Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


(19) 



Europfilsches Patentamt 
European Patent Office 
Office europeen des brevets 



(12) 



(ID EP 0 688 869 B1 

EUROPEAN PATENT SPECIFICATION 



(45) Date of publication and mention 
of the grant of the patent: 
19.03.2003 Bulletin 2003/12 

(21) Application number: 95111771.2 

(22) Date of filing: 30.06.1987 



(51) mtci7: C12N 15/12, C07K 14/51, 
C12N5/10, C12N 1/21, 
A61K38/18 



(54) Novel osteoinductive compositions 

Osteoinduktive Zusammensetzungen 
Compositions osteoinductrices 



(84) Designated Contracting States: 

AT BE CH DE FR GB IT LI LU NL SE 

(30) Priority: 01.07.1986 US 880776 

17.12.1986 US 943332 

20.03.1987 US 28285 
26.03.1987 US 31346 

(43) Date of publication of application: 
27.12.1995 Bulletin 1995/52 

(60) Divisional application: 
02014841.7/1 254 956 

(62) Document number(s) of the earlier application(s) in 
accordance with Art. 76 E PC: 
87905023.5/0 313 578 



m 

a> 

CO 
00 

00 
00 
CO 

o 

LU 



(73) Proprietor: Genetics Institute, LLC 
Cambridge, MA 02140 (US) 

(72) Inventors: 

• Wang, Elizabeth A. 
Carlisle, MA 01741 (US) 

• Wozney, John M. 
Hudson, MA 01749 (US) 

• Rosen, Vicki A. 
Boston, MA 02116 (US) 

(74) Representative: VOSSIUS & PARTNER 
Postfach 86 07 67 

81634 MOnchen (DE) 

(56) References cited: 
US-A- 4 455 256 



00 

rn 

i 

r~ 
m 

O 
O 
~o 



Note: Within nine months from the publication of the mention of the grant of the European patent, any person may -give 
notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in 
a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 
99(1 ) European Patent Convention). 



Printed by Jouve. 75001 PARIS (FR) 



EP 0 688 869 B1 



Description 

[0001] The present invention relates to novel proteins and processes for obtaining them. These proteins are capable 
of inducing cartilage and bone formation. 

5 

Background 

[0002] Bone is a highly specialized tissue characterized by an extensive matrix structure formed of fibrous bundles 
of the protein collagen, and proteoglycans, noncollagenous proteins, lipids and acidic proteins. The processes of bone 

10 formation and renewal/repair of bone tissue, which occur continuously throughout life, are performed by specialized 
cells. Normal embryonic long bone development is preceded by formation of a cartilage model. Bone growth is pre- 
sumably mediated by "osteoblasts" (bone-forming cells), while remodeling of bone is apparently accomplished by the 
joint activities of bone-resorbing cells, called "osteoclasts" and osteoblasts. A variety of osteogenic, cartilage-inducing 
and bone inducing factors have been described, See, e.g. European patent applications 148,155 and 169,016 for 

15 discussions thereof. 

Brief Description of the Invention 

[0003] The present invention provides a novel protein in purified form BMP-3, wherein BMP is bone morphogenic 
20 protein. This protein is characterized by peptide sequences the same as or substantially homologous to amino acid 
sequences illustrated in Tables IV A+B below. It is capable of inducing bone formation at a predetermined site. This 
bone inductive factor is further characterized by biochemical and biological characteristics including activity at a con- 
centration of 1 0 to 1 0OOng/gram of bone in an in vivo rat bone formation assay described below. Proteins of this invention 
may be encoded by the DNA sequences depicted in the Tables or by sequences capable of hybridizing thereto unden 
25 strigent conditions and coding for polypeptides with bone growth factor biological properties or other variously modified 
sequences demonstrating such properties. 

[0004] The pone inductive factor of the invention, BMP-3, is represented by the bovine homolog bBMP-3. bBMP-3 
is characterized by the DNA sequence and amino acid sequence of Table IV A and B which represents the bovine 
genomic sequence. It is characterized by at least a portion of a peptide sequence the same or substantially the same 

30 as amino acid #1 through amino acid #175 of Table IV A and B. BMP-3 is further characterized by the ability to induce 
bone formation. The bovine factor may be employed as a tool for obtaining the analogous human BMP-3 protein or 
other mammalian bone inductive proteins. The proper characterization of this bovine bone inductive factor provides 
the essential "starting point" for the method employing this sequence. The method, employing techniques known to 
those skilled in the art of genetic engineering, involves using the bovine DNA sequence as a probe to screen a human 

35 genomic or cDNA library; and identifying the DNA sequences which hybridize to the probes. A clone with a hybridizable 
sequence is plaque purified and the DNA isolated therefrom, subcloned and subjected to DNA sequence analysis. 
Thus another aspect of this invention is a human protein hBMP-3, produced by this method. 
[0005] Another aspect of the invention provides pharmaceutical compositions containing a therapeutically effective 
amount of the bone growth factor polypeptide according to the invention in a pharmaceuticalty acceptable vehicle.. 

40 These compositions may further include other therapeutically useful agents. They may also include an appropriate 
matrix for delivering the proteins to the site of the bone defect and for providing a structure for'bone growth. These 
compositions may be employed in methods for treating a number of bone defects and periodontal disease. These 
methods, according to the invention, entail administering to a patient needing such bone formation an effective amount 
of the novel protein BMP-3 as described herein. 

45 [0006] Still a further aspect of the invention are DNA sequences coding on expression for a human or bovine polypep- 
tide having the ability to induce bone formation. Such sequences include the sequence or nucleotides in a 5' to 3' 
direction illustrated in Tables IV A+B. Alternatively, a DNA sequence which hybridizes under stringent conditions with 
the DNA sequences of Tables IV A+B and which codes on expression for a protein having at least one bone growth 
factor biological property are included in the present invention. Finally, allelic or other variations of the sequences of 

so Tables IV A+B, whether such nucleotide changes result in changes in the peptide sequence or not, are also included 
in the present invention. 

[0007] Still a further aspect of the invention is a vector containing a DNA sequence as described above in operative 
association with an expression control sequence. Such vector may be employed in a novel process for producing a 
bone growth factor polypeptide in which a cell line transformed with a DNA sequence encoding expression of a bone 
55 growth factor polypeptide in operative association with an expression control sequence therefor, is cultured. This 
claimed process may employ a number of known cells as host cells for expression of the polypeptide. Presently pre- 
ferred cell lines are mammalian cell lines and bacterial cells. 

[0008] Other aspects and advantages of the present invention will be apparent upon consideration of the following 



2 



EP 0 688 869 B1 



detailed description and preferred embodiments thereof. 

Detailed Description of the Invention 

5 [0009] The protein of the present invention is characterized by amino acid sequences or portions thereof the same 
as or substantially homologous to the sequences shown in Tables IV A+B below. This protein is also characterized by 
the ability to induce bone formation. 

[0010] The bone growth factors provided herein also include factors encoded by the sequences similar to those of 
Tables IV A+B, but into which modifications are naturally provided (e.g. allelic variations in the nucleotide sequence 

10 which may result in amino acid changes in the polypeptide) or deliberately engineered. For example, synthetic polypep- 
tides may wholly or partially duplicate continuous sequences of the amino acid residues of Tables IV A+B. These 
sequences, by virtue of sharing primary, secondary, or tertiary structural and conformational characteristics with the 
bone growth factor polypeptide of Tables IV A+B may possess bone growth factor biological properties in common 
therewith. Thus : they may be employed as biologically active substitutes for naturally-occurring bone growth factor 

15 polypeptides in therapeutic processes. 

[0011] Other specific mutations of the sequences of the bone growth factor described herein involve modifications 
of one or both of the glycosylation sites. The absence of glycosylation or only partial glycosylation results from amino 
acid substitution or deletion at one or both of the asparagine-linked glycosylation recognition sites present in the se- 
quences of the bone growth factor shown in Tables IV A+B. The asparagine-linked glycosylation recognition sites 

20 comprise tripeptide sequences which are specifically recognized by appropriate cellular glycosylation enzymes. These 
tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine, where X is usually any amino acid. A 
variety 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 n on -glycosylation at the modified tripeptide 
sequence. 

25 [0012] The present invention also encompasses the novel DNA sequences, free of association with DNA sequences 
encoding other proteinaceous materials, and coding on expression for bone growth factors. These DNA sequences 
include those depicted in Tables IV A+B in a 5' to 3' direction and those sequences which hybridize under stringent 
hybridization conditions [see, T Maniatis et al , Molecular Cloning (A Laboratory Manual) , Cold Spring Harbor Laboratory 
(1 982), pages 387 to 389] to the DNA sequences of Tables IV A+B. 

30 [0013] Similarly, DNA sequences which code for bone growth factor polypeptides coded for by the sequences of 
Tables IV A+B, but which differ in codon sequence due to the degeneracies of the genetic code or allelic variations 
(naturally-occurring base changes in the species population which may or may not result in an amino acid change) 
also encode the novel growth factors described herein. Variations in the DNA sequences of Tables IV A+B which are 
caused by point mutations or by induced modifications to enhance the activity, half-life or production of the polypeptides 

35 encoded thereby are also encompassed in the invention. 

[0014] Another aspect of the present invention provides a novel method for producing the novel osteoinductive fac- 
tors. The method of the present invention involves culturing a suitable cell or cell line, which has been transformed 
with a DNA sequence coding on expression for a novel bone growth factor polypeptide of the invention, under the 
control of known regulatory sequences. Suitable cells or cell lines may be rriammalian cells, such as Chinese hamster 

40 ovary cells (CHO). The selection of suitable mammalian host cells and methods for transformation, culture, amplifica- 
tion, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 
293:620-625 (1 981 ), or alternatively, Kaufman et al, Mol. Cell. Biol. , 5(7):1 750-1 759 (1 985) or Howley et al, U.S. Patent 
4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey 
COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line. 

45 [0015] Bacterial cells are suitable hosts. For example, the various strains of E.coli (e.g., HB101, MC1 061) are well- 
known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like 
may also be employed in this method. 

[0016] Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of 
the polypeptides of the present invention. Additionally where desired, insect cells may be utilized as host cells in the 
50 method of the present invention. See, e.g. Miller et al, Genetic Engineering , 8:277-298 (Plenum Press 1986) and 
references, cited therein. 

[0017] Another aspect of the present invention provides vectors for use in the method of expression of these novel 
osteoinductive polypeptides. Preferably the vectors contain the full novel DNA sequences described above which code 
for the novel factors of the invention. Additionally the vectors also contain appropriate expression control sequences 
55 permitting expression of the bone inductive protein sequences. Alternatively, vectors incorporating modified sequences 
as described above are also embodiments of the present invention and useful in the production of the bone inductive 
proteins. The vectors may be employed in the method of transforming cell lines and contain selected regulatory se- 
quences in operative association with the DNA coding sequences of the invention which are capable of directing the 



3 



EP 0 688 869 B1 



replication and expression thereof in selected host celts. Useful regulatory sequences for such vectors are known to 
one of skill in the art and may be selected depending upon the selected host cells. Such selection is routine and does 
not form part of the present invention. 

[0018] A protein of the present invention, which induces bone growth in circumstances where bone is not normally 
5 formed, has application'^ the healing of bone fractures. An osteogenic preparation employing is protein of the invention 
may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial 
joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital , trauma induced, 
or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery. An osteogenic factor 
of the invention may be valuable in the treatment of periodontal disease, and in other tooth repair processes. Such 
10 agents may provide an environment to attract borie-forming cells, stimulate growth of bone-forming ceils or induce 
differentiation of progenitors of bone-forming cells. Of course, the proteins of the invention may have other therapeutic 
uses. 

[0019] A further aspect of the invention is a therapeutic method and composition for repairing fractures and other 
conditions related to bone defects or periodontal diseases. Such a composition comprises a therapeutically effective 

is amount of the bone inductive factor protein of the invention. The bone inductive factor according to the present invention 
may be present in a therapeutic composition in admixture with a pharmaceutically acceptable vehicle or matrix. Further 
therapeutic methods and compositions of the invention comprise a therapeutic amount of a bone inductive factor of 
the invention with a therapeutic amount of at least one of the other bone inductive factors of the invention. Additionally, 
the protein according to the present invention may be co-administered with one or more different osteoinductive factors 

20 with which it may interact. Further, the bone inductive protein may be combined with other agents beneficial to the 
treatment of the bone defect in question. Such agents include, but are not limited to various growth factors. The prep- 
aration of such physiologically acceptable protein compositions, having due regard to pH ; isotonicity, stability and the 
like, is within the skill of the art. 

[0020] The therapeutic method includes locally administering the composition as an implant or device. When admin- 

25 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 
bone damage. Preferably, the bone growth inductive factor composition would include a matrix capable of delivering 
the bone inductive factor to the site of bone damage, providing a structure for the developing bone and cartilage and 
optimally capable of being resorted into the body. Such matrices may be formed of other materials presently in use 

30 for other implanted medical applications. 

[0021] The choice of material is based on, for example, biocompatibility, biodegradability, mechanical properties, 
cosmetic appearance and interface properties. Similarly, the application of the osteoinductive factors will define the 
appropriate formulation. Potential matrices for the osteoinductive factors may be biodegradable and chemically defined, 
such as, but not limited to calcium sulfate, tricalciumphosphate, hydroxy apatite, polylactic acid, polyan hydrides; bio- 

35 degradable and biologically well defined, such as bone or dermal collagen, other pure proteins or extracellular matrix 
components; nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other 
ceramics; or combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite 
or collagen and tricalciumphosphate. The bioceramics might also be altered in composition, such as in calcium-alumi- 
nate-phosphate and processing to alter for example, pore size, particle size, particle shape, and biodegradability. 

40 [0022] The dosage regimen will be determined by the attending physician considering various factors which modify 
the action of such a growth factor, e.g. amount of bone weight desired to be formed, the site of bone damage, the 
condition of the damaged bone, the patient's age, sex, and diet, the severity of any infection, time of administration 
and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution. The addition of other 
known growth factors, such as IGF 1 {insulin like growth factor 1 ), to the final composition, may also effect the dosage. 

« Generally, the dosage regimen should be in the range of approximately 10 to 10 6 nanograms of protein per gram of 
bone weight desired. Progress can be monitored by periodic assessment of bone growth and/or repair, e.g. x-rays. 
Such therapeutic compositions are also presently valuable for veterinary applications due to the lack of species spe- 
cificity in bone inductive factors. Particularly domestic animals and thoroughbred horses in addition to humans are 
desired patients for such treatment with the bone inductive factors of the present invention. 

so [0023] The following examples illustrate practice of the present invention in recovering and characterizing the bovine 
proteins and employing them to recover the human proteins, obtaining the human proteins and in expressing the 
proteins via recombinant techniques. 

EXAMPLE I 

55 

Isolation of Bovine Bone Inductive Factor 

[0024] Ground bovine bone powder (20-120 mesh, Helitrex) is prepared according to the procedures of M. R. Urist 



4 



EP 0 688 869 B1 



et al., Proc. Natl Acad. Sci USA, 70:3511 (1973) with elimination of some extraction steps as identified below. Ten kgs 
of the ground powder is demineralized in successive changes of 0.6N HCI at 4°C over a 48 hour period with vigorous 
stirring. The resulting suspension is extracted for 1 6 hours at 4°C with 50 liters of 2M CaCI 2 and 1 0mM ethylenediamine- 
tetraacetic acid [EDTA], and followed by extraction for 4 hours in 50 liters of.0.5M EDTA. The residue is washed three 
s times with distilled water before its resuspension in 20 liters of 4M guanidine hydrochloride [GuCI], 20mM Tris (pH 7.4), 
1mM N-ethylmaleimide, 1mM iodoacetamide, 1mM phenylmethylsulfonyl fluorine as described in Clin. Orthop. Rel. 
Res. , 171: 213 (1982). After 16 to 20 hours. the supernatant is removed and replaced with another 10 liters of GuCI 
buffer. The residue is extracted for another 24 hours. 

[0025] The crude GuCI extracts are combined, concentrated approximately 20 times on a Pellicon apparatus with a 
10 10,000 molecular weight cut-off membrane, and then dialyzed in 50mM Tris t 0.1 M NaCI, 6M urea (pH7.2), the starting 
buffer for the first column. After extensive dialysis the protein is loaded on a 4 liter DEAF cellulose column and the 
unbound fractions are collected. 

[0026] The unbound fractions are concentrated and dialyzed against 50mM NaAc, 50mM NaCI (pH 4.6) in 6M urea. 

The unbound fractions are applied to a carboxymethyl cellulose column. Protein not bound to the column is removed 
15 by extensive washing with starting buffer, and the bone inductive factor containing material desorbed from the column 

by 50mM NaAc, 0.25mM NaCI, 6M urea (pH 4.6). The protein from this step elution is concentrated 20- to 40- fold, 

then diluted 5 times with 80mM KP0 4 , 6M urea (pH6.0). The pH of the solution is adjusted to 6.0 with 500mM K 2 HP0 4 . 

The sample is applied to an hydroxylapatite column (LKB) equilibrated in 80mM KP0 4 , 6M urea (pH6.0) and all unbound 

protein is removed by washing the column with the same buffer. Bone inductive factor activity is eluted with 100mM 
20 KP0 4 (pH7.4) and 6M urea. 

[0027] The protein is concentrated approximately 1 0 times, and solid NaCI added to a final concentration of 0.1 5M. 

This material is applied to a heparin - Sepharose column equilibrated in 50mM KP0 4 , 150mM NaCI, 6M urea (pH7.4). 

After extensive washing of the column with starting buffer, a protein with bone inductive factor activity is eluted by 

50mM KP0 4 , 700mM NaCI, 6M urea (pH7.4). This fraction is concentrated to a minimum volume, and 0.4ml aliquots 
25 are applied to Superose 6 and Superose 1 2 columns connected in series, equilibrated with 4M GuCI, 20mM Tris (pH7.2) 

and the columns developed at a flow rate of 0.25ml/min. The protein demonstrating bone inductive factor activity has 

a relative migration corresponding to approximately 30,000 dalton protein. 

[0028] The above fractions are pooled, dialyzed against 50mM NaAc, 6M urea (pH4.6), and applied to a Pharmacia 
MonoS HR column. The column is developed with a gradient to 1.0M NaCI, 50mM NaAc, 6M urea (pH4.6). Active 

30 fractions are pooled and brought to pH3.0 with 1 0% trifluoroacetic acid (TFA). The material is applied to a 0.46 x 25cm 
Vydac C4 column in 0.1% TFA and the column developed with a gradient to 90% acetonitrile, 0.1% TFA (31 .5% ace- 
tonitrile, 0.1% TFA to 49.5% acetonitrile, 0.1% TFA in 60 minutes at 1ml per minute). Active material is eluted at ap- 
proximately 40-44% acetonitrile. Aliquots of the appropriate fractions are iodinated by one of the following methods: 
P. J. McConahey et al, Int. Arch. Allergy , 29:185-189 (1966); A. E. Bolton et al, Biochem J ., 133:529 (1973); and D. F. 

35 Bowen-Pope, J. Biol. Chem. , 237:51 61 (1 982). The iodinated proteins present in these fractions are analyzed by SDS 
gel electrophoresis and urea Triton X 1 00 isoelectric focusing. At this stage, the bone inductive factor is estimated to 
be approximately 10-50% pure. 

EXAMPLE II 

40 

Characterization of Bovine Bone Inductive Factor 
A. Molecular Weight 

45 [0029] Approximately 20ug protein from Example I is lyophilized and redissolved in 1X SDS sample buffer. After 15 
minutes of heating at 37°C, the sample is applied to a 15% SDS polyacrylamide gel and then electrophoresed with 
cooling. The molecular weight is determined relative to prestained molecular weight standards (Bethesda Research 
Labs). Immediately after completion, the gel lane containing bone inductive factor is sliced into 0,3cm pieces. Each 
piece is mashed and 1 .4m! of 0.1% SDS is added. The samples are shaken gently overnight at room temperature to 

50 elute the protein. Each gel slice is desalted to prevent interference in the biological assay. The supernatant from each 
sample is acidified to pH 3.0 with 10% TFA, filtered through a 0.45 micron membrane and loaded on a 0.46cm x 5cm 
C4 Vydac column developed with a gradient of 0.1% TFA to 0.1% TFA, 90%*CH 3 CN. The appropriate bone inductive 
factor - containing fractions are pooled and reconstituted with 20mg rat matrix. In this gel system, the majority of bone 
inductive factor fractions have the mobility of a protein having a molecular weight of approximately 28,000 - 30,000 

55 daltons. 



5 



EP 0 688 869 B1 

B. Isoelectric Focusing 

[0030] The isoelectric point of bone inductive factor activity is determined in a denaturing isoelectric focusing system. 
The Triton X1 00 urea gel system (Hoeffer Scientific) is modified as follows: 1 ) 40% of the ampholytes used are Servalyte 

5 3/10; 60% are Servalyte 7-9. 2) The catholyte used is 40mM NaOH. Approximately 20ug of protein from Example I is 
lyophilized, dissolved in sample buffer and applied to the isoelectrofocusing gel. The gel is run at 20 watts, 10°C for 
approximately 3 hours. At completion the lane containing bone inductive factor is sliced into 0.5 cm slices. Each piece 
is mashed in 1 .0ml 6M urea, 5mM Tris (pH 7.8) and the samples agitated at room temperature. The samples are 
acidified, filtered, desalted and assayed as described above. The major portion of activity as determined in the assay 

10 described in Example III migrates in a manner consistent with a pi of 8.8 - 9.2, 

C. Subunit Characterization 

[0031] The subunit composition of bone inductive factor is also determined. Pure bone inductive factor is isolated 
*5 from a preparative 15% SDS gel as described above. A portion of the sample is then reduced with 5mM DTT in sample 
buffer and re-electrophoresed on a 15% SDS gel. The approximately 30kd protein yields two major bands at approx- 
imately 20kd and 18kd, as well as a minor band at 30kd. The broadness of the two bands indicates heterogeneity 
caused most probably by glycosylation, other post translational modification, proteolytic degradation or carbamylation. 

20 EXAMPLE III 

Biological Activity of Bone Inductive Factor 

[0032] A rat bone formation assay according to the general procedure of Sampath and Reddi, Proc. Natl. Acad. Sci. 

25 U.S.A. , 80:6591 -6595 (1 983) is used to evaluate the osteogenic activity of the bovine bone inductive factor of the 
present invention obtained in Example I. This assay can also be used to evaluate bone inductive factors of other 
species. The ethanol precipitation step is replaced by dialyzing the fraction to be assayed against water. The solution 
or suspension is then redissolved in a volatile solvent, e.g. 0.1 - 0.2 % TFA, and the resulting solution added to 20mg 
of rat matrix. This material is frozen and lyophilized and the resulting powder enclosed in #5 gelatin capsules. The 

30 capsules are implanted subcutaneousty in the abdominal thoracic area of 21 - 49 day old male long Evans rats. The 
implants are removed after 7-14 days. Half of each implant is used for alkaline phosphatase analysis [See, A. H. Reddi 
et al., Proc. Natl Acad Sci. , 69:1601 (1972)] and half is fixed and processed for histological analysis. Routinely, lum 
glycolmethacrylate sections are stained with Von Kossa and acid fuschin to detect new bone mineral. Alkaline phos- 
phatase, an enzyme produced by chondroblasts and osteoblasts in the process of matrix formation, is also measured. 

35 New cartilage and bone formation often correlates with alkaline phosphatase levels. Table I below illustrates the dose 
response of the rat matrix samples including a control not treated with bone inductive factor. 



TABLE 1 



40 



45 



Protein* Implanted ug 


Cartilage 


Alk. Phos.u/I 


7.5 


2 


Not done 


2.5 


3 


445.7 


0.83 


3 


77.4 


0.28 


0 


32.5 


0.00 


0 


31.0 



♦At this stage the bone inductive factor is approximately 10-15% pure. 



[0033] The bone or cartilage formed is physically confined to the space occupied by the matrix. Samples are also 
5Q analyzed by SDS gel electrophoresis and isoelectric focusing as described above, followed by autoradiography. Anal- 
ysis reveals a correlation of activity with protein bands at 28 - 30kd and a pi 9.0. An extinction coefficient of 1 OD/mg- 
cm is used as an estimate for protein and approximating the purity of bone inductive factor in a particular fraction. In 
the in vivo rat bone formation assays on dilutions as described above, the protein is active in vivo at 10 to 200ng protein/ 
gram bone to probably greater than lug protein/gram bone. 

55 



6 



EP 0 688 869 B1 



EXAMPLE IV 

Bovine Bone Inductive Factor Protein Composition 

5 [0034] The protein composition of Example II A of molecular weight 28 - 30kd is reduced as described in Example 
IIC and digested with trypsin. Eight tryptic fragments are isolated by standard procedures having the following amino 
acid sequences: 

Fragment 1:AAFLGDI ALDEEDLG 
io Fragment 2:AFQVQQAADL 

Fragment 3: NYQDMVVEG 

Fragment 4:STPAQDVSR 

Fragment 5: N Q E A L R 

Fragment 6:LSEPDPSHTLEE 
15 Fragment 7: F D A Y Y 

Fragment 8:LKPSN?ATIQSIVE 

[0035] A less highly purified preparation of protein from bovine bone is prepared according to a purification scheme 
similar to that described in Example I. The purification basically varies from that previously described by omission of 

20 the DE-52 column, the CM cellulose column and the mono S column, as well as a reversal in the order of the hydrox- 
ylapatite and heparin sepharose columns. Briefly, the concentrted crude 4 M extract is brought to 85% final concen- 
tration of ethanol at 4 degrees. The mixture is then centrifuged, and the precipitate redissolved in 50 mM Tris, 0.15 M 
NaCI, 6.0 M urea. This material is then fractionated on Heparin Sepharose as described. The Heparin bound material 
is fractionated on hydroxyapatite as described. The active fractions are pooled, concentrated, and fractionated on a 

25 high resolution gel filtration (TSK 30000 in 6 M guanidinium chloride, 50 mM Tris, pH 7.2). The active fractions are 
pooled, diatyzed against 0.1% TFA, and then fractionated on a C4 Vydac reverse phase column as described. The 
preparation is reduced and etectrophoresed on an acrylamide gel. The protein corresponding to the 1 8K band is eluted 
and digested with trypsin. Tryptic fragments are isolated having the following amino acid sequences: 

30 Fragment 9:SLKPSNH ATIQS? V 

Fragment 10: S F D A Y Y C S ? A 
Fragment 11:VYPNMTVESCA 
Fragment 12: VD FAD I? W 

35 [0036] Tryptic Fragments 7 and 8 are noted to be substantially the same as Fragments 1 0 and 9, respectively. 

A. bBMP-3 

[0037] Probes consisting of pools of oligonucleotides are designed on the basis of the amino acid sequences of the 
to tryptic Fragments 9 (Probe #3), 10 (Probe #2), and 11 (Probe #1), and synthesized on an automated DNA synthesizer. 

Probe. #1: ACNGTCAT [A/G] T T N G G [A/G] T A 



Probe #2: C A [A/G] T A [A/G] T A N G C "[A/G] T C [A/G] A A 
Probe #3: T G [A/G/T] ATNGTNGC [A/G] T G {A/G] T T 

50 

[0038] A bovine genomic recombinant library is constructed as follows: Bovine liver DNA is partially digested with 
the restriction endonuclease enzyme Sau 3A and sedimented through a sucrose gradient. Size fractionated DNA in 
the range of 15-30kb is then iigated to the bacteriophage Bam HI vector EMBL3 [Frischauf et al, J. Mol. Biol., 170: 
827-842 (1983)]. The library is plated at 8000 recombinants per plate. Duplicate nitrocellulose replicas of the plaques 
55 are made and amplified according to a modification of the procedure of Woo et al, Proc. Natl. Acad. Sci. USA , 75: 
3688-91 (1978). 

[0039] This recombinant bovine genomic library constructed in EMBL3 is screened by the TMAC hybridization pro- 
cedure, i.e. kyloridized in 3M tetramethylammonium chloride (TMAC), 0.1 M sodium phosphate pH6.5, 1mM EDTA, 5X 



7 



EP 0 688 869 B1 



Denhardts, 0.6% SDS, 100ug/ml salmon sperm DNA at 48 degrees C, and washed in 3M TMAC, 50mM Tris pH8.0 at 
50 degrees C. These conditions minimize the detection of mismatches to the 1 7 mer probe pool [see, Wood et al, Proc, 
Natl. Acad, Sci, U.S.A., 82:1585-1588 (1985)]. 400,000 recombinants are screened in duplicate with Probe #1 which 
has been labeled with 32 P. All recombinants which hybridized to this probe are replated for secondaries. Triplicate 
nitrocellulose replicas are made of the secondary plates, and amplified as described. The three sets of filters, are 
hybridized to Probes #1 , #2 and #3, again under TMAC conditions. One clone, lambda bP-819, hybridizes to all three 
probes and is plaque purified and DNA is isolated from a plate lysate. Bacteriophage lambda bP-819 was deposited 
with the ATCC on June 16, 1987 under accession number 40344. This bP-819 clone~encodes the bovine bone growth 
factor designated bBMP-3. 

[0040] The region of bP-81 9 which hybridizes to Probe #2 is localized and. sequenced. The partial DNA and derived 
amino acid sequences of this region are shown in Table IVA. The amino acid sequences corresponding to tryptic 
Fragments 10 and 12 are underlined. The first underlined sequence corresponds to Fragment 12 while the second 
corresponds to Fragment 10. This region of bP-819, therefore, which hybridizes to Probe #2 encodes at least 111 
amino acids. This amino acid sequence is encoded by the DNA sequence from nucleotide #414 through #746. 



TABLE IV. A. 

383 393 403 413 (1) 428 

GAGGAGGAAG CGGTCEkCGG GGGIXXTIUT GCCTCTCCAG AAC AAT GAG CIT CCT GGG GCA 

Asn Asn Glu Leu Fro Gly Ala . 

443 458 473 488 

GAA TAT CAG TAC AAG GAG GAT GAA GTA TCG GAG GAG AGG AAG OCT TAC AAG ACT 
Glu Tyr Gin Tyr Lys Glu Asp Glu Val Tip Glu Glu Arg Lys Pro Tyr Lys Thr 

503 518 . 533 

CTT CAG ACT CAG CCC CCT GAT AAG AGT AAG AAC AAA AAG AAA CAG AGG AAG GGA 
Leu Gin !Dir Gin Pro Pro Asp Lys Ser Lys Asn lys lys lys Gin Arg Lys Gly 

548 563 578 593 

CCT CAG CAG AAG ACT CAG AOS CTC CAG ITT GAT GAA CAG ACC CIG AAG AAG GGA 
Pro Gin Gin lys Ser Gin Bir Leu Gin Fhe Asp Glu Gin Shr Leu Lys Lys Ala 

608 623 638 

AGA AGA AAG CAA TCG ATT GAA COC CGG AAT TCT GCC AGA CGG TAC CTT AAA GIG ' 
Arg Arg lys Gin Trp lie Glu Pro Arg Asn Cys Ala Arg Arg Tyr Leu Lys Val 

653 668 683 698 

GAC OTC GCA GAT ATT GGC TGG AGO GAA TGG ATT AIT TCC OCC AAG TOC TTC GAT 

Asp Fhe Ala Asp lie Glv Tro Ser Glu Trp He He Ser Pro Lys Ser Rie Aso 

713 728 743 (111) 756 

GCC TAT TAC TCC TOC GGA GOG TCC CAG TIC CCC AIG CCA AAG GIAGCCATTG 
Ala Tvr Tvr Cys Ser Glv Ala cys Gin Hie Pro MET Pro lys 

766 776 786 

TITITIGTCC 'IGIUITIOUC ATTTGCATAG 



[0041] The region of bP-819 which hybridizes to Probe #1 and #3 is localized and sequenced. The partial DNA and 
derived amino acid sequences of this region are shown in Table IVB. The amino acid sequences corresponding to 
tryptic Fragments 9 and 11 are underlined. The first underlined sequence corresponds to Fragment 9 while the second 
underlined sequence corresponds to Fragment 1 1 . The peptide sequence of this region of bP-81 9 which hybridizes to 
Probe #1 and #3 is 64 amino acids in length encoded by nucleotide #305 through #493 of Table IVB. The arginine 



8 



EP 0 688 869 B1 



residue encoded by the AGA triplet is presumed to be the carboxy-terminus of the protein based on the presence of 
a stop codon (TAA) adjacent to it. The nucleic acid sequence preceding the couplet TC (positions 305-306) is presumed 
to be an intron (non-coding sequence) based on the presence of a consensus acceptor sequence (i.e. a pyrimidine- 
rich stretch, TTCTCCC mm CGTTCCT, followed by AG) and the presence of a stop rather than a basic residue in the 
appropriate position of the derived amino acid sequence. 

[0042] bBMP-3 is therefore characterized by the DNA and amino acid sequence of Table IV A and Table IV B. The 
peptide sequence of this clone is 175 amino acids in length and is encoded by the DNA sequence from nucleotide 
#414 through nucleotide #746 of Table IV A and nucleotide #305 through nucleotide #493 of Table IV B. 



TABLE IV.. B. 



284 294 304 (112) 319* 

CTAAOCTCTC TICTCCCTIT TOGTTCCIAG TCT TIG AAG OCA TCA AAT CAC GCT AOC 

Ser Leu Lvs Pro Ser Asn His Ala Thr 



334 


349 








364 










379 




ATC CAG ACT ATA GIG 


AGA 


GCT 


GIG 


GGG 


GTC GTC 


OCT 


GGA ATC 


ccc 


GAG 


OCT TGC 


lie Gin Ser lie Val 


Arg 


Ala 


Val 


Gly 


Val Val 


Pro 


Gly 


He 


Pro 


GlU 


Pro cys 


394 






409 








424 








439 


TGI GIG OCA GAA AAG 


ATC 


TOC 


TCA 


CTC 


AGO ATC 


TEA 


TTC 


TIT 


GAT 


GAA 


AAC AAG 


cys Val Pro Glu lys 


MET 


Ser 


Ser 


leii 


Ser. lie 


Leu 


Hie. 


Rie 


Asp 


Glu 


Asn lys 


454 










469 








484 




(175) 


AAT GIG CTA CTT AAA 


CTA 


TAT 


OCA 


AAC 


ATC ACA 


GEA 


GAG 


TCT 


TCT 


GCT 


TGC AGA 


Asn Val Val Leu Lys 


Val 


Tyr 


Pro 


Asn 


MET Tnr 


Val 


GlU 


Ser 


Cys 


Ala 


cys Arg 



503 513 523 533 

TAACCTGGTC AAGAACTCAT CIGGATCCIT AACTCAATOG 



EXAMPLE V 
Human BMP-3 

[0043] The sequences of BMP-3 as shown in Tables IV A+B have significant homology to the beta (B) and beta (A) 
subunits of the inhtbins. The inhibins are a family of hormones which are presently being investigated for use in con- 
traception. See, A. J. Mason et al, Nature, 31 8:659-663 (1 985). To a lesser extent they are also homologous to Mullerian 
inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Mullerian duct during development 
of the male embryo and transforming growth factor-beta (TGF-b) which can inhibit or stimulate growth of cells or cause 
them to differentiate. 

[0044] Because bovine and human bone growth factor genes are presumed to be significantly homologous, oligo- 
nucleotide probes which have been shown to hybridize to the bovine DNA sequence of Table IV.A and IV.B are used 
to screen a human genomic library. A human genomic library (Toole et al.; supra) is screened using these probes, and 
presumptive positives are isolated and DNA sequence obtained as described above. Evidence that this recombinant 
encodes a portion of the human bone inductive factor molecule relies on the bovine/human protein and gene structure 
homologies. 

[0045] Once- a recombinant bacteriophage containing DNA encoding a portion of the human BMP-3 molecule is 
obtained the human coding sequence is used as a probe as described in Example V (A) to identify a human cell line 
or tissue which synthesizes BMP-3. mRNA is selected by oligo (dT) cellulose chromatography and cDNA is synthesized 
and cloned in lambda gt1 0 by established techniques (Toole et al., supra ). 

[0046] Alternatively, the entire gene encoding this human bone inductive factor can be identified and obtained in 
additional recombinant clones if necessary. Additional recombinants containing further 3' or 5' regions of this human 



9 



EP 0 688 869 B1 

bone inductive factor gene can be obtained by identifying unique DNA sequences at the end<s) of the original clone 
and using these as probes to rescreen the human genomic library. The gene can then be reassembled in a single 
plasmid by standard molecular biology techniques and amplified in bacteria. The entire human BMP-3 factor gene can 
then be transferred to an appropriate expression vector The expression vector containing the gene is then transfected 

5 into a mammalian cell, e.g. monkey COS cells, where the human gene is transcribed and the RNA correctly spliced. 
Media from the transfected cells are assayed for bone inductive factor activity as described herein as an indication 
that the gene is complete. mRNA is obtained from these cells and cDNA synthesized from this mRNA source and 
cloned. The procedures described above may similarly be employed to isolate other species' bone inductive factor of 
interest by utilizing the bovine bone inductive factor and/or human bone inductive factor as a probe source. Such other 

10 species' bone inductive factor may find similar utility in, inter alia, fracture repair. 

EXAMPLE VI 

Expression of Bone Inductive Factors. 

15 

[0047] In order to produce bovine, human or other mammalian bone inductive factors, the DNA encoding it is trans- 
ferred into an appropriate expression vector and introduced into mammalian cells by conventional genetic engineering 
techniques. 

[0048] One skilled in the art can construct mammalian expression vectors by employing the sequence of Tables IV 

20 a+B or other modified sequences and known vectors, such as pCD [Okayama et al., Mol. Cell Biol., 2:161-170 (1982)] * 
and pJL3, pJL4 [Gough et al., EM BO J., 4:645-653 (1 985)]. The transformation of these vectors into appropriate host 
cells can result in expression of osteoinductive factors. One skilled in the art could manipulate the sequences of Tables 
IV A+B by eliminating or replacing the mammalian regulatory sequences flanking the coding sequence with bacterial 
sequences to create bacterial vectors for intracellular or extracellular expression by bacterial cells. For example, the 

25 coding sequences could be further manipulated (e.g.. ligated to other known linkers or modified by deleting non-coding ~ 
sequences there-from or altering nucleotides therein by other known techniques). The modified bone inductive factor 
coding sequence could then be inserted into a known bacterial vector using procedures such as described in T. Tan- 
iguchi et al., Proc. Natl Acad. Sci. USA , 77:5230-5233 (1980). This exemplary bacterial vector could then be trans- 
formed into bacterial host cells and bone inductive factor expressed thereby. For a strategy for producing extracellular 

30 expression of bone inductive factor in bacterial cells., see, e.g. European patent application EPA 177,343. 

[0049] Similar manipulations can be performed for the construction of an insect vector [See. e.g. procedures de- 
scribed in published European patent application 155 : 476] for expression in insect cells. A yeast vector could also be 
constructed employing yeast regulatory sequences for intracellular or extracellular expression of the factors of the 
present invention by yeast cells. [See, e.g., procedures described in published PCT application WO86/00639 and 

35 European patent application EPA 123,289]. 

[0050] A method for producing high levels of an osteoinductive factor of the invention from mammalian cells involves 
the construction of cells containing multiple copies of the heterologous bone inductive factor gene. The heterologous 
gene can be linked to an amplifiable marker, e.g. the dihydrofolate reductase (DHFR) gene for which cells containing 
increased gene copies can be selected for propagation in increasing concentrations of methotrexate <MTX) according 

40 to the procedures of Kaufman and Sharp, J. Mol. Biol., 159:601-629 (1982). This approach can be employed with a 
number of different cell types. 

[0051] For example, a plasmid containing a DNA sequence for a bone inductive factor of the invention in operative 
association with other plasmid sequences enabling expression thereof and the DHFR expression plasmid pAdA26SV 
(A)3 [Kaufman and Sharp, Mol. Cell. Biol. , 2:1304 (1982)] can be co-introduced into DHFR-deficient CHO cells, 

45 DUKX-BII, by calcium phosphate coprecipitation and transfection. DHFR expressing transformants are selected for 
growth in alpha media with dialyzed fetal calf serum, and subsequently selected for amplification by growth in increasing 
concentrations of MTX (sequential steps in 0.02, 0.2, 1 .0 and 5uM MTX) as described in Kaufman et al., Mol Cell Biol. , 
5:1750 (1983). Transformants are cloned, and biologically active bone inductive factor expression is monitored by rat 
bone formation assay. Bone inductive factor expression should increase with increasing levels of MTX resistance. 

50 Similar procedures can be followed to produce other bone inductive factors. 

[0052] Alternatively, the human gene is expressed directly, as described above. Active bone inductive factor may be 
produced in bacteria or yeast cells. However the presently preferred expression system for biologically active recom- 
binant human bone inductive factor is stably transformed CHO cells. 

55 



10 



EP 0 688 869 B1 

Example VII 

Biological Activity of Expressed Bone Inductive Factor 

5 [0053] To measure the biological activity of the expressed bone inductive factor obtained in Example VI above. The 
factor is partially purified on a Heparin Sepharose column. 4 ml of transfection supernatant from one 100 mm dish is 
concentrated approximately 10 fold by ultrafiltration on a YM 10 membrane and then dialyzed against 20mM Tris, 0.15 
M NaCI, pH 7.4 (starting buffer). This material is then applied to a 1 .1 ml Heparin Sepharose column in starting buffer. 
Unbound proteins are removed by an 8 ml wash of starting buffer, and bound proteins are desorbed by a 3-4 ml wash 

10 of 20 mM Tris, 2.0 M NaCI, pH 7.4. 

[0054] The proteins bound by the Heparin column are concentrated approximately 10-fold on a Centricon 10 and 
the salt reduced by diafiltration with 0.1% trifluoroacetic acid. The appropriate amount of this solution is mixed with 20 
mg of rat matrix and then assayed for in vivo bone and cartilage formation as previously described in Example III. A 
mock transfection supernatant fractionation is used as a control. 

15 [0055] The implants containing rat matrix to which specific amounts of human BMP-3 have been added are removed 
from rats after seven days and processed for histological evaluation. Representative sections from each implant are 
stained for the presence of new bone mineral with von Kossa and acid fuschin, and for the presence of cartilage- 
specific matrix formation using toluidine blue. The types of cells present within the section, as well as the extent to 
which these cells display phenotype are evaluated. 

20 [0056] The procedures described above may be employed to isolate other bone inductive factors of interest by uti- 
lizing the bovine bone inductive factors and/or human bone inductive factors as a probe source. Such other bone 
inductive factors may find similar utility in, inter alia, fracture repair. 



25 Claims 



Claims for the following Contracting States : BE, CH, LI, DE, FR, GB, IT, LU, NL, SE 

30 1 . A gene encoding bovine BMP-3 comprising the following DNA sequence: 



11 



EP 0 688 869 B1 



10 



15 



20 



25 



30 



3S3 3=3 403 413 423 

gaggaggaag cctciscsg GGGiccncr gccicigcag ■ aac aat gag err ccr ggg gca 

Asn Asn GIu Leu Pro Gly Ala 

<43 453 473 433 

GAA TAT CAG TA.C AAG GAG GAT GAA GZA TGG GAG GAG AGG AAG CCT TAC AAG ACT 
Glu Tyr Gin ivr Lys Glu Asp Glu Val Tr? GIu Glu Arg Lys Pro Tyr Lys T>ir 

503 513 533 

CIT CAG ACT CAG CCC CCT GAT AAG ACT AAG AAC AAA AAG AAA CAG AGG AAG GGA 
Leu Gin Sir Gin Pro Pro Asp Lys Ser Lys Asr. Lys Lys Lys Gin Arg Lys Gly 

548 563 573 533 

CCT CAG CAG AAG AGT CAG ACS CTC CAG TIT GAT GAA CAG ACC CIG AAG AAG GCA 
Pro Gin Gin Lys Ser Gin Thr Ian Gin Pr.e As? Glu Gin Sir I^u Lys Lys Ala 

603 ■ 623 633 

AGA AGA AAG CAA TGG ATT GAA CCC CSG A~T 1GT GCC AGA CGG TAC CIT AAA GTG 
Arg Arg Lys Gin Tr? lie Glu Pro Arg Asn Cys Ala Arg Arg Tyr leu Lys .Val 

653 ■ 653* 533 633 

GAG TIC GCA GAT ATT GGC TGG AGC GAA TGG ATT ATT TCC CCC AAG TCC TIC GAT 

Asp Sfcfi Ala Asp lie Gly TTp Ser Glu Tr?* lie lie Ser Pro Lys Ser rte Asp 

713 723 743 755 766 

GCC TAT TAG TCC TCC GGA GCG TGC CAG TTC CCT ATS OCA AAG GTAGCCATTG TTITTTGTOC 
Ala Tyr Tyr Cys Ser Gly Ala cys Gin Phe Pro !<ZT Pro Lys 

776 736 
'iCrlUJi'Ica: ATITCCAZAG ;. arxi 



234 294 304 319 

35 csgACCTCic - Ticicccrrr tcgttcciag tct ire- aag cca tca aat cac gct acc 

Ser Lsu Lys Pro Ser Asn Mis A2a T~r 

334 349 354 375 

ATC CAG 'AGT ATA GIG AGA GCT GIG GGG GIC GIC CCT GGA ATC CCC GAG CCT TGC 
4 o lie Gin Ser lis Val Arg Ala Val Gly val Vai' Pro Gly lis Pro Glu Pro Cys 

394 "409 424 439 

TGT GIG CCA GAA AAG ATG TCC TCA CTC AGC ATC- TTA TTC TTT 'GAT GAA AAC AAG 
Cys Vai Pro Glu Lys KIT Ser Ser Lsu Ser lie Leu Phe. P*ie Asp Glu Asn Lys 

45 



2. A gene encoding bovine BMP-3 having the amino acid sequence given in claim 1 . 

so 3. A gene encoding a protein exhibiting at least the property of BMP-3 to indues the formation of bone and comprising 
a DNA sequence: 

(a) which differs from a DNA sequence of claim 1 in codon sequence due to the degeneracy of the genetic code; 

(b) which hybridizes with a DNA sequence of claim 1 or section (a), above, under stringent hybridization con- 
55 ditions; or 

(c) which represents a fragment, or allelic variation of a DNA sequence of claim. 1 . 



12 



EP 0 688 869 B1 



454 • 455 434 

liT- GTC- GIVCIT J£A GEL 1ST CC?. AAC £TG kCk <ZSk GAG TCT 1GT GCT TGC 
Asn Vai val Lad Lys Val Tyr Pro Asr. MET Thr Val Glu Ser Cys lit C/s Arc 

503 . 513 523 533 

■Z^ACCIGGIG >ASWjCICS CISGnTOCIT ,-ACICmSCS. 



4. The DNA sequence of claim 3, which encodes human BMP-3. 

5. The DNA sequence of claim 3 or 4, which is a genomic DNA sequence. 

6. The DNA sequence of claim 3 or 4, which is a cDNA sequence. 

7. A vector containing the gene or DNA sequence of any one of claims 1 to 6 in operative association with an ex- 
pression control sequence. 

8. A cell transformed with a vector of claim 7. 

9. The cell of claim 8 which is a mammalian cell, a bacterial cell, an insect cell or a yeast cell. 

10. The cell of claim 9 which is a CHO cell. 

1 1 . A protein exhibiting properties of BMP-3 which is encoded by the gene or DNA sequence of any one of claims 1 to 6. 

12. A protein exhibiting properties of BMP-3 which is produced by the steps of culturing in a suitable culture medium 
a cell transformed with an expression vector comprising a gene or a DNA sequence of any one of claims 1 to 6, 
and recovering said protein from said culture medium. 

13. A process for producing the protein of claim 11 or 12, comprising the steps of culturing in a suitable culture medium 
the cell of claim 9 and isolating said protein from said culture medium. 

14. A pharmaceutical composition comprising the protein of claim 11 or 12 and a pharmaceutical^ acceptable vehicle. 

15. The pharmaceutical composition of Claim 14, further comprising a matrix capable of delivering the composition to 
the site of the bone or cartilage defect and providing a structure for inducing bone or cartilage formation. 

16. The pharmaceutical composition of claim 15, wherein said matrix comprises hydroxyapatite, collagen, polylactic 
acid or tricalcium phosphate. 

17. Use of a protein of claim 11 or 12 for the preparation of a pharmaceutical composition for inducing bone or cartilage 
formation. 



Claims for the following Contracting State : AT 

1 . A method for the preparation of a gene encoding bovine BMP-3 comprising the following DNA sequence: 



13 



EP 0 688 869 B1 



333 393 403 413 423 

GAGSMSAAG CT-CTCZACSG GGGICCnCT GCCTCTGCAG • AAC AAT GAG CTT OCT GGG GCA 

Asn Asn Glu Lai Pre Gly Ale 

443 4=3 473 433 

GAA TAT CAG ISC AAG GAG GAT GAA G2A TGG G-G GAG AGG AAG CCT TAC AAG ACT 
Glu ivr Gin ivr Lys Glu Asp Glu Val Trp Giu Glu Arg Lys Pro Tyr Lys 'I>>r 

503 513 533 

CTT GAG ACT CAG CCC OCT GAT AAG AGT AAG AAC AAA AAG AAA CAG AGG AAG GGA 

Lsu Gin &r C-L^. Pro Pro Asp Lys Sar Lys Asn Lys Lys Lys Gin Arg Lys Gly 

548 '563 573 593 

OCT CAG CAG AAG AGT CAG ACG CIC CAG TTT GAT GAA CAG ACC CIG AAG AAG CCA 
Pro Gin Gin Lys Ser Gin T^r L~u Gin H?.e As? Glu Glr* TMr isu Lys Lys Ala 

60S * 623 633 

AGA AGA AAG CAA TGG ATT GAA CCC CGG AAT TGT GCC AGA CCG TAC CTT 'AAA GIG 
Azg Arg Lys Gin Tro lie Giu Pro Arg Asn cys Ala Arg* Arg Tyr Leu Lys .Val 

653 ■ 653' 633 693 

GAC TTC GCA GAT ATT GGC TGG AGC GAA TGG ATT ATT TCC CCC AAG TCC TIC GAT 

As? Hie Ala Asp Ha Gly Ttrp Sar Giu Trp* tie Lie Ser Pro Lys Ser Phs Aso 

713 723 743 755 766 

GCC TAT TAC TGC TCC GGA. GCG TGC CAG TTC CCC "ATG CC\ AAG GIAGCCATTG TZTTTTGTCG-* 
Ala Tyr IVr Cys Ser Gly Ala Ofs Gin Phe Pro MTT Pro Lys 

776 736 . .* 

TGICCTICCC ATTICCA2AG ar*i 



234 294 304 

CTAACCIGIG TICICCCITT TCGTTCCTAG 



319 ... 

TCT TSC- AAG CCA TCA AAT CAC GCT ACC 
Ser Leu Lys Pro Ser Asn sis Ala Tnr 



334 349 354 375 

ATC CAG "ACT ATA GIG AGA GCT GIG GGG CIC CIC CTT GCA ATC CCC GAG OCT TGC 

lie Gin Ser lie Val Ar? Ala Val Gly Val VaT Pro Gly lis Pro Glu Pro Cys 

394 409 424 439 

TCT GIG CCA GAA AAG ATG TCC TCA CIC AGC ATC TEA. TTC TIT GAT GAA. AAC AAG 
cys Val Pro* Glu Lys KTT Ser Ser Leu Ser lis Leu Phe. Phe Asp Glu Asn Lys 



The method of claim 1 , wherein the gene encoding the BMP-3 has the amino acid sequence given in claim 1 . 

The method of claim 1 , wherein a gene encoding a protein exhibiting at least the property of BMP-3 to induce the 
formation of bone comprises a DNA sequence: 

(a) which differs from a DNA sequence obtained according to the method of claim 1 in codon sequence due 
to the degeneracy of the genetic code; 

(b) which hybridises with a DNA sequence obtained according to the method of cfaim 1 or section (a) : above 
under stringent hybridisation conditions; or 

(c) which represents a fragment, or allelic variation of a DNA sequence obtained according to the method of 
claim 1. 



14 



EP 0 688 869 B1 



; 454 . 4€9 Z*Z _ ^ 

' »t err- gza err jua gz?. t*t ca a?c as aca gsa ec ^ ^ ;~ 
^ v£ vll Leu Lvs Val Tvr 3te> Asr. ^ -ffir Val clu ser C/s .^a C-s 



=03 513 523 533 

^ccresw msucics csggsigctt ^rrojaxc. 



15 



said method comprising screening of a recombinant bovine genomic library with probes consisting of pools of 
oligonucleotides designed on the basis of partial amino acid sequences and isolating a positive clone. 

4. The method of claim 3, wherein the DNA sequence encodes human BMP-3. 

5. The method of claim 3 or 4, wherein the DNA sequence is a genomic DNA sequence. 

6. The method of claim 3 or 4, wherein the DNA sequence is a cDNA sequence. 

20 7. A method for the preparation of a recombinant vector comprising inserting the gene or DNA sequence obtained 
according to a method of any one of claims 1 to 6 in operative association with an expression control sequence 
in a suitable vector. 

8. A cell transformed with a vector prepared according to the method of claim 7. 

25 

9. The cell of claim 8 which is a mammalian cell, a bacterial cell, an insect cell or a yeast cell. 

10. The cell of claim 9 which is a CHO cell. 

30 11. a process for producing a protein exhibiting properties of BMP-3 comprising the steps of culturing in a suitable 
culture medium the cell of claim 9 and isolating said protein from said culture medium. 

12. A method for the preparation of a pharmaceutical composition comprising combining the protein produced accord- 
ing to the method of claim 11 with a pharmaceutical ly acceptable vehicle. 

35 

1 3. The method of claim 1 2, wherein the pharmaceutical composition further comprises a matrix capable of delivering 
the composition to the site of the bone or cartilage defect and providing a structure for inducing bone or cartilage 
formation. 

^0 14. The method of claim 13, wherein said matrix of said pharmaceutical composition comprises hydroxyapatite, col- 
lagen, poly lactic acid or tricalcium phosphate. 

15. Use of a protein of claim 11 for the preparation of a pharmaceutical composition for inducing bone or cartilage 
formation. 

45 

16. A gene encoding bovine BMP-3 comprising the following DNA sequence: 



50 



55 



15 



EP 0 688 869 B1 



10 



15 



30 



35 



40 



3S3 '393 403 413 423 

gaggaggaag cc<n?cF--czc~ cccnccrrcr gccicit-c^ * aac aat g^g err ccr ggg gca 

Asn Asn Glu Leu Pro Gly .Ue 



443 453 473 

GAA TAT OjG TAC AAG GAS GAT G-A GZA TGG GAG GAG AGG AAG CCT TAC AAG ACT 
Glu Tyr Gin ivr Lys Glu Asp Glu Val Tr? Glu Glu Arg Lys Fro Tyr Lys J>u: 

503 513 , 533 

err og act c~g ccc ccr gat aag agt aag aac aaa aag aaa c-r- ac-g aag gga 
Leu Gin dr Gin Pro Asp Lys Ser Lys Asn Lys Lys Lys Gin Argr Lys Gly 

54.3 '563 573 533 

CCT CAG CAG AAG AGT CAG ACS CTC CAG TTT G2? GAX CAG ACC CTC- AAG AAG GCA 
Pro Gin Gin Lys Ser Gin Ttir Lsu Gin Hie As? Glu Gin Sir Lsu Lys Lys Ala 



60S 623 ' 633 

AGA A^A AAG CAA TCG AST GAA CCC CSG AAT TGI GCC AGA C3G dC CTT 'AAA 

20 Arg Arg Lys Gin Tr? He Glu Pro Arg Asn cys Ala Ar? Arr Tyr Leu Lys .Val 



S53 ' 653" 533 653 

GAC TTC GCA G?T AST GGC TCG AGC GAA TCG AST ATT ICC CCC AAG TCC TIC GAT 

As? Hie Ala As? lis Gly Tr? Ser Glu 1r?- lie lie Ser Pro Lys Ser Hie As? 

25 713 723 743 755 766 

: GCC TAT TAC TCC TCC GGA GCC- TCC CAG TIC CCC ATC CCA AAG GZZGGGSZG TTITITCTCC*^' 
Ala Tyr tyr Cys Ser Gly Ala cys Glr. Fhe ?ro Pro Lys- 



776 736 

tctccitccc mejxtmm ;. erd 



234 254 304 ~ 313 ..... 

: CZ3SCCICTG TICTCCCTIT TC3ITCC3SS TCI TTC AAC- CCA TCA AAT CAC GCI ACC 

Ser leu Lys Pro Ser Asn His Ala Sr 

334 349 354 37S 

ATC CAG ACT ASA GIG AGA GCI GIG GGG GIC GIC OCT GGA AXC CCC GAG CCT TCC 

lie Gin Ser He Val Ar^r Ala Val Gly -Val Val' ?ra Gly lis ?ro Glu Fro Cys 

394 409 424 439 

TCT GIG CCA GAA AAG ATG TCC TCA CTC AGC ATC TTA TTC TTT GAT GAA AAC AAG 
Cys Val Pro* Glu Lys MET Ser Ser Leu Ser He Leu She. Phe As? Glu Asn Lys 



45 17. A gene encoding bovine BMP-3 having the amino acid sequence given in claim 16. 

1 8. A gene encoding a protein exhibiting at least the property of BMP-3 to induce the formation of bone and comprising 
a DNA sequence: 

so (a) which differs from a DNA sequence of claim 16 in codon sequence due to the degeneracy of the genetic 

code; 

(b) which hybridises with a DNA sequence of claim 16 or section (a), above, under stringent hybridisation 
conditions; or 

(c) which represents a fragment, or allelic variation of a DNA sequence of claim 16. 

55 



16 



EP 0 688 869 B1 



«4 *** 



- ^ . — — rrrr CI?. TST CCA ^-.C SZ5 ACS. <^£- TCT IS OCT TC-C iSA 
S.S 7£ UrI tys" val Tvtr ?ro Asn MET Tl" Val Glu Ssr cys Aia C/s tar 

503 513 523 . 533 

rs&ccrssiG >^^cro2 ctsgmhch smicakecs. 

to 

19. The DNA sequence of claim 18, which encodes human BMP-3. 

20. The DNA sequence of claim 18 or 19, which is a genomic DNA sequence. 

15 21. The DNA sequence of claim 18 or 19, which is a cDNA sequence. 

22. A vector containing the gene or DNA sequence of any one of claims 16 to 21 in operative association with an 
expression control sequence. 

20 23. A cell transformed with a vector of claim 22. 

24. The cell of claim 23 which is a mammalian cell, a bacterial cell, an insect cell or a yeast cell. 

25. The cell of claim 24, which is a CHO cell. 

25 

26. A protein exhibiting properties of BMP-3 which is encoded by the gene or DNA sequence of any one of claims 1 
to 21. 

27. A protein exhibiting properties of BMP-3 which is produced by the steps of culturing in a suitable culture medium 
30 a cell transformed with an expression vector comprising a gene or a DNA sequence of any one of claims 1 to 21 

and recovering said protein from said culture medium, 

28. A pharmaceutical composition comprising the protein of claim 26 or 27 and a pharmaceutically acceptable vehicle. 

35 29. The pharmaceutical composition of claim 29, further comprising a matrix capable of delivering the composition to 
the site of the bone or cartilage defect and providing a structure for inducing bone or cartilage formation. 

30. The pharmaceutical composition of claim 29, wherein said matrix comprises hydroxyapatite, collagen, polylactic 
acid or tricalcium phosphate. 

40 

Patentanspriiche 

45 Patentanspruche fur folgende Vertragsstaaten : BE, CH, LI, DE, FR, GB, IT, LU, NL, SE 

1. Gen, das bovines BMP-3 codiert, welches die folgende DNA-Sequenz umfasst: 

50 



55 



17 



EP 0 688 869 B1 



3S3 ' 3=3 403 413 

g-£GA.£GA?g gcctcscsg cc<n:ccrrcr cccrcrcor- * aac aat g-£ err ccr ggg cca 

Asn Asn Giu Lsu ?rc c-ly Ala 



443 453 473 

GAA *E2 OC TAC AAG GAG GAT GAA CIA ?GG d^T- GAG AGG AAG CCT T?jC AA£ ACT 
Giu Tyr Gin ryr Lys Glu As? GIu Val irp Giu Glu Azg Lys ?ro Tyr Lys *I>ir 

503 £13 533 

err act cvg ccc ccr gat aag act aag aac aaa aag aaa c-j2 asg aac gga 
Lsu Gin 2r Girt Pro Pro Asp Lys Sar Lys Asn Lys Lys Lys Gin Arg Lys Gly 

54S '363 573 593 

CCT CAG C?£ AAG AST CAG AGS CTC G«3 'ITT GAT GAA. CAG ACC CIG AAG AAG GCA 
Pro Gin Gin Lys Ser Gin 1>!r Lsu Gin Kia Asp Glu Gin Thr Lsu Lys Lys Ala 



623 - 633 
AGA ASA AAG CAA TGG AST «A CTC CGG AAT TGT GCC AHA CGG CTT AAA GIG 
Arg Arg Lys Gin Trp lie Glu Pro Arg A-^i 0,-5 Ala Arg Arg Tyr Lsu Lys ,v=l 

553 * 653' 633.. . . 653 

GAC TTC GCA GrT ACT GGC TGG AGC GAA TGG ATT AST TCC CCC AAG ICC TIC GAT 

Asp Alia Asp Ila Gly Trp Sar Glu Trp- Xia lie Ser Pro Lys Ser His Asp 

713 -723 743 756 766 

CCC ET TAC TGC TCC GGA GCG TGC Gu TTC CCC ATG CCA AAG GTAGCX^TTG TTTI^rG^CC- 
Ala Tyr TVr Cfs Ser Gly Ala Cys Gin SSie Pro MTT Pro LVS 

776 ,736 
1L-±XXZI<JZZ AT2TCC22SG ;. arid 

234 294 304 " 313* 

crAACcrciG ricicccrrr tcticcsg tci tig aag cca tc\ aat cac gct-acc 

Sar Lsu Lys Pro Ser Asn His Ala T-r 



334 349 354 379 

ATC <3£ 'AGT ATA GIG AGA GCT GIG GGG GTC GIC CX2T GGA ATC CCC GAG CCT TGC 

lie Gin Ser lis Val Arg Ala Val Gly Val Vai Pro Gly lis Pro Glu Pro Cys 

354 409 424 439 

TGT GIG CCA GAA AAG ATG TCC TCA CTC AGC ATC TTA TIC TIT GAT GAA AAC AAG 

Cys Val Pro- Glu Lys KET Ser Sar Lsu Ser lis Lsu Fr.« PSxs Asp Giu Ajst. Lys 



4S4 - <£5 

aat gis gza czt aaa gta t?t cca aac atg aca gta gac- rcr tgt gct igc aga 
Asn v^l val Leu Lys Val r^-r pro Asr. ?£ET n-ir Val Giu Ser Cys .AI2. cys Arc 

503 513 523 533 

rAA-CCIGGIG A.-jGnAC3TGA2 CraGATOCIT AACICAATCG. 



2. Gen, das bovines BMP-3 codiert, welches die Aminosauresequenz aufweist, die in Anspruch 1 angegeben ist. 

3. Gen, das ein Protein codiert, das mindestens die Eigenschaft von BMP-3 aufweist, die Bildung von Knochen zu 
induzieren, und eine DNA-Sequenz umfasst: 

(a) die sich von einer DNA-Sequenz nach Anspruch 1 in der Codonsequenz auf Grund der Degeneriertheit 
des genetischen Codes unterscheidet; 



16 



EP 0 688 869 B1 



(b) die mit einer DNA-Sequenz nach Anspruch 1 Oder Absatz (a) oben unter stringenten Hybridisierungsbe- 
dingungen hybridisiert; oder 

(c) die ein Fragment Oder eine allelische Variation einer DNA-Sequenz nach Anspruch 1 darstellt. 

4. DNA-Sequenz nach Anspruch 3, die menschliches BMP-3 codiert. 

5. DNA-Sequenz nach Anspruch 3 oder 4, die eine genomische DNA-Sequenz ist. 

6. DNA-Sequenz nach Anspruch 3 oder 4, die eine cDNA-Sequenz ist. 

7. Vektor, der das Gen Oder die DNA-Sequenz nach einem der Anspruche 1 bis 6 in funktioneller Verknupfung mit 
einer Expressionskontrollsequenz enthalt. 

8. Zelle, transformiert mit einem Vektor nach Anspruch 7. 

9. Zelie nach Anspruch 8, die eine Saugerzeile, eine Bakterienzelle, eine Insektenzelle oder eine Hefezelle ist. 

10. Zelle nach Anspruch 9, die eine CHO-Zelle ist. 

11. Protein, das Eigenschaften von BMP-3 aufweist, das von dem Gen oder der DNA-Sequenz nach einem der An- 
spruche 1 bis 6 codiert wird. 

12. Protein, das Eigenschaften von BMP-3 aufweist, das durch die Schritte des Zuchtens in einem geeigneten Kul- 
turmedium von einer Zelle, die mit einem Expressionsvektor transformiert ist, der ein Gen oder eine DNA-Sequenz 
nach einem der Anspruche 1 bis 6 umfasst, und des Gewinnens des Proteins aus dem Kulturmedium hergestellt 
wird. 

13. Verfahren zur Herstellung des Proteins nach Anspruch 11 oder 12, umfassend die Schritte des Zuchtens der Zelle 
nach Anspruch 9 in einem geeigneten Kulturmedium und des Isolierens des Proteins aus dem Kulturmedium. 

14. Arzneimittel, umfassend das Protein nach Anspruch 11 oder 12 und ein pharmazeutisch vertragliches Vehikel. 

15. Arzneimittel nach Anspruch 14, das zusatzlich eine Matrix umfasst, die das Mittel an die Stelle des Knochen- oder 
Knorpeldefekts bringen und eine Struktur zum Induzieren von Knochen- oder Knorpelbitdung zur Verfugung stellen 
kann. 

1 6. Arzneimittel nach Anspruch 1 5, wobei die Matrix Hydroxyapatit, Kollagen, Polymilchsaure oderTricatciumphosphat 
umfasst. 

17. Verwendung eines Proteins nach Anspruch 11 oder 12 fur die Herstellung eines Arzneimittels zum Induzieren von 
Knochen- oder Knorpelbildung. 



Patentanspruche fur folgenden Vertragsstaat : AT 

1. Verfahren zur Herstellung eines Gens, das bovines BMP-3 codiert, welches die folgende DNA-Sequenz umfasst: 



19 



EP 0 688 869 B1 



333 3=3 403 413 425 

GrCSSSAAG CSCTCmCSG GGGXCCnCT GCCICTCCAS * AAC GAG CTT OCT GGS C-CA 

Asn Asn Glu Lsu Pro Gly Ale 

443 453 473 433 

GA TAT CAG TAC AA3 GAS GAT GAA GZA TGG GAG G-C ACG AAG CCT IAC AAC ACT 
Glu Tyr Gin 2Vr Lys Glu As? GIu Val Tr? Glu Glu Arg Lys Pro Tyr Lys TMr 

503 513 533 

err ess act cr-r- ccc 'ccr aac agt aag aac saa aac aaa og ac-g aac gga 
Lsu Gin Irzr GL^. Pro Pro Asp Lys Sar Lys Asn Lys Lys Lys Gin Arg Lys Gly 

543 "563 573 533 

CCT C=C CSC AAG AST GG ACS CIC CSC TIT GAT GAA GC ACC CIC AAG AAG GCA 
Pro Gin Gin Lys Sar Gin Bur Lsu Gin Hie As? Glu Gin Sir Lsu Lys Lys Ala 

60S - * 623 533 

AGA ASA AAG CAA TGG AST GAA CCC CGG A^T TGT GCC AGLA CSG TAC CTT "AAA GIG- 
Arg Arg Lys Gin Tr? He Glu Pro Arg Asn Cys AJLa Arg Arg Tyr I^u Lys ( Vai 

653 - 653* 533 653 

GAC TIC GCA GAT AFT GGC TGG ACC G-A TGG ATT ATT TCC CCC AAG TCC TIC G£T 

As? Ala As? lis Gly Tr? Sar Glu Tr?* lie He S^ar Pro Lys S«r P?,e Asp 

.* 

713 723 743 755 765 

GCC TAC TC-C TCC GGA GCG TCC CAS TIC CCC ATG CCA AAG <35GCSaTG TITTTIGTCG- 
Ala Tyr Tyr Cys Sar Gly Ala Cys Gin ssae Pro MET Pro Lys 

776 73S 

TGiccrrccc atticcvtag ;. arc. 



234 234 304 ' 313 

CTAACCTGTG TTCICCCTTI TCGTTCCTAG TCT TIG AAG CCA TCA AAT CAC GCT ACC 

Sar Lau Lv-s Pro Sar Asn -is Ala *Tlnr 



334 349 354 379 

>ZC C-C AST ATA GIG AGA GCT GIG GGG GIC CIC CCT GGA ATC CCC G=C CCT TSC 

He GLn Sar lis Val Arg Ala Val Gly Val Val' Pro Gly Lie Pro Glu Pro O/s 

3S4 4C9 424 439 

TGT GIG CCA GAA AAG AUG TCC 1C. CIC AC-C ATC TTA TtC TIT GAT GAA AAC AAG 

Q/s Val Pro' Glu Lys MTI Sar Sar Lsu Sar lis Lau Fns. Phe As? Glu Asn Lys 

454 45r 434 

.-AT GTT- GIA CTT AAA CIA TAT CCA AAC ATS ACA GLA G-C TCT TST GCT TGC .^GA 
Asn Val Val Lsu Lys Val Tyr Pro Asn ■•ET Thr Val Glu Sar Cys Ala Cys Arg 

503 513 523 533 

TTACCIGGTG AACAACTC32 CIC<LAIt:CIT AACICA.-TCS . 



wobei das Verfahren das Durchmustern einer rekombinanten bovinen genomischen Bibliothek mit Sonden, die 
aus Pools von Oligonucleotiden bestehen : die auf der Grundlage von partiellen Aminosauresequenzen hergestellt 
wurden, und das Isolieren eines positiven Klons umfasst. 

2. Verfahren nach Anspruch 1 , wobei das Gen, das BMP-3 codiert, die Aminosauresequenz aufweist, die in Anspruch 
1 angegeben ist. 

3. Verfahren nach Anspruch 1 , wobei ein Gen, das ein Protein codiert, welches mindestens dieEigenschaft von BMP- 



20 



EP 0 688 869 B1 



3 zeigt, die Bildung von Knochen zu induzieren, eine DNA-Sequenz umfasst: 

(a) die sich von einer DNA-Sequenz, die gemaB dem Verfahren nach Anspruch 1 erhalten wurde, in der Co- 
donsequenz auf Grund der Degeneriertheit des genetischen Codes unterscheidet; 

(b) die mit einer DNA-Sequenz, die gemaB dem Verfahren nach Anspruch 1 Oder Absatz (a) oben erhalten 
wurde, unter stringenten Hybridisierungsbedingungen hybridisiert; Oder 

(c) die ein Fragment oder eine allelische Variation einer DNA-Sequenz darstellt, die gemaB dem Verfahren 
nach Anspruch 1 erhalten wurde. 

4. Verfahren nach Anspruch 3, wobei die DNA-Sequenz menschliches BMP-3 codiert. 

5. Verfahren nach Anspruch 3 oder 4, wobei die DNA-Sequenz eine genomische DNA-Sequenz ist. 

6. Verfahren nach Anspruch 3 oder 4, wobei die DNA-Sequenz eine cDNA-Sequenz ist. 

7. Verfahren zur Herstellung eines rekombinanten Vektors, umfassend das Einfugen des Gens oder der DNA-Se- 
quenz, die gemaB einem Verfahren nach einem der Anspriiche 1 bis 6 erhalten wurde, in funktioneller Verkniipfung 
mit einer Expressibnskontrollsequenz in einen geeigneten Vektor. 

8. Zelle, transformiert mit einem Vektor, der gemaB dem Verfahren nach Anspruch 7 hergestellt wurde. 

9. Zelle nach Anspruch 8, die eine Saugerzelle, eine Bakterienzelle, eine Insektenzelle oder eine Hefezelle ist. 

10. Zelle nach Anspruch 9, die eine CHO-Zelle ist. 

11. Verfahren zur Herstellung eines Proteins, das Eigenschaften von BMP-3 aufweist, umfassend die Schritte des 
Zuchtens der Zelle nach Anspruch 9 in einem geeigneten Kulturmedium und des Isolierens des Proteins aus dem 
Kulturmedium. 

12. Verfahren zur Herstellung eines Arzneimittels, umfassend das Kombinieren des Proteins; das gemaB dem Ver- 
fahren nach Anspruch 11 hergestellt wurde, mit einem pharmazeutisch vertraglichen Vehikel. 

13. Verfahren nach Anspruch 12, wobei das Arzneimittel zusatzlich eine Matrix umfasst, die das Mitt el an die Stelle 
des Knochen- oder Knorpeldefekts bringen und eine Struktur fur das Induzieren von Knochen- oder Knorpelbildung 
zur Verfugung stellen kann. 

14. Verfahren nach Anspruch 13, wobei die Matrix des Arzneimittels Hydroxyapatit, Kollagen, Polymilchsaure oder 
Tricalciumphosphat umfasst. 

15. Verwendung eines Proteins nach Anspruch 1 1 fur die Herstellung eines Arzneimittels z urn Induzieren von Knochen- 
oder Knorpelbildung. 

16. Gen, das bovines BMP-3 codiert, welches die folgende DNA-Sequenz umfasst: 



21 



EP 0 688 869 B1 



3S3 *3S3 403 413 423 

cssicsex- Greener cccictgog . aac aac gac ctt cct ggg gca 

Asn Asn Giu Leu ?rc Gly Ala 

443 453 473 423 

GA-A Or- TAC AAC- GAS GAT GAA GTA TGG GAG GAS AGG AM CCT TAC AAS ACT 
Glu Tyr Gin Tyr Lys Glu Asp Giu Val Tr? Giu Glu Arg Lys Tyr Lys TJir 

303 513 533 

CTT Off ACT OG CCC CCT GAT AAC- AGT AA£ AAC AAA AAG AAA CA« AC-G A?iS GGA 
I~u Gin T??r Gin r^c tTo A.sp Lys Sar Lys £sn Lys Lys Lys Gin Ar^ Lys Gly 

543 "563 57c 553 

CCT GG OC- AAG AG? CAG ACT- CIC CAT TIT GAT GAA CAG ACC CIC- AAC- AAG GCA 
i>ro Gin Gin Lys Ser Gin T^ Lau Gin ?iie Asp Glu Gin Tnr lau Lys Lys Ala 

60S • * 613 €33 

AGA ATA AAG OA TGG ATT GAA CCC CSG AAT 1 TGT GCC AGLA CT-G TAC CTT "AAA GIG 
A-Tw Ar^ Lys Gin Trp lie Glu ?td Arg Asn Cys Ala Ar~ Ajrg Tyr Lau Lys ,Vai 

653 ■ 653* £53 6=3 

GAC TIC GO. GAT ATT GGC TGG AGC GAA TGG AST ATT TCC CCC AAG TCC ITC GAJT 

Asp 3>:s A-la Asp lie Gly lip Sar Glu Trp- lie lie Sar Lys £«=- Shs A.sp 

.* 

713 723 743 755 765 

GCC XAC 1GC TCC GGA, GCG TGC OG TTC CCC ATS CCA AAC- GtAGCCACTG T FxxiJ G X GG-- 
AJLa Tyr Tyr Cys Ser Gly Ala Cys Gin ?Me Pro • / CTT Pro Lys 

776 736 
TGICCTTCCC ArTTCOEG ;. arsi 

234 254 304 319 

cgLA cc i ui c - Ttcicccrrr tcgttcceajg tct Tic- aac- cca tca aat cac c-cr acc 

Sar Lau Lys ?rs Sar Asn His Ala Tl— 

334 349 354 37? 

ATC OG AjGT A2A GTG AGA GOT GIG GGG GTC GTC CCT GGA ATC CCC GAG CCT TGC 

lie Gin Ser lis Val Arg Ala Val GlyVal VaT Pro Gly Ha Pro Giu Pre Cys 

354 4G9 424 439 

1CT GIG CCA GAA AA£ ASS TCC TCA CIC AiSC ATC TEA. TTC TIT GAX GAA. AAC AAG 

cys Val Pro Giu Lys Mil Ser S«r Lau Sar lis Lau Fha_ riie Asp Giu Asp. Lys 

454 - 45r 454 

:-JT GTG GIA CIT AAA CIA CCA AAC AIG AO. GTA C=C- TC3 TGT C-TT IGC AGA 
A-sn Val Val Lire Lys Val Tyr r^a ?sr, . l -ET Tnr Val Glu 5ar C : - A2a Cys Arc 

;03 513 523 533 

TAA.CCTGGIG AAjGAA.CTCAT CIC-SS^GCTT AACICAA5TCG. 



17. Gen, das bovines BMP-3 codiert, wetches die Aminosauresequenz aufweist, die in Anspruch 16 angegeben ist. 

18. Gen, das ein Protein codiert, das mindestens die Eigenschaft von BMP-3 aufweist, die Bildung von Knochen zu 
induzieren, und eine DNA-Sequenz umfasst: 

(a) die sich von einer DNA-Sequenz nach Anspruch 16 in derCodonsequenz auf Grund der Degeneriertheit 
des genetischen Codes unterscheidet; 

(b) die mit einer DNA-Sequenz nach Anspruch 16 oder Absatz (a) oben unter stringenten Hybridisierungsbe- 



22 



EP 0 688 869 B1 



dingungen hybridisiert; Oder 

(c) die ein Fragment oder eine allelische Variation einer DNA-Sequenz nach Anspruch 16 darstellt. 

19. DNA-Sequenz nach Anspruch 18, die menschliches BMP-3 codtert. 

20. DNA-Sequenz nach Anspruch 18 oder 19, die eine genomische DNA-Sequenz ist. 

21. DNA-Sequenz nach Anspruch 18 oder 19, die eine cDNA-Sequenz ist. 

22. Vektor, der das Gen oder die DNA-Sequenz nach einem der Anspriiche 1 6 bis 21 in funktioneller Verknupfung mit 
einer Expressionskontrollsequenz enthalt. 

23. Zelle, transformiert mit einem Vektor nach Anspruch 22. 

24. Zelle nach Anspruch 23, die eine Saugerzelle, eine Bakterienzelle, eine Insektenzelle oder eine Hefezelle ist. 

25. Zelle nach Anspruch 24, die eine CHO-Zetle ist. 

26. Protein, das Eigenschaften von BMP-3 aufweist, das von dem Gen oder der DNA-Sequenz nach einem der An- 
spriiche 1 bis 21 codiert wird. 

27. Protein, das Eigenschaften von BMP-3 aufweist, das durch die Schritte des Zuchtens in einem geeigneten Kul- 
turmedium von einer Zelle, die mit einem Expressionsvektor transformiert ist, der ein Gen oder eine DNA-Sequenz 
nach einem der Anspriiche 1 bis 21 umfasst, und des Gewinnens des Proteins aus dem Kulturmedium hergestellt 
wird. 

28. Arzneimittel, das das Protein nach Anspruch 26 oder 27 und ein pharmazeutisch vertragliches Vehikel umfasst. 

29. Arzneimittel nach Anspruch 29, das zusatzlich eine Matrix umfasst. die das Mittel an die Stelle des Knochen- oder 
Knorpeldefekts bringen und eine Struktur zum Induzieren von Knochen- oder Knorpelbildung zur Verfugung stellen 
kann. 

30. Arzneimittel nach Anspruch 29, wobei die Matrix Hydroxyapatit, Kollagen, Polymilchsaure oderTricalciumphosphat 
umfasst. 



Revendications 



Revendications pour les Etats contractants sulvants : BE, CH, LI, DE, FR, GB, IT, LU, NL, SE 

1. Gene codant pour la BMP-3 bovine comprenant la sequence d'ADN suivante : 



23 



EP0 688 869 B1 



3S3 333 433 ft 13 

GAGGAGGA-r, csgtc^cgs csroxircr cccictgcag . iic aat cm ci? err- ggs cca 

A=n Asr. Glu Lsu Giy .Us 



443 *=3 <73 

GAA GAG AW GAG «Z GA\ GE\ TCC- d-£ GAS Am AAS CCT IAC AAG ACT 
Giu Tyr Gin Tyr Lys Glu Asp GLu Vai *Erp Glu Glu Arg Lys ?ro Tyr Lys -Thr 

503 515 533 

CTT CC ACT CCC CTT GAT *ST AAG A*C AAA AAG AAA C£ .*£G GGA 
La: Gin Gin ?ro ?r= Asp Lys Ser Lys Asr. ^ys Lys Lys Gin Arr Ly* ciy 



563 . 573 533 

CCT Cw AAG AGT G£ ACS CTC Or- TIT GAT CAA CAS AOC CIG AAG AF£ GCA 
iro Gin Gin Lys Ser Gin Z^ir Leu sin ?Si* As? Glu Gin 1?ir L£U Lys Lys Ala 

€03 * 623 eaa 

AGA ASA *AG C?A TGG GAA CCC C3G AAff «T GCC A£A CGC- ZAC CTT'j^A 
Ar? Arg Lys Gin Trc Ha Glu 'Pro Ars Ash Cy* Alt Ars- Arg Tyr Lsu Lys .v*i 

551 653 533 €53 

GAC TTC CO. J£T GGC 1GG AGC GAA TC5 AT! ATT TCC CCC AAG TCG TIC «3» 

Asp Site Ala. Asp II* Gly itrp S&r Glu Crp- He ris S«sr ?rt Lys Ser Asp 

713 723 743 756 765 

GGC K -rXC TCC TCC GGA GCG TK TIC C2G AIG CCA AAG OfiCSfflS TTZTTIGICC 
A1& ivr Tyr Cys S*r Giy Aia Cys c-Lr. sue ?ra ?<TT L^s 

776 735 

^ citAjriiu: a itzc^a sag ; et 



234 234 304 319 

ce?accxg5g Trcrcccm Tasirccr-r; icr t+g aajg cca tca aaj cac ccr acc 

Ser Lau Lv-s Ser Asn Kia AU S'X 
334 3^9* 35^ 37= 

>5c ';^r >jT-. gig aga gct gig ggg crc gtc cct gg.v aic ore gag err igc 

He Gin Ser Ha V&l Arj Ala Vai Gly Vai vai' Pro Giv* lis Glu «td O/s 

3S4 4C9 <3< 439 

GOT CCa AAG ASG TCC TCA. CZC AGC >JC T^A TTC TIT GAX C?A >J^jC W^C 

cys Vai ?ro Glu i^-s WTI Ser Ser Ls*-i sar lis Sfca. Pie Asp Glu Asr. Lys 



«24 ^35 

A : r gtg czk err ctia cn^, aac aca gia rcr tgi gh tgc a^.- 

■ast, Vai Vtl Lsu Lys Vai ?rc i=r. ^ v ai c-i- S^r Cys ;ii cys Ary 

203 513 523 23: 

CAXCTTGCTG >A^ACTCT-w? CICGATGCTI AACTCT-A^w. 



Gene codant pour la BMP-3 bovine ayant la sequence d'acides amines donnee a la revendication 1 . 

Gene codant pour une proteine montrant au moins les proprietes de la BMP-3 pour indutre la formation osseuse 
et comprenant une sequence d'ADN : 

(a) qui d iff ere d'une sequence d'ADN de la revendication 1 dans la sequence de codons du fait de la dege- 
nerescence du code genetique ; 



24 



EP 0 688 869 B1 



10 



15 



20 



(b) qui s'hybride avec une sequence d'ADN de la revendication 1 ou du paragraphe (a) ci-dessus dans des 
conditions d'hybridation stringentes; ou 

(c) repr£sente un fragment ou une variation allelique d'une sequence d'ADN de la revendication 1 . 

4. Sequence d'ADN suivant la revendication 3, qui code pour la BMP-3 humaine. 

5. Sequence d'ADN suivant la revendication 3 ou 4, qui est une sequence d'ADN genomique. 

6. Sequence d'ADN suivant la revendication 3 ou 4, qui est une sequence d'ADNc. 

7. Vecteur contenant le gene ou la sequence d'ADN suivant Tune quelconque des revendications 1 a 6, en association 
operationnelle avec une sequence de controle d'expression. 

8. Cellule transformed avec un vecteur de la revendication 7. 

9. Cellule suivant la revendication 8, qui est une cellule mammifere, une cellule bacterienne, une cellule d'insecte 
ou une cellule de levure. 

10. Cellule suivant la revendication 9, qui est une cellule CHO. 

11 . Protelne montrant des proprietes de la BMP-3, qui est codeepar le gene ou la sequence d'ADN de Tune quelconque 
des revendications 1 a 6. 

12. Proteine montrant des proprietes de la BMP-3, qui est produite par les Stapes de culture dans un milieu de culture 
25 approprie d'une cellule transformee avec un vecteur d'expression comprenant un gene ou une sequence d'ADN 

de Tune quelconque des revendications 1 a 6, et de recuperation de ladite proline du milieu de culture pr£cite. 

13. ProcedS de production de la proteine suivant Tune ou Pautre des revendications 11 ou 12, comprenant les etapes 
de culture dans un milieu de culture approprie de la cellule de la revendication 9 et d'isolement de ladite p rote in e 

30 du milieu de culture prdcite. 

14. Composition pharmaceutique comprenant la prot£ine suivant Tune ou I'autre des revendications 11 ou 12 et un 
vehicule pharmaceutiquement acceptable. 

35 15. Composition pharmaceutique suivant la revendication 14, comprenant en outre une matrice pouvant distribuer la 
composition au site de I'anomalie osseuse ou cartilagineuse et fournir une structure pour induire une formation 
osseuse ou cartilagineuse. 

16. Composition pharmaceutique suivant la revendication 15, dans laquelle ladite matrice comprend de I 1 hydroxy a- 
40 patite, du collagene, de I'acide polylactique ou du phosphate tricalcique. 

17. Utilisation d'une proteine suivant Tune ou I'autre des revendications 11 ou 12 pour la preparation d'une composition 
pharmaceutique pour induire une formation osseuse ou cartilagineuse. 



45 



50 



Revendications pour I'Etat contractant suivant : AT 

1. Procede de preparation d'un gene codant pour le BMP-3 bovin comprenant la sequence d'ADN sutvante ; 



55 



25 



EP 0 688 869 B1 



3c3 393 403 413 423 

(l^ggaggaag cx^raex, c-GGrccrrcr c-cciciccac- • aac aajt gas err cc? ggs c-ca 

Asn Asn Glu Leu Pre civ Ale 



GAA 
Glu 


r-.r 

ryr 


443 
GAG 

Gin 


TAG 

rvr 


a*g 

LV3 


G2£ 
GlU 


GAS? 
Asp 


433 
GAA 
GlU 


GIA TGG 
val irp 


GAG 
GlU 


GAG 
GlU 


473 

agg 


PAG 

Lys 


ccr 
Pro 


•S.C AAG 
Tyr Lys 


433 
ACT 

Ear 


err 


Gin 


act 


cag 
Gin 


303 
CCC 


ccr 
?ro 


GAT 
Asp 




51= 

ACT AAG 

Sar Lys 


AAC 

Asn 


AAA 


aag 


AAA 
lys 


533 
cag 
Gin 


agg aag 

Arr Lys 


gga 

Gly 


err 

PT3 


£43 

CAG 

Gin 


Gin 


■» ^ 

Lys 


Star 


C-A^: 

Gin 


563 
Sir 


ere 


Gin P*s 


GAT 
Asp 


57c 
G.--r. 
GlU 


Gin 


^ **** 

n~ 


Civ? 
LSU 


393 
AAG AAG 
lys Lys 


GCA 

Ala- 


AGA 


AGA 


aag 


60S 

Cr-A 

Gln 


TGG 

Trp 


ATT 
lie 


GA 
Glu 


CCC 


CGG ?A— 

Arc A=n 


TGT 


GCC 

Ala 


aga 

to? 


cr-G 

AC? 


TAG 
TVT 


err "AAA GIG 
Leu lys ,Vsl 


553 
GAC 


rtc 

PIS 


GCA 

aa 


GAT 
A3? 


in 

lie 


655* 
GGC 
Gly 


igg 


mc 
Ssr 


SS3 

GAA TGG "ATT 
Glu rrp* lie 


ATT 

lis 


TCC 
s~r 


CCC 

Pro 


AAG 

Lys 


6=3 

rcc nc gat 

S«ar P?.e Asp 






713 










72S 








743 






73= 





ccc sac rc-e rcc gga gcc- tgc cag tic ecc azc- cca aag gtascc^ig r:.-^. 
Ala ryr TVr Cys S^r Gly Ala cys Gin Sh- Pro >*rr Pro Lys 

776 756 

tgiccitccc aiticc^jg 



234 254 304 "313 

cta-.cctgig rrercccrrr rcrrrccz^is rcr Tie- A^r- cca tca a^t c-.c gct acc 

Ssr L-u Lv-s rro Ser r=n :-^.s Ala *r.ir 

334 349 3=4 37= 

AIC CAG ".-GT A-HA GTG AGA GCI GIG GGG GIC GIC CCT GGA ASC CCC GAG CCT TGC 

Il£ Gin £er lis Val Arj Ala Val Gly Val Val* Prr: Gly lis ?rc Glu Pro Cys 

3S4 4C9 424 435 

TGT GIG CCA GAA AJ^G ATG TCC TOA CIC i^-C .^C- TTA 1TC TTT G^T GAA. AAC 
O/s Val Pro- Glu Lys S^r Ser Lsu Ser lis Lsu Fns. Pr.s Asp Glu A-sr. Lys 

434 A5r 4*4 

A-r GTG GTA GIT AAA GIA TAT CCA AAC ATG AC-. GIA d-G TCI TGT GCT IC-C ALA 
i=n Val Val Ltj Lys Val Tyr Pre Asr. Tnr Val Glu Ser Cys Ala Cys .^rc 

503 513 523 533 

^ACCIGGIG CIC-GAIGCTT AACICAAICG , 



(edit procede comprenant le criblage d'une banque genomique recombinante de bovin avec des sondes 
comprenant des pools d'oligonucleotides concues sur la base des sequences d'acides amines partielles et I'iso- 
iement d'un clone positif. 

2. Procede selon la revendication 1 , dans lequel le gene codant pour le BMP-3 a la sequence d'acides amines donnee 
dans la revendication 1 . 

3. Procede selon la revendicacton 1 , dans lequel un gene codant pour une proteine presentant au mois la proprtete 



26 



EP 0 688 869 B1 



du BMP-3 conduction de la formation de Pos comprend une sequence d'ADN : 

a) qui d iff 6 re d'une sequence d'ADN obtenue selon la procede de la revendication 1 par la sequence de codons 
du fait de la degenerescence du code genetique ; 

b) qui s'hybride avec une sequence d'ADN obtenue selon le procede de la revendication 1 ou la section a) ci- 
dessus dans des conditions d'hybridation stringentes ; ou 

c) represente un fragment ou une variation allelique d'une sequence d'ADN obtenue selon le proced6 de la 
revendication 1 , 

4. Procede selon la revendication 3, dans lequel la sequence d'ADN code pour le BMP-3 humain. 

5. Procede selon la revendication 3 ou 4, dans lequel la sequence d'ADN est une sequence d'ADN genomique. 

6. Procede selon la revendication 3 ou 4, dans lequel la sequence d'ADN est une sequence d'ADNc. 

7. Procede de preparation d'un vecteur recombinant comprenant insertion du gene ou de la sequence d'ADN ob- 
tenue selon un procede de Tune quelconque des revendications 1 a 6 en association operatoire avec une sequence 
de co nt role d'expression dans un vecteur adapte. 

8. Cellule transformed avec un vecteur prepare selon le procede de la revendication 7. 

9. Cellule selon la revendication 8, qui est une cellule mammifere. une cellule bacterienne, une cellule d'insecte ou 
une cellule de levure. 

10. Cellule selon la revendication 9, qui est une cellule CHO, 

11. Procede de production d'une proteine presentant les proprietes du BMP-3, comprenant les etapes de culture dans 
un milieu de culture adapte de la cellule de la revendication 9 et d'isolement de ladite proteine dudit milieu de culture. 

12. Proc6d6 de preparation d'une composition pharmaceutique comprenant la combinaison de la proteine produite 
selon le proced6 de la revendication 11 avec un vehicule pharmaceutiquement acceptable. 

13. Proced6 selon la revendication 12, dans lequel la composition pharmaceutique comprend en outre une matrice 
capable de delivrer la composition sur le site du defaut de Pos ou du cartilage et la fourniture d'une structure pour 
induire la formation d'os ou de cartilage. 

14. Proced6 selon la revendication 1 3, dans lequel ladite matrice de ladite composition pharmaceutique comprend de 
I'hydroxyapatite, du collagene, de I'acide polylactique ou du phosphate tricalcique. 

15. Utilisation d'une proteine selon la revendication 11 pour la preparation d'une composition pharmaceutique pour 
induire la formation d'os ou de cartilage. 

16. Gene codant pour le BMP-3 bovin comprenant la sequence d'ADN suivante : 



27 



EP 0 688 869 B1 



10 



15 



353 " 353 403 413 433 

^ cz^icr?,cz-z gggicttict gcctcigcat - aac aat g^g err* ccr gcg gca 

^ Asn Aj=H GlU LSU Src Giy 

443 453 473 4co 

ta^ cag tag aag gag gxp g-a gta tgc- 'gag agg aag ccr -tag aag act 
Glu Tyr Gin Tyr Lys Giu Asp Giu val Trp Glu c-Iu Arg Lys Fro Tyr Lys Thr 

=03 513 533 

c^ 1 or- act cag ccc ccr gat aag agt aag aag aaa aag aaa cag ask aag gga 
tIm Gin. Est Gir. >3?roPi? Lys Ser Lys £sn Lys Lys Lys Gin Arc Lys Giy 

zs S '5=3 57c 5S3 

CTT C--- C-G .-AG AGT CAG ACG CIC CAG TIT GAT GAA CAG ACC CIS AAG AAG GCA 
-^o Gin Gin LV3 Ser Gin Gar Leu Gin Fne Ajrp Glu Gin Ear leu Lys Lys Ala 



30 



40 



45 



50 



20 60S 623 £3S 

AG.-. ASA. AAG GA CC-G ATT GAA. CCC CCG AAT TGT GCC .i^A CGC- T-.C CTT "AAA GIC- 
Arg Arg Lys C-lr. Trj: lie Glu ?ro Arr Aen Cys Ala Arr Arg Tyr Leu Lys .Val 

£33 ■ S« 633 .6=3 

25 GAC TIG GCA GAT .-IT GGC TGG AGC G-A CGG ATT ATT TCC CCC AAG TCC TIC CAT 

Asp xrjz Ala Aj-rr Ha C-Iy Cr? Ser Glu Cr?- lis lis Ser Pre Lys Ser Trje .Asp 

713 ' 72= 743 75£ 76 c 

GCC TAT T?.C CGC TCC GG-, GCC- TGC CAG TIC CCC ATG CCA AAG GTAGCCATTG TITTTIGrcC- 
Ale Tyr Tyr Cys Ser Giy Ala Cys Glr. *S:e >ro ><rr ^ro Lys 

776 7S6 

tgtccttccc agiicl^ag ;. et 

35 234 254 . 304 " 315 . 

dAA.CX.TGIG TTCT-CCCm TCGTTCCIAG TCI TTG AAG CCA TCA AAT C-G GCT ACC 

Ser Leu Lys ?rc Ser Aen >3is A-la rir 



334 349* ' 3 = 4 379 

AGC C-G AGT ASA GIG AGA. GCT GIG CGG GIC GTC CCI GGA AGC CCC G^G CCT TGC 

lie Gin Ser Us Val Arg Ala Val GiyVel val Pro Giy II a ?rc Glu ?rc Cys 

3=4 405. 424 433 

TGT GIG OCX GP-A ArG >TG TCC TC-. CIC AT-C TI>. TIC TTT G-T GAA. AA-C AAG 

Cys Vel Pro Glu ufs KTT Ser S=r Leu £ar lis Leu Fna. rt:s Asp Glu Asr. Lys 

^54 4" 434 

AAT GIG GIA CTT AAA. GXA TAT CIA AA.C ATG AC-. GTA G-G TCI TGI GCI TGC AGA. 
Asn Vci val L^u Lv-s Val T> ^ro A=r ; MTT Tnr Vel Giu Ser Cys Ale Cys Arr 

=03 513 523 533 

IG AAGAA-CTCAT CICGAIGCTT AACICAATCC. 



55 17. G6ne codant pour le BMP-3 bovin ayant la sequence d'acides amines donn6e dans la revendlcation 16. 

18. G6ne codant pour une protdine pr6sentant au moins la propri6t6 du BMP-3 d'induction de la formation de I'es et 
comprenant une sequence d'ADN : 



28 



EP 0 688 869 B1 



a) qui diff&re d'une sequence d'ADN de la revendication 16 par la sequence de codons du fait de la d6g£n6- 
rescence du code genetique ; 

b) qui s'hybride avec une sequence d'ADN de la revendication 16 ou de la section a) ci-dessus dans des 
conditions d'hybridation stringentes ou 

c) represente un fragment ou une variation allelique d'une sequence d'ADN selon la revendication 16. 

19. Sequence d'ADN selon la revendication 18, qui code pour le BMP-3 humain. 

20. Sequence d'ADN selon la revendication 18 ou 19, qui est une sequence d'ADN genomtque. 

21. Sequence d'ADN selon la revendication 18 ou 19, qui est une sequence d'ADNc. 

22. Vecteur contenant le gene ou la sequence d'ADN selon I'une quelconque des revendications 1 6 a 21 en association 
operatoire avec une sequence de controle d'expression. 

23. Cellule transformee avec un vecteur selon la revendication 22. 

24. Cellule selon la revendication 23, qui est une cellule mammifere, une cellule bacterienne, une cellule d'insecte ou 
une cellule de levure, 

25. Cellule selon la revendication 24, qui est une cellule CHC. 

26. Proteine presentant les proprietes du BMP-3 qui est encodee par le gene ou la sequence d'ADN selon Tune quel- 
conque des revendications 1 a 21 . 

27. proteine presentant les proprietes du BMP-3 qui est produite par les etapes de culture dans un milieu de culture 
adapte d'une cellule transformee avec un vecteur d'expression comprenant un gene ou la sequence d'ADN selon 
I'une quelconque des revendications 1 a 21 1 et de recuperation de ladite proteine dudit milieu de culture. 

28. Composition pharmaceutique comprenant la proteine selon la revendication 26 ou 27 et un vehicule pharmaceu- 
tiquement acceptable. 

29. Composition pharmaceutique selon la revendication 29, comprenant en outre une matrice capable de deiivrer la 
composition sur le site du defaut de Pos ou du cartilage et la fourniture d'une structure pour induire la formation 
d'os ou de cartilage. 

30. Composition pharmaceutique selon la revendication 29, dans laquelle ladite matrice comprend de Phydroxyapatite, 
du coliagene, de I'acide polyiactique ou du phosphate tricalcique. 



29 



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.