Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


per 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International Patent Classification 5 : 

C07K 15/06, C12N 15/12 
A61K 37/02 



Al 



(11) International Publication Number: 
(43) International Publication Date: 



WO 92/07004 

30 April 1992(30.04.92) 



(21) Internationa] Application Number: PCT/US9 1/07654 

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



(30) Priority data: 
600,024 



18 October 1990 (18.10.90) US 



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

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

(72) Inventors: OZKAYNAK, Engin ; 44 Purdue Drive, Mil- 

ford, MA 01757 (US). OPPERMANN, Hermann ; 25 
Summer Hill Road, Medway, MA 02053 (US). KUBER- 
AS AM PATH, Thangavel ; 6 Spring Street, Medway, MA 
02053 (US). RUEGER, David, C. ; 150 Edgemere Road, 
Apt. 4, West Roxbury, MA 02132 (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 PROTEIN 



(57) Abstract 



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


MG 


Madagascar 


AU 


Australia 


Fl 


Finland 


ML 


Mali 


BB 


Barbados 


FR 


France 


MN 


Mongolia 


BE 


Belgium 


CA 


Gabon 


MR 


Mauritania 


BF 


Burkina Fa*o 


GB 


United Kingdom 


MW 


Malawi 


BG 


Bulgaria 


GN 


Guinea 


NL 


Netherlands 


BJ 


Benin 


GR 


Greece 


NO 


Norway 


BR 


Brazil 


HU 


Hungary 


PL 


Poland 


CA 


Canada 


IT 


Italy 


RO 


Romania 


CF 


Central African Republic 


JP 


Japan 


SD 


Sudan 


CG 


Congo 


KP 


Democratic People's Republic 


SB 


Sweden 


CH 


Switzerland 




of Korea 


SN 


Senegal 


CI 


C3die d'lvoirc 


KR 


Republic of Korea 


SU + 


Soviet Union 


CM 


Camcroon 


LI 


Liechtenstein 


ID 


Chad 


CS 


Czechoslovak i j 


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 92/07004 ^ PCT/US91/07654 



Osteogenic Protein 

Background of the Invention 

This invention relates to a novel polypeptide chain 
and to osteogenic proteins comprising this polypeptide 
chain which are capable of inducing osteogenesis in 
mammals, to a gene encoding the polypeptide chain, to 
methods for its production using recombinant DNA 
techniques, and to bone and cartilage repair procedures 
using the dosteogenic proteins. 

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

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

The developmental cascade of bone differentiation 
25 consists of recruitment of mesenchymal cells, 

proliferation of progenitor cells, calcification of 
cartilage, vascular invasion, bone formation, 
remodeling, and finally marrow differentiation (Reddi 
(1981) Collagen Rel. Res . 1^:209-226). 

30 

Though the precise mechanisms underlying these 
phenotypic transformations are unclear, it has been 
shown that the natural endochondral bone 



WO 92/07004 



PCT/US91/07654 



2 



dissociatively extracted and reconstituted with 
inactive residual collagenous matrix to restore full 
bone induction activity (Sampath and Reddi, (1981) 
Proc. Natl. Acad. Sci. USA 78:7599-7603). This 

05 provides an experimental method for assaying protein 
extracts for their ability to induce endochondral bone 
in vivo. Several species of mammals produce closely 
related protein as demonstrated by cross species 
implant experiments (Sampath and Reddi (1983) Proc. 

10 Natl. Acad. Sci. USA 80:6591-6595). 

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

The observed properties of these protein fractions 
have induced an intense research effort in various 
laboratories directed to isolating and identifying the 

2o pure factor or factors responsible for osteogenic 

activity. The current state of the art of purification 
of osteogenic protein from mammalian bone is disclosed 
by Sampath et al. (1987) Proc. Natl. Acad* Sci. USA 84 : 
7109-7113. Urist et al. (1984) Proc. Soc. Exp. Biol. 

25 Med. 173 : 194-199 disclose a human osteogenic protein 
fraction which was extracted from demineralized 
cortical bone by means of a calcium chloride-urea 
inorganic-organic solvent mixture, and retrieved by 
differential precipitation in guanidine-hydrochloride 

30 and preparative gel electrophoresis. The authors 
report that the protein fraction has an amino acid 
composition of an acidic polypeptide and a molecular 
weight in a range of 17-18 kD. 



WO 92/07004 PCT/US91/07654 

- 3 - 



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

05 authors reported that the protein was present in a 
fraction separated by hydroxyapatite chromatography, 
and that it induced bone formation in mouse hindquarter 
muscle and bone regeneration in trephine defects in rat 
and dog skulls. Their method of obtaining the extract 

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 

15 human origin. One of the proteins, designated by the 
inventors as a P3 protein having a molecular weight of 
22-24 kD, is said to have been purified to an 
essentially homogeneous state. This material is 
reported to induce bone formation when implanted into 

20 animals. 

International Application No. PCT/087/01537 , 
published January 14, 1988, discloses an impure 
fraction from bovine bone which has bone induction 
qualities. The named applicants also disclose putative 

25 "bone inductive factors" produced by . recombinant DNA 
techniques. Four DNA sequences were retrieved from 
human or bovine genomic or cDNA libraries and expressed 
in recombinant host cells. While the applicants stated 
that the expressed proteins may be bone morphogenic 

30 proteins, bone induction was not demonstrated, 
suggesting that the recombinant proteins are not 
osteogenic. The same group reported subsequently 



WO 92/07004 



PCT/US91/07654 



- 4 - 



(Science 242:1528, Dec, 1988) that three of the four 
factors induce cartilage formation, and postulate that 
bone formation activity "is due to a mixture of 
regulatory molecules" and that "bone formation is most 
05 likely controlled ... by the interaction of these 

molecules." Again, no bone induction was attributed to 
the products of expression of the cDNAs • See also 
Urist et al., EP0,212,474 entitled Bone Morphogenic 
Agents . 

10 Wang et al. (1988) Proc. Nat. Acad. Sci. USA 85; 

9484-9488 discloses the purification of a bovine bone 
morphogenetic protein from guanidine extracts of 
demineralized bone having cartilage and bone formation 
activity as a basic protein corresponding to a 

15 molecular weight of 30 kD determined from gel elution. 
Purification of the protein yielded proteins of 30, 18 
and 16 kD which, upon separation, were inactive. In 
view of this result, the authors acknowledged that the 
exact identity of the active material had not been 

20 determined. 

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

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



WO 92/07004 



PCT/US91/07654 



- 5 - 



impure mixture P3 OF 31-34 is assayed for osteogenic 
activity. The activity of the individual proteins is 
neither assessed nor discussed. 

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

15 These and other objects and features of the 

invention will be apparent from the description, 
drawings, and claims which follow. 

Summary of the Invention 

This invention provides novel polypeptide chains 
20 useful as either one or both subunits of dimeric 
osteogenic proteins which, when implanted in a 
mammalian body in association with a matrix, can induce 
at the locus of the implant the full developmental 
cascade of endochondral bone formation and bone marrow 
2 5 di f f erent iation . 

A key to these developments was the elucidation of 
amino acid sequence and structure data of native bovine 
osteogenic protein. A protocol was developed which 
results in retrieval of active, substantially pure 
30 osteogenic protein from bovine bone having a half- 



WO 92/07004 



PCT/US91/07654 



- 6 - 



maximum bone forming activity of about 0.8 to 1.0 ng 
per mg of implant. The availability of the material 
enabled the inventors to elucidate key structural 
details of the protein necessary to achieve bone 
Q5 formation. Knowledge of the protein's amino acid 
sequence and other structural features enabled the 
identification and cloning of native genes in the human 
genome • 

Consensus DNA sequences based on partial sequence 
10 data and observed homologies with regulatory proteins 
disclosed in the literature were used as probes for 
extracting genes encoding osteogenic protein from human 
genomic and cDNA libraries. One of the consensus 
sequences was used to isolate a previously unidentified 
15 gene which, when expressed, encoded a protein 

comprising a region capable of inducing endochondral 
bone formation when properly modified, incorporated in 
a suitable matrix, and implanted as disclosed herein. 
The gene, called "hOPl" or "OP-l" , is described in 
20 greater detail in U.S. 660,162, filed 27-SEP-91 the 
disclosure of which is herein incorporated by 
reference. 

Fragments of the hOPl DNA sequence subsequently 
were used to probe a mouse embryo cDNA library in 

25 search of additional genes encoding osteogenic 
proteins. This process isolated a heretofore 
unidentified DNA sequence which encodes a polypeptide 
chain referred to herein as mOPl protein. Mouse OP1 
(mOP-1) protein shares significant amino acid sequence 

30 homology with human hOPl protein, particularly in the 
region encoding the mature protein. Based on detailed 
structural and physical analyses of hOPl protein and 
the high degree of amino acid sequence homology between 



WO 92/07004 



PCT/US91/07654 



- 7 - 



the hOPl and mOP-1 proteins / homodimers of mOPl 
proteins and heterodimers comprising mOPl protein are 
believed to be capable of inducing endochondral bone 
formation, when the protein is dispersed in a suitable 
05 matrix, and implanted as disclosed herein* 

The sequence of what is believed to be the mature 
form of the murine protein, designated herein raOPl-S, 
is (residues 292-430 of Seq. ID No. 1). The amino acid 
sequence of the full length protein, mOPl-PP (the 
10 "prepro" form, see infra), and the cDNA sequence 
encoding it are set forth in Seq. ID No. 1. 

The invention provides recombinant dimeric 
proteins, and osteogenic devices comprising these 
proteins, wherein the subunits of the osteogenic dimers 
15 comprise an amino acid sequence described by Seq. ID 
No. 1, including allelic and biosynthetically mutated 
variants thereof. 

Mouse OP1 can be expressed from intact or truncated 
cDNA or from synthetic DNAs in procaryotic or 

20 eucaryotic host cells, and then purified, cleaved, 
refolded, dimerized, and implanted in experimental 
animals. Currently preferred host cells include E. 
coli, or mammalian cells, such as CHO, COS or BSC 
cells. The osteogenic protein of the invention may 

25 include forms having varying glycosylation patterns, 
varying N-termini, a family of related proteins having 
regions of amino acid sequence homology, and active 
truncated or mutated forms of native or biosynthetic 
protein, produced by expression of recombinant DNA in 

„ host cells. 



WO 92/07004 



PCT/US91/07654 



- 8 



Thus, in view of this disclosure, skilled genetic 
engineers can isolate genes from cDNA or genomic 
libraries of various different species which encode 
appropriate amino acid sequences/ or construct DNAs 
05 from oligonucleotides, and then can express them in 

various types of host cells, including both procaryotes 
and eucaryotes, to produce large quantities of active 
proteins capable of inducing bone formation in mammals 
including humans. 

10 In view of this disclosure, and using standard 

immunology techniques well known in the art, those 
skilled in the art also may raise polyclonal or 
monclonal antibodies against all or part of the 
polypeptide chains described herein. Useful protocols 

15 for antibody production may be found, for example, in 
Molecular Cloninq-A Laboratory Manual ( Sambrook et al . , 
eds.) Cold Spring Harbor Press 2nd ed. 1989). See 
Book 3, Section 18. 

The osteogenic proteins are useful in clinical 
20 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 
25 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- 
20 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 



WO 92/07004 



PCT/US91/07654 



- 9 - 



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

05 xenogenic bone powder matrices also may be treated with 
proteases such as trypsin and/or fibril modifying 
agents to increase the intraparticle intrusion volume 
and surface area. Useful agents include solvents such 
as dichloromethane, trichloroacetic acid, acetonitrile 

10 and acids such as trifluoroacetic acid and hydrogen 
fluoride. Alternatively, the matrix may be treated 
with a hot aqueous medium having a temperature within 
the range of about 37 °C to 75°C, including heated 
acidic aqueous medium. Other potentially useful matrix 

15 materials comprise collagen, homopolymers and 
copolymers of glycolic acid and lactic acid, 
hydroxyapatite, tricalcium phosphate and other calcium 
phosphates . 

The osteogenic proteins and implantable osteogenic 
20 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 
25 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 
30 application is in cartilage repair, for example, in the 
treatment of osteoarthritis. 



WO 92/07004 



PCT/US91/07654 



- 10 - 



Brief Description of the Drawing 

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

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

- 10 Description 

Purification protocols first were developed which 
enabled isolation of the osteogenic protein present in 
crude protein extracts from mammalian bone. (See PCT 
US 89/01453, and U.S. Serial No. 179,406 filed April 8, 

15 1988, now U.S. Patent No. 4,968,950). The development 
of the procedure, coupled with the availability of 
fresh calf bone, enabled isolation of substantially 
pure bovine osteogenic protein (bOP). bOP was 
characterized significantly; its ability to induce 

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

25 extracts. This dose dependent and highly specific 
activity was present whether or not the protein was 
glycosylated (see (1990) J. Biol. Chem. 265 : 13198- 
13205). Sequence data obtained from the bovine 
materials suggested probe designs which were used to 

30 isolate human genes. The OP human counterpart proteins 
have now been expressed and extensively characterized. 



WO 92/07004 



PCT/US91/07654 



These discoveries enabled preparation of DNAs 
encoding totally novel, non-native protein constructs 
which individually as homodimers and combined with 
other species as heterodimers are capable of producing 

05 true endochondral bone (see PCT WO 89/09788, published 
19-OCT-89 and US Serial No. 315,342, filed 23-FEB-89, 
now U.S. Patent No. 5,011,691.) They also permitted 
expression of the natural material, truncated forms, 
muteins, analogs, fusion proteins, and various other 

10 variants and constructs, from cDNAs and genomic DNAs 
retrieved from natural sources or from synthetic DNA 
produced using the techniques disclosed herein and 
using automated, commercially available equipment. The 
DNAs may be expressed using well established molecular 

15 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 
20 genomic and cDNA libraries encoded a previously 

unidentified gene, referred to herein as hOPl. The 
protein encoded by the isolated DNA was identified 
originally by amino acid homology with proteins in the 
TGF-p family. Consensus splice signals were found 
25 where amino acid homologies ended, designating exon- 

intron boundaries. Three exons were combined to obtain 
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: 
30 pp. 2085-2093). The DNA also is referred to in related 
applications as "OP1 and "OP-l". 



WO 92/07004 



PCT/US91/07654 



- 12 - 



In its native form, hOPl expression yields an 
immature translation product ( "hOPl-PP" , where "PP" 
refers to "prepro form") of about 400 amino acids that 
subsequently is processed to yield a mature sequence of 

05 139 amino acids ("OP1-18"). The active region 

(functional domain) of the protein comprises the C- 
terminal 97 amino acids of the hOPl sequence, "OPS", 
which includes a conserved six cysteine skeleton. A 
longer active sequence is 0P7, comprising the C- 

10 terminal 102 amino acids, and which includes a 
conserved seven cysteine skeleton. 

The full length cDNA sequence for hOPl, and its 
encoded "prepro" form hOFl-PP, which includes an N- 
terminal signal peptide sequence, are disclosed in 
-15 Seq. ID No. 3 (residues 1-431). The mature form of 
hOPl protein expressed in mammalian cells, designated 
herein 0P1-18, is indicated by residues 293-431 of Seq. 
ID No. 3. 

cDNA sequences encoding the "prepro" form, of the 
20 protein and the mature form, as well as various 

truncated forms of the gene, and fused genes, have been 
expressed in E. coli (see, for example, U.S. Serial No. 
422, 699) and numerous mammalian cells (See, for 
example, PCT WO 91/05802, published 2-MAY-91, and all 
25 have been shown to have osteogenic activity when 

implanted in a mammal in association with a suitable 
matrix . 

Given the foregoing amino acid and DNA sequence 
information, various nucleic acids (RNAs and DNAs) can 
3Q be constructed which encode at least the active region 
of an OP1 protein (e.g., OPS or OP7, amino acid 
residues 335-431 or 330-431, respectively, of Seq. ID 



WO 92/07004 



PCT/US91/07654 



- 13 - 



No. 3) and various analogs thereof, as well as fusion 
proteins, truncated forms of the mature proteins, and 
similar constructs. Moreover, DNA hybridization probes 
can be constructed from fragments of the hOPl DNA or 
05 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 
10 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 
j 5 Model 8600 Synthesizer, and purified by polyacrylamide 
gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer. 
The DNA may then be electroeluted from the gel. 
Overlapping oligomers may be phosphorylated by T4 
polynucleotide kinase and ligated into larger blocks 
20 which may also be purified by PAGE. 

DNAs for use as hybridization probes may be 
labelled (e.g., as with a radioisotope, by nick 
translation) and used to identify clones in a given 
library containing DNA to which the probe hybridizes, 

25 following techniques well known in the art. The 
libraries may be obtained commercially or they may 
constructed de novo using conventional molecular 
biology techniques. Further information on DNA library 
construction and hybridization techniques can be found 

2q in numerous texts known to those skilled in the art. 
See, for example, F.M. Ausubel, ed., Current Protocols 
in Molecular Bioloqy-Vol. l f (1989). In particular, 



WO 92/07004 



PCT/US91/07654 



- 14 - 



see unit 5, "Construction of Recombinant DNA Libraries" 
and Unit 6, "Screening of Recombinant Libraries." 

Appropriately identified clones then can be 
sequenced using any of a number of techniques well 

05 known in the art. A DNA fragment containing the 
sequence of interest then can be subcloned into an 
expression vector and trans fected into an appropriate 
host cell for protein expression and further 
characterization. The host may be a procaryotic or 

10 eucaryotic cell since the former's inability to 

glycosylate protein will not destroy the protein's 
osteogenic activity. Useful host cells include E. 
coli , Saccharomyces , the insect/baculovirus cell 
system, myeloma cells, and various mammalian cells. 

15 The vector additionally may encode various sequences to 
promote correct expression of the recombinant protein, 
including transcription promoter and termination 
sequences, enhancer sequences, preferred ribosome 
binding site sequences, preferred mRNA leader 

20 sequences, preferred signal sequences for protein 

secretion, and the like. The DNA sequence encoding the 
gene of interest also may be manipulated to remove 
potentially inhibiting sequences or to minimize 
unwanted secondary structure formation. The 

25 recombinant osteogenic protein also may be expressed as 
a fusion protein. After being translated, the protein 
may be purified from the cells themselves or recovered 
from the culture medium. All biologically active 
protein forms comprise dimeric species joined by 

30 disulfide bonds or otherwise associated, produced by 
oxidizing and refolding one or more of the various 
recombinant proteins within an appropriate eucaryotic 
cell or in vitro after expression of individual 
subunits. A detailed description of osteogenic protein 



WO 92/07004 



PCT/US91/07654 



- 15 - 



expressed from recombinant DNA in E. coli is disclosed 
in U.S. Serial No. 660,162, the disclosure of which has 
been incorporated by reference, supra. A detailed 
description of osteogenic protein expressed from 
05 recombinant DNA in numerous different mammalian cells 
is disclosed in PCT WO 91/05802. 

Exemplification 

In an effort to identify additional DNA sequences 
encoding osteogenic proteins, a hybridization probe 
10 specific to the C-terminus of the DNA of mature hOPl 
was prepared using a StuI-EcoRl digest fragment of hOPl 
(base pairs 1034-1354 in Seq. ID No. 3), and labelled 

• 3 2 

with P by nick translation, as described in the art. 
The C-terminus of the protein encodes a key functional 

15 domain e.g., the "active region" for osteogenic 

activity. The C-terminus also is the region of the 
protein whose amino acid sequence shares specific amino 
acid sequence homology with particular proteins in the 
TGF-/3 super-family of regulatory proteins and which 

20 includes the conserved cysteine skeleton. 

Approximately 7 x 10 5 phages of an oligo (dT) 
primed 17.5 days p.c. mouse embryo 5' stretch cDNA 
(gtlO) library (Clontech, Inc., Palo Alto, CA) was 
screened with the labelled probe. The screen was 
25 performed using the following stringent hybridization 
conditions: 40% formamide, 5 x SSPE, 5 x Denhardt's 
solution, 0.1% SDS, at 37°C overnight, and washing in 
0.1 x SSPE, 0.1% SDS, at 50°C. 

Five recombinant phages were purified over three 
30 rounds of screening. Phage DNA was prepared from all 
five phages, subjected to an EcoRl digest, subcloned 



WO 92/07004 



PCT/US91/07654 



- 16 - 



into the EcoRl site of common pUC-type plasmid modified 
to allow single strand sequencing, and sequenced using 
means well known in the art. 

Two different DNA sequences were identified by this 
05 procedure. One DNA, referred to herein as mOP2, is 
described in detail in copending USSN 599,543, filed 
18-Oct-90. A second DNA, referred to herein as mOPl, 
is described below. 

The cDNA and encoded amino acid sequence for the 

10 full length mOPl protein is depicted in Seq. ID No. 1. 
The full-length form of the protein is referred to as 
the prepro form of mOP-1 ( "mOPl-PP" ) , and includes a 
signal peptide sequence at its N-terminus. The amino 
acid sequence Ser-Ala-Leu-Ala-Asp (amino acid residues 

15 26-30 in Seq. ID No. 1) is believed to constitute the 
cleavage site for the removal of the signal peptide 
sequence, leaving an intermediate form of the protein, 
the "pro" form, to be secreted from the expressing 
cell. The amino acid sequence Arg-Ser-Ile-Arg-Ser 

20 (amino acid residue nos. 288-292 in Seq. ID No. 1) is 
believed to constitute the cleavage site that produces 
the mature form of the protein, herein referred to as 
"mOPl-S" and described by amino acid residues 292-430 
of Seq. ID No. 1. The region of the mOPl amino acid 

25 sequence corresponding to the conserved six cysteine 
skeleton is described by residues 334-430 of Seq. ID 
No. 1. The region corresponding to the conserved seven 
cystein skeleton is described by residues 329-430 of 
Seq. ID No. 1. 

30 Figure 1 compares the amino acid sequence homology 

of the mature hOPl and mOPl proteins (OP1-18 and 
mOPl-S). Amino acid identity is indicated by three 



WO 92/07004 



PCT/US91/07654 



- 17 - 



dots (...)• As can be seen in Figure 1, the mature 
form of mOPl, mOPl-S shows significant sequence 
homology with OP-1-18 (98%), differing at only three 
positions in this region. Like OP-1-18 , mOPl-S has a 

05 seven cysteine functional domain (residues 38-139 of 
Fig. 1). The prepro form of the mOPl protein shares 
substantially less amino acid sequence homology with 
that of OP1-PP. The high degree of homology of the 
mature domains is not surprising as the amino acid 

10 sequences of the mature forms of TGF-p-like proteins 
generally also have been found to be highly conserved 
across different animal species (e.g., compare Vgr and 
Vgl, two related genes from mouse and Xenopus, 
respectively, see U.S. Pat. No. 5,011,691). The high 

^ degree of amino acid sequence homology exhibited 

between the mature forms of the two animal species of 
OP1 proteins identified suggests that the mOP-1 protein 
will purify essentially as the human OP1 protein does, 
or with only minor modifications of the protocols 

20 disclosed for human OP1 protein. Similarly, purified 
mOPl-S is predicted to have an apparent molecular 
weight of about 36 kD as a glycosylated oxidized 
homodimer, and about 18 kD as a reduced single subunit, 
as determined by comparison with molecular weight 

25 standards on an SDS-polyacrylamide electrophoresis gel. 
There appear to be three potential N glycosylation 
sites in the mature mOPl protein. The unglycosylated 
homodimer (e.g., one expressed from E. coli) is 
predicted to have a molecular weight of about 27 kD. 

30 MATRIX PREPARATION 

A. General Consideration of Matrix Properties 



WO 92/07004 PCT/US91/07654 

- 18 - 



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 
05 matrix (e.g., hydroxylapatite (HAP), collagen, 

tricalcium phosphate or polylactic acid, polyglycolic 
acid and various copolymers thereof.) 

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

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

A successful carrier for osteogenic protein 
30 must perform several important functions. It must bind 
osteogenic protein and act as a slow release delivery 
system, accommodate each step of the cellular response 



WO 92/07004 PCT/US91/07654 

- 19 - 



during bone development, and protect the osteogenic 
protein from nonspecific proteolysis. In addition, 
selected materials must be biocompatible in vivo and 
preferably biodegradable; the carrier must act as a 

05 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 
vary according to whether the implant is placed in 

j q cortical or trabecular bone. 

Matrix geometry, particle size, the presence 
of surface charge, and the degree of both intra-and- 
inter-particle porosity are all important to successful 
matrix performance. It is preferred to shape the 
j 5 matrix to the desired form of the new bone and to have 
dimensions which span non-union defects. Rat studies 
show that the new bone is formed essentially having the 
dimensions of the device implanted. 



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

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

produces an implantable material useful in a variety of 
clinical settings. In addition to its use as a matrix 



20 



25 



WO 92/07004 



PCT/US91/07654 



- 20 - 



for bone formation in various orthopedic, periodontal, 
and reconstructive procedures, the matrix also may be 
used as a sustained release carrier, or as a 
collagenous coating for implants. The matrix may be 

05 shaped as desired in anticipation of surgery or shaped 
by the physician or technician during surgery. Thus, 
the material may be used for topical, subcutaneous, 
intraperitoneal, or intramuscular implants; it may be 
shaped to span a nonunion fracture or to fill a bone 

10 defect. In bone formation or conduction procedures, 
the material is slowly absorbed by the body and is 
replaced by bone in the shape of or very nearly the 
shape of the implant. 

Various growth factors, hormones, enzymes, 
15 therapeutic compositions, antibiotics, and other body 
treating agents also may be absorbed onto the carrier 
material and will be released over time when implanted 
as the matrix material is slowly absorbed. Thus, 
various known growth factors such as EGF, PDGF, IGF, 
20 FGF, TGF-cl, and TGF-B 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 

25 Demineralized bone matrix, preferably bovine 

bone matrix, is prepared by previously published 
procedures (Sampath and Reddi (1983) Proc. Natl. Acad. 
Sci. USA 80 ;6591-6595) . Bovine diaphyseal bones (age 
1-10 days) are obtained from a local slaughterhouse and 

3q used fresh. The bones are stripped of muscle and fat, 



WO 92/07004 



PCT/US91/07654 



- 21 - 



cleaned of periosteum, demarrowed by pressure with cold 
water, dipped in cold absolute ethanol, and stored at 
-20°C. They are then dried and fragmented by crushing 
and pulverized in a large mill* Care is taken to 
05 prevent heating by using liquid nitrogen. The 

pulverized bone is milled to a particle size in the 
range of 70-850 pm, preferably 150-420 /jm, and is 
defatted by two washes of approximately two hours 
duration with three volumes of chloroform and methanol 
10 (3:1). The particulate bone is then washed with one 
volume of absolute ethanol and dried over one volume of 
anhydrous ether yielding defatted bone powder. The 
defatted bone powder is then demineralized by four 
successive treatments with 10 volumes of 0.5 N HC1 at 
15 4°C for 40 min. Finally, neutralizing washes are done 
on the demineralized bone powder with a large volume of 
water . 

2 . Guanidine Extraction 

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

3. Matrix Treatments 

The major component of all bone matrices is 
Type-I collagen. In addition to collagen, 
demineralized bone extracted as disclosed above 
30 includes non-collagenous proteins which may account for 
5% of its mass. In a xenogenic matrix, these 



WO 92/07004 



PCT/US91/07654 



- 22 - 



noncollagenous components may present themselves as 
potent antigens / and may constitute immunogenic and/or 
inhibitory components. These components also may 
inhibit osteogenesis in allogenic implants by 

Q5 interfering with the developmental cascade of bone 
differentiation. It has been discovered that 
treatment of the matrix particles with a collagen 
fibril-modifying agent extracts potentially unwanted 
components from the matrix, and alters the surface 

10 structure of the matrix material. Useful agents 
include acids, organic solvents or heated aqueous 
media. Various treatments are described below. A 
detailed physical analysis of the effect these fibril- 
modifying agents have on demineralized, quanidine- 

15 extracted bone collagen particles is disclosed in 

copending U.S. Patent Application Serial No. 483,913, 
filed February 22, 1990. 

After contact with the fibril -modifying agent, 

the treated matrix is washed to remove any extracted 

20 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 

25 stir at room temperature (RT) for 30 minutes 
(sufficient time to neutralize the pH); 

2. Centrifuge and repeat wash step; and 

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

30 3.1 Acid Treatments 



WO 92/07004 



PCT/US91/07654 



- 23 - 



1. Trif luoroacetic acid. 

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

05 Bovine bone residue prepared as described 

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

2. Hydrogen Fluoride. 

Like trif luoroacetic acid, hydrogen fluoride 
is a strong acid and swelling agent, and also is 
capable of altering intraparticle surface structure. 
Hydrogen fluoride is also a known deglycosylating 
agent. As such, HF may function to increase the 
osteogenic activity of these matrices by removing the 
antigenic carbohydrate content of any glycoproteins 
still associated with the matrix after guanidine 
extraction. 

Bovine bone residue prepared as described above is 
sieved, and particles of the appropriate size are 
25 collected. The sample is dried in vacuo over ^^S' 
transferred to the reaction vessel and exposed to 
anhydrous hydrogen fluoride (10-20 ml/g of matrix) by 
distillation onto the sample at -70 °C. The vessel is 
allowed to warm to 0°C and the reaction mixture is 



15 



20 



WO 92/07004 



PCTAJS91/07654 



- 24 - 



stirred at this temperature for 120 minutes. After 
evaporation of the hydrogen fluoride in vacuo, the 
residue is dried thoroughly in vacuo over KOH pellets 
to remove any remaining traces of acid. Extent of 

05 deglycosylation can be determined from carbohydrate 
analysis of matrix samples taken before and after 
treatment with hydrogen fluoride, after washing the 
samples appropriately to remove non-covalently bound 
carbohydrates. SDS-extracted protein from HF-treated 

10 material is negative for carbohydrate as determined by 
Con A blotting. 

The deglycosylated bone matrix is next washed 
twice in TBS (Tris-buf f ered saline) or UTBS, water- 
washed, and then lyophilized. 

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

3.2 Solvent Treatment 

1 . Dichloromethane . 

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

Bovine bone residue, prepared as described 
above, is sieved, and particles of the appropriate size 



15 



20 



WO 92/07004 



PCT/US91/07654 



- 25 - 



are incubated in 100% DCM or, preferably, 99.9% 
DCM/0.1% TFA. The matrix is incubated with the 
swelling agent for one or two hours at 0°C or at room 
temperature. Alternatively, the matrix is treated with 
05 the agent at least three times with short washes (20 
minutes each) with no incubation. 

2. Acetonitrile. 

Acetonitrile (ACN) is an organic solvent, 
capable of denaturing proteins without affecting their 
10 primary structure. It is a common reagent used in 

high-performance liquid chromatography, and is used to 
elute proteins from silica-based columns by perturbing 
hydrophobic interactions. 

Bovine bone residue particles of the 
15 appropriate size, prepared as described above, are 
treated with 100% ACN (1.0 g/30 ml) or, preferably, 
99.9% ACN/0.1% TFA at room temperature for 1-2 hours 
with constant stirring. The treated matrix is then 
water-washed, or washed with urea buffer, or 4 M NaCl 
2 q and lyophili2ed. Alternatively, the ACN or ACN/TFA 
treated matrix may be lyophilized without wash. 

3. Isopropanol. 

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



Bovine bone residue particles of the 
appropriate size prepared as described above are 
treated with 100% isopropanol (1.0 g/30 ml) or, 



WO 92/07004 PCT/US91/07654 

- 26 - 



preferably, in the presence of 0.1% TFA, at room 
temperature for 1-2 hours with constant stirring. The 
matrix is then water-washed or washed with urea buffer 
or 4 M NaCl before being lyophilized. 

05 4 . Chloroform 

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

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

3.3 Heat Treatment 

The currently most preferred agent is a heated 
aqueous fibril-modifying medium such as water, to 
increase the matrix particle surface area and porosity. 
The currently most preferred aqueous medium is an 
acidic aqueous medium having a pH of less than about 
4.5, e.g., within the range of pH 2 - pH 4 . which may 
help to "swell" the collagen before heating. 0.1% 
acetic acid, which has a pH of about 3, currently is 
preferred. 0.1 M acetic acid also may be used. 

Various amounts of delipidated, demineralized 
guanidine-extracted bone collagen are heated in the 
aqueous medium ( lg matrix/30ml aqueous medium) under 
25 constant stirring in a water jacketed glass flask, and 
maintained at a given temperature for a predetermined 
period of time. Preferred treatment times are about 
one hour, although exposure times of between about 0.5 
to two hours appear acceptable. The temperature 



15 



20 



WO 92/07004 



PCT/US91/07654 



- 27 - 



employed is held constant at a temperature within the 
range of about 37°C to 75°C. The currently preferred 
heat treatment temperature is within the range of 45°C 
to 60°C. 

05 After the heat treatment, the matrix is filtered, 

washed, lyophilized and used for implant. Where an 
acidic aqueous medium is used, the matrix also is 
preferably neutralized prior to washing and 
lyophilization. A currently preferred neutralization 

10 buffer is a 200mM sodium phosphate buffer, pH 7.0, To 
neutralize the matrix, the matrix preferably first is 
allowed to cool following thermal treatment, the acidic 
aqueous medium (e.g., 0.1% acetic acid) then is removed 
and replaced with the neutralization buffer and the 

15 matrix agitated for about 30 minutes. The 

neutralization buffer then may be removed and the 
matrix washed and lyophilized (see infra). 

The matrix also may be treated to remove 
contaminating heavy metals, such as by exposing the 

20 matrix to a metal ion chelator. For example, following 
thermal treatment with 0.1% acetic acid, the matrix may 
be neutralized in a neutralization buffer containing 
EDTA (sodium ethylenediaminetetraacetic acid), e.g., 
200 mM sodium phosphate, 5mM EDTA, pH 7.0. 5 mM EDTA 

25 provides about a 100-fold molar excess of chelator to 
residual heavy metals present in the most contaminated 
matrix tested to date. Subsequent washing of the 
matrix following neutralization appears to remove the 
bulk of the EDTA. EDTA treatment of matrix particles 

30 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- 



WO 92/07004 



PCT/US91/07654 



- 28 - 



treated matrices indicate that treatment with the metal 
ion chelator does not inhibit bone inducing activity. 

The collagen matrix materials preferably take 
the form of a fine powder, insoluble in water, 

05 comprising nonadherent particles. It may be used 

simply by packing into the volume where new bone growth 
or sustained release is desired, held in place by 
surrounding tissue. Alternatively, the powder may be 
encapsulated in, e.g., a gelatin or polylactic acid 

10 coating, which is adsorbed readily by the body. The 
powder may be shaped to a volume of given dimensions 
and held in that shape by interadhering the particles 
using, for example, soluble, species-biocompatible 
collagen. The material may also be produced in sheet, 

j 5 rod, bead, or other macroscopic shapes. 

FABRICATION OF OSTEOGENIC DEVICE 

The naturally sourced and recombinant protein as 
set forth above, and other constructs, can be combined 
and dispersed in a suitable matrix preparation using 
2Q any of the methods described below. In general, 50-100 
ng of active protein is combined with the inactive 
carrier matrix (e.g., 25 mg for rat bioassays). 
Greater amounts may be used for large implants. 

1. Ethanol Precipitation 

25 Matrix is added to osteogenic protein 

dissolved in guanidine-HCl. Samples are vortex-ed 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 



WO 92/07004 



PCT/US91/07654 



- 29 - 



and incubated, preferably for 30 minutes at -20°C. 
After centrifugation (microfuge, high speed) the 
supernatant is discarded. The reconstituted matrix is 
washed twice with cold concentrated ethanol in water 
05 (85% EtOH) and then lyophilized. 

2. Acetonitrile Trif luoroacetic 

Acid Lyophilization 

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

15 3. Urea Lyophilization 

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

4. Buffered Saline Lyophilization 

CP1 preparations in physiological saline may 
also be vortexed with the matrix and lyophilized to 
produce osteogenically active material. 

25 These procedures also can be used to adsorb 

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



WO 92/07004 



PCT/US91/07654 



- 30 - 



BIOASSAY 

The functioning of the various proteins and 
devices of this invention can be evaluated with an in 
vivo bioassay. Studies in rats show the osteogenic 

05 effect in an appropriate matrix to be dependent on the 
dose of osteogenic protein dispersed in the matrix. No 
activity is observed if the matrix is implanted alone. 
In vivo bioassays performed in the rat model also have 
shown that demineralized, guanidine-extracted xenogenic 

10 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 

15 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. 

20 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 

25 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 

30 under sterile conditions in the skin over the thoracic 



WO 92/07004 



PCT/US91/07654 



- 31 - 



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 

05 designated as day one of the experiment. Implants were 
removed on day 12. The heterotropic site allows for 
the study of bone induction without the possible 
ambiguities resulting from the use of orthotropic 
sites. As disclosed herein, both allogenic (rat bone 

10 matrix) and xenogenic (bovine- bone matrix) implants 
were assayed. 

2. Cellular Events 

Successful implants exhibit a controlled 
progression through the stages of protein-induced 

15 endochondral bone development, including: (1) transient 
infiltration by polymorphonuclear leukocytes on day 
one; (2) mesenchymal cell migration and proliferation 
on days two and three; (3) chondrocyte appearance on 
days five and six; (4) cartilage matrix formation on 

20 day seven; (5) cartilage calcification on day eight; 
(6) vascular invasion, appearance of osteoblasts, and 
formation of new bone on days nine and ten; (7) 
appearance of osteoblastic and bone remodeling and 
dissolution of the implanted matrix on days twelve to 

25 eighteen; and (8) hematopoietic bone marrow 

differentiation in the ossicle on day twenty-one. The 
results show that the shape of the new bone conforms to 
the shape of the implanted matrix. 

3. Histological Evaluation 

30 Histological sectioning and staining is 

preferred to determine the extent of osteogenesis in 



WO 92/07004 



PCT/US91/07654 



- 32 - 



the implants. Implants are fixed in Bouins Solution, 
embedded in paraffin, and cut into 6-8 //m sections. 
Staining with toluidine blue or hemotoxylin/eosin 
demonstrates clearly the ultimate development of 
05 endochondral bone. Twelve day implants are usually 
sufficient to determine whether the implants contain 
newly induced bone. 

4. Biological Markers 

Alkaline phosphatase activity may be used as a 
10 marker for osteogenesis. The enzyme activity may be 
determined spectrophotometrically after homogenization 
of the implant. The activity peaks at 9-10 days in 
vivo and thereafter slowly declines. Implants showing 
no bone development by histology have little or no 
15 alkaline phosphatase activity under these assay 

conditions. The assay is useful for quantitation and 
obtaining an estimate of bone formation quickly after 
the implants are removed from the rat. Alternatively, 
the amount of bone formation can be determined by 
2o measuring the calcium content of the implant. 

The invention may be embodied in other 
specific forms without departing from the spirit or 
essential characteristics thereof. The present 
embodiments are therefore to be considered in all 

25 respects as illustrative and not restrictive, the scope 
of the invention being indicated by the appended claims 
rather than by the foregoing description, and all 
changes which come within the meaning and range of 
equivalency of the claims are therefore intended to be 

30 embraced therein. 



WO 92/07004 



PCT/US91/07654 



- 33 - 



SEQUENCE LISTING 



(1) GENERAL INFORMATION: 

(i) APPLICANT: 

OZKAYNAK, ENGIN 
05 OPPERMANN, HERMANN 

KUBERASAMPATH, THANGAVEL 
RUEGER, DAVID C. 

(ii) TITLE OF INVENTION: OSTEOGENIC DEVICES 

(iii) NUMBER OF SEQUENCES: 4 

10 (iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: TESTA, HURWITZ & THIBEAULT 

(B) STREET: 53 STATE STREET 

(C) CITY: BOSTON 

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

(F) ZIP: 02109 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBH PC compatible 

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

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

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: 

(B) FILING DATE: 
25 (C) CLASSIFICATION: 

(viii) ATTORNEY/AGENT INFORMATION: 

(A) NAME: PITCHER, EDMUND R. 

(B) REGISTRATION NUHBER: 27,829 

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

30 (ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: 617/248-7000 

(B) TELEFAX: 617/248-7100 

(2) INFORMATION FOR SEQ ID NO:l: 

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

(B) TYPE: nucleic acid 

(C) STRAND EDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 



WO 92/07004 



PCT/US91/07654 



- 34 - 



(vi) ORIGINAL SOURCE: 

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

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

(B) LOCATION: 104.. 1393 

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

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

CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60 

CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC 115 

Met His Val Arg 
1 

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

CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TTC AGC CTG GAC AAC GAG 211 
Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu 
20 25 30 35 

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

GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 307 
25Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg Pro 
55 60 65 

CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GCG CCC ATG TTC ATG TTG 355 
Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Met Phe Met Leu 
70 75 80 

30GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AGC GGG CCG GAC GGA CAG 403 
Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gin 
85 90 95 100 

GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC CCC CCT 451 
Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 
35 105 110 115 

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



ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AAA GAA TTC TTC CAC CCT 



547 



WO 92/07004 



PCT/US91/076S4 



- 35 - 



05 



Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro 
135 140 145 

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



595 



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

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

185 190 195 

CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 739 
Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser 
200 205 210 

1 5 CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 787 
Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp He Thr 
215 220 225 

GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CGG CAC AAC CTG GGC TTA 835 
Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu 
20 230 235 240 

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

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

265 270 275 

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

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

CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GAA AAC AGC AGC AGT GAC 1075 
Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp 
35 310 315 320 

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

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



WO 92/07004 



PCT/US91/07654 



- 36 - 



345 350 355 

TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AAC TCG TAC ATG AAC GCC 1219 
lyv Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala 
360 365 370 

05 ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC AAC CCA GAC 1267 
Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn Pro Asp 
375 380 385 

ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CAG CTC AAC GCC ATC TCT 1315 
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He Ser 
!0 390 395 400 

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

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

425 430 



20 



ACCTTTGCGG 


GGCCACACCT 


TTCCAAATCT 


TCGATGTCTC ACCATCTAAG 


TCTCTCACTG 


1473 


CCCACCTTGG 


CGAGGAGAAC 


AGACCAACCT 


CTCCTGAGCC TTCCCTCACC 


TCCCAACCGG 


1533 


AAGCATGTAA 


GGGTTCCAGA 


AACCTGAGCG 


TGCAGCAGCT GATGAGCGCC 


CTTTCCTTCT 


1593 


GGCACGTGAC 


GGACAAGATC 


CTACCAGCTA 


CCACAGCAAA CGCCTAAGAG 


CAGGAAAAAT 


1653 


GTCTGCCAGG 


AAAGTGTCCA 


GTGTCCACAT 


GGCCCCTGGC GCTCTGAGTC 


TTTGAGGAGT 


1713 


AATCGCAAGC 


CTCGTTCAGC 


TGCAGCAGAA 


GGAAGGGCTT AGCCAGGGTG 


GGCGCTGGCG 


1773 


TCTGTGTTGA 


AGGGAAACCA 


AGCAGAAGCC 


ACTGTAATGA TATGTCACAA 


TAAAACCCAT 


1833 


GAATGAAAAA 


AAAAAAAAAA 


AAAAAAAAAA 


AAAAGAATTC 




1873 



25 (2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 430 amino acids 

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

30 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(D) OTHER INFORMATION: /products "mOPl-PP' 

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



WO 92/07004 



PCT/US91/07654 



- 37 - 



05 



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

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

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

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

Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 
10 65 70 75 80 

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

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

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

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

Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser 
20 145 150 155 160 

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

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

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

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

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

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

Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys 
260 265 270 

35 Gin Pro Phe Het Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 
275 280 285 



30 



WO 92/07004 



PCT/US91/07654 



- 38 - 



05 



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

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

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

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

Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 
10 355 360 365 

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

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

15 Asn Ala He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu 

405 410 415 

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

(2) INFORMATION FOR SEQ ID NO: 3: 

20 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1822 base pairs 

(B) TYPE: nucleic acid 

(C) STRAND EDNESS: single 

(D) TOPOLOGY: linear 

25 (ii) MOLECULE TYPE: cDNA 

(iii) HYPOTHETICAL: NO 

(iv) ANTI- SENSE: NO 

(vi) ORIGINAL SOURCE: 

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

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 49.. 1341 

(C) IDENTIFICATION METHOD: experimental 

35 (D) OTHER INFORMATION: /function* "OSTEOGENIC PROTEIN" 

/product* "hOPl-PP n 
/evidence* EXPERIMENTAL 



WO 92/07004 



PCT/US91/07654 



- 39 - 

/standard_name= "hOPl" 

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

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 

Met His Val 

05 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 



10 



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

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

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

CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TCG GCA CCC ATG TTC ATG 297 
Pro Arg Pro His Leu Gin .ly Lys His Asn Ser Ala Pro Het Phe Het 
20 70 75 80 

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

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

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

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

CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TTT GAT CTT TCC AAG ATC 537 
His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He 
35 150 155 160 

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

TAC ATC CGG GAA CGC IIC GAC AAT GAG ACG TTC CGG ATC AGC GTT TAT 633 



WO 92/07004 



PCT/US91/07654 



- 40 - 



10 



Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He Ser Val Tyr 
180 185 190 195 

CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681 
Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu 
05 200 205 210 

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

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

GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 825 
Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro 
245 250 255 

15 AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG CAG CCC 873 

Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro 
260 265 270 275 

TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GTC CAC TTC CGC AGC ATC 921 
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser He 
20 280 285 290 

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

AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GTG GCA GAG AAC AGC AGC 1017 
25 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 
325 330 335 

30 CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GAA GGC TAC GCC 1113 
Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala 
340 345 350 ■ 355 

GCC TAC TAC TGT GAG GGG GAG TGT -GCC TTC CCT CTG AAC TCC TAC ATG 1161 
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 
35 360 365 370 

AAC GCC ACC AAC CAC GCC ATC <»TG 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 
40 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala 



WO 92/07004 



PCT/US91/07654 



- 41 - 



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 Lvs 
405 410 415 

TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351 
Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 

TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 

CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 

CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 

(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 431 amino acids 

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

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

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

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

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

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

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

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



WO 92/07004 



PCT/US91/076S4 



- 42 - 



05 



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

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

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



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

Asp Ala Asp Het Val Het Ser Phe Val Asn Leu Val Glu His Asp Lys 
10 130 135 140 

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

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

15 Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg lie 
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 
20 210 215 220 

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 

25 He Asn Pro Lys Leu Ala Gly Leu He Gly Arg His £ly Pro Gin Asn 
260 265 270 

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



Arg Ser He Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser 
30 290 295 300 

Lys Thr Pro Lys Asn Gin Glu Ala Leu Arg Het 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 

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



WO 92/07004 PCT/US91/07d54 

- 43 - 



Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Fhe 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 

05 Fhe lie Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin 
385 390 395 400 

Leu Asn Ala lie Ser Val Leu Tyr Fhe Asp Asp Ser Ser Asn Val lie 
405 410 415 

Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
10 420 «5 430 



WO 92/07004 



PCT/US91/07654 



- 44 - 



What is claimed is: 

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

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

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

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

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

said polypeptide chain comprising an amino 
acid sequence described by residues 334-430 of 
Seq. ID No. 1, including allelic variants 
thereof. 

6. The polypeptide chain of claim 5 wherein said 
polypeptide chain comprises the amino acid 
sequence described by residues 292-430 of Seq. 
ID No. 1, including allelic variants thereof.: 



WO 92/07004 
7. 

05 

9. 

10. 

10 ii. 

12. 
13. 

15 

14. 

20 

15. 

25 



PCT/US91/07654 

- 45 - 



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

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

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

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

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

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

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

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

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



WO 92/07004 



PCT/US91/07654 



- 46 - 



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



WO 92/07004 



PCT/US9 1/07654 



hOP-1 Ser Thr Gly Ser Lys Gin Arg Ser Gin 

mOP-1 Gly 

1 5 



hOP-1 Asn Arg Ser Lys Thr Pro Lys Asn Gin 

mOP-1 , 

10 15 



hOP-1 Glu Ala Leu Arg Met Ala Asn Val Ala 

mOP-1 Ser 

20 25 

hOP-1 Glu Asn Ser Ser Ser Asp Gin Arg Gin 

mOP-1 

30 35 

hOP-1 Ala Cys Lys Lys His Glu Leu Tyr Val 

mOP-1 

AO 45 



hOF-1 Ser Phe Arg Asp Leu Gly Trp Gin Asp 

raOP-1- 

50 



hOP-1 Trp He He Ala Pro Glu Gly Tyr Ala 

mOP-1 

55 60 



FIG. 1.1 



WO 92/07004 



PCT/US91/07654 



<6 



hOP-l Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 

mOP-1 

65 70 



hOP-1 Phe Pro Leu Asn Ser Tyr Met Asn Ala 

mOP-1 

75 80 



hOP-1 Thr Asn His Ala He Val Gin Thr Leu 

mOP-1 

85 90 

hOP-1 Val His Phe He Asn Pro Glu Thr Val 

mOP-1 Asp 

95 

hOP-1 Pro Lys Pro Cys Cys Ala Pro Thr Gin 

mOP-1 

100 105 



hOP-1 Leu Asn Ala He Ser Val Leu Tyr Phe 

mOP-1 

110 115 



hOP-1 Asp Asp Ser Ser Asn Val He Leu Lys 

mOP-1 Asp 

120 125 



FIG. 1.2 



WO 92/07004 



PCT/US91/07654 



hOP-1 Lys Tyr Arg Asn Met Val Val Arg 

mOP-1 

130 135 



hOP-1 Ala Cys Gly Cys His 
mOP-1 



FIG. 1.3 



INTERNATIONAL SEARCH REPORT 

Internattcr.s! Application No PCT/US 91/07654 



1. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) 6 


According to international Patent Classification (IPC) or to both National Classification and IPC 

IPC5: C 07 K 15/06, C 12 N 15/12, A 61 K 37/02 


IK FIELDS SEARCHED 


Minimum Documentation Searched 7 


Classification System 


Classification Symbols 


IPC5 


C 07 K; C 12 N; A 61 K 


Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are Included in Fields Searched 9 




III. DOCUMENTS CONSIDERED TO BE RELEVANT 9 


Category " 


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


Relevant to Claim No. 13 


p»x 


EP, A2, 0416578 (TAKEDA CHEMICAL INDUSTRIES, 
LTD.ET AL) 13 March 1991, 
see e.g. fig. 2, fig. 4-3 


1-3,5- 
16 


P,X 


Proc. Natl. Acad. Sci. USA, vol. 87, December 1990, 
A.J. Celeste et al: "Identification of 
transforming growth factor Beta family members 
present in bone- inductive protein purified from 
bovine bone 11 , see page 9843 - page 9847 
see fig.l 


1-16 


X 


WO, Al, 9011366 (GENETICS INSTITUTE, INC.) 
4 October 1990, 
see especially table V 


1-16 


" Special categories of cited documents: 10 

'A' document defining the general slate of the art which Is not 
considered to he of particular relevance 

*E* earlier document but published on or after tha international 
filing date 

*L" document which may throw doubts on priority claimfs) or 
which is cited to establish the publication dale 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 ftlfrs date bul 
later than the priority date claimed 


T* later.dnpumsnt published after the international f J lino date 
or priority dale 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 w .-ch docu- 
ments, such combination being obvious to a person skilled 
in the art 

1 *&* document member of the same patent family 


IV. CERTIFICATION 


Date of the Actual Completion of the International Search 

20th February 1992 


Date of Mailing of this International Search Report 

*l 01S2 


International Searching Authority 

EUROPEAN PATENT OFFICE 


Signature of Authorized Officer 





orm PCT/ISA/210 (second sheet) (January 1985) 



International Application No. PCT/US 91/07654 



111. DOCUMENTS CONSIDERED TO BE RELEVANT {CONTINUED FROM THE SECOND SHEET) 


Category • 


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


Relevant to Claim No 


A 


WO, Al, 9003733 (INTERNATIONAL GENETIC 
ENGINEERING, INC.) 19 April 1990, 
see the whole document 


1-16 


X 


WO, Al, 8909787 (CREATIVE BI0M0LECULES, INC.) 
19 October 1989, 
see especially claim 23 


1-2,5,7- 
14,16 


X 


Chemical Abstracts, volume 113, no. 19, 5 November 
1990, (Columbus, Ohio, US), Ozkaynak Engin et 
al: "OP-l cDNA encodes an osteogenic protein in 
the TGF-beta family", see page 181, abstract 
166493q, & EMBO J. 1990, 9( 7), 2085-2093 


1-16 



For* PCT/XSA/210 tsxtra shut) C January 1985) 



This annex 
The members 



ANNEX TO THE INTERNATIONAL SEARCH REPORT 
ON INTERNATIONAL PATENT APPLICATION NO.PCT/US 91/07654 

SA 53533 

lists (he patent family members relating to the patent documents cited in the above-mentioned international search report, 
i ne memoers are as contained in the European Patent Office EDP file on 30/12/91 
The European Patent office is in no way liable for thesepariicufars which are merely given for the purpose of information. 





Patent document 


Publication 


Patent family 


Publication 


cueo in search report 


date 




memberts) 


data 


EP-A2- 


0416578 


13/03/91 


NONE 






WO^Al- 


9011366 


04/10/90 


AU-D- 


. - , , , , 

5357790 


_ 

22/10/90 








EP-A- 


0429570 


05/06/91 


W0-A1- 


9003733 


19/04/90 


AU-B- 


615810 


10/10/91 








AU-D- 


4488689 


01/05/90 








CA-A- 


2000498 


11/04/90 








EP-A- 


0394418 


31/10/90 


W0-A1- 


8909787 


19/10/89 


AU-D- 


3444989 


03/11/89 








AU-D- 


3530589 


03/11/89 








EP-A- 


0362367 


11/04/90 








EP-A- 


0372031 


13/06/90 








JP-T- 


3500655 


14/02/91 








JP-T- 


3502579 


13/06/91 








US-A- 


4968590 


06/11/90 








WO-A- 


89/09788 


19/10/89 








US-A- 


5011691 


30/04/91 








AU-D- 


5174790 


26/09/90 








EP-A- 


0411105 


06/02/91 








JP-T- 


3504736 


17/10/91 








US-A- 


4975526 


04/12/90 








W0-A- 


90/10018 


07/09/90 



For more details about this annex : see Official Journal of the European patent Office, Ho. 12/82 



EPO FORM P0478 



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

Defective images within this document are accurate representations of the original 
documents submitted by the applicant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
Of FADED TEXT OR DRAWING 
[^BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

□ REFERENCE(S) OR EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: . 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.