US 200400.58321A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0058321 A1 BrunkOW et al. (43) Pub. Date: Mar. 25, 2004 (54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data INCREASING BONE MINERALIZATION (63) Continuation of application No. 09/449,218, filed on (75) Inventors: Mary E. Brunkow, Seattle, WA (US); Nov. 24, 1999, now Pat. No. 6,395,511. David J. Galas, Claremont, CA (US); Brian Kovacevich, Renton, WA (US); (60) Provisional application No. 60/110,283, filed on Nov. John T. Mulligan, Seattle, WA (US); 27, 1998. Bryan Paeper, Seattle, WA (US); Jeffrey Van Ness, Claremont, CA (US); Publication Classification David G. Winkler, Seattle, WA (US) (51) Int. Cl. ............................ C12O 1/68; CO7H 21/04; Correspondence Address: A61K 39/395; C12P 21/02; SEED INTELLECTUAL PROPERTY LAW C12N 5/06; CO7K 16/22 GROUP PLLC (52) U.S. Cl. ......................... 435/6; 435/69.1; 435/320.1; 701 FIFTHAVE 435/325; 530/388.25; 424/145.1; SUTE 6300 536/23.5 SEATTLE, WA 98104-7092 (US) (57) ABSTRACT (73) Assignee: Darwin Discovery Ltd., Slough (GB) A novel class or family of TGF-B binding proteins is (21) Appl. No.: 10/095,248 disclosed. Also disclosed are assays for Selecting molecules for increasing bone mineralization and methods for utilizing (22) Filed: Mar. 7, 2002 Such molecules. Patent Application Publication Mar. 25, 2004 Sheet 1 of 6 US 2004/0058321 A1 Common Cysteine Backbone 1. 50 human-gremlin.pro human-Cerberus pro MHLLLFOLLY LLPLGKTTRH ODGRONOSSL SPYLLPRNOR ELPTGNHEEA human-dan pro re-asawwara reserwrwarrara's swarara-as-a-Wiswe sawsWawswaas awaawawa-a-a-ay human-beer pro 51 100 human-gremlin.pro MSRTAYTVGALLLLLGTLLPA AEGKKKGSOG human-CerberuS. pro EEKPDLFVAY PHLVAT.SPAGEGOROREKMLSRFGRFWKK PEREMHPSRD human-dan-pro human-beer pro M M M MMOLPLA LCLYCLLVHT 101 150 human-gremlin.pro AI.PPPDKAQ HNDSEQTOSP OQPGSRNRGR GOGRGTAMPG EEYLESSQEA human-CerberuS. pro SDSEPFPPGT OSLIOPIDG MKMEKSPLRE EAKKFWHHFM FRKTPASQGV human-dan-pro MRVLVGAYL PAMLAAPPP human-beer.pro AFRYVEGOGW QAFKNDATEI IPELGEYPEP PPELENNKTM NRAENGGRPP 151 Y 200 human-gremlin.pro HVTERKYLK RDWCKTOPLK GTIHEEGCNS RIINRF.CY GOCNSFYIPR human-CerberUS. pro ILPIKSHEVH WETCRTYPFS QTITHEGCEK VVVONNLCF GKCGSVHFP. human-dan.pro INKLALFPDK SAWCEAKNIT QIVGHSGCEA KSIONRACL GOCFSYSVPN human-beer.pro HHPFETKDVS EYSCRELHFTRYVTDGPCRS AKPVTELVCS GOCGPARLLP 201 250 human-gremlin.pro HIRKEEGSFQ SCSF.CKP KKF TMMVT NCPELOPPTK K.KRVTRYKQ human-CerberuS. pro GAAQHSHT SCSH. CLP AKFTIMHLPL NCTELSSVIK V.VMLVEE human-dan-pro TFPOSTESLV HCDS. CMP AOSMWEIVTL ECPGHEEVPR YDKLVEKILH human-beer pro NAIGRGKWWR PSGPDFRCIP DRYRAORVOLLCPGGEAPRA RKVRLYAS. s 300 human-gremlin.pro CRC, ISIDLD Aawawawawa.AMAa human-Cerberus. pro COCKYKTEHE DGHILHAGSO DSFIPGVSA- ~~~~~~~~~~~ human-dan-pro CSCOACGKEP SHEGLSYYYO GEDGPGSOPG THPHPHPHPHPGGOTPEPED human-beer pro CKCKRLTRFH NOSELKDFGT EAARPOKGRK PRPRARSAKA NOAELENAY 301 314 human-gremlin, pro human-CerberuS. pro Awarwaw-w human-dan, pro PPGAPHTEEE GAED human-beer, pro ww.waw Fig. 1 Patent Application Publication Mar. 25, 2004 Sheet 2 of 6 US 2004/0058321 A1 Human Beer Gene Expression by RT-PCR BEER beta-actin ig. 2 Patent Application Publication Mar. 25, 2004 Sheet 3 of 6 US 2004/0058321 A1 Patent Application Publication Mar. 25, 2004 Sheet 4 of 6 US 2004/0058321 A1 Antibody Selectivity Anti Anti Anti Beer Don Gremlin Co BEER Protein S3 3se seS S3 seS s (75 ng/lane) Fig. 4A 50 kD 34 kD 28 kD 20 kD Anti Anti Anti Gremlin Beer Dan O Gremlin Protein S3 3s S S S S3 (75 ng/lane) : * : * : : 34 kD Fig.f 4B 28 kD 20 kD Anti Anti Anti Don Beer Gremlion . S s S 8 S 8 Dan Protein 3 S S 3 Se 3 (75 ng/lane) 54 kD 28 kD 20 kD Patent Application Publication Mar. 25, 2004 Sheet 5 of 6 US 2004/0058321 A1 07-dW80+-+ -1998++---•• –snuºqu30-++…-, : s g: ?ae?**¿ 3:is . Patent Application Publication Mar. 25, 2004 Sheet 6 of 6 US 2004/0058321 A1 BMP-5/Beer Dissociation Constant Characterization 75 15 7.5 15 50 60 120 nM. BMP-5 i. *Anti-FLAG immunoprecipitation Anti-BMP-5 Western blot lonic Disruption of BMP-5/Beer Binding NaCI(mlM) 500 150 150 lo Beer - A. BMP-5 *Anti-FLAG immunoprecipitation Anti-BMP-5 Western blot Fig. 6 US 2004/0058321 A1 Mar. 25, 2004 COMPOSITIONS AND METHODS FOR patient pool in the United States). The increased fragility and INCREASING BONE MINERALIZATION Susceptibility to fracture of skeletal bone in the aged is aggravated by the greater risk of accidental falls in this CROSS-REFERENCE TO RELATED population. More than 1.5 million osteoporosis-related bone APPLICATIONS fractures are reported in the United States each year. Frac tured hips, wrists, and vertebrae are among the most com 0001. This application claims priority from U.S. Provi mon injuries associated with Osteoporosis. Hip fractures in sional Application No. 60/110,283 filed Nov. 27, 1998, particular are extremely uncomfortable and expensive for which application is incorporated by reference in its entirety. the patient, and for women correlate with high rates of TECHNICAL FIELD mortality and morbidity. 0002 The present invention relates generally to pharma 0006 Although osteoporosis has been defined as an ceutical products and methods and, more specifically, to increase in the risk of fracture due to decreased bone mass, methods and compositions Suitable for increasing the min none of the presently available treatments for skeletal dis eral content of bone. Such compositions and methods may orders can Substantially increase the bone density of adults. be utilized to treat a wide variety of conditions, including for There is a Strong perception among all physicians that drugs example, osteopenia, Osteoporosis, fractures and other dis are needed which could increase bone density in adults, orders in which low bone mineral density are a hallmark of particularly in the bones of the wrist, Spinal column and hip the disease. that are at risk in Osteopenia and osteoporosis. 0007 Current strategies for the prevention of osteoporo BACKGROUND OF THE INVENTION sis may offer Some benefit to individuals but cannot ensure resolution of the disease. These Strategies include moderat 0003. Two or three distinct phases of changes to bone ing physical activity (particularly in weight-bearing activi mass occur over the life of an individual (see Riggs, West J. ties) with the onset of advanced age, including adequate Med. 154:63-77, 1991). The first phase occurs in both men calcium in the diet, and avoiding consumption of products and women, and proceeds to attainment of a peak bone mass. containing alcohol or tobacco. For patients presenting with This first phase is achieved through linear growth of the clinical osteopenia or Osteoporosis, all current therapeutic endochondral growth plates, and radial growth due to a rate drugs and Strategies are directed to reducing further loSS of of perioSteal apposition. The Second phase begins around bone mass by inhibiting the process of bone absorption, a age 30 for trabecular bone (flat bones such as the vertebrae natural component of the bone remodeling process that and pelvis) and about age 40 for cortical bone (e.g., long occurs constitutively. bones found in the limbs) and continues to old age. This phase is characterized by Slow bone loSS, and occurs in both 0008 For example, estrogen is now being prescribed to men and women. In Women, a third phase of bone loSS also retard bone loSS. There is, however, Some controversy over occurs, most likely due to postmenopausal estrogen defi whether there is any long term benefit to patients and ciencies. During this phase alone, women may lose an whether there is any effect at all on patients over 75 years additional 10% of bone mass from the cortical bone and 25% old. Moreover, use of estrogen is believed to increase the from the trabecular compartment (see Riggs, Supra). risk of breast and endometrial cancer. 0004 Loss of bone mineral content can be caused by a 0009 High doses of dietary calcium, with or without wide variety of conditions, and may result in Significant Vitamin D has also been Suggested for postmenopausal medical problems. For example, Osteoporosis is a debilitat Women. However, high doses of calcium can often have ing disease in humans characterized by marked decreases in unpleasant gastrointestinal Side effects, and Serum and uri skeletal bone mass and mineral density, Structural deterio nary calcium levels must be continuously monitored (see ration of bone including degradation of bone microarchitec Khosla and Rigss, Mayo Clin. Proc. 70:978-982, 1995). ture and corresponding increases in bone fragility and SuS 0010. Other therapeutics which have been suggested ceptibility to fracture in afflicted individuals. Osteoporosis include calcitonin, bisphosphonates, anabolic Steroids and in humans is preceded by clinical osteopenia (bone mineral Sodium fluoride. Such therapeutics however, have undesir density that is greater than one Standard deviation but leSS able side effects (e.g., calcitonin and Steroids may cause than 2.5 standard deviations below the mean value for young nausea and provoke an immune reaction, bisphosphonates adult bone), a condition found in approximately 25 million and Sodium fluoride may inhibit repair of fractures, even people in the United States. Another 7-8 million patients in though bone density increases modestly) that may prevent the United States have been diagnosed with clinical their usage (see Khosla and RigSS, Supra). osteoporosis (defined as bone mineral content greater than 2.5 Standard deviations below that of mature young adult 0011 No currently practiced therapeutic strategy bone). Osteoporosis is one of the most expensive diseases involves a drug that Stimulates or enhances the growth of for the health care System, coSting tens of billions of dollars new bone mass. The present invention provides composi annually in the United States. In addition to health care tions and methods which can be utilized to increase bone related costs, long-term residential care and lost working mineralization, and thus may be utilized to treat a wide days add to the financial and Social costs of this disease.