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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT> 



(51) International patent Classification 
C07K 1MK), A61L 27/00, C12N 154)0 
A61K3S32,C12P 21/02 



A2 



(11) International Publication Number: WO 89/09787 

(43) International Publication Date: 19 October 1989 (19.10.89) 



(21) International Application Number: PCT/US89/01453 

(22) Internationa! Filing Date : 7 April 1989 (07.04.89) 



(30) Priority data: 
179,406 
232,630 
315,342 



8 April 1988 (08.04.88) 
15 August 1988 (15.08.88) 
23 February 1989 (23.02.89) 



US 
US 
US 



(60) Parent Applications or Grants 
(63) Related by Continuation 
US 

Filed on 
US 

Filed on 
US 

Filed on 



179,406 (CIP) 
8 April 1988(08.04.88) 
232,630 (CIP) 
15 August 1988(15.08.88) 
315,342 (CIP) 
23 February 1989 (23.02.89) 



(71) Applicant (for all designated States except US): CREATIVE 
BIOMOLECULES, INC. [US/US]; 35 South Street, 
Hopkinton, MA 01748 (US). 

(75) In^entors/Appli (for US only) ; KUBERASAMPATH, 
Thangavel [IN/US]; 6 Spring Street, Medway, MA 
02053 (US). OPPERMANN, Hermann [US/US]; 25 
Summer Hill Road, Medway, MA 02053 (US). RUE- 
GER, David, C. [US/US]; 



150 Edgemere Road, Apt. 4, West Roxbury, MA 02132 (US). 
OZKAYNAK, Engin [TR/US]; 44 Purdue Dnve, Milford, MA 
01757 (US). 

(74) Agent: PITCHER, Edmund, R.; Lahive & Cockfleld, 60 
State Street, Boston, MA 02109 (US). 

(81) Designated States: AT (European patent), AU, BB, BE 
(European patent), BF (OAPI patent), BG, BJ (OAPI 
patent), BR, CF (OAPI patent), CG (OAPI patent), CH 
(European patent), CM (OAPI patent), DE (European 
patent), DK, FI, FR (European patent), GA (OAPI pa- 
tent), GB (European patent), HU, IT (European patent), 
JP, KP, KR, LK, LU (European patent), MC, MG, ML 
(OAPI patent), MR (OAPI patent), MW, NL (European 
patent), NO, RO, SD, SE (European patent), SN (OAPI 
patent), 5U, TD (OAPI patent), TG (OAPI patent), US, 



Published , ... . , 

Without international search report and to be republished 
upon receipt of that report. 



(54) Title: OSTEOGENIC DEVICES 



(57) Abstract 



Disclosed are 1) osteogenic devices comprising a matrix containing osteogenic protein and methods of inducing endochon- 
dral bone growth in mammals using the devices; 2) amino acid sequence data, amino acid composition solubility properties, 
structural features, homologies and various other data characterizing osteogenic proteins, and 3) methods of producing osteogen- 
ic proteins using recombinant DNA technology. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AT Austria 

AU Australia 

BB Barbados 

BE Belgium 

BG Bulgaria 

BJ Benin 

BR Brazil 

CF Central African Republic 

CG Congo 

CH Switzerland 

CM Cameroon 

DE -Germany, Federal Republic of 

DK Denmark 

FT Finland 



FR France 

GA Gabon 

GB United Kingdom 

HU Hungary 

IT Italy 

JP Japan 

KP Democratic People's Republic 

of Korea' 

KR Republic of Korea 

LI Liechtenstein 

LK Sri Lanka 

LU Luxembourg 

MC Monaco 

MG Madagascar 



ML 


Mali 


MR 


Mauritania 


MW 


Malawi 


NX 


Netherlands 


NO 


Norway 


RO 


Romania 


SD 


Sudan 


SE 


Sweden 


SN 


'Senegal 


su 


Soviet Union 


TD 


Chad 


TG 


Togo 


US 


United "States of America 



WO 89/09787 



PCT/US89/014S3 



-1- 

OSTEOGENIC PEVICEg 



This invention relates to osteogenic 
devices, to genes encoding proteins which can induce 
osteogenesis in mammals and methods for their 
production using recombinant DNA techniques, to a 
method of reproducibly purifying osteogenic protein 
from mammalian bone, and to bone and cartilage repair 
procedures using the osteogenic device. 

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

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



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PCT/US89/01453 



- 2 - 

Though the precise mechanisms underlying 
these phenotypic transformations are unclear, it has 
been shown that the natural endochondral bone 
differentiation activity of bone matrix can be . 
dissociatively extracted and reconstituted with 
inactive residual collagenous matrix to restore full 
bone induction activity (Sampath and Reddi, (1981) 
Proc. Natl. Acad. Sci. USA 7J$.:7599-7603) . This 
provides an experimental method for assaying protein 
extracts for their ability to induce endochondral 
bone in vivo . 

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

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

The observed properties of these protein 
fractions have induced an intense research effort in 
various laboratories directed to isolating and 
identifying the pure factor or factors responsible 
for osteogenic activity. The current state of the 
art of purification of osteogenic protein from 



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PCT/US89/01453 



mammalian bone is disclosed by Sampath et al. (PrpQ. 
Natl , Acad. Sci. USA (1987) M) • Urist et al. (Proc. 
Soc. Exp. Biol. Med. (1984) 173:194-199) disclose a 
human osteogenic protein fraction which was extracted 
from demineralized cortical bone by means of a 
calcium chloride-urea inorganic-organic solvent 
mixture, and retrieved by differential precipitation 
in guanidine-hydrochloride and preparative gel 
electrophoresis. The authors report that the protein 
fraction has an amino acid composition of an acidic 
polypeptide and a molecular weight in a range of 
17-18 kD. 

Urist et al. (Proc. Natl. Acad. Sci. USA 
(1984) £1:371-375) disclose a bovine bone 
morphogenetic protein extract having the properties 
of an acidic polypeptide and a molecular weight of 
approximately 18 kD. The authors reported that the 
protein was present in a fraction separated by 
hydroxyapatite chromatography, and that it induced 
bone formation in mouse hindquarter muscle and bone 
regeneration in trephine defects in rat and dog 
skulls. Their method of obtaining the extract from 
bone results in ill-defined and impure preparations, 

European Patent Application Serial No. 
148,155, published October 7, 1985, purports to 
disclose osteogenic proteins derived from bovine, 
porcine, and human origin. One of the proteins, 
designated by the inventors as a P3 protein having a 
molecular weight of 22-24 kD, is said to have been 



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PCT/US89/01453 



purified to an essentially homogeneous state. This 
material is reported to induce bone formation when 
implanted into animals. 

International Application No. PCT/087/01537, 
published January 14/ 1988, discloses an impure 
fraction from bovine bone which has bone induction 
qualities. The named applicants also disclose 
putative bone inductive factors produced by 
recombinant DNA techniques. Four DNA sequences were 
retrieved from human or bovine genomic or cDNA 
libraries and apparently expressed in recombinant 
host cells. While the applicants stated that the 
expressed proteins may be bone morphogenic proteins, 
bone induction was not demonstrated. See also Urist 
et al., EP 0,212,474 entitled Bone Morphogenic Agents. 

Wang et al. (Proc. Nat. Acad. Sci. USA 
(1988) £5.: 9484-9488) discloses the purification of a 
bovine bone morphogenetic protein from guanidine 
extracts of demineralized bone having cartilage and 
bone formation activity as a basic protein 
corresponding to a molecular weight of 30 kD 
determined from gel elution. Purification of the 
protein yielded proteins of 30, 18 and 16 kD which, 
upon separation, were inactive. In view of this 
result, the authors acknowledged that the exact 
identity of the active material had not been 
determined. 



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PCT/US89/014S3 



5 



Wozney et al. (Science (1988) 2A2: 
1528-1534) discloses the isolation of full-length 
cDNA's encoding the human equivalents of three 
polypeptides originally purified from bovine bone. 
The authors report that each of the three 
recombinantly expressed human proteins are 
independently or in combination capable of inducing 
cartilage formation. No evidence of bone formation 
is reported. 

It is an object of this invention to provide 
osteogenic devices comprising matrices containing 
dispersed osteogenic protein capable of bone 
induction in allogenic and xenogenic implants. 
Another object is to provide a reproducible method of 
isolating osteogenic protein from mammalian bone 
tissue. Another object is to characterize the 
protein responsible for osteogenesis. Another object 
is to provide natural and recombinant osteogenic 
proteins capable of inducing endochondral bone 
formation in mammals, including humans. Yet another 
object is to provide genes encoding osteogenic 
proteins and methods for their production using 
recombinant DNA techniques. Another object is to 
provide methods for inducing cartilage formation. 

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



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PCT/US89/01453 



Summary of the Invention 

This invention involves osteogenic devices 
which, when implanted in a mammalian body, can induce 
at the locus of the implant the full developmental 
cascade of endochondral bone formation and bone 
marrow differentiation. Suitably modified as 
disclosed herein, the devices also may be used to 
induce cartilage formation. The devices comprise a 
carrier material, referred to herein as a matrix, 
having the characteristics disclosed below, 
containing dispersed osteogenic protein either in its 
native form as purified from natural sources or 
produced using recombinant DNA techniques. 

Key to these developments was the successful 
development of a protocol which results in retrieval 
of active, substantially pure osteogenic protein from 
mammalian bone, and subsequent elucidation of amino 
acid sequence and structure data of native osteogenic 
protein. The protein has a half -maximum bone forming 
activity of about 0.8 to 1.0 ng per mg of implant. 
The protein is believed to be a dimer. It appears 
not to be active when reduced. Various combinations 
of species of the proteins may exist as heterodimers 
or homodimers. 

The invention provides native forms of 
osteogenic protein, extracted from bone or produced 
using recombinant DNA techniques. The substantially 



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PCT/US89/01453 



pure osteogenic protein may include forms having 
varying glycosylation patterns, varying N-termini, a 
family of related proteins having regions of amino 
acid sequence homology, and active truncated or 
mutated forms of native protein, no matter how 
derived. The naturally sourced osteogenic protein in 
its native form is glycosylated and has an apparent 
molecular weight of about 30 kD as determined by 
SDS-PAGE. When reduced, the 30 kD protein gives rise 
to two glycosylated polypeptide chains having 
apparent molecular weights of about 16 kD and 18 kD. 
In the reduced state, the 30 kD protein has no 
detectable osteogenic activity. The deglycosylated 
protein, which has osteogenic activity, has an 
apparent molecular weight of about 27 kD. When 
reduced, the 27 kD protein gives rise to the two 
deglycosylated polypeptides have molecular weights of 
about 14 kD to 16 kD. 

Analysis of intact molecules and digestion 
fragments indicate that the native 30 kD osteogenic 
protein contains the following amino acid sequences 
(question marks indicate undetermined residues) : 

( 1 ) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K ; 

(2) S-L-K-P-S-N-Y-A-T-I-Q-S-I-V; 

(3) A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K; 

(4) M-S-S-L-S-I-L-F-F-D-E-N-K; 

(5) S-Q-E-L-Y-V-D-F-Q-R; 

(6) F-L-H-C-Q-F-S-E-R-N-S; 

(7) T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y; 



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PCT/US89/01453 



(8) L-Y-D-P-M-V-V; 

(9) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E ; 

(10) V-D-F-A-D-I-G; 

(11) V-P-K-P-C-C-A-P-T ; 

(12) I-N-I-A-N-Y-L; 

( 13 ) D-N-H-V-L-T-M-F-P-I-A-I-N ; 

( 14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-7-P; 

(15) D-I-G-?-S-E-W-I-I-?-P; 

( 16) S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-?-V; 

(17) D-7-I-V-A-P-P-Q-Y-H-A-F-Y; 

(18) D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E ; 

( 19) S-Q-T-L-Q-F-D-E-Q-T-L-K-7-A-R-7-K-Q; 

( 20 ) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-E-P-R-N-? -A-R-R-Y-L ; 

(21) A-R-R-K-Q-W-I-E-P-R-N-? -A-? -R-Y-? -? -V-D ; and 

(22) R-? -Q-W-I -E-P-? -N-? -A-? -? -Y-L-K-V-D-? -A-? -? -G . 

The availability of the protein in 
substantially pure form, and knowledge of its amino 
acid sequence and other structural features, enable 
the identification, cloning, and expression of native 
genes which encode osteogenic proteins. When 
properly modified after translation, incorporated in 
a suitable matrix, and implanted as disclosed herein, 
these proteins are operative to induce formation of 
cartilage and endochondral bone. 



Consensus DNA sequences designed as 
disclosed herein based on partial sequence data and 
observed homologies with regulatory proteins 
disclosed in the literature are useful as probes for 
extracting genes encoding osteogenic protein from 
genomic and cDNA libraries. One of the consensus 



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PCT/US89/014S3 



sequences has been used to isolate a heretofore 
unidentified genomic DNA sequence, portions of which 
when ligated encode a protein having a region capable 
of inducing endochondral bone formation. This 
protein, designated OP1, has an active region having 
the sequence set forth below. 

1 10 20 30 40 

OP1 LYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFI NPET- VPKPCC APTQLNA 

80 90 100 

I S VLY FDD S SNV I LKKYRNMWRAC GCH 

A longer active sequence is: 

-5 
HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100 

I SVL YFDDS SNV I LKKYRNMWRACGCH 

Fig. 1A discloses the genomic DNA sequence of OP1. 



The probes have also retrieved the DNA sequences 
identified in PCT/087/01537, referenced above, 
designated therein as BMPII(b) and BMPIII. The 
inventors herein have discovered that certain 
subparts of these genomic DNAs, and BMPIIa, from the 
same publication, when properly assembled, encode 
proteins (CBMPIIa, CBMPIIb, and CBMPIII) which have 
true osteogenic activity, i.e., induce the full 
* cascade of events when properly implanted in a mammal 



WO 89/09787 



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PCT/US89/01453 



leading to endochondral bone formation. These 
sequences are: 



1 10 20 30 40 

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-K-IPKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQDMWEGCGCR 

1 10 20 30 40 

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD 

50 60 70 

HLNSTN — H-A1VQTLVNSVNS-S-IPKACCVPTELSA 

80 90 100 

I SMLYLDEYDKWLKNYQEMWEGCGCR 



1 10 20 30 40 

CBMP-3 CARRYLKVDFA-DIGWSEWI ISPKSFDAYYCSGACQFPMPK 

50 60 70 

SLKPSN — H-ATIQSIVRAVGWPGIPEPCCVPEKMSS 

80 90 100 

LSI LFFDENKNWLKVYFNMTVE S CACR 



Thus, in view of this disclosure, skilled 
genetic engineers can isolate genes from cDNA or 
genomic libraries which encode appropriate amino 
acid sequences, 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. 



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PCT/US89/01453 



The substantially pure osteogenic 
proteins (i.e., naturally derived or recombinant 
proteins free of contaminating proteins having no 
osteoinductive activity) are useful in clinical 
applications in conjunction with a suitable 
delivery or support system (matrix). The matrix 
is made up of particles or porous materials. The 
pores must be of a dimension to permit progenitor 
cell migration and subsequent differentiation and 
proliferation. The particle size should be within 
the range of 70 - 850 ym, preferably 70 - 420 ym. 
It may be fabricated by close packing particulate 
material into a shape spanning the bone defect, or 
by otherwise structuring as desired a material 
that is biocompatible (non-inflammatory) and, 
biodegradable in vivo to serve as a "temporary 
scaffold" and substratum for recruitment of 
migratory progenitor cells, and as a base for 
their subsequent anchoring and proliferation. 
Currently preferred carriers include particulate, 
demineralized, guanidine extracted, 
species-specific (allogenic) bone, and 
particulate, deglycosylated (or HF treated), 
protein extracted, demineralized, xenogenic bone. 
Optionally, such xenogenic bone powder matrices 
also may be treated with proteases such as 
trypsin. Other useful matrix materials comprise 
collagen, homopolymers and copolymers of glycolic 
acid and lactic acid, hydroxyapatite, tricalcium 
phosphate and other calcium phosphates. 



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PCT/US89/0I453 



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 periodontal applications where bone 
formation is required. Another potential clinical 
application is in cartilage repair, for example, 
in the treatment of osteoarthritis. 



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PCT/US89/01453 



Brief Desc ription of the Drawing 

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

FIGURE 1A represents the nucleotide sequence 
of the genomic copy of osteogenic protein "OPl" 
gene. The unknown region between 1880 and 1920 
actually represents about 1000 nucleotides; 

FIGURE IB is a representation of the 
hybridization of the consensus gene/probe to the 
osteogenic protein "OP1" gene; 

FIGURE- 2 is a collection of plots of protein 
concentration (as indicated by optical absorption) vs 
elution volume illustrating the results of bovine 
osteogenic protein (BOP) fractionation during 
purification on heparin-Sepharose-I; HAP-Ultragel ; 
sieving gel (Sephacryl 300); and heparin-Sepharose-II ; 

FIGURE 3 is a photographic reproduction of a 
Coomassie blue stained SDS polyacrylamide gel of the 
osteogenic protein under non-reducing (A) and 
reducing (B) conditions; 

FIGURE 4 is a photographic reproduction of a 
Con A blot of an SDS polyacrylamide gel showing the 
carbohydrate component of oxidized (A) and reduced 
(B) 30 kD protein; 



89/09787 



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PCT/US89/01453 



FIGURE 5 is a photographic reproduction of 
an autoradiogram of an SDS polyacrylamide gel of 
125 I-labelled glycosylated (A) and deglycosylated (B) 
osteogenic protein under non-reducing (1) and 
reducing (2) conditions; 

FIGURE 6 is a photographic reproduction of 
an autoradiogram of an SDS polyacrylamide gel of 
peptides produced upon the digestion of the 30 kD 
osteogenic protein with V-8 protease (B) , Endo Lys C 
protease (C), pepsin (D) , and trypsin (E) . (A) is 
control; 

FIGURE 7 is a collection of HPLC 
chromatograms of tryptic peptide digestions of 30 kD 
BOP (A) , the 16 kD subunit (B) , and the 18 kD subunit 
<C); 

FIGURE 8 is an HPLC chromatogram of an 
elution profile on reverse phase C-18 HPLC of the 
samples recovered from the second heparin-Sepharose 
chromatography step (see FIGURE 2D), Superimposed is 
the percent bone formation in each fraction; 

FIGURE 9 is a gel permeation chromatogram of 
an elution profile on TSK 3000/2000 gel of the C-18 
purified osteogenic peak fraction. Superimposed is 
the percent bone formation in each fraction; 

FIGURE 10 is a collection of graphs of 
protein concentration (as indicated by optical 
absorption) vs. elution volume illustrating the 
results of human protein fractionation on 



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PCT/US89/01453 



heparin-Sepharose I (A), HAP-Ultragel <B), TSK 
3000/2000 (C), and heparin-Sepharose II (D) . Arrows 
indicate buffer changes; 

FIGURE 11 is a graph showing representative 
dose response curves for bone-inducing activity in 
samples from various purification steps including 
reverse phase HPLC on C-18 (A), Heparin-Sepharose II 
(B) , TSK 3000 (C), HAP-ultragel (D) , and 
Heparin-Sepharose I (E); 

FIGURE 12 is a bar graph of 
radiomorphometric analyses of feline bone defect 
repair after treatment with an osteogenic device (A) , 
carrier control (B) , and demineralized bone (C) ; 

FIGURE 13 is a schematic representation of 
the DNA sequence and corresponding amino acid 
sequence of a consensus gene/probe for osteogenic 
protein (COPO); 

FIGURE 14 is a graph of osteogenic activity 
vs. increasing molecular weight showing peak bone 
forming activity in the 30 kD region of an SDS 
polyacrylamide gel; 

FIGURE 15 is a photographic representation 
of a Coomassie blue stained SDS gel showing gel 
purified subunits of the 30 kD protein; 

FIGURE 16 is a pair of HPLC chromatograms of 
Endo Asp N proteinase digests of the 18 kD subunit 
(A) and the 16 kD subunit (B); 



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PCT/US89/01453 



FIGURE 17 is a photographic representation 
of the histological examination of bone implants in 
the rat model: carrier alone (A); carrier ana 
glycosylated osteogenic protein (B) ; and carrier and 
deglycosylated osteogenic protein (C) . Arrows 
indicate osteoblasts; 

FIGURE 18 is a graph illustrating the 
activity of xenogenic matrix (deglycolylated bovine 
matrix) ; and 

FIGURES -19A and 19B are bar graphs showing 
the specific activity of naturally sourced OP before 
and after gel elution as measured by calcium content 
vs. increasing concentrations of proteins (dose 
curve, in ng) . 



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PCT/US89/01453 



Description 

Purification protocols have been developed 
which enable isolation of the osteogenic protein 
present in crude protein extracts from mammalian 
bone. While each of the separation steps constitute 
a known separation technique, it has been discovered 
that the combination of a sequence of separations 
exploiting the protein's affinity for heparin and for 
hydroxyapatite (HAP) in the presence of a denaturant 
such as urea is key to isolating the pure protein 
from the crude extract. These critical separation 
steps are combined with separations on hydrophobic 
media, gel exclusion chromatography, and elution form 
SDS PAGE. 

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



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PCT/US89/014S3 



it is active in deglycosylated form. It has been 
partially sequenced. Its primary structure includes 
the amino acid sequences set forth herein. 

Elucidation of the amino acid sequence of 
BOP enables the construction of pools of nucleic acid 
probes encoding peptide fragments. Also, a consensus 
nucleic acid sequence designed as disclosed herein 
based on the amino acid sequence data, inferred 
codons for the sequences, and observation of partial 
homology with known genes, also has been used as a 
probe. The probes may be used to isolate naturally 
occuring cDNAs which encode active mammalian 
osteogenic proteins (OP) as described below using 
standard hybridization methodology. The mRNAs are 
present in the cytoplasm of cells of various species 
which are known to synthesize osteogenic proteins. 
Useful cells harboring the mRNAs include, for 
example, osteoblasts from bone or osteosarcoma, 
hypertrophic chondrocytes, and stem cells. The mRNAs 
can be used to produce cDNA libraries. 
Alternatively, relevant DNAs encoding osteogenic 
protein may be retrieved from cloned genomic DNA 
libraries from various mammalian species. 

These discoveries enable the construction of 
DNAs encoding totally novel, non-native protein 
constructs which individually, and combined are 
capable of producing true endochondral bone. They 
also permit expression of the natural material, 
truncated forms, muteins, analogs, fusion proteins, 



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PCT/US89/01453 



and various other variants and constructs, from cDNAs 
retrieved from natural sources or synthesized using 
the techniques disclosed herein using automated, 
commercially available equipment. The DNAs may be 
expressed using well established recombinant DNA 
technologies in procaryotic or eucaryotic host cells, 
and may be oxidized and refolded in vit.ro. if 
necessary for biological activity. 

The isolation procedure for obtaining the 
protein from bone, the retrieval of an osteogenic 
protein gene, the design and production of 
recombinant protein, the nature of the matrix, and 
other material aspects concerning the nature, 
utility, how to make, and how to use the subject 
matter claimed herein will be further understood from 
the following, which constitutes the best method 
currently known for practicing the various aspects of 
the invention. 



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PCT/US89/01453 



A - PURIFICATION OF BOP 

Al. Preparation of Demineralized Bone 

Demineralized bovine bone matrix is prepared 
by previously published procedures (Sampath and Reddi 
(1983) Proc. Natl. Acad. Sci. USA £0: 6591-6595) . 
Bovine diaphyseal bones (age 1-10 days) are obtained 
from a local slaughterhouse and used fresh. The 
bones are stripped of muscle and fat, cleaned of 
periosteum, demar rowed by pressure with cold water, 
dipped in cold absolute ethanol, and stored at 
-20°C. They are then dried and fragmented by 
crushing and pulverized in a large mill. Care is 
taken to prevent heating by using liquid nitrogen. 
The pulverized bone is milled to a particle size 
between 70-420 ym and is defatted by two washes of 
approximately two hours duration with three volumes 
of chloroform and methanol (3:1). The particulate 
bone is then washed with one volume of absolute 
ethanol and dried over one volume of anhydrous 
ether. The defatted bone powder (the alternative 
method is to obtain Bovine Cortical Bone Powder 
(75-425 ym) from American Biomaterials) is then 
demineralized with 10 volumes of 0.5 N HC1 at 4°C for 
40 min. , four times. Finally, neutralizing washes 
are done on the demineralized bone powder with a 
large volume of water. 



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A2, Dissociative Extraction and Etha nol Precipitation 

Demineralized bone matrix thus prepared is 
dissociatively extracted with 5 volumes of 4 M 
guanidine-HCl (Gu-HCl), 50mM Tris-HCl, pH 7.0, 
containing protease inhibitors (5 mM benzamidine, 44 
mM 6-aminohexanoic acid, 4.3 mM N-ethylmaleimide, 
0.44 mM phenylmethylsulfonyf luoride) for 16 hr. at 
4°C. The suspension is filtered. The supernatant is 
collected and concentrated to one volume using an 
ultrafiltration hollow fiber membrane (Amicon, 
YM-10). The concentrate is centrifuged (8,000 x g 
for 10 min. at 4°C), and the supernatant is then 
subjected to ethanol precipitation. To one volume of 
concentrate is added five volumes of cold (-70°C) 
absolute ethanol (100%), which is then kept at -70°C 
for 16 hrs. The precipitate is obtained upon 
centrifugation at 10,000 x g for 10 min. at 4°C. The 
resulting pellet is resuspended in 4 1 of 85% cold 
ethanol incubated for 60 min. at -70°C and 
recentrifuged. The precipitate is again resuspended 
in 85% cold ethanol (2 1), incubated at -70°C for 60 
min. and centrifuged. The precipitate is then 
lyophilized. 

A3. Hftnarin-sepharose Chromatography I 

The ethanol precipitated, lyophilized, 
extracted crude protein is dissolved in 25 volumes of 
6 M urea, 50 mM Tris-HCl, pH 7.0 (Buffer A) 
containing 0.15 M NaCl, and clarified by 
centrifugation at 8,000 x g for 10 min. The 



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heparin-Sepharose is column-equilibrated with Buffer 
A* The protein is loaded onto the column and after 
washing with three column volume of initial buffer 
(Buffer A containing 0.15 M NaCI), protein is eluted 
with Buffer A containing 0.5 M NaCI. The absorption ^ 
of the eluate is monitored continuously at 280 nm. 
The pool of protein eluted by 0.5 M NaCI * 
(approximately 1 column volumes) is collected and 
stored at 4°C. 

As shown in FIGURE 2A, most of the protein 
(about 95%) remains unbound. Approximately 5% of the 
protein is bound to the column. The unbound fraction 
has no bone inductive activity when bioassayed as a 
whole or after a partial purification through 
Sepharose CL-6B. 

A4. Hydroxvapaptite-Ultrogel Chromatography 

The volume of protein eluted by Buffer A 
containing 0.5 M NaCI from the heparin-Sepharose is 
applied directly to a column of hydroxyapaptite- 
ultrogel (HAP-ultrogel) (LKB Instruments), 
equilibrated with Buffer A containing 0.5 M NaCI. 
The HAP-ultrogel is treated with Buffer A containing 
500 iriM Na phosphate prior to equilibration. The 
unadsorbed protein is collected as an unbound 
fraction, and the column is washed with three column 
volumes of Buffer A containing 0.5 M NaCI. The 
column is subsequently eluted with Buffer A 
containing 100 mM Na Phosphate (FIGURE 2B) . 



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The eluted component can induce endochondral 
bone as measured by alkaline phosphatase activity and 
histology. As the biologically active protein is 
bound to HAP in the presence of 6 M urea and 0.5 M 
NaCl, it is likely that the protein has an affinity 
for bone mineral and may be displaced only by 
phosphate ions. 

A5 . Rp.phanrvl S-300 Gel Exclusi on Chromatography 

Sephacryl S-300 HR (High Resolution, 5 cm x 
100 cm column) is obtained from Pharmacia and 
equilibrated with 4 M guanidine-HCl, 50 mM Tris-HCl, 
pH 7.0. The bound protein fraction from HA-ultrogel 
is concentrated and exhanged from urea to 4 M 
guanidine-HCl, 50 mM Tris-HCl, pH 7.0 via an Arnicon 
ultrafiltration YM-10 membrane. The solution is then 
filtered with Schleicher and Schuell CENTREX 
disposable microf ilters . A sample aliquot of 
approximately 15 ml containing approximately 400 mg 
of protein is loaded onto the column and then eluted 
with 4 M guanidine-HCl, 50 mM Tris-HCl, pH 7.0, with 
a flow rate of 3 ml/min; 12 ml fractions are 
collected over 8 hours and the concentration of 
protein is measured at A 2 80 nm (FIGURE 2C) . An 
aliquot of the individual fractions is bioassayed for 
bone formation. Those fractions which have shown 
bone formation and migrate with an apparent molecular 
weight of less than 35 kD are pooled and concentrated 
via an Amicon ultrafiltration system with YM-10 
membrane. 



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A6. Heparin-Sepharose Chromatography-!! 

The pooled osteo-inductive fractions 
obtained from gel exclusion chromatography are 
dialysed extensively against distilled water (dH2<> 
and then against 6 M urea, 50 iriM Tris-HCl, pH 7.0 
(Buffer A) containing 0.1 M NaCl. The dialysate is 
then cleared through centrifugation. The sample is 
applied to the heparin-sepharose column (equilibrated 
with the same buffer). After washing with three 
column volumes of initial buffer, the column is 
developed sequentially with Buffer B containing 0.15 
M NaCl, and 0.5 M NaCl (FIGURE 2D). The protein 
eluted by 0.5 M NaCl is collected and dialyzed 
extensively against distilled water. It is then 
dialyzed against 30% acetonitrile, 0.1% TFA at 4°C. 

A7. Reverse Phase HPLC 

The protein is further purified by C-18 
Vydac silica-based HPLC column chromatography 
(particle size 5 ym; pore size 300 A). The 
osteoinductive fraction obtained from 
heparin-sepharose-II chromatograph is loaded onto the 
column, and washed in 0.1% TFA, 10% acetonitrile for 
five min. As shown in FIGURE 8, the bound proteins 
are eluted with a linear gradient of 10-30% 
acetonitrile over 15 min., 30-50% acetonitrile over 
60 min, and 50-70% acetonitrile over 10 min at 22°C 
with a flow rate of 1.5 ml/min and 1.4 ml samples are 
collected in polycarbonate tubes. Protein is 



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monitored by absorbance at A214 nm. Column fractions 
are tested for the presence of osteoinductive 
activity, and concanavalin A-blottable proteins. 
These fractions are then pooled, and characterized 
biochemically for the presence of 30 kD protein by 
autoradiography, concanavalin A blotting, and 
Coomassie blue dye staining. They are then assayed 
for in vivo osteogenic activity. Biological activity 
is not found in the absence of 30 kD protein. 

A8 . fiftl Elution 

The glycosylated or deglycosylated protein 
is eluted from SDS gels (0.5 mm thickness) for 
further characterization. 125i_i a belled 30 kD 
protein is routinely added to each preparation to 
monitor yields. TABLE 1 shows the various elution 
buffers that have been tested and the yields of 
125i_i a belled protein. 

TABLE 1 

pim-ion of 30 kD P rnfftin from SDS Gel 

Buffer 

(1) dH 2 0 

(2) 4 M Guanidine-HCl, Tris-HCl, pH 7.0 

(3) 4 M Guanidine-HCl, Tris-HCl, pH 7.0, 
0.5% Triton x 100 

(4) 0.1% SDS, Tris-HCl, pH 7.0 



% Eluted 
22 

2 

93 

98 



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TABLE 2 lists the steps used to isolate the 
30 kD or deglycosylated 27 kD gel-bound protein. The 
standard protocol uses diffusion elution using 4M 
guanidine-HCl containing 0.5% Triton x 100 in 
Tris-HCl buffer or in Tris-HCl buffer containing 0.1% 
SDS to achieve greater than 95% elution of the 
protein from the 27 or 30 kD region of the gel for 
demonstration of osteogenic activity in vivo as 
described in later section. 



TABLE 2 

Preparati on of Gel Eluted Protein 

(C-18 Pool or deglycoslated protein plus 
l2 5l-labelled 30 kD protein) 

Dry using vacuum centrifugation; 
Wash pellet with H2O; 

Dissolve pellet in gel sample buffer (no reducing 
agent) ; 

Electrophorese on pre-electrophoresed 0.5 mm mini 
gel; 

Cut out 27 or 30 kD protein; 

Elute from gel with 0.1% SDS, 50mM Tris-HCl, pH 
7.0; 

Filter through Centres membrane; 
Concentrate and wash with water in Centricon 
tube (10 kD membrane) . 



1. 
2. 
3. 

4. 

5. 
6. 

7. 
8. 



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The overall yield of labelled 30 kD protein 
from the gel elution protocol is 50 - 60% of the 
loaded sample. Most of the loss occurs in the 
electrophoresis step, due to protein aggregation 
and/or smearing. 

The yield is 0.5 to 1.0 pg substantially 
pure osteogenic protein per kg of bone. 

A9. isolation r>f the 16 kD and IB kP Species 

TABLE 3 summarizes the procedures involved 
in the preparation of the subunits. Approximately 10 
v g of gel eluted 30 kD protein (FIGURE 3) is 
carboxymethylated and electrophoresed on an SDS-gel. 
The sample contains ^Sj-iabel to trace yields and to 
use as an indicator for slicing the 16 kD and 18 kD 
regions from the gel. FIGURE 15 shows a Coomassie 
blue stained gel of gel-purified 16 kD and 18 kD 
proteins. 



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TABLE 3 

Isolation of the Subunits of the 30 kD protein 
(C-18 pool plus 125 I-labeled 30 kD protein) 



1. Electrophorese on SDS gel. 

2. Cut out 30 kD protein, 

3. Elute with 0.1% SDS, 50 iriM Tris-HCl, pH 7.0. 

4. Concentrate and wash with H2O in Centricon 
tube (10 kD membranes). 

5. Electrophorese reduced sample- on SDS gel. 

6. Cut out the 16 kD and 18 kD subunits. 

7. Elute with 0.1% SDS, 50 mM Tris-HCl, pH 7.0. 

8. Concentrate and wash with H2O in Centricon 
tubes. 

9. Reduce and carboxymethylate in 1% SDS, 0.4 M 
Tris-HCl, pH 8.5. 

10. Concentrate and wash with H2O in Centricon 
tube. 



B. Biological Characterization of BOP 



Bl. Gel Slicing: 



Gel slicing experiments confirm that the 
isolated 30 kD protein is the protein responsible for 
osteogenic activity* 



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Gels from the last step of the purification 
are. sliced. Protein in each fraction is extracted in 
15 mM Tris-HCl, pH 7.0 containing 0.1% SDS or in 
buffer containing 4 M guanidine-HCl, 0.5% non-ionic 
detergent (Triton x 100), 50 mM Tris-HCl. The 
extracted proteins are desalted, concentrated, and 
assayed for endochondral bone formation activity. 
The results are set forth in FIGURE 14. From this 
figure it is clear that the majority of osteogenic 
activity is due to protein at 30 kD region of the 
gel. Activity in higher molecular weight regions is 
apparently due to protein aggregation. These protein 
aggregates, when reduced, yields the 16 kD and 18 kD 
species discussed above. 

B2. rnn A-Sep harose Chromatography; 

A sample containing the 30 kD protein is 
solubilized using 0.1% SDS, 50 mM Tris-HCl, and is 
applied to a column of concanavalin A (Con 
A)-Sepharose equilibrated with the same buffer. The 
bound material is eluted in SDS Tris-HCl buffer 
containing 0.5 M alpha-methyl mannoside. After 
reverse phase chromatography of both the bound and 
unbound fractions, Con A-bound materials, when 
implanted, result in extensive bone formation. 
Further characterization of the bound materials show 
a Con A-blottable 30 kD protein. Accordingly, the 30 
kD glycosylated protein is responsible for the bone 
forming activity. 



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B3. Gel Permeation Chromatography: 

TSK-3000/2000 gel permeation chromatography 
in guanidine-HCl alternately is used to achieve 
separation of the high specific activity fraction 
obtained from C-18 chromatography (FIGURE 9), The 
results demonstrate that the peak of bone inducing 
activity elutes in fractions containing substantially 
pure 30 kD protein by Coomassie blue staining. When 
this fraction is iodinated and subjected to 
autoradiography, a strong band at 30 kD accounts for 
90% of the iodinated proteins. The fraction induces 
bone formation in vivo at a dose of 50 to 100 ng per 
implant. 

B4. Structural Requirements for Biological Activity 

B4-1 Activity after Digestion 

Although the role of 30 kD osteogenic 
protein is clearly established for bone induction, 
through analysis of proteolytic cleavage products we 
have begun to search for a minimum structure that is 
necessary for activity in vivo . The results of 
cleavage experiments demonstrate that pepsin 
treatment fails to destroy bone inducing capacity, 
whereas trypsin or CNBr completely abolishes the 
activity. 



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An experiment is performed to isolate and 
identify pepsin digested product responsible for 
biological activity. The sample used for pepsin 
digestion was 20% - 30% pure. The buffer used is 
0.1% TFA in water. The enzyme to substrate ratio is 
1:10. A control sample is made without enzyme. The 
digestion mixture is incubated at room temperature 
for 16 hr. The digested product is then separated in 
4 M guanidine-HCl using gel permeation 
chromatography/ and the fractions are prepared for in 
vivo assay. The results demonstrate that active 
fractions from gel permeation chromotography of the 
pepsin digest correspond to peptides having an 
apparent molecular weight range of 8 kD - 10 kD. 

B4-2 TTnolvcosylated P rotein is Active 

In order to understand the importance of the 
carbohydrates moiety with respect to osteogenic 
activity, the 30 kD protein has been chemically 
deglycosylated using HF (see below). After analyzing 
an aliquot of the reaction product by Con A blot to 
confirm the absence of carbohydrate, the material is 
assayed for its activity in vivo. The bioassay is 
positive (i.e., the deglycosylated protein produces a 
bone formation response as determined by histological 
examination shown in FIGURE 17C) , demonstrating that 
exposure to HF did not destroy the biological 
function of the protein, and thus that the OP does 
not require carboyhdrate for biological activity. In 
addition, the specific activity of the deglycosylated 
protein is approximately the same as that of the 
native glycosylated protein. 



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B5. Specific Activity of BOP 

Experiments were performed 1) to determine 
the half maximal bone-inducing activity based on 
calcium content of the implant; 2) to estimate 
proteins at nanogram levels using a gel scanning 
method; and 3) to establish dose for half maximal 
bone inducing activity for gel eluted 30 kD BOP. The 
results demonstrate that gel eluted substantially 
pure 30 kD osteogenic protein induces bone at less 
than 5 ng per implant and exhibits half maximal bone 
differentiation activity at 20 ng per implant 
(approx. 25 mg) . The purification data suggest that 
osteogenic protein has been purified from bovine bone 
to 367,307 fold after the final gel elution step with 
a specific activity of 47,750 bone forming units per 
mg of protein. 

B5(a)Half Maximal Bone Differentiation Activity 

The bone inducing activity is determined 
biochemically by the specific activity of alkaline 
phosphatase and calcium content of the day 12 
implant. An increase in the specific activity of 
alkaline phosphatase indicates the onset of bone 
formation. Calcium content, on the other hand, is 
proportional to the amount of bone formed in the 
implant. The bone formation is therefore 
calculated by determining calcium content of the 
implant on day 12 in rats and expressed as bone 
forming units, which represent the amount that 
exhibits half maximal bone inducing activity compared 
to rat demineralized bone matrix. Bone induction 



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exhibited by intact demineralized rat bone matrix is 
considered to be the maximal bone-differentiation 
activity for comparison. 

B5(b) Protein Estimation Using Gel Scanning Techniques 

A standard curve is developed employing 
known amounts of a standard protein, bovine serum 
albumin. The protein at varying concentration 
(50-300 ng) is loaded on a 15% SDS gel, 
electrophoresed, stained in comassie and destained. 
The gel is scanned at predetermined settings using a 
gel scanner at 580 nm. The area covered by the 
protein band is calculated and a standard curve 
against concentrations of protein is constructed. A 
sample with an unknown protein concentration is 
electrophoresed with BSA as a standard. The lane 
containing the unknown sample is scanned, and the 
concentration of protein is determined from the area 
under the curve. 

B5(c)Gel Elution and Specific Activity 

An aliquot of C-18 highly purified active 
fraction is subjected to SDS gel and sliced according 
to molecular weights described in FIGURE 14. 
Proteins are eluted from the slices in 4 M 
guanidine-HCl containing 0.5% Triton X-100, desalted, 
concentrated and assayed for endochondral bone 
forming activity as determined by calcium content. 
The C-18 highly active fractions and gel eluted 



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substantially pure 30 kD osteogenic protein are 
implanted in varying concentrations in order to 
determine the half maximal bone inducing activity. 

FIGURE 14 shows that the bone inducing 
activity is due to proteins eluted in the 28-34 kD 
region. The recovery of activity after the gel 
elution step is determined by calcium content. 
FIGURES 19A and 19 B represent the bone inducing 
activity for the various concentrations of 30 kD 
protein before and after gel elution as estimated by 
calcium content. The data suggest that the half 
maximal activity for 30 kD protein before gel elution 
is 69 ng per 25 mg implant and is 21 ng per 25 mg 
implant after elution. TABLE 4 describes the yield, 
total specific activity, and fold purification of 
osteogenic protein at each step during purification. 
Approximately 500 ug of heparin sepharose I fraction, 
130-150 ug of the HA ultrogel fraction, 10-12 ug of 
the gel filtration fraction, 4-5 ug of the heparin 
sepharose II fraction, 0.4-0.5 ug of the C-18 highly 
purified fraction, and 20-25 ng of the gel eluted, 
substantially purified fraction is needed per 25 mg 
of implant for unequivocal bone formation for half 
maximal activity. Thus, 0.8-1.0 ng purified 
osteogenic protein per mg. of implant is required to 
exhibit half maximal bone differentiation activity in 
vivo- 



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TABLE 4 
PURIFICATION OF BOP 

Purification Protein Biological Specific Purification 

Steps <mg.) Activity Activity Fold 

Units* Units/mg. 

Ethanol 

Precipitate** 30,000# 4,000 0.13 1 
Heparin 

Sepharose I 1,200# 2,400 2.00 15 

HA-Ultrogel 300# 2,307 7.69 59 

Gel filtration 20# 1,600 80.00 6i5 
Heparin 

Sepharose II 5# 1,000 200.00 1,538 

C-18 HPLC 0.070@ 150 2,043.00 15,715 

Gel elution 0.004S 191 47,750.00 367,307 



Values are calculated from 4 kg of bovine bone matrix 
(800 g of demineralized matrix). 

* One unit of bone forming activity is defined as the 
amount that exhibits half maximal bone 
differentiation activity compared to rat 
demineralized bone matrix, as determined by calcium 
content of the implant on day 12 in rats. 

# Proteins were measured by absorbance at 280 nm. 



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@ Proteins were measured by gel scanning method 
compared to known standard protein, bovine serum 
albumin. 

** Ethanol-precipitated guanidine extract of bovine 
bone is a weak inducer of bone in rats, possibly due 
to endogenous inhibitors. This precipitate is 
subjected to gel filtration and proteins less than 50 
kD were separated and used for bioassay. 



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C. CHEMICAL CHARACTER I Z AT I ON OF BOP 

CI. Molecular Weight and Structure 

Electrophoresis of the proteins after the 
final purification step on non-reducing SDS 
polyacrylamide gels reveals a diffuse band at about 
30 kD as detected by both Coomassie blue staining 
(FIGURE 3A) and autoradiography. 

In order to extend the analysis of BOP, the 
protein was examined under reducing conditions. 
FIGURE 3B shows an SDS gel of BOP in the presence of 
dithiothreitol. Upon reduction, 30 kD BOP yields two 
species which are stained with Coomassie blue dye: a 
16 kD species and an 18 kD species. Reduction causes 
loss of biological activity. The two reduced BOP 
species have been analyzed to determine if they are 
structurally related. Comparison of the amino acid 
composition and peptide mapping of the two species 
(as disclosed below) shows little differences, 
indicating that the native protein may comprise two 
chains having significant homology, 

C2. Presence of Carbohydrate 

The 30 kD protein has been tested for the 
presence of carbohydrate by Con A blotting after 
SDS-PAGE and transfer to nitrocellulose paper. The 
results demonstrate that the 30 kD protein has a high 
affinity for Con A, indicating that the protein is 
glycosylated (FIGURE 4A) . In addition, the Con A 



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blots provide evidence for a substructure in the 30 
kD region of the gel, suggesting heterogeneity due to 
varying degrees of glycosylation. After reduction 
(FIGURE 4B), Con A blots show evidence for two major 
components at 16 kD and 18 kD. In addition, it has 
been demonstrated that no glycosylated material 
remains at the 30 kD region after reduction. 

In order to confirm the presence of 
carbohydrate and to estimate the amount of 
carbohydrate attached, the 30 kD protein is treated 
with N-glycanase, a deglycosylating enzyme with a 
broad specificity. Samples of the 125 I-labelled 30 
kD protein are incubated with the enzyme in the 
presence of SDS for 24 hours at 37 °C. As observed by 
SDS-PAGE, the treated samples appear as a prominent 
species at about 27 kD (FIGURE 5A) . Upon reduction, 
the 27 kD species is reduced to species having a 
molecular weight of about 14 kD - 16 kD (FIGURE 5B) . 

To ensure complete deglycosylation of the 
30KD protein, chemical cleavage of the carbohydrate 
moieties using hydrogen fluoride (HF) is performed. 
Active osteogenic protein fractions pooled from the 
C-18 chromatography step are dried in vacuo over P2O5 
in a polypropylene tube, and 50 yl freshly distilled 
anhydrous HF at -70 °C is added. After capping the 
tube tightly, the mixture is kept at 0°C in an 
ice-bath with occasional agitation for 1 hr. The HF 
is then evaporated using a continuous stream of dry 
nitrogen gas. The tube is removed from the ice bath 
and the residue dried in vacuo over P2O5 and KOH 
pellets. 



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Following drying, the samples are dissolved 
in 100 pi of 50% acetonitrile/0.1% TFA and aliquoted 
for SDS gel analysis, Con A binding, and biological 
assay. Aliquots are dried and dissolved in either 
SDS gel sample buffer in preparation for SDS gel 
analysis and Con a blotting or 4 M guanidine-HCl, 50 
mM Tris-HCl, pH 7.0 for biological assay. 

The results show that samples are completely 
deglycosylated by the HF treatment: Con A blots after 
SDS gel electrophoreses and transfer to Immobilon 
membrane showed no binding of Con A to the treated 
samples, while untreated controls were strongly 
positive at 30 kD. Coomassie gels of treated samples 
showed the presense of a 27 kD band instead of the 30 
kD band present in the untreated controls. 

C3. C hpmical and Enzvf ^ir Cleavage 

Cleavage reactions with CNBr are analyzed 
using Con A binding for detection of fragments 
associated with carbohydrate. Cleavage reactions are 
conducted using trif luoroacetic acid (TFA) in the 
presence and absence of CNBr. Reactions are 
conducted at 37°C for 18 hours, and the samples are 
vacuum dried. The samples are washed with water, 
dissolved in SDS gel sample buffer with reducing 
agent, boiled and applied to an SDS gel. After 
electrophoresis, the protein is transferred to 
immobilon membrane and visualized by Con A binding, 
in low concentrations of acid (1%), CNBr cleaves the 



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majority of 16 kb and 18 kD species to one product/ a 
species about 14 kD. In reactions using 10% TFA, a 
14 kD species is observed both with and without CNBr. 

Four proteolytic enzymes are used in these 
experiments to examine the digestion products of the 
30 kD protein: 1) V-8 protease; 2) Endo Lys C 
protease; 3) pepsin; and 4) trypsin. Except for 
pepsin, the digestion buffer for the enzymes is 0.1 M 
ammonium bicarbonate, pH 8.3. The pepsin reactions 
are done in 0.1% TFA. The digestion volume is 100 jil 
and the ratio of enzyme to substrate is 1:10. 
125 I-labelled 30 kD osteogenic protein is added for 
detection. After incubation at 37°C for 16 hr., 
digestion mixtures are dried down and taken up in gel 
sample buffer containing dithiothreitol for 
SDS-PAGE. FIGURE 6 shows an autoradiograph of an SDS 
gel of the digestion products. The results show that 
under these conditions/ only trypsin digests the 
reduced 16 kD/18 kD species completely and yields a 
major species at around 12 kD. Pepsin digestion 
yields better defined, lower molecular weight 
species. However, the 16 kD/18 kD fragments were not 
digested completely. The V-8 digest shows limited 
digestion with one dominant species at 16 kD. 

C4. Protein Seouencino 

To obtain amino acid sequence data, the 
protein is cleaved with trypsin or Endoproteinase 
Asp-N (EndoAsp-N) . The tryptic digest of reduced and 
carboxymethylated 30 kD protein (approximately 10 jig) 



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is fractionated by reverse-phase HPLC using a C-8 
narrowbore column (13 cm x 2.1 mm ID) with a 
TFA/acetonitrile gradient and a flow rate of 150 
pl/min. The gradient employs (A) 0*06% TFA in water 
and (B) 0.04% TFA in water and acetonitrile (1:4; 
v:v). The procedure was 10% B for five min., 
followed by a linear gradient for 70 min. to 80% B, 
followed by a linear gradient for 10 min. to 100% B. 
Fractions containing fragments as determined from the 
peaks in the HPLC profile (FIGURE 7A) are 
rechromatographed at least once under the same 
conditions in order to isolate single components 
satisfactory for sequence analysis. 

The HPLC profiles of the similarly digested 
16 kD and 18 kD subunits are shown in FIGURES 7B and 
7C, respectively. These peptide maps are similar 
suggesting that the subunits are identical or are 
closely related. 

The 16 kD and 18 kD subunits are digested 
with EndoAsp-N proteinase. The protein is treated 
with 0.5 pg EndoAsp-N in 50 mM sodium phosphate 
buffer, pH 7.8 at 36°C for 20 hr. The conditions for 
fractionation are the same as those described 
previously for the 30 kD, 16 kD, and 18 kD digests. 
The profiles obtained are shown in FIGURES 16A and 
16B. 



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Various peptide fragments produced using the 
foregoing procedures have been analyzed in an 
automated amino acid sequencer (Applied Biosystems 
470A with 120A on-line PTH analysis) . The following 
sequence data has been obtained: 

(1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K; 

(2) S-L-K-P-S-N-Y-A-T-I-Q-S-I-V; 

( 3 ) A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K ; 

(4) M-S-S-L-S-I-L-F-F-D-E-N-K; 

( 5 ) S-Q-E-L-Y-V-D-F-Q-R ; 

(6) F-L-H-C-Q-F-S-E-R-N-S ; 

(7) T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y; 

(8) L-Y-D-P-M-V-V; 

( 9 ) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E ; 

(10) V-D-F-A-D-I-G; 

(11) V-P-K-P-C-C-A-P-T ; 

(12) I-N-I-A-N-Y-L; 

(13) D-N-H-V-L-T-M-F-P- 1 -A- 1 -N ; 

( 14 ) D-E-Q-T-L-K-K-A-R-R-K-Q-W- I-?-P; 

(15) D-I-G-?-S-E-W-I-I-?-P; 

(1 6 ) S-I-V-R-A-V-G-V-P-G-I-P-E-P-? -? -V ; 

( 17) D-? -I-V-A-P-P-Q-Y-H-A-F-Y ; 

(18) D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E ; 

(19) S-Q-T-L-Q-F-D-E-Q-T-L-K-? -A-R-? -K-Q ; 

(20) D-E-Q-T-L-K-K-A-R-R-K-Q-W- I-E-P-R-N-? -A-R-R-Y 
-L; 

(21) A-R-R-K-Q-W- 1 -E-P-R-N-7 -A-? -R-Y-? -? -V-D ; and 

(22) R_?_Q_W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-?-G 



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C5. Amino Aci d Analysis 

Samples of oxidized (30 kD) and "reduced (16 
kD and 18 kD) BOP are electrophoresed on a gel and 
transferred to Immobilon for hydrolysis and amino 
acid analysis using conventional, commercially 
available reagents to derivatize samples and HPLC 
using the PicO Tag (Millipore) system. The 
composition data generated by amino acid analyses of 
30 kD BOP is reproducible, with some variation in the 
number of residues for a few amino acids, especially 
cysteine and isoleucine. 



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Composition data obtained are shown in TABLE 

5. 



TABLE 5 
BOP Amino Acid Analyses 

Amino Acid 30 kD 16 kD 18 kD 



Aspartic Acid/ 


22 


14 


15 


Asparagine 








Glutamic Acid/ 


24 


14 


16 


Glutamine 








Serine 


24 


16 


23 


Glycine 


29 


18 


26 


Histidine 


5 


* 


4 


Arginine 


13 


6 


6 


Threonine 


11 


6 


7 


Alanine 


18 


11 


12 


Proline 


14 


6 


6 


Tyrosine 


11 


3 


3 


Valine 


14 


8 


7 


Methionine 


3 


0 


2 


Cysteine** 


16 


14 


12 


Isoleucine 


15 


14 


10 


Leucine 


15 


8 


9 


Phenylalanine 


7 


4 


4 


Tryptophan 


ND 


ND 


ND 


Lysine 


12 


6 


6 



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*This result is not integrated because histidine is 
present in low quantities. 

"Cysteine is corrected by percent normally recovered 
from performic acid hydrolysis of the standard 
> protein. 

The results obtained from the 16 kD and 18 
kD subunits, when combined/ closely resemble the 
numbers obtained from the native 30 kD protein. The 
high figures obtained for glycine and serine are most 
likely the result of gel elution. 



D. PURIFICATION OF HUMAN OSTEOGENIC PROTEIN 

Human bone is obtained from the Bone Bank, 
(Massachusetts General Hospital, Boston, MA), and is 
milled, defatted, demarrowed and demineralized by the 
procedure disclosed above. 320 g of mineralized bone 
matrix yields 70 - 80 g of demineralized bone 
matrix. Dissociative extraction and ethanol 
precipitation of the matrix gives 12.5 g of 
guanidine-HCl extract. 

One third of the ethanol precipitate (0.5 g) 
is used for gel filtration through 4 M guanidine-HCl 
(FIGURE 10A) . Approximately 70-80 g of ethanol 
precipitate per run is used. In vivo bone inducing 
activity is localized in the fractions containing 
proteins in the 30 kD range. They are pooled and 
equilibrated in 6 M urea, 0.5 M NaCl buffer, and 
applied directly onto a HAP column; the bound protein 



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is eluted stepwise by using the same buffer 
containing 100 iriM and 500 iriM phosphate (FIGURE 10B) . 
Bioassay of HAP bound and unbound fractions 
demonstrates that only the fraction eluted by 100 mM 
phosphate has bone inducing activity in vivo . The 
biologically active fraction obtained from HAP 
chromatography is subjected to heparin-Sepharose 
affinity chromatography in buffer containing low 
salt; the bound proteins are eluted by 0.5 M NaCl 
(FIGURE 10C). Assaying the heparin-Sepharose 
fractions shows that the bound fraction eluted by 0.5 
M NaCl have bone-inducing activity. The active 
fraction is then subjected to C-18 reverse phase 
chromatography. (FIGURE 10D) . 

The active fraction can then be subjected to 
SDS-PAGE as noted above to yield a band at about 30 
kD comprising substantially pure human osteogenic 
protein. 

E. BIO SYNTHET I C PROBES FOR ISOLATION OF GENES 
ENCODING NATIVE OSTEOGENIC PROTEIN 

E-l PROBE DESIGN 

A synthetic consensus gene shown in FIGURE 
13 was designed as a hybridization probe based on 
amino acid predictions from homology with the 
TGF-beta gene family and using human codon bias as 
found in human TGF-beta. The designed concensus 
sequence was then constructed using known techniques 
involving assembly of oligonucleotides manufactured 
in a DNA synthesizer. 



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Tryptic peptides derived from BOP and 
sequenced by Edman degradation provided amino acid 
sequences that showed strong homology with the 
nrosophila DPP protein sequence (as inferred from the 
gene), the Xenopus VG1 protein, and somewhat less 
homology to inhibin and TGF-beta, as demonstrated 
below in TABLE 6. 





TABLJS 6 




protein 


f.mino acifl seauence 


homoloav 


(POP) 


or JJAx itDynCyr iro 
***** * * ** 


(9/15 matches) 


(BEE) 


GYDAYYCHGKCPFFL 




(BOP) 


SFDAYYCSGACQFPS 
***** * 


(6/15 matches) 


<V3l> 


GYMANYCYGECPYPL 




(BOP) 


SFDAYYCSGACQFPS 
***** 


(5/15 matches) 


f.inhibin) 


GYHANYCEGECPSHI 




(BOP) 


SFDAYYCSGACQFPS 
* * * * 


(4/15 matches) 


/TGF-betai 


GYHANFCLGPCPYIW 




(1QP) 


K/RACCVPTELSAI SMLYLDEN 
***** * **** * * 


(12/20 matches) 


(val) 


LPCCVPTKMSPI SMLFYDNN 





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( BOP ) K/RACCVPTEL S A I SMLYLDEN 

* ***** * **** * (12/20 matches) 
( inhibin ) KSCCVPTKLRPMSMLYYDDG 



( BOP ) K/RACCVPTELSAISMLYLDE 

**** * * (6/19 matches) 

( TGF-beta ) APCCVPQALEPLPIVYYVG 



(SQP) K/RACCVPTELSA I SMLYLDEN 

******* * **** (12/20 matches) 
( DPP ) KACCVPTQLDSVAMLYLNDQ 



( BOP ) LYVDF 

***** (5/5 matches) 

( DPP ) LYVDF 



( BOP ) LYVDF 

*** * (4/5 matches) 

( Vol ) LYVEF 



( BOP ) LYVDF 

** ** (4/5 matches) 

( TGF-beta ) LYIDF 



( BOP ) LYVDF 

* * (2/5 matches) 

( inhibin ) FFVSF 



*-match 



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

One purpose of the originally designed 
synthetic consensus gene sequence, designated COPO, 
(see FIGURE 13), was to serve as a probe to isolate 
natural genes. For this reason the DNA was designed 
using human codon bias. Alternatively, probes may be 
constructed using conventional techniques comprising 
a group of sequences of nucleotides which encode any 
portion of the amino acid sequence of the osteogenic 
protein produced in accordance with the foregoing 
isolation procedure. Use of such pools of probes 
also will enable isolation of a DNA encoding the 
intact protein. 



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E-2 Retrieval of Genes Encoding Osteogenic 

Protein from Geno mic Library 

A human genomic library (Maniatis-library) 
carried in lambda phage (Charon 4A) was screened 
using the COPO consensus gene as probe. The initial 
screening was of 500,000 plaques (10 plates of 50,000 
each) . Areas giving hybridization signal were 
punched out from the plates, phage particles were 
eluted and plated again at a density of 2000-3000 
plaques per plate. A second hybridization yielded 
plaques which were plated once more, this time at a 
density of ca 100 plaques per plate allowing 
isolation of pure clones. The probe (COPO) is a 300 
base pair BamHI-PstI fragment restricted from an 
amplification plasmid which was labeled using alpha 
32 dCTP according to the random priming method of 
Feinberg and Vogelstein (1984) Anal. Biochem. 137 : 
266-267. Prehybridization was done for 1 hr in 5x 
SSPE, lOx Denhardfs mix, 0.5% SDS at 50°C. 
Hybridization was overnight in the same solution as 
above plus probe. The washing of nitrocellulose 
membranes was done, once cold for 5 min. in lx SSPE 
with 0.1% SDS and twice at 50°C for 2 x 30 min. in 
the same solution. Using this procedure, twenty-four 
positive clones were found. Two contained a gene 
never before reported designated OPl, osteogenic 
protein-1 described below. Two others yielded the 
genes corresponding to BMP-2b, one yielded BMP- 3 (see 
PCT US 87/01537). 



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Southern blot analysis of lambda #13 DNA 
showed that an approximately 3kb BamHI fragment 
hybridized to the probe, (See FIGURE IB). This 
fragment was isolated and subcloned into a bluescript 
vector (at the BamHI site). The clone was further 
analyzed by Southern blotting and hybridization to 
the COPO probe. This showed that a 1 kb (approx.) 
EcoRI fragment strongly hybridized to the probe. 
This fragment was subcloned into the EcoRI site of a 
bluescript vector, and sequenced. Analysis of this 
sequence showed that the fragment encoded the carboxy 
terminus of a protein, named osteogenic protein-1 
(OP1) . The protein was identified by amino acid 
homology with the TGF-beta family. For this 
comparison cysteine patterns were used and then the 
adjacent amino acids were compared. Consensus splice 
signals were found where amino acid homologies ended, 
designating exon intron boundaries. Three exons were 
combined to obtain a functional TGF-beta-like domain 
containing seven cysteines. Two introns were deleted 
by looping out via primers bridging the exons using 
the single stranded mutagenesis method of Kunkel. 
Also, upstream of the first cysteine, an EcoRI site 
and an asp-pro junction for acid cleavage were 
introduced, and at the 3' end a PstI site was added 
by the same technique. Further sequence information 
(penultimate exon) was obtained by sequencing the 
entire insert. The sequencing was done by generating 
a set of unidirectionally deleted clones (Ozkaynak, 
E., and Putney, S. (1987) Biotechniques, 1:770-773). 
The obtained sequence covers about 80% of the 
TGF-beta-like region of OP1 and is set forth in FIGURE 



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1A. The complete sequence of the TGF-beta like 
region was obtained by first subcloning all EcoRI 
generated fragments of lambda clone #13 DNA and 
sequencing a 4 kb fragment that includes the first 
portion of the TGF-beta like region (third exon 
counting from end) as well as sequences characterized 
earlier/ The gene on an EcoRI to PstI fragment was 
inserted into an coli expression vector controlled 
by the trp promoter-operator to produce a modified 
trp LE fusion protein with an acid cleavage site. 
The OP1 gene encodes amino acids corresponding 
substantially to a peptide found in sequences of 
naturally sourced material. The amino acid sequence 
of what is believed to be its active region is set 
forth below: 

1 10 20 30 40 

OP1 LYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMN ATN — H- AI VQTLVHF I NPET-VPKPCCAPTQLN A 

80 90 100 

I S VLYFDDS SNVI LKKYRNMWRACGCH 



A longer active sequence is: 

-5 
HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100 

I SVLYFDDSSNVILKKYRNMWRACGCH 



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The amino acid sequence of what is 
believed to be the active regions encoded by the 
other three native genes retrieved using the 
consensus probe are: 

! 10 20 30 40 

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-K-IPKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQDMWEGCGCR 

1 10 20 30 40 

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD 

50 60 70 

HLNSTN— H-AIVQTLVNSVNS-S-IPKACCVPTELSA 

80 90 100 

I SMLYLDEYDKWLKNYQEMWEGCGCR 

1 10 20 30 40 

CBMP-3 CARRYLKVDFA-D I GWSEWI I SPKSFDAYYCSGACQFPMPK 

50 60 70 

SLKPSN— H-ATIQSIVRAVGWPGIPEPCCVPEKMSS 

80 90 100 

LSILFFDENKNWLKVYPNMTVESCACR 



E-3 frnhina cP WA Library 

Another example of the use of pools of 
probes to enable isolation of a DNA encoding the 



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intact protein is shown by the following. Cells 
known to express the protein (e.g., osteoblasts or 
osteosarcoma) are extracted to isolate total 
cytoplasmic RNA. An oligo-dT column can be used 
to isolate mRNA. This mRNA can be size 
fractionated by, for example, gel 
electrophoresis. The fraction which includes the 
mRNA of interest may be determined by inducing 
transient expression in a suitable host cell and 
testing for the presence of osteogenic protein 
using, for example, antibody raised against 
peptides derived from the tryptic fragments of 
osteogenic protein in an immunoassay. The mRNA 
fraction is then reverse transcribed to single 
stranded cDNA using reverse transcriptase; a 
second complementary DNA strand can then be 
synthesized using the cDNA as a template. The 
double-standard DNA is then ligated into vectors 
which are used to transfect bacteria to produce a 
cDNA library. 

The radiolabelled consensus sequence, 
portions thereof, and/or synthetic deoxy 
oligonucleotides complementary to codons for the 
known amino acid sequences in the osteogenic 
protein may be used to identify which of the DNAs 
in the cDNA library encode the full length 
osteogenic protein by standard DNA-DNA 
hybridization techniques. 



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The cDNA may then be integrated in an 
expression vector and transfected into an 
appropriate host cell for protein expression. The 
host may be a prokaryotic or eucaryotic cell since 
the former's inability to glycosylate osteogenic 
protein will not effect the protein's enzymatic 
activity. Useful host cells include 
Saccharomvces , E. coli , and various mammalian cell 
cultures. The vector may additionally encode 
various signal sequences for protein secretion 
and/or may encode osteogenic protein as a fusion 
protein. After being translated, protein may be 
purified from the cells or recovered from the 
culture medium. 

E4 . flene Pre paration 

Natural gene sequences and cDNAs 
retrieved as described above may be used for 
expression. The genes above may also be produced 
by assembly of chemically synthesized 
oligonucleotides. 15-100mer oligonucleotides may 
be synthesized on a Biosearch DNA Model 8600 
Synthesizer, and purified by polyacrylamide gel 
electrophoresis -<PA<3E) in Tris-Borate-EDTA buffer 
(TBE) • The DNA is then electroeluted from the 
gel. Overlapping oligomers may be phosphorylated 
by T4 polynucleotide kinase and ligated into 
larger blocks which may also be purifed by PAGE. 



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£5. Expression 

The genes can be expressed in appropriate 
prokaryotic hosts such as various strains of E*. 
coli * and also in bacillus, yeasts, and various 
animal cells such as CHO, myeloma, etc. 
Generally, expression may be achieved using many 
cell types and expression systems well known to 
those skilled in the art. For example, if the 
gene is to be expressed in Ej- coli . an expression 
vector based on pBR322 and containing a synthetic 
trp promoter operator and the modified trp LE 
leader may be used. The vector can be opened at 
the EcoRI and PSTI restriction sites, and, for 
example, an OP gene fragment can be inserted 
between these two sites. The OP protein is joined 
to the leader protein via a hinge region having 
the sequence Asp-Pro. This hinge permits chemical 
cleavage of the fusion protein with dilute acid at 
the Asp-Pro site. 

E6. Production of Active Proteins 

The following procedure may be followed 
for production of active recombinant proteins. |L_ 
coli cells containing the fusion proteins are 
lysed. The fusion proteins are purified by 
differential solubilization. Cleavage is 
conducted with dilute acid, and the resulting 
cleavage products are passed through a 
Sephacryl-200HR or SP Trisacyl column to separate 
the cleaved proteins. The reduced OP fractions 
are then subjected to HPLC on a semi-prep C-18 
column. 



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Conditions for refolding of OP were at pH 
8.0 using 50 mM Tris-HCl and 6M Gu-HCl. Samples 
were refolded for 18 hours at 4°C. 

These procedures have been used to 
express in E_s. coli on the active protein 
designated OP1 having the amino acid sequence set 
forth above (longer species). 

Refolding may not be required if the 
proteins are expressed in animal cells. 

MATRIX PREPARATION 

A . r,PTiPral Consideratio n r>f Matrix Properties 

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

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



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modification abolishes the inductive response. 
Particle size influences the quantitative response of 
new bone; particles between 75 and 420 ym elicit the 
maximum response. Contamination of the matrix with 
bone mineral will inhibit bone formation. Most 
importantly, the procedures used to formulate 
osteogenic protein onto the matrix are extremely 
sensitive to the physical and chemical state of both 
the osteogenic protein and the matrix. 

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

A successful carrier for osteogenic protein 
must perform several important functions. It must 
bind osteogenic protein and act as a slow release 
delivery system, accommodate each step of the 
cellular response during bone development, and 
protect the osteogenic protein from nonspecific 
proteolysis. In addition, selected materials must be 
biocompatible in vivo and biodegradable; the carrier 
must act as_ja temporary scaffold until replaced 
completely by new bone. Biocompatibility requires 
that the matrix not induce significant inflammation 
when implanted and not be rejected by the host 



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animal. Biodegradability requires that the matrix be 
slowly absorbed by the body of the host during 
development of new bone or cartilage. 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 
cortical or trabecular bone. 

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

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



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B. Preparation of Biologically Active A llogenic 

Matrix 

Demineralized bone matrix is prepared from 
the dehydrated diaphyseal shafts of rat femur and 
tibia as described herein to produce a bone particle 
size which pass through a 420 y sieve- The bone 
particles are subjected to dissociative extraction 
with 4 M guanidine-HCl. Such treatment results in a 
complete loss of the inherent ability of the bone 
matrix to induce endochondral bone differentiation. 
The remaining insoluble material is used to fabricate 
the matrix. The material is mostly collagenous in 
nature, and upon implantation, does not induce 
cartilage and bone. All new preparations are tested 
for mineral content and false positives before use. 
The total loss of biological activity of bone matrix 
is restored when an active osteoinductive protein 
fraction or a pure protein is reconstituted with the 
biologically inactive insoluble collagenous matrix. 
The osteoinductive protein can be obtained from any 
vertebrate, e.g., bovine, porcine, monkey, or human, 
or produced using recombinant DNA techniques. 

C. Preparation of Dealvcos Ylated Bone Matrix 

for Use in Xenooenic Implant 

When osteogenic protein is reconstituted 
with collagenous bone matrix from other species and 
implanted in rat, no bone is formed. This suggests 
that while the osteogenic protein is xenogenic (not 



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species specific)/ the matrix is species specific and 
cannt>t be implanted cross species perhaps due to 
intrinsic immunogenic or inhibitory components. 
Thus, heretofore, for bone-based matrices, in order 
for the osteogenic protein to exhibit its full bone 
inducing activity, a species specific collagenous 
bone matrix was required. 

The major component of all bone matrices is 
Type I collagen. In addition to collagen, extracted 
bone includes non-collagenous proteins which may 
account for 5% of its mass. Many non-collagenous 
components of bone matrix are glycoproteins. 
Although the biological significance of the 
glycoproteins in bone formation is not known, they 
may present themselves as potent antigens by virtue 
of their carbohydrate content and may constitute 
immunogenic and/or inhibitory components that are 
present in xenogenic matrix. 

It has now been discovered that a 
collagenous bone matrix may be used as a carrier to 
effect bone inducing activity in xenogenic implants, 
if one first removes the immonogenic and inhibitory 
components from the matrix. The matrix is 
deglycosglated chemically using, for example, 
hydrogen fluoride to achieve this purpose. 



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Bovine bone residue prepared as described 
above is sieved, and particles of the 74-420 \M are 
collected. The sample is dried in vacuo over P2O5, 
transferred to the reaction vessel and' anhydrous 
hydrogen fluoride (HF) (10-20 ml/g of matrix) is then 
distilled 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 min. After 
evaporation of the HF in vacuo , the residue is dried 
thoroughly in vacuo over KOH pellets to remove any 
remaining traces of acid. 

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

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

1) suspend in TBS (Tris-buf f ered Saline) 
lg/200 ml and stir at 4°C for 2 hrs or 
in 6 M urea, 50 mM Tris-HCl, 500 mM 
NaCI, pH 7.0 (UTBS), and stir at RT for 
30 min. ; 

2) centrifuge and wash with TBS or UTBS as 
in step 1); and 

3) centrifuge; discard supernatant; water 
wash residue; and then lyophilize. 



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FABRICATION OF OSTEOGENIC DEVICE 

Fabrication of osteogenic devices using any 
of the matrices set forth above with any of the 
osteogenic proteins described above may be performed 
as follows. 

A. f+hanol p rcrinitation 

In this procedure, matrix was added to 
osteogenic protein in guanidine-HCl . Samples were 
vortexed and incubated at a low temperature. Samples 
were then further vortexed. Cold absolute ethanol 
was added to the mixture which was then stirred and 
incubated. After centrifugation (microfuge high 
speed) the. supernatant was discarded. The 
reconstituted matrix was washed with cold 
concentrated ethanol in water and then lyophilized. 

B# Arftfconitri la Trif Inoroacetic Acid 

Lvonhilization 

In this procedure, osteogenic protein in an 
acetonitrile trif luroacetic acid (ACN/TFA) solution 
was added to the carrier. Samples were vigorously 
vortexed many times and then lyophilized. 



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C. Urea Lvoohilization . 

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

IN VIVO RAT BIOASSAY 

Substantially pure BOP, BOP-rich extracts 
comprising protein having the properties set forth 
above, and several of the recombinant proteins have 
been incorporated in matrices to produce osteogenic 
devices, and assayed in rat for endochondral bone. 
Studies in rats show the osteogenic effect to be 
dependent on the dose of osteogenic protein dispersed 
in the osteogenic device. No activity is observed if 
the matrix is implanted alone. The following sets 
forth guidelines for how the osteogenic devices 
disclosed herein might be assayed for determining 
active fractions of osteogenic protein when employing 
the isolation procedure of the invention, and 
evaluating protein constructs and matrices for 
biological activity. 

A. Subcutane ous Implantation 

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



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reference, is used to monitor the purification 
protocols for endochondral bone differentiation 
activity. This assay consists of implanting the test 
samples in subcutaneous sites in allogeneic recipient 
rats under ether anesthesia. Male Long-Evans rats, 
aged 28-32 days, were used. A vertical incision (1 
cm) is made under sterile conditions in the skin over 
the thoraic region, and a pocket is prepared by blunt 
dissection. Approximately 25 mg of the test sample 
is implanted deep into the pocket and the incision is 
closed with a metallic skin clip. The day of 
implantation is designated as day of the experiment. 
Implants were removed on day 12. The heterotropic 
site allows for the study of bone induction without 
the possible ambiguities resulting from the use of 
orthotopic sites. 

B. fTAlliilar Events 

The implant model in rats exhibits a 
controlled progression through the stages of matrix 
induced endochondral bone development including: (1) 
transient infiltration by polymorphonuclear 
leukocytes on day one; (2) mesenchymal cell migration 
and proliferation on days two and three; (3) 
chondrocyte appearance on days five and six; (4) 
cartilage matrix formation on day seven; (5) 
cartiliage calcification on day eight; (6) vascular 
invasion, appearance of osteoblasts, and formation of 
new bone on days nine and ten; (7) appearance of 
osteoblastic and bone remodeling and dissolution of 



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the implanted matrix on days twelve to eighteen; and 
(8) hematopoietic bone marrow differentiation in the 
ossicle on day twenty-one. The results show that the 
shape of the new bone conforms to the shape of the 
implanted matrix. 

C. Histological Evaluation 

Histological sectioning and staining is 
preferred to determine the extent of osteogenesis in 
the implants. Implants are fixed in Bouins Solution, 
embedded in parafilm, cut into 6-8 mm sections. 
Staining with toluidine blue or hemotoxylin/eosin 
demonstrates clearly the ultimate development of 
endochondrial bone. Twelve day implants are usually 
sufficient to determine whether the implants show 
bone inducing activity. 

D. Biological Markers 

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



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Implants containing osteogenic protein at 
several levels of purity have been tested to 
determine the most effective dose/purity level, in 
order to seek a formulation which could be produced 
on a commercial scale. The results are measured by 
specific acivity of alkaline phosphatase, calcium 
content, and histological examination. As noted 
previously, the specific activity of alkaline 
phosphatase is elevated during onset of bone 
formation and then declines. On the other hand, 
calcium content is directly proportional to the total 
amount of bone that is formed. The osteogenic 
activity due to osteogenic protein is represented by 
"bone forming units". For example, one bone forming 
unit represents the amount of protein that is needed 
for half maximal bone forming activity as compared to 
rat demineralized bone matrix as control and 
determined by calcium content of the implant on day 
12. 

E. Results 

Dose curves are constructed for bone 
inducing activity in vivo at each step of the 
purification scheme by assaying various 
concentrations of protein. FIGURE 11 shows 
representative dose curves in rats as determined by 
alkaline phosphatase. Similar results are obtained 
when represented as bone forming units. 
Approximately 10-12 ug of the TSK-f raction, 3-4 



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yg of heparin-Sepharose-II fraction, 0.4-0.5 pg of 
the C-18 column purified fraction, and 20-25 ng of 
gel eluted highly purified 30 kD protein is needed 
for unequivocal bone formation (half maximum 
activity). 20-25 ng of the substantially pure 
protein per 25 mg of implant is normally sufficient 
to produce endochondral bone* Thus, 1-2 ng 
osteogenic protein per mg of implant is a reasonable 
dosage, although higher dosages may be used* (See 
section IBS on specific activity of osteogenic 
protein.) 

OP1 expressed as set forth above (longer 
version), when assayed for activity histologically, 
induced cartilage and bone formation as evidenced by 
the presence of numerous chondrocytes in many areas 
of the implant and by the presence of osteoblasts 
surrounding vascular endothelium forming new matrix. 

Deglycosylated xenogenic collagenous bone 
matrix (example: bovine) has been used instead of 
allogenic collagenous matrix to prepare osteogenic 
devices (see previous section) and bioassayed in rat 
for bone inducing activity in vivo . The results 
demonstrate that xenogenic collagenous bone matrix 
after chemical deglycosylation induces successful 
endochondral bone formation (FIGURE 19). As shown by 
specific activity of alkaline phosphotase, it is 
evident that the deglycosylated xenogenic matrix 
induced bone whereas untreated bovine matrix did not. 



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Histological evaluation of implants suggests 
that the deglycosylated bovine matrix not only has 
induced bone in a way comparable to the rat residue 
matrix but also has advanced the developmental stages 
that are involved in endochondral bone 
differentiation. Compared to rat residue as control, 
the HF treated bovine matrix contains extensively 
remodeled bone. Ossicles are formed that are already 
filled with bone marrow elements by 12 days. This 
profound action as elicited by deglycosylated bovine 
matrix in supporting bone induction is reproducible 
and is dose dependent with varying concentration of 
osteogenic protein. 

ANIMAL EFFICACY STUDIES 

Substantially pure osteogenic protein from 
bovine bone (BOP), BOP-rich osteogenic fractions 
having the properties set forth above, and several 
recombinant proteins have been incorporated in 
matrices to produce osteogenic devices. The efficacy 
of bone-inducing potential of these devices was 
tested in cat and rabbit models, and found to be 
potent inducers of osteogenesis, ultimately resulting 
in formation of mineralized bone. The following sets 
forth guidelines as to how the osteogenic devices 
disclosed herein might be used in a clinical setting. 

A. -fa line Model 

The purpose of this study is to establish a 
large animal efficacy model for the testing of the 



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osteogenic devices of the invention, and to 
characterize repair of massive bone defects and 
simulated fracture non-union encountered frequently 
in the practice of orthopedic surgery. The study is 
designed to evaluate whether implants of osteogenic 
protein with a carrier can enhance the regeneration 
of bone following injury and major reconstructive 
surgery by use. of this large mammal model. The first 
step in this study design consists of the surgical 
preparation of a femoral osteotomy defect which, 
without further intervention, would consistently 
progress to non-union of the simulated fracture 
defect. The effects of implants of osteogenic 
devices into the created bone defects were evaluated 
by the following study protocol. 

A-l. Procedure 

Sixteen adult cats weighing less than 10 
lbs. undergo unilateral preparation of a 1 cm bone 
defect in the right femur through a lateral surgical 
approach* In other experiments, a 2 cm bone defect 
was created. The femur is immediately internally 
fixed by lateral placement of an 8-hole plate to 
preserve the exact dimensions of the defect. There 
are three different types of materials implanted in 
the surgically created feline femoral defects: group 
I (n = 3) is a control group which undergo the same 
plate fixation with implants of 4 M 
guanidine-HCl-treated (inactivated) feline 
demineralized bone matrix powder (Gu-HCl-DBM) (360 
mg); group II (n - 3) is a positive control group 



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implanted with biologically active feline 
demineralized bone matrix powder (DBM) (3-60 mg); and 
group III (n = 10) undergo a procedure identical to 
groups 1-11/ with the addition of osteogenic protein 
onto each of the Gu-HCl-DBM carrier samples. To 
summarize, the group III osteogenic protein-treated 
animals are implanted with exactly the same* material 
as the group I animals, but with the singular 
addition of osteogenic protein. 

All animals are allowed to ambulate 
libitum within their cages post-operatively . All 
cats are injected with tetracycline (25 mg/kg SQ each 
week for four weeks) for bone labelling. All but 
four group III animals are sacrificed four months 
after femoral osteotomy. 

A- 2 . Radiomorphometrics 

In vivo radiomorphometric studies are 
carried out immediately post-op at 4, 8, 12 and 16 
weeks by taking a standardized x-ray of the lightly 
anesthesized animal positioned in a cushioned x-ray 
jig designed to consistently produce a true 
anterio-posterior view of the femur and the osteotomy 
site. All x-rays are taken in exactly the same 
fashion and in exactly the same position on each 
animal. Bone repair is calculated as a function of _ 
mineralization by means of random point analysis. A 
final specimen radiographic study of the excised bone 
is taken in two planes after sacrifice. X-ray 



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PCT/US89/014S3 



results are shown in FIGURE 12, and displaced as 
percent of bone defect repair. To summarize, at 16 
weeks, 60% of the group III femors are united with 
average 86% bone defect regeneration. By contrast, 
the group I Gu-HCl-DMB negative-control implants 
exhibit no bone growth at four weeks, less than 10% 
at eight and 12 weeks, and 16% (+ 10%) at 16 weeks 
with one of the five exhibiting a small amount of 
bridging bone. The group II DMB positive-control 
implants exhibited 18% ( + 3%) repair at four weeks, 
35% at eight weeks, 50% (+10%) at twelve weeks and 
70% (± 12%) by 16 weeks, a statistical difference of 
p <0.01 compared to osteogenic protein at every 
month. One of the three (33%) is united at 16 weeks. 

A-3 . * Biomechanics 

Excised test and normal femurs are 
immediately studied by bone densitometry, wrapped in 
two layers of saline-soaked towels, placed in two 
sealed plastic bags, and stored at -20° G until 
further study. Bone repair strength, load to 
failure, and work to failure are tested by loading to 
failure on a specially designed steel 4-point bending 
jig attached to an Instron testing machine to 
quantitate bone strength, stiffness, energy absorbed 
and deformation to failure. The study of test femurs 
and normal femurs yield the bone strength (load) in 
pounds and work to failure in joules. Normal femurs 
exhibit a strength of 96 (± 12) pounds, osteogenic 
protein-implanted femurs exhibited 35 (±4) pounds, 
but when corrected for surface area at the site of 



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PCT/US89/014S3 



fracture (due to the -hourglass" shape of the bone 
defect repair) this correlated closely with normal 
bone strength. Only one demineralized bone specimen 
was available for testing with a strength of 25 
pounds, but, again, the strength correlated closely 
with normal bone when corrected for fracture surface 
area. 

A-4. Histomorphometry /Histology 

Following biomechanical testing the bones 
are immediately sliced into two longitudinal sections 
at the defect site, weighed, and the volume 
measured. One-half is fixed for standard calcified 
bone histomorphometrics with fluorescent stain 
incorporation evaluation, and one-half is fixed for 
decalcified hemotoxylin/eos in stain histology 
preparation. 

A-5. Biochemistry 

Selected specimens from the bone repair site 
(n=6> are homogenized in cold 0.15 M NaCl, 3 mM 
NaHC0 3 , pH 9.0 by a Spex freezer mill. The alkaline 
phosphatase activity of the supernatant and total 
calcium content of the acid soluble fraction of 
sediment are then determined. 

A-6. Histopathology 

The final autopsy reports reveal no unusual 
or pathologic findings noted at necropsy of any of 
the animals studied. Portion of all major organs are 



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PCT/US89/01453 



preserved for further study. A histopathological 
evaluation is performed on samples of the following 
organs: heart, lung, liver, both kidneys, spleen, 
both adrenals, . lymph nodes, left and right quadriceps 
muscles at mid-femur (adjacent to defect site in 
experimental femur) . No unusual or pathological 
lesions are seen in any of the tissues. Mild lesions 
seen in the quadriceps muscles are compatible with 
healing responses to the surgical manipulation at the 
defect site. Pulmonary edema is attributable to the 
euthanasia procedure. There is no evidence of any 
general systemic effects or any effects on the 
specific organs examined. 

A-7. Feline Study Summary 

The 1 cm and 2 cm femoral defect cat studies 
demonstrate that devices comprising, a matrix 
containing disposed osteogenic protein can: (1) 
repair a weight-bearing bone defect in a large 
animal; (2) consistently induces bone formation 
shortly following (less than two weeks) implantation; 
and (3) induce bone by endochondral ossification, 
with a strength equal to normal bone, on a volume for 
volume basis. Furthermore, all animals remained 
healthy during the study and showed no evidence of 
clinical or histological laboratory reaction to the 
implanted device. In this bone defect model, there 
was little or no healing at control bone implant 
sites. The results provide evidence for the 
successful use of osteogenic devices to repair large, 
non-union bone defects. 



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B. Rabbit Model; 

Bl. Procedure and Results 

The purpose of this study is to establish a 
model in which there is minimal or no bone growth in 
the control animals, so that when bone induction is 
tested, only a strongly inductive substance will 
yield a positive result. Defects of 1.5 cm are 
created in the ulnae of rabbits with implantation of 
osteogenic devices or no implant. 

Eight mature (greater than 10 lbs) New 
Zealand White rabbits with epiphyseal closure 
documented by X-ray were studied. Of these eight 
animals (one animal each was sacrificed at one and 
two weeks), 11 ulnae defects are followed for the 
full course of the eight week study. In all cases (n 
= 7) following osteo- 
periosteal bone resection, the no implant animals 
establish no radiographic union by eight weeks. All 
no implant animals develop a thin "shell" of bone 
growing from surrounding bone present at four weeks 
and, to a slightly greater degree, by eight weeks. 
In all cases (n = 4)., radiographic union with marked 
bone induction is established in the osteogenic 
protein-implanted animals by eight weeks. As opposed 
to the no implant repairs, this bone repair is in the 
site of the removed bone. 



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PCT/US89/01453 



Radiomorphometric analysis reveal 90% 
osteogenic protein-implant bone repair and 18% 
no-implant bone repair at sacrifice at eight weeks. 
At autopsy , the osteogenic protein bone appears 
normal, while "no implant" bone sites have only a 
soft fibrous tissue with no evidence of cartilage or 
bone repair in the defect site. 

B-2. Allograft Device 

In another experiment , the marrow cavity of 
the 1.5 cm ulnar defect is packed with activated 
osteogenic protein rabbit bone powder and the bones 
are allografted in an intercalary fashion. The two 
control ulnae are not healed by eight weeks and 
reveal the classic "ivory" appearance. In distinct 
contrast, the osteogenic protein-treated implants 
"disappear" radiographically by four weeks with the 
start of remineralization by six to eight weeks. 
These allografts heal at each end with mild 
proliferative bone formation by eight weeks. 

This type of device serves to accelerate 
allograph repair. 

B-3. Summary 

These studies of 1.5 cm osteo-periosteal 
defects in the ulnae of mature rabbits show that: (1) 
it is a suitable model for the study of bone growth; 
(2) "no implant" or Gu-HCl negative control implants 
yield a small amount of periosteal-type bone, but not 



WO 89/09787 PCT/US89/01453 

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medullary or cortical bone growth; (3) osteogenic 
protein-implanted rabbits exhibited proliferative 
bone growth in a fashion highly different from the 
control groups; (4) initial studies show that the 
bones exhibit 50% of normal bone strength (100% of 
normal correlated vol: vol) at only eight weeks after 
creation of the surgical defect; and (5) osteogenic 
protein-allograft studies reveal a marked effect upon 
both the allograft and bone healing. 

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



What is claimed is: 



89/09787 



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PCT/US89/01453 



Claims 

1. An osteogenic device for implantation in a 
mammal, said device comprising: 

a biocompatible, in vivo biodegradable matrix 
defining pores of a dimension sufficient to 
permit influx, proliferation and differentiation 
of migratory progenitor cells from the body of 
said mammal; and 

substantially pure osteogenic protein capable of 
inducing endochondral bone formation in said 
mammal disposed in said matrix and accessible to 
said cells. 

2. Substantially pure osteogenic protein capable of 
inducing endochondral bone formation in a mammal when 
disposed within a matrix implanted in said mammal, 

3. The device of claim 1 wherein said matrix 
comprises close-packed particulate matter having a 
particle size within the range of 70-420 ym. 

4. The device of claim 1 wherein said matrix 
comprises demineralized, protein-extracted, 
particulate, allogenic bone. 

5. The device of claim 1 wherein said matrix 
comprises collagen, hydroxyapatite, tricalcium 
phosphate, polymers comprising lactic acid monomer 
units, polymers comprising glycolic acid monomer 
units, demineralized, guanidine-extracted allogenic 
bone, or a mixture thereof. 



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6. The device of claim 1 wherein said matrix is 
shaped to span a non-union fracture in said mammal. 

7. The device of claim 1 disposed within the marrow 
cavity of allogenic bone. 

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

9. The device of claim 8 wherein said matrix is 
treated with a protease. 

10. The invention of claim 1 or 2 wherein said 
osteogenic protein is unglycosylated . 

11. The invention of claim 10 wherein said 
osteogenic protein has an apparent molecular weight 
of about 27 kD when oxidized as determined by 
comparison to molecular weight standards in 
SDS-polyacrylamide gel electrophoresis. 

12. The invention of claim 1 or 2 wherein said 
osteogenic protein is glycosylated. 

13. The invention of claim 12 wherein said 
osteogenic protein has an apparent molecular weight 
of about 30 kD when oxidized as determined by 
comparison to molecular weight standards in 
SDS-polyacrylamide gel electrophoresis. 



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14. The invention of claim 1 or 2 wherein said 
osteogenic protein comprises a pair of polypeptide 
chains . 

15. The invention of claim 14 wherein one chain 
of said pair of polypeptide chains has an apparent 
molecular weight of about 14 kD and the other has an 
apparent molecular weight of about 16 kD, both as 
determined after reduction by comparison to molecular 
weight standards in SDS-polyacrylamide gel 
electrophoresis . 

16. The invention of claim 14 wherein one chain 
of said pair of polypeptide chains has an apparent 
molecular weight of about 16 kD and the other has an 
apparent molecular weight of about 18 kD, both as 
determined after reduction by comparison to molecular 
weight standards in SDS-polyacrylamide gel 
electrophoresis . 

17. The invention of claim 1 or 2 wherein said 
osteogenic protein has the approximate amino acid 
composition set forth below: 



Amino acid 
residue 



Rel. no. 
res ./molec. 



Amino a-cid 
rgsidyj* 



Rel. no, 
res. /molec. 



Aspartic acid/ 22 
Asparagine 
Glutamic acid/ 24 
Glut amine 

Serine 24 



Tyrosine 
Valine 
Methionine 
Cysteine 
Iso leucine 



11 
14 
3 
16 
15 



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PCT/US89/014S3 



Glycine 
Histidine 
Arginine 
Threonine 
Alanine 
- Lysine 

18. The invention of claim 1 or 2 wherein said 
osteogenic protein comprises the amino acid sequence: 

VPKPCCAPT . 

19. The invention of claim 1 or 2 wherein the 
half maximum bone inducing activity of said protein 
is 0.8 to 1.0 ng per mg of said matrix. 

20. A method of inducing local cartilage or bone 
formation in a mammal comprising the step of 
implanting the device of claim 1 in said mammal at a 
locus accessible to migratory progenitor cells of 
said mammal. 

21. A method of inducing endochondral bone 
formation in a mammal comprising the step of 
implanting the device of claim 1 in said mammal at a 
locus accessible to migratory progenitor cells of 
said mammal. 

22. A method of inducing endochondral bone 
formation in a non-union fracture in a mammal 
comprising the step of implanting in the fracture in 
said mammal the device of claim 6. 



29 Leucine 15 

5 Proline 14 

13 Phenylalanine 7 

11 Tryptophan ND 
18 
12 



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PCT/US89/01453 



23. The invention of claim 1 or 2 wherein the 

protein comprises the sequence: 

1 10 20 30 40 

OP1 LYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H- A I VQTL VHF I NPET- VPKPCC APTQLNA 

80 90 100 

I SVLYFDD S SNVI LKKYRNMWRACGCH 



24. The device of claim 1 or 2 wherein the 

protein comprises the sequence: 

-5 
HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DL<3WQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H- A I VQTLVHF I NPET- VPKPCC APTQLNA 

80 90 100 

I SVLYFDDSSNVTLKKYRNMVVRACGCH 



25. The device of claim 1 or 2 wherein the 

protein comprises the sequence: 

1 10 20 30 40 

CBMP-2 a CKRHFLYVDFS-DVGWNDWI VAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-K-IPKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQDMWEGCGCR 



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PCT/US89/01453 



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

1 10 20 30 40 

CBMP-2D CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-S-IPKACCVPTELSA 

80 90 100 

I SMLYLDEYDKWLKNYQEMWEGCGCR 

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

1 10 20 30 40 

CBMP-3 CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK 

50 60 70 

SLKPSN— H-ATIQSIVRAVGWPGIPEPCCVPEKMSS 

80 90 100 

LSI LFFDENKNVVLKVYPNMTVESCACR 

28. A DNA sequence encoding an amino acid 
sequence sufficiently duplicative of that of the 
sequence encoded by the gene of FIGURE 1A such 
that said encoded sequence induces bone or 
cartilage formation when implanted in a mammal in 
association with a matrix. 

29. The DNA of claim 28 encoding the same 
amino acid sequence as the gene set forth in 
FIGURE 1A. 



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30. The DNA sequence of claim 28 encoding: 

1 10 20 30 40 

0P1 LYVSFR-DLGWQDWI I APEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100 

I SVLYFDDS SNVILKKYRNMVVRACGCH 

31. The DNA sequence of claim 28 encoding: 

-5 
HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DLGWQDWI I APEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN— H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100 

ISVLYFDDSSNVILKKYRNMWRACGCH 

32. A cell line engineered to express the 
protein of claim 2. 



33. The device of claim 1 wherein said matrix 

comprises demineralized, protein extracted, 
particulate xenogenic bone treated with HF. 



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1 / 19 



PCT/US89/014S3 




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WO 89/09787 



PCI7US89/01453 



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WO 89/09787 



PCT/US89/01453 



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PCI7US89/01453 



A / 19 




<2 
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WO 89/09787 



PCT/US89/014S3 



5/19 




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SUBSTITUTE SHEET 



6/19 



PCI7US89/01453 



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SUBSTITUTE SHEET 



WO 89/09787 



7 / 19 



PCT/US89/01453 



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WO 89/09787 



8/19 



PCT/US89/01453 




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WO 89/09787 



9/ 19 



PCT/US89/01453 



FIG. 3A FIG. 3B FIG.4A FIG. 4B 




FIG. 5A FIG. 5B 



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DEGLY 

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«fe DEGLY 



WO 89/09787 



PCT/US89/01453 



10/19 



FIG. 6 A FIG.6B FIG.6C FIG. 6 D FIG. 6 E 



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FIG. 15 




18K SUBUNIT 

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WO 89/09787 



PCI7US89/01453 



11/19 




40 
TIME(MIN) 



60 70 

FIG. 7A 




40 
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60 70 

FIG. 7C 



SUBSTITUTE SHEET 



WO 89/09787 



12 / 19 



PCT/US89/01453 



PERCENT BONE FORMATION 



ABSORSANCE (2l4nm) 




35 



40 



45 50 55 

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60 



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100 



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ABSORBANCE (21 4 nm) 




0.005 



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FIG. 9 



qhr^titiitf CV4CET 



WO 89/09787 



13 / 19 



PCI7US89/01453 




QUBSTITUTE SHEET 



WO 89/09787 



PCT/US89/01453 



14/ 19 



OOSE CURVES 

PERCENT BONE FORMATION 




POST-OP TIMEMEEKS)-*- 4 8 12 "6 4 8 12 16 4 8 12 16 

A 8 C 

FIG. 12 



SUBSTITUTE SHEET 



WO 89/09787 PCT/US89/014S3 

15/19 



FIG. 13 

10 20 30 40 50 

GATCCrAATGGCCTGTACGTGGACTTCCAGCGCGACGTGGGCTGGGACGA 
DPHCLYVDPQRDVGWDD 

60 70 80 90 100 

CTGGATCATCGCCCCCGTCGACTTCGACGCCTACTACTGCTCCGGAGCCT 
W I IAPVDF DAYYCSG A 

HO 120 130 140 150 

GCCAGTTCCCCTCTGCGGATCACTTCAACAGCACCAACCACGCCGTGGTG 
CQFPSADHFNSTNHAVV 

160 170 180 190 200 

CAGACCCTGGTGAACAACATGAACCCCGGCAAGGTACCCAAGCCCTGCTG 
QTLVNMMNPGKVPKPCC 

210 220 230 240 250 

CGTGCCCACCGAGCTGTCCGCCATCAGCATGCTGTACCTGGACGAGAATT 
VPTELSA ISMLYLDEN 

260 270 280 290 300 

CCACCGTGGTGCTGAAGAACTACCAGGAGATGACCGTGGTGGGCTGCGGC 
STVVLKHYQEMTVVCCG 

310 

TGCCGCTAACTGCAG 
C R * 



e.lOCTlTUTE SHEET 



WO 89/09787 



16/ 19 



PCT/US89/01453 



SDS GEL ELUTION OF OSTEOGENIC ACTIVITY 
CALCIUM CONTENT (uq/mg tissue) 



30- 



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I 



-£=^> t^J <7T^i 



CI8Prepool >67 67-42 42-34 34-28 28-25 25-21 21-18 < 18 

MOLECULAR WEIGHTS (KOa) pjQ 



ALKALINE PHOSPHATASE (U/mg protein) 




2.5 



5.0 10.0 20.0 

CONCENTRATION OF BOP PROTEIN Olg) 



3O.0 



RAT MATRIX 



BOVINE MATRIX 



DEGLY. BOVINE MATRIX 



FIG. 18 



eT'TIITS SHEET 



WO 89/09787 



17/19 



PCT/US89/01453 



HPLC PROFILE 

ENDO ASP-N DIGEST - PREPOOL I6K OP SUBUNIT 



cn 

LL 

o 
< 
m 
o 




20 



40 

TIME (MIN) 



60 80 

FIG. I6A 



HPLC PROFILE 

ENDO ASP-N DIGEST - PREPOOL I8K OP SUBUNIT 



CO 
U- 

< 

in 
O 

6 




J L 



J I I I 



20 



40 
TIME(MIN) 



€0 



-80 



FIG. 168 



WO 89/09787 



PCT/US89/01453 



18/19 



WO 89/09787 PCT/US89/01453 

19/19 



% 



CALCIUM CONTENT (uq/mg TISSUE ) 



40 



20 



BEFORE GEL ELUTION 




1 



H 



I 



RATDBM 18.7 37.5 75 150 

CONCENTRATION OF 30K BOP(ng) 



300 



FIG. I9A 



CALCIUM CONTENT (uq/mg) 



40- 



20- 



AFTER GEL ELUTION 



I 



i 

I 



i 



I 



RATDBM 5£5 105 210 315 560 
CONCENTRATION OF 30KB0P(ng) 



FIG. I9B 



BSTITUTE SHEET 



per 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International patent Classification 4: 
C07K 13/00, A61L 27/00, C12N 15/00 
A61K 35/32, C12P 21/02 



A3 



(11) International Publication Number: WO 89/09787 

(43) International Publication Date: 19 October 1989 (19.10.89) 



(21) International Application Number: PCT/US89/01453 

(22) International Filing Date: 7 April 1989 (07.04.89) 



(30) Priority data: 
179,406 
232,630 
315,342 



8 April 1988 (08.04.88) US 
15 August 1988 (15.08.88) US 
23 February 1989 (23.02.89) US 



(60) Parent Applications or Grants 

(63) Related by Continuation 
US 

Filed on 
US 

Filed on 
US 

Filed on 



179,406 (CIP) 
8 April 1988 (08.04.88) 
232,630 (CIP) 
15 August 1988 (15.08.88) 
315,342 (CIP) 
23 February 1989(23.02.89) 



(71) Applicant (for all designated States except US): CREATIVE 

BIOMOLECULES, INC. [US/US]; 35 South Street, 
Hopkinton, MA 01748 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only) : KUBERASAMPATH, 
Thangavel [IN/US]; 6 Spring Street, Medway, MA 
02053 (US). OPPERMANN, Hermann [US/US]; 25 
Summer Hill Road, Medway, MA 02053 (US). RUE- 
GER, David, C. IUS/US]; 



150 Edgemere Road, Apt. 4, West Roxbury, MA 02132 (US). 
OZKAYNAK, Engin [TR/USJ; 44 Purdue Drive, Milford, MA 
01757 (US). 

(74) Agent: PITCHER, Edmund, R.; Lahive & Cockfield, 60 
State Street, Boston, MA 02109 (US). 

(81) Designated States: AT (European patent), AU, BB, BE 
(European patent), BF (OAPI patent), BG, BJ (OAPI 
patent), BR, CF (OAPI patent), CG (OAPI patent), CH 
(European patent), CM (OAPI patent), DE (European 
patent), DK, FI, FR (European patent), GA (OAPI pa- 
tent), GB (European patent), HU, IT (European patent). 
JP, KP, KR, LK, LU (European patent), MC, MG, ML 
(OAPI patent), MR (OAPI patent), MW, NL (European 
patent), NO, RO, SD, SE (European patent), SN (OAPI 
patent), SU, TD (OAPI patent), TG (OAPI patent), US. 

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. 

(88) Date of publication of the international search report: 

8 February 1990 (08.02.90) 



(54) Title: OSTEOGENIC DEVICES 



(57) Abstract 



Disclosed are 1) osteogenic devices comprising a matrix containing osteogenic protein and methods of inducing endochon- 
dral bone growth in mammals using the devices; 2) amino acid sequence data, amino acid composition, solubility properties, 
structural features, homologies and various other data characterizing osteogenic proteins, and 3) methods of producing osteogen- 
ic proteins using recombinant DNA technology. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States 
applications under the PCT. 

AT Austria 

AU Aintxalb 

BB Barbados 

BE Belgium 

BF Burkina Fasso 

B6 Bulgaria 

BJ Benin 

BR Brazil 

CA Canada 

CF Central African RepubBc 

CG Congo 

CH Switzerland 

CM Cameroon 

DE Germany. Federal Republic of 

DK Denmark 



to the PCT on the front pages 



ES 


Spain 


n 


Finland 


FR 


France 


GA 


Gabon 


GB 


United Kingdom 


HU 


Hungary 


rr 


Italy 


jp 


Japan 


KP 


Democratic People's Republic 




of Korea 


KR 


Republic of Korea 


u 


Liechtenstein 


LK 


Sri Lanka 


US 


Luxembourg 


MC 


Monaco 



of pamphlets publishing international 



MG 


Madagascar 


ML 


Mali 


MR 


Mauritania 


MW 


Malawi 


NL 


Netherlands 


NO 


Norway 


RO 


Romania 


9) 


Sudan 


S£ 


Sweden 


SN 


Senegal 


SU 


Soviet Union 


TO 


Chad 


TG 


Togo 


US 


United States of America 



INTERNATIONAL SEARCH REPORT 

International Application No ■ 



PCT/US 89/01453 



_CLASSiriCATION Of SUBJECT MATTIR (it ,ever»l cl»«,f, c3l,on sylnt> ol, apply, indleat. 

^CCCTdino tO lnt*rn*«i n ..i d.« * j*i ' — ■ — ■ ■■■ — 



* ■ ' *" *" ciaasHicaiion symoois appl y, indie 

According to International Patent Clarification (IPC) or to both National CIa.aific.tion and IPC ~ 

IPC 4 ; g 07 K 13/00, A 61 L 27/00, C 12 N.15/00, A 61 K 35/32, 



H. FIELDS SEARCHED 



Classification System 



IPC 



Minimum Documentation Searched * 

Classification Symbol* 



C 07 K, C 12 N, A 61 K, A 61 L, C 12 P 



Documentation Searched other than Minimum Documentation 
to the Extent that auch Documents are Included In the Fields Searched * 



111. DOCUMENTS CONSIDERED TO BE RELEVANT * 



Catepory * | Citation ot Document, " with Indication, where appropriate/of the relevant passages w 



Relevant to Claim No. " 



WO, A, 88/00205 (GENETICS INSTITUTE) 
14 January 1988 

see pages 1-12,15-17,22-24,49; 
pages 61-73, claims 1-23 

The Journal of Cell Biolgoy, volume 97 
December 1983, The Rockefeller 
University Press, 
S.M. Seyedin et al.: "In vitro 
induction of cartilage-specific 
macromolecules by a bone extract' 1 , 
pages 1950-1953 

see the whole article, especially 
page 1952, right-hand column 

EP, A, 0182483 (COLLAGEN CORP.) 
28 May 1986 

see the whole document, especially 
page 6, first paragraph; page 7, 
lines 10-18 



1-16,25,, 

26,28,32, 

33 



1-16,25, 

26,28,32, 

33 



1-16,25, 

26,28,32, 

33 



* Special categorise of ctted documents: to 

" A " ^2s■ , Zfl^^tal^ h •s.• n f B, °* ***** *• »« 

considered to be ot particular relevance 
" E " "in? dSte CUm * m bUt pub " ,n,d 0B or »*•' *ntematiorml 

Siaiion «f 1^.1 .!!? b i,h tne P UDll ««on date of another 
citation or other special reason (as specified) 

"°" ot°hVr ,n m2n r I <,,rrlB8 t0 ** oml di » e,0 «°™« «hlblllon or 
MP " &r7h.^ «!». Out 



T later document published after the International filing date 
or priority date and not in conflict with the application but 
lnvantion Und * f>Und principh 01 tnt0f y unaertying the 

-X* document of particular relevance; the claimed invention 
cannot be constdsred novel or cannot be considsred to 
Involve an inventive atap 

"Y" document of particular relevance;' the claimed Invention 
cannot be conaldered to Involve an Inventive step when the 
document is combined with one or more other auch docu- 
ments, such combination being obvious to a person skilled 
in the art* 

*eV document member of the same patent family 



IV. CERTIFICATION 



»ate of the Actual Completion of the International Search 

13th November 1989 

International Searching Authority 

EUROPEAN PATENT OFFICE 



Date of Mailing of this International Search Report 
1 




T.K. WILLIS 



form PCT/ISA/210 (aecond sheet) (January 1685) 



International Application No. PCT/US 89/01453 



III. OOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) 



Calagory* . 



CiUtron of Oocument. with indication, wrwe aooropnata. oi in* rotevant ptssaQfl* 



r Rttavant to Claim No 



I 



WO, A, 85/05274 (R.F. OLIVERS) , 
5 December 1985 I 
see the whole document j 

Analytical Biochemistry, volume 146, j 

1985, Academic press, Inc., j 

C.A. Olson et al. : "Deglycosylation j 

of chondroitin sulfate proteoglycan j 

by hydrogen fluoride in pyridine" , | 

pages 232-237 j 

see "Discussion" on page 236 J 

Trends in Biochem. Sci. (TIBS), volume 9, . 
1984, Elsevier Science Publishers B.V.,j 
(Amsterdam, NL) , . : 
E. Simpson: "Growth factors which j 
affect bone", pages 527-530 
see the whole article J 

EP, A, 0148155 (DOW CHEMICAL CO.) \ 
10 July 1985 ! 
cited in the application j 

WO, A, 86/00526 (A.I. CAPLAN) j 
30 January 1986 

US, A, 4394370 (S.R. JEFFERIES) 
19 July 1983 

US, A, 456348? (M.R. URIST) 
7 January "1986 

S.P. Colowick et al.: "Methods in 
Enzymology", volume 146, Peptide 
Growth Factors, part A, edited by 
David Barnes et al., Academic Press 
Inc., 

M.R. Urist et al.: "Preparation and 
bioassay of bone morphogenetic protein 
and polypeptide fragments", pages 294- 
312 

EP, A, 0169016 (COLLAGEN CORP.) 
22 January 1986 



1-19,23-33 



Form PCT ISA310 (aitra ehaat) (Jtntory 1M5) 



International Application No. PCT/US 



FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET 



i 



V.QOBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUNO UNSEARCHABLE 



Th£ Internationa, aearch^repor. ha. not been eat.Wi.hed In re.pect of certain «l.lm. under Article 17,2) (a) tor the follows reaeone: 
1.|2J dm number. bec.uae they relate 10 aubject matter not required to be ..arched by thl. Authority, namely: 

** Claim numbers 20-22: 
See PCT Rule 39.1(iv) 

Methods for treatment of the human or animal body 
by surgery or therapy, as well as diagnostic methods. 

** Claim numbers 1-16,19,32,33 

See Article 6 PCT, 17(2) (a) (ii) PCT 

./. 

3 Q ^STT*""" °* y m de P <WOent "* im * ,n0 '™ not drafted in accordance with the aecono and third Mntences of 

PCT rtUW 6.4(a). 

Vl-B OBSERVATIONS WHERE UNITY OF INVENTION 18 LACKIMC > ~~ 



Thla International Searehln B Authority found multiple Invention. In thl. Inl.rnellon.l application ,. follow.: 

Please see Form PCT/ISA/206 dated 14-8-89 

lS oMh'e^n?^ 

lD -"S^^^ - ...r, ,. Mrt cover, enly 

lD --.ir^^ - , M „«.- , e 



U *S!a^lt?ffi^l^^ , * ,,l ,Umint " • <Witi0M ' ""•"-t'on.l S.archm, Au.hor.fy 

Remark on Protest 

ED The additional search feet were accompanied by applicant's protest 
Q No protest accompanied the payment of additional search fees. 

Form PCT/1SA/210 (supplemental sheet (2)) (January 1985) . 



-2- 

Internaticnal Application No PCT / US 89/01453 



further information continued from pct/ISA/210 (supplemental sheet (2)) 



The present application is admittedly NOT the first to 
describe the phenomenon of bone^ inducing proteins, or 
devices containing them. It is therefore necessarily 
drafted to the individual compounds (on well-known 
devices) . 

In claims 1-16 , however, proteins (on a device) are 
claimed, which are only defined by their biological 
activity and/or their molecular weight, which is 
clearly not sufficient for a full characterization of 
individual compounds . Despite this broad scope, the 
biological activity has only been demonstrated for one 
single compound (BOP 30K, which is covered by subject 1). 

The sea rch has therefore been restricted to the embodiments 
of claim s 1-19 and 23-33, in as far as the proteins (and 
DNAs) correspond to the definitions given in the cl aims 
17, 18 . and 23-31. 

This searchable subject matter has been regrouped according 
to the non-unity specification, in order to establish 
conceptually individual subjects, each of which now 
constitutes a potential selection invention. 



form PCT;iSA/ 



^KJ£™ E INTERNATIONAL SEARCH REPORT 

ON INTERNATIONAL PATENT APPLICATION NO. US 8901453 



SA 28156 



m"mbe^a™ i ^ i ! y .T^"" """l" 8 ,0 ,he pattnt *"-««» cited in the above-mentioned international search report, 
ine members are as contained in the European Patent Office EDP file on 19/12/89 

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



Patent document 
cited in search report 


rUDHCatlOD 

date 


Patent family 
members) 


Publication 

Hot* 

tunc 


WfWA— oorinonc 

WU A" OOUUxUO 


14-01-88 


AU-A- 


7783587 


29-01-88 






EP-A- 


0313578 


03-05-89 


EP-A- 0182483 


28-05-86 


US-A- 


4563350 


07-01-86 






AU-B- 


585268 


15-06-89 






AU-A- 


4900585 


01-05-86 






JP-A- 


62016421 


24-01-87 


WO-A- 8505274 


05-12-85 


AU-B- 


575660 ' 


04-08-88 






AU-A- 


4405285 


13-12-85 






CA-A- 


1261760 


26-09-89 






EP-A- 


0182842 


04-06-86 






JP-T- 


61502588 


13-11-86 


EP-A- 0148155 


10-07-85 


AU-B- 


580975 


09-02-89 






AU-A- 


3722184 


11-07-85 






CA-A- 


. 1241641 


06-09-88 






JP-A- 


60226814 


12-11-85 






US-A- 


4804744 


14-02-89 


WO-A- 8600526 


30-01-86 


US-A- 


4620327 


04-11-86 






AU-A- 


4601485 


10-02-86 






EP-A- 


0188552 


30-07-86 


US-A- 4394370 


19-07-83 


US-A- 


4472840 


25-09-84 


US-A- 4563489 


07-01-86 


None 






EP-A- 0169016 


22-01-86 


AU-A- 


4501585 


23-01-86 






CA-A- 


1261549 


26-09-89 






JP-A- 


61036223 


20-02-86 






US-A- 


4774322 


27-09-88 






US-A- 


4774228 


27-09-88 






US-A- 


4810691 


07-03-89 






US-A- 


4843063 


27-06-89 





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



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