TW201034685A - Rheumatoid arthritis treatment agent - Google Patents

Abstract

By properly combining complementarity determining region (CDR) amino acid sequence modification, variable region amino acid sequence modification, and constant region amino acid sequence modification, a treatment agent for rheumatoid arthritis, juvenile chronic arthritis, and Castleman's disease was successfully created which has, as an active ingredient, an IL-6 receptor antibody which has improved material properties, safety, immunogenicity, pharmacokinetics (retentivity in blood plasma), and antigen neutralization which are superior to tocilizumab.

Description

201034685 ' VI. EMBODIMENT OF THE INVENTION: TECHNICAL FIELD The present invention relates to a therapeutic agent for rheumatoid arthritis, chronic arthritis or Kastoman disease in children containing an anti-IL-6 receptor antibody as an active ingredient. [Prior Art]

IL-6 is a cytokine with diverse functions, which can be composed of tau cells, B fine I mononuclear cells, fibroblasts, osteoblasts, keratinocytes, endothelial cell kidneys, 'mesangium cells, synovium The various cells of the synovial cell are produced (Non-patent literature, IL_6 and 1L_6 are combined, and the IL-6/IL-6 receptor complex and gpl30 are used to transmit information in the cell. (Non-Patent Document 3). The IL-6 receptor is present in both the membrane and the gluten-type, and the soluble IL-6 receptor can also form an IL 6/IL 6 steroid complex, which binds to gpl3〇. To convey the message. A rheumatism is the same as inflammation (10)) with multiple arthritis and progressive joint destruction as the main symptoms, extra-articular symptoms are lung, kidney shouting 'subcutaneous tissue and other lesions, for the unexplained Wang Sexual inflammatory disease . Although there are various types of systemic symptoms, the nature of RA is persistent synovitis, which is the result of synovitis and subsequent joint destruction of cartilage destruction and bone erosion, which is a result of patient characteristics. RA increases the blood vessels of the joints. White blood cells such as lymphocytes and macrophages move from the inside of the tube to the synovial tissue of the joint. In the joint, the immune response is caused by the action of cytokines produced by lymphocytes or macrophages, and the cartilage and hard bone are destroyed. Canine RA is thought to have hypererythrocyte sedimentation, increased CRP, increased blood count, increased multi-strain immunoglobulin, or rheumatoid factor, and these 3 201034685 have been taught to be associated with IL-6. The il_6 concentration in the serum of the RA patient and the joint showed a high value 'the degree of IL-6 is known to be associated with RA disease activity (Non-Patent Documents 4 to 6). Furthermore, it has been confirmed that the production of IL_6 in the synovial tissue is also advanced (Non-Patent Document 7). Further, it is known that in the presence of a soluble il_6 receptor, the promotion of osteoclast precursor cells into osteoclasts is also associated with bone resorption (Non-Patent Document 8). These suggest that IL_6 and soluble il_6 receptors are associated with joint destruction. In fact, the concentration of soluble IL-6 receptor in the joint fluid of patients with RA also shows a high value 'including lL-6, the concentration of which induces osteoclasts in vitro. A sufficient amount (Non-Patent Document 9). As described above, il_6 is associated with various effects such as antibody production, lymphocyte infiltration, pannus formation, joint destruction, acute phase reaction, and anemia in the pathological state of RA (Non-Patent Document 10). Recently, the combination of membrane-type IL-6R and soluble il-6 receptor inhibits the IL-6 signaling of humanized IL-6 antibody (the white matter-6 receptor antagonist; T0CILIZUMAB) is excellent in RA. As a result of clinical trials, it has been found that 'inhibiting the IL-6 receptor shows a method which is extremely effective for the treatment of RA (Non-Patent Document 10). Further, T0CILIZUMAB is known to be effective in the treatment of childhood chronic arthritis and Cas11 eman di sease in addition to RA. Further, the prior art documents of the present invention are as follows. [Prior Art Document] [Non-Patent Document] [Non-Patent Document 1] Kishimoto T. The biology of interlukin-6. Blood 1989; 74: 1-10. [Non-Patent Document 2] GuernePA, ZurawBL, Vaughan JH, Garson t 201034685 DA, Lotz M. Synovium as a source of interlukin 6 in vitro. Contribution to local and systemic manifestations of arthritis. J Clin Invest 1989; 83:585-92. [Non-Patent Document 3] NishimotoN, KishimotoT· Interlukin 6: from bench To beside. Nature Clinical Practice Rheumatology 2006; 11:19-26. [Non-Patent Document 4] HiranoT, MatsudaT, Turner M, Miyasaka ® N, Bouchan G, Tang B, Sato K, Shimizu M, Maini R, F e1dmann M , Ki shimoto T : Excessive Production of interlukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. European Journal of Immuniology 1988; 18:1797-1801. [Non-Patent Document 5] Houssiau FA, Devogelaer JP, Van Damme J, De Deuxchaisnes CN, Van Snick J: Interlukin-6 in q synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides.

Arthritis & Rheumatism. 1988; 31:784-788. [Non-Patent Document 6] Madhok R, Crilly A, Capell HA. Serum interleukin 6 levels in rheumatoid arthritis: correlations with clinical and laboratory indices of disease activity. Ann Rheum Dis 1993 52: 232-4. [Non-Patent Document 7] Sack U, Kinne RW, Marx T, Heppt P, Bender S, Emmrich F: Interlukin-6 in synovial fluid 5 201034685 is closely associated with chronic synovitis in rheumatoid arthritis. Rheumatology International 1993; 13:45-51. [Non-Patent Document 8] Tamura T, Udagawa N, Takahashi N, Miyaura C, Tanaka S, Yamada Y, Koishihara Y, Ohsugi Y, Kumki K, Taga T, Kishimoto T, Suda T : Soluble interlukin-6 receptor triggers osteoclast formation by inter1ukin 6. Proceedings of the National Academy of Sciences of the United States of America USA 1 993; 90: 11924-11928. [Non-Patent Document 9] Kotake S, Sato K, Kim KJ , Takahashi N, Udagawa N, Nakamura I, Yamaguchi A, Kishimoto T, Suda T, Kashiwazaki S: Interlukin-6 and soluble Interlukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation. Journal of Bone & Mineral Research 1996; 11: 88-95. [Non-patent document l〇] Inhibiting interlukin-6 in rheumatoid arthritis [Cleaning content] The present invention is directed to the provision of anti-IL- The antibody of 6 receptors is used as an active ingredient for rheumatoid arthritis, a therapeutic agent for children 201034685 inflammation or Kastoman disease. More system by 2 wet joints I children chronic arthritis or treatment of Kastoman's disease: agent: T〇ClH-amino acid replacement, providing antigen neutralization energy, material dynamics (retention in blood accumulation) ), immunogenicity, safety, and physical properties are improved by the use of & IL-6 receptor antibody as an active ingredient in rheumatoid arthritis, childhood chronic arthritis or Kastoman's disease therapeutic agent. [Means for solving the problem]

The inventors of the present invention replaced the amino acid of TQeiliz(10)ab as a therapeutic agent for rheumatoid arthritis and childhood chronic joint disease, @^原 neutralizing & force' drug dynamics (retention in plasma), immunogenicity The improved anti-IL_6 receptor antibody was obtained from female sex and physical properties. Therefore, there are 3 antibodies to the IL-6 receptor as active ingredients, and for the treatment of rheumatoid joints, chronic arthritis in children or Kastoman disease. The present invention more specifically provides the following [1] to [3]. [1] A therapeutic agent for rheumatoid arthritis, childhood chronic sclerotherapy or Catosman's disease containing the following antibodies as an active ingredient: (a) an antibody comprising a heavy chain comprising the sequence number: 1 ( CDR1 of the amino acid sequence of CDR1) of VH3-M73, CDR2 of amino acid sequence having SEQ ID NO: 2 (CDR2 of VH3-M73), amino acid having SEQ ID NO: 3 (CDR3 of VH3-M73) CDR3 of the sequence, (b) an antibody comprising a light chain comprising the CDR1 of the amino acid sequence having the sequence number: 4 (CDR1 of VL3), the amino acid sequence having the sequence number: 5 (CDR2 of VL3) CDR2, CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 of VL3 7 201034685), (c) an antibody comprising a heavy chain and a light chain, the heavy chain comprising the CDR1 having the sequence number: 1 (VH3-M73) CDR1 of the amino acid sequence of amino acid, CDR2 of the amino acid sequence having SEQ ID NO: 2 (CDR2 of VH3-M73), CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 of VH3-M73) The light chain has the CDR1 of the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), and the CDR2 of the amino acid sequence having the SEQ ID NO: 5 (CDR2 of VL3) CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 of Vl3). [2] A therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Catosman's disease containing the following antibodies as an active ingredient: (a) an antibody comprising a heavy chain comprising the sequence number: 7 (VH3) - a heavy chain variable region of the amino acid sequence of the variable region of M73, (b) an antibody comprising a light chain comprising an amino acid sequence having the sequence number: 8 (variable region of vl3) a light chain variable region, (an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy bond variable region having the amino acid sequence of SEQ ID NO: 7 (variable region of VH3 M73) and the light chain A light-bond variable region comprising an amino acid sequence having the sequence number: 8 (variable region of VL3). [3] containing the following antibodies as rheumatoid arthritis with a knife, or spastic arthritis in children A therapeutic agent for Katosmann's disease: (a) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), 201034685 (b) an antibody comprising a light chain, the light chain An amino acid sequence having the sequence number: i〇(VL3), (C) an antibody comprising a heavy chain and a light chain, the heavy chain The amino acid sequence having the sequence number: 9 (VH3-M73), and the amino acid sequence of the light chain having the sequence number: 10 (VL3). Further, the present invention provides the following: [4] comprising the following antibodies The steps of rheumatoid arthritis, childhood slow q arthritis or treatment of Katosmann's disease: (a) an antibody comprising a heavy chain comprising the sequence number: 1 (CDR1 of VH3-M73) CDR1 of the amino acid sequence, CDR2 of the amino acid sequence of SEQ ID NO: 2 (CDR2 of VH3-M73), CDR3 of amino acid sequence having SEQ ID NO: 3 (CDR3 of VH3-M73), (b An antibody comprising a light chain comprising the CDR of the amino acid sequence having the sequence number: 4 (CDR1 of VL3), the CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR3 Ο CDR2) having the sequence CDR3 of the amino acid sequence of number 6 (CDR3 of VL3), (c) an antibody comprising a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having the sequence number: 1 (CDR1 of VH3-M73) CDR1, CDR2 of amino acid sequence having SEQ ID NO: 2 (CDR2 of VH3-M73), amine having SEQ ID NO: 3 (CDR3 of VH3-M73) CDR3 of the acid sequence, the CDR1 of the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), CDR2 of the amino acid sequence having SEQ ID NO: 5 (CDR2 of VL3), with sequence coding > :6 (CDR3 of VL3) 9 CDR3 of the amino acid sequence of 201034685. [5] Treatment of rheumatoid arthritis, chronic arthritis or Catosman's disease in a step of administering the following antibodies: (a) An antibody comprising a heavy chain comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 7 (variable region of VH3-M73), (b) an antibody comprising a light chain, the light chain comprising SEQ ID NO: 8 (the variable region of vL3) the light chain variable region of the amino acid sequence, (c) an antibody comprising a heavy chain and a light chain, the heavy chain comprising the sequence number: 7 (VH3-M73 The heavy chain variable region of the amino acid sequence of the variable region, and the light chain comprises the light chain variable region of the amino acid sequence having the sequence number: 8 (variable region of VL3). m includes a method for treating rheumatoid arthritis, chronic arthritis or Catosman's disease such as a step of administering the following antibodies: u) an antibody comprising a heavy chain having an amino group of SEQ ID NO: 9 (VH3_m73) An acid sequence, (b) an antibody comprising a light chain having an amino acid sequence of SEQ ID NO: vl3, the heavy chain having a sequence, and the light chain having a sequence of chronic arthritis or Cato (c) an antibody comprising a heavy chain and a light chain, No. 9 (VH3-M73) amino acid sequence number: 1 0 (VL3) amino acid sequence [7] The following antibodies in rheumatoid arthritis, Use in the manufacture of therapeutic agents for the disease: (a) The antibody-containing heavy chain comprising the heavy chain comprises the CDR1 of the amino acid sequence having the CDR1 of SEQ ID NO: 201034685: 1CVH3-M73, having the SEQ ID NO: 2 (VH3) CDR2 of the amino acid sequence of CDR2) of M73) CDR3 of the amino acid sequence of SEQ ID NO: 3 (CDR3 of VH3_M73), (b) Antibody comprising a light chain comprising the SEQ ID NO: 4 ( The CDR of the amino acid sequence of CDR1) of VL3 has the CDR of the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3) 2. CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 of VL3), (c) an antibody comprising a heavy chain and a light chain, the heavy chain comprising an amine having the SEQ ID NO: 1 (CDR1 of VH3-M73) CDR1 of the acid sequence, CDR2 of the amino acid sequence having the sequence number: 2 (CDR2 of VH3-M73), CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 of VH3-M73), the light chain has SEQ ID NO: 4 (CDR1 of VL3) amino acid sequence of the amino acid sequence CDR2 of SEQ ID NO: 5 (cdr2 of VL3), amino acid sequence having SEQ ID NO: 6 (CDR3 of VL3) CDR3. [8] Use of the following antibodies in the manufacture of therapeutic agents for rheumatoid arthritis, chronic arthritis in children or Katosmann's disease: (a) An antibody comprising a heavy chain comprising the sequence number: 7 (VH3) - a variable region of the amino acid sequence of the variable region of -M73) (b) an antibody comprising a light chain comprising an amino acid sequence having the sequence number: 8 (variable region of vl3) a light chain variable region, (c) an antibody comprising a heavy chain and a light chain comprising a heavy chain variable region having the amino acid sequence of Sequence No. 11 201034685: 7 (variable region of VH3-M73) And the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 8 (variable region of VL3). [9] Use of the following antibodies in the manufacture of therapeutic agents for rheumatoid arthritis in children with chronic arthritis or Cattosman disease: (a) Antibody containing heavy chain 'The heavy chain has the sequence number: 9 (VH3_M73) An amino acid sequence, (b) an antibody comprising a light chain having the SEQ ID NO: 1 〇 (VL3) amino acid sequence, (c) an antibody comprising a heavy chain and a light chain, the heavy chain having a sequence Number: 9 (¥ 113 - 73) amino acid sequence, and the light chain has the amino acid sequence of SEQ ID NO: 10 (VL3). [1 〇] The following antibodies for use in the treatment of rheumatoid arthritis, chronic arthritis in children, or Cattosman disease: (a) Antibody containing heavy chain 'The heavy chain contains the sequence number: 1 (VH3 CDR1 of the amino acid sequence of CDR1) of M73, CDR3 of amino acid sequence having SEQ ID NO: 2 (CDR2 of VH3-M73), amino acid sequence having SEQ ID NO: 3CVH3-M73 CDR3) (b) an antibody comprising a light chain comprising: CDIU having an amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), CDR2 of an amino acid sequence having SEQ ID NO: 5 (CDR2 of VL3) CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 of VL3), (c) an antibody comprising a heavy chain and a light chain, the heavy chain comprising the sequence of SEQ ID NO: 12 201034685: 1 (CDR1 of VH3-M73) CDR1 of the amino acid sequence, CDR2 of the amino acid sequence having the sequence number: 2 (CDR2 of VH3-M73), CDR3 of the amino acid sequence having the sequence number: 3 (CDR3 of VH3-M73), the light chain The CDR2 of the amino acid sequence having the sequence number: 4 (CDR1 of VL3), the CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3) SEQ ID NO: CDR3 6 (VL3 of CDR3) of the amino acid sequences. [Π] The following antibodies for use in the treatment of rheumatoid arthritis, childhood chronic arthritis or Cattosman disease: (a) An antibody comprising a heavy chain comprising the sequence number: 7 (VH3- a heavy chain variable region of the amino acid sequence of the variable region of M73 (b) an antibody comprising a light chain comprising a light amino acid sequence having the sequence number: 8 (variable region of VL3) a variable region of a chain, (c) an antibody comprising a heavy bond and a light chain, the heavy chain comprising a heavy chain variable region of the amino acid sequence having the sequence number: 7 (variable region of VH3_M73) and the light The chain comprises a light chain variable region of the amino acid sequence having the sequence number: 8 (variable region of VL3). [12] The following antibodies are used in the treatment of rheumatoid arthritis, chronic arthritis in children or Catotsman's disease: (a) Antibodies containing heavy bonds 'The heavy chain has the sequence number: 9 (VH3_M73) Amino acid sequence, (b) an antibody comprising a light bond 'The light chain has the amino acid sequence of SEQ ID NO: 10 (VL3), 13 201034685 The heavy chain has a sequence and the light chain has a sequence (C) comprising Antibody for heavy and light chains, amino acid sequence number: 9 (VH3-M73) SEQ ID NO: 10 (VL3) amino acid sequence. [Effects of the Invention] The effective treatment of 'immunization' can include an antibody against the IL-6 receptor obtained according to the present invention as a rheumatoid arthritis, a chronic arthritis or a Katosman therapeutic agent, antigen Neutralizing ability, drug dynamics (when staying in blood accumulation:) The originality, safety, and physical properties are improved and the therapeutic effect of the drug administration frequency can be reduced. [Embodiment] The present invention relates to a therapeutic agent for rheumatoid joint, childhood chronic arthritis or Catosman's disease which comprises the following antibodies as an active ingredient: (a) an antibody comprising a heavy chain, the heavy chain comprising The CDR of the amino acid sequence of SEQ ID NO: 1 (CDR1 of VH3-M73) has the CDR2 of the amino acid sequence of SEQ ID NO: 2 (CDR2 of VH3-M73) having the SEQ ID NO: 3 (CDR3 of VH3-M73) a CDR3 of the amino acid sequence, (b) an antibody comprising a light chain comprising the CDR of the amino acid sequence having the sequence number j (CDR1 of VL3) having the sequence number: 5 (CDR2 of VL3) CDR2 of the amino acid sequence, CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 of VL3), (c) an antibody comprising a heavy chain and a light chain, the heavy chain comprising the sequence number: 1 (VH3-M73) CDR1 of the amino acid sequence of CDR1), amino acid sequence of SEQ ID NO: 2 (CDR2 of VH3-M73) 14 201034685 CDR2, amino acid sequence having SEQ ID NO: 3 (CDR3 of VH3-M73) CDR3 'The light chain has the CDIU of the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), with SEQ ID NO: 5 (VL3) CDR2 CDR2) of the amino acid sequences, having SEQ ID NO: CDR3 6 (VL3 of CDR3) of the amino acid sequences. Further, the present invention relates to a therapeutic agent for rheumatoid arthritis, chronic arthritis or cattosine disease in children containing the following antibodies as an active ingredient: 0 (a) an antibody comprising a heavy chain comprising a sequence Number: 7 (the variable region of VH3-M73) the heavy chain variable region of the amino acid sequence, (b) an antibody comprising a light bond comprising the sequence number: 8 (variable region of vL3) a light chain variable region of an amino acid sequence, (c) an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain having an amino acid sequence of SEQ ID NO: 7 (variable region of VH3-M73) The variable region 'and the light chain comprise a light chain variable region having an amino acid sequence of SEQ ID NO: 8 (variable region of VL3). Progressively, the present invention relates to a therapeutic agent comprising the following antibody as an active ingredient = rheumatoid arthritis, childhood chronic arthritis or Catosman's disease: the heavy chain has the sequence number: 9 (VH3-M73) the light chain An amine having the SEQ ID NO: 10 (VL3) (a) an amino acid sequence of an antibody comprising a heavy chain, 仏) an antibody-based acid sequence comprising a light chain, (C) an antibody comprising a heavy chain and a light chain, the heavy chain The amino acid sequence having the sequence code 15 201034685··9 (VH3-M73) is ^月^^ μ and the light chain has the amino acid sequence of SEQ ID NO: 10 (VL3). Further, the present invention provides that, in the antibody of the present invention having the above amino acid sequence, 'having one or more amino acids (usually within 3q amino acids) preferably within 10 amino acids Further preferably, within 5 amino acids, more preferably within 3 amino acids, the antibody is altered (substituted, deleted, affixed and/or inserted, etc.) or modified amino acid sequence. The antibody whose amino acid sequence is altered or modified preferably has an activity (antigen-binding activity, antigen-neutralizing activity, etc.) equivalent to that of the original antibody. Further, the antibody used in the antibody of the present invention may be a dual specific antibody. By dual specific antibody is meant an antibody having variable regions that recognize different epitopes within the same antibody molecule. In the present invention, the dual specific antibody may also be a dual specific antibody that recognizes different epitopes on the IL-6 receptor molecule, wherein one antigen binding site recognizes the IL_6 receptor's other antigen binding site recognizes other substances. Specific antibodies. The antigen to which another antigen-binding site of the dual-specific antibody formed by the antibody of the present invention which recognizes the IL-6 receptor binds, for example, IL-6, TNFa, TNFR1, TNFR2, CD80, CD86, CD28, CD20, CD19 , IL-1 a, IL-/5, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxin a, lymph Toxin; 5, LIGHT ligand, LIGHT, VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL-32, IL-21, IL-21R, GM-CSF, GM-CSFR, M-CSF, M-CSFR, IFN-a, VEGF, VEGFR, EGF, EGFR 'CCR5, APRIL 'APRILR, etc. 16 201034685 The FR used for the antibody of the present invention is not particularly limited and can be selected and selected by the skilled artisan. Although not particularly limited, it is preferred to use FR derived from humans. FR can also be a change from the amino acid of the native sequence. The region to be used for the antibody of the present invention is not particularly limited, and can be suitably selected from the technical field of the art. Although it is not particularly limited, it is preferable to use an original self-contained region. The constant quinone region can also be altered by the amino acid of the native sequence. 0 The above-mentioned antibody of the present invention is an antibody against the 丨1-6 receptor which is excellent in antigen neutralization ability, drug dynamics (remaining in plasma), safety and/or physical property, and is very useful as a rheumatoid joint. A therapeutic agent for inflammation, chronic arthritis in children, or Kastoman's disease. The antibodies of the invention can be made by methods known to those skilled in the art. For example, a gene encoding an antibody of the present invention is produced, the gene is assembled into a suitable vector, and the vector is introduced into a host, and the gene can be produced by genetic recombination techniques (for example, see Borrebaeck C. A. K. and Larrick J. w · THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Thus, the present invention provides a method of producing an antibody of the present invention comprising the step of culturing a host cell containing a gene encoding the antibody of the present invention into a vector. More specifically, a method of producing the antibody of the present invention comprising the following steps is provided. 17 201034685 (a) A step of culturing a host cell into which a gene encoding the antibody of the present invention is introduced into a vector, and (b) a step of obtaining an antibody encoded by the gene. /, body 5, when using mammalian cells, by using a commonly used promoter, the gene to be expressed, the 3, the downstream side, the DNA functionally binding to the P0ly A message or the vector containing the DNA Behave. For example, as a promoter/enhancer, a human cytomegalovirus immediate early promoter/enhancer can be cited. Furthermore, other promoters/k hadrons which can be used for antibody expression in the present invention can use retrovirus, polyomavirus, advenovirus, simian viral 4 () ( Simiari viruS 40) A promoter/enhancer derived from a mammalian cell such as a viral promoter/enhancer or human elongation factor 1 a (HEF1 α ). For example, when the SV40 promoter/enhancer is used, it can be easily carried out according to the method of Mulligan et al. (MuUigan, R.c. 纣al., Nature (1979) 277, 108-114), or using the HEFla promoter/enhancement. The sub-day guard, can be implemented according to the method of Mizushime et al. (Mizushima, S. and Nagata, S. Nucleic Acids Res. (1 990) 18, 5322). When Escherichia coli is used, the commonly used useful promoter, the message sequence for antibody secretion is functionally bound to the gene expressing the antibody, and for example, the promoter can be exemplified by the 1 acZ promoter and the araB promoter. When using the 1acZ promoter, the method according to Ward et al. (Ward, ES 18 201034685 et al., Nature (1989) 341, 544-546; Ward, ES et al FASEB J. (1 992) 6, 2422-2427 When using the araB promoter, it can be according to the method of Better et al. (Beter, M. et al., Science (1988) 240, 1041-1043). The message sequence for antibody secretion, generated from the periplasm of E. coli, can be used with the peiB message sequence (Lei, SP et al., J. Bacteriol· (1 987) 1 69, 4379-4383) . The antibody produced in the intercellular membrane region is isolated and appropriately refolded (for example, see WO96/30394). As the origin of replication, SV40, polyoma virus, adenovirus, bovine papi 1 l〇ma virus (bpv), etc. may be used, and further, in order to expand the number of genes of the host cell, it may be contained as Amin〇glyc〇side ph〇Ph〇transferase (APH) gene, thiamine kinase (TK) gene; jaundice-guanine guanine phosphoribosyltransferase 〇^ xanthine_Suamne phosphoribosy 1 transferase; Ecogpt) gene, monofolate reductase (dhfr) gene. For the manufacture of the antibodies used in the present invention, any production system can be used. A production system for producing antibodies, which has an in vitro and in vivo production system. The extracorporeal production system 'is a production system using eukaryotic cells or a production system using prokaryotic cells. When eukaryotic cells are used, there are systems for producing animal cells, plant cells or fungal cells. As animal cells, there are known (1) mammalian cells such as CH〇, C〇S, myel〇ma 'BHK (hamster kidney, baby hamster·201034685 kidney), HeLa, Vero, etc., (2) amphibian cells. For example, African clawed frog oocytes or (3) insect cells, such as sf9, Sf21, Tn5 and the like. As a scorpion cell, a cell derived from Nicotiana tabacum is known, and a culture of its callus can also be used. As fungal cells, for example, SaC Char〇 myces (e.g., Sacchar〇 myces cerevisiae), filamentous fungi such as Aspergius (e.g., Aspergillus niger), and the like are known. When using prokaryotic cells, there is a system for producing bacterial cells. As bacterial cells, Escherichia coli (E. c〇1 i ) and Bacillus subtilis are known. In these cells, the antibody is transformed by introduction of the antibody gene of interest, and the antibody can be obtained by culturing the transformed cell in vitro. The cultivation can be carried out according to a conventional method. For example, DMEM, MEM, RPMI 1 640, IMDM may be used as the culture solution, and a serum supplement such as fetal bovine serum (FCS) may be used in combination, and the cells into which the antibody gene is introduced may be transplanted into the abdominal cavity of the animal. Produces antibodies in living organisms. On the other hand, as a system for producing a living body, a system for producing an animal or a system for producing a plant can be cited. When animals are used, mammals, insect production systems, and the like are used. As a mammal, goats, pigs, sheep, mice, cows, etc. can be used.

Glaser, SPECTRUM Biotechnology Application, 1 9 9 3). Furthermore, silkworms can be used as insects. When plants are used, for example, tobacco can be used. The antibody gene is introduced into the animal or plant, and the antibody is produced in the animal or plant 20 201034685 and recovered. For example, a fusion gene is modulated by inserting an antibody gene into a gene encoding a protein inherently produced in goat casein milk. A DNA fragment containing the fusion gene into which the antibody gene was inserted was injected into the goat embryo I, and the embryo was introduced into the female goat. The desired antibodies can be obtained from the milk produced by the goats that receive the embryos from the goats or their progeny. In order to increase the amount of milk containing the desired antibody produced by genetically-transferred goats, suitable hormones can also be used in genetically-transferred goats (Ebert, κ. m. et al., Bio/Technology (1994) 12, 699/702 ). Further, when a silkworm is used, a baculovirus in which an antibody gene of interest is inserted is infected with a silkworm, and a desired antibody can be obtained from the body fluid of the silkworm (Maeda, S. et al., Nature (1985) 315, 592 -594). Further, using tobacco%, a plant expression vector for the antibody of interest, for example, PMON53〇 is inserted, and the vector is introduced into a bacterium of the genus Agr〇bacteri· tumefaciens. The bacterial infection such as tabaCUm to grass, by the desired antibody available in the grass industry (juHan, Ο Κ C. Ma, et al., Eur. J. Immunol. (1 994) 24, 131-138) . When the antibody production is carried out in vitro or in vivo as described above, the dm encoding the heavy chain (H chain) or the light chain (1 chain) of the antibody can be individually assembled into the expression vector while the host form is transformed. Alternatively, the DNA encoding the Η chain and the L chain may be assembled into a single-expression vector to convert the host shape (refer to International Patent Application Publication No. WO 〇 94_1 1523). The antibody produced and expressed in the above manner can be purified by being uniformly separated from the inside and outside of the cell and by the host. Isolation of antibodies used in the present invention, 21 201034685 Hardcover hunting by affinity chromatography - afflnity chromatography. For the column of the affinity layer chromatography, for example, a Pr〇tein A column and a Protein G column can be cited. For the support used in the Protein A column, for example, D, P_s, and SepharQseF.F # can be cited. ^^ I use the separation and purification method used for the general protein f without any limitation. For example, it is suitable to select and combine the above-mentioned affinity chromatography, chromatography, filtration, ultrafiltration, salting out, dialysis, etc. The antibody used in the present invention is purified. Examples of the layer include ion exchange chromatography, hydrophobic chromatography, gel filtration, and the like. These chromatography can be applied to 9 muscles for high performance liquid chromatography, High Perf0rmance Hquid chr〇mat〇graphy). Furthermore, reverse phase HPLC can also be used. The concentration of the antibody obtained above can be measured by measurement of absorbance or ELISA. Namely, when the absorbance was measured, the absorbance at 28 〇 nm was measured by appropriately diluting with pBs (_), and the calculation was carried out at 135 〇 d at 1 mg/ml. Furthermore, it can be measured by the following method by elisa. That is, goat anti-drug diluted to 0.1 M sodium bicarbonate buffer (ρΗ9·6) to 1//g/ml

Human IgG (manufactured by TAG) was added to a 96-well plate (manufactured by Nunc), and after incubation at 4 ° C overnight, the antibody was immobilized. After blocking, a sample of the antibody used in the present invention, or a standard human IgG (manufactured by CAPPEL), which was appropriately diluted, was added for 1 hour at room temperature. After washing, add 10 0 / 1 of the 5,000-fold diluted alkaline phosphatase

(alkaline phosphatase) labeled anti-human igG (manufactured by BI0 SOURCE) was incubated at room temperature for 1 hour. After washing, the substrate solution was cultured, and the absorbance at 405 nm was measured using a MICROPLATE READER Model 3550 (manufactured by Bio-Rad) at 22, 2010, 685, and the concentration of the antibody of interest was calculated. The IL-6 signaling inhibitory activity of the antibody used in the present invention can be evaluated according to a conventional method generally used by those skilled in the art. For example, 'culturing an IL-6-dependent human bone marrow tumor strain (S6B45, KPMM2), human Lennert's T lymphoma cell line KT3 or IL-6-dependent cell MH60.BSF2, adding IL-6 thereto, by coexisting at the same time The IL-6 inhibitor can be used to measure 3H-thymidine uptake in IL-6 dependent cells. Further, a method of culturing IL-6 receptor-expressing cell U266, adding a 125 y tag, and simultaneously adding an IL-6 inhibitor, and measuring IL-1 which binds to the 1251 marker of the jL_6 receptor-expressing cell can be determined. In the above analysis system, in addition to the group in which the IL-6 inhibitor was present, a negative control group containing no inhibitor was provided, and the results obtained by the two were compared to evaluate the IL-6 inhibitory activity of the IL-6 inhibitor. The antibody used in the present invention is not limited to the user of the present invention, and may be a fragment of an antibody or a modification thereof. For example, as the antibody fragment, an early chain Fv (scFv), sc(Fv) 2 or the like in which 〇 Fab, F(ab,) 2, Fv or an F chain of an H chain and an L chain are linked by an appropriate linker can be cited. Further, as the antibody used in this month, an antibody which binds to various molecules such as polyethylene glycol (PEG) as an antibody modification can also be used. The "antibody" of the present invention also includes such modifications. Such modifications are obtained by chemically modifying the resulting antibody. These methods have been established in the field of this technology. T〇ciHzumab, a humanized antibody against human IL-6 receptor, has been recognized as rheumatoid arthritis and chronic arthritis in children in Japan (for example, on 23 201034685 multiple joint active adolescents sudden Arthritis, systemic adolescent sudden arthritis), treatment of Kastoman's disease. The antibody of the present invention is an antibody which is improved in antigen neutralization ability, immunogenicity, safety, and physical properties by changing the amino acid of T〇ciHzumab. Therefore, the above-mentioned antibody of the present invention certainly has the same therapeutic effect as 111 zumab, and can be used as a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Kasman disease. The therapeutic agent of the present invention can be administered in the form of a pharmaceutical product, and can be administered systemically or locally by oral administration. For example, an intravenous injection, intra-abdominal injection, subcutaneous injection, or suppository method can be selected. Efficient administration of the drug and selection of appropriate investment. The effective technique is usually selected at 1 _ Γ for each time relative to the body. Or two: each patient can be selected: those who are in the technical field should appropriately choose 2 = the amount of music. The preferred dosage can be the carrier 2 allowed by this day and month rfr! The material for the above therapeutic effect may be added by a preparation such as a preservative or a stabilizer; each carrier means that it may be a material which has the same therapeutic effect as the above-mentioned therapeutic agent, and which can be "administered simultaneously". Furthermore, it is not a matter of ancient or human materials, but also a material that has a therapeutic effect by burial "(4)", and has a multiplicative or phase effect by using it in combination with an antibody. The sub-addition of the custom agent is insulting > u water, stabilizer,:: wide material / for example, sterilized or physiological salt mixture (10) "), binding agent, etc. ^ interface active agent 'cultivation 24 201034685 Ο Ο In the month of the month, the interface activity may be exemplified by a nonionic surfactant, and 2, for example, sorbitol succinic acid S, sorbitan monolaurate, sorbitol = monopalmitic acid vinegar, etc. Glycerol monocapry vinegar, glycerol fatty acid esters such as early, phthalate, glyceryl monostearate; decaglycerin prestearate, decaglyceryl distearate, decaglycerin monolinoleic oil Polyglycerol fatty acid such as vinegar and vinegar; polyoxyethylene ethyl sorbitol needle lauric acid vinegar, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitol liver monostearate, Polyoxyethylene exoethyl sorbitol singular palm sulphate, polyoxynexene ethyl sorbitan trioleate, polyoxyethylene sorbitan tristearate = polyoxyethylene sorbitol Alkyd fatty acid ester; polyoxyethylene ethyl sorbitol tetrahardy month: acid ester, polyoxyethylene ethyl sorbitol tetraoleate Ethyl sorbitol sulphate vinegar; polyoxyethylene glycerol monostearic acid vinegar, polyoxyethylene ethyl glycerol fatty acid vinegar; polyethylene glycol distearate vinegar and other polyethylene glycol fatty acid ester; Ethoxylated ethyl lauryl hydrazine and other polyoxyalkylene ketone vinegar; polyoxyethylene ethyl polyoxypropyl propylene glycol polyoxyethylene ethoxypropyl propyl propyl ether, polyoxyethyl ether Polyoxynyl propyl hexyl sulphate and other polyoxo-ethyl polyoxy-propion propyl base; poly-extension ethyl phenyl phenyl ether and other polyoxyethyl phenyl ether; polyoxygen Kewan sesame oil, polyoxy-extension ethyl hard sulphur oil (polyoxy-extension ethyl hydroquinone oil) and other polyoxy-extension ethyl hardened lotus oil; polyoxy-extension ethyl sorbitol honey and other polyoxy-extension ethyl preserve derived Polyoxyethylene ethyl lanolin derivatives such as polyoxyethylene ethyl lanolin; polyoxyethylene ethyl stearylamine, etc. Typical examples of #HU6 S. Further, 'interfacial surfactants can be listed as anionic interfacial activity. The agent may, for example, be an alkyl sulfate having a carbon 25 201034685 atomic number ίο to 18 alkyl group such as sodium hexyl sulfate, sodium lauryl sulfate or sodium oleate; polyoxyethylene ethyl laurel The average addition molar number of the epoxy sulphate such as sodium sulphate is 2 to 4. The number of polyoxyalkylene sulfonate sulfates with a sub-number of 10 to 18, · Laurel-based acid-based sorrel The number of carbon atoms in the base of the hospital is 8 to 18, the base acid group, the acid salt, the natural surfactant, such as lecithin, glycerophospholipid; the phospholipid (sPhingQeinyiin) (iv) fat f (sphingQUn iipid) A typical example of a sucrose fatty acid ester of a fatty acid having an atomic number of 12 to 18, etc. In the therapeutic agent of the present invention, one or a combination of two or more of these surfactants may be added. The preferred surfactants used in the present invention are polysorbate 2 〇, 4 〇, 60 or 80 polyoxyethyl sorbitol hydroxy fatty acid vinegar, particularly preferably polysorbate 20 and 80. Further, a polyoxyethylidene propylene monohydric alcohol represented by pol〇xamer (PU1_K F-68 (registered trademark) or the like) is also preferred. The interface is added to the polysorbate 2 depending on the type of surfactant used. Or polysorbate; in the case of 70XAMER 188, it is generally 〇. With ΐ (W/W)', it is preferably (4) 1 to ^ and further preferably U05 to 3%. Examples of the buffering agent in the present invention include a dish acid, a citric acid buffer solution, a citric acid, a tartaric acid, an acid, a lactic acid, a (tetra), a glucose: an acid: an acid, a bile acid, a salicylic acid, a triethanolamine, a fumaric acid, and the like. There are saliva buffers and the like. ... Buffer, histidine buffer, and sodium may also be prepared by a conventional aqueous buffer solution dissolved in the field of solution preparation. The concentration of the buffer is generally i to plan, preferably 26 201034685 is 5 to loomM, and more preferably ι to 2 〇. Further, the therapeutic agent of the present invention may contain other low molecular weight polypeptides, proteins such as serum albumin, collagen or immunoglobulin, saccharides such as amino acid 'polysaccharides and monosaccharides, or carbohydrates and sugar alcohols. In the present invention, examples of the amino acid include a basic amino acid such as arginine, lysine, histidine, ornithamine, or the like: or a salt (preferably a salt) In the form of an acid salt or a phosphate, that is, an amino acid phosphate. When the free amino acid is used, the preferred value can be adjusted by the addition of a suitable physiologically acceptable buffer, such as a mineral acid, particularly hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, formic acid or the like. In this case, the use of phosphate is particularly advantageous in terms of the point at which a stable freeze-dried product can be obtained. The preparation can be used to be substantially free of organic acids, such as malic acid, tartaric acid, citric acid, succinic acid, fumaric acid, etc., or corresponding anions (phosphoric anions, tartaric acid anions, succinic anions, fumaric acid) It is particularly advantageous in the case where an anion or the like does not exist. Preferred amino acids are arginine, lysine, histidine or ornithine. Further, acidic amino acids such as glutamic acid and aspartic acid, and salts thereof (preferably sodium salts) or neutral amino acids such as isoleucine, leucine, may also be used. Glycine, serine, threonine, valine, methionine, cysteine or alanine, or an aromatic amino acid such as phenylalanine, lysine, tryptophan or a derivative N-acetyl leucine. In the present invention, examples of the saccharide or carbohydrate such as a polysaccharide or a monosaccharide include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and galactose. 27 201034685 In the present invention, as the sugar alcohol, for example, an alcohol, an inositol or the like. Listed mannitol, sorbose The agent of the present invention is water for injection, water, glucose or other isotonic agent (for example, physiological salt, D-mannitol, sodium chloride) isotonic solution 714 sugar alcohol, D - nectar can also be combined with a suitable transfer adjuvant (4): 5. Further, the aqueous alcohol, PEG, etc., nonionic (ethanol, etc.), and polyhydric alcohol (such as propylene) are used in combination. ^Mountain® Ester 80, HC0-50) Further winter according to needs

Adjusting agent, benefit pain and heat, a thinner, dissolution aid, pH painless agent, sulfur-containing reducing agent 'antioxidant, etc. In the present invention, as the sulfur-containing reducing agent, the MJ can be exemplified by N-acetylcysteine, N-ethylglycosyl homosalicylate, cow's ethanolamine, thioglycerol, thiosorbitol: ::·, sodium thiosulfate, gluten, and carbon (4) and their salts, thiosulfate. & thiophene acid having 1 to 7 atomic number, etc., as an antioxidant, and examples thereof include an anti-ascorbic acid, a monobutyl permethythine, a butyl-based benzoyl bond, a tocopherol, and an acetic acid. Tocopherol, L-ascorbic acid and its salt, L_ascorbic acid standard vinegar, ascorbic acid hard fat, sodium bisulfite, sodium sulfite, gallic acid triacetin, wi1 acid propyl vinegar or ethylenediamine tetraacetic acid Chelating agents such as EDTA, sodium pyrophosphate, and sodium metaphosphate. In addition, if necessary, encapsulate microcapsules (hydroxymethylcellulose, gelatin, poly [methacrylic acid methyl methacrylate] and other microcapsules), which can be a colloidal drug delivery system (microlipid, albumin, micro-emulsification , nanoparticle and nanocapsules 28 201034685, etc. (refer to Remington's Pharmaceutical Science 16th edition), Oslo Ed. 1 980, etc.) Further, methods for using a pharmaceutical agent as a sputum agent are also known, and Suitable for use in the present invention (Unger et al., J. Biomed. Mater. Res. 1981, 15: 162-277;

Langer, Chem. Tech. 1982, 12: 98-105; U.S. Patent No. 3,773,91 9; European Patent Application Publication (£1), No. 58,48i;

Sidman et al·, Biop〇lymers 1983, 22: 547-556; EP Ο

133’ 988). Further, hyaluronic acid may be added or mixed in the medicament to increase the amount of the subcutaneous administration (for example, ff〇2〇〇4/〇78i4〇, etc.). The carrier to be used in the preparation to be used may be appropriately selected from the above depending on the dosage form, but is not limited thereto. The present invention relates to a method of administering a therapeutic agent of the present invention to a subject for treating rheumatoid arthritis, chronic arthritis in children, and Kastoman disease. The present invention t'"subject" means administration of a therapeutic agent of the present invention = an organism or a part of the living body. The organism is not particularly limited and includes animals (e.g., humans, livestock species, and wild animals). The above "part of the living body" is not particularly limited. In the present invention, "administration" includes oral or parenteral administration. The oral administration can be exemplified by administration in the form of an oral preparation, and the oral preparation can be selected from granules, powders, troches, capsules, solvents, emulsions or suspensions. (4) The administration of the oral administration may be exemplified by administration in the form of an injection, and the design of the residence may be enumerated as: a primary injection, an intramuscular injection or an intraperitoneal injection. Furthermore, the gene containing oligonucleoside acid is introduced into a living body by a gene therapy method, and the effect of the method of the invention can be achieved. Further, the agent of the present invention, 29 201034685 may also be administered by the local administration of the present invention from the area where the treatment is performed. For example, administration of a host in the middle of a bureau, the use of a catheter, or the delivery of a gene encoding the DNA of a peptide of the moon. The therapeutic agent of the present invention can be administered to a subject, and the symptoms of the disease can be manifested, and the preventive administration can be performed before the symptoms appear. Further, the anti-system of the present invention relates to the manufacture of a therapeutic agent for rheumatoid arthritis, chronic arthritis in children or Kastoman disease. Furthermore,

It is also used in the treatment of rheumatoid arthritis, chronic arthritis in children or Kastoman disease. Further, the amino acid contained in the amino acid sequence of the present invention is also subjected to post-translational modification (for example, by the pyrochlorination of the guanamine of the guanidine to the pyrophyllin) Modifications well known to those skilled in the art, where the amino acids are modified after translation, are included in the amino acid sequence of the invention.

Further, it may be any structure that incorporates a sugar chain structure. The sugar chain of Eu number (9) 7 may also be any sugar chain structure (preferably fucosylated sugar chain) or may not bind to a sugar chain (for example, when produced in Escherichia coli, or EU No. 297 does not bind a sugar chain). Way to change). All prior art documents cited in this "children's book are incorporated herein by reference. [Embodiment] The present invention will be described more specifically by way of examples, but the invention is not limited to the embodiments. [Example 1] Monkey anti-human IL_6 receptor-antibody pK/pD assay 30 201034685 T0CILIZUMAB (H chain WT-IgGl/SEQ ID NO: 13, L chain WT- /c / SEQ ID NO: 14), and aims to improve TOC ILIZUMAB antigen-neutralizing ability, blood dynamics, immunogenicity, safety and physical properties, and introducing Fv4_M73 in amino acid substitution in T0CILIZUMAB (H chain VH3-M73/SEQ ID NO: 9 'L-chain VL3-/C / SEQ ID NO: 10), performance and purification are carried out by a method known in the art (for reference, refer to Reference Example), and as a therapeutic drug for rheumatoid arthritis, the following review is carried out.

T0CILIZUMAB and Fv4-M73 were administered intravenously in a single dose of 1 mg/kg to cynomolgus monkeys to evaluate changes in blood-slurry concentration (methods refer to Reference Examples). The plasma concentration changes after intravenous administration of T0CILIZUMAB and FV4-M73 are shown in Fig. 1. As a result, Fv4-M73 significantly improved drug dynamics in cynomolgus monkeys compared to t〇ciuzumab. In order to evaluate the effect of the degree of neutralization of the membrane type receptors of cynomolgus monkeys, from the 6th to the 18th day of the administration (about the 3rd to the 10th of the T〇CIUzumab), at the waist The IL-6 of cynomolgus monkey 5#g/kg was administered subcutaneously on the back, and the CRp concentration of each body after 24 hours was measured (the method is referred to the reference example). The change in CRP concentration at the time of administration of each antibody is shown in Fig. 2. In order to evaluate the degree of neutralization of the soluble IL_6 receptor in cynomolgus monkeys, the concentration of non-binding cynomolgus monkeys in the blood collection of cynomolgus monkeys was determined, and the unconjugated solubles were calculated. Type IL_6 receptor ratio (method please > test case) Non-binding soluble form at the time of administration of each antibody. 6 The change in the total ratio is shown in Fig. 3. also

W4-M73 and T0CIUZUMAB 4m flat, Wen Feng continued to neutralize the membrane type 1L'6 receptor of the monkey. Furthermore, Fv4-M73 compared with T0CILIZUMAB phase 31 201034685' more consistently neutralized the soluble IL-6 receptor of cynomolgus monkeys and inhibited the increase of non-binding cynomolgus soluble IL-6 receptors for a long time. The persistence of neutralization of these membrane-type IL 6 receptors and soluble IL-6 receptors was found

The Fv4-M73 system is superior to T0CILIZUMAB. [Example 2] MonoCyte chemoattractant protein (MCP)-1, which is known to be involved in cell infiltration of mononuclear sphere-tau cells - NK cells - basophil. It has been reported that MCP-1 is a manifestation of sputum in menstrual tissue and synovial fluid of RA patients (j CHn Invest. 1992Sep; ❹ 90(3): 722-9), and the pathological state associated with ra (Inf lamm) Allergy Drug Targets. 2008 Mar; 7(1): 53-66). Furthermore, VEGF is a potent angiogenic factor known to be produced by macrophages-fibroblasts-synovial cells in the synovium of patients with ra (j

Rheumatol. 1995 Sep; 22(9): 1 624-30). Furthermore, serum VEGF concentrations in RA patients are associated with disease activity or radiographic progression (Arthritis Rheum. g, 2003 Jun; 48(6): 152b, Arthritis Rheum. 2001 Sep;

44(9)·· 2055-64), because the VEGF concentration in serum is decreased by treating patients with RA with anti-il-6 antibody TOCILIZUMAB, VEGF also plays an important role in the pathology of RA (M〇d Rheumato 1. 2009; 19(1): 12-9 'Mediators Inflamm. 2008; 2008: 129873). Therefore, 'TOCILIZUMAB and Fv4-M73 can inhibit the production of MCP-1 and VEGF in synoviocytes derived from human RA patients by sIL-6R and IL-6 stimulation, and review the following methods. 32 201034685 Synovial cells (τ〇γ〇Β〇) derived from human RA patients were seeded in a 96-well plate in a C〇2 incubator at 2xl〇V〇.05mL/well in IMDM medium containing 5% FCS (37 (:, 5% C〇2) was allowed to stand for 90 minutes. 〇. 05mL of the appropriate dilution concentration of T0CILIZUMAB and Fv4-M73, after standing for 15 minutes, add 0. 05mL of soluble il-6 receptor ( SR344: Prepared according to the method of the reference example) and allowed to stand for another 30 minutes, and then add 〇. 〇 5 mL of IL-6 (TORAY) (the final concentration of soluble IL-6 receptor and il-6 is 50 ng/mL each) After 2 days of culture, the culture supernatant was recovered, and the concentration of MCP-1 and VEGF in the culture supernatant was determined using an ELISA kit (Bi〇s〇urce and pierce).

Biotechnology) assay. The results are shown in Figures 4 and 5. TOCILIZUMAB and Fv4-M73 inhibited the production of Mcp-丨 and VEGF derived from synoviocytes of human RA patients in a concentration-dependent manner by soluble IL-6 receptor and IL_6. From the above, it can be seen that 'Fv4-M73 acts as an anti-il-6 receptor neutralizing antibody (blocking the message of membrane-type IL-6 receptor and soluble IL-6 receptor bound to 1L-6 receptor) The persistence is superior to TOCILIZUMAB. Compared with TOC ILIZUMAB, the frequency of administration and dosage can be greatly reduced. Further, since Fv4-M73 inhibits the production of CP 1 and VEGF from synoviocytes of human RA patients, Fv4_M73 is shown to be an extremely useful therapeutic agent for RA. <Reference Example> Recombination, Modulation of the Gluten I-6 Receptor The recombinant soluble il_6 of the human IL-6 receptor of the antigen was prepared by the system as follows. j. Biochem. 1 〇 8, 673 676 (199 〇) has reported human IL_6 receptors formed by the amino acid sequence of No. 1 to No. 344 on the N-terminal side of 33 201034685 (Yamasaki et al., Science 1988; 241 825-828 (GenBank # X1 2830)) production of stable strains of CH0 cells. The culture supernatant obtained by expressing CH0 cells by SR344 was subjected to column chromatography with Blue Sepharose 6 FF column chromatography, and column chromatography affinity chromatography with gel-filter column chromatography. Tube chromatography, purification of soluble IL-6 receptor. The dissolution of the main peak is used as the final refined product. Modulation of recombinant soluble cynomolgus monkey IL-6 receptor (CIL-6R) to disclose the IL-6 receptor gene sequence of Macaca mulatta

Column (Birney et al, Ensembl 2006, Nucleic Acids Res· 2006 Jan 1; 34 (Database issue): D556-61) to make the original oligo DNA primer, the CDNA prepared from the pancreas of the cynomolgus monkey (Macaca fascicularis) as a template, A DNA fragment encoding a full-length cynomolgus il-6 genus gene was modulated by PCR using primers. The resulting DNA fragment was inserted into an animal cell expression vector to prepare a CH0 stable expression strain (cyn〇.sIL_6R to produce CH0 cells). The culture medium in which cyno.sIL-6R-producing CH〇 cells was purified by HisTrap column (GE Healthcare Bi〇sciences), concentrated using Amic〇n Ultra 15 U1 trace 1-1 Ok (Mi 11ipore), and then

Superdex 20° 16/6 〇 Gel filtration (GE Healthcare Biosciences) was purified as the final refined product of the soluble cynomolgus monkey R_6 receptor (hereinafter referred to as cIL-6R). Preparation of recombinant cynomolgus monkey IL-6 (cIL-6) The cynomolgus monkey IL-6 was prepared as follows. The base sequence of 212 amino acids encoded by 201034685 SW0ISSPR0T Accession No. P79341 was cloned into an animal expression vector and introduced into CHO cells to produce a stable expression cell line (cyno. IL-6 producing CH0 cells). The culture medium in which cyno. IL-6 produces CH0 cells was purified by SP-S/FF column (GE Healthcare Biosciences), using Amicon Ultra-15

Ultracel-5k (Millipore) concentrated 're-purified with Superdex 75pg26/60 gel column (GE Healthcare Biosciences), concentrated with Amicon Ultra-15 Ultracel-5k (Millip〇re), as f) cynomolgus IL The final refined product below -6C is called cIL-6). Preparation, expression, and purification of a mutant of T0CILIZUMAB The plastid fragment encoding the antibody sequence of interest is inserted into an animal cell expression vector to produce a coding target Η chain expression vector and an l chain expression vector. The base sequence of the resulting expression vector can be determined by a method known to those skilled in the art. The expression of the antibody was carried out by the following method. A 293 Η cell line (Invitrogen) derived from human fetal kidney cancer cells was suspended in DMEM medium (lnvitr〇gen) containing 10% fetal calf (Inv itrogen) at a cell density of 5 to 6 χ 1 〇 5 cells/mL. 1 mL each was inoculated into each dish of a Petri dish for attachment cells (10 cm in diameter, CORNING), and cultured in a c〇2 incubator (37 ° C, 5% C 2 ) for a day and night, and then the medium was removed by suction. 6.9 mL of CHO-S-SFM-Il (Invitrogen) medium was added. The prepared plastid is introduced into the cell by the Lipofection method. After recovering the obtained supernatant culture solution, the cells were removed by centrifugation (about 2000 g, 5 minutes, room temperature), and then sterilized by a 0.222# in filter-MILLEX(R)-GV (Millipore).

The supernatant is cultured. The culture supernatant obtained was obtained using Pr〇tein A 35 201034685

SepharoseTM Fast Flow (Amersham Biosciences) is used to purify antibodies in accordance with conventional methods in the art. The purified antibody concentration was measured for absorbance at 28 nm using a spectrophotometer. The obtained value was calculated by the PACE method and the antibody concentration was calculated using the absorption coefficient (pr〇tein).

Science 1 995; 4: 241 Bu 2423). Determination of the concentration of the antibody in the monkey PK/PD test, the concentration of crp, and the concentration of the non-binding soluble IL-6 receptor. The concentration of the plasma in the cynomolgus monkey is determined by the EL 丨sA method. Know the method of determination. The CRP concentration was determined by SCI AS R CRP (Kanato Chemical Co., Ltd. using an automatic analyzer (TBA-120FR, TOSHIBA Medical Systems Corporation). The non-binding type soluble cynomolgus IL-6 in cynomolgus monkey plasma was subjected to The body concentration is measured by the following method: Adding cynomolgus plasma 30/zL' to a 0.22/zm filter cup (Mi 1 lipore), and drying the appropriate amount of rpr〇tein a Sepharose Fast Flow (GE Healthcare) resin To make all the sputum-type antibodies (the cynomolgus IgG, the anti-human IL-6 receptor antibody and the anti-human condition -6 receptor antibody) in the plasma can be combined with the cynomolgus monkey 1 L-6 receptor. Soaked in protein A. After that, it spins down in a high-speed centrifuge to recover the passed solution. Since the solution passed does not contain the antibody against human IL-6 receptor bound to Protein A - soluble crab In the complex of monkey IL-6, the concentration of non-binding "i IL 6 receptor" can be determined by measuring the concentration of soluble cynomolgus IL-6 receptor in pr〇teinA ^ over two liquids. Soluble crab I ^ - β receptor concentration system, I soluble soluble cynomolgus IL-6 receptor (clL-6R) as the standard 'to this item The method of measuring the human IL-6 receptor by the conventional method is determined by the method of measuring the human IL-6 receptor. The ratio of the non-binding type soluble IL_6 receptor is calculated according to the following formula: (uncombined soluble after antibody administration) Type IL_6 receptor concentration + soluble IL-6 receptor concentration before antibody administration) X 1〇〇 [Simplified description of the figure] Figure 1 shows that TOCILIZUMAB and FV4-M73 are administered in igma when img/kg Fig. 2 is a graph showing changes in plasma concentration of CRP when TOCILIZUMAB and Fv4-M73 are administered at 1 mg/kg in cynomolgus monkeys. Fig. 3 shows TOCILIZUMAB and Fv4- A graph of the change in plasma concentration of non-binding soluble IL-6 receptors when M73 was administered to cynomolgus monkeys at img/kg. & Figure 4 shows patients with human RA from TOCILIZUMAB and Fv4-M73 Inhibition of MCP_i production by synoviocytes. Figure 5 is a graph showing the inhibition of VEGF production by synoviocytes derived from human sputum RA patients with T0CILIZUMAB and fv4-M73. Explanation of symbols] No 37 201034685 Sequence Listing &lt;110> Chinese and foreign pharmaceutical companies have Company &lt; 120 &gt; rheumatoid arthritis therapeutic agent

&lt;130&gt; C1-A0903-TW &lt;150&gt; JP 2009-067358 &lt;151> 2009-03-19 &lt;160&gt; 14 &lt;170&gt; Patentln version 3.4 &lt;210&gt; 1 &lt;211&gt; 6 &lt;212&gt; PRT &lt; 213 &gt; 213 &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

His Asp His Ala Trp Ser 1 5 &lt;21〇&gt; 2 &lt;211&gt; 16 &lt;212&gt; PRT &lt;213&gt;Manual &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu Gin Gly 15 10 15 &lt;210&gt; 3 &lt;211&gt; 10 201034685 &lt;212&gt; PRT &lt;213&gt; Labor &lt;22〇&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt; 3

Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr 15 10

&lt;210&gt; 4 &lt;211&gt; 11 &lt;212&gt; PRT &lt; 213 &gt; 213 &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Gin Ala Ser Thr Asp lie Ser Ser His Leu Asn 15 10 &lt;210&gt; 5 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213>manual &lt;22〇&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;; 5

Tyr Gly Ser His Leu Leu Ser 1 5 &lt;210&gt; 6 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Labor 2 201034685 &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Gly Gin Gly Asn Arg Leu Pro Tyr Thr 1 5 &lt;210&gt; 7 &lt;211&gt; 119 &lt;212&gt; PRT &lt;213&gt;Labor&lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;4〇〇&gt; 1

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30

His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60

Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 201034685

Thr Leu Val Thr Val Ser Ser 115 &lt;21〇&gt; 8 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213&gt;manual &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Ser Val Thr lie Thr Cys Gin Ala Ser Thr Asp lie Ser Ser His 20 25 30

Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45

Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 〇 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu 100 105 &lt;210&gt; 9 4 201034685 &lt;211&gt; 443 &lt;212&gt; PRT &lt; 213 &gt; 213 &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;; 9

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser lie Ser His Asp 20 25 30

His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 lie Gly Phe lie Ser Tyr Ser Gly lie Thr Asn Tyr Asn Pro Ser Leu 50 55 60

Gin Gly Arg Val Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 5 201034685

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190

Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu 210 215 220

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 245 250 255 o

Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285

Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300

Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 6 201034685

Val Ser Asn Lys Gly Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys 325 330 335

Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser 340 345 350

Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys 355 360 365

Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala 420 425 430

His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 &lt;210&gt; 10 &lt;211&gt; 214 &lt;212&gt; PRT &lt;213&gt;Manual &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 201034685 15 10

Asp Ser Val Thr lie Thr Cys Gin Ala Ser Thr Asp lie Ser Ser His 20 25 30

Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Glu Leu Leu lie 35 40 45

Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Glu Ala 65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gin Gly Asn Arg Leu Pro Tyr 85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Glu Arg Thr Val Ala Ala 100 105 110

Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 8 201034685 180 185 190

Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210

&lt;210&gt; 11 &lt;211&gt; 324 &lt;212&gt; PRT &lt;213&gt; J\X &lt;22〇&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr 65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 9 201034685

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125

Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140

Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205

G

Ala Pro work Le Glu Lys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220

Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn 225 230 235 240

Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie 245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 260 265 270 10 201034685

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys 290 295 300

Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gin Lys Ser Leu 305 310 315 320

Ser Leu Ser Pro &lt;210&gt; 12 &lt;211&gt; 107 &lt;212&gt; PRT &lt;213>manual &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15

Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30

Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45

Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 11 201034685

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser 85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 &lt;210&gt; 13 &lt;211&gt; 449 &lt;212&gt; PRT &lt;213&gt;

&lt;220> &lt;223&gt; Synthetic polypeptide sequence &lt;40〇&gt; 13

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser lie Thr Ser Asp 20 25 30

His Ala Trp Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 lie Gly Tyr lie Ser Tyr Ser Gly lie Thr Thr Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gin Phe Ser 65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 12 201034685

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110

Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190

Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser 245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 13 201034685

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300

Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335 o

Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu 370 375 380

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

Lys 14 201034685 &lt;21〇&gt; 14 &lt;211&gt; 214 &lt;212&gt; PRT &lt;213>manual &lt;220&gt;&lt;223&gt; Synthetic polypeptide sequence &lt;400&gt;

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp lie Ser Ser Tyr 20 25 30

Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Thr Leu Pro Tyr 85 90 95

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala 100 105 110

Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 15 201034685 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210

16

Claims (1)

201034685 VII. Patent application scope: 1 · A therapeutic agent for rheumatoid arthritis, chronic arthritis in children or Cast leman desease contains the following antibodies as active ingredients: (a) Antibodies containing heavy chains , the heavy chain comprises CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 of VH3-M73) having the sequence number: 3 CDR3 of the amino acid sequence of (CDR3 of VH3_M73); (b) Antibody comprising a light chain comprising the CDR1 of the amino acid sequence having the sequence number: 4 (CDR1 of 3) having the sequence number: 5 ( CDR2 of the amino acid sequence of CDR2) of VL3) CDR3 of amino acid sequence having SEQ ID NO: 6 (CDR3 of 3), (c) Antibody comprising heavy chain and light chain 'The heavy chain contains the sequence number CDR1 of the amino acid sequence of 1 (CDR1 of VH3-M73), CDR2 of the amino acid sequence of SEQ ID NO: 2 (CDR2 of VH3-M73) having SEQ ID NO: 3 (CDR3 of VH3-M73) a CDR3 of an amino acid sequence comprising an amino acid sequence having the sequence number: 4 (CDR1 of VL3) cj) Ri, having SEQ ID NO: cdR2 5 (VL3 of CDR2) of the amino acid sequences, having SEQ ID NO: CDR3 6 (VL3 of CDR3) of the amino acid sequences. 2. A therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castlemandesease containing the following antibodies as active ingredients: (a) an antibody comprising a heavy chain comprising the sequence number: Heavy chain variable region of the amino acid sequence of 7 (variable region of VH3-M73), 201034685 (b) an antibody comprising a light chain comprising an amine having the sequence number: 8 (accessible region of ια3) a light chain variable region of a base acid sequence, (C) an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable having an amino acid sequence of SEQ ID NO: 7 (variable region of VH3-M73) The region, and the light chain comprises a light chain variable region of the amino acid sequence having the sequence number: 8 (variable region of VL3). 3. A therapeutic agent for rheumatoid arthritis, chronic arthritis in children or Castlemandesease containing the following antibodies as effective U components: (a) An antibody comprising a heavy chain having a sequence number: 9("3^ Amino acid sequence, (b) an antibody comprising a light chain" The light chain has the sequence number: amino acid sequence of 丨〇 (3), (c) an antibody comprising a heavy chain and a light bond The heavy chain has the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and the light chain has the amino acid sequence of sequence Q: 10 (VL3).