WO1992007004A1 - Osteogenic protein - Google Patents

Abstract

Disclosed are 1) the cDNA and amino acid sequence for a murine polypeptide chain, mOP-1, useful in dimeric osteogenic proteins, 2) methods of producing osteogenic proteins using recombinant technology, 3) methods of producing osteogenic devices comprising mOP-1 dispersed in xenogenic bone matrices, and 4) use of the osteogenic devices to mimic the natural course of endochondral bone formation in mammals.

Description

Osteogenic Protein

Background of the Invention

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).

Though the precise mechanisms underlying these phenotypic transformations are unclear, it has been shown that the natural endochondral bone dissociatively extracted and reconstituted with inactive residual collagenous matrix to restore full bone induction activity (Sampath and Reddi, (1981) Proc. Natl. Acad. Sci. USA 78:7599-7603). This provides an experimental method for assaying protein extracts for their ability to induce endochondral bone in vivo. Several species of mammals produce closely related protein as demonstrated by cross species implant experiments (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA 80:6591-6595).

The potential utility of these proteins has been recognized widely. It is contemplated that the availability of the protein would revolutionize orthopedic medicine, certain types of plastic surgery, and various periodontal and craniofacial reconstructive procedures.

The observed properties of these protein fractions have induced an intense research effort in various laboratories directed to isolating and identifying the pure factor or factors responsible for osteogenic activity. The current state of the art of purification of osteogenic protein from mammalian bone is disclosed by Sampath et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7109-7113. Urist et al. (1984) Proc. Soc. Exp. Biol. Med. 173: 194-199 disclose a human osteogenic protein fraction which was extracted from demineralized cortical bone by means of a calcium chloride-urea inorganic-organic solvent mixture, and retrieved by differential precipitation in guanidine-hydrochloride and preparative gel electrophoresis. The authors report that 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.

European Patent Application Serial No. 148,155, published October 7, 1985, purports to disclose osteogenic proteins derived from bovine, porcine, and human origin. One of the proteins, designated by the inventors as a P3 protein having a molecular weight of 22-24 kD, is said to have been purified to an essentially homogeneous state. This material is reported to induce bone formation when implanted into animals.

International Application No. PCT/087/01537, published January 14, 1988, discloses an impure fraction from bovine bone which has bone induction qualities. The named applicants also disclose putative "bone inductive factors" produced by. recombinant DNA techniques. Four DNA sequences were retrieved from human or bovine genomic or cDNA libraries and expressed in recombinant host cells. While the applicants stated that the expressed proteins may be bone morphogenic proteins, bone induction was not demonstrated, suggesting that the recombinant proteins are not osteogenic. The same group reported subsequently (Science 242:1528, Dec, 1988) that three of the four factors induce cartilage formation, and postulate that bone formation activity "is due to a mixture of regulatory molecules" and that "bone formation is most likely controlled ... by the interaction of these molecules." Again, no bone induction was attributed to the products of expression of the cDNAs. See also Urist et al., EP0,212,474 entitled Bone Morphogenic Agents.

Wang et al. (1988) Proc. Nat. Acad. Sci. USA 85: 9484-9488 discloses the purification of a bovine bone morphogenetic protein from guanidine extracts of demineralized bone having cartilage and bone formation activity as a basic protein corresponding to a molecular weight of 30 kD determined from gel elution. Purification of the protein yielded proteins of 30, 18 and 16 kD which, upon separation, were inactive. In view of this result, the authors acknowledged that the exact identity of the active material had not been determined.

Wang et al. (1990) Proc. Nat. Acad. Sci. USA JT7: 2220-2227 describes the expression and partial purification of one of the cDNA sequences described in PCT 87/01537. Consistent cartilage and/or bone formation with their protein requires a minimum of 600 ng of 50% pure material.

International Application No. PCT/89/04458 published April 19, 1990 (Int. Pub. No. WO90/003733) , describes the purification and analysis of a family of osteogenic factors called "P3 OF 31-34". The protein family contains at least four proteins, which are characterized by peptide fragment sequences. The impure mixture P3 OF 31-34 is assayed for osteogenic activity. The activity of the individual proteins is neither assessed nor discussed.

It is an object of this invention to provide novel polypeptide chains useful as subunits of dimeric osteogenic proteins capable of endochondral bone formation in allogenic and xenogenic implants in mammals, including humans. Another object is to provide genes encoding these polypeptide chains and to provide methods for the production of osteogenic proteins comprising these polypeptide chains using recombinant DNA techniques, as well as to provide antibodies capable of binding specifically to epitopes on these proteins.

These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.

Summary of the Invention

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.

A key to these developments was the elucidation of amino acid sequence and structure data of native bovine osteogenic protein. A protocol was developed which results in retrieval of active, substantially pure osteogenic protein from bovine bone having a half- maximum bone forming activity of about 0.8 to 1.0 ng per mg of implant. The availability of the material enabled the inventors to elucidate key structural details of the protein necessary to achieve bone formation. Knowledge of the protein's amino acid sequence and other structural features enabled the identification and cloning of native genes in the human genome.

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.

Fragments of the 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. Based on detailed structural and physical analyses of hOPl protein and the high degree of amino acid sequence homology between the hOPl and mOP-1 proteins, 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.

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. Currently preferred 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. Thus, in view of this disclosure, 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.

In view of this disclosure, and using standard immunology techniques well known in the art, those skilled in the art also may raise polyclonal or monclonal antibodies against all or part of the polypeptide chains described herein. Useful protocols for antibody production may be found, for example, in Molecular Cloninq-A Laboratory Manual (Sambrook et al. , eds.) Cold Spring Harbor Press 2nd ed. 1989). See Book 3, Section 18.

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. Currently preferred carriers include particulate, demineralized, guanidine extracted, species-specific (allogenic) bone, and specially treated particulate, protein extracted, demineralized, xenogenic bone. Optionally, such 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. Alternatively, 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

The foregoing and other objects of this invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which:

FIGURE 1 compares the amino acid sequences of the mature hOPl and mOPl polypeptide chains: OP1-18 and mOPl-S.

Description

Purification protocols first were developed which enabled isolation of the osteogenic protein present in crude protein extracts from mammalian bone. (See PCT US 89/01453, and U.S. Serial No. 179,406 filed April 8, 1988, now U.S. Patent No. 4,968,950). The development of the procedure, coupled with the availability of fresh calf bone, enabled isolation of substantially pure bovine osteogenic protein (bOP). bOP was characterized significantly; its ability to induce cartilage and ultimately endochondral bone growth in cat, rabbit, and rat were demonstrated and studied; it was shown to be able to induce the full developmental cascade of bone formation previously ascribed to unknown protein or proteins in heterogeneous bone extracts. This dose dependent and highly specific activity was present whether or not the protein was glycosylated (see (1990) J. Biol. Chem. 265: 13198- 13205). Sequence data obtained from the bovine materials suggested probe designs which were used to isolate human genes. The OP human counterpart proteins have now been expressed and extensively characterized. These discoveries enabled preparation of DNAs encoding totally novel, non-native protein constructs which individually as homodimers and combined with other species as heterodimers are capable of producing true endochondral bone (see PCT WO 89/09788, published 19-OCT-89 and US Serial No. 315,342, filed 23-FEB-89, now U.S. Patent No. 5,011,691.) They also permitted expression of the natural material, truncated forms, muteins, analogs, fusion proteins, and various other variants and constructs, from cDNAs and genomic DNAs retrieved from natural sources or from synthetic DNA produced using the techniques disclosed herein and using automated, commercially available equipment. The DNAs may be expressed using well established molecular biology and recombinant DNA techniques in procaryotic or eucaryotic host cells, and may be oxidized and refolded in vitro if necessary, to produce biologically active protein.

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-β family. Consensus splice signals were found where amino acid homologies ended, designating exon- intron boundaries. Three exons were combined to obtain a functional TGF-β 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". In its native form, 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"). The active region

(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.

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). The mature form of hOPl protein expressed in mammalian cells, designated herein OP1-18, is indicated by residues 293-431 of Seq. ID No. 3.

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.

Given the foregoing amino acid and DNA sequence information, various nucleic acids (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. Moreover, 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.

Exemplification

In an effort to identify additional DNA sequences encoding osteogenic proteins, 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-β super-family of regulatory proteins and which includes the conserved cysteine skeleton.

Approximately 7 x 10⁵ 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

Five recombinant phages were purified over three rounds of screening. 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.

Two different DNA sequences were identified by this procedure. One DNA, referred to herein as mOP2, is described in detail in copending USSN 599,543, filed 18-Oct-90. 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 (amino acid residues 26-30 in Seq. ID No. 1) 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. ID No. 1) is believed to constitute the cleavage site that produces the mature form of the protein, herein referred to as "mOPl-S" and described by amino acid residues 292-430 of Seq. ID No. 1. 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 (...). 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-β-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. Similarly, 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.

MATRIX PREPARATION

A. General Consideration of Matrix Properties 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.)

Studies have shown that surface charge, particle size, the presence of mineral, and the methodology for combining matrix and osteogenic protein all play a role in achieving successful bone induction. Perturbation of the charge by chemical modification abolishes the inductive response. Particle size influences the quantitative response of new bone; particles between 75 μm and 420 μm elicit the maximum response.

Contamination of the matrix with bone mineral will inhibit bone formation. Most importantly, the procedures used to formulate OP onto the matrix are extremely sensitive to the physical and chemical state of both the osteogenic protein and the matrix.

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. In addition, 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. In addition to its use as a matrix for bone formation in various orthopedic, periodontal, and reconstructive procedures, 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. Thus, 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. In bone formation or conduction procedures, 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. Thus, various known growth factors such as EGF, PDGF, IGF, FGF, TGF-α, and TGF-β may be released in vivo. The material can be used to release chemotherapeutic agents, insulin, enzymes, or enzyme inhibitors.

B. Bone-Derived Matrices

1. Preparation of Demineralized Bone

Demineralized bone matrix, preferably 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 μm, preferably 150-420 μm, 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.

2. Guanidine Extraction

Demineralized bone matrix thus prepared is extracted with 5 volumes of 4 M guanidine-HCl, 50mM

Tris-HCl, pH 7.0 for 16 hr. at 4°C. 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.

3. Matrix Treatments

The major component of all bone matrices is Type-I collagen. In addition to 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. It has been discovered that 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.

After contact with the fibril-modifying agent, the treated matrix is washed to remove any extracted 0 components, following a form of the procedure set forth below:

1. Suspend in TBS (Tris-buffered saline) lg/200 ml and stir at 4°C for 2 hrs; or in 6 M urea, 50 mM Tris-HCl, 500 mM NaCl, pH 7.0 (UTBS) or water and 5 stir at room temperature (RT) for 30 minutes (sufficient time to neutralize the pH);

2. Centrifuge and repeat wash step; and

3. Centrifuge; discard supernatant; water wash residue; and then lyophilize.

0 3.1 Acid Treatments 1. Trifluoroacetic acid.

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.

2. Hydrogen Fluoride.

Like trifluoroacetic acid, 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 P₂°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.

Other acid treatments are envisioned in addition to HF and TFA. TFA is a currently preferred acidifying reagent in these treatments because of its volatility. However, it is understood that other, potentially less caustic acids may be used, such as acetic or formic acid.

3.2 Solvent Treatment

1. Dichloromethane.

Dichloromethane (DCM) 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.

2. Acetonitrile.

Acetonitrile (ACN) 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, prepared as described above, 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. Alternatively, the ACN or ACN/TFA treated matrix may be lyophilized without wash.

3. Isopropanol.

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.

4. Chloroform

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 .

3.3 Heat Treatment

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.

Various amounts of delipidated, demineralized guanidine-extracted bone collagen are heated in the aqueous medium (lg matrix/30ml aqueous medium) under constant stirring in a water jacketed glass flask, and maintained at a given temperature for a predetermined period of time. 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.

After the heat treatment, the matrix is filtered, washed, lyophilized and used for implant. Where an acidic aqueous medium is used, 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. To neutralize the matrix, 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. 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 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. Alternatively, 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.

FABRICATION OF OSTEOGENIC DEVICE

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. In general, 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.

1. Ethanol Precipitation

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.

2. Acetonitrile Trifluoroacetic Acid Lyophilization

In this procedure, 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.

3. Urea Lyophilization

For those osteogenic proteins that are pre Ted in urea buffer, the protein is mixed with the matrix material, vortexed many times, and then lyophilized. The lyophilized material may be used "as is" for implants.

4. Buffered Saline Lyophilization

OPl preparations in physiological saline may also be vortexed with the matrix and lyophilized to produce osteogenically active material.

These procedures also can be used to adsorb other active therapeutic drugs, hormones, and various bioactive species to the matrix for sustained release purposes. BIOASSAY

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 following sets forth various procedures for preparing osteogenic devices from the proteins and matrix materials prepared as set forth above, and for evaluating their osteogenic utility.

^A- Rat Model

1. Implantation

The bioassay for bone induction as described by Sampath and Reddi ((1983) Proc. Natl. Acad. Sci. USA 80 6591-6595), herein incorporated by reference, 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.

2. Cellular Events

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.

3. Histological Evaluation

0 Histological sectioning and staining is preferred to determine the extent of osteogenesis in the implants. Implants are fixed in Bouins Solution, embedded in paraffin, and cut into 6-8 μm 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.

4. Biological Markers

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.

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all 5 respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be 0 embraced therein. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

OZKAYNAK, ENGIN OPPERMANN, HERMANN UBERASAMPATH, THANGAVEL RUEGER, DAVID C.

(ii) TITLE OF INVENTION: OSTEOGENIC DEVICES

(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: TESTA, HURWITZ & THIBEAULT

(B) STREET: 53 STATE STREET

(C) CITY: BOSTON

(D) STATE: MASSACHUSETTS (E) COUNTRY: U.S.A.

(F) ZIP: 02109

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patent In Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: PITCHER, EDMUND R.

(B) REGISTRATION NUMBER: 27,829

(C) REFERENCE/DOCKET NUMBER: CRP-001PC5 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617/248-7000

(B) TELEFAX: 617/248-7100

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1873 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: MURIDAE (F) TISSUE TYPE: EMBRYO

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 104..1393

(D) OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "mOPl-PP" /note= "mOPl (CDNA)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60

CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC 115

Met His Val Arg 1 TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GTG GCG CTC TGG GCG CCT 163 Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro 5 10 15 20

CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TTC AGC CTG GAC AAC GAG 211 Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu 25 30 35

GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG CGG 259 Val His Ser Ser Phe He His Arg Arg Leu Arg Ser Gin Glu Arg Arg 40 45 50

GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 307 Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg Pro 55 60 65

CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GCG CCC ATG TTC ATG TTG 355 Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Met Phe Met Leu 70 75 80 GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AGC GGG CCG GAC GGA CAG 403 Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gin 85 90 95 100

GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC CCC CCT 451 Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 105 110 115

TTA GCC AGC CTG CAG GAC AGC CAT TTC CTC ACT GAC GCC GAC ATG GTC 499 Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp Met Val 120 125 130

ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AAA GAA TTC TTC CAC CCT 547 Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro 135 140 145

CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CTT TCC AAG ATC CCC GAG 595 Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He Pro Glu 150 155 160

GGC GAA GCG GTG ACC GCA GCC GAA TTC AGG ATC TAT AAG GAC TAC ATC 643 Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp Tyr He 165 170 175 180

CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG TGG 691 Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin He Thr Val Tyr Gin Trp

185 190 195

CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 739 Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser 200 205 210 CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 787 Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp He Thr 215 220 225

GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CGG CAC AAC CTG GGC TTA 835 Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu 230 235 240

CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AGC ATC AAC CCC AAG TTG 883 Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro Lys Leu 245 250 255 260

GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG AAC AAG CAA CCC TTC ATG 931 Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro Phe Met

265 270 275

GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CTC CGT AGT ATC CGG TCC 979 Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser He Arg Ser 280 285 290

ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TCC AAG ACG CCA AAG AAC 1027 Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys Asn 295 300 305

CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GAA AAC AGC AGC AGT GAC 1075 Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp 310 315 320

CAG AGG CAG GCC TGC AAG AAA CAT GAG CTG TAC GTC AGC TTC CGA GAC 1123 Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp 325 330 335 340

CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GAA GGC TAT GCT GCC TAC 1171 Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala Tyr 345 350 355

TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AAC TCC TAC ATG AAC GCC 1219 Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala 360 365 370 ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC AAC CCA GAC 1267 Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn Pro Asp 375 380 385

ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CAG CTC AAC GCC ATC TCT 1315 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He Ser 390 395 400

GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC GAC CTG AAG AAG TAC AGA 1363 Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu Lys Lys Tyr Arg 405 410 415 420

AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCTTCC TGAGACCCTG 1413 Asn Met Val Val Arg Ala Cys Gly Cys His

425 430

ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACTG 1473

CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCGG 1533

AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593 GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653

GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAGT 1713

AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GGCGCTGGCG 1773

TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCAT 1833

GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC 1873

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 430 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(ix) FEATURE:

(D) OTHER INFORMATION: /product= "mOPl-PP"

( i) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15

Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30 Leu Asp Asn Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser 35 40 45

Gin Glu Arg Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu 50 55 60

Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 65 70 75 80

Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly 85 90 95

Pro Asp Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr 100 105 110 Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp 115 120 125

Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu 130 135 140

Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser 145 150 155 160

Lys He Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr 165 170 175

Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin He Thr 180 185 190

Val Tyr Gin Trp Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe 195 200 205

Leu Leu Asp Ser Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val 210 215 220

Phe Asp He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His 225 230 235 240

Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He 245 250 255 sn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys 260 265 270 Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 275 280 285 Ser He Arg Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys 290 295 300

Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn 305 310 315 320

Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val 325 330 335

Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly 340 345 350

Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 355 360 365

Tyr Met Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe 370 375 380

He Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu 385 390 395 400 Asn Ala He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu

405 410 415

Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1822 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

( i) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS (F) TISSUE TYPE: HIPPOCAMPUS

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 49..1341

(C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN"

/product= "hOPl-PP" /evidence= EXPERIMENTAL /standard_name= "hOPl" ( i) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57

Met His Val 1

CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105 Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala 5 10 15

CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153

Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn

20 25 30 35

GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 201 Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser Gin Glu Arg 40 45 50 CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG CCC CAC CGC 249 Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg 55 60 65

CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TCG GCA CCC ATG TTC ATG 297 Pro Arg Pro His Leu Gin ,ly Lys His Asn Ser Ala Pro Met Phe Met 70 75 80

CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC GGC 345 Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 85 90 95

GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC 393 Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly

100 105 110 115

CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TTC CTC ACC GAC GCC GAC 441 Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp 120 125 130

ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CAT GAC AAG GAA TTC TTC 489 Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe 135 140 145

CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TTT GAT CTT TCC AAG ATC 537 His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He 150 155 160

CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CGG ATC TAC AAG GAC 585 ^•o Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp 165 170 175

TAC ATC CGG GAA CGC 12C GAC AAT GAG ACG TTC CGG ATC AGC GTT TAT 633 Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He Ser Val Tyr 180 185 190 195

CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681 Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu 200 205 210

GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 729 Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp 215 220 225

ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG 777 He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu 230 235 240

GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 825 Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro 245 250 255 AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG CAG CCC 873 Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro 260 265 270 275

TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GTC CAC TTC CGC AGC ATC 921 Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser He 280 285 290

CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AAC CGC TCC AAG ACG CCC 969 Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro 295 300 305

AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GTG GCA GAG AAC AGC AGC 1017 Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 310 315 320

AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GAG CTG TAT GTC AGC TTC 1065 Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 325 330 335 CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GAA GGC TAC GCC 1113 Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala 340 345 350 ^■ 355

GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CCT CTG AAC TCC TAC ATG 1161 Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 360 365 370

AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CTG GTC CAC TTC ATC AAC 1209 Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn 375 380 385

CCG GAA ACG GTG CCC AAG CCC TGC TGT GCG CCC ACG CAG CTC AAT GCC 1257 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala 390 395 400

ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AAC GTC ATC CTG AAG AAA 1305 He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys 405 410 415

TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351

Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651

CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 431 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(D) OTHER INFORMATION: /Product="hOPl-PP"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15

Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30

Leu Asp Asn Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser 35 40 45

Gin Glu Arg Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu 50 55 60 Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 65 70 75 80

Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly 85 90 95

Gly Pro Gly Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 100 105 110

Thr Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr 115 120 125

Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135 140

Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 145 150 155 160

Ser Lys He Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He 165 170 175 Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He

180 185 190

Ser Val Tyr Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu 195 200 205

Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 210 215 220

Val Phe Asp He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 225 230 235 240

His Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser 245 250 255 He Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn

260 265 270

Lys Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 275 280 285

Arg Ser He Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser 290 295 300

Lys Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu 305 310 315 320

Asn Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr 325 330 335

Val Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu 340 345 350 Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 355 360 365

Ser Tyr Met Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His 370 375 380 phe He Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin 385 390 395 400

Leu Asn Ala He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He 405 410 415

Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430

Claims

What is claimed is:

1. A polypeptide chain comprising an amino acid sequence described by residues 334-430 of Seq. ID No. 1.

2. The polypeptide chain of claim 1 comprising an amino acid sequence described by residues 329- 430 of Seq. ID No. 1.

3. The polypeptide chain of claim 2 comprising an amino acid sequence described by residues 292- 430 of Seq. ID No. 1.

4. The polypeptide chain of claim 3 comprising an amino acid sequence described by residues 1- 430 of Seq. ID No. 1.

5. A polypeptide chain useful as a subunit of a dimeric osteogenic protein, said protein being capable of inducing endochondral bone formation when implanted in a mammal in association with a matrix;

said polypeptide chain comprising an amino acid sequence described by residues 334-430 of

Seq. ID No. 1, including allelic variants thereof.

6. The polypeptide chain of claim 5 wherein said polypeptide chain comprises the amino acid sequence described by residues 292-430 of Seq.

ID No. 1, including allelic variants thereof.:

7. The polypeptide chain of claim 1 or 5 produced by expression of recombinant DNA in a host cell.

8. The polypeptide chain of claim 7 wherein said host cell is a eucaryotic host cell.

9. The polypeptide chain of claim 8 wherein said eucaryotic host cell is a mammalian cell.

10. The polypeptide chain of claim 7 wherein said host cell is a procaryotic host cell.

11. The polypeptide chain of claim 10 wherein said procaryotic host cell is E.coli.

12. The polypeptide chain of claim 1 or 5 that is glycosylated.

13. A nucleic acid encoding the polypeptide chain of claim 1 or 5.

14. An osteogenic protein capable of inducing endochondral bone formation when implanted in a mammal in association with a matrix; said protein comprising a dimeric species having two oxidized subunits, the amino acid sequence of each said subunit comprising the amino acid sequence described by residues 334-430 of Seq. ID No.l, including allelic variants thereof.

15. The osteogenic protein of claim 14 wherein said amino acid sequence comprises the sequence described by residues 292-430 of Seq. ID No. 1, including allelic variants thereof.

16. An antibody capable of binding to an epitope on a protein comprising the amino acid sequence described by residues 334-430 of Seq. ID No. 1, including allelic variants thereof.