MXPA99009387A - Osteoprotegerin binding proteins and receptors - Google Patents

Abstract

A novel polypeptide, osteoprotegerin binding protein, involved in osteoclast maturation has been identified based upon its affinity for osteoprotegerin. Nucleic acid sequences encoding the polypeptide, or a fragment, analog or derivative thereof, vectors and host cells for production, methods of preparing osteoprotegerin binding protein, and binding assays are also described. Compositions and methods for the treatment of bone diseases such as osteoporosis, bone loss due to arthritis or metastasis, hypercalcemia, and Paget's disease are also provided. Receptors for osteoprotegerin binding proteins are also described. The receptors, and agonists and antagonists thereof, may be used to treat bone diseases.

Description

PROTEINS THAT JOIN OSTEOPROTEGERINE AND RECEPTORS THEREOF FIELD OF THE INVENTION The present invention relates to polypeptides which are involved in osteoclast differentiation. More particularly, the invention relates to proteins that bind to osteoprotegerin, nucleic acids encoding proteins, expression vectors and host cells for the production of proteins, and binding assays. Also described are compositions and methods for the treatment of bone diseases such as osteoporosis, bone loss by arthritis, Paget's disease and hypercalcemia. The invention also relates to receptors for proteins that bind to osteoprotegerin and to methods and compositions for the treatment of bone diseases using the receptors.

BACKGROUND OF THE INVENTION Living bone tissue shows a dynamic balance between bone deposition and desorption. These processes are mainly medicated by two types of cells: REF .: 31463 osteoblasts, which secrete molecules that comprise the organic matrix of bone; and osteoclasts, which promote the dissolution of the bone matrix and the solubilization of bone salts. In young individuals with growing bones, the rate of bone deposition exceeds the rate of bone resolution, while in older individuals, the rate of resorption may exceed the deposition. In this situation, an increased breakdown of bone leads to reduced bone mass and strength, which increases the risk of fractures and incomplete loss or repair of broken bones. Osteoclasts are phagocytic multinucleated cells which are formed from hematopoietic precursor cells in the bone marrow. Although the growth and formation of mature functional osteoclasts has not been well understood, it is considered that osteoclasts mature along the monocyte / macrophage cell line in response to exposure to various growth promoting factors. The early development of pre-bone marrow precursor cells to pre-osteoclasts is considered to be mediated by soluble factors such as "tumor necrosis factor-a (TNF-a), tumor necrosis factor-β (TNF-β), inteleukin-1 (IL-). 1), interleukin-4 (IL-4), interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) .In culture, preosteoclasts are based on presence of colony-stimulating factors. of macrophages that is added (-CSF). These factors act mainly in the early stages of osteoclast development. The relationship of polypeptide factors in the terminal stages of osteoclast formation has not been reported extensively. However, it has been reported that the parathyroid hormone stimulates the formation and activity of osteoclasts and that calcitonin has the opposite effect, although to a lesser extent. Recently, a new polypeptide factor, called osteoprogeterin (OPG), has been described which negatively regulates the formation of osteoclasts in vitro and in vivo (see the application of the co-pending United States and in co-ownership Nos. 08 / 577,788 presented. on December 22, 1995, 08 / 706,945 filed September 3, 1996 and 08 / 771,777 filed December 20, 1996, incorporated herein by reference, and PCT Application No. W096 / 26271). OPG markedly increases bone density in txansgenic mice expressing the OPG polypeptide and reduces the rate of bone loss when administered in ovarioctamized rats. An analysis of OPG activity in osteoclast formation in vitro shows that OPG does not interfere with the growth and differentiation of monoclonal / macrophage precursors, but that it is more likely to block osteoclast differentiation from small tube precursors / macrophage. Thus, OPG seems to present a specificity for the regulation of the degree of osteoclast formation. OPG comprises two polypeptide domains that have different structural and functional properties. The aminoterminal domain encompasses approximately residues 22-194 of the full-length polypeptide (N-terminal methionine is termed residue 1) and shows homology to other members of the tumor necrosis factor receptor (TNFR) family, especially with TNFR-2, through the conservation of cysteine-rich domains characteristic of the TNFR family. The carboxy terminal domain encompasses residues 194-401 and has no important homology with any known sequence. Unlike numerous additional members of the TNFR family, OPG appears to be exclusively a secreted protein and does not appear to be synthesized as a membrane-associated form. Based on its activity as a negative regulator of osteoclast formation, it is postulated that OPG can bind to a polypeptide factor involved in the differentiation of osteoclasts _and in this way block one or more terminal stages that_ lead to the formation of osteoclasts. mature. Therefore, an objective of the invention is to identify polypeptides which interact with OPG. Polypeptides can play a role in the maturation of osteoclasts and may be useful in the treatment of bone diseases.

BRIEF DESCRIPTION OF THE INVENTION A novel member of the tumor necrosis factor family expressed Jen COS cells analyzed using the OPG-Fc recombinant fusion protein as an affinity probe has been identified from a mouse cDNA library. The new Tpolipeptide is a transmembrane protein that binds to OPG 'which is predicted to be 316 amino acids in length, and has an aminoterminal cytoplasmic domain, transmembrane domain and a carboxy terminal extracellular domain. The OPG-binding proteins of the invention may be membrane-associated or may be in soluble form. The invention provides nucleic acid encoding a protein that binds OPG, vectors and host cells that express the polypeptide, and a method for producing recombinant protein that binds OPG. Antibodies or fragments thereof are also provided which bind specifically to the protein that binds OPG. Proteins that are zoned to OPG can be used in assays to quantify OPG concentrations in samples biological, identify cells and tissues that show protein that binds to OPG, and identify new members of OPG and family members of proteins that bind to OPG. Methods for identifying compounds which interact with protein that binds to OPG are also provided. Such compounds include nucleic acids, peptides, proteins, carbohydrates, lipids or low molecular weight organic molecules and can act as agonists or antagonists of the activity of the OPG binding protein. The proteins that bind to OPG are involved in the differentiation of osteoclasts and the level of activity of osteoclasts in turn modulates bone resorption. Protein agonists and antagonists that bind to OPG modulate osteoclast formation and bone resorption and can be used to treat bone diseases characterized by changes in bone resorption, such as osteoporosis, hypercalcemia, bone loss due to arthritis, metastasis, immobilization of periodental disease, Paget's disease, osteoporosis, prosthetic loss and the like. Pharmaceutical compositions comprising proteins that bind to OPG and protein agonists and antagonists that bind to OPG are also included within the invention. Receptors have also been identified for OPG-binding proteins from a mouse cDNA library constructed from bone marrow cells The proteins bind to a protein that binds to OPG, fluorescently labeled. The receptors can be used to identify agonists and antagonists of interactions with the protein that binds OPG with its receptor, which can be used to treat bone diseases.

DESCRIPTION OF THE FIGURES Figure 1. Structure and sequence of the protein that binds to OPG that codes for the insert 32D-F3. The predicted transmembrane domain and the sites for asparagine-linked carbohydrate chains are underlined. Figure 2. Expression of protein that binds to OPG in COS-7 cells transfected with pcDNA / 32D-F3. Cells are lipofected with pcDNA / 32D-F3 DNA, tested for binding to goat antibody conjugate against human IgGl and alkaline phosphatase (secondary only), OPG [22-201] -Fc human plus (OPG-Fc), secondary, or an extracellular FAC condominium fusion protein ATARTquimeric (sATAR-Fc). ATAR is a new member of the TNFR superfamily, and the sATAR-Fc fusion protein serves as a control both for the binding of the human IgGl Fc domain and for the generic TNFR related protein, which binds to 32D cell surface molecules.

Figure 3. Expression of the protein that binds OPG in human tissues. Northern blot analysis of human tissue mRNA (Clontech) using a radiolabeled 32D-F3 derived hybridization probe. The relative molecular mass is indicated on the left in pairs of kilobases (kb). The tip of the arrow on the right side indicates the migration of a transcript of approximately 2.5 kb detected in lymph node mRNA. A very light band of the same mass in fetal liver is also detected. Figure 4. Structure and sequence of the insert of cDNA / hu OPGpb 1.1 that codes for the human protein that binds to OPG. The predicted transmembrane domain and the site for, the chains of carbohydrates United to asparagine are underlined. Figure 5. Stimulation of osteoclast development in vitro from bone marrow macrophages and cocultures of ST2 cells treated with recombinant mouse protein that binds to OPG [158-316]. The cultures are treated with varying concentrations of mouse protein that binds to OPG ranging from 1.6 to 500 ng / ml. After 8-10 days, the cultures are lysed, and the TRAP activity is measured by solution assay. In addition, some cultures were treated simultaneously with 1, 10, 100, 500 and 1000 ng / ml of recombinant mouse OPG protein [22-401] -Fc. The mouse protein that binds to OPG induces a dose-dependent stimulation in osteoclast formation, while OPG [22-401] -Fc inhibits osteoclast formation. Figure 6. Stimulation of the development of osteoclasts from bone marrow precursors in vitro in the presence of M-CSF and mouse protein that binds to OPG [158-316]. Mouse bone marrow is harvested and cultured in the presence of 250, 500, 1000 and 2000 U / ml of M-CSF. The variable concentrations of OPG-binding protein [158-316], which vary from 1.6 to 500 ng / ml, are added to these same cultures. The development of osteoclasts is measured by TRAP solution assay. Figure 7. Osteoclasts derived from bone marrow cells in the presence of both M-CSF and protein that binds to OPG [158-316] resorb bone in vitro. Bone marrow cells treated either with M-CSF or with protein that binds to OPG, or with both factors combined, are seeded in plaques on bone plates in culture cells, and allowed to develop into mature osteoclasts. The resulting cultures are then stained with toluidine blue (left column), or histochemically to detect the enzymatic activity of TRAP (right column). In cultures that receive both factors, mature osteoclasts are formed that are capable of eroding the bone judging from the presence of blue-stained spots on the bone surface. This correlates with the presence of large and multiple multimucleated TRAP positive cells. Figure 8. Graph showing the total blood ionized calcium (iCa) concentrations of mice injected with the protein that binds to OPG, 51 hours after the first injection, and in mice that also receive concurrent administration with OPG. The protein that binds OPG significantly and dose-dependently increases iCa concentrations. OPG (1 mg / kg / day) completely blocks the increase in iCa at a dose of protein that binds to OPG of 5 μg / day, and partially blocks _ the increase at a dose of protein that binds to OPG of_25 μg / day day. (*), different from the control treated with vehicle (p <0.05). (#), Concentration of iCa treated with OPG that is significantly different from the level in mice receiving that dose of protein that binds to OPG alone (p <0.05). Figure 9. Radiographs of the left and tibia fermur in mice treated with 0, 5, 25, or 100 μg / day of protein that binds to OPG for 3.5 ~~ days. There is a dose-dependent decrease in bone density evident most clearly in metastasis of the proximal tibia of these mice, and that it is profound at a dose of 100 μg / day. Figure 10. Mouse ODAR cDNA sequence and protein sequence. The nucleic acid sequence of -2.1 kb of the cDNA clone is shown, and the translation of the Open reading frame of 625 residues long indicated above. The hydrophobic signal peptide is underlined, and the transmembrane hydrojphobic sequence (residues 214-234) is in bold. The cysteine residues that constitute the repeated cysteine-rich motifs in the extracellular domain are in bold. Figure 11. Immunofluorescent staining of ODAR-Fc that binds to the protein that binds to OPG in transfected cells. COS-7 cells transfected with the expression plasmid are incubated for the protein that binds to OPG with IgG Fc (upper panel), ODAR-FcT (middle panel) or OPG-Fc (lower panel). A goat antibody against human IgG Fc, labeled with FITC, is used as a secondary antibody. The positive binding cells are examined by confocal microscopy. Figure 12. Effects of ODAR-Fc on the generation of osteoclasts of mouse bone marrow in vitro. Mouse bone marrow cultures are established as in Example 8 and exposed to protein that binds OPG (5 ng / ml) and CSF-1 (30 ng / ml). Various concentrations of ODAR-Fc ranging from 1500 ng / ml to 65 ng / ml are added. The formation of osteoclasts is determined by TRAP cytometry and the TRAP solution test after 5 days_ in culture. Figure 13. Density of bone mineral in mice after treatment for 4 days with ODAR-Fc at variable doses. The mice received ODAR-Fc for injection daily subcutaneous in a saline vehicle buffered with phosphate. The mineral density was determined from bones fixed in ETOH 70% in the proximal tibial metaphysis of mice by peripheral quantitative computed tomography (pQCT) (XCT-960M, Norland Medical Systems, Ft Atkinson, Wl). Two cross sections of 0.5 mm bone, 1.5 mm and 2.0 mm were analyzed from the proximal end of the tibia (XMICE 5.2, Stratec, Germany) to determine total bone mineral density in the metaphysis. A woven separation threshold was used: _soft of 1500 to define the bone limit in metaphysis. 0DAR-Fc produced a significant increase in bone mineral density in the proximal tibial metaphysis in a dose-dependent manner. Group n = 4. - DETAILED DESCRIPTION OF THE INVENTION The invention provides a polypeptide that is referred to as a protein that binds to OPG, which binds specifically to OPG and is involved in the differentiation of osteoclasts. A cDNA clone encoding the murine form of the polypeptide is identified from a library prepared from the 32D line of mouse myelomonocytic cells and which is transfected into COS cells. The transfectants are analyzed to determine their capacity to bind to an OPG fusion polypeptide [22-201] -Fc (example 1). The nucleic acid sequence reveals that the OPG-binding protein is a novel member of the TNF family and is closely related to AGP-1, a polypeptide previously described in the co-pending and co-owned US application. series 08 / 660,562, filed June 7, 1996. (A polypeptide identical to AGP-1 and designated TRAIL is described in Wiley et al., Immufiity 3, 673-682 (1995).) The binding protein is predicted to be a OPG is a type II transmembrane protein that has a site-plasmatic domain in the amino-terminal part, a transmembrane domain, and a carboxy-terminal extracellular domain (see Figure 1). The amino terminal-site plasmatic domain encompasses approximately residues 1-48, the Transmembrane domain covers approximately residues 49-69 and the extracellular domain encompasses approximately residues 70-316, "as shown in Figure 1 (SEQ ID NO: 2) .Membrane associated protein binds specifically to OPG (see Figure 2). Therefore, the protein that binds to OPG and OPG share many characteristics of a receptor-ligand pair, although it is possible that there are other receptors that occur naturally for the protein that binds to OPG. - The DNA clone encoding the human protein that binds to OPG is isolated from a cDNA library of lymphatic node The human sequence (see Figure 4) is homologous to the murine sequence.The purified soluble murine ~ OPG binding protein stimulates the formation of osteoclasts in vitro and induces hypercalcemia and bone resorption in vivo. "OPG" refers to a polypeptide having a protein amino acid sequence that binds mammalian OPG, or a fragment, analogue, or derivative thereof and having at least the activity of unir_OPG. , the protein that binds OPG is of murine or human origin In another embodiment, the protein that binds OPG is a soluble protein that has, in one form, an isolated extracellular domain separated from the transmembrane cytoplasmic domains. - OPG is involved in the differentiation of osteoclasts and in the speed and degree of bone resorption, and is found to stimulate the formation of osteoclasts and stimulate bone resorption.

Nucleic acids The invention provides isolated nucleic acids coding for OPG binding proteins. As used herein, the term "nucleic acid" comprises cDNA, genomic DNA, total or partially synthetic DNA and RNA.

The nucleic acids of the invention are selected from the group consisting of: a) the nucleic acids shown in Figure 1 (SEQ ID NO: 1) and Figure 4 (SEQ ID NO. 3); b) Nucleic acids which hybridize to the polypeptide coding regions of the nucleic acids shown in Figure 1 (SEQ ID NO: 1) and Figure 4 (SEQ ID NO: "3) and remain hybridized to nucleic acids under conditions of high restriction, and c) nucleic acids which degenerate to nucleic acids of (a) or (b). "Nucleic acid hybridizations typically involve a multi-step process comprising a first step of hybridization to form double strands of nucleic acid from single strands followed by a second step of hybridization that is carried out under more stringent conditions to selectively retain the double strands of nucleic acid having the desired homology. The conditions of the first hybridization step are generally not crucial, with the proviso that they do not have a greater restriction than the second hybridization step. Generally, the second stage of hybridization was carried out under conditions of high restriction, where the "conditions of" high restriction "refers to conditions of temperature and saline concentration which approximately 12-20 ° C lower than the melting temperature (Tra) of a perfect hybrid or part or all of the compelling chains corresponding to Figure 1 (SEQ ID NO: 2) and Figure 4 (SEQ ID NO: 4) In one embodiment, the conditions of "high restriction" refer to conditions of approximately 65 ° C and not greater than about 1 M Na ". It is understood that the salt concentration, the temperature and / or the incubation duration can vary in any of the first or second hybridization step so that one obtains the hybridizing nucleic acid molecules according to the invention. Conditions for nucleic acid hybridization and T ^ calculations for nucleic acid hybrids are described in Sambrook et al. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, New York (1989). The nucleic acids of the invention can hybridize to part or all of the polypeptide coding regions of the OPG-binding protein, as shown in Figure 1_ (SEQ ID NO: 2) and Figure 4 (FIG. SEQ ID NO: 4); and therefore may be truncated sequences or extensions of the nucleic acid sequences shown herein. The truncated or extended nucleic acids which are encompassed by the invention with the condition that they retain at least the property that they join OPG. In one embodiment, the nucleic acid will encode zzun polypeptide of at least about 10 amino acids. In another embodiment, the nucleic acid will encode for a polypeptide of at least about 20 amino acids. In another additional embodiment, the nucleic acid will code for a polypeptide of at least about 50 amino acids. Hybridizing nucleic acids may also include non-coding sequences that are located 5 'and / or 3' to the coding regions of the OPG-binding protein. The non-coding sequences include regulatory regions involved in expression of the OPG binding protein, such as promoters, elongation regions, reductive initiation sites, transcription termination sites and the like. In preferred embodiments, the nucleic acids of the invention encode a protein that binds to mouse or human OPG. Nucleic acids can encode a membrane-bound form of protein that binds to OPG or soluble forms which lack a functional transmembrane region. The predicted transmembrane region for the protein that binds murine OPG includes amino acid residues 49-69 inclusive, as shown in Figure 1 (SEQ ID NO: 1). The transmembrane region predicted for the protein that binds to human OPG includes the residues 49-69, as shown in Figure 4 (SEQ ID NO: 3). Substitutions which substitute for hydrophobic amino acid residues in this region with neutral or hydrophilic amino acid residues are expected to disrupt the membrane association "and result in a soluble protein that binds to OPG, in addition, the supersions of part or all of from the transmembrane region are also expected to produce soluble forms of OPG-binding protein.Nucleic acids encoding Iso amino acid residues 70-316 as shown in "Figure 1 (SEQ ID NO: 1) or the fragments and analogs thereof, encompass the soluble proteins that bind to OPG. Also included are nucleic acids encoding truncated human soluble proteins that bind to OPG. Soluble forms include residues 69-317 as shown in Figure 4 (SEQ ID NO: 3) and truncated forms thereof. In one embodiment, the N terminal truncated forms generate polypeptides from residues 70-317, 71-317 and 72-317 and so on, in another embodiment, nucleic acids encode soluble OPGbp comprising residues 69 ^ 317 and residues. truncated N-terminal forms thereof to OPGbp [158-317], or alternatively to OPGbp [T66-317]. The plasmid phuOPGbp 1.1 in E. coli strain DH10 that codes for the human protein that binds to OPG was deposited before the American Type Culture Collection, Rockville, MD on June 13, 1997. The nucleic acid sequences of the invention can be used for the detection of sequences that encode "the protein that binds to OPG in biological samples. The sequences can be used to analyze cDNAs and genomic libraries for sequences related to the protein that binds to OPG, especially from other species.Nucleic acids are also useful for modulating the levels of protein that bind OPG through Antisense technology or gene expression in vivo The development of transgenic animals expressing the OPG-binding protein is useful for the production of the polypeptide and for the study of biological activity in vivo.

Vectors and Guest Cells The nucleic acids of the invention will be linked with DNA sequences so as to express the biologically active OPG binding protein. The sequences necessary for expression are known to those familiar with the art and include promoters and lengthening sequences for initiation of RNA synthesis, transcription termination sites, ribosome-binding sites for the initiation of protein and sequence synthesis leader for secretion.

The sequences that direct secretion expressions of the OPG-binding protein can be homologous, ie, the sequences are identical or similar to those sequences in the genome that are involved in the expression and secretion of the protein that binds to OPG, or they can be heterologous. Various plasmid vectors are available for expression of the OPG-binding protein in host cells (see for example, Methods in Enzymology v. 185, Goeddel D.V., ed., Academic Press (1990)). For expression in mammalian host cells, a preferred embodiment is the pDSRa plasmid described in PCT Application No. 90/14363. For "expression in bacterial host cells, preferred embodiments include plasmids harboring the lux promoter (see co-pending and co-owned US application No." of series 08 / 577,778, filed December 22, 1995). In addition, vectors are available for tissue-specific expression of the OPG-binding protein in transgenic animals. Gene transfer vectors based on retroviruses and adenoviruses can also be used for the expression of the OPG binding protein in human cells for in vivo therapy (see PCT Application No. 86/00922). Prokaryotic and eukaryotic host cells expressing the protein that binds OPG are also provided by the invention. The host cells include bacterial, yeast, vegetable, insect or mammalian cells. The protein that binds to OPG can also be produced in transgenic animals such as mice or goats. Plasmids and vectors containing the nucleic acids of. invention are introduced into appropriate host cells using known transfection or transformation techniques by those familiar in the art. The host cells may contain DNA sequences encoding the protein that binds OPG as shown in Figure 1, or with a portion thereof, such as the extracellular domain or the siteplasmic domain. The nucleic acids encoding the OPG-binding proteins can be modified by codon substitution which allows optimal expression in a given host. At least part of the codons may be codons of the so-called preferential ones which do not alter the amino acid sequence and are frequently found in genes that are expressed in large measure.However, codon alterations for Optimizing expression are not restricted to the introduction of codons of preference. Examples of preferred mammalian host cells for expression of the OPG-binding protein include, but are not limited to COS, CHOd-, 293 and 3T3 cells. A preferred bacterial host cell is Escherichia coli. .- Polypeptides The invention also provides a protein that binds to OPG as the prokaryotic or eukaryotic expression product of an "exogenous DNA" sequence, i.e., protein that binds to OPG which is a recombinant protein that binds to "OPG. Exogenous DNA sequences include cDNA, genomic DNA and synthetic DNA sequences. The protein that binds to OPG can be the product of expression in bacterial, yeast, plant, insect or mammalian cells, or cell-free translation systems. The protein that binds to OPG produced in bacterial cells will have an N-terminal methionine residue. The invention also provides a process for making OPG-binding protein comprising growing prokaryotic or eukaryotic host cells transformed or transfected with nucleic acids encoding the OPG-binding protein and isolating polypeptide expression products from nucleic acids. . The invention encompasses polypeptides which are mammalian proteins that "bind OPG or fragments, analogs or derivatives thereof In a preferred embodiment, the protein that binds OPG is a human protein that binds OPG. A protein fragment that binds to OPG refers to a polypeptide having a deletion of one or more amino acids so as to result in a polypeptide having at least the property of binding to OPG. Such fragments will have deletions that originate from the amino terminal end, the carboxy terminus and the internal regions of the polypeptide. The protein fragments that bind to OPG are of at least about 10 amino acids, at least about 20 amino acids, or at least about 50 amino acids in length. In preferred embodiments, the OPG-binding protein will have a deletion of one or more amino acids from the transmembrane region (amino acid residues 49-69 as shown in FIG. 1), or alternatively, one or more amino acids from the amino terminal part and / o that includes the transmembrane region (amino acid residues 1-49 as shown in Figure 1). In another embodiment, the protein that binds to OPG is a soluble protein comprising, for example, amino acid residues 69-316 or 70-316, or the N terminal or C terminal truncated forms thereof, which retain activity that is unites OPG. The protein that binds to OPG is also a soluble human protein, as shown in Figure 4, which comprises residues 69-317 as shown in Figure 4 and the forms truncated in the N-terminal part thereof, by Examples 70-517, 71-517, 71-317, 72-317 and so on, in a preferred embodiment, the soluble human protein that binds to OPG comprises residues 69-317 in the section truncated N terminal thereof to OPGbp [158-317], or alternatively to OPG [166-317]. An analogue of a protein linking OPG is related to a polypeptide having a substitution or addition of one or more = F amino acids so that the resulting polypeptide has at least the property of binding OPG. Such analogs will have substitutions or additions anywhere along the polypeptide. Preferred analogs include those of soluble proteins that bind OPG. The fragments or analogs may be presented naturally, for example as a polypeptide product of an allelic variant or a splice variant of mRNA or they may be constructed using available techniques, for a person familiar with the "technique for manipulating and synthesizing acids". The polypeptides may or may not have an amino-terminal methionine residue. Also included in the invention are derivatives of OPG-binding protein which are polypeptides that have undergone post-translational modifications (eg, addition of N-linked or 0-linked carbohydrates, N-terminal or C-terminal processing), binding of chemical portions to the main amino acid structure, chemical modifications of N-linked or O-linked carbohydrate chains, and addition of a N-terminal methymn residue as a result of the expression of the prokaryotic host cell. In particular, chemically modified derivatives of the OPG binding protein which provide additional advantages such as increased stability, longer circulation time or decreased immunogenicity are contemplated. Of particular use is modification with water soluble polymers, such as polyethylene glycol and derivatives thereof (see for example U.S. Patent No. 4,179,337). The chemical portions for derivatization "(formation of derivatives) can be selected from water-soluble polymers such as polyethylene glycol, and ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. Polypeptides can be modified at random positions within the molecule, or at predetermined positions within the molecule and can include 1, 2, 3, or more chemically linked portions Polypeptides can also be modified at predetermined positions in the polypeptide, such as the amino terminal part, or in a lysine residue or argenine selects within the polypeptide Other chemical modifications provided include a detectable label, such as an enzymatic, fluorescent, isotopic or affinity tag to allow the detection and isolation of the protein.

OPG-binding protein chimeras comprising part or all of an amino acid sequence of a protein that binds OPG fused to a heterologous amino acid sequence are also included. The heterologous sequence can be any sequence which allows the resulting fusion protein to retain at least the activity that binds to OPG. In a preferred embodiment, the extracellular carboxy terminal domain of the OPG ^ binding protein is fused to a heterologous sequence. Such sequences include heterologous plasmatic domains that allow for alternative intracellular signaling events, which sequence promotes oligomerization such as the Fc region of IgG, sequence of enzymes which provide a tag for the polypeptide and sequence which provide affinity probes, such as an "antigen-antibody recognition." The polypeptides of the invention are isolated and purified from tissues and cell lines which express the OPT binding protein, either extracted from lysates or from conditioned growth media, and host cells. Transformed expressing protein that binds to OPG - Protein that binds to OPG can be obtained from murine myelomonocyte 32D cell line (ATCC accession no CRL-11346) The protein that binds to OPG, or nucleic acids which code for it, can be isolated from human lymph node or from fetal liver tissue. The protein that binds OPG isolated is free to associate with human proteins and other cellular constituents! A method for the purification of protein that binds OPG from natural sources (eg, tissues and cell lines which normally express the OPG-binding protein) and from transfected host cells is also encompassed by the invention . The purification process can use one or more standard protein purification steps in an appropriate order to obtain the purified protein "Chromatography steps" can include ion exchange, geJL filtration, hydrophobic interaction, - reverse phase, chromatofocusing, affinity chromatography using a protein antibody that binds against OPG or a biotin-streptavidin affinity complex and the like.

Antibodies Antibodies that bind specifically to polypeptides of the invention are also encompassed by the invention. Antibodies can be reduced by immunization with the full-length protein that binds to OPG ", or soluble forms of the protein that binds to OPG, or a fragment of them. The antibodies of the invention can be polyclonal or monoclonal, or they can be recombinant antibodies such as chimeric antibodies wherein the murine constant regions on the light and heavy chains are replaced by human sequences, or antibodies inserted with CDR where only the regions of determination of complementarity are of murine origin. The antibodies of the invention can also be human antibodies prepared, for example, by immunization of transgenic animals capable of producing human antibodies (see, for example, PCT Application No. W093 / 12227). The antibodies are useful for detecting the protein that binds OPG in biological samples, thus allowing the identification of cells or tissues which produce the protein. In addition, the antibodies which bind to the protein that binds OPG and block the interaction with other binding compounds may have therapeutic use in the of Osteoclast Differentiation and Osseous Resorption "7" Antibodies to the OPG-binding protein may be useful in the treatment of bone diseases such as osteoporosis and Paget's disease. The antibodies can be tested for binding to the protein that binds to OPG in the absence or presence of .OPG and can be examined for their ability to inhibit osteoclastogenesis and / or Bone resorption mediated by ligands (protein that binds to OPG). It is also anticipated that the peptides themselves may act as an antagonist of the ligand: receptor interaction and inhibit ligand-mediated osteoclastogenesis, and the peptides of the OPG-binding protein will also be explored for this purpose.

Compositions "The invention also provides pharmaceutical compositions comprising a therapeutically effective amount of the OPG-binding protein of the invention together with a pharmaceutically acceptable carrier, solubilizer, emulsifier, preservative and / or adjuvant. The invention also provides pharmaceutical compositions comprising a therapeutically effective amount of a OPG agonist or antagonist binding protein. The term "therapeutically effective amount" means an amount which provides a therapeutic effect for a specified condition and route of administration. The compositions may be in liquid or lyophilized form and comprise a diluent (tris, acetate or phosphate buffer) having various pH values and ionic strengths, solubilizers such as Tween. or polysorbate, carriers such as human serum alumina or gelatin, preservatives such as thimerosal or Benzyl alcohol and antioxidants such as ascorbic acid or sodium metabisulfite. The selection of the particular composition will depend on numerous factors, including the condition to be treated and the route of administration and the desired pharmacokinetic parameters. A more extensive review of the components suitable for pharmaceutical compositions is found in Reington's Pharmaceutical Sciences, 18th of. A.R. Gennaro, ed. Mack, Easton, PA (1980.). ' T. In a preferred embodiment, compositions comprising soluble proteins that bind to OPG are also provided. Also encompassed are compositions comprising soluble protein that binds to-OPG modified with water-soluble polymers to increase solubility, stability, plasma half-life and bioavailability. The compositions may also comprise the incorporation of soluble protein which is linked to OPG in liposomes, microemulsions, miscelas or vesicles for controlled delivery over a prolonged period of time. It can be formulated to the soluble protein that binds to OPG in microparticles suitable for pulmonary administration. The compositions of the invention can be administered by injection, either "subcutaneously, intravenously or intramuscularly, or by oral, nasal, pulmonary or rectal administration." The route of administration will finally be chosen and it will depend on many factors which can be determined by a person familiar with the art. The invention also provides pharmaceutical compositions comprising a therapeutically effective amount of the nucleic acids of the invention together with a pharmaceutically acceptable adjuvant. The nucleic acid compositions will be suitable for the delivery of part or the. whole of the coding region of the OPG binding protein and / or flanking regions to cells and tissues as part of an "antisense" therapy regimen.

Methods of Use The OPG-binding proteins can be used in various assays to detect OPG and to characterize interactions with OPG. In general, the assay comprises incubating its protein that binds OPG with a biological sample containing OPG under conditions which allow the binding of OPG to the protein that binds to OPG, and measure the extent of binding, OPG can be purified, or it occurs in mixtures, for example as in body fluids or culture media. ~ Tests can be developed which are qualitative or quantitative, the latter are useful for determining the binding parameters (affinity and kinetic constants) of OPG to prostein that binds to OPG and to quantify the levels of Biologically active OPG and mixtures. The assays can also be used to evaluate the binding of OPG to fragments, analogs and protein derivatives that bind to OPG, and to identify new members z of the OPG family and of proteins that bind to OPG. The binding of OPG to the OPG binding protein can be carried in various formats, including cell-based assays, membrane binding assays, solution phase assays and immunoassays. traces of OPG labeled with protein samples that were stained to OPG for a specified period of time followed by measurement of OPG bound by filtration, electrochemiluminescent assays (ECL, system originating by IGEN) or cell-based immunoassays. for radioactivity (SPA, Amersham) and time-separated fluorescence (HTRF, Packard) can also be implemented.The binding is detected by labeling OPG or an antibody against OPG with radioactive isotopes (1251, 35S, 3H), fluorescent dyes (fluorosein) ), lanthanides (E3 +), chelates or cryptates, orbipyridyl-ruthenium complexes (Ru2 +). It is understood that the choice of a labeled probe will depend on the detection system used. Alternatively, OPG can be modified with an unlabeled epitope tag (eg, biotin, peptides, His6, myc) and protein binding such as streptavidin, antibodies against peptide or against protein which have a detectable label as described above. In an alternative method, the OPG-binding protein can be tested directly using polyclonal or monoclonal antibodies to proteins that bind to OPG in one assay. Additional forms of OPG-binding proteins that contain epitope tags as described above can be used in solutions and immunoassays. Methods for identifying compounds which interact with the OPG-binding protein are also encompassed by the invention. The method comprises incubating OPG-binding protein with a compound under conditions which allow the binding of the compound to OPG-binding protein and "mend the extent of binding." The compound can be substantially purified or it can be present in a untreated mixture The binding compounds can be nucleic acids, proteins, peptides, carbohydrates, lipids or small molecular weight organic compounds The compounds can also be characterized by their ability to increase or decrease the activity of the protein that binds OPG in order to determine whether they act as an agonist or an antagonist The proteins that bind to OPG are also useful for the identification of intracellular zproteins which interact with the plasmatic domain by a Two-hybrid yeast analysis process. As an example, hybrid constructs comprising DNA encoding the fifty amino acids in the N-terminal part of an OPG binding protein fused to a yeast GAL4 DNA binding domain can be used as a plasmid of two hybrid primers. The positive clones that arise from the analysis can also be characterized to identify the proteins that interact. This information can help elucidate an intracellular signaling mechanism associated with the protein that binds OPG and provides intracellular targets for new drugs that modulate bone resorption. The protein that binds zOPG can be used to treat conditions characterized by excessive bone density. The most common condition is osteopetrosis in which a genetic defect results in a high and usually deadly bone mass in the first years of life. Osteopetrosis is preferably treated by administration of soluble protein that binds to OPG. The invention also encompasses modulators (agonists and antagonists) of OPG binding protein and the method to obtain it.A modulator for OPG binding protein can increase or decrease at least one activity associated with OPG binding protein, such as ability to bind OPG or some other molecule that interacts or to regulate the Osteoclast maturation. Typically, an agonist or antagonist can be a cofactor, such as a protein, peptide, carbohydrate, lipid or small molecular weight molecule, which interacts with the OPG binding protein to regulate its activity. Potential polypeptide antagonists include antibodies which react with either the soluble or membrane-bound forms of the OPG-binding protein, and the soluble forms of OPG-binding protein which comprise part or all of the extracellular protein domain. The molecules that regulate the expression of the protein that binds OPG typically include nucleic acids -which are complementary to nucleic acids that encode- for OPG-binding protein and which act as antisense regulators of expression. u e- OPG is involved in the control of the formation of mature osteoclasts, the type of primary cell involved in bone resorption.An increase in the rate of bone resorption (with respect to bone formation) can lead to various bone disorders called collectively as osteopenia and include osteoporosis, osteomyelitis, hypercalcemia, osteopenia that was performed by surgery or administration of steroids, Paget's disease, osteonecrosis, bone loss due to rheumatoid arthritis, periodental bone loss, immobilization, prosthetic loss and metastasis-zosteolitic. Conversely, a decrease in the rate of bone resorption can lead to osteopretosis, a condition marked by excessive bone density. The protein agonists and antagonists that bind to OPG -modulate the formation of osteoclasts and can be administered to patients suffering from bone disorders. The OPG protein agonists and antagonists used for the treatment of osteopenia can be administered alone or in combination with a therapeutically effective amount of a bone growth promoting agent and including bone morphogenic factors called BMP-1 through BMP-12, factor of transforming growth ß and members of the TGF-β family, fibroblast growth factors FGF-1 to FGF-1"0, interleukin 1 inhibitors, TNFa inhibitors, parathyroid hormone, prostaglandin E series, bisphosphonates and mineral quef The bone agonists such as fluoride "ro" and calcium are agonists of the OPG-binding proteins can be particularly useful in the treatment of osteopenia.

Receptors for Proteins that bind Osteoproteserin The invention also provides receptors which interact with OPG binding proteins. More particularly, the invention provides a receiver for osteoclast differentiation and activation (ODAR). ODAR is a transmembrane polypeptide. which shows the highest degree of homology for CD40", a member of the TNF receptor family.The frequency of murine ODAR nucleic acids and the modified peptide is shown in Figure 10. The human homologue of murine ODAR can be easily isolate by hybridization analysis of human cDNA or a genomic library with the nucleic acid concentration of Figure 10. Methods for cloning human ODAR are similar to those "described in Example 5 for cloning human OPG-binding proteins. The human homolog of the polypeptide shown in "FIG. 10 has appeared in Anderson et al., (Nature 390, 175-179 (1997)) and is referred to herein as" RANK. RANK is characterized as a type I transmembrane protein that has homology to members of the TNF receptor family and is involved in dendritic cell function. In example 13 we have evidence for the interaction of ODAR and the protein that binds to OPG. A soluble form of ODAR (ODAR-Fc fusion protein) prevents osteoclast modulation in zyitro (see Figure 12) and increases "bone density in normal mice after subcutaneous injection (see Figure 13). The results are consistent with the protein that binds to OPG that interacts with and that activates ODAR to promote the maturation of osteoclasts. The development of osteoclasts and the speed and extent of bone resorption are regulated by the interaction of the protein that binds to OPG and ODAR. Compounds which decrease or block the interaction of the protein that binds to OPG and ODAR are potential antagonists of the activity of the protein that binds to OPG and can interrupt the development of osteoclasts leading to decreased bone resorption. Alternatively, the compounds which increase the interaction of the binding protein - OPG and ODAR are potential agonists which promote the development of osteoclasts and improve bone resorption. Various assays can be used to measure the interaction of the protein that binds to OPG and ODAR in vitro using purified proteins. These assays can be used to analyze compounds for their ability to increase or decrease the elicit or extent that ODAR binds to the protein that binds to OPG.In one type of assay, the ODAR protein can be immobilized by binding to the bottom of the wells of another microtitre plate The protein that binds OPG, radiolabeled (for example protein that binds to iodinated OPG) and the test compound or compounds can be added either one at a time (in any order) or simultaneously to the wells. After incubation, the wells can be washed and counted using a scintillation counter for radioactivity, in order to determine the extesion that binds to ODAR for the protein that binds to OPG in the presence of the test compound. Typically, the compound will be tested over a range of concentrations, and a series of control wells lacking one or more elements of the test assays can be used for precision in the evaluation of the results. An alternative to this method involves inverting the "positions" of the proteins, i.e., immobilizing the protein that binds OPG to the wells of the microtitre plates, incubating with the radiolabeled ODAR and test compound, and determining the extent of the protein. ODAR binding (see, for example, chapter 18 of Current Protocols in Molecular Biology, Ausubel et al., Eds., - John Wiley &Sons, New York, NY. [1995]). As an alternative to radiolabelling, the protein that binds to OPG or ODAR can be conjugated to biotin and then the presence of biotinylated protein can be detected using streptavidin bound to an enzyme, for example horseradish peroxidase [HRP] or alkaline phosphatase [ AP], which can be detected colorimetrically or by streptavidin fluorescent labeling. An antibody directed to protein that binds to OPG or ODAR that is conjugated to biotin and can be detected after incubation with enzyme bound streptavidin, ~~ bound to AP or HRP. The protein that is cured to OPG and ODAR can also be immobilized by attachment to sucrose spheres, acrylic spheres or other types of such inert substrates. The substrate-protein complex can be placed in a solution containing the complementary protein and the test compound; After incubation, the spheres can be precipitated by centrifugation, and the amount of binding between the protein that "binds to OPG and ODAR using the methods described above can be determined.Alternatively, the substrate-protein complex can be immobilized in a column and the test molecule and the complementary protein are passed over the column.The formation of a complex between the protein that binds OPG and ODAR can then be determined using any of the techniques set forth above, ie radiolabelling, Binding of antibodies or the like Another type of in vitro assay that is useful for identifying a compound which increases or decreases the formation of an ODAR / protein complex that binds to OPG is a surface plasmon resonance detector system such as "the Biacore assay system (Pharmacia, Piscataway, NJ). The Biacre system can be carried out using the manufacturer's protocol. This assay essentially involves the covalent attachment of both the protein that binds to OPG and of ODAR to a sensor chip coated with dextran which is located in a detector. The test compound and the other complementary protein can be injected into the chamber containing the sensor chip either simultaneously or sequentially and the amount of complementary protein that binds based on the change in the molecular mass which can be determined can be determined. it is physically associated with the dextran coated side of the sensor chip, the change in molecular mass can be measured by the detector system In some cases, it may be desirable to evaluate two or more test compounds together for use in the increasing formation or In these cases, the assays set forth above can be easily modified by adding such a compound or such additional test compounds, either simultaneously or subsequently to the first test compound. The rest of the steps in the test are as stated in the above.In vitro tests such as those described above can be used advantageously It is important to quickly analyze large amounts of compounds to determine their effects on the formation of complexes by ODAR and OPG-binding protein. The assays can be automated to analyze compounds generated in phage display, synthetic peptides and chemical synthesis libraries.

Compounds which increase or decrease the protein complex formation that binds to OPG and ODAR can also be analyzed in cell culture using cells and cell lines having ODAR. Cells and cell lines can be obtained from any mammal, but preferably from human or other primate, canine or rodent sources. ODAR-containing cells such as osteoclasts can be enriched from other cell types by "affinity chromatography using publicly available products." The binding of the protein that binds OPG to cells that exhibit ODAR is evaluated in the presence or absence of the test compounds and the binding extension 7 is determined, for example, by cytometry of. flow using a biotinylated antibody to a protein that binds OPG. Alternatively, a culture of human or mouse osteoclasts can be established as described in example 8 and the test compounds can be evaluated to determine their. ability to block the maturation of osteoclasts stimulated by the addition of CSF-1 and protein that binds to OPG. The cell culture assays can be advantageously used to further evaluate compounds that provide a positive score in the protein binding assays described above. The compounds which increase or decrease the interaction of the protein that binds OPG with ODAR also they can be evaluated for their in vivo activity by administering the compounds to mice followed by bone density measurements - using bone scan or radiography densitometry. "Methods for measuring bone density are described in PCT publication W097 / 23614 and in Example 13. The invention provides compounds which diminish or block the interaction of the OPG-ODAR-binding protein and are antagonists of osteoclast formation Such compounds are generally found in two groups. which are derived from the protein that binds to OPG or which interact with the protein that_ binds to OPG.These have been • described in the above.A second group includes those compounds which are derived from ODAR or which interact with ODAR Examples of compounds which are ODAR antagonists include nucleic acids, proteins, peptides, carbohydrates idratos, lipids or organic compounds of small molecular weight. The "ODAR antagonists can compounds which bind at or near one or more binding sites for OPGbp in the extracellular ODAR domain and decrease or completely block complex formation." Those regions in ODAR that are involved The formation of complexes with the protein that binds to OPG can be identified by analogy with the structure of the TNFβ / TNF-R55 homologous complex which has been described in Banner et al., (Cell, 73., 431-445 (1993")) For example, the structure of the TNFβ / TNF-R55 complex It can be used to identify regions of the protein that is cloned to OPG and ODAR that are involved in complex formation.The compounds can then be designed which bind preferentially to the regions involved in the formation of the complex and act as antagonists. In an approach set forth in Example 11, peptide antigens are designed for use in the generation of antibodies against the protein that binds to OPG and which act as antagonists.These antibodies are expected to bind to the protein that is bind OPG and block complex formation with ODAR In a similar approach, peptide antigens based on ODAR structure can be used to generate antibodies against ODAR that act as antagonists. ODAR antagonists can also bind to ODAR at positions other than the binding site for OPGbp and include conformational changes in the ODAR polypeptide resulting in decreased or nonproductive complex formation with the I OPG binding proteins. In one embodiment, an antagonist is a soluble form of ODAR that lacks the functional transmembrane domain. The soluble ODAR forms can have a deletion of one or more amino acids in the transmembrane domain (amino acids 214-234, as shown in Figure 10). The soluble ODAR polypeptides may have part or all of the extracellular domain and be capable of binding protein that binds to OPG. Optionally, soluble ODAR can be part of a chimeric protein where part or all of the extracellular domain of ODAR is fused to a heterologous amino acid sequence. In one embodiment, the heterologous amino acid sequence is an Fc region from human IgG. Modulators (agonists and antagonists) of ODAR can be used to prevent or treat osteopenia, which includes osteoporisis, osteomyelitis, malignant hypercalcemia, osteopenia that comes from surgery or administration of spheroids, Paget's disease, osteonecrosis, bone loss due to rheumatoid arthritis , periodontal bone loss, immobilization, prosthetic loss and osteolytic metastasis. The ODAR agonists and antagonists used for the treatment of osteopenias can be administered alone or in combination with a therapeutically effective amount of a bone growth promoting agent that includes bone morphogenic factors termed BMP-1 through BMP-12, transforming growth factor β and the members of the TGF-β family, fibroblast growth factors FGF-1 to FGF-10, inhibitors of interleukin-1 inhibitors of TNF, hormone parathyroid, prostaglandins ~ of the E series, bisphosphonates, estrogens, SERM and minerals -bone enhancers such as fluoride and calcium. "ODAR antagonists are particularly useful in the treatment of osteopenia." The following examples are provided to more fully illustrate the invention, but are not constructed as limiting the scope thereof.

Example 1 Identification of a Fuenté de Líneas Celular for a Protein that binds to OPG Osteoprotegerin - (OPG) negatively regulates genesis in vitro and in vivo. Since OPG is a protein related to TNFR, it is likely that it interacts with a family member related to TNF and at the same time mediates its effects. With one exception, all known members of the TNF superfamily are transmembrane type II proteins expressed on the cell surface. To identify a source for protein that binds to OPG, recombinant OPG-Fc fusion proteins are used as immunobands to perform an analysis to determine OPG-binding proteins located on the surface of various cell lines and primary hematopoietic cells. Cell lines growing as in vitro adherent cultures are treated using the following methods: the cells are seeded in plates, in 24-well tissue culture plates (Falcon), and then allowed to grow to approximately 80% confluence . The growth medium is then removed, and the adherent cultures are washed with phosphate-buffered saline (PBS) (Gibco) containing 1% fetal bovine serum (FCS). OPG fusion proteins [22-194] -Fc of mouse and recombinant human OPG [22-201] -Fc (see US Serial No. 08 / 706,945, filed September 3, 1996) are individually diluted to 5 μg / ml in PBS containing FCS 1%, then added to the cultures and allowed to cultivate for 45 min at 0 ° C. The OPG-Fc fusion protein solution is discarded and the cells are washed in PBS-FCS solution as described above. The cultures are then exposed to goat secondary antibody F (ab ') against human IgG, conjugated to phycoerythrin (Southern Biotechnology Associates Cat. # 2043-09) diluted in PBS-FCS After an incubation of 30-45 min at 0 C, the solution is discarded and the cultures are washed as described above. zan by immunofluorescent microscopy to detect cell lines which express on the cell surface protein that binds OPG. __ The cell suspension cultures are analyzed in a similar way with the following modifications: the diluent and the wash buffer consist of phosphate buffered saline "free of calcium and magnesium containing 1% FCS." The cells are harvested from cultures exponentially duplicated in growth medium, sedimented by centrifugation and then resuspended at 1 X 101 cells / ml in a 96-well microtiter tissue culture dish (Falcon) .The cells are sequentially exposed to recombinant OPG-Fc fusion proteins, and then to the secondary antibody that has been described above, and the "cells are washed by centrifugation between each step. Subsequently the cells are analyzed by fluorescence activated cell plasticization (FACS) using a FACscan Becton Dickinson. By using this approach, it is found that the 32D line of murine myelomonocytic cells (ATCC-access CRL-11346) expresses a surface molecule which can be detected with both OPG [22-194] -Fc mouse like. OPG [22-201] -Fc human. The secondary antibody alone does not bind to the surface of 32D cells nor to purified human IgGl Fc, indicating that the binding of the OPG-Fc fusion proteins is due to the OPG portion. This binding can compete in a dose-dependent manner by the addition of murine or human recombinant OPG protein [22-401]. Therefore, the OPG region necessary for biological activity is capable of specifically binding to a surface molecule derived from 32D.

Example 2 Expression Cloning of a Murine Protein that Joins OPG A cDNA library is prepared from 32D mRNA and ligated into the mammalian expression vector pcDNA3.1 (+) (Invitrogen, San Diego, CA). 32D cells are grown exponentially in the presence of recombinant interleukin-3 and harvested, and the total cellular RNA is purified by guanidinium thiocyanate acid-phenol-chloroform thiocyanate (Chomczynski and Sacchi, Anal. Biochem. 162, 156-159 (1987)). The poly (A +) mRNA fraction is obtained from the preparation of total RNA by adsorption to, and elution from, Dynabeads Oligo (dT) 25 (Dynal Corp) using the procedures recommended by the manufacturer. A directional oligo-dT primed cDNA library is prepared using the SuperScript Plasmid system (Gibco BRL, Gaithersburg, Md) using the recommended procedures by the manufacturer. the resulting cDNA is digested to completion with the restriction endonucleases Sal I and Not I, and then fractionated by size exclusion gel chromatography. The highest molecular weight fractions are selected and then ligated into the polylinker region of plasmid vector pcDNA3.1 (+) (Invitrogen, San Diego, CA). This vector contains the CMV promoter towards, the 5 'end of the multiple cloning site, and directs the elevated expression level in "eukaryotic cells." The library is then electroporated into competent E. coli (ElectroMAX DH10B, Gibco, NY) and it is titrated on LB agar containing 100 μg / ml ampicillin.The library is then placed in segregated pools containing approximately 1000.clones / accumulated and cultures of 1.0 ml of each accumulated are grown for 16-20 h at 37 ° C. The plasmid DNA of each culture is prepared using the Qiagen Qiawell 96 Ultra Plasmid Kit (catalog # 16191) following the procedures recommended by the manufacturer. The accumulated pools of the 32D cDNA expression library are individually lipofected in COS-7 cultures, and then assays are performed for the acquisition of a protein that binds to OPG on the cell surface. To do this, COS-7 cells are seeded at a density of 1 X 106 per ml in six-well tissue culture plates (Costar) and then cultured the night in DMEM (Gibco) containing FCS 10%. Approximately 2 μg of plasmid DNA from each well are diluted in 0.5 ml of serum-free DMEM, then sterilized by centrifugation through a "! Spin-X 0.2 μm (Costar) column." Simultaneously, 10 μl of Lipofectamine (Life) is added. Technologies Cat # 18324-012) a_ a separate tube containing 0.5 ml of serum-free DMEM The DNA and Lipofectamine solutions are mixed and allowed to incubate at room temperature for 30 min COS-7 cell cultures after they are washed with serum-free DMEM, and the DNA-Lipofectamine complexes are exposed to the cultures for 2-5 h "at 37 ° C. After this period, the medium is removed, and replaced with DMEM containing 10% FCS. The cells are then cultured for 48_h at 37 ° C. To detect cultures expressing a protein that binds to OPG, the growth medium is removed, and the cells are washed with PBS-FCS solution. To each well is added 1.0 ml by volume of PBS-FCS containing 5 μg / ml of the human OPG fusion protein [22-201] -Fc and incubated at RT for 1 h. The cells were lev ¥ n three times with a solution of PBS-FCS and then fixed in PBS containing 2% formaldehyde and 0.2% glutaraldehyde in PBS at RT for 5 min. The cultures are washed once with PBS-FCS, and then incubated for 1 h at 65 ° C while immersing in a PBS-FCS solution. The crops are allowed to cool and the PBS-FCS solution is aspirated. The cultures are then incubated with a goat antibody against human IgG (specific for Fc) conjugated with alkaline phosphatase (SIGMA product # A-9544) at RT for 30 min., and then washed 3 times with 20 mM Tris-Cl (pH 7.6_) and 137 mM NaCl. The immune complexes that are formed during these steps are detected by assay for alkaline phosphatase activity using the Fast Red TR / AS-MX Substrate kit (Pierce, Cat. # 34034) following the procedures recommended by the manufacturer. __ Using this approach, a total of approximately 3007,000 independent 32D cDNA clones are analyzed, represented by 300 transfected accumulations of 10.00 clones each. A unique well is identified that contains cells which acquire the ability to be decorated specifically by the OPG-Fc fusion protein. This .. accumulated is subdivided by sequential rounds of sib selection, which provides a single plasmid clone 32D-F3 (see Figure 1). The plasmid DNA of 32D-F3 is then transfected into COS-7 cells, which are immunostained with goat-only secondary antibody against human IgG, conjugated with FITC, the OPG fusion protein [22-201] -Fc human plus secondary antibody or with the fusion protein ATAR-Fc (ATAR is also known as HVEM; Montgomery et al., Cell 82, 427-436 (1996)) (see Figure 2). Secondary antibody only does not bind to COS-7 / 32D-F3 cells, nor the fusion protein ATAR-Fc. Only, the OPG Fc fusion protein bound to the COS-7 / 32D-F3 cells indicates that 32D-F3 codes for an OPG-binding protein displayed on the surface of cells expressing them.

Example 3 Sequence of Protein that joins OPG The previously isolated 32D-F3 clone contains a cDNA insert of approximately 2.3 kb (see Figure 1), which is sequenced in both directions in an Applied Biosystems 373A automated DNA sequencer using primer-driven Taq dye terminator reactions. (Applied Biosystems) following the procedures recommended by the manufacturer. The resulting nucleotide sequence obtained is compared to the DNA sequence database using the FASTA program (GCG, University of Wisconsin) "and analyzed for the presence of long open reading frames (LORF) using the application" Six-way open reading frame "(Fra is) (GCG, University of Wisconsin). An LORF of 316 amino acid residues (aa) is detected beginning in methionine, in the appropriate orientation, and is preceded by a 5 'region. translated from approximately 150 bp. The 5 'untranslated region contains a codon of detection in frame towards the 5' end of the predicted start codon. This indicates that the structure of plasmid 32D-F3 is consistent with its ability to utilize the CMV promoter region to direct the expression of a 316 aa product in mammalian cells. The predicted protein sequence that binds to OPG is then compared to the existing database in known protein sequences using a modified version of the FASTA program "(Pearson, Meth., Enzymol, 183, 63-98" (1990)). The amino acid sequences are also analyzed for the presence of specific motifs conserved in all known members of the tumor necrosis factor (TNF) superfamily using the sequence profile method of (Gribskov et al., Proc. Nati. Acad. Sci., USA 83, 4355-4359 (1987)), modified by Luethy et al., Protein Sci. 3., 139-146 (1994)). There seems to be significant homology through the protein that binds OPG with several members of the TNF superfamily. The mouse protein that binds to OPG appears to be more closely related to the mouse and human homologs of TRAIL and the CD40 ligand. An additional analysis of the sequence of the protein that binds to OPG indicates a strong coincidence with the TNF superfamily, with a highly significant Z score of 19.46.

The amino acid sequence in the protein that binds to OPG contains a transmembrane hydrophobic domain likely starting in M49 and extending to L69. Based on - in this configuration relative to the methionine start codon, the OPG-binding protein is predicted to be a transmembrane type II protein, with a short N-terminal intracellular domain and a larger C-terminal extracellular domain (cf. figure 4). This would be similar to all known TNF family members, with the exception of alpha lymphotoxin (Nagata and Golstein, Science 267, * 1449-1456 (1995)).

Example 4 Expression of mRNA for Human Protein that joins OPG Northern blots of human multiple tissue (Clontech, Palo Alto, CA) were probed with a 32D-F3 restriction fragment labeled with 32 P-dCTP to detect the size of the human transcript and to determine expression patterns. Northern blots were prehybridized in 5X SSPE, 5% formamide, Denhart 5X solution, 0.5% SDS and 100 μg / ml denatured salmon sperm DNA for 2-4 h at 42 ° C. The spots are then hybridized in 5X SSPE, formamide 50%, Denhart 2X solution, SDS 0.1%, 100 μg / ml of denatured salmon sperm DNA and 5 ng / ml of labeled probe for 18-24 h at 42 ° C. The spots are then washed in 2X SSC for 10 min at RT, SSC IX for 10 ^ min at 50 ° C and then in 0.5X SSC for 10-15 min. Using a probe derived from mouse cDNA and hybridization under restriction conditions, lymph nodes are detected as a predominant mRNA species with a relative molecular mass of approximately 2.5 kb (see figure "3) .Also a light signal is detected at the same relative molecular mass in fetal hepatic mRNA. protein that binds to OPG in the other tissues examined.The data suggest that the expression of mRNA for the protein that binds to OPG is markedly restricted in human tissues.The data also indicate that the isolated cDNA clone is very close to the size of the native transcript, suggesting that 32D-F3 is a full-length clone.

Example 5 Molecular Cloning of Human Protein that Joins OPG The human homologue of the protein that binds to OPG is expressed as an mRNA of approximately 2.5 kb in human peripheral lymph nodes and is detected by hybridization with "a mouse cDNA probe under restricted hybridization conditions." The DNA encoding the human protein that binds to OPG is obtained by analysis of the human lymph node cDNA library either by a "bacteriophage" plate. recombinant or by a transformed bacterial colony, and with hybridization methods (Sambrook et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press, New York (1989)). For this the phage or the plasmid cDNA library is analyzed using radioactively labeled probes derived from the murine protein that binds to OPG, clone 32D-F3. The probes are used to analyze high nitrocellulose filters from a library sown in plates. These filters are prehybridized and then hybridized using conditions specified in Example 4, which finally results in purified clones of human cDNA for the protein that binds to OPG. The "insertions that are obtained from any of the human protein clones that bind to OPG can be sequenced and analyzed as described in" Example 3. _ A poly (A +) RNA is analyzed from human lymph node (Clontech, Inc. ., Palo. Alta, CA) to determine the presence of transcripts of _.OPG-bp as previously indicated in the document of the United States no. series 08 / 577,788, filed on December 22, 1995. A Northern blot of one. sample of this probed RNA low. Restriction conditions with a 32P tagged OPG-bp probe indicates the presence of human OPG-bp transcripts. A cDNA library primed with olive dT is then synthesized from lymph node mRNA using SuperScript equipment (GIBCO Ufe technologies, Gaithersberg, MD) as described in example 2. The resulting cDNA is selected in terms of size and High molecular fraction is ligated to the plasmid vector pcDNA3.1 (+) (Invitrogen, San Diego, CA). DH10 electrocompetent E. coli (GIBCO life technologies, Gaithersberg, MD) is transformed and the "transformants resistant to 1 X 10d of ampicillin are analyzed by colony hybridization using a mouse protein probe that binds to" OPG labeled with 2P . A plasmid clone of human protein cDNA that binds to putative OPG, phuOPGbp-1.1, is isolated and contains an insert of 2.3 kp. The resulting nucleotide sequence of the phuOPGbp-1.1 insert is approximately 80-85% homologous to the protein cDNA sequence that binds to mouse OPG. Translation of the insert DNA indicates the presence of a large open reading frame predicted to encode a 317 aa polypeptide (see Figure 4). The comparison of the OPG-bp polypeptides of mouse and human shows that they are -87% identical, which indicates that this protein is highly conserved during evolution.

The DNA of the human protein that binds to OPG and the protein sequences are not present in Genbank, and there are no homologous EST sequences. As with the murine homologue, the human protein that binds to OPG shows strong sequence similarity to all members of the TNFOI superfamily of cytokines.

Example 6 Cloning and bacterial expression of the protein that binds to OPG PCR amplification using the primer pairs and templates described below is used to generate various forms of murine proteins that bind to OPG. One of the primers of each pair introduces a TAA stop codon and a unique Xhol or SacII site after the carboxy terminal part of the gene. The other primer of each pair introduces a unique Ndel site, an N-terminal methionine, and codons optimized for the amino terminal portion of the gene. PCR and thermocycling are carried out using standard recombinant DNA methodology. The PCR products are purified, digested by restriction, and inserted into the unique Ndel and Xhol or SacII sites of the pAMG21 vector (ATCC accession No. 98113) and transformed into prototype 3, 33 or 339 E. coli. Others Commonly used E. coli expression vectors and host cells are also suitable for expression. After transformation, the clones are selected, the plasmid DNA is isolated and the sequence of the OPG-binding protein insert is confirmed. pAMG21-murine protein that binds to OPG 1 * 75-3161 This construction is engineered to have a length of 242 amino acids and have the following N terminal and C terminal residues, NH2-Met (75) -Asp-Pro-Asn-Arg-Gln-Asp-Iile-Asp (316) - COOH. The template to be used for PCR is cDNA / 32D-F3 and oligonucleotides # 1581-72 and # 1581-76 where the primer pair to be used for PCR and cloning of this gene construct. 1581-72: 5 '-GTTCTCCTCATATGGATCCAAACCGTATTTCTGAAGACAGCACTCACTGCTTT-3' (SEQ ID NO: 5) 1581-76: "- 5 '-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID NO: 6) pAMG21-pro murine eine that binds to OPG I "95-3161 This construction is engineered to have a length of 223 amino acids and has the following N terminal and C terminal residues, NH, -Met-His (95) -Glu-Asn-Ala-Gly Gln-Asp-Ile-Asp ( 316) -COOH. The template used for "PCR is cDNA / 32D-F3 and oligonucleotides # 1591-90 and # 1591-95 where the primer pair- used for PCR and cloning of this gene construct. 1591-90: 5 '-ATTTGATTCTAGAAGGAGGAATAACATATGCATGAAAACGCAGGTCTGCAG-3' (SEQ ID NO: 7) 1591-95: 5 '-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3' (SEQ ID NO: 8) pAMG21-murine protein that binds to OPG 1 * 107-3161 This construction is engineered to be 211 amino acids in length and have the following N-terminal and C-terminal residues, NH2-Met-Ser (107) -Glu-Asp-Thr-Leu -Gln-Asp-Ile-Asp (316) -COOH. The template used for PCR is cDNA / 32D-F3 and oligonucleotides # 1591-93 and # 1591-95 where the primer pair used for PCR and cloning of this gene construct. 1591-93: 5 '-ATTTGATTCTAGAAGGAGGAATAACATATGTCTGAAGACACTCTGCCGGACTCC-3' (SEQ ID NO: 9) 1591-95: 5 '-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3' (SEQ ID NO: 10) pAMG21-murine protein that binds to OPG "" 118-3161 - This construction is "engineered to be 199 amino acids in length and have the following N-terminal and C-terminal residues NH2-Met-Ser (118) -Lys-Gln-Ala-Phe-Gln-Gln-Asp-Ile-Asp (316) -COOH The template used to PCR e: s cDNA / 32D-F3 and oligonucleotides # 1591-94 and # 1591-95 where the primer pair used for PCR and cloning of this gene construct. 1591-94: 5 '-ATTTGATTCTAGAAGGAGGAATAACATATGAAACAAGCTTTTCAGGGG-3' (SEQ ID NO: 11) 1591-95: 5 '-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3' (SEQ ID NO: 12) pAMG21-murine protein that binds to OPG "" 128-3161 This construction is -engineered to be 190 amino acids in length and have the following N terminal and C terminal residues, NH2-Metz-Ser (128) -Glu-Leu-Gln-His -Gln-Asp-Ile-Asp (316) -COOH. The template used for PCR is cDNA / 32D-F3 and oligonucleotides # 1591-91 and # 1591-95 where the primer pair used for PCR and cloning of this gene construct. 1591-91: 5 '-ATTTGATTCTAGAAGGAGGAATAACATATGAAAGAACTGCAGCACATTGTG-3' (SEQ ID NO: 13) 1591-95: 5 '-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3' (SEQ ID NO: 14) pAMG21-murine protein that binds to OPG "" 137-3161 This construction is engineered to be 181 amino acids in length and have the following N-terminal and C-terminal residues, NH ~ -Met-Ser (137) -Arg-Phe Ser-Gly -Gln-Asp-Ile-Asp (316) -COOH. The "template used for PCR is cDNA / 32D-F3 and oligonucleotides # 1591-92 wherein the primer pair used for PCR and cloning of this gene construct. 1591 ^ 92: 5 '-ATTTGATTCTAGAAGGAGGAATAACATATGCAGCGTTTCTCTGGTGCTCCA-3' (SECT DE IDENT N0: 15) 1591-95: 5 '-TATCCGCGGATCCTCGAGTTAGTCTATGTCCTGAACTTTGAA-3' (SEQ ID NO: 16) T pAMG21-murine protein that binds to OPG "" 146-3161 _ This construction is engineered to be 171 amino acids in length and have the following N-terminal and C-terminal residues, NH2-Met- (146) -Glu-Gly-Ser-Trp Gln-Asp-Ile-Asp (316) -COOH. The template used for PCR is pAMG21-murine protein that binds to OPG [75-316] described above and oligonucleotides # 1600-98 and # 1581-76 where the pair zr primer used for PCR and cloning of this gene construct . _ 1600-98: 5 '-GTTCTCCTCATATGGAAGGTTCTTGGTTGGATGTGGCCCA-3' (SEQ ID NO: 17) 1581-76: - 5 '-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID NO: 18) pAMG21-murine protein that binds to OPG "" 156-3161 This construction is engineered to be 162 amino acids in length and have the following N-terminal and C-terminal residues, NH2-Met-Arg (156) -Gly-Lys-Pro Gln-Asp-Ile-Asp (316) -COOH. The template used for PCR is pAMG21-murine protein that binds to OPG [75-316] described above and oligonucleotides # 1619-86 and # 1581-76 where the primer pair used for PCR and cloning of this gene construct . 1619-86: 5 '-GTTCTCCTCTATGCGTGGTAAACCTGAAGCTCAACCATTTGCA-3 (SEQ ID NO: 19) 1581-76: 5' -TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3 '(SEQ ID NO: 20) pAMG21-murine protein that binds to OPG Í158-3161 This construction is "engineered to be 160 amino acids in length and have the following N-terminal and C-terminal residues, NH, -Met-Lys (158) -Pro-Glu-Ala Gln-Asp-Ile-Asp (316) - COOH The template used for PCR is CDNA / 32D-F3 and oligonucleotides # 1581-73 and # 1581-76 where the primer pair used for PCR and cloning of this gene construct. 1581-73: 5 '-GTTCTCCTCATATGAAACCTGAAGCTCAACCATTTGCACACCTCACCATCAAT-3' (SEQ ID NO: 21) 1581-76: 5 '-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID NO: 22) pAMG21-murine protein that binds to OPG "" 166-3161 This construction is "engineered to be 152 amino acids in length and have the following N-terminal and C-terminal residues, NH, -Met-His (166) -Leu-Thr-Ile Gln-Asp-Ile-Asp (316) - COOH The template used for PCR is cDNA / 32D-F3 and oligonucleotides # 1581-75 and # 1581-76 where the primer pair used for PCR and cloning of this gene construct. 1581-75: 5 '-GTTCTCCTCATATGCATTTAACTATTAACGCTGCATCTATCCCAT CGGGTTCCCATAAAGTCACT-3' _ 1581-76: '-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA3' (SEQ ID NO: 24) pAMG21-murine protein that binds to OPG "" 168-3161 - - This construct is engineered to be 150 amino acids in length and have the following N terminal and C terminal residues, NH2-Met-Thr (168) -Ile -Asn-Ala-Gln-Asp-Ile-Asp (316) -COOH. The template used for PCR is cDNA / 32D-F3 and oligonucleotides # 1581-74 and # 1581-76 where. the primer pair used for PCR and cloning of this gene construct. 1581-74: 5'- - GTTCTCCTCATATGACTATTAACGCTGCATCTATCCCATCGGGTTCCCATAAAGTCACT-3 ' (SEQ ID NO: 25) 1581-76: 5 '-TACGCACTCCGCGGTTAGTCTATGTCCTGAACTTTGA-3' (SEQ ID NO: 26) It is understood that the above constructions are examples and that a person familiar with the art can easily obtain other forms of protein that binds to OPG using the general methodology presented herein.

The recombinant bacterial constructs pAMG21-murine protein that binds to .OPG [75-316], [95-316], [107-316], - [118-316], [128-316], [137-316] , and [158-316] have been cloned, their has been confirmed. DNA sequence and the expression levels of the recombinant gene product subsequent to the induction have been examined. All constructs produce levels of recombinant gene product which is easily visible after electrophoresis in SDS and polyacrylamide gel and coomalssie staining of untreated lysates. The growth of transformed E. coli 393 or ^ 2596, the induction of protein expression that binds to OPG and the isolation of inclusion bodies that contain protein that bind, OPG are performed according to "procedures described in PCT W097 / 23614. The purification of proteins that bind to OPG from bodies of __ inclusion requires the solubilization and renaturation of protein - which binds to OPG using procedures It is found that the murine protein that binds to recombinant OPG [158-316] is produced mainly insoluble, but about 40% is found in the soluble fraction.The recombinant protein is purified of the soluble fraction as described below and its bioactivity is examined.

Example 7 Purification of recombinant murine protein that binds to OPG [158-316] ~ Frozen bacterial cells harboring the murine protein that binds to OPG, expressed (158-316) is reheated and resuspended in 20 mM Tris-HCl, pH 7.0, EDTA 10 mM. The cell suspension (20% w / v) is then homogenized by three passes through a microfluidizer. The suspension of used cells is centrifuged in a JA14 rotor at 10,000 rpm for 45 minutes. Analysis by SDS-PAGE shows a band of approximately 18 kd molecular weight present in both inclusion bodies and in the supernatant. The soluble fraction is then applied to a Pharmacia SP Sepharose 4FF column-equilibrated with 10 mM MES, pH 6.0. The protein that binds to OPG is eluted with 20 column volumes of the gradient of 0-0.4M NaCl in MES, pH 6.0. The fractions containing the protein that binds to OPG are then applied to an ABX Bakerbond column equilibrated with 20 mM MES, pH 6.0. The protein that binds to OPG is eluted with a gradient of 15 CV of 0-0.5M NaCl in MES, pH 6.0. The final product is more than 95% homogeneous, by SDS-PAGE. Sequencing of the N-terminal part provides the following sequence: Met-Lys-Pro-Glu-Ala-Gln-Pro-Phe-Ala-His, which is identified with that predicted for a polypeptide starting "at residue 158 (with a methionine initiator.) The relative molecular weight of the protein during SDS-PAGE does not change upon subjecting it to reduction.

- Example 8 In vitro bioactivity of recombinant soluble protein that binds to OPG It has previously been shown that the recombinant OPG protein blocks bone marrow-dependent vitamin D3-dependent osteoclast formation and bone precursors in an osteoclast formation assay, as described in US Serial No. 08 / 577,788. Since the pfoteine that binds OPG binds it to OPG, and is a novel member of the TNF ligand family, and is a potential target for OPG bioactivity. The soluble and recombinant protein that binds to OPG (158-316), represents the minimum core domain similar to TNFa, and is tested for its ability to determine osteoclast differentiation from osteoclast precursors. Bone marrow cells from adult mouse femurs are treated with M-CSF.The non-adherent fraction is co-cultured with ST2 cells in the presence and absence of both vitamin D3 and dexamethasone.

Osteoclasts develop "only from cultures containing stromal cells (ST2), vitamin D3, and dexamethasone.The recombinant soluble protein that binds to OPG is added at varying concentrations ranging from 0.16 to 500 ng / ml and the maturation "of osteoclasts by TRAP solution assay and by visual observation. The protein that binds to OPG strongly stimulates the differentiation and maturation of osteoclasts in a dose-dependent manner, with half-maximal effects in the range of 1-2 ng / ml, suggesting that it acts as a potent inducer of osteoclastosterone "in vitro (see figure 5). The effect of the OPG-binding protein is blocked by recombinant OPG (see Figure "6"). To determine whether the OPG-binding protein can replace the stroma and the aggregated steroids, cultures are established using M-CSF at varying concentrations to promote the growth of osteoclast precursors and various amounts of OPG-binding protein are also added. As shown in Figure 6, the protein that binds to OPG stimulates the activity of TRAP in a dose-dependent manner, and the magnitude of the stimulation depends on the level of M-CSF added, which suggests that these Two factors "together are pivotal for the development of osteoclasts. To confirm the biological importance of this last observation, they were established cultures in cuts of bovine cortical bone and the effects of M-CSF and the protein that binds OPG were tested, either alone or together. As shown in Figure 7, the protein that binds OPG in the presence of M-CSF stimulates the formation of large positive TRAP osteoclasts that are eroded from the bone surface resulting in n cavities. Therefore, the protein that binds OPG acts "as a osteoclastogenesis stimulating factor (differentiation)." This suggests that OPG blocks the development of osteoclasts by sequestering the protein that binds to OPG.

Example 9 In vivo soluble r.ecombinant protein that binds to OPG Based on in vitro studies, the recombinant murine protein that binds to OPG [158-316] produced in E. coli is a potent inducer of the development of osteoclasts from myeloid precursors. To determine their effects in vivo, male BDF1 rats aged 4-5 weeks (Charles River Laboratories) received subcutaneous injections of OPG binding protein [158-316] twice a day for three days and on the morning of the fourth day ( days 0, 1, 2, and 3). Five groups of mice (n = -) received only carrier, or 1, 5, 25 or 100 μg / of the OPG-binding protein [158-316] per day. 5 groups Additional mice (n = 4) received the above doses of carrier or protein that bind OPG [158-316] and additionally received human Fc-OPG [22-194] at 1 mg / kg / day (approximately 20 μg / day ) by single daily subcutaneous injection. The ionized calcium in whole blood was determined before treatment on day 0 and 3-4 hours after the first daily injection of the protein that binds OPG [158-316] on days -1, 2 and 3. Four hours later After the last injection on day 3, the mice were sacrificed and x-rays were taken. The recombination of OPG-binding protein [158-316] produces a significant increase in blood ionized calcium after two days of treatment at a dose of 5 μg / day and higher (see Figure 8). The severity of hypercalcemia indicates a potent induction of osteoclast activity resulting in increased bone resorption. The concurrent administration of OPG limits hypercalcemia to doses of OPG binding protein [158-316] of 5 and 25 μg / day, but not to 100 μg / day. "These same animals were analyzed by radiography to determine if there were effects of any kind on bone mineral density visible by X-rays (see Figure 9). The recombination of OPG binding protein [158-316] injected during 3 days decreases bone density in the proximal tibia of mice in a dose-dependent manner.The reduction in bone density is Particularly evident in mice receiving 100 μg / d confirms that the profound hypercalcemia in these animals occurred from an increased bone resorption and the resulting release of calcium from the skeleton. These data clearly indicate that the protein that binds OPG [158-316] acts "in vivo to promote bone resorption, leading to systemic hypercalcemia, and recombinant OPG cancels these effects." . "" Example 10 Cloning and expression of soluble protein that binds OPG in dezmamífero cells - The full length clone of murine and human protein that binds OPG that can express in mammalian cells as _. is described previously in Example 2. Alternatively, DNA clones can be modified to encode secreted forms of the protein when expressed in "mammalian" cells To "do this, the natural 5 'end of codon encoding cDNA of initiation, and extending approximately through the first 69 amino acids of the protein, which includes the region encompassing the transmembrane, can be substituted with a leader signal peptide sequence. For example, "the DNA sequences that encode for the initiation codon of the signal peptide of a known gene can be cut and spliced to the cDNA sequence of OPG-binding protein, being anywhere after the region coding for amino acid residue 68. "The recombinant clones- The resulting proteins are predicted to produce secreted forms of protein that bind to OPB in mammalian cells, and must undergo posttranslational modifications which normally occur in the extralular domain in the "C-terminal" part of the OPG-binding protein, such as glycosylation. . Using this strategy, a secreted form of protein that binds OPG is constructed which has at its 5 'end the murine peptide signal for OPG, and in its 3 'end the Fc domain of IgGl. The plasmid vector pCEP / muOPG [22-401] -Fc as described in United States Patent Serial No. 08 / 577,788, filed on December 22, 1995, is digested with Notl to -separate the 3 'end of OPG and the gene for Fc. The linearized DNA is then partially digested with Xmnl to separate only residues 23 and 24 of OPG leaving a blunt end. The restriction digests are then dephosphorylated with CIP and the vector portion of this digest (which includes residues 1-23 of OPG and Fc) are gel purified. The cDNA region for the murine protein that binds OPG "coding for amino acid residues 93-316 is PCR amplified using Pfu polymerase (Stratagene, San Diego, CA) from the plasmid template using primers with the following oligonucleotides: 1602-61: CCT CTA GGC CTG TAC jTTT CGA GCG CAG ATG (SEQ ID NO: 27) ) 1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT TG (SEQ ID NO: 28) Oligonucleotide 1602-61 amplifies the 5 'end of the gene and contains an artificial Stul site. Primer 1602-59 amplifies the 3 'end of the gene containing an artificial Notl site. The resultant PCR product obtained is digested with Notl and Stul, and then gel purified. The purified PCR product is ligated with the vector, then used to transform the E cells. cb1i DH10B electrocompetent. The resulting clone is sequenced to confirm the integrity of the amplified sequence and the junctions of the restriction sites. This plasmid is then used to transfect human fibroblasts 293, and the protein that binds to OPG-Fc fusion protein is harvested from the culture medium as previously described in US Serial No. 08 / 577,788, filed on December 22, 1995. By utilizing a similar strategy, an expression vector is designed that is capable of expressing an N-terminal truncated sequence fused to the Fc domain of human IgGl. This construct consists of the signal peptide for OPG of mouse (amino acid residues 1-21), fused in frame with residues 158-316 of murine protein binding OPG, followed by a frame fusion to the Fc domain of human IgGl. To do this, the murine pCEP4 / 0PG plasmid vector [22-401] (U.S. Serial No. 08 / 577,788, filed December 22, 1995) is digested with HindIII and NotI to remove the entire framework reading for OPG. The murine protein binding OPG, residues 158-316 is amplified by PCR using pCDNA / 32D-F3 plasmid template using the following primers: 1616-144: CCT CTC TCG AGT GGA CA CCC AGA AGC CTG AGG CCC AGC CAT TTG C (SEQ ID NO: 29) 1602-59: CCT CTG CGG CCG CGT CTA TGT CCT GAA CTT TG (SEQ ID NO: 30) "" 1616-44 amplifies the protein that binds OPG starting at residue 158 as well as that containing residues 16-21 of the muOPG signal peptide with an artificial Xhol site. 1602-59 amplifies the 3 'end of the gene and adds a Notl site in frame. The PCR product is digested with Notl and Xhol and then purified in gel. The following complementary primers align themselves to form an adapter encoding the murine peptide OPG signal and the Kozak sequence surrounding the translation start site: 1616- "41: AGC TTC CAC CAT GAA CA GTG GCT GTG CTG CGC ACT CCT GGT __GCT CCT GGA CAT CA (SEQ ID NO: 31) 1616-42: TCG ATG ATG TCC AGG AGC ACC AGG AGT GCG CAG CAC AGC CAC TTG TTC ATG GTG GA (SEQ ID NO: 32) These primers are aligned, generating 5 'pendant parts compatible with HindIII at the 5' end and Xhol at the 3 'end. The digested vector obtained above, the aligned oligonucleotides and the digested PCR fragment are ligated together and electroporated into DH10B cells. The resulting clone is = sequence to confirm the authentic reconstruction of the binding between the signal peptide, the protein fragment binds to "OPG coding for residues 158-316 and the Fc domain of IgGl." The recombinant plasmid is purified, transfected into 293 human fibroblasts and ~ is expressed as a product of conditioned medium as described above.Full-length human and mouse cDNAs are cloned into the expression vector pCEP4 (Invitrogen, San Diego, CA) and then transfected into fibroblast cultures human 293 as "described in Example 1. Cell cultures are selected with hygrogrhythmy as described above and serum-free conditioned medium is prepared." Conditioned media is exposed to an OPG column immobilized recombinant, and the scattered forms of recombinant mouse OPG and de-human bp are affinity purified. Analysis of the N-terminal sequence of the purified soluble proteins that bind OPG indicates that the murine proton is preferentially separated before phenylalanine., and that the "human protein is preferentially separated before the homologous residue, and isoleucine 140. In addition, the human protein is also preferentially separated before the glycine 145. This suggests that the naturally occurring soluble forms of human protein that binds to OPG have amino acid residues in either isoleucine at position 140 or glycine at position 145.

Example 11 Peptides of the protein que_une OPG and preparation of polyclonal and "monoclonal antibodies to the protein "Antibodies to specific regions of the OPG-binding protein can be obtained by immunization with peptides from the OPG-binding protein.These peptides can be used alone, oz., Conjugated forms of the peptide can be used for immunization.

The crystal structure of mature TNF has been described [E.Y. Jones, D.I. Stuart, and N.P.C. Walker (1990) J. Cell .1 Sci. Suppl. 13, 11-18] and "the monomeric forms and an interposed form of ß-antiparallel folded sheet with a topology of rolled cake." There are 10 antiparallel ß chains in this crystalline structure and form an interposition ß with a ß sheet " consists of B'BIDG chains and another of the C'CHEF JE chains. Y. Jones et al., Ibid. ] Two curls of mature TNFα have been implicated from mutagenesis studies to establish contacts with the receptor, these are the curls formed between the beta B and B 'chain and the loop between the beta E and F chains [C.R. Goh, C-S. Loh, and A.G. Porter (1991) Protein Engineering 4, 785-791]. The crystal structure of the complex formed between TNFβ and the extracellular domain of the 55 kd TNF receptor (TNF-R55) has been resolved and receptor-ligand contacts have been described [D.W. Banner, A. D'Arcy, W. Janes, R. Gentz, H-J. Schoenfeld, C. Broger, H. Loetscher, and W. Lesslauer (1993) Cell 73, 431-445]. In accordance with the mutagenesis studies described above [C.R. Goh et al., Ibid.] The corresponding curls BB 'and EF of the TNFβ ligand are found to establish most of the contacts with the receptor in the resolved crystal structure of the TNFb: TNF-R55 complex. The amino acid sequence of the murine protein that binds OPG is compared to the amino acid sequences of TNFα and TNFβ. The regions of Murine protein binding OPGs corresponding to curls BB 'and EF. are predicted based on this comparison and the peptides have been designated and have been described subsequently. A. Antigen or antigens: Murine protein binding OPG [158-316] has been used as an antigen (ag) for immunization of animals as described below, and serum has been examined using approaches described below. Peptides of the putative curls BB 'and EF of the murine protein that binds OPG have been synthesized and will be used for immunization; These peptides are: BB 'curl peptide: NH2-NAASIPSGSHKVTLSSWYHDRGWAKIS-COOH (SEQ ID NO: 33) cyss peptide BB' NH2 - NAASIPSGSHKVTLSSWYHDRGWAKICSC-C00H (SEQ ID NO: 34) EF curl peptide: NH2-VYWKTSIKIPSSHNLM-COOH (SEQ ID NO: 35) EF-Cys curl peptide: NH2-VYWKTSIKIPSSHNLMC-COOH (SEQ ID NO: 35) Peptides with a carboxy terminal cysteine residue have been used for conjugation using the approaches described in section B below, and have been used for -immunization. = - B. Conjugation of keyhole limpet hemocyanin or bovine serum albumin: The selected peptides or protein fragments can be conjugated to keyhole limpet hemocionin (KLH) in order to increase their immunogenicity in animals. In addition, peptides conjugated to bovine serum albumin (BSA) or protein fragments can be used in the EIA protocol. Activated KLH is reconstituted with Imject maleimide or BSA (Pierce Chemical Company, Rockford, IL) in dH20 to a final concentration of 10 mg / ml. The peptide or protein fragments are dissolved in phosphate buffer and then mixed with an equivalent mass of KLH or BSA. Sé. Allows the conjugation to react for 2 hours at room temperature (rt) with gentle agitation. The solution is then passed over a salt removal column or dialyzed against PBS overnight. The peptide conjugate is stored at -20 ° C until used in immunizations or in EIA.

C. Immunization: Balb / c mice (Charles Rivers Laboratories, Wilmington, MA) Lou rats, New Zealánd white rabbits are injected subcutaneously (SQI) with ag (50 μg, 150 μg and 100 μg, respectively) emulsified in complete Freund's adjuvant (CFA, 50% vol / vol; Difco Laboratories, Detroit, MI). Rabbits are then boosted two or three times at 2 week intervals with antigen similarly prepared in incomplete Freund's adjuvant (ICFA, Difco Laboratories, Detroit, MI). "Mice and rats are boosted approximately every 4 weeks Seven days after the second booster, blood samples are taken for testing and the spherical antibody titers are determined.When a titer is developed in rabbits, blood samples are taken from weekly production.l "of 50 ml for 6 consecutive weeks. Mice and rats are selected for hybridoma production based on serum titer levels; Animals with semi-maximal titers greater than 5000 are used. The adjustments to this protocol can be applied to a person familiar with the art. For example, several types of immunomodulators are now available and can be incorporated into this protocol.

D. Enzyme-Linked Immunosorbent Assay (EIA): EIAs will be performed to determine the serum (ab) antibody titers of individual animals, and subsequently for an analysis of potential hybridomas. EIA / RIA microtitre plates of 96 well, flat-bottomed microtitre wells (Costar Corporation, Cambridge, MA) are coated with purified recombinant protein or with protein fragments (antigen, ag) at 5 μg per ml "in buffer carbonatebicarbonate, pH 9.2 (Na2C03 0 ^ 015 M, 0.035 M NaHCO3) The protein fragments can be conjugated to bovine serum albumin (BSA) if necessary. 15 μl of ag are added to each well. The plates are then covered with acetate film (ICN Biomedicals, Inc., Costa Mesa, CA) and incubated at room temperature (rt) on an oscillating platform for 2 hours or overnight at 4 ° C. Plates will be blocked for 30 minutes at rt with 250 μl per well of a 5% BSA solution prepared by mixing 1 part BSA diluent / blocking solution concentration (Kirkegaard and Perry Laboratories, Inc., "Gaithersburg, MD) with 1 part of deionized water (dH20) The blocking solution has been discarded, 50 μl of double dilutions of serum (1: 100 to 1: 12,800) or culture supernatants of hybridoma tissue will be added to each well. BSA 1% (diluent BSA 10% / concentrate of blocking solution diluted 1:10 in saline buffered with Dulbecco's phosphate, D-PBS; Gibco-BRL, Grand Island, NY)). While the hybridoma supernatants are tested undiluted. In the case of hybridoma analysis, one well remains as a conjugate control, and the second well is one. ab positive control. The plates are again incubated at rt, oscillating for 1 hour, and then washed 4 times using an ix preparation of 20x concentrated wash solution (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) in dH20. Then secondary conjugate was conjugated with horseradish peroxidase (Boeringer Mannheim Biochemicals, Indianapolis, IN) diluted in 1% BSA in each well for 30 minutes. The plates are washed as above, dried in spot, and the unique ABTS peroxidase component substrate (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) is added.The absorbance is read at 405 nm for each well, using a Microplate EL310 vector (Bio-tek Instruments, Inc., Winooski, VT) The semi-maximal titre of serum antibody is calculated by plotting log10 of serum dilution versus optical density at 405, and then extrapolating at the 50% point of the Maximum optical density obtained for that serum Hybridomas are selected as positive if the optical density ratings are greater than 5 times greater than the background The settings for this protocol can be applied, for example, secondary antibody can be chosen conjugate for specificity or for nullity of cross-reactivity. " Cell fusion: The animal that is selected for hybridoma production is injected intravenously with 50 to 100 μg of ag in PBS. Four days later, the animal is sacrificed with carbon dioxide and its spleen is extracted under sterile conditions in which it contains 200 U / ml of penicillin G, 200 μg / ml of streptomycin sulfate and 4 mM glutamine (2x P7S / G DMEM) . The spleen is trimmed by removing the excess of fatty tissue, and then it is moistened through 4 containers of DMEM 2x P / S / G clean. It is then transferred to a sterile digester bag (Tekmar, Cincinnati, OH) containing 10 ml of DMEM 2x P / S / G and digested in a single cell suspension using a Stomacher Lab Blender 80 mixer (Sewárd Laboratory UAC House, London , England). As the cells are released from the spleen capsule into the medium, they are removed from the capsule and transferred to a 50 ml sterile conical centrifuge tube (Becton Dickinson and Company, Lincoln Park, NJ). Fresh media is added to the bag and the process is repeated until the entire spleen cell content is released. "These splenocytes are washed 3 times by centrifugation at 225 x g for 10 minutes.

Concurrently, logarithmic phase cultures of myeloma cells, Sp2 / 0-Agl4 or Y3-Agl.2.3 for fusions of mouse or rat splenocytes, respectively (American Type Culture Collection, Rockville, MD) growing in medium Complete (DMEM, 10% inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 10 mM HEPES buffer, Gibco Laboratories, Grand Island, NY) are washed in a similar manner. Splenocytes combine with the myeloma cells and settle once more. The medium is aspirated from the cell pellet and 2 ml of polyethylene glycol 1500 (PEG 1500, Boehringer Mannheim Biochemicals, Indianapolis, IN) are gently mixed in the cells for 1 minute, then an equal volume of DMEM 2x P / S is slowly added. / G. Cells are allowed to fuse at 37 ° C "for 2 minutes, and then add an additional 6 ml of DMEM 2x P / S / G. The cells are pelleted again at 37 ° C for 3 minutes. Finally, 35 ml of DMEM 2x P / S / G are added to the cell suspension, and the cells are pelleted by "centrifugation." The medium is aspirated from the pellet and the cells are gently resuspended in complete medium. on 96 well flat bottom tissue culture plates (Becton Dickison Labware; Lincoln Park, NJ) for single drops of a 5 mlT pipette The plates are incubated overnight under humidified conditions at 37 ° C, CO, at 5% - The next day, it adds to each well an equal volume_ of the selection medium. The selection consists of hypoxanthine 0.1 mM, aminopterin 4 x 10"4 m" M and thymidine 1.6 x 10 ~ 2 mM "in complete medium.The fusion plates are incubated for 7 days followed by two changes of medium during the following 3 days The HAT Z selection medium is used after each fluid change. "Tissue culture supernatants are taken 3 to 4 days after the last fluid change for each well containing hybrid and tested pro EIA for specific antibody reactivity. . This protocol has been modified by Hudson and Hay, "Practical Immunology, Second Edition", Blackwell Scientific Publications.

Example 12 Cloning of a protein receptor that binds OPG expressed in hematopoietic precursor cells The recombinant, biologically active murine protein, which binds OPG [158-316] to fluoroacetic-isotecyanate (FITC), is conjugated to generate a fluorescent probe. Fluorescent labeling is performed by incubation of recombinant murine protein binding OPG [158-316] with 6-fluorescein-5- (and 6) carboxamidohexanoic acid succinimidyl ester) (Molecular Probes, Eugene, OR) in a molar ratio 1: 6 for 12"h at 4 ° C. The OPG-binding protein, labeled with FITC [158-316] is further purified by gel filtration chromatography, mouse bone marrow cells are isolated and incubated culture in the presence of CSF-1 and OPG-binding protein [158-316] _ as described in Example 10. Mouse bone marrow cells are cultured overnight in CSF-1 (30 ng / ml) and protein that binds OPG [158-316] (20 ng / ml) The non-adherent cells are removed first and stored on ice, and the remaining adherent cells are removed by incubation with cell dissociation buffer (Sigma Chemicals, St. Louis, MO ), and accumulates with the nonadherent population, and then stained with FITC-protein that binds to OPG, as described above.After washing and resuspending in PBS with 0.5% BSA, the cells are exposed to FITC- protein that binds OPG, are washed, and then sorted by FACS.The population of cells that are for staining with the protein that binds to FITC-OPG are harvested and the mRNA is isolated as described in Example 2. This mRNA preparation is used to make a cDNA library following the procedures described in Example 2. The cDNA library produced from this source is used for a random EST sequence analysis as previously described in PCT publication W097 / 23614 and in Simonet et al. (Cell 8j3, 309-319 (1977)). Using In this method, a -2.1 kb cDNA encoding the novel protein related to TNFR is detected. The long open reading frame of the murine ODAR cDNA encodes a protein of 625 amino acid residues and contains the remarkable characteristics of the TNFR-related proteins: a hydrophobic signal peptide in its N-terminal part, four repeated sequences rich in cysteine in battery, a hydrophobic transmembrane domain and a cytoplasmic signaling domain. The homology of this protein with other members of the TNF receptor family and its expression in bone marrow cells that bind OPG-tagged OPG binding protein suggest that it is a potential receptor for the OPG-binding protein related to TNF. This protein is called ODAR or osteoclast differentiation and activation receptor. The = nucleic acid sequence and the predicted amino acid sequence of murine ODAR is shown in Figure 10. Recent analysis of the sequences in publicly available databases indicates that this protein is the murine homolog of a known human protein related to TNFR. as RANK (Anderson et al., Nature 390, 175-179 (1997)).

Example 13 Production of ODAR protein. recombinant in mammalian cells A soluble ODAR extracellular domain fused to the Fc region of human IgGx is produced using methods for the construction and expression of Fc fusion proteins as previously described in W097 / 23614 and Simonet et al., Supra. Shovel generate a soluble ODAR protein in mammalian cells, "the cDNA encoding the extracellular domain of murine ODAR (amino acids 27-211) is amplified by PCR with the following set of oligonucleotide primers: 'TCT CCA AGC TTG TGA CTC TCC * AGG TCA CTC C-3' (SEQ ID NO: 37) 5 'TCT CCG CGG CCG CGT AAG CCT_ GGG CCT CAT TGG GTG-3' (SEC1 DE IDENT. : 38) PCR reactions are carried out in a volume of 50 μl with 1 unit of DNA polymerase vent (New England Biolabs) in 20 mM Tris-HCl pH 8.8, 10 mM KCl, (NH4) 2S04 mM, Triton-XlOO 0.1%, and 10 μM of each of dNTP, 1 μM of each primer and 10 ng of the cDNA template for ODAR.

The reactions are performed at 94 ° C for 30 s, 55 ° C for 30 s "and 72 ° C for 1 min, for a total of 16 cycles.The PCR fragment is isolated by electrophoresis. Hind III restriction at the 5 'end and a restriction site N.ot I at the 3' end.The fragment of "PCR digested with Hind III-Not I is then subcloned in frame into a pCEP4-Fc vector modified against the Human IgG-? l heavy chain sequence as previously described in W097 / 23614 and in Simonet et al. supra). A linker is introduced which codes for 2 unrelated amino acids that span the junction between the extracellular ODAR domain and the Fc region of IgG. The construction is then digested with Nhe I and Hind III and "the following aligned oligonucleotide pair coding for the OPG signal peptide (amino acids 1-21) is inserted into the frame: 'CTA GCA CCA TGA AC AGT GGC T.GT GCT GCG CAC TCC TGG TGC TCC TGG AC TCA TTG AAT GGA CA CCC. AGA-3 '(SEQ ID NO: 39) 5' GC TTC TGG GTT GTC CAT TCA ATG ATG TCC AGG AGC ACC AGG AGT GCG CAG CAC AGC CAC TTG TTC ATG GTG-3 '(SEQ ID NO. : 40) A linker is introduced which encodes two unrelated amino acids between the OPG signal peptide and the ODAR sequences. The final construction under engineering (0DAR-Fc / pCEP4) codes for a fusion protein that contains from the amino terminal part to the carboxy terminal: the signal peptide of OPG (amino acids 1-21) -linker (LysLeu) -ODAR (amino acids 27-211) and a Linker (AlaAla) -IgG Fc human. The construct is transferred into 293-EBNA-1 cells by the calcium phosphate method as described (Ausubel et al., Curr. Prot. Mol. Biol. "1, 9.1.1-9.1.3, (1994). The cells transfected after they are selected in 200 μg / ml of hygromycin (GibcoBRL) and the resulting mass cultures resistant to the drug accumulate and grow to confluence. The cells are washed in PBS once and then cultured in serum-free medium for 72 h. The conditioned medium is collected. The ~ ODAR-Fc fusion protein in the medium is detected by Western blot analysis with human IgG Fc antibody. The "Fc fusion protein is purified by protein A column chromatography (Pierce) using the procedures recommended by the manufacturer, then 50 pmol of the purified protein is subjected to N terminal sequence analysis by automated Edman degradation as described essentially by J. Biol. Chem. 262, 10-35 (1987)). The following amino acid sequence is read after 10 cycles: NH, K L V T L Q V T-_P-CO, H.

The binding activity of ODAR-Fc with the OPG binding protein was examined by immunofluorescent staining of cultures of transfected COS-7 cells as described in Example 2. CQS-7 cells are lipofected with 1 μg of a vector of expression that contains DNA that codes for the murine protein that binds to OPG. After 48 h of incubation, the cells are subsequently incubated in a solution of PBS-FBS containing 10 mg / μl of IgG Fc huaman, ODAR-Fc or protein OPG-Fc at "4 ° C for 1 h. they are washed with PBS twice and then incubated in a PBS-FBS solution containing 20 μg / ml goat anti-human IgG, labeled with FITC (Southern Biotech Associates) for another hour. Cells are examined with focal microscopy (ACAS, Ultima, Insight Biomedical Imaging, Inc., Okemos, MI). Both ODAR-Fc and. OPG-Fc bind to "COS-7 cells transfected with OPGL (see Figure 11). .

Example 14 In vitro biological activity of recombinant soluble ODAR "" The ability of ODAR to inhibit the stimulation of osteoclast formation by the OPG binding protein is determined in a mouse bone marrow culture in the presence of CSF-1 (30 ng / ml) and OPG binding protein (5). ng / ml). Methods for the use of mouse bone marrow cultures to study osteoclast maturation are described in W097 / 23614 and Example 8. The ODAR-Fc fusion protein produced as described in Example 12 is Add at concentrations of 65 to 1500 ng / ml. The formation of osteoclasts is determined by "tartrate-resistant alkaline phosphatase cytochemistry" (TRAP) and the TRAP solution assay after five days in culture. A dose-dependent inhibition in the formation of osteoclasts is observed by the fusion of ODAR-Fc both by cytochemistry and by TRAP activity (see Figure 12). The ODAR-Fc fusion protein inhibits the formation of osteoclasts with an ED50 of about 10-50 ng / ml.

Example 15 In vivo biological activity of recombinant soluble ODAR Rapidly growing male BDF1 mice, 3 ^ 4 week olds receive variable dose of the ODAR-Fc fusion protein by single daily subcutaneous injection in one carrier (PBS / 0.1% BSA) for four days. The mice were subjected to X-rays on day 5. The doses of ODAR-Fc fusion protein used were 0.5, 1.5 and 5 mg / kg / day. For each treatment, all mice in this group and in the control group that received PBS / BSA 0.1% were subjected to X-rays in a single film.The metaphyseal region of the proximal tibia was compared between pairs of control and treated tibiae and they were "rated" + "if the tibia treated is more dense by visual determination than control by providing the 8 ratings shown below. An arbitrary rating of 5/8 is required for a "positive" result.

(The dose is in mg / kg / day). (n = 4). After sacrifice, the right tibia of each animal is removed and the bone density in proximal tibia metaphysis is measured by peripheral quantitative computer tomography (pQCT) (Stratec, Germany). Two transverse sections of 0.5 mm bone, 1.5 mm and 2.0 mm were analyzed from the proximal end of the tibia (XMICE 5.2, Stratec, Germany) to determine total bone mineral density in metaphysics. A soft tissue separation threshold of 1500 was used to define the limit of metaphyseal bone. The administration of ODAR-Fc in young growing mice inhibits bone resorption in the proximal tibial growth plate which produces a region of increased bone density that is visually evident on radiographs. The radiographic changes are evident at a dose of 1.5 mg / kg / day and above, in the two experiments (see Table 1). The measurement of bone density by pQCT in the samples from the second experiment in a similar region of the tibia confirms the dose-dependent increase in bone density in these mice (see Figure 13).

Table 1 Inhibition of bone resorption by the ODAR-Fc fusion protein Experiment # 1 Dose Factor 1 2 3 4 5 6 7 8 Result ODAR.Fc 5.0 + + + + + + + + Positive 8/8 ODAR.Fc 1.5 - + + - + + + + Positive 6/8 ODAR-Fc 0.5 - - - - - - - - Negative 0/8 Dose Factor 1 2 3 4 5 6 7 8 Result ODAR-Fc 0.15 - - - - - - - - Negative 0/8 Dose Factor 1 2 3 4 5 6 7 8 Result ODAR.Fc 5.0 + + + + + + + + Positive 8/8 ODAR.Fc 1.5 + + + + + + + + Positive 8/8 ODAR-Fc 0.5 - - - + - - - - Negative 1/8 Although the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those familiar with the art. Therefore, it is intended that the appended claims cover all equivalent variations. which are within the scope of the invention as claimed.It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, "is the conventional one for the manufacture of the objects or products to which it refers. - "

Claims (42)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated nucleic acid encoding an osteoprotegerin binding protein that is selected from the group consisting of: a) the nucleic acid sequence of FIG. 1 (SEQ ID NO: 1) and Figure 4 (SEQ ID NO: 3); b) Nucleic acids - which hybridize with the polypeptide coding regions as shown in Figure 1 (SEQ ID NO: 2) and Figure 4 (SEQ ID NO: 4) and which remain hybridized under conditions of high restriction; and c) nucleic acids which are degenerate to nucleic acids of subsections (a) or (b).

2. The nucleic acid according to claim 1, characterized in that it is cDNA, genomic DNA, synthetic DNA or RNA.

3. A polypeptide encoded by the nucleic acid according to claim 1.

4. The nucleic acid according to claim 1, characterized in that it includes one or more preferred codons for expression in Escherichia coli.

5. The nucleic acid according to claim 1, characterized in that it has a detectable label attached thereto.

6. A nucleic acid encoding a polypeptide comprising the amino acid sequence of residues 1-316 and residues -70-316, as shown in Figure 1.

7. A nucleic acid encoding a polypeptide comprising the amino acid sequence of residues 1-317 and residues 69-317, as shown in Figure 4 (SEQ ID NO: 3).

8. A nucleic acid that codes for a soluble protein that binds to osteoprotegerin.

9. The nucleic acid according to claim 8, characterized in that it encodes "a polypeptide comprising residues 69-317 as shown in figure .4 (SEQ ID NO: 3) and truncated sections thereof.

10. An expression vector characterized in that it comprises the nucleic acid according to claims 1 and 9.

11. The expression vector according to claim 10, characterized in that the nucleic acid comprises the polypeptide-coding region as shown in Figure 1 (SEQ ID NO: 2) and Figure 4 (SEQ ID NO. NO: 4)

12. A host cell characterized in that it is transformed or transfected with the expression vector according to claim 10.

13. The host cell according to claim 12, characterized in that it is a eukaryotic or prokaryotic cell.

14. The host cell according to claim 13, characterized in that it is Escherichia coli.

15. A process for the production of a protein that binds osteoprotegerin, characterized in that it comprises: growing under suitable nutritional conditions host cells transformed or transfected with the nucleic acid according to claim 1; and isolating the polypeptide product from the expression of the nucleic acid.

16. A polypeptide, characterized in that it is produced by the process according to claim 15.

17. A purified and isolated protein, which binds to osteoprotegerin, or a fragment, analog or derivative thereof.

18. The protein according to claim 17, characterized in that it is a human osteoprotegerin.

19. The protein according to claim 17, characterized in that it has the amino acid sequence as shown in Figure 1 (SEQ ID NO: 2) and Figure 4 (SEQ ID NO: 4)

20. The protein according to claim 17, characterized in that it has been covalently modified with a water-soluble polymer.

21. The protein - according to claim 20, characterized in that the polymer is polyethylene glycol.

22. The protein "- according to claim 17, characterized in that it is a soluble protein that binds to osteoprotegerin.

23. The protein according to claim 22, characterized in that it comprises the amino acid sequence of residues 70-316 inclusive, as shown in Figure 1 (SEQ ID NO: 2), or a fragment, analog or derivative thereof. the same.

24. The protein according to claim 22, characterized in that it comprises the amino acid sequence of residues 69-317 inclusive, as shown in Figure 4 (SEQ ID NO: 4) and truncated sections thereof.

25. An antibody or fragment thereof, characterized in that it binds specifically to a protein that binds osteoprotegerin.

26. The antibody according to claim 25, characterized in that it is a monoclonal antibody.

27. A method for detecting the presence of a protein that binds osteoprotegerin in a biological sample, characterized in that it comprises: incubating the sample with the antibody according to claim 25 under conditions that allow the binding of the antibody to the protein that binds to osteoprotegerin; and detect the bound antibody.

28. A method for detecting the presence of osteoprotegerin in a biological sample, characterized in that it comprises: incubating the sample with a protein that binds osteoprotegerin under conditions that allow the binding of the protein to osteoprotegerin; and measuring the protein that binds osteoprotegerin that binds.

29. A method for determining the ability of a candidate compound to bind to a protein zoning osteoprotegerin, characterized in that it comprises: incubating the protein that binds osteoprotegerin with the candidate compound under conditions that allow binding; and measure the bound compound.

30. The method according to claim 29, characterized in that the compound is an agonist or an antagonist of a protein that binds osteoprotegerin.

31. A method for regulating the expression of an osteoprotegerin-binding protein in an animal, characterized in that it comprises administering the animal in nucleic acid complementary to the nucleic acids as shown in Figure 1 (SEQ ID NO: 1) and FIG. 4 (SEQ ID NO: 3).

32. A pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of a protein that binds osteoprotegerin in a pharmaceutically acceptable carrier, adjuvant, stabilizing solubilizer and / or antioxidant.

33. The composition according to claim 32, characterized in that the osteoprotein protein is a protein that binds human osteoprotegerin.

34. A method for preventing or treating bone diseases in a mammal, characterized in that it comprises administering a therapeutically effective amount of. a modulator of a protein that binds osteoprotegerin.

35. The method "" according to claim 34, characterized in that the modulator is a soluble formula of a protein that binds osteoprotegerin.

36. The method according to claim 35, characterized in that the modulator is an antibody, or fragment thereof, which binds specifically to a protein that binds osteoprotegerin.

37. The "protein" according to claim 22, characterized in that it comprises the amino acid sequence of residues 140-316 inclusive, as shown in Figure 4 (SEQ ID NO: 4) or a fragment, analog or derived from it.

38. The protein according to claim 22, characterized in that it comprises the amino acid sequence of residues -145 = 316 inclusive as shown in Figure 4 (SEQ ID NO: 4) or a fragment, analog or derivative thereof.

39. A method for preventing or treating bone diseases "in a mammal characterized in that it comprises administering a therapeutically effective amount of a modulator of an osteoclast differentiation and activation receptor.

40. The method . according to claim 39, characterized in that the modulator is a soluble form of a osteoclast differentiation and activation receptor.

41. The method "according to claim 39, characterized in that the modulator is an antibody, or a fragment thereof, which specifically binds a" differentiation factor "and osteoclast activation.

42. A method for determining the ability of a test compound to increase or decrease the binding of - 1CT8 - a protein that binds osteoprotegerin to ODAR, characterized in that it comprises: incubating protein that binds to osteoprotegerin, ODAR and optionally the test compound under conditions that allow the binding of the protein that binds to osteoprotegerin with ODAR; and measuring the binding of the osteoprotegerin binding protein with ODAR in the absence and presence of the test compound.