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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 5 : 

C12N 15/00, O07K 7/10, 13/00 
A61K 37/02, 27/00 



Al 



(1 1) International Publication Number: WO 92/07073 

(43) International Publication Date: 30 April 1992 (30.04.92) 



(21) International Application Number: PCT/US9 1/07635 

(22) International Filing Date: 18 October 1991 (18.10.91) 



(30) Priority data: 
599,543 



1 8 October 1990(18.1 0.90) US 



(71) Applicant: CREATIVE BIOMOLECULES, INC. [US/ 

US1; 35 South Street, Hopkinton, MA 01748 (US). 

(72) Inventors: OPPERMANN, Hermann ; 25 Summer Hill 

Road, Medway, MA 02053 (US). OZKAYNAK, Engin ; 
44 Purdue Drive, Milford, MA 01757 (US). RUEGER, 
David, C. ; 150 Edgemere Road, Apt. 4, West Roxbury, 
MA 02132 (US). KUBERASAMPATH, "Inangavel ; 6 
Spring Street, Medway, MA 02053 (US). 



(74) Agent: PITCHER, Edmund, R.; Testa, Hurwitz & Thi 
beault, Exchange Place, 53 State Street, Boston, MA 
02109-2809 (US). 

(81) Designated States: AT (European patent), AU, BE (Euro- 
pean patent), CA, CH (European patent), DE (Euro- 
pean patent), DK (European patent), ES (European pa- 
tent), FR (European patent), GB (European patent), GR 
(European patent), IT (European patent), JP, LU (Euro- 
pean patent), NL (European patent), SE (European pa 
tent). 



Published 

With international search report. 
Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: OSTEOGENIC PEPTIDES 



(57) Abstract 



Disclosed are 1) the cDNA and amino acid sequences for novel polypeptide chains useful as subunits of dimeric osteogen- 
ic proteins, 2) osteogenic devices comprising these proteins in association with an appropriate carrier matrix, 3) methods of prod- 
ucing the polypeptide chains using recombinant DNA technology, and 4) methods of using the osteogenic devices to mimic the 
natural course of endochondral bone formation in mammals. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


ES 


Spain 


MC 


Madagascar 


AU 


Australia 


Fl 


Finland 


ML 


Mali 


BB 


Barbados 


FR 


France 


MN 


Mongolia 


BE 


Belgium 


GA 


Gabon 


MR 


Mauritania 


BF 


Burkina Faso 


CB 


United Kingdom 


MW 


Malawi 


6G 


Bulgaria 


CN 


Guinea 


NL 


Netherlands 


BJ 


Benin 


CR 


Greece 


NO 


Norway 


BR 


Brazil 


HU 


Hungary 


PL 


Poland 


CA 


(Canada 


IT 


Italy 


RO 


Romania 


CF 


(Antral African Republic 


JP 


Japan 


SO 


Sudan 


CG 


Congo 


KP 


Democratic People Is Republic 


SE 


Sweden 


CH 


Switzerland 




of Korea 


SN 


Senegal 


CI 


Cote d'lvoirc 


KR 


Republic of Korea 


su* 


Soviet Union 


CM 


Cameroon 


LI 


Liechtenstein 


TO 


Chad 


CS 


Chechoslovakia 


LK 


Sri Lanka 


TC 


Togo 


DE* 


Germany 


LU 


Luxembourg 


US 


United States of America 


DK 


Denmark 


MC 


Monaco 







+ Any designation of "SU" has effect in the Russian Federation. It is not yet known whether 
any such designation has effect in other States of the former Soviet Union. 



WO 92/07073 



PCTAJS91/07635 



Osteogenic peptides 

Background of the Invention 

5 This invention relates to novel polypeptide chains 

and to osteogenic proteins comprising these polypeptide 
chains which are capable of inducing osteogenesis in 
mammals; to genes encoding the polypeptide chains; to 
methods for their production using recombinant DNA 
10 techniques , and to bone and cartilage repair procedures 
using the osteogenic proteins. 

Mammalian bone tissue is known to contain one or 
more proteinaceous materials, presumably active during 

15 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 

20 protein, bone inductive protein, osteogenic protein, 
osteogenin, or osteoinductive protein. 

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

30 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 



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inactive residual collagenous matrix to restore full 
bone induction activity (Sampath and Reddi, (1981) 
Proc. Natl. Acad. Sci. USA 78:7599-7603) . This 
provides an experimental method for assaying protein 
05 extracts for their ability to induce endochondral bone 
in vivo. Several species of mammals produce closely 
related protein as demonstrated by cross species 
implant experiments (Sampath and Reddi (1983) Proc. 
Natl. Acad. Sci. USA 80:6591-6595) . 

10 The potential utility of these proteins has been 

recognized widely- It is contemplated that the 
availability of the protein would revolutionize . 
orthopedic medicine, certain types of plastic surgery, 
and various periodontal and craniofacial reconstructive 

15 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 

20 activity. The current state of the art of purification 
of osteogenic protein from mammalian bone is disclosed 
by Sampath et al. ((1987) Proc. Natl. Acad. Sci. USA 
84; 7109-7113). Urist et al. (1984) Proc. Soc. Exp. 
Biol. Med. 173 : 194-199 disclose a human osteogenic 

25 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 

30 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. 



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Urist et al. (1984) Prnr. Natl. Arari. Sci. USA 81: 
371-375 disclose a bovine bone morphogenetic protein 
extract having the properties of an acidic polypeptide 
and a molecular weight of approximately 18 kD. Tne 

05 authors reported that the protein was present in a 
fraction separated by hydroxyapatite chromat °^P^; 
and that it induced bone formation in m0 ^% hl f q ^ r ^ 
raU scle and bone regeneration in trephine defects in rat 
and dog skulls. Their method of obtaining the extract 

. 10 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 

15human 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 

20 animals . 

international Application Ho. PCT/087/01537 , 
published January 14, 1988, discloses an impure 
fraction from bovine bone which has bone 
qualities. The named applicants also disclose putative 

26 -bone inductive factors- produced by recombinant DHA 
techniques. Four DHA sequences were retrieved from 
human or bovine genomic or cDHA libraries and expressed 
in recombinant host cells. While the applicants stated 
that the expressed protein, may be bone morphogeny 

,„proteins, bone induction was not demonstrated, 
suggesting that the recombinant proteins are not 
osteogenic. The same group reported 
(Science, 242.1528, Dec, 1988) that three of the four 



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factors induce cartilage formation , and postulate that 
bone formation activity "is due to a mixture of 
regulatory molecules" and that "bone formation is most 
likely controlled . . . by the interaction of these 
05 molecules. " Again, no bone induction was attributed to 
the products of expression of the cDNAs. See also 
Urist et al., EP0,212 / 474 entitled Bone Morphogenic 
Agents . 

Wang et al. (1988) Froc. Nat. Acad. Sci. USA 85: 
10 9484-9488 discloses the purification of a bovine bone 
morphogenetic protein from guanidine extracts of 
demineralized bone having cartilage and bone formation 
activity as a basic protein corresponding to a 
molecular weight of 30 kD determined from gel elution. 
15 Purification of the protein yielded proteins of 30, 18 
and 16 kD which, upon separation, were inactive. In 
view of this result, the authors acknowledged that the 
exact identity of the active material had not been 
determined. 

20 wang et al. (1990) Froc. Nat. Acad. Sci. USA 87: 
2220-2227 describes the expression and partial 
purification of one of the cDNA sequences described in 
PCT 87/01537. Consistent cartilage and/or bone 
formation with their protein requires a minimum of 600 

25 ng of 50% pure material. 

International Application No. PCT/89/04458 
published April 19, 1990 (Int. Pub. No. WO90/003733) , 
describes the purification and analysis of a family of 
osteogenic factors called "P3 OF 31-34". The protein 
30 family contains at least four proteins, which are 
characterized by peptide fragment sequences. The 
impure mixture P3 OF 31-34 is assayed for osteogenic 



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05 activity. The activity of the individual proteins is 
neither assessed nor discussed. 

It is an object of this invention to provide novel 
polypeptide chains useful as subunits of diraeric 
osteogenic proteins capable of endochondral bone 

10 formation in allogenic and xenogenic implants in 
mammals, including humans. Another object is to 
provide genes encoding these polypeptide chains and 
methods for the production of osteogenic proteins 
comprising these polypeptide chains using recombinant 

15 DNA techniques, as well as to provide antibodies 
capable of binding specifically to these proteins. 

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



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Summary of the Invention 

This invention provides novel polypeptide chains 
useful as either one or both subunits of dimeric 
osteogenic proteins which/ when implanted in a 
05 mammalian body in association with a matrix , can induce 
at the locus of the implant the full developmental 
cascade of endochondral bone formation and bone marrow 
differentiation. 

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

Consensus DNA sequences based on partial sequence 
data and observed homologies with regulatory proteins 
disclosed in the literature were used as probes for 

25 extracting genes encoding osteogenic protein from human 
genomic and cDNA libraries. One of the consensus 
sequences was used to isolate a previously unidentified 
gene which, when expressed, encoded a protein 
comprising a region capable of inducing endochondral 

3° bone formation when properly modified, incorporated in 
a suitable matrix, and implanted as disclosed herein. 
The gene, called "hOPl" or "OP-1" (human OP-1), is 



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described in greater detail in copending U.S. 422,699, 
the disclosure of which is herein incorporated by 
reference. 

In its native form, hOPl expression yields an 
05 immature translation product ("hOPl-PP", where "PP" 
refers to "prepro form") of about 400 amino acids that 
subsequently is processed to yield a mature sequence of 
139 amino acids ("OP1-18"). The active region 
(functional domain) of the protein comprises the 
10 C-terminal 97 amino acids of the hOPl sequence ("OPS"). 
A long active sequence is OP7 (comprising the 
C-terminal 102 amino acids). 

Further probing of mammalian cDNA libraries (human 
and mouse) with sequences specific to hOPl also has 

15 identified novel OPl-like sequences herein referred to 
as "OP2" ("hOP2" or "mOP2"). The OP2 proteins share 
significant amino acid sequence homology, approximately 
74%, with the active region of the OPl proteins (e.g., 
OP7 ) , and less homology with the intact mature form 

20 (e.g., OP1-18, 58% amino acid homology). 

The amino acid sequence of the osteogenic proteins 
disclosed herein also share significant homology with 
various of the regulatory proteins on which the 
consensus probe was modeled. In particular, the 

25 proteins share significant homology in their C-terminal 
sequences, which comprise the active region of the 
osteogenic proteins. (Compare, for example, OP7 with 
DPP from Drosophila and Vgl from Xenopus. See, for 
example, U.S. Pat. No. 5,011,691). In addition, these 

30 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 



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the different proteins.) See, for example, OP7, whose 
sequence defines the seven cysteine skeleton, or OPS, 
whose sequence defines the six cysteine skeleton. The 
OP2 proteins also contain an additional cysteine 
05 residue within this region. 

Thus, in one preferred aspect, the invention 
comprises osteogenic proteins comprising a polypeptide 
chain comprising an amino acid sequence described by 
Seq. ID No. 3 or 5, including allelic and species 

10 variants thereof, and naturally-occurring or 

biosynthetic mutants, such that a dimeric protein 
comprising this polypeptide chain has a conformation 
capable of inducing endochondral bone formation when 
implanted in a mammal in association with a suitable 

15 matrix. Useful proteins include the full-length 
protein, mature proteins and truncated proteins 
comprising the functional domain described by the 
C-terminal . 

in addition, the invention is not limited to thse 
20 specific constructs. Thus, the osteogenic proteins of 
this invention comprising any of these polypeptide 
chains may include forms having varying glycosylation 
patterns, varying N-termini, a family of related 
proteins having regions of amino acid sequence homology 

25 which may be naturally occurring or biosynthetically 
derived, and active truncated or mutated forms of the 
native amino acid sequence, produced by expression of 
recombinant DNA in procaryotic or eucaryotic host 
cells. Active squances useful as osteogenic proteins 

30 of this invention are envisioned to include proteins 
capable of inducing endochondral bone formation when 
implanted in a mammal in association wiht a matrix and 
having at lest a 70% sequence homology, preferably at 



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least 80%, with the amino acid sequence of OPS. This 
includes longer forms of a given protein, as well as 
allelic variants and rauteins, including addition and 
deletion mutants, such as those which may alter the 
05 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. 

10 The novel polypeptide chains and the osteogenic 
\roteins they comprise can be expressed from intact or 
truncated cDNA or from synthetic DNAs in procaryotic or 
eucaryotic host cells, and then purified, cleaved, 
refolded, dimerized, and implanted in experimental 

15 animals. Currently preferred host cells include E^olx 
or mammalian cells, such as CHO, COS or BSC cells. The 
osteogenic protein of the invention may include forms 
having varying glycosylation patterns, varying N- 
termini, a family of related proteins having regions of 

20 amino acid sequence homology, and active truncated or 
mutated forms of native or biosynthetic proteins, 
produced by expression of recombinant DNA in host 
cells. 

Thus, in view of this disclosure, skilled genetic 
25 engineers can isolate genes from cDNA or genomic 

libraries of various different species which encode 
appropriate amino acid sequences, or construct DNAs 
from oligonucleotides, and then can express them in 
various types of host cells, including both procaryotes 
30 and eucaryotes, to produce large quantities of active 
proteins capable of inducing bone formation in mammals 
including humans. In view of this disclosure, those 
skilled in the art, using standard immunology 



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techniques also may create antibodies capable of 
binding specifically to the osteogenic proteins 
disclosed herein, including fragments thereof. 

The osteogenic proteins are useful in clinical 
05 applications in conjunction with a suitable delivery or 
support system (matrix). The matrix is made up of 
particles of porous materials- The pores must be of a 
dimension to permit progenitor cell migration and 
subsequent differentiation and proliferation. The 
10 particle size should be within the range of 70 - 850 
mm, preferably 150mm - 420mm. It may be fabricated by 
close packing particulate material into a shape 
spanning the bone defect, or by otherwise structuring 
as desired a material that is biocompatible (non- 
15 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, 
20 guanidine extracted, species-specific (allogenic) bone, 
and specially treated particulate, protein extracted, 
demineralized, xenogenic bone. Optionally, such 
xenogenic bone powder matrices also may be treated with 
proteases such as trypsin and/or fibril modifying 
25 agents to increase the intraparticle intrusion volume 
and surface area. Useful agents include solvents such 
as dichloromethane, trichloroacetic acid, acetonitrile 
and acids such as trif luoroacetic acid and hydrogen 
fluoride. Alternatively, the matrix may be treated 
30 with a hot aqueous medium having a temperature within 
the range of about 37°C to 75°C, including a heated 
acidic aqueous medium. Other potentially useful matrix 
materials comprise collagen, homopolymers and 
copolymers of glycolic acid and lactic acid/ 



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05 



10 



hydroxyapatite, tricalcium phosphate and other calcium 
phosphates . 

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

Brief Description of t he Drawing 

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

FIGURE 1 compares the amino acid sequences of the 
mature mOP-2 and hOP-2 polypeptide chains: hOP2-A and 
mOP2-A; and 

25 FIGURE 2 compares the amino acid sequences 

of the mature OP1 and OP2 polypeptide chains: OP1-18, 
mOPl-S, hOP2-A and mOP2-A. 



15 



20 



Description 



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Purification protocols first were developed which 
enabled isolation of the osteogenic protein present in 
crude protein extracts from mammalian bone. (See PCT 
WO 89/09787, published 19-OCT-89, and U.S. Serial No. 

05 179,406 filed April 8, 1988, now U.S. Patent No. 

4,968,950). The development of the procedure, coupled 
with the availability of fresh calf bone, enabled 
isolation of substantially pure bovine osteogenic 
protein (bOP). bOP was characterized significantly; 

10 its ability to induce cartilage and ultimately 

endochondral bone growth in cat, rabbit, and rat were 
demonstrated and studied; it was shown to be able to 
induce the full developmental cascade of bone formation 
previously ascribed to unknown protein or proteins in 

15 heterogeneous bone extracts. This dose dependent and 
highly specific activity was present whether or not the 
protein was glycosylated (see Sampath et al., (1990) J± 
Biol. Chem . 265 ; 13198-13205). Sequence data obtained 
from the bovine materials suggested probe designs which 

20 were used to isolate human genes. The OP human 
counterpart proteins have now been expressed and 
extensively characterized. 

These discoveries enabled preparation of DNAs 
encoding totally novel, non-native protein constructs 

25 which individually as homodimers and combined with 

other species as heterodimers are capable of producing 
true endochondral bone (see PCT WO 09788, published 19- 
OCT-89, and US Serial No. 315,342, filed 23-FEB-89, now 
U.S. Patent No. 5,011,691). They also permitted 

30 expression of the natural material, truncated forms, 
muteins, analogs, fusion proteins, and various other 
variants and constructs, from cDNAs and genomic DNAs 
retrieved from natural sources or from synthetic DNA 
produced using the techniques disclosed herein and 



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05 



- 13 - 



using automated, commercially available equipment. The 
DNAs may be expressed using well established molecular 
biology and recombinant DNA techniques in procaryotic 
or eucaryotic host cells, and may be oxidized and 
refolded in vitro if necessary, to produce biologically 
active protein. 



One of the DNA sequences isolated from human 
genomic and cDNA libraries encoded a previously 
unidentified gene, referred to herein as OP1. The 

10 protein encoded by the isolated DNA was identified 

originally by amino acid homology with proteins in the 
TGF-p family. Consensus splice signals were found 
where amino acid homologies ended, designating exon- 
intron boundaries. Three exons were combined to obtain 

15 a functional TGF-p-like domain containing seven 
cysteines. (See, for example, U.S. Patent No. 
5,011,691, or Ozkaynak, E. et al., (1990) EMBO. 9: 
2085-2093) . 

The full-length cDNA sequence for hOPl, and its 
20 encoded "prepro" form "hOPl-PP," which includes an N- 
terminal signal peptide sequence, are disclosed in Seq. 
ID No. 1 (residues 1-431). The mature form of the hOPl 
protein expressed in mammalian cells, "0P1-18", is 
described by amino acid residues 293-431 of Seq. ID 
25 No. 1. The full length form of hOPl, as well as 

various truncated forms of the gene, and fused genes, 
have been expressed in E. coli and numerous mammalian 
cells (see, for example, published PCT application WO 
91/05802, published 2-MAY-91) and all have been shown 
30 to have osteogenic activity when implanted in a mammal 
in association with a suitable matrix. 



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Given the foregoing amino acid and DNA sequence 
information, various nucleic acids (RNAs and DNAs) can 
be constructed which encode at least the active region 
of the hOPl protein (e.g., OPS or OP7) and various 

05 analogs thereof (including allelic and species variants 
and those containing genetically engineered mutations), 
as well as fusion proteins, truncated forms of the 
mature proteins, and similar constructs. Moreover, DNA 
hybridization probes can be constructed from fragments 

10 of the hOPl DNA or designed de novo based on the hOPl 
DNA or amino acid sequence. These probes then can be 
used to screen different genomic and cDNA libraries to 
identify additional osteogenic proteins. 

The DNAs can be produced by those skilled in the 
15 art using well known DNA manipulation techniques 

involving genomic and cDNA isolation, construction of 
synthetic DNA from synthesized oligonucleotides, and 
cassette mutagenesis techniques. 15-100mer 
oligonucleotides may be synthesized on a Biosearch DNA 
20 Model 8600 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 
25 which may also be purified by PAGE. 

DNAs used as hybridization probes may be labelled 
(e.g., as with a radioisotope, by nick- translation) and 
used to identify clones in a given library containing 
DNA to which the probe hybridizes, following techniques 
30 well known in the art. The libraries may be obtained 
commercially or they may constructed de novo using 
conventional molecular biology techniques. Further 
information on DNA library construction and 



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hybridization techniques can be found in numerous texts 
known to those skilled in the art. See, for example, 
F.M. Ausubel., ed., Current Protocols in Molecular 
Bioloqv-Vol. 1 , (1989). In particular, see unit 5, 
05 "Construction of Recombinant DNA Libraries" and Unit 6, 
"Screening of Recombinant Libraries." 

The DNA from appropriately identified clones then 
can be isolated, subcloned (preferably into an 
expression vector), and sequenced. Plasmids containing 

10 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 

15 protein's osteogenic activity. Useful host cells 

include E. coli, Saccharomyces , the insect/baculovirus 
cell system, myeloma cells, and various mammalian 
cells. The vector additionally may encode various 
sequences to promote correct expression of the 

20 recombinant protein, including transcription promoter 
and termination sequences, enhancer sequences, 
preferred ribosome binding site sequences, preferred 
raRNA leader sequences, preferred signal sequences for 
protein secretion, and the like. The DNA sequence 

25 encoding the gene of interest also may be manipulated 
to remove potentially inhibiting sequences or to 
minimize unwanted secondary structure formation. The 
recombinant osteogenic protein also may be expressed as 
a fusion protein. After being translated, the protein 

30 may be purified from the cells themselves or recovered 
from the culture medium. All biologically active 
protein forms comprise dimeric species joined by 
disulfide bonds or otherwise associated, produced by 
oxidizing and refolding one or more of the various 



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recombinant polypeptide chains within an appropriate 
eucaryotic cell or in vitro after expression of 
individual subunits. A detailed description of 
osteogenic protein expressed from recombinant DNA in E. 
05 cgli is disclosed in U.S. Serial No. 660,162, filed 27- 
FEB-91, the disclosure of which incorporated by 
reference herein. A detailed description of osteogenic 
protein expressed from recombinant DNA in numerous 
different mammalian cells is disclosed in PCT 
10 WO91/05802, also incorporated herein by reference. 

Finally, in view of the disclosure made herein, and 
using standard methodologies known in the art, persosn 
skilled inthe art can raise polyclonal and monoclonal 
antibodies against all or part of a polypeptide chain 
disclosed herein, such that the antibodies are capable 
of binding specifically to an epitope on the 
polypeptide chain. Useful protocols can be found in, 
for example, Molecular C loina-A Laboratory Manual 
(Sambrook et al. eds., Cold Spring Harbor Press 2nd ed. 
1989). See Book 3, Section 18. 



15 



20 



25 



30 



Exemplification 

in an effort to identify additional DNA sequences 
encoding osteogenic proteins, a hybridization probe 
specific to the C-terminus of the DNA of mature OP-1 
was prepared using a StuI-EcoRl digest fragment of OP-1 
(base pairs 1034-1354 in Sequence ID No. 1), and 
labelled with 32 P by nick translation, as described in 
the art. As disclosed supra, the OPl C-terminus 
encodes a key functional domain, e.g., the "active 
region" for osteogenic activity. The C-terminus also 
is the region of the protein whose amino acid sequence 
shares specific amino acid sequence homology with 



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particular proteins in the TGF-f3 super- family of 
regulatory proteins, and which includes the conserved 
cysteine skeleton. 

Approximately 7 x 10 5 phages of an oligo(dT) primed 
05 17.5 days p.c. mouse embryo 5* stretch cDNA (gtlO) 

library (Clonetech, Inc., Palo Alto, CA) was screened 
with the labelled probe. The screen was performed 
using the following stringent hybridization conditions: 
40% formamide, 5 x SSPE, 5 x Denhart's solution, 0.1% 
10 SDS, at 37°C overnight, and washing in 0.1 x SSPE, 0.1% 
SDS at 50 °C. 

Five recombinant phages were purified over three 
rounds of screening. Phage DNA was prepared from all 
five phages, subjected to an EcoRl digest, subcloned 
15 into the EcoRl site of a common pUC-type plasmid 
modified to allow single strand sequencing, and 
sequenced using means well known in the art. 

Two different DNAs were identified by this 
procedure. One DNA, referred to herein as mOPl, has 

20 substantial homology to the mature form of OP1 (about 
98%), and is described in detail in copending USSN 
600,024, filed 18-Oct-90. A second DNA, encoding the 
C-terminus of a related gene and referred to herein as 
mOP2, also was identified by this procedure. The 

25 N-terminus of the gene encoding mOP2 was identified 
subsequently by screening a second mouse cDNA library 
(Mouse PCC4 cDNA (ZAP) library, Stratagene, Inc., La 
Jolla, CA) . 



30 



Mouse OP2 (mOP2) protein shares significant amino 
acid sequence homology with the amino acid sequence of 
the hOPl active region, e.g., OPS or OP7, about 74% 



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homology , and less homology with the intact mature 
form, e.g., OP1-18, about 58% homology. The cDNA 
sequence, and the encoded amino acid sequence, for the 
full length mOP-2 protein is depicted in Sequence ID 

05 No. 3. The full-length form of the protein is referred 
to as the prepro form of mOP-2 ("mOP2-PP"), and 
includes a signal peptide sequence at its N-terminus. 
The amino acid sequence Leu- Ala-Leu -Cys-Ala-Leu (amino 
acid residues 13-18 of Sequence ID No. 3) is believed 

10 to constitute the cleavage site for the removal of the 
signal peptide sequence, leaving an intermediate form 
of the protein, the "pro" form, to be secreted from the 
expressing cell. The amino acid sequence Arg-Ala-Pro- 
Arg-Ala (amino acid residues 255-259 of Sequence ID 

15 No. 3) is believed to constitute the cleavage site that 
produces the mature form of the protein, herein 
referred to as ,! mOP2-A", and described by residues 259- 
397 of Seq. ID No. 3. Residues 301-397 of Seq. ID 
No. 3 correspond to the region defining the conserved 

20 six cysteine skeleton. Residues 296-397 of Seq. ID 
No. 3 correspond to the region defining the conserved 
seven cysteine skeleton. 

Using a probe prepared from the pro region of mOP2 
(an EcoRl-BamHl digest fragment, bp 467-771 of Sequence 
25 ID No. 3), a human hippocampus library was screened 
(human hippocampus cDNA lambda (ZAP II library 
Stratagene, Inc., La Jolla, CA) following essentially 
the same procedure as for the mouse library screens. 
The procedure identified the N-terminus of a novel DNA 
2 0 encoding an amino acid sequence having substantial 
homology with mOP2. The C-terminus of the gene 
subsequently was identified by probing a human genomic 
library (in lambda phage EMBL-3, Clonetech, Inc., Palo 
Alto, CA) with a labelled fragment from the novel human 



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DNA in hand. The novel polypeptide chain encoded by 
this DNA is referred to herein as hOP2 protein, and 
shares almost complete amino acid identity (about 92% 
amino acid sequence homology) with mOP2-A (see Fig. 1 
05 and infra ) . 

The cDNA sequencer and the encoded amino acid 
sequence, for the prepro form of hOP2, "hOP2-PP", is 
described in Sequence ID No. 5. This full-length form 
of the protein also includes a signal peptide sequence 

10 at its N- terminus. The amino acid sequence Leu-Ala- 
Leu-Cys-Ala-Leu (amino acid residues 13-18 of Sequence 
ID No. 5) is believed to constitute the cleavage site 
for the removal of the signal peptide sequence, leaving 
an intermediate form of the protein, the "pro" form, to 

15 be secreted from the expressing cell. The amino acid 
sequence Arg-Thr-Pro-Arg-Ala (amino acid residues 257- 
261 of Sequence ID No. 5) is believed to constitute the 
cleavage site that produces what is believed to be the 
mature form of the protein, herein referred to as 

20 hOP2-A" and described by residues 261-399 of Seq. ID 
No. 5. 

Additional mature species of hOP2 thought to be 
active include truncated sequences, "hOP2-P" (described 
by residues 264-399 of Seq. ID No. 5) and "hOP2-R" 

25 (described by residues 267-399 of Seq. ID No. 5), and a 
slightly longer sequence ("hOP2-S", described by 
residues 240-399 of Seq. ID No. 5). Residues 303-399 
of Seq. ID No. 5 correspond to the region defining the 
conserved six cysteine skeleton. Residues 297-399 of 

30 Seq. ID No. 5 correspond to the region defining the 
conserved seven cystein skeleton. 



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It should be noted that the nucleic acid sequence 
encoding the N-terminus of the prepro form of both mOP2 
and hOP2 is rich in guanidine and cytosine base pairs. 
As will be appreciated by those skilled in the art, 

05 sequencing such a "G-C rich" region can be problematic, 
due to stutter and/or band compression. Accordingly, 
the possibility of sequencing errors in this region can 
not be ruled out. However, the definitive amino acid 
sequence for these and other, similarly identified 

10 proteins can be determined readily by expressing the 
protein from recombinant DNA using, for example, any of 
the means disclosed herein, and sequencing the 
polypeptide chain by conventional peptide sequencing 
methods well known in the art. 

15 Figure 1 compares the amino acid sequences of 
mature mOP2 and hOP2. Identity is indicated by three 
dots (...) in the mOP2 sequence. As is evident from 
the figure, the amino acid sequence homology between 
the mature forms of these two proteins is substantial 

20 (92% homology between the mature sequences, about 95% 
homology within the C-terminal active region (e.g., 
residues 38-139 or 42-139 of Fig. 1.) 

Fig. 2 compares the amino acid sequences for 
the mature forms of all four species of OPl and OP2 

25 proteins. Here again, identity is indicated by three 
dots (...)• Like the mOP2 protein, the hOP2 protein 
shares significant homology (about 74%) with the amino 
acid sequence defining the OPl active region (OPS or 
OP7, residues 43-139 and 38-139, respectively, in 

30 Fig. 2), and less homology with OP1-18 (about 58% 

homology). Both OP2 proteins share the conserved seven 
cysteine skeleton seen in the OPl proteins. In 



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addition, the OP2 proteins comprise an eighth cysteine 
residue within this region (see position 78 in FIG. 2). 

A preferred generic amino acid sequence useful 
5 as a subunit of a dimeric osteogenic protein capable of 
inducing endochondral bone or cartilage formation when 
implanted in a mammal in association with a matrix, and 
which incorporates the maximum homology between the 
identified OP1 and OP2 proteins, can be described by 
10 the sequence referred to herein as "OPX", described 
below and in Seq. No, 7. 

Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe 
1 5 10 

15 Xaa Asp Leu Gly Trp Xaa Asp Trp Xaa He 

15 20 
Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys 

25 30 
Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser 
20 35 40 

Xaa Met Asn Ala Thr Asn His Ala He Xaa 

45 50 
Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa 

55 60 
25 Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr 

65 70 
Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 

75 80 
Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys 
30 85 90 

Xaa Arg Asn Met Val Val Xaa Ala Cys Gly 

95 100 

Cys His, 



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and wherein Xaa at res. 2 = (Lys or Arg); Xaa at 
res. 3 = (Lys or Arg); Xaa at res. 9 = (Ser or Arg); 
Xaa at res. 11 = (Arg or Gin); Xaa at res. 16 - (Gin or 
m Leu); Xaa at res. 19 - (He or Val); Xaa at res. 23 « 
5 (Glu or Gin); Xaa at res. 26 = (Ala or Ser); Xaa at 
res. 35 = (Ala or Ser); Xaa at res. 39 ■ (Asn or Asp); 
Xaa at res. 41 = (Tyr or Cys); Xaa at res. 50 = (Val or 
Leu); Xaa at res. 52 - (Ser or Thr); Xaa at res. 56 = 
(Phe or Leu); Xaa at res. 57 * (He or Met); Xaa at 

10 res. 58 = (Asn or Lys); Xaa at res. 60 = (Glu, Asp or 
Asn); Xaa at res. 61 = (Thr, Ala or Val); Xaa at 
res. 65 = (Pro or Ala); Xaa at res. 71 = (Gin or Lys); 
Xaa at res. 73 = (Asn or Ser); Xaa at res. 75 = (He or 
Thr); Xaa at res. 80 = (Phe or Tyr); Xaa at res. 82 = 

15 (Asp or Ser); Xaa at res. 84 = (Ser or Asn); Xaa at 
res. 87 = (He or Asp); Xaa at res. 89 = (Lys or Arg); 
Xaa at res. 91 = (Tyr, Ala or His); and Xaa at res. 97 
= (Arg or Lys). 



20 The high degree of homology exhibited between the 

various OP1 and OP2 proteins suggests that the novel 
osteogenic proteins identified herein will purify 
essentially as OP1 does, or with only minor 
modifications of the protocols disclosed for OP1. 

25 Similarly, the purified mOPl, mOP2, and hOP2 proteins 
are predicted to have an apparent molecular weight of 
about 18 kDa as reduced single subunits, and an 
apparent molecular weight of about 36 kDa as oxidized 
dimers, as determined by comparison with molecular 

30 weight standards on an SDS-polyacrylamide 

electrophoresis gel. Unglycosylated dimers (e.g., 
proteins produced by recombinant expression in E. coli) 
are predicted to have an apparent molecular weight of 
about 27 kDa. There appears to be one potential N 



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10 



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glycosylation site in the mature forms of the mOP2 and 
hOF2 proteins. 

The identification of osteogenic proteins having an 
active region comprising eight cysteine residues also 
allows one to construct osteogenic polypeptide chains 
patterned after either of the following template amino 
acid sequences, or to identify additional osteogenic 
proteins having this sequence. The template sequences 
contemplated are "OPX-7C", comprising the conserved six 
cysteine skeleton plus the additional cysteine residue 
identified in the OP2 proteins, and "OPX-8C", 
comprising the conserved seven cysteine skeleton plus 
the additional cysteine residue identified in the OP2 
proteins. The OPX-7C and OPX-8C sequences are 
15 described below and in Seq. ID Nos. 8 and 9, 

respectively. Each Xaa in these template sequences 
independently represents one of the 20 naturally- 
occurring L-isomer, a-amino acids, or a derivative 
thereof. Biosynthetic constructs patterned after this 
20 template readily are constructed using conventional DNA 
synthesis or peptide synthesis techniques well known in 
the art. Once constructed, osteogenic proteins 
comprising these polypeptide chains can be tested as 
disclosed herein. 

25 "OPX-1C (Sequence ID No. 8): 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

1 5 10 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

15 20 

30 xaa Xaa Cys Xaa Xaa xaa Cys xaa xaa Xaa Xaa 

25 30 

Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa xaa Xaa Xaa 



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35 40 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
45 50 55 

Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa 
05 60 65 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

70 " 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
80 85 
10 xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 

90 95 

"OPX-8C" (Sequence ID No. 9 comprising additional five 
residues at the N-terminus, including a conserved 
cysteine residue): 

15 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

1 5 10 

Xaa xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

15 20 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 
20 25 30 

Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 
35 40 45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

50 55 
xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 



25 



30 



60 *5 
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

80 85 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

90 95 
Xaa Cys Xaa 



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05 



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100 

MATRIX PREPARATION 
A. General Consideration of Matrix Properties 

The currently preferred carrier material is a 
xenogenic bone-derived particulate matrix treated as 
disclosed herein. This carrier may be replaced by 
either a biodegradable-synthetic or synthetic- inorganic 
matrix (e.g., hydroxy lapatite (HAP), collagen, 
tricalcium phosphate or polylactic acid, polyglycolic 
10 acid and various copolymers thereof.) 

Studies have shown that surface charge, particle 
size, the presence of mineral, and the methodology for 
combining matrix and osteogenic protein all play a role 
in achieving successful bone induction. Perturbation 

15 of the charge by chemical modification abolishes the 
inductive response. Particle size influences the 
quantitative response of new bone; particles between 
75 fjm and 420 /um elicit the maximum response. 
Contamination of the matrix with bone mineral will 

20 inhibit bone formation. Most importantly, the 

procedures used to formulate OP onto the matrix are 
extremely sensitive to the physical and chemical state 
of both the osteogenic protein and the matrix. 



25 



The sequential cellular reactions in the 
interface of the bone matrix/osteogenic protein 
implants are complex. The multistep cascade includes: 
binding of fibrin and fibronectin to implated matrix, 
chemotaxis of cells, proliferation of fibroblasts, 
differentiation into chondroblasts , cartilage 



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formation, vascular invasion, bone formation, 
remodeling, and bone marrow differentiation. 

A successful carrier for osteogenic protein 
must perform several important functions. It must bind 

05 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 

10 preferably 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 in vivo . In bones, the dissolution rates can 

15 vary according to whether the implant is placed in 
cortical or trabecular bone. 

Matrix geometry, particle size, the presence 
of surface charge, and the degree of both intra-and- 
inter-particle porosity are all important to successful 
20 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. 



25 



30 



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 allogenic bone implants can act as a carrier for 
the matrix if their marrow cavities are cleaned and 



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packed with particle and the dispersed osteogenic 
protein. 

The preferred matrix material, prepared from 
xenogenic bone and treated as disclosed herein, 

05 produces an implantable material useful in a variety of 
clinical settings. In addition to its use as a matrix 
for bone formation in various orthopedic, periodontal, 
and reconstructive procedures, the matrix also may be 
used as a sustained release carrier, or as a 

10 collagenous coating for implants. The matrix may be 
shaped as desired in anticipation of surgery or shaped 
by the physician or technician during surgery. Thus, 
the material may be used for topical, subcutaneous, 
intraperitoneal, or intramuscular implants; it may be 

15 shaped to span a nonunion fracture or to fill a bone 
defect. In bone formation or conduction procedures, 
the material is slowly absorbed by the body and is 
replaced by bone in the shape of or very nearly the 
shape of the implant. 

20 Various growth factors, hormones, enzymes, 

therapeutic compositions, antibiotics, and other body 
treating agents also may be absorbed onto the carrier 
material and will be released over time when implanted 
as the matrix material is slowly absorbed. Thus, 

25 various known growth factors such as EGF, PDGF, IGF, 
FGF, TGF-a, and TGF-fl may be released in vivo . The 
material can be used to release chemotherapeutic 
agents, insulin, enzymes, or enzyme inhibitors. 

B. Bone-Derived Matrices 

1. Preparation of Demineralized Bone 



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Demineralized bone matrix, preferably bovine 
bone matrix, is prepared by previously published 
procedures (Sampath and Reddi (1983) Proc. Natl. Acad. 
Sci. USA 80:6591-6595). Bovine diaphyseal bones (age 
05 1-10 days) are obtained from a local slaughterhouse and 
used fresh. The bones are stripped of muscle and fat, 
cleaned of periosteum, demarrowed by pressure with cold 
water, dipped in cold absolute ethanol, and stored at 
-20 °C. They are then dried and fragmented by crushing 
10 and pulverized in a large mill. Care is taken to 
prevent heating by using liquid nitrogen. The 
pulverized bone is milled to a particle size in the 
range of 70-850 fjm, preferably 150-420 fjm, and is 
defatted by two washes of approximately two hours 
15 duration with three volumes of chloroform and methanol 
(3:1). The particulate bone is then washed with one 
volume of absolute ethanol and dried over one volume of 
anhydrous ether yielding defatted bone powder. The 
defatted bone powder is then demineralized by four 
20 successive treatments with 10 volumes of 0.5 N HC1 at 
4°C for 40 min. Finally, neutralizing washes are done 
on the demineralized bone powder with a large volume of 
water. 



2. Guanidine Extraction 

Demineralized bone matrix thus prepared is 
extracted with 5 volumes of 4 M guanidine-HCl, 50mM 
Tris-HCl, pH 7.0 for 16 hr. at 4°C. The suspension is 
filtered. The insoluble material is collected and used 
to fabricate the matrix. The material is mostly 
collagenous in nature. It is devoid of osteogenic or 
chondrogenic activity. 



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3. Matrix Treatments 

The major component of all bone matrices is 
Type-I collagen. In addition to collagen, 
demineralized bone extracted as disclosed above 

05 includes non-collagenous proteins which may account for 
5% of its mass. In a xenogenic matrix, these 
noncollagenous components may present themselves as 
potent antigens, and may constitute immunogenic and/or 
inhibitory components. These components also may 

10 inhibit osteogenesis in allogenic implants by 

interfering with the developmental cascade of bone 
differentiation. It has been discovered that 
treatment of the matrix particles with a collagen 
fibril-modifying agent extracts potentially unwanted 

15 components from the matrix, and alters the surface 
structure of the matrix material. Useful agents 
include acids, organic solvents or heated aqueous 
media. Various treatments are described below. A 
detailed physical analysis of the effect these fibril- 

20 modifying agents have on demineralized, quanidine- 
extracted bone collagen particles is disclosed in PCT 
WO 90/10018, published 7-SEP-90. 

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

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



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2. Centrifuge and repeat wash step; and 

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

3.1 Acid Treatments 

°5 1. Trifluoroacetic acid. 

Trifluoroacetic acid is a strong non-oxidizing 
acid that is a known swelling agent for proteins, and 
which modifies collagen fibrils. 

Bovine bone residue prepared as described 
10 above is sieved, and particles of the appropriate size 
are collected. These particles are extracted with 
various percentages (1.0% to 100%) of trifluoroacetic 
acid and water (v/v) at 0°C or room temperature for 1-2 
hours with constant stirring. The treated matrix is 
15 filtered, lyophilized, or washed with water/salt and 
then lyophilized. 

2. Hydrogen Fluoride. 

Like trifluoroacetic acid, hydrogen fluoride 
is a strong acid and swelling agent, and also is 

20 capable of altering intraparticle surface structure. 
Hydrogen fluoride is also a known deglycosylating 
agent. As such, HF may function to increase the 
osteogenic activity of these matrices by removing the 
antigenic carbohydrate content of any glycoproteins 

25 still associated with the matrix after guanidine 
extraction. 



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Bovine bone residue prepared as described above is 
sieved, and particles of the appropriate size are 
collected. The sample is dried in vacuo over P 2 °5' 
transferred to the reaction vessel and exposed to 

05 anhydrous hydrogen fluoride (10-20 ml/g of matrix) by 
distillation onto the sample at -70°C. The vessel is 
allowed to warm to 0°C and the reaction mixture is 
stirred at this temperature for 120 minutes. After 
evaporation of the hydrogen fluoride in vacuo, the 

10 residue is dried thoroughly in vacuo over ROH pellets 
to remove any remaining traces of acid. Extent of 
deglycosylation can be determined from carbohydrate 
analysis of matrix samples taken before and after 
treatment with hydrogen fluoride, after washing the 

15 samples appropriately to remove non-covalently bound 
carbohydrates. SDS-extracted protein from HF-treated 
material is negative for carbohydrate as determined by 
Con A blotting. 

The deglycosylated bone matrix is next washed 
20 twice in TBS (Tr is -buffered saline) or UTBS, water- 
washed, and then lyophilized. 

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

3.2 Solvent Treatment 



1 . Dichloromethane . 



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Dichloromethane (DCM) is an organic solvent 
capable of denaturing proteins without affecting their 
primary structure. This swelling agent is a common 
reagent in automated peptide synthesis, and is used in 
05 washing steps to remove components. 

Bovine bone residue, prepared as described 
above, is sieved, and particles of the appropriate size 
are incubated in 100% DCM or, preferably, 99.9% 
DCM/0.1% TFA. The matrix is incubated with the 
10 swelling agent for one or two hours at 0°C or at room 
temperature. Alternatively, the matrix is treated with 
the agent at least three times with short washes (20 
minutes each) with no incubation. 



2 . Acetonitrile . 



15 Acetonitrile (ACN) is an organic solvent, 

capable of denaturing proteins without affecting their 
primary structure. It is a common reagent used in 
high-performance liquid chromatography, and is used to 
elute proteins from silica-based columns by perturbing 

20 hydrophobic interactions. 

Bovine bone residue particles of the 
appropriate size, prepared as described above, are 
treated with 100% ACN (1.0 g/30 ml) or, preferably, 
99.9% ACN/0.1% TFA at room temperature for 1-2 hours 
25 with constant stirring. The treated matrix is then 
water-washed, or washed with urea buffer, or 4 M NaCl 
and lyophilized. Alternatively, the ACN or ACN/TFA 
treated matrix may be lyophilized without wash. 

3. Isopropanol. 



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Isopropanol is also an organic solvent capable 
of denaturing proteins without affecting their primary 
structure. It is a common reagent used to elute 
proteins from silica HPLC columns. 

05 

Bovine bone residue particles of the 
appropriate size prepared as described above are 
treated with 100% isopropanol (1.0 g/30 ml) or, 
preferably, in the presence of 0.1% TFA, at room 
temperature for 1-2 hours with constant stirring. The 
io matrix is then water-washed or washed with urea buffer 
or 4 M NaCl before being lyophilized. 

4 . Chloroform 

Chloroform also may be used to increase 
surface area of bone matrix like the reagents set forth 
15 above, either alone or acidified. 

Treatment as set forth above is effective to 
assure that the material is free of pathogens prior to 
implantation. 

3.3 Heat Treatment 

20 The currently most preferred agent is a heated 

aqueous fibril -modifying medium such as water, to 
increase the matrix particle surface area and porosity. 
The currently most preferred aqueous medium is an 
acidic aqueous medium having a pH of less than about 

25 4.5, e.g., within the range of pH 2 - pH 4 . which may 
help to "swell" the collagen before heating. 0.1% 
acetic acid, which has a pH of about 3, currently is 
preferred. 0.1 M acetic acid also may be used. 



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Various amounts of delipidated, demineralized 
guanidine-extracted bone collagen are heated in the 
aqueous medium (lg matrix/30ml aqueous medium) under 
constant stirring in a water jacketed glass flask, and 

05 maintained at a given temperature for a predetermined 
period of time. Preferred treatment times are about 
one hour, although exposure times of between about 0.5 
to two hours appear acceptable. The temperature 
employed is held constant at a temperature generally 

10 within the range of about 37 °C to 75 °C. The currently 
preferred heat treatment temperature is within the 
range of 45°C to 60°C. 

After the heat treatment, the matrix is filtered, 
washed, lyophilized and used for implant. Where an 

15 acidic aqueous medium is used, the matrix also is 
preferably neutralized prior to washing and 
lyophilization. A currently preferred neutralization 
buffer is a 200mM sodium phosphate buffer, pH 7.0. To 
neutralize the matrix, the matrix preferably first is 

20 allowed to cool following thermal treatment, the acidic 
aqueous medium (e.g., 0.1% acetic acid) then is removed 
and replaced with the neutralization buffer and the 
matrix agitated for about 30 minutes. The 
neutralization buffer then may be removed and the 

25 matrix washed and lyophilized (see infra). 

The matrix also may be treated to remove 
contaminating heavy metals, such as by exposing the 
matrix to a metal ion chelator. For example, following 
thermal treatment with 0.1% acetic acid, the matrix may 
30 be neutralized in a neutralization buffer containing 
EDTA (sodium ethylenediaminetetraacetic acid), e.g., 
200 mM sodium phosphate, 5mM EDTA, pH 7.0. 5 mM EDTA 
provides about a 100-fold molar excess of chelator to 



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residual heavy metals present in the most contaminated 
matrix tested to date. Subsequent washing of the 
matrix following neutralization appears to remove the 
bulk of the EDTA. EDTA treatment of matrix particles 
05 reduces the residual heavy metal content of all metals 
tested (Sb, As, Be, Cd, Cr, Cu, Co, Pb, Hg, Ni, Se, Ag, 
Zn, Tl) to less than about 1 ppm. Bioassays with EDTA- 
treated matrices indicate that treatment with the metal 
ion chelator does not inhibit bone inducing activity. 

10 The collagen matrix materials preferably take 

the form of a fine powder, insoluble in water, 
comprising nonadherent particles. It may be used 
simply by packing into the volume where new bone growth 
or sustained release is desired, held in place by 

15 surrounding tissue. Alternatively, the powder may be 
encapsulated in, e.g., a gelatin or polylactic acid 
coating, which is adsorbed readily by the body. The 
powder may be shaped to a volume of given dimensions 
and held in that shape by interadhering the particles 

20 using, for example, soluble, species-biocompatible 
collagen. The material may also be produced in sheet, 
rod, bead, or other macroscopic shapes. 

Demineralized rat bone matrix used as an 
allogenic matrix in certain of the experiments 

25 disclosed herein, is prepared from several of the 

dehydrated diaphyseal shafts of rat femur and tibia as 
described herein to produce a bone particle size which 
passes through a 420 pm sieve. The bone particles are 
subjected to dissociative extraction with 4 M 

30 guanidine-HCl. Such treatment results in a complete 
loss of the inherent ability of the bone matrix to 
induce endochondral bone differentiation. The 
remaining insoluble material is used to fabricate the 



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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 osteogenic activity before use. The total 
loss of biological activity of bone matrix is restored 
when an active osteoinductive protein fraction or a 
pure osteoinductive protein preparation is 
reconstituted with the biologically inactive insoluble 
collagenous matrix. 

FABRICATION OF OSTEOGENIC DEVICE 



The naturally sourced and recombinant protein as 
set forth above, and other constructs, can be combined 
and dispersed in a suitable matrix preparation using 
any of the methods described below. In general, 50-100 
15 ng of active protein is combined with the inactive 
carrier matrix (e.g., 25 mg for rat bioassays). 
Greater amounts may be used for large implants. 

1. Ethanol Precipitation 

Matrix is added to osteogenic protein 
20 dissolved in guanidine-HCl. Samples are vortexed and 
incubated at a low temperature (e.g., 4°C). Samples 
are then further vortexed. Cold absolute ethanol (5 
volumes) is added to the mixture which is then stirred 
and incubated, preferably for 30 minutes at -20°C. 
25 After centrifugation (microfuge, high speed) the 

supernatant is discarded. The reconstituted matrix is 
washed twice with cold concentrated ethanol in water 
(85% EtOH) and then lyophilized. 

2. Acetonitrile Trifluoroacetic 



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Acid Lyophilization 

In this procedure, osteogenic protein in an 
acetonitrile trif luroacetic acid (ACN/TFA) solution is 
added to the carrier material. Samples are vigorously 
05 vortexed many times and then lyophilized. This method 
is currently preferred, and has been tested with 
osteogenic protein at varying concentrations and 
different levels of purity. 

3. Urea Lyophilization 

10 For those osteogenic proteins that are 

prepared in urea buffer, the protein is mixed with the 
matrix material, vortexed many times, and then 
lyophilized. The lyophilized material may be used "as 
is" for implants. 

15 4. Buffered Saline Lyophilization 

OP1 and OP2 preparations in physiological 
saline may also be vortexed with the matrix and 
lyophilized to produce osteogenically active material. 

These procedures also can be used to adsorb 

20 

other active therapeutic drugs, hormones, and various 
bioactive species to the matrix for sustained release 
purposes . 

BIOASSAY 

The functioning of the various proteins and 
25 devices of this invention can be evaluated with an in 
vivo bioassay. Studies in rats show the osteogenic 
effect in an appropriate matrix to be dependent on the 



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05 



10 



dose of osteogenic protein dispersed in the matrix. No 
activity is observed if the matrix is implanted alone. 
In vivo bioassays performed in the rat model also have 
shown that demineralized, guanidine-extracted xenogenic 
bone matrix materials of the type described in the 
literature are ineffective as a carrier, fail to induce 
bone, and produce an inflammatory and immunological 
response when implanted unless treated as disclosed 
above. In certain species (e.g., monkey) allogenic 
matrix materials also apparently are ineffective as 
carriers. The following sets forth various procedures 
for preparing osteogenic devices from the proteins and 
matrix materials prepared as set forth above, and for 
evaluating their osteogenic utility. 

15 A. Rat Model 
1 • Implantation 

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

20 used to monitor endochondral bone differentiation 
activity. This assay consists of implanting test 
samples in subcutaneous sites in recipient rats under 
ether anesthesia. Male Long-Evans rats, aged 28-32 
days, were used. A vertical incision (1 cm) is made 

25 under sterile conditions in the skin over the thoracic 
region, and a pocket is prepared by blunt dissection. 
Approximately 25 mg of the test sample is implanted 
deep into the pocket and the incision is closed with a 
metallic skin clip. The day of implantation is 

30 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 



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ambiguities resulting from the use of brthotropic 
sites. As disclosed herein, both allogenic (rat bone 
matrix) and xenogenic (bovine bone matrix) implants 
were assayed. 

05 2. Cellular Events 

Successful implants exhibit a controlled 
progression through the stages of protein- induced 
endochondral bone development, including: (1) transient 
infiltration by polymorphonuclear leukocytes on day 

10 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) cartilage calcification on day eight; 
(6) vascular invasion, appearance of osteoblasts, and 

15 formation of new bone on days nine and ten; (7) 
appearance of osteoblastic and bone remodeling and 
dissolution of the implanted matrix on days twelve to 
eighteen; and (8) hematopoietic bone marrow 
differentiation in the ossicle on day twenty-one. The 

20 results show that the shape of the new bone conforms to 
the shape of the implanted matrix. 

3. Histological Evaluation 

Histological sectioning and staining is 
preferred to determine the extent of osteogenesis in 

25 the implants. Implants are fixed in Bouins Solution, 
embedded in paraffin, and cut into 6-8 pra sections. 
Staining with toluidine blue or hemotoxylin/eosin 
demonstrates clearly the ultimate development of 
endochondral bone. Twelve day implants are usually 

30 sufficient to determine whether the implants contain 
newly induced bone. 



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4. Biological Markers 

Alkaline phosphatase activity may be used as a 
marker for osteogenesis. The enzyme activity may be 
determined spectrophotometrically after homogenization 

05 of the implant. The activity peaks at 9-10 days in 
vivo and thereafter slowly declines. Implants showing 
no bone development by histology have little or no 
alkaline phosphatase activity under these assay 
conditions. The assay is useful for quantitation and 

10 obtaining an estimate of bone formation quickly after 
the implants are removed from the rat. Alternatively, 
the amount of bone formation can be determined by 
measuring the calcium content of the implant. 

The invention may be* embodied in other specific 
15 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 
20 than by the foregoing description, and all changes 

which come within the meaning and range of equivalency 
of the claims are therefore intended to be embraced 
therein. 



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SEQUENCE LISTING 



(1) GENERAL 



INFORMATION: 



(i) 



APPLICANT: OPPERMANN, HERMANN 
OZKAYNAK, ENGIN 
KUBERASAMPATH , THANGAVEL 
RUEGER, DAVID C. 



05 



(ii) TITLE OF INVENTION: OSTEOGENIC DEVICES 
(iii) NUHBER OF SEQUENCES: 9 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: TESTA, HURWITZ & THIBEAULT 

(B) STREET: 53 STATE STREET 

(C) CITY: BOSTON 

(D) STATE: MASSACHUSETTS 

(E) COUNTRY: U.S.A. 

(F) ZIP: 02109 

(v) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

(C) OPERATING SYSTEM: PC-DOS/MS-DOS 

(D) SOFTVARE: Patentln Release #1.0, Version #1.25 

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUHBER: 

(B) FILING DATE: 

(C) CLASSIFICATION: 

(viii) ATTORNEY/AGENT INFORMATION: 

(A) NAME: PITCHER, EDMUND R. 

(B) REGISTRATION NUMBER: 27,829 

(C) REFERENCE/DOCKET NUMBER: CRR056PC 

(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 617/248-7000 

(B) TELEFAX: 617/248-7100 



(2) INFORMATION FOR SEQ ID N0:1: 

(i) SEQUENCE CHARACTERISTICS: 
. (A) LENGTH: 1822 base pairs 

(B) TYPE: nucleic acid 

(C) STRAND EDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 



(iii) HYPOTHETICAL: NO 



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10 



(iv) ANTI- SENSE: NO 

(vi) ORIGINAL SOURCE: 

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

05 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 49.. 1341 

(C) IDENTIFICATION METHOD: experimental 

(D) OTHER INFORMATION: /function- "OSTEOGENIC PROTEIN" 
/product- "hOPl-PP" 

/evidence- EXPERIMENTAL 
/standard_name= "hOPl" 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 
15 Met His Val 

1 

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

20 CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153 
Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 
20 25 30 35 

GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 201 
Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser Gin Glu Arg 
25 40 45 50 

CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG CCC CAC CGC 249 
Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg 
55 60 65 

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

CTG GAC CTG TAC AAC GCC ATG -GCG GTG GAG GAG GGC «5C GGG CCC GGC 345 
Leu Asp Leu Tyr Asn Ala Het Ala Val Glu Glu Gly Gly Gly Pro Gly 
85 90 95 

35 GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC 393 
Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly 
100 105 110 115 

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



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120 



125 



130 



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

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



489 



537 



CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CGG ATC TAC AAG GAC 585 
Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp 
10 165 170 175 

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

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

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

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

GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 
Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro 
25 245 250 255 

AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG CAG CCC 
Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro 
260 265 270 275 

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

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

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

AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GAG CTG TAT GTC AGC TTC 1065 
Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 
40 325 330 335 



633 



681 



729 



777 



825 



873 



921 



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05 CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GAA GGC TAC GCC 1113 
Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala 
3*0 345 350 7 355 

GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CCT CTG AAC TCC TAC ATG 1161 
Ala Tyr. Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 
10 360 365 370 

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

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

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

20 TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351 
Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
«0 425 430 

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 

25 TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 

CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 

30 CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 

(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 

(ix) FEATURE: 



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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

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

Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 
10 20 25 30 

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

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

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

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

Gly Pro Gly Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 
20 100 105 110 

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

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

25 Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 
1« 150 155 160 

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

Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He 
30 180 185 190 

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

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

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

His Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser 
245 250 255 

He Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn 



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05 260 265 270 

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

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

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

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

Val Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu 
15 340 345 350 

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

Ser Tyr Met Asn Ala Thr Asn His Ala lie Val Gin Thr Leu Val His 
370 375 380 

20 Phe He Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin 
385 390 395 400 

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

Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
25 420 425 430 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1929 base pairs 

(B) TYPE: nucleic acid 
30 (C) STRANDEDNESS : single 

(D TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(ix) FEATURE: 

(A) NAME/KEY: COS 

(B) LOCATION: 103.. 1293 

(D) OTHER INFORMATION: /function* "osteogenic protein" 
/product^ "m0P2-PP" 
/notes n m0P2 cDNA" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

GAATTCCGCT GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC 60 



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05 CCGACCAGCT ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT 114 

Met Ala Met Arg 
1 

CCC GGG CCA CTC TGG CTA TTG GGC CTT GCT CTG IGC GCG CTG GGA GGC 162 
Pro Gly Fro Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly 
10 5 10 15 20 

GGC CAC GGT CCC GGT CCC CCG CAC ACC TGT CCC CAG CGT CGC CTG GGA 210 
Gly His Gly Pro Gly Pro Pro His Thr Cys Pro Gin Arg Arg Leu Gly 
25 30 35 

GCG CGC GAC CGG GAC ATG CAG CGT GAA ATC CTG CCG GTG CTC GGG CTA 258 
15 Ala Arg Asp Arg Asp Met Gin Arg Glu lie Leu Pro Val Leu Gly Leu 
40 45 50 

CCG GGA CGC CCC GAC CCC GTG CAC AAC CCG CCG CTG CCC GGC ACG CAG 306 
Pro Gly Arg Pro Asp Pro Val His Asn Pro Pro Leu Pro Gly Thr Gin 
55 60 65 

20 CGT GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GCC ATG ACC GAT GAC 354 
Arg Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Met Thr Asp Asp 
70 75 80 

GAC GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CGT GCC GAC CTG GTC 402 
Asp Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg Ala Asp Leu Val 
25 85 90 95 100 

ATG AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT ACC CTG GGC TAC CAG 450 
Met Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr Leu Gly Tyr Gin 
105 110 115 

GAG CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA ACC CAG ATC CCT GCT 498 
30 Glu Pro His Trp Lys Glu Phe His Phe Asp Leu Thr Gin lie Pro Ala 
120 125 130 

GGG GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TAC AAA GAA CCC AGC 546 
Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Glu Pro Ser 
135 140 145 

ACC CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC ATG TTC GAA GTG GTC 594 
Thr His Pro Leu Asn Thr Thr Leu His He Ser Met Phe Glu Val Val 
150 155 160 

CAA GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG 642 
Gin Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin 
165 170 175 180 

ACG CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CTG GAC ATC ACA GCA 690 
Thr Leu Are Ser Gly Asp Glu Gly Trp Leu Val Leu Asp He Thr Ala 
185 190 195 

GCC AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GAC CTG GGA CTC CGC 738 



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05 Ala Ser Asp Arg Trp Leu Leu Asn His His Lys Asp Leu Gly Leu Arc 
200 205 210 

CTC TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GAT CCT GGC CTG GCT 786 
Leu Tyr Val Glu Thr Ala Asp Gly His Ser Met Asp Pro Gly Leu Ala 
215 220 225 

10 GGT CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CAG CCT TTC ATG GTA 834 

y ^il Leu Gly Ar 2 Gln na Pro Ar 8 Ser Ar 8 Gin Pro Phe Met Val 
230 235 240 

ACC TTC TTC AGG GCC AGC CAG AGT CCT GTG GGG GCC CCT CGG GCA GCG 882 

I,r Phe Phe Arg Ala Ser Gln Ser Pro Val Ar 8 Ala Pro Arg Ala Ala 
!5 245 250 255 260 

AGA CCA CTG AAG AGG AGG CAG CCA AAG AAA ACG AAC GAG CTT CCG CAC 930 
Arg Pro Leu Lys Arg Arg Gln Pro Lys Lys Thr Asn Glu Leu Pro His 
265 270 275 

CCC AAC AAA CTC CCA GGG ATC TTT GAT GAT GGC CAC GGT TCC CGC GGC 978 
Pro Asn Lys Leu Pro Gly lie Phe Asp Asp Gly His Gly Ser Arc Gly 
280 285 290 

AGA GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AGA TTC CGT GAC CTT 1026 
Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Arg Phe Arg Asp Leu 
295 300 305 

GGC TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TAC TCT GCC TAT TAC 1074 
Gly Trp Leu Asp Trp Val lie Ala Pro Gln Gly Tyr Ser Ala Tyr Tvr 
310 315 320 



20 



25 



30 



35 



TGT 
Cys 
325 


GAG 
Glu 


GGG 
Gly 


GAG 
Glu 


TGT 
Cys 


GCT TTC 
Ala Phe 
330 


CCA 
Pro 


CTG 
Leu 


GAC 
Asp 


TCC TGT ATG 
Ser Cys Met 
335 


AAC GCC ACC 
Asn Ala Thr 
340 


1122 


AAC 
Asn 


CAT 
His 


GCC 
Ala 


ATC 
He 


TTG 
Leu 
345 


CAG TCT 
Gln Ser 


CTG 
Leu 


GTG 
Val 


CAC 
His 
350 


CTG ATG AAG 
Leu Met Lys 


CCA GAT GTT 
Pro Asp Val 
355 


1170 


GTC 
Val 


CCC 
Pro 


AAG 
Lys 


GCA 
Ala 
360 


TGC 
Cys 


TGT GCA 
Cys Ala 


CCC 
Pro 


ACC 
Thr 
365 


AAA 
Lys 


CTG AGT GCC 
Leu Ser Ala 


ACC TCT GTG 
Thr Ser Val 
370 


1218 


CTG 
Leu 


TAC 
Tyr 


TAT 
Tyr 
375 


GAC 
Asp 


AGC 
Ser 


AGC AAC 
Ser. Asn 


AAT 
Asn 
380 


GTC 
Val 


ATC 
lie 


CTG CGT AAA 
Leu Arg Lys 
385 


CAC CGT AAC 
His Arg Asn 


1266 


ATG 
Met 


GTG 
Val 
390 


GTC 
Val 


AAG 
Lys 


GCC 
Ala 


TGT GGC 
Cys Gly 
395 


TGC 
Cys 


CAC 
His 


TGAGGCCCCG CCCAGCATCC 


1313 



TGCTTCTACT ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT 1373 



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TATCATAGCT CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA 1433 

AAATTCTGGT CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC 1493 

CTCTCCATCC TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA 1553 

ACTGAGAGGT CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC 1613 

CTCAGCCCAC AATGGCAAAT TCTGGATGGT CTAAGAAGCC CTGGAATTCT AAACTAGATG 1673 

ATCTGGGCTC TCTGCAGCAT TCATTGTGGC AGTTGGGACA TTTTTAGGTA TAACAGACAC 1733 

ATACACTTAG ATCAATGCAT CGCTGTACTC CTTGAAATCA GAGCTAGCTT GTTAGAAAAA 1793 

GAATCAGAGC CAGGTATAGC GGTGCATGTC ATTAATCCCA GCGCTAAAGA GACAGAGACA 1853 

GGAGAATCTC TGTGAGTTCA AGGCCACATA GAAAGAGCCT GTCTCGGGAG CAGGAAAAAA 1913 

AAAAAAAACG GAATTC 1929 

(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 397 amino acids 

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

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(D) OTHER INFORMATION: /Product* n mOP2-PP n 

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

Met Ala Met Arg Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
15 10 15 

Ala Leu Gly Gly Gly His Gly Pro Gly Pro Pro His Thr Cys Pro Gin 



20 



25 



30 



Arg Arg 



Leu Gly Ala Arg Asp Arg Asp Met Gin Arg Glu He Leu Pro 
35 40 45 



Val Leu 
50 



Gly Leu Pro Gly Arg Pro Asp Pro Val His Asn Pro Pro Leu 



55 60 



Pro Gly 
65 



Thr Gin Arg Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala 
70 75 80 



Met Thr 



Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg 
85 90 95 



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- 50 - 



05 Ala Asp Leu Val Met Ser Phe Val Asn Het Val Glu Arg Asp Arg Thr 
100 105 110 

Leu Gly Tyr Gin Glu Pro His Trp Lys Glu Phe His Phe Asp Leu Thr 
115 120 125 

Gin He Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Are He Tyr 
10 130 135 140 



15 



Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His He Ser Het 
1« 150 155 160 

Phe Glu Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe 
165 170 175 

Leu Asp Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu 
180 185 190 

Asp lie Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His Lys Asp 
195 200 205 

Leu Gly Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser Het Asp 
20 210 215 220 

Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser Arg Gin 
225 230 235 240 

Pro Phe Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala 
245 250 255 

25 Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn 
260 265 270 

Glu Leu Pro His Pro Asn Lys Leu Pro Gly He Phe Asp Asp Gly His 
275 280 285 

Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Arg 
30 290 295 300 

Phe Arg Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr 
305 310 315 320 

Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys 
325 330 335 

35 Het Asn Ala Thr Asn His Ala He Leu Gin Ser Leu Val His Leu Het 
340 345 350 

Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser 
355 360 365 

Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg 
370 375 380 



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05 Lys His Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
385 390 395 

(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS : 
(A) LENGTH: 1941 base pairs 
10 (B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

15 (vi) ORIGINAL SOURCE: 

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

(ix) FEATURE: 

(A) NAME/KEY: CDS 
20 (B) LOCATION: 507.. 1703 

(D) OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" 
/product= "h0P2-PP" 
/note= "hOP2 (CDNA)" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

25 GGAATTCCGG CCACAGTGGC GCCGGCAGAG CAGGAGTGGC TGGAGGAGCT GTGGTTGGAG 60 

CAGGAGGTGG CACGGCAGGG CTGGAGGGCT CCCTATGAGT GGCGGAGACG GCCCAGGAGG 120 

CGCTGGAGCA ACAGCTCCCA CACCGCACCA AGCGGTGGCT GCAGGAGCTC GCCCATCGCC 180 

CCTGCGCTGC TCGGACCGCG GCCACAGCCG GACTGGCGGG TACGGCGGCG ACAGAGGCAT 240 

TGGCCGAGAG TCCCAGTCCG CAGAGTAGCC CCGGCCTCGA GGCGGTGGCG TCCCGGTCCT 300 

30 CTCCGTCCAG GAGCCAGGAC AGGTGTCGCG CGGCGGGGCT CCAGGGACCG CGCCTGAGGC 360 

CGGCTGCCCG CCCGTCCCGC CCCGCCCCGC CGCCCGCCGC CCGCCGAGCC CAGCCTCCTT 420 

GCCGTCGGGG CGTCCCCAGG CCCTGGGTCG GCCGCGGAGC CGATGCGCGC CCGCTGAGCG 480 

CCCCAGCTGA GCGCCCCCGG CCTGCC ATG ACC GCG CTC CCC GGC CC<5 CTC TGG 533 

Met Thr Ala Leu Pro Gly Pro Leu Trp 
35 15 

CTC CTG GGC CTG GCG CTA TGC GCG CTG GGC GGG GGC GGC CCC GGC CTG 58 i 

Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gly Leu 
10 15 20 25 

CGA CCC CCG CCC GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GAC CGG 629 



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05 Arg Pro Pro Pro Gly Cys Pro Gin Arg Arg Leu Gly Ala Arg Asp Arg 

30 35 40 

GAC GTG CAG CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GGG CGG CCC 
Asp Val Gin Arg Glu lie Leu Ala Val Leu Gly Leu Pro Gly Arg Pro 
45 50 55 

10 CGG CCC CGC GCG CCA CCC GCC GCC TCC CGG CTG CCC GCG TCC GCG CCG 
Arg Pro Arg Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro 
60 65 70 



30 



35 



40 



CTC TTC ATG CTG GAC CTG TAC CAC CGC ATG GCC GGC GAC GAC GAC GAG 
Leu Phe Het Leu Asp Leu Tyr His Arg Met Ala Gly Asp Asp Asp Glu 
15 o 80 85 

GAC GGC GCC GCG GAG GCC CTG GGC CGC GCC GAC CTG GTC ATG AGC TTC 
'* y Ala Ala Glu Ala Leu G1 y Ar S Ala Asp Leu Val Met Ser Phe 

50 95 100 105 

on M tiS ?? rf CGA GAC CGT GCC 600 CAG GAG CCC CAT 
20 Val Asn Met Val Glu Arg Asp Arg Ala Leu Gly His Gin Glu Pro His 

H° 115 120 

TGG AAG GAG TTC CGC TTT GAC CTG ACC CAG ATC CCG GCT GGG GAG GCG 
Trp Lys Glu Phe Arg Phe Asp Leu Thr Gin lie Pro Ala Gly Glu Ala 
125 130 135 

25 Sf i£t GCT ? CG ^ G £ C CGG ATT TAC GTG C CC AGC ATC CAC CTG 
Val Thr Ala Ala Glu Phe Arg He Tyr Lys Val Pro Ser He His Leu 

140 145 150 



CTC AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GTG GTC CAG GAG CAG 
Leu Asn Arg Thr Leu His Val Ser Met Phe Gin Val Val Gin Glu Gin 
155 160 165 

TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG CTC CGA 
Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr Leu Arg 
1/0 175 180 185 

GCT GGA GAC GAG GGC TGG CTG GTG CTG GAT GTC ACA GCA GCC AGT GAC 
Ala Gly Asp Glu Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp 
190 195 200 

TGC TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CTC CGC CTC TAT GTG 
Cys Trp Leu Leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val 
205 210 215 

GAG ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CTG GCC GGC CTG CTG 
Glu Thr Glu Asp Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu 
220 225 230 

GGT CAA CGG GCC CCA CGC TCC CAA CAG CCT TTC GTG GTC ACT TTC TTC 
Gly Gin Arg Ala Pro Arg Ser Gin Gin Pro Phe Val Val Thr Phe Phe 



677 



725 



773 



821 



869 



917 



965 



1013 



1061 



1109 



1157 



1205 



1253 



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05 235 240 245 

AGG GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GCA GTG AGG CCA CTG 1301 
Arg Ala Ser Pro Ser Pro He Arg Thr Pro Arg Ala Val Arg Pro Leu 
250 255 260 265 

AGG AGG AGG CAG CCG AAG AAA AGC AAC GAG CTG CCG CAG GCC AAC CGA 1349 
10 Arg Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg 

270 275 280 

CTC CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CAC GGC CGG CAG GTC 1397 
Leu Pro Gly He Phe Asp Asp Val His Gly Ser His Gly Arg Gin Val 
285 290 295 

15 TGC CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GAC CTC GGC TGG CTG 1445 
Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu 
300 305 310 

GAC TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TAT TAC TGT GAG GGG 1493 
Asp Trp Val lie Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly 
20 315 320 325 

GAG TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT GCC ACC AAC CAC GCC 1541 
Glu Cys Ser Phe Pro Leu Asp Ser Cys Het Asn Ala Thr Asn His Ala 
330 335 340 345 

ATC CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA AAC GCA GTC CCC AAG 1589 
25 He Leu Gin Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys 

350 355 360 

GCG TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC TCT GTG CTC TAC TAT 1637 
Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr 
365 370 375 

30 GAC AGC AGC AAC AAC GTC ATC CTG CGC AAA GCC CGC AAC ATG GTG GTC 1685 
Asp Ser Ser Asn Asn Val He Leu Arg Lys Ala Arg Asn Met Val Val 
380 385 390 

AAG GCC TGC GGC TGC CAC TGAGTCAGCC CGCCCAGCCC TACTGCAGCA 1733 
Lys Ala Cys Gly Cys His 
35 395 

ATTCACTGGC CGTCGTTTTA CAACGTGTGA CTGGGAAAAC CCTGGCGTTA CCCAACTTAA 1793 

TCGCCTTGCA GCACATCCCC CTTTCGCCAG CTGGCTAATA GCGAAGAGGC CCCGCACCGA 1853 

TCGCCCTTCC CAACAGTTGC GCCCCAGTGA ATGGCGAATG GCAAATTGTA AGCGTTAATA 1913 

TTTTGTTAAA ATTCGCGTTA AATTTTTT 1941 



(2) INFORMATION FOR SEQ ID NO: 6: 



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05 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 399 amino acids 

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

(ii) MOLECULE TYPE: protein 

10 (ix) FEATURE: 

(D) OTHER INFORMATION: /product- "hOP2-PP" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
15 10 15 

i5 Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro 
20 25 30 

Gin Arg Arg Leu Gly Ala Arg Asp Arg Asp Val Gin Arg Glu lie Leu 
35 40 45 

Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Pro Pro Ala 
20 50 55 60 

Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr 
65 70 75 80 

His Arg Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Ala Glu Ala Leu 
85 90 95 

25 Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val Glu Arg Asp 
100 105 110 

Arg Ala Leu Gly His Gin Glu Pro His Trp Lys Glu Phe Arg Phe Asp 
115 120 125 

Leu Thr Gin He Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg 
30 130 135 140 

He Tyr Lys Val Pro Ser He His Leu Leu Asn Arg Thr Leu His Val 
145 150 155 160 

Ser Met Phe Gin Val Val Gin Glu Gin Ser Asn Arg Glu Ser Asp Leu 
165 170 175 

35 Phe Phe Leu Asp Leu Gin Thr Leu Arg Ala Gly Asp Glu Gly Trp Leu 
180 185 190 

Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu Lys Arg His 
195 200 205 

Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp Gly His Ser 
210 215 220 



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05 Val Asp Pro Gly Leu Ala Gly Leu Leu Gly Gin Arg Ala Pro Are Ser 
225 230 235 240 

Gin Gin Pro Phe Val Val Thr Phe Phe Arg Ala Ser Pro Ser Pro lie 

245 250 255 

Arg Thr Pro Arg Ala Val Arg Pro Leu Arg Arg Arg Gin Pro Lys Lys 
10 260 265 270 

Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu Pro Gly He Phe Asp Asp 
275 280 285 

Val His Gly Ser His Gly Arg Gin Val Cys Arg Arg His Glu Leu Tyr 
290 295 300 

15 Val Ser Phe Gin Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin 
305 310 315 320 

Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp 
325 330 335 

Ser Cys Met Asn Ala Thr Asn His Ala lie Leu Gin Ser Leu Val His 
20 340 345 350 

Leu Met Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys 
355 360 365 

Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He 
370 375 380 

25 Leu Arg Lys Ala Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
385 390 395 

(2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 
30 (A) LENGTH: 102 amino acids 

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

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 
35 (A) NAME/KEY: Protein 

(B) LOCATION: 1..102 
(D) OTHER INFORMATION: /label- OPX 

/note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED 
FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS 
AS DEFINED IN THE SPECIFICATION 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 



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Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 
15 10 15 

Asp Trp Xaa He Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 
5 20 25 30 

Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 
35 40 45 

10 He Xaa Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 

50 55 60 



15 



Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 
65 70 75 80 

Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 
85 90 95 



Xaa Ala Cys Gly Cys His 
20 100 

(2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 
25 (A) LENGTH: 97 amino acids 

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



30 



(ii) MOLECULE TYPE: protein 



(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..97 

35 (D) OTHER INFORMATION: /label- 0PX-7C 

/note* "WHEREIN EACH XAA INDEPENDENTLY INDICATES 
ONE OF THE 20 NATURALLY-OCCURRING L- ISOMER, 
A-AMINO ACIDS, OR A DERIVATIVE THEREOF." 

40 

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

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
15 10 15 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 
20 25 30 

Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
50 35 40 45 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa 
50 55 60 



45 



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05 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

65 70 75 80 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 
85 90 95 

Xaa 

10 (2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

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

15 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /labels PROTEIN 
20 /note« "WHEREIN EACH XAA INDEPENDENTLY INDICATES 

ONE OF THE 20 NATURALLY-OCCURRING L- ISOMER A-AHINO 
AICDS, OR A DERIVATIVE THEREOF." 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
25 1 5 10 15 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 
20 25 30 

Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
35 40 45 

30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

50 55 60 

Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 70 75 80 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
85 90 95 

Xaa Xaa Cys Xaa Cys Xaa 
100 



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05 What is claimed is: 

1. A polypeptide chain comprising an amino acid 
sequence described by residues 303-399 of Seq. ID 
No. 5. 

2. The polypeptide chain of claim 1 comprising an 
10 amino acid sequence described by residues 297-399 of 

Seq. ID No. 5. 

3. The polypeptide chain of claim 2 comprising of 
amino acid sequence described by residues 267-399 of 
Seq. ID No. 5. 

15 4. The polypeptide chain of claim 3 comprising an 

amino acid sequence described by residues 264-399 of 
Seq. ID No. 5. 

5. The polypeptide chain of claim 4 comprising an 
amino acid sequence described by residues 240-399 of 

20 Seq. ID No. 5. 

6. The polypeptide chain of claim 5 comprising an 
amino acid sequence described by residues 1-399 of Seq. 
ID No. 5. 

7. A polypeptide chain comprising an amino acid 

25 sequence described by residues of 301-397 of Seq. ID 
No. 3. 

8. The polypeptide chain of claim 7 comprising an 
amino acid sequence described by residues 296-397 of 
Seq. ID No. 3. 



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- 59 - 



05 9. The polypeptide chain of claim 8 comprising an 

amino acid sequence described by residues 259-397 of 
Seg. ID No. 3. 

10. The polypeptide chain of claim 9 comprising an 
amino acid described by residues 1-397 of Seq. ID 

10 No. 3. 

11. A polypeptide chain useful as a subunit of a 
dimeric osteogenic protein comprising a pair of 
disulf ide-bonded polypeptide chains , said polypeptide 
chain having an amino acid sequence described by 

15 residues 303-399 of Seq. ID No. 5, including allelic 
and species variants thereof, such that the dimeric 
osteogenic protein comprising said polypeptide chain 
has a conformation capable of inducing endochondral 
bone formation when implanted in a mammal in 

20 association with a matrix. 

12. The polypeptide chain of claim 11 wherein said 
amino acid sequence comprises residues 261-399 of Seq. 
ID 5. 

13. The polypeptide chain of claim 11 wherein the 

25 amino acid sequence comprises residues 301-397 of Seq. 
ID No. 3. 

14. The polypeptide chain of claim 13 wherein said 
amino acid sequent comprises residues 259-397 of Seq. 
ID No. 3. 

30 15. A dimeric osteogenic protein capable of inducing 
endochondral bone formation in a mammal when implanted 
in said mammal in association with a matrix; 



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05 said protein comprising a pair of disul fide-bonded 

polypeptide chains constituting a dimeric species, 
wherein each said polypeptide chain is the polypeptide 
chain of claim 11. 

16. The polypeptide chain of claim 3 or 11 produced by 
10 expression of recombinant DNA in a host cell. 

17. The polypeptide chain of claim 16 wherein said 
host cell is a procaryotic host cell. 

18. The polypeptide chain of claim 16 wherein said 
host cell is a mammalian cell. 

15 19. The polypeptide of claim 1, 3 or 11 that is 
glycosylated. 

20. A nucleic acid encoding the polypeptide chain of 
claim 1, 3, or 11. 

21. A dimeric protein comprising a pair of polypeptide 
20 chains expressed from a DNA sequence described by ID 

No. 3 or ID No. 5, including allelic and species 
variants thereof, such that, when said polypeptide 
chains are oxidized to produce a disul fide-bonded 
dimeric species, the dimeric species has a conformation 
25 that is capable of inducing endochondral bone or 

cartilage formation when disposed within a matrix and 
implanted in a mammal. 



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1/5 



PCT/US91/07635 



hOP2 Ala Val Arg Pro Leu Arg Arg Arg 
mOP2 ... Ala Lys ..7 ..7 

5 



hOP2 Gin Pro Lys Lys Ser Asn Glu Leu 

mOP2 Thr 

10 15 



hOP2 Pro Gin Ala Asn Arg Leu Pro Gly 

mOP2 ... His Pro ... Lys ~ 

20 



hOP2 He Phe Asp Asp Val His Gly Ser 

mOP2 . . . Gly . ... 

25 30 



hOP2 His Gly Arg Gin Val Cys Arg Arg 
mOP2 Arg 7 Glu ... ... ..7 ..7 

35 40 



hOP2 His Glu Leu Tyr Val Ser Phe Gin 

mOP2 Arg . . . Arg 

45 



hOP2 Asp Leu Gly Trp Leu Asp Trp Val 
mOP2 . ... ... 

50 55 



hOP2 He Ala Pro Gin Gly Tyr Ser Ala 
mOP2 ... ... ... 

60 



hOP2 Tyr Tyr Cys Glu Gly Glu Cys Ser 

mOP2 . ... ... Ala 

65 70 



Fig. 1.1 



SUBSTITUTE SHEET 



WO 92/07073 



2/5 



PCT/US91/07635 



h 2£? Phe Pro Leu As P Ser Cys Met Asn 
mOP2 J 

75 80 



hOP2 Ala Thr Asn His Ala He Leu Gin 
mOP2 

85 



hOP2 Ser Leu Val His Leu Met Lys Pro 
mOP2 ■* 

90 95 

hOP2 Asn Ala Val Pro Lvs Ala Cys Cys 
mOP2 Asp Val 

100 

hOP2 Ala Pro Thr Lys Leu Ser Ala Thr 
mOP2 

105 iio 

hOP2 Ser Val Leu Tyr Tyr Asp Ser Ser 

mOP2 ".. 

115 ' i20 

hOP2 Asn Asn Val He Leu Arg Lys Ala 

mOP2 7 ... His 

125 



hOP2 Arg Asn Met Val Val Lys Ala Cys 
mOP2 ... J 

130 135 



hOP2 Gly Cys His 
mOP2 



Fig. 1.2 

SUBSTITUTE SHEET 



WO 92/07073 



3/5 



PCT/US91/07635 



hOPl 
mOPl 
hOP2 
mOP2 



hOP2 
mOP2 



Ser Thr Gly Ser Lys Gin Arg Ser Gin 

Gly 

Ala Val Arg Pro Leu Arg . . . Arg . . . 
Ala Ala Arg Pro Leu Lys . . . Arg '. . . 
1 5 



hOPl Asn Arg Ser Lys Thr Pro Lys Asn Gin 
mOPl 

hOP2 Pro Lys Lys Ser Asn Glu Leu Pro Gin 
mOP2 Pro Lys Lys Thr Asn Glu Leu Pro His 
10 15 



hOPl Glu Ala Leu Arg Met Ala Asn Val Ala 
mOPl Ser 

Ala Asn Arg Leu Pro Gly lie Phe Asp 
Pro Asn Lys Leu Pro Gly lie Phe Asp 
20 25 



hOPl Glu Asn Ser Ser Ser Asp Gin Arg Gin 

mOPl 

hOP2 Asp Val His Gly .' ! His Gly ! .' 

mOP2 Asp Gly His Gly . . . Arg Gly ... Glu 

30 35 



hOPl Ala Cys Lys Lys His Glu Leu Tyr Val 
mOPl 

hOP2 Val ... Arg Arg . . ! !.' .' ! ! \ [ 
mOP2 Val . . . Arg Arg 

40 45 



Fig. 2.1 



SUBSTITUTE SHEET 



WO 92/07073 4/5 PCT/US91/07635 



hOPl Ser Phe Arg Asp Leu Gly Trp Gin Asp 
mOPl #i . # ; # . /; 

hOP2 Gin ... ... Leu 

mOP2 Arg Leu ... 

10 



Trp He He Ala Pro Glu Gly Tyr Ala 



hOPl 
mOPl 

hOP2 ... val Gin Ser 

mOP2 ... Val Gin Ser 

55 60 



hOPl Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 

mOPl 

hop2 ; 

mOP2 



Ser 

65 70 



hOPl Phe Pro Leu Asn Ser Tyr Met Asn Ala 

mOPl 

hOP2 Asp Cys 

mOP2 



Asp . . . Cys 

75 80 



hOPl Thr Asn His Ala He Val Gin Thr Leu 
mOPl 

h °P2 Leu . . . Ser . . 

mOP2 Leu ... Ser ... 

85 90 



Fig. 2.2 



SUBSTITUTE SHEET 



WO 92/07073 



5/5 



PCT/US91/07635 



hOPl Val His Phe He Asn Pro Glu Thr Val 
mOPl Asp 

h0p 2 Leu Met Lys . . ! Asn Ala ! ! ! 

mOP2 Leu Met Lys . . . Asp Val . . . 

95 

hOPl Pro Lys Pro Cys Cys Ala Pro Thr Gin 

mOPl 

hOP2 Ala . . Lys 

niOP2 Ala Lvs 

100 105 

hOPl Leu Asn Ala lie Ser Val Leu Tyr Phe 
mOPl * 

hOP2 ... Ser ... Thr ... Tyr 
mOP2 . . . Ser . . . Thr Tyr 

110 115 

hOPl Asp Asp Ser Ser Asn Val He Leu Lys 

mOPl Asp 

hOP2 . . . Ser . . . Asn '. . Arg 

mOP2 . . . Ser . . . Asn Arg 

120 125 

hOPl Lys Tyr Arg Asn Met Val Val Arg 

mOPl . ... 

hOP2 ... Ala Lys 

mOP2 ... His Lys 

130 

hOPl Ala Cys Gly Cys His 

mOPl ... 

hOP2 ... 

mOP2 

135 

Fig. 2.3 

SUflRTITlltir cueer 



INTERNATIONAL SEARCH REPORT 

International Application No PCT/US 91/07(j>ft5 



1. CLASSinCATlON OF SUBJECT MATTER (if several cla ssification symbols apply, indicate alii* 
According to International Patent Classification (IPC) or to both National Classification and IPC 

Int. CI. 5 C 12 N 15/00 C 07 K 7/10 C 07 K 13/00 

A 61 K 37/02 A 61 K 27/00 



II. FIELDS SEARCHED 



Minimum Documentation Searched 7 



Classification System 



Classification Symbols 



Int.C1.5 



C 07 K 



A 61 K 



Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are Included in the Fields Searched 1 



HI. DOCUMENTS CONSIDERED TO BE RELEVANT 9 



Category 0 



Citation of Document, 11 with indication, where appropriate, of the relevant passages 12 



Relevant to Claim No. 13 



WO,A,8909788 (CREATIVE BI0M0LECULES) 
19 October 1989, see the whole dcument 



WO, A, 9011366 (GENETICS INSTITUTE) 4 
October 1990, see the whole document 



EMBO Journal, volume 9, no. 7, 1990, Oxford 
University Press (Eynsham, Oxford, GB) E. 
Ozkaynak et al . : "OP-l cDNA encodes an osteogenic 
protein in the TGF-beta familiy", pages 
2085-2093, see the whole article 



11,15- 
21 

1-10,12 
-14 

11,15- 
21 

11,15- 
21 

11,15- 
21 



6 Special categories of cited documents : 10 

'A* document defining the general state of the an which is not 
considered to be of particular relevance 

~E" earlier document but published on or after the International 
filing date 

"L* document which may throw doubts on priority daimfs) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

'O' document referring to an oral disclosure, use, exhibition or 
other means 

*P* document published prior to the international filing date but 
later than the priority date claimed 



T* later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

*X* document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step 

*Y* document of particular relevance; the claimed invention 
cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

*A* document member of the same patent family 



IV. CERTIFICATION 



Date of the Actual Completion of the International Search 

31-01-1992 



Date of Mailing of this International Search Report 




International Searching Authority 

EUROPEAN PATENT OFFICE 



Signature of Authorized Offi 



i PCT/JSA/210 <uco*d sheet) (Ja 



f IMS) 



ANNEX TO THE INTERNATIONAL SEARCH REPORT 

ON INTERNATIONAL PATENT APPLICATION NO. US 9107635 

SA 53017 

This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. 
The members are as contained in the European Patent Office EDP file on 12/02/92 

Toe European Patent Office is in no way liable for these particulars which are merely oven for the purpose of information. 



Patent document 


Publication 


Patent 


Family 


Publication 


cited in exarch rranrt 


date 


members) 


date 


m 

W0-A- 8909788 


19-10-89 


US-A- 


4968590 


06-11-90 






US-A- 


5011691 


30-04-91 






AU-A- 


3444989 


03-11-89 






AU-A- 


3530589 


03-11-89 






EP-A- 


0372031 


13-06-90 






EP-A- 


0362367 


11-04-90 






JP-T- 


3500655 


14-02-91 






JP-T- 


3502579 


13-06-91 






W0-A- 


8909787 


19-10-89 






AU-A- 


5174790 


26-09-90 






EP-A- 


0411105 


06-02-91 






JP-T- 


3504736 


17-10-91 






W0-A- 


9010018 


07-09-90 






US-A- 


4975526 


04-12-90 



WO-A- 9011366 04-10-90 AU-A- 5357790 22-10-90 

CA-A- 2030518 29-09-90 
EP-A- 0429570 05-06-91 



For more details about this annex : see Official Journal of the European Patent Office, No. 12/82 



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