CA2138270C - Prosthetic devices having enhanced osteogenic properties - Google Patents

Abstract

A prosthetic device comprising a prosthesis coated with substantially pure osteogenic protein is disclosed. A method for biologically fixing prosthetic devices in vivo is also disclosed. In this method, a prosthesis is implanted in an individual in contact with a substantially pure osteogenic protein, enhancing the strength of the bond between the prosthesis and the existing bone at the joining site.

Description

:.21 38270 PROSTHETIC DEVICES HAVING ENHANCED
OSTEOGENIC PROPERTIES
Background of the Invention Regeneration of skeletal tissues is thought to be regulated by specific protein factors that are naturally present within bone matrix. When a bone is damaged, these factors stimulate cells to i:orm new cartilage and bone tissue which replaces or repairs lost or damaged bone.
Regeneration of bane is particularly important where prosthetic implants are used without bonding cement to replace diseased bone, as in hip replacement. In these cases, formation of a tight bond between the prosthesis and the existing bone is very important, and successful function depends on the interaction between the implant and the bone tissue at the interface.
c . 21 38270 .- 2 -Bone healing can be stimulated by one or more osteogenic proteins which can induce a developmental cascade of cellular events resulting ire endochondral bone formation.
Proteins stimulating bone growth have been referred to in the literature as bone morphogenic proteins, bone inductive proteins, osteogenic proteins, osteogenin or osteoinductive proteins.
U.S. 4,968,590 (November' 6, 1990) discloses the purification of "substantially pure" osteogenic protein from bone, capable of inducing endochondral bone formation in a mammal when implanted in the mammal in association with a matrix, and having a half maximum activity of at least about 25 to 50 nanograms per 25 milligrams of implanted matrix.
Higher activity subsequently has been shown for this protein, e.g., 0.8-1.0 ng of osteogenic protein per mg of implant matrix, as disclosed in U.S. Patent 5,011,691. This patent also disclosed a consensus DNA sequence probe useful for identifying genes encoding osteogenic proteins, and a number of human genes encoding osteogenic proteins identified using the consensus probe, including a previously unidentified gene referred to therein as "OP1" (osteogenic protein-1). The consensus probe also identified DNA

WO 93/25246 ~ ~ PCT/US93/05446 sequences corresponding to sequences termed BMP-2 Class I
and Class II ("BMP2" and "BMP4" respectively) and BMP3 in International Appl. No. PCT/US87/01537. The osteogenic proteins encoded by these sequences are referred to herein as "CBMP2A," "CBMP2B", and "CBMP~3", respectively. U.S.
5,011,691 also defined a consensus "active region" required for osteogenic activity and described several novel biosynthetic constructs using this consensus sequence which were capable of inducing cartilage or bone formation in a mammal in association with a matrix.
These and other researchers have stated that successful implantation of the osteogenic factors for endochondral bone formation requires that the proteins be associated with a suitable carrier material or matrix which maintains the proteins at the site of application. Bone collagen particles which remain after demineralization, guanidine extraction and delipidation of pulverized bone have been used for this purpose. Many osteoinductive proteins are useful cross-species. However, demineralized, delipidated, guanidine-extracted xenogenic collagen matrices typically have inhibited bone induction in vivo. Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA, 80: 6591-6594. Recently, however, Sampath et al. have described a method for treating demineralized guanidine-extracted bone powder to create a matrix useful for xenogenic implants. See, U.S. 4,975,526 (December 4, 1990). Other useful matrix materials include for example, collagen; homopolymers or copolymers of glycolic acid, lactic acid, and butryic acid, including derivatives thereof; and ceramics, such as hydroxyapatite, tricalcium phosphate and other calcium phosphates.
Combinations of these matrix materials also may be usef~~l.
Orthopedic implants have traditionally been attached to natural bone using bone cement. More recently, cementless prostheses have been used, in which the portion of the prosthesis that contacts the natural bone is coated with a porous material. M. Spector, J. Arthroplasty, 2(2):163-176 (1987); and Cook et al., Clin. Orthoped. and Rel. Res., 232:
225-243 (1988). Cementless fixation is preferred because biological fixation of the prosthesis is stronger when osseointegration is achieved. The porous coatings reportedly stimulate bone ingrowth resulting in enhanced biological fixation of the prosthesis. However, there are several problems with porous-coated prostheses. For example, careful prosthetic selection is required to obtain a close fit with the bone to ensure initial mechanical stabilization of the device, and surgical precision is required to ensure initial implant-bone contact to promote bone ingrowth. Porous coated implants have not resulted in bone ingrowth in some instances, for example, in porous coated tibial plateaus used in knee replacements. A
prosthetic implant that results in significant bone ingrowth and forms a strong bond with the natural bone at the site of the join would be very valuable.
The current state of the art for the anchoring of embedded implants such as dental implants also is unsatisfactory. Typically, dental implant fixation first requires preparing a tooth socket in the jawbone of an individual for prosthesis implantation by allowing bone ingrowth into the socket void to fill in the socket. This preparatory step alone can take several months to complete.
The prosthesis then is threaded into the new bone in the socket and new bone is allowed to regrow around the threaded portion of the implant embedded in the socket. The interval between tooth extraction and prosthetic restoration therefore can take up to eight months. In addition, threading the prosthesis into bone can damage the integrity of the bone. Prosthetic dental implants that can improve osseointegration and reduce the time and effort for fixation would be advantageous.

Summary of the Invention The present invention relates to a method of enhancing the growth of bone at the site of implantation of a prosthesis to form a bond between the prosthesis and the existing bone. As used herein, a prosthesis is understood to describe the addition of an artificial part to supply a defect in the body. The method involves coating or otherwise contacting all or a portion of the prosthesis that will be in contact with bone with a substantially pure osteogenic protein. The prosthesis first may be coated with the osteogenic protein and then implanted in the individual at a site wherein the bone tissue and the surface of the prosthesis are maintained in close proximity for a time sufficient to permit enhanced bone tissue growth between the tissue and the implanted prosthea is. Alternatively, the site of implantation first may be treated with substantially pure osteogenic protein and the prosthesis then implanted at the treated site such that all or a portion of the prosthesis is in contact with the osteogenic protein at the site, and the prosthesis, the os;teogenic protein and the existing bone tissue are maintained in close proximity to one another for a time sufficient to permit enhanced bone tissue growth between the tissue: and the prosthesis. The osteogenic protein associated with the implanted prosthesis stimulates bone growth around the prosthesis and causes a stronger bond to form between the prosthesis and the existing bone than would form beaween the prosthesis and the bone in the absence of the protE:in.
In a preferred embodiment oi= the present method a prosthetic device, such as an artificial hip replacement device, e.g., a metallic device made from titanium, for example, is first coated with an osteogenic material which induces bone ingrowth. When the device is subsequently implanted into the individual, bone growth around the site of the implant is enhanced, cau:aing a strong bond to form between the implant and the existing bone. The present method results in enhanced biological fixation of the prosthesis in the body, which is particularly important for weight bearing prostheses. Prostheses defining a microporous surface structure are locked in place as bone formation occurs within the micropores. The metal or ceramic prosthesis may itself define such a structure, or the prosthesis may be coated to provide an adherent porous surface. Materials useful for this purpose include, for example, collagen, homopolymers of glycolic acid, lactic acid, and butyric acid, including derivatives thereof; and ceramics such as hydroxyapatite, tricalcium phosphate or other calcium phosphates. Combinations of these materials may be used. A substantially pure osteogenic protein is then bound to the uncoated or coated prosthesis.
Alternatively, the osteogenic protein can be mixed with the coating material, and the mixture adhered onto the surface of the prosthesis.
In another embodiment of the present invention, osteogenic protein combined with a matrix material is packed into an orifice prepared to receive the prosthetic implant.
The surface of the implant also may be coated with osteogenic protein, as described above. The implant has a shape defining one or more indentations to permit bone ingrowth. The indentations are preferably transverse to the longitudinal axis of the implant. In general, the longitudinal axis of the implant will be parallel to the longitudinal axis of the bone which has been treated to receive the implant. New bone grows into the indentations thereby filling them, integrates with the surface of the implant as described above, and integrates with existing bone. Thus, the prosthesis can be more tightly fixed into the orifice, and "latched" or held in place by bone growing into the indentations, and by osseointegration of new bone with the surface of the implant, both of which are stimulated by the osteogenic protein.

WO 93/25246 ~ ~ PCT/US93/05446 In a specific embodiment, a dental implant is used to replace missing teeth. The implant typically comprises a threaded portion which is fixed into the jawbone and a tooth portion configured to integrate with the rest of the patient's teeth. The implant i;s coated with osteogenic protein (with or without a matrix or carrier) and threaded or screwed into a tooth socket in the jawbone prepared to receive it (e.g., bone has been allowed to grow into and fill the socket void.) In a particularly preferred embodiment, the socket is prepared to receive the implant by packing the void with a bone growth composition composed of osteogenic protein dispersed in a suitable carrier material.
The combination of osteogenic protein and carrier is referred to herein as an "osteogenic device." The osteogenic protein promotes osseointegration of the implant into the jawbone without first requiring bone growth to fill the socket, and without requiring that the prosthesis be threaded into existing bone, which may weaken the integrity of the the existing bone. Accordingly, the time interval between tooth extraction and prosthetic restoration is reduced significantly. It is anticipated that prosthetic restoration may be complete in as little time as one month.
In addition, the ability of the osteogenic protein to promote osseointegration of the prosthesis will provide a superior anchor.
A prosthetic device coated with the above osteogenic protein also is the subject of the present invention. All or a portion of the device may be coated with the protein.
Generally, only the portion of the device which will be in contact with the existing bone will be coated.
The present method and device results in enhanced biological fixation of the prosthesis. A strong bond is formed between the existing bone and the prosthesis, resulting in improved mechanical strength at the joining _ - Z? 3820 site. Higher attachment strength means that the prosthesis will be more secure and perttianent, and therefore will be more comfortable and durable for the patient.
In a preferred aspect of the present invention, the matrix material is selected from the group comprising collagen, hydroxyapatite, homopolymers or copolymers of glycolic acid, lactic acid or butyric acid and derivatives thereof, tricalcium phosphate or other calcium phosphates;
metal oxides, demineralized, guanidine extracted bone and mixtures thereof.
In another preferred aspect of the present invention, the surface of said prosthetic device is coated with a material selected from the croup comprising collagen, homopolymers or copolymers of glycolic acid, lactic acid or butyric acid and derivative~~ thereof, tricalcium phosphate or other calcium phosphates, metal oxides, demineralized, guanidine extracted bone and mixtures thereof.
In yet another preferrs~d aspect of the present invention, at least one of the polypeptide chains of the osteogenic protein is selected from the group comprising OP2, Vgr-1, CBMP2A, CBMP2B, BMP3, BMPS, BMP6, COPS, COP7, DPP, Vgl and variants thereof.
In yet another preferred aspect of the present invention, one or both of the polypeptide chains of the osteogenic protein is selected from the group comprising OP1, OP2, Vgr-1, BMPS, BMP6 and variants thereof.
In yet another preferrf~d aspect of the present invention, the dimeric species is a heterodimer.
In yet another preferrs~d aspect of the present invention, the osteogenic protein is provided in combination with one or more dimeric species of OP2, Vgr-1, CBMP2A, CBMP2B, BMP3, BMPS, BMP6, COPS, COP7, DPP, Vgl and variants thereof.

_ g _.
Brief Description of the Drawing The sole Figure of the drawing schematically depicts a cross-sectional view of a portion of a prosthesis implanted in a femur and illustrates the latching action of bone ingrowth in accordance with an embodiment of the invention.

~.aaa~ o --Detailed Description of the Invention The present invention relates to a method for enhancing osseointegration between a prosthesis and natural bone in an individual at the site of implantation of the prosthesis.
The method involves providing a prosthesis to a site of implantation together with substantially pure osteogenic protein such that the osteogenic protein is in contact with all or a portion of the implanted prosthesis. The protein promotes osseointegration of the prosthesis and the bone, resulting in a strong bond having improved tensile strength.
Osteogenic proteins which are useful in the present invention are substantially pure osteogenically active dimeric proteins. As used herein "substantially pure" means substantially free of other contaminating proteins having no endochondral bone formation activity. The protein can be either natural-sourced protein derived from mammalian bone or recombinantly produced proteins, including biosynthetic constructs. The natural-sourced proteins are characterized by having a half maximum activity of at least 25 to 50 ng per 25 mg of demineralized protein extracted bone powder, as compared to rat demineralized bone powder.
The natural-sourced osteogenic protein in its mature, native form is a glycosylated dimer having an apparent molecular weight of about 30 kDa as determined by SDS-PAGE.
When reduced, the 30 kDa protein gives rise to two glycosylated peptide subunits having apparent molecular weights of about 16 kDa and 18 kDa. In the reduced state, the protein has no detectable osteogenic activity. The unglycosylated protein, which also has osteogenic activity, has an apparent molecular weight of about 27 kDa. When reduced, the 27 kDa protein gives rise to two unglycosylated polypeptides having molecular weights of about 14 kDa to 16 kDa. The recombinantly-produced osteogenic protein describes a class of dimeric proteins capable of inducing endochondral bone formation in a mammal comprising a pair of ..
-~~1 - ~2t 38~~~
polypeptide chains, each of which has an amino acid sequence sufficiently duplicative of the sequence.of the biosynthetic constructs or COP-5 Or COP-7, (SEQ. ID NOS.3 and 4), such .. that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species is capable of inducing endochondral bone formation in a mammal. As defined herein, "sufficiently duplicative" is understood to describe the class of proteins having endochondral bone activity as dimeric proteins implanted in a mammal in association with a .matrix, each of the subunits having at least 60% amino acid sequence homology in the C-terminal cysteine-rich region with the_sequence of OPS (residues 335 to 431, SEQ. ID
No. 1). "Homology" is defined herein as amino acid sequence identity or conservative amino acid changes within the sequence, as defined by Dayoff, et al., Atlas of. Protein Sequence and. Structure; vol.5, Supp.3, pp.345-362, (M.O.~
Dayoff, ed. Nat'1 Biomed. Research Fdn., Washington, D.C., 1979.). Useful sequences include those comprising the C-terminal sequences of DPP (from~Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse), the OP1 and OP2 proteins, the CBMP2, CBMP3, and CBMP4 proteins (see U.S. .Pat. No.
5,011,691 and U.S. Patent No. 5,266,683, as well as the proteins referred to as BMP5 and BMP6 (see W090/11366, PCT/US90/01630.) A number of these proteins also are described in W088/00205, U.S. Patent No. 5,013,649 and W091/18098. Table I provides a list of the preferred members of this family of osteogenic proteins.
TABLE I - OSTEOGENI~C PROTEIN SEQUENCES
hOPl - DNA sequence encoding human OP1 protein (Seq.
ID No. l~or 3). Also referred to i~n related applications as "OP1", "hOP-1" and"OP-1".
AMENDED SHEFT

WO 93/25246 PCT/L'S93/05446 OP1 - Refers generically 'to the family of osteogenically active proteins produced by expression of part ~or all of the hOPl gene.
Also referred to in related applications as "OPI" and OP-1".
hOPl-PP - Amino acid sequence of human OP1 protein (prepro form), Seq. ID No. 1, residues 1-431.
Also referred to in related applications as "OP1-PP" and "OPP".
OP1-l8Ser - Amino acid sequence of mature human OP1 protein, Seq. ID No. 1, residues 293-431.
N-terminal amino acid is serine. Originally identified as migrating at 18 kDa on SDS-PAGE
in COS cells. Depending on protein glycosylation pattern in different host cells, also migrates at 23kDa, l9kDa and l7kDa on SDS-PAGE. Also referred to in related applications as "OP1-18".
OPS - Human OP1 protein species defining the conserved 6 cysteine skeleton in the active region (97 amino acids, Seq. ID No. 1, residues 335-431). "S" stands for "short".
OP7 - Human OP1 protein species defining the conserved 7 cysteine skeleton in the active region (102 amino acids, Seq. ID No. 1, residues 330-431).
OP1-l6Ser - N-terminally truncated mature human OP1 protein species. (Seq. ID No. 1, residues 300-431). N-terminal amino acid is serine;
protein migrates at l6kDa or lSkDa on _. 21382'T~

SDS-PAGE, depending on glycosylation pattern.
Also referred to in related applications as "OP-16S".
OP1-l6Leu - N-terminally truncated mature human OP1 protein species, Seq. ID No. 1, residues 313-431. N-terminal amino acid is leucine;
protein migrates at 16 or l5kDa on SDS-PAGE, depending on glycosylation pattern. Also referred to in related applications as "OP-16L".
OP1-l6Met - N-terminally truncated mature human OP1 protein species, Seq. ID No. 1, residues 315-431. N-terminal amino acid is methionine;
protein migrates at 16 or lSkDa on SDS-PAGE, depending on glycosylation pattern. Also referred to in related applications as "OP-16M".
OP1-l6Ala - N-terminally truncated mature human OP1 protein species, Seq. ID No. 1, residues 316-431. N-terminal amino acid is alanine, protein migrates at 16 or 15 kDa on SDS-PAGE, depending on glycosylation pattern. Also referred to in related applications as "OP-16A".
OP1-16Va1 - N-terminally truncated mature human OP1 protein species, Seq. ID No. 1, residues 318-431. N-terminal amino acid is valine; protein migrates at 16 or 15 kDa on SDS-PAGE, depending on glycosylation pattern. Also referred to in related applications as "OP-16V".

mOPl - DNA encoding mouse OP1 protein, Seq. ID No. 8.
Also referred to in related applications as "mOP-1".
mOPl-PP - Prepro form of mouse protein, Seq. ID No. 8, residues 1-430. Also referred to in related applications as "mOP-1-PP".
mOPl-Ser - Mature mouse OP1 protein species (Seq. ID No.
8, residues 292-430). N-terminal amino acid is serine. Also referred to in related applications as "mOPl" and "mOP-1".
mOP2 - DNA encoding mouse OP2 protein, Seq. ID No.
12. Also referred to in related applications as "mOP-2".
mOP2-PP - Prepro form of mOP2 protein, Seq. ID No. 12, residues 1-399. Also referred to in related applications as "mOP-2-PP".
mOP2-Ala - Mature mouse OP2 protein, Seq. ID No. 12, residues 261-399. N-terminal amino acid in alanine. Also referred to in related applications as "mOP2" and "mOP-2".
hOP2 - DNA encoding human OP2 protein, Seq. ID No.
10. Also referred to in related applications as "hOP-2".
hOP2-PP - Prepro form of human OP2 protein, Seq. ID No.
10, res. 1-402). Also referred to in related applications as "hOP-2-PP".

WO 93/25246 ~ ~i PCT/US93/05446 hOP2-Ala - Possible mature human OP2 protein species:
Seq. ID No. 10, residues 264-402. Also referred to in related applications as "hOP-2".
hOP2-Pro - Possible mature human OP2 protein species:
Seq. ID No. 10, residues 267-402. N-terminal amino acid is proli;ne. Also referred to in related applications as "hOP-2P".
hOP2-Arg - Possible mature human OP2 protein species:
Seq. ID No. 10, res. 270-402. N-terminal amino acid is arginine. Also referred to in related applications as "hOP-2R".
hOP2-Ser - Possible mature human OP2 protein species:
Seq. ID No. 10, res. 243-402. N-terminal amino acid is serin~e. Also referred to in related applications as "hOP-2S".
Vgr-1-fx C-terminal 102 amino acid residues of the murine "Vgr-1" protein (Seq. ID No. 7).
CBMP2A C-terminal 101 amino acid residues of the human BMP2A protein. (Residues 296-396 of Seq. ID No. 14).
CBMP2B C-terminal 101 amino acid residues of the human BMP2B protein. (Seq. ID No. 18).
BMP3 Mature human BMP3 (;partial sequence, Seq. ID
No. 16. See U.S. 5,011,691 for C-terminal 102 residues, "CBMP3.") BMPS-fx C-terminal 102 amino acid residues of the human BMP5 protein. (Seq ID No. 20).

BMP6-fx C-terminal 102 amino acid residues of the human BMP6 protein. (Seq ID No. 21).
COP5 Biosynthetic ostegenic 96 amino acid sequence (Seq. ID No. 3).
COP7 Biosynthetic osteogenic 96 amino acid sequence (Seq. ID No. 4).
DPP-fx C-terminal 102 amino acid residues of the Drosophila "DPP" protein (Seq. ID No. 5).
Vgl-fx C-terminal 102 amino acid residues of the Xenopus "Vgl" protein (Seq. ID No. 6).
The members of this family of proteins share a conserved six or seven cysteine skeleton in this region (e.g., the linear arrangement of these C-terminal cysteine residues is conserved in the different proteins.) See, for example, OPS, whose sequence defines the six cysteine skeleton, or OP7, a longer form of OP1, comprising 102 amino acids and whose sequence defines the seven cysteine skeleton.) In addition, the OP2 proteins contain an additional cysteine residue within this region.
This family of proteins includes longer forms of a given protein, as well as species and allelic variants and biosynthetic mutants, including addition and deletion mutants and variants, such as those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration still allows the protein to form a dimeric species having a conformation capable of inducing bone formation in a mammal when implanted in the mammal in association with a matrix. In addition, the osteogenic proteins useful in devices of this invention may include forms having varying glycosylation patterns and varying ~ ~. 3 8~ ~'~ (~

N-termini, may be naturally occurring or biosynthetically derived, and may be produced by expression of recombinant DNA in procaryotic or eucaryotic host cells. The proteins are active as a single species (e.g., as homodimers), or combined as a mixed species.
A particularly preferred embodiment of the proteins useful in the prosthetic devices of this invention includes proteins whose amino acid sequence in the cysteine-rich C-terminal domain has greater than 60% identity, and preferably greater than 65% identity with the amino acid sequence of OPS.
In another preferred aspect, the invention comprises osteogenic proteins comprising species of polypeptide chains having the generic amino acid sequence herein referred to as "OPX" which accommodates the homologies between the various identified species of the osteoge:nic OP1 and OP2 proteins, and which is described by the amino acid sequence of Sequence ID No. 22.
In still another preferred aspect, the invention comprises nucleic acids and the osteogenically active polypeptide chains encoded by these nucleic acids which hybridize to DNA or RNA sequences. encoding the active region of OP1 or OP2 under stringent hybridization conditions. As used herein, stringent hybridization conditions are defined as hybridization in 40% formamide:, 5 X SSPE, 5 X Denhardt's Solution, and 0.1% SDS at 37°C overnight, and washing in 0.1 X SSPE, 0.1%.SDS at 50°C.
The invention further comprises nucleic acids and the osteogenically active polypeptide: chains encoded by these nucleic acids which hybridize to the "pro" region of the OP1 or OP2 proteins under stringent hybridization conditions.
As used herein, "osteogenically active polypeptide chains"
is understood to mean those polypeptide chains which, when dimerized, produce a protein species having a conformation such that the pair of polypeptide: chains is capable of WO 93/25246 PCf/US93/05440 21 ;38270 inducing endochondral bone formation in a mammal when implanted in a mammal in association with a matrix or carrier.
Given the foregoing amino acid and DNA sequence information, the level of skill in the art, and the disclosures of U.S. Patent 5,011,691 and published PCT
specification US 89/01469, published October 19, 1989, as International publication No. W089/09788 various DNAs can be constructed which encode at least the active domain of an osteogen:ic protein useful in the devices of this invention, and various analogs thereof (including species and allelic variants and those containing genetically engineered mutations), as well as fusion proteins, truncated forms of the mature proteins, deletion and addition mutants, and similar constructs. Moreover, DNA
hybridization probes can be constructed from fragments of any of these proteins, or de~~igned de novo from the generic sequence. These probes then can be used to screen different genomic and cDNA libraries to identify additional osteogenic proteins useful in the prosthetic devices of this invention.
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 oligonucleot.ides, and cassette mutagenesis techniques. 15-100mer oligon.ucleotides may be synthesized on a DNA synthesizer, and purified by polyacrylamide gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer. The DNA
then may 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.
The DNA from appropriately identified clones then can be isolated, subcloned (preferably into an expression vector), and sequenced. Plasmids containing sequences of interest then can be 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 morphogenic activity. Useful host cells include E. coli, Saccharomyces, the insect/baculovirus cell system, myeloma cells, CHO cells and various other mammalian cells.
The vectors additionally may encode various sequences to promote correct expression o:f 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 pots=ntially inhibiting sequences or to minimize unwanted secondary structure formation. The recombinant osteogenic protean 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 folding and oxidizing one or more of the various recombinant polypeptide chains within an appropriate eucaryotic cell or in vitro after expression of individual subunits. A detailed description of osteogenic proteins expressed from recombinant DNA in E.
co ' is disclosed in International Publication W091/05802 on May 2, 1991. A detailed de:acription of osteogenic proteins expressed from recombinant I)NA in numerous different mammalian cells is disclosed in W091/05802.
Alternatively, osteogenic polypeptide chains can be synthesized chemically using conventional peptide synthesis techniques well known to those having ordinary skill in the e. 21 38270 art. For example, the proteins may be synthesized intact or in parts on a solid phase peptide synthesizer, using standard operating procedure.;. Completed chains then are deprotected and purified by FiPLC (high pressure liquid chromatography). If the protein is synthesized in parts, the parts may be peptide bonded using standard methodologies to form the intact protein. In general, the manner in which the osteogenic proteins are made can be conventional and does not form a part of this invention.
The osteogenic proteins useful in the present invention are proteins which, when implanted in a mammalian body, induce the developmental cascade of endochondral bone formation including recruitment and proliferation of mesenchymal cells, differentiation Of progenitor cells, cartilage formation, calcification of cartilage, vascular invasion, bone formation, remodeling and bone marrow differentiation. The osteopenic protein in contact with the present prostheses can induce the full developmental cascade of endochondral bone formation at the site of implantation essentially as it occurs in natural bone healing.
Prostheses which can be used with the present method include porous or non-porous orthopedic prostheses of the types well known in the art. Such prostheses are generally fabricated from rigid materials such as metals, including for example, stainless steel, titanium, molybdenuun, cobalt, chromium and/or alloys or oxides of these metals. Such oxides typically comprise a thin, stable, adherent metal oxide surface coating. The prostheses are preferably formed from or coated with porous metals to permit infiltration of the bone, but non-porous materials also can be used. Porous metallic materials for use in prostheses are described, for example, by Spector in J. Arthroplasty, 2(21:163-176 (1987), and by Cook et al. in Clin. O:rthoped. and Rel. Res., 232:225-243 (1988), C

prostheses may be used for major :bone or joint replacement and for repairing non-union fractures, for example, where the existing bone has been destroyed by disease or injury.
In a preferred embodiment of the present device and method, the prosthesis is coated with a material which enhances bone ingrowth and fixation, in addition to the protein. Materials which are useful for this purpose are biocompatible, and preferably _in vivo biodegradable and non-immunogenic. Such materials include, for example, collagen, hydroxyapatite, homopolymers or copolymers of g.lycolic acid lactic acid, and butyric acid and derivatives thereof, tricalcium phosphate or other calcium phosphates, metal oxides, (e. g., titanium oxide), and demineralized, guanidine extracted bone.
The present coated prostheses are prepared by applying a solution of the protein, and optionally, hydroxylapatite or other material to all or a portia~n of the prosthesis. The protein can be applied by any convenient method, for example, by dipping, brushing, immersing, spraying or freeze-drying. Hydroxylapatite i.s preferably applied by a plasma spraying process. The protein is preferably applied by immersing the prostheses in a solution of the protein under conditions appropriate to induce binding or precipitation of the protein from solution onto the implant.
The amount of protein which is applied to the implant should be a concentration sufficient to induce endochondral bone formation when the prosthesis is implanted in the recipient.
Generally a concentration in the range of at least 5Ng protein per 3.4cmz surface area is sufficient for this purpose. If hydroxylapatite or other carrier material is used, it is applied to the prosthesis in an amount required to form a coating of from about :15N to about 60N thick. A
layer about 25N thick of hydroxy:lapatite has been used to improve implant fixation, as shown in the exemplification.

i-._ ~ ~ ~ _ : = ~ :. . 2~ 38270 - 22: -In one aspect, the prosthesis comprises a device corifigured for insertion into an orifice prepared to receive . the~prosthesis. In this embodiment, as illustrated in the Figure,~the interior of a bones 10 is hollowed out in preparation for insertion of t:he implant 12. The implant has a contoured surface design 14 defining plural indentations 16 to permit ingrowth of bone into the indentations. The indentations are preferably transverse to the longitudinal axis 18 of the implant. The contoured portion to be inserted in the orifice may be coated with osteogenic protein as described above. Osteogenic protein combined with a matrix materiel 20 is packed into the orifice with the prosthetic implant, thereby surrounding it.
_ ~ Stimulated by the osteogenic protein, new bone grows into the indentations 16 and becomes integrated with the surface of the implant. 12 and with preexisting bone 10 as described above. Thus, the prosthesis is both mechanically and biologically fixed in place, and axial movement of the implant relative to the bone requires shearing Qf bone tissue. Matrix. material 20 can be any of the materials described above for coating the prosthesis for enhancing bone growth and fixation, e.g., collagen, hydroxyapatite, homopolymers or copolymers of glycolic acid lactic acid, and butyric acid and derivatives thereof, tricalcium phosphate or other calcium phosphates, metal oxides and deminera~lized, guanidine extracted bone. Matrix materials for use with osteogenic proteins which can be used in the present embodiment are those described, for example, in U.S. Patent 5,011,691 and U.S. Patent No. 5,266,683.
The prothesis illustrated in the Figure is particularly useful for dental and other innplants where at last part of the prosthesis is to be embedded into bone tissue. Packing the orifice, e.g., tooth sockEa, with an "osteogenic ~4NiENDED SHEE

WO 93/25246 ~ ~, PCT/US93/05446 device," e.g., osteogenic protein in combination with a matrix material, provides a solid material in which to embed the prosthesis without requiring 'that the device be threaded into existing bone. Moreover, the osteogenic protein stimulates endochondral bone formation within the socket and into and around the implant, thereby obviating the previously required step of first allowing bone ingrowth into the socket in order to provide a suitable surface into which to implant the prosthesis. Accordingly, using the method and devices of the invention, strong fixation of an implanted prosthesis may be achieved in a fraction of the time previously required, significantly shortening the time interval between tooth extraction and prosthetic restoration. In addition, this treatment may expand the use of implant therapy and enhance success rates by eliminating a surgical procedure, reducing the amount of bone lost following tooth extraction, permitting the insertion of longer implants and minimizing prosthetic compromises necessitated by alveolar ridge resorption.
The invention will be further illustrated by the following Exemplification which is not intended to be limiting in any way.
EXEMPLIFICATION
Example 1 Metal Implant Fixation Cylindrical implants 18mm in length and 5.95 + 0.05mm in diameter were fabricated from spherical Co-Cr-Mo particles resulting in a pore size of 250-300Nm and a volume porosity of 38-40%. A highly crystalline, high density and low porosity hydroxylapatite (HA) coating was applied by plasma spray process to one-half of the length of each of the implants. The coating thickness was 25 Nm and did not alter the porous coating morphology.

WO 93/25246 ~ ,~ ~ ~ ~ ~ ~ PCT/US93/05446 In the initial study, three implants were treated with a partially purified bovine OF~ (bOP) preparation. The bOP was naturally sourced OP extracted from cortical bone and partially purified through t:he Sephacryl-300* HR step in the purification protocol as described in Sampath et al. (1990), J. Hiol. Chem., 265: 13198-13205. 200N1 aliquots of 4 M
guanidine-HC1, 50 mM Tris-HC:1, pH 7.0, containing approximately 80 Ng bOP were added to each implant in an eppendorf tube. After overnight incubation at 4°C the protein was precipitated and. the implant washed with 80~
ethanol. The implants were subsequently freeze dried. Two implants without bOP served as the controls.
The implants were evaluated in one skeletally mature adult mongrel dog (3-5 years old, 20-25Kg weight) using the femoral transcortical model. Standard surgical techniques were used such that the animal received the five implants in one femur. At three weeks the dog was sacrificed and the femur removed.
The harvested femur was sectioned transverse to the long axis such that each implant was isolated. Each implant was sectioned in half to yield one HA-coated and one uncoated push-out sample. Interface attachment strength was determined using a specifically designed test fixture. The implants were pushed to failure with a MTS test machine at a displacement rate of 1.27 mm./minute. After testing, all samples were prepared for standard undecalcified histologic and microradiographic analyses. The sections (4 sections from each implant) were qualitatively examined for the type and quality of tissue ingrovrth, and quantitatively evaluated for % bone ingrowth with a computerized image analysis system. The mechanical and quantitative histological data is shown in Table II.
* Trade Mark ! 21 3 270 - 2.5 -TABLE II

METAL IMPLANTS
- bOP

HA-Coated Uncoated Interface Shear Strength, MPa Control 9.70 3.40 (n=2) (n=2) Protein 10.75 4.08 n=3 (n=3) (bOP) ( percent: Bone Ingrowth Control 42.56 37.82 (ns4) (n=4) Protein 51.66 46.38 (bOP) (n=4) (n=4) Both the mechanical and histological data suggested that bOP enhanced osseointegration of the implants. Both the HA-coated and uncoated implants showed an increase of shear strength and bone ingrowth compared with untreated controls.
Moreover, the HA-coated implants appeared to show significant enhancement compared to the uncoated implant. The histological sections direct7.y showed a greater number of cells between the metal pores.
The positive results of t:he initial implant study prompted a more detailed study. Twenty-seven implants were treated with a recombinant human OPl protein. The OP1 protein was produced by transformed CHO cells. The protein was purified to contain as the major species the protein designated OP1-l8Ser (Seq. ID No. 1, residues 293-431), and about 30%
truncated forms of OP1 (e. g., OP1-l6Ser, OP1-l6Leu, OP1-l6Met, OP1-l6Ala and OP1-16'Val). The protein was greater than 90% pure. The impants were immersed for 30 minutes in C

200 N1 50% ethanol/O.Olo TFA containing 5 Ng recombinant protein and the solution frozen in an ethanol/dry ice bath while the formulation tube was rolled. The tubes were subsequently freeze dried. Nineteen implants were also prepared by treatment with ethan~ol/TFA without the OP1 protein by the same procedure.
In test implants, it was found that OP1 could be extracted from treated implants with 8M urea, 1% Tween 80, 50mM Tris, pH
8.0 and analyzed by HPLC. By this method, it was shown that all of the OP1 in the formulation tubes bound to the implant under the conditions employed. Furthermore, since the test implants were half coated with H.A, additional implants were obtained to independently evaluate the binding of OP1 to each of these surfaces. Initial binding studies showed that the OP1 binds more readily to the HA than to the uncoated metal.
The implants for the second study were evaluated in skeletally mature adult mongrel dogs using the femoral transcortical model. Standard aseptic surgical techniques were used such that each animal received five implants bilaterally. Implantation periods of three weeks were used.
The mechanical and quantitative histological data are shown in Table III. Three HA-coated and uncoated configurations were evaluated: controls (no treatment), precoat samples (formulated without OP1) and the OP1 samples.

WO 93/25246 2 ~ ~ $ ~~ ~ ~ PCT/US93/05446 TABLE III

INTERFACE SHEAR
ATTACHMENT STRENGTH, MPA PERCENT BONE INGROWTH
3 Weeks: 3 Weeks:
HA-coated Uncoated HA-coated Uncoated Control 7.59+2.99 6.47+1.23 44.98+12.57 41.66+11.91 (n=10) (n=10) (n=24) (n=24) Precoat 7.85+3.43 6.49+2.20 40.73+16.88 39.14+16.18 (n=9) (n=9) (n=24) (n=24) Protein 8.69+3.17 6.34+3.04 48.68+16.61 47.89+11.91 (hOP-1) (n=17) (n=17) (n=24) (n=24) Mechanical testing results demonstrated enhanced attachment strength for the HA-coated samples as compared to the uncoated samples. At three weeks the greatest fixation was observed with the HA-coated implant with protein.
Histologic analysis demonstrated greater bone ingrowth for all HA-coated versus uncoated samples although the differences were not significant. The percent bone ingrowth was greatest for the HA-coated and uncoated implants with the protein present. Linear regression analysis demonstrated that attachment strength was predicted by amount of bone growth into the porous structure, presence of HA coating, and presence of protein.
Example 2 Titanium frequently is used to fabricate metal prostheses.
The surface of these prostheses comprise a layer of titanium oxide. Therefore, titanium oxide itself was evaluated for its ability to serve as a carrier for OP-1 and in general for its biocompatibility with the bone formation process. The in vivo biological activity of implants containing a combination of titanium oxide and OP-1 (Sequence ID No. 1, residues 293-431) ~f~~'~ ~~ 28 was examined in rat subcutaneous and intramuscular assays.
Implants contained 0, 6.25, 12.5, 25 or 50 Ng of OP-1 formulated onto 30 mg of titanium oxide.
Implants were formulated by a modification of the ethanol/TFA freeze-drying method. Titanium oxide pellets were milled and sieved to a particle size of 250-420 microns. 30 mg of these particles were mixed with 50 N1 aliquots of 450 ethanol, 0.090 trifluoroacetic acid containing no OP-1 or various concentrations of OP-1. After 3 hours at 4 °C, the samples were frozen, freeze-dried and implanted into rats.
After 12 days in vivo the implants were removed and evaluated for bone formation by alkaline phosphatase specific activity, calcium content and histological evidence. The results showed that OP-1 induced the formation of bone at each concentration of OP-1 at both the subcutaneous and intramuscular implant sites. No bone formed without OP-1 added to the titanium oxide. The amount of bone as quantitated by calcium content of the implants was similar to that observed using bone collagen carriers. Therefore titanium is a useful carrier for osteogenic proteins and is biocompatible with the bone formation process.
Example 3 The efficacy of the method of this invention on standard dental prosthesis may be assessed using the following model and protocol. Maxillary and mandibular incisor and mandibular canine teeth are extracted from several (e. g., 3) male cynomolgus (Macca fascularis) monkeys (4-6 kilograms) under ketamine anesthesia and local infiltration of lidocaine.
Hemostasis is achieved with pressure.
The resultant toothless sockets are filled either with (a) collagen matrix (CM), (b) with collagen matrix containing osteogenic protein, such as the recombinantly produced OP1 protein used in Example 1, above (e. g., an ostegenic device) or c) are left untreated. Titanium, self-tapping, oral, __ ~1~82~~

endosseous implants (Nobelpharma, Chicago, I11.) are inserted into all of the sockets by minimally engaging the self-tapping tip. The mucoperiosteal flap is released from the underlying tissue and used to obtain primary wound closure using standard surgical procedures known in the ;medical art.
The animals are sacrificed after three weeks by lethal injection of pentobarbital and perfusion with paraformaldehyde-glutaraldehyde. The jaws then are dissected and the blocks containing the appropriate sockets are resected, further fixed in neutral buffered forznalin, decalcified in formic acid and sodium citrate, embedded in plastic and stained with basic Fuchsin and toluidine blue.
Sections then are analyzes by light microscopy. Preferably, computer assisted histomorphometric analysis is used to evaluate the new tissue, e.g., using Image 1.27 and Quick CaptureR (Data Translation, Inc. :Marlboro, MA 07152).
It is anticipated that sockets which contain the osteogenic device will induce the formation of new bone in close apposition to the threaded surface of the titanium implants within 3 weeks. By contrast, sockets treated only with collagen matrix or sockets receiving neither collagen matrix nor the osteogenic device should show no evidence of new bone formation in close apposition to the implant surface.

Equivalents One skilled in the art will be able to ascertain, using no more than routine experimentation, many equivalents to the subject matter described herein. Such equivalents are intended to be encompassed by the following claims.

WO 93/25246 ~ ~ ~ PCT/US93/05446 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Creative BioMolecules, Inc.
(B) STREET: 35 South Street (C) CITY: Hopkinton (D) STATE: Massachusetts (E) COUNTRY: United States (F) POSTAL CODE (ZIP): 01748 (G) TELEPHONE: 1-508-435-9001 (H) TELEFAX: 1-508-435-0454 (I) TELE%:
(A) NAME: Stryker Biotech (B) STREET: One Apple Hill (C) CITY: Natick (D) STATE: Massachusetts (E) COUNTRY: United States (F) POSTAL CODE (ZIP): 017(0 (G) TELEPHONE: 1-508-653-2280 (H) TELEFAX: 1-508-653-2770 (I) TELE%:
(ii) TITLE OF INVENTION: PROSTHETIC DEVICES HAVING ENHANCED
OSTEOGENIC PROPERTIES
(iii) NUMBER OF SEQUENCES: 22 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Creative BioMolecules, Inc.
(B) STREET: 35 South Street (C) CITY: Hopkinton (D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 01748 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy dish (B) COMPUTER: IBM PC compatj.ble (C) OPERATING SYSTEM: PC-DOf>/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version ~~1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATIQN:
(A) NAME: PITCHER ESQ, EDMUND R
(B) REGISTRATION NUMBER: 27,829 (C) REFERENCE/DOCKET NUMBER: STK-057 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617/248-7000 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1822 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) 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= "OP1"
/evidence= EXPERIMENTAL
/standard name= "OP1"

WO 93/25246 ~ PCT/US93/05446 (xi)SEQUENCE SEQID
DESCRIPTION: N0:1:

GGTGCGGGCC GGAGCCCGG GCGCGTAGAG CCGGCGCG ATGCACGTG

C AGCCCGGGTA

MetHisVal ArgSer LeuArgAla AlaAlaPro HisSerPh~eValAla LeuTrpAla ProLeu PheLeuLeu ArgSerAla LeuAlaAs;pPheSer LeuAspAsn 20 25 3~0 35 GluVal HisSerSer PheIleHis ArgArgLeu ArgSer GlnGluArg ArgGlu HetGlnArg GluIleLeu SerIleLeu GlyLeu ProHisArg ProArg ProHisLeu GlnGlyLys HisAsnSer AlaPro MetPheMet LeuAsp LeuTyrAsn AlaMetAla ValGluGl.uGlyGly GlyProGly GGCCAG GGCTTCTCC TACCCCTAC AAGGCCGT'CTTCAGT ACCCAGGGC 393 GlyGln GlyPheSer TyrProTyr LysAlaVal PheSer ThrGlnGly 100 105 11.0 115 Pro ProLeuAla SerLeuGln AspSerHis PheLeuThr AspAlaAsp Met ValMetSer PheValAsn LeuValGlu HisAspLys GluPhePhe His ProArgTyr HisHisArg GluPheArg PheAspLeu SerLysIle Pro GluGlyGlu AlaValThr AlaAlaGlu PheArgIle TyrLysAsp Tyr IleArgGlu ArgPheAsp AsnGluThr PheArgIle SerValTyr Gln ValLeuGln GluHisLeu GlyArgGlu SerAspLeu PheLeuLeu Asp SerArgThr LeuTrpAla SerGluGlu GlyTrpLeu ValPheAsp Ile ThrAlaThr SerAsnHis TrpValVal AsnProArg HisAsnLeu Gly LeuGlnLeu SerValGlu ThrLeuAsp GlyGlnSer IleAsnPro Lys LeuAlaGly LeuIleGly ArgHisGly ProGlnAsn LysGlnPro Phe MetValAla PhePheLys AlaThrGlu ValHisPhe ArgSerIle Arg SerThrGly SerLysGln ArgSerGln AsnArgSer LysThrPro Lys AsnGlnGlu AlaLeuArg MetAlaAsn ValAlaGlu AsnSerSer - 35 ~-AGG GCC AAG
AAG
CAC
GAG

SerAsp Gln Gln Cys Lys His Leu Tyr Val Ser Phe Arg Ala Lye Glu GGC CAG TGG GCG

ArgAsp Leu Trp Asp Ile Ile Pro Glu Gly Tyr Ala Gly Gln Trp Ala TGT GGG TGT CCT

AlaTyr Tyr Glu Glu Ala Phe Leu Asn Ser Tyr Met Cys Gly Cys Pro AAC GCC GTG CTG

AsnAla Thr His Ile Gln Thr Val His Phe Ile Asn Asn Ala Val Leu GTG AAG TGC CCC

ProGlu Thr Pro Pro Cys Ala Thr Gln Leu Asn Ala Val Lys Cys Pro CTC TTC GAC AAC

IleSer Val Tyr Asp Ser Ser Val Ile Leu Lys Lys Leu Phe Asp Asn ATG GTC GCC TGC

TyrArg Asn Val Arg Cys Gly His Met Val Ala Cys GAGAATTCAG TCTGGA'.~CCTCCATTGCTCG CCTTGGCCAG1411 ACCCTTTGGG
GCCAAGTTTT

ACCAACTGCC
TTTTGTGAGA

TGTGAGAGTA ATGGCT'.~TTGATCAGTTTTT CAGTGGCAGC1531 TTAGGAAACA
TGAGCAGCAT

CAAGATCCTA
CAAGCTGTGC

GCATAAAGAA TGGCTG(~GAAGTCTCAGCCA TGCACGGACT1651 AAATGGCCGG
GCCAGGTCAT

CGTTTCCAGA AGCCAG(iCCACCCAGCCGTG GGAGGAAGGG1711 GGTAATTATG
AGCGCCTACC

GGGGTGGGCA
CATTGGTGTC

TGTCACAATA
AAACGAATGA

(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 431 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly g5 90 95 Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn a~.3~2'l~

Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu 305 310 31.'i 320 Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Ly,s Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Tr;p Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cy,s Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Va.l Gln Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..96 (D) OTHER INFORMATION: /note= "COP-5"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro WO 93/25246 PCf/US93/05446 Leu Ala Asp His Phe Asn Ser Thr Asn His Ala Val Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..96 (D) OTHER INFORMATION: /note= "COP-7"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Val Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Yal Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: DROSOPHILA MEIrANOGASTER
(ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..101 (D) OTHER INFORMATION: /label= DPP-FX
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly Lys Cys Pro Phe Pro Leu Ala Asp His Phe Assn Ser Thr Asn His Ala Yal Val Gln Thr Leu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys Ala Cys Cys Val Pro Thr Gln Leu Asp Ser Val Ala Met Leu Tyr Leu Asn Asp Gln Ser Thr Val Val Leu Lys Asn Tyr Gln Glu Met Thr Val Val Gly Cys Gly Cys Arg (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein _ 40 _ (vi) ORIGINAL SOURCE:
(A) ORGANISM: XENOPUS
(ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..102 (D) OTHER INFORMATION: /label= VG1-FR
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gln Asn Trp Val Ile Ala Pro Gln Gly Tyr Met Ala Asn Tyr Cys Tyr Gly Glu Cys Pro Tyr Pro Leu Thr Glu Ile Leu Asn Gly Ser Asn His Ala Ile Leu Gln Thr Leu Val His Ser Ile Glu Pro Glu Asp Ile Pro Leu Pro Cys Cys Val Pro Thr Lys Met Ser Pro Ile Ser Met Leu Phe Tyr Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val Asp Glu Cys Gly Cys Arg (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: MURIDAE
(ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..102 (D) OTHER INFORMATION: /label= VGR-1-FX

WO 93/25246 ~ g ~ ~ ~ PCl'/US93/05446 (xi) SEQUENCE DESCRIPTION: SEQ II) N0:7:
Cys Lys Lys His Gly Leu Tyr Val Ser Phe G:ln Asp Val Gly Trp Gln Asp Trp Ile Ile Ala Pro Xaa Gly Tyr Ala A:la Asn Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Val Met Asn Pro Glu Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Val Asn Ala I:Le Ser Val Leu Tyr Phe 65 70 7.'i 80 Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1873 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: si- ?.e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(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= "MOP1"
/note= "MOP1 (CDN.A)"

_ 42 _ (xi)SEQUENCE SEQ ID
DESCRIPTION: N0:8:

CTGCAGCAAG CTGCCCTGCC CCCTCCGCTG

TGACCTCGGG CCACCTGGGG
TCGTGGACCG

CCGGTGCCCC
GGATCGCGCG

Met HisValArg SerLeu ArgAlaAla AlaProHis SerPhe ValAlaLeu TrpAlaPro LeuPhe LeuLeuArg SerAlaLeu AlaAsp PheSerLeu AspAsnGlu ValHis SerSerPhe IleHisArg ArgLeu ArgSerGln GluArgArg GluMet GlnArgGlu IleLeuSer IleLeu GlyLeuPro HisArgPro ArgPro HisLeuGln GlyLysHis AsnSer AlaProMet PheMetLeu AspLeu TyrAsnAla MetAlaVal GluGlu SerGlyPro AspGlyGln GlyPhe SerTyrPro TyrLysAla ValPhe SerThrGln GlyProPro LeuAla SerLeuGln AspSerHis PheLeu ThrAspAla AspMetVal HetSer PheValAsn LeuValGlu HisAsp LysGluPhe PheHisPro ArgTyr HisHisArg GluPheArg PheAsp LeuSerLys IleProGlu Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Tyr Ile WO 93/25246 ''~ ~ PCT/US93/05446 ArgGluArg PheAsp AsnGluThr PheGlnIle ThrValTyr GlnVal LeuGlnGlu HisSer GlyArgGlu SerAspLeu PheLeuLeu AspSer ArgThrIle TrpAla SerGluGlu GlyTrpLeu ValPheAsp IleThr GCCACCAGC AACCAC TGGGTGGTC AACCCTCGI,;CACAACCTG GGCTTA 835 AlaThrSer AsnHis TrpValVal AsnProAr;~HisAsnLeu GlyLeu GlnLeuSer ValGlu ThrLeuAsp GlyGlnSe:rIleAsnPro LysLeu 245 250 25:5. 260 AlaGlyLeu IleGly ArgHisGly ProGlnAssnLysGlnPro PheMet ValAlaPhe PheLys AlaThrGlu ValHisLeu ArgSerIle ArgSer ThrGlyGly LysGln ArgSerGln AsnArgSer LysThrPro LysAsn GlnGluAla LeuArg HetAlaSer ValAlaGlu AsnSerSer SerAsp GlnArgGln AlaCys LysLysHis GluLeuTyr ValSerPhe ArgAsp LeuGlyTrp GlnAsp TrpIleIle AlaProGlu GlyTyrAla AlaTyr TyrCysGlu GlyGlu CysAlaPhe ProLeuAsn SerTyrMet AsnAla ACCAACCAC GCCATC GTCCAGACA CTGGTTCA.CTTCATCAAC CCAGAC 1267 ThrAsnHis AlaIle ValGlnThr LeuValHis PheIleAsn ProAsp CCC ACC CAG GCC

Thr Val Lys Pro Cys Cys Ala Pro Leu Asn Ile Ser Pro Thr Gln Ala TAC GTC GAC AAG

Val Leu Phe Asp Asp Ser Ser Asn Leu Lys Tyr Arg Tyr Val Asp Lys GTG CAC TAGCTCTTCC TGAGACCCTG

Asn Met Val Arg Ala Cys Gly Cys Val His (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 430 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro 65 70 7.5 80 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al,a Val Glu Glu Ser Gly Pro Asp Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Va:l Glu His Asp Lys Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser 145 150 15.'5 160 Lys Ile Pro Glu Gly Glu Arg Val Thr Ala Al;a Glu Phe Arg Ile Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln Ile Thr Val Tyr Gln Val Leu Gln Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Va:1 Val Asn Pro Arg His 225 230 23.'i 240 Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser Ile Arg Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn 305 310 31'i 320 Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly _ 46 _ Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1723 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (F) TISSUE TYPE: HIPPOCAMPUS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 490..1696 (D) OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN"
/product= "hOP2-PP"
/note= "hOP2 (cDNA)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:

WO 93/25246 1 ~ ~'~ PCT/US93/05446 ATG
ACC
GCG
CTC
CCC
GGC
CCG
CTC
'.EGG
CTC
CTG
GGC
CTG

Met Thr Ala Leu Pro Gly Pro Leu '.~rp Leu Leu Gly Leu AlaLeu CysAla LeuGlyGly GlyGlyPro GlyLeuArg ProProPro GGCTGT CCCCAG CGACGTCTG GGCGCGCGC GAI;CGCCGG GACGTGCAG 624 GlyCys ProGln ArgArgLeu GlyAlaArg GluArgArg AspValGln 30 35 41) 45 CGCGAG ATCCTG GCGGTGCTC GGGCTGCCT GGI;,CGGCCC CGGCCCCGC 672 ArgGlu IleLeu AlaValLeu GlyLeuPro G1;~ArgPro ArgProArg AlaPro ProAla AlaSerArg LeuProAla Se:rAlaPro LeuPheHet LeuAsp LeuTyr HisAlaMet AlaGlyAsp As;pAspGlu AspGlyAla ProAla GluArg ArgLeuGly ArgAlaAsp LeuValMet SerPheVal AsnMet ValGlu ArgAspArg AlaLeuGly HisGlnGlu ProHisTrp LysGlu PheArg PheAspLeu ThrGlnIle ProAlaGly GluAlaVal ThrAla AlaGlu PheArgIle TyrLysVal ProSerIle HisLeuLeu AsnArg ThrLeu HisValSer MetPheGln ValValGln GluGlnSer AsnArg GluSer AspLeuPhe PheLeuAsp LeuGlnThr LeuArgAla _ 48 _ GlyAsp GluGlyTrp LeuValLeu AspValThr AlaAlaSer AspCys TrpLeu LeuLysArg HisLysAsp LeuGlyLeu ArgLeuTyr ValGlu ThrGlu AspGlyHis SerValAsp ProGlyLeu AlaGlyLeu LeuGly GlnArg AlaProArg SerGlnGln ProPheVal ValThrPhe PheArg AlaSer ProSerPro IleArgThr ProArgAla ValArgPro LeuArg ArgArg GlnProLys LysSerAsn GluLeuPro GlnAlaAsn ArgLeu ProGly IlePheAsp AspValHis GlySerHis GlyArgGln ValCys ArgArg HisGluLeu TyrValSer PheGlnAsp LeuGlyTrp LeuAsp TrpVal IleAlaPro GlnGlyTyr SerAlaTyr TyrCysGlu GlyGlu CysSer PheProLeu AspSerCys MetAsnAla ThrAsnHis AlaIle LeuGln SerLeuVal HisLeunet LysProAsn AlaValPro LysAla CysCys AlaProThr LysLeuSer AlaThrSer ValLeuTyr TyrAsp SerSer AsnAsnVal IleLeuArg LysAlaArg AsnMetVal ValLys WO 93/25246 ~ PCT/US93/05446 Ala Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 402 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro Gln Arg Arg Leu Gly Ala Arg Glu Arg Arg As;p Val Gln Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Ar,g Pro Arg Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leo Phe Met Leu Asp Leu 65 70 7:5 80 Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Leu Val Met Se:r Phe Val Asn Met Val Glu Arg Asp Arg Ala Leu Gly His Gln Glu Pro His Trp Lys Glu Phe Arg Phe Asp Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Val Pro Ser Ile His Leu Leu Asn Arg Thr 145 150 15.'i 160 Leu His Val Ser Met Phe Gln Val Val Gln Glu Gln Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr Leu Arg Ala Gly Asp Glu Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly Gln Arg Ala Pro Arg Ser Gln Gln Pro Phe Val Val Thr Phe Phe Arg Ala Ser Pro Ser Pro Ile Arg Thr Pro Arg Ala Val Arg Pro Leu Arg Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu Pro Gln Ala Asn Arg Leu Pro Gly Ile Phe'Asp Asp Val His Gly Ser His Gly Arg Gln Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala Ile Leu Gln Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys Ala Arg Asn Met Val Val Lys Ala Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1926 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE:
(A) ORGANISM: MURIDAE
(F) TISSUE TYPE: EMBRYO

2~38~~?0 (ix) FEATURE:
(A) NAME/KEY:
CDS

(B) LOCATION:
93..1289 (D) OTHERINFORMATION:/funC'tlon= "OSTEOGENIC PROTEIN"

/product=
"mOP2-PP"' /note= "mOP2cDNA"

(xi)SEQUENCE SEQID
DESCRIPTION: N0:12:

GGTGCGCCGT CCGACCAGCT
CTGGTCCTCC
CCGTCTGGCI;

ACCAGTGGAT ATGGC'.TATGCGT CCCGGGCCA 113 GCGCGCCGGC
TGAAAGTCCG
AG

MetAlaMetArg ProGlyPro CTCTGG CTA TTG GGC GCT CTG GCGCTIiGGAGGC GGCCACGGT 161 CTT TGC

LeuTrp Leu Leu Gly Ala Leu AlaLeuGlyGly GlyHisGly Leu Cys ACC CAG

ProArg Pro Pro His Cys Pro ArgArgLeuGly AlaArgGlu Thr Gln CGCCGC GAC ATG CAG GAA ATC GCGGT1;CTCGGG CTACCGGGA 257 CGT CTG

ArgArg Asp Met Gln Glu Ile AlaVa:LLeuGly LeuProGly Arg Leu GCA GCC

ArgPro Arg Pro Arg Gln Pro AlaAlaArgGln ProAlaSer Ala Ala AlaProLeuPhe MetLeuAsp LeuTyrHis AlaMetThr AspAsp Asp GACGGCGGGCCA CCACAGGCT CACTTAGGC CG'.fGCCGAC CTGGTC ATG 401 AspGlyGlyPro ProGlnAla HisLeuGly ArgAlaAsp .LeuVal Met AGCTTCGTCAAC ATGGTGGAA CGCGACCGT ACI:CTGGGC TACCAG GAG 449 SerPheValAsn MetValGlu ArgAspArg ThrLeuGly TyrGln Glu CCACACTGGAAG GAATTCCAC TTTGACCTA ACI:CAGATC CCTGCT GGG 497 ProHisTrpLys GluPheHis PheAspLeu ThrGlnIle ProAla Gly GAGGCTGTCACA GCTGCTGAG TTCCGGATC TAI:AAAGAA CCCAGC ACC 545 GluAlaValThr AlaAlaGlu PheArgIle TyrLysGlu ProSer Thr _ 52 _ HisProLeuAsn ThrThr LeuHisIle SerMetPhe GluValVal Gln GluHisSerAsn ArgGlu SerAspLeu PhePheLeu AspLeuGln Thr LeuArgSerGly AspGlu GlyTrpLeu ValLeuAsp IleThrAla Ala SerAspArgTrp LeuLeu AsnHisHis LysAspLeu GlyLeuArg Leu TyrValGluThr AlaAsp GlyHisSer HetAspPro GlyLeuAla Gly LeuLeuGlyArg GlnAla ProArgSer ArgGlnPro PheMetVal Thr PhePheArgAla SerGln SerProVal ArgAlaPro ArgAlaAla Arg ProLeuLysArg ArgGln ProLysLys ThrAsnGlu LeuProHis Pro AsnLysLeuPro GlyIle PheAspAsp GlyHisGly SerArgGly Arg GluValCysArg ArgHis GluLeuTyr ValSerPhe ArgAspLeu Gly TrpLeuAspTrp ValIle AlaProGln GlyTyrSer AlaTyrTyr Cys GluGlyGluCys AlaPhe ProLeuAsp SerCysMet AsnAlaThr Asn HisAlaIleLeu GlnSer LeuValHis LeuMetLys ProAspVal Val WO 93/25246 ~ PCT/US93/05446 CCC ACC AG'T

Pro Lys Ala Cys Cys Ala Lys Leu Ala Thr Ser Val Leu Pro Thr Ser AAT GTC CG'T

Tyr Tyr Asp Ser Ser Asn Ile Leu Lys His Arg Asn Met Asn Val Ar,g TGC CAC CCCAGCATCC
TGCTTCTACT

Val Val Lys Ala Cys Gly Cys His CAGACAGGGG CAATGGGAGG CCCTTCACTTCCCCTGGCC~ACTTCCTGCTA AAATTCTGGT1439 AATGGCAAAT TCTGGATGGT CTAAGAAGGCCCTGGAATT1:TAAACTAGAT GATCTGGGCT1679 CTCTGCACCA TTCATTGTGG CAGTTGGGACATTTTTAGG'.fATAACAGACA CATACACTTA1739 GATCAATGCA TCGCTGTACT CCTTGAAATCAGAGCTAGC'.~TGTTAGAAAA AGAATCAGAG1799 CCAGGTATAG CGGTGCATGT CATTAATCCCAGCGCTAAA(iAGACAGAGAC AGGAGAATCT1859 (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 amino acids (B) TYPE:. amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Het Ala Met Arg Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gln ~a~~Q _ Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Met Gln Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Gln Pro Ala Ala Ala Arg Gln Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Met Thr Asp Asp Asp Asp Gly Gly Pro Pro Gln Ala His Leu g5 90 95 Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Lys Glu Phe His Phe Asp Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His Ile Ser Met Phe Glu Val Val Gln Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser Met Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gln Ala Pro Arg Ser Arg Gln Pro Phe Met Val Thr Phe Phe Arg Ala Ser Gln Ser Pro Val Arg Ala Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gln Pro Lys Lys Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly His Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gln WO 93/25246 ~ ~ PCT/US93/05446 Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala Ile Lesu Gln Ser Leu Val His Leu Met Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His Arg Asn Met Val Val Lys Al.a Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1260 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 9..1196 (D) OTHER INFORMATION: /funcaion= "OSTEOGENIC PROTEIN"
/product= "BMP2A"
/note= "BMP2A (CDNA)"
(xi) SEQUENCE DESCRIPTION: SEQ II) N0:14:
GGTCGACC ATG GTG GCC GGG ACC CGC TGT CTT (:TA GCG TTG CTG CTT CCC 50 Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro CAG GTC CTC CTG GGC GGC GCG GCT GGC CTC G'.~T CCG GAG CTG GGC CGC 98 Gln Val Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg AAG

ArgLys PheAlaAla AlaSerSer GlyArgPro SerSer GlnProSer AspGlu ValLeuSer GluPheGlu LeuArgLeu LeuSer MetPheGly LeuLys GlnArgPro ThrProSer ArgAspAla ValVal ProProTyr MetLeu AspLeuTyr ArgArgHis SerGlyGln ProGly SerProAla ProAsp HisArgLeu GluArgAla AlaSerArg AlaAsn ThrValArg SerPhe HisHisGlu GluSerLeu GluGluLeu ProGlu ThrSerGly LysThr ThrArgArg PhePhePhe AsnLeuSer SerIle ProThrGlu GluPhe IleThrSer AlaGluLeu GlnValPhe ArgGlu GlnMetGln AspAla LeuGlyAsn AsnSerSer PheHisHis ArgIle AsnIleTyr GluIle IleLysPro AlaThrAla AsnSerLys PhePro ValThrSer LeuLeu AspThrArg LeuValAsn GlnAsnAla SerArg TrpGluSer PheAsp ValThrPro AlaValMet ArgTrpThr AlaGln GlyHisAla AsnHis GlyPheVal ValGluVal AlaHisLeu GluGlu LysGlnGly GTCTCC AAGAGA CATGTTAGG ATAAGCAGG TCT'TTGCAC CAAGATGAA 770 ValSer LysArg HisValArg IleSerArg SerLeuHis GlnAspGlu CACAGC TGGTCA CAGATAAGG CCATTGCTA GTA.ACTTTT GGCCATGAT 818 HisSer TrpSer GlnIleArg ProLeuLeu Val.ThrPhe GlyHisAsp GGAAAA GGGCAT CCTCTCCAC AAAAGAGAA AAA,CGTCAA GCCAAACAC 866 GlyLys GlyHis ProLeuHis LysArgGlu Lys;ArgGln AlaLysHis AAACAG CGGAAA CGCCTTAAG TCCAGCTGT AAGiAGACAC CCTTTGTAC 914 LysGln ArgLys ArgLeuLys SerSerCys LysArgHis ProLeuTyr GTGGAC TTCAGT GACGTGGGG TGGAATGAC TG(~ATTGTG GCTCCCCCG 962 ValAsp PheSer AspValGly TrpAsnAsp TrpIleVal AlaProPro GGGTAT CACGCC TTTTACTGC CACGGAGAA TG(:CCTTTT CCTCTGGCT 1010 GlyTyr HisAla PheTyrCys HisGlyGlu CysProPhe ProLeuAla GATCAT CTGAAC TCCACTAAT CATGCCATT GT'.fCAGACG TTGGTCAAC 1058 AspHis LeuAsn SerThrAsn HisAlaIle Va:lGlnThr LeuValAsn 335 340 34li 350 TCTGTT AACTCT AAGATTCCT AAGGCATGC TG'.fGTCCCG ACAGAACTC 1106 SerVal AsnSer LysIlePro LysAlaCys Cy,sValPro ThrGluLeu AGTGCT ATCTCG ATGCTGTAC CTTGACGAG AA'fGAAAAG GTTGTATTA 1154 SerAla IleSer MetLeuTyr LeuAspGlu AsnGluLys ValValLeu AAGAAC TATCAG GATATGGTT GTGGAGGGT TG'TGGGTGT CGC 1196 LysAsn TyrGln AspMetVal ValGluGly CysGlyCys Arg TAGTACAGCA AAATTAAATA TATATTTTAG

CATAAATATA AAAAAAGAAA
TATATATAT.A

(2)INF ORMATION FORSEQID NO:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear _ _ (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys Gln Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp His Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Ser Leu Leu Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp Val Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys Gly His Pro Leu His Lys Arg Glu Lys Arg Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Gys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Va:l Ala Pro Pro Gly Tyr 305 310 31!i 320 His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln Th;r Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg 385 390 39li (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 574 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) HOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..327 (D) OTHER INFORMATION: /product= "MATURE hBMP3 (PARTIAL)"
/note= "THIS PARTIAL SEQUENCE OF THE MATURE HUMAN

THE CONSERVED 7 CY:iTEINE SKELETON. SEE U.S. PAT.
NO. 5,011,691 FOR :L02 C-TERMINAL SEQUENCE (CBMP3.)"
(ix) FEATURE:
(A) NAME/KEY: intron (B) LOCATION: 328..574 _ 0 _ (xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:

ATA AAG

ArgAlaSer LysIleGlu TyrGlnTyr LysLysAsp GluValTrp Glu GluArgLys ProTyrLys ThrLeuGln GlySerGly ProGluLys Ser LysAsnLys LysLysGln ArgLysGly ProHisArg LysSerGln Thr LeuGlnPhe AspGluGln ThrLeuLys LysAlaArg ArgLysGln Trp IleGluPro ArgAsnCys AlaArgArg TyrLeuLys ValAspPhe Ala AspIleGly TrpSerGlu TrpIleIle SerProLys SerPheAsp Ala TyrTyrCys SerGlyAla CysGlnPhe ProMetPro Lys (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Arg Ala Ser Lys Ile Glu Tyr Gln Tyr Lys Lys Asp Glu Val Trp Glu Glu Arg Lys Pro Tyr Lys Thr Leu Gln Gly Ser Gly Pro Glu Lys Ser Lys Asn Lys Lys Lys Gln Arg Lys Gly Pro His Arg Lys Ser Gln Thr Leu Gln Phe Asp Glu Gln Thr Leu Lys Lys Ala Arg Arg Lys Gln Trp Ile Glu Pro Arg Asn Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp Ser Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala g5 90 95 Tyr Tyr Cys Ser Gly Ala Cys Gln Phe Pro Met Pro Lys (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1788 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(F) TISSUE TYPE: HIPPOCAMPUS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 403..1626 (C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /funcaion= "OSTEOGENIC PROTEIN"
/product= "BMP2B"
/evidence= ERPERII"~ENTAL
/note= "BMP2B (CDNA)"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:

AGTATCTAGC TTGTCTCCCC GATGGGATTC CCGTCCAAC~C TATCTCGAGC CTGCAGCGCC 180 _ _ ACAGTCCCCG CAACCGTTCA GAGGTCCCCA

GCCCTCGCCC GGAGCTGCTG
AGGTTCACTG

CTGGCGAGCC GAGCCATTCC GTAGTGCCAT

CGCTACTGCA CCCGAGCAAC
GGGACCTATG

GCACTGCTGC GCAAGTTTGT TCAAGATTGG

AGCTTCCCTG CTGTCAAGAA
AGCCTTTCCA

TTATTATATG ATG
CCTTGTTTTC ATT
CCT
GGT

Met Ile Pro Gly AsnArgMet LeuMetVal ValLeu LeuCysGlnVal LeuLeuGly Gly AlaSerHis AlaSerLeu IlePro GluThrGlyLys LysLysVal Ala GluIleGln GlyHisAla GlyGly ArgArgSerGly GlnSerHis Glu LeuLeuArg AspPheGlu AlaThr LeuLeuGlnMet PheGlyLeu Arg ArgArgPro GlnProSer LysSer AlaValIlePro AspTyrMet Arg AspLeuTyr ArgLeuGln SerGly GluGluGluGlu GluGlnIle His SerThrGly LeuGluTyr ProGlu ArgProAlaSer ArgAlaAsn Thr GTGAGGAGC TTCCACCAC.GAAGAA CATCTGGAGAAC ATCCCAGGG ACC 798 ValArgSer PheHisHis GluGlu HisLeuGluAsn IleProGly Thr SerGluAsn SerAlaPhe ArgPhe LeuPheAsnLeu SerSerIle Pro GluAsnGlu ValIleSer SerAla GluLeuArgLeu PheArgGlu Gln GTGGAC CAGGGCCCT GATTGG GAA GGC TTC:CACCGT ATA ATT 942 AGG AAC

ValAsp GlnGlyPro AspTrp GluArgGly Phe~HisArg IleAsnIle 165 170 1?_'i 180 TATGAG GTTATGAAG CCCCCA GCAGAAGTG GTC~CCTGGG CACCTCATC 990 TyrGlu ValMetLys ProPro AlaGluVal Va7.ProGly HisLeuIle ACACGA CTACTGGAC ACGAGA CTGGTCCAC CA(;AATGTG ACACGGTGG 1038 ThrArg LeuLeuAsp ThrArg LeuValHis HisAsnVal ThrArgTrp GluThr PheAspVal SerPro AlaValLeu Ark;TrpThr ArgGluLys GlnPro AsnTyrGly LeuAla IleGluVal ThrHisLeu HisGlnThr CGGACC CACCAGGGC CAGCAT GTCAGGATT AG(;CGATCG TTACCTCAA 1182 ArgThr HisGlnGly GlnHis ValArgIle SerArgSer LeuProGln 245 250 25_'i 260 GGGAGT GGGAATTGG GCCCAG CTCCGGCCC CT(:CTGGTC ACCTTTGGC 1230 GlySer GlyAsnTrp AlaGln LeuArgPro LeuLeuVal ThrPheGly CATGAT GGCCGGGGC CATGCC TTGACCCGA CG(:CGGAGG GCCAAGCGT 1278 HisAsp GlyArgGly HisAla LeuThrArg Ark;ArgArg AlaLysArg SerPro LysHisHis SerGln ArgAlaArg LysLysAsn LysAsnCys CGGCGC CACTCGCTC TATGTG GACTTCAGC GA'.fGTGGGC TGGAATGAC 1374 ArgArg HisSerLeu TyrVal AspPheSer AspValGly TrpAsnAsp TrpIle ValAlaPro ProGly TyrGlnAla PheTyrCys HisGlyAsp 325 330 33.'i 340 CysPro PheProLeu AlaAsp HisLeuAsn Se:rThrAsn HisAlaIle GTGCAG ACCCTGGTC AATTCT GTCAATTCC AG'TATCCCC AAAGCCTGT 1518 ValGln ThrLeuVal AsnSer ValAsnSer SerIlePro LysAlaCys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg CACAGACTGC TTCCTTATAG CTGGACTTTT ATTTAAAAAA A~?,AAAAAAAA AAACCCGAAT 1786 (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 408 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Met Ile Pro Gly Asn Arg Met Leu Met Val Val Leu Leu Cys Gln Val Leu Leu Gly Gly Ala Ser His Ala Ser Leu Ile Pro Glu Thr Gly Lys Lys Lys Val Ala Glu Ile Gln Gly His Ala Gly Gly Arg Arg Ser Gly Gln Ser His Glu Leu Leu Arg Asp Phe Glu Ala Thr Leu Leu Gln Met Phe Gly Leu Arg Arg Arg Pro Gln Pro Ser Lys Ser Ala Val Ile Pro Asp Tyr Met Arg Asp Leu Tyr Arg Leu Gln Ser Gly Glu Glu Glu Glu Glu Gln Ile His Ser Thr Gly Leu Glu Tyr Pro Glu Arg Pro Ala Ser Arg Ala Asn Thr Val Arg Ser Phe His His Glu Glu His Leu Glu Asn WO 93/25246 ~ ~ ~ PCT/US93/05446 Ile Pro Gly Thr Ser Glu Asn Ser Ala Phe Ark; Phe Leu Phe Asn Leu Ser Ser Ile Pro Glu Asn Glu Val Ile Ser Ser Ala Glu Leu Arg Leu 145 150 155. 160 Phe Arg Glu Gln Val Asp Gln Gly Pro Asp Trp Glu Arg Gly Phe His Arg Ile Asn Ile Tyr Glu Val Met Lys Pro Pro Ala Glu Val Val Pro Gly His Leu Ile Thr Arg Leu Leu Asp Thr Arg; Leu Val His His Asn Val Thr Arg Trp Glu Thr Phe Asp Val Ser Pro Ala Val Leu Arg Trp Thr Arg Glu Lys Gln Pro Asn Tyr Gly Leu Alai Ile Glu Val Thr His 225 230 235. 240 Leu His Gln Thr Arg Thr His Gln Gly Gln His. Val Arg Ile Ser Arg Ser Leu Pro Gln Gly Ser Gly Asn Trp Ala Gln Leu Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Arg Gly His Alai Leu Thr Arg Arg Arg Arg Ala Lys Arg Ser Pro Lys His His Ser Glri Arg Ala Arg Lys Lys Asn Lys Asn Cys Arg Arg His Ser Leu Tyr Val. Asp Phe Ser Asp Val 305 310 315. 320 Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Lys; Asn Tyr Gln Glu Met '~,,'~, 'j' - 6 6 -Val Val Glu Gly Cys Gly Cys Arg (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..102 (D) OTHER INFORMATION: /note= "BHPS"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val. Ile Leu Lys Lys Tyr Arg Asn Met Val Yal Arg Ser Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..102 (D) OTHER INFORMATION: /note= "BHP6"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gln. Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala. Asn Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn. Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile~ Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Trp Met Val Val Arg Ala Cys Gly Cys His (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAHE/KEY: Protein (B) LOCATION: 1..102 (D) OTHER INFORMATION: /label.= OPX
/note= "WHEREIN XAA AT EACH POS'N IS INDEPENDENTLY
SELECTED FROM THE RESIDUES OCCURRING AT THE
CORRESPONDING POS'N IN THE C-TERMINAL SEQUENCE OF HOUSE
OR HUMAN OP1 OR OP2 (SEE. SEQ. ID NOS. 1,8,10 AND 12.)"

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys Raa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val Xaa Ala Cys Gly Cys His

Claims (50)

-69-

1. Use of a substantially pure osteogenic protein on a surface of a prosthetic device for promoting in vivo osseointegration of said device for implanting in a mammal at a site wherein bone tissue and said surface are maintained at least partially it contact for a time sufficient to permit enhanced bone tissue growth between said tissue and said device, wherein said osteogenic protein is a basic protein comprising a pair of polypeptide chains, one of said polypeptide chains comprising an amino acid sequence sharing greater than 60% identity with the amino acid sequence defined by residues 335 to 431 of Seq. ID
No. 1 (OPS) such that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species, has a conformation capable of inducing endochondral bone formation when disposed in a matrix and implanted in a mammal.

2. Use of a substantially pure osteogenic protein or a surface of a prosthetic device prior to its implantation for repairing the skeletal system of a mammal, thereby to promote enhanced bone tissue growth into said device and to improve the tensile strength of the junction between the bone and said device, wherein said protein is an osteogenically active protein, characterized in that the protein is a basic protein comprising a pair of polypeptide chains, one of said polypeptide chains comprising an amino acid sequence sharing greater than 60%
identity with the amino acid sequence defined by residues 335 to 431 of Seq. ID No. 1 (OPS) such that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species, has a conformation capable of inducing endochondral bone formation when disposed in a matrix and implanted in a mammal.

3 Use according to claims 1 or 2 wherein said surface of said prosthetic device further comprises hydroxylapatite, collagen, homopolymers or copolymers of glycolic acid, lactic acid or butyric acid and derivatives thereof, tricalcium phosphate or other calcium phosphate, metal oxides or combinations thereof.

4. Use according to claims 1 or 2 wherein the prosthetic device comprises a porous metallic material.

5. Use according to claims 1 or 2 wherein said dimeric osteogenic protein is produced by recombinant DNA in a host cell and is isolated therefrom.

6. Use according to claims 1 or 2 wherein the osteogenic protein is an osteogenically active dimeric protein expressed from recombinant DNA in a host cell, further characterized in that the protein comprises a pair of oxidized subunits disulfide bonded to produce a dimeric species, one of said subunits having an amino acid sequence encoded by a nucleic acid capable of hybridizing to a nucleic acid encoding OPS (residues 335 to 341 of Seq. ID No. 1) under stringent hybridization conditions, such that the disulfide bonded dimeric species comprising said subunit has a conformation capable of inducing endochondral bone formation in a mammal when disposed on the surface of said device.

7. Use according to claim 5 wherein said dimeric osteogenic protein is unglycosylated.

8. Use according to claim 1 or 2 wherein each said polypeptide chain of said protein comprises an amino acid sequence sharing greater than 65% identity with an amino acid sequence comprising OPS.

9. Use according to claim 8 wherein the amino acid sequence of one of said polypeptide chains comprises the amino acid sequence defined by residues 335-431 of Seq. ID No. 1 (OPS).

10. Use according to claim 8 wherein both said polypeptide chairs comprise the amino acid sequence defined by residues 335-431 of Seq. ID No.1 (OPS.)

11. Use according to claim 10 wherein both said polypeptide chains comprise the amino acid sequence of residues 318-431 of Seq. ID No. 1 (OP1-16Val).

12. An improved prosthetic device for repairing mammalian skeletal defects, injuries, or anomalies comprising a rigid prosthetic implant having a porous or non-porous surface region for implantation adjacent bone tissue, wherein the improvement comprises:
substantially pure osteogenically active osteogenic protein disposed on said surface region in an amount sufficient to promote enhanced bone tissue growth into said surface; wherein said protein is an osteogenically active protein, characterized in that the protein is a basic protein comprising a pair of polypeptide chains, one of said polypeptide chains comprising an amino acid sequence sharing greater than 60% identity with the amino acid sequence defined by residues 335 to 431 of Seq. ID
No. 1 (OPS) such that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species, has a conformation capable of inducing endochondral bone formation when disposed in a matrix and implanted in a mammal.

13. The device of claim 12 wherein said surface of said prosthetic device further comprises hydroxylapatite.

14. The device of claim 12 wherein said dimeric osteogenic protein is produced by recombinant DNA in a host cell and is isolated therefrom.

15. The device of claim 14 wherein said dimeric osteogenic protein is unglycosylated.

16. The device of claim 12 wherein one of said polypeptide chains comprises an amino acid sequence encoded by a nucleic acid capable of hybridizing to a nucleic acid encoding OPS (residues 335-431 of Seq. ID No. 1), such that the disulfide bonded dimeric species comprising said polypeptide chain has a conformation capable of inducing endochondral bone formation in a mammal when disposed on the surface of said device.

17. The device of claim 12 wherein each of said polypeptide.
chains comprises an amino acid sequence sharing greater than 65%
identity with the amino acid sequence defined by residues 335 to 431 of Seq. ID No. 1 (OPS).

18. The device of claim 17 wherein one of said polypeptide chains of said protein comprises residues 335-431 of Seq. ID
No. 1 (OPS).

19. The device of claim li wherein both said polypeptide chains comprise the amino acid sequence defined by residues 335-431 of Seq. ID No. 1 (OPS).

20. The device of claim 19 wherein both said polypeptide chains comprise the amino acid sequence defined by residues 318-431 of Seq. ID No.1 (OP1-16Val.)

21. The device of claim 12 wherein the prosthesis comprises a porous metallic material.

22. The device of claim 12 wherein the prosthesis comprises a countoured implantable portion for in section into an orifice having plural indentations transverse to its longitudinal axis.

23. The device of claim 22 comprising a dental implant.

24. Use of a prosthetic device having a contoured implantable portion for promoting in vivo osseointegration of the prosthetic device into an orifice of a bone, said contoured portion having plural indentations transverse to its longitudinal axis, wherein said contoured portion of the prosthetic device is coated with a bone growth composition comprising a substantially pur a osteogeric protein combined with a matrix material which induces bone growth in said indentations, osseointegration between the bone and the prosthetic device, and.osseointegration of new bone induced by said composition and said bone; wherein said protein is an osteogenically active protein, characterized in that the protein is a basic protein comprising a pair of polypeptide chains, one of said polypeptide chains comprising an amino acid sequence sharing greater than 60% identity with the amino acid sequence defined by residues 335 to 431 of Seq. ID No.
1 (OPS) such that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species, has a conformation capable of inducing endochondral bone formation when disposed in a matrix and implanted in a mammal.

25. Use according to claim 24 wherein the contoured portion comprises a porous metallic material.

26. Use according to claim 25 wherein the osteogenic protein enhances bone ingrowth into said pores.

27. A device for promoting in vivo osseointegration of a prosthesis into an orifice of a bone, comprising a rigid prosthetic implant having a contoured portion for insertion into said orifice, said contoured portion raving plural indentations transverse to its longitudinal axis, and a bone growth composition comprising a substantially pure osteogenic protein combined with a matrix material which induces bone growth in said indentations, osseointegration between the bone and the prosthetic implant and osseointegration of new bone induced by said composition and said bone; wherein said protein is an osteogenically active protein characterized in that the protein is a basic, dimeric protein and comprises a pair of polypeptide chains, one of said polypeptide chairs having an amino acid sequence sharing greater than 60% identity with the amino acid sequence defined by residues 335 to 431 of Seq. ID
No. 1 (OPS) such that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species, has a conformation capable of inducing endochondral bone formation in association with said contoured portion of said prosthesis when implanted in a mammal.

28. The device of claim 27 wherein the contoured portion comprises a porous metallic material.

29. The device of claim 28 wherein the osteogenic protein enhances bone ingrowth into said pores.

30. The device of claim 27 wherein said matrix material is selected from the group consisting of hydroxylapatite, collagen, polymers or copolymers of glycolic acid, lactic acid or butyric acid, tricalcium phosphate or other calcium phosphates, metal oxides, demineralized guanidine extracted bone and combinations thereof.

31. The device of claim 27 comprising a dental implant.

32. The device of claim 24 or 27 wherein said protein is produced by recombinant DNA in a host and is isolated therefrom.

33. The device of claim 24 wherein said protein is unglycosylated.

34. Use according to claims 1, 2 or 24 wherein the prosthetic device comprises stainless steel, titanium, molybdenum, cobalt, chromium or alloys or oxides of these metals.

35. Use according to claims 1 or 2 wherein the osteogenic protein is combined with a matrix material.

36. Use according to claims 24 or 35 wherein the matrix material is selected from the group comprising collagen, hydroxyapatite, homopolymers or copolymers of glycolic acid, lactic acid or butyric acid and derivatives thereof, tricalcium phosphate, or other calcium phosphates, metal oxides, demineralized, guanidine extracted bone and mixtures thereof.

37. The device of claim 12 wherein said surface of said prosthetic device is coated with a material selected from the group comprising collagen, homopolymers or copolymers of glycolic acid, lactic acid or butyric acid and derivatives thereof, tricalcium phosphate or other calcium phosphates, metal oxides, demineralized, guanidine extracted bone and mixtures thereof.

38. The device of claim 12 wherein the prosthetic device comprises stainless steel, titanium, molybdenum, cobalt, chromium or alloys or oxides of these metals.

39. The device of claim 12 wherein the osteogenic protein is combined with a matrix material.

40. The device of claim 39 wherein the matrix material is selected from the group comprising collagen, hydroxyapatite, homopolymers or copolymers of glycolic acid, lactic acid or butyric acid and derivatives thereof, tricalcium phosphate or other calcium phosphates, metal oxides, demineralized, guanidine extracted bone, and mixtures thereof.

41. The device of claim 27 wherein the device comprises stainless steel, titanium, molybdenum, cobalt, chromium or alloys or oxides of these metals.

42. The device of claim 27 wherein the matrix material is selected from the group comprising collagen, homopolymers or copolymers of glycolic acid, lactic acid or butyric acid and derivatives thereof, tricalcium phosphate or other calcium phosphates, metal oxides, demineralized, guanidine extracted bone and mixtures thereof.

43. Use according to claims 1, 2 or 24 wherein at least one of said polypeptide chains is selected from the group comprising OP2, Vgr-1, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, COP5, COP7, DPP, Vgl and variants thereof.

44. Use according to claims 1, 2 or 24 wherein one or both of said polypeptide chains is selected from the group comprising OP1, OP2, Vgr-1, BMPS, BMP6 and variants thereof.

45. Use according to claim 44 wherein said dimeric species is a heterodimer.

46. Use according to claim 1, 2 or 24 wherein said osteogenic protein is provided in combination with one or more dimeric species of OP2, Vgr-1, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, COPS, COP7, DPP, Vgl and variants thereof.

47. The device of claims 12 or 27 wherein at least one of said polypeptide chains is selected from the group comprising OP2, Vgr-1, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, COP5, COP7, DPP, Vgl and variants thereof.

48. The device of claims 12 or 27 wherein one or both of said polypeptide chains is selected from the group comprising OP1, OP2, Vgr-1, BMP5, BMP6 and variants thereof.

49. The device of claim 48 wherein said dimeric species is a heterodimer.

50. The device of claims 12 or 27 wherein said osteogenic protein is provided in combination with one or more dimeric species of OP2, Vgr-1, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, COP5, COP7, DPP, Vgl and variants thereof.