AP856A - A novel homodimer protein used in pharmaceutical preparation for treating cartlage and bone diseases. - Google Patents

Abstract

A protein having amino acid sequence in SEQ ID No.:l of the Sequence Listing derived from.human MP52, and a dimer protein thereof. A homodimer protein described above can be obtained by constructing a plasmid containing DNA coding "amino acid sequence in SEQ ID No.:1 of the Sequence Listing with a methionine at the N-terrninus, introducing the plasmid into E. coli for transformation, solubilizing'inclusion bodies obtained by culturing the transformant, purifying the monomer protein from the solubilized solution, refolding the monomer protein into a dimer protein and purifying the same. The homodimer protein described above is useful as a pharmaceutical composition for treating cartilage and bone diseases.

Description

Title of the Invention

A novel protein and process for preparing the same

Background of the Invention

1. Field of the Invention

This invention relates to a protein having amino acid sequence in SEQ ID No.:1 of the Sequence Listing derived from MP52. The invention also relates to a homodimer protein of said protein and a pharmaceutical composition for treating cartilage and bone diseases containing the dimer protein as an active ingredient. The invention also relates to a process for preparing the above described protein in a large amount and with a high purity by culturing E. coli which was transformed with a plasmid containing a DNA sequence capable of expressing the above described protein. The invention further relates to a method for treating cartilage and bone diseases, which comprises administering to a human a pharmaceutical composition containing, as an active . ingredient, an effective amount of the homodimer protein.

. Description of the Prior Art

Pharmaceutical compositions comprising vitamin D3, calcitonin, estrogen or their derivatives as well as bisphosphonate derivatives have been used in clinical practice for preventing and treating bone diseases. Recently, bone morphogenetic protein (BMP hereinafter), the TGF-β gene superfamily comprising BMP-2 through BMP-9 and related proteins, have been reported to have bone morphogenetic activity.

AP/P/97/0 1 136

AP Ο Ο Ο 8 5 6

Furthermore, the bone morphogenetic activity of one of those proteins called MP52 has been also reported (WO 93/16099 and WO 95/04819). A mature region of MP52 protein is considered to be a protein consisting of 120 amino acid residues having the N-terminal alanine, and its amino acid sequence is described in these publications.

A protein called GDF-5, having an analogous amino acid sequence with MP52, is also described in Nature, vol. 368, p.

639-643 (1994) and WO 94/15949.

However, these proteins can not be easily prepared in a purified form on an industrial scale.

Mammalian cell lines such as L-cells have been tried on for producing MP52 with genetic engineering technology.

However, it has been found not easy to prepare MP52 in a purified form and in a high yield with the expression systems.

Detailed Description of the Invention_

The present inventors have tried to prepare MP52 using E. coli on a large scale by genetic engineering technology.

Briefly, the inventors have tried to prepare MP52 using E.

coli by adding a codon encoding methionine to the DNA encoding mature region of MP52 which starts from alanine. The resultant product was not MP52 only but a mixture of MP52, a protein of 121 amino acid residues having the N-terminal methionine and a protein of 119 amino acid residues having the

N-terminal alanine detached and starting from proline. It was extremely difficult to isolate pure MP52 at least with the mature region from the mixture.

AP/P/ 97 /01138

AP O 0 0 8 5 6

JThe inventors have found that a protein in SEQ ID No.:1 of the Sequence Listing starting from proline at the N-terminus can be selectively produced in an extremely high yield by constructing a plasmid wherein a codon encoding methionine was connected to the DNA sequence encoding amino acid sequence in SEQ ID No.:1 of the Sequence Listing consisting of 119 amino acid residues with elimination of the N-terminal alanine of MP52, and by using the obtained plasmid-introduced E. coli for expression. Moreover, the homodimer of the protein ^! +10 described in SEQ ID No.:1 in the Sequence Listing was confirmed to have a cartilage and bone morphogenetic activity, and thus the invention was completed.

An object of the invention is to provide a protein having amino acid sequence shown in SEQ ID No.:1 of the Sequence

Listing. The protein consists of 119 amino acid residues, and

I corresponds to one in which the N-terminal alanine is eliminated from human MP52 which is regarded as a mature region consisting of 120 amino acid residues. The protein acL' cording to the invention is soluble in water. Moreover, the 20 protein is low toxic itself because it is derived from human.

Another object of the invention is to provide a-pharmaceutical composition for treating cartilage and/or bone diseases, which comprises as an active ingredient a homodimer of the protein having the amino acid sequence shown in SEQ ID

No.:1 of the Sequence Listing. More in detail, the invention relates to a pharmaceutical composition for preventing and treating osteoporosis, osteoarthritis such as gonarthritis deformans and malum coxae deformans, or arthrosteitis,

AP/P/ 9 7/01 138

APOΟ Ο 8 56 cartίlagineous lesion such as articular meniscus lesion, reconstruction in the defective parts of bone and cartilage caused by injury and oncoectomy, defect of bone and cartilage, bone fracture, congenital cartilage and bone diseases such as chondrodysplasia, chondrohypoplasia, achondrogenesis, palatoschisis and osteodysplasia, and furthermore radicular and arvecular defects, since the homodimer· protein according to the invention has a cartilage and bone morphogenetic activity. Furthermore, the homodimer protein can be applied to a treatment of bone grafting in cosmetic surgery. These treatments also include those in the area of veterinary surgery .

A further object of the invention is to provide a process for preparing a protein consisting of 119 amino acid residues derived from human MP52 shown in SEQ ID No.:1 of the Sequence

Listing using E. coli.

In particular, the invention relates to the construction of a plasmid containing a DNA sequence encoding the amino acid, sequence consisting of 119 amino acid residues shown in SEQ ID

No. :1 of the Sequence Listing with an additional methionine at the N-terminus. Only the mature region of human MP52 cDNA was amplified by polymerase chain reaction (PCR method) by using, as a template DNA, a plasmid vector containing cDNA described in WO 93/16099. The PCR method referred to herein generally means to multiply a very small amount of fragments of DNA or

RNA by the method described in U.S. Patent 4,683,195.

It is necessary for preparing the protein of the invention to construct appropriate expression vectors containing

AP/P/ 97 / 0 1138

AP Ο Ο Ο 8 5 6

DNA encoding the protein, which are then introduced into desirable E. coli host strains by genetic engineering technology. The following two improved processes were applied for a large scale production of the protein;

1) A process for increasing the productivity of target proteins by increasing the translation efficiency as reported by M. Nobuhara et al. (Agric. Biol. Chem., 52 (6), 1331-1338,

1988}, viz. the method of increasing the AT content around the ATG initiation codon, and

... 10 2) A process for increasing an average copy number of plasmids per cell, viz. the method of replacing ori region for the replication origin of pBR vector by that of pUC vector. Further, the expression vector (pKOT245) of the invention was constructed by direcr ligation of the promoter region with the

DNA sequence encoding amino acid sequence in SEQ ID No.:1 of the Sequence Listing with an additional methionine in its Nterminus. The E. coli containing said vector was deposited (Accession No. 3IK0KEN-KI P-14895) at National Institute of Bioscience and Human-Technology, Agency of Industrial Science

(.••-IO and Technology which is located at 1-3, Higashi 1-chome,

Yatake-cho, Tsukuba-shi, Ibaraki-ken, 305 Japan on April 14, 1995 and transferred to a deposit (Accession No. BIKOKEN-KI BP-5499) on April 10, 1996 according to Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

This invention relates to a process for preparing monomer proteins comprising the steps of:

constructing a plasmid containing DNA encoding amino acid sequence in SEQ ID No.:1 of the Sequence Listing with a methiAP/P/ 9 7/0113«

ΑΡΟ Ο Ο 8 5 6 onine at its N-terminus, introducing the plasmid into £. coli for transformation, cultivating the E. coli to obtain inclusion bodies, solubilizing and purifying said inclusion bodies to ob5 tain monomer proteins, and to a process for preparing homodimer proteins of the protein in SEQ ID No.:1 of the Sequence Listing by refolding and purifying the monomer proteins obtained in the above. Briefly, the proteins of the invention were prepared by solubilizing the E. coli inclusion bodies followed by loading on SP-Sepharose FF column and Sephacryl S-200 -column to obtain purified sulfonated MP52 monomers, which were subjected to refolding and isoelectric precipitation, then to RESOURCE RPC column of reverse-phase HPLC to obtain the purified dimer fractions of the proteins. The physicochemical properties of the proteins were analyzed on the basis of N-terminal amino acid sequence and amino acid composition and by electrophoresis.

This invention further relates to a process for culturing

E. coli which” were introduced with the expression vectors of ,-20 the invention under the conditions of culture medium at 2834'C, pH 6-8 and ”a dissolved oxygen concentration of 20-50%.

This invention further relates to a method for treating cartilage and bone diseases, which comprises administering to a human a pharmaceutical· composition containing, as an active ingredient, an effective amount of the homodimer protein.

Biological activities of the homodimer protein were determined by analysis of soft X-ray radiographs, analysis of tissue-staining and analysis of time-course of ectopic cartiAP/P/ 97 / 0 1138

AP Ο Ο Ο 8 5 6 lage/bone formation. Furthermore, from the results of the effect on the intramembranous ossification, the effect on the regeneration of articular cartilage and the effect on the healing of bone fracture and defects, the homodimer protein of the present invention is proved to be beneficial to the therapies of cartilage and/or bone regeneration.

The homodimer protein of the invention can be administered in systemic by intravenous, intramuscular or intra-peritoneal injection. In case of intravenous administration, an intrave— 10 nous drip infusion can also be used, in addition to conventional intravenous injections.

Injectable preparations can be formulated, for example, in the form of injectable powders. In that case, the powders can be prepared by adding one or more of suitable water-soluble excipients such as mannitol, sucrose, lactose, maltose, glucose, fructose and the like, to an active ingredient, dissolving the mixture in water, dividing it into vials or ampoules followed by lyophilizing and hermetically sealing.

In the case of local administration, the homodimer protein ¢,=/0 can be coated on the surface of cartilage, bone or tooth to be treated with collagen paste, fibrin glue or other adhering materials. In the case of bone grafting, both natural bone and conventional artificial bone can be used. Artificial bone means the bone made of metal, ceramics, glass, and other natural or artificial inorganic substance. Hydroxyapatite is cited as preferable artificial substance. For example, artificial bone can be constructed by steel as dense material in the inner part and hydroxyapatite as porous material in outer part. Moreover,

AP/P/ 9 7/01 138

AP Ο Ο Ο 8 5 6 it is beneficial to apply the homodimer protein to the. part from which cancerous bone tissue is removed in order to accelerate the reconstruction of bone. It can also be applied, to the cartilage grafting.

The dose may be varied depending upon various factors influencing the activity of the protein such as weight of bone and cartilage to be reconstructed, injured site of bone and cartilage and the symptoms, age and sex of patients, severity of infection, administration intervals and other clinical factors.

The dose can also be varied depending upon types of carriers to be used for restructuring with the dimer protein. In general, the dose is in the range of about 10-10θ ng of the homodimer. protein per wet weight of desired bone and cartilage when administered as a composition containing a'carrier. In the case of local and systemic administration by injection, it is preferable to administer 0.1-10^ μg in a frequency of from once a week to once a day.

A synergetic effect can be expected by administering the homodimer protein simultaneously with known growth factors, for example, insulin-like growth factor-I for regeneration of bone and cartilage.

There has never been reported of a process for preparing the protein of the invention on an industrial scale and in a purified- form as described above, and the homodimer protein is useful as a medical composition for treating cartilage and bone diseases since it has a cartilage and bone morphogenetic activity. Further, the process of preparing the protein of the present invention can be applicable for the preparation of other

AP/P/ 97/01138

APO Ο Ο 8 5 6 proteins of the above-described TGF-β superfamily members, all of which were only successful so far to prepare by using mammalian cell lines .

This invention is further illustrated by the following 5 examples. However, it should not be construed that the invention is limited to these specific examples.

Example

Example .1 Construction of expression vector ’10 (1) Isolation of a mature region of MP52

A mature region of human MP52 cDNA was PCR-amplified using the plasmid vector (pSK52s) containing cDNA described in WO 93/16099 as a template DNA.

In accordance with the process for increasing a produc- tivity of the target proteins reported by M. Nobuhara, et al.

{Agric. Biol. Chem., 52 (6), 1331-1338, 1988}, a part of DNA of the mature region of MP52 gene was substituted to increase the

AT content around the ATG initiation codon.

The mutagenesis was introduced by PCR method using the 20 designed upstream PCR primer encompassing the mutation of SEQ ID

No. :2 of the Sequence Listing. For the DNA sequence of the PCR primers were used the DNA in the SEQ ID No.:2 as an upstream primer, and the DNA in SEQ ID No.:3 of the Sequence Listing as a downstream -primer.

The PCR was performed by adding the template DNA (10 ng), pmols each of the PCR primers in an order direction and in a reverse direction, dNTP (0.2 mmol) and MgCl2 (1.5 mmol) in the same test tube, together with Taq DNA polymerase (5 U) .

AP/P/ 9 7 /0 1 1 36

AP Ο Ο Ο 8 5 6

Thirty cycles of PCR were performed; the conditions of each cycle were 94'C for a minute for denaturation, 55'C for a minute for primer annealing, and 72’C for 2 minutes for primer extension .

The products obtained from the PCR was isolated by electrophoresis in 1.5% low melting point agarose (purchased from FMC), and the fragments of about 360 bp were isolated (Fragment 1).

(2) Construction of E. coll expression vector for the 10 protein of the invention

In order to increase a copy number of the plasmid per bacteria, the orl region for replication origin was changed from that of pBR to pUC vector. The E. coll expression vector pKK223-3 available in the market (purchased from Pharmacia 15 Biotech) was used to isolate tac promoter region by digestion with restriction endonucleases SspI and EcoRI, and also to isolate rrnBt]_t2 terminator region by using Sail and SspI. A

DNA fragment of tac promoter region which had been treated with

Mung Bean Nuclease (Takara Shuzo Co., Ltd.) was ligated by T4 20 DNA ligase with Fragment 1 which was obtained above. The resultant DNA fragment was digested by Sail and re-ligated with the rrnBt]_t2 region. The DNA fragment was ligated into the Smal site of pUC18 vector to construct the expression vector {pKOT245 (Accession No. BIKOKEN-KI P-14895)} (Fig. 1) for the production 25 of the protein. The length of pKOT245 DNA is 3.7 kb. The nucleotide sequence of the expression vector constructed for the protein was analyzed by Pharmacia ALF DNA sequencer.

AP/P/ 97/0 1T38

AP Ο Ο Ο 8 5 6 (3) 'Transformation

Transformation was performed according to the rubidium chloride transformation method by Kushner et al. (Genetic Engineering, p. 17, Elsevier, 1978). Briefly, pKOT245 was used to transform the host strain E. coli W3110M according to the method described above to produce Ξ. coli transformants for the production of the protein.

Example 2 Cultivation .10 (1) Cultivation

The E. coli expressing the protein of the invention was precultured in the modified SOC medium (Bacto tryptone 20 g/1, Bacto yeast extract 5 g/1, NaCl 0.5 g/1, MgCl2'6H2O 2.03 g/1, Glucose 3.6 g/1). 100 ml of the bacteria suspension was used- to inoculate 5 1 of the production medium (Bacto tryptone 5 g/1,

Citric acid 4.3 g/1, K₂HPO₄ 4.675 g/1, KH₂PO₄ 1.275 g/1, NaCl 0.865 g/1, FeS0₄-7H₂0 100 mg/1, CuSO₄-5H₂O 1 mg/1, MnSO₄-nH₂O 0.5 mg/1, CaCl₂-2H₂O 2 mg/1, Na₂B₄O₇·10H₂O 0.225 mg/1, ·' (NH₄)₆Mo₇0₂₄-4H₂0 0.1 mg/1, ZnSO₄-7H₂O 2.25 mg/1, CoCl₂·6H₂O 6 if mg/1, MgSO47H₂O 2.2 g/1, Thiamine HC1 5.0 mg/1, Glucose 3 g/1), which was cultured in a 10-liter fermentor with aeration-agitation, and then upon reaching the early stage of logarithmic growth phase (OD55o=5.0), isopropyl-h-D-thio-galactopyranoside at a final concentration of 1 mM was added and the cultivation was continued until reaching OD55o=150. During the cultivation, temperature was kept at 32 C, and pH value of 7.15 by adding ammonia. In order to prevent lowering of a dissolved oxygen concentration, an agitation was sped up to keep the dissolved

AP/P/ 9 7/01138

AP Ο Ο Ο 8 5 6 oxygen- concentration at 50% of air saturation. The cultivation was proceeded by adding 50% glucose solution at a level of 0.2% to obtain a high cell density, with an indication of abrupt increase of the dissolved oxygen concentration.

♦ (2) Preparation of E. coli inclusion bodies

The culture broth obtained by the method described above was centrifuged to harvest the cells, which were then suspended in 25 mM Tris-HCl buffer containing 10 mM ethylene diamine .'10 tetraacetic acid (pH 7.3) . The cells were disrupted by passing through a homogenizer (made by APV Gaulin Inc.) and centrifuged again to harvest the precipitate containing the inclusion bodies .

Example 3 Purification (1) Solubilization of E. coli inclusion bodies

After washing with l%..Triton X-100 three times, the E. coli inclusion bodies were centrifuged at 3,000 x g for 30 minutes at 4’C, and then the resultant precipitate was solubilized by ’20 sonication in 20 mM Tris-HCl buffer, pH 8.3, 8 M urea, 10 mM

DTT, and 1 mM EDTA.

(2) Preparation of monomers

The solubilized solution was centrifuged at 20,000 x g for 25 30 minutes at 4 ” C and the resultant supernatant was collected.

The obtained supernatant was subjected to SP-Sepharose FF (Pharmacia AB) equilibrated with 20 mM Tris-HCl buffer pH 8.3, 6

M urea, and 1 mM EDTA, and then, after washing with the same

AP/P/97/0 1 13«

AP Ο Ο Ο 8 5 6 solution, it was eluted with the same solution containing 0.5 M NaCl. The protein in the eluate were sulfonated by adding NagSOg and Na2S40g to read the final concentration respectively at 111 mM and 13 mM and by incubating at 4'C for 15 hours. The sulfonated solution was gel-filtrated on Sephacryl S-200 HR (Pharmacia AB) equilibrated with 20 mM Tris-HCl buffer, pH 8.3,

M urea, 0.2 M NaCl, and 1 mM EDTA to obtain purified sulfonated monomers of the protein of the invention.

.-.:¾ 10 (3) Refolding

The solution of the sulfonated monomers was added into a 9 times volume of 50 mM Na-Glycine buffer pH 9.8, 0.2 M NaCl, 16 mM CHAPS, 5 mM EDTA, 2 mM GSH (reduction type glutathione), and 1 mM GSSG (oxydation type glutathione) with stirring, and then incubated for 24 hours at 4'C to oxidize and refold the protein of the invention.

(4) Preparation of homodimers

The refolding solution was diluted with the same volume of +3-20 purified water and then by adding 6 N NaCl adjusted pH value to approximately 7.4 and placed to isoelectric precipitation. The precipitates collected by centrifugation at 3,000 x g for 20 minutes were solubilized in a solution with 30% acetonitrile containing 0.1% TEA. The solution was diluted with the same 25 volume of purified water and loaded on RESOURCE RPC column (Pharmacia AB) of a reverse-phase HPLC preequilibrated with 25% acetonitrile containing 0.05% TFA, and then eluted with a linear gradient of 25-45% acetonitrile containing 0.05% TFA. The

AP/P/ 9 7 /0 1 1 3β· eluate was monitored at 280 nm absorbance. The purified homodimer protein fractions were collected and lyophilized by

SpeedVac Concentrator (Servant Co.)

AP/P/97/0 1 1 3« (5) Determination of physicochemical properties of the purified protein of the invention

a) Analysis of N-terminal amino acid sequence

Analysis of the N-terminal amino acid sequence for the purified proteins was performed using an amino acid sequencer Model 47 6A (Applied Biosystems Inc.) to confirm the amino acid sequence from the N-terminal to the 30th amino acid as shown in SEQ ID No.:1 of the Sequence Listing.

b) Analysis of amino acid composition

The analysis of amino acid composition of the purified proteins obtained above was performed by an amino acid sequencer (PICO TAG Systems, Waters). The result was shown in Table 1.

The number described in Table 1 indicates the number of amino acid residue per a monomer protein.

Table 1

Amino acid

Asx

Glx

Ser

Gly

His

Arg

Thr

Ala

Pro

Tyr

Val

Met ¹/2Cys lie

Leu

Phe

Practical number Exoected number

11.5 12

10.9 11

8.4 9

4.3 4

4.0- 4

7.7 7

5.4 6

7.3 7

10.2 10

2.9 3

5.7 7

5.1 . 4

2.6 7

4.9 6

10.0 10

4.0 4

APO Ο Ο 8 5 6

- Lys 5.9 6

Typ - 2 length of the sequence_119_

-: undetectable

c) Analysis by electrophoresis

Molecular weight of the purified proteins obtained above was confirmed to be about 28 KDa on SDS-PAGE 'under non-reducing condition .

From the results shown in the above a), b) and c), it is found that the protein of the invention comprises 119 amino acid residues starting from the N-terminal Pro singly.

Example 4 Determination of biological activities (1) Activity in ectopic bone formation in mice.

About 500 gg of the homodimer protein obtained in Example 3 was dissolved and diluted in 50 gl of 10 mM hydrochloric acid, and 1 gg/10 μΐ, 10 gg/10 gl, and 100 gg/10 gl concentrations of the solution were prepared. Ten μΐ of each solution was mixed with 150 μΐ porcine tendon type-I collagen solution (Koken, 0.5%, pH 3, I-AC), neutralized, lyophilized, and the resultant mixture was implanted into pockets created in the thigh muscles of 8-week-old male ICR mice. At day 21 from implantation, the animals were sacrificed and the thighs were excised. After peeling skins off, the incidence of calcified tissues was evaluated by soft X-ray radiography. As shown in Table 2, the implantation of 1 gg/site or more of the dimer protein induced calcified tissue in part of the group of the mice, and 10 and more doses

AP Ο Ο Ο 8 5 6 induced calcified tissue in all mice used.

Table 2

Dose	of the homodimer protein	Incidence of calcified tissue
Control (Type-I collagen alone)	0/4
1	μ7/3ίίβ	3/4 '
10	μ9/3^θ	4/4
100	μg/site	4/4

Figure 2 shows typical examples of soft X-ray radiographs of calcified tissue induced by different doses of MP52 protein. Figures 2A, 23 and 2C show examples of soft Xray radiographs of 1 hg the homodimer protein/site-, 10 ^g/site- and 100 jig/site-implanted mice thighs, respectively.

These radiographs indicate that the homodimer protein induced <

calcified tissue in the mouse thigh and increased it in a dose-dependent manner. In order to verify if the formed calcified tissues were cartilage or bone, the sections of the ' /20 fixed mouse thighs into which 10 μ9/3ίίε the homodimer protein ·^;· was implanted were stained with von Kossa, Alcian blue or

Hematoxylin-eosin.

Figure 3 shows light-microscopic photographs of the sections stained with the respective staining methods . In

Figure 3A (von Kossa staining), areas indicated by ct and cc show calcified tissue and calcified chondrocytes, respectively. In Figure 3B (Alcian blue staining), an area indicated by rc shows remaining cartilage tissue. In Figure 3C (Hematoxylin-eosin staining), elements indicated by ad, bm,

AP/P/97/01 13«

APO Ο Ο 8 5 6 lb, ob and wb are an adipocyte, bone marrow cells, lamellae bone, osteoblasts, and woven bone, respectively. Thus, it is evident that the implantation of the homodimer protein with Type-I collagen into mouse thighs induces calcified chondrocytes, osteoblasts, and bone marrow cells at the.sites.

Thus, the homodimer protein was demonstrated to possess activity in ectopic cartilage and bone formation.

(2) Analysis of time-course of ectopic bone formation in mice ’10 The dimer protein (3 gg) obtained in Example 3 was mixed with Type—I collagen solution and neutralized as described in Example 4 (1), and the lyophilized materials were implanted into the male ICR mouse thighs. At days 3, 7, 10, 14, 21, and 28 from implantation, the thighs were excised and fixed in 10 % formalin, and then, sections were stained with Hematoxylineosin or von Kossa, Figure 4 shows the light-microscopic photographs of the sections stained.

:. At day 3 (Figure 4A, Hematoxy-lin-eosin staining) ,

Ktr undifferentiated mesenchymal cells (me) including morphologically fibrous connective cells appeared in the space between collagen fibers (co) implanted and muscle cells (m).

At between days 7 and 10 (Figures 4B and 4C, respectively, Hematoxylin-eosin staining) , the space was filled with undifferentiated mesenchymal cells (me) and these cells were hypertrophied and differentiated into precartilagenous tissue.

At day 14 (Figure 4D, Hematoxylin-eosin staining and Figure 4E, von Kossa staining), calcified cartilage tissue (cc) and bone tissue (b) were observed. At day 21 (Figure 4D,

AP/P/97/0 1 138

AP Ο Ο Ο 8 5 6

Hematoxylin-eosin staining and Figure 4Ξ, von Kossa staining) , calcified cartilage tissue was not observed at all, and the tissue observed at day 14 appeared to be replaced into bone (b) with bone marrow (bm). At day 28 (Figure 4H, Hematoxylin5 eosin staining), there were a large mount of bone marrow cells and formed bone appeared to be under a resorptive process.

Thus, it is evident that the homodimer protein induces endochondral ossification through cartilage formation at ectopic sites, as reported by using other BMPs.

vid _1Q (3) Effect on the intramembranous ossification

The homodimer protein obtained in Example 3 was dissolved in phosphate-buffered saline (pH 3.4 ) containing 0.01% human serum albumin, and 0.01 μς/20 μΐ-, 0.1 μg/20 μΐ-, and 1 μς/2015 μΐ-concentrations of solutions were prepared- Twenty μΐportion of each solution was injected 12 times once a day onto the periosteum of neonatal rat parietal bone by using a microsyringe from day 1 after birth. The same volume of the vehicle was injected onto the counter-side of parietal bone of each rat. The same volume of the vehicle was also injected onto both sides of parietal bones of control rats. At day 1 from the final injection, the rats were sacrificed and the both sides of parietal bones were excised and fixed, and then, the decalcified sections stained with Hematoxylin-eosin were prepared to measure the thickness of the parietal bones at the injected sites on microscopic photographs. The ratio of the homodimer protein-injected site/vehicle-injected site in the parietal bone thickness of each rat was calculated. As shown

AP/P/ 97/01138

AP Ο Ο Ο 8 5 6 in Table 3, the homodimer protein increased parietal bone thickness in a dose-dependent manner. A typical example of microscopic photographs of the section at a homodimer protein 0.1 gg/site-injected site is shown in Figure 5B in comparison with that of the counter-side of vehicle-injected site (Figure

5A) . The injection of the homodimer protein induced the activation and proliferation of periosteal cells (p), and activated osteoblasts (ob) were observed in and on the parietal bone (b). These results indicate that the homodimer g|l0 protein stimulated intramembranous ossification when locally injected, and that the homodimer protein is beneficial to the therapies of osteoporosis, bone fracture, and alveolar ridge and periodontal defects .

Table 3

AP/P/ 9 7/01 138

Dose cf homodimer procein ?;r.e~3i bore criicxness (μ.·π)_ Ratio orotein fua/site/cav) Vehicle-i.-.’eccea site (A) XP52-iniecced site (3)(B/A)

	0 (vehicle)	123 ± 7	141 ± 20	1.10 ±	0.16
20	0.01	134 ± 9	167 ± 30	1.27 ±	0.33
	0.1	119 ± 19	190 ± 29	1.60 ±	0.10*
	1	132 ± 9	225 ± 25	1.70 ±	0.14**
	Values represent	means ± SD (n=4),	*p<0.05,	**p<0.01 vs .	ratio
	of the group in	which vehicle was	injected	into both the	sites

(Williams' test) .

(4) Effect on the regeneration of articular cartilage

Six 12-week-old male New Zealand White rabbits were used for this study. Right knee skin and articular capsule were cut and a 5 x 5 mm full thickness osteochondral defect was created in the patellar groove using a dental burr so as not to damage surrounding tendons. The defects were filled with

AP Ο Ο Ο 8 5 6 either lyophilized Type-I collagen sponge or with lyophilized Type-I collagen sponge containing 10 p.g homodimer protein, prepared as described in Example 4 (1), and then, the cut articular capsule and skin were sutured.» Three weeks post5 operatively, the rabbits were sacrificed and the femoral heads were excised and fixed in 10% formalin, and then, decalcified sections were stained with Alcian blue. Typical examples of microscopic photographs of the sections were shown in Figure

6. The dimer protein treated defects (Figures 6C and 6D) demonstrated the regeneration of chondrocytes (ch) with extracellular matrices which were stained intensively with Alcian blue, as compared to the Type-I collagen sponge implanted control defects (Figures 6A and 6B) which were filled with fibrous tissue (f) . The cartilage tissue induced15 by the dimer protein showed zonal structure including resting chondrocytes, growing chondrocytes and hypertrophied chondrocytes, like that of normal articular cartilage. The chondroinduct-ion by the MP52 protein were observed in the

-ΐ defects of all rabbits used (n=3) . These results indicate that the dimer protein is effective to the repair of .damaged cartilage tissue in patients such as osteoarthritis.

(5) Effect on the healing of bone fracture and defects

Thirty male Sprague-Dawley rats (about 15-week-old) were used for this study. Using a lateral approach to the femur, all muscle and periosteal tissue were stripped from the diaphysis. A 5 mm-segmental bone defect was created in the middle region of the right femur shaft with use of dental

AP/P/97/0 1 138

APO Ο Ο 8 5 6 burr, 'and then, a special-made polyethylene plate was fixed with stainless screws along the cortex of the femur. Type-I collagen sponges containing 0, 1, 10, and 100 gg of the homodimer protein were prepared as described in Example 4 (1), and implanted into the segmental bone defects and then, the wound was sutured. Just after operation and '12 weeks postoperatively, the defects were evaluated by soft X-ray radiography. As shown in Figure 7, 10 and 100 gg/site of the homodimer protein stimulated callus (cs) formation in the

510 defects and formed bony unions, but the effect at 1 gg/site was not clear as compared to the control collagen implanted defect in which only marginal endosteal bone formation was observed. Twelve weeks post-operatively, rats were sacrificed, and the femur with a defect was excised and bone15 mineral' content (accumulated one of mid-three scannings in the defect) was measured by dual energy X-ray absorptiometry (Aloka, Model DCS-600) in a mode with 1 mm scanning width after removing the polyethylene plate. Both ends of the femur with the resin were fixed, then, maximum torsional strength to break the union of specimens were measured by bone strainer system (Malto, model MZ-500D) in a routing speed with 180*/min (Table 4). It shows that the homodimer protein increases both bone mineral content and bone strength at the rat femur defect in which the. protein is implanted, and indicates the efficacy of the present protein for fracture healing and bone reconstruction of the defect.

APIPI 9 7/01 138

AP Ο Ο Ο 8 5 6

Table -4

Dose of homodimer protein (μς/είϋβ)	Sone Mineral Concent in rac femur defect (mg)	Maximum Torsional Strength (Kgf*cm)	Number
collagen alone	120.2 ± 24.5	2.92 ± 0.09	6
1	176.9 ± 36.4	6.24 ± 1.00	8
10	277.4 ± 63.9	9.35 ± 3.14	8
100	374.8 ± 67.1*	40.34 ± 7.64*	8

Values represent means ± SE, *p<0.05 vs. collagen alone group (Student's t-test).

From the results in Example 4, the homodimer protein of the invention was found to have a cartilage and bone morphogenetic act ivity.

The protein composed of a homodimer of the protein having an amino acid sequence in SEQ ID No. :1 of the Sequence Listing has a cartilage and bone morphogenetic activity and is useful as a pharmaceutical composition for treating cartilage and bone diseases. Furthermore, the protein of the invention can be prepared on an industrial scale and in a pure form by a gene engineering process culturing E. coli transformed with a higher copy number expression vector for said protein.

AP/P/· 9 7/01138

AP 0 0 0 8 5 6

Sequence Listing”

SEQ ID No . :1

Sequence length :119

Sequence type:amino acid

Topology:linear

Molecular Type:peptide

Type of Fragment:N-terminal fragment

Original Source

Organism:homo sapiens

Name of tissue:fetus Features :

Other information: From 383'to 501 amino acid sequence of MP52 amino acid sequence Sequence Description :SEQ ID No .:1:

CCA

CTG

GCC

ACT

CGC

CAG

GGC

AAG

CGA

CCC

AGC

AAG

AAC

CTT

AAG

GCT

48

Pro

Leu

Ala

Thr

Arg

Gin

Gly

Lys

Arg

Pro

Ser

Lys

Asn

Leu

Lys

Ala

5

10

15

CGC

TGC

AGT

CGG

AAG

GCA

CTG

CAT

GTC

AAC

TTC

AAG

GAC

ATG

GGC

TGG

96

Arg

Cys

Ser

Arg

Lys

Ala

Leu

His .

.Val

Asn

Phe

Lys

Asp

Met

Gly

Trp

20

25

30

GAC

GAC

TGG

ATC

ATC

GCA

CCC

CTT

GAG

TAC

GAG

GCT

TTC

CAC

TGC

GAG

144-

Asp

Asp

Trp

lie

He

Ala

Pro

Leu

Glu

Tvr

Glu

Ala

Phe

His

Cys

Glu

35

40

45

GGG

CTG

TGC

GAG

TTC

CCA

TTG

CGC

TCC

CAC

CTG

GAG

CCC

ACG

AAT

CAT

192

Gly

Leu

Cys

Glu

Phe

Pro

Leu

Arg

Ser

His

Leu

Glu

Pro

Thr

Asn

His

50

55

60

GCA

GTC

ATC

CAG

ACC

CTG

ATG

AAC

TCC

ATG

GAC

CCC

GAG

TCC

ACA

CCA

240

Ala

Val

He

Gin

Thr

Leu

Met

Asn

Ser

Met

Asp

Pro

Glu

Ser

Thr

Pro

65

70

75

80

CCC

ACC

TGC

TGT

GTG

CCC

ACG

CGA

CTG

AGT

CCC

ATC

AGC

ATC

CTC

TTC

288

Pro

Thr

Cys

Cys

Val

Pro

Thr

Arg

Leu

Ser

Pro

He

Ser

He

Leu

Phe

90 95

AP/P/87/0 1 136

ΑΡΟ Ο Ο 8 5 6

ATT

GAC

TCT

GCC

AAC

AAC

GTG

GTG

TAT

AAG

CAG

TAT

GAG

GAC

ATG

GTC

336

lie

Asp

Ser

Ala

Asn

Asn

Val

Val

Tyr

Lys

Gin

Tyr

Glu

Asp

Met

Val

100

105

110

GTG

GAG

TCG

TGT

GGC

TGC

AGG

357

Val

Glu

Ser

Cys

Gly

Cys

Arg

115

SEQ

! ID

NO

. : 2

Sequence length:27

Sequence type:nucleic acid

Strandness: single Topology : linear

Molecular Type:other nucleic acid Original Source:none

Organism:none

Strain : none

Features:PCR upstream primer of MP52 mature type Sequence Description :SEQ ID No .:2:

ATAATGCCAC TAGCAACTCG TCAGGGC 27

SEQ ID No.:3 Sequence length:26 Sequence type:nucleic acid Strandness: single

Topology: linear

Molecular Type:other nucleic acid Original Source:none

Organism:none Name of tissue:none

Features:

Other information:PCR downstream primer of MP52 mature type Sequence Description :SEQ ID No .:3:

CGTCGACTAC CTGCAGCCAC ACGACT 26

AP/P/9 7 /0.1 1 38

4

AP Ο Ο Ο 8 5 6

Brief Explanation of Drawings

Fig. 1 shows a plasmid map of the expression vector (pKOT245) for the protein of the invention obtained in Example 1 (2) .

Fig. 2 shows a soft X-ray radiograph of the calcified tissue induced in mouse thigh in Example 4 (1).

Fig. 3 shows a light-microscopic photograph of the calcified tissue stained in mouse thigh in Example 4 (1).

Fig. 4 shows a light-microscopic photograph of the timecoursed calcified tissue stained in mouse thigh in Example 4 (2) .

Fig. 5 shows a light-microscopic photograph of rat parietal bone stained in Example 4 (3) .

Fig. 6 shows a light-microscopic photograph of articular cartilage defects stained in the rabbit femoral head in Example 4 (4) .

Fig. 7 shows a soft X-ray radiograph of the bone formation in -the bone defects of” the rat femurs in Example_ 4

Claims (14)

1. What is claimed is:

   1. A protein having amino acid sequence in SEQ ID No.:1 of the Sequence Listing.

   5
2. 2. A homodimer protein according to claim 1.
3. 3. A pharmaceutical composition for treating cartilage and bone diseases comprising a therapeutically effective amount of the protein according to claim 2 and a pharmaceutical carrier.

   10
4. 4. The pharmaceutical composition according to claim 3 wherein cartilage and bone diseases are osteoporosis.
5. 5. The pharmaceutical composition according to claim 3 wherein cartilage and bone diseases are osteoarthritis or arthrosteitis.

   15
6. 6. The pharmaceutical composition according to claim 3 wherein cartilage and bone diseases are bone fracture and bone defect.
7. 7. The pharmaceutical composition according to claim 3 wherein cartilage and bone diseases are radicular and

   20 arvecular defects.
8. 8. A process for preparing the protein of claim 1 which comprises culturing £. coli transformed with a plasmid containing DNA sequence which is capable of expressing said protein .

   25
9. 9. The process according to claim 8 wherein the plasmid contains a DNA coding amino acid sequence in SEQ ID No.:1 of the Sequence Listing with a methionine at the

   N-terminus.

   AP/P/ 97/0 1 13«

   -
10. 10.

    AP 0 0 0 8 5 6

    A process for preparing the dimer protein according to claim 2 which comprises the steps of;

    constructing a plasmid containing DNA coding amino acid sequence in SEQ ID No.:1 of the Sequence Listing with a 5 methionine at the N-terminus, introducing the plasmid into E. coli for transformation, solubilizing inclusion bodies obtained by culturing said

    E. coli, purifying the monomer protein from the solubilized 10 solution, refolding the monomer protein into a dimer protein and purifying the same.
11. 11. A method for treating cartilage and bone diseases, which comprises administering to a human a pharmaceutical

    15 composition containing, as an active .ingredient, an effective amount of the homodimer protein as claimed in claim 2.
12. 12. The method for treating cartilage and bone diseases as claimed in claim 11, wherein said disease is osteoporosis.
13. 13. The method for treating cartilage and bone diseases '20 as claimed in claim 11, wherein said disease is osteoarthritis or arthrosteitis.
14. 14. The method for treating cartilage and bone diseases as claimed in claim 11, wherein said disease is bone fracture and bone defect.

    25 15. The method for treating cartilage and bone diseases as claimed in claim 11, wherein said disease is radicular and arvecular defects .