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WO2005060627A2 - Methodes permettant d'evaluer un risque de fracture osseuse non traumatique - Google Patents

Methodes permettant d'evaluer un risque de fracture osseuse non traumatique Download PDF

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Publication number
WO2005060627A2
WO2005060627A2 PCT/US2004/041489 US2004041489W WO2005060627A2 WO 2005060627 A2 WO2005060627 A2 WO 2005060627A2 US 2004041489 W US2004041489 W US 2004041489W WO 2005060627 A2 WO2005060627 A2 WO 2005060627A2
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rankl
level
bone
mammal
risk
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PCT/US2004/041489
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WO2005060627A3 (fr
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Georg Schett
Stefan Kiechl
Johann Willeit
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Auxeris Therapeutics, Inc.
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Publication of WO2005060627A2 publication Critical patent/WO2005060627A2/fr
Publication of WO2005060627A3 publication Critical patent/WO2005060627A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • 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.
  • 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,
  • RANK ligand 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.
  • RANKL nuclear factor kappaB ligand
  • FIG. 1 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).
  • OPG RANK osteoprotegerin
  • 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.
  • 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.
  • bone loss therapy is primarily anti-resorptive, in that they inhibit bone resorption processes, rather than enhance bone formation.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -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.
  • RANKL soluble uncomplexed RANKL
  • 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 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.
  • 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 ⁇ -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.
  • 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, cortic
  • 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 ⁇ -adrenergic antagonist, particularly a ⁇ 2- selective adrenergic antagonist, and a RANK ligand (RANKL), including an osteogenic form of RANKL such as GST-RANKL or other oligomerized form of RANKL.
  • a bisphosphonate a calcitonin, a calcitriol
  • an estrogen selective estrogen receptor modulators
  • 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.
  • the body fluid is serum
  • the soluble uncomplexed RANKL is purified by binding to a RANKL binding protein or agent
  • the quantitating is performed using a labeled anti-RANKL antibody.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 is contemplated using any of various diagnostic techniques, including immunoassays, such as a radioimmunoassay.
  • the assessment or monitoring of RANKL levels utilizes an antibody to RANKL that has been labeled, for instance by radioactive addition, radioiodination, biotinylation.
  • 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.
  • the labeled material or its binding partner(s) may be examined by known techniques, which may vary with the nature of the label attached.
  • a radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, 5l Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 1, 131 I, and 186 Re are used, known currently available counting procedures may be utilized.
  • 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.
  • 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.
  • a RANKL binding protein or agent for instance RANK or OPG
  • an anti-RANKL antibody their agonists and/or antagonists
  • 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.
  • 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.
  • FIGURE 1 depicts the role of receptor-activator of nuclear factor kappa B ligand (RANKL) in bone remodeling.
  • RANKL nuclear factor kappa B ligand
  • 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.
  • OPG osteoprotegerin
  • RANKLRANK binding on osteoblasts induces bone formation, whereas binding on osteoclasts induces bone resorption.
  • OPG osteoprotegerin
  • 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).
  • non-traumatic bone fracture means one resulting from a fall from standing height or less, or manifesting itself without any trauma.
  • RANK refers to Receptor Activator of Nuclear Factor (NF)-kappa B .
  • RNKL or “RANK ligand” refers to Receptor Activator of
  • RANKL 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.
  • 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.
  • soluble uncomplexed RANKL means RANKL which is not bound to RANK or OPG and which is not membrane bound.
  • OPG refers to osteoprotegerin.
  • OPGL refers to osteoprotegerin ligand.
  • 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.
  • the subject is a mammal.
  • 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.
  • 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,
  • 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.
  • 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.
  • PEG polyethylene glycol
  • 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.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic
  • 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.
  • pg means picogram
  • ng means nanogram
  • ⁇ g means microgram
  • ⁇ g means milligram
  • ul means 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.
  • 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.
  • 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.
  • 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 RANKL
  • 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 ⁇ -crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25- hydroxy-vitamin-D.
  • the comparison or reference population is age- matched, i.e. contains individuals who are at least in the fifth decade in age.
  • 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.
  • 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.
  • 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.
  • the statistic may be one standard deviation of the population higher than the mean.
  • the statistic is two standard deviations of the population from the mean.
  • the statistic may be two standard deviations lower than the mean.
  • the statisitic may be two standard deviations higher than the mean.
  • 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.
  • 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.
  • medication(s) for instance hormone replacement therapy, birth control pills, calcium supplements
  • 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.
  • 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.
  • 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.
  • OPG including a chimeric OPG-Fc protein
  • the second RANKL binding protein or binding partner is a RANKL specific antibody, particularly a detectably labeled RANKL specific antibody.
  • the RANKL can be detected in any suitable immunoassay.
  • 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.
  • 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).
  • ELISA enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • FACS fluorescent activated cell sorting
  • 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.
  • 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.
  • 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.
  • 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
  • An example of a protocol for a direct radioimmunoassay 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.
  • 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.
  • 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.
  • the solid support is a surface plasmon resonance sensor chip.
  • the surface plasmon resonance sensor chip can have pre-immobilized streptavidin.
  • One example of a surface plasmon resonance sensor chip is a BIAcore7m chip.
  • 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.
  • 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.
  • 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.
  • body fluid particularly serum
  • 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
  • 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 ⁇ -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.
  • 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, cortic
  • 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, PGE 2 agonists, a statin, a ⁇ -adrenergic antagonist, particularly a ⁇ 2- selective adrenergic antagonist, and a RANK ligand (RANKL), including an osteogenic form of RANKL such as GST-RANKL or other oligomerized form of RANKL.
  • a bisphosphonate a calcitonin, a calcitriol
  • an estrogen selective estrogen receptor modulators
  • PTH
  • RANKL particularly soluble uncomplexed RANKL
  • 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.
  • RANKL examples include, but are not limited to, antibodies available commercially from R&D Systems (Minneapolis, MN), Imgenix (San Diego, CA) and Active Motif (Carlsbad,CA).
  • R&D Systems Minneapolis, MN
  • Imgenix San Diego, CA
  • 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.
  • RANKL can be detected in a fluid sample (e.g., blood or serum) by means of a two-step sandwich assay.
  • a capture reagent e.g., a RANKL binding partner or binding protein, for instance OPG
  • the capture reagent can optionally be immobilized on a solid phase.
  • a directly or indirectly labeled detection reagent is used to detect the captured RANKL.
  • the detection reagent is a labelled anti-RANKL antibody.
  • an antibody when 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 can be screened for various properties; i.e., isotype, epitope, affinity, specificity, etc.
  • monoclonal antibodies that specifically bind soluble RANKL or interact with the RANK binding site or region of RANKL.
  • 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
  • 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).
  • 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.
  • 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.
  • 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
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 C1, 5I Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 1, 131 L and 186 Re.
  • 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, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -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.
  • 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.
  • a detectable marker e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.
  • a diagnostic kit 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.
  • a labelled e.g., radioactive, fluorescent, enzymatic etc., anti-RANKL antibody
  • a solid phase such as a reagent strip
  • 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.
  • 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.
  • 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.
  • 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.
  • 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(
  • kits comprising a nucleic acid probe capable of hybridizing to RNA encoding RANKL.
  • 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 Q ⁇ replicase, cyclic probe reaction, or other methods known in the art.
  • primers e.g., each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25-hydroxy-vitamin-D) can be used for diagnosis.
  • 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 ⁇ -crosslaps, levels of osteocalcin, levels of parathyroid hormone, and levels of 25-hydroxy-vitamin-D
  • risk factors related to bone metabolism including, for instance, but not limited to age (greater than the 5
  • 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.
  • 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.
  • 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.
  • 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.
  • detection of RANKL may optionally be combined with detection of one or more additional risk factors or bone metabolism indicators.
  • 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.
  • 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.).
  • 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.
  • 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.
  • 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.
  • biological libraries include 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).
  • 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.
  • agents that affect RANKL levels are identified in a cell-based assay system.
  • 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.
  • 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.
  • 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.
  • 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.
  • a radioactive label such as P, S or I
  • a fluorescent label such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde or fluorescamine
  • 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.
  • RANKL nuclear factor kappaB ligand
  • FIG. 1 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).
  • RANKL/RANK interaction and an increased formation of osteoclasts (17).
  • RANKL 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.
  • RANKL emerged as a significant risk predictor of non-traumatic fracture.
  • 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.
  • 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.
  • 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
  • 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).
  • Body mass index was calculated as weight divided by height squared (kg/m 2 ).
  • 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).
  • 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, ⁇ -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.
  • 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%.
  • 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 metabolism 5 Multivariate.Cox model** 9.4 (2.2-40.1) 3.9 (0.8-18.6) 1.0 ⁇ 0.001 ⁇ 0.001
  • 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).
  • RANKL is relevant to physiological bone remodeling
  • low levels of RANKL may unfavorably affect bone microarchitecture and enhance fracture risk.
  • 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 ⁇ -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.
  • Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165-76.
  • Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/ RANKL. Proc Natl Acad Sci U S A. 1998;95:3597-602.
  • TNF-alpha tumor necrosis factor- alpha
  • Kiechl S Willeit J, Rungger G, Egger G, Oberhollenzer F, Bonora E. Alcohol consumption and atherosclerosis: What is the relation? Prospective results from the Bruneck Study. Stroke 1998;29:900-7.
  • Kiechl S Lorenz E, Reindl M, et al. Toll-like receptor 4 polymorphisms and atherogenesis in humans. N Engl J Med 2002;347: 185-92.

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Abstract

L'invention concerne l'identification d'un ligand RANK (RANKL) associé à un marqueur et utilisé comme marqueur de conditions métaboliques osseuses, et plus particulièrement la prédiction d'un risque de fractures osseuses non traumatiques. L'invention concerne également des dosages, des méthodes et des kits comprenant RANKL et/ou des anticorps contre celui-ci à des fins de criblage, de diagnostic, de prédiction et de surveillance. L'invention concerne enfin des méthodes permettant de déterminer un risque de fracture osseuse non traumatique chez un sujet, qui consistent à déterminer le niveau de RANKL chez ce sujet, en particulier, le niveau d'un RANKL soluble non complexé.
PCT/US2004/041489 2003-12-10 2004-12-10 Methodes permettant d'evaluer un risque de fracture osseuse non traumatique WO2005060627A2 (fr)

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US10143747B2 (en) 2010-09-22 2018-12-04 Imba—Institut Für Molekulare Biotechnologie Gmbh Breast cancer therapeutics
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WO2012038505A1 (fr) 2010-09-22 2012-03-29 Imba - Institut Für Molekulare Biotechnologie Gmbh Diagnostic du cancer du sein
CN103238067A (zh) * 2010-09-22 2013-08-07 分子生物技术院有限公司 乳腺癌诊断学
JP2013542419A (ja) * 2010-09-22 2013-11-21 イーエムベーアー−インスティテュート フュール モレクラレ バイオテクロノジー ゲゼルシャフト ミット ベシュレンクテル ハフツング 乳癌の診断
US10143747B2 (en) 2010-09-22 2018-12-04 Imba—Institut Für Molekulare Biotechnologie Gmbh Breast cancer therapeutics
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