WO2010107108A1

WO2010107108A1 - Rheumatoid arthritis treatment agent

Info

Publication number: WO2010107108A1
Application number: PCT/JP2010/054763
Authority: WO
Inventors: 智之井川; 慎也石井; 敦彦前田; 実香櫻井; 哲郎小嶋; 橘　達彦; 宙丈白岩; 角田　浩行; 義信樋口
Original assignee: 中外製薬株式会社
Priority date: 2009-03-19
Filing date: 2010-03-19
Publication date: 2010-09-23
Also published as: TW201034685A; JP4809930B2; KR101314880B1; RU2011142183A; RU2524152C2; TWI457134B; JPWO2010107108A1; KR20110139734A; JP2011219478A

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

Rheumatoid arthritis treatment

The present invention relates to a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing an anti-IL-6 receptor antibody as an active ingredient.

IL-6 is a cytokine with various functions, and is produced from various types of cells such as T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells, and synovial cells. (Non-Patent Documents 1 and 2). IL-6 binds to the IL-6 receptor, and the IL-6 / IL-6 receptor complex binds to gp130, whereby a signal is transmitted into the cell (Non-patent Document 3). There are two types of IL-6 receptor, membrane type and soluble type, but soluble IL-6 receptor can also form an IL-6 / IL-6 receptor complex and binds to gp130. It is possible to transmit a signal.

Rheumatoid arthritis (RA) is a systemic inflammatory disease of unknown cause whose main symptoms are frequent arthritis and progressive joint destruction, and as extra-articular symptoms spread to the lungs, kidneys, subcutaneous tissues and the like. Although there are various systemic signs, the essence of RA is persistent synovitis, and synovitis that causes cartilage destruction and bone erosion and subsequent destruction of joint function are characteristic features of the disease . RA increases joint blood vessels, and leukocytes such as lymphocytes and macrophages migrate from the blood vessels to the synovial tissue. An immune response occurs locally in the joint, and an inflammatory reaction is caused by the action of cytokines produced by lymphocytes and macrophages, and cartilage and bone destruction progress.

In RA, increased blood sedimentation, increased CRP, increased platelet count, increased polyclonal immunoglobulin, or rheumatoid factor, suggesting the involvement of IL-6. It has been reported that IL-6 concentrations in serum and joint fluid of RA patients show high values, and this IL-6 level correlates with RA disease activity (Non-patent Documents 4-6). It has also been confirmed that IL-6 production from synovial tissue is also enhanced (Non-patent Document 7). Furthermore, it has been reported that IL-6 promotes the differentiation of osteoclast precursor cells into osteoclasts in the presence of soluble IL-6 receptor and is also involved in bone resorption (Non-patent Document 8). These suggest that IL-6 and soluble IL-6 receptor are involved in joint destruction, and in fact, soluble IL-6 receptor concentration is also high in the joint fluid of RA patients. The concentration including 6 is sufficient to induce osteoclasts in vitro (Non-patent Document 9). As described above, IL-6 is considered to be involved in various effects such as antibody production, lymphocyte infiltration, pannus formation, joint destruction, acute phase reaction, anemia in the pathology of RA (non-patent literature). 10). Recently, RA has demonstrated superior humanized anti-IL-6R antibody tocilizumab (TOCILIZUMAB) that can bind to both membrane IL-6R and soluble IL-6 receptor and inhibit IL-6 signaling The test results have been clarified and it has been shown that inhibiting IL-6 receptor is a very effective means for the treatment of RA (Non-patent Document 10).

In addition to RA, TOCILIZUMAB is also known to be effective in the treatment of childhood chronic arthritis and Castleman's disease.

Prior art document information related to the invention of the present application is shown below.

The present invention has been made in view of such circumstances, and an object thereof is to provide a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing an anti-IL-6 receptor antibody as an active ingredient. It is. More specifically, by substituting the amino acid of Tocilizumab, which is used as a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease, antigen neutralizing ability, pharmacokinetics (retention in plasma), immunogenicity The present invention provides a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease comprising an anti-IL-6 receptor antibody with improved safety and physical properties as an active ingredient.

By substituting the amino acid of Tocilizumab, which has been used as a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease, the present inventors have made it possible to neutralize antigens, pharmacokinetics (retention in plasma), immunogenicity. An anti-IL-6 receptor antibody with improved safety and physical properties was obtained. The inventors have found that a drug containing the anti-IL-6 receptor antibody as an active ingredient is useful for the treatment of rheumatoid arthritis, childhood chronic arthritis or Castleman's disease.

More specifically, the present invention provides the following [1] to [3].
[1] A therapeutic agent for rheumatoid arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibody as an active ingredient;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence of M73 CDR3),
(b) CDR1 having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3), SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence of:
(c) CDR1 having the amino acid sequence described in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence described in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- A heavy chain comprising CDR3 having the amino acid sequence described in CDR3) of M73, and CDR1, having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), and the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3).
[2] A therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease comprising the following antibody as an active ingredient;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73);
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3) An antibody comprising a light chain comprising a chain variable region.
[3] A therapeutic agent for rheumatoid arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibody as an active ingredient;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),
(c) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73) and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

Furthermore, the present invention provides the following.
[4] A method for treating rheumatoid arthritis, childhood chronic arthritis, or Castleman's disease, comprising a step of administering the following antibody;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence of M73 CDR3),
(b) CDR1 having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3), SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence of:
(c) CDR1 having the amino acid sequence described in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence described in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- A heavy chain comprising CDR3 having the amino acid sequence described in CDR3) of M73, and CDR1, having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), and the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3).
[5] A method for treating rheumatoid arthritis, childhood chronic arthritis, or Castleman's disease, comprising a step of administering the following antibody;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73);
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3) An antibody comprising a light chain comprising a chain variable region.
[6] A method for treating rheumatoid arthritis, pediatric chronic arthritis or Castleman's disease, comprising the step of administering the following antibody;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),
(c) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73) and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).
[7] Use of the following antibody in the manufacture of a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence of M73 CDR3),
(b) CDR1 having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3), SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence of:
(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- A heavy chain comprising CDR3 having the amino acid sequence described in CDR3) of M73, and CDR1, having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), and the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3).
[8] Use of the following antibodies in the manufacture of a therapeutic agent for rheumatoid arthritis, pediatric chronic arthritis or Castleman's disease;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73);
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3) An antibody comprising a light chain comprising a chain variable region.
[9] Use of the following antibodies in the manufacture of a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis, or Castleman's disease;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),
(c) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73) and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).
[10] The following antibody for use in a method for treating rheumatoid arthritis, childhood chronic arthritis or Castleman's disease;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence of M73 CDR3),
(b) CDR1 having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3), SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence of:
(c) CDR1 having the amino acid sequence described in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence described in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- A heavy chain comprising CDR3 having the amino acid sequence described in CDR3) of M73, and CDR1, having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), and the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3).
[11] The following antibody for use in a method for treating rheumatoid arthritis, childhood chronic arthritis or Castleman's disease;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73);
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3) An antibody comprising a light chain comprising a chain variable region.
[12] The following antibody for use in a method for treating rheumatoid arthritis, childhood chronic arthritis or Castleman's disease;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),
(c) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73) and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

The therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing the anti-IL-6 receptor antibody obtained by the present invention as an active ingredient is antigen neutralizing ability, pharmacokinetics (retention in plasma), immunogen The frequency of administration can be reduced by improving the safety, safety and physical properties, and the therapeutic effect can be exerted continuously.

It is a figure which shows the plasma concentration transition of an antibody when TOCILIZUMAB and Fv4-M73 are administered to a cynomolgus monkey at 1 mg / kg. It is a figure which shows the plasma concentration transition of CRP when TOCILIZUMAB and Fv4-M73 are administered to a cynomolgus monkey at 1 mg / kg. It is a figure which shows the plasma concentration transition of the non-binding soluble IL-6 receptor rate when TOCILIZUMAB and Fv4-M73 are administered to cynomolgus monkeys at 1 mg / kg. It is a figure which shows the MCP-1 production inhibitory effect from the human RA patient-derived synoviocytes by TOCILIZUMAB and Fv4-M73. It is a figure which shows the VEGF production inhibitory effect from the human RA patient origin synovial cell by TOCILIZUMAB and Fv4-M73.

The present invention relates to a therapeutic agent for rheumatoid arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibody as an active ingredient.
(a) CDR1, having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73),
CDR2, having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73),
CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (CDR3 of VH3-M73)
An antibody comprising a heavy chain comprising
(b) CDR1, having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3),
CDR2, having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3),
CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3)
An antibody comprising a light chain comprising, or
(c) CDR1, having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73),
CDR2, having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73),
A heavy chain comprising CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (CDR3 of VH3-M73), and CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3),
CDR2, having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3),
An antibody comprising a light chain comprising CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3).

Furthermore, the present invention relates to a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing the following antibody as an active ingredient.
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73);
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (VL3 variable region), or
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3) An antibody comprising a light chain comprising a chain variable region.

Furthermore, the present invention relates to a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing the following antibody as an active ingredient.
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3), or
(c) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73) and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

Furthermore, the present invention provides the antibody of the present invention having the amino acid sequence described above, wherein one or more amino acids (usually within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, more preferably within 3 amino acids). Antibodies having amino acid sequences that are altered (such as substitutions, deletions, additions and / or insertions) or modified are provided. Such an antibody having an altered or modified amino acid sequence preferably has the same activity (antigen binding activity, antigen neutralization activity, etc.) as the original antibody.

The antibody used in the antibody of the present invention may be a bispecific antibody. Bispecific antibodies refer to antibodies that have variable regions that recognize different epitopes within the same antibody molecule. In the present invention, the bispecific antibody may be a bispecific antibody that recognizes different epitopes on the IL-6 receptor molecule, or one antigen binding site recognizes the IL-6 receptor and the other Bispecific antibodies whose antigen-binding sites recognize other substances can also be used. Antigens to which the other antigen-binding site of the bispecific antibody comprising the antibody of the present invention that recognizes the IL-6 receptor binds include, for example, IL-6, TNFα, TNFR1, TNFR2, CD80, CD86, CD28, CD20 , CD19, IL-1α, IL-β, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxinα, lymphotoxinβ, 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- Examples include CSF, M-CSFR, IFN-alpha, VEGF, VEGFR, EGF, EGFR, CCR5, APRIL, and APRILR.

The FR used for the antibody of the present invention is not particularly limited, and can be appropriately selected by those skilled in the art. Although not particularly limited, human-derived FR is preferably used. In FR, amino acids may be modified from the natural sequence.

The constant region used for the antibody of the present invention is not particularly limited and can be appropriately selected by those skilled in the art. Although not particularly limited, a human-derived constant region is preferably used. The constant region may be altered in amino acid from the natural sequence.

The above-described antibody of the present invention is an anti-IL-6 receptor antibody excellent in antigen neutralizing ability, pharmacokinetics (retention in plasma), immunogenicity, safety and / or physical properties, rheumatoid arthritis, chronic pediatric It is very useful as a therapeutic agent for arthritis or Castleman's disease.

The antibody of the present invention can be produced by methods known to those skilled in the art.
For example, a gene encoding the antibody of the present invention can be prepared, the gene can be incorporated into an appropriate vector, introduced into a host, and produced using a gene recombination technique (for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

Accordingly, the present invention provides a method for producing the antibody of the present invention, comprising the step of culturing a host cell containing a vector into which a gene encoding the antibody of the present invention has been introduced.
More specifically, a method for producing the antibody of the present invention comprising the following steps is provided.
(a) culturing a host cell containing a vector into which a gene encoding the antibody of the present invention has been introduced,
(b) A step of obtaining an antibody encoded by the gene.

Specifically, in the case of production using mammalian cells, a commonly used useful promoter, an antibody gene to be expressed, a DNA having a poly A signal operably linked to the 3 ′ downstream thereof, or a vector containing the same Can be expressed. For example, the promoter / enhancer includes human cytomegalovirus early promoter / enhancer (human cytomegalovirus immediate-promoter / enhancer).

In addition, other promoters / enhancers that can be used for the expression of antibodies used in the present invention include retrovirus, polyomavirus, adenovirus, simian virus 40 (SV40) and other viral promoters / enhancers and human elongation factor 1α (HEF1α). It is possible to use promoters / enhancers derived from mammalian cells such as

For example, when the SV40 promoter / enhancer is used, the method of Mulligan et al. (Mulligan, R. C. et al., Nature (1979) 277, 108-114), and when the HEF1α promoter / enhancer is used, Mizushima et al. This method can be easily implemented according to the method (Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18, 5322).

In the case of production using Escherichia coli, a commonly used useful promoter, a signal sequence for antibody secretion, and an antibody gene to be expressed can be functionally linked for expression. For example, examples of the promoter include lacZ promoter and araB promoter. When using the lacZ promoter, the method of Ward et al. (Ward, E. S. et al., Nature (1989) 341, 544-546; Ward, E. S. et al. FASEB J. (1992) 6, 2422) When using the araB promoter, the method of Better et al. (Better, M. et al. Science (1988) 240, 1041-1043) may be used.

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

As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can be used. Furthermore, for amplification of gene copy number in the host cell system, the expression vector is used as a selection marker. An aminoglycoside phosphotransferase (APH) gene, a thymidine kinase (TK) gene, an E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene and the like can be included.

Any production system can be used for the production of the antibodies used in the present invention.
Production systems for antibody production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems that use eukaryotic cells and production systems that use prokaryotic cells.

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

When using prokaryotic cells, there is a production system using bacterial cells. Known bacterial cells include E. coli and Bacillus subtilis.

An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro. Culture is performed according to a known method. For example, DMEM, MEM, RPMI1640, and IMDM can be used as the culture medium, and serum supplements such as fetal calf serum (FCS) can be used in combination. Alternatively, antibodies may be produced in vivo by transferring cells into which the antibody gene has been introduced to the abdominal cavity of animals.

On the other hand, examples of production systems for in vivo include production systems that use animals and production systems that use plants. When animals are used, there are production systems using mammals and insects.

As mammals, goats, pigs, sheep, mice, cows, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). In addition, silkworms can be used as insects. When using a plant, for example, tobacco can be used.

An antibody gene is introduced into these animals or plants, and antibodies are produced and collected in the animals or plants. For example, an antibody gene is inserted into the middle of a gene encoding a protein inherently produced in milk such as goat β casein to prepare a fusion gene. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its progeny. In order to increase the amount of milk containing the desired antibody produced from the transgenic goat, hormones may be used in the transgenic goat as appropriate (Ebert, KM et al., Bio / Technology (1994) 12, 699- 702).

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

As described above, when an antibody is produced in an introvitro or in 上述 vivo production system, DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) are separately incorporated into an expression vector to simultaneously transform the host. Alternatively, the host may be transformed by incorporating DNAs encoding the H and L chains into a single expression vector (see International Patent Application Publication No. WO 94-11523).

The antibody produced and expressed as described above can be isolated from the inside and outside of the cell and from the host and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed by affinity chromatography. Examples of the column used for affinity chromatography include a protein A column and a protein G column. Examples of the carrier used for the protein A column include HyperD, POROS, Sepharose F.F. and the like. In addition, it is only necessary to use a separation and purification method used in ordinary proteins, and the method is not limited at all.

For example, the antibodies used in the present invention can be separated and purified by appropriately selecting and combining chromatography, filters, ultrafiltration, salting out, dialysis and the like other than the affinity chromatography. Examples of chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, and the like. These chromatographies can be applied to HPLC (High-performance liquid-chromatography). Further, reverse phase HPLC (reverse phase HPLC) may be used.

The antibody concentration obtained above can be measured by measuring absorbance, ELISA, or the like. That is, in the case of measuring the absorbance, after appropriately diluting with PBS (−), the absorbance at 280 nm is measured, and 1 mg / ml is calculated as 1.35 μOD. In the case of ELISA, the measurement can be performed as follows. That is, 100 μl of goat anti-human IgG (manufactured by TAG) diluted to 1 μg / ml with 0.1 μM bicarbonate buffer (pH 9.6) was added to a 96-well plate (manufactured by Nunc) and incubated overnight at 4 ° C. Solidify. After blocking, 100 μl of appropriately diluted antibody used in the present invention or a sample containing the antibody or human IgG (manufactured by CAPPEL) as a standard is added and incubated at room temperature for 1 hour.

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

The IL-6 signaling inhibition activity of the antibody used in the present invention can be evaluated by a method commonly used by those skilled in the art. For example, an IL-6-dependent human myeloma line (S6B45, KPMM2), a human Rennelt T lymphoma cell line KT3, or an IL-6-dependent cell MH60.BSF2 is cultured, and IL-6 is added thereto, and IL- 6 Incorporation of ³ H-thymidine uptake by IL-6-dependent cells can be measured by coexisting with an inhibitor. In addition, by culturing U266, which is an IL-6 receptor-expressing cell, adding ¹²⁵ I-labeled IL-6, and simultaneously adding an IL-6 inhibitor, ¹²⁵ I-labeled cells bound to IL-6 receptor-expressing cells A method for measuring IL-6 may also be used. In the above assay system, there is a negative control group that does not contain an IL-6 inhibitor in addition to the group in which an IL-6 inhibitor is present. Activity can be evaluated.

The antibody used in the present invention may be an antibody fragment or a modified product thereof as long as it can be suitably used in the present invention. For example, antibody fragments include Fab, F (ab ′) 2, Fv, or single chain Fv (scFv), sc (Fv) 2 obtained by linking Fv of H chain and L chain with an appropriate linker. .

Furthermore, as the antibody used in the present invention, an antibody bound to various molecules such as polyethylene glycol (PEG) can be used as a modified antibody. The “antibody” referred to in the present invention includes these modified antibodies. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.

Tocilizumab, a humanized anti-human IL-6 receptor antibody, is present in rheumatoid arthritis, childhood chronic arthritis (eg, juvenile idiopathic arthritis active in multiple joints, systemic juvenile idiopathic arthritis), Castleman's disease It is approved in Japan as a therapeutic drug.

The above-mentioned antibody of the present invention is an antibody whose antigen neutralizing ability, blood kinetics, immunogenicity, safety and physical properties are improved by modifying the amino acid of Tocilizumab. Accordingly, the above-described antibody of the present invention naturally has the same therapeutic effect as Tocilizumab, and can be used as a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis, and Castleman's disease.

The therapeutic agent of the present invention can be administered in the form of a pharmaceutical, and can be administered systemically or locally orally or parenterally. For example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent and the like can be selected, and the administration method can be appropriately selected depending on the age and symptoms of the patient. The effective dose is usually selected in the range of 0.01 mg / kg to 100 mg / kg body weight. Alternatively, a dose of 1 to 1000 mg, preferably 50 to 250 mg per patient can be selected. A preferable dose can be appropriately selected by those skilled in the art.

The therapeutic agent of the present invention may contain a pharmaceutically acceptable carrier such as a preservative and a stabilizer. The pharmaceutically acceptable carrier may itself be a material having the above therapeutic effect or a material having no such therapeutic effect, and is a material that can be administered together with the above therapeutic agent. means. Moreover, although it is a material which does not have a therapeutic effect, the material which has a synergistic or additive stabilization effect by using together with an antibody may be sufficient.

Examples of materials that are acceptable for formulation include sterilized water and physiological saline, stabilizers, excipients, buffers, preservatives, surfactants, chelating agents (EDTA, etc.), binders, and the like. .

In the present invention, examples of the surfactant include nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate; glycerin monocaprylate, glycerin monomyristate. Glycerin fatty acid esters such as glyceryl monostearate; polyglycerin fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate; polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate , Polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene Polyoxyethylene sorbitan fatty acid esters such as sorbitan tristearate; Polyoxyethylene sorbite fatty acid esters such as polyoxyethylene sorbit tetrastearate and polyoxyethylene sorbit tetraoleate; Polyoxyethylene glycerin such as polyoxyethylene glyceryl monostearate Fatty acid ester; polyethylene glycol fatty acid ester such as polyethylene glycol distearate; polyoxyethylene alkyl ether such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxy Polyoxyethylene polyoxypropylene alkyl such as propylene cetyl ether Ether; Polyoxyethylene alkylphenyl ether such as polyoxyethylene nonylphenyl ether; Polyoxyethylene hydrogenated castor oil such as polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogen castor oil); Polyoxyethylene sorbit Typical examples include polyoxyethylene beeswax derivatives such as beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides such as polyoxyethylene stearic acid amides and the like having HLB6-18. be able to.

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

In the therapeutic agent of the present invention, one or more of these surfactants can be added in combination. Preferred surfactants for use in the formulations of the present invention are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60 or 80, with polysorbates 20 and 80 being particularly preferred. Polyoxyethylene polyoxypropylene glycol represented by poloxamer (such as Pluronic F-68 (registered trademark)) is also preferable.

The amount of the surfactant to be added varies depending on the type of the surfactant to be used. In the case of polysorbate 20, polysorbate 80, or poloxamer 188, it is generally 0.0001 to 10% (w / v), preferably about 0.1. 001 to 5%, more preferably 0.005 to 3%.

In the present invention, as the buffering agent, phosphoric acid, citric acid buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, caprylic acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid Other organic acids, etc., or carbonate buffer, Tris buffer, histidine buffer, imidazole buffer and the like can be mentioned.

Alternatively, a solution formulation may be prepared by dissolving in an aqueous buffer known in the field of solution formulation. The concentration of the buffer is generally 1 to 500 μmM, preferably 5 to 100 μmM, and more preferably 10 to 20 μmM.

The therapeutic agent of the present invention may contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, saccharides such as amino acids, polysaccharides and monosaccharides, carbohydrates, and sugar alcohols.

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

In the present invention, examples of saccharides and carbohydrates such as polysaccharides and monosaccharides include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.

In the present invention, examples of the sugar alcohol include mannitol, sorbitol, inositol and the like.

When the drug of the present invention is used as an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride) Can be mixed. The aqueous solution may be used in combination with an appropriate solubilizing agent (for example, alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG etc.), nonionic surfactant (polysorbate 80, HCO-50) etc.).

If desired, it may further contain a diluent, a solubilizer, a pH adjuster, a soothing agent, a sulfur-containing reducing agent, an antioxidant and the like.

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

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

In addition, if necessary, it can be enclosed in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) ) and can also be ^{( "Remington's Pharmaceutical Science 16 th} edition", Oslo Ed., see 1980, etc.). Furthermore, a method of making a drug a sustained-release drug is also known and can be applied to the present invention (Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech 1982, 12: 98-105; US Pat. No. 3,773,919; European Patent Application Publication (EP) 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; EP 133,988). Furthermore, it is also possible to increase the amount of liquid administered subcutaneously by adding or mixing hyaluronidase to this agent (for example, WO2004 / 078140).

The pharmaceutically acceptable carrier to be used is selected appropriately or in combination from the above depending on the dosage form, but is not limited thereto.

The present invention relates to a method for treating rheumatoid arthritis, childhood chronic arthritis, and Castleman's disease, comprising the step of administering the therapeutic agent of the present invention to a subject. In the present invention, the “subject” refers to an organism to which the therapeutic agent of the present invention is administered and a part of the organism. Organisms include, but are not limited to, animals (eg, humans, domestic animal species, wild animals). The “part of the living body” is not particularly limited.

In the present invention, “administering” includes administering orally or parenterally. Oral administration can include administration in the form of an oral agent, and as the oral agent, a dosage form such as a granule, powder, tablet, capsule, solvent, emulsion, or suspension is selected. be able to.

Parenteral administration can include administration in the form of injections, and examples of injections include intravenous injections, subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. Moreover, the effect of the method of the present invention can be achieved by introducing a gene containing an oligonucleotide to be administered into a living body using a gene therapy technique. In addition, the agent of the present invention can be locally administered to an area where treatment is desired. For example, it can be administered by local injection during surgery, the use of a catheter, or targeted gene delivery of DNA encoding a peptide of the invention.

The administration of the therapeutic agent of the present invention to the subject may be performed after the symptoms of the disease appear, or may be administered prophylactically before the symptoms appear.

Furthermore, the present invention relates to the use of the antibody of the present invention in the manufacture of a therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease. The present invention also relates to the antibody of the present invention for use in a method for treating rheumatoid arthritis, childhood chronic arthritis or Castleman's disease.

The amino acids contained in the amino acid sequences described in the present invention are modified after translation (for example, modification to pyroglutamic acid by pyroglutamylation of N-terminal glutamine is a modification well known to those skilled in the art). In some cases, even if the amino acid is post-translationally modified as such, it is naturally included in the amino acid sequence described in the present invention.

The structure of the sugar chain to be bonded may be any structure. The 297th sugar chain of EU numbering may have any sugar chain structure (preferably fucosylated sugar chain), and sugar chains may not be bound (for example, produced in E. coli, or EU numbering) It is possible to modify the sugar chain so that it does not bind to the 297th position).

Note that all prior art documents cited in the present specification are incorporated herein by reference.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

[Example 1] Monkey PK / PD test of anti-human IL-6 receptor antibody TOCILIZUMAB (H chain WT-IgG1 / SEQ ID NO: 13, L chain WT-kappa / SEQ ID NO: 14) and TOCILIZUMAB antigen neutralizing ability Fv4-M73 (H chain VH3-M73 / SEQ ID NO: 9, L chain VL3-kappa / sequence) introduced with TOCILIZUMAB for the purpose of improving blood dynamics, immunogenicity, safety and physical properties No. 10) was expressed and purified by methods known to those skilled in the art (see Reference Examples for methods), and their effects as therapeutic agents for rheumatoid arthritis were examined as follows.

TOCILIZUMAB and Fv4-M73 were administered to cynomolgus monkeys at a dose of 1 mg / kg intravenously and evaluated for changes in plasma concentrations (see Reference Examples for methods). The change in plasma concentration after intravenous administration of TOCILIZUMAB and Fv4-M73 is shown in FIG. As a result, Fv4-M73 significantly improved pharmacokinetics in cynomolgus monkeys compared to TOCILIZUMAB.

Cynomolgus monkey IL-6 5μg from day 6 to day 18 of antibody administration (from day 3 to day 10 for TOCILIZUMAB) to evaluate the efficacy of neutralization of cynomolgus membrane IL-6 receptor / kg was subcutaneously administered daily to the back of the lumbar region, and the CRP concentration of each individual was measured 24 hours later (see Reference Examples for methods). The change in CRP concentration at the time of administration of each antibody is shown in FIG. In order to evaluate the degree of neutralization of cynomolgus monkey soluble IL-6 receptor, the concentration of unbound cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma was measured, and unbound The percentage of soluble IL-6 receptor was calculated (see Reference Example for method). The transition of the unbound soluble IL-6 receptor rate at the time of administration of each antibody is shown in FIG.

Fv4-M73 neutralized cynomolgus monkey membrane-type IL-6 receptor more persistently than TOCILIZUMAB and suppressed the increase in CRP for a long period of time. In addition, Fv4-M73 neutralized cynomolgus monkey-soluble IL-6 receptor more persistently than TOCILIZUMAB, and suppressed the increase of unbound cynomolgus monkey-soluble IL-6 receptor for a long period of time. From this, it was found that Fv4-M73 is superior to TOCILIZUMAB with respect to the persistence of neutralization of membrane IL-6 receptor and soluble IL-6 receptor.

[Example 2]
Monocyte chemoattractant protein (MCP) -1 is known to be involved in cell infiltration of monocytes, T cells, NK cells, and basophil. MCP-1 has been reported to be highly expressed in the synovial tissue and synovial fluid of RA patients (J Clin Invest. 1992 Sep; 90 (3): 772-9) and is involved in the pathology of RA (Inflamm Allergy Drug Targets. 2008 Mar; 7 (1): 53-66.).

VEGF is a potent angiogenic factor and is known to be produced from macrophages, fibroblasts, synovial cells, etc. in the synovium of RA patients (J Rheumatol. 1995 Sep; 22 (9) : 1624-30.) In addition, VEGF levels in RA patients' sera correlated with disease activity and radiographic progression (Arthritis Rheum. 2003 Jun; 48 (6): 1521-9., Arthritis Rheum. 2001 Sep; 44 (9): 2055-64 .), Treatment of RA patients with anti-IL-6R antibody TOCILIZUMAB reduces serum VEGF levels, and VEGF is also considered to play an important role in the pathology of RA (Mod Rheumatol 2009; 19 (1): 12-9, Mediators Inflamm. 2008; 2008: 129873).

Therefore, the following method was used to examine whether TOCILIZUMAB and Fv4-M73 could suppress MCP-1 and VEGF production from human RA patient-derived synovial cells by sIL-6R and IL-6 stimulation.

Human RA patient-derived synoviocytes (TOYOBO) were seeded in 2 × 10 ⁴ /0.05 mL / well in 96% plate in IMDM medium containing 5% FCS, and in a CO ₂ incubator (37 ° C, 5% CO ₂ ) Left for 90 minutes. Add 0.05 mL of appropriately diluted concentrations of TOCILIZUMAB and Fv4-M73, let stand for 15 minutes, add 0.05 mL of soluble IL-6 receptor (SR344: prepared according to the method of Reference Example) and let stand for another 30 minutes. Furthermore, 0.05 mL of IL-6 (TORAY) was added (the final concentrations of soluble IL-6 receptor and IL-6 were 50 ng / mL each). After culturing for 2 days, the culture supernatant was collected, and MCP-1 and VEGF concentrations in the culture supernatant were measured using ELISA kit (Biosource and Pierce Biotechnology). The results are shown in FIGS. TOCILIZUMAB and Fv4-M73 inhibited MCP-1 and VEGF production from human RA patient-derived synoviocytes induced by soluble IL-6 receptor and IL-6 in a concentration-dependent manner.

Based on these results, Fv4-M73 has a long-lasting effect as an anti-IL-6 receptor neutralizing antibody (binding to IL-6 receptor and blocking membrane IL-6 receptor and soluble IL-6 receptor signals). Is superior to TOCILIZUMAB, and can significantly reduce the administration frequency and dosage compared to TOCILIZUMAB, and Fv4-M73 is an MCP-1 from human RA patient-derived synoviocytes And suppression of VEGF production, Fv4-M73 was shown to be a very useful therapeutic agent for RA.

<Reference example>
Preparation of recombinant soluble human IL-6 receptor Recombinant soluble human IL-6 receptor of human IL-6 receptor, which is an antigen, was prepared as follows. J. Biochem. 108, 673-676 (1990) reported soluble human IL-6 receptor consisting of the amino acid sequence from the 1st to 344th N-terminal side (Yamasaki et al., Science 1988; 241: 825-828 (GenBank # X12830)) CHO cell constant expression strain was prepared. From the culture supernatant obtained from SR344-expressing CHO cells, a soluble type was obtained by three column chromatography: Blue Sepharose 6 FF column chromatography, affinity chromatography with a column immobilized with a specific antibody against SR344, and gel filtration column chromatography. Human IL-6 receptor was purified. The fraction eluted as the main peak was used as the final purified product.

Preparation of recombinant soluble cynomolgus monkey IL-6 receptor (cIL-6R) Published rhesus monkey IL-6 receptor gene sequence (Birney et al, Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): D556 Based on -61.), An oligo DNA primer was prepared, and a DNA fragment encoding the full length of the cynomolgus IL-6 receptor gene was prepared by PCR using the cDNA prepared from cynomolgus monkey pancreas as a template. The obtained DNA fragment was inserted into an animal cell expression vector, and a CHO constant expression strain (cyno.sIL-6R-producing CHO cell) was produced using this. After purifying the culture fluid of cyno.sIL-6R-producing CHO cells using HisTrap column (GE Healthcare Bioscience), it is concentrated using Amicon Ultra-15 Ultracel-10k (Millipore), and Superdex200pg16 / 60 gel filtration column (GE Health) The product was further purified by Care Bioscience, and used as a final purified product of soluble cynomolgus monkey IL-6 receptor (hereinafter cIL-6R).

Preparation of recombinant cynomolgus monkey IL-6 (cIL-6) Cynomolgus monkey IL-6 was prepared as follows. A base sequence encoding 212 amino acids registered in SWISSPROT Accession No.P79341 was created, cloned into an animal cell expression vector, and introduced into CHO cells to produce a constant expression cell line (cyno.IL-6 production) CHO cells). After purifying the culture fluid of cyno.IL-6 producing CHO cells with SP-Sepharose / FF column (GE Healthcare Bioscience), using Amicon Ultra-15 Ultracel-5k (Millipore) and concentrating, Superdex75pg26 / 60 gel filtration Further purification was performed using a column (GE Healthcare Bioscience), and concentration was performed using Amicon Ultra-15 Ultracel-5k (Millipore) to obtain a final purified product of cynomolgus monkey IL-6 (hereinafter cIL-6).

Preparation, expression and purification of TOCILIZUMAB mutants Plasmid fragments encoding the desired antibody sequences were inserted into animal cell expression vectors to prepare the desired H chain expression vectors and L chain expression vectors. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art. Antibody expression was carried out using the following method. HEK293H derived from human fetal kidney cancer cells (Invitrogen) is suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and the dish (diameter) is 5-6 x 10 ⁵ cells / mL. 10 mL to each dish of 10 cm, CORNING) After culturing overnight in a CO ₂ incubator (37 ° C, 5% CO ₂ ), the medium is removed by suction and CHO-S-SFM-II (Invitrogen) medium 6.9 mL was added. The prepared plasmid was introduced into cells by the lipofection method. After collecting the obtained culture supernatant, the cells are removed by centrifugation (approximately 2000 g, 5 minutes, room temperature), and further sterilized through a 0.22 μm filter MILLEX (R) -GV (Millipore). Got. The antibody was purified from the obtained culture supernatant by a method known to those skilled in the art using rProtein A Sepharose ™ Fast Flow (Amersham Biosciences). The purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

Measurement of Antibody Plasma Concentration, CRP Concentration, and Unbound Soluble IL-6 Receptor by Monkey PK / PD Test Cynomolgus monkey plasma concentration was measured by ELISA using a method known to those skilled in the art. CRP concentration was measured with Sias R CRP (Kanto Chemical Co., Inc.) using an automatic analyzer (TBA-120FR, Toshiba Medical Systems Co., Ltd.). The concentration of unbound soluble cynomolgus monkey IL-6 receptor in cynomolgus monkey plasma was measured as follows. By adding 30 μL of cynomolgus monkey plasma to an appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 μm filter cup (Millipore), all IgG type antibodies (cynomolgus IgG, anti-human) IL-6 receptor antibody and anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6 receptor complex) were adsorbed to Protein A. Then, it spin-down with the high-speed centrifuge and collect | recovered pass solutions. Since the path solution does not contain the anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6 receptor complex bound to protein A, the concentration of soluble cynomolgus IL-6 receptor in the protein A path solution should be measured. Can measure the concentration of unbound soluble IL-6 receptor. Soluble cynomolgus monkey IL-6 receptor concentration was measured by a method known to those skilled in the art to measure human IL-6 receptor concentration using the soluble cynomolgus monkey IL-6 receptor (cIL-6R) prepared above as a standard. did. The non-binding soluble IL-6 receptor ratio was calculated by the following formula.

(Unbound soluble IL-6 receptor concentration after antibody administration / soluble IL-6 receptor concentration before antibody administration) × 100

Claims

A therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing the following antibody as an active ingredient;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence of M73 CDR3),
(b) CDR1 having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3), SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence of:
(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 (VH3- A heavy chain comprising CDR3 having the amino acid sequence described in CDR3) of M73, and CDR1, having the amino acid sequence described in SEQ ID NO: 4 (CDR1 of VL3), and the amino acid sequence described in SEQ ID NO: 5 (CDR2 of VL3) An antibody comprising a light chain comprising CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3).
A therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing the following antibody as an active ingredient;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73);
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3) An antibody comprising a light chain comprising a chain variable region.
A therapeutic agent for rheumatoid arthritis, childhood chronic arthritis or Castleman's disease containing the following antibody as an active ingredient;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),
(c) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73) and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).