HK1185535B - Preventive or remedy for inflammatory disease - Google Patents

Description

Preventive or therapeutic agent for inflammatory diseases

This application is a divisional application of patent applications having a filing date of 2007, 6/8.2007, a filing number of 200780029602.8 (international application number of PCT/JP2007/061625), and an invention name of "preventive or therapeutic agent for inflammatory diseases".

Technical Field

The present invention relates to a novel prophylactic or therapeutic agent for inflammatory diseases, which comprises an NR10 antagonist as an active ingredient. The invention also relates to methods of preventing or treating inflammatory diseases using NR10 antagonists.

Background

As liquid factors involved in the activation of the functions of cells that proliferate, differentiate, or mature in various cells, various cytokines are known. Cells stimulated by cytokines in the body produce additional cytokines, forming a network from multiple cytokines. The homeostasis is kept in delicate balance by the mutual regulation of the network. Various immunoinflammatory diseases are thought to be caused by the disruption of these cytokine networks, and monoclonal antibody-based anti-cytokine therapies are of great interest. For example, anti-TNF antibodies and anti-IL-6 receptor antibodies have been shown to be clinically superior. However, in actual pathological conditions, since the compensatory pathway acts, a therapeutic effect cannot be obtained only by blocking one cytokine such as IL-4, and thus there are many cases where the therapeutic effect fails.

The present inventors succeeded in isolating a novel cytokine receptor NR10 having high homology with gp130, a signaling receptor for IL-6 (patent document 1). NR10 forms a heterodimer with oncostatin M receptor (OSMR), and functions as an IL-31 receptor (non-patent document 1). Zymogenetics reports that transgenic mice overexpressing IL-31 naturally develop pruritic dermatitis (patent document 2).

However, it has not been possible to assert that forced expression of cytokines in mice or high cytokine concentrations in the blood of diseased mice are the actual causes. It is not known at all whether a therapeutic effect is obtained when the signal is blocked by an antibody. For example, transgenic mice that overexpress IL-18 in keratinocytes develop pruritic dermatitis. In addition, the blood IL-18 concentration in the natural onset model NC/Nga mice of atopic dermatitis increased with the development of the disease state. Based on the above findings, it is presumed that overexpression of IL-18 is causative. However, the therapeutic effect of administration of a neutralizing antibody has not been actually confirmed (non-patent document 2).

As described above, in diseases in which the expression of cytokines is increased, even if the function of the cytokines is inhibited, a therapeutic effect is not necessarily obtained, and it is difficult to estimate a disease in which a therapeutic effect is actually obtained from the expression level of the cytokines. Therefore, it is important to find diseases that actually achieve therapeutic effects by blocking the signal transmission of target cytokines.

The prior art documents of the present invention are as follows.

Patent document 1: WO 00/75314;

patent document 2: WO 03/060090;

non-patent document 1: IL-31 is associated with cutaneous lymphocyte antigen-positive skin cells with atopic dermatitis T cells in atopic dermatitis patients, J Allergy Clin Immunol.2006 Feb; 117(2): 418-25;

non-patent document 2: administration of anti-interleukin 18 antibody tissues of inflammation in atopic dermatitis-model mice NC/Nga (Administration of anti-interleukin 18 antibody does not inhibit the development of dermatitis in atopic dermatitis model mice NC/Nga), British journal of Dermatology 149: 39-45, 2003.

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made in view of the above background, and an object of the present invention is to provide an anti-cytokine therapy for treating inflammatory diseases using a cytokine receptor antagonist. More specifically, the present invention addresses the problem of finding inflammatory diseases that can be treated with an anti-NR 10 neutralizing antibody, and providing a novel therapeutic approach for these diseases. Another object of the present invention is to provide a neutralizing antibody against human NR10, which can be used clinically in humans.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above problems. The present inventors have attempted to evaluate the efficacy of an anti-mouse NR10 neutralizing antibody against various disease model mice. The results showed that the neutralizing antibody showed a significant symptom-inhibiting effect in an atopic dermatitis model mouse using NC/Nga mice and a chronic dermatitis model mouse produced by repeatedly applying picryl chloride, and it was found that the neutralizing antibody can be practically used as a therapeutic agent. In addition, the effect of suppressing symptoms was also confirmed in collagen arthritis, a model of rheumatism, and collagenase arthritis, a model of osteoarthritis. The above results show that: the NR10 neutralizing antibodies of the present application are useful for the prevention or treatment of chronic inflammation.

Furthermore, the present inventors have succeeded in obtaining a human NR 10-neutralizing antibody. Namely, the present invention provides the following [1] to [17 ].

[1] A prophylactic or therapeutic agent for inflammatory diseases, which comprises an NR10 antagonist as an active ingredient.

[2] [1] the prophylactic or therapeutic agent according to, wherein the NR10 antagonist is an antibody having a NR10 neutralizing activity.

[3] [2] the prophylactic or therapeutic agent according to any one of the above, wherein the antibody is a monoclonal antibody.

[4] [2] the prophylactic or therapeutic agent according to any one of the above, wherein the antibody is a monoclonal antibody having a neutralizing activity against human NR10.

[5] The prophylactic or therapeutic agent according to any one of [2] to [4], wherein the antibody is a recombinant antibody.

[6] [5] the prophylactic or therapeutic agent according to any one of the above aspects, wherein the recombinant antibody is a chimeric antibody, a humanized antibody or a human antibody.

[7] The prophylactic or therapeutic agent according to any one of [2] to [6], which comprises a fragment of an antibody having NR10 neutralizing activity and/or a modified product thereof as an active ingredient.

[8] The prophylactic or therapeutic agent according to any one of [1] to [7], wherein the inflammatory disease is atopic dermatitis.

[9] The prophylactic or therapeutic agent according to any one of [1] to [7], wherein the inflammatory disease is chronic dermatitis.

[10] The prophylactic or therapeutic agent of any one of [1] to [7], wherein the inflammatory disease is rheumatism.

[11] The prophylactic or therapeutic agent according to any one of [1] to [7], wherein the inflammatory disease is osteoarthritis.

[12] An antibody having NR10 neutralizing activity.

[13] [12] the antibody according to [1], which is a monoclonal antibody.

[14] [12] the antibody according to, wherein NR10 is human NR10.

[15] The antibody according to any one of [12] to [14], which is a recombinant antibody.

[16] [15] the antibody according to any one of the above, wherein the recombinant antibody is a chimeric antibody, a humanized antibody or a human antibody.

[17] A fragment of the antibody according to any one of [12] to [16], and/or a modified product thereof.

[18] A method of preventing or treating an inflammatory disease, the method comprising the step of administering an NR10 antagonist to a patient having an inflammatory disease.

[19] Use of an NR10 antagonist in the manufacture of a medicament for the prevention or treatment of an inflammatory disease.

Brief Description of Drawings

FIG. 1 is a graph showing the results of observing the cell growth inhibitory effect of BM095 when BM095 is added to an IL-31-dependent Ba/F3 cell growth assay system. The horizontal axis represents the estimated concentration of mIL-31 in the measurement system, and the vertical axis (OD450 (absorbance at 450 nm)) represents the cell mass. All the examples show the addition amount of BM095 (unit: ng/mL). The cell growth inhibitory effect was observed depending on the amount of BM095 added.

FIG. 2 is a graph showing the therapeutic effect of an anti-NR 10 antibody administered to an atopic dermatitis model mouse. A significant inflammation inhibitory effect was confirmed in the anti-NR 10 antibody-administered group compared to the negative control group (vehicle).

Fig. 3 is a graph showing the change in body weight with time when an anti-NR 10 antibody was administered to atopic dermatitis model mice. Although weight loss was observed in the existing anti-inflammatory drug-administered group, no weight change occurred in the anti-NR 10-administered group, and the safety of the anti-NR 10 antibody was confirmed.

Fig. 4 is a graph showing the therapeutic effect of the anti-NR 10 antibody administered to chronic dermatitis model mice. A significant auricle swelling inhibitory effect was confirmed in the anti-NR 10 antibody administration group.

FIG. 5 is a photograph showing immunohistological staining of auricles of mice as models of chronic dermatitis. As in human, the expression of mouse NR10 was increased in the thickened epidermis.

FIG. 6 is a graph showing the effect of inhibiting the onset of arthritis when an anti-NR 10 antibody is administered to a collagen-induced arthritis model mouse.

Fig. 7 is a graph showing the relationship between the administration concentration of BM095 and AUC in the collagenase-induced arthritis (osteoarthritis) model. The difference in width between the right and left knee joints is used as a value indicating swelling of the right knee joint, and AUC means the area under the curve in which the difference in width changes.

FIG. 8 is a graph showing the correlation between the concentration of a human NR10 neutralizing antibody (purified antibody) and the cell proliferation inhibitory activity in the presence of IL-31. Antibody 1, antibody 2 and antibody 3 showed high NR10 neutralizing activity.

FIG. 9 is a graph showing the correlation between the concentration of the chimeric antibody NA633 against human NR10 in the presence of IL-31 and the cell proliferation inhibitory activity. NA633 showed high NR10 neutralizing activity.

FIG. 10 is a diagram comparing the amino acid sequences of cynomolgus monkey (cynomol) NR10 with human NR10. The double underlined sequence indicates the transmembrane domain.

FIG. 11 is a graph showing cell proliferation inhibitory activity of the chimeric NA633 antibody in human IL-31-stimulated cynomolgus monkey NR 10/human OSMR/BaF cell line. NA633 also showed neutralizing activity against cynomolgus NR10.

FIG. 12 is a graph showing the rate of change of body weight in mice that are models of DSS colitis.

Fig. 13 is a graph showing the transition of changes in the thickness of a pinna in the acute picryl chloride contact dermatitis model.

Best Mode for Carrying Out The Invention

The present invention relates to a prophylactic or therapeutic agent for inflammatory diseases, which comprises an NR10 antagonist as an active ingredient. The present invention is based on the finding by the present inventors that an NR10 antagonist (e.g., an anti-NR 10 neutralizing antibody) significantly suppresses its symptoms in model mice suffering from atopic dermatitis, chronic dermatitis, rheumatism, osteoarthritis, and the like.

NR10 is a protein that forms a heterodimer with the oncostatin M receptor (OSMR) and functions as an IL-31 receptor. NR10 is also known under the names glm-r (J Biol Chem 277, 16831-6, 2002), GPL (J Biol Chem 278,49850-9, 2003), IL-31RA (Nat Immunol 5, 752-60, 2004) and the like, and NR10 of the present invention also includes proteins referred to by the above names. The NR10 of the present invention also includes NR10 derived from a human, mouse or other mammal, and preferred examples of NR10 include, but are not particularly limited to: NR10 from human or mouse. As NR10 derived from human, various splice variants are known (WO 00/075314). Among the above splice variants, NR10.1 is characterized by comprising 662 amino acids and having a transmembrane domain. And NR10.2 is a soluble receptor-like protein comprising 252 amino acids and no transmembrane domain. On the other hand, NR10 splice variants that function as transmembrane type receptor proteins are known as NR10.3 and IL-31RAv 3. The human NR10 in the present invention is not particularly limited as long as it is a protein that forms a heterodimer with oncostatin M receptor (OSMR) and functions as an IL-31 receptor, but preferred examples of NR10 include: NR10.3 (also known as ILRAv4 (Nat Immunol 5, 752-60, 2004)) and IL-31RAv 3. NR10.3 (IL-31RAv4) comprises 662 amino acids (WO00/075314, Nat Immunol 5, 752-60, 2004), whereas IL-31RAv3 comprises 732 amino acids (GenBank accession NM-139017). The amino acid sequence of IL-31RAv4 is shown in SEQ ID No. 6, and the amino acid sequence of IL-31RAv3 is shown in SEQ ID No. 7. On the other hand, examples of mouse-derived NR10 are: a protein comprising the amino acid sequence shown in SEQ ID No. 5.

In the present invention, an NR10 antagonist refers to a substance that blocks intracellular signaling based on NR10 activation by binding to NR10, so that the physiological activity of a cell is lost or inhibited. Examples of physiological activities are: the production-inducing activity, production-inhibiting activity, secretion-promoting activity, secretion-inhibiting activity, proliferation-inducing activity, survival activity, differentiation-inducing activity, transcription activity, membrane transport activity, binding activity, proteolytic activity, phosphorylation/dephosphorylation activity, redox activity, transfer activity, nucleolytic activity, dehydration activity, cell death-inducing activity, apoptosis-inducing activity, and the like of a physiologically active substance (for example, a chemokine, an inflammatory cytokine, and the like), but are not limited thereto.

Determination of whether an antagonistic activity is present can be carried out using methods known to those skilled in the art. For example, the test compound may be contacted with NR10 expressed on the cell surface in the presence of a ligand to determine whether or not intracellular signaling is generated as an indicator of NR10 activation. The determination can be carried out, for example, according to the method described in "Dillon SR et al, Interleukin 31, a cytokine produced by activated T cells, and indeces dermatitin mice (cytokine-Interleukin 31 produced by activated T cells induces mouse dermatitis),. Nat Immunol 2004Jul; 5(7): 752-60". Compounds that block intracellular signaling in response to ligand stimulation are considered antagonists of NR10.

The antagonists of the present invention may be natural or artificial compounds. The antagonist of the present invention may be any known antagonist. Novel compounds determined to have antagonistic activity by the above-described method can also be used.

As one form of the NR10 antagonist in the present invention, an antibody having a NR10 neutralizing activity can be exemplified. The "antibody having NR10 neutralizing activity" in the present invention refers to an antibody having an effect of inhibiting physiological activity based on NR10. The "antibody having NR10 neutralizing activity" in the present invention may be either a polyclonal antibody or a monoclonal antibody, but is preferably in the form of a monoclonal antibody.

A monoclonal antibody having NR10 neutralizing activity can be obtained as follows: an anti-NR 10 monoclonal antibody is prepared by a known method using NR10 derived from a mammal such as a human or a mouse or a fragment peptide thereof as an immunogen, and then an antibody having NR10 neutralizing activity is selected from the obtained anti-NR 10 monoclonal antibody. That is, a desired antigen or a cell expressing a desired antigen is used as a sensitizing antigen, and the immunization is performed according to a usual immunization method. The obtained immune cells are fused with known parental cells by a general cell fusion method, and monoclonal antibody-producing cells (hybridomas) are screened by a general screening method to produce an anti-NR 10 monoclonal antibody. The animals to be immunized can be, for example: mammals such as mice, rats, rabbits, sheep, monkeys, goats, donkeys, cows, horses, and pigs. The antigen can be produced by a known method, for example, a method using baculovirus (WO 98/46777) using a known NR10 gene sequence. When an antibody having a NR10 neutralizing activity is screened, for example, in the examples, whether or not the antibody is an antibody having a NR10 neutralizing activity can be determined by observing the growth inhibitory effect of an IL-31-dependent cell line when a candidate antibody is added to the IL-31-dependent cell line, as described below. In the above-mentioned method, it is considered that the antibody inhibiting the proliferation of the IL-31-dependent cell line is an antibody having NR10 neutralizing activity.

Hybridomas can be prepared, for example, by the method of Milstein et al (Kohler, G., and Milstein, C., Methods enzymol. (1981) 73: 3-46). When the immunogenicity of an antigen is low, immunization can be carried out by binding the antigen to a large immunogenic molecule such as albumin.

Examples of preferred forms of the antibody having NR10 neutralizing activity of the present invention are: a monoclonal antibody having neutralizing activity against human NR10. The immunogen used for producing a monoclonal antibody having human NR10 neutralizing activity is not particularly limited as long as an antibody having human NR10 neutralizing activity can be produced. For example, although there are known a plurality of variants in human NR10, any variant may be used as an immunogen as long as an antibody having human NR10 neutralizing activity can be produced. Alternatively, under the same conditions, a fragment peptide of NR10 or a sequence obtained by introducing artificial mutation into a natural NR10 sequence may be used as an immunogen. Human NR10.3 is one of the preferred immunogens in making antibodies of the invention with NR10 neutralizing activity.

The antibody of the present invention is not particularly limited as long as it has NR10 neutralizing activity, and includes recombinant antibodies such as chimeric antibodies, humanized antibodies, and human antibodies. A chimeric antibody is an antibody comprising the variable regions of the heavy and light chains of a mammalian, e.g., mouse, antibody, except human, and the constant regions of the heavy and light chains of a human antibody. The chimeric antibody can be produced by a known method. For example, chimeric ANTIBODIES can be produced by cloning antibody genes from hybridomas, inserting them into an appropriate vector, and introducing them into a host (see, e.g., Carl, A.K., Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom byMACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA of the antibody variable region (V region) is synthesized from mRNA of the hybridoma using reverse transcriptase. When a DNA encoding the V region of the antibody of interest is obtained, it is ligated with a DNA encoding the constant region (C region) of the desired human antibody, and inserted into an expression vector. Alternatively, a DNA encoding the V region of an antibody may be inserted into an expression vector including a DNA of the C region of a human antibody. For expression under the regulation of expression regulatory regions such as enhancers, promoters, the DNA is inserted into an expression vector. Subsequently, the expression vector can be used to transform a host cell and express the chimeric antibody.

A humanized antibody, also called a reshaped human antibody, is an antibody obtained by grafting Complementarity Determining Regions (CDRs) of a non-human mammal, for example, a mouse antibody, to the complementarity determining regions of a human antibody, and a general genetic recombination method thereof is also known. Specifically, a DNA sequence designed to link CDRs of a mouse antibody and Framework Regions (FRs) of a human antibody is synthesized by PCR from a plurality of oligonucleotides whose terminal portions have overlapping portions. Humanized antibodies can be obtained as follows: the resulting DNA is ligated with a DNA encoding a human antibody constant region, and then inserted into an expression vector, which is introduced into a host to produce an antibody (see European patent application publication No. EP239400, International patent application publication No. WO 96/02576). FRs of human antibodies connected via CDRs are screened for FRs whose complementarity determining regions form good antigen binding sites. If necessary, amino acids in the framework regions of the antibody variable regions may be substituted so that the complementarity determining regions of the reshaped human antibody form appropriate antigen-binding sites (Sato, K., et al, Cancer Res. (1993) 53, 851 856).

In addition, methods for obtaining human antibodies are also known. For example, a desired human antibody having an antigen-binding activity can be obtained by sensitizing human lymphocytes with a desired antigen or cells expressing a desired antigen in vitro and fusing the sensitized lymphocytes with human myeloma cells such as U266 (see Japanese examined patent publication (JP-B) No. 1-59878). Alternatively, a desired human antibody can be obtained by immunizing a transgenic animal having a complete human antibody gene bank with a desired antigen (see International patent application publication Nos. WO93/12227, WO92/03918, WO94/02602, WO94/25585, WO96/34096, WO 96/33735).

Also, a technique for obtaining a human antibody by a screening method using a human antibody phage library is known. For example, phage display can be used to express the variable region of a human antibody as a single chain antibody (scFv) on the surface of a phage, and phage that bind to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector having the sequence can be prepared, and a human antibody can be obtained. The above-mentioned methods are widely known and can be referred to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388 and the like.

The amino acid sequence of the heavy chain variable region or the light chain variable region may be one in which one or more amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of the heavy chain variable region or the light chain variable region of an antibody confirmed to have NR10 neutralizing activity, as long as the amino acid sequence has NR10 neutralizing activity. As a method well known to those skilled in the art for preparing an amino acid sequence of a heavy chain variable region or a light chain variable region of an antibody having NR10 neutralizing activity by substituting, deleting, adding and/or inserting one or more amino acids in the amino acid sequence of the heavy chain variable region or the light chain variable region, a method for introducing a mutation into a protein is known. For example, one skilled in The art can determine The expression level of a Gene by using The site-specific mutagenesis method (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y and Nakagawa, M. (1995) An Oligonucleotide-oxidized dual-plasmid method for site-directed mutagenesis. Gene 152, 271-fragment of DNA fragments cloned into M13 vectors (Oligonucleotide site-directed mutagenesis of DNA fragments cloned into M13 vector.) Methods Enzyl. 100, 468-fragment 500, Hkramer, W, Drsaratio, V, HW, Jatamer, B, Pflukibe, Pflukipedia, DNA fragment of DNA fragments cloned into M13 vector. DNA encoding DNA, 19832-fragment, DNA encoding DNA, 1985, Hphosphor, W, D, P, 350-; kunkel, TA (1985) Rapid and effective site-specific mutagenesis with phenotyping selection (a Rapid and efficient site-specific mutagenesis method not requiring phenotypic selection), Proc Natl Acad Sci USA 82, 488-cohun 492), etc. by introducing an appropriate mutation into the amino acid sequence of the heavy chain variable region or the light chain variable region of an antibody having NR10 neutralizing activity, a mutant having the same function as that of the heavy chain variable region or the light chain variable region of an antibody having NR10 neutralizing activity can be prepared. Thus, a heavy chain variable region or a light chain variable region of an antibody having a mutation of one or more amino acids in the heavy chain variable region or the light chain variable region and having NR10 neutralizing activity is also included in the heavy chain variable region or the light chain variable region of the present invention.

When an amino acid residue is changed, it is preferably mutated to another amino acid that retains the property of an amino acid side chain. Examples of the properties of the amino acid side chain include: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (all of which are marked by one letter of amino acids in parentheses). Substitutions of amino acids within the above groups are referred to as conservative substitutions. Polypeptides comprising amino acid sequences modified by deletion, addition of one or more amino acid residues and/or substitution with other amino Acids are known to maintain their original biological activity (Mark, D. F. et al, Proc. Natl. Acad. Sci. USA (1984) 81: 5662-6; Zoller, M. J. and Smith, M., Nucleic Acids Res. (1982) 10: 6487-500; Wang, A. et al, Science (1984) 224: 1431-3; Dalbadie-McFaand, G. et al, Proc. Natl. Acad. Sci. USA (1982) 79: 6409-13). The above mutant has at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence homology with the amino acid sequence of the heavy chain variable region or the light chain variable region of the present invention. In the present specification, sequence homology is defined as: the ratio of residues identical to those of the amino acid sequence of the original heavy chain variable region or light chain variable region after aligning the sequences as necessary and introducing gaps as appropriate to maximize the sequence homology. The homology of amino acid sequences can be determined by the methods described above.

In addition, the amino acid sequence of the heavy chain variable region or light chain variable region in which one or more amino acids are substituted, deleted, added, and/or inserted and which has NR10 neutralizing activity in the amino acid sequence of the heavy chain variable region or light chain variable region may also be obtained from a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region or light chain variable region. As stringent hybridization conditions for isolating a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a nucleotide sequence encoding an amino acid sequence for a heavy chain variable region or a light chain variable region, there can be exemplified: 6M urea, 0.4% SDS, 0.5XSSC, 37 ℃ or the same strict hybridization conditions. When more stringent conditions are used, for example, 6M urea, 0.4% SDS, 0.1XSSC, 42 ℃ conditions, it is expected that nucleic acids having higher homology will be isolated. The sequence of the isolated nucleic acid can be determined by a known method described later. Regarding the homology of the isolated nucleic acids, the homology has a sequence homology of at least 50% or more, more preferably 70% or more, and still more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, 99% or more) in the entire nucleotide sequence.

In addition to the methods using the hybridization techniques described above, a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region or the light chain variable region can also be isolated using a gene amplification method using primers synthesized based on the nucleotide sequence information of the amino acid sequence encoding the heavy chain variable region or the light chain variable region, for example, a Polymerase Chain Reaction (PCR) method.

The homology of nucleotide sequences and amino acid sequences can be determined by the algorithm BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90, 5873-7). Programs called BLASTN and BLASTX were developed based on this algorithm (Altschul et al, J. mol. biol. (1990) 215, 403-10). When nucleotide sequences are analyzed by BLASTN according to BLAST, the parameters are set as follows: score (score) =100, word length (wordlength) = 12. When an amino acid sequence is analyzed by BLASTX according to BLAST, the following parameters are set: score =50, word length = 3. When BLAST and Gapped BLAST programs are used, the default parameters for each program are used. Specific methods for the above analysis are widely known (see the website of BLAST (basic Localalignment Search tool) of NCBI (national Center for Biotechnology information); http:// www.ncbi.nlm.nih.gov)

The antibody of the present invention may be a low molecular weight antibody. The low molecular weight antibody of the present invention includes an antibody fragment in which a part of a whole antibody (for example, whole IgG) is deleted, and is not particularly limited as long as it has NR10 neutralizing activity. The low molecular weight antibody of the present invention is not particularly limited as long as it is a part of a whole antibody, but preferably includes a heavy chain variable region (VH) or a light chain variable region (VL), and particularly preferably includes both VH and VL.

The low molecular weight antibody of the present invention is preferably smaller than the molecular weight of the whole antibody, but may form multimers such as dimers, trimers, tetramers, and the like, and may have a larger molecular weight than the whole antibody.

One of the low molecular weight antibodies of the present invention is, for example, an scFv antibody. scFv antibody refers to a single-chain polypeptide antibody obtained by ligating a heavy chain variable region ([ VH ]) and a light chain variable region ([ VL ]) via a linker or the like (Huston, J. S. et al, Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879. sup. 5883; Plickthun "the pharmaceutical of Monoclonal Antibodies" 113, edited by Resenburg and Moore, Springer Verlag, New York, pp. 269, 315, (1994)). The order of the heavy chain variable region and the light chain variable region to be linked is not particularly limited, and the sequences may be arranged in any order, for example, the following sequences.

[ VH ] linker [ VL ]

[ VL ] linker [ VH ]

The amino acid sequence of the heavy chain variable region or the light chain variable region may be substituted, deleted, added, and/or inserted. In addition, when the heavy chain variable region is associated with the light chain variable region, a part thereof may be deleted or another polypeptide may be added as long as it has an antigen binding activity. And the variable regions may be chimeric or humanized.

In the present invention, the linker for linking the antibody variable region may be used: any peptide linker or linker of synthetic compounds which can be introduced by genetic Engineering, for example linkers as disclosed in Protein Engineering, 9(3), 299-305, 1996.

Preferred linkers of the invention are peptide linkers. The length of the peptide linker is not particularly limited, and may be appropriately selected by those skilled in the art according to the purpose, but is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and particularly preferably 12 to 18 amino acids (for example, 15 amino acids).

Examples of the amino acid sequence of the peptide linker are the following sequences.

Ser

Gly•Ser

Gly•Gly•Ser

Ser•Gly•Gly

Gly•Gly•Gly•Ser (SEQ ID No:8)

Ser•Gly•Gly•Gly (SEQ ID No:9)

Gly•Gly•Gly•Gly•Ser (SEQ ID No:10)

Ser•Gly•Gly•Gly•Gly (SEQ ID No:11)

Gly•Gly•Gly•Gly•Gly•Ser (SEQ ID No:12)

Ser•Gly•Gly•Gly•Gly•Gly (SEQ ID No:13)

Gly•Gly•Gly•Gly•Gly•Gly•Ser (SEQ ID No:14)

Ser•Gly•Gly•Gly•Gly•Gly•Gly (SEQ ID No:15)

(Gly•Gly•Gly•Gly•Ser (SEQ ID No:10))n

(Ser•Gly•Gly•Gly•Gly (SEQ ID No:11))n

Examples thereof include [ n is an integer of 1 or more ].

Synthetic compound linkers (chemical crosslinkers) are crosslinkers commonly used for peptide crosslinking, such as: n-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl suberate) (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene glycol bis (sulfosuccinimidyl succinate) (Sulfo-EGS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (Sulfo-DST), bis [2- (succinimidyloxycarbonyloxy) ethyl ] sulfone (BSOCOES), bis [2- (sulfosuccinimidyloxycarbonyloxy) ethyl ] sulfone (Sulfo-BSOCOES), and the like, and the above-mentioned crosslinking agents are commercially available.

The method for producing the fusion protein may be carried out by, for example, ligating a polynucleotide encoding the antibody of the present invention with a polynucleotide encoding another peptide or polypeptide, introducing the resulting construct into an expression vector, and expressing the construct in a host, by a method known to those skilled in the art, and other peptides or polypeptides fused with the antibody of the present invention may be expressed, for example, by using FLAG (Hopp, T. P. et al., Biotechnology (1988) 6, 1204-1210), 6 XHis comprising 6 His (histidine) residues, 10 XHis, influenza Hemagglutinin (HA), human C-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, microtubule tag, α -tag fragment, B-tag, a fragment of a protein, a polypeptide encoding a protein of the present invention, a polypeptide encoding a protein of the polypeptide of the present invention, a commercially available polypeptide such as a polypeptide, a polypeptide encoding a glutathione-transferase, a polypeptide of the present invention, a glutathione-polypeptide, a commercially available polypeptide, a polypeptide of the present invention, a polypeptide of the present invention, a glutathione-galactosidase-tag, a polypeptide of the present invention, a commercially available polypeptide of the present invention, a variety of the present invention, a variety of.

The antibody of the present invention differs in amino acid sequence, molecular weight, isoelectric point, or whether it has a sugar chain or conformation depending on the cell or host producing the antibody or the purification method described later. However, the antibody obtained is included in the present invention as long as it has the same function as the antibody of the present invention. For example, when the antibody of the present invention is expressed in a prokaryotic cell such as E.coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original antibody. The antibodies of the invention also include such antibodies.

The antibody of the present invention can be prepared by a method known to those skilled in the art. For example, the antibody can be prepared by gene recombination techniques known to those skilled in the art based on the sequence of the antibody recognizing NR10. Specifically, a polynucleotide encoding an antibody is constructed based on the sequence of an antibody recognizing NR10, introduced into an expression vector, and then expressed in an appropriate host cell (see, for example, Co, M. S. et al, J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986)121, 652-663; Rousseaux, J. et al, Methods Enzymol. (1986)121, 663-669; Bird, R. E. and Walker, B. W. Trench. (1991).

Examples of the vector include M13-based vector, pUC-based vector, pBR322, pBluescript, pCR-Script, etc. for subcloning and excision of cDNA, in addition to the above-mentioned vector, for example, pGEM-T, pDIRECT, pT7, etc. when a vector is used for producing the antibody of the present invention, the expression vector is particularly useful, and when a vector is used for expression in E.coli, for example, the vector must have a promoter capable of being expressed at high efficiency in E.coli hosts such as JM109, DH5 α, HB101, XL 1-and the like, for example, lacZ promoter (Ward et al, Nature (1989) 341, 544 and 546; FASEB J. (1992) 6, 2422 and 2427), araB promoter (Better et al, Science (1988) 240, 1041 393) or T10456 promoter, and when the vector includes pGEX-5-PCR polymerase (QI) and pGEX-5, QI-4625, as such a promoter.

The vector may also include a signal sequence for antibody secretion. As a signal sequence for antibody secretion, the pelB signal sequence can be used when a protein is secreted into the periplasm of E.coli (Lei, S. P. et al, J. Bacteriol. (1987) 169, 4379). For example, the vector can be introduced into a host cell by the calcium chloride method or the electroporation method.

In addition to E.coli, vectors for producing the antibody of the present invention include, for example: expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen Co., Ltd.) or pEF-BOS (Nucleic acids. Res. 1990, 18(17), page 5322), pEF, pCDM8), Expression vectors derived from insect cells (for example, "Bac-to-BAC baculovirus Expression system" (manufactured by Gibco-BRL), pBacPAK8), Expression vectors derived from plants (for example, pMH1, pMH2), Expression vectors derived from animal viruses (for example, pHSV, pMV, pAdexLcw), Expression vectors derived from retroviruses (for example, pZIPneo), Expression vectors derived from yeasts (for example, "Pichia Expression Kit" (manufactured by Invitrogen), pNV11, SP-Q01), Expression vectors derived from Bacillus (for example, pPL608, pKTH 50).

In order to express in animal cells such as CHO cells, COS cells, and NIH3T3 cells, the vector must have a promoter necessary for intracellular expression, for example, SV40 promoter (Mullgan et al, Nature (1979) 277, 108), MMLV-LTR promoter, EF1 α promoter (Mizushima et al, Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and the vector preferably has a gene for screening transformed cells (for example, a drug resistance gene distinguishable by a drug (neomycin, G418, etc.). examples of vectors having the above properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc.

In order to stably express a gene and to amplify the copy number of the gene in a cell, a method may be mentioned in which a vector having a DHFR gene (for example, pSV2-DHFR (Molecular Cloning second edition, Cold Spring Harbor Laboratory Press, (1989)) for compensating the pathway is introduced into a CHO cell lacking the nucleic acid synthesis pathway, and the vector is amplified with methotrexate Thymidine Kinase (TK) gene, E.coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc. as selection markers.

The antibody of the present invention thus obtained can be isolated from the inside or outside of the host cell (such as a culture medium) and purified to a substantially pure and homogeneous antibody. The antibody can be isolated and purified by a method of isolation and purification generally used for purification of an antibody, but is not limited thereto. For example, the antibody can be separated and purified by appropriately selecting and combining a chromatography column, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric electrophoresis, dialysis, recrystallization, and the like.

Examples of chromatography include: affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and chromatography (Protein Purification and Characterization guide): A Laboratory Course Manual, Ed Daniel R. Marshark et al, Cold spring harbor Laboratory Press, 1996). The above chromatography can be performed using liquid chromatography such as HPLC, FPLC, and the like. The affinity chromatography column has: protein A column and protein G column. Examples of protein a columns are: hyper D, POROS, Sepharose FF (GEAmersham Biosciences), and the like. The present invention also encompasses an antibody highly purified by the above-described purification method.

The present invention also provides a prophylactic or therapeutic agent for inflammatory diseases, which comprises the above-described fragment of the antibody of the present invention and/or a modified product thereof as an active ingredient. The fragment of the antibody of the present invention and/or a modified product thereof includes an antibody fragment in which a part of the antibody of the present invention (a whole antibody (e.g., a whole IgG), a recombinant antibody (e.g., a chimeric antibody, a humanized antibody, a human antibody, etc.), or a low-molecular antibody (e.g., an scFv antibody, etc.)) is deleted, and is not particularly limited as long as it has an antigen binding ability. The antibody fragment of the present invention is not particularly limited as long as it is a part of a whole antibody, but preferably includes a heavy chain variable region (VH) and/or a light chain variable region (VL). The amino acid sequence of VH or VL may comprise substitutions, deletions, additions and/or insertions. Also, VH and/or VL may be partially deleted as long as it has an antigen binding ability. The variable regions may also be chimeric or humanized. Specific examples of antibody fragments are: fab, Fab ', F (ab') 2, Fv, etc. In order to obtain the above-mentioned antibody fragment, it is sufficient to construct a gene encoding the above-mentioned antibody fragment, introduce it into an expression vector, and express it in an appropriate host cell (for example, refer to Co, M. S. et al, J.Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986)121, 652-663; Rousseaux, J. et al, Methods Enzymol. (1986)121, 663-669; R. E. and lker, W. Trendhnol. (1991) 132).

The antibody of the present invention may be a conjugated antibody obtained by binding various molecules such as polyethylene glycol (PEG), radioactive substances, fluorescent substances, luminescent substances, enzymes, and toxins. The above-mentioned conjugated antibody can be obtained by chemically modifying the obtained antibody. It is to be noted that methods for modifying antibodies have been established in the art (e.g., US 5057313, US 5156840). The term "antibody" in the present invention also includes the above-mentioned conjugated antibody.

The measurement of the NR10 binding activity of an antibody can be carried out by a method known to those skilled in the art. For example, a method for measuring the antigen-binding activity of an antibody can employ: ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method. For example, when an enzyme immunoassay method is used, a sample containing an antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added to an antigen-coated plate. The antigen binding activity can be evaluated by adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, washing the plate with a warm bath, adding an enzyme substrate such as p-nitrophenyl phosphate to the washed plate, and measuring the absorbance.

The measurement of the NR10 neutralizing activity of the antibody can be carried out, for example, by observing the growth inhibitory effect of the IL-31-dependent cell line as described in examples.

The NR10 antagonist or the antibody having NR10 neutralizing activity of the present invention can be used in a prophylactic or therapeutic agent for inflammatory diseases. The present inventors confirmed that: when the mouse NR10 neutralizing antibody is administered to various inflammation model animals, a remarkable treatment effect is achieved. In addition, it has been reported that the expression of human NR10 is increased in the hypertrophic epidermis of atopic dermatitis patients (non-patent document 1). On the other hand, the present inventors have confirmed that the expression of mouse NR10 is increased in the thickened epidermis as in the case of human when the above-mentioned auricles of the chronic dermatitis model mice are subjected to immunohistostaining (example 5). The above situation shows that: NR10 is also involved in inflammatory diseases in all animal species, and it is considered that a human NR10 neutralizing antibody is effective for the prevention or treatment of various inflammatory diseases as well as a mouse NR10 neutralizing antibody. And NR10 antagonists other than antibodies are also considered to have therapeutic effects on various inflammatory diseases as seen in the present example.

In the present invention, the inflammatory disease refers to a disease associated with a histological reaction of cytological ∙ occurring in affected blood vessels and adjacent tissues due to damage or abnormal stimulation caused by a physically, chemically or biologically acting substance, accompanied by a pathology (Stedman medical major dictionary, 5 th edition, MEDICAL VIEW co., 2005). Examples of common inflammatory diseases are: dermatitis (atopic dermatitis, chronic dermatitis, etc.), inflammatory bowel disease (colitis, etc.), asthma, arthritis (rheumatoid arthritis, osteoarthritis, etc.), bronchitis, Th-2 type autoimmune disease, systemic lupus erythematosus, myasthenia gravis, chronic GVHD, Crohn's disease, ankylosing spondylitis, lumbago, gout, inflammation after surgical trauma, relief of swelling, neuralgia, pharyngolaryngitis, cystitis, hepatitis (non-alcoholic fatty liver, alcoholic hepatitis, etc.), hepatitis b, hepatitis c, arteriosclerosis, etc.

Preferred examples of inflammatory diseases to be the subject of the present invention include: atopic dermatitis, chronic dermatitis, rheumatism, osteoarthritis, and chronic asthma.

The inventors of the present invention found that: the NR10 antagonist antibody has therapeutic effects on atopic dermatitis, chronic dermatitis, rheumatism, and osteoarthritis. On the other hand, the NR10 antagonist antibody was found to have no therapeutic effect in models of acute contact dermatitis and DSS acute colitis.

The prophylactic or therapeutic agent for inflammatory diseases of the present invention is characterized in that: contains the above-mentioned NR10 antagonist or an antibody having a NR10 neutralizing activity as an active ingredient. The phrase "comprising an NR10 antagonist as an active ingredient" means that at least one of NR10 antagonists is contained as an active ingredient, and the content ratio thereof is not limited. The prophylactic or therapeutic agent for inflammatory diseases of the present invention may contain an NR10 antagonist and other components that promote the prevention or treatment of inflammatory diseases.

The NR10 antagonist of the present invention can be formulated in accordance with a conventional method (e.g., Remington's pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). Further, a pharmaceutically acceptable carrier and/or additive may be contained as required. For example, it may contain: surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, fragrances, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, glidants, flavoring agents, etc. However, the preventive or therapeutic agent for inflammatory diseases of the present invention is not limited thereto, and may contain other commonly used carriers as appropriate. Specific examples are: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like. May further comprise: other low molecular weight polypeptides, serum albumin, gelatin, and immunoglobulin proteins; and amino acids such as glycine, glutamine, asparagine, arginine and lysine. When prepared as an aqueous injection solution, the NR10 antagonist is dissolved in an isotonic solution containing, for example, physiological saline, glucose, or other adjuvants. Examples of adjuvants are: d-sorbitol, D-mannose, D-mannitol, sodium chloride, and may be used in combination with an appropriate cosolvent such as alcohol (ethanol, etc.), polyol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), and the like.

The NR10 antagonist may also be encapsulated in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, polymethylmethacrylate, etc.) or formulated into colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) as required (see, for example, Remington's Pharmaceutical Science, 16 th edition, Oslo Ed. (1980), etc.). Also, methods are known for the formulation of drugs into sustained release formulations, which are applicable to NR10 antagonists (Langer et al, J. biomed. Mater. Res. (1981) 15, 167-277; Langer, chem. Tech. (1982) 12, 98-105; U.S. Pat. No. 3,773,919; European patent application publication (EP) No. 58,481; Sidman et al, Biopolymers (1983) 22, 547-56; EP 133,988).

The preventive or therapeutic agent for inflammatory diseases of the present invention may be administered orally or parenterally, but parenteral administration is preferred. Specifically, the injection and transdermal administration are performed to the patient. Examples of the injection form include systemic or local administration by intravenous injection, intramuscular injection, subcutaneous injection, or the like. It can be injected locally, especially intramuscularly, at or around the site where inflammation is to be inhibited. The appropriate administration method can be selected according to the age and symptoms of the patient. The dose can be selected, for example, from 0.0001 mg to 100 mg per 1 kg body weight of the active ingredient per dose. Alternatively, for example, when administered to a patient, the amount of the NR10 antagonist to be administered per person may be selected in the range of 0.0001 to 1000 mg/kg body weight as the active ingredient, and preferably, the amount of the NR10 antagonist is about 0.01 to 50 mg/kg body weight per administration. However, the prophylactic or therapeutic agent for inflammatory diseases of the present invention is not limited to the above-mentioned dose.

The present invention also provides an antibody having NR10 neutralizing activity (including a fragment of an antibody having NR10 neutralizing activity and/or a modification thereof. The antibody having NR10 neutralizing activity of the present invention can be used as an active ingredient in the prophylactic or therapeutic agent for the above-mentioned inflammatory diseases. The antibody having NR10 neutralizing activity of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. The monoclonal antibody having NR10 neutralizing activity can be obtained by the above-described method. In addition, the monoclonal antibody of the present invention has a neutralizing activity against NR10 derived from a mammal or a fragment thereof, preferably against NR10 derived from a human or a mouse or a fragment thereof, and more preferably against NR10 derived from a human or a fragment thereof. In order to make a monoclonal antibody having neutralizing activity to human NR10, human NR10 or a fragment peptide thereof may be used as an immunogen. The antibody having NR10 neutralizing activity of the present invention may be a recombinant antibody. The recombinant antibody having NR10 neutralizing activity of the present invention is not limited thereto, and examples thereof are: chimeric antibodies, humanized antibodies, human antibodies, low molecular weight antibodies.

In the present invention, examples of the antibody having NR10 neutralizing activity are: an anti-mouse NR10 antibody having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID No. 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID No. 3.

The present invention also includes monoclonal antibodies having neutralizing activity against human NR10. As described above, the monoclonal antibody having human NR10 neutralizing activity of the present invention can be prepared by using human NR10 or a fragment peptide thereof as an immunogen, and then screening an antibody having human NR10 neutralizing activity from the above-mentioned anti-human NR10 monoclonal antibody by using the above-mentioned measurement system using an IL-31-dependent cell line.

Examples of the antibody having a human NR10 neutralizing activity in the present invention are: an antibody according to any one of the following (a) to (d).

(a) An antibody having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID No. 18 as CDR1, the amino acid sequence shown in SEQ ID No. 19 as CDR2, and the amino acid sequence shown in SEQ ID No. 20 as CDR 3.

(b) An antibody having a light chain variable region comprising the amino acid sequence of SEQ ID No. 21 as CDR1, the amino acid sequence of SEQ ID No. 22 as CDR2, and the amino acid sequence of SEQ ID No. 23 as CDR 3.

(c) An antibody having the heavy chain variable region of (a) and the light chain variable region of (b).

(d) An antibody recognizing the same epitope as the antibody according to any one of (a) to (c).

Whether an antibody recognizes the same epitope as other antibodies can be confirmed by competition of the two for the epitope. Competition between antibodies can be assessed by competition binding assays, which include: ELISA, fluorescence energy transfer assay (FRET), fluorescence microassay technique (FMAT (registered trademark)), and the like. The amount of this antibody that binds to the antigen is indirectly related to the binding energy of the candidate competitor antibody (test antibody), which competitively binds to the same epitope. That is, the greater the amount or affinity of a test antibody that binds to the same epitope, the lower the amount of the antibody that binds to the antigen, and the greater the amount of the test antibody that binds to the antigen. Specifically, the antibody and the antibody to be evaluated are added to the antigen together with an appropriate label, and the bound antibody is detected by the label. The amount of the antibody bound to the antigen can be easily determined by labeling the antibody in advance. The labeling method is not particularly limited, and the labeling method is selected according to the detection method. Specific examples of the labeling method include: fluorescent labels, radioactive labels, enzymatic labels, and the like.

For example, the antibody labeled with fluorescence and the antibody not labeled or a test antibody are added simultaneously to an animal cell expressing NR10, and the labeled antibody is detected by a fluorescence microassay technique.

The term "antibody recognizing the same epitope" as used herein means that the concentration of the test antibody is IC of the unlabeled antibody₅₀An antibody that is capable of reducing the amount of binding of the labeled antibody to the epitope by at least 50% at 10-fold. Wherein the IC₅₀The concentration (IC) is the concentration at which the binding amount of the labeled antibody to the epitope is reduced by 50% by the binding of the unlabeled antibody₅₀)。

The antibody having human NR10 neutralizing activity used in the present invention preferably further has cynomolgus monkey NR10 binding activity, and further preferably has cynomolgus monkey NR10 neutralizing activity. For the determination of the binding activity or neutralizing activity against cynomolgus monkey NR10, cynomolgus monkey NR10 comprising the amino acid sequence shown in SEQ ID No. 24 can be used.

The invention also provides the methods of treatment shown below. Among them, NR10, antagonists, monoclonal antibodies, recombinant antibodies, inflammatory diseases and the like are as described above.

(1) A method for the prophylaxis or treatment of an inflammatory disease, which method comprises the step of administering an NR10 antagonist to a patient suffering from an inflammatory disease.

(2) The method of (1), wherein the NR10 antagonist is an antibody having a NR10 binding activity.

(3) The method of (2), wherein the antibody is a monoclonal antibody.

(4) The method of (2), wherein the antibody is a monoclonal antibody that binds to human NR10.

(5) The method of any one of (2) to (4), wherein the antibody is a recombinant antibody.

(6) The method of (5), wherein the recombinant antibody is a chimeric antibody, a humanized antibody or a human antibody.

(7) The method according to any one of (2) to (6), which comprises a fragment of an antibody having a NR10 neutralizing activity and/or a modified product thereof as an active ingredient.

(8) The method according to any one of (1) to (7), wherein the inflammatory disease is atopic dermatitis.

(9) The method of any one of (1) to (7), wherein the inflammatory disease is chronic dermatitis.

(10) The method of any one of (1) to (7), wherein the inflammatory disease is rheumatism.

(11) The method according to any one of (1) to (7), wherein the inflammatory disease is osteoarthritis.

The present invention also provides the following inventions. Among them, NR10, antagonists, monoclonal antibodies, recombinant antibodies, inflammatory diseases and the like are as described above.

(1) Use of an NR10 antagonist in the manufacture of a medicament for the prevention or treatment of an inflammatory disease.

(2) The use as described in (1), wherein the NR10 antagonist is an antibody having a NR10 binding activity.

(3) The use of (2), wherein the antibody is a monoclonal antibody.

(4) The use of (2), wherein the antibody is a monoclonal antibody that binds to human NR10.

(5) The use according to any one of (2) to (4), wherein the antibody is a recombinant antibody.

(6) The use of (5), wherein the recombinant antibody is a chimeric antibody, a humanized antibody or a human antibody.

(7) The use according to any one of (2) to (6), which comprises a fragment of an antibody having a NR10 neutralizing activity and/or a modified product thereof as an active ingredient.

(8) The use according to any one of (1) to (7), wherein the inflammatory disease is atopic dermatitis.

(9) The use according to any one of (1) to (7), wherein the inflammatory disease is chronic dermatitis.

(10) The use according to any one of (1) to (7), wherein the inflammatory disease is rheumatism.

(11) The use according to any one of (1) to (7), wherein the inflammatory disease is osteoarthritis.

All prior art documents cited in the present specification are incorporated herein by reference.

Examples

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

[ example 1]Establishment of a Ba/F3 cell line expressing NR10 and OSMR

Human NR10 cDNA (WO 0075314 SEQ ID No:1) was inserted into expression vector pCOS1 (biochem Biophys Res Commun. 228, pages 838-45, 1996) to prepare pCosNR10.3. The oncoprotein M receptor cDNA (OSMR, GenBank accession No. NM003999) was isolated from a human placenta library by PCR, and an expression vector pCos1-hOSMR was constructed in the same manner. Each 10 was electroporatedμg vector was simultaneously introduced into cell line Ba/F3 derived from mouse IL-3-dependent pro-B cells (BioRad Gene Pulser, 960)μF, 0.33 kV). After introduction, human IL-31 was added thereto and cultured to obtain a cell line that proliferated depending on IL-31. Similarly, mouse IL-31-dependent cell lines were prepared from Ba/F3 cells expressing mouse NR10 and mouse OSMR genes.

All cell lines showed an ED50 of several ng/mL, resulting in a well-propagated cell line. The human IL-31-dependent cell line did not react with mouse IL-31, and proliferation was inhibited by the addition of human NR10 protein (extracellular domain). Mouse IL-31-dependent cell lines did not react with human IL-31 and proliferation was not inhibited by addition of the mouse NR10 protein (extracellular domain).

[ example 2]Preparation of the NR10 protein (extracellular domain)

Using human NR10 cDNA as a template, only the extracellular domain was amplified by PCR, and a FLAG tag sequence was added to the C-terminus, which was inserted into an expression vector pCXND3 (WO2005/005636) (pCXND3-NR 10-FLAG). Using electroporation method to mix the mixture 10μThe vector was introduced in a linear form into Chinese hamster ovary cell line DG44 (BioRad Gene Pu)lserII; 25μF, 1.5kV), cell lines showing high expression were obtained. Purified samples were obtained from culture supernatants obtained by mass-culturing the cell lines by an anti-FLAG antibody column (manufactured by SIGMA) and a gel filtration method, and were subjected to the following experiments. Mouse NR10 (extracellular domain) having a FLAG tag sequence added to the C-terminal thereof was also prepared in the same manner.

[ example 3]Isolation of scFv having neutralizing Activity against mouse NR10 and preparation of chimeric IgG-Egged BM095

Specifically, the present inventors have used a biotinylated mouse NR10 protein (extracellular domain) to narrow the range of candidate clones from a human antibody phage library by a screening method. Secretory scFv was purified from these clones and added to the IL-31-dependent Ba/F3 cell proliferation assay system described in example 1. As a result, clone BM095 showing strong proliferation inhibitory activity was successfully obtained.

The human H chain variable region sequence (VH) of BM095 was ligated to the mouse IgG2a constant region (after CH 1) and the human L chain variable region sequence (VL) to the mouse lambda chain constant region by PCR to construct an expression vector. In this case, the amino acid sequence of VH is shown in SEQ ID No. 1, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID No. 2. The amino acid sequence of VL is shown in SEQ ID No. 3, and the nucleotide sequence for coding the amino acid sequence is shown in SEQ ID No. 4. The linear expression vectors were simultaneously introduced into the strain DG44, and cell lines expressing chimeric IgG at high levels were selected. Purified samples were obtained from the culture supernatant obtained by mass-culturing the cell line using a protein A (rProtein A Sepharose fast flow, manufactured by GE Amersham Biosciences) column and a cation exchange (SP-TOYOPEARL 650M, manufactured by TOSOH) column chromatography. Then, Acticlean Etox (Sterogen) resin was used to reduce pyrogen to below the detection limit, and the resultant was subjected to the following animal experiments. The results of adding BM095 to the above measurement system are shown in FIG. 1.

[ example 4]]Study of drug efficacy Using model of atopic dermatitis in NC/Nga mice

A dermatitis model was made by repeated weekly applications of picryl chloride (PiCl). I.e., with 5% Picl (150)μl) sensitizing the abdomen,After 4 days on the toes, 0.8% PiCl (150) was administered weeklyμl) applying to the back and auricle once, and inducing dermatitis by repeated application. Symptoms were observed 2 times per week from week 3 to week 6, and scores were made from 0 to 3 points for each of 5 aspects ((1) pruritus, (2) redness and bleeding, (3) edema, (4) lesions and tissue damage, (5) crust formation and dryness). The antibody was administered intraperitoneally at 10 mg/kg 6 times a day before sensitization and each induction (BM095 group), or 3 times a day before each induction after week 3 (V/B group). Dexamethasone (DEX group) was given daily at 1 mg/kg per mouth after week 3 as a positive control. 10 NC/Nga male mice were used per group.

As shown in fig. 2, a significant inhibitory effect was confirmed in the antibody-administered group compared to the solvent-administered group. At this time, as shown in fig. 2, the DEX group confirmed significant weight loss, but the weight of the antibody-administered group did not change, indicating that the antibody is a very safe drug.

[ example 5]Drug efficacy was investigated using a chronic dermatitis model produced by repeated application of picryl chloride

Right ear spread 20 in 6 week old female BALB/c miceμSensitization was carried out with 0.5% of picryl chloride (acetone/olive oil (1:4, v/v) solution). After sensitization, the right ear was coated with 20 every 1 day on day 8μl 0.25% of picryl chloride (acetone/olive oil (1:4, v/v)), dermatitis was induced by repeated applications. The BM095 prophylactic effect evaluation group was administered intraperitoneally at 10 mg/kg once a week from the day before sensitization, while the BM095 therapeutic effect evaluation group was administered intraperitoneally at 10 mg/kg once a week from the 20 th day after induction. Auricular swelling was evaluated by measuring the thickness of the right ear with a watch disk thickness gauge over time.

The results are shown in fig. 4, and a significant inhibitory effect was observed in the antibody-administered group. It has been reported that human NR10 expression is enhanced in the hypertrophic epidermis of atopic dermatitis patients (non-patent document 1). When the auricles of the mouse model of chronic dermatitis were subjected to immunohistological staining, as in the case of human, the expression of mouse NR10 was increased in the hypertrophic epidermis (fig. 5. BM095 in which the constant region was changed to human IgG was prepared in example 3 and used for immunohistological staining; dark brown portion was NR10 expression site). The present finding shows that: NR10 is also involved in inflammatory diseases in humans and mice.

[ example 6]Drug effect study by using collagen-induced arthritis (rheumatism) model

The preparation of the model mouse and the evaluation of the drug effect were carried out as follows.

9-week-old female DBA/1JN mice were intradermally administered 140 consisting of bovine-derived collagen II 0.3% mixed with complete adjuvant H37Ra in equal amounts to the caudal rootμl collagen gel (day 0, sensitization). The same prepared collagen gel was administered into the back skin 3 weeks later to induce the onset of arthritis (day 21, induction). 16 mice were divided into 2 groups of 8 mice each based on the body weight two days (day-2) before sensitization, and set as a solvent administration group and a BM095 administration group. 8 mice in the BM 095-administered group were intravenously administered at 10 mg/kg body weight one day (day-1) before sensitization with the test substance BM095 diluted 6-fold with 20 mmol/L acetate buffer (pH5.5) (200 mmol/L NaCl) as a solvent. While 8 mice in the solvent administration group as a control were administered the same dose of solvent on Day-1. The swelling of the limbs was observed every 2 to 3 days from the day before induction (day 20), and was evaluated by scoring (0 to 4 points per limb: 16 points full).

The results are shown in fig. 6, and the fraction of limb swelling decreased after day 20 in the BM 095-administered group, indicating that BM095 has an effect of inhibiting the onset of arthritis.

[ example 7]Drug effect study by using collagenase induced arthritis (osteoarthritis) model

The preparation of the model mouse and the evaluation of the drug effect were carried out as follows.

Male C57BL/6J Jcl mice (manufactured by CLEA, japan) 8 weeks old were intravenously administered solvent control (20 mmol/L acetate buffer diluted 6-fold with PBS, ph5.5, 200 mmol/L NaCl, n =5), BM 0952 mg/kg (n =5), BM 09520 mg/kg (n =6) (5 mL/kg) from the tail vein. Then under 3% isoflurane inhalation anesthesia, toRight knee joint intracavity injection 6μl through 0.45μm.Filter (MILLIPORE corporation) and 1.5% collagenase solution (TypeII, Sigma) (Am J Pathol 1989; 135(6): 1001-14).

The width of the left and right knee joints was measured with a caliper (manufactured by Mitsutoyo) immediately before administration of collagenase and 3,7, and 14 days after administration, and the difference between the left and right was calculated. The difference was used as a value indicating swelling of the right knee joint, and the area under the transition curve (AUC) was calculated by the trapezoidal rule as a pharmacodynamic index. The AUC between the solvent control group and the CNP administration group was subjected to the student's t-test using statistical analysis software (SAS Institute corporation) (significant difference was found when p < 0.05). The results are shown in FIG. 7, where the AUC of BM095 administration group was significantly lower than that of the solvent control group in all the amounts. From the results, it is found that: BM095 inhibits arthritis in the osteoarthritis model.

[ example 8]Acquisition of neutralizing antibody against human NR10

Mice were immunized with human NR10 protein (extracellular domain) [ described in example 2], and hybridomas were prepared according to the conventional method. The neutralizing activity of the above hybridoma culture supernatants was evaluated using the human IL-31-dependent cell line (hNR10/hOSMR/BaF3 cells) shown in example 1, resulting in several clones having high NR10 neutralizing activity (FIG. 8).

[ example 9]Production of human chimeric antibody

The amino acid sequence of the heavy chain variable region of NA633 with the highest activity in the neutralizing antibody is shown in SEQ ID No. 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 17. Furthermore, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of NA633 are shown in SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID No. 21, SEQ ID No. 22 and SEQ ID No. 23, respectively.

A chimeric antibody having the mouse variable region and the human constant region (H chain: IgG1 type, L chain: kappa type) was prepared by a conventional method, and the neutralizing activity was measured. As shown in fig. 9, the chimeric NA633 antibody showed strong neutralizing activity at low concentration.

[ example 10]Isolation of cynomolgus monkey NR10

Since the preclinical safety evaluation is considered to be important for the cynomolgus monkey cross reactivity and the neutralizing activity, the present inventors tried to isolate the cynomolgus monkey NR10 gene. Primers were designed based on published rhesus monkey genome information and the like, and the NR10 gene was successfully amplified from cynomolgus monkey organs by PCR. The alignment of the amino acid sequences of isolated cynomolgus monkey NR10 and human NR10 is shown in FIG. 10. Furthermore, the amino acid sequence of the cynomolgus monkey NR10 is shown in SEQ ID No. 24. As described in example 1, the cynomolgus monkey NR10 gene was introduced into Ba/F3 cells together with the human OSMR gene, and a cell line showing human IL-31-dependent proliferation was established. When the neutralizing activity of the chimeric antibody NA633 of example 9 was examined using this cell line, it was found that this antibody also exhibited strong neutralizing activity in cynomolgus monkeys (fig. 11).

[ reference example 1]Drug effect of BM095 on induction of colitis by Dextran Sodium Sulfate (DSS)

A DSS-induced colitis model reported as a pathologic model of Inflammatory Bowel Disease (IBD) was prepared (J Immunol (2003); 171: 5507-5513), and the effect of the anti-mouse NR10 neutralizing antibody, BM095, was examined. Using a warp of 0.22μA5% (w/v) aqueous solution of dextran sodium sulfate (Wako pure chemical industries, Ltd.) was prepared by filtering sterilized distilled water with a m filter (Millipore Co., Ltd.), and a 6-week-old male Balb/cAnN Crj mouse (Japanese CHARLES RIVER Co., Ltd.) was allowed to freely take the above solution through a water bottle for 7 days, and the body weight was measured, and the rate of change in body weight from the day of initiation of DSS administration was used as an index for evaluating the drug effect.

Using this model, the anti-mouse NR10 neutralizing antibody BM095 was administered intravenously at a dose of 10 mg/kg the day before DSS administration to evaluate weight loss (n =10) to examine whether the pathology was improved by neutralization of IL-31 signaling. A group (solvent group, n =10) to which a solvent (mixed solution of acetate buffer [20 mmol/L sodium acetate, 20 mmol/L sodium chloride ] and phosphate-buffered saline [ PBS, manufactured by GIBCO ] mixed at a volume ratio of 1: 5) was intravenously administered one day before DSS administration was set as a solvent control group. In addition, in order to grasp the transition of the rate of change in body weight of normal mice, the transition of the rate of change in body weight of Balb/cAnN Crj mice of the same week age and sex as those of the DSS-administered group was also observed (n = 1).

The body weight shift is shown in FIG. 12. The solvent group had a reduced rate of body weight change by administration of DSS. On the other hand, although the BM 095-administered group also showed the same weight shift as the solvent group, the rate of change in body weight of the BM095 group was significantly reduced after 4 days and 5 days after DSS administration, compared to the solvent group. From the above results, it was confirmed that the administration of BM095 did not produce a therapeutic effect on colitis in this model.

It has been reported that the expression of IL-31RA is accelerated in this model (WO2004/003140), but the above experimental results show that: homomolecular neutralizing antibodies had no therapeutic effect on colitis in this model.

[ reference example 2]Drug effect of BM095 on acute picryl chloride contact dermatitis model

Dermatitis which was reported as an acute contact dermatitis model (Clin Immunol (2003); 108: 257- & 262) and which was sensitized and induced by picrylchloride application and resulted in delayed type allergic reaction results was prepared, and the effect of the anti-mouse NR10 neutralizing antibody, BM095, was investigated. 50 pieces of abdominal skin of 8-week-old female Balb/cAnN Crj mice (manufactured by Japan CHARLES RIVER)μL7% solution of picryl chloride (nacalai tesque, Inc.) was sensitized (ethanol: acetone =3:1, v/v), and 5 days later, 20 days later, was applied to the right auricle skinμL1% solution of picryl chloride (acetone: olive oil =1:4, v/v), induced contact dermatitis (induction). The skin of the left auricle of the same mouse was applied with 20 coatsμL solvent (acetone: olive oil =1:4, v/v) as a control for evaluating the effect of solvent on the thickness of the auricle (positive control group, n = 6). The thicknesses of the right and left ears were measured with a dial thickness gauge (manufactured by Kawasaki corporation) immediately before and 24, 48, and 72 hours after the induction, and the change in the thickness of the auricle immediately before the induction was used as an evaluation index of the drug effect.

In the setting of sensitization, ethanol-acetone mixture (3:1, v/v) without picryl chloride is applied on abdominal skin, and after 5 days, the mixture is applied on right auricle skin for 20 daysμL1% solution of picryl chloride in waterAuricle skin coating 20μA group of L solvents (acetone: olive oil =1:4, v/v) (negative control group, n =6) was used as a control group for evaluating establishment of a pathological condition.

In order to evaluate the effect of the anti-NR 10 antibody administration in this disease state, a group (BM095 group, n =6) in which acute contact dermatitis was induced by the method of the above-described positive control group and 10 mg/kg of BM095 was intravenously administered on the day before sensitization and the day before induction and a group (solvent group, n =5) in which a solvent (mixed solution of acetate buffer [20 mmol/L sodium acetate, 20 mmol/L sodium chloride ] and phosphate-buffered saline [ PBS, manufactured by GIBCO ] at a volume ratio of 1: 5) was administered at the same timing were established.

Changes in pinna thickness up to 72 hours after induction are shown in figure 13. At 24, 48, and 72 hours after induction, auricles of the positive control group were significantly thicker than those of the negative control group, indicating that a pathological condition was established. At this time, the BM095 group showed the same change in the pinna thickness as the solvent group, and no significant inhibition was observed.

The results show that: administration of BM095 did not produce a therapeutic effect on the acute contact dermatitis observed in this model.

Industrial applicability

According to the present invention, a novel prophylactic or therapeutic agent for inflammatory diseases can be provided. The prophylactic or therapeutic agent for inflammatory diseases provided by the present invention contains an NR10 antagonist, more preferably an antibody having a NR10 neutralizing activity, as an active ingredient thereof.

In recent years, anti-cytokine therapy using monoclonal antibodies has been attracting attention. However, in many cases in actual pathological conditions, even if one cytokine is blocked, it acts to compensate the pathway, so that it is difficult to obtain a therapeutic effect. Furthermore, when blocking a target cytokine, it is difficult to predict in which disease a therapeutic effect will be obtained. Under the above circumstances, the present inventors have found that: the anti-NR 10 neutralizing antibody can significantly suppress the symptoms in model mice suffering from atopic dermatitis, chronic dermatitis, rheumatism, osteoarthritis, etc.

The present inventors also succeeded in obtaining a human NR 10-neutralizing antibody. A prophylactic or therapeutic agent for inflammatory diseases, which contains the human NR10 neutralizing antibody as an active ingredient, can be clinically applied to humans.

Claims (10)

1. Use of an anti-NR 10 monoclonal IgG antibody in the manufacture of a medicament for inhibiting or treating an inflammatory disease associated with IL-31 signalling, wherein the antibody has human NR10 and cynomolgus monkey NR10 neutralising activity, the antibody being directed against the extracellular domain of the NR10 protein and being selected from the group consisting of:

(i) an antibody having a heavy chain variable region comprising CDR1, CDR2 and CDR3 consisting of the amino acid sequences of SEQ ID NOs 18, 19 and 20, respectively, and a light chain variable region comprising CDR1, CDR2 and CDR3 consisting of the amino acid sequences of SEQ ID NOs 21, 22 and 23, respectively; and

(ii) (ii) an antibody that binds to the same epitope as the antibody of (i),

and wherein the inflammatory disease is: (A) atopic dermatitis, wherein the antibody inhibits at least one symptom of atopic dermatitis selected from the group consisting of: (1) pruritus, (2) redness and bleeding, (3) edema, (4) lesions and tissue damage, (5) crust formation and dryness; (B) chronic dermatitis; (C) rheumatism; or (D) osteoarthritis.

2. The use according to claim 1, wherein NR10 is human NR10 which forms a heterodimer with the oncostatin M receptor (OSMR) and functions as an IL-31 receptor.

3. The use of claim 1, wherein the human NR10 is NR10.1 comprising the amino acid sequence encoded by the cDNA of SEQ ID No. 25 and the cynomolgus monkey NR10 is NR10 comprising the amino acid sequence of SEQ ID No. 24.

4. The use of claim 1, wherein the antibody is a recombinant antibody.

5. The use of claim 4, wherein the recombinant antibody is a chimeric, humanized or human antibody.

6. Use of a fragment of an anti-NR 10 monoclonal IgG antibody according to claim 1 in the manufacture of a medicament for the inhibition or treatment of an inflammatory disease associated with IL-31 signal transduction, wherein said inflammatory disease is atopic dermatitis, chronic dermatitis, rheumatism or osteoarthritis, and wherein said fragment is Fab, Fab ', F (ab')₂Or Fv fragment and has human NR10 and cynomolgus NR10 neutralizing activity.

7. An anti-NR 10 monoclonal IgG antibody having neutralizing activity to human NR10 and cynomolgus monkey NR10, wherein the antibody (a) comprises a heavy chain variable region of CDR1, CDR2 and CDR3 consisting of the amino acid sequences of SEQ ID NOs 18, 19 and 20, respectively, and (b) comprises a light chain variable region of CDR1, CDR2 and CDR3 consisting of the amino acid sequences of SEQ ID NOs 21, 22 and 23, respectively.

8. An anti-NR 10 monoclonal IgG antibody having neutralizing activity of human NR10 and cynomolgus monkey NR10, wherein the antibody is an anti-NR 10 monoclonal IgG antibody comprising a heavy chain variable region of SEQ ID NO:16 and a light chain variable region of SEQ ID NO: 17.

9. The antibody of claim 7 or 8, wherein the antibody is a recombinant antibody.

10. The antibody of claim 9, wherein the recombinant antibody is a chimeric, humanized, or human antibody.