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 Sse 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. variety of conditions where it is desired to increase bone Worldwide approximately 75 million people are at risk for mass. Further, the present invention provides other, related Osteoporosis. advantages. 0005 The frequency of osteoporosis in the human popu SUMMARY OF THE INVENTION lation increases with age, and among Caucasians is pre 0012. As noted above, the present invention provides a dominant in women (who comprise 80% of the osteoporosis novel class or family of TGF-beta binding-proteins, as well US 2004/0058321 A1 Mar. 25, 2004 as assays for Selecting compounds which increase bone examples of Suitable promoters include tissue-specific pro mineral content and bone mineral density, compounds which moters, and viral-based promoters (e.g., CMV-based pro increase bone mineral content and bone mineral density and moters such as CMV I-E, SV40 early promoter, and Mul V methods for utilizing Such compounds in the treatment or LTR). Expression vectors may also be based upon, or prevention of a wide variety of conditions. derived from Viruses (e.g., a “viral vector”). Representative examples of Viral vectors include herpes simplex viral 0013 Within one aspect of the present invention, isolated vectors, adenoviral vectors, adenovirus-associated Viral vec nucleic acid molecules are provided, wherein Said nucleic tors and retroviral vectors. Also provided are host cells acid molecules are Selected from the group consisting of: (a) containing or comprising any of above-noted vectors an isolated nucleic acid molecule comprising Sequence ID (including for example, host cells of human, monkey, dog, NoS. 1, 5, 7, 9, 11, 13, or, 15, or complementary Sequence rat, or mouse origin). thereof; (b) an isolated nucleic acid molecule that specifi cally hybridizes to the nucleic acid molecule of (a) under 0017 Within other aspects of the present invention, conditions of high Stringency; and (c) an isolated nucleic methods of producing TGF-beta binding-proteins are pro acid that encodes a TGF-beta binding-protein according to Vided, comprising the Step of culturing the aforementioned (a) or (b). Within related aspects of the present invention, host cell containing vector under conditions and for a time isolated nucleic acid molecules are provided based upon sufficient to produce the TGF-beta binding protein. Within hybridization to only a portion of one of the above-identified further embodiments, the protein produced by this method Sequences (e.g., for (a) hybridization may be to a probe of may be further purified (e.g., by column chromatography, at least 20, 25, 50, or 100 nucleotides selected from nucle affinity purification, and the like). Hence, isolated proteins otides 156 to 539 or 555 to 687 of Sequence ID No. 1). As which are encoded by the above-noted nucleic acid mol should be readily evident, the necessary Stringency to be ecules (e.g., Sequence ID NOS. 2, 4, 6, 8, 10, 12, 14, or 16) utilized for hybridization may vary based upon the size of may be readily produced given the disclosure of the Subject the probe. For example, for a 25-mer probe high Stringency application. conditions could include: 60 mM Tris pH 8.0, 2 mM EDTA, 0018. It should also be noted that the aforementioned 5xDenhardt's, 6xSSC. 0.1% (w/v) N-laurylsarcosine, 0.5% proteins, or fragments thereof, may be produced as fusion (w/v) NP-40 (nonidet P-40) overnight at 45 degrees C., proteins. For example, within one aspect fusion proteins are followed by two washes with with 0.2xSSC/0.1% SDS at provided comprising a first polypeptide Segment comprising 45-50 degrees. For a 100-mer probe under low stringency a TGF-beta binding-protein encoded by a nucleic acid conditions, Suitable conditions might include the following: molecule as described above, or a portion thereof of at least 5xSSPE, 5xDenhardt's, and 0.5% SDS overnight at 42-50 10, 20, 30, 50, or 100 amino acids in length, and a second degrees, followed by two washes with 2xSSPE (or 2xSSC)/ polypeptide Segment comprising a non-TGF-beta binding 0.1% SDS at 42-50 degrees. protein. Within certain embodiments, the Second polypep 0.014 Within related aspects of the present invention, tide may be a tag Suitable for purification or recognition isolated nucleic acid molecules are provided which have (e.g., a polypeptide comprising multiple anionic amino acid homology to Sequence ID Nos. 1, 5, 7, 9, 11, 13, or 15, at residues-see U.S. Pat. No. 4,851,341), a marker (e.g., a 50%, 60%, 75%, 80%, 90%, 95%, or 98% level of green fluorescent protein, or alkaline phosphatase), or a homology utilizing a Wilbur-Lipman algorithm. Represen toxic molecule (e.g., ricin). tative examples of Such isolated molecules include, for 0019. Within another aspect of the present invention, example, nucleic acid molecules which encode a protein antibodies are provided which are capable of Specifically comprising Sequence ID NOS. 2, 6, 10, 12, 14, or 16, or have binding the above-described class of TGF-beta binding homology to these sequences at a level of 50%, 60%, 75%, proteins (e.g., human BEER). Within various embodiments, 80%, 90%, 95%, or 98% level of homology utilizing a the antibody may be a polyclonal antibody, or a monoclonal Lipman-Pearson algorithm. antibody (e.g., of human or murine origin). Within further 0.015 Isolated nucleic acid molecules are typically less embodiments, the antibody is a fragment of an antibody than 100 kb in size, and. within certain embodiments, less which retains the binding characteristics of a whole antibody than 50 kb, 25 kb, 10 kb, or even 5 kb in size. Further, (e.g., an F(ab'), F(ab), Fab', Fab, or FV fragment, or even isolated nucleic acid molecules, within other embodiments, a CDR). Also provided are hybridomas and other cells which do not exist in a “library” of other unrelated nucleic acid are capable of producing or expressing the aforementioned molecules (e.g., a Subclone BAC Such as described in antibodies. GenBank Accession No. ACOO3098 and EMB No. 0020. Within related aspects of the invention, methods AQ171546). However, isolated nucleic acid molecules can are provided detecting a TGF-beta binding protein, com be found in libraries of related molecules (e.g., for shuffling, prising the Steps of incubating an antibody as described Such as is described in U.S. Pat. Nos. 5,837,458; 5,830,721; above under conditions and for a time Sufficient to permit and 5,811.238). Finally, isolated nucleic acid molecules as said antibody to bind to a TGF-beta binding protein, and described herein do not include nucleic acid molecules detecting the binding. Within various embodiments the which encode Dan, Cerberus, Gremlin, or SCGF (U.S. Pat. antibody may be bound to a Solid Support to facilitate No. 5,780,263). washing or separation, and/or labeled, (e.g., with a marker 0016. Also provided by the present invention are cloning Selected from the group consisting of enzymes, fluorescent vectors which contain the above-noted nucleic acid mol proteins, and radioisotopes). ecules, and expression vectors which comprise a promoter 0021 Within other aspects of the present invention, iso (e.g., a regulatory Sequence) operably linked to one of the lated oligonucleotides are provided which hybridize to a above-noted nucleic acid molecules. Representative nucleic acid molecule according to Sequence ID NOS. 1, 3, US 2004/0058321 A1 Mar. 25, 2004

5, 7, 9, 11, 13, 15, 17, or 18 or the complement thereto, under one or more nucleic acid analogs, ribonucleic acids, or conditions of high stringency. Within further embodiments, deoxyribonucleic acids. Further, the oligonucleotide may be the oligonucleotide may be found in the Sequence which modified by one or more linkages, including for example, encodes Sequence ID Nos. 2, 4, 6, 8, 10, 12, 14, or 16. covalent linkage Such as a phosphorothioate linkage, a Within certain embodiments, the oligonucleotide is at least phosphotriester linkage, a methyl phosphonate linkage, a 15, 20, 30, 50, or 100 nucleotides in length. Within further methylene(methylimino) linkage, a morpholino linkage, an embodiments, the oligonucleotide is labeled with another amide linkage, a polyamide linkage, a short chain alkyl molecule (e.g., an enzyme, fluorescent molecule, or radio interSugar linkage, a cycloalkyl interSugar linkage, a short isotope). Also provided are primers which are capable of chain heteroatomic interSugar linkage and a heterocyclic Specifically amplifying all or a portion of the above-men interSugar linkage. One representative example of a chi tioned nucleic acid molecules which encode TGF-beta bind meric oligonucleotide is provied in U.S. Pat. No. 5,989,912. ing-proteins. AS utilized herein, the term "specifically ampli fying should be understood to refer to primers which 0026. Within yet another aspect of the present invention, amplify the aforementioned TGF-beta binding-proteins, and methods are provided for increasing bone mineralization, not other TGF-beta binding proteins such as Dan, Cerberus, comprising introducing into a warm-blooded animal an effective amount of the ribozyme as described above. Within Gremlin, or SCGF (U.S. Pat. No. 5,780,263). related aspects, Such methods comprise the Step of intro 0022 Within related aspects of the present invention, ducing into a patient an effective amount of the nucleic acid methods are provided for detecting a nucleic acid molecule molecule or vector as described herein which is capable of which encodes a TGF-beta binding protein, comprising the producing the desired ribozyme, under conditions favoring Steps of incubating an oligonucleotide as described above transcription of the nucleic acid molecule to produce the under conditions of high Stringency, and detecting hybrid ribozyme. ization of Said oligonucleotide. Within certain embodiments, the oligonucleotide may be labeled and/or bound to a Solid 0027. Within other aspects of the invention transgenic, Support. non-human animals are provided. Within one embodiment a transgenic animal is provided whose germ cells and Somatic 0023. Within other aspects of the present invention, cells contain a nucleic acid molecule encoding a TGF-beta ribozymes are provided which are capable of cleaving RNA binding-protein as described above which is operably linked which encodes one of the above-mentioned TGF-beta bind to a promoter effective for the expression of the gene, the ing-proteins (e.g., Sequence ID NOS. 2. 6, 8, 10, 12, 14, or gene being introduced into the animal, or an ancestor of the 16). Such ribozymes may be composed of DNA, RNA animal, at an embryonic Stage, with the proviso that Said (including 2'-O-methyl ribonucleic acids) nucleic acid ana animal is not a human. Within other embodiments, trans logs (e.g., nucleic acids having phosphorothioate linkages) genic knockout animals are provided, comprising an animal or mixtures thereof. Also provided are nucleic acid mol whose germ cells and Somatic cells comprise a disruption of ecules (e.g., DNA or cDNA) which encode these ribozymes, at least one allele of an endogenous nucleic acid molecule and vectors which are capable of expressing or producing which hybridizes to a nucleic acid molecule which encodes the ribozymes. Representative examples of Vectors include a TGF-binding protein as described herein, wherein the plasmids, retrotransposons, cosmids, and viral-based vectors disruption prevents transcription of messenger RNA from (e.g., viral vectors generated at least in part from a retrovi Said allele as compared to an animal without the disruption, rus, adenovirus, or, adeno-associated virus). Also provided with the proviso that the animal is not a human. Within are host cells (e.g., human, dog, rat, or mouse cells) which various embodiments, the disruption is a nucleic acid dele contain these vectors. In certain embodiments, the host cell tion, Substitution, or, insertion. Within other embodiments may be stably transformed with the vector. the transgenic animal is a mouse, rat, sheep, pig, or dog. 0024. Within further aspects of the invention, methods 0028. Within further aspects of the invention, kits are are provided for producing ribozymes either Synthetically, or provided for the detection of TGF-beta binding-protein gene by in vitro or in vivo transcription. Within further embodi expression, comprising a container that comprises a nucleic ments, the ribozymes So produced may be further purified acid molecule, wherein the nucleic acid molecule is Selected and/or formulated into pharmaceutical compositions (e.g., from the group consisting of (a) a nucleic acid molecule the ribozyme or nucleic acid molecule encoding the comprising the nucleotide sequence of SEQ ID NOs: 1, 3, 5, ribozyme along with a pharmaceutically acceptable carrier 7, 9, 11, 13, or 15; (b) a nucleic acid molecule comprising or diluent). Similarly, the antisense oligonucleotides and the complement of the nucleotide sequence of (a); (c) a antibodies or other Selected molecules described herein may nucleic acid molecule that is a fragment of (a) or (b) of at be formulated into pharmaceutical compositions. least 15, 2030, 50, 75, or, 100 nucleotides in length. Also w provided are kits for the detection of a TGF-beta binding 0.025. Within other aspects of the present invention, anti protein which comprise a container that comprise one of the Sense oligonucleotides are provided comprising a nucleic TGF-beta binding protein antibodies described herein. acid molecule which hybridizes to a nucleic acid molecule according to Sequence ID NOS. 1, 3, 5, 7, 9, 11, 13, or 15, 0029. For example, within one aspect of the present or the complement thereto, and wherein Said oligonucleotide invention methods are provided for determining whether a inhibits the expression of TGF-beta binding protein as Selected molecule is capable of increasing bone mineral described herein (e.g., human BEER). Within various content, comprising the steps of (a) mixing one or more embodiments, the oligonucleotide is 15, 20, 25, 30, 35, 40, candidate molecules with TGF-beta-binding-protein or 50 nucleotides in length. Preferably, the oligonucleotide encoded by the nucleic acid molecule according to claim 1 is less than 100, 75, or 60 nucleotides in length. As should and a selected member of the TGF-beta family of proteins be readily evident, the oligonucleotide may be comprised of (e.g., BMP 5 or 6), (b) determining whether the candidate US 2004/0058321 A1 Mar. 25, 2004

molecule alters the signaling of the TGF-beta family mem home to the bone. Within a further embodiment, the cells ber, or alters the binding of the TGF-beta binding-protein to which home to bone are Selected from the group consisting the TGF-beta family member. Within certain embodiments, of CD34+ cells and osteoblasts. the molecule alters the ability of TGF-beta to function as a positive regulator of mesenchymal cell differentiation. 0035. Within other aspects of the present invention mol Within this aspect of the present invention, the candidate ecules are provided (preferably isolated) which inhibit the molecule(s) may alter Signaling or binding by, for example, binding of the TGF-beta binding-protein to the TGF-beta either decreasing (e.g., inhibiting), or increasing (e.g., Super-family of proteins. enhancing) signaling or binding. 0036 Within further embodiments, the molecules may be 0.030. Within yet another aspect, methods are provided provided as a composition, and can further comprise an for determining whether a Selected molecule is capable of inhibitor of bone resorption. Representative examples of increasing bone mineral content, comprising the Step of Such inhibitors include calcitonin, estrogen, a bisphospho determining whether a selected molecule inhibits the bind nate, a growth factor having anti-resorptive activity and ing of TGF-beta binding-protein to bone, or an analogue tamoxifen. thereof. Representative examples of bone or analogues 0037 Representative examples of molecules which may thereof include hydroxyapatite and primary human bone be utilized in the afore-mentioned therapeutic contexts Samples obtained via biopsy. include, e.g., ribozymes, ribozyme genes, antisense mol 0031. Within certain embodiments of the above-recited ecules, and/or antibodies (e.g., humanized antibodies). Such methods, the Selected molecule is contained within a mix molecules may depending upon their Selection, used to alter, ture of molecules and the methods may further comprise the antagonize, or agonize the Signalling or binding of a TGF Step of isolating one or more molecules which are functional beta binding-protein family member as described herein within the assay. Within yet other embodiments, TGF-beta 0038. Within various embodiments of the invention, the family of proteins is bound to a Solid Support and the binding above-described molecules and methods of treatment or of TGF-beta binding-protein is measured or TGF-beta bind prevention may be utilized on conditions Such as Osteoporo ing-protein are bound to a Solid Support and the binding of sis, osteomalasia, periodontal disease, Scurvy, Cushings TGF-beta proteins are measured. Disease, bone fracture and conditions due to limb immobi 0.032 Utilizing methods such as those described above, a lization and Steroid usage. wide variety of molecules may be assayed for their ability to 0039 These and other aspects of the present invention increase bone mineral content by inhibiting the binding of will become evident upon reference to the following detailed the TGF-beta binding-protein to the TGF-beta family of description and attached drawings. In addition, various proteins. Representative examples of Such molecules references are set forth herein which describe in more detail include proteins or peptides, organic molecules, and nucleic certain procedures or compositions (e.g., plasmids, etc.), and acid molecules. are therefore incorporated by reference in their entirety. 0.033 Within other related aspects of the invention, meth ods are provided for increasing bone mineral content in a BRIEF DESCRIPTION OF THE DRAWINGS warm-blooded animal, comprising the Step of administering to a warm-blooded animal atherapeutically effective amount 0040 FIG. 1 is a schematic illustration comparing the of a molecule identified from the assays recited herein. amino acid Sequence of Human Dan; Human Gremlin; Within another aspect, methods are provided for increasing Human Cerberus and Human Beer. Arrows indicate the bone mineral content in a warm-blooded animal, comprising Cysteine backbone. the Step of administering to a warm-blooded animal a 0041 FIG. 2 Summarizes the results obtained from Sur therapeutically effective amount of a molecule which inhib veying a variety of human tissues for the expression of a its the binding of the TGF-beta binding-protein to the TGF-beta binding-protein gene, Specifically, the Human TGF-beta Super-family of proteins, including bone morpho Beer gene. A Semi-quantitative Reverse Transcription-Poly genic proteins (BMPs). Representative examples of suitable molecules include antisense molecules, ribozymes, merase Chain Reaction (RT-PCR) procedure was used to ribozyme genes, and antibodies (e.g., a humanized antibody) amplify a portion of the gene from first-strand cDNA which specifically recognize and alter the activity of the synthesized from total RNA (described in more detail in TGF-beta binding-protein. EXAMPLE 2A). 0042 FIG.3 summarizes the results obtained from RNA 0034. Within another aspect of the present invention, in Situ hybridization of mouse embryo Sections, using a methods are provided for increasing bone mineral content in a warm-blooded animal, comprising the Steps of (a) intro cRNA probe that is complementary to the mouse Beer ducing into cells which home to the bone a vector which transcript (described in more detail in EXAMPLE 2B). directs the expression of a molecule which inhibits the Panel A is a transverse section of 10.5 dpc embryo. Panel B binding of the TGF-beta binding-protein to the TGF-beta is a Sagittal Section of 12.5 dpc embryo and panels C and D family of proteins and bone morphogenic proteins (BMPs), are Sagittal Sections of 15.5 dpc embryos. and (b) administering the vector-containing cells to a warm 0043 FIG. 4 illustrates, by western blot analysis, the blooded animal. As utilized herein, it should be understood specificity of three different polyclonal antibodies for their that cells “home to bone' if they localize within the bone respective antigens (described in more detail in EXAMPLE matrix after peripheral administration. Within one embodi 4). FIG. 4A shows specific reactivity of an anti-H. Beer ment, Such methods further comprise, prior to the Step of antibody for H. Beer antigen, but not H. Dan or H. Gremlin. introducing, isolating cells from the marrow of bone which FIG. 4B shows reactivity of an anti-H. Gremlin antibody for US 2004/0058321 A1 Mar. 25, 2004

H. Gremlin antigen, but not H. Beer or H. Dan. FIG. 4C phosphotransferase or hygromycin phosphotransferase. shows reactivity of an anti-H. Dan antibody for H. Dan, but Additionally, depending on the host cell chosen and the not H. Beer or H. Gremlin. vector employed, other genetic elements Such as an origin of 0044 FIG. 5 illustrates, by western blot analysis, the replication, additional nucleic acid restriction sites, enhanc selectivity of the TGF-beta binding-protein, Beer, for ers, Sequences conferring inducibility of transcription, and BMP-5 and BMP-6, but not BMP-4 (described in more Selectable markers, may also be incorporated into the vectors detail in EXAMPLE 5). described herein. 0053 An "isolated nucleic acid molecule' is a nucleic 004.5 FIG. 6 demonstrates that the ionic interaction acid molecule that is not integrated in the genomic DNA of between the TGF-beta binding-protein, Beer, and BMP-5 an organism. For example a DNA molecule that encodes a has a dissociation constant in the 15-30 nM range. TGF-binding protein that has been separated from the DETAILED DESCRIPTION OF THE genomic DNA of a eukaryotic cell is an isolated DNA molecule. Another example of an isolated nucleic acid INVENTION molecule is a chemically-Synthesized nucleic acid molecule Definitions that is not integrated in the genome of an organism. The isolated nucleic acid molecule may be genomic DNA, 0.046 Prior to setting forth the invention in detail, it may cDNA, RNA, or composed at least in part of nucleic acid be helpful to an understanding thereof to set forth definitions analogs. of certain terms and to list and to define the abbreviations 0054 An "isolated polypeptide' is a polypeptide that is that will be used hereinafter. essentially free from contaminating cellular components, 0047. “Molecule' should be understood to include pro Such as carbohydrate, lipid, or other proteinaceous impuri teins or peptides (e.g., antibodies, recombinant binding ties associated with the polypeptide in nature. Within certain partners, peptides with a desired binding affinity), nucleic embodiments, a particular protein preparation contains an acids (e.g., DNA, RNA, chimeric nucleic acid molecules, isolated polypeptide if it appears nominally as a single band and nucleic acid analogues Such as PNA); and organic or on SDS-PAGE gel with Coomassie Blue staining. “Iso inorganic compounds. lated”, when referring to organic molecules means that the 0048 “TGF-beta’ should be understood to include any compounds are greater than 90 percent pure utilizing meth known or novel member of the TGF-beta Super-family, ods which are well known in the art (e.g., NMR, melting which also includes bone morphogenic proteins (BMPs). point). 0049) “TGF-beta receptor” should be understood to refer 0055 “Sclerosteosis' Sclerosteosis is a term that was to the receptor Specific for a particular member of the applied by Hansen (1967) (Hansen, H. G., Sklerosteose In: TGF-beta Super-family (including bone morphogenic pro Opitz. H.; Schmid, F., Handbuch der Kinderheilkunde. Ber lin: Springer (pub.) 6 1967. Pp. 351-355) to a disorder teins (BMPs)). Similar to Van Buchem hyperoStosis corticalis generalisata 0050 “TGF-beta binding-protein” should be understood but possibly differing in radiologic appearance of the bone to refer to a protein with Specific binding affinity for a changes and in the presence of asymmetric cutaneous Syn particular member or subset of members of the TGF-beta dactyly of the indeX and middle fingers in many cases. The Super-family (including bone morphogenic proteins jaw has an unusually Square appearance in this condition. (BMPs)). Specific examples of TGF-beta binding-proteins include proteins encoded by Sequence ID Nos. 1, 5, 7, 9, 11, 0056 “Humanized antibodies” are recombinant proteins 13, and 15. in which murine complementary determining regions of monoclonal antibodies have been transferred from heavy 0051). Inhibiting the “binding of the TGF-beta binding and light variable chains of the murine immunoglobulin into protein to the TGF-beta family of proteins and bone mor a human variable domain. phogenic proteins (BMPs)” should be understood to refer to molecules which allow the activation of TGF-beta or bone 0057. As used herein, an “antibody fragment” is a portion morphogenic proteins (BMPs), or allow the binding of of an antibody such as F(ab'), F(ab), Fab', Fab, and the TGF-beta family members including bone morphogenic like. Regardless of Structure, an antibody fragment binds proteins (BMPs) to their respective receptors, by removing with the Same antigen that is recognized by the intact or preventing TGF-beta from binding to TGF-binding-pro antibody. For example, an anti-TGF-beta binding-protein tein. Such inhibition may be accomplished, for example, by monoclonal antibody fragment binds with an epitope of molecules which inhibit the binding of the TGF-beta bind TGF-beta binding-protein. ing-protein to specific members of the TGF-beta Super 0058. The term “antibody fragment” also includes any family. Synthetic or genetically engineered protein that acts like an 0.052 “Vector” refers to an assembly which is capable of antibody by binding to a specific antigen to form a complex. directing the expression of desired protein. The vector must For example, antibody fragments include isolated fragments include transcriptional promoter elements which are oper consisting of the light chain variable region, “Fv' fragments ably linked to the gene(s) of interest. The vector may be consisting of the variable regions of the heavy and light composed of either deoxyribonucleic acids (“DNA”), ribo chains, recombinant Single chain polypeptide molecules in nucleic acids (“RNA”), or a combination of the two (e.g., a which light and heavy variable regions are connected by a DNA-RNA chimeric). Optionally, the vector may include a peptide linker (“SFv proteins”), and minimal recognition polyadenylation Sequence, one or more restriction sites, as units consisting of the amino acid residues that mimic the well as one or more Selectable markerS Such as neomycin hyperVariable region. US 2004/0058321 A1 Mar. 25, 2004

0059) A “detectable label” is a molecule or atom which shows 100% penetrance. There are anecdotal reports of can be conjugated to an antibody moiety to produce a increased of bone mineral density in heterozygotes with no molecule useful for diagnosis. Examples of detectable labels associated pathologies (Syndactyly or skull overgrowth). include chelators, photoactive agents, radioisotopes, fluo 0066. It appears at the present time that there is no rescent agents, paramagnetic ions, enzymes, and other abnormality of the pituitary-hypothalamus axis in Scleros marker moieties. teosis. In particular, there appears to be no over-production 0060 AS used herein, an “immunoconjugate” is a mol of growth hormone and cortisone. In addition, SeX hormone ecule comprising an anti-TGF-beta binding-protein anti levels are normal in affected individuals. However, bone body, or an antibody fragment, and a detectable label. An turnover markers (osteoblast specific alkaline phosphatase, immunoconjugate has roughly the Same, or only slightly osteocalcin, type 1 procollagen C propeptide (PICP), and reduced, ability to bind TGF-beta binding-protein after total alkaline phosphatase, (see Comier, C., Curr: Opin. in conjugation as before conjugation. Rheu. 7:243, 1995) indicate that there is hyperosteoblastic 0061 Abbreviations: TGF-beta-"Transforming Growth activity associated with the disease but that there is normal Factor-beta; TGF-bBP “Transforming Growth Factor to slightly decreased osteoclast activity as measured by beta binding-protein” (one representative TGF-bBP is des markers of bone resorption (pyridinoline, deoxypryridino ignated “H. Beer"); BMP “bone morphogenic protein'; line, N-telopeptide, urinary hydroxyproline, plasma tartrate PCR-"polymerase chain reaction'; RT-PCR-PCR pro resistant acid phosphatases and galactosyl hydroxylysine cess in which RNA is first transcribed into DNA at the first (see Comier, Supra)). step using reverse transcriptase (RT); clDNA-any DNA 0067 Sclerosteosis is characterized by the continual made by copying an RNA sequence into DNA form. deposition of bone throughout the skeleton during the life 0.062. As noted above, the present invention provides a time of the affected individuals. In homozygotes the con novel class of TGF-beta binding-proteins, as well as meth tinual deposition of bone mineral leads to an Overgrowth of ods and compositions for increasing bone mineral content in bone in areas of the Skeleton where there is an absence of warm-blooded animals. Briefly, the present inventions are mechanoreceptors (skull, jaw, cranium). In homozygotes based upon the unexpected discovery that a mutation in the with Sclerosteosis, the overgrowth of the bones of the skull gene which encodes a novel member of the TGF-beta leads to cranial compression and eventually to death due to binding-protein family results in a rare condition (Scleros excessive hydroStatic pressure on the brain stem. In all other teosis) characterized by bone mineral contents which are parts of the Skeleton there is a generalized and diffuse one- to four-fold higher than in normal individuals. Thus, as Sclerosis. Cortical areas of the long bones are greatly thick discussed in more detail below this discovery has led to the ened resulting in a Substantial increase in bone Strength. development of assays which may be utilized to Select Trabecular connections are increased in thickness which in molecules which inhibit the binding of the TGF-beta bind turn increases the Strength of the trabecular bone. Sclerotic ing-protein to the TGF-beta family of proteins and bone bones appear unusually opaque to X-rayS. morphogenic proteins (BMPs), and methods of utilizing 0068. As described in more detail in Example 1, the rare Such molecules for increasing the bone mineral content of genetic mutation that is responsible for the Sclerosteosis warm-blooded animals (including for example, humans). Syndrome has been localized to the region of human chro mosome 17 that encodes a novel member of the TGF-beta Discussion of the Disease Known as Sclerosteosis binding-protein family (one representative example of which is designated “H. Beer”). As described in more detail 0.063 Sclerosteosis is a term that was applied by Hansen below, based upon this discovery, the mechanism of bone (1967) (Hansen, H. G., Sklerosteose, in: Opitz, H.; Schmid, mineralization is more fully understood, allowing the devel F., Handbuch der Kinderheilkunde. Berlin: Springer (pub.) 6 opment of assays for molecules which increase bone min 1967. Pp. 351-355) to a disorder similar to van Buchem eralization, and use of Such molecules to increase bone hyperoStosis corticalis generalisata but possibly differing in mineral content, and in the treatment or prevention of a wide radiologic appearance of the bone changes and in the number of diseases. presence of asymmetric cutaneous Syndactyly of the index and middle fingers in many cases. TGF-beta Super-Family 0.064 Sclerosteosis is now known to be an autosomal Semi-dominant disorder which is characterized by widely 0069. The Transforming Growth Factor-beta (TGF-beta) disseminated Sclerotic lesions of the bone in the adult. The Super-family contains a variety of growth factors that share common Sequence elements and structural motifs (at both condition is progressive. Sclerosteosis also has a develop the Secondary and tertiary levels). This protein family is mental aspect which is associated with Syndactyly (two or known to exert a wide spectrum of biological responses on more fingers are fused together). The Sclerosteosis Syn a large variety of cell types. Many of them have important drome is associated with large Stature and many affected functions during the embryonal development in pattern individuals attain a height of six feet or more. The bone formation and tissue specification; in adults they are mineral content of homozygotes can be 1 to 6 fold over involved, e.g., in wound healing and bone repair and bone normal individuals and bone mineral density can be 1 to 4 remodeling, and in the modulation of the immune System. In fold above normal values (e.g., from unaffected siblings). addition to the three TGF-beta's, the Super-family includes 0065. The Sclerosteosis Syndrome occurs primarily in the Bone Morphogenic Proteins (BMPs), Activins, Inhibins, Afrikaaners of Dutch descent in South Africa. Approxi Growth and Differentiation Factors (GDFs), and Glial-De mately 1/140 individuals in the Afrikaaner population are rived Neurotrophic Factors (GDNFs). Primary classification carriers of the mutated gene (heterozygotes). The mutation is established through general Sequence features that bin a US 2004/0058321 A1 Mar. 25, 2004

Specific protein into a general Sub-family. Additional Strati functions deploying molecular isoforms with minor varia fication within the subfamily is possible due to stricter tion in amino acid motifs within highly conserved carboxy Sequence conservation between members of the Smaller terminal regions. group. In certain instances, such as with BMP-5, BMP-6 and BMP-7, this can be as high as 75 percent amino acid BMP Antagonism homology between members of the Smaller group. This level 0074 The BMP and Activin sub-families are subject to of identity enables a single representative Sequence to illus Significant post-translational regulation. An intricate extra trate the key biochemical elements of the Sub-group that cellular control System exists, whereby a high affinity Separates it from other members of the larger family. antagonist is Synthesized and exported, and Subsequently 0070 TGF-beta signals by inducing the formation of complexes selectively with BMPs or activins to disrupt their hetero-oligomeric complexes of type I and type II receptors. biological activity (W. C. Smith (1999) TIG 15(1) 3-6). A The crystal structure of TGF-beta2 has been determined. number of these natural antagonists have been identified, The general fold of the TGF-beta2 monomer contains a and based on Sequence divergence appear to have evolved Stable, compact, cysteine knotlike Structure formed by three independently due to the lack of primary Sequence conser disulphide bridges. Dimerization, stabilized by one disul vation. There has been no structural work to date on this phide bridge, is antiparallel. class of proteins. Studies of these antagonists has high lighted a distinct preference for interacting and neutralizing 0071 TGF-beta family members initiate their cellular BMP-2 and BMP-4. Furthermore, the mechanism of inhi action by binding to receptors with intrinsic Serine/threonine kinase activity. This receptor family consists of two Sub bition seems to differ for the different antagonists (S. Iemura families, denoted type I and type II receptors. Each member et al. (1998) Proc Natl AcadSci USA 95 9337-9342). of the TGF-beta family binds to a characteristic combination Novel TGF-beta Binding-proteins of type I and type II receptors, both of which are needed for Signaling. In the current model for TGF-beta receptor acti 0075 1. Background Re: TGF-beta Binding-proteins vation, TGF-beta first binds to the type II receptor (TbR-II), 0076. As noted above, the present invention provides a which occurs in the cell membrane in an oligomeric form novel class of TGF-beta binding-proteins that possess a with activated kinase. Thereafter, the type I receptor (TbR nearly identical cysteine (disulfide) scaffold when compared I), which can not bind ligand in the absence of TbR-II, is to Human DAN, Human Gremlin, and Human Cerberus, and recruited into the complex. ThR-II then phosphorylates SCGF (U.S. Pat. No. 5,780,263) but almost no homology at TbR-I predominantly in a domain rich in glycine and Serine the nucleotide level (for background information, see gen residues (GS domain) in the juxtamembrane region, and erally Hsu, D. R., Economides, A. N., Wang, X., Eimon, P. thereby activates TbR-I. M., Harland, R. M., “The Xenopus Dorsalizing Factor 0.072 Thus far seven type I receptors and five type II Gremlin Identifies a Novel Family of Secreted Proteins that receptors have been identified. Antagonize BMP Activities."Molecular Cell 1:673-683, 1998). Bone Morphogenic Proteins (BMPs) are Key 0077 One representative example of the novel class of Regulatory Proteins in Determining Bone Mineral TGF-beta binding-proteins is disclosed in Sequence ID Nos. Density in Humans 1, 5, 9, 11, 13, and 15. Representative members of this class 0073. A major advance in the understanding of bone of binding proteins should also be understood to include formation was the identification of the bone morphogenic variants of the TGF-beta binding-protein (e.g., Sequence ID proteins (BMPs), also known as osteogenic proteins (OPs), Nos. 5 and 7). As utilized herein, a “TGF-beta binding which regulate cartilage and bone differentiation in Vivo. protein variant gene’ refers to nucleic acid molecules that BMPS/OPs induce endochondral bone differentiation encode a polypeptide having an amino acid Sequence that is through a cascade of events which include formation of a modification of SEQ ID Nos: 2, 10, 12, 14 or 16. Such cartilage, hypertrophy and calcification of the cartilage, variants include naturally-occurring polymorphisms or vascular invasion, differentiation of Osteoblasts, and forma allelic variants of TGF-beta binding-protein genes, as well tion of bone. As described above, the BMPs/OPs (BMP as Synthetic genes that contain conservative amino acid 2-14, and osteogenic protein 1 and -2. OP-1 and OP-2) are Substitutions of these amino acid Sequences. Additional members of the TGF-beta Super-family. The striking evolu variant forms of a TGF-beta binding-protein gene are tionary conservation between members the BMP/OP Sub nucleic acid molecules that contain insertions or deletions of family Suggests that they are critical in the normal devel the nucleotide sequences described herein. TGF-beta bind opment and function of animals. Moreover, the presence of ing-protein variant genes can be identified by determining multiple forms of BMPS/OPs raises an important question whether the genes hybridize with a nucleic acid molecule about the biological relevance of this apparent redundancy. having the nucleotide sequence of SEQ ID Nos: 1, 5, 7, 9, In addition to postfetal chondrogenesis and Osteogenesis, the 11, 13, or 15 under stringent conditions. In addition, TGF BMPS/OPS play multiple roles in skeletogenesis (including beta binding-protein variant genes should encode a protein the development of craniofacial and dental tissues) and in having a cysteine backbone. embryonic development and organogenesis of parenchyma 0078. As an alternative, TGF-beta binding-protein vari tous organs, including the kidney. It is now understood that ant genes can be identified by Sequence comparison. AS used nature relies on common (and few) molecular mechanisms herein, two amino acid Sequences have "100% amino acid tailored to provide the emergence of Specialized tissues and Sequence identity’ if the amino acid residues of the two organs. The BMP/OP Super-family is an elegant example of amino acid Sequences are the Same when aligned for maxi nature parSimony in programming multiple specialized mal correspondence. Similarly, two nucleotide Sequences US 2004/0058321 A1 Mar. 25, 2004 have “100% nucleotide sequence identity” if the nucleotide the remaining impurities by Selective precipitation with residues of the two nucleotide Sequences are the same when lithium chloride (see, for example, Ausubel et al. (eds.), aligned for maximal correspondence. Sequence compari Short Protocols in Molecular Biology, 3rd Edition, pages Sons can be performed using Standard Software programs 4-1 to 4-6 (John Wiley & Sons 1995) “Ausubel (1995)'); Such as those included in the LASERGENE bioinformatics Wu et al., Methods in Gene Biotechnology, pages 33-41 computing Suite, which is produced by DNASTAR (Madi (CRC Press, Inc. 1997) “Wu (1997)”). son, Wis.). Other methods for comparing two nucleotide or amino acid Sequences by determining optimal alignment are 0084. Alternatively, total RNA can be isolated by extract well-known to those of skill in the art (see, for example, ing ground tissue with guanidinium isothiocyanate, extract Peruski and Peruski. The Internet and the New Biology. ing with organic Solvents, and Separating RNA from con Tools for Genomic and Molecular Research (ASM Press, taminants using differential centrifugation (see, for example, Inc. 1997), Wu et al. (eds.), “Information Superhighway and Ausubel (1995) at pages 4-1 to 4-6, Wu (1997) at pages Computer Databases of Nucleic Acids and Proteins,” in 33-41). Methods in Gene Biotechnology, pages 123-151 (CRC 0085. In order to construct a cDNA library, poly(A)" Press, Inc. 1997), and Bishop (ed.), Guide to Human RNA must be isolated from a total RNA preparation. Genome Computing, 2nd Edition (Academic Press, Inc. Poly(A) RNA can be isolated from total RNA by using the 1998)). Standard technique of oligo(dT)-cellulose chromatography 0079 A variant TGF-beta binding-protein should have at (see, for example, Ausubel (1995) at pages 4-11 to 4-12). least a 50% amino acid sequence identity to SEQ ID NOS: 0086 Double-stranded cDNA molecules are synthesized 2, 6, 10, 12, 14 or 16 and preferably, greater than 60%, 65%, from poly(A) RNA using techniques well-known to those 70%, 75%, 80%, 85%, 90%, or 95% identity. Alternatively, in the art. (see, for example, Wu (1997) at pages 41-46). TGF-beta binding-protein variants can be identified by hav Moreover, commercially available kits can be used to Syn ing at least a 70% nucleotide sequence identity to SEQ ID thesize double-stranded cDNA molecules. For example, NOs: 1, 5, 9, 11, 13 or 15. Moreover, the present invention Such kits are available from Life Technologies, Inc. (Gaith contemplates TGF-beta binding-protein gene variants hav ersburg, Md.), CLONTECH Laboratories. Inc. (Palo Alto, ing greater than 75%, 80%, 85%, 90%, or 95% identity to Calif.), Promega Corporation (Madison, Wis.) and Strat SEQ ID NO:1. Regardless of the particular method used to agene Cloning Systems (La Jolla, Calif.). identify a TGF-beta binding-protein variant gene or variant TGF-beta binding-protein, a variant TGF-beta binding-pro 0087. The basic approach for obtaining TGF-beta bind tein or a polypeptide encoded by a variant TGF-beta bind ing-protein cDNA clones can be modified by constructing a ing-protein gene can be functionally characterized by, for subtracted cDNA library which is enriched in TGF-binding example, its ability to bind to and/or inhibit the Signaling of protein-Specific cDNA molecules. Techniques for construct a selected member of the TGF-beta family of proteins, or by ing subtracted libraries are well-known to those of skill in its ability to bind specifically to an anti-TGF-beta binding the art (See, for example, Sargent, “Isolation of Differentially protein antibody. Expressed Genes,” in Meth. Enzymol. 152:423, 1987, and Wu et al. (eds.), “Construction and Screening of Subtracted 0080. The present invention includes functional frag and Complete Expression cDNA Libraries,” in Methods in ments of TGF-beta binding-protein genes. Within the con Gene Biotechnology, pages 29-65 (CRC Press, Inc. 1997)). text of this invention, a “functional fragment” of a TGF-beta binding-protein gene refers to a nucleic acid molecule that 0088 Various cloning vectors are appropriate for the encodes a portion of a TGF-beta binding-protein polypep construction of a cDNA library. For example, a cDNA tide which either (1) possesses the above-noted function library can be prepared in a vector derived from bacterioph activity, or (2) specifically binds with an anti-TGF-beta age, Such as a 2gt10 vector (See, for example, Huynh et al., binding-protein antibody. For example, a functional frag “Constructing and Screening cDNA Libraries in 2gt10 and ment of a TGF-beta binding-protein gene described herein 2gt11,” in DNA Cloning. A Practical Approach Vol. 1, comprises a portion of the nucleotide Sequence of SEQ ID Glover (ed.), page 49 (IRL Press, 1985); Wu (1997) at pages Nos: 1, 5, 9, 11, 13 or 15. 47-52). 0.081 2. Isolation of the TGF-beta Binding-protein Gene 0089 Alternatively, double-stranded cDNA molecules can be inserted into a plasmid vector, Such as a pBlueScript 0082 DNA molecules encoding a binding-protein gene vector (Stratagene Cloning Systems: La Jolla, Calif.), a can be obtained by Screening a human cDNA or genomic LambdaGEM-4 (Promega Corp.; Madison, Wis.) or other library using polynucleotide probes based upon, for commercially available vectors. Suitable cloning vectors example, SEQ ID NO:1. also can be obtained from the American Type Culture 0.083 For example, the first step in the preparation of a Collection (Rockville, Md.). cDNA library is to isolate RNA using methods well-known 0090. In order to amplify the cloned cDNA molecules, to those of skill in the art. In general, RNA isolation the cDNA library is inserted into a prokaryotic host, using techniques must provide a method for breaking cells, a Standard techniques. For example, a cDNA library can be means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and introduced into competent E. coli DH5 cells, which can be polysaccharide contaminants. For example, total RNA can obtained from Life Technologies, Inc. (Gaithersburg, Md.). be isolated by freezing tissue in liquid nitrogen, grinding the 0091. A human genomic DNA library can be prepared by frozen tissue with a mortar and pestle to lyse the cells, means well-known in the art (See, for example, Ausubel extracting the ground tissue with a Solution of phenol/ (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327). chloroform to remove proteins, and Separating RNA from Genomic DNA can be isolated by lysing tissue with the US 2004/0058321 A1 Mar. 25, 2004 detergent Sarkosyl, digesting the lysate with proteinase K, 0098. As an alternative, a TGF-beta binding-protein gene clearing insoluble debris from the lysate by centrifugation, can be obtained by Synthesizing DNA molecules using precipitating nucleic acid from the lysate using isopropanol, mutually priming long oligonucleotides and the nucleotide and purifying resuspended DNA on a cesium chloride den Sequences described herein (See, for example, Ausubel sity gradient. (1995) at pages 8-8 to 8-9). Established techniques using the polymerase chain reaction provide the ability to Synthesize 0092 DNA fragments that are suitable for the production DNA molecules at least two kilobases in length (Adang et of a genomic library can be obtained by the random shearing al., Plant Molec. Biol. 21:1131, 1993; Bambot et al., PCR of genomic DNA or by the partial digestion of genomic Methods and Applications 2:266, 1993; Dillon et al., “Use of DNA with restriction endonucleases. Genomic DNA frag the Polymerase Chain Reaction for the Rapid Construction ments can be inserted into a vector, Such as a bacteriophage of Synthetic Genes,” in Methods in Molecular Biology, Vol. or coSmid vector, in accordance with conventional tech niques, Such as the use of restriction enzyme digestion to 15: PCR Protocols. Current Methods and Applications, provide appropriate termini, the use of alkaline phosphatase White (ed.), pages 263-268, (Humana Press, Inc. 1993); treatment to avoid undesirable joining of DNA molecules, Holowachuk et al., PCR Methods Appl. 4:299, 1995). and ligation with appropriate ligases. Techniques for Such 0099 3. Production of TGF-beta Binding-protein Genes manipulation are well-known in the art (See, for example, 0100 Nucleic acid molecules encoding variant TGF-beta Ausubel (1995) at pages 5-1 to 5-6, Wu (1997) at pages binding-protein genes can be obtained by Screening various 307-327). cDNA or genomic libraries with polynucleotide probes 0093. Nucleic acid molecules that encode a TGF-beta having nucleotide sequences based upon SEQ ID NO:1, 5, binding-protein gene can also be obtained using the poly 9, 11, 13, or 15, using procedures described above. TGF-beta merase chain reaction (PCR) with oligonucleotide primers binding-protein gene variants can also be constructed Syn having nucleotide Sequences that are based upon the nucle thetically. For example, a nucleic acid molecule can be otide Sequences of the human TGF-beta binding-protein devised that encodes a polypeptide having a conservative gene, as described herein. General methods for Screening amino acid change, compared with the amino acid Sequence libraries with PCR are provided by, for example, Yu et al., of SEQ ID NOS: 2, 6, 8, 10, 12, 14, or 16. That is, variants “Use of the Polymerase Chain Reaction to Screen Phage can be obtained that contain one or more amino acid Libraries,” in Methods in Molecular Biology, Vol. 15: PCR Substitutions of SEQ ID NOS: 2, 6, 8, 10, 12, 14 or 16, in Protocols. Current Methods and Applications, White (ed.), which an alkyl amino acid is Substituted for an alkyl amino pages 211-215 (Humana Press, Inc. 1993). Moreover, tech acid in a TGF-beta binding-protein amino acid Sequence, an niques for using PCR to isolate related genes are described aromatic amino acid is Substituted for an aromatic amino by, for example, Preston, "Use of Degenerate Oligonucle acid in a TGF-beta binding-protein amino acid Sequence, a otide Primers and the Polymerase Chain Reaction to Clone Sulfur-containing amino acid is Substituted for a Sulfur Gene Family Members,” in Methods in Molecular Biology, containing amino acid in a TGF-beta binding-protein amino Vol. 15: PCR Protocols. Current Methods and Applications, acid Sequence, a hydroxy-containing amino acid is Substi White (ed.), pages 317-337 (Humana Press, Inc. 1993). tuted for a hydroxy-containing amino acid in a TGF-beta binding-protein amino acid Sequence, an acidic amino acid 0094. Alternatively, human genomic libraries can be is Substituted for an acidic amino acid in a TGF-beta obtained from commercial Sources Such as Research Genet binding-protein amino acid Sequence, a basic amino acid is ics (Huntsville, Ala.) and the American Type Culture Col substituted for a basic amino acid in a TGF-beta binding lection (Rockville, Md.). protein amino acid Sequence, or a dibasic monocarboxylic 0.095 A library containing cDNA or genomic clones can amino acid is Substituted for a dibasic monocarboxylic be Screened with one or more polynucleotide probes based amino acid in a TGF-beta binding-protein amino acid upon SEQ ID NO:1, using standard methods (see, for Sequence. example, Ausubel (1995) at pages 6-1 to 6-11). 0101 Among the common amino acids, for example, a 0.096 Anti-TGF-beta binding-protein antibodies, pro “conservative amino acid substitution' is illustrated by a duced as described below, can also be used to isolate DNA Substitution among amino acids within each of the following Sequences that encode TGF-beta binding-protein genes from groups: (1)glycine, alanine, Valine, leucine, and isoleucine, cDNA libraries. For example, the antibodies can be used to (2) phenylalanine, tyrosine, and tryptophan, (3) Serine and Screen ogt 11 expression libraries, or the antibodies can be threonine, (4) aspartate and glutamate, (5) glutamine and used for immunoScreening following hybrid Selection and asparagine, and (6) lysine, arginine and histidine. In making translation (see, for example, Ausubel (1995) at pages 6-12 Such Substitutions, it is important to, where possible, main to 6-16; Margolis et al., “Screening expression libraries tain the cysteine backbone outlined in FIG. 1. with antibody and protein probes,” in DNA Cloning 2: 0102 Conservative amino acid changes in a TGF-beta Expression Systems, 2nd Edition. Glover et al. (eds.), pages binding-protein gene can be introduced by Substituting 1-14 (Oxford University Press 1995)). nucleotides for the nucleotides recited in SEQ ID NO:1. 0097. The sequence of a TGF-beta binding-protein Such “conservative amino acid' variants can be obtained, cDNA or TGF-beta binding-protein genomic fragment can for example, by oligonucleotide-directed mutagenesis, be determined using Standard methods. Moreover, the iden linker-Scanning mutagenesis, mutagenesis using the poly tification of genomic fragments containing a TGF-beta bind merase chain reaction, and the like (see Ausubel (1995) at ing-protein promoter or regulatory element can be achieved pages 8-10 to 8-22; and McPherson (ed.), Directed using well-established techniques, Such as deletion analysis Mutagenesis: A Practical Approach (IRL Press 1991)). The (see, generally, Ausubel (1995)). functional ability of Such variants can be determined using US 2004/0058321 A1 Mar. 25, 2004

a Standard method, Such as the assay described herein. a polypeptide that can be characterized by its functional Alternatively, a variant TGF-beta binding-protein polypep activity, or by the ability to bind specifically to an anti-TGF tide can be identified by the ability to specifically bind beta binding-protein antibody. More specifically, variant anti-TGF-beta binding-protein antibodies. TGF-beta binding-protein genes encode polypeptides which 0103 Routine deletion analyses of nucleic acid mol exhibit at least 50%, and preferably, greater than 60, 70, 80 ecules can be performed to obtain “functional fragments of or 90%, of the activity of polypeptides encoded by the a nucleic acid molecule that encodes a TGF-beta binding human TGF-beta binding-protein gene described herein. protein polypeptide. As an illustration, DNA molecules 0108) 4. Production of TGF-beta Binding-protein in Cul having the nucleotide sequence of SEQ ID NO:1 can be tured Cells digested with Bal31 nuclease to obtain a series of nested 0109 To express a TGF-beta binding-protein gene, a deletions. The fragments are then inserted into expression nucleic acid molecule encoding the polypeptide must be vectors in proper reading frame, and the expressed polypep operably linked to regulatory Sequences that control tran tides are isolated and tested for activity, or for the ability to Scriptional expression in an expression vector and then bind anti-TGF-beta binding-protein antibodies. One alterna introduced into a host cell. In addition to transcriptional tive to exonuclease digestion is to use oligonucleotide regulatory Sequences, Such as promoters and enhancers, directed mutagenesis to introduce deletions or Stop codons expression vectors can include translational regulatory to specify production of a desired fragment. Alternatively, Sequences and a marker gene which is Suitable for Selection particular fragments of a TGF-beta binding-protein gene can of cells that carry the expression vector. be Synthesized using the polymerase chain reaction. 0110 Expression vectors that are suitable for production 0104 Standard techniques for functional analysis of pro of a foreign protein in eukaryotic cells typically contain (1) teins are described by, for example, Treuter et al., Molec. prokaryotic DNA elements coding for a bacterial replication Gen. Genet. 240: 113, 1993; Content et al., “Expression and origin and an antibiotic resistance marker to provide for the preliminary deletion analysis of the 42 kDa 2-5A synthetase growth and Selection of the expression vector in a bacterial induced by human interferon,” in Biological Interferon host: (2) eukaryotic DNA elements that control initiation of Systems, Proceedings of ISIR-TNO Meeting on Interferon transcription, Such as a promoter; and (3) DNA elements that Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Her control the processing of transcripts, Such as a transcription schman, “The EGF Receptor,” in Control of Animal Cell termination/polyadenylation Sequence. Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199 0111 TGF-beta binding-proteins of the present invention (Academic Press 1985); Coumailleau et al., J. Biol. Chem. are preferably expressed in mammalian cells. Examples of 270:29270, 1995; Fukunaga et al., J. Biol. Chem. mammalian host cells include African green monkey kidney 270:25291, 1995; Yamaguchi et al., Biochem. Pharmacol. cells (Vero; ATCC CRL 1587), human embryonic kidney 50:1295, 1995; and Meisel et al., Plant Molec. Biol. 30:1, cells (293-HEK; ATCC CRL 1573), baby hamster kidney 1996. cells (BHK-21; ATCC CRL 8544), canine kidney cells 0105 The present invention also contemplates functional (MDCK; ATCC CCL 34), Chinese hamster ovary cells fragments of a TGF-beta binding-protein gene that have (CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rathepatoma cells conservative amino acid changes. (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey 0106 A TGF-beta binding-protein variant gene can be kidney cells (COS-1; ATCC CRL 1650) and murine embry identified on the basis of structure by determining the level onic cells (NIH-3T3; ATCC CRL 1658). of identity with nucleotide and amino acid Sequences of 0112 For a mammalian host, the transcriptional and SEQ ID NOs: 1, 5, 9, 11, 13, or, 15 and 2, 6, 10, 12, 14, or translational regulatory Signals may be derived from viral 16, as discussed above. An alternative approach to identi Sources, Such as adenovirus, bovine papilloma virus, Simian fying a variant gene on the basis of Structure is to determine Virus, or the like, in which the regulatory Signals are asso whether a nucleic acid molecule encoding a potential variant ciated with a particular gene which has a high level of TGF-beta binding-protein gene can hybridize under Strin expression. Suitable transcriptional and translational regu gent conditions to a nucleic acid molecule having the latory Sequences also can be obtained from mammalian nucleotide sequence of SEQ ID Nos: 1, 5, 9, 11, 13, or, 15, genes, Such as actin, collagen, myosin, and metallothionein or a portion thereof of at least 15 or 20 nucleotides in length. geneS. AS an illustration of Stringent hybridization conditions, a nucleic acid molecule having a variant TGF-beta binding 0113 Transcriptional regulatory Sequences include a pro protein Sequence can bind with a fragment of a nucleic acid moter region Sufficient to direct the initiation of RNA molecule having a sequence from SEQ ID NO:1 in a buffer Synthesis. Suitable eukaryotic promoters include the pro containing, for example, 5xSSPE (1xSSPE=180 mM moter of the mouse metallothionein I gene Hamer et al., J. Sodium chloride, 10 mM sodium phosphate, 1 mM EDTA Molec. Appl. Genet. 1:273, 1982), the TK promoter of (pH 7.7), 5xDenhardt's solution (100xDenhardt's 2% (w/v) Herpesvirus McKnight, Cell 31:355, 1982), the SV40 early bovine serum albumin, 2% (w/v) Ficoll, 2% (w/v) polyvi promoter Benoist et al., Nature 290:304, 1981), the Rous nylpyrrolidone) and 0.5% SDS incubated overnight at sarcoma virus promoter Gorman et al., Proc. Nat'l Acad. 55-60° C. Post-hybridization washes at high stringency are Sci. USA 79:6777, 1982), the cytomegalovirus promoter Foecking et al., Gene 45: 101, 1980), and the mouse mam typically performed in 0.5xSSC (1xSSC=150 mM sodium mary tumor virus promoter (See, generally, Etcheverry, chloride, 15 mM trisodium citrate) or in 0.5xSSPE at 55-60 “Expression of Engineered Proteins in Mammalian Cell C. Culture,” in Protein Engineering. Principles and Practice, 0107 Regardless of the particular nucleotide sequence of Cleland et al. (eds.), pages 163-181 (John Wiley & Sons, a variant TGF-beta binding-protein gene, the gene encodes Inc. 1996)). US 2004/0058321 A1 Mar. 25, 2004

0114. Alternatively, a prokaryotic promoter, Such as the Volume 7: Gene Transfer and Expression Protocols, Murray bacteriophage T3 RNA polymerase promoter, can be used to (ed.), pages 147-168 (The Humana Press, Inc. 1991), by control TGF-beta binding-protein gene expression in mam Patel et al., “The baculovirus expression system,” in DNA malian cells if the prokaryotic promoter is regulated by a Cloning 2: Expression Systems, 2nd Edition, Glover et al., eukaryotic promoter (Zhou et al., Mol. Cell. Biol. 10:4529, (eds.), pages 205-244 (Oxford University Press 1995), by 1990; Kaufman et al., Nucl. Acids Res. 19:4485, 1991). Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (The Humana 0115 TGF-beta binding-protein genes may also be Press, Inc. 1995), and by Lucknow, “Insect Cell Expression expressed in bacterial, yeast, insect, or plant cells. Suitable Technology,” in Protein Engineering. Principles and Prac promoters that can be used to express TGF-beta binding protein polypeptides in a prokaryotic host are well-known to tice, Cleland et al. (eds.), pages 183-218 (John Wiley & those of skill in the art and include promoters capable of Sons, Inc. 1996). recognizing the T4, T3, Sp6 and T7 polymerases, the PR and 0119 Promoters for expression in yeast include promot P. promoters of bacteriophage lambda, the trip, recA, heat ers from GAL1 (galactose), PGK (phosphoglycerate Shock, lacUV5, tac, lipp-lacSpr, phoA, and lac7, promoters of kinase), ADH (alcoholdehydrogenase), AOX1 (alcohol oxi E. coli, promoters of B. Subtilis, the promoters of the dase), HIS4 (histidinol dehydrogenase), and the like. Many bacteriophages of Bacillus, Streptomyces promoters, the int yeast cloning vectors have been designed and are readily promoter of bacteriophage lambda, the bla promoter of available. These vectors include YIp-based vectors, such as pBR322, and the CAT promoter of the chloramphenicol YIp5, YRp vectors, such as YRp17, YEp vectors such as acetyl transferase gene. Prokaryotic promoters have been YEp13 and YCp vectors, such as YCp19. One skilled in the reviewed by Glick, J. Ind. Microbiol. 1:277, 1987, Watson art will appreciate that there are a wide variety of Suitable et al., Molecular Biology of the Gene, 4th Ed. (Benjamin vectors for expression in yeast cells. Cummins 1987), and by Ausubel et al. (1995). 0120 Expression vectors can also be introduced into 0116 Preferred prokaryotic hosts include E. coli and plant protoplasts, intact plant tissues, or isolated plant cells. Bacillus Subtilus. Suitable strains of E. coli include General methods of culturing plant tissues are provided, for BL21(DE3), BL21(DE3)pLyss, BL21 (DE3)pLysE, DH1, example, by Miki et al., “Procedures for Introducing Foreign DH4I, DH5, DH5I, DH5IF, DH5IMCR, DH10B, DH1OB/ DNA into Plants,” in Methods in Plant Molecular Biology p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC K38, RR1, Y1088, Y1089, CSH 18, ER 1451, and ER1647 Press, 1993). (see, for example, Brown (Ed.), Molecular Biology Labfax 0121 An expression vector can be introduced into host (Academic Press 1991)). Suitable strains of Bacillus Subtilus cells using a variety of Standard techniques including cal include BR151, YB886, MI119, MI120, and B170 (see, for cium phosphate transfection, liposome-mediated transfec example, Hardy, “Bacillus Cloning Methods,” in DNA Clon tion, microprojectile-mediated delivery, electroporation, and ing: A Practical Approach. Glover (Ed.) (IRL Press 1985)). the like. Preferably, the transfected cells are selected and 0117 Methods for expressing proteins in prokaryotic propagated to provide recombinant host cells that comprise hosts are well-known to those of skill in the art (see, for the expression vector Stably integrated in the host cell example, Williams et al., “Expression of foreign proteins in genome. Techniques for introducing vectors into eukaryotic E. coli using plasmid vectors and purification of Specific cells and techniques for Selecting Such stable transformants polyclonal antibodies,” in DNA Cloning 2: Expression Sys using a dominant Selectable marker are described, for tems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford example, by Ausubel (1995) and by Murray (ed.), Gene University Press 1995); Ward et al., “Genetic Manipulation Transfer and Expression Protocols (Humana Press 1991). and Expression of Antibodies,” in Monoclonal Antibodies. Methods for introducing expression vectors into bacterial, Principles and Applications, page 137 (Wiley-Liss, Inc. yeast, insect, and plant cells are also provided by Ausubel 1995); and Georgiou, “Expression of Proteins in Bacteria.” (1995). in Protein Engineering. Principles and Practice, Cleland et 0.122 General methods for expressing and recovering al. (eds.), page 101 (John Wiley & Sons, Inc. 1996)). foreign protein produced by a mammalian cell System is provided by, for example, Etcheverry, “Expression of Engi 0118. The baculovirus system provides an efficient means neered Proteins in Mammalian Cell Culture.” in Protein to introduce cloned TGF-beta binding-protein genes into Engineering. Principles and Practice, Cleland et al. (eds.), insect cells. Suitable expression vectors are based upon the pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for Autographa Californica multiple nuclear polyhedrosis virus recovering protein produced by a bacterial System is pro (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, vided by, for example, Grisshammer et al., “Purification of Autographa Californica nuclear polyhedrosis virus immedi over-produced proteins from E. coli cells,” in DNA Cloning ate-early gene promoter (ie-1) and the delayed early 39K 2: Expression Systems, 2nd Edition, Glover et al. (eds.), promoter, baculovirus p10 promoter, and the Drosophila pages 59-92 (Oxford University Press 1995). Established metallothionein promoter. Suitable insect host cells include methods for isolating recombinant proteins from a baculovi cell lines derived from IPLB-Sf-21, a Spodoptera frugiperda rus system are described by Richardson (ed.), Baculovirus pupal ovarian cell line, such as Sf9 (ATCC CRL 1711), Expression Protocols (The Humana Press, Inc., 1995). Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, 0123. More generally, TGF-beta binding-protein can be Calif.), as well as Drosophila Schneider-2 cells. Established isolated by Standard techniques, Such as affinity chromatog techniques for producing recombinant proteins in baculovi raphy, size exclusion chromatography, ion eXchange chro rus systems are provided by Bailey et al., “Manipulation of matography, HPLC and the like. Additional variations in Baculovirus Vectors,” in Methods in Molecular Biology, TGF-beta binding-protein isolation and purification can be US 2004/0058321 A1 Mar. 25, 2004

devised by those of skill in the art. For example, anti-TGF techniques. Such engineered versions include those created beta binding-protein antibodies, obtained as described for example from natural antibody variable regions by below, can be used to isolate large quantities of protein by insertions, deletions or changes in or to the amino acid immunoaffinity purification. Sequences of the natural antibodies. Particular examples of 0.124 5. Production of Antibodies to TGF-beta Binding this type include those engineered variable region domains proteins containing at least one CDR and optionally one or more framework amino acids from one antibody and the remain 0.125 Antibodies to TGF-beta binding-protein can be der of the variable region domain from a Second antibody. obtained, for example, using the product of an expression vector as an antigen. Particularly useful anti-TGF-beta bind 0129. The variable region domain may be covalently ing-protein antibodies “bind specifically” with TGF-beta attached at a C-terminal amino acid to at least one other binding-protein of Sequence ID Nos. 2, 6, 10, 12, 14, or 16, antibody domain or a fragment thereof. Thus, for example but not to other TGF-beta binding-proteisn. Such as Dan, where a V domain is present in the variable region domain Cerberus, SCGF, or Gremlin. Antibodies of the present this may be linked to an immunoglobulin C1 domain or a invention (including fragments and derivatives thereof) may fragment thereof. Similarly a V domain may be linked to a be a polyclonal or, especially a monoclonal antibody. The C. domain or a fragment thereof. In this way for example antibody may belong to any immunoglobulin class, and may the antibody may be a Fab fragment wherein the antigen binding domain contains associated V and V. domains be for example an IgG, for example IgG, IgG, IgG, IgG, covalently linked at their C-termini to a CH1 and C domain IgE., IgM, or IgA antibody. It may be of animal, for example respectively. The CH1 domain may be extended with further mammalian origin, and may be for example a murine, rat, amino acids, for example to provide a hinge region domain human or other primate antibody. Where desired the anti as found in a Fab' fragment, or to provide further domains, body may be an internalising antibody. such as antibody CH2 and CH3 domains. 0.126 Polyclonal antibodies to recombinant TGF-beta binding-protein can be prepared using methods well-known 0.130. Another form of an antibody fragment is a peptide to those of skill in the art (See, for example, Green et al., coding for a Single complementarity-determining region “Production of Polyclonal Antisera,” in Immunochemical (CDR). CDR peptides (“minimal recognition units”) can be Protocols (Manson, ed.), pages 1-5 (Humana Press 1992); obtained by constructing genes encoding the CDR of an Williams et al., “Expression of foreign proteins in E. coli antibody of interest. Such genes are prepared, for example, using plasmid vectors and purification of Specific polyclonal by using the polymerase chain reaction to Synthesize the antibodies,” in DNA Cloning 2: Expression Systems, 2nd variable region from RNA of antibody-producing cells (see, Edition, Glover et al. (eds.), page 15 (Oxford University for example, Larrick et al., Methods. A Companion to Press 1995)). Although polyclonal antibodies are typically Methods in Enzymology 2:106, 1991; Courtenay-Luck, raised in animals. Such as rats, mice, rabbits, goats, or sheep, “Genetic Manipulation of Monoclonal Antibodies,” in an anti-TGF-beta binding-protein antibody of the present Monoclonal Antibodies. Production, Engineering and Clini invention may also be derived from a Subhuman primate cal Application, Ritter et al. (eds.), page 166 (Cambridge antibody. General techniques for raising diagnostically and University Press 1995); and Ward et al., “Genetic Manipu therapeutically useful antibodies in baboons may be found, lation and Expression of Antibodies,” in Monoclonal Anti for example, in Goldenberg et al., international patent pub bodies. Principles and Applications, Birch et al., (eds.), page lication No. WO 91/11465 (1991), and in Losman et al., Int. 137 (Wiley-Liss, Inc. 1995)). J. Cancer 46:310, 1990. 0131) Antibodies for use in the invention may in general 0127. The antibody should comprise at least a variable be monoclonal (prepared by conventional immunisation and region domain. The variable region domain may be of any cell fusion procedures) or in the case of fragments, derived Size or amino acid composition and will generally comprise therefrom using any Suitable Standard chemical e.g. reduc at least one hyperVariable amino acid Sequence responsible tion or enzymatic cleavage and/or digestion techniques, for for antigen binding embedded in a framework Sequence. In example by treatment with pepsin. general terms the variable (V) region domain may be any 0132) More specifically, monoclonal anti-TGF-beta bind Suitable arrangement of immunoglobulin heavy (V) and/or ing-protein antibodies can be generated utilizing a variety of light (V) chain variable domains. Thus for example the V techniques. Rodent monoclonal antibodies to specific anti region domain may be monomeric and be a V or V. domain gens may be obtained by methods known to those skilled in where these are capable of independently binding antigen the art (see, for example, Kohler et al., Nature 256:495, with acceptable affinity. Alternatively the V region domain 1975; and Coligan et al. (eds.), Current Protocols in Immu may be dimeric and contain V-V, V-V, or V-V, nology, 1:2.5.1-2.6.7 (John Wiley & Sons 1991) “Coli dimers in which the V and V chains are non-covalently gan”; Picksley et al., “Production of monoclonal antibodies associated (abbreviated hereinafter as F). Where desired, against proteins expressed in E. coli, in DNA Cloning 2: however, the chains may be covalently coupled either Expression Systems, 2nd Edition. Glover et al. (eds.), page directly, for example via a disulphide bond between the two 93 (Oxford University Press 1995)). variable domains, or through a linker, for example a peptide 0.133 Briefly, monoclonal antibodies can be obtained by linker, to form a single chain domain (abbreviated herein injecting mice with a composition comprising a TGF-beta after as ScF). binding-protein gene product, Verifying the presence of 0128. The variable region domain may be any naturally antibody production by removing a Serum Sample, removing occuring variable domain or an engineered version thereof. the Spleen to obtain B-lymphocytes, fusing the B-lympho By engineered version is meant a variable region domain cytes with myeloma cells to produce hybridomas, cloning which has been created using recombinant DNA engineering the hybridomas, Selecting positive clones which produce US 2004/0058321 A1 Mar. 25, 2004 antibodies to the antigen, culturing the clones that produce molecular biology and/or chemistry procedures may be used antibodies to the antigen, and isolating the antibodies from to Sequence and manipulate the DNA, for example, to the hybridoma cultures. introduce codons to create cysteine residues, to modify, add 0134. In addition, an anti-TGF-beta binding-protein anti or delete other amino acids or domains as desired. body of the present invention may be derived from a human 0.139. From here, one or more replicable expression vec monoclonal antibody. Human monoclonal antibodies are tors containing the DNA may be prepared and used to obtained from transgenic mice that have been engineered to transform an appropriate cell line, e.g. a non-producing produce Specific human antibodies in response to antigenic myeloma cell line, Such as a mouse NSO line or a bacterial, challenge. In this technique, elements of the human heavy e.g. E. coli line, in which production of the antibody will and light chain locus are introduced into Strains of mice occur. In order to obtain efficient transcription and transla derived from embryonic Stem cell lines that contain targeted tion, the DNA sequence in each vector should include disruptions of the endogenous heavy chain and light chain appropriate regulatory Sequences, particularly a promoter loci. The transgenic mice can Synthesize human antibodies and leader Sequence operably linked to the variable domain Specific for human antigens, and the mice can be used to Sequence. Particular methods for producing antibodies in produce human antibody-Secreting hybridomas. Methods this way are generally well known and routinely used. For for obtaining human antibodies from transgenic mice are example, basic molecular biology procedures are described described, for example, by Green et al., Nature Genet. 7:13, by Maniatis et al (Molecular Cloning, Cold Spring Harbor 1994: Lonberg et al., Nature 368:856, 1994; and Taylor et Laboratory, New York, 1989); DNA sequencing can be al., Int. Immun. 6:579, 1994. performed as described in Sanger et al (PNAS 74, 5463, 0135 Monoclonal antibodies can be isolated and purified (1977)) and the Amersham International plc sequencing from hybridoma cultures by a variety of well-established handbook, and Site directed mutagenesis can be carried out techniques. Such isolation techniques include affinity chro according to the method of Kramer et al (Nucl. Acids Res. matography with Protein-A Sepharose, size-exclusion chro 12,9441, (1984)) and the Anglian-Biotechnology Ltd hand matography, and ion-exchange chromatography (see, for book. Additionally, there are numerous publications, detail example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1- ing techniques Suitable for the preparation of antibodies by 2.9.3; Baines et al., “Purification of Immunoglobulin G manipulation of DNA, creation of expression vectors and (IgG).” in Methods in Molecular Biology, Vol. 10, pages transformation of appropriate cells, for example as reviewed by Mountain A and Adair, J R in Biotechnology and Genetic 79-104 (The Humana Press, Inc. 1992)). Engineering Reviews (ed. Tombs, MP, 10, Chapter 1, 1992, 0136. For particular uses, it may be desirable to prepare Intercept, Andover, UK) and in International Patent Speci fragments of anti-TGF-beta binding-protein antibodies. fication No. WO 91/O9967. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments 0140. Where desired, the antibody according to the can be obtained by pepsin or papain digestion of whole invention may have one or more effector or reporter mol antibodies by conventional methods. AS an illustration, ecules attached to it and the invention extends to Such antibody fragments can be produced by enzymatic cleavage modified proteins. The effector or reporter molecules may be of antibodies with pepsin to provide a 5S fragment denoted attached to the antibody through any available amino acid F(ab'). This fragment can be further cleaved using a thiol Side-chain, terminal amino acid or, where present carbohy reducing agent to produce 3.5S Fab' monovalent fragments. drate functional group located in the antibody, always pro Optionally, the cleavage reaction can be performed using a vided of course that this does not adversely affect the blocking group for the Sulfhydryl groups that result from binding properties and eventual usefulness of the molecule. cleavage of disulfide linkages. AS an alternative, an enzy Particular functional groups include, for example any free matic cleavage using pepsin produces two monovalent Fab amino, imino, thiol hydroxyl, carboxyl or aldehyde group. fragments and an Fc fragment directly. These methods are Attachment of the antibody and the effector and/or reporter described, for example, by Goldenberg, U.S. Pat. No. 4,331, molecule(s) may be achieved via Such groups and an appro 647, Nisonoff et al., Arch Biochem. Biophys. 89:230, 1960, priate functional group in the effector or reporter molecules. Porter, Biochem. J. 73:119, 1959, Edelman et al., in Methods The linkage may be direct or indirect, through spacing or in Enzymology 1:422 (Academic Press 1967), and by Coli bridging groups. gan at pages 2.8.1-2.8.10 and 2.10.-2.10.4. 0.141. Effector molecules include, for example, antine 0.137 Other methods of cleaving antibodies, such as oplastic agents, toxins (Such as enzymatically active toxins Separation of heavy chains to form monovalent light-heavy of bacterial or plant origin and fragments thereof e.g. risin chain fragments, further cleavage of fragments, or other and fragments thereof) biologically active proteins, for enzymatic, chemical or genetic techniques may also be used, example enzymes, nucleic acids and fragments thereof, e.g. So long as the fragments bind to the antigen that is recog DNA, RNA and fragments thereof, naturally occurring and nized by the intact antibody. Synthetic polymers e.g. polysaccharides and polyalkylene polymerS Such as poly(ethylene glycol) and derivatives 0138 Alternatively, the antibody may be a recombinant thereof, radionuclides, particularly radioiodide, and chelated or engineered antibody obtained by the use of recombinant metals. Suitable reporter groups include chelated metals, DNA techniques involving the manipulation and re-expres fluorescent compounds or compounds which may be sion of DNA encoding antibody variable and/or constant detected by NMR or ESR spectroscopy. regions. Such DNA is known and/or is readily available from DNA libraries including for example phage-antibody 0.142 Particular antineoplastic agents include cytotoxic libraries (see Chiswell, DJ and McCafferty, J. Tibtech. 10 and cytostatic agents, for example alkylating agents, Such as 80-84 (1992)) or where desired can be synthesised. Standard nitrogen mustards (e.g. chlorambucil, melphalan, mechlo US 2004/0058321 A1 Mar. 25, 2004 rethamine, cyclophosphamide, or uracil mustard) and capable of increasing bone density. For example, within one derivatives thereof, triethylenephosphoramide, triethyl aspect of the present invention methods are provided for enethiophosphor-amide, buSulphan, or cisplatin; antime determining whether a Selected molecule is capable of tabolites, Such as methotrexate, fluorouracil, floXuridine, increasing bone mineral content, comprising the Steps of (a) cytarabine, mercaptopurine, thioguanine, fluoroacetic acid mixing a selected molecule with TGF-beta binding protein or fluorocitric acid, antibiotics, Such as bleomycins (e.g. and a selected member of the TGF-beta family of proteins, bleomycin Sulphate), doxorubicin, daunorubicin, mitomy (b) determining whether the Selected molecule stimulates cins (e.g. mitomycin C), actinomycins (e.g. dactinomycin) signaling by the TGF-beta family of proteins, or inhibits the plicamycin, calichaemicin and derivatives thereof, or espe binding of the TGF-beta binding protein to the TGF-beta ramicin and derivatives thereof, mitotic inhibitors, Such as etoposide, Vincristine or vinblastine and derivatives thereof; family of proteins. Within certain embodiments, the mol alkaloids, Such as ellipticine, polyols Such as taxicin-I or ecule enhances the ability of TGF-beta to function as a taxicin-II; hormones, such as androgens (e.g. dromo positive regulator of mesenchymal cell differentiation. Stanolone or testolactone), progestins (e.g. megestrol acetate 0149. Within other aspects of the invention, methods are or medroxyprogesterone acetate), estrogens (e.g. dimethyl provided for determining whether a Selected molecule is Stilbestrol diphosphate, polyestradiol phosphate or estra capable of increasing bone mineral content, comprising the mustine phosphate) or antiestrogens (e.g. tamoxifen); Steps of (a) exposing a selected molecule to cells which anthraquinones, Such as mitoxantrone, ureas, Such as express TGF-beta binding-protein and (b) determining hydroxyurea; hydrazines, Such as procarbazine, or imida whether the expression (or activity) of TGF-beta binding Zoles, Such as dacarbazine. protein from Said exposed cells decreases, and therefrom 0143 Particularly useful effector groups are calichaemi determining whether the compound is capable of increasing cin and derivatives thereof (see for example South African bone mineral content. Within one embodiment, the cells are Patent Specifications Nos. 85/8794, 88/8127 and 90/2839). Selected from the group consisting of the Spontaneously transformed or untransformed normal human bone from 0144 Chelated metals include chelates of di- or triposi bone biopsies and rat parietal bone osteoblasts. Such meth tive metals having a coordination number from 2 to 8 ods may be accomplished in a wide variety of assay formats inclusive. Particular examples of Such metals include tech including, for example, Countercurrent Immuno-Electro netium (Tc), rhenium (Re), cobalt (Co), copper (Cu), gold phoresis (CIEP), Radioimmunoassays, Radioimmunopre (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), cipitations, Enzyme-Linked Immuno-Sorbent ASSayS gallium (Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), (ELISA), Dot Blot assays, Inhibition or Competition assays, and scandium (Sc). In general the metal is preferably a and sandwich assays (see U.S. Pat. Nos. 4,376,110 and radionuclide. Particular radionuclides include '"Te, Re, 4,486,530; see also Antibodies. A Laboratory Manual, 188Re, 58Co., OCo, 7Cu, 195Au, 19°Au, ''Ag, 208Pb, 206Bi, Supra). 207Bi, 11 In, 7Ga, Ga, 88Y. 90Y, 160Tb, 153Gd and 7Sc. 0145 The chelated metal may be for example one of the 0150 Representative embodiments of Such assays are above types of metal chelated with any Suitable polydentate provided below in Examples 5 and 6. Briefly, a family chelating agent, for example acyclic or cyclic polyamines, member of the TGF-beta Super-family or a TGF-beta bind polyethers, (e.g. crown ethers and derivatives thereof); ing protein is first bound to a Solid phase, followed by polyamides, porphyrins; and carbocyclic derivatives. addition of a candidate molecule. The labeled family mem ber of the TGF-beta Super-family or a TGF-beta binding 0146 In general, the type of chelating agent will depend protein is then added to the assay, the Solid phase washed, on the metal in use. One particularly useful group of and the quantity of bound or labeled TGF-beta Super-family chelating agents in conjugates according to the invention, member or TGF-beta binding protein on the solid support however, are acyclic and cyclic polyamines, especially determined. Molecules which are Suitable for use in increas polyaminocarboxylic acids, for example diethylenetriamine ing bone mineral content as described herein are those pentaacetic acid and derivatives thereof, and macrocyclic molecules which decrease the binding of TGF-beta binding amines, e.g. cyclic tri-aza and tetra-aza derivatives (for protein to a member or members of the TGF-beta Super example as described in International Patent Specification family in a Statistically significant manner. Obviously, No. WO92/22583); and polyamides, especially desferriox assays Suitable for use within the present invention should amine and derivatives thereof. not be limited to the embodiments described within 0147 Thus for example when it is desired to use a thiol Examples 2 and 3. In particular, numerous parameters may group in the antibody as the point of attachment this may be be altered, such as by binding TGF-beta to a solid phase, or achieved through reaction with a thiol reactive group present by elimination of a Solid phase entirely. in the effector or reporter molecule. Examples of Such groups include an a-halocarboxylic acid or ester, e.g. iodoac 0151. Within other aspects of the invention, methods are etamide, an imide, e.g. maleimide, a vinyl Sulphone, or a provided for determining whether a Selected molecule is disulphide. These and other Suitable linking procedures are capable of increasing bone mineral content, comprising the generally and more particularly described in International Steps of (a) exposing a selected molecule to cells which Patent Specifications Nos. WO 93/06231, WO 92/22583, express TGF-beta and (b) determining whether the activity WO 90/091195 and WO 89/O1476. of TGF-beta from said exposed cells is altered, and there from determining whether the compound is capable of ASSays for Selecting Molecules which Increase increasing bone mineral content. Similar to the above Bone Density described methods, a wide variety of methods may be 0148 AS discussed above, the present invention provides utilized to assess the changes of TGF-beta binding-protein methods for Selecting and/or isolating compounds which are expression due to a Selected test compound. US 2004/0058321 A1 Mar. 25, 2004

0152 For example, within one aspect of the present 0160 Representative examples of such combinatorial invention methods are provided for determining whether a chemical libraries include those described by Agrafiotis et Selected molecule is capable of increasing bone mineral al., "System and method of automatically generating chemi content, comprising the Steps of (a) mixing a selected cal compounds with desired properties,” U.S. Pat. No. molecule with TGF-beta-binding-protein and a selected 5,463,564; Armstrong, R. W., “Synthesis of combinatorial member of the TGF-beta family of proteins, (b) determining arrays of organic compounds through the use of multiple whether the Selected molecule up-regulates the Signaling of component combinatorial array syntheses.” WO95/02566; the TGF-beta family of proteins, or inhibits the binding of Baldwin. J. J. et al., “Sulfonamide derivatives and their use,” the TGF-beta binding-protein to the TGF-beta family of WO95/24186; Baldwin, J. J. et al., “Combinatorial dihy proteins. Within certain embodiments, the molecule drobenzopyran library,” WO95/30642; Brenner, S., “New enhances the ability of TGF-beta to function as a positive kit for preparing combinatorial libraries,” WO95/16918; regulator of mechemchymal cell differentiation. Chenera, B. et al., “Preparation of library of resin-bound aromatic carbocyclic compounds,” WO95/16712; Ellman, 0153. Similar to the above described methods, a wide J. A., “Solid phase and combinatorial synthesis of benzodi variety of methods may be utilized to assess Stimulation of aZepine compounds on a Solid Support, U.S. Pat. No. TGF-beta due to a selected test compound. One such rep 5,288,514; Felder, E. et al., “Novel combinatorial compound resentative method is provided below in Example 6 (See also libraries,” WO95/16209; Lerner, R. et al., “Encoded com Durham et al., Endo. 136:1374-1380. binatorial chemical libraries,” WO93/20242; Pavia, M. R. et 0154 Within yet other aspects of the present invention, al., “A method for preparing and Selecting pharmaceutically methods are provided for determining whether a Selected useful non-peptide compounds from a structurally diverse molecule is capable of increasing bone mineral content, universal library,” WO95/04277; Summerton, J. E. and D. comprising the Step of determining whether a Selected D. Weller, “Morpholino-Subunit combinatorial library and molecule inhibits the binding of TGF-beta binding-protein method,” U.S. Pat. No. 5,506,337; Holmes, C., “Methods for to bone, or an analogue thereof. AS utilized herein, it should the Solid Phase Synthesis of Thiazolidinones, Metathiaza be understood that bone or analogues thereof refers to nones, and Derivatives thereof.” WO96/00148; Phillips, G. hydroxyapatite, or a Surface composed of a powdered form B. and G. P. Wei, “Solid-phase Synthesis of Benzimidazoles, of bone, crushed bone or intact bone. Similar to the above 'Tet. Letters 37:4887-90, 1996; Ruhland, B. et al., “Solid described methods, a wide variety of methods may be supported Combinatorial Synthesis of Structurally Diverse utilized to assess the inhibition of TGF-beta binding-protein B-Lactans, J. Amer: Chem. Soc. 111:253-4, 1996; Look, G. localization to bone matrix. One Such representative method C. et al., “The Indentification of Cyclooxygenase-1 Inhibi is provided below in Example 7. tors from 4-Thiazolidinone Combinatorial Libraries,BioOrg and Med. Chem. Letters 6:707-12, 1996. 0155. It should be noted that while the methods recited herein may refer to the analysis of an individual test mol 0161) 2. Proteins and Peptides ecule, that the present invention should not be So limited. In 0162. A wide range of proteins and peptides may likewise particular, the Selected molecule may be contained within a be utilized as candidate molecules for inhibitors of the mixture of compounds. Hence, the recited methods may binding of TGF-beta binding-protein to a TGF-beta family further comprise the Step of isolating a molecule which member. inhibits the binding of TGF-beta binding-protein to a TGF beta family member. 0163 a. Combinatorial Peptide Libraries 0.164 Peptide molecules which are putative inhibitors of Candidate Molecules the binding of TGF-beta binding-protein to a TGF-beta 0156 A wide variety of molecules may be assayed for family member may be obtained through the Screening of their ability to inhibit the binding of TGF-beta binding combinatorial peptide libraries. Such libraries may either be protein to a TGF-beta family member. Representative prepared by one of skill in the art (see e.g., U.S. Pat. Nos. examples which are discussed in more detail below include 4,528,266 and 4,359,535, and Patent Cooperation Treaty organic molecules, proteins or peptides, and nucleic acid Publication Nos. WO 92/15679, WO 92/15677, WO molecules. Although it should be evident from the discus 90/07862, WO 90/02809, or purchased from commercially Sion below that the candidate molecules described herein available sources (e.g. New England Biolabs Ph.D.T.M. Phage may be utilized in the assays described herein, it should also Display Peptide Library Kit). be readily apparent that Such molecules can also be utilized in a variety of diagnostic and therapeutic Settins. 0165) b. Antibodies 0166 Antibodies which inhibit the binding of TGF-beta 0157 1. Organic Molecules binding-protein to a TGF-beta family member may readily 0158 Numerous organic molecules may be assayed for be prepared given the disclosure provided herein. Within the their ability to inhibit the binding of TGF-beta binding context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, protein to a TGF-beta family member. anti-idiotypic antibodies, antibody fragments (e.g., Fab, and 0159 For example, within one embodiment of the inven F(ab'), F variable regions, or complementarity determining tion Suitable organic molecules may be selected from either regions). AS discussed above, antibodies are understood to a chemical library, wherein chemicals are assayed individu be specific against TGF-beta binding-protein, or against a ally, or from combinatorial chemical libraries where mul specific TGF-beta family member, if they bind with a K of tiple compounds are assayed at once, then deconvoluted to greater than or equal to 107M, preferably greater than or determine and isolate the most active compounds. equal to 10M, and do not bind to other TGF-beta binding US 2004/0058321 A1 Mar. 25, 2004

proteins, or, bind with a K, of less than or equal to 10°M. (JRH BioSciences, Lenexa, Kans.), as well as additional Furthermore, antibodies of the present invention should ingredients, Such as fetal bovine Serum (FBS, i.e., from block or inhibit the binding of TGF-beta binding-protein to Hyclone, Logan, Utah, or JRH Biosciences). Additionally, a TGF-beta family member. the medium should contain a reagent which Selectively allows for the growth of fused Spleen and myeloma cells 0167 The affinity of a monoclonal antibody or binding Such as HAT (hypoxanthine, aminopterin, and thymidine) partner, as well as inhibition of binding can be readily (Sigma Chemical Co., St. Louis, Mo.). After about seven determined by one of ordinary skill in the art (see Scatchard, days, the resulting fused cells or hybridomas may be Ann. N.Y. Acad. Sci. 51:660-672, 1949). Screened in order to determine the presence of antibodies 0168 Briefly, polyclonal antibodies may be readily gen which are reactive against TGF-beta binding-protein erated by one of ordinary skill in the art from a variety of (depending on the antigen used), and which block or inhibit warm-blooded animals. Such as horses, cows, various fowl, the binding of TGF-beta binding-protein to a TGF-beta rabbits, mice, or rats. Typically, the -TGF-beta binding family member. protein or unique peptide thereof of 13-20 amino acids 0173 A wide variety of assays may be utilized to deter (preferably conjugated to keyhole limpet hemocyanin by mine the presence of antibodies which are reactive against cross-linking with glutaraldehyde) is utilized to immunize the proteins of the present invention, including for example the animal through intraperitoneal, intramuscular, intraocu countercurrent immuno-electrophoresis, radioimmunoas lar, or Subcutaneous injections, along with an adjuvant Such says, radioimmunoprecipitations, enzyme-linked immuno as Freund's complete or incomplete adjuvant. Following Sorbent assays (ELISA), dot blot assays, western blots, Several booster immunizations, Samples of Serum are col immunoprecipitation, inhibition or competition assays, and lected and tested for reactivity to the protein or peptide. sandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486, Particularly preferred polyclonal antisera will give a signal 530; see also Antibodies. A Laboratory Manual, Harlow and on one of these assays that is at least three times greater than Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). background. Once the titer of the animal has reached a Following Several clonal dilutions and reassays, a hybri plateau in terms of its reactivity to the protein, larger doma producing antibodies reactive against the desired quantities of antisera may be readily obtained either by protein may be isolated. weekly bleedings, or by eXSanguinating the animal. 0.174. Other techniques may also be utilized to construct 0169 Monoclonal antibodies may also be readily gener monoclonal antibodies (see William D. Huse et al., “Gen ated using conventional techniques (see U.S. Pat. Nos. RE eration of a Large Combinational Library of the Immuno 32,011, 4,902,614, 4,543,439, and 4,411,993 which are globulin Repertoire in Phage Lambda,'Science 246:1275 incorporated herein by reference, See also Monoclonal Anti 1281, December 1989; see also L. Sastry et al., “Cloning of bodies, Hybridomas. A New Dimension in Biological Analy the Immunological Repertoire in Escherichia coli for Gen ses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), eration of Monoclonal Catalytic Antibodies: Construction of 1980, and Antibodies: A Laboratory Manual, Harlow and a Heavy Chain Variable Region-Specific cDNA Library, Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, "Proc. Natl. Acad. Sci. USA 86:5728-5732, August 1989; see which are also incorporated herein by reference). also Michelle Alting-Mees et al., “Monoclonal Antibody 0170 Briefly, within one embodiment a subject animal Expression Libraries: A Rapid Alternative to Hybridomas, such as a rat or mouse is immunized with TGF-beta binding "Strategies in Molecular Biology 3: 1-9, January 1990). protein or portion thereof as described above. The protein These references describe a commercial System available may be admixed with an adjuvant Such as Freund's complete from Stratagene (La Jolla, Calif.) which enables the produc or incomplete adjuvant in order to increase the resultant tion of antibodies through recombinant techniques. Briefly, immune response. Between one and three weeks after the mRNA is isolated from a B cell population, and utilized to initial immunization the animal may be reimmunized with create heavy and light chain immunoglobulin cDNA expres another booster immunization, and tested for reactivity to sion libraries in the ImmunoZap(H) and ImmunoZap(L) the protein utilizing assays described above. Once the ani vectors. These vectors may be screened individually or mal has reached a plateau in its reactivity to the injected co-expressed to form Fab fragments or antibodies (see Huse protein, it is Sacrificed, and organs which contain large et al., Supra; See also Sastry et al., Supra). Positive plaques numbers of B cells Such as the Spleen and lymph nodes are may Subsequently be converted to a non-lytic plasmid which harvested. allows high level expression of monoclonal antibody frag ments from E. coli. 0171 Cells which are obtained from the immunized animal may be immortalized by infection with a virus Such 0.175 Similarly, portions or fragments, such as Fab and as the Epstein-Barr virus (EBV) (see Glasky and Reading, Fv fragments, of antibodies may also be constructed utiliz Hybridoma 8(4):377-389, 1989). Alternatively, within a ing conventional enzymatic digestion or recombinant DNA preferred embodiment, the harvested Spleen and/or lymph techniques to incorporate the variable regions of a gene node cell Suspensions are fused with a Suitable myeloma cell which encodes a specifically binding antibody. Within one in order to create a “hybridoma” which secretes monoclonal embodiment, the genes which encode the variable region antibody. Suitable myeloma lines include, for example, from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the Vari NS-1 (ATCC No. TIB 18), and P3X63 - Ag 8,653 (ATCC able region. These primerS may be Synthesized by one of No. CRL 1580). ordinary skill in the art, or may be purchased from com 0172 Following the fusion, the cells may be placed into mercially available Sources. Stratagene (La Jolla, Calif.) culture plates containing a Suitable medium, Such as RPMI Sells primers for mouse and human variable regions includ 1640, or DMEM (Dulbecco's Modified Eagles Medium) ing, among others, primers for VHa, VI, VHe, Vid, CH, VL US 2004/0058321 A1 Mar. 25, 2004

and C regions. These primerS may be utilized to amplify from the primary translation products using the hydropho heavy or light chain variable regions, which may then be bicity plot function of, for example, P/C Gene or Intellige inserted into vectors Such as ImmunoZAPTM H or Immu netics Suite (Intelligenetics, Mountain View, Calif.), or noZAPTM L (Stratagene), respectively. These vectors may according to the methods described by Kyte and Doolittle (J. then be introduced into E. coli, yeast, or mammalian-based Mol. Biol. 157:105-132, 1982). Systems for expression. Utilizing these techniques, large amounts of a Single-chain protein containing a fusion of the 0182 Proteins of the present invention may be prepared V and V domains may-be produced (see Bird et al., in the form of acidic or basic Salts, or in neutral form. In Science 242:423-426, 1988). In addition, such techniques addition, individual amino acid residues may be modified by may be utilized to change a “murine' antibody to a “human” oxidation or reduction. Furthermore, various Substitutions, antibody, without altering the binding Specificity of the deletions, or additions may be made to the amino acid or antibody. nucleic acid Sequences, the net effect of which is to retain or further enhance or decrease the biological activity of the 0176) Once suitable antibodies have been obtained, they mutant or wild-type protein. Moreover, due to degeneracy in may be isolated or purified by many techniques well known the genetic code, for example, there may be considerable to those of ordinary skill in the art (see Antibodies. A variation in nucleotide Sequences encoding the same amino Laboratory Manual, Harlow and Lane (eds.), Cold Spring acid Sequence. Harbor Laboratory Press, 1988). Suitable techniques include 0183. Other derivatives of the proteins disclosed herein peptide or protein affinity columns, HPLC or RP-HPLC, include conjugates of the proteins along with other proteins purification on protein A or protein G columns, or any or polypeptides. This may be accomplished, for example, by combination of these techniques. the synthesis of N-terminal or C-terminal fusion proteins 0177) c. Mutant TGF-beta Binding-proteins which may be added to facilitate purification or identifica 0.178 As described herein and below in the Examples tion of proteins (see U.S. Pat. No. 4,851,341, see also, Hopp (e.g., Examples 8 and 9). altered versions of TGF-beta et al., Bio/Technology 6:1204, 1988.) Alternatively, fusion binding-protein which compete with native TGF-beta bind proteins Such as Flag/TGF-beta binding-protein be con ing-protein's ability to block the activity of a particular Structed in order to assist in the identification, expression, TGF-beta family member should lead to increased bone and analysis of the protein. density. Thus, mutants of TGF-beta binding-protein which 0.184 Proteins of the present invention may be con bind to the TGF-beta family member but do not inhibit the Structed using a wide variety of techniques described herein. function of the TGF-beta family member would meet the Further, mutations may be introduced at particular loci by criteria. The mutant versions must effectively compete with Synthesizing oligonucleotides containing a mutant Sequence, the endogenous inhibitory functions of TGF-beta binding flanked by restriction Sites enabling ligation to fragments of protein. the native Sequence. Following ligation, the resulting recon 0179 d. Production of Proteins Structed Sequence encodes a derivative having the desired 0180 Although various genes (or portions thereof) have amino acid insertion, Substitution, or deletion. been provided herein, it should be understood that within the 0185. Alternatively, oligonucleotide-directed site-spe context of the present invention, reference to one or more of cific (or segment specific) mutagenesis procedures may be these genes includes derivatives of the genes that are Sub employed to provide an altered gene having particular Stantially similar to the genes (and, where appropriate, the codons altered according to the Substitution, deletion, or proteins (including peptides and polypeptides) that are insertion required. Exemplary methods of making the alter encoded by the genes and their derivatives). AS used herein, ations set forth above are disclosed by Walder et al. (Gene a nucleotide Sequence is deemed to be “Substantially simi 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik lar” if: (a) the nucleotide sequence is derived from the (BioTechniques, January 1985, 12-19); Smith et al. (Genetic coding region of the above-described genes and includes, for Engineering. Principles and Methods, Plenum Press, 1981); example, portions of the Sequence or allelic variations of the and Sambrook et al. (Supra). Deletion or truncation deriva Sequences discussed above, or alternatively, encodes a mol tives of proteins (e.g., a Soluble extracellular portion) may ecule which inhibits the binding of TGF-beta binding also be constructed by utilizing convenient restriction endo protein to a member of the TGF-beta family, (b) the nucle nuclease Sites adjacent to the desired deletion. Subsequent to otide Sequence is capable of hybridization to nucleotide restriction, overhangs may be filled in, and the DNA reli Sequences of the present invention under moderate, high or gated. Exemplary methods of making the alterations Set very high Stringency (see Sambrook et al., Molecular Clon forth above are disclosed by Sambrook et al. (Molecular ing: A Laboratory Manual, 2nd ed., Cold Spring Harbor Cloning. A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) the DNA sequences are Laboratory Press, 1989). degenerate as a result of the genetic code to the DNA 0186 Mutations which are made in the nucleic acid Sequences defined in (a) or (b). Further, the nucleic acid molecules of the present invention preferably preserve the molecule disclosed herein includes both complementary and reading frame of the coding Sequences. Furthermore, the non-complementary Sequences, provided the Sequences oth mutations will preferably not create complementary regions erwise meet the criteria set forth herein. Within the context that could hybridize to produce secondary mRNA structures, of the present invention, high Stringency means Standard Such as loops or hairpins, that would adversely affect trans hybridization conditions (e.g., 5xSSPE, 0.5% SDS at 65°C., lation of the mRNA. Although a mutation site may be or the equivalent). predetermined, it is not necessary that the nature of the 0181. The structure of the proteins encoded by the mutation per Se be predetermined. For example, in order to nucleic acid molecules described herein may be predicted Select for optimum characteristics of mutants at a given Site, US 2004/0058321 A1 Mar. 25, 2004 random mutagenesis may be conducted at the target codon Chang et al., Nature 275:615, 1978), the T7 RNA poly and the expressed mutants Screened for indicative biological merase promoter (Studier et al., Meth. Enzymol. 185:60-89, activity. Alternatively, mutations may be introduced at par 1990), the lambda promoter (Elvin et al., Gene 87: 123-126, ticular loci by Synthesizing oligonucleotides containing a 1990), the trp promoter (Nichols and Yanofsky, Meth. in mutant Sequence, flanked by restriction sites enabling liga Enzymology 101: 155, 1983) and the tac promoter (Russellet tion to fragments of the native Sequence. Following ligation, al., Gene 20:231, 1982). Representative selectable markers the resulting reconstructed Sequence encodes a derivative include various antibiotic resistance markerS Such as the having the desired amino acid insertion, Substitution, or kanamycin or amplicillin resistance genes. Many plasmids deletion. Suitable for transforming host cells are well known in the art, 0187 Nucleic acid molecules which encode proteins of including among others, pBR322 (see Bolivar et al., Gene the present invention may also be constructed utilizing 2:95, 1977), the pUC plasmids pUC18, puC19, puC118, techniques of PCR mutagenesis, chemical mutagenesis pUC119 (see Messing, Meth. in Enzymology 101:20-77, (Drinkwater and Klinedinst, PNAS 83:3402-3406, 1986), by 1983 and Vieira and Messing, Gene 19:259-268, 1982), and forced nucleotide misincorporation (e.g., Liao and Wise pNH8A, pNH16a, pNH18a, and Bluescript M13 (Strat Gene 88: 107-111, 1990), or by use of randomly agene, La Jolla, Calif.). mutagenized oligonucleotides (Horwitz et al., Genome 0.192 Yeast and fungi host cells suitable for carrying out 3:112-117, 1989). the present invention include, among others, Saccharomyces 0188 The present invention also provides-for the pombe, Saccharomyces cerevisiae, the genera Pichia or manipulation and expression of the above described genes Kluyveromyces and various Species of the genus Aspergillus by culturing host cells containing a vector capable of (McKnight et al., U.S. Pat. No. 4,935,349). Suitable expres expressing the above-described genes. Such vectors or vec Sion vectors for yeast and fungi include, among others, tor constructs include either synthetic or cDNA-derived YCp50 (ATCC No. 37419) for yeast, and the amdS cloning nucleic acid molecules encoding the desired protein, which vector pV3 (Turnbull, Bio/Technology 7: 169, 1989), YRp7 are operably linked to Suitable transcriptional or transla (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, tional regulatory elements. Suitable regulatory elements 1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), may be derived from a variety of Sources, including bacte plDB249 and plDB219 (Beggs, Nature 275:104-108, 1978) rial, fungal, Viral, mammalian, insect, or plant genes. Selec and derivatives thereof. tion of appropriate regulatory elements is dependent on the 0193 Preferred promoters for use in yeast include pro host cell chosen, and may be readily accomplished by one of moters from yeast glycolytic genes (Hitzeman et al., J. Biol. ordinary skill in the art. Examples of regulatory elements Chem. 255:12073-12080, 1980; Alber and Kawasaki. J. include: a transcriptional promoter and enhancer or RNA Mol. Appl. Genet. 1:419-434, 1982) or alcohol dehydroge polymerase binding Sequence, a transcriptional terminator, nase genes (Young et al., in Genetic Engineering of Micro and a ribosomal binding Sequence, including a translation organisms for Chemicals, Hollaender et al. (eds.), p. 355, initiation signal. Plenum, New York, 1982; Ammerer, Meth. Enzymol. 0189 Nucleic acid molecules that encode any of the 101:192-201, 1983). Examples of useful promoters for fungi proteins described above may be readily expressed by a vectors include those derived from Aspergillus nidulans wide variety of prokaryotic and eukaryotic host cells, glycolytic genes, Such as the adh3 promoter (McKnight et including bacterial, mammalian, yeast or other fungi, Viral, al., EMBO.J. 4:2093-2099, 1985). The expression units may insect, or plant cells. Methods for transforming or transfect also include a transcriptional terminator. An example of a ing Such cells to express foreign DNA are well known in the suitable terminator is the adh3 terminator (McKnight et al., art (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362; Hinnen ibid., 1985). et al., Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978; 0194 AS with bacterial vectors, the yeast vectors will Murray et al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. generally include a Selectable marker, which may be one of Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950; any number of genes that exhibit a dominant phenotype for Axel et al., U.S. Pat. No. 4.399.216; Goeddelet al., U.S. Pat. which a phenotypic assay exists to enable transformants to No. 4,766,075; and Sambrook et al. Molecular Cloning. A be selected. Preferred selectable markers are those that Laboratory Manual, 2nd ed., Cold Spring Harbor Labora complement host cell auxotrophy, provide antibiotic resis tory Press, 1989: for plant cells see CZako and Marton, Plant tance or enable a cell to utilize specific carbon Sources, and Physiol. 104:1067-1071, 1994; and Paszkowski et al., Bio include leu2 (Broach et al., ibid.), ura3 (Botstein et al., Gene tech. 24:387-392, 1992). 8:17, 1979), or his3 (Struhl et al., ibid.). Another suitable 0.190 Bacterial host cells suitable for carrying out the Selectable marker is the cat gene, which conferS chloram present invention include E. coli, B. Subtilis, Salmonella phenicol resistance on yeast cells. typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, as well as 0.195 Techniques for transforming fungi are well known in the literature, and have been described, for instance, by many other bacterial Species well known to one of ordinary Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA skill in the art. Representative examples of bacterial host 75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. cells include DH5C. (Stratagene, LaJolla, Calif.). USA 81:1740-1747, 1984), and Russell (Nature 301:167 0191 Bacterial expression vectors preferably comprise a 169, 1983). The genotype of the host cell may contain a promoter which functions in the host cell, one or more genetic defect that is complemented by the Selectable marker Selectable phenotypic markers, and a bacterial origin of present on the expression vector. Choice of a particular host replication. Representative promoters include the B-lacta and selectable marker is well within the level of ordinary mase (penicillinase) and lactose promoter System (see skill in the art. US 2004/0058321 A1 Mar. 25, 2004

0196. Protocols for the transformation of yeast are also Also contained in the expression vectorS is a polyadenyla well known to those of ordinary skill in the art. For example, tion signal located downstream of the coding Sequence of transformation may be readily accomplished either by interest. Suitable polyadenylation signals include the early preparation of spheroplasts of yeast with DNA (see Hinnen or late polyadenylation signals from SV40 (Kaufman and et al., PNAS USA 75:1929, 1978) or by treatment with Sharp, ibid.), the polyadenylation signal from the Adenovi alkaline Salts Such as LiCl (See Itoh et al., J. Bacteriology rus 5 E1B region and the human growth hormone gene 153:163, 1983). Transformation of fungi may also be carried terminator (DeNoto et al., Nuc. Acids Res. 9:3719-3730, out using polyethylene glycol as described by Cullen et al. 1981). The expression vectors may include a noncoding (Bio/Technology 5:369, 1987). Viral leader Sequence, Such as the Adenovirus 2 tripartite 0.197 Viral vectors include those which comprise a pro leader, located between the promoter and the RNA splice moter that directs the expression of an isolated nucleic acid Sites. Preferred vectors may also include enhancer molecule that encodes a desired protein as described above. Sequences, Such as the SV40 enhancer. Expression vectors A wide variety of promoters may be utilized within the may also include Sequences encoding the adenovirus VA context of the present invention, including for example, RNAS. Suitable expression vectors can be obtained from promoters such as MoMLV LTR, RSV LTR, Friend Mul V commercial Sources (e.g., Stratagene, La Jolla, Calif.). LTR, adenoviral promoter (Ohno et al., Science 265:781 0200 Vector constructs comprising cloned DNA 784, 1994), neomycin phosphotransferase promoter/en Sequences can be introduced into cultured mammalian cells hancer, late parvovirus promoter (Koering et al., Hum. Gene by, for example, calcium phosphate-mediated transfection Therap. 5:457-463, 1994), Herpes TK promoter, SV40 pro (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, moter. metallothionein IIa gene enhancer/promoter, cytome Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, galovirus immediate early promoter, and the cytomegalovi Virology 52:456, 1973), electroporation (Neumann et al., rus immediate late promoter. Within particularly preferred EMBO J. 1:841-845, 1982), or DEAE-dextran mediated embodiments of the invention, the promoter is a tissue transfection (Ausubel et al. (eds.), Current Protocols in specific promoter (see e.g., WO 91/02805; EP 0.415,731; Molecular Biology, John Wiley and Sons, Inc., NY, 1987). and WO 90/07936). Representative examples of suitable To identify cells that have stably integrated the cloned DNA, tissue Specific promoters include neural Specific enolase a Selectable marker is generally introduced into the cells promoter, platelet derived growth factor beta promoter, bone along with the gene or cDNA of interest. Preferred selectable morphogenic protein promoter, human alpha1-chimaerin markers for use in cultured mammalian cells include genes promoter, Synapsin I promoter and Synapsin II promoter. In that confer resistance to drugs, Such as neomycin, hygro addition to the above-noted promoters, other viral-specific mycin, and methotrexate. The Selectable marker may be an promoters (e.g., retroviral promoters (including those noted amplifiable selectable marker. Preferred amplifiable select above, as well as otherS Such as HIV promoters), hepatitis, able markers are the DHFR gene and the neomycin resis herpes (e.g., EBV), and bacterial, fungal or parasitic (e.g., tance gene. Selectable markers are reviewed by Thilly malarial)-specific promoters may be utilized in order to (Mammalian Cell Technology, Butterworth Publishers, target a specific cell or tissue which is infected with a virus, Stoneham, Mass., which is incorporated herein by refer bacteria, fungus or parasite. ence). 0198 Mammalian cells suitable for carrying out the 0201 Mammalian cells containing a suitable vector are present invention include, among others COS, CHO, SaOS, allowed to grow for a period of time, typically 1-2 days, to osteosarcomas, KS483, MG-63, primary osteoblasts, and begin expressing the DNA sequence(s) of interest. Drug human or mammalian bone marrow Stroma. Mammalian Selection is then applied to Select for growth of cells that are expression vectors for use in carrying out the present inven expressing the Selectable marker in a stable fashion. For tion will include a promoter capable of directing the tran cells that have been transfected with an amplifiable, Select scription of a cloned gene or cDNA. Preferred promoters able marker the drug concentration may be increased in a include Viral promoters and cellular promoters. Bone spe Stepwise manner to Select for increased copy number of the cific promoters include the bone Sialo-protein and the pro cloned Sequences, thereby increasing expression levels. moter for Osteocalcin. Viral promoters include the cytome Cells expressing the introduced Sequences are Selected and galovirus immediate early promoter (Boshart et al., Cell Screened for production of the protein of interest in the 41:521-530, 1985), cytomegalovirus immediate late pro desired form or at the desired level. Cells that satisfy these moter, SV40 promoter (Subramani et al., Mol. Cell. Biol. criteria can then be cloned and Scaled up for production. 1:854-864, 1981), MMTV LTR, RSV LTR, metallothionein 0202 Protocols for the transfection of mammalian cells 1, adenovirus Ela. Cellular promoters include the mouse are well known to those of ordinary skill in the art. Repre metallothionein-1 promoter (Palmiter et al., U.S. Pat. No. Sentative methods include calcium phosphate mediated 4.579,821), a mouse V promoter (Bergman et al., Proc. transfection, electroporation, lipofection, retroviral, aden Natl. Acad. Sci. USA 81: 7041-7045, 1983; Grant et al., Nucl. Oviral and protoplast fusion-mediated transfection (see Sam Acids Res. 15:5496, 1987) and a mouse V promoter (Loh brook et al., Supra). Naked vector constructs can also be et al., Cell 33:85-93, 1983). The choice of promoter will taken up by muscular cells or other Suitable cells Subsequent depend, at least in part, upon the level of expression desired to injection into the muscle of a mammal (or other animals). or the recipient cell line to be transfected. 0203) Numerous insect host cells known in the art can 0199 Such expression vectors may also contain a set of also be useful within the present invention, in light of the RNA splice sites located downstream from the promoter and Subject Specification. For example, the use of baculoviruses upstream from the DNA sequence encoding the peptide or as Vectors for expressing heterologous DNA sequences in protein of interest. Preferred RNA splice sites may be insect cells has been reviewed by Atkinson et al. (Pestic. Sci. obtained from adenovirus and/or immunoglobulin genes. 28:215-224, 1990). US 2004/0058321 A1 Mar. 25, 2004 20

0204 Numerous plant host cells known in the art can also TGF-beta binding-protein binding to a member of the TGF be useful within the present invention, in light of the subject beta family. For example, within one embodiment antisense Specification. For example, the use of Agrobacterium rhizo oligonucleotide molecules are provided which specifically genes as vectors for expressing genes in plant cells has been inhibit expression of TGF-beta binding-protein nucleic acid reviewed by Sinkar et al. (J. BioSci. (Bangalore) 11:47-58, Sequences (see generally, Hirashima et al. in Molecular 1987). Biology of RNA. New Perspectives (M. Inouye and B. S. 0205 Within related aspects of the present invention, Dudock, eds., 1987 Academic Press, San Diego, p. 401); proteins of the present invention may be expressed in a Oligonucleotides. Antisense Inhibitors of Gene Expression transgenic animal whose germ cells and Somatic cells con (J. S. Cohen, ed., 1989 MacMillan Press, London); Stein and tain a gene which encodes the desired protein and which is Cheng, Science 261:1004-1012, 1993; WO95/10607; U.S. operably linked to a promoter effective for the expression of Pat. No. 5,359,051; WO 92/06693; and EP-A2-612844). the gene. Alternatively, in a similar manner transgenic Briefly, Such molecules are constructed Such that they are animals may be prepared that lack the desired gene (e.g., complementary to, and able to form Watson-Crick base pairs “knock-Out' mice). Such transgenics may be prepared in a with, a region of transcribed TGF-beta binding-protein variety of non-human animals, including mice, rats, rabbits, mRNA sequence. The resultant double-stranded nucleic acid sheep, dogs, goats and pigs (see Hammer et al., Nature interferes with subsequent processing of the mRNA, thereby 315:680-683, 1985, Palmiter et al., Science 222:809-814, preventing protein Synthesis (see Example 10). 1983, Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438 0210. Within other aspects of the invention, ribozymes 4442, 1985, Palmiter and Brinster, Cell 41:343-345, 1985, are provided which are capable of inhibiting the TGF-beta and U.S. Pat. Nos. 5,175,383, 5,087,571, 4,736,866, 5,387, binding-protein binding to a member of the TGF-beta fam 742, 5,347,075, 5,221,778, and 5,175,384). Briefly, an ily. As used herein, “ribozymes' are intended to include expression vector, including a nucleic acid molecule to be RNA molecules that contain anti-Sense Sequences for Spe expressed together with appropriately positioned expression cific recognition, and an RNA-cleaving enzymatic activity. control Sequences, is introduced into pronuclei of fertilized The catalytic Strand cleaves a specific Site in a target RNA eggs, for example, by microinjection. Integration of the at greater than Stoichiometric concentration. A wide variety injected DNA is detected by blot analysis of DNA from of ribozymes may be utilized within the context of the tissue samples. It is preferred that the introduced DNA be present invention, including for example, the hammerhead incorporated into the germ line of the animal So that it is ribozyme (for example, as described by Forster and Symons, passed on to the animal's progeny. TiSSue-specific expres Cell 48:211-220, 1987; Haseloff and Gerlach, Nature Sion may be achieved through the use of a tissue-specific 328:596-600, 1988; Walbot and Bruening, Nature 334:196, promoter, or through the use of an inducible promoter, Such 1988; Haseloff and Gerlach, Nature 334:585, 1988); the as the metallothionein gene promoter (Palmiter et al., 1983, hairpin ribozyme (for example, as described by Haseloff et ibid), which allows regulated expression of the transgene. al., U.S. Pat. No. 5,254,678, issued Oct. 19, 1993 and 0206 Proteins can be isolated by, among other methods, Hempel et al., European Patent Publication No. 0360 257, culturing Suitable host and vector Systems to produce the published Mar. 26, 1990); and Tetrahymena ribosomal recombinant translation products of the present invention. RNA-based ribozymes (see Cech et al., U.S. Pat. No. Supernatants from Such cell lines, or protein inclusions or 4.987,071). Ribozymes of the present invention typically whole cells where the protein is not excreted into the consist of RNA, but may also be composed of DNA, nucleic Supernatant, can then be treated by a variety of purification acid analogs (e.g., phosphorothioates), or chimerics thereof procedures in order to isolate the desired proteins. For (e.g., DNA/RNA/RNA). example, the Supernatant may be first concentrated using 02.11 4. Labels commercially available protein concentration filters, Such as an Amicon or Millipore Pellicon ultrafiltration unit. Follow 0212. The gene product or any of The candidate mol ing concentration, the concentrate may be applied to a ecules described above and below, may be labeled with a Suitable purification matrix Such as, for example, an anti variety of compounds, including for example, fluorescent protein antibody bound to a Suitable Support. Alternatively, molecules, toxins, and radionuclides. Representative anion or cation eXchange resins may be employed in order examples of fluorescent molecules include fluorescein. Phy to purify the protein. As a further alternative, one or more cobili proteins, Such as phycoerythrin, rhodamine, Texas red reverse-phase high performance liquid chromatography, and luciferase. Representative examples of toxins include (RP-HPLC) steps may be employed to further purify the ricin, abrin diphtheria toxin, cholera toxin, gelonin, protein. Other methods of isolating the proteins of the pokeweed antiviral protein, tritin, Shigella toxin, and present invention are well known in the skill of the art. Pseudomonas eXotoxin A. Representative examples of 0207. A protein is deemed to be “isolated” within the radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, context of the present invention if no other (undesired) Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, protein is detected pursuant to SDS-PAGE analysis followed Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 by Coomassie blue staining. Within other embodiments, the and Bi-212. In addition, the antibodies described above may desired protein can be isolated Such that no other (undesired) also be labeled or conjugated to one partner of a ligand protein is detected pursuant to SDS-PAGE analysis followed binding pair. Representative examples include avidin-biotin, by Silver Staining. and riboflavin-riboflavin binding protein. 0213 Methods for conjugating or labeling the molecules 0208. 3. Nucleic Acid Molecules described herein with the representative labels set forth 0209. Within other aspects of the invention, nucleic acid above may be readily accomplished by one of ordinary skill molecules are provided which are capable of inhibiting in the art (see Trichothecene Antibody Conjugate, U.S. Pat. US 2004/0058321 A1 Mar. 25, 2004

No. 4,744,981; Antibody Conjugate, U.S. Pat. No. 5,106, cine, McGraw-Hill, Inc.). Representative examples of dis 951; Fluorogenic Materials and Labeling Techniques, U.S. eases that may be treated included dysplasias, wherein there Pat. No. 4,018,884; Metal Radionuclide Labeled Proteins for is abnormal growth or development of bone. Representative Diagnosis and Therapy, U.S. Pat. No. 4,897.255; and Metal examples of Such conditions include achondroplasia, Radionuclide Chelating Compounds for Improved Chelation cleidocranial dysostosis, enchondromatosis, fibrous dyspla Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Methods sia, Gauchers, hypophosphatemic rickets, Marfan's, mul In Enzymology, Vol. 34, Afinity Techniques, Enzyme Puri tiple hereditary exotoses, neurofibromatosis, osteogenesis fication. Part B, Jakoby and Wilchek (eds.), Academic imperfecta, Osteopetrosis, osteopoikilosis, Sclerotic lesions, Press, New York, p. 30, 1974; see also Wilchek and Bayer, fractures, periodontal disease, pseudoarthrosis and pyogenic “The Avidin-Biotin Complex in Bioanalytical Applications, Osteomyelitis. 'Anal. Biochem. 171:1-32, 1988). 0218. Other conditions which may be treated or pre Pharmaceutical Compositions vented include a wide variety of causes of Osteopenia (i.e., a condition that causes greater than one Standard deviation 0214. As noted above, the present invention also provides of bone mineral content or density below peak skeletal a variety of pharmaceutical compositions, comprising one of mineral content at youth). Representative examples of Such the above-described molecules which inhibits the TGF-beta conditions include anemic States, conditions caused Steroids, binding-protein binding to a member of the TGF-beta family conditions caused by heparin, bone marrow disorders, along with a pharmaceutically or physiologically acceptable Scurvy, malnutrition, calcium deficiency, idiopathic carrier, excipients or diluents. Generally, Such carriers Osteoporosis, congenital Osteopenia or osteoporosis, alco should be nontoxic to recipients at the dosages and concen holism, chronic liver disease, Senility, postmenopausal State, trations employed. Ordinarily, the preparation of Such com oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, positions entails combining the therapeutic agent with buff hyperthyroidism, Cushing's disease, acromegaly, hypogo ers, antioxidants Such as ascorbic acid, low molecular nadism, immobilization or disuse, refleX Sympathetic dyS weight (less than about 10 residues) polypeptides, proteins, trophy Syndrome, transient regional Osteoporosis and Osteo amino acids, carbohydrates including glucose, Sucrose or malacia. dextrins, chelating agents Such as EDTA, glutathione and 0219. Within one aspect of the present invention, bone other stabilizers and excipients. Neutral buffered saline or mineral content or density may be increased by administer Saline mixed with nonspecific Serum albumin are exemplary ing to a warm-blooded animal a therapeutically effective appropriate diluents. amount of a molecule which inhibits the TGF-beta binding 0215. In addition, the pharmaceutical compositions of the protein binding to a TGF-beta family member. Examples of present invention may be prepared for administration by a warm-blooded animals that may be treated include both variety of different routes. In addition, pharmaceutical com vertebrates and mammals, including for example horses, positions of the present invention may be placed within cows, pigs, sheep, dogs, cats, rats and mice. Representative containers, along with packaging material which provides examples of therapeutic molecules include ribozymes, instructions regarding the use of Such pharmaceutical com ribozyme genes, antisense oligonucleotides and antibodies positions. Generally, Such instructions will include a tan (e.g., humanized antibodies). gible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of 0220. Within other aspects of the present invention, excipient ingredients or diluents (e.g., water, Saline or PBS) methods are provided for increasing bone density, compris which may be necessary to reconstitute the pharmaceutical ing the Step of introducing into cells which home to bone a composition. vector which directs the expression of a molecule which inhibits the TGF-beta binding-protein binding to a member of the TGF-beta family, and administering the vector con Methods of Treatment taining cells to a warm-blooded animal. Briefly, cells which 0216) The present invention also provides methods for home to bone may be obtained directly from the bone of increasing the mineral content and mineral density of bone. patients (e.g., cells obtained from the bone marrow Such as Briefly, numerous conditions result in the loss of bone CD34+, osteoblasts, osteocytes, and the like), from periph mineral content, including for example, disease, genetic eral blood, or from cultures. predisposition, accidents which result in the lack of use of 0221) A vector which directs the expression of a molecule bone (e.g., due to fracture), therapeutics which effect bone that inhibits the TGF-beta binding-protein binding to a resorption, or which kill bone forming cells and normal member of the TGF-beta family is introduced into the cells. aging. Through use of the molecules described herein which Representative examples of Suitable vectors include viral inhibit the TGF-beta binding-protein binding to a TGF-beta vectorS Such as herpes viral vectors (e.g., U.S. Pat. No. family member Such conditions may be treated or prevented. 5,288,641), adenoviral vectors (e.g., WO 94/26914, WO AS utilized herein, it should be understood that bone mineral 93/9191; Kolls et al., PNAS 91(1):215-219, 1994; Kass content has been increased, if bone mineral content has been Eisler et al., PNAS 90(24):11498–502, 1993; Guzman et al., increased in a statistically significant manner (e.g., greater Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. than one-half standard deviation), at a selected site. 73(6):1202-1207, 1993: Zabner et al., Cell 75(2):207-216, 0217. A wide variety of conditions which result in loss of 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993; Cail bone mineral content may be treated with the molecules laudet al., Eur. J. Neurosci. 5(10:1287-1291, 1993; Vincent described herein. Patients with such conditions may be et al., Nat. Genet. 5(2): 130-134, 1993; Jaffe et al., Nat. identified through clinical diagnosis utilizing well known Genet. 1(5):372-378, 1992; and Levrero et al., Gene techniques (see, e.g., Harrison's Principles of Internal Medi 101(2):195-202, 1991), adeno-associated viral vectors (WO US 2004/0058321 A1 Mar. 25, 2004 22

95/13365; Flotte et al., PNAS 90(22):10613-10617, 1993), EXAMPLES baculovirus vectors, parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457-463, 1994), pox virus vectors Example 1 (Panicali and Paoletti, PNAS 79:4927-4931, 1982; and Ozaki et al., Biochem. BiophyS. Res. Comm. 193(2):653 Sclerosteosis Maps to the Long Arm of Human 660, 1993), and retroviruses (e.g., EP 0.415,731; WO Chromosome 17 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 0227 Genetic mapping of the defect responsible for 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO Sclerosteosis in humans localized the gene responsible for 93/10218). Viral vectors may likewise be constructed which this disorder to the region of human chromosome 17 that contain a mixture of different elements (e.g., promoters, encodes a novel TGF-beta binding-protein family member. envelope Sequences and the like) from different viruses, or In Sclerosteosis, Skeletal bone displays a Substantial increase non-viral Sources. Within various embodiments, either the in mineral density relative to that of unafflicted individuals. viral vector itself, or a viral particle which contains the viral Bone in the head displays overgrowth as well. Sclerosteosis vector may be utilized in the methods and compositions patients are generally healthy although they may exhibit described below. variable degrees of Syndactyly at birth and variable degrees 0222. Within other embodiments of the invention, of cranial compression and nerve compression in the Skull. nucleic acid molecules which encode a molecule which 0228 Linkage analysis of the gene defect associated with inhibits the TGF-beta binding-protein binding to a member Sclerosteosis was conducted by applying the homozygosity of the TGF-beta family themselves may be administered by mapping method to DNA samples collected from 24 South a variety of techniques, including, for example, administra African Afrikaaner families in which the disease occurred tion of asialoosomucoid (ASOR) conjugated with poly-L- (Sheffield et al., 1994, Human Molecular Genetics 3:1331 lysine DNA complexes (Cristano et al., PNAS92122-92126, 1335. “Identification of a Bardet-Biedl syndrome locus on chromosome 3 and evaluation of an efficient approach to 1993), DNA linked to killed adenovirus (Curiel et al., Hum. homozygosity mapping”). The Afrikaaner population of Gene Ther. 3(2): 147-154, 1992), cytofectin-mediated intro South Africa is genetically homogeneous, the population is duction (DMRIE-DOPE, Vical, Calif.), direct DNAinjection descended from a small number of founders who colonized (AcSadiet al., Nature 352:815-818, 1991); DNA ligand (Wu the area Several centuries ago, and it has been isolated by et al., J. of Biol. Chem. 264:16985-16987, 1989); lipofection geographic and Social barrierS Since the founding. Scleros (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, teosis is rare everywhere in the world outside the Afrikaaner 1989); liposomes (Pickering et al., Circ. 89(1):13-21, 1994; community, which Suggests that a mutation in the gene was and Wang et al., PNAS 84:7851-7855, 1987); microprojec present in the founding population and has Since increased tile bombardment (Williams et al., PNAS 88:2726-2730, in numbers along with the increase in the population. The 1991); and direct delivery of nucleic acids which encode the use of homozygosity mapping is based on the assumption protein itself either alone (Vile and Hart, Cancer Res. 53: that DNA mapping markers adjacent to a recessive mutation 3860-3864, 1993), or utilizing PEG-nucleic acid complexes. are likely to be homozygous in affected individuals from 0223 Representative examples of molecules which may consanguineous families and isolated populations. be expressed by the vectors of present invention include 0229. A set of 371 microsatellite markers (Research ribozymes and antisense molecules, each of which are Genetics, Set 6) from the autosomal chromosomes was discussed in more detail above. Selected to type pools of DNA from Sclerosteosis patient 0224 Determination of increased bone mineral content samples. The DNA samples for this analysis came from 29 may be determined directly through the use of X-rays (e.g., Sclerosteosis patients in 24 families, 59 unaffected family Dual Energy X-ray Absorptometry or “DEXA”), or by members and a set of unrelated control individuals from the inference through bone turnover markers (osteoblast specific Same population. The pools consisted of 4-6 individuals, alkaline phosphatase, osteocalcin, type 1 procollagen C either affected individuals, affected individuals from con propeptide (PICP), and total alkaline phosphatase; see Sanguineous families, parents and unaffected Siblings, or Comier, C., Curr. Opin. in Rheu. 7:243, 1995), or markers of unrelated controls. In the pools of unrelated individuals and bone resorption (pyridinoline, deoxypryridinoline, N-te in most of the pools with affected individuals or family lopeptide, urinary hydroxyproline, plasma tartrate-resistant members analysis of the markers showed Several allele sizes acid phosphatases and galactosyl hydroxylysine; See for each marker. One marker, D17S1299, showed an indi Comier, Supra). The amount of bone mass may also be cation of homozygosity: one band in Several of the pools of calculated from body weights, or utilizing other methods affected individuals. (see Guinness-Hey, Metab. Bone Dis. and Rel. Res. 5:177 0230 All 24 sclerosteosis families were typed with a total 181, 1984). of 19 markers in the region of D17S1299 (at 17q12-q21). 0225. As will be evident to one of skill in the art, the Affected individuals from every family were shown to be amount and frequency of administration will depend, of homozygous in this region, and 25 of the 29 individuals course, on Such factors as the nature and Severity of the were homozygous for a core haplotype; they each had the indication being treated, the desired response, the condition same alleles between D17S1787 and D17S930. The other of the patient, and So forth. Typically, the compositions may four individuals had one chromosome which matched this be administered by a variety of techniques, as noted above. haplotype and a Second which did not. In Sum, the data compellingly Suggested that this 3 megabase region con 0226. The following examples are offered by way of tained the Sclerosteosis mutation. Sequence analysis of most illustration, and not by way of limitation. of the exons in this 3 megabase region identified a nonsense US 2004/0058321 A1 Mar. 25, 2004 mutation in the novel TGF-beta binding-protein coding tin, Tex.). In-Situ hybridization was performed according to sequence (C>Tmutation at position 117 of Sequence ID No. the protocols of Lyons et al. (J. Cell Biol. 111:2427-2436, 1 results in a stop codon). This mutation was shown to be 1990). unique to Sclerosteosis patients and carriers of Afrikaaner 0237) The mouse Beer cRNA probe detected a specific descent. The identity of the gene was further confirmed by message expressed in the neural tube, limb buds, blood identifying a mutation in its intron (A>Tmutation at position vessels and OSSifying cartilages of developing mouse +3 of the intron) which results in improper mRNA process embryos. Panel A in FIG. 3 shows expression in the apical ing in a single, unrelated patient with diagnosed Sclerosteo ectodermal ridge (aer) of the limb (1) bud, blood vessels (bv) SS. and the neural tube (nt). Panel B shows expression in the 4" ventricle of the brain (4). Panel C shows expression in the Example 2 mandible (ma) cervical vertebrae (cv), occipital bone (oc), palate (pa) and a blood vessel (bv). Panel D shows expres Tissue-specificity of TGF-beta Binding-protein sion in the ribs (r) and a heart valve (va). Panel A is a Gene Expression transverse section of 10.5 dpc embryo. Panel B is a sagittal 0231 A. Human Beer Gene Expression by RT-PCR: Section of 12.5 dpc embryo and panels C and D are Sagittal sections of 15.5 dpc embryos. ba-branchial arch, h=heart, 0232 First-strand cDNA was prepared from the follow te=telencephalon (forebrain), b=brain, f=frontonasal mass, ing total RNA samples using a commercially available kit g=gut, h=heart, j=jaw, li=liver, lu=lung, ot=otic Vesicle, ao-, (“Superscript Preamplification System for First-Strand Sc=Spinal cord, Skm=Skeletal muscle, nS=nasal Sinus, cDNA Synthesis”, Life Technologies, Rockville, Md.): th=thymus, to=tongue, fl=forelimb, di-diaphragm human brain, human liver, human Spleen, human thymus, human placenta, human skeletal muscle, human thyroid, Example 3 human pituitary, human osteoblast (NHOst from Clonetics Corp., San Diego, Calif.), human osteosarcoma cell line Expression and Purification of Recombinant Beer (Saos-2, ATCC# HTB-85), human bone, human bone mar Protein row, human cartilage, Vervet monkey bone, Saccharomyces cerevisiae, and human peripheral blood monocytes. All 0238 A. Expression in COS-1 Cells: RNA samples were purchased from a commercial Source (Clontech, Palo Alto, Calif.), except the following which 0239). The DNA sequence encoding the full length human were prepared in-house: human osteoblast, human osteosa Beer protein was amplified using the following PCR oligo rcoma cell line, human bone, human cartilage and Vervet nucleotide primers: The 5' oligonucleotide primer had the monkey bone. These in-house RNA samples were prepared sequence 5'-AAGCTTGGTACCATGCAGCTCCCAC-3' using a commercially available kit (“TRI Reagent”, Molecu (SEQID NO:23) and contained a HindIII restriction enzyme site (in bold) followed by 19 nucleotides of the Beer gene lar Research Center, Inc., Cincinnati, Ohio). Starting 6 base pairs prior to the presumed amino terminal 0233 PCR was performed on these samples, and addi start codon (ATG). The 3' oligonucleotide primer had the tionally on a human genomic Sample as a control. The Sense Sequence 5'-AAGCTTCTACTTGTCATCGTCGTCCT Beer oligonucleotide primer had the sequence 5'-CCG TGTAGTCGTAGGCGTTCTCCAGCT-3' (SEQ ID NO:24) GAGCTGGAGAACAACAAG-3' (SEQ ID NO:19). The and contained a HindIII restriction enzyme site (in bold) antisense Beer oligonucleotide primer had the Sequence followed by a reverse complement stop codon (CTA) fol 5'-GCACTGGCCGGAGCACACC-3' (SEQ ID NO:20). In lowed by the reverse complement of the FLAG epitope addition, PCR was performed using primers for the human (underlined, Sigma-Aldrich Co., St. Louis, Mo.) flanked by beta-actin gene, as a control. The Sense beta-actin oligo the reverse complement of nucleotides coding for the car nucleotide primer had the sequence 5'-AGGCCAACCGC boxy terminal 5 amino acids of the Beer. The PCR product GAGAAGATGA CC-3' (SEQ ID NO:21). The antisense was TA cloned (“Original TA Cloning Kit”, Invitrogen, beta-actin oligonucleotide primer had the Sequence Carlsbad, Calif.) and individual clones were screened by 5'-GAAGTCCAGGGCGACGTAGCA-3' (SEQID NO:22). DNA sequencing. A sequence-verified clone was then PCR was performed using standard conditions in 25 ul digested by HindIII and purified on a 1.5% agarose gel using reactions, with an annealing temperature of 61 degrees a commercially available reagents ("QIAquick Gel Extrac Celsius. Thirty-two cycles of PCR were performed with the tion Kit, Qiagen Inc., Valencia, Calif.). This fragment was Beer primers and twenty-four cycles were performed with then ligated to HindIII digested, phosphatase-treated the beta-actin primerS. pcDNA3.1 (Invitrogen, Carlsbad, Calif.) plasmid with T4 DNA ligase. DH 10B E. coli were transformed and plated on 0234. Following amplification, 12 ul from each reaction LB, 100 lug/ml amplicillin plates. Colonies bearing the were analyzed by agarose gel electrophoresis and ethidium desired recombinant in the proper orientation were identified bromide staining. See FIG. 2A. by a PCR-based Screen, using a 5" primer corresponding to 0235 B. RNA In-situ Hybridization of Mouse Embryo the T7 promoter/priming site in pcDNA3.1 and a 3' primer Sections: with the sequence 5'-GCACTGGCCGGAGCACACC-3' (SEQ ID NO:25) that corresponds to the reverse comple 0236. The full length mouse Beer cDNA (Sequence ID ment of internal BEER sequence. The sequence of the No. 11) was cloned into the pCR2.1 vector (Invitrogen, cloned fragment was confirmed by DNA sequencing. Carlsbad, Calif.) in the antisense and Sense direction using the manufacturer's protocol. S-alpha-GTP-labeled cRNA 0240 COS-1 cells (ATCC# CRL-1650) were used for Sense and antisense transcripts were Synthesized using in transfection. 50 lug of the expression plasmid pcDNA-Beer vitro transcription reagents Supplied by Ambion, Inc (Aus Flag was transfected using a commercially available kit US 2004/0058321 A1 Mar. 25, 2004 24 following protocols supplied by the manufacturer (“DEAE was extracted from the cell pellet using a high Salt extraction Dextran Transfection Kit', Sigma Chemical Co., St. Louis, buffer (1.5M NaCl, 50 mM Tris pH 7.5). The extract (20 ml Mo.). The final media following transfection was DMEM (Life Technologies, Rockville, Md.) containing 0.1% Fetal per 300 ml culture) was clarified by centrifugation, dialyzed Bovine Serum. After 4 days in culture, the media was three times against four liters of Tris buffered saline (150 removed. Expression of recombinant BEER was analyzed mM NaCl, 50 mM Tris pH 7.5), and clarified by centrifu by SDS-PAGE and Western Blot using anti-FLAG M2 gation again. This high Salt fraction was applied to Hitrap monoclonal antibody (Sigma-Aldrich Co., St. Louis, Mo.). Heparin (Pharmacia; 5 ml bed volume), washed extensively Purification of recombinant BEER protein was performed using an anti-FLAG M2 affinity column (“Mammalian Tran with HEPES buffered saline (25 mM HEPES 7.5, 150 mM Sient Expression System', Sigma-Aldrich Co., St. Louis, Nacl) and bound proteins were eluted with a gradient from Mo.). The column profile was analyzed via SDS-PAGE and 150 mM NaCl to 1200 mM NaCl. Beer elution was observed Western Blot using anti-FLAG M2 monoclonal antibody. at aproximately 800 mM NaCl. Beer containing fractions 0241 B. Expression in SF9 Insect Cells: were supplemented to 10% glycerol and 1 mM DTT and frozen at -80 degrees C. 0242. The human Beer gene sequence was amplified using PCR with standard conditions and the following primers: Example 4 Preparation and Testing of Polyclonal Antibodies to (SEQ ID NO: 26) Beer, Gremlin, and Dan Sense primer: 5'-GTCGTCGGATCCATGGGGTGGCAGGCGTT CAAGAATGAT-3' 0247 A. Preparation of Antigen: (SEQ ID NO: 27) Antisense primer: 5'-GTCGTCAAGCTTCTACTTGTCATCGTCCT 0248. The DNA sequences of Human Beer, Human TGTAGTCGTAGGCGTTCTCCAGCTCGGC-3' Gremlin, and Human Dan were amplified using Standard PCR methods with the following oligonucleotide primers:

H. Beer Sense: 5'-GACTTGGATCCCAGGGGTGGCAGGCGTTC-3' (SEQ ID NO:28) Antisense 5'-AGCATAAGCTTCTAGTAGGCGTTCTCCAG-3' (SEQ ID NO : 29)

H. Gremlin Sense: 5'-GACTTGGATCCGAAGGGAAAAAGAAAGGG-3' (SEQ ID NO:30) Antisense: 5'-AGCATAAGCTTTTAATCCAAATCGATGGA-3' (SEQ ID NO:31)

HDan Sense: 5'-ACTACGAGCTCGGCCCCACCACCCATCAACAAG-3' (SEQ ID NO:32) Antisense: 5'-ACTTAGAAGCTTTCAGTCCTCAGCCCCCTCTTCC-3' (SEQ ID NO:33)

0243 The resulting cDNA contained the coding region of 0249. In each case the listed primers amplified the entire Beer with two modifications. The N-terminal secretion sig coding region minus the Secretion Signal Sequence. These nal was removed and a FLAG epitope tag (Sigma) was fused include restriction Sites for Subcloning into the bacterial in frame to the C-terminal end of the insert. BamHI and expression vector pGE-30 (Qiagen Inc., Valencia, Calif.) at sites BamHI/HindIII for Beer and Gremlin, and sites SacI/ HindIII cloning sites were added and the gene was Sub HindIII for Dan. pOE30 contains a coding sequence for a cloned into pMelBac vector (Invitrogen) for transfer into a 6xHis tag at the 5' end of the cloning region. The completed baculoviral expression vector using Standard methods. constructs were transformed into E. coli strain M-15/pRep (Qiagen Inc) and individual clones verified by Sequencing. 0244 Recombinant baculoviruses expressing Beer pro Protein expression in M-15/pRep and purification (6xHis tein were made using the Bac-N-Blue transfection kit (Invit affinity tag binding to Ni-NTA coupled to Sepharose) were rogen) and purified according to the manufacturers instruc performed as described by the manufacturer (Qiagen, The tions. QIAexpressionist). 0245 SF9 cells (Invitrogen) were maintained in 0250) The E. coli-derived Beer protein was recovered in TNM FH media (Invitrogen) containing 10% fetal calf Significant quantity using Solubilization in 6 M guanidine and dialyzed to 2-4M to prevent precipitation during Stor Serum. For protein expression, SF9 cultures in Spinner flaskS age. Gremlin and Dan protein were recovered in higher were infected at an MOI of greater than 10. Samples of the quantity with Solubilization in 6M guanidine and a post media and cells were taken daily for five days, and Beer purification guanidine concentration of 0.5M. expression monitored by Western blot using an anti-FLAG M2 monoclonal antibody (Sigma) or an anti-Beer rabbit 0251 B. Production and Testing of Polyclonal Antibod polyclonal antiserum. ies: 0252 Polyclonal antibodies to each of the three antigens 0246. After five days the baculovirus-infected SF9 cells were produced in rabbit and in chicken hosts using Standard were harvested by centrifigation and cell associated protein protocols (R & R Antibody, Stanwood, Wash.; standard US 2004/0058321 A1 Mar. 25, 2004 protocol for rabbit immunization and antisera recovery; sample buffer. The proteins were resolved by SDS PAGE Short Protocols in Molecular Biology. 2nd edition. 1992, and Beer associated BMP-5 was detected by western blot 11.37- 11.41. Contributors Helen M. Cooper and Yvonne using anti-BMP-5 antiserum (Research Diagnostics, Inc) PaterSon; chicken antisera was generated with Strategic (see FIG. 5). Biosolutions, Ramona, Calif.). 0257 BEER Ligand Binding Assay: 0253 Rabbit antisera and chicken egg Igy fraction were screened for activity via Western blot. Each of the three 0258 FLAG-Beer protein (20 ng) is added to 100 ul antigens was separated by PAGE and transferred to 0.45 um PBS/O.2% BSA and adsorbed into each well of 96 well nitrocellulose (Novex, San Diego, Calif.). The membrane microtiter plate previously coated with anti-FLAG mono was cut into Strips with each Strip containing approximately clonal antibody (Sigma; St Louis Mo.) and blocked with 75 ng of antigen. The strips were blocked in 3% Blotting 10% BSA in PBS. This is conducted at room temperature for Grade Block (Bio-Rad Laboratories, Hercules, Calif.) and 60 minutes. This protein solution is removed and the wells washed 3 times in 1xTris buffer saline (TBS)/0.02% TWEEN buffer. The primary antibody (preimmunization are washed to remove unbound protein. BMP-5 is added to bleeds, rabbit antisera or chicken egg IgY in dilutions each well in concentrations ranging from 10 pM to 500 nM ranging from 1:100 to 1:10,000 in blocking buffer) was in PBS/O.2% BSA and incubated for 2 hours at room incubated with the Strips for one hour with gentle rocking. temperature. The binding Solution is removed and the plate A Second Series of three washes 1XTBS/O.02% TWEEN was washed with three times with 200 ul volumes of PBS/O.2% followed by an one hour incubation with the secondary BSA. BMP-5 levels are then detected using BMP-5 anti antibody (peroxidase conjugated donkey anti-rabbit, Amer serum via ELISA (F. M. Ausubel et al (1998) Current Sham Life Science. Piscataway, N.J., or peroxidase conju Protocols in Mol Biol. Vol 2 11.2.1-11.2.22). Specific bind gated donkey anti-chicken. Jackson ImmunoResearch. West ing is calculated by Subtracting non-specific binding from Grove, Pa.). A final cycle of 3x washes of 1xTBS/0.02% total binding and analyzed by the LIGAND program (Mun TWEEN was performed and the strips were developed with son and Podbard, Anal. Biochem., 107, p220-239, (1980). Lumi-Light Western Blotting Substrate (Roche Molecular Biochemicals, Mannheim, Germany). 0259. In a variation of this method, Beer is engineered and expressed as a human Fc fusion protein. Likewise the 0254 C. Antibody Cross-reactivity Test: ligand BMP is engineered and expressed as mouse Fc fusion. 0255 Following the protocol described in the previous These proteins are incubated together and the assay con Section, nitrocellulose strips of Beer, Gremlin or Dan were ducted as described by Mellor et all using homogeneous time incubated with dilutions (1:5000 and 1:10,000) of their resolved fluorescence detection (G. W. Mellor et al., J of respective rabbit antisera or chicken egg IgY as well as to Biomol Screening, 3(2) 91-99, 1998). antisera or chicken egg Igy (dilutions 1:1000 and 1:5000) made to the remaining two antigens. The increased levels of Example 6 nonmatching antibodies was performed to detect low affinity binding by those antibodies that may be seen only at Screening Assay for Inhibition of TGF-beta increased concentration. The protocol and duration of devel Binding-protein opment is the same for all three binding events using the protocol described above. There was no antigen croSS Binding to TGF-beta Family Members reactivity observed for any of the antigens tested. 0260 The assay described above is replicated with two Example 5 exceptions. First, BMP concentration is held fixed at the Kd determined previously. Second, a collection of antagonist Interaction of Beer with TGF-beta Super-family candidates is added at a fixed concentration (20 uM in the Proteins case of the Small organic molecule collections and 1 uM in antibody Studies). These candidate molecules (antagonists) 0256 The interaction of Beer with proteins from different of TGF-beta binding-protein binding include organic com phylogenetic arms of the TGF-B superfamily were studied pounds derived from commercial or internal collections using immunoprecipitation methods. Purified TGFB-1, representing diverse chemical Structures. These compounds TGFB-2, TGFB-3, BMP4, BMP-5, BMP-6 and GDNF were are prepared as stock solutions in DMSO and are added to obtained from commerical sources (R&D systems; Minne assay wells at is 1% of final Volume under the Standard assay apolis, Minn.). A representative protocol is as follows. conditions. These are incubated for 2 hours at room tem Partially purified Beer was dialyzed into HEPES buffered perature with the BMP and Beer, the solution removed and saline (25 mM HEPES 7.5, 150 mM NaCl). Immunopre the bound BMP is quantitated as described. Agents that cipitations were done in 300 ul of IP buffer (150 mM NaCl, inhibit 40% of the BMP binding observed in the absence of 25 mM Tris pH 7.5, 1 mM EDTA, 1.4 mM B-mercaptoet compound or antibody are considered antagonists of this hanol, 0.5% triton X 100, and 10% glycerol). 30 ng recom interaction. These are further evaluated as potential inhibi binant human BMP-5 protein (R&D systems) was applied to tors based on titration studies to determine their inhibition 15ul of FLAG affinity matrix (Sigma; St Louis Mo.)) in the constants and their influence on TGF-beta binding-protein presence and absence of 500 ng FLAG epitope-tagged Beer. binding affinity. Comparable specificity control assays may The proteins were incubated for 4 hours (a 4 C. and then also be conducted to establish the selectivity profile for the the affinity matrix-associated proteins were washed 5 times identified antagonist through Studies using assays dependent in IP buffer (1 ml per wash). The bound proteins were eluted on the BMP ligand action (e.g. BMP/BMP receptor compe from the affinity matrix in 60 microliters of 1xSDS PAGE tition study). US 2004/0058321 A1 Mar. 25, 2004 26

Example 7 Research Genetics, Huntsville, Ala.) was used to determine the complete Sequence of the mouse Beer gene and its 5' and Inhibition of TGF-beta Binding-protein 3' flanking regions. A 41 kb Sal fragment, containing the Localization to Bone Matrix entire gene body, plus ~17 kb of 5" flanking and ~20 kb of 3' flanking sequence was sub-cloned into the BamHI site of 0261) Evaluation of inhibition of localization to bone the SuperCosI cosmid vector (Stratagene, La Jolla, Calif.) matrix (hydroxyapatite) is conducted using modifications to and propagated in the E. coli strain DH10B. From this the method of Nicolas (Nicolas, V. Calcif Tissue Int 57:206, cosmid construct, a 35 kb Mlul-Avil restriction fragment 1995). Briefly, 'I-labelled TGF-beta binding-protein is (Sequence No. 6), including the entire mouse Beer gene, as prepared as described by Nicolas (Supra). Hydroxyapatite is well as 17 kb and 14 kb of 5" and 3’ flanking sequence, added to each well of a 96 well microtiter plate equipped respectively, was then gel purified, using conventional with a polypropylene filtration membrane (Polyfiltroninc, means, and used for microinjection of mouse Zygotes (DNX Weymouth Mass.). TGF-beta binding-protein is added to Transgenics; U.S. Pat. No. 4,873,191). Founder animals in 0.2% albumin in PBS buffer. The wells containing matrix are which the cloned DNA fragment was integrated randomly washed 3 times with this buffer. Adsorbed TGF-beta bind into the genome were obtained at a frequency of 5-30% of ing-protein is eluted using 0.3M NaOH and quantitated. live-born pups. The presence of the transgene was ascer 0262 Inhibitor identification is conducted via incubation tained by performing Southern blot analysis of genomic of TGF-beta binding-protein with test molecules and apply DNA extracted from a Small amount of mouse tissue, Such ing the mixture to the matrix as described above. The matrix as the tip of a tail DNA was extracted using the following is washed 3 times with 0.2% albumin in PBS buffer. protocol: tissue was digested overnight at 55 C. in a lysis Adsorbed TGF-beta binding-protein is eluted using 0.3 M buffer containing 200 mM NaCl. 100 m Tris pH8.5, 5 mM NaOH and quantitated. Agents that inhibit 40% of the EDTA, 0.2% SDS and 0.5 mg/ml Proteinase K. The follow TGF-beta binding-protein binding observed in the absence ing day, the DNA was extracted once with phenol/chloro of compound or antibody are considered bone localization form (50:50), once with chloroform/isoamylalcohol (24:1) inhibitors. These inhibitors are further characterized through and precipitated with ethanol. Upon resuspension in TE (10 dose response Studies to determine their inhibition constants mM Tris pH7.5, 1 mM EDTA) 8-10 ug of each DNA sample and their influence on TGF-beta binding-protein binding were digested with a restriction endonuclease, Such as affinity. EcoRI, Subjected to gel electrophoresis and transferred to a charged nylon membrane, Such as HyBondN+ (Amersham, Example 8 Arlington Heights, Ill.). The resulting filter was then hybrid ized with a radioactively labelled fragment of DNA deriving Construction of TGF-beta Binding-protein Mutant from the mouse Beer gene locus, and able to recognize both a fragment from the endogenous gene locus and a fragment 0263. A. Mutagenesis: of a different size deriving from the transgene. Founder 0264. A full-length TGF-beta binding-protein cDNA in animals were bred to normal non-transgenic mice to gener pBlueScript SK Serves as a template for mutagenesis. ate Sufficient numbers of transgenic and non-transgenic Briefly, appropriate primers (see the discussion provided progeny in which to determine the effects of Beer gene above) are utilized to generate the DNA fragment by poly overexpression. For these Studies, animals at various ages merase chain reaction using Vent DNA polymerase (New (for example, 1 day, 3 weeks, 6 weeks, 4 months) are England Biolabs, Beverly, Mass.). The polymerase chain Subjected to a number of different assays designed to ascer reaction is run for 23 cycles in buffers provided by the tain groSS Skeletal formation, bone mineral density, bone manufacturer using a 57 C. annealing temperature. The mineral content, osteoclast and Osteoblast activity extent of product is then exposed to two restriction enzymes and after endochondral OSSification, cartilage formation, etc. The tran isolation using agarose gel electrophoresis, ligated back into Scriptional activity from the transgene may be determined by pRBP4-503 from which the matching sequence has been extracting RNA from various tissues, and using an RT-PCR removed by enzymatic digestion. Integrity of the mutant is assay which takes advantage of Single nucleotide polymor verified by DNA sequencing. phisms between the mouse Strain from which the transgene 0265 B. Mammalian Cell Expression and Isolation of is derived (129Sv/J) and the strain of mice used for DNA Mutant TGF-beta Binding-protein: microinjection (C57BL5/JxSJL/J)F2). 0266 The mutant TGF-beta binding-protein cDNAS are Animal Models-II transferred into the pcDNA3.1 mammalian expression vec tor described in EXAMPLE 3. After verifying the sequence, Disruption of the Mouse Beer Gene by the resultant constructs are transfected into COS-1 cells, and Homologous Recombination secreted protein is purified as described in EXAMPLE 3. 0268 Homologous recombination in embryonic stem Example 9 (ES) cells can be used to inactivate the endogenous mouse Beer gene and Subsequently generate animals carrying the loSS-of-function mutation. A reporter gene, Such as the E. Animal Models-I coli 3-galactosidase gene, was engineered into the targeting Generation of Transgenic Mice Overexpressing the vector So that its expression is controlled by the endogenous Beer gene's promoter and translational initiation signal. In Beer Gene this way, the Spatial and temporal patterns of Beer gene 0267 The -200 kilobase (kb) BAC clone 15G5 isolated expression can be determined in animals carrying a targeted from the CITB mouse genomic DNA library (distributed by allele. US 2004/0058321 A1 Mar. 25, 2004 27

0269. The targeting vector was constructed by first clon which the construct has integrated by a non-homologous ing the drug-Selectable phosphoglycerate kinase (PGK) pro event (U.S. Pat. No. 5,464,764). The RSVLTR-HSVTK moter driven neomycin-resistance gene (neo) cassette from cassette was amplified from pPS1337 using primers that pGT-N29 (New England Biolabs, Beverly, Mass.) into the allow Subsequent cloning into the FSeI and AScI sites of the cloning vector pSP72 (Promega, Madson, Wis.). PCR was “long arm'-TA vector plasmid. For this PCR, the sequence used to flank the PGKneo cassette with bacteriophage P1 of the sense primer was 5'-ATTACGGCCGGCCGCAAAG loxP sites, which are recognition sites for the P1 Cre GAATTCAAGATCTGA-3' (SEQ ID NO:40); the sequence recombinase (Hoess et al., PNAS USA, 79:3398, 1982). This allows Subsequent removal of the neo-resistance of the anti-sense primer was 5'-ATTACGGCGCGCCCCTC marker in targeted ES cells or ES cell-derived animals (U.S. ACAGGCCGCACCCAGCT-3' (SEQ ID NO:41). Pat. No. 4,959,317). The PCR primers were comprised of 0273. The final step in the construction of the targeting the 34 nucleotide (ntd) loxP sequence. 15-25 ntd comple vector involved cloning the 8.8 kb Sgr AI-AscI fragment mentary to the 5' and 3' ends of the PGKneo cassette, as well containing the “long arm” and RSVLTR-HSVTK gene into as restriction enzyme recognition sites (BamHI in the Sense primer and EcoRI in the anti-sense primer) for cloning into the Sgr AI and AscI sites of the pSP72-“short arm'-Bgal pSP72. The sequence of the sense primer was 5'-AATCTG PGKneo plasmid. This targeting vector was linearized by GATCCATAACTTCGTATAGCATACAT. digestion with either AscI or PacI before electroporation into TATACGAAGTTATCTGCAG GATTCGAGGGCCCCT-3' ES cells. (SEQ ID NO:34); sequence of the anti-sense primer was 5'-AATCTGAATTCCACCGGTGTTAAT Example 10 TAAATAACTTCGT ATAATGTATGCTATACGAAGT TATAGATCTAGAG TCAGCTTCTGA-3' (SEQ ID AntiSense-mediated Beer Inactivation NO:35). 0274) 17-nucleotide antisense oligonucleotides are pre 0270. The next step was to clone a 3.6 kb XhoI-HindIII pared in an overlapping format, in Such a way that the 5' end fragment, containing the E. coli 3-galactosidase gene and of the first oligonucleotide overlaps the translation initiating SV40 polyadenylation signal from pSVB (Clontech, Palo AUG of the Beer transcript, and the 5' ends of successive Alto, Calif.) into the pSP72-PGKneo plasmid. The “short oligonucleotides occur in 5 nucleotide increments moving in arm' of homology from the mouse Beer gene locus was the 5' direction (up to 50 nucleotides away), relative to the generated by amplifying a 2.4 kb fragment from the BAC Beer AUG. Corresponding control oligonucleotides are clone 15G5. The 3' end of the fragment coincided with the designed and prepared using equivalent base composition translational initiation site of the Beer gene, and the anti but redistributed in Sequence to inhibit any significant sense primer used in the PCR also included 30 ntd comple hybridization to the coding mRNA. Reagent delivery to the mentary to the 5' end of the B-galactosidase gene So that its test cellular System is conducted through cationic lipid coding region could be fused to the Beer initiation site delivery (P. L. Felgner, Proc. Natl. Acad. Sci. USA 84:7413, in-frame. The approach taken for introducing the “short 1987). 2 ug of antisense oligonucleotide is added to 100 ul arm' into the pSP72-fgal-PGKneo plasmid was to linearize of reduced serum media (Opti-MEMI reduced serum media; the plasmid at a Site upstream of the B-gal gene and then to Life Technologies, Gaithersburg Md.) and this is mixed with co-transform this fragment with the “short arm’ PCR prod Lipofectin reagent (6 ul) (Life Technologies, Gaithersburg uct and to select for plasmids in which the PCR product was Md.) in the 100 ul of reduced serum media. These are mixed, integrated by homologous recombination. The Sense primer allowed to complex for 30 minutes at room temperature and for the “short arm” amplification included 30 ntd comple the mixture is added to previously seeded MC3T3E21 or mentary to the pSP72 vector to allow for this recombination KS483 cells. These cells are cultured and the mRNA recov event. The sequence of the sense primer was 5'-ATTTAG ered. Beer mRNA is monitored using RT-PCR in conjunc GTGACACT ATAGAACTCGAGCAGCTGAAGCT tion with Beer Specific primers. In addition, Separate experi TAACCACATGGTGGCTCACAACCAT3' (SEQ ID mental wells are collected and protein levels characterized NO:36) and the sequence of the anti-sense primer was through western blot methods described in Example 4. The 5'-AACGACGGCCAGTGAATCCGTA ATCATGGTCAT. cells are harvested, resuspended in lysis buffer (50 mM Tris GCTGCCAGGTGGAGGAGGGCA-3' (SEQ ID NO:37). pH 7.5, 20 mM NaCl, 1 mM EDTA, 1% SDS) and the 0271 The “long arm” from the Beer gene locus was soluble protein collected. This material is applied to 10-20% generated by amplifying a 6.1kb fragment from BAC clone gradient denaturing SDS PAGE. The separated proteins are 15G5 with primers which also introduce the rare-cutting transferred to nitrocellulose and the western blot conducted restriction enzyme sites Sgr AI, FSeI, AScI and PacI. Spe as above using the antibody reagents described. In parallel, cifically, the sequence of the sense primer was 5'-ATTAC the control oligonucleotides are added to identical cultures CACCGGTGACACCCGCTTCCTGACAG-3 (SEQ ID and experimental operations are repeated. Decrease in Beer NO:38); the sequence of the anti-sense primer was 5'-AT mRNA or protein levels are considered significant if the TACTTAATTAAACATGGCGCGCCAT ATGGCCGGC treatment with the antisense oligonucleotide results in a 50% CCCTAATTGCGGCGCATCGTTAATT-3' (SEQ ID change in either instance compared to the control Scrambled NO:39). The resulting PCR product was cloned into the TA oligonucleotide. This methodology enables Selective gene vector (Invitrogen, Carlsbad, Calif.) as an intermediate Step. inactivation and Subsequent phenotype characterization of the mineralized nodules in the tissue culture model. 0272. The mouse Beer gene targeting construct also included a Second Selectable marker, the herpes simplex 0275 From the foregoing, it will be appreciated that, virus I thymidine kinase gene (HSVTK) under the control of although Specific embodiments of the invention have been rous Sarcoma virus long terminal repeat element (RSV described herein for purposes of illustration, various modi LTR). Expression of this gene renders mammalian cells fications may be made without deviating from the Spirit and Sensitive (and inviable) to gaincyclovir; it is therefore a Scope of the invention. Accordingly, the invention is not convenient way to Select against neomycin-resistant cells in limited except as by the appended claims.

US 2004/0058321 A1 Mar. 25, 2004 29

-continued gctgtacata togctgaga aa citgcagagca taatagotgc caccolaaaaa totttittgaa 1980 aatcatttcc agacaaccitc titactittctg totagitttitt aattgttaaa aaaaaaaagt 20 40 tittaalacaga agcacatgac atatgaaagc ctdcagg act ggtogtttitt ttggcaattic 2100 titccacgtgg gacittgtc.ca caagaatgaa agtag toggtt tittaaagagt taagttacat 216 O atttatttitc. tcacttaagt tatttatgca aaagtttittc ttgtagagaa tdacaatgtt 2220 aatattgctt tatgaattaa cagtctgttc titccagag to cagagacatt gttaataaag 228O acaatgaatc atgaccgaaa g 2301

<210> SEQ ID NO 2 <211& LENGTH 213 &212> TYPE PRT <213> ORGANISM: Homo sapien <400 SEQUENCE: 2 Met Gln Leu Pro Leu Ala Lieu. Cys Lieu Val Cys Lieu Lleu Val His Thr 1 5 10 15 Ala Phe Arg Val Val Glu Gly Glin Gly Trp Glin Ala Phe Lys Asn Asp 2O 25 30 Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 Glu Lieu Glu Asn. Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 8O Arg Glu Leu. His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Lieu Lleu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Glin Arg Val 130 135 1 4 0 Glin Leu Lieu. Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 145 15 O 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg Lieu. Thr Arg Phe His Asn Glin 1.65 170 175 Ser Glu Lieu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Glin Lys Gly 18O 185 190 Arg Llys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Glin Ala Glu 195 200 2O5 Leu Glu Asn Ala Tyr 210

<210> SEQ ID NO 3 &2 11s LENGTH 2301 &212> TYPE DNA <213> ORGANISM: Homo sapien <400 SEQUENCE: 3 agagcctgtg citactggaag gtggcgtgcc citccitctggc tiggtaccatg cagotcc cac 60 tggcc.ctgtg totcgtctgc citgctggtac acacago citt cogtgtagt g gagggctagg 120

US 2004/0058321 A1 Mar. 25, 2004 32

-continued acco at agcc atgttittaaa gtcaccittcc gaagagaagt gaaaggttca agg acactogg 1740 ccittgcaggc cc gagggagc agc catcaca aactcacaga ccago acatc ccttittgaga 1800 caccgccttctg.cccaccac toacggacac atttctgcct agaaaacago ttcttactg.c 1860 tottacatgt gatggcatat cittacactaa aagaatatta ttgggggaaa alactacaagt 1920 gctgtacata togctgaga aa citgcagagca taatagotgc caccolaaaaa totttittgaa 1980 aatcatttcc agacaaccitc titactittctg totagitttitt aattgttaaa aaaaaaaagt 20 40 tittaalacaga agcacatgac atatgaaagc ctdcagg act ggtogtttitt ttggcaattic 2100 titccacgtgg gacittgtc.ca caagaatgaa agtag toggtt tittaaagagt taagttacat 216 O atttatttitc. tcacttaagt tatttatgca aaagtttittc ttgtagagaa tdacaatgtt 2220 aatattgctt tatgaattaa cagtctgttc titccagag to cagagacatt gttaataaag 228O acaatgaatc atgaccgaaa g 2301

<210> SEQ ID NO 6 <211& LENGTH 213 &212> TYPE PRT <213> ORGANISM: Homo sapien <400 SEQUENCE: 6 Met Gln Leu Pro Leu Ala Lieu. Cys Lieu. Ile Cys Lieu Lleu Val His Thr 1 5 10 15 Ala Phe Arg Val Val Glu Gly Glin Gly Trp Glin Ala Phe Lys Asn Asp 20 25 30 Ala Thr Glu Ile Ile Arg Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 Glu Lieu Glu Asn. Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 8O Arg Glu Leu. His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Lieu Lleu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Glin Arg Val 130 135 1 4 0 Glin Leu Lieu. Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 145 15 O 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg Lieu. Thr Arg Phe His Asn Glin 1.65 170 175 Ser Glu Lieu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Glin Lys Gly 18O 185 190 Arg Llys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Glin Ala Glu 195 200 2O5 Leu Glu Asn Ala Tyr 210

<210 SEQ ID NO 7 &2 11s LENGTH 2301 &212> TYPE DNA

US 2004/0058321 A1 Mar. 25, 2004 34

-continued atttatttitc. tcacttaagt tatttatgca aaagtttittc ttgtagagaa tdacaatgtt 2220 aatattgctt tatgaattaa cagtctgttc titccagag to cagagacatt gttaataaag 228O acaatgaatc atgaccgaaa g 2301

<210 SEQ ID NO 8 <211& LENGTH 213 &212> TYPE PRT <213> ORGANISM: Homo sapien <400 SEQUENCE: 8 Met Gln Leu Pro Leu Ala Lieu. Cys Lieu Val Cys Lieu Lleu Val His Thr 1 5 10 15 Ala Phe Arg Val Val Glu Gly Glin Gly Trp Glin Ala Phe Lys Asn Asp 2O 25 30 Ala Thr Glu Ile Ile Arg Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 Glu Lieu Glu Asn. Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 8O Arg Glu Leu. His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Lieu Lleu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Glin Arg Val 130 135 1 4 0 Glin Leu Lieu. Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 145 15 O 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg Lieu. Thr Arg Phe His Asn Glin 1.65 170 175 Ser Glu Lieu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Glin Lys Gly 18O 185 190 Arg Llys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Glin Ala Glu 195 200 2O5 Leu Glu Asn Ala Tyr 210

<210 SEQ ID NO 9 <211& LENGTH 642 &212> TYPE DNA <213> ORGANISM: Cercopithecus pygerythrus <400 SEQUENCE: 9 atgcagctcc cactggcc ct gtgtc.ttgtc tocctgctgg tacacgcago citt.ccgtgta 60 gtggagggcc aggggtggca ggc ctitcaag aatgatgcca cqgaaatcat coccgagctic 120 ggagagtacc cc.gagcctico accggagctg gagaacaa.ca agaccatgaa cc.ggg.cggag 18O aatggagggc ggc citccc.ca ccaccc.ctitt gagaccaaag acgtgtc.cga gtacagotgc 240 cgagagctgc actitcaccc.g. citacgtgacc gatggg.ccgt gcc.gcagogc caa.gc.ca.gtc 3OO accgagttgg totgcticcgg ccagtgcggc ccggcacgcc toc tocc caa cqc catcggc 360 US 2004/0058321 A1 Mar. 25, 2004 35

-continued cgcggcaagt ggtggcgc.cc gagtggg.ccc gacitt.ccgct gcatcc.ccga cc.gctaccgc 420 gcgcagcgtg togCagctgct gtgtc.ccggt ggtgcc.gc.gc cqC gC gC gCg Caaggtocgc 480 citggtggcct c gtgcaagtg caa.gc.gc.citc accc.gct tcc acaac cagtic ggagctdaag 540 gactitcggtc. cc.gaggcc.gc. tcggcc.gcag aaggg.ccgga agcc.gcggCC cc.gc.gc.ccgg 600 ggggccaaag ccaatcaggc cqagctggag aacgc.ctact ag 642

<210> SEQ ID NO 10 <211& LENGTH 213 &212> TYPE PRT <213> ORGANISM: Cercopithecus pygerythrus <400 SEQUENCE: 10 Met Gln Leu Pro Leu Ala Lieu. Cys Lieu Val Cys Lieu Lleu Val His Ala 1 5 10 15 Ala Phe Arg Val Val Glu Gly Glin Gly Trp Glin Ala Phe Lys Asn Asp 2O 25 30 Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 Glu Lieu Glu Asn. Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 8O Arg Glu Leu. His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Lieu Lleu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Glin Arg Val 130 135 1 4 0 Glin Leu Lieu. Cys Pro Gly Gly Ala Ala Pro Arg Ala Arg Lys Val Arg 145 15 O 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg Lieu. Thr Arg Phe His Asn Glin 1.65 170 175 Ser Glu Lieu Lys Asp Phe Gly Pro Glu Ala Ala Arg Pro Glin Lys Gly 18O 185 190 Arg Llys Pro Arg Pro Arg Ala Arg Gly Ala Lys Ala Asn Glin Ala Glu 195 200 2O5 Leu Glu Asn Ala Tyr 210

<210> SEQ ID NO 11 &2 11s LENGTH 638 &212> TYPE DNA <213> ORGANISM: Mus musculus

<400 SEQUENCE: 11 atgcagocct cactag cocc gtgccitcatc tacctacttg tdcacgctgc cittctgtgct 60 gtggagggcc aggggtggca agc ctitcagg aatgatgcca cagaggtoat cocagggctt 120 ggagagtacc cc.gagcctico toc to agaac alaccagacca toga accgggc ggagaatgga 18O ggcagaccitc cccaccatcc citatgacgcc aaaggtotgt cc.gagtacag citgcc.gc gag 240

US 2004/0058321 A1 Mar. 25, 2004 37

-continued agaatgatgc cacagaaatc atc.ccgggac toaga gagta cccagagcct cotcaggaac 18O tagagaacaa cca gaccato aaccq gg.ccg agaacggagg cag accoccc cac catccitt 240 atgacaccaa agacgtgtcc gagtacagct gcc.gc gagct gcact acacic cqctt.cgtga 3OO cc.gacggc.cc gtgcc.gcagt gccaag.ccgg to accgagtt gotgtgcto g g gccagtgcg 360 gcc.ccgc.gcg gctgctg.ccc aacgc.catcg ggcgc.gtgaa gtggtgg.cgc cc.gaacggac 420 cc.gactitc.cg citgcatcc.cg gatcgctacc gcgc.gcagog ggtgcagot g c totgcc.ccg. 480 gcgg.cgcggc gcc.gc.gct cq C gCaaggtgc gtctggtggC Ctcgtgcaag togcaa.gc gCC 540 to acco gott coacalaccag toggagctica agg actt.cgg acctgaga.cc gcgcggcc.gc 600 agaagggtcg Caag.ccg.cgg CCCC gcgCCC ggggagccala agcCaac Cag gcggagctgg 660 agaacgccita citag 674

<210> SEQ ID NO 14 <211& LENGTH 213 &212> TYPE PRT <213> ORGANISM: Rattus norvegicus <400 SEQUENCE: 14 Met Gln Leu Ser Leu Ala Pro Cys Lieu Ala Cys Lieu Lleu Val His Ala 1 5 10 15 Ala Phe Val Ala Val Glu Ser Glin Gly Trp Glin Ala Phe Lys Asn Asp 2O 25 30 Ala Thr Glu Ile Ile Pro Gly Leu Arg Glu Tyr Pro Glu Pro Pro Gln 35 40 45 Glu Lieu Glu Asn. Asn Glin Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Tyr Asp Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 8O Arg Glu Leu. His Tyr Thr Arg Phe Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Lieu Lleu Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn 115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Glin Arg Val 130 135 1 4 0 Glin Leu Lieu. Cys Pro Gly Gly Ala Ala Pro Arg Ser Arg Lys Val Arg 145 15 O 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg Lieu. Thr Arg Phe His Asn Glin 1.65 170 175 Ser Glu Lieu Lys Asp Phe Gly Pro Glu Thir Ala Arg Pro Glin Lys Gly 18O 185 190 Arg Llys Pro Arg Pro Arg Ala Arg Gly Ala Lys Ala Asn Glin Ala Glu 195 200 2O5 Leu Glu Asn Ala Tyr 210

<210 SEQ ID NO 15 &2 11s LENGTH 532 &212> TYPE DNA <213> ORGANISM Bos torus

US 2004/0058321 A1 Mar. 25, 2004 49

-continued tgacaacacc tocgactgct citctggtagc ccttgttggca atacgtgttt cotttgaaaa 22.980 gtoacattca toctittccitt togcaaacctg gctcitcattc cccagotggg to atcgtcat 23040 accotcacco cagoctocct ttagctgacc actictocaca citgtctitcca aaagtgcacg 23100 tittcaccqag coagttccct ggtocagg to atcccattgc ticcitccttgc ticcag accot 23160 totcccacaa agatgttcat citcccactcc atcaag.cccc agtgg ccct g c ggctatocc 23220 tgtctottca gttagctgaa totacttgct gacaccacat gaatticcittc ccctgtctta 23280 aggttcatgg aactcittgcc toccc.ctgaa cct tccagga citgtc.ccago gttctgatgtg 23340 toctotctot totaaag.ccc cacco cacta tittgattccc aattctagat cittcccttgt 23400 to attcctitc acgggatagt gtc.tcatctg gccaagttcct gcttgatatt goggataaatg 23460 caaag.ccaag tacaattgag gaccagttca to attgg gcc aagctttittcaaaatgtgaa 23520 ttttacacct atagaagtgt aaaag.cctitc caaag cagag goaatgcct g g citctitcctt 23580 caa.catcagg gcticcitgctt tatgggtotg gtgggg tagt acatt cataa accoa acact 23640 aggggtgttga aag caagatg attgggagtt cqaggccaat cittggctato aggcc citgtc. 23700 tdaaccitcto citccctcc ct coagg gttitt gttttgttitt gtttittittga tittgaaactg. 23760 caacactitta aatccagtca agtgcatctt tag.cgtgaggg gaactictato cotaatataa 23820 gctt.ccatct to atttgttgt atgtgcacac togggggttga acct g g gcct ttgtacct gc 23880 cgggcaa.gct citctactgct citaaaccoag cccitcactgg citttctgttt caactcc caa 23940 tgaatticccc taaatgaatt atcaatatoa totctittgaa aaataccatt gag togctgct 24000 ggtgtc.cctg toggttccaga titcCaggaag gacttitt cag ggaatcCagg catcc tdaag 24060 aatgtc.ttag agcaggaggc catggaga.cc ttggc.ca.gcc ccaca aggca gtgtggtgca 24 120 gagggtgagg atggaggcag gottgcaatt gaagctdaga caggg tactic aggattaaaa 241.80 agctitccccc aaaacaattic caagatcagt toctagtact tccacctgtt cagotatgca 24240 gag.cccagtg ggcataggtg aag acaccgg ttgtactgtc atgtactaac totgcttcag 24.300 agcc.gg caga gacaaataat gttatggtga ccc.cagggga cagtgattoc agaaggaac a 24360 cagaagagag togctgctaga ggctgcct ga aggagaaggg gtc.ccagact citctaag caa 24 420 agactic cact cacataaaga cacaggctga gcagagctgg cc.gtggatgc agg gag.ccca 24480 to caccatcc tittagcatgc ccttgtatto coatcacatg ccagg gatga ggggcatcag 245 40 agagtccaag tdatgcc.caa accolaaacac accitagg act togctttctgg gacagacaga 24 600 tgcaggagag act aggttgg gctgtgatcc cattaccaca aagagggaala aaacaaaaaa 24 660 caaacaaaca aacaaaaaaa aacaaaacaa aacaaaaaaa aaccolaaggit coaaattgta 24.720 gg to aggtta gagtttattt atggaaagtt atattotacic to catggggit citaca aggct 24780 ggcgcc catc agaaagaa.ca aacaa.caggc tigatctggga ggggtggtac totatgg cag 24840 ggag cacgtg togcttggggit acago cagac acggggcttg tattaatcac agggcttgta 24900 ttaataggct gagagt caag cagacagaga gacagaagga alacacacaca cacacacaca 24960 cacacacaca cacacacaca catgcacaca ccactcactt citcactcgaa gag coccitac 25020 ttacattcta agaacaaacc attccitccitc ataaag.gaga caaagttgca gaaaccoaaa 25080 agagccacag g g tocc cact citctttgaaa tacttggac ttgttgcagg galagacagag 25 140 gggtotgcag aggctitcc to ggtgacccag agccacagac actgaaatct ggtgctgaga 25200

US 2004/0058321 A1 Mar. 25, 2004 51

-continued ccagtc.tc.to citacagatgg gag acagagg cqagagatga atggtoaggg gaggagt cag 275 40 agaaaggaga gggtgaggca gagaccaaag gagggaaa.ca cittgtgctot acago tact g 27 600 actgagtacc agctg.cgtgg cagacago.ca atgccaaggc ticggctgatc atggcacct c 27660 gtgggacitcc tagcc.cagtg citggcagagg g gagtgctga atggtgcatg gtttggatat 27720 gatctgaatg togtocagoc citagtttcct tccagttgct gggataaag.c accotgacca 27780 aagctactitt tttgtttgtt tattittggitt taggittttgtt tagtttittcg agg cagg gtt 27840 totctgitatc accotagotg toctoggaact cactctgtag accaggctgg cctogaactic 27900 agaaatcc cc citgcctctgc citcctaagtg citggaattaa aggccitacgc caccactgcc 27960 ggcc caaag.c tactittaaga gagagagagg aatgtataag tattataatt coaggittata 28020 gttcattgct gtagaattgg agtcttcata titccaggitaa totcc cacag acatgccaca 28080 aaacaacctg titctacgaaa totctdatgg actcc ctitcc ccagtaattic taaactgtgt 28140 caaatctaca agaaatagtg acagt cacag totctaacgt tittgggcatg agtctgaagt 28200 citcattgcta agtactggga agatgaaaac tttacctagt gtcago attt gag cagagc 28260 citttgggatt tagatgg to ttittgcagag citcctaatgg citacatggag agagggggcc 28320 tgggagagac ccatacacct tittgctgcct tatgtcacct gacct gctoc ttgggaagct 28380 citagcaagaa gocctt.ccct g gatcaccca ccaccittgca cct coagaac toaga.gc.cala 28440 attaaactitt cittgttact.g. tcgtoaaag.c acagtcgg to tgg gttgtat cactgtcaat 28500 gggaaacaga cittgcctgga tiggataactt gtacattgca taatgtctag aaatgaaaag 285 60 to citatagag aaaaagaaaa ttagctggca cacagataga gg.ccctggag gaggctggct 28 620 ttgtc.citc.cc cqaggagg to go.gagtaagg totaaatgtt catggatgta aatggg.ccca 28680 tatatgaggg totggggtaa caagaaggcc tdtgaatata aag cactgaa gotiatgtcta 28740 gtotggagaa got cactaca gagagttcto caacticagtg cccatacaca cacacacaca 28800 cacacacaca cacacacaca cacacacaca ccacaaagaa aaaaaggaag aaaaatctga 28.860 gagcaagtac agtacttaaa attgttgttgat tdtgttgttgttg actctgatgt cacatgcto a 28920 tott gcc.cta tagttgaaa accalaatggc ccctgagagg cataacaa.cc acactgttgg 28980 citgttgttgcto acgtttittct taaag.cgtct gtctggitttg citgctag cat caggcag act 29040 tgcagoagac tacatatgct cagcc ctdaa gttcctitctag ggtgcatgtc. tcttcagaat 291.00 ttcagaaagt catctgtggc ticcaggaccg cct gcactct coctotgcc g c gaggctgca 29160 gactictaggc tiggggtggaa goaacgctta cct citgggac aagtataa.ca tottggctitt 29220 totttcccitc tdtggctoca acctggacat aaaatagatg caagctgttgt aataaatatt 29280 toctoccg to cacttagttc. tcaacaataa citactctgag agcacttatt aataggtggc 29340 ttag acataa gotttggcto attcc cccac tagctcittac ttctittaact citttcaaacc 29.400 attctgttgtc titccacatgg ttagttacct citcctitccat cotggttc.gc titctitcctitc 2.9460 gagtc.gc.cct cagtgtc.tct aggtgatgct totaagatat totttctaca aagct gagag 29520 tggtgg cact citgggagttcaaag.ccagcc tdatctacac agcaa.gctoc aggatat coa 295.80 gggcaatgtt gogaaaacct ttctoaaa.ca aaaagagggg titcagttgtc. aggaggagac 29 640 ccatgggitta agaagttctag acgagccatg gtgatgcata cctitt catcc aag cacttag 297.00 gaggcaaaga aaggtgaaac totttgacitt taggccago taggittacat agtgataccc 29760 US 2004/0058321 A1 Mar. 25, 2004 52

-continued tgcttagtgt gtgtgttgttgt gtgttgttgttgt gtgttgttgttgt gtgtgtaatt taaaagttcta 29820 aaaatgcatt cittittaaaaa tatgtataag tatttgcctg. cacatatgta totatgtatg 29880 tataccatgt gtgtgtctgg togctdaagga citagg catag acticcictaga act agagtca 29940 tag acagttg tdacactccc caaccoccca ccatgtgggit gcttgaagct aaactcctgt 30000 cctttgtaaa goagcaggtg totatgaacc ctgaaccatc. tctocagtct coagatgtgc 30 060 attctoaaag aggagtccitt catatttccc taaactgaac atccittatca gtgag catcc 30 120 togagtcacc aaagctactg caaaccotct tagggaacat tcactatto a cittctacttg 30 180 gctcatgaaa cittaagtaca cacacacaaa cacacacaca cacacagagt catgcactca 30240 caaaag catg catgtacacc attcttatta gactatoctit tactaaaaga cittitcctaga 30300 tactittaaaa catcacttct gccttittggit g g g caggttc caagattggit actgg.cgitac 30360 tggaaact ga acaaggtaga gatctagaaa toacago agg to agaagggc cagoctotac 30420 aagaga gagt to cacaccitt coaggaacac tag cagggg gctgg gacct tocct citcag 30480 cccaagaaac tagtg.cgttt cotgitatgca tacctcitcag agattccata agatctgcct 30540 totgccataa gatctoctoc atccagacaa goctagggga agttgagagg citgcctgagt 30 600 citct cocaca ggc.cccittct toccitggcag tatttittitta totggaggag aggaatcagg 30 660 gtgg gaatga toaaatacaa ttatcaagga aaaagtaaaa alacatatata tatatatatt 30720 aact gatcta gggagctggc ticago agitta agagttctgg citgcc cittgc titcagat citt 30780 gctittgatto coag caccca catgatggct ttcaactgta totctgctitc caggggatcc 30840 aacagoctot totgacctcc atagacaaga cctagtocto tgcaagagca ccaaatgctc. 30900 ttatctgttg atccatctot ctagoctoat gccagatcat ttaaaactac tagg acactgt 30960 cccattttac gaagatgtca ctg.cccagtc atttgccatg agtggatatt tog attctitt 31020 citatgttcto accottgcaa tittataagaa agatatotgc atttgttctoc tdagagaaca 31080 aagggtggag ggctactgag atggctotag g g g taaaggit gcttgccaca aaatctgaca 311 40 acttaagttt gotcittggaa tocacatggt ggagaga gag aagagattoc cqtaagttgt 31.200 cctdaaactt cocacacatg togctgtggct tatgttgtaac cocaataagt aaagatagitt 31260 ttaalacacta catalaggtag ggtttctt.ca to accocaag gaatgatgcc cct gatagag 31320 cittatgct ga aacco catct coattgttgcc atctggaaag agacaattgc atc.ccggaaa 31380 cagaatctitc atgaatggat taatgagcta ttaagaaagt ggcttggitta ttgcacatgc 31440 tggcgg.cgta atgaccitcca ccatogatgtt atc.ca.gcatg aaggtocto a ccagaagtca 31500 tacaaatctt cittaggctitc cagagtcgtg agcaaaaaaa goacaccitct aaataaatta 31560 actago citca ggtagittaac caccgaaaat galaccalaggc agttctaata caaaaccact 31 620 toccittccct gttcaaacca cagtgcccita ttatctaaaa gataaacttic aag coaagct 31680 tittaggittgc cagtatttat gtaacaacaa gg.ccc.gttga cacacatctg taactcc tag 31740 tactgggcct cagggg caga gacaggtgga gcc citggagt ttgaatticca ggttctgtga 31800 gaaactctgt citgaaaagac aatatggtga gtgaccc.ggg aggatat citg atattgacitt 31860 citggccaaca cacago catc. tctgcacatc totagttgca agccttittgc actaagtttg 31920 gccagagtica gagtttgcaa gtgtttgttgg actgaatgca cqtgttgct g g to atctaca 3 1980 aagttcaccct cottctdaag ctagoagcac taggctitcggc cagotgctoa ttcaagcctc. 32040 US 2004/0058321 A1 Mar. 25, 2004 53

-continued tittgcagagt catcacgggg atgggggagc agg gcc.ccitc cctagaacac caa.gc.ctgtg 32100 gttgtttatt caggacatta ttgagggcca agatgacaga taactictato acttggccaa 32160 cagtcgggtg ttgcggtgtt aggttatttctgttgttctgca gaaaa.ca.gtg caacctggac 32220 aaaagaaata aatgat atca tttitt cattc aggcaac tag attcc.gtggt acaaaaggct 32280 ccctggggaa cqagg.ccggg acagogcggc ticcitgagtcg citattitcc.gt ctotcaactt 32340 citctaatcto ttgattitcct coctotgtct gtttcctitcc tottgctggg gcc.cagtgga 32400 gtotgtgtac to acagg gag gagggtggca aagcc citggit cotcitacggg citgggggaag 32460 gggggaagct gtcgg.cccag tdacttitttc ccctittctot ttittcttaga aaccagtcto 32520 aatttaag at aatgagtcto citcattcacg tatgctoact attcataggg acttatccac 32580 cccc.gc.cctg. tcaatctggc taagtaagac aagtcaaatt taaaagg gaa cqtttittcta 32640 aaaatgtggc tigg accgtgt gcc.gg cacga aaccagg gat gg.cggtotaa gttacatgct 32700 citctgccago cocggtgcct titt cotttcg gaaaggagac ccggaggtaa aac gaagttg 32760 ccaacttittg atgatggtgt gcgc.cgggtg actictittaaa atgtcatcca tacctgggat 32820 agggaaggct cittcagg gag to atctagoc citcc.cittcag gaaaagattic cactt.ccggt 32880 ttagttagct tccaccitggit cocittatcc.g. citgtctotgc ccactag toc to atccatcc 32940 ggittitcc.gcc citcatccacc ttgcc cttitt agttcctaga aag cago acc gtagtottgg 33000 caggtgggcc attggtoact cogctaccac tottaccato gocaccalagg totcatttaa 33060 atatgagctc actgagtcct gcgggatggc titggttggta atatgcttgc tigcaaaatcg 33120 tgagaactgg agttcaattic ccago acatg gatgtatttc cago acctgg aaggcaggga 331.80 gcagagat.ct taaagctcct ggc.ca.gacag cccagoctaa ttagtaatca gtgagagacc 33240 citgtctdaag aaacaagatg gaacatcaaa ggtoaaccto ttgttctocac acacacaaat 33300 acacacatgc acatacatcc acacacaggc aaacacatgc acacaccitga acaccctcca 33360 caaatacata cataaaaaaa taaatacata cacacataca tacatacacc alacattccct 33 420 citccittagtc. tcctggctac gotcttgtca ccc.ccactaa ggcttcaact tcttctattt 33480 cittcatcttg acticcitctgt actittgcatg cctttitccag caaaggcttt totttaaatc. 33540 tocgtoattc ataaactccc totaaattitc titcccctg.cc cittittctittc. tctotaggga 33600 gataaagaca cacactacaa agtcaccgtg g gaccagttt attcacccac ccacccctgc 33660 ttctgttcat coggccagot aagtag toca accitctotgg tactgtaccc tag accotgg 33.720 cittcaccaca gctcctccat gctacccago cotgcaaacc titcagoctag cctotggttc. 33780 to caaccago acagg.cccag totggcttct atgtc.ctaga aatctoctitc attctotcca 33840 tittcccitcct gaatctacca ccttctttct cocttctoct gacctctaat gtcttgg to a 33900 aacgattaca aggaagccaa toaaattagc agtttggggit accitcagagt cagoagggga 33960 gctgggatga attcacattt coaggcctitt gctittgctoc ccggattctg acagg cagtt 34020 cc.gaagct ga gtc.caggaag citgaatttaa aatcacactc. cagotgggitt citgaggcago. 34 080 ccitaccacat cagotggc.cc tdactgagct gtgtctgggt ggcagtggtg citggtggtoc 34140 tggtggtgct ggtggtggtg gtggtggtgg toggtggtggt ggtggtggtg togtgtgtgtg 34200 ttittctgctt ttacaaaact tittctaattic titatacaaag gacaaatctg. cctoatatag 34260 gcagaaagat gacittatgcc tatataagat ataaagatga citt tatgc.ca cittattagca 34320

US 2004/0058321 A1 Mar. 25, 2004 59

-continued

SEQUENCE: 19 cc.ggagctgg agaacaacaa g 21

SEQ ID NO 20 LENGTH 19 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: PRimer for PCR

<400 SEQUENCE: 20 gcactggc.cg gag cacacc 19

SEQ ID NO 21 LENGTH 23 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 21 aggcca accg cq agaagatg acc 23

SEQ ID NO 22 LENGTH 21 TYPE DNA ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 22 galagto: Cagg gcgacgtagc a 21

SEQ ID NO 23 LENGTH 25 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 23 aagcttggta ccatgcagot cocac 25

SEQ ID NO 24 LENGTH 50 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 24 aagcttctac ttgtcatcgt cqtccttgta gtcgtagg.cg ttctocagot 5 O

SEQ ID NO 25 LENGTH 19 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 25 gcactggc.cg gag cacacc 19 US 2004/0058321 A1 Mar. 25, 2004 60

-continued

<210> SEQ ID NO 26 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 26 gtogtoggat coatggggtg gcaggcgttcaagaatgat 39

<210 SEQ ID NO 27 &2 11s LENGTH 57 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 27 gtogtoaa.gc titctacttgt catcgtoctit gtagt cqtag gogttcticca gctcqgc 57

<210> SEQ ID NO 28 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 28 gacittggatc ccaggggtgg caggc gttc 29

<210 SEQ ID NO 29 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 29 agcataagct tctagtaggc gttcticcag 29

<210 SEQ ID NO 30 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 30 gactitggatc. Cqaagggaaa aagaaaggg 29

<210> SEQ ID NO 31 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 31 agcataagct tittaatccaa atc gatgga 29

<210> SEQ ID NO 32 &2 11s LENGTH 33 US 2004/0058321 A1 Mar. 25, 2004 61

-continued

&212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 32 actacgagct c gg.ccccacc accolatcaac aag 33

<210 SEQ ID NO 33 <211& LENGTH: 34 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 33 acttagaagc tittcagtcct cago.ccccitc titcc 34

<210> SEQ ID NO 34 &2 11s LENGTH 66 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 34 aatctggatc catalactitcg tatagdatac attatacgaa gttatctgca ggattic gagg 60 gcc.cct 66

<210 SEQ ID NO 35 &2 11s LENGTH 82 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 35 aatctgaatt coaccggtgt taattaaata actitcgtata atgitatgcta tacgaagtta 60 tagatctaga gtcagottct ga 82

<210 SEQ ID NO 36 &2 11s LENGTH 62 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 36 atttaggtga cactatagaa citcgagcago toaa.gcttaa ccacatggtg gct caca acc 60 at 62

<210 SEQ ID NO 37 &2 11s LENGTH 54 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 37 aacgacggcc agtgaatc.cg taatcatggit catgctocca ggtggaggag ggca 54 US 2004/0058321 A1 Mar. 25, 2004 62

-continued <210 SEQ ID NO 38 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 38 attaccaccg gtgacaccc.g. cittcc tdaca g 31

<210 SEQ ID NO 39 &2 11s LENGTH: 61 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 39 attacittaat taalacatggc gcgc.catatg gcc.ggcc cct aattgcggcg catcgittaat 60 t 61

<210> SEQ ID NO 40 <211& LENGTH: 34 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 40 attacggc.cg gcc.gcaaagg aattcaagat citga 34

<210> SEQ ID NO 41 <211& LENGTH: 34 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer for PCR

<400 SEQUENCE: 41 attacggcgc gcc cct caca gg.ccgcaccc agct 34

<210> SEQ ID NO 42 <211& LENGTH 184 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 42 Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Lieu Lleu Lleu Lleu Lieu Gly 1 5 10 15 Thr Lieu Lieu Pro Ala Ala Glu Gly Lys Lys Lys Gly Ser Glin Gly Ala 2O 25 30 Ile Pro Pro Pro Asp Lys Ala Gln His Asn Asp Ser Glu Glin Thr Glin 35 40 45 Ser Pro Glin Glin Pro Gly Ser Arg Asn Arg Gly Arg Gly Glin Gly Arg 50 55 60 Gly Thr Ala Met Pro Gly Glu Glu Val Leu Glu Ser Ser Glin Glu Ala 65 70 75 8O Lieu. His Val Thr Glu Arg Lys Tyr Lieu Lys Arg Asp Trp Cys Lys Thr 85 90 95 Glin Pro Leu Lys Glin Thr Ile His Glu Glu Gly Cys Asn. Ser Arg Thr US 2004/0058321 A1 Mar. 25, 2004 63

-continued

100 105 110

Ile Ile Asn Arg Phe Gly Glin Cys Asn Ser Phe Tyr Ile Pro 115 120 125

Arg His Ile Arg Glu Glu Gly Ser Phe Glin Ser Ser Phe Cys 130 135 1 4 0

Lys Pro Lys Phe Thr Thr Met Met Wall Thr Teu Asn Pro Glu 145 15 O 155 160

Teu Glin Pro Pro Thr Arg Wall Thr Arg Wall Glin Cys 1.65 170 175

Arg Ile Ser Ile Asp Teu Asp 18O

SEQ ID NO 43 LENGTH 267 TYPE PRT ORGANISM Homo sapiens

<400 SEQUENCE: 43

Met His Telu Telu Teu Phe Glin Telu Telu Wall Teu Teu Pro Teu Gly Lys 1 5 10 15

Thr Thr Arg His Glin Asp Gly Glin Asn Glin Ser Ser Teu Ser Pro 25 30

Wall Telu Telu Pro Arg Asn Glin Arg Glu Telu Pro Thr Gly Asn His Glu 35 40 45

Glu Ala Glu Glu Pro Asp Leu Phe Wall Ala Wall Pro His Leu Wall 50 55 60

Ala Thr Ser Pro Gly Glu Gly Glin Glin Arg Glu Met Telu 65 70 75

Ser Arg Phe Gly Phe Trp Pro Glu Arg Glu Met His Pro 90 95

Ser Arg Asp Ser Asp Ser Glu Pro Phe Pro Pro Gly Thr Glin Ser Telu 100 105 110

Ile Glin Pro Ile Asp Gly Met Lys Met Glu Ser Pro Teu Glu 115 120 125

Glu Ala Phe Trp His His Phe Met Phe Arg Thr Pro Ala 130 135 1 4 0

Ser Glin Gly Wall Ile Teu Pro Ile Ser His Glu Wall His Trp Glu 145 15 O 155 160

Thr Arg Thr Wall Pro Phe Ser Glin Thr Ile Thr His Glu Gly Cys 1.65 170 175

Glu Wall Wall Wall Glin Asn Asn Telu Cys Phe Gly Lys Cys Gly Ser 18O 185 190

Wall His Phe Pro Gly Ala Ala Glin His Ser His Thr Ser Ser His 195 200

Telu Pro Ala Phe Thr Thr Met His Teu Pro Teu Asn Cys Thr 210 215 220

Glu Telu Ser Ser Wall Ile Wall Wall Met Teu Wall Glu Glu Cys Glin 225 230 235 240

Wall Thr Glu His Glu Asp Gly His Ile Teu His Ala Gly 245 250 255

Ser Glin Asp Ser Phe Ile Pro Gly Wall Ser Ala 260 265 US 2004/0058321 A1 Mar. 25, 2004 64

-continued

<210> SEQ ID NO 44 &2 11s LENGTH 18O &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 44 Met Leu Arg Val Lieu Val Gly Ala Val Lieu Pro Ala Met Leu Lieu Ala 1 5 10 15 Ala Pro Pro Pro Ile Asn Lys Lieu Ala Lieu Phe Pro Asp Llys Ser Ala 2O 25 30 Trp Cys Glu Ala Lys Asn. Ile Thr Glin Ile Val Gly His Ser Gly Cys 35 40 45 Glu Ala Lys Ser Ile Glin Asn Arg Ala Cys Lieu Gly Glin Cys Phe Ser 50 55 60 Tyr Ser Val Pro Asn Thr Phe Pro Glin Ser Thr Glu Ser Leu Val His 65 70 75 8O Cys Asp Ser Cys Met Pro Ala Glin Ser Met Trp Glu Ile Val Thr Leu 85 90 95 Glu Cys Pro Gly His Glu Glu Val Pro Arg Val Asp Lys Lieu Val Glu 100 105 110 Lys Ile Lieu. His Cys Ser Cys Glin Ala Cys Gly Lys Glu Pro Ser His 115 120 125 Glu Gly Leu Ser Val Tyr Val Glin Gly Glu Asp Gly Pro Gly Ser Glin 130 135 1 4 0 Pro Gly. Thr His Pro His Pro His Pro His Pro His Pro Gly Gly Glin 145 15 O 155 160 Thr Pro Glu Pro Glu Asp Pro Pro Gly Ala Pro His Thr Glu Glu Glu 1.65 170 175 Gly Ala Glu Asp 18O

<210> SEQ ID NO 45 <211& LENGTH 642 &212> TYPE DNA <213> ORGANISM: Homo sapiens &220s FEATURE <221 NAME/KEY: CDS <222> LOCATION: (1) . . (639) <400 SEQUENCE: 45 atg cag ctic coa citg gcc ctd tot citc gtc toc citg citg gta cac aca 48 Met Gln Leu Pro Leu Ala Lieu. Cys Lieu Val Cys Lieu Lleu Val His Thr 1 5 10 15 gcc titc cqt gta gtg gag ggc cag ggg togg cag gog titc aag aat gat 96 Ala Phe Arg Val Val Glu Gly Glin Gly Trp Glin Ala Phe Lys Asn Asp 2O 25 30 gcc acg gala atc atc ccc gag citc gga gag tac ccc gag cot coa cog 144 Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 gag citg gag aac aac aag acc atgaac cqg gC9 gag aac gga ggg cqg 192 Glu Lieu Glu Asn. Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 5 O 55 60 cct coc cac cac ccc titt gag acc aaa gac gtg toc gag tac agc toc 240 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 8O cgc gag citg cac titc acc cqc tac gitg acc gat ggg ccg togc cqc agc 288 US 2004/0058321 A1 Mar. 25, 2004

-continued Arg Glu Leu. His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 gcc aag cog gtc. acc gag citg gtg toc toc ggc cag togc ggc cc g gC g 336 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 cgc citg citg ccc aac goc atc ggc cqc ggc aag togg togg cqa cot agt 384 Arg Lieu Lleu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 ggg ccc gac ttic cqc toc atc ccc gac cqc tac cqc gcg cag cqc gtg 432 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Glin Arg Val 130 135 1 4 0 Cag Ctg Ctg togt CCC ggt ggit gag gC g ccg cqC gCg C gC aag gtg CGC 480 Glin Leu Lieu. Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 145 15 O 155 160 citg gtg gCC to g togc aag toc aag cqc ctic acc cqc titc. cac aac cag 528 Leu Val Ala Ser Cys Lys Cys Lys Arg Lieu. Thr Arg Phe His Asn Glin 1.65 170 175 tog gag Ct c aag gaC titc ggg acc gag gCC gct C gg CCG cag aag ggC 576 Ser Glu Lieu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Glin Lys Gly 18O 185 190

Cgg aag cc g cqg CCC cqC gcc cqg agc gcc aaa gcc aac Cag gCC gag 624 Arg Llys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Glin Ala Glu 195 200 2O5 citg gag aac goc tac tag 642 Leu Glu Asn Ala Tyr 210

We claim: 8. An expression vector, comprising a promoter operably 1. An isolated nucleic acid molecule Selected from the linked to a nucleic acid molecule according to any one of group consisting of claims 1 to 7. 9. The expression vector according to claim 8 wherein (a) an isolated nucleic acid molecule comprising Sequence Said promoter is Selected from the group consisting of CMV ID Nos., 1, 5, 9, 11, 13, or, 15, or complementary I-E promoter, SV40 early promoter and Mul V LTR. Sequence thereof; 10. The expression vector according to claim 8 wherein (b) an isolated nucleic acid molecule that specifically Said promoter is a tissue-specific promoter. hybridizes to the nucleic acid molecule of (a) under 11. A method of producing a TGF-beta binding protein, conditions of high Stringency; and comprising, culturing a cell which contains a vector accord ing to claim 8 under conditions and for a time Sufficient to (c) an isolated nucleic acid that encodes a TGF-beta produce Said protein. binding-protein according to (a) or (b). 12. The method according to claim 11, further comprising 2. The isolated nucleic acid molecule according to claim the Step of purifying Said protein. 1 wherein Said nucleic acid molecule encodes a protein 13. A viral vector capable of directing the expression of a comprising the protein of Sequence ID NO. 2. nucleic acid molecule according to any one of claims 1 to 7. 14. The viral vector according to claim 13 wherein said 3. The isolated nucleic acid molecule according to claim vector is Selected from the group consisting of herpes 1 wherein Said nucleic acid molecule encodes a protein Simplex viral vectors, adenoviral vectors, adenovirus-asso comprising the protein of Sequence ID NO. 6. ciated viral vectors and retroviral vectors. 4. The isolated nucleic acid molecule according to claim 15. A host cell carrying a vector according to any one of 1 wherein Said nucleic acid molecule encodes a protein claims 8 to 14. comprising the protein of Sequence ID NO. 10. 16. The host cell according to claim 15 wherein said cell 5. The isolated nucleic acid molecule according to claim is Selected from the group consisting of a human cell, dog 1 wherein Said nucleic acid molecule encodes a protein cell, monkey cell, rat cell and mouse cell. comprising the protein of Sequence ID NO. 12. 17. An isolated protein, comprising a TGF-beta binding 6. The isolated nucleic acid molecule according to claim protein encoded by the nucleic acid molecule according to 1 wherein Said nucleic acid molecule encodes a protein any one of claims 1 to 7. comprising the protein of Sequence ID NO. 14. 18. An antibody which specifically binds to the protein 7. The isolated nucleic acid molecule according to claim according to claim 17. 1 wherein Said nucleic acid molecule encodes a protein 19. The antibody according to claim 18 wherein said comprising the protein of Sequence ID NO. 16. antibody is a monoclonal antibody. US 2004/0058321 A1 Mar. 25, 2004

20. The antibody according to claim 19 wherein said 42. The ribozyme according to claim 33 wherein said monoclonal antibody is a murine or human antibody. ribozyme is composed of a mixture of deoxyribonucleic 21. The antibody according to claim 18 wherein said acids and ribonucleic acids. antibody is selected from the group consisting of F(ab'), 43. The ribozyme according to claim 33 wherein said F(ab), Fab', Fab, and Fv. ribozyme is composed of nucleic acids having phosphothio 22. A hybridoma which produces an antibody according ate linkages. to claim 19. 44. A nucleic acid molecule comprising a nucleic acid 23. A fusion protein, comprising a first polypeptide Seg Sequence which encodes a ribozyme according to claim 33. ment comprising a TGF-beta binding-protein encoded by the 45. The nucleic acid molecule of claim 44, wherein the nucleic acid molecule according to any one of claims 1 to 7, nucleic acid is DNA or cDNA. or a portion thereof of at least 10 amino acids in length, and 46. The nucleic acid molecule of claim 44, under the a Second polypeptide Segment comprising a non-TGF-beta control of a promoter to transcribe the nucleic acid. binding-protein. 47. A host cell comprising the ribozyme of claim 33. 24. The fusion protein according to claim 23 wherein Said 48. A vector, comprising the nucleic acid molecule of first polypeptide Segment is at least 20 amino acids in length. claim 44. 25. The fusion protein according to claim 23 wherein said 49. The vector of claim 54, wherein the vector is a first polypeptide Segment is at least 50 amino acids in length. plasmid, a virus, retrotransposon or a cosmid. 26. The fusion protein according to claim 23 wherein Said 50. The vector of claim 49 wherein said virus is selected Second polypeptide comprises multiple anionic amino acid from the group consisting of retroviruses, adenoviruses, and residues. adeno-associated viruses. 27. An isolated oligonucleotide which hybridizes to a 51. A host cell containing the vector according to any one nucleic acid molecule according to Sequence ID NOS. 1, 3, of claims 48 to 50. 5, 7, 9, 11, 13, or 15, or the complement thereto, under 52. The host cell according to claim 51 wherein said host conditions of high Stringency. cell is stably transformed with said vector. 28. The isolated oligonucleotide according to claim 27 53. The host cell according to claim 51 wherein the host wherein Said oligonucleotide is at least 20 nucleotides in cell is a human cell. length. 54. A method for producing a ribozyme, comprising 29. The isolated oligonucleotide according to claim 27 providing DNA encoding the ribozyme according to claim wherein Said oligonucleotide is at least 30 nucleotides in 33 under the transcriptional control of a promoter, and length. transcribing the DNA to produce the ribozyme. 30. The isolated oligonucleotide according to claim 27 55. The method of claim 54 wherein the ribozyme is wherein Said oligonucleotide is at least 50 nucleotides in produced in vitro. length. 56. The method of claim 54, further comprising purifying 31. The isolated oligonucleotide according to claim 27 the ribozyme. wherein said oligonucleotide is between 50 to 100 nucle 57. A method for increasing bone mineralization, com otides in length. prising introducing into a warm-blooded animal an effective 32. A pair of primers which Specifically amplifies all or a amount of the ribozyme according to any one of claims 33 portion of a nucleic acid molecule according to any one of to 43. claims 1 to 7. 58. A method of increasing bone mineralization, compris 33. A ribozyme which cleaves RNA encoding a protein ing introducing into a patient an effective amount of the nucleic acid molecule of claim 44, under conditions favoring according to claim 17. transcription of the nucleic acid molecule to produce a 34. The ribozyme according to claim 33 wherein said ribozyme. protein comprises the protein of Sequence ID NO. 2. 59. A pharmaceutical composition, comprising the 35. The ribozyme according to claim 33 wherein said ribozyme according to any one of claims 33 to 43, and a protein comprises the protein of Sequence ID NO. 6. pharmaceutically acceptable carrier or diluent. 36. The ribozyme according to claim 33 wherein said 60. A pair of primers capable of Specifically amplifying all RNA encodes a protein comprising the protein of Sequence or a portion of a nucleic acid molecule according to any one ID NO. 10. claims 1 to 7. 37. The ribozyme according to claim 33 wherein said 61. A method for detecting a nucleic acid molecule which RNA encodes a protein comprising the protein of Sequence encodes a TGF-beta binding protein, comprising incubating ID NO. 12. an oligonucleotide according to any one of claims 27 to 31 38. The ribozyme according to claim 33 wherein said under conditions of high Stringency, and detecting hybrid RNA encodes a protein comprising the protein of Sequence ization of Said oligonucleotide. ID NO. 14. 62. The method according to claim 61 wherein said 39. The ribozyme according to claim 33 wherein said oligonucleotide is labeled. RNA encodes a protein comprising the protein of Sequence 63. The method according to claim 61 wherein said ID NO. 16. oligonucleotide is bound to a Solid Support. 40. The ribozyme according to claim 33 wherein said 64. A method for detecting a TGF-beta binding protein, ribozyme is composed of ribonucleic acids. comprising incubating an antibody according to any one of 41. The ribozyme according to claim 40 wherein one or claims 18 to 21 under conditions and for a time Sufficient to more of said ribonucleic acids are 2'-O-methyl ribonucleic permit said antibody to bind to a TGF-beta binding protein, acids. and detecting Said binding. US 2004/0058321 A1 Mar. 25, 2004

65. The method according to claim 64 wherein said 76. The method according to claim 75 wherein said antibody is bound to a Solid Support. analogue of bone is hydroxyapatite. 66. The method according to claim 64 wherein said 77. A kit for detection of TGF-beta binding-protein gene antibody is labeled. expression, comprising a container that comprises a nucleic 67. The method according to claim 66 wherein said antibody is labeled with a marker Selected from the group acid molecule, wherein Said nucleic acid molecule is consisting of enzymes, fluorescent proteins, and radioiso Selected from the group consisting of (a) a nucleic acid topes. molecule comprising the nucleotide Sequence of SEQ ID 68. Atansgenic animal whose germ cells and Somatic cells NO: 1, 5, 7, 9, 11, 13, or 15; (b) a nucleic acid molecule contain a nucleic acid molecule encoding a TGF-beta bind comprising the complement of the nucleotide Sequence of ing-protein according to claim 1 which is operably linked to (a); (c) a nucleic acid molecule that is a fragment of (a) or a promoter effective for the expression of Said gene, Said (b) of at least 20 nucleotides in length. gene being introduced into Said animal, or an ancestor of 78. A kit for detection of TGF-beta binding-protein, Said animal, at an embryonic Stage, with the proviso that Said comprising a container that comprises an antibody according animal is not a human. to any one of claims 18 to 21. 69. The transgenic animal according to claim 68 wherein 79. An antisense oligonucleotide, comprising a nucleic TGF-beta binding-protein is expressed from a vector accord acid molecule which hybridizes to a nucleic acid molecule ing to any one of claims 8 to 10. according to Sequence ID NOS. 1, 3, 5, 7, 9, 11, 13, or 15, 70. A transgenic knockout animal, comprising an animal or the complement thereto, and wherein Said oligonucleotide whose germ cells and Somatic cells comprise a disruption of inhibits the expression of TGF-beta binding protein accord at least one allele of an endogenous nucleic acid molecule ing to claim 17. which hybridizes to the nucleic acid molecule according to 80. The oligonucleotide according to claim 79 wherein claim 1, wherein Said disruption prevents transcription of Said oligonucleotide is 15 nucleotides in length. messenger RNA from Said allele as compared to an animal without Said disruption, with the proviso that Said animal is 81. The oligonucleotide according to claim 79 wherein not a human. Said oligonucleotide is 20 nucleotides in length. 71. The transgenic animal according to claim 70 wherein 82. The oligonucleotide according to claim 79 wherein Said disruption is a nucleic acid deletion, Substitution, or, Said oligonucleotide is 50 nucleotides in length. insertion. 83. The oligonucleotide according to claim 79, wherein 72. The transgenic animal according to claim 68 or 70 Said oligonucleotide is comprised of one or more nucleic wherein the animal is selected from the group consisting of acid analogs. a mouse, a rat and a dog. 84. The oligonucleotide according to claim 79, wherein 73. A method for determining whether a candidate mol Said oligonucleotide is comprised of one or more ribonucleic ecule is capable of increasing bone mineral content, com acids. prising: 85. The oligonucleotide according to claim 79, wherein (a) mixing one or more candidate molecules with TGF Said oligonucleotide is comprised of one or more deoxyri beta-binding-protein encoded by the nucleic acid mol bonucleic acids. ecule according to any one of claims 1 to 7 and a 86. The oligonucleotide according to claim 79 wherein selected member of the TGF-beta family of proteins; Said oligonucleotide Sequence comrpises one or more modi (b) determining whether the candidate molecule alters the fied covalent linkages. signaling of the TGF-beta family member, or alters the 87. The oligonucleotide according to claim 86 wherein binding of the TGF-beta binding-protein to the TGF Said modified covalent linkage is Selected from the group beta family member. consisting of a phosphorothioate linkage, a phosphotriester 74. The method according to claim 73 wherein said linkage, a methyl phosphonate linkage, a methylene(meth member of the TGF-beta family of proteins is BMP6. ylimino) linkage, a morpholino linkage, an amide linkage, a 75. A method for determining whether a candidate mol polyamide linkage, a short chain alkyl interSugar linkage, a ecule is capable of increasing bone mineral content, com cycloalkyl interSugar linkage, a short chain heteroatomic prising: determining whether a candidate molecule inhibits interSugar linkage and a heterocyclic interSugar linkage. the binding of TGF-beta binding-protein to bone, or an analogue thereof.