WO2005060627A2

WO2005060627A2 - Methods of assessing the risk of non-traumatic bone fracture

Info

Publication number: WO2005060627A2
Application number: PCT/US2004/041489
Authority: WO
Inventors: Georg Schett; Stefan Kiechl; Johann Willeit
Original assignee: Auxeris Therapeutics, Inc.
Priority date: 2003-12-10
Filing date: 2004-12-10
Publication date: 2005-07-07
Also published as: WO2005060627A3

Abstract

The present invention relates to the identification of RANK ligand (RANKL) as associated with and useful as a marker of bone metabolism conditions and particularly of predicting risk of non-traumatic bone fractures. The invention also relates to assays, methods and kits comprising RANKL and/or antibodies thereto for screening, diagnosis, prediction and monitoring. This invention provides methods for determining a subject's risk of non-traumatic bone fracture, comprising determining the subject's level of RANKL, particularly of soluble uncomplexed RANKL.

Description

METHODS OF ASSESSING THE RISK OF NON-TRAUMATIC BONE FRACTURE

FIELD OF THE INVENTION

The present invention relates generally to the identification of proteins, particularly RANK ligand (RANKL), that are associated with and useful as markers of bone metabolism conditions and particularly of predicting risk of non-traumatic bone fractures. The invention also relates to assays, methods and kits comprising RANKL and/or antibodies thereto or binding agents thereof for screening, diagnosis, prediction and monitoring.

BACKGROUND OF THE INVENTION

Bone is a specialized connective tissue and is constantly undergoing remodeling. Bone matrix is formed by osteoblast cells located at or near the surface of existing bone matrix. Bone is resorbed (eroded) by a cell known as an osteoclast, which is a type of macrophage, by secreting acids which dissolve bone minerals, and hydrolases, which digest its organic components. Thus, bone formation and remodeling is a dynamic process involving an ongoing interplay between the creation and erosion activities of osteoblasts and osteoclasts (Alberts, et al., Molecular Biology of the Cell,

Garland Publishing, N.Y. (3rd ed. 1994), pρ.1182-1186). Normal bone remodeling requires that the processes of bone resorption and bone formation take place in a coordinated fashion, with the orderly development of osteoclasts and activation of osteoblasts. A key development in the field of bone cell biology was the discovery that RANK ligand (RANKL), also known as osteoprotegerin ligand (OPGL), TNF-related activation induced cytokine (TRANCE), and osteoclast differentiation factor (ODF), expressed on stromal cells, osteoblasts, activated T-lymphocytes and mammary epithelium, is essential for differentiation of macrophages into osteoclasts (Lacey, et al. (1998) Cell 93: 165-176) The cell surface receptor for RANKL is RANK, Receptor Activator of Nuclear Factor (NF)-kappa B.

RANKL is a type-2 transmembrane protein with an intracellular domain of less than about 50 amino acids, a transmembrane domain of about 21 amino acids, and an extracellular domain of about 240 to 250 amino acids. RANKL exists naturally in transmembrane and soluble forms. The deduced amino acid sequence for the murine, rat and human forms of RANKL are known (see e.g., Anderson, et al., U.S. Pat. No. 6,017,729, Boyle, U.S. Pat. No. 5,843,678, and Xu J. et al., J. Bone Min. Res. (2000/15:2178) which are incorporated herein by reference). RANKL (OPGL) has been identified as a potent inducer of bone resorption and as a positive regulator of osteoclast development. Lacey et al., supra. In addition to its role as a factor in osteoclast differentiation and activation, RANKL has been reported to induce human dendritic cell (DC) cluster formation (Anderson et al., U.S. Pat. No. 6,017,729) and mammary epithelium development (Fata, J. et al. (2000) Cell, 103:41-50). More recently, it has become evident that RANKL plays a role in anabolic bone formation processes. PCT International Application No. PCT/US02/09271, filed March 22, 2002, published as WO 02/080955 on October 17, 2002 further describes the stimulation of osteogenesis using oligomerized RANKL.

Recently, essential physiological interactions between osteoblasts and osteoclasts have been unraveled. Receptor activator of nuclear factor kappaB ligand (RANKL) was proposed to be a key player in this scenario. RANKL expression by bone marrow stromal cells and osteoblasts contributes to a suitable microenvironment for osteoclastogenesis (10-12) and, according to novel investigations, may also stimulate bone formation due to direct activation of osteoblasts (13) (FIGURE 1). RANKL acts through binding to the transmembrane receptor molecule RANK, a member of the tumor necrosis factor receptor superfamily (14). Knockout mice deficient of RANKL (12) or its receptor RANK (15) develop severe osteopetrosis due to blockage of osteoclastogenesis. A naturally occurring decoy receptor of RANK osteoprotegerin (OPG) blocks RANKL/RANK interaction at the ligand/receptor level (16) and mice deficient of OPG are osteoporotic due to deregulation of RANKIJRANK interaction and an increased formation of osteoclasts (17). Apart from bone cells, RANKL is expressed on activated T lymphocytes, which represents a new link between the immune and skeletal systems (18) and occurs as a soluble molecule in circulation (19) suitable for laboratory assessment. Efficient bone turnover requires proper coupling of osteoblast to osteoclast function. RANKL is essential for osteoclast and possibly also osteoblast activity, and may represent a key link between bone formation and resorption.

Bone turnover is a continuous remodeling process allowing optimal adaptation of bone microarchitecture to individual demands (Mundy GR. Bone remodelling. In

Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, 4^th edition, ed. Favus MJ; 1999. Lippincott Williams & Wilkins. Pp. 30-39). Bone resorption is physiologically coupled to and usually in balance with bone formation.

Bone quality and resistance against fracture do not exclusively rely on bone mass but also on bone remodeling (2). Focus on estimates of fracture risk in osteoporosis to date, however, have focused primarily on bone mass. Established risk predictors like high age, postmenopausal status and impaired neuromuscular function all are associated with a loss in bone mass (3-7). In addition, genetic markers of low bone mass and an increased susceptibility to fracture have been identified including polymorphism in the estrogen receptor alpha- and collagen type-1 locus (8,9). By contrast, risk predictors with a focus on bone remodeling and quality remain to be identified.

Similarly, prevention of non-traumatic fracture has emphasized prevention of bone loss. Present forms of bone loss therapy are primarily anti-resorptive, in that they inhibit bone resorption processes, rather than enhance bone formation. Among the agents which have been used or suggested for treatment of osteoporosis because of their claimed ability to inhibit bone resorption are estrogen, selective estrogen receptor modulators (SERM's), calcium, calcitriol, calcitonin (Sambrook, P. et al., N.Engl.J.Med. 328:1747-1753), alendronate (Saag, K. et al., N.EngW.Med. 339:292299) and other bisphosphonates (Luckman et al., J. Bone Min. Res. 13, 581 (1998)). However, anti-resorptives fail to correct the low bone formation rate frequently involved in net bone loss, and may have undesired effects relating to their impact on the inhibition of bone resorption/remodeling or other unwanted side effects.

Despite recent advances in our understanding of RANKL and OPG function in knockout mouse models, their significance in physiological bone metabolism remains to be clarified. In. addition, a need exists in the art for estimates of fracture risk and for a marker or markers for increased susceptibility of fracture. Therefore, in view of the aforementioned deficiencies attendant with prior art methods of predicting risk, it should be apparent that there still exists a need in the art for methods and markers for predicting risk of bone^' fracture and extent or existence of bone disease, particularly with a focus on bone remodeling and quality.

The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INNENTION

This invention is based on the discovery that a low level of RANKL, particularly soluble uncomplexed RANKL, is a risk factor for non-traumatic bone fracture. It was hypothesized that RANKL is relevant to human bone turnover, and would qualify as a useful marker of fracture risk. It is herein demonstrated that the level of RANKL is a risk factor for non-traumatic bone fracture that is independent of bone density, i.e. a person with normal bone density but low RANKL is at risk for fractures, and is also independent of other known risk factors and bone metabolism markers. Other known risk factors and bone metabolism markers include, but are not limited to, age (greater than the 5^th decade), sex (female), menopausal status, diabetes, body mass index, hormone replacement therapy, levels of osteoprotegerin, levels of β-crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25-hydroxy-vitamin-D.

The invention provides a method for determining the existence or severity of bone disease, particularly bone metabolism disease, in a mammal comprising quantitating the level of RANKL, particularly soluble uncomplexed RANKL, in said mammal.

The invention provides a method for determining a subject's risk of non-traumatic bone fracture comprising quantitating the subject's level of RANKL, particularly soluble uncomplexed RANKL. Thus, it is an object of the invention to provide a method for determining the risk of non-traumatic bone fracture in a mammal comprising quantitating the level of soluble uncomplexed RANKL in said mammal.

The invention therefore provides a method for determining in a mammal whether said mammal is at risk for non-traumatic bone fracture comprising: isolating body fluid from said mammal and quantitating the level or amount of soluble uncomplexed

RANKL in the body fluid of said mammal. In a particularly preferred embodiment, the body fluid is serum. A level or amount of soluble uncomplexed RANKL in said mammal of less than 0.8 pmol/L in said serum indicates that said mammal is at risk for non-traumatic bone fracture.

It is an object of the present invention to provide a method for determining in a mammal whether said mammal is likely to suffer a non-traumatic bone fracture comprising: isolating body fluid from said mammal and quantitating the level or amount of soluble uncomplexed RANKL in the body fluid of said mammal, wherein a level or amount of soluble uncomplexed RANKL in said mammal of less than 0.8 pmol/L in said body fluid indicates that said mammal is likely to suffer a nontraumatic bone fracture. In a particularly preferred method the invention provides a method for determining in a mammal whether said mammal is likely to suffer a non- traumatic bone fracture comprising: isolating serum from said mammal and quantitating the level or amount of soluble uncomplexed RANKL in the serum of said mammal, wherein a level or amount of soluble uncomplexed RANKL in said mammal of less than 0.8 pmol L in said serum indicates that said mammal is likely to suffer a non-traumatic bone fracture.

The diagnostic utility of the present invention extends to the use of the present method in assays to identify subjects at risk for non-traumatic bone fracture, to monitor the risk level of a subject, and to assess the effect on risk of non-traumatic bone fracture of potential therapeutic agents.

A mammal or subject for whom or for which the risk of non-traumatic bone fracture is to be determined includes, but is not limited to, a mammal or subject who demonstrates other known risk factors or bone metabolism markers. Other known risk factors and bone metabolism markers include, but are not limited to, age (greater than the 5^th decade), sex (female), menopausal status, diabetes, body mass index, hormone replacement therapy, levels of osteoprotegerin, levels of β-crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25-hydroxy-vitamin-D. A mammal or subject may be suffering from bone disease, including for instance, but not limited to, osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease of bone, bone loss due to rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid treatment, metastatic bone diseases, periodontal bone loss, bone loss due to cancer, age-related loss of bone mass, other forms of osteopenia, as well as bone fractures and bone defects. Potential therapeutic agents for which the effect on the risk of non-traumatic bone fracture can ' be assessed include anti-resorptive or anabolic compounds for treatment of bone disease, including but not limited to, a bisphosphonate, a calcitonin, a calcitriol, an estrogen, selective estrogen receptor modulators (SERM's) and a calcium source, a supplemental bone formation agent parathyroid hormone (PTH) or its derivative or fragments thereof, PTH related protein (PTHrp), a bone morphogenetic protein, osteogenin, NaF, PGF^ agonists, a statin, a β-adrenergic antagonist, particularly a β2- selective adrenergic antagonist, and a RANK ligand (RANKL), including an osteogenic form of RANKL such as GST-RANKL or other oligomerized form of RANKL. It is an object of the present invention to provide a method for detecting the level or amount of RANKL, particularly soluble uncomplexed RANKL, in a mammal in which increased risk of non-traumatic bone fracture is possible or suspected to be present. In a particular embodiment, said method comprises isolating body fluid from said mammal, purifying soluble uncomplexed RANKL from said body fluid and quantitating the level of soluble uncomplexed RANKL in said body fluid. In one embodiment the body fluid is serum, the soluble uncomplexed RANKL is purified by binding to a RANKL binding protein or agent, and the quantitating is performed using a labeled anti-RANKL antibody. In one embodiment the body fluid is serum, the soluble uncomplexed RANKL is purified by binding to OPG, and the quantitating is performed using a labeled anti-RANKL antibody. In one embodiment the body fluid is serum, the soluble uncomplexed RANKL is purified by binding to RANK, and the quantitating is performed using a labeled anti-RANKL antibody.

The invention provides a method for determining a subject's risk of non-traumatic fracture by measuring the level of RANKL in said subject, in a particular embodiment by measuring the level of soluble uncomplexed RANKL. Determination of a subject's RANKL level allows determination of their relative risk of non-traumatic bone fracture. In a particular embodiment, a subject having soluble uncomplexed

RANKL level less than 1.0 pmol/L, particularly less than 0.8 pmol/L, most particularly less than 0.6 pmol/L is at significant risk of non-traumatic bone fracture.

In a particular embodiment, the invention provides a method for determining a subject's risk of non-traumatic bone fracture by measuring the level of soluble uncomplexed RANKL in said subject, wherein a subject having soluble uncomplexed RANKL level less than 1.0 pmol L is at risk of non-traumatic bone fracture. In a further particular embodiment, the invention provides a method for determining a subject's risk of non-traumatic bone fracture by measuring the level of soluble uncomplexed RANKL in said subject, wherein a subject having soluble uncomplexed

RANKL level less than 0.8 pmol L is at significant risk of non-traumatic bone fracture. In a still further particular embodiment, the invention provides a method for determining a subject's risk of non-traumatic bone fracture by measuring the level of soluble uncomplexed RANKL in said subject, wherein a subject having soluble uncomplexed RANKL level less than 0.6 pmol L is at significant risk of nontraumatic bone fracture.

In a particularly preferred method, the risk of non-traumatic bone fracture in a mammal is determined comprising quantitating the level of soluble uncomplexed RANKL in said mammal, wherein a soluble uncomplexed RANKL level which is reduced relative to the level found in a reference population indicates increased risk of non-traumatic bone fracture in said mammal versus the reference population. The invention provides a method of comparing a subject's risk of non-traumatic bone fracture with that of a reference population, comprising: determining the subject's level of soluble uncomplexed RANKL; determining a statistic characteristic of the reference population's serum level of soluble uncomplexed RANKL; and comparing the subject's level and the statistic, wherein a subject's level that is lower than the statistic indicates that the subject is at greater risk than the fraction of the reference population with a level at or above the statistic.

The assessment or monitoring of RANKL levels, particularly soluble uncomplexed RANKL levels, is contemplated using any of various diagnostic techniques, including immunoassays, such as a radioimmunoassay. In one embodiment, the assessment or monitoring of RANKL levels utilizes an antibody to RANKL that has been labeled, for instance by radioactive addition, radioiodination, biotinylation.

In an immunoassay, a control quantity of the antagonists or antibodies to RANKL, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular or serum/fluid sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached. In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ^5lCr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵1, ¹³¹I, and ¹⁸⁶Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative determination of RANKL, particularly soluble uncomplexed RANKL. The invention further includes an assay system which may be prepared in the form of a test kit to screen or identify drugs or other agents that alter RANKL levels. In a further embodiment, an assay system may be prepared in the form of a test kit to monitor RANKL levels, particularly soluble uncomplexed RANKL levels, in subjects undergoing therapy for bone disease or subjects who are participating in clinical trials or testing of agents for treatment of bone disease, including osteoporosis, diseases of bone loss or altered bone metabolism, or of bone cancer or bone metastases. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to a RANKL binding protein or agent (for instance RANK or OPG) or to an anti-RANKL antibody, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized RANKL binding protein or agent.

The invention includes an assay system for screening of potential drugs effective to modulate RANKL levels, particularly levels of soluble uncomplexed RANKL, in mammalian cells. In one instance, the test drug could be administered to a cellular sample with a RANKL binding protein or agent, or with RANKL, to determine its effect upon the levels of RANKL, particularly of soluble uncomplexed RANKL, by comparison with a control.

The assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to RANKL, either soluble RANKL and/or membrane bound RANKL, thereby altering the levels of RANKL, particularly RANKL available for binding, most particularly soluble uncomplexed RANKL. The assay system could further be adapted to identify drugs or other entities that are capable of binding to RANK, thereby altering the levels of RANKL, particularly of soluble uncomplexed RANKL. Such an assay would be useful in the development of drugs that would alter a subject's risk of fracture, particularly non-traumatic fracture or to treat osteoporosis or other bone diseases and pathologies, including bone cancer and bone metastases.

Other objects and advantages will become apparent to those skilled in the art from a review of the following description which proceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 depicts the role of receptor-activator of nuclear factor kappa B ligand (RANKL) in bone remodeling. RANKL is produced by bone marrow stromal cells, osteoblasts and activated T cells. It is synthesized as a transmembrane and as a soluble molecule and binds to receptor-activator of nuclear factor kappa B (RANK) expressed on osteoclasts and osteoblasts. This interaction is blocked by osteoprotegerin (OPG) a decoy receptor of RANK. RANKLRANK binding on osteoblasts induces bone formation, whereas binding on osteoclasts induces bone resorption. The data presented in this manuscript support the concept that high serum level of .RANKL is of benefit for bone. Sufficient bone turnover, warranted by an adequate coupling of bone formation and resorption may be relevant to bone quality and resistance against fracture.

FIGURE 2 depicts baseline distribution of serum concentration of RANKL. IQR denotes inter quartile range. FIGURE 3 depicts regression-adjusted rates of non-traumatic fractures according to sex, menopausal status and age. Calculations are based on multivariate analysis as detailed in TABLE 2.

FIGURE 4 depicts the distribution of results when dividing the population into tertiles, quartiles and quintiles.

FIGURE 5 depicts the amino acid sequence of human RANKL. The type II membrane protein of human RANKL consists of amino acids 1 through 317, MW 35.5 kD (SEQ ID NO: 1).

FIGURE 6 depicts the nucleic acid sequence of human RANKL (SEQ ID NO: 2).

DETAILED DESCRIPTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-DI [Ausubel, R. M., ed. (1994)]; "Cell Biology: A

Laboratory Handbook" Volumes I-DI [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-DI [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & SJ. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And

Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Therefore, if appearing herein, the following terms shall have the definitions set out below. As used herein, "non-traumatic bone fracture" means one resulting from a fall from standing height or less, or manifesting itself without any trauma.

As used herein "RANK" refers to Receptor Activator of Nuclear Factor (NF)-kappa B . As used herein, "RANKL" or "RANK ligand" refers to Receptor Activator of

Nuclear Factor (NF)-kappa B Ligand. RANKL includes two isoforms, a membrane bound form and a secreted or soluble form. Both forms are capable of binding or otherwise interacting with RANK. The membrane bound form of human RANKL comprises 317 amino acids and comprises the sequence set out in FIGURE 5, SEQ DO NO: 1. Soluble or secreted human RANKL is not membrane bound and therefore is capable of circulating. Soluble or secreted RANKL lacks the cytoplasmic and transmembrane domains. One example or embodiment of soluble RANKL comprises the C-terminal region of RANKL following the transmembrane domain, comprising approximately 244 amino acids. Soluble or secreted RANKL can be generated by alternative splicing of RANKL encoding RNA and/or by cleavage or proteolytic digestion of RANKL. RANKL, particularly human RANKL, refers to proteinaceous material including single or multiple proteins, and extends to those proteins RANKL and soluble RANKL including those polypeptides having the amino acid sequence data described herein and presented in FIGURE 5 (SEQ ID NO:l), and the profile of activities and diagnostic utilities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. RANKL proteins which are isoforms and are allelic variants, particularly including naturally occurring isoforms and allelic variants which are found or can be identified in a population or distinctly isolated RANKL forms, are therefore contemplated and included herein. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of RANKL. Sequences, nucleic acid and polypeptide, of RANKL, including human RANKL, are known and include those provided in Genbank Accession No. NM_003701. Lacey et al describes RANKL (OPGL) sequence and predicted circulating forms (Lacey, DL et al (1998) Cell 93(2): 165-176). Also, the terms "RANKL," "soluble RANKL" and "RANK ligand" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

As used herein, "soluble uncomplexed RANKL" means RANKL which is not bound to RANK or OPG and which is not membrane bound.

As used herein, "OPG" refers to osteoprotegerin. As used herein "OPGL" refers to osteoprotegerin ligand.

As used herein, " subject "may include any animal, including mammals, capable of suffering from bone fracture. The subjects include but are not limited to a human being, a primate, an equine, an opine, an avian, a bovine, a porcine, a canine, a feline or a mouse. The animals include but are not limited to mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates. In a preferred embodiment, the subject is a mammal. In a particularly preferred embodiment, the subject is a human being.

"Diagnosis" refers to diagnosis, prognosis, monitoring, characterizing, selecting patients, including participants in clinical trials, and identifying patients at risk for or having a particular disorder or clinical event or those most likely to respond to a particular therapeutic treatment, or for assessing or monitoring a patient's response to a particular therapeutic treatment.

"Body fluid" refers to any collectable or isolateable fluid or liquid, with or without cells, which can be collected or otherwise derived from a patient. The term body fluid includes but is not limited to blood, serum, plasma, urine, semen and saliva. Serum refers to the supernatant fluid produced by clotting and centrifugal sedimentation of a blood sample. Plasma refers to the supernatant fluid produced by inhibition of clotting (for example, by citrate or or a chelating agent such as EDTA) and centrifugal sedimentation of a blood sample. Blood refers to whole blood as collected, without particular separation on centrifugal sedimentation and includes any and/or all particular cellular, protein and lipid components of blood as well as serum. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chi eric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen,

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v), which portions are preferred for use in the methods described herein.

Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Patent No.4,493,795 to Nestor et al. A monoclonal antibody, typically containing Fab and/or F(ab')₂ portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies - A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with RANK, or RANKL. Splenocytes may be fused with myeloma cells using for instance polyethylene glycol (PEG) 6000. Fused hybrids are then selected. Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the RANKL and their ability to inhibit RANK or OPG binding activity in target cells Fab and F(ab')₂ portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al. Fab' antibody molecule portions are also well-known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.

The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic

DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques" (1980); Hammerling et al., "Monoclonal Antibodies And T- cell Hybridomas" (1981); Kennett et al., "Monoclonal Antibodies" (1980); see also U.S. Patent Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

Antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. Monoclonal antibodies may be produced by mammalian cell culture in hydridoma or recombinant cell lines such as Chinese hamster ovary cells or murine myeloma cell lines. Such methods are well- known to those skilled in the art. Bacterial, yeast, and insect cell lines can also be used to produce monoclonal antibodies or fragments thereof. In^' addition, methods exist to produce monoclonal antibodies in transgenic animals or plants (Pollock et al. J. Immunol. Methods, 231:147, 1999; Russell, Curr.Top. Microbiol. Immunol. 240:119, 1999).

As used herein, "pg" means picogram, "ng" means nanogram, "μg" or "ug" mean microgram, "mg" means milligram, "ul" or "μl" mean microliter, "ml" means milliliter, "1" means liter.

The present invention relates to a variety of diagnostic applications relating to the risk of non-traumatic bone fracture in a subject whereby RANKL levels in said subject are quantitated. The invention includes methods for determining the existence or relative extent of risk of non-traumatic bone fracture in a mammal, by reference to the amount of RANKL, particularly of soluble uncomplexed RANKL, in said mammal.

This invention provides a method of comparing a subject's risk of non-traumatic bone fracture with that of a reference population, comprising: determining the subject's serum level of soluble uncomplexed RANKL; determining a statistic characteristic of the reference population's serum level of soluble uncomplexed RANKL; and comparing the subject's level and the statistic, wherein a subject's level that is lower than the statistic_^ indicates that the subject is at greater risk than the fraction of the reference population with a level at or above the statistic.

In one embodiment of the method of the invention, the risk of non-traumatic bone fracture in a mammal is determined comprising quantitating the level of soluble uncomplexed RANKL in said mammal, wherein a soluble uncomplexed RANKL level which is reduced relative to the level found in a reference population indicates increased risk of non-traumatic bone fracture in said mammal versus the reference population. The invention provides a method of comparing a subject's risk of nontraumatic bone fracture with that of a reference population, comprising: determining the subject's level of soluble uncomplexed RANKL; determining a statistic characteristic of the reference population's serum level of soluble uncomplexed

RANKL; and comparing the subject's level and the statistic, wherein a subject's level that is lower than the statistic indicates that the subject is at greater risk than the fraction of the reference population with a level at or above the statistic.

In one embodiment for practice of this method of the invention, the comparison population can be similar to the population described herein, which is outlined in TABLE 1, and which is age matched and wherein some individuals possess identified independent risk factors such as that of bone metabolism disease, including for instance, age (greater than the 5^th decade), sex (female), menopausal status, diabetes, body mass index, hormone replacement therapy, levels of osteoprotegerin, levels of β-crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25- hydroxy-vitamin-D. In particular, the comparison or reference population is age- matched, i.e. contains individuals who are at least in the fifth decade in age. Alternatively, those skilled in the art can develop reference populations that are more closely matched in age, life-style and genetic makeup to the clinical population of interest.

In one embodiment, the population's statistic is a percentile of the population. The percentiles include but are not limited to the following: the median (50%); the 33^rd percentile; the 25th percentile; and the 20th percentile. As used herein, when referring to a specific percentile, e.g. the 33^rd percentile, this is in reference to the high. In one embodiment, the statistic is the mean. The statistic may be one standard deviation of the population from the mean. The statistic may be one standard deviation of the population lower than the mean. Alternatively, the statistic may be one standard deviation of the population higher than the mean. In another embodiment, the statistic is two standard deviations of the population from the mean. The statistic may be two standard deviations lower than the mean. Alternatively, the statisitic may be two standard deviations higher than the mean. For the population studied herein described in TABLE 1, the standard deviation is 1.15.

The risk for non-traumatic bone fracture in a subject can be due to (i.e. the direct result of) and/or correlated to low serum RANKL. Low serum RANKL can be an amount which would place the subject within a lower portion of a population when comparing the subject's RANKL level with the population's level of RANKL (or each member of the population's level). Such portions include but are not limited to the lowest half of a population, the lowest fertile of a population, the lowest quartile of a population, and the lowest quintle of a population. In one embodiment, a low serum RANKL is an amount less than 1.0 pmol L, particularly less than 0.8 pmol/L, most particularly less than 0.6 pmol/L.

The subject's body fluid, particularly serum, sample may be obtained after a time period in which the subject is or has fasted, i.e. abstaining from the consumption pf food and/or drink. The subject's serum sample may be obtained after a time period in which the subject has abstained from taking medication(s) (for instance hormone replacement therapy, birth control pills, calcium supplements), using tobacco products, alcohol, or other drugs or agents. The length and timing of the fasting period, and abstention from using medications, tobacco products, alcohol, or other drugs or agents may depend on when the subject's serum sample is obtained.

As one skilled in the art will appreciate, the RANKL being assessed in accordance with the present invention can be qualitatively or quantitatively detected by any method known to those skilled in the art, including but not limited to the sandwich type assay described herein, enzyme assays, binding assays and other functional assays, immunoassays, and western blotting, including based on the present description. In one embodiment, RANKL is assessed by sandwich type assay, wherein soluble uncomplexed RANKL is quantitatively detected by isolating or extracting soluble uncomplexed or free RANKL from a sample using a first RANKL binding protein or binding partner, for instance osteoprotegerin (OPG), and the isolated or extracted RANKL is then detected and quantitated using a second RANKL binding protein or binding partner, which is capable of binding the RANKL in the presence of the first RANKL binding protein or binding partner. In a particular such embodiment, OPG, including a chimeric OPG-Fc protein, is used as the first RANKL binding protein. In one embodiment, the second RANKL binding protein or binding partner is a RANKL specific antibody, particularly a detectably labeled RANKL specific antibody.

In one embodiment, the RANKL can be detected in any suitable immunoassay. For example, an immunoassay is performed by contacting a sample with an anti-RANKL antibody under conditions such that immunospecific binding can occur if the RANKL is present and/or soluble or unbound, and detecting or measuring the amount of any immunospecific binding by the antibody. Those skilled in the art, based on the present description, will understand how to select any suitable conditions for any desired assay or binding. One skilled in the art, based on the present description, can generate additional antibodies by using the RANKL itself for the generation of such antibodies.

Any suitable immunoassay can be used to detect RANKL, including, without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISAs (enzyme linked immunosorbent assays),

"sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays, based on the present description.

A variety of protocols for detecting and measuring the expression of a molecule as well as functional derivatives thereof, using either polyclonal or monoclonal antibodies specific for the protein are well-known in the art. Examples include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using mAbs to two non-interfering epitopes may be employed. Well- known competitive binding techniques may also be employed (see e.g., Hampton, R. et al. (1990), Serological Methods - a Laboratory Manual, APS Press, St. Paul Minn.; Maddox, D.E., et al, J. Exp. Med. 158:1211). The antibodies, or reactive fragments thereof, used in such assays may be detectable, such as being labeled with a suitable detectable marker such as a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, an enzyme, or other antibody label molecules using techniques known in the art. Such methodology is further described in Abbas et al (1994), Cellular and Molecular Immunology, 2^nd Edition, W.B. Saunders Company, which describes protocols for direct radioimmunoassay, competitive ELISA or RIA, and sandwich ELISA or RIA.

An example of a protocol for a direct radioimmunoassay (RIA) is as follows. An antibody or binding agent is affixed, such as to a well of a microtiter plate. A constant amount of labeled antigen is mixed with varying amounts of unlabeled antigen or test sample and the unbound antigen (both labeled and unlabeled) is then removed, such as by washing. The amount of label that is bound is measured. In this instance, the amount of labeled antigen bound decreases as the concentration of competing unlabeled antigen is increased, allowing quantification of unlabeled antigen. The subject invention may use any detectable label known to one skilled in the art, including a label which is radioactive, enzymatic, biotin, fluorescent or other detectable marker to facilitate detection.

An example of the protocol for a competitive assay, such as either an ELISA or a RIA, is as follows. An antigen or binding agent is affixed, such as to a microtiter plate. A constant amount of labeled antibody and varying amounts of unlabeled soluble antigen are added and the unbound antibody and unbound soluble antigen are then removed, such as by washing. The amount of labeled antibody that is bound is measured. In this instance, the amount of labeled antibody bound decreases as the concentration of unlabeled antigen (competitive inhibitor) is increased, allowing quantification of unlabeled antigen. An example of a protocol for a sandwich assay, such as either an ELISA or a RIA, is as follows. An antibody is affixed, such as to a well of a microtiter plate. Varying amounts of antigen are added and unbound antigen is removed, such as by washing. A labeled second antibody, specific for non-overlapping epitopes of the antigen, is added. Unbound labeled second antibody is removed, such as by washing. The amount of the second antibody bound is measured. Li this instance, the binding of a second, labeled antibody increases as the concentration of antigen increases, allowing quantification of antigen.

In a method or assay or kit of the present method, the binding agent or antibody may be immobilized. Immobilization may be to a solid support. The solid support may be any solid support known in the art to which the agent can be operably affixed. Solid supports include, by way of example, natural or synthetic polymers. Synthetic polymers include, by way of example, polystyrene, polyethylene and polypropylene. Natural polymers include, by way of example, latex. The solid support may be selected, for example, from the group consisting of a bead, a plate (for instance but not limited to a tissue culture or microtiter plate), a chip, a membrane, a slide, a receptacle, and a filter. Solid supports in the form of beads are widely used and readily available to those skilled in the art. Beads include, for example, latex and polystyrene beads. Solid supports in the form of filters are widely used and readily available to those skilled in the art. Filters include, for example, polyester filters (e.g., polyester leukofiltration devices) and cellulose acetate filters. The solid support can be a microtiter plate well. The solid support can be a PVDF membrane. The solid support can be a bead. In one embodiment, the solid support is a surface plasmon resonance sensor chip. In a particular embodiment, the surface plasmon resonance sensor chip can have pre-immobilized streptavidin. One example of a surface plasmon resonance sensor chip is a BIAcore7m chip.

Those skilled in the art will appreciate that the signal obtained upon analyzing fluid from subjects, including those at risk to have a non-traumatic bone fracture relative to the signal obtained upon analyzing fluid from subjects not likely to have or at relatively low risk to have a non-traumatic bone fracture will depend upon the particular analytical protocol and detection technique that is used. Accordingly, those skilled in the art will understand that a laboratory, based on the present description, can establish a suitable reference range for RANKL, particularly soluble uncomplexed RANKL, in subjects at the age where there may be a risk of non- traumatic bone fracture according to the analytical protocol and detection technique in use. In particular, in a preferred embodiment, at least one positive control sample containing a RANKL level which is indicative of significantly increased risk of nontraumatic bone fracture or at least one negative control sample containing a RANKL level which is normal or relatively elevated and which is not associated with significantly increased risk of non-traumatic bone fracture (and more preferably both positive and negative control samples) are included in each batch of test samples analyzed. In one embodiment, the amount of RANKL, particularly of soluble uncomplexed RANKL, is determined relative to a background value or a set of background values, for instance and particularly as set out in TABLE 1, which is defined as the amount of RANKL associated with each of increased risk and no relative risk for non-traumatic bone fracture.

The methods and assays of the invention can be used, for example, for detection, treatment, diagnosis, or for drug development. In one embodiment of the invention, body fluid (particularly serum) or other sample from a subject (e.g., a subject having a suspected risk of non-traumatic bone fracture by virtue of a risk factor, including for instance age, sex, pre- or post-menopausal status) is analysed for quantitative detection of RANKL, particularly of soluble uncomplexed RANKL. A decreased abundance of RANKL, particularly of soluble uncomplexed RANKL, from the subject relative to serum from a subject or subjects determined to be at reduced or not at significantly increased relative risk for non-traumatic bone fracture indicates the presence of increased risk of non-traumatic bone fracture.

A mammal or subject for whom or for which the risk of non-traumatic bone fracture is to be determined includes, but is not limited to, a mammal or subject who demonstrates other known risk factors or bone metabolism markers. Other known risk factors and bone metabolism markers include, but are not limited to, age (greater than the 5 decade), sex (female), menopausal status, diabetes, body mass index, hormone replacement therapy, levels of osteoprotegerin, levels of β-crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25-hydroxy-vitamin-D. A mammal or subject may be suffering from bone disease, including for instance, but not limited to, osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease of bone, bone loss due to rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid treatment, metastatic bone diseases, periodontal bone loss, bone loss due to cancer, age-related loss of bone mass, other forms of osteopenia, as well as bone fractures and bone defects. Potential therapeutic agents for which the effect on the risk of non-traumatic bone fracture can be assessed include anti-resorptive or anabolic compounds for treatment of bone disease, including but not limited to, a bisphosphonate, a calcitonin, a calcitriol, an estrogen, selective estrogen receptor modulators (SERM's) and a calcium source, a supplemental bone formation agent parathyroid hormone (PTH) or its derivative or fragments thereof, PTH related protein (PTHrp), a bone morphogenetic protein, osteogenin, NaF, PGE₂ agonists, a statin, a β-adrenergic antagonist, particularly a β2- selective adrenergic antagonist, and a RANK ligand (RANKL), including an osteogenic form of RANKL such as GST-RANKL or other oligomerized form of RANKL.

In accordance with the invention, RANKL, particularly soluble uncomplexed RANKL can be detected in an immunoassay. In one embodiment, an immunoassay is performed by contacting a sample with an anti-RANKL antibody under conditions such that immunospecific binding can occur if the RANKL is present, and particularly if it is not bound to its binding partner RANK or OPG, and detecting or measuring the amount of any immunospecific binding by the antibody. Anti-RANKL antibodies, including polyclonal and monoclonal antibodies, can be produced by methods and techniques well known to those of skill in the art. Examples of such antibodies known in the art which have been reported to recognize RANKL include, but are not limited to, antibodies available commercially from R&D Systems (Minneapolis, MN), Imgenix (San Diego, CA) and Active Motif (Carlsbad,CA). The skilled artisan can readily assess and determine the ability of the anti-RANKL antibody to recognize or bind to RANKL and the specificity of such binding or recognition, based on the present description. For example, and as utilized in the Example embodiment provided herein, RANKL can be detected in a fluid sample (e.g., blood or serum) by means of a two-step sandwich assay. In the first step, a capture reagent (e.g., a RANKL binding partner or binding protein, for instance OPG) is used to capture the RANKL, particularly soluble unbound RANKL. The capture reagent can optionally be immobilized on a solid phase. In the second step, a directly or indirectly labeled detection reagent is used to detect the captured RANKL. In one embodiment, the detection reagent is a labelled anti-RANKL antibody.

In the assays described herein, when an antibody is used to measure the free RANKL, it is preferred that such antibody is a RANKL antibody which binds to an epitope which is only accessible on free uncomplexed RANKL, i.e. the epitope would not be accessible when the RANKL is bound to OPG or RANK.

Panels of monoclonal antibodies produced against RANKL, including or particularly soluble RANKL, can be screened for various properties; i.e., isotype, epitope, affinity, specificity, etc. Of particular interest are monoclonal antibodies that specifically bind soluble RANKL or interact with the RANK binding site or region of RANKL. Such monoclonals can be readily identified in RANK or OPG binding assays. High affinity antibodies are also useful when immunoaffinity purification or quantitative purification for detection of RANKL, particularly soluble uncomplexed

RANKL, is desired.

Preferably, the anti-RANKL antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb). In addition, the anti-RANKL antibody molecules used herein may be in the form of Fab, Fab', F(ab')₂ or F(v) portions of whole antibody molecules. The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure, Procedure A, is described in U.S. Patent Nos. 3,654,090 and 3,850,752. Procedure C, the "sandwich" procedure, is described in U.S. Patent Nos.

RE 31,006 and 4,016,043. Still other procedures are known such as the "double antibody," or "DASP" procedure. In each instance, the RANKL forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. The RANKL or its binding partner(s) (for instance OPG) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ^5ICr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵1, ¹³¹L and ¹⁸⁶Re. Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods. The invention also provides diagnostic kits, comprising an anti-RANKL antibody. In addition, such a kit may optionally comprise one or more of the following: (1) instructions for using the anti-RANKL antibody for diagnosis, prognosis, therapeutic monitoring or any suitable combination of these applications; (2) a labelled, e.g., radioactive, fluorescent, enzymatic etc., binding partner to the antibody; (3) a solid phase (such as a reagent strip) upon which the anti-RANKL antibody is immobilized; and (4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any suitable combination thereof. If no labeled binding partner to the antibody is provided, the anti-RANKL antibody itself can be labeled with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.

In an additional embodiment, a diagnostic kit is provided comprising a RANKL binding protein or agent and one or more of the following: (1) instructions for using the RANKL binding protein or agent for diagnosis, prognosis, therapeutic monitoring or any suitable combination of these applications; (2) a labelled, e.g., radioactive, fluorescent, enzymatic etc., anti-RANKL antibody; (3) a solid phase (such as a reagent strip) upon which the RANKL binding protein or agent is immobilized; and (4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any suitable combination thereof.

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence or more particularly the amount of RANKL, particularly soluble uncomplexed RANKL. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled RANKL or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive," "sandwich," "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of the presence or amount of RANKL, particularly soluble uncomplexed RANKL, comprising: (a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of RANKL or a specific binding partner thereto, to a detectable label; (b) other reagents; and (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise: (a) a known amount of RANKL as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag; (b) if necessary, other reagents; and (c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive," "sandwich," "double antibody," etc.), and comprises: (a) a labeled component which has been obtained by coupling the RANKL or a RANKL binding protein or agent (for instance RANK or OPG) to a detectable label; (b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of: (i) a ligand capable of binding with the labeled component (a); (ii) a ligand capable of binding with a binding partner of the labeled component (a); (iii) a ligand capable of binding with at least one of the component(s) to be determined; and (iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and (c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between RANKL and a specific binding partner thereto. The invention also provides kits comprising a nucleic acid probe capable of hybridizing to RNA encoding RANKL. In a specific embodiment, a kit comprises in one or more containers a pair of primers (e.g., each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides) that under appropriate reaction conditions can prime amplification of at least a portion of a nucleic acid encoding RANKL, such as by polymerase chain reaction (see, e.g., Innis et al., 1990, PCR Protocols, Academic Press, Inc., San Diego, CA), ligase chain reaction (see EP 320,308), use of Qβ replicase, cyclic probe reaction, or other methods known in the art.

The diagnostic methods and compositions of the present invention can assist in monitoring a clinical study, e.g. to evaluate therapies for increased risk of nontraumatic bone fractures, osteoporosis, brittle bones, reduced bone mass, or related conditions. In one embodiment, chemical compounds are tested for their ability to restore RANKL levels, particularly soluble uncomplexed RANKL levels, in a subject having relatively low levels of RANKL or one of the conditions of interest herein to levels found in subjects relatively free from or not likely to have such condition(s) to preserve RANKL levels at or near levels seen in subjects at relatively low or reduced risk of non-traumatic bone fracture.

In another embodiment, the methods and assays of the present invention are used to screen individuals for entry into a clinical study to identify individuals at higher risk for non-traumatic bone fracture, etc.; individuals at high risk of such condition(s) can then be placed accordingly depending upon the goals of any given study, e.g. individuals can be excluded from the study or can be placed in a separate cohort for treatment or analysis.

In a further embodiment, the methods and compositions of the present invention are used to evaluate patients and select the appropriate cohort or group(s) of patients for clinical assessment or for evaluation in a clinical trial. The methods and compositions may be utilized to identify patients most at risk for having a disease event, such as, non-traumatic bone fracture. In accordance with the present invention, suitable test samples, e.g., of body fluid or tissue, obtained from a subject at risk of having a non-traumatic bone fracture or a subject having recognized risk factors related to bone metabolism (including, for instance, but not limited to age (greater than the 5^th decade), sex (female), menopausal status, diabetes, body mass index, hormone replacement therapy, levels of osteoprotegerin, levels of β-crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25-hydroxy-vitamin-D) can be used for diagnosis. In one embodiment, a decreased abundance of RANKL, particularly of soluble uncomplexed RANKL, in a test sample relative to a control sample (from a subject or subjects at relatively low risk of having the condition being predicted) or a previously determined reference range (for instance having RANKL levels in the third tertile as demonstrated herein) indicates the existence of an increased risk of non-traumatic bone fracture. In another embodiment, the relative abundance of RANKL in a test sample compared to a control sample or a previously determined reference range may be indicative independently of a significant risk of non-traumatic bone fracture. In a particular embodiment, a decreased abundance of RANKL is an amount in a test sample less than 1.0 pmol/L, particularly less than 0.8 pmol/L, most particularly less than 0.6 pmol L. In yet another embodiment, the relative abundance of RANKL in a test sample relative to a control sample or a previously determined reference range indicates the degree or severity of risk of non-traumatic bone fracture. In any diagnostic method, detection of RANKL may optionally be combined with detection of one or more additional risk factors or bone metabolism indicators.

In a further embodiment, a decreased abundance of mRNA encoding RANKL in a test sample relative to a control sample or a previously determined reference range indicates the risk of non-traumatic bone fracture. In a still further embodiment, the relative abundance of an mRNA encoding RANKL indicates the existence of an altered bone metabolism and the risk of non-traumatic bone fracture. Any suitable hybridization assay can be used to detect RANKL expression by detecting and/or visualizing mRNA encoding the RANKL (e.g., Northern assays, dot blots, in situ hybridization, RT-PCR, etc.). In another embodiment of the invention, labeled antibodies, derivatives and analogs thereof, which specifically bind to RANKL can be used for diagnostic purposes, e.g., to detect, diagnose, or monitor a bone metabolism disease or the risk of non-traumatic bone fracture, to predict the likely onset of a non-traumatic bone fracture, etc.

Preferably, the conditions or impending events are detected in an animal, more preferably in a mammal and most preferably in a human.

The invention provides methods for identifying agents (e.g., chemical compounds, proteins, or peptides) that have a stimulatory or inhibitory effect on the expression or activity of RANKL, e.g. that effect RANKL levels, particularly levels of soluble uncomplexed RANKL. Examples of agents, candidate compounds or test compounds include, but are not limited to, chemical agents, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules and other drugs. Agents can be obtained using any of the numerous suitable approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring decon volution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145; U.S. Patent No. 5,738,996; and U.S. Patent No. 5,807,683).

In accordance with the above, an assay system for screening potential drugs effective to modulate the level of RANKL, particularly soluble uncomplexed RANKL, may be prepared. The cells expressing RANKL or fluid therefrom may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture or test system, and the culture thereafter examined to observe any changes in the level or amount of RANKL, particularly soluble RANKL.

In one embodiment, agents that affect RANKL levels are identified in a cell-based assay system. In accordance with this embodiment, cells expressing RANKL, soluble or secreted RANKL, an active fragment thereof, or a RANKL fusion protein are contacted with a candidate compound or a control compound and the ability of the candidate compound to alter RANKL levels, e.g. to increase RANKL levels, particularly to increase soluble RANKL levels, is determined. If desired, this assay may be used to screen a plurality (e.g. a library) of candidate compounds. The cell, for example, can be of prokaryotic origin (e.g., E. coli) or eukaryotic origin (e.g., yeast, insect or mammalian). Further, the cells can express RANKL, an active fragment thereof, or a RANKL fusion protein endogenously or be genetically engineered to express RANKL, an active fragment thereof, or a RANKL fusion protein. In some embodiments, the RANKL, an active fragment thereof, or a RANKL fusion protein or the candidate compound is labeled, for example with a radioactive label (such as P, S or I) or a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde or fluorescamine) to enable detection of RANKL or a candidate compound. The ability of the candidate compound to alter levels of RANKL, an active fragment thereof, or a RANKL fusion protein can be determined by methods known to those of skill in the art.

The present invention also provides assays for use in discovery of pharmaceutical products to identify or verify the efficacy of compounds for treatment of bone metabolism or bone diseases and for reduction of the risk of non-traumatic bone fracture. Agents can be assayed for their ability to substantially restore RANKL levels in a subject having low RANKL levels and at risk for non-traumatic bone fracture towards normal or higher levels found in subjects not at risk for nontraumatic bone fracture or to produce similar changes in experimental animal models of altered bone metabolism or osteoporosis. RANKL levels and/or expression can be assayed by immunoassays, gel electrophoresis followed by visualization, detection of RANK or OPG binding, or any other method taught herein or known to those skilled in the art. Such assays can be used to screen candidate drugs, in clinical monitoring or in drug development, where abundance of RANKL can serve as a surrogate marker or biomarker for clinical disease. The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however,- as limiting the broad scope of the invention.

EXAMPLE 1

Bone underlies a continuous remodeling process allowing optimal adaptation of bone microarchitecture to individual demands (1). Bone resorption is physiologically coupled to and usually in balance with bone formation, and vice versa. It is commonly assumed that bone quality and resistance against fracture do not exclusively rely on bone mass but also on bone remodeling (2). So far, estimation of fracture risk was a clear domain of the assessment of bone mass (osteoporosis). Established risk predictors like high age, postmenopausal status and impaired neuromuscular function all are considered to go along with a loss in bone mass (3-7).

In addition, genetic markers of low bone mass and an increased susceptibility to fracture have been identified including polymorphism in the estrogen receptor alpha- and collagen type-1 locus (8,9). Risk predictors with a focus on bone remodeling and quality remain to be identified.

Recently, essential physiological interactions between osteoblasts and osteoclasts have been unraveled. Receptor activator of nuclear factor kappaB ligand (RANKL) was proposed to be a key player in this scenario. RANKL expression by bone marrow stromal cells and osteoblasts contributes to a suitable microenvironment for osteoclastogenesis (10-12) and, according to novel investigations, may also stimulate bone formation due to direct activation of osteoblasts (13) (FIGURE 1). RANKL acts by binding to the transmembrane receptor molecule RANK, a member of the tumor necrosis factor receptor superfamily (14). Knockout mice deficient of RANKL (12) or its receptor RANK (15) develop severe osteopetrosis due to blockage of osteoclastogenesis. Osteoprotegerin (OPG), a naturally occurring decoy receptor of RANK, blocks RANKL/RANK interaction at the ligand receptor level (16) and knockout mice deficient of OPG are osteoporotic due to deregulation of

RANKL/RANK interaction and an increased formation of osteoclasts (17). Apart from mesenchymal cells, RANKL is expressed on activated T lymphocytes, representing a new link between the immune and skeletal systems (18). Since RANKL occurs in circulation as a soluble molecule (19), it is suitable for laboratory assessment.

Despite recent advances in understanding of the significance of RANKL in experimental animal models, its relevance in physiological bone metabolism of humans remains to be clarified. In the present study, we investigated whether RANKL is relevant for human bone remodeling and may qualify as a useful laboratory marker of fracture risk. This hypothesis was tested in a large prospective population study.

Levels of RANKL did not differ between genders and were not related to age, menopausal status, life-style characteristics and bone density at the heel. However,

RANKL emerged as a significant risk predictor of non-traumatic fracture. In pooled logistic regression analysis the relative risks (95%CI) of non-traumatic fracture in the lowest and middle versus highest tertile group of RANKL were 10 (2.3-43.1) and 3.9 (0.8-19) (P<0.001), respectively. Remarkably, subjects in the highest tertile group appeared to be protected against fractures even in the presence of other prominent risk factors, whereas in women aged 60 or over the regression-adjusted five-year rate of non-traumatic fracture exceeded 7 percent. All associations were independent of osteoprotegerin levels.

We demonstrate and conclude that low level of RANKL is a highly significant and independent risk predictor of non-traumatic fracture. This finding is consistent with a crucial role of RANKL in human bone turnover and may gain relevance in the routine assessment of fracture risk.

METHODS

As part of the prospective population-based Bruneck Study we recorded all fractures which occurred between 1990 and 2000 and classified them as traumatic or nontraumatic (n=31). Serum levels of RANKL, osteoprotegerin and various characteristics of bone metabolism and life-style were assessed in 1990 and every five years during follow-up.

Study subjects: The Bruneck Study is a prospective population-based survey of the epidemiology and pathogenesis of atherosclerosis and disorders of the brain and bone (20-24). The study protocol was reviewed and approved by the appropriate ethics committees, and all study subjects gave their written informed consent. At the 1990 baseline the study population was recruited as a random sample, stratified according to sex and age, of all inhabitants of Bruneck (125 women and 125 men in each the 5- th to 8-th decades of age). A total of 93.6 percent participated with data assessment completed in 919 subjects. Reevaluations were performed after 5 (1995) and 10 years (2000) (23). Blood samples for measurement of RANKL and other parameters were available from 919 (1990), 826 (1995, 96.5 percent of those alive) and 700 subjects

(2000, 97.7 percent of those alive), respectively. To form the cohort for the current analysis one subject with a pathologic fracture due to bone metastasis and 12 who had experienced non-traumatic fractures before baseline were excluded. In the remaining 906 subjects determination of clinically apparent non-traumatic fractures was nearly complete (> 99 percent) during the follow-up between 1990 and 2000.

Clinical history taking and examination: Lifetime peripheral and clinically apparent vertebral fractures were carefully recorded for all study participants thereby utilizing two sources of information, the subject's self -report and a standardized reevaluation of all radiographs ever taken on study subjects. The situation in Bruneck is unique in that (1) the only X-ray facility in the whole district is located at the hospital and all radiographs were available for review, (2) it is convenient to perform radiographs in virtually all cases of injury and moderate-to-severe or long-lasting back pain, and (3) population mobility in the survey area was extremely low over the last decade. None of the fracture reported occurred outside the Bruneck area. For all radiologically confirmed fractures localization, date of occurrence and associated circumstances were recorded. Fractures were classified as non-traumatic if resulting from a fall from standing height or less or manifesting without any trauma (25). Other fractures especially all those of fingers, toes, skull, face, cervical vertebrae and chest sternum were considered traumatic (26). Vertebral fractures were radiologically defined by a decrease of at least 20 percent and 4 mm of anterior, medial or posterior vertebral height (compared to the posterior margin of the same vertebra or, if reduced, above vertebra) in lateral thoracic and lumbar spine X-rays (segments T4 to L5 (27).

All life-style variables were assessed in 1990, 1995 and 2000. Body mass index was calculated as weight divided by height squared (kg/m²). Smoking status and alcohol consumption were recorded as detailed previously (21,22). The activity score was composed of the scores for work (three categories) and sports/leisure activities (0, <2, >2 hours per week) (20). Socioeconomic status was defined with a three-category scale (low, medium, high) based on information about occupational status and educational level of the person with the highest income in the household. Diabetes was diagnosed according to standard WHO criteria. Bone density was assessed in 2000 at the heel bones using an ultrasonic bone densitometer (SAHARA, Hologic Inc., USA).

Laboratory methods:

Blood samples were drawn in 1990, 1995 and 2000 after an overnight fast and 12 hours of abstinence from smoking (20). Serum was immediately frozen and stored at - 70°C without any cycle of thawing-freezing until analysis. Serum levels of osteocalcin, parathyroid hormone, and β-crosslaps were measured by electrochemiluminescence immunoassay (ECLIA, Roche Diagnostics, Mannheim,

Germany). 25-hydroxyvitamin D was analyzed by automated chemiluminescence assay (Nichols Advantage, Nichols Institute Diagnostics, San Juan Capistrano, CA). Intra-assay coefficients of variation for osteocalcin, parathyroid hormone, β-crosslaps and 25-hydroxyvitamin D testing were low at 0.6%, 1.4% and 3.1% and 3.7% respectively. Serum levels of soluble uncomplexed RANKL were measured by a sandwich type assay. Chimaeric OPG-Fc protein (R&D Systems, Minneapolis, MN was coated on microtiter plates and used to extract free RANKL from the samples. Li a second step, RANKL captured by OPG was detected by a specific affinity-purified and biotinylated polyclonal rabbit anti-RANKL antibody (Leinco Technologies, St. Louis, MI) followed by incubation with streptavidin peroxidase and visualization with tetramethylbenzidin. Biosynthetic RANKL (Peprotech, Rocky Hill, NJ) diluted in stripped human serum was used as a standard, Litra- and inter-assay coefficients of variation were 6% and 8%. Lower detection limit of the test was 0.1 pmol/L. OPG was measured using a sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN) Recombinant OPG from Research Diagnostics Inc. (Flanders, NJ) served as a standard. Intra- and inter-assay coefficients of variation were below 10%.

Statistics:

Person-years of follow-up for each participant were accrued from the 1990 baseline until diagnosis of non-traumatic fracture, death or August 1, 2000, whichever came first. Subjects who suffered non-traumatic fractures were censored with respect to subsequent follow-up. Participants were divided into three groups according to tertiles of RANKL. Relative risks were estimated with rate ratios comparing the incidence of non-traumatic fractures in each tertile with that of the highest tertile (referent tertile) using pooled logistic regression (28,29). This technique treated each observation period (1990-1995 and 1995-2000) as a mini-follow-up study in which current risk factor measurements are employed to predict fracture risk in given intervals. Observations over both periods were pooled into a single sample. This approach has been shown to be asymptotically equivalent to the Cox regression model with time-dependent covariates given short intervals between reevaluations and low rates of events (28,29). Multivariate models were adjusted for age (years), sex (men, premenopausal women, postmenopausal women), follow-up period (1990- 1995, 1995-2000), social status (low, medium, high), smoking (no, yes), alcohol consumption (g day), physical activity (score), diabetes (no, yes), body mass index (kg/m²), creatinine levels (mg/dL), hormone replacement therapy (no, yes), and facultatively other types of medication and parameters of bone metabolism. We performed tests for linear trend by treating the medians in each category of RANKL as a continuous variable. Regression-adjusted risks of non-traumatic fractures according to tertiles of RANKL, age, sex and menopausal status were calculated with the marginal method of the regression adjustment technique (24). All reported P values are two-sided.

Results

During 8087 person-years of follow-up 31 cases of non-traumatic and 115 cases of traumatic fractures were documented. Baseline levels of RANKL and main descriptive characteristics are given in FIGURE 2. Age, sex and menopausal state were equally distributed among tertile groups of RANKL (TABLE 1). Subjects with lower levels of RANKL tended to be of lower social status, less active and were more likely to be current smokers and diabetic. However, none of these trends achieved significance once considering the performance of multiple comparisons. Furthermore, serum levels of RANKL were unrelated to bone density assessed at the heel and various parameters of bone metabolism except for OPG (modest inverse relation, P<0.001).

TABLE 1

Distribution of Demographic and Life-Style Parameters, Lidicators of Bone Metabolism, Bone Mineral Density and Medication According to Tertile Group for Receptor Activator for Nuclear Factor KB Ligand (RANKL) at the Baseline 1990.

TERTILE GROUPFORRANKL VARIABLES* 1 2 3

No. (%) of subjects 310 (34.2%) 292 (32.2%) 304 (33.6%) RANKL (pmol L) 0.60 1.00 1.60 Median Range 0.10-0.80 0.81-1.29 1.30-16.95

Demographic variables 59.6 59.2 58.2 Age (years) Sex (%) 53.2 Men 51.0 47.1 Premenopausal women 10.7 14.7 18.4 Postmenopausal women 36.1 34.3 34.5

Social status (%) Low 66.8 61.6 57.9 Medium 18.0 21.2 23.0 High 15.2 17.2 19.1

Life-style variables Smoking (%) 30.3 20.5 22.4 Alcohol consumption (g/day) 31.3 28.4 25.0 Physical activity (score) 4.2 4.3 4.5 Diabetes mellitus (%) 9.7 6.8 4.9 Body-mass index (kg/m²) 24.8 25.2 24.8

Bone metabolism / renal function 27.3 28.1 27.4 Osteocalcin (ng/mL) 3.9 3.7 3.6 Osteoprotegerin (pmol/L) 0.47 0.46 0.46 β-Crosslaps (ng/mL) 55.3 49.5 47.8 Parathyroid hormone 31.1 30.5 32.3 (pg mL) 0.50 0.50 0.50 25-hydroxy- vitamin D 0.94 0.91 0.90 (ng/mL) Bone mineral density (unit)^1, Creatinine (mg/dL) Medication Corticosteroids (%) 1.0 0.0 0.7 Cumarine (%) 0.3 0.3 0.0 Biphosphonates (%) 0.0 0.0 0.0 Statins (%) 0.0 0.0 0.0 Hormone replacement 11.7 9.1 11.2 therapy*(%)

* Values presented are unadjusted means or percentages. Factors to convert conventional units into SI units are as follows: osteocalcin [ng/mL] x 0.171 [pmol/L], β-crosslaps [ng/mL] x 8.857 [pmol/L], parathyroid hormone [pg/mL] x 1.0 [ng L], 25-hydroxy-vitamin D [ng/mL] x 2.496 [nmol/L], creatinine [mg/dL] x 88.4 [μmol/L]. t Bone mineral density was measured in 2000 at the left and right heel (n=683). Values presented are the means of both measurements. If the categorization of RANKL tertiles was based on RANKL measurements during the

2000 follow-up examination means of bone mineral density in the three categories were as follows: 0.49, 0.49 and 0.50. t Percentages of women on hormone replacement therapy.

The incidence of non-traumatic fractures varied from 0.7 per 1000 person-years in the highest to 8.1 per 1000 person-year in the lowest tertile group of RANKL (TABLE 2). In pooled logistic regression analyses adjusted for age, sex, menopausal status and period of follow-up (n=1712) the 5-year risk of non-traumatic fracture continuously increased with decreasing tertiles of RANKL (relative risk, 3.9 and 10.0 as compared with the risk in the highest tertile group, P for trend <0.001, TABLE 2). In multivariate analyses, after simultaneous control for a variety of demographic and life-style variables, creatinine concentrations and hormone replacement therapy, results did not change appreciably (relative risk, 4.0 and 9.7, P for trend <0.001). In this model, the relative risk of non-traumatic fractures was 1.4 (0.9-2.3, P=0.15) for a 10-year increase in age, and 4.2 (0.4-47.3, P=0.25) and 9.7 (2.8-33.3, P<0.001) for pre- and postmenopausal women versus men. Additional adjustment for levels of OPG and other parameters of bone metabolism and/or concomitant medication had no further effect (TABLE 2). As expected, corresponding Cox regression models with time-dependent covariates yielded results very similar to those of the pooled logistic regression models (TABLE 2). There was no evidence for a differential association between RANKL and non-traumatic fracture in men and women (interaction term, P=0.66), different age groups (interaction term, P=0.66) and the findings emerged consistent in analyses separately focusing on hip and vertebral fractures.

TABLE 2

Relative Risk of Nontraumatic Fractures According to Tertile Group for Receptor Activator for Nuclear Factor KB Ligand (RANKL)

TERTILE GROUP FOR RANKL P FOR TREND VARIABLES 1 2 3

RANKL (pmol/L) Median 0.60 1.00 1.60 Range 0.10-0.80 0.81-1.29 1,30-16.95 No.of nontraumatic 22 7 2 • fractures 2726 2545 2816 Person-yr of follow-up* 8.1 2.8 0.7 Incidence (events per 1000 person years) Relative risk (95% confidence interval) Type of analysis Age- and sex-adjusted* 10.0 (2.3-43.1) 3.9 (0.8-19.0) 1.0 <0.001 Multivariate* 9.7 (2.2-42.1) 4.0 (0.8-19.7) 1.0 <0.001 Multivariate, including 9.4 (2.2-40.8) 3.8 (0.8-18.6) 1.0 <0.001 parameters of bone metabolism⁵ Multivariate.Cox model** 9.4 (2.2-40.1) 3.9 (0.8-18.6) 1.0 <0.001

• *Cut-off values for tertile groups were defined based on the baseline distribution of RANKL. Allocation of person-time to each tertile group was based on 1990 level of RANKL for the first 5-year period (1990-1995) and 1995 level of RANKL for the second 5-year period (1995-2000). t The model included variables for age (years), period during the study (two five-year periods) and sex menopausal status (men, premenopausal women, postmenopausal women). $The multivariate relative risk was adjusted for age (years), period during the study (two five-year periods), sex/menopausal status (men, premenopausal women, postmenopausal women), smoking (0,1), alcohol consumption (g/day), physical activity (score), diabetes (0,1), body mass index (kg/m²), creatinine levels (unit) and hormone replacement therapy. §This model was additionally adjusted for levels of osteoprotegerin, osteocalcin, β-crosslaps, parathyroid hormone, and 25-hydroxy-vitamin D^ II Cox regression analysis with time-dependent covariates. Next, we calculated regression-adjusted rates of non-traumatic fractures in subgroup according to age, sex and menopausal statues (FIGURE 3). In brief, subjects in the highest tertile group for RANKL faced a low risk of non-traumatic fracture irrespective of age and sex. On the other hand, fracture risk steeply increased from the highest to the lowest tertile group for RANKL especially in postmenopausal women. Li contrast to non-traumatic fractures, traumatic fractures were not related to RANKL in multivariate pooled logistic regression models (relative risk in the lowest and middle versus highest tertile for RANKL, 1.1 (0.7-1.8) and 1.0 (0.6-1.7), P for trend = 0.72) (adjustment see TABLE 2).

As anticipated, the relation between RANKL and non-traumatic fracture was independent of serum OPG. OPG per se was associated with the risk of non-traumatic fracture (relative risk in the lowest and middle versus highest tertile group for OPG, 0.2 (0.1-0.6) and 0.4 (0.2-0.9), P for trend = 0.001) but this relation disappeared after adjustment for age (relative risk, 0.5 (0.2-1.8) and 0.6 (0.2-1.6), P for trend = 0.23) and other variables (relative risk, 0.5 (0.2-1.8) and 0.6 (0.2-1.5), P for trend = 0.20) due to a high correlation between OPG and age (Spearman correlation coefficient r = 0.52, P <0.001).

Level of RANKL did not differ between genders and were not related to age, menopausal status, life-style characteristics and bone density at the heel. However, RANKL emerged as a significant risk predictor of non-traumatic fracture. In pooled logistic regression analysis the relative risks (95%CI) of non-traumatic fracture in the lowest and middle versus highest tertile group of RANKL were 10 (2.3-43.1) and 3.9

(0.8-19) (P<0.001). Remarkably, subjects in the highest tertile group appeared to be protected against fractures even in the presence of other prominent risk conditions, whereas in women aged 60 or over the regression-adjusted five-year rate of nontraumatic fracture exceeded 7 percent. All associations were independent of osteoprotegerin levels. Thus, low level of RANKL is a highly significant and independent risk predictor of non-traumatic fracture. This finding is consistent with a crucial role of RANKL in human bone turnover and may gain relevance in the routine assessment of fracture risk.

Discussion

RANKL is a recently discovered protein with structure homology to tumor necrosis factor alpha. It is expressed by osteoblasts, bone marrow stromal cells and activated T cells and acts through binding to the transmembrane receptor RANK (10-12). For several reasons RANKL has been proposed to be a key factor in physiological bone remodeling: 1) RANKL stimulates osteoclastogenesis and induces osteoclast activation (). 2) Recent data suggest that RANKL directly activates osteoblasts and triggers bone formation at concentrations well below those necessary to induce osteoclastogenesis (13). 3) RANKL is involved in coupling since its expression on osteoblasts stimulates osteoclastogenesis (). 4) Juvenile Paget's disease, a rare genetic condition of very high bone turnover, is associated with excessively elevated serum levels of RANKL (>10 times those measured in healthy individuals) (30).

Given the concept that RANKL is relevant to physiological bone remodeling, low levels of RANKL may unfavorably affect bone microarchitecture and enhance fracture risk. Actually, our prospective study demonstrates that a low serum level of RANKL is a highly significant risk predictor for non-traumatic fractures in the general population independent of age, sex, menopausal status, levels of OPG and life-style characteristics. This finding adds strong epidemiological support to a role of RANKL in bone remodeling and extends previous experimental findings (11-13). Lack of an association of RANKL with osteocalcin and β-crosslaps in our population is not in contradiction to the concept because the process of a continuous adaptation of bone microarchitecture putatively occurs within a physiological range of bone turnover. As a further interesting observation, serum level of RANKL was unrelated to bone density, which again fits well into the concept that RANKL affects bone quality rather than bone mass. Owing to the fact that bone density was assessed at the heel and standard radiological measurements of bone mass at predilection sites of osteoporosis were not available, this observation should be regarded as preliminary.

Due to increasing life expectancy non-traumatic fractures are a steadily growing health burden in industrialized countries with important medical, social and financial dimensions (31-33). In the past years drugs have become available which prove highly efficacious in reducing fracture risk. This brings along the challenge of accurately identifying subjects with the highest benefit from such therapy (33). Currently, risk assessment is a clear domain of quantitative measures of bone density. Although some laboratory parameters such as β-crosslaps were reported to predict the risk of hip fracture in subgroups of patients (34) no blood test has so far found broad access to the routine estimation of fracture risk. RANKL is a promising candidate to close this gap. Notably, in our study subjects in the highest tertile of RANKL appeared to enjoy excellent protection against non-traumatic fractures (incidence < 1 per 1000 person-years) irrespective of the presence or absence of other prominent risk predictors, whereas women of 60 or older with a serum RANKL within the lowest tertile faced a regression-adjusted 5-year rate of fracture exceeding 7 percent. Strengths of our study including its representative nature for the general community (near complete participation and follow-up), the broad age range studied (40 to 89 years) and the high degree of accuracy in assessing and classifying fractures in the setting of a population study. Although, clinically inapparent vertebral fractures where not assessed in our study, such events are of little relevance from a clinical viewpoint, and assessment of minor disease phenotypes may be expected to weaken evident relations rather than to create spurious ones.

Our study identifies low serum level of RANKL as a novel and significant risk predictor of non-traumatic fractures unrelated to age, gender, menopausal status and bone density at the heel. This finding is consistent with a crucial role of RANKL in human bone turnover and may gain relevance in the routine assessment of fracture risk.

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8. Salmen T, Heikkinen AM, Mahonen A, et al. The protective effect of hormone- replacement therapy on fracture risk is modulated by estrogen receptor alpha genotype in early postmenopausal women.J Bone Miner Res. 2000;15:2479-86.

9. Uitterlinden AG, Burger H, Huang Q, et al. Relation of alleles of the collagen type Ialphal gene to bone density and the risk of osteoporotic fractures inpostmenopausal women. N Engl J Med. 1998;338:1016-21.

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12. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. 1999;397:315-23

13. Lam J, Ross FP, Teitelbaum SL. RANK ligand stimulates anabolic bone formation. J Bone Min Res 2001; 16 (SI): 1053.

14. Hsu H, Lacey DL, Dunstan CR, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A. 1999;96:3540-5.

15. Dougall WC, Glaccum M, Charrier K, et al. RANK is essential for osteoclast and lymph node development. Genes Dev. 1999;13:2412-24. 16. Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997;89:309-19. 17. Bucay N, Sarosi I, Dunstan CR, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998;12:1260-8. 18. Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature. 1999;402:304-9. 19. Lum L, Wong BR, Josien R, et al. Evidence for a role of a tumor necrosis factor- alpha (TNF-alpha)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival. J Biol Chem. 1999;274:13613-8.

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30. Whyte MP, Obrecht SE, Finnegan PM, et al. Osteoprotegerin deficiency and juvenile Pagefs disease.N Engl J Med. 2002;347:175-84. 31. Cauley JA, Thompson DE, Ensrud KC, Scott JC, Black D. Risk of mortality following clinical fractures. Osteoporos Lit. 2000;11:556-61. 32. Center JR, Nguyen TV, Schneider D, Sambrook PN, Eisman JA. Mortality after all major types of osteoporotic fracture in men and women: an observational study. Lancet. 1999;353:878-82. 33. Brown JP, Josse RG. 2002 clinical practice guidelines for the diagnosis and management of osteoporosis in Canada. CMAJ. 2002;167:Sl-34. 34. Chapurlat RD, Garnero P, Breart G, Meunier PJ, Delmas PD. Serum type I collagen breakdown product (serum CTX) predicts hip fracture risk in elderly women: the EPJOOS study. Bone. 2000;27:283-6.

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrate and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. A method for determining the risk of non-traumatic bone fracture in a mammal comprising quantitating the level of RANKL in said mammal.

2. The method of Claim 1 wherein the level of soluble uncomplexed RANKL is quantitated in said mammal.

3. The method of Claim 2 wherein the level of soluble uncomplexed RANKL is less than 1.0 pmol/L and said mammal is thereby determined to have significant risk of non-traumatic bone fracture.

4. The method of Claim 2 wherein the level of soluble uncomplexed RANKL is less than 0.8 pmol/L and said mammal is thereby determined to have significant risk of non-traumatic bone fracture.

5. The method of Claim 2 wherein the level of soluble uncomplexed RANKL is less than 0.6 pmol/L and said mammal is thereby determined to have significant risk of non-traumatic bone fracture.

6. The method of Claim 1 wherein the level of RANKL is quantitated by an immunoassay.

7. The method of Claim 1 wherein the level of RANKL is quantitated in body fluid of said mammal by binding soluble uncomplexed RANKL to a RANKL binding protein or agent.

8. The method of Claim 7 wherein the RANKL binding protein or agent is labeled.

9. The method of Claim 7 wherein the RANKL binding protein or agent is selected from the group of anti-RANKL antibody, RANK and OPG.

10. A method for determining in a mammal whether said mammal is likely to suffer a non-traumatic bone fracture comprising:

(a) isolating serum from said mammal; and

(b) quantitating the level or amount of soluble uncomplexed RANKL in the serum of said mammal.

11. The method of Claim 10, wherein a level or amount of soluble uncomplexed RANKL in said mammal of less than 0.8 pmol/L indicates that said mammal is likely to suffer a non-traumatic bone fracture.

12. The method of Claim 10 wherein quantitating the level or amount of soluble uncomplexed RANKL is determined by purifying soluble uncomplexed RANKL by binding to a first RANKL binding protein or agent and quantitating purified RANKL using a second RANKL binding protein or agent.

13. The method of Claim 12 wherein the first and second RANKL binding proteins or agents are distinct and are selected from the group of anti-RANKL antibody, RANK and OPG.

14. The method of Claim 12 wherein the second RANKL binding protein or agent is labeled.

15. The method of Claim 12 wherein the first RANKL binding protein or agent is immobilized.

16. A method for detecting the level or amount of soluble uncomplexed RANKL in a mammal in which increased risk of non-traumatic bone fracture is possible or suspected to be present comprising:

(a) isolating body fluid from said mammal;

(b) purifying soluble uncomplexed RANKL from said body fluid, and;

(c) quantitating the level of soluble uncomplexed RANKL in said body fluid.

17. The method of Claim 16 wherein the body fluid is serum, the soluble uncomplexed RANKL is purified by binding to a RANKL binding protein or agent, and the quantitating is performed using a labeled anti-RANKL antibody.

18. The method of Claim 17 wherein the soluble uncomplexed RANKL is purified by binding to OPG.

19. The method of Claim 17 wherein the soluble uncomplexed RANKL is purified by binding to a RANK.

20. The method of Claim 17 wherein the agent is immobilized.

21. The method of Claim 16 wherein purifying and quantitating is performed by an immunoassay.

22. A method for determining the risk of non-traumatic bone fracture in a mammal comprising quantitating the level of soluble uncomplexed RANKL in said mammal, wherein a soluble uncomplexed RANKL level which is determined to be reduced relative to the level found in a reference population indicates increased risk of non-traumatic bone fracture in said mammal versus the reference population.

23. The method of Claim 22 wherein the determination of whether RANKL is reduced relative to the level found in a reference population comprises:

(a) determining a statistic characteristic of the reference population's serum level of soluble uncomplexed RANKL; and

(b) comparing the subject's level and the statistic, wherein a subject's level that is lower than the statistic indicates that the subject is at greater risk than the fraction of the reference population with a level at, or above the statistic.

24. The method of Claim 23 wherein the statistic is a percentile of the population or is the mean.

25. The method of Claim 22 wherein the reference population is a population of individuals including individuals with other known risk factors and bobe matabolism markers associated with osteoporosis or with non-traumatic bone fractures.

26. The method of Claim 22 wherein quantitating is performed by an immunoassay.

27. A test kit to monitor soluble uncomplexed RANKL levels in subjects undergoing therapy for bone disease or subjects who are participating in clinical trials or testing of agents for treatment of bone disease comprising a labeled RANKL binding protein or agent and one or more additional immunochemical reagents

28. The test kit of Claim 27 wherein at least one of the additional immunochemical reagents is a free or immobilized RANKL binding protein or agent.

29. The test kit of Claim 27 wherein the RANKL binding protein or agent is selected from the group of anti-RANKL antibody, RANK and OPG.

30. An assay system for screening or identifying drugs or other agents that alter soluble uncomplexed RANKL levels comprising a labeled RANKL binding protein or agent and one or more additional immunochemical reagents.

31. The assay system of Claim 30 wherein the drug or other agent is administered to a cellular sample with a RANKL binding protein or agent, or with RANKL, to determine its effect upon the levels of soluble uncomplexed RANKL, by comparison with a control.