JP2001039892A - MEDICINAL COMPOSITION CONTAINING ANTI-Fas ANTIBODY - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicinal composition capable of reducing the tolerance in the patient's body against the anti-Fas antibody and administering for a long period by including a specific anti-Fas antibody and a such specific compound as to enhance effect of the anti-Fas antibody. SOLUTION: This medicinal composition comprises, as active ingredients, (A) antihuman Fas antibody having an activity of inducing apoptosis and (B) a compound having an antagonism to folic acid or an effect of inhibiting dihydrofolate reductase. The component A is preferably monoclonal antibody CH11 or its humanized antibody, etc., especially preferably antihuman Fas antibody HFA7A which mouse-mouse hybridoma HFE7A (FERM BP-5828) produces or its humanized antibody. The component B is preferably one or two or more compounds selected from methotrexate, edatrexate, epiroprim, etc. Further, the composition is useful as preventives or remedies for autoimmune diseases or rheumatoid arthritis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel pharmaceutical composition which is effective for preventing or treating an autoimmune disease or rheumatoid arthritis.

[0002]

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

[0003] Apoptosis plays a role in the differentiation of lymphocytes (T cells and B cells) to eliminate cells that recognize self antigens. It is believed that the onset of so-called autoimmune diseases is caused by the generation of autoreactive lymphocytes due to apoptosis in lymphocyte differentiation (see Keiichi Nakayama et al. (1995) Mebio 12 (10), 79-86).

As a molecule involved in such apoptosis, Fas [Yonehara, S. et al. (1989) J. Exp.
ed. 169, 1747-1756], Tumor Necrosis Factor Receptor [Loet
scher, H. et al. (1990) Cell 61, 351-359], C
D40 [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 present on the cell surface. When a protein called Fas ligand binds to its extracellular region, it induces apoptosis in the cell.

[0005] Some anti-Fas monoclonal antibodies include F
Similar to the as ligand, it is reported that some of them have cytotoxic activity to induce cell apoptosis, and thus can be used as a therapeutic agent for autoimmune diseases, AIDS and tumors (JP-A-2-237935 and JP-T-Hei. 5-50
No. 3281).

On the other hand, rheumatism, especially rheumatoid arthritis,
A disease based on synovial cell proliferation with various immunological abnormalities caused by internal and external factors, which is considered to be a disorder of synovial cell proliferation accompanied by inflammatory cell infiltration and bone erosion. Have been. Tissue destruction mainly in affected joints in rheumatoid arthritis is considered to be caused by abnormal production of cytokines from inflammatory synovial cells. Examination of the joint condition of patients with rheumatism reveals abnormal synovial cell proliferation, including increased synovial villi and multilayered synovial cells (Daniel J. McCarty (1985)
in "Arthritisand allied conditions, A textbook of
rheumatology "10th Edition, Lea & Febiger).
Currently, drug therapy for rheumatism mainly uses anti-inflammatory drugs such as steroids or immunomodulators, but if such abnormal synovial cell proliferation can be suppressed by a drug, the drug is not used. It is considered useful as a therapeutic agent for rheumatism.

[0007] By the way, it is known that proliferation of synovial cells in rheumatism does not occur indefinitely, but suppresses spontaneously (Daniel J. McCarty (1985) in "Arthrit").
is and allied conditions, A textbook of rheumatolo
gy "10th Edition, Lea & Febiger). More recently, it was revealed that apoptosis occurs in synovial cells of rheumatic patients and that Fas antigen is expressed on the synovial cell membrane. Nakajima et al. T., et al. (1995)
Arthritis Rheum. 38, 485-491) and Aono et al. (See the 38th Annual Meeting of the Japanese Society of Rheumatology (1994), p. 487, and the 1994 Annual Meeting of the Japanese Cancer Society (1994), p. 338) have cytotoxic activity. Examination of whether apoptosis is induced in synovial cells by adding anti-human Fas antibody to abnormally proliferating synovial cells from rheumatic patients showed that abnormally proliferating synovial cells in rheumatic patients were not from rheumatic patients. Apoptosis occurs at a higher rate than that of synovial cells. Therefore, the anti-human Fas antibody can selectively lead not only lymphocytes but also abnormally proliferating synovial cells to apoptosis, and is considered to be useful as a therapeutic agent for rheumatism.

Several kinds of anti-human Fas mouse monoclonal antibodies have already been obtained (Yonehara, S., et al (1989) J. Am.
Exp. Med. 169 , 1747-1756, (1989); SCIENCE, 245 ,
301-305, (1989)) and, as mentioned above,
It has also been reported that the antibody induces apoptosis in synovial cells of rheumatic patients in vitro (38th Annual Meeting of the Japanese Association for Rheumatology (1994), p. 487, and the 1994 General Meeting of the Japanese Cancer Society (1994)). See page 338). Furthermore, an anti-Fas antibody showing its therapeutic effect and safety in autoimmune disease model animals and rheumatoid arthritis model animals has also been found (see European Patent Publication No. 0909816).

On the other hand, it is known that the combined use of a monoclonal antibody cA2 against tumor necrosis factor α (TNFα) and methotrexate increases the therapeutic effect on rheumatoid arthritis patients (Calden, 7th International Rheumatic Symposium (1998)). 12-13), but there is no known synergy between an anti-Fas antibody and methotrexate.

[0010]

PROBLEM TO BE SOLVED BY THE INVENTION Anti-Fas useful as an agent for preventing or treating autoimmune diseases and rheumatoid arthritis
If there is a compound that enhances the effect of the antibody, the amount of the anti-Fas antibody used can be suppressed by using the compound in combination with the anti-Fas antibody. As a result, the possibility that resistance due to the expression of the anti-Fas antibody in the body of the anti-Fas antibody or the like can be reduced, and the development of an excellent prophylactic or therapeutic agent that can withstand long-term administration can be expected. An object of the present invention is to provide a novel pharmaceutical composition useful as an agent for preventing or treating an autoimmune disease.

[0011]

Means for Solving the Problems The present invention provides (1) a pharmaceutical composition comprising, as active ingredients, an anti-human Fas antibody having an apoptosis-inducing activity and a compound having a folate antagonism or a dihydrofolate reductase inhibitory activity. 2)
The pharmaceutical composition according to (1), wherein the anti-human Fas antibody having apoptosis-inducing activity is a monoclonal antibody CH11, HFE7A or a humanized antibody thereof, (3) an anti-human Fs having apoptosis-inducing activity.
(4) the pharmaceutical composition according to (1) or (2), wherein the anti-human Fas antibody having apoptosis-inducing activity is a mouse-mouse hybridoma; (1) or (2), which is an anti-human Fas monoclonal antibody HFE7A produced by HFE7A (FERM BP-5828) or a humanized antibody thereof.
The pharmaceutical composition according to (5), wherein the compound having a folate antagonistic action or a dihydrofolate reductase inhibitory action is methotrexate, edatrexate, epiloprim, iometrexol, pyritrexime, 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-1,4,5,6,7,8-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] benzoyl acid, N- [4-
[3- (2,4-diamino-1H-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- (1-methylethyl) -N '[3- (2,4,5-trichlorophenoxy) propoxy] imide dicarbonimidic diamide hydro The pharmaceutical composition according to any one of (1) to (4), which is selected from the group consisting of chloride (PS15), (6) having a folate antagonistic action or a dihydrofolate reductase inhibitory action. (7) The pharmaceutical composition according to any one of (1) to (5), wherein the compound has methotrexate.
The pharmaceutical composition according to any one of (1) to (6), which is an agent for preventing or treating an autoimmune disease or rheumatoid arthritis.

The present inventors have found that the effect of an anti-Fas monoclonal antibody having an activity of inducing apoptosis is increased by coexisting a compound having an antifolate activity or a dihydrofolate reductase inhibitory activity. Completed.

In the present invention, the term "apoptosis-inducing activity" refers to an activity capable of inducing apoptosis of cells expressing Fas on the cell membrane surface by binding to the Fas.

The anti-human Fas antibody used as the first active ingredient of the pharmaceutical composition of the present invention may be any one that can specifically bind to human Fas and has apoptosis-inducing activity. Such an anti-human Fas antibody is preferably an anti-human Fas monoclonal antibody CH1.
1 and mouse-mouse hybridoma HFE7A (F
ERM BP-5828) produced by the anti-human Fas monoclonal antibody HFE7A or a humanized antibody thereof (European Patent Publication No. 0909816), but the present invention is not limited thereto. Recombinants of these anti-human Fas monoclonal antibodies also have the same effect as these monoclonal antibodies, and are included in the anti-human Fas monoclonal antibody of the present invention. Further, in the present invention, a so-called humanized antibody that has been modified by genetic recombination technology to reduce the immunogenicity to humans without impairing the binding ability to Fas or the apoptosis-inducing activity of the anti-Fas monoclonal antibody described above is used. It can also be used.

In the present invention, "a compound having an antifolate action or an inhibitory action of dihydrofolate reductase" refers to a pharmacological compound having an activity of antagonistically inhibiting folate metabolism, which is an essential process in DNA synthesis in cells. Refers to compounds that are acceptable. Suitably, the following compounds may be mentioned: Methotrexate (formula (I) below)

[0016]

Embedded image Edatrexate (GB 2058770)
B, the following formula (II))

[0017]

Embedded image Epiloprim (see International Patent Application WO 92/8461, the following formula (III))

[0018]

Embedded image Iometrexol (International Patent Application WO86 / 5181)
Reference, the following formula (IV))

[0019]

Embedded image Pyritrexim (see European Patent No. 21292, the following formula (V))

[0020]

Embedded image Trimetrexate (GB 134550
2, see the following formula (VI))

[0021]

Embedded image Brodimoprim (see GB 1449387, the following formula (VII))

[0022]

Embedded image MX-68 (see International Patent Application WO 97/34606, the following formula (VIII))

[0023]

Embedded image N- [4- [3- (2,4-diamino-6,7-dihydro-5H-cyclopenta
[d] pyrimidin-5-yl) propyl] benzoyl] -L-glutamic acid (J. Med. Chem. (1994) 37, 1616-1624, Formula (IX) below)

[0024]

Embedded image 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 (EP 34380)
No. 1, refer to the following formula (X))

[0025]

Embedded image N-((2,4-diamino-3,4,5,6,7,8-hexahydropyrido [2,
3-d] pyrimidin-6-yl) ethyl) -2-thienylcarbonyl
-L-glutamic acid (American Cancer Research Association 88th Annual Meeting (1997)
(See abstract number 660, the following formula (XI))

[0026]

Embedded image (S) -2-[[[4-carboxy-4-[[4-[[(2,4-diamino-6-pteridinyl) methyl] amino] benzoyl] amino] butyl] amino] carbonyl] benzoylic acid (Europe Patent 345308
Reference, the following formula (XII))

[0027]

Embedded image N- [4- [3- (2,4-diamino-1H-pyrrolo [2,3-d] pyrimidine-5
-Yl) propyl] benzoyl] -L-glutamic acid (see EP 334636, the following formula (XIII))

[0028]

Embedded image 2,4-diamino-6- (N- (4- (phenylsulfonyl) benzyl)
Methylamino) quinazoline (American Cancer Research Society Annual Meeting (1992)
(See Abstract No. 2458, Formula (XIV) below)

[0029]

Embedded image 2,4-diamino-5- [4- [3- (4-aminophenyl-4-sulfonylphenylamino) propoxy] -3,5-dimethoxybenzyl] pyrimidine (see EP 231888, the following formula (XV) )

[0030]

Embedded image N- [4- [4- (2,4-diamino-5-pyrimidinyl) butyl] benzoyl] -L-glutamic acid (International Patent Application WO 95/984)
No. 5, JP-A-5, the following formula (XVI))

[0031]

Embedded image N- [4- [3- (2,4-diamino-5-pyrimidinyl) propyl] benzoyl] -L-glutamic acid (the following formula (International Patent Application WO9
No. 5/9845, XVII))

[0032]

Embedded image N- [4- [2- (2,4-diamino-6-pteridinyl) ethyl] benzoyl] -4-methylene-DL-glutamic acid (International Patent Application WO
91/10666, the following formula (XIII))

[0033]

Embedded image And N- (1-methylethyl) -N ′ [3- (2,4,5-trichlorophenoxy) propoxy] imidodicarbonimidic diamide hydrochloride (PS15) (International Patent Application WO 93
/ 16037 gazette, the following formula (XIX))

[0034]

Embedded image All of these compounds are known to have a folate antagonistic action or a dihydrofolate reductase inhibitory action.
Of the above compounds, the most suitable compound to be included in the pharmaceutical composition of the present invention is methotrexate.

[0035]

BEST MODE FOR CARRYING OUT THE INVENTION The pharmaceutical composition of the present invention comprises Fas
Can be obtained by formulating an appropriate mixture of an anti-Fas monoclonal antibody having an activity of inducing apoptosis in cells expressing the compound and a compound having an antifolate action or a dihydrofolate reductase inhibitory action. it can.

The anti-Fas monoclonal antibody can be produced by a method well known in the art, for example, using a molecule containing the extracellular region of human Fas as an antigen. For example, a monoclonal antibody HFE7A, which is one of the preferred anti-Fas monoclonal antibodies contained in the pharmaceutical composition of the present invention, is obtained by immunizing a Fas knockout mouse with human Fas and fusing spleen cells of the mouse with mouse myeloma cells. By culturing the hybridoma obtained by the above, it can be obtained. Specifically, the method described below is used.

In producing a 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 an antibody-producing cell after immunizing the animal by injecting the antigen with an antigen, collecting blood, assaying the antibody titer thereof, determining the timing of spleen extraction, and (c) myeloma cells (Hereinafter referred to as "myeloma"), (d) cell fusion between antibody-producing cells and myeloma, (e) selection of hybridomas producing the desired antibody, and (f) division into single cell clones (cloning). ), (G) cultivation of hybridomas to produce monoclonal antibodies in large quantities, or breeding of animals transplanted with hybridomas, (h) biological activity of monoclonal antibodies thus produced, and recognition thereof Examination of specificity,
Alternatively, it is a test of characteristics as a labeling reagent.

Hereinafter, a method for preparing an anti-Fas monoclonal antibody will be described in detail along the above steps, but the method for preparing the antibody is not limited thereto. For example, antibody-producing cells other than splenocytes and myeloma may be used. it can.

(A) Purification of antigen As antigens, 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], a recombinant protein obtained by introducing and expressing a phFas-AIC2 fusion protein into a monkey-derived cell line COS-1 and partially purifying the same (hereinafter referred to as “recombinant human Fas”). ) Is valid. 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 DNA, vector, host and the like encoding Fas are not limited thereto.

Specifically, phFas-AIC2 uses CO
A transformant obtained by transforming S-1 cells is cultured, and human Fas and mouse I produced in the culture supernatant are cultured.
Recombinant Fas can be partially purified by performing ion exchange chromatography on the L3R fusion protein using a Resource Q column (trade name; manufactured by Pharmacia).

In addition, F present on the cell membrane of a human cell line
Purified as itself can also be used as the antigen. Furthermore, the primary structure of Fas is known [I
toh, N., et al. (1991) Cell 66, 233-243], and a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing was chemically synthesized by a method well known to those skilled in the art. This can also be used as an antigen.

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

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

The immunization can be performed once or repeatedly at appropriate intervals (preferably at intervals of one to five weeks). Thereafter, the antibody titer to the antigen in the serum of the immunized animal is measured, and the effect of the subsequent operation can be enhanced by using the animal whose antibody titer is sufficiently high as a source of the antibody-producing cells. Generally, 3 to 3 days after the last immunization
It is preferable to use antibody-producing cells derived from an animal 5 days later for cell fusion later.

The antibody titers used herein include radioisotope immunoassay (hereinafter referred to as "RIA"), solid-phase enzyme immunoassay (hereinafter referred to as "ELISA"), fluorescent antibody assay, and passive immunoassay. Although various known techniques such as a hemagglutination reaction method can be mentioned, the RIA method or the ELISA method is more preferable from the viewpoints of detection sensitivity, speed, accuracy, and possibility of automation of the operation.

In the present invention, the measurement of the antibody titer
According to the LISA method, it can be performed according to the procedure described below. First, purified or partially purified Fa
s is adsorbed on a solid phase surface of a 96-well plate for ELISA or the like, and a solid phase surface on which no antigen is adsorbed is added to a protein irrelevant to the antigen, for example, bovine serum albumin (hereinafter referred to as “B
After washing the surface, the sample is contacted with a serially diluted sample (for example, mouse serum) as the first antibody, and the anti-Fas antibody in the sample is bound to the antigen. Further, an antibody against an enzyme-labeled mouse antibody is added as a second antibody, and the antibody is bound to the mouse antibody.After washing, a substrate of the enzyme is added, and a change in absorbance due to color development based on the decomposition of the substrate is measured. calculate.

(C) Myeloma Preparation Step As a myeloma, a cell line generally obtained from a mouse, for example, an 8-azaguanine-resistant mouse (BALB)
/ 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
(653) [Kearney, JF et al. J. Immunology, 12
3, 1548-1550 (1979)], P3X63Ag8 (X63) [Horibata,
K. and Harris, AW Nature, 256, 495-497 (1975)]
It is preferable to use such as. These cell lines are grown in a suitable medium, for example, 8-azaguanine medium [RPMI-16
A medium in which 8-azaguanine is added to a medium in which glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter, referred to as “FCS”) are added to 40 media.
, Iscove's Modified Dulbecco's Medium
ulbecco's Medium (hereinafter referred to as "IMDM") or Dulbecco's Modified Eagle's medium (Dulbecco's Modified)
Eagle Medium (hereinafter referred to as "DMEM"), but 3-4 days before cell fusion, a normal medium [eg, 10
% FCS-containing ASF104 medium (manufactured by Ajinomoto Co., Inc.)], and secure 2 × 10 ⁷ or more cells on the day of fusion.

(D) Cell fusion Antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any site of an individual. In general, spleen, lymph node, peripheral blood, Alternatively, it can be obtained from a combination of these as appropriate, and spleen cells are most commonly used.

After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. Currently, 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 simple fusion operation. This method includes, for example, the following procedure.

The spleen cells and the myeloma were thoroughly washed with a serum-free medium (for example, RPMI 1640) or a phosphate buffered saline (hereinafter referred to as “PBS”), and the ratio of the number of spleen cells to the number of myeloma was 5: 1 to 1: 1. Mix to about 10: 1 and centrifuge. The supernatant was removed, and the precipitated cell group was thoroughly loosened. Then, 1 ml of 50% (w
/ V) polyethylene glycol (molecular weight 1000 to 40)
00) is dropped. Then 10ml
And slowly centrifuge. The supernatant was discarded again, and the precipitated cells were placed in a normal medium (hereinafter, referred to as “HAT”) containing an appropriate amount of hypoxanthine / aminopterin / thymidine (hereinafter, referred to as “HAT”) solution and mouse interleukin-2 (hereinafter, referred to as “IL-2”). Culture plate (hereinafter referred to as "plate")
And incubate at 37 ° C. for about 2 weeks in the presence of 5% carbon dioxide. HAT medium is supplemented as needed on the way.

(E) Selection of hybridoma group When the myeloma cells are 8-azaguanine-resistant strains, that is, when they are hypoxanthine guanine phosphoribosyltransferase (HGPRT) -deficient strains, the unfused myeloma cells , And fusion cells of myeloma cells cannot survive in a HAT-containing medium. On the other hand, a fusion cell between antibody-producing cells or a hybridoma of an antibody-producing cell and a myeloma cell can survive, but a fusion cell between antibody-producing cells has a life span. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma between the antibody-producing cell and the myeloma cell survives, and as a result, the hybridoma can be selected.

For the hybridomas that have grown in a colony, the medium is replaced with a medium obtained by removing aminopterin from the HAT medium (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the anti-Fas antibody titer is measured, for example, by ELISA. However, when the above fusion protein is used as an antigen for ELISA, it is necessary to remove the clone so as not to select a clone that produces an antibody that specifically binds to the extracellular region of the mouse IL3 receptor. . The presence or absence of such a clone can be confirmed, for example, by ELISA using the mouse IL3 receptor or its extracellular region as an antigen.

Although the method using an 8-azaguanine-resistant cell line has been described above, other cell lines can be used according to the selection method of the hybridoma, and in that case, the composition of the medium used also changes. (F) Cloning A hybridoma that has been found to produce a specific antibody by measuring the antibody titer in the same manner as described in step (b) is transferred to another plate and cloned. As the cloning method, a limiting dilution method in which one hybridoma is diluted and cultured so that one hybridoma is contained in one well of the plate, a soft agar method in which the colony is collected by culturing in a soft agar medium, and a micromanipulator one by one. Method of removing and culturing cells from a cell sorter
A "sort clone" for separating individual cells may be mentioned, but the limiting dilution method is simple and often used.

For the wells in which the antibody titer has been recognized, for example, cloning by the limiting dilution method is repeated 2 to 4 times, and those having a stable antibody titer are selected as anti-Fas monoclonal antibody-producing hybridoma strains. further,
By selecting a hybridoma producing an antibody that binds to mouse Fas in the same manner, an anti-Fas monoclonal antibody-producing cell is obtained. The mouse Fas used at this time includes, for example, an expression plasmid vector pME18 for 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] was introduced into cultured animal cells and expressed.
or cells expressing mouse Fas on the cell surface can be used.

The mouse-mouse hybridoma HFE7 which is a cell producing anti-Fas monoclonal antibody suitable as an active ingredient to be contained in the pharmaceutical composition of the present invention.
A: February 19, 1997
Deposited internationally under accession number FERM BP-58
28 is attached. Therefore, for example, when preparing an antibody using the mouse-mouse hybridoma HFE7A, the preparation of the antibody can be performed from the step (g) described below, omitting the step (f).

(G) Preparation of Monoclonal Antibody by Hybridoma Culture After completion of cloning, the culture medium was HT
The medium is replaced with a normal medium and cultured. Large-scale culture is performed by rotary culture using a large culture bottle or spinner culture. The supernatant in this large-scale culture is purified using a method well known to those skilled in the art, such as gel filtration, so that the anti-Fas containing the preventive or therapeutic agent of the present invention as an active ingredient.
A monoclonal antibody can be obtained. In addition, mice of the same strain (for example, BALB / c described above) or N
By growing the hybridoma in the abdominal cavity of a u / Nu mouse, ascites containing a large amount of an anti-Fas monoclonal antibody containing the prophylactic or therapeutic agent of the present invention as an active ingredient can be obtained. As a simple method of purification,
A commercially available monoclonal antibody purification kit (for example, MAb
Trap GII kit; manufactured by Pharmacia) and the like can also be used.

The monoclonal antibody thus obtained is
It has high antigen specificity for 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, Ouchterlony (Ouchterlon
y) method, ELISA method, or RIA method.
The Otterlony method is simple, but requires a concentration operation when the concentration of the monoclonal antibody is low. on the other hand,
When the ELISA method or the RIA method is used, the culture supernatant is allowed to react with the antigen-adsorbed solid phase as it is, and various immunoglobulin isotypes and antibodies corresponding to subclasses are used as secondary antibodies, thereby isolating the monoclonal antibody isotype. It is possible to identify subclasses. Further, as a simpler method, a commercially available identification kit (for example, mouse typer kit; manufactured by Bio-Rad) 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].

Next, the DNA encoding the heavy chain or light chain of the anti-Fas monoclonal antibody suitable as an active ingredient to be contained in the pharmaceutical composition of the present invention was obtained from the hybridoma cell producing the anti-Fas monoclonal antibody by mR
NA is prepared, the mRNA is converted to cDNA with reverse transcriptase, and then the DN encoding the heavy or light chain of the antibody is prepared.
A is obtained by isolating each.

For the extraction of mRNA, the guanidine thiocyanate hot phenol method, the guanidine thiocyanate-guanidine hydrochloric acid method and the like can be employed, but the guanidine thiocyanate cesium chloride method is preferred. Preparation of mRNA from cells involves first
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 NA purification or a method of directly purifying from a cell lysate using the carrier. As a method for preparing total RNA, alkaline sucrose density gradient centrifugation [Dougherty, WG and Hiebert,
E. (1980) Viology 101, 466-474], a guanidine thiocyanate / phenol method, a guanidine thiocyanate / trifluorocesium method, a phenol / SDS method and the like can be employed, but a method using guanidine thiocyanate and cesium chloride [Chirgwin, JM, et al. (197
9) Biochemistry 18, 5294-5299].

The poly (A) ⁺ R obtained as described above
Single-stranded cDN by reverse transcriptase reaction using NA as a template
After the synthesis of A, the double-stranded cDN
A can be synthesized. This method includes the S1 nuclease method [Efstratiadis, A., et al. (1976) Cel17,
279-288], the Gubler-Hoffman method [Gubler, U. a.
nd Hoffman, BJ (1983) Gene 25, 263-269],
The Okayama-Berg method [Okayama, H. and Berg, P. (198
2) Mol. Cell. Biol. 2, 161-170], but in the present invention, the polymerase chain reaction (hereinafter referred to as “PCR”) using single-stranded cDNA as a template [Saik
i, RK, et al. (1988) Science 239, 487-49]
The so-called RT-PCR method is preferred.

The double-stranded cDNA thus obtained is incorporated into a cloning vector, the resulting recombinant vector is introduced into a microorganism such as Escherichia coli, and transformed, and transformation is performed using tetracycline resistance or ampicillin resistance as an index. You can choose the body. Transformation of E. coli is performed by the Hanahan method [Hanahan, D. (1983) J. Mol. Bio.
l. 166, 557-580], that is, by adding the recombinant DNA vector to competent cells prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. When a plasmid is used as a vector, it is necessary to have the above-mentioned drug resistance gene. It is also possible to use cloning vectors other than plasmids, for example, lambda phage and the like.

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

(1) Method Using Polymerase Chain Reaction When the whole or a part of the amino acid sequence of the target protein has been elucidated, oligonucleotide primers of sense strand and antisense strand corresponding to a part of the amino acid sequence are synthesized. And combining them, the polymerase chain reaction [Saiki, RK, et al. (1988) Science
239, 487-49] to amplify a DNA fragment encoding the desired anti-human Fas antibody heavy or light chain subunit. As the template DNA used here, for example, mRN of a hybridoma (FERM BP-5828) producing an anti-human Fas monoclonal antibody HFE7A is used.
CDNA synthesized from A by reverse transcriptase reaction can be used.

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

For example, a monoclonal antibody HFE7A suitable as an active ingredient to be contained in the pharmaceutical composition of the present invention.
Means immunoglobulin G1 (hereinafter referred to as "IgG1")
It is a molecule and is a complex composed of each subunit of a heavy chain (γ1 chain) and a 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) A method of analyzing the N-terminal amino acid sequence of each subunit using PPSQ-10) or the like is preferable.

From the target transformant obtained as described above, a cDNA encoding each subunit of the anti-human Fas monoclonal antibody protein can be collected by a known method [Maniatis, T., et al. (1982) in "Mo
lecular Cloning A LaboratoryManual "Cold Spring Ha
rbor Laboratory, NY.]. For example, it can be carried out by separating a fraction corresponding to a vector DNA from a cell, and cutting out a DNA region encoding a subunit of interest from the plasmid DNA.

(2) Screening Method Using Synthetic Oligonucleotide Probe When the entire or partial amino acid sequence of the target protein has been elucidated (if the sequence is a specific sequence consisting of a plurality of consecutive Region), and synthesize an oligonucleotide corresponding to the amino acid sequence (in this case, any of a nucleotide sequence deduced with reference to codon usage or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences) Can be used, and in the latter case, the type can be reduced by including inosine), which is used as a probe (labeled with ³² P, ³⁵ S or biotin, etc.) to obtain D
Hybridization is performed with a nitrocellulose filter in which NA is modified and fixed, and the obtained positive strain is selected.

The sequence of the DNA thus obtained can be determined, for example, by 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 [Messing, J. and Vieira, J. (1982) Gene 1
9, 269-276].

In recent years, automatic base sequence determination systems using fluorescent dyes have become widespread (for example, a sequence robot “CATALYST 800” manufactured by PerkinElmer Japan, and a model 373A DNA sequencer).

By using such a system, DN
The A nucleotide sequencing operation can be performed efficiently and safely. From the nucleotide sequence of the DNA of the present invention thus determined and the N-terminal amino acid sequence data of the heavy and light chains, the entire amino acid sequence of the heavy and light chains of the monoclonal antibody of the present invention is determined. Can be.

In preparing a variant in which any one or more amino acids in the amino acid sequence are deleted, a cassette mutation method [Toshimitsu Kishimoto, "Shinsei Kagaku Kenkyusho 2 Nucleic Acid III Recombinant DNA Technology" See 242-251].

Such various DNAs can be prepared, for example, by the phosphite triester method [Hunkapiller, M., et al.
984) See Nature 310, 105-111] and the like, and can be produced by chemical synthesis of nucleic acid. The codon for the desired amino acid is known per se, and its selection may be arbitrarily determined. For example, the codon for the desired amino acid may be determined according to a conventional method in consideration of the codon usage of the host to be used. These nucleotide sequence codons can be partially modified by a site-directed mutagenesis method (cytospecific mutagenesis) using a primer comprising a synthetic oligonucleotide encoding the desired modification [Mark, D.
F., et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5
662-5666].

Further, a certain DNA is used in the pharmaceutical composition of the present invention.
Anti-Fas monochrome suitable as an active ingredient to be contained
DNA encoding the heavy or light chain of a null antibody and a hive
Whether or not redising is determined, for example, by randomizing the DNA
Primer method [Feinberg, A.P. and Vogelstein, B. (1
983) Anal. Biochem. 132, 6-13] and Nick Tran
Slation method [Maniatis, T., et al. (1982) in "Mo
lecular Cloning A laboratory Manual "Cold Spring H
arbor Laboratory, NY.] [α- ³²P] d
Using probe DNA labeled with CTP, etc.,
You can find out by conducting an experiment like
You.

First, the DNA to be examined is adsorbed on, for example, a nitrocellulose membrane or a nylon membrane, and if necessary, treated with alkali denaturation or the like, and then solidified by heating or ultraviolet rays. This film was subjected to 6 × SSC
(1 × SSC is 0.15M sodium chloride, 0.01
5M trisodium citrate solution), 5% Denhardt's solution, and 0.1% sodium dodecyl sulfate (SDS). Pre-hybridization solution, and incubate at 55 ° C for 4 hours or more. 1 × 1 final specific activity in prehybridization solution
It added to a 0 ⁶ cpm / ml, incubated overnight at 60 ° C.. Thereafter, the operation of washing the membrane with 6 × SSC at room temperature for 5 minutes is repeated several times, and further washed with 2 × SSC for 20 minutes, followed by autoradiography.

Using the above method, any cD
From the NA library or the genomic library, it is possible to isolate DNA that hybridizes with DNA encoding the heavy or light chain of an anti-Fas monoclonal antibody suitable as an active ingredient to be contained in the pharmaceutical composition of the present invention [ Maniatis, T., et al. (1982) in "Molecular
Cloning A laboratory Manual "Cold Spring Harbor La
boratory, NY.].

By incorporating each DNA obtained as described above into an expression vector, it is possible to introduce the gene into prokaryotic or eukaryotic host cells and to express each gene in those host cells. .

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
) And the like. In order 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, i.e., a plasmid vector containing an origin of replication and a promoter sequence such as lacUV5. . The vector preferably has a sequence capable of imparting selectivity based on the phenotypic trait in the transformed cell. For example, Escherichia coli includes E. coli.
The JM109 strain derived from E. coli K12 strain and the like are often used, and as the vector, pBR322 and pUC-based plasmids are generally often used, but are not limited thereto, and any of 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 Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 [Ohmura,
K., et al. (1984) J. Biochem. 95, 87-93] and the like, but are not limited thereto. As the promoter, a regulatory sequence of the Bacillus subtilis α-amylase gene is often used, and if necessary, a DNA sequence encoding the signal peptide sequence of α-amylase can be ligated to allow extracellular secretion. .

Eukaryotic host cells include cells such as vertebrates and yeast. Examples of vertebrate cells include COS-1 [Gluzman, Y. (1981) C, a monkey-derived cell line.
ell23, 175-182]. Yeasts include baker's yeast (Saccharomycescerevisiae), alcohol-assimilating yeast (Pichia pastoris), and fission yeast (Schizos
saccharomyces pombe) is used. Cells such as those exemplified here are commonly used as host cells, but are not limited thereto.

As a vertebrate cell expression vector, those having a promoter, an RNA splicing site, a polyadenylation site, a transcription termination sequence, etc. which are usually located upstream of the gene to be expressed can be used. Further, a replication origin may be provided if necessary. Examples of such expression vectors include pSV2dhfr with the SV40 early promoter [Subramani, S., et al. (198
1) Mol. Cell. Bio. 1, 854-864], but are not limited thereto.

Examples of yeast expression vectors include, for example, an alcohol dehydrogenase gene promoter [Bennetzen,
JL and Hall, BD (1982) J. Biol. Chem. 257, 3
018-3025] and the promoter of a galactose metabolism enzyme gene (such as gal10) [Ichikawa, K.,
et al. (1993) Biosci. Biotech. Biochem. 57, 1686-1.
690], and if necessary, by linking a DNA sequence encoding the signal peptide sequence of the yeast gene, extracellular secretion becomes possible, but is not limited thereto.

For example, when COS-1 is used as a host cell, the expression vector has an SV40 origin of replication and is capable of autonomous growth in COS-1.
In addition, a transcription promoter, a transcription termination signal, and R
An expression vector having an NA splice site can be used. The expression vector is prepared by the DEAE-dextran method [Luthman, H. and Magnusson, G. (1983) Nucleic A
cids Res. 11, 1295-1308], calcium phosphate-
DNA coprecipitation method [Graham, FL and van der Eb, AJ
(1973) Virology 52, 456-457] and electroporation [see Neumann, E., et al. (1982) EMBO J. 1, 841-845].
Thus, a desired transformed cell can be obtained.

In particular, an expression vector comprising a DNA encoding the heavy chain and a DNA encoding the light chain
By co-transfecting COS-1 with an expression vector comprising the above, these DNAs can be simultaneously expressed, 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. For example, a single expression vector capable of retaining both DNAs encoding each of the heavy chain and the light chain and expressing them simultaneously is constructed. 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 culture allows the recombinant anti-Fas to be produced inside or outside the transformant cells.
Antibodies are produced. As a medium used for the culture,
Various conventional ones can be appropriately selected depending on the host cell employed. For example, in the case of COS-1, the RPMI16
A medium obtained by adding a serum component such as fetal calf serum (FCS) to a medium such as 40 medium or Dulbecco's modified Eagle medium (DMEM), if necessary, can be used.

The culture temperature at the time of culturing the transformant is not particularly limited as long as it does not remarkably reduce the protein synthesis ability in cells, but is preferably 32 to 42 ° C., and most preferably 37 ° C. Incubate with Also, if necessary, 1-1
The cells 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 obtained by various known protein separation procedures using the physical properties and chemical properties of the protein. Can be separated and purified. Specific examples of such a method include treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC). ), Various types of chromatography, dialysis methods, combinations thereof, and the like.

According to the above method, a recombinant anti-Fas antibody can be easily produced with high yield and high purity.

The recombinant anti-Fas thus produced
As a method for confirming that an antibody specifically binds to Fas, for example, an ELISA method similar to the case of evaluating an antibody titer at the time of immunization with a mouse is suitable.

On the other hand, compounds having the folate antagonistic activity or dihydrofolate reductase inhibitory activity, for example, those of the above formulas (I) to (X)
Compounds represented by IX) can be obtained as described in the literature referenced in the description of each compound. Whether or not other compounds have folate antagonistic activity or dihydrofolate reductase inhibitory activity can also be examined using the method for confirming folate antagonistic activity or dihydrofolate reductase inhibitory activity used in those documents. .

The pharmaceutical composition of the present invention can be used as a prophylactic or therapeutic agent for an autoimmune disease or rheumatoid arthritis patient as an active ingredient. Such a prophylactic or therapeutic agent can be administered in various forms, such as tablets, capsules,
Oral administration by granules, powders, syrups, etc., or
Parenteral administration by injections, drops, suppositories and the like can be mentioned.

The anti-human F in the pharmaceutical composition of the present invention
The dose of the as antibody and the compound having a folate antagonism or dihydrofolate reductase inhibitory action may vary depending on each antibody used, the strength of the action specific to the compound, and the like. For example, when CH11 or HFE7A is used as an anti-human Fas antibody and methotrexate is used as a compound having folate antagonism or dihydrofolate reductase inhibitory activity, the pharmaceutical composition of the present invention preferably contains CH11 or HFE7A.
Alternatively, the solution is prepared as a solution containing 0.1 to 100 ng / ml of HFE7A and 0.05 to 5 nM of methotrexate, but is not limited to this range when another antibody or compound is used.

The dose varies depending on symptoms, age, body weight, etc., but usually, the amount of anti-human Fas antibody is 1%.
0.1 mg to 1000 mg may be administered by subcutaneous injection, intramuscular injection or intravenous injection.

The effectiveness of the pharmaceutical composition of the present invention prepared as described above in the treatment of autoimmune diseases or rheumatoid arthritis is determined by measuring the presence of cells (for example, human lymphocytes) in a medium supplemented with the pharmaceutical composition of the present invention. Spheroid cell line HPB-ALL
(Morikawa, S., et al. (1978) Int. J. Cancer 21, 1
66-170), Jurkat (American Type Culture)
No. TIB-152), synovial cells from rheumatoid arthritis patients, etc.) were cultured, and the survival rate was measured by MTT assay (Green,
LM, et al. (1984) J. Immunological Methods 70,
257-268) and the XTT assay described in Examples below.
The pharmaceutical composition of the present invention administers only an anti-Fas antibody to autoreactive lymphocytes, which is one of the main causes of autoimmune diseases, and synovial cells that are abnormally proliferating in the affected area of rheumatoid arthritis. Apoptosis can be induced with a lower dose of anti-Fas antibody than is effective in treating these autoimmune diseases or rheumatoid arthritis. European Patent Publication 0909 discloses that a composition containing an anti-Fas antibody, which has been confirmed to be effective in experiments using cultured cells, is also effective in actual autoimmune disease symptoms and rheumatoid arthritis.
No. 816 discloses an experimental result using an animal experimental model.

[0096]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 As an anti-Fas antibody, an anti-human Fas monoclonal antibody HFE7A described in European Patent Publication No. 0909816 was used.
Alternatively, CH11 (manufactured by Medical Biology Laboratories) was used.

The human lymphocyte cell line HPB-ALL (Mori
kawa, S., et al. (1978) Int. J. Cancer 21, 166-170
5% in RPMI1640 medium containing 10% FCS (manufactured by Gibco BRL) at 37 ° C.
Culture for 3 days in the presence of carbon dioxide (2.5 × 10 ⁵ cells /
50 μl) was dispensed into each well of a 96-well microplate (manufactured by Sumitomo Bakelite Co., Ltd.) by 50 μl.
Next, methotrexate (manufactured by Sigma) and anti-Fas
Antibody (CH11 or HFE7A, 0.001 mg / m
50 μl of RPMI medium containing 3 times serial dilution from 1)
Was added to each well and cultured at 37 ° C.

The plate to which CH11 was added as an anti-Fas antibody was cultured at 37 ° C. overnight. On the other hand, for the plate to which HFE7A was added, culture 1
After a lapse of time, the cells were washed with an RPMI medium, and then 100 μl / well of an anti-mouse IgG antibody (manufactured by Biosource) adjusted to 1 μg / ml with the RPMI medium was added. After culturing at 37 ° C. for 1 hour, washing the cells with a serum-free RPMI medium,
After adding 100 μl / well of RPMI medium containing 0.05 nM methotrexate, the cells were cultured at 37 ° C. overnight.

Thereafter, 1 mg / ml of XTT (2,3-
Bis [2-methoxy-4-nitro-5-sulfophenyl] -2H-tetrazolium-5-carboxanilide
25 μM PMS containing an inner salt (manufactured by Sigma)
(Phenazine methosulfate: Sigma) 50 μl
/ Well (final concentration 250 μg / ml XTT, 5 μM
PMS) was added. After culturing at 37 ° C. for 3 hours, the absorbance at 450 nm of each well was measured, and the cell viability was calculated using the mitochondrial reducing ability as an index.

The survival rate of the cells in each well was calculated by the following equation: survival rate (%) = 100 × (ab) / (c−b) (where a represents the measured value of the test well) , B represents the measured value of the cell-free well, and c represents the measured value of the well without the antibody).

As a result, it was revealed that the apoptosis-inducing activity of CH11 was significantly increased by adding methotrexate of 0.05 nM or more (FIG. 1.
In the figure, “MTX” represents methotrexate. Hereinafter the same). The apoptosis-inducing activity of HFE7A was also increased by the addition of 0.05 nM methotrexate (FIG. 2). Not adding anti-Fas antibody,
Under the condition of adding methotrexate alone, the cell viability hardly changed (FIG. 3).

The above results indicate that the addition of methotrexate enhances the apoptosis-inducing activity of the anti-Fas antibody, and the administration of a lower dose of the anti-Fas antibody can reduce the number of cells causing autoimmune disease. .

Formulation Examples The prophylactic or therapeutic agent of the present invention is obtained by dissolving an anti-human Fas antibody and a compound having an antifolate action or an inhibitor of dihydrofolate reductase in water or another pharmacologically acceptable solution. Served as an ampoule of a sterile solution or suspension. Specifically, for example, 0.5 mg of anti-human Fas antibody and methotrexate in a final amount of 0.05 nM are dissolved in 1 liter of water for injection, and then dispensed aseptically into ampoules and sealed.

Also, a sterile powder preparation (preferably freeze-dried with an anti-human Fas antibody and a compound having an antifolate action or a dihydrofolate reductase inhibitory action) is filled in an ampoule and pharmacologically used at the time of use. It may be diluted with an acceptable solution.

[0106]

According to the present invention, a novel pharmaceutical composition useful as an agent for preventing or treating an autoimmune disease is provided.
According to the present invention, the amount of the anti-Fas antibody used can be reduced by using a compound having a folate antagonistic action or a dihydrofolate reductase inhibitory action in combination with the anti-human Fas antibody. Thereby, the anti-Fas in the patient body
The pharmaceutical composition of the present invention is excellent in fascinating rheumatoid arthritis including long-term administration because it can reduce the possibility of manifestation of resistance due to the expression of the antibody against the antibody.
-It is useful as an agent for preventing or treating autoimmune diseases caused by abnormal Fas ligand system.

[Brief description of the drawings]

FIG. 1 shows the synergistic effect of the anti-human Fas monoclonal antibody CH11 and methotrexate on cell apoptosis induction.

FIG. 2: Anti-human Fas monoclonal antibody HFE7A
Showing the synergistic effect of cell and methotrexate on the induction of cell apoptosis

FIG. 3 is a diagram showing cell viability in the presence of methotrexate.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (A61K 39/395 31: 519) (A61K 39/395 31: 506) (A61K 39/395 31: 517) ( A61K 39/395 31: 505) (A61K 39/395 31: 155)

Claims (7)

[Claims] 1. An anti-human F having an apoptosis-inducing activity
A pharmaceutical composition comprising, as an active ingredient, an as antibody and a compound having a folate antagonistic action or a dihydrofolate reductase inhibitory action. 2. An anti-human F having apoptosis-inducing activity
The pharmaceutical composition according to claim 1, wherein the as antibody is a monoclonal antibody CH11, HFE7A or a humanized antibody thereof. 3. An anti-human F having an apoptosis-inducing activity
The pharmaceutical composition according to claim 1 or 2, wherein the as antibody is the monoclonal antibody CH11 or a humanized antibody thereof. 4. An anti-human F having apoptosis-inducing activity
as antibody is mouse-mouse hybridoma HFE7A
(FERM BP-5828) produces anti-human Fas
The pharmaceutical composition according to claim 1 or 2, which is a monoclonal antibody HFE7A or a humanized antibody thereof. 5. A compound having an antifolate action or an inhibitory action of dihydrofolate reductase, wherein methotrexate, edatrexate, epiloprim, iometrexol, pyritrexime, trimetrexate, brodimoprim, M
X-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-1,4,5,6,7,8-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] pyrimidine-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] benzoyl acid, N -[4- [3- (2,4-diamino-1H-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- (1-methylethyl) -N '[3- (2,4,5-trichlorophenoxy) propoxy] imidodicarbonimidic diamide hydrochloride (PS15) 1 selected from the group consisting of
Or the pharmaceutical composition according to any one of claims 1 to 5, wherein the composition is two or more compounds. 6. The pharmaceutical composition according to claim 1, wherein the compound having an antifolate action or an inhibitory action of dihydrofolate reductase is methotrexate. 7. The pharmaceutical composition according to claim 1, which is an agent for preventing or treating an autoimmune disease or rheumatoid arthritis.