WO2003020768A1

WO2003020768A1 - A method for the treatment or prevention of bone erosion

Info

Publication number: WO2003020768A1
Application number: PCT/JP2002/008630
Authority: WO
Inventors: Yukie Ogawa; Chie Fukuda; Masahiko Ohtsuki
Original assignee: Sankyo Company, Limited
Priority date: 2001-08-28
Filing date: 2002-08-27
Publication date: 2003-03-13
Also published as: EP1421118A1; PE20030340A1; PA8553901A1; AR036305A1; CA2458993A1

Abstract

There is provided a method for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts (e.g. rheumatoid arthritis) comprising administering to a patient an anti-human Fas monoclonal antibody HFE7A or a humanized version thereof, as well as a composition for use in such a method. There is also provided a method for testing a substance for the treatment or prevention of bone erosion comprising: transplanting pieces of dentine and synovial tissue from a patient suffering from a disease that activates osteoclasts to immunodeficient non-human mammals; administering the substance to be tested to at least one of the test animals and a control to at least one different test animal and breeding them; and then extracting said pieces of dentine from the test animals and counting the number of resorption pits formed on the surface of each piece that was in contact with said sample of synovial tissue.

Description

A Method for the Treatment or Prevention of Bone Erosion

Technical Field

The present invention relates to a method for treating or preventing bone erosion caused by a disease that activates osteoclasts (e.g. rheumatoid arthritis), to a composition for treating or preventing bone erosion caused by a disease that activates osteoclasts and to a method for testing an agent to determine its potential for use in the treatment and prevention of bone erosion caused a disease that activates osteoclasts.

Background to the Invention

Rheumatoid arthritis is a systemic inflammatory autoimmune disease characterized by chronic inflammation of the synovium at many of the joints. One of the major pathological changes in rheumatoid arthritis is the formation of pannus, leading to joint erosion with cartilage and bone erosion. This will generally result in a major reduction in the function of the affected joint as well as causing considerable pain. There is also hyperplasia of the cells of the synovial lining which is thought to be due to the influx of type A synoviocytes.

Although the use of anti-inflammatory drugs (e.g. steroids such as hydrocortisone and prednisalone and non-steroidal anti-inflammatory drugs such as diclofenac and loxoprofen sodium) has been successful in controlling inflammation and pain in the joints of an increasing number of patients suffering from rheumatoid arthritis, in most cases this is unfortunately not accompanied by any control of the progress of bone erosion. As a result, in many cases where the damage is sufficiently serious the only option available is to perform orthopedic surgery on the damaged joint.

Activation of osteoclasts is the most important factor in the mechanism of bone resorption. Osteoclasts have been found at the pannus/bone interface in joints affected by rheumatoid arthritis. These cells increase in number and activity early on in the progress of the disease. It is known that osteoclast precursor cells which belong to the monocyte - macrophage system are differentiated and activated to give activated osteoclasts as a result of stimulus from a macrophage colony stimulating factor (M-CSF) and an osteoclast differentiation factor (RANKL/ODF) expressed by osteoblast/stromal cells [see Yasuda H. et al. (1999) Bone 25, 109 - 113]. In rheumatoid arthritis, it is suggested that bone resorption is accelerated by differentiation and activation of the osteoclasts through the action of RANKL according to three separate mechanisms. Namely, the RANKL-RANK signal is activated by: (1) production of RANKL by osteoblasts stimulated by the cytokine IL-17 or the like produced by proliferated T cells [see Kotake, S. et al. (1999) J. Clin. Invest. 103, 1345 -1352); (2) production of RANKL by proliferated synovial fibroblasts themselves [see Takayanagi, H. et al. (2000) Arthritis Rheum. 43, 259 -269]; and (3) production of RANKL by activated T cells themselves [see Kong, Y. et al. (1999) Nature 402, 304 -309]. The production of RANKL by these different mechanisms triggers the RANKL-RANK signal that appears to be essential in triggering differentiation of osteoclast progenitors into osteoclasts. However, it is not yet known whether inhibiting any one of these mechanisms can lead to control of bone erosion.

Physiological death of cells as a result of normal alternation of cells in a living organism is called apoptosis, and is distinguished from the pathological death of cells, i.e. necrosis [cf. Kerr et al., (1972), Br. J. Cancer, 26, 239]. Apoptosis is a kind of so-called programmed cell death, which is observed in certain cells that are programmed, in advance, to die in a living organism. Apoptosis is characterized by a curved cell surface, condensed nuclear chromatin and fragmented chromosomal DNA, amongst others.

Apoptosis plays a role in the differentiation of lymphocytes (T cells and B cells) by eliminating cells that recognize an autoantigen. It is believed that a cause of autoimmune diseases, including rheumatoid arthritis, is the presence of auto-reactive lymphocytes generated due to failure of apoptosis in differentiation of lymphocytes [cf. Nakayama et al., (1995), Mebio, 12 (10), 79-86].

Various molecules have been identified as being involved in apoptosis, including: Fas [cf. Yonehara. S., et al., (1989), J. Exp. Med., 169, 1747-1756]; tumor necrosis factor receptor [cf. Loetscher. H., et al., (1990), Cell, 61, 351-359]; CD40 [cf. Tsubata, T, et al., (1993), Nature, 364, 645-648]; and perforin/granzyme A [cf. Jenne. D. E., et al., (1988), Immunol. Rev. 103, 53-71]. Fas is a transmembrane protein present on the cellular surface, and binding of its extracellular domain to a protein called "Fas ligand" induces apoptosis in the cell.

It has been shown that anti-human Fas monoclonal antibodies such as HFE7A and humanized versions thereof have activity in treating rheumatoid arthritis, by eliminating self-reactive immunocytes that have survived because apotosis has not been induced due to abnormality of the Fas-Fas ligand system (e.g. see EP-A-0909816, EP-A-0990663 and Japanese Patent Application laid open (kokai) No. 2001-342148). However, there has never previously been a disclosure of the action of anti-Fas monoclonal antibodies on the function of osteoclasts nor has there been any disclosure of their having any effect on bone erosion caused by rheumatoid arthritis.

Methods for investigating the function of osteoclasts in vitro are known. For example, one method involves the culturing of osteoclasts on a slice of dentine such as ivory and the number of resorption pits produced on the dentine slice is counted [see Tamura, T. et al, (1993), J. Bone Miner. Res., Aug 8(8), 953-960]. In an alternative in vitro method, synovial cells obtained from a patient suffering from rheumatoid arthritis are cultured on a slice of dentine such as ivory and the number of resorption pits produced on the dentine slice is counted [see Fujikawa, Y. et al, (1996), Br. J. Rheumatol., 35, 213-7)]. However, the effect in a living organism of substances that affect the function of osteoclasts on cells other than said osteoclasts cannot be determined by these methods. An in vivo test method which is adapted to the actual onset mechanism of bone erosion in human rheumatoid arthritis would address this problem and provide a much more effective means of testing substances for their ability to affect the function of osteoclasts and treat or prevent bone resorption. However, such a mechanism has not previously been disclosed. The ability to test substances for their ability to affect the function of osteoclasts and treat or prevent bone resoφtion would be greatly enhanced if such a method could be developed. Furthermore, it is highly desirable to develop a novel pharmaceutical composition that is useful for the treatment or prevention of bone erosion and that has an excellent safety profile.

Objects of the Invention

It is an object of the present invention to provide a pharmaceutical composition useful for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis and a method of treating or preventing bone erosion caused by a disease that activates osteoclasts.

It is a further object of the present invention to provide a method for testing in vivo the effectiveness of a substance as an agent for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts.

Summary of the Invention

The inventors of the present invention have found that when synovial tissue from an affected part of a patient with rheumatoid arthritis and a piece of dentine are transplanted simultaneously to an immunodeficient mouse, bone resorption is caused by osteoclasts present in the transplanted synovial tissue in contact with the piece of dentine. Thus, the inventors of the present invention have provided an in vivo test method for investigating the effect of a substance on osteoclasts and the effectiveness of the substance as an agent for the treatment or prevention of bone resorption caused by rheumatoid arthritis. Furthermore, it has been found that a humanized anti-human Fas monoclonal antibody HFE7A controls bone resorption extremely well in vivo in this test model, thus providing the desired composition for treating or preventing bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis.

(1) In a first aspect of the present invention there is provided a pharmaceutical composition for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising a pharmaceutically acceptable carrier or diluent and an anti-human Fas monoclonal antibody HFE7A or a humanized anti-human Fas monoclonal antibody HFE7A.

(2) Preferred is a pharmaceutical composition for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising a pharmaceutically acceptable carrier or diluent and a humanized anti-human Fas monoclonal antibody HFE7A.

(3) In a second aspect of the present invention there is provided use of an anti-human Fas monoclonal antibody HFE7A or a humanized anti-human Fas monoclonal antibody HFE7Ain the manufacture of a medicament for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts.

(4) Preferred is use of a humanized anti-human Fas monoclonal antibody HFE7A in the manufacture of a medicament for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts.

(5) In a third aspect of the present invention, there is provided a method for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising administering to a patient suffering therefrom an effective amount of an anti-human Fas monoclonal antibody HFE7A or a humanized anti-human Fas monoclonal antibody HFE7A.

(6) Preferred is a method for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising administering to a patient suffering therefrom an effective amount of a humanized anti-human Fas monoclonal antibody HFE7A.

(7) In a fourth aspect, there is provided a method for testing in vivo the effectiveness of a substance as an agent for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising the following steps: (i) transplanting hypodermically pieces of dentine and synovial tissue obtained from an affected part of a patient suffering from a disease that activates osteoclasts to more than one immunodeficient non-human mammal so that each test animal has a piece of dentine and a sample of said synovial tissue that are in mutual contact;

(ii) administering the substance to be tested to at least one of the test animals prepared in step (i), and breeding said test animal for a certain period;

(iii) administering either a control substance or nothing to at least one of the test animals prepared in step (i) which is different from the animal in step (ii), and breeding it under the same conditions as the test animal in step (ii); and

(iv) extracting said pieces of dentine from the test animals at the end of steps (ii) and (iii), and counting and comparing the number of resorption pits formed on each piece of dentine on the surface of said piece that was in contact with said sample of synovial tissue.

(8) In a preferred method according to (7), the immunodeficient non-human mammal is a mouse.

In each of (1) to (8) above, the disease that activates osteoclasts is preferably rheumatoid arthritis.

Detailed Description of the Invention

The pharmaceutical composition of the present invention can be obtained by formulating the anti-human Fas monoclonal antibody HFE7A or a humanized anti-human Fas monoclonal antibody HFE7A using standard pharmaceutical formulation techniques, as is described in greater detail below.

The anti-human Fas monoclonal antibody HFE7A and the humanized anti-human Fas monoclonal antibody HFE7A for use in the composition and the method of prevention or treatment of bone erosion of the present invention may be any such monoclonal antibody or humanized monoclonal antibody that specifically binds to human Fas and has activity in preventing or treating bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis. Suitable examples include anti-human Fas monoclonal antibody HFE7A produced by the mouse-mouse hybridoma HFE7A (FERM BP-5828) and humanized versions thereof produced, for example, by the methods described in EP-A-0909816, EP-A-0990663 or Japanese patent application laid open (kokai) No.2001-342148.

The anti-human Fas monoclonal antibody HFE7A can be produced by methods known in the art using, for example, a molecule containing an extracellular domain of human Fas as an antigen. For example, the monoclonal antibody HFE7A can be obtained by immunizing a Fas knock-out mouse with human Fas, subsequently fusing the spleen cells from the mouse with mouse myeloma cells, and culturing the resultant hybridoma. Specifically, it can be obtained according to the following method.

Preparation of a monoclonal antibody involves at least the following steps:

(a) purification of a biomacromolecule for use as the antigen;

(b) preparation of antibody producing cells, after immunizing an animal using injections of the antigen, bleeding the animal and assaying the antibody titer, in order to determine when to remove the spleen;

(c) preparation of myeloma cells;

(d) fusing the antibody producing cells and myeloma cells;

(e) selecting a hybridoma producing an antibody of interest;

(f) preparing a single cell clone (cloning);

(g) optionally, culturing the hybridoma cells, or growing animals into which the hybridoma cells have been transplanted, for large scale preparation of the monoclonal antibody; and

(h) testing the biological activities and the specificity, or assaying marker agent properties, of the monoclonal antibody thus prepared.

The method for the preparation of an anti-Fas monoclonal antibody is described below more in detail, in line with the above described steps. However, the method fci preparing the antibody is not limited thereto. Other antibody-producing cells than spleen cells and myeloma can also be used.

(a) Purification of antigen

Arecombinant protein (hereinafter referred to as "recombinant human Fas"), effective as the antigen, can be obtained by transfecting the monkey cell line COS-1 with the expression vector phFAS-AIC2, which encodes a fusion protein comprising the extracellular domain of human Fas and the extracellular domain of the mouse interleukin-3 receptor (hereinafter referred to as IL3R), [e.g. Nishimura, Y, et al., (1995), J. Immunol., 154, 4395-4403] to express it, and collecting and partially purifying the expression product. The plasmid phFas-AIC2 was constructed by inserting DNA encoding a human Fas and mouse IL3R fusion protein into pME18S, which is an expression vector for animal cells. As noted above, the materials used, such as the DNA encoding Fas, the vector and the host, are not restricted to those mentioned.

For example, the human Fas and mouse IL3R fusion protein produced in the culture supernatant of the transformed COS-1 cells transfected with the plasmid phFas-AIC2 may be partially purified by ion-exchange chromatography using a Resource Q column (tradename; manufactured by Pharmacia). Purified Fas obtained from the cell membranes of human cell lines can also be used as the antigen. Furthermore, since the primary structure of Fas is known [e.g. Itoh, N, et al., (1991), Cell, 66, 233-243], a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the Sequence Listing of EP-A-1180369, may be chemically synthesized by a method well known in the art, and used as the antigen.

(b) Preparation of antibody producing cells

The immunogen produced in step (a) is mixed with an adjuvant, such as Freund's complete or incomplete adjuvant and alum, and an experimental animal is immunized therewith. A suitable experimental animal may be a Fas knock-out mouse, which may be produced by the method of Senju et al. [Senju, S., et al., (1996), International Immunology, 8, 423].

Suitable administration routes to immunize the mouse include the subcutaneous, intraperitoneal, intravenous, intradermal and intramuscular injection routes, with subcutaneous and intraperitoneal injections being preferred.

Immunization can be by a single dose or, by several repeated doses at appropriate intervals (preferably 1 to 5 weeks). Immunized animals are monitored for antibody titer in their sera, and an animal with a sufficiently high antibody titer is selected as the source of antibody producing cells. Selecting an animal with a high titer makes the subsequent process more efficient. Cells for the subsequent fusion are generally harvested from the animal 3 to 5 days after the final immunization.

Methods for assaying antibody titer include various well known techniques such as radioimmunoassay (hereinafter, referred to as RIA), solid-phase enzyme immunoassay (hereinafter, referred to as ELISA), fluorescent antibody assay and passive hemagglutination assay, with RIA and ELISA preferred for reasons of detection sensitivity, rapidity, accuracy and potential for automation.

Determination of antibody titer may be performed, for example, by ELISA, as follows. First, purified or partially purified Fas is adsorbed onto the surface of a solid phase, such as a 96-well ELISA plate, followed by blocking any remaining surface, to which Fas has not been bound, with a protein unrelated to the antigen, such as bovine serum albumin (hereinafter referred to as BSA). After washing, the well surfaces are contacted with serially diluted samples of the first antibody (for example, mouse serum) to enable binding of the anti-Fas antibody in the samples to the antigen. An enzyme-labeled, anti-mouse antibody, as the secondary antibody, is added to be bound to the mouse antibody. After washing, the substrate for the enzyme is added, and antibody titer can then be estimated by determining absorbance change due to color development caused by the decomposed substrate or the like.

(c) Preparation of myeloma cells

In general, cells from established mouse cell lines serve as the source of myeloma cells, for example, 8-azaguanine resistant mouse (derived from BALB/c) myeloma strains P3X63Ag8U.l (P3-U1) [Yelton, D. E., et al., Current Topics in Microbiology and Immunology, 81, 1-7, (1978)], P3 NSI/l-Ag4-l(NS-l) [Kohler, G, et al., European J. Immunology, 6, 511-519 (1976)], Sp2/0-Agl4 (SP-2) [Shulman, M., et al., Nature, 276, 269-270 (1978)], P3X63Ag8.653 (653) [Kearney, J. R, et al., J. Immunology, 123, 1548-1550 (1979)] and P3X63Ag8 (X63) [Horibata, K. and Harris, A. W., Nature, 256, 495-497 (1975)]. The cell line selected is subcultured in an appropriate medium, such as 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, fetal calf serum (hereinafter referred to as FCS), and 8-azaguanine], Iscove's Modified Dulbecco's Medium (hereinafter referred to as IMDM) or Dulbecco's Modified Eagle Medium (hereinafter referred to as DMEM). The cells are then subcultured in a normal medium, such as ASF104 medium (Ajinomoto, K. K.) containing 10% FCS, 3 to 4 days prior to fusion, in order to ensure that at least 2 x 10⁷ cells are available on the day of fusion.

(d) Cell fusion

The antibody producing cells to be used are plasma cells and lymphocytes which are their precursor cells, which may be obtained from any suitable part of the animal. Typical areas are spleen, lymph nodes, peripheral blood, or any appropriate combination thereof, spleen cells most commonly being used.

After the last booster injection, tissue in which antibody-producing cells are present, such as the spleen, is enucleated from a mouse having the predetermined antibody titer to prepare antibody-producing cells such as spleen cells. The currently favored technique for fusion of the spleen cells with the myeloma cells prepared in step (c), employs polyethylene glycol, which has relatively low cytotoxicity and the fusion procedure using it is simple. An example of this technique is as follows.

The spleen and myeloma cells are washed well with serum-free medium (such as RPMI 1640) or phosphate buffered saline (hereinafter referred to as PBS) and then mixed, so that the number ratio of spleen cells to myeloma cells is approximately between 5 : 1 and 10 : 1, and then centrifuged. After the supernatant has been discarded and the pelleted cells sufficiently loosened, 1 ml of serum-free medium containing 50% (w/v) polyethylene glycol (m.w. 1,000 to 4,000) is added dropwise with stirring. Subsequently, 10 ml of serum-free medium is slowly added and then the mixture centrifuged. The supernatant is discarded again, and the pelleted cells are suspended in an appropriate amount of HAT medium containing a solution of hypoxanthin, aminopterin and thymidine (hereinafter referred to as "HAT" ) and mouse interleukin-2 (hereinafter referred to as IL-2). The suspension is then dispensed into the wells of culture plates (hereinafter referred to as "plates") and incubated in the presence of 5% v/v CO₂ at 37°C for about 2 weeks, with the supplementary addition of HAT medium as appropriate.

(e) Selection of hybridomas

When the myeloma strain used is resistant to 8-azaguanine, i.e., it is deficient in the hypoxanthin guanine phosphoribosyl transferase (HGPRT) enzyme, any unfused myeloma cells and any myeloma-myeloma fusions are unable to survive in HAT medium. On the other hand, fusions of antibody-producing cells with each other, as well as hybridomas of antibody producing-cells with myeloma cells can survive, the former only having a limited life. Accordingly, continued incubation in HAT medium results in selection of only the desired hybridomas.

The resulting hybridomas grown up into colonies are then transferred into HAT medium without aminopterin (hereinafter referred to as "HT medium"). Thereafter, aliquots of the culture supernatant are collected to determine anti-Fas antibody titer by, for example, ELISA. When the above-mentioned fusion protein is used as the ELISA antigen, it is also necessary to eliminate clones producing an antibody which specifically binds to the extracellular domain of the mouse IL3 receptor. The presence or absence of such a clone may be verified, for example, by ELISA using the mouse IL3 receptor, or its extracellular domain, as the antigen.

Although the above selection procedure is exemplified using an 8-azaguanine resistant cell line, it will be appreciated that other cell lines may be used with appropriate modifications to the media used.

(f) Cloning

Hybridomas which have been shown to produce specific antibodies, using a method similar to that described in the step (b) to determine antibody titer, are then transferred to another plate for cloning. Suitable cloning methods include: the limiting dilution method, in which hybridomas are diluted to contain one cell per well of a plate and then cultured; the soft agar method in which colonies are recovered after culturing in soft agar medium; a method of using a micromanipulator to separate a single cell for culture; and "sort-a-clone", in which single cells are separated by a cell sorter. Limiting dilution is generally the most simple and is commonly used.

The cloning procedure according to, for example, the limiting dilution method is repeated 2 to 4 times for each well demonstrating an antibody titer, and clones having stable antibody titers are selected as anti-Fas monoclonal antibody producing hybridomas. Hybridomas producing an anti mouse Fas antibody are selected by a similar method to obtain an anti-Fas monoclonal antibody producing cell line. A mouse Fas useful for this purpose, for example, is the fusion protein expressed by culturing animal cells transfected with the expression vector pME18S-mFas-AIC. This plasmid has DNA encoding a fusion protein comprising the extracellular domain of mouse Fas and the extracellular domain of the mouse IL3 receptor [e.g. Nishimura. Y, et al., (1995). J. Immunol., 154, 4395-4403]. Other sources of murine Fas include purified mouse Fas and cells which express mouse Fas on their surface.

The mouse-mouse hybridoma HFE7Athat produces the anti-human Fas monoclonal antibody HFE7A was deposited with the National Institute of Bioscience and Human-Technology at 1-3, Higashi 1-chome, Tsukuba, Ibaraki, Japan on February 20, 1997, in accordance with the Budapest Treaty on the Deposition of Microorganisms, and was accorded the accession number FERM BP-5828. Accordingly, when preparing an antibody using the mouse-mouse hybridoma HFE7A, the preparation may be performed by following a procedure starting from the step (g) below, with the steps (a) to (f) omitted.

(g) Culture of hybridoma to prepare monoclonal antibody

The hybridoma obtained by the cloning is then cultured in normal medium, not in HT medium. Large-scale culture can be performed by roller bottle culture, using large culture bottles, or by spinner culture. The supernatant from the large-scale culture is purified by a suitable method, such as gel filtration, which is well known to those skilled in the art, to obtain an anti-Fas monoclonal antibody which the prophylactic or therapeutic agent of the present invention contains. The hybridoma may also be grown intraperitoneally in a syngeneic mouse, such as a BALB/c mouse or a Nu/Nu mouse, to obtain a large quantity of ascites containing an anti-Fas monoclonal antibody which the prophylactic or therapeutic agent of the present invention contains. Purification can also be conducted through use of commercially available monoclonal antibody purification kits (for example, MAbTrap Gil Kit; Pharmacia).

Monoclonal antibodies prepared as above have a high specificity to human and mouse Fas.

(h) Assay of monoclonal antibody

Determination of the isotype and the subclass of the monoclonal antibody thus obtained may be performed as follows. Suitable identification methods include the Ouchterlony method, ELISA and RIA. The Ouchterlony method is simple, but requires concentration of the solution when the concentration of the monoclonal antibody is low. When ELISA or RIA is used, the culture supernatant can be reacted directly with an antigen adsorbed on a solid phase and with secondary antibodies having specificities for the various immunoglobulin isotypes and subclasses to identify the isotype and subclass of the monoclonal antibody. A method of using a commercial kit for identification, such as a Mouse Typer Kit (manufactured by Bio-Rad Laboratories, Inc.) is more simple.

Quantification of protein may be performed by the Folin-Lowry method, or by calculation based on the absorbance at 280 nm [1.4 (OD 280) = Immunoglobulin 1 mg/ml].

DNA encoding the heavy and light chains of the anti-human Fas monoclonal antibody HFE7Amay be obtained by preparing mRNAfrom hybridoma cells producing the anti-Fas monoclonal antibody, converting the mRNAinto cDNAwith reverse transcriptase, and then isolating the DNA encoding the heavy and/or light chains of the antibody, respectively.

Extraction of mRNA can be performed by the guanidinium thiocyanate-hot phenol method, the guanidinium thiocyanate-guanidinium HC1 method, or the like, but the guanidinium thiocyanate-cesium chloride method is preferred. Preparation of mRNAfrom cells is generally performed by first preparing total RNA and then purifying mRNAfrom the total RNA by using a poly(A)⁺ RNA purification matrix, such as oligo(dT) cellulose and oligo (dT) latex beads. Alternatively, mRNAmay be prepared directly from a cell lysate using such a matrix. Methods for preparing total RNA include: alkaline sucrose density gradient centrifugation [e.g. Dougherty, W. G. and Hiebert, E. (1980), Virology, 101, 466-474]; the guanidinium thiocyanate-phenol method; the guanidinium thiocyanate-trifluorocesium method; and the phenol-SDS method. The method using guanidinium thiocyanate and cesium chloride [e.g. Chirgwin, J. M., et al., (1979), Biochemistry, 18, 5294-5299] is preferable.

The thus obtained poly(A)⁺ RNA can be used as the template in a reverse transcriptase reaction to prepare single-strand cDNA which can then be converted to double stranded cDNA. Suitable methods therefor include the SI nuclease method [e.g. Efstratiadis. A., et al., (1976), Cell, 7, 279-288], the Gubler-Hoffman method [e.g. Gubler. U. and Hoffman, B. J., (1983), Gene, 25, 263-269] and the Okayama-Berg method [e.g. Okayama. H. and Berg, P., (1982). Mol. Cell. Biol, 2, 161-170]. However, the preferred method involves the polymerase chain reaction [hereinafter referred to as PCR, e.g. Saiki, R. K., et al., (1988), Science, 239, 487-49] using single-strand cDNAas the template, namely "RT PCR". The double-strand cDNA obtained above may then be integrated into a cloning vector and the resulting recombinant vector can then be used to transform a micro-organism, such as E. coli. The transformant can be selected using tetracycline resistance or ampicillin resistance. If E. coli is used, then transformation may be effected by the Hanahan method [e.g. Hanahan, D., (1983), J. Mol. Biol., 166, 557-580]. Namely, the recombinant vector may be introduced into competent cells prepared by co-exposure to calcium chloride, magnesium chloride or rubidium chloride. If a plasmid is used as a vector, it is necessary that the plasmid harbors a drug-resistant gene, such as mentioned above. It is also possible to use other cloning vehicles, such as lambda phages.

In order to select transformants for those which carry cDNA encoding a subunit of an anti-Fas antibody of interest, various methods, such as those described below, can be used. When the cDNAof interest is specifically amplified by the above-mentioned RT-PCR, these steps may be omitted.

(1) Method by polymerase chain reaction

If all or part of the amino acid sequence of the desired protein has been elucidated, then sense and antisense oligonucleotide primers corresponding to parts of the amino acid sequence can be synthesized, and used in the polymerase chain reaction technique [e.g. Saiki, R. K., et al. (1988), Science, 239, 487-49] to amplify the desired DNA fragment encoding the anti-human Fas monoclonal antibody light chain subunit and heavy chain subunit. The template DNA may be, for example, cDNA synthesized by reverse transcription from mRNA of the hybridoma producing the anti-human Fas monoclonal antibody HFE7A(FERM BP-5828).

The DNA fragment thus synthesized may either be directly integrated into a plasmid vector by using a commercially available kit or the like, or may be labelled with, for example, P, S or biotin, and then used as a probe for colony hybridization or plaque hybridization to obtain the desired clone.

Anti-human Fas monoclonal antibody HFE7A is an immunoglobulin Gl (hereinafter referred to as "IgGl") comprising a heavy chain (γl chain) subunit and a light chain (K chain) subunit. The partial amino acid sequence of each of subunits mentioned above can be determined preferably by isolating each subunit by a well known method such as electrophoresis and column chromatography, and sequencing the N-terminal amino acid sequence of each subunit with an auto protein sequencer (for example, PPSQ-10 by Shimadzu Seisakusho, Corp.).

Harvesting of cDNA encoding each subunit of anti-human Fas monoclonal antibody from the appropriate transformants obtained above may be performed by well known techniques [e.g. Maniatis, T., et al., in "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory, NY, (1982)]. For example, the region of DNA encoding the desired subunit may be excised from plasmid DNA after separating the fraction corresponding to the vector DNA from cells.

(2) Screening using a synthetic oligonucleotide probe

If all or part of the amino acid sequence of the desired protein has been elucidated (the sequence can be that in any region of the protein, provided that is specific and contains continuous amino acids), oligonucleotides corresponding thereto may be synthesized, and used as a probe (after labelling with "P, S, biotin or the like), namely, hybridized with DNA from the transformant which has been immobilized on a nitrocellulose filter to select positive strains. As the probe, there can be used one oligonucleotide which is designed considering the frequency of codons in a host, or a mixture of possible oligonucleotides. In the latter case, the number of oligonucleotides to be used can be reduced by using inosine.

DNA thus obtained may be sequenced by, for example, the Maxam-Gilbert chemical modification technique [e.g. Maxam, A. M. and Gilbert. W. (1980) in "Methods in Enzymology" 65, 499-576], the dideoxy chain termination method [e.g. Messing J. and Vieira J. (1982) Gene, 19, 269-276] or the like.

In recent years, automated DNA sequencers using a fluorogenic dye have been widely used, for example Sequence robot "CATALYST 800" and the model 373A DNA Sequencer, manufactured by Perkin-Elmer Japan, Inc. By using systems such as those described above, determination of the DNA sequence can be performed efficiently and safely. Based on the data of the nucleotide sequences of the DNA of the present invention thus determined and the data of the N-terminal amino acid sequences of the heavy chain and the light chain thereof, the entire amino acid sequences of the heavy chain and the light chain of a monoclonal antibody of the present invention can be determined.

Construction of a mutant wherein one or more amino acids in an amino acid sequence is deleted may be performed, for example, by cassette mutagenesis [e.g. Toshimitsu Kishimoto, "Shin-Seikagaku Jikken Kouza 2: Kakusan III Kumikae DNA Gijutsu", 242-251].

Such DNA sequences may be prepared by chemical synthesis using a conventional method, such as the phosphite triester method [e.g. Hunkapiller, M., et al., (1984), Nature, 310, 105-111]. Codons for each amino acid themselves are known, and a specific codon for a desired amino acid may be selected arbitrarily, or by taking a frequency of a given codon in a host into account. Partial modification of the nucleotide sequence can be accomplished by conventional techniques, such as site-specific mutagenesis utilizing synthetic oligonucleotide primers encoding the desired modifications [e.g. Mark, D. E, et al., (1984), Proc. Natl. Acad. Sci. USA, 81, 5662-5666].

Whether DNA can be hybridized with DNA encoding the light chain or the heavy chain of the anti-Fas monoclonal antibody HFE7A can be determined, for example, by using a DNA probe labelled with (α-³²P)dCTP or the like, for example, by the random primer method [e.g. Feinberg, A. P. and Vogelstein, B. (1983), Anal. Biochem., 132, 6-13], by the nick translation method [e.g. Maniatis, T, et al., (1982), in "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory, NY] or the like. A suitable technique is as follows.

First, the DNA to be determined is adsorbed onto a nitrocellulose membrane or a nylon membrane, for example, being subjected to alkaline treatment if necessary, and then being fixed on the membrane by heating or UV irradiation. The membrane is next immersed in prehybridisation solution containing 6 x SSC (1 x SSC is a solution of 0.15 M NaCl and 0.015 M tri-sodium citrate), 5% v/v Denhardt solution and 0.1% v/v sodium dodecyl sulfate (SDS), and incubated at 55°C for 4 hours or more. Then, the probe previously prepared is added in similar prehybridisation solution to a final specific activity of 1 x 10⁶ cpm/ml, followed by incubation at 60°C overnight. Subsequently, the membrane is washed at room temperature by repeated washing with 6 x SSC for 5 minutes and further with 2 x SSC for 20 minutes, and is then subjected to autoradiography.

By using the above method, DNAhybridisable with the DNA encoding the heavy or light chain of the anti-Fas monoclonal antibody HFE7A can be isolated from any cDNA library or genomic library [e.g. Maniatis, T, et al., (1982), "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory, NY].

Integration of DNA thus obtained into an expression vector allows transformation of prokaryotic or eukaryotic host cells, thereby allowing the DNA to be expressed in the host cell.

Suitable prokaryotic host cells include, for example, Escherichia coli, Bacillus subtilis, and the like. In order to express the gene of interest in such host cells, these host cells may be transformed with a plasmid vector containing a replicon derived from a species compatible with the host, namely an origin of replication and a promoter sequence, such as lac UV5. These vectors preferably have sequences capable of conferring a selection phenotype on the transformed cell. A suitable strain of E. coli is strain JM109 derived from E. coli K12. Suitable vectors include pBR322 and the pUC series plasmids, without being limited thereto. Other known strains and vectors can also be utilized. Suitable promoters include the tryptophan (trp) promoter, the lactose promoter (lac), the tryptophan lactose promoter (tac), the lipoprotein promoter (lpp), the lambda (λ) PL promoter derived from bacteriophage, and the polypeptide chain elongation factor Tu (tufB) promoter, without being limited thereto.

A preferred strain of Bacillus subtilis is strain 207-25, and a preferred vector is pTUB228 [e.g. Ohmura, K., et al., (1984), J. Biochem., 95, 87-93], without being limited thereto. A suitable promoter is the regulatory sequence of the Bacillus subtilis α-amylase gene. If desired, the DNA sequence encoding the signal peptide sequence of α-amylase may be linked to the DNA of the present invention to enable extracellular secretion.

Eukaryotic hosts include cells from vertebrate and yeast species. An example of vertebrate cells used is the monkey COS-1 cell line [e.g. Gluzman, Y, (1981), Cell, 23, 175-182]. Suitable yeast cell hosts include baker's yeast (Saccharomyces cerevisiae), methylotrophic yeast (Pichia pastoris) and fission yeast (Schizosaccharomyces pombe). The cells mentioned above are generally used as the host cell, but the host cell to be used is not limited thereto.

In general, the requirements for suitable expression vectors for vertebrate cells are that they comprise: a promoter, usually located upstream of the gene to be expressed; an RNA splicing site; a polyadenylatiori site; and a transcription termination sequence; and an origin of replication if necessary. A suitable plasmid is, for example, pSV2dhfr containing the SV40 early promoter [e.g. Subramani, S., et. al. (1981), Mol. Cell. Bio., 1, 854-864], without being limited thereto.

Suitable expression vectors for yeasts contain, for example, the promoter of the alcohol dehydrogenase gene [e.g. Bennetzen, J. L. and Hall, B. D., (1982), J. Biol. Chem., 257, 3018-3025) or the promoter of a galactose metabolic enzyme (for example, gal 10) [e.g. Ichikawa, K., et. al. (1993), Biosci. Biotech. Biochem., 57, 1686-1690], without being limited thereto. If desired, the DNA sequence encoding the signal peptide sequence of a yeast gene may be linked to the DNA to be expressed in order to enable extracellular secretion.

When COS-1 cells are used as a host cell, expression vectors suitably comprise the SV40 replication origin, enabling autonomous replication, a transcription promoter, a transcription termination signal and an RNA splicing site. The said expression vectors can be used to transform the COS-1 cells by any suitable method, such as the DEAE-dextran method [e.g. Luthman. H, and Magnusson. G (1983), Nucleic Acids Res., 11, 1295-1308], the phosphate calcium-DNA co-precipitation method [e.g. Graham, F. L. and Van der Eb, A. J., (1973), Virology, 52, 456-457] and the electric pulse electroporation method [e.g. Neumann, E., et. al., (1982), EMBO J, 1, 841-845]. A desired transformant can be obtained by these methods.

Preferably, COS-1 cells are co-transfected with two expression vectors: one containing DNA encoding the heavy chain and the other containing DNA encoding the light chain, to provide a transformant producing the recombinant anti-Fas antibody. However, the method of producing the recombinant anti-Fas antibody is not limited thereto. For example, it is also possible to construct only one expression vector containing both the DNA encoding the heavy chain and the DNA encoding the light chain, which is expressed simultaneously, and to transfect COS-1 cells therewith.

Desired transformants obtained by the above methods may be cultured using conventional methods, the recombinant anti-Fas antibody being expressed either intra- or extra- cellularly. Suitable culture media include various commonly used media, depending on the host chosen. For example, suitable media for COS-1 cells include RPMI-1640 and Dulbecco's Modified Eagle Medium (DMEM), which can be supplemented with, as desired, fetal calf serum (FCS).

The culture temperature for culturing the transformant may be any suitable temperature which does not markedly depress the protein synthesis capability of the cell, and is preferably in the range of 32 to 42°C, most preferably 37°C. If desired, culture may be effected in an atmosphere containing 1 to 10% (v/v) carbon dioxide.

The fraction containing the anti-Fas antibody protein produced intra- or extra-cellularly by the transformants as described above may be isolated and purified by various well known methods of separation according to the physical and chemical properties of the protein. Suitable specific methods of separation include: treatment with commonly used precipitating agents for protein; various methods of chromatography such as ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography (HPLC), dialysis and combinations thereof.

In order to produce humanized anti-human Fas monoclonal antibody HFE7A, known procedures such as those disclosed in EP-A-0909816, EP-A-0990663 or Japanese patent application laid open (kokai) No.2001-342148 can be used.

According to the methods described above, the highly pure recombinant, anti-human Fas monoclonal antibody HFE7A and humanized anti-human Fas monoclonal antibody HFE7A can be readily produced in high yields.

In order to confirm that a recombinant anti-Fas antibody prepared by the above methods specifically binds to Fas, ELISA may be preferably performed in a manner similar to that described above for the evaluation of antibody titers in immunized mice.

The anti-human Fas monoclonal antibody of the present invention also includes recombinants of these anti-human Fas monoclonal antibodies, which have an effect equivalent to these monoclonal antibodies. Moreover, as stated above, there can also be used a so-called humanized antibody modified using gene recombination technology so that the immunogenicity to humans may be reduced, without deteriorating the binding ability of the above-mentioned anti-Fas monoclonal antibody to bind to Fas.

Of these, humanized anti-human Fas monoclonal antibody HFE7A prepared as disclosed in the examples of EP-A-0990663 and Japanese patent application laid open (kokai) No. 2001-342148 are preferred for use in the pharmaceutical composition and method of prevention or treatment of bone erosion of the present invention, and particularly preferred is the humanized anti-human Fas monoclonal antibody "h-HFE7A" manufactured using recombinant technology from a humanized light chain [SEQ ID number 107 described in EP-A-0990663 and Japanese patent application laid open (kokai) No. 2001-342148] and a humanized heavy chain [SEQ ID number 117 described in EP-A-0990663 and Japanese patent application laid open (kokai) No. 2001-342148] obtainable from the recombinant plasmids carried by transformed strains E. coli pHSHH5 SANK 70398 and E. coli pgHPDHV3 SANK 70298 respectively that were deposited under the Budapest Treaty on 26 February 1998 in Kogyo Gijutsuin Seimei-Kogaku Kogyo Gijutsu Kenkyujo (National Institute of Advanced Industrial Science and Technology, International Patent Depositary) at 1-1-3, Higashi-cho, Tsukuba-shi, Ibaraki-ken, Japan and accorded the accession numbers FΕRM BP-6274 and FΕRM BP-6273 respectively.

The anti-human Fas monoclonal antibody HFΕ7A and the humanized anti-human Fas monoclonal antibody HFE7A used in the pharmaceutical composition and method of treatment or prevention of bone erosion of the present invention do not induce liver disorders and cause few side effects. This is particularly true of humanized versions of anti-human Fas monoclonal antibody HFE7Athat have reduced immunogenicity to humans. Because of this, the pharmaceutical composition and method of treatment or prevention of the present invention may use the anti-human Fas monoclonal antibody HFE7A or the humanized anti-human Fas monoclonal antibody HFE7A in combination with other anti-inflammatory agents used in the treatment or prevention of bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis. Suitable examples include non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, piroxicam, indomethacin, ibuprofen and naproxen, and disease-modifying antirheumatic drugs (DMARDS) such as methotrexate, chloroquine, hydrochloroquine, cyclosporin, penicillamine, sulphasalazine, azathioprine and leflunomide.

Agents that can reinforce the activity of the anti-human Fas monoclonal antibody HFE7A or the humanized anti-human Fas monoclonal antibody HFE7A can also favorably be used therewith, e.g. by incorporating said agents into the pharmaceutical composition of the present invention or by administering them simultaneously with said pharmaceutical composition of the present invention. By using such an agent, the amount of the anti-human Fas monoclonal antibody HFE7A or the humanized anti-human Fas monoclonal antibody HFE7Ato be used can be decreased. In this way, the possibility that a patient becomes tolerant to the anti-human Fas monoclonal antibody HFE7A or the humanized anti-human Fas monoclonal antibody HFE7Acan be decreased. Suitable examples of agents that can be used to reinforce the activity of the anti-human Fas monoclonal antibody HFE7A or the humanized anti-humaή Fas monoclonal antibody HFE7A include interferon-γ and compounds having a folate antagonist activity or a dihydrofolate reductase inhibiting activity, preferred examples being selected from the group consisting of: methotrexate, edatrexate, epiroprim, iometrexol, pyritrexim, trimetrexate, brodimoprim, MX-68, N-[4-[3-(2,4-diamino-6,7-dihydro-5H- cyclopenta[d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid, N-[[5-[2-(2-amino- l,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]-pyrimidin-6-yl)ethyl-2-thienyl]carbonyl]- L-glutamic acid, (R)-N-[[5-[2-(2-amino-l,4,5,6,7,8-hexahydro-4-oxopyrido- [2,3-d]pyrimidin-6-yl)ethyl-2-thienyl]carbonyl]-L-glutamic acid, N-((2,4-diamino- 3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-6-yl)-ethyl)-2-thienylcarbonyl-L- glutamic acid, (S)-2- [[ [4-carboxy-4- [ [4- [ [(2,4-diamino-6-pteridinyl)methyl] - amino]benzoyl]amino]butyl]amino]carbonyl]benzoic acid, N-[4-[3-(2,4-diamino-lH- pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid, 2,4-diamino-6-(N-(4- (phenylsulfonyl)benzyl)methylamino)quinazoline, 2,4-diamino-5-[4-[3-(4- aminophenyl-4-sulfonylphenylamino)propoxy-3,5-dimethoxybenzyl]-pyrimidine, N-[4-[4-(2,4-diamino-5-pyrimidinyl)butyl]benzoyl]-L-glutamic acid, N-[4-[3-(2,4-diamino-5-pyrimidinyl)propyl]benzoyl]-L-glutamic acid, N-[4-[2-(2,4-diamino-6-pteridinyl)ethyl]benzoyl]-4-methylene-DL-glutamic acid and N-(l-methylethyl)-N'[3-(2,4,5-trichlorophenoxy)propoxy]imidodicarbonimidic diamide hydrochloride (PS 15). Of these, methotrexate is particularly preferred.

The pharmaceutical composition of the present invention can be used as a therapeutic or preventive agent for bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis. The pharmaceutical composition can be administered in various forms. Where the pharmaceutical composition comprises the anti-human Fas monoclonal antibody HFE7A or the humanized anti-human Fas monoclonal antibody HFE7A in combination with another agent such as a compound having folate antagonist activity or a dihydrofolate reductase inhibiting activity then the active agents can be administered in admixture, separately but simultaneously or separately and successively. The pharmaceutical composition may be administered in various forms. Examples of such forms include oral administration, with tablets, capsules, granules, powders, syrups or the like, and parenteral administration, with injection, dropping injection, suppositories or the like. The pharmaceutical composition can be prepared according to conventional methods using known additives which are generally used in the field of the preparation of pharmaceuticals such as excipients, binding agents, disintegrants, lubricants, flavoring agents, solubilizing agents, suspension agents and coating agents.

When formulated as an injection, a solution or a suspension of said formulation is preferably sterilized, and is made isotonic with blood. In preparation of the solution, emulsion or suspension, a variety of diluents known in the art can be used, including, for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In this case, sodium chloride, glucose, or glycerol in an amount enough to maintain the isotonicity may be added to the pharmaceutical composition. Furthermore, solubilizing agents, buffers, soothing agents or the like may also be added thereto.

Moreover, if needed, the preparation may contain coloring agents, preservatives, perfumes, flavoring agents, sweeteners or other medicines, or the like.

The amount of anti-human Fas monoclonal antibody HFE7A in the pharmaceutical composition of the present invention will vary according to factors such as the additives that are used and the dosage form. The does of said antibody will vary depending on factors such as the condition, age and body weight of the patient, but usually it may be administered in an amount of from 1 mg to 100 mg of the antibody which may, for example, be administered in a single dose by a subcutaneous, intramuscular or intravenous injection.

Confirmation that the pharmaceutical composition of the present mvention prepared as described above is useful as a therapeutic or preventive agent for bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis can be provided by a novel in vivo test method that involves transplanting synovial tissue originating from an affected part of a patient suffering from said disease and a piece of dentme into a primary immunodeficient non-human animal (e.g. a mouse such as a SCID mouse), and observing suppression of the formation of resorption pits on the pieces of the transplanted dentine when the pharmaceutical composition of the present invention that is being tested has been administered to the test animal. More details on how this novel in vivo test for a therapeutic or preventive agent for bone erosion can be carried out are described below.

In the first step of the test method, a piece of dentine (e.g. ivory) and a piece of synovial tissue originating from an affected part of a patient suffering from a disease that activates osteoclasts such as rheumatoid arthritis are transplanted to an immunodeficient non-human laboratory animal so that said piece of dentine and said piece of synovial tissue are kept in mutual contact in the test animal. Suitable immunodeficient animals that can be used as the test animals include primary immunodeficient mice of the SCID substrain such as CB-17/Icr Crj-scid, BALB/cA-scid and BALB/cA-bg, scid (obtainable, for example, from Nihon Charles River Co., Ltd.), and CB-17/Icr Crj-scid mice are preferred without being limited thereto.

The size and shape of the piece of dentine to be used is not particularly limited, but it should have a size and shape that makes it possible for it to be transplanted into the test animal to be used, and it should be suitable for observation under a microscope of any resorption pits that are formed during the test. It should have at least one plane suitable for observation of the resorption pits. For example, when transplanting to an immunodeficient mouse such as a CB-17/ Icr Crj-scid mouse, the piece of dentine is preferably a sheet with a thickness of 100 μm - 250 μm, either in the shape of a disk with a diameter of 4-8 mm or a rectangle with a length of 4-8 mm.

After transplantation of the piece of dentine into the test animal, the substance to be tested for its effectiveness as an agent for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts (e.g. rheumatoid arthritis) is administered to the animal to which the piece of dentine and the synovial tissue have been transplanted as described above. Administration of said test substance can be varied according to the characteristics of the compound to be tested. For example, administration can be via the oral, intravenous, subcutaneous or intramuscular route or it can be performed by embedding the test substance in the part of the animal where the dentine and synovial tissue are transplanted or a part close to the transplant site. Administration conditions, such as the dosage, interval and frequency of administration of the substance to be tested, are set up as is suitable for the particular test to be performed. For comparison, either a control substance such as a known medicine or the same solvent as is used for dissolving the substance to be tested, or nothing at all is administered to at least one further animal into which another piece of dentine and synovial cells have been transplanted in identical manner and at the same time as the first test animal described above.

After breeding the animals treated as described above for the period of the test, the pieces of dentine are retrieved from the test animals, washed and stained (e.g. using hematoxylin), and the number of resorption pits formed on the surface that has been in contact with the synovial tissue for each of the dentine pieces is counted by observing under a microscope. The numbers of resorption pits are compared between the control animals and the animals to which the test substance has not been administered or the control medicine has been administered. The test substances that suppress the formation of resorption pits are selected as candidate substances for use as an agent for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis.

Using the above mentioned method, it has been possible to confirm that the anti-human Fas monoclonal antibody HFE7A and humanized anti-human Fas monoclonal antibody HFE7A are useful as active ingredients of agents for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis.

Brief Description of the Drawings

Figure 1 is a graph showing the suppressing effect of a humanized anti-Fas monoclonal antibody HFE7A on the formation of resorption pits using IgG as a control.

Figure 2 is a graph showing the suppressing effect of a humanized anti-Fas monoclonal antibody HFE7A on the formation of resorption pits using PBS as a control. Best Mode for Carrying Out the Invention

The present invention will now be illustrated by the following Examples. It will be understood that the scope of the present invention is not limited by these Examples.

Example 1 Preparation of a Humanized Anti-Human Fas Monoclonal Antibody HFE7A

According to the procedures described in the examples of EP-A-0990663 and Japanese patent application laid open (kokai) No. 2001-342148, a mammalian cell line prepared from COS-1 cells derived from a monkey kidney that produced a humanized light chain [SEQ ID number 107 described in the sequence listing of EP-A-0990663 and Japanese patent application laid open (kokai) No. 2001-342148] and a humanized heavy chain [SEQ ID number 117 described in the sequence listing of EP-A-0990663 and Japanese patent application laid open (kokai) No. 2001-342148] of the anti-human Fas antibody HFE7A was manufactured by using recombinant DNA technology, said light and heavy chains being obtained from the recombinant plasmids carried by transformed strains E. coli pHSHH5 SANK 70398 and E. coli pgHPDHV3 SANK 70298 respectively that were deposited under the Budapest Treaty on 26 February 1998 in Kogyo Gijutsuin Seimei-Kogaku Kogyo Gijutsu Kenkyujo (National Institute of Advanced Industrial Science and Technology, International Patent Depositary) at 1-1-3, Higashi-cho, Tsukuba-shi, Ibaraki-ken, Japan and accorded the accession numbers FERM BP-6274 and FERM BP-6273 respectively. A humanized anti-human Fas monoclonal antibody HFE7A (hereinafter referred to as "h-HFE7A") was isolated and purified from this cell line as described in the examples of EP-A-0990663 and Japanese patent application laid open (kokai) No. 2001-342148. Example 2

Test for the Effectivness of a Humanized Anti-Human Fas Monoclonal

Antibody HFE7A as an Agent for the Treatment or Prevention of Bone

Erosion Caused by Rheumatoid Arthritis Using IgG as a Control

Synovial tissue was surgically removed from an affected joint of a patient suffering from rheumatoid arthritis. The synovial tissue was wrapped in a piece of gauze that was moistened with minimal essential medium and then stored at 4°C until it was needed.

Two five-week old male CB-17/Icr Crj-scid mice (hereinafter referred to as "SCID mouse", purchased from Nihon Charles River Co., Ltd.) were allowed to acclimatize to the laboratory conditions for one week. At the end of this time, each SCID mouse was anesthetized with diethyl ether and then given an incision hypodermically on its back with a pair of sterilized scissors. An ivory piece, prepared as described below, was covered with a portion of the synovial tissue (about 0.5 g) obtained as described above from a patient suffering from rheumatoid arthritis and then, inserted into the incision so that the synovial tissue and one face of the ivory piece were in mutual contact. After the transplant, the incised skin on the back of each mouse was closed using surgical sutures.

The ivory piece transplanted into each animal was prepared as follows. A piece of ivory was obtained by slicing an ivory block (purchased from Shin-nihon-zougesha) using a precision low-speed cutting machine (Isomet, manufactured by Bular Co., Ltd.) so as to give a slice having a thickness of 150 to 200 μm. This slice was then pressed for several days so that it was completely plain, punched to give a disk having a diameter of 6 mm using an eyelet punch (manufactured by TOHO Co., Ltd.) and then sterilized by ultrasonic cleaning in 70% ethanol (using Sonifier 450 manufactured by Branson Co., Ltd., 10 minute x 3 times). The ivory slice was dipped and washed in physiological saline before the synovial tissue was placed thereon and transplanted into the mouse.

Concentrated stock of h-HFE7A (4.63 mg/ml), prepared as described in Example 1 above, was diluted using phosphate buffered saline (PBS) to give a solution containing lmg/ml of h-HFE7A. This solution was administered intravenously to one of the mice (the test mouse) into which an ivory slice and synovial tissue had been transplanted at a level of 200 μg/mouse of h-HFE7Aper administration, administration being once a week and 3 times in total (namely, on the day of the transplantation, and then on the 8th day and the 15th day therefrom). To the other mouse (the control mouse), 200 μg/mouse of human IgG (obtained from COSMOBIO, catalogue number OBMOHPOIO; the IgG was diluted with PBS from a stock solution having a concentration of 1.39 mg/ml to a concentration of 1 mg/ml) were administered in the same manner and on the same days as for the test mouse. At the end of the third week (i.e. on the 21st day after transplantation), the test mouse and the control mouse were sacrificed by draining blood from the hearts of the animals under anesthesia. The transplanted synovial tissue and the ivory slice were then removed from each animal using a pair of scissors and a pincette, taking care to ensure that no mouse tissue became mixed therewith.

Each ivory slice was put into a well of a 96-well plate. Distilled water was added to each well, and both sides of each slice were washed twice, for 5 seconds each time, using a hand-held motor (available from Harayoshi Shouten) before dipping the slice in physiological saline (manufactured by Otsuka Pharmaceutical). Subsequently, each ivory slice was stained for 13 minutes in the acid hematoxylin (manufactured by Sigma Corporation), and then both sides thereof were again washed twice, for 5 seconds each time, using the hand-held motor.

The formation of resorption pits on each ivory slice due to osteoclasts present in the synovial tissue that was in contact with one face of said ivory slice was observed under a microscope, and the number of the resorption pits that were stained purple-blue in colour was counted. The averaged counts for the total number of pits on the ivory slices recovered from the test mouse and the control mouse are shown in Figure 1. It was found that an average of 73.3 resorption pits were formed on the ivory slice in the control mouse to which human IgG was administered, whereas the number of resorption pits was suppressed to an average of 3.8 per ivory slice in the test mouse to which h-HFE7A was administered. Example 3

Test for the Effectivness of a Humanized Anti-Human Fas Monoclonal

Antibody HFE7A as an Agent for the Treatment or Prevention of Bone

Erosion Caused by Rheumatoid Arthritis Using PBS as a Control

This Example was conducted in a very similar manner to Example 2 above. The only differences are as follows:

1. Instead of having just one mouse in each of the test and control groups, the experiment was conducted three times, the first time with 5 animals in each of the test and control groups, the second time with 2 animals in each of the test and control groups and the third time with 5 animals in the test group and 4 animals in the control group.

2. The dose of h-HFE7A for each administration to the test group was 10 mg/kg body weight of each test mouse instead of 200 μg/mouse.

3. In the control group, instead of administering IgG as the control, each mouse of the control group was administered PBS (10 mg/kg body weight intravenously).

The averaged results for the numbers of resorption pits counted on the ivory slices recovered from the mice in the three test groups and the three control groups are shown in Figure 2. It was found that an average of 367.5 resorption pits were formed on the ivory slice in each control mouse to which PBS was administered, whereas the number of resorption pits was suppressed to an average of 58.6 per ivory slice in each test mouse to which h-HFE7A was administered.

From Examples 2 and 3, it can be seen that anti-human monoclonal antibody HFE7A, and especially humanized versions thereof have excellent activity in preventing the formation by osteoclasts of resorption pits in bone, thus demonstrating that it has excellent potential as an agent for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis. Formulation Example

The anti-human Fas monoclonal antibody HFE7A and humanized anti-human Fas monoclonal antibody HFE7Ain the preventive or therapeutic agent of the present invention can be prepared as an ampoule of a sterile solution or suspension wherein it is dissolved or suspended in water or another pharmaceutically acceptable solvent. For example, 0.5 mg of humanized anti-human Fas antibody HFE7A can be dissolved in 1 liter of water, the resulting solution sterilely filled into an ampoule and sealed. Alternatively, a sterile powder preparation (which is preferably obtained by freeze-drying the anti-human Fas monoclonal antibody HFE7A or humanized anti-human Fas monoclonal antibody HFE7A contained as an active ingredient in the preventive or therapeutic agent of the present invention) may be put in an ampoule, so that the powder at the time of use is dissolved or suspended in a pharmaceutically acceptable solvent.

Advantages of the Invention

The present invention provides a novel pharmaceutical composition useful for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts such as rheumatoid arthritis. Furthermore, it also provides a method for testing in vivo the effectiveness of a substance as an agent for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts.

Claims

1. A pharmaceutical composition for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising a pharmaceutically carrier acceptable or diluent and an anti-human Fas monoclonal antibody HFE7A or a humanized anti-human Fas monoclonal antibody HFE7A.

2. A pharmaceutical composition according to claim 1, wherein said monoclonal antibody is a humanized anti-human Fas monoclonal antibody HFE7A.

3. A pharmaceutical composition according to claim 2, wherein said humanized anti- human Fas monoclonal antibody HFE7A is manufactured using recombinant technology from a humanized light chain and a humanized heavy chain obtainable from the recombinant plasmids carried by transformed strains E. coli pHSHH5 SANK 70398 (FERM BP-6274) and E. coli pgHPDHV3 SANK 70298 (FERM BP-6273) respectively.

4. A pharmaceutical composition according to any one of claims 1 to 3 which further comprises at least one anti-inflammatory agent selected from non-steroidal anti-inflammatory drugs and disease-modifying antirheumatic drugs.

5. A pharmaceutical composition according to claim 4, wherein said non-steroidal anti- inflammatory drugs are selected from the group consisting of diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, piroxicam, indomethacin, ibuprofen and naproxen, and said disease-modifying antirheumatic drugs are selected from the group consisting of methotrexate, chloroquine, hydrochloroquine, cyclosporin, penicillamine, sulphasalazine, azathioprine and leflunomide.

6. A pharmaceutical composition according to any one of claims 1 to 5 which further comprises at least one compound selected from the group consisting of interferon-γ and compounds having a folate antagonist activity or a dihydrofolate antagonist activity.

7. A pharmaceutical composition according to claim 6, wherein said compounds having a folate antagonist activity or a dihydrofolate antagonist activity are selected from the group consisting of methotrexate, edatrexate, epiroprim, iometrexol, pyritrexim, trimetrexate, brodimoprim, MX-68, N-[4-[3-(2,4-diamino-6,7- dihydro-5H-cyclopenta[d]pyrimidin-5- yl)propyl]benzoyl] -L-glutamic acid, N- [ [5 - [2-(2-amino- 1,4,5 ,6,7, 8-hexahydro-4- oxopyrido[2,3-d]-pyrimidin-6-yl)ethyl-2-thienyl]carbonyl]- L-glutamic acid, (R)-N-[[5-[2-(2- amino-l,4,5,6,7,8-hexahydro- 4-oxopyrido-[2,3-d]pyrimidin-6-yl)ethyl-2-thienyl]carbonyl]- L-glutamic acid, N-((2,4-diamino-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-6-yl)-ethyl)- 2- thienylcarbonyl-L- glutamic acid, (S)-2-[[[4-carboxy-4-[[4-[[(2,4-diamino-6- pteridinyl)methyl]amino]benzoyl]amino]butyl]amino]carbonyl]benzoic acid, N-[4-[3-(2,4- diamino-lH- pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid, 2,4-diamino-6- (N-(4- (phenylsulfonyl)benzyl)methylamino)quinazoline, 2,4-diamino-5-[4-[3-(4- aminophenyl-4-sulf onylphenylamino)propoxy-3,5 - dimethoxybenzyl]pyrimidine, N- [4- [4- (2,4-diamino-5-pyrimidinyl)butyl]benzoyl]-L- glutamic acid, N-[4-[3-(2,4-diamino-5- pyrimidinyl)propyl]benzoyl]-L- glutamic acid, N-[4-[2-(2,4-diamino-6- pteridinyl)ethyl]benzoyl]-4-methylene-DL-glutamic acid and N-(l-methylethyl)-N'[3-(2,4,5- trichlorophenoxy)propoxy]imidodicarbonimidic diamide hydrochloride (PS15).

8. A pharmaceutical composition according to claim 6, wherein said compound having a folate antagonist activity or a dihydrofolate antagonist activity is methotrexate.

9. A pharmaceutical composition according to any one of claims 1 to 8, wherein said disease that activates osteoclasts is rheumatoid arthritis.

10. Use of an anti-human Fas monoclonal antibody HFE7A or a humanized anti-human Fas monoclonal antibody HFE7A in the manufacture of a medicament for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts.

11. Use according to claim 10, wherein said monoclonal antibody is a humanized anti- human Fas monoclonal antibody HFE7A.

12. Use according to claim 11, wherein said humanized anti-human Fas monoclonal antibody HFE7A is manufactured using recombinant technology from a humanized light chain and a humanized heavy chain obtainable from the recombinant plasmids carried by transformed strains E. coli pHSHH5 SANK 70398 (FERM BP-6274) and E. coli pgHPDHV3 SANK 70298 (FERM BP-6273) respectively.

13. Use according to any one of claims 10 to 12, wherein said medicament further comprises at least one anti-inflammatory agent selected from non-steroidal anti-inflammatory drugs and disease-modifying antirheumatic drugs.

14. Use according to claim 13, wherein said non-steroidal anti-inflammatory drugs are selected from the group consisting of diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, piroxicam, indomethacin, ibuprofen and naproxen, and said disease- modifying antirheumatic drugs are selected from the group consisting of methotrexate, chloroquine, hydrochloroquine, cyclosporin, penicillamine, sulphasalazine, azathioprine and leflunomide.

15. Use according to any one of claims 10 to 14, wherein said medicament further comprises at least one compound selected from the group consisting of interferon-γ and compounds having a folate antagonist activity or a dihydrofolate antagonist activity.

16. Use according to claim 15, wherein said compounds having a folate antagonist activity or a dihydrofolate antagonist activity are selected from the group consisting of methotrexate, edatrexate, epiroprim, iometrexol, pyritrexim, trimetrexate, brodimoprim, MX-68, N-[4-[3- (2,4-diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidin- 5-yl)propyl]benzoyl]-L-glutamic acid, N-[[5-[2-(2-amino-l,4,5,6,7,8-hexahydro-4- oxopyrido[2,3-d]-pyrimidm-6-yl)efhyl-2- thienyljcarbonyl]- L-glutamic acid, (R)-N-[[5-[2-(2-amino-l,4,5,6,7,8-hexahydro-4- oxopyrido-[2,3-d]pyrimidin-6- yl)ethyl-2-thienyl]carbonyl] -L-glutamic acid, N-((2,4- diamino-3,4,5, 6,7,8- hexahydropyrido[2,3-d]pyrimidin-6-yl)-ethyl)-2- thienylcarbonyl-L- glutamic acid, (S)-2-[[[4-carboxy-4-[[4-[[(2,4-diamino-6- pteridinyl)methyl]amino]benzoyl] amino]- butyl] amino] carbonyljbenzoic acid, N-[4-[3-(2,4- diamino-lH-pyrrolo[2,3- d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid, 2,4-diamino-6- (N-(4- (phenylsulfonyl)benzyl)methylamino)quinazoline, 2,4-diamino-5-[4-[3- (4- aminophenyl-4-sulfonylphenylamino)propoxy-3,5- dimethoxybenzyl]pyrimidine, N-[4-[4- (2,4-diamino-5-pyrimidinyl)butyl]benzoyl]-L- glutamic acid, N-[4-[3-(2,4-diamino-5- pyrimidinyl)propyl]benzoyl]-L-glutamic acid, N-[4-[2-(2,4-diamino-6- pteridinyl)ethyl]benzoyl]-4-methylene-DL-glutamic acid and N-(l-methylethyl)-N'[3-(2,4,5- trichlorophenoxy)propoxy]imidodicarbonimidic diamide hydrochloride (PS15).

17. Use according to claim 15, wherein said compound having a folate antagonist activity or a dihydrofolate antagonist activity is methotrexate.

18. Use according to any one of claims 10 to 17, wherein said disease that activates osteoclasts is rheumatoid arthritis.

19. A method for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising administering to a patient suffering from bone erosion an effective amount of an anti-human Fas monoclonal antibody HFE7A or a humanized anti- human Fas monoclonal antibody HFE7A.

20. A method according to claim 19, wherein said monoclonal antibody is a humanized anti-human Fas monoclonal antibody HFE7A.

21. A method according to claim 20, wherein said humanized anti-human Fas monoclonal antibody HFE7A is manufactured using recombinant technology from a humanized light chain and a humanized heavy chain obtainable from the recombinant plasmids carried by transformed strains E. coli pHSHH5 SANK 70398 (FERM BP-6274) and E. coli pgHPDHV3 SANK 70298 (FERM BP-6273) respectively.

22. A method according to any one of claims 19 to 21, which further comprises administering to said patient at least one anti-inflammatory agent selected from non-steroidal anti-inflammatory drugs and disease-modifying antirheumatic drugs.

23. A method according to claim 22, wherein said non-steroidal anti-inflammatory drugs are selected from the group consisting of diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, piroxicam, indomethacin, ibuprofen and naproxen, and said disease- modifying antirheumatic drugs are selected from the group consisting of methotrexate, chloroquine, hydrochloroquine, cyclosporin, penicillamine, sulphasalazine, azathioprine and leflunomide.

24. A method according to any one of claims 19 to 23, which further comprises administering to said patient at least one compound selected from the group consisting of interferon-γ and compounds having a folate antagonist activity or a dihydrofolate antagonist activity.

25. A method according to claim 24, wherein said compounds having a folate antagonist activity or a dihydrofolate antagonist activity are selected from the group consisting of methotrexate, edatrexate, epiroprim, iometrexol, pyritrexim, trimetrexate, brodimoprim, MX- 68, N-[4-[3-(2,4-diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidin- 5-yl)propyl]benzoyl]-L- glutamic acid, N-[[5-[2-(2-amino-l,4,5,6,7,8-hexahydro-4- oxopyrido[2,3-d]-pyrimidin-6- yl)ethyl-2-thienyl]carbonyl]- L-glutamic acid, (R)-N-[[5-[2-(2-amino-l,4,5,6,7,8-hexahydro- 4-oxopyrido-[2,3-d]pyrimidin-6- yl)ethyl-2-thienyl]carbonyl] -L-glutamic acid, N-((2,4- diamino-3,4,5,6,7,8- hexahydropyrido[2,3-d]pyrimidin-6-yl)-ethyl)-2- thienylcarbonyl-L- glutamic acid, (S)-2-[[[4-carboxy-4-[[4-[[(2,4-diamino-6- pteridinyl)methyl]amino]benzoyl] amino]- butyι]amino]carbonyl]benzoic acid, N-[4-[3-(2,4- diamino-lH-pyrrolo[2,3- d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid, 2,4-diamino-6- (N-(4- (phenylsulfonyl)benzyl)methylamino)quinazoline, 2,4-diamino-5-[4-[3- (4- aminophenyl-4-sulfonylphenylamino)propoxy-3,5- dimethoxybenzyl]pyrimidine, N-[4-[4- (2,4-diamino-5-pyrimidinyl)butyl]benzoyl]-L- glutamic acid, N-[4-[3-(2,4-diamino-5- pyrimidinyl)propyl]benzoyl] -L-glutamic acid, N-[4-[2-(2,4-diamino-6- pteridinyl)ethyl]benzoyl]-4-methylene-DL-glutamic acid and N-(l-methylethyl)-N'[3-(2,4,5- trichlorophenoxy)propoxy]imidodicarbonimidic diamide hydrochloride (PS15).

26. A method according to claim 24, wherein said comound having a folate antagonist activity or a dihydrofolate antagonist activity is methotrexate.

27. A method according to any one of claims 19 to 26, wherein said disease that activates osteoclasts is rheumatoid arthritis.

28. A method for testing in vivo the effectiveness of a substance as an agent for the treatment or prevention of bone erosion caused by a disease that activates osteoclasts comprising the following steps:

(i) transplanting hypodermically pieces of dentine and synovial tissue obtained from an affected part of a patient suffering from a disease that activates osteoclasts to more than one immunodeficient non-human mammal so that each test animal has a piece of dentine and a sample of said synovial tissue that are in mutual contact; (ii) administering the substance to be tested to at least one of the test animals prepared in step (i), and breeding said test animal for a certain period;

29. A method according to claim 28, wherein said immunodeficient non-human mammal is an immunodeficient mouse.

30. A method according to claim 29, wherein said immunodeficient mouse is selected from the group consisting of a CB-17/Icr Crj-scid mouse, a BALB/cA-scid mouse and a BALB/cA-bg, scid mouse.

31. A method according to any one of claims 28 to 30, wherein said piece of dentine is a piece of ivory.