CA1338663C

CA1338663C - Biosynthetic osteogenic proteins and osteogenic devices containing them

Info

Publication number: CA1338663C
Application number: CA000596144A
Authority: CA
Inventors: Hermann Oppermann; Thangavel Kuberasampath; David C. Rueger; Engin Ozkaynak
Original assignee: Stryker Corp
Current assignee: Stryker Corp
Priority date: 1988-04-08
Filing date: 1989-04-07
Publication date: 1996-10-22
Anticipated expiration: 2013-10-22
Also published as: JPH08336390A; JPH08322570A; EP0723013A2; EP0362367B1; JPH03500655A; DE68929453T2; ATE231523T1; EP0714665A2; EP0372031B1; JP2869381B2; EP0723013B1; US20030124169A1; JPH03502579A; DE68925773D1; EP0362367A1; AU3444989A; ATE142644T1; EP0714665A3; WO1989009788A3; EP1225225A2

Abstract

Disclosed are 1) osteogenic devices comprising a matris containing osteogenic protein and methods of inducing endochondral bone growth and cartilage in mammals using the devices; 2) amino acid sequence data, amino acid composition, solubility properties, structural features, homologies and various other data characterizing osteogenic proteins, 3) methods of producing osteogenic proteins using recombinant DNA technology, and 4) osteogenically and chondrogenically active synthetic protein constructs.

Description

~ I ~ 1 33 8 6 63 OSTEOGENIC DEVICES CONTAINING THEM

This invention relates to osteogenic devices, to synthetic genes encoding proteins which can induce osteogenesis in mammals and methods for their production using recombinant DNA techniques, to synthetic forms of osteogenic protein, and to bone and cartilage repair procedures using osteogenic device comprising the synthetic proteins.

Mammalian bone tissue is known to contain one or more proteinaceous materials, presumably active during growth and natural bone healing, which can induce a developmental cascade of cellular events resulting in endochondral bone formation. This active factor (or factors) has variously been referred to in the literature as bone morphogenetic or morphogenic protein, bone inductive protein, osteogenic protein, osteogenin, or osteoinductive protein.

The developmental cascade of bone differentiation consists of recruitment of mesenchymal cells, proliferation of progenitor cells, calcification of cartilage, vascular invasion, bone formation, remodeling, and finally marrow differentiation (Reddi (1981) Collagen Rel. Res.
1:209-226).
.L

1 Though the precise mechanisms underlying these phenotypic transformations are unclear, it has been shown that the natural endochondral bone differentiation activity of bone matrix can be dissociatively extracted and reconstituted with inactive residual collagenous matris to restore full bone induction activity (Sampath and Reddi, (1981) Proc. Natl. Acad. Sci. USA 78:7599-7603). This provides an experimental method for assaying protein extracts for their ability to induce endochondral bone in vivo.

This putative bone inductive protein has been shown to have a molecular mass of less than 50 kilodaltons (kD). Several species of mammals produce closely related protein as demonstrated by cross species implant experiments (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA 80:6591-6595).

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

The observed properties of these protein fractions have induced an intense research effort in various laboratories directed to isolating and identifying the pure factor or factors responsible for osteogenic activity. The current state of the art of purification of osteogenic protein from _ _ 3 _ 1 3 3 8 6 6 3 l mammalian bone is disclosed by Sampath et al. (Proc.
Natl. Acad. Sci. USA (1987) 80). Urist et al. (Proc.
Soc. Exp. Biol. Med. (1984) 173:194-199) disclose a human osteogenic protein fraction which was extracted from demineralized cortical bone by means of a calcium chloride-urea inorganic-organic solvent mixture, and retrieved by differential precipitation in guanidine-hydrochloride and preparative gel electrophoresis. The authors report that the protein fraction has an amino acid composition of an acidic polypeptide and a molecular weight in a range of 17-18 kD.

Urist et al. (Proc. Natl. Acad. Sci. USA
(1984) 81:371-375) disclose a bovine bone morphogenetic protein extract having the properties of an acidic polypeptide and a molecular weight of approximately 18 kD. The authors reported that the protein was present in a fraction separated by hydroxyapatite chromatography, and that it induced bone formation in mouse hindguarter muscle and bone regeneration in trephine defects in rat and dog skulls. Their method of obtaining the extract from bone results in ill-defined and impure preparations.
European Patent Application Serial No.
148,155, published October 7, 1985, purports to disclose osteogenic proteins derived from bovine, porcine, and human origin. One of the proteins, designated by the inventors as a P3 protein having a molecular weight of 22-24 kD, is said to have been purified to an essentially homogeneous state. This material is reported to induce bone formation when implanted into animals.

-1 International Application No. PCT/087/01537, published January 14, 1988, discloses an impure fraction from bovine bone which has bone induction qualities. The named applicants also disclose putative bone inductive factors produced by recombinant DNA techniques. Four DNA sequences were retrieved from human or bovine genomic or cDNA
libraries and apparently expressed in recombinant host cells. While the applicants stated that the expressed proteins may be bone morphogenic proteins, bone induction was not demonstrated, suggesting that the recombinant proteins are not osteogenic. See also Urist et al., EP 0,212,474 entitled Bone Morphogenic Agents.
Wang et al. ~Proc. Nat. Acad. Sci. USA
(1988) 85: 9484-9488) discloses the purification of a bovine bone morphogenetic protein from guanidine estracts of demineralized bone having cartilage and bone formation activity as a basic protein corresponding to a molecular weight of 30 kD
determined from gel elution. Purification of the protein yielded proteins of 30, 18 and 16 kD which, upon separation, were inactive. In view of this result, the authors acknowledged that the esact identity of the active material had not been determined.

Wozney et al. (Science (1988) 242:

3~ 1528-1534) discloses the isolation of full-length cDNA's encoding the human equivalents of three - _ 5 1338~3 1 polypeptides originally purified from bovine bone.
The authors report that each of the three recombinantly expressed human proteins are independently or in combination capable of inducing S cartilage formation. No evidence of bone formation is reported.

It is an object of this invention to provide osteogenic devices comprising matrices containing dispersed osteogenic protein capable of bone induction in allogenic and xenogenic implants.
Another object is to provide synthetic osteogenic proteins capable of inducing endochondral bone formation in mammals, including humans. Yet another object is to provide genes encoding non-native osteogenic proteins and methods for their production using recombinant DNA techni~ues. Another object is to provide novel biosynthetic forms of osteogenic proteins and a structural design for novel, functional osteogenic proteins. Another object is to provide methods for inducing cartilage formation.

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

1 Summary of the Invention This invention involves osteogenic devices which, when implanted in a mammalian body, can induce at the locus of the implant the full developmental cascade of endochondral bone formation and bone marrow differentiation. Suitably modified as disclosed herein, the devices also may be used to induce cartilage formation. The devices comprise a carrier material, referred to herein as a matrix, having the characteristics disclosed below, containing dispersed osteogenic protein in the form of a biosynthetic construct.

A key to these developments was the elucidation of amino acid sequence and structure data of native osteogenic protein. A protocol was developed which results in retrieval of active, substantially pure osteogenic protein from mammalian bone. Investigation of the properties and structure of the native form osteogenic protein then permitted the inventors to develop a rational design for non-native forms, i.e., forms never before known in nature, capable of inducing bone formation. As far as applicants are aware, the constructs disclosed herein constitute the first instance of the design of a functional, active protein without preexisting knowledge of the active region of a native form nucleotide or amino acid sequence.

_ 7 _ 1 338663 1 A series of consensus DNA sequences were designed with the goal of producing an active osteogenic protein. The sequences were based on partial amino acid sequence data obtained from the natural source product and on observed homologies with unrelated genes reported in the literature, or the sequences they encode, having a presumed or demonstrated developmental function. Several of the biosynthetic consensus sequences have been expressed as fusion proteins in procaryotes, purified, cleaved, refolded, combined with a matrix, implanted in an established animal model, and shown to have endochondral bone-inducing activity. The currently preferred active proteins comprise sequences designated COP5, COP7, COP16, and OPl. The amino acid sequences of these proteins are set forth below.

HFNSTN--H-A W QTLVNSVNSKI--PKACW ~ SA

ISMLYLD~:N~:KVV~KNYQEMVVEGcGcR

HLNSTN--H-A W QTLVNSVNSKI--PKAC~vPl~LSA

ISMLYLD~:N~KVv~KNYQEMVvEGcGcR

PKHHSQRARKKNKN

HFNSTN--H-A WQTLVNSVNSKI--PKAC~VPL~:~SA

ISMLYLD~:N~:KVV~KNYQEMVVEGCGCR

-s HQRQA

OPl ~KK~LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS

YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA

ISVLYFDDSSNVILKKYRNMVVRACGCH

In these sequences and all other amino acid sequences disclosed herein, the dashes (-) are used as fillers only to line up comparable sequences in related proteins, and have no other function. Thus, amino acids 45-50 of COP7, for example, are NHAW.
Also, the numbering of amino acids is selected solely for purposes of facilitating comparisons between sequences. Thus, for example, the DF residues numbered at 9 and lO of COP5 and COP7 may comprise residues, e.g., 35 and 36, of an osteogenic protein embodying the invention.

Thus, in one aspect, the invention comprises a protein having an amino acid sequence sufficiently duplicative of the sequence of COP5, COP7, COP16, or OPl such that it is capable of inducing endochondral bone formation when properly folded and implanted in a mammal in association with a matris. Some of these sequences induce cartilage, but not bone. Also, the bone forming materials may be used to produce cartilage if implanted in an avascular locus, or if an inhibitor to full bone development is implanted together with the active protein. Thus, in another aspect, the invention comprises a protein less than 1 3386b3 1 about 200 amino acids long for each chain including a sequence sufficiently duplicative of the sequence of COP5, COP7, COP16, or OPl such that it is capable at least of cartilage formation when properly folded and implanted in a mammal in association with a matris.

In one preferred aspect, these proteins comprise species of the generic amino acid sequences:

L~v~c~xL)xGwxx~xxx~xGxxAxycxGx~ ~xx~xxxNllAxx QXXVXXXNXXXX~XXCCXPXXX~XXXXLXXXXXXXVXLXXYX~XXCXCX
or ~X XX XhXVX ~X~XGWXXWXXXPXGXXAXYCXGX~XXPXXXXXXXXN~AXX

QXXVXXXNXXXX~XXC~XPXXXXXXXXLXXXXXXXVXhXXYXX~XVXXCXCX

where the letters indicate the amino acid residues of standard single letter code, and the Xs represent amino acid residues. Preferred amino acid sequences within the foregoing generic sequences are:

LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV
K S S L QE VIS E FD Y E A AY MPESMKAS VI
F E K I DN L N S Q ITK F P TL
A S K

QTLVNsVNPGKIPKAc(;v~ LSAISMLYLD~:N~:NVVLKNYQDMVVEGCGCR
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H
RF T S K DPV V Y N S H RN RS
N S K P E

1 and CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV
RRRS K S S L QE VIS E FD Y E A AY MPESMKAS VI
KE F E K I DN L N S Q ITK F P TL
Q A S K

QTLvNsvNpGKIpKAc~;v~~ sAIsMLyLD~N~:NvvL~KNyQDM wEGcGcR
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H
RF T S K DPV V Y N S H RN RS
N S K P E
wherein each of the amino acids arranged vertically at each position in the seguence may be used alternatively in various combinations. Note that these generic seguences have 6 and preferably 7 cysteine residues where inter- or intramolecular disulfide bonds can form, and contain other critical amino acids which influence the tertiary structure of the proteins. These generic structural features are found in previously published sequences, none of which have been described as capable of osteogenic activity, and most of which never have been linked with such activity.

Particular useful sequences include:

vg1 CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE

ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKMSP

ISMLFYDNNDNVVLRHYENMAVDECGCR

DPPCRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD

HFNSTN--H-A W QTLVNNNN~GK-VPKACCVPTQLDS

VAMLYLNDQSTWLKNYQEMTWGCGCR

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD

HLNSTN--H-AIVQTLVNSVNS-K-IPKACCv~-l~L.SA

ISMLYLD~:N ~ KV VL,KNYQDMWEGCGCR

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD
HLNSTN--H-AIVQTLVNSVNS-S-IPKAC(;v~l~;L.SA

ISMLYLDEYDKWLKNYQEMWEGCGCR

SLKPSN---H-ATIQSIVRAVGWPGIPEPCCVPEKMSS

LsILFFD~;NKNvvhKvy~NrlTvEscAcR

COPl LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD

HFNSTN---H-AWQTLVNNMNPGK-VPKPCW~ll~:l.SA

ISMLYLDENSTWLKNYQEMTWGCGCR

HFNSTN--H-A W QTLvNNr.N~GK-VPKPC~;v~ .SA

ISMLYLDl':N~;KV v~KNYQEMWEGCGCR

HFNSTN--H-AWQTLVNNMNPGK-VPRPCCv~l~LSA

IsMLyLD~:N~KvvL~KNyQEMwEGcGcE~

1 Vgl is a known Xenopus sequence heretofore not associated with bone formation. DPP is an amino acid sequence encoded by a drosophila gene responsible for development of the dorsoventral pattern. OPl is a S region of a natural sequence encoded by exons of a genomic DNA sequence retrieved by applicants. The CBMPs are amino acid sequences comprising subparts of mammalian proteins encoded by genomic DNAs and cDNAs retrieved by applicants. The COPs are totally biosynthetic protein sequences expressed by novel consensus gene constructs, designed using the criteria set forth herein, and not yet found in nature.

lS These proteins are believed to dimerize during refolding. They appear not to be active when reduced. Various combinations of species of the proteins, i.e., heterodimers, have activity, as do homodimers. As far as applicants are aware, the COP5, COP7, COP16, and OPl constructs constitute the first instances of the design of a bioactive protein without preexisting knowledge of the active region of a native form nucleotide or amino acid sequence.

The invention thus provides synthetic osteogenic protein produced using recombinant DNA
techniques. The protein may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native protein, no matter how 1 derived. In view of this disclosure, skilled genetic engineers can design and synthesize genes which encode appropriate amino acid sequences, and then can e2press them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active synthetic proteins comprising truncated analogs, muteins, fusion proteins, and other constructs mimicking the biological activity of the native forms and capable lo of inducing bone formation in mammals including humans.

The synthetics are useful in clinical applications in conjunction with a suitable delivery or support system (matrix). The matri2 is made up of particles or porous materials. The pores must be of a dimension to permit progenitor cell migration and subsequent differentiation and proliferation. The particle size should be within the range of 70 - 850 ~m, preferably 70 - 420 ~m. It may be fabricated by close packing particulate material into a shape spanning the bone defect, or by otherwise structuring as desired a material that is biocompatible (non-inflammatory) and, biodegradable in vivo to serve as a ~temporary scaffold~ and substratum for recruitment of migratory progenitor cells, and as a base for their subsequent anchoring and proliferation. Currently preferred carriers include particulate, demineralized, guanidine extracted, species-specific (allogenic) bone, and particulate, deglycosglated, protein extracted, demineralized, 2enogenic bone. Optionally, such 2enogenic bone 1 powder matrices also may be treated with proteases such as trypsin. Other useful matrix materials comprise collagen, homopolymers and copolymers of glycolic acid and lactic acid, hydroxyapatite, tricalcium phosphate and other calcium phosphates.

The osteogenic proteins and implantable osteogenic devices enabled and disclosed herein will permit the physician to obtain optimal predictable bone formation to correct, for example, acquired and congenital craniofacial and other skeletal or dental anomalies (Glowacki et al.
(1981) Lancet 1:959-963). The devices may be used to induce local endochondral bone formation in non-union fractures, and in other clinical applications including periodontal applications where bone formation is required. Another lS potential clinical application is in cartilage repair, for example, in the treatment of osteoarthritis.

In another aspect, the present invention provides a process for producing a synthetic polypeptide chain having less than 200 amino acids and capable of inducing cartilage and endochondral bone formation in a mammal when disulfide bonded with a second polypeptide chain to produce a dimeric species, the method comprising the steps of:
(a) providing a generic amino acid sequence, the generic sequence comprising:

CxxxxT ~xvxFxDxGwxxwxxxpxGxxAxycxGxcxx~ x x x x x x x x ~ Axx QxxvxxxNxxxxpxxccxpxxxxxxxxnxxxxxxxvxLxxyxxMxvxxcxcx or LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPxxxxxxxx~HAXX

-- 14a -QXXVXXXNXXXXPXXCCXPXxxxxxxxT.xxxXXXXVXLXXYXXNXVXXCxCX

wherein, in each position where an X occurs, said X can be any naturally occurring amino acid residue;
(b) deriving an amino acid sequence from said consensus sequence by independently selecting an amino acid residue for each position where an X occurs in said consensus sequence, such that, when a polypeptide chain comprising said amino acid sequence is combined with a second polypeptide chain to produce a dimeric species, said dimeric species is capable of inducing cartilage and endochondral bone formation.

In yet another aspect, the present invention provides a process for producing active synthetic osteogenic protein comprising the steps of:
(a) assembling a synthetic nucleic acid molecule comprising a nucleotide sequence encoding a synthetic polypeptide chain having fewer than 200 amino acids and having the same number of cysteines placed in the same relative positions as COP5, COP7, COP16 or OP1 such that said polypeptide chain, when disulfide bonded with a second polypeptide chain to produce a dimeric species, is capable of inducing cartilage and endochondral bone formation when implanted in a mammal;
(b) introducing said nucleic acid molecule into a 25 host cell; and (c) expressing said nucleotide sequence under conditions sufficient to produce said synthetic polypeptide chain.

1 3386~3 1 Brief Description of the Drawinq The foregoing and other objects of this invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which:

FIGURE 1 is a comparison of the amino acid sequence of various osteogenic proteins to those of the TGF-beta family. COPl, COP3, COP4, COP5, and COP7 are a family of analogs of synthetic osteogenic proteins developed from the consensus gene that was joined to a leader protein via a hinge region having the sequence D-P-N-G that permitted chemical cleavage at the D-P site (by acid) or N-G (by hydroxylamine) resulting in the release of the analog protein; VGI
is a Xenopus protein, DPP is a Drosophila protein;
OPl is a native osteogenic protein; CBMP2a and 2b, and CBMP3 are subparts of proteins disclosed in PCT
application 087/01537; MIS is Mullerian inhibitory substance; and ~consensus choices~ represent various substitutions of amino acids that may be made at various positions in osteogenic proteins;
FIGURE 2A is an E. coli expression vector containing a gene of an osteogenic protein fused to a leader protein;

FIGURE 2B is the DNA sequence comprising a modified trp-LE leader, two Fb domains of protein A, an ASP-PRO cleavage site, and the COP5 sequence;

1 Figures 3A and 3B are photomicrographs of implants showing the histology (day 12) of COP5 active recombinant protein. A is a control (rat matrix alone, 25 mg). B is rat matrix plus COP5, showing +++ cartilage formation and ++ bone formation (see key infra). Similar results are achieved with COP7.

FIGURE 4 is a schematic representation of the DNA sequence and corresponding amino acid sequence of a consensus gene for osteogenic protein ( COPO);

- 17 - 1 3386~3 1 Description Purification protocols have been developed which enable isolation of the osteogenic protein present in crude protein extracts from mammalian bone. The isolation procedure enables the production of significant quantities of substantially pure osteogenic protein from any mammalian species, provided sufficient amounts of fresh bone from the species is available. The empirical development of the procedure, coupled with the availability of fresh calf bone, enabled isolation of substantially pure bovine osteogenic protein (BOP). BOP has been characterized significantly; its ability to induce cartilage and ultimately endochondral bone growth in cat, rabbit, and rat have been studied; it has been shown to be able to induce the full developmental cascade of bone formation previously ascribed to unknown protein or proteins in heterogeneous bone extracts; and it may be used to induce formation of endochondral bone in orthopedic defects including non-union fractures. In its native form it is a glycosylated, dimeric protein. However, it is active in deglycosylated form. It has been partially sequenced.

Elucidation of the amino acid sequence of BOP enabled the construction of a consensus nucleic acid sequence designed as disclosed herein based on the sequence data, inferred codons for the sequences, and observation of partial homology with known genes.

1 3~ 3 1 These consensus sequences have been refined by comparison with the sequences present in certain regulatory genes from drosophila, senopus, and human followed by point mutation, expression, and assay for activity. This approach has been successful in producing several active totally synthetic constructs not found in nature (as far as applicants are aware) which have true osteogenic activity.

These discoveries enable the construction of DNAs encoding totally novel, non-native protein constructs which individually and combined are capable of producing true endochondral bone. The DNAs may be espressed using well established recombinant DNA technologies in procaryotic or eucaryotic host cells, and the expressed proteins may be oxidized and refolded in vitro if necessary for biological activity.

The design and production of such biosynthetic proteins, the nature of the matrix, and other material aspects concerning the nature, utility, how to make, and how to use the subject matter claimed herein will be further understood from the following, which constitutes the best method currently known for practicing the various aspects of the invention.

CONSENSUS SEQUENCE DESIGN
A synthetic consensus gene shown in FIGURE 4 was designed to encode a consensus protein based on amino acid predictions from homology with the 1 338~63 1 TGF-beta gene family. The designed concensus sequence was then constructed using known techniques involving assembly of oligonucleotides manufactured in a DNA synthesizer.

Tryptic peptides derived from Bovine Osteogenic Protein isolated by applicants and sequenced by Edman degradation provided amino acid sequences that showed stronq homology with the o Drosophila DPP protein sequence (as inferred from the gene), the Xenopus VGl protein, and somewhat less homology to inhibin and TGF-beta, as demonstrated below in TABLE 1.

~ 338663 proteinamino acid sequencehomology (BOP)SFDAYYCSGACQFPS
*~*** * * **(9/15 matches) (DPP)GYDAYYCHGKCPFFL

(BOP)SFDAYYCSGACQFPS
* ** * * * (6/15 matches) (Val)GYMANYCYGECPYPL

(BOP)SFDAYYCSGACQFPS
* ** * * (5/15 matches) (inhibin) GYHANYCEGECPSHI

(BOP)SFDAYYCSGACQFPS
* * * * (4/15 matches) (TGF-beta) GYHANFCLGPCPYIW

(BOP)K/RAC~v~l~SAISMLYLDEN
***** * **** * * (12/20 matches) (Val) LPCw ~lK~SPISMLFYDNN

(BOP) K/RAC~v~L~SAISMLYLDEN
* ***** * **** * (12/20 matches) (inhibin) KSCCv~LK~RPMSMLYYDDG

(BOP) K/RAC~v~l~:LSAISMLYLDE
**** * * (6/19 matches) (TGF-beta) APCCVPQALEPLPIVYYVG

(BOP)K/RAC~v~l~SAISMLYLDEN
******* * ****(12/20 matches) (DPP)KACCVPTQLDSVAMLYLNDQ

1 (BOP)LYVDF
***** (5/5 matches) (DPP)LYVDF

(BOP)LYVDF
*~ * (4/5 matches) (V~l)LYVEF

(BOP)LYVDF
** ** (4/5 matches) (TGF-beta) LYIDF

(BOP)LYVDF
* * (2/4 matches) (inhibin)FFVSF

*-match In determining an appropriate amino acid sequence of an osteogenic protein (from which the nucleic acid sequence can be determined), the following points were considered: (1) the amino acid sequence determined by Edman degradation of natural source osteogenic protein tryptic fragments is ranked highest as long as it has a strong signal and shows homology or conservative changes when aligned with the other members of the gene family; (2) where the sequence matches for all four proteins, it is used in the synthetic gene seguence; (3) matching amino acids in DPP and Vgl are used; (4) If Vgl or DPP diverged but either one were matched by inhibin or by TGF-beta, this matched amino acid is chosen; (5) where all sequences diverged, the DPP sequence is initially chosen, with a later plan of creating the 1 Vgl sequence by mutaqenesis kept as a possibility.
In addition, the consensus sequence is designed to preserve the disulfide crosslinking and the apparent structural homology among the related proteins.

RECOMBINANT OSTEOGENIC
PROTEIN CONSTRUCTS

This approach resulted in the production of novel recombinant proteins capable of inducing formation of cartilage and endochondral bone comprising a protein structure analogous to or duplicative of the functional domain of the naturally sourced material. The amino acid sequences encoded by the consensus DNA sequences were derived from a family of natural proteins implicated in tissue development. These gene products~proteins are known to exist in active form as dimers and are, in general, processed from a precursor protein to produce an active C-terminal domain of the precursor.

The recombinant osteogenic/chondrogenic proteins are ~novel~ in the sense that, as far as applicants are aware, they do not e~ist in nature or, if they do exist, have never before been associated with bone or cartilage formation. The approach to design of these proteins is to employ amino acid sequences, found in the native OP isolates, in polypeptide structures are patterned after certain proteins reported in the literature, or the amino acid sequences inferred from DNAs reported in the literature. Thus, using the design criteria set 1 forth above, and refining the amino acid sequence as more protein sequence information was learned, a series of synthetic proteins were designed with the hope and intent that they might have osteogenic or chondrogenic activity when tested in the bioassay system disclosed below.

It was noted, for example, that DPP from drosophila, VGl from Xenopus, the TGF beta family of proteins, and to a lesser extent, alpha and beta inhibins, had significant homologies with certain of the sequences derived from the naturally sourced OP
product. (Fig. 1.) Study of these proteins led to the realization that a portion of the sequence of each had a structural similarity observable by analysis of the positional relationship of cysteines and other amino acids which have an important influence on three dimensional protein conformation.
It was noted that a region of these sequences had a series of seven cysteines, placed very nearly in the same relative positions, and certain other amino acids in sequence as set forth below:

~xxx~Xvx~x~XGWXXhxxx~GXXAXYCXGX~xx~xxxxxxxh~AXX

QXXVXX.XNXX~X~'XXCC;X~X~UiXX~XXLXXX~X~VXl~CXCX
wherein each X independently represents an amino acid. Expression experiments of two of these constructs demonstrate activity. Expression experiments with constructs patterned after this template amino acid sequence with a shorter sequence having only six cysteines also show activity:

- 24 - ~ 3 3 8 6 63 LXVX~XL)XGWXXWXXXPXGXXAXYCXGX(;xx~xX ~ NHAXX

QxxvxxxNxxxx~xxC~x~Xxx~xx~Lxxxxxxxv~hxxYxx~XVXXCXCX

wherein each X independently represents an amino acid. Within these generic structures are a multiplicity of specific sequences which have osteogenic or chondrogenic activity. Preferred structures are those having the amino acid sequence:

CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV
RKRS K S S L QE VIS E FD Y E A AY MPESMKAS VI
KE F E K I DN L N S Q I TK F P TL
Q A S K

QTLVNSVNPGKIPKAC~;v~l~LSAISMLYLDI~N~;NvvL.KNYQDMVVEGCGCR
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H
RF T S K DPV V Y N S H RN RS
N S K P E

wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be used. Novel active proteins also are defined by amino acid sequences comprising an active domain beginning at residue number 6 of this sequence, i.e, omitting the N terminal CXXXX, or omitting any of the preferred specific combinations such as CKRHP, CRRKQ, CKRHE, etc, resulting in a construct having only six cysteine residues. After this work, PCT 87/01537 was published, and it was observed that the proteins there identified as BMPII a and b and BMPIII each included a region embodying this generic structure.
These proteins were not demonstrated to be osteogenic in the published application. However, applicants ~ 25 - 1 338663 1 discovered that a subpart of the amino acid sequence of these proteins, properly folded, and implanted as set forth herein, is active. These are disclosed herein as CBMPIIa, CBMPIIb, and CBMPIII. Also, applicants retrieved a previously unreported gene by probing a human DNA library with COPO. This protein was designated OPl. It comprises a region exhibiting the same generic structure.

Thus, the preferred osteogenic proteins are expressed from recombinant DNA and comprise amino acid sequences including any of the following sequences:

Vgl CKKRHL-yv~;~K-DvGwQNwvIApQGyMANycyGEcpypLTE

ILNGSN--H-AILQTLVHSIEPED-IPLPC~v~-LKMSP

ISMLFYDNNDNWLRHYENMAVDECGCR

DPP CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD

HFNSTN--H-AWQTLVNNNN~GK-VPKACCVPTQLDS

VAMLYLNDQSTWLKNYQEMTVVGCGCR

OPl LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS

YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA

ISVLYFDDSSNVILKKYRNMVVRACGCH

- 26 - l 338663 HQRQA

OPlC~CKH~:~.YVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS

YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA

ISVLYFDDSSNVILKKYRNMVVRACGCH

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD
HLNSTN--H-AIVQTLVNSVNS-K-IPKACC:v~~ .SA

ISMLYLD~ N~;KvvhKNyQDMvvEGcGcR

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAEYCHGDCPFPLAD

HLNSTN--H-AIVQTLVNSVNS-S-IPKAC(;v~l~hSA

ISMLYLDEYDKVVLKNYQEMWEGCGCR

SLKPSN--H-ATIQSIVRAVGWPGIPEPCCv~ :KrISS

LsILFFD~:NKNvvLKvypNMTvEscAcR
l 10 20 30 40 COPl LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD

HFNSTN--H-AWQTLVNNMNPGK-VPKPC~;v~l~hSA

ISMLYLDENSTWLKNYQ~ vvGCGCR

l 10 20 30 40 HFNSTN--H-AWQTLvNNMN~GK-VPKPCCv~L~hSA

ISMLYLD ~ N ~:KV vhKNYQEMVVEGCGCR

HFNSTN--H-A WQTLVNNMNPGK-VPKPC~;v~L~;hSA

IsMLyLD~:N l4 KV VhKNYQEMVVEGCGCR

HFNSTN--H-AWQTLVNSVNSKI--PKAC(;v~ LSA

ISMLYLD~:N~;KVVLKNYQEMVVEGCGcR

HLNSTN--H-AWQTLVNSVNSKI--PKAC~;v~ hSA

ISMLYLD ~:N ~ KV VLRNYQEMWEGCGcR

PKHHSQRARKKNKN

HFNSTN--H-AWQTLVNSVNSKI--PKAC~;v~L~:LSA

ISMLYLD~N~KvvhKNYQEMVVEGCGCR

As shown in FIGURE 1, these sequences have considerable homology with the alpha and beta inhibins, three forms of TGF beta, and MIS.

1 Gene Prepartaion 1 3 3 8 6 ~ 3 The synthetic genes designed as described above preferably are produced by assembly of chemically synthesized oligonucleotides. 15-lOOmer oligonucleotides may be synthesized on a Biosearch *

DNA Model 8600 Synthesizer, and purified by polyacrylamide gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer (TBE). The DNA is then electroeluted from the gel. Overlapping oligomers may be phosphorylated by T4 polynucleotide kinase and ligated into larger blocks which may also be purifed by PAGE. Natural gene sequences and cDNAs also may be used for expression.
Expression The genes can be expressed in appropriate prokaryotic hosts such as various strains of E.
coli. For example, if the gene is to be expressed in E. coli, it must first be cloned into an expression vector. An expression vector (FIG. 2A) based on pBR322 and containing a synthetic trp promoter operator and the modified trp LE leader can be opened 2S at the EcoRI and PSTI restriction sites, and a FB-FB
COP gene fragment (FIG. 2B) can be inserted between these sites, where FB is fragment B of Staphylococcal Protein A. The expressed fusion protein results from attachment of the COP gene to a fragment encoding FB. The COP protein is joined to the leader protein via a hinge region having the sequence asp-pro-asn-gly. This hinge permits chemical cleavage of the fusion protein with dilute acid at * Trade mark ~S~

_ - 29 -1 the asp-pro site or cleavage at asn-gly with hydroxylamine, resulting in release of the COP
protein.

Production of Active Proteins The following procedure was followed for production of active recombinant proteins. E. coli cells containing the fusion proteins were lysed. The fusion proteins were purified by differential solubilization. In the case of the COP 1, 3, 4, 5, and 7 fusion proteins, cleavage was with dilute acid, and the resulting cleavage products were passed through a Sephacryl-200HR column. The Sephacryl column separated most of the uncleaved fusion products from the COP 1, 3, 4, 5, and 7 analogs. In the case of the COP 16 fusion protein, cleavage was with a more concentrated acid, and an SP-Trisacryl column was used to separate COP 16, the leader protein, and the residual fusion protein. The COP
fractions from any of the COP analogs were then subjected to HPLC on a semi-prep C-18 column. The HPLC column primarily separated the leader proteins and other minor impurities from the COP analogs.
Initial conditions for refolding of COP
analogs were at pH 8.0 using Tris, GuHCl, dithiothreitol. Final conditions for refolding of COP analogs were at pH 8.0 using Tris, oxidized glutathione, and lower amounts of GuHCl and dithiothreitol.
* Trade mark ~ .
I . ~

1 Production of Antisera Antisera to COP 7 and COP5 were produced in New Zealand white rabbits. Western blots demonstrate that the antisera react with COP 7 and COP5 preparations. Antisera to COP 7 has been tested for reactivity to naturally sensed bovine osteogenic protein samples. Western blots show a clear reaction with the 30kD protein and, when reduced, with the 16kD subunit. The im~unoreactive species appears as a closely-spaced doublet in the 16K subunit region, similar to the 16K doublet seen in Con A blots.

MATRIX PREPARATION

General Consideration of Matrix Properties The carrier described in the bioassay section, infra, may be replaced by either a biodegradable-synthetic or synthetic-inorganic matrix te.g., HAP, collagen, tricalcium phosphate, or polylactic acid, polyglycolic acid and various copolymers thereof). Also xenogeneic bone may be used if pretreated as described below.

Studies have shown that surface charge, particle size, the presence of mineral, and the methodology for combining matrix and osteogenic protein all play a role in achieving successful bone induction. Perturbation of the charge by chemical modification abolishes the inductive response.
Particle size influences the quantitative response of new bone; particles between 75 and 420 ~m elicit 1 the maximum response. Contamination of the matrix with bone mineral will inhibit bone formation. Most importantly, the procedures used to formulate osteogenic protein onto the matrix are extremely sensitive to the physical and chemical state of both the osteogenic protein and the matris.

The sequential cellular reactions at the interface of the bone matrix/OP implants are 1~ complex. The multistep cascade includes: binding of fibrin and fibronectin to implanted matrix, chemotaxis of cells, proliferation of fibroblasts, differentiation into chondroblasts, cartilage formation, vascular invasion, bone formation, remodeling, and bone marrow differentiation.

A successful carrier for osteogenic protein must perform several important functions. It must bind osteogenic protein and act as a slow release delivery system, accommodate each step of the cellular response during bone development, and protect the osteogenic protein from nonspecific proteolysis. In addition, selected materials must be biocompatible in vivo and biodegradable; the carrier must act as a temporary scaffold until replaced completely by new bone. Polylactic acid (PLA), polyglycolic acid (PGA), and various combinations have different dissolution rates n v vo. In bones, the dissolution rates can vary according to whether the implant is placed in cortical or trabecular bone.

1 Matrix geometry, particle size, the presence of surface charge, and porosity or the presence of interstices among the particles of a size sufficient to permit cell infiltration, are all important to successful matrix performance. It is preferred to shape the matrix to the desired form of the new bone and to have dimensions which span non-union defects.
Rat studies show that the new bone is formed essentially having the dimensions of the device implanted.

The matrix may comprise a shape-retaining solid made of loosely adhered particulate material, e.g., with collagen. It may also comprise a molded, porous solid, or simply an aggregation of close-packed particles held in place by surrounding tissue. Masticated muscle or other tissue may also be used. Large allogeneic bone implants can act as a carrier for the matrix if their marrow cavities are cleaned and packed with particles and the dispersed osteogenic protein.

Preparation of 8iologicallY Active Allogenic Matrix Demineralized bone matris is prepared from the dehydrated diaphyseal shafts of rat femur and tibia as described herein to produce a bone particle size which pass through a 420 ~m sieve. The bone particles are subjected to dissociative extraction with 4 M guanidine-HCl. Such treatment results in a complete loss of the inherent ability of the bone matrix to induce endochondral bone differentiation.

1 The remaining insoluble material is used to fabricate the matrix. The material is mostly collagenous in nature, and upon implantation, does not induce cartilage and bone. All new preparations are tested for mineral content and false positives before use.
The total loss of biological activity of bone matrix is restored when an active osteoinductive protein fraction or a pure protein is reconstituted with the biologically inactive insoluble collagenous matri~.
The osteoinductive protein can be obtained from any vertebrate, e.g., bovine, porcine, monkey, or human, or produced using recombinant DNA techniques.

PreParation of Deqlycosylated Bone Matrix for Use in Xenogenic Implant When osteogenic protein is reconstituted with collagenous bone matrix from other species and implanted in rat, no bone is formed. This suggests that while the osteogenic protein is xenogenic (not species specific), while the matris is species specific and cannot be implanted cross species perhaps due to intrinsic immunogenic or inhibitory components. Thus, heretofore, for bone-based matrices, in order for the osteogenic protein to eshibit its full bone inducing activity, a species specific collagenous bone matrix was required.

The major component of all bone matrices is Type I collagen. In addition to collagen, extracted bone includes non-collagenous proteins which may account for 5~ of its mass. Many non-collagenous components of bone matrix are glycoproteins.

1 Although the biological significance of the glycoproteins in bone formation is not known, they may present themselves as potent antigens by virtue of their carbohydrate content and may constitute immunogenic and/or inhibitory components that are present in xenogenic matrix.

It has now been discovered that a collagenous bone matrix may be used as a carrier to effect bone inducing activity in xenogenic implants, if one first removes the immunogenic and inhibitory components from the matrix. The matrix is deglycosglated chemically using, for example, hydrogen fluoride to achieve this purpose.
Bovine bone residue prepared as described above is sieved, and particles of the 74-420 ~M are collected. The sample is dried in vacuo over P205, transferred to the reaction vessel and anhydrous hydrogen fluoride (HF) (10-20 ml/g of matrix) is then distilled onto the sample at -70C.
The vessel is allowed to warm to 0 and the reaction mixture is stirred at this temperature for 60 min.
After evaporation of the HF in vacuo, the residue is dried thoroughly n vacuo over KOH pellets to remove any remaining traces of acid.

Extent of deglycosylation can be determined from carbohydrate analysis of matrix samples taken before and after treatment with HF, after washing the samples appropriately to remove non-covalently bound carbohydrates.

1 The deglycosylated bone matrix is next treated as set forth below:

1) suspend in TBS (Tris-buffered Saline) lg/200 ml and stir at 4C for 2 hrs;

2) centrifuge then treated again with TBS, lg/200 ml and stir at 4C overnight; and 3) centrifuged; discard supernatant; water wash residue; and then lyophilized.

FABRICATION OF DEVICE
lS
Fabrication of osteogenic devices using any of the matrices set forth above with any of the osteoqenic proteins described above may be performed as follows.
A. Ethanol ~recipitation In this procedure, matrix is added to osteogenic protein in guanidine-HCl. Samples are vortexed and incubated at a low temperature. Samples are then further vortexed. Cold absolute ethanol is added to the mixture which is then stirred and incubated. After centrifugation (microfuge high speed) the supernatant is discarded. The reconstituted matrix is washed with cold concentrated ethanol in water and then lyophilized.

-1 B. Acetonitrile Trifluoroacetic Acid LYophilization In this procedure, osteogenic protein in an acetonitrile trifluroacetic acid (ACN/TFA) solution is added to the carrier. Samples are vigorously vortexed many times and then lyophilized.

C. Urea Lyophilization For those proteins that are prepared in urea buffer, the protein is mised with the matrix, vortexed many times, and then lyophilized. The lyophilized material may be used ~as is~ for implants.

IN VIVO RAT BIOASSAY

Several of the synthetic proteins have been incorporated in matrices to produce osteogenic devices, and assayed in rat for endochondral bone.
Studies in rats show the osteogenic effect to be dependent on the dose of osteogenic protein dispersed in the osteogenic device. No activity is observed if the matrix is implanted alone. The following sets forth guidelines for how the osteogenic devices disclosed herein can be assayed for evaluating protein constructs and matrices for biological activity.

~ 37 ~ 1 338663 1 A. Subcutaneous ImPlantation The bioassay for bone induction as described by Sampath and Reddi ~Proc. Natl. Acad. Sci. USA
(1983) 80: 6591-6595), is used to assess endochondral bone differentiation activity. This assay consists of implanting the test samples in subcutaneous sites in allogenic recipient rats under ether anesthesia.
Male Long-Evans rats, aged 28-32 days, were used. A
vertical incision (1 cm) is made under sterile conditions in the skin over the thoraic region, and a pocket is prepared by blunt dissection.
Approximately 25 mg of the test sample is implanted deep into the pocket and the incision is closed with a metallic skin clip. The day of implantation is designated as day one of the experiment. Implants were removed on day 12. The heterotropic site allows for the study of bone induction without the possible ambiguities resulting from the use of orthotopic sites.

B. Cellular Events The implant model in rats exhibits a controlled progression through the stages of matrix induced endochondral bone development including: (1) transient infiltration by polymorphonuclear leukocytes on day one; (2) mesenchymal cell migration and proliferation on days two and three; (3) chondrocyte appearance on days five and six; (4) cartilage matrix formation on day seven; (5) 1 cartiliage calcification on day eight; (6) vascular invasion, appearance of osteoblasts, and formation of new bone on days nine and ten; (7) appearance of osteoblastic and bone remodeling and dissolution of the implanted matris on days twelve to eighteen; and (8) hematopoietic bone marrow differentiation in the ossicle on day twenty-one. The results show that the shape of the new bone conforms to the shape of the implanted matrix.
C. Histological Evaluation Histological sectioning and staining is preferred to determine the extent of osteogenesis in the implants. Implants are fixed in Bouins Solution, embedded in parafilm, cut into 6-8 mm sections.
Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of endochondrial bone. Twelve day implants are usually sufficient to determine whether the implants show bone inducing activity.

D. Biological Markers Alkaline phosphatase activity may be used as a marker for osteogenesis. The enzyme activity may be determined spectrophotometrically after homogenization of the implant. The activity peaks at 9-10 days in vivo and thereafter slowly declines.
Implants showing no bone development by bistology should have little or no alkaline phosphatase activity under these assay conditions. The assay is 1 useful for quantitation and obtaining an estimate of bone formation very quickly after the implants are removed from the rat. Alternatively the amount of bone formation can be determined by measuring the calcium content of the implant.

The osteogenic activity due to osteogenic protein is represented by ~bone forming units~. One bone forming unit represents the amount of protein that is needed for half maximal bone forming activity as compared to rat demineralized bone matrix as control and determined by calcium content of the implant on day 12.

Devices that contained only rat carrier show complete absence of new bone formation. The implant consists of carrier rat matrix and surrounding mesenchymal cells. Again, the devices that contained rat carrier and not correctly folded (or biologically inactive) recombinant protein also showed complete absence of bone formation. These implants are scored as cartilage formation (-) and bone formation (-).
The endochondral bone formation activity is scored as zero percent (0~). (FIGURE 3A) Implants included biologically active recombinant protein, however, showed evidence of endochondral bone formation. Histologically they showed new cartilage and bone formation.

1 The cartilage formation is scored as (+) by the presence of metachromatically stained chondrocytes in the center of the implant, as (++) by the presence of numerous chondrocytes in many areas of the implant and as (~++) by the presence of abundant chondrocytes forming cartilage matrix and the appearance of hypertrophied chondrocytes accompanying cartilage calcification (FIGURE 3B).

The bone formation is scored as (+) by the presence of osteoblast surrounding vascular endothelium forming new matrix, as (++) by the formation of bone due to osteoblasts (as indicated by arrows) and further bone remodeling by the appearance of osteoclasts in opposition to the newly formed bone matrix. Vascular invasion is evident in these implants (FIGURE 3B). Formation is scored as (+++) by the presence of extensive remodeled bone which results in the formation of ossicles.
The overall bone inducing activity due to recombinant protein is represented as percent response of endochondral bone formation (see Table 2 below).

3n - 41 - 1 33 8 5 ~3 HISTOLOGICAL EVALUATION OF RECOMBINANT BONE INDUCTIVE
PROTEINS

Sample ImPlantedCartilage ,Bone No. ProteinFormation Formation 260-54 COP-5 +++ ++
279-5 COP-5 ++ +
285-13 COP-5 +++ ++
277-7 COP-7 +++ ++
277-8 COP-7 +++ ++
277-9 COP-7 ++ +
285-14 COP-7 +++ ++
285-24 COP-7 ++ +
285-25 COP-7 ++ ++
314-6 COP-16 +++ +++
314-15 COP-16 ++ +
314-16 COP-16 ++ +
314-12 OP-l ++ +

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all _ _ - 42 - 1 338563 1 changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

What is claimed is:

Claims

1. An osteogenic device for implantation in a mammal, said device comprising:
a biocompatible matrix defining pores of a dimension sufficient to permit influx, proliferation and differentiation of migratory progenitor cells from the body of said mammal; and a protein, produced by expression of recombinant DNA in a host cell, comprising a pair of polypeptide chains, each of said chains having less than 200 amino acids and an amino acid sequence having the same number of cysteines in the same relative positions as occurs in COP5, COP7, COP16 or OP1, and that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species, is capable of inducing cartilage and endochondral bone formation in association with said matrix when implanted in a mammal.

2. The device of claim 1 wherein the sequence comprises:

wherein each X independently represents an amino acid.

3. The device of claim 1 wherein the sequence comprises:

wherein each X independently represents an amino acid.

4. The device of claim 1 wherein the sequence comprises:

wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be in that position.

5. The device of claim 1 wherein the sequence comprises:

wherein, in each position where more than one amino acid is shown, any one of the amio acids shown may be in that position.

6. The device of claim 1 wherein the sequence comprises:

7. The device of claim 1 wherein the sequence comprises:

8. The device of claim 1 wherein the sequence comprises:

9. The device of claim 1 wherein the sequence comprises:

10. The device of claim 1 wherein the sequence comprises:

11. The device of claim 1 wherein the sequence comprises:

12. The device of claim 1 wherein the sequence comprises:

13. The device of claim 1 wherein the sequence comprises:

14. The device of claim 1 wherein the sequence comprises:

15. The device of claim 1 wherein the sequence comprises:

16. The device of claim 1 wherein the sequence comprises:

17. The device of claim 1 wherein the sequence comprises:

18. The device of claim 1 wherein the sequence comprises:

19. A protein produced by expression of recombinant DNA
in a host cell, comprising a pair of polypeptide chains, each of said chains having less than 200 amino acids and an amino acid sequence having the same number of cysteines in the same relative positions as occurs in COP5, COP7, COP16 or OP1, and that said pair of polypeptide chains, when disulfide bonded to produce a dimeric species, is capable of inducing cartilage and endochondral bone formation in association with a matrix when implanted in a mammal.

20. The protein of claim 19 further characterized by being unglycosylated.

21. The protein of claim 19 comprising the amino acid sequences:

wherein each X independently represents an amino acid.

22. The protein of claim 19 comprising the amino acid sequences:

wherein each X independently represents an amino acid.

23. The protein of claim 19 comprising the amino acid sequences:

wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be in that position.

24. The protein of claim 19 comprising the amino acid sequences:

wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be in that position.

25. The protein of claim 19 comprising the amino acid sequences:

26. The protein of claim 19 comprising the amino acid sequences:

27. The protein of claim 19 comprising the amino acid sequence:

28. The protein of claim 19 comprising the amino acid sequences:

29. The protein of claim 19 comprising the amino acid sequences:

30. The protein of claim 19 comprising the amino acid sequences:

31. The protein of claim 19 comprising the amino acid sequences:

32. The protein of claim 19 comprising the amino acid sequences:

33. The protein of claim 19 comprising the amino acid sequences:

34. The protein of claim 19 comprising the amino acid sequences:

35. The protein of claim 19 comprising the amino acid sequences:

36. The protein of claim 19 comprising the amino acid sequences:

37. The protein of claim 19 comprising the amino acid sequences:

38. The protein of claim 19 comprising the product of expression of DNA in a procaryotic cell.

39. A cell line engineered to express the protein of claim 19.

40. The protein of claim 19 having a half maximum bone forming activity of about 20 - 25 ng per 25mg of implant.

41. Antibodies reactive with an epitope of the protein of claim 19.

42. The device of claim 1 wherein said matrix comprises demineralized, deglycosylated, protein extracted, particulate, xenogenic bone.

43. The device as claimed in claim 1 wherein said matrix comprises a member selected from the group consisting of collagen, hydroxyapatite, tricalcium phosphate, polymers comprising lactic acid monomer units, polymers comprising glycolic acid monomer units, demineralized, guanadine-extracted allogenic bone, or a mixture thereof.

44. The device as claimed in claim 1 wherein said matrix comprises close-packed particulate matter having a particle size within the range of 70-850 µm.

45. The device as claimed in claim 1 wherein said matrix comprises close-packed particulate matter having a particle size within the range of 70-420 µm.

46. The device as claimed in claim 1 wherein said matrix is shaped to span a non-union fracture in said mammal.

47. The device as claimed in claim 1 adapted to be disposed within the marrow cavity of allogenic bone.

48. The device as claimed in claim 1 wherein said matrix is treated with a protease.

49. The device as claimed in claim 1 wherein said protein is unglycosylated.

50. A device as claimed in claim 1 for use in periodontal or dental reconstructive procedures.

51. A device as claimed in claim 1 for use in craniofacial reconstructive procedures.

52. A process for producing a synthetic polypeptide chain having less than 200 amino acids and capable of inducing cartilage and endochondral bone formation in a mammal when disulfide bonded with a second polypeptide chain to produce a dimeric species, the method comprising the steps of:
(a) providing a generic consensus amino acid sequence, the generic sequence comprising:

or wherein, in each position where an X occurs, said X can be any naturally occurring amino acid residue;
(b) deriving an amino acid sequence from said consensus sequence by independently selecting an amino acid residue for each position where an X occurs in said consensus sequence, such that, when a polypeptide chain comprising said amino acid sequence is combined with a second polypeptide chain to produce a dimeric species, said dimeric species is capable of inducing cartilage and endochondral bone formation.

53. The process according to claim 52 comprising the additional steps of:

(c) producing a dimeric protein species having a polypeptide chain comprising said amino acid sequence; and (d) assaying said dimeric protein for cartilage and endochondral bone formation activity.

54. The process according to claim 52 wherein said step of producing said dimeric protein species comprises the steps of: .
(b) constructing a synthetic nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of step (b);
(c) introducing said synthetic nucleic acid molecule into a host cell; and (d) expressing said nucleotide sequence under conditions sufficient to produce said synthetic polypeptide chain.

55. The process according to any one of claims 52-54 comprising the additional step of replacing one or more of the amino acids in the sequence derived in step (b) with a different amino acid to enhance the activity of said dimeric protein.

56. The process according to claim 52 or 55 wherein one or more of said amino acids independently selected in step (b) are selected from the among the amino acids occurring at the corresponding position in any of the sequences: Vg1; DPP; OP1;
CBMP2A; CBMP2B, CBMP3, COP1, COP3, COP4, COP5, COP7 or COP16.

57. A process for producing active synthetic osteogenic protein comprising the steps of:
(a) assembling a synthetic nucleic acid molecule comprising a nucleotide sequence encoding a synthetic polypeptide chain having fewer than 200 amino acids and having the same number of cysteines placed in the same relative positions as COP5, COP7, COP16, or OP1 such that said polypeptide chain, when disulfide bonded with a second polypeptide chain to produce a dimeric species, is capable of inducing cartilage and endochondral bone formation when implanted in a mammal;
(b) introducing said nucleic acid molecule into a host cell; and (c) expressing said nucleotide sequence under conditions sufficient to produce said synthetic polypeptide chain.

58. The process of claim 54 or 57 wherein the host cell is a prokaryotic host cell.

59. The process of claim 58, wherein the prokaryotic host cell is E.coli..

60. Synthetic ostenogenic protein produced by the process of any one of claims 52 to 57.

61. The device of any one of claims 1 or 42-45 for use in therapy for inducing one or both of local cartilage and endochondral bone formation in a mammal by implanting the device in a mammal at a locus accessible to migratory progenitor cells.

62. The device of any one of claims 1 or 42-45 disposed within a marrow cavity of allogenic bone.

63. The protein of any one of claims 19 or 60 for use in inducing local cartilage repair or for treating osteoarthritis.