. These two loops have been randomized and products 30 selected for antigen binding, but thus far the framework appears to have somewhat limited utility due to solubility problems. Another framework used to display loops has been tendamistat, a small protein inhibitor of α-amylase, which contains a 74 residue, six-strand beta sheet sandwich held together by two disulfide bonds and forms 3 CDR-like loops (McConnell and Hoess, J. Mol. Biol. 250:460, 1995). [0006] Other proteins have been tested as frameworks and have been used to display randomized residues on alpha 35 helical surfaces (Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loops between alpha helices in alpha helix bundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfide bridges, such as those of the small protease inhibitors (Markland et al., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058, 1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol. Chem. 270:12250, 1995). 40 [0007] Koide A et al.: "The fibronectin type III domain as a scaffold for novel binding proteins", Journal Of Molecular Biology, vol. 284, no. 4, 11 December 1998 (1998-12-11), pages 1141-1151 describe preparation of a phage display library of FN3 in which residues in two surface loops were randomized. Mutant FN3s were selected that bound a test ligand, ubiquitin, with significant affinities; the wild-type FN3 showed no measurable affinity. [0008] Batori V et al.: "Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of 45 a fibronectin type III domain", Protein Engineering, vol. 15, no. 12, 1 January 2002, pages 1015-1020, describe devel- opment of small antibody mimics, ’monobodies’, using the tenth fibronectin type III domain of human fibronectin (FNfn10) as a scaffold. Initially, alterations were made in two loops of FNfn10 that were structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. To assess the possibility of utilizing other loops in FNfn10 for target binding, the effects of the elongation of each loop on the conformational stability of FNfn10 was investigated. The results 50 suggested that all loops, except for the EF loop, could be used for engineering a binding site. [0009] Koide Akiko et al.: "Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain", Methods in Molecular Biology, vol. 352, 1 January 2007, pages 95-109, described the use of the 10th fibronectin type III domain of human fibronectin (FNfn10) as a scaffold to display multiple surface loops for target binding. [0010] Karatan E et al.: "Molecular Recognition Properties of FN3 Monobodies that Bind the Src SH3 Domain", Chem- 55 istry and Biology, Current Biology, vol. 11, no. 6, 1 June 2004, pages 835-844, described construction of a phage- displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyse the recognition properties of selected binders. 3 EP 2 215 246 B1 [0011] WO 2005/056764 A2 describes single domain polypeptides that bind to vascular endothelial growth factor receptor 2 (VEGFR-2). [0012] Gill D S et al.: "Biopharmaceutical drug discovery using novel protein scaffolds", Current Opinion In Biotech- nology, vol. 17, no. 6, 1 December 2006, pages 653-658, reviewed the development of novel protein scaffold technologies, 5 including single-domain antibodies, small modular immunopharmaceuticals, tetranectins, AdNectins, A-domain proteins, lipocalins and ankyrin repeat proteins. [0013] US 6 818 418 B1 (Lipovsek et al.) discloses proteins that include a fibronectin type III domain having at least one randomized loop. [0014] Thus, there is a need to develop small, stable, artificial antibody-like molecules for a variety of therapeutic and 10 diagnostic applications. [0015] Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention. 3. SUMMARY OF THE INVENTION 15 [0016] The present invention provides a recombinant polypeptide scaffold comprising: I. seven beta strand domains designated A, B, C, D, E, F, and G; II.
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