JP2010095445A - Therapeutic agent for inflammatory myopathy containing il-6 antagonist as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an excellent therapeutic agent for inflammatory myopathy which can remarkably suppress symptoms of inflammatory myopathy such as abnormal inflammatory cellular infiltration in muscle fibers does not have disadvantages of existing medicines such as a general decrease of immunocompetence. <P>SOLUTION: The therapeutic agent for inflammatory myopathy contains an IL-6 antagonist as an active ingredient. In the therapeutic agent for inflammatory myopathy, the IL-6 antagonist is preferably an antibody against IL-6 receptor, a monoclonal antibody, or the like. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for inflammatory muscle disease comprising an IL-6 antagonist as an active ingredient.

  IL-6 is a cytokine also called B cell stimulating factor 2 (BSF2) or interferon β2. IL-6 was discovered as a differentiation factor involved in the activation of B lymphocyte cells (Non-Patent Document 1), and subsequently became a multifunctional cytokine that affects the functions of various cells. (Non-patent document 2). IL-6 has been reported to induce maturation of T lymphocyte cells (Non-patent Document 3).

IL-6 transmits its biological activity via two proteins on the cell. One is IL-6 receptor, which is a ligand-binding protein having a molecular weight of about 80 kD to which IL-6 binds (Non-patent Documents 4 and 5). The IL-6 receptor exists as a soluble IL-6 receptor mainly composed of the extracellular region in addition to the membrane-bound type that penetrates the cell membrane and is expressed on the cell membrane.
The other is a membrane protein gp130 having a molecular weight of about 130 kD involved in non-ligand binding signaling. IL-6 and IL-6 receptor form an IL-6 / IL-6 receptor complex and then bind to gp130, thereby transmitting the biological activity of IL-6 into the cell (non- Patent Document 6).

An IL-6 antagonist is a substance that inhibits the transmission of biological activity of IL-6. So far, antibodies against IL-6 (anti-IL-6 antibody), antibodies against IL-6 receptor (anti-IL-6 receptor antibody), antibodies against gp130 (anti-gp130 antibody), IL-6 variants, IL -6 or IL-6 receptor partial peptides are known.
There are several reports regarding anti-IL-6 receptor antibodies (Non-patent Documents 7 and 8, Patent Documents 1 to 3). A humanized PM-1 antibody obtained by transplanting a complementarity determining region (CDR) of the mouse antibody PM-1 (Non-Patent Document 9), which is one of these, into a human antibody is known. (Patent Document 4).

  By the way, polymyositis is a disease in which extensive inflammatory changes of skeletal muscles are recognized, and basic symptoms such as loss of power, fatigue, and muscle pain. Characteristic skin such as heliotrophic eruption When accompanied by symptoms, it is called dermatomyositis. Although details regarding the relationship between such inflammatory myopathy and IL-6 are not clear, there have been cases of polymyositis in which mononuclear cells expressing IL-6 are present in inflammatory muscle tissue. (Non-Patent Document 10). However, in this report, the expression level is rather small compared to other inflammatory cytokines such as IL-1. Although it has been reported that IL-6 is expressed in myoblasts, its expression has not been confirmed from mature myocytes (Non-patent Document 11). On the other hand, it has become clear that IL-6 is necessary for inducing Th17 cells, which are recently considered to be a major cell group of autoreactive T cells (Non-patent Documents 12 and 13). . However, the role of Th17 cells in autoimmune myositis is unclear. Thus, although there is a report suggesting a relationship between inflammatory myopathy and IL-6, whether inhibition of expression or function of IL-6 has a therapeutic effect on inflammatory myopathy. Was still unknown.

Prior art document information related to the invention of the present application is shown below.
Hirano, T .; et al. , Nature (1986) 324, 73-76. Akira, S.A. et al. , Adv. in Immunology (1993) 54, 1-78 Lotz, M.M. et al. , J .; Exp. Med. (1988) 167, 1253-1258 Taga, T .; et al. , J .; Exp. Med. (1987) 166, 967-981 Yamazaki, K .; et al. , Science (1988) 241, 825-828. Taga, T .; et al. Cell (1989) 58, 573-581. Novick, D.M. et al. , Hybridoma (1991) 10, 137-146. Huang, Y. et al. W. et al. , Hybridoma (1993) 12, 621-630. Hirata, Y. et al. et al. , J .; Immunol. (1989) 143, 2900-2906 Lundberg, I.D. et al. Arthritis Rheum (1997) 40 (5), 865-874. De Rossi, M .; et al. Int Immunol (2000) 12 (9), 1329-1335. Yoichiro, I. et al. and Harumichi, I .; , The Journal of Clinical Investigation (2006) 116 (5) 1218-1222. Marc, V.M. et al. , Immunity (2006) 24, 179-189 International Publication No. 95-09873 Pamphlet French Patent Application No. 2694767 US Pat. No. 5,216,128 International Publication No. 92-19759 Pamphlet

  In the past, corticosteroids and immunosuppressive drugs have been used to treat inflammatory myopathy such as polymyositis and dermatomyositis. There are various side effects such as diabetes, abnormal bone metabolism, gastric ulcer, muscle atrophy, and cataract, and the latter can also be a fatal side effect of infection caused by a strong and general decline in immunity. An object of the present invention is to provide a novel therapeutic agent for inflammatory muscle disease that does not have the disadvantages of the previous period by targeting a single molecule.

As a result of intensive studies on searching for a molecule having a therapeutic effect using a model mouse of polymyositis / dermatomyositis, the present inventors have found that a molecule that suppresses the function of IL-6 is abnormal in muscle fibers. The inventors have found that symptoms of inflammatory muscle diseases such as inflammatory cell infiltration can be remarkably suppressed, and have completed the present invention.
That is, the present invention relates to a therapeutic agent for inflammatory myopathy targeting IL-6, more specifically,
(1) A therapeutic agent for inflammatory muscle disease, characterized by comprising an IL-6 antagonist as an active ingredient,
(2) The therapeutic agent for inflammatory muscle disease according to (1), wherein the IL-6 antagonist is an antibody to IL-6 receptor or an antibody to IL-6,
(3) The therapeutic agent for inflammatory muscle disease according to (2), wherein the antibody is a monoclonal antibody,
(4) The therapeutic agent for inflammatory muscle disease according to (2), wherein the antibody is a recombinant antibody,
(5) The therapeutic agent for inflammatory muscle disease according to (2), wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody,
(6) The therapeutic agent for inflammatory myopathy according to any one of (1) to (5), wherein the inflammatory myopathy is polymyositis or dermatomyositis,
(7) The therapeutic agent for inflammatory muscle disease according to any one of (1) to (6), which has an action of suppressing abnormal inflammatory cell infiltration in muscle fibers.

  In model mice with polymyositis / dermatomyositis, inflammatory cell infiltration in muscle fibers was remarkably suppressed by suppressing the function of IL-6. Therefore, the IL-6 antagonist including the anti-IL-6 receptor monoclonal antibody according to the present invention is useful as a new therapeutic agent for inflammatory myopathy having an action of suppressing abnormal inflammatory cell infiltration in muscle.

(Inflammatory muscle disease treatment)
The present invention provides a therapeutic agent for inflammatory muscle disease comprising an IL-6 antagonist as an active ingredient.

<IL-6 antagonist>
In the present invention, an “IL-6 antagonist” is a substance that blocks signal transduction by IL-6 and inhibits the biological activity of IL-6. The IL-6 antagonist is preferably a substance having an inhibitory action on binding of any of IL-6, IL-6 receptor, and gp130.
Examples of the IL-6 antagonist in the present invention include, for example, anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-gp130 antibody, IL-6 variant, soluble IL-6 receptor variant, or IL-6 or Examples include IL-6 receptor partial peptides, and proteins exhibiting the same activity as these (for example, C326 Avimer; Nature Biotechnology Vol. 23, Number 12, December 2005) and low molecular weight substances. It is not a thing. Among them, the IL-6 antagonist in the present invention preferably includes an antibody that recognizes an IL-6 receptor (anti-IL-6 receptor antibody).
The origin of the antibody in the present invention is not particularly limited, but is preferably derived from a mammal, more preferably a human-derived antibody.

-Anti-IL-6 antibody-
The anti-IL-6 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-IL-6 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. This antibody binds to IL-6, thereby blocking the binding of IL-6 to the IL-6 receptor and blocking the intracellular transmission of IL-6 biological activity.
Examples of such antibodies include MH166 (Matsuda, T. et al., Eur. J. Immunol. (1988) 18, 951-956) and SK2 antibody (Sato, K. et al., 21st Japan Immunological Society). General meeting, academic record (1991) 21, 166).

Anti-IL-6 antibody-producing hybridomas can be basically produced using known techniques as follows. That is, using IL-6 as a sensitizing antigen, this is immunized according to a normal immunization method, the resulting immune cells are fused with a known parent cell by a normal cell fusion method, and by a normal screening method, It can be produced by screening monoclonal antibody-producing cells.
Specifically, the anti-IL-6 antibody can be prepared as follows. For example, human IL-6 used as a sensitizing antigen for antibody acquisition is described in Eur. J. et al. Biochem (1987) 168, 543-550; Immunol. (1988) 140, 1534-1541, or Agr. Biol. Chem. (1990) 54, 2685-2688, obtained by using the IL-6 gene / amino acid sequence.
An IL-6 gene sequence is inserted into a known expression vector system to transform an appropriate host cell, and then the target IL-6 protein is obtained from the host cell or culture supernatant by a known method. The purified IL-6 protein may be purified and used as a sensitizing antigen. Moreover, you may use the fusion protein of IL-6 protein and another protein as a sensitizing antigen.

-Anti-IL-6 receptor antibody-
The anti-IL-6 receptor antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-IL-6 receptor antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. This antibody binds to the IL-6 receptor, thereby inhibiting the binding of IL-6 to the IL-6 receptor and blocking the intracellular transmission of IL-6 biological activity.
Examples of such antibodies include MR16-1 antibody (Tamura, T. et al. Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928) and PM-1 antibody (Hirata, Y. et al. , J. Immunol. (1989) 143, 2900-2906), AUK12-20 antibody, AUK64-7 antibody, or AUK146-15 antibody (WO92-19759 pamphlet). Among these, a preferred monoclonal antibody against human IL-6 receptor is exemplified by PM-1 antibody, and a preferred monoclonal antibody against mouse IL-6 receptor is MR16-1 antibody.

An anti-IL-6 receptor monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, IL-6 receptor is used as a sensitizing antigen, this is immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. Can be prepared by screening monoclonal antibody-producing cells.
Specifically, the anti-IL-6 receptor antibody can be prepared as follows. For example, a human IL-6 receptor used as a sensitizing antigen for obtaining an antibody is disclosed in European Patent Application Publication No. 325474, and a mouse IL-6 receptor is disclosed in Japanese Patent Application Laid-Open No. 3-15595. It is obtained by using the disclosed IL-6 receptor gene / amino acid sequence.
IL-6 receptor protein is expressed on the cell membrane and separated from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676). The soluble IL-6 receptor antibody is substantially composed of the extracellular region of IL-6 receptor bound to the cell membrane, and the cell transmembrane region or the cell membrane transmembrane region and the intracellular region are deficient. And different from membrane-bound IL-6 receptor. Any IL-6 receptor may be used as the IL-6 receptor protein as long as it can be used as a sensitizing antigen for producing the anti-IL-6 receptor antibody used in the present invention.
After inserting the gene sequence of IL-6 receptor into a known expression vector system and transforming an appropriate host cell, the target IL-6 receptor protein is transferred from the host cell or culture supernatant. The purified IL-6 receptor protein may be used as a sensitizing antigen after purification by a known method. Alternatively, cells expressing IL-6 receptor or a fusion protein of IL-6 receptor protein and another protein may be used as the sensitizing antigen.

-Anti-gp130 antibody-
The anti-gp130 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-gp130 antibody used in the present invention, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. This antibody binds to gp130, thereby inhibiting the binding of IL-6 / IL-6 receptor complex to gp130 and blocking the transmission of IL-6 biological activity into cells.
Examples of such antibodies include AM64 antibody (Japanese Patent Laid-Open No. 3-219894), 4B11 antibody and 2H4 antibody (US Pat. No. 5,571,513), B-S12 antibody and B-P8 antibody (Japanese Patent Laid-Open No. 8-291199). Publication).

An anti-gp130 monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, using gp130 as a sensitizing antigen, this is immunized according to a normal immunization method, the obtained immune cells are fused with a known parent cell by a normal cell fusion method, and a monoclonal antibody is obtained by a normal screening method. It can be produced by screening production cells.
Specifically, the monoclonal antibody can be produced as follows. For example, gp130 used as a sensitizing antigen for obtaining an antibody can be obtained by using the gp130 gene / amino acid sequence disclosed in European Patent Application No. 411946.
After the gp130 gene sequence is inserted into a known expression vector system to transform an appropriate host cell, the desired gp130 protein is purified from the host cell or culture supernatant by a known method. A purified gp130 protein may be used as a sensitizing antigen. Further, a cell expressing gp130 or a fusion protein of gp130 protein and another protein may be used as a sensitizing antigen.

-Antibody production-
The mammal immunized with each sensitizing antigen as described above is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Rodent animals such as mice, rats, hamsters and the like are used.
In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if desired, and emulsified. Thereafter, the mammal is preferably administered several times every 4 to 21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.
After immunizing in this way and confirming that the desired antibody level rises in the serum, the antiserum may be used as a polyclonal antibody as it is, or if a monoclonal antibody is to be prepared, a suckling is used. Immune cells are removed from the animal and subjected to cell fusion. Spleen cells are particularly preferred as preferable immune cells to be subjected to cell fusion.

  Mammalian myeloma cells as the other parental cells to be fused with the immune cells have already been known in various known cell lines such as P3X63Ag8.653 (Kearney, JF et al. J. Immunol. (1979). ) 123, 1548-1550), P3X63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Travebridge, I.S. J. Exp. Med. (1978) 148, 313-323), R210. (Galfre G.et al., Nature (1979) 277,131-133) such as are known, they are used as appropriate.

The cell fusion between the immune cells and myeloma cells is basically performed by a known method such as the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It can be done according to this.
More specifically, the cell fusion is performed, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used. If desired, an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency.

  The ratio of the immune cells and myeloma cells used is preferably, for example, 1 to 10 times as many immune cells as the myeloma cells. As the culture solution used for the cell fusion, for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this type of cell culture can be used. Serum replacement fluid such as fetal calf serum (FCS) can be used in combination.

  In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution and pre-warmed to about 37 ° C., for example, PEG having an average molecular weight of about 1,000 to 6,000. A target fused cell (hybridoma) is formed by adding the solution at a concentration of usually 30 to 60% (w / v) and mixing. Subsequently, cell fusion agents and the like that are undesirable for hybridoma growth can be removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

  The hybridoma is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin, and thymidine). Culturing with the HAT culture solution is continued for a time sufficient for the cells other than the target hybridoma (non-fused cells) to die, usually several days to several weeks. Subsequently, a normal limiting dilution method is performed, and screening and cloning of the hybridoma producing the target antibody are performed.

  In addition to immunizing animals other than humans to obtain the above hybridomas, human lymphocytes are sensitized with a desired antigen protein or antigen-expressing cells in vitro, and sensitized B lymphocytes are human myeloma cells, for example, It is also possible to obtain a desired human antibody having a binding activity to a desired antigen or an antigen-expressing cell by fusing with U266 (see Japanese Patent Publication No. 1-59878). Furthermore, antigens or antigen-expressing cells may be administered to a transgenic animal having a repertoire of human antibody genes, and desired human antibodies may be obtained according to the method described above (WO 93/12227, WO 92). No. 03918, Pamphlet of International Publication No. 94/02602, Pamphlet of International Publication No. 94/25585, Pamphlet of International Publication No. 96/34096, Pamphlet of International Publication No. 96/33735).

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible therewith to proliferate, and its ascites The method obtained as follows is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

  For example, the production of an anti-IL-6 receptor antibody-producing hybridoma can be performed by the method disclosed in JP-A-3-139293. A PM-1 antibody-producing hybridoma is injected into the abdominal cavity of a BALB / c mouse to obtain ascites, and the PM-1 antibody is purified from this ascites, or the hybridoma is treated with an appropriate medium such as 10% fetal bovine serum, Culture in 5% BM-Condimed H1 (Boehringer Mannheim) -containing RPMI1640 medium, hybridoma SFM medium (GIBCO-BRL), PFHM-II medium (GIBCO-BRL), etc., and use the PM-1 antibody from the culture supernatant. It can be performed by a purification method.

Further, in the present invention, as a monoclonal antibody, an antibody gene is cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host, and a recombinant antibody produced using a gene recombination technique is used. (See, for example, Borrebaeck C.A.K. and Larrick J.W. THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom MACMILLAN PUBLISHERS LTD, 19).
Specifically, mRNA encoding the variable (V) region of an antibody is isolated from a cell that produces the target antibody, for example, a hybridoma. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159), etc., and mRNA is prepared using mRNA Purification Kit (manufactured by Pharmacia). Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).

Antibody V region cDNA is synthesized from the obtained mRNA using reverse transcriptase. The synthesis of cDNA can be performed using AMV Reverse Transcrispase First-strand cDNA Synthesis Kit or the like. In order to synthesize and amplify cDNA, 5'-Ampli FINDER RACE Kit (Clontech) and 5'-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). The target DNA fragment is purified from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA is confirmed by a known method such as the deoxy method.
If DNA encoding the V region of the target antibody is obtained, it is ligated with DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector. Alternatively, DNA encoding the V region of the antibody may be incorporated into an expression vector containing DNA of the antibody C region.

  In order to produce the antibody used in the present invention, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter, as described later. Next, host cells can be transformed with this expression vector to express the antibody.

In the present invention, a genetically modified antibody artificially modified for the purpose of reducing the heterologous antigenicity to humans, for example, a chimeric antibody, a humanized antibody, or a human antibody is used. it can. These modified antibodies can be produced using known methods.
A chimeric antibody is obtained by ligating the DNA encoding the antibody V region obtained as described above with DNA encoding the human antibody C region, incorporating it into an expression vector, introducing it into a host, and producing it (Europe). (See Patent Application Publication No. 125023, International Publication No. 92-19759 Pamphlet). Using this known method, a chimeric antibody useful in the present invention can be obtained.

  A humanized antibody is also called a reshaped human antibody or a humanized antibody, and a complementarity determining region (CDR) of a non-human mammal, for example, a mouse antibody, is transplanted to the complementarity determining region of a human antibody. The general genetic recombination technique is also known (see European Patent Application Publication No. 125023, Pamphlet of International Publication No. 92-19759). Specifically, several oligonucleotides were prepared so that a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody had an overlapping portion at the end. It is synthesized from nucleotides by PCR. The obtained DNA is obtained by ligating with the DNA encoding the human antibody C region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication No. 239400, International Publication). No. 92-19759 pamphlet).

  As the FR of a human antibody linked through CDR, a complementarity determining region that forms a favorable antigen binding site is selected. If necessary, amino acid in the framework region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen binding site (Sato, K. et al., Cancer). Res. (1993) 53, 851-856).

The human antibody C region is used for the chimeric antibody and the humanized antibody. Examples of the human antibody C region include Cγ, and for example, Cγ1, Cγ2, Cγ3, or Cγ4 can be used. In addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production.
A chimeric antibody is composed of a variable region of a non-human mammal-derived antibody and a C region derived from a human antibody, and a humanized antibody is a complementarity determining region of a non-human mammal-derived antibody, a framework region and a C region derived from a human antibody Since the antigenicity in the human body is reduced, it is useful as an antibody used in the present invention.
A preferred specific example of the humanized antibody used in the present invention includes a humanized PM-1 antibody (see International Publication No. 92-19759).

  In addition to the above-described methods for obtaining human antibodies, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the variable region of a human antibody can be expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector can be prepared from the sequence to obtain a human antibody. These methods are already known, and are described in WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/11. The 19172 pamphlet, the international publication 95/01438 pamphlet, and the international publication 95/15388 pamphlet can be referred to.

  The antibody gene constructed as described above can be expressed and obtained by a known method. In the case of mammalian cells, it can be expressed by a commonly used useful promoter, an antibody gene to be expressed, a DNA in which a poly A signal is operably linked to the 3 'downstream side thereof, or a vector containing the same. For example, as a promoter / enhancer, human cytomegalovirus early promoter / enhancer (human cytomegalovirus immediate early promoter / enhancer) can be mentioned.

In addition, other promoters / enhancers that can be used for expression of antibodies used in the present invention include viral promoters / enhancers such as retrovirus, polyoma virus, adenovirus, simian virus 40 (SV40), and human elongation factor 1α (HEF1α). Promoters / enhancers derived from mammalian cells such as
For example, when using the SV40 promoter / enhancer, the method of Mulligan et al. (Mulligan, RC et al., Nature (1979) 277, 108-114), and when using the HEF1α promoter / enhancer, Mizushima et al. (Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18, 5322).

In the case of Escherichia coli, it can be expressed by functionally combining a commonly used useful promoter, a signal sequence for antibody secretion, and an antibody gene to be expressed. For example, examples of the promoter include lacZ promoter and araB promoter. When the lacZ promoter is used, the method of Ward et al. (Ward, ES et al., Nature (1989) 341, 544-546; Ward, ES et al. FASEB J. (1992) 6, 2422). -2427), when using the araB promoter, the method of Better et al. (Better, M. et al. Science (1988) 240, 1041-1043) may be followed.
As a signal sequence for antibody secretion, the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379-4383) may be used in the case of production in the periplasm of E. coli. After separating the antibody produced in the periplasm, the structure of the antibody is appropriately refolded and used (see, for example, WO 96/30394).
As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. Furthermore, for amplification of gene copy number in the host cell system, the expression vector is used as a selection marker. An aminoglycoside phosphotransferase (APH) gene, a thymidine kinase (TK) gene, an E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene and the like can be included.

Any production system can be used for the production of the antibodies used in the present invention. Production systems for antibody production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.
When eukaryotic cells are used, there are production systems using animal cells, plant cells, or fungal cells. Examples of animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, etc. (2) amphibian cells such as Xenopus oocytes or (3) insects Cells such as sf9, sf21, Tn5, etc. are known. As plant cells, cells derived from Nicotiana tabacum are known, and these may be cultured in callus. Known fungal cells include yeasts such as Saccharomyces (Saccharomyces) genus, for example, Saccharomyces cerevisiae (Saccharomyces cerevisiae), filamentous fungi such as Aspergillus (Aspergillus) genus, for example, Aspergillus niger (Aspergillus niger) are known.

When prokaryotic cells are used, there are production systems using bacterial cells. Known bacterial cells include Escherichia coli ( E. coli ) and Bacillus subtilis.
An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro. Culture is performed according to a known method. For example, DMEM, MEM, RPMI 1640, and IMDM can be used as a culture solution, and a serum supplement such as fetal calf serum (FCS) can be used in combination. Alternatively, the antibody may be produced in vivo by transferring the cell into which the antibody gene has been introduced to the abdominal cavity of an animal.

On the other hand, examples of in vivo production systems include production systems using animals and production systems using plants. When animals are used, there are production systems using mammals and insects.
As mammals, goats, pigs, sheep, mice, cows and the like can be used (Vicki Glasser, SPECTRUM Biotechnology Applications, 1993). In addition, silkworms can be used as insects. When using a plant, for example, tobacco can be used.
An antibody gene is introduced into these animals or plants, and antibodies are produced in the body of the animals or plants and recovered. For example, an antibody gene is inserted into the middle of a gene encoding a protein inherently produced in milk such as goat β casein to prepare a fusion gene. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its progeny. In order to increase the amount of milk containing the desired antibody produced from the transgenic goat, hormones may be used in the transgenic goat as appropriate. (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).

When silkworms are used, silkworms are infected with baculovirus into which the antibody gene of interest is inserted, and desired antibodies are obtained from the body fluids of these silkworms (Maeda, S. et al., Nature (1985) 315, 592-594). ). Further, when tobacco is used, the target antibody gene is inserted into a plant expression vector such as pMON530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens . This bacterium is infected with tobacco, for example Nicotiana tabacum, and the desired antibody is obtained from the leaves of this tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138). .

  As described above, when an antibody is produced in an in vitro or in vivo production system, DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) are separately incorporated into an expression vector and simultaneously transformed into the host. Alternatively, the host may be transformed by incorporating DNAs encoding H and L chains into a single expression vector (see WO94-11523).

The antibody used in the present invention may be an antibody fragment or a modified product thereof as long as it can be suitably used in the present invention. For example, antibody fragments include Fab, F (ab ′) 2, Fv, or single chain Fv (scFv) in which the H chain and the L chain Fv are linked by an appropriate linker.
Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed and introduced into an expression vector, and then appropriate. Expressed in host cells (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Pureckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousesex. Me thds in Enzymology (1989) 121, 663-66, Bird, RE et al., TIBTECH (1991) 9, 132-137).

scFv is obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. US A. (1988) 85, 5879-5883). The H chain V region and the L chain V region in scFv may be derived from any of those described as the above antibody. As the peptide linker that links the V regions, for example, any single chain peptide consisting of amino acid residues 12 to 19 is used.
The DNA encoding the scFv is a DNA encoding the H chain or H chain V region of the antibody and a DNA encoding the L chain or L chain V region as a template, and a desired amino acid sequence of those sequences. The DNA part encoding the DNA is amplified by PCR using a primer pair defining both ends thereof, and then the DNA encoding the peptide linker part and both ends thereof are connected to the H chain and the L chain, respectively. It is obtained by amplifying by combining a specified primer pair.
Moreover, once a DNA encoding scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method. ScFv can be obtained according to

These antibody fragments can be produced by the host by obtaining and expressing the gene in the same manner as described above. The term “antibody” as used in the present invention encompasses these antibody fragments.
As a modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used. The “antibody” referred to in the present invention includes these modified antibodies. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.

The antibody produced and expressed as described above can be separated from the inside and outside of the cell and from the host and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed, for example, by affinity chromatography. Examples of the column used for affinity chromatography include a protein A column and a protein G column. Examples of the carrier used for the protein A column include HyperD, POROS, Sepharose F., and the like. F. Etc. In addition, it is only necessary to use a separation and purification method used in ordinary proteins, and the method is not limited at all.
For example, the antibody used in the present invention can be separated and purified by appropriately selecting and combining chromatography other than the affinity chromatography, filter, ultrafiltration, salting out, dialysis and the like. Examples of chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, and the like. These chromatographies can be applied to HPLC (High performance liquid chromatography). Moreover, you may use reverse phase HPLC (reverse phase HPLC).

The antibody concentration obtained above can be measured by measuring absorbance, ELISA, or the like. That is, in the case of measuring the absorbance, after appropriately diluting with PBS (−), the absorbance at 280 nm is measured, and 1 mg / ml is calculated as 1.35 OD. In the case of ELISA, the measurement can be performed as follows. That is, 100 μl of goat anti-human IgG (manufactured by TAG) diluted to 1 μg / ml with 0.1 M bicarbonate buffer (pH 9.6) was added to a 96-well plate (manufactured by Nunc) and incubated at 4 ° C. overnight. Is immobilized. After blocking, 100 μl of appropriately diluted antibody used in the present invention or a sample containing the antibody, or human IgG (manufactured by CAPPEL) as a standard is added, and incubated at room temperature for 1 hour.
After washing, 100 μl of 5,000-fold diluted alkaline phosphatase-labeled anti-human IgG (BIO SOURCE) is added and incubated at room temperature for 1 hour. After washing, a substrate solution is added, and after incubation, the absorbance at 405 nm is measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad), and the concentration of the target antibody is calculated.

-IL-6 variant, soluble IL-6 receptor variant-
The IL-6 variant used in the present invention is a substance that has a binding activity to the IL-6 receptor and does not transmit the biological activity of IL-6. That is, the IL-6 variant binds to IL-6 competitively with IL-6, but does not transmit the biological activity of IL-6 and thus blocks signal transduction by IL-6.
IL-6 variants are produced by introducing mutations by substituting amino acid residues in the amino acid sequence of IL-6. The origin of IL-6, which is a variant of IL-6, is not limited, but human IL-6 is preferable in consideration of antigenicity and the like.
Specifically, the secondary structure of the amino acid sequence of IL-6 is predicted using a known molecular modeling program such as WHATIF (Vriend et al., J. Mol. Graphics (1990) 8, 52-56). Further, it is carried out by evaluating the influence on the entire amino acid residue to be substituted. After determining an appropriate substituted amino acid residue, using a vector containing a nucleotide sequence encoding the human IL-6 gene as a template, a mutation is introduced so that the amino acid is substituted by a commonly performed PCR method. A gene encoding 6 variants is obtained. This can be incorporated into an appropriate expression vector as necessary, and an IL-6 variant can be obtained according to the expression, production and purification methods of the recombinant antibody.
Specific examples of IL-6 variants include Brakenoff et al. , J .; Biol. Chem. (1994) 269, 86-93, and Savino et al. , EMBO J. et al. (1994) 13, 1357-1367, WO 96-18648 pamphlet, and WO 96-17869 pamphlet.
In addition, the soluble IL-6 receptor variant used in the present invention is a substance that has a binding activity to IL-6 and does not transmit the biological activity of IL-6. That is, soluble IL-6 receptor variants bind to IL-6 competitively with IL-6 receptor, but do not transmit the biological activity of IL-6, thus blocking IL-6 signaling. To do.
The soluble IL-6 receptor variant can be appropriately produced according to, for example, the method for producing an IL-6 variant described above.

-IL-6 partial peptide, IL-6 receptor partial peptide-
The IL-6 partial peptide or IL-6 receptor partial peptide used in the present invention has a binding activity to IL-6 receptor or IL-6, respectively, and transmits the biological activity of IL-6. It is a substance that does not. That is, IL-6 partial peptide or IL-6 receptor partial peptide binds to IL-6 receptor or IL-6, and captures them to specifically bind IL-6 to IL-6 receptor. Inhibit it. As a result, it does not transmit the biological activity of IL-6, thus blocking signal transmission by IL-6.
IL-6 partial peptide or IL-6 receptor partial peptide is a part or all of amino acids in the region related to the binding of IL-6 to IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor A peptide consisting of a sequence. Such a peptide consists of 10-80 amino acid residues normally, Preferably it is 20-50, More preferably, it consists of 20-40 amino acid residues.

The IL-6 partial peptide or IL-6 receptor partial peptide specifies a region related to the binding between IL-6 and IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor. Part or all of the amino acid sequences can be prepared by a generally known method such as a genetic engineering method or a peptide synthesis method.
In order to prepare an IL-6 partial peptide or an IL-6 receptor partial peptide by a genetic engineering technique, a DNA sequence encoding a desired peptide is incorporated into an expression vector, and the recombinant antibody is expressed, produced and purified. It can obtain according to.
In order to prepare an IL-6 partial peptide or IL-6 receptor partial peptide by a peptide synthesis method, a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method can be used.
Specifically, it may be carried out according to the method described in the follow-up drug development, Volume 14, Peptide Synthesis Supervision Yajima Haruaki Yodogawa Shoten 1991. As the solid-phase synthesis method, for example, an amino acid corresponding to the C-terminus of the peptide to be synthesized is bound to a support that is insoluble in an organic solvent, and the α-amino group and the side chain functional group are protected with an appropriate protecting group. By alternately repeating the reaction of condensing the amino acids one amino acid at a time in the order from the C-terminal to the N-terminal and the reaction of removing the protecting group of the α-amino group of the amino acid or peptide bound on the resin, the peptide A method of extending the chain is used. Solid phase peptide synthesis methods are roughly classified into Boc method and Fmoc method depending on the type of protecting group used.

After synthesizing the target peptide in this way, a deprotection reaction and a cleavage reaction from the support of the peptide chain are performed. For the cleavage reaction with the peptide chain, hydrogen fluoride or trifluoromethanesulfonic acid can be usually used in the Boc method, and TFA can be usually used in the Fmoc method. In the Boc method, for example, the protected peptide resin is treated in the presence of anisole in hydrogen fluoride. Next, the protecting group is removed and cleaved from the support to recover the peptide. This is freeze-dried to obtain a crude peptide. On the other hand, in the Fmoc method, for example, a deprotection reaction and a cleavage reaction from a support of a peptide chain can be performed in the same manner as described above in TFA.
The obtained crude peptide can be separated and purified by application to HPLC. For the elution, a water-acetonitrile solvent usually used for protein purification may be used under optimum conditions. The fraction corresponding to the peak of the obtained chromatographic profile is collected and lyophilized. The peptide fraction thus purified is identified by molecular weight analysis by mass spectrum analysis, amino acid composition analysis, amino acid sequence analysis or the like.

  Specific examples of the IL-6 partial peptide and IL-6 receptor partial peptide are described in JP-A-2-188600, JP-A-7-324097, JP-A-8-311098 and US Pat. No. 5210075. It is disclosed.

  The antibody used in the present invention may be a conjugated antibody bound to various molecules such as polyethylene glycol (PEG), radioactive substances, and toxins. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field. The “antibody” in the present invention includes these conjugated antibodies.

  The IL-6 signaling inhibitory activity of the IL-6 antagonist used in the present invention can be evaluated by a commonly used method. Specifically, an IL-6-dependent human myeloma line (S6B45, KPMM2), a human Rennelt T lymphoma cell line KT3, or an IL-6-dependent cell MH60. What is necessary is just to measure 3H-thymidine uptake of IL-6-dependent cells by culturing BSF2, adding IL-6 thereto, and simultaneously coexisting an IL-6 antagonist. Furthermore, 125I-labeled IL-6 bound to IL-6 receptor-expressing cells was obtained by culturing U266, which is an IL-6 receptor-expressing cell, and adding 125I-labeled IL-6 and simultaneously adding an IL-6 antagonist. Measure. In the above assay system, in addition to the group in which the IL-6 antagonist is present, a negative control group not containing the IL-6 antagonist is placed, and the IL-6 inhibitory activity of the IL-6 antagonist is evaluated by comparing the results obtained with both. can do.

<Therapeutic agent>
The therapeutic agent for inflammatory muscle disease of the present invention has an action of suppressing abnormal inflammatory cell infiltration in muscle fibers in a subject suffering from inflammatory muscle disease by administration. The inflammatory myopathy is preferably polymyositis or dermatomyositis. The subject to be administered is a mammal, and the mammal is preferably a human.
The therapeutic agent for inflammatory muscle disease of the present invention can be administered in the form of a pharmaceutical, and can be administered systemically or locally orally or parenterally. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent, etc. can be selected, and the administration method should be selected appropriately depending on the age and symptoms of the patient Can do. The effective dose is selected in the range of 0.01 mg to 100 mg per kg body weight at a time. Alternatively, a dose of 1 to 1,000 mg, preferably 5 to 50 mg per patient can be selected. For example, in the case of an anti-IL-6 receptor antibody, a preferable dose and administration method are effective doses in such an amount that free antibodies are present in the blood. Specific examples are as follows: 0.5 mg to 40 mg, preferably 1 mg to 20 mg per month (4 weeks), divided into 1 to several times, for example, 2 times / week, 1 time / week, 1 time / 2 weeks, 1 time / For example, it may be administered by a method such as intravenous injection such as infusion or subcutaneous injection in an administration schedule such as 4 weeks. The dosing schedule is 2 times / week, or once / week to once / 2 weeks, once / 3 weeks, once / 4 weeks, etc. while observing the post-dose status and observing the trend of blood test values It is also possible to adjust such as extending the administration interval.
In addition, the therapeutic agent for inflammatory muscle disease of the present invention may be administered therapeutically or may be administered prophylactically.

In the present invention, a therapeutically acceptable carrier such as a preservative or a stabilizer may be added to the therapeutic agent for inflammatory muscle disease. “Pharmaceutically acceptable” may be a material that itself has the above-mentioned myositis-inhibiting effect or may be a material that does not have the above-mentioned inhibitory effect. Means acceptable material. Moreover, even if it is a material which does not have a myositis inhibitory effect, the material which has a synergistic or additive stabilization effect by using together with an IL-6 antagonist may be sufficient.
Examples of materials that are acceptable for formulation include sterilized water and physiological saline, stabilizers, excipients, buffering agents, preservatives, surfactants, chelating agents (such as EDTA), and binders. .

  In the present invention, examples of the surfactant include nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate; glycerin monocaprylate, glycerin monomylate. Glycerin fatty acid esters such as glyceryl monostearate; polyglycerin fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate; polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate , Polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene Polyoxyethylene sorbitan fatty acid ester such as bitane tristearate; Polyoxyethylene sorbite fatty acid ester such as polyoxyethylene sorbite tetrastearate and polyoxyethylene sorbite tetraoleate; Polyoxyethylene glycerin such as polyoxyethylene glyceryl monostearate Fatty acid ester; polyethylene glycol fatty acid ester such as polyethylene glycol distearate; polyoxyethylene alkyl ether such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxy Polyoxyethylene polyoxypropylene alkyl ester such as propylene cetyl ether Poly; polyoxyethylene alkyl phenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hydrogenated castor oil such as polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogen castor oil); polyoxyethylene Typical examples include polyoxyethylene beeswax derivatives such as sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides such as polyoxyethylene stearamide having HLB 6-18, etc. Can be mentioned.

  Examples of the surfactant include an anionic surfactant, for example, an alkyl sulfate having an alkyl group having 10 to 18 carbon atoms such as sodium cetyl sulfate, sodium lauryl sulfate, and sodium oleyl sulfate; polyoxyethylene Polyoxyethylene alkyl ether sulfates having an average added mole number of ethylene oxide of 2 to 4 and an alkyl group of 10 to 18 carbon atoms such as sodium lauryl sulfate; carbon atoms of the alkyl group such as sodium lauryl sulfosuccinate Typical examples include alkylsulfosuccinic acid ester salts having 8 to 18 numbers; natural surfactants such as lecithin, glycerophospholipid; fingophospholipids such as sphingomyelin; and sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms. Can be mentioned.

One or a combination of two or more of these surfactants can be added to the inflammatory muscle disease therapeutic agent of the present invention. Preferred surfactants for use in the formulations of the present invention are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60 or 80, with polysorbates 20 and 80 being particularly preferred. Further, polyoxyethylene polyoxypropylene glycol represented by poloxamer (such as Pluronic F-68 (registered trademark)) is also preferable.
The amount of surfactant to be added varies depending on the type of surfactant to be used, but in the case of polysorbate 20 or polysorbate 80, it is generally 0.001 to 100 mg / mL, preferably 0.003 to 50 mg / mL. More preferably, it is 0.005 to 2 mg / mL.

In the present invention, as the buffering agent, phosphoric acid, citric acid buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, caprylic acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid Other organic acids, etc., or carbonate buffer, Tris buffer, histidine buffer, imidazole buffer and the like can be mentioned.
Alternatively, a solution formulation may be prepared by dissolving in an aqueous buffer known in the field of solution formulation. The concentration of the buffer is generally 1 to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 20 mM.

  The therapeutic agent for inflammatory muscle disease of the present invention contains other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, saccharides such as amino acids, polysaccharides and monosaccharides, carbohydrates, and sugar alcohols. Also good.

  Examples of amino acids in the present invention include basic amino acids such as arginine, lysine, histidine, ornithine and the like, or inorganic salts of these amino acids (preferably in the form of hydrochloride or phosphate, that is, phosphate amino acids). it can. When free amino acids are used, the preferred pH value is adjusted by the addition of suitable physiologically acceptable buffer substances, such as inorganic acids, in particular hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, formic acid or salts thereof. In this case, the use of phosphate is particularly advantageous in that a particularly stable lyophilizate is obtained. If the preparation is substantially free of organic acids such as malic acid, tartaric acid, citric acid, succinic acid, fumaric acid, etc., or the corresponding anion (malate ion, tartaric acid ion, citrate ion, succinic acid ion, This is particularly advantageous when no fumaric acid ion or the like is present. Preferred amino acids are arginine, lysine, histidine, or ornithine. Furthermore, acidic amino acids such as glutamic acid and aspartic acid, and their salt forms (preferably sodium salts) or neutral amino acids such as isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine or alanine, or aromatic Amino acids such as phenylalanine, tyrosine, tryptophan, or the derivative N-acetyltryptophan can also be used.

In the present invention, examples of sugars and carbohydrates such as polysaccharides and monosaccharides include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.
In the present invention, examples of the sugar alcohol include mannitol, sorbitol, inositol, and the like.

When the therapeutic agent for inflammatory muscle disease of the present invention is used as an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol. Sodium chloride, and may be used in combination with a suitable solubilizer such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc. Good.
The therapeutic agent for inflammatory muscle disease of the present invention may further contain a diluent, a solubilizing agent, a pH adjuster, a soothing agent, a sulfur-containing reducing agent, an antioxidant and the like, if desired.

  In the present invention, examples of the sulfur-containing reducing agent include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, and thiosulfuric acid. Examples include sodium, glutathione, and those having a sulfhydryl group such as thioalkanoic acid having 1 to 7 carbon atoms.

  In the present invention, examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L-ascorbic acid steer. Examples thereof include chelating agents such as rate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

  In addition, the therapeutic agent for inflammatory muscle disease of the present invention may contain an active ingredient in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or a colloid drug delivery system (liposomes) as necessary. , Albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) (see “Remington's Pharmaceutical Science 16th edition”, Oslo Ed., 1980, etc.). Furthermore, a method of making a drug a sustained-release drug is also known and can be applied to the present invention (Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech). 1982, 12: 98-105; U.S. Pat. No. 3,773,919; European Patent Application Publication No. 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; 133,988). The pharmaceutically acceptable carrier to be used is appropriately or in combination selected from the above depending on the dosage form, but is not limited thereto.

<Treatment method>
The invention also relates to a method of treating inflammatory muscle disease in a subject comprising administering an IL-6 antagonist to the subject who has developed inflammatory muscle disease. In the present invention, the “subject” refers to an organism to which the therapeutic agent for inflammatory muscle disease of the present invention is administered, and a part of the organism. Organisms include, but are not limited to, animals (eg, humans, domestic animal species, wild animals). In the present invention, “administering” includes administering orally or parenterally. Oral administration can include administration in the form of oral preparations, and as oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions or suspensions are selected. be able to. Parenteral administration can include administration in the form of injection, and injection includes intravenous injection such as infusion, subcutaneous injection, intramuscular injection, or intraperitoneal injection. it can. Moreover, the effect of the method of the present invention can be achieved by introducing a gene containing an oligonucleotide to be administered into a living body using a gene therapy technique. In addition, the agent of the present invention is administered at the same time or at intervals of known treatments for inflammatory myopathy, such as rehabilitation, physical therapy, administration of corticosteroid hormones, administration of azathioprine, methoxerate, etc. Also good.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

[Example 1: Induction and clinical evaluation of protein C-induced myositis (CIM)]
C57BL / 6J mice (8 weeks old, female) used in this example were purchased from Charles River Japan. Prior to treatment, the mice were bred in an SPF (specific pathogen-free) experimental animal facility of the Research Center for Allergy and Immunology (RIKEN).
Protein C used in this example was prepared as follows. A complementary DNA (cDNA) of protein C derived from fast mouse muscle fibers was amplified by PCR from a cDNA library derived from mouse skeletal muscle. The sequences of the primers used were 5′-GGGA GGATCC GACCTCTCCTCTCAAGTGG-3 ′ (SEQ ID NO: 1), 5′-ATTA AAGCTT AGCCAGGTAGCATGGG-3 ′ (SEQ ID NO: 2) (underlined part is pQE30 vector (Qiagen)) (Restriction enzyme recognition site used for subcloning). The protein C expression vector was introduced into TOP10F ′ E. coli strain (Invitrogen), and the recombinant protein C protein fragment was purified according to the instruction manual. The soluble fraction of the recombinant protein C protein fragment was dialyzed using pH 7.4, PBS buffer (containing 0.5 M arginine, 2 mM reduced glutathione, 0.2 mM oxidized glutathione).
Using the mouse and protein C, CIM was induced in the mouse as follows. The mice were subcutaneously administered with 200 μg protein C protein fragment-containing complete Freund's adjuvant (CFA) and immunized (CFA contains 100 μg of heat-treated Mycobacterium butyricum (Defco)). The antigen was introduced into a plurality of locations on the back and footpads, and at the same time, 2 μg pertussis toxin (Seikagaku Corporation) dissolved in PBS was injected intraperitoneally. 21 days after immunization, proximal muscles (knee flexor and quadriceps) collected from mice were paraffin sections (10 μm) and stained with hematoxylin & eosin (H & E). Mononuclear cells in muscle fibers The presence or absence of infiltration and necrosis was observed histologically. The histological severity (histological score) of inflammation in each muscle block was evaluated by dividing it into the following four categories.
<Histological score>
Inflammatory cell infiltration is observed in muscle fibers of grade 1: 1 to less than 5.
Grade 2: Inflammatory cell infiltration is observed in 5 to 30 muscle fibers.
Grade 3: Inflammatory cell infiltration is observed in the muscle bundle.
Grade 4: Diffuse extensive inflammatory cell infiltration is observed.
(If multiple inflammatory cell infiltrations are observed in the muscle block, add 0.5 to the grade.)

[Example 2: Effect of anti-mouse IL-6 receptor monoclonal antibody on serum amyloid A (SAA) concentration in myositis model mice]
The anti-mouse IL-6 receptor monoclonal antibody (rat IgG1) used in this example was purified from the MR16-1 hybridoma cell line. Anti-dinitrophenol monoclonal antibody (rat IgG1) was purified from the KH-5 hybridoma cell line. Both hybridoma cell lines were provided by Chugai Pharmaceutical Co., Ltd.
Protein C immunization was performed as in Example 1, and on the same day, 4 mg or 2 mg of each monoclonal antibody (anti-mouse IL-6 receptor monoclonal antibody (IL-6R mAb), or anti-dinitrophenol monoclonal antibody (control mAb) as a control thereof. ) Was intraperitoneally administered to the mice, and blood was collected from the facial vein 5 days later, and the SAA concentration was measured. The concentration of SAA was measured using a mouse SAA ELISA kit (BioSource) according to the attached instruction manual.
As a result, the SAA concentration in the group administered with 4 mg of the anti-mouse IL-6 receptor monoclonal antibody simultaneously with the immunization treatment was found to be statistically significantly reduced compared to the group administered with the control antibody 4 mg. The SAA concentration in the group administered with 2 mg of anti-mouse IL-6 receptor monoclonal antibody was not significantly different from the group administered with 2 mg of control antibody, but a decreasing tendency was observed (FIG. 1).

[Example 3: Therapeutic effect of anti-mouse IL-6 receptor monoclonal antibody in CIM (1)]
Mice were immunized with protein C as in Example 1, and 4 mg or 2 mg of each monoclonal antibody (anti-mouse IL-6 receptor monoclonal antibody (IL-6R mAb) on the same day, or anti-dinitrophenol monoclonal antibody as a control thereof (Control mAb)) was administered intraperitoneally, and then 0.1 mg of each monoclonal antibody was administered twice a week for 3 weeks after immunization.
As a result, histological myositis was confirmed on the 21st day after immunization in all mice in the untreated group, the control antibody administration group, and the anti-mouse IL-6 receptor monoclonal antibody administration group. The average histological score is 1.92 ± 0.38 (± SD) in the untreated group, 2.1 ± 0.22 in the control antibody 4 mg group, and 1.93 ± 0.24 in the 2 mg group. there were. On the other hand, the anti-mouse IL-6 receptor monoclonal antibody 4 mg administration group had 1.13 ± 0.32, and the 2 mg administration group had 1.55 ± 0.54. In the 4 mg group administered with the anti-mouse IL-6 receptor monoclonal antibody, the histological score was significantly reduced as compared to the 4 mg group administered with the control antibody (Table 1, FIG. 2).

[Example 4: Therapeutic effect of anti-mouse IL-6 receptor monoclonal antibody in CIM (2)]
The present inventors have confirmed that CIM has already developed on the 7th day after the immunization by observing CIM repeatedly, and therefore, each monoclonal antibody was administered on the 7th day after the immunization as follows. The effectiveness of the treatment was verified.
Protein C immunization was performed as in Example 1, and on the 7th day after immunization, 4 mg or 2 mg of each monoclonal antibody (anti-mouse IL-6 receptor monoclonal antibody (IL-6R mAb), or anti-dinitrophenol monoclonal as a control thereof) Antibody (control mAb)) was intraperitoneally administered, and then 0.1 mg of each monoclonal antibody was administered twice a week until the third week after immunization.
As a result, histological myositis was confirmed on the 21st day after immunization in all mice in the untreated group, the control antibody administration group, and the anti-mouse IL-6 receptor monoclonal antibody administration group. In the group administered with 4 mg of anti-mouse IL-6 receptor monoclonal antibody, CIM expression was 40%. The average histological score was 1.25 ± 0.71 in the untreated group, 1.43 ± 0.38 in the control antibody 4 mg group, and 1.65 ± 0.29 in the 2 mg group. On the other hand, the anti-mouse IL-6 receptor monoclonal antibody 4 mg group was 0.2 ± 0.27, and the 2 mg group was 0.75 ± 0.43. In the group administered with 2 mg or 4 mg of anti-mouse IL-6 receptor monoclonal antibody, the histological score was significantly decreased as compared with the group administered with the control antibody (Table 2, FIG. 3).

  From the results of the above Examples, it was possible to confirm the therapeutic effect of the anti-mouse IL-6 receptor monoclonal antibody in CIM.

  The present invention is a drug for treating inflammatory myopathy targeting IL-6, and is useful as a drug capable of effectively suppressing abnormal inflammatory cell infiltration in muscle fibers.

FIG. 1 is a diagram showing the results of ELISA. In mice 5 days after protein C immunization, a decrease in SAA concentration was observed with administration of 4 mg or 2 mg of anti-mouse IL-6 receptor monoclonal antibody. All groups n = 3, * p <0.05. FIG. 2 is a diagram showing the results of histologically confirming the onset of CIM 21 days after immunization by administering anti-mouse IL-6 receptor monoclonal antibody simultaneously with immunization. Table 1 is rewritten into a graph, and * p <0.05. FIG. 3 is a diagram showing the results of histological confirmation of the onset of CIM on day 21 after immunization by administering anti-mouse IL-6 receptor monoclonal antibody on day 7 after immunization. Table 2 is rewritten into a graph, and * p <0.05.

Claims (7)

  A therapeutic agent for inflammatory myopathy characterized by comprising an IL-6 antagonist as an active ingredient.   The therapeutic agent for inflammatory muscle disease according to claim 1, wherein the IL-6 antagonist is an antibody against IL-6 receptor or an antibody against IL-6.   The therapeutic agent for inflammatory muscle disease according to claim 2, wherein the antibody is a monoclonal antibody.   The therapeutic agent for inflammatory muscle disease according to claim 2, wherein the antibody is a recombinant antibody.   The therapeutic agent for inflammatory muscle disease according to claim 2, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.   The therapeutic agent for inflammatory myopathy according to any one of claims 1 to 5, wherein the inflammatory myopathy is polymyositis or dermatomyositis.   The therapeutic agent for inflammatory myopathy according to any one of claims 1 to 6, which has an action of suppressing abnormal inflammatory cell infiltration in muscle fibers.