JP2003146903A - Therapeutic or prophylactic agent composition for osteoclasia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new pharmaceutical composition useful for therapy or prophylaxis of osteoclasia in rheumatoid arthritis. SOLUTION: This pharmaceutical composition for the therapy or prophylaxis of the osteoclasia in the rheumatoid arthritis comprises a humanized antibody of an antihuman Fas monoclonal antibody HFE7A produced by a mouse-mouse hybridoma HFE7A (FERM BP-5828).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel pharmaceutical composition for treating or preventing bone destruction in diseases in which osteoclasts are activated (eg, rheumatoid arthritis).

[0002]

BACKGROUND OF THE INVENTION Rheumatoid arthritis is an inflammatory autoimmune disease in which various synovial membranes are the main lesions, and the progression of the disease causes destruction of articular cartilage and bone, leading to deterioration of joint function. . The number of patients who can obtain good control of inflammation and pain of rheumatoid arthritis by drug treatment with steroids or non-steroidal anti-inflammatory drugs is increasing, but it may not be effective in changing the natural course of bone destruction. In many cases, orthopedic surgery has been commonly performed in severe cases.

Among the bone destruction mechanisms, the activation of osteoclasts is the most important factor. In joints affected by rheumatoid arthritis, osteoclasts infiltrate the interface between synovial tissue and bone tissue, and the number of cells and bone resorption ability increase as the disease progresses. The monocyte / macrophage osteoclast precursor cells include macrophage colony stimulating factor (M-CSF) and osteoclast differentiation factor (RAN) expressed by osteoblasts / stromal cells and the like.
It is known that it is differentiated into activated osteoclasts and activated by stimulation from KL / ODF (Yasuda, H. et al.
(1999) Bone 25, 109-133). In rheumatoid arthritis, RANKL has the following three mechanisms of action.
It has been suggested that bone resorption is promoted by the differentiation and activation of osteoclasts mediated by. That is, RAN by osteoblasts stimulated by IL-17 produced by expanded T cells
Production of KL (Kotake, S. et al. (1999) J. Clin. Inves
t. 103, 1345-1352), and production of RANKL by proliferating synovial fibroblasts themselves (Takayanagi, H. et al.
(2000) Arthritis Rheum. 43, 259-269), production of RANKL by activated T cells themselves (Kong, Y. et.
al. (1999) Nature 402, 304-309), etc.
The ANKL-RANK signal acts to cause osteoclast differentiation and activation. However, it is not known whether bone destruction can be suppressed by inhibiting any one of these action mechanisms.

In vivo, physiological cell death caused by normal cell replacement is called apoptosis and is distinguished from pathological cell death, necrosis (K).
err, et al. (1972) Br. J. Cancer 26, 239). So-called programmed cell death is cell death found in certain cells that have been programmed in advance to die in the living body, and apoptosis is one of them. In apoptosis, phenomena such as cell surface curvature, nuclear chromatin condensation, and chromosomal DNA fragmentation are characteristically observed.

Apoptosis plays a role in the differentiation of lymphocytes (T cells and B cells) by eliminating cells that recognize self-antigens. In fact, it is known that 95% or more of the cells, such as cells that react with self, die in the thymus during the maturation process of T lymphocytes (see Shigekazu Nagata (1993) Protein Nucleic Acid Enzyme 38, 2208-2218). The onset of so-called autoimmune diseases is thought to be due to the generation of autoreactive lymphocytes due to the failure of apoptosis in lymphocyte differentiation (Nakayamaet).
al. (1995) Mebio, 12, 79-86).

Fas (Yonehara, S. et al. (1989) J. Exp. Me is one of the molecules involved in apoptosis.
d. 169, 1747-1756) and Tumor Necrosis Factor Receptor (Loetsch
er, H. et al. (1990) Cell 61, 351-359), CD4
0 (see Tsubata, T. et al. (1993) Nature 364, 645-648) and perforin / granzyme A (Jenne, DE et.
al. (1988) Immunol. Rev. 103, 53-71)). Fas is a transmembrane protein existing on the cell surface and induces apoptosis in the cell when a protein called Fas ligand binds to its extracellular region (Nagata et al. (1995) Science, 267,
1449-1456). When the Fas / Fas ligand system is abnormal, cells that should be eliminated by apoptosis, which adversely affect maintenance of homeostasis, remain unremoved, or conversely, cells essential for maintenance of homeostasis undergo apoptosis. Due to being induced, various disorders occur.

Anti-human Fas monoclonal antibody HFE7
Regarding A and its humanized antibody, rheumatoid arthritis, which is a type of autoimmune disease, is eliminated by eliminating self-reactive immune cells that have not been eliminated because apoptosis is not induced by abnormalities in the Fas-Fas ligand system. Are known to have therapeutic effects (Japanese Patent Application Laid-Open Nos. 11-171900 and 2000-1).
66573 or JP 2000-166574 A or JP 2001-342148 A). However, the action of anti-human Fas monoclonal antibody on the function of osteoclasts is not known, and the effect on bone destruction in rheumatoid arthritis is not known.

As a method for examining the function of osteoclasts in vitro, a method of culturing osteoclasts on an ivory slice and measuring the number of resorption cavities formed on the ivory slice is known (T
amura, T. et al. (1993) J. Bone Miner. Res., 8, 95
3-960). Similarly, a method of culturing synovial cells derived from a rheumatoid patient on an ivory slice and measuring a resorption pit is also known (Fujikawa, Y. et al, (1996) Br. J. Rheumato).
However, it is not possible to know the effect of substances that suppress the function of osteoclasts on cells other than osteoclasts in vivo in these methods. However,
The test method in experimental animals that matches the pathogenic mechanism of bone destruction in actual human rheumatoid arthritis has not been known.
Once such a method was established, it was expected that it would be possible to test substances capable of regulating the function of osteoclasts and treating or preventing bone destruction. Further, it has been desired to find a novel pharmaceutical composition which is useful for the treatment or prevention of bone destruction and is excellent in safety by establishing such a method.

[0009]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel pharmaceutical composition, a novel method, and a treatment for bone destruction useful for the treatment or prevention of bone destruction that causes joint deformation caused by rheumatoid arthritis and the like. Another object is to provide a new test method for determining the preventive effect.

[0010]

[Means for Solving the Problems] The present inventors have simultaneously transplanted an affected synovial tissue of a patient with rheumatoid arthritis and an ivory piece into an immunodeficient mouse, thereby rupturing the synovial tissue in contact with the ivory piece. Found that bone cells cause bone resorption,
A test method for investigating the function of osteoclasts in experimental animals was established. Furthermore, using this test method, it was found that a humanized antibody of anti-human Fas monoclonal antibody HFE7A or the like remarkably suppressed bone resorption in experimental animals, and completed the present invention.

That is, the present invention provides (1) treatment or prevention of bone destruction in a disease involving activation of osteoclasts, which comprises an anti-human Fas monoclonal antibody having apoptosis-inducing activity or a humanized antibody thereof as an active ingredient. (2) A pharmaceutical composition according to (1), which comprises, as an active ingredient, a humanized antibody of an anti-human Fas monoclonal antibody having apoptosis-inducing activity.
(3) The anti-human Fas monoclonal antibody having an apoptosis-inducing activity is mouse-mouse hybridoma H.
From the pharmaceutical composition according to (1) or (2), which is an antibody produced from FE7A (FERM-5828), (4) a nonsteroidal anti-inflammatory drug and a disease-modifying antirheumatic drug The pharmaceutical composition according to (1) to (3), further containing at least one selected anti-inflammatory agent, (5) the nonsteroidal anti-inflammatory agent is diclofenac, loxoprofen sodium, celecoxib,
Selected from the group consisting of etodolac, meloxicam, rofecoxib, indomethacin, ibuprofen, naproxen, and the disease-modifying antirheumatic drug is selected from the group consisting of methotrexate, chloroquine, hydrochloroquine, cyclosporine, penicillamine, sulfasalazine, azathioprine, leflunomide,
The pharmaceutical composition according to (4), (6) further comprising at least one component selected from the group consisting of interferon γ and a folate antagonist or a dihydrofolate reductase inhibitor, (1) to (5) (7) A folate antagonist or a dihydrofolate reductase inhibitor is methotrexate, edatrexat
e, epiroprim, iometrexol, py
ritrexim, trimetrexate, bro
dimoprim, MX-68, N- [4- [3- (2,4-
diamino-6,7-dihydro-5H-cyc
lopenta [d] pyrimidin-5-yl)
Propyl] benzoyl] -L-glutami
c acid, N-[[5- [2- (2-amino-1,
4,5,6,7,8-hexahydro-4-oxopyr
ido [2,3-d] -pyrimidin-6-yl)
Ethyl-2-thienyl] carbonyl]
-L-glutamic acid, (R) -N-
[[5- [2- (2-amino-1,4,5,6,7,8-h
exahydro-4-oxopyrido- [2,3-
d] pyrimidin-6-yl) ethyl-2-t
hienyl] carbonyl] -L-glutam
ic acid, N-((2,4-diamino-3,
4,5,6,7,8-hexahydropyrido [2,
3-d] pyrimidin-6-yl) -ethyl)
-2-thienylcarbonyl-L-glut
atomic acid, (S) -2-[[[4-carb
oxy-4-[[4-[[(2,4-diamino-6-
pteridinyl) methyl] amino]
benzoyl] amino] butyl] amin
o] carbonyl] benzoic acid, N
-[4- [3-(2,4-diamino-1H-pyrr
olo [2,3-d] pyrimidin-5-yl) p
ropyl] benzoyl] -L-glutamic
acid, 2,4-diamino-6- (N- (4-
(Phenylsulfonyl) benzyl) me
thylamino) quinazoline, 2, 4-
diamino-5- [4- [3-(4-aminophe
nyl-4-sulfonyphenylamine
o) propoxy-3,5-dimethyoxybe
nzyl]] pyrimidine, N- [4- [4-
(2,4-diamino-5-pyrimidiny
l) butyl] benzoyl] -L-glutam
ic acid, N- [4- [3- (2,4-diamin
o-5-pyrimidinyl) propyl] be
nzoyl] -L-glutamic acid, N-
[4- [2- (2,4-diamino-6-pterid
inyl) ethyl] benzoyl] -4-met
hyrene-DL-glutamic acid, N
-(1-methylethyl) -N '[3- (2,4,5)
-Trichlophenoxy) propox
y] imidodicarbonimic dia
(6) The pharmaceutical composition according to (6), which is selected from the group consisting of middle hydrochloride (PS15), (8) The pharmaceutical composition according to (6), wherein the folate antagonist or dihydrofolate reductase inhibitor is methotrexate, 9) The pharmaceutical composition according to (1) to (8), wherein the disease associated with activation of osteoclasts is rheumatoid arthritis, (10) an anti-human Fas monoclonal antibody having apoptosis-inducing activity, or a humanized antibody thereof. A method for treating or preventing bone destruction in a disease involving activation of osteoclasts, which comprises administering to a patient, (1)
1) administering a humanized antibody of an anti-human Fas monoclonal antibody having an apoptosis-inducing activity,
The method according to (10), (12) the anti-human Fas monoclonal antibody having an apoptosis-inducing activity is used as a mouse-mouse hybridoma HFE7A (FERM-582).
(8) is an antibody produced from (1),
0) or (11), (13) further administering at least one anti-inflammatory drug selected from non-steroidal anti-inflammatory drugs and disease-modifying anti-rheumatic drugs,
(10) to (12), (14) the nonsteroidal anti-inflammatory drug is selected from the group consisting of diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, indomethacin, ibuprofen, naproxen, and disease modifying Antirheumatic drugs include methotrexate, chloroquine,
The method according to (13), which is selected from the group consisting of hydrochloroquine, cyclosporine, penicillamine, sulfasalazine, azathioprine, leflunomide,
(15) further administering at least one component selected from the group consisting of interferon γ and a folate antagonist or a dihydrofolate reductase inhibitor,
(10) to (14), (16) The antifolate or dihydrofolate reductase inhibitor is methotrexate, edrexate, epiropri
m, iometrexol, pyritrexim, t
rimetrexate, brodimoprim, M
X-68, N- [4- [3- (2,4-diamino-6,7
-Dihydro-5H-cyclopenta [d]
pyrimidin-5-yl) propyl] ben
zoyl] -L-glutamic acid, N-
[[5- [2- (2-amino-1,4,5,6,7,8-h
exahydro-4-oxopyrido [2,3-
d] -pyrimidin-6-yl) ethyl-2-
thienyl] carbonyl]-L-glut
atomic acid, (R) -N-[[5--2- (2-
amino-1,4,5,6,7,8-hexahydro-
4-oxopyrido- [2,3-d] pyrimid
in-6-yl) ethyl-2-thienyl] ca
rbonyl] -L-glutamic acid, N
-((2,4-diamino-3,4,5,6,7,8-he
xahydropyrido [2,3-d] pyrimi
din-6-yl) -ethyl) -2-thienyl
carbonyl-L-glutamic acid,
(S) -2-[[[4-carbox-4-[[4-
[[(2,4-diamino-6- pteridiny
l) methyl] amino] benzoyl] am
ino] butyl] amino] carbonyl]
benzic acid, N- [4- [3- (2,4-d
iamino-1H-pyrrolo [2,3-d] py
rimidin-5-yl) propyl] benzo
yl] -L-glutamic acid, 2,4-di
amino-6- (N- (4- (phenylsulfo
nyl) benzyl) methylamino) qu
inazoline, 2,4-diamino-5- [4-
[3- (4-aminophenyl-4-sulfon
ylphenylamino) propoxy-3,5-
dimethyoxybenzyl]] pyrimid
ine, N- [4- [4- (2,4-diamino-5-
pyrimidinyl) butyl] benzoy
l] -L-glutamic acid, N- [4- [3
-(2,4-diamino-5-pyrimidiny
l) propyl] benzoyl] -L-gluta
mic acid, N- [4- [2- (2,4-diami
no-6-pteridinyl) ethyl] ben
zoyl] -4-methylene-DL-glut
atomic acid, N- (1-methyl
l) -N '[3- (2,4,5-trichlorophe
noxy) propoxy] imidodicarbo
nimidic diamide hydrochlo
(15) The method according to (15), wherein the antifolate or dihydrofolate reductase inhibitor is methotrexate selected from the group consisting of Ride (PS15), (18) osteoclasts (10) to (17), wherein the disease associated with activation of rheumatoid arthritis is rheumatoid arthritis,
For bone destruction in diseases involving activation of osteoclasts,
Method for determining therapeutic or preventive effect of test substance: 1) Subcutaneous dentin piece having at least one plane and immunosynaptic synovium separated from affected part of rheumatoid arthritis patient Tissue is transplanted so that the dentin piece and the synovial tissue contact each other; 2) At least one of the animals in 1) above is administered with the substance to be tested and kept for a certain period of time. 3) Among the animals of 1) above, at least one animal different from 2) above is administered with a control drug or a solvent alone, or nothing is administered under the same conditions as the animals of 2) above. 4) Remove dentin pieces from the animals of 2) and 3) above,
The number of resorption pits formed on the plane of each dentin piece is measured and compared.

(20) The method according to (19), wherein the immunodeficient non-human mammal is an immunodeficient mouse, (21) the immunodeficient mouse is CB-17 / Icr
Crj-scid mouse, BALB / cA-scid
The method according to (20), which is selected from the group consisting of mice, BALB / cA-bg, and scid mice.
(22) The dentin piece is a dentin piece, (19) to (2)
The method according to 1) is provided.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The pharmaceutical composition of the present invention can be obtained by formulating an anti-human Fas monoclonal antibody or a humanized antibody thereof, and preferred examples include the anti-human Fas monoclonal antibody HFE7A. You can

Anti-human Fas monoclonal antibody HFE7
A is, for example, Japanese Unexamined Patent Publication No. 11-171900, Japanese Unexamined Patent Publication No.
000-166573, JP-A-2000-1665.
According to the method described in Japanese Patent Publication No. 74-74 or JP 2001-342148 A, it can be obtained by immunizing a Fas knockout mouse with human Fas and culturing a hybridoma produced by fusing spleen cells of the mouse and mouse myeloma cells. .

In producing the monoclonal antibody,
At least the following work steps are required. That is, (a) purification of a biopolymer used as an antigen,
(B) a step of preparing antibody-producing cells after immunizing an animal by injecting the antigen, collecting blood, assaying the antibody titer to determine the time of splenectomy, and (c) myeloma cells (Hereinafter referred to as "myeloma"), (d) cell fusion between antibody-producing cells and myeloma, (e) selection of hybridoma group producing the desired antibody, (f) division into single cell clones (cloning) ), (G) in some cases, culturing the hybridoma for producing a large amount of monoclonal antibody, or raising an animal transplanted with the hybridoma, (h) physiological activity of the monoclonal antibody thus produced, and recognition thereof Examination of specificity,
Alternatively, it is an assay of characteristics as a labeling reagent.

The method for producing the anti-Fas monoclonal antibody will be described in detail below with reference to the above steps. However, the method for producing the antibody is not limited thereto, and for example, antibody-producing cells other than splenocytes and myeloma may be used. it can.

(A) Purification of antigen As the antigen, the extracellular region of human Fas and the extracellular region of mouse interleukin 3 receptor (hereinafter referred to as "IL3R") (Nishimura, Y. et al. (1995) J. Immunol. 15
4, 4395-4403), and a recombinant protein (hereinafter referred to as “recombinant human Fas”) obtained by introducing the expression vector phFas-AIC2 of the fusion protein with the cell line COS-1 derived from monkey to express it and partially purifying it. ) Is effective. phFas-AIC2 can be prepared by incorporating a DNA encoding a fusion protein of human Fas and mouse IL3R into an expression vector pME18S for animal cells. However, the Fas-encoding DNA, vector, host, etc. are not limited to these.

Specifically, phFas-AIC2 is used for CO
The transformant obtained by transforming S-1 cells was cultured, and human Fas and mouse I produced in the culture supernatant were cultured.
Recombinant Fas can be partially purified by subjecting the L3R fusion protein to ion exchange chromatography using a resource Q column (trade name; manufactured by Pharmacia).

Further, F existing on the cell membrane of a human cell line
Purified as itself can also be used as an antigen. Furthermore, the primary structure of Fas is known (Ito
h, N., et al. (1991) Cell 66, 233-243).
By a method well known to those skilled in the art, a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing can be chemically synthesized and used as an antigen.

(B) Preparation of antibody-producing cells The antigen obtained in step (a) is mixed with Freund's complete or incomplete adjuvant or an auxiliary agent such as potassium alum to immunize an experimental animal as an immunogen. To do. Fas knockout mice are most preferably used as experimental animals, and such mice are described in Senju et al. (Senj).
u, S., et al (1996) International Immunology 8, 42
It can be produced by the method described in (3).

The immunogen administration method for immunizing mice may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.

Immunization can be performed once or repeatedly at appropriate intervals (preferably at 1 to 5 week intervals). After that, the antibody titer against the antigen in the serum of the immunized animal is measured, and if the animal having a sufficiently high antibody titer is used as a source of antibody-producing cells, the effect of the subsequent operation can be enhanced. Generally, 3 to 3 after the final immunization
It is preferable to use the antibody-producing cells derived from the animal after 5 days for the subsequent cell fusion.

The antibody titer used here is measured by radioisotope immunoassay (hereinafter referred to as "RIA method"), solid-phase enzyme immunoassay (hereinafter referred to as "ELISA method"), fluorescent antibody method, passive method. There are various known techniques such as the hemagglutination method, but the RIA method or the ELISA method is more preferable from the viewpoints of detection sensitivity, rapidity, accuracy, and possibility of automation of operation.

The antibody titer in the present invention is measured by, for example, E
According to the LISA method, the procedure described below can be used. First, purified or partially purified F
As is adsorbed on a solid phase surface such as a 96-well plate for ELISA, and the solid phase surface on which the antigen is not adsorbed is covered with a protein unrelated to the antigen, for example, bovine serum albumin (hereinafter referred to as "BSA"), and the surface is covered. After washing, the sample is brought into contact with a serially diluted sample (eg, mouse serum) as the first antibody, and the anti-Fas antibody in the sample is bound to the antigen. Furthermore, an antibody against an enzyme-labeled mouse antibody is added as a second antibody to bind to the mouse antibody, and after washing, a substrate for the enzyme is added, and the change in absorbance due to color development based on substrate decomposition is measured to determine the antibody titer. calculate.

(C) Step of preparing myeloma As a myeloma, a cell line generally obtained from a mouse, for example, 8-azaguanine resistant mouse (BALB) is used.
/ C)) Myeloma strain P3X63Ag8U.1 (P3-U1) (Yelton,
DE et al. Current Topics in Microbiology and Imm
unology, 81, 1-7 (1978)), P3 / NSI / 1-Ag4-1 (NS-1)
(Kohler, G. et al. European J. Immunology, 6, 511-
519 (1976)), Sp2 / O-Ag14 (SP-2) (Shulman, M. et
al. Nature, 276, 269-270 (1978)), P3X63Ag8.653 (6
53) (Kearney, JF et al. J. Immunology, 123, 154
8-1550 (1979)), P3X63Ag8 (X63) (Horibata, K. and Ha
rris, AW Nature, 256, 495-497 (1975)) is preferably used. These cell lines are prepared by adding 8-azaguanine to an appropriate medium such as 8-azaguanine medium [RPMI-1640 medium to which glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as "FCS") are added. Added medium], Iscove's Modified Dulbecco's M
edium; hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle Mediu
m; hereinafter referred to as "DMEM"), but subcultured in a normal medium [eg 10% FCS] 3-4 days before cell fusion.
In ASF104 medium (manufactured by Ajinomoto Co., Inc.) containing a) and subcultured to secure a cell number of 2 × 10 ⁷ or more on the day of fusion.

(D) Cell fusion antibody-producing cells are plasma cells and their precursor cells, lymphocytes, which may be obtained from any part of the individual, and are generally spleen, lymph nodes, peripheral blood, Alternatively, splenocytes are most commonly used although they can be obtained by appropriately combining these.

After the final immunization, the site where antibody-producing cells are present, for example, the spleen is excised from the mouse in which a predetermined antibody titer has been obtained, and splenocytes, which are antibody-producing cells, are prepared. The most commonly used means for fusing the spleen cells with the myeloma obtained in step (c) is a method using polyethylene glycol, which has relatively low cytotoxicity and is easy to fuse. This method consists of the following procedures, for example.

Spleen cells and myeloma were thoroughly washed with a serum-free medium (for example, RPMI1640) or phosphate buffered saline (hereinafter referred to as "PBS"), and the ratio of the number of splenocytes to myeloma cells was from 5: 1. Mix to about 10: 1 and centrifuge. After removing the supernatant and thoroughly loosening the precipitated cells, 1 ml of 50% (w
/ V) polyethylene glycol (molecular weight 1000-40
00) containing serum-free medium is added dropwise. Then 10 ml
Slowly add the serum-free medium of, and then centrifuge. The supernatant was discarded again, and the precipitated cells were treated with a proper amount of hypoxanthine / aminopterin / thymidine (hereinafter referred to as “HAT”) solution and mouse interleukin-2 (hereinafter referred to as “IL-2”) in a normal medium (hereinafter referred to as “HAT”). Culture plate suspended in "medium" (hereinafter referred to as "plate")
To each well and cultured at 37 ° C. for about 2 weeks in the presence of 5% carbon dioxide. On the way, supplement HAT medium appropriately.

(E) Selection of hybridoma group If the myeloma cell is an 8-azaguanine resistant strain, that is, if it is a hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient strain, the myeloma cell that has not been fused , And fused cells of myeloma cells cannot survive in HAT-containing medium. On the other hand, fused cells of antibody-producing cells or hybridomas of antibody-producing cells and myeloma cells can survive, but fused cells of antibody-producing cells have a life span. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma of the antibody-producing cell and the myeloma cell survives, and as a result, the hybridoma can be selected.

The hybridomas that have grown in the form of colonies are replaced with a medium in which aminopterin is removed from the HAT medium (hereinafter referred to as "HT medium"). After that, a part of the culture supernatant is collected, and the anti-Fas antibody titer is measured by, for example, the ELISA method. However, when the above fusion protein is used as an antigen for ELISA, it is necessary to perform an operation to exclude a clone that produces an antibody that specifically binds to the extracellular region of mouse IL3 receptor, so as not to select the clone. . The presence or absence of such a clone can be confirmed by, for example, ELISA using mouse IL3 receptor or its extracellular region as an antigen.

Although the method using the 8-azaguanine-resistant cell line has been exemplified above, other cell lines can also be used depending on the selection method of the hybridoma, and in that case, the medium composition used also changes. (F) Cloning step By measuring the antibody titer in the same manner as described in (b), the hybridoma that is found to produce a specific antibody is transferred to another plate and cloned.
For this cloning method, 1 per well of the plate
Limiting dilution method of culturing by diluting so that each hybridoma is contained, soft agar method of culturing in soft agar medium and collecting colonies, method of taking out cells one by one by micromanipulator, culturing by cell sorter "Sortah clone" for separating the cells of E. coli is mentioned, but the limiting dilution method is simple and often used.

With respect to wells for which an antibody titer has been recognized, for example, cloning by the limiting dilution method is repeated 2 to 4 times, and those with a stable antibody titer are selected as an anti-Fas monoclonal antibody producing hybridoma strain. further,
By selecting a hybridoma that produces an antibody that binds to mouse Fas by the same method, anti-Fas monoclonal antibody-producing cells are obtained. The mouse Fas used at this time is, for example, an expression plasmid vector pME18 of a DNA encoding a fusion protein of the extracellular region of mouse Fas and the extracellular region of mouse IL3 receptor.
S-mFas-AIC (Nishimura, Y. et al. (1995)
J. Immunol. 154, 4395-4403) introduced into cultured animal cells and expressed therein, and purified mouse Fa.
s, or cells expressing mouse Fas on the cell surface can be used.

Mouse-mouse hybridoma HFE7A, which is a cell producing anti-Fas monoclonal antibody, which is the basis for producing the antibody of the present invention (that is, serves as a donor for humanization), is a biotechnology institute of industrial technology. It has been deposited internationally at the Industrial Research Institute (now the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center) on February 20, 1997, and is assigned the deposit number FERM BP-5828. Therefore, for example, when preparing an antibody using mouse-mouse hybridoma HFE7A,
Steps (g) described below, omitting steps (f)
The antibody can be prepared from

(G) Preparation of Monoclonal Antibody by Hybridoma Culture Hybridomas that have been cloned are cultured in HT.
The medium is replaced with a normal medium and the cells are cultured. Large-scale culture is performed by spin culture using a large culture bottle or spinner culture. The supernatant in this large-scale culture can be purified by a method known to those skilled in the art such as gel filtration to obtain an anti-Fas monoclonal antibody which is a basis for producing the antibody of the present invention. In addition, a mouse of the same strain (for example, BALB / c described above) or Nu /
By proliferating the hybridoma in the abdominal cavity of Nu mouse, ascites containing a large amount of anti-Fas monoclonal antibody, which is the basis for producing the antibody of the present invention, can be obtained. As a simple method for purification, a commercially available monoclonal antibody purification kit (for example, MAbTrap) is used.
GII kit; manufactured by Pharmacia) or the like can also be used.

The monoclonal antibody thus obtained is
It has high antigen specificity to human and mouse Fas.

(H) Assay of Monoclonal Antibody The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. First, as an identification method, Ouchterlon
y) method, ELISA method, or RIA method.
The octerlonie method is simple, but when the concentration of the monoclonal antibody is low, a concentration operation is necessary. on the other hand,
When the ELISA method or the RIA method is used, the culture supernatant is allowed to react with the antigen-adsorbing solid phase as it is, and further various immunoglobulin isotypes and antibodies corresponding to subclasses are used as the secondary antibody to detect the isotype of the monoclonal antibody, It is possible to identify subclasses. Further, as a simpler method, a commercially available kit for identification (for example, mouse typer kit; manufactured by Bio-Rad) or the like can be used.

Further, the protein was quantified by the Foreign Lowry method and the absorbance at 280 nm [1.4
(OD280) = immunoglobulin 1 mg / ml].

Identification of the recognition epitope of the monoclonal antibody can be performed as follows. First, various partial structures of the molecule recognized by the monoclonal antibody are prepared. In the production of the partial structure, a method for producing various partial peptides of the molecule using a known oligopeptide synthesis technique, and a DNA sequence encoding the target partial peptide using a gene recombination technique is used as a suitable expression plasmid. There are methods such as integration and production inside and outside a host such as Escherichia coli, etc., but for the above-mentioned purpose, it is general to use both in combination. For example, a series of polypeptides sequentially shortened from the C-terminal or N-terminal of an antigen protein by an appropriate length to prepare a series of them using a gene recombination technique well known to those skilled in the art, and then the reactivity of monoclonal antibodies against them is examined. , Determine a rough recognition site.

Then, further finely, variously synthesizing oligopeptides of the corresponding portions, mutants of the peptides and the like by using oligopeptide synthesis techniques well known to those skilled in the art,
Anti-Fa as a basis for producing the antibody of the present invention
Epitopes are limited by examining the binding properties of the monoclonal antibodies to these peptides, or by examining the competitive inhibitory activity of the peptides for the binding of the monoclonal antibody to the antigen. As a convenient method for obtaining various oligopeptides, a commercially available kit (for example, SPO
It is also possible to use a Ts kit (manufactured by Genosys Biotechnology), a series of multi-pin peptide synthesis kits (manufactured by Chiron) using the multi-pin synthesis method, and the like.

Next, for the DNA encoding the heavy chain or light chain of the anti-Fas monoclonal antibody, which is the basis for preparing the antibody of the present invention, mRNA is prepared from hybridoma cells producing the above-mentioned anti-Fas monoclonal antibody, It can be obtained by converting the mRNA into cDNA with reverse transcriptase and then isolating the DNAs encoding the heavy or light chains of the antibody, respectively.

For the extraction of mRNA, the guanidine / thiocyanate / hot / phenol method, the guanidine / thiocyanate / guanidine / hydrochloric acid method and the like can be adopted, but the guanidine / thiocyanate / cesium chloride method is preferable. Preparation of mRNA from cells begins with total RN
A is prepared, and poly (A) ⁺ R such as oligo (dT) cellulose or oligo (dT) latex beads is prepared from the total RNA.
It can be carried out by a method of purifying using a carrier for purifying NA or a method of directly purifying from a cell lysate using the carrier. As a method for preparing total RNA, an alkaline sucrose density gradient centrifugation method (Dougherty, WG and Hiebert,
E. (1980) Viology 101, 466-474), guanidine thiocyanate / phenol method, guanidine thiocyanate / trifluorocesium method, phenol / SDS method and the like can be adopted, but a method using guanidine thiocyanate and cesium chloride (Chirgwin, JM, et al. (197
9) Biochemistry 18, 5294-5299) is preferable.

Poly (A) ⁺ R obtained as described above
Single-stranded cDNA by reverse transcriptase reaction using NA as a template
After synthesizing A, the double-stranded cDNA was converted from this single-stranded cDNA.
A can be synthesized. As this method, S1 nuclease method (Efstratiadis, A., et al. (1976) Cel17,
279-288), Gubler-Hoffman method (Gubler, U. and
Hoffman, BJ (1983) Gene 25, 263-269), Okayama, H. and Berg, P. (1982) M
ol. Cell. Biol. 2, 161-170), etc.
In the present invention, the polymerase chain reaction (hereinafter referred to as “PCR”) using a single-stranded cDNA as a template (Saiki, R. et al.
K., et al. (1988) Science 239, 487-49), so-called RT-PCR, is preferred.

The double-stranded cDNA thus obtained is incorporated into a cloning vector, and the obtained recombinant vector is introduced into a microorganism such as Escherichia coli for transformation, and transformed with tetracycline resistance or ampicillin resistance as an index. You can choose your body. Transformation of E. coli was carried out by the Hanahan method (Hanahan, D. (1983) J. Mol.
l. 166, 557-580), that is, the method of adding the recombinant DNA vector to competent cells prepared in the coexistence of calcium chloride, magnesium chloride or rubidium chloride. In addition, when a plasmid is used as a vector, it is necessary to have the above drug resistance gene. It is also possible to use cloning vectors other than plasmids, such as lambda phages.

From the transformant obtained as described above, cDNA encoding each subunit of the anti-Fas antibody of interest
As a method of selecting a strain having, for example, the following various methods can be adopted. When the target cDNA is specifically amplified by the above RT-PCR, these operations can be omitted.

(1) Method using polymerase chain reaction When all or part of the amino acid sequence of the target protein has been elucidated, oligonucleotide primers for the sense strand and antisense strand corresponding to a part of the amino acid sequence are synthesized. Then, the polymerase chain reaction (Saiki, RK, et al. (1988) Scien
ce 239, 487-491) to obtain the desired anti-human Fas
DNA encoding antibody heavy chain or light chain subunit
Amplify the fragment. The template DNA used here is, for example, m of a hybridoma (FERM BP-5828) producing the anti-human Fas monoclonal antibody HFE7A.
CDNA synthesized from RNA by reverse transcriptase reaction can be used.

The DNA fragment thus prepared can be directly incorporated into a plasmid vector by using a commercially available kit or the like, or the fragment is labeled with ³² P, ³⁵ S or biotin and used as a probe. It is also possible to select a desired clone by performing colony hybridization or plaque hybridization using it.

For example, the anti-Fas monoclonal antibody HFE7A which is the basis for preparing the antibody of the present invention
Is immunoglobulin G1 (hereinafter referred to as "IgG1")
It is a molecule and is a complex composed of each subunit of heavy chain (γ1 chain) and light chain (κ chain). As a method for examining the partial amino acid sequence of each of these subunits, each subunit is isolated using a well-known method such as electrophoresis or column chromatography, and then an automatic protein sequencer (for example, manufactured by Shimadzu Corporation) is used. A method of analyzing the N-terminal amino acid sequence of each subunit using PPSQ-10) or the like is preferable.

A method for collecting cDNA encoding each subunit of anti-human Fas monoclonal antibody protein from the target transformant obtained as described above is a known method (Maniatis, T., et al. (1982) in "Mol
ecular Cloning A Laboratory Manual "Cold Spring Har
bor Laboratory, NY.). For example, it can be carried out by separating a fraction corresponding to vector DNA from cells and cutting out a DNA region encoding a desired subunit from the plasmid DNA.

(2) Screening Method Using Synthetic Oligonucleotide Probe When all or part of the amino acid sequence of the target protein has been elucidated (if the sequence is a specific sequence consisting of a plurality of consecutive specific sequences, Region) and synthesize an oligonucleotide corresponding to the amino acid sequence (in this case, a nucleotide sequence deduced with reference to the frequency of codon usage, or a plurality of nucleotide sequences combining possible nucleotide sequences) (In the latter case, inosine can be included to reduce the number of species), and this can be used as a probe (labeled with ³² P, ³⁵ S, biotin, etc.) to transform the transformant D.
NA is hybridized with a denaturing and fixed nitrocellulose filter, and the obtained positive strain is selected.

The sequence of the DNA thus obtained can be determined by, for example, the Maxam-Gilbert chemical modification method (Maxa
m, AM and Gilbert, W. (1980) in "Methods in Enz
ymology "65, 499-576) and dideoxynucleotide chain termination method (Messing, J. and Vieira, J. (1982) Gene 1
9, 269-276) and so on.

In recent years, automatic base sequence determination systems using fluorescent dyes have become widespread (eg, Perkin Elmer Japan Co., Ltd. sequence robot "CATALYST 800" and model 373A DNA sequencer).

By using such a system, DN
It is also possible to carry out the A nucleotide sequencing operation efficiently and safely. DNA encoding the heavy chain or light chain of the anti-Fas monoclonal antibody, which is the basis for producing the antibody of the present invention thus determined
The complete amino acid sequences of the heavy and light chains of the monoclonal antibody can be determined from the respective nucleotide sequences of A. and the N-terminal amino acid sequence data of the heavy and light chains.

In order to prepare a variant in which any one or two or more amino acids in the amino acid sequence are deleted, the cassette mutation method (Toshimitsu Kishimoto, “Shinsei Chemistry Experimental Course 2 Nucleic Acid III Recombinant DNA Technology” “See 242-251) and so on.

Such various kinds of DNA can be obtained by, for example, the phosphite triester method (Hunkapiller, M., et al. (19
84) Nature 310, 105-111) and the like, and can also be produced by chemical synthesis of nucleic acid. The codon for the desired amino acid is known per se, and its selection may be arbitrary. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host to be used. Partial modification of these nucleotide sequence codons, according to a conventional method,
Site-directed mutagenesis using a primer consisting of a synthetic oligonucleotide that encodes the desired modification (site-specific mutagenesis) (Mark, DF, e
t al. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-56
66) and so on.

Whether or not a certain DNA hybridizes with the DNA of the present invention is determined by, for example, using the random primer method (Feinberg, AP and Vogelstein, B.
(1983) Anal. Biochem. 132, 6-13) and Nick translation method (Maniatis, T., et al. (1982) in "M
olecular Cloning A laboratory Manual "Cold SpringH
arbor Laboratory, NY.) [α- ³² P] d
The probe DNA labeled with CTP or the like can be used to perform the experiment described below.

First, the DNA to be investigated is adsorbed on, for example, a nitrocellulose membrane or a nylon membrane, and if necessary, subjected to treatment such as alkali denaturation, and then solidified by heating or ultraviolet rays. This membrane is 6 × SSC
(1 × SSC is 0.15M sodium chloride, 0.01
5M trisodium citrate solution), 5% Denhardt's solution, and 0.1% sodium dodecylsulfate (SDS) in a prehybridization solution, and incubate at 55 ° C for 4 hours or longer. 1x1 final specific activity for prehybridization solution
Add ⁶ Cpm / ml and incubate at 60 ° C overnight. Then, the membrane is washed with 6 × SSC at room temperature for 5 minutes, repeated several times, further washed with 2 × SSC for 20 minutes, and then subjected to autoradiography.

By using the above method, an arbitrary cd
DNAs that hybridize with the DNAs encoding the heavy or light chains of the anti-Fas monoclonal antibody on which the antibodies of the present invention are based can be isolated from NA or genomic libraries (Maniatis, T., et. al.
(1982) in "Molecular Cloning A laboratory Manual"
Cold Spring Harbor Laboratory, NY.).

Each DNA obtained as described above can be introduced into a prokaryotic or eukaryotic host cell by incorporating it into an expression vector, and each gene can be expressed in those host cells. .

Examples of the host for prokaryotic cells include Escherichia coli and Bacillus subtilis.
) And the like. To express the gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and a promoter sequence such as lac UV5. . Further, the vector preferably has a sequence capable of imparting selectivity by the phenotypic trait in the transformed cell. For example, as E. coli, E.
The JM109 strain and the like derived from E. coli K12 strain are often used, and as the vector, pBR322 and pUC type plasmids are commonly used, but the vector is not limited thereto, and various known strains and vectors can be used. As a promoter, in E. coli,
Tryptophan (trp) promoter, lactose (la
c) Promoter, tryptophan lactose (tac
) Promoter, lipoprotein (lpp) promoter, bacteriophage-derived lambda (λ) PL promoter, polypeptide chain elongation factor Tu (tufB) promoter and the like, but are not limited thereto.

As the Bacillus subtilis, for example, strain 207-25 is preferable, and as the vector, pTUB228 (Ohmura,
K., et al. (1984) J. Biochem. 95, 87-93) and the like are used, but not limited thereto. As a promoter, a regulatory sequence of the Bacillus subtilis α-amylase gene is often used, and if necessary, a DNA sequence encoding a signal peptide sequence of α-amylase can be ligated to enable extracellular secretion. .

Eukaryotic host cells include cells such as vertebrates and yeasts. Examples of vertebrate cells include COS-1 (Gluzman, Y. (1981) Ce, which is a cell line derived from monkey.
ll 23, 175-182) etc. are used. As for yeast,
Baker's yeast (Saccharomyces cerevisiae), alcohol-utilizing yeast (Pichia pastoris), fission yeast (Schizossacc
haromyces pombe) and the like are used. Although cells such as those exemplified here are commonly used as host cells, the cells are not limited thereto.

As an expression vector for vertebrate cells, one having a promoter, an RNA splicing site, a polyadenylation site, a transcription termination sequence and the like, which are usually located upstream of the gene to be expressed, can be used. Further, it may have a replication origin if necessary. As an example of the expression vector, pSV2dhfr (Subramani, S., et al. (198) having an SV40 early promoter is used.
1) Mol. Cell. Bio. 1, 854-864), etc., but not limited thereto.

Examples of yeast expression vectors include the promoter of the alcohol dehydrogenase gene (Bennetzen, J. et al.
L. and Hall, BD (1982) J. Biol. Chem. 257, 301
8-3025) and galactose metabolism enzyme gene (ga
promoters (Ichikawa, K., et al.
(1993) Biosci. Biotech. Biochem. 57, 1686-1690), etc., and if necessary, by ligating a DNA sequence encoding the signal peptide sequence of the yeast gene, extracellular secretion is also possible. , But is not limited to this.

Taking COS-1 as a host cell as an example, the expression vector has an SV40 origin of replication and is capable of autonomous growth in COS-1.
Furthermore, a transcription promoter, a transcription termination signal, and R
An expression vector with an NA splice site can be used. The expression vector is the DEAE-dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Ac
ids Res. 11, 1295-1308), calcium phosphate-D
NA coprecipitation method (Graham, FL and van der Eb, AJ (19
73) Virology 52, 456-457) and electroporation (Neu
Mann, E., et al. (1982) EMBO J. 1, 841-845) and the like, and can be incorporated into COS-1, and thus desired transformed cells can be obtained.

In particular, an expression vector comprising a heavy chain-encoding DNA and a DNA encoding the same light chain
By co-transfecting COS-1 with an expression vector comprising the following, these DNAs can be expressed simultaneously and a transformant producing a recombinant anti-Fas antibody can be obtained. However, recombinant anti-Fa
The method for producing the s antibody is not limited to the above, and for example, a single expression vector can be constructed that holds both the heavy chain and the light chain-encoding DNAs and can express these simultaneously. A method of transforming COS-1 cells with a vector can also be used.

The desired transformant obtained above can be cultured according to a method well known to those skilled in the art, and the recombinant anti-Fas can be cultured inside or outside the transformant cell by the culture.
Antibodies are produced. The medium used for the culture,
Various commonly used ones can be appropriately selected according to the host cell used, and for example, in the case of the above COS-1, RPMI16
For example, a medium such as 40 medium or Dulbecco's modified Eagle medium (DMEM) to which a serum component such as fetal calf serum (FCS) is added can be used.

The culture temperature for culturing the transformant may be any temperature as long as it does not markedly reduce the intracellular protein synthesizing ability, but is preferably 32 to 42 ° C., most preferably 37 ° C. Culture at. If necessary, 1 to 1
It can be cultured in air containing 0% (v / v) carbon dioxide.

The fraction containing the anti-Fas antibody protein produced intracellularly or extracellularly of the transformant as described above can be subjected to various known protein separation manipulation methods utilizing the physical properties and chemical properties of the protein. Can be separated and purified. Specific examples of such methods include treatment with conventional protein precipitants, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC). ) Etc., various chromatographies, dialysis methods, and combinations thereof can be adopted.

The anti-human Fas monoclonal antibody or its humanized antibody is described in, for example, JP-A-11-171900, JP-A-2000-166573, and JP-A-2000.
It can be obtained by the method described in JP-A-166574, JP-A-2001-342148 or 2001-342149. Above all, JP-A-2001-34
The humanized antibody disclosed in Examples of 2148 is preferable as an active ingredient of the pharmaceutical composition of the present invention. Mouse-mouse hybridoma H, which is a cell producing an anti-Fas monoclonal antibody suitable as a base antibody for producing the humanized anti-Fas antibody of the present invention.
FE7A was deposited internationally at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center) on February 20, 1997, with the deposit number FERM BP-5828. Is attached. In addition, E. coli strain E. coli transformed with a recombinant DNA vector for expressing DNA encoding heavy chain or light chain of anti-Fas monoclonal antibody produced by HEF7A in animal cells. coli pME-H and E. coli. coli pME
-L has been internationally deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology and the Patent Biological Depository Center) on March 12, 1997, with the deposit number FERM. BP-5868 and FE
RM BP-5867 is attached. Also, HEF7
A humanized anti-Fas monoclonal antibody produced by A, and three transformants in which DNA encoding the variable region of the light chain of the anti-Fas antibody was incorporated, coli pHSGM
M6 SANK 73697 strain, E. coli pHSG
HM17 SANK 73597 strain and E. coli p
HSGHH7 SANK 73497 strain, and DNA encoding the variable region of the heavy chain of humanized anti-Fas antibody
A transformant incorporating E. coli pgHSL7
The A62 SANK73397 strain was purchased in 1997 (1997).
Internationally deposited on August 22nd at the Institute of Biotechnology, Institute of Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology and the Patent Biological Deposit Center), each with the deposit number FERM.
BP-6071, FERM BP-6072 and FER
M BP-6073 and FERM BP-6074 are attached. Furthermore, anti-Fas produced by HEF7A
Two types of transformants in which a DNA encoding the variable region of the light chain of an anti-Fas antibody obtained by humanizing a monoclonal antibody was incorporated, E. coli pHSHM2 SANK 7019
8 strains and E. coli pHSHH5 SANK 703
Strain 98, and a transformant incorporating a DNA encoding the variable region of the heavy chain of a humanized anti-Fas antibody, E.
The coli pgHPDHV3 SANK 70298 strain was internationally deposited on February 26, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (now National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center). Accession number FERM BP-6272 and FERM
BP-6274 and FERM BP-6273 are attached.

Further, another anti-F which is suitable as a base antibody for preparing the humanized anti-Fas antibody of the present invention
CH11 can be mentioned as an as monoclonal antibody. The CH11 producing hybridoma is Yonehara et al.
ra, S., et al. (1989) J. Exp. Med. 169, 1747)
According to the above, it was obtained by cell fusion of mouse lymphocytes obtained by immunization with human diploid fibroblast FS-7 and mouse myeloma cells. The hybridoma-derived CH11 itself is commercially available from Institute of Medical Biology. DNA encoding the heavy or light chain of CH11
E. coli strain transformed with a recombinant DNA vector expressing E. coli in animal cells. E. coli pCR3-H123 and E. coli. coli pCR3-L103 is the
1996) It was deposited internationally at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center) on February 28, with the deposit number FERM B.
P-5427 and FERM BP-5428 are attached. For the process of humanizing CH11, see JP-A-200
No. 0-154149. Four transformants, E. coli, which incorporate DNA encoding the variable region of the light chain of a humanized antibody of CH11. coli pHκKY
2-58 strain, E. E. coli pHκKF2-19 strain,
E. E. coli pHκRY2-10 strain and E. coli. coli
pHκRF2-52 strain and a DNA encoding the variable region of the heavy chain of a humanized antibody of CH11 were incorporated, 2
Seed transformants, E. E. coli pHμH5-1 strain and E. coli. E. coli pHμM1-1 strain was prepared in 1997 (199
(7 years) Internationally deposited on March 11th at the Institute of Biotechnology, Institute of Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center), each with the deposit number FERM.
BP-5861, BP-5860, BP-5859 and BP-5862, and BP-5863 and FER
MBP-5864 is attached.

Therefore, the protein of the anti-human Fas antibody produced by the hybridoma or the humanized antibody thereof is isolated by a method such as isolation of a plasmid from the deposited strain or PCR using an extract from the deposited strain as a template. It is possible to obtain the DNA encoding the subunit of.

In the present invention, diseases associated with activation of osteoclasts include primary osteoporosis (senile osteoporosis, postmenopausal osteoporosis and idiopathic juvenile osteoporosis), endocrine osteoporosis (hyperthyroidism, hyperparathyroidism). , Cushing's syndrome and acromegaly), osteoporosis associated with hypogonadism (hypopituitarism, Klinefelter's syndrome and Turner's syndrome), hereditary and congenital forms of osteoporosis (osteodystrophy, homocystinuria, Menkes' syndrome and (Reilly-Day syndrome), osteopenia due to reduction of gravity load or fixation or immobilization of limbs, Paget's disease, osteomyelitis, infectious lesion due to bone loss, solid tumor (breast cancer, lung cancer, kidney cancer,
Prostate cancer, etc.), hematological malignancies (multiple myeloma, lymphoma and leukemia), idiopathic hypercalcemia, hypercalcemia associated with hyperthyroidism or renal insufficiency, Due to various types of rheumatism such as osteopenia associated with renal insufficiency, surgery, digestive system diseases (small bowel disorder, large bowel disorder, chronic hepatitis, gastrectomy, primary biliary cirrhosis and cirrhosis), rheumatoid arthritis, etc. Osteopenia, bone destruction and joint destruction due to various types of rheumatism such as rheumatoid arthritis, mutilance type rheumatism, osteoarthritis, periodontal bone loss, bone metastasis of cancer (osteolytic metastasis), traumatic injury, Gaucher disease, Osteonecrosis or bone cell death associated with sickle cell anemia, systemic lupus erythematosus or non-traumatic injury, osteodystrophy such as renal osteodystrophy, hypoalkaline phosphataseemia, and bone loss associated with diabetes Disease, osteopenia accompanying nutritional disorder or eating disorders, and other osteopenia and the like.

Testing of a therapeutic or preventive agent for bone destruction of rheumatoid arthritis can be carried out by the method described below.

First, an ivory piece and an affected synovial tissue derived from a human rheumatoid arthritis patient are transplanted into an experimental animal exhibiting immunodeficiency so that the ivory piece and the synovial tissue contact each other. As the immunodeficient animal used here, CB-1
7 / Icr Crj-scid, BALB / cA-scid, BALB / cA-bg, scid, etc.
Congenital immunodeficient mice of SCID mice are preferably used, and CB-17 / Icr Crj-scid mice are more preferable, but not limited thereto. Further, the ivory piece may have any shape and size which can be transplanted into an animal to be used and which is suitable for observing the resorption pit under a microscope, but at least 1 piece for observing the resorption pit. It only has to have two planes. For example, when it is transplanted to a CB-17 / Icr Crj-scid mouse, it has a thickness of 100 μm to 250 μm and a circle with a diameter of 4 to 8 mm or a rectangular plane with a vertical and horizontal length of 4 to 8 mm, respectively. It is preferably in the form of a thin plate.

Next, the properties of the compound to be tested were tested for the substance to be tested for its effect as a therapeutic or preventive agent for bone destruction in rheumatoid arthritis in animals into which ivory pieces and synovial tissue were transplanted as described above. Depending on the route, it may be administered by oral, intravenous, subcutaneous, intramuscular, or implantation at or near the transplant site. The conditions such as the dose of test substance, the frequency of administration, the number of administrations, etc. are set appropriately. For comparison, another individual transplanted with ivory pieces and synovial cells as described above is administered with the same solvent as that used for dissolving the control drug or test substance, or with nothing. Don't do it.

After the animals thus treated are bred for a certain period of time, ivory pieces are excised from the animals, washed, stained and observed under a microscope to measure the number of resorption pits formed on the surface of the ivory pieces. . The number of resorption pits was compared between the control group to which the test substance was not administered or the control drug was administered and the group to which the test substance was administered. Selected as a candidate substance for a therapeutic or preventive agent for destruction.

By the above method, it can be confirmed that the anti-human Fas monoclonal antibody HFE7A or its humanized antibody is useful as an active ingredient of a therapeutic or preventive agent for bone destruction of rheumatoid arthritis.

Since the anti-human Fas monoclonal antibody HFE7A or its humanized antibody contained as an active ingredient in the pharmaceutical composition of the present invention does not induce liver injury and has few side effects, the pharmaceutical composition of the present invention is It may also be combined with other anti-inflammatory agents in the treatment or prevention of bone destruction in osteoclast activated diseases such as rheumatoid arthritis. Examples of suitable anti-inflammatory agents that may be combined include non-steroidal anti-inflammatory agents such as diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, piroxicam, indomethacin, ibuprofen, naprocene or methotrexate, chloroquine, hydrochloroquine, cyclosporine,
Mention may be made of disease-modifying antirheumatic drugs such as penicillamine, sulfasalazine, azathioprine, leflunomide.

Furthermore, an agent capable of enhancing the action of the anti-human Fas monoclonal antibody HFE7A or its humanized antibody is also contained in the pharmaceutical composition of the present invention or is administered at the same time as the pharmaceutical composition of the present invention. , Anti-human Fas monoclonal antibody HFE7A or its humanized antibody. If the dose of the anti-human Fas monoclonal antibody HFE7A or its humanized antibody can be reduced by such combination, the possibility that the patient will be resistant to the antibody can be reduced.
Suitable agents that can enhance the action of the anti-human Fas monoclonal antibody HFE7A or its humanized antibody include interferon γ, a folate antagonist, and a dihydrofolate reductase inhibitor. Methotrexate, edatrexate, epiropri as folate antagonists or dihydrofolate reductase inhibitors
m, iometrexol, pyritrexim, t
rimetrexate, brodimoprim, M
X-68, N- [4- [3- (2,4-diamino-6,7
-Dihydro-5H-cyclopenta [d]
pyrimidin-5-yl) propyl] ben
zoyl] -L-glutamic acid, N-
[[5- [2- (2-amino-1,4,5,6,7,8-h
exahydro-4-oxopyrido [2,3-
d] -pyrimidin-6-yl) ethyl-2-
thienyl] carbonyl]-L-glut
atomic acid, (R) -N-[[5--2- (2-
amino-1,4,5,6,7,8-hexahydro-
4-oxopyrido- [2,3-d] pyrimid
in-6-yl) ethyl-2-thienyl] ca
rbonyl] -L-glutamic acid, N
-((2,4-diamino-3,4,5,6,7,8-he
xahydropyrido [2,3-d] pyrimi
din-6-yl) -ethyl) -2-thienyl
carbonyl-L-glutamic acid,
(S) -2-[[[4-carbox-4-[[4-
[[(2,4-diamino-6- pteridiny
l) methyl] amino] benzoyl] am
ino] butyl] amino] carbonyl]
benzic acid, N- [4- [3- (2,4-d
iamino-1H-pyrrolo [2,3-d] py
rimidin-5-yl) propyl] benzo
yl] -L-glutamic acid, 2,4-di
amino-6- (N- (4- (phenylsulfo
nyl) benzyl) methylamino) qu
inazoline, 2,4-diamino-5- [4-
[3- (4-aminophenyl-4-sulfon
ylphenylamino) propoxy-3,5-
dimethyoxybenzyl]] pyrimid
ine, N- [4- [4- (2,4-diamino-5-
pyrimidinyl) butyl] benzoy
l] -L-glutamic acid, N- [4- [3
-(2,4-diamino-5-pyrimidiny
l) propyl] benzoyl] -L-gluta
mic acid, N- [4- [2- (2,4-diami
no-6-pteridinyl) ethyl] ben
zoyl] -4-methylene-DL-glut
atomic acid, N- (1-methyl
l) -N '[3- (2,4,5-trichlorophe
noxy) propoxy] imidodicarbo
nimidic diamide hydrochlo
It can be selected from the group consisting of Ride (PS15), and of these, the combination with methotrexate is preferred.

The pharmaceutical composition of the present invention can be used as a therapeutic or prophylactic agent for bone destruction in diseases in which osteoclasts are activated such as rheumatoid arthritis. Also,
It can be used as a mixture with other agents such as dihydrofolate reductase. Furthermore, the pharmaceutical composition of the present invention and the other drug can be used in combination by separately or simultaneously administering them. Such therapeutic or prophylactic agent can be administered in various forms. Examples of the dosage form include oral preparations such as tablets, capsules, granules, powders and syrups. Further, as a more preferable administration form, parenteral administration such as injection, drip, suppository and the like can be mentioned. These various preparations are usually used in the technical field of pharmaceutical preparation such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It is possible to formulate with known auxiliary agents.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood, and when formed into a solution, emulsion and suspension, they are diluted. All the agents conventionally used in this field can be used as agents, such as water, ethanol, propylene glycol, ethoxylated isostearyl alcohol,
Examples thereof include polyoxylated isostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. May be added.

If desired, colorants, preservatives, fragrances, flavors, sweeteners and other pharmaceuticals may be incorporated.

As described above, the pharmaceutical composition of the present invention prepared as described above is useful as a therapeutic or preventive agent for bone destruction, as described above, in immunodeficient non-human mammals (eg, SCID mice, etc.). (Congenital immunodeficient mouse) of a human chronic rheumatoid arthritis patient-derived affected part synovial tissue and ivory pieces are transplanted, and when the pharmaceutical composition of the present invention is administered,
This can be confirmed by observing the suppression of the formation of resorption cavities in the transplanted ivory pieces.

Anti-human F in the pharmaceutical composition of the present invention
The dose of the as antibody HFE7A may vary depending on the compounding agent used and the administration form. The dose varies depending on symptoms, age, body weight and the like, but usually, the amount of anti-human Fas antibody can be 1 mg to 100 mg once, for example, subcutaneous injection, intramuscular injection or intravenous injection.

[0085]

EXAMPLES The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto. Example 1. Anti-human Fas monoclonal antibody HFE7A
Of humanized antibody of JP-A-11-171900 or 2001-3
According to the method described in Japanese Patent No. 42148, the humanized HFE7A light chain of SEQ ID NO: 1 in the sequence listing and the SEQ ID NO: 2 in the sequence listing
Mammalian cells producing human HFE7A heavy chain were produced using recombinant DNA technology and used to produce humanized HFE
7A (hereinafter referred to as "h-HFE7A") was produced and purified. A transformant incorporating the DNA encoding the variable region of the light chain of the above-mentioned humanized anti-Fas antibody,
E. E. coli pHSHH5 SANK 70398 strain,
And a transformant incorporating a DNA encoding the variable region of the heavy chain of a humanized anti-Fas antibody, E. coli
pgHPDHV3 SANK70298 strain is
International Depositary on February 26, 1998 (Institute of Industrial Science and Technology, Institute of Biotechnology, Institute of Biotechnology (now National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center), with deposit numbers FERM BP-6274 and FERM, respectively. BP-6
273 is attached. Example 2. Examination of Bone Destruction Inhibitory Effect of Humanized Antibody of Anti-Human Fas Monoclonal Antibody HFE7A Using Human IgG as a Control The effect of anti-Fas antibody on bone destruction in rheumatoid arthritis was examined according to the method described below.

Two 5-week-old male CB-17 / Icr Crj-scid mice (hereinafter referred to as “SCID mice”; purchased from Charles River Japan, Inc.) were acclimatized for 1 week. SCI
The D mouse was anesthetized with ether, and the back of the mouse was slightly injured with sterile scissors, and then a synovial tissue (about 0.5 g) collected from an affected part of a human rheumatoid arthritis patient was transplanted with wrapped ivory sections ( 1 transplanted per mouse).
After the transplantation, the incised back skin was sutured using a surgical suture. The ivory section was sliced into an ivory block (purchased from New Japan Ivory Co., Ltd.) with a precision low-speed cutting machine (ISOMET, manufactured by Buehler) to a thickness of 150 to 200 μm.
After pressing and flattening for several days, it is punched into a disk shape with a diameter of 6 mm by an eyelet punch (manufactured by TOHO Co., Ltd.) and molded, and ultrasonically cleaned in 70% ethanol (using Branson Sonifer 450). 10 minutes x 3 times)
What was sterilized by doing was used. At the time of the experiment, this ivory slice was immersed in physiological saline and washed before use.

H-HFE7A prepared according to Example 1
The solution (4.63 mg / ml) was phosphate b
1 mg / m with oversized saline (PBS)
diluted to 1. The diluted h-HFE7A was intravenously administered once per week at a dose of 200 μg / individual to a total of three times (on the day of transplantation, on the 8th and 15th day of transplantation) to one mouse transplanted with synovial tissue and ivory. 200 μ for another mouse
g / individual human IgG (Cosmo Bio, 1.39 m
g / ml solution diluted to 1 mg / ml and used) h-H
The same dose and schedule as those of the FE7A-administered mouse were administered intravenously three times in total. On the 3rd week (21st day) after the transplantation, SCID mice were killed by cardiac blood sampling under ether anesthesia, and then using scissors and tweezers, be careful not to mix the transplanted synovial tissue and the ivory section with the mouse tissue. I took it out. The extracted ivory slice was placed in a well of a 96-well plate, distilled water was added to the well, and both sides were washed with a hand motor (Harayoshi Shoten) (5 seconds x 2
After that, it was immersed in a physiological saline solution (Otsuka Pharmaceutical Factory Co., Ltd.). Then, the ivory slices were stained with Acid Hematoxylin (manufactured by Sigma) for 13 minutes, and again washed in distilled water for 5 seconds with a hand motor for 5 seconds on both sides. The formation of resorption pits on the ivory slices due to osteoclasts in the synovial cells was observed with a microscope, and the number of resorption pits stained blue-violet was measured.
The measurement was performed twice for each ivory piece by different persons, and the average value of the measured values was obtained. The results are shown in Fig. 1.
In the control group administered with human IgG, an average of 73.3 resorption cavities were formed per ivory slice, whereas h
In the group administered with -HFE7A, the number of ivory slices was suppressed to 3.8 on average. Example 3. Examination of Bone Destruction Inhibitory Effect of Humanized Antibody of Anti-Human Fas Monoclonal Antibody HFE7A Using PBS as a Control This example was carried out in the same manner as in Example 2 except for the following points. 1) Synovial tissues were obtained from three patients with rheumatoid arthritis, and three independent experiments were performed. The first time is h-HFE7A
Five mice each were used as a treatment group and a control group. In the second time, two mice each were used as the h-HFE7A administration group and the control group. The third time is h-
Five mice were used as the HFE7A administration group and four mice were used as the control group. 2) In the h-HFE7A administration group, 1 mg / ml h-HFE7A solution was intravenously administered so that the body weight was 10 mg / kg. 3) PB was adjusted to 10 ml / kg body weight in the control group.
S was administered intravenously.

The results of averaging the number of resorption cavities on the ivory pieces in the three experiments are shown in FIG. In the control group administered with PBS, an average of 367.5 resorption cavities were formed per ivory section, whereas in the group administered with h-HFE7A, the average was suppressed to 58.6 per ivory section. It had been.

Formulation Example The humanized antibody of anti-human Fas monoclonal antibody HFE7A contained in the prophylactic or therapeutic agent of the present invention as an active ingredient is a sterile solution prepared by dissolving it in water or other pharmacologically acceptable solution. Used as an ampoule of suspension. For example, 0.5 mg of the humanized antibody of anti-human Fas monoclonal antibody HFE7A can be dissolved in 1 ml of sterile water, injected into an ampoule and sealed. In addition, a sterile powder preparation (preferably freeze-dried with a molecule contained in the preventive or therapeutic agent of the present invention as an active ingredient) is filled in an ampoule and diluted with a pharmacologically acceptable solution at the time of use. Good.

[0090]

EFFECTS OF THE INVENTION The present invention provides a novel pharmaceutical composition useful for treatment or prevention of bone destruction in diseases associated with activation of osteoclasts such as rheumatoid arthritis. Further provided is a method of testing in a laboratory animal the effect of a therapeutic or prophylactic agent on bone destruction in diseases involving activation of osteoclasts.

[0091]

[Sequence list]                                SEQUENCE LISTING        <110> Sankyo Company, Limited <120> A composition for treatment or prevention of bone erosion. <130> 2002082SM <140> <141> <150> JP2001 / 257436 <151> 2001-8-28 <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed light       chain of humanized anti-Fas antibody <400> 1 Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20 -15 -10 -5 Gly Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser              -1 1 5 10 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser          15 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro      30 35 40 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser  45 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr                  65 70 75 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys              80 85 90 Gln Gln Ser Asn Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu          95 100 105 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro     110 115 120 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 125 130 135 140 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn                 145 150 155 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser             160 165 170 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala         175 180 185 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly     190 195 200 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 215 <210> 2 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed heavy       chain of humanized anti-Fas antibody <400> 2 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly                 -15 -10 -5 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys          -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe      15 20 25 Thr Ser Tyr Trp Met Gln Trp Val Lys Gln Ala Pro Gly Gln Gly Leu  30 35 40 45 Glu Trp Met Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn                  50 55 60 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Thr Ser              65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val          80 85 90 Tyr Tyr Cys Ala Arg Asn Arg Asp Tyr Ser Asn Asn Trp Tyr Phe Asp      95 100 105 Val Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 110 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly                 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro             145 150 155 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr         160 165 170 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val     175 180 185 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 190 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro                 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu             225 230 235 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp         240 245 250 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp     255 260 265 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 270 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn                 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp             305 310 315 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro         320 325 330 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu     335 340 345 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 350 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile                 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr             385 390 395 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys         400 405 410 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys     415 420 425 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 430 435 440 445 Ser Leu Ser Pro Gly Lys                 450

[0092]

[Brief description of drawings]

FIG. 1 is a view showing the inhibitory effect of humanized anti-Fas antibody on the formation of resorption pits using human IgG as a control.

FIG. 2 is a view showing the inhibitory effect on the formation of resorption pits of a humanized anti-Fas antibody using PBS as a control.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI Theme code (reference) A61P 43/00 105 A61P 43/00 105 121 121 121 // C12N 5/10 C12N 15/00 C 15/02 5 / 00 B (72) Inventor Masahiko Otsuki 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. F-term in the company (reference) 4B024 AA01 AA11 BA44 GA03 GA23 4B065 AA91X AA93Y AB05 BA06 BA08 CA25 CA44 4C084 AA19 AA22 MA02 NA14 ZA96 ZB11 ZB15 ZB21 ZC75 4C085 AA14 BB11 BB17 CC02 CC05 CC07 CC08 CC21 CC32 DD23 DD62 DD63 EE01 EE03

Claims (22)

[Claims] 1. An anti-human F having an apoptosis-inducing activity.
A pharmaceutical composition comprising as monoclonal antibody or a humanized antibody thereof as an active ingredient for the treatment or prevention of bone destruction in diseases associated with activation of osteoclasts. 2. An anti-human F having an apoptosis inducing activity.
The pharmaceutical composition according to claim 1, comprising a humanized antibody of an as monoclonal antibody as an active ingredient. 3. Anti-human F having apoptosis-inducing activity
3. The pharmaceutical composition according to claim 1 or 2, wherein the as monoclonal antibody is an antibody produced from a mouse-mouse hybridoma HFE7A (FERM-5828). 4. The pharmaceutical composition according to claim 1, which further comprises at least one anti-inflammatory agent selected from non-steroidal anti-inflammatory agents and disease-modifying anti-rheumatic agents. 5. The non-steroidal anti-inflammatory drug is selected from the group consisting of diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, indomethacin, ibuprofen, naproxen, and the disease-modifying antirheumatic drug is methotrexate, chloroquine, hydrochloroquine. , Cyclosporine,
The pharmaceutical composition according to claim 4, which is selected from the group consisting of penicillamine, sulfasalazine, azathioprine and leflunomide. 6. The pharmaceutical composition according to claim 1, further comprising at least one component selected from the group consisting of interferon γ and a folate antagonist or a dihydrofolate reductase inhibitor. 7. A folate antagonist or dihydrofolate reductase inhibitor is methotrexate, edatrexat.
e, epiroprim, iometrexol, py
ritrexim, trimetrexate, bro
dimoprim, MX-68, N- [4- [3- (2,4-
diamino-6,7-dihydro-5H-cyc
lopenta [d] pyrimidin-5-yl)
Propyl] benzoyl] -L-glutami
c acid, N-[[5- [2- (2-amino-1,
4,5,6,7,8-hexahydro-4-oxopyr
ido [2,3-d] -pyrimidin-6-yl)
Ethyl-2-thienyl] carbonyl]
-L-glutamic acid, (R) -N-
[[5- [2- (2-amino-1,4,5,6,7,8-h
exahydro-4-oxopyrido- [2,3-
d] pyrimidin-6-yl) ethyl-2-t
hienyl] carbonyl] -L-glutam
ic acid, N-((2,4-diamino-3,
4,5,6,7,8-hexahydropyrido [2,
3-d] pyrimidin-6-yl) -ethyl)
-2-thienylcarbonyl-L-glut
atomic acid, (S) -2-[[[4-carb
oxy-4-[[4-[[(2,4-diamino-6-
pteridinyl) methyl] amino]
benzoyl] amino] butyl] amin
o] carbonyl] benzoic acid, N
-[4- [3-(2,4-diamino-1H-pyrr
olo [2,3-d] pyrimidin-5-yl) p
ropyl] benzoyl] -L-glutamic
acid, 2,4-diamino-6- (N- (4- (p
henylsulfonyl) benzyl) meth
ylamino) quinazoline, 2,4-di
amino-5- [4- [3-(4-aminopheny
l-4-sulfonyphenylamino) p
ropoxy-3,5-dimethoxybenzy
l]] pyrimidine, N- [4- [4- (2,4-
diamino-5-pyrimidinyl) but
yl] benzoyl] -L-glutamic ac
id, N- [4- [3- (2,4-diamino-5-p
yrimidinyl) propyl] benzoy
l] -L-glutamic acid, N- [4- [2-
(2,4-diamino-6-pteridinyl)
[Ethyl] benzoyl] -4-methylen
e-DL-glutamic acid, N- (1-m
ethylethyl) -N '[3- (2,4,5-tri
chlorophenoxy) propoxy] imi
dodicalbonimidic diamide
The pharmaceutical composition according to claim 6, which is selected from the group consisting of hydrochloride (PS15). 8. The pharmaceutical composition according to claim 6, wherein the antifolate or dihydrofolate reductase inhibitor is methotrexate. 9. The pharmaceutical composition according to claim 1, wherein the disease associated with activation of osteoclasts is rheumatoid arthritis. 10. A method for treating or preventing bone destruction in a disease involving activation of osteoclasts, which comprises administering an anti-human Fas monoclonal antibody having apoptosis-inducing activity or a humanized antibody thereof to a patient. 11. The method according to claim 10, which comprises administering a humanized antibody of an anti-human Fas monoclonal antibody having an apoptosis-inducing activity. 12. The anti-human Fas monoclonal antibody having apoptosis-inducing activity is an antibody produced from a mouse-mouse hybridoma HFE7A (FERM-5828), which is characterized in that:
The method described in. 13. The method according to claim 10, further comprising administering at least one anti-inflammatory agent selected from non-steroidal anti-inflammatory agents and disease-modifying anti-rheumatic agents. 14. The nonsteroidal anti-inflammatory drug is selected from the group consisting of diclofenac, loxoprofen sodium, celecoxib, etodolac, meloxicam, rofecoxib, indomethacin, ibuprofen, naproxen, and the disease-modifying antirheumatic drug is methotrexate, chloroquine, hydrochloroquine. , Cyclosporine, penicillamine, sulfasalazine, azathioprine, leflunomide,
The method according to claim 13. 15. The method according to claim 10, further comprising administering at least one component selected from the group consisting of interferon γ and a folate antagonist or a dihydrofolate reductase inhibitor. 16. A folate antagonist or dihydrofolate reductase inhibitor is methotrexate, edatrexat.
e, epiroprim, iometrexol, py
ritrexim, trimetrexate, bro
dimoprim, MX-68, N- [4- [3- (2,4-
diamino-6,7-dihydro-5H-cyc
lopenta [d] pyrimidin-5-yl)
Propyl] benzoyl] -L-glutami
c acid, N-[[5- [2- (2-amino-1,
4,5,6,7,8-hexahydro-4-oxopyr
ido [2,3-d] -pyrimidin-6-yl)
Ethyl-2-thienyl] carbonyl]
-L-glutamic acid, (R) -N-
[[5- [2- (2-amino-1,4,5,6,7,8-h
exahydro-4-oxopyrido- [2,3-
d] pyrimidin-6-yl) ethyl-2-t
hienyl] carbonyl] -L-glutam
ic acid, N-((2,4-diamino-3,
4,5,6,7,8-hexahydropyrido [2,
3-d] pyrimidin-6-yl) -ethyl)
-2-thienylcarbonyl-L-glut
atomic acid, (S) -2-[[[4-carb
oxy-4-[[4-[[(2,4-diamino-6-
pteridinyl) methyl] amino]
benzoyl] amino] butyl] amin
o] carbonyl] benzoic acid, N
-[4- [3-(2,4-diamino-1H-pyrr
olo [2,3-d] pyrimidin-5-yl) p
ropyl] benzoyl] -L-glutamic
acid, 2,4-diamino-6- (N- (4-
(Phenylsulfonyl) benzyl) me
thylamino) quinazoline, 2, 4-
diamino-5- [4- [3-(4-aminophe
nyl-4-sulfonyphenylamine
o) propoxy-3,5-dimethyoxybe
nzyl]] pyrimidine, N- [4- [4-
(2,4-diamino-5-pyrimidiny
l) butyl] benzoyl] -L-glutam
ic acid, N- [4- [3- (2,4-diamin
o-5-pyrimidinyl) propyl] be
nzoyl] -L-glutamic acid, N-
[4- [2- (2,4-diamino-6-pterid
inyl) ethyl] benzoyl] -4-met
hyrene-DL-glutamic acid, N
-(1-methylethyl) -N '[3- (2,4,5)
-Trichlophenoxy) propox
y] imidodicarbonimic dia
16. The method according to claim 15, which is selected from the group consisting of a middle hydrochloride (PS15). 17. The method of claim 15, wherein the antifolate or dihydrofolate reductase inhibitor is methotrexate. 18. The method according to claim 10, wherein the disease associated with activation of osteoclasts is rheumatoid arthritis. 19. A method for determining the therapeutic or prophylactic effect of a test substance on bone destruction in a disease involving activation of osteoclasts, which comprises the following steps: 1) Subcutaneous injection of a plurality of immunodeficient non-human mammals A dentin piece having at least one plane and a synovial tissue separated from an affected area of a patient with rheumatoid arthritis so that the dentin piece and the synovial tissue are in contact with each other; 2) The substance to be tested is administered to at least one of the animals of 1) above and kept for a certain period of time; 3) At least one animal of the above 1) other than 2) is used as a control. The animals are raised under the same conditions as the animal of 2) above with or without administration of the drug or solvent alone; 4) excision of dentin pieces from the animals of 2) and 3) above,
The number of resorption pits formed on the plane of each dentin piece is measured and compared. 20. The method according to claim 19, characterized in that the immunodeficient non-human mammal is an immunodeficient mouse. 21. The immunodeficient mouse is CB-17 / Icr.
Crj-scid mouse, BALB / cA-scid
21. The method of claim 20, selected from the group consisting of mice, BALB / cA-bg, scid mice. 22. The dentin piece is a dentin piece.
22. The method according to any one of 21 to 21.