(19) TZZ _ _T (11) EP 2 681 242 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 16/18 (2006.01) A61K 39/00 (2006.01) 24.01.2018 Bulletin 2018/04 C07K 16/22 (2006.01) (21) Application number: 12708472.1 (86) International application number: PCT/US2012/027154 (22) Date of filing: 29.02.2012 (87) International publication number: WO 2012/118903 (07.09.2012 Gazette 2012/36) (54) SCLEROSTIN AND DKK-1 BISPECIFIC BINDING AGENTS SCLEROSTIN- UND DKK-1-BISPEZIFISCHE BINDEMITTEL AGENTS LIANTS BISPÉCIFIQUEMENT SCLÉROSTINE ET DKK-1 (84) Designated Contracting States: (74) Representative: J A Kemp AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 14 South Square GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Gray’s Inn PL PT RO RS SE SI SK SM TR London WC1R 5JJ (GB) Designated Extension States: BA ME (56) References cited: WO-A1-2009/047356 WO-A2-2006/015373 (30) Priority: 01.03.2011 US 201161448089 P WO-A2-2006/119062 WO-A2-2009/149189 05.05.2011 US 201161482979 P WO-A2-2012/058393 (43) Date of publication of application: • HOLT L J ET AL: "Domain antibodies: proteins 08.01.2014 Bulletin 2014/02 for therapy", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, (73) Proprietor: Amgen Inc. vol. 21, no. 11, 1 November 2003 (2003-11-01), Thousand Oaks, California 91320-1799 (US) pages 484-490, XP004467495, ISSN: 0167-7799, DOI: 10.1016/J.TIBTECH.2003.08.007 (72) Inventors: • DAVIES J ET AL: "Affinity improvement of single • FLORIO, Monica antibody VH domains: residues in all three Thousand Oaks, CA 91362 (US) hypervariable regions affect antigen binding", • ARORA, Taruna IMMUNOTECHNOLOGY, ELSEVIER SCIENCE Thousand Oaks, CA 91320 (US) PUBLISHERS BV, NL, vol. 2, no. 3, 1 September • PASZTY, Christopher, J., R. 1996 (1996-09-01), pages 169-179, XP004070292, Ventura, CA 93003 (US) ISSN: 1380-2933, DOI: • RICHARDS, William, G. 10.1016/S1380-2933(96)00045-0 Thousand Oaks, CA 91362-4921 (US) • TILMAN D RACHNER ET AL: "Osteoporosis: now and the future", THE LANCET, vol. 377, no. 9773, 1 April 2011 (2011-04-01), pages 1276-1287, XP55031012, ISSN: 0140-6736, DOI: 10.1016/S0140-6736(10)62349-5 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 2 681 242 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 681 242 B1 Description Field of the Invention 5 [0001] The invention relates to bispecific anti-sclerostin/anti-DKK1 binding agents and combinations of anti-sclerostin and anti-DKK1 binding agents, and related treatments. Background of the Invention 10 [0002] Two or three distinct phases of changes to bone mass occur over the life of an individual (see Riggs, West J. Med. 154:63 77 (1991)). The first phase occurs in both men and women and proceeds to attainment of a peak bone mass. This first phase is achieved through linear growth of the endochondral growth plates and radial growth due to a rate of periosteal apposition. The second phase begins around age 30 for trabecular bone (flat bones such as the vertebrae and pelvis) and about age 40 for cortical bone (e.g., long bones found in the limbs) and continues to old age. 15 This phase is characterized by slow bone loss and occurs in both men and women. In women, a third phase of bone loss also occurs, most likely due to postmenopausal estrogen deficiencies. During this phase alone, women may lose additional bone mass from the cortical bone and from the trabecular compartment (see Riggs, supra). [0003] Loss of bone mineral content can be caused by a wide variety of conditions and may result in significant medical problems. For example, osteoporosis is a debilitating disease in humans and is characterized by marked decreases in 20 skeletal bone mass and mineral density, structural deterioration of bone, including degradation of bone microarchitecture and corresponding increases in bone fragility (i.e., decreases in bone strength), and susceptibility to fracture in afflicted individuals. Osteoporosis in humans is generally preceded by clinical osteopenia (bone mineral density that is greater than one standard deviation but less than 2.5 standard deviations below the mean value for young adult bone), a condition found in approximately 25 million people in the United States. Another 7 8 million patients in the United States have 25 been diagnosed with clinical osteoporosis (defined as bone mineral content greater than 2.5 standard deviations below that of mature young adult bone). The frequency of osteoporosis in the human population increases with age. Among Caucasians, osteoporosis is predominant in women who, in the United States, comprise 80% of the osteoporosis patient pool. The increased fragility and susceptibility to fracture of skeletal bone in the aged is aggravated by the greater risk of accidental falls in this population. Fractured hips, wrists, and vertebrae are among the most common injuries associated 30 with osteoporosis. Hip fractures in particular are extremely uncomfortable and expensive for the patient, and for women, correlate with high rates of mortality and morbidity. [0004] Although osteoporosis has been regarded as an increase in the risk of fracture due to decreased bone mass, few of the presently available treatments for skeletal disorders can increase the bone density of adults, and most of the presently available treatments work primarily by inhibiting further bone resorption rather than stimulating new bone 35 formation. Estrogen is now being prescribed to retard bone loss. However, some controversy exists over whether patients gain any long term benefit and whether estrogen has any effect on patients over 75 years old. Moreover, use of estrogen is believed to increase the risk of breast and endometrial cancer. Calcitonin, osteocalcin with vitamin K, or high doses of dietary calcium, with or without vitamin D, have also been suggested for postmenopausal women. High doses of calcium, however, often have undesired gastrointestinal side effects, and serum and urinary calcium levels must be 40 continuously monitored (e.g., Khosla and Riggs, Mayo Clin. Proc. 70:978982, 1995). [0005] Other current therapeutic approaches to osteoporosis include bisphosphonates (e.g., Fosamax™, Actonel™, Bonviva™, Zometa™, olpadronate, neridronate, skelid, bonefos), parathyroid hormone, calcilytics, calcimimetics (e.g., cinacalcet), statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. Such therapeutics, however, are often associated with undesirable side effects (see Khosla and Riggs, supra). 45 [0006] Sclerostin, the product of the SOST gene, is absent in sclerosteosis, a skeletal disease illustrated by bone overgrowth and strong dense bones (Brunkow et al., Am. J. Hum. Genet., 68:577 589, 2001; Balemans et al., Hum. Mol. Genet., 10:537 543, 2001). Inhibitors of sclerostin have been shown to increase the rate of bone mineralization, and thus bone mineral density (Padhi et al., J Bone Miner Res. 2010 Jun; e-published ahead of print). WO 2009/047356 discloses compositions and methods for use of antibodies against sclerostin. Likewise, Dkk-1 has been shown to be 50 involved in the regulation of bone formation, particularly in bone fracture repair, and its role in various other diseases that are associated with bone loss (e.g., cancer and diabetes) (Komatsu et al., J. Orthop. Res. 2010 Jul;28(7):928-36; Gavriatolpoulou et al., Expert Opin. Ther. Targets. 2009 Jul;13(7):839-48). [0007] Dickkopf-1 (Dkk-1) is a secreted protein that participates in embryonic head induction and antagonizes Wnt (Glinka et al., Nature 391: 357-362 (1998)). The amino acid sequence of human Dkk-1 and nucleotides encoding it have 55 been described (U.S. Pat. Nos. 6,344,541; 6844422; 7,057,017; Published Patent Application No. 20050069915; Krup- nick et al., Gene 238: 301-313(1999)). Expression of Dkk-1 in human was thought to be restricted to placenta, suggesting a role for Dkk-1 in embryonic development (Krupnick et al., supra). Allen and colleagues (U.S. Published Patent Appli- cation No. 20040038860) describe assays relating to the interaction between LRP5, HBM or LRP6 with Dkk-1. Antibodies 2 EP 2 681 242 B1 that bind Dkk-1 have been described in the aforementioned patents and patent applications and in U.S. Published patent Application Nos. 20050079173 and 20060127393. [0008] Human Dkk-1 is a member of a Dickkopf gene family which includes Dkk-1, Dkk-2, Dkk-3, and Dkk4 (Krupnick et al., supra). Although Dkk-1 and Dkk-4 have been shown to suppress Wnt-induced secondary axis induction in Xenopus 5 embryos, neither block axis induction triggered by Xenopus Dishevelled or Frizzled, suggesting that their Wnt inhibitory activity is upstream of Frizzled in the Wnt signaling pathway (Krupnick et al., supra). It has been suggested that Dkk-1 might have an inhibitory effect on bone formation, making them potential targets for the prevention or treatment of osteoporosis (Patel and Karensky, N. Eng. J. Med. 346: 1572-1573 (2002); Boyden et al., N. Eng. J. Med. 346: 1513-1521 (2002)). 10 [0009] Wu et al., (mAbs 1:4, 339-347 (2009)) discloses molecular construction and optimization of antihuman IL-1 α/β dual variable domain immunoglobulin (DVD-Ig™) molecules. SUMMARY 15 [0010] Disclosed herein are novel inhibitors that are effective in treating conditions requiring increased bone building, for example, fracture repair or bone loss associated with pathological conditions, such as multiple myeloma.