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WO1998007032A1 - Procede et trousse d'evaluation de la resorption osseuse - Google Patents

Procede et trousse d'evaluation de la resorption osseuse Download PDF

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Publication number
WO1998007032A1
WO1998007032A1 PCT/US1997/014434 US9714434W WO9807032A1 WO 1998007032 A1 WO1998007032 A1 WO 1998007032A1 US 9714434 W US9714434 W US 9714434W WO 9807032 A1 WO9807032 A1 WO 9807032A1
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Prior art keywords
tgf
level
bone
latent
resoφtion
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PCT/US1997/014434
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English (en)
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David J. Grainger
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Metra Biosystems, Inc.
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Priority to AU41520/97A priority Critical patent/AU4152097A/en
Publication of WO1998007032A1 publication Critical patent/WO1998007032A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention relates to methods for detecting screening for and monitoring bone metabolism abnormalities, particularly those relating to elevated bone reso ⁇ tion
  • the invention also relates to reagents and kits for use in such methods
  • a variety of disease conditions in humans are characterized by an elevated level of bone reso ⁇ tion or by an abnormal imbalance between bone reso ⁇ tion and bone formation.
  • these conditions are osteoporosis, osteoarthritis. hyperthyroidism, hype ⁇ arathyroidism, osteomalacia, osteosarcomas, and Paget's disease (Epstein, 1986; Van
  • Osteoporosis is particularly problematic, affecting upwards of twenty million people in the United States alone. Osteoporosis is characterized by pathologically low bone density, often first presenting clinically with fractures. Both the deposition of bone, which is mediated by osteoblasts, and bone reso ⁇ tion, which is mediated by osteoclasts, occur in an ongoing recycling process of the bone matrix throughout the lifetime of humans.
  • agents have been found to affect the rate of deposition of inorganic matrix by cultured osteoblasts, either by affecting the rate of proliferation of these cells or by increasing their differentiation status. Similarly, agents have been identified which modulate the rate of reso ⁇ tion by osteoclasts in bone explant cultures. The in vivo roles of many of these agents remain to be clarified, and the signalling pathways which mediate deposition and reso ⁇ tion are not well understood.
  • a number of serum and/or urinary substances have been proposed as possible indicators for bone degradation.
  • Exemplary substances which have been studied include hydroxyproline, tartrate-resistant acid phosphatase, galactosyl hydroxylysine, various collagen peptides, and peptide-free pyridinium species (e.g. , Taylor, 1994).
  • the invention includes a method of assessing the status of bone reso ⁇ tion in a mammalian subject, based on measuring the level of total TGF-B3, or a selected subform thereof, in a body fluid sample from the subject.
  • a level of total TGF-B3 in the sample is determined.
  • the determined level is compared with a predetermined threshold or level characteristic of normal subjects, wherein a determined level that is above a level characteristic of normal subjects is an indication that the subject has a bone reso ⁇ tion disorder.
  • a level below a selected threshold which may be different from the first threshold, may indicate that the subject's level of bone reso ⁇ tion is within normal limits.
  • the invention includes a method of assessing the status of bone reso ⁇ tion in a mammalian subject
  • the levels of (1) non-latent TGF- ⁇ and (n) the sum of small latent TGF- ⁇ plus mature TGF- ⁇ 1 dimer are determined in a body fluid from the subject
  • the determined levels are compared with each other, where a determined ratio of non-latent TGF- ⁇ versus small latent TGF- ⁇ plus mature ⁇ l dimer (non-latent/(small latent TGF- ⁇ + mature ⁇ l)) above a selected threshold is an indication that a bone reso ⁇ tion disorder may be present in the subject
  • a ratio below a selected threshold which may be different from the first threshold, may be used as an indication that the subject's level of bone reso ⁇ tion is within normal limits
  • the methods of the invention are useful in the context of detecting or aiding in the diagnosis of a variety of disorders associated with bone metabolism abnormalities (bone reso ⁇ tion disorders), such as osteoporosis, osteoarth ⁇ tis, hype ⁇ arathyroidism rheumatoid arthritis, Paget's disease, or a malignant tumor or metastatic cancer in bone
  • bone reso ⁇ tion disorders such as osteoporosis, osteoarth ⁇ tis, hype ⁇ arathyroidism rheumatoid arthritis, Paget's disease, or a malignant tumor or metastatic cancer in bone
  • the methods of the invention may be used as a screening tool to identify subjects from the general population who are at increased risk for elevated bone loss or bone fracture, where a level, ratio or difference of TGF- ⁇ species above a selected threshold is an indication of increased risk of developing a bone reso ⁇ tion disorder, such as osteoporosis, or of suffering a bone fracture
  • the methods of the invention may also be used in combination with other diagnostic methods, such as radiographic techniques for measuring bone density, to further define a patient's condition That is, if an elevated level, ratio, or difference of an analyte or analytes of the invention is detected, indicating the presence of a bone reso ⁇ tion disorder or an increased risk for such a disorder or bone fracture, the subject is then further tested using an independent technique, such as lumbar spine bone mineral density (LS-BMD) measurements, to further characterize the health status of the subject
  • LS-BMD lumbar spine bone mineral density
  • the methods of the invention may also include determining the level of an indicator of bone formation, such as bone alkaline phosphatase, tartrate resistant acid phosphatase (TRAP), Type HI procollagen, or the like, to assess the balance between bone formation and bone reso ⁇ tion in the subject
  • an indicator of bone formation such as bone alkaline phosphatase, tartrate resistant acid phosphatase (TRAP), Type HI procollagen, or the like
  • the invention includes a method of monitoring bone degradation in a subject, for detecting changes in the bone degradation status of the subject over time
  • the level, ratio or difference of TGF- ⁇ species discussed above is measured at least twice in a body fluid from the subject over a period of time, where the measured level, ratio or difference is used to assess whether bone reso ⁇ tion has increased, decreased, or stayed the same
  • the invention includes a substantially pure OP-TGF- ⁇ polypeptide having an apparent molecular weight of between 10 and 15 kDa, as measured by gel filtration chromatography under non-denaturing conditions, and which is characterized by the elution and
  • TGF- ⁇ activity properties shown in Figs 3B and detailed in Example 7
  • the polypeptide is particularly useful as a standard cahbrant in the OP-TGF- ⁇ assay above
  • the analytes of the invention are measured by any analytical method capable of quantitatively measuring the analyte(s) of interest without significant interference from other components in the sample fluid
  • suitable methods may include chromatographic, electrophoretic, and immunoassay methods, and combinations thereof
  • the analyte(s) are measured by immunoassay techiques using polyclonal or monoclonal antibodies, or art-recognized equivalents thereof, which are specific for the analyte(s) of interest
  • the invention also contemplates kits and reagents for use in carrying out the methods described above
  • Fig 1A illustrates an assay format for measuring small latent TGF- ⁇ species plus mature ⁇ l dimer
  • Fig. IB illustrates an assay format for measuring non-latent TGF- ⁇ species
  • Figs 2A and 2B show serum TGF- ⁇ 3 levels measured in normal (2A) and osteoporotic subjects (2B), Fig 3A shows chromatograms of TGF- ⁇ -contaimng fractions (assay in accordance with
  • Fig 4 shows serum TGF- ⁇ 3 levels measured in four groups of patients (only individuals with TGF-B3 concentrations > 0 3 ng/mL are shown) group 1 28 women with normal bone density, group 2 24 osteoporotic women presenting with vertebral fracture, group 3 1 1 osteoporotic women with a family history of osteoporosis, and group 4 26 osteoporotic patients with osteoporosis of the hip
  • body fluid includes any human body fluid suitable for diagnostic assay for assessing the level of bone reso ⁇ tion in accordance with the present invention
  • body fluids include whole blood and blood fractions, such as serum, plasma, and platelet-poor plasma, urine, saliva, sweat, synovial fluid, tear fluid, cerebral spinal fluid, and liquid extracts of bone tissues (e g , bone biopsy)
  • the body fluid is a blood fraction or urine, and more preferably is serum or plasma
  • TGF- ⁇ and active, mature TGF- ⁇ refer to the homodime ⁇ c form of TGF- ⁇ comprising ammo acid residues 279 to 390 of the pre-pro polypeptide form of TGF- ⁇
  • active TGF- ⁇ or “non-latent TGF- ⁇ ” encompasses monome ⁇ c and homodime ⁇ c forms of TGF- ⁇ comprising amino acid residues 279 to 390 of the pre-pro polypeptide form of TGF- ⁇ , or a subset thereof, and which is not complexed with LAP or LTBP
  • “Latent-associated peptide” and “LAP” refer to a polypeptide chain spanning residues 30 to 278 of the pre-pro polypeptide form of TGF- ⁇
  • Distal latent TGF- ⁇ refers to a 1 10 kDa dimer containing a mature TGF- ⁇ dimer complexed noncovalently with two LAP chains
  • Plate large latent complex refers to a 220-240 kDa complex containing a mature TGF- ⁇ dimer, two LAP chains, and an LTBP chain
  • Latent TGF- ⁇ binding protein and LTBP refer to the 130 kDa polypeptide which associates covalently with latent TGF- ⁇ to form the 220-240 kDa large latent complex, as described in Miyazono et al (1988)
  • Latent TGF- ⁇ and “latent forms of TGF- ⁇ ” refer to proteins or protein complexes which contain a mature TGF- ⁇ dimer and at least one other associated polypeptide, and which are unable to interact with cellular surface receptors for TGF- ⁇
  • Exemplary latent forms of TGF- ⁇ include small latent TGF- ⁇ and the large latent complex
  • TGF- ⁇ isoform refers to mature TGF- ⁇ 1 , ⁇ 2, 153, ⁇ 4, and/or ⁇ 5, a polypeptide derived therefrom, or a polypeptide having greater than 95 % homology therewith
  • total TGF- ⁇ 3 The phrases “total TGF- ⁇ 3" .
  • total TGF- ⁇ 3 species and “total TGF- ⁇ 3 antigens” refer to the total amount of mature TGF-153 species (and fragments thereof) measurable after release of any latent proteins from the TGF- ⁇ 3 species present in the sample
  • total TGF- ⁇ 3 can be prepared by treating the sample with acidic urea, as described below
  • proteolyt-ic treatments may be used, for example, to selectively destroy associated structure of the TGF-B3 polypeptides to be measured
  • test subject is intended to have its conventional meaning, and includes man, horse, cow, sheep, rabbit, dog, cat, rat, and mouse, for example
  • test subject is a human subject
  • TGF- ⁇ Transforming growth factor- ⁇
  • TGF- ⁇ is a plu ⁇ potent cytokme which has been found to regulate cell growth in a variety of tissues Reported activities include suppression of thymocytes and lymphocytes, stimulation or inhibition of osteoblast function, mediation of the formation of extracellular matrices, and more generally, inhibition or stimulation of proliferation and differentiation of a host of cell types (e g , Sporn et al , 1992, 1993, Ammann et al , 1992) TGF- ⁇ has also been proposed for use in wound repair (Derynck et al , 1989), for promoting bone growth (Ammann et al , 1992), and for immunosuppression and septic shock (Burnier et al , 1991 ) To date, at least five isoforms of TGF- ⁇ have been identified in mammals, designated
  • TGF- ⁇ l through TGF- ⁇ 5 Of these, only TGF- ⁇ l , ⁇ 2, and ⁇ 3 have been found in humans The five isoforms share high sequence similarities and overlapping biological activities, although some differences in activity have been reported (R&D Systems 1994 Catalog, Minneapolis, MN, pp 4-6) In humans, TGF- ⁇ l is found in highest concentrations m platelets and bone, TGF-B2 is found in bone and other tissues, and TGF-B3 is found mostly in cells of esenchymal origin Properties of the TGF- ⁇ proteins have been reviewed (Sporn et al , 1992, 1993)
  • TGF- ⁇ is synthesized as a 390 residue pre pro polypeptide which dime ⁇ zes to form a homodimer
  • the pre-pro form contains a 29 residue signal sequence which is cleaved mtracellularly to form a pro-TGF- ⁇ l dimer of 261 -residue chains
  • pro-TGF- ⁇ l is further cleaved to produce a 1 10 kDa nicked dimer in which the polypeptide chains of the dimer have been cleaved between Arg278 and Ala279
  • the newly created C-terminal chains, spanning residues 279 to 390 from the pre- pro polypeptide, constitute the polypeptide chains
  • Additional proteins may associate with latent TGF- ⁇ , either covalently or non-covalently to form additional forms of TGF- ⁇
  • a complex has been isolated from human platelets containing LAP linked to an additional 130 kDa protein (known as the latent TGF- ⁇ binding protein, or LTBP) by a single disulphide bond (Miyazono et al , 1988, 1991)
  • This 220-240 kDa complex which includes a mature (25 kDa) TGF- ⁇ dimer, two associated LAP chains, and an LTBP chain, has been termed the "large latent complex '
  • Active, mature TGF- ⁇ can be formed in vitro by exposing latent TGF- ⁇ or the large latent complex to strong acid, strong base, or chaotropic agents Such conditions presumably disrupt the non-covalent interactions between the mature TGF- ⁇ dimer core and the non-covalently attached polypeptides (LAP and LTBP) to produce active TGF- ⁇ Formation of active TGF- ⁇ in vivo may occur through the action of proteases which degrade the LAP but not the TGF- ⁇ core Plasmm has been implicated as the physiological activator under some circumstances (Grainger et al , 1994, 1995), but other proteases may also be involved (Kojima et al , 1993 Dennis et al , 1991) At present, the actual in vivo mechanism for forming active TGF- ⁇ from its latent forms is not known III Reso ⁇ tion Assay Methods
  • the present invention is directed generally to a method of assessing the status of bone reso ⁇ tion in a subject, based on the measurement of certain TGF- ⁇ species in a body fluid, particularly serum, plasma or urine
  • a measured level, ratio, or difference, of the selected TGF- ⁇ species that is at or above a selected threshold value is an indication that an elevated level of bone resorption is present in the subject
  • the method may also be configured such that a measured level, ratio or difference, of the selected TGF- ⁇ species that is at or below a selected threshold value can be used as an indication that bone reso ⁇ tion is within normal limits
  • the level of true positives detected must be weighed against the number of false positives as well as false negatives For example, a higher threshold will lead to a lower rate of false positives, but at the expense of reduced
  • the method of the invention may be used in a monitoring mode, where at least two measurements are made in body fluid samples from a subject at different times, and the change in the measured level, if any, is used to determine whether the level of bone reso ⁇ tion has increased, decreased, or remained the same
  • the invention may also be used in prognostic applications, for predicting the likelihood or risk of developing a bone reso ⁇ tion abnormality such as discussed above, or for predicting the likelihood or risk of suffering a bone fracture
  • an increased risk may be indicated by a level of selected TGF- ⁇ species that is above a selected threshold, where the threshold has been selected on the basis of clinical trials, for example, or by a level which is a selected percentage above a baseline level established for a particular individual
  • the methods of the invention may be used in a screening capacity for the general public, or may be targeted to certain patients for whom such testing is deemed appropriate
  • the present reso ⁇ tion assessment methods may be used in conjunction with other medical techniques, such as bone density measuring methods or diagnostic assays based on other markers
  • TGF- ⁇ -containing species may be detected using antibodies which are lmmunoreactive with one or more TGF- ⁇ species, allowing highly sensitive detection of low concentrations of the species of interest by conventional immunoassay techniques
  • Antibodies raised against mature TGF- ⁇ l , ⁇ 2, or ⁇ 3, and having high binding affinity for TGF- ⁇ l are useful for detecting the OP-TGF- ⁇ protein of the present invention
  • Such antibodies are readily available from commercial sources (e g , R&D Systems. Minneapolis. MN, R&D Systems, Oxford, United Kingdom, Genzyme Diagnostics, Cambridge, MA) or may be prepared by standard methods (e g , Harlow, 1988)
  • Section A describes an aspect of the invention based on measuring total TGF-B3, or a subform thereof, such as active ⁇ 3 dimer
  • TGF-B3 species were not known to be present in significant amounts in plasma or serum
  • Section B describes an aspect of the invention based on measuring the levels of (I) non- latent TGF- ⁇ and (u) the sum of small latent TGF- ⁇ plus mature TGF- ⁇ l dimer are determined in a body fluid from the subject The ratio or difference of these measured levels is used as an indicator of the level of bone reso ⁇ tion in the subject
  • Section C describes an aspect of the invention based on the measurement of a novel protein designated herein as OP-TGF- ⁇
  • the level of total TGF- ⁇ 3, or alternatively, mature TGF- ⁇ 3 dimer, in a body fluid is measured, where a measured level above a selected threshold level is an indication that a bone metabolism abnormality associated with elevated bone resorption may be present in the subject Similarly, a level below a selected threshold, which may be different from the first threshold, may indicate that the subject's level of bone reso ⁇ tion is within normal limits
  • TGF-B3 may be measured using any analytical technique available in the art, and is conveniently measured using immunoassay techniques
  • Example 4 describes an exemplary assay format which is useful in this aspect of the invention
  • the assay includes an immobilized TGF- ⁇ Type II receptor such as described in Example 1 , for binding the TGF- ⁇ 3 dimer to the support for subsequent detection
  • the assay further includes an antibody which is specific for the ⁇ l isoforms of TGF, to bind to and sequester the ⁇ l isoforms, to prevent ⁇ l monomer or dimer from competing with ⁇ 3 dimer for binding to the immobilized receptor This allows low levels of TGF- ⁇ 3 to be detected even in the presence of a high concentration of TGF- ⁇ l species, when the binding partner is cross reactive with both ⁇ 1 and ⁇ 3 isoforms
  • the serum samples are treated with acidic urea to release the active, dime ⁇ c forms of TGF- ⁇ 3 from their latent forms
  • the samples are mixed with a neutralization solution and then incubated with a ⁇ l-specific antibody (BDA19) to sequester ⁇ l species in the samples
  • BDA19 ⁇ l-specific antibody
  • the samples are then contacted with the immobilized TGF- ⁇ receptors to bind the TGF-B3 to the solid support
  • Primary, non-immobilized antibody is then added to bind to the immobilized TGF- ⁇ 3, followed by addition of peroxidase-labeled antibody for detection
  • the non-latent forms of TGF-B3 can be measured without treating the sample to convert latent to non-latent forms
  • the subjects with low bone density exhibited a mean TGF-B3 level of 6 4 .+ . 0 6 ng/mL, compared with 2 7 +_ 0 6 ng/mL for the control group
  • more than half of the control individuals showed no detectable TGF-B3
  • all but two of the osteoporosis subjects showed detectable TGF-B3 levels greater than about 2 ng/mL
  • Fig 4 shows additional data illustrating the usefulness of the method
  • Serum samples were collected from 28 women with normal bone density (group 1) and 61 women with osteoporosis (groups 2-4) Serum TGF- ⁇ 3 levels were measured as described in Example 4 Results ( 1 ) Of the 28 women with normal bone density, 5 had detectable serum TGF-B3 levels ( > 0 1 ng/mL), but only one had a level greater than 2 ng/mL (2 2 ng/mL) (2) Of the 61 osteoporotic women (groups 2-4 together).
  • Sample Storage Serum samples containing TGF- ⁇ 3 can be subjected to repeated freeze-thaw cycles with only a slight reduction in the measured amount of TGF-B3 Thus, repeated freeze-thawing of human serum samples does not significantly affect TGF- ⁇ 3 measurements However, it is preferable to store samples at -20°C until measurement of TGF- ⁇ 3, without thawing until testing
  • the applicant has found that red blood cells contain about 800 molecules of TGF- ⁇ 3 per cell, equivalent to about 100 ng of TGF- ⁇ 3 per mL of blood Thus, hemolysis should be avoided to obtain an accurate measure of serum TGF-B3 species
  • the degree of hemolysis in a test sample is less than 2% , e g , when a threshold of 2 ng/mL is used to discriminate between normal and "diseased" or at- ⁇ sk subjects This level of hemolysis can be readily detected visually by eye from the red color of the sample, indicating that another sample should probably be taken from the subject
  • hemolysis is generally not a significant problem
  • Studies by the applicant have shown that hemolysis of at least 3 2 % (as measured by assaying serum hemoglobin concentration after drawing blood through a fine gauge needle a variable number of times prior to clotting and collection of serum) is generally
  • TGF- ⁇ 3 Levels and Drug Treatment There is some variation in the effects of hormone-mimetic drugs on TGF- ⁇ 3 levels measured in women For example, prophyllactic hormone replacement therapy (HRT) was found to lead to mildly increased levels of TGF-B3 during treatment
  • HRT prophyllactic hormone replacement therapy
  • Serum and Plasma The assay described in Example 4 detected the same levels of total TGF- ⁇ 3 in serum and platelet-poor plasma prepared from the same blood samples Thus, testing either serum or plasma samples should provide similar results
  • TGF- ⁇ + mature ⁇ l dimer are measured, and either the ratio (i) (n) or the difference (l - n) is used as an indicator of the level of bone reso ⁇ tion in the subject
  • the measurement of non-latent TGF- ⁇ , and the sum of small latent TGF- ⁇ + mature ⁇ l dimer, may be carried out using any suitable analytical approaches known in the art for measuring these species
  • non-latent TGF- ⁇ can be measured by immunoassay using lmmunological binding partners which are lmmunospecific for both desired TGF- ⁇ species, and which do not significantly react with the large latent complex
  • the assay may be homogeneous or heterogeneous, and may utilize a direct (e g , sandwich) or competitive format in accordance with assay configurations well known in the art (e g , Harlow)
  • FIG. 1 A One suitable assay format for measuring non-latent TGF- ⁇ species is illustrated in Figure 1 A In this assay, an immunological binding partner, 12a, 12b, 12c, 12d, here shown as a type
  • TGF- ⁇ receptor is immobilized on a solid support 40, for binding to non-latent monomenc and dimeric forms of TGF- ⁇ , particularly those containing TGF- ⁇ l and TGF- ⁇ 3 chains
  • the assay format also includes antibodies 30a, 30b, for binding to the target TGF- ⁇ species, and reporter-labeled antibodies 32a, 32b, for binding to the primary antibodies 30a, 30b to produce a measurable signal
  • the sample to be tested may contain any or all of the following TGF- ⁇ species (0 a monomenc form of TGF- ⁇ ( 14 in the figure), such as the OP-TGF- ⁇ protein described further below, (n) a mature TGF- ⁇ dimer 16, (in) small latent TGF- ⁇ 20, which contains a TGF- ⁇ dimer 16 and two LAP chains 18, and/or (IV) a large latent complex 24 which contains a mature TGF- ⁇ dimer 16, two LAP chains 18, and an LTPB chain 22
  • TGF- ⁇ species a monomenc form of TGF- ⁇ ( 14 in the figure), such as the OP-TGF- ⁇ protein described further below, (n) a mature TGF- ⁇ dimer 16, (in) small latent TGF- ⁇ 20, which contains a TGF- ⁇ dimer 16 and two LAP chains 18, and/or (IV) a large latent complex 24 which contains a mature TGF- ⁇ dimer 16, two LAP chains 18, and an LTPB chain 22
  • TGF- ⁇ monomenc species 14 is bound by immobilized receptor 12a
  • the immobilized TGF- ⁇ monomer is then detected by binding to a first antibody 30a which binds directly to monomer 14, followed by the binding of reporter- labeled antibody 32a
  • the TGF- ⁇ is then detected by virtue of the reporter-labeled antibody immobilized on the solid support
  • TGF- ⁇ dimer 16 is likewise detected when bound to immobilized receptor 12b, by virtue of binding with primary antibody 30b which in turn is bound by reporter labeled antibody 32b Since receptor 12c does not bind latent forms of TGF- ⁇ , small latent form 20 and large latent form 24 remain free from the support, and thus cannot serve to anchor a reporter labeled antibody 32 to the support Details of an exemplary assay format in accordance with the above are provided in Examples 1 and 2
  • the primary antibody 30a does not bind significantly to the latent forms of TGF- ⁇ , it is not important whether the primary antibody 30a can bind the latent forms as well as the non-latent forms, provided the primary and reporter-labeled antibodies are present in excess relative to the TGF- ⁇ which could potentially be bound
  • immobilized binding partner 12 can be selected to bind to both non-latent as well as latent forms of TGF- ⁇
  • the specificity of the assay for the non- latent forms is achieved by selecting the primary antibody (or other suitable binding partner) to be lmmunospecific for the non-latent forms
  • the immobilized binding partner 12 can be an antibody which binds with TGF- ⁇ species 14, 16, 20, and 24, and antibody 30 could be replaced with a type II TGF- ⁇ receptor which recognizes only non-latent TGF- ⁇ forms
  • the sum of small latent TGF- ⁇ + mature ⁇ l dimer can be measured using an assay format such as illustrated in Fig IB and detailed in Example 3 In the format of Fig I B, receptor 12a from Fig 1A has been replaced with an immunological binding partner 42 (shown schematically as an antibody) which can bind mature ⁇ l dimer (16) and small latent TGF- ⁇ (20), but not monomer 14 or large latent complex 24 Dimer 16 and small latent form 14 may
  • Reporter R is any entity which is effective to produce a detectable signal for the pu ⁇ ose of detecting or quantitating the TGF- ⁇ analyte of interest
  • exemplary reporters include enzymes, such as alkaline phosphatase and horse radish peroxidase, radioactive labels such as l25 I, spin labels, etc
  • binding partner 30 does not bind significantly with monomer 14 or large latent complex 24 m order to avoid competition between species 14 and 24 and species 16 and 20 for binding to binding partner 30 Otherwise, there might be an insufficient amount of binding partner 30 available to bind with and detect species 14 and 16
  • blood samples were collected from 39 women with osteoporosis and 41 women having normal bone density The samples were tested using the non-latent TGF- ⁇ assay of Example 3, and the small latent + mature TGF- ⁇ dimer assay described in Example 2 The results were as follows
  • the average level of small latent TGF + mature dimer was 7 0 _+ 1 6 ng/mL, whereas the average for the osteoporosis subjects was 4 3 +_ 0 8 ng/mL
  • the average level was 5 4 +_ 1 6 ng/mL
  • the average was 10 7 +_ 2 7 ng/mL
  • the average ratio [non- latent] /[small latent + mature dimer) was about 0 8
  • the average ratio for the osteoporosis subjects was about 2 5
  • only 10-15 % of the women with normal bone density had a ratio > 1 0, whereas > 80% of the osteoporosis subjects had such a ratio ( > 1 0)
  • results show that the measured ratio provides a useful indication of the status of bone reso ⁇ tion in human subjects Furthermore, the results show how a threshold can be selected to discriminate between subjects likely to have elevated bone reso ⁇ tion levels, and subjects who are either healthy or whose bone reso ⁇ tion status is uncertain For example, subjects showing ratios greater than 1 0, as just discussed, would be likely candidates for elevated bone reso ⁇ tion levels, although some false positives would also occur Conversely, a ratio of less than 1 0 would be a likely indication that the subject has normal bone reso ⁇ tion, although false negatives on the order of 10-20% might occur It will be appreciated that other threshold ratios could be selected to improve the accuracy of the assay, depending on the desired level of tolerance for false positives or false negatives
  • the difference between the levels of non-latent TGF- ⁇ and small latent TGF- ⁇ + mature ⁇ l dimer is determined, where a difference that is above a selected threshold is an indication that a bone reso ⁇ tion disorder may be present in the subject, and a difference that is below a selected threshold may be used as an indication of normal bone reso ⁇ tion
  • the level of a protein designated OP-TGF- ⁇ in a body fluid is measured, where a measured level above a selected threshold level is an indication that a bone metabolism abnormality associated with elevated bone reso ⁇ tion may be present in the subject
  • Example 7 describes a study showing certain physical properties of the TGF- ⁇ -OP protein of the invention
  • serum samples were obtained from two normal, healthy male subjects (45 and 54 years old, respectively), and from a woman diagnosed with osteoporosis Each sample was applied to an anion exchange column and fractionated using a sodium chloride salt gradient in 10 itiM sodium phosphate, pH 6 8
  • One mL fractions were collected and assayed for TGF- ⁇ l -containing proteins using a commercial ELISA kit (Quantikine ELISA kit, R&D Systems), after an initial acidic urea pre-treatment step to release mature TGF- ⁇ from latent forms
  • the two healthy subjects showed qualitatively similar profiles (Fig 3A) Specifically, the sample from the 45 year old subject showed TGF-prote peaks at elution volumes of about 12, 24, and 30 mL, corresponding to the mature TGF- ⁇ dimer (A), the platelet large latent complex (P), and the small latent form (L), respectively In addition, this sample showed a peak at about 19 mL, which appears to correspond to a previously uncharac- te ⁇ zed form of TGF- ⁇ The sample from the 54 year old showed peaks at about 24 mL and 30 mL, corresponding to the large latent complex and small latent complex, respectively, but only a weak peak at 14 mL for the mature dimer form
  • the osteoporotic subject on the other hand, exhibited a large new peak eluting at about 35 L (indicated by arrow and labeled OP-TGF- ⁇ ), in addition to the peaks typically observed in normal subjects
  • the new peak constitutes a new form of TGF- ⁇ which is clearly distinguishable from other known forms of TGF- ⁇ , particularly mature TGF- ⁇ , small latent TGF- ⁇ , and the 220-240 kDa large latent complex
  • the OP-TGF- ⁇ protein When chromatographed on a gel filtration column, the OP-TGF- ⁇ protein exhibits an elution volume consistent with a molecular weight of about 10 to 15 kDa, suggesting that OP- TGF- ⁇ consists of a mature TGF- ⁇ chain in monomenc form, or a truncated TGF- ⁇ monomer
  • studies conducted in support of the invention indicate that the OP-TGF- ⁇ protein is recognized by (I) a type II TGF- ⁇ receptor and (n) a antibodies which recognized mature TGF- ⁇ , but that the protein is not bound by antibodies which recognize latent forms of TGF- ⁇ , consistent with the OP-TGF- ⁇ protein being a mature TGF- ⁇ monomer
  • the invention includes a method of purifying an OP-TGF- ⁇ polypeptide from a liquid sample, comprising the steps of (I) contacting the liquid sample with a solid support having immobilized thereon a binding partner which is lmmunospecific for mature TGF- ⁇ l and OP-TGF- ⁇ , under conditions effective to specifically bind the polypeptide to the immobilized
  • the purification method is conducted using an immobilized binding partner which is immunospecific for OP-TGF- ⁇ . to the exclusion of mature dimeric TGF- ⁇ l or any latent form thereof, such that the OP-TGF- ⁇ polypeptide is eluted in substantially pure form
  • the invention includes an immunological binding partner which is immunospecific for OP-TGF- ⁇ , and which does not bind mature dimeric TGF- ⁇ or any latent form thereof
  • BDA19 antibody is a chicken polyclonal IgY antiserum
  • BDA47 is an affinity-purified rabbit polyclonal IgG antibody
  • BDA19 binds the mature dimer and small latent forms of TGF- ⁇ l
  • TGF- ⁇ 3 BDA47 binds both the mature dimer and small latent forms of TGF- ⁇ l
  • TGF-B3 BDA48 is a polyclonal antiserum specific for the B3 isoform of TGF
  • TGF- ⁇ l and TGF- ⁇ 3 were obtained from R&D Systems or Amersham International
  • peripheral venous blood samples (10 L) were collected from human subjects and transferred immediately to tubes containing 1 1 mL of sterile 3 8% (w/v) t ⁇ sodium citrate in MilhQ water at room temperature
  • the samples were cent ⁇ fuged to remove red blood cells (250 x g, 15 mm)
  • Apyrase (Sigma) was added to the resulting platelet-rich plasma to a final concentration of 100 mg/L, to prevent platelet degranulation PMSF (1 mmol/L) and aprotinm (1 mg/L) were added to prevent proteolytic activation or degradation of TGF- ⁇
  • the samples were then cent ⁇ fuged (700 x g, 15 mm), and the supernatant platelet-poor plasma was separated from the platelet pellet
  • the platelet-poor plasma was kept at room temperature until assay by ELISA within 2 h of preparation or was stored frozen in 0 5-mL aliquots at -80°C
  • peripheral venous blood samples from human subjects were dispensed immediately into polypropylene tubes and allowed to clot at room temperature for 2 h
  • the collected samples were centrifuged (1000 x g, 4 min), after which the serum was removed and either stored on ice until assay within 2 h or stored at -80 °C for later assay
  • the extracellular domain of the TGF- ⁇ type II receptor was amplified from the vector H2 3FF, as described in Lin et al ( 1992)
  • the vector DNA was linearized with NotI, precipitated, and resuspended at 10 ng/ ⁇ L
  • Amplification by polymerase chain reaction (PCR) was carried out in a 50 ⁇ L reaction volume containing vector DNA (2 5 ⁇ L), lOx TAQ buffer (5 ⁇ L) (LKB Pharmacia, Uppsala, Sweden), 250 ng ofeach ohgonucleotide primer (5'-GAATTCCCATGGGTCGGGGGCTGCTC and GAATTCGTCAGGATTGCTGGTGTT), TAQ polymerase ( 1 U) and a mixture of dAI P, dTTP, dCTP and dGTP to give a final concentration of 200 ⁇ M for each nucleotide
  • the sample was overlaid with 50 ⁇ L paraffin oil -The reaction was carried out using a thermal cycler for 30 cycles (denaturing at 94°C for 1 min, annealing at 55 °C for 2 min, and elongation at 72 °C for 2
  • the 450 bp product was purified by eiectrophoresis in low gel temperature agarose, digested with EcoRI, and cloned into the glutathione-S-transferase fusion vector pGEX 2T (LKB Pharmacia) Vectors carrying inserts in the required orientation were identified by plasmid mapping The sequence of the insert was checked by subcloning the 450 bp EcoRI fragment from the chosen clone (pGTIC) into Bluesc ⁇ pt KS + , followed by double strand sequencing The sequence showed a single base change (C to A at position + 13 from the initiation codon) compared with the published sequence (Lin et al , 1992), which introduces a Leu to Met mutation in the expressed receptor protein
  • the plates were washed with a wash buffer consisting of TBS, 3 % FAF-BSA, and 0 1 % Triton X-100, and incubated with 20 ⁇ L detection antibody (BDA47) at 1 ⁇ g/mL in wash buffer for 1 h
  • the plates were rinsed with wash buffer (3 3 mm) and incubated with an antibody against rabbit IgG conjugated to horseradish peroxidase at 1 2500 dilution in wash buffer for 1 h
  • wash buffer 3 x 3 min
  • the plates were incubated for 15 min with o-phenylenediamine according to the manufacturer's instructions (Sigma Chem Co )
  • the reaction was stopped by addition of an equal volume of 3 M HC1, and the absorbance values were read on an ELISA platereader (Titerteck Multiscan, Flow Laboratories, High Wycombe, UK) within 15 min of stopping the reaction
  • TGF-B3 stock recombinant human TGF-B3 (2 ⁇ g) was dissolved in 4 mM HC1 (400 ⁇ l) containing 1 mg/mL fatty acid-free bovine serum albumin (Sigma, A-6003) to give a final concentration of 5 ⁇ g/mL
  • an initial standard solution of TGF-153 was prepared by adding 2 ⁇ l of TGF- ⁇ 3 stock (5 ⁇ g/mL) to an Eppendorf tube containing 100 ⁇ l BDA19 working solution (40 ⁇ g/mL BDA19) and 300 ⁇ l RD6M diluent, giving a final BDA19 concentration of 10 ⁇ g/mL and a final TGF- ⁇ 3 concentration of 25 ng/mL
  • this initial TGF- ⁇ 3/BDA19 solution was diluted serially 1 1 into six successive Eppendorf tubes each containing 150 ⁇ l RD6M diluent and 50 ⁇ l working BDA19 solution
  • the seven TGF-B3 standard tubes thus contained 10 ⁇ g/mL BDA19 and from 25 ng/mL to 0 39 ng/mL TGF- ⁇ 3
  • an additional tube containing 150 ⁇ l RD6M diluent and 50 ⁇ l working BDA19 solution was also prepared without TGF-B3
  • TGF- ⁇ 3/BDA19 and TGF-free control were each loaded into separate wells (200 ⁇ l each) in a Quantikine ELISA strip (R&D Systems)
  • 75 ⁇ l neutralisation buffer and 75 ⁇ l BDA19 working solution The contents of the sample tubes were mixed, and 200 ⁇ l ahquots of each were transferred to separate wells on the ELISA strip
  • the strips were covered with adhesive film and incubated for 1 hour at room temperature on a microtitre plate shaker (500 ⁇ ) After incubation, the wells were aspirated by inversion and washed 3 times with wash buffer (R&D Systems, 1 25 dilution) To ensure liquid removal, the strips were blotted on a clean paper towel between wash steps
  • 50 ⁇ l BDA48 working solution 100 ⁇ g/mL
  • Serum samples were obtained as described in the Material and Methods section from two normal, healthy male subjects and (45 and 54 years old, respectively), and from a female patient diagnosed with osteoporosis
  • Each sample (1 L) was diluted 1 3 with 10 mM sodium phosphate, pH 6 8, and then applied to a 1-mL ResourceQ disposable MonoQ column (Pharmacia) on an FPLC system (Pharmacia) equilibrated in 10 mM sodium phosphate pH 6 8 (buffer A)
  • the column was washed with buffer A (flow rate 1 mL/min) until the absorbance at 280 nm had returned to baseline
  • TGF- ⁇ proteins were assayed for TGF- ⁇ proteins as follows A quots of the fractions were diluted 1 1 with 2 5 M acetic acid containing 10 M urea for 10 mn at room temperature to release mature 25-kDa TGF- ⁇ 1 dimer Samples were then neutralized by adding the same volume again of 2 7 M NaOH/1 M HEPES Neutralized samples were assayed immediately for TGF- ⁇ l using a Quantikine ELISA kit (R&D Systems) in accordance with the manufacturer's instructions Fractions containing TGF- ⁇ (typically fractions 20 to 29) were combined, diluted 1 4 with 200 mM sodium phosphate pH 6 8, and concentrated to 200 ⁇ L using an Amicon cell with a 3,000 kDa cut-off membrane The concentrated sample was then loaded onto a Superose 12 gel filtration column (Pharmacia) equilibrated in 200 mM sodium phosphate pH 6 8 on an FPLC system Fractions (0 5 mL) were

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Abstract

La présente invention se rapporte, de manière générale, à un procédé permettant d'évaluer la qualité de la résorption osseuse chez un patient en se basant sur la mesure de l'espèce TGF-β3 dans un liquide organique, en particulier le sérum sanguin. Si le niveau mesuré de l'espèce TGF-β3 choisie est égal ou supérieur à une valeur limite déterminée, il constitue une indication utile que le patient présente un niveau élevé de résorption osseuse. Si le niveau mesuré de l'espèce TGF-β3 choisie est égal ou inférieur à une valeur limite déterminée, il peut être utilisé comme une indication que le patient présente une résorption osseuse dans les limites de la normale. On décrit également une trousse et des réactifs à utiliser dans ledit procédé.
PCT/US1997/014434 1996-08-16 1997-08-15 Procede et trousse d'evaluation de la resorption osseuse WO1998007032A1 (fr)

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AU41520/97A AU4152097A (en) 1996-08-16 1997-08-15 Method and kit for assessing bone resorption

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US60/024,084 1996-08-16

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000013024A1 (fr) * 1998-08-26 2000-03-09 Medvet Science Pty Ltd. Evaluation predictive de certaines affections du squelette
US6300534B1 (en) 1998-07-01 2001-10-09 Nippon Petrochemicals Company, Limited Process for producing dehydrogenated compounds of m-ethyldiphenylalkane

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5262319A (en) * 1985-04-19 1993-11-16 Oncogene Science, Inc. Method for obtaining bone marrow free of tumor cells using transforming growth factor β3
US5393739A (en) * 1990-11-30 1995-02-28 Celtrix Pharmaceuticals, Inc. Use of bone morphogenetic protein in synergistic combination with TGF-β for bone repair

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5262319A (en) * 1985-04-19 1993-11-16 Oncogene Science, Inc. Method for obtaining bone marrow free of tumor cells using transforming growth factor β3
US5393739A (en) * 1990-11-30 1995-02-28 Celtrix Pharmaceuticals, Inc. Use of bone morphogenetic protein in synergistic combination with TGF-β for bone repair

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6300534B1 (en) 1998-07-01 2001-10-09 Nippon Petrochemicals Company, Limited Process for producing dehydrogenated compounds of m-ethyldiphenylalkane
WO2000013024A1 (fr) * 1998-08-26 2000-03-09 Medvet Science Pty Ltd. Evaluation predictive de certaines affections du squelette

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