MXPA06005875A

MXPA06005875A - Remedy for angitis

Info

Publication number: MXPA06005875A
Application number: MXPA/A/2006/005875A
Authority: MX
Inventors: Nishimoto Norihiro; Kishimoto Tadamitsu; Nakahara Hideko
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2003-12-19
Filing date: 2006-05-24
Publication date: 2006-10-17

Abstract

It is intended to provide a preventive and/or a remedy for angitis such as nodal polyarteritis, aortitis syndrome and angitis accompanying immunopathy which contains an antagonist to interleukin-6 (IL-6) such as an antibody against IL-6 receptor (IL-6R).

Description

A PREVENTIVE AGENT FOR VASCUL1T1S Technical Field The present invention relates to a preventive and / or therapeutic agent for vasculitis. BACKGROUND OF THE ART Vasculitis is one of the intractable pathological conditions commonly observed in autoimmune diseases, and many cases thereof are refractory to conventionally used therapeutic methods, such as spheroids and immunosuppressants and, therefore, new methods have been sought. therapeutic In the vasculitis syndrome, inflammation occurs in arteries of different sizes, and may develop fever, pain in muscles and joints, vascular obstruction, skin ulcer and multiple mononeuritis. Vasculitis includes intractable vasculitis syndromes such as polyarteritis nodosa and aortitis syndrome. The lesions of polyarteritis nodosa are characterized by necrotic inflammations of the media and the adventitia, and aortitis in general develops inflammations of the intima, media and adventitia. Aortitis is also called Takayasu's arteritis. It was suggested that the pathology of vasculitis is associated with IL-6. For example, Noris et al. have reported that blood levels of IL-6 are increased in patients with Takayasu arteritis in the active stage of the pathology compared to normal healthy people (Circulation 1999, July 6; 100 (1); 55-60). However, this article also reports that the serum concentration of RANTES, one of the chemokines, also increases. Noris et. to suggest the possibility that these cytokines are responsible for vascular lesions in patients suffering from Takayasu arteritis. Description of the Invention However, the article does not mention at any time the possibility that Takayasu's arteritis can be treated by inhibiting IL-6. Therefore, the present invention provides preventive and / or therapeutic agents for vasculitis that comprise an IL-6 antagonist as an active component. After intensive and extensive research, the inventors of the present demonstrated that IL-6 is indispensable in the pathology for vasculitis and that an IL-6 antagonist possesses a therapeutic effect for vasculitis. More surprisingly, in the study performed by the present inventors, when the binding of IL-6 to its receptor was inhibited by means of an antibody of the IL-6 receptor, IL-6 per was reduced in blood. Therefore, it was shown that IL-6 inhibition therapy not only has an anti-inflammatory effect on vasculitis per se, but also treats vasculitis per se by acting on the nucleus of vasculitis. Therefore, the present invention provides a preventive and / or therapeutic agent for vasculitis, said agent comprising an interleukin 6 (IL-6) antagonist as an active component. The present invention provides a preventive and / or therapeutic agent for vasculitis that is resistant to spheroids and / or immunosuppressants, said agent comprises an interleukin 6 (IL-6) antagonist as an active component. Said vasculitis for example polyarteritis nodosa, the aortitis syndrome, or a vasculitis that is associated with immunological abnormalities. The aortitis syndrome is also called Takayasu arteritis. As vasculitis associated with immunological abnormalities, for example, vasculitis associated with rheumatoid and vasculitis associated with systemic lupus erythematosus (SLE) can be mentioned. Said IL-6 antagonist is, for example, an antibody against IL-6 or an antibody against the IL-6 receptor, preferably a monoclonal antibody against the IL-6 receptor. Said antibody against the IL-6 receptor more preferably is a monoclonal antibody against the human IL-6 receptor, for example, PM-1 antibody, or a monoclonal antibody against the mouse IL-6 receptor, for example. , MR16-1 antibody. Said antibody against the IL-6 receptor is preferably a recombinant antibody. Said antibody against the IL-6 receptor can be a chimeric antibody, humanized antibody or human antibody. As used herein, the most preferred antibody is a humanized PM-1 antibody. The present invention may also comprise the following aspects: (1) The use of the interleukin 6 (IL-6) antagonist for the manufacture of a preventive and / or therapeutic agent for vasculitis. (2) The use of the interleukin 6 (IL-6) antagonist for the manufacture of a preventive and / or therapeutic agent for vasculitis that possesses resistance to spheroids and / or immunosuppressants. (3) Use according to the previous (1) or (2) in which said vasculitis is polyarteritis nodosa. (4) Use according to the above (1) or (2) in which said vasculitis is the aortitis syndrome. (5) Use according to the above (1) or (2) in which said vasculitis is vasculitis associated with immunological abnormalities. (6) The use according to any of the foregoing (1) to (5) in which said IL-6 antagonist is an antibody against the IL-6 receptor. (7) The use according to the above (6) in which said antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor. (8) The use according to the above (6) in which said antibody against the IL-6 receptor is a monoclonal antibody against the human IL-6 receptor. (9) The use according to the preceding (6) in which said antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor. (10) The use according to any of the above (6) to (9) in which said antibody against the IL-6 receptor is a recombinant antibody. (11) The use according to the above (8) in which said monoclonal antibody against the human IL-6 receptor is PM-1 antibody. (12) The use according to the above (9) in which said monoclonal antibody against the mouse IL-6 receptor is an antibody of MR16-1. (13) The use according to any of the above (6) to (12) in which said antibody against the IL-6 receptor is a chimeric antibody, a humanized antibody or a human antibody. (14) The use according to the above (13) in which said humanized antibody against the IL-6 receptor is a humanized antibody of PM-1. (15) A method for preventing and / or treating vasculitis which comprises administering an interleukin 6 (IL-6) antagonist. (16) A method for preventing and / or treating vasculitis that possesses resistance to spheroids and / or immunosuppressants comprising administering an interleukin 6 (IL-6) antagonist. (17) The method according to the previous (15) or (16) in which said vasculitis is polyarteritis nodosa. (18) The method according to the previous (15) or (16) in which said vasculitis is the aortitis syndrome. (19) The method according to the above (15) or (16) in which said vasculitis is vasculitis associated with immunological abnormalities. (20) The method according to any of the above (15) to (19) in which said IL-6 antagonist is an antibody against the IL-6 receptor. (21) The method according to the preceding (20) in which said antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor. (22) The method according to the preceding (20) in which said antibody against the IL-6 receptor is a monoclonal antibody against the human IL-6 receptor. (23) The method according to the preceding (20) in which said antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor. (24) The method according to any of the foregoing (20) to (23) wherein said antibody against the IL-6 receptor is a recombinant antibody. (25) The method according to the previous one (22) in which said monoclonal antibody against the human IL-6 receptor is PM-1 antibody. (26) The method according to the preceding (23) in which said monoclonal antibody against the mouse IL-6 receptor is MR1 antibody. (27) The method according to any of the above (20) to (26) in which said antibody against the IL-6 receptor is a chimeric antibody, humanized antibody, or a human antibody antibody against the IL-6 receptor. (28) The method according to the previous one (27) in which said humanized antibody against the IL-6 receptor is a humanized antibody of PM-1. Brief Explanation of the Drawings Fig. 1 is a photograph showing the result of CT in the treatment of vasculitis syndrome with humanized antibody of 1L-6R, in which the upper arrow indicates the ascending aorta and the lower arrow indicates the descending aorta. Fig. 2 is a photograph showing the result of CT in the treatment of the vasculitis syndrome with humanized IL-6R antibody, in which the arrow indicates the aortic arch. Fig. 3 is a photograph showing the result of CT in the treatment of vasculitis syndrome with humanized IL-6R antibody, in which the upper arrow indicates the ascending aorta and the lower arrow indicates the descending aorta. Fig. 4 is a photograph showing the result of CT in the treatment of vasculitis syndrome with humanized IL-6R antibody, in which the upper arrow indicates the ascending aorta and the lower arrow indicates the descending aorta. Fig. 5 is a photograph showing the result of CT in the treatment of vasculitis syndrome with humanized IL-6R antibody, in which the arrow indicates the aortic arch. FIG. 6 is a photograph showing the result of CT in the treatment of vasculitis syndrome with humanized IL-6R antibody, in which the upper arrow indicates the ascending aorta and the lower arrow indicates the descending aorta. BEST MODE FOR CARRYING OUT THE INVENTION! L-6 is a cytokine that is also called stimulation factor 2 of the B cell (BSF2) or interleukin R2. IL-6 was discovered as a differentiation factor involved in the activation of lymphatic B cells (Hirano, T. et al., Nature (1986) 324, 73-76). Next, it was discovered that a multifunctional cytokine influences several cell functions (Akira, S. et al., Adv.

Immunology (1993) 54, 1-78). It was reported that IL-6 induces the maturation of lymphatic T cells (Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1259).

IL-6 transmits its biological activity through two types of proteins in the cell. One of them 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). The IL-6 receptor occurs not only in the membrane-bound form that penetrates and is expressed in the cell membrane but also as a soluble IL-6 receptor consisting mainly of the extracellular region. The other is a gp130 protein bound to the membrane that has a molecular weight of about 130KD that is involved in the transduction of non-ligand binding signal. IL-6 and the IL-6 receptor form the IL-6 / IL-6 receptor complex, which after binding to gp130 transmits its biological activity to the cell. (Taga, T. et al., Cell (1989) 58, 573-581). An IL-6 antagonist is a substance that inhibits the transduction of the biological activity of IL-6. As the IL-6 antagonist, antibodies directed against IL-6 (anti-IL-6 antibody), antibody directed against the IL-6 receptor (anti-IL-6 receptor antibody), and antibody were known up to now. directed against gp130 (anti-gp130 antibody), altered IL-6, partial peptides of IL-6 or IL-6 receptor and the like. The anti-IL-6 receptor antibody has been described in several reports (Novick D. et al., Hybridoma (1991) 10, 137-146, Huang, Y. W. et al., Hybridoma. (1993) 12, 621-630, International Patent Publication WO 95-09873, French Patent Application FR 2694767, US Patent US 521628). It was known that a humanized antibody of PM-1 was obtained by transplantation of the region that determines the complementarity (CDR) of one of them, a mouse PM-1 antibody (Hirata, Y. at al., J. Immunology ( 1989) 143, 2900-2506), to a human antibody (International Patent Application WO 92-19759). The above IL-6 antagonist is preferably an antibody against the IL-6 receptor, preferably a monoclonal antibody against the human IL-6 receptor or a monoclonal antibody against the mouse IL-6 receptor. As the above monoclonal antibody against the human IL-6 receptor, PM-1 antibody can be illustrated, and like the above monoclonal antibody against the mouse IL-6 receptor, the MR16-1 antibody can be screened . The above antibody is preferably a chimeric antibody, humanized antibody or a human antibody, for example a humanized antibody of PM-1. The IL-6 antagonist for use in the present invention can be of any origin, any type and any form, as long as they are useful as an active component for a preventive or therapeutic effect for vasculitis. The IL-6 antagonist blocks signal transduction by IL-6 and inhibits the biological activity of IL-6. The IL-6 antagonists are preferably substances possessing an inhibition activity of any of IL-6, IL-6 receptor and gp130. As antagonists of IL-6, there may be mentioned for example anti-IL-6 antibody, anti-IL-6 receptor antibody, anti-gp130 antibody, altered IL-6, altered soluble receptor of IL-6, a IL-6 partial peptide or IL-6 receptor, and low molecular weight substances that have the same activity as these. Anti-IL-6 antibodies for use in the present invention can be obtained as polyclonal or monoclonal antibodies using a known method. As the anti-IL-6 antibodies in the present invention, monoclonal antibodies of, in particular, mammalian origin are preferred. Monoclonal antibodies of mammalian origin include those that are produced by a hybridoma and recombinant antibody produced by a host that was transformed with an expression vector containing engineered genes of the antibody. These antibodies, by binding with IL-6, they block the binding of IL-6 to the IL-6 receptor and therefore block the signal transduction of the biological activity of IL-6 in the cell. Examples of such antibodies include MH166 (Matsuda et al., Eur. J. Immunol. (1988) 18, 951-956) and SK2 antibody (Sato, K. et al., The 21 st Nihon Menekigakkai Soukai (General Assembly of the Japanese Society of Immunology), Academic Record (1991) 21, 166) and the like. An anti-IL-6 antibody that produces hybridoma can be constructed basically using a known method, as described below. Therefore, IL-6 can be used as a sensitizing antigen and is immunized in the conventional method of immunization. The immune cells obtained in this way are fused with known progenitor cells in the conventional cell fusion process and the cells that produce monoclonal antibodies are screened by the conventional screening method to prepare the desired hybridoma. Specifically, the anti-IL-6 antibody can be obtained in the following manner. For example, a human UL-6 to be used as the sensitizing antigen to obtain the antibody can be obtained using the IL-6 gene / amino acid sequence disclosed in Eur. J. Biochem (1987) 168, 543-550, J. Immunol. (1988) 140, 1534-1541, or Argic. Biol. (1990) 54, 2685-2688. After a suitable host cell is transformed by the insertion of the IL-6 gene sequence into a known system of the expression vector, the IL-6 protein of interest is purified from the host cell or the supernatant of the host cell. culture thereof, and the purified IL-6 protein can be used as the sensitizing antigen. Alternatively, one fusion protein of the IL-6 protein and another protein can be used as the sensitizing antigen. Anti-IL-6 receptor antibodies for use in the present invention can be obtained as polyclonal or monoclonal antibodies using a known method. As the anti-IL-6 antibodies for use in the present invention, monoclonal antibodies of, in particular, mammalian origin are preferred. Monoclonal antibodies of a mammalian origin include those produced by a hybridoma and those produced by a host that has been transformed with an expression vector containing engineered genes of the antibody. The antibodies, by binding to the IL-6 receptor, inhibit the binding of IL-6 to the IL-6 receptor, and thereby block the transduction of the biological activity of IL-6 in the cell. Examples of said antibodies include MR16-1 antibody (Tamura, T., et al., Proc. Nati Acad. Sci. USA (1993) 90, 11924-11928), PM-1 antibody (Hirata, et al., J. Immunol. (1989) 143, 2900-2906), or antibody of AUK12-20, antibody of AUK64- 7 or antibody of AUK146-15 (International Patent Publication WO 92-19759), and the like. Among them, the PM-1 antibody is more preferred. Incidentally, the hybridoma cell line that produces the PM-1 antibody has been deposited internationally according to the provisions of the Budapest Treaty as PM-1 on July 12, 1995 with the Patent Microorganism Depository of the National Institute of Industrial Science and Technology (Deposit of Patents of Microorganisms of the National Institute of Sciences and Industrial Technology), of Chuo 6, 1 -1, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, as FERM BP-2998. The hybridoma cell line that produces the MR16-1 antibody has been deposited internationally according to the provisions of the Budapest Treaty as MR16-1 on March 13, 1997, with the Patent Microorganism Depository of the National Institute of Industrial Science and Technology, from Chuo 6, 1-1, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, as FERM BP-5875. Hybridomas producing the anti-IL-6 receptor monoclonal antibody can be prepared basically by using a known method as described below. Therefore, the IL-6 receptor is used as a sensitizing antigen and is immunized according to the conventional method of immunization. The immune cells that were obtained in that manner were fused with known progenitor cells in the conventional cell fusion process and then the cells that produce monoclonal antibodies can be screened by the conventional screening method to prepare the desired hybridoma. Specifically, the anti-IL-6 receptor antibody can be prepared in the following manner. For example, the human IL-6 receptor that is used as the sensitizing antigen to obtain the antibody can be obtained by using the den sequence of the IL-6 receptor / amino acid sequence disclosed in the European Patent Application EP 325474, and the mouse IL-6 receptor can be obtained by using the IL-6 receptor gene disclosed in Japanese Unexamined Patent Publication (Kokai) 3-155795. There are two types of IL receptor proteins. -6: IL-6 receptor expressed on the cell membrane, and the IL-6 receptor separated from the cell membrane (soluble receptor of IL-6) (Yasukawa et al., J. Biochem. (1990) ) 108, 673-676). The IL-6 soluble receptor antibody is essentially composed of the extracellular region of the IL.6 receptor bound to the cell membrane, and therefore is different to the IL-6 receptor bound to the membrane in that the former it does not possess the transmembrane region or both the transmembrane region and the intracellular region. As the IL-6 receptor protein, any IL-6 receptor can be used, as long as it can be used as a sensitizing antigen for the production of the IL-6 receptor antibody for use in the present invention.

After the gene sequence of the IL-6 receptor is inserted into a system of known expression vectors to transform an appropriate host cell, the desired protein of the IL-6 receptor can be purified from the host cell or a supernatant of the culture thereof using a known method, and the purified protein of the IL-6 receptor purified in that manner can be used as the sensitizing antigen. Alternatively, cells expressing the IL-6 receptor or a fusion protein of the IL-6 receptor protein and another protein can be used as a sensitizing antigen. E. coli possesses a plBIBSF2R plasmid containing cDNA encoding the human receptor of IL-6 deposited intemationally according to the provisions of the Budapest Treaty as HB101-plBIBSF2R on January 9, 1989 with the Patent Microorganism Depository of the National Institute of Industrial Science and Technology, from Chuo 6, 1-1, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, as FERM BP-2232. Anti-gp130 antibodies for use in the present invention can be obtained as polyclonal or monoclonal antibodies using a known method. As with anti-gp130 antibodies for use in the present invention, monoclonal antibodies of mammalian origin are especially preferred. Monoclonal antibodies of mammalian origin include those produced by a hybridoma and those produced by a host that was transformed with an expression vector containing genes of engineered antibodies. The antibodies, by binding to gp130, inhibit the binding of the IL-6 / IL-6 receptor complex to gp130, and thereby block the transduction of the biological activity of IL-6 in the cell. Examples of said antibodies include AM64 antibody (Publication of Unexamined Japanese Patent (Kokai) 3-219894), antibody 4B11 and antibody 2H4 (US5571513), antibody B-S12 and antibody B-P8 (Japanese Unexamined Patent Publication (Kokai) 8-291199). Basically, a hybridoma that produces anti-gp130 monoclonal antibodies can be created using a method as described below. Therefore, gp130 can be used as a sensitizing antigen and is immunized in the conventional method of immunization. The immune cells that were obtained in that manner are fused with known progenitor cells and then the hybridomas that produce monoclonal antibodies are screened by the conventional screening method to prepare the desired hybridoma. Specifically, the monoclonal antibody can be obtained in the following manner. For example, gp130 is used as the sensitizing antigen for the generation of antibodies can be obtained using the sequence of the gp130 gene / amino acid sequence disclosed in the European Patent Application EP 411946. After a host cell is transformed by inserting the gp130 gene sequence into a known expression vector system, the gp130 protein of interest is purified from the host cell or from the culture supernatant thereof. The purified gp130 receptor protein can be used as the sensitizing antigen. Alternatively, a fusion protein of the gp130 protein and another protein can be used as the sensitizing antigen. Although the mammals to be immunized with the sensitizing antigen are not specifically limited, they are preferably selected considering their compatibility with the progenitor cell for use in cell fusion. In general they include rodents such as mice, rats, hamsters and the like. Immunization of animals with a sensitizing antigen is carried out using a known method. A general method, for example, comprises an intraperitoneal or subcutaneous administration of a mammalian sensitizing antigen. Specifically, a sensitizing antigen that is diluted and suspended in an appropriate amount of Ph-regulated phosphate salt (PBS) or physiological saline, etc., is mixed as desired with an appropriate amount of a common adjuvant, for example Freund's complete adjuvant.

After the emulsion, it is preferably administered to a mammal several times every 4 to 21 days. Alternatively, a suitable vehicle can be used at the time of immunization of the sensitizing antigen. After immunization and confirmation of the increase in the desired antibody levels in the serum, the immune cells are removed from the mammal and subjected to cell fusion. Preferred immune cells that will undergo cell fusion include, in particular, spleen cells. The cells of the myeloma mammal, like the other progenitor cells that fuse with the immune cells mentioned above, preferably include several 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 (Kohier, 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 (from St. Groth, SF et al., J. Immunol. Methods (1980) ; 1-21), S194 (Trowbridge, I.S., J. Exp. Med. (1978) 148; 313-323), R210 (Galfre, G. et al., Nature (1979) 277; 131-133) and the like. The cell fusion between the above immune cells and the myeloma cells can be carried out essentially in accordance with a known method as described in Miistein et al. (Kohier, G. and Milstein, C, Methods Enzymol. (1981) 73; 3-46) and the like. More specifically, the above cell fusion is carried out in a nutrient broth in the presence of, for example, a cell fusion accelerator. Like the cell fusion accelerator, polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used and, in addition, an adjuvant such as dimethyl sulfoxide, etc., can be added as desired to improve the efficiency of the fusion. The preferred ratio of immune cells and myeloma cells to be used is, for example, 1 to 10 times more immune cells than myeloma cells. Examples of the culture medium to be used for the above cell fusion include RPMI1640 medium and MEM culture medium suitable for the growth of the previous myeloma cell lines and the conventional culture medium used for this type of cell culture, and can also be used add a serum supplement such as fetal bovine serum (FCS). In cell fusion, the predetermined amounts of the above immune cells and the myeloma cells are mixed well in the above culture, to which is added PEG solution previously heated to about 37 ° C, for example, a solution of PEG with a molecular weight of the average of about 1000 to 6000, at a concentration of 30 to 60% (w / v) and mixed to obtain the desired fusion cells (hybridoma). Then, by repeating the addition in sequences of a suitable culture liquid and centrifugation to remove the supernatant, the cell fusion agents etc. can be removed. which are not desirable for the growth of the hybridoma. Said hybridoma is selected by means of culture in the conventional selection medium, for example, the HAT culture medium (a culture liquid containing hypoxanthine, aminopterin and thymidine). The culture in said HAT culture medium is continued, in general, for a period of time sufficient to kill the cells instead of the desired hybridoma (non-fusion cells), in general, for several days to several weeks. The limiting method of dilution is carried out in which the hybridomas that produce the desired antibody are screened and cloned monoclonally. In addition to obtaining the above hybridoma by means of immunizing an animal instead of a human with an antigen, it is also possible to sensitize human lymphocytes in vitro with a desired antigen or cells expressing desired antigens., and the resulting sensitized B lymphocytes are fused with human myeloma cells, e.g., U266, to obtain the desired human antibody possessing the activity of binding the desired antigen or desired antigen expressing cells (see Japanese Patent Publication subsequently examined (Kokoku) No. 1-59878). In addition, a transgenic animal that possesses a repertoire of all human antibody genes can be immunized with the antigen or cells expressing antigens to obtain the desired human antibody in the method described above (see International Patent Publication WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735). The monoclonal hybridoma that produces antibodies that are constructed in this way can be subcultured in the conventional culture liquid, or can be stored for a prolonged period in liquid nitrogen. To obtain monoclonal antibodies to said hybridoma, a method can be used in which said hybridoma is cultured in the conventional method and antibodies are obtained as the supernatant, or a method in which the hybridoma is administered and cultured in a compatible mammal. with said hybridoma and the antibodies are obtained as the ascites. The above method is suitable for obtaining high purity antibodies, while the latter is suitable for a large scale production of antibodies. For example, a hybridoma that produces antibodies to the anti-IL-6 receptor can be prepared using the method disclosed in Japanese Unexamined Patent Publication (Kokai) 3-139293. It can be carried out by a method in which the hybridoma antibody that produces PM-1 that was deposited intemationally according to the provisions of the Budapest Treaty as FERM BP-2998 on July 12, 1989 with the Patent Microorganism Depository of the National Institute of Industrial Science and Technology, Chuo 6, 1-1, Higashi 1- chome, Tsukuba city, Ibaraki pref., Japan, is injected intraperitoneally with BALB / c mice to obtain the ascites from which the antibody is purified. PM-1, or a method in which said hybridoma is cultured in a suitable culture medium such as RPMI1640 medium containing 10% fetal bovine serum and 5% MB-Condimed Hi (manufactured by Boehringer Mannheim), the medium of SFM of the hybridoma (manufactured by GIBCO-BRL), the medium of PFHM-II (manufactured by GIBCO-BRL) and the like, and the PM-1 antibody can be purified from the supernatant. In the present invention, a recombinant antibody that was produced by recombinant gene technology in which an antibody gene was cloned from the hybridoma and integrated into a suitable vector which was then introduced into a host (see, for example, , Borrebaeck CAK, and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD., 1990). Specifically, the mRNA encoding the variable region (V) of the desired antibody is isolated from the cells that produce antibodies such as a hybridoma. The isolation of mRNA is carried out by means of preparing total RNA using, for example, a known method such as the ultracentrifugal guanidine method (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), the method of AGPC (Chomczynski, P. et al., Anal, Biochem. (1987) 162, 156-159), and then the mRNA of the total RNA is purified using the Purification of mRNA (manufactured by Pharmacia) and the like. Alternatively, the mRNA can be prepared directly using the Quick Prep mRNA Purification kit (manufactured by Pharmacia). The cDNA of the V region of the antibody can be synthesized from the mRNA that was obtained in that way using a reverse transcriptase. The cDNA can be synthesized using the AMV Transcriptase Reverse Synthesis Kit of first strand cDNA and the like. Alternatively, for the synthesis and amplification of the cDNA, the 5'-Ampli FINDER RACE equipment can be used. (manufactured by Clontech) and the 5'-RACE method (Frohman, M.A. et al., Proc. Nati.

Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al, Nucleic Acids Res. (1989) 17, 2919-2932) employing the polymerase chain reaction (PCR). The desired DNA fragment is purified from the obtained PCR product and can be ligated to the vector DNA. In addition, the recombinant vector is prepared from them and then introduced into E. coli, etc., from which colonies are selected to prepare the desired recombinant vector. The base sequence of The desired DNA can be confirmed by a known method such as the dideoxy method. Once the DNA encoding the V region of the desired antibody is obtained, it can be ligated to the DNA encoding the constant region (C region) of the desired antibody, which is then integrated into an expression vector. Alternatively, the DNA encoding the V region of the antibody can be integrated into an expression vector containing DNA encoding the C region of the antibody. To produce the antibody for use in the present invention, the antibody gene is integrated as described below into an expression vector so that it is expressed under the control of the expression regulatory region, eg, an enhancer and / or a promoter. Subsequently, the expression vector can be converted into a host cell and then the antibody can be expressed therein. In accordance with the present invention, the artificially altered recombinant antibody such as chimeric antibody, humanized antibody and human antibody can be used in order to reduce the heterologous antigenicity against humans. These altered antibodies can be produced using known methods. The chimeric antibody can be obtained by ligating the DNA obtained thereby coding for the V region of the antibody to DNA encoding the C region of the human antibody, which is then integrated into an expression vector and introduced into a host for the production of the antibody therein (see European Patent Application EP 125023, and Patent Publication ; 10 International WO 92-19759). Using this known method, you can get the -: chimeric antibody useful for the present invention. - For example, a plasmid containing DNA encoding the L chain V region or the H chain V region of the chimeric antibody PM-1 was designated:; as pPM-k3 or pPM-h1, respectively, and E. coli that possesses the plasmid has been deposited internationally according to the provisions of the Budapest Treaty as NCIMB 40366 and NCIMB 40362, respectively, on February 12, 1991 with National Collections of Industrial and Marine Bacteria Limited (23 St Machar Drive, Aberdeen, Scotland, AB2 1 RY, United Kingdom and Northern Ireland). The humanized antibody which is also referred to as the human antibody reformed was prepared by transplanting the region that determines the - complementarity (CDR) of the antibody of a non-human mammal, for example the mouse antibody, in the CDR of the human antibody. The - General recombinant technology for the preparation of said antibodies is also known (see European Patent Application EP 125023 and Publication - 25 of International Patent WO 92-19759). Specifically, a DNA sequence that was designated to bind the CDR of the mouse antibody with frame region (FR) of the human antibody is synthesized from various split oligonucleotides having sections superimposed on each other at the ends thereof. The DNA thus obtained is ligated to the DNA encoding the C region of the human antibody and then integrated into an expression vector, which is introduced into a host for the production of antibodies (see European Patent Application EP 239400 and International Patent Publication WO 92-19759). For the FR of the human body bound through the CDR, the region that determines the complementarity that forms a favorable site of antigen binding is selected. When desired, the amino acids in the framework region of the variable region of the antibody can be substituted so that the region determining complementarity of the human reformed antibody can form an appropriate antigen binding site (Sato, K. et al. , Cancer Res. (1993) 53, 851-856). For example, for a chimeric antibody or a humanized antibody, the C region of the human antibody is used. As the C region of the human antibody, one can for example mention Cy and C? 1, C? 2, C? 3 and C? 9. The C region of the human antibody can be modified to improve the stability of the antibody or the production thereof. The chimeric antibody consists of the variable region of the antibody that is derived from a non-human mammal, and the C region that is derived from the human antibody, while the humanized antibody consists of the region that determines the complementarity of the antibody derived from a mammal which is not a human and the framework region and the C region of the antibody that is derived from a human antibody. Accordingly, the antigenicity thereof in the human body was reduced so that they are useful as antibodies for use in the present invention. As a preferred embodiment of the humanized antibody for use in the present invention, the humanized antibody of PM-1 can be mentioned (see International Patent Publication WO 92-19759). Furthermore, as a method for obtaining human antibodies, a technology is known that employs panning with a human antibody library, in addition to those described above, for example, the variable region of the human antibody is expressed on the surface of a phage by the phage sample method as a single chain antibody (scFv) to select a phage that binds to the antigen. By analyzing the selected phage gene, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the scFv DNA sequence that binds to the antigen is clarified, it is possible to prepare an appropriate expression vector containing said sequence and then obtain a human antibody. These methods are already known and can be found in WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388. The genes of the antibodies as described above can be expressed and obtained in a known method. In the case of mammalian cells, the expression can be achieved by using a vector containing a useful promoter commonly used, the gene of the antibody to be expressed, the DNA in which the polypeptide A has been operatively linked at the 3-position downstream thereof or a vector containing said DNA. Examples of the promoter / enhancer include immediate early promoters / enhancers of the human cytomegalovirus. Additionally, as the promoter / enhancer that can be used for the expression of the antibody for use in the present invention, viral promoters / enhancers such as retroviruses, polyoma viruses, adenoviruses and simian viruses (SV40), and promoters can be used. / breeders derived from mammalian cells such as factor 1 to human extension (HEF1).

For example, the expression can be easily achieved by the method of Mulligan et al. (Mulligan, R. O et al., Nature (1979) 277, 108-114) when the SV40 enhancer promoter is used, or by the method of Mizushima et al.

(Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18, 5322) when using the HEF1a promoter / enhancer. In the case of E. coli, expression can be carried out by operatively joining a commonly used useful promoter, a signal sequence for the secretion of antibodies or the gene of the antibody to be expressed, followed by the expression thereof. As the promoter, for example, the promoter 1 acZ and the promoter araB can be mentioned. The method of Ward et al. (Ward, E.S. et al., Nature (1098) 341, 544-546; Ward, E.S. et al., FASEB J. (1992) 6, 2422-2427) can be used when using the acZ promoter 1 and the method of Better et al. (Better, M. et al., Science (1988) 240, 1041-1043) can be used when using the araB promoter. As the signaling sequence for secretion of the antibody, when produced in the periplasm of E. coli, the signaling sequence pe1B can be used (Lei, SP et al., J. Bacteriol. (1987) 169, 4379-4383 ). After separating the antibody produced in the periplasm, the structure of the antibody is appropriately folded before use (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. In addition, to extend the number of genes in the host cell system, expression vectors can include, as selectable markers, the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the gene for xanthine guanine phosphorylboyl transferase from E. coli (Ecogpt), the dihydrofolate reductase (dhfr) gene and the like. For the production of the antibody for use in the present invention, any production system can be used. The production system for the preparation of the antibody comprises the in vivo production system or the in vitro production system. As the in vitro production system, we can mention a production system that uses eukaryotic cells and the production system that uses prokaryotic cells. When eukaryotic cells are used, there are production systems that employ animal cells, plant cells or fungal cells. Known animal cells include (1) mammalian cells such as CHO cells, COS cells, myeloma cells, hamster calf kidney cells (BHK), HeLa cells and Vero cells, (2) amphibian cells such as oocytes. of Xenopus or (3) insect cells such as sf9, sf21, and Tn5. Known plant cells include, for example, those derived from nicotine tobacco, which can be subjected to callus culture. Known fungal cells include yeasts such as those of the genus Saccharomyces, more specifically Saccharomyces cereviceae, or filamentous fungi such as the genus Aspergillus, more specifically Aspergillus niger. When prokaryotic cells are used, there are production systems that use bacteria cells. Bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis. By introducing by means of the transformation of the desired antibody gene into these cells and by culturing the cells transformed in vitro, the antibody can be obtained. The cultivation is carried out according to known methods. For example, DMEM, MEM, RPM11640 and IMDM can be used as the culture liquid, and serum supplements such as fetal bovine serum (FCS) can be used in combination. In addition, the antibodies can be produced in vivo by means of implanting cells in which the antibody gene was introduced into the abdominal cavity of an animal or the like. As in vivo production systems, those that employ animals and those that use vegetables can be mentioned. When animals are used, are the production systems that use mammals and 5 insects. As mammals, goats, pigs, sheep, mice and cows can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Also as an insect, silkworms can be used. When vegetables are used, tobacco, for example, can be used. 1 ° The genes of the antibodies are introduced into these animals or plants and the antibodies are produced in said animals or plants and recovered. For example, an antibody gene is inserted into the medium of a protein encoding genes that is inherently produced in milk such as goat casein to prepare fusion genes. The DNA fragments that contain the gene The fusion in which the antibody gene was 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 that comes from the goat that received the embryo or its offspring. To increase the amount of milk that contains the desired antibody produced by the transgenic goat, it can be give hormones to the transgenic goat as appropriate. (Ebert, K.M. et al., Bio / Technology (1994) 12, 699-702). When silkworms are used, baculovirus is infected in which the desired antibody gene is inserted into the silkworm, and the desired antibody can be obtained from the body fluid of the silkworm (Maeda, S. et al., Nature I "25 (1985) 315, 592-594.) In addition, when tobacco is used, the desired antibody gene is inserted into an expression vector for plants, for example, pMON 530, and then the vector is introduced into a bacterium. such as Agrobacterium tumefaciens, then the bacterium is infected to tobacco such as Nicotiana tabacum to obtain the desired antibody from the tobacco leaves (Julián, K.-C. Ma et al., Eur. J. Immunol. (1994 ) 24, 131-138) When the antibody is produced in vitro or in vitro production systems alive, as described above, the DNA encoding the heavy chain • (H chain) or the light chain (L) of the antibody, can be integrated separately into an expression vector and the hosts are transformed simultaneously, or the DNA encoding the H chain and the L chain can be integrated into a vector of unique expression, and the host transforms with the , (see International Patent Publication WO 94-11523). Antibodies for use in the present invention can be antibody fragments or modified versions thereof, provided they are used 1, preferably. For example, as antibody fragments, they can be : to mention Fab, F (ab ') 2, Fv or Fv (scFc) of simple chain in whose Fv of chain H - 15 and L chain were linked by an appropriate link. _-_ Specifically, the antibodies were treated with an enzyme, for example, papain or pepsin, to produce antibody fragments or I. they build genes that encode these antibody fragments and then enter an expression vector that is expressed in a host cell (see, for example, Co, M.S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A.H., Methods, Enzymology (1989) 178, 476-496; Plueckthun, A. and Skerra, A., Methods in Enzymology (1989) 178, 476-496; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-66; Bird, R.E. et al., TIBTECH (1991) 9, 132-137). 25 scFv can be obtained by ligating the V chain region H and the. V region of L chain of the antibody. In scFv, the V region of H chain and the region V of L chain are preferably linked by a link, preferably a peptide link (Huston, J.S. et al., Proc. Nati, Acad. Sci. USA (1988) 85, 5879-5883). The H chain V region and the L chain V region in scFv can be derived from any of the aforementioned antibodies. As the peptide linkage for ligating the V regions, any single chain peptide comprising, for example, 12-19 amino acid residues can be used. The DNA encoding the scFv can be obtained using DNA encoding the H chain or the H chain V region of the above antibody and the DNA encoding the L chain or the L chain V region of the above antibody as the model by means of extend the part of DNA that encodes the desired amino acid sequence between the previous sequences by means of the PCR technique with the pair of lighters that specifies both ends thereof, and also by means of extending the combination of the DNA encoding the part of the link of the peptide and the pair of lighters that defines that the two ends of said DNA are linked to the H chain and the L chain, respectively. Once scFv-encoding DNAs were constructed, an expression vector containing them and a host transformed with said expression vector can be obtained by conventional methods and scFv can be obtained using the resulting host by conventional methods. These antibody fragments can be produced by obtaining the gene thereof in a manner similar to that mentioned above and by allowing it to be expressed in a host. "Antibody" as used herein also comprises these antibody fragments. As modified antibodies, antibodies associated with various molecules such as polyethylene glycol (PEG) can be used. "Antibody", as used herein, comprises these modified antibodies. These modified antibodies can be obtained by chemically modifying the antibodies that were obtained in that way. These methods have already been established in the art. The antibodies produced and expressed as described above can be separated from the interior or exterior of the host cell and then can be purified until homogeneity is achieved. The separation and purification of the antibody 5 for use in the present invention can be achieved by affinity chromatography. As the column used for said affinity chromatography, you can use the Protein A column and the Protein G column. Examples of the vehicles used in the Protein A column are Hyper D, POROS, 1. Sepharose F. F. and the like. Alternatively, they can be used without limitation methods for separation and purification conventionally used for - proteins. - The separation and purification of antibodies for use in the present invention can be achieved by combining, as appropriate, I chromatography, other than affinity chromatography mentioned above, filtration, ufiltration, salt precipitation, dialysis and the like. Chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration and the like. These chromatographies can be applied in high performance liquid chromatography (HPLC). Alternatively, reverse phase HPLC can be used. 20 The concentration of the antibody obtained in the previous one can be determined by measuring the absorbance or by the test ZT is an enzyme-linked immunosorbent (ELISA) and the like. Thus, - •• When the absorbance measurement is used, a sample is diluted in a 7 appropriate with PBS (-) and then the absorbance at 280 nm is measured, followed by the i; 25 calculation using the absorption coefficient of 1.35 OD at 1 mg / ml. When the ELISA method is used, the measurement is carried out in the following manner. Therefore, 100 μl of goat antihuman IgG (manufactured by TAG) diluted at 14g / ml in 0.1 M bicarbonate buffer, pH 9.6, is added to a 96-well plate (manufactured by Nunc), and incubate until the next day at 4 ° C to immobilize the antibody. After blocking, add 100 μl of each of the appropriately diluted antibodies of the present invention or a sample containing the antibody, or 100 μl of human IgG (manufactured by CAPPEL) as the standard, and incubate at room temperature environment for 1 hour. After washing, 100 μl of alkaline phosphatase-labeled anti-human IgG antibody diluted 5000 fold (manufactured by BIO SOURCE) is added and incubated at room temperature for 1 hour. After washing, the substrate solution is added and incubated, followed by measurement of the absorbance at 905 nm using the MICROPLATE READER model 3550 (manufactured by Bio-Rad) to calculate the concentration of the desired antibody. The altered IL-6 for use in the present invention possesses an IL-6 receptor binding activity and does not transmit the biological activity of IL-6. Therefore, altered IL-6, although competing with IL-6 for binding to the IL-6 receptor, does not transmit the biological activity of IL-6 and thereby blocks signal transduction through IL-6. 6 The altered IL-6 can be prepared through the introduction of mutation by the replacement of amino acid residues of the amino acid sequence of IL-6. IL-6, the source of altered IL-6, can be of any origin, but when antigenicity is considered, it is preferably human IL-6. Specifically, the secondary structure of IL-6 is predicted using a molecular modeling program of the amino acid sequence, for example, WHATIF (Vriend et al., J. Mel. Graphics (1990), 0, 52-56), and the total effects on the amino acid residue to be replaced are evaluated. After determining an appropriate residue, the mutation is introduced to effect replacement of the amino acid by means of the commonly used polymerase chain reaction (PCB) method using a vector containing the base sequence encoding the human IL-6 gene as a model by it to obtain a gene that encodes an altered IL-6. Then, this is integrated, as desired, into an appropriate expression vector from which the altered IL-6 can be obtained. - agreement with the methods of expression, production and purification of said zz recombinant antibody. "Specific examples of altered IL-6 are disclosed in Brakenhoff et al., J.

"'Biol. Chem. (1994) 269, 86-93, and Savino et al., EMBO J. (1994) 13, 1357-1367, ~; 10 WO 96-18648, and WO 96-17869. The partial peptide of IL-6 or the partial peptide of the IL-6 receptor for use in the present invention possesses a binding activity to the IL-6 or IL-6 receptor, respectively and does not transmit the biological activity of IL-6. . Therefore, the partial peptide of IL-6 or the partial peptide of the IL-6 receptor specifically inhibits the binding of IL-6 to the L-6 receptor by binding to the IL-6 or IL-6 receptor, respectively, and in that way capturing it. As -_ result, these do not transmit the biological activity of IL-6, and therefore - block the signal transduction of IL-6. rz The partial peptide of IL-6 or the partial peptide of the IL-6 receptor is a - • 20 peptide comprising some or all of the amino acid sequences of the region involved in binding to IL-6 and the IL-6 receptor in the 7 amino acid sequence of IL-6 or the IL-6 receptor. Said peptide generally comprises -. 10-60, preferably 20-50, more preferably 20-40 amino acid residues. The partial peptide of IL-6 or the partial peptide of the IL-6 receptor can be prepared by specifying the region involved in the binding to IL-6 and the IL-6 receptor in the amino acid sequence of IL-6. 6 or of the IL-6 receptor and by means of producing some or all of the sequences by a conventional method such as genetic engineering technology or a peptide synthesis method. To prepare the partial peptide of IL-6 or the partial peptide of the IL-6 receptor by a genetic engineering technology, the DNA sequence encoding the desired peptide is integrated into an expression vector, from which the peptide can be obtained by methods of expression, production and purification of said recombinant antibody. The preparation of the partial peptide of IL-6 or the partial peptide of the L-6 receptor by the method of peptide synthesis can be carried out using a method commonly employed in the synthesis of peptides such as solid phase synthesis or the synthesis of liquid phase. Specifically, the method described in Zokulyakuhin no Kaihatsu (Sequei to Development of Pharmaceuticals), Vol. 14, Peutido Gousei (Peptide Synthesis), edited by Haruaki Yajima, Hirokawa can be used.

Shoten, 1991. The solid phase synthesis method employed includes, for example, a reaction in which an amino acid corresponding to the C-terminus of the peptide to be synthesized is coupled to a support that is soluble in organic solvents and then amino acid. in which an a-amino group or a side chain functional group is protected with an appropriate protecting group an amino acid is condensed at a time from the C-terminal direction to the N-terminal direction and a reaction in which said protective group of the Amino amino acid group or peptide coupled to the resin is removed are repeated alternately to prolong the chain of peptides. The solid phase peptide synthesis methods are divided into the Boc method and the Fmoc method depending on the type of protective group to be used. After the synthesis of the desired peptide is complete, a deprotection reaction and a reaction to cleave the protein chain are carried out. - support peptide. For the cleavage of the peptide chain, in general, ZZ "employs hydrogen fluoride or trifluoromethanesulfonic acid in the Boc method - and TRFA in the Fmoc method. In the Boc method, for example the resin of The above peptide is treated in hydrogen fluoride in the presence of anisole. z: Subsequently, the protective group is removed and the peptide is recovered by means of the "-" cleavage of the support, by lyophilization of this, crude peptide can be obtained, on the other hand, in the Fmoc method, the deprotection reaction and the cleavage reaction of the support peptide can be performed in TFA, for example, in a procedure similar to the previous one.The crude peptide obtained in this manner can be applied to HPLC for separation and purification. carried out in a water-acetonitrile solvent system that is commonly used for ZZ protein purification under an optimal condition. The lyophilization was collected and lyophilized - 15 fraction corresponding to the peak of the chromatography profile obtained. The fraction of the peptide that was purified in this manner is identified by means of subjecting it to the analysis of the molecule weight by means of analysis of mass z: spectroscopic, the analysis of the amino acid composition or the analysis of the sequence of amino acids and the like. Specific examples of the partial peptide of 1L-6 or of the partial peptide of the IL-6 receptor are disclosed in Japanese Unexamined Patent Publication (Kokai) 2-188600, Japanese Unexamined Patent Publication (Kokai) 7-324097, Japanese Unexamined Patent Publication (Kokai) B-311098, and US Patent Publication US 5210075. - 25 IL-6 Antagonist Activity for Use in the Present invention of Z7 block signal transduction of IL-6 can be evaluated using a known method. Specifically, the human myeloma cell line dependent on IL-6 (56545, XPMM2), human lymphoma cell line of Lennert's KT3, or the IL-6 dependent cell MH60.BSF2 is grown, to which IL- is added. 6 and the activity can be assessed using the incorporation of 3H-thymidine in the IL-6 dependent cell in the coexistence of the IL-6 antagonist. Alternatively, 0266 can be cultured, a cell expressing the IL-6 receptor to which 125 I-labeled IL-6 is added and an IL-6 antagonist is added at the same time, and then IL-6 is determined. labeled with 125 μl bound to the cell expressing the IL-6 receptor. In the above assay system, a negative control group that does not contain IL-6 antagonists, furthermore, is prepared from the group in which an L-6 receptor antagonist is present, and the results obtained for they are compared to evaluate the inhibition activity of IL-6 of the IL-6 receptor antagonist. As described in the following Example, the anti-IL-6 receptor antibody showed a therapeutic effect of vasculitis, suggesting that IL-6 antagonists such as IL-6 receptor antibodies are effective as therapeutic agents for vasculitis. The subject to be treated in the present invention is a mammal. The mammalian subject to be treated is, preferably, a human being. The preventive or therapeutic agents of the present invention can be administered, either orally or parenterally, systemically or locally. For example, intravenous injection such as drip infusion, intramuscular injection, intrapereal injection, subcutaneous injection, supposy, intestinal lavage, enteric oral coated tablets, and the like may be selected, and the method of administration may be chosen, as appropriate, depending of the age and the conditions of the patient. The effective dosage is chosen from the range of 0.01 to 100 mg per kg of body weight per administration. Alternatively, the dosage can be chosen in the range of 1 to 20, preferably 2 to 6 mg per patient. Preferred dosages and preferred methods of administration are such that, in the case of the anti-IL-6 receptor antibody, the amounts in which the free antibody is present in the blood in effective dosages. In specific examples, 1 mg to 20 mg per kg of body weight, preferably 2 mg to 8 mg, per month (4 weeks) is administered in one to several doses, for example, twice a week, once a week. , once every two weeks, once every four weeks and the like. The preventive or therapeutic agents for vasculitis of the present invention may contain acceptable carriers for pharmaceutical use or additives depending on the route of administration. Examples of such vehicles or additives include water, an organic solvent acceptable for pharmaceutical use, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (used), mann, sorb, lactose, or surfactants acceptable for pharmaceutical use and the like. The additives used are chosen, but not limited to, the above or combinations thereof depending on the dosage form. EXAMPLES The present invention will be explained below in greater detail with reference to the operative examples and the reference examples. Nevertheless, it should be noted that the present invention is not limited by them in any way. Operative Example 1 Method: Patients suffering from the incurable vasculitis syndrome (polyarteritis nodosa, aortitis syndrome) refractory to conventional therapy were treated with humanized anti-IL-6 receptor antibody. With the authorization of the Advanced Medical Treatment Committee of Osaka University Hospital, two patients received the humanized antibody to PM-1 (MRA) which is a humanized antibody to the anti-IL-6 receptor. The final point was the improvement in the evaluation of images by means of magnetic resonance imaging (MRI) and computed tomography (CT), improvement of the conditions of the skin, improvement in inflammatory markers such as C-reactive protein (CRP), and improvement in QOL (pain, arthralgia, discomfort). Likewise, peripheral blood cell counts, general biochemistry, homeostatic function, IL-6, soluble IL-6 receptor, humanized antibody concentration of anti-IL-6 receptor in the blood, necrosis factor were evaluated. of tumor a (TNFa, interleukin-1b (IL-1b) and vascular epidermal growth factor (VEGF) Result: Case 1: A 19-year-old woman diagnosed as suffering from the aortitis syndrome in 1996 With a complication of ulcerative colitis, prednisolone (PSL) 60 mg / day was started.Not even the combined use of ciclosporin could reduce PSL to 20 mg / day or less.In 1998, in response to aggravation, 150 mg was used Cyclophosphamide day in addition to methyl prednisolone pulse therapy (mPSL), but could not be reduced to 30 mg / day or less, 1 mg / day of betamethasone was added and then leukophoresis was performed seven times, but without In 2000, and after, mPSL d pulse therapy was used. intermittently and 100 mg / day of azathioprine, 2 g / day of mycophenolate mofetil and 17.5 mg / week of methotrexate were used in combination, but without effect. As shown in Figs. 1 to 6, CT revealed prominent hypertrophy of the wall of blood vessels in the ascending aorta, trifurcation of the aortic arch and descending aorta. Stenosis was observed in the subclavian artery (1t.

SCA). Severe inflammation was observed with CRP 12.6 mg / dl, and due to severe persistent chest pain, there was a reduction in body weight of 5 kg per month, and attacks of loss of consciousness, drip infusion was used of 200 mg / week of MRA. About two weeks later, CRP became negative. One month later, the chest pain improved, and as seen in Fig. 1-6, improvements were observed in the hypertrophy of the blood vessel wall and in the ascending aorta, trifurcation of the aortic arch and the descending aorta and the lengthening of the lumen of the blood vessels, and also improved blood flow in the carotid artery. Likewise, the fecal hemoglobin also became negative and the symptoms of ulcerative colitis disappeared. During MRA therapy, blood levels of TNFa were elevated, but without any aggravation of symptoms. The improvement in the hypertrophy of the aortic wall and the lengthening of the lumen of the blood vessels was maintained even in two years after the MRA therapy. Takayasu's arteritis is also referred to as the pulseless disease, and in this patient the pulse could not be felt, but after treatment, the pulsation of the radius and ulna arteries was felt in the wrist. Due to the prolonged use of MRA blood levels of L-6 decreased from 1720 mg / ml to 100 pg / ml. Case 2: A 42-year-old man. In 1986, he developed knotty arteritis (skin type) and despite treatments with PSL, azathiopurine, colchicine and anticoagulants, repeated remission and aggravation. In 1995 and after, cyclophosphamide was used in combination with mPSL pulse therapy, but without effect. Beginning in 1997, bolus administration of azathiopurine, cyclophosphamide and d-globulin began, and since 2000, cyclophosphamide pulse therapy and leukophoresis were initiated, but without effect. The skin graft was performed on the ulcer of the skin due to vasculitis, but without effect, and due to the aggravation, the removal of the right fibula and the amputation of B-K were performed. The necrotic vasculitis became more aggravated and the ulcer spread in the lower extremity. After starting the treatment with 200 mg / week of a humanized anti-IL-6 receptor antibody, the fever and erythema of the skin and the muscular pains that were previously observed improved. Although removal of the right thigh could not be avoided, the leukocyte counts were normalized with the reduction of IL-6 in the blood and a worsening in the skin ulcer was not subsequently observed. Discussion: Because MAR was effective for patients with incurable vasculitis that could not be controlled by conventional treatments, it was suggested that the IL-6 inhibition treatment could provide a novel method of treatment for vasculitis. This means that IL-6 is essential to determine the pathology of vasculitis. Also, as the reduction per se of IL-6 was observed in all cases, this demonstrated that the inhibition treatment of IL-6 not only has an anti-inflammatory effect but also acts on the essential nature of vasculitis. Reference Example 1. Preparation of the soluble human receptor of IL-6 The soluble receptor of IL-6 was prepared by the PCR method using a plasmid pBSF2R.236 containing cDNA encoding the IL-6 receptor that was obtained according to with the method of Yamasaki et al., (Yamasaki, K. et al., Science (1988) 241, 825-828). The plasmid pBSF2R.236 was digested with a restriction enzyme Sph I to obtain the I L-6 receptor cDNA, which was then inserted into mp18 (manufactured by Amersham). Using a synthetic oligoenceptor designed to introduce a stop codon into the cDNA of the IL-6 receptor, a mutation was introduced into the cDNA of the IL-6 receptor by means of the PGA method using the in vitro Mutagenesis System (manufactured by Amersham). The procedure resulted in the introduction of a stop codon to the amino acid at position 345 and gave cDNA encoding the soluble receptor of IL-6. To express the cDNA of the soluble receptor of IL-6 in CHO cells, it was ligated to the pSV plasmid (manufactured by Pharmacia) to obtain plasmid pSVL344.

The IL-6 soluble receptor cDNA that was cleaved with Hind ill-Sal I was inserted into the plasmid pECEdhfr344 containing the dhfr cDNA to obtain the plasmid pECEdhfr344 which can be expressed in the CHO cells. 10 g of the pECEdhfr344 plasmid was transfected into the dhfr-CHO DXB-11 cell line (Orlaub G. et al., Proc. Nati, Acad. Sci. USA (1980) 77, 4216-4220) by the precipitation method of Calcium phosphate (Chen C. et al., Mal. Cell, Biol. (4987) 7, 2745-27511.Changed CHO cells were cultured for 3 weeks in a MEM-free selection medium of nucleosides containing 1 mM of glutamine, 10% of dialyzed FCS, 100 U / ml of penicillin and 100 μg / ml of streptomycin The selected CHO cells were screened by the limiting dilution method to obtain a simple CHO cell clone. of CHO was expanded by 20 nM - 200 nM methotrexate (MTX) to obtain a CHO 5E27 cell line that produces soluble human L-6 receptor The CHO 5E27 cell line was cultured in Dulbecco's Iscov modified medium (IMDM, manufactured by Gibco) that contains 5% FBS. The culture supernatant was collected and the concentration of soluble IL-6 receptor in the culture supernatant was determined by ELISA. The result confirmed that the soluble receptor of L-6 is present in the culture supernatant. Reference Example 2. Preparation of the human IL-6 antibody 10 g of the recombinant IL-6 were immunized (Hirano et al., Immunol. Lett., (1988) 17, 41) for BALB / c mice together with Freund's complete adjuvant, and this was repeated every week until the anti-IL-6 antibody could be detected in the serum. Immune cells were extracted from the local lymph node and then fused with a myeloma P301 cell line using polyethylene glycol 1500. Hybridomas were selected according to the method of Oi et al. (Selective Methods: Cellular Immunology, W.H. Freeman and Co., San Francisco, 351, 1980) using the HAT medium and the hybridoma which produces anti-human IL-6 antibody was established. The hybridoma producing the anti-human IL-6 antibody was subjected to the L-6 binding assay in the following manner. Therefore, a 96 microliter well plate made of flexible polyvinyl (manufactured by Dynatech Laboratories, Inc., Alexandria, VA) was coated with 100 μl goat anti-mouse Ig (10 μl / ml, manufactured by Cooper Biomedical , Inc., Malvern, PA) until the next day at 4 ° C. Subsequently, the plate was treated with 100 μl of PBS containing 1% bovine serum albumin (BSA) at room temperature for 2 hours. After washing it in PBS, 100 μl of the culture supernatant of the hybridoma was added to each well and then incubated overnight at 4 ° C. The plate was washed, 125I labeled recombinant IL-6 was added to each well at a concentration of 2000 cpm / 0.5 ng / well and then the radioactivity of each well was determined after each wash by a gamma counter (Beckman Gamma 9000 , Beckman Instruments, Fullerton, CA). Of 216 clones of the hybridoma, 32 were positive in the IL-6 binding assay. From these clones, stable MH166.BSF2 was finally obtained. The anti-IL-6 antibody MH166 produced by said hybridoma possesses a subtype of IgG1 K. Then, the clone MH60.BSF2 of IL-6-dependent mouse hybridoma was used to examine a neutralizing activity with respect to the growth of the hybridoma by the MH166 antibody. MH60.BSF cells were distributed at 1 x 104/200 μl / well and samples containing the MH166 antibody were added thereto, cultured for 48 hours, 0.5 μCi / well 3H-thymidine was added (New England Nuclear , Boston, MA) and the culture was continued for another 6 hours. The cells were placed on a glass filter paper and treated by the automatic harvester (Lebo Mash Science Co., Tokyo, Japan). As the control, rabbit anti-IL-6 antibody was used. As a result, the MH166 antibody inhibited, in a dose-dependent manner, the incorporation of 3H-thymidine from MH60.BSF2 cells induced by IL-6. This revealed that the MH166 antibody neutralizes the activity of IL-6. Reference Example 3. Preparation of the human anti-IL-6 receptor antibody The anti-IL-6 receptor MT18 prepared by the method of Hirata et al. (Hirata, Y. et al. J. Immunol., (1989) 143, 2900-2906) was linked to Sepharose 4B activated by CNBr (manufactured by Pharmacia Fine Chemicals, Piscataway, NJ) according to the adjunct regimen and the IL-6 receptor was purified (Yamasaki, K. et al., Science (1988) 241, 925-828). A human myeloma U266 cell line was solubilized with 1 mM p-para-aminophenyl methane sulfonyl fluoride hydrochloride (manufactured by Wako Chemicals) (digitonin buffer) containing 1% digitonin (manufactured by Wake Chemicals), 10 mM triethanolamine (pH 7.8) and 0.15 M NaCl, and mixed with MT18 antibody bound to Sepharose 4B beads. Then, the beads were washed six times with digitonin buffer to prepare the partially purified IL-6 receptor to be used in the immunization. BALB / c mice were immunized four times every ten days with the partially purified IL-6 receptor obtained from 3 x 103 U266 cells and then a hybridoma was prepared using a standard method. The supernatant of the hybridoma culture of the positive growth well was analyzed for its IL-6 receptor binding activity according to the method described below. The U266 5 x 107 cells were labeled with 35S-methionine (2.5 mCi) and solubilized with the previous digitopin buffer. The solubilized U266 cells were mixed with a volume of 0.04 ml of the MT18 antibody bound to beads of Sepharose 4B, and then washed six times with digitonin buffer. The labeled IL-6 receptor of 39S-methionine was eluted with 0.25 ml of the digitonin buffer (pH 3.4) and neutralized in 0.025 ml of 1 M Tris (pH 7.4). 0.05 ml of the culture supernatant of the hybridoma was mixed with 0.01 ml of Protein G Sepharose (manufactured by Pharmacia). After washing, the Sepharose was incubated with 0.005 ml of 35S-labeled IL-6 receptor solution prepared as described above, the immunoprecipitate was analyzed by SDS-PAGE to investigate the culture supernatant of the hybridoma which reacts with the IL-6 receptor. As a result, clone PM-1 of the positive reaction hybridoma (FERM BP-2996) was established. The antibody that is produced from the hybridoma PM-1 possesses a subtype of IgGI K. The inhibitory activity of IL-6 binding of the antibody produced by the hybridoma PM-1 with the human receptor of IL-6 was studied using the line U266 cellular of human myeloma. A recombinant human IL-6 was prepared from E. Coli (Hirano et al., Immunol.Lett, (1988) 17, 41-45), and labeled with 125I using the Bolton-Hunter reagent (New England Nuclear , Boston, MA) (Taga, T. et al., J. Exp. Med. (1987) 166, 967-981).

ZZ The U266 4 x 105 cells were cultured with the culture supernatant of 70% (v / v) of the hybridoma PM-1 together with 14,000 cpm of IL-6 labeled with 125 l for one hour. Seventy μl of the sample was layered on 300 μl of FCS in a 400 μl microfuge polyethylene tube. After centrifugation, determined the radioactivity of the cell. The result revealed that the antibody produced by the hybridoma PM-1 inhibits the binding of IL-6 to the IL-6 receptor. ZZ Reference Example 4. ZZZ Preparation of the anti-IL-6 receptor mouse antibody. A monoclonal antibody directed against the IL-6 receptor was prepared. - mouse according to the method described in Saito, et al., S. Immunol. (1991) 147,? 168-173. Z CHO cells that produce the soluble receptor of mouse IL-6 were cultured in the IMDM culture liquid containing 10% FCS. From the supernatant of the culture, the soluble mouse IL-6 receptor was purified using an affinity column in which the RS12 antibody of the anti-mouse IL-6 receptor (see Saito, et al., Supra) was fixed to Affigel 10 gel (manufactured by Biorad). The soluble receptor of mouse IL-6 (50 μg) which was obtained in this manner _ - was mixed with Freund's complete adjuvant, which was then injected into the Wistar rats abdomen. As of 2 weeks after administration, ZZ animals were elevated with incomplete Freund's adjuvant. On day 45, - harvested the spleen cells from the rat and about 2 × 10 8 cells of the same were fused with 1 × 107 cells of mouse myeloma P3U1 using a 50% ZT PEG1500 (manufactured by Boehringer Mannheim) according to the method , and then they were explored through the HAT culture medium. After the culture supernatant of the hybridoma was added to the plate coated with anti-rat IgG rabbit antibody (manufactured by Cappel), the soluble receptor of the IL-6 receptor was reacted. Subsequently, using mouse anti-IL-6 receptor antibody and alkaline IgG labeled with anti-rabbit sheep phosphatase, hybridomas that produce antibody directed against the soluble receptor of mouse IL-6 by ELISA were screened. After the production of the antibody was confirmed, the hybridoma clones were subexplored twice to obtain a single hybridoma clone. The clone was named MR16-1. The neutralizing activity of the antibody produced by the hybridoma on the signal transduction of mouse IL-6 was examined by the incorporation of 3H-thymidine using MH60.BSF2 cells (Matsuda, T. et al., J. limnunol. (1988) 18, 951-956). For a 96-well plate, MH60.BSF2 cells were prepared at 1 x 10 4 cells / 200 μl / well. 10 pg / ml of mouse IL-6 and MR16-1 antibody or RS12 antibody were added to the plate at 12.3-1000 nq / ml, and then cultured at 37 ° C and 5% C02 for 44 hours , and then 1 μCi / well of 3H-thymidine was added. After 4 hours, the incorporation of 3H-thymidine was measured.

As a result, the MR16-1 antibody suppressed 3H-thymidine incorporation by the MH60.BSF2 cells. Therefore, it was shown that the antibody produced by hybridoma MR16-1 (FERM BP-5875) inhibits the binding of IL-6 to the IL-6 receptor.

Claims

- CLAIMS - 5 Having thus specially described and determined the nature of the ZZ present invention and the way it has to be put into practice, it is claimed to claim as property and exclusive right. A preventive and / or therapeutic agent for vasculitis, said agent comprising an interleukin 6 (IL-6) antagonist as an active component.
2. A preventive and / or therapeutic agent for vasculitis that possesses resistance to spheroids and / or immunosuppressants, said agent comprising an interleukin 6 (IL-6) antagonist as an active component.
3. The preventive and / or therapeutic agent according to claim 1 or 2 wherein said vasculitis is polyarteritis nodosa.
4. The preventive and / or therapeutic agent according to claim 1 or 2 wherein the vasculitis is the aortitis syndrome.
5. The preventive and / or therapeutic agent according to claim 1 or 2 wherein the vasculitis is vasculitis associated with immunological abnormalities.
6. The preventive and / or therapeutic agent according to any of claims 1 to 5 wherein said IL-6 antagonist is an antibody against the IL-6 receptor.
The preventive and / or therapeutic agent according to claim 6 wherein said antibody against the L-6 receptor is a monoclonal antibody against the I L-6 receptor.
8. The preventive and / or therapeutic agent according to claim 6 wherein said antibody against the IL-6 receptor is a mono-ionic antibody against the human IL-6 receptor.
9. The preventive and / or therapeutic agent according to claim 6 wherein said antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor.
10. The preventive and / or therapeutic agent according to any of claims 6 to 9 wherein said antibody against the IL-6 receptor is a recombinant antibody.
11. The preventive and / or therapeutic agent according to claim 6 wherein said monoclonal antibody against the human IL-6 receptor is the PM-1 antibody.
12. The preventive and / or therapeutic agent according to claim 9, wherein said monoclonal antibody against the mouse IL-6 receptor is the MR16-1 antibody
13. The preventive and / or therapeutic agent according to the invention. with any of claims 6 to 12 wherein said antibody against the IL-6 receptor is a Chimeric antibody, a humanized antibody, or a human antibody against the I L-6 receptor.
14. The preventive and / or therapeutic agent according to claim 13 wherein said humanized antibody against the mouse IL-6 receptor is the humanized antibody PM-1.
15. The use of an interleukin 6 (L-6) antagonist for the manufacture of a preventive and / or therapeutic agent for vasculitis.
16. The use of an interleukin 6 (IL-6) antagonist for the manufacture of a preventive and / or therapeutic agent for vascuitis that possesses resistance to spheroids and / or immunosuppressants.
17. The use according to claim 15 or 16 wherein said vasculitis is polyarteritis nodosa.
18. The use according to claim 15 or 16 wherein said vasculitis is the aortitis syndrome.
19. The use according to claim 15 or 16 wherein said vasculitis is vasculitis associated with immunological abnormalities.
20. The use according to any of claims 15 to 19 wherein said IL-6 antagonist is an antibody against the IL-6 receptor.
21. The use according to claim 20 wherein said antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor.
22. The use according to claim 20 wherein said antibody against the IL-6 receptor is a monoclonal antibody against the human IL-6 receptor.
23. The use according to claim 20 wherein said antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor.
24. The use according to any of claims 20 to 23 wherein said antibody against the IL-6 receptor is a recombinant antibody.
25. The use according to claim 22 wherein said monoclonal antibody against the human IL-6 receptor is the PM-1 antibody.
26. The use according to claim 23 wherein said monoclonal antibody against the mouse IL-6 receptor is the MR16-1 antibody.
27. The use according to any of claims 20 to 26 wherein said antibody against the IL-6 receptor is a chimeric antibody, a humanized antibody or a human antibody.
28. The use according to claim 27 wherein said humanized antibody against the IL-6 receptor is a humanized antibody PM-1.
29. A method for preventing and / or treating vasculitis which comprises administering an interleukin 6 (IL-6) antagonist to a subject in need thereof.
30. A method for preventing and / or treating vasculitis that possesses resistance to spheroids and / or immunosuppressants which comprises administering an antagonist of interleukin 6 (IL-6) to a subject in need thereof.
31. The method according to claim 29 or 30 wherein said vasculitis is polyarteritis nodosa.
32. The method according to claim 29 or 30 wherein said vasculitis is the aortitis syndrome.
33. The method according to claim 29 or 30 wherein said vasculitis is vasculitis associated with immunological abnormalities.
34. The method according to any of claims 29 to 33 wherein said IL-6 antagonist is an antibody against the IL-6 receptor.
35. The method according to claim 34 wherein said antibody against the IL-6 receptor is a monoclonal antibody against the IL-6 receptor.
36. The method according to claim 34, wherein said antibody against the IL-6 receptor is a monoclonal antibody against the human L-6 receptor.
37. The method according to claim 34 wherein said antibody against the IL-6 receptor is a monoclonal antibody against the mouse IL-6 receptor.
38. The method according to any of claims 34 to 37 wherein said antibody against the IL-6 receptor is a recombinant antibody.
39. The method according to claim 36 wherein said monoclonal antibody against the human IL-6 receptor is the PM-1 antibody.
40. The method according to claim 37 wherein said monoclonal antibody against the mouse IL-6 receptor is the MR1 antibody.
41. The method according to any of claims 34 to 40 wherein the antibody against the IL-6 receptor is a chimeric antibody, a humanized antibody or a human antibody against the IL-6 receptor.
42. The method according to claim 41 wherein said humanized antibody against the IL-6 receptor is a humanized antibody PM-1. - 10 15 20 25