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WORLD INTELLECTUAL PROPER.JY ORGANIZATION 
InternationaJbBurLau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International patent Classification 4 
C07K 1MK), C12N ISW. C12P 21/02 
A6IL 27/00, A61K 37/36// A61K 3*32 



A2 



(11) International Publication Number: WO 89/09788 

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



(21) International Application Number: PCT/US89/01469 

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



(30) Priority data: 

179,406- 



232,630 
315,342 



JApril 1988 (08.04.88) US 

15 Augusn988 (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 1988 (23.02.88) 



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

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

(72) Inventors; and 

(75)Tnventbrs/AppHcants ^?r-US-Ort/^;_OPPERMANN, Her- 
mann [US/US]; 25 Summer Hill Road, MedwayrMA- 
02053 (US). KUB ERAS AM PATH, Thangavel [IN/US]; 
6 Spring Street, Medway, MA 02053 (US). RUEGER, 
David, C. [US/US]; 



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

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



(81) Designated StatesfAT (European-patent),-AU,-BB f _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 

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



(54) Title: BIOSYNTHET1C OSTEOGENIC PROTEINS AND OSTEOGENIC DEVICES CONTAINING THEM 



(57) Abstract 

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



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 


BR 


France 


ML 


Mali 


AU 


Australia 


GA 


Gabon 


MR 


Mauritania 


BB 


Barbados 


GB 


United Kingdom 


MW 


Malawi 


BE 


Belgium 


HU 


Hungary 


NL 


Netherlands 


BG 


Bulgaria 


rr 


Italy 


NO 


Norway 


BJ 


Benin 


jp 


Japan 


RO 


Romania 


BR 


Brazil 


KP 


Democratic People's Republic 


SD 


Sudan 


CF 


Central African Republic 




of Korea" 


SE 


Sweden 


CG 


Congo 


KR 


Republic of Korea 


SN 


Senegal 


CH 


Switzerland 


u 


Liechtenstein 


SU 


Soviet Union 


CM 


Cameroon 


LK 


Sri Lanka 


TO 


Chad 


DE 


Germany, Federal Republic of 


LU 


Luxembourg 


TG 


Togo 


DK 


Denmark 


MC 


Monaco 


US 


United States of America 


FI 


Finland 


MG 


Madagascar 







WO 89/09788 



1 



PCT/US89/01469 



BIOSYNTHFTIC QSTEO ^MTC PROTEINS AND 
OSTEOGENIC DEVTCF g CONTAINING THEM 



This invention relates to osteogenic 
^vices~to-synt-h«tic-^enes._en^oaing_ proteins which 
can induce osteogenesis in mammals and methods~for 
their production using recombinant DNA techniques, to 
synthetic forms of osteogenic protein, and to bone 
and cartilage repair procedures using osteogenic 
device comprising the synthetic proteins. 

Mammalian bone tissue is known to contain 
one or more proteinaceous materials, presumably 
active during growth and natural bone healing, which 
can induce a developmental cascade of cellular events 
resulting in endochondral bone formation. This 

acti_v_e_f actor (or factors) has variously been 

referred to in the~riterTture~as bone-mo rpho.gene tic _ 
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). 



WO 89/09788 



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PCT/US89/01469 



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" ^ollagen^ 

bone induction activity (Sampath and Reddi, (1981) 
Proc. Natl* Acad. Sci. USA 2&: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) 
Pr oc ._Nat 1 ^Acad . Sci_. USA M: 6591-6595) . 



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

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



WO 89/09788 



- 3 - 



PCI7USS9/01469 



mammalian bone is disclosed by Sampath et al. ( Proc . 
Natl . Acad. Sci. USA (1987) 8fi> . 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 

ca Icium chlor Tde^ur ear~i~nor gani-c-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— 1.8_kDs_ _The^ authors reported that the 

protein was present in a fraction separated by 
hydroxyapatite chromatography, and that it induced 
bone formation in mouse hindguarter muscle and bone 
regeneration in trephine defects in rat and dog 
skulls. Their method of obtaining the extract from 
bone results in ill-defined and impure preparations. 

European Patent Application Serial No. 
148,155, published October 7, 1985, purports to 
disclose osteogenic proteins derived from bovine, 
porcine, and human origin. One of the proteins, 
designated by the • inventors as a P3 protein having a 
molecular weight of 22-24 kD, is said to have been 
purified to an essentially homogeneous state. This 
material is reported to induce bone formation when 
implanted into animals. 



WO 89/09788 PCT/US89/01469 

- 4 - 



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 

put at ive~Bone~"induct i ve- f actor s-produced__bji_ 

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) ££: 9484-9488) discloses the purification of a 
bovine bone morphogenetic protein from guanidine 

extr.act_s_^^^minerali2ed bone having cartilage and 

bone formation activity as a basi"c protein r— 

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. 



Wozney et al. (Science (1988) 242 : 
1528-1534) discloses the isolation of full-length 
cDNA's encoding the human equivalents of three 



WO 89/09788 



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PCT/US89/01469 



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 
Ts~ reported-; 

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

proteins and a^Cfuctur"al~ design- for— novels 

functional osteogenic proteins. Another object is to 
provide methods for inducing cartilage formation. 

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



WO 89/09788 



- 6 - 



PCT/US89/01469 



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_ end<^cho^r^3^bone formation and bone 

marrow differentiation. Suitably modifiedas 
disclosed herein, the devices also may be used to 
induce cartilage formation. The devices comprise a 
carrier material,, referred to herein as a matrix, 
having the characteristics disclosed below, 
containing dispersed osteogenic protein in the form 
of a biosynthetic construct. 

Key to these developments was the successful 
preparation of substantially pure osteogenic protein 
by purification from bone, the elucidation of amino 
acid sequence and structure data of the native 
o s t eog en i c ~ p r o t e in-and-i n s i g h t s -i nvo lv i ng__s_t udy_ o f__ 
the DNA and amino acid sequences of the natural 
source product. A protocol was developed which 
results in retrieval of active, substantially pure 
osteogenic protein from mammalian bone. 
Investigation of the properties and structure of the 
native form osteogenic protein then permitted the 
inventors to develop a rational design for non-native 
forms, i.e., forms never before known in nature, 
capable of inducing bone formation. As far as 
applicants are aware, the constructs disclosed herein 
constitute the first instance of the design of a 
functional, active protein without preexisting 
knowledge of the active region of a native form 
nucleotide or amino acid sequence. 



A series of consensus DNA sequences were 
designed with the goal of producing an active 
osteogenic protein. The sequences were based on 
partial amino acid sequence data obtained from the 
naturally sourced product and on observed homologies 
"~ with :-unxel-a-t-ed-g«nes_xep^.ted_J.n the literature, or 
the sequences they encode, having a presumed" or 
demonstrated developmental function. Several of the 
biosynthetic consensus sequences have been expressed 
as fusion proteins in procaryotes, purified, cleaved, 
refolded, combined with a matrix, implanted in an 
established animal model, and shown to have 
endochondral bone-inducing activity. The currently 
preferred active proteins comprise sequences 
designated COPS, COP7, COP16, and OP1. The amino 
acid sequences of these proteins are set forth below. 



1 10 20 30 40 

COP5 L-YVDFS-DVGWDDWI^ 

50 60 70- 

HFNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



1 10 20 30 40 

COP7 LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

ISMLYLDENEKWLKNYQEMWEGCGCR 

-10 

PKHHSQRARKKNKN 
1 10 20 30 40 

COP16 CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



WO 89/09788 



- 8 - 



PCT/US89/01469 



-5 

HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100- 

. I SVLYFDDSSNVILKKYRNMWRACGCH " "~" ~" 

Ii\ these sequences and all other amino acid 
sequences disclosed herein, the dashes (-) are used 
as fillers only to line up comparable sequences in 
related proteins, and have no other function. Thus, 
amino acids 45-50 of COP7, for example, are NHAW. 
Also, the numbering of amino acids is selected solely 
for purposes of facilitating comparisons between 
sequences. Thus, for example, the DF residues 
numbered at 9 and 10 of COPS and COP7 may comprise 
— — residues, __e..g. j_ 35^ and 36, of an osteogenic protein 

embodying the invention. Various "leader" of~"trai~ler 

sequences may be attached to the operative active 
region provided the osteogenic or chondrogenic 
activity of the protein is not destroyed. 

Thus, in one aspect, the invention comprises 
a protein comprising an amino acid sequence 
sufficiently duplicative of the sequence of COPS, 
COP7, COP16, or 0P1 such that it is capable of 
inducing endochondral bone formation when properly 
folded and implanted in a mammal in association with 
" a matrix. Some of these sequences induce cartilage, 



WO 89/09788 



- 9 - 



PCT/US89/01469 



but not bone. Also, the bone forming materials may- 
be used to produce cartilage if implanted in an 
avascular locus, or if an inhibitor to full bone 
development is implanted together with the active 
protein. Thus, in another aspect, the invention 
"c omp r is e s —a- p r-o t e-i-a- 1 e s.s_ _th a n__a b pu t _ 200 amino acids 
long (for each chain) including a sequence 
sufficiently duplicative of the sequence of COPS, 
COP7, COP16, or OP1 such that it is capable at least 
of cartilage formation when properly folded and 
implanted in a mammal in association with a matrix. 
The phrase "sufficiently duplicative", as used 
herein, is used to describe proteins having a degree 
of homology with the specific sequences disclosed 
herein and other, different amino acids but which 
nevertheless exhibit osteogenic or chondrogenic 
activity. 

In one pref er"fea~aspect ,— these— proteins 

comprise species of the generic amino acid sequences: 

1 10 20 30 40 50 

LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 
. 60 70 80 90 100 

QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX 

or 



1 10 20 30 40 50 

CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 
60 70 80 90 100 

QXXVXXXNXXXXPXX CCX PXXXXXXXXLXXXXXXXVXLXX YXXMX VXXCX CX 



WO 89/09788 



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PCT/US89/01469 



where the letters indicate the amino acid residues of 
standard single letter code/ and the Xs each 
represent any one of the 22 naturally occurring amino 
acid residues. Preferred amino acid sequences within 
~" — -the-f oregoing generic^sequences are : 



1 10 20 30 40 50 

LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 
K S S L QE VIS E FD Y E A AY MPESMKAS VI 
FEKIDN L NS Q ITK F P TL 

AS K 
60 70 80 90 100 

QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR 
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H 
RF T S K DPV V Y N S H RN RS 

N S K P E 



and 



1 TO "20 • 30 40__ 50 

CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 
RRRS K S S L QE VIS E FD Y E A AY MPESMKAS VI 
KEFEKIDN L .NS Q ITK F P TL 

Q A S K 

60 70 80 90 100 

QTLVNSVNPGKI PKACCVPTELS AI SMLYLDENENWLKNYQDMWEGCGCR 
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H 
RF T S K DPV V Y N S H RN RS 

N S K P E 



WO 89/09788 



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PCT/US89/01469 



wherein each of the amino acids arranged vertically 
at each position in the sequence may be used 
alternatively in various combinations. Note that 
these generic sequences have 6 and preferably 7 
cysteine residues where inter- or intramolecular 
disulfide bonds " c an~f o rn»7~ and-con tain- o t her-_c ri ti c al 
amino acids which influence the tertiary structure of 
the proteins. These generic structural features are 
found in previously published sequences, none of 
which have been described as capable of osteogenic 
activity, and most of which never have been linked 
with such activity. 

Particular useful sequences include: 



1 10 20 30 40 

Vg 1 CKKRHLYVEFK-DVGWQNWVI APQGYMANYCYGECPYPLTE 

50 60 70 

ILNGSN—HrAII^TLVHSIEPED-IPLPCCVPTKMSP 

80 90 100 

I SMLFYDNNDNWLRHYENMAVDECGCR 

1 10 20 30 40 

DPP CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNNNNPGK-VPKACCVPTQLDS 

80 90 100 

VAMLYLNDQSTWLKNYQEMTWGCGCR 

1 10 20 30 40 

CBMP-2 a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-K-IPKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQDMWEGCGCR 



WO 89/09788 



PCT/US89/01469 



- 12 - 



1 10 20 30 40 

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD 

50 60 70 
HLKSTN — H-AIVQTLVNSVNS-S-IPKACCVPTELSA 
80 90 100 
ISMLYLDEYDKWLKNYQEMWEGCGCR 



1 10 20 30 40 

CBMP-3 CARRYLKVDFA-DI GWSEWI I SPKSFDAYYCSGACQFPMPK 

50 60 70 

SLKPSN — H-ATIQSIVRAVGWPGIPEPCCVPEKMSS 

80 90 100 

LSI LFFDENKNVVLKVYPNMTVE SCACR 

. 1 10 20 30 40 

COP1 LYVDFQRDVGWDDWI IAPVDFDAYYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENSTWLKNYQEMTWGCGCR 

1 10 20 30 40 

COP3 LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD 

5 0 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSX 

80 90 100 

I SMLYLDENEKWLKNYQEMWE GCGCR 

1 10 20 30 40 

COP4 LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



f 

WO 89/09788 PCT/US89/01469 

- 14 - 



derived. In view of this disclosure, skilled genetic 
engineers can design and synthesize genes which 
encode appropriate amino acid sequences, and then can 

_ express them in various types of host cells, 

includingTEoth "procaryot es-and -eucaryo.tes ,_jto_ produce 
large quantities of active synthetic proteins 
comprising truncated analogs, muteins, fusion 
proteins, and other constructs mimicking the 
biological activity of the native forms and capable 
of inducing bone formation in mammals including 
humans . 

The synthetic proteins 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 L_of^ _d intension to permit progenitor cell 

migration and subsequent^Tf^rentratioir-and 

proliferation. The particle size should be within 
the range of 70. - 850 um, preferably 70 - 420 um. 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 viae, to serve as a "temporary scaffold" and 
substratum for recruitment of migratory progenitor 
cells, and as a base for their subsequent anchoring 
and proliferation. Currently preferred carriers 
include particulate, demineralized, guanidine • 
extracted, species-specific (allogenic) bone, and 
particulate, deglycosglated (or HF treated), protein 
extracted, demineralized, xenogenic bone. 



WO 89/09788 



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PCT/US89/01469 



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- ea-lcium-pho_sph_ates. 



The osteogenic proteins and implantable 
osteogenic devices enabled and disclosed herein will 
permit the physician to obtain optimal predictable 
bone formation to correct, for example, acquired and 
congenital craniofacial and other skeletal or dental 
anomalies (Glowacki et al. (1981) Lancet 1:959-963). 
The devices may be used to induce local endochondral 
bone formation in non-union fractures, and in other 
clinical applications including periodontal 
applications where bone formation is required. 
Another potential clinical application is in 
cartilage "repafi r, ~ for ex amp 1 e ,— i n_ t he_t re a tme n t o f 
osteoarthritis. 



WO 89/09788 



PCT/US89/01469 



- 16 - 

Brief Description of the Drawing 

The foregoing and other objects of this 
invention, the various features thereof, as well as 
th^^invent ion— itself may_be_more^ fully understood 
from the following description, when read together ~~ 
with the accompanying drawings, in which: 

FIGURE 1 is a comparison of the amino acid 
sequence of various osteogenic proteins to those of 
the TGF-beta family. COP1, COP3, COP4, COPS, and 
COP7 are a family of analogs of synthetic osteogenic 
proteins developed from the consensus gene that was 
joined to a leader protein via a hinge region having 
the sequence D-P-N-G that permitted chemical cleavage 
at the D-P site (by acid) or N-G (by hydroxyl amine) 
Resulting in the release of the analog protein; VGI 

is a Xenopus v r o t e '£nT "DPP ~ is~ a ~ D r o s o ph-i-1 a -pro t e i n : L 

OP1 is a native osteogenic protein; CBMP2a and 2b, 
and CBMP3 are subparts of proteins disclosed in PCT 
application 087/01537; MIS is Mullerian inhibitory 
substance; and "consensus choices" represent various 
substitutions of amino acids that may be made at 
various positions in osteogenic proteins; 

FIGURE 2A is an coli expression vector 
containing a gene of an osteogenic protein fused to a 
leader protein; 

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



WO 89/09788 



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PCT/US89/01469 



FIGURES 3A and 3B are photomicrographs of 
implants showing the histology (day 12) of COPS 
active recombinant protein. A is a control (rat 

— — matr.ix_.a_l one, _ 25 mg)^ B is rat matrix plus COP5/ 

showing +++ cartilage formation and T+~fcrone~ format-ion — 
(see key infra). Similar results are achieved with 
COP 7; and 

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



WO 89/09788 



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PCT/US89/01469 



Description 

Purification protocols have been developed 
which enable isolation of the osteogenic protein 
present in crude protein extracts from mammalian 
bone^ — The-4-s-ql : atlon--pxo£^ the production 

of significant quantities of substantially pure 
osteogenic protein from any mammalian species / 
provided sufficient amounts of fresh bone from the 
species is available. The empirical development of 
the procedure, coupled with the availability of fresh 
calf bone, enabled isolation of substantially pure 
bovine osteogenic protein (BOP) . BOP has been 
characterized significantly; its ability to induce 
cartilage and ultimately endochondral bone growth in 
cat, rabbit, and rat have been studied; it has been 
shown to be able to induce the full developmental 
.cascade of bone formation previously ascribed to 

unknown protein or pro^eins"~in^heterogeneous-bone ~ 

extracts; and it may be used to induce formation of 
endochondral bone in orthopedic defects including 
non-union fractures. In its native form it is a 
glycosylated, dimeric protein. However, it is active 
in deglycosylated form. It has been partially 
sequenced. 

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



f 



WO 89/09788 



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PCT/US89/01469 



These consensus sequences have been refined 
by comparison with the sequences present in certain 
regulatory genes from drosophila, xenopus, and human 
followed by point mutation, expression, and assay for 
activity. This approach has been successful in 
p r oducing severa I~~a~ct i ve~t o t a Hy— synthet ic._corist^uc t s 
not found in nature (as far as applicants are aware) 
which have true osteogenic activity. 

These discoveries enable the construction of 
DNAs encoding totally novel, non-native protein 
constructs which individually and combined are 
capable of producing true endochondral bone. The 
DNAs may be expressed using well established 
recombinant DNA technologies in procaryotic or . 
eucaryotic host cells, and the expressed proteins may 
be oxidized and refolded in vitro if necessary for 
b i o 1 o g i c al _a c_t iylty^ 



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

CONSENSUS SEQUENCE DESIGN 

A synthetic consensus gene shown in FIGURE 4 
was designed to encode a consensus protein based on 
amino acid predictions from homology with the 



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TGF-beta gene family. The designed- concensus 
sequence was then constructed using known techniques 
involving assembly of oligonucleotides manufactured 
in a DNA synthesizer. 



Tr yp t i c pep ti~de s~ d~e r i ved -f -r om-Bo vi ne 

Osteogenic Protein isolated by applicants and 
sequenced by Edman degradation provided amino acid 
sequences that showed strong homology with the 
Drosophila 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 1. 



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PCT/US89/01469 



TABLE 1 



protein amino acid sequence 

( BOP ) SFDAYYCSGACQFPS 
***** * * ** 

— -(£££> GYDAYYCHGKCPFFL 



homology 
(9/15 matches) 



(BOP) 
(Ysl) 



SFDAYYCSGACQFPS 
* ** * * * 

GYMANYCYGECPYPL 



(6/15 matches) 



( BOP ) 
( inhibin ) 



SFDAYYCSGACQFPS 
* ** * * 

GYHANYCEGECPSHI 



(5/15 matches) 



SFDAYYCSGACQFPS 
* * * * 



(1Q£) 

( TGF-beta ) GYHANFCLGPCPYIW 



(4/15 matches) 



(BO£) 

(Yal) 



K/RACCVPTELSAI SMLYLDEN 

***** * **** * * (12/20 matches) 
LPCCVPTKMSPI SMLFYDNN 



( BOP ) K/RACCVPTEL S A I SMLYLDEN 

* ***** * **** * (12/20 matches) 

( inhibin ) KSCCVPTKLRPMSMLYYDDG 



( BOP ) K/RACCVPTEL SAISMLYLDE 

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

( TGF-beta ) APCCVPQALEPLPIVYYVG 



( BOP ) 
( DPP ) 



K/RACCVPTEL SAI SMLYLDEN 

******* * **** (12/20 matches) 

KACCVPTQLDSVAMLYLNDQ 



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PCT/US89/01469 



(BOP) LYVDF 

***** (5/5 matches) 

(EPP) LYVDF 



(BOP)^ LYVDF 

-***-*- __(4/5 matches) 

( Vol ) LYVEF 



(BOP) LYVDF 

** ** (4/5 matches) 

( TGF-beta l LYIDF 



(BOP) 
(inhibin) 



*-match 

In determining an ap~pr^^l~at:e~amino-acid — 

sequence of an osteogenic protein (from which the 
nucleic acid sequence can be determined) , the 
following points were considered: (1) the amino acid 
sequence determined by Edman degradation of natural 
source osteogenic protein tryptic fragments is ranked 
highest as long as it has a strong signal and shows 
homology or conservative changes when aligned with 
the other members of the gene family; (2) where the 
sequence matches for all four proteins, it is used in 
the synthetic gene 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 



LYVDF 

* * (2/5 matches) 

FFVSF 



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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 among the related proteins. 

RECOMBTnANT "OSTEOGENIC _ 

PROTEIN CONSTRUCTS 

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

The recombinant osteogenic/chondrogenic 
proteins are "novel" in the sense that/ as far as 
applicants are aware, they do not exist in nature or, 
if they do exist, have never before been associated 
with bone or cartilage formation. The approach to 
design of these proteins is to employ amino acid 
sequences, found in the native OP isolates, in 
polypeptide structures are patterned after certain 
proteins reported in the literature, or the amino 
acid sequences inferred from DNAs reported in the 
literature. Thus, using the design criteria set 



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forth above/ and refining the amino acid sequence as 
more protein sequence information was learned, a 
. series of synthetic proteins were designed with the 
hope and intent that they might have osteogenic or 
chondrogenic activity when tested in the bioassay 
— — system^di s_closed_ below . 

It was noted, for example, that DPP from 
drosophila, VG1 from Xenopus , the TGF beta family of 
proteins, and to a lesser extent, alpha and beta 
inhibins, had significant homologies with certain of 
the sequences derived from the naturally sourced OP 
product. (FIGURE 1.) Study of these proteins led to 
the realization that a portion of the sequence of 
each had a structural similarity observable by 
analysis of the positional relationship of cysteines 
and other amino acids which have an important 

__ influence on three dimensional protein conformation. 

1 1 wa sHaoted" ~t ;h"a~fc~a~ r eg ion— of -thes e— sequences had a 
series of seven cysteines, placed very nearly in the 
same relative positions, and certain other amino 
acids in sequence as set forth below: 

10 20 30 40 50 

CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 

60 70 80 90 100 

QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXL^ 

wherein each X independently represents an amino 
acid. Expression experiments of two of. these 
constructs demonstrate activity. Expression 
experiments with constructs patterned after this 
template amino acid sequence with a shorter sequence 
having only six cysteines also show activity: 



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10 20 30 40 50 

LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXP»OCXXXXXNHAXX 
60 70 80 90 100 

QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX 



"wherein- each- -X-i-ndependentAy__r_e£resents an amino 
acid. Within these generic structures are a 
multiplicity of specific sequences which have 
osteogenic or chondrogenic activity. Preferred 
structures are those having the amino acid sequence: 



10 20 30 40 50 

CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 
RKRS K S S L QE VIS E FD Y E A AY MPESMKAS VI 
KEFEKIDN L NS Q ITK F P TL 

Q A S K 

60 70 80 90 100 

QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENWLKNYQDMWEGCGCR 
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H 
RF T S K DPV V Y N S H RN RS 

N S K P E 



wherein, in each position where more than one amino 
acid is shown, any one of the amino acids shown may 
be used. Novel active proteins also are defined by 
amino acid sequences comprising an active domain 
beginning at residue number 6 of this sequence/ i.e, 
omitting the N terminal CXXXX, or omitting any of the 
preferred specific combinations such as CKRHP, CRRKQ, 
CKRHE, etc, resulting in a construct having only six 
cysteine residues. After this work, PCT 87/01537 was 
published, and it was observed that the proteins 
there identified as BMPII a and b and BMPIII each 
included a region embodying this generic structure. 
These proteins were not demonstrated to be osteogenic 
in the published application. However, applicants 



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discovered that a subpart of the amino acid sequence 
of these proteins, properly folded, and implanted as 
set forth herein, is active. These are disclosed 
herein as CBMPIIa, CBMPIIb, and CBMPIII. Also, 
applicants retrieved a previously unreported gene by 
probing -a-human T -genomic_PNA_J.^b^ary_with COPO. This 
protein was designated OP1. It comprises a region ~ 
exhibiting the same generic structure. 

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



1 10 20 30 40 

Vgl CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE 

50 60 70 
ILNGSN — H-AILQTLVHSIEPED-IPLPCCVPTKMSP 
80 90 100 
ISMLFYDNNDNWLRHYENMAVDECGCR 



1 10 20 30 40 

DPP CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNNNNPGK-VPKACCVPTQLDS 

80 90 100 

VAMLYLNDQSTVVLKNYQEMTVVGCGCR 

1 10 20 30 40 

OP1 LYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 .90 100 

I S VLYFDD S SNVI LKKYRNMWRACGCH 



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-5 

HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 
80 90 100 

I SVLYFDDSSNVItKKYRNMWRACGCH 



1 10 20 30 40 

CBMP-2 a CKRHPLYVDFS-DVGWNDWI VAPPGYHAFYCHGECPFPLAD 

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 GARRYLKVDFA=DI.GWSEWIISPKSFDAYYCSGACQFPMPK 

50 60 70 

SLKPSN — H-ATIQSIVRAVGWPGIPEPCCVPEKMSS 

80 90 100 

L S I LFFDENKNWLKVYPNMTVESCACR 



1 10 20 30 40 

COP1 LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENSTWLKNYQEMTWGCGCR 



1 10 20 30 40 

COP3 LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA • 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



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PCT/LJS89/01469 



1 10 20 30 40 

COP4 LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD 

50 60 70 
HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 
80 90 100 
ISMLYLDENEKVVLKNYQEMVVEGCGCR 



1 10 20 30 40 

COP5 LYVDFS-DVGWDDWI VAPPGYQAFYCHGECPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

ISMLYLDENEKVVLKNYQEMVVEGCGCR 



1 10 20 30 40 

COP7 LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AWQTLVNSVNSKI --PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



10 

PKHHSQRARKKNKN 

1 10 20 30 40 

COP 1 6 CRRHSLYVDFS -DVGWNDWIVAPPGYQAFYCHGECPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



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



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PCT/US89/01469 



Gene Preparation 

The synthetic genes designed to express the 
proteins as described above preferably are produced 
by assembly of chemically synthesized 

oligonucleotides. lSjlO^Omer oligonucleotides may be 

synthesized on a Biosearch DNA Model 8600 — 
Synthesizer, and purified by polyacrylamide gel 
electrophoresis (PAGE) in Tris-Borate-EDTA buffer 
(TBE) • The DNA is then elect roeluted from the gel. 
Overlapping oligomers may be phosphorylated by T4 
polynucleotide kinase and ligated into larger blocks 
which may also be purifed by PAGE. Natural gene 
sequences and cDNAs also may be used for expression. 

Ex pression 

The genes can be expressed in appropriate 
pr^^ryoticf hb~sts~~such-as -various— st.rain^^f^SL^ colj- 
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. If the gene is to be expressed in EL. coli . it 
must first be cloned into an expression vector. An 
expression vector (FIGURE 2A) based on pBR322 and 
containing a synthetic trp promoter operator and the 
modified trp LE leader can be opened at the EcoRI and 
PSTI restriction sites, and a FB-FB COP1, COP3, COPS, 
and COP7 gene fragments (FIGURE 2B) can be inserted 
between these sites, where FB is fragment B of 
Staphylococcal Protein A. The expressed fusion 



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PCT/US89/01469 



protein results from attachment of the COP gene to a 
fragment encoding FB. The COP protein is joined to 
the leader protein via a hinge region having the 
sequence Asp-Pro-Asn-Gly. This hinge permits 
chemical cleavage of the fusion protein with dilute 
~acid— at— fehe-asp-pro__site_or_cleavage at Asn-Gly with 
hydroxy lamine, resulting in release of the COP 
protein. For COP 16 and OP1, the proteins are 
expressed as fusion products, using the modified 
trp-LE as a leader. 

Producti on of Active Proteins 

The following procedure was followed for 
production of active recombinant proteins* L. coli 
cells containing the fusion proteins were lysed. The 
fusion proteins were purified by differential 
solubilization. In the case of the COP1, 3/ 4, 5, 
and 7 fusion prdt^ins^~cleayage was -with -dilute— acid^ 
and the resulting cleavage products were passed 
through a Sephacryl-200HR column. The Sephacryl 
column separated most of the uncleaved fusion 
products from the COP1, 3, 4, 5, and 7 analogs. In 
the case of the COP16 or OP1 fusion protein, cleavage 
was with a more concentrated acid, and an 
SP-Trisacryl column was used as an additional 
purification step. The COP or OP fractions were then 
subjected to HPLC on a semi-prep C-18 column. 

Initial conditions for refolding of COP 
analogs or OP1 were at pH 8-. 0 using Tris, Gu-HCl, 
dithiothreitol. Final conditions for refolding of 



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PCT/US89/01469 



COP analogs were at pH 8.0 using Tris, oxidized 
glutathione, and lower amounts of Gu-HCl and 
dithiothreitol. Alternatively, the COP or OP1 
proteins are suspended in 50 mM HC1, 6 M 
guanidine-HCl, pH 8.0, for 18 hours at 4°C. 

Ref olding- may^not.ie required if the proteins are 

expressed in animal cells. 

Production of Antisera 

Antisera to COP7 and COPS were produced in 
New Zealand white rabbits. Western blots demonstrate 
that the antisera react with COP7 and COPS 
preparations. Antisera to COP7 has been tested for 
reactivity to naturally sourced bovine osteogenic 
protein samples. Western blots show a clear reaction 
with the 30 kD protein and, when reduced, with the 16 
kD subunit. The immunoreactive species appears as a 
c 1 o s eTy- s p ac ed iioubl-et;-i-n---tfae^ , 
similar to the 16 kD doublet seen in Con A blots. 

MATRIX PREPARATION 

General Consideration of Matrix Properties ' 

The carrier described in the bioassay 
section, infra, may be replaced by either a 
biodegradable-synthetic or synthetic-inorganic matrix 
(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. 



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PCT/US89/01469 



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

induction.. Perturbation of the charge by chemical 

modification abolishes the inductive "~fesp"onser; — 
Particle size influences the quantitative response of 
new bone; particles between 75 and 420 pm 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. 

a 

The sequential cellular reactions at the 
interface of the bone matrix/OP implants are 

complex. ~~Tfie~^nurtistep-caseade -includes : finding of 

fibrin and fibronectin to implanted matrix, 
chemotaxis of cells, proliferation of fibroblasts, 
differentiation into chondroblasts , cartilage 
formation, vascular invasion, bone formation, 
remodeling, and bone marrow differentiation. 

A successful carrier for osteogenic protein 
must perform several important functions. It must 
bind osteogenic protein and act as a slow release 
delivery system, accommodate each step of the 
. cellular response during bone development, and 
protect the osteogenic protein from nonspecific 
proteolysis. In addition, selected materials must be 



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PCT/US89/01469 



biocompatible in vivo and biodegradable; the carrier 
must act as a 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 
animal-. — Bi-odegradabi.li±y^ 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"." rt~i~s~~pref erred— 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 



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PCT/US89/01469 



carrier for the matrix if their marrow cavities are 
cleaned and packed with particles and the dispersed 
osteogenic protein. 

Preparation of Biologically Active Allogenic 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 pm 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 f alse^c^xtfi~ves~bef ore- 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. 



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Preparation of Dealvcosvlated Bone Matrix for Use in 
Xenogenic Implant 

When osteogenic protein is reconstituted 

with collagenous bone matrix from other species and 

implanted in rat/ ~ncTT56ne~ "is -formed. — This^sugsejsts 

that while the osteogenic protein is xenogenic (not 
species specific), while the matrix is species 
specific and cannot be implanted cross species 
perhaps due to intrinsic immunogenic or inhibitory 
components. Thus, heretofore , for bone-based 
matrices/ in order for the osteogenic protein to 
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. Iri addition to collagen/ extracted 
bone^i-nel-udes-nonj^o^lagenous proteins which may 
account for 5% of its mass. Many non^colTagenous— — — 
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 immunogenic and inhibitory 



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PCT/US89/01469 



components from the matrix. The matrix is 
deglycosglated chemically using, for example, 
hydrogen fluoride to achieve this purpose. 

Bovine bone residue prepared as described 
^S6ve"~i's" "si-eved-r-a-na— par 7 ticle_^_of_jthe_J74-4^ yM 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 rnin. 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 
__frpm carbohydrate analysis of matrix samples taken 
■before and after treatm^iT^with'HF—af -fcer— 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; 



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PCI7US89/01469 



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

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

FABRICATION OF 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. Ethanol precipitation 

In this procedure, matrix is added to 
— osteogenic protein in guanidine-HCl . Samples are 

vortexed and incubated at a low temperature". Samples — 
are then further vortexed. Cold absolute ethanol is 
added to the mixture which is then stirred and 
incubated. After centrifugation (microfuge high 
speed) the supernatant is discarded. The 
reconstituted matrix is washed with cold concentrated 
ethanol in water and then lyophilized. 

B. Acetonitrile Trif luoroacetic Acid 
. Lvophilization 

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



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C. Urea Lvophilizafr i nn 

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 

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

A. Subcutaneous Implantation 

The bioassay for bone induction as described 
by Sampath and Reddi (Proc. Natl. Acad. Sci. USA 
(1983) HO: 6591-6595), herein incorporated by 
reference, is used to assess endochondral bone 
differentiation activity. This assay consists of 
implanting the test samples in subcutaneous sites in 



f 



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PCT/US89/01469 



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

B. Cellular Events 

The implant model in rats exhibits a 
controlled progression through the stages of matrix 
i nduced end^hondra 1 ~bo ne~deveio pment- inc lud ingj_J 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) vascul-ar 
invasion, appearance of osteoblasts, and formation of 
new bone on days nine and ten; (7) appearance of 
osteoblastic and bone remodeling and dissolution of 
the implanted matrix on days twelve to 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. 



f 

WO 89/09788 PCI7US89/01469 

• 40 - 

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 

7". be_ deternan^d_sp^trop^otometrically after 

homogenization of the implant. Th^~ac^iVity~ peaks" ~a~tf 

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. 



WO 89/09788 



- 41 - 



PCT/US89/01469 



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

Devices that contained only rat carrier show 
complete absence of new bone formation. The implant 
consists of carrier rat matrix and surrounding 
mesenchymal cells. Again, the devices that contained 
rat carrier and not correctly folded (or biologically 
inactive) recombinant protein also showed complete 
absence of bone formation. These implants are scored 
as cartilage formation (-) and bone formation (-) . 
The endochondral bone formation activity is scored as 
zero" peFcentf~< 0%)- (-F-IGURE -3A)..__ 



Implants included biologically active 
recombinant protein, however, showed evidence of 
endochondral bone formation. Histologically they 
showed new cartilage and bone formation. 

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



WO 89/09788 



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PCT/US89/01469 



The bone formation is scored as (+) by the 
presence of osteoblast surrounding vascular 
endothelium forming new matrix, , as (++) by the 
formation of bone due to osteoblasts (as indicated by 
arrows) and further bone remodeling by the appearance 
of osteoclasts in opposition to the newly formed bone 
matrix. Vascular invasion is evident in these 
implants (FIGURE 3B) . Formation is scored as (+++) 
by the presence of extensive remodeled bone which 
results in the formation of ossicles. 

The overall bone inducing activity due to 
recombinant protein is represented as percent 
response of endochondral bone formation (see TABLE 2 
below) . 



WO 89/09788 



- 43 - 



PCI7US89/01469 



TABLE 2 



HISTOLOGICAL EVALUATION OF" RECOMBINANT BONE INDUCTIVE 

PROTEINS 



Sample Implanted Cartilage Bone 

No. Protein Formation Formation 

260-54 COP-5 +++ * ++ 

279-5 COP-5 ++ + 

285-13 COP-5 +++ ++ 

277-7 COP-7 +++ ++ 

277-8 COP-7 +++ ++ 

277-9 COP-7 ++ + 

-285--14 COP^7 +++ ++ 

285-24 COP-7 ~++ +" 

285-25 COP-7 ++ ++ 

314-6 COP^-16 +++ +++ 

314-15 COP-16 ++ + 

314-16 COP-16 ++ + 

314-12 OP-1 ++ + 

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 



WO 89/09788 PCT/US89/01469 

- 44 - 

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



WO 89/09788 



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PCT/US89/01469 



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 

a protein, produced by expression of recombinant 
DNA in a host cell, comprising one or more 
polypeptide chains, each of which comprises an 
amino acid sequence sufficiently duplicative of 
the sequence of COPS, COP7, COP16, or OP1 such 
that said protein is capable of inducing 

— endochondral _bone_f ormation in association with 

said matrix when implanted in a mammals — — — 

2. A 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 prog-enitor cells from the body of 
said mammal; and 



WO 89/09788 



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PCT/US89/01469 



a protein, produced by expression of recombinant 
DNA in a host cell, comprising one or more 
polypeptide chains, each of which has less than 
about 200 amino acids, and each of which 
comprises a sequence sufficiently duplicative of 
the sequence of COPS, COP7, COP16, or OP1 such 
that said protein is capable of inducing 
cartilage formation in association with said 
matrix when implanted in a mammal. 

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

10 20 30 40 50 

CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 
* 60 '■ 70 80 90 100 

QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX 

wherein each X independently represents an amino acid. 

4. The device of claim 1 or 2 wherein the 

sequence comprises: 

10 20 30 40 50 

LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 
60 70 80 90 100 

QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXC 

wherein each X independently represents an amino acid. 



WO 89/09788 



- 47 - 



PCT/US89/01469 



5. The device of claim 1 or 2 wherein the 

sequence comprises: 

10 20 30 40 50 

CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 

RKRS K S S L QE VIS E FD Y E A AY MPESMKAS VI 
KEFEKIDN L N S Q ITK F P TL 

Q AS K 

60 70 80 90 100 

QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENWLKNYQDMVVEGCGCR 
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H 
RF T S K DPV V Y N S H RK RS 

N S K P E 

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



6. The device of claim 1 or 2 wherein the 

sequence comprises: 

10 20 30 40 50 

LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 

" k" S~S~lr- QE-V-I S-E -FD JY E A jAY MPESMKAS V I 

F E K I DN L N S Q ITK -F P TL 

A S K 
60 70 80 90 100 

QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENWLKNYQDMWEGCGCR 
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H 
RF T S K DPV V Y N S H RN RS 

N S K P E 

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



WO 89/09788 



PCT/US89/01469 



- 48 - 



7. The device of claim 1 or 2 wherein the 

sequence comprises: 

1 10 20 30 40 

' Vgl CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE 

50 60 70 

ILNGSN — H-AILQTLVHSIEPED-IPLPCCVPTKMSP 

80 90 100 

I SMLFYDNNDNWLRHYENMAVDECGCR 



8. The device of claim 1 or 2 wherein the 

sequence comprises: 

1 10 20 30 40 

DPP CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNNNNPGK-VPKACCVPTQLDS 

80 90 100 

VAMLYLNDQSTWLKNYQEMTWGCGCR 



9. The device of claim 1' or 2 wherein the 

sequence comprises: 



1 10 20 30 40 

OP1 JLYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100 

ISVLYFDDSSKVILKKYRNMWRACGCH 



WO 89/09788 



- 49 - 



PCT/US89/01469 



10. The device of claim 1 or 2 wherein the 

sequence comprises: 

-5 
HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100 

I SVLYFDD SSNVI LKKYRNMWRACGCH 



11. The device of claim 1 or 2 wherein the 

sequence comprises: 

1 10 20 30 40 

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 ''O 

HLNSTN — H-AIVQTLVNSVNS-K-IPKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQDMWEGCGCR 



12. The device of claim 1 or 2 wherein the 
~ : — sequence-comprises : 

1 10 ~20 :'3"0 40 

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-S-IPKACCVPTELSA 

80 90 100 

I SMLYLDE YDKWLKNYQEMWE G CGCR 



WO 89/09788 



- 50 - 



PCT/US89/01469 



13. The device of claim 1 or 2 wherein the 

sequence comprises: 

1 10 20 30 40 

CBMP-3 CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK 

50 60 70 

SLKPSN — H-ATIQSIVRAVGWPGIPEPCCVPEKMSS 

80 90 100 

LS ILFFDENKNWLKVYPNMTVESCACR 



14. The device of claim 1 or 2 wherein the 

sequence comprises : 

1 10 20 30 40 

COP1 LYVDFQRDVGWDDWI IAPVDFDAYYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AVVQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENSTWLKNYQEMTWGCGCR 



15. The device of claim 1 or 2 wherein the 

sequence comprises: 

1 10 20 30 40 

-GOP3- - - __. JLYVDFQRDVGVropWIVAPPGYQAFYCSGACQFPSAD 

50 60- — 70 

HFNSTN — H-AVVQTLVNNMNPGK-VFKPCCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



16. The device of claim 1 or 2 wherein the 

sequence comprises: 

1 10 20 30 40 

COP4 LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENEKVVLKNYQEMVVEGCGCR 



WO 89/09788 



- 51 - 



PCT/US89/01469 



17. The device of claim 1 or 2 wherein the 

sequence comprises: 

1 10 20 30 40 

COP5 LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



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

1 10 20 30 40 

COP7 LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 

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

10 

PJCHHSQRARKKNKN 

1 ' 10 20 30 " -40 

COP16 CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 

20. The device of claim 1 or 2 wherein the 
osteogenics protein comprises a pair of separate 
polypeptide chains. 



WO 89/09788 



- 52 - 



PCT/US89/01469 



21. Osteogenic protein, produced by 
expression of recombinant DNA in a host cell/ 
comprising one or more polypeptide chains each of 
which comprises a sequence sufficiently 
duplicative of the sequence of COPS, COP7, COP16, 
or OP1 such that said protein is capable of 
inducing endochondral bone formation in 
association with a matrix when implanted in a 
mammal. 

22. A protein, produced by expression of 
recombinant DNA in a host cell, comprising one or 
more polypeptide chains less than about 200 amino 
acids long, and comprising a sequence sufficiently 
duplicative of the sequence of COP5, COP7, COPI6, 
or OP1 such that said protein is capable of 

— inducing_cartllage formation in association with a 

matrix when implanted in a mammal". — : — _ 

23. The protein of claim 21 or 22 further 
characterized by being unglycosylated. 

24. The protein of claim 21 or 22 comprising 
a pair of separate polypeptide chains. 

25. The protein of claim 21 or 22 comprising the 
amino acid sequences: 

10 20 30 40 50 

CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 
60 70 80 90 100 

QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX 



WO 89/09788 



- 53 - 



PCT/US89/01469 



wherein each X independently represents an amino acid. 



26. The protein of claim 21 or 22 comprising the 

amino acid sequences: 

10 20 30 40 50 

LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 
60 70 80 90 100 

QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX 



wherein each X independently represents an amino acid. 



27. The protein of claim 21 or 22 comprising the 

amino acid sequences: 

10 20 30 40 50 

CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 
RKRS K S S L QE VIS E FD Y E A AY' MPESMKAS VI 
KEFEKIDN L NS Q ITK F P TL 

Q _A S K 

60 70 80 9_0 100 

QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR 

SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H 
RF T S K DPV V Y N S H RN RS 

N S K P E 



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



WO 89/09788 



- 54 - 



PCI7US89/01469 



28. The protein of claim 21 or 22 comprising the 

amino acid sequences : 

10 20 30 40 50 

LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 
K S S L QE VIS E FD Y E A AY MPESMKAS VI 
F-EKIDN L NS Q ITK F P TL 

ASK 
60 70 80 90 100 

QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENWLKNYQDMWEGCGCR 
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H 
RF T S K DPV V Y N S H RN RS 

N S K P E 



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



29. The protein of claim 21 or 22 comprising the 

amino acid sequences : 

1 10 20 30 40 

Vg 1 CKKRHLYVEFK-DVGWQNWVI APQGYMANYCYGECPYPLTE 

50 60 ,70 

-I-LNGSN — H-AILQTLViISIEPED-IPLP_Cj^PTKMSP 
80 90 * 100 

I SMLFYDNNDNWLRHYENMAVDECGCR 



30. The protein of claim 21 or 22 comprising the 

amino acid sequences : 

1 10 20 30 40 

DPP CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNNNNPGK-VPKACCVPTQLDS 

80 90 100 

VAMLYLNDQSTWLKNYQEMTWGCGCR 



WO 89/09788 



- 55 - 



PCT/US89/01469 



31. The protein of claim 21 or 22 comprising the 
amino acid sequence: 

1 10 '20 30 40 

OP1 LYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H-AIVQTLVHFINPET-VPKPCCAPTQLNA 

80 90 100 

I SVLYFDDSSNVILKKYRNMWRACGCH 

32. The protein of claim 21 or 22 comprising 
the amino acid sequences: 

-5 
HQRQA 

1 10 20 30 40 

OP1 CKKHELYVSFR-DLGWQDWI IAPEGYAAYYCEGECAFPLNS 

50 60 70 

YMNATN — H- A I VQTLVHF I NPET-VPKPCCAPTQLN A 

80 90 100 

I SVLYFDDSSNVILKKYRNMWRACGCH 



33. The protein of ~craim~21-or-2-2- -eompr-ising 

the amino acid sequences: 

1 10 20 30 40 

CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-K-IPKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQDMWEGCGCR 



WO 89/09788 



- 56 - 



PCT/US89/01469 



34. The protein of claim 21 or 22 comprising 

the amino acid sequences: 

1 10 20 30 40 

CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD 

50 60 70 

HLNSTN — H-AIVQTLVNSVNS-S-IPKACCVPTELSA 

80 90 100 

ISMLYLDEYDKWLKNYQEMWEGCGCR 



35. The protein of claim 21 or 22 comprising 

the amino acid sequences: 

1 10 20 30 40 

CBMP-3 CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK 

50 60 70 

SLKPSN — H-ATIQSIVRAVGWPGIPEPCCVPEKMSS 

80 90 100 

LSI LFFDENKNWLKVYPNMTVE S C ACR 



36. The protein of claim 21 or 22 comprising 

the amino acid sequences: 

1 10 20 30 40 

COP 1 LYVDFQRDVGWDDWI I AP VDFD AYYC S G ACQ FP SAD 

_5CL__ 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENSTWLKNYQEMTWGCGCR 



37. The protein of claim 21 or 22 comprising 

the amino acid sequences: 

1 10 20 30 40 

COP3 LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



WO 89/09788 



- 57 - 



PCT/US89/01469 



38. The protein of claim 21 or 22 comprising 
the amino acid sequences: 

1 10 20 30 40 

C0P4 LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD 

50 60 70 

HFNSTN — H-AWQTLVNNMNPGK-VPKPCCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 

39. The protein of claim 21 or 22 comprising 
the amino acid sequences: 

1 10 20 30 40 

cop5 LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD 

50 60 70 

HFNSTN — H- AWQTLVNSVNSK I — PKACCVPTEL SA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 

40. The protein of claim 21 or 22 comprising 
the amino acid sequences: 

1 10 20 30 40 

_ GO p 7 _ LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 

50— -6.0 70 

HLNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 

41. The protein of claim 21 or 22 comprising 
the amino acid sequences: 

-10 

PKHHSSRARKKNKN 
1 10 20 30 40 

COP1 6 CRRHSLYVDFS-DVGWNDWI VAPPGYQAFYCHGECPFPLAD 

50 60 70 

HFNSTN — H-AWQTLVNSVNSKI — PKACCVPTELSA 

80 90 100 

I SMLYLDENEKWLKNYQEMWEGCGCR 



WO 89/09788 



- 58 - 



PCT/US89/01469 



42. The protein of claim 21 or 22 .comprising 

the product of expression of a DNA in a 
procaryotic cell. 



43. A cell line engineered to express the 
protein of claim 21 or 22. 

44. The protein of claim 21 having a half 
maximum bone forming activity of about 20 - 25 ng 
per 25 mg of implant. 

45. Antibodies reactive with an epitope of 
the protein of claim 21 or 22. 

46. The device of claim 1 or 2 wherein said 

ma t r ix corrip f ise s ""demine r a Hz ed -deg ly co sy 1 a ted / 

protein extracted, particulate, xenogenic bone. 

47. The device of claim 1 or 2 wherein said 
matrix comprises demineralized, protein-extracted, 
particulate, xenogenic bone treated with HF. ' 



WO 89/09788 



PCT/US89/01469 



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PCT/US89/01469 



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WO 89/09788 



PCT/US89/01469 



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3689, SphI 
3606, PstI 



4733,EcoRI-killed 
4658, Aatll 

4542, Sspl 
4501, Hpal 
4314, Smal 
4287,EcoRI 
4247, Mlul 

rp p/o 



leader-peptide 




Clal.20 



BamHI, 372 

,559 



COP-5- 
4733 bp 




FIG. 2A 



PvuII, 2063 
Ndel, 2294 



WO 89/09788 



PCI7US89/01469 



6/10 



FIG.2B-1 

COP-5 fusion protein 

10 20 30 40 50 

ATGAAAGCAATTTTCGTACTGAAAGGTTCACTGGACAGAGATCTGGACTC 
MKAIFVLKGSLDRDLDE 

60 70 80 90 100 

TCGTCTGGATCTGGACGTTCGTACCGACCACAAAGACCTGTCTGATCACC 
RLDLDVRTDHKDLSDH 

110 120 130 1«0 150 

TGGTTCTGGTCGACCTGGCTCGTAACGACCTGGCTCGTATCGTTACTCCC 

L V LVD -LARNDLARIVT P 
Sail SBa 

ISO 170 180 190 200 

GGGT CTCGTTACGTTG CGG ATCTG G AATTCATGG CTG A CAACAAATTCAA 
GSRYVADLEFHADNKFN 

j EcoRI 

210 220 230- 240 250 

CAAGGAACAGCAGAACGCGTTCTACGAGATCTTGCACCTGCCG'AACCTGA— 
KEQQNAFYEILHLPKL 
Mlul Bglll _ BspMI+ 

260 270 280 290 300 

ACGAAGAGCAGCGTAACGGCTTCATCCAAAGCTTGAAGGATGAGCCCTCT 
NEEQRNGFIQSLKDEPS 

Hindlll 

310 320 330 340 350 

CAGTCTG CG AATCTG CTAG CGG ATG C CAAG AAA CTG AA CG ATG CG C AGG C 
QSANLLADAKKLNDAQA 

Nhel fspl 

360 370 380 390 400 

ACCGAAATCGGATCAGGGGCAATTCATGGCTGACAACAAATTCAACAAGG 

PKSDQ GQFMADNKFNK 

410 420 430 440 450 

AACAGCAGAACGCGTTCTACGAGATCTTGCACCTGCCGAACCTGAACGAA 

EQQNAFYEI LHLPNLNE 
MlUl Bglll BspMI+ 

460 470 480 490 500 

GAGCAGCGTAACGGCTTCATCCAAAGCTTGAAGGATGAGCCCTCTCAGTC 
£Q RKGFIQSLKDEPSQS 

Hindlll 

SUBSTITUTE SHEET 



WO 89/09788 



PCI7US89/01469 



If 10 



510 520 530 540 550 

TGCGAATCTGCTAGCGGATGCCAAGAAACTGAACGATGCGCAGGCACCGA 

ANLLADAKKLN D A Q A P 
Nhel FspI 



560 570 



580 590 600 



AGGATCCTAATGGGCTGTACGTCGACTTCAGCGACGTGGGCTG^ 

BanHI Sal1 

Sin 620 630 640 650 

TGGATTGTGGCCCCACCAGGCTACCAGGCCTTCTACTGCCATGGCGAATG 
WIVAPPGYQAFYCHGE C 
— StuI Ncol Bsml+ 



€60 670 680 690 700 

CCCTTTCCCGCTAGCGGATCACTTCAACAGCACCAACCACGCCGTGGTGC 
PFPLADHFNSTNHAVV 
NhoT - Dralll 
NheI PflMI 

710 720 730 740 750 

AGACCCTGGTGAACTCTGTCAACTCCAAGATCCCTAAGGCTTGCTGCGTG 
OTLVKSVNSKIPKACCV 
u Mstll 

760 770 780 790 800 

CCC* CCGAGCTGTCCGCCATCAGCATGCTGTACCTGGACGAGAATGAGAA 
PTELSAISMLYLDENEK 

SphI 

810 820 830 840 850 

GGTGGTGCTGAAGAACTACCAGGAGATGGTAGTAGAGGGCTGCGGCTGCC 
VVLKNYQEMVVEGCGC 

PflMI 

860 
GCTAACTGCAG 

R * 

PstI 



SUBSTITUTE SHEET 



WO 89/09788 



8/10 



PCT/US89/01469 



FIG. 3A 




WO 89/09788 



9/10 




WO 89/09788 



PCT/US89/01469 



10/ 10 



FIG. 4 



10 20 30 40 50 

GATCCTAATGGGCTGTACGTGGACTTCCAGCGCGACGTGGGCTGGGACGA 
DPNGLYVDFQRDVGWDD 

60 70 80 90 100 

CTGGATCATCGCCCCCGTCGACTTCGACGCCTACTACTGCTCCGGAGCCT 
W IIAPVDFDAYYCSGA 

HO 120 130 140 150 

-GeeAGT-TCCCCTCTGCGGATCACTTCAACAGCACCAACCACGCCGTGGTG 

C Q . F P S A D H F — N— S T_JL_? L V __ V 

160 170 180 190 200 

CAGACCCTGGTGAACAACATGAACCCCGGCAAGGTACCCAAGCCCTGCTG 
QTLV NNMNPGKVPKPCC 

210 220 230 240 250 

CGTGCCCACCGAGCTGTCCGCCATCAGCATGCTGTACCTGGACGAGAATT 
VPTELSAISMLY LD'EN 

260 270 280 290 300 

CCACCGTGGTGCTGAAGAACTACCAGGAGATGACCGTGGTGGGCTGCGGC 
STVVLKNYQEMTVVGCG 

310 

TGCCGCTAACTGCAG 
C R * 



SUBSTITUTE SHEET 




PIT WORLD INTELLECTUAL PROPERTY ORGANIZATION 

X V^l International Bureau 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International patent Classification 4; 
C12N 15/00, C12P 21/02, A61L 27/00 
C07K 13/00,A61K 37/36, //A61K 35/32 



A3 



(11) International Publication Number: WO 89/09788 

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



(21) International Application Number: PCT/US89/01469 

(22) International Filing Date : 7 April 1 989 (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 1988 (23.02.88) 



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

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

(72) Inventors ; and 

(75) Inventors/ Applicants (for US only) ; OPPERMANN, Her- 
mann [US/US]; 25 Summer Hill Road, Medway, MA 
02053 (US). KUB ERAS AM PATH, Thangavel [IN/USJ; 
6 Spring Street, Medway, MA 02053 (US). RUEGER, 
David, C. [US/US]; 



150 Edgemere Road, Apt. 4, West Roxbury, MA 02132 (US). 
OZKAYNAK, Engin [TR/US]; 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 (OAP1 
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 ihe receipt of 
amendments. 

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

8 February 1990 (08.02.90) 



(54) Title: BIOSYNTHETIC OSTEOGENIC PROTEINS AND OSTEOGENIC DEVIGES CONTAINING THEM^ __ __ 
(57) Abstract 

Disclosed are 1) osteogenic devices comprising a matrix containing osteogenic protein and methods of inducing endochon- 
dral bone growth and cartilage in mammals using the devices; 2) amino acid sequence data, amino acid composition, solubility 
properties, structural features, homologies and various other data characterizing osteogenic proteins, 3) methods of producing 
osteogenic proteins using recombinant DN A technology, and 4) osteogenically and chondrogenicaliy active synthetic protein con- 
structs. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AT Austria 

AU Australia 

BB Rarhrtas 

BE Belgium 

BF Burkina Fasso 

BG Bulgaria 

RJ Benin 

BR Brazfl 

CA Canada 

CF Central African Republic 

CC Congo 

Q{ Switzerland 

CM Cameroon 

DE Germany; Federal Rcpubfic of 

DK Denmark 



ES Spain 

H 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 

UC Sri Lanka 

LU Luxembourg 

MC Monaco 



MG 


Madagascar 


ML 


MaU 


MR 


Mauritania 


MW 


Malawi 


NL 


Netherlands 


NO 


Norway 


RO 


Romania 


SD 


Sudan 


SE 


Sweden 


SN 


Senegal 


SU 


Soviet Union 


TO 


Chad 


TG 


Togo 


US 


United States of America 



INTERNATIONAL SEARCH REPORT 

■ ^ international Application No PCT/US P.Q/014fi9 



1 CLASSIFICATION OP SUBJECT MATTER fit several r-i««*fn^«tJnn ivmbols sddIv Indicate all) * 


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

IPC 4\ C 12 N 15/00, C 12 P 21/02, A 61 'L 2.7/00, C 07 K 13/00, 
* A 61 K 37/36' // A 61 -K 35^32 


II. FIELDS SEARCHED 


Minimum Documentation Searched 7 


Classification System 


Classification Symbols . 




IPC 4 


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


Documentation Searched other than Minimum Documentation 
to the Extent that auch Documents are Included In the Flelda Searched • 




III. DOCUMENTS CONSIDERED TO IE RELEVANT* 


Category * [ Citation of Document. " with Indication, where appropriate, of the relevant paaaagaa ** 


Relevant to Claim No. " 


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

see pages 1-12,15,16,17,22-24,49.; 
pages 61-73, claims 1-23 
cited in the application 


1-6,11,12, 

in no a~i 

43,46,47 

7-10,13- 
19,29-32, 
36-41,44, 
45 


Y j Cell ~w lume~5T~, ~4-December- 1 9.87_,^ . _Cel 1 

1 Press, 

j D.L. Weeks et al.: 11 A maternal mRNA 
localized to .the vegetal hemisphere 
j in Xenopus eggs codes for a growth 
| factor related to TGF-A", pages 861- 
867 

I see the whole article, especially 
figure 3 


7-10,13- 

19v29-32-,- 

36-41 


Y j Nature, volume 325, 1 January 1987, 
j R.W. Padgett et al.: "A transcript 
| from a Drosophila pattern gene •/. 


7-10,13- 
19,29-32, 
36-41 • 


* Special categories of cited documenta: »o 

"A" document defining the oeneral atate oi tha art which la not 
conaidarad to bo of particular relevance 

M E" eerlier document but pub lie had on or attar tha International 
filing data 

document which may throw doubta on priority clalm(a) or 
which Is cited to eatabllah tha publication data of another 
citation or other apacial raaaon (as specified) 

M 0" document referring to an oral disclosure, use, exhibition or 
othar mean a 

"P" document published prior to tha International filing data but 
later than tha priority data claimed 


T later document published after tha International filing data 
or priority data and not In conflict with the application but 
citad to underatand tha principle or theory underlying tha 
invention j 

"X" document of particular relevance; tha claimed Invention 
cannot ba conaidarad noval or cannot be conaidarad to 
Involve an Inventive atep 

."Y" document of particular relevance;* the claimed Invention 
cannot ba considered to Involve an Inventive step whan tha 
document is combined with one or more other such docu- 
menta, such combination being obvious to a paraon skilled 
In tha art 

"A" document member of the earn* patent family 


IV. CERTIFICATION 


Data of the Actual Completion of the International Search 


Data of Mailing of this International Search Report 


8th November 1989 


1 5. 01. 9Q 


International Searching Authority 




EUROPEAN PATENT OFFICE 


J.K. WILLIS^ 



Form PCT/ISA/210 (second sheet) (January 1M5) 



Intirnitlonat AooUcation No. 



PCT/US 89/01469 



III. 



DOCUMENTS CONSIDERED TO SE RELEVANT (CONTINUED FROM THE SECOND SHEET) 



Caiaoory * t 



Citation of Document, with indication. wt*r« topropniti. o* maw»*«vant passages 



Rtfavant to Claim No 



P,X: 



predicts a protein homologous to 
the transforming growth factor- p 
family", pages 81-84 
see figure 2 on page 82; figure 3, 
on page 83; figure 4 on page 84 

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

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

US, A, 4563350 (NATHAN et al.) 
7 January 1986 

see column 3, lines 30-40; column 5, 
lines 5-10; column 7, lines 45-60; 
column 10, lines 45-50 

EP, A, 0128041 (BAYLINK) 
12 December 1984 
see pages 27-28, claims 



EP, A, 0169016 (COLLAGEN ' CORPT)— — 

22 January 1986 

see pages 19-20, claims 1-7 

EP, A, 0212474 (UNIVERSITY OF CALIFORNIA) 
4 March 1987 

Science, volume 217, 27 August 1982, 
AAAS, 

T.H. Maugh II: "Human skeletal 
growth factor isolated", page 819 

S.P. Colowick et al. : "Methods in 

Enzymology", volume 146, "Peptide 
Growth Factors", part A, edited by 
D. Barnes et al. , published by 
Academic Press, Inc., 
M. R. Urist et al.: "Preparation and 
bioassay of bone morphogenetic 
protein and polypeptide fragments", 
pages 294-312 

Science, volume 242, 16 December 1988, 

J.M. Wozney et al.: "Novel regulators 
of bone formation: molecular clones 
and activities", pages 1528-1534 

./.. 



21-41 



1,2,21,22, 

42,43,46, 

47 



1,2,45 



1,2,21,22, 
42,43,46V 
47 



1-6,11,12, 

20-28,42, 

43,46,47 



Form PCT ISA.W (aitra «h«t) (January 1M5) 



International Application No. 



PCT/US 89/01469 



III. DOCUMENTS CONSIDERED TO SI RELEVANT (CONTINUED FROM THE SECOND SHEET) 



Category • , Citation. of Document, with indication, wtmt aooropnate. oftne ratovant pessaoea i Relevant to Claim No 



L * 



see the whole article 
cited in the application 

World Patent Information, volume 11, no. 1/ 1-47 
1989, Pergamon Orbit InfoLine Inc., 
(GB), 

K.E.H. Gohring et al.: "A giant step 
for mankind?", pages 5-10 
see the whole article 
*(in connection with the declaration of incomplete 
search, see PCT/ISA/210 (Supplemental sheet 2)) 



Proceedings of the Montreux 1989 

International Chemical - Information 
Conference, Montreux, CH, 26-28 
September 1989, edited by Harry R. 
Collier, published by Infonortics Ltd, 
(Calne) 

C. Suhr: "Hypertrophic generic 
structures in patent claims: an 
extravagance and a remedy for it"*, 
pages 131-139 
see the whole article 
*(in connection with the declaration of inccniplete 
search, see PCT/Isa/210 (Supplemental sheet 2) 



1-47 



Form PCT IS A. -210 (aitra aheet) (January 19*9) 



International Appiicat»on No. PCT/US 89/01469 



FURTHER INFORMATION CONTINUEO FROM THE SECOND SHEET 



V. jg) OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE * 

Thi. international aearch report has not been established In respect of certain claim, under Article 17(2) (a) for the following reasons: 
1.Q Claim number. because they relate to subject matter not required to be searched by this Authority, n.mely: 

pis. see attached sheet 



i Claim number. because they relate to parts of the International application that do not comply wllh the prescribed require- 

' ments to such an extent that no meaningful International search can be carried out. specifically: 



3 .TH Claim number* — 
PCT RuJe 6.4(a). 



, because they are dependent 



claims and a/a not drafted In accordance wrtti me second and third aatittncaaof 



VI.Q3 OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING ' 



This International Searching Authority found multiple Inventions In this IntemaUonar application as follows: 



28 and 42 - 47 partially 
28 and 42 - 47 partially 
6, 20-28 and 42 - 47 partially 
6, 20-28 and 42 - 47 partially 



1. claims 7 and 29 totally; 1 - 6, 20 - 

2. claims 8 and 30 totally; 1-6, 20 

3. claims 9, 10 and 31, 32 totally; 1 

4. claims 11,12 and 33, 34 totally; 1 

5- claims 13 and 35 totally; 1 - 6, 20 - 28 and 42 - 47 partially 

IJGJ As all required additional aearch fee. were timely paid by the applicant, thlt International aearch report eovera all searchable clalmt 

^ of the International application. ' 
2Q As only soma of the r^uired additional aearch fees war. timely paid by tha applicant, this International search report eovera only 
those claims of tha International application for which fees war* paid, specifically claima: 

aQ No required additional aearch fees were timely paid by the applicant Consequently, this International search report la restricted to 
the Invention first mentioned In tha claims; It Is covered by claim numbers: 

4.n As all searchable claima could be searched without effort Juatlfyino «n additional fee, the Internetional Searching Authority did not 
" Invito payment of any additional fee. 

Remark on Protest 

The additional aearch fees were accompanied by applicants protest. 

n No protest accompanied tha payment of additional search fees. 



form PCT/ISAfflO (supplemental ahaet (3)) (January IMS) 



International Application No PCT/US 89/01469 



further information continued from PCT/ISA/2 10 ( 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 claim 3, for example, a protein (on a device) is claimed, 
which comprises 103 amino acids, whereof 68 are completely 
unspecified. Even if it is assumed that "amino acid" means 
"natural alpha amino acid", and that "comprises" means 
"consisting of exactly", the formula would still cover 20 68 
different compounds. The scope of the claims 1 and 2 is even 
vaster. (Despite this broad. scope, the biological activity 
has only been demonstrated for the compounds COPS, C0P7, 
COP16, and OP1, corresponding -to subjects 3 and 7 of the 
non unity declaration!). 

Formulas of the type of claim 3 cannot validly be regarded 
as a concise description of the matter for which protection 
is sought,. all the more as the few defined amino acids are 
totally dispersed in the hypothetical peptide chain. The 
search has therefore been restricted to the embodiments of 
claims 1-47, in as far as the protein sequences correspond 
to the sequences disclosed in the claims 7-16 (or 29*41). 



This searchable sub~j ectr matterHia-s--been-xeg.r.o.uped_ according 
to the non-unity specification, in order to establish 
conceptually individual subjects, each of which now 
constitutes a potential selection invention. 
(Please see also the citations "A giant step..." and 
"Hypertrophic Patent Claims..."). 



xx 6. claims 14 - 16 and 36 - 38 totally; 1 - 6, 20 - 28 and 42 - 47 partia, 
7. claims 17 - 19 and 39 - 41 totally; 1 - 6, 20 - 28 and 42 - 47 partia! 



iy 
iy 



Form PCT/ISA/ 



ANNEX TO THE INTERNATIONAL SEARCH REPO RT 

ON INTERNATIONAL PATENT APPLICATION NO. US 8901469 

SA 28080 

This annex fa. the patent family members relating to «•» above-mentioned international search report. 

The members are as contained in the European Patent Office EDP file on 19/iV» of information. 

The European Patent Office is in no way liable for these particulars which are merely given for the purpose 01 intormanon. 



Patent document 
cited in search report 


Publication 
date 


Patent family 
members) 


Publication 
date 







WO-A- 8800205 1W1-88 • AU-A- . 7783|87 J£0£g 

EP-A- 0182483 28-05-86 

JP-A- 62016421 24-01-87 

JP-A- 61036223 20-02-86 

US-A- 4774322 27-09-88 

US-A- 4774228 27-09-88 

US-A- 4810691 07-03-89 

USrAr 4843063 27-06-89 

"eP-A-"o212474~ 04-03-87 US-A- 4795804 03-01-89 

hP A u*m/<r jp _ A _ 62in933 22-05-87 



C 

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



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




per 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International patent Classification 4 : 
C12N 15/00, C12P 21/02, A61L 27/00 
C07K 13/00,A61K 37/36, //A61K 35/32 



A3 



(11) International Publication Number: WO 89/09788 

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



(21) International Application Number: PCT/US89/01469 

(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 1988 (23.02.88) 



(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) : OPPERMANN, Her- 
mann [US/US]; 25 Summer Hill Road, Medway, MA 
02053 (US). KUBERASAMPATH, Thangavel [IN/US]; 
. 6 Spring Street, Medway, MA 02053 (US). RUEGER, 
David, C. [US/US]; 



150 Edgemere Road, Apt. 4, West Roxbury, MA 02132 (US). 
OZKAYNAK, Engin [TR/US]; 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: BIOSYNTHETIC OSTEOGENIC PROTEINS AND OSTEOGENIC DEVICES CONTAINING THEM 



(57) Abstract 

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



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States 
applications under the PCT. 

AT Austria 

AU Australia 

BB Barbados 

BE Belgium 

BF Burkina Fasso 

BG Bulgaria 

Rf Benin 

BR Brazil 

CA Canada 

CF Central African Republic 

CG Congo 

CH Switzerland 

CM Cameroon 

DE Germany. Federal Republic of 

DK Denmark 



party to the PCT on the front pages 



ES 


Spain 


FT 


Finland 


FR 


France 


CA 


Gabon 


GB 


United Kingdom 


HU 


Hungary 


rx 


Italy 


jp 


Japan 


KP 


Democratic People 1 * Republic 




of Korea 


KR 


Republic of Korea 


U 


Liechtenstein 


IX 


Sri Lanka 


LU 


Luxembourg 


MC 


Monaco 



of pamphlets publishing international 



MG 


Madagascar 


ML 


Mali 


MR 


Mauritania 


MW 


Malawi 


NL 


Netherlands 


NO 


Norway 


RO 


Romania 


S> 


Sudan 


SE 


Sweden 


ss 


Senegal 


SU 


Soviet Union 


TD 


Chad 


TG 


Togo 


US 


United States of America 



INTERNATIONAL SEARCH REPORT 

International Application No 



I. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) * 



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

,PC4 : C 12 N mi , l^l/O^A 61 L 27/00, C 07 K 13/00, 



II. FIELDS SEARCHED 



Minimum Documentation Searched 7 



Classification System f 



Classification Symbols 



IPC' 



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



Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are included In the Fields Searched * 



III. DOCUMENTS CONSIDERED TO BE RELEVANT 1 



Category • | 



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



Relevant to Claim No. " 



X 



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

see pages 1-12,15,16,17,22-24,49; 
pages 61-73, claims 1-23 
cited in the application 



Cell, volume 51, 4 December 1987", "CelT~ 
Press, 

D.L. Weeks et al.: "A maternal mRNA 
localized to .the vegetal hemisphere 
in Xenopus eggs codes for a growth 
factor related to TGF-A" , pages 861- 
867 ' 
see the whole article, especially 
figure 3 

Nature, volume 325, 1 January 1987, 

R.W. Padgett et al.: "A transcript 
from a Drosophila pattern gene 



1-6,11,12, 

20-28,42, 

43,46,47 



7-10,13- 
19,29-32, 
36-41,44, 
45 

7--l-0- r l-3--- 

19,29-32, 
36-41 



7-10,13- 

19,29-32, 

36-41 



* Special categories of cited documents: « 

"A" document defining the general state of the art which Is not 
considered to be of particular relevance 

**E* earlier document but published on or after the International 
filing data 

M L" document which may threw doubts on priority clalm(s) or 
which Is cited to establish the pub licet I on date of anothsr 
citation or other special reason (as specified) 

"O" document referring to an oral disclosure, use, eihlbltlon or 
other means 

"P M document published prior to the international filing data but 
later than the priority data claimed 



M T" later document publiahed after the International filing date 
or priority dete and not In conflict with the application but 
cited to understand the principle or theory underlying the 
invention a 

"X** document of particular relevance; the claimed invention 
cannot bs considered novel or cannot be considered to 
Involvo an Inventive step 

"Y" document of particular relevance;' the elalmed invention 
cannot be considered to Involve an Inventive step when the 
document ia combined with one or more other auch docu- 
ments* such combinstion being obvious to a person skilled 
In the art. 

M «7* document member of the same patent family 



IV. CERTIFICATION 



Data of the Actual Completion of the International Search 



8th November 1989 



Date of Mailing of thla International Search Report 

1 5. 01. 9Q 



International Searching Authority 

EUROPEAN PATENT OFFICE 



Signature of Authorlied Officer 



\K. WILLI 



Form PCT/ISA/210 (second shoot) (January 10*5) 



International AoDlleation No. 



PCT/US 89/01469 



-2 



III. OOCUMENT8 CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) 



CiMgory * i 



A 



P,X: 



Citaucn of Oocumem. witn inoicaiwn. wrma aooropnatt. of tna'rafcvam oasaag** 



. Ratavant to Claim No 



predicts a protein homologous to 
the transforming growth factor-^ 
family", pages 81-84 
see figure 2 on page 82; figure 3, 
on page 83; figure 4 on page 84 

The Journal of Cell Biology, volume 97, 
December 1983, The Rockefeller 
University Press, 
S.M. Seyedin et al.: "In vitro 
induction of cartilage-specific 
macromolecules by a bone extract 11 , 
pages 1950-1953 

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

US, A, 4563350 (NATHAN et al.) 
7 January 1986 

see column 3, lines 30-40; column 5, 
lines 5-10; column 7, lines 45-60; 
column 10, lines 45-50 

EP, A, 0128041 (BAYLINK) 
12 December 1984 
see pages 27-28, claims 



EP, 



A, 0169 016 (COLLAGEN~CORP7-)- 

22 January 1986 

see pages 19-20, claims 1-7 



EP, A, 0212474 (UNIVERSITY OF CALIFORNIA) 
4 March 1987 

Science, volume 217, 27 August 1982, 
AAAS, 

T.H. Maugh II: "Human skeletal 
growth factor isolated", page 819 

S.P. Colowick et al. : "Methods in 

Enzymology", volume 146, "Peptide 
Growth Factors", part A, edited by 
D. Barnes et al. , published by 
Academic Press , Inc . , 
M. R. Urist et al.: "Preparation and 
bioassay of bone morphogenetic 
protein and polypeptide fragments", 
pages 294-312 

Science, volume 242, 16 December 1988, 

J.M. Wozney et al. : "Novel regulators 
of bone formation: molecular clones 
and activities", pages 1528-1534 

./.. 



21-41 



1,2,21,22, 

42,43,46, 

47 



1,2,45 



_1,2,_2_1,_22, 
42,43,46, " 
47 



1-6,11,12, 

20-28,42, 

43,46,47 



Form PCT ISA.-210 (aitra ahMt) (January 1**9> 



International Application No. 



PCT/US 89/01469 



in. 



DOCUMENTS CONSIDERED TO M RELEVANT (CONTINUED FROM THI SECOND SHEET) 



Catagory * 



Citation of Document, with indication, wfwt aooropnatt. of'tft* '•fcvam passages 



Ralavant to Claim No 



I 



see the whole article 
cited in the application 

L *! World Patent Information, volume 11, no. 1, 
! 1989, Pergamon Orbit InfoLine Inc., 

: (GB), 

K.E.H. Gohring et al. : "A giant step 
for mankind?' 1 , pages 5-10 
see the whole article 
*(in connection with the declaration of incomplete 
search, see PCT/ISA/210 (Supplemental sheet 2)) 

L * Proceedings of the Montreux 1989 

International Chemical Information 
Conference, Montreux, CH, 26-28 
September 1989, edited by Harry R. 
Collier, published by Infonortics Ltd, 
(Calne) 

C. Suhr: "Hypertrophic generic 
structures in patent claims: an 
extravagance and a remedy for it", 
i pages 131-139 

I see the whole article 

*(in connection with the declaration of incomplete 
search, see PCT/ISA/210 (Supplemental sheet 2) 



1-47 



1-47 



Form PCT IS A. -2 10 (aitra ahaat) (January IMS) 



International Application No. PCT/US 89/01469 



FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET 



V.jX] OBSERVATI ONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE ' 

This International search report has not bean established In respect of certain claims under Article 17(2) (a) for the following reasons: 
1.Q Claim numbers . because they relate to subject mattsr not required to be searched by this Authority, namely: 

pis. see artached sheet 



fcQ Claim numbers because they relate to parts of the Internstlonal application that do not comply wllh the prescribed require- 
ments to auch an enent that no meaningful International search ean be carried out specifically: 



3.fH Claim numbers— 
PCTRuto 6.4(e). 



, because) may an* cfcpandent 



claims and en not drafted In accordance with the) second and tWrd sentences of 



VI.Q§ OBSERVATIONS WHERE UNITY OF INVENTION IB LACKING * 



This International Searching Authority found multiple Inventions in this International application aa follows: 



1. claims 7 and 29 totally; 1 - 6, 20 

2. claims 8 and 30 totally; 1-6, 20 

3. claims 9, 10 and 31, 32 totally; 1 

4. claims 11,12 and 33, 34 totally; 1 

5. claims 13 and 35 totally; 1 - 6, 20 



28 and 42 - 47 partially 
28 and 42 - 47 partially 
6, 20 - 28 and 42 - 47 partially 
6, 20 - 28 and 42 - 47 partially 
28 and 42 - 47 partially ./• 



iQ As ail required additional search fees were timely paid by the applicant this International search report covers all sesrehsble claims 

™ of the International application. f 
2.Q As only some of the required additional search feee were timely paid by the applicant, this International search report eovera only 
those claims of the International application for which fees were paid, specifically claims: 



3. Q No required additional aeareh fess were timely paid by the applicant Consequently, this International search rspcrt la restricted to 

the Invention first mentioned In the claims; It la covered by claim numbers: 

4. n As all aeercheble claims could be sesrched without effort Justifying an additional fee. the International Searching Authority did not 
„ LJ Invite payment of any additional fee. 

Remark on Protest 

The additional eesrch fees were accompanied by epplleant'a protest 

f~| No protsst accompanied the payment of additional search feee. 



Form PCT/ISA/210 (supplemental sheet (2)) (January 1965) 



International Application No PCT/US 89/01469 



further information continued from PCT/ISA/2 10 ( 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 claim 3, for example, a protein (on a device) is claimed, 
which comprises 103 amino acids, whereof 68 are completely 
unspecified. Even if it is assumed that "amino acid" means 
"natural alpha amino acid", and that "comprises" means 
"consisting of exactly", the formula would still cover 20 68 
different compounds* The scope of the claims 1 and 2 is even 
vaster. (Despite this broad scope, the biological activity 
has only been demonstrated for the compounds COPS, COP7, 
COP16, and OP1, corresponding to subjects 3 and 7 of the 
non unity declaration!). 

Formulas of the type of claim 3 cannot validly be regarded 
as a concise description of the matter for which protection., 
is sought, .all the more as the few defined amino acids are 
totally dispersed in the hypothetical peptide chain. The 
search has therefore been restricted to the embodiments of 
- claims 1-47, in as far as the protein sequences correspond 
— to— the-sequences disclosed in the claims 7-16 (or 29-41). 

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. 
(Please see also the citations "A giant step..." and 
"Hypertrophic Patent Claims ..."). 



xx 6, claims 14 - 16 and 36 - 38 totally; 1 - 6, 20 - 28 and 42 - 47 partiaMy 



7. claims 17 - 19 and 39 - 41 totally; 1-6, 20 - 28 and 42 - 47 partia 



ly 



Form PCT/ISA/ 



J) ANNEX TO THE INTERNATIONAL SEARCH REPORT 
ON iftt^RNATIONAL PATENT APPLICATION NO- us 8901469 
4) SA 28080 

This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. 

The members are as contained in the European Patent Office EDP file on 19/12/89 , ^ . fl|1 

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 



Publication 
date 



Patent family 
member($) 



publication 
date 



W0-A- 


8800205 


14-01-88 ' 


A 1 1_ A _ 

AU-A" 


/ /OODO/ 






tr A 




03-05-89 


US-A- 


4563350 


07-01-86 


AU-B- 


585268 


15-06-89 




AU-A- 


4900585 


01-05-86 








EP-A- 


0182483 


28-05-86 








JP-A- 


62016421 


24-01-87 


EP-A- 


0128041 


12-12-84 


CA-A- 


1229789 


01-12-87 




JP-A- 


60069020 


19-04-85 


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 



EP-A- 0212474 



04-03-87 



US-A- 
JP-A- 



4795804- 
62111933 



03-01-89 
22-05-87 



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