(19) TZZ Z¥_T (11) EP 2 205 639 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 16/40 (2006.01) A61K 39/395 (2006.01) 23.12.2015 Bulletin 2015/52 A61P 3/06 (2006.01) (21) Application number: 08841231.7 (86) International application number: PCT/US2008/081311 (22) Date of filing: 27.10.2008 (87) International publication number: WO 2009/055783 (30.04.2009 Gazette 2009/18) (54) ANTI-PCSK9 AND METHODS FOR TREATING LIPID AND CHOLESTEROL DISORDERS ANTI-PCSK9 UND VERFAHREN ZUR BEHANDLUNG VON STÖRUNGEN DES LIPID- UND CHOLESTERINHAUSHALTES ANTI-PCSK9 ET MÉTHODES DE TRAITEMENT DE TROUBLES DU MÉTABOLISME LIPIDIQUE ET DU CHOLESTÉROL (84) Designated Contracting States: (74) Representative: Horgan, James Michael Frederic AT BE BG CH CY CZ DE DK EE ES FI FR GB GR et al HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT Merck & Co., Inc. RO SE SI SK TR European Patent Department Designated Extension States: Hertford Road AL BA MK RS Hoddesdon EN11 9BU (GB) (30) Priority: 26.10.2007 US 982922 P (56) References cited: (43) Date of publication of application: WO-A2-2008/057457 WO-A2-2008/057458 14.07.2010 Bulletin 2010/28 WO-A2-2008/057459 WO-A2-2008/125623 (73) Proprietor: Merck Sharp & Dohme Corp. • LAGACE THOMAS A ET AL: "Secreted PCSK9 Rahway, NJ 07065-0907 (US) decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice" (72) Inventors: JOURNAL OF CLINICAL INVESTIGATION, • HEDRICK, Joseph A. AMERICAN SOCIETY FOR CLINICAL South River INVESTIGATION, US,vol. 116, no. 11, 1 November New Jersey 08882 (US) 2006 (2006-11-01), pages 2995-3005, • MONSMA, Frederick James, Jr. XP002493243 ISSN: 0021-9738 Summit • BENJANNET SUZANNE ET AL: "The proprotein New Jersey 07901 (US) convertase (PC) PCSK9 is inactivated by furin • CHURAKOVA, Tatyana and/or PC5/6A: functional consequences of Sunnyvale natural mutations and post-translational California 94086 (US) modifications" JOURNAL OF BIOLOGICAL • HOLLENBAUGH, Diane CHEMISTRY, AMERICAN SOCIETY OF Mountain View BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,; California 94040 (US) US, vol. 281, no. 41, 13 October 2006 (2006-10-13), pages 30561-30572, XP002508324 ISSN: 0021-9258 [retrieved on 2006-08-15] 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 2 205 639 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 205 639 B1 • CAYMAN CHEMICAL COMPANY: "Product • HORTON ET AL: "Molecular biology of PCSK9: Information PCSK9(murine) Polyclonal Antibody its role in LDL metabolism" TRENDS IN Catalog No. 10008811" INTERNET CITATION 12 BIOCHEMICAL SCIENCES, ELSEVIER, December 2007 (2007-12-12), pages 1-2, HAYWARDS, GB, vol. 32, no. 2, 6 February 2007 XP002517273 Retrieved from the Internet: URL: (2007-02-06), pages 71-77, XP005876225 ISSN: http://www.caymaneurope.com/pdfs/10008 0968-0004 811.pdf> [retrieved on 2009-02-27] • "Chapter 1: Antibodies binding antigens" In: Tai • CAYMAN CHEMICAL COMPANY: "Product Te Wu: "Analytical molecular biology", 2001 Information PCSK9 Polyclonal Antibody Catalog pages 1-36, No. 10007185" INTERNET CITATION 10 • TOMLINSON I M ET AL: "The repertoire of human December 2007 (2007-12-10), pages 1-2, germlinevH sequences reveals about fifty groups XP002517274 Retrieved from the Internet: URL: of VH segments with different hypervariable http://www.caymaneurope.com/pdfs/10007 loops", JOURNAL OF MOLECULAR BIOLOGY, 185.pdf> [retrieved on 2009-02-27] ACADEMIC PRESS, UNITED KINGDOM, vol. 227, • CAYMAN CHEMICAL COMPANY: "Material no.3, 5 October 1992 (1992-10-05), pages 776-798, Safety Data Sheet PCSK9 (murine) Polyclonal XP024020607, ISSN: 0022-2836, DOI: Antibody" INTERNET CITATION 5 September 10.1016/0022-2836(92)90223-7 [retrieved on 2007 (2007-09-05), pages 1-4, XP002517275 1992-10-05] Retrieved from the Internet: URL:http: • CHANG B ET AL: "A sequence analysis of human //www.caymaneurope.com/msdss/1000 germline Ig VH and VL genes. The CDR1s of a 8811m.pdf> [retrieved on 2009-02-27] major proportion of VH, but not VL, genes display • CAYMAN CHEMICAL COMPANY: "Material a high inherent susceptibility to amino acid Safety Data Sheet PCSK9 (human) Polyclonal replacement.", ANNALS OF THE NEW YORK Antibody" INTERNET CITATION 26 July 2007 ACADEMY OF SCIENCES 29 SEP 1995, vol. 764, (2007-07-26), pages 1-3, XP002517276 Retrieved 29 September 1995 (1995-09-29), pages 170-179, from the Internet: URL:http: ISSN: 0077-8923 //www.caymaneurope.com/msdss/1000 7185m.pdf> [retrieved on 2009-02-27] 2 EP 2 205 639 B1 Description Field of the Invention 5 [0001] The field of the present invention relates to methods and compositions for treating disorders of cholesterol homeostasis by administering an anti-PCSK9 antibody or antigen-binding fragment thereof. Background of the Invention 10 [0002] Atheroscleroticcoronary heart disease(CHD) represents themajor cause for death and cardiovascular morbidity in the western world. Risk factors for atherosclerotic coronary heart disease include hypertension, diabetes mellitus, family history, male gender, cigarette smoke, high serum cholesterol, high low density lipoprotein (LDL) cholesterol levels and low high density lipoprotein (HDL) cholesterol levels. In general, a total cholesterol level in excess of about 225-250 mg/dl is associated with significant elevation of risk of CHD. 15 [0003] A variety of clinical studies have demonstrated that elevated levels of total cholesterol or LDL cholesterol promote human atherosclerosis. Epidemiologic investigations have established that cardiovascular morbidity and mor- tality vary directly with the level of total cholesterol and LDL cholesterol. [0004] One method for lowering LDL cholesterol levels is by administration of HMG-CoA reductase inhibiting drugs. These drugs antagonize HMG-CoA reductase and cholesterol synthesis in the liver and increase the number of hepatic 20 LDL receptors on the cell-surface to enhance uptake and catabolism of LDL. A drawback of such an approach is that these drugs commonly suffer from a disadvantageous side-effect profile, including, for example, liver toxicity. An alternate approach is to modulate the LDL receptor pathway directly. [0005] PCSK9 (proprotein convertase subtilisin/kexin type 9) is a serine protease family member that binds to and regulates LDL receptor expression on the surface of cells. Inhibition of the LDL receptor-PCSK9 interaction is an attractive 25 approach to the treatment of cholesterol disorders. Inhibition of interactions between large proteins ( i.e., protein-protein interactions or PPI) by the use of antibodies or small molecule inhibitors is, however, generally regarded as being particularly difficult and challenging. Large proteins such as PCSK9, with a molecular weight of about 74 KDa, and LDLR, with a molecular weight of about 160 KDa (glycosylated on cell surface; 115 KDa in immature form), are likely to exhibit extensive intermolecular contacts over a large area. The existence of extensive contacts makes it unlikely that a given 30 antibody or small molecule inhibitor will successfully block their binding. WO2008/057457 discloses several neutralizing anti-PCSK9 antibodies. Summary of the Invention 35 [0006] The present invention surprisingly has overcome the technical difficulties associated with the blocking of inter- molecular interactions between large proteins and has demonstrated that blockage of the PCSK9-LDLR interaction with an antibody or peptide is possible. As discussed in detail herein, this, in turn, provides a novel method by which to treat cholesterol disorders. [0007] The present invention discloses in part, a method for reducing total cholesterol level, low density lipoprotein 40 cholesterol level, apolipoprotein B level, total cholesterol/high density lipoprotein ratio or low density lipoprotein/high density lipoprotein ratio, in a subject (e.g., a human), comprising administering, to said subject, a therapeutically effective amount of an antibody or antigen-binding fragment thereof ( e.g., monoclonal antibody, polyclonal antibody or recombinant antibody) or EGF-A polypeptide that binds specifically to PCSK9 which antibody or fragment or polypeptide inhibits binding between PCSK9 and LDL receptor; optionally in association with a further chemotherapeutic agent(e.g., 45 ezetimibe and/or simvastatin). In an embodiment of the invention, the antibody or fragment or EGF-A polypeptide binds specifically to a PCSK9 catalytic domain or to a domain of PCSK9 which interacts with an LDL receptor EGF-A domain. [0008] The present invention further discloses in part, a method for treating or preventing hypercholesterolemia, hy- perlipidemia, hypertriglyceridaemia, sitosierolemia, atherosclerosis, arteriosclerosis, coronary heart disease, vascular inflammation or xanthoma, in an subject, comprising administering, to said subject, a therapeutically effective amount 50 of an antibody or antigen-binding fragment thereof (e.g., monoclonal antibody, polyclonal antibody or recombinant an- tibody) or EGF-A polypeptide that binds specifically to PCSK9, which antibody or fragment or polypeptide inhibits binding between PCSK9 and LDL receptor; optionally in association with a further therapeutic agente.g ( ., ezetimibe