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WO2007083759A1 - Inhibiteur de la destruction osseuse comprenant un anticorps anti-ccl20 - Google Patents

Inhibiteur de la destruction osseuse comprenant un anticorps anti-ccl20 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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Prior art keywords
ccl20
antibody
ccr6
bone destruction
monoclonal antibody
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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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Eisai R&D Management Co Ltd
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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

L'invention concerne un produit pharmaceutique qui permet d'inhiber efficacement la destruction osseuse. Elle concerne ainsi un inhibiteur de la destruction osseuse qui comprend comme principe actif un anticorps monoclonal réactif au CCL20 ou un fragment de cet anticorps monoclonal.
PCT/JP2007/050823 2006-01-19 2007-01-19 Inhibiteur de la destruction osseuse comprenant un anticorps anti-ccl20 Ceased WO2007083759A1 (fr)

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JP2006011525A JP2009096716A (ja) 2006-01-19 2006-01-19 抗ｃｃｌ２０抗体による自己免疫疾患の治療
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JP2014502154A (ja) * 2010-11-19 2014-01-30 俊夫 今井 中和抗ｃｃｌ２０抗体

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JP5864564B2 (ja) * 2010-06-28 2016-02-17 ユニベルシテーツクリニクム フライブルグ 線維性疾患および癌における治療選択肢としてのｃｃｒ６を介するｃｃｌ１８シグナル伝達の遮断
JP6220222B2 (ja) * 2013-10-28 2017-10-25 シスメックス株式会社 関節リウマチの診断を補助するための方法、システム及びコンピュータプログラム製品

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014502154A (ja) * 2010-11-19 2014-01-30 俊夫 今井 中和抗ｃｃｌ２０抗体
EP2640744A4 (fr) * 2010-11-19 2014-05-28 Eisai R&D Man Co Ltd Neutralisation d'anticorps anti-ccl20
US9133273B2 (en) 2010-11-19 2015-09-15 Eisai R&D Management Co., Ltd. Nucleic acids encoding neutralizing anti-CCL20 antibodies
JP2016093195A (ja) * 2010-11-19 2016-05-26 エーザイ・アール・アンド・ディー・マネジメント株式会社 中和抗ｃｃｌ２０抗体
US9809647B2 (en) 2010-11-19 2017-11-07 Eisai R&D Management Co., Ltd. Neutralizing anti-CCL20 antibodies
CN102540784A (zh) * 2010-12-20 2012-07-04 Asml荷兰有限公司 更新校准数据的方法和器件制造方法

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