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© 



J 



Europaisches Patentamt 
European Patent Office 
Office europeen des brevets 



© Publication number: 



0169 016 B1 



© 



EUROPEAN PATENT SPECIFICATION 



© Date of publication of patent specification: 04.10.95 © Int. CI. 6 : C07K 14/00, A61 K 38/00, 

A61L 27/00 

© Application number: 85304848.6 
© Date of filing: 08.07.85 



© Polypeptide cartilage-inducing factors found in bone. 



® 


Priority: 16.07.84 US 630938 




formed cells" 




Date of publication of application: 


© 


Proprietor: CELTRIX PHARMACEUTICALS, INC. 




22.01.86 Bulletin 86/04 




2500 Faber Place 








Palo Alto 


© 


Publication of the grant of the patent: 




CA 94303 (US) 




04.10.95 Bulletin 95/40 










© 


Inventor: Seyedin, Saeid 


© 


Designated Contracting States: 




645 Cheshire Way 




AT BE CH DE FR GB IT LI LU NL SE 




Sunnyvale 








California 94087 (US) 




References cited: 




Inventor: Thomas, Thomas 




EP A- 0 182 483 




3000 Cowell Boulevard, No. 275 




WO-A-84/01106 




Davis 




US-A- 4 434 094 




California 95616 (US) 




US-A- 4 440 750 








PROCEEDNGS OF THE NATIONAL ACADEMY 


© 


Representative: Harrison, David Christopher 




OF SCIENCES OF THE USA, vol. 80, Novem- 




et al 




ber 1983, pages 6591-6595, National Acad- 




MEWBURN ELLIS 




emy of sciences of the USA, Washington, 




York House 




D.C. US; T.K.SAMPATH et al. 




23 Kingsway 








London WC2B 6HP (GB) 




NATURE, vol. 316, 22nd August 1985, pages 








701-705, Macmillan Journals Ltd,London, GB; 








R.DERYNCK et al.: " Human transforming 








growth factor-betacomplementary DNA se- 








quence and expression in normal and trans- 







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). 



Rank Xerox (UK) Business Services 

(3.10/3.0S/3.3.3> 



EPO 169 016 B1 



Description 

The present invention relates to protein chemistry. More particularly, it relates to two proteins that are 
found in bone, are co-factors for inducing cartilage formation, and are also active in the beta type 

5 transforming growth factor (TGF-/3) assay. These polypeptides are sometimes referred to herein as 
cartilage-inducing factors (CIFs). 

Human platelet/human placenta/bovine kidney-derived TGF-£s are described in international Patent 
Application WO-A-84/01 1 06 and in EP-A-01 28849. 

U.S. 4,434,094 reports the partial purification of a bone generation-stimulating, bone-derived protein 

70 factor 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 characterized as having a molecular weight below about 30,000 daltons 
and as tracking the purification process described. The proteins of the current invention were purified to 
homogeneity using a purification procedure that is similar in part to that disclosed in U.S. 4,434,094. 

75 EF^A-105014 describes another TGF-/J. The structure of one such, there designated TGF-/81 is 
elucidated and it is suggested that the TGF-0's could be used for wound healing. TGF-01 is the 
polypeptide designated CIFA in the present application. 

The invention provides a process for obtaining these polypeptides in substantially pure form from bone. 
Both CIFs are also active when combined with epidermal growth factor (EGF) in the TGF-/S assay for in vitro 

20 induction of anchorage-independent growth of normal rat kidney (NRK) cells in soft agar. This assay is 
sometimes referred to herein as the TGF-j9 assay. In this regard the presence in bone of proteins having 
activity in the TGF-0 assay has not been reported previously. One of the CIFs, designated CIF-A, has a 
partial (30 amino acids) N-terminal sequence that is identical to that reported in the literature for human 
placenta-derived TGF-/S. The other CIF, designated CIF-B, has a partial N-terminal sequence that is 

25 different from the human placenta-derived TGF-0 sequence and is claimed per se, and its structure 
elucidated, in the present invention. 

Accordingly, one aspect of the invention is a polypeptide cartilage-inducing factor, which factor: 

(a) is found in mammalian bone; 

(b) is a co-factor for inducing cartilage formation; 
30 (c) has activity in the TGF-0 assay; 

(d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS-PAGE; 

(e) is isolatable by a process as claimed in claim 1 or claim 2; and 

(f) does not have the N-terminal sequence: 

35 Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser (Ser) Thr-Glu-Lys- 

Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys- 

Asp-Leu-Gly-Trp- . 

AO 

The process for isolating the two factors from bone is characterized by the following steps: 
(a) treating demineralized bone (DMB) with a chaotropic (dissociative) extractant that solubilizes non- 
fibrous proteins; 

45 (b) subjecting the extract from step (a) to gel filtration to recover a fraction containing proteins of 
molecular weight 10,000-40,000 daltons; 

(c) adsorbing the fraction from step (b) onto a carboxymethyl cellulose cation exchanger at approxi- 
mately pH 4.5-5.5 under denaturing conditions; 

(d) eluting the adsorbed fraction from the cation exchanger with a sodium chloride gradient; 

so (e) subjecting the portion of the eluate from step (d) eluting at approximately 150 to 250 mM NaCI to 
reverse phase high performance liquid chromatography (RP-HPLC) or a nondenaturing gel elec- 
trophoresis; and 

(f) recovering the factors from the RP-HPLC or gel electrophoresis. 

An implant composition for inducing chondrogenesis/osteogenesis is characterized in that it contains an 
55 effective amount of CIFB, or both of the above-described CIFs, optionally together with a chon- 
drogenic/osteogenic co-factor. 

An implant composition for promoting connective tissue deposition is characterized in that it contains an 
effective amount of at least CIFB and is substantially free of any activating agent or chondrogenic co-factor. 



2 



EP0 169 016 B1 



Brief Description of the Drawings 
In the drawings: 

Figure 1 is a graph of the optical densities (absorbances) (280 nm) and in vitro chondrogenic activities of 
6 the gel filtration fractions of the example (section C), infra; 

Figure 2 is a graph of the optical densities (280 nm) of eluate fractions from the preparative ion 
exchange chromatography of the example (section D), infra; 

Figure 3 is a graph of the UV absorbance and electrophoretic profiles of peaks A (CIF-A) and B (CIF-B) 
of the preparative RP-HPLC of the example (section E), infra; 
70 Figure 4 is a graph of the results of the enzyme-linked immunosorbent assays (ELISAs) for in vitro 
chondrogenic activity of the CIF-A and CIF-B obtained from the RP-HPLC of the example (section E), 
infra; 

Figure 5 is a graph of the results of the ELISAs of the acid-urea gel electrophoresis fractions (section F) 
of the example, infra; and 

75 Figure 6 is a graph of the results of the TGF-0 assays described in section I of the example, infra. 

Modes for Carrying Out the Invention 

The polypeptides of the invention were isolated from bone. The polypeptides have been only partially 
20 sequenced at this time. In view of this and since the complete amino acid sequence of TGF-0 has not been 
reported, the primary structure relationships between the CIFs of the invention and TGF-0 are not known 
completely. 

The polypeptides of the invention are co-factors for inducing cartilage formation. In view of their 
chondrogenic activity and the mode of endochondral bone formation, they are also expected to play a role 

25 in osteogenesis. The polypeptides are also active in the TGF-£ assay and have been found to promote 
connective tissue deposition independently of association with TGF-0 activating agents. 

In view of the showings that bone inductive proteins from human, monkey, bovine and rat are 
nonspecies-specific in their abilities to produce endochondral bone in xenogeneic implants (Sampath, T. K., 
et al, Proc Natl Acad Sci (USA) (1983) 80:6591) and that human platelet/human placenta/bovine kidney- 

30 derived TGF-0 is nonspecies-specific between rodents, cattle and humans, it is believed that the polypep- 
tides of this invention have been highly conserved among mammalian species (i.e., polypeptides from 
different mammalian species have amino acid sequences that vary, if at all, in one or more amino acid 
residue additions, deletions, or substitutions that do not affect the nonspecies-specific activity of the 
molecule adversely). In this regard the term "substantially equivalent" as used to describe a polypeptide is 

35 intended to mean polypeptides, whether native or synthetic and regardless of species or derivation, that 
have the same amino acid sequence as a CIF, and polypeptides of substantially homologous but different 
amino acid sequence, which difference^) does not affect nonspecies-specific activity adversely. Accord- 
ingly, the polypeptides may be derived from bone and perhaps other tissue of diverse animal origin or 
made by recombinant DNA technology. Porcine or bovine long bone are preferred native sources of the 

40 CIFs because of the ready availability of such bone and the high levels of the polypeptides in bone. 

The procedure for isolating CIF from bone is as follows. The bone is first cleaned using mechanical or 
abrasive techniques, fragmented, and further washed with, for example, dilute aqueous acid preferably at 
low temperature, and then defatted by extraction with a lipophilic solvent such as ether or ethyl acetate. The 
bone is then demineralized by removal of the calcium phosphates in their various forms, usually by 

45 extraction with stronger acid. The resulting preparation, a demineralized bone, is the starting material for the 
preparation of the polypeptides of the invention. 

The initial extraction is designed to remove the non-fibrous (e.g., non-col lagenous) proteins from the 
demineralized bone. This can be done with the use of chaotropic agents such as guanidine hydrochloride 
(at least about 4 molar), urea (8 molar) plus salt, or sodium dodecylsulfate (at least about 1% by volume). 

so The extraction is preferably carried out at reduced temperatures in the presence of a protease inhibitor to 
reduce the likelihood of digestion or denaturation of the extracted protein. Examples of protease inhibitors 
that may be included are phenylmethylsulfonylfluoride (PMSF) sodium azide, N-ethyl maleimide (NEM), 
benzamidine, and 6-aminohexanoic acid. The pH of the medium depends upon the extractant used. The 
process of extraction generally takes on the order of about 4 hr to one day. 

55 After extraction, the extractant may be removed by suitable means such as dialysis against water, 
preceded by concentration by ultrafiltration if desired. Salts can also be removed by controlled elec- 
trophoresis or by molecular sieving. It is also preferred to maintain a low temperature during this process so 
as to minimize denaturation of the proteins. Alternatively, the extractant need not be removed, but rather the 

3 



EP0 169 016 B1 



solution need only be concentrated, for example, by ultrafiltration. 

The extract, dissolved or redissolved in chaotropic agent, is subjected to gel filtration to obtain fractions 
of molecular weight below about 40,000 daltons, thus resulting in a major enhancement of purity. Gel sizing 
is done using standard techniques, preferably on a Sephacryl column at room (10-25 # C) temperature. The 

5 low molecular weight fraction is then subjected to ion exchange chromatography using carboxymethyl 
cellulose (CMC) at approximately pH 4.5-5.5, preferably about 4.8, in the presence of a nonionic chaotropic 
agent such as urea. Other cation exchangers may be used, including those derived from polyacrylamide 
and cross-linked dextran; however cellulosic cation exchangers are preferred. Of course, as in any ion 
exchange procedure, the solution must be freed of competing ions before application to the column, and is 

w eluted in an increasing salt concentration gradient as is understood in the art. The fraction eluting from CMC 
at about 150 to 250 mM NaCI contains the CIFs. 

The eluate fraction from the cation exchange chromatography is then subjected to RP-HPLC or a 
nondenaturing gel electrophoresis for final purification. Standard RP-HPLC techniques and gel elec- 
trophoresis techniques are used. Exemplified below is a commercially available RP-HPLC column using a 

75 commercially prescribed RP-HPLC protocol. This final purification yields the two polypeptides in substan- 
tially pure form. "Substantially pure" means that a polypeptide contains less than about 5% by weight 
contaminants. 

Example 

20 

The following example is intended to illustrate the process for purification as applied to a particular 
sample. It is not intended to limit the invention. 

A. Preparation of Demineralized Bone 

25 

Bovine metatarsal bone was obtained fresh from the slaughterhouse and transported on dry ice. The 
bones were cleaned of marrow and non-bone tissues, broken in fragments smaller than 1 cm diameter, and 
pulverized in a mill at 4 ° C. The pulverized bone was washed twice with 9.4 liters of double distilled water 
per kg of bone for about 15 min each, and then washed overnight in 0.01 N HCI at 4°C. Washed bone was 
so defatted using 3X3 volumes ethanol, followed by 3 X 3 volumes diethylether, each washed for 20 min, and 
all at room temperature. The resulting defatted bone powder was then demineralized in 0.5 N HCI (25 l/kg 
defatted bone) at 4°C; The acid was decanted and the resulting DMB washed until the wash pH was 
greater than 4, and the DMB dried on a suction filter. 

35 B. Extraction of Noncollagenous Proteins 

The DMB as prepared in paragraph A was extracted with 3.3 I of 4 M guanidine-HCI, 10 mM 
ethylenediaminetetraacetic acid (EDTA), pH 6.8, 1 mM PMSF, 10 mM NEM per kg for 16 hrs, the 
suspension suction filtered and the non-soluble material extracted again for 4 hrs. The soluble fractions 
40 were combined and concentrated at least 5-fold by ultrafiltration using an Amicon ultrafiltration (10K) unit, 
and the concentrate dialyzed against 6 changes of 35 volumes cold deionized water over a period of 4 
days, and then lyophilized. All of the procedures of this paragraph were conducted at 4°C except the 
lyophilization which was conducted under standard lyophilization conditions. 

45 C. Gel Filtration 

The extract from paragraph B, redissolved in 4 M guanidine-HCI, was fractionated on a Sephacryl S-200 
column equilibrated in 4 M guanidine-HCI, 0.02% sodium azide, 10 mM EDTA, pH 6.8. Fractions were 
assayed by their absorbance at 280 nm and chondrogenic activity by ELISA (described below) and the 
so fractions were combined as shown in Figure 1 . Fraction F2 of Figure 1 , constituting a low molecular weight 
(LMW, 10,000-40,000 daltons) protein fraction possessing the greatest activity was dialyzed against 6 
changes of 180 volumes of deionized water and lyophilized. All operations except lyophilization and dialysis 
(4 • C) were conducted at room temperature. 

55 D. Ion Exchange Chromatography 

Fraction F2 from paragraph C was dissolved in 6 M urea, 10 mM NaCI, 1 mM NEM, 50 mM sodium 
acetate, pH 4.8 and centrifuged at 10,000 rpm for 5 min. The supernatant was fractionated on a CM52 (a 

4 



EP0 169 016 B1 



commercially available CMC) column equilibrated in the same buffer. Bound proteins were eluted from the 
column using a 10 mM to 400 mM NaCI gradient in the same buffer, and a total volume of 350 ml at a flow 
rate of 27 ml/hr. Three major fractions, designated CM-1, CM-2, and CM-3, were collected as shown in 
Figure 2. CM-2 and CM-3 were eluted at about 150 to 250 mM NaCI. Each fraction was dialyzed against 6 
5 changes of 110 volumes of deionized water for 4 days and lyophilized. All of the foregoing operations were 
conducted at room temperature except dialysis (4°C). 

E. RP-HPLC 

70 The lyophilized fractions CM-2 and CM-3 from HD were combined and dissolved in 0.1% trifluoroacetic 
acid (TFA) and aliquots of the solutions loaded onto a Vydac C18 RP-HPLC column (4.6 mm ID x 25 cm) 
and washed with 0.1% TFA for 5 min at 1 ml/min. The eluting solvent was a 0%-60% acetonitrile gradient in 
0.1% TFA at a rate of 2%/min. 

Two peaks were obtained from the RP-HPLC of combined CM-2 and CM-3--peak A at about 29.5 min 
75 and peak B at about 31.2 min. Figure 3 shows the absorbance and electrophoretic profiles (reduced and 
nonreduced) of peaks A and B. The proteins of these peaks were designated CIF-A and CIF-B, respectively. 
The proteins were stored in 0.1% TFA/acetonitrile eluting solution at -20 *C until used. 

F. Alternate Purification by Gel Electrophoresis 

20 

The combined lyophilized fractions CM-2 and CM-3 were fractionated by electrophoresis on an acetic 
acid-urea gel using the general procedure of Paynim, S. and Chalkley, R., Arch Bioch Biophys (1969) 
130:337-346. 

25 G. Assay for In Vitro Chondrogenic Activity 

The presence of the desired protein in fractions during purification was confirmed using an in vitro 
assay for the production of cartilage-specific proteoglycans (PG), the identity of which was confirmed by 
ELISA. This assay is an agarose gel culture model using mesenchymal cells isolated from rat fetal muscle. 
30 It assesses the ability of the samples to induce the production of PG. The correlation between in vitro 
cartilage induction and in vivo bone formation has been shown by Seyedin, S., et al, J Cell Biol (1983) 
97:1950-1953. 

The cell culture was prepared by removing muscle tissue aseptically from the upper limbs of nineteen- 
day-old Sprague Dawley rat fetuses, mincing the tissue and culturing it in Eagle's . Minimum Essential 

35 Medium (MEM) with 10% fetal bovine serum (FBS) and 50 units penicillin, 50 ug streptomycin per ml. 
Cellular outgrowth usually reached confluency within one week, whereupon cells were trypsinized, split 1:2 
and used for experimentation within the first three passages. 

The cells were placed in agarose gel cultures either with control medium or with samples to be tested. 
The procedure was basically that of Benya, et al, Cell (1982) 30:215. Briefly, the cell monolayers were 

40 harvested by trypsinization, counted on a hemocytometer, and resuspended at two times the final cell 
concentration in the medium with or without the protein fraction to be tested. The control medium was either 
Hams F-12, Dulbecco's Minimum Essential Medium (DMEM) or CMRL 1066 (Gibco) each containing 10% 
FBS and antibiotics. The test protein fractions in 0.01 N HCI were diluted directly to the desired 
concentration of test protein diluted with an ^qual volume with 1% low melting agarose (Bio-Rad, #162- 

45 0017) in F-12, and 0.2 ml of the dilution was plated on 17 mm wells coated with 0.15 ml of 1% high melting 
(Bio-Rad, #162-0100) agarose. The resulting cultures were incubated at 37 *C for 5 min, chilled at 4'C for 
10 min, and then overlayed with 1 ml of the corresponding medium (control or test protein). The cells were 
then cultured in a humidified atmosphere of 5% CO2, 95% air and fed every 3-4 days thereafter by a 
complete change with control medium. After 7 days the cultures were frozen and stored at -80 *C before 

50 assay. 

The cultures were assayed by thawing at 4*C, homogenizing in 4 M guanidine-HCI with 50 nM Na 
acetate, 13 mM EDTA, 6 mM NEM, and 3 nM PMSF at pH 5.8, and extracting by shaking overnight at 4*C. 
The supernatant fraction from centrifugation at 25,000 X g for 40 min at 4 • C was dialyzed overnight at 4 • C 
against 50 volumes 0.2 M NaCI, 50 mM Tris, pH 7.4. The supernatant was assayed for PG by ELISA as 
55 described by Renard, et al, Anal Biochem (1980) 104:205, and in U.S. 4,434,094. 

Briefly, for the ELISA, antiserum to PG was raised in rabbits using standard techniques which showed 
no cross-reactivity with hyaluronic acid or PG extracted from rat bone. Purified PG (Seyedin, S., et al, 
supra) from Swarm rat chondrosarcoma tissue was used as standard antigen. The dialyzed samples were 



5 



EP0 169 016 B1 



diluted 1 :1 (v/v) in phosphate-buffered saline (PBS) with 0.05% Tween 20, 1 mg/ml bovine serum albumin 
(BSA), pH 7.2 for assay. Horseradish peroxidase conjugated goat anti-rabbit IgG (Tago) was the second 
antibody with o-phenylenediamine as substrate. 

The results of the ELISAs of CIF-A and CIF-B purified by RP-HPLC are shown in Figure 4. As indicated 
5 there, the sensitivity of the assay is within 1 to 5 ng/ml of culture media. The results of the ELISAs on the 
gel slices of section F are shown in Figure 5. These results are comparable to the results for CIF-A and 
CIF-B (corresponding to gel slices 7 and 6) from the RP-HPLC. 

H. Characterization of Purified CIF-A and CIF-B 

70 

CIF-A was shown to be a 25,800 dalton protein which on reduction, yielded a 14,800 dalton polypeptide 
by measurements of the mobilities of the proteins in a 15% Laemmli polyacrylamide gel in SDS (Figure 3) 
as described by Laemmli, U. K., et al, Nature (1970) 227:680. It Is well understood that molecular weights 
so determined are approximate and their values are dependent on the method used. The conformation of 

75 the protein affects its mobility in this system, and, therefore, the molecular weights obtained will be similar, 
but not necessarily identical when determined by other procedures. The presence of a single band in the 
profile of the reduced protein indicates the protein is probably a dimer composed of two polypeptide chains 
having substantially equivalent amino acid sequences (i.e., it is a homodimer). The discrepancy between the 
measured weights of dimer and the individual chains is an artifact of the procedure. 

20 CIF-A maintained its activity in the ELISA assay of paragraph G above even after heating for 3 min at 
100 °C in PBS, after treatment with collagenase for 2 hrs at 37 *C in 0.1 M Tris, pH 7.4, 5 mM CaCI 2 , 0.02 
mM PMSF with a ratio of collagenase to protein of 400 units/mg protein, and after treatment with trypsin for 
2 hrs at 37 *C in 50 mM Tris, pH 7.4, 10 mM CaCfe with a ratio of trypsin to protein of 100 units/mg of 
protein. However, the protein lost activity after treatment for 1 hr at room temperature in PBS containing 5 

25 mM dithiothreitol (DTT), which would effect reduction of disulfide linkages. Similarly, SDS treatment or 
fractionation on SDS-PAGE resulted in inactivation of the protein, presumably due to denaturation or 
complexing by the SDS. The partial amino acid composition of CIF-A is shown in Table 1. 

Table 1 

30 



Amino Acid 


Mols/100 Mols Recovered 


Asp 


9.2 


Glu 


9.2 


Ser 


7.0 


His 


2.7 


Gly 


16.5 


Thr 


2.7 


Arg 


5.9 


Ala 


6.6 


Tyr 


3.2 


Met 


0.0 


Val 


7.5 


Phe 


3.0 


lie 


3.9 


Leu 


8.6 


Lys 


13.9 


Pro 


ND 


Cys 


ND 


Trp 


ND 



55 



6 



EPO 169 016 B1 



Amino acid sequence analysis of CIF-A showed that it has the following single N-terminal sequence: 

15 10 
5 Ala-Leu-Aep-Thr-Asn-Tyr-Cys-Phe-Ser (Ser )Thr-Glu-Ly6-Asn- 

15 20 25 

to -Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-ABp-Phe-Arg-Lys-Asp-Leu- 

30 

-Gly-Trp-. 

75 

( ) = suspected 

This N-terminal sequence is identical to that reported for human placenta-derived TGF-/?. 

CIF-B had a slightly different molecular weight (26,000) as measured by the same procedure. This 
20 difference may be caused by the procedure. Accordingly, both proteins are considered to have a molecular 
weight of approximately 26,000 daltons as measured by SDS-PAGE. On reduction the protein of peak B 
showed a single band at approximately 14,200 daltons indicating that it, too, is probably a homodimer. It 
has the amino acid composition set forth in Table 2. 

25 Table 2 



Amino Acid 


Mols/100 Mols Recovered 


Asp 


12.0 


Glu 


8.5 


Ser 


10.6 


His 


0.9 


Gly 


22.0 


Thr 


0.0 


Arg 


4.3 


Ala 


6.7 


Tyr 


1.9 


Met 


0.0 


Val 


2.4 


Phe 


3.0 


lie 


2.2 


Leu 


8.2 


Lys 


17.3 


Pro 


ND 


Cys 


ND 


Trp 


ND 



7 



EPO 169 016 B1 



Amino acid sequence analysis indicated CIF-B has a single N-terminal sequence as follows: 

15 10 
5 Ala-Leu- Asp-Ala- Ala-Tyr-Cye-Phe-Arg-Asn-Val-Glu-Asp-Afin- 

15 20 25 

10 -Cye-Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lyfi-Arg-Asp-Leu- 

30 

-Gly-Trp- . 

76 

Its other properties, as qualitatively assessed, were similar to those set forth above for CIF-A. 
I. Assay for TGF-ff Activity 

20 

CIF-A and CIF-B were tested in the TGF-/5 bioassay. The assay was performed as described in 
Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture (1984) 
pp 181-194, Alan R. Liss, Inc. The results of the assay are shown in Figure 6. As depicted, both proteins 
exhibit a clear dose-response in the assay and require the presence of an activating agent (EGF) to be 

25 active. The levels of activity are comparable to the reported levels of activity of human platelet/human 
placenta/bovine kidney-derived TGF-0. 

The ability of the CIFs to withstand treatment with trypsin without losing activity may make it possible to 
isolate them from demineralized bone powder by means of enzymatic digestion. In such a process the 
demineralized bone powder is digested with an aqueous solution of trypsin and/or other proteases that do 

30 not degrade the proteins of interest under conditions at which such enzymes are active. This treatment 
digests the majority of other protein components in the powder. The proteins of interest may be purified 
from the resulting digest using one or more of the fractionation techniques described above (gel filtration, 
ion exchange chromatography, RP-HPLC or nondenaturing gel electrophoresis). Depending upon the extent 
to which the CIFs are released from the bone matrix and not complexed with other materials, use of 

35 soiubilizing agents may be avoided. In this regard the pure proteins are substantially soluble in water. 

The CIFs are useful for inducing cartilage/bone growth for repairing, replacing or augmenting car- 
tilage/bone tissue in animals, including humans. Chondrogenically/osteogenically effective amounts of the 
proteins may be combined with chondrogenic/osteogenic co-factors found in bone and formulated with 
pharmacologically and physiologically acceptable fluid or solid carriers such as purified collagen for 

40 implantation. The weight ratio of active protein to carrier will typically be in the range of 1:50 to 1:1000. The 
implants may be placed at a predetermined site in the patient by conventional surgical techniques, 
including injection as an active ingredient. Collagenous implants containing only CIFB as an active 
ingredient (i.e., free of any activating agent or co-factor) at CIF to carrier weight ratios above about 1 :6000 
promoted collagenous connective tissue deposition. 

45 The CIFs may also be used in the same manner as human platelet/human placenta/bovine kidney- 
derived TGF-/9 to promote (provoke and sustain) non-species specific cellular proliferation. In such 
application one or both of the CIFs is combined in approximately stoichiometric proportions with a TGF-0 
activating agent such as an EGF or a TGF-o. Clinical applications of the cell proliferation activity of these 
compositions include topical administration for burn or wound healing, implantation for tissue augmentation, 

so and systemic administration for internal wound healing. In such uses the CIF and activating agent will be 
formulated in amounts sufficient to induce cell proliferation with pharmaceutical^ acceptable carriers that 
are adapted for the particular mode of administration. Topical dosage forms will typically be formulated as 
sprays, gels, ointments, or salves. Implants will be formulated as injectables. Systemic dosage forms may 
be formulated for enteral administration (e.g., liquids, pills, tablets) or for parenteral injection. The dosages 

55 used in such applications cannot be specified because of the nature of cell proliferation and the variability in 
wounds and other traumata. 



8 



EPO 169 016 B1 



Claims 

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

1. A process for isolating a polypeptide cartilage-inducing factor from bone, which factor: 
5 (a) is found in mammalian bone; 

(b) is a co-factor for inducing cartilage formation; 

(c) has activity in the TGF-0 assay; and 

(d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS- 
PAGE; 

70 the process comprising: 

(i) treating demineralized bone with a chaotropic extractant that solubilizes nonfibrous proteins; 

(ii) subjecting the extract from step (i) to gel filtration to recover a fraction containing proteins of 
molecular weight 10,000-40,000 daltons; 

(iii) adsorbing the fraction from step (ii) onto a carboxymethyl cellulose cation exchanger at 
75 approximately pH 4.5-5.5 under denaturing conditions; 

(iv) eluting the adsorbed fraction from the cation exchanger with a sodium chloride gradient; 

(v) subjecting the portion of the eluate of (iv) that elutes at approximately 150 to 250 mM sodium 
chloride to RP-HPLC or a nondenaturing gel electrophoresis; and 

(vi) recovering said factor from the RP-HPLC or nondenaturing gel electrophoresis. 

20 

2. A process according to claim 1 wherein the bone is bovine bone. 

3. A process according to claim 1 or 2 wherein each chain of the dimer has an N-terminat sequence 
substantially as follows: 

25 



Ala- 


Leu- 


-Asp- 


-Thr-Asn- 


-Tyr- 


■Cys- 


Phe-Ser (Ser) Thr-Glu- 


-Lys- 


Asn- 


CyS' 


-Cys- 


-Val-Arg- 


-Gln- 


-Leu- 


Tyr-Ile-Asp-Phe-Arg- 


-Lys- 


Asp- 


■Leu 


-Gly- 


-Trp- . 











A polypeptide cartilage-inducing factor, which factor: 

(a) is found in mammalian bone; 

(b) is a co-factor for inducing cartilage formation; 

(c) has activity in the TGF-/9 assay; 

(d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS- 
PAGE; 

(e) is isolatable by a process according to claim 1 or claim 2; and 

(f) does not have the N-terminal sequence 



Ala- 


-Leu- 


-Asp- 


-Thr-Asn- 


-Tyr- 


Cys 


-Phe- 


Ser (Ser) Thr-Glu- 


■Lys 


Asn- 


-Cys- 


-Cys- 


-Val-Arg- 


-Gln- 


Leu 


-Tyr- 


Ile-Asp-Phe-Arg- 


-Lys 


Asp- 


-Leu 


-Gly- 


-Trp- . 













50 

5. A factor according to claim 4 wherein the bone of (a) is bovine bone. 

6. An implant composition for inducing chondrogenesis/osteogenesis which contains at least one factor 
55 according to claim 4 or 5. 

7. An implant composition for promoting connective tissue deposition which contains at least one factor 
according to claims 4 or 5 and which is substantially free of any activating agent or co-factor. 



9 



EP0 169 016 B1 



8. A composition for inducing chondrogenesis or osteogenesis which contains 

(a) at least one factor according to claim 4 or 5; and 

(b) a chondrogenic or osteogenic co-factor. 

9. Use of a factor of claim 4 or claim 5 in the manufacture of an implant composition for inducing 
chondrogenesis/osteogenesis or for promoting connective tissue deposition. 

Claims for the following Contracting State : AT 

1. A process for isolating a polypeptide cartilage-inducing factor from bone, which factor: 

(a) is found in mammalian bone; 

(b) is a co-factor for inducing cartilage formation; 

(c) has activity in the TGF-0 assay; and 

(d) is a dimer having an approximate molecular weight of 26,000 daltons as determined by SDS- 
PAGE; 

the process comprising: 

(i) treating demineralized bone with a chaotropic extractant that solubilizes nonfibrous proteins; 

(ii) subjecting the extract from step (i) to gel filtration to recover a fraction containing proteins of 
molecular weight 10,000-40,000 daltons; 

(iii) adsorbing the fraction from step (ii) onto a carboxymethyl cellulose cation exchanger at 
approximately pH 4.5-5.5 under denaturing conditions; 

(iv) eluting the adsorbed fraction from the cation exchanger with a sodium chloride gradient; 

(v) subjecting the portion of the eluate of (iv) that elutes at approximately 150 to 250 mM sodium 
chloride to RP-HPLC or a nondenaturing gel electrophoresis; and 

(vi) recovering said factor from the RP-HPLC or nondenaturing gel electrophoresis. 

2. A process according to claim 1 wherein the bone is bovine bone. 

3. A process according to claim 1 or 2 wherein each chain of the dimer has an N-terminal sequence 
substantially as follows: 

Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser (Ser) Thr-Glu-Lys- 
Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys- 
Asp-Leu-Gly-Trp- . 



4. A process according to claim 1 or 2 wherein each chain of the dimer has not got the N-terminal 
sequence substantially as follows: 

Al a - Leu - Asp - Thr - Asn - Ty r - Cy s - Phe - Ser ( Se r ) Thr - Glu - Ly s - 
Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys- 
Asp-Leu-Gly-Trp- . 



5. A process for producing an implant composition for inducing chondrogenesis/osteogenesis, which 
composition contains at least one factor produced according to claim 4. 

6. A process for producing an implant composition for promoting connective tissue deposition, which 
comprises forming the composition to contain at least one factor produced according to claim 4 but to 
be substantially free of any activating agent or co-factor. 



10 



EP0 169 016 B1 



7. A process for producing a composition for inducing chondrogenesis or osteogenesis comprising 
forming the composition to contain: 

(a) at least one factor produced according to claim 4; and 

(b) a chondrogenic or osteogenic co-factor. 

5 

8. Use of a factor produced according to claim 4 in the manufacture of an implant composition for 
inducing chondrogenesis/osteogenesis or for promoting connective tissue deposition. 

PatentanspriJche 

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

1. Verfahren zum Isolieren eines knorpel-induzierenden Polypeptid-Faktors aus Knochen, wobei der Faktor 

(a) in Saugetierknochen anzutreffen ist; 

(b) ein Co-Faktor zum Induzieren von Knorpelbildung ist; 
75 (c) im TGF-0-Assay Aktivitat zeigt; und 

(d) ein Dimer mit einem ungefahren Molekulargewicht von 26.000 Dalton ist, wie durch SDS-PAGE 
bestimmt; 

wobei das Verfahren umfaBt: 

(i) das Behandeln von entmineralisierten Knochen mit einem chaotropen Extraktionsmittel, das 
20 nicht-fibrose Proteine solubilisiert; 

(ii) das Unterwerfen des Extrakts aus Schritt (i) einer Gelfiltration, urn eine Fraktion zu gewinnen, 
die Proteine mit einem Molekulargewicht von 10.000 - 40.000 Dalton enthalt; 

(iii) das Adsorbieren der Fraktion aus Schritt (ii) an einen Carboxymethylzellulose-Kationenaustau- 
scher bei pH = 4,5 - 5,5 unter denaturierenden Bedingungen; 

25 (iv) das Eluieren der adsorbierten Fraktion vom Kationenaustauscher mittels eines NaCI-Gradien- 

ten; 

(v) das Unterziehen jenes Anteils des Eluats aus (iv), der bei etwa 150 - 250 mMol NaCI eluiert, 
einer RP-HPLC oder einer nicht-denaturierenden Gelelktrophorese; und 

(vi) das Gewinnen des Faktors aus der RP-HPLC oder der nicht-denaturierenden Gelelktrophore- 
30 se. 

2. Verfahren nach Anspruch 1 , worin die Knochen Rinderknochen sind. 

3. Verfahren nach Anspruch 1 oder 2, worin jede der Dimer-Ketten eine N-terminale Sequenz im 
35 wesentlichen wie folgt aufweist: 

Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser (Ser)Thr-Glu-Lys- 
Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys- 

40 

Asp-Leu-Gly-Trp- . 



45 4. Knorpel-induzierender Polypeptid-Faktor, der 

(a) in Saugetierknochen anzutreffen ist; 

(b) ein Co-Faktor zum Induzieren von Knorpelbildung ist; 

(c) im TGF-jS-Assay Aktivitat zeigt; 

(d) ein Dimer mit einem ungefahren Molekulargewicht von 26.000 Dalton ist, wie durch SDS-PAGE 
50 bestimmt; 

(e) nach einem Verfahren nach Anspruch 1 oder 2 isolierbar ist; und 



55 



11 



EP0 169 016 B1 



<f) nicht die N-terminale Sequenz 



Ala 



Leu 



Asp 



Thr 



Asn - Tyr - Cys - Phe - Ser ( Ser ) Thr -Glu - Lys - 



Asn 



Cys 



Cys 



Val 



Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys- 



Asp 



Leu 



Gly 



Trp 



aufweist. 



5. Faktor nach Anspruch 4, worin die Knochen unter (a) Rinderknochen sind. 

6. Implantat-Zusammensetzung zur Induktion von Chondrogenese/Osteogenese, die zumindest einen 
Faktor nach Anspruch 4 oder 5 enthSlt. 

7. Implantat-Zusammensetzung zur Forderung von Bindegewebe-Ablagerung, die zumindest einen Faktor 
nach Anspruch 4 oder 5 enthalt und im wesentlichen frei von jeglichem Aktivator oder Co-Faktor ist. 

8. Zusammensetzung zur Induktion von Chondrogenese oder Osteogenese, die enthalt: 

(a) zumindest einen Faktor nach Anspruch 4 oder 5; und 

(b) einen Chondrogenese- Oder Osteogenese-Co-Faktor. 

9. Verwendung eines Faktors nach Anspruch 4 oder 5 zur Herstellung einer Implantat-Zusammensetzung 
zur Induktion von Chondrogenese/Osteogenese oder zur Forderung von Bindegewebe-Ablagerung. 

Patentanspruche fur folgenden Vertragsstaat : AT 

1. Verfahren zum Isolieren eines knorpel-induzierenden Polypeptid-Faktors aus Knochen, wobei der Faktor 

(a) in Saugetierknochen anzutreffen ist; 

(b) ein Co-Faktor zum induzieren von Knorpelbildung ist; 

(c) im TGF-0-Assay Aktivitat zeigt; und 

(d) ein Dimer mit einem ungefahren Molekulargewicht von 26.000 Dalton ist wie durch SDS-PAGE 
bestimmt; 



wobei das Verfahren umfaBt: 

(i) das Behandeln von entmineralisierten Knochen mit einem chaotropen Extraktionsmittel, das 
nicht-fibrose Proteine solubilisiert; 

(ii) das Unterwerfen des Extrakts aus Schritt (i) einer Gelfiltration, urn eine Fraktion zu gewinnen, 
die Proteine mit einem Molekulargewicht von 10.000 - 40.000 Dalton enthalt; 

(iii) das Adsorbieren der Fraktion aus Schritt (ii) an einen Carboxymethylzellulose-Kationenaustau- 
scher bei pH = 4,5 - 5,5 unter denaturierenden Bedingungen; 

(iv) das Eluieren der adsorbierten Fraktion vom Kationenaustauscher mittels eines NaCI-Gradien- 
ten; 

(v) das Unterziehen jenes Anteils des Eluats aus (iv), der bei etwa 150 - 250 mMol NaCI eluiert, 
einer RP-HPLC oder einer nicht-denaturierenden Gelelktrophorese; und 

(vi) das Gewinnen des Faktors aus der RP-HPLC oder der nicht-denaturierenden Gelelktrophore- 
se. 



2. Verfahren nach Anspruch 1 , worin die Knochen Rinderknochen sind. 

3. Verfahren nach Anspruch 1 oder 2, worin jede der Dimer-Ketten eine N-terminale Sequenz im 
wesentlichen wie folgt aufweist: 



12 



EPO 169 016 B1 



Ala- 


-Leu- 


-Asp- 


-Thr 


- Asn - Tyr - Cys - Phe - 


Ser (Ser) Thr-Glu-Lys- 


Asn- 


•Cys 


-Cys- 


-Val 


-Arg-Gln-Leu-Tyr- 


He - Asp - Phe - Arg- Lye - 


Asp- 


■Leu- 


-Gly- 


■Trp 







10 4. Verfahren nach Anspruch 1 Oder 2, worin keine der Dimer-Ketten eine N-terminale Sequenz im 
wesentlichen wie folgt aufweist: 





Ala- 


-Leu- 


-Asp- 


Thr -As ri- 


•Tyr- 


Cys -Phe - 


Ser (Ser ) Thr-Glu-Lys- 


75 


Asn- 


-Cys 


-Cys- 


val -Arg- 


■Gln- 


• Leu -Tyr - 


Ile-Asp-Phe-Arg-Lys- 




Asp 


-Leu 


-Gly- 


•Trp- - 









20 

5. Verfahren zur Herstellung einer implantat-Zusammensetzung zur Induktion von Chondrogene- 
se/Osteogenese, die zumindest einen nach Anspruch 4 hergestellten Faktor enthalt. 

6. Verfahren zur Herstellung einer Implantat-Zusammensetzung zur Forderung von Bindegewebe-Ablage- 
25 rung, das die Bildung der Zusammensetzung umfafit, die zumindest einen nach Anspruch 4 hergestell- 
ten Faktor enthalt und im wesentlichen frei von jeglichem Aktivator Oder Co-Faktor ist. 

7. Verfahren zur Herstellung einer Zusammensetzung zur Induktion von Chondrogenese Oder Osteogene- 
se, das die Bildung der Zusammensetzung umfafit, die enthalt: 

30 (a) zumindest einen nach Anspruch 4 hergestellten Faktor; und 

(b) einen Chondrogenese- Oder Osteogenese-Co-Faktor. 

8. Verwendung eines nach Anspruch 4 hergestellten Faktors zur Herstellung einer Implantat-Zusammen- 
setzung zur Induktion von Chondrogenese/Osteogenese Oder zur Forderung von Bindegewebe-Ablage- 

35 rung. 

Revendicatfons 

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

40 1. Procede d'isolement d'un facteur polypeptidique, induisant le cartilage, de Pos, lequel facteur : 

(a) est trouv6 dans I'os mammalien ; 

(b) est un co-facteur pour induire la formation de cartilage ; 
c) a une activity dans le dosage de TGF-/8 ; et 

(d) est un dimere ayant un poids moleculaire approximatif de 26 000 daltons comme determine par 
45 SDS-PAGE ; 

le proc£d6 consistant a : 

(i) traiter I'os demoralise avec un agent d'extraction chaotrope qui solubiltse les prolines non- 
fibreuses ; 

(ii) soumettre Pextrait de I'etape (i) a une filtration sur gel pour nScup^rer une fraction contenant 
so des proteines de poids moleculaire de 10 000 a 40 000 daltons ; 

(iii) adsorber la fraction de I'etape (ii) sur un echangeur de cations de carboxymethylcellulose a 
un pH d'approximativement 4,5 a 5,5 en conditions d§naturantes ; 

(iv) ^luer la fraction adsorb^e de I'^changeur de cations avec un gradient de chlorure de sodium ; 

(v) soumettre la portion de l'4luat de (iv) qui s'6lue a approximativement 150 a 250 mM de 
55 chlorure de sodium a une RP-HPLC ou a une £lectrophorese sur gel non-d£naturante ; et 

(vi) r£cup§rer ledit facteur de la RP-HPLC ou de l'£lectrophor&se sur gel non-d^naturante. 

2. Proc£d£ selon la revendication 1 dans lequel I'os est de Pos bovin. 



13 



EPO 169 016 B1 



3. Proceed selon la revendication 1 ou 2 dans lequel chaque chaTne du dimere a une sequence N- 
terminale substantiellement comme suit : 



Ala- 


Leu< 


-Asp- 


-Thr- 


-Asn- 


-Tyr- 


Cys- 


-Phe- 


Ser (Ser) Thr- 


-Glu 


-Lys- 


Asn- 


•Cys 


-Cys- 


-Val- 


-Arg- 


-Gln- 


■Leu- 


-Tyr- 


Ile-Asp-Phe- 


-Arg 


-Lys- 


Asp- 


■Leu 


-Gly- 


-Trp 

















4. Facteur polypeptidique induisant du cartilage, lequel facteur : 

(a) est trouv£ dans I'os mammalien ; 

(b) est un co-facteur pour induire la formation de cartilage ; 

(c) a une activity dans le dosage de TGF-0 ; 

(d) est un dimere ayant un poids moleculaire d'approximativement 26 000 daltons comme d6termin6 
par SDS-PAGE ; 

(e) est isolable par un procede selon la revendication 1 ou la revendication 2 ; et 

(f) n'a pas la sequence N-terminale 

Ala-Leu-Asp-Thr-Asn-Tyr-Cys-Phe-Ser (Ser) Thr-Glu-Lys- 
Asn-Cys-Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys- 

Asp-Leu-Gly-Trp- . 

5. Facteur selon la revendication 4 dans lequel I'os de (a) est un os bovin. 

6. Composition d'implant pour induire une chondrogenese/osteogSnese qui contient au moins un facteur 
selon la revendication 4 ou 5. 

7. Composition d'implant pour provoquer un dǤpot de tissu conjonctif qui contient au moins un facteur 
selon les revendications 4 ou 5 et qui est substantiellement exempte de tout agent ou co-facteur 
activateur. 

8. Composition pour induire la chondrogenese ou I'osteogenese qui contient 

(a) au moins un facteur selon ia revendication 4 ou 5 ; et 

(b) un co-facteur chondrogene ou osteogene. 

9. Utilisation d*un facteur selon la revendication 4 ou ia revendication 5 dans la fabrication d'une 
composition d'implant pour induire la chondrog£nese/ost6og£nese ou pour provoquer un d§p6t de tissu 
conjonctif. 

Revendications pour i'Etat contractant suivant : AT 

1. Proced£ d'isolement d'un facteur polypeptidique induisant le cartilage de I'os, lequel facteur : 

(a) est trouve dans Cos mammalien ; 

(b) est un co-facteur pour induire la formation de cartilage ; 
c) a une activite dans le dosage de TGF-0 ; et 

(d) est un dimere ayant un poids moldculaire approximatif de 26 000 daltons comme d£termin£ par 
SDS-PAGE ; 

le proc6d§ consistant a : 

(i) traiter I'os d6min£ralis£ avec un agent d'extraction chaotrope qui solubilise les prolines non- 
fibreuses ; 

(ii) soumettre I'extrait de l'£tape (i) a une filtration sur gel pour recuperer une fraction contenant 
des prolines de poids moleculaire de 10 000 a 40 000 daltons ; 



14 



EP 0 169 016 B1 



(iii) adsorber la fraction de I'etape (ii) sur un echangeur de cations de carboxym^thylceliulose a 
un pH d'approximativement 4,5 a 5,5 en conditions d^naturantes ; 

(iv) £luer la fraction adsorb^e de I'echangeur de cations avec un gradient de chlorure de sodium ; 

(v) soumettre la portion de l^luat de (iv) qui s*£lue a approximativement 150 a 250 mM de 
5 chlorure de sodium a une RP-HPLC ou a une Slectrophorese sur gel non-denaturante ; et 

(vi) r£cup§rer ledit facteur de la RP-HPLC ou de l'6lectrophorese sur gel non-d6naturante. 

2. Proc£d6 selon la revendication 1 dans lequel Cos est de I'os bovin. 

70 3. Proced6 selon la revendication 1 ou 2 dans lequel chaque chaTne du dimere a une sequence N- 
terminale substantiellement comme suit : 



75 



20 



25 



30 



Ala- 


-Leu- 


-Asp- 


Thr- 


-Asn- 


-Tyr- 


Cys- 


-Phe- 


Ser (Ser) 


Thr- 


-Glu- 


-Lys- 


Asn- 


-Cys 


-Cys- 


■Val- 


-Arg- 


-Gln- 


Leu- 


-Tyr- 


Ile-Asp- 


-Phe- 


-Arg- 


-Lys- 


Asp- 


-Leu 


-Gly- 


-Trp- 



















4. Proc6d6 selon la revendication 1 ou 2 dans lequel chaque chaTne du dimere n'a pas la sequence N- 
terminale substantiellement comme suit : 



Ala- 


LeU' 


-Asp- 


Thr- 


-Asn- 


-Tyr- 


-Cys- 


-Phe- 


Ser {Ser) Thr- 


-Glu- 


-Lys- 


Asn- 


Cys 


-Cys- 


Val 


-Arg- 


-Gln- 


-Leu- 


-Tyr- 


Ile-Asp-Phe 


-Arg- 


-Lys- 


Asp- 


■Leu 


-Gly- 


•Trp 

















5. Proc6d£ de production d'une composition d'implant pour induire une chondrog6nese/ost£og6nese, 
laquelie composition contient au moins un facteur produit selon la revendication 4. 

35 6. Procede de production d'une composition d'implant pour provoquer un depot de tissu conjonctif, qui 
comprend la formation de la composition pour contenir au moins un facteur produit selon la revendica- 
tion 4 mais pour etre substantiellement libre de tout agent ou co-facteur activateur. 

7, Proc6d§ de production d'une composition pour induire une chondrog^nese ou une ost£og6n£se 
40 comprenant la formation de la composition pour contenir : 

(a) au moins un facteur produit selon la revendication 4 ; et 

(b) un co-facteur chondrogene ou osteogene. 

8. Utilisation d'un facteur produit selon la revendication 4 dans la fabrication d'une composition d'implant 
45 pour induire une chondrog§nese/ost6og§nese ou pour provoquer un d6pdt de tissu conjonctif. 



50 



55 



15 



EP0 169 016 B1 




16 



EP 0 169 016 B1 




EP 0 169 016 B1 




18 



EP 0 169 016 B1 



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19 



EP 0 169 016 B1 




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EP0169 016 B1 



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21 



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