CA2282302A1 - Morphogenic proteins - Google Patents

Abstract

b57 proteins and related nucleic acids are provided. Included are natural DAN
and b57 homologs from several species and proteins comprising a DAN or b57 domain having specific activity, particularly the ability to antagonize a bone morphogenic protein. The proteins may be produced recombinantly from transformed host cells with the subject nucleic acids. Also provided are isolated hybridization probes and primers capable of specifically hybridizing with the disclosed genes, specific binding agents and methods of making and using the subject compositions.

Description

MORPHOGENIC PROTEINS
This International Application claims priority of United States Provisional Application No. 60/038,279 filed February 19, 1997. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
INTRODUCTION
The field of this invention is proteins which regulate cell function, and in particular, antagonize bone morphogenic proteins.
Natural regulators of cellular growth, differentiation and function have provided important pharmaceuticals, clinical and laboratory tools, and targets for therapeutic intervention. A variety of such regulators have been shown to 15 have profound effects on basic cellular differentiation and developmental pathways. For example, the recently cloned cerberus protein induces the formation of head structures in anterior endoderm of vertebrate embryos.
Similarly, the noggin protein induces head structures in vertebrate embryos, and can redirect mesodermal fates from ventral fates, such as blood and zo mesenchyme, to dorsal fates such as muscle and notochord and can redirect epidermal fates to anterior neural fates. The activities of chordin are similar to those of noggin, reflecting a common mechanism of action - namely antagonizing bone morphogenic proteins (BMP) and thereby preventing their function. BMPs have diverse biological activities in different biological z5 contexts, including the induction of cartilage, bone and connective tissue, and roles in kidney, tooth, gut, skin and hair development.
Different members of the TGF(3 superfamily can instruct cells to follow different fates, for example TGF(3 induces neural crest to form smooth muscle, ~~hile 3o BMP2 induces the same cells to became neurons. In Xeno~us experiments, dissociated animal cap cells (prospective ectoderm) become epidermis in WO 98/37195 PCTlUS98/03283 response to BMP4 but become mesoderm in response to activin.
Since the sequence identity between activin and BMP4 is low, it is not surprising that they induce different fates. It is more surprising that members of the BMP subfamily, which are quite closely related in sequence, can induce distinct fates_ A striking example results from implantation of a matrix impregnated with a BMP into muscle; when the effects are monitored histologically, BMP2, BMP4 and BMP7 induce endochondral bone formation, whereas a related molecule BMP12/GDF7 induces connective tissue similar to tendon. Similarly, BMP4 can induce cell death in the hindbrain neural crest, 1o while the related protein dorsalin does not.
Since different BMP family members can induce different fates, then BMI' antagonists that have specificity in blocking subsets of BMPs could change the balance of BMPs that are presented to a cell, thus altering cell fate. In view of ~5 the importance of relative BMP expression in human health and disease, regulators of cellular function and BMP function in particular, such as noggin and cerberus, provide valuable reagents with a host of clinical and biotechnological applications. The present invention relates to a new family of regulators of cellular function.
Relevant Literature Bouwmeester, et al. (1996) Nature 382: 595-601 describe the cloning of Xenopus cerberus gene. PCT International Publication No. WO 94/05791 published 17 March 1994 entitled Dorsal Tissue Affecting Factor and Compositions; Lamb, T.
z5 M., et al. (1993) Science 262: 713-718; Smith, W.' C., et al. (1992) Cell 70: 829-840;
Smith, W. C., et al. (1993) Nature 361: 547-549; and Zimmerman, L. B., et al.
(1996) Cell 86: 599-606 describe the isolation and function of the noggin nucleic acid and protein. Piccolo, S., et al. (1996) Cell 86: 589-598; Sasai, Y., et al. (1995) Nature 376: 333-336; and Sasai, Y., et al. (1994) Cell 79: 779-790 relate to the 3o chordin protein. Enomoto et al. (1994) Oncogene 9: 2785-2791 and Ozaki, et al.
(1996) Jpn. J. Cancer Res. 87: 58-61 describe human and marine homologs of the DAN gene.

SUMMARY OF THE INVENTION
The in~~ention provides methods and compositions relating to DAN
s (Differential-screening-selected gene Aberrative in Neuroblastoma) and b57 proteins and related nucleic acids. Included are natural DAN and b57 homologs from different species, as well as proteins comprising a DAN or b57 domain and having DAN or b57-specific activity, particularly the ability to antagonize a bone morphogenic protein such as BMP2 or BMP4. The proteins may be produced 1o recombinantly from transformed host cells with the subject nucleic acids.
The invention provides isolated hybridization probes and primers capable of specifically hybridizing with the disclosed genes, specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g., genetic hybridization screens for b57 transcripts), therapy (e.g., 15 gene therapy to modulate b57 gene expression) and in the biopharmaceutical industry (e.g., reagents for screening chemical libraries for lead pharmacological agents).
Preferred applications of the subject DAN and b57 proteins include modifying 20 the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DAN or b57 protein under conditions whereby the added protein specifically interacts with a component of the medium and/or the extracellular surface to effect a change in the physiology of the cell. Also preferred are methods for screening for biologically active 2s agents, which methods involve incubating a DAN or b57 protein in the presence of an extracelluiar DAN or b57 protein-specific binding target and a candidate agent, under conditions whereby, but for the presence of the agent, the protein specifically binds the binding target at a reference affinity;
detecting the binding affinity of the protein to the binding target to determine an agent-3o biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the binding of the protein to the binding target.

BRIEF DESCRIPTION OF THE FIGURES
FIGURES 1A - 1B - Demonstration that human b57 binds to BMP2 and BMP4.
FIGURE 2 - Demonstration that human b57 blocks BMP2 biological activity.
FIGURE 3 - Xenopus b57 (also referred to as Gremlin) blocks the activity of BMP2. BMP2 at 78pM, 156pM, 313 pM, 625 pM, 1.25 nM, 2.5 nM or 5 nM was to preincubated with a Gremlin COS supernatant at final concentration of 83nM
or 21 nM Gremlin, mock-transfected media, or fresh DMEM prior to addition to cells. Alkaline phosphatase activity was assayed 24 hours later. Approx. 83nM
Gremlin completely blocks BMP2 activity. Approx. 2lnM Gremlin partially blocks BMP2 doses tested.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides DAN and b57 proteins which include natural DAN
2o and b57 proteins and recombinant proteins comprising a DAN or b57 amino acid sequence, or a functional DAN or b57 protein domain thereof having an assay-discernable DAN or b57-specific activity. Accordingly, the proteins may be deletion mutants of the disclosed natural DAN and b57 proteins and may be provided as fusion products, e.g., with non-b57 polypeptides. The subject DAN
and b57 protein domains have DAN or b57-specific activity or function and are functionally distinct from each other and from cerberus and noggin homologs.
Such domains include at least 6 and preferably at least 8 consecutive residues of a natural DAN or b57 protein (See DAN sequence reported by Enomoto, et al.
(1994) Oncogene 9: 2785-2791 and human b57 sequence disclosed herein).
3o Preferred b57 proteins comprise a b57 sequence conserved across species.
Note that contrary to prior art teachings which state that DAN is an _ ~__ __ intracellular zinc finger protein, applicants disclose that the natural DAN
protein is structurally and functionally related to b57 and that both it and DAN
proteins as described herein are extracellularly active as antagonists of certain morphogenic proteins such as BMPs. DAN or b57-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays -e.g., in vitro binding assays, cell culture assays, in animals (e.g., immune response, gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the specific molecular interaction of a DAN or b57 protein with a binding target is evaluated. The binding target may be a natural binding target such as a TGF(3 1o protein, a morphogenic protein, preferably a bone morphogenic protein such as BMP2 or BMP4, chaperone, or other regulator that directly modulates DAN or b57 activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a DAN or b57 specific agent such as those identified in assays described below. Generally, binding specificity is assayed by bioassay (e.g., the ability to induce neuronal tissue from injected embryonic ectoderm), TGFj3 protein binding equilibrium constants (usually at least about 10~ M-~, preferably at least about 208 M-~, more preferably at least about 109 Mr), by the ability of the subject protein to function as negative mutants in DAN or b57-expressing cells, to elicit DAN or b57 specific antibody 2o in a heterologous host (e.g., a rodent or rabbit), etc.
The claimed proteins may be isolated or pure - an "isolated" protein is one that is no longer accompanied by some of the material with which it is associated in its natural state, and that preferably constitutes at least about 0.5°/", and more preferably at least about 5% by weight of the total protein in a given sample;
a "pure" protein constitutes at least about 90'%, and preferably at least about 99"/"
by weight of the total protein in a given sample. The subject proteins and protein domains may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are 3o available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g., Molecular Cloning, A Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY). An exemplary method for isolating natural DAN and b57 proteins involves expressing a cDNA library (e.g., one derived from Xenopus ovarian s cells) and assaying expression products for embryonic axis formation. This method and other suitable bioassays amenable to detecting DAN and b57 proteins have been described by Lemaire, P., et al., (1995) Cell 81:85-94;
Smith, W. C., and Harland, R. M. (1992) Cell 70: 829-40; Smith, W. C., and Harland, R.
M. (1991) Cell 67: 753-765; Piccolo, S., et al., (1996) Cell 86: 589-98; and 1o Zimmerman, L. B., et al., (1996) Cell 86: 599-606.
The subject proteins find a wide variety of uses including use as immunogens, targets in screening assays, bioactive reagents for modulating cell growth, differentiation and/or function, etc. For example, the invention provides 15 methods for modifying the physiology of a cell comprising an extracellular surface by contacting the cell or medium surrounding the cell with an exogenous DAN or b57 protein under conditions whereby the added protein specifically interacts with a component of the medium and/or the extracellular surface to effect a change in the physiology of the cell. According to these 2o methods, the extracellular surface includes plasma membrane-associated receptors; the exogenous DAN or b57 refers to a protein not made by the cell or, if so, expressed at non-natural levels, times or physiologic locales; and suitable media include in vitro culture media and physiological fluids such as blood, synovial fluid, etc. Effective administrations of subject proteins can be used to 25 reduce undesirable (e.g., ectopic) bone formation, inhibit the growth of cells that require a morphogenic protein (e.g., BMP-dependent neuroblastomas and gliomas), alter morphogen-dependent cell fate/differentiation in culture, such as with cells for transplantation or infusion, etc. The proteins may be may be introduced, expressed, or repressed in specific populations of cells by anv 3o convenient way such as microinjection, promoter-specific expression of recombinant enzyme, targeted delivery of lipid vesicles, etc.

~_ r ____ _...__ The in~~ention provides natural and non-natural DAN and b57-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. DAN or b57-specific binding agents include b57-specific ligands such as BMPs, and receptors, such as somatically recombined protein receptors like specific antibodies or T-cell antigen receptors (See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and also includes other natural binding agents identified with assays such as one-, two- and three-hybrid screens, and non-natural binding agents identified in screens of chemical 1o libraries such as described below. Agents of particular interest modulate DAN
or b57 function.
The invention provides b57 and DAN nucleic acids, which find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc., as well as use in detecting the presence of DAN and b57 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional DAN and b57 homologs and structural analogs. For example, Xenopus and chick b57 sequence data was used to search the EST database of the LM.A.G.E. consortium and a human cDNA
2o clone 272074 was discerned to contain homologous sequence homologous by DNA sequencing. The insert was cloned into CS105, a suitable vector for synthesis of synthetic mRNA (Turner, D. L., and Weintraub, H. (1994) Genes Dev 8, 1434-47; Baker, J. C., and Harland, R. M. (1996) Genes & Development 10).
b57-specific function of the human gene product was determined by injection of purified, synthetic transcripts of cDNA clones' into Xeno~us embryos. This assay provides a bioassay for antagonists of BMP activity, as exemplified by the induction of ectopic body axes or enlarged heads. Embryos injected with transcript from the human b57 clone had enlarged heads and partial ectopic body axes similar to the Xenopus b57 injected embryos, indicating a b57 specific 3o biological function for the human gene product.
Similarly, Xenopus cerberus sequence data was used to search the EST database of the LM.A.G.E. consortium and human cDNA clone 272074 was discerned to contain homologous sequence. This clone was obtained from Genome Systems, Inc. (St. Louis, MO) and sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The nucleotide sequence encoding human b57 is set forth herein as SEQ. NO. 1 and the deduced amino acid sequence of human b57 is set forth herein as SEQ. NO. 2.
The subject nucleic acids are of synthetic/non-natural sequences and/or are 1o isolated, i.e., no longer accompanied by some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5°/o by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotides) other than that which it is joined to on a natural chromosome. Nucleic acids comprising the nucleotide sequence of SEQ. NO. 1 or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that to which it is joined on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is immediately 2o flanked by a sequence other than that to which it is joined on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The amino acid sequences of the disclosed DAN and b57 proteins are used to back translate DAN or b57 protein-encoding nucleic acids optimized for selected expression systems (Holler, et al. (1993) Gene 136: 323-328; Martin, et al.
(1995) Gene 154: 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural b57 encoding nucleic acid sequences ("GCG" software, Genetics Computer Group, Inc., Madison, WI). DAN and b57 encoding nucleic acids may be part of expression vectors and may be incorporated into recombinant host cells, e.g., for expression and screening, for WO 98!37195 PCT/CTS98/03283 transgenic animals, for functional studies such as the efficacy of candidate drugs for disease associated with b57 mediated signal transduction, etc. Expression systems are selected and/or tailored to effect b57 protein structural and functional variants through alternative post-translational processing.
The invention also provides for nucleic acid hybridization probes and replication/amplification primers having a DAN or b57 cDNA specific sequence and sufficient to effect specific hybridization with SEQ. NO. 1. Demonstrating specific hybridization generally requires stringent conditions, for example, lU hybridizing in a buffer comprising 30°/'~ formamide in 5 x SSPE
(0.18 M NaCI, 0.01 M NaP04, pl-~7.7, 0.001 M EDTA) buffer at a temperature of 42°C
and remaining bound when subject to washing at 42°C with 0.2 x SSPE;
preferably hybridizing in a buffer comprising 50'% formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C
with 0.2x SSPE buffer at 42°C. DAN and b57 cDNA homologs can also be distinguished from other protein using alignment algorithms, such as BLASTX
(Altschul, et al. (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215:

410).
2U DAN and b57 hybridization probes find use in identifying wild-type and mutant alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. DAN and b57 nucleic acids are also used to modulate cellular expression or intracellular concentration or availability of active DAN or b57.
DAN and b57 inhibitory nucleic acids are typically antisense - single stranded sequences comprising complements of the disclosed natural b57 coding sequences. Antisense modulation of the expression of a given DAN or b57 protein may employ antisense nucleic acids operably linked to gene regulatory sequences. Cells are transfected with a vector comprising a DAN or b57 3U sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous DAN or b57 encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a given DAN or b57 protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein. An enhancement in DAN or b57 expression is effected by introducing into the targeted cell type DAN or b57 nucleic acids which increase the functional expression of the corresponding gene products.
1o Such nucleic acids may be DAN or b57 expression vectors, vectors which upregulate the functional expression of an endogenous allele, or replacement vectors for targeted correction of mutant alleles. Techniques for introducing the nucleic acids into viable cells are known in the art and include retroviral-based transfection, viral coat protein-liposome mediated transfection, etc.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of DAN or b57 modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate DAN or b57 interaction with a natural DAN or 2o b57 binding target. A wide variety of assays for binding agents are provided including protein-protein binding assays, immunoassays, cell based assays, etc.
Preferred methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds.
In vitro binding assays employ a mixture of components including a DAN or b57 protein, which may be part of a fusion product with another peptide or polypeptide, e.g., a tag for detection or anchoring, etc. The assay mixtures comprise a natural DAN or b57 binding target, e.g., a TGF~3 protein such as a BMP. While native binding targets may be used, it is frequently preferred to use portions thereof as long as the portion provides binding affinity and avidity to the subject DAN or b57 conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds, preferably small organic compounds, and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A
variety of other reagents such as salts, buffers, neutral proteins, e.g., albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may also be included. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. The mixture is incubated under to conditions whereby, but for the presence of the candidate pharmacological agent, the DAN or b57 specifically binds the cellular binding target, portion or analog with a reference binding affinity. Incubation periods are chosen for optimal binding but are also minimized to facilitate rapid, high throughput screening.
After incubation, the agent-biased binding between the DAN or b57 and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation, immobilization, etc., 2o followed by washing by, e.g., membrane filtration or gel chromatography.
For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc., or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label anci other assay components, e.g., through optical or electron density, radiative emissions, nonradiative energy transfers, or indirectly detected with antibody conjugates, etc. A difference in the binding affinity of the DAN or b57 protein to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the DAN or b57protein to the corresponding binding target. A difference, as used herein, is statistically significant and preferably represents at least a 50°/«, more preferably at least a 90°ra difference.
The invention provides for a method for modifying the physiology of a cell comprising an extracellular surface in contact with a medium, said method comprising the step of contacting said medium with an exogenous DAN or b57 protein under conditions whereby said protein specifically interacts with at least one of a component of said medium and said extracellular surface to effect a change in the physiology of said cell.
The invention further provides for a method for screening for biologically active agents, said method comprising the steps of a) incubating a DAN or b57 protein in the presence of an extracellular DAN or b57 protein specific binding target and a candidate agent, under conditions whereby, but for the presence of said agent, said protein specifically binds said binding target at a reference affinity; b) detecting the binding affinity of said protein to said binding target to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that said agent modulates the binding of said protein to said binding target.
2o One embodiment of the invention is an isolated b57 protein comprising the amino acid sequence as set forth in SEQ NO. 2 or a fragment thereof having b57-specific activity.
Another embodiment of the invention is a recombinant nucleic acid encoding b57 protein comprising the amino acid sequence as set forth in SEQ NO. 2 or a fragment thereof having b57- specific activity.
Still another embodiment is an isolated nucleic acid comprising a nucleotide sequence as set forth in SEQ NO. 1 or a fragment thereof having at least 18 consecutive bases of SEQ NO. 1 and sufficient,to specifically hybridize with a nucleic acid having the sequence of SEQ NO. 1 in the presence of natural DAN
and cerberus cDNA.

_. ____ _ _ _ _- _____ The present invention also provides for antibodies to the b57 protein described herein which are useful for detection of the protein in, for example, diagnostic applications. For preparation of monoclonal antibodies directed toward this b57 protein, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the 1o EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc.
pp.
77-96) and the like are within the scope of the present invention.
The monoclonal antibodies for diagnostic or therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (~ Teng et al., 1983, Proc. Natl. Acad.
Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79;
Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be 2o prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).
Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of the b57 protein described herein. For the production of antibody, various host animals can be immunized by injection with the b57 protein, or a fragment or derivative thereof, including but not limited to rabbits, mice and rats. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
A molecular clone of an antibody to a selected b57 protein epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.
io The present invention provides for antibody molecules as well as fragments of such antibody molecules. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')., fragment which can bc:
produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab'), fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Antibody molecules may be purified by known techniques, ~, immunoabsorption or immunoaffinity 2o chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.
The invention further provides for a method of using a DAN or b57 protein or fragment thereof as an antagonist of the activity of a bone morphogenic protein (BMP). Preferably, the invention provides for a method of antagonizing the function of a Bone Morphogenic Protein (BMP) which comprises contacting said BMP with b57. The method of the invention is carried out under conditions whereby the b57 binds to the BMP. In a preferred embodiment of the invention, the b57 is mammalian b57, and more preferably, human b57, or a 3o fragment thereof capable of binding to the BMP. In further preferred embodiments of the invention, the human b57 is used to antagonize the _ _ _--~ __ 1 __ _.... T _ function of BMP2 or BMP4.
Antagonists to BMP's may be useful for preventing and treating BMP-related disorders of animals, especially of humans. It was, therefore, an object of this invention to identify substances which effectively antagonize the function of BMP's in disease states in animals, preferably mammals, especially in humans.
It was another object of this invention to prepare novel compounds which inhibit BMP. It was still another object of this invention to develop a method of antagonizing the functions of BMP's in disease states in mammals. It was 1o also an object of this invention to develop a method of preventing or treating disorders relating to the function of BMP's.
In addition to their roles in normal bone formation, the BMPs appear to be involved in diseases in which they promote abnormal bone growth. For example, BMPs have been reported to play a causative role in the disease known as Fibrodysplasia Ossificans Progressiva (FOP), in which patients grow an abnormal "second skeleton" that prevents any movement.
Therefore, an object of the present invention is to provide a novel molecule for 2o the treatment of diseases or disorders including, but not limited to, Fibrodysplasia Ossificans Progressiva (FOP). Since b57 is a Mocker of BMP's, it offers hope as a therapeutic agent for this disease. Additionally, abnormal bone growth can occur after hip replacement surgery and thus ruin the surgical outcome. This is a more common example of pathological bone growth and a situation in which Mockers of BMP's such as b57 may be therapeutically useful.
b57 may be useful as well as for treating other forms of abnormal bone growth, such as the pathological growth of bone following trauma, burns or spinal cord injury. In addition, b57 may be useful for treating or preventing the undesirable actions of BMP's associated with the abnormal bone growth seen in 3o connection with metastatic prostate cancer or osteosarcoma.
In additional embodiments, the b57 nucleic acids, proteins, and peptides of the invention may be used to block BMP activity in mammals.
The present invention also provides for compositions comprising a b57 molecule, as described herein and a suitable carrier. The active ingredient, which may comprise the b57, should be formulated in a suitable carrier for systemic or local administration in vivo by any appropriate route including, but not limited to injection (e.~., intravenous, intraperitoneal, intramuscular, subcutaneous, endoneural, perineural, intraspinal, intraventricular, intravitreal, intrathecal etc.), by absorption through epithelial or 1o mucocutaneous linings (e.~., oral mucosa, rectal and intestinal mucosa, etc.); or by a sustained release implant, including a cellular or tissue implant.
Depending upon the mode of administration, the active ingredient may be formulated in a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms.
The concentration of the active ingredient used in the formulation will depend upon the effective dose required and the mode of administration used. The 2o dose used should be sufficient to achieve circulating plasma concentrations of active ingredient that are efficacious. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
In addition, the invention further provides for the use of yon Willebrand factor to regulate or modulate the activity of a' BMP. By aligning the carboxy-terminal domains of chicken (chB57), human (hB57) and Xenopus b57 (xB57), human and mouse DAN, mouse and Xenopus cerberus and human yon Willebrand factor (VWF), (see Table 1) applicants have discovered a striking homology among these various proteins, including the conservation of nine 3o separate cysteine residues. Given this striking homology, it is expected that yon Willebrand factor may also be useful for regulating or modulating the activity of a BMP.

_ T ___ t _ .. _._ T

The following protocol may be used for a high throughput human b57 - BMP
binding assay:
A. Reagents:
- Neutralite Avidin: 20 ug/ml in PBS.
Blocking buffer: 5'%'~ BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.
- Assay Buffer: 100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1'%
glycerol, 0.5% NP-40, 50 mM ~i-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.
- s3P human b57 protein lOx stock: 10-A - 10-6M "cold" human b57 supplemented with 200,000-250,000 cpm of labeled human b57 (Beckman counter). Place in the 4°C microfridge during screening.
- Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB #
109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB # 917575), and 2mM NaV03 (Sigma # S-6508) in 10 ml of PBS.
- BMP: 10-~ - 10-4M biotinylated BMP in PBS.
B. Preparation of assay plates:
- Coat with 120 ~l of stock N-Avidin per well overnight at 4°C.
- Wash 2 times with 200 ~1 PBS.
- Block with 150 Ill of blocking buffer.
- Wash 2 times with 200 Ill PBS.
C. Assay:
- Add 40 ftl assay buffer/well.
- Add 10 ul compound or extract - .Add 10 ftl ~~P- b57 protein (20-25,000 cpm/0.1-10 pmoles/well =10-'~- 10-~

M final conc).
- Shake at 25°C for 15 minutes.
- Incubate additional 45 minutes at 25°C.
- Add 40 ~tl biotinylated BMP (0.1-10 pmoles/40 ~! in assay buffer) - Incubate 1 hour at room temperature.
- Stop the reaction by washing 4 times with 200 ~l PBS.
- Add 150 ~tl scintillation cocktail.
- Count in Topcount.
to D. Controls for all assays (located on each plate):
i. Non-specific binding ii. Soluble (non-biotinylated b57} at 80"/° inhibition.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
1. Sequencing of Human b57 clone As stated previously, Xenopus cerberus sequence data was used as a probe to z5 search the EST database of the LM.A.G.E. consortium and human cDNA clone 272074 was discerned to contain homologous sequence. This clone ~n~as obtained from Genome Systems, Inc. (St. Louis, MO) and sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The nucleotide sequence encoding 3o human b57 is set forth as SEQ. NO. 1 and the deduced amino acid sequence of human b57 is set forth as SEQ. NO. 2.

T _ ___ _ T __- i SEQ. NO. 1 - Nucleotide Sequence encoding human b57 ATGAGCCGCA.CAGCTTACAC GGTGGGAGCC CTGCTTCTCC TCTTGGGGAC CCTGCTGCCG

GCTGCTGAAG GGAAAAAGAA AGGGTCCCAA GGTGCCATCC CCCCGCCAGA CAAGGCCCAG

CACAATGACT CAGAGCAGAC TCAGTCGCCC CAGCAGCCTG GCTCCAGGAA CCGGGGGCGG

GGCCAAGGGC GGGGCACTGC CATGCCCGGG GAGGAGGTGC TGGAGTCCAG CCAAGAGGCC

*
CTGCATGTGA CGGAGCGCAA ATACCTGAAG CGAGACTGGT GCAAAACCCA GCCGCTTAAG

CAGACCATCC ACGAGGAAGG CTGCAACAGT CGCACCATCA TCAACCGCTT CTGTTACGGC

* * * * *
CAGTGCAACT CTTTCTACAT CCCCAGGCAC ATCCGGAAGG AGGAAGGTTC CTTTCAGTCC

* *
TGCTCCTTCT GCAAGCCCAA GAAATTCACT ACCATGATGG TCACACTCAA CTGCCCTGAA

CTACAGCCAC CTACCAAGAA GAAGAGAGTC ACACGTGTGA AGCAGTGTCG TTGCATATCC

ATCGATTTGG ATTAR

SEQ. NO. 2 - Deduced Amino Acid Sequence of human b57 MetSerArgThrAlaTyrThrValGlyAla LeuLeuLeuLeuLeuGlyThrLeuLeuPro AlaAlaGluGlyLysLysLysGlySerGln GlyAlaIleProProProAspLysAlaGln HisAsnAspSerGluGlnThrGlnSerPro GlnGlnProGlySerArgAsnArgGlyArg GlyGlnGlyArgGlyThrAlaMetProGly GluGluValLeuGluSerSerGlnGluAla LeuHisValThrGluArgLysTyrLeuLys ArgAspTrpCysLysThrGlnProLeuLys GlnThrIleHisGluGluGlyCysAsnSer ArgThrIleIleAsnArgPheCysTyrGly GlnCysAsnSerPheTyrIleProArgHis IleArgLysGluGluGlySerPheGlnSer CysSerPheCysLysProLysLysPheThr ThrMetMetValThrLeuAsnCysProGlu LeuGlnProProThrLysLysLysArgVal ThrArgValLysGlnCysArgCysIleSer IleAspLeuAsp _._ __T T __- _ __ T

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Human b57 belongs to a family of proteins that includes cerberus (Bouwmeester et al., 1996) and DAN (Enomoto et al., 1994) - see Table 1. These b57 relatives have been postulated to function as antagonists for different members of the bone morphogenetic protein (BMP) family. The BMP family has many different members displaying varying degrees of homology to each other and it includes not only the BMPs, but also the growth differentiation factors (GDFs), transforming growth factor beta and its homologues (TGF(3s), the activins, the inhibins, the dorsalins, as well as nodal, vegetal, vegetal-related, and several new members (Furuta et ai., 1997). BMPs have been shown to play to important role in many different biological processes and thus the existence of naturally occurring antagonists of their activity is of great interest and pharmacological potential.
Other than the cerberus/b57/DAN family of BMP antagonists, several other antagonists to BMPs are known namely noggin (Smith and Harland, 1992;
Zimmerman et al., 1996), chordin (Piccolo et al., 1996), and follistatin (Hemmati-Brivanlou et al., 1994). These do not belong to the cerberus/b57/DAN family, and they also do not share any homology with each other. Nonetheless, noggin and chordin have been shown to bind to BMP2 and 2o BMP4 and inhibit their biological actions by blocking the interaction with the BMP receptors.
We describe here the expression of human b57 using mammalian expression systems and have generated, using standard laboratory techniques (See, e.g., z5 Molecular Cloning, A Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory), several tagged forms of hb57, such as hb57-Fc~Cl, hb57-FLAG, and hb57myc3. hb57 has also been expressed in E. cofi and refolded, and rabbit anti-b57 polyclonal antisera have been raised against it (Antisera # Q-1523-1 and Q-1523-2 prepared under contract by Quality Controlled Biochemicals, Inc., 3o Hopkiaton, Massachusetts, 01748 USA). Using recombinant hb57 proteins, we have tested hb57 for binding to hBMP2 and hBMP4, and the ability of noggin to __ t T _ I

antagonize this interaction. We have also tested the ability of hb57 to block the biological activity of hBMP2 in a cell-based assay.
2. Construction of hb57 expression plasmid pRG622 A DNA fragment encoding the gene for human b57 (hb57) was PCR amplified from an EST clone using the primers N1-hb57 (5'-GAGAGTCATGAAAAAGAAAGGGTCCCAAGGTGC-3') and io C1-hb57 (5'-GAGAGGCGGCCGCTCATTAATCCAAATCGATGGATATGCAAC-3'). The resulting 509 by fragment was digested with BspH 1 and Not 1 then ligated into the Nco 1-Not I sites of pRG536. A clone was identified and named pRG622, then transformed into E. coli strain RFJ143. The construct was confirmed by DNA sequence analysis.
3. Purification of hb57 E. coli strain RFJ143 containing pRG622 was grown in LB medium and 2o expression of hb57 was induced by the addition of 1 mM IPTG. Induced cells were collected by centrifugation, resuspended in 10 volumes of 100 mM Tris-HCI, pH 8.5, 20 mM EDTA, and lysed by passage through a Niro-Soave Panda cell disrupter. The cell lysate was centrifuged and the pellet was resuspended in 10 volumes of 9 M urea, 50 mM Tris-HCI, pH 8.5, 1 mM EDTA, 100 mM
z5 Na2S03, 10 mM Na2S4O6 and stirred for 16 hr at room temperature. The solubilized inclusion bodies were fractionated on a Sephacryl S-300 column equilibrated in 8 M urea, 20 mM MES, pH 6.0, 200 mM NaCI, 1 mM EDTA.
Fractions containing hb57 were pooled and diluted 10-fold into 1 M urea, 50 mM Tris-HCI, pH 8.0, 2 M NaCI, 0.1 mM EDTA, 0.5 mM cysteine. After 1-2 days 3o incubation at 4°C, the refolded hb57 was purified by reverse phase chromatography on a Jupiter C5 column. Properly refolded protein was eluted from the column by a 1.3"/«/min. gradient from 30'%'~ to 50'%« acetonitrile in 0.1'%'~

TFA. Fractions containing hb57 c~~ere pooled, dried under vacuum, and resuspended in 20 mM Tris-HCI, pH 8.0, 150 mM NaCI, 0.1 mM EDTA.
Demonstration That hb57 Binds to BMP2 and BMP4 In one example, hb57myc3 (1 ml of COS7-derived serum-free conditioned media) was co-incubated with hBMP2 (1 ~tg/ml) or hBMP4 (1 ~tg/ml) in the absence or in the presence of human noggin protein (hNG~B2, 10 llg/ml). The formation of a stable complex between hb57 and the BMPs was determined by 1o immunoprecipitating hb57 and associated proteins using an anti-myc monoclonal antibody (9E10; 1 ~g/ml) bound to Protein G-Sepharose beads (Pharmacia). The binding reaction was carried out in the serum-free conditioned media after it was made 20 mM Tris pl-~ 7.6, 150 mM NaCI, 0.1%
Tween 20 (TBST), 1 mg/ml bovine serum albumin (BSA), by addition of a 10x concentrate of these reagents. Binding was allowed to proceed for 1 hour, at 25°C, in a reaction volume of I.1 ml, with continuous mixing to keep the Protein G-Sepharose in suspension, after which point the beads were spun down, washed once with TBST, moved to new eppendorf tubes, and washed 3 more times with TBST. Proteins bound to the beads were solubilized by 2o addition of 25 ~l of Laemli SDS-PAGE sample buffer and loaded onto ~ to 12%
NuPAGE/MES gradient gels (Novex), which were run under reducing conditions. The proteins were subsequently transferred on Immobilon P and western blotted for the presence of BMP2 or BMP4 using polvclonal antisera raised against the respective proteins.
As can be seen in Figures lA-1B, hb57myc3 binds to both hBMP2 (Fig. lA, lane 1) and hBMP4 (figure 1B, lane 1) . This interaction appears to be stable in 1 M
NaCI (Fig. 1A, lane 2; and Fig. 1B, lane 2), although some reduction of binding is seen under those conditions. Addition of 10 ~g hNG completely blocks this so interaction (Fig. lA, lane 3; and Fig. 1B, lane 3), presumably by binding to hBMP2 or hBMP4 and blocking their ability to bind to hb57myc3. Further more, there was no binding of hBMP2 or hBMP4 to the beads if hb57myc3 was omitted from the reaction (Fig. 1A, lane 6; and Fig. 1B, lane 6), indicating that there is no non-specific binding of hBMP2 or hBMP4 to the beads and that the observed binding is hb57-dependent. It should be noted that identical results have been obtained using different tagged forms of hb57, and also using a different buffer system containing 20 mM Tris pH 7.6, 200 mM KCI, 0.1 '%. Nonidet P-40, 1 mg/ml bovine serum albumin (BSA). For comparison and as a positive control, hBMP2 and hBMP4 were also tested for their ability to bind to hNG~B2Fc (an Fc-tagged form of the hNG mutein hNGOB2}. Both hBMP2 and hBMP4 bound to hNGOB2Fc (Fig. lA, lane 4; and Fig. 1B, lane 4), in agreement to with results obtained previously (Zimmerman et al., 1996), and the interaction was blocked by the addition of untagged hNG (Fig. lA, lane 5; and Fig. 1B, lane 5). Taken together, these results indicate that the epitope recognized by hb57 on hBMP2 and hBMP4 is the same or overlaps with the epitope recognized by noggin, or alternatively that binding of noggin to BMP2 and BMP4 sterically hinders the binding of hb57.
5. Demonstration That hb57 Blocks BMP2 Biological Activity In another example, hb57 that had expressed in E. coli and refolded, was tested 2o for its ability to block the biological activity of hBMP2 in the C2C12 mouse pluripotent mesenchymal precursor cell line. The C2C12 cells have been shown to respond to BMP2 and BMP4 (Katagiri et al., 1994). One of the hallmarks of the response is upregulation of expression of Alkaline Phoshatase, the activity of which can easily be measured in cells or cell lysates using a colorimetric substrate. As shown in figure 2, C2C12 cells respond to hBMP2 with a maximal response obtained at 200 ng/ml hBMP2, and a minimal response obtained with ~10 ng/ml hBMP2, with an apparent EC50 of ~70 ng/ml.
The ability of hb57 to block this response was tested by co-incubating different amounts of hb57 (3 ~g/ml, or 1 ~g/ml, or 0.3 ~tg/ml) while performing a dose 3o response with hBMP2 starting at i ~g/ml. As can be seen in figure 2, inclusion of 3 ~tg/ml hb57 leads to complete blocking of the hBMP2 response when used at 0.5 ~g/ml. With hb57 at 1 ~g/ml, there is ~-50°/'> inhibition of the hBMP2 response when hBMP2 is at ~ 0.3 ug/ml and complete inhibition when hBMP2 is at 0.12 ~tg/ml. In order to make certain that the blocking of Alkaline Phosphatase induction in BMP2-stimulated C2C12 was not due to inhibition of their proliferation by b57, an identically-treated plate was subjected to an MTT
assay (Mosmann, 1983) which measures the proliferation of cells. There was no effect on cell proliferation by the hb57 treatment, indicating that inhibition of the Alkaline Phosphatase expression is due to blocking of hBMP2 activity in this assay. Thus, hb57 is a potent antagonist of BMP2 activity, and it appears to mediate this effect by directly binding to BMP2 and blocking its biological 1o actions. Taken together with the binding results, it is postulated that hb57 should also block the activity of hBMP4.
Xenopus b57 (Gremlin) was also examined for its ability to antagonize the activity of purified BMP-2 in a cytokine assay. The murine bone marrow stromal cell line W-20-17 provides a direct, quantitative bioassay for BMP
activity by induction of alkaline phosphatase in response to BMP treatment.
(Thies, et al. Endocrinology 130: 1318-24 (1992}). Preincubation of purified BMP-2 with a Gremlin COS supernatant at a final concentration of approx. 83nM
Gremlin completely blocked BMP-2 activity at doses from 78pM to 5nM. At approx. 2lnM Gremlin, BMP-2 activity was reduced, but not eliminated (see Figure 3). Mock-transfected COS supernatant had no effect. Similar results were obtained with BMP-4.
6. Tissue Expression of hb57 We have examined the expression of human b57 by analysis of RNA prepared from different adult human tissues. Table 2 lists the tissues tested and the level of expression of hb57 detected in these tissues. The expression of hb57 in so many different tissues indicates that it may play important biological roles.
One piece of evidence that supports this hypothesis is that the expression of drm (Topol et al., 1997), which is the rat homolog of hb57, is down-regulated in _ T _ _~

transformed cells. A similar observation has been made for DAN (Ozaki et al., 1995; Ozaki et al., 1996; Enomoto et al., 1994), which is related by homology to b57.
_ TABLE 2 - Tissue Expression of hb57 Tissue Relative Level of Expression heart very low brain medium placenta undetectable lung undetectable liver low skeletal muscle low kidney low ~5 pancreas low spleen undetectable thymus undetectable prostate low testis very low ovary very low small intestine high colon (mucosa lining) high peripheral blood leukocytes undetectable stomach high thyroid very low spinal chord medium lymph node high trachea low adrenal gland low bone marrow very low 7. Materials and Methods For Examples 2 through 6 a. Bindin assays:
Bindings were carried out in 20 mM Tris pH 7.6, 150 mM NaCI, 0.1'%. Tween 20 (TBST), 1 mg/ml bovine serum albumin (BSA}, at 25°C, with continuous mixing. Protein G-Sepharose {G-Se) was used to capture either the anti-myc 9E10 monoclonal antibody or the Fc-tagged hNGOB2Fc. Non-specifically bound proteins were removed from the beads by washing once with TBST, then moving the G-Se beads to new tubes and washing three more times with TBST.
1o Bound hBMP2 and hBMP4 were visualized by western blotting with anti-hBMP2 or anti-hBMP4 polyclonal antisera.
b. Anti-hBMP2 and anti-hBMP4 westerns:
The antibodies were derived from rabbits immunized with recombinant hBMP2 or recombinant hBMP4. The antibody preparation was total serum from the bled rabbits.
1. Block the filters in 5°/'> non-fat dry milk (NFDM) in 20 mM Tris pH
7.6, 150 mM NaCI, 0.1% Tween-20 (TBST) for 1 hour or more.
2. Probe with anti-BMP2 @ 1:20,000 or with the anti-hBMP4 antibody ~ 1:10,000 2o dilution in 2.5% NFDM/TBST for 1 hour.
3. Wash three times with TBST, 10 min. each time.
=I. Probe with the anti-rabbit IgG~HRPO conjugated 2° (Rockland, Inc.) at 50 ng/ml {1:20,000 dilution) in 2.5% NFDM/TBST for 1 hour.
5. Wash three times with TBST, 10 min. each time.
6. Wash three times with TBS (without Tween-20), 5 to 10 min. each time.
7. Perform ECL (Pierce).
c. C2C12 bioassa,~pro~ (adapted from (Katagiri et al., 1994)):
1. Seed C2C12 ~ 500 cells/well in 96-well plate, in DMEM + 15"/« FBS +
Pen/Str.
+ Glutamine. Important: Cells must be monodispersed during trypsinization and prior to plating. Clumps of cells will give erroneous results and variability in the response.

2. The following day add BMPs and other factors to each well.
Important points:
a. C2C12 will respond to BMP2 and to BMP4. If the cells are incubated with these factors for three days a maximal response can be obtained at 1 to 2 ug/ml.
A response above background is seen at 10 ng/ml. If the cells are incubated with the BMPs for 4 days, a response to as little as 1 ng/ml can be seen, and a maximal response is obtained at 300 ng/ml. No change of medium is required during the 4 day incubation period.
b. Incubation to both BMP2 and BMP4 together does not lead to an additive response above that expected for the equivalent amount of each factor alone.
c. Dilution of BMP2 or BMP4 must be made either directly into the assay media (as long as the concentration is kept below 10 ~tg/ml).
d. The response to BMP2 and BMP4 can be blocked by addition of an equimolar amount of human noggin.
3. Incubate for 3 to 4 days, depending on levels of BMPs used.
4. Aspirate media, wash once with PBS, add 0.05 ml of ddH20 per well. Freeze C~-20°C until next day or proceed to step 5.
5. Freeze-thaw three times to lyse the cells. A dry ice tray may be used for this.
6. Add 0.05 ml 2x Alk-Phos substrate/buffer mix (Sigma N2720).
7. Follow development - usually it takes about 40 minutes. Stop development by bringing the pH in each well to 14, by using a 50% w/v solution of NaOH.
8. Measure A405.
d. Northern Analysis:
I. Probe preparation:
1. Restrict 20 Bg of plasmid pCAE304 (pMT21.hb57) with NgoM I and Bgl II.
2. Gel-purify the 486 by fragment (about 2 ug), and elute in 100 ~1 ddH~O.
3. Label the probe using the Prime-It II Random Primer Labeling Kit (Stratagene), according to the following protocol:
3o a. Mix 4 Itl of the purified hb57 fragment + 20 ~l HBO + 10 ~tl random oligonucleotide primers in a tube.

WO 9.8/37195 PCT/US98103283 b. Heat the reaction tube in a boiling water bath for 5 minutes, then leave at room temperature.
c. Add 10 ~l of 5x dCTP primer buffer + 5 ~ti of [a-32P] dCTP + 1 ~l Exo(-) Klenow enzyme (5 u/~1) to the tube.
d. Incubate the tube at 37-40°C for 30 minutes.
=1. Purify the labeled probe with MicroSpin Column (Pharmacia Biotech).
II. Northern Blotting of Human Multiple Tissue Northern (MTN) Blots (CLONTECH):
1. Prehybridize the blots in 20 ml of PreHyb solution + ssDNA (10 ~l/ml) at 65°C
for more than 2 hours, using Hybridization oven.
2. Denature the labeled probe at 95°C for 5 minutes, immediately put the tube on ice.
3. Add the probe directly to the PreHyb solution, hybridize the blots at 65°C, overnight.
4. Wash the blots twice with 100 ml of 2x SSC + 0.1'% SDS at 65°C, 1 hour each time.
5. Expose the blots on film.
e. References:
Bouwmeester, T., Kim, S. H., Sasai, Y., Lu, B., and De, R. E. M. (1996).
Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature 382, 595-601.
Enomoto, H., Ozaki, T., Takahashi, E., Nomura, N., Tabata, S., Takahashi, H., Ohnuma, N., Tanabe, M., Iwai, J., Yoshida, H., and et, a. (1994).
Identification of human DAN gene, mapping to the putative neuroblastoma tumor suppressor locus. Oncogene 9, 2785-91.
3o Furuta, Y., Piston, D. W., and Hogan, B. L. (1997). Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development I?~, 2203-12.
Hemmati-Brivanlou, A., Kelly, O. G., and Melton, D. A. (1994). Follistatin, an antagonist of.activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 77, 283-95.
Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda, T. {1994). Bone 1o morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128(4):following 713]. Journal of Cell Biology 127, 1755-66.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival:
15 application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63.
Ozaki, T., Ma, J., Takenaga, K., and Sakiyama, S. (1996). Cloning of mouse DAN
cDNA and its down-regulation in transformed cells. Japanese Journal of Cancer 20 Research 87, 58-61.
Ozaki, T., Nakamura, Y., Enomoto, H., Hirose, M., and Sakiyama, S. (1995).
Overexpression of DAN gene product in normal rat fibroblasts causes a retardation of the entry into the S phase. Cancer Research 55, 895-900.
Piccolo, S., Sasai, Y., Lu, B., and De, R. E. M. (1996). Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of chordin to BMP-4.
Cell 86, 589-598.
3o Smith, W. C., and Harland, R. M. (1992). Expression cloning of noggin a new dorsalizing factor localized to the spemann organizer in xenopus embryos. Cell 70, 829-840.

Topol, L. Z., Marx, M., Laugier, D., Bogdanova, N. N., Boubnov, N. V., Clausen, P. A., Calothy, G., and Blair, D. G. (1997). Identification of drm, a novel gene whose expression is suppressed in transformed cells and which can inhibit growth of normal but not transformed cells in culture. Molecular & Cellular Biology 17, 4801-10.
Zimmerman, L. B., Jesus, E. J. M. D., and Harland, R. M. (1996). The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4.
to Cell 86, 599-606.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be ~5 readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (31)

-33-

1. An isolated nucleic acid molecule encoding mammalian b57.

2. An isolated nucleic acid molecule according to claim 1, having a sequence selected from the group consisting of:
(a) the nucleotide sequence comprising the coding region of human b57 as set forth in SEQ ID NO. 1;
(b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) and which encodes a molecule having biological activity to antagonise the bone morphogenic proteins BMP2 and BMP4; or (c) a nucleotide sequence which, but for the degeneracy of the genetic code would hybridize to a nucleotide sequence of (a) or (b), and which encodes a molecule having biological activity to antagonise the bone morphogenic proteins BMP2 and BMP4.

3. A vector which comprises a nucleic acid molecule of claim 1 or 2.

4. A vector according to claim 3, wherein the nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a host cell.

5. A vector according to claim 3 or 4, which is a plasmid.

6. An isolated protein encoded by a nucleic acid according to claim 1 or 2.

7. Isolated human b57 protein, having the amino acid sequence as set forth in SEQ NO. 2 or a fragment thereof having biological activity to antagonise the bone morphogenic proteins BMP2 and BMP4.

8. A host-vector system which comprises a vector according to claim 3 or 4, in a host cell.

9. A host-vector system according to claim 8, wherein the host cell is a bacterial, yeast, insect or mammalian cell.

10. A method of producing a protein according to claim 6 or 7 which comprises growing cells of a host-vector system of claim 8 or 9, under conditions permitting production of the protein, and recovering the protein so produced.

11. A nucleic acid probe or primer capable of specifically hybrising under stringent conditions to a nucleic acid according to claim 1 or 2.

12. An antibody which specifically binds the protein of claim 6 or 7.

13. An antibody according to claim 12 which is a monoclonal antibody.

14. An antibody according to claim 12 which is a polyclonal antibody.

15. A pharmaceutical composition comprising a protein according to claim 6 or 7, and a carrier.

16. A pharmaceutical composition comprising an antibody according to claim 12, 13 or 14 and a carrier.

17. A protein produced by the method of claim 10.

18. A ligandbody which comprises a protein according to claim 6 or 7 fused to an immunoglobulin constant region.

19. A ligandbody according to claim 18, wherein the immunoglobulin constant region is the Fc portion of human IgG1.

20. A protein according to claim 6 or 7, an antibody according to claim 12, 13 or 14, a pharmaceutical composition according to claim 15 or 16, or a ligandbody according to claim 18 or 19, for use in a method of treatment of the human or animal body, or in a method of diagnosis.

21. Use of a protein according to claim 6 or 7 in the manufacture of a medicament for use in a method of antagonizing the function of a Bone Morphogenic Protein (BMP).

22. Use according to claim 21 wherein the protein is human b57.

23. Use according to claim 21 or 22 wherein the BMP is BMP2 or BMP4.

24. Use according to any one of claims 21 to 23 for use in preventing or treating BMP-related disorders of animals.

25. Use according to claim 24 for treatment of a human.

26. Use according to claim 25 wherein the BMP-related disorder is abnormal bone growth.

27. Use according to claim 25 wherein the BMP-related disorder is Fibrodysplasia Ossificans Progressiva (FOP).

28. Use according to claim 25 wherein the abnormal bone growth occurs following hip replacement surgery.

29. Use according to claim 25 wherein the abnormal bone growth occurs following trauma, a burn or a spinal cord injury or in connection with metastatic prostate cancer or osteosarcoma.

30. A method of antagonising the function of a Bone Morphogenic Protein (BMP) which comprises contacting said BMP with a protein according to claim 6 or 7.

31. A method of antagonising the function of a Bone Morphogenic Protein (BMP) which comprises contacting said BMP with b57.