JP4698652B2 - A prophylactic or therapeutic agent for inflammatory bowel disease comprising an IL-6 antagonist as an active ingredient - Google Patents

Description

  The present invention relates to a preventive or therapeutic agent for inflammatory bowel disease comprising an interleukin-6 (IL-6) antagonist as an active ingredient. The present invention also relates to a preventive or therapeutic agent for Crohn's disease or ulcerative colitis 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 lymphoid cells (Hirano, T. et al., Nature (1986) 324, 73-76) and subsequently affected the function of various cells. (Akira, S. et al., Adv. In Immunology (1993) 54, 1-78). IL-6 has been reported to induce maturation of T lymphoid cells (Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1258).

  IL-6 transmits its biological activity through two proteins on the cell. One is the IL-6 receptor, a ligand-binding protein with a molecular weight of about 80 kD to which IL-6 binds (Taga, T. et al., J. Exp. Med. (1987) 166, 967-981, Yamasaki, K. et al., Science (1987) 241, 825-828). 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 with 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 (Taga , T. et al., Cell (1989) 58, 573-581).
An IL-6 antagonist is a substance that inhibits the transmission of the 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 peptide and the like are known.

  There are several reports on anti-IL-6 receptor antibodies (Novick, D. et al., Hybridoma (1991) 10, 137-146, Huang, YW et al., Hybridoma (1993) 12, 621- 630, international patent application publication number WO 95-09873, French patent application publication number FR 2694767, US patent number US 521628). One of them, mouse antibody PM-1 (Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906) transplantation of complementarity determining region (CDR) to human antibody A humanized PM-1 antibody obtained by doing so is known (International Patent Application Publication No. WO 92-19759).

  Inflammatory bowel disease (IBD) is a nonspecific inflammation typified by ulcerative colitis and Crohn's disease. Regarding the establishment of the disease, the involvement of immunological abnormalities has been deeply suggested, but the etiology has not yet been elucidated. However, it is thought that monocytes and lymphocytes gathered at the lesion site are related to mucosal damage, and inflammatory mediators produced from these cells, especially cytokines (IL1β, TNFα, IL-6, etc.) Attention has been paid.

  Of these inflammatory mediators, IL-6 has been focused on the relationship with disease activity, that is, whether it is an IBD-specific indicator. First, serum IL-6 levels increase in both Crohn's disease and ulcerative colitis, which correlates with disease activity (Holtkamp, W. et al., J. Clin. Gastroenterology (1995) 20 , 123-126, Niederau, C. et al., Hepato-Gastroenterology (1997) 44, 90-107). In addition, when the amount of IL-6 mRNA in the tissue was detected as a PCR (polymerase chain reaction) product, it was shown that both ulcerative colitis and Crohn's disease increased in good correlation with activity (Stevens, C et al., Dig. Dis. Sci. (1992) 37, 818-826). The mechanism of IL-6 production in active IBD has been analyzed, and it is clear that the production of mononuclear cells in the lamina propria stimulated with Pokeweed mitogen correlates with disease activity. (Reinecker, H.-C. et al., Clin. Exp. Immunol. (1993) 94, 174-181).

  Subsequently, there was a correlation between IL-6 production and disease activity in both unstimulated monolayers derived from the lamina propria and tissue cultures of patient mucosa. In the former, the number of IL-6 producing cells in the mucosal tissue Was also shown to increase. Among these mononuclear cells, the most important IL-6 producing cells are macrophages, and there are many CD68 positive macrophages producing IL-6 actively in the lamina propria of active IBD patients. It has been confirmed (Kusugami, K et al., Dig. Dis. Sci. (1995), 40, 949-959).

  IL-6 production has been shown to correlate with endoscopic observations in patients with Crohn's disease. (Reimund, J.-M. et al., Gut (1996) 39, 684-689). There is also a report that serum soluble IL-6 receptor concentration and disease activity correlate with not only IL-6 (Mitsuyama. K. et al., Gut (1995) 36, 45-49).

In inflammatory mediators other than IL-6, it has been observed that IL-1β production correlates with disease activity in the same manner as IL-6. On the other hand, the production amount of TNFα does not necessarily correlate, and the production amount tends to be high even in a low activity state (Reinecker, H.-C. et al., Clin. Exp. Immunol. (1993 94, 174-181, Reimund, J.-M. et al., Gut (1996) 39, 684-689).
Current IBD treatment consists of a combination of diet and drug therapy, and salazosulfapyridine, glucocorticoids, etc. are prescribed. However, these drugs have intolerance patients due to side effects, and there is a problem with long-term continuous casting.

  On the other hand, attempts are being made to improve the disease state by inhibiting cytokine activity as a new treatment for IBD. Its main targets are IL-1 and TNFα (Van Deventer, SJH Gut (1997) 40, 443-448), and IL-1 receptor antagonists (Cominelli F etal., Gastroenterology (1992) 103, 65-71) and CGP47969A (Casini-Raggi et al., Gastroenterology (1995) 109, 812-818), which is an IL-1 inhibitor, has been studied at the clinical or experimental animal level. In addition, a specific monoclonal antibody was administered to patients with Crohn's disease for TNFα, and 8 out of 10 patients with Crohn's disease who were resistant to other treatments showed reduced activity and healing of ulcers. (Van Dullemen, HM et al., Gastroenterology (1995) 109, 129-135). However, it has not been known to show therapeutic effects on IBD by specifically suppressing IL-6 biological activity using IL-6 antagonists.

The present invention is intended to provide a preventive or therapeutic agent for inflammatory bowel disease that does not have the above-mentioned drawbacks.
That is, the present invention provides (1) a preventive or therapeutic agent for inflammatory bowel disease containing an IL-6 antagonist as an active ingredient.
The present invention also provides (2) a prophylactic or therapeutic agent for inflammatory bowel disease comprising an antibody against IL-6 receptor as an active ingredient.
The present invention also provides (3) a preventive or therapeutic agent for inflammatory bowel disease comprising a monoclonal antibody against IL-6 receptor as an active ingredient.

The present invention also provides (4) a preventive or therapeutic agent for inflammatory bowel disease comprising a monoclonal antibody against human IL-6 receptor as an active ingredient. The monoclonal antibody against human IL-6 receptor is preferably PM-1 antibody.
The present invention also provides (5) a preventive or therapeutic agent for inflammatory bowel disease comprising a monoclonal antibody against mouse IL-6 receptor as an active ingredient. The monoclonal antibody against mouse IL-6 receptor is preferably MR16-1 antibody.
The present invention also provides (6) a preventive or therapeutic agent for inflammatory bowel disease comprising a recombinant antibody against IL-6 receptor as an active ingredient. It has a recombinant antibody against IL-6 receptor, preferably a human antibody constant region (C region).

The present invention also provides (7) a prophylactic or therapeutic agent for inflammatory bowel disease comprising a chimeric antibody or humanized antibody against IL-6 receptor as an active ingredient.
The present invention also provides (8) a prophylactic or therapeutic agent for inflammatory bowel disease comprising humanized PM-1 antibody as an active ingredient.
The present invention also provides (9) a preventive or therapeutic agent for Crohn's disease or ulcerative colitis comprising the IL-6 antagonist described in (1) to (8) as an active ingredient.
The present invention also provides (10) a weight loss inhibitor in inflammatory bowel disease comprising the IL-6 antagonist described in (1) to (8) as an active ingredient.
The present invention further provides (11) an agent for inhibiting weight loss in inflammatory bowel disease, comprising as an active ingredient the antibody to the IL-6 receptor described in (3) to (8) above.

The IL-6 antagonist used in the present invention is not limited in its origin, type and shape as long as it exhibits an effect of preventing or treating inflammatory bowel disease or an effect of suppressing weight loss in inflammatory bowel disease.
An IL-6 antagonist is a substance that blocks IL-6 signaling and inhibits IL-6 biological activity. The IL-6 antagonist is preferably a substance having an inhibitory action on the binding of any of IL-6, IL-6 receptor and gp130. Examples of IL-6 antagonists include anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-gp130 antibody, IL-6 variant, soluble IL-6 receptor variant or IL-6 or IL-6 receptor And low molecular weight substances exhibiting the same activity as these partial peptides.

  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 transmission of IL-6 biological activity into the cell.

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 Japanese Society for Immunology). General meeting, academic records (1991) 21, 166).
An anti-IL-6 antibody-producing hybridoma can be basically produced using a known technique as follows. That is, using IL-6 as a sensitizing antigen, immunizing it according to a normal immunization method, fusing the resulting immune cells with a known parent cell by a normal cell fusion method, 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 Eur. J. Biochem (1987) 168, 543-550, J. Immunol. (1988) 140, 1534-1541, or Agr. Biol. It is obtained by using the IL-6 gene / amino acid sequence disclosed in Chem. (1990) 54, 2685-2688.
After inserting the gene sequence of IL-6 into a known expression vector system and transforming an appropriate host cell, the target IL-6 protein is purified from the host cell or culture supernatant by a known method. The purified IL-6 protein may be used as a sensitizing antigen. Further, a fusion protein of IL-6 protein and another protein may be used as a sensitizing antigen.

  The anti-IL-6 receptor antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using a 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 blocking the binding of IL-6 to the IL-6 receptor and blocking the transmission of IL-6 biological activity into the cell.

  Examples of such antibodies include MR16-1 antibody (Tamura, T. et al. Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-1 antibody (Hirata, Y. et al. , J. Immunol. (1989) 143, 2900-2906), AUK12-20 antibody, AUK64-7 antibody or AUK146-15 antibody (International Patent Application Publication No. WO 92-19759). Among these, PM-1 antibody is mentioned as a particularly preferable antibody.

  The PM-1 antibody-producing hybridoma cell line is designated as PM-1 in the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki, Japan 305-5466, Japan). On July 12, 1989, FERM BP-2998 was deposited internationally under the Budapest Treaty. In addition, the MR16-1 antibody-producing hybridoma cell line is Rat-mouse hybridoma MR16-1, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-5 Higashi 1-chome, Tsukuba, Ibaraki, Japan 305-5466, Japan) 6), on March 13, 1997, FERM BP-5875 was deposited internationally under the Budapest Treaty.

  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, immunized according to a normal immunization method, and the obtained immune cells are fused with a known parent cell by a normal cell fusion method. Can be prepared by screening monoclonal antibody-producing cells.

  Specifically, an anti-IL-6 receptor antibody can be produced as follows. For example, a human IL-6 receptor used as a sensitizing antigen for obtaining an antibody was disclosed in European Patent Application Publication No. EP 325474, and a mouse IL-6 receptor was disclosed in Japanese Patent Application Publication No. JP-A-3-155795. It is obtained by using IL-6 receptor gene / amino acid sequence.

  IL-6 receptor protein is expressed on the cell membrane and detached from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676). Soluble IL-6 receptor antibody is composed essentially of the extracellular region of IL-6 receptor bound to the cell membrane, and lacks the transmembrane region or the transmembrane region and the intracellular region. 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 known from the host cell or culture supernatant. The purified IL-6 receptor protein may be used as a sensitizing antigen. Further, cells expressing IL-6 receptor or fusion proteins of IL-6 receptor protein and other proteins may be used as the sensitizing antigen.

  E. coli containing the plasmid pIBIBSF2R containing the cDNA encoding the human IL-6 receptor was inaugurated on January 9, 1989, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center ( HB101-pIBIBSF2R has been deposited internationally under the Budapest Treaty as HB101-pIBIBSF2R in Tsukuba City, Ibaraki, Japan, 305-5466, Japan.

  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 blocking 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 (JP-A-3-219894), 4B11 antibody and 2H4 antibody (US 5571513) B-S12 antibody and B-P8 antibody (JP-A-8-291199).
An anti-gp130 monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, gp130 is used as a sensitizing antigen and 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 antibodies can be obtained by using the gp130 gene / amino acid sequence disclosed in European Patent Application Publication No. EP 411946.
After inserting the gene sequence of gp130 into a known expression vector system and transforming an appropriate host cell, the target gp130 protein is purified from the host cell or the culture supernatant by a known method. A gp130 receptor 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.

The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. 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 into a mammal intraperitoneally or subcutaneously. Specifically, the sensitized antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, etc. and mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. Preferably, it is administered to mammals several times every 4-21 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, immune cells are removed from the mammal 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 are already known in various cell lines such as P3X63Ag8.653 (Kearney, JF et al. J. Immnol. (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 etal., 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 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) are used as appropriate.

The cell fusion between the immunocytes 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. For example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used as a fusion promoter, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.

  The usage ratio of immune cells and myeloma cells is preferably, for example, 1-10 times that of immune cells relative to myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture can be used. Serum supplements such as fetal calf serum (FCS) can be used in combination.

  For cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution pre-warmed to about 37 ° C., for example, a PEG solution having an average molecular weight of about 1000-6000 is usually used. The target fused cell (hybridoma) is formed by adding and mixing at a concentration of 30-60% (w / v). Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma can be removed by adding an appropriate culture solution successively 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 a non-human animal with an antigen to obtain the above hybridoma, human lymphocytes are sensitized with a desired antigen protein or antigen-expressing cells in vitro, and sensitized B lymphocytes are human myeloma cells such as U266. And a desired human antibody having binding activity to a desired antigen or antigen-expressing cell can be obtained (see Japanese Patent Publication No. 1-59878). Further, antigens or antigen-expressing cells may be administered to a transgenic animal having a repertoire of human antibody genes, and a desired human antibody may be obtained according to the aforementioned method (International Patent Application Publication Nos. WO 93/12227, WO 92 / 03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 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. Incorporated by National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st, 1st, 1st, Tsukuba, Ibaraki, Japan 305-5466, Japan) on July 12, 1989 as FERM BP-2998 The PM-1 antibody-producing hybridoma deposited internationally based on this was injected into the peritoneal cavity of BALB / c mice to obtain ascites, and the PM-1 antibody was purified from this ascites, or the hybridoma was treated with an appropriate medium, for example, Culture with 10% fetal bovine serum, RPMI1640 medium containing 5% BM-Condimed H1 (Boehringer Mannheim), hybridoma SFM medium (GIBCO-BRL), PFHM-II medium (GIBCO-BRL), etc. The method can be carried out by purifying the PM-1 antibody.

  In the present invention, as a monoclonal antibody, a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing it into a host, and producing it using a gene recombination technique can be used. (See, for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

  Specifically, mRNA encoding the variable (V) region of the antibody is isolated from cells producing the antibody of interest, such as 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 (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) etc., and then prepare mRNA using mRNA Purification Kit (manufactured by Pharmacia) etc. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

  From the obtained mRNA, cDNA of the antibody V region is synthesized using reverse transcriptase. cDNA synthesis can be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit or the like. In addition, 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 desired 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 recombinant antibody that has been artificially modified for the purpose of reducing the heterologous antigenicity to humans, for example, a chimeric antibody or a humanized antibody can be used. These modified antibodies can be produced using known methods.
A chimeric antibody can be 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). Patent application publication number EP 125023, International patent application publication number WO 92-19759). Using this known method, a chimeric antibody useful in the present invention can be obtained.

For example, plasmids containing DNA encoding the V regions of the L chain and H chain of the chimeric PM-1 antibody are named pPM-k3 and pPM-h1, respectively, and Escherichia coli having this plasmid is National Collections of Industrial and Marine. Internationally deposited with Bacteria Limited on February 12, 1991 as NCIMB 40366 and NCIMB40362, respectively, under the Budapest Treaty.
A humanized antibody is also referred to as a reshaped human antibody, and is obtained by transplanting the complementarity determining region (CDR) of a non-human mammal such as a mouse antibody into the complementarity determining region of a human antibody. General gene recombination techniques are also known (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92-19759).

  Specifically, several oligo oligonucleotides were prepared so that the DNA sequence designed to link the CDR of the mouse antibody and the framework region (FR) of the human antibody had an overlapping portion at the end. Synthesize from nucleotides by PCR. The obtained DNA is ligated with DNA encoding the human antibody C region, then incorporated into an expression vector, and introduced into a host for production (European Patent Application Publication Number EP 239400, International Patent Application Publication Number). See WO 92-19759).

As the FR of the human antibody to be ligated via CDR, the one in which the complementarity determining region 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, and 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 derived from a human antibody, and a C region. Since it is composed of regions and has reduced antigenicity in the human body, it is useful as an antibody used in the present invention.
A preferred specific example of the humanized antibody used in the present invention is a humanized PM-1 antibody (see International Patent Application Publication No. WO 92-19759).

  The antibody gene constructed as described above can be expressed and obtained by a known method. In the case of a mammalian cell, it can be expressed by a commonly used useful promoter, an antibody gene to be expressed, a DNA operably linked with a poly A signal at the 3 ′ downstream thereof, or a vector containing the same. For example, examples of promoter / enhancer include human cytomegalovirus immediate early promoter / enhancer.

In addition, other promoters / enhancers that can be used for expression of antibodies used in the present invention include retrovirus, polyoma virus, adenovirus, simian virus 40 (SV 40) and other promoters / enhancers and human elongation factor 1 α ( A promoter / enhancer derived from mammalian cells such as HEF1α) may be used.
For example, when the SV 40 promoter / enhancer is used, the method of Mulligan et al. (Mulligan, RC et al., Nature (1979) 277, 108-114), and when the HEF1α promoter / enhancer is used, the method of 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 using the lacZ promoter, 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), araB promoter Can be used according to the method of Better et al. (Better, M. et al. Science (1988) 240, 1041-1043).

  As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379-4383) may be used when the periplasm of E. coli is produced. After separating the antibody produced in the periplasm, the structure of the antibody is appropriately refolded and used (see, for example, WO96 / 30394).

As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can be used. Furthermore, the expression vector serves as a selection marker for gene copy number amplification in the host cell system. Aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene and the like.
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 that use eukaryotic cells and production systems that use prokaryotic cells.

  When eukaryotic cells are used, there are production systems using animal cells, plant cells, and fungal cells. Animal cells include: (1) mammalian cells, such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, (2) amphibian cells, such as Xenopus oocytes, or (3) insect cells For example, sf9, sf21, and Tn5 are known. As plant cells, cells derived from Nicotiana tabacum are known, and these may be cultured in callus. As fungal cells, yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, and fungi such as the genus Aspergillus, such as Aspergillus niger, are known.

When prokaryotic cells are used, there are production systems using bacterial cells. Known bacterial cells include 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, RPMI1640, IMDM can be used as a culture solution, and serum supplements such as fetal calf serum (FCS) can be used in combination. Alternatively, antibodies may be produced in vivo by transferring cells 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, and cows can be used (Vicki Glaser, 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, K.M. 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 pMON 530, 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 to simultaneously transform the host. Alternatively, the host may be transformed by incorporating DNAs encoding H and L chains into a single expression vector (see International Patent Application Publication No. WO 94-11523).
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 H chain and 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 a gene encoding these antibody fragments is constructed and introduced into an expression vector, and then an appropriate host cell. (E.g., Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Plueckthun, A. & Skerra, A. Methodsin Enzymology (1989) 178, 476-496, Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663 -669, Bird, RE et al., TIBTECH (1991) 9, 132-137).

  scFv can be 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. USA (1988) 85, 5879. -5883). The H chain V region and 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 12-19 is used.

  The scFv-encoding DNA is a DNA sequence encoding the H chain or H chain V region of the antibody and a DNA encoding the L chain or L chain V region, and a desired amino acid sequence of those sequences. The DNA part encoding the DNA is amplified by PCR using a primer pair that defines both ends of the DNA, and then the DNA that encodes the peptide linker part and both ends thereof are ligated to the H and L chains, respectively. Obtained by combining and amplifying primer pairs.

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, and the host is used according to a conventional method. , ScFv can be obtained.
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 claims of the present application 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 claims of the present application 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 isolated 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 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 Hyper D, POROS, Sepharose F.F. 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 high performance liquid chromatography (HPLC). Moreover, you may use reverse phase HPLC (reverse phase HPLC).

  The concentration of the antibody obtained above can be measured by measuring absorbance or ELISA. That is, in the case of measuring absorbance, after appropriately diluting with PBS (-), the absorbance at 280 nm is measured, and 1 mg / ml is calculated as 1.35 OD. When using ELISA, the measurement can be performed as follows. That is, 100 μl of goat anti-human IgG (manufactured by TAGO) 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. Solidify. 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 sample is added and incubated at room temperature for 1 hour.

  After washing, add 100 μl of alkaline phosphatase-labeled anti-human IgG (BIO SOURCE) diluted 5000 times and incubate at room temperature for 1 hour. After washing, the substrate solution is added, and after incubation, the absorbance at 405 nm is measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate the concentration of the target antibody.

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 competitively binds IL-6 to the IL-6 receptor, but does not transmit IL-6 biological activity, and therefore 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, does not matter, but human IL-6 is preferred 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 performed by evaluating the influence on the whole of the amino acid residue to be substituted. After determining the appropriate substitution amino acid residue, IL- is introduced by introducing a mutation so that the amino acid is substituted by a usual PCR method using a vector containing a nucleotide sequence encoding the human IL-6 gene as a template. 6 Gene encoding the variant is obtained. This is 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 Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, and Savino et al., EMBO J. (1994) 13, 1357-1367, WO 96-18648, WO96-17869.
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 these to specifically bind IL-6 to IL-6 receptor. To inhibit. As a result, it does not transmit the biological activity of IL-6, and therefore blocks signal transduction 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 and IL-6 receptor in the amino acid sequence of IL-6 or IL-6 receptor A peptide consisting of a sequence. Such a peptide usually consists of 10 to 80, preferably 20 to 50, more preferably 20 to 40 amino acid residues.
IL-6 partial peptide or IL-6 receptor partial peptide specifies the region involved in the binding of 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 IL-6 receptor partial peptide by genetic engineering techniques, a DNA sequence encoding the 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 peptide synthesis, a method generally 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 1991 follow-up drug development, Volume 14, Peptide Synthesis Supervision, Haraaki Yajima, 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 amino acids one by one in the order from the C-terminal to the N-terminal and the reaction of removing the protecting group of the amino acid or peptide α-amino group bound on the resin, A method of stretching is used. Solid phase peptide synthesis methods are roughly classified into Boc method and Fmoc method according to 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 hydrogen fluoride in the presence of anisole. The peptide is then recovered by removing the protecting group and cleaving from the support. This is freeze-dried to obtain a crude peptide. On the other hand, in the Fmoc method, for example, the deprotection reaction and the cleavage reaction from the support of the 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 IL-6 partial peptide and IL-6 receptor partial peptide are disclosed in JP-A-2-188600, JP-A-7-324097, JP-A-8-311098 and US Pat.

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, IL-6-dependent cells MH60.BSF2 were cultured, IL-6 was added to the cells, and at the same time, IL-6 antagonists were coexisted to measure 3H-thymidine incorporation in IL-6-dependent cells. do it. In addition, by culturing U266, which is an IL-6 receptor-expressing cell, adding ^125I -labeled IL-6 and simultaneously adding an IL-6 antagonist, ¹²⁵ I-labeled IL- Measure 6. In the above assay system, there is a negative control group that does not contain IL-6 antagonist in addition to the group in which IL-6 antagonist is present, and the IL-6 inhibitory activity of IL-6 antagonist can be evaluated by comparing the results obtained with both. can do.

In order to confirm the effect of the present invention, an IL-6 antagonist used in the present invention is administered to an animal that has developed inflammatory bowel disease by transferring CD4-positive and CD45RB-positive cells (CD4 ⁺ CD45RB ^high cells). It can be performed by evaluating the effect of suppressing weight loss and improving the inflammatory bowel disease score. Other effects of the present invention include anorexia suppression effect, abdominal pain reduction effect, diarrhea reduction effect, or inflammatory bowel disease recurrence prevention effect in inflammatory bowel disease.

CD4 ⁺ CD45RB ^high cells transferred to an animal with an IL-6 antagonist can be isolated, for example, by using the method described in the Examples below. Further, the animal from which the CD4 ⁺ CD45RB ^high cells are derived may be an animal that is usually used in an experiment, such as a mouse or a rat.
As shown in the examples described later, administration of anti-IL-6 receptor antibody showed suppression of weight loss and improvement of bowel disease score in experimental animals that developed inflammatory bowel disease. IL-6 antagonists such as IL-6 receptor antibodies have been shown to have a therapeutic effect on inflammatory bowel disease.
The subject of treatment in the present invention is a mammal. The mammal to be treated is preferably a human.

  The preventive or therapeutic agent of the present invention can be administered orally or parenterally systemically or locally. 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. Alternatively, a dose of 1-1000 mg, preferably 5-50 mg per patient can be selected.

  The preventive or therapeutic agent of the present invention may contain both pharmaceutically acceptable carriers and additives depending on the administration route. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble dextran, Sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, Examples include sorbitol, lactose, and surfactants acceptable as pharmaceutical additives. The additive to be used is selected appropriately or in combination from the above depending on the dosage form, but is not limited thereto.

  The disease to be treated in the present invention is inflammatory bowel disease. Inflammatory bowel disease includes ulcerative colitis and Crohn's disease. These diseases are all chronic diseases that are prevalent in young people around 20 years old, and the mechanisms of antigens and inflammatory pathologies that cause it have not been elucidated. However, research to elucidate this has been actively conducted using cells and cytokines as clues.

In recent years, analysis of intestinal disease-causing cells has progressed mainly using mouse systems. That is, it has been clarified that the pathological condition of chronic intestinal disease can be induced by transplanting purified CD4 positive CD45RB strong positive cells (CD4 ⁺ CD45RB ^high ) into immunodeficient mice (SCID mice) (Morrissey, PJ et al. , J. Exp. Med. (1993) 178, 237-244, Leach, MW et al., Am. J. Pathol. (1996) 148, 1503-1515, Aranda, R. et al., J. Immunol. (1997) 158, 3464-3473).

On the other hand, CD4 positive CD45RB weakly positive cells (CD4 ⁺ CD45RB ^low ) have not been shown to induce intestinal disease, but rather have been shown to suppress intestinal disease induction by CD4 positive CD45RB strongly positive cells (Powrie, FR et al., J. Exp. Med. (1994) 179, 589-600). The correlation between CD45RB expression level and cytokine production pattern is known. That is, CD4 positive CD45RB strongly positive cells are type 1 helper cells (Th1) that produce IFN-γ, TNFα, etc., whereas CD4 positive CD45RB weakly positive cells produce IL-4, IL-10, etc. Type 2 helper cells (Th2) -like (Lee, W. et al., J. Immunol. (1990) 144, 3288-3295).

Therefore, it is highly likely that the onset of IBD is related to the imbalance between Th1 and Th2, which resulted in deletion of the IL-10 gene, which suppresses Th1 function, by gene targeting. This is supported by the development of chronic enteritis in mice that resembles human ulcerative colitis (Kuhn, R. et al., Cell (1993) 75, 263-274).
The model animals used in this example are very similar in histological characteristics of the large intestine to patients with ulcerative colitis and Crohn's disease (Leach, MW et al., Am. J. Pathol. (1996) 148, 1503-1515). In ulcerative colitis, many lesions are seen continuously and widely from the rectum, and the mucosal epithelium is specifically damaged. In this model, the lesion is mainly limited to the large intestine, but it extends over a wide range, and the crypt extends, which is similar to clinical pathology.

  On the other hand, Crohn's disease is a full-thickness inflammation that occurs everywhere in the digestive tract from the oral cavity to the anus and is not limited to the mucosa. Histologically, it is characterized by non-caseous granulomatous inflammation. In this model, inflammation that is not confined to the mucosal layer was found. Accumulation of macrophages, lymphocytes, multinucleated giant cells, etc., often granuloma-like morphology, and the absence of crypt abscesses were found in Crohn's disease. It is similar.

  Thus, cases in which characteristics of ulcerative colitis and Crohn's disease coexist have been reported clinically (Tanaka, M et al., Hepato-gastroenterology (1990) 37, 18-31). Moreover, in inflammatory bowel disease, expression of major histocompatibility antigen class II is found to be enhanced in epithelial cells (Trejdosiewicz, LK et al., Dig. Dis. Sci. (1989) 34, 1449-1456) The same applies to this model. In this model, thickening of epithelial tissue appears characteristically, which is an increase in cell proliferation found in patients with ulcerative colitis (Serafini, EP etal., Gut (1981) 22, 648-652). It seems to be related.

  This model is highly similar to clinical inflammatory bowel disease and causes weight loss in severe cases. The experiments using this model showed that histological damage was significantly reduced and weight loss was not observed. IL-6 antagonist was effective for treating inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Is shown.

EXAMPLES Hereinafter, although an Example, a reference example, and an experiment example demonstrate this invention concretely, this invention is not limited to these.
Example
The spleen was aseptically collected from BALB / c male mice, crushed, and then pipetted well to obtain a single cell suspension. Next, in order to remove erythrocytes, the cell pellet is treated with Lysis solution (0.16M NH ₄ Cl mixed with 0.17M Tris buffer solution at pH 7.2 in a ratio of 9: 1), and phosphate buffered saline. After washing twice with water, mouse spleen cells were obtained.

The washed mouse spleen cells were suspended in 2% FCS-added RPMI1640 medium and the number of cells was counted, and then prepared to 1.1 × 10 ⁸ / ml. To this was added 1/9 volume of anti-mouse CD4 antibody (L3T4 Microbeads, Miltenyi Biotec) and allowed to bind to the cells for 15 minutes under ice temperature (cell density 1 × 10 ⁸ / ml). Furthermore, a CD4 positive cell fraction was fractionated by column operation using Mini MACS separation system (Miltenyi Biotec). After counting the number of cells, the cells were suspended in 2% FCS-added phosphate buffered saline, and the cell density was adjusted to 4 × 10 ⁷ / ml.

  1/100 volume PE-labeled rat anti-mouse CD4 (L3T4) antibody (0.2 mg / ml, clone RM4-5, Pharmingen) and 1/100 volume FITC-labeled rat anti-mouse CD45RB antibody in CD4-positive cell suspension (0.5 mg / ml, clone 16A, manufactured by Pharmingen) was added. The antibody was bound by allowing it to stand at ice temperature for 20 minutes. The labeled cells were washed by adding RPMI1640 medium supplemented with 2% FCS and centrifuging, resuspended in phosphate buffered saline containing 2% FCS, and kept in a cool dark place.

From the labeled CD4 positive cells, CD4 positive and CD45RB strongly positive cell groups (CD4 ⁺ CD45RB ^high ) were collected using a flow cytometer (FACS Vantage, manufactured by Becton Dickenson). This cell group corresponds to about 50% of the CD4 and CD45RB positive cells with the highest expression level of CD45RB. The obtained cells were spun down and suspended in phosphate buffered saline so as to be 4 × 10 ⁶ / ml. The purity of the cells was 97% as CD45RB strongly positive cells, and the survival rate was 98%.

An inflammatory bowel disease model was prepared by transplanting 4 × 10 ⁵ cells (100 μl each of 4 × 10 ⁶ / ml) of highly purified cells into the abdominal cavity of CB-17 scid mice (Leach, MW et al., Am. J. Pathol. (1996) 148, 1503-1515, Aranda, R. et al., J. Immunol. (1997) 158, 3464-3473). The experiment was conducted in the following three groups according to the treatment method. (1) Cell transplantation / anti-IL-6 receptor antibody non-administration group, 5 mice, (2) Cell transplantation / anti-IL-6 receptor antibody administration group, 3 mice, (3) Cell non-transplantation group, 3 mice.

  The anti-IL-6 receptor antibody MR16-1 was administered as follows. First, 100 μl / mouse of 20 mg / ml in phosphate buffered saline was administered intraperitoneally 15-30 minutes before transplanting the cells. One week later, 10 μl / ml of phosphate buffered saline was administered intraperitoneally at 100 μl per mouse, and this was repeated weekly until 8 weeks later. For the antibody non-administered group, phosphate buffered saline was administered in the same manner.

  After measuring the body weight of the mice 8 to 9 weeks after cell transplantation, the large intestine tissue (the descending colon part) was collected, immersed in OCT compound, cooled to −80 ° C. and frozen. A 6 μm frozen section was prepared using a cryostat, and then fixed with a 10% formalin solution. The fixed tissue sections were subjected to heavy staining by the hematoxylin-eosin method and analyzed histologically.

  The evaluation of drug efficacy was performed by the change in body weight (expressed as a ratio to the former) and histological analysis before inducing inflammatory bowel disease by cell transplantation and 8-9 weeks after cell transplantation. In the histological analysis, each mouse tissue was evaluated by classification into the following four grades of inflammatory bowel disease score (hereinafter also referred to as bowel disease score) (Ito, H. et al., J. Autoimmunity ( 1997) 10, 455-459).

Inflammatory bowel disease score
Grade 0 (non): Indistinguishable from normal BALB / c mice
Grade 1 (minimal): Mild epithelial tissue thickening is observed.
Grade 2 (moderate): intermediate between Grade 1 and 3
Grade 3 (severe): Significant epithelial thickening with extensive inflammatory infiltration and goblet cell loss.

Table 1 shows the results of weight change and inflammatory bowel disease score.
Mice transplanted with CD4 + CD45 strongly positive cells developed inflammatory bowel disease, and histologically significant inflammation was observed. In addition, she became weak with the onset, and her body weight decreased by an average of 11%. On the other hand, in the group administered with the anti-IL-6 receptor antibody, statistically significant suppression of weight loss was observed, and the body weight was maintained at the same level as before cell transplantation. In addition, the histological inflammatory bowel disease score revealed that inflammatory bowel disease was reduced.

In addition, for the change in body weight, first the ANOVA method (Analysis of variance, SPSS for windows ver.6, SPSS Inc.) was used to confirm the significance, and then multiple comparisons using the Bonferroni method were used. Significance was observed at% or less.
This model is highly similar to human inflammatory bowel disease, and thus it is clear that anti-IL-6 receptor antibody is effective as a preventive or therapeutic agent for inflammatory bowel disease such as ulcerative colitis or Crohn's disease. became.

Reference Example 1. Preparation of human soluble IL-6 receptor
Soluble by PCR using plasmid pBSF2R.236 containing cDNA encoding IL-6 receptor obtained according to the method of Yamasaki et al. (Yamasaki, K. et al., Science (1988) 241, 825-828) IL-6 receptor was created. Plasmid pBSF2R.236 was digested with restriction enzyme Sph I to obtain IL-6 receptor cDNA, which was inserted into mp18 (Amersham). Using a synthetic oligo primer designed to introduce a stop codon into the IL-6 receptor cDNA, a mutation was introduced into the IL-6 receptor cDNA by PCR using an in vitro mutagenesis system (manufactured by Amersham). By this operation, a stop codon was introduced at the position of amino acid 345, and a cDNA encoding a soluble IL-6 receptor was obtained.

  In order to express soluble IL-6 receptor cDNA in CHO cells, it was ligated with plasmid pSV (manufactured by Pharmacia) to obtain plasmid pSVL344. The soluble IL-6 receptor cDNA cleaved with Hind III-Sal I was inserted into the plasmid pECEdhfr containing the dhfr cDNA to obtain a CHO cell expression plasmid pECEdhfr344.

  10 μg of plasmid pECEdhfr344 was transferred to dhfr-CHO cell line DXB-11 (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA (1980) 77, 4216-420) by calcium phosphate precipitation (Chen, C et al., Mol. Cell. Biol. (1987) 7, 2745-2751). Transfected CHO cells were cultured for 3 weeks in a nucleoside-free αMEM selective culture medium containing 1 mM glutamine, 10% dialyzed FCS, 100 U / ml penicillin and 100 μ / ml streptomycin.

  Selected CHO cells were screened by limiting dilution to obtain a single CHO cell clone. This CHO cell clone was amplified with methotrexate at a concentration of 20 nM-200 nM to obtain a human soluble IL-6 receptor-producing CHO cell line 5E27. CHO cell line 5E27 was cultured in Iscove modified Dulbecco medium (IMDM, Gibco) containing 5% FBS. The culture supernatant was collected, and the concentration of soluble IL-6 receptor in the culture supernatant was measured by ELISA. As a result, it was confirmed that soluble IL-6 receptor was present in the culture supernatant.

Reference Example 2 Preparation of anti-human IL-6 antibody
10 μg of recombinant IL-6 (Hirano, T. et al., Immunol. Lett., 17:41, 1988) was immunized with Freund's complete adjuvant in BALB / c mice. This was continued every week until detected. Immune cells were removed from local lymph nodes and fused with myeloma cell line P3U1 using polyethylene glycol 1500. Hybridomas were selected according to the method of Oi et al. Using a HAT culture solution (Selective Methods in Cellular Immunology, WHFreeman and Co., San Francisco, 351, 1980) to establish hybridomas producing anti-human IL-6 antibodies.

  Hybridomas producing anti-human IL-6 antibodies were subjected to IL-6 binding assay as follows. Specifically, a 96-well microplate made of flexible polyvinyl (Dynatech Laboratories, Inc., Alexandria, VA) was washed with 100 μl of goat anti-mouse Ig (10 μl / ml, 0.1 M carbonate-hydrogen carbonate buffer (pH 9.6)). Cooper Biomedical, Inc. Malvern, PA) and coated overnight at 4 ° C. The plates were then treated with 100 μl of 1% bovine serum albumin (BSA) in PBS for 2 hours at room temperature.

  After washing with PBS, 100 μl of hybridoma culture supernatant was added to each well and incubated at 4 ° C. overnight. The plate was washed, and 125I-labeled recombinant IL-6 was added to each well so as to obtain 2000 cpm / 0.5 ng / well. After washing, the radioactivity in each well was measured using a gamma counter (Beckman Gamma 9000, Beckman Instruments, Fullerton, CA). Of the 216 hybridoma clones, 32 hybridoma clones were positive by IL-6 binding assay. Of these clones, stable MH166.BSF2 was finally obtained. The anti-IL-6 antibody MH166 produced by the hybridoma has an IgG1κ type subtype.

Subsequently, the neutralizing activity of the MH166 antibody on the growth of the hybridoma was examined using the IL-6-dependent mouse hybridoma clone MH60.BSF2. MH60.BSF2 cells dispensed so minute a 1 × 10 ⁴ / 200μl / well, to which was added a sample containing MH166 antibody were cultured for 48 hours, 0.5 [mu] Ci / well of ³ H-thymidine (New England Nuclear, Boston , MA) was added, and the culture was further continued for 6 hours. Cells were placed on glass filter paper and treated with an automatic harvester (Labo Mash Science Co., Tokyo, Japan). Rabbit anti-IL-6 antibody was used as a control.

As a result, MH166 antibody inhibited uptake of ³ H thymidine in MH60.BSF2 cells induced by IL-6 in a dose-dependent manner. This revealed that MH166 antibody neutralizes IL-6 activity.

Reference Example 3 Preparation of anti-human IL-6 receptor antibody
Sepharose 4B (manufactured by Pharmacia Fine Chemicals) in which anti-IL-6 receptor antibody MT18 prepared by the method of Hirata et al. (Hirata, Y. et al. J. Immunol., 143: 2900-2906, 1989) was activated with CNBr. And IL-6 receptor (Yamasaki, K. et al., Science (1988) 241, 825-828). Human myeloma cell line U266 was converted to 1% digitonin (manufactured by Wako Chemicals), 1 mM p-paraaminophenylmethanesulfonyl fluoride hydrochloride (manufactured by Wako Chemicals) containing 10 mM triethanolamine (pH 7.8) and 0.15 M NaCl (digitonin buffer) And mixed with MT18 antibody conjugated with Sepharose 4B beads. Thereafter, the beads were washed 6 times with digitonin buffer to obtain partially purified IL-6 receptor for immunization.

BALB / c mice were immunized 4 times every 10 days with the partially purified IL-6 receptor obtained from 3 × 10 ⁹ U266 cells, and then a hybridoma was prepared by a conventional method. The hybridoma culture supernatant from the positive growth hole was examined for binding activity to IL-6 receptor by the following method. 5 × 10 ⁷ U266 cells were labeled with ³⁵ S-methionine (2.5 mCi) and solubilized with the above digitonin buffer. Solubilized U266 cells were mixed with 0.018 ml volume of Sepharose 4B beads-bound MT18 antibody, then washed 6 times with digitonin buffer and ³⁵ S-methionine with 0.25 ml digitonin buffer (pH 3.4). The labeled IL-6 receptor was drained and neutralized with 0.025 ml of 1M Tris (pH 7.4).

0.05 ml of the hybridoma culture supernatant was mixed with 0.01 ml of Protein G Sepharose (manufactured by Pharmacia). After washing, Sepharose was incubated with 0.005 ml of ³⁵ S-labeled IL-6 receptor solution prepared above. Immunoprecipitates were analyzed by SDS-PAGE to examine hybridoma culture supernatants that react with IL-6 receptor. As a result, a reaction-positive hybridoma clone PM-1 was established. The antibody produced from hybridoma PM-1 has an IgG1κ type subtype.

The inhibitory activity of IL-6 binding to the human IL-6 receptor of the antibody produced by hybridoma PM-1 was examined using the human myeloma cell line U266. Human recombinant IL-6 was prepared from E. coli (Hirano, T. et al., Immunol. Lett. (1988) 17, 41-45) and ¹²⁵ by Bolton-Hunter reagent (New England Nuclear, Boston, MA). I-labeled (Taga, T. et al., J. Exp. Med. (1987) 166, 967-981). 4 × 10 ⁵ U266 cells were cultured for 1 hour with 70% (v / v) hybridoma PM-1 culture supernatant and 14000 cpm of ¹²⁵ I-labeled IL-6. 70 μl of the sample was layered on 400 μl of microfuge polyethylene tube on 300 μl of FCS, and after centrifugation, the radioactivity on the cells was measured.
As a result, it was revealed that the antibody produced by hybridoma PM-1 inhibits the binding of IL-6 to the IL-6 receptor.

Reference Example 4 Preparation of anti-mouse IL-6 receptor antibody
A monoclonal antibody against mouse IL-6 receptor was prepared by the method described in Saito, T. et al., J. Immunol. (1991) 147, 168-173.
CHO cells producing mouse soluble IL-6 receptor are cultured in IMDM medium containing 10% FCS, and anti-mouse IL-6 receptor antibody RS12 (see Saito, T. et al above) is cultured from the culture supernatant. Mouse soluble IL-6 receptor was purified using an affinity column immobilized on Affigel 10 gel (Biorad).

The obtained mouse soluble IL-6 receptor (50 μg) was mixed with Freund's complete adjuvant and injected into the abdomen of Wistar rats. Two weeks later, booster immunization was performed with Freund's incomplete adjuvant. On day 45, rat spleen cells were collected, and 2 x 10 ⁸ cells were fused with 1 x 10 ⁷ mouse myeloma cells P3U1 and 50% PEG1500 (manufactured by Boehringer Mannheim) in a conventional manner. Hybridomas were screened in the medium.

  Hybridoma culture supernatant was added to a plate coated with rabbit anti-rat IgG antibody (Cappel), and then mouse soluble IL-6 receptor was reacted. Subsequently, hybridomas producing antibodies against mouse soluble IL-6 receptor were screened by ELISA using rabbit anti-mouse IL-6 receptor antibody and alkaline phosphatase-labeled sheep anti-rabbit IgG. Hybridoma clones in which antibody production was confirmed were sub-screened twice to obtain a single hybridoma clone. This clone was named MR16-1.

The neutralizing activity of the antibody produced by this hybridoma in the signal transduction of mouse IL-6 was measured by ³ H using MH60.BSF2 cells (Matsuda, T. et al., J. Immunol. (1988) 18, 951-956). Investigated by thymidine incorporation. MH60.BSF2 cells were prepared in a 96-well plate at 1 × 10 ⁴ cells / 200 μl / well. Add 10 pg / ml mouse IL-6 and MR16-1 antibody or 12.12 ng / ml to this plate and incubate for 44 hours at 37 ° C, 5% CO ₂ , then add 1 µCi / well of ³ H thymidine. It was. After 4 hours, ³ H thymidine incorporation was measured. As a result, MR16-1 antibody suppressed ³ H thymidine incorporation in MH60.BSF2 cells.
Therefore, it was revealed that the antibody produced by the hybridoma MR16-1 inhibits the binding of IL-6 to the IL-6 receptor.

EFFECT OF THE INVENTION According to the present invention, it was shown that IL-6 antagonists such as anti-IL-6 receptor antibodies have a therapeutic effect on inflammatory bowel disease. Therefore, it was revealed that IL-6 antagonist is useful as a therapeutic agent for Crohn's disease or ulcerative colitis.

Claims (6)

  A prophylactic or therapeutic agent for inflammatory bowel disease comprising an anti-gp130 antibody that blocks signal transduction by IL-6 as an active ingredient.   A prophylactic or therapeutic agent for inflammatory bowel disease comprising an anti-IL-6 antibody that blocks signal transduction by IL-6 as an active ingredient. Inflammatory intestine containing, as an active ingredient, an IL-6 variant that blocks IL-6 signal transduction and is produced by introducing a mutation by substituting an amino acid residue in the amino acid sequence of IL-6 A preventive or therapeutic agent for diseases. A prophylactic or therapeutic agent for inflammatory bowel disease comprising an IL-6 partial peptide consisting of 10 to 80 amino acid residues and blocking IL-6 signaling as an active ingredient. A prophylactic or therapeutic agent for inflammatory bowel disease comprising an IL-6 receptor partial peptide consisting of 10 to 80 amino acid residues and blocking IL-6 signal transduction as an active ingredient. The preventive or therapeutic agent according to any one of claims 1 to 5 , wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.