Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


PCT 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
Internationa] Bureau 




INTERNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ j 
A61K 38/18 



Al 



(11) International Publication Number: 



WO 96/39170 



(43) 



International Publication Date: 12 December 1996 (12,12.96) 



(21) International Application Number: PCT/US96/07803 

(22) International Filing Date: 28 May 1996 (28.05.96) 



(30) Priority Data: 
08/467,110 
08/646.193 



6 June 1995 (06.06.95) US 

7 May 1996 (07.05.96) US 



(71) Applicant: GENEHCS INSTITUTE, INC. [USAJS]; 87 Cam- 
bridgcPark Drive, Cambridge, MA 02140 (US). 

(72) Inventors: HATTERSLEY, Gaiy; 10 Rogers Street #303, 

Cambridge, MA 02142 (US). WOLFMAN. Neil. M.; 30 
Rolling Lane, Dover. MA 02030 (US). MORRIS, Elizabeth, 
A.; 167 Southville Road, Southboro. MA 01772 (US). 
ROSEN, Vicki, A.; 2 Cedar Road, Chesmut Hill. MA 02167 
(US). 

(74) Agent: LAZAR. Steven, R.; Genetics Institute. Inc.. 87 
CambridgcPaik Drive, Cambridge. MA 02140 (US). 



(81) Designated States: AU. CA, CN. R HU. JP. KR, NO. RU. 
European patent (AT. BE. CH. DE. DK, ES, H. ¥R GB 
GR, IE. IT, LU, MC. NL. PT, SE). OAPI patent (BF. BJ. 
CF CG, CI, CM, GA, GN. ML. MR, NE, SN. TD. TG). 



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. 



O 
5 



(54) Titte: CARTILAGE INDUCTION BY BONE MORPHOGENETIC PROTEINS 
(57) Abstract 

Compositions of proteins with cartilaginous tissue inducing and maintenance activity are disclosed. IHe compositions are useful in 
the treatment of osteoarthritis, cartilage defects and in related tissue repair. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States 
applications under the PCT. 



AM 


Annenia 


AT 


Awtrie 


AU 


Ausmlia 


BB 


Barbados 


BE 


Bclgiun) 


BF 


Butkiiu Paso 


BG 


Bulgaria 


BJ 


Benin 


BR 


Brazil 


BY 


Belanis 


CA 


Canada 


CF 


Cenoal African Republic 


CG 


Congo 


CH 


Switzerland 


CI 


Cfite d'ivoiit 


CM 


CamcTDOD 


CN 


China 


CS 


Czechoslovakia 


CZ 


Czech Republic 


DE 


Gam any 


DK 


Denmark 


EE 


Bstomia 


ES 


Spain 


n 


Finland 


FR 


I^vnoe 


GA 


Gabon 



party to the PCT m the front pages 



GB 


United Kingdom 


GE 


Georgia 


GN 


Guinea 


GR 


Greece 


HU 


Hungary 


IE 


Ireland 


n 


hafy 


IP 


Japan 


KE 


Kenya 


KG 


Kyigystan 


KP 


Deraocntic People's Republic 




of Kofca 


KR 


Republic of Korea 


KZ 


Kazakhstan 


U 


Ltechtenstem 


LK 


Sri Lanka 


LR 


Liberia 


LT 


Lithuania 


LU 


Luxembourg 


LV 


Latvia 


MC 


Monaco 


MD 


Republic of Motdova 


MG 


Madagascar 


ML 


Malt 


MN 


Mongolia 


MR 


Maur^ia 



pamphlets publishing international 



MW 


Malacwi 


MX 


Mexico 


N£ 


Niger 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


PT 


Portutgal 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


5G 


Singapore 


SI 


Slovenia 


SK 


Skyvakia 


SN 


Senegal 


sz 


Swaziland 


TD 


Chad 


TG 


Togo 


TJ 


Tajikistan 


TT 


Trinklad and Tobogo 


VA 


Ukraine 


UG 


Uganda 


US 


United States of America 


uz 


Uzbekistan 


VN 


<VietNam 



wo 96/39170 



PCT/US96/07803 



TTTI TT OF THE INVENTION 
CARTILAGE INDUCTION BY BONE MORPHOGENETIC PROTEINS 
5 VTPJTi OF TP F INVENTION 

The present invention relates to a novel family of purified proteins, and 
compositions containing such proteins, which compositions axe useful for tiie 
induction of cartilaginous tissue formation, wound healing and cartilage and otiier 
tissue repair. These proteins may also be used in compositions for augmenting the 
10 activity of bone morphogenetic proteins. In particular, the present application also 
relates to the use of the above proteins for the induction of cartilaginous tissue, such 

as articular cartilage. 

BArKGROUNP r>F THE INVENTION 

The search for the molecule or molecules responsible for formation of bone, 
15 cartilage, tendon and other tissues present in bone and other tissue extracts has led 
to the discovery of a novel set of molecules called the Bone Morphogenetic Proteins 
(BMPs). The structures of several proteins, designated BMP-1 through BMP-13, 
have previously been elucidated. The unique inductive activities of these proteins, 
along with their presence in bone, suggests that they are important regulators of bone 
20 repair processes, and may be involved in the normal maintenance of bone tissue. 
There is a need to identify additional proteins which play a role in forming other 
vital tissues. Recently, the BMP- 12-related subfamily of proteins, including BMP- 
13, was shown to have tendon/ligament-like tissue inducing activity, and to be useful 
in Compositions for the induction of tendon/ligament-like tissue formation and repair. 
25 Surprisingly, the present inventors have found that members of this subfamily are 
also effective for the induction of cartilaginous tissue, and thus are useful for the 
treatment of diseases or defects of cartilaginous tissue. In particular, the inventors 
have found that BMP-13 or VL-1 is particulariy effective for the induction of 
cartilaginous tissue. In addition, the inventors have found that BMP-9 is useful for 
30 increasing proteoglycan matrix synthesis, and is therefore useful for the maintenance 
of cartilaginous tissue. 



wo 96/39170 



PCTAJS96/07803 



2 

SUMMARY OF THE INVENTION 
The present invention relates to the use of proteins in the BMP- 12 related 
subfamily of proteins for the induction of cartilaginous tissue, such as articular 
cartilage, the meniscus, and the articular surfaces of developing bone, or for the 
5 treatment of diseases or defects of cartilaginous tissue. In a preferred embodiment 
of the present invention, the protein used is BMP-13 or VL-1 . This murine version 
of this protein has also been described as GDF-6. 

BMP- 12 related proteins are a subset of the BMP/TGF-jS/Vg-l family of 
proteins, including BMP- 12 and BMP- 13, which have previously been shown to have 

10 tendon/ligament-like tissue inducing ability, and which are encoded by DNA 
sequences which have been cloned and identified, e.g., using PGR. This subfamily 
also includes MP52, which is described in WO93/16099. The BMP-12-related 
family of proteins, the DNA sequences encoding them, vectors, host cells, 
compositions and methods of making the proteins have all been extensively described 

15 in WO95/16035, as well as serial numbers 08/362,670, filed on December 22, 1994; 
08/333,576, filed on November 2, 1994, and 08/217,780, filed March 25, 1994; all 
of these applications are continuations-in-part of 08/164,103, filed on December 7, 
1993, presently abandoned. The disclosures of these applications are hereby 
incorporated herein by reference, 

20 In the present invention, compositions containing a BMP- 12 related protein, 

preferably BMP- 13 or VL-1 , are administered to a patient in need of cartilage repair, 
or having a disease or defect involving cartilaginous tissue, such as osteoarthritis. 

The DNA molecules preferably have a DNA sequence encoding the BMP- 13 
protein, the sequence of which is provided in W095/ 16035, The DNA molecules 

25 encoding BMP- 13 protein, host cells and vectors and methods of making BMP- 13 
are also described in WO95/16035. The disclosure of that document is hereby 
incorporated by reference herein. 

BMP- 12, and other proteins in the related subfamily, such as BMP- 13, have 
previously been characterized by the ability to induce the formation of 

30 tendon/ligament-like tissue. The present inventors have shown that members of the 
BMP-12-related family of proteins, preferably BMP-13, may further be characterized 
by the ability to induce formation of cartilaginous tissue. In a preferred 



wo 96/39170 



PCT/US96/07803 



3 

embodiment, the purified polypeptide may be in the form of a dimer comprised of 
two subunits, each with the amino acid sequence of BMP-13. 

In another embodiment, the present invention comprises compositions 
comprising an effective amount of the above-described BMP-12 related proteins, 
5 preferably BMP-13. In the compositions, the protein may be admixed with a 
pharmaceutically acceptable vehicle. In a particular embodiment, the composition 
may additionally include one or more additional transforming growth factor-^ 
proteins or BMPs proven to be osteogenic, preferably BMP-2, -4, -5, -6 and/or 
BMP-7; more preferably BMP-2, -4 or BMP-7. The composition comprising both 
10 a BMP-12 related protein and another TGF-j8 or BMP may be useful for the 
regeneration of multiple tissue types, for example, at the interface or junction 
between tissues. The composition comprising both a protein which is useful for the 
induction of chondrocytes and cartilaginous tissue, such as BMP-12, BMP-13 or 
MP52 and a protein useful for the induction of osteocytes and bone tissue, such as 
15 BMP-2, -4,-5, -6 or BMP-7 may be especially useful for the treatment of articular 
cartilage, in which the articular surface, cartilage, subchondral bone and/or tidemark 
interface between cartilage and bone may need to be repaired. A good source of 
osteocytes and chondrocytes for repair of the subchondral bone, tidemark interface 
and cartilage tissue may be obtained using the combination of factors. The most 
20 preferred of such embodiments is a composition combining BMP-2 and BMP-13. 

In another preferred embodiment of the present invention, the composition 
comprises both a protein which is useful for the induction of chondrocytes or 
cartilaginous tissue, such as BMP-12, MP52 or BMP-13, together with a protein 
which is useful for the maintenance of chondrocytes, or cartilaginous tissue, such 
25 as BMP-9 and, to a lesser degree, BMP-2, BMP-4 and BMP-7. The composition 
comprising BMP-13 and BMP-9 may be especially useful for the induction and 
maintenance of cartilaginous tissue at a site in need of cartilage repair, such as an 
articular cartilage defect. BMP-9 has been shown to be useful in the maintenance 
of mature chondrocytes. Thus, in a preferred embodiment of the present invention, 
30 a protein useful for the induction of chondrocytes or cartilaginous tissue, such as 
BMP-13, BMP-12 or MP52 may be applied first, in order to induce the formation 
of chondrocytes, and BMP-9 or other suitable factor, such as BMP-2, BMP-4 or 



wo 96/39170 



4 



PCTAJS96/07803 



BMP-7, administered at a later time, in order to maintain the chondrocytes and 
cartilage tissue thus formed. The BMP- 13 and BMP-9 may also be administered in 
a single composition. Such a composition is preferably administered in a form 
which allows for release of BMP- 13 prior to release of BMP-9, so that the cartilage- 

5 induction effect of BMP- 13 precedes the cartilage-maintenance effect of BMP-9. 

In another preferred embodiment, the composition comprises at least one 
protein which is useful for the induction of cartilaginous tissue, such as BMP-13, 
BMP- 12 or MP-52; one protein which is able to induce formation of subchondral 
bone tissue, such as BMP-2, -4 or -7; and one protein useful for the maintenance 

10 of cartilaginous tissuie, such as BMP-9. The most preferred of such compositions 
comprises BMP-13, BMP-2 and BMP-9; or BMP-13, BMP-7 and BMP-9, 

The present invention also includes methods for cartilaginous tissue healing 
and tissue repair, for treating osteoarthritis, or other cartilage defects, and for 
inducing cartilaginous tissue formation in a patient in need of same, comprising 

15 administering to said patient an effective amount of the above composition. The 
invention also includes heterodimeric protein molecules comprising one monomer 
having the amino acid sequence of a protein which is useful for the induction of 
chondrocytes or cartilaginous tissue, preferably BMP-13, and one monomer having 
the amino acid sequence of another protein of the TGF-jS subfamily. 

20 Finally, the present invention comprises methods for inducing cartilaginous 

tissue formation in a patient in need of same comprising administering to said patient 
an effective amount of a composition comprising a protein which exhibits the ability 
to induce formation of cartilaginous tissue, such as BMP-13, BMP-12 and MP-52, 
most preferably BMP-13. In a preferred embodiment, this method comprises 

25 administering to said patient simultaneously or subsequently an effective amount of 
a composition protein selected from the group consisting of comprising BMP-9, 
BMP-2, BMP-4 or BMP-7, most preferably BMP-9. 

Detailed Description of the Invention 
30 The methods of inducing the formation of cartilaginous tissue are described 

further below. The DNA sequences further useful for isolating and cloning further 
DNA sequences encoding BMP-12 related proteins with similar activity. These 



wo 96/39170 



PCT/US96/07803 



5 

BMP- 12 related proteins may be homologues from other species, or may be related 
proteins within the same species. 

As described previously, BMP-12 related proteins are a subset of the 
BMP/TGF-^/Vg-1 family of proteins, including BMP-12 and BMP-13, which have 

5 been previously been characterized by their tendon/ligament-like tissue inducing 
proteins encoded by DNA sequences which can be cloned and identified, e.g., using 
PGR, using BMP-12 specific primers reduced stringency conditions. In the present 
invention, it has also been shown that members of tiie BMP-12 related protein 
subfamily, particularly BMP-13 or CDF-6, are able to induce cartilaginous tissue 

10 formation. 

The DNA encoding and amino acid sequences of BMP-12-related proteins, 
including BMP-13, are disclosed in WO95/16035, tiie disclosure of which is 
incorporated herein. The human MP52 DNA is described in WO93/16099, the 
disclosure of which is incorporated herein by reference. The MP52 protein is related 

15 to BMP-12 and BMP-13. However, W093/ 16099 does not disclose the ability of 
the protein to form cartilaginous or tendon/ligament-like tissue, or its use in 
compositions for induction of cartilage or tendon/ligament-like tissue. Human MP52 
was originally isolated using RNA from human embryo tissue. It is contemplated 
herein that MP52 may be useful in the compositions and methods of the present 

20 invention. 

The DNA encoding and amino acid sequences of BMP-9 are disclosed in 
WO93/00432, the disclosure of which is incorporated herein by reference. 

The method of the present invention employs proteins which are able to 
induce cartilaginous tissue or other tissue formation in circumstances where such 
25 tissue is not normally formed, and has application in the healing of cartilage, for 
example articular cartilage tears, deformities and other cartilage defects in humans 
and other animals. Such a preparation employing a cartilaginous tissue inducing 
protein may have prophylactic use in preventing damage to cartilaginous tissue, as 
well as use in the improved fixation of cartilage to bone or other tissues, and in 
30 repairing defects to cartilage tissue. De novo cartilaginous tissue formation induced 
by a composition of the pfesem invention contributes to the repair of congeniuL 
trauma induced, or other cartilage defects of other origin, and is also useful in 



wo 96/39170 



FCTAJS96/07803 



6 

surgery for attachment or repair of cartilage. The compositions of the invention may 
also be useful in the treatment of arthritis and other cartilage defects. The 
compositions of the present invention can. also be used in other indications wherein 
it is desirable to heal or regenerate cartilage tissue. Such indications include, 
5 without limitation, regeneration or repair of injuries to the articular cartilage. The 
compositions of the present invention may provide an environment to attract 
cartilage-forming cells, stimulate growth of cartilage-forming cells or induce 
differentiation of progenitors of cartilage-forming cells. 

By cartilaginous tissue, it is meant chondrocytes, and tissue which is fonned 

10 by chondrocytes, which demonstrate the histological and compositional 
characteristics of cartilage. For example, tissue which exhibits the marker proteins 
characteristic of cartilage and/or chondrocytes, which are described further herein, 
such as type II collagen and aggrecan, also known as proteoglycan core protein. 

The proteins useful in the methods of the present invention are capable of 

15 inducing the formation of cartilaginous tissue. These proteins may be further 
characterized by the ability to demonstrate cartilaginous tissue formation activit>^ in 
the assays described below. It is contemplated that these proteins may have ability 
to induce the formation of other types of tissue, such as tendon and ligament, as 
well. 

20 The cartilaginous tissue-inducing proteins provided herein also include factors 

encoded by the sequences similar to those of naturally-occurring BMP-12 related 
proteins, such as BMP- 13, but into which modifications are naturally provided (e.g. 
allelic variations in the nucleotide sequence which may result in amino acid changes 
in the polypeptide) or deliberately engineered. Similarly, the cartilaginous tissue- 

25 maintaining proteins provided herein also include factors encoded by the sequences 
similar to those of naturally-occurring BMP-9 protein, but into which modifications 
are naturally provided (e.g. allelic variations in the nucleotide sequence which may 
result in amino acid changes in the polypeptide) or deliberately engineered. For 
example, synthetic polypeptides may wholly or partially duplicate continuous 

30 sequences of the amino acid residues of BMP- 13 and/or BMP-9. These sequences, 
by virtue of sharing primary, secondary, or tertiary structural and conformational 
characteristics with cartilaginous tissue growth or maintenance factor polypeptides 



wo 96/39170 



PCTAJS96/07803 



of naturally-occurring BMP- 13 or BMP-9 may possess cartilaginous or other tissue 
growth or maintenance factor biological properties in common therewith. Thus, they 
may be employed as biologically active substitutes for naturally-occurring 
cartilaginous tissue inducing polypeptides, and cartilaginous tissue maintenance 

5 polypeptides in therapeutic compositions and processes. 

Other specific mutations of the sequences of cartilaginous tissue inducing 
proteins described herein involve modifications of glycosylation sites. These 
modifications may involve 0-linked or N-linked glycosylation sites. For instance, 
the absence of glycosylation or only partial glycosylation results from amino acid 

10 substitution or deletion at asparagine-linked glycosylation recognition sites. The 
asparagine-linked glycosylation recognition sites comprise tripeptide sequences which 
are specifically recognized by appropriate cellular glycosylation enzymes. These 
tripeptide sequences may be asparagine-X-threonine, asparagine-X-serine or 
asparagine-X-cysteine, where X is usually any amino acid except proline. A variety 

15 of amino acid substitutions or deletions at one or both of the first or third amino acid 
positions of a glycosylation recognition site (and/or amino acid deletion at the second 
position) results in non-glycosylation at the modified tripeptide sequence. 
Additionally, bacterial expression of protein will also result in production of a non- 
glycosylated protein, even if the glycosylation sites are left unmodified. 

20 The compositions for inducing cartilaginous tissue formation of the present 

invention may comprise an effective amount of a cartilaginous tissue inducing 
protein, wherein said protein comprises the amino acid sequence of BMP- 13, as well 
as mutants and/or variants of BMP- 13, which exhibit the ability to form cartilaginous 
tissue. Compositions of the present invention may further comprise additional 

25 proteins, such as additional members of the TGF-iS superfamily of proteins, such as 
activins. Another aspect of the invention provides pharmaceutical compositions 
containing a therapeutically effective amount of a cartilaginous tissue-inducing 
protein, such as BMP- 13 or VL-1, in a pharmaceutically acceptable vehicle or 
carrier. These compositions may be used to induce the formation of cartilaginous 

30 tissue or other tissue. It is contemplated that such compositions may also be used 
for articular cartilage repair, wound healing and other tissue repair, such as skin 
repair. It is further contemplated that proteins of the invention may increase 



wo 96/39170 



PCTAJS9<W07803 



8 

neuronal survival and therefore be useful in transplantation and treatment of 
conditions exhibiting a decrease in neuronal survival. Compositions of the invention 
may further include at least one other therapeutically useful agent, such as the BMP 
proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed 
5 for instance in United States Patents 5,108,922; 5,013,649; 5,116,738; 5,106,748; 
5,187,076; and 5,141,905; BMP-8, disclosed in PCT pubUcation WO91/18098; 
BMP-9, disclosed in PCT publication WO93/00432; BMP-10, disclosed in PCT 
application W094/26893; and BMP-11 , disclosed in PCT application W094/26892. 
The disclosure of the above documents are hereby incorporated by reference herein. 

10 In one particular embodiment of the invention, the compositions comprising 

a therapeutically effective amount of a cartilaginouis tissue-inducing protein, such as 
BMP- 13 or VL-1, together with a therapeutically effective amount of a cartilaginous 
tissue-maintaining protein, such as BMP-9. In such a composition, the BMP-9 
protein is preferably encapsulated, or otherwise administered in a manner which 

15 allows for the cartilaginous tissue-maintaining activity of BMP-9 to begin 
simultaneously with and continue subsequent to the cartilaginous tissue-inducing 
activity. For example, the BMP-9 component may be encapsulated in a resorbable 
polymer delivery system, such as polylactic acid, polyglycolic acid or copolymers 
thereof, polyorthoesters, polyorthocarbonates, and other polymers. Suitable 

20 polymers are disclosed for example in EP 0145240, the disclosure of which is hereby 
incorporated by reference. Alternatively, BMP-9 may be encapsulated in liposomes 
for delivery simultaneously with BMP-13. For example, liposome delivery of TGF- 
0 protein is described in United States Patent 5,206,023, 5,270,300; and 5,368^858, 
the disclosure of each of which are hereby incorporated by reference. Both of these 

25 delivery systems may be modified to provide for release of BMP-9 at a lat^r time, 
or over a more sustained time period, allowing for the beneficial effects of BMP-9 
on chondrocyte and cartilage maintenance to act complementary to the beneficial 
effects of BMP-9 on induction of chondrocytes and cartilaginous tissue. 

The proteins or compositions of the present invention may also be useful for 

30 treating cell populations, such as embryonic cells or stem cell populations, to 
enhance or enrich the growth, differentiation and/or maintenance of the cells. The 
treated cell populations may be useful for gene therapy applications. 



wo 96/39170 



PCTAJS96/07803 



9 

The compositions of the invention may comprise, in addition to a 
cartilaginous tissue-inducing protein such as BMP-13 or VL-1, other therapeutically 
useful agents including MP52, parathyroid hormone-related peptide (PTHrP); 
epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived 

5 growth factor (PDGF), transforming growth factors (TGF-a and TGF-jS), and 
fibroblast growth factor-4 (FGF-4), parathyroid hormone (PTH), leukemia inhibitory 
factor (LIF/HILDA/DIA), insulin-like growth factors (IGF-l and IGF-II). Portions 
of these agents may also be used in compositions of the present invention. For 
example, a composition comprising both BMP-2 and BMP-13 implanted together 

10 may give rise to both bone and cartilaginous tissue. Such a composition may be 
useful for treating defects of the junction between cartilage, and bone form 
simultaneously at contiguous anatomical locations, and may be useful for 
regenerating tissue at the site of cartilage attachment to bone. Compositions 
including PTHrP may also be of particular interest because it has been found that 

15 this factor is useful in maintaining chondrocytic phenotype of cells. See co-pending 

patent application serial number , filed on March 26, 1996, the disclosure 

of which is hereby incorporated by reference. Thus, the compositions of the present 
invention include combinations of PTHrP with the cartilage-inducing and/or 
cartilage-maintaining proteins of the present invention. 

20 It is contemplated th^'t the compositions of the invention may also be used 

in wound healing, such as skin healing and related tissue repair. The types of 
wounds include, but are not limited to bums, incisions and ulcers. (See, e.g. PCT 
Publication WO84/01 106 for discussion of wound healing and related tissue repair). 
It is expected that the proteins of the invention may act in concert with or 

25 perhaps synergistically with other related proteins and growth factors. Further 
therapeutic methods and compositions of the invention therefore comprise a 
therapeutic amount of at least one protein of the invention with a therapeutic amount 
of at least one of the BMP proteins described above. Such compositions may 
comprise separate molecules of the BMP proteins or heteromolecules compri-sed of 

30 different BMP moieties. For example, a method and composition of the invention 
may comprise a disulfide linked dimer comprising a BMP- 12 related protein subunit 
and a subunit from one of the "BMP" proteins described above. Thus, the present 



wo 96/39170 



PCT/US96/07803 



10 

invention includes compositions comprising a purified BMP- 12 related polypeptide 
which is a heterodimer wherein one subunit comprises the amino acid sequence of 
BMP-13, and one subunit comprises an amino acid sequence for a bone 
moiphogenetic protein selected from the group consisting of BMP- 1, BMP-2, BMP- 
5 3. BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP- 10 and BMP-11. A 
further embodiment may comprise a heterodimer of two disulfide bonded 
cartilaginous tissue inducing moieties such as BMP-12, VL-1 (BMP-13) or MP52, 
For example the heterodimer may comprise one subunit comprising the amino acid 
sequence of BMP-13, and the other subunit may comprise the amino acid of BMP- 

10 12. Further, compositions of the present invention may be combined with other 
agents beneficial to the treatment of the defect, wound, or tissue in question. 

The preparation and formulation of such physiologically acceptable protein 
compositions, having due regard to pH, isotonicity, stability and the like, is within 
the skill of the art. The therapeutic compositions are also presently valuable for 

15 veterinary applications due to the lack of species specificity in TGF-j3 proteins. 
Particularly domestic animals and thoroughbred horses in addition to humans are 
desired patients for such treatment with the compositions of the present invention. 

The therapeutic method includes administering the composition topically, 
systemically, or locally as an injectable and/or implant or device. When admin- 

20 istered, the therapeutic composition for use in this invention is, of course, in a 
pyrogen-free, physiologically acceptable form. Further, the composition may 
desirably be encapsulated or injected in a viscous form for delivery to the site of 
tissue damage. Topical administration may be suitable for wound healing and tissue 
repair. Therapeutically useful agents other than the proteins which may also 

25 optionally be included in the composition as described above, may alternatively or 
additionally, be administered simultaneously or sequentially with the composition 
in the methods of the invention. In addition, the compositions of the present 
invention may be used in conjunction with presently available treatments for cartilage 
injuries, such as suture (e.g., vicryl sutures or surgical gut sutures, Ethicon Inc., 

30 Somerville, NJ) or cartilage allograft or autograft, in order to enhance or accelerate 
the healing potential of the suture or graft. For example, the suture, allograft or 
autograft may be soaked in the compositions of the present invention prior to 



wo 96/39170 



PCT/US96/07803 



1 1 

implantation. It may also be possible to incorporate the protein or composition of 
the invention onto suture materials, for example, by freeze-drying. 

The compositions may include an appropriate matrix and/or sequestering 
agent as a carrier. For instance, the matrix may support the composition or provide 
5 a surface for cartilaginous tissue formation and/or other tissue formation. The 
matrix may provide slow release of the protein and/or the appropriate environment 
for presentation thereof. The sequestering agent may be a substance which aids in 
ease of administration through injection or other means, or may slow the migration 
of protein from the site of application. 
10 The choice of a carrier material is based on biocompatibility, 

biodegradability, mechanical properties, cosmetic appearance and interface 
properties. The particular application of the compositions will define the appropriate 
formulation. Potential matrices for the compositions may be biodegradable and 
chemically defined. Further matrices are comprised of pure proteins or extracellular 
15 matrix components. Other potential matrices are non -biodegradable and chemically 
defined. Preferred matrices include collagen-based materials, including sponges, 
such as Helistat* (Integra LifeSciences, Plainsboro, N.J.), or collagen in an injectable 
form, as well as sequestering agents, which may be biodegradable, for example 
hyaluronic acid derived. Biodegradable materials, such as cellulose films, or 
20 surgical meshes, may also serve as matrices. Such materials could be sutured into 
an injury site, or wrapped around the cartilage. 

Another preferred class of carrier are polymeric matrices, including polymers 
of poly (lactic acid), poly(glycolic acid) and copolymers of lactic acid and glycolic 
acid. These matrices may be in the form of a sponge, or in the form of porous 
25 particles, and may also include a sequestering agent. Suitable polymer matrices are 
described, for example, in WO93/00050, the disclosure of which is incorporated 
herein by reference. 

Preferred families of sequestering agents include blood, fibrin clot and/or 
cellulosic materials such as alkylcelluloses (including hydroxyalkylcellutoses), 
30 including methylcellulose, elhylcellulose, hydroxyethylcellulose, 
hydroxypropylcellulose, hydroxypropyl-melhyiceUulose. and carboxymethylcellulose, 
the most preferred being caiionic salts of carboxymethylcellulose (CMC). Other 



wo 96/39170 



PCTAJS96/07803 



12 

preferred sequestering agents include hyaluronic acid, sodium alginate, poly (ethylene 
glycol), polyoxyethylene oxide, carboxy vinyl polymer and poly(vinyl alcohol). The 
amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% 
based on total formulation weight, which represents the amount necessary to prevent 

5 desorbtion of the protein from the polymer matrix and to provide appropriate 
handling of the composition, yet not so much that the progenitor cells are prevented 
from infiltrating the matrix, thereby providing the protein the opportunity to assist 
the activity of the progenitor cells. 

Additional optional components useful in the practice of the subject 

10 application include, e.g. cryogenic protectors such as mannitol, sucrose, lactose, 
glucose, or glycine (to protect the protein from degradation during lyophilization), 
antimicrobial preservatives such as methyl and propyl parabens and benzyl alcohol; 
antioxidants such as EDTA, citrate and BHT (butylated hydroxy toluene); and 
surfactants such as poly(sorbates) and poly(oxyethylenes); etc. 

15 As described above, the compositions of the invention may be employed in 

methods for treating a number of cartilage defects, such as the regeneration of 
cartilaginous tissue in areas of cartilage damage, to assist in repair of tears of 
cartilage tissue, and various other types of tissue defects or wounds. These methods, 
according to the invention, entail administering to a patient needing such 

20 cartilaginous tissue or other tissue repair, a composition comprising an effective 
amount of a cartilaginous tissue inducing protein, such as described in WO95/16035, 
the disclosure of which is hereby incorporated by reference. These methods may 
also entail the administration of a cartilaginous tissue inducing protein in conjunction 
with at least one of the BMP proteins described above. 

25 In another embodiment, the methods may entail administration of a 

heterodimeric protein in which one of the monomers is a cartilaginous tissue 
inducing polypeptide, such as BMP-12, VL-1 (BMP-13) or MP52, and the second 
monomer is a member of the TGF-/3 superfamily of growth factors. In addition, 
these methods may also include the administration of a cartilaginous tissue inducing 

30 protein with other factors including PTHrP, EGF, FGF, TGF-a, TGF-/J, and IGF. 

Thus, a further aspect of the invention is a therapeutic method and 
composition for repairing cartilaginous tissue, for repairing cartilage as well as 



wo 96/39170 



PCTAJS96/07803 



13 

treating arthrids and other conditions related to arthritis defects. Such compositions 
comprise a therapeutically effective amount of one or more cartUaginous tissue 
inducing proteins, such as BMP-13, in admixture with a pharmaceutically acceptable 

vehicle, carrier or matrix. 
5 The dosage regimen will be determined by the attending physician considering 

various factors which modify the action of the composition, e.g., amount of 

cartilaginous tissue desired to be formed, the site of cartilaginous tissue damage, the 

condition of the damaged cartilaginous tissue, the size of a wound, type of damaged 

tissue, the patient's age, sex, and diet, the severity of any infection, time of 

10 administration and other clinical factors. The dosage may vary with the type of 

matrix used in the reconstitution and the types of additional proteins in the 

composition. The addition of other known growth factors, such as K3F-I (insulin 

like growth factor I), to the final composition, may also affect the dosage. In 

general, the amount of recombinant BMP protein useful for inducing formation of 

15 cartilaginous tissue will be in an amount of about 1 to about 100 ug for a defect of 

approximately 3 mm x 3 mm in size. In general, the amount of recombinant BMP 

protein useful for inducing maintenance of cartilaginous tissue will be in an amount 

of about 1 to about 1000 ng per ml of solution. 

Progress can be monitored by periodic assessment of cartilaginous tissue 

20 formation, or cartilaginous tissue growth and/or repair. The progress can be 

monitored by methods known in the art, for example, X-rays, arthroscopy, 

histomorphometric determinations and tetracycline labeling. 

The following examples illustrate practice of the present invention in 

recovering and characterizing human cartilaginous tissue inducing protein and 

25 employing them to recover the other cartilaginous tissue inducing proteins, obtaining 

the human proteins, expressing the proteins via recombinant techniques, and 

demonstration of the ability of the compositions of the present invention to form 

cartilaginous tissue in an in yiye model. Although the examples demonstrate the 

invention with respect to BMP-13 as the cartilage-inducing protein, with minor 

30 modifications within the skill of the art, the same results may be attainable with other 

proteins with the effect of inducing formation of cartilaginous tissue, particulariy 

BMP- 12 or MP52. Although the examples demonstrate the invention with respect 



wo 96/39170 



PCT/US96/07803 



to BMP-2 as the osteogenic protein, with minor modifications within the skill of the 
art, the same results may be attainable with other proteins with the effect of inducing 
the formation of osteocytes and bone tissue, particularly BMP-4 or BMP-7. 
Example 1: Localization of BMF-13 mRNA During Embryogenesis 

5 BMP- 13 specific ribonucleotide probes were used to localize the expression 

of BMP- 13 mRNA transcripts during mouse embryogenesis. When compared to the 
osteogenic protein, BMP-2, BMP-13 mRNAs exhibit a more restricted pattern of 
expression. In the developing limb, BMP-13 transcripts are observed only in the 
region between developing articular cartilage surfaces, and not in the cartilage 

10 condensations wherie transcripts for BMP-2 receptors are evident, or in the 
interdigital mesenchyme where BMP-2 and BMP-4 mRNA transcripts are abundant. 

Example 2: Cartilage Induction Using BMP-13 

BMP-13 was tested for its effect on cell lines derived from E13 mouse limb 
15 buds. Cells are grown to confluence on medium containing DME supplemented with 

I % fetal calf serum. The cells are treated with BMP-13 in varying doses from less 
than 1 ng/ml up to about 5.0 x 10^ ng/ml. Effects are seen with doses of at least 
about 0.1 ng/ml. After 10 days of treatment with BMP-13, a chondroblast-like 
phenotype with an accumulation of proteoglycan-rich matrix around the cells. 

20 Northern analysis of mRNA from BMP-13 treated limb bud cells revealed expression 
of both type II collagen and proteoglycan core protein, but not osteocalcin. Type 

II collagen and proteoglycan core protein are known markers for chondroblasts, or 
cartilaginous tissue. 

As a control, similar treatment with BMP-2, a known osteogenic protein, 
25 results in the expression of a bone phenotype characterized by the production of 
osteocalcin, alkaline phosphatase and type I collagen. Osteocalcin, alkaline 
phosphatase and type 1 collagen are known markers for osteoblasts, or bone tissue. 

These observations, combined with the localization of BMP-13 during limb 
development support the conclusion that BMP-13 is involved in induction of 
30 cartilaginous tissue, and more particularly in cartilage formation that occurs at the 
articular surfaces of developing bones. 



wo 96/39170 



PCTAJS96/07803 



Example 3: Northern Analysis 

Using Northern analysis, BMP-13 and other proteins can be tested for their 
effects on various cell lines. Suitable cell lines include cell lines derived from E13 
mouse limb buds. After 10 days of treatment with BMP-13 or other protein, the cell 
5 phenotype is examined histologically for indications of tissue differentiation. In 
addition, Northern analysis of mRNA from BMP-13 or other protein treated cells 
can be performed for various markers including one or more of the following 
markers for bone, cartilage and/or tendon/ligament, as described in Table I: 

10 Table I 

Marker Bone Cartilage Tendon/Ligament 

Osteocalcin -f - - 

Alkaline Phosphatase 
Proteoglycan Core Protein 
15 Collagen Type I 
Collagen Type II 
Decorin 
Elastin 

1- Marker seen early, marker not seen as mature bone tissue forms 
20 2- Marker depends upon site of tendon; strongest at bone interface 
3- Marker seen at low levels 

Example 4: Full Thickness Articular Cartilage Repair Model 

A full thickness articular cartilage defect model in the femoral-patellar joint 
25 of adult rabbits is used to evaluate the ability of the BMPs to affect cartilage and 
bone repair. Adult New Zealand White rabbits are anesthetized and prepared for 
sterile surgery, A 3 x 3 mm defect through articular cartilage and into underlying 
subchondral bone is drilled into the patellar groove of the knee joint. The defect 
is either left empty (empty control), filled with collagen sponge {collagen control), 
30 or with collagen sponge soaked with rhBMP-13 protein, another BMP protein, or 
a combination of BMP-13 and other BMPs (experimental). The incision is closed 
and animals are allowed free movement within their cages for 4 weeks. After 4 
weeks the animals are humanely euthanized and the articular cartilage/subchondral 



+ 

+ + + 

+ + + 



wo 96/39170 



PCT/US96/07803 



bone defect is evaluated histologically for tissue architecture, quantity and quality 
of repair tissue. 

In one experiment, defects were filled with (1) 10 ug of rhBMP-13; (2) 5 ug 
rhBMP-2; or (3) 2.5 ug rhBMP-2 plus 5 ug rhBMP-13 combined on a collagen 

5 sponge [rhBMP-2/13]. Four weeks post-operatively, repair tissues were evaluated 
histologically using a grading system modified from Wakatani et al., J. Bone and 
Joint Surg. , 76-A:4 (1994). Total histological scores demonstrated significant 
differences between all BMP treated groups relative to empty or collagen controlis. 
Morphologically, the repair cartilage resulting from treatment with rhBMP-13 or 

10 rhBMP-2/13 was different than that resulting from treatment with rhBMP-2 alone. 
Thirty percent of the defects containing rhBMP-13 had an organization of 
chondrocytes in the repair cartilage similar to normal hyaline articular cartilage 
radial zone architecture, a phenotype not seen with rhBMP-2 treatment at 4 weeks. 
Defects treated with rhBMP-2 alone repaired with fibrocartilage rather than hyaline- 

15 like cartilage. However, this cartilage consistently lacked the holes and fissures 
regularly observed in empty or collagen controls. This improved repair may reflect 
the rapid and reproducible subchondral bone healing in defects exposed to rhBMP-2. 
Subchondral bone repair was significantly improved (p < 0.05) in defects treated with 
rhBMP-2 and rhBMP-2/13 (88% and 90% replacement, respectively) as compared 

20 to empty or collagen controls, or defects treated with rhBMP-13 alone (53%, 55%, 
and 51% replacement, respectively). These results demonstrate the differential 
effects of rhBMP-2 and rhBMP-13 on the early repair of full-thickness articular 
cartilage defects. The beneficial effects of rhBMP-2 on cartilage repair appear to 
stem from its ability to rapidly and consistently reproduce the subchondral bone 

25 plate, resulting in a stable site for chondrogenesis, while the beneficial effects of 
rhBMP-13 are more directly related to repairing articular cartilage with little 
osteogenic effect, and results in a cartilage which more closely resembles normal 
hyaline articular cartilage in its cellular organization. The combined beneficial 
effects of these two proteins results in consistent subchondral bone repair with a 

30 significant percentage of repair tissue demonstrating the hyaline -cartilage-like 
phenotype. 



wo 96/39170 



PCTAJS96/07803 



Example 5: Modulation of Matrix Synthesis by BMPs 

Articular cartilage was shaved from calf carpal joints and digested in 0.2% 
coUagenase to release the chondrocytes. The chondrocytes were maintained in DME 
with 50 ug/mL ascorbate^ 6 mM glutamine, antibiotics, and supplemented with 10% 

5 FCS for21 days. The initial cell density was 0:125 x 10** ceUs/mL. Cytokines were 
added to the cultures at concentrations previously shown to induce near maximal 
response. Total DNA and proteoglycan content were measured using 
spectrophotometric assays on Day 1, 2, 6, 8, 10, 12, 14, and 21 cultures. Histology 
of the cell layers was performed on Day 14 samples and the slides were stained for 

10 proteoglycan content using Safranin-0. 

Results: rhBMP-2, rhBMP-9 and TGF-)31 stimulated proteoglycan synthesis 
significantly above control levels after 14 days in culture (p<0,05). Further, after 
21 days in culture, rhBMP-9 treatment increased proteoglycan levels significantly 
more than control, rhBMP-2 or TGF-^1 treatment. rhBMP-9 and TGF-/31 

15 significantly increased the rate of increase of cell number, as measured by DNA 
content, when compared to empty control cultures (p<0.05). Histologically, the 
empty cultures showed minimal extracellular matrix synthesis by Safranin-O staining . 
The TGF-jSl treated cultures contained more cells, but they were not 
morphologically similar to chondrocytes. Safranin-0 staining demonsti-ated a marked 

20 increase in proteoglycan production in rhBMP-9 treated cultures, with rhBMP-2 
treatment increasing proteoglycan synthesis of a sub-population of cells. 

The observation that rhBMP-2 and rhBMP-9 stimulate proteoglycan synthesis 
in culture indicate that these factors play a role in adult articular cartilage 
metabolism. The ability of these cytokines to stimulate matrix synthesis by articular 

25 chondrocytes and maintain chondrocyte phenotype suggest important applications 
including cartilage defect repair and prevention/reversal of osteoarthritis, 
chondrocyte phenotype. These studies suggest that these BMPs may be particularly 
useful for cartilage differentiation, growth, maintenance and repair in conjunction 
with rhBMP-13, which has shown the ability to cause chondrocyte differentiation. 



30 



wo 96/39170 



PCT/US96/07803 



Example 6: Stimulation of Articular CartUage Metabolism by rhBMP-9 and 
rhBMP-2 

5 X 1mm diameter cartilage discs were cut from the metacarpophalangeal 
joints of 7-10 day old calves immediately after euthanasia. After three days of 

5 equilibration, the explants were incubated in serum free medium with increasing 
doses of TGF-i31 , rhBMP-2 or rhBMP-9. Medium and cytokine were changed daily. 
After three days in culture, 10/xCi"SO4/ml was added for eight hours. Explants 
were harvested and the radiolabeled proteoglycans quantified after column 
chromatography of tissue digests. Newly synthesized proteoglycan was normalized 

10 to the DNA content of the explant. Total proteoglycan accumulation in the cartilage 
matrix was also evaluated. Explants were allowed to incubate with or without 
cytokine for 14 days and evaluated for total proteoglycan and DNA content. 
Proteoglycan content was evaluated using a spectrophotometric assay and DNA 
content using the Hoechst dye binding assay. All results are the mean of six trials. 

15 Results: Explants treated with 10, 100 and 1,000 ng rhBMP-9/ml showed a 
significant increase in proteoglycan synthesis after three days in culture. 100 or 
1,000 ng rhBMP-2/ml also increased proteoglycan synthesis. In contrast, 
proteoglycan synthesis by explants treated with TGF-/31 did not increase above 
untreated levels. In fact, 100 ng/ml TGF-j31 significantly inhibited proteoglycan 

20 synthesis below that of untreated explants. Total proteoglycan content of rhBMP-9 
treated explants increased to 1.45-2.1 times untreated explants after 14 days in 
culture, with a maximal increase at a dose of 10 ng/ml. Content was significantly 
higher than untreated explants at all does (p<0.05). rhBMP-2 increased total 
proteoglycan content 1.46-1.91 limes untreated explants, with maximal increase at 

25 1 ,000 ng/ml. Content was significantly higher than untreated explants at 10 to 1 ,000 
ng/ml (p < 0.05). Proteoglycan content of TGF-/3 1 treated explants did not change 
during the culture period. DNA content of the rhBMP-9 and rhBMP-2 treated 
explants increased in a manner parallel to. but less pronounced than, the 
proteoglycan content. 

30 The above results demonstrate that rhBMP-9 and rhBMP-2 increased 

proteoglycan synthetic rale and total matrix proteoglycan accumulation in bovine 
articular cartilage explants. This contrasted with the effects of TGF-)S1 which 



wo 96/39170 



PCT/US96/07803 



inhibited proteoglycan synthesis. DN A content of the rhBMP-9 and rhBMP-2 treated 
explants also increased. These findings suggest both an increase in cell number and 
rate of synthesis per cell may be responsible for the increase in proteoglycan content. 
In all experiments, rhBMP-9 had a significant effect at lower doses than rhBMP-2. 
5 These results demonstrate that rhBMP-9 and rhBMP-2, particularly rhBMP-9, have 
the ability to increase DNA synthesis in differentiated chondrocytic cells, and thus 
may be effective inducers of chondrocyte or cartilaginous tissue maintenance. 

Example 7: Induction of Aggrecan Gene Expression and Synthesis in Articular 
10 Cartilage Explants by rhBMP-P- 

Articular cartilage was harvested from the carpometacarpal joints of fr-eshly 
killed calves and maintained in explant culture in DMEM supplemented with 1- 
glutamine, ascorbate, and 0. 1 % BSA. The cartilage was allowed to equilibrate for 
48 hours, and to determine the dose-dependent response, the explants were then 

15 cultured, in triplicate, in the presence or absence of recombinant human BMP-9 
(rhBMP-9)(0, 1, 10, 100, and 1,000 ng/ml) or IL-1 (10 ng/ml) for five days. To 
determine the time-dependent response, cartilage explants were cultured in the 
presence of 1 ,000 ng/ml for three, five, and seven days. For mRNA determinations, 
cartilage explants were then weighed and placed into 0.5 ml of Promega Total RNA 

20 Isolation System denaturing buffer [Chomczynski el al., Anal. Biochem. . 162:156 
(1987)], RNA was isolated, then purified (RNeasy RNA isolation kit), and reverse 
transcription was performed using oligo-dT primers [Re et al., Anal. Biochem. 
225:357 (1995)]. Non-competitive quantitative PCR was used to determine the 
mRNA levels for aggrecan rid.l . The primers used were designed to amplify intron- 

25 spanning regions. The PCR products were analyzed by agarose gel electrophoresis 
with ethidium bromide staining, and band intensity was determined by image 
analysis. Quantitation was done by using standards consisting of known copy 
numbers of a plasmid containing, as insert, the specific PCR product. Results were 
expressed as copies per /ig total RNA. For ^"^S-sulfate incorporation studies, on day 

30 5 explants were pulsed with 10 fcCi ^''S-sulfate for 12 hours. The cartilage was then 
digested with papain, and ^-^S-sulfate incorporation into glycosaminoglycan chains 
was determined. Statistical analysis was by ANOVA and Dunnens. 



wo 96/39170 



PCT/US96/07803 



)Results: The response of the cartilage explants to BMP-9 was dose-dependent. 
Control explants showed a level of aggrecan mRNA of 2.8 ± 0.4 x 10* copies per 
lig RNA, With increasing concentrations of BMP-9. there was a dose-dependent 
increase in the aggrecan mRNA levels. No change in aggrecan mRNA levels was 

5 detected at 1 and 10 ng/ml, but at 100 ng/ml and 1,000 ng/nil BMP-9 there was a 
significant stimulation of aggrecan mRNA levels (p< 0.05). At 100 ng/ml there was 
a 1.9 fold increase in aggrecan mRNA levels, and at 1,000 ng/ml there was an 
increase of 6.5 fold compared to control levels. A time course study showed that 
maximal stimulation with 1 ,000 ng/ml was achieved within three days of incubation, 

10 and was maintained 6ver seven days. ^^S-sulfate incorporation was also increased 
by culture in the presence of BMP-9, and at 100 ng/ml the ^''S-sulfate incorporate 
was increased by 2.7 fold. In contrast, explants cultured in the presence of 10 ng/ml 
IL-1 were found to have dramatically suppressed aggrecan mRNA levels, to < 10% 
of controls (p<0.05). ^^S-sulfate incorporation was also reduced, to 27% of the 

15 control incorporation. 

Aggrecan is a major component of the extracellular matrix of articular 
cartilage, and is synthesized by chondrocytes. These results demonstrate that BMP-9 
stimulates both the gene expression and synthesis of aggrecan in cartilage. Thus, 
BMP-9 may be effective for repair and maintenance of cartilage where stimulation 

20 of the synthesis of cartilage-specific matrix components is important. 



PCTAJS96/07803 

WO 96/39170 

21 

What is claimed is: 

1 . A composition for inducing cartilaginous tissue formation and maintenance 

comprising: 

(a) a cartilage formation-inducing protein; and 
5 (b) a cartilage maintenance-inducing protein. 

2. A method for inducing formation and/or maintenance of chondrocytes or 
cartilaginous tissue in a patient in need of same, said method comprising 
administering to said patient an effective amount of the composition of claim 1. 

3. A method for treating arthritis, or other cartilaginous tissue defect in a 
10 patient in need of same, said method comprising administering to said patient an 

effective amount of the composition of claim L 

4. A method for treating articular cartilage defects or damage in a patient 
in need of same, said method comprising administering to said patient an effective 
amount of the composition of claim 1 . 

15 5. The composition of claim 1, further comprising an osteogenic protein. 

6. The composition of claim 5, wherein the osteogenic protein is selected 
from the group consisting of BMP-2, BMP-4 and BMP-7. 

7. The composition of claim 1, wherein the cartilage formation-inducing 
protein is selected from the group consisting of BMP-13, BMP-12 and MP-52, 

20 8. The composition of claim 7, wherein the cartilage maintenance-inducing 

protein is BMP-9. 

9. The composition of claim 8, further comprising an osteogenic protein. 

10. The composition of claim 9, wherein the osteogenic protein is selected 
from the group consisting of BMP-2, BMP-4 and BMP-7. 

25 11. The composition of claim 10, wherein the cartilage formation-inducing 

protein is BMP-13. 

12. The composition of claim 10, wherein the cartilage formation-inducing 

protein is BMP-12. 

13. The composition of claim 10, wherein the cartilage formation-inducing 

30 protein is MP52. 

14. The composition of claim 10, wherein the osteogenic protein is BMP-2 . 

15. The composition of claim 10, wherein the osteogenic protein is BMP--;. 



wo 96/39170 PCT/US96/07803 

22 

16. The composition of claim 10, wherein the osteogenic protein is BMP-7. 

17. A composition effective for the formation and maintenance of 
chondrocytes and cartilaginous tissue comprising: 

(a) BMP-13 protein; and 
5 (b) BMP-9 protein. 

18. A composition effective for the formation and maintenance of 
cartilaginous tissue and associated subchondral bone comprising: 

(a) BMP-13 protein; 

(b) BMP-9 protein; and 
10 (c) BMP-2 protein. 

19. A composition effective for the formation and maintenance of 
cartilaginous tissue and associated subchondral bone comprising: 

(a) BMP-13 protein; 

(b) BMP-9 protein; and 
15 (c) BMP-7 protein. 



INTERNATIONAL SEARCH REPORT 



I A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 A61K38/18 



Inter -mal Application No 

PC I /US 96/07803 



Intetnational Patent gasificaticn (IPQ or to both national c iasafication and IPC 

SEARCHED 
, cumcntation searched 

IPC 6 C07K A61K 



I According to 

I R FIE LDS SEARCHED 

Minimum documentation searched (classification system followed by dassification symbols) 



I Documentation seawhed otticr than minimum documentation 



to the extent that such documents art included in the fields searched 



I Elccironic data base consulted during the 



international search (name of data base and. where practical, search lenns used) 



I C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category ' 



x,p 



Qtttion of document, wi* indieation, where appropriite, of «tt relevant p»B«ge« 



W0,A,95 16035 (GENETICS INST -.HARVARD 

COLLEGE (US)) 15 June 1995 

cited in the application 

see page 9, line 15 - line 31 

see page 13, line 18 - page 18, line 31 

W0,A,93 00432 (GENETICS INST) 7 January 
1993 

cited in the application 

see page 8, line 15 - page 10, line 3Z 

W0,A,92 09697 (CELTRIX UB INC) 11 June 
1992 

see page 6, line 1 - page 9, line 17 
see page 13, line 14 - page 15, line 29 

-/" 



Relevant to claim No. 



1-19 



pjlQ Further documents are listed in the continuation of box C. 

i ' Special categories of cited documents : 

•A* document defining the general rtate of the ait which is not 

considered to be of particular relevance 
•E' eariier document but publiAed on or after the international 

filing date 

•L' documem which may throw doubte on pritm^daiii^^ 
^ch is cited to establish the piiWicatocn date of another 
citation or other special itason (as specified) 
•O- document referring to an oral disclosure, use, exhibition or 
other means 

•p- document published prior to the inwmational filing date but 
later than the priori^ date claimed ^^^^ 



Pitent family members are listed in annex. 



•r Uter document published after JiemtcmationalfiU^^ 

iiriority date and not in conflict with the appU«tioi^^ 
SSvSiS^rtand the princii^e or ttieory undertymg the 
invention 

•X- documem of particular itla^anoe;*wdain^^ 

cannot be coKidered novd or cannotbe cOTfflde^^ 
involve an inventive step when toe document is taken alone 

•Y' document of particiilarrelevancr. the daimed invention 

^ cSS^ooSdertdtoinyrfveanin^^^ 

documert is combined with one or inoie other sudi docu- 

SSS^A combination being obvious to a person stalled 

in the art 

•A" document member of the same patent fannly 



"bate of the actiial completion of the international iea«* 

15 October 1996 ^ 

I Name and mailing address of the ISA 

European Patent Office, P.B. 5818 Patentlaan 2 
NL • 2280 HV Rijswijk 
Td.(-^ 31-70) 340-2040. Til 31 651 eponl. 
Far (+31-70)340-3016 

Fotm PCT/ISA/aiO ineooA sbMi) »«) 



Date of maiUng of the international search report 

25. ia96 



Autbofized officer 



Rempp, G 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



IbW •mu) Applictlion No 

PC I /US 96/07803 



C^Contiimation) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * Gtotion of document, with indication, where appropriate, of the relevant passages 



Relevant to claim Na 



Y.P 



W0,A,95 33830 (GENETICS INST ;ROSEN VICKI 
A (US) : WOZNEY JOHN M (US) ; CELESTE ANT) 
14 December 1995 

see page 7, line 33 - page 10, line 11 



2 



Fotm PCTfSSAmt <aa«tinttatteA of tmta ttw«g (July 1993) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



Imcmationi] apptication No. 

PCT/US 96/ 07803 



Bex I Obsmalion* where certain daims were found unsearchable (Continuation of item I oflirrtahed) 

TWi in«rn«io«l mr* «port h» not b««n «UbliriKd in rctpcct of muin d»i>> under Artdr 17(JX.) for the following re-ont 

® SSw**!^ relate to nibject matter not req»Bred to be leiiched by this Authority, nimelr 

Remark: Although clalns 2 - 4 are directed to a method of treatment 

of the human/animal body, the search has been carried out and based on 
the alleged effects of the compound/composition. 

O SS« *l?y reUte to part, of the international ^.plication that do not «on?plywith the prtwribed requbonentt to tuch 
an extent that no meaningful international (earch can be carried out, ipedTieally: 



□ 



SS^w'S^y .re dependent daims «id are not drafted in a«ordance with the »cond and third sentences of Rule 6,4(a). 



Box II ObservAlions where unity of invention i$ lacking (Contintifttifin of hem 2 of fir*! sheet) 



This InternaUonal Seardung Authority found multiple inventions in this international ^pUcation. as follows: 



1. rn As aU required additional seardi fees were timely paid by the appUcanl, this international search report covers aU 
searchable datms. 

2, ri As alt searchable daims could be seardies without effort justifying an additional fee. tins Authority ifid not invite payment 
of any additioMl lee. 



3 I 1 As only some of the required additional search fees were timely paid by the applicant, 
' ' — ' covers only those claims for which fees were paid, spedfically daims Nos,: 



ttus international search report 



4. n No reqinred additional search fees were timely paid by <he.«PptonL<^»^^ 
^ resttteted to the invention first mentioned in the daims; tt is covered by daans Nos.. 



this interiuoional search report is 



I [ The addiuonal search fees were accompanied by the ^pficani's protest 
j~j f^Q protest accompanied the payment of additional search fees. 



Form PCT/lSA/210 {continuation of first iheei ^l)) (July W2) 



INTERNATIONAL SEARCH REPORT 



Patent document 
died in search report 

WO-A-9516035 



iifonnation on patent family ment^Krs 



Publication 
date 



15-06-95 



Inter 'onal AppUcaiion No 

PC I /US 96/07803 



Patent family 
member (s) 



AU-A- 
CA-A- 
EP-A- 
FI-A- 
NO-A- 



1301395 
2176942 
0733109 
962350 
962304 



Publication 
date 

27-06^5 

15- 06-95 
25-09-96 

16- 07-96 
04-06-96 



WO-A-9300432 



07-01-93 



AU-B- 
AU-A- 
CA-A- 
EP-A- 
JP-T- 



652472 
2269992 
2108770 
0592562 
6508990 



25-08-94 

25- 01-93 

26- 12-92 
20-04-94 
13-10-94 



WO-A-9209697 



11-06-92 



AU-B- 
AU-A- 
CA-A- 
EP-A- 
US-A- 



651421 
9141991 
2071912 
0513334 
5393739 



21-07-94 
25-06-92 
31-05-92 
19-11-92 
28-02-95 



WO-A-9533830 14-12-95 AU-A- 2817095 



04-01-96 



Fonn rCTflSA/a« (pM»l W"* 



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

BEST AVAILABLE IMAGES 

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

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

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□f FADED TEXT OR DRAWING 

BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

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

□ OTHER: — ^ — 

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