WO2007083759A1 - Bone destruction inhibitor comprising anti-ccl20 antibody - Google Patents

Bone destruction inhibitor comprising anti-ccl20 antibody Download PDF

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Publication number
WO2007083759A1
WO2007083759A1 PCT/JP2007/050823 JP2007050823W WO2007083759A1 WO 2007083759 A1 WO2007083759 A1 WO 2007083759A1 JP 2007050823 W JP2007050823 W JP 2007050823W WO 2007083759 A1 WO2007083759 A1 WO 2007083759A1
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ccl20
antibody
ccr6
bone destruction
monoclonal antibody
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PCT/JP2007/050823
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French (fr)
Japanese (ja)
Inventor
Kenzo Muramoto
Tatsuo Horizoe
Toshio Imai
Miyuki Nishimura
Keiko Mizuno
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Eisai R & D Management Co., Ltd.
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Publication of WO2007083759A1 publication Critical patent/WO2007083759A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/108—Osteoporosis

Definitions

  • the present invention relates to suppression of bone destruction and treatment of bone destruction by inhibiting the interaction between the chemokines CCL20 and CCR6.
  • the invention also relates to antibodies against CCL20 and their pharmaceutical and diagnostic uses.
  • Chemokines are the main cell migration factors in vivo, and control tissue infiltration of immune cells through enhancement of cell motility and activation of cell adhesion molecules. Chemokines are classified into four sub families, CC, CXC, C, and CXXXC, based on the sequence of the first two cysteine residues. Members of CC, CXC, and C chemokines are secreted proteins of about 70 amino acids, and they are not active as adhesion molecules but can induce cell adhesion. The secreted chemokine binds to the 7-transmembrane receptor on the target cell surface, activates integrins via the trimeric G protein, and induces cell adhesion and migration. In recent years, chemokines and their receptors have been reported to be central site power-ins that control immune cell infiltration, and the actions of chemokine systems in autoimmune diseases are closely related to their pathogenesis. It is becoming clear.
  • the CCR6 CCL20 (LARC (Liver— and activation-regulated chemokine) / MIP 3 a Zexodus) system is used to place rods, memory T cells, and mature B cells involved in acquired immunity at sites exposed to foreign antigens. It is thought to play an important role in concentrating and quickly initiating the immune response (Cytokine & Growth Factor Reviews, 14; 409-42 6, 2003). Regulates migration and localization of gut-directed memory T cells, na ⁇ ve and memory B cells (Immunity, 12; 495-503, 2000., J Immunol, 162; 186-194, 1999., Blood, 96; 2338-2345, 2000).
  • Intestinal epithelial cells and Peyer's follicle-related epithelial cells show constitutive expression of CCL20, which is enhanced by LPS, TNF a, and IL 1 j8 (Gut, 51; 818-826, 2002, Eur J Immunol, 29, 633-642, 1999., J Exp Med, 191; 1381-1394 "Int Immunol, 13; 1255-63, 2001).
  • Keratinocytes Keratinocytes, tracheal branch epithelial cells, vascular endothelial cells, lymphatic endothelial cells, Also in synovial fibroblasts, CCL20 expression is induced by inflammatory stimulation (Cytokine & Growth Factor Reviews, 14; 409-426, 2003, J Exp Med, 194; 797-808, 2001., J Exp Med, 190; 1755-1768, 1999).
  • the CCL20 receptor CCR6 is expressed on rods, T cells, B cells, etc. Expression in T cells is limited to memory T cells, and expression is observed in both ⁇ 4 ⁇ 7-positive intestinal-directed sputum cells and CLA + skin-directed sputum cells (J Immunol, 162; 186-194, 1999., J Immunol, 164, 6621-6632, 2000). Recently, it has become clear that CCR6 is also expressed in inhibitory T cells in the memory type (Blood, 105; 2877-2886, 2005). In B cells, it is selectively expressed in naive B cells in peripheral blood and memory B cells that have undergone antigen stimulation, and CCL20 is highly expressed in order to recognize antigens and produce antibodies quickly.
  • CD34-positive precursor cells are differentiated in the presence of SCF, GM-CSF, and TNFa
  • peripheral blood monocytes are differentiated in the presence of GM-CSF, IL-4, and TGF ⁇ . It is expressed in so-called immature rod cells such as immature rod cells and epidermal Langerhans cells (J Exp Med, 186; 825-835, 1997., J Immunol, 163, 1737-1741, 1999, J Exp Med, 192; 705-718, 2000).
  • CCL20 produced by constitutive and inflammatory stimuli from the mucosal epidermis and skin epithelium allows immature dendritic cells to be placed on the surface of the body cavity, enabling efficient antigen uptake and triggering acquired immunity.
  • CCR6 is expressed in human neutrophils stimulated with TNF ⁇ (Blood, 96; 3958-3963, 2000). More recently, it has been reported that CCR6-positive immature rod cells are taken up by cancer neovascularization and separated into vascular endothelial cells (Nat Med, 10; 950-958, 2004).
  • CDla-positive immature rod cells are separated into osteoclasts by synovial fluid from patients with rheumatoid arthritis.
  • Bood, 104; 4029-4037, 2004 the function of the CCR6-CCL20 system in CDla-positive immature cells as osteoclast precursor cells, its involvement in rheumatoid arthritis, and when this pathway is blocked. The effect is not mentioned at all.
  • CCL20 expression is low in synovial fluid from patients with osteoarthritis. In the synovial tissue of patients with rheumatoid arthritis, CCL20 expression is observed on the outermost layer and around the blood vessel, and CDla-positive immature rod cells are present in the vicinity of the CCL20 expression site (J Immunol, 168; 5333-5341, 2002). In addition, stimulation of synovial cells from rheumatoid arthritis patients with IL-1 ⁇ , TNFa, IL-17, etc. induces CCL20 production (J Immu nol, 167; 6015-6020, 2001). However, since the expression of various chemokines is observed in the intra-articular lavage fluid, the contribution of CCL20 to pathogenesis is not clear.
  • JP-A-2002-187856 and WO2002Z32456 disclose that a LARC inhibitor is used as a therapeutic agent for rheumatoid arthritis.
  • LARC should be suppressed because LARC is highly expressed in patients with rheumatoid arthritis, and no therapeutic effect has been demonstrated.
  • chemokines are expressed in various diseases, and if they are suppressed, they may have a therapeutic effect, but conversely, if they are promoted, they may have a therapeutic effect.
  • chemokine CCL2 is known to be highly expressed in rheumatoid arthritis.
  • the present inventors have surprisingly found that anti-inhibition of the migration of CCR6-expressing cells to CCL20. It was found that the body has an anti-inflammatory action and an anti-bone destruction action on a collagen-induced arthritis model. The present invention is based on this finding.
  • a bone destruction inhibitor comprising an antibody or fragment thereof reactive to CCL20 as an active ingredient.
  • test substance is a substance useful for the treatment of bone destruction disease.
  • a step of measuring the degree of binding of CCL20 to CCR6 in each condition in the presence and absence of the test substance, the degree of binding in the absence of the test substance, and the test substance A method for screening a substance useful for the treatment of bone destruction comprising the step of comparing the degree of action in the presence of
  • test substance is a substance useful for treatment of bone destruction when binding is suppressed.
  • a step of contacting a cell membrane containing CCR6 or a cell containing the same with CCL20 and measuring cell stimulating activity in the following, cell stimulating activity in the absence of the test substance, and presence of the test substance The method for screening according to (9) or (10), comprising the step of comparing the cell stimulating activity below.
  • Evaluation of the action of CCL20 on CCR6 is carried out by comparing the CCL20 inhibitory action of an antibody or fragment thereof reactive to CCL20, or the CCR6 inhibitory action of an antibody or fragment thereof reactive to CCR6.
  • a method for detecting bone destruction and a method for diagnosing bone destruction comprising a step of detecting the degree of expression of CCL20 in a test tissue.
  • the degree of expression of CCL20 in the test tissue further comprises the step of determining that bone destruction occurs or suffers from bone destruction when the degree of expression of CCL20 in normal tissue is exceeded.
  • An agent for detecting bone destruction and a diagnostic agent for bone destruction comprising an antibody reactive with CCL20 or a fragment thereof.
  • a bone destruction detection kit and a bone destruction diagnosis kit comprising an antibody having reactivity with CCL20 or a fragment thereof.
  • FIG. 1 shows inhibition of cell migration of CCR6-expressing cells to mouse CCL20 protein by anti-CCL20 antibody 2F5-5.
  • FIG. 2 shows CCL20 mRNA expression in foot and foot in a collagen-induced arthritis model.
  • FIG. 4 is a diagram showing changes in appearance and body weight over time in a collagen-induced arthritis model with incomplete Freund's adjuvant and complete Freund's adjuvant administered with anti-CCL20 antibody.
  • Triangle anti-CCL20 monoclonal antibody
  • square IgG (control).
  • FIG. 5 is a graph showing the effect of anti-CCL 20 antibody administration on serum amyloid A concentration in plasma of a collagen-induced arthritis model. (a) When incomplete Freund's adjuvant is used; (b) When complete Freund's adjuvant is used.
  • FIG. 7 is a graph showing the effect of anti-CCL20 antibody administration on the COMP concentration in plasma of a collagen-induced arthritis model.
  • FIG. 8 is a graph showing the effect of anti-CCL20 antibody administration on plasma active TRAP concentration in a collagen-induced arthritis model.
  • FIG. 9 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of inflammation-related molecule mRNA in the foot and foot in a collagen-induced arthritis model.
  • Open bars normal, black bars: IgG (control), hatched bars: anti-CCL20 monoclonal antibody.
  • FIG. 10 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of chemokine's chemokine receptor mRNA in the foot and foot in a collagen-induced arthritis model. Open bars: normal, black bars: IgG (control), hatched bars: anti-CCL20 monoclonal antibody.
  • FIG. 11 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of cell marker mRNA in the foot and foot in a collagen-induced arthritis model. Open bars: normal, black bars: IgG (control), hatched bars: anti-CCL20 monoclonal antibody.
  • FIG. 12 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of osteoclast-related molecule mRNA in the foot and foot in a collagen-induced arthritis model.
  • Open bars normal, black bars: IgG (control), hatched bars: anti-C CL20 monoclonal antibody.
  • FIG. 13 (a) shows changes over time in appearance in a collagen arthritis model in which administration of anti-CCL20 antibody was started after onset.
  • Triangle anti-CCL20 monoclonal antibody
  • square IgG (control).
  • the antigen CCL20 may be purified according to a known method, but it can also be prepared by genetic engineering.
  • CCL20 protein The amino acid sequence of CCL20 protein and the DNA encoding it have been reported. For example, in the case of mouse GenBank accession number: NM 058656, human field GenBank accession number: NM 004582 and mouse GenBank accession number: NM 016960, human case GenBank accession number: NM 004591 etc. have been reported.
  • the CCL20 protein used in the present invention can specifically identify the amino acid sequence or the DNA encoding it based on such known information.
  • the CCL20 protein in the present invention consists of a polypeptide selected from the group consisting of the following (A) to (E):
  • amino acid sequence of (A) one or more (preferably one or several, more preferably 1, 2, or 3) amino acids are substituted, deleted, inserted, and Z or added.
  • a polypeptide comprising a defined amino acid sequence and having substantially the same activity as the CCL20 protein;
  • (C) a polypeptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence of (A);
  • (D) A polypeptide encoded by a polynucleotide that is hybridized under stringent conditions with a polynucleotide that also has a nucleotide sequence that encodes the amino acid sequence of (A), and is substantially the same as the CCL20 protein.
  • CCL20 protein used in the present invention "a polypeptide having a certain amino acid sequence ability, which is specified by the aforementioned accession number" is preferable.
  • accession number any one amino acid sequence specified by the above-mentioned accession number is specified from a predetermined publicly-known database by the accession number listed above. It is an amino acid sequence.
  • the phrase “the polypeptide has substantially the same activity as the CCL20 protein” includes any polypeptide that induces a bone destruction action. Specifically, it means that it has a migratory action directly or indirectly on CCR6.
  • substantially the same means that the activities are homogeneous in nature. That is, in order to have “substantially the same activity as the CCL20 protein”, the activity is equivalent (for example, about 0.01 to: LOO times, preferably 0.05 to 20 times, more preferably 0.5 to 2 times). About these activity, it can measure according to a conventional method, for example, it can measure according to the method as described in the Example mentioned later.
  • the polypeptide (B) (hereinafter sometimes referred to as "modified polypeptide”) has an amino acid sequence ability according to the accession number described above.
  • modified polypeptide has an amino acid sequence ability according to the accession number described above.
  • “conservative substitution” means one or more (preferably several, more preferably 1, 2, or 3) amino acids so as not to substantially alter the activity of the peptide. It means replacing the residue with another chemically similar amino acid residue. For example, when one hydrophobic residue is substituted with another hydrophobic residue, one polar residue is substituted with another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, parin, isoleucine, leucine, proline, tryptophan, ferrolanine and methionine.
  • Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • Examples of positively charged (basic) amino acids include algin, histidine, and lysine.
  • Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the number of amino acids that may be deleted, substituted and Z or added is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to: LO, and still more preferably 1 to Five, particularly preferably 1-2.
  • the modified polypeptide contains a salt of the modified polypeptide, and those having a disulfide bond, those not having a disulfide bond, those phosphorylated and those not phosphorylated, and sugars Both those having no chain and those having a sugar chain are included. Therefore, as long as these conditions are satisfied, the origin of the modified polypeptide is not limited to humans.
  • Mutations due to amino acid deletions, substitutions, and Zs or attachments are performed, for example, on the DNA encoding the site-directed mutagenesis (for example, Nucleic Acid Research, Vol. 10 , No. 20, p. 6487-6500, 1982).
  • site-directed mutagenesis for example, Nucleic Acid Research, Vol. 10 , No. 20, p. 6487-6500, 1982.
  • “one or more amino acids” means as many amino acids as can be deleted, substituted and Z or added by site-directed mutagenesis.
  • a method for deleting, inserting, substituting, or adding one or more amino acids in a protein while maintaining the antigenicity of the original protein is known.
  • a polynucleotide encoding a mutant protein by parts position-specific mutagenesis is prepared, can be obtained by suitable Yichun expression (Molecular Cloning.A Laboratory Manual 2 nd ed , Cold Spring Harbor Press (1989); Current Protocols in Molecular Biology, John Wiley & Sons, (1987-1997), Section 8.1-8.5; Hashimoto- Goto et al. (1995) Gene 152: 271--5; Kinkel (1985) Proc. Natl. Acad Sci. USA 82: 488-92; Kramer and Fritz (1987) Method.
  • o Site-directed mutagenesis is, for example, Other than the specific mismatch that is the desired mutation, it can be performed as follows using a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated. That is, the above-mentioned synthetic oligonucleotide is used as a primer to synthesize a strand complementary to the phage, and a host cell is transformed with the obtained double-stranded DNA. Transformed bacterial cultures are plated on agar to form single-cell force plaques containing phage.
  • the above-mentioned site-directed mutagenesis is used as a method of performing deletion, substitution and Z or addition of one or more amino acids that do not lose its activity to the amino acid sequence of the biologically active peptide of CCL20 protein.
  • Other methods include treating the gene with a mutagen and selectively cleaving the gene, then deleting, substituting and appending or adding selected nucleotides, and then ligating.
  • deletion includes those in which an amino acid residue is deleted from the end of the amino acid sequence and amino acid residues in the middle of the amino acid sequence are deleted.
  • Additional includes those in which an amino acid residue is added to the end of the amino acid sequence and those in which an amino acid residue is added in the middle of the amino acid sequence.
  • any amino acid sequence identified by the aforementioned accession number preferably GenBank accession number: NP-058656 for mice, GenBank accession number: NP-004 582 for humans
  • any DNA encoding the enzyme active part is included in the scope of the present invention.
  • the polynucleotide encoding the CCL20 protein used in the present invention is any nucleotide sequence (preferably, a human sequence) identified by the aforementioned accession number.
  • GenBank accession number: NM 004591 Hybridizes with a strong polynucleotide under stringent conditions, and the force also encodes a polypeptide having substantially the same activity as the CCL20 protein.
  • a polynucleotide having a sequence power other than the nucleotide sequence specified by the accession number, and in the case of a mouse, GenBank accession number: NM 016960 (derived from a mouse) is a polynucleotide. .
  • the altered polypeptide may further include those in which the N-terminus (amino terminus) and C-terminus (carboxyl terminus) are altered or modified.
  • C-terminal force ruboxyllucacarboxylate one COO—
  • amide one CONH
  • ester — COOR
  • examples of R include a linear, branched or cyclic C alkyl group, C aryl group and the like.
  • the N-terminal amino group is
  • Those protected by a protective group for use can also be included in the modified polypeptide.
  • polypeptide (B) examples include non-human organisms [eg, non-human mammals (eg, mouse, rat, hamster, pig, Inu, etc.), birds, reptiles, amphibians, fish, insects. Etc.] derived from CCL20 protein or a variant thereof.
  • non-human mammals eg, mouse, rat, hamster, pig, Inu, etc.
  • NP_058656 derived from a mouse
  • a polypeptide having a strong force can be mentioned.
  • the polypeptide of (C) (hereinafter sometimes referred to as "homologous polypeptide") is particularly limited as long as it comprises an amino acid sequence having 80% or more identity with respect to the amino acid sequence of CCL20 protein.
  • the CCL20 protein has an identity of 85% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more.
  • An amino acid sequence consisting of amino acid sequences having the same homology as described above, and having a force substantially the same as that of the CCL20 protein.
  • any value of "identity” may be any value calculated using a homology search program known to those skilled in the art.
  • NCBI National Biotechnology Information Center
  • the homology algorithm BLAST Basic local alignment search tool
  • http://www.ncbi.nlm.nih.gov/BLAST/ can be calculated using the default (initial setting) parameter 1 it can.
  • the homologous polypeptide includes a salt of the homologous polypeptide, which is disulfide-bonded to a disulfide-bonded one! /, Those that are not phosphated, those that are not phosphated, and those that do not have sugar chains and those that have sugar chains.
  • the origin of the homologous polypeptide is not limited to humans.
  • a CCL20 protein derived from a non-human organism eg, a non-human mammal (eg, mouse, rat, mouse, muster, pig, Inu, etc.)] or a variant thereof is included.
  • examples of the homologous polypeptide of (C) above include a polypeptide comprising GenBank accession number: NP_058656 (derived from mouse) in the case of a mouse.
  • a variant means "variation", that is, an individual difference found in the same polypeptide within the same species, or a difference found in several homologous polypeptides. To do.
  • a partial polypeptide of CCL20 protein (that is, CCL20 protein, modified polypeptide, homologous polypeptide) used in the present invention can be used as long as it has substantially the same activity as CCL20 protein.
  • the number of amino acids ⁇ to configure the partial polynucleotide or, CCL20 Tanno click protein amino acid number of 90 0/0 of, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5%.
  • the "polynucleotide hybridizing under stringent conditions" in the polynucleotide having the nucleotide sequence ability encoding the amino acid sequence of (D) specifically includes FASTA, BLAST, Smith-Waterman [Meth. Enzym , 164, 765 (1988)], etc., using the default (default) parameters, for example, for humans, GenBank accession number: NM 004591 and at least 70% or more 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Nucleotides are mentioned.
  • f column is“ 2 X SSC, 0.1% SDS, 50 ° C ”,“ 2 X SSC, 0.1% SDS, 42.C ”,“ 1 X SSC, 0.1% SDS, 37.C ”, more stringent end conditions include, for example,“ 2 X SSC, 0.1% SDS, 65.C ”,“ 0.5 X SSC, 0.1% SDS, 42.C "," 0.2 X SSC, 0.1% SDS, 65 ° C "can be mentioned.
  • Rap id-hyb buffer As a method using Rap id-hyb buffer (Amersham Life Science), after pre-hybridization at 68 ° C for 30 minutes or more, add the probe to 68 ° C for 1 hour or more. Keep hybridized, then wash 3 times in 2 X SSC, 0.1% SDS, 20 min at room temperature, 1 X SSC, 0.1% SDS, 37. It is conceivable to perform 3 20 minute washes at C, and finally 2 washes for 20 minutes at 50 ° C in 1 X SSC, 0.1% SDS.
  • the temperature of the prehybridization and the hybridization can be 60 ° C
  • the stringent condition can be 68 ° C.
  • the conditions such as the salt concentration and temperature of the koffer take into account other conditions such as the probe concentration, probe length, reaction time, etc. It is possible to set conditions for obtaining genetic variants and corresponding genes from other species.
  • polynucleotide to be hybridized for example, at least 50%, preferably 70%, more preferably 80%, and still more preferably 90% of the nucleotide sequence containing the base of GenBank accession number: NM 004591 (for example, a polynucleotide containing a nucleotide sequence having an identity of 95% or more, and further 99%) can be mentioned. This identity is similar to the BLAST algorithm described above (Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 58 73-7).
  • BLASTX Altschul et al. (1990) J. Mol. Biol. 215: 403-10 is a program for determining the identity of amino acid sequences based on this algorithm. Etc. have been developed and are available. For specific analysis methods, refer to http://www.ncbi.nlm.nih.gov. Etc. as mentioned above.
  • the base sequence of the polynucleotide can be confirmed by sequencing by a conventional method. For example, it can be confirmed by the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463). It is also possible to analyze the sequence using an appropriate DNA sequencer.
  • the polynucleotide encoding the CCL20 protein used in the present invention may be derived from, for example, a natural source or a totally synthesized one. Furthermore, it can also be synthesized by using a part of the natural origin.
  • a typical method for obtaining a polynucleotide encoding the CCL20 protein used in the present invention is, for example, a method commonly used in the field of genetic engineering from, for example, a commercially available library or cDNA library, such as a partial polynucleotide sequence. Examples include screening methods using appropriate DNA probes created based on information.
  • polynucleotide encoding the CCL20 protein used in the present invention "a polynucleotide having the nucleotide sequence identified by the above-mentioned accession number (for example, GenBank accession number: NM 004591 in the case of humans)" Is preferred.
  • the CCL20 protein for obtaining the antibody of the present invention includes at least 6 amino acid residues of the CCL20 protein in addition to a polypeptide having the full-length amino acid sequence of the CCL20 protein (for example, 6, 8 , 10, 12 or 15 amino acid residues or more) and polypeptide fragments (sometimes referred to as “fragments”).
  • the fragment of the CCL20 protein in the present specification may be any fragment as long as it has the antigenicity of the CCL20 protein.
  • Preferable fragments include, for example, fragments such as amino terminal and carboxyl terminal of CCL20 protein.
  • the antigenic determinant of the polypeptide is a method of analyzing hydrophobic Z hydrophilicity on the amino acid sequence of the protein (Kyte-Doolittle (1982) J. Mol. Biol. 157: 105-22), a method of analyzing secondary structure (Chou- Fasman (1978) Ann. Rev. Biochem. 47: 2 51-76) and further computer program (Anal. Biochem. 151: 540-6 (1985) ) Or short, it can be confirmed by a method such as PEPSCAN method (Japanese Patent Publication No. 60-500684) which synthesizes a peptide and confirms its antigenicity.
  • the antibody according to the present invention includes a monoclonal antibody obtained by immunizing a mammal such as a mouse with the CCL20 protein as an antigen, a chimeric monoclonal antibody and a human monoclonal antibody produced using a gene recombination technique, and Human monoclonal antibodies produced using human antibody-producing transgenic animals and the like are included.
  • a human monoclonal antibody is desirable from the viewpoint of side effects.
  • the “antibody” in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody, preferably a monoclonal antibody.
  • the “antibody” of the present invention is a natural antibody obtained by immunizing a mammal such as those described below with an antigen (eg, a natural antigen, a genetically modified antigen, an antigen-expressing cell, etc.).
  • an antigen eg, a natural antigen, a genetically modified antigen, an antigen-expressing cell, etc.
  • chimeric monoclonal antibodies and human monoclonal antibodies that can be produced using gene recombination techniques, and human monoclonal antibodies that can also be produced using human antibody-producing transgenic animals and the like are included.
  • CCL20 protein or a fragment thereof can be used as the immunizing antigen.
  • the antigen may be used as a complex with the carrier protein.
  • Various condensing agents can be used for the preparation of the antigen-carrier protein complex, such as dartal aldehyde, carbodiimide, maleimide active ester, and the like.
  • the carrier protein is a commonly used one such as bovine serum albumin, thyroglobulin, hemocyanin and the like. Usually, a method of coupling at a ratio of 1 to 5 times is used.
  • Mammals to be immunized preferably mice, rats, mice, mussels, guinea pigs, rabbits, cats, dogs, pigs, goats, horses or rushes, more preferably mice, rats, hamsters, Guinea pigs or rabbits can be used.
  • Inoculation methods include subcutaneous, intramuscular or intraperitoneal administration. When administered, it may be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant, usually once every 2 to 5 weeks. Immunized The resulting antibody-producing cells are fused with myeloma cells and isolated as a hyperidoma.
  • the myeloma cells are derived from mammals such as mice, rats, and humans, and are preferably derived from the same species as the antibody-producing cells! / But sometimes it is possible.
  • the antibody according to the present invention can specifically recognize CCL20 protein. Therefore, the CCL20 protein for obtaining the antibody according to the present invention has only to have the antigenicity of CCL20.
  • One or more amino acid residues are deleted, inserted, substituted or added in the amino acid sequence of the CCL20 protein. Proteins having different amino acid sequences are included. It is known that such mutant proteins maintain the same biological activity as the original protein (Mark et al. (1984) Proc. Natl. Acad. Sci. USA 81: 5662-6; Zoller and Smith (1982) Nucleic Acids Res. 10: 6487—500; Wang et al. (1984) Science 224: 1431—3; Dalbad ie-McFarland et al. (1982) Proc. Natl. Acad. Sci. USA 79: 6409- 13).
  • the antibody according to the present invention also includes an antibody specific for a part of the CCL20 protein.
  • the antibody according to the present invention is preferably an antibody that affects the function of the CCL20 protein.
  • the effect of CCL20 protein is, for example, that the antibody binds to CCL20 protein, thereby inhibiting the binding between CCL20 protein and CCR6, or the antibody binds to CCL20 protein. This is to inhibit cell migration through the activity of CCR6.
  • the “polyclonal antibody” in the present invention can be produced by an existing general production method. That is, for example, an antigen as described above is added to a mammal, preferably a mouse, a rat, a nomster, a guinea pig, a rabbit, a cat, a dog, a pig, a pig, a goat, together with Freund's Adjuvant as necessary. Serum strength obtained by immunizing a immunized animal by immunizing mouse, rat, mouse, more preferably mouse, rat, mouse, muster, guinea pig or rabbit.
  • the “monoclonal antibody” in the present invention can be specifically produced as follows. That is, an antigen as described above is used as an immunogen, and the immunogen is Along with Freund's Adjuvant, mammals, preferably mice, rats, rats, mustards, guinea pigs, usagis, cats, nu, pigs, goats, horses or tusks, more preferably mice , Rats, mice, guinea pigs, or rabbits (including transgenic animals engineered to produce antibodies from other animals such as human antibody-producing transgenic mice described below), muscle Immunization is carried out by injecting or transplanting once, several times, intraperitoneally, intravenously, into the food pad or into the abdominal cavity.
  • an antigen as described above is used as an immunogen
  • the immunogen is Along with Freund's Adjuvant, mammals, preferably mice, rats, rats, mustards, guinea pigs, usagis, cats, nu, pigs, goats, horses or tusks, more preferably mice
  • the initial immunity is also immunized 1 to 4 times about every 1 to 14 days, and the antibody-producing cells that are immunized about 1 to 5 days after the final immunization are obtained.
  • the number of times of immunization and the time interval can be appropriately changed depending on the nature of the immunogen used.
  • Monoclonal antibodies according to the present invention can be obtained using methods well known to those skilled in the art (for example, Current Protocols in Molecular Biology JQohn Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Labora tory (1988)) 0
  • "Neubridoma" secreting monoclonal antibodies in the present invention can be prepared according to the method of Köhler and Milstein et al. (Nature, 256, 495, 1975) and a modification method according thereto. That is, the spleen and lymph nodes obtained from a mammal immunized as described above, preferably a mouse, rat, hamster, guinea pig, rabbit, cat, Inu, butterfly, goat, horse or rush.
  • Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3Z X63-AG8. 653 (653), P3 / NSl / l-Ag4-l (NS-l), P3 / X63-Ag8.
  • U1 P3U1
  • SP2Z0 Agl4
  • PAI PAI
  • FO! /
  • BW5147 rat-derived myeloma 210RCY3— Ag.
  • human-derived myeloma U—266AR1, GM15 00-6TG-A1 -2, UC729-6, CEM-AGR, D1R11, CEM-T15, etc. can be used.
  • Examples of fusion promoters include polyethylene glycol and Sendai virus.
  • the ratio is usually about 1: 1 to 10: 1, and cell fusion can be carried out by reacting for about 1 to 10 minutes.
  • the screening of hybridoma clones producing monoclonal antibodies is carried out by culturing the hybridomas in, for example, a microtiter plate, and reacting the culture supernatant of the wells in which proliferation is observed with the immunizing antigen used in the immunization described above.
  • the sex can be measured by, for example, immunochemical methods such as RIA and ELIS A.
  • immunochemical methods such as RIA and ELIS A.
  • ELISA method using microplate coated with CCL20 protein EIA method using microplate coated with anti-immunoglobulin antibody
  • electrophoresis of sample containing CCL20 protein and nitrocellulose transfer membrane Examples include the immunoblot method used.
  • screening can be performed based on whether or not the antibody strength affects the function of the SCCL20 protein. For example, when the antibody binds to the CCL20 protein, the binding of the CCL20 protein to CCR6 is inhibited, and when the antibody binds to the CCL20 protein, the cell is mediated by the activity of CCR6.
  • Antibody-producing hybridomas can be screened depending on whether or not migration is inhibited.
  • an antibody that affects the function of the CCL20 protein which is a preferred embodiment of the antibody of the present invention, can be selected.
  • this screening may be performed as a secondary screening performed after the immunochemical method screening that is selected based on the ability to produce an antibody that binds to the CCL20 protein.
  • Further cloning from such a well can be performed, for example, by limiting dilution to obtain a clone.
  • Selection and breeding of hypridoma is usually performed in a medium for animal cells (eg, RPMI1640) containing 10-20% fetal calf serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hypoxanthine, aminopterin, thymidine
  • the clones thus obtained can be transplanted into the abdominal cavity of SCID mice pre-administered with pristane, and ascites containing a high concentration of monoclonal antibody can be collected 10-14 days later and used as a raw material for antibody purification.
  • the clone It is also possible to culture and use the culture as a raw material for antibody purification.
  • Hypridoma in vitro, or in mammals, preferably mice, rats, mice, guinea pigs, rabbits, cats, dogs, pigs, goats, mice.
  • mammals preferably mice, rats, mice, guinea pigs, rabbits, cats, dogs, pigs, goats, mice.
  • mouse, rat, etc. preferably mouse, rat, hamster or guinea pig, more preferably mouse ascites, etc., and isolating from the obtained culture supernatant or mammalian ascites It can be carried out.
  • Examples of basic medium include low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium, MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium.
  • the basic medium can contain, for example, serum, hormones, cytosine and Z, various inorganic or organic substances, etc., depending on the purpose.
  • Monoclonal antibodies can be isolated and purified by using the above-mentioned culture supernatant or ascites fluid, saturated ammonium sulfate, euglobulin precipitation method, force proic acid method, force prillic acid method, ion exchange chromatography (DEAE Or DE52, etc.), affinity column chromatography such as an anti-immunoglobulin column or protein A column.
  • monoclonal antibodies can be purified by using known methods for immunoglobulin purification such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, use of anion exchanger, Using CCL20 protein, it can be easily achieved by means such as affinity chromatography.
  • the patent biological deposit center (National Institute of Advanced Industrial Science and Technology) (T 305 8566 Tsukuba Ibaraki 1-chome 1 1 6) includes monoclonal antibodies produced by hybridomas commissioned under FERM BP-10445.
  • the “chimeric monoclonal antibody” in the present invention can be a recombinant monoclonal antibody produced by genetic engineering.
  • the variable region is a non-human mammal.
  • a chimeric monoclonal antibody such as a mouse Z human chimeric monoclonal antibody characterized in that it is a variable region derived from an immunoglobulin of (mouse, rat, hamster, etc.), and the constant region is a constant region derived from human immunoglobulin. Means.
  • the antibody is immunized with a mouse, and the antibody variable region (V region) that binds to the antigenic power of the mouse monoclonal antibody is excised and combined with the antibody constant region (C region) gene derived from human bone marrow to produce a chimeric monoclonal antibody.
  • V region antibody variable region
  • C region antibody constant region
  • the constant region derived from human immunoglobulin is a recombinant chimera having a unique amino acid sequence depending on isotypes such as IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE.
  • the constant region of the monoclonal antibody may be a constant region of human immunoglobulin belonging to a different isotype. Preferably, it is a constant region of human IgG.
  • the chimeric monoclonal antibody of the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
  • a mouse Z human chimeric monoclonal antibody is prepared with reference to experimental medicine (special issue), No. 1.6, No. 10, 1988, and Japanese Patent Publication No. 3-73280. Can do. That is, human immunoglobulin downstream of an active VH gene (rearranged VDJ gene encoding a heavy chain variable region) obtained from DNA encoding the mouse monoclonal antibody isolated from a hybridoma producing a mouse monoclonal antibody.
  • an active VH gene rearranged VDJ gene encoding a heavy chain variable region
  • the CH gene (C gene encoding the heavy chain constant region) obtained from the DNA encoding
  • Each CL gene (C gene encoding the light chain constant region) obtained from DNA encoding human immunoglobulin is expressed downstream of the active VL gene (rearranged VJ gene encoding the light chain variable region). It can be prepared by arranging as possible and inserting it into one or a separate expression vector, transforming a host cell with the expression vector, and culturing the transformed cell.
  • DNA is extracted from a mouse monoclonal antibody-producing hybridoma by a conventional method, and then the DNA is digested with an appropriate restriction enzyme (eg, EcoRI, Hindlll, etc.) and subjected to electrophoresis.
  • an appropriate restriction enzyme eg, EcoRI, Hindlll, etc.
  • electrophoresis For example, use a 0.7% agarose gel
  • attach the marker wash the gel twice, and soak in 0.25 M HC1 solution for 15 minutes. Then, soak in 0.4N NaOH solution for 10 minutes and gently shake during that time. Transfer to the filter in the usual manner, collect the filter after 4 hours, and wash twice with 2 X SSC.
  • the filter After the filter is sufficiently dried, perform baking (75 ° C, 3 hours). After baking, the filter is placed in a 0.1 X S SC / 0.1% SDS solution and treated at 65 ° C for 30 minutes. It is then immersed in 3 X SSC / 0.1% SDS solution. The obtained filter is placed in a beer bag together with the prehybridization solution and treated at 65 ° C. for 3 to 4 hours.
  • VDJ and VJ genes that code for the H and L chains of the mouse monoclonal antibody are identified.
  • the region containing the identified DNA fragment is fractionated by sucrose density gradient centrifugation and incorporated into a phage vector (for example, Charon 4 A, Charon 28, ⁇ EMBL3, ⁇ EMBL4, etc.). LE392, NM539, etc.) to produce a genomic library.
  • a phage vector for example, Charon 4 A, Charon 28, ⁇ EMBL3, ⁇ EMBL4, etc.
  • the human CH gene and human CL gene used for chimerization are isolated separately.
  • a chimeric antibody with HI HgGl is prepared, one gene that is a CH gene and a C ⁇ gene that is a CL gene are isolated.
  • These genes utilize the high homology of the nucleotide sequences of the mouse immunoglobulin gene and the human immunoglobulin gene to produce the mouse Cy 1 gene and mouse C ⁇ gene corresponding to the human Cy 1 gene and human C ⁇ gene. And can be obtained by isolating from a human genomic library.
  • mouse VH gene and mouse VL gene isolated as described above, and the human CH gene and human CL gene the promoter region and the enzyme region are taken into consideration and the downstream of the mouse VH gene.
  • the human CH gene and the human CL gene downstream of the mouse VL gene are incorporated into an expression vector such as pSV2gpt or pSV2neo using an appropriate restriction enzyme and DNA ligase according to a conventional method.
  • the chimeric gene of mouse VH gene / human CH gene and mouse VL gene / human CL gene may be arranged simultaneously in one expression vector, or may be arranged in separate expression vectors.
  • the chimeric gene insertion expression vector thus prepared was used for prototyping myeloma cells that did not produce antibodies such as P3X63 'Ag8' 653 cells or SP210 cells. Introduced by the plast fusion method, DEAE-dextran method, calcium phosphate method or electroporation. Transformed cells are selected by culturing in a drug-containing medium corresponding to the drug resistance gene introduced into the expression vector to obtain the desired chimeric monoclonal antibody-producing cells.
  • the target chimeric monoclonal antibody is obtained from the culture supernatant of the antibody-producing cells thus selected.
  • the "human monoclonal antibody” refers to an antigen binding site (CDR) of a mouse antibody.
  • the complementarity determining region of the hypervariable region is the complementarity determining region of the hypervariable region derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.) It means a human monoclonal antibody characterized in that the framework region of the variable region is a framework region of a variable region derived from human immunoglobulin, and the constant region is a constant region derived from human immunoglobulin.
  • the complementarity-determining regions of the hypervariable region are the three regions (Complementarity-deter mining) that are present in the hypervariable region of the variable region of the antibody and directly bind complementarily to the antigen. residue; CDR1, CDR2, CDR3) and the variable region framework region is four relatively conserved regions intervening before and after the three complementarity determining regions (Framework; FR 1, FR2, FR3, FR4).
  • a monoclonal antibody in which all regions other than part or all of the complementarity determining region of the hypervariable region of a monoclonal antibody derived from a non-human mammal are replaced with the corresponding region of human immunoglobulin. means.
  • the constant region derived from human immunoglobulin has a unique amino acid sequence depending on isotypes such as IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD and IgE.
  • the constant region may be a constant region of human immunoglobulin belonging to any isotype. Preferably, it is a constant region of human IgG.
  • the framework region of the variable region derived from human immunoglobulin is not limited. Yes.
  • the human monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
  • a recombinant human monoclonal antibody derived from a mouse monoclonal antibody can be prepared by genetic engineering with reference to JP-A-4 506458 and JP-A-62-296890. . That is, at least one mouse heavy chain CDR gene and at least one mouse light chain CDR gene corresponding to the mouse heavy chain CDR gene are isolated from the hybridoma producing the mouse monoclonal antibody, and the human immunoglobulin gene power is also increased.
  • the isolated mouse H chain CDR gene and the human H chain gene can be introduced into an appropriate expression vector so that they can be expressed, and the mouse L chain CDR gene and the human L chain gene can be expressed in the same manner.
  • the mouse H chain CDR gene Z human H chain gene and the mouse L chain CDR gene Z human L chain gene can also be introduced so that they can be expressed in the same expression vector.
  • the "human monoclonal antibody” in the present invention refers to all regions including the variable region of the heavy chain and the constant region of the heavy chain, and the variable region of the light chain and the constant region of the light chain that constitute immunoglobulin.
  • the polyclonal antibody described above can be obtained by immunizing a transgenic animal prepared by incorporating at least a human immunoglobulin gene into the locus of a mammal other than human, such as a mouse, with an antigen. Alternatively, it can be produced in the same manner as the monoclonal antibody production method.
  • transgenic mouse producing a human monoclonal antibody is described in Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; Hei 4-50 4365 gazette; JP 7-509137 gazette; Nikkei Science, June, pp. 40-50, 195; International application publication W094Z25585 gazette; Nature, Vol.368, p. 856-859, 1994; and JP-T 6-500233.
  • the term "monoclonal antibody” may include the above-described chimeric monoclonal antibody, human monoclonal antibody and human monoclonal antibody.
  • the "antibody fragment” in the present invention means a partial region of the monoclonal antibody, chimeric monoclonal antibody, human monoclonal antibody, and human monoclonal antibody as described above, specifically F ( ab ′) 2, Fab ′, Fab, Fv (variable fragment of antibody), disulfide-binding Fv, single chain antibody (scFv), and polymers thereof.
  • Fv, dsFv disulfide stabilised Fv
  • single domain antibody single domain antibody
  • chobi ⁇ & is produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin or nopain, This refers to an antibody fragment that is produced by digestion before and after the disulfide bond existing between two heavy chains in the hinge region.
  • antibodies reactive to CCL20 and fragments thereof are useful for the treatment and prevention of bone destruction, and are used as bone destruction inhibitors. can do.
  • bone destruction is used to include one or both of bone destruction and cartilage destruction.
  • the antibody reactive to CCL20 of the present invention can be used as an agent for preventing, diagnosing or treating autoimmune diseases (particularly rheumatoid arthritis).
  • treatment generally means obtaining a desired pharmacological and / or physiological effect.
  • the effect is therapeutic in terms of partial or complete cure of the disease and / or the adverse effects caused by the disease.
  • treatment includes any treatment of diseases of mammals, particularly humans, and includes, for example, the following treatments (a) and (b):
  • treatment may include “prevention” (including prevention of the occurrence of a disease or symptom in a patient).
  • bone comprising the step of administering to a mammal in need thereof a therapeutically effective amount of a CCL20 reactive antibody and fragment thereof.
  • an antibody having CCL20 reactivity and a fragment thereof for the manufacture of a bone destruction inhibitor and a bone destruction therapeutic agent.
  • cell migration of CCR6-expressing cells to CCL20 was remarkably suppressed by an antibody having reactivity with CCL20. Therefore, an antibody having reactivity with CCL20 can be used as a cell migration inhibitor.
  • cell migration means cell migration of CCR6-expressing cells with respect to CCL20 protein.
  • an antibody having reactivity to CCL20 can be used as a bone destruction inhibitor, a bone destruction therapeutic agent, and a cell migration inhibitor, those containing this antibody can be used as a pharmaceutical composition. it can.
  • the administration mode of the antibody according to the present invention is not particularly limited, and is any one of oral administration and parenteral administration (for example, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, topical administration). Although it can be administered to mammals including humans by the route, parenteral administration, particularly intravenous injection, is preferred.
  • Dosage forms for oral administration and parenteral administration and methods for producing the same are well known to those skilled in the art.
  • a conventional method is obtained.
  • Dosage forms for parenteral administration include injectable preparations (eg, instillation, intravenous injection, muscle injection, subcutaneous injection, intradermal injection), and external preparations (eg, ointment, And suppositories, ophthalmic preparations, eye ointments, nasal drops, ear drops, and ribosomes.
  • injectable preparations eg, instillation, intravenous injection, muscle injection, subcutaneous injection, intradermal injection
  • external preparations eg, ointment, And suppositories, ophthalmic preparations, eye ointments, nasal drops, ear drops, and ribosomes.
  • a preparation for injection is usually prepared by dissolving the antibody according to the present invention in distilled water for injection.
  • solubilizers, buffers, pH adjusters, isotonic agents, soothing agents, preservatives, stabilizers and the like can be added as necessary. It can also be made into a freeze-dried preparation for business preparation.
  • the dosage forms for oral administration are solid or liquid dosage forms, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions. Agents, injections, troches and the like.
  • the pharmaceutical composition according to the present invention may further contain other therapeutically effective drugs, and if necessary, blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins Ingredients such as amino acids, humectants and keratolytic agents can also be added.
  • the ratio of the active ingredient to the carrier at this time can be varied between 1 to 90% by weight.
  • Carriers used for formulating these preparations include, for example, commonly used excipients, binders, disintegrants, lubricants, colorants, flavoring agents, and if necessary, stabilizers, emulsifiers, absorptions.
  • Non-toxic components that can be used include, for example, animal and vegetable oils such as soybean oil, beef tallow and synthetic glycerides; hydrocarbons such as liquid paraffin, squalene and solid paraffin; for example, otatildodecyl myristate, isopropyl myristate, etc.
  • Ester oils of higher alcohols such as cetostearyl alcohol and beh- ol alcohol; silicone oils; silicone oils such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, Surfactants such as polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene block copolymer; for example, hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol Water-soluble polymers such as ethanol, polyvinyl pyrrolidone and methyl cellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols (polyols) such as glycerin, propylene glycol, dipropylene glycol, sorbitol and polyethylene glycol; Sugars such as sucrose; inorganics such as caustic anhydride, aluminum magnesium silicate, aluminum silicate, etc. Powder; inorganic salt such
  • excipient examples include lactose, fructose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, silicon dioxide, and the like
  • binder examples include polyvinyl alcohole, polyvinylinoleate, methyl. Noresenorelose, ethinoresenorelose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropyl pillcellulose, polyvinylpyrrolidone, polypropylene glycol.Polyoxyethylene block polymer, medalmin, etc.
  • starch starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, calcium quenate, dextrin, pectin, carboxymethyl cellulose 'calcium, etc.
  • Powers that are permitted to be added to pharmaceuticals are flavourants. , Hatsu power oil, Borneolum, cinnamon powder, etc. are used.
  • the above component may be a salt thereof or a hydrate thereof.
  • an oral preparation is prepared by adding an excipient, and further, for example, a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, and the like to an active ingredient, and then, for example, a powder by a conventional method.
  • sugar coating or other appropriate coating may be used if necessary.
  • pH adjusters, solubilizers, tonicity agents, etc., and if necessary, solubilizing agents, stabilizers, etc. are added and formulated in conventional manner.
  • the production method is not particularly limited, and it can be produced by a conventional method.
  • the base material to be used various raw materials usually used for pharmaceuticals, quasi drugs, cosmetics, etc. can be used.
  • animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fats examples include raw materials such as acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric alcohols, water-soluble polymers, clay minerals, purified water, and pH adjusters and antioxidants as necessary.
  • Agents, chelating agents, antiseptic / antifungal agents, coloring agents, fragrances and the like can be added.
  • components such as blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, and keratolytic agents can be blended as necessary.
  • the ratio of the active ingredient to the carrier at this time can be varied between 1 to 90% by weight.
  • Compounds used in the present invention, the present invention When the peptides used in the present invention or the polynucleotides used in the present invention are used for the treatment, they are purified to at least 90% or more, preferably 95% or more, more preferably 98% or more, and further preferably 99% or more. It is preferable to use those prepared.
  • the dose of the antibody according to the present invention is, for example, a drug such as administration route, disease type, symptom severity, patient age, sex, body weight, disease severity, pharmacokinetics and toxicological characteristics. It can be determined by the clinician based on a variety of factors, including physical findings, whether drug delivery systems are used, and whether they are administered as part of a combination of other drugs.
  • a drug such as administration route, disease type, symptom severity, patient age, sex, body weight, disease severity, pharmacokinetics and toxicological characteristics. It can be determined by the clinician based on a variety of factors, including physical findings, whether drug delivery systems are used, and whether they are administered as part of a combination of other drugs.
  • Per 60 kg) 1 to 5000 gZ5000 for oral administration, preferably 10 to 2000 gZ days, more preferably 50 to 2000 gZ days, 1 to 5000 g / day, preferably 5 to 2000 gZ days for injection administration, More preferably, 50 to 2000 gZ days can be administered in
  • a method for screening a substance that alters the interaction between CCR6 and CCL20 which comprises using CCL20 and a cell membrane containing CCR6 or a cell containing the same.
  • this method comprises contacting CCL20 with a cell membrane containing CCR6 or a cell containing the same in the presence of a test substance and in the absence of the test substance, and then measuring cell stimulating activity. And comparing the measurement results in the presence of the test substance and in the absence of the test substance.
  • the method determines the substance when there is a difference in the result between the presence of the test substance and the absence of the test substance. It further comprises the step of determining that it is a substance that changes the cell stimulating activity via CCR6.
  • a test substance (or test compound) can be screened by distinguishing the ability to promote or inhibit the function (migration) of CCR6.
  • this screening method can screen for substances that alter the interaction (binding ability) between CCL20 and CCR6.
  • compounds that affect the activation of CCR6 and CCR6 More specifically, compounds that inhibit the binding of CCL20 to CCR6
  • There is a substance that alters cell-stimulating activity mediated by glycine more specifically, a substance that promotes the function of CCR6 (agonist) !, a substance that inhibits the function (migration) of CCR6 (antagonist) Can be screened.
  • the agonist promotes the function (migration) of CCR6, and therefore induces a disease model of bone destruction, and thus can be used for the preparation of the disease model.
  • the present invention produces an antibody that suppresses the migration of CCR6-expressing cells to CCL20, and the antibody against the protein has a therapeutic effect on a collagen-induced arthritis model in which bone destruction is observed (especially inflammation suppression, bone destruction). It is preferable to screen for substances (antagonists) that inhibit the function (migration) of CCR6. Antagost suppresses the function (migration) of CCR6 and is therefore useful for suppressing bone destruction and treating bone destruction.
  • the test substance can be determined as a substance that promotes the function of CCR6 (CCR6 agonist).
  • test substance When the cell stimulation activity in the presence of the test substance decreases compared to the cell stimulation activity in the absence of the test substance (preferably, when it decreases by about 0.9 times, more preferably about 0.5 times
  • the test substance can be determined to be a substance that inhibits the function of CCR6 (CCR6 antagonist).
  • the concentration of intracellular cAMP that decreases due to inhibition of adenylate cyclase activity, or the concentration of intracellular calcium that increases is measured by a known method.
  • the compound can be screened by distinguishing its ability to promote or inhibit the function of CCR6. This mode is the intracellular signal transduction caused by the action of CCL20 on CCR6, that is, the activity of adenylate cyclase, one of the cell stimulating activities of CCR6, and the effect of increasing intracellular calcium concentration. Is to be used.
  • CAMP concentration decreases and intracellular Ca 2+ concentration increases.
  • an adenylate cyclase activator, CCL20, and a test substance may be added to the screening cells.
  • the amount of cAMP produced is reduced by the action of CCL20 compared to the case where the adenylate cyclase activator is added alone, but the amount of cAMP produced is reduced when antagonizing the action of test substance SCCL20. Suppress.
  • a test substance is contacted. Select compounds that suppress the increase in intracellular Ca 2+ concentration. In this case, the test substance can be selected as a compound that inhibits the function of CCR6.
  • Examples of the method for measuring the amount of intracellular cAMP include immunoassay and the like, and a commercially available cAMP quantification kit can also be used.
  • the measurement of the cell stimulating activity is carried out by measuring the reporter gene translation by the generation of a signal transducing substance 'the force measured by a reporter assay system that detects a change in the transcription amount, or Intracellular calcium ion release, adenylate cyclase activity, intracellular cAMP production, intracellular cGMP production, arachidonic acid release, acetylcholine release , Inositol phosphate production, cell membrane potential fluctuation, phosphorylation or activation of intracellular protein, pH lowering fluctuation activity, phosphorylation or activation of MAP kinase, c-fos activation, glycerol production activity, lipolysis Activity and adrenocortical hormone secretion activity can be performed by measuring a parameter selected from the group consisting of:
  • CCR6 is also expressed on the cell membrane (preferably, an expression vector containing CCR6 is introduced and overexpressed), and the cAMP response element (CRE) is 5 'upstream.
  • a reporter gene for example, alkaline phosphatase gene, luciferase gene, beta-lactamase gene, nitroreductase gene, chloramphene-coal cetyltransferase gene, beta-galactosidase gene, or fluorescent protein gene such as GFP (Green Fluorescent Protein)) Etc.
  • screening cells compounds can be screened by distinguishing the ability to promote or inhibit the function of CCR6. In this case, it is utilized that the generation of the above-mentioned cAMP increases, and as a result, the transcription of the reporter gene having the CRE introduced into the screening cell in the promoter region is promoted.
  • the CRE introduced into the screening cell is a nucleotide sequence that is commonly present in the transcriptional regulatory region of a gene group (cAMP-inducible gene) whose expression increases when the intracellular cAMP concentration increases. It is. Therefore, when an adenylate cyclase active agent (eg, FSK) is added to screening cells, the intracellular cAMP concentration increases, resulting in an increase in the expression level of the reporter gene located downstream of the CRE. To do. Furthermore, the expression level of this reporter gene is also increased by a signal transduction system due to an increase in intracellular Ca 2+ concentration.
  • adenylate cyclase active agent eg, FSK
  • the expression level of the reporter gene product is measured by measuring the luminescence derived from the amount of the luminescent substance that reacts with the reporter gene product and generates substrate power, or the fluorescence derived from the fluorescent protein produced as the reporter gene. Therefore, it is possible to measure easily.
  • test substance is a force through CCR6.
  • a screening cell ie, a cell that expresses CCR6 on the cell membrane and contains a reporter gene in which CRE is located 5 ′ upstream.
  • control cells for example, cells that contain a reporter gene in which CRE is located 5 ′ upstream, but do not express CCR6 on the cell membrane.
  • the method for screening for a substance that alters the interaction (binding ability) between CCL20 and CCR6 comprises: CCR6 in the presence of the test substance and in the absence of the test substance. A cell membrane containing or a cell containing the same and CCL20, and then measuring the amount of CCL20 bound to the cell membrane containing CCR6 or the cell containing the same, in the presence of the test substance Comparing to the absence of the substance.
  • a test compound can be screened without distinguishing the ability to promote or inhibit the function of CCR6. That is, when the method of this embodiment is applicable, it is possible to screen a substance that changes the interaction between CCL20 and CCR6.
  • the binding property of CCL20 to CCR6 can be screened. More specifically, a compound having the ability to promote or inhibit the function of CCR6 can be screened.
  • CCR6 and labeled CCL20 were brought into contact with each other under the absence of the test substance and in the presence of the test substance, and specific binding of CCL20 to CCR6 under the above conditions
  • the test substances are CCL20 and CCR6. More specifically, it is a compound that changes the binding of CCL20 to CCR6. More specifically, it is a CCR6 antagonist or CCR6 antagonist.
  • CCL20 can be labeled.
  • the label include a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance.
  • the radioisotope for example, [ 3 H], [ 14 C], [ 125 I], [ 35 S] and the like can be used.
  • the enzyme for example, j8-galactosidase, alkaline phosphatase, peroxidase and the like can be used.
  • the fluorescent material for example, fluorescein isothiocyanate, B ODIPY or the like can be used. Luciferin, lucigenin, etc. can be used as the luminescent substance.
  • the test substance used in the present invention may be any compound.
  • the present invention evaluates the ability to inhibit CCR6-containing cell migration induced by CCL20, or CCL20 forcedly expressed in bacteria, yeast, mammalian cells, or insect cells. By doing so, screening for therapeutic agents for bone destruction can be performed.
  • the CCL20 used may be CCL20 purified from the mammalian body, CCL20 in cultured cells, and CCL20 in the culture supernatant as long as it is a substance that retains the biological activity of CCL20. .
  • the evaluation of the action of CCL20 on CCR6 should be compared with the CCL20 inhibitory action of an antibody reactive to CCL20 or the CCR6 inhibitory action of an antibody reactive to CCR6.
  • the screening method using an antibody is, for example, an ability to inhibit cell migration induced by CCL20 protein or CCL20 protein forcedly expressed in bacteria, yeast, mammalian cells, or insect cells.
  • (Degree of action or binding) genetically engineered CCR6 cells, B lymphocytes, memory T lymphocytes, immature rod cells, and rod precursor cells , CCR6 inhibition of anti-CCR6 antibody Perform by evaluating the ability to inhibit the action (degree of action or binding), or by comparing and evaluating the degree of action or binding of CCL20 protein expressed as described above and CCR6 expressed as described above be able to.
  • Evaluation of the comparison can be made by comparing the degree of action or binding between CCL20 and CCR6 in the absence of the test substance and the degree of action or binding between CCL20 and CCR6 in the presence of the test substance. It can also be evaluated using cell stimulation activity as an index. Cell stimulation activity can be measured by reporter gene translation due to the generation of signal transmitters. Reporter detection of changes in transcription level.
  • the expression of CCL20 stimulates cell migration of CCR6-expressing cells and thereby promotes bone destruction. Therefore, by detecting or quantifying the degree of CCL20 expression, Bone destruction can be detected. Therefore, according to the present invention, there is provided a method for detecting bone destruction and a method for diagnosing bone destruction disease, which comprises the step of detecting the degree of CCL20 expression.
  • a step of comparing the level of CCL20 expression in normal tissues with the level of CCL20 expression in test tissues may be further included.
  • an expression exceeding the expression level of CCL20 in a normal tissue preferably about 1.1-fold expression, more preferably about 1.5-fold expression
  • bone destruction may occur. It can be determined that it is occurring or is a bone destruction disease.
  • body force such as blood, synovial fluid, or synovial tissue
  • site force such as veins or joints.
  • CCL20 should be reactive.
  • Antibodies and fragments thereof can be used to detect or quantify the extent of CCL20 expression. Detection and quantification by an antibody is well known to those skilled in the art, and is not particularly limited as long as the degree of expression of CCL20 can be detected and quantified.
  • immunoassay can be preferably used.
  • immunoassay refers to the detection of an antigen contained in a sample (eg, a body fluid sample such as plasma, a culture supernatant or a centrifugal supernatant) based on the principle of an antigen-antibody reaction.
  • a sample eg, a body fluid sample such as plasma, a culture supernatant or a centrifugal supernatant
  • monoclonal antibody or antibody fragment thereof having reactivity with the antibody force CCL20 in the antigen-antibody reaction is meant.
  • one antibody solid phase method two antibody liquid phase method, as described in enzyme immunoassay (3rd edition, edited by Yuji Ishikawa et al., Published by Medical School, 1987).
  • Two-antibody solid phase method Santotsuchi method, EMIT method (Enzyme multiplied immunoassay technique; Enzyme channeling immunoassay), Enzyme modulator mediated enzyme immunoassay ⁇ EMMIA Enzyme inhibitor immunoassay, Imunoenzymometric assay, Enzyme enhanced immunoassay, Proximal inhibitor immunoassay (Proximal inhibitor immunoassay) linkage im munoassay), one-pot method (Japanese Patent Publication No. 2-39747), and the like.
  • an immunoassay can be appropriately selected and used depending on the purpose. However, it is easy to operate and has Z or economical convenience, particularly clinical versatility.
  • the sandwich method it is preferable to use the sandwich method, the one-pot method, the single-antibody solid-phase method or the dual-antibody liquid-phase method, more preferably the one-pot method, which is immobilized on beads or balls.
  • This is a one-pot method using a fixed monoclonal antibody or a fixed antibody fragment and a labeled monoclonal antibody or labeled antibody fragment labeled with an enzyme or piotin.
  • This particularly preferred embodiment is, as a specific example, according to the embodiment, the hybridoma (FERM BP-10445) -producing monoclonal antibody of the present invention or their F (ab,) or
  • the first one-pot method is an immunoassay method including at least the following steps (a) and (b).
  • the second is an immunoassay method including at least the following steps (a) and (b).
  • the third is an immunoassay method including at least the following step (a).
  • the first method includes the following process power.
  • Step 1 Immobilized monoclonal antibody (FERM BP-1044 5) -producing monoclonal antibody of the present invention having reactivity with CCL20 is immobilized on beads, and immobilized monoclonal antibody (monoclonal antibody-immobilized beads) Producing
  • Step 2 Place the monoclonal antibody-immobilized beads and human plasma samples together with the buffer in a container with an internal volume such as a test tube, plate, tube, etc.
  • Step 3 Step of removing the inner solution in the container and washing the monoclonal antibody immobilized beads.
  • Step 4 A step of producing a labeled monoclonal antibody by labeling a monoclonal antibody produced by the hyperidoma of the present invention (FERM BP-10445) having reactivity to CCL20 with an enzyme such as piotin or peroxidase;
  • Step 5 Antigen antibody formed by adding labeled monoclonal antibody to the container containing the monoclonal antibody-immobilized beads washed in step 3, and reacting the immobilized monoclonal antibody with CC L20 contained in the sample Reacting the conjugate with a labeled monoclonal antibody;
  • Step 6 Step of removing the internal solution in the container and washing the monoclonal antibody fixed beads to remove unreacted labeled monoclonal antibody from the antigen-antibody complex;
  • Step 7 A container containing the monoclonal antibody-fixed beads washed in Step 6 contains avidin or an enzyme-modified avidin in Step 4, and peroxidase etc. in Step 4.
  • an enzyme-labeled monoclonal antibody labeled with any of the above enzymes depending on the method used to measure the enzyme activity, various substrates are added together with a chromogenic material as necessary to react with the labeled material on the labeled monoclonal antibody.
  • a step of causing; (Step 8) When enzyme-modified avidin is added in Step 7, depending on the method for measuring enzyme activity, various substrates are added, and the enzyme bound to avidin is reacted with the substrate;
  • Step 9 A step of adding a reaction stop solution to the reaction system of Step 7 or Step 8 to stop the enzymatic reaction and the color development reaction; and (Step 10) a step of measuring the colorimetric intensity, fluorescence intensity or emission intensity.
  • the second method has the following process power.
  • Step 1 A step of producing a labeled monoclonal antibody by labeling a monoclonal antibody produced by the hyperpridoma (FERM BP-1044 5) of the present invention having reactivity to CCL20 with an enzyme such as piotin or peroxidase;
  • Step 2 A step of adding a labeled monoclonal antibody and a sample such as human plasma together with a buffer to a container having an internal volume such as a test tube, plate or tube, and reacting the labeled monoclonal antibody with the sample;
  • Step 3 Immobilize the monoclonal antibody produced by the high-pridoma of the present invention (FERM BP—1044 5), which is reactive to CCL20, on the beads to produce the immobilized monoclonal antibody (monoclonal antibody-immobilized beads).
  • Step 4 Add monoclonal antibody-immobilized beads to the reaction system of Step 2, and add the immobilized monoclonal antibody to the antigen-antibody complex formed by the reaction between the labeled monoclonal antibody and CCL20 contained in the sample. Reacting;
  • Step 5 Step of removing the inner solution in the container and washing the monoclonal antibody immobilized beads to remove unreacted labeled monoclonal antibody;
  • Step 6 When the biotin-labeled monoclonal antibody used in Step 1 is used in the container containing the monoclonal antibody-fixed beads washed in Step 5, avidin is present, enzyme modified avidin, and peroxidase etc. in Step 1.
  • an enzyme-labeled monoclonal antibody labeled with any of the above enzymes depending on the method used to measure the enzyme activity, various substrates are added together with a chromogenic material as necessary to react with the labeled material on the labeled monoclonal antibody.
  • Step 7 When enzyme-modified avidin is added in Step 6, depending on the method for measuring enzyme activity, various substrates are added, and the enzyme bound to avidin is reacted with the substrate;
  • Step 8) A step of adding a reaction stop solution to the reaction system of Step 6 or Step 7 to stop the enzymatic reaction and the color reaction; and
  • Step 9) a step of measuring the colorimetric intensity, fluorescence intensity, or luminescence intensity.
  • the third method also has the following process capability.
  • Step 1 Immobilizing the monoclonal antibody produced by the high-pridoma of the present invention (FERM BP—1044 5), which is reactive to CCL20, on the beads, and immobilizing the monoclonal antibody (monoclonal antibody-immobilized beads).
  • Step 2 A step of producing a labeled monoclonal antibody by labeling a monoclonal antibody produced by the present invention having high reactivity with CCL20 (FERM BP-10445) with an enzyme such as piotin or peroxidase;
  • Step 3 Monoclonal antibody-immobilized beads prepared in Step 1, labeled monoclonal antibody prepared in Step 2, and human plasma together with a buffer in a container having an internal volume such as a test tube, plate or tube And the like, and the step of reacting the immobilized monoclonal antibody, the labeled monoclonal antibody, and the sample simultaneously;
  • Step 4 Step of removing the inner solution in the container and washing the monoclonal antibody immobilized beads to remove unreacted labeled monoclonal antibody;
  • Step 5 If the container containing the monoclonal antibody-immobilized beads washed in Step 4 is used with a piotin-labeled monoclonal antibody in Step 2, avidin is present, enzyme-modified avidin, and in Step 2, peroxidase, etc.
  • an enzyme-labeled monoclonal antibody labeled with any of the above enzymes depending on the method used to measure the enzyme activity, various substrates are added together with a chromogenic material as necessary to react with the labeled material on the labeled monoclonal antibody.
  • Step 6 When enzyme-modified avidin is added in Step 5, depending on the method for measuring enzyme activity, various substrates are added, and the enzyme bound to avidin is reacted with the substrate;
  • Step 7 A step of adding a reaction stop solution to the reaction system of Step 5 or Step 6 to stop the enzymatic reaction and the color reaction; and (Step 8) a step of measuring the colorimetric intensity, fluorescence intensity, or luminescence intensity.
  • the Hyperidoma (FERM BP 10445)
  • the production monoclonal antibody and another monoclonal antibody or polyclonal antibody can be used for the detection and quantification method of the present invention.
  • Antibodies or labeled antibody fragments can be used.
  • An immunoassay method such as a one-antibody solid phase method or a two-antibody liquid phase method is also effective for the detection / quantification method of the present invention.
  • affinity chromatography refers to CCL20 contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, or a centrifugal supernatant) by using an antigen-antibody reaction. It means a method of separation or purification. Specifically, (1) a filter which is an insoluble carrier is immobilized on a membrane or the like after the hybridoma (FERM BP-10445) -producing monoclonal antibody or antibody fragment of the present invention having reactivity to CCL20 is immobilized.
  • a filter which is an insoluble carrier is immobilized on a membrane or the like after the hybridoma (FERM BP-10445) -producing monoclonal antibody or antibody fragment of the present invention having reactivity to CCL20 is immobilized.
  • the high-pridoma (FERM) of the present invention having reactivity to CCL20 on an insoluble carrier such as a polymer, a polyvinyl alcohol carrier, a polyamino acid carrier or a porous silica carrier.
  • BP-10445 produced monoclonal antibody or antibody fragment is immobilized by a conventional method (physical adsorption, polymerization by crosslinking, sealing in a matrix or immobilization by noncovalent bonding, etc.), and the insoluble carrier is made of glass.
  • the sample is packed in a plastic or stainless steel column, and the sample (eg, a columnar column) is eluted through a sample (eg, a body fluid sample such as plasma, a culture supernatant, or a centrifugal supernatant). Separation of CCL20 contained in a sample is a method for purification.
  • the latter method (2) is particularly referred to as affinity column chromatography.
  • any insoluble carrier capable of immobilizing the monoclonal antibody or antibody fragment of the present invention can be used.
  • immobilized monoclonal antibody and “immobilized antibody fragment” refer to a monoclonal antibody and an antibody fragment that are supported on an insoluble carrier by physical adsorption or chemical binding. Each means.
  • These fixed monoclonal antibodies and fixed antibody fragments detect, quantify, separate or purify CCL20 contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, or a centrifugal supernatant).
  • a sample for example, a body fluid sample such as plasma, a culture supernatant, or a centrifugal supernatant.
  • an immobilized antibody monoclonal antibody or immobilized antibody fragment immobilized on the insoluble carrier can be used.
  • a plastic plate having many wells such as a 96-well microtiter plate.
  • the immobilized monoclonal antibody or the immobilized antibody fragment immobilized on the filter or membrane described in (1) above or the insoluble carrier listed in (2) above is used. Can be used.
  • an antibody having reactivity with CCL20 may be labeled with "a labeling substance capable of producing a detectable signal by reacting alone or with another substance". They are used to detect their presence by binding to monoclonal antibodies or antibody fragments by physicochemical binding or the like.
  • a labeling substance capable of producing a detectable signal by reacting alone or with another substance They are used to detect their presence by binding to monoclonal antibodies or antibody fragments by physicochemical binding or the like.
  • labeling substances are specifically enzymes, fluorescent substances, chemiluminescent substances, piotin, avidin, or radioisotopes, and more specifically, peroxidase, alkaline phosphatase, j8 D galactosidase, glucose oxidase.
  • Gnolecose 6-phosphate dehydrogenase alcohol dehydrogenase, malate dehydrogenase, percylinase, catalase, apoglucosoxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, Fi co pyridinium protein, rare earth metal chelates, Danshiruku port ride or fluorescent substances such as tetramethylrhodamine isothiocyanate Xia sulfonates, 3 ⁇ 4, "C, 125 1 or 131 radioactive isotope, such as 1, Piochin, avidin or chemiluminescence, Quality, and the like.
  • the radioisotope and the fluorescent substance alone can provide a detectable signal.
  • enzymes, chemiluminescent substances, piotin, and avidin alone cannot produce a detectable signal, and therefore react with one or more other substances to produce a detectable signal.
  • an enzyme at least a substrate is required, and various substrates are used depending on the method for measuring enzyme activity (colorimetric method, fluorescence method, bioluminescence method, chemiluminescence method, etc.).
  • there is at least avidin but it is common to react with enzyme-modified avidin.
  • Various color-developing substances depending on the substrate are used as necessary.
  • labeled monoclonal antibody and labeled antibody fragment mean a monoclonal antibody and an antibody fragment each labeled with a labeling substance. These labeled monoclonal antibodies and labeled antibody fragments can be used to detect or quantify CCL20 contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, a centrifugal supernatant, or the like).
  • a sample for example, a body fluid sample such as plasma, a culture supernatant, a centrifugal supernatant, or the like.
  • labeling with piotin is preferable.
  • Kits and diagnostic agents [0161] Kits and diagnostic agents
  • kits used for carrying out the method for detecting bone fracture and the method for diagnosing bone fracture according to the present invention are necessary for measuring or quantifying an antibody antigen reaction that may contain an antibody reactive with CCL20 or a fragment thereof. Including additional necessary reagents and instruments.
  • the antibody reactive with CCL20 and the fragment thereof may be immobilized or labeled according to the measurement method.
  • a bone destruction detection agent and a bone destruction diagnosis agent comprising an antibody reactive with CCL20 and a fragment thereof.
  • an antibody reactive with CCL20 or a fragment thereof for the production of a bone destruction detection agent and a bone destruction diagnosis agent.
  • Anti-mouse CCL20 antibody was prepared as follows. After mixing mouse CCL20 (manufactured by R & D) and TiterMax TM Gold adjuvant, Armenian hamster (obtained from Oriental Bio) was immunized several times, and final immunization was performed with mouse CCL20 alone. The antibody titer in the serum was measured by ELISA using mouse CCL20 with solid phase, and lymphocytes were separated from the armored hamster with an increased antibody titer, so that the ratio of lymphocytes to P3 myeloma cells was 1: 1. And cell fusion was performed using a PEG 1500 solution (Boehringer).
  • Hypridoma was cultured on a plate for 1 week using RPMI-1640 / 10% FCS / HAT / 10% Origen HCF (manufactured by ISGN). Hybridomas were selected with HAT medium (Invitrogen), and the culture supernatant of the obtained hybridoma was screened by sandwich ELISA using mouse CCL20-AP chimeric protein. As a result, 42 positive wells were obtained. Next, the inhibition of the binding of mouse CCL20-AP to mouse CCR6-expressing cells was used as an index to obtain a well 2F5 showing strong binding inhibitory activity. Hyperidoma producing anti-mouse CCL 20 antibody was cloned by two limiting dilutions.
  • Monoclonal antibodies were purified using protein A columns from SCID and pristane-administered ascites prepared by inoculating Hypridoma in nude mice.
  • the neutralization activity of the obtained antibody was measured using inhibition of migration of mouse CCR6-expressing cells to mouse CCL20 as an index, and 2F5-5 antibody having neutralizing activity was obtained.
  • mice CCL20- diluted to a concentration of 6 nM in a binding solution (RPM—1640 containing 20 mM HEPES (pH 7.4), 1% BSA, 0.02% sodium azide) in 100 ⁇ l of culture supernatant of Hypridoma Add SEAP solution 25 ⁇ 1 and react at room temperature for 10 minutes, then mix 2 ⁇ 10 5 cells with binding solution ⁇ 25 1 mouse CCR6 expression ⁇ 300. 19 cells 25 ⁇ 1 mix and react at 16 ° C for 1 hour It was.
  • the resulting hybridoma producing anti-mouse CCL20 monoclonal antibody 2F5-5 was inoculated into the abdominal cavity of nude mice to obtain ascites, and the antibody was purified using a Protein A column.
  • the activity of suppressing the migration of mouse CCR6-expressing cells to mouse CCL20 was measured using the purified antibody, it became clear that 1 ⁇ gZml showed almost 100% neutralizing activity (FIG. 1).
  • the hybridoma producing the anti-mouse CCL20 monoclonal antibody (CCL20 mAb) 2F5-5 was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under FERM BP-10445.
  • the mouse CCR6 gene expression vector was prepared as follows.
  • mCCR6-Sail— F CGCGTCGACGCCACCATGAATTCCACAGAGTCCTA (sequence number 1)
  • mCCR6-Notl-R GCGGGCGGCCGCCATGGTAAAGGACGATGCAT (SEQ ID NO: 2)
  • PCR Mouse spleen quick-clone cDNA (Clontech) was used in a saddle shape.
  • PCR is the following Reaction solution thread (10 X buffer 51, 2.5 mM dNTP 41, Pyrobest polymer ase (manufactured by TAKARA) 0.5 1, 100 100 primer 0.5 1 each, cDNA 1 1, distilled water 38. 5 Performed in 1).
  • PCR was performed at 94 ° C for 3 minutes, followed by 40 cycles of reaction at 94 ° C for 30 seconds, 65 ° C for 30 seconds and 72 ° C for 3 minutes, and finally at 72 ° C for 3 minutes. .
  • the amplified cDNA was cloned into pSPORTl (manufactured by GIBCO) and the base sequence was confirmed using ABI3100 sequence analyzer.
  • the obtained cDNA fragment was inserted into an expression vector pM XII EGFPN (Oncogene (2000) 19 (27): 3050-3058) to prepare an mCCR6 gene expression vector.
  • a recombinant retrovirus was produced as follows.
  • Transfusion solution is expressed in 600 ml of OPTI-MEM (GI BCO BRL) and 24 ⁇ 1 of TransIT LT1 (TaKaRa) mixed in a 5 ml tube and allowed to stand for 5 minutes at room temperature.
  • the vector 9 ⁇ g and the packaging vector pCL-Eco (manufactured by Imgenex) 9 ⁇ g were added and the mixture was allowed to stand at room temperature for 5 minutes. After 48 hours, the culture supernatant was collected and filtered through 0.45 / zm to obtain a recombinant virus solution.
  • This recombinant virus was infected with the B300.19 cell line (EMBO J. (1984) 3: 1209-121 9) as follows to prepare mCCR6 gene-expressing cells.
  • B300. 2 2 ⁇ 10 5 cells were added to a 1.5 ml tube, centrifuged at 3000 rpm for 2 minutes at room temperature, and the culture supernatant was removed by aspiration.
  • Mouse CCR6-expressing cells were obtained by isolating cells that migrated against mouse CCL20.
  • Neo vector was constructed as follows [0173] The endogenous Sail site of pCDNA3.1 (+) — Neo vector (manufactured by Invitrogen) was deleted by digestion with Sail and smoothing. SEAP (His) cDNA fragment is
  • Amplification was performed by PCR using 5 'primer with Hindlll and 3' primer with Xhol.
  • the obtained cDNA fragment was digested with Hindlll and Xhol and then inserted into PCDNA3.1 (+) — Neo vector from which the Sail site was deleted.
  • mMIP-3 ⁇ -Sail- F CGCGTCGACGCCACCATGGCCTGCGGTGGCAAGCG
  • mMIP-3 a Notl— R: GCGGGCGGCCGCCATCTTCTTGACTCTTAGGC (sequence number 4)
  • mouse CCL20 The extracellular region of mouse CCL20 consists of a single-stranded cDNA synthesized from total RNA from the BALB / c mouse small intestine using RNA PCR kit (TAKARA), with a sail attached, and a primer. Amplification was performed by PCR using (mMIP-3 ⁇ -Sail-F) and a primer (mMIP-3a-Notl-R) with Notl attached.
  • TAKARA RNA PCR kit
  • PCR was carried out using the following reaction mixture (10 X buffer 51, 2.5 mM dNTP 41, Pyrobest polymerase (TAKARA) 0.5 1, 100 ⁇ primer 0.5 1 each, cDNA 1 1, Distilled water 38.5 1) PCR was performed at 94 ° C for 3 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 3 minutes. .
  • the amplified cDNA was cloned into pBlueScriptl SK (—) (Stratagene), and the nucleotide sequence was confirmed using ABI3100 sequence analyzer.
  • the obtained cDNA fragment was digested with Sail and Notl, and inserted into the above pcDNA3.1 (+) — SEAP (His) —Neo vector to obtain mouse CCL20—
  • AP expression vector was prepared.
  • AP chimeric protein a secreted chimeric protein (hereinafter referred to as AP chimeric protein) The present is possible.
  • the obtained AP chimeric protein expression vector was introduced into 293 / EBNA-1 cell line using TransIT LT1 (manufactured by TAKARA), and cultured for 4 days for 5 days.
  • AP chimera protein secreted into the culture supernatant is collected by centrifugation, filtered through a 0.22 m filter, and then Hepes (pH 7.4) and sodium azide, respectively. The final concentration was 20 mM and was stored at 4 ° C. so as to be 0.02%. The concentration of the AP chimeric protein was calculated by measuring the alkaline phosphatase activity using Aurora AP chemiluminesce reporter gene assay (ICN).
  • ICN Aurora AP chemiluminesce reporter gene assay
  • Example 2 Pile treatment effect of pile CCL20 pile in collagen-induced pakistritis model
  • the Collagen-Induced Arthritis (CIA) model is a disease model of rheumatoid arthritis with bone destruction, and CD4 + T cells and antibodies that react with type II collagen are detected. It is thought to cause arthritis. In the CIA model, it is said that MHC class II is restricted.
  • CCL20 is a cell migration molecule expressed in activated epidermal cells and induces cell migration of immune cells such as rod cells, T cells, and B cells via CCR6. Therefore, by inhibiting the interaction with CCR6 with anti-CCL20 antibody, it was possible to suppress the immune response involving rod cells, T cells, and B cells.
  • the anti-CCL20 antibody may be able to enhance the immune response by releasing the immunosuppressive effect involving inhibitory T cells. Therefore, the therapeutic effect of anti-mouse CCL20 antibody in a collagen-induced arthritis model was examined.
  • the collagen-induced arthritis model was prepared by mixing an equal volume of 3% type II collagen solution (made by collagen technology training company) derived from ushi joint and complete Freund's adjuvant (made by Difco). 100 / ⁇ per DBAZU mouse (obtained from Charles River Company) -Immunization was performed in the buttocks on day 21 (primary immunization) and day 0 (boost immunization). From the third day after the booster immunization, body weight measurement and appearance evaluation were performed over time. Appearance findings were evaluated by scoring as follows.
  • 0 Non-onset
  • 1 Erythema and mild swelling at the ankle or ankle joint
  • 2 Erythema and ankle strength, mild swelling at the ankle
  • 3 Erythema and moderate swelling from the ankle to the midfoot
  • 4 Erythema and severe swelling of the ankles, feet and fingers.
  • Emulsion was prepared by mixing an equal volume of a 3% solution of cocoon-type collagen derived from tussock joints (manufactured by a collagen technology training company) and complete Freund's adjuvant (manufactured by Difco).
  • a 5-week old DBAZ 1J mouse 100 1 (150 ⁇ gZ) per animal was immunized in the buttocks skin on day -21 (primary immunization) and day 0 (boost immunization).
  • Anti-mouse CCL20 antibody 2F5-5 was administered to the tail vein twice a week at 500 gZl from the booster immunization.
  • the booster immunity was also measured from the third day on body weight measurement and appearance (sometimes referred to as “macroscopic”).
  • SAA serum serum amyloid A
  • COMP Cartilage Oligomeric Matrix Protein
  • COMP Cartilage Oligomeric Matrix Protein
  • Type II collagen which is a major component of the cartilage matrix, and has a function of stabilizing the collagen network. It is known that when cartilage is damaged in an inflammatory disease, a part of matrix constituent proteins flows into the joint fluid. COMP flows into the synovial fluid prior to Type II collagen due to the destruction of articular cartilage, and then moves into the blood, the amount of which is an indicator of cartilage destruction.
  • the concentration of SAA in plasma was measured using an ELISA kit (Biosource) after diluting plasma 12,000 times with 2000 power.
  • the antibody titer against collagen in plasma was measured as follows. First, 50 g of a type II collagen solution derived from a ushi joint was added to a 96-well ELISA plate (manufactured by Nunc) one by one and allowed to stand at 4 ° C for solid phase immobilization. After washing with T-PBS (0.02% Tween20ZPBS), non-specific binding sites were blocked with 1% BSAZPBS. After washing with T-PBS three times, 50-fold diluted plasma with T-PBS was placed in each well and placed at room temperature for 2 hours.
  • RNA expression analysis related to inflammation, cell infiltration, and bone destruction was performed in the foot-and-mouth of collagen-induced arthritis using incomplete Freund's adjuvant for booster immunization.
  • Total RNA (500 ng) purified from the foot and foot using TorizoKlnvitrigen) and RNeasy mini kit (Qiagen) was reverse-transcribed using AMV Reverse transcriptase (TAKARA) and randam hexamer (TAK ARA).
  • TAKARA AMV Reverse transcriptase
  • TAK ARA randam hexamer
  • PCR For real-time PCR, prepare a reaction mixture of various primers, QuantiTect SYBR Green PCR kit (Qiagen) and Uracil-DNA-glycosylase (Invitrogen), then AB I PRISM 7700 Sequence Detector (Applied Biosystems) It was performed using. PCR was performed at 50 ° C. for 2 minutes and at 95 ° C. for 15 minutes, and then at 95 ° C. for 15 seconds and at 60 ° C. for 1 minute for 35 sites.
  • the set of primers used is as follows.
  • TRAP Sterate-Resistant Acid Phosphatase
  • concentration in blood is considered to be a useful indicator of bone destruction.
  • mouse T An ARP activity measurement kit manufactured by SBA Sciences
  • enzyme-linked immunosorbent enzyme assay only fresh active TRAP type-5 produced by osteoclast force was detected, and TRAP type_5b was inactivated and fragmented over time after production. Is not detected.
  • concentration of active TRAP in the plasma was measured by diluting plasma 4 times.
  • COX-2 sense 5,-CTCCCTGAAGCCGTACACAT-3 '(SEQ ID NO: 5) COX— 2 anti-sense 5'-CCCCAAAGATAGCATCTGGA-3 '(SEQ ID NO: 6) MMP9 sense 5'-AGACGACATAGACGGCATCC- 3 '(SEQ ID NO: 7) MMP9 anti -sense 5, -GTGGTTCAGTTGTGGTGGTG-3 '(SEQ ID NO: 8) IL-1 j8 sense 5'-GCTGAAAGCTCTCCACCTCA -3 '(SEQ ID NO: 9) IL-1 j8 anti-sense 5'-AGGCCACAGGTATTTTGTCG -3 '(SEQ ID NO: 10) IL ⁇ 6 sense 5 '-CAAAGCCAGAGTCCTTCAGAG-3' (SEQ ID NO: 11) IL ⁇ 6 anti-sense 5 '-GCCACTCCTTCTGTGACTCC-3' (SEQ ID NO: 12) KC sense 5
  • M-CSFR sense 5,-CGACTTCTTCAAGTGACTCCTTC -3, (SEQ ID NO: 31
  • Collagen arthritis was induced as described in (3), and solids that developed 6 days after the booster were selected and used for the experiment. Apparent findings were scored in the same manner as in (3), and anti-mouse CCL20 antibody 2F5-5 was administered to the tail vein every other day at 500 ⁇ gZl from 6 days after booster immunization. The animals were sacrificed on the 17th day after the booster immunization, and soft X-ray images of the limbs were scored as follows according to the evaluation method described in Agents Actions, 39; 187-194, 1993 to evaluate bone destruction. 0: Non-onset, 1: mild bone destruction, 2: moderate bone destruction, 3: severe bone destruction. As a result, it became clear that anti-mouse CCL20 antibody started administration after onset suppresses not only the appearance (Fig. 13 (a)) but also the development of bone destruction (Fig. 13 (b)).

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Abstract

The object is to provide a pharmaceutical which is effective for the inhibition of bone destruction. Thus, disclosed is a bone destruction inhibitor which comprises a monoclonal antibody responsive to CCL20 or a fragment of the monoclonal antibody as an active ingredient.

Description

明 細 書  Specification
抗 CCL20抗体を含む骨破壊抑制剤  Bone destruction inhibitor containing anti-CCL20 antibody
発明の分野  Field of Invention
[0001] 本発明は、ケモカイン CCL20と CCR6との相互作用を阻害することによる骨破壊の 抑制および骨破壊の治療に関する。本発明はまた、 CCL20に対する抗体並びにそ の医薬および診断用途に関する。  [0001] The present invention relates to suppression of bone destruction and treatment of bone destruction by inhibiting the interaction between the chemokines CCL20 and CCR6. The invention also relates to antibodies against CCL20 and their pharmaceutical and diagnostic uses.
背景技術  Background art
[0002] ケモカインは生体内での主たる細胞遊走因子であり、細胞運動の亢進や細胞接着 分子の活性化を介して、免疫細胞の組織浸潤を制御している。ケモカインは、その最 初の 2つのシスティン残基の配列により、 CC、 CXC、 C、 CXXXCの 4つのサブフアミ リーに分類される。 CC、 CXC、 Cケモカインのメンバーは、約 70アミノ酸力もなる分泌 タンパク質であり、それ自身には接着分子としての活性はないが、細胞接着を誘導す ることができる。分泌されたケモカインは、標的細胞表面上の 7回膜貫通型受容体に 結合して、三量体 Gタンパク質を介してインテグリンを活性ィ匕し、細胞の接着や遊走を 誘導する。近年、ケモカインとその受容体が免疫細胞の浸潤を制御する中心的なサ イト力インであることが報告されたことをはじめ、自己免疫疾患におけるケモカインシス テムの働きがその病態形成に密接に関係していることが明らかになってきている。  [0002] Chemokines are the main cell migration factors in vivo, and control tissue infiltration of immune cells through enhancement of cell motility and activation of cell adhesion molecules. Chemokines are classified into four sub families, CC, CXC, C, and CXXXC, based on the sequence of the first two cysteine residues. Members of CC, CXC, and C chemokines are secreted proteins of about 70 amino acids, and they are not active as adhesion molecules but can induce cell adhesion. The secreted chemokine binds to the 7-transmembrane receptor on the target cell surface, activates integrins via the trimeric G protein, and induces cell adhesion and migration. In recent years, chemokines and their receptors have been reported to be central site power-ins that control immune cell infiltration, and the actions of chemokine systems in autoimmune diseases are closely related to their pathogenesis. It is becoming clear.
[0003] CCR6 CCL20 (LARC(Liver— and activation-regulated chemokine)/MIP 3 a Zexodus)系は、獲得免疫に関与する榭状細胞、メモリー T細胞、成熟 B細胞を 外来抗原に暴露された部位に集結させ、免疫応答を迅速に開始するために重要な 役割を担っていると考えられている（Cytokine & Growth Factor Reviews, 14; 409-42 6, 2003) o腸管免疫ではミエロイド系榭状細胞、腸管指向性メモリー T細胞、ナイ一 ブおよびメモリー B細胞の移動と局在を制御している（Immunity, 12; 495-503, 2000., J Immunol, 162; 186-194, 1999., Blood, 96; 2338-2345, 2000)。皮膚免疫ではラン ゲルハンス細胞と皮膚指向性メモリー T細胞の移動を制御している（J Exp Med, 190; 1755-1768, 1999., J Immunol, 164, 6621-6632, 2000)。また、上皮系組織では炎症 の開始や再燃時に CCL20の発現が誘導されることが知られている（Cytokine & Gro wth Factor Reviews, 14; 409-426, 2003)。腸管上皮細胞やパイエル板の濾胞関連 上皮細胞等では、 CCL20の構成的な発現が認められ、 LPSや TNF a、 IL 1 j8に より発現が増強する（Gut, 51 ; 818-826, 2002, Eur J Immunol, 29, 633-642, 1999., J Exp Med, 191 ; 1381-1394" Int Immunol, 13; 1255-63, 2001)。ケラチノサイト、気管 支上皮細胞、血管内皮細胞、リンパ管内皮細胞、滑膜線維芽細胞等でも、炎症性刺 激により CCL20の発現が誘導される（Cytokine & Growth Factor Reviews, 14; 409- 426, 2003, J Exp Med, 194; 797-808, 2001. , J Exp Med, 190; 1755-1768, 1999)。 [0003] The CCR6 CCL20 (LARC (Liver— and activation-regulated chemokine) / MIP 3 a Zexodus) system is used to place rods, memory T cells, and mature B cells involved in acquired immunity at sites exposed to foreign antigens. It is thought to play an important role in concentrating and quickly initiating the immune response (Cytokine & Growth Factor Reviews, 14; 409-42 6, 2003). Regulates migration and localization of gut-directed memory T cells, naïve and memory B cells (Immunity, 12; 495-503, 2000., J Immunol, 162; 186-194, 1999., Blood, 96; 2338-2345, 2000). Skin immunity controls the migration of Langerhans cells and skin-oriented memory T cells (J Exp Med, 190; 1755-1768, 1999., J Immunol, 164, 6621-6632, 2000). In addition, it is known that CCL20 expression is induced in epithelial tissues when inflammation begins and relapses (Cytokine & Gro wth Factor Reviews, 14; 409-426, 2003). Intestinal epithelial cells and Peyer's follicle-related epithelial cells show constitutive expression of CCL20, which is enhanced by LPS, TNF a, and IL 1 j8 (Gut, 51; 818-826, 2002, Eur J Immunol, 29, 633-642, 1999., J Exp Med, 191; 1381-1394 "Int Immunol, 13; 1255-63, 2001). Keratinocytes, tracheal branch epithelial cells, vascular endothelial cells, lymphatic endothelial cells, Also in synovial fibroblasts, CCL20 expression is induced by inflammatory stimulation (Cytokine & Growth Factor Reviews, 14; 409-426, 2003, J Exp Med, 194; 797-808, 2001., J Exp Med, 190; 1755-1768, 1999).
CCL20の受容体 CCR6は、榭状細胞、 T細胞、 B細胞等で発現して!/、る。 T細胞 での発現は、メモリー T細胞に限定され、 α 4 β 7陽性腸管指向性 Τ細胞、 CLA+皮 膚指向性 Τ細胞のいずれにも発現が認められる（J Immunol, 162; 186-194, 1999., J Immunol, 164, 6621-6632, 2000)。最近では、 CCR6が抑制性 T細胞でもェフエクタ 一メモリータイプに発現していることが明ら力となった（Blood, 105; 2877-2886, 2005) 。 B細胞においては、末梢血中のナイーブ B細胞、抗原刺激を経験したメモリー B細 胞に選択的に発現しており、抗原を認識して迅速に抗体産生を行う為に、 CCL20の 発現が多いパイエル板や炎症上皮組織に移行すると考えられている（Blood, 96; 233 8-2345, 2000., J Exp Med, 191 ; 1303-1318, 2000)。榭状細胞では、 CD34陽性前 駆細胞を SCF、 GM— CSF、 TNF a存在下に分化させた未熟榭状細胞、末梢血単 球を GM— CSF、 IL— 4、 TGF β存在下に分化させた未熟榭状細胞、表皮ランゲル ハンス細胞等、いわゆる未熟な榭状細胞に発現している（J Exp Med, 186; 825-835, 1997. , J Immunol, 163, 1737-1741 , 1999, J Exp Med, 192; 705-718, 2000)。粘膜上 皮や皮膚上皮からの構成的および炎症刺激により産生される CCL20により、未熟榭 状細胞が体腔表層に配置されることで抗原の効率よい取り込みが可能となり、獲得 免疫が始動されると考えられている（Cytokine & Growth Factor Reviews, 14; 409-42 6, 2003, J Exp Med, 190; 1755-1768, 1999)。 TNF αで刺激されたヒト好中球で CC R6が発現しているとの報告もある (Blood, 96; 3958-3963, 2000)。さらに最近では、 C CR6陽性未熟榭状細胞が、ガン新生血管に取り込まれて血管内皮細胞に分ィ匕する ことが報告されている（Nat Med, 10; 950-958, 2004)。一方で、 CDla陽性未熟榭状 細胞が関節リウマチ患者由来の滑膜液によって破骨細胞に分ィ匕することが記載され ているが（Blood, 104; 4029-4037, 2004)、破骨前駆細胞としての CDla陽性未熟榭 状細胞における CCR6— CCL20系の機能や関節リウマチへの関与、およびこの経 路を遮断した際の効果にっ 、ては全く言及されて 、な 、。 The CCL20 receptor CCR6 is expressed on rods, T cells, B cells, etc. Expression in T cells is limited to memory T cells, and expression is observed in both α4β7-positive intestinal-directed sputum cells and CLA + skin-directed sputum cells (J Immunol, 162; 186-194, 1999., J Immunol, 164, 6621-6632, 2000). Recently, it has become clear that CCR6 is also expressed in inhibitory T cells in the memory type (Blood, 105; 2877-2886, 2005). In B cells, it is selectively expressed in naive B cells in peripheral blood and memory B cells that have undergone antigen stimulation, and CCL20 is highly expressed in order to recognize antigens and produce antibodies quickly. It is thought to migrate to Peyer's patches and inflammatory epithelial tissues (Blood, 96; 233 8-2345, 2000., J Exp Med, 191; 1303-1318, 2000). As for rod-shaped cells, CD34-positive precursor cells are differentiated in the presence of SCF, GM-CSF, and TNFa, and peripheral blood monocytes are differentiated in the presence of GM-CSF, IL-4, and TGF β. It is expressed in so-called immature rod cells such as immature rod cells and epidermal Langerhans cells (J Exp Med, 186; 825-835, 1997., J Immunol, 163, 1737-1741, 1999, J Exp Med, 192; 705-718, 2000). CCL20 produced by constitutive and inflammatory stimuli from the mucosal epidermis and skin epithelium allows immature dendritic cells to be placed on the surface of the body cavity, enabling efficient antigen uptake and triggering acquired immunity. (Cytokine & Growth Factor Reviews, 14; 409-42 6, 2003, J Exp Med, 190; 1755-1768, 1999). There are reports that CCR6 is expressed in human neutrophils stimulated with TNFα (Blood, 96; 3958-3963, 2000). More recently, it has been reported that CCR6-positive immature rod cells are taken up by cancer neovascularization and separated into vascular endothelial cells (Nat Med, 10; 950-958, 2004). On the other hand, it is described that CDla-positive immature rod cells are separated into osteoclasts by synovial fluid from patients with rheumatoid arthritis. (Blood, 104; 4029-4037, 2004), the function of the CCR6-CCL20 system in CDla-positive immature cells as osteoclast precursor cells, its involvement in rheumatoid arthritis, and when this pathway is blocked. The effect is not mentioned at all.
[0005] ところで、関節リウマチでは、患者の滑膜液中に CCL20が認められて 、る (Clin Exp  [0005] By the way, in rheumatoid arthritis, CCL20 is found in the synovial fluid of patients (Clin Exp
Immunol, 125; 155-161 , 2001)。変形性関節炎患者の滑膜液中で CCL20の発現 は低い。関節リウマチ患者の滑膜組織では、最表層および血管周囲に CCL20の発 現が見られ、最表層には CCL20発現部位の近傍に CDla陽性未熟榭状細胞が存在 している（J Immunol, 168; 5333-5341 , 2002)。また、関節リウマチ患者由来の滑膜細 胞を IL— 1 β、 TNF a、 IL— 17等で刺激すると CCL20の産生が誘導される (J Immu nol, 167; 6015-6020, 2001)。しかしながら、関節内洗浄液中には種々のケモカイン の発現が認められるため、 CCL20の病態形成への寄与度は明らかではな 、。  Immunol, 125; 155-161, 2001). CCL20 expression is low in synovial fluid from patients with osteoarthritis. In the synovial tissue of patients with rheumatoid arthritis, CCL20 expression is observed on the outermost layer and around the blood vessel, and CDla-positive immature rod cells are present in the vicinity of the CCL20 expression site (J Immunol, 168; 5333-5341, 2002). In addition, stimulation of synovial cells from rheumatoid arthritis patients with IL-1β, TNFa, IL-17, etc. induces CCL20 production (J Immu nol, 167; 6015-6020, 2001). However, since the expression of various chemokines is observed in the intra-articular lavage fluid, the contribution of CCL20 to pathogenesis is not clear.
[0006] 一方、特開 2002— 187856号公報および WO2002Z32456号公報には LARC 阻害物質を関節リウマチの治療剤等に用いるとの記載は存在する。しかし、当該文 献は単に LARCが関節リウマチ患者において高発現をしているから LARCを抑制す ればよいと記載されているだけであり、治療効果の実証は何らなされていない。また、 ケモカインは、各種疾患にお 、て発現して 、るものを抑制すれば治療効果がある場 合もあるが、逆に発現を促進すれば治療効果がある場合もある。例えば、関節リウマ チでは、ケモカイン CCL2が高発現していることが知られている。そこで、遺伝的に C CR2を欠損させる、もしくは CCR2を薬剤で阻害すると、コラーゲン関節炎モデルの 発症や病態進行を抑制すると考えられていた。し力しながら、 CCR2欠損マウスでは 野生型マウスよりコラーゲン誘導関節炎の発症が促進し、より重篤な症状を呈する (J. Clin. Invest., 113: 856-866, 2004)。また、 CCR2に対する中和抗体を投与すると、初 回感作時に抗体を投与した場合は症状を軽減することができる力 追加免疫時に抗 体を投与した場合は、関節炎の臨床的、組織学的病態像は顕著に悪ィ匕するという報 告がある (J. Immunol., 172: 890-898, 2004)。従って、高発現している LARCの抑制 が関節リウマチ等の治療に有効であるとは考えられていな力つた。  [0006] On the other hand, JP-A-2002-187856 and WO2002Z32456 disclose that a LARC inhibitor is used as a therapeutic agent for rheumatoid arthritis. However, this document merely describes that LARC should be suppressed because LARC is highly expressed in patients with rheumatoid arthritis, and no therapeutic effect has been demonstrated. Further, chemokines are expressed in various diseases, and if they are suppressed, they may have a therapeutic effect, but conversely, if they are promoted, they may have a therapeutic effect. For example, chemokine CCL2 is known to be highly expressed in rheumatoid arthritis. Therefore, it was thought that genetically deleting CCR2 or inhibiting CCR2 with a drug would suppress the onset and progression of the collagen arthritis model. However, the development of collagen-induced arthritis is promoted in CCR2-deficient mice more severely than in wild-type mice (J. Clin. Invest., 113: 856-866, 2004). In addition, when neutralizing antibody against CCR2 is administered, the ability to relieve symptoms when the antibody is administered during the first sensitization. When the antibody is administered during booster immunization, the clinical and histological pathology of arthritis. There are reports that the image is significantly worse (J. Immunol., 172: 890-898, 2004). Therefore, suppression of LARC, which is highly expressed, has not been considered effective for the treatment of rheumatoid arthritis.
発明の概要  Summary of the Invention
[0007] 本発明者らは、意外にも、 CCR6発現細胞の CCL20に対する遊走を抑制する抗 体がコラーゲン誘導関節炎モデルに対して炎症抑制作用および骨破壊抑制作用を 有することを見出した。本発明はこの知見に基づくものである。 [0007] The present inventors have surprisingly found that anti-inhibition of the migration of CCR6-expressing cells to CCL20. It was found that the body has an anti-inflammatory action and an anti-bone destruction action on a collagen-induced arthritis model. The present invention is based on this finding.
[0008] 本発明によれば以下の発明が提供される。  [0008] According to the present invention, the following inventions are provided.
[0009] (1) CCL20に反応性を有する抗体またはそのフラグメントを有効成分として含んでな る、骨破壊抑制剤。  [0009] (1) A bone destruction inhibitor comprising an antibody or fragment thereof reactive to CCL20 as an active ingredient.
(2)抗体が、モノクローナル抗体である、（1)に記載の骨破壊抑制剤。  (2) The bone destruction inhibitor according to (1), wherein the antibody is a monoclonal antibody.
(3)抗体が、キメラモノクローナル抗体、ヒト型モノクローナル抗体、ヒトモノクローナル 抗体、およびこれらの抗体フラグメントからなる群力 選択される、（1)に記載の骨破 壊抑制剤。  (3) The bone destruction inhibitor according to (1), wherein the antibody is selected from the group consisting of a chimeric monoclonal antibody, a human monoclonal antibody, a human monoclonal antibody, and an antibody fragment thereof.
(4)モノクローナル抗体が、 FERM BP— 10445の受託番号のもと寄託されたハイ プリドーマにより産生される抗体である、（2)または（3)に記載の骨破壊抑制剤。 (4) The bone destruction inhibitor according to (2) or (3), wherein the monoclonal antibody is an antibody produced by a hyperprideoma deposited under a deposit number of FERM BP-10445.
(5) FERM BP— 10445の受託番号のもと寄託されたハイブリドーマ。 (5) FERM BP—Hybridoma deposited under the accession number 10445.
(6)被検物質の存在下および非存在下のそれぞれの条件において、 CCL20の CC R6への作用の程度を測定する工程と、被験物質の非存在下での作用の程度と、被 験物質の存在下での作用の程度とを比較する工程とを含んでなる、骨破壊疾患の治 療に有用な物質のスクリーニング方法。  (6) The step of measuring the degree of action of CCL20 on CCR6 in the presence and absence of the test substance, the degree of action in the absence of the test substance, and the test substance A method for screening a substance useful for the treatment of bone destruction disease, comprising the step of comparing the degree of action in the presence of
(7)被検物質の非存在下よりも被検物質の存在下にお 、て CCL20の CCR6への作 用が抑制される場合に、被検物質を骨破壊疾患の治療に有用な物質であると判定 する工程を更に含んでなる、 (6)に記載のスクリーニング方法。  (7) When the action of CCL20 on CCR6 is suppressed in the presence of the test substance rather than in the absence of the test substance, the test substance is a substance useful for the treatment of bone destruction disease. The screening method according to (6), further comprising a step of determining that there is.
(8) CCR6を含む細胞膜またはそれを含む細胞と CCL20とを接触させ、次 ヽで細胞 刺激活性を測定する工程と、被験物質の非存在下での細胞刺激活性と、被験物質 の存在下での細胞刺激活性とを比較する工程とを含んでなる、（6)または（7)に記載 のスクリーニング方法。  (8) The step of contacting CCL20 with a cell membrane containing CCR6 or a cell containing the same, measuring cell stimulating activity in the next step, cell stimulating activity in the absence of the test substance, and in the presence of the test substance The method for screening according to (6) or (7), comprising the step of comparing the cell stimulating activity of.
(9)被検物質の存在下および非存在下のそれぞれの条件において、 CCL20の CC R6への結合の程度を測定する工程と、被験物質の非存在下での結合の程度と、被 験物質の存在下での作用の程度とを比較する工程とを含んでなる、骨破壊の治療に 有用な物質のスクリ一ユング方法。  (9) A step of measuring the degree of binding of CCL20 to CCR6 in each condition in the presence and absence of the test substance, the degree of binding in the absence of the test substance, and the test substance A method for screening a substance useful for the treatment of bone destruction comprising the step of comparing the degree of action in the presence of
(10)被検物質の非存在下よりも被検物質の存在下において CCL20の CCR6への 結合が抑制される場合に、被検物質を骨破壊の治療に有用な物質であると判定する 工程を更に含んでなる、 (9)に記載のスクリーニング方法。 (10) CCL20 to CCR6 in the presence of the test substance rather than in the absence of the test substance The screening method according to (9), further comprising a step of determining that the test substance is a substance useful for treatment of bone destruction when binding is suppressed.
(11) CCR6を含む細胞膜またはそれを含む細胞と CCL20とを接触させ、次 、で細 胞刺激活性を測定する工程と、被験物質の非存在下での細胞刺激活性と、被験物 質の存在下での細胞刺激活性とを比較する工程とを含んでなる、（9)または（10)に 記載のスクリーニング方法。  (11) A step of contacting a cell membrane containing CCR6 or a cell containing the same with CCL20 and measuring cell stimulating activity in the following, cell stimulating activity in the absence of the test substance, and presence of the test substance The method for screening according to (9) or (10), comprising the step of comparing the cell stimulating activity below.
(12) CCL20の CCR6への作用の評価を、 CCL20に反応性を有する抗体またはそ のフラグメントの CCL20阻害作用、あるいは CCR6に反応性を有する抗体またはそ のフラグメントの CCR6阻害作用との比較により行う工程を更に含んでなる、（6)〜（1 1)のいずれか一項に記載のスクリーニング方法。  (12) Evaluation of the action of CCL20 on CCR6 is carried out by comparing the CCL20 inhibitory action of an antibody or fragment thereof reactive to CCL20, or the CCR6 inhibitory action of an antibody or fragment thereof reactive to CCR6. The screening method according to any one of (6) to (11), further comprising a step.
(13)被験組織における CCL20の発現の程度を検出する工程を含んでなる、骨破 壊の検出方法および骨破壊の診断方法。  (13) A method for detecting bone destruction and a method for diagnosing bone destruction, comprising a step of detecting the degree of expression of CCL20 in a test tissue.
(14) CCL20に反応性を有する抗体またはそのフラグメントを使用して CCL20の発 現の程度を検出する、（13)に記載の方法。  (14) The method according to (13), wherein the degree of expression of CCL20 is detected using an antibody reactive with CCL20 or a fragment thereof.
(15)正常組織における CCL20の発現の程度と被験組織における CCL20の発現の 程度を比較する工程を更に含んでなる、（13)または（14)に記載の方法。  (15) The method according to (13) or (14), further comprising a step of comparing the level of expression of CCL20 in a normal tissue with the level of expression of CCL20 in a test tissue.
(16)被検組織における CCL20の発現の程度力 正常組織における CCL20の発現 の程度を超える場合に、骨破壊が生じて 、るまたは骨破壊に罹って 、ると判定する 工程を更に含んでなる、（15)に記載の方法。  (16) The degree of expression of CCL20 in the test tissue The method further comprises the step of determining that bone destruction occurs or suffers from bone destruction when the degree of expression of CCL20 in normal tissue is exceeded. The method according to (15).
(17) CCL20に反応性を有する抗体またはそのフラグメントを含んでなる、骨破壊検 出剤および骨破壊診断剤。  (17) An agent for detecting bone destruction and a diagnostic agent for bone destruction, comprising an antibody reactive with CCL20 or a fragment thereof.
(18) CCL20に反応性を有する抗体またはそのフラグメントを含んでなる、骨破壊検 出キットおよび骨破壊診断キット。  (18) A bone destruction detection kit and a bone destruction diagnosis kit comprising an antibody having reactivity with CCL20 or a fragment thereof.
図面の簡単な説明 Brief Description of Drawings
[図 1]抗 CCL20抗体 2F5— 5による CCR6発現細胞のマウス CCL20タンパク質への 細胞遊走の阻害を示した図である。 FIG. 1 shows inhibition of cell migration of CCR6-expressing cells to mouse CCL20 protein by anti-CCL20 antibody 2F5-5.
[図 2]コラーゲン誘導関節炎モデルにおける足蹄での CCL20 mRNAの発現を示し た図である。 圆 3]抗 CCL20抗体を投与されたコラーゲン誘導関節炎モデルにおける外見的所 見（a)および体重の経時的変化 (b)を示した図である。三角形：抗 CCL20モノクロ一 ナル抗体；四角形： IgG (コントロール）。 FIG. 2 shows CCL20 mRNA expression in foot and foot in a collagen-induced arthritis model. (3) Appearance findings (a) and changes in body weight over time (b) in a collagen-induced arthritis model administered with anti-CCL20 antibody. Triangle: anti-CCL20 monoclonal antibody; square: IgG (control).
[図 4]抗 CCL20抗体を投与された不完全フロイントアジュバントおよび完全フロイント アジュバントによるコラーゲン誘導関節炎モデルにおける外見的所見および体重の 経時的変化を示した図である。 (a)不完全フロイントアジュバントを使用した場合の外 見的所見；（b)不完全フロイントアジュバントを使用した場合の体重変化；（c)完全フ ロイントアジュバントを使用した場合の外見的所見；（d)完全フロイントアジュバントを 使用した場合の体重変化。三角形:抗 CCL20モノクローナル抗体；四角形: IgG (コ ントロール）。  FIG. 4 is a diagram showing changes in appearance and body weight over time in a collagen-induced arthritis model with incomplete Freund's adjuvant and complete Freund's adjuvant administered with anti-CCL20 antibody. (a) Appearance findings when using incomplete Freund's adjuvant; (b) Body weight change when using incomplete Freund's adjuvant; (c) Appearance findings when using complete Freund's adjuvant; d) Changes in body weight when complete Freund's adjuvant is used. Triangle: anti-CCL20 monoclonal antibody; square: IgG (control).
[図 5]コラーゲン誘導関節炎モデルの血漿中の血清アミロイド A濃度に対する抗 CCL 20抗体投与の効果を示した図である。 (a)不完全フロイントアジュバントを使用した場 合；（b)：完全フロイントアジュバントを使用した場合。  FIG. 5 is a graph showing the effect of anti-CCL 20 antibody administration on serum amyloid A concentration in plasma of a collagen-induced arthritis model. (a) When incomplete Freund's adjuvant is used; (b): When complete Freund's adjuvant is used.
圆 6]コラーゲン誘導関節炎モデルの血漿中の抗コラーゲン抗体価に対する抗 CCL 20抗体 (CCL20 mAb)投与の効果を示した図である。（a) :IgGlクラス抗コラーゲ ン抗体価； (b) :IgG2aクラス抗コラーゲン抗体価。 圆 6] It is a figure showing the effect of anti-CCL 20 antibody (CCL20 mAb) administration on anti-collagen antibody titer in plasma of a collagen-induced arthritis model. (A): IgGl class anti-collagen antibody titer; (b): IgG2a class anti-collagen antibody titer.
[図 7]コラーゲン誘導関節炎モデルの血漿中の COMP濃度に対する抗 CCL20抗体 投与の効果を示した図である。  FIG. 7 is a graph showing the effect of anti-CCL20 antibody administration on the COMP concentration in plasma of a collagen-induced arthritis model.
[図 8]コラーゲン誘導関節炎モデルの血漿中の活性型 TRAP濃度に対する抗 CCL2 0抗体投与の効果を示した図である。  FIG. 8 is a graph showing the effect of anti-CCL20 antibody administration on plasma active TRAP concentration in a collagen-induced arthritis model.
[図 9]コラーゲン誘導関節炎モデルにおける足蹄での炎症関連分子の mRNAの発 現に対する抗 CCL20抗体投与の効果をリアルタイム PCRにより解析した結果を示し た図である。白抜きバー：正常、黒塗りバー: IgG (コントロール）、斜線バー:抗 CCL2 0モノクローナル抗体。  FIG. 9 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of inflammation-related molecule mRNA in the foot and foot in a collagen-induced arthritis model. Open bars: normal, black bars: IgG (control), hatched bars: anti-CCL20 monoclonal antibody.
[図 10]コラーゲン誘導関節炎モデルにおける足蹄でのケモカイン'ケモカイン受容体 の mRNAの発現に対する抗 CCL20抗体投与の効果をリアルタイム PCRにより解析 した結果を示した図である。白抜きバー：正常、黒塗りバー: IgG (コントロール）、斜線 バー：抗 CCL20モノクローナル抗体。 [図 11]コラーゲン誘導関節炎モデルにおける足蹄での細胞マーカーの mRNAの発 現に対する抗 CCL20抗体投与の効果をリアルタイム PCRにより解析した結果を示し た図である。白抜きバー：正常、黒塗りバー: IgG (コントロール）、斜線バー:抗 CCL2 0モノクローナル抗体。 FIG. 10 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of chemokine's chemokine receptor mRNA in the foot and foot in a collagen-induced arthritis model. Open bars: normal, black bars: IgG (control), hatched bars: anti-CCL20 monoclonal antibody. FIG. 11 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of cell marker mRNA in the foot and foot in a collagen-induced arthritis model. Open bars: normal, black bars: IgG (control), hatched bars: anti-CCL20 monoclonal antibody.
[図 12]コラーゲン誘導関節炎モデルにおける足蹄での破骨細胞関連分子の mRNA の発現に対する抗 CCL20抗体投与の効果をリアルタイム PCRにより解析した結果を 示した図である。白抜きバー：正常、黒塗りバー: IgG (コントロール）、斜線バー:抗 C CL20モノクローナル抗体。  FIG. 12 shows the results of real-time PCR analysis of the effect of anti-CCL20 antibody administration on the expression of osteoclast-related molecule mRNA in the foot and foot in a collagen-induced arthritis model. Open bars: normal, black bars: IgG (control), hatched bars: anti-C CL20 monoclonal antibody.
[図 13] (a)発症後から抗 CCL20抗体の投与を開始したコラーゲン関節炎モデルにお ける外見的所見の経時変化を示した図である。三角形：抗 CCL20モノクローナル抗 体、四角形: IgG (コントロール)。（b)骨破壊の評価のための軟 X線所見を示した図 である。  FIG. 13 (a) shows changes over time in appearance in a collagen arthritis model in which administration of anti-CCL20 antibody was started after onset. Triangle: anti-CCL20 monoclonal antibody, square: IgG (control). (B) Soft X-ray findings for evaluation of bone destruction.
発明の具体的な説明  Detailed Description of the Invention
[0011] 以下、本発明を詳細に説明する。以下の記述は、本発明を説明するための例示で あり、本発明を記述された実施形態にのみ限定する趣旨ではない。本明細書中で使 用される全ての技術的用語、科学的用語および専門用語は、本発明が属する技術 分野の通常の当業者により一般的に理解されるのと同じ意味を有し、単に特定の態 様を説明することを目的として用いられ、限定することを意図したものではない。本発 明は、その要旨を逸脱しない限り、さまざまな形態で実施をすることができる。本明細 書において引用された全ての先行技術文献および公開公報、特許公報その他の特 許文献は、参照として本明細書に組み入れられ、本発明の実施のために用いること ができる。  Hereinafter, the present invention will be described in detail. The following description is an example for explaining the present invention, and is not intended to limit the present invention to the described embodiments. All technical, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are simply It is used for the purpose of explaining specific aspects and is not intended to be limiting. The present invention can be implemented in various forms without departing from the gist thereof. All prior art documents and publications, patent publications and other patent documents cited herein are hereby incorporated by reference and can be used to practice the present invention.
[0012] [抗原および抗体]  [0012] [Antigen and antibody]
舰  舰
本発明で使用する抗体を製造する場合、抗原である CCL20は公知の方法に従つ て精製したものであってもよいが、遺伝子工学的に調製することも可能である。  When the antibody used in the present invention is produced, the antigen CCL20 may be purified according to a known method, but it can also be prepared by genetic engineering.
[0013] CCL20タンパク質は、そのアミノ酸配列およびそれをコードする DNAが報告され ている。例えば、マウスの場合 GenBankァクセッション番号： NM 058656、ヒトの場 合 GenBankァクセッション番号： NM 004582およびマウスの場合 GenBankァクセ ッシヨン番号： NM 016960、ヒトの場合 GenBankァクセッション番号： NM 004591 等が報告されている。本発明において用いる CCL20タンパク質は、このような公知の 情報に基づいてそのアミノ酸配列またはそれをコードする DNAを具体的に特定する ことができる。 [0013] The amino acid sequence of CCL20 protein and the DNA encoding it have been reported. For example, in the case of mouse GenBank accession number: NM 058656, human field GenBank accession number: NM 004582 and mouse GenBank accession number: NM 016960, human case GenBank accession number: NM 004591 etc. have been reported. The CCL20 protein used in the present invention can specifically identify the amino acid sequence or the DNA encoding it based on such known information.
[0014] 具体的には、本発明における CCL20タンパク質は下記 (A)〜(E)からなる群より 選択されるポリペプチドからなる：  [0014] Specifically, the CCL20 protein in the present invention consists of a polypeptide selected from the group consisting of the following (A) to (E):
(A) 前述のァクセッション番号： NM 004591によりコードされるアミノ酸配列を含 む、ポリペプチド；  (A) a polypeptide comprising the amino acid sequence encoded by the aforementioned accession number: NM 004591;
(B) 前記 (A)のアミノ酸配列において、 1または複数個（好ましくは 1または数個、よ り好ましくは 1、 2、または 3個）のアミノ酸が置換、欠失、挿入、および Zまたは付加さ れたアミノ酸配列を含んでなり、かつ、 CCL20タンパク質と実質的に同じ活性を有す る、ポリペプチド；  (B) In the amino acid sequence of (A), one or more (preferably one or several, more preferably 1, 2, or 3) amino acids are substituted, deleted, inserted, and Z or added. A polypeptide comprising a defined amino acid sequence and having substantially the same activity as the CCL20 protein;
(C) 前記 (A)のアミノ酸配列に対して 80%以上の同一性を有するアミノ酸配列か らなる、ポリペプチド；  (C) a polypeptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence of (A);
(D) 前記 (A)のアミノ酸配列をコードする塩基配列力もなるポリヌクレオチドとストリ ンジヱントな条件でノヽイブリダィズするポリヌクレオチドによりコードされてなるポリぺプ チドであって、 CCL20タンパク質と実質的に同じ活性を有する、ポリペプチド;および  (D) A polypeptide encoded by a polynucleotide that is hybridized under stringent conditions with a polynucleotide that also has a nucleotide sequence that encodes the amino acid sequence of (A), and is substantially the same as the CCL20 protein. A polypeptide having activity; and
(E) 前記 (A)のアミノ酸配列をコードする塩基配列に対して 80%以上 (好ましくは 8 5%以上、より好ましくは 90%以上、さらに好ましくは 95%以上、さらにより好ましくは 98%以上、特に好ましくは 99%以上）の同一性を有する塩基配列力もなるポリヌクレ ォチドによりコードされてなるポリペプチドであって、 CCL20タンパク質と実質的に同 じ活性を有する、ポリペプチド。 (E) 80% or more (preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more) with respect to the base sequence encoding the amino acid sequence of (A). A polypeptide encoded by a polynucleotide having a nucleotide sequence ability having an identity of 99% or more, and having substantially the same activity as that of CCL20 protein.
[0015] 本発明に使用する CCL20タンパク質としては、「前述のァクセッション番号により特 定される 、ずれかのアミノ酸配列力 なるポリペプチド」が好まし 、。  [0015] As the CCL20 protein used in the present invention, "a polypeptide having a certain amino acid sequence ability, which is specified by the aforementioned accession number" is preferable.
[0016] ここで、「前述のァクセッション番号により特定されるいずれかのアミノ酸配列」とは 前記に列記したァクセッション番号により所定の公知のデータベースより特定される アミノ酸配列のことである。 Here, “any one amino acid sequence specified by the above-mentioned accession number” is specified from a predetermined publicly-known database by the accession number listed above. It is an amino acid sequence.
[0017] ここで、ポリペプチドが「CCL20タンパク質と実質的に同じ活性を有する」とは、その ポリペプチドが、骨破壊作用を誘発するものであればいかなるものも含まれる。具体 的には、 CCR6に直接的または間接的に遊走ィ匕作用を有することを意味する。また 実質的に同じとは、活性が性質的に同質であることを意味する。すなわち、「CCL20 タンパク質と実質的に同じ活性を有する」ためには、前記活性が同等 (例えば、約 0. 01〜： LOO倍、好ましくは 0. 05〜20倍、より好ましくは 0. 5〜2倍）であることが好まし い。これらの活性については、慣用の方法にしたがって測定することができ、例えば、 後述する実施例に記載の方法にしたがって測定することができる。  Here, the phrase “the polypeptide has substantially the same activity as the CCL20 protein” includes any polypeptide that induces a bone destruction action. Specifically, it means that it has a migratory action directly or indirectly on CCR6. In addition, substantially the same means that the activities are homogeneous in nature. That is, in order to have “substantially the same activity as the CCL20 protein”, the activity is equivalent (for example, about 0.01 to: LOO times, preferably 0.05 to 20 times, more preferably 0.5 to 2 times). About these activity, it can measure according to a conventional method, for example, it can measure according to the method as described in the Example mentioned later.
[0018] 本発明の好ま 、態様によれば、前記 (B)のポリペプチド（以下にぉ 、て「改変ポリ ペプチド」と言うことがある）は、そのアミノ酸配列力 前述のァクセッション番号により 特定されるいずれかのアミノ酸配列（好ましくは、ヒトの場合 GenBankァクセッション 番号: NP— 004582)を含むポリペプチドにおいて 1または複数個（好ましくは 1また は数個、より好ましくは 1、 2、または 3個）の保存的置換を有するアミノ酸配列であつ て、し力も CCL20タンパク質と実質的に同じ活性を有するポリペプチドであることが できる。  [0018] According to a preferred aspect of the present invention, the polypeptide (B) (hereinafter sometimes referred to as "modified polypeptide") has an amino acid sequence ability according to the accession number described above. One or more (preferably one or several, preferably 1, 2, or more) in a polypeptide comprising any amino acid sequence specified (preferably GenBank accession number: NP-004582 for humans) Or an amino acid sequence having 3) conservative substitutions, and can also be a polypeptide having substantially the same activity as the CCL20 protein.
[0019] 本願明細書において「保存的置換」とは、ペプチドの活性を実質的に改変しないよ うに、 1または複数個（好ましくは数個、より好ましくは 1、 2、または 3個）のアミノ酸残 基を、別の化学的に類似したアミノ酸残基で置換えることを意味する。例えば、ある疎 水性残基を別の疎水性残基によって置換する場合、ある極性残基を同じ電荷を有す る別の極性残基によって置換する場合等が挙げられる。このような置換を行うことが できる機能的に類似のアミノ酸は、アミノ酸毎に当該技術分野において公知である。 具体例を挙げると、非極性 (疎水性)アミノ酸としては、ァラニン、パリン、イソロイシン、 ロイシン、プロリン、トリプトファン、フエ-ルァラニン、メチォニン等が挙げられる。極性 (中性）アミノ酸としては、グリシン、セリン、スレオニン、チロシン、グルタミン、ァスパラ ギン、システィン等が挙げられる。陽電荷をもつ (塩基性)アミノ酸としては、アルギ- ン、ヒスチジン、リジン等が挙げられる。また、負電荷をもつ（酸性)アミノ酸としては、 ァスパラギン酸、グルタミン酸等が挙げられる。 [0020] ここで、欠失、置換および Zまたは付加されてもよいアミノ酸の数は、例えば 1〜30 個、好ましくは 1〜20個、より好ましくは 1〜： LO個、さらに好ましくは 1〜5個、特に好ま しくは 1〜2個である。なお、前記改変ポリペプチドには、改変ポリペプチドの塩が含 まれ、ジスルフイド結合をしたものとジスルフイド結合をしていないもの、リン酸化され たものとリン酸ィ匕されていないもの、さらには糖鎖を有しないものと糖鎖を有するもの との両方が含まれる。したがって、これらの条件を満たす限り、前記改変ポリペプチド の起源は、ヒトに限定されない。 [0019] As used herein, "conservative substitution" means one or more (preferably several, more preferably 1, 2, or 3) amino acids so as not to substantially alter the activity of the peptide. It means replacing the residue with another chemically similar amino acid residue. For example, when one hydrophobic residue is substituted with another hydrophobic residue, one polar residue is substituted with another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, parin, isoleucine, leucine, proline, tryptophan, ferrolanine and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include algin, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid. [0020] Here, the number of amino acids that may be deleted, substituted and Z or added is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to: LO, and still more preferably 1 to Five, particularly preferably 1-2. The modified polypeptide contains a salt of the modified polypeptide, and those having a disulfide bond, those not having a disulfide bond, those phosphorylated and those not phosphorylated, and sugars Both those having no chain and those having a sugar chain are included. Therefore, as long as these conditions are satisfied, the origin of the modified polypeptide is not limited to humans.
[0021] アミノ酸の欠失、置換および Zまたは付カ卩による変異体は、例えばそれをコードす る DNAに例えば、周知技術である部位特異的変異誘発（例えば、 Nucleic Acid Rese arch, Vol.10, No.20, p.6487-6500, 1982参照）を施すことにより作成できる。本明細 書において「1または複数のアミノ酸」とは、部位特異的変異誘発法により欠失、置換 および Zまたは付加できる程度の数のアミノ酸を意味する。  [0021] Mutations due to amino acid deletions, substitutions, and Zs or attachments are performed, for example, on the DNA encoding the site-directed mutagenesis (for example, Nucleic Acid Research, Vol. 10 , No. 20, p. 6487-6500, 1982). As used herein, “one or more amino acids” means as many amino acids as can be deleted, substituted and Z or added by site-directed mutagenesis.
[0022] あるタンパク質において、元のタンパク質の抗原性を維持した状態で、 1若しくは複 数個のアミノ酸を欠失、挿入、置換または付加させる方法は公知である。例えば、部 位特異的変異誘発法により変異タンパク質をコードするポリヌクレオチドを調製し、適 宜発現させて得ることができる (Molecular Cloning.A Laboratory Manual 2nd ed., Cold Spring Harbor Press(1989);Current Protocols in Molecular Biology, John Wiley & So ns,(1987— 1997),Section8.1— 8.5;Hashimoto— Goto et al.(1995)Gene 152:271— 5;Kinke l(1985)Proc.Natl.Acad.Sci.USA 82 :488-92 ;Kramer and Fritz(1987)Method.Enzymol 154:350-67;Kunkel(1988)Method.Enzymol.85:2763-6)o部位特異的変異誘発法は 、例えば、所望の変異である特定の不一致の他は、変異を受けるべき一本鎖ファー ジ DNAに相補的な合成オリゴヌクレオチドプライマーを用いて次のように行うことが できる。即ち、プライマーとして上記合成オリゴヌクレオチドを用いてファージに相補 的な鎖を合成させ、得られた二重鎖 DNAで宿主細胞を形質転換する。形質転換さ れた細菌の培養物を寒天にプレートし、ファージを含有する単一細胞力 プラークを 形成せしめる。そうすると、理論的には 50%の新コロニーが一本鎖として変異を有す るファージを含有し、残りの 50%が元の配列を有する。上記所望の変異を有する DN Aと完全に一致するものとはハイブリダィズする力 元の鎖を有するものとはハイプリ ダイズしな 、温度にぉ 、て、得られたプラークをキナーゼ処理により標識した合成プ ローブとハイブリダィズさせる。次に該プローブとハイブリダィズするプラークを拾!、、 培養し DNAを回収する。 [0022] A method for deleting, inserting, substituting, or adding one or more amino acids in a protein while maintaining the antigenicity of the original protein is known. . For example, a polynucleotide encoding a mutant protein by parts position-specific mutagenesis is prepared, can be obtained by suitable Yichun expression (Molecular Cloning.A Laboratory Manual 2 nd ed , Cold Spring Harbor Press (1989); Current Protocols in Molecular Biology, John Wiley & Sons, (1987-1997), Section 8.1-8.5; Hashimoto- Goto et al. (1995) Gene 152: 271--5; Kinkel (1985) Proc. Natl. Acad Sci. USA 82: 488-92; Kramer and Fritz (1987) Method. Enzymol 154: 350-67; Kunkel (1988) Method. Enzymol. 85: 2763-6) o Site-directed mutagenesis is, for example, Other than the specific mismatch that is the desired mutation, it can be performed as follows using a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated. That is, the above-mentioned synthetic oligonucleotide is used as a primer to synthesize a strand complementary to the phage, and a host cell is transformed with the obtained double-stranded DNA. Transformed bacterial cultures are plated on agar to form single-cell force plaques containing phage. Then, theoretically 50% of new colonies contain phages with mutations as single strands, and the remaining 50% have the original sequence. A DNA having the desired mutation is completely identical to a DNA having the desired mutation. Without soy, the resulting plaques are hybridized with a synthetic probe labeled by kinase treatment under temperature. Next, plaques that hybridize with the probe are picked up and cultured to collect DNA.
[0023] 尚、 CCL20タンパク質の生物活性ペプチドのアミノ酸配列にその活性を喪失せし めない 1または複数のアミノ酸の欠失、置換および Zまたは付加を施す方法としては 、上記の部位特異的変異誘発の他にも、遺伝子を変異源で処理する方法および遺 伝子を選択的に開裂し、次に選択されたヌクレオチドを欠失、置換および Zまたは付 加し、次いで連結する方法もある。  [0023] In addition, as a method of performing deletion, substitution and Z or addition of one or more amino acids that do not lose its activity to the amino acid sequence of the biologically active peptide of CCL20 protein, the above-mentioned site-directed mutagenesis is used. Other methods include treating the gene with a mutagen and selectively cleaving the gene, then deleting, substituting and appending or adding selected nucleotides, and then ligating.
[0024] 本明細書にぉ 、て、「欠失」には、アミノ酸配列の端からアミノ酸残基を欠失したも のおよびアミノ酸配列の途中のアミノ酸残基が欠失したものも含まれる。  As used herein, “deletion” includes those in which an amino acid residue is deleted from the end of the amino acid sequence and amino acid residues in the middle of the amino acid sequence are deleted.
[0025] 「付加」には、アミノ酸配列の端にアミノ酸残基を付加したものおよびアミノ酸配列の 途中にアミノ酸残基を付加したものも含まれる。  [0025] "Addition" includes those in which an amino acid residue is added to the end of the amino acid sequence and those in which an amino acid residue is added in the middle of the amino acid sequence.
[0026] 1つのアミノ酸をコードするコドンは複数存在する。従って、前述のァクセッション番 号により特定される 、ずれかのアミノ酸配列（好ましくは、マウスの場合 GenBankァク セッション番号： NP— 058656、ヒトの場合 GenBankァクセッション番号： NP— 004 582)またはその酵素活性部分をコードするいずれの DNAも本発明の範囲に含まれ る。  [0026] There are a plurality of codons encoding one amino acid. Therefore, any amino acid sequence identified by the aforementioned accession number (preferably GenBank accession number: NP-058656 for mice, GenBank accession number: NP-004 582 for humans) Alternatively, any DNA encoding the enzyme active part is included in the scope of the present invention.
[0027] 本発明の別の一つの態様によれば、本発明に使用する CCL20タンパク質をコード するポリヌクレオチドは、前述のァクセッション番号により特定されるいずれかの塩基 配列（好ましくは、ヒトの場合 GenBankァクセッション番号： NM 004591)力らなるポ リヌクレオチドとストリンジェントな条件下でハイブリダィズし、し力も、前記の CCL20タ ンパク質と実質的に同じ活性を有するポリペプチドをコードしてなるものである。具体 的には、前記ァクセッション番号により特定される塩基配列以外の配列力 なるポリヌ クレオチドであって、マウスの場合 GenBankァクセッション番号： NM 016960 (マウ ス由来)力 なるポリヌクレオチドが挙げられる。  [0027] According to another embodiment of the present invention, the polynucleotide encoding the CCL20 protein used in the present invention is any nucleotide sequence (preferably, a human sequence) identified by the aforementioned accession number. GenBank accession number: NM 004591) Hybridizes with a strong polynucleotide under stringent conditions, and the force also encodes a polypeptide having substantially the same activity as the CCL20 protein. Is. Specifically, a polynucleotide having a sequence power other than the nucleotide sequence specified by the accession number, and in the case of a mouse, GenBank accession number: NM 016960 (derived from a mouse) is a polynucleotide. .
[0028] この改変ポリペプチドには、さらにその N末端 (ァミノ末端)および C末端 (カルボキ シル末端）が改変または修飾されているものも包含されてよい。例えば、 C末端の力 ルボキシルカ カルボキシレート（一 COO— )、アミド（一 CONH )またはエステル（ — COOR)とされていてもよい。なおここで前記 Rは、例えば直鎖、分岐鎖もしくは環 状の C アルキル基、 C ァリール基等が挙げられる。また N末端のァミノ基が慣[0028] The altered polypeptide may further include those in which the N-terminus (amino terminus) and C-terminus (carboxyl terminus) are altered or modified. For example, C-terminal force ruboxyllucacarboxylate (one COO—), amide (one CONH) or ester ( — COOR). Here, examples of R include a linear, branched or cyclic C alkyl group, C aryl group and the like. The N-terminal amino group is
1-6 6- 12 1-6 6-12
用の保護基により保護されたもの等も改変ポリペプチドに包含され得る。  Those protected by a protective group for use can also be included in the modified polypeptide.
[0029] 前記 (B)のポリペプチドの例としては、ヒト以外の生物 [例えば非ヒト哺乳動物（例え ばマウス、ラット、ハムスター、ブタ、ィヌ等）、鳥類、爬虫類、両生類、魚類、昆虫類等 ]由来の CCL20タンパク質もしくはその変異体が挙げられる。具体的には例えば、マ ウスの場合 GenBankァクセッション番号： NP_058656 (マウス由来）力 なるポリぺプ チドが挙げられる。 [0029] Examples of the polypeptide (B) include non-human organisms [eg, non-human mammals (eg, mouse, rat, hamster, pig, Inu, etc.), birds, reptiles, amphibians, fish, insects. Etc.] derived from CCL20 protein or a variant thereof. Specifically, for example, in the case of a mouse, GenBank accession number: NP_058656 (derived from a mouse), a polypeptide having a strong force can be mentioned.
[0030] 前記 (C)のポリペプチド（以下において「相同ポリペプチド」と言うことがある）は、 C CL20タンパク質のアミノ酸配列に関して 80%以上の同一性を有するアミノ酸配列か らなる限り、特に限定されるものではないが、好ましくは、 CCL20タンパク質に関して 、同一性が 85%以上、より好ましくは 90%以上、さらに好ましくは 95%以上、さらによ り好ましくは 98%以上、特に好ましくは 99%以上の相同性を有するアミノ酸配列から なるアミノ酸配列であって、し力も CCL20タンパク質と実質的に同じ活性を有するポ リペプチドである。  [0030] The polypeptide of (C) (hereinafter sometimes referred to as "homologous polypeptide") is particularly limited as long as it comprises an amino acid sequence having 80% or more identity with respect to the amino acid sequence of CCL20 protein. Preferably, however, the CCL20 protein has an identity of 85% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more. An amino acid sequence consisting of amino acid sequences having the same homology as described above, and having a force substantially the same as that of the CCL20 protein.
[0031] 本願明細書において、「同一性」の数値はいずれも、当業者に公知の相同性検索 プログラムを用いて算出される数値であればよぐ例えば全米バイオテクノロジー情 報センター（NCBI)の相同性アルゴリズム BLAST (Basic local alignment search tool ) http://www.ncbi.nlm.nih.gov/BLAST/にお!/、てデフォルト（初期設定）のパラメータ 一を用いることにより、算出することができる。なお、前記相同ポリペプチドには、相同 ポリペプチドの塩が含まれ、ジスルフイド結合をしたものとジスルフイド結合をして！/、な いもの、リン酸ィ匕されたものとリン酸ィ匕されていないもの、さらには糖鎖を有しないもの と糖鎖を有するものとの両方が含まれる。したがって、これらの条件を満たす限り、前 記相同ポリペプチドの起源は、ヒトに限定されない。例えばヒト以外の生物 [例えば非 ヒト哺乳動物（例えばマウス、ラット、ノ、ムスター、ブタ、ィヌ等）]由来の CCL20タンパ ク質もしくはその変異体が含まれる。  [0031] In the present specification, any value of "identity" may be any value calculated using a homology search program known to those skilled in the art. For example, the National Biotechnology Information Center (NCBI) The homology algorithm BLAST (Basic local alignment search tool) http://www.ncbi.nlm.nih.gov/BLAST/ can be calculated using the default (initial setting) parameter 1 it can. The homologous polypeptide includes a salt of the homologous polypeptide, which is disulfide-bonded to a disulfide-bonded one! /, Those that are not phosphated, those that are not phosphated, and those that do not have sugar chains and those that have sugar chains. Therefore, as long as these conditions are satisfied, the origin of the homologous polypeptide is not limited to humans. For example, a CCL20 protein derived from a non-human organism [eg, a non-human mammal (eg, mouse, rat, mouse, muster, pig, Inu, etc.)] or a variant thereof is included.
[0032] 具体的には、前記（C)の相同ポリペプチドとしては、例えば、マウスの場合 GenBa nkァクセッション番号： NP_058656 (マウス由来）からなるポリペプチドが挙げられる。 [0033] なお、本明細書において、変異体とは、「variation」、すなわち、同一種内の同一 ポリペプチドにみられる個体差、あるいは、数種間の相同ポリペプチドにみられる差 異を意味する。 [0032] Specifically, examples of the homologous polypeptide of (C) above include a polypeptide comprising GenBank accession number: NP_058656 (derived from mouse) in the case of a mouse. [0033] In the present specification, a variant means "variation", that is, an individual difference found in the same polypeptide within the same species, or a difference found in several homologous polypeptides. To do.
[0034] さらに、本発明に使用する CCL20タンパク質 (すなわち、 CCL20タンパク質、改変 ポリペプチド、相同ポリペプチド)の部分ポリペプチドも、 CCL20タンパク質と実質的 に同じ活性を有する限り使用することができる。この場合、部分ポリヌクレオチドを構 成するアミノ酸数 ίま、 CCL20タンノ ク質のアミノ酸数の 900/0、 80%, 70%, 60%, 5 0%、 40%、 30%、 20%、 10%または 5%である。 Furthermore, a partial polypeptide of CCL20 protein (that is, CCL20 protein, modified polypeptide, homologous polypeptide) used in the present invention can be used as long as it has substantially the same activity as CCL20 protein. In this case, the number of amino acids ί to configure the partial polynucleotide or, CCL20 Tanno click protein amino acid number of 90 0/0 of, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5%.
[0035] 前記 (D)のアミノ酸配列をコードする塩基配列力もなるポリヌクレオチドにおける「スト リンジヱントな条件でハイブリダィズするポリヌクレオチド」とは、具体的には、 FASTA 、 BLAST, Smith- Waterman [Meth. Enzym., 164, 765 (1988)〕等の相同性検索 ソフトウェアにより、デフォルト (初期設定)のパラメーターを用いて計算したときに、例 えばヒトの場合 GenBankァクセッション番号： NM 004591と少なくとも 70%以上、 好ましくは 80%以上、より好ましくは 85%以上、さらに好ましくは 90%以上、さらによ り好ましくは 95%以上、特に好ましくは 98%以上、そして最も好ましくは 99%以上の 同一性を有するポリヌクレオチドが挙げられる。また、「ストリンジ工ントなハイブリダィ ゼーシヨン条件としては、 f列えば「2 X SSC、 0. 1%SDS、 50°C」、「2 X SSC、 0. 1 %SDS、 42。C」、「1 X SSC、 0. 1%SDS、 37。C」、よりストリンジエンドな条件として は、例えば「2 X SSC、 0. 1%SDS、 65。C」、「0. 5 X SSC、 0. 1%SDS、 42。C」、「 0. 2 X SSC、 0. 1%SDS、 65°C」等の条件を挙げることができる。より詳細には、 Rap id-hyb buffer(Amersham Life Science)を用いた方法として、 68°Cで 30分以上プレハ イブリダィゼーションを行つた後、プローブを添加して 1時間以上 68°Cに保つてハイ ブリツド形成させ、その後、 2 X SSC、 0. 1%SDS中、室温で 20分の洗浄を 3回、 1 X SSC、 0. 1%SDS中、 37。Cで 20分の洗浄を 3回、最後に、 1 X SSC、 0. 1%SD S中、 50°Cで 20分の洗浄を 2回行うことも考えられる。その他、例えば Expresshyb Hy bridization Solution (CLONTECH)中、 55°Cで 30分以上プレハイブリダィゼーシヨンを 行い、標識プローブを添カ卩し、 37〜55°Cで 1時間以上インキュベートし、 2 X SSC、 0. 1%SDS中、室温で 20分の洗浄を 3回、 1 X SSC、 0. 1%SDS中、 37。Cで 20分 の洗浄を 1回行うこともできる。ここで、例えば、プレノ、イブリダィゼーシヨン、ハイブリ ダイゼーシヨンや 2度目の洗浄の際の温度を上げることにより、よりストリンジェントな条 件とすることがでさる [0035] The "polynucleotide hybridizing under stringent conditions" in the polynucleotide having the nucleotide sequence ability encoding the amino acid sequence of (D) specifically includes FASTA, BLAST, Smith-Waterman [Meth. Enzym , 164, 765 (1988)], etc., using the default (default) parameters, for example, for humans, GenBank accession number: NM 004591 and at least 70% or more 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Nucleotides are mentioned. In addition, “As for the stringent hybridization condition, f column is“ 2 X SSC, 0.1% SDS, 50 ° C ”,“ 2 X SSC, 0.1% SDS, 42.C ”,“ 1 X SSC, 0.1% SDS, 37.C ”, more stringent end conditions include, for example,“ 2 X SSC, 0.1% SDS, 65.C ”,“ 0.5 X SSC, 0.1% SDS, 42.C "," 0.2 X SSC, 0.1% SDS, 65 ° C "can be mentioned. More specifically, as a method using Rap id-hyb buffer (Amersham Life Science), after pre-hybridization at 68 ° C for 30 minutes or more, add the probe to 68 ° C for 1 hour or more. Keep hybridized, then wash 3 times in 2 X SSC, 0.1% SDS, 20 min at room temperature, 1 X SSC, 0.1% SDS, 37. It is conceivable to perform 3 20 minute washes at C, and finally 2 washes for 20 minutes at 50 ° C in 1 X SSC, 0.1% SDS. In addition, for example, in Expresshyb Hybridization Solution (CLONTECH), prehybridize at 55 ° C for 30 minutes or more, add the labeled probe, incubate at 37-55 ° C for 1 hour or more, and 2 X Wash 3 times for 20 minutes at room temperature in SSC, 0.1% SDS, 37 in 1 X SSC, 0.1% SDS. 20 minutes on C Can be washed once. Here, for example, it is possible to make the conditions more stringent by increasing the temperature at the time of preno, hybridization, hybridization, and the second washing.
。例えば、プレハイブリダィゼーシヨンおよびハイブリダィゼーシヨンの温度を 60°C、さ らにストリンジェントな条件としては 68°Cとすることができる。当業者であれば、このよう なノッファーの塩濃度、温度等の条件に加えて、その他のプローブ濃度、プローブ の長さ、反応時間等の諸条件を加味し、 CCL20タンパク質のァイソフォーム、ァレリ ック変異体、および対応する他種生物由来の遺伝子を得るための条件を設定するこ とがでさる。  . For example, the temperature of the prehybridization and the hybridization can be 60 ° C, and the stringent condition can be 68 ° C. Those skilled in the art, in addition to the conditions such as the salt concentration and temperature of the koffer, take into account other conditions such as the probe concentration, probe length, reaction time, etc. It is possible to set conditions for obtaining genetic variants and corresponding genes from other species.
[0036] ハイブリダィゼーシヨン法の詳細な手順にっ 、ては、『Molecular Cloning, A Labora tory Manual 2nd ed.J(Cold Spring Harbor Press (1989);特に Section9.47- 9.58)、『C urrent Protocols in Molecular Biology』(John Wiley & Sons (1987- 1997);特に Section 6.3-6.4)、『DNA Cloning 1: Core Techniques, A Practical Approach 2nd ed.』(Oxfor d University (1995);条件については特に Section2.10)等を参照することができる。ノヽ イブリダィズするポリヌクレオチドとしては、例えば GenBankァクセッション番号： NM 004591の塩基を含む塩基配列に対して少なくとも 50%以上、好ましくは 70%、さら に好ましくは 80%、より一層好ましくは 90% (例えば、 95%以上、さらには 99%)の 同一性を有する塩基配列を含むポリヌクレオチドが挙げられる。このような同一性は、 上述の相同性の決定と同様に BLASTアルゴリズム (Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 58 73-7)によって決定することができる。上述の塩基配列についてのプログラム BLASTN の他に、このアルゴリズムに基づいたアミノ酸配列についての同一性を決定するプロ グラムとして BLASTX(Altschul et al. (1990) J. Mol. Biol. 215: 403- 10)等が開発され ており、利用可能である。具体的な解析方法については先に挙げたように、 http://w ww.ncbi.nlm.nih.gov.等を参照することがでさる。  [0036] For detailed procedures of the hybridization method, see Molecular Cloning, A Labora tory Manual 2nd ed. J (Cold Spring Harbor Press (1989); especially Sections 9.47-9.58), C urrent Protocols in Molecular Biology (John Wiley & Sons (1987-1997); especially Section 6.3-6.4), DNA Cloning 1: Core Techniques, A Practical Approach 2nd ed. (Oxfor d University (1995); In particular, refer to Section 2.10). As a polynucleotide to be hybridized, for example, at least 50%, preferably 70%, more preferably 80%, and still more preferably 90% of the nucleotide sequence containing the base of GenBank accession number: NM 004591 ( For example, a polynucleotide containing a nucleotide sequence having an identity of 95% or more, and further 99%) can be mentioned. This identity is similar to the BLAST algorithm described above (Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 58 73-7). In addition to the above-mentioned program BLASTN for the base sequence, BLASTX (Altschul et al. (1990) J. Mol. Biol. 215: 403-10) is a program for determining the identity of amino acid sequences based on this algorithm. Etc. have been developed and are available. For specific analysis methods, refer to http://www.ncbi.nlm.nih.gov. Etc. as mentioned above.
[0037] その他、遺伝子増幅技術 (PCR) (Current Protocols in Molecular Biology, John Wi ley & Sons (1987) Section 6.1- 6.4)により、 CCL20タンパク質のァイソフォームゃァレ リック変異体等を、ヒト、マウス、ラット、ゥサギ、ノヽムスター、 -ヮトリ、ブタ、ゥシ、ャギ、 ヒッジ等の cDNAライブラリーおよびゲノムライブラリーから、ヒトの場合 GenBankァク セッション番号： NM 004591、マウスの場合 GenBankァクセッション番号： NM 01 6960等を基に設計したプライマーを利用して得ることができる。 [0037] In addition, by means of gene amplification technology (PCR) (Current Protocols in Molecular Biology, John Wiley & Sons (1987) Section 6.1-6.4), glycoform mutants of CCL20 protein, Mouse, rat, usagi, nomstar, -bird, pig, rush, goat, It can be obtained from the cDNA library and genomic library of Hedge etc. using primers designed based on GenBank succession number: NM 004591 for humans and GenBank succession number: NM 01 6960 for humans. it can.
[0038] ポリヌクレオチドの塩基配列は、慣用の方法により配列決定して確認することができ る。例えば、ジデォキシヌクレオチドチェーンターミネーシヨン法（Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463)等による確認が可能である。また、適当な DNA シークェンサ一を利用して配列を解析することも可能である。 [0038] The base sequence of the polynucleotide can be confirmed by sequencing by a conventional method. For example, it can be confirmed by the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463). It is also possible to analyze the sequence using an appropriate DNA sequencer.
[0039] 本発明に使用する CCL20タンパク質をコードするポリヌクレオチドは、例えば天然 由来のものであることもできるし、または全合成したものであることもできる。さらには、 天然由来のものの一部を利用して合成を行なったものであることもできる。本発明に 使用する CCL20タンパク質をコードするポリヌクレオチドの典型的な取得方法として は、例えば市販のライブラリーまたは cDNAライブラリーから、遺伝子工学の分野で 慣用されて 、る方法、例えば部分ポリヌクレオチド配列の情報を基にして作成した適 当な DNAプローブを用いてスクリーニングを行なう方法等を挙げることができる。  [0039] The polynucleotide encoding the CCL20 protein used in the present invention may be derived from, for example, a natural source or a totally synthesized one. Furthermore, it can also be synthesized by using a part of the natural origin. A typical method for obtaining a polynucleotide encoding the CCL20 protein used in the present invention is, for example, a method commonly used in the field of genetic engineering from, for example, a commercially available library or cDNA library, such as a partial polynucleotide sequence. Examples include screening methods using appropriate DNA probes created based on information.
[0040] 本発明に使用する CCL20タンパク質をコードするポリヌクレオチドとしては、「前述 のァクセッション番号により特定される塩基配列（例えば、ヒトの場合 GenBankァクセ ッシヨン番号： NM 004591)力 なるポリヌクレオチド」が好ましい。  [0040] As the polynucleotide encoding the CCL20 protein used in the present invention, "a polynucleotide having the nucleotide sequence identified by the above-mentioned accession number (for example, GenBank accession number: NM 004591 in the case of humans)" Is preferred.
[0041] また、本発明の抗体を得るための CCL20タンパク質には、 CCL20タンパク質の全 長アミノ酸配列を有するポリペプチドに加えて、 CCL20タンパク質の少なくとも 6アミ ノ酸残基以上 (例えば、 6、 8、 10、 12または 15アミノ酸残基以上）と同一の配列を有 するポリペプチド断片（「フラグメント」ということもある。）も含まれる。本明細書におけ る CCL20タンパク質のフラグメントは、 CCL20タンパク質の抗原性さえ有すればど のようなフラグメントであってもよ 、。  [0041] The CCL20 protein for obtaining the antibody of the present invention includes at least 6 amino acid residues of the CCL20 protein in addition to a polypeptide having the full-length amino acid sequence of the CCL20 protein (for example, 6, 8 , 10, 12 or 15 amino acid residues or more) and polypeptide fragments (sometimes referred to as “fragments”). The fragment of the CCL20 protein in the present specification may be any fragment as long as it has the antigenicity of the CCL20 protein.
[0042] 好まし!/、フラグメントとしては、 CCL20タンパク質のァミノ末端、カルボキシル末端 等のフラグメントを挙げることができる。ポリペプチドの抗原決定部位は、タンパク質の アミノ酸配列上の疎水性 Z親水性を解析する方法 (Kyte-Doolittle(1982)J.Mol.Biol. 157: 105- 22)、二次構造を解析する方法 (Chou- Fasman(1978)Ann.Rev.Biochem. 47:2 51-76)により推定し、さらにコンピュータープログラム (Anal.Biochem. 151:540-6(1985) )または短 、ペプチドを合成しその抗原性を確認する PEPSCAN法 (特表昭 60 - 50 0684号公報)等の手法により確認することができる。 [0042] Preferable fragments include, for example, fragments such as amino terminal and carboxyl terminal of CCL20 protein. The antigenic determinant of the polypeptide is a method of analyzing hydrophobic Z hydrophilicity on the amino acid sequence of the protein (Kyte-Doolittle (1982) J. Mol. Biol. 157: 105-22), a method of analyzing secondary structure (Chou- Fasman (1978) Ann. Rev. Biochem. 47: 2 51-76) and further computer program (Anal. Biochem. 151: 540-6 (1985) ) Or short, it can be confirmed by a method such as PEPSCAN method (Japanese Patent Publication No. 60-500684) which synthesizes a peptide and confirms its antigenicity.
[0043] [0043]
本発明による抗体には、 CCL20タンパク質を抗原として、該抗原をマウス等の哺乳 動物に免疫して得られるモノクローナル抗体、遺伝子組換え技術を用いて製造され るキメラモノクローナル抗体およびヒト型モノクローナル抗体、並びにヒト抗体産生トラ ンスジエニック動物等を用いて製造されるヒトモノクローナル抗体が含まれる。本発明 による抗体を医薬としてヒトに投与する場合は、副作用の観点から、ヒトモノクローナ ル抗体が望ましい。  The antibody according to the present invention includes a monoclonal antibody obtained by immunizing a mammal such as a mouse with the CCL20 protein as an antigen, a chimeric monoclonal antibody and a human monoclonal antibody produced using a gene recombination technique, and Human monoclonal antibodies produced using human antibody-producing transgenic animals and the like are included. When the antibody according to the present invention is administered as a medicament to a human, a human monoclonal antibody is desirable from the viewpoint of side effects.
[0044] 本発明における「抗体」とは、ポリクローナル抗体（抗血清)あるいはモノクローナル 抗体を意味し、好ましくはモノクローナル抗体である。また、本発明の「抗体」は、抗 原 (例えば、天然型抗原、遺伝子組換抗原、抗原を発現している細胞等)を後述に 挙げるような哺乳動物に免疫して得られる天然型抗体、遺伝子組換技術を用いて製 造され得るキメラモノクローナル抗体およびヒト型モノクローナル抗体、並びにヒト抗 体産生トランスジエニック動物等も用いて製造され得るヒトモノクローナル抗体を包含 する。  The “antibody” in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody, preferably a monoclonal antibody. The “antibody” of the present invention is a natural antibody obtained by immunizing a mammal such as those described below with an antigen (eg, a natural antigen, a genetically modified antigen, an antigen-expressing cell, etc.). In addition, chimeric monoclonal antibodies and human monoclonal antibodies that can be produced using gene recombination techniques, and human monoclonal antibodies that can also be produced using human antibody-producing transgenic animals and the like are included.
[0045] 免疫抗原としては、 CCL20タンパク質またはそのフラグメントを用いることができる。  [0045] As the immunizing antigen, CCL20 protein or a fragment thereof can be used.
あるいは、上記抗原のアミノ酸配列に基づき合成したものを用いることができる。抗原 はキャリアタンパク質との複合体として用いてもょ 、。抗原とキャリアタンパク質の複合 体の調製には種々の縮合剤を用いることができる、例えばダルタルアルデヒド、カル ポジイミド、マレイミド活性エステル等が使用できる。キャリアタンパク質は牛血清アル ブミン、サイログロブリン、へモシァニン等の常用されているものでよぐ通常 1〜5倍 量の割合でカップリングさせる方法が用いられる。  Or what was synthesize | combined based on the amino acid sequence of the said antigen can be used. The antigen may be used as a complex with the carrier protein. Various condensing agents can be used for the preparation of the antigen-carrier protein complex, such as dartal aldehyde, carbodiimide, maleimide active ester, and the like. The carrier protein is a commonly used one such as bovine serum albumin, thyroglobulin, hemocyanin and the like. Usually, a method of coupling at a ratio of 1 to 5 times is used.
[0046] 免疫される哺乳動物、好ましくは、マウス、ラット、ノ、ムスター、モルモット、ゥサギ、ネ コ、ィヌ、ブタ、ャギ、ゥマあるいはゥシ、より好ましくはマウス、ラット、ハムスター、モル モットまたはゥサギ等が挙げられ、接種方法は皮下、筋肉あるいは腹腔内の投与が 挙げられる。投与に際しては完全フロイントアジュバンドや不完全フロイントアジュバ ンドと混和して投与してもよぐ投与は通常 2〜5週毎に 1回ずつ行われる。免疫され た動物の脾臓あるいはリンパ節力 得られた抗体産生細胞は骨髄腫細胞と細胞融合 させ、ハイプリドーマとして単離される。骨髄腫細胞としては哺乳動物由来、例えばマ ウス、ラット、ヒト等由来のものが使用され、抗体産生細胞と同種由来のものであること が好まし!/、が、異種間にお！/、ても可能な場合もある。 [0046] Mammals to be immunized, preferably mice, rats, mice, mussels, guinea pigs, rabbits, cats, dogs, pigs, goats, horses or rushes, more preferably mice, rats, hamsters, Guinea pigs or rabbits can be used. Inoculation methods include subcutaneous, intramuscular or intraperitoneal administration. When administered, it may be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant, usually once every 2 to 5 weeks. Immunized The resulting antibody-producing cells are fused with myeloma cells and isolated as a hyperidoma. The myeloma cells are derived from mammals such as mice, rats, and humans, and are preferably derived from the same species as the antibody-producing cells! / But sometimes it is possible.
[0047] 本発明による抗体は、 CCL20タンパク質を特異的に認識することができる。従って 、本発明による抗体を得るための CCL20タンパク質は、 CCL20の抗原性を有して いればよぐ CCL20タンパク質のアミノ酸配列において 1若しくは複数個のアミノ酸残 基が欠失、挿入、置換または付加されたアミノ酸配列を有するタンパク質が包含され る。このような変異タンパク質が元のタンパク質と同じ生物学的活性が維持されること は公知である (Mark et al.(1984)Proc.Natl.Acad.Sci.USA 81:5662- 6;Zoller and Smith (1982)Nucleic Acids Res. 10:6487— 500;Wang et al.(1984)Science 224:1431— 3;Dalbad ie-McFarland et al.(1982)Proc.Natl.Acad.Sci.USA 79:6409-13)。また、本発明による 抗体には、 CCL20タンパク質の一部に対して特異的な抗体も含まれる。  [0047] The antibody according to the present invention can specifically recognize CCL20 protein. Therefore, the CCL20 protein for obtaining the antibody according to the present invention has only to have the antigenicity of CCL20. One or more amino acid residues are deleted, inserted, substituted or added in the amino acid sequence of the CCL20 protein. Proteins having different amino acid sequences are included. It is known that such mutant proteins maintain the same biological activity as the original protein (Mark et al. (1984) Proc. Natl. Acad. Sci. USA 81: 5662-6; Zoller and Smith (1982) Nucleic Acids Res. 10: 6487—500; Wang et al. (1984) Science 224: 1431—3; Dalbad ie-McFarland et al. (1982) Proc. Natl. Acad. Sci. USA 79: 6409- 13). The antibody according to the present invention also includes an antibody specific for a part of the CCL20 protein.
[0048] 本発明による抗体は、好ましくは CCL20タンパク質の機能に影響を与える抗体で ある。 CCL20タンパク質の機能に影響を与えるとは、例えば、該抗体が CCL20タン ノ ク質と結合することにより、 CCL20タンパク質と CCR6との結合を阻害すること、ま たは該抗体が CCL20タンパク質と結合することにより、 CCR6の活性ィ匕を介した細 胞遊走を阻害することである。  [0048] The antibody according to the present invention is preferably an antibody that affects the function of the CCL20 protein. The effect of CCL20 protein is, for example, that the antibody binds to CCL20 protein, thereby inhibiting the binding between CCL20 protein and CCR6, or the antibody binds to CCL20 protein. This is to inhibit cell migration through the activity of CCR6.
[0049] ポリクローナル杭体  [0049] Polyclonal pile
本発明における「ポリクローナル抗体」は、既存の一般的な製造方法によって製造 することができる。即ち、例えば、前述のような抗原を、必要に応じてフロイントアジュ バント（Freund's Adjuvant)とともに、哺乳動物、好ましくは、マウス、ラット、ノヽムスター 、モルモット、ゥサギ、ネコ、ィヌ、ブタ、ャギ、ゥマあるいはゥシ、より好ましくはマウス、 ラット、ノ、ムスター、モルモットまたはゥサギに免疫することで該免疫感作動物力 得 た血清力も取得することができる。  The “polyclonal antibody” in the present invention can be produced by an existing general production method. That is, for example, an antigen as described above is added to a mammal, preferably a mouse, a rat, a nomster, a guinea pig, a rabbit, a cat, a dog, a pig, a pig, a goat, together with Freund's Adjuvant as necessary. Serum strength obtained by immunizing a immunized animal by immunizing mouse, rat, mouse, more preferably mouse, rat, mouse, muster, guinea pig or rabbit.
[0050] モノクローナル抗体  [0050] Monoclonal antibody
また、本発明における「モノクローナル抗体」は、具体的には下記のようにして製造 することができる。即ち、前述のような抗原を免疫原とし、該免疫原を、必要に応じて フロイントアジュバント（Freund's Adjuvant)とともに、哺乳動物、好ましくは、マウス、ラ ット、ノ、ムスター、モルモット、ゥサギ、ネコ、ィヌ、ブタ、ャギ、ゥマあるいはゥシ、より好 ましくはマウス、ラット、ノ、ムスター、モルモットまたはゥサギ (後述するヒト抗体産生トラ ンスジヱニックマウスのような他の動物由来の抗体を産生するように作出されたトラン スジエニック動物を含む）の皮下内、筋肉内、静脈内、フッドパッド内あるいは腹腔内 に 1〜数回注射するかあるいは移植することにより免疫感作を施す。通常、初回免疫 力も約 1〜14日毎に 1〜4回免疫を行って、最終免疫より約 1〜5日後に免疫感作さ れた該哺乳動物力 抗体産生細胞が取得される。免疫を施す回数および時間的ィ ンターバルは、使用する免疫原の性質等により、適宜変更することができる。 Further, the “monoclonal antibody” in the present invention can be specifically produced as follows. That is, an antigen as described above is used as an immunogen, and the immunogen is Along with Freund's Adjuvant, mammals, preferably mice, rats, rats, mustards, guinea pigs, usagis, cats, nu, pigs, goats, horses or tusks, more preferably mice , Rats, mice, guinea pigs, or rabbits (including transgenic animals engineered to produce antibodies from other animals such as human antibody-producing transgenic mice described below), muscle Immunization is carried out by injecting or transplanting once, several times, intraperitoneally, intravenously, into the food pad or into the abdominal cavity. Usually, the initial immunity is also immunized 1 to 4 times about every 1 to 14 days, and the antibody-producing cells that are immunized about 1 to 5 days after the final immunization are obtained. The number of times of immunization and the time interval can be appropriately changed depending on the nature of the immunogen used.
[0051] 本発明によるモノクローナル抗体は、当業者に周知方法を用いて得ることができる（ ί列えば、『Current Protocols in Molecular BiologyJQohn Wiley & Sons(1987))、 Antib odies:A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Labora tory(1988)) 0 [0051] Monoclonal antibodies according to the present invention can be obtained using methods well known to those skilled in the art (for example, Current Protocols in Molecular Biology JQohn Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Labora tory (1988)) 0
[0052] さらに、本発明におけるモノクローナル抗体を分泌する「ノヽイブリドーマ」の調製は、 ケーラーおよびミルシュタインらの方法（ネイチヤー (Nature) , 256,495, 1975)および それに準じる修飾方法に従って行うことができる。即ち、前述の如く免疫感作された 哺乳動物、好ましくは、マウス、ラット、ハムスター、モルモット、ゥサギ、ネコ、ィヌ、ブ タ、ャギ、ゥマあるいはゥシから取得される脾臓、リンパ節、骨髄あるいは扁桃等、好 ましくは脾臓に含まれる抗体産生細胞と、好ましくはマウス、ラット、モルモット、ハムス ター、ゥサギまたはヒト等の哺乳動物、より好ましくはマウス、ラットまたはヒト由来の自 己抗体産生能のないミエローマ細胞を細胞融合させることにより調製される。  [0052] Furthermore, "Neubridoma" secreting monoclonal antibodies in the present invention can be prepared according to the method of Köhler and Milstein et al. (Nature, 256, 495, 1975) and a modification method according thereto. That is, the spleen and lymph nodes obtained from a mammal immunized as described above, preferably a mouse, rat, hamster, guinea pig, rabbit, cat, Inu, butterfly, goat, horse or rush. Antibody-producing cells contained in the spleen, preferably bone marrow or tonsil, and preferably mammals such as mice, rats, guinea pigs, hamsters, rabbits or humans, more preferably self derived from mice, rats or humans. It is prepared by cell fusion of myeloma cells having no antibody-producing ability.
[0053] 細胞融合に用いられるミエローマ細胞としては、例えばマウス由来ミエローマ P3Z X63-AG8. 653 (653) , P3/NSl/l -Ag4- l (NS - l) , P3/X63-Ag8. U1 (P3U1)、 SP2Z0— Agl4 (Sp2Z〇、 Sp2)、 PAI、 FOある!/、 ίま BW5147、ラッ ト由来ミエローマ 210RCY3— Ag. 2. 3. 、ヒト由来ミエローマ U— 266AR1、 GM15 00-6TG-A1 - 2, UC729-6, CEM—AGR、 D1R11あるいは CEM—T15等 を使用することができる。  [0053] Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3Z X63-AG8. 653 (653), P3 / NSl / l-Ag4-l (NS-l), P3 / X63-Ag8. U1 ( P3U1), SP2Z0—Agl4 (Sp2Z〇, Sp2), PAI, FO! /, Ί or BW5147, rat-derived myeloma 210RCY3— Ag. 2. 3., human-derived myeloma U—266AR1, GM15 00-6TG-A1 -2, UC729-6, CEM-AGR, D1R11, CEM-T15, etc. can be used.
[0054] 融合促進剤としてはポリエチレングリコールやセンダイウィルス等が挙げられ、通常 には、 20〜50%程度の濃度のポリエチレングリコール（平均分子量 1000〜4000) を用いて 20〜40°C、好ましくは 30〜37°Cの温度下、抗体産生細胞数と骨髄腫細 胞数の比は通常 1： 1〜10： 1程度、約 1〜10分間程度反応させることにより細胞融合 を実施することができる。 [0054] Examples of fusion promoters include polyethylene glycol and Sendai virus. The number of antibody-producing cells and the number of myeloma cells at a temperature of 20-40 ° C, preferably 30-37 ° C, using polyethylene glycol (average molecular weight 1000-4000) at a concentration of about 20-50%. The ratio is usually about 1: 1 to 10: 1, and cell fusion can be carried out by reacting for about 1 to 10 minutes.
[0055] モノクローナル抗体を産生するハイブリドーマクローンのスクリーニングは、ハイブリ ドーマを、例えばマイクロタイタープレート中で培養し、増殖の見られたゥエルの培養 上清の前述の免疫感作で用いた免疫抗原に対する反応性を、例えば RIAや ELIS A等の免疫化学的方法によって測定することにより行うことができる。具体的には、例 えば、 CCL20タンパク質をコートしたマイクロプレートを用いる ELISA法、抗免疫グ ロブリン抗体をコートしたマイクロプレートを用いる EIA法、 CCL20タンパク質を含む サンプルを電気泳動後、ニトロセルロース転写膜を用いるィムノブロット法等が挙げら れる。 [0055] The screening of hybridoma clones producing monoclonal antibodies is carried out by culturing the hybridomas in, for example, a microtiter plate, and reacting the culture supernatant of the wells in which proliferation is observed with the immunizing antigen used in the immunization described above. The sex can be measured by, for example, immunochemical methods such as RIA and ELIS A. Specifically, for example, ELISA method using microplate coated with CCL20 protein, EIA method using microplate coated with anti-immunoglobulin antibody, electrophoresis of sample containing CCL20 protein, and nitrocellulose transfer membrane Examples include the immunoblot method used.
[0056] 抗体産生ノ、イブリドーマのスクリーニングには、また、上記免疫化学的方法に代え て、該抗体力 SCCL20タンパク質の機能に影響を与える力否かによりスクリーニングす ることができる。例えば、該抗体が CCL20タンパク質と結合することにより、 CCL20タ ンパク質と CCR6との結合が阻害される力否力 および該抗体が CCL20タンパク質 と結合することにより、 CCR6の活性ィ匕を介した細胞遊走を阻害するか否かにより、抗 体産生ハイブリドーマをスクリーニングすることができる。このスクリーニング方法により 、本発明の抗体の好ましい様態である CCL20タンパク質の機能に影響を与える抗 体を選択することができる。またこのスクリーニングは、 CCL20タンパク質と結合する 抗体を産生する力否かにより選択する上記免疫化学的方法スクリーニングに次いで 行う、 2次スクリーニングとして行っても良い。  [0056] For the screening of antibody-producing cells and hybridomas, instead of the immunochemical method described above, screening can be performed based on whether or not the antibody strength affects the function of the SCCL20 protein. For example, when the antibody binds to the CCL20 protein, the binding of the CCL20 protein to CCR6 is inhibited, and when the antibody binds to the CCL20 protein, the cell is mediated by the activity of CCR6. Antibody-producing hybridomas can be screened depending on whether or not migration is inhibited. By this screening method, an antibody that affects the function of the CCL20 protein, which is a preferred embodiment of the antibody of the present invention, can be selected. In addition, this screening may be performed as a secondary screening performed after the immunochemical method screening that is selected based on the ability to produce an antibody that binds to the CCL20 protein.
[0057] このようなゥエルから更に、例えば限界希釈法によってクローニングを行い、クロー ンを得ることができる。ハイプリドーマの選別、育種は、通常、 HAT (ヒポキサンチン、 アミノプテリン、チミジン）を添加して、 10〜20%牛胎児血清を含む動物細胞用培地 (例、 RPMI1640)で行われる。このようにして得られたクローンはあらかじめプリスタ ンを投与した SCIDマウスの腹腔内へ移植し、 10〜14日後にモノクローナル抗体を 高濃度に含む腹水を採取し、抗体精製の原料とすることができる。また、該クローンを 培養し、その培養物を抗体精製の原料とすることもできる。 [0057] Further cloning from such a well can be performed, for example, by limiting dilution to obtain a clone. Selection and breeding of hypridoma is usually performed in a medium for animal cells (eg, RPMI1640) containing 10-20% fetal calf serum with the addition of HAT (hypoxanthine, aminopterin, thymidine). The clones thus obtained can be transplanted into the abdominal cavity of SCID mice pre-administered with pristane, and ascites containing a high concentration of monoclonal antibody can be collected 10-14 days later and used as a raw material for antibody purification. . In addition, the clone It is also possible to culture and use the culture as a raw material for antibody purification.
[0058] ハイプリドーマからのモノクローナル抗体の製造は、ハイプリドーマをインビトロ、ま たは哺乳動物、好ましくは、マウス、ラット、ノ、ムスター、モルモット、ゥサギ、ネコ、ィヌ 、ブタ、ャギ、ゥマあるいはゥシ等、好ましくはマウス、ラット、ハムスターまたはモルモ ット、より好ましくはマウスの腹水中等でのインビボで行い、得られた培養上清、また は哺乳動物の腹水から単離することにより行うことができる。  [0058] The production of monoclonal antibodies from Hypridoma can be achieved by using Hypridoma in vitro, or in mammals, preferably mice, rats, mice, guinea pigs, rabbits, cats, dogs, pigs, goats, mice. By conducting in vivo in mouse, rat, etc., preferably mouse, rat, hamster or guinea pig, more preferably mouse ascites, etc., and isolating from the obtained culture supernatant or mammalian ascites It can be carried out.
[0059] インビトロで培養する場合には、培養する細胞種の特性および培養方法等の種々 条件に合わせて、ハイプリドーマを増殖、維持および保存させ、培養上清中にモノク ローナル抗体を産生させるために用いられるような既知の栄養培地あるいは既知の 基本培地カゝら誘導調製されるあらゆる栄養培地を用いて実施することが可能である。  [0059] When cultivating in vitro, in order to grow, maintain, and store hyperpridoma according to various conditions such as the characteristics of the cell type to be cultured and the culture method, and to produce a monoclonal antibody in the culture supernatant. It is possible to carry out using any known nutrient medium such as that used in the above, or any nutrient medium that is derived from a known basic medium.
[0060] 基本培地としては、例えば、 Ham' F12培地、 MCDB153培地あるいは低カルシゥ ム MEM培地等の低カルシウム培地および MCDB104培地、 MEM培地、 D— ME M培地、 RPMI1640培地、 ASF104培地あるいは RD培地等の高カルシウム培地 等が挙げられ、該基本培地は、 目的に応じて、例えば血清、ホルモン、サイト力イン および Zまたは種々無機あるいは有機物質等を含有することができる。  [0060] Examples of basic medium include low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium, MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium. The basic medium can contain, for example, serum, hormones, cytosine and Z, various inorganic or organic substances, etc., depending on the purpose.
[0061] モノクローナル抗体の単離、精製は、上述の培養上清あるいは腹水を、飽和硫酸 アンモ-ゥム、ユーグロブリン沈澱法、力プロイン酸法、力プリル酸法、イオン交換クロ マトグラフィー（DEAEまたは DE52等）、抗ィムノグロブリンカラムあるいはプロテイン Aカラム等のァフィ二ティカラムクロマトグラフィーに供すること等により行うことができ る。具体的には、モノクローナル抗体の精製は免疫グロブリンの精製法として既知の 方法を用いればよぐたとえば、硫安分画法、 PEG分画法、エタノール分画法、陰ィ オン交換体の利用、さらに CCL20タンパク質を用 、るァフィユティークロマトグラフィ 一等の手段により容易に達成することができる。  [0061] Monoclonal antibodies can be isolated and purified by using the above-mentioned culture supernatant or ascites fluid, saturated ammonium sulfate, euglobulin precipitation method, force proic acid method, force prillic acid method, ion exchange chromatography (DEAE Or DE52, etc.), affinity column chromatography such as an anti-immunoglobulin column or protein A column. Specifically, monoclonal antibodies can be purified by using known methods for immunoglobulin purification such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, use of anion exchanger, Using CCL20 protein, it can be easily achieved by means such as affinity chromatography.
[0062] 本発明によるモノクローナル抗体の具体例としては、 2005年 11月 8日付で独立行 政法人産業技術総合研究所特許生物寄託センター ( T 305 8566茨城県つくば 巿東 1丁目 1番地 1中央第 6)に FERM BP— 10445のもと受託されたハイブリドー マが産生するモノクローナル抗体が挙げられる。  [0062] As a specific example of the monoclonal antibody according to the present invention, the patent biological deposit center (National Institute of Advanced Industrial Science and Technology) (T 305 8566 Tsukuba Ibaraki 1-chome 1 1 6) includes monoclonal antibodies produced by hybridomas commissioned under FERM BP-10445.
[0063] 従って、本発明のハイプリドーマの具体例としては、 2005年 11月 8日付で独立行 政法人産業技術総合研究所特許生物寄託センター ( T 305 8566茨城県つくば 巿東 1丁目 1番地 1中央第 6)に FERM BP— 10445のもと寄託されたハイブリドー マ（@mLARC # 2F5— 5)が挙げられる。 [0063] Therefore, as a specific example of the hyperidoma of the present invention, an independent Hybridoma (@mLARC # 2F5—5) deposited under the FERM BP-10445 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (T305 8566, Tsukuba 1-chome, 1-chome, Ibaraki, 1-chome, 1st, 6th) Is mentioned.
[0064] 遣伝子組換え技術を用いて製造されるモノクローナル抗体 [0064] Monoclonal antibody produced using gene recombination technology
本発明における「キメラモノクローナル抗体」は、遺伝子工学的に作製される組換え モノクローナル抗体であることができ、具体的には、その可変領域が、非ヒト哺乳動物 The “chimeric monoclonal antibody” in the present invention can be a recombinant monoclonal antibody produced by genetic engineering. Specifically, the variable region is a non-human mammal.
(マウス、ラット、ハムスター等）のィムノグロブリン由来の可変領域であり、かつその定 常領域がヒトイムノグロブリン由来の定常領域であることを特徴とするマウス Zヒトキメ ラモノクローナル抗体等のキメラモノクローナル抗体を意味する。また、抗原をマウス に免疫し、そのマウスモノクローナル抗体の遺伝子力 抗原と結合する抗体可変部（ V領域)を切り出し、ヒト骨髄由来の抗体定常部 (C領域)遺伝子と結合して、キメラモ ノクローナル抗体を作製することができる。例えば、特公平 3— 73280号公報に記載 の方法を参照して作製することができる。 A chimeric monoclonal antibody such as a mouse Z human chimeric monoclonal antibody characterized in that it is a variable region derived from an immunoglobulin of (mouse, rat, hamster, etc.), and the constant region is a constant region derived from human immunoglobulin. Means. In addition, the antibody is immunized with a mouse, and the antibody variable region (V region) that binds to the antigenic power of the mouse monoclonal antibody is excised and combined with the antibody constant region (C region) gene derived from human bone marrow to produce a chimeric monoclonal antibody. Can be produced. For example, it can be produced with reference to the method described in Japanese Patent Publication No. 3-73280.
[0065] ヒトイムノグロブリン由来の定常領域は、 IgG (IgGl、 IgG2、 IgG3、 IgG4)、 IgM、 I gA、 IgDおよび IgE等のアイソタイプにより各々固有のアミノ酸配列を有する力 本発 明における組換キメラモノクローナル抗体の定常領域は 、ずれのアイソタイプに属す るヒトイムノグログリンの定常領域であってもよい。好ましくは、ヒト IgGの定常領域であ る。 [0065] The constant region derived from human immunoglobulin is a recombinant chimera having a unique amino acid sequence depending on isotypes such as IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of the monoclonal antibody may be a constant region of human immunoglobulin belonging to a different isotype. Preferably, it is a constant region of human IgG.
[0066] 本発明におけるキメラモノクローナル抗体は、例えば以下のようにして製造すること ができる。しかしながら、そのような製造方法に限定されるものでないことは言うまでも ない。  [0066] The chimeric monoclonal antibody of the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
[0067] 例えば、マウス Zヒトキメラモノクローナル抗体は、実験医学（臨時増刊号)、第 1. 6 卷、第 10号、 1988年および特公平 3— 73280号公報等を参照しながら作製するこ とができる。即ち、マウスモノクローナル抗体を産生するハイブリドーマから単離した 該マウスモノクローナル抗体をコードする DNAから取得した活性な VH遺伝子 (H鎖 可変領域をコードする再配列された VDJ遺伝子）の下流に、ヒトイムノグロムリンをコ ードする DNAから取得した CH遺伝子 (H鎖定常領域をコードする C遺伝子）を、また 該ハイブリドーマから単離したマウスモノクローナル抗体をコードする DNAから取得 した活性な VL遺伝子（L鎖可変領域をコードする再配列された VJ遺伝子)の下流に ヒトイムノグロムリンをコードする DNAから取得した CL遺伝子 (L鎖定常領域をコード する C遺伝子）を、各々発現可能なように配列して 1つ又は別々の発現ベクターに挿 入し、該発現べクタ一で宿主細胞を形質転換し、該形質転換細胞を培養すること〖こ より作製することができる。 [0067] For example, a mouse Z human chimeric monoclonal antibody is prepared with reference to experimental medicine (special issue), No. 1.6, No. 10, 1988, and Japanese Patent Publication No. 3-73280. Can do. That is, human immunoglobulin downstream of an active VH gene (rearranged VDJ gene encoding a heavy chain variable region) obtained from DNA encoding the mouse monoclonal antibody isolated from a hybridoma producing a mouse monoclonal antibody. Obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma, the CH gene (C gene encoding the heavy chain constant region) obtained from the DNA encoding Each CL gene (C gene encoding the light chain constant region) obtained from DNA encoding human immunoglobulin is expressed downstream of the active VL gene (rearranged VJ gene encoding the light chain variable region). It can be prepared by arranging as possible and inserting it into one or a separate expression vector, transforming a host cell with the expression vector, and culturing the transformed cell.
[0068] 具体的には、まず、マウスモノクローナル抗体産生ハイブリドーマから常法により D NAを抽出後、該 DNAを適切な制限酵素（例えば EcoRI、 Hindlll等）を用いて消化 し、電気泳動に付して (例えば 0. 7%ァガロースゲル使用）サザンプロット法を行う。 泳動したゲルを例えばェチジゥムブロマイド等で染色し、写真撮影後、マーカーの位 置を付し、ゲルを 2回水洗し、 0. 25M HC1溶液に 15分間浸す。次いで、 0. 4Nの N aOH溶液に 10分間浸し、その間緩やかに振盪する。常法により、フィルターに移し、 4時間後フィルターを回収して 2 X SSCで 2回洗浄する。フィルターを十分乾燥した 後、べィキング（75°C、 3時間）を行う。べィキング終了後に、該フィルターを 0. 1 X S SC/0. 1%SDS溶液に入れ、 65°Cで 30分間処理する。次いで、 3 X SSC/0. 1 %SDS溶液に浸す。得られたフィルターをプレハイブリダィゼーシヨン液と共にビ- ール袋に入れ、 65°Cで 3〜4時間処理する。  [0068] Specifically, first, DNA is extracted from a mouse monoclonal antibody-producing hybridoma by a conventional method, and then the DNA is digested with an appropriate restriction enzyme (eg, EcoRI, Hindlll, etc.) and subjected to electrophoresis. (For example, use a 0.7% agarose gel) and perform the Southern plot method. Stain the electrophoresed gel with, for example, ethidium bromide, etc. After photography, attach the marker, wash the gel twice, and soak in 0.25 M HC1 solution for 15 minutes. Then, soak in 0.4N NaOH solution for 10 minutes and gently shake during that time. Transfer to the filter in the usual manner, collect the filter after 4 hours, and wash twice with 2 X SSC. After the filter is sufficiently dried, perform baking (75 ° C, 3 hours). After baking, the filter is placed in a 0.1 X S SC / 0.1% SDS solution and treated at 65 ° C for 30 minutes. It is then immersed in 3 X SSC / 0.1% SDS solution. The obtained filter is placed in a beer bag together with the prehybridization solution and treated at 65 ° C. for 3 to 4 hours.
[0069] 次に、この中に32 P標識したプローブ DNAおよびハイブリダィゼーシヨン液を入れ、 65°Cで 12時間程度反応させる。ハイブリダィゼーシヨン終了後、適切な塩濃度、反 応温度および時間（例えば、 2 X SSC-0. 1%SDS溶液、室温、 10分間）のもとで、 フィルターを洗う。該フィルターをビニール袋に入れ、 2 X SSCを少量加え、密封し、 オートラジオグラフィーを行う。 [0069] Next, 32 P-labeled probe DNA and a hybridization solution are put into this and reacted at 65 ° C for about 12 hours. After hybridization, wash the filter under appropriate salt concentration, reaction temperature and time (eg, 2 X SSC-0. 1% SDS solution, room temperature, 10 minutes). Place the filter in a plastic bag, add a small amount of 2 X SSC, seal, and perform autoradiography.
[0070] 上記サザンブロット法により、マウスモノクローナル抗体の H鎖および L鎖を各々コ ードする再配列された VDJ遺伝子および VJ遺伝子を同定する。同定した DNA断片 を含む領域をショ糖密度勾配遠心にて分画し、ファージベクター（例えば、 Charon 4 A、 Charon 28、 λ EMBL3、 λ EMBL4等）に組み込み、該ファージベクターで大腸 菌（例えば、 LE392、 NM539等） を形質転換し、ゲノムライブラリーを作製する。その ゲノムライブラリーを適当なプローブ (H^J遺伝子、 L鎖（ K )J遺伝子等)を用いて、 例えばベントンデイビス法（サイエンス (Science)ゝ第 196卷、第 180〜第 182頁、 197 7年）に従って、プラークハイブリダィゼーシヨンを行い、再配列された VDJ遺伝子あ るいは VJ遺伝子を各々含むポジティブクローンを得る。得られたクローンの制限酵素 地図を作製し、塩基配列を決定し、 目的とする再配列された VH (VDJ)遺伝子ある!/ヽ は VL (VJ)遺伝子を含む遺伝子が得られて!/ヽることを確認する。 [0070] By the Southern blotting method described above, rearranged VDJ and VJ genes that code for the H and L chains of the mouse monoclonal antibody are identified. The region containing the identified DNA fragment is fractionated by sucrose density gradient centrifugation and incorporated into a phage vector (for example, Charon 4 A, Charon 28, λ EMBL3, λ EMBL4, etc.). LE392, NM539, etc.) to produce a genomic library. Using the genomic library with appropriate probes (H ^ J gene, L chain ( K ) J gene, etc.), for example, the Benton Davis method (Science ゝ 196, 180-182, 197 7), plaque hybridization is performed to obtain positive clones each containing a rearranged VDJ gene or VJ gene. A restriction enzyme map of the obtained clone is prepared, the nucleotide sequence is determined, and the target rearranged VH (VDJ) gene is! / ヽ is obtained as a gene containing the VL (VJ) gene! / ヽTo verify that.
[0071] 一方、キメラ化に用いるヒト CH遺伝子およびヒト CL遺伝子を別に単離する。例えば 、ヒ HgGl とのキメラ抗体を作製する場合には、 CH遺伝子である 1遺伝子と CL 遺伝子である C κ遺伝子を単離する。これらの遺伝子はマウス免疫 グロブリン遺伝 子とヒト免疫グロブリン遺伝子の塩基配列の高い相同性を利用してヒト C y 1遺伝子 およびヒト C κ遺伝子に相当するマウス C y 1遺伝子およびマウス C κ遺伝子をプロ ーブとして用い、ヒトゲノムライブラリーから単離することによって得ることができる。  On the other hand, the human CH gene and human CL gene used for chimerization are isolated separately. For example, when a chimeric antibody with HI HgGl is prepared, one gene that is a CH gene and a Cκ gene that is a CL gene are isolated. These genes utilize the high homology of the nucleotide sequences of the mouse immunoglobulin gene and the human immunoglobulin gene to produce the mouse Cy 1 gene and mouse C κ gene corresponding to the human Cy 1 gene and human C κ gene. And can be obtained by isolating from a human genomic library.
[0072] 具体的には、例えば、クローン Igl46 (Proc. Natl. Acad. Sci. USA,第 75卷、第 47 09〜第 4713頁、 1978年）からの 3kbの Hindlll— BamHI断片とクローン MEP10 ( Proc. Natl. Acad. Sci. USA,第 78卷、第 474〜第 478頁、 1981年）力らの 6. 8kbの EcoRI断片をプローブとして用い、ヒトのラムダ Charon 4Aの Haelll— Alulゲノムライ ブラリー（（Cell、第 15卷、第 1157〜第 1174頁、 1978年）中から、ヒト C κ遺伝子を 含み、ェンノヽンサ一領域を保持している DNA断片を単離する。また、ヒト 1遺伝 子は、 例えばヒト胎児肝細胞 DNAを Hindlllで切断し、ァガロースゲル電気泳動で 分画した後、 5. 9kbのバンドをえ 788に挿入し、前記のプローブを用いて単離する。  [0072] Specifically, for example, a 3 kb Hindlll-BamHI fragment from clone Igl46 (Proc. Natl. Acad. Sci. USA, 75th, 4709-4713, 1978) and clone MEP10 ( Proc. Natl. Acad. Sci. USA, Vol. 78, pp. 474-478, 1981) Using the 6.8 kb EcoRI fragment as a probe, the human lambda Charon 4A Haelll—Alul genomic library ( (Cell, 15th, 1157 to 1174, 1978) Isolate a DNA fragment containing the human Cκ gene and retaining the genomic region. For example, human fetal liver cell DNA is cleaved with Hindlll and fractionated by agarose gel electrophoresis, and then a 5.9 kb band is inserted into 788 and isolated using the above probe.
[0073] このようにして単離されたマウス VH遺伝子とマウス VL遺伝子、およびヒト CH遺伝 子とヒト CL遺伝子を用いて、プロモーター領域およびェンノヽンサ一領域等を考慮し ながらマウス VH遺伝子の下流にヒト CH遺伝子を、またマウス VL遺伝子の下流にヒト CL遺伝子を、適切な制限酵素および DNAリガーゼを用いて、例えば pSV2gptある いは pSV2neo等の発現ベクターに常法に従って組み込む。この際、マウス VH遺伝 子/ヒト CH遺伝子とマウス VL遺伝子/ヒト CL遺伝子のキメラ遺伝子は、一つの発現 ベクターに同時に配置されてもよいし、各々別個の発現ベクターに配置することもで きる。  [0073] Using the mouse VH gene and mouse VL gene isolated as described above, and the human CH gene and human CL gene, the promoter region and the enzyme region are taken into consideration and the downstream of the mouse VH gene. The human CH gene and the human CL gene downstream of the mouse VL gene are incorporated into an expression vector such as pSV2gpt or pSV2neo using an appropriate restriction enzyme and DNA ligase according to a conventional method. At this time, the chimeric gene of mouse VH gene / human CH gene and mouse VL gene / human CL gene may be arranged simultaneously in one expression vector, or may be arranged in separate expression vectors.
[0074] このようにして作製したキメラ遺伝子挿入発現ベクターを、例えば P3X63 'Ag8 ' 653 細胞あるいは SP210細胞といった、自らは抗体を産生していない骨髄腫細胞にプロト プラスト融合法、 DEAE—デキストラン法、リン酸カルシウム法あるいは電気穿孔法等 により導入する。形質転換細胞は、発現ベクターに導入された薬物耐性遺伝子に対 応する薬物含有培地中での培養により選別し、 目的とするキメラモノクローナル抗体 産生細胞を取得する。 [0074] The chimeric gene insertion expression vector thus prepared was used for prototyping myeloma cells that did not produce antibodies such as P3X63 'Ag8' 653 cells or SP210 cells. Introduced by the plast fusion method, DEAE-dextran method, calcium phosphate method or electroporation. Transformed cells are selected by culturing in a drug-containing medium corresponding to the drug resistance gene introduced into the expression vector to obtain the desired chimeric monoclonal antibody-producing cells.
[0075] このようにして選別された抗体産生細胞の培養上清中から目的のキメラモノクロ一 ナル抗体を取得する。  [0075] The target chimeric monoclonal antibody is obtained from the culture supernatant of the antibody-producing cells thus selected.
[0076] 本発明における「ヒト型モノクローナル抗体」は、マウス抗体の抗原結合部位 (CDR [0076] In the present invention, the "human monoclonal antibody" refers to an antigen binding site (CDR) of a mouse antibody.
；相補性決定領域)の遺伝子配列だけをヒト抗体遺伝子に移植 (CDRグラフティング; Transplantation of only the gene sequence of the complementarity determining region) into human antibody genes (CDR grafting
)した組換え抗体であり、例えば、特表平 4— 506458号公報および特開昭 62— 29For example, Japanese Patent Application Laid-Open No. 4-506458 and Japanese Patent Laid-Open No. 62-29.
6890号公報等に記載の方法を参照して作製することができる。具体的には、その超 可変領域の相補性決定領域の一部または全部が非ヒト哺乳動物 (マウス、ラット、ハ ムスター等）のモノクローナル抗体に由来する超可変領域の相補性決定領域であり、 その可変領域の枠組領域がヒトイムノグロブリン由来の可変領域の枠組領域であり、 かつその定常領域がヒトイムノグロブリン由来の定常領域であることを特徴とするヒト 型モノクローナル抗体を意味する。 It can be produced by referring to the method described in Japanese Patent No. 6890. Specifically, part or all of the complementarity determining region of the hypervariable region is the complementarity determining region of the hypervariable region derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.) It means a human monoclonal antibody characterized in that the framework region of the variable region is a framework region of a variable region derived from human immunoglobulin, and the constant region is a constant region derived from human immunoglobulin.
[0077] ここで、超可変領域の相補性決定領域とは、抗体の可変領域中の超可変領域に存 在し、抗原と相補的に直接結合する部位である 3つの領域 (Complementarity- deter mining residue ;CDR1, CDR2、 CDR3)を指し、また可変領域の枠組領域とは、該 3 つ相補性決定領域の前後に介在する比較的保存された 4つの領域 (Framework； FR 1、 FR2、 FR3、 FR4)を指す。  Here, the complementarity-determining regions of the hypervariable region are the three regions (Complementarity-deter mining) that are present in the hypervariable region of the variable region of the antibody and directly bind complementarily to the antigen. residue; CDR1, CDR2, CDR3) and the variable region framework region is four relatively conserved regions intervening before and after the three complementarity determining regions (Framework; FR 1, FR2, FR3, FR4).
[0078] 換言すれば、非ヒト哺乳動物由来のモノクローナル抗体の超可変領域の相補性決 定領域の一部または全部以外の全ての領域力 ヒトイムノグロブリンの対応領域と置 き代わったモノクローナル抗体を意味する。  [0078] In other words, a monoclonal antibody in which all regions other than part or all of the complementarity determining region of the hypervariable region of a monoclonal antibody derived from a non-human mammal are replaced with the corresponding region of human immunoglobulin. means.
[0079] ヒトイムノグロブリン由来の定常領域は、 IgG (IgGl, IgG2, IgG3, IgG4)、 IgM、 IgA 、 IgDおよび IgE等のアイソタイプにより各々固有のアミノ酸配列を有する力 本発明 におけるヒト型モノクローナル抗体の定常領域はいずれのアイソタイプに属するヒトイ ムノグログリンの定常領域であってもよい。好ましくは、ヒト IgGの定常領域である。ま た、ヒトイムノグロブリン由来の可変領域の枠組領域についても限定されるものではな い。 [0079] The constant region derived from human immunoglobulin has a unique amino acid sequence depending on isotypes such as IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD and IgE. The constant region may be a constant region of human immunoglobulin belonging to any isotype. Preferably, it is a constant region of human IgG. In addition, the framework region of the variable region derived from human immunoglobulin is not limited. Yes.
[0080] 本発明におけるヒト型モノクローナル抗体は、例えば以下のようにして製造すること ができる。しかしながら、そのような製造方法に限定されるものでないことは言うまでも ない。  [0080] The human monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
[0081] 例えば、マウスモノクローナル抗体に由来する組換ヒト型モノクローナル抗体は、特 表平 4 506458号公報および特開昭 62— 296890号公報等を参照して、遺伝子 工学的に作製することができる。即ち、マウスモノクローナル抗体を産生するハイプリ ドーマから、少なくとも 1つのマウス H鎖 CDR遺伝子と該マウス H鎖 CDR遺伝子に対 応する少なくとも 1つのマウス L鎖 CDR遺伝子を単離し、またヒトイムノグロブリン遺伝 子力も前記マウス H鎖 CDRに対応するヒト H鎖 CDR以外の全領域をコードするヒト H 鎖遺伝子と、前マウス L鎖 CDRに対応するヒト L鎖 CDR以外の全領域をコードするヒ ト L鎖遺伝子を単離する。  [0081] For example, a recombinant human monoclonal antibody derived from a mouse monoclonal antibody can be prepared by genetic engineering with reference to JP-A-4 506458 and JP-A-62-296890. . That is, at least one mouse heavy chain CDR gene and at least one mouse light chain CDR gene corresponding to the mouse heavy chain CDR gene are isolated from the hybridoma producing the mouse monoclonal antibody, and the human immunoglobulin gene power is also increased. A human H chain gene encoding the entire region other than the human H chain CDR corresponding to the mouse H chain CDR, and a human L chain gene encoding the entire region other than the human L chain CDR corresponding to the previous mouse L chain CDR. Isolate.
[0082] 単離した該マウス H鎖 CDR遺伝子と該ヒト H鎖遺伝子を発現可能なように適当な発 現ベクターに導入し、同様に該マウス L鎖 CDR遺伝子と該ヒト L鎖遺伝子を発現可能 なように適当なもう 1つの発現ベクターに導入する。または、該マウス H鎖 CDR遺伝 子 Zヒト H鎖遺伝子とマウス L鎖 CDR遺伝子 Zヒト L鎖遺伝子を同一の発現ベクター に発現可能なよう〖こ導入することもできる。このようにして作製された発現ベクターで 宿主細胞を形質転換することによりヒト型モノクローナル抗体産生形質転換細胞を得 、該形質転換細胞を培養することにより培養上清中から目的のヒト型モノクローナル 抗体を得る。  [0082] The isolated mouse H chain CDR gene and the human H chain gene can be introduced into an appropriate expression vector so that they can be expressed, and the mouse L chain CDR gene and the human L chain gene can be expressed in the same manner. Into another appropriate expression vector. Alternatively, the mouse H chain CDR gene Z human H chain gene and the mouse L chain CDR gene Z human L chain gene can also be introduced so that they can be expressed in the same expression vector. By transforming host cells with the expression vector thus prepared, human-type monoclonal antibody-producing transformed cells are obtained, and by culturing the transformed cells, the desired human-type monoclonal antibody is obtained from the culture supernatant. obtain.
[0083] 本発明における「ヒトモノクローナル抗体」とは、ィムノグロブリンを構成する H鎖の可 変領域および H鎖の定常領域並びに L鎖の可変領域および L鎖の定常領域を含む すべての領域がヒトイムノグロブリンをコードする遺伝子に由来するィムノグロブリンと なっている抗体を意味し、ヒト抗体遺伝子をマウス等の動物に導入して作製されたヒト 抗体産生トランスジエニック動物を用いて作製することができる。例えば、 Nature Gen etics, Vol.7, p.13- 21, 1994; Nature Genetics, Vol.15, p.146-156, 1997 ;特表平 4 504365号公報；特表平 7— 509137号公報； WO 94,25585号公報； Nature, Vol .368, p.856-859, 1994;および特表平 6— 500233号公報に記載の方法を参照して 作製することができる。具体的には、例えば、少なくともヒトイムノグロブリン遺伝子を マウス等のヒト以外の哺乳動物の遺伝子座中に組込むことにより作製されたトランス ジェニック動物を、抗原で免疫感作することにより、前述したポリクローナル抗体ある いはモノクローナル抗体の作製法と同様にして製造することができる。 [0083] The "human monoclonal antibody" in the present invention refers to all regions including the variable region of the heavy chain and the constant region of the heavy chain, and the variable region of the light chain and the constant region of the light chain that constitute immunoglobulin. An antibody that is an immunoglobulin derived from a gene encoding human immunoglobulin, and is produced using a human antibody-producing transgenic animal produced by introducing a human antibody gene into an animal such as a mouse. Can do. For example, Nature Genetics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; Tokuheihei 504365 Publication; Tokuheiheihei 7-509137 Publication; See the method described in WO 94,25585; Nature, Vol. 368, p.856-859, 1994; and JP-T 6-500233. Can be produced. Specifically, for example, the polyclonal antibody described above can be obtained by immunizing a transgenic animal prepared by incorporating at least a human immunoglobulin gene into the locus of a mammal other than human, such as a mouse, with an antigen. Alternatively, it can be produced in the same manner as the monoclonal antibody production method.
[0084] 例えば、ヒトモノクローナル抗体を産生するトランスジエニックマウスは、 Nature Gene tics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997 ;特表平 4— 50 4365号公報；特表平 7-509137号公報；日経サイエンス、 6月号、第 40〜第 50頁、 19 95年；国際出願公開 W094Z25585号公報； Nature, Vol.368, p.856- 859, 1994; および特表平 6— 500233号公報等に記載の方法に従って作製することができる。  [0084] For example, a transgenic mouse producing a human monoclonal antibody is described in Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; Hei 4-50 4365 gazette; JP 7-509137 gazette; Nikkei Science, June, pp. 40-50, 195; International application publication W094Z25585 gazette; Nature, Vol.368, p. 856-859, 1994; and JP-T 6-500233.
[0085] また、昨今開発された技術であるトランスジエニックなゥシゃブタのミルク中力もヒト 由来タンパク質を製造する方法を適用することも可能である（日経サイエンス、 1997 年 4月号、第 78頁乃至 84頁)。  [0085] Moreover, it is also possible to apply the method of producing human-derived protein to the milk neutrality of Transgenic Usa pig, which is a recently developed technology (Nikkei Science, April 1997, No. 1). 78 to 84).
[0086] 以下に単にモノクローナル抗体と記載した場合、上述のキメラモノクローナル抗体、 ヒト型モノクローナル抗体およびヒトモノクローナル抗体を含む場合もある。  [0086] In the following description, the term "monoclonal antibody" may include the above-described chimeric monoclonal antibody, human monoclonal antibody and human monoclonal antibody.
[0087] 本発明における「抗体のフラグメント」とは、前述のようなモノクローナル抗体、キメラ モノクローナル抗体、ヒト型モノクローナル抗体、およびヒトモノクローナル抗体の一部 分の領域を意味し、具体的には F(ab')2 、 Fab'、 Fab 、 Fv (variable fragment of antibo dy)、ジスルフイド結合 Fv、一本鎖抗体 (scFv)、およびこれらの重合体等が挙げられ る。 sFv、 dsFv (disulphide stabilised Fv)あ ヽ
Figure imgf000028_0001
(single domain antibody)等を 意味する（Exp. Opin. Ther. Patents,第 6卷，第 5号，第 441〜456頁， 1996年）。 [0087] The "antibody fragment" in the present invention means a partial region of the monoclonal antibody, chimeric monoclonal antibody, human monoclonal antibody, and human monoclonal antibody as described above, specifically F ( ab ′) 2, Fab ′, Fab, Fv (variable fragment of antibody), disulfide-binding Fv, single chain antibody (scFv), and polymers thereof. sFv, dsFv (disulphide stabilised Fv)
Figure imgf000028_0001
(single domain antibody) and the like (Exp. Opin. Ther. Patents, No. 6, No. 5, pp. 441-456, 1996).
[0088] ここで、「？(& ）2」ぉょび^& 」とは、ィムノグロブリン（モノクローナル抗体）を、蛋 白分解酵素であるペプシンあるいはノ パイン等で処理することにより製造され、ヒンジ 領域中の 2本の H鎖間に存在するジスルフイド結合の前後で消化されて生成される 抗体フラグメントを意味する。例えば、 IgGをパパインで処理すると、ヒンジ領域中の 2 本の H鎖間に存在するジスルフイド結合の上流で切断されて VL (L鎖可変領域）とじ L (L鎖定常領域)力 なる L鎖、および VH (H鎖可変領域)と CH y 1 (H鎖定常領域 中の γ 1領域）とからなる Η鎖フラグメントが C末端領域でジスルフイド結合により結合 した相同な 2つの抗体フラグメントを製造することができる。これら 2つの相同な抗体フ ラグメントを各々 Fab'という。また IgGをペプシンで処理すると、ヒンジ領域中の 2本の H鎖間に存在するジスルフイド結合の下流で切断されて前記 2つの Fab'がヒンジ領 域でつながったものよりやや大き!/、抗体フラグメントを製造することができる。この抗 体フラグメントを F(ab，）2と 、う。 [0088] Here, "? (&) 2" chobi ^ &"is produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin or nopain, This refers to an antibody fragment that is produced by digestion before and after the disulfide bond existing between two heavy chains in the hinge region. For example, when IgG is treated with papain, the L chain that is cleaved upstream of the disulfide bond existing between the two H chains in the hinge region and binds to the VL (L chain variable region) L (L chain constant region) force, And two homologous antibody fragments in which the long chain fragment consisting of VH (H chain variable region) and CH y 1 (γ 1 region in the H chain constant region) is linked by disulfide bond at the C-terminal region. it can. These two homologous antibody files Each ragment is called Fab '. In addition, when IgG is treated with pepsin, it is cleaved downstream of the disulfide bond existing between the two H chains in the hinge region, so that the two Fab's are slightly larger than those connected in the hinge region! /, Antibody fragment Can be manufactured. This antibody fragment is called F (ab,) 2.
[0089] [抗体の用途および医薬組成物]  [0089] [Use of antibody and pharmaceutical composition]
骨破壊の予防、診断または '治療蓉  Prevention, diagnosis or 'treatment of bone destruction
後記実施例に記載したように、コラーゲン誘導関節炎モデルにおいて認められる骨 破壊に対し、 CCL20に反応性を有する抗体が実際に治療効果を有することが確認 された。従って、 CCL20に反応性を有する抗体およびそのフラグメント（以下、単に「 CCL20に反応性を有する抗体」ということがある）は、骨破壊の治療および予防に有 用であり、骨破壊抑制剤として使用することができる。ここで「骨破壊」とは、骨破壊お よび軟骨破壊の ヽずれか一方または両方を含む意味で用いられる。  As described in Examples below, it was confirmed that an antibody having reactivity with CCL20 actually has a therapeutic effect on bone destruction observed in a collagen-induced arthritis model. Therefore, antibodies reactive to CCL20 and fragments thereof (hereinafter sometimes referred to simply as “antibodies reactive to CCL20”) are useful for the treatment and prevention of bone destruction, and are used as bone destruction inhibitors. can do. Here, the term “bone destruction” is used to include one or both of bone destruction and cartilage destruction.
なお、本発明の CCL20に反応性を有する抗体は、自己免疫疾患 (特に、関節リウ マチ)の予防、診断、または治療剤として使用できる。  The antibody reactive to CCL20 of the present invention can be used as an agent for preventing, diagnosing or treating autoimmune diseases (particularly rheumatoid arthritis).
[0090] ここで、「治療」とは、一般的に、所望の薬理学的効果および/または生理学的効果 を得ることを意味する。効果は、疾病および/または疾病に起因する悪影響の部分的 または完全な治癒という点では治療的である。本明細書において「治療」とは、哺乳 動物、特にヒトの疾病の任意の治療を含み、例えば以下の（a)および (b)の治療を含 む：  [0090] Here, "treatment" generally means obtaining a desired pharmacological and / or physiological effect. The effect is therapeutic in terms of partial or complete cure of the disease and / or the adverse effects caused by the disease. As used herein, “treatment” includes any treatment of diseases of mammals, particularly humans, and includes, for example, the following treatments (a) and (b):
ω疾病症状を阻害する、即ち、その進行を阻止または遅延すること；  inhibit omega disease symptoms, ie prevent or delay its progression;
(b)疾病症状を緩和すること、即ち、疾病または症状の後退、または症状の進行の逆 転を引き起こすこと。  (b) To alleviate disease symptoms, that is, cause regression of disease or symptoms, or reverse the progression of symptoms.
[0091] また、上記効果については、疾病および Zまたは症状を完全にまたは部分的に防 止する点では予防的であり（疾病または症状の素因を持ちうるが、まだ持っていると 診断されて 、な 、患者にぉ 、て、疾病または症状が起こることを予防することも含む) 、この意味においては、「治療」に「予防」が含まれる場合もある。  [0091] In addition, the above effects are preventive in terms of completely or partially preventing the disease and Z or symptoms (which may have a predisposition to the disease or symptoms but are still diagnosed as having In this sense, “treatment” may include “prevention” (including prevention of the occurrence of a disease or symptom in a patient).
[0092] 本発明の別の態様によれば、治療上有効な量の CCL20に反応性を有する抗体お よびそのフラグメントをそれを必要とする哺乳動物に投与する工程を含んでなる、骨 破壊の抑制方法および骨破壊の治療方法が提供される。 [0092] According to another aspect of the present invention, bone comprising the step of administering to a mammal in need thereof a therapeutically effective amount of a CCL20 reactive antibody and fragment thereof. Methods for inhibiting destruction and methods for treating bone destruction are provided.
[0093] 本発明の更に別の態様によれば、骨破壊抑制剤および骨破壊治療剤の製造のた めの、 CCL20に反応性を有する抗体およびそのフラグメントの使用が提供される。  [0093] According to still another aspect of the present invention, there is provided use of an antibody having CCL20 reactivity and a fragment thereof for the manufacture of a bone destruction inhibitor and a bone destruction therapeutic agent.
[0094] 細朐游走阻害剤  [0094] Fine streak inhibitor
後記実施例に記載したように、 CCL20に反応性を有する抗体により、 CCR6発現 細胞の CCL20に対する細胞遊走が顕著に抑制された。従って、 CCL20に反応性 を有する抗体は、細胞遊走阻害剤として用いることができる。  As described later in Examples, cell migration of CCR6-expressing cells to CCL20 was remarkably suppressed by an antibody having reactivity with CCL20. Therefore, an antibody having reactivity with CCL20 can be used as a cell migration inhibitor.
[0095] ここで「細胞遊走」とは、 CCL20タンパク質に対する CCR6発現細胞の細胞遊走を 意味する。本発明の細胞遊走阻害剤を用いて、 CCR6陽性の免疫細胞の CCL20タ ンパク質に対する細胞遊走を阻害することにより、免疫細胞の集積によって生じる免 疫応答、例えば、榭状細胞および T細胞の活性化、増殖、分化、サイト力イン'ケモカ イン産生を抑制することができる。  Here, “cell migration” means cell migration of CCR6-expressing cells with respect to CCL20 protein. By inhibiting cell migration of CCR6-positive immune cells to CCL20 protein using the cell migration inhibitor of the present invention, an immune response caused by immune cell accumulation, for example, activity of rod cells and T cells , Proliferation, differentiation, and site force-in chemokine production can be suppressed.
[0096] 医蓉組成物  [0096] Medical composition
CCL20に反応性を有する抗体は、骨破壊抑制剤、骨破壊治療剤、および細胞遊 走阻害剤として使用することができることから、この抗体を含むものは、医薬組成物と して使用することができる。  Since an antibody having reactivity to CCL20 can be used as a bone destruction inhibitor, a bone destruction therapeutic agent, and a cell migration inhibitor, those containing this antibody can be used as a pharmaceutical composition. it can.
[0097] 本発明による抗体の投与形態は、特に制限されず、経口投与、非経口投与 (例え ば静脈注射、筋肉注射、皮下投与、直腸投与、経皮投与、局所投与)のいずれかの 投与経路でヒトを含む哺乳類に投与することができるが、非経口投与、特に静脈注射 、が好ましい。  [0097] The administration mode of the antibody according to the present invention is not particularly limited, and is any one of oral administration and parenteral administration (for example, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, topical administration). Although it can be administered to mammals including humans by the route, parenteral administration, particularly intravenous injection, is preferred.
[0098] 経口投与および非経口投与のための剤形およびその製造方法は当業者に周知で あり、本発明による抗体を、薬学的に許容される坦体等と混合等することにより、常法 に従って製造することができる。  [0098] Dosage forms for oral administration and parenteral administration and methods for producing the same are well known to those skilled in the art. By mixing the antibody according to the present invention with a pharmaceutically acceptable carrier or the like, a conventional method is obtained. Can be manufactured according to.
[0099] 非経口投与のための剤型は、注射用製剤 (例えば、点滴注射剤、静脈注射剤、筋 肉注射剤、皮下注射剤、皮内注射剤）、外用剤 (例えば、軟膏剤、パップ剤、ローショ ン剤）、坐剤吸入剤、眼剤、眼軟膏剤、点鼻剤、点耳剤、リボソーム剤等が挙げられ る。  [0099] Dosage forms for parenteral administration include injectable preparations (eg, instillation, intravenous injection, muscle injection, subcutaneous injection, intradermal injection), and external preparations (eg, ointment, And suppositories, ophthalmic preparations, eye ointments, nasal drops, ear drops, and ribosomes.
[0100] 例えば、注射用製剤は、通常、本発明による抗体を注射用蒸留水に溶解して調製 するが、必要に応じて溶解補助剤、緩衝剤、 pH調整剤、等張化剤、無痛化剤、保存 剤、安定化剤等を添加することができる。また、用事調製用の凍結乾燥製剤とするこ とちでさる。 [0100] For example, a preparation for injection is usually prepared by dissolving the antibody according to the present invention in distilled water for injection. However, solubilizers, buffers, pH adjusters, isotonic agents, soothing agents, preservatives, stabilizers and the like can be added as necessary. It can also be made into a freeze-dried preparation for business preparation.
[0101] 経口投与のための剤型は、固体または液体の剤型、具体的には錠剤、被覆錠剤、 丸剤、細粒剤、顆粒剤、散剤、カプセル剤、シロップ剤、乳剤、懸濁剤、注射剤、トロ ーチ剤等が挙げられる。  [0101] The dosage forms for oral administration are solid or liquid dosage forms, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions. Agents, injections, troches and the like.
[0102] 本発明による医薬組成物は、治療上有効な他の薬剤を更に含有していてもよぐま た、必要に応じて血流促進剤、殺菌剤、消炎剤、細胞賦活剤、ビタミン類、アミノ酸、 保湿剤、角質溶解剤等の成分を配合することもできる。このときの有効成分の担体に 対する割合は、 1〜90重量％の間で変動され得る。  [0102] The pharmaceutical composition according to the present invention may further contain other therapeutically effective drugs, and if necessary, blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins Ingredients such as amino acids, humectants and keratolytic agents can also be added. The ratio of the active ingredient to the carrier at this time can be varied between 1 to 90% by weight.
[0103] これらの製剤の製剤化に用いる担体には、例えば通常用いられる賦形剤、結合剤 、崩壊剤、滑沢剤、着色剤、矯味矯臭剤や、必要により安定化剤、乳化剤、吸収促 進剤、界面活性剤、 pH調整剤、防腐剤、抗酸化剤、増量剤、湿潤化剤、表面活性化 剤、分散剤、緩衝剤、保存剤、溶解補助剤、無痛化剤等を使用することができ、一般 に医薬品製剤の原料として用いられる成分を配合して常法により製剤化することが可 能である。使用可能な無毒性のこれらの成分としては、例えば大豆油、牛脂、合成グ リセライド等の動植物油；例えば流動パラフィン、スクヮラン、固形パラフィン等の炭化 水素；例えばミリスチン酸オタチルドデシル、ミリスチン酸イソプロピル等のエステル油 ；例えばセトステアリルアルコール、ベへ-ルアルコール等の高級アルコール；シリコ ン榭脂；シリコン油；例えばポリオキシエチレン脂肪酸エステル、ソルビタン脂肪酸ェ ステル、グリセリン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、ポ リオキシエチレン硬化ひまし油、ポリオキシエチレン ポリオキシプロピレンブロックコ ポリマー等の界面活性剤；例えばヒドロキシェチルセルロース、ポリアクリル酸、カル ボキシビニルポリマー、ポリエチレングリコール、ポリビニルピロリドン、メチルセルロー ス等の水溶性高分子；例えばエタノール、イソプロパノール等の低級アルコール；例 えばグリセリン、プロピレングリコール、ジプロピレングリコール、ソルビトール、ポリエ チレングリコール等の多価アルコール（ポリオール）；例えばグルコース、ショ糖等の糖 ；例えば無水ケィ酸、ケィ酸アルミニウムマグネシウム、ケィ酸アルミニウム等の無機 粉体;塩ィ匕ナトリウム、リン酸ナトリウム等の無機塩;精製水等が挙げられる。 [0103] Carriers used for formulating these preparations include, for example, commonly used excipients, binders, disintegrants, lubricants, colorants, flavoring agents, and if necessary, stabilizers, emulsifiers, absorptions. Use accelerators, surfactants, pH adjusters, preservatives, antioxidants, extenders, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, etc. In general, it is possible to formulate by a conventional method by mixing ingredients used as raw materials for pharmaceutical preparations. Non-toxic components that can be used include, for example, animal and vegetable oils such as soybean oil, beef tallow and synthetic glycerides; hydrocarbons such as liquid paraffin, squalene and solid paraffin; for example, otatildodecyl myristate, isopropyl myristate, etc. Ester oils of higher alcohols such as cetostearyl alcohol and beh- ol alcohol; silicone oils; silicone oils such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, Surfactants such as polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene block copolymer; for example, hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol Water-soluble polymers such as ethanol, polyvinyl pyrrolidone and methyl cellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols (polyols) such as glycerin, propylene glycol, dipropylene glycol, sorbitol and polyethylene glycol; Sugars such as sucrose; inorganics such as caustic anhydride, aluminum magnesium silicate, aluminum silicate, etc. Powder; inorganic salt such as sodium chloride sodium phosphate and sodium phosphate; purified water and the like.
[0104] 賦形剤としては、例えば乳糖、果糖、コーンスターチ、白糖、ブドウ糖、マン-トール 、ソルビット、結晶セルロース、二酸化ケイ素等が、結合剤としては、例えばポリビニル ァノレコーノレ、ポリビニノレエーテノレ、メチノレセノレロース、ェチノレセノレロース、アラビアゴ ム、トラガント、ゼラチン、シェラック、ヒドロキシプロピルメチルセルロース、ヒドロキシプ 口ピルセルロース、ポリビニルピロリドン、ポリプロピレングリコール.ポリオキシエチレン •ブロックポリマー、メダルミン等力 崩壊剤としては、例えば澱粉、寒天、ゼラチン末、 結晶セルロース、炭酸カルシウム、炭酸水素ナトリウム、クェン酸カルシウム、デキスト リン、ぺクチン、カルボキシメチルセルロース 'カルシウム等力 滑沢剤としては、例え ばステアリン酸マグネシウム、タルク、ポリエチレングリコール、シリカ、硬化植物油等 力 着色剤としては医薬品に添加することが許可されているものが、矯味矯臭剤とし ては、ココア末、ノヽッ力脳、芳香散、ハツ力油、竜脳、桂皮末等が、ぞれぞれ用いられ る。上記の成分は、その塩またはその水和物であってもよい。  [0104] Examples of the excipient include lactose, fructose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, silicon dioxide, and the like, and examples of the binder include polyvinyl alcohole, polyvinylinoleate, methyl. Noresenorelose, ethinoresenorelose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropyl pillcellulose, polyvinylpyrrolidone, polypropylene glycol.Polyoxyethylene block polymer, medalmin, etc. For example, starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, calcium quenate, dextrin, pectin, carboxymethyl cellulose 'calcium, etc. For example, magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, etc. Powers that are permitted to be added to pharmaceuticals are flavourants. , Hatsu power oil, Borneolum, cinnamon powder, etc. are used. The above component may be a salt thereof or a hydrate thereof.
[0105] 例えば経口製剤は、有効成分に、賦形剤、さらに必要に応じて例えば結合剤、崩 壊剤、滑沢剤、着色剤、矯味矯臭剤等を加えた後、常法により例えば散剤、細粒剤、 顆粒剤、錠剤、被覆錠剤、カプセル剤等とする。錠剤'顆粒剤の場合には、例えば糖 衣、その他必要により適宜コーティングすることはもちろん差支えない。シロップ剤や 注射用製剤等の場合は、例えば pH調整剤、溶解剤、等張化剤等と、必要に応じて 溶解補助剤、安定化剤等とを加えて、常法により製剤化する。また、外用剤の場合は 、特に製法が限定されず、常法により製造することができる。使用する基剤原料として は、医薬品、医薬部外品、化粧品等に通常使用される各種原料を用いることが可能 であり、例えば動植物油、鉱物油、エステル油、ワックス類、高級アルコール類、脂肪 酸類、シリコン油、界面活性剤、リン脂質類、アルコール類、多価アルコール類、水溶 性高分子類、粘土鉱物類、精製水等の原料が挙げられ、必要に応じ、 pH調整剤、抗 酸化剤、キレート剤、防腐防黴剤、着色料、香料等を添加することができる。さらに、 必要に応じて血流促進剤、殺菌剤、消炎剤、細胞賦活剤、ビタミン類、アミノ酸、保湿 剤、角質溶解剤等の成分を配合することもできる。この時の有効成分の担体に対す る割合は、 1〜90重量％の間で変動され得る。本発明に使用する化合物類、本発明 に使用するペプチド類または本発明に使用するポリヌクレオチド類を前記治療に使 用する場合は、少なくとも 90%以上、好ましくは 95%以上、より好ましくは 98%以上 、さらに好ましくは 99%以上に精製されたものを使用するのが好ましい。 [0105] For example, an oral preparation is prepared by adding an excipient, and further, for example, a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, and the like to an active ingredient, and then, for example, a powder by a conventional method. , Fine granules, granules, tablets, coated tablets, capsules, etc. In the case of tablets and granules, for example, sugar coating or other appropriate coating may be used if necessary. In the case of syrups or injectable preparations, for example, pH adjusters, solubilizers, tonicity agents, etc., and if necessary, solubilizing agents, stabilizers, etc., are added and formulated in conventional manner. In the case of an external preparation, the production method is not particularly limited, and it can be produced by a conventional method. As the base material to be used, various raw materials usually used for pharmaceuticals, quasi drugs, cosmetics, etc. can be used. For example, animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fats Examples include raw materials such as acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric alcohols, water-soluble polymers, clay minerals, purified water, and pH adjusters and antioxidants as necessary. Agents, chelating agents, antiseptic / antifungal agents, coloring agents, fragrances and the like can be added. Furthermore, components such as blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, and keratolytic agents can be blended as necessary. The ratio of the active ingredient to the carrier at this time can be varied between 1 to 90% by weight. Compounds used in the present invention, the present invention When the peptides used in the present invention or the polynucleotides used in the present invention are used for the treatment, they are purified to at least 90% or more, preferably 95% or more, more preferably 98% or more, and further preferably 99% or more. It is preferable to use those prepared.
[0106] 本発明による抗体の投与量は、例えば、投与経路、疾患の種類、症状の程度、患 者の年齢、性別、体重、疾患の重篤度、薬物動態および毒物学的特徴等の薬理学 的知見、薬物送達系の利用の有無、並びに他の薬物の組合せの一部として投与さ れるか、など様々な因子を元に、臨床医師により決定することができるが、通常、成人 (体重 60kg)あたり、経口投与では 1〜5000 gZ曰、好ましくは 10〜2000 gZ 日、さらに好ましくは 50〜2000 gZ日を、注射投与では 1〜5000 g/日、好ま しくは 5〜2000 gZ日、さら〖こ好ましくは 50〜2000 gZ日を、 1回または数回に 分けて投与することができる。小児に投与される場合は、用量は成人に投与される量 よりも少ない可能性がある。実際に用いられる投与法は、臨床医師の判断により大幅 に変動することもあり、上記の投与範囲力 逸脱することがある。  [0106] The dose of the antibody according to the present invention is, for example, a drug such as administration route, disease type, symptom severity, patient age, sex, body weight, disease severity, pharmacokinetics and toxicological characteristics. It can be determined by the clinician based on a variety of factors, including physical findings, whether drug delivery systems are used, and whether they are administered as part of a combination of other drugs. Per 60 kg), 1 to 5000 gZ5000 for oral administration, preferably 10 to 2000 gZ days, more preferably 50 to 2000 gZ days, 1 to 5000 g / day, preferably 5 to 2000 gZ days for injection administration, More preferably, 50 to 2000 gZ days can be administered in one or several divided doses. When administered to children, the dose may be less than that administered to adults. The administration method actually used may vary greatly depending on the judgment of the clinician, and may deviate from the above-mentioned dose range.
[0107] スクリーニング方法  [0107] Screening method
本発明によれば、 CCR6を含む細胞膜またはそれを含む細胞と、 CCL20とを用い ることを特徴とする、 CCR6と CCL20との相互作用を変化させる物質のスクリーニン グ方法が提供される。好ましくは、この方法は、被検物質存在下および被検物質非 存在下のそれぞれにおいて、 CCR6を含む細胞膜またはそれを含む細胞と、 CCL2 0とを接触させ、次いで、細胞刺激活性を測定して、被検物質存在下の場合と被検 物質非存在下の場合とにおける測定結果を比較することを含んでなる。  According to the present invention, there is provided a method for screening a substance that alters the interaction between CCR6 and CCL20, which comprises using CCL20 and a cell membrane containing CCR6 or a cell containing the same. Preferably, this method comprises contacting CCL20 with a cell membrane containing CCR6 or a cell containing the same in the presence of a test substance and in the absence of the test substance, and then measuring cell stimulating activity. And comparing the measurement results in the presence of the test substance and in the absence of the test substance.
[0108] 本発明のより好ましい態様によれば、前記方法は、被検物質存在下の場合と被検 物質非存在下の場合との間で、結果に相違が出た場合に、その物質を CCR6を介し た細胞刺激活性を変化させる物質であると決定する工程をさらに含んでなる。  [0108] According to a more preferred embodiment of the present invention, the method determines the substance when there is a difference in the result between the presence of the test substance and the absence of the test substance. It further comprises the step of determining that it is a substance that changes the cell stimulating activity via CCR6.
[0109] このスクリーニング方法によれば、 CCR6の機能 (遊走)を促進もしくは阻害する能 力を区別して被検物質 (または被検化合物）をスクリーニングすることができる。すな わち、このスクリーニング方法では、 CCL20と CCR6との相互作用（結合能）を変化さ せる物質をスクリーニングすることができ、具体的には、 CCR6の活性化に影響を与 える化合物や CCR6と CCL20の結合を阻害する化合物を、より具体的には、 CCR6 を介した細胞刺激活性を変化させる物質、さらに具体的には、 CCR6の機能を促進 する物質 (ァゴ二スト)ある!、は CCR6の機能 (遊走)を阻害する物質 (アンタゴ-スト） を、スクリーニングすることができる。 [0109] According to this screening method, a test substance (or test compound) can be screened by distinguishing the ability to promote or inhibit the function (migration) of CCR6. In other words, this screening method can screen for substances that alter the interaction (binding ability) between CCL20 and CCR6. Specifically, compounds that affect the activation of CCR6 and CCR6 More specifically, compounds that inhibit the binding of CCL20 to CCR6 There is a substance that alters cell-stimulating activity mediated by glycine, more specifically, a substance that promotes the function of CCR6 (agonist) !, a substance that inhibits the function (migration) of CCR6 (antagonist) Can be screened.
[0110] 本発明において、ァゴニストは CCR6の機能 (遊走)を促進するため、骨破壊の疾 患モデルを誘発することとなるので、当該疾患モデルの調製に使用することができる [0110] In the present invention, the agonist promotes the function (migration) of CCR6, and therefore induces a disease model of bone destruction, and thus can be used for the preparation of the disease model.
[0111] 本発明は、 CCR6発現細胞の CCL20に対する遊走を抑制する抗体を作製し、該 タンパク質に対する抗体が骨破壊の所見が認められるコラーゲン誘導関節炎モデル に対して治療効果 (特に炎症抑制、骨破壊抑制）を有することを見出したことから、 C CR6の機能 (遊走)を阻害する物質 (アンタゴニスト）を、スクリーニングすることが好ま しい。アンタゴ-ストは CCR6の機能 (遊走)を抑制するため、骨破壊の抑制や骨破 壊の治療に有用である。 [0111] The present invention produces an antibody that suppresses the migration of CCR6-expressing cells to CCL20, and the antibody against the protein has a therapeutic effect on a collagen-induced arthritis model in which bone destruction is observed (especially inflammation suppression, bone destruction It is preferable to screen for substances (antagonists) that inhibit the function (migration) of CCR6. Antagost suppresses the function (migration) of CCR6 and is therefore useful for suppressing bone destruction and treating bone destruction.
[0112] したがって、被検物質非存在下における細胞刺激活性に対して、被検物質存在下 における細胞刺激活性が上昇する場合 (好ましくは、約 1. 1倍上昇する場合、より好 ましくは、約 1. 5倍上昇する場合）または CCL20の非存在下において CCL20の代 わりに細胞刺激活性が上昇する場合 (好ましくは、約 1. 1倍上昇する場合、より好ま しくは、約 1. 5倍上昇する場合）には、その被検物質は、 CCR6の機能を促進する物 質 (CCR6ァゴニスト）と決定することができる。被検物質非存在下における細胞刺激 活性に対して、被検物質存在下における細胞刺激活性が低下する場合 (好ましくは 、約 0. 9倍に低下する場合、より好ましくは、約 0. 5倍に低下する場合）には、その被 検物質は、 CCR6の機能を阻害する物質 (CCR6アンタゴ-スト）と決定することがで きる。  [0112] Therefore, when the cell stimulating activity in the presence of the test substance increases compared to the cell stimulating activity in the absence of the test substance (preferably, when the cell stimulating activity increases by about 1.1 times, more preferably , About 1.5-fold increase) or when cell-stimulating activity increases instead of CCL20 in the absence of CCL20 (preferably about 1.1-fold increase, more preferably about 1.5 In the case of a doubling), the test substance can be determined as a substance that promotes the function of CCR6 (CCR6 agonist). When the cell stimulation activity in the presence of the test substance decreases compared to the cell stimulation activity in the absence of the test substance (preferably, when it decreases by about 0.9 times, more preferably about 0.5 times The test substance can be determined to be a substance that inhibits the function of CCR6 (CCR6 antagonist).
[0113] 本発明においては、例えば CCR6の活性ィ匕によって、アデ二ル酸シクラ一ゼの活 性抑制により減少する細胞内 cAMPの濃度、あるいは上昇する細胞内カルシウム濃 度を公知の手法で測定すればよぐ CCR6の機能を促進もしくは阻害する能力を区 別して化合物をスクリーニングすることができる。この態様は、 CCR6に CCL20が作 用することにより生じる細胞内シグナル伝達、すなわち、 CCR6の細胞刺激活性の一 つであるアデ二ル酸シクラーゼの活性抑制および細胞内カルシウム濃度の上昇作用 を利用するものである。 [0113] In the present invention, for example, by measuring the activity of CCR6, the concentration of intracellular cAMP that decreases due to inhibition of adenylate cyclase activity, or the concentration of intracellular calcium that increases, is measured by a known method. The compound can be screened by distinguishing its ability to promote or inhibit the function of CCR6. This mode is the intracellular signal transduction caused by the action of CCL20 on CCR6, that is, the activity of adenylate cyclase, one of the cell stimulating activities of CCR6, and the effect of increasing intracellular calcium concentration. Is to be used.
[0114] 例えば CCR6を細胞膜上に発現 (好ましくは、 CCR6を含む発現ベクターを導入し 過剰に発現)した哺乳動物由来細胞 (例えば HEK— 293細胞もしくは CHO細胞）に CCL20を作用させると、細胞内の cAMPの濃度が減少、および細胞内 Ca2+濃度が 上昇する。 [0114] For example, when CCL20 is allowed to act on a mammal-derived cell (for example, HEK-293 cell or CHO cell) in which CCR6 is expressed on a cell membrane (preferably, an expression vector containing CCR6 is introduced and overexpressed) CAMP concentration decreases and intracellular Ca 2+ concentration increases.
[0115] CCR6の機能を促進する化合物をスクリーニングする場合には、このスクリーニング 系で CCR6を介して細胞刺激活性を惹起しうる物質 (例えば CCL20)に代わり、被検 物質を単独で接触させて細胞内の cAMPの濃度が減少する、あるいは細胞内 Ca2+ 濃度が上昇する化合物を選択するか、または CCR6の機能を促進する化合物をスク リー-ングする場合には、このスクリーニング系で CCR6を介して細胞刺激活性を惹 起しうる物質 (例えば CCL20)にカ卩えて、被検物質を接触させて細胞内の cAMPの 濃度がさらに減少する、あるいは細胞内 Ca2+濃度がさらに上昇する化合物を選択す ると良い。 [0115] When screening for a compound that promotes the function of CCR6, in this screening system, instead of a substance that can induce cell stimulating activity via CCR6 (for example, CCL20), a test substance is contacted alone and the cell is contacted. When screening for compounds that reduce intracellular cAMP concentration or increase intracellular Ca 2+ concentration or to screen for compounds that promote CCR6 function, this screening system may be mediated by CCR6. In this way, compounds that can induce cell stimulating activity (for example, CCL20) are contacted with a test substance to further reduce the intracellular cAMP concentration or increase the intracellular Ca 2+ concentration. Choose it.
[0116] CCR6の機能を阻害する化合物をスクリーニングする場合には、アデ二ル酸シクラ ーゼ活性化剤、 CCL20、および被検物質をスクリーニング用細胞に添加するとよい 。アデ-ル酸シクラーゼ活性化剤を単独で添加した場合に比べて、 CCL20の作用 で cAMPの生成量が減少するが、被検物質力 SCCL20の作用に拮抗する場合には、 cAMP生成量の減少を抑制する。または、 CCR6の機能を抑制する化合物をスクリ 一二ングする場合には、このスクリーニング系で CCR6を介して細胞刺激活性を惹起 しうる物質 (例えば CCL20)に加えて、被検物質を接触させて細胞内 Ca2+濃度の上 昇を抑制する化合物を選択する。このときには、前記被検物質は CCR6の機能を阻 害する化合物として選択できる。 [0116] When screening for a compound that inhibits the function of CCR6, an adenylate cyclase activator, CCL20, and a test substance may be added to the screening cells. The amount of cAMP produced is reduced by the action of CCL20 compared to the case where the adenylate cyclase activator is added alone, but the amount of cAMP produced is reduced when antagonizing the action of test substance SCCL20. Suppress. Alternatively, when screening for a compound that suppresses the function of CCR6, in addition to a substance that can induce cell stimulating activity via CCR6 in this screening system (for example, CCL20), a test substance is contacted. Select compounds that suppress the increase in intracellular Ca 2+ concentration. In this case, the test substance can be selected as a compound that inhibits the function of CCR6.
[0117] 細胞内 cAMP量を測定する方法としては、例えばィムノアッセィ等が挙げられるが 、市販の cAMP定量キットを使用することもできる。  [0117] Examples of the method for measuring the amount of intracellular cAMP include immunoassay and the like, and a commercially available cAMP quantification kit can also be used.
[0118] 本発明の好ま 、態様によれば、細胞刺激活性の測定は、シグナル伝達物質生成 によるレポーター遺伝子の翻訳'転写量の変化を検出するレポーターアツセィ系によ り測定する力、または、細胞内カルシウムイオン遊離、アデ二ル酸シクラーゼの活性 ィ匕、細胞内 cAMP生成、細胞内 cGMP生成、ァラキドン酸遊離、アセチルコリン遊離 、イノシトールリン酸産生、細胞膜電位変動、細胞内タンパク質のリン酸化もしくは活 性化、 pHの低下変動活性、 MAPキナーゼのリン酸化もしくは活性化、 c—fosの活 性化、グリセロール生成活性、脂肪分解活性、および、副腎皮質ホルモン分泌活性 力 なる群より選択されるパラメーターを測定することによって行うことができる。 [0118] According to a preferred embodiment of the present invention, the measurement of the cell stimulating activity is carried out by measuring the reporter gene translation by the generation of a signal transducing substance 'the force measured by a reporter assay system that detects a change in the transcription amount, or Intracellular calcium ion release, adenylate cyclase activity, intracellular cAMP production, intracellular cGMP production, arachidonic acid release, acetylcholine release , Inositol phosphate production, cell membrane potential fluctuation, phosphorylation or activation of intracellular protein, pH lowering fluctuation activity, phosphorylation or activation of MAP kinase, c-fos activation, glycerol production activity, lipolysis Activity and adrenocortical hormone secretion activity can be performed by measuring a parameter selected from the group consisting of:
[0119] 本発明においては、また、 CCR6を細胞膜上に発現 (好ましくは、 CCR6を含む発 現ベクターを導入し過剰に発現)し、しカゝも、 cAMP応答配列（CRE)が 5'上流に位 置するレポーター遺伝子 (例えばアルカリフォスファターゼ遺伝子、ルシフェラーゼ遺 伝子、ベータラクタマーゼ遺伝子、ニトロレダクターゼ遺伝子、クロラムフエ-コールァ セチルトランスフェラーゼ遺伝子、ベータガラクトシダーゼ遺伝子等、または GFP (Gr een Fluorescent Protein)等の蛍光タンパク質遺伝子等）を含有する細胞（以下、「ス クリーニング用細胞」と称することもある）を用いることにより、 CCR6の機能を促進もし くは阻害する能力を区別して化合物をスクリーニングすることができる。この場合は、 前述の cAMPの生成が増加するとその結果、前記スクリーニング用細胞に導入され ている CREをプロモーター領域に有するレポーター遺伝子の転写が促進されること を利用している。 [0119] In the present invention, CCR6 is also expressed on the cell membrane (preferably, an expression vector containing CCR6 is introduced and overexpressed), and the cAMP response element (CRE) is 5 'upstream. A reporter gene (for example, alkaline phosphatase gene, luciferase gene, beta-lactamase gene, nitroreductase gene, chloramphene-coal cetyltransferase gene, beta-galactosidase gene, or fluorescent protein gene such as GFP (Green Fluorescent Protein)) Etc.) (hereinafter also referred to as “screening cells”), compounds can be screened by distinguishing the ability to promote or inhibit the function of CCR6. In this case, it is utilized that the generation of the above-mentioned cAMP increases, and as a result, the transcription of the reporter gene having the CRE introduced into the screening cell in the promoter region is promoted.
[0120] すなわち、前記スクリーニング用細胞に導入されている CREは、細胞内の cAMP の濃度が上昇すると発現が亢進する遺伝子群 (cAMP誘導性遺伝子)の転写調節 領域に共通して存在する塩基配列である。したがって、アデ二ル酸シクラーゼの活性 ィ匕剤（例えば FSK)をスクリーニング用細胞に添加すると、細胞内の cAMPの濃度が 上昇し、その結果、 CREの下流に位置するレポーター遺伝子の発現量が増加する。 さらにこのレポーター遺伝子は、細胞内 Ca2+濃度の上昇によるシグナル伝達系によ つても発現量が増加する。レポーター遺伝子産物の発現量は、レポーター遺伝子産 物と反応し基質力 生成した発光物質の量に由来する発光を測定することにより、も しくはレポーター遺伝子として産生された蛍光タンパク質由来の蛍光を測定すること により、容易に測定することが可能である。 [0120] That is, the CRE introduced into the screening cell is a nucleotide sequence that is commonly present in the transcriptional regulatory region of a gene group (cAMP-inducible gene) whose expression increases when the intracellular cAMP concentration increases. It is. Therefore, when an adenylate cyclase active agent (eg, FSK) is added to screening cells, the intracellular cAMP concentration increases, resulting in an increase in the expression level of the reporter gene located downstream of the CRE. To do. Furthermore, the expression level of this reporter gene is also increased by a signal transduction system due to an increase in intracellular Ca 2+ concentration. The expression level of the reporter gene product is measured by measuring the luminescence derived from the amount of the luminescent substance that reacts with the reporter gene product and generates substrate power, or the fluorescence derived from the fluorescent protein produced as the reporter gene. Therefore, it is possible to measure easily.
[0121] 被検物質による作用が、 CCR6を介した作用である力否かは、簡単に確認すること ができる。例えばスクリーニング用細胞 (すなわち、 CCR6を細胞膜上に発現し、しか も、 CREが 5'上流に位置するレポーター遺伝子を含有する細胞）を用いた前記試験 と並行して、コントロール用細胞（例えば CREが 5'上流に位置するレポーター遺伝 子を含有するものの、 CCR6を細胞膜上に発現して 、な 、細胞）を用いて同様の試 験を実施する。その結果、前記被検物質による作用が、 CCR6に対する結合による 作用でない場合には、スクリーニング用細胞およびコントロール用細胞でレポーター 遺伝子産物の発現量に関して同じ現象が観察されるのに対して、前記被検物質によ る作用が、 CCR6に対する結合による作用である場合には、スクリーニング用細胞と コントロール用細胞とでレポーター遺伝子産物の発現量に関して異なる現象が観察 される。 [0121] It is possible to easily confirm whether the action of the test substance is a force through CCR6. For example, the above test using a screening cell (ie, a cell that expresses CCR6 on the cell membrane and contains a reporter gene in which CRE is located 5 ′ upstream). In parallel, a similar test is performed using control cells (for example, cells that contain a reporter gene in which CRE is located 5 ′ upstream, but do not express CCR6 on the cell membrane). As a result, when the effect of the test substance is not the effect of binding to CCR6, the same phenomenon is observed with respect to the expression level of the reporter gene product in the screening cell and the control cell, whereas the test substance When the effect of the substance is due to the binding to CCR6, a different phenomenon is observed regarding the expression level of the reporter gene product between the screening cell and the control cell.
[0122] 本発明の別の態様の CCL20と CCR6との相互作用（結合能）を変化させる物質を スクリーニング方法は、被検物質存在下および被検物質非存在下のそれぞれにお ヽ て、 CCR6を含む細胞膜またはそれを含む細胞と、 CCL20とを接触させ、次いで、 C CR6を含む細胞膜またはそれを含む細胞への CCL20の結合量を測定して、被検物 質存在下の場合と被検物質非存在下の場合とを比較することを含んでなる。このスク リー-ング方法によれば、 CCR6の機能を促進もしくは阻害する能力を区別せずに 被検化合物をスクリーニングすることができる。すなわち、この態様の方法が適用可 能な場合、この方法により、 CCL20と CCR6との相互作用を変化させる物質をスクリ 一-ングすることができ、具体的には、 CCL20の CCR6への結合性を変化させる化 合物を、より具体的には、 CCR6の機能を促進もしくは阻害する能力を有する化合物 を、スクリーニングすることができる。  [0122] In another embodiment of the present invention, the method for screening for a substance that alters the interaction (binding ability) between CCL20 and CCR6 comprises: CCR6 in the presence of the test substance and in the absence of the test substance. A cell membrane containing or a cell containing the same and CCL20, and then measuring the amount of CCL20 bound to the cell membrane containing CCR6 or the cell containing the same, in the presence of the test substance Comparing to the absence of the substance. According to this screening method, a test compound can be screened without distinguishing the ability to promote or inhibit the function of CCR6. That is, when the method of this embodiment is applicable, it is possible to screen a substance that changes the interaction between CCL20 and CCR6. Specifically, the binding property of CCL20 to CCR6 can be screened. More specifically, a compound having the ability to promote or inhibit the function of CCR6 can be screened.
[0123] 具体的には、被検物質非存在下および被検物質存在下の各条件下において、 C CR6と、標識した CCL20とを接触させ、前記条件下における CCR6への CCL20の 特異的結合量を比較することにより、 CCR6の機能を促進もしくは阻害する能力を区 別せずに化合物をスクリーニングすることができる。すなわち、被検物質非存在下に おける CCR6への CCL20の特異的結合量に対して、被検物質存在下における前記 特異的結合量が低下する場合には、その被検物質は、 CCL20と CCR6との相互作 用を変化させる物質であると、具体的には、 CCL20の CCR6への結合性を変化させ る化合物であると、より具体的には、 CCR6ァゴ-ストあるいは CCR6アンタゴ-ストで あると、決定することができる。 [0124] 結合量を測定する場合、 CCL20は、標識することができる。前記標識としては、例 えば、放射性同位元素、酵素、蛍光物質、発光物質等が用いられる。放射性同位元 素としては、例えば、 [3H]、 [14C]、 [125I]、 [35S]等を用いることができる。前記酵素 としては、例えば j8—ガラクトシダーゼ、アルカリフォスファターゼ、パーォキシダーゼ 等を用いることができる。蛍光物質としては、例えばフルォレセイソチオシァネート、 B ODIPY等を用いることができる。発光物質としてはルシフェリン、ルシゲニン等を用 いることがでさる。 [0123] Specifically, CCR6 and labeled CCL20 were brought into contact with each other under the absence of the test substance and in the presence of the test substance, and specific binding of CCL20 to CCR6 under the above conditions By comparing the amounts, compounds can be screened without distinguishing their ability to promote or inhibit CCR6 function. In other words, when the specific binding amount in the presence of the test substance decreases relative to the specific binding amount of CCL20 to CCR6 in the absence of the test substance, the test substances are CCL20 and CCR6. More specifically, it is a compound that changes the binding of CCL20 to CCR6. More specifically, it is a CCR6 antagonist or CCR6 antagonist. It can be determined that [0124] When measuring the amount of binding, CCL20 can be labeled. Examples of the label include a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance. As the radioisotope, for example, [ 3 H], [ 14 C], [ 125 I], [ 35 S] and the like can be used. As the enzyme, for example, j8-galactosidase, alkaline phosphatase, peroxidase and the like can be used. As the fluorescent material, for example, fluorescein isothiocyanate, B ODIPY or the like can be used. Luciferin, lucigenin, etc. can be used as the luminescent substance.
[0125] 本発明に使用する被検物質は、どのような化合物であってもよいが、例えば遺伝子 ライブラリーの発現産物、合成低分子化合物ライブラリー、核酸 (オリゴ DNA、オリゴ RNA)、合成ペプチドライブラリー、抗体、細菌放出物質、細胞 (微生物、植物細胞、 動物細胞)抽出液、細胞 (微生物、植物細胞、動物細胞)培養上清、精製または部分 精製ポリペプチド、海洋生物、植物または動物由来の抽出物、土壌、ランダムファー ジペプチドディスプレイライブラリーを挙げることができる。  [0125] The test substance used in the present invention may be any compound. For example, an expression product of a gene library, a synthetic low molecular compound library, a nucleic acid (oligo DNA, oligo RNA), a synthetic peptide Library, antibody, bacterial release material, cell (microbe, plant cell, animal cell) extract, cell (microbe, plant cell, animal cell) culture supernatant, purified or partially purified polypeptide, marine organism, plant or animal origin Extract, soil, and random phage peptide display library.
[0126] さらに具体的には、本発明は、 CCL20、もしくは細菌、酵母、哺乳類由来の細胞、 昆虫由来の細胞において強制的に発現させた CCL20により誘導された CCR6含有 細胞遊走に対する阻害能を評価することにより骨破壊の治療剤のスクリーニングを行 うことができる。用いる CCL20は CCL20の生物学的活性を保持する物質であれば、 その種類および由来を問わず、哺乳類の体内から精製された CCL20、培養細胞内 およびその培養上清中の CCL20であってもよい。  [0126] More specifically, the present invention evaluates the ability to inhibit CCR6-containing cell migration induced by CCL20, or CCL20 forcedly expressed in bacteria, yeast, mammalian cells, or insect cells. By doing so, screening for therapeutic agents for bone destruction can be performed. The CCL20 used may be CCL20 purified from the mammalian body, CCL20 in cultured cells, and CCL20 in the culture supernatant as long as it is a substance that retains the biological activity of CCL20. .
[0127] 本発明によるスクリーニング方法の実施に当たっては、 CCL20の CCR6への作用 の評価を、 CCL20へ反応性を有する抗体の CCL20阻害作用または CCR6へ反応 性を有する抗体の CCR6阻害作用と比較することによって、行うことができる。具体的 には以下のようにして実施することができる。  [0127] In carrying out the screening method according to the present invention, the evaluation of the action of CCL20 on CCR6 should be compared with the CCL20 inhibitory action of an antibody reactive to CCL20 or the CCR6 inhibitory action of an antibody reactive to CCR6. Can be done. Specifically, it can be implemented as follows.
[0128] 抗体を用いたスクリーニング法は、例えば、 CCL20タンパク質、もしくは細菌、酵母 、哺乳類由来の細胞、昆虫由来の細胞において強制的に発現させた CCL20タンパ ク質により誘導された細胞遊走に対する阻害能 (作用または結合の程度)を評価する こと、遺伝子工学的に CCR6を強制的に発現させた細胞、 Bリンパ球、メモリー Tリン パ球、未熟榭状細胞、榭状細胞の前駆細胞を用いて、抗 CCR6抗体の CCR6阻害 作用に対する阻害能 (作用または結合の程度)を評価すること、または、上記のとおり に発現させた CCL20タンパク質および上記のとおり発現させた CCR6の作用または 結合の程度を比較し評価することによって実施することができる。比較の評価は、被 検物質の非存在下の CCL20と CCR6の作用または結合の程度と被検物質の存在 下の CCL20と CCR6の作用または結合の程度を比較することで判断できる。また、 細胞刺激活性を指標として評価することもできる。細胞刺激活性の測定は、シグナル 伝達物質生成によるレポーター遺伝子の翻訳 ·転写量の変化を検出するレポーター アツセィ系により測定する力 または、細胞内カルシウムイオン遊離、アデ-ル酸シク ラーゼの活性変動、細胞内 cAMP濃度変動、細胞内 cGMP濃度変動、ァラキドン酸 遊離、アセチルコリン遊離、イノシトールリン酸産生、細胞膜電位変動、細胞内タンパ ク質のリン酸化もしくは活性化、 pHの変動活性、 MAPキナーゼのリン酸化もしくは活 性化からなる群より選択されるノ メーターを測定することによって行うことができる。 [0128] The screening method using an antibody is, for example, an ability to inhibit cell migration induced by CCL20 protein or CCL20 protein forcedly expressed in bacteria, yeast, mammalian cells, or insect cells. (Degree of action or binding), genetically engineered CCR6 cells, B lymphocytes, memory T lymphocytes, immature rod cells, and rod precursor cells , CCR6 inhibition of anti-CCR6 antibody Perform by evaluating the ability to inhibit the action (degree of action or binding), or by comparing and evaluating the degree of action or binding of CCL20 protein expressed as described above and CCR6 expressed as described above be able to. Evaluation of the comparison can be made by comparing the degree of action or binding between CCL20 and CCR6 in the absence of the test substance and the degree of action or binding between CCL20 and CCR6 in the presence of the test substance. It can also be evaluated using cell stimulation activity as an index. Cell stimulation activity can be measured by reporter gene translation due to the generation of signal transmitters. Reporter detection of changes in transcription level. Force measured by an assay system or intracellular calcium ion release, adenylate cyclase activity change, cell CAMP concentration fluctuation, intracellular cGMP concentration fluctuation, arachidonic acid release, acetylcholine release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation or activation, pH fluctuation activity, MAP kinase phosphorylation or This can be done by measuring a meter selected from the group consisting of activation.
[0129] 骨破璩の檢出および骨破璩疾唐、の診断  [0129] Diagnosis of bone rupture and bone rupture
後記実施例に記載したように、 CCL20の発現により CCR6発現細胞の細胞遊走が 刺激され、それにより骨破壊が促進されることから、 CCL20の発現の程度を検出また は定量することにより骨破壊や骨破壊を検出することができる。従って、本発明によ れば、 CCL20発現の程度を検出する工程を含んでなる、骨破壊の検出方法および 骨破壊疾患の診断方法が提供される。  As described in Examples below, the expression of CCL20 stimulates cell migration of CCR6-expressing cells and thereby promotes bone destruction. Therefore, by detecting or quantifying the degree of CCL20 expression, Bone destruction can be detected. Therefore, according to the present invention, there is provided a method for detecting bone destruction and a method for diagnosing bone destruction disease, which comprises the step of detecting the degree of CCL20 expression.
[0130] 本発明による検出方法および診断方法の実施に当たっては、正常組織における C CL20の発現の程度と被験組織における CCL20の発現の程度を比較する工程を更 に含んでいてもよい。この場合、正常組織における CCL20の発現量を超える発現（ 好ましくは、約 1. 1倍の発現、より好ましくは約 1. 5倍の発現)が被験組織において 認められた場合には、骨破壊が生じている、あるいは骨破壊疾患である、と判定する ことができる。  [0130] In carrying out the detection method and the diagnostic method according to the present invention, a step of comparing the level of CCL20 expression in normal tissues with the level of CCL20 expression in test tissues may be further included. In this case, if an expression exceeding the expression level of CCL20 in a normal tissue (preferably about 1.1-fold expression, more preferably about 1.5-fold expression) is observed in the test tissue, bone destruction may occur. It can be determined that it is occurring or is a bone destruction disease.
[0131] 正常組織および被験組織力ゝらの試料は、例えば、血液、滑液または滑膜組織等の 体液を採血または切除術等の方法により、静脈または関節等の部位力 得ることが できる。  [0131] For samples of normal tissue and test tissue force, for example, body force such as blood, synovial fluid, or synovial tissue can be obtained by using a method such as blood collection or excision to obtain site force such as veins or joints.
[0132] 本発明による検出方法および診断方法の実施に当たっては、 CCL20に反応性を 有する抗体およびそのフラグメントを使用して CCL20の発現の程度を検出または定 量することができる。抗体による検出および定量は当業者に周知であり、 CCL20の 発現の程度が検出および定量できる限り特に限定されるものではないが、例えば、ィ ムノアツセィを好ましくは用いることができる。 [0132] In carrying out the detection method and the diagnostic method according to the present invention, CCL20 should be reactive. Antibodies and fragments thereof can be used to detect or quantify the extent of CCL20 expression. Detection and quantification by an antibody is well known to those skilled in the art, and is not particularly limited as long as the degree of expression of CCL20 can be detected and quantified. For example, immunoassay can be preferably used.
[0133] 本発明における「ィムノアツセィ」とは、抗原抗体反応の原理に基づき、試料 (例え ば、血漿等の体液試料、培養上清あるいは遠心上清等）中に含まれる抗原の検出あ るいは定量を行う方法を意味し、本発明においては、該抗原抗体反応における抗体 力 CCL20に反応性を有する前記モノクローナル抗体若しくはその抗体フラグメント[0133] In the present invention, "immunoassay" refers to the detection of an antigen contained in a sample (eg, a body fluid sample such as plasma, a culture supernatant or a centrifugal supernatant) based on the principle of an antigen-antibody reaction. In the present invention, the monoclonal antibody or antibody fragment thereof having reactivity with the antibody force CCL20 in the antigen-antibody reaction is meant.
、前記固定ィ匕モノクローナル抗体若しくは固定ィ匕抗体フラグメント、または前記標識 モノクローナル抗体若しくは標識抗体フラグメントから選ばれる一つ以上の該モノクロ ーナル抗体または抗体フラグメントであること、および抗原が CCL20であること以外 は、これまでに知られて!/ヽるィムノアツセィをも適用することができる。 Except that it is at least one monoclonal antibody or antibody fragment selected from the above-mentioned immobilized monoclonal antibody or immobilized antibody fragment, or the labeled monoclonal antibody or labeled antibody fragment, and the antigen is CCL20. , So far known! / I can also apply Imunoatsusei.
[0134] 具体的には、酵素免疫測定法 (第 3版、石川榮治ら編集、医学書院発行、 1987年 )に記載されているような、例えば、一抗体固相法、二抗体液相法、二抗体固相法、 サントづツチ法、 EMIT法 (Enzyme multiplied immunoassay technique;、ェンザ ムテ ャネリングアツセィ（Enzyme channeling immunoassay)、酵素活性修飾物質標識ィムノ ツセィ (Enzyme modulator mediated enzyme immunoassay^ EMMIA)、酵素！ 告 物質標識ィムノアッセィ（Enzyme inhibitor immunoassay)、ィムノエンザィムメトリックァ ッセ (Immunoenzymometricassay)、酵素活'性増強 ムノアッセ Enzyme enhanced immunoassay)め レヽ ίまプロキシマーノレリンケーンィムノアッセィ (Proximal linkage im munoassay)、ワンポット法（特公平 2— 39747号公報）等を挙げることができる。  [0134] Specifically, for example, one antibody solid phase method, two antibody liquid phase method, as described in enzyme immunoassay (3rd edition, edited by Yuji Ishikawa et al., Published by Medical School, 1987). , Two-antibody solid phase method, Santotsuchi method, EMIT method (Enzyme multiplied immunoassay technique; Enzyme channeling immunoassay), Enzyme modulator mediated enzyme immunoassay ^ EMMIA Enzyme inhibitor immunoassay, Imunoenzymometric assay, Enzyme enhanced immunoassay, Proximal inhibitor immunoassay (Proximal inhibitor immunoassay) linkage im munoassay), one-pot method (Japanese Patent Publication No. 2-39747), and the like.
[0135] 本発明においては、このようなィムノアッセィを、目的に応じて適宜選択して用いる ことができるが、操作上の簡便性および Zまたは経済的な利便性、とりわけ臨床上で の汎用性の点を考慮すると、サンドイッチ法、ワンポット法、一抗体固相法またはニ抗 体液相法を用いるのが好ましぐより好ましくは、ワンポット法であり、ビーズまたはボ ールに固定ィ匕された固定ィ匕モノクローナル抗体または固定ィ匕抗体フラグメントと、酵 素あるいはピオチンにより標識された標識モノクローナル抗体または標識抗体フラグ メントとを用いるワンポット法である。 [0136] この特に好ま 、態様にぉ 、て具体的な一例を挙げるならば、本発明のハイブリド 一マ（FERM BP— 10445)産生モノクローナル抗体またはそれらの F (ab，）若しく [0135] In the present invention, such an immunoassay can be appropriately selected and used depending on the purpose. However, it is easy to operate and has Z or economical convenience, particularly clinical versatility. In view of the above, it is preferable to use the sandwich method, the one-pot method, the single-antibody solid-phase method or the dual-antibody liquid-phase method, more preferably the one-pot method, which is immobilized on beads or balls. This is a one-pot method using a fixed monoclonal antibody or a fixed antibody fragment and a labeled monoclonal antibody or labeled antibody fragment labeled with an enzyme or piotin. [0136] This particularly preferred embodiment is, as a specific example, according to the embodiment, the hybridoma (FERM BP-10445) -producing monoclonal antibody of the present invention or their F (ab,) or
2 は Fab'をマイクロプレートに固定ィ匕した固定ィ匕モノクローナル抗体、固定化 F (ab' )  2 is an immobilized monoclonal antibody in which Fab 'is immobilized on a microplate, immobilized F (ab')
2 若しくは固定化 Fab'と本発明のハイプリドーマ（FERM BP— 10445)産生モノクロ ーナル抗体またはその F (ab， ) 若しくは Fab，を酵素またはピオチンで標識した標識  2 or immobilized Fab ′ and the monoclonal antibody produced by the hyperpridoma (FERM BP-10445) of the present invention or its F (ab,) or Fab labeled with an enzyme or piotin
2  2
モノクローナル抗体また標識 F (ab， ) 若しくは Fab，との組合わせによるワンポット法  One-pot method by combination with monoclonal antibody or labeled F (ab,) or Fab
2  2
である。  It is.
[0137] 以下に、ワンポット法、サンドイッチ法、一抗体固相法および二抗体液相法にっ ヽ て詳述する。  [0137] Hereinafter, the one-pot method, the sandwich method, the one-antibody solid phase method and the two-antibody liquid phase method will be described in detail.
[0138] ワンポット法は、第 1は、少なくとも下記 (a)および (b)の工程を含むィムノアツセィ法 である。  [0138] The first one-pot method is an immunoassay method including at least the following steps (a) and (b).
[0139] (a)本発明の固定ィ匕モノクローナル抗体または固定ィ匕抗体フラグメントに試料を反 応せしめる工程。  [0139] (a) A step of reacting a sample with the immobilized antibody monoclonal antibody or immobilized antibody antibody fragment of the present invention.
[0140] (b)該固定ィ匕モノクローナル抗体または該固定ィ匕抗体フラグメントと試料中に含ま れる CCL20との結合により形成される抗原抗体複合体に本発明の標識モノクローナ ル抗体または標識抗体フラグメントを反応せしめる工程。  [0140] (b) The labeled monoclonal antibody or labeled antibody fragment of the present invention formed on the antigen-antibody complex formed by the binding of the immobilized monoclonal antibody or the immobilized antibody fragment to CCL20 contained in the sample The process of reacting.
[0141] 第 2は、少なくとも下記 (a)および (b)の工程を含むィムノアッセィ法である。 [0141] The second is an immunoassay method including at least the following steps (a) and (b).
[0142] (a)本発明の標識モノクローナル抗体または標識抗体フラグメントに試料を反応せ しめる工程。 [0142] (a) A step of reacting a sample with the labeled monoclonal antibody or labeled antibody fragment of the present invention.
[0143] (b)該標識モノクローナル抗体または該標識抗体フラグメントと試料中に含まれる C (B) The labeled monoclonal antibody or the labeled antibody fragment and C contained in the sample
CL20との結合により形成される抗原抗体複合体に本発明の固定ィ匕モノクローナル 抗体または固定ィ匕抗体フラグメントを反応せしめる工程。 A step of reacting the immobilized antibody monoclonal antibody or immobilized antibody antibody fragment of the present invention with an antigen-antibody complex formed by binding to CL20.
[0144] 第 3は、少なくとも下記 (a)の工程を含むィムノアッセィ法である。 [0144] The third is an immunoassay method including at least the following step (a).
[0145] (a)本発明の固定ィ匕モノクローナル抗体または固定ィ匕抗体フラグメント、本発明の 標識モノクローナル抗体もしくは標識抗体フラグメント、および試料を含む混合物を 反応せしめる工程。 [0145] (a) A step of reacting a mixture containing the immobilized monoclonal antibody or immobilized antibody fragment of the present invention, the labeled monoclonal antibody or labeled antibody fragment of the present invention, and a sample.
[0146] 上記第 1〜第 3の方法を、本発明に即して、特に一般的である酵素あるいはビォチ ンを標識物質として用いる方法について具体的に説明すると、例えば下記のようなェ 程により構成されるが、該具体例のみに限定されるものではない。なお、以下で使用 する「固定ィ匕モノクローナル抗体」と「モノクローナル抗体固定ィ匕ビーズ」は、同一物 を意味する。 [0146] The above-mentioned first to third methods will be specifically described in accordance with the present invention, particularly for a method using an enzyme or biotin as a labeling substance, which is generally common. However, the present invention is not limited to the specific example. In the following, “immobilized monoclonal antibody” and “monoclonal antibody immobilized bead” mean the same thing.
[0147] 第 1の方法は、下記のような工程力も構成される。  [0147] The first method includes the following process power.
[0148] (工程 1) CCL20に反応性を有する本発明のハイプリドーマ（FERM BP— 1044 5)産生モノクローナル抗体をビーズに固定ィ匕し、固定ィ匕モノクローナル抗体 (モノク ローナル抗体固定ィ匕ビーズ)を作製する工程；  [0148] (Step 1) Immobilized monoclonal antibody (FERM BP-1044 5) -producing monoclonal antibody of the present invention having reactivity with CCL20 is immobilized on beads, and immobilized monoclonal antibody (monoclonal antibody-immobilized beads) Producing
(工程 2)試験管、プレートあるいはチューブ等のような内容積を有する容器に緩衝 液とともにモノクローナル抗体固定ィ匕ビーズとヒト血漿等の試料をカ卩え、固定化モノク ロー  (Step 2) Place the monoclonal antibody-immobilized beads and human plasma samples together with the buffer in a container with an internal volume such as a test tube, plate, tube, etc.
ナル抗体と試料を反応させる工程；  Reacting a sample with a null antibody;
(工程 3)容器中の内溶液の除去およびモノクローナル抗体固定ィ匕ビーズを洗浄す る工程；  (Step 3) Step of removing the inner solution in the container and washing the monoclonal antibody immobilized beads.
(工程 4) CCL20に反応性を有する本発明のハイプリドーマ（FERM BP— 1044 5)産生モノクローナル抗体をピオチンあるいはペルォキシダーゼ等の酵素により標 識し、標識モノクローナル抗体を作製する工程；  (Step 4) A step of producing a labeled monoclonal antibody by labeling a monoclonal antibody produced by the hyperidoma of the present invention (FERM BP-10445) having reactivity to CCL20 with an enzyme such as piotin or peroxidase;
(工程 5)工程 3で洗浄されたモノクローナル抗体固定ィ匕ビーズを含有する容器に、 標識モノクローナル抗体を加え、固定ィ匕モノクローナル抗体と試料中に含まれる CC L20が反応して形成される抗原抗体複合体に標識モノクローナル抗体を反応させる 工程；  (Step 5) Antigen antibody formed by adding labeled monoclonal antibody to the container containing the monoclonal antibody-immobilized beads washed in step 3, and reacting the immobilized monoclonal antibody with CC L20 contained in the sample Reacting the conjugate with a labeled monoclonal antibody;
(工程 6)容器中の内溶液の除去およびモノクローナル抗体固定ィ匕ビーズを洗浄し 、未反応の標識モノクローナル抗体を抗原抗体複合体から取り除く工程；  (Step 6) Step of removing the internal solution in the container and washing the monoclonal antibody fixed beads to remove unreacted labeled monoclonal antibody from the antigen-antibody complex;
(工程 7)工程 6で洗浄されたモノクローナル抗体固定ィ匕ビーズを含む容器に、工程 4でピオチン標識モノクローナル抗体を用いた場合にはアビジンある 、は酵素修飾ァ ビジンを、また工程 4でペルォキシダーゼ等の酵素で標識した酵素標識モノクローナ ル抗体を用いた場合には酵素活性を測定する方法に依存して種々の基質を、必要 に応じて発色物質とともに加え、標識モノクローナル抗体上の標識物質と反応させる 工程； (工程 8)工程 7で酵素修飾アビジンを加えた場合には、酵素活性を測定する方法 に依存して種々の基質を加え、アビジンに結合した酵素と基質を反応させる工程；(Step 7) A container containing the monoclonal antibody-fixed beads washed in Step 6 contains avidin or an enzyme-modified avidin in Step 4, and peroxidase etc. in Step 4. When using an enzyme-labeled monoclonal antibody labeled with any of the above enzymes, depending on the method used to measure the enzyme activity, various substrates are added together with a chromogenic material as necessary to react with the labeled material on the labeled monoclonal antibody. A step of causing; (Step 8) When enzyme-modified avidin is added in Step 7, depending on the method for measuring enzyme activity, various substrates are added, and the enzyme bound to avidin is reacted with the substrate;
(工程 9)工程 7または工程 8の反応系に反応停止液を加え、酵素反応および発色 反応を停止させる工程；および (工程 10)比色強度、蛍光強度あるいは発光強度を 測定する工程。 (Step 9) A step of adding a reaction stop solution to the reaction system of Step 7 or Step 8 to stop the enzymatic reaction and the color development reaction; and (Step 10) a step of measuring the colorimetric intensity, fluorescence intensity or emission intensity.
[0149] 第 2の方法は、下記のような工程力も構成される。 [0149] The second method has the following process power.
[0150] (工程 1) CCL20に反応性を有する本発明のハイプリドーマ（FERM BP— 1044 5)産生モノクローナル抗体をピオチンあるいはペルォキシダーゼ等の酵素により標 識し、標識モノクローナル抗体を作製する工程；  [0150] (Step 1) A step of producing a labeled monoclonal antibody by labeling a monoclonal antibody produced by the hyperpridoma (FERM BP-1044 5) of the present invention having reactivity to CCL20 with an enzyme such as piotin or peroxidase;
(工程 2)試験管、プレートあるいはチューブ等のような内容積を有する容器に緩衝 液とともに標識モノクローナル抗体とヒト血漿等の試料を加え、標識モノクローナル抗 体と試料を反応させる工程；  (Step 2) A step of adding a labeled monoclonal antibody and a sample such as human plasma together with a buffer to a container having an internal volume such as a test tube, plate or tube, and reacting the labeled monoclonal antibody with the sample;
(工程 3) CCL20に反応性を有する本発明のハイプリドーマ（FERM BP— 1044 5)産生モノクローナル抗体をビーズに固定ィ匕し、固定ィ匕モノクローナル抗体 (モノク ローナル抗体固定ィ匕ビーズ)を作製する工程；  (Step 3) Immobilize the monoclonal antibody produced by the high-pridoma of the present invention (FERM BP—1044 5), which is reactive to CCL20, on the beads to produce the immobilized monoclonal antibody (monoclonal antibody-immobilized beads). Process;
(工程 4)工程 2の反応系に、モノクローナル抗体固定ィ匕ビーズを加え、標識モノクロ ーナル抗体と試料中に含まれる CCL20が反応して形成される抗原抗体複合体に固 定ィ匕モノクローナル抗体を反応させる工程；  (Step 4) Add monoclonal antibody-immobilized beads to the reaction system of Step 2, and add the immobilized monoclonal antibody to the antigen-antibody complex formed by the reaction between the labeled monoclonal antibody and CCL20 contained in the sample. Reacting;
(工程 5)容器中の内溶液の除去およびモノクローナル抗体固定ィ匕ビーズを洗浄し 、未反応の標識モノクローナル抗体を取り除く工程；  (Step 5) Step of removing the inner solution in the container and washing the monoclonal antibody immobilized beads to remove unreacted labeled monoclonal antibody;
(工程 6)工程 5で洗浄されたモノクローナル抗体固定ィ匕ビーズを含む容器に、工程 1でビォチン標識モノクローナル抗体を用いた場合にはアビジンある 、は酵素修飾ァ ビジンを、また工程 1でペルォキシダーゼ等の酵素で標識した酵素標識モノクローナ ル抗体を用いた場合には酵素活性を測定する方法に依存して種々の基質を、必要 に応じて発色物質とともに加え、標識モノクローナル抗体上の標識物質と反応させる 工程；  (Step 6) When the biotin-labeled monoclonal antibody used in Step 1 is used in the container containing the monoclonal antibody-fixed beads washed in Step 5, avidin is present, enzyme modified avidin, and peroxidase etc. in Step 1. When using an enzyme-labeled monoclonal antibody labeled with any of the above enzymes, depending on the method used to measure the enzyme activity, various substrates are added together with a chromogenic material as necessary to react with the labeled material on the labeled monoclonal antibody. A step of causing;
(工程 7)工程 6で酵素修飾アビジンを加えた場合には、酵素活性を測定する方法 に依存して種々の基質を加え、アビジンに結合した酵素と基質を反応させる工程； (工程 8)工程 6または工程 7の反応系に反応停止液を加え、酵素反応および発色 反応を停止させる工程；および (工程 9)比色強度、蛍光強度あるいは発光強度を測 定する工程。 (Step 7) When enzyme-modified avidin is added in Step 6, depending on the method for measuring enzyme activity, various substrates are added, and the enzyme bound to avidin is reacted with the substrate; (Step 8) A step of adding a reaction stop solution to the reaction system of Step 6 or Step 7 to stop the enzymatic reaction and the color reaction; and (Step 9) a step of measuring the colorimetric intensity, fluorescence intensity, or luminescence intensity.
[0151] 第 3の方法は、下記のような工程力も構成される。  [0151] The third method also has the following process capability.
[0152] (工程 1) CCL20に反応性を有する本発明のハイプリドーマ（FERM BP— 1044 5)産生モノクローナル抗体をビーズに固定ィ匕し、固定ィ匕モノクローナル抗体 (モノク ローナル抗体固定ィ匕ビーズ)を作製する工程；  [0152] (Step 1) Immobilizing the monoclonal antibody produced by the high-pridoma of the present invention (FERM BP—1044 5), which is reactive to CCL20, on the beads, and immobilizing the monoclonal antibody (monoclonal antibody-immobilized beads). Producing
(工程 2) CCL20に反応性を有する本発明のハイプリドーマ（FERM BP— 1044 5)産生モノクローナル抗体をピオチンあるいはペルォキシダーゼ等の酵素により標 識し、標識モノクローナル抗体を作製する工程；  (Step 2) A step of producing a labeled monoclonal antibody by labeling a monoclonal antibody produced by the present invention having high reactivity with CCL20 (FERM BP-10445) with an enzyme such as piotin or peroxidase;
(工程 3)試験管、プレートあるいはチューブ等のような内容積を有する容器に緩衝 液とともに、工程 1で作製されたモノクローナル抗体固定ィ匕ビーズ、工程 2で作製され た標識モノクローナル抗体、およびヒト血漿等の試料を加え、固定ィ匕モノクローナル 抗体、標識モノクローナル抗体、および試料を同時に反応させる工程；  (Step 3) Monoclonal antibody-immobilized beads prepared in Step 1, labeled monoclonal antibody prepared in Step 2, and human plasma together with a buffer in a container having an internal volume such as a test tube, plate or tube And the like, and the step of reacting the immobilized monoclonal antibody, the labeled monoclonal antibody, and the sample simultaneously;
(工程 4)容器中の内溶液の除去およびモノクローナル抗体固定ィ匕ビーズを洗浄し 、未反応の標識モノクローナル抗体を取り除く工程；  (Step 4) Step of removing the inner solution in the container and washing the monoclonal antibody immobilized beads to remove unreacted labeled monoclonal antibody;
(工程 5)工程 4で洗浄されたモノクローナル抗体固定ィ匕ビーズを含む容器に、工程 2でピオチン標識モノクローナル抗体を用いた場合にはアビジンある 、は酵素修飾ァ ビジンを、また工程 2でペルォキシダーゼ等の酵素で標識した酵素標識モノクローナ ル抗体を用いた場合には酵素活性を測定する方法に依存して種々の基質を、必要 に応じて発色物質とともに加え、標識モノクローナル抗体上の標識物質と反応させる 工程；  (Step 5) If the container containing the monoclonal antibody-immobilized beads washed in Step 4 is used with a piotin-labeled monoclonal antibody in Step 2, avidin is present, enzyme-modified avidin, and in Step 2, peroxidase, etc. When using an enzyme-labeled monoclonal antibody labeled with any of the above enzymes, depending on the method used to measure the enzyme activity, various substrates are added together with a chromogenic material as necessary to react with the labeled material on the labeled monoclonal antibody. A step of causing;
(工程 6)工程 5で酵素修飾アビジンを加えた場合には、酵素活性を測定する方法 に依存して種々の基質を加え、アビジンに結合した酵素と基質を反応させる工程； (Step 6) When enzyme-modified avidin is added in Step 5, depending on the method for measuring enzyme activity, various substrates are added, and the enzyme bound to avidin is reacted with the substrate;
(工程 7)工程 5または工程 6の反応系に反応停止液を加え、酵素反応および発色 反応を停止させる工程；および (工程 8)比色強度、蛍光強度あるいは発光強度を測 定する工程。 (Step 7) A step of adding a reaction stop solution to the reaction system of Step 5 or Step 6 to stop the enzymatic reaction and the color reaction; and (Step 8) a step of measuring the colorimetric intensity, fluorescence intensity, or luminescence intensity.
[0153] サンドイッチ法によるィムノアツセィ法を使用するには、ハイプリドーマ（FERM BP 10445)産生モノクローナル抗体と別のモノクローナル抗体またはポリクローナル 抗体を用いて本発明の検出 ·定量方法に使用することができる。サンドイッチ法では 、 96穴マイクロプレートに代表されるような多数のゥエルを有するマイクロプレートに 固定化された固定ィ匕モノクローナル抗体または固定ィ匕抗体フラグメントと、酵素ある いはピオチンにより標識された標識モノクローナル抗体または標識抗体フラグメントと を用いることができる。 [0153] In order to use the Imno Atssay method by the sandwich method, the Hyperidoma (FERM BP 10445) The production monoclonal antibody and another monoclonal antibody or polyclonal antibody can be used for the detection and quantification method of the present invention. In the sandwich method, a immobilized monoclonal antibody or immobilized antibody fragment immobilized on a microplate having a large number of wells such as a 96-well microplate, and a labeled monoclonal labeled with an enzyme or piotin. Antibodies or labeled antibody fragments can be used.
[0154] 一抗体固相法や二抗体液相法等によるィムノアツセィ法も本発明の検出 ·定量方 法には有効である。  [0154] An immunoassay method such as a one-antibody solid phase method or a two-antibody liquid phase method is also effective for the detection / quantification method of the present invention.
[0155] 本発明における「ァフィ-ティークロマトグラフィー」とは、抗原抗体反応を利用する ことにより試料 (例えば、血漿等の体液試料、培養上清あるいは遠心上清等）中に含 まれる CCL20を分離または精製する方法を意味する。具体的には、（1)不溶性担体 であるフィルターある 、はメンブレン等に CCL20に反応性を有する本発明のハイブリ ドーマ（FERM BP— 10445)産生モノクローナル抗体あるいは抗体フラグメントを 固定ィ匕した後、該フィルターあるいはメンブレンと試料を接触させることにより該試料 中に含まれる CCL20を分離する方法、および（2)前述のようなセルロース系担体、 ァガロース系担体、ポリアクリルアミド系担体、デキストラン系担体、ポリスチレン系担 体、ポリビニルアルコール系担体、ポリアミノ酸系担体あるいは多孔性シリカ系担体等 のような不溶性担体上に CCL20に反応性を有する本発明のハイプリドーマ (FERM [0155] In the present invention, "affinity chromatography" refers to CCL20 contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, or a centrifugal supernatant) by using an antigen-antibody reaction. It means a method of separation or purification. Specifically, (1) a filter which is an insoluble carrier is immobilized on a membrane or the like after the hybridoma (FERM BP-10445) -producing monoclonal antibody or antibody fragment of the present invention having reactivity to CCL20 is immobilized. A method for separating CCL20 contained in a sample by contacting the filter or membrane with the sample, and (2) a cellulose-based carrier, agarose-based carrier, polyacrylamide-based carrier, dextran-based carrier, polystyrene-based carrier as described above. The high-pridoma (FERM) of the present invention having reactivity to CCL20 on an insoluble carrier such as a polymer, a polyvinyl alcohol carrier, a polyamino acid carrier or a porous silica carrier.
BP— 10445)産生モノクローナル抗体あるいは抗体フラグメントを常法により固定 ィ匕 (物理的吸着、架橋による高分子化、マトリックス中への封印あるいは非共有結合 等による固定化）し、該不溶性担体をガラス製、プラスチック製あるいはステンレス製 等のカラムに充填し、該カラム (例えば、円柱状カラム）に、試料 (例えば、血漿等の 体液試料、培養上清あるいは遠心上清等)を通じて溶出させることにより、該試料中 に含まれる CCL20を分離ある 、は精製する方法である。後者（2)の方法を特にァフ ィニティーカラムクロマトグラフィーと 、う。 BP-10445) produced monoclonal antibody or antibody fragment is immobilized by a conventional method (physical adsorption, polymerization by crosslinking, sealing in a matrix or immobilization by noncovalent bonding, etc.), and the insoluble carrier is made of glass. The sample is packed in a plastic or stainless steel column, and the sample (eg, a columnar column) is eluted through a sample (eg, a body fluid sample such as plasma, a culture supernatant, or a centrifugal supernatant). Separation of CCL20 contained in a sample is a method for purification. The latter method (2) is particularly referred to as affinity column chromatography.
[0156] 該ァフィ-ティーカラムクロマトグラフィーに用いられる前記不溶性担体としては、本 発明のモノクローナル抗体あるいは抗体フラグメントを固定ィ匕でき得るものであれば どのような不溶性担体でも使用できる力 例えば、市販品である、フアルマシア (Pharm acia)社 (製)の Sepharose 2B、 Sepharose 4B、 Sepharose 6B、 CNBr— activated Sepharo se 4B、 AH— Sepharose 4B、 CH— Sepharose 4B、 Activated CH— Sepharose 4B、 Epoxy- activated Sepharose 6B、 Activated thiol— Sepharose 4B、 Sephadex、 CM— Sephadex、 E CH— Sepharose 4B、 EAH— Sepharose 4B、 NHS— activated Sepharoseある ヽは Tmoprop yl Sepharose 6B等、バイオラッド (Bio- Rad)社（製）の Bio- Gel Aゝ Cellex, Cellex AE、 C ellex— CM、 Cellex PABゝ Bio-Gel P、 Hydrazide Bio-Gel P、 Aminoethyl Bio-Gel P、 Bi o-Gel CM、 Affi-Gel 10、 Affi— Gel 15、 Affi— Prep 10、 Affi— Gel Hzゝ Affi— Prep Hzゝ Affi— Gel 102、 CMBio-Gel A、 Affi-Gel heparin, Affi-Gel 501あるいは Affi- Gel 601等、和 光純薬工業社（製）のクロマゲル A、クロマゲル P、ェンザフィックス P-HZ、ェンザフィ ックス P-SHあるいはェンザフィックス P- AB等、セルノ (Serva)社（製）の AE- Cellurose 、 CM- Celluroseあるいは PAB Cellurose等を挙げることができる。 [0156] As the insoluble carrier used in the affinity column chromatography, any insoluble carrier capable of immobilizing the monoclonal antibody or antibody fragment of the present invention can be used. For example, commercially available products Hurmasia (Pharm acia) Sepharose 2B, Sepharose 4B, Sepharose 6B, CNBr— activated Sepharo se 4B, AH— Sepharose 4B, CH— Sepharose 4B, Activated CH— Sepharose 4B, Epoxy-activated Sepharose 6B, Activated thiol— Sepharose 4B , Sephadex, CM— Sephadex, E CH— Sepharose 4B, EAH— Sepharose 4B, NHS— activated Sepharose, etc., such as Tmoprop yl Sepharose 6B, Bio-Gel A ゝ Cellex, manufactured by Bio-Rad Cellex AE, C ellex— CM, Cellex PAB ゝ Bio-Gel P, Hydrazide Bio-Gel P, Aminoethyl Bio-Gel P, Bio-Gel CM, Affi-Gel 10, Affi— Gel 15, Affi— Prep 10, Affi — Gel Hz ゝ Affi— Prep Hz ゝ Affi— Gel 102, CMBio-Gel A, Affi-Gel heparin, Affi-Gel 501 or Affi-Gel 601, etc., Chromagel A, Chromagel P, manufactured by Wako Pure Chemical Industries, Ltd. Enzafix P-HZ, Enzafix P-SH or Enzafix P-AB, such as AE-Cellurose, CM- from Serva (made by Serva) Cellurose or PAB Cellurose can be mentioned.
[0157] 本発明において「固定ィ匕モノクローナル抗体」および「固定ィ匕抗体フラグメント」とは 、不溶性担体に物理的吸着あるいは化学的結合等によって坦持された状態にあるモ ノクローナル抗体および抗体フラグメントを各々意味する。これらの固定ィ匕モノクロ一 ナル抗体および固定ィ匕抗体フラグメントは、試料 (例えば、血漿等の体液試料、培養 上清あるいは遠心上清等）中に含まれる CCL20を検出、定量、分離または精製する ために用いることができる。該検出または定量の目的においては、該不溶性担体に 固定化された固定ィ匕モノクローナル抗体または固定ィ匕抗体フラグメントを用いること ができ、とりわけ定量に用いる不溶性担体としては、操作の簡便性および多数検体の 同時処理の観点を考慮すると、例えば 96穴マイクロタイタープレート等の多数のゥェ ルを有するプラスチックプレートを用いるのが好ましい。また、該分離または精製の目 的においては、前記（1)に挙げたフィルター若しくはメンブレンまたは前記（2)に挙 げた不溶性担体に固定化された固定ィ匕モノクローナル抗体または固定ィ匕抗体フラグ メントを用いることができる。 [0157] In the present invention, "immobilized monoclonal antibody" and "immobilized antibody fragment" refer to a monoclonal antibody and an antibody fragment that are supported on an insoluble carrier by physical adsorption or chemical binding. Each means. These fixed monoclonal antibodies and fixed antibody fragments detect, quantify, separate or purify CCL20 contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, or a centrifugal supernatant). Can be used for For the purpose of the detection or quantification, an immobilized antibody monoclonal antibody or immobilized antibody fragment immobilized on the insoluble carrier can be used. In view of the simultaneous processing, it is preferable to use a plastic plate having many wells such as a 96-well microtiter plate. For the purpose of the separation or purification, the immobilized monoclonal antibody or the immobilized antibody fragment immobilized on the filter or membrane described in (1) above or the insoluble carrier listed in (2) above is used. Can be used.
[0158] 本発明においては CCL20に反応性を有する抗体を「単独または他の物質と反応 することにより検出可能なシグナルをもたらすことができる標識物質」で標識してもよく 、この標識物質は前記モノクローナル抗体または抗体フラグメントに物理化学的結合 等により結合させることによりそれらの存在を検出可能にするために用いられる。この ような標識物質は、具体的には、酵素、蛍光物質、化学発光物質、ピオチン、ァビジ ンあるいは放射性同位体等であり、さらに具体的には、ペルォキシダーゼ、アルカリ フォスファターゼ、 j8 D ガラクトシダーゼ、グルコースォキシダーゼ、グノレコース 6—ホスフェートデヒドロゲナーゼ、アルコール脱水素酵素、リンゴ酸脱水素酵素、ぺ -シリナーゼ、カタラーゼ、アポグルコースォキシダーゼ、ゥレアーゼ、ルシフェラー ゼ若しくはアセチルコリンエステラーゼ等の酵素、フルォレスセインイソチオシァネー ト、フィコピリタンパク質、希土類金属キレート、ダンシルク口ライド若しくはテトラメチル ローダミンイソチオシァネート等の蛍光物質、 ¾、 "C、 1251若しくは1311等の放射性同 位体、ピオチン、アビジン、または化学発光物質が挙げられる。 [0158] In the present invention, an antibody having reactivity with CCL20 may be labeled with "a labeling substance capable of producing a detectable signal by reacting alone or with another substance". They are used to detect their presence by binding to monoclonal antibodies or antibody fragments by physicochemical binding or the like. this Such labeling substances are specifically enzymes, fluorescent substances, chemiluminescent substances, piotin, avidin, or radioisotopes, and more specifically, peroxidase, alkaline phosphatase, j8 D galactosidase, glucose oxidase. , Gnolecose 6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, percylinase, catalase, apoglucosoxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, Fi co pyridinium protein, rare earth metal chelates, Danshiruku port ride or fluorescent substances such as tetramethylrhodamine isothiocyanate Xia sulfonates, ¾, "C, 125 1 or 131 radioactive isotope, such as 1, Piochin, avidin or chemiluminescence, Quality, and the like.
[0159] ここで、放射性同位体および蛍光物質は単独で検出可能なシグナルをもたらすこと ができる。一方、酵素、化学発光物質、ピオチンおよびアビジンは、単独では検出可 能なシグナルをもたらすことができないため、さらに 1種以上の他の物質と反応するこ とにより検出可能なシグナルをもたらす。例えば、酵素の場合には少なくとも基質が 必要であり、酵素活性を測定する方法 (比色法、蛍光法、生物発光法あるいは化学 発光法等）に依存して種々の基質が用いられる。また、ピオチンの場合には少なくと もアビジンある 、は酵素修飾アビジンを反応させるのが一般的である力 この限りで はな 、。必要に応じてさらに該基質に依存する種々の発色物質が用いられる。  [0159] Here, the radioisotope and the fluorescent substance alone can provide a detectable signal. On the other hand, enzymes, chemiluminescent substances, piotin, and avidin alone cannot produce a detectable signal, and therefore react with one or more other substances to produce a detectable signal. For example, in the case of an enzyme, at least a substrate is required, and various substrates are used depending on the method for measuring enzyme activity (colorimetric method, fluorescence method, bioluminescence method, chemiluminescence method, etc.). In the case of piotin, there is at least avidin, but it is common to react with enzyme-modified avidin. Various color-developing substances depending on the substrate are used as necessary.
[0160] 本発明において「標識モノクローナル抗体」および「標識抗体フラグメント」とは、各 々標識物質で標識されたモノクローナル抗体および抗体フラグメントを意味する。こ れらの標識モノクローナル抗体および標識抗体フラグメントは、試料 (例えば、血漿等 の体液試料、培養上清あるいは遠心上清等）中に含まれる CCL20を検出または定 量するために用いることができる。本発明においては、上記のいずれの標識物質をも 使用可能である力 検出感度あるいは定量感度の高さおよび操作の利便性の点を 考慮すると、ピオチンで標識するのが好ましい。  [0160] In the present invention, "labeled monoclonal antibody" and "labeled antibody fragment" mean a monoclonal antibody and an antibody fragment each labeled with a labeling substance. These labeled monoclonal antibodies and labeled antibody fragments can be used to detect or quantify CCL20 contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant, a centrifugal supernatant, or the like). In the present invention, in view of the high force detection sensitivity or quantitative sensitivity that can use any of the above-described labeling substances and the convenience of operation, labeling with piotin is preferable.
[0161] キットおよび診断剤  [0161] Kits and diagnostic agents
本発明によれば、本発明による骨破壊の検出法や骨破壊の診断方法の実施に用い られるキットが提供される。本発明によるキットは、 CCL20に反応性を有する抗体や そのフラグメントを含んでいてもよぐ抗体抗原反応を測定または定量するために必 要な試薬や器具を更に含んで 、てもよ 、。 CCL20に反応性を有する抗体やそのフ ラグメントは、測定方法に従って、固相化や標識化がなされていてもよい。 According to the present invention, there is provided a kit used for carrying out the method for detecting bone fracture and the method for diagnosing bone fracture according to the present invention. The kit according to the present invention is necessary for measuring or quantifying an antibody antigen reaction that may contain an antibody reactive with CCL20 or a fragment thereof. Including additional necessary reagents and instruments. The antibody reactive with CCL20 and the fragment thereof may be immobilized or labeled according to the measurement method.
[0162] 本発明の別の態様によれば、 CCL20に反応性を有する抗体やそのフラグメントを 含んでなる骨破壊検出剤および骨破壊診断剤が提供される。 [0162] According to another aspect of the present invention, there are provided a bone destruction detection agent and a bone destruction diagnosis agent comprising an antibody reactive with CCL20 and a fragment thereof.
[0163] 本発明の更に別の態様によれば、骨破壊検出剤および骨破壊診断剤の製造のた めの、 CCL20に反応性を有する抗体やそのフラグメントの使用が提供される。 [0163] According to still another aspect of the present invention, there is provided use of an antibody reactive with CCL20 or a fragment thereof for the production of a bone destruction detection agent and a bone destruction diagnosis agent.
実施例  Example
[0164] mn マウス ccuoに針する杭体の作 現解析  [0164] mn mouse Analysis of the pile body that needles ccuo
(1)マウス CCL20に対するモノクローナル抗体の作製  (1) Production of monoclonal antibody against mouse CCL20
抗マウス CCL20抗体は、以下のようにして作製した。マウス CCL20 (R&D社製）と T iterMax™Goldアジュバンドを混合した後、アルメニアハムスター（オリエンタルバイオ 社より入手）に複数回免疫し、さらに最終免疫をマウス CCL20のみで行った。血清中 の抗体価を固相化したマウス CCL20を用いた ELISAで測定し、抗体価が上昇したァ ルメユアハムスターからリンパ球を分離し、リンパ球： P3ミエローマ細胞の比率力 :1に なるように混合し、 PEG1500溶液 (ベーリンガー社製)を用いて細胞融合を行った。 ハイプリドーマは、 RPMI-1640/10% FCS/HAT/10% Origen HCF (ISGN社製)を用い て、プレートで 1週間培養した。 HAT培地（Invitrogen社製）でハイブリドーマを選択し 、得られたノヽイブリドーマの培養上清は、マウス CCL20— APキメラタンパク質を用い たサンドイッチ ELISAによるスクリーニングを行った。その結果、 42個の陽性ゥエル が得られた。つぎにマウス CCL20— APのマウス CCR6発現細胞に対する結合を抑 制することを指標にして、強い結合阻害活性を示すゥエル 2F5を得た。抗マウス CCL 20抗体を産生するハイプリドーマは、 2回の限界希釈によりクローユングを行った。モ ノクローナル抗体は、プリスタンを投与した SCIDおよびヌードマウスにハイプリドーマ を接種して作製した腹水から、 Protein Aカラムを用いて精製した。得られた抗体の中 和活性は、マウス CCR6発現細胞のマウス CCL20に対する遊走を抑制することを指 標にして測定して、中和活性を有する 2F5-5抗体を得た。  Anti-mouse CCL20 antibody was prepared as follows. After mixing mouse CCL20 (manufactured by R & D) and TiterMax ™ Gold adjuvant, Armenian hamster (obtained from Oriental Bio) was immunized several times, and final immunization was performed with mouse CCL20 alone. The antibody titer in the serum was measured by ELISA using mouse CCL20 with solid phase, and lymphocytes were separated from the armored hamster with an increased antibody titer, so that the ratio of lymphocytes to P3 myeloma cells was 1: 1. And cell fusion was performed using a PEG 1500 solution (Boehringer). Hypridoma was cultured on a plate for 1 week using RPMI-1640 / 10% FCS / HAT / 10% Origen HCF (manufactured by ISGN). Hybridomas were selected with HAT medium (Invitrogen), and the culture supernatant of the obtained hybridoma was screened by sandwich ELISA using mouse CCL20-AP chimeric protein. As a result, 42 positive wells were obtained. Next, the inhibition of the binding of mouse CCL20-AP to mouse CCR6-expressing cells was used as an index to obtain a well 2F5 showing strong binding inhibitory activity. Hyperidoma producing anti-mouse CCL 20 antibody was cloned by two limiting dilutions. Monoclonal antibodies were purified using protein A columns from SCID and pristane-administered ascites prepared by inoculating Hypridoma in nude mice. The neutralization activity of the obtained antibody was measured using inhibition of migration of mouse CCR6-expressing cells to mouse CCL20 as an index, and 2F5-5 antibody having neutralizing activity was obtained.
[0165] (2)マウス CCL20に対するモノクローナル抗体の中和活性の同定 [0165] (2) Identification of neutralizing activity of monoclonal antibodies against mouse CCL20
抗マウス CCL20のモノクローナル抗体を産生するハイブリドーマの培養上清をカロ えた場合のマウス CCR6発現 B300. 19細胞へのマウス CCL20— SEAPの結合量 の変化を調べた。ハイプリドーマの培養上清 100 μ 1に結合溶液 (20mM HEPES ( pH 7. 4) , 1% BSA, 0. 02% アジィ匕ナトリウムを含む RPMI— 1640)で 6nMと なるように希釈したマウス CCL20— SEAP溶液 25 μ 1を加え室温で 10分間反応させ た後に、結合溶液で 2χ105個 Ζ25 1に調整したマウス CCR6発現 Β300. 19細胞 2 5 μ 1を混合して 16°Cで 1時間反応させた。結合溶液 150 μ 1で 5回洗浄した後、細胞 を 50 1の 1% Triton X— 100を含む 10mM Tris— HCl (pH8. 0)で溶解した 。次に細胞に由来するフォスファターゼを 65°C10分間の処理で不活ィ匕した後、細胞 溶解液中の AP活性を測定することで、マウス CCR6に結合したマウス CCL20— SE APの量を検出した。結合活性は、抗マウス CCL20抗体非存在下での結合量を 100 %として計算した。 42サンプルのうち、 2ゥエルが強い結合阻害活性を示した。強い 結合阻害活性を示す 2つのゥエルからクローユングを行 、、 1種類のクローン（2F5— 5)を得た。得られた抗マウス CCL20モノクローナル抗体 2F5— 5を産生するハイブリ ドーマをヌードマウスの腹腔に接種し腹水を得て、 Protein Aカラムを用いて抗体を 精製した。精製した抗体を用いて、マウス CCR6発現細胞のマウス CCL20に対する 遊走の抑制活性を測定したところ、 1 μ gZmlでほぼ 100%の中和活性を示すことが 明ら力となった（図 1)。抗マウス CCL20モノクローナル抗体（CCL20 mAb) 2F5- 5を産生するハイブリドーマは、 FERM BP— 10445のもと独立行政法人産業技術 総合研究所特許生物寄託センターに寄託された。 The culture supernatant of the hybridoma producing the monoclonal antibody against anti-mouse CCL20 The change in the amount of mouse CCL20—SEAP binding to mouse CCR6 expression B300.19 cells was examined. Mouse CCL20- diluted to a concentration of 6 nM in a binding solution (RPM—1640 containing 20 mM HEPES (pH 7.4), 1% BSA, 0.02% sodium azide) in 100 μl of culture supernatant of Hypridoma Add SEAP solution 25 μ1 and react at room temperature for 10 minutes, then mix 2 × 10 5 cells with binding solution Ζ25 1 mouse CCR6 expression Β300. 19 cells 25 μ1 mix and react at 16 ° C for 1 hour It was. After 5 washes with 150 μl binding solution, cells were lysed with 10 mM Tris-HCl (pH 8.0) containing 50 1 of 1% Triton X-100. Next, phosphatase derived from cells was inactivated by treatment at 65 ° C for 10 minutes, and then the amount of mouse CCL20-SEAP bound to mouse CCR6 was detected by measuring AP activity in the cell lysate. . The binding activity was calculated assuming that the binding amount in the absence of anti-mouse CCL20 antibody was 100%. Of 42 samples, 2well showed strong binding inhibitory activity. Cloning was performed from two wells showing strong binding inhibitory activity, and one type of clone (2F5-5) was obtained. The resulting hybridoma producing anti-mouse CCL20 monoclonal antibody 2F5-5 was inoculated into the abdominal cavity of nude mice to obtain ascites, and the antibody was purified using a Protein A column. When the activity of suppressing the migration of mouse CCR6-expressing cells to mouse CCL20 was measured using the purified antibody, it became clear that 1 μgZml showed almost 100% neutralizing activity (FIG. 1). The hybridoma producing the anti-mouse CCL20 monoclonal antibody (CCL20 mAb) 2F5-5 was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under FERM BP-10445.
[0166] (3)マウス CCR6遺伝子発現細胞の作製  [0166] (3) Production of mouse CCR6 gene expression cells
マウス CCR6遺伝子発現ベクターの作製は、以下のように行った。  The mouse CCR6 gene expression vector was prepared as follows.
[0167] プライマーは、 GenBankァクセッション番号： NM— 009835の配列をもとに設計し た。  [0167] Primers were designed based on the sequence of GenBank accession number: NM-009835.
[0168] mCCR6 - Sail— F： CGCGTCGACGCCACCATGAATTCCACAGAGTCCTA (配 列番号 1)  [0168] mCCR6-Sail— F: CGCGTCGACGCCACCATGAATTCCACAGAGTCCTA (sequence number 1)
mCCR6 - Notl-R: GCGGGCGGCCGCCATGGTAAAGGACGATGCAT (配列番 号 2)  mCCR6-Notl-R: GCGGGCGGCCGCCATGGTAAAGGACGATGCAT (SEQ ID NO: 2)
マウス脾臓 quick-clone cDNA (Clontech社製)を铸型に用いた。 PCRは、以下の 反応液糸且成（10 Xバッファー 5 1、2. 5mM dNTP 4 1、 Pyrobest polymer ase (TAKARA社製） 0. 5 1、 100 Μプライマー 各 0. 5 1、 cDNA 1 1、蒸 留水 38. 5 1)で行った。 PCRは、 94°Cで 3分間処理した後、 94°Cで 30秒、 65°C で 30秒、 72°Cで 3分の反応を 40サイクル行い、最後に 72°Cで 3分間反応した。増幅 した cDNAを pSPORTl (GIBCO社製）にクローユングして、 ABI3100シーケンス アナライザーを用いて塩基配列を確認した。得られた cDNA断片を発現ベクター pM XII EGFPN (Oncogene (2000) 19 (27): 3050-3058)に挿入して、 mCCR6遺伝子 発現ベクターを作製した。 Mouse spleen quick-clone cDNA (Clontech) was used in a saddle shape. PCR is the following Reaction solution thread (10 X buffer 51, 2.5 mM dNTP 41, Pyrobest polymer ase (manufactured by TAKARA) 0.5 1, 100 100 primer 0.5 1 each, cDNA 1 1, distilled water 38. 5 Performed in 1). PCR was performed at 94 ° C for 3 minutes, followed by 40 cycles of reaction at 94 ° C for 30 seconds, 65 ° C for 30 seconds and 72 ° C for 3 minutes, and finally at 72 ° C for 3 minutes. . The amplified cDNA was cloned into pSPORTl (manufactured by GIBCO) and the base sequence was confirmed using ABI3100 sequence analyzer. The obtained cDNA fragment was inserted into an expression vector pM XII EGFPN (Oncogene (2000) 19 (27): 3050-3058) to prepare an mCCR6 gene expression vector.
[0169] 組換えレトロウイルスの作製は以下のように行った。 [0169] A recombinant retrovirus was produced as follows.
[0170] 293/EBNA- 1細胞株（Invitrogen社製） 3 X 106個を培地（D—MEMZlO%F BS)に懸濁して 10cmディッシュに播き、 COインキュベーターで 24時間培養した。 [0170] 293 / EBNA-1 cell line (manufactured by Invitrogen) 3 X 10 6 cells were suspended in a medium (D-MEMZlO% FBS), seeded in a 10 cm dish, and cultured in a CO incubator for 24 hours.
2  2
翌日、培地を交換し、以下のように調製したトランスフエクシヨン溶液を加えてトランス フエクシヨンを行った。トランスフエクシヨン溶液は、 5mlチューブに OPTI— MEM (GI BCO BRL社製）を 600 μ 1と TransIT LT1 (TaKaRa社製） 24 μ 1をカ卩えて混合して 室温 5分静置した後、発現ベクター 9 μ gとパッケージングベクターである pCL—Ec o (Imgenex社製） 9 μ gを加えて室温 5分静置して調製した。 48時間後に、培養上清 を回収して 0. 45 /z mのフィルターろ過を行い、組換えウィルス液を得た。  On the next day, the medium was changed, and transfection was performed by adding the transfection solution prepared as follows. Transfusion solution is expressed in 600 ml of OPTI-MEM (GI BCO BRL) and 24 μ 1 of TransIT LT1 (TaKaRa) mixed in a 5 ml tube and allowed to stand for 5 minutes at room temperature. The vector 9 μg and the packaging vector pCL-Eco (manufactured by Imgenex) 9 μg were added and the mixture was allowed to stand at room temperature for 5 minutes. After 48 hours, the culture supernatant was collected and filtered through 0.45 / zm to obtain a recombinant virus solution.
[0171] この組換えウィルスを以下のように B300. 19細胞株（EMBO J. (1984)3: 1209-121 9)に感染させ、 mCCR6遺伝子発現細胞を作製した。 B300. 19細胞 2 X 105個を 1. 5mlチューブに加え、 3000rpm、室温で 2分間遠心後、培養上清を吸引除去し た。細胞に、組換えウィルス液 1mlに polybrene ( lOmgZml) 1 μ 1と 55 ^ Μ 2 メルカプトエタノール 1 1をカ卩えて混合した溶液をカ卩え、 24ゥエルプレートに移し 、 C02インキュベーターで 8時間培養して感染を行った。さらに、 1mlの培地 (RPMI - 1640/10%FBS/55uM 2—メルカプトエタノール）をカ卩ぇー晚培養した後に 組換えウィルス液を除去し、培地を加えて培養を行った。マウス CCL20に対して遊 走する細胞を分離することでマウス CCR6発現細胞を得た。 [0171] This recombinant virus was infected with the B300.19 cell line (EMBO J. (1984) 3: 1209-121 9) as follows to prepare mCCR6 gene-expressing cells. B300. 2 2 × 10 5 cells were added to a 1.5 ml tube, centrifuged at 3000 rpm for 2 minutes at room temperature, and the culture supernatant was removed by aspiration. Prepare a mixture of polybrene (lOmgZml) 1 μ 1 and 55 ^ Μ 2 mercaptoethanol 1 1 in 1 ml of recombinant virus solution, transfer to a 24-well plate, and incubate for 8 hours in a C02 incubator And infected. Further, 1 ml of a medium (RPMI-1640 / 10% FBS / 55uM 2-mercaptoethanol) was cultured in a cake, and then the recombinant virus solution was removed, and the medium was added for cultivation. Mouse CCR6-expressing cells were obtained by isolating cells that migrated against mouse CCL20.
[0172] (4)マウス CCL20の細胞外領域と SEAPキメラタンパク質の作製  [0172] (4) Mouse CCL20 extracellular region and SEAP chimeric protein
まず、 pcDNA3. 1 (+ ) -SEAP (His) — Neoベクターを、以下のように作製した [0173] pCDNA3. 1 ( + )— Neoベクター（Invitrogen社製）の内在性の Sailサイトは、 Sail で消化した後、平滑ィ匕を行うことにより欠損させた。 SEAP (His) の cDNA断片は、 First, pcDNA3.1 (1) -SEAP (His) — Neo vector was constructed as follows [0173] The endogenous Sail site of pCDNA3.1 (+) — Neo vector (manufactured by Invitrogen) was deleted by digestion with Sail and smoothing. SEAP (His) cDNA fragment is
10  Ten
pDREF- SEAP His — Hyg (J. Biol. Chem., 1996, 271 , 21514— 21521)を铸型と  pDREF- SEAP His — Hyg (J. Biol. Chem., 1996, 271, 21514— 21521)
6  6
して、 Hindlllを付カ卩した 5 'プライマーと Xholを付カ卩した 3 'プライマーを用いて PCR で増幅した。得られた cDNA断片を Hindlllと Xholで消化した後、 Sailサイトを欠損 させた PCDNA3. 1 ( + )—Neoベクターに挿入した。  Amplification was performed by PCR using 5 'primer with Hindlll and 3' primer with Xhol. The obtained cDNA fragment was digested with Hindlll and Xhol and then inserted into PCDNA3.1 (+) — Neo vector from which the Sail site was deleted.
[0174] プライマーは、 GenBankァクセッション番号： NM— 016960の配列をもとに設計し た。 [0174] Primers were designed based on the sequence of GenBank accession number: NM-016960.
[0175] mMIP— 3 α— Sail— F : CGCGTCGACGCCACCATGGCCTGCGGTGGCAAGCG  [0175] mMIP-3 α-Sail- F: CGCGTCGACGCCACCATGGCCTGCGGTGGCAAGCG
(配列番号 3)  (SEQ ID NO: 3)
mMIP - 3 a— Notl— R : GCGGGCGGCCGCCATCTTCTTGACTCTTAGGC (配 列番号 4)  mMIP-3 a— Notl— R: GCGGGCGGCCGCCATCTTCTTGACTCTTAGGC (sequence number 4)
マウス CCL20の細胞外領域は、 BALB/cマウス小腸よりの全 RNAから RNA PCR kit (TAKARA社製）を用いて 1本鎖 cDNAを合成して铸型として、 Sailを付カ卩した 5 ，プライマー（mMIP - 3 α - Sail - F)と Notlを付カ卩した 3，プライマー（mMIP - 3 a— Notl— R)を用いて PCRで増幅した。 PCRは、以下の反応液組成（10 Xバッフ ァー 5 1、2. 5mM dNTP 4 1、 Pyrobest polymerase (TAKARA社製） 0. 5 1、 100 μ Μプライマー 各 0. 5 1、 cDNA 1 1、蒸留水 38. 5 1)で行った。 PCRは、 94°Cで 3分間処理した後、 94°Cで 30秒、 60°Cで 30秒、 72°Cで 3分の反応 を 40サイクル行い、最後に 72°Cで 3分間反応した。増幅した cDNAを pBlueScriptl I SK (—）（Stratagene社製）にクローユングして、 ABI3100シーケンスアナライザー を用いて塩基配列を確認した。得られた cDNA断片を Sailと Notlで消化した後、上 記 pcDNA3. 1 ( + )— SEAP (His) —Neoベクターに挿入して、マウス CCL20—  The extracellular region of mouse CCL20 consists of a single-stranded cDNA synthesized from total RNA from the BALB / c mouse small intestine using RNA PCR kit (TAKARA), with a sail attached, and a primer. Amplification was performed by PCR using (mMIP-3α-Sail-F) and a primer (mMIP-3a-Notl-R) with Notl attached. PCR was carried out using the following reaction mixture (10 X buffer 51, 2.5 mM dNTP 41, Pyrobest polymerase (TAKARA) 0.5 1, 100 μΜ primer 0.5 1 each, cDNA 1 1, Distilled water 38.5 1) PCR was performed at 94 ° C for 3 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 3 minutes. . The amplified cDNA was cloned into pBlueScriptl SK (—) (Stratagene), and the nucleotide sequence was confirmed using ABI3100 sequence analyzer. The obtained cDNA fragment was digested with Sail and Notl, and inserted into the above pcDNA3.1 (+) — SEAP (His) —Neo vector to obtain mouse CCL20—
10  Ten
AP発現ベクターを作製した。  AP expression vector was prepared.
[0176] これにより、マウス CCL20の細胞外領域と分泌型ヒト胎盤性アルカリフォスファタ一 ゼが 3個のアミノ酸リンカ一 (Ala - Ala - Ala)で結合され、さらに C末端に 10個のヒ スチジンタグ (His) のつ 、た分泌キメラタンパク質（以下 APキメラタンパク質）の発 現が可能となる。得られた APキメラタンパク質発現ベクターは、 TransIT LT1 (TA KARA社製）を用いて 293/EBNA— 1細胞株へ導入し、 4力も 5日間培養を行った 。培養上清中に分泌された APキメラタンパク質は、遠心分離により培養上清を回収 し、 0. 22 mのフィルターでろ過した後、 Hepes (pH 7. 4)とアジ化ナトリウムをそ れぞれ最終濃度が 20mM、 0. 02%となるようにカ卩えて 4°Cで保存した。 APキメラタ ンパク質の濃度は、アルカリフォスファターゼ活性を Aurora AP chemiluminesce nt reporter gene assay (ICN社製）を用いて測定して算出した。 [0176] As a result, the extracellular region of mouse CCL20 and the secreted human placental alkaline phosphatase were linked by a 3 amino acid linker (Ala-Ala-Ala), and 10 histidine tags at the C-terminus. (His), a secreted chimeric protein (hereinafter referred to as AP chimeric protein) The present is possible. The obtained AP chimeric protein expression vector was introduced into 293 / EBNA-1 cell line using TransIT LT1 (manufactured by TAKARA), and cultured for 4 days for 5 days. AP chimera protein secreted into the culture supernatant is collected by centrifugation, filtered through a 0.22 m filter, and then Hepes (pH 7.4) and sodium azide, respectively. The final concentration was 20 mM and was stored at 4 ° C. so as to be 0.02%. The concentration of the AP chimeric protein was calculated by measuring the alkaline phosphatase activity using Aurora AP chemiluminesce reporter gene assay (ICN).
[0177] 「実施例 2Ί コラーゲン誘導閣節炎モデルにおける杭 CCL20杭体の'治療効果  [0177] "Example 2: Pile treatment effect of pile CCL20 pile in collagen-induced pakistritis model"
コラーゲン誘導関節炎（Collagen-Induced Arthritis: CIA)モデルは骨破壊を伴う関 節リウマチの疾患モデルであり、 II型コラーゲンに反応する CD4陽性 T細胞および抗 体が検出されることから、両者が協同して関節炎を惹起すると考えられている。また、 CIAモデルでは、 MHCクラス IIに拘束があると言われている。 CCL20は、活性化上 皮細胞等に発現する細胞遊走分子であり、また CCR6を介して榭状細胞、 T細胞、 B 細胞等の免疫細胞の細胞遊走を誘導する。したがって、抗 CCL20抗体により CCR6 との相互作用を阻害することで、榭状細胞、 T細胞、 B細胞の関与する免疫応答を抑 制する可能性が考えられた。一方で、 CCR6は抑制性 T細胞にも発現しているため、 抗 CCL20抗体により抑制性 T細胞の関与する免疫抑制効果が解除されることで免 疫応答が増強する可能性も考えられた。そこで、コラーゲン誘導関節炎モデルにお ける抗マウス CCL20抗体の治療効果を検討した。  The Collagen-Induced Arthritis (CIA) model is a disease model of rheumatoid arthritis with bone destruction, and CD4 + T cells and antibodies that react with type II collagen are detected. It is thought to cause arthritis. In the CIA model, it is said that MHC class II is restricted. CCL20 is a cell migration molecule expressed in activated epidermal cells and induces cell migration of immune cells such as rod cells, T cells, and B cells via CCR6. Therefore, by inhibiting the interaction with CCR6 with anti-CCL20 antibody, it was possible to suppress the immune response involving rod cells, T cells, and B cells. On the other hand, since CCR6 is also expressed on inhibitory T cells, the anti-CCL20 antibody may be able to enhance the immune response by releasing the immunosuppressive effect involving inhibitory T cells. Therefore, the therapeutic effect of anti-mouse CCL20 antibody in a collagen-induced arthritis model was examined.
[0178] コラーゲン誘導関節炎モデルは、ゥシ関節由来の II型コラーゲン 3%溶液 (コラーゲ ン技術研修会社製)と完全フロイントアジュバント (Difco社製)を等量混合してェマル ジョンを作製し、 5週齢の DBAZUマウス（チャールズリバ一社より入手）に一匹あた り 100 /ζ
Figure imgf000052_0001
—21日目（初回免疫）と 0日目（追加免疫）に臀部皮内 に免疫した。追加免疫から 3日目より体重測定と外見的評価を経時的に行った。外 見的所見は、以下の様にスコア化して評価した。 0 :非発症、 1 :紅斑と足根もしくは足 首の関節に軽い腫脹、 2 :紅斑と足首力 足根にかけて軽い腫脹、 3 :紅斑と足首から 中足の関節にかけての中程度の腫脹、 4 :紅斑と足首、足、指全体に重度の腫脹。 [0178] The collagen-induced arthritis model was prepared by mixing an equal volume of 3% type II collagen solution (made by collagen technology training company) derived from ushi joint and complete Freund's adjuvant (made by Difco). 100 / ζ per DBAZU mouse (obtained from Charles River Company)
Figure imgf000052_0001
-Immunization was performed in the buttocks on day 21 (primary immunization) and day 0 (boost immunization). From the third day after the booster immunization, body weight measurement and appearance evaluation were performed over time. Appearance findings were evaluated by scoring as follows. 0: Non-onset, 1: Erythema and mild swelling at the ankle or ankle joint, 2: Erythema and ankle strength, mild swelling at the ankle, 3: Erythema and moderate swelling from the ankle to the midfoot, 4 : Erythema and severe swelling of the ankles, feet and fingers.
[0179] (1)コラーゲン誘導関節炎マウスの足蹄での CCL20mRNAの発現誘導 コラーゲン誘導関節炎マウスの足蹄から RNAを回収して、 CCL20mRNAの発現を 解析した。解析は後述のリアルタイム PCR法に従って実施した。その結果、コラーゲ ン誘導関節炎を発症したマウスの足蹄で CCL20mRNAの発現が認められた（図 2) 。したがって、 CCL20 mRNAは、イン 'ビボにおける正常時の足蹄ではほとんど発 現していないが、発症時には足蹄に発現していることが明ら力となり、 CCL20はコラ 一ゲン誘導関節炎の発症に関与していることが示唆された。 [0179] (1) Induction of CCL20 mRNA expression in the foot and foot of collagen-induced arthritis mice RNA was collected from the foot and foot of collagen-induced arthritis mice and analyzed for the expression of CCL20 mRNA. The analysis was performed according to the real-time PCR method described later. As a result, expression of CCL20 mRNA was observed in the foot and foot of mice that developed collagen-induced arthritis (Fig. 2). Therefore, CCL20 mRNA is rarely expressed in the normal foot and foot in vivo, but it is evident that it is expressed in the foot and foot at the time of onset, and CCL20 is involved in the development of collagen-induced arthritis. It was suggested that
[0180] (2)抗マウス CCL20抗体によるコラーゲン誘導関節炎の抑制効果  [0180] (2) Inhibitory effect of collagen-induced arthritis by anti-mouse CCL20 antibody
ゥシ関節由来の Π型コラーゲン 3%溶液 (コラーゲン技術研修会社製)と完全フロイ ントアジュバント（Difco社製）を等量混合してェマルジヨンを作製し、 5週齢の DBAZ 1Jマウス（チャールズリバ一社より入手）に一匹あたり 100 1(150 μ gZ匹）を、—21 日目（初回免疫）と 0日目（追加免疫）に臀部皮内に免疫した。抗マウス CCL20抗体 2F5— 5は、追加免疫時から 500 gZl回で週 2回、尾静脈に投与した。追加免疫 力も 3日目より体重測定と外見的（「肉眼的」ということもある。）評価を行った。 13日目 に屠殺した。外見的所見は、以下の様にスコア化して評価した。 0 :非発症、 1 :紅斑 と足根もしくは足首の関節に軽い腫脹、 2 :紅斑と足首力 足根にかけて軽い腫脹、 3 ：紅斑と足首力 中足の関節にかけての中程度の腫脹、 4 :紅斑と足首、足、指全体 に重度の腫脹。その結果、抗マウス CCL20抗体投与により、外見的所見において明 らかな症状の軽減（図 3(a))と、体重減少の抑制が認められた（図 3(b))。  Emulsion was prepared by mixing an equal volume of a 3% solution of cocoon-type collagen derived from tussock joints (manufactured by a collagen technology training company) and complete Freund's adjuvant (manufactured by Difco). A 5-week old DBAZ 1J mouse 100 1 (150 μgZ) per animal was immunized in the buttocks skin on day -21 (primary immunization) and day 0 (boost immunization). Anti-mouse CCL20 antibody 2F5-5 was administered to the tail vein twice a week at 500 gZl from the booster immunization. The booster immunity was also measured from the third day on body weight measurement and appearance (sometimes referred to as “macroscopic”). Slaughtered on day 13. Appearance findings were scored and evaluated as follows. 0: No onset, 1: Erythema and mild swelling in the ankle or ankle joint, 2: Erythema and ankle strength, mild swelling over the ankle, 3: Erythema and ankle strength, moderate swelling over the midfoot joint, 4: Erythema and severe swelling of the entire ankle, foot and fingers. As a result, the administration of anti-mouse CCL20 antibody showed a clear reduction in appearance (Fig. 3 (a)) and suppression of weight loss (Fig. 3 (b)).
[0181] (3)抗マウス CCL20抗体による不完全フロイントアジュバントを用いた追加免疫によ るコラーゲン誘導関節炎の抑制効果  [0181] (3) Suppression of collagen-induced arthritis by booster immunization with incomplete Freund's adjuvant using anti-mouse CCL20 antibody
0日目（追加免疫）をゥシ関節由来の II型コラーゲン 3%溶液 (コラーゲン技術研修 会社製）と完全フロイントアジュバントあるいは不完全フロイントアジュバント（Difco社 製)を等量混合してェマルジヨンを作製し、臀部皮内に免疫した。抗マウス CCL20抗 体 2F5— 5は、追加免疫時から 500 gZl回で週 3回、尾静脈に投与した。追加免 疫から 3日目より体重測定と外見的評価を行い、 13日目に屠殺した。外見的所見は 、以下の様にスコア化して評価した。 0 :非発症、 1 :指に限定された浮腫、 2 :四肢の 軽度の浮腫、 3 :四肢の重度の浮腫。その結果、抗マウス CCL20抗体投与により、不 完全フロイントアジュバント（図 4(a)、（b))および完全フロイントアジュバント（図 4(c)、 (d) )を追加免疫に用いたいずれのコラーゲン誘導関節炎においても、肉眼的所見にお いて明らかな症状の軽減（図 4(a)、（c))と、体重減少の抑制が認められた（図 4(b)、 (d) )。コラーゲン誘導関節炎発症抑制効果は、不完全フロイントアジュバント追加免疫 群のほうが強かった（図 4(a))。 On day 0 (boost immunization), an emulsion of type II collagen 3% solution (manufactured by Collagen Technology Training Company) derived from a Cushion joint and equal volume of complete Freund's adjuvant or incomplete Freund's adjuvant (Difco) was prepared. Immunized in the buttocks skin. Anti-mouse CCL20 antibody 2F5-5 was administered to the tail vein 3 times a week at 500 gZl times from the booster immunization. From the third day after additional immunization, body weight measurements and appearance evaluation were performed, and the animals were sacrificed on day 13. Apparent findings were scored and evaluated as follows. 0: No onset, 1: Finger-limited edema, 2: Mild edema of limbs, 3: Severe edema of limbs. As a result, administration of anti-mouse CCL20 antibody resulted in incomplete Freund's adjuvant (Fig. 4 (a), (b)) and complete Freund's adjuvant (Fig. 4 (c), (d) ) In any collagen-induced arthritis used for booster immunity, the symptoms were clearly reduced by macroscopic findings (Figs. 4 (a) and (c)) and weight loss was suppressed (Fig. 4). (b), (d)). The effect of collagen-induced arthritis onset was stronger in the incomplete Freund's adjuvant immunization group (Fig. 4 (a)).
[0182] また、屠殺したマウスの腹部大静脈よりへノ^ン採血をおこない、血漿中の血清アミ ロイド A (Serum Amyloid A: SAA)濃度、コラーゲンに対する抗体価、 COMP (C artilage Oligomeric Matrix Protein)濃度を測定した。 SAAは炎症刺激により産生さ れたサイト力インの作用により、肝細胞で産生される血漿タンパク質であり、血漿中の SAA濃度は炎症の指標となる。コラーゲンに対する抗体価は抗原特異的液性免疫 応答の指標となる。 COMP(Cartilage Oligomeric Matrix Protein)は、関節軟骨中に 存在するタンパク質であり、軟骨マトリクスの主要成分である Type IIコラーゲンと結合 し、コラーゲンネットワークの安定化機能を有している。炎症性疾患において軟骨が 損傷すると、マトリクス構成蛋白の一部が関節液中に流出してくることが知られている 。 COMPは、関節軟骨の破壊によって Type IIコラーゲンより先に関節液中に流出し、 ついで血中に移行し、その量は、軟骨破壊の指標となる。  [0182] In addition, blood was collected from the abdominal vena cava of a slaughtered mouse, and serum serum amyloid A (Serum Amyloid A: SAA) concentration, antibody titer against collagen, COMP (Cartilage Oligomeric Matrix Protein) Concentration was measured. SAA is a plasma protein produced in hepatocytes by the action of cytodynamic force produced by inflammatory stimuli, and the concentration of SAA in plasma is an indicator of inflammation. The antibody titer against collagen is an index of antigen-specific humoral immune response. COMP (Cartilage Oligomeric Matrix Protein) is a protein present in articular cartilage and binds to Type II collagen, which is a major component of the cartilage matrix, and has a function of stabilizing the collagen network. It is known that when cartilage is damaged in an inflammatory disease, a part of matrix constituent proteins flows into the joint fluid. COMP flows into the synovial fluid prior to Type II collagen due to the destruction of articular cartilage, and then moves into the blood, the amount of which is an indicator of cartilage destruction.
[0183] 血漿中の SAAの濃度は、血漿を 2000力ら 16000倍希釈し、 ELISAキット（バイオ ソース社製)を用いて測定した。血漿中のコラーゲンに対する抗体価の測定は、以下 のように行った。まず、 96穴 ELISAプレート（Nunc社製）に 5 gZmlのゥシ関節由 来の II型コラーゲン溶液を 50 1ずつ加え、 4°Cでー晚静置して固相化した。 T-PB S (0. 02% Tween20ZPBS)で洗浄後、 1% BSAZPBSで非特異的結合部位 をブロックした。 T— PBSで 3回洗浄後、 T—PBSで 10および 50万倍希釈した血漿 を 50 1ずつ各穴にカ卩え、室温で 2時間静置した。 T— PBSで 3回洗浄後、ピオチン 化抗マウス IgGl (BD社製）、ピオチン化抗マウス IgG2a (BD社製）を T—PBSで 100 0倍希釈して 50 1ずつ各穴に加え、室温で 2時間静置した。 T—PBSで 3回洗浄後 、 HRP標識ストレプトアビジン（Pierce社製）を T— PBSで 5000倍希釈して 50 μ 1ず つ各穴に加え、室温で 30分間静置した。その後、発色基質を用いて発色させた。血 漿中の COMPの濃度は、血漿を 20倍希釈し、 ELISAキット（AnaMar社製）を用いて 測定した。 [0184] その結果、血漿中の SAAの濃度は、不完全フロイントアジュバントを追加免疫に用 いたコラーゲン誘導関節炎において抗マウス CCL20抗体の投与により減少する傾 向が見られた（図 5(a))。したがって、不完全フロイントアジュバントを追加免疫に用い た場合、抗マウス CCL20抗体の投与は、炎症反応を抑制する傾向にあることが明ら カゝとなった。 [0183] The concentration of SAA in plasma was measured using an ELISA kit (Biosource) after diluting plasma 12,000 times with 2000 power. The antibody titer against collagen in plasma was measured as follows. First, 50 g of a type II collagen solution derived from a ushi joint was added to a 96-well ELISA plate (manufactured by Nunc) one by one and allowed to stand at 4 ° C for solid phase immobilization. After washing with T-PBS (0.02% Tween20ZPBS), non-specific binding sites were blocked with 1% BSAZPBS. After washing with T-PBS three times, 50-fold diluted plasma with T-PBS was placed in each well and placed at room temperature for 2 hours. After washing 3 times with T-PBS, dilute piotinized anti-mouse IgGl (BD) and piotinylated anti-mouse IgG2a (BD) 1000 times with T-PBS and add 50 1 to each well at room temperature. And left for 2 hours. After washing 3 times with T-PBS, HRP-labeled streptavidin (Pierce) was diluted 5000-fold with T-PBS, added to each 50 μl, and allowed to stand at room temperature for 30 minutes. Thereafter, color was developed using a chromogenic substrate. The concentration of COMP in the plasma was measured by diluting plasma 20 times and using an ELISA kit (AnaMar). [0184] As a result, the concentration of SAA in plasma tended to decrease with the administration of anti-mouse CCL20 antibody in collagen-induced arthritis using incomplete Freund's adjuvant for booster immunization (Fig. 5 (a)). . Therefore, when incomplete Freund's adjuvant was used for booster immunization, it became clear that administration of anti-mouse CCL20 antibody tends to suppress the inflammatory response.
[0185] 一方、血漿中の抗コラーゲン抗体価は、抗マウス CCL20抗体の投与により変化が 認められなかった（図 6(a)、 (b)) 0したがって、抗マウス CCL20抗体の投与は、コラー ゲン特異的液性免疫応答に影響を及ぼさないことが示唆された。 [0185] On the other hand, anti-collagen antibody titer in plasma varies with the administration of anti-mouse CCL20 antibody was observed (FIG. 6 (a), (b) ) administration of 0 Accordingly, an anti-mouse CCL20 antibody, Koller It was suggested that it does not affect the gene-specific humoral immune response.
[0186] 興味深いことに、血漿中の COMPの濃度は、不完全フロイントアジュバントおよび完 全フロイントアジュバントを追加免疫に用 ヽた 、ずれのコラーゲン誘導関節炎にお ヽ ても、抗マウス CCL20抗体の投与により顕著な減少が認められた（図 7)。したがって 、 CCR6— CCL20系を遮断することにより、関節軟骨の破壊を抑制できることが明ら カゝとなった。  [0186] Interestingly, the concentration of COMP in plasma was also increased by the administration of anti-mouse CCL20 antibody, even in cases of collagen-induced arthritis, where incomplete Freund's adjuvant and complete Freund's adjuvant were used for booster immunization. A significant decrease was observed (Figure 7). Therefore, it became clear that blocking the CCR6-CCL20 system can suppress the destruction of articular cartilage.
[0187] さらに、不完全フロイントアジュバントを追加免疫に用いたコラーゲン誘導関節炎の 足蹄における、炎症、細胞浸潤、骨破壊に関与する mRNA発現解析を行った。足蹄 より TorizoKlnvitrigen社製）と RNeasy mini kit (キアゲン社製)を用いて精製した total RNA(500 ng)を AMV Reverse transcriptase (TAKARA社製)と randam hexamer (TAK ARA社製)を用いて逆転写して得た cDNAをテンプレートとして、リアルタイム PCRに解 祈した。リアルタイム PCRは、各種プライマーと QuantiTect SYBR Green PCR kit (Qia gen社製)、 Uracil- DNA-glycosylase (Invitrogen社製)を混合した反応液を調製し、 AB I PRISM 7700 Sequence Detector (Applied Biosystems社製)を用いて行った。 PCRは 、 50°C 2分、 95°C 15分の反応を行った後、 95°C 15秒および 60°C 1分の反応を 35サ イタル実施した。使用したプライマーのセットは以下のとおりである。  [0187] Furthermore, mRNA expression analysis related to inflammation, cell infiltration, and bone destruction was performed in the foot-and-mouth of collagen-induced arthritis using incomplete Freund's adjuvant for booster immunization. Total RNA (500 ng) purified from the foot and foot using TorizoKlnvitrigen) and RNeasy mini kit (Qiagen) was reverse-transcribed using AMV Reverse transcriptase (TAKARA) and randam hexamer (TAK ARA). Using the cDNA obtained as a template, we prayed for real-time PCR. For real-time PCR, prepare a reaction mixture of various primers, QuantiTect SYBR Green PCR kit (Qiagen) and Uracil-DNA-glycosylase (Invitrogen), then AB I PRISM 7700 Sequence Detector (Applied Biosystems) It was performed using. PCR was performed at 50 ° C. for 2 minutes and at 95 ° C. for 15 minutes, and then at 95 ° C. for 15 seconds and at 60 ° C. for 1 minute for 35 sites. The set of primers used is as follows.
[0188] (4)コラーゲン誘導関節炎モデルにおける抗 CCL20抗体の骨破壊抑制効果  [0188] (4) Inhibition of bone destruction by anti-CCL20 antibody in a collagen-induced arthritis model
(3)で調整した血漿を用いて、活性型 TRAP (Tartrate- Resistant Acid Phosphatase )濃度を測定した。 TRAPは、骨吸収を担当する破骨細胞で産生され、血液中に放出 される。従って、血液中の TRAP濃度は、骨破壊の有用な指標となると考えられている 。骨破壊を正確に測定するために、酵素免疫固相化酵素活性測定法によるマウス T ARP活性測定キット (SBA Sciences社製)を用いた。酵素免疫固相化酵素活性測定 法では、破骨細胞力ゝら産生された新鮮な活性型 TRAP type-5のみを検出し、産生さ れてから時間が経って不活化および断片化した TRAP type_5bは検出されない。血 漿中の活性型 TRAPの濃度は、血漿を 4倍希釈して測定した。その結果、血漿中の 活性型 TRAP濃度は、不完全フロイントアジュバントおよび完全フロイントアジュバント を追加免疫に用いた 、ずれのコラーゲン関節炎にお 、ても、抗マウス CCL20抗体 の投与により顕著な減少が認められた（図 8)。したがって、 CCR6— CCL20系を遮 断することにより、炎症による関節の骨破壊を抑制できることが明らかとなった。 Using the plasma prepared in (3), the concentration of active TRAP (Tartrate-Resistant Acid Phosphatase) was measured. TRAP is produced by osteoclasts responsible for bone resorption and released into the blood. Therefore, TRAP concentration in blood is considered to be a useful indicator of bone destruction. To accurately measure bone destruction, mouse T An ARP activity measurement kit (manufactured by SBA Sciences) was used. In the enzyme-linked immunosorbent enzyme assay, only fresh active TRAP type-5 produced by osteoclast force was detected, and TRAP type_5b was inactivated and fragmented over time after production. Is not detected. The concentration of active TRAP in the plasma was measured by diluting plasma 4 times. As a result, the active TRAP concentration in plasma was significantly reduced by the administration of anti-mouse CCL20 antibody even in the case of misaligned collagen arthritis using incomplete Freund's adjuvant and complete Freund's adjuvant for booster immunization. (Figure 8). Therefore, it was clarified that blocking the CCR6-CCL20 system can suppress joint bone destruction caused by inflammation.
プライマー名 Primer name
COX-2 sense 5，- CTCCCTGAAGCCGTACACAT-3 ' (配列番号 5) COX— 2 anti-sense 5 ' - CCCCAAAGATAGCATCTGGA-3 ' (配列番号 6) MMP9 sense 5 ' - AGACGACATAGACGGCATCC- 3 ' (配列番号 7) MMP9 anti-sense 5，- GTGGTTCAGTTGTGGTGGTG-3 ' (配列番号 8) IL-1 j8 sense 5 ' - GCTGAAAGCTCTCCACCTCA -3 ' (配列番号 9) IL-1 j8 anti-sense 5 ' - AGGCCACAGGTATTTTGTCG -3 ' (配列番号 10) IL~6 sense 5 ' - CAAAGCCAGAGTCCTTCAGAG-3 ' (配列番号 11) IL~6 anti-sense 5 ' - GCCACTCCTTCTGTGACTCC-3 ' (配列番号 12) KC sense 5 ' - CTTGAAGGTGTTGCCCTCAG-3 ' (配列番号 13) KC anti-sense 5 ' - TGGGGACACCTTTTAGCATC-3 ' (配列番号 14) MIP-2 sense 5，- TCCAGAGCTTGAGTGTGACG-3 ' (配列番号 15) MIP-2 anti-sense 5，- GCCTTGCCTTTGTTCAGTATC-3 ' (配列番号 16) MIP-1 sense 5 ' - ACCATGACACTCTGCAACCA- 3 ' (配列番号 17) MIP-1 a anti-sense 5， - GATGAATTGGCGTGGAATCT- 3 ' (配列番号 18) MIP-1 β sense 5，- CCCACTTCCTGCTGTTTCTC- 3， （配列番号 19) MIP-1 β anti-sense 5， - CTCACTGGGGTTAGCACAGA- 3， （配列番号 20) CCL20 sense 5 ' - CTTGCTTTGGCATGGGTACT- 3 ' (配列番号 21) CCL20 anti-sense 5 ' - CTTCATCGGCCATCTGTCTT- 3， （配列番号 22) CCR5 sense 5， - GCCAGAGGAGGTGAGACATC- 3 ' (配列番号 23) CCR5 anti-sense 5' - GCCAGAGGAGGTGAGACATC- 3' (配列番号 24) CCR6 sense 5' - CCTGCCTGGGGAATGAATTC-3 ' (配列番号 25) CCR6 anti-sense 5' - TGGCACAAATACCTTGGTGA-3 ' (配列番号 26) CD45 sense 5' - TGACTCATGTGCTCCAGCTA -3' (配列番号 27) CD45 anti-sense 5，- AGCCTTTTCTTTTGGTGTGC -3' (配列番号 28) CD4 sense 5' - GGGCTGTGGCAGTGTCTACT -3' (配列番号 29) CD4 anti-sense 5 ' - CTGGTTCACCCCTCTGGATA -3 ' (配列番号 30COX-2 sense 5,-CTCCCTGAAGCCGTACACAT-3 '(SEQ ID NO: 5) COX— 2 anti-sense 5'-CCCCAAAGATAGCATCTGGA-3 '(SEQ ID NO: 6) MMP9 sense 5'-AGACGACATAGACGGCATCC- 3 '(SEQ ID NO: 7) MMP9 anti -sense 5, -GTGGTTCAGTTGTGGTGGTG-3 '(SEQ ID NO: 8) IL-1 j8 sense 5'-GCTGAAAGCTCTCCACCTCA -3 '(SEQ ID NO: 9) IL-1 j8 anti-sense 5'-AGGCCACAGGTATTTTGTCG -3 '(SEQ ID NO: 10) IL ~ 6 sense 5 '-CAAAGCCAGAGTCCTTCAGAG-3' (SEQ ID NO: 11) IL ~ 6 anti-sense 5 '-GCCACTCCTTCTGTGACTCC-3' (SEQ ID NO: 12) KC sense 5 '-CTTGAAGGTGTTGCCCTCAG-3' (SEQ ID NO: 13) KC anti -sense 5 '-TGGGGACACCTTTTAGCATC-3' (SEQ ID NO: 14) MIP-2 sense 5,-TCCAGAGCTTGAGTGTGACG-3 '(SEQ ID NO: 15) MIP-2 anti-sense 5,-GCCTTGCCTTTGTTCAGTATC-3' (SEQ ID NO: 16) MIP- 1 sense 5 '-ACCATGACACTCTGCAACCA- 3' (SEQ ID NO: 17) MIP-1 a anti-sense 5,-GATGAATTGGCGTGGAATCT- 3 '(SEQ ID NO: 18) MIP-1 β sense 5,-CCCACTTCCTGCTGTTTCTC-3, (SEQ ID NO: 19) MIP-1 β anti-sense 5,-CTCACTGGGGTTAG CACAGA-3, (SEQ ID NO: 20) CCL20 sense 5 '-CTTGCTTTGGCATGGGTACT-3-3 (SEQ ID NO: 21) CCL20 anti-sense 5'-CTTCATCGGCCATCTGTCTT-3, (SEQ ID NO: 22) CCR5 sense 5,-GCCAGAGGAGGTGAGACATC-3 '(sequence (Number 23) CCR5 anti-sense 5 '-GCCAGAGGAGGTGAGACATC- 3' (SEQ ID NO: 24) CCR6 sense 5 '-CCTGCCTGGGGAATGAATTC-3' (SEQ ID NO: 25) CCR6 anti-sense 5 '-TGGCACAAATACCTTGGTGA-3' (SEQ ID NO: 26) CD45 sense 5 ' -TGACTCATGTGCTCCAGCTA -3 '(SEQ ID NO: 27) CD45 anti-sense 5,-AGCCTTTTCTTTTGGTGTGC -3' (SEQ ID NO: 28) CD4 sense 5 '-GGGCTGTGGCAGTGTCTACT -3' (SEQ ID NO: 29) CD4 anti-sense 5 '-CTGGTTCACCCCTCTGGATA -3 '(SEQ ID NO: 30
) )
M-CSFR sense 5，- CGACTTCTTCAAGTGACTCCTTC -3， （配列番号 31  M-CSFR sense 5,-CGACTTCTTCAAGTGACTCCTTC -3, (SEQ ID NO: 31
5' - CTACGTCCCGGTGGATGC -3' (配列番号 32) - TGCCTCCCTGACTTTCAAAT -3' (配列番号 33)5 '-CTACGTCCCGGTGGATGC -3' (SEQ ID NO: 32)-TGCCTCCCTGACTTTCAAAT -3 '(SEQ ID NO: 33)
F4/80 anti-sense ' - TGGCATTGCTGTATCTGCTC -3' (配列番号 34) Ly6 sense - GATGGATTTTGCGTTGCTCT -3' (配列番号 35) Ly6 anti-sense ' - GTCCAGAGTAGTGGGGCAGA -3' (配列番号 36) RANKL sense ，- CATTTGCACACCTCACCATC -3， （配列番号 37) RANKL anti-sense 5，- TCCGTTGCTTAACGTCATGT -3' (配列番号 38) RANK sense ' - CGGCGTTTACTACAGGAAGG-3 ' (配列番号 39) RANK anti-sense 5，- TTCTTGCTGACTGGAGGTTG-3 ' (配列番号 40) TRAP sense - GCTGGAAACCATGATCACCT -3， （配列番号 41) TRAP anti-sense GGTAGTAAGGGCTGGGGAAG -3' (配列番号 42) Cathepsin K sense ' - CAGTGTTGGTGGTGGGCTAT -3， （配列番号 43) Cathepsin K anti-sense 5，- CCGAGCCAAGAGAGCATATC -3， （配列番号 4F4 / 80 anti-sense '-TGGCATTGCTGTATCTGCTC -3' (SEQ ID NO: 34) Ly6 sense-GATGGATTTTGCGTTGCTCT -3 '(SEQ ID NO: 35) Ly6 anti-sense'-GTCCAGAGTAGTGGGGCAGA -3 '(SEQ ID NO: 36) RANKL sense,-CATTTGCACACCTCACCATC- 3, (SEQ ID NO: 37) RANKL anti-sense 5,-TCCGTTGCTTAACGTCATGT -3 '(SEQ ID NO: 38) RANK sense'-CGGCGTTTACTACAGGAAGG-3 '(SEQ ID NO: 39) RANK anti-sense 5,-TTCTTGCTGACTGGAGGTTG-3' (SEQ ID NO: 38) 40) TRAP sense-GCTGGAAACCATGATCACCT -3, (SEQ ID NO: 41) TRAP anti-sense GGTAGTAAGGGCTGGGGAAG -3 '(SEQ ID NO: 42) Cathepsin K sense'-CAGTGTTGGTGGTGGGCTAT -3, (SEQ ID NO: 43) Cathepsin K anti-sense 5,-CCGAGCCAAGAGAGCATATC -3, (SEQ ID NO: 4
4) Four)
抗 CCL20抗体で発症抑制効果が認められたマウスでは、炎症（COX— 2(図 9(a)) 、 IL 1 18 (図 9(b》、 MMP9(図 9(c))、 IL 6(図 9(d)))、細胞浸潤を主に制御するケ モカイン *ケモカイン受容体 (KC (図 10(a))、 MIP— 1 α (図 10(b))、 CCR5(図 10(c))、 MIP— 2(図 10(d))、 MIP— 1 j8 (図 10(e》， CCR6(図 10(D) , CCL20(図 10(g)))、免 疫細胞マーカー（CD45(図 l l(a))、 M— CSFR (図 l l(b))、 CD4(図 l l(c))、 F4/80( 図 l l(d))、 Ly6G (図 11(e)))の足蹄における mRNAの発現がコントロール抗体投与 群では上昇し、抗 CCL20抗体によりその発現が抑制される傾向が認められた。さら に、骨破壊に関係する遺伝子 (RANKL (図 12(a))、 TRAP (図 12(b))、 RANK (図 12( c》、 Cathepsin K (図 12(d)))の発現が顕著に抑制されていることが明ら力となった。 In mice in which the onset suppression effect was observed with anti-CCL20 antibody, inflammation (COX-2 (Fig. 9 (a)), IL 118 (Fig. 9 (b), MMP9 (Fig. 9 (c))), IL 6 (Fig. 9 (d))), chemokines that mainly control cell invasion * chemokine receptors (KC (Fig. 10 (a)), MIP-1α (Fig. 10 (b)), CCR5 (Fig. 10 (c)) , MIP-2 (Fig. 10 (d)), MIP-1 j8 (Fig. 10 (e), CCR6 (Fig. 10 (D), CCL20 (Fig. 10 (g))), Epidemic cell markers (CD45 (Fig. Ll (a)), M—CSFR (Fig. Ll (b)), CD4 (Fig. Ll (c)), F4 / 80 (Fig. Ll (d)), Ly6G (Fig. 11 (e The expression of mRNA in the foot and foot of))) increased in the control antibody-administered group, and it was observed that the expression was suppressed by anti-CCL20 antibody. Furthermore, the expression of genes related to bone destruction (RANKL (Fig. 12 (a)), TRAP (Fig. 12 (b)), RANK (Fig. 12 (c), Cathepsin K (Fig. 12 (d))) is remarkable. It became clear that it was restrained by.
[0190] (5)抗マウス CCL20抗体の発症後投与によるコラーゲン関節炎における骨破壊抑制 効果 [0190] (5) Inhibition of bone destruction in collagen arthritis by post-onset administration of anti-mouse CCL20 antibody
コラーゲン関節炎を (3)の記載と同様に惹起し、追加免疫から 6日後に発症した固 体を選抜して実験に供した。外見的所見は（3)と同様にスコア化し、抗マウス CCL2 0抗体 2F5— 5は追加免疫から 6日後より 500 μ gZl回で一日おきに尾静脈に投与 した。追加免疫から 17日目に屠殺し、 Agents Actions, 39; 187-194, 1993に記載の 評価法に従って四肢の軟 X線画像を以下のようにスコア化し骨破壊を評価した。 0： 非発症、 1 :軽度な骨破壊、 2 :中等度の骨破壊、 3 :重度の骨破壊。その結果、発症 後に投与を開始した抗マウス CCL20抗体により外見的所見（図 13 (a) )のみならず 骨破壊（図 13 (b) )の進展を抑制することが明らかとなった。  Collagen arthritis was induced as described in (3), and solids that developed 6 days after the booster were selected and used for the experiment. Apparent findings were scored in the same manner as in (3), and anti-mouse CCL20 antibody 2F5-5 was administered to the tail vein every other day at 500 μgZl from 6 days after booster immunization. The animals were sacrificed on the 17th day after the booster immunization, and soft X-ray images of the limbs were scored as follows according to the evaluation method described in Agents Actions, 39; 187-194, 1993 to evaluate bone destruction. 0: Non-onset, 1: mild bone destruction, 2: moderate bone destruction, 3: severe bone destruction. As a result, it became clear that anti-mouse CCL20 antibody started administration after onset suppresses not only the appearance (Fig. 13 (a)) but also the development of bone destruction (Fig. 13 (b)).
[0191] 以上の結果から、 CCR6-CCL20経路がコラーゲン誘導関節炎モデルにおいて重 要な役割を果たすことが明らかとなった。その作用は細胞浸潤を抑制することにより 炎症反応を軽減することに加え、骨 (軟骨)破壊に関与する細胞の足蹄への浸潤や 分ィ匕を顕著に抑制することにより、組織障害を軽減しているものと考えられる。 [0191] The above results revealed that the CCR6-CCL20 pathway plays an important role in the collagen-induced arthritis model. In addition to reducing the inflammatory response by suppressing cell infiltration, the action reduces tissue damage by significantly suppressing the invasion and separation of cells involved in bone (cartilage) destruction. It is thought that.

Claims

請求の範囲 The scope of the claims
[1] CCL20に反応性を有する抗体またはそのフラグメントを有効成分として含んでなる 、骨破壊抑制剤。  [1] An agent for inhibiting bone destruction, comprising an antibody or fragment thereof having reactivity with CCL20 as an active ingredient.
[2] 抗体が、モノクローナル抗体である、請求項 1に記載の骨破壊抑制剤。  [2] The bone destruction inhibitor according to claim 1, wherein the antibody is a monoclonal antibody.
[3] 抗体が、キメラモノクローナル抗体、ヒト型モノクローナル抗体、ヒトモノクローナル抗 体、およびこれらの抗体フラグメントからなる群力 選択される、請求項 1に記載の骨 破壊抑制剤。  [3] The bone destruction inhibitor according to claim 1, wherein the antibody is selected from the group consisting of a chimeric monoclonal antibody, a human monoclonal antibody, a human monoclonal antibody, and an antibody fragment thereof.
[4] モノクローナル抗体力 FERM BP— 10445の受託番号のもと寄託されたハイブ リドーマにより産生される抗体である、請求項 2または 3に記載の骨破壊抑制剤。  [4] Monoclonal antibody potency The bone destruction inhibitor according to claim 2 or 3, which is an antibody produced by a hybridoma deposited under a deposit number of FERM BP-10445.
[5] FERM BP— 10445の受託番号のもと寄託されたハイブリドーマ。  [5] FERM BP—Hybridoma deposited under the deposit number 10445.
[6] 被検物質の存在下および非存在下のそれぞれの条件において、 CCL20の CCR6 への作用の程度を測定する工程と、被験物質の非存在下での作用の程度と、被験 物質の存在下での作用の程度とを比較する工程とを含んでなる、骨破壊の治療に有 用な物質のスクリ一ユング方法。  [6] A step of measuring the degree of action of CCL20 on CCR6 in each condition in the presence and absence of the test substance, the degree of action in the absence of the test substance, and the presence of the test substance A method for screening a substance useful for the treatment of bone destruction comprising the step of comparing the degree of action below.
[7] 被検物質の非存在下よりも被検物質の存在下にお!/、て CCL20の CCR6への作用 が抑制される場合に、被検物質を骨破壊の治療に有用な物質であると判定する工程 を更に含んでなる、請求項 6に記載のスクリ一ユング方法。  [7] When the action of CCL20 on CCR6 is suppressed in the presence of the test substance rather than in the absence of the test substance, the test substance is a substance useful for the treatment of bone destruction. The screening method according to claim 6, further comprising a step of determining that there is.
[8] CCR6を含む細胞膜またはそれを含む細胞と CCL20とを接触させ、次いで細胞刺 激活性を測定する工程と、被験物質の非存在下での細胞刺激活性と、被験物質の 存在下での細胞刺激活性とを比較する工程とを含んでなる、請求項 6または 7に記載 のスクリーニング方法。  [8] A step of contacting CCL20 with a cell membrane containing CCR6 or a cell containing the cell membrane, and then measuring cell stimulating activity, cell stimulating activity in the absence of the test substance, and in the presence of the test substance The screening method according to claim 6 or 7, comprising a step of comparing the cell stimulating activity.
[9] 被検物質の存在下および非存在下のそれぞれの条件において、 CCL20の CCR6 への結合の程度を測定する工程と、被験物質の非存在下での結合の程度と、被験 物質の存在下での作用の程度とを比較する工程とを含んでなる、骨破壊の治療に有 用な物質のスクリ一ユング方法。  [9] The step of measuring the degree of binding of CCL20 to CCR6 in each condition in the presence and absence of the test substance, the degree of binding in the absence of the test substance, and the presence of the test substance A method for screening a substance useful for the treatment of bone destruction comprising the step of comparing the degree of action below.
[10] 被検物質の非存在下よりも被検物質の存在下にお!/、て CCL20の CCR6への結合 が抑制される場合に、被検物質を骨破壊の治療に有用な物質であると判定する工程 を更に含んでなる、請求項 9に記載のスクリ一ユング方法。 [10] When the binding of CCL20 to CCR6 is suppressed in the presence of the test substance rather than in the absence of the test substance, the test substance is a substance useful for the treatment of bone destruction. 10. The screening method according to claim 9, further comprising a step of determining that there is.
[11] CCR6を含む細胞膜またはそれを含む細胞と CCL20とを接触させ、次 、で細胞刺 激活性を測定する工程と、被験物質の非存在下での細胞刺激活性と、被験物質の 存在下での細胞刺激活性とを比較する工程とを含んでなる、請求項 9または 10に記 載のスクリーニング方法。 [11] A step of contacting CCL20 with a cell membrane containing CCR6 or a cell containing the same, and then measuring cell stimulating activity, cell stimulating activity in the absence of the test substance, and in the presence of the test substance The screening method according to claim 9 or 10, which comprises a step of comparing the cell stimulating activity in the method.
[12] CCL20の CCR6への作用の評価を、 CCL20に反応性を有する抗体またはそのフ ラグメントの CCL20阻害作用、あるいは CCR6に反応性を有する抗体またはそのフ ラグメントの CCR6阻害作用との比較により行う工程を更に含んでなる、請求項 6〜1[12] The effect of CCL20 on CCR6 is evaluated by comparing it with the CCL20 inhibitory action of an antibody reactive with CCL20 or its fragment, or with the CCR6 inhibitory action of an antibody reactive with CCR6 or its fragment. The method of claim 6 further comprising the steps of:
1のいずれか一項に記載のスクリーニング方法。 The screening method according to any one of 1.
[13] 被験組織における CCL20の発現の程度を検出する工程を含んでなる、骨破壊の 検出方法および骨破壊の診断方法。 [13] A method for detecting bone destruction and a method for diagnosing bone destruction, comprising a step of detecting the degree of expression of CCL20 in a test tissue.
[14] CCL20に反応性を有する抗体またはそのフラグメントを使用して CCL20の発現の 程度を検出する、請求項 13に記載の方法。 [14] The method according to claim 13, wherein the level of expression of CCL20 is detected using an antibody or fragment thereof reactive to CCL20.
[15] 正常組織における CCL20の発現の程度と被験組織における CCL20の発現の程 度を比較する工程を更に含んでなる、請求項 13または 14に記載の方法。 [15] The method according to claim 13 or 14, further comprising a step of comparing the degree of expression of CCL20 in a normal tissue with the degree of expression of CCL20 in a test tissue.
[16] 被検組織における CCL20の発現の程度力 正常組織における CCL20の発現の 程度を超える場合に、骨破壊が生じているまたは骨破壊に罹っていると判定するェ 程を更に含んでなる、請求項 15に記載の方法。 [16] The degree of CCL20 expression in the test tissue further comprises the step of determining that bone destruction has occurred or is suffering from bone destruction if the degree of CCL20 expression in normal tissue is exceeded. 16. A method according to claim 15.
[17] CCL20に反応性を有する抗体またはそのフラグメントを含んでなる、骨破壊検出 剤および骨破壊診断剤。 [17] An agent for detecting bone destruction and a diagnostic agent for bone destruction, comprising an antibody having CCL20 reactivity or a fragment thereof.
[18] CCL20に反応性を有する抗体またはそのフラグメントを含んでなる、骨破壊検出キ ットおよび骨破壊診断キット。 [18] A bone destruction detection kit and a bone destruction diagnosis kit comprising an antibody reactive with CCL20 or a fragment thereof.
PCT/JP2007/050823 2006-01-19 2007-01-19 Bone destruction inhibitor comprising anti-ccl20 antibody WO2007083759A1 (en)

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