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WO1992007004A1 - Proteine osteogenique - Google Patents

Proteine osteogenique Download PDF

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Publication number
WO1992007004A1
WO1992007004A1 PCT/US1991/007654 US9107654W WO9207004A1 WO 1992007004 A1 WO1992007004 A1 WO 1992007004A1 US 9107654 W US9107654 W US 9107654W WO 9207004 A1 WO9207004 A1 WO 9207004A1
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WIPO (PCT)
Prior art keywords
leu
ser
ala
arg
protein
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PCT/US1991/007654
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English (en)
Inventor
Engin Ozkaynak
Hermann Oppermann
Thangavel Kuberasampath
David C. Rueger
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Creative Biomolecules, Inc.
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Application filed by Creative Biomolecules, Inc. filed Critical Creative Biomolecules, Inc.
Publication of WO1992007004A1 publication Critical patent/WO1992007004A1/fr

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    • 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
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • This invention relates to a novel polypeptide chain and to osteogenic proteins comprising this polypeptide chain which are capable of inducing osteogenesis in mammals, to a gene encoding the polypeptide chain, to methods for its production using recombinant DNA techniques, and to bone and cartilage repair procedures using the dosteogenic proteins.
  • Mammalian bone tissue is known to contain one or more proteinaceous materials, presumably active during growth and natural bone healing, which can induce a developmental cascade of cellular events resulting in endochondral bone formation.
  • This active factor (or factors) has variously been referred to in the literature as bone morphogenetic or morphogenic protein, bone inductive protein, osteogenic protein, osteogenin, or osteoinductive protein.
  • the developmental cascade of bone differentiation consists of recruitment of mesenchymal cells, proliferation of progenitor cells, calcification of cartilage, vascular invasion, bone formation, remodeling, and finally marrow differentiation (Reddi (1981) Collagen Rel. Res. .1:209-226).
  • the protein fraction has an amino acid composition of an acidic polypeptide and a molecular weight in a range of 17-18 kD.
  • Urist et al. (1984) Proc. Natl. Acad. Sci. USA 81: 371-375 disclose a bovine bone morphogenetic protein extract having the properties of an acidic polypeptide and a molecular weight of approximately 18 kD.
  • the authors reported that the protein was present in a fraction separated by hydroxyapatite chromatography, and that it induced bone formation in mouse hindquarter muscle and bone regeneration in trephine defects in rat and dog skulls. Their method of obtaining the extract from bone results in ill-defined and impure preparations.
  • This invention provides novel polypeptide chains useful as either one or both subunits of dimeric osteogenic proteins which, when implanted in a mammalian body in association with a matrix, can induce at the locus of the implant the full developmental cascade of endochondral bone formation and bone marrow differentiation.
  • Consensus DNA sequences based on partial sequence data and observed homologies with regulatory proteins disclosed in the literature were used as probes for extracting genes encoding osteogenic protein from human genomic and cDNA libraries.
  • One of the consensus sequences was used to isolate a previously unidentified gene which, when expressed, encoded a protein comprising a region capable of inducing endochondral bone formation when properly modified, incorporated in a suitable matrix, and implanted as disclosed herein.
  • the gene, called “hOPl” or "OP-1” is described in greater detail in U.S. 660,162, filed 27-SEP-91 the disclosure of which is herein incorporated by reference.
  • hOPl DNA sequence subsequently were used to probe a mouse embryo cDNA library in search of additional genes encoding osteogenic proteins.
  • This process isolated a heretofore unidentified DNA sequence which encodes a polypeptide chain referred to herein as mOPl protein.
  • Mouse 0P1 (mOP-1) protein shares significant amino acid sequence homology with human hOPl protein, particularly in the region encoding the mature protein.
  • homodimers of mOPl proteins and heterodimers comprising mOPl protein are believed to be capable of inducing endochondral bone formation, when the protein is dispersed in a suitable matrix, and implanted as disclosed herein.
  • mOPl-S The sequence of what is believed to be the mature form of the murine protein, designated herein mOPl-S, is (residues 292-430 of Seq. ID No. 1).
  • the amino acid sequence of the full length protein, mOPl-PP (the "prepro” form, see infra), and the cDNA sequence encoding it are set forth in Seq. ID No. 1.
  • the invention provides recombinant dimeric proteins, and osteogenic devices comprising these proteins, wherein the subunits of the osteogenic dimers comprise an amino acid sequence described by Seq. ID No. 1, including allelic and biosynthetically mutated variants thereof.
  • Mouse OP1 can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and then purified, cleaved, refolded, dimerized, and implanted in experimental animals.
  • host cells include E. coli, or mammalian cells, such as CHO, COS or BSC cells.
  • the osteogenic protein of the invention may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic protein, produced by expression of recombinant DNA in host ⁇ _,ells.
  • skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different species which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins capable of inducing bone formation in mammals including humans.
  • the osteogenic proteins are useful in clinical applications in conjunction with a suitable delivery or support system (matrix).
  • the matrix is made up of particles of porous materials.
  • the pores must be of a dimension to permit progenitor cell migration and subsequent differentiation and proliferation.
  • the particle size should be within the range of 70 - 850 mm, preferably 150mm - 420mm. It may be fabricated by close packing particulate material into a shape spanning the bone defect, or by otherwise structuring as desired a material that is biocompatible (non ⁇ inflammatory) and, biodegradable iji vivo to serve as a "temporary scaffold" and substratum for recruitment of migratory progenitor cells, and as a base for their subsequent anchoring and proliferation.
  • xenogenic bone powder matrices also may be treated with proteases such as trypsin and/or fibril modifying agents to increase the intraparticle intrusion volume and surface area.
  • useful agents include solvents such as dichloromethane, trichloroacetic acid, acetonitrile and acids such as trifluoroacetic acid and hydrogen fluoride.
  • the matrix may be treated with a hot aqueous medium having a temperature within the range of about 37°C to 75°C, including heated acidic aqueous medium.
  • Other potentially useful matrix materials comprise collagen, homopolymers and copolymers of glycolic acid and lactic acid, hydroxyapatite, tricalcium phosphate and other calcium phosphates.
  • the osteogenic proteins and implantable osteogenic devices enabled and disclosed herein will permit the physician to obtain optimal predictable bone formation to correct, for example, acquired and congenital craniofacial and other skeletal or dental anomalies (Glowacki et al. (1981) Lancet 1; 959-963).
  • the devices may be used to induce local endochondral bone formation in non-union fractures as demonstrated in animal tests, and in other clinical applications including dental and periodontal applications where bone formation is required.
  • Another potential clinical application is in cartilage repair, for example, in the treatment of osteoarthritis. Brief Description of the Drawing
  • FIGURE 1 compares the amino acid sequences of the mature hOPl and mOPl polypeptide chains: OP1-18 and mOPl-S.
  • hOPl One of the DNA sequences isolated from human genomic and cDNA libraries encoded a previously unidentified gene, referred to herein as hOPl.
  • the protein encoded by the isolated DNA was identified originally by amino acid homology with proteins in the TGF- ⁇ family. Consensus splice signals were found where amino acid homologies ended, designating exon- intron boundaries. Three exons were combined to obtain a functional TGF- ⁇ like domain containing seven cysteines. (See, for example, U.S. Patent No. 5,011,691 or Ozkaynak, E. et al., (1990) EMBO. : pp. 2085-2093).
  • the DNA also is referred to in related applications as "OP1 and "OP-1".
  • hOPl expression yields an immature translation product ("hOPl-PP", where "PP” refers to "prepro form") of about 400 amino acids that subsequently is processed to yield a mature sequence of 139 amino acids (“OPl-18").
  • PP immature translation product
  • (functional domain) of the protein comprises the C- ter inal 97 amino acids of the hOPl sequence, "OPS", which includes a conserved six cysteine skeleton.
  • a longer active sequence is OP7, comprising the C- terminal 102 amino acids, and which includes a conserved seven cysteine skeleton.
  • hOPl-PP The full length cDNA sequence for hOPl, and its encoded "prepro" form hOPl-PP, which includes an N- terminal signal peptide sequence, are disclosed in Seq. ID No. 3 (residues 1-431).
  • cDNA sequences encoding the "prepro" form, of the protein and the mature form, as well as various truncated forms of the gene, and fused genes, have been expressed in E. coli (see, for example, U.S. Serial No. 422, 699) and numerous mammalian cells (See, for example, PCT WO 91/05802, published 2-MAY-91, and all have been shown to have osteogenic activity when implanted in a mammal in association with a suitable matrix.
  • RNAs and DNAs can be constructed which encode at least the active region of an OP1 protein (e.g., OPS or OP7, amino acid residues 335-431 or 330-431, respectively, of Seq. ID No. 3) and various analogs thereof, as well as fusion proteins, truncated forms of the mature proteins, and similar constructs.
  • DNA hybridization probes can be constructed from fragments of the hOPl DNA or designed de novo based on the hOPl DNA or amino acid sequence. These probes then can be used to screen different genomic and cDNA libraries to identify additional osteogenic proteins.
  • the DNAs can be produced by those skilled in the art using well known DNA manipulation techniques involving genomic and cDNA isolation, construction of synthetic DNA from synthesized oligonucleotides, and cassette mutagenesis techniques.
  • 15-100mer oligonucleotides may be synthesized on a Biosearch DNA Model 8600 Synthesizer, and purified by polyacrylamide gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer. The DNA may then be electroeluted from the gel.
  • Overlapping oligomers may be phosphorylated by T4 polynucleotide kinase and ligated into larger blocks which may also be purified by PAGE.
  • DNAs for use as hybridization probes may be labelled (e.g., as with a radioisotope, by nick translation) and used to identify clones in a given library containing DNA to which the probe hybridizes, following techniques well known in the art.
  • the libraries may be obtained commercially or they may constructed de novo using conventional molecular biology techniques. Further information on DNA library construction and hybridization techniques can be found n numerous texts known to those skilled in the art. See, for example, F.M. Ausubel, ed., Current Protocols in Molecular Bioloqy-Vol. 1, (1989). In particular, see unit 5, "Construction of Recombinant DNA Libraries” and Unit 6, "Screening of Recombinant Libraries.”
  • Appropriately identified clones then can be sequenced using any of a number of techniques well known in the art.
  • a DNA fragment containing the sequence of interest then can be subcloned into an expression vector and transfected into an appropriate host cell for protein expression and further characterization.
  • the host may be a procaryotic or eucaryotic cell since the former's inability to glycosylate protein will not destroy the protein's osteogenic activity.
  • Useful host cells include E. coli, Saccharomyces, the insect/baculovirus cell system, myeloma cells, and various mammalian cells.
  • the vector additionally may encode various sequences to promote correct expression of the recombinant protein, including transcription promoter and termination sequences, enhancer sequences, preferred ribosome binding site sequences, preferred mRNA leader sequences, preferred signal sequences for protein secretion, and the like.
  • the DNA sequence encoding the gene of interest also may be manipulated to remove potentially inhibiting sequences or to minimize unwanted secondary structure formation.
  • the recombinant osteogenic protein also may be expressed as a fusion protein. After being translated, the protein may be purified from the cells themselves or recovered from the culture medium.
  • All biologically active protein forms comprise dimeric species joined by disulfide bonds or otherwise associated, produced by oxidizing and refolding one or more of the various recombinant proteins within an appropriate eucaryotic cell or i i vitro after expression of individual subunits.
  • a detailed description of osteogenic protein expressed from recombinant DNA in E. coli is disclosed in U.S. Serial No. 660,162, the disclosure of which has been incorporated by reference, supra.
  • a detailed description of osteogenic protein expressed from recombinant DNA in numerous different mammalian cells is disclosed in PCT WO 91/05802.
  • a hybridization probe specific to the C-terminus of the DNA of mature hOPl was prepared using a StuI-EcoRl digest fragment of hOPl (base pairs 1034-1354 in Seq. ID No. 3), and labelled with P by nick translation, as described in the art.
  • the C-terminus of the protein encodes a key functional domain e.g., the "active region" for osteogenic activity.
  • the C-terminus also is the region of the protein whose amino acid sequence shares specific amino acid sequence homology with particular proteins in the TGF- ⁇ super-family of regulatory proteins and which includes the conserved cysteine skeleton.
  • Approximately 7 x 10 5 phages of an oligo (dT) primed 17.5 days p.c. mouse embryo 5' stretch cDNA (gtlO) library (Clontech, Inc., Palo Alto, CA) was screened with the labelled probe.
  • the screen was performed using the following stringent hybridization conditions: 40% formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SDS, at 37°C overnight, and washing in 0.1 x SSPE, 0.1% SDS, at 50°C
  • Phage DNA was prepared from all five phages, subjected to an EcoRl digest, subcloned into the EcoRl site of common pUC-type plas id modified to allow single strand sequencing, and sequenced using means well known in the art.
  • mOP2 Two different DNA sequences were identified by this procedure.
  • mOP2 One DNA, referred to herein as mOP2
  • mOPl A second DNA, referred to herein as mOPl, is described below.
  • the cDNA and encoded amino acid sequence for the full length mOPl protein is depicted in Seq. ID No. 1.
  • the full-length form of the protein is referred to as the prepro form of mOP-1 ("mOPl-PP"), and includes a signal peptide sequence at its N-terminus.
  • the amino acid sequence Ser-Ala-Leu-Ala-Asp is believed to constitute the cleavage site for the removal of the signal peptide sequence, leaving an intermediate form of the protein, the "pro" form, to be secreted from the expressing cell.
  • the amino acid sequence Arg-Ser-Ile-Arg-Ser (amino acid residue nos. 288-292 in Seq.
  • mOPl-S cleavage site that produces the mature form of the protein
  • the region of the mOPl amino acid sequence corresponding to the conserved six cysteine skeleton is described by residues 334-430 of Seq. ID No. 1.
  • the region corresponding to the conserved seven cystein skeleton is described by residues 329-430 of Seq. ID No. 1.
  • Figure 1 compares the amino acid sequence homology of the mature hOPl and mOPl proteins (OP1-18 and mOPl-S). Amino acid identity is indicated by three dots white. As can be seen in Figure 1, the mature form of mOPl, mOPl-S shows significant sequence homology with OP-1-18 (98%), differing at only three positions in this region. Like OP-1-18, mOPl-S has a seven cysteine functional domain (residues 38-139 of Fig. 1). The prepro form of the mOPl protein shares substantially less amino acid sequence homology with that of OP1-PP.
  • the high degree of homology of the mature domains is not surprising as the amino acid sequences of the mature forms of TGF- ⁇ -like proteins generally also have been found to be highly conserved across different animal species (e.g., compare Vgr and Vgl, two related genes from mouse and Xenopus, respectively, see U.S. Pat. No. 5,011,691).
  • the high degree of amino acid sequence homology exhibited between the mature forms of the two animal species of OPl proteins identified suggests that the mOP-1 protein will purify essentially as the human OPl protein does, or with only minor modifications of the protocols disclosed for human OPl protein.
  • purified mOPl-S is predicted to have an apparent molecular weight of about 36 kD as a glycosylated oxidized homodimer, and about 18 kD as a reduced single subunit, as determined by comparison with molecular weight standards on an SDS-polyacrylamide electrophoresis gel. There appear to be three potential N glycosylation sites in the mature mOPl protein.
  • the unglycosylated homodimer (e.g., one expressed from E_. coli) is predicted to have a molecular weight of about 27 kD.
  • the currently preferred carrier material is a xenogenic bone-derived particulate matrix treated as disclosed herein.
  • This carrier may be replaced by either a biodegradable-synthetic or synthetic-inorganic matrix (e.g., hydroxylapatite (HAP), collagen, tricalcium phosphate or polylactic acid, polyglycolic acid and various copolymers thereof.)
  • HAP hydroxylapatite
  • collagen e.g., hydroxylapatite (HAP), collagen, tricalcium phosphate or polylactic acid, polyglycolic acid and various copolymers thereof.
  • the sequential cellular reactions in the interface of the bone matrix/osteogenic protein implants are complex.
  • the multistep cascade includes: binding of fibrin and fibronectin to implated matrix, chemotaxis of cells, proliferation of fibroblasts, differentiation into chondroblasts, cartilage formation, vascular invasion, bone formation, remodeling, and bone marrow differentiation.
  • a successful carrier for osteogenic protein must perform several important functions. It must bind osteogenic protein and act as a slow release delivery system, accommodate each step of the cellular response during bone development, and protect the osteogenic protein from nonspecific proteolysis.
  • selected materials must be biocompatible n vivo and preferably biodegradable; the carrier must act as a 5 temporary scaffold until replaced completely by new bone.
  • Polylactic acid (PLA), polyglycolic acid (PGA), and various combinations have different dissolution rates mi vivo. In bones, the dissolution rates can vary according to whether the implant is placed in Q cortical or trabecular bone.
  • Matrix geometry, particle size, the presence of surface charge, and the degree of both intra-and- inter-particle porosity are all important to successful matrix performance. It is preferred to shape the matrix to the desired form of the new bone and to have dimensions which span non-union defects. Rat studies show that the new bone is formed essentially having the dimensions of the device implanted.
  • the matrix may comprise a shape-retaining o solid made of loosely adhered particulate material, e.g., with collagen. It may also comprise a molded, porous solid, or simply an aggregation of close-packed particles held in place by surrounding tissue. Masticated muscle or other tissue may also be used. 5 Large allogenic bone implants can act as a carrier for the matrix if their marrow cavities are cleaned and packed with particle and the dispersed osteogenic protein.
  • the preferred matrix material prepared from 0 xenogenic bone and treated as disclosed herein, produces an implantable material useful in a variety of clinical settings.
  • the matrix also may be used as a sustained release carrier, or as a collagenous coating for implants.
  • the matrix may be shaped as desired in anticipation of surgery or shaped by the physician or technician during surgery.
  • the material may be used for topical, subcutaneous, intraperitoneal, or intramuscular implants; it may be shaped to span a nonunion fracture or to fill a bone defect.
  • the material is slowly absorbed by the body and is replaced by bone in the shape of or very nearly the shape of the implant.
  • Various growth factors, hormones, enzymes, therapeutic compositions, antibiotics, and other body treating agents also may be absorbed onto the carrier material and will be released over time when implanted as the matrix material is slowly absorbed.
  • various known growth factors such as EGF, PDGF, IGF, FGF, TGF- ⁇ , and TGF- ⁇ may be released in vivo.
  • the material can be used to release chemotherapeutic agents, insulin, enzymes, or enzyme inhibitors.
  • Demineralized bone matrix preferably bovine bone matrix
  • bovine bone matrix is prepared by previously published procedures (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA 80:6591-6595).
  • Bovine diaphyseal bones (age 1-10 days) are obtained from a local slaughterhouse and used fresh. The bones are stripped of muscle and fat, cleaned of periosteum, demarrowed by pressure with cold water, dipped in cold absolute ethanol, and stored at -20°C. They are then dried and fragmented by crushing and pulverized in a large mill. Care is taken to prevent heating by using liquid nitrogen.
  • the pulverized bone is milled to a particle size in the range of 70-850 ⁇ m, preferably 150-420 ⁇ m, and is defatted by two washes of approximately two hours duration with three volumes of chloroform and methanol (3:1).
  • the particulate bone is then washed with one volume of absolute ethanol and dried over one volume of anhydrous ether yielding defatted bone powder.
  • the defatted bone powder is then demineralized by four successive treatments with 10 volumes of 0.5 N HC1 at 4°C for 40 min. Finally, neutralizing washes are done on the demineralized bone powder with a large volume of water.
  • Demineralized bone matrix thus prepared is extracted with 5 volumes of 4 M guanidine-HCl, 50mM
  • the suspension is filtered.
  • the insoluble material is collected and u?ed to fabricate the matrix.
  • the material is mostly collagenous in nature. It is devoid of osteogenic or chondrogenic activity.
  • the major component of all bone matrices is Type-I collagen.
  • demineralized bone extracted as disclosed above includes non-collagenous proteins which may account for 5% of its mass. In a xenogenic matrix, these noncollagenous components may present themselves as potent antigens, and may constitute immunogenic and/or inhibitory components. These components also may inhibit osteogenesis in allogenic implants by 5 interfering with the developmental cascade of bone differentiation.
  • treatment of the matrix particles with a collagen fibril-modifying agent extracts potentially unwanted components from the matrix, and alters the surface o structure of the matrix material.
  • Useful agents include acids, organic solvents or heated aqueous media. Various treatments are described below. A detailed physical analysis of the effect these fibril- modifying agents have on demineralized, quanidine- 5 extracted bone collagen particles is disclosed in copending U.S. Patent Application Serial No. 483,913, filed February 22, 1990.
  • the treated matrix is washed to remove any extracted 0 components, following a form of the procedure set forth below:
  • TBS Tris-buffered saline
  • UTBS Tris-buffered saline
  • RT room temperature
  • Trifluoroacetic acid is a strong non-oxidizing acid that is a known swelling agent for proteins, and which modifies collagen fibrils.
  • Bovine bone residue prepared as described above is sieved, and particles of the appropriate size are collected. These particles are extracted with various percentages (1.0% to 100%) of trifluoroacetic acid and water (v/v) at 0°C or room temperature for 1-2 hours with constant stirring. The treated matrix is filtered, lyophilized, or washed with water/salt and then lyophilized.
  • hydrogen fluoride is a strong acid and swelling agent, and also is capable of altering intraparticle surface structure. Hydrogen fluoride is also a known deglycosylating agent. As such, HF may function to increase the osteogenic activity of these matrices by removing the antigenic carbohydrate content of any glycoproteins still associated with the matrix after guanidine extraction.
  • Bovine bone residue prepared as described above is sieved, and particles of the appropriate size are collected.
  • the sample is dried iri vacuo over P 2 °5' transferred to the reaction vessel and exposed to anhydrous hydrogen fluoride (10-20 ml/g of matrix) by distillation onto the sample at -70°C.
  • the vessel is allowed to warm to 0°C and the reaction mixture is stirred at this temperature for 120 minutes.
  • After evaporation of the hydrogen fluoride in vacuo the residue is dried thoroughly in vacuo over KOH pellets to remove any remaining traces of acid.
  • Extent of deglycosylation can be determined from carbohydrate analysis of matrix samples taken before and after treatment with hydrogen fluoride, after washing the samples appropriately to remove non-covalently bound carbohydrates.
  • SDS-extracted protein from HF-treated material is negative for carbohydrate as determined by Con A blotting.
  • the deglycosylated bone matrix is next washed twice in TBS (Tris-buffered saline) or UTBS, water- washed, and then lyophilized.
  • TBS Tris-buffered saline
  • UTBS Tris-buffered saline
  • TFA is a currently preferred acidifying reagent in these treatments because of its volatility.
  • other, potentially less caustic acids may be used, such as acetic or formic acid.
  • Dichloromethane is an organic solvent capable of denaturing proteins without affecting their primary structure.
  • This swelling agent is a common reagent in automated peptide synthesis, and is used in washing steps to remove components.
  • Bovine bone residue prepared as described above, is sieved, and particles of the appropriate size are incubated in 100% DCM or, preferably, 99.9% DCM/0.1% TFA.
  • the matrix is incubated with the swelling agent for one or two hours at 0°C or at room temperature. Alternatively, the matrix is treated with the agent at least three times with short washes (20 minutes each) with no incubation.
  • Acetonitrile is an organic solvent, capable of denaturing proteins without affecting their primary structure. It is a common reagent used in high-performance liquid chromatography, and is used to elute proteins from silica-based columns by perturbing hydrophobic interactions.
  • Bovine bone residue particles of the appropriate size are treated with 100% ACN (1.0 g/30 ml) or, preferably, 99.9% ACN/0.1% TFA at room temperature for 1-2 hours with constant stirring.
  • the treated matrix is then water-washed, or washed with urea buffer, or 4 M NaCl and lyophilized.
  • the ACN or ACN/TFA treated matrix may be lyophilized without wash.
  • Isopropanol is also an organic solvent capable of denaturing proteins without affecting their primary structure. It is a common reagent used to elute proteins from silica HPLC columns.
  • Bovine bone residue particles of the appropriate size prepared as described above are treated with 100% isopropanol (1.0 g/30 ml) or. preferably, in the presence of 0.1% TFA, at room temperature for 1-2 hours with constant stirring. The matrix is then water-washed or washed with urea buffer or 4 M NaCl before being lyophilized.
  • Chloroform also may be used to increase surface area of bone matrix like the reagents set forth above, either alone or acidified.
  • Treatment as set forth above is effective to assure that the material is free of pathogens prior to implantatio .
  • the currently most preferred agent is a heated aqueous fibril-modifying medium such as water, to increase the matrix particle surface area and porosity.
  • the currently most preferred aqueous medium is an acidic aqueous medium having a pH of less than about 4.5, e.g., within the range of pH 2 - pH 4. which may help to "swell" the collagen before heating. 0.1% acetic acid, which has a pH of about 3, currently is preferred. 0.1 M acetic acid also may be used.
  • aqueous medium lg matrix/30ml aqueous medium
  • Preferred treatment times are about one hour, although exposure times of between about 0.5 to two hours appear acceptable.
  • the temperature employed is held constant at a temperature within the range of about 37°C to 75°C.
  • the currently preferred heat treatment temperature is within the range of 45°C to 60°C.
  • the matrix is filtered, washed, lyophilized and used for implant.
  • the matrix also is preferably neutralized prior to washing and lyophilization.
  • a currently preferred neutralization buffer is a 200mM sodium phosphate buffer, pH 7.0.
  • the matrix preferably first is allowed to cool following thermal treatment, the acidic aqueous medium (e.g., 0.1% acetic acid) then is removed and replaced with the neutralization buffer and the matrix agitated for about 30 minutes. The neutralization buffer then may be removed and the matrix washed and lyophilized (see infra).
  • the matrix also may be treated to remove contaminating heavy metals, such as by exposing the matrix to a metal ion chelator.
  • a metal ion chelator For example, following thermal treatment with 0.1% acetic acid, the matrix may be neutralized in a neutralization buffer containing EDTA (sodium ethylenediaminetetraacetic acid), e.g., 200 mM sodium phosphate, 5mM EDTA, pH 7.0. 5 mM EDTA provides about a 100-fold molar excess of chelator to residual heavy metals present in the most contaminated matrix tested to date. Subsequent washing of the matrix following neutralization appears to remove the bulk of the EDTA.
  • EDTA sodium ethylenediaminetetraacetic acid
  • EDTA treatment of matrix particles reduces the residual heavy metal content of all metals tested (Sb, As, Be, Cd, Cr, Cu, Co, Pb, Hg, Ni, Se, Ag, Zn, Tl) to less than about 1 pp .
  • Bioassays with EDTA- treated matrices indicate that treatment with the metal ion chelator does not inhibit bone inducing activity.
  • the collagen matrix materials preferably take the form of a fine powder, insoluble in water, comprising nonadherent particles. It may be used simply by packing into the volume where new bone growth or sustained release is desired, held in place by surrounding tissue.
  • the powder may be encapsulated in, e.g., a gelatin or polylactic acid coating, which is adsorbed readily by the body.
  • the powder may be shaped to a volume of given dimensions and held in that shape by interadhering the particles using, for example, soluble, species-biocompatible collagen.
  • the material may also be produced in sheet, rod, bead, or other macroscopic shapes.
  • the naturally sourced and recombinant protein as set forth above, and other constructs, can be combined and dispersed in a suitable matrix preparation using any of the methods described below.
  • 50-100 ng of active protein is combined with the inactive carrier matrix (e.g., 25 mg for rat bioassays). Greater amounts may be used for large implants.
  • Matrix is added to osteogenic protein dissolved in guanidine-HCl. Samples are vortexed and incubated at a low temperature (e.g., 4°C). Samples are then further vortexed. Cold absolute ethanol (5 volumes) is added to the mixture which is then stirred and incubated, preferably for 30 minutes at -20°C. After centrifugation (microfuge, high speed) the supernatant is discarded. The reconstituted matrix is washed twice with cold concentrated ethanol in water (85% EtOH) and then lyophilized.
  • osteogenic protein in an acetonitrile trifluroacetic acid (ACN/TFA) solution is added to the carrier material.
  • Samples are vigorously vortexed many times and then lyophilized. This method is currently preferred, and has been tested with osteogenic protein at varying concentrations and different levels of purity.
  • the protein is mixed with the matrix material, vortexed many times, and then lyophilized.
  • the lyophilized material may be used "as is" for implants.
  • OPl preparations in physiological saline may also be vortexed with the matrix and lyophilized to produce osteogenically active material.
  • BIOASSAY Procedures also can be used to adsorb other active therapeutic drugs, hormones, and various bioactive species to the matrix for sustained release purposes.
  • the functioning of the various proteins and devices of this invention can be evaluated with an in vivo bioassay.
  • Studies in rats show the osteogenic effect in an appropriate matrix to be dependent on the dose of osteogenic protein dispersed in the matrix. No activity is observed if the matrix is implanted alone.
  • In vivo bioassays performed in the rat model also have shown that demineralized, guanidine-extracted xenogenic bone matrix materials of the type described in the literature are ineffective as a carrier, fail to induce bone, and produce an inflammatory and immunological response when implanted unless treated as disclosed above. In certain species (e.g., monkey) allogenic matrix materials also apparently are ineffective as carriers.
  • the bioassay for bone induction as described by Sampath and Reddi may be used to monitor endochondral bone differentiation activity.
  • This assay consists of implanting test samples in subcutaneous sites in recipient rats under ether anesthesia. Male Long-Evans rats, aged 28-32 days, were used. A vertical incision (1 cm) is made under sterile conditions in the skin over the thoracic region, and a pocket is prepared by blunt dissection. Approximately 25 mg of the test sample is implanted deep into the pocket and the incision is closed with a metallic skin clip. The day of implantation is 5 designated as day one of the experiment. Implants were removed on day 12. The heterotropic site allows for the study of bone induction without the possible ambiguities resulting from the use of orthotropic sites. As disclosed herein, both allogenic (rat bone o matrix) and xenogenic (bovine bone matrix) implants were assayed.
  • Successful implants exhibit a controlled progression through the stages of protein-induced 5 endochondral bone development, including: (1) transient infiltration by polymorphonuclear leukocytes on day one; (2) mesenchymal cell migration and proliferation on days two and three; (3) chondrocyte appearance on days five and six; (4) cartilage matrix formation on 0 day seven; (5) cartilage calcification on day eight; (6) vascular invasion, appearance of osteoblasts, and formation of new bone on days nine and ten; (7) appearance of osteoblastic and bone remodeling and dissolution of the implanted matrix on days twelve to 5 eighteen; and (8) hematopoietic bone marrow differentiation in the ossicle on day twenty-one.
  • the results show that the shape of the new bone conforms to the shape of the implanted matrix.
  • Implants are fixed in Bouins Solution, embedded in paraffin, and cut into 6-8 ⁇ m sections. Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of 5 endochondral bone. Twelve day implants are usually sufficient to determine whether the implants contain newly induced bone.
  • Alkaline phosphatase activity may be used as a 0 marker for osteogenesis.
  • the enzyme activity may be determined spectrophoto etrically after homogenization of the implant. The activity peaks at 9-10 days in vivo and thereafter slowly declines. Implants showing no bone development by histology have little or no 5 alkaline phosphatase activity under these assay conditions.
  • the assay is useful for quantitation and obtaining an estimate of bone formation quickly after the implants are removed from the rat. Alternatively, the amount of bone formation can be determined by o measuring the calcium content of the implant.
  • CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60
  • AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593 GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653

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Abstract

L'invention concerne 1) l'ADNc ainsi que la séquence d'acides aminés d'une chaîne polypeptidique murine, mOP-1, utilisés dans des protéines ostéogéniques dimères, 2) des procédés de production de protéines ostéogéniques par génie génétique, 3) des procédés de production de dispositifs ostéogéniques comprenant mOP-1 dispersée dans des matrices d'os xénogéniques, et 4) l'emploi des dispositifs ostéogéniques pour mimer le cours naturel de la formation d'os endochondral chez les mammifères.
PCT/US1991/007654 1990-10-18 1991-10-18 Proteine osteogenique WO1992007004A1 (fr)

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US5661007A (en) * 1991-06-25 1997-08-26 Genetics Institute, Inc. Bone morphogenetic protein-9 compositions
US5688678A (en) * 1990-05-16 1997-11-18 Genetics Institute, Inc. DNA encoding and methods for producing BMP-8 proteins
US6034061A (en) * 1991-06-25 2000-03-07 Genetics Institute, Inc. BMP-9 compositions
US6034062A (en) * 1997-03-13 2000-03-07 Genetics Institute, Inc. Bone morphogenetic protein (BMP)-9 compositions and their uses
US6984623B2 (en) 1993-12-07 2006-01-10 Genetics, Institute Institute, LLC. Tendon-inducing compositions
US7091007B2 (en) 1993-09-17 2006-08-15 Genetics Institute, Llc DNA molecules encoding BMP receptor proteins
US7189392B1 (en) 1999-10-15 2007-03-13 Genetics Institute, Llc Injectable carrier formulations of hyaluronic acid derivatives for delivery of osteogenic proteins
US7217691B2 (en) 1986-07-01 2007-05-15 Genetics Institute, Llc Methods of treatment of periodontal disease
US7226587B2 (en) 2001-06-01 2007-06-05 Wyeth Compositions and methods for systemic administration of sequences encoding bone morphogenetic proteins
US7323445B2 (en) 1999-02-01 2008-01-29 Genetics Institute, Llc Methods and compositions for healing and repair of articular cartilage
US7378392B1 (en) 1990-05-16 2008-05-27 Genetics Institute, Llc Bone and cartilage inductive proteins
US7413753B2 (en) 2001-06-08 2008-08-19 Wyeth Calcium phosphate delivery vehicles for osteoinductive proteins
US8952130B2 (en) 2009-02-24 2015-02-10 The Salk Institute For Biological Studies Designer ligands of TGF-β superfamily

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WO1989009787A2 (fr) * 1988-04-08 1989-10-19 Creative Biomolecules, Inc. Dispositifs osteogeniques
WO1990003733A1 (fr) * 1988-10-11 1990-04-19 International Genetic Engineering, Inc. Facteurs osteogeniques
WO1990011366A1 (fr) * 1989-03-28 1990-10-04 Genetics Institute, Inc. Compositions osteoinductrices
EP0416578A2 (fr) * 1989-09-06 1991-03-13 Takeda Chemical Industries, Ltd. Protéine, ADN et leur utilisation

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WO1989009787A2 (fr) * 1988-04-08 1989-10-19 Creative Biomolecules, Inc. Dispositifs osteogeniques
WO1990003733A1 (fr) * 1988-10-11 1990-04-19 International Genetic Engineering, Inc. Facteurs osteogeniques
WO1990011366A1 (fr) * 1989-03-28 1990-10-04 Genetics Institute, Inc. Compositions osteoinductrices
EP0416578A2 (fr) * 1989-09-06 1991-03-13 Takeda Chemical Industries, Ltd. Protéine, ADN et leur utilisation

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Proc. Natl. Acad. Sci. USA, Vol. 87, December 1990 A.J. Celeste et al: "Identification of transforming growth factor Beta family members present in bone-inductive protein purified from bovine bone ", *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7217691B2 (en) 1986-07-01 2007-05-15 Genetics Institute, Llc Methods of treatment of periodontal disease
US7300772B2 (en) 1986-07-01 2007-11-27 Genetics Institute, Llc BMP products
US5688678A (en) * 1990-05-16 1997-11-18 Genetics Institute, Inc. DNA encoding and methods for producing BMP-8 proteins
US7378392B1 (en) 1990-05-16 2008-05-27 Genetics Institute, Llc Bone and cartilage inductive proteins
US6287816B1 (en) 1991-06-25 2001-09-11 Genetics Institute, Inc. BMP-9 compositions
US5661007A (en) * 1991-06-25 1997-08-26 Genetics Institute, Inc. Bone morphogenetic protein-9 compositions
US6034061A (en) * 1991-06-25 2000-03-07 Genetics Institute, Inc. BMP-9 compositions
US7091007B2 (en) 1993-09-17 2006-08-15 Genetics Institute, Llc DNA molecules encoding BMP receptor proteins
US6984623B2 (en) 1993-12-07 2006-01-10 Genetics, Institute Institute, LLC. Tendon-inducing compositions
US7365052B2 (en) 1993-12-07 2008-04-29 Genetics Institute, Llc. Tendon-inducing methods
US6034062A (en) * 1997-03-13 2000-03-07 Genetics Institute, Inc. Bone morphogenetic protein (BMP)-9 compositions and their uses
US7323445B2 (en) 1999-02-01 2008-01-29 Genetics Institute, Llc Methods and compositions for healing and repair of articular cartilage
US7189392B1 (en) 1999-10-15 2007-03-13 Genetics Institute, Llc Injectable carrier formulations of hyaluronic acid derivatives for delivery of osteogenic proteins
US7226587B2 (en) 2001-06-01 2007-06-05 Wyeth Compositions and methods for systemic administration of sequences encoding bone morphogenetic proteins
US7413753B2 (en) 2001-06-08 2008-08-19 Wyeth Calcium phosphate delivery vehicles for osteoinductive proteins
US8952130B2 (en) 2009-02-24 2015-02-10 The Salk Institute For Biological Studies Designer ligands of TGF-β superfamily

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