Classifications

• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4743—Insulin-like growth factor binding protein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to bone metabolism, and more particularly to bone metabolic processes which are mediated by a novel insulin-like growth factor binding protein (IGFBP) isolated from human bone. More specifically, the invention relates to a IGFBP termed human bone derived IGFBP (hBD-IGFBP) which potentiates the effect of insulin-like growth factor-II (IGF-II) on bone cell proliferation.
• IGFBP insulin-like growth factor binding protein
• IGF-I and IGF-II the two most abundant growth factors present in human plasma, constitute a family of polypeptides that resemble proinsulin in structure and have both anabolic and acute insulin-like activities in numerous tissues (Daughaday, et al., Endocrine Rev.. 10:68-91 (1989)).
• IGF-I the primary IGF
• IGF-II being a fetal hormone
• recent findings have pointed to an important role for IGF-II in adult human bone metabolism.
• IGF-II has been found to be the most abundant growth factor present in human bone, and is the most abundant growth factor produced by human bone cells. Further, IGF-II is one of the few growth factors which is mitogenic to human bone cells.
• IGFBP-4 a recently purified inhibitory IGFBP, termed IGFBP-4, was found to inhibit basal bone cell proliferation by about 40% in serum free conditions, suggesting that endogenous production of IGFs contributed substantially to cell proliferation in the absence of added growth factors.
• IGF-II receptor blocking antibodies have been shown to inhibit basal bone cell proliferation, suggesting that IGF-II is a key bone cell growth factor (Mohan, et al., Growth, Genetics and Hormones, 6:1-9 (1990) and Mohan, et al., Clin. Orthopedics & Rel. Res. , 263:30-48 (1990)).
• IGFBP-l has been isolated from various sources including amniotic fluid, placental membranes, decidua and HEP G2 hepatoma cells. The N-terminal amino acid sequences of the IGFBP-l proteins isolated from these different sources have been found to be identical. The cloning and complete sequence of cDNA encoding IGFBP-l from HEP G2, human uterus and human placental cDNA libraries have been reported. IGFBP-2 has been purified from conditioned medium collected from rat liver cells (BRL-3A) and from Madin-Darby bovine kidney cells. The gene encoding IGFBP-2 has been cloned from BRL-3A and human fetal liver cDNA libraries.
• IGFBP-3 is found in serum as a 150 kilodalton ternary complex between IGF-I or IGF-II, an acid labile glycoprotein of about 85 kilodaltons, and the IGFBP-3 molecule, which is an acid stable glycoprotein of 53 kilodaltons.
• IGFBP-3 has been purified to homogeneity from human serum and the cloning and sequencing of the cDNA encoding IGFBP-3 has been reported.
• IGFBP-4 was originally purified from human bone cell conditioned medium as inhibitory IGFBP and from rat serum. The cloning and sequencing of IGFBP-4 cDNA clone isolated from human bone cell cDNA library and liver cDNA library have been recently reported.
• IGFBPs In addition to these four classes of IGFBPs, Martin et al., J. Biol. Chem.. 265:4124-4130 (1990), Roghani et al. , FEBS Lett.. 255:253-258 (1989), and Zapf et al., J. Biol. Chem.. 265:14892-14898 (1990), report the partial purification of IGFBP from human cerebrospinal fluid, from culture medium conditioned by AG 2804 transformed fibroblasts and from hypoglycemic serum respectively, which exhibit strong affinity for IGF-II over IGF-I.
• IGFBP-l IGFBP-3 and IGFBP-4 bind both IGF-I and IGF-II with nearly equal affinity
• IGFBP-2 and IGFBP purified from amniotic fluid, fibroblast cells and human serum bind IGF-II with higher affinity than IGF-I.
• IGFBP-l has been shown to both inhibit and potentiate the proliferative action of IGFBP-l in choriocarcinoma cells and in human fibroblasts (Elgin, et al., J. Biol. Chem..
• IGFBP-3 has been shown to inhibit or stimulate IGF-I actions depending on culture conditions in fibroblasts (De Mellow, et al., Biochem. Biophys. Res. Comm.. 156:199-204 (1988)).
• IGFBP-4 has only been shown to inhibit IGF-I and IGF- II actions in bone cells (Mohan, et al., Proc. Natl. Acad. Sci. USA, 86:8338-8342 (1989)).
• IGFBP-l production is modulated by insulin while IGFBP-3 production is modulated by growth hormone (Baxter, et al.. Prog. Growth Factor Res.. 1:49- 68 (1989)).
• IGFBPs IGF-I and IGF-II functions in bone metabolism via IGFBPs, and thus there is a need to identify the IGFBPs produced by bone cells and present in human bone matrix.
• IGFBPs are likely to be involved in regulating bone metabolism and can be used in clinical assays to provide information in the diagnosis of defects in bone metabolism.
• IGFBPs which potentiate the IGF- dependent growth of bone would be particularly useful in therapeutic applications for treatment of metabolic bone diseases such as osteoporosis.
• the present invention fulfills these and other related needs.
• the present invention provides methods and compositions for clinical diagnosis and treatment of metabolic disorders related to IGF-mediated bone formation and cell proliferation. More particularly, the invention provides human bone derived IGF binding protein (hBD-IGFBP) .
• hBD-IGFBP human bone derived IGF binding protein
• the hBD-IGFBP acts synergistically with IGF-II to potentiate IGF-II mediated cell proliferation under conditions where both IGF-II and IGFBP are administered simultaneously.
• the purified hBD-IGFBP also binds to hydroxyapatite with strong affinity and thus provides a reagent to target molecules specifically to bone, such as IGF-II and/or IGF-I, other growth factors or drugs which may affect bone resorption or formation, and thus is useful in treatment of, e.g., bone fractures and bone diseases such as osteoporosis or osteosarcoma.
• hBD-IGFBP may also be used in the treatment of wound healing and in skin repair.
• hBD-IGFBP may be used diagnostically as a marker of bone formation rate, such as during treatment of bone disorders with therapeutic agents.
• hBD-IGFBP may also be used as a reagent for clinical evaluation of IGF levels in samples from patients with bone metabolism and other disorders.
• Fig. 1 Comparison of N-terminal amino acid sequence of hBD-IGFBP with that of other known IGFBPs. The sequences are aligned to give maximum identity.
• Fig. 2. Competitive binding curves of hBD-IGFBP. The sample was assayed for binding protein activity using labeled IGF-II in the presence or absence of unlabeled IGF-I and IGF- II.
• Fig. 3. Protein profile (A) and IGFBP activity profile (B) of the human bone extract in the FPLC Mono Q chromatography step. Three 50 ml aliquots of HA bound fraction pools were applied to the IGF-II affinity column. The resulting three affinity bound fractions were pooled and run on the Mono Q anion-exchange column. Protein profile is monitored by absorbance at 280 nm. Two ml, 2 min fractions were collected. Aliquots of the fractions were diluted 10 fold and assayed for binding protein activity. IGFBP activity is expressed in amounts of specifically bound labeled IGF-II.
• Fig. 4 Ligand blot analysis of hBD-IGFBP at different stages of purification. 50 ⁇ l of the sample were run on SDS-PAGE (3-27% gradient) , transferred to nitrocellulose membranes, blotted with labeled IGF-II, and autoradiographed. Lane a, HA bound fraction of human bone extract; Lane b, IGF-II affinity bound fraction; Lane c. Mono Q IGFBP peak A; Lane d. Mono Q IGFBP peak B; Lane e. Mono Q IGFBP peak C and Lane f, Mono Q IGFBP peak D.
• the invention provides purified and isolated human bone derived IGFBP which binds specifically to IGF-II with greater affinity than to IGF-I.
• the protein may be purified to homogeneity as desired from proteins extracted from, e.g., human bone preparations, human bone cell conditioned medium, or human serum.
• Substantially pure hBD-IGFBP of at least about 50% is preferred, at least about 70-80% more preferred, and 95-99% or more homogeneity most preferred, particularly for pharmaceutical uses.
• the hBD-IGFBP may then be used diagnostically, as an immunogen, therapeutically, etc.
• the hBD-IGFBP produced according to the present invention may be purified by hydroxyapatite apatite chromatography followed by affinity chromatography on a column with IGF-II and finally for additional purity by Mono Q anion exchange chromatography using an FPLC system.
• Apparent homogeneity of the purified protein is demonstrated by, e.g., its migration as a single band on SDS-PAGE and by the production of a single amino acid sequence upon N-terminal sequence analysis.
• Affinity chromatography on an antibody column using antibodies specifically directed against hBD-IGFBP can also be used in a purification scheme.
• Additional purification may be achieved by conventional chemical purification means, such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others.
• Methods of protein purification are known in the art (see generally. Scopes, R. , Protein Purification. Springer-Verlag, NY (1982), which is incorporated herein by reference) and may be applied to the purification of the hBD-IGFBP described herein.
• hBD-IGFBP specific binding of hBD-IGFBP to IGF-II and IGF-I is demonstrated by a polyethylene glycol precipitation assay.
• the invention provides a purified protein which is useful in structure-function studies of the determinants of IGFs which allow binding to specific receptors as well as to hBD-IGFBP. Further utility of the purified hBD-IGFBP of the invention is disclosed in the descriptions of other aspects of the invention below.
• the purified IGFBP of the invention is unique and distinct from all previously identified IGFBPs.
• Human IGFBP-l, IGFBP-2, IGFBP-3 and IGFBP-4 are characterized by amino acid sequences which have been published and which are distinct from the amino acid sequence of hBD-IGFBP.
• the N-terminal amino acid sequence reported for the IGFBP purified from cerebrospinal fluid, fibroblast cell conditioned medium and human serum is also distinct from that of hBD-IGFBP.
• the homogeneous human IGFBP of the invention is characterized by an N-terminal amino acid sequence identical to, or substantially identical to that shown in Fig. 1.
• an N-terminal amino acid sequence substantially identical to that shown in Fig. 1 is understood to mean an amino acid sequence identical to that of Fig. 1 except for the presence of conservative amino acid substitutions or other amino acid substitutions, insertions or deletions which do not materially affect the binding of the substantially identical protein to IGF-I or IGF-II, or otherwise materially alter its function in the applications set forth below.
• the gene which encodes the hBD-IGFBP of the present invention is cloned and expressed by insertion in a suitable expression vector which in turn is used to transform or transfect appropriate host cells for expression of recombinant hBD-IGFBP polypeptide.
• a suitable expression vector which in turn is used to transform or transfect appropriate host cells for expression of recombinant hBD-IGFBP polypeptide.
• One or more synthetic olig ⁇ nucleotide probes reflecting at least a portion of the amino terminal sequence of purified hBD- IGFBP, typically from about 14 to about 25 nucleotides are used to screen a human bone cell cDNA library.
• the positive clones containing the longest insert is sequenced according to standard procedures.
• the deduced amino acid sequence is compared with the N-terminal amino acid sequence of the purified protein, and the predicted molecular weight and amino acid composition based on the deduced amino acid sequence are compared with those observed for purified hBD-IGFBP.
• the clone is then used to produce recombinant hBD-IGFBP by using standard procedures, as generally described in, e.g., Sa brook et al., Molecular Cloning. A Laboratory Manual- 1989 Cold Spring Harbor Press, NY, which is incorporated herein by reference.
• the invention concerns polypeptides and fragments of hBD-IGFBP.
• Polypeptides and fragments of hBD-IGFBP may be isolated from recombinant expression systems or may be synthesized by the solid phase method of Merrifield, Fed. Proc. 21:412 (1962), Merrifield, i . Am. Chem. Soc. 85:2149 (1963), or Barany and Merrifield, in The Peptides. vol. 2, pp. 1-284 (1979) Academic Press, NY, each of which are incorporated herein by reference, or by use of an automated peptide synthesizer.
• polypeptides is meant a sequence of at least about 3 amino acids, typically 6 or more, up to 100-200 amino acids or more, including entire proteins.
• the portion(s) of hBD-IGFBP protein which bind hydroxyapatite and/or IGF-II may be identified by a variety of methods, such as by treating purified hBD-IGFBP with a protease or a chemical agent to fragment it and determine which fragment is able to bind to labeled IGF-II or hydroxyapatite. Polypeptides may then be synthesized and used as antigen, to inhibit IGF-II or hydroxyapatite-hBD-IGFBP interaction, etc. It should be understood that as used herein, reference to hBD- IGFBP is meant to include the proteins, polypeptides, and fragments thereof unless the context indicates otherwise.
• the invention provides means for regulating aspects of the hydroxyapatite/hBD-IGFBP/IGF-II interaction, and thus treating, therapeutically and/or prophylactically, a disorder which can be linked directly or indirectly to hBD-IGFBP or to its ligands, such as IFG-II.
• a disorder which can be linked directly or indirectly to hBD-IGFBP or to its ligands, such as IFG-II.
• agonists or antagonists may be identified which stimulate or inhibit the interaction of IGF-II, hydroxyapatite or other ligand with a hBD-IGFBP. With either agonists or antagonists the metabolism and reactivity of cells in response to hBD-IGFBP or IGF-II are controlled, thereby providing a means to abate or in some instances prevent the disease of interest.
• the invention provides screening procedures for identifying agonists or antagonists of events mediated by the ligand/hBD-IGFBP interaction.
• screening assays may employ a wide variety of formats, depending to some extent on which aspect of the ligand/binding protein interaction is targeted. For example, such assays may be designed to identify compounds which bind to the binding protein and thereby block or inhibit interaction with the IGF-II or hydroxyapatite. Other assays can be designed to identify compounds which can substitute for hBD-IGFBP. Yet other assays can be used to identify compounds which inhibit or facilitate the association of IGF to hBD-IGFBP and thereby mediate the cellular response to IGF.
• the invention provides a protein which binds IGF-II with selective affinity over IGF-I.
• Fig. 2 shows the competitive binding curve of the hBD-IGFBP [ 125 I]IGF- II as ligand and IGF-I and IGF-II as competitors.
• To displace 50% of bound [ 125 I]IGF-II from the IGFBP about 10 ng/ml of IGF-I and 1 ng/ml of IGF-II was needed, indicating that IGF-II was 10 times more potent than IGF-I in displacing tracer.
• [ 125 I]IGF-I was used as tracer, IGF-II was still more potent (4 times) than IGF-I in displacing tracer.
• the IGFBP binds IGF-II with greater affinity than IGF-I.
• the binding affinity of hBD-IGFBP for IGF-II will range from about 10 ⁇ 9 M up to about 10 ⁇ 12 M or more, and more likely in the range of at least about 10 ⁇ 10 to 10 "11 M, whereas the binding affinity of hBD-IGFBP for IGF-I ranges about 10 times less, i.e., from about 10 ⁇ 8 M up to about 10 ⁇ 10 M.
• the selective affinity of hBD-IGFBP for IGF-II over IGF-I could explain why IGF-II is 10-15 times more abundant than IGF-I in human bone.
• the invention provides therapeutic and pharmaceutical compositions of hBD-IGFBP which take advantage of hBD-IGFBP's strong binding affinity to hydroxyapatite, which is at least about 10 ⁇ 9 M up to about 10 "11 M or more.
• the hBD-IGFBP of the invention binds to hydroxyapatite even in the presence of strong denaturing agents, such as 4M guanidine HCl, while purified IGF-II does not bind to hydroxyapatite.
• the hBD-IGFBP provides a means or vehicle to fix or target IGF-II in or to bone.
• IGF-II can be chemically coupled to hBD-IGFBP via conjugation means that will be readily apparent to those of skill in the art, but should not substantially diminish the desired activity of either protein.
• the linkage of hBD-IGFBP to another molecule which is to be targeted to bone tissue or cells, such as the IGFs or other molecules as set forth hereinbelow, can be produced by chemical conjugation using well known laboratory procedures, such as by employing cross-linking reagents.
• chemically linked is meant that the protein molecules are linked, typically one to another, typically by covalent bonds.
• a preferred method of conjugation is the formation of at least one covalent bond between the hBD-IGFBP/IGF-II molecules.
• the linkage may be direct, which includes linkages containing a synthetic linking group, or indirect, by which is meant a link having an intervening moiety, such as a protein or peptide, e.g., plasma albumin, or other spacer molecule.
• the linkage may be by way of heterobifunctional or homobifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde, N-succinimidyl 3-(2-pyridydithio) propionate (SPDP) and derivatives, bis-maleimide, 4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC), cross-linking without exogenous cross-linkers by means of groups reactive with the individual molecules, such as carbohydrate, disulfide, carboxyl or amino groups via oxidation or reduction of the native protein, or treatment with an enzyme or the like.
• heterobifunctional or homobifunctional cross-linkers e.g., carbodiimide, glutaraldehyde, N-succinimidyl 3-(2-pyridydithio) propionate (SPDP) and derivatives, bis-maleimide, 4-(N- maleimidomethyl)cyclohexan
• Hybrid, chimeric or fusion protein molecules of IGF- II and hBD-IGFBP or portions thereof can also be prepared by recombinant DNA techniques, as described in, for example, U.S. Patent 4,859,609, and Sambrook et al., supra. incorporated herein by reference.
• the deposition of the IGF-II/hBD-IGFBP complex in bone allows, during bone resorption and the dissolution of hydroxyapatite, the complex to be released and initiate new bone formation by stimulating the proliferation of osteoblasts in the vicinity of the resorption site.
• this invention thus provides therapeutic agents and compositions which are useful to, inter alia, target IGF-II and/or IGF-I specifically to bone.
• novel hBD-IGFBP, hBD-IGFBP/IGF-II and other conjugates, antibodies to hBD-IGFBP and antagonists thereof, and pharmaceutical compositions prepared therefrom are particularly useful for administration for treatment of a wide variety of hBD-IGFBP and IGF-II related disease.
• the pharmaceutical compositions can be administered parenterally, i.e., subcutaneously, intramuscularly or intravenously, or topically, orally, via aerosol, intranasal delivery and the like.
• compositions for parenteral administration which comprise a solution of the hBD-IGFBP, hBD-IGFBP/IGF-II and other conjugates, antibodies to hBD-IGFBP and antagonists thereof or a cocktail of hBD-IGFBP and IGF-II dissolved in an acceptable carrier, preferably an aqueous carrier.
• an acceptable carrier preferably an aqueous carrier.
• aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like.
• These compositions may be sterilized by conventional, well known sterilization techniques.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
• concentration of the desired hBD-IGFBP, hBD-IGFBP/IGF-II, or antibodies to hBD-IGFBP or other antagonists thereof in these formulations can vary widely, i.e., from less than about 0.00001%, usually at or at least about 0.001%, to as much as about 0.05 to 0.1% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected, the condition being treated, e.g., fracture repair, osteoporosis, surgical or traumatic wound repair, tumors such as osteosarco a or breast carcinomas, etc., and the subject being treated, i.e., an adult, child or neonate.
• a typical pharmaceutical composition for intravenous infusion to treat an adult suffering from moderate osteodegenerative disease could be made up to contain 250 ml of sterile Ringer's solution, and about 50 mg to 5 grams of hBD- IGFBP or hBD-IGFBP/IGF-II.
• Actual methods for preparing parenterally or orally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example. Remington's Pharmaceutical Science. 16th ed.. Mack Publishing Company, Easton, PA (1982), which is incorporated herein by reference.
• the compositions containing the present hBD-IGFBP or hBD-IGFBP/IGF-II or cocktails thereof can be administered for prophylactic and/or therapeutic treatments.
• compositions are administered to a patient already suffering from an hBD-IGFBP or IGF-II related disease, in an amount sufficient to cure or at least partially arrest the disease and its complications.
• An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend on the disease, i.e., bone degeneration as in osteoporosis, fracture, wound, tumor, etc. , and its severity, the age of the patient and the general state of the patient's health.
• the amounts will range from about 1.0 to about 500 ⁇ g of hBD-IGFBP or hBD-IGFBP/IFG-II per kilogram of body weight per hour of infusion, with dosages of from 10 to 50 ⁇ g of hBD-IGFBP or hBD- IGFBP/IFG-II per kilogram per hour of infusion being more commonly used.
• dosages of from 10 to 50 ⁇ g of hBD-IGFBP or hBD- IGFBP/IFG-II per kilogram per hour of infusion being more commonly used.
• the materials of the present invention may be employed in serious disease states, in view of the minimization of extraneous substances and the absence of foreign substance responses, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these pharmaceutical compositions.
• compositions containing the present hBD-IGFBP or hBD-IGFBP/IFG-II or cocktails thereof are administered to a patient not already in a disease state to enhance the patient's resistance to the disease.
• Such an amount is defined to be a "prophylactically effective dose.”
• the precise amounts again depend on the patient's state of health, etc. but generally range from 1 to 500 ⁇ g per kilogram per hour of infusion, especially 10 to 50 ⁇ g per kilogram per hour.
• a preferred prophylactic use is for treatment of patients at risk of severe osteodegenerative diseases.
• Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.
• the pharmaceutical formulations should provide a quantity of hBD- IGFBP or hBD-IGFBP/IGF-II, for example, sufficient to treat the patient.
• the invention provides an agent which is effective in stimulating bone cell proliferation in response to IGF-II.
• IGF-II Exogenous addition of IGF-II to bone cells in serum free conditions increases their proliferation.
• This proliferative effect of IGF-II is potentiated by the addition of hBD-IGFBP in conjunction with IGF-II.
• This synergistic action of the combination of hBD-IGFBP and IGF-II that is, greater than the additive effect achieved by combining the results obtained with either agent individually, has not been reported for other IGFBPs in any cell type.
• the pharmaceutical compositions will comprise the hBD-IGFBP and, if desired, IGF, with physiologically acceptable carriers and or excipients.
• This invention also provides an agent that can be used in general wound healing and in skin repair to increase the potency and half life of IGFs, as hBD-IGFBP may increase the half-life of IGFs by protecting them from proteases, act to target IGFs specifically to bone, and/or potentiate the proliferative action of IGFs.
• hBD-IGFBP + IGF can be prepared in a variety of ways, e.g., by incubating concentrations of purified hBD-IGFBP and IGF at neutral pH overnight prior to adminis ⁇ tration.
• concentrations of hBD-IGFBP and IGF in the composition can vary widely, but preferably are approximately equimolar. Other formulations will be apparent to the skilled artisan from the context of the present disclosure.
• the compositions of hBD-IGFBP can be administered separately or simultaneously with the IGF-II compositions.
• the hBD- IGFBP will be administered first, followed by the IGF-II.
• Such compositions can be administered in specific areas to stimulate local bone formation (e.g., fracture repair) or administered systemically to increase general bone formation, as in the treatment of bone disorders such as osteoporosis.
• the present invention provides for the preparation of more potent IGF molecules.
• Structure- function analysis of IGFs and hBD-IGFBP can identify region(s) of IGFs that is involved in binding to hBD-IGFBP. It is possible to produce modified IGF molecules by amino acid substitutions, insertions or deletions such that the modified molecules bind to IGF receptors and the hBD-IGFBP with high affinity but not to inhibitory IGFBPs, such as IGFBP-4 and IGFBP-3.
• Such modified IGF molecules can serve as potent anabolic agents in promoting general wound healing and in skin repair.
• fragments of the hBD-IGFBP are produced. The fragments will typically have a desired function, such as the ability to bind IGF-II or hydroxyapatite, while eliminating other portions of the molecule which are not essential for this function. Fragments may be used individually or joined together.
• the hBD-IGFBP of the invention are useful in targeting other molecules of interest to the bone.
• the molecules may affect the formation or resorption of bone directly or indirectly.
• agents can be targeted to bone tissue in this manner.
• Representative examples include those which stimulate bone formation, such as bone morphogenic protein (BMP), TGFo, fibroblast growth factor (FGF) , platelet-derived growth factor (PDGF) , agents which decrease bone formation as may be desired in certain cancers, such as glucocorticoid or 1, 25-dihydroxyvitamin D 3 , those compounds which increase bone resorption, such as macrophage colony stimulating factor (M-CSF) and interleukins, and compounds which decrease bone resorption, such as bis- phosphonate and calcitonin, for example.
• BMP bone morphogenic protein
• FGF fibroblast growth factor
• PDGF platelet-derived growth factor
• agents which decrease bone formation as may be desired in certain cancers such as glucocorticoid or 1, 25-dihydroxyvitamin D 3
• M-CSF macrophage colony stimulating factor
• the compounds can be joined to the hBD-IGFBP of the invention in a variety of ways, including conjugation means set forth above as well
• the invention provides a diagnostic marker to evaluate bone formation in clinical samples taken from patients who have metabolic bone disease or bone neoplasia. Based on the findings that hBD-IGFBP is an important modulator of IGF-II actions and that IGF-II is an important human bone growth factor, levels of hBD-IGFBP can be used to monitor disorders of bone metabolism, where aberrant levels of hBD-IGFBP represent present of the disorder. Thus the invention also provides reagents for a clinical diagnostic bone formation marker to monitor bone formation during treatment of bone disorders with therapeutic agents.
• the compositions of hBD-IGFBP or antibodies thereto can be used for the detection and quantitation of hBD-IGFBP in a biological fluid, such as human plasma, serum or urine.
• hBD-IGFBP is quantified directly by measuring the binding of antibodies to hBD-IGFBP, which antibodies are then detected by, e.g., labeled anti-IgG, IgM and/or IgA human antibodies.
• a patient's hBD-IGFBP can be measured by competing with labeled or unlabeled hBD-IGFBP, for binding.
• labels may be employed, such as radionuclides, particles (e.g., gold, ferritin, magnetic particles, red blood cells), fluors, enzymes, enzyme substrates, enzyme cofactors enzyme inhibitors, ligands (particularly haptens) , chemiluminescers, etc., but preferably radionuclides.
• particles e.g., gold, ferritin, magnetic particles, red blood cells
• fluors e.g., gold, ferritin, magnetic particles, red blood cells
• enzymes e.g., enzyme substrates, enzyme cofactors enzyme inhibitors, ligands (particularly haptens) , chemiluminescers, etc.
• ligands particularly haptens
• chemiluminescers chemiluminescers, etc.
• kits can also be supplied for use in detecting the hBD-IGFBP, where the hBD-IGFBP and/or antibodies thereto may be provided, usually in lyophilized form, in a ID container, either alone or in conjunction with additional reagents, labels, and/or anti-antibodies, and the like.
• hBD-IGFBP polypeptide and antibodies which may be conjugated to a label, or unconjugated, and are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum, albumin, or the like. Frequently it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% of the total composition.
• Antibodies for diagnostic or therapeutic uses which bind hBD-IGFBP polypeptides of the invention can be produced by a variety of means. The production of non-human monoclonal antibodies, e.g., murine, is well known and may be accomplished by, for example, immunizing the animal with a recombinant or synthetic hBD-IGFBP molecule or a selected portion thereof
• Antibody producing cells obtained from the immunized animals are immortalized and screened, or screened first for, e.g., the production of antibody which binds the hBD-IGFBP, and then immortalized.
• Bones were cut using a band saw and ground to fine particles in a Wiley mill for hBD-IGFBP extraction. Bone proteins were extracted by demineralization of the femoral head bone powder with 10% ethylenediaminetetraacetate (EDTA) in the presence of 4M guanidine HCl and protease inhibitors (Guanidine EDTA extract) after an initial extraction with water and 4M guanidine HCl as described in Mohan, et al., .Biochem. Biophvs. Acta, 884:234-242 (1986)). Guanidine EDTA extract was then concentrated in an Amicon using YM5 (5 kilodaltons molecular weight cut-off) membrane and used for the purification of hBD- IGFBP.
• YM5 5 kilodaltons molecular weight cut-off
• hBD-IGFBP Human bone extract was subjected to hydroxyapatite (HA) chromatography in 4 M guanidine HCl as described in Mohan, et al., ibid.
• An IGF-II affinity column was constructed by coupling 250 ⁇ g of IGF-II purified from human bone to cyanogen bromide-activated Sepharose 4B beads.
• a pool of HA bound fractions was concentrated in an Amicon cell using a YM5 mem ⁇ brane to about 300 ml and dialyzed against 20 times volume of potassium phosphate buffer (10 mM potassium phosphate, pH 6.0) containing protease inhibitors (100 mM epsilon aminocaproic acid, 5 mM benzamidine, 1 mM phenyl ethylsulfonyl fluoride) .
• the IGF-II affinity column was equilibrated with the potassium phosphate buffer after which a 50 ml aliquot (approximately 3-4 mg total protein/ml) of the dialyzed HA bound fraction pool of the human bone extract was loaded into the column.
• the column was then extensively washed with potassium phosphate buffer to completely remove the unbound proteins.
• the bound proteins were eluted with 20-25 ml of 30 mM tris-acetate (pH 7.2)/4 M guanidine-HCl.
• the affinity bound fraction was concentrated in an Amicon cell using a YM 5 membrane and then dialyzed against 20 mM tris-HCl (pH 8.0) buffer.
• the dialyzed affinity bound fraction was applied to a Pharmacia FPLC Mono Q anion exchange column previously equilibrated with the tris-HCl buffer.
• the bound proteins were eluted with a linear gradient from 0-1 M NaCl in 20 mM tris-HCl buffer in 100 min.
• hBD-IGFBP activity was determined by a polyethylene glycol precipitation method. Briefly, 50 ⁇ l of sample to be assayed was incubated with 25,000 to 50,000 cpm of 1 5 I-labeled IGF-I or IGF-II for 60 minutes at room temperature in 250 ⁇ l of 0.1M HEPES/0.1% bovine serum albumin/0.1% Triton X100/44 mM Na 2 CO3/0.02% NaN3, pH 6.0. To this mixture was added 100 ⁇ l of 2% immune serum globulin and 500 microliters of 25% polyethylene glycol, followed by centrifugation.
• IGFBP ligand blot analysis was carried out using 125 I-IGF-II as a tracer.
• 50 ⁇ l of samples was electrophoresed under non-reducing conditions on precast 3-27% SDS polyacrylamide slab gels.
• the nitrocellulose membrane was incubated with radiolabeled IGF-II.
• the membrane was subjected to autoradiography as described in Hossenloop, et al., (Anal. Biochem.. 154:138-143 (1986)).
• Fig. 3 shows the protein profile and IGFBP activity profile of the affinity-bound fraction pool in the Mono Q chromatography step. There was neither a protein absorbance peak nor a IGFBP activity peak in the region where authentic IGFBP-4 elutes (Fractions 9-15, 0.1M NaCl), thus suggesting that the bone derived IGFBP is not IGFBP-4.
• IGFBPs present in human bone extract ligand blotting and [ 125 I]IGF-II affinity labeling were used.
• Fig. 4 shows ligand blots in which the HA bound, IGF-II bound and Mono Q protein peaks were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with [ 125 I]IGF-II tracer.
• the major IGFBP present in HA bound and IGF-II bound fractions had an apparent molecular weight of 29 kDa. In addition, these fractions also exhibited a broad, less intense band between the 68 and 43 kDa molecular weight markers.
• the major 29 kDa IGFBP was separated from the higher molecular weight IGFBP by Mono Q chromatography.
• Mono Q peak A showed a major band at 29 kDa and a minor band at 24 kDa.
• Mono Q peak B showed a broad diffuse band between the 68 and 43 kDa markers and a minor band at 29 kDa.
• Mono Q peak C (represents the major protein absorbance peak) and D showed only a weak band at 29 kDa.
• the 29 kDa IGFBP has been designated human bone derived IGFBP (hBD-IGFBP) .
• hBD-IGFBP human bone derived IGFBP
• major sequence Leu-Gly-Phe-Phe-Val-X-Val-Glu-Pro-Asp-Asp-Lys- Ala-Ala-Leu
• hBD-IGFBP human bone derived IGFBP
• the purified hBD- IGFBP appeared to be susceptible to proteolytic cleavage since storage of purified 29 kDa IGFBP at 5°C for overnight led to the disappearance of 29 kDa band and appearance of a major band at 24 kDa and several low molecular weight minor bands.
• Mono Q peak B appeared to have a different amino acid composition and did not potentiate the action of IGF-II on bone cells. Our attempts to sequence peak B proved unsuccessful. The sequence determination of the first few cycles of the major protein peak (Fraction 45, peak C) , yielded multiple amino acids with no readable sequence. Thus, Mono Q peaks C and D which contained very little IGFBP activity may represent the degradation products of the 29 kDa IGFBP.
• hBD-IGFBP may have the sequence of L-G-F-F-V-X-C-E-P-C-D-K-A-A-L in one embodiment.
• An alternative sequence for hBD-IGFBP which also takes into account the foregoing considerations is: L-G-S-F-V-H-C-E-P-C-D-E-K-A-L, which sequence is similar to the BP-5 sequence of Kiefer et al. , Biochem. Biophvs. Res. Com .
• the sequence may be subject to possible variations based on native or introduced substitutions, additions or deletions, which variations may be allelic variants or produced through particular sequence techniques employed herein. It will be recognized that using the methods described herein, the protein may be isolated and purified and the sequence determined by a variety of well known methods. Further, the N-terminal sequence allows for the construction of degenerate oligonucleotide probes for the cloning of the gene which encodes the hBD-IGFBP of the invention.
• IGFBP-3 has been shown to increase IGF-I action only when added to cultures several hours prior to the addition of IGF-I, (Mohan, et al., Proc. Natl. Acad. Sci. USA. 86:8338-8342 (1989) and De Mellow, et al., Biochem. Biophvs. Res. Comm. , 156:199-204 (1988)) and no other IGFBPs are believed to have been shown to potentiate the actions of IGFs under conditions where both an IGF and an IGFBP are added simultaneously. These data suggest that the hBD- IGFBP is not merely a passive carrier for the IGFs but also positively regulates the action of the IGFs.
• hBD-IGFBP potentiates IGF-II stimulated [ 3 H]thymidine incorporation
• the hBD- IGFBP targets the IGF-II to the cell membrane for easy access of the IGF-II to its receptor (perhaps through an RGD sequence as in the case of IGFBP-l) .
• the hBD- IGFBP may increase the affinity of IGF-II to its receptor by virtue of its binding to IGF-II, and/or increase the half-life of IGF-II by protecting it from proteases.
• Serum free cultures of mouse osteoblastic cell line, MC3T3-E1 were incubated for 18 hrs with the effectors prior to the addition of [ 3 H]thymidine.
• the final concentrations of IGF-II and hBD-IGFBP were 3 and 10 ng/ml respectively. Values are Mean + SD of 6 replicate wells.
• [ H]thymidine incorporation in bovine serum albumin (BSA) treated control cultures were 1404 ⁇ 217, 237 + 53 and 730 + 91 respectively in the three experiments.
• bone cell conditioned medium is concentrated in an Amicon using YM5 (5 kilodalton molecular weight cut-off) , acidified with acetic acid to a final concentration of IM and subjected to Sephadex G- 100 gel filtration to separate IGFs from IGFBPs.
• the proteins are eluted with IM acetic acid.
• the fractions containing IGFBPs are pooled, lyophilized, reconstituted with phosphate buffered saline and subjected to an IGF-II affinity column.
• the affinity bound proteins are then subjected to FPLC Mono Q anion exchange chromatography to separate hBD-IGFBP from other IGFBPs.
• EXAMPLE V Quantitative diagnostic assay for hBD-IGFBP hBD-IGFBP purified from human bone or expressed by recombinant means is used for polyclonal and/or monoclonal antibody production, which antibodies are then used in quantitative assays for hBD-IGFBP. Briefly, hBD- IGFBP is mixed with complete Freund's adjuvant and injected into rabbits, guinea pigs, rats or mice following established protocols for antibody production. Animals are subsequently injected with hBD-IGFBP mixed with incomplete Freund's adjuvant every 3-4 weeks.
• the animals are bled after 3-4 injections and the antibody titer to hBD-IGFBP determined using radioimmunoassay or other means.
• Monoclonal antibodies are produced by immortalizing antibody producing cells obtained from the immunized animals using well known techniques. Purified hBD-IGFBP is radiolabeled and used as the signal-producing tracer. The monoclonal antibody or antiserum with high titer is then used for development of hBD-IGFBP radioimmunoassay for measurement of hBD-IGFBP levels in the serum urine and other biological fluids.
• hBD-IGFBP In general, the production of hBD-IGFBP is increased by treatment of bone cells with agents which increase bone cell proliferation.
• hBD-IGFBP can be used as diagnostic marker for disease states associated -with bone cell proliferation, such as osteoporosis. Accordingly, low serum hBD-IGFBP indicates osteoporosis associated with low bone formation. Since hBD-IGFBP potentiates IGF-II action, a high serum hBD-IGFBP may also be associated with some cancers.
• EXAMPLE VI Quantitative diagnostic assay for IGFs using hBD-IGFBP As described in this Example, recombinant or purified hBD-IGFBP can also be used to quantify levels of IGFs in a biological sample, such as serum.
• hBD-IGFBP 2-5 ng of purified hBD-IGFBP was incubated with 40,000 cpm of 1 5 ⁇ - IGF in the presence or absence of unlabeled competitor. IGF standards or sample containing unknown amounts of IGF were used as competitors. After 60 minutes of incubation at room temperature, hBD-IGFBP-IGF complex was precipitated by adding polyethylene glycol in the presence of bovine gamma globulin. After a 30 minute centrifugation at 1185 X g, an aliquot of the supernatant was counted in a gamma counter. A standard curve was set up with different concentrations of unlabeled IGF and the amount of IGF in the unknown sample was calculated using the standard curve. Thus the amount of biologically active free IGFs was determined by this assay utilizing purified hBD-IGFBP that has high affinity for IGFs.
• Human bone contains a relatively large amount of IGF-II.
• IGF-II 125 ⁇ labeled IGF-II by itself does not specifically bind to hydroxyapatite (the Table shows only non-specific binding which is less than 10% of total counts added) or to collagen.
• labeled hBD-IGFBP exhibited specific binding to hydroxyapatite. The binding of hBD-IGFBP to hydroxyapatite was specific since the major serum binding protein, i.e., IGFBP-3 had no similar activity and since hBD-IGFBP did not bind to collagen, the other major constituent of bone.
• the stimulatory effect of progesterone on bone cell proliferation could be mediated not only by increased IGF production, but also by an increase in IGF receptor expression, an increase in hBD-IGFBP (potentiates IGF action) , and a decreased production of the inhibitory IGFBP-4.

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