WO1997006254A1

WO1997006254A1 - Human mp52 arg protein

Info

Publication number: WO1997006254A1
Application number: PCT/EP1996/003427
Authority: WO
Inventors: Michio Kimura; Tomoaki Matsumoto; Mikiko Takahashi; Shinji Kawai; Yukio Fujino
Original assignee: Biopharm Gesellschaft Zur Biotechnologischen Entwicklung Von Pharmaka Mbh
Priority date: 1995-08-03
Filing date: 1996-08-02
Publication date: 1997-02-20
Also published as: PL324822A1; ZA966489B; NO980375L; MX9800801A; AU6789196A; CN1192237A; CA2227204A1; HUP9900362A2; KR19990036081A; JP2000511155A; IL122817A0; NO980375D0; EP0842274A1; AU699708B2; AR003974A1; BR9609983A

Abstract

This invention relates to a human MP52 Arg and a pharmaceutical medical composition inter alia for promoting cartilage and bone morphogenation comprising human MP52 Arg. In particular, the medical composition is useful for treating bone diseases caused by abnormal bone metabolism such as osteoporosis, for treating bone fracture and for the purpose of orthopedic reconstruction, bone transplantation, cosmetic surgery and dental therapeutics. Further, it is useful for treating cartilage disorders.

Description

HUMAN MP52 ARG PROTEIN

This invention relates to a new compound human MP52 Arg and a pharmaceutical medical composition inter alia for promoting cartilage and bone morphogenation comprising human MP52 Arg. In particular, the medical composition is useful for treating bone diseases caused by abnormal bone metabolism such as osteoporosis, for treating bone fracture and for the purpose of orthopedic reconstruction, bone transplantation, cosmetic surgery and dental therapeutics. Further, it is useful for treating cartilage disorders.

Pharmaceutical compositions including vitamin D3, calcitonin, estrogen and bisphosphonate derivatives have been used in clinical practice for treating bone diseases. Their therapeutic results, however, are not entirely satisfactory, and a better pharmaceutical composition is highly desired.

The TGF-β family of growth factors comprising BMP, TGF, and inhibin related proteins have been reported to be useful for wound healing and tissue repair. The bone morphogenetic activity of some of those proteins has been also known. PCT patent application WO 93/16099 and WO 25/04819 disclose DNA sequences encoding human TGF-β-like proteins, and as a preferred protein human MP52.

E. E. Storm et al . reported in Nature, 1994, vol.368, p.639-643 that mutations of mouse growth/differentiation factor 5 (GDF5) gene, a new member of the TGF-ß superfamily, cause mice brachypodism. Mouse GDF5 has the same amino acid sequence of the predicted mature form as that of human MP52 except one amino acid. However, there is no suggestion in this publication to use those proteins for the treatment of bone diseases.

It was therefore the object of the present invention to provide a further growth factor that is useful as an agent for the stimulation of bone or cartilage formation. One embodiment of the present invention is therefore protein human MP52 Arg comprising amino acids 1 to 121 of SEQ ID NO: 1.

In fact, it has surprisingly been found by the invention that there are cell lines which, when expressing a suitable DNA sequence, form human MP52 Arg or a mixture of human MP52 and human MP52 Arg. For the first time the invention succeeded in providing human MP52 Arg which has bone morphogenetic activity and is useful for preventing and/or treating bone diseases.

It has also been confirmed that human MP52 Arg induces formation of cartilage from undifferentiated mesenchymal cell and stimulates the differentiation and maturation of osteoblasts. Therefore, human MP52 Arg is effective for preventing and/or treating bone diseases caused by abnormal bone metabolism such as osteoporosis. It also accelerates the healing process of bone fractures. Moreover, it is useful for orthopedic reconstructions, bone transplantations and dental therapeutics because of its bone morphogenetic activity. Furthermore, MP52 Arg is effective for preventing and/or treating cartilage disorders caused by abnormal cartilage metabolism.

A further object of the present invention is a process for the production of human MP52 Arg, wherein at least a part of a DNA sequence as shown in Sequence ID NO:1 is introduced into a suitable host cell under conditions favouring expression of the DNA sequence and protein formation, followed by isolation of said protein from other proteins produced by said host cell.

Within the framework of the present invention the DNA sequence depicted in SEQ ID NO:1 may be used for producing human MP52 Arg, however also shorter portions thereof, provided they still encode human MP52 Arg and expression of the DNA sequence in a suitable vector/host cell system is possible. Suitable expression systems are known to a person skilled in the art and it can easily be determined by routine experimentation what the minimum requirements for the length of the DNA sequence of SEQ ID NO:1 are.

Subsequent to protein formation the proteins are recovered from the host cell by methods known per se and finally human MP52 Arg is isolated therefrom. In particular the isolation of human MP52 and MP52 Arg, which differ from one another only in respect of one amino acid, can be carried out using very precisely differentiating separation methods which are known to a person skilled in the art. One example is the electrophoretic separation of MP52 Arg performed according to the method of Davis (Ann. NY Acad. Sci., 121, 404-427, 1964) with small modifications like addition of 0.1 % Nonidet P-40 and 6 M urea. After electrophoresis the separated MP52 Arg is electroeluted from the gel pieces in the same buffer.

A further object of the present invention is a pharmaceutical composition containing human MP52 Arg. Optionally, this composition may also include usual carrier substances, auxiliary substances, diluents and/or fillers. The pharmaceutical composition according to the invention is useful for promoting bone morphogenation, treatment or prevention of damage to bone, cartilage, connective tissues, skin, mucous membranes, epithelium or teeth, for application in dental implants and for application in wound-healing and tissue regeneration processes.

For the treatment of bone diseases caused by abnormal bone metabolism, human MP52 Arg is administered in systemic by injection such as intravenous injection, muscle injection and intraperitoneal injection, oral administration, non-oral administration such as suppository, and other any conventional methods.

For the treatment of bone fracture, it is administered in systemic and locally by injection, oral and non-oral administration. Also matrix containing human MP52 Arg is preferably implanted in the area close to the fractured bone. Suitable matrixes are natural polymers such as collagen and fibrin clot, and artificial polymers degradable in living body such as polylactated glycolic acid.

In case of orthopedic reconstruction, cosmetic surgery, bone transplantation and dental implantation, human MP52 Arg for example can be coated on the surface of bone and tooth to be implanted by means of collagen paste, fibrin glue and other adhering materials. It can also be applied to the tissue, the bone or alveolar bone around which the bone and tooth is transplanted. In case of bone transplantation it can be used for both natural and artificial bone. As the material of artificial bone and tooth, conventional materials such as metals, ceramics, glass and other natural or artificial inorganic substance are used. Hydroxyapatite is a preferred artificial substance. Artificial bone can be constructed by dense material in the inner part and porous material in the other part. For example, dense steel covered porous steal can be cited. Porous hyroxyapatite is one of the materials to produce artificial bone. When such porous material is used, human MP52 Arg can be penetrated into it. The surface of artificial bone can also be roughened to keep human MP52 Arg on the surface.

It is also beneficial to apply human MP52 Arg to the part from which cancerous bone tissue is removed in order to accelerate the reconstruction of bone.

The dose of human MP52 Arg to be obtained is decided depending upon the purpose and the method of application. In general, when it is administered in systemic, the dose is from 1 μg to 100 μg/kg. When it is used for implantation, the preferred dose is from 30 μg to 30 mg per site.

This purified human MP52 Arg can be formulated in any conventional form such as injection liquid, pills, capsules and suppository. For local administration human MP52 Arg can be included in a matrix such as collagen, fibrin glue and poly lactated glycolic acid. For the use of implantation and transplantation it is applied to the surface or in the porous part of bone and tooth.

This invention is illustrated by the examples.

Example 1

Construction of expression vector for MP52 Arg

The pSK52s plasmid (WO 95/04819) was digested with Hind III and the DNA fragment containing the cDNA comprising the complete coding region for MP52 was isolated by extraction from 0.8 % low melting agarose gels and ligated into the Hind III site of pABstop vector, which is supplied by Dr. Gerd Zettlmeißl of Behringwerke AG. The structure of the resulting MP52 expression vector, pMSS99 (5.0 kb), was confirmed by the DNA sequencing and the restriction enzyme mapping. The genetic elements of pMSS99 are schematically shown in Fig.1. The MP52 sequence in pMSS99 comprises the nucleotides 576-2279 in SEQ ID NO:1 of the Sequence Listing.

Example 2

Establishment of CHO clones producing MP52 Arg

Dhfr-deficient CHO cells CHO-DUKX-B11 (Urlaub, G. and Chasin, L.A. (1980) Proc.Natl.Acad.Sci, USA, 77, pp.4216-4220) were cotransfected with the expression plasmid for MP52 (pMSS99) and pSVOAdhfr (Zettlmeiβl, G. et al., (1987) Bio/Technology 5 , 720-725) by calcium phosphate-mediated DNA transfer method. Then high producer clones of MP52 Arg were established by a gene amplification protocol using methotrexate (MTX) .

Briefly, 10 μg of pMSS99 and 2 μg of pSVOAdhfr were dissolved in 1 ml of 25 mM HEPES-140 mM NaCl-0.75 mM Na₂HPO₄ (pH 7.05), then mixed with 50 μl of 2.5 M CaCl₂. The resultant precipitates were overlaid to CHO-DUKX-B11 cells and incubated at room temperature for 30 min. Then MEM ALPHA medium with ribo- and deoxyribonucleosides (MEMα⁺) containing 10 % fetal bovine serum (FBS) was added to the cell layer to incubate in CO₂ incubator for 4-6 h. After a treatment with 10 % glycerol in MEMoJ containing 10 % FBS at room temperature for 3 min, the cells were cultured in MEMα⁺ containing 10 % FBS for 2 days. Then the cells were placed in MEM ALPHA medium without ribo- and deoxyribo-nucleosides (MEMαT) containing 10 % dialyzed FBS to select the transformants. The transformant clones were isolated and assayed for the expression of MP52 Arg by Western blot analysis as described in the next section.

Then high producer clones of MP52 Arg were established by a gene amplification protocol using methotrexate (MTX). The MP52 Arg producing clones were further selected stepwisely in increasing concentrations of methotrexate (MTX) to amplify the MP52 gene in accordance with the pSVOAdhfr gene. Several clones were obtained which produced 1-3 μg of mature MP52 Arg/10⁶ cells/24 h at 400 nM MTX.

Example 3

Detection of MP52 Arg in the culture supernatants

Clones were examined for the expression of MP52 Arg by Western blot analysis as follows: The culture supernatants (1-15 μl) were applied on SDS-PAGE (15-25 % polyacrylamide gradient gel, Daiichi Pure Chemicals) under reducing conditions, then the proteins were transferred to a PVDF membrane (Clear Blot Membrane-P, ATTO). The membrane was blocked with Block Ace (Dai-Nihon Seiyaku) for 1 h, rinsed with Tris-buffered saline (TBS), then treated with 10 μg/ml of chicken antibodies to MP52 Arg in 10-times diluted Block Ace overnight. After washing the membrane with 0,1 % Tween 20 in TBS (TTBS), the membrane was treated with rabbit anti-chicken IgG-ALP conjugate (Sigma A 9171) in 10-times diluted Block Ace for 1 h. The membrane was washed with TTBS and then reacted with Alkaline phosphatase Conjugate Substrate Kit (BIO-RAD) to visualize the bands corresponding to MP52 Arg.

Example 4

Cell culture of the MP52 Arg-producing CHO cell line

The CHO cell line with the highest productivity of MP52 Arg and MP52, MC-2 (deposited under No.FERM BP-5142 on June 21, 1995 with the National Institute of Bioscience and Human Technology, Japan) was grown with roller bottles containing MEMα⁺ supplemented with 10 % FBS, 400 nM MTX, 100 U/ml Penicillin and 100 μg/ml Streptomycin. After the MC-2 cells had grown to confluency, they were washed with serum-free MEMα-n and then cultured in serum-free DME/F12 medium supplemented with 10 mM HEPES (pH 7.3), 10 KIE/ml Aprotinin, 1 mM sodium butyrate, 6 μg/ml sodium selenate, 5 μg/ml transferrin, 18 μg/ml ethanol amine, 9 μg/ml insulin, 100 U/ml Penicillin and 100 μg/ml Streptomycin. The conditioned medium was collected every day for a week.

Example 5

Purification

One liter of the culture supernatants were mixed with 0.1 volume of 0.2 M sodium phosphate buffer, pH 6.0, and applied to a POROS HS column (10 ml, PerSeptive Biosystems). The elution was performed by a linear gradient of NaCl from 0.3 to 2 M. The eluate containing MP52 Arg was applied to reverse-phase column (RESOURCE RPC, Pharmacia). The elution was performed by linear gradient of acetonitrile, containing 0.05 % TFA and MP52 Arg was eluted at about 35 % acetonitrile. The N-terminal amino acid sequence analysis for purified MP52 Arg was performed using a pulse liquid gas phase sequencer (Applied Biosystems model 476). The result is shown in Table 1. It is indicated that MP52 Arg is processed proteolytically at Arg(380)-Arg(381) (amino acid positions -1 and +1 of Sequence ID NO:1) from its precursor. However, the precursor can also be processed at Arg(381)-Ala(382) (amino acid positions +1 and +2 of Sequence ID NO:1).

Example 6 Biological activity

Osteoprogenitor-like ROB-C26 cells (Calcif. Tissue Int. vol. 49, p. 221-225, 1991) were plated onto 48-well multi-well plates (Coaster) at a density of 1.5 x 10* cells/well and pre-incubated for 3 days in MEMα- containing 10% fetal bovine serum (FBS). After the removal of culture medium, fresh MEMα- containing 10% FBS and serially diluted MP52 Arg in 10mM HCl (2μl/ml) was added to the cultures and incubated for 6 days with changing the medium and the additives on day 3. Cell layers were washed with phosphate-buffered saline, and extracted with 0.2% Nonidet containing 1 mM MgCl₂. Alkaline phosphatase (ALP) activities were determined according to the procedure of Takuwa et al. (Am. J. Physiol. vol. 257, p. E797-E803, 1989). As shown in Table 2, the treatment of ROB-C26 cells with MP52 Arg increased total ALP activities per well concentration-dependently.

Claims

1. A protein comprising amino acids 1 to 121 of SEQ ID NO.1 of the enclosed Sequence Listing.

2. Process for the production of a biologically active protein according to claim 1, wherein at least a part of a DNA- sequence as shown in SEQ ID NO:1 is introduced into a suitable host cell under conditions that allow expression of the DNA-sequence and protein formation, followed by isolation of said protein from other proteins produced by said host cell.

3. Pharmaceutical composition, containing as the active substance a protein according to claim 1, if desired together with usual carrier substances, auxiliary substances, diluents and fillers.

4. Use of a pharmaceutical composition according to claim 3 for promoting bone morphogenation, treatment or prevention of damage to bone, cartilage, connective tissues, skin, mucous membranes, epithelium or teeth, for application in dental implants and for application in wound-healing and tissue regeneration processes.

5. Use of a pharmaceutical composition as claimed in claim 3 for treating osteoporosis or bone fracture.

6. A parmaceutical composition as claimed in claim 3 for the use of orthopedic reconstruction, bone transplantation, cosmetic surgery or dental implantation.