Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


per 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
Internationa] Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International Patent Classification 5 : 

C12P 21/00, C12N 15/00, 5/00 
A61K 37/12, O07K7/00 



Al 



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



WO 92/09697 

11 June 1992(11.06.92) 



(21) Internationa] Application Number: PCT/US91 /08953 

(22) Internationa] Filing Date : . 27 November 1 991 (27. 1 1.91) 



(30) Priority data: 
620,142 



30 November 1990 (30.1 1.90) US 



(71) Applicant: CELTRIX LABORATORIES, INC. [US/USJ; 

2500 Faber Place, Palo Alto, CA 94303 (US). 

(72) Inventors: BENTZ, Hanne ; 36125 Toulouse Street, Ne- 

wark, CA 94560 (US). THOMPSON, Andrea, Y. ; 221 
Easy Street, Mountain View, CA 94043 (US). ARM- 
STRONG, Rosa ; 2106 Louis Road, Palo Alto, CA 
94303 (US). ROSEN, David, M. ; 2981 Nieman Boule- 
vard #430, San Jose, CA 95148 (US). 



(74) Agents: BENZ, William, H. et al.; Morrison & Foerster, 
545 Middlefield Road, Suite 200, Menlo Park, CA 94025 
(US). 



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



Published 

With international search report. 



(54) Title: USE OF A BONE MORPHOGENETIC PROTEIN IN SYNERGISTIC COMBINATION WITH TGF-B FOR 
BONE REPAIR 



(57) Abstract 

Bone morphogenetic proteins (BMPs) work in synergistic combination with TGF-ps to provide compositions with in- 
creased osteogenic activity. Methods of treating bone defects, inducing bone growth and increasing bone marrow cell production 
using these compositions are also disclosed. 



FOR JOE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front 
applications under the PCT. 



pages of pamphlets publishing international 



AT Austria 

AU Australia 

BB Barbados 

BE Belgium 

BP Burkina Faao 

BG Bulgaria 

BJ Benin 

BR Brazil 

CA Canada 

CF Central African Republic 

CG Congo 

CH Switzerland 

CI Cole d'lvoire . 

CM Cameroon 

CS Czechoslovakia 

DE* Germany 

DK Denmark 



ES Spain 

Fl Finland 

FR France 

CA Gabon 

GB , United Kingdom 

CN Guinea 

GR Greece 

HU Hungary 

IT Italy 

JP iapan 

KP Democratic People's Republic 

of Korea 

KR Republic or Korea 

U Liechtenslein 

LK Sri Lanka 

LU Luxembourg 

MC Monaco 



MC 

ML 

MN 

MR 

MW 

NL 

NO 

PL 

RO 

SD 

SE 

SN 

su + 

TO 
TC 

US 



Mongolia 

Mauritania 

Malawi 

Netherlands 

Norway 

Poland 

Romania 

Sudan 

Sweden 

Senegal 

Soviet Union 

Chad 

Togo 

United States of America 



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



WO 92/09697 



PCT/US91/08953 



5 USE OF A BONE MORPHOGENETIC PROTEIN IN SYNERGISTIC 

COMBINATION WITH TGF-S FOR BONE REPAIR 

Technical Field 

The present invention relates to osteoplasty* 

10 More particularly, it relates to a combination of 

proteins that induce bone growth, pharmaceutical composi- 
tions containing that combination, methods for promoting 
bone growth using such compositions, methods for 
stimulating bone marrow progenitor cells to divide and 

15 differentiate into bone marrow cells, and methods for 

treating diseases associated with dysfunction/malfunction 
of bone generation and/ or bone resorption, such as 
osteoporosis . 

20 Background Art 

It has been established that bone contains 
materials which can stimulate the formation of new bone 
when placed in contact with living systems. (Urist, M. 
R. , Clin Orthop (1968) 56:37; Science (1965) 1S0:893; 

25 Reddi, A. H., et al., Proc Natl Acad Sci (USA) (1972) 
69:1601.) Attempts have been made to purify whatever 
factors are responsible for this activity. A "bone 
morphogenetic protein" (BMP) was extracted from 
demineralized bone using urea or guanidine hydrochloride 

30 and reprecipitated according to the disclosures in U.S. 
Patents Nos. 4,294,753 and 4,455,256 to Urist. Urist 
subsequently reported (Urist, M. R., Clin Orthop Rel Res 
(1982) 162:219) that ion exchange purification of this 
crude protein mixture yielded an activity which was 

35 



WO 92/09697 



PCT/US91/08953 



unadsorbed to carboxymethyl cellulose resin (CMC) at 
pH 4.8. Urist's reports in Science (1983) 220:680-685 
and Proc Natl Acad Science (USA) (1984) 81:371-375 
describe BMPs having molecular weights of 17,500 and 
5 18,500 daltons. Urist's patent publication, EPA Publi- 
cation No. 0212474, describes BMP fragments of 4,000 to 
7,000 daltons obtained by limited proteolysis of BMP. 

U.S. Patent No. 4,608,199 describes a 
bone -derived protein of 30,000-32,000 daltons. The 

10 protein is described as being water soluble and having no 
affinity for concanavalin A (ConA) . 

WO 88/00205 reports four proteins, designated 
BMP-l, BMP- 2 Class I ("BMP-2"), BMP -3 , and BMP -2 Class II 
( n BMP-4 n ), that are alleged to have osteogenic activity. 

15 It is not known whether these BMPs have osteogenic 

activity by themselves or require combination with other 
factors . 

J.M. Wozney, in Growth Factor Research. Vol. 1 
(1989) , pp. 267-280, describes three additional BMP 

20 proteins closely related to BMP-2, and which have been 
designated BMP- 5, BMP- 6 and BMP- 7. 

WO 89/09787 and 89/09788 describe a protein 
called "OP-l", now known to be BMP-7. The cloning of 
BMP- 7 is described in E. Ozkaynak et al., EMBO Journal 

25 (1990) 9:2085-2093, and the purification of BMP-7 is 
described in T.K. Sampath et al., J Biol Chem (1990) 
265:13198-13205. 

U.S. 4,434,094 to Seyedin and Thomas reported 
the partial purification of a bone generation- 

30 stimulating, bone -derived protein by extraction with 
chaotropic agents, fractionation on anion and cation 
exchange columns, and recovery of the activity from a 
fraction adsorbed to CMC at pH 4.8. This new protein 
fraction was termed "osteogenic factor" (OF) and was 



35 



WO 92/09697 PCT/US91/08953 



-3- 

characterized as having a molecular weight below about 
30,000 daltons. 

Commonly owned U.S. Patent No. 4,774,332 
describes two bone-derived proteins that were purified to 
5 homogeneity by extraction with chaotropic agents, and ion 
exchange column fractionation. These two proteins were 
originally called cartilage- inducing factor (CIF) A and 
CIF 6 . CIF A was subsequently found to be identical to a 
previously identified protein called transforming growth 

10 factor beta (TGF-£) . CIF B has been found to be a novel 
form of TGF-S and is now known as TGF-S2, while CIF A is 
known as TGF-El. 

Additional TGFs have also been described. U.S. 
Patent No. 4,886,747 to Derynck et al., describes the 

15 identification of TGF-S3 and its nucleotide sequence, and 
describes a method for recovery of TGF-S3 from 
recombinant cell cultures. S.B. Jakowlew et al., Molec 
Endocrinol (1988) 2:1186-1195 describes TGF-64 and its 
nucleotide sequence, identified by cDNA characterization. 

20 A.B. Roberts et al., Growth Factors. Vol. 2 (1990), pp. 
135-147, describes the purification of TGF-S5 from 
Xenopus - conditioned medium. 

A novel glycoprotein preparation from bovine 
bone designated osteoinductive factor (OIF) , based on the 

25 ectopic osteoinductive activity used to follow its 

purification, has also been reported (Bentz, H., et al., 
J. Biol. Chem. (1989) 264:20805-20810) ... It was first 
thought that the osteoinductive activity of these "OIF 
preparations" could be substantially enhanced by either 

30 TGF-fil or 2 (Bentz, H., et al., J. Biol. Chem. (1989) 
264 :20805-20810; Bentz, H., et al., Development and 
Diseases of -Cartilage and Bone Matrix (A. Sen and T. 
Thornhill eds.) pp. 137-147, Alan R. Liss, Inc. (1987) 
New York) . However, it was later found that the factors 

35 



WO 92/09697 



PCT/US91/08953 



-4- 

believed to have OIF activity did not in fact have that 
activity. 

It is not known whether bone- inducing activity 
in isolated preparations is attributable to a single 
5 protein or a plurality of proteins acting in concert. 
Identification of the protein (s) responsible for 
bone- inducing activity is complicated by the large number 
of proteins extracted from bone (estimated to be several 
hundred) , and the lack of a conclusive in vitro assay for 
10 bone- inducing activity. 



Summary of the Invention 

The invention results from two unpredicted 
discoveries. The first is that the bone generation - 
15 stimulating activity in the partially purified "OIF 

preparation" from bovine bone is apparently due to the 
presence of BMPs in the preparation, including BMP-2, 
BMP-3, BMP-4 and BMP- 7. The second is that the TGF-Ss 
have a cell -proliferative effect such that when co- 
20 administered with the BMPs # including BMP-2, BMP-3, BMP- 
4 and BMP- 7, a synergistic effect in osteogenic activity 
is obtained. 

Accordingly, it is a primary object of this 
invention to provide a composition for treating cartilage 
25 and/or bone defects containing a BMP and a TGF-fi. 

It is a further object of this invention to 
provide a method of treating bone defects in vivo at a 
predetermined site in a living mammal. 

It is another object of this invention to 
30 provide a method of inducing bone marrow cell production 
in a living mammal. 

Additional objects, advantages and novel 
features of the invention will be set forth in part in 
the description which follows , and in part will become 



WO 92/09697 



PCT/US91/089S3 



apparent to those skilled in the art upon examination of 
the following, or may be learned by practice of the 
invention. 

In one aspect of the invention, a composition 
5 for treating cartilage and/or bone defects in vivo is 
provided comprising (a) an effective tissue- growth - 
inducing amount of a bone morphogenetic protein (BMP) and 
a transforming growth factor beta (TGF-S) combined with 
(b) a pharmaceutical^ acceptable carrier. 

10 In another aspect of the invention, a method of 

treating cartilage and/or bone defects in vivo at a 
desired site is provided, comprising implanting the above 
described composition in a mammal at said site. 

In yet another aspect of the invention, a 

15 method of inducing bone marrow cell production in a 

living mammal is provided, comprising administering an 
effective tissue-growth inducing amount of the above- 
described composition systemically to the mammal. 

20 Brief Description of the Figures 

Figure 1 shows the RP-HPLC chromatographic 
fractionation of an osteoinductive factor- containing 
fraction. 

Figure 2 shows the results of SDS-PAGE on 
25 several fractions obtained in the process of Figure 1. 

Figure 3 shows the RP-HPLC fractionation of 
material from the first peak of Figure 1. 

Figure 4 compares peptide sequences of peak 1 
proteins with BMP-2 and BMP-3 sequences. 
30 Figure 5 show the bone -inductive properties of 

BMP-2+3 implants with TGF-S (•) and without TGF-S (O) . 
Implants were assayed for alkaline phosphatase (ALPase) 
activity, and evaluated histologically for bone (B) and 
cartilage (C) on a scale of 0-5 (highest). 

35 



WO 92/09697 



PCT/US91/089S3 



Detailed Description of t he Invention 
Definitions: 

As used herein, "bone morphogenetic protein" 
5 (BMP) refers to a class of bone -derived proteins capable 
of inducing bone formation, as measured by activity in an 
in vivo rat bone formation assay- BMPs include BMP-1, 
BMP- 2, BMP- 3, BMP-4, BMP- 5, BMP- 6 and BMP- 7, and 
fragments, deletions, additions, substitutions, mutations 

10 and modifications thereof which retain the biological 
characteristics of the natural BMP. The BMP may be of 
human, bovine, or other species origin. 

As used herein, "transforming growth factor 
beta" (TGF-fi) refers to a family of polypeptides capable 

15 of inducing cell proliferation including TGF-B1, TGF-S2, 
TGF-S3, TGF-S4, and TGF-S5, and to fragments, deletions, 
additions, substitutions, mutations and modifications 
thereof which retain the biological characteristics of 
the naturally occurring TGF-B. The TGF-fi may be of 

20 human, bovine, or other species origin. 

A "mutation" in a protein alters its primary 
structure (relative to the commonly occurring protein) 
due to changes in the nucleotide sequence of the DNA 
which encodes it. These mutations include genetically 

25 engineered variants as well as allelic variants. A 

"modified" protein differs from the commonly occurring 
protein as a result of post-translational events which - 
change the glycosylation or lipidation pattern, or the 
primary, secondary, or tertiary structure of the protein. 

30 Changes in the primary structure of a protein can also 
result from delations, additions or substitutions. A 
"deletion" is defined as a polypeptide in which one or 
more internal amino acid residues are absent. An 
"addition" is defined as a polypeptide which has one or 

35 



WO 92/09697 



PCT/US91/089S3 



more additional internal amino acid residues as compared 
to the wild type. A "substitution" results from the 
replacement of one or more amino acid residues by other 
residues . A protein "fragment" is a polypeptide 
5 consisting of a primary amino acid sequence which is 
identical to a portion of the primary sequence of the 
protein to which the polypeptide is related. 

Preferred "substitutions" are those which are 
conservative, i.e., wherein a residue is replaced by 

10 another of the same general type. As is well understood, 
naturally- occurring amino acids can be subclassif ied as 
acidic, basic, neutral polar, or neutral nonpolar. 
Furthermore, three of the naturally- occurring amino acids 
are aromatic. It is generally preferred that encoded 

15 peptides differing from the natural BMPs and TGF-Ss 

contain substituted codons for amino acids which are from 
the same group as that of the amino acid replaced. Thus, 
in general, the basic amino acids Lys, Arg, and His are 
interchangeable; the acidic amino acids aspartic acid and 

20 glutamic acid are interchangeable; the neutral polar 
amino acids Ser, Thr, Cys, Gin, and Asn are 
interchangeable; the nonpolar aliphatic acids Gly, Ala, 
Val, lie, and Leu are conservative with respect to each 
other (but because of size, Gly and Ala are more closely 

25 related and Val, lie and Leu are more closely related), 
and the aromatic amino acids Phe, Trp, and Thr are 
interchangeable. While proline is a nonpolar neutral 
amino acid, it creates difficulties because of its 
effects on conformation, and substitutions by or for 

30 proline are not preferred, except when the same or 

similar conformational results can be obtained. Polar 
amino acids which represent conservative changes include 
Ser, Thr, Gin, Asn; and, to a lesser extent, Met. In 
addition, although classified in different categories, 



WO 92/09697 



PCT/US91/08953 



-8- 

Ala, Gly, and Ser seem to be interchangeable, and Cys 
additionally fits into this group, and may be 
alternatively classified with the polar neutral amino 
acids . 

5 It should further be noted that if either the 

BMP or the TGF-S or both is made synthetically, 
substitutions by amino acids which are not encoded by a 
naturally- occurring gene may also be made. Alternative 
residues include, for example, the w-amino acids of the 

10 formula H 2 N(CH 2 ) n COOH wherein n is 2-6. These are 

neutral, nonpolar amino acids, as are sarcosine, t -butyl 
alanine, t- butyl glycine, N-methyl-isoleucine, and 
norleucine. Other amino acids such as phenyl glycine, for 
example, can be substituted for Trp, Tyr or Phe as an 

15 aromatic neutral amino acid; citrulline and methionine 
sulfoxide are neutral polar, cyclohexyl alanine is 
neutral nonpolar, cysteic acid is acidic, and ornithine 
is basic- The conformation- conferring properties of 
substituted proline residues may be retained if one or 

20 more of these is substituted with hydroxyproline. 

The biological "characteristics" of a protein 
refer to the structural or biochemical function of the 
protein in the normal biological processes of the 
organism in which the protein naturally occurs. Examples 

25 of biological characteristics of a BMP include its 

specific antigenicity or immunogenicity, and/or its bone- 
inducing activity. Biological characteristics of a 
TGF-S include its specific antigenicity or immuno- 
genicity, and/or -its ability to mediate inflammatory and 

30 chemotactic responses in vivo . 

The term "effective amount" as used herein 
intends that quantity of a therapeutic agent that, when 
administered to a patient, is required to provide the 

35 



WO 92/09697 



PCI7US91/08933 



desired or intended beneficial effect without intoler- 
able side effects, such as toxicity . 
The Composition : 

There are two active components in the 
5 compositions of this invention: a BMP protein and a 
TGF-S protein. Together, these components work 
synergistically with respect to the osteogenic properties 
of the composition. 

Preferably, the BMP component includes BMP-2, 
10 BMP- 3, BMP- 4, BMP -7 or mixtures thereof. Most 

preferably, the BMP component is a mixture of BMP-2 and 
BMP-3. Preferably, the TGF-S component includes TGF-fcl 
or TGF-62 or both, most preferably TGF-S2 ♦ 

Both components may be prepared in one of three 
15 ways: (l) preparative methods of isolation and 

purification of the naturally occurring product; (2) 
synthetic methods; and (3) recombinant methods. 

Preparative Methods. Isolation and 
purification of BMP-1, BMP-2, BMP-3 and BMP -4 is 
20 described in U.S. Patent No. 4,877,864, the disclosure of 
which is incorporated herein by reference. The isolation 
and purification of BMP-7 is described in T.K. Sampath et 
al., J Biol Chem (1990) 265:13198-13205. 

Isolation and purification of TGF-S1 and TGF- 
25 £2 is described in U.S. Patent No. 4,774,332, the 

disclosure of which is incorporated herein by reference. 
The isolation and purification of TGF-S5 is described in 
A.B. Roberts et al., .Growth Factors. Vol. 2 (1990), pp. 
135-147. 

30 Synthetic Methods. The BMPs and TGF-6s of the 

present invention can be synthesized chemically by means 
well known in the art such as, e.g., solid-phase peptide 
synthesis. Their amino acid sequences are known or 
deduced from their art -known nucleotide sequences. The 



35 



WO 92/09697 



PCT/US91/08953 



-10- 

synthesis is commenced from the carboxy- terminal end of 
the peptide using an a- amino protected amino acid, 
t- Butyl oxycarbonyl (Boc) protective groups can be used 
for all amino groups even though other protective groups 
5 are suitable. For example, Boc-Lys-OH or Boc-Arg-OH 
(i.e., BMP- like carboxy- terminal amino acids) can be 
esterified to chloromethylated polystyrene resin 
supports. The polystyrene resin support is preferably a 
copolymer of styrene with about 0.5 to 2% divinyl benzene 

10 as a cross -linking agent which causes the polystyrene 
polymer to be completely insoluble in certain organic 
solvents. See Stewart, et al., Solid- Ph ase Peptide 
Synthesis (1969), W.H. Freeman Co., San Francisco, and 
Merrifield, J. Am. Chem. Soc. (1963) 85:2149-2154. These 

15 and other methods of peptide synthesis are also 

exemplified by U.S. Patent Nos. 3,862,925; 3,842,067; 
3,972,859; and 4,105,602. 

The synthesis may use manual techniques or 
automatically employing, for example, an Applied 

20 Biosys terns 43 OA Peptide Synthesizer (Foster City, 
California) or Biosearch SAM II automatic peptide 
synthesizer (Biosearch, Inc., San Rafael, California), 
following the instructions provided in the instruction 
manual supplied by the manufacturer. 

25 Of course, since automated synthesis also 

permits control of the sequence, additions, substitutions 
and deletions in the amino acid sequence are available 
using this method of synthesis. In addition, it is not 
necessary that a substituted amino acid be encoded by a 

30 gene. Therefore, the D- forms or £- or w-amino acids can 
be substituted for those natively present. 

Recombinant Methods. The BMPs and TGF-Ss of 
the present invention may also be made, using their 
identified and isolated nucleotide sequences, and using 



35 



WO 92/09697 



PCT/US91/089S3 



-11- 

conventional techniques of molecular biology, 
microbiology, recombinant DNA, and immunology, which are 
within the skill of the art. Such techniques are 
explained fully in the literature. See, e.g., Maniatis, 
5 Fritsch and Sambrook, Molecular Cloning: A Laboratory 

Manual (1982); DNA Cloning . Volumes I and II (D.N. Glover 
ed. 1985) ; Oligonucleotide Synthesis (M.J. Gait ed. 
1984); Nucleic Acid Hybridization (B.D. Hames and S.J. 
Higgins eds. 1984); Transcription and Translation (B.D. 

10 Hames and S.J. Higgins eds. 1984); Animal Cell Culture 
(R.K. Freshney ed. 1986); Immobilized Cells and Enzymes 
(IRL press, 1986); Perbal, B., A Practical Guide to 
Molecular Cloning (1984); Gene Transfer Vectors for 
Mammalian Cells (J.H. Miller and M.P. Calos eds, Cold 

15 Spring Harbor Laboratory, 1987); the series, Methods in 
Enzymology (S. Colowick and N. Kaplan eds., Academic 
Press, Inc.) , and Handbook of Experimental Immunology , 
Volumes I-IV (D.M. Weir and C.C. Blackwell eds., 1986, 
Blackwell Scientific Publications) . 

20 Recombinantly produced modified forms of a BMP 

or TGF-E may be obtained by mutagenesis of a sequence 
encoding a BMP or TGF-S, for example, by site directed 
mutagenesis, as well as by deletions or insertions of 
sequences. Techniques for causing mutations are known to 

25 those of skill in the art. See, for example, Maniatis et 
al . , supra . , Perbal , B . , supra . , Methods in Enzymology . 
supra., and Gene Transfer Vectors for Mammalian Cells , 
supra . 

The foregoing methods to obtain the BMP and 
30 TGF-S polypeptides of the invention are not intended to 
be limiting, and those components of the invention may be 
prepared in any convenient manner. 

In view of the showing that bone- inductive 
proteins from human, monkey, bovine and rat sources are 



WO 92/09697 



PCT/US91/08953 



-12- 

nonspecies- specific in their ability to produce endo- 
chondral bone in xenogeneic implants (Sampath, T.K., et 
al., Proc Natl Arad Sci (USA) (1983) 80:6591) it is 
believed that the BMPs and TGF-Ss described herein are 
5 highly conserved among mammalian species- -i.e. , corres- 
ponding bone- inducing proteins from different mammalian 
species (herein called "species analogs") will have 
substantially homologous amino acid sequences that vary 
from, e.g., the bovine protein, if at all, in one or more 

10 amino acid residue additions, deletions or substitutions 
and/or substantially similar glycosylation patterns that 
do not affect the nonspecies -specif ic ability of the 
molecule to induce bone formation. In this regard, the 
terms "substantially equivalent" and "substantially 

15 homologous" are intended to mean proteins, regardless of 
species or method of preparation, that have the same 
amino acid sequence as the bovine osteogenic proteins 
described in the examples and proteins of similar but 
different amino acid sequence, which difference (s) does 

20 not affect nonspecies -specific endochondral bone- inducing 
activity adversely. The amino acid sequences of such 
"substantially homologous" proteins will usually be at 
least 50%. homologous, more usually at least 80% homolo- 
gous, and preferably at least 90% homologous to the 

25 bovine proteins described herein. Accordingly, such 

proteins may be derived from bone of diverse mammalian 
origin or synthesized using recombinant DNA procedures. 

The BMP and TGF-S proteins of the invention, 
depending on the pH of their environment if suspended or 

30 in solution, or of their environment when crystallized or 
precipitated if in solid form, may be in the form of 
pharmaceutically acceptable salts or may be in neutral 
form. The free amino groups of the proteins are capable 
of forming acid addition salts with, for example, 



35 



WO 92/09697 



PCI7US91/08953 



•13- 



inorganic acids such as hydrochloric, phosphoric, or 
sulfuric acid; or with organic acids such as, for 
example, acetic, glycolic, succinic, or mandelic acid. 
The free carboxyl groups are capable of forming salts 
5 with bases, including inorganic bases such as sodium, 

potassium, or calcium hydroxides, and such organic bases 
such as piperidine, glucosamine, t rime thy lamine, choline, 
and caffeine. In addition, the proteins may be modified 
by combination with other biological materials such as 
10 lipids and saccharides, or by side chain modification 

such as acetylation of amino groups, phosphorylation or 
hydroxyl side chains, or oxidation of sulfhydryl groups. 
All of these modifications are included within the scope 
of the definition. 

15 

Modes of Administration : 

The osteogenic compositions of the invention 
may be used to induce de novo bone formation in 
circumstances where bone is not normally formed. The 

20 composition may thus also be used to treat a variety of 
bone defects: it may be used prophylactically to reduce 
the likelihood of fracture, to improve fixation of 
artificial joints, to repair congenital or trauma- induced 
bone defects, or in cosmetic plastic surgery. The 

25 composition may also be used to enhance bone formation in 
instances where bone is normally formed, such as in 
fracture repair, replacement of surgically removed bone, 
or repair of bone damaged by periodontal disease or in 
other tooth or alveolar ridge repair processes. In such 

30 uses, the composition will be administered locally, such 
as by implantation, at the desired site of bone 
formation. 

The composition may also be administered 
systemically, such as intravenously, to treat indications 

35 



WO 92/09697 



PCT/US91/08953 



■14- 



associated with insufficient bone formation and/or un- 
desirable levels of bone resorption such as localized, 
regionalized or generalized osteoporosis or to stimulate 
bone marrow progenitor cells in the treatment of mal- 
5 functions or dysfunctions of the hematopoietic system 
such as chronic and acute mylocytic leukemia and other 
cancers of the hematopoietic system or in post- 
irradiation treatment to stimulate bone marrow stem cells 
to divide and differentiate. 
10 The osteogenic composition of the invention 

will normally be formulated in osteogenically effective 
amounts with pharmaceutically acceptable solid or fluid 
carriers, for local injection or implantation at the 
desired site of activity or systemic administration by 
15 conventional parenteral routes. Preferably the 

formulations for local administration include a matrix 
material that is capable of presenting the protein at the 
desired site of activity as well as providing a structure 
for developing bone and cartilage. Potential matrices 
20 may be biodegradable or nonbiodegradable and be 

chemically or biologically defined. Examples of such 
materials are calcium sulfate, hydroxyapatite , trical- 
ciumphosphate, polyorthoesters , polylactic- polyglycolic 
acid copolymers, collagen, bioglass, and the like. 
25 Formulations for systemic administration will typically 
involve liquid vehicles that are commonly used for 
parenteral administration of proteinaceous therapeutics. 

The osteogenic composition of the invention may 
be conjugated with other molecules to increase its 
water-solubility, increase its half-life, or enhance its 
ability to bind to bone. For instance, the proteins may 
be conjugated to polyethylene glycol to increase their 
water solubility or to bone -binding molecules such as 
bisphosphonates (e.g. i-hydroxyethylidene-l,l-bis- 



30 



35 



WO 92/09697 



PCI7US91/08953 



-15- 

phosphonic acid, dichloromethylene bisphosphonic acid, 
and 3-amino-l-hydroxypropylidene-l, 1- bisphosphonic acid) 
and f luorochromes (e.g. tetracyclines, calcein blue, 
xylenol orange, calcein green, and alizarin complexone 
5 red) to target the proteins to bony sites. Various 
agents for conjugating molecules to proteins are well 
known in the art and include aldehydes, carbodiimides, 
and other bifunctional moieties. 

The amount of the osteogenic composition 

10 administered may vary depending upon the carrier used, 

the patient (age, sex, medical history, species) and the 
site and condition being treated. For local implanta- 
tion, the weight ratio of both BMP and TGF-S to carrier 
in the formulation will typically be in the range of 

15 about 1:5,000 to 1:50,000. The weight ratio of BMP to 
TGF-S in the composition will usually be in the range of 
10:1 to 1:10. The implant may be placed at a predeter- 
mined site in the patient by conventional surgical 
techniques, such as implantation or injection. 

20 For systemic administration the amount of total 

BMP and TGF-S will usually range between 20 /zg/kg body 
weight and 2 mg/kg body weight. In addition it may be 
desirable to combine the BMP and TGF-S proteins with 
other therapeutics, such as, in the case of osteoporosis, 

25 fluoride, calcitonin, vitamin D metabolites, and 
parathyroid hormone. Because the composition is 
nonspecies- specific in its activity it may be used to 
treat mammals in general including sport, pet, farm 
animals and humans. 

30 The following example is intended to illustrate 

but not limit the invention. 



35 



WO 92/09697 



PCT/US91/08953 



-16- 
Bxample 

A. Purification of BM P« from an OTF Preparation 

OIF preparations were obtained from a 4M 
guanidine-HCl extract of demineralized bovine bone by 
5 sequential application of five chromatographic steps, the 
last consisting of RP-HPLC using an acetonitrile gradient 
(sees Bentz, H., et al., .T. Biol. Chem. (1989) 2£4:20805- 
20810) . 

For further purification of the OIF 

10 preparations, fractions containing the bulk of the OIF 
were pooled, diluted with two volumes of 0.1% TFA, and 
rechromatographed on the same column using an n-propanol 
gradient. Solvent A was 0.1% TFA and solvent B was 90% 
n-propanol in solvent A. Proteins were eluted from the 

15 column with a linear gradient of 20-45% solvent B in 
solvent A at 0.4%/min and a flow rate of 1.2 ml/min. 

Two major peaks were, obtained as shown in 
Figure l. Protein concentration was determined by the 
BCA Pierce assay (Smith, P.K., et al., Anal, piochem. 

20 (1985) l£0:76-8613) , and by comparing peak area 

(absorbance at 230 nm) with the peak area of a standard 
sample of bovine serum albumin. Proteins were analyzed 
by electrophoresis on 15% polyacrylamide gels by the 
method of U.K. Laemmli ( Nature (1970) 212:680-685), as 

25 modified by P.W. Studier f.T. Mol. Biol. (1973) 2£:237- 
248) . Gels were stained with silver and western blot 
analysis was performed using a monoclonal antibody 
specific for OIF (Dasch, J.R., et al., J. Bone tyin. Res f 
(1989) 4_:S286 (abstr.)). 

30 The later eluted peak (Peak 2) contained OIF as 

characterized by its gel electrophoretic properties and 
immunochemical staining (Bentz, H. , et al., J T Bjol, 
Chem. (1989) 2£4.:20805-20810) . Peak 1 represented 10- 



WO 92/09697 



PCT/US91/08953 



-17- 

40% of total protein in various samples as measured by 
peak area. 

When fractions 40-49 in Figure 1 were analyzed 
by SDS-PAGE (lanes 1-10 shown in Figure 2), peak 1 
5 material (lanes 2-4) was found to contain several 

components migrating in the mass range of 28-34 kDa, 
which on reduction were converted to components of 30, 18 
and 16 kDa in relative amounts of 1, 3 and 4, 
respectively. This behavior is characteristic of BMP 

10 preparations as reported by Wang et al. ( Proc. Natl. 

Acad. Sci USA (1988) 85:9484-9488) and Luyten et al. {J*. 
Biol. Chem. (1989) 264:13377-13380). 

To confirm this identification and determine 
the BMP species, peak l proteins were subjected to 

15 peptide analysis. Reduction, S-pyridylethylation, 

cleavage with endoproteinase Lys-C (Boehringer Mannheim) , 
and peptide purification were performed essentially as 
described in H. Bentz et al., J. Biol. Chem. (1990) 
265:5024-5029. The peptide mixture resulting from the 

20 proteolytic cleavage of peak 1 proteins was fractionated 
by RP-HPLC on a Vydak C18 column, 2.1 x 150 mm 
(Figure 3) . Solvent A was 0.1% TFA and solvent C was 90% 
acetonitrile in solvent A. The column was eluted with a 
linear gradient of 0-20% solvent C in solvent A at 1%/min 

25 followed by a 20-54% linear gradient of the same solvents 
at 0.4%/min and a flow rate of 0.25 ml/min. 

Selected fractions from this separation were 
then sequenced. Peptides were sequenced by automated 
Edman degradation on a model 4 75 A microprotein sequencer 

30 (Applied Biosystems) equipped with a model 120A on-line 
microbore phenyl t hi ohydantoin derivative analyzer 
(Applied Biosystems) . The sequences (LI through L10, 
shown in Figure 4) were compared to the known sequences 
of BMP- 1-4 (Wozney, J.M., et al., Science (19€fi) 



WO 92/09697 



PCT/US91/089S3 



-18- 



242:1528-1534; Wozney, J.M. , et al. f Prog, Growth Factor 
Res . (1990) 1:267-280). Although the peptides were not 
pure, as shown by secondary residue assignments at some 
sites and some residues that did not match, it is 
5 apparent that the peptides could only have come from a 
mixture of BMP -2 and BMP-3. 

B. nHt-.aninductHvft Acti v i ty of Peak 1 Proteins and the 
fiynpraisfir Effect nf TGF-fis. 

10 The osteoinductive activity of BMP-2 and BMP-3 

(together "BMP- 2+3"), as represented by peak 1 material, 
and of OIF, as represented by peak 2 material, was 
measured in the following rat subcutis model. 

Peak 1 or 2 material was. mixed with a carrier 

15 of porous particulate hydroxyapatite/tricalcium phosphate 
(Zimmer, Warsaw, IN) and bovine dermal collagen 
(Vitrogen, Collagen Corporation) , lyophilized, hydrated 
with phosphate -buffered saline, and compacted into 
pellets weighing about 50 mg each (dry weight) . The 

20 pellets contained 120 ng peak 1 or 2 material per mg. 
Some pellets also contained 140 ng TGF-S2 per mg. 
Pellets, usually four per group, were implanted into the 
subcutaneous tissue of 34- to 40-day-old male Sprague- 
Dawley rats as previously described (Bentz, H. , et al., 

25 -t B-inl. Chem. (1989) 214:20805-20810) . After 14 days, 
pellets were explanted and evaluated for mineralization 
by assaying for alkaline phosphatase activity and for 
bone and cartilage by histological methods as previously 
described (Bentz, H., et al., J. Bjpl, Chem,.. (1989) 

30 264:20805-20810) . TGF-£l and 2 were isolated from bovine 
bone as previously described (Seyedin, S.M. , et al., 
Prnr , M atl Sei. USA (1985) 82:2267-2271; Seyedin, 

S.M., et al., .t. Biol. Chem. (1986) 261:5693-5695; 



35 



WO 92/09697 



PCI7US91/08953 



-19- 



Seyedin, S.M., et al. f J- Biol. Chem. (1987) 262:1946- 
1949). 

As shown in Table 1, at 14 days BMP -2 +3 (peak 1 
material) induced extensive bone formation and increased 
5 alkaline phosphatase activity both with and without TGF- 
S2 in this experiment. Cartilage induction by BMP-2+3, 
on the other hand, was markedly stimulated by TGF-S2. 
OIF (peak 2 material) without TGF-S2 was completely 
inactive. With TGF-S2, OIF explants occasionally showed 
10 both cartilage and bone as illustrated in Table 1. This 
finding suggests that some BMP contamination remained in 
some OIF samples. OIF added to BMP-2+3 had no effect 
with or without TGF-S2. 

Table 1 

15 In vivo osteoinductive activity of BMP-2+3 and OIF 

Material Cartilage Hist. a Bone Hist. a ALPase b 
- TGF-S2 

BMP-2+3 trace 4.0 15.4 

OIF 0 0 2.3 

20 BMP-2+3/OIF trace 3.25 11.8 

+ TGF-S2 

BMP-2+3 3.25 4.5 12.5 

OIF 0.75 c 0.75° 4.6 C 

BMP-2+3/OIF 3.0 4.75 10.7 

25 a evaluated on a scale of 0-5 (highest) 

b expressed as /xmoles p-nitrophenol/min/g wet weight 
Represents activity in one of four samples. 

To more adequately show the effect of TGF-S2 on 
modulation of the osteoinductive activity of BMP-2+3, 
BMP-2+3 dose-response curves with and without TGF-32 were 
determined again at 14 days. As shown in Figure 5, the 
effect of TGF-E2 in potentiating both cartilage and bone 
formation is clearly evident. Without TGF-S2, BMP-2+3 



30 



35 



WO 92/09697 



PCT/US91/089S3 



-20- 

was essentially inactive at levels up to 25 ng/mg implant 
and cartilage was absent at all levels. With TGF-S2, 
potentiation was evident at levels of BMP -2 +3 at and 
above 12.5 ng/mg implant and cartilage was present at and 
5 above 25 ng/mg implant. 

The data at this single time point in 
endochondral bone formation induced by BMP- 2+3, 
represented by the two experiments described here, 
strongly suggest that TGF-S2 increases early cartilage 
10 formation and delays the onset of cartilage degeneration 
and replacement by bone. TGF-E1 in other experiments 
gave similar results. 



WO 92/09697 



PCI7US91/08953 



-21- 
Claims 

What is claimed is: 

1. A composition for treating cartilage and/or 
bone defects comprising: at least one bone morphogenetic 
protein (BMP) and at least one transforming growth factor 
(TGF) together in a tissue -growth -inducing amount; and a 
pharmaceutically acceptable carrier or excipient. 

2 . The composition of claim 1 wherein the BMP 
comprises BMP-2. 

3 - The composition of claim 1 wherein the BMP 
15 comprises BMP-3. 

4. The composition of claim l wherein the BMP 
comprises BMP-2 and BMP- 3. 

20 5. The composition of claim 1 wherein the BMP 

comprises BMP -4 . 

6. The composition of claim 1 wherein the BMP 
comprises BMP- 7. 

25 

7. The composition of claim 1 wherein the TGF 
comprises TGF- El. 



5 



10 



8. The. composition of claim 1 wherein the TGF 
30 comprises TGF-S2 . 

9. The composition of claim 4 wherein the TGF 
comprises TGF-S2. 



35 



WO 92/09697 



PCT/US91/08953 



-22- 

10. A osteogenic composition for inducing bone 
growth comprising: at least one bone morphogenetic 
protein (BMP) and at least one transforming growth factor 
(TGF) together in a bone -growth -inducing amount; and a 

5 pharmaceutical^ acceptable carrier or excipient. 

11. The composition of claim 10 wherein the 
BMP comprises BMP-2. 

10 12. The composition of claim 10 wherein the 

BMP comprises BMP -3 . 

13 . The composition of claim 10 wherein the 
BMP comprises BMP-2 and BMP-3. 

15 

14. The composition of claim 10 wherein the 
BMP comprises BMP- 4. 

15. The conqposition of claim 10 wherein the 
20 BMP comprises BMP-7. 

16. The composition of claim 10 wherein the 
TGF comprises TGF -SI. 

25 17. The composition of claim 10 wherein the 

TGF comprises TGF- £2 . 

18. The conqaosition of -claim 13 wherein the 
TGF comprises TGF-B2. 

30 



35 



WO 92/09697 



PCT/US91/089S3 



-23- 

19. A method for treating an individual having 
a cartilage or bone defect comprising: 

administering to the individual a composition 
comprising at least one bone morphogenetic protein (BMP) 
5 and at least one transforming growth factor {TGF) 
together in a tissue -growth- inducing amount, and a 
pharmaceutically acceptable carrier or excipient. 

20. The method of claim 19 wherein the BMP 
10 comprises BMP- 2. 

21. The method of claim 19 wherein the BMP 
comprises BMP- 3. 

15 22. The method of claim 19 wherein the BMP 

comprises BMP -2 and BMP-3. 

23. The method of claim 19 wherein the BMP 
comprises BMP- 4. 

20 

24. The method of claim 19 wherein the BMP 
comprises BMP- 7. 



25. The method of claim 19 wherein the TGF 
25 comprises TGF-Sl. 

26. The method of claim 19 wherein the TGF 
comprises TGF -62. 

30 27. The method of claim 22 wherein the TGF 

comprises TGF-S2. 



35 



28. A method for inducing bone marrow cell 
production in an individual comprising: 



WO 92/09697 



PCT/US91/089S3 



-24- 

administering to the individual a composition 
comprising at least one bone morphogenetic protein (BMP) 
and at least one transforming growth factor (TGF) 
together in a bone marrow cell growth- inducing amount, 
5 and a pharmaceutical^ acceptable carrier or excipient. 

29. The method of claim 28 wherein the BMP 
comprises BMP -2 . 

0 30. The method of claim 28 wherein the BMP 

comprises BMP- 3. 

31. The method of claim 28 wherein the BMP 
BMP -2 and BMP-3. 

32. The method of claim 28 wherein the BMP 
BMP -4 • 

33 . The method of claim 28 wherein the BMP 
20 comprises BMP- 7. 

34. The method of claim 28 wherein the TGF 
comprises TGF-Sl. 

25 35. The method of claim 28 wherein the TGF 

comprises TGF- £2 . 

36. The method of claim 31 wherein the TGF 
TGF- £2. 



comprises 

15 

comprises 



comprises 
30 



35 



WO 92/09697 



PCI7US91/08953 




0 20 40 60 

MINUTES 



FIG. 1 



SUBSTITUTE SHEET 



WO 92/09697 PCT/US91/08953 

2/5 



17% SDS-PAGE 




1 2 345678 9 10 

FIG. 2 



SUBSTITUTE SHEET 



WO 92/09697 



PCT/US91/089S3 




0 20 40 60 80 100 120 

MINUTES 



FIG. 3 



WO 92/09697 PCT/US91 /089S3 

4/5 

BMP-2 . RESIDUES 295-396 

SCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV 

L9: HPLYVDFSDVGLND-IVAP — 

G 

L 1 0 : -PLYVDFSDVGWNDWIVAP-GYHAFY — GE 
IVNSFQG 
YVTL 

OTLVNSVNSKIPKACCVPTELSAISMLYLDENEKWLKNYODMWEGCGCR 

L6: A-NVPTELSAISMLYLDE L2: NYQDMWEG-GEN 
DPA DQP L AN 

BMP-3 , RESIDUES 363-472 

OWIEPRNCARRYLKVDFADIGWSEWIISPKSFDAYYCSGACOFPMPKSLKPSNHA 

L3: — IEPRN-ATRYLK — DP-G-D L4: SFDAY — SGA — QF — D 
PA D EL F E I 

L8: VDFADIG-SE-ILSPKF — P L5: PSNPA 
S L V F V M ALKVS 

TIOSIVRAVGVVPGIPEPCCVPEKMSSLSILFFDENKNVVLKVYPNMTVESCACR 

TIQSIVRAVGVVPGIPEP-E L1 : VYP-fdTVES-A-R 

NEATAQSIL AG I QVALK ANV E 

L7: MS-LSILFFDENKL 
P DPPT L 

FIG. 4 



SUBSTITUTE SHEET 



WO 92/09697 



PCT/US91/08953 



5/5 





20 1 




15- 






CO 
D 


10- 


Q. 




-J 
< 






5- 



o- 




FIG. 5A 




FIG. 5B 



g 4,0 

co 3.0 



3 



« 2. OH 



1.0- 



0.0 




FIG. 5C 



0 6.25 12.5 25 50 100 
BMP-2+3, ng/mg IMPLANT 



SUBSTITUTE SHEET 



INTERNATIONAL SEARCH REPORT 

International Application No. PCT/US9 1/08953 



I. CLASSIFICATION OP SUBJECT MATTER (if several classification symbols apply, indicate all) 3 
According to International Patent Classification (IPC) or to both National Classification and IPC 

IPC (S) : C12P 21/00; C12N 15/00,5/00; A61K 37/12; C07K 7/00 
US CL : 514/2,14,21 



It. FIELDS SEARCHED 



Minimum Documentation Searched 4 



Classification System 



Classification Symbols 



U.S. 



514/2,14,21 



Documentation Searched other than Minimum Documentation 
to the extent that such Documents are included in the Fields Searched 6 



III. DOCUMENTS CONSIDERED TO BE RELEVANT 14 



Category 



Citation of Document 16 with indication, where appropriate, of the relevant passages 17 



Relevant to Claim No. 18 



US, A, 4,886,747 {DERYNCK ET AL) 12 DECEMBER 1989, SEE 
ENTIRE DOCUMENT. 

US, A, 4,774,322 (S EYED IN ET AL) 27 SEPTEMBER 1988, SEE 
ENTIRE DOCUMENT. 

US, A, 4,806,523 (BENTZ ET AL) 21 FEBRUARY 1989, SEE 
ENTIRE DOCUMENT. 

US, A, 4,877,864 (WANG ET AL) 31 OCTOBER 1989, SEE 
ENTIRE DOCUMENT. 

US, A, 4,563,489 (URIST) 07 JANUARY 1986, SEE ENTIRE 
DOCUMENT. 

US, A, 4,789,732 (URIST) 06 DECEMBER 1988, SEE ENTIRE 
DOCUMENT. 

US, A, 4,455,256 (URIST) 19 JUNE 1984, SEE ENTIRE 
DOCUMENT. 

US, A, 4,795,804 (URIST) 03 JANUARY 1989, SEE ENTIRE 
DOCUMENT. 

US, A, 4,294,753 (URIST) 13 OCTOBER 1981, SEE ENTIRE 
DOCUMENT. 



1-36 
1-36 
1-36 
1-36 
1-36 
1-36 
1-36 
1-36 
1-36 



Special categories of cited documents: ' 6 
\ m document defining the general state of the art which is 

not considered to oe of particular relevance 
"E* earlier document but published on or after the 

international filing data 
L" document which may throw doubts on priority daim(s) 

or which is cited to establish the publication data of 

another citation or other special reason <es specified) 
O" document referring to an oral disclosure, use, exhibition 

or other means 

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



later document published after the international filing 
date or priority dote and not in conflict with the 
application but cited to understand the principle or 
theory underlying the invention 
document of particular relevance; the claimed 
invention cannot be considered novel or cannot be 
considered to involve an inventive step 
document of particular relevance; the claimed 
invention cannot be considered to i nvol ve an 
inventive step when the document is combined with 
one or more other such documents, such combination 
being obvious to a person skilled in the art 
document member of the same patent family 



IV. CERTIFICATION 



Date of the Actual Completion of the International Search 2 
09 MARCH 1992 



Date of Mailt 



Search Report 2 



International Searching Authority 1 
ISA/US 



Signature, of Authorized Officer 20 f\ 
HOWARD S CHAIN 7 V 



Form PCT/ISA/210 (second sheetXMay 1986) 6 



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 
□TfADED TEXT OR DRAWING 

EZf 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.