WO2013005649A1 - Anticorps anti-ccr6 humain - Google Patents

Anticorps anti-ccr6 humain Download PDF

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WO2013005649A1
WO2013005649A1 PCT/JP2012/066594 JP2012066594W WO2013005649A1 WO 2013005649 A1 WO2013005649 A1 WO 2013005649A1 JP 2012066594 W JP2012066594 W JP 2012066594W WO 2013005649 A1 WO2013005649 A1 WO 2013005649A1
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antibody
ccr6
human
amino acid
seq
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PCT/JP2012/066594
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Japanese (ja)
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裕也 磯田
真美 小山
神田　豊
山野　和也
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協和発酵キリン株式会社
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    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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

Definitions

  • the present invention includes an antibody that binds to the extracellular region of sulfated human CCR6 and the antibody fragment, a hybridoma that produces the antibody, a DNA encoding the antibody, a vector containing the DNA, and a vector obtained by introducing the vector.
  • the present invention relates to a transformant obtained, a method for producing an antibody or antibody fragment using the hybridoma or the transformant, and a therapeutic agent and a diagnostic agent containing the antibody or antibody fragment.
  • the human CC chemokine receptor 6 (CC chemokine receptor 6 or chemokine, CC motif, receptor 6) (hereinafter referred to as CCR6) is also known as chemokine receptor-like 3 (CKR-L3), YTL6PR, YTL4PR BN-1, DCCR2, DCR2, surface antigen classification (hereinafter referred to as CD) 196 (CD196), G protein-coupled receptor 29 (G protein-coupled receptor 29; GPR29), CMKBR6, CC-CKR- Also known as 6.
  • Human CCR6 was isolated and identified as orphan receptor CKR-L3 in 1996 by Pharmacia Upjohn (Non-patent Document 1). On the other hand, a gene encoding human CCR6 protein was cloned in 1997 as STRL22 (Non-patent Document 2). Further, the DNA sequence and amino acid sequence of human CCR6 are published on public databases and can be referred to from accession numbers such as NM_004367, NM_031409, and NP_004358, for example. However, the three-dimensional structure of human CCR6 protein has not yet been clarified.
  • Human CCR6 is a G protein-coupled receptor (GPCR) having a seven-transmembrane structure, and is a membrane protein consisting of a molecular weight of about 65 kDa and a total length of 374 amino acids.
  • GPCR G protein-coupled receptor
  • the structural feature of human CCR6 is that an extracellular loop (hereinafter referred to as ECL) 1 and an extracellular loop 2 (ECL2) are intermolecular disulfide bonds, as is commonly recognized in many GPCRs.
  • ECL extracellular loop
  • ECL2 extracellular loop 2
  • Nt The N-terminal extracellular domain containing the 1st to 46th amino acid sequences of human CCR6 has 11 serine residues (Ser) and 1 threonine residue (Ser) and 1 threonine residue (Ser) and 1 threonine residue (Ser) and 1 threonine residue (Ser) and 1 threonine residue (Ser) and 1 threonine residue (Ser) and 1 threonine residue (Ser) and 1 threonine residue (Ser) and 1 threonine residue). It is suggested from the results of in silico prediction experiments that there is a possibility that O-type sugar chains may be added to these Ser and Thr residues. In addition, there are two potential sites for N-linked sugar chain addition in Nt, and one site each in ECL1 and ECL3. However, in reality, N-linked sugar chains are added in vivo. Whether or not it is still unknown.
  • Non-Patent Document 4 the possibility of the 18th tyrosine residue present in Nt being sulfated is suggested from the results of in silico prediction experiments.
  • Non-Patent Document 5 the possibility of the 18th tyrosine residue present in Nt being sulfated is suggested from the results of in silico prediction experiments.
  • Non-Patent Document 5 the possibility of the 18th tyrosine residue present in Nt being sulfated is suggested from the results of in silico prediction experiments.
  • Non-Patent Document 5 the possibility of the 18th tyrosine residue present in Nt being sulfated is suggested from the results of in silico prediction experiments.
  • Non-Patent Document 5 the possibility of the 18th tyrosine residue present in Nt being sulfated is suggested from the results of in silico prediction experiments.
  • sulfation of Tyr residues existing in the N-terminal extracellular region has been confirmed in vitro, and these sulfated Tyr
  • IL interleukin
  • Non-patent Documents 7 and 8 As ligands that bind to human CCR6, chemokine types Mip-3 ⁇ (LARC / CCL20) and defensin ⁇ (defensin- ⁇ ) have been reported (Non-patent Documents 7 and 8). It has been revealed that macrophage inflammatory protein (Macrophage inflammatory protein 3 ⁇ ; Mip-3 ⁇ ) is secreted from macrophages and hepatocytes, and defensin ⁇ is secreted from epithelium, skin, oral cavity and trachea, respectively. It is known to be involved in the defense of infection by attracting to the local site of inflammation and also involved in the induction of inflammation.
  • Macrophage inflammatory protein 3 ⁇ is secreted from macrophages and hepatocytes
  • defensin ⁇ is secreted from epithelium, skin, oral cavity and trachea, respectively. It is known to be involved in the defense of infection by attracting to the local site of inflammation and also
  • Th17 cells Human CCR6 is expressed in Th17 cells.
  • Th1 cells involved in cellular immunity
  • Th2 cells involved in humoral immunity.
  • the Th17 cell is a helper T cell newly discovered in recent years as one of the subsets of helper T cells having different properties from Th1 cells and Th2 cells.
  • Th17 cells occupy about several percent of CD4 positive helper T cells in human blood. Th17 cells have the ability to produce inflammatory cytokines IL-17, IL-21 and IL-22 (Non-Patent Document 9), rheumatoid arthritis (RA), multiple sclerosis (MS), It has been revealed that it is closely involved in the pathogenesis of autoimmune diseases such as inflammatory bowel disease (IBD), asthma, psoriasis and systemic lupus erythematosus (SLE) (Non-patent Documents 10-14).
  • IBD inflammatory bowel disease
  • SLE systemic lupus erythematosus
  • Th17 cells are currently attracting worldwide attention as new target cells for therapeutic drugs for autoimmune diseases. It has been revealed that most Th17 cells in human peripheral blood express CCR6 (Non-patent Documents 15-19). So far, human CCR6 has been used in various studies as a specific marker for Th17 cells.
  • Th17 cells have been implicated in HIV infection, graft-versus-host disease (GVHD), periodontal disease, and the like (Non-patent Documents 20-22).
  • Human CCR6 is also expressed in Th1 / 17 cells, ⁇ T cells, Th22 cells, memory B cells, naive B cells, some CD8 cells and some Th1 cells in addition to Th17 cells. Have also been reported to be associated with autoimmune diseases (Non-Patent Documents 23-29). Furthermore, it is known that human CCR6 is also expressed in a cell group (population) having IL-17 production ability among regulatory T cells (regulatory T cell; Treg) (Non-patent Document 30). In recent years, cells of the innate immune system called Innate Lymphoid cells (innate immunity lymphoid cells; ILC) have been revealed (Non-patent Document 31), and some of them (ILC17 or ILC2 etc.) are human CCR6. Has been reported to be associated with the expression of cerebral dysfunction and disease state (IBD or sinusitis) (Non-patent Documents 32-34).
  • CCR6 expression has also been confirmed in cancers such as liver cancer, pancreatic cancer, prostate cancer, and colon cancer, and is considered to be related to cancer growth, invasion, and metastasis (Non-patent Document 35-). 38).
  • anti-CCR6 antibody examples include 53103 (R & D systems), 11A9 (BD Pharmingen), 4C6 (ATGen), R6H1 (eBioscience), TG7 (BioLegend) or MM (abcam). It has been known.
  • the present invention relates to an antibody that binds to sulfated human CCR6 and the antibody fragment, a hybridoma that produces the antibody, a DNA encoding the antibody, a vector containing the DNA, and a trait obtained by introducing the vector. It is an object of the present invention to provide a transformant, a method for producing an antibody or the antibody fragment using the hybridoma or the transformant, and a therapeutic agent and a diagnostic agent using the antibody or the antibody fragment.
  • CCR6 CC chemokine receptor 6
  • the antibody and the antibody fragment according to (1) or (2), wherein the epitope to which the antibody and the antibody fragment bind includes at least the 18th Tyr of the amino acid sequence represented by SEQ ID NO: 2.
  • CDRs Antibody variable regions comprising CDR1 to chain variable regions 1 to 3
  • the antibody variable region comprising CDR1 to CDR3 of the antibody light chain variable region, and the CDR1 to CDR3 of the antibody heavy chain variable region comprising the amino acid sequence represented by SEQ ID NOs: 80 to 82 and represented by SEQ ID NOs: 83 to 85, respectively.
  • An antibody comprising any one antibody variable region selected from antibody variable regions comprising CDR1 to CDR3 of an antibody light chain variable region comprising an amino acid sequence.
  • B An antibody that competitively reacts with any one antibody selected from the antibodies represented by (a) and the antibody fragment.
  • C An antibody that reacts with an epitope including an epitope with which any one antibody selected from the antibodies represented by (a) reacts, and the antibody fragment.
  • (F) An antibody that competitively reacts with any one antibody selected from the antibodies represented by (d) above and the antibody fragment.
  • G An antibody that reacts with an epitope including an epitope with which any one antibody selected from the antibodies represented by (d) reacts, and the antibody fragment.
  • H An antibody that reacts with the same epitope as the epitope with which any one antibody selected from the antibodies represented by (d) reacts, and the antibody fragment.
  • antibody fragment from a peptide comprising Fab, Fab ′, (Fab ′) 2 , single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv) and CDR The antibody fragment according to any one of (1) to (9), which is selected.
  • (11) A DNA encoding the antibody or the antibody fragment thereof according to any one of (1) to (10).
  • a transformant obtained by introducing the vector containing the DNA described in (11) is cultured in a medium, and the antibody or the antibody fragment described in any one of 1 to 10 is produced in the culture.
  • a diagnostic agent for a disease involving human CCR6-positive cells comprising the antibody or the antibody fragment according to any one of (1) to (10) as an active ingredient.
  • the diagnostic agent according to (14), wherein the disease involving human CCR6-positive cells is a disease selected from cancer and autoimmune diseases.
  • the diagnostic agent according to (14) or (15), wherein the human CCR6-positive cell is at least one cell selected from naive / memory B cells, Th1 / 17 cells, Th17 cells, and Th22 cells.
  • a therapeutic agent for a disease involving human CCR6-positive cells comprising the antibody or the antibody fragment thereof according to any one of (1) to (10) as an active ingredient.
  • the therapeutic agent according to (17), wherein the disease involving human CCR6-positive cells is a disease selected from cancer and autoimmune diseases.
  • the therapeutic agent according to (17) or (18), wherein the human CCR6-positive cells are at least one cell selected from naive / memory B cells, Th1 / 17 cells, Th17 cells and Th22 cells.
  • a method for diagnosing a disease involving human CCR6-positive cells comprising detecting or measuring human CCR6-positive cells using the antibody or the antibody fragment thereof according to any one of (1) to (10).
  • (21) Use of the antibody or antibody fragment according to any one of (1) to (10) for producing a diagnostic agent for a disease involving human CCR6-positive cells.
  • (22) Use of the antibody or antibody fragment thereof according to any one of (1) to (10) for producing a therapeutic agent for a disease involving human CCR6-positive cells.
  • an antibody that binds to sulfated human CCR6 and the antibody fragment a hybridoma that produces the antibody, a DNA encoding the antibody, a vector containing the DNA, and a transformant obtained by introducing the vector , A method for producing an antibody or antibody fragment using the hybridoma or the transformant, and a therapeutic agent and a diagnostic agent using the antibody or antibody fragment.
  • FIG. 1 shows a schematic diagram of the cloning of the human CCR6 gene.
  • FIG. 2 (a) shows a schematic diagram of the construction of the human CCR6 expression vector pKANTEX-hCCR6.
  • FIG. 2 (b) shows a schematic diagram of the construction of Tc26-hCCR6.
  • FIGS. 3 (a) and (b) show the results of FCM analysis of the CCR6 expression level on the cell surface of human CCR6-expressing cells CHO-hCCR6 [FIG. 3 (a)] and CHOK1-hCCR6 [FIG. 3 (b)]. Show.
  • FIG. 3 (c) shows the result of FCM analysis for the hCCR6-His tag expressed in CHO-hCCR6.
  • FIG. 4 (a) and 4 (b) show the results of FCM analysis of the CCR6 expression level on the cell surface of human CCR6-expressing cells U937-CCR6 [FIG. 4 (a)] and CCR6G16 [FIG. 4 (b)].
  • the vertical axis represents the number of cells and the horizontal axis represents the fluorescence intensity [FIG. 4 (a) is FL1, FIG. 4 (b) is FL3].
  • FIG. 5 shows a schematic diagram of the construction of various human mouse chimeric CCR6 expression vectors.
  • FIG. 5 shows a schematic diagram of the construction of various human mouse chimeric CCR6 expression vectors.
  • FIGS. 7 (a) to (g) show the results of analyzing the expression level of human mouse chimeric CCR6 on the cell surface of various cells by FCM using an anti-human CCR6 monoclonal antibody.
  • Parent strains CHO / DG44 [FIG. 7 (a)] Various human mouse chimeric CCR6-expressing cells [CHO-mECL2hCCR6 [FIG. 7 (b)], CHO-mNthCCR6 [FIG. 7 (c)], CHO-mECL3hCCR6 [FIG. 7 (d) )], CHO-mECL1hCCR6 [FIG.
  • FIG. 7 (e) CHO-hCCR [FIG. 7 (f)] and CHO-mNt-hCCR6 [FIG. 7 (g)]
  • the expression level of human mouse chimeric CCR6 on the cell surface The result analyzed by FCM using an anti-human CCR6 monoclonal antibody is shown.
  • the vertical axis represents the number of cells and the horizontal axis represents the fluorescence intensity [FIG. 7 (a) to FIG. 7 (f) is FL1, FIG. 7 (g) is FL2].
  • 8A to 8D show various human mouse chimeric CCR6-expressing cells [CHO-mNthCCR6 [FIG. 8 (a)], CHO-mECL1hCCR6 [FIG.
  • FIG. 10 shows a schematic diagram of the construction of a human CCR6 N-terminal extracellular domain expression vector pKANTEX / NthCCR6-IgG4Fc fused with the Fc region of human IgG4.
  • FIG. 11 shows the results of SDS-PAGE analysis of human CCR6 N-terminal extracellular domain Fc fusion protein NthCCR6-IgG4Fc digested with glycosidase.
  • FIG. 10 shows a schematic diagram of the construction of a human CCR6 N-terminal extracellular domain expression vector pKANTEX / NthCCR6-IgG4Fc fused with the Fc region of human IgG4.
  • FIG. 11 shows the results of SDS-PAGE analysis of human CCR6 N-terminal extracellular domain Fc fusion protein NthCCR6-IgG4Fc digested with glycosidase.
  • FIG. 12 shows the result of binding ELISA of anti-human CCR6 monoclonal antibody against human CCR6 N-terminal extracellular domain Fc fusion protein NthCCR6-IgG4Fc.
  • the vertical axis shows the binding activity (OD415 / 490), and the horizontal axis shows the type of protein reacted.
  • FIG. 13 shows the results of SDS-PAGE analysis of purified anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704 and KM4705.
  • FIG. 14 shows information on instructions for commercially available anti-human CCR6 monoclonal antibodies.
  • FIGS. 15 (a)-(d) show anti-human CCR6 monoclonal antibodies KM4592 [FIG.
  • the vertical axis represents binding activity (mean fluorescence intensity; MFI), and the horizontal axis represents antibody concentration ( ⁇ g / mL).
  • FIGS. 16 (a) to (c) show anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704 and KM4705 and commercially available anti-human CCR6 monoclonal antibodies, human CCR6-expressing cells CHO-hCCR6, various human mouse chimeric CCR6-expressing cells CHO-mNthCCR6.
  • the result of having analyzed the binding activity with respect to CHO-mECL1hCCR6, CHO-mECL2hCCR6 and CHO-mECL3hCCR6 by FCM is shown.
  • the vertical axis represents the binding activity (mean fluorescence intensity; MFI), and the horizontal axis represents each CCR6-expressing cell.
  • FIG. 17 (a) and (b) show the N-terminal extracellular domain amino acid sequence of human CCR6 and the synthetic peptide used in the binding ELISA.
  • 18 (a) and (b) show the results of binding ELISA analysis of the binding activity of anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704, KM4705 and commercially available anti-human CCR6 monoclonal antibodies to various human CCR6 N-terminal partial peptides. Show.
  • FIG. 18 (a) shows the results of anti-human CCR6 monoclonal antibodies KM4592 and KM4705 and commercially available anti-human CCR6 monoclonal antibodies, and FIG.
  • 18 (b) shows the results of anti-human CCR6 monoclonal antibodies KM4703 and KM4704 antibodies.
  • the vertical axis represents the binding activity (OD415 / 490), and the horizontal axis represents the type of peptide reacted.
  • 19 (a)-(h) show the commercially available anti-human CCR6 monoclonal antibody 53103 [FIG. 19 (a)], R6H1 [FIG. 19 (b)], 11A9 [FIG. 19 (d)] and MM [FIG. d)], and the NthCCR6-IgG4Fc protein of the anti-human CCR6 monoclonal antibodies KM4592 [FIG. 19 (e)], KM4703 [FIG.
  • FIGS. 19 (a) to 19 (h) show typical sensorgrams of the respective antibodies obtained from the Biacore analysis results.
  • the vertical axis represents antibody reactivity (RU), and the horizontal axis represents time (sec) after antibody injection.
  • FIG. 20 (a) shows a schematic diagram of a calcium assay system for measuring an increase in intracellular calcium by a human CCR6-specific ligand Mip-3 ⁇ -dependent human CCR6 signal using human CCR6-expressing nalwa cells.
  • FIG. 21 shows a schematic diagram of cloning of a gene encoding the KM4705 antibody variable region of the anti-human CCR6 monoclonal antibodies KM4592, KM4703, and KM4704 and preparation of an anti-human CCR6 chimeric antibody expression vector.
  • 22 (a)-(d) show the anti-human CCR6 chimeric antibody KM4592 chimera [FIG.
  • FIG. 22 (d) shows the results of FCM analysis of the binding activity to human CCR6-expressing cell CHO-hCCR6 and cynomolgus CCR6-expressing cell CHO-cynomolgus CCR6.
  • the vertical axis represents binding activity (mean fluorescence intensity; MFI), and the horizontal axis represents antibody concentration ( ⁇ g / mL).
  • FIG. 23 shows anti-human CCR6 chimeric antibodies KM4592 chimera, KM4703 chimera, KM4704 chimera and KM4705 chimera human CCR6-expressing cell CHO-hCCR6, various human mouse chimeric CCR6-expressing cells CHO-mNthCCR6, CHO-mECL1hCCR6, CHO-mECL2hCCR6 and CHO-
  • the vertical axis represents the binding activity (mean fluorescence intensity; MFI), and the horizontal axis represents each CCR6-expressing cell.
  • MFI mean fluorescence intensity
  • FIG. 24 shows the result of binding ELISA analysis of the binding activity of anti-human CCR6 chimeric antibodies KM4592 chimera, KM4703 chimera, KM4704 chimera, and KM4705 chimera to various human CCR6 N-terminal partial peptides.
  • the vertical axis represents the binding activity (OD415 / 490), and the horizontal axis represents the peptide used.
  • FIG. 25 shows the neutralizing activity of the anti-human CCR6 chimeric antibody in the Mip-3 ⁇ -dependent intracellular calcium increase assay system using human CCR6-expressing namalwa cells.
  • the vertical axis shows the increase in intracellular calcium (%), and the horizontal axis shows the anti-human CCR6 chimeric antibody added.
  • 26 (a) and 26 (b) show effector cells obtained by isolating PBMCs isolated from healthy human peripheral blood [donor number # 11-067 [FIG. 26 (a)] and # 11-068 [FIG. 26 (b)]].
  • 2 shows the antibody-dependent cytotoxic activity (ADCC activity) of the anti-CCR6 antibody against human CCR6-expressing CHO cells (CHO-hCCR6) used as.
  • the horizontal axis represents the concentration (ng / mL) of each antibody, and the vertical axis represents the cytotoxic activity rate (%).
  • FIGS. 27 (a) and (b) show anti-T and B cells in PBMC isolated from peripheral blood of healthy individuals [donor number # 45 [FIG. 27 (a)] and 95 [FIG. 27 (b)]].
  • the removal activity of CCR6 antibody is shown.
  • the ratio of the number of various living cells is shown on the vertical axis when the number of memory T cells, naive T cells or B cells in the sample treated with the anti-DNP antibody is 100%.
  • the horizontal axis indicates the name of the treated antibody.
  • the present invention relates to an antibody that binds to sulfated human CCR6 and the antibody fragment.
  • the antibody and the antibody fragment of the present invention include an antibody that binds to the extracellular region of sulfated human CCR6 and the antibody fragment.
  • the extracellular region of human CCR6 preferably contains the 1st to 46th amino acids of the amino acid sequence represented by SEQ ID NO: 2.
  • the epitope to which the antibody of the present invention and the antibody fragment bind preferably contains at least the 18th Tyr of the amino acid sequence represented by SEQ ID NO: 2, and the ninth to 23rd amino acid sequences represented by SEQ ID NO: 2 More preferably, the amino acid is included.
  • the 18th Tyr of the amino acid sequence represented by SEQ ID NO: 2 is preferably sulfated.
  • an antibody of the present invention specifically, an antibody containing the 18th Tyr in the amino acid sequence represented by SEQ ID NO: 2 of sulfated human CCR6 as an epitope and a protein represented by sulfated SEQ ID NO: 2 of human CCR6 And an antibody containing the 18th Tyr of the amino acid sequence as an epitope.
  • the antibody of the present invention has a high affinity for human CCR6 and binds to an epitope containing the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2.
  • An antibody having a sum activity is mentioned.
  • the antibody of the present invention has a high affinity for human CCR6 and binds to an epitope containing the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2. And antibodies having antibody-dependent cytotoxic activity (ADCC activity).
  • the antibodies of the present invention include antibodies that bind to sulfated human CCR6 and not to unsulfated human CCR6, specifically bind to human CCR6 containing sulfated amino acid residues, and An antibody that does not bind to human CCR6 containing an unsulfated amino acid residue, and specifically binds to an epitope containing the 18th Tyr of the amino acid sequence represented by SEQ ID NO: 2 of human CCR6 and Examples thereof include an antibody that does not bind to an epitope containing the 18th Tyr of the amino acid sequence represented by SEQ ID NO: 2 that is not sulfated.
  • antibody of the present invention include antibodies selected from the following (a) to (d).
  • CDR Complementarity determining regions
  • VH sequences of antibody heavy chain variable regions
  • V regions Antibody variable regions
  • VL comprising CDR1 to CDR3 of an antibody light chain variable region (hereinafter referred to as VL) comprising the amino acid sequences represented by numbers 59 to 61, represented by SEQ ID NOs: 68 to 70, respectively.
  • VH CDRs 1 to 3 containing the amino acid sequences shown below and V regions containing CDRs 1 to 3 of the VL containing amino acid sequences represented by SEQ ID NOs: 71 to 73, respectively, and amino acid sequences represented by SEQ ID NOs: 80 to 82, respectively.
  • VH CDR1-3 and VL C comprising the amino acid sequences represented by SEQ ID NOs: 83-85, respectively Antibodies comprising any one of V regions selected from V region containing the R1 ⁇ 3.
  • B An antibody that competitively reacts with any one antibody selected from the antibodies represented by (a) above.
  • C An antibody that reacts with an epitope including an epitope to which any one antibody selected from the antibodies represented by (a) reacts.
  • D An antibody that reacts with the same epitope as that to which any one antibody selected from the antibodies represented by (a) reacts.
  • an antibody selected from the following (e) to (h) can be mentioned.
  • KM4592 is an example of an anti-human CCR6 monoclonal antibody comprising a V region comprising the amino acid sequence represented by SEQ ID NO: 52 and a V region comprising VL comprising the amino acid sequence represented by SEQ ID NO: 55. It is done.
  • One embodiment of an anti-human CCR6 monoclonal antibody comprising a V region comprising the amino acid sequence represented by SEQ ID NO: 64 and a V region comprising VL comprising the amino acid sequence represented by SEQ ID NO: 67 is KM4703.
  • an anti-human CCR6 monoclonal antibody comprising a V region comprising the amino acid sequence represented by SEQ ID NO: 76 and a V region comprising VL comprising the amino acid sequence represented by SEQ ID NO: 79 is KM4704.
  • One embodiment of the anti-human CCR6 monoclonal antibody comprising a V region comprising the amino acid sequence represented by SEQ ID NO: 88 and a V region comprising VL comprising the amino acid sequence represented by SEQ ID NO: 91 is KM4705.
  • the antibody of the present invention includes an antibody that binds to both sulfated human CCR6 and non-sulfated CCR6, and a human in which the 18th Tyr of the amino acid sequence represented by SEQ ID NO: 2 is sulfated.
  • the antibody of the present invention includes an antibody that binds to the 1st to 23rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2, and the 9th to 17th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2.
  • Antibodies that bind to epitopes comprising are also included. Specifically, anti-human CCR6 monoclonal antibodies KM4705, an antibody that reacts with an epitope containing an epitope to which KM4705 reacts, an antibody that reacts with the same epitope as that to which KM4705 reacts, and the like.
  • An antibody that reacts with an epitope including the epitope to which the antibody of the present invention reacts has the same or a part of the same epitope (also referred to as an antigenic determinant) as the antibody of the present invention in human CCR6, and binds to the epitope refers to an antibody.
  • An antibody that reacts with the same epitope as that to which the antibody of the present invention reacts refers to an antibody that can recognize and bind to the same amino acid residue on human CCR6 recognized by the antibody of the present invention.
  • the antibody of the present invention includes an antibody that specifically binds to the N-terminal region of human CCR6 and has neutralizing activity of human CCR6, an antibody that specifically binds to the N-terminal region of human CCR6 and has ADCC activity, sequence An antibody that binds to amino acids 9 to 23 of the amino acid sequence represented by No. 2 and has human CCR6 neutralizing activity and an epitope comprising amino acids 9 to 23 of the amino acid sequence represented by SEQ ID No. 2 Antibodies that bind and have ADCC activity are also included.
  • human CCR6 comprises a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2. And a polypeptide having the function of human CCR6, and a polypeptide having 60% or more, preferably 80% or more, more preferably 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2, most preferably 95 And a polypeptide having an amino acid sequence having a homology of at least% and having the function of human CCR6.
  • a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 is obtained by site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold. Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)] etc., for example, can be obtained by introducing a site-specific mutation into DNA encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2.
  • the number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example 1 to 20, more preferably 1 to several, for example 1 to 5 amino acids. It is.
  • Examples of the gene encoding human CCR6 include the nucleotide sequence represented by SEQ ID NO: 1, the nucleotide sequence represented by SEQ ID NO: 3, NCBI accession number NM_004367 or NM_031409. Consisting of a base sequence represented by SEQ ID NO: 1, a base sequence represented by SEQ ID NO: 3, a base sequence of NCBI accession number NM_004367 or NM_031409, wherein one or more bases are deleted, substituted or added, And a gene comprising a DNA encoding a polypeptide having the function of human CCR6, a base sequence represented by SEQ ID NO: 1, a base sequence represented by SEQ ID NO: 3, NCBI accession number NM_004367 or NM_031409 and at least 60 base sequences
  • a polypeptide comprising a human CCR6 function comprising a base sequence having a homology of at least%, preferably a base sequence having a homology of at least 80%, more preferably a base
  • a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, the nucleotide sequence represented by SEQ ID NO: 3, NCBI accession number NM_004367 or NM_031409 is used as a probe.
  • a hybridizable DNA obtained by a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, a DNA microarray method, etc. specifically, derived from a hybridized colony or plaque Hybridization at 65 ° C in the presence of 0.7 to 1.0 mol / L sodium chloride using a filter or slide glass on which DNA or a PCR product or oligo DNA having the sequence is immobilized Shon method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, After Oxford University, (1995)], the SSC solution with a concentration of 0.1 to 2 times (the composition of the SSC solution with a concentration of 1 consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate) The used, mention may be made of DNA that can be identified by washing the filter or slide
  • the DNA capable of hybridizing is the base sequence represented by SEQ ID NO: 1, the base sequence represented by SEQ ID NO: 3, DNA having at least 60% homology with the base sequence of NCBI accession number NM_004367 or NM_031409, preferably Can include DNA having a homology of 80% or more, more preferably DNA having a homology of 95% or more.
  • the gene used in the present invention includes a gene in which a small-scale mutation has occurred in the nucleotide sequence due to such polymorphism, and is also included in the gene encoding human CCR6 of the present invention.
  • the numerical value of homology in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art unless otherwise specified, but the base sequence may be BLAST [J. Mol. Biol. , 215, 403 (1990)], for amino acid sequences such as numerical values calculated using default parameters, BLAST2 [Nucleic Acids Res. , 25, 3389 (1997), Genome Res. , 7, 649 (1997), http: // www. ncbi. nlm. nih. gov / Education / BLASTinfo / information3. numerical value calculated using default parameters in htmL].
  • the default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, and 1 if it is an amino acid sequence.
  • -Q (Penalty for nucleotide mismatch) is -3
  • -r (reward for nucleotide match) is 1
  • -e (expect value) is 10
  • 11 residues when -W (wordsize) is a base sequence
  • -y [Dropoff (X) for blast extensions in bits] is 20 if blastn, 7 for programs other than blastn
  • -X X dropoff value f If the r aligned alignment in bits) is 15 and -Z (final X dropoff value for gapd alignment in bits) is blastn, it is 25 for programs other than blastn (http: // www. gov / blast / htmL / blastcgihelp.
  • a polypeptide containing a partial sequence of the amino acid sequence represented by SEQ ID NO: 2 can be prepared by a method known to those skilled in the art. For example, a part of DNA encoding the amino acid sequence represented by SEQ ID NO: 2 is prepared. It can be produced by culturing a transformant that has been deleted and into which an expression vector containing the deletion has been introduced. Further, based on the polypeptide or DNA prepared by the above method, an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 by the same method as described above. Polypeptides containing can be obtained.
  • a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in FIG. 1 is fluorenylmethyloxycarbonyl (Fmoc). Or a chemical synthesis method such as t-butyloxycarbonyl (tBoc) method.
  • the amino acid sequence represented by SEQ ID NO: 2 is converted into a known transmembrane region prediction program SOSUI (http://bp.nuap.nagaya-u.ac.jp/SOSUI). /SOSUI_submit.html), TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), TMHMM ver. 2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) or ExPAsy Proteomics Server (http://Ca.expasy.org/) It is done.
  • the N-terminal extracellular domain from the N-terminal which is the extracellular domain predicted in TMpred, is from the N-terminal extracellular domain
  • the 100-th to 117-th are extracellular loop (hereinafter abbreviated as ECL) 1, 178-th.
  • ECL2 extracellular loop
  • ECL3 extracellular loop
  • ECL3 extracellular loop
  • the extracellular region of human CCR6 in the present invention may be any structure as long as the extracellular region of human CCR6 containing the amino acid sequence represented by SEQ ID NO: 2 has a structure equivalent to a structure that can be taken in the natural state. Good.
  • the structure that the extracellular region of human CCR6 can take in the natural state refers to the natural three-dimensional structure of human CCR6 expressed on the cell membrane.
  • That the antibody of the present invention or an antibody fragment thereof specifically recognizes the natural three-dimensional structure of the extracellular region of human CCR6 and binds to the extracellular region is a radioimmunoassay using a solid phase sandwich method or the like, Alternatively, a known immunological detection method for cells expressing human CCR6 using an enzyme immunoassay (ELISA) or the like, preferably a binding property of a cell expressing a specific antigen such as a fluorescent cell staining method and an antibody to the specific antigen Can be examined.
  • ELISA enzyme immunoassay
  • FCM flow cytometry
  • Biacore Biacore
  • the cell expressing human CCR6 may be any cell as long as it expresses human CCR6.
  • a cell naturally existing in the human body a cell line established from a cell naturally existing in the human body, Or the cell obtained by the gene recombination technique etc. are mentioned.
  • Examples of the cells naturally present in the human body include cells in which the polypeptide is expressed in the human body.
  • cells in which the polypeptide is expressed in the human body include cells in which the polypeptide is expressed in the human body.
  • Th17 cells Th1 / 17 cells, ⁇ T cells, Th22 cells, memory / naive B cells, Some CD8 cells, some Th1 cells and some ILCs.
  • human CCR6 As a cell line established from cells naturally existing in the human body, among cell lines obtained by establishing cells expressing the human CCR6 obtained from the human body, the human CCR6 is expressed.
  • Cell lines For example, human liver cancer cell line HepG2, human colon cancer cell line Cano-2, human colon cancer cell line HT-29, human T cell leukemia cell lines HUT102 and MT-2, which are cell lines established from humans, etc. Can be mentioned.
  • cells obtained by gene recombination techniques specifically express human CCR6 obtained by introducing an expression vector containing cDNA encoding human CCR6 into insect cells or animal cells, for example. Cell and the like.
  • sulfated human CCR6 refers to CCR6 in which a sulfate molecule is bound to the OH substituent of a tyrosine residue (Tyr residue) present in the amino acid sequence of human CCR6.
  • Figure 3 shows human CCR6 containing at least one sulfated Tyr residue.
  • the Tyr residue to be sulfated may be any Tyr residue in the amino acid sequence of human CCR6, and preferably includes a Tyr residue present in the extracellular region of human CCR6.
  • human CCR6 examples include the 1st to 50th N-terminal domains of the amino acid sequence represented by SEQ ID NO: 2, ECL1, ECL2 and ECL3, preferably the N-terminal domain.
  • sulfated human CCR6 examples include human CCR6 in which the 18th Tyr residue of the amino acid sequence represented by SEQ ID NO: 2 is sulfated.
  • TPSTs tyrosyl protein sulfotransferases
  • Examples of the monoclonal antibody in the present invention include an antibody produced by a hybridoma or a recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.
  • a monoclonal antibody is an antibody that is secreted by a single clone of antibody-producing cells, recognizes only one epitope (also called an antigenic determinant), and has a uniform amino acid sequence (primary sequence) constituting the monoclonal antibody.
  • epitopes include a single amino acid sequence that is recognized and bound by a monoclonal antibody, a three-dimensional structure composed of amino acid sequences, an amino acid sequence modified by post-translational modification, and a three-dimensional structure composed of the amino acid sequences.
  • amino acid sequences modified by post-translational modification include, for example, an O-linked sugar chain bonded to Tyr and Ser having an OH substituent, and an N-linked sugar bonded to Gln and Asn having an NH 2 substituent.
  • An amino acid sequence in which a sulfate group or the like in which a chain and a sulfate molecule are bonded to Tyr having an OH substituent is bound.
  • the epitope to which the antibody of the present invention binds includes an epitope containing a sulfated Tyr residue contained in the extracellular region of human CCR6.
  • a sulfated Tyr residue contained in the 1st to 46th amino acids of the amino acid sequence represented by SEQ ID NO: 2 is preferable, and is contained in the 9th to 46th amino acids of the amino acid sequence represented by SEQ ID NO: 2.
  • a sulfated Tyr residue is more preferred, and a sulfated Tyr residue contained in the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2 is more preferred, and the 18th amino acid sequence represented by SEQ ID NO: 2 Most preferred are sulfated Tyr residues.
  • examples of the epitope to which the antibody of the present invention binds include an epitope containing a sulfated Tyr residue and an epitope containing a non-sulfated Tyr residue contained in the extracellular region of human CCR6.
  • the epitope to which the antibody of the present invention binds is a deletion in which a partial domain of human CCR6 is deleted, or a mutant in which a domain derived from another protein is substituted, and a partial peptide fragment of human CCR6, etc. This can be determined by conducting a binding experiment.
  • the epitope recognized by the antibody of the present invention specifically recognizes sulfated Tyr in the N-terminal extracellular region of human CCR6, and has a different amino acid sequence (including post-translational modifications) or three-dimensional structure from that of commercially available anti-human CCR6. Structure. Specific recognition of sulfated Tyr in the N-terminal extracellular region of human CCR6 means that human CCR6 that binds to human CCR6 whose N-terminal extracellular region is sulfated and whose N-terminal extracellular region is not sulfated. Means not to be combined.
  • epitope recognized by the antibody of the present invention include an epitope containing the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2.
  • An epitope containing the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2 is represented by SEQ ID NO: 2 when binding of the antibody to a partial peptide containing the human CCR6N-terminal extracellular region is confirmed. It has high binding activity to the partial peptide containing amino acids 9 to 23 of the amino acid sequence represented by SEQ ID NO: 2 in which the 18th tyrosine of the amino acid sequence is sulfated, and is represented by SEQ ID NO: 2 that is not sulfated. This means that the binding activity to the partial peptide containing the 9th to 23rd amino acids in the amino acid sequence is reduced or absent.
  • the epitope recognized by the antibody of the present invention also includes an epitope comprising the first to 23rd amino acids, particularly the ninth to 17th amino acids of the amino acid sequence represented by SEQ ID NO: 2.
  • human CCR6 functions include CCR6 activation dependent on human CCR6-specific ligand, activation of G protein (G protein), increase of intracellular calcium, influx of extracellular calcium, cell Examples include proliferation, cell migration, and cell differentiation.
  • G protein G protein
  • neutralizing activity refers to activity that inhibits any of the functions of human CCR6 described above.
  • human CCR6-specific ligands macrophage inductive protein-3 ⁇ (Mip-3 ⁇ ), CC chemokine receptor ligand 20 (CCL20), and defensin- ⁇ are known.
  • the neutralizing activity of the antibody of the present invention was obtained by simultaneously adding a human CCR6-specific ligand such as Mip-3 ⁇ and an antibody to a CCR6-expressing cell into which a human CCR6 gene was introduced or a cell line naturally expressing CCR6.
  • a human CCR6-specific ligand such as Mip-3 ⁇
  • an antibody to a CCR6-expressing cell into which a human CCR6 gene was introduced or a cell line naturally expressing CCR6.
  • FDSS manufactured by Hamamatsu Photonics
  • FLIPR manufactured by Molecular Devices
  • NOVOstar manufactured by BMG LABTECH
  • FlexSTATION manufactured by Molecular Devices
  • x ViewL PerkinElmer, IN Cell analyzer (GE Healthcare) or ArrayScan (Thermo Scientific) can be used for measurement.
  • high neutralizing activity means stronger neutralizing activity than known anti-human CCR6 antibodies or commercially available anti-human CCR6 antibodies, and is 2 times, 3 times, 4 times stronger than existing antibodies.
  • affinity means the affinity with which the antibody of the present invention binds to human CCR6, and is measured by kinetic analysis.
  • Biacore T100 manufactured by GE Healthcare
  • affinity more preferably 1.5 ⁇ 10 9 (mol / L) or less is mentioned.
  • the antibody that binds to human CCR6 extracellular domain with high affinity a antibody having sufficient affinity as therapeutic antibodies, existing dissociation constant K D of 1.5 ⁇ 10 9 (mol / L ) (M) refers to binding to human CCR6 with the following affinity.
  • an antibody having high ADCC activity refers to a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)], an ADCC activity higher than that of a commercially available anti-human CCR6 antibody when simultaneously measuring the ADCC activity of a plurality of antibodies.
  • Antibody molecules are also referred to as immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM isotypes according to the difference in molecular structure. Is done. IgG1, IgG2, IgG3, and IgG4 having relatively high amino acid sequence homology are collectively referred to as IgG.
  • Antibody molecules are composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (hereinafter referred to as L chains).
  • H chains is an H chain variable region (also expressed as VH)
  • H chain constant region also expressed as CH
  • L chain is also expressed as an L chain variable region (VL) from the N terminal side.
  • CL Each region of the L chain constant region (also expressed as CL).
  • CH has known ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains for each subclass.
  • CH is further composed of each domain of the CH1 domain, hinge domain, CH2 domain, and CH3 domain from the N-terminal side.
  • a domain refers to a functional structural unit constituting each polypeptide of an antibody molecule.
  • the CH2 domain and the CH3 domain are collectively referred to as an Fc region or simply Fc.
  • CL C ⁇ chain and C ⁇ chain are known.
  • the CH1 domain, hinge domain, CH2 domain, CH3 domain, and Fc region are EU indexes [Kabat et al. , Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] can be specified by the number of amino acid residues from the N-terminus.
  • CH1 is an amino acid sequence of EU indexes 118 to 215
  • hinge is an amino acid sequence of EU indexes 216 to 230
  • CH2 is an amino acid sequence of EU indexes 231 to 340
  • CH3 is an EU index 341 to 447. Each amino acid sequence is identified.
  • antibodies of the present invention include gene sets such as human chimeric antibodies (hereinafter also simply referred to as “chimeric antibodies”), humanized antibodies (also referred to as complementarity determining regions (CDR) transplanted antibodies), and human antibodies. Replacement antibodies are also included.
  • chimeric antibodies hereinafter also simply referred to as “chimeric antibodies”
  • CDR complementarity determining regions
  • the chimeric antibody means an antibody composed of VH and VL of an antibody other than an animal (non-human animal) and CH and CL of a human antibody.
  • non-human animal any mouse, rat, hamster, rabbit or the like can be used as long as it can produce a hybridoma.
  • a hybridoma is a cell that produces a monoclonal antibody having a desired antigen specificity obtained by fusing a B cell obtained by immunizing a non-human animal with a myeloma cell derived from a mouse or the like.
  • the variable region constituting the antibody produced by the hybridoma consists of the amino acid sequence of a non-human animal antibody.
  • Human chimeric antibodies are obtained by obtaining cDNAs encoding VH and VL from non-human animal cell-derived hybridomas producing monoclonal antibodies, and expressing them as expression vectors for animal cells having DNAs encoding human antibodies CH and CL, respectively.
  • a human chimeric antibody expression vector is constructed by insertion and can be expressed and produced by introducing it into animal cells.
  • a humanized antibody refers to an antibody obtained by grafting the amino acid sequences of CDRs of VH and VL of a non-human animal antibody into CDRs corresponding to VH and VL of a human antibody.
  • An area other than the CDRs of VH and VL is referred to as a framework area (hereinafter referred to as FR).
  • the humanized antibody is composed of a cDNA encoding a VH amino acid sequence consisting of a VH CDR amino acid sequence of a non-human animal antibody and a VH FR amino acid sequence of any human antibody, and a VL CDR amino acid of a non-human animal antibody.
  • a cDNA encoding the amino acid sequence of VL consisting of the sequence and the amino acid sequence of FR of VL of any human antibody is constructed, and inserted into an expression vector for animal cells having DNA encoding CH and CL of human antibody, respectively.
  • An antibody expression vector can be constructed and introduced into animal cells for expression and production.
  • a human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody-producing transgene prepared by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from transgenic animals.
  • a human antibody can be obtained by immunizing a mouse carrying a human immunoglobulin gene (Tomizuka K. et. Al., Proc Natl Acad Sci USA. 97, 722-7, 2000.) with a desired antigen. I can do it. Further, by using a phage display library obtained by amplifying antibody genes from human-derived B cells, a human antibody can be obtained without immunization by selecting a human antibody having a desired binding activity ( Winter G. et.al., Annu Rev Immunol. 12: 433-55.1994). Furthermore, by immortalizing human B cells using EB virus, cells that produce human antibodies having a desired binding activity can be produced and human antibodies can be obtained (Rosen A. et. Al.,). Nature 267, 52-54.1977).
  • the antibody present in the human body can be cultured, for example, by immortalizing lymphocytes isolated from human peripheral blood by infecting EB virus or the like and then cloning the lymphocytes, The antibody can be purified from the culture.
  • the human antibody phage library is a phage library in which antibody fragments such as Fab and scFv are expressed on the surface by inserting antibody genes prepared from human B cells into the phage genes. From the library, phages expressing antibody fragments having a desired antigen-binding activity can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.
  • a human antibody-producing transgenic animal is an animal in which a human antibody gene is integrated into the chromosome of a host animal.
  • a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, and then transplanting the ES cells into early embryos of other mice and then generating them.
  • a human antibody production method from a human antibody-producing transgenic animal is obtained by obtaining and culturing a human antibody-producing hybridoma by a hybridoma production method performed in a normal non-human mammal. Production can be accumulated.
  • the VH and VL amino acid sequences of the antibodies of the present invention include human antibody VH and VL amino acid sequences, non-human animal antibody VH and VL amino acid sequences, or non-human animal antibody CDRs as the human antibody framework. Any of the amino acid sequences of the transplanted humanized antibody may be used. Specific examples include VH and VL amino acid sequences of non-human animal antibodies produced by hybridomas, humanized antibody VH and VL amino acid sequences, human antibody VH and VL amino acid sequences, and the like.
  • the amino acid sequence of CL in the antibody of the present invention may be either an amino acid sequence of a human antibody or an amino acid sequence of a non-human animal antibody, but C ⁇ or C ⁇ of an amino acid sequence of a human antibody is preferable.
  • the CH of the antibody of the present invention may be any as long as it belongs to immunoglobulin, but preferably any of subclass belonging to IgG class, ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3) and ⁇ 4 (IgG4) Can also be used.
  • the effector activity refers to antibody-dependent activity caused through the Fc region of an antibody.
  • Antibody-dependent cellular cytotoxicity (ADCC activity), complement-dependent cytotoxicity (Complement-Dependent) Cytotoxicity (CDC activity) or antibody-dependent phagocytosis (ADP activity) by phagocytic cells such as macrophages or dendritic cells is known.
  • ADCC activity and CDC activity are measured using a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)].
  • ADCC activity means that an antibody bound to an antigen on a target cell binds to an Fc receptor of an immune cell through the Fc region of the antibody, thereby activating an immune cell (natural killer cell, etc.) and damaging the target cell. Activity.
  • the Fc receptor (hereinafter also referred to as FcR) is a receptor that binds to the Fc region of an antibody, and induces various effector activities by the binding of the antibody.
  • FcR corresponds to an antibody subclass, and IgG, IgE, IgA, and IgM specifically bind to Fc ⁇ R, Fc ⁇ R, Fc ⁇ R, and Fc ⁇ R, respectively.
  • Fc ⁇ R has Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and Fc ⁇ RIII (CD16) subtypes, and Fc ⁇ RIA, Fc ⁇ RIB, Fc ⁇ RIIC, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIIC, Fc ⁇ RIIIA, and Fc ⁇ RIIIB are present. To do. These different Fc ⁇ Rs are present on different cells [Annu. Rev. Immunol. 9: 457-492 (1991)].
  • Fc ⁇ RIIIB is specifically expressed in neutrophils, and Fc ⁇ RIIIA is expressed in monocytes, Natural Killer cells (NK cells) and some T cells. Antibody binding via Fc ⁇ RIIIA induces NK cell-dependent ADCC activity.
  • CDC activity refers to an activity in which an antibody bound to an antigen on a target cell activates a series of cascades (complement activation pathways) composed of complement-related proteins in the blood and damages the target cell.
  • cascades complement activation pathways
  • migration and activation of immune cells can be induced by protein fragments generated by complement activation.
  • the cascade of CDC activity begins when C1q, which has a binding domain with the Fc region of an antibody, binds to the Fc region and binds to two serine proteases, C1r and C1s, to form a C1 complex.
  • the antibodies of the present invention include VH complementary chain determining regions CDR1 to CDR3 comprising the amino acid sequences represented by SEQ ID NOs: 56 to 58 and VL comprising the amino acid sequences represented by SEQ ID NOs: 59 to 61 respectively.
  • V region comprising CDR1-3, VH CDR1-3 comprising amino acid sequences represented by SEQ ID NOs: 68-70, respectively, and V comprising CDR1-3, VL comprising amino acid sequences represented by SEQ ID NOs: 71-73, respectively Region, V region comprising VH CDR1 to 3 each comprising the amino acid sequence represented by SEQ ID NOs: 80 to 82 and V region comprising CDR 1 to 3 comprising the amino acid sequence represented by SEQ ID NOs: 83 to 85, respectively, and SEQ ID NO: 92, respectively. CDRs 1 to 3 of VH containing the amino acid sequences represented by ⁇ 94 and amino acid sequences represented by SEQ ID NOs: 95 to 97, respectively.
  • Antibodies comprising any one of V regions selected from V region comprising CDRl ⁇ 3 of VL comprising the like.
  • the antibody of the present invention is represented by the anti-human CCR6 monoclonal antibody KM4592 containing VH containing the amino acid sequence represented by SEQ ID NO: 52 and VL containing the amino acid sequence represented by SEQ ID NO: 55, SEQ ID NO: 64.
  • One antibody is mentioned.
  • the antibodies of the present invention include antibodies that compete with the above-described antibodies and bind to human CCR6, antibodies that react with the epitope to which the above-mentioned antibody reacts, and antibodies that react with the same epitope as the above-mentioned antibody reacts with. It is done.
  • the antibody of the present invention is fused with an Fc fusion protein in which Fc and an antibody fragment are bound, an Fc fusion protein in which Fc and a naturally occurring ligand or receptor are bound (also referred to as immunoadhesin), and a plurality of Fc regions. Also included are Fc fusion proteins and the like. An Fc region containing an amino acid residue modification that has been modified to enhance or lack the effector activity of the antibody, stabilize the antibody, and control the blood half-life is also included in the antibody of the present invention. Can be used.
  • Examples of the antibody fragment in the present invention include Fab, Fab ′, F (ab ′) 2 , scFv, diabody, dsFv, and a peptide containing a plurality of CDRs.
  • Fab is a fragment obtained by treating an IgG antibody with the proteolytic enzyme papain (cleaved at the amino acid residue at position 224 of the H chain), and about half of the N-terminal side of the H chain and the entire L chain are disulfide bonded. It is an antibody fragment having an antigen binding activity of about 50,000 molecular weight bound by (SS bond).
  • F (ab ′) 2 is a fragment obtained by treating IgG with proteolytic enzyme pepsin (which is cleaved at the 234th amino acid residue of the H chain), and Fab is linked via an SS bond in the hinge region.
  • Antibody fragment having an antigen-binding activity with a molecular weight of about 100,000, which is slightly larger than those bound together.
  • Fab ′ is an antibody fragment having an antigen binding activity of about 50,000 molecular weight obtained by cleaving the SS bond in the hinge region of F (ab ′) 2 .
  • scFv uses an appropriate peptide linker (P) such as a linker peptide in which one VH and one VL are connected to an arbitrary number of linkers (G4S) consisting of four Gly and one Ser residue.
  • P peptide linker
  • G4S linkers
  • VH-P-VL or VL-P-VH polypeptide which is an antibody fragment having antigen-binding activity.
  • Diabody is an antibody fragment in which scFv having the same or different antigen binding specificity forms a dimer, and is an antibody fragment having a bivalent antigen-binding activity for the same antigen or a bispecific antigen-binding activity for different antigens.
  • DsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are bound via an SS bond between the cysteine residues.
  • the peptide containing CDR is configured to contain at least one region of CDR of VH or VL.
  • a peptide containing a plurality of CDRs can be linked to each other directly or via an appropriate peptide linker.
  • a DNA encoding the CDRs of the modified antibody VH and VL of the present invention is constructed, the DNA is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector is introduced into a prokaryotic or eukaryotic organism It can be expressed and manufactured by doing.
  • the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc method.
  • the monoclonal antibody of the present invention specifically recognizes the extracellular region of human CCR6 of the present invention and binds to the extracellular region with a radioisotope, a low molecular weight drug, a macromolecule. Or a derivative of an antibody obtained by chemically or genetically linking a drug, protein, or antibody drug.
  • the derivative of the antibody in the present invention specifically recognizes the extracellular region of human CCR6 of the present invention and binds to the extracellular region on the N-terminal side of the H chain or L chain of the monoclonal antibody or antibody fragment thereof, Chemistry of radioisotopes, low molecular weight drugs, high molecular weight drugs, immunostimulants, proteins, antibody drugs, or nucleic acid drugs on the C-terminal side, appropriate substituents or side chains in antibody molecules, and sugar chains Can be produced by a conventional method [Introduction to Antibody Engineering, Jinjinshokan (1994)].
  • DNA encoding a monoclonal antibody or antibody fragment that specifically recognizes the extracellular region of human CCR6 of the present invention and binds to the extracellular region is linked to DNA encoding the protein or antibody drug to be bound. And then inserted into an expression vector, introduced into an appropriate host cell, and expressed by a genetic engineering technique.
  • radioisotope examples include 111 In, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, and 211 At.
  • the radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance that chelates a radioisotope may be bound to the antibody.
  • the chelating agent include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).
  • low molecular weight drugs examples include alkylating agents, nitrosourea agents, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P glycoprotein inhibitors, platinum Anti-cancer agents such as complex derivatives, M-phase inhibitors and kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroidal agents such as hydrocortisone and prednisone, non-steroidal agents such as aspirin and indomethacin, gold thiomalate, Anti-inflammatory agents such as immunomodulators such as penicillamine, cyclophosphamide and azathioprine, and antihistamines such as chlorpheniramine maleate and clemacytin [Inflammation and anti-inflammatory therapy, Ishiyaku Publishing Co., Ltd. ( 1982)].
  • anticancer agents include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), epirubicin, gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, pepromycin, estramustine, paclitaxel, paclitaxel, paclitaxel Taxotea), aldesleukin, asparaginase, buoy Rufan, carboplatin, oxaliplatin, nedaplatin, cladribine
  • Examples of a method for binding a low molecular weight drug to an antibody include, for example, a method of binding a drug and an amino group of an antibody via glutaraldehyde, and a drug amino group and an antibody carboxyl group via a water-soluble carbodiimide. And the like.
  • polymer drug examples include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer, and hydroxypropyl methacrylamide.
  • PEG polyethylene glycol
  • albumin dextran
  • polyoxyethylene polyoxyethylene
  • styrene maleic acid copolymer polyoxyethylene
  • polyvinyl pyrrolidone polyvinyl pyrrolidone
  • pyran copolymer examples include hydroxypropyl methacrylamide.
  • Examples of a method of binding PEG and an antibody include a method of reacting with a PEGylation modifying reagent [Bioconjugate pharmaceutical, Yodogawa Shoten (1993)].
  • Examples of the PEGylation modifying reagent include a modifier for the ⁇ -amino group of lysine (Japanese Patent Laid-Open No. 61-178926), a modifier for the carboxyl group of aspartic acid and glutamic acid (Japanese Patent Laid-Open No. 56-23587). And a modifier for guanidino group of arginine (Japanese Patent Laid-Open No. 2-117920).
  • the immunostimulant may be a natural product known as an immunoadjuvant, and specific examples include ⁇ (1 ⁇ 3) glucan (lentinan, schizophyllan) and ⁇ -galactosylceramide (KRN7000). Is mentioned.
  • proteins include cytokines or growth factors that activate immunocompetent cells such as NK cells, macrophages, or neutrophils, and toxin proteins.
  • cytokines or growth factors examples include interferon (hereinafter referred to as INF) - ⁇ , INF- ⁇ , INF- ⁇ , interleukin (hereinafter referred to as IL) -2, IL-12, IL-15, IL- 18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF).
  • INF interferon
  • IL interleukin
  • IL-12 interleukin
  • IL-15 interleukin
  • IL-15 interleukin
  • IL-15 interleukin- 18, IL-21
  • IL-23 granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte / macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • toxin protein examples include ricin, diph
  • Examples of the antibody drug include an antigen against which apoptosis is induced by antibody binding, an antigen involved in tumor pathogenesis or an antigen that regulates immune function and an antigen involved in angiogenesis of a lesion site.
  • antigens whose apoptosis is induced by antibody binding include, for example, cluster of differentiation (hereinafter referred to as CD) 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA) -Class II, and Epidemial GrowthFact. EGFR) and the like.
  • CD cluster of differentiation
  • antigens involved in tumor pathogenesis or antibodies that regulate immune function include CD4, CD40, CD40 ligand, B7 family molecules (eg, CD80, CD86, CD274, B7-DC, B7-H2, B7- H3 or B7-H4), ligands of B7 family molecules (eg, CD28, CTLA-4, ICOS, PD-1 or BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF) receptor family Molecule (eg DR4, DR5, TNFR1 or TNFR2), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecule, receptor family of TRAIL family molecules ( RAIL-R1, TRAIL-R2, TRAIL-R3, or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor and cytokine [eg IL-1 ⁇ , IL-1 ⁇
  • antigens for antibodies that inhibit angiogenesis of lesion sites include, for example, vascular electrical growth factor (VEGF), anangiopoietin, fibroblast growth factor (FGF), EGF, hepatocyte growthGF (hepatocytes). Insulin-like growth factor (IGF), erythropoietin (EPO), TGF ⁇ , IL-8, ephrin, angiopoietin, SDF-1, or a receptor thereof.
  • VEGF vascular electrical growth factor
  • FGF fibroblast growth factor
  • EGF hepatocyte growthGF
  • IGF Insulin-like growth factor
  • EPO erythropoietin
  • TGF ⁇ IL-8
  • ephrin angiopoietin
  • SDF-1 a receptor thereof.
  • a fusion antibody with a protein or antibody drug comprises linking a cDNA encoding a protein to a cDNA encoding a monoclonal antibody or antibody fragment, constructing a DNA encoding the fusion antibody, and expressing the DNA for prokaryote or eukaryote It is inserted into a vector and expressed by introducing the expression vector into a prokaryotic or eukaryotic organism to produce a fusion antibody.
  • the nucleic acid drug include a drug containing a nucleic acid such as small interference ribonucleic acid (siRNA) or microRNA that acts on a living body by controlling the function of a gene.
  • siRNA small interference ribonucleic acid
  • microRNA microRNA
  • the extracellular region of human CCR6 of the present invention is specifically recognized and bound to the extracellular region.
  • the agent that binds to the monoclonal antibody or the antibody fragment thereof include a label used in a usual immunological detection or measurement method.
  • the label include enzymes such as alkaline phosphatase, peroxidase and luciferase, luminescent materials such as acridinium ester and lophine, and fluorescent materials such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (RITC). Can be mentioned.
  • the present invention also relates to a therapeutic agent for a disease involving human CCR6 positive cells which specifically recognize the extracellular region of human CCR6 and which contains a monoclonal antibody or an antibody fragment thereof which binds to the extracellular region as an active ingredient. About.
  • the disease involving human CCR6-positive cells may be any disease involving human CCR6-positive cells, and examples thereof include autoimmune diseases.
  • autoimmune diseases include autoimmune diseases involving human CCR6 positive T cells and / or B cells and / or ILC, human CCR6 positive T cells and / or B cells and / or cytokines derived from ILC. Or autoimmune diseases involving cells controlled by human CCR6-positive T cells and / or B cells and / or ILC-derived cytokines.
  • autoimmune disease examples include rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma, psoriasis, sinusitis, and systemic lupus erythematosus.
  • human CCR6-positive T cells include Th17 cells, Th1 / 17 cells, and Th22 cells.
  • human CCR6-positive B cells include naive / memory B cells.
  • the antibody of the present invention can inhibit CCR6-positive T cells and / or B cells, it simultaneously suppresses a plurality of cells such as Th1 / 17, Th17, Th22 and naive / memory B cells, and is used in autoimmune diseases. Inhibition of T-cell cytokine production, which is considered a problem, and autoantibody production by B cells can also be inhibited. Further, the antibody of the present invention can inhibit CCR6-positive ILC.
  • the therapeutic agent containing the antibody or the antibody fragment or derivative thereof of the present invention may contain only the antibody or the antibody fragment or derivative thereof as an active ingredient. It is desirable to provide a pharmaceutical formulation prepared by any method known in the pharmaceutical arts, mixed with one or more pharmaceutically acceptable carriers.
  • the route of administration is preferably the most effective for treatment, and includes oral administration, parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular or intravenous, and intravenous administration. Is preferred.
  • parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular or intravenous, and intravenous administration. Is preferred.
  • the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments and tapes.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 ⁇ g / kg to 10 mg / kg per day for an adult.
  • the present invention relates to an immunological detection or measurement method for human CCR6, an immunological detection for human CCR6, or an antibody that binds to an extracellular region of sulfated human CCR6 or an antibody fragment thereof as an active ingredient.
  • the present invention relates to a reagent for measurement, an immunological detection or measurement method for cells expressing human CCR6, and a diagnostic agent for diseases involving human CCR6-positive cells.
  • any known method can be used as a method for detecting or measuring the amount of human CCR6.
  • Examples include immunological detection or measurement methods.
  • the immunological detection or measurement method is a method of detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • immunological detection or measurement methods include radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (Western immunoassay), Western Examples include blotting or physicochemical techniques.
  • a disease associated with human CCR6 can be diagnosed.
  • Known immunological detection methods can be used to detect cells expressing the polypeptide, but immunoprecipitation, fluorescent cell staining, immunohistochemical staining, or immunohistochemical staining, Preferably used.
  • fluorescent antibody staining methods such as FMAT8100HTS system (Applied Biosystems) can also be used.
  • biological samples to be used for detecting or measuring human CCR6 in the present invention include tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid, and human CCR6 is expressed. There is no particular limitation as long as it may contain cells.
  • the diagnostic agent containing the monoclonal antibody of the present invention or an antibody fragment thereof, or a derivative thereof may contain a reagent for performing an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method.
  • a reagent for performing the antigen-antibody reaction include a buffer and a salt.
  • detection reagent examples include reagents used in usual immunological detection or measurement methods such as a labeled secondary antibody that recognizes the monoclonal antibody or antibody fragment or derivative thereof, and a substrate corresponding to the label. Is mentioned.
  • the antibody production method, disease treatment method, and disease diagnosis method of the present invention will be specifically described below.
  • Antibody Production Method Preparation of Antigen Human CCR6 or human CCR6 to be used as an antigen is expressed in Escherichia coli, yeast, insect cells, or an expression vector containing cDNA encoding human CCR6 full length or a partial length thereof. It can be obtained by introducing it into animal cells. Moreover, human CCR6 can be purified and obtained from various human tumor cultured cells, human cells, human tissues, etc. that express human CCR6 in large quantities. Further, the cultured tumor cells or the tissue can be used as an antigen as it is. Furthermore, a synthetic peptide having a partial sequence of human CCR6 can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method and used as an antigen.
  • the human CCR6 used in the present invention is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols InMolecular 7 in Current Protocols in 1989.
  • the DNA encoding human CCR6 can be expressed in a host cell and produced by the following method.
  • a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding human CCR6 downstream of a promoter of an appropriate expression vector.
  • a DNA fragment of an appropriate length containing a polypeptide-encoding portion prepared based on the full-length cDNA may be used.
  • a transformant producing the polypeptide can be obtained by introducing the obtained recombinant vector into a host cell suitable for the expression vector.
  • Any expression vector can be used as long as it can autonomously replicate in the host cell to be used or can be integrated into the chromosome, and contains an appropriate promoter at a position where DNA encoding the polypeptide can be transcribed. Can do.
  • host cells include microorganisms belonging to the genus Escherichia such as Escherichia coli, yeast, insect cells and animal cells, and any can be used as long as it can express the target gene.
  • the recombinant vector When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in a prokaryote, and at the same time a promoter, a ribosome binding sequence, DNA containing a portion encoding human CCR6, and a transcription termination sequence. It is preferable that the vector contains The recombinant vector does not necessarily require a transcription termination sequence, but it is preferable to place the transcription termination sequence immediately below the structural gene. Furthermore, the recombinant vector may contain a gene that controls the promoter.
  • the recombinant vector it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also referred to as SD sequence), which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • SD sequence also referred to as SD sequence
  • start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • the base sequence of the DNA encoding the human CCR6 can be substituted so that the codon is optimal for expression in the host, thereby improving the production rate of the target human CCR6. Can do.
  • Any expression vector can be used as long as it can function in the host cell to be used.
  • pBTrp2, pBTac1, pBTac2 from Roche Diagnostics
  • pKK233-2 Pharmacia
  • pSE280 Invitrogen
  • pGEMEX-1 Promega
  • pQE-8 Qiagen
  • PKYP10 Japanese Unexamined Patent Publication No. 58-110600
  • pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)]
  • pLSA1 Agric. Biol. Chem. , 53, 277 (1989)
  • pGEL1 Proc. Natl. Acad. Sci.
  • any promoter can be used as long as it can function in the host cell to be used.
  • promoters derived from Escherichia coli or phages such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter and T7 promoter can be mentioned.
  • artificially designed and modified promoters such as a tandem promoter, tac promoter, lacT7 promoter, or let I promoter in which two Ptrps are connected in series are also included.
  • Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, E. coli DH5 ⁇ and the like.
  • Any method can be used for introducing a recombinant vector into a host cell as long as it is a method for introducing DNA into the host cell to be used.
  • a method using calcium ions Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].
  • any expression vector can be used as long as it can function in animal cells.
  • Any promoter can be used as long as it can function in animal cells.
  • a cytomegalovirus (CMV) immediate early (IE) gene promoter for example, a cytomegalovirus (CMV) immediate early (IE) gene promoter, an SV40 early promoter, a retroviral promoter. , Metallothionein promoter, heat shock promoter, SR ⁇ promoter, or Moloney murine leukemia virus promoter and enhancer.
  • CMV cytomegalovirus
  • IE immediate early
  • IE SV40 early promoter
  • a retroviral promoter e.g., a promoter for example, a cytomegalovirus (CMV) immediate early (IE) gene promoter, an SV40 early promoter, a retroviral promoter.
  • Metallothionein promoter e.g., Metallothionein promoter, heat shock promoter, SR ⁇ promoter, or Moloney murine leukemia virus promoter and enhancer.
  • host cells examples include human leukemia cells Namalwa cells, monkey cells COS cells, Chinese hamster ovary cells CHO cells [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc . Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci. , 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp.
  • any method for introducing a recombinant vector into a host cell any method can be used as long as it introduces DNA into animal cells.
  • electroporation method [Cytotechnology, 3, 133 (1990)]
  • calcium phosphate method Japanese Patent Laid-Open No. 2-227075
  • lipofection method Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • a transformant derived from a microorganism or animal cell having a recombinant vector incorporating a DNA encoding human CCR6 obtained as described above is cultured in a medium, and the human CCR6 is produced and accumulated in the culture. Then, human CCR6 can be produced by collecting from the culture.
  • the method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
  • human CCR6 When expressed in cells derived from eukaryotes, human CCR6 with an added sugar or sugar chain can be obtained.
  • an inducer may be added to the medium as necessary.
  • cultivating a microorganism transformed with a recombinant vector using the lac promoter cultivate a microorganism transformed with isopropyl- ⁇ -D-thiogalactopyranoside or the like using a recombinant vector using the trp promoter.
  • indole acrylic acid or the like may be added to the medium.
  • a medium for culturing a transformant obtained using an animal cell as a host a commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122 , 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med. 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, or a medium obtained by adding fetal bovine serum (FBS) or the like to these mediums.
  • RPMI 1640 medium The Journal of the American Medical Association, 199, 519 (1967)]
  • Eagle's MEM medium Science, 122 , 501 (1952)]
  • Dulbecco's modified MEM medium Virology, 8, 396 (1959)]
  • 199 medium Proc. Soc. Exp. Biol. Med. 73, 1
  • the culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and the presence of 5% CO 2 .
  • Examples of the method for producing human CCR6 include a method for producing it in a host cell, a method for secreting it outside the host cell, and a method for producing it on the outer cell membrane.
  • the host cell to be used or the human CCR6 to be produced is produced. By changing the structure, an appropriate method can be selected.
  • human CCR6 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Laid-Open No. 2-227075).
  • the obtained human CCR6 can be isolated and purified as follows, for example.
  • the cells When human CCR6 is expressed in a dissolved state in cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, French press, Manton Gaurin homogenizer, or dynomill. Use to crush the cells to obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing or isoelectric focusing. Use alone or in combination, Rukoto can.
  • a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical)
  • human CCR6 When human CCR6 is expressed in the form of an insoluble substance in the cell, the cell is recovered and crushed in the same manner as described above, and centrifuged to collect the insoluble substance of human CCR6 as a precipitate fraction.
  • the recovered insoluble body of human CCR6 is solubilized with a protein denaturant. By diluting or dialyzing the solubilized solution, the human CCR6 is returned to a normal three-dimensional structure, and then a purified polypeptide preparation can be obtained by the same isolation and purification method as described above.
  • the derivative such as human CCR6 or a sugar modification product thereof can be recovered from the culture supernatant.
  • a soluble fraction can be obtained by treating the culture by a method such as centrifugation as described above, and a purified preparation can be obtained from the soluble fraction by using the same isolation and purification method as described above. it can.
  • human CCR6 used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • Chemical synthesis using peptide synthesizers such as Advanced Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecel-Vega, Perceptive or Shimadzu You can also be produced.
  • Immunization is carried out by administering the antigen subcutaneously or intravenously or intraperitoneally in the animal together with an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine.
  • an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine.
  • the antigen is a partial peptide
  • a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole limpet hemocyanin) is prepared and used as an immunogen.
  • BSA bovine serum albumin
  • KLH Keyhole limpet hemocyanin
  • the antigen is administered 5 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the antibody titer of the serum is measured using an enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. An animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody producing cells for fusion.
  • tissues containing antibody-producing cells such as the spleen are removed from the immunized animal, and antibody-producing cells are collected.
  • the spleen is shredded and loosened, and then centrifuged, and the erythrocytes are removed to obtain antibody producing cells for fusion.
  • a cell line obtained from a mouse is used.
  • the myeloma cells are subcultured in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine], and subcultured to normal medium 3 to 4 days before cell fusion. On the day of fusion, secure a cell count of 2 ⁇ 10 7 or more.
  • MEM Minimum Essential Medium
  • a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethyl sulfoxide is added at 37 ° C. with stirring.
  • the cells are gently suspended in the HAT medium [normal medium containing hypoxanthine, thymidine, and aminopterin] to the antibody-producing cells for fusion. This suspension is cultured for 7-14 days at 37 ° C. in a 5% CO 2 incubator.
  • a part of the culture supernatant is extracted, and a cell group that reacts with an antigen containing human CCR6 and does not react with an antigen not containing human CCR6 is selected by a hybridoma selection method such as a binding assay described later.
  • cloning was repeated twice by the limiting dilution method (first time was HT medium (medium obtained by removing aminopterin from HAT medium), second time normal medium was used), and a stable and strong antibody titer was observed.
  • One is selected as a monoclonal antibody-producing hybridoma.
  • the monoclonal antibody-producing hybridoma obtained in (4) is cultured in an RPMI1640 medium supplemented with 10% FBS, the supernatant is removed by centrifugation, and the suspension is suspended in a Hybridoma SFM medium and cultured for 3 to 7 days. .
  • the obtained cell suspension is centrifuged, and purified using a protein A-column or protein G-column from the resulting supernatant, and the IgG fraction is collected to obtain a purified monoclonal antibody.
  • 5% Digo GF21 can also be added to the Hybridoma SFM medium.
  • the antibody subclass is determined by enzyme immunoassay using a sub-clustering kit.
  • the amount of protein is quantified by the Raleigh method or absorbance at 280 nM.
  • the monoclonal antibody is selected by a binding assay by the enzyme immunoassay shown below and a kinetic analysis by Biacore. In addition to these methods, selection can also be made by identifying a target antigen of an antibody by a known method [The Prostate, 67, 1163 (2007)].
  • (6-a) Binding assay As an antigen, a gene-transferred cell or a recombinant obtained by introducing an expression vector containing cDNA encoding human CCR6 obtained in (1) into E. coli, yeast, insect cells, animal cells, or the like. A protein or a purified polypeptide or partial peptide obtained from human tissue is used. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA or KLH is prepared and used.
  • a carrier protein such as BSA or KLH
  • a test substance such as serum, hybridoma culture supernatant or purified monoclonal antibody is dispensed as the first antibody and allowed to react.
  • an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like is dispensed and reacted as a second antibody.
  • a reaction is performed according to the labeling substance of the second antibody, and a monoclonal antibody that specifically reacts with the immunogen is selected.
  • a monoclonal antibody that competes with the anti-human CCR6 monoclonal antibody of the present invention and binds to human CCR6 can be obtained by adding a test antibody to the above-described binding assay system and allowing it to react. That is, by screening for an antibody that inhibits the binding of the monoclonal antibody when the test antibody is added, a monoclonal antibody that competes with the acquired monoclonal antibody for binding to the human CCR6 extracellular region can be obtained.
  • an epitope that is recognized by a monoclonal antibody that binds to the extracellular region of human CCR6 of the present invention and an antibody that binds to the same epitope are identified by identifying the epitope of the antibody obtained by the above-described binding assay system. These can be obtained by preparing and immunizing a partial synthetic peptide, a synthetic peptide mimicking the three-dimensional structure of an epitope, or the like.
  • human CCR6, the partial peptide, and a conjugate of the partial peptide and a carrier protein are immobilized on a sensor chip by, for example, an amine coupling method, and then a purified monoclonal antibody having a plurality of known concentrations is allowed to flow, bind and Measure dissociation.
  • the obtained data is subjected to kinetics analysis using a positive binding model using software attached to the device, and various parameters are acquired.
  • the binding activity to cells expressing human CCR6 is determined by fluorescence using an antibody conjugated with an appropriate fluorescent substance FITC, PE, Cy5, GFP, Alexa488, Alexa647, etc.
  • Cell binding ELISA, FCM (Guava EasyCyte Mini flow cytometry system, manufactured by Millipore), cell sorter FACSAria (registered trademark) (manufactured by BD Pharmigen), FMAT8100HTS system (manufactured by Applied Biosystems), ABI 8200 cell (Applied Bio) and the like can be used.
  • a recombinant antibody expression vector is an animal cell expression vector in which DNAs encoding human antibodies CH and CL are incorporated, and the animal cell expression vector is human. It can be constructed by cloning DNAs encoding antibody CH and CL, respectively.
  • Any human antibody CH and CL can be used for the C region of a human antibody.
  • ⁇ 1 subclass CH and ⁇ class CL of human antibodies are used.
  • cDNA is used for DNA encoding CH and CL of human antibodies
  • chromosomal DNA consisting of exons and introns can also be used.
  • Any animal cell expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody.
  • promoters and enhancers include SV40 early promoter [J. Biochem. , 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun. 149, 960 (1987)], or an immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and an enhancer [Cell, 33, 717 (1983)].
  • Recombinant antibody expression vectors balance the ease of construction of recombinant antibody expression vectors, the ease of introduction into animal cells, and the balance of expression levels of antibody H and L chains in animal cells.
  • a vector for expressing a recombinant antibody of a type (tandem type) in which the antibody H chain and L chain are present on the same vector [J. Immunol. Methods, 167, 271 (1994)]
  • pKANTEX93 International Publication No. 97/10354
  • pEE18 Hybridoma, 17, 559 (1998)
  • MRNA is extracted from hybridoma cells producing non-human antibodies, and cDNA is synthesized.
  • the synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library.
  • a recombinant phage or recombinant plasmid having cDNA encoding VH or VL is isolated from the library using DNA encoding the C region portion or V region portion of the mouse antibody as a probe.
  • the entire base sequence of VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, respectively, and the total amino acid sequence of VH or VL is estimated from the base sequence.
  • non-human animals that produce non-human antibody-producing hybridoma cells include mice, rats, hamsters, and rabbits, and any animal can be used as long as hybridoma cells can be produced. it can.
  • RNA easy kit manufactured by Qiagen
  • oligo (dT) -immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989)], or Oligo-dT> Sur (Sup.
  • a kit such as a registered trademark Kit (manufactured by Takara Bio Inc.) is used.
  • mRNA can also be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation (registered trademark) Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification (registered trademark) Kit (manufactured by Pharmacia).
  • any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma cell as a template is incorporated.
  • ZAP Express [Stratesies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], ⁇ ZAPI II (manufactured by Stratagene), ⁇ gt10, Clgt11A: Clgt11 DNA: Approach, I, 49 (1985)], Lambda BlueMid (Clontech), ⁇ ExCell, pT7T3-18U (Pharmacia), pCD2 [Mol. Cell. Biol. 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985)].
  • Escherichia coli into which a cDNA library constructed by a phage or plasmid vector is introduced can be used as long as the cDNA library can be introduced, expressed and maintained.
  • cDNA clones encoding non-human antibody VH or VL from a cDNA library for example, colony hybridization using an isotope or fluorescently labeled probe, or plaque hybridization [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] and the like are used.
  • a primer is prepared, and cDNA or cDNA library synthesized from mRNA is used as a template, Polymerase Chain Reaction method (hereinafter referred to as PCR method, Molecular Cloning, A Laboratory Manual, Second Edition, Spring Spring Harbor). (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)] can also be used to prepare cDNA encoding VH or VL.
  • PCR method Polymerase Chain Reaction method
  • the selected cDNA is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method. .
  • nucleotide sequence analysis methods include the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], etc., followed by ABI PRISM 3700 (manufactured by PE Biosystems) and A.P. L. F. Examples thereof include a base sequence automatic analyzer such as a DNA sequencer (Pharmacia).
  • VH and VL complete amino acid sequences for example, BLAST method [J. Mol. Biol. , 215, 403 (1990)], and the like, it can be confirmed whether the complete amino acid sequences of VH and VL are novel.
  • a human chimeric antibody expression vector VH or VL of a non-human antibody is respectively upstream of each gene encoding CH or CL of the human antibody of the recombinant antibody expression vector obtained in (1).
  • a human chimeric antibody expression vector can be constructed by cloning each of the encoded cDNAs.
  • the base sequence of the linking portion encodes an appropriate amino acid, and VH and VL cDNAs designed to be appropriate restriction enzyme recognition sequences are prepared.
  • the prepared VH and VL cDNAs are expressed in an appropriate form upstream of each gene encoding the human antibody CH or CL of the human CDR-grafted antibody expression vector obtained in (1). Each is cloned to construct a human chimeric antibody expression vector.
  • a cDNA encoding the non-human antibody VH or VL is amplified by a PCR method using a synthetic DNA having a recognition sequence of an appropriate restriction enzyme at both ends, and the recombinant antibody expression vector obtained in (1) Can also be cloned.
  • a cDNA encoding VH or VL of human CDR-grafted antibody can be constructed as follows.
  • the amino acid sequence of the VH or VL of the human antibody for grafting the amino acid sequence of the CDR of the VH or VL of the non-human antibody is selected. Any amino acid sequence can be used as long as it is derived from a human antibody.
  • FR amino acid sequences of human antibodies registered in databases such as Protein Data Bank, or common amino acid sequences of each subgroup of FRs of human antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] are used.
  • an FR amino acid sequence having the highest homology (at least 60% or more) with the FR amino acid sequence of the VH or VL of the original antibody is selected.
  • the amino acid sequence of the CDR of the original antibody is transplanted to the amino acid sequence of VH or VL of the selected human antibody, respectively, and the amino acid sequence of VH or VL of the human CDR-grafted antibody is designed.
  • Frequency of codon usage of the designed amino acid sequence in the base sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Considering Health and Human Services (1991)]
  • the DNA sequence is converted into a DNA sequence, and the DNA sequence encoding the VH or VL amino acid sequence of the human CDR-grafted antibody is designed.
  • the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the nucleotide sequence is determined by the same method as described in (2), and the desired human CDR graft A plasmid having a DNA sequence encoding the amino acid sequence of the antibody VH or VL is obtained.
  • a plasmid such as pBluescript SK (-) (manufactured by Stratagene)
  • the nucleotide sequence is determined by the same method as described in (2), and the desired human CDR graft
  • a plasmid having a DNA sequence encoding the amino acid sequence of the antibody VH or VL is obtained.
  • a product obtained by synthesizing the full length VH and the full length VL as one long chain DNA based on the designed DNA sequence can be used in place of the PCR amplification product.
  • cDNA encoding the human CDR-grafted antibody VH or VL can be obtained for expression of the human-type CDR-grafted antibody obtained in (1). Can be easily cloned into a vector.
  • a human CDR-grafted antibody can be obtained by transplanting only non-human antibody VH and VL CDRs into human antibody VH and VL FRs. The binding activity is reduced compared to the original non-human antibody [BIO / TECHNOLOGY, 9,266 (1991)].
  • amino acid residues that are directly involved in binding to the antigen amino acid residues that interact with the CDR amino acid residues, and Decreased by maintaining the three-dimensional structure of the antibody, identifying amino acid residues indirectly involved in antigen binding, and substituting those amino acid residues with the amino acid residues of the original non-human antibody Antigen binding activity can be increased.
  • the amino acid residues of FR of human antibody VH and VL can be modified by performing the PCR described in (4) using the synthetic DNA for modification.
  • the base sequence is determined by the method described in (2), and it is confirmed that the target modification has been performed.
  • a recognition sequence for an appropriate restriction enzyme at the 5 ′ end of the synthetic DNA located at both ends are introduced into the human CDR-grafted antibody expression vector obtained in (1), and cloned so as to be expressed in an appropriate form upstream of each gene encoding CH or CL of the human antibody.
  • Transient expression of recombinant antibodies Transient expression of recombinant antibodies using the recombinant antibody expression vectors obtained in (3) and (6) or modified expression vectors
  • any cell can be used as long as it can express the recombinant antibody.
  • COS-7 cells American Type Culture Collection (ATCC) number: CRL1651] [Methods in Nucleic Acids Res. , CRC press, 283 (1991)].
  • an expression vector For introduction of an expression vector into COS-7 cells, the DEAE-dextran method [Methods in Nucleic Acids Res. , CRC press (1991)], or lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant are measured by enzyme immuno-antibody method [Monoclonal Antibodies-Principles and practices, Academic Press (1996), Antibodies-A Laboratories, 1996]. Cold Spring Harbor Laboratory (1988), monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like.
  • Any host cell capable of expressing a recombinant antibody can be used as a host cell into which the recombinant antibody expression vector is introduced.
  • CHO-K1 ATCC CCL-61
  • DUKXB11 ATCC CCL-9096
  • Pro-5 ATCC CCL-1781
  • CHO-S Life Technologies, Cat # 11619
  • mice 20 also referred to as ATCC number: CRL1662 or YB2 / 0
  • mouse myeloma cell NS0 mouse myeloma cell SP2 / 0-Ag14
  • mouse P3X63-Ag8.653 cells ATCC number: CRL1580
  • dihydro CHO cells deficient in folate reductase gene Dihydroformate Reductase, hereinafter referred to as dhfr
  • a sugar chain modification in which the 1-position of fucose is ⁇ -linked to the 6-position of the reducing terminal of a protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the N-glycoside-linked complex sugar chain
  • a host cell having a decreased or deleted activity such as a protein involved in the transport of an intracellular sugar nucleotide GDP-fucose to the Golgi apparatus, such as an ⁇ 1,6-fucose transferase gene
  • Examples include CHO cells (International Publication No. 2005/035586, International Publication No. 02/31140), Lec13 [Somatic Cell and Molecular genetics, 12, 55 (1986)] that has acquired lectin resistance.
  • a transformant that stably expresses the recombinant antibody is selected by culturing in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japan).
  • G418 sulfate hereinafter referred to as G418, (Japan).
  • Examples of the medium for animal cell culture include RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM medium ( Invitrogen) or a medium obtained by adding various additives such as FBS to these mediums.
  • RPMI1640 medium manufactured by Invitrogen
  • GIT medium manufactured by Nippon Pharmaceutical
  • EX-CELL301 medium manufactured by JRH
  • IMDM medium manufactured by Invitrogen
  • Hybridoma-SFM medium Invitrogen
  • the recombinant antibody is purified from the culture supernatant of the transformant using a protein A-column [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-A LaboratoryLaboratoryLaboratoryLaboratoryLaboratory. (1988)]. It is also possible to combine methods used in protein purification such as gel filtration, ion exchange chromatography and ultrafiltration.
  • the molecular weight of the purified recombinant antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies-Principles and practicies, Third]. edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  • Activity evaluation of purified monoclonal antibody or antibody fragment thereof The activity evaluation of the purified monoclonal antibody or antibody fragment thereof of the present invention can be carried out as follows.
  • the binding activity to the human CCR6-expressing cell line is measured by the surface plasmon resonance method using the binding assay described in 1- (6a) and the Biacore system described in (6b). In addition, the fluorescent antibody method [Cancer Immunol. Immunother. , 36, 373 (1993)].
  • CDC activity or ADCC activity against an antigen-positive cultured cell line is measured by a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)].
  • Method for controlling effector activity of antibody As a method for controlling the effector activity of the human CCR6 monoclonal antibody of the present invention, for example, the 297th asparagine (Fragment, crystallizable, hereinafter referred to as Fc region) of an antibody A method for controlling the amount of fucose (also referred to as core fucose) ⁇ -1,6-linked to N-acetylglucosamine (GlcNAc) present at the reducing end of an N-linked complex sugar chain that binds to Asn) (International Publication No. 2005 / No. 035586, International Publication No. 2002/31140, International Publication No. 00/61739) and a method of controlling by modifying amino acid residues in the Fc region of an antibody.
  • the effector activity can be controlled by any method using the human CCR6 monoclonal antibody of the present invention.
  • Effector activity refers to antibody-dependent activity caused through the Fc region of an antibody.
  • antibody-dependent cytotoxic activity ADCC activity
  • complement-dependent cytotoxic activity CDC activity
  • antibody-dependent phagocytosis antibody-dependent phagocytosis, ADP activity
  • the effector activity of the antibody can be increased or decreased by controlling the content of fucose added to N-acetylglucosamine at the reducing end of the N-linked complex sugar chain on the Fc region.
  • the antibody is expressed using CHO cells deficient in the ⁇ 1,6-fucose transferase gene. An antibody to which fucose is not bound can be obtained.
  • Antibodies without fucose binding have high ADCC activity.
  • the antibody is expressed using a host cell into which an ⁇ 1,6-fucose transferase gene has been introduced.
  • an antibody to which fucose is bound can be obtained.
  • An antibody to which fucose is bound has a lower ADCC activity than an antibody to which fucose is not bound.
  • ADCC activity and CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of the antibody.
  • the CDC activity of an antibody can be increased by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165.
  • ADCC activity or CDC activity can be increased or decreased.
  • an antibody with controlled effector activity of an antibody can be obtained by using the above method in combination with one antibody.
  • the monoclonal antibody or antibody fragment of the present invention can be used for the treatment of diseases involving human CCR6-positive cells.
  • the therapeutic agent containing the monoclonal antibody of the present invention or an antibody fragment thereof, or a derivative thereof may contain only the antibody or the antibody fragment, or a derivative thereof as an active ingredient. It is provided as a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics, mixed with one or more physically acceptable carriers.
  • Examples of the administration route include oral administration and parenteral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular and intravenous.
  • Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments and tapes.
  • Preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders and granules.
  • Liquid preparations such as emulsions or syrups include water, sugars such as sucrose, sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid It is produced using an antiseptic such as esters, or a flavor such as strawberry flavor or peppermint as an additive.
  • Capsules, tablets, powders or granules include excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose or gelatin, a surfactant such as fatty acid ester, or a plasticizer such as glycerin is used as an additive.
  • preparations suitable for parenteral administration include injections, suppositories, and sprays.
  • Injection is manufactured using a carrier made of a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are produced using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the propellant is manufactured using a carrier that does not irritate the recipient's oral cavity and airway mucosa, disperses the monoclonal antibody of the present invention or an antibody fragment thereof as fine particles, and facilitates absorption.
  • a carrier for example, lactose or glycerin is used. It can also be produced as an aerosol or dry powder.
  • Method for diagnosing disease using anti-human CCR6 monoclonal antibody or antibody fragment thereof of the present invention By detecting or measuring human CCR6 or cells expressing human CCR6 using the monoclonal antibody or the antibody fragment of the present invention, Diseases associated with CCR6 can be diagnosed.
  • the diagnosis of an autoimmune disease which is one of the diseases associated with human CCR6, can be performed, for example, by detecting or measuring human CCR6 as follows. Diagnosis can be made by detecting human CCR6 expressed in cells in a patient using an immunological technique such as flow cytometry.
  • the immunological technique is a method of detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • a radioactive substance labeled immunoassay method an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blot method, a physicochemical method and the like can be mentioned.
  • the radioactive substance-labeled immunoantibody method is, for example, a method in which an antigen of the present invention or an antibody fragment thereof is reacted with an antigen or an antigen-expressing cell and further reacted with a radiolabeled anti-immunoglobulin antibody or binding fragment. Measure with a scintillation counter.
  • an antigen or a cell expressing the antigen is reacted with the antibody of the present invention or the antibody fragment, and further, a labeled anti-immunoglobulin antibody or binding fragment is reacted, and then a coloring dye. Is measured with an absorptiometer. For example, a sandwich ELISA method is used.
  • an enzyme label known in the art [enzyme immunoassay, Medical School (1987)] can be used.
  • alkaline phosphatase label for example, alkaline phosphatase label, peroxidase label, luciferase label, or biotin label is used.
  • the sandwich ELISA method is a method of binding an antibody to a solid phase, trapping an antigen to be detected or measured, and reacting a second antibody with the trapped antigen.
  • the ELISA method two types of antibodies or antibody fragments recognizing an antigen to be detected or measured and having different antigen recognition sites are prepared, and the first antibody or antibody fragment is pre-plated on a plate (for example, 96 Next, the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin. After the cells adsorbed with the antibody are reacted with cells or a lysate thereof, tissue or a lysate thereof, cell culture supernatant, serum, pleural effusion, ascites or ocular fluid, the label is obtained. The detected monoclonal antibody or antibody fragment is reacted, and a detection reaction according to the labeling substance is performed.
  • the antigen concentration in the test sample is calculated from a calibration curve prepared by diluting antigens with known concentrations stepwise.
  • an antibody used in the sandwich ELISA method either a polyclonal antibody or a monoclonal antibody may be used, and an antibody fragment such as Fab, Fab ′, or F (ab) 2 may be used.
  • the combination of two types of antibodies used in the sandwich ELISA method may be a combination of monoclonal antibodies or antibody fragments recognizing different epitopes, or a combination of polyclonal antibodies and monoclonal antibodies or antibody fragments.
  • the fluorescence immunoassay is measured by the method described in the literature [Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)].
  • a label used in the fluorescence immunoassay a fluorescent label known in the art [fluorescent antibody method, Soft Science (1983)] can be used.
  • FITC or RITC is used.
  • the luminescent immunoassay is measured by the method described in the literature [Bioluminescence and chemiluminescence, clinical examination 42, Yodogawa Shoten (1998)].
  • Examples of the label used in the luminescence immunoassay include known phosphor labels, and acridinium ester, lophine, and the like are used.
  • Western blotting involves fractionating an antigen or cells expressing the antigen with SDS (sodium dodecyl sulfate) -PAGE (polyacrylamide gel) [Antibodies-A Laboratory Manual Spring Harbor Laboratory (1988)], and then separating the gel. Blotting was performed on a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose membrane, and an antibody or antibody fragment that recognizes the antigen was reacted with the membrane, and further, a fluorescent substance such as FITC, an enzyme label such as peroxidase, or a biotin label was applied. After reacting with the anti-mouse IgG antibody or binding fragment, the label is measured by visualization.
  • SDS sodium dodecyl sulfate
  • PAGE polyacrylamide gel
  • the monoclonal antibody of the present invention was reacted, washed with PBS containing 0.05 to 0.1% Tween-20 (hereinafter referred to as Tween-PBS), and peroxidase-labeled goat anti-mouse IgG was washed at room temperature. React for 2 hours.
  • the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 is detected by washing with Tween-PBS and detecting the band to which the monoclonal antibody is bound using ECL Western Blotting Detection Reagents (manufactured by Amersham).
  • ECL Western Blotting Detection Reagents manufactured by Amersham.
  • an antibody used for detection by Western blotting an antibody capable of binding to a polypeptide that does not have a natural three-dimensional structure is used.
  • the physicochemical method is performed by, for example, forming an aggregate by binding human CCR6, which is an antigen, to the monoclonal antibody of the present invention or an antibody fragment thereof, and detecting the aggregate.
  • a physicochemical method a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetric method, or a latex immunoturbidimetric method [Presentation of clinical test method, Kanbara Publishing (1998)] can be used.
  • Latex immunoturbidimetry is a method in which an antibody or antigen-sensitized carrier such as polystyrene latex having a particle size of about 0.1 to 1 ⁇ m is used to cause an antigen-antibody reaction with the corresponding antigen or antibody. Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integrating sphere turbidity, the antigen concentration or the like in the test sample is measured.
  • an antibody or antigen-sensitized carrier such as polystyrene latex having a particle size of about 0.1 to 1 ⁇ m
  • a known immunological detection method can be used, but preferably, immunoprecipitation method, immune cell staining method, immunohistochemical staining method or fluorescent antibody staining method Etc. are used.
  • cells expressing human CCR6 are reacted with the monoclonal antibody of the present invention or an antibody fragment thereof, and then a carrier having specific binding ability to immunoglobulin such as protein G-sepharose is added to the antigen antibody. Allow the complex to settle.
  • a carrier having specific binding ability to immunoglobulin such as protein G-sepharose is added to the antigen antibody. Allow the complex to settle.
  • the following method can be used.
  • the above-described monoclonal antibody of the present invention or an antibody fragment thereof is immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS.
  • the antibody When the antibody is in an unpurified state, such as a hybridoma culture supernatant, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein-A or protein-G is immobilized on a 96-well plate for ELISA in advance. After blocking with BSA-PBS, the hybridoma culture supernatant is dispensed and bound.
  • an unpurified state such as a hybridoma culture supernatant, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein-A or protein-G is immobilized on a 96-well plate for ELISA in advance. After blocking with BSA-PBS, the hybridoma culture supernatant is dispensed and bound.
  • Immunoprecipitates are extracted from the well-washed plate with SDS-PAGE sample buffer and detected by Western blotting as described above.
  • the immune cell staining method or the immunohistochemical staining method is a method in which cells or tissues expressing an antigen are treated with a surfactant or methanol to improve the passage of the antibody in some cases, and then reacted with the monoclonal antibody of the present invention. And then reacting with a fluorescent label such as FITC, an enzyme label such as peroxidase or a biotin label, or an anti-immunoglobulin antibody or a binding fragment thereof, then the label is visualized and microscopically observed .
  • a fluorescent label such as FITC
  • an enzyme label such as peroxidase or a biotin label
  • a fluorescent antibody staining method in which a fluorescently labeled antibody is reacted with a cell and analyzed with a flow cytometer [Monoclonal Antibodies-Principles and Practice, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific Fick (1987)].
  • the monoclonal antibody of the present invention or an antibody fragment thereof, which binds to the extracellular region of human CCR6, can detect cells expressing a natural three-dimensional structure by fluorescent antibody staining.
  • the formed antibody-antigen complex and the free that is not involved in the formation of the antibody-antigen complex can be measured without separating the antibody or antigen.
  • Example 1 Construction of CHO cell expressing human CCR6 on cell membrane (1) Cloning of human CCR6 gene A gene encoding human CCR6 was isolated from cDNA derived from human leukocytes by the following procedure (SEQ ID NOs: 1 and 3).
  • a 24 mer region within the open reading frame (hereinafter abbreviated as ORF) from the CCR6 start codon A forward primer (Human-CCR6-f) (SEQ ID NO: 5) that binds to and a reverse primer (Human-CCR6-r) (SEQ ID NO: 6) that binds to the 21 mer within the ORF up to the CCR6 stop codon was designed.
  • DNA polymerase KOD-Plus (TOYOBO) was used, and 50 ⁇ L of a reaction solution containing human leukocyte-derived cDNA as a template [10 ⁇ Buffer for KOD-Plus, 0.2 mmol / L dNTPs, 0. 2 ⁇ mol / L
  • PCR In PCR, after heating at 94 ° C. for 5 minutes, 30 cycles of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 2 minutes were performed.
  • the reaction solution was subjected to agarose gel electrophoresis, and an amplified DNA fragment of about 1.1 kbp was recovered using QIAEXII (QIAGEN) according to the attached manual.
  • the recovered DNA fragment was subjected to ligation reaction using pCR4 Blunt-TOPO vector (Invitrogen) according to the attached manual.
  • the reaction solution was used to transform E. coli DH5 ⁇ strain (Competent Quick DH5 ⁇ , TOYOBO), and vector DNA was isolated from the resulting ampicillin resistant clone using a vector extractor PI-50 (Kurabo).
  • the base sequence of DNA inserted into each vector was determined according to the attached manual using PRISM3730x1 (Applied Biosystems) and Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Amplified fragments derived from two independent PCRs were subcloned, and the base sequences of 6 clones in which no PCR error was observed were determined.
  • the human CCR6 expression vector pKANTEX-hCCR6 (isoform 1) was constructed by the following procedure (FIGS. 1 and 2).
  • a synthetic oligo DNA (CCR6MunI-f) (SEQ ID NO: 7) in which a restriction enzyme MunI recognition sequence and a Kozak sequence are added to the 5 ′ end of the forward primer that binds to the 28mer region in the ORF from the human CCR6 start codon, and CCR6 termination Synthetic oligo DNA (CCR6BamHI-) with a histidine tag (His-tag) sequence, a stop codon, and a restriction enzyme BamHI recognition sequence added to the 5 'end of the reverse primer that binds to the 3' 25 base region in the ORF without codons r) (SEQ ID NO: 8) was designed.
  • DNA polymerase KOD-Plus (TOYOBO) was used, and 50 ⁇ L of a reaction solution [10 ⁇ Buffer for KOD-plus, 0.2 mmol containing the TOPO-hCCR6 (isoform1) vector obtained above as a template. / L dNTPs, 0.2 ⁇ mol / L
  • the above two primers, KOD-plus 1U, 1 mmol / LmgSO 4 ] were prepared.
  • PCR was performed at 94 ° C. for 2 minutes, followed by 30 cycles of 94 ° C. for 15 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 1.5 minutes.
  • a vector DNA fragment TOPO-hCCR6MunI-BamHI was prepared in the same manner as in Example 1 (1).
  • the target vector was treated with the restriction enzyme BamHI (Takara Bio) for 2 hours, and then treated with the restriction enzyme MunI (Takara Bio) for 2 hours.
  • the pKANTEX93 vector described in International Publication No. 97/10354 was subjected to enzyme treatment with restriction enzymes EcoRI (Bio labs) and BamHI (Takara Bio) for 3 hours.
  • Each reaction solution is subjected to agarose electrophoresis, and about 1.1 kbp hCCR6 DNA fragment and about 9.1 kbp pKANTEX93-EcoRI-BamHI DNA fragment are collected using QIAEXII (QIAGEN) according to the attached manual. did.
  • the CCR6 DNA fragment and pKANTEX93-EcoRI-BamHI DNA fragment obtained above were mixed at a molar ratio of 16: 1, and Ligation High ver. 2 (manufactured by TOYOBO) was added in an equal volume, and a binding reaction was performed overnight at 16 ° C.
  • the hCCR6 expression vector pKANTEX-hCCR6 (containing a restriction enzyme MunI site and a Kozak sequence at the N-terminus of isoform 1 and a His-tag sequence and a restriction enzyme BamHI site at the C-terminus) isoform1) was produced [FIG. 2 (a)].
  • the human CCR6-expressing Tol2 transposon vector Tc26_hCCR6 was constructed according to the following procedure.
  • pKTABEX_TC26 an expression vector (hereinafter referred to as pKTABEX_TC26) excluding the antibody constant region of the Tol2 transposon expression vector (International Publication No. 2010/143698) is described. ) was digested with the restriction enzyme BglII, and the blunt ends were blunted using a Blunting high kit (TOYOBO).
  • CHO / DG44 cells are Iscove's Modified Dulbecco's Medium (IMDM medium) supplemented with 10% fetal bovine dialysis serum (Invitrogen), 50 ⁇ g / mL Gentamycin (Nacalai Tesque) and 1 ⁇ HT supplement (Invitrogen). (Invitrogen) (hereinafter referred to as a basic medium).
  • the cultured CHO / DG44 cells were 137 mmol / L KCl, 2.7 mmol / L NaCl, 8.1 mmol / L Na2HPO4, 1.5 mmol / L KH 2 PO 4 , 4.0 mmol / LmgCl 2 (hereinafter referred to as K-PBS buffer).
  • a cell suspension of 8 ⁇ 10 6 cells / mL was prepared.
  • 200 ⁇ L cell suspension (1.6 ⁇ 10 6 cells) and 10 ⁇ g pKANTEX-hCCR6 (isoform 1) linearized vector were mixed and added to Gene PuLser Cuvette (distance between electrodes 2 mm) (BIO-RAD) for gene transfer.
  • Gene transfer was performed using the apparatus Gene Pulser (BIO-RAD) under the conditions of a pulse voltage of 0.35 KV and an electric capacity of 250 ⁇ F.
  • the cell suspension is mixed with 10 mL of a basic medium not added with HT (hereinafter referred to as HT-medium), seeded in a 75 cm 2 adherent cell flask (manufactured by Greiner), and incubated at 37 ° C., 5% CO 2 incubator. Culturing was performed in the inside. After culturing for 3 days, the culture medium is replaced with an HT-basic medium (hereinafter referred to as 50 nM / IMDM medium) containing 50 nM methotrexate (hereinafter referred to as MTX) and cultured to proliferate cells resistant to MTX.
  • HT-medium a basic medium not added with HT
  • 50 nM / IMDM medium an HT-basic medium
  • MTX 50 nM methotrexate
  • the HT-basal medium hereinafter, 200 nM MTX / IMDM medium
  • 200 nM MTX / IMDM medium containing 200 nM MTX was replaced and cultured to obtain a 200 nM MTX resistant strain.
  • the obtained MTX resistant strain was named CHO-hCCR6 (isoform 1).
  • the human CCR6-expressing Tol2 transposon vector Tc26_hCCR6 prepared above was introduced into CHO-K1 cells as follows. 4.0 ⁇ 10 6 floating-conditioned CHO-K1 cells (RIKEN cell bank: RCB0285) were collected in a centrifuge tube, centrifuged at 1000 rpm for 5 minutes to remove the supernatant, and then washed with cold PBS ( ⁇ ). did.
  • the washed treated cells were suspended in the included 100 [mu] L Solution_V the Nucleofector TM kit V (amaxa), 2 ⁇ g human CCR6 expression Tol2 transposon vector per cuvette, and 5 [mu] g Tol2 transposase expression vector (WO 2010/143698) (hereinafter, after adding abbreviated as TPEX_pMug), transferred a total volume of Nucleofector dedicated cuvette, under conditions of program U024 of Nucleofector TM, were transgenic. 20 mL of subculture medium was placed in the reservoir, and the pulsed cells were immediately added and suspended. After seeding in a 96-well plate for culture at 200 ⁇ L / well, the cells were cultured at 37 ° C. under 5% CO 2 conditions.
  • CHX / IMDM basic medium
  • CHX basic medium
  • CHX / IMDM a basic medium
  • CHX cycloheximide
  • BSA-PBS bovine serum albumin
  • the cells were centrifuged, the supernatant was removed, and the cells were washed 3 times with BSA-PBS. After washing, the cells were suspended in 400 ⁇ L of FCM buffer, passed through a cell strainer mesh, and the fluorescence intensity was analyzed using a flow cytometer (FACSCalibur (registered trademark), Becton Dickinson).
  • FACSCalibur registered trademark
  • Becton Dickinson Becton Dickinson
  • the anti-human CCR6 monoclonal antibody did not react with the parental strains CHO / DG44 and CHO-K1, but introduced the hCCR6 expression vector pKANTEX-hCCR6 (isoform 1) or Tc26-hCCR6, respectively. It reacted to both DG44 cells and CHO-K1 cells [FIGS. 3 (a) and 3 (b)].
  • hCCR6-His-tag protein For CHO-hCCR6, the expression of hCCR6-His-tag protein was confirmed using an anti-His tag body.
  • Cells were prepared in the same manner as described above, treated with ethanol, and subjected to cell membrane permeabilization. 100 ⁇ L of 1 ⁇ g / mL anti-His tag antibody (QIAGEN) was added to these cells, reacted at 4 ° C. for 1 hour, and Goat F (ab ′) 2 polyclonal anti-mouse-FITC (Dako) diluted 50 times as a secondary antibody. Analysis was performed in the same manner as described above except that 100 ⁇ L was added and reacted at 4 ° C. for 1 hour.
  • the anti-His tag antibody did not react with the parent strain CHO / DG44, but reacted with CHO / DG44 cells into which the hCCR6 expression vector pKANTEX-hCCR6 (isoform 1) was introduced [FIG. c)]. Therefore, it was confirmed that CHO / DG44 cells introduced with the hCCR6 expression vector pKANTEX-hCCR6 (isoform 1) highly express hCCR6 fused with a His tag.
  • Example 2 Construction of human monocytic cells expressing human CCR6
  • the hCCR6 expression vector prepared in Example 1 (2) Using pKANTEX-hCCR6 (isoform 1), gene transfer was performed to U937 cells in the same manner as in Example 1 (3). Drug resistance selection was performed under culture conditions containing 0.5 mg / mL G418. As a result, U937 cells U937-CCR6 expressing hCCR6 were obtained.
  • Example 3 Construction of cells expressing human CCR6 for calcium assay A cell for detecting a signal from human CCR6 (hCCR6) by a calcium assay was constructed. According to a known method [Analytical Biochemistry 400 (2010) 163-172], an inducible expression cell of hCCR6 was constructed using KJMGER8 cells (a cell line derived from Namalwa cells) as a host. Specifically, an assay cell (hereinafter referred to as CCR6G16) capable of detecting a signal from hCCR6 by a calcium assay by co-introducing an hCCR6 (isoform 1) induction plasmid and a G ⁇ 16 expression plasmid into KJMGER8 cells is constructed. did.
  • CCR6G16 an assay cell capable of detecting a signal from hCCR6 by a calcium assay by co-introducing an hCCR6 (isoform 1) induction plasmid and a G ⁇ 16 expression plasmid into KJMGER8
  • the CCR6-induced expression plasmid was prepared according to a known method [Analytical Biochemistry 400 (2010) 163-172].
  • the G ⁇ 16 expression plasmid was prepared by incorporating human G ⁇ 16 DNA into the expression vector pAMoh (International Publication No. 030887366).
  • pAMoh International Publication No. 030887366
  • SIGMA 10 nM ⁇ -estradiol
  • DNA fragments encoding various types of chimeric CCR6 are sense megaprimes and antisense megaprimers of about 150 bases (Human-1-162-f, Human-138-288-r, Human-264-413-f, Human-394). -540-r, Human-519-677-f, Human-649-781-r, Human-759-914-f, Human-890-1020-r, Human-999-1125-f, MouseNt-1-162 -F, MouseL1-264-413-f, MouseL2-519-677-f and MouseL3-759-914-f) (SEQ ID NOs: 9-21) and restriction enzyme added primers (MouseCCR6Mun1-f, CCR6Mun1 and CCR6Bam) 1) (SEQ ID NO: 22-24), was prepared subjected to PCR.
  • the expression vector TOPO-mNtCCR6 containing a DNA fragment encoding chimeric CCR6 whose N-terminal extracellular region is the amino acid sequence of a mouse is used, and the extracellular loop (ECL) 1 is a mouse.
  • Expression vector TOPO-mECL2hCCR6 which contains a DNA fragment encoding chimeric CCR6, wherein ECL2 is the amino acid sequence of a mouse, is an amino acid sequence of mouse
  • An expression vector TOPO-mECL3hCCR6 containing a DNA fragment encoding a certain chimeric CCR6 was prepared.
  • the produced expression vector was subjected to enzyme treatment for 2 hours using the restriction enzyme BamHI (Takara Bio). Next, the enzyme treatment was performed for 2 hours using the restriction enzyme MunI (Takara Bio).
  • the DNA encoding the target chimeric CCR6 was obtained using the EcoRI-BamHI-pKANTEX93 vector fragment treated with the restriction enzymes EcoRI and BamHI and the DNA fragment encoding each chimeric CCR6.
  • Expression vectors pKANTEX-mNthCCR6, pKANTEX-mECL1hCCR6, pKANTEX-mECL2hCCR6 and pKANTEX-mECL3hCCR6 were prepared.
  • CHO / DG44 cells CHO-mNthCCR6, CHO-mECL1hCCR6, CHO-mECL2hCCR6 and CHO-mECL3hCCR6 expressing each chimeric CCR6 were prepared.
  • the anti-human CCR6 monoclonal antibody reacted with CHO-mECL1hCCR6, CHO-mECL2hCCR6 and CHO-mECL3hCCR6, but did not react with CHO-mNthCCR6 and the parental CHO / DG44 [FIG. 7 (a) to (f) ].
  • the anti-mouse CCR6 monoclonal antibody reacted with CHO-mNthCCR6 [FIG. 7 (g)].
  • Each isotype control antibody did not react with any cells [FIGS. 7 (a) to (g)].
  • Example 5 Construction of CHO cells expressing cynomolgus monkey CCR6 (1) Cloning of cynomolgus monkey CCR6 gene In order to examine the reactivity of anti-human CCR6 antibody to cynomolgus monkey CCR6, a cynomolgus monkey CCR6 expression vector was prepared. Since cynomolgus CCR6 cDNA sequence has not been reported so far, cynomolgus CCR6 cDNA was cloned by the following procedure.
  • rhesus monkey CCR6 ORF accesion No. AF508730
  • rhesus monkey chromosome 4 accesion No. NC — 007861
  • the nucleotide sequences located upstream and downstream of the rhesus monkey CCR6 ORF were determined.
  • cynomolgus monkey CCR6 cloning primers Macacaf-CCR6-f and Macacaf-CCR6- r
  • an expression vector TOPO-mfCCR6-MunI-BamHI containing a gene encoding cynomolgus monkey CCR6 was prepared from cynomolgus monkey leukocyte-derived cDNA in the same manner as in Example 1 (1), and the gene sequence was identified. did.
  • cynomolgus monkey CCR6 has two isoforms (SEQ ID NOs: 114 and 116), which differ in base sequence by 4 bases, and in terms of amino acid sequence, the 177th amino acid residue of the cynomolgus monkey CCR6 intracellular domain.
  • Val or Ile SEQ ID NOs: 115, 117.
  • 177Val type cynomolgus CCR6 was 11 clones out of 15 clones
  • 177Ile type cynomolgus CCR6 was 4 clones out of 15 clones.
  • the vector in which isoform 1 was cloned was TOPO-cynomolgus CCR6 (isoform 1), and the vector in which isoform 2 was cloned was TOPO-mfCCR6 (isoform 2).
  • a cynomolgus monkey CCR6 expression vector pKANTEX-mfCCR6 (isoform1) was constructed in the same manner as in Example 1 (2) except that TOPO-mfCCR6 (isoform1) was used as a template.
  • the DNA fragment encoding the human CCR6 N-terminal extracellular domain is a primer [CCR6 (1) containing a restriction enzyme NotI recognition sequence or a restriction enzyme BamHI recognition sequence using pKANTEX-hCCR6 (isoform1) prepared in Example 1 (2) as a template. -28) -f and CCR6 (118-138) -BamHI-r] (SEQ ID NO: 29, 30) were used for PCR amplification.
  • DNA polymerase KOD-Plus (TOYOBO) was used, and 50 ⁇ L of the reaction solution [10 ⁇ Buffer for KOD-Plus, 0.2 mmol containing TOPO-hCCR6 (isoform1) prepared in Example 1 as a template. / L dNTPs, 0.2 ⁇ mol / L The above two primers, KOD-plus 1U, 1 mmol / LmgSO 4 ] were prepared.
  • PCR was performed at 94 ° C. for 2 minutes, followed by 25 cycles of 94 ° C. for 15 seconds, 62 ° C. for 30 seconds, and 68 ° C. for 1 minute.
  • the resulting vector was named TOPO-NthCCR6-IgG4Fc.
  • TOPO-NthCCR6-IgG4Fc and pKANTEX CD27-IgG4Fc described in International Publication No. 2010/001908 were subjected to enzyme treatment using restriction enzymes NotI and BamHI, and fragments of about 250 bp and about 9.5 kbp were obtained and ligated. .
  • NthCCR6-IgG4Fc for expressing NthCCR6-Fc was obtained.
  • the gene sequence of NthCCR6-Fc encoded by this vector is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 32.
  • NthCCR6-IgG4Fc Production of human CCR6 N-terminal extracellular domain Fc fusion protein (NthCCR6-IgG4Fc) Expression of NthCCR6-IgG4Fc in the same manner as in Example 1 (3) using the expression vector prepared in Example 6 (1) above Cells were made. Cell culture was performed using serum-free medium EXCELL302 (manufactured by JRH Bioscience) for one week, and then the culture supernatant was collected and purified by the method described below.
  • EXCELL302 manufactured by JRH Bioscience
  • the culture supernatant was centrifuged at 3000 rpm, 4 ° C. for 10 minutes, and after collecting the supernatant, the supernatant was filtered using 0.22 ⁇ m pore size PES membrane (manufactured by Asahi Techno Glass).
  • a 0.8 cm pore diameter column is filled with 0.5 mL of Mab Select (Amersham Pharmacia Biotech), 3.0 mL of purified water is added, and 2.0 mL of 0.1 M citrate buffer and 2 M borate-0.15 M NaCl are added. Equilibration was performed with 1.5 mL of a buffer solution (pH 7.5) (hereinafter referred to as borate buffer).
  • the culture supernatant was passed through a column and then washed with 10 mL of borate buffer. After washing, the protein adsorbed on the carrier was eluted using 2.25 mL of 0.1 M citrate buffer (pH 3.5). The elution fraction was obtained by dividing it into 3 fractions each of 250 ⁇ L, 1 mL ⁇ 2. Next, the absorbance (OD 280 ) of 280 nM of each of the obtained fractions was measured with an absorptiometer (SHIMADZU UV-1700) to identify the eluted fraction containing the target protein.
  • SHIMADZU UV-1700 an absorptiometer
  • the buffer for the target protein elution fraction was changed to 0.01 M sodium citrate-0.15 M NaCl buffer (pH 6.0) using Econo-Pac 10DG (BIO-RAD).
  • the NthCCR6-IgG4Fc solution was sterile filtered using a 0.22 ⁇ m pore size Millex GV (MILLIPORE) and stored at 4 ° C.
  • NthCCR6-IgG4Fc protein It was confirmed to exist as a dimer. Moreover, since the molecular weight of NthCCR6-IgG4Fc inferred from the amino acid sequence is about 30 kDa, the possibility that a sugar chain was added to the NthCCR6-IgG4Fc protein was considered.
  • the obtained NthCCR6-IgG4Fc purified protein was subjected to enzyme treatment using O-Glycosidase and N-Glycosidase, and SDS-PAGE analysis of the digest was performed.
  • the O-glycosidase-treated sample band was at the same 100 kDa and 50 kDa positions as the enzyme-untreated sample band, but the N-glycosidase-treated sample and O The bands of the samples treated with -glycosidase + N-glycosidase shifted to low molecular weights of 70 kDa and 35 kDa (FIG. 11).
  • NthCCR6-IgG4Fc Since two sugar chains of about 1.5 kDa are bound to the Fc site of the antibody, it is considered that the produced NthCCR6-IgG4Fc has more sugar chains in the NthCCR6 site. In addition, it was revealed that the produced NthCCR6-IgG4Fc protein has an N-linked sugar chain bound thereto instead of an O-linked sugar chain.
  • NthCCR6-IgG4Fc human CCR6 N-terminal extracellular domain Fc fusion protein
  • tween-PBS PBS containing 0.05% tween 20
  • BSA-PBS PBS containing 0.05% tween 20
  • mouse IgG2b, kappa isotype control (BD Bioscience) was added at 50 ⁇ L / well and allowed to react at room temperature for 1 hour.
  • DAKO peroxidase-labeled anti-mouse immunoglobulin rabbit antibody
  • the substrate was washed three times with tween-PBS, and a substrate solution containing a chromogenic substrate ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] [1 mmol / L ABTS-0.1 mol / L citrate buffer (pH 4.2), 0.1% H 2 O 2 ] was added at 50 ⁇ L / well to perform a color reaction. After the reaction, the absorbance at 415 nM (OD 415 ) was measured using a plate reader (Emax; Moleclar Devices).
  • the anti-human CCR6 antibody bound to the NtCCR6-IgG4Fc protein.
  • almost no binding was observed in wells to which isotype control mouse IgG2b antibody or BSA-PBS was added (FIG. 12). Therefore, it was revealed that the produced NthCCR6 protein fused with human IgG4Fc has the N-terminal region of human CCR6 to which the anti-human CCR6 monoclonal antibody specifically reacts.
  • Example 7 Production of monoclonal antibody against human CCR6 (1) Preparation of immunogen (a) Cellular immunization CHOK1-hCCR6 produced in Example 1 and U937CCR6 produced in Example 2 were added at 1 ⁇ 10 7 cells / 500 ⁇ L. The number of cells was adjusted to 4 weeks, 4 weeks old to SD rats, NZB mice, (NZB ⁇ NZW) F1 mice, BXSB mice (Japan SLC Co., Ltd.) or Balb / c mice (Claire Japan), a total of 4 Administered once.
  • KLH, THY or BSA is dissolved in PBS to a concentration of 10 mg / mL, and 1/10 volume of 25 mg / mL MBS [N- (m-Leimidobenzoyl-oxy) -succinimide] or SMCC [4- (N- Maleimidomethyl) cyclohexane- 1-carboxylicacid N-hydroxysuccinimidester] was added dropwise and allowed to react with stirring for 30 minutes.
  • KLH-MBS, THY-SMCC, or BSA-SMCC were prepared by removing unreacted MBS or SMCC using a Sephadex G-25 column pre-equilibrated with PBS, respectively.
  • each carrier protein was mixed with 1 mg of peptide dissolved in 0.1M sodium phosphate buffer (pH 7.0), stirred at room temperature for 3 hours, dialyzed with PBS after reaction, Used as a source.
  • mice immunized with cells or peptides were partially blood collected from the tail vein after the final immunization, and the binding activity of the obtained antiserum was measured by a fluorescent cell staining method using a flow cytometer (Becton Dickinson).
  • MEM medium Minimum Essential Medium medium
  • loosened with tweezers and centrifuged (1200 rpm).
  • Red blood cells were removed by adding Tris-ammonium chloride buffer (pH 7.6) to the resulting precipitate fraction and treating for 1 to 2 minutes.
  • the obtained precipitate fraction (cell fraction) was washed 3 times with MEM medium and used for cell fusion.
  • U937-CCR6 cells and U937 cells prepared in Example 2 were used as assay cells. After washing the cells passaged in RPMI medium supplemented with 500 ng / mL G418 and GlutaMAX-1 (manufactured by GIBCO) or RPMI medium supplemented with only GlutaMAX-1 (hereinafter abbreviated as subculture medium), 0. A cell suspension was prepared by suspending in BSA-PBS (hereinafter abbreviated as assay buffer) containing 2 mg / mL human IgG and 0.05% sodium azide.
  • BSA-PBS hereinafter abbreviated assay buffer
  • Example 7 (2) Flow cytometer analysis As in Example 7 (2), 1 to 5 ⁇ 10 5 assay cells were suspended in 100 ⁇ L assay buffer, and the hybridoma supernatant was dispensed at 50 ⁇ L / well as the primary antibody. And allowed to react for 30 minutes at ice temperature. After the reaction, the cells were washed twice with BSA-PBS, and Alexa488-labeled anti-rat immunoglobulin G (H + L) (manufactured by Invitrogen) diluted with FCM buffer 500 times as a secondary antibody was added at 50 ⁇ L / well. In addition, the reaction was allowed to proceed for 30 minutes in the dark at light temperatures.
  • H + L Alexa488-labeled anti-rat immunoglobulin G
  • P2 CCR6-2S (B) peptide (SEQ ID NO: 33) -THY conjugate body
  • B peptide (SEQ ID NO: 33) -THY conjugate body
  • P2-BSA was used as a positive antigen
  • control peptide-BSA conjugate was used as a negative antigen.
  • P2-THY was used as a positive antigen, and a control peptide-THY conjugate was used as a negative antigen.
  • P3 CCR6-2 (B) peptide (SEQ ID NO: 40) -THY conjugate body
  • P3-BSA was used as a positive antigen
  • control peptide-BSA was used as a negative antigen.
  • P3-THY was used as a positive antigen and control peptide-THY negative antigen was used.
  • the plate on which the antigen was solidified was washed with PBS, and 1% BSA-PBS was added at 100 ⁇ L / well to block active groups remaining for 1 hour at room temperature. After standing, 1% BSA-PBS was discarded, and the immunized animal antiserum or hybridoma culture supernatant was dispensed at 50 ⁇ L / well as a test sample and allowed to stand at room temperature for 2 hours.
  • Tween-PBS After washing with 0.05% polyoxyethylene (20) sorbitan monolaurate (ICI trademark Tween 20 equivalent) -PBS (hereinafter referred to as Tween-PBS), HRP-labeled rabbit anti-mouse IgG (H + L) as a secondary antibody A polyclonal antibody or an HRP-labeled rabbit anti-rat IgG (H + L) polyclonal antibody was added at 50 ⁇ L / well and left at room temperature for 1 hour.
  • polyoxyethylene (20) sorbitan monolaurate ICI trademark Tween 20 equivalent
  • ABTS 2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmol / L ABTS, 0.1 mol / L citrate buffer (PH 4.2), 0.1% H 2 O 2 ] was added to cause color development.
  • 50 ⁇ L / well of 5% SDS was added to stop the reaction, and the absorbance (OD415 nM-OD490 nM) at a sample wavelength of 415 nM and a reference wavelength of 490 nM was measured using a plate reader (SPECTRA max; Molecular Devices).
  • mice 8-Azagnine resistant mouse myeloma cell line P3X63Ag8U. 1 (P3-U1: purchased from ATCC) was cultured in RPMI 1640 medium (manufactured by Invitrogen) containing 10% FCS (hereinafter referred to as normal medium), and 2 ⁇ 10 7 cells or more were secured at the time of cell fusion. Used for production fusion partner.
  • HAT medium RPMI1640 medium (manufactured by Invtrogen) [suspended in 100 mL.
  • the suspension was dispensed into a 96-well culture plate at 200 ⁇ L / well and cultured for 8 days at 37 ° C. in a 5% CO 2 incubator. The culture supernatant was aspirated and fresh HAT medium was dispensed at 200 ⁇ L / well.
  • Example 7 For peptide-immunized individuals, the binding ELISA assay described in Example 7 (4) was performed, and wells that reacted with positive subjects but did not react with negative subjects were selected. Cloning from cells contained in selected wells by limiting dilution using s-cloning medium (manufactured by Sanko Junyaku Co., Ltd.) containing 2-mercaptoethanol, FCS and gentamicin (hereinafter referred to as medium for hybridoma culture)] This was performed twice to obtain a single cell clone.
  • s-cloning medium manufactured by Sanko Junyaku Co., Ltd.
  • FCS 2-mercaptoethanol
  • FCS gentamicin
  • anti-human CCR6 rat monoclonal antibodies KM4703 and KM4704 were obtained from individuals immunized with SD rats with CHOK1-hCCR6, anti-human CCR6 mouse monoclonal antibody KM4705 was obtained from individuals immunized with NZB mice with CHOK1-hCCR6, and sulfated Tyr residues.
  • Anti-human CCR6 mouse monoclonal antibody KM4592 was established from balb / c mice immunized with partial peptide P2 of human CCR6 containing CCR6-2S (B). These monoclonal antibodies that specifically bind to human CCR6 were obtained by conducting a screening assay on a scale of about 24,000.
  • Example 7 (7) Purification of monoclonal antibody
  • the hybridoma obtained in Example 7 (6) above is expanded and cultured in a hybridoma culture medium until the number of cells becomes 1 ⁇ 10 7 cells, and centrifuged (1200 rpm, 4 ° C., 5 minutes). After that, the suspension was suspended in 100 mL of a medium in which 5% Ultra-Low IgG FCS (Invitrogen) was added to Hybridoma SFM (Invitrogen) and cultured for 7 days.
  • Ultra-Low IgG FCS Invitrogen
  • the culture supernatant was centrifuged (1200 rpm, 4 ° C., 5 minutes), and after collecting the supernatant, the supernatant was filtered using 0.22 ⁇ m pore size PES membrane (manufactured by Asahi Techno Glass). Purification of the monoclonal antibody and SDS-PAGE were performed in the same manner as in Example 6 (2).
  • the mouse antibody was purified using ProSepG (Millipore) resin, and the rat antibody was purified using Mabselect (Millipore) resin.
  • the subclass of the monoclonal antibody was determined by a binding ELISA using a sub cluster ipting kit (manufactured by DS Pharma Biomedical).
  • the anti-human CCR6 mouse monoclonal antibodies KM4592 and KM4705 are mouse IgG2b subclass
  • the anti-human CCR6 rat monoclonal antibodies KM4703 and KM4704 are rat IgG2b subclass.
  • Example 8 Examination of properties of anti-CCR6 monoclonal antibody
  • the anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704, KM4705 and commercially available anti-human CCR6 obtained in Example 7 (7) were used.
  • the activity of the monoclonal antibody was evaluated. Information on instructions for commercially available anti-human monoclonal antibodies is shown in FIG.
  • Each 5 ⁇ 10 5 cells suspended in FCM buffer were reacted with 0.0001 to 10 ⁇ g / mL anti-human CCR6 monoclonal antibody (KM4592, KM4703, KM4704, KM4705) as the primary antibody.
  • anti-human CCR6 monoclonal antibody KM4592, KM4703, KM4704, KM4705
  • Alexa Fluoro 488-labeled anti-mouse immunoglobulin G (H + L) antibody or Alexa Fluoro 488-labeled anti-rat immunoglobulin G (H + L) antibody both manufactured by Biolegend was used.
  • Measurement was carried out by measuring 510 to 530 nM fluorescence excited by a laser beam of 488 nM argon with a flow cytometer (Cytomics FC500 MPL, manufactured by Beckman Colter).
  • the anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704 and KM4705 of the present invention all have a cross reaction with cynomolgus monkey CCR6.
  • the anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704, KM4705 and commercially available anti-human CCR6 mouse monoclonal antibodies of the present invention all bind to CHO-hCCR6, CHO-mECL1hCCR6, CHO-mECL2hCCR6 and CHO-mECL3hCCR6, It did not bind only to CHO-mNt hCCR6 [FIGS. 16 (a) to (c)].
  • the epitopes of the anti-CCR6 monoclonal antibodies KM4592, KM4703, KM4704, KM4705 and the commercially available anti-human CCR6 mouse monoclonal antibody of the present invention are all N-terminal regions containing the 1st to 46th amino acids of human CCR6. It was revealed.
  • any synthetic peptide introduced a Cys residue on the N-terminal side or C-terminal side.
  • 11 polyethylene glycol (PEG) polymers were introduced on the C-terminal side of CCR6-1 and the N-terminal side of CCR6-4.
  • the 7th Cys residue of CCR6-4 was changed to a Ser residue to suppress binding between peptides.
  • the BSA-PBS was discarded, and 10 ⁇ g / mL anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704, KM4705 and a commercially available anti-human CCR6 mouse monoclonal antibody were dispensed at 50 ⁇ L / well as primary antibodies on the plate.
  • the reaction was performed for 2 hours. Further, as a positive control for an antibody that specifically reacts with a sulfated Tyr residue, an anti-sulfated Tyr antibody (Millipore) diluted to 10 ⁇ g / mL was used.
  • any anti-human CCR6 monoclonal antibody binds to an epitope present in the extracellular domain of the human CCR6 N-terminal region (amino acids 1 to 46 of the amino acid sequence represented by SEQ ID NO: 2). became. Further, since the anti-sulfated Tyr-specific antibody partially bound to the NthCCR6-IgG4Fc protein produced in CHO cells, it was revealed that the NthCCR6-IgG4Fc protein contains a sulfated Tyr residue [FIG. a) and Table 1].
  • Both of the commercially available anti-human CCR6 monoclonal antibodies 11A9 and R6H1 bind strongly to the CCR6-1 peptide containing the first to 17th amino acids of the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 2 CCR6 containing the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2 containing a sulfated Tyr residue, although partially binding to the CCR6-2 peptide containing the 9th to 23rd amino acids of It did not bind to the -2S peptide [FIG. 18 (a) and Table 1].
  • the anti-human CCR6 monoclonal antibodies KM4592, KM4703 and KM4704 of the present invention are all strong against the CCR6-2S peptide containing the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2 containing the sulfated Tyr residue.
  • the anti-human CCR6 monoclonal antibodies KM4592, KM4703 and KM4704 all bind to the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2, and the 18th amino acid sequence represented by SEQ ID NO: 2 It has been shown to bind to epitopes containing sulfated Tyr.
  • the anti-human CCR6 monoclonal antibody KM4705 of the present invention binds strongly to the CCR6-1 peptide containing the first to 17th amino acids of the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 2
  • the CCR6-2 peptide containing the 9th to 23rd amino acids and the CCR6-2S peptide containing the 9th to 23rd amino acids of the amino acid sequence represented by SEQ ID NO: 2 containing the sulfated Tyr residue [ FIG. 18 (a) and Table 1].
  • the anti-human CCR6 monoclonal antibody KM4705 bound to human CCR6 regardless of the presence or absence of sulfation of the 18th Tyr of the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 2 It binds to the 1st to 23rd amino acid sequences, and the epitope does not include the 18th Tyr side chain of the amino acid sequence represented by SEQ ID NO: 2. It was revealed to bind to an epitope containing the 17th amino acid sequence.
  • An anti-human IgG antibody was immobilized on a CM5 sensor chip (manufactured by GE Healthcare Bioscience) using a Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience) according to the attached protocol.
  • the NthCCR6-IgG4Fc protein obtained in Example 6 was injected into a flow cell in which anti-human IgG antibody was immobilized, and was captured so as to have about 10 resonance units (hereinafter abbreviated as RU).
  • the anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704, KM4705 and commercially available anti-human CCR6 monoclonal antibodies which were diluted 2-fold stepwise from 27000 ng / mL to a concentration of 10 points, were supplied to the flow cell at a flow rate of 30 ⁇ L / min.
  • the sensorgram for each antibody concentration was obtained by injection.
  • the acquired sensorgram was analyzed using Biacore T100 Evaluation software (manufactured by GE Healthcare Bioscience), and the affinity of each antibody for human CCR6 was calculated.
  • each dissociation constant K D of 2.45 nm was the affinity of 4.97NM, 53103 or 11A9, respectively dissociation constant, K D, Of 70.9 nM and 20 nM, and a low affinity of 1/4 or less compared to MM or R6H1.
  • anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704 and KM4705 of the invention each dissociation constant K D of 0.38 nM, 1.15 nm, a 0.67nM or 6.52NM, any antibodies 6.5nM It had the following affinity. Particularly KM4592, KM4703 and KM4704 the dissociation constant, K D, is less high affinity 1.2 nM, had a superior high affinity as compared to existing anti-human CCR6 monoclonal antibodies.
  • CCR6G16 cells in which human CCR6 (hCCR6) was induced and expressed were prepared.
  • Mip-3 ⁇ a human CCR6-specific ligand
  • GPCR G-protein coupled receptor
  • CCR6G16 cells constructed in Example 3 were cultured in the presence of 10 nM ⁇ -estradiol (manufactured by SIGMA) for 24 to 48 hours to induce and express hCCR6.
  • CCR6G16 cells in which hCCR6 was inducibly expressed were suspended at 4 ⁇ 10 5 cells / mL with 10 mm HEPES (Invitrogen) -HBSS (GIBCO).
  • the value obtained by subtracting the AUC of the well not added with the ligand from the AUC of each well added with the ligand was defined as a calcium reaction.
  • the calcium reaction was shown with the calcium reaction at the time of addition of ligand 1 nM (no antibody) as 100% [FIG. 20 (b)].
  • anti-human CCR6 monoclonal antibodies KM4703 and KM4704 strongly inhibited Mip-3 ⁇ -dependent intracellular Ca 2+ increase compared to commercially available anti-human CCR6 monoclonal antibodies.
  • anti-human CCR6 monoclonal antibodies KM4592 and KM4705 hardly inhibited the increase in Mip-3 ⁇ -dependent intracellular Ca 2+ .
  • Example 9 Isolation and analysis of cDNA encoding variable region of anti-human CCR6 monoclonal antibody (1) Preparation of mRNA from anti-human CCR6 antibody-producing hybridoma Each hybridoma KM4592, KM4703, KM4704 obtained in Example 7 and MRNA was prepared from 5 ⁇ 10 7 to 1 ⁇ 10 8 cells of KM4705 using ISOGEN (Nippon Gene). Using the prepared mRNA as a template, cDNA of each anti-human CCR6 monoclonal antibody was prepared using SMART RACE cDNA Amplification Kit (Clontech) according to the attached instruction manual.
  • VL light chain variable region
  • primers specific to mouse IgG1 (mouse-IgG1-a1 and mouse-IgG1-a2) using the cDNA obtained in Example 9 (1) as a template (SEQ ID NOs: 46 and 47) PCR was carried out using and the VH cDNA fragment of each antibody was amplified.
  • PCR was carried out using mouse Ig ( ⁇ ) specific primers (mouse-kappa-a1 and mouse-kappa-a2) (SEQ ID NOs: 48 and 49) instead of the antibody-specific subclass-specific primers.
  • the VL cDNA fragment was amplified. Thereafter, cloning was performed in the same manner as in Example 1 (1).
  • a vector containing a full-length VH cDNA and an expression vector containing a VL cDNA pCR / KM4592VL PCR, pCR / KM4592VH PCR, pCR / KM4703VL PCR, pCR / KM4703VH PCR, pCR / KM4704VL PCR, pCR / KM4704VH PCR, pCR / KM4705VL PCR and pCR / KM4705VH PCR were obtained.
  • a schematic diagram of the cloning is shown in FIG.
  • the base sequence and amino acid sequence of the variable region contained in the obtained anti-human CCR6 monoclonal antibody expression vector are shown below.
  • VH and VL base sequences of the anti-human CCR6 monoclonal antibody KM4592 are SEQ ID NOs: 50 and 53
  • amino acid sequences deduced from the sequences are SEQ ID NOs: 51 and 54
  • VH and VL amino acid sequences of the secreted antibody are SEQ ID NO: 52 and 55.
  • CDR1 to CDR3 of VH are shown in SEQ ID NOs: 56 to 58
  • CDR1 to CDR of VL are shown in SEQ ID NOs: 59 to 61.
  • VH and VL nucleotide sequences of the anti-human CCR6 monoclonal antibody KM4703 are SEQ ID NOs: 62 and 65
  • the amino acid sequences deduced from the sequences are SEQ ID NOs: 63 and 66
  • the VH and VL amino acid sequences of the secreted antibody are SEQ ID NO: 64 and 67.
  • the amino acid sequences of CDR1 to CDR3 of VH are shown in SEQ ID NOs: 68 to 70, respectively
  • amino acid sequences of CDR1 to CDR3 of VL are shown in SEQ ID NOs: 71 to 73, respectively.
  • VH and VL nucleotide sequences of the anti-human CCR6 monoclonal antibody KM4704 are SEQ ID NOs: 74 and 77
  • the amino acid sequences deduced from the sequences are SEQ ID NOs: 75 and 78
  • the VH and VL amino acid sequences of the secreted antibody are SEQ ID NO: 76 and 79.
  • the amino acid sequences of CDR1 to CDR3 of VH are shown in SEQ ID NOs: 80 to 82, respectively
  • amino acid sequences of CDR1 to CDR3 of VL are shown in SEQ ID NOs: 83 to 85, respectively.
  • VH and VL base sequences of the anti-human CCR6 monoclonal antibody KM4705 are shown in SEQ ID NOs: 86 and 89, the amino acid sequences deduced from the sequences are shown in SEQ ID NOs: 87 and 90, and the VH and VL amino acid sequences of the secreted antibody are shown in SEQ ID NO: 88 and 91.
  • the amino acid sequences of CDR1 to CDR3 of VH are shown in SEQ ID NOs: 92 to 94, respectively, and the amino acid sequences of CDR1 to CDR3 of VL are shown in SEQ ID NOs: 95 to 97, respectively.
  • Example 10 Preparation of anti-CCR6 chimeric antibody (1) Construction of anti-human CCR6 chimeric antibody expression vector The chimeric antibody prepared in the present invention was prepared by using VH of the anti-human CCR6 monoclonal antibody obtained in Example 9 (2) and It is a chimeric antibody in which the H chain constant region and human kappa chain constant region of human IgG1 disclosed in WO 97/10354 are linked to VL.
  • KM4592VL primer KM4592K pKTABEX F and KM4592K pKTABEX R (SEQ ID NO: 98, 99), KM4592VH primer KM4592H pKTABEX92 and KM4592F, respectively, using pCR vectors containing DNA fragments encoding VL or VH of each antibody as templates.
  • H pKTABEX R (SEQ ID NO: 100, 101), KM4703VL primer KM4703 K pKTABEX F and KM4703 K pKTABEX R (SEQ ID NO: 102, 103), KM4703VH primer KM4703H pKTABEX F and KM4703Hp 105 , KM4704VL primer KM4704 pKTABEX F and KM4704 K pKTABEX R (SEQ ID NO: 106, 107), KM4704VH primer KM4704 H pKTABEX F and KM4704 H pKTABEX R (SEQ ID NO: 108, 109), KM4705VL primer KM4705KXFK 110, 111) and KM4705VH primers KM4705H pKTABEX F and KM4705H pKTABEX R (SEQ ID NOS: 112, 113) were used to amplify DNA fragments
  • PCR is 0.5 ⁇ g / mL pCR / VH or VL vector, 2 ⁇ KOD-FX buffer 25 ⁇ L, 0.4 mm dNTP, KOD-FX polymerase (manufactured by TOYOBO) 1 ⁇ L, each containing 50 ⁇ L of total 0.3 ⁇ M primer.
  • a solution was prepared, heated at 94 ° C. for 2 minutes, and then subjected to 30 cycles of 94 ° C. for 20 seconds and 68 ° C. for 40 seconds.
  • a pCR vector containing DNA fragments encoding VH and VL was prepared.
  • the DNA fragment encoding VL was digested with restriction enzymes BglII (manufactured by New England Biolabs) and BsiWI (manufactured by New England Biolabs) to obtain a BglII-BsiWI fragment of VL. Further, the DNA fragment encoding VH was digested with restriction enzymes SalI (manufactured by New England Biolabs) and NheI (manufactured by New England Biolabs) to obtain a SalI-NheI fragment of VH.
  • the obtained DNA fragment encoding the VH or VL is inserted into an appropriate position of the human pKTABEX-Tc26.2 expression vector, and the anti-human CCR6 monoclonal antibodies KM4592, KM4703, KM4704 or KM4705 are used.
  • Anti-human CCR6 chimeric antibody expression vectors pKTABEX-Tc26.2 / KM4592, pKTABEX-Tc26.2 / KM4703, pKTABEX-Tc26.2 / KM4704 and pKTABEX-Tc26.2 / KM4705 containing DNA encoding the variable region of .
  • Example 10 (2) Expression and purification of anti-human CCR6 chimeric antibody using animal cells
  • the anti-human CCR6 chimeric antibody expression vector prepared in Example 10 (1) was expressed in the same manner as in Example 1 (3).
  • the fucosyltransferase (FUT8) gene was introduced into CHO-K1 cells knocked out (hereinafter referred to as CHO-K1 FUT8KO), and anti-human CCR6 chimeric antibody expressing cells CHO / KM4592 (Fu-), CHO / KM4703 (Fu-), CHO, / KM4704 (Fu-) and CHO / KM4705 (Fu-) were prepared.
  • an anti-dinitrophenylazine (DNP) human antibody (Motoki K et. Al., Clin. Cancer Res. 11, 3126-3135, 2005) was prepared.
  • the cells were cultured in CHX / IMDM medium for 1 week in the same manner as in Example 6 (2).
  • a purified antibody was obtained from the obtained culture supernatant using Mabselect (manufactured by Millipore) in the same manner as in Example 6 (2) above.
  • the purified antibody was subjected to SDS-PAGE analysis to confirm the antibody protein.
  • Example 11 Evaluation of activity of anti-human CCR6 chimeric antibody
  • the anti-human CCR6 chimeric antibody KM4592 chimera, KM4703 chimera, KM4704 chimera, and KM4705 obtained in Example 10 (2) The activity of the chimera was evaluated.
  • Example 1 Analysis of binding activity of anti-human CCR6 chimeric antibody to human and cynomolgus CCR6 by fluorescent cell staining method (FCM analysis)
  • FCM analysis Anti-human CCR6 chimeric antibodies KM4592 chimera, KM4703 chimera, KM4704 chimera and KM4705 chimera were prepared in Example 1.
  • the binding activity to human CCR6-expressing CHO cells (CHO-hCCR6) and cynomolgus monkey CCR6-expressing CHO cells (CHO-cynomolgus CCR6) prepared in Example 5 was measured by the same method as in Example 8 (1).
  • Alexa Fluor 488 goat anti-human IgG (H + L) manufactured by Invitrogen, cat. A11013 was used as the secondary antibody.
  • the anti-human CCR6 chimeric antibody KM4592 chimera [FIG. 22 (a)], KM4703 chimera [FIG. 22 (b)], and KM4704 chimera [FIG. c)] and KM4705 chimera [FIG. 22 (d)] reacted to both CHO-hCCR6 and CHO-cynomolgus CCR6 in an antibody concentration-dependent manner (FIG. 22).
  • the reactivity of any antibody to human CCR6 and cynomolgus monkey CCR6 was equivalent, and had the same binding activity as the parent antibody.
  • the anti-human CCR6 chimeric antibody KM4592 chimera, KM4703 chimera, KM4704 chimera and KM4705 chimera of the present invention all bind to CHO-hCCR6, CHO-mECL1hCCR6, CHO-mECL2hCCR6 and CHO-mECL3hCCR6, and to CHO-mNtCCR6. It did not bind only to (FIG. 23). Moreover, the anti-DNP antibody did not bind to any cells.
  • the epitope of the anti-human CCR6 chimeric antibody KM4592 chimera, KM4703 chimera, KM4704 chimera and KM4705 chimera of the present invention is the same as that of each parent antibody, and all antibodies have amino acids 1 to 46 of human CCR6. It became clear that it reacts with the epitope of the containing N terminal region.
  • all of the anti-human CCR6 chimeric antibodies KM4592 chimera, KM4703 chimera and KM4704 chimera contain CCR6 containing the 9th to 23rd amino acid sequences of human CCR6 containing a sulfated Tyr residue as compared with the negative control anti-DNP antibody. It bound strongly to the -2S peptide. Further, it did not bind at all to the CCR6-1 peptide containing the 1st to 17th amino acid sequences of human CCR6 and the CCR6-2 peptide containing the 9th to 23rd amino acid sequences of human CCR6 (FIG. 24).
  • the anti-human CCR6 chimeric antibodies KM4592 chimera, KM4703 chimera and KM4704 chimera all bind to the amino acid sequence from 9th to 23rd, and in particular to the epitope containing the 18th sulfated Tyr residue of human CCR6. Became clear.
  • the anti-human CCR6 chimeric antibody KM4705 chimera of the present invention binds strongly to the CCR6-1 peptide containing the first to 17th amino acid sequences of human CCR6 and contains CCR6 containing the 9th to 23rd amino acid sequences of human CCR6. It also bound to the CCR6-2S peptide containing the 9th to 23rd amino acid sequences of human CCR6 containing the -2 peptide and sulfated Tyr residues (FIG. 24).
  • the anti-human CCR6 chimeric antibody KM4705 chimera binds to human CCR6 regardless of the presence or absence of sulfation of the 18th Tyr of human CCR6, so the 1st to 23rd amino acids not containing the 18th Tyr side chain It has been shown that it binds to a sequence and in particular to an epitope comprising the amino acid sequence from 9th to 17th of human CCR6.
  • FIG. 25 shows the amount of intracellular Ca 2+ increase (%) in each antibody-added well when the value obtained by subtracting the AUC of the well not added with ligand from the AUC of the well added with 1 nM of ligand is 100%. Shown in
  • anti-CCR6 chimeric antibodies KM4703 chimera and KM4704 chimera strongly inhibited Mip-3 ⁇ -dependent intracellular Ca 2+ increase compared to commercially available anti-human CCR6 monoclonal antibody. Further, the anti-human CCR6 chimeric antibodies KM4592 chimera and KM4705 chimera hardly inhibited the increase in Mip-3 ⁇ -dependent intracellular Ca 2+ .
  • the anti-human CCR6 chimeric antibodies KM4703 chimera and KM4704 chimera exhibit strong neutralizing activity. Further, like the binding activity of each chimeric antibody confirmed in Examples 11 (1) to (3), the neutralizing activity of any anti-human CCR6 chimeric antibody was equivalent to that of each parent antibody.
  • PBMC Peripheral blood mononuclear cells
  • Lymphoprep (Axis-Shield) dispensed 12.5 mL into a 50 mL tube (Greiner), 25 mL of diluted peripheral blood was gently layered and centrifuged for 20 minutes at 840 g, brake off, and room temperature. Separated, the mononuclear cell layer was separated. The mononuclear cell fraction thus obtained was washed twice with an ADCC medium, adjusted to an optimal cell density with the same medium, and used as an effector cell suspension.
  • each antibody solution was given a final concentration of 10000 ng / mL, 100 ng / mL, 1 ng / mL, 0.1 ng / mL,.
  • the target cell suspension prepared in (5) -1 was then dispensed at 1 ⁇ 10 4 cells / 50 ⁇ L / well, and finally prepared in (5) -2.
  • the effector cell suspension was dispensed at 2.5 ⁇ 10 5 cells / 50 ⁇ L / well to a total volume of 150 ⁇ L and reacted at 37 ° C. under 5% CO 2 for 4 hours.
  • anti-human CCR6 chimeric antibodies KM4592 chimera, KM4703 chimera, KM4704 chimera, and KM4705 chimera showed ADCC activity against human CCR6-expressing CHO cells CHO-hCCR6 [FIGS. 26 (a) and (b)].
  • Flow Count manufactured by BECKMAN COULTER
  • Flow Count as an internal standard was added at 100 ⁇ L / well, centrifuged, and the supernatant was removed. Further, after washing the cells with 1 to 2 mL of FACS buffer, 1 mL of FACS buffer and 1 ⁇ L LIVE / DEAD yellow (manufactured by Invitrogen) were added, and the mixture was allowed to react at room temperature for 30 minutes in the dark.
  • the cells were washed with a FACS buffer, 100 ⁇ L of PBS containing 0.2 mg / mL human IgG (manufactured by Sigma) was added, and reacted at 4 ° C. for 1 hour in the dark.
  • the cells were washed with FACS buffer, and various staining antibodies (FITC-labeled anti-CD3 antibody, AF700-labeled anti-CD45RO antibody, PerCPCy5.5 standard anti-CD45RA antibody for T cell staining, and PerCPCy5.5 label for B-cell staining) 100 ⁇ L FACS buffer containing an appropriate amount of anti-CD19 antibody was added and reacted in the dark for 20 minutes at 4 ° C., then the cells were washed with FACS buffer, and 100 ⁇ L PFA (para form aldehyde) was added. And allowed to react at room temperature for 10 minutes.
  • various staining antibodies FITC-labeled anti-CD3 antibody, AF700-labeled anti-CD45RO antibody, PerCPCy5.5 standard anti-CD45RA antibody for T cell staining, and PerCPCy5.5 label for B-cell staining
  • the cells were washed with FACS buffer, suspended in 300 ⁇ L of FACS buffer, and the fluorescence intensity was measured using a flow cytometer (LSRIIFlow cytometer (registered trademark), Becton Dickinson). The data measurement was completed when the number of Flow Count Beads used as an internal standard reached 50000 counts.
  • LSRIIFlow cytometer registered trademark
  • Becton Dickinson The data measurement was completed when the number of Flow Count Beads used as an internal standard reached 50000 counts.
  • the anti-human CCR6 chimeric antibodies KM4592 chimera, KM4703 chimera, KM4704 chimera, and KM4705 chimera exhibited cytotoxic activity against CD19 + B cells and CD3 + CD45RO + CD45RA-Memory T cells, while cytotoxic against CD3 + CD45RO-CD45RA + Native T cells. It was confirmed that no activity was shown [FIGS. 27 (a) and (b)].
  • SEQ ID NO: 5 base sequence of Human-CCR6-f primer
  • SEQ ID NO: 6 base sequence of Human-CCR6-r primer
  • SEQ ID NO: 7 base sequence of CCR6MunI-f primer
  • SEQ ID NO: 8 base sequence sequence number of CCR6BamHI-r primer 9: nucleotide sequence of Human-1-162-f primer
  • SEQ ID NO: 10 nucleotide sequence of Human-138-288-r primer
  • SEQ ID NO: 11 nucleotide sequence of Human-264-413-f primer
  • SEQ ID NO: 12 Human-394 -540-r primer nucleotide sequence
  • SEQ ID NO: 13 Human-519-677-f primer nucleotide sequence
  • SEQ ID NO: 14 Human-649-781-r primer nucleotide sequence
  • SEQ ID NO: 15 Human-759-914-f primer
  • Nucleotide sequence number 16 nucleotide sequence of Human 890-1020-r primer

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Abstract

La présente invention concerne : un anticorps qui peut se lier au CCR6 humain avec une affinité élevée et a une activité de neutralisation, et un fragment de l'anticorps ; un anticorps qui peut se lier au CCR6 humain sulfaté et un fragment de l'anticorps ; un hybridome qui peut produire l'anticorps ; de l'ADN qui code pour l'anticorps ; un vecteur qui porte l'ADN ; un transformant qui est produit par l'introduction du vecteur ; un procédé de production d'un anticorps ou d'un fragment de l'anticorps qui utilise l'hybridome ou le transformant ; et un agent thérapeutique et un agent de diagnostic, chacun desquels étant produit à l'aide d'un anticorps ou d'un fragment de l'anticorps.
PCT/JP2012/066594 2011-07-01 2012-06-28 Anticorps anti-ccr6 humain WO2013005649A1 (fr)

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WO2016059253A1 (fr) * 2014-10-17 2016-04-21 Glenmark Pharmaceuticals S.A. Anticorps qui se lient au ccr6 et leurs utilisations
WO2019017401A1 (fr) 2017-07-18 2019-01-24 協和発酵キリン株式会社 Anticorps monoclonal anti-ccr1 humain
WO2020067541A1 (fr) 2018-09-28 2020-04-02 協和キリン株式会社 Composition d'anticorps
WO2021201236A1 (fr) 2020-04-01 2021-10-07 協和キリン株式会社 Composition d'anticorps
WO2023125793A1 (fr) * 2021-12-29 2023-07-06 Nanjing Immunophage Biotech Co., Ltd Anticorps anti-ccr6 et leurs utilisations
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US9809647B2 (en) 2010-11-19 2017-11-07 Eisai R&D Management Co., Ltd. Neutralizing anti-CCL20 antibodies
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
EP2640744A2 (fr) * 2010-11-19 2013-09-25 Toshio Imai Neutralisation d'anticorps anti-ccl20
JP2017536101A (ja) * 2014-10-17 2017-12-07 グレンマーク ファーマシューティカルズ， エセ．アー． Ｃｃｒ６と結合する抗体およびその使用
CN107108742A (zh) * 2014-10-17 2017-08-29 格兰马克药品股份有限公司 结合ccr6的抗体和它们的用途
WO2016059253A1 (fr) * 2014-10-17 2016-04-21 Glenmark Pharmaceuticals S.A. Anticorps qui se lient au ccr6 et leurs utilisations
US10640564B2 (en) 2014-10-17 2020-05-05 Ichnos Sciences SA Antibodies that bind to CCR6 and their uses
WO2019017401A1 (fr) 2017-07-18 2019-01-24 協和発酵キリン株式会社 Anticorps monoclonal anti-ccr1 humain
WO2020067541A1 (fr) 2018-09-28 2020-04-02 協和キリン株式会社 Composition d'anticorps
WO2021201236A1 (fr) 2020-04-01 2021-10-07 協和キリン株式会社 Composition d'anticorps
WO2023125793A1 (fr) * 2021-12-29 2023-07-06 Nanjing Immunophage Biotech Co., Ltd Anticorps anti-ccr6 et leurs utilisations
CN117129668A (zh) * 2023-10-27 2023-11-28 江西赛基生物技术有限公司 一种用于化学发光免疫分析的清洗液及其制备方法和应用
CN117129668B (zh) * 2023-10-27 2024-01-09 江西赛基生物技术有限公司 一种用于化学发光免疫分析的清洗液及其制备方法和应用

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