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WO2017126587A1 - Anti-human ccr1 monoclonal antibody - Google Patents

Anti-human ccr1 monoclonal antibody Download PDF

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Publication number
WO2017126587A1
WO2017126587A1 PCT/JP2017/001687 JP2017001687W WO2017126587A1 WO 2017126587 A1 WO2017126587 A1 WO 2017126587A1 JP 2017001687 W JP2017001687 W JP 2017001687W WO 2017126587 A1 WO2017126587 A1 WO 2017126587A1
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amino acid
antibody
acid sequence
seq
human
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French (fr)
Japanese (ja)
Inventor
正之 甲斐
誠 武藤
健二 河田
秀世 平位
義治 坂井
平 前川
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Kyowa Kirin Co Ltd
Kyoto University NUC
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Kyowa Hakko Kirin Co Ltd
Kyoto University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention binds to the extracellular region of human CC chemokine receptor 1 (CC chemokine receptor 1, hereinafter referred to as CCR1) and activates CCR1 by human CC chemokine ligand (hereinafter referred to as CCL) 15.
  • CCR1 human CC chemokine receptor 1
  • CCL human CC chemokine ligand
  • the present invention relates to a method for producing the antibody or the antibody fragment to be used, a therapeutic agent and a diagnostic agent containing the antibody or the antibody fragment, and a method for treating and diagnosing a CCR1-related disease using the antibody or the antibody fragment.
  • CCR1 has aliases such as surface antigen classification (cluster of differentiation, CD) 191, CKR-1, HM145, Macrophage inflammatory protein 1 ⁇ receptor (MIP1 ⁇ R), CMKBR1 or SCYAR1.
  • CD surface antigen classification
  • CKR-1 CKR-1
  • HM145 CKR-1
  • MIP1 ⁇ R Macrophage inflammatory protein 1 ⁇ receptor
  • SCYAR1 SCYAR1.
  • Non-patent Document 1 A gene encoding human CCR1 was identified in 1993 (Non-patent Document 1).
  • the cDNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of human CCR1 are publicly available.
  • the cDNA sequence can be referred to NM_001295 and the protein amino acid sequence can be referred to NP_001286.
  • the mouse CCR1 cDNA sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) are also disclosed.
  • the cDNA sequence can be referred to NM_009912
  • the protein amino acid sequence can be referred to NP_034042.
  • CCR1 is a G protein-coupled receptor (hereinafter referred to as GPCR) having a seven-transmembrane structure, and is a membrane protein consisting of a total length of 355 amino acids.
  • GPCR G protein-coupled receptor
  • Human CCR3, CCL5, CCL8, CCL14, CCL15, CCL16 and CCL23 have been reported as ligands for human CCR1 (Non-patent Document 2).
  • mouse CCL3, CCL5, CCL7 and CCL9 have been reported as ligands for mouse CCR1 (Non-patent Document 3).
  • Human CCL15 is a ligand contained in the CC chemokine family and consists of a total of 92 amino acids.
  • CCR1 and CCR3 are known to function as CCL15 receptors. It is known that CCL15 exhibits a stronger activity by being decomposed at the N-terminus by the action of a proteolytic enzyme and becomes an activated form of around 68 amino acids (Non-patent Document 4).
  • Step 1 involves interaction of the chemokine (ligand) with the N-terminal extracellular region of the receptor.
  • the N-terminal region of the chemokine interacts with the extracellular loop region of the receptor, and as a result of the structural change of the receptor, a signal is transmitted into the cell.
  • G proteins ⁇ , ⁇ , and ⁇ trimers associated with the C-terminus of GPCR are activated in response to structural changes in GPCR caused by ligand binding, and ⁇ subunit is ⁇ Dissociate from the complex.
  • the ⁇ subunit acts on further downstream factors and activates signal transduction pathways.
  • phospholipase C phospholipase C, following PLC
  • phosphatidylinositol (4,5) 2-phosphate [phosphatidylinositol (4,5) bisphosphate, PIP 2] is decomposed, inositol triphosphate Acid (Inositol triphosphate, IP 3 ) and diacylglycerol (DAG) are produced.
  • IP 3 acts on the endoplasmic reticulum, releases calcium ions (Ca 2+ ) into cells, and causes various cellular responses via calmodulin.
  • This increase in intracellular calcium concentration can be measured using a fluorescent calcium indicator or the like, and can be used as an index of GPCR activation.
  • CCR1 it is also possible to measure the activation of intracellular signals by this method.
  • Non-patent Documents 11 and 12 The expression of human CCR1 in various blood cells such as neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, NK cells, T cells or B cells has been reported so far (non-) Patent Documents 6 to 10).
  • iMC immature myelocytes
  • MDSCs bone marrow-derived immunosuppressor cells
  • CCR1 is expressed (Non-patent Documents 11 and 12).
  • Non-patent Document 13 the expression in iMC and MDSC described above suggests that CCR1 contributes to the progression and progression of cancer.
  • Non-patent Documents 11 and 12 disclose mutations of SMAD4, which is a tumor suppressor gene, or disappearance of SMAD4 protein is seen at a certain frequency, and deficiency of SMAD4 is considered to be a poor prognostic factor.
  • deficiency of SMAD4 has been a factor that draws CCR1-positive iMC or MDSC into the tumor environment through increased expression of CCL15, and these cells are also a matrix metalloprotease (MMP).
  • MMP matrix metalloprotease
  • Non-Patent Document 14 CP481,715 (Pfizer), MLN3897 (Millennium), BX-471 (Berlex), CCX-354 (Chemocentryx), and the like. These small molecule inhibitors have been tested in patients with autoimmune or inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and chronic obstructive pulmonary disease. (Non-Patent Document 14).
  • Non-patent Document 15 Non-patent Document 15
  • 53504 R & D Systems
  • MAB145 Non-patent Document 16
  • 2D4 Melennium
  • Neote Kuldeep, et al. "Molecular cloning, functional expression, and signaling characteristics of a CC chemokine receptor.” Cell 72.3 (1993): 415-425. Mannhold, Raimund, Hugo Kubinyi, and Gerd Folkers. Chemokine receptors as drug targets. Eds. Martine J. Smit, Sergio A. Lira, and Rob Leurs. Vol. 46. John Wiley & Sons Ono, Santa Jeremy, et al. "Chemokines: roles in leukocyte development, trafficking, and effector function.” Journal of allergy and clinical immunology 111.6 (2003): 1185-1199. Berahovich, Robert D., et al.
  • CCR CC chemokine receptor
  • MIP-1 ⁇ utilizes both CCR1 and CCR5 to induce osteoclast formation and increase adhesion of myeloma cells to marrow stromal cells.
  • the present invention provides a monoclonal antibody or antibody fragment that binds to the extracellular region of human CCR1 and inhibits activation of CCR1 by human CCL15, a hybridoma that produces the antibody, a base encoding the antibody or the antibody fragment A nucleic acid having a sequence, a transformed cell comprising a vector comprising the nucleic acid, a method for producing the antibody or the antibody fragment using the hybridoma or the transformed cell, a therapeutic agent and a diagnostic agent comprising the antibody or the antibody fragment, and It is an object of the present invention to provide a method for treating and diagnosing a CCR1-related disease using the antibody or the antibody fragment.
  • the present invention provides a human CCR1 monoclonal antibody that binds to the extracellular region of human CCR1 and inhibits activation of human CCR1 by human CCL15.
  • the present invention relates to the following (1) to (21).
  • the monoclonal antibody or the antibody fragment thereof according to (1) which inhibits migration of human CCR1-expressing cells induced by human CCL15.
  • the monoclonal antibody or the antibody fragment thereof according to (1) or (2) which binds to at least one amino acid residue in the amino acid sequence of the extracellular loop 2 region of human CCR1.
  • 71, and the amino acid sequences of CDR1 to CDR3 of the light chain variable region are SEQ ID NOs: 72, 73 and 73, respectively.
  • An antibody having an amino acid sequence comprising the amino acid sequence described in 74.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 75, 76, and 77, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 78, respectively. 79.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 81, 82 and 83, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 84 , 85 and 86, an antibody having an amino acid sequence comprising the amino acid sequence described in 85 and 86.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 87, 88, and 89, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 90 , 91 and 92, an antibody having an amino acid sequence comprising the amino acid sequence described in 91 and 92.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 93, 94 and 95, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 96, respectively.
  • the antibody which is an amino acid sequence containing the amino acid sequence described in 97 and 98.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 99, 100 and 101, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 102. , 103 and 104.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 105, 106 and 107, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 108 , 109 and 110, an antibody having an amino acid sequence comprising the amino acid sequence described in 109 and 110.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 111, 112, and 113, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 114, respectively. , 115 and 116, an antibody having an amino acid sequence comprising the amino acid sequence described in 115 and 116.
  • the amino acid sequences of CDRs 1 to 3 of VH are amino acid sequences including the amino acid sequences described in SEQ ID NOs: 117, 118, and 119, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 120, respectively.
  • a transformed cell comprising a vector comprising the nucleic acid according to (10).
  • the hybridoma according to (9) or the transformed cell according to (11) is cultured, and the monoclonal antibody or the antibody fragment according to any one of (1) to (8) is collected from the culture solution A method for producing the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
  • a reagent for detecting or measuring human CCR1, comprising the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
  • a diagnostic agent for human CCR1-related diseases comprising the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
  • the diagnostic agent according to (14), wherein the human CCR1-related disease is cancer, autoimmune disease or inflammatory disease.
  • a therapeutic agent for a human CCR1-related disease comprising the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8) as an active ingredient.
  • the therapeutic agent according to (16), wherein the human CCR1-related disease is cancer, autoimmune disease or inflammatory disease.
  • (20) Use of the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8) for producing a diagnostic agent for a human CCR1-related disease.
  • the monoclonal antibody or the antibody fragment of the present invention binds to the extracellular region of human CCR1 and inhibits various reactions associated with human CCR1 activation. Therefore, the monoclonal antibody or the antibody fragment of the present invention can be used as a therapeutic agent and a diagnostic agent for human CCR1-related diseases.
  • FIG. 1 (a) and FIG. 1 (b) are the results of measuring the activity of anti-human CCR1 antibody to inhibit THP-1 migration by activated human CCL15.
  • the vertical axis in FIGS. 1 (a) and 1 (b) shows THP-1 cell migration (%). When DPBS and activated CCL15 were added, the number of cells that migrated to the lower layer of Transwell was 100%. did.
  • the horizontal axis of FIG. 1 (a) and FIG. 1 (b) shows the antibody and ligand added to THP-1 cells and their concentrations.
  • the sample to which DPBS is added is DPBS
  • the sample to which activated human CCL15 is not added is No ligand
  • the sample to which activated human CCL15 is added is hCCL15 (68aa) It was described.
  • anti-human CCR1 antibodies KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody were used.
  • FIG. 2 shows the results of measuring the activity of anti-human CCR1 antibody to inhibit THP-1 migration by activated human CCL15.
  • the vertical axis of the figure represents the amount of luminescence (relative light unit; RLU) when the number of cells that have moved to the lower layer of Transwell is measured by CellTiter-Glo.
  • the horizontal axis of FIG. 2 shows the antibodies, ligands and their concentrations added to THP-1 cells.
  • the sample to which DPBS is added is described as DPBS
  • the sample to which activated human CCL15 is not added is referred to as No ligand
  • the sample to which activated human CCL15 is added is described as hCCL15 (68aa).
  • As the anti-human CCR1 antibody 2D4 antibody (Millennium), 53504 antibody (R & D Technologies), 141-2 antibody (MBL, # D063-3), KM5908 antibody and KM5916 antibody were used.
  • the present invention relates to a monoclonal antibody or an antibody fragment thereof that binds to the extracellular region of human CCR1 and inhibits activation of CCR1 by human CCL15.
  • CCR1 is also called CD191, CKR-1, HM145, Macrophage infrastructure protein 1 ⁇ receptor (MIP1 ⁇ R), CMKBR1, SCYAR1, and the like.
  • CCR1 is a GPCR having a seven-transmembrane structure, and is a membrane protein consisting of a total length of 355 amino acids.
  • the GPCR on the cell surface is activated by binding of a ligand, and the receptor-dependent signal is transmitted into the cell, and at the same time, the calcium ion concentration in the cell increases. To do. As a result, it is known that cell migration, chemokine production, matrix metalloprotease MMP production, and the like occur in the cells.
  • a CCR1-dependent signal is transmitted into the cell, and at the same time, the calcium ion concentration in the cell increases.
  • Examples include cell migration, chemokine production, or MMP production.
  • human CCR1 ligands include human CCL3, CCL5, CCL8, CCL14, CCL15, CCL16, and CCL23.
  • mouse CCR1 ligand examples include mouse CCL3, CCL5, CCL7, and CCL9.
  • Human CCL15 is a ligand contained in the CC chemokine family and consists of a total of 92 amino acids. Human CCL15 is degraded at the N-terminus by the action of a proteolytic enzyme and becomes an activated form of about 68 amino acids (hereinafter referred to as activated human CCL15 or hCCL15 (68aa) in the present invention), so that full-length CCL15 ( Hereinafter, it is known that the present invention exhibits a stronger activity than the full length CCL15).
  • Examples of the antibody of the present invention include antibodies that inhibit various reactions associated with human CCR1 activation by human CCL15.
  • Specific examples of the antibody of the present invention include, for example, CCR1-dependent signaling in human CCR1-expressing cells by human CCL15, activation of PLC, increase in intracellular calcium ion concentration, activation of NF- ⁇ B and CCR1.
  • Examples thereof include an antibody that inhibits at least one reaction selected from the migration of expressed cells.
  • the antibody of the present invention is preferably an antibody that inhibits migration of human CCR1-expressing cells induced by human CCL15.
  • the antibody of the present invention is preferably 5% or more, 10% or more, or 20% or more, compared to the control in which only CCL15 is added and no antibody is added, for the above-mentioned reaction accompanying human CCR1 activation by human CCL15. , 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more.
  • the concentration of human CCL15 can be appropriately adjusted to a concentration at which the activity of the above reaction when human CCL15 is added becomes a maximum value depending on the measurement system. For example, when the migration of CCR1-expressing cells is measured by the method described in the Examples of the present application, the concentration of CCL15 is preferably 1 ng / mL.
  • the concentration of the antibody of the present invention can also be adjusted as appropriate by the measurement system.
  • the antibody concentration of the present invention is 0.3 ⁇ g / mL or more, preferably 1 ⁇ g / mL or more, more preferably 3 ⁇ g / mL or more, Most preferably, it is 10 ⁇ g / mL or more.
  • human CCL15 may be either full-length CCL15 or activated human CCL15 as long as it activates CCR1.
  • the human CCR1-expressing cell may be any cell as long as it expresses human CCR1, and examples thereof include human cells, human cell lines, and human CCR1 forced expression lines.
  • human cells expressing human CCR1 include neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, NK cells, T cells, B cells, immature myelocytes (iMC). ) And bone marrow-derived immunosuppressive cells (MDSC).
  • the extracellular region of human CCR1 includes an N-terminal region containing the 1st to 31st amino acid sequence from the N-terminal of the amino acid sequence of human CCR1, an extracellular loop 1 region containing the 97th to 103rd amino acid sequence, and 172nd to 195th And the extracellular loop 2 region containing the amino acid sequence of 266 to 278 [Cell 72.3 (1993): 415-425].
  • extracellular loop 1 region As the N-terminal region, extracellular loop 1 region, extracellular loop 2 region and extracellular loop 3 region, specifically, the 1st to 31st positions, the 97th to 103rd positions, and the 172th to 195th positions in the amino acid sequence of SEQ ID NO: 2, respectively And amino acid sequences from 266 to 278.
  • the antibody of the present invention may be any antibody that binds to the extracellular region of human CCR1 described above, but binds to at least one amino acid residue in the amino acid sequence of the extracellular loop 2 region of human CCR1. It is preferable that it is an antibody. Examples of such an antibody include an antibody that binds to at least one amino acid residue in the amino acid sequence of positions 172 to 195 of the amino acid sequence of SEQ ID NO: 2.
  • the antibody of the present invention includes any one antibody selected from the following (a) to (l).
  • the amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 69, 70 and 71, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 72
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 75, 76, and 77, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 78, respectively. 79.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 81, 82 and 83, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 84 , 85 and 86, an antibody having an amino acid sequence comprising the amino acid sequence described in 85 and 86.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 87, 88, and 89, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 90 , 91 and 92, an antibody having an amino acid sequence comprising the amino acid sequence described in 91 and 92.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 93, 94 and 95, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 96, respectively.
  • the antibody which is an amino acid sequence containing the amino acid sequence described in 97 and 98.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 99, 100 and 101, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 102. , 103 and 104.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 105, 106 and 107, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 108 , 109 and 110, an antibody having an amino acid sequence comprising the amino acid sequence described in 109 and 110.
  • the amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 111, 112, and 113, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 114, respectively. , 115 and 116, an antibody having an amino acid sequence comprising the amino acid sequence described in 115 and 116.
  • the amino acid sequences of CDRs 1 to 3 of VH are amino acid sequences including the amino acid sequences described in SEQ ID NOs: 117, 118, and 119, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 120, respectively.
  • the antibody of the present invention shows 90% or more homology with the VH CDR1 to 3 and VL CDR1 to 3 amino acid sequences of any one of the antibodies described in (a) to (i) above. It includes an antibody having the amino acid sequences of CDR1-3 of VH and CDR1-3 of VL.
  • the 90% or higher homology specifically includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology.
  • one embodiment of the antibodies described in the above (a) to (i) includes mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, and KM5954 antibody, respectively.
  • KM5955 antibody and KM5956 antibody are examples of the antibodies described in (a) to (i) above.
  • the antibody (j) of the present invention refers to a second antibody that inhibits the binding between the first antibody and human CCR1 when the antibodies described in (a) to (i) above are used as the first antibody. .
  • the antibody of (k) of the present invention is the antibody described in (a) to (i) above when the first antibody is used, and the epitope to which the first antibody binds is the first epitope.
  • the antibody of (1) of the present invention refers to the antibody described in (a) to (i) above when the first antibody is used, and the epitope to which the first antibody binds is used as the first epitope.
  • the antibody of the present invention also specifically includes any one antibody selected from the following (a) to (i).
  • the antibody of the present invention includes the VH and VL amino acid sequences of any one of the antibodies described in (a) to (i) above, and the VH and VL amino acid sequences of antibodies that exhibit 90% or more homology, respectively.
  • An antibody having The 90% or higher homology specifically includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology.
  • one embodiment of the antibodies described in the above (a) to (i) includes mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, and KM5954 antibody, respectively.
  • KM5955 antibody and KM5956 antibody includes mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, and KM5954 antibody, respectively.
  • An example is the chKM5956 antibody.
  • human CCR1 is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286, one in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286.
  • a polypeptide having the amino acid sequence in which the above amino acids are deleted, substituted or added and having the function of human CCR1, or the amino acid sequence described in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286, and 60% or more Preferred examples include a polypeptide having an amino acid sequence having a homology of 80% or more, more preferably 90% or more, and most preferably 95% or more, and having the function of human CCR1.
  • a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown by NCBI accession number NP — 001286 is a site-directed mutagenesis method [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 (tl), Proc. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)]
  • 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 CCR1 include the nucleotide sequence set forth in SEQ ID NO: 1 and the nucleotide sequence of NCBI accession number NM_001295.
  • a colony hybridization method As DNA that hybridizes under stringent conditions, a colony hybridization method, a plaque hybridization method, a Southern blotting method using a DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence of NM_001295 as a probe, This refers to hybridizable DNA obtained by a hybridization method or a DNA microarray method.
  • 0.7 to 1.0 mol / L of sodium chloride is present using a hybridized colony or plaque-derived DNA or a filter or slide glass on which a PCR product or oligo DNA having the sequence is immobilized.
  • hybridization method at 65 ° C [Molecular lon Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), DNA Cloningique 1: , A Practical Approach, Second Edition, Oxford University, (1995)]
  • 0.1 to 2 times concentrated SSC solution the composition of 1 times concentrated SSC solution is 150 mmol / L sodium chloride, 15 mmol / L Wash the filter or glass slide at 65 ° C using sodium citrate
  • the DNA which can be identified by doing is mentioned.
  • the hybridizable DNA is DNA having at least 60% homology with the nucleotide sequence of SEQ ID NO: 1 or NM_001295, preferably DNA having 80% or more homology, more preferably 95% DNA having the above homology can be mentioned
  • 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 included in the gene encoding human CCR1 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, but for the base sequence, 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.htmL], numerical values calculated using default parameters.
  • 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 when blastn, 7 for programs other than blastn
  • -X X dropoff value for If the grouped alignment in bits is 15 and -Z (final X dropoff value for grouped alignment in bits) is blastn, it is 50, and 25 for programs other than blastn (http: //www.ncbi.nlmnih.vlm. /blast/htm
  • a polypeptide containing a partial sequence of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286 can be prepared by methods known to those skilled in the art. Specifically, it can be prepared by deleting a part of DNA encoding the amino acid sequence of SEQ ID NO: 2 and culturing a transformant into which an expression vector containing the DNA is introduced.
  • a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286 can be obtained by the same method as described above. it can.
  • one or more amino acids are deleted in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286, or the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286.
  • a polypeptide having a substituted or added amino acid sequence can also be produced by a chemical synthesis method such as the fluorenylmethyloxycarbonyl (Fmoc) method or the t-butyloxycarbonyl (tBoc) method.
  • the antibody of the present invention includes any of polyclonal antibodies, monoclonal antibodies and oligoclonal antibodies.
  • Polyclonal antibody refers to a population of antibody molecules secreted by antibody-producing cells of different clones.
  • a monoclonal antibody is an antibody that is secreted by an antibody-producing cell of a single clone, recognizes only one epitope (also called an antigenic determinant), and has a uniform amino acid sequence (primary sequence) that constitutes a monoclonal antibody.
  • An oligoclonal antibody refers to a population of antibody molecules in which a plurality of different monoclonal antibodies are mixed.
  • 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.
  • 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.
  • the amino acid sequence modified by post-translational modification includes an O-linked sugar chain bonded to Tyr and Ser having an OH substituent, an N-linked sugar chain bonded to Gln and Asn having an NH 2 substituent, and Examples thereof include an amino acid sequence in which a sulfate group or the like in which a sulfuric acid molecule is bonded to Tyr having an OH substituent.
  • the binding of the antibody of the present invention to the extracellular region of human CCR1 was confirmed by measuring the binding of the antibody of the present invention to human CCR1-expressing cells using ELISA, flow cytometry, surface plasmon resonance, etc. can do. Also known immunological detection methods [Monoclonal-Antibodies-Principles-and-practice, Third-edition, Academic-Press- (1996), Antibodies-A-Laboratory-Manual, Cold-Spring-Harbor-Laboratory- (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] etc. can also be confirmed.
  • the amino acid residue or epitope of human CCR1 to which the antibody of the present invention binds is a deletion in which a partial domain of human CCR1 is deleted, a mutant in which a domain derived from another protein is substituted, and a partial peptide of human CCR1 It can be determined by conducting antibody binding experiments using fragments or the like. Antibody binding experiments can also be performed using cells expressing the above-mentioned mutants or mutants.
  • the amino acid residue or epitope of human CCR1 to which the antibody of the present invention binds can be epitope mapped using a known mass spectrometry method by adding the antibody of the present invention to a peptide fragment of human CCR1 digested with a proteolytic enzyme. It can also be determined by performing.
  • That the antibody of the present invention inhibits the activation of human CCR1 by human CCL15 is, for example, CCR1-dependent signaling in human CCR1-expressing cells, activation of PLC, increase in intracellular calcium ion concentration, NF- ⁇ B activation or migration of human CCR1-expressing cells can be confirmed as an index.
  • Cell migration can be measured using the chemotaxis assay described below.
  • human CCR1-expressing cells are added to the upper part of the chemotaxis assay chamber, 1) a negative control such as a medium or DPBS, 2) human CCL15 and 3) human CCL15 and the antibody of the present invention are added to the lower part of the chamber, After culturing for a certain time, the number of human CCR1-expressing cells present in the lower part of the chamber is measured by an appropriate method.
  • the number of cells when the human CCL15 and the antibody of the present invention were added under the condition that the number of cells when the human CCL15 was added was larger than the number of cells when the medium was added. If the number of cells is smaller than that when CCL15 is added, it can be determined that the antibody of the present invention inhibits the activation of human CCR1 by human CCL15.
  • the inhibition of the activation of human CCR1 by human CCL15 by the antibody of the present invention can be confirmed by using a change in the calcium ion concentration in human CCR1-expressing cells as an index.
  • the change in intracellular calcium concentration can be measured by a known method, for example, using an intracellular Ca measurement kit (manufactured by Wako) and the like, and can be measured according to the attached protocol.
  • intracellular calcium ion concentration when human CCL15 and the antibody of the present invention are added under conditions where the intracellular calcium ion concentration when human CCL15 is added is higher than the intracellular calcium ion concentration when medium is added.
  • the antibody of the present invention can be determined to inhibit the activation of human CCR1 by human CCL15.
  • 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 (light chains).
  • the H chain is composed of VH and H chain constant regions (also referred to as CH) from the N-terminal side
  • the L chain is composed of VL and L chain constant regions (also referred to as CL) from the N-terminal side. Is done.
  • CH ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains are known 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.
  • CH1 domain, hinge domain, CH2 domain, CH3 domain and Fc region in the present invention are identified from the N-terminus by the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. It can be specified by the amino acid residue number. Specifically, 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, and CH3 is an EU index 341 to 447. Each amino acid sequence is identified.
  • Examples of the antibody of the present invention include a recombinant mouse antibody, a recombinant rat antibody, a recombinant rabbit antibody, a human chimeric antibody (hereinafter simply abbreviated as a chimeric antibody), a humanized antibody (human) prepared in particular by genetic engineering. Type complementarity determining region CDR-grafted antibody) and recombinant antibodies such as human antibodies are also included.
  • 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.
  • a human chimeric antibody is obtained from a hybridoma derived from a non-human animal cell that produces a monoclonal antibody, and obtains cDNA encoding the VH and VL of the monoclonal antibody, and for animal cells having DNA encoding the human antibody CH and CL.
  • a human chimeric antibody expression vector can be constructed by inserting each into an expression vector and introduced into an animal cell for expression and production.
  • 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 is obtained by immunizing a mouse carrying a human immunoglobulin gene (Tomizuka K. et. Al., Proc Natl Acad Sci U S A. 97, 722-7, 2000). 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 obtained by, for example, immortalizing lymphocytes isolated from human peripheral blood by infecting EB virus or the like and then cloning to obtain lymphocytes that produce the antibody.
  • the antibody can be purified from the culture in which the lymphocytes are cultured.
  • 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.
  • VH and VL amino acid sequences of the antibody of the present invention the VH and VL amino acid sequences of a human antibody, the VH and VL amino acid sequences of a non-human animal antibody, or the CDR of a non-human animal antibody are used. Any of the VH and VL amino acid sequences of the humanized antibody transplanted to the workpiece may be used.
  • the amino acid sequence of CL in the antibody of the present invention may be any amino acid sequence or non-human animal antibody amino acid sequences of a human antibody, preferably C kappa or C lambda amino acid sequences of a human antibody.
  • 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 antibodies of the present invention include 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.
  • Fc fusion proteins and the like that have been made are also encompassed by the present invention.
  • an Fc region with a modified amino acid residue can also be used in the antibody of the present invention.
  • the antibody of the present invention or the antibody fragment includes an antibody containing any amino acid residue modified after translation.
  • Post-translational modifications include, for example, deletion of a lysine residue at the C-terminus of the H chain [lysine clipping] or conversion of a glutamine residue at the N-terminus of a polypeptide to pyroglutin (pyroGlu). [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].
  • an antibody fragment is an antibody fragment having an antigen-binding activity that binds to the extracellular region of human CCR1 and inhibits activation of human CCR1 by human CCL15.
  • the antibody fragment in the present invention include Fab, Fab ′, F (ab ′) 2 , scFv, diabody, dsFv, or 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 having a molecular weight of about 50,000, 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.
  • 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 specific 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 includes a radioisotope, a low molecular drug, a high molecular drug, a protein or an antibody drug chemically or genetically engineered to the monoclonal antibody or antibody fragment thereof that binds to human CCR1 of the present invention. Includes derivatives of conjugated antibodies.
  • the derivative of the antibody is added to the N-terminal side or C-terminal side of the H chain or L chain of the monoclonal antibody or antibody fragment thereof binding to human CCR1 of the present invention, an appropriate substituent in the antibody molecule, the side chain or sugar chain, etc. , Radioisotopes, low molecular weight drugs, high molecular weight drugs, immunostimulants, proteins, antibody drugs, or nucleic acid drugs, etc., are combined by chemical techniques [Introduction to Antibody Engineering, Jinshokan (1994)]. be able to.
  • the DNA encoding the monoclonal antibody or antibody fragment thereof that binds to human CCR1 of the present invention and the DNA encoding the protein or antibody drug to be bound are linked and inserted into an expression vector, and the expression vector is inserted into a suitable host. It can be produced by genetic engineering techniques that are introduced into cells and expressed.
  • 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 Anticancer agents such as complex derivatives, M-phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroidal agents such as hydrocortisone or prednisone, non-steroidal agents such as aspirin or indomethacin, gold thiomalate or Anti-inflammatory agents such as immunomodulators such as penicillamine, immunosuppressive agents such as cyclophosphamide or azathioprine, or antihistamines such as chlorpheniramine maleate or clemacytin [Inflammation and anti-inflammatory therapy, Ishiyaku Shuppan Co., Ltd. (1982) ]] Etc., and the like.
  • 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 and an antibody include, for example, a method in which a drug and an amino group of an antibody are bonded via glutaraldehyde, or an amino group of a drug and a carboxyl group of an antibody via a water-soluble carbodiimide. The method of combining is mentioned.
  • 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.
  • a method for linking PEG and an antibody a method of reacting with a PEGylation modifying reagent and the like can be mentioned [Bioconjugate pharmaceutical, Yodogawa Shoten (1993)].
  • 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 (eg, lentinan or schizophyllan) or ⁇ -galactosylceramide (KRN7000). ) And the like.
  • proteins include cytokines, growth factors or toxin proteins that activate immunocompetent cells such as NK cells, macrophages or neutrophils.
  • cytokines or growth factors examples include interferon (hereinafter referred to as IFN) - ⁇ , IFN- ⁇ , IFN- ⁇ , 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) or macrophage colony stimulating factor (M-CSF).
  • IFN interferon
  • IL interleukin
  • IL-12 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
  • examples of the toxin protein include ricin, diphtheria toxin, ON
  • Examples of the antibody drug include an antigen against which apoptosis is induced by antibody binding, an antigen associated with tumor pathogenesis, an antigen that regulates immune function, or an antigen associated with 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 or Epidemial GrowthFactor ) 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 ( For example, TRAIL-R1, TRAIL-R2, TRAIL-R3 or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor, cytokine [eg 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, SDF-1, or their receptors.
  • VEGF vascular electrical growth factor
  • FGF fibroblast growth factor
  • EGF fibroblast growth factor
  • EGF hepatocyte growthGF
  • IGF Insulin-like growth factor
  • EPO erythropoietin
  • TGF ⁇ IL-8
  • ephrin ephrin
  • SDF-1 ephrin
  • a fusion antibody with a protein or antibody drug is obtained by linking a cDNA encoding an antibody contained in a protein or antibody drug to a cDNA encoding a monoclonal antibody or antibody fragment, constructing a DNA encoding the fusion antibody, and
  • a fusion antibody can be produced by inserting into an expression vector for organisms or eukaryotes and expressing the expression vector by introducing the vector into prokaryotes or eukaryotes.
  • nucleic acid drugs include pharmaceuticals containing nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA that act on a living body by controlling the function of the gene.
  • nucleic acid drugs include pharmaceuticals containing nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA that act on a living body by controlling the function of the gene.
  • siRNA small interference ribonucleic acid
  • microRNA that act on a living body by controlling the function of the gene.
  • a conjugate with a nucleic acid drug that suppresses the master transcription factor ROR ⁇ t of Th17 cells is conceivable.
  • the drug that binds to the antibody includes a label used in usual immunological detection or measurement methods.
  • the label include an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).
  • FITC fluorescein isothiocyanate
  • RVC tetramethylrhodamine isothiocyanate
  • the present invention also includes a composition containing a monoclonal antibody that binds to human CCR1 or an antibody fragment thereof as an active ingredient.
  • the present invention also relates to a therapeutic agent for human CCR1-related diseases, which contains a monoclonal antibody that binds to human CCR1 or an antibody fragment thereof as an active ingredient.
  • the present invention also relates to a method for treating human CCR1-related diseases, comprising administering a monoclonal antibody that binds to human CCR1 or an antibody fragment thereof.
  • the human CCR1-related disease may be any disease involving human CCR1 or a human CCR1 ligand, and examples thereof include cancer, autoimmune diseases and inflammatory diseases.
  • cancer diseases include diffuse large B cell lymphoma, follicular lymphoma, B cell lymphoma, T cell lymphoma, plasma cell myeloma, acute myeloid leukemia, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy Cell leukemia, mantle cell lymphoma, follicular marginal zone lymphoma, small lymphocytic lymphoma, multiple myeloma, hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, oral squamous cell carcinoma, ovarian cancer, prostate cancer, breast cancer, Examples include glioma or osteosarcoma.
  • autoimmune diseases or inflammatory diseases include rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, lupus nephritis, asthma, atopic dermatitis, inflammatory bowel disease, Crohn's disease or Behcet's disease Is mentioned.
  • the therapeutic agent containing the antibody or the antibody fragment of the present invention may contain only the antibody or the antibody fragment as an active ingredient, but usually one or more pharmacologically acceptable carriers and They are preferably mixed together and provided as pharmaceutical preparations produced by any method known in the pharmaceutical arts.
  • the administration route is preferably the most effective for treatment, and includes oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular or intravenous, preferably intravenous. Internal administration can be mentioned.
  • oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular or intravenous, preferably intravenous.
  • Internal administration can be mentioned.
  • the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or tapes.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age and weight, but is usually 10 ⁇ g / kg to 10 mg / kg per day for an adult.
  • the present invention relates to a reagent for detecting or measuring CCR1 containing a monoclonal antibody that binds to human CCR1 or the antibody fragment, or a method for detecting or measuring CCR1 using the monoclonal antibody that binds to human CCR1 or the antibody fragment.
  • any known method may be used as a method for detecting or measuring human CCR1. 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.
  • Examples of the immunological detection or measurement method include radioactive substance-labeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (Western immunoassay), Western Examples include blotting or physicochemical techniques.
  • the present invention includes a diagnostic agent for a CCR1-related disease, comprising a monoclonal antibody that binds to human CCR1 or the antibody fragment, or detection or measurement of CCR1 using a monoclonal antibody that binds to human CCR1 or the antibody fragment.
  • the present invention relates to a method for diagnosing CCR1-related diseases. By using the monoclonal antibody or the antibody fragment of the present invention to detect or measure a cell in which human CCR1 is expressed according to the above method, a disease associated with human CCR1 can be diagnosed.
  • Examples of biological samples to be detected or measured for human CCR1 in the present invention include human CCR1 or human CCR1 expressed in tissues, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.
  • the cell is not particularly limited as long as it may contain cells.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present invention may contain a reagent for conducting an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method.
  • the reagent for performing the antigen-antibody reaction include a buffer and a salt.
  • the detection reagent include a reagent used in a usual immunological detection or measurement method such as a labeled secondary antibody that recognizes the monoclonal antibody or an antibody fragment thereof, or a substrate corresponding to the label.
  • the present invention also relates to the use of an anti-human CCR1 monoclonal antibody or the antibody fragment for the manufacture of a therapeutic or diagnostic agent for CCR1-related diseases.
  • 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 CCR1 or human CCR1-expressing cells to be used as an antigen are expressed in Escherichia coli, yeast, insect cells, animal cells, or the like using an expression vector containing cDNA encoding the full length of human CCR1 or a partial length thereof. It can be obtained by introduction. Human CCR1 can also be obtained by purifying human CCR1 from various human cell lines, human cells, human tissues, etc. that express human CCR1 in large amounts. In addition, these human cell lines, human cells, human tissues, and the like can be used as antigens as they are.
  • a synthetic peptide having a partial sequence of human CCR1 can be prepared by a chemical synthesis method such as Fmoc method or tBoc method and used as an antigen.
  • a known peptide such as FLAG or His may be added to the C-terminus or N-terminus of the synthetic peptide having human CCR1 or a partial sequence of human CCR1.
  • Human CCR1 used in the present invention is a method described in MolecularMCloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), etc. Can be produced by expressing the DNA encoding human CCR1 in a host cell by the following method, for example.
  • a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding human CCR1 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.
  • any microorganisms belonging to the genus Escherichia such as Escherichia coli, yeast, insect cells or animal cells can be used so long as they 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 the prokaryote, and at the same time a promoter, a ribosome binding sequence, DNA containing a portion encoding human CCR1, 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 DNA encoding the human CCR1 can be substituted so that the codon is optimal for expression in the host, thereby improving the production rate of the target human CCR1. Can do.
  • Any expression vector can be used as long as it can function in the host cell to be used.
  • pBTrp2, pBTac1, pBTac2 hereinafter, Roche Diagnostics
  • pKK233-2 Pharmacia
  • pSE280 manufactured by Invitrogen
  • pGEMEX-1 manufactured by Promega
  • pQE-8 manufactured by Qiagen
  • pKYP10 Japanese Patent Laid-Open Publication No. 58-110600
  • pKYP200 Agricultural Biobiological Chemistry, 48, 669 (1984)]
  • pLSA1 Agric. Biol. Chem., 53, 277 (1989)
  • pGEL1 Proc.
  • any promoter can be used as long as it can function in the host cell to be used.
  • promoters derived from Escherichia coli or phage such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter or T7 promoter.
  • Terp trp promoter
  • lac promoter lac promoter
  • PL promoter PL promoter
  • PR promoter PR promoter
  • T7 promoter trp promoter
  • 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 can be mentioned.
  • 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 or E. coli DH5 ⁇ .
  • any method for introducing DNA into a host cell to be used can be used.
  • a method using calcium ion [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.
  • pcDNAI, pCDM8 (manufactured by Funakoshi), pAGE107 Japanese Unexamined Patent Publication No. 3]. No. 22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen) , PcDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J.
  • 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 or 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 or 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); Chromosoma, 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, sAppendix I, II (pp.
  • CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) (CHO / DG44 cells) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC) CCL-1781), CHO-S (Life Technologies, Cat # 11619), Pro-3, rat myeloma cells YB2 / 3HL. P2. G11.16 Ag.
  • dhfr dihydrofolate reductase gene
  • mouse myeloma cell NSO mouse myeloma cell SP2 / 0-Ag14
  • Syrian hamster cell BHK or HBT5637 Japanese Unexamined Patent Publication No. 63-000299
  • 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 [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 CCR1 obtained as described above is cultured in a medium, and the human CCR1 is produced and accumulated in the culture solution.
  • Human CCR1 can be produced by collecting from the culture medium.
  • the method of culturing the transformant in a medium can be carried out according to a usual method used for culturing a host.
  • human CCR1 When expressed in cells derived from eukaryotes, human CCR1 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 animal cells as a host for example, 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)] or Iscove's Modified Examples include a Dulbecco's Medium (IMDM) medium or a medium obtained by adding fetal bovine serum (FBS) or the like to these mediums.
  • IMDM Dulbecco's Medium
  • FBS fetal bovine serum
  • the culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO 2 .
  • Examples of the method for producing human CCR1 include a method for producing it in a host cell, a method for producing it secreted outside the host cell, and a method for producing it on the host cell outer membrane. By changing the structure, an appropriate method can be selected.
  • Human CCR1 When human CCR1 is produced in or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 1989 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci , USA, 86, 19898227 (1989), Genes Develop., 4, 1288 (1990)], Japanese Patent Application Laid-Open No. 05-336963, International Publication No. 94/23021, etc. Human CCR1 can be actively secreted outside the host cell. In addition, the production amount of human CCR1 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Application Laid-Open No. 2-227075).
  • the obtained human CCR1 can be isolated and purified as follows, for example.
  • human CCR1 When human CCR1 is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, and then used with an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, or a dynomill. The cells are disrupted to obtain a cell-free extract.
  • an ordinary 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 hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing To obtain purified preparations It is possible.
  • human CCR1 When human CCR1 is expressed in the form of an insoluble substance in the cell, the cell is collected and disrupted in the same manner as described above, and centrifuged to collect the insoluble substance of human CCR1 as a precipitate fraction.
  • the recovered insoluble body of human CCR1 is solubilized with a protein denaturant.
  • the solubilized solution is diluted or dialyzed to return the human CCR1 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 CCR1 or a modified sugar 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 CCR1 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 performed by administering the antigen subcutaneously, intravenously or intraperitoneally to 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 passaged in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine] and passaged to normal medium 3-4 days prior to cell fusion. On the day of fusion, secure a cell count of 2 ⁇ 10 7 or more.
  • MEM Minimum Essential Medium
  • PBS dimethyl phosphate 1.
  • HAT medium normal medium containing hypoxanthine, thymidine, and aminopterin
  • a part of the culture supernatant is extracted, and a cell group that reacts with an antigen containing human CCR1 and does not react with an antigen not containing human CCR1 is selected by a hybridoma selection method such as a binding assay described later.
  • cloning is performed by a limiting dilution method, and a stable and strong antibody titer recognized is selected as a monoclonal antibody-producing hybridoma.
  • the supernatant is removed by centrifugation, 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 calculated from the Raleigh method or absorbance at 280 nm.
  • the monoclonal antibody is selected by measuring the binding of the antibody to human CCR1-expressing cells using flow cytometry, as shown below.
  • the human CCR1-expressing cells may be any cells as long as human CCR1 is expressed on the cell surface, and examples thereof include human cells, human cell lines, and human CCR1 forced expression cell lines obtained in (1).
  • a test substance such as serum, hybridoma culture supernatant or purified monoclonal antibody is dispensed as a first antibody and allowed to react.
  • the cells after the reaction are thoroughly washed with PBS containing 1 to 10% bovine serum albumin (BSA) (hereinafter referred to as BSA-PBS), and then anti-immunoglobulin antibody labeled with a fluorescent reagent or the like as a second antibody. Dispense and react.
  • BSA-PBS bovine serum albumin
  • a monoclonal antibody that specifically reacts with human CCR1-expressing cells is selected by measuring the amount of fluorescence of the labeled antibody using a flow cytometer.
  • an antibody that competes with the antibody of the present invention and binds to human CCR1 can be obtained by adding a test antibody to the above-described measurement system using flow cytometry and reacting. That is, by screening for an antibody that inhibits the binding between the antibody of the present invention and human CCR1 when a test antibody is added, it competes with the antibody of the present invention for binding to the amino acid sequence of human CCR1 or its three-dimensional structure. Monoclonal antibodies can be obtained.
  • an antibody that binds to an epitope containing an epitope to which a monoclonal antibody that binds to human CCR1 of the present invention binds is identified by a known method in which the epitope of the antibody obtained by the screening method described above is synthesized.
  • a peptide or a synthetic peptide that mimics the three-dimensional structure of an epitope can be prepared and obtained by immunization.
  • the epitope that the monoclonal antibody that binds to human CCR1 of the present invention binds to the same epitope is identified by identifying the epitope of the antibody obtained by the screening method described above, and a partial synthetic peptide of the identified epitope Alternatively, a synthetic peptide or the like that mimics the three-dimensional structure of an epitope is prepared and immunized.
  • Recombinant Antibody As a production example of a recombinant antibody, a method for producing a human chimeric antibody and a humanized antibody is shown below. Recombinant mouse antibodies, rat antibodies, rabbit antibodies, and the like can be prepared by the same method.
  • 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. For example, pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci.
  • 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 animal for producing a hybridoma cell producing a non-human antibody
  • mouse, rat, hamster, rabbit or the like is used, but any animal can be used as long as it can produce a hybridoma cell.
  • RNA easy kit for preparation of total RNA from hybridoma cells.
  • oligo (dT) immobilized cellulose column method [MolecularMCloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]
  • Oligo-dT30 ⁇ Super> mRNA Purification
  • 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 [Strategies, 5, 58 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)]
  • ⁇ ZAPIII (manufactured by Stratagene)
  • Lambda BlueMid manufactured by Clontech
  • pT7T3-18U manufactured by Pharmacia
  • pCD2 Mol. Cell. Biol., 3, 280 (1983)
  • pUC18 Gene , 33, 103 (1985)
  • Any 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.
  • XL1-Blue MRF ′ [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)] or JM105 [Gene, 38, 275 (1985)] or the like is used.
  • PCR method Molecular® Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989 ), Current Protocols in Molecular Molecular Biology, Supplements 1, John Wiley & Sons (1987-1997)
  • PCR method Molecular® Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989 ), Current Protocols in Molecular Molecular Biology, Supplements 1, John Wiley & Sons (1987-1997)
  • cDNA encoding VH or VL can also be prepared.
  • 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.
  • the base sequence analysis method includes, for example, a dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], ABI PRISM 3700 (PE Biosystems) or A.I. L. F. An automatic base sequence analyzer such as a DNA sequencer (Pharmacia) is used.
  • the total amino acid sequences of VH and VL are estimated from the determined nucleotide sequences, respectively, and compared with the entire amino acid sequences of known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] Thus, it is confirmed whether the obtained cDNA codes for the complete amino acid sequence of VH and VL of the antibody including the secretory signal sequence. Compare the complete amino acid sequence of the VH and VL of the antibody containing the secretory signal sequence with the entire amino acid sequence of the known antibody VH and VL [Sequences of Proteins of Immunological Interest, US Dept.
  • the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and further, the subgroup to which they belong can be known.
  • the amino acid sequences of CDRs of VH and VL are also found by comparing with the VH and VL amino acid sequences of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Can do.
  • the base sequence of the linking portion encodes an appropriate amino acid
  • VH and VL cDNAs designed to be appropriate restriction enzyme recognition sequences are prepared.
  • the prepared VH and VL cDNAs are respectively expressed so that they are expressed in an appropriate form upstream of each gene encoding the human antibody CH or CL of the recombinant antibody expression vector obtained in (1). Cloning 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.
  • 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.
  • human antibody FR amino acid sequences registered in databases such as Protein Data Bank, or common amino acid sequences of each subgroup of human antibody FRs [Sequencesenceof Proteins of Immunological Interest, estUS Dept.eptHealth and Human Services ( 1991)].
  • 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 CDR of the original antibody is transplanted to the amino acid sequence of FR of VH or VL of the selected human antibody, respectively, and the amino acid sequence of VH or VL of the humanized antibody is designed respectively.
  • the designed amino acid sequence is converted into a DNA sequence in consideration of the frequency of codon usage [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].
  • a DNA sequence encoding the amino acid sequence of VH or VL is designed.
  • the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene), the base sequence is determined by the same method as described in (2), and the desired humanized antibody is obtained.
  • a plasmid having a DNA sequence encoding the amino acid sequence of 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.
  • the cDNA encoding the humanized antibody VH or VL can be easily converted into the recombinant antibody expression vector obtained in (1). Can be cloned into.
  • a humanized antibody can only have its antigen-binding activity by transplanting only the VH and VL CDRs of a non-human antibody into the VH and VL FRs of a human antibody. [BIO / TECHNOLOGY, 9, 266 (1991)].
  • the amino acid residues of FR of human antibody VH and VL can be modified by performing the PCR reaction 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.
  • the gene is cloned so that it is expressed in an appropriate form upstream of each gene encoding CH or CL of the human antibody of the recombinant antibody expression vector obtained in (1).
  • Any host cell capable of expressing a recombinant antibody can be used as a host cell into which an expression vector is introduced.
  • COS-7 cells American Type Culture Collection (ATCC) number: CRL1651] are used. [Methods in Nucleic Acids Res., CRC press, 283 (1991)].
  • the expression vector is introduced into COS-7 cells by DEAE-dextran method [Methods in Nucleic Acids Res., CRC press (1991)] or lipofection method [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 immunoassay [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, 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 (ATCC number: also referred to as CRL1662 or YB2 / 0), mouse myeloma cell NS0, mouse myeloma cell SP2 / 0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cells (ATCC number: CRL1580), dihydro CHO cells (CHO / DG44 cells) lacking a folate reductase gene (Dihydroformate Reductase, hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] are used.
  • dhfr dihydroformate Reductase
  • a protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or a sugar chain modification in which the 1-position of fucose is ⁇ -linked to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex sugar chain
  • 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).
  • 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 manufactured by Invitrogen
  • FBS FBS
  • a medium obtained by adding various additives such as FBS By culturing the obtained transformant in a medium, the recombinant antibody is expressed and accumulated in the culture supernatant.
  • the expression level and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA method or the like.
  • the expression level of the recombinant antibody produced by the transformed strain can be improved using a dhfr gene amplification system (Japanese Patent Laid-Open No. 2-257891).
  • 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 Laboratory Manual, Cold Spring Harbor Laboratory (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 practice, Third edition, “Academic” Press (1996), Antibodies—A Laboratory, Manual, Cold Spring, 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 of the antibody of the present invention or the antibody fragment to human CCR1 is measured using the flow cytometry described in 1- (6) above. Further, it can be measured using a fluorescent antibody method [Cancer Immunol. Immunolother, 36, 373 (1993)].
  • the activity of inhibiting the migration of human CCR1-expressing cells by human CCL15 of the antibody of the present invention or the antibody fragment thereof can be measured using the chemotaxis assay described above.
  • CDC activity or ADCC activity against human CCR1-expressing cells can be measured by a known assay method [Cancer Immunol. Immunother., 36, 373 (1993); Current protocols in Immunology, Chapter 7 Immunologic studies in humans, Editor, John ., John Wiley & Sons, Inc., (1993)].
  • Method for controlling the effector activity of the antibody As a method for controlling the effector activity of the monoclonal antibody of the present invention, the N-linked complex type sugar chain binding to the 297th asparagine (Asn) of the Fc region of the antibody is used. Methods for controlling the amount of fucose (also referred to as core fucose) that binds ⁇ 1,6 to N-acetylglucosamine (GlcNAc) present (International Publication No. 2005/035586, International Publication No. 2002/31140, International Publication No. 00/61739) No.), or 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 monoclonal antibody of the present invention.
  • fucose also referred to as core fucose
  • GlcNAc N-acetylglucosamine
  • the effector activity refers to antibody-dependent activity caused through the Fc region of an antibody, and includes ADCC activity, CDC activity, or antibody-dependent phagocytosis by phagocytic cells such as macrophages or dendritic cells (Antibody-dependent phagocytosis). , ADP activity) and the like are known.
  • effector activity for example, inflammatory cells as targets, human peripheral blood mononuclear cells (PBMC) as effectors, and inflammatory cell-specific antibodies are mixed and incubated for about 4 hours, and then an indication of cytotoxicity Lactate dehydrogenase (LDH) released as can be measured.
  • PBMC peripheral blood mononuclear cells
  • LDH cytotoxicity Lactate dehydrogenase
  • an antibody that recognizes a blood cell-specific antigen such as CD20 is added to human whole blood, and after incubation, a decrease in the number of blood cells to be targeted can be measured as an effector activity.
  • the effector activity can be measured by a free LDH method, a free 51 Cr method, a flow cytometry method, or the like.
  • the effector activity of the antibody can be increased or decreased.
  • expressing the antibody 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 or 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.
  • the antibody of the present invention is described in, for example, Japanese Patent Application Publication No. 2013-165716 or Japanese Patent Application Publication No. 2012-021004 according to amino acid modification or sugar chain modification in the antibody constant region described above. Also included are antibodies whose half-life in blood is controlled by controlling the reactivity to Fc receptors by performing the amino acid modification of.
  • Method for treating diseases using anti-human CCR1 monoclonal antibody or antibody fragment thereof of the present invention can be any one of CCR1-related diseases such as human CCR1-dependent cell migration and lesions. Can be used to treat human CCR1-related diseases.
  • the therapeutic agent containing the monoclonal antibody or antibody fragment thereof of the present invention may contain only the antibody or the antibody fragment as an active ingredient, but usually one or more pharmacologically acceptable carriers And a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics.
  • Examples of the administration route include oral administration and parenteral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular or intravenous.
  • Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or tapes.
  • Preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders or 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 Manufactured using preservatives such as esters or flavors such as strawberry flavor or peppermint as additives.
  • 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.
  • Suitable formulations for parenteral administration include injections, suppositories or sprays.
  • An injection is produced using a carrier comprising a salt solution or 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.
  • the components exemplified as additives in preparations suitable for oral administration can also be added.
  • Method for diagnosing disease using anti-human CCR1 monoclonal antibody or antibody fragment thereof of the present invention By detecting or measuring human CCR1 or cells expressing human CCR1 using the monoclonal antibody or the antibody fragment of the present invention, CCR1-related diseases can be diagnosed.
  • cancer diseases, autoimmune diseases, and inflammatory diseases which are human CCR1-related diseases
  • diagnosis can be performed, for example, by detecting or measuring human CCR1 present in a patient by an immunological technique.
  • diagnosis can be performed by detecting human CCR1 expressed in cells in a patient using an immunological technique such as flow cytometry.
  • An immunological technique is a method for detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • a radioactive substance-labeled immunoantibody method an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blot method or a physicochemical method is used.
  • the antibody of the present invention or the antibody fragment thereof is reacted with an antigen or an antigen-expressing cell, and the anti-immunoglobulin antibody or the antibody fragment with a radiolabel is further reacted. Then, 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 reacted with an anti-immunoglobulin antibody or binding fragment labeled with an enzyme or the like.
  • the substrate is added and the absorbance of the reaction solution is measured with an absorptiometer.
  • 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, 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.
  • two types of antibodies or antibody fragments that recognize an antigen to be detected or measured and that have different antigen recognition sites are prepared, of which the first antibody or antibody fragment is pre-plated (for example, 96 Next, the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, biotin, or the like.
  • the above-mentioned antibody-adsorbed plate was labeled after reacting cells or its lysate, tissue or its lysate, cell culture supernatant, serum, pleural effusion, ascites or ocular fluid, etc., separated from the living body.
  • a 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 for 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.
  • Fluorescence immunoassay is measured by the method described in the literature [Monoclonal Antibodies-Principles and Practice, A 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.
  • Luminescent immunoassay is performed 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 or lophine is used.
  • Western blotting involves fractionating the antigen or antigen-expressing cells with SDS (sodium dodecyl sulfate) -PAGE (polyacrylamide gel) [Antibodies-A Laboratory Manual Cold Spring Harbor Laboratory (1988)], Blotting on a polyvinylidene fluoride (PVDF) membrane or nitrocellulose membrane, causing the membrane to react with an antibody or antibody fragment that recognizes an antigen, and further applying a fluorescent substance such as FITC, an enzyme label such as peroxidase, or a biotin label It is measured by visualizing the label after reacting with mouse IgG antibody or binding fragment.
  • SDS sodium dodecyl sulfate
  • PAGE polyacrylamide gel
  • the polypeptide having the amino acid sequence of 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 CCR1 as an antigen and the monoclonal antibody of the present invention or an antibody fragment thereof, and detecting the aggregate.
  • 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.
  • immunological detection methods For detection or measurement of cells expressing human CCR1, known immunological detection methods can be used. Among them, immunoprecipitation, immunocytostaining, immunohistochemical staining, fluorescent antibody staining, etc. can be used. It is preferable to use it.
  • cells expressing human CCR1 are reacted with the monoclonal antibody of the present invention or an antibody fragment thereof, and then a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added. Allow the complex to settle.
  • a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added. 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. Next, after discarding BSA-PBS and thoroughly washing with PBS, a lysate of cells or tissues expressing human CCR1 is reacted. Immunoprecipitates are extracted from the well-washed plate with SDS-PAGE sample buffer and detected by Western blotting as described above.
  • 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 superna
  • 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
  • fluorescent antibody staining methods that react with fluorescently labeled antibodies and cells and analyze with a flow cytometer [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific Fick (1987)] can be used for detection.
  • the monoclonal antibody of the present invention or an antibody fragment thereof that binds to human CCR1 can detect cells expressing and retaining 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 Preparation of expression vectors for human and mouse CCR1 (1) Preparation of each CCR1 gene DNA encoding human or mouse CCR1 or CCR1-CCR3 chimeric receptors 1 to 7 below was synthesized (Genscript) Japan). In the synthesis, restriction enzyme sites (BamHI and NotI) for incorporation into each vector and a Kozak sequence were added. 1. CDNA sequence encoding human CCR1 (hereinafter, hCCR1) (SEQ ID NO: 1) 2. CDNA sequence encoding mouse CCR1 (hereinafter, mCCR1) (SEQ ID NO: 3) 3. CDNA sequence encoding human CCR3 (hereinafter hCCR3) (SEQ ID NO: 5) 4).
  • CDNA sequence (SEQ ID NO: 6) encoding a chimeric receptor (hereinafter referred to as NC3-hCCR1) in which the amino acid sequence from human 31 to human CCR1 is substituted with the corresponding N-terminal amino acid sequence of human CCR3 5).
  • CDNA sequence (SEQ ID NO: 7) encoding a chimeric receptor (hereinafter, NC3-mCCR1) in which the first to 31st amino acid sequences of mouse CCR1 are replaced with the corresponding N-terminal amino acid sequences of human CCR3 6).
  • expression vectors were constructed (hereinafter, mCCR1 / Tn-pMug-Hygro, hCCR3 / Tn-pMug-Hygro, NC3-hCCR1 / Tn-pMug-Hygro, NC3-mCCR1 / Tn-pMug-Hygro, hCCR3_EL2hCCR1 / Tn-T pMug-Hygro, represented as hCCR3_EL2mCCR1 / Tn-pMug-Hygro).
  • Example 2 Preparation of CCR1-expressing cell line (1) Preparation of hCCR1-expressing cell hCCR1 / Tn-pMug-Hygro and Tol2 transposase expression vector TPEX_pMug (International Publication No. 2013/005649) prepared in Example 1 ) was co-introduced into CHO-S (Thermo Fisher Scientific) to produce an expression cell line. Gene transfer was performed using Fugene HD (Promega) as follows.
  • CHO-S-mCCR1 CHO-S-hCCR3, CHO-S-NC3-hCCR1, CHO-S-NC3-mCCR1, CHO-S-hCCR3_EL2hCCR1 and CHO-S-hCCR3_EL2mCCR1, respectively.
  • Cells prepared to 1 ⁇ 10 5 cells / mL were seeded in 2 mL each in a 6-well plate, and a mixture of 2.5 ⁇ g of plasmid DNA and 5 ⁇ L of Lipofectamine LTX was added to the medium. 72 hours after the addition, 1 mg / mL hygromycin was added, and drug selection was performed for about 2 weeks. Cells that acquired drug resistance were collected and analyzed for expression by flow cytometry. As a result, the expression of the introduced hCCR1 was confirmed. Hereinafter, this cell line is designated as RL33-hCCR1.
  • RL33-mCCR1 cells The mCCR1 / pCAG-IRES-neo produced in Example 1 (4) was introduced into RL-33 in the same manner as in (3) above to produce an expression cell line. Drug selection was performed with 0.5 mg / mL G418. Hereinafter, this cell line is designated as RL33-mCCR1.
  • Example 3 Preparation of anti-CCR1 rabbit polyclonal antibody An anti-CCR1 rabbit polyclonal antibody was prepared by the following method. Human CCR1 N-terminal peptide (SEQ ID NO: 10) was synthesized, and two rabbits (New Zealand White) were immunized 5 times every 2 weeks. Immunization was carried out by subcutaneous injection at multiple locations on the back using Complete Freund's Adjuvant (CFA) only for the first time and Incomplete Freund's Adjuvant (IFA) for the second and subsequent times. Sera were collected from individuals whose antibody titers increased after immunization, and IgG was purified by affinity purification using a Protein A column (GE Healthcare). The anti-CCR1 rabbit polyclonal antibody thus prepared is designated E5971.
  • CFA Complete Freund's Adjuvant
  • IFA Incomplete Freund's Adjuvant
  • Example 4 Expression analysis by flow cytometry (1) Confirmation of CCR1 expression The CCR1-expressing cell line prepared in Example 2 was stained with the anti-CCR1 rabbit polyclonal antibody E5971 prepared in Example 3, and the flow site CCR1 expression was confirmed by measurement (FCM). FCM analysis was performed as follows. Cells were seeded in 96-well plates at 2 ⁇ 10 5 cells / well and stained buffer [3% FBS (Thermo Fisher Scientific) / DPBS (Nacalai Tesque) /0.1% sodium azide (Nacalai Tesque)] Washed with.
  • FBS Thermo Fisher Scientific
  • the cells were treated with 10 ⁇ g / mL E5971 for 1 hour on ice, washed with staining buffer, and then added with secondary antibody Alexa Fluor 647 goat Anti-Rabbit IgG (Thermo Fisher Scientific) at a final concentration of 1 ⁇ g / mL. Treated for 30 minutes at room temperature. The cells were washed again with a staining buffer, suspended in the staining buffer, and analyzed using BD FACSCalibur (BD Biosciences). This confirmed that the introduced CCR1 was expressed in the produced CCR1-expressing cell line.
  • Example 5 Production of monoclonal antibody using CCR1 knockout mouse To obtain a mouse cross-linking antibody, commercially available CCR1 knockout (KO) mouse (B6.129S4-Ccr1 tm1Gao N10 + N5) (Non-patent Document 17) (Taconic) Were used to prepare monoclonal antibodies. Antibody production was performed according to the following procedure.
  • CHO-S-hCCR1, CHO-S-mCCR1, RL33-hCCR1, and RL33-mCCR1 prepared in Example 2 were used. 1 ⁇ 10 7 cells / mouse were used per immunization. 5-9 week old CCR1 KO mice were treated with Alum gel (ELS) (80 ⁇ L / mouse) and pertussis vaccine (Nacalai Tesque) (1 ⁇ 10 7 cells / mouse) as adjuvants only at the time of the first immunization. In addition, immunization was performed by intraperitoneal administration. All immunizations were prepared with PBS so that the dose was 500 ⁇ L / animal. The second immunization was performed 2 weeks after the first immunization, and the third immunization was further performed 1 week later, and partial blood collection was performed 3 days later.
  • ELS Alum gel
  • pertussis vaccine Nacalai Tesque
  • FCM Antiserum evaluation
  • Serum collected from the immunized animal as a test sample was diluted with 1% BSA-PBS (0.02% EDTA, 0.05% NaN 3) so that the final concentrations were 200-fold dilution, 1000-fold dilution, and 5000-fold dilution. ) And dispensed at 50 ⁇ L / well and left at 4 ° C. for 30 minutes. After centrifugation (2000 rpm, 2 minutes), the supernatant was aspirated, and the cell pellet was broken with a plate shaker.
  • escron cloning medium supplemented with HAT SUPPLEMENT (Thermo Fisher Scientific). Suspended to a cell concentration of 5 ⁇ 10 7 cells / 9 mL.
  • a 96-well culture plate was previously dispensed with HAT-added cloning medium at 100 ⁇ L / well, and the cell suspension was dispensed at 100 ⁇ L / well into a CO 2 incubator (5% CO 2 , 37 ° C.). ) For 8-10 days.
  • Subcloning of hybridoma was performed on cells in the wells that were positive in the screening, and the cells were cultured in a cloning medium for about 7 to 10 days.
  • the subclass of each antibody was determined by FCM using a subclass-specific secondary antibody. The procedure for staining and measurement was the same as (2) above. Hybridoma culture supernatant was used as a test sample.
  • the detection antibodies include Alexa Fluor 488 goat anti-mouse IgG (H + L) (Thermo Fisher Scientific), and each subclass-specific antibody (Alexa Fluor 488 goat anti-mouse IgG 8 (Thermo Fisher 8). (Thermo Fisher Scientific), Alexa Fluor 488 goat anti-mouse IgG2b (Thermo Fluor 488 goat anti-mouse IgG used)
  • Antibodies were purified from the hybridoma culture supernatants cloned as described above.
  • Protein G Sepharose 4Fast Flow (GE Healthcare) was used. The culture supernatant was centrifuged to remove the precipitate and filtered through a filter. The column was packed with 400 ⁇ L of carrier and the buffer was replaced with DPBS. The culture supernatant was added, and the antibody was adsorbed to the simple substance, followed by washing twice with 10 mL of DPBS.
  • IgG Elution Buffer (Thermo Fisher Scientific) was added and eluted, and immediately neutralized with 0.1 mL of 1 M Tris-HCl (Nippon Gene) pH 8.6. Desalting and buffer replacement with DPBS were performed using a NAP column (GE Healthcare) and used for the subsequent analysis. Table 1 shows the clone name, origin, and subclass of the prepared antibody.
  • THP-1 migration (chemotaxis) assay The human monocytic leukemia cell line THP-1 is known as a human cell line expressing CCR1. This cell is known to exhibit chemotaxis to a concentration gradient of CCR1 ligand such as CCL3, CCL5, CCL15 or CCL23, and a migration assay using THP-1 is widely used as an evaluation system for CCR1 inhibitors. System. Therefore, the anti-human CCR1 antibody obtained in Example 5 was also evaluated using this experimental system to inhibit the activation of human CCR1 by human CCL15.
  • THP-1 cells were obtained from ATCC. THP-1 cells were cultured for 3 days in the presence of 5 ⁇ M All-trans-retinoic acid (ATRA) (Wako Pure Chemical Industries, Ltd.), induced to differentiate, recovered, and incubated at 37 ° C. [1% FBS ( (Thermo Fisher Scientific) / RPMI1640 (Nacalai Tesque)] and then resuspended in the same medium. 1 ⁇ 10 6 cells / mL were prepared, and cells were dispensed at 100 ⁇ L / well on the upper layer of Transwell (Corning, # 3421) having a pore size of 5 ⁇ m.
  • ATRA All-trans-retinoic acid
  • the lower layer is filled with assay medium supplemented with 1 ng / mL recombinant human CCL15 (68aa) (R & D technologies, # 628-LK) as a chemoattractant, and 4-fold in a 5% CO 2 incubator at 37 ° C. After culturing for 6 hours, the number of cells migrated to the lower layer was quantified with Celltiter-Glo (Promega).
  • FIG. 1 (a) and FIG. 1 (b) The obtained results are shown in FIG. 1 (a) and FIG. 1 (b).
  • the mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody obtained in Example 5 Both KM5955 antibody and KM5956 antibody inhibited THP-1 migration induced by activated CCL15 in a concentration-dependent manner.
  • the mouse anti-human CCR1 monoclonal antibody of the present invention is an antibody that inhibits the activation of human CCR1 by human CCL15.
  • Example 7 Determination of human CCR1 binding region of anti-human CCR1 antibody
  • the human CCR1 binding region of the mouse anti-human CCR1 monoclonal antibody obtained in Example 5 was subjected to FCM using cells expressing CCR1-CCR3 chimeric receptor. Examined. The measurement was performed in the same manner as in Example 4.
  • CHO-S-hCCR3, CHO-S-NC3-hCCR1, CHO-S-NC3-mCCR1, and CHO-S-hCCR3_EL2hCCR1 prepared in Example 2 were used as CCR1-CCR3 chimeric receptor-expressing cells. Moreover, CHO-S was used as a negative control.
  • the fluorescence intensity when a certain cell was stained with a certain test antibody (each hybridoma culture supernatant, 53504 antibody or 444-11 antibody) and a secondary antibody was measured when the cell was stained only with the secondary antibody.
  • a certain test antibody each hybridoma culture supernatant, 53504 antibody or 444-11 antibody
  • a secondary antibody was measured when the cell was stained only with the secondary antibody.
  • the obtained numerical value is 10 or more, it is determined that the test antibody binds to the cell, and when it is less than 10, it is determined that the test antibody does not bind to the cell. It showed in.
  • mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody do not bind to CHO-S-hCCR3.
  • the column was packed with 400 ⁇ L of carrier and the buffer was replaced with DPBS.
  • the culture supernatant was added to the column, and the antibody was adsorbed onto the carrier, followed by washing twice with 10 mL of DPBS.
  • 0.4 mL of IgG Elution Buffer (Thermo Scientific) was added to the column to elute the antibody, and the antibody solution was immediately neutralized with 0.1 mL of 1 M Tris-Cl (Nacalai Tesque) pH 8.6. .
  • the antibody solution was desalted and buffered with DPBS using a NAP column (GE Healthcare) and used for the subsequent analysis.
  • the purified 2D4 antibody was subjected to SDS-PAGE under reducing conditions by a conventional method, and it was confirmed that the antibody was purified.
  • the binding activity of the 2D4 antibody to human CCR1 was confirmed by FCM according to the method described in Example 4.
  • the 2D4 antibody was reacted at 0.1 and 1 ⁇ g / mL, and the cells used were CHO-S-hCCR1 as human CCR1-expressing cells and CHO-S as a negative control.
  • the 2D4 antibody did not bind to CHO-S and bound to CHO-S-hCCR1 in a concentration-dependent manner. Therefore, it was confirmed that the purified 2D4 antibody has a binding property to human CCR1 similarly to the commercially available 2D4 antibody.
  • mice Anti-human CCR1 monoclonal antibody KM5908 antibody and KM5916 antibody inhibited the migration of the cells in a concentration-dependent manner.
  • Example 6 all of the anti-human CCR1 antibodies obtained in Example 5 were THP-1 cells induced by activated CCL15 in an antibody concentration-dependent manner under the same experimental conditions as in this Example. Migration was inhibited [FIGS. 1 (a) and 1 (b)].
  • the existing anti-human CCR1 antibody does not inhibit the activation of human CCR1 by human CCL15
  • the mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 obtained in Example 5
  • Antibodies, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody were all shown to be antibodies that inhibit activation of human CCR1 by human CCL15.
  • Example 9 Production of recombinant antibody (1) Cloning and sequencing of antibody variable region gene Total RNA was extracted from the hybridoma cloned in Example 5 using Trizol (Life Technologies), and 5'-RACE. Antibody genes were amplified by the method. A SMARTER RACE Kit (Clontech) was used for the synthesis of RACE cDNA. Antibody variable region fragments are amplified by PCR using primers specific to the sequence added in the RACE cDNA synthesis process and a mouse Ig gamma chain or kappa chain amplification primer (SEQ ID NO: 11-14), and cloned. The base sequence of the DNA fragment was confirmed.
  • Example 5 For each anti-human CCR1 antibody obtained in Example 5, the base sequence encoding the amino acid sequence of the variable region of the heavy chain and the light chain, the amino acid sequence deduced from the base sequence, and the signal sequence were removed from the amino acid sequence Table 3 shows SEQ ID NOs representing the amino acid sequences. Further, Table 4 shows SEQ ID NOs representing the amino acid sequences of CDRs of the respective antibodies of the present invention.
  • N5KG4PE R409K vector N5KG1 vector (US Pat. No. 6,001,358).
  • the sequence was ligated to a vector (hereinafter referred to as N5KG4PE R409K vector) in which the base sequence encoding the constant region of mutant human IgG4 containing the above-mentioned amino acid modification was substituted to prepare an expression vector for the chimeric antibody.
  • N5KG4PE R409K vector a vector in which the base sequence encoding the constant region of mutant human IgG4 containing the above-mentioned amino acid modification was substituted to prepare an expression vector for the chimeric antibody.
  • the experimental procedure followed the manual that came with the kit.
  • plasmid DNA and Expifectamine 293 Reagent were added to Opti-MEM (Thermo Fisher Scientific), and allowed to stand for 30 minutes, and then the plasmid solution was added to the above cell solution. Further, after overnight culture, Expifectamine 293 Transfection Enhancer was added to the cell solution (total culture volume was 50 mL). After culturing the cell solution for 7 to 10 days, the culture supernatant was recovered.
  • Protein G Sepharose 4Fast Flow (GE Healthcare) was used for antibody purification.
  • the collected culture supernatant was centrifuged, and the obtained culture supernatant was filtered with a filter.
  • the column was packed with 400 ⁇ L of carrier and the buffer was replaced with DPBS.
  • the culture supernatant was added to the column, and the antibody was adsorbed to the single body, and then the column was washed twice with 10 mL of DPBS.
  • the antibody was eluted by adding 0.4 mL of IgG Elution Buffer (Thermo Scientific) to the column, and immediately 0.1 mL of 1 M Tris-Cl pH 8.6 was added to the antibody solution for neutralization.
  • the antibody solution was desalted using a NAP column (GE Healthcare) and used for the subsequent analysis.
  • mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody chimeric antibody were chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, respectively.
  • ChKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and chKM5956 antibody were chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, respectively.
  • Example 10 Evaluation of Chimeric Antibody Binding Chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and chKM5956 antibody prepared in Example 9 Binding to mouse CCR1 was measured by FCM according to the method described in Example 4. As human CCR1-expressing cells and mouse CCR1-expressing cells, CHO-S-hCCR1 and CHO-S-mCCR1 prepared in Example 2 were used, respectively. As a result, the chKM5955 antibody was shown to bind to human CCR1. Other chimeric antibodies have been shown to bind to human and mouse CCR1.
  • Example 11 Chemotaxis assay using chimeric antibody
  • the activity of inhibiting human CCR1 activation was measured.
  • all of the chimeric antibodies were shown to inhibit THP-1 migration by activated human CCL15.

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Abstract

The present invention relates to: a monoclonal antibody which can bind to an extracellular domain of human CC chemokine receptor 1 (CCR1) and can inhibit the activation of CCR1 with human CC chemokine ligand 15 (CCL15), or an antibody fragment of the monoclonal antibody; a hybridoma which can produce the antibody; a nucleic acid which has a nucleotide sequence encoding the antibody or the antibody fragment; a transformed cell which contains a vector carrying the nucleic acid; a method for producing the antibody or the antibody fragment using the hybridoma or the transformed cell; a therapeutic agent and a diagnostic agent, each of which contains the antibody or the antibody fragment; and a method for treating a CCR1-related disease and a method for diagnosing a CCR1-related disease, in each of which the antibody or the antibody fragment is used.

Description

抗ヒトＣＣＲ１モノクローナル抗体Anti-human CCR1 monoclonal antibody

　本発明は、ヒトＣＣケモカイン受容体１（ＣＣ　ｃｈｅｍｏｋｉｎｅ　ｒｅｃｅｐｔｏｒ　１、以下、ＣＣＲ１と記載する）の細胞外領域に結合し、ヒトＣＣケモカインリガンド（以下、ＣＣＬと記載する）１５によるＣＣＲ１の活性化を阻害するモノクローナル抗体または該抗体断片、該抗体を産生するハイブリドーマ、該抗体または該抗体断片をコードする塩基配列を有する核酸、該核酸を含むベクターを含む形質転換細胞、該ハイブリドーマまたは該形質転換細胞を用いる該抗体または該抗体断片の製造方法、該抗体または該抗体断片を含む治療薬および診断薬、並びに該抗体または該抗体断片を用いたＣＣＲ１関連疾患の治療方法および診断方法に関する。 The present invention binds to the extracellular region of human CC chemokine receptor 1 (CC chemokine receptor 1, hereinafter referred to as CCR1) and activates CCR1 by human CC chemokine ligand (hereinafter referred to as CCL) 15. Inhibiting monoclonal antibody or antibody fragment, hybridoma producing the antibody, nucleic acid having a base sequence encoding the antibody or antibody fragment, transformed cell containing a vector containing the nucleic acid, hybridoma or transformed cell The present invention relates to a method for producing the antibody or the antibody fragment to be used, a therapeutic agent and a diagnostic agent containing the antibody or the antibody fragment, and a method for treating and diagnosing a CCR1-related disease using the antibody or the antibody fragment.

　ＣＣＲ１は、表面抗原分類（ｃｌｕｓｔｅｒ　ｏｆ　ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ、ＣＤ）１９１、ＣＫＲ－１、ＨＭ１４５、Ｍａｃｒｏｐｈａｇｅ　ｉｎｆｌａｍｍａｔｏｒｙ　ｐｒｏｔｅｉｎ　１α　ｒｅｃｅｐｔｏｒ（ＭＩＰ１α　Ｒ）、ＣＭＫＢＲ１またはＳＣＹＡＲ１等の別名がある。 CCR1 has aliases such as surface antigen classification (cluster of differentiation, CD) 191, CKR-1, HM145, Macrophage inflammatory protein 1α receptor (MIP1α R), CMKBR1 or SCYAR1.

　ヒトＣＣＲ１をコードする遺伝子は、１９９３年に同定されている（非特許文献１）。ヒトＣＣＲ１のｃＤＮＡ配列（配列番号１）およびアミノ酸配列（配列番号２）は公開されており、例えばＮａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）では、ｃＤＮＡ配列はＮＭ＿００１２９５、タンパク質のアミノ酸配列はＮＰ＿００１２８６から参照できる。マウスＣＣＲ１のｃＤＮＡ配列（配列番号３）およびアミノ酸配列（配列番号４）も公開されており、ＮＣＢＩでは、ｃＤＮＡ配列はＮＭ＿００９９１２、タンパク質のアミノ酸配列はＮＰ＿０３４０４２から参照できる。 A gene encoding human CCR1 was identified in 1993 (Non-patent Document 1). The cDNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of human CCR1 are publicly available. For example, in National Center for Biotechnology Information (NCBI), the cDNA sequence can be referred to NM_001295 and the protein amino acid sequence can be referred to NP_001286. The mouse CCR1 cDNA sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) are also disclosed. In NCBI, the cDNA sequence can be referred to NM_009912, and the protein amino acid sequence can be referred to NP_034042.

　ＣＣＲ１は、７回膜貫通型の構造を有するＧタンパク質共役型受容体（Ｇ　ｐｒｏｔｅｉｎ－ｃｏｕｐｌｅｄ　ｒｅｃｅｐｔｏｒ；以下ＧＰＣＲ）であり、全長３５５アミノ酸からなる膜タンパク質である。ヒトＣＣＲ１のリガンドとしてヒトＣＣＬ３、ＣＣＬ５、ＣＣＬ８、ＣＣＬ１４、ＣＣＬ１５、ＣＣＬ１６およびＣＣＬ２３が報告されている（非特許文献２）。また、マウスＣＣＲ１のリガンドとしてマウスＣＣＬ３、ＣＣＬ５、ＣＣＬ７およびＣＣＬ９が報告されている（非特許文献３）。 CCR1 is a G protein-coupled receptor (hereinafter referred to as GPCR) having a seven-transmembrane structure, and is a membrane protein consisting of a total length of 355 amino acids. Human CCR3, CCL5, CCL8, CCL14, CCL15, CCL16 and CCL23 have been reported as ligands for human CCR1 (Non-patent Document 2). Moreover, mouse CCL3, CCL5, CCL7 and CCL9 have been reported as ligands for mouse CCR1 (Non-patent Document 3).

　ヒトＣＣＬ１５はＣ－Ｃケモカインファミリーに含まれるリガンドであり、全長９２アミノ酸からなる。ＣＣＲ１とＣＣＲ３がＣＣＬ１５の受容体として機能することが知られている。ＣＣＬ１５はタンパク質分解酵素の作用によってＮ末端が分解され、６８アミノ酸前後の活性化型となることでより強力な活性を発揮することが知られている（非特許文献４）。 Human CCL15 is a ligand contained in the CC chemokine family and consists of a total of 92 amino acids. CCR1 and CCR3 are known to function as CCL15 receptors. It is known that CCL15 exhibits a stronger activity by being decomposed at the N-terminus by the action of a proteolytic enzyme and becomes an activated form of around 68 amino acids (Non-patent Document 4).

　ＣＣＲ１を含むケモカイン受容体の活性化は、以下の２つのステップを経て起こると考えられている（非特許文献５）。ステップ１としてケモカイン（リガンド）と、受容体のＮ末端細胞外領域との相互作用が生じる。ステップ２としてケモカインのＮ末端領域が、受容体の細胞外ループ領域と相互作用し、受容体の構造変化が起こった結果、細胞内にシグナルが伝達される。 The activation of chemokine receptors including CCR1 is considered to occur through the following two steps (Non-patent Document 5). Step 1 involves interaction of the chemokine (ligand) with the N-terminal extracellular region of the receptor. As a step 2, the N-terminal region of the chemokine interacts with the extracellular loop region of the receptor, and as a result of the structural change of the receptor, a signal is transmitted into the cell.

　ＧＰＣＲの細胞内シグナル伝達においては、リガンドの結合によって生じたＧＰＣＲの構造変化に応じて、ＧＰＣＲのＣ末端に会合したＧタンパク質α、β、γ三量体が活性化され、αサブユニットがβγ複合体から解離する。αサブユニットはさらに下流の因子に作用し、シグナル伝達経路を活性化する。αサブユニットの活性化によりホスホリパーゼＣ（ｐｈｏｓｐｈｏｌｉｐａｓｅ　Ｃ、以下ＰＬＣ）が活性化すると、ホスファチジルイノシトール（４，５）２リン酸［ｐｈｏｓｐｈａｔｉｄｙｌｉｎｏｓｉｔｏｌ（４，５）ｂｉｓｐｈｏｓｐｈａｔｅ、ＰＩＰ２］が分解され、イノシトール３リン酸（Ｉｎｏｓｉｔｏｌ　ｔｒｉｐｈｏｓｐｈａｔｅ、ＩＰ３）とジアシルグリセロール（ｄｉａｃｙｌｇｌｙｃｅｒｏｌ、ＤＡＧ）が産生される。 In intracellular signal transduction of GPCRs, G proteins α, β, and γ trimers associated with the C-terminus of GPCR are activated in response to structural changes in GPCR caused by ligand binding, and α subunit is βγ Dissociate from the complex. The α subunit acts on further downstream factors and activates signal transduction pathways. activated by phospholipase C (phospholipase C, following PLC) of the α-subunit When activated, phosphatidylinositol (4,5) 2-phosphate [phosphatidylinositol (4,5) bisphosphate, PIP 2] is decomposed, inositol triphosphate Acid (Inositol triphosphate, IP 3 ) and diacylglycerol (DAG) are produced.

　ＩＰ３は小胞体に作用し、カルシウムイオン（Ｃａ２＋）を細胞内に放出させ、カルモジュリンを介して種々の細胞反応を引き起こす。この細胞内カルシウム濃度の上昇は、蛍光カルシウムインジケーター等を使用して測定することができ、ＧＰＣＲの活性化の指標とすることができる。ＣＣＲ１についてもこの方法で細胞内シグナルの活性化を測定することが可能である。 IP 3 acts on the endoplasmic reticulum, releases calcium ions (Ca 2+ ) into cells, and causes various cellular responses via calmodulin. This increase in intracellular calcium concentration can be measured using a fluorescent calcium indicator or the like, and can be used as an index of GPCR activation. For CCR1, it is also possible to measure the activation of intracellular signals by this method.

　好中球、好酸球、好塩基球、単球、マクロファージ、樹状細胞、ＮＫ細胞、Ｔ細胞またはＢ細胞等、種々の血球細胞におけるヒトＣＣＲ１の発現がこれまでに報告されている（非特許文献６～１０）。さらに近年、癌微小環境中に存在し、癌の進展を促進する未成熟骨髄球（ｉｍｍａｔｕｒｅ　ｍｙｅｌｏｉｄ　ｃｅｌｌ、以下ｉＭＣ）、骨髄由来免疫抑制性細胞（ｍｙｅｌｏｉｄ　ｄｅｒｉｖｅｄ　ｓｕｐｐｒｅｓｓｏｒ　ｃｅｌｌ、以下ＭＤＳＣ）と呼ばれる細胞群がＣＣＲ１を発現していることが報告されている（非特許文献１１および１２）。 The expression of human CCR1 in various blood cells such as neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, NK cells, T cells or B cells has been reported so far (non-) Patent Documents 6 to 10). In recent years, a group of cells called immature myelocytes (iMC) and bone marrow-derived immunosuppressor cells (MDSCs), which exist in the cancer microenvironment and promote the progression of cancer, are called “myeloid derived suppressor cells” (MDSCs). It has been reported that CCR1 is expressed (Non-patent Documents 11 and 12).

　ＣＣＲ１は関節リウマチ、多発性硬化症、慢性閉塞性肺疾患等、種々の自己免疫性疾患、炎症性疾患への関与が示唆されている（非特許文献１３）。また、上記のｉＭＣおよびＭＤＳＣに発現していることから、癌の進展および増悪過程へのＣＣＲ１の寄与が示唆されている（非特許文献１１および１２）。 CCR1 has been suggested to be involved in various autoimmune diseases and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and chronic obstructive pulmonary disease (Non-patent Document 13). In addition, the expression in iMC and MDSC described above suggests that CCR1 contributes to the progression and progression of cancer (Non-Patent Documents 11 and 12).

　例えば、ヒト大腸癌においては、ある頻度で、癌抑制遺伝子であるＳＭＡＤ４の変異またはＳＭＡＤ４タンパク質の消失が見られることが知られており、ＳＭＡＤ４の欠損は予後不良因子であると考えられている。近年、ＳＭＡＤ４の欠損が、ＣＣＬ１５の発現上昇を介して、腫瘍環境中にＣＣＲ１陽性のｉＭＣまたはＭＤＳＣ等を引き込む要因となっていること、さらに、これらの細胞がマトリクスメタロプロテアーゼ（ｍａｔｒｉｘ　ｍｅｔａｌｌｏｐｒｏｔｅａｓｅ、ＭＭＰ）の分泌や免疫抑制作用によって癌の浸潤または転移を補助し、患者の予後を悪化させるといった機序が明らかにされつつある（非特許文献１１および１２）。 For example, in human colorectal cancer, it is known that mutation of SMAD4, which is a tumor suppressor gene, or disappearance of SMAD4 protein is seen at a certain frequency, and deficiency of SMAD4 is considered to be a poor prognostic factor. In recent years, deficiency of SMAD4 has been a factor that draws CCR1-positive iMC or MDSC into the tumor environment through increased expression of CCL15, and these cells are also a matrix metalloprotease (MMP). The mechanism of assisting the invasion or metastasis of cancer by the secretion and immunosuppressive action and worsening the prognosis of patients is being clarified (Non-patent Documents 11 and 12).

　既存の低分子ＣＣＲ１阻害剤としてはＣＰ４８１，７１５（Ｐｆｉｚｅｒ社）、ＭＬＮ３８９７（Ｍｉｌｌｅｎｎｉｕｍ社）、ＢＸ－４７１（Ｂｅｒｌｅｘ社）、ＣＣＸ－３５４（Ｃｈｅｍｏｃｅｎｔｒｙｘ社）等を挙げることができる。これら低分子阻害剤については、関節リウマチ、多発性硬化症、慢性閉塞性肺疾患等の自己免疫性または炎症性疾患の患者に対する臨床試験が行われたが、いずれも有効性を示すことができていない（非特許文献１４）。 Existing low molecular weight CCR1 inhibitors include CP481,715 (Pfizer), MLN3897 (Millennium), BX-471 (Berlex), CCX-354 (Chemocentryx), and the like. These small molecule inhibitors have been tested in patients with autoimmune or inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and chronic obstructive pulmonary disease. (Non-Patent Document 14).

　既存の抗ＣＣＲ１抗体のうち、文献等でＣＣＲ１活性化の阻害効果が報告されているものとしては、１４１－２（ＭＢＬ社、＃Ｄ０６３－３）（非特許文献１５）、５３５０４（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社、＃ＭＡＢ１４５）（非特許文献１６）および２Ｄ４（Ｍｉｌｌｅｎｎｉｕｍ社）（特許文献１）が挙げられる。 Among the existing anti-CCR1 antibodies, those that have been reported to inhibit CCR1 activation in the literature include 141-2 (MBL, # D063-3) (Non-patent Document 15), 53504 (R & D Systems) , # MAB145) (Non-patent Document 16) and 2D4 (Millennium) (Patent Document 1).

米国特許第６，７５６，０３５号明細書US Pat. No. 6,756,035

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　上記の非特許文献１５、非特許文献１６および特許文献１等に記載の既存の抗ＣＣＲ１抗体には医薬品として開発されたものはなく、抗体医薬品としての性能に関する情報は十分ではない。したがって、本発明は、ヒトＣＣＲ１の細胞外領域に結合し、ヒトＣＣＬ１５によるＣＣＲ１の活性化を阻害するモノクローナル抗体または該抗体断片、該抗体を産生するハイブリドーマ、該抗体または該抗体断片をコードする塩基配列を有する核酸、該核酸を含むベクターを含む形質転換細胞、該ハイブリドーマまたは該形質転換細胞を用いる該抗体または該抗体断片の製造方法、該抗体または該抗体断片を含む治療薬および診断薬、並びに該抗体または該抗体断片を用いたＣＣＲ１関連疾患の治療方法および診断方法を提供することを目的とする。 None of the existing anti-CCR1 antibodies described in Non-Patent Document 15, Non-Patent Document 16, Patent Document 1, etc. have been developed as pharmaceuticals, and information on the performance as antibody pharmaceuticals is not sufficient. Accordingly, the present invention provides a monoclonal antibody or antibody fragment that binds to the extracellular region of human CCR1 and inhibits activation of CCR1 by human CCL15, a hybridoma that produces the antibody, a base encoding the antibody or the antibody fragment A nucleic acid having a sequence, a transformed cell comprising a vector comprising the nucleic acid, a method for producing the antibody or the antibody fragment using the hybridoma or the transformed cell, a therapeutic agent and a diagnostic agent comprising the antibody or the antibody fragment, and It is an object of the present invention to provide a method for treating and diagnosing a CCR1-related disease using the antibody or the antibody fragment.

　上記課題を解決するための手段として、本発明はヒトＣＣＲ１の細胞外領域に結合し、ヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害するヒトＣＣＲ１モノクローナル抗体を提供する。 As a means for solving the above problems, the present invention provides a human CCR1 monoclonal antibody that binds to the extracellular region of human CCR1 and inhibits activation of human CCR1 by human CCL15.

　すなわち、本発明は以下の（１）～（２１）に関する。
（１）ヒトＣＣＲ１の細胞外領域に結合し、ヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害するモノクローナル抗体または該抗体断片。
（２）ヒトＣＣＬ１５により誘導されるヒトＣＣＲ１発現細胞の遊走を阻害する（１）に記載のモノクローナル抗体または該抗体断片。
（３）ヒトＣＣＲ１の細胞外ループ２領域のアミノ酸配列のうち、少なくとも１つのアミノ酸残基に結合する、（１）または（２）に記載のモノクローナル抗体または該抗体断片。
（４）モノクローナル抗体が、下記（ａ）～（ｌ）から選ばれるいずれか一つの抗体である、（１）～（３）のいずれか１つに記載のモノクローナル抗体または該抗体断片。
（ａ）重鎖可変領域（ｈｅａｖｙ　ｃｈａｉｎ　ｖａｒｉａｂｌｅ　ｒｅｇｉｏｎ；以下、ＶＨと略記する）の相補性決定領域（ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ；以下ＣＤＲと略記する）１～３のアミノ酸配列が、それぞれ配列番号６９、７０および７１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつ軽鎖可変領域（ｌｉｇｈｔ　ｃｈａｉｎ　ｖａｒｉａｂｌｅ　ｒｅｇｉｏｎ；以下、ＶＬと略記する）のＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７２、７３および７４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｂ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７５、７６および７７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７８、７９および８０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｃ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８１、８２および８３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８４、８５および８６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｄ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８７、８８および８９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９０、９１および９２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｅ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９３、９４および９５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９６、９７および９８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｆ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９９、１００および１０１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０２、１０３および１０４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｇ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０５、１０６および１０７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０８、１０９および１１０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｈ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１１、１１２および１１３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１４、１１５および１１６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｉ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１７、１１８および１１９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１２０、１２１および１２２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｊ）前記（ａ）～（ｉ）に記載の少なくとも一つの抗体と、ヒトＣＣＲ１への結合について競合する抗体。
（ｋ）前記（ａ）～（ｉ）に記載のいずれか一つの抗体が結合するエピトープを含むエピトープに結合する抗体。
（ｌ）前記（ａ）～（ｉ）に記載のいずれか一つの抗体が結合するエピトープと同じエピトープに結合する抗体。
（５）モノクローナル抗体が、下記（ａ）～（ｉ）から選ばれるいずれか一つの抗体である、（１）～（４）のいずれか１つに記載のモノクローナル抗体または該抗体断片。
（ａ）ＶＨのアミノ酸配列が配列番号５１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｂ）ＶＨのアミノ酸配列が配列番号５３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｃ）ＶＨのアミノ酸配列が配列番号５５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｄ）ＶＨのアミノ酸配列が配列番号５７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｅ）ＶＨのアミノ酸配列が配列番号５９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｆ）ＶＨのアミノ酸配列が配列番号６１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｇ）ＶＨのアミノ酸配列が配列番号６３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｈ）ＶＨのアミノ酸配列が配列番号６５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｉ）ＶＨのアミノ酸配列が配列番号６７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（６）モノクローナル抗体が遺伝子組換え抗体である（１）～（５）のいずれか１つに記載のモノクローナル抗体または該抗体断片。
（７）遺伝子組換え抗体が、ヒト型キメラ抗体、ヒト化抗体およびヒト抗体から選ばれる１の遺伝子組換え抗体である、（６）に記載のモノクローナル抗体または該抗体断片。
（８）Ｆａｂ、Ｆａｂ’、（Ｆａｂ’）２、一本鎖抗体（ｓｃＦｖ）、二量体化Ｖ領域（ｄｉａｂｏｄｙ）、ジスルフィド安定化Ｖ領域（ｄｓＦｖ）およびＣＤＲを含むペプチドから選ばれる１の抗体断片である、（１）～（７）のいずれか１つに記載の抗体断片。
（９）（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片を産生するハイブリドーマ。
（１０）（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片をコードする塩基配列を有する核酸。
（１１）（１０）に記載の核酸を含むベクターを含む形質転換細胞。
（１２）（９）に記載のハイブリドーマまたは（１１）に記載の形質転換細胞を培養し、培養液から（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片を採取することを含む、（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片の製造方法。
（１３）（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片を含む、ヒトＣＣＲ１の検出または測定用試薬。
（１４）（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片を含む、ヒトＣＣＲ１関連疾患の診断薬。
（１５）ヒトＣＣＲ１関連疾患が、癌、自己免疫疾患または炎症性疾患である、（１４）に記載の診断薬。
（１６）（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片を有効成分として含有する、ヒトＣＣＲ１関連疾患の治療薬。
（１７）ヒトＣＣＲ１関連疾患が、癌、自己免疫疾患または炎症性疾患である、（１６）に記載の治療薬。
（１８）（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片を用いたヒトＣＣＲ１関連疾患の診断方法。
（１９）（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片を用いたヒトＣＣＲ１関連疾患の治療方法。
（２０）ヒトＣＣＲ１関連疾患の診断薬を製造するための、（１）～（８）のいずれか１つに記載のモノクローナル抗体または該抗体断片の使用。
（２１）ヒトＣＣＲ１関連疾患の治療薬を製造するための、（１）～（８）のいずれか１項に記載のモノクローナル抗体または該抗体断片の使用。 That is, the present invention relates to the following (1) to (21).
(1) A monoclonal antibody or an antibody fragment thereof that binds to the extracellular region of human CCR1 and inhibits activation of human CCR1 by human CCL15.
(2) The monoclonal antibody or the antibody fragment thereof according to (1), which inhibits migration of human CCR1-expressing cells induced by human CCL15.
(3) The monoclonal antibody or the antibody fragment thereof according to (1) or (2), which binds to at least one amino acid residue in the amino acid sequence of the extracellular loop 2 region of human CCR1.
(4) The monoclonal antibody or the antibody fragment thereof according to any one of (1) to (3), wherein the monoclonal antibody is any one antibody selected from the following (a) to (l):
(A) The amino acid sequences of complementarity determining regions (complementary determining regions; hereinafter abbreviated as CDR) 1 to 3 of heavy chain variable regions (hereinafter abbreviated as VH) are SEQ ID NOs: 69 and 70, respectively. And 71, and the amino acid sequences of CDR1 to CDR3 of the light chain variable region (hereinafter abbreviated as VL) are SEQ ID NOs: 72, 73 and 73, respectively. 74. An antibody having an amino acid sequence comprising the amino acid sequence described in 74.
(B) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 75, 76, and 77, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 78, respectively. 79. An antibody having an amino acid sequence comprising the amino acid sequence described in 79 and 80.
(C) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 81, 82 and 83, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 84 , 85 and 86, an antibody having an amino acid sequence comprising the amino acid sequence described in 85 and 86.
(D) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 87, 88, and 89, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 90 , 91 and 92, an antibody having an amino acid sequence comprising the amino acid sequence described in 91 and 92.
(E) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 93, 94 and 95, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 96, respectively. The antibody which is an amino acid sequence containing the amino acid sequence described in 97 and 98.
(F) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 99, 100 and 101, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 102. , 103 and 104. An antibody having an amino acid sequence comprising the amino acid sequence described in 103 and 104.
(G) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 105, 106 and 107, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 108 , 109 and 110, an antibody having an amino acid sequence comprising the amino acid sequence described in 109 and 110.
(H) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 111, 112, and 113, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 114, respectively. , 115 and 116, an antibody having an amino acid sequence comprising the amino acid sequence described in 115 and 116.
(I) The amino acid sequences of CDRs 1 to 3 of VH are amino acid sequences including the amino acid sequences described in SEQ ID NOs: 117, 118, and 119, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 120, respectively. , 121 and 122, which is an amino acid sequence comprising the amino acid sequence described in 121 and 122.
(J) An antibody that competes with at least one antibody described in (a) to (i) for binding to human CCR1.
(K) An antibody that binds to an epitope including the epitope to which any one of the antibodies (a) to (i) binds.
(L) An antibody that binds to the same epitope as that to which any one of the antibodies (a) to (i) binds.
(5) The monoclonal antibody or the antibody fragment thereof according to any one of (1) to (4), wherein the monoclonal antibody is any one antibody selected from the following (a) to (i):
(A) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 51, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 52.
(B) An antibody wherein the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 53, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 54.
(C) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 55, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 56.
(D) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 57, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 58.
(E) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 59, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 60.
(F) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 61, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 62.
(G) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 63, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 64.
(H) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 65, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 66.
(I) An antibody wherein the amino acid sequence of VH is an amino acid sequence containing the amino acid sequence set forth in SEQ ID NO: 67, and the amino acid sequence of VL is an amino acid sequence containing the amino acid sequence set forth in SEQ ID NO: 68.
(6) The monoclonal antibody or the antibody fragment thereof according to any one of (1) to (5), wherein the monoclonal antibody is a gene recombinant antibody.
(7) The monoclonal antibody or the antibody fragment thereof according to (6), wherein the recombinant antibody is one recombinant antibody selected from a human chimeric antibody, a humanized antibody and a human antibody.
(8) One selected from peptides including 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 (7), which is an antibody fragment.
(9) A hybridoma that produces the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
(10) A nucleic acid having a base sequence encoding the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
(11) A transformed cell comprising a vector comprising the nucleic acid according to (10).
(12) The hybridoma according to (9) or the transformed cell according to (11) is cultured, and the monoclonal antibody or the antibody fragment according to any one of (1) to (8) is collected from the culture solution A method for producing the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
(13) A reagent for detecting or measuring human CCR1, comprising the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
(14) A diagnostic agent for human CCR1-related diseases comprising the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
(15) The diagnostic agent according to (14), wherein the human CCR1-related disease is cancer, autoimmune disease or inflammatory disease.
(16) A therapeutic agent for a human CCR1-related disease comprising the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8) as an active ingredient.
(17) The therapeutic agent according to (16), wherein the human CCR1-related disease is cancer, autoimmune disease or inflammatory disease.
(18) A method for diagnosing a human CCR1-related disease using the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
(19) A method for treating a human CCR1-related disease using the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8).
(20) Use of the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8) for producing a diagnostic agent for a human CCR1-related disease.
(21) Use of the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (8) for producing a therapeutic agent for a human CCR1-related disease.

　本発明のモノクローナル抗体または該抗体断片は、ヒトＣＣＲ１の細胞外領域に結合し、ヒトＣＣＲ１活性化に伴う種々の反応を阻害する。それゆえ、本発明のモノクローナル抗体または該抗体断片は、ヒトＣＣＲ１関連疾患の治療薬および診断薬として利用できる。 The monoclonal antibody or the antibody fragment of the present invention binds to the extracellular region of human CCR1 and inhibits various reactions associated with human CCR1 activation. Therefore, the monoclonal antibody or the antibody fragment of the present invention can be used as a therapeutic agent and a diagnostic agent for human CCR1-related diseases.

図１（ａ）および図１（ｂ）は、抗ヒトＣＣＲ１抗体について、活性化ヒトＣＣＬ１５によるＴＨＰ－１の遊走を阻害する活性を測定した結果である。図１（ａ）および図１（ｂ）の縦軸は、ＴＨＰ－１細胞の遊走（％）を示し、ＤＰＢＳと活性化ＣＣＬ１５を添加した時に、Ｔｒａｎｓｗｅｌｌの下層に移動した細胞数を１００％とした。図１（ａ）および図１（ｂ）の横軸は、ＴＨＰ－１細胞に添加した抗体、リガンドおよびこれらの濃度を示している。図１（ａ）および図１（ｂ）中には、ＤＰＢＳを添加したサンプルをＤＰＢＳ、活性化ヒトＣＣＬ１５を添加していないサンプルをＮｏ　ｌｉｇａｎｄ、活性化ヒトＣＣＬ１５を添加したサンプルをｈＣＣＬ１５（６８ａａ）と記載した。抗ヒトＣＣＲ１抗体としては、ＫＭ５９０７抗体、ＫＭ５９０８抗体、ＫＭ５９０９抗体、ＫＭ５９１１抗体、ＫＭ５９１５抗体、ＫＭ５９１６抗体、ＫＭ５９５４抗体、ＫＭ５９５５抗体およびＫＭ５９５６抗体を使用した。FIG. 1 (a) and FIG. 1 (b) are the results of measuring the activity of anti-human CCR1 antibody to inhibit THP-1 migration by activated human CCL15. The vertical axis in FIGS. 1 (a) and 1 (b) shows THP-1 cell migration (%). When DPBS and activated CCL15 were added, the number of cells that migrated to the lower layer of Transwell was 100%. did. The horizontal axis of FIG. 1 (a) and FIG. 1 (b) shows the antibody and ligand added to THP-1 cells and their concentrations. In FIG. 1 (a) and FIG. 1 (b), the sample to which DPBS is added is DPBS, the sample to which activated human CCL15 is not added is No ligand, and the sample to which activated human CCL15 is added is hCCL15 (68aa) It was described. As anti-human CCR1 antibodies, KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody were used. 図２は、抗ヒトＣＣＲ１抗体について、活性化ヒトＣＣＬ１５によるＴＨＰ－１の遊走を阻害する活性を測定した結果である。図の縦軸は、Ｔｒａｎｓｗｅｌｌの下層に移動した細胞数をＣｅｌｌＴｉｔｅｒ－Ｇｌｏで測定した際の発光量（ｒｅｌａｔｉｖｅ　ｌｉｇｈｔ　ｕｎｉｔ；ＲＬＵ）を表わす。図２の横軸は、ＴＨＰ－１細胞に添加した抗体、リガンドおよびこれらの濃度を示している。図２中には、ＤＰＢＳを添加したサンプルをＤＰＢＳ、活性化ヒトＣＣＬ１５を添加していないサンプルをＮｏ　ｌｉｇａｎｄ、活性化ヒトＣＣＬ１５を添加したサンプルをｈＣＣＬ１５（６８ａａ）と記載した。抗ヒトＣＣＲ１抗体としては、２Ｄ４抗体（Ｍｉｌｌｅｎｎｉｕｍ社）、５３５０４抗体（Ｒ＆Ｄ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）、１４１－２抗体（ＭＢＬ社、＃Ｄ０６３－３）、ＫＭ５９０８抗体およびＫＭ５９１６抗体を使用した。FIG. 2 shows the results of measuring the activity of anti-human CCR1 antibody to inhibit THP-1 migration by activated human CCL15. The vertical axis of the figure represents the amount of luminescence (relative light unit; RLU) when the number of cells that have moved to the lower layer of Transwell is measured by CellTiter-Glo. The horizontal axis of FIG. 2 shows the antibodies, ligands and their concentrations added to THP-1 cells. In FIG. 2, the sample to which DPBS is added is described as DPBS, the sample to which activated human CCL15 is not added is referred to as No ligand, and the sample to which activated human CCL15 is added is described as hCCL15 (68aa). As the anti-human CCR1 antibody, 2D4 antibody (Millennium), 53504 antibody (R & D Technologies), 141-2 antibody (MBL, # D063-3), KM5908 antibody and KM5916 antibody were used.

　本発明は、ヒトＣＣＲ１の細胞外領域に結合し、ヒトＣＣＬ１５によるＣＣＲ１の活性化を阻害するモノクローナル抗体または該抗体断片に関する。 The present invention relates to a monoclonal antibody or an antibody fragment thereof that binds to the extracellular region of human CCR1 and inhibits activation of CCR1 by human CCL15.

　ＣＣＲ１は、ＣＤ１９１、ＣＫＲ－１、ＨＭ１４５、Ｍａｃｒｏｐｈａｇｅ　ｉｎｆｌａｍｍａｔｏｒｙ　ｐｒｏｔｅｉｎ　１α　ｒｅｃｅｐｔｏｒ（ＭＩＰ１αＲ）、ＣＭＫＢＲ１およびＳＣＹＡＲ１等ともいう。ＣＣＲ１は、７回膜貫通型の構造を有するＧＰＣＲであり、全長３５５アミノ酸からなる膜タンパク質である。 CCR1 is also called CD191, CKR-1, HM145, Macrophage infrastructure protein 1α receptor (MIP1αR), CMKBR1, SCYAR1, and the like. CCR1 is a GPCR having a seven-transmembrane structure, and is a membrane protein consisting of a total length of 355 amino acids.

　ＣＣＲ１を含むＧＰＣＲにおいて、細胞表面上のＧＰＣＲは、リガンドが結合することにより活性化され、該細胞内に該受容体依存的なシグナルが伝達されると同時に、該細胞内のカルシウムイオン濃度が上昇する。その結果、該細胞について、細胞遊走、ケモカインの産生、マトリックスメタロプロテアーゼＭＭＰの産生などが生じることが知られている。 In the GPCR containing CCR1, the GPCR on the cell surface is activated by binding of a ligand, and the receptor-dependent signal is transmitted into the cell, and at the same time, the calcium ion concentration in the cell increases. To do. As a result, it is known that cell migration, chemokine production, matrix metalloprotease MMP production, and the like occur in the cells.

　すなわち、ＣＣＲ１の機能としては、リガンドが細胞表面上のＣＣＲ１に結合することにより、該細胞内にＣＣＲ１依存的なシグナルが伝達されると同時に、該細胞内のカルシウムイオン濃度が上昇した結果、該細胞について細胞遊走、ケモカインの産生またはＭＭＰの産生などを起こすことなどが挙げられる。 That is, as a function of CCR1, as a result of the ligand binding to CCR1 on the cell surface, a CCR1-dependent signal is transmitted into the cell, and at the same time, the calcium ion concentration in the cell increases. Examples include cell migration, chemokine production, or MMP production.

　ヒトＣＣＲ１のリガンドとしては、例えば、ヒトＣＣＬ３、ＣＣＬ５、ＣＣＬ８、ＣＣＬ１４、ＣＣＬ１５、ＣＣＬ１６およびＣＣＬ２３などが挙げられる。マウスＣＣＲ１のリガンドとしては、例えば、マウスＣＣＬ３、ＣＣＬ５、ＣＣＬ７およびＣＣＬ９などが挙げられる。 Examples of human CCR1 ligands include human CCL3, CCL5, CCL8, CCL14, CCL15, CCL16, and CCL23. Examples of the mouse CCR1 ligand include mouse CCL3, CCL5, CCL7, and CCL9.

　ヒトＣＣＬ１５はＣ－Ｃケモカインファミリーに含まれるリガンドであり、全長９２アミノ酸からなる。ヒトＣＣＬ１５はタンパク質分解酵素の作用によってＮ末端が分解され、６８アミノ酸前後の活性化型［以下、本発明では活性化ヒトＣＣＬ１５またはｈＣＣＬ１５（６８ａａ）と記載する］になることで、全長のＣＣＬ１５（以下、本発明では全長ＣＣＬ１５と記載する）よりも強力な活性を発揮することが知られている。 Human CCL15 is a ligand contained in the CC chemokine family and consists of a total of 92 amino acids. Human CCL15 is degraded at the N-terminus by the action of a proteolytic enzyme and becomes an activated form of about 68 amino acids (hereinafter referred to as activated human CCL15 or hCCL15 (68aa) in the present invention), so that full-length CCL15 ( Hereinafter, it is known that the present invention exhibits a stronger activity than the full length CCL15).

　ヒトＣＣＬ１５が細胞表面上のヒトＣＣＲ１に結合し、該受容体が活性化されると、該細胞内にＣＣＲ１依存的なシグナルが伝達され、ホスホリパーゼＣ（ＰＬＣ）の活性化、細胞内のカルシウムイオン濃度の上昇またはｎｕｃｌｅａｒ　ｆａｃｔｏｒ－κＢ（ＮＦ－κＢ）の活性化等が起こる。その結果、該細胞について、細胞遊走などが生じる。 When human CCL15 binds to human CCR1 on the cell surface and the receptor is activated, a CCR1-dependent signal is transmitted into the cell, activation of phospholipase C (PLC), intracellular calcium ion Increase in concentration or activation of nuclear factor-κB (NF-κB) occurs. As a result, cell migration or the like occurs for the cell.

　本発明の抗体としては、ヒトＣＣＬ１５によるヒトＣＣＲ１活性化に伴う種々の反応を阻害する抗体が挙げられる。本発明の抗体として具体的には、例えば、ヒトＣＣＬ１５によるヒトＣＣＲ１発現細胞内でのＣＣＲ１依存的なシグナル伝達、ＰＬＣの活性化、細胞内カルシウムイオン濃度の上昇、ＮＦ－κＢの活性化およびＣＣＲ１発現細胞の遊走から選ばれる少なくとも一つの反応を阻害する抗体などが挙げられる。このうち、本発明の抗体としては、ヒトＣＣＬ１５により誘導されるヒトＣＣＲ１発現細胞の遊走を阻害する抗体であることが好ましい。 Examples of the antibody of the present invention include antibodies that inhibit various reactions associated with human CCR1 activation by human CCL15. Specific examples of the antibody of the present invention include, for example, CCR1-dependent signaling in human CCR1-expressing cells by human CCL15, activation of PLC, increase in intracellular calcium ion concentration, activation of NF-κB and CCR1. Examples thereof include an antibody that inhibits at least one reaction selected from the migration of expressed cells. Among these, the antibody of the present invention is preferably an antibody that inhibits migration of human CCR1-expressing cells induced by human CCL15.

　本発明の抗体としては、ヒトＣＣＬ１５によるヒトＣＣＲ１活性化に伴う上記の反応について、ＣＣＬ１５のみを添加し、抗体を添加しないコントロールと比べて、好ましくは、５％以上、１０％以上、２０％以上、３０％以上、４０％以上、５０％以上、６０％以上、７０％以上、８０％以上、９０％以上阻害する抗体が挙げられる。ヒトＣＣＬ１５の濃度は、測定系に応じてヒトＣＣＬ１５添加時の上記反応の活性が最大値となる濃度に適宜調整することができる。例えば本願実施例に記載する方法でＣＣＲ１発現細胞の遊走を測定する場合には、ＣＣＬ１５の濃度は１ｎｇ／ｍＬであることが好ましい。また、本発明の抗体の濃度についても測定系により適宜調整することができる。例えば本実施例に記載する方法でＣＣＲ１発現細胞の遊走を測定する場合には、本発明の抗体濃度は、０．３μｇ／ｍＬ以上、好ましくは１μｇ／ｍＬ以上、より好ましくは３μｇ／ｍＬ以上、最も好ましくは１０μｇ／ｍＬ以上であることが挙げられる。 The antibody of the present invention is preferably 5% or more, 10% or more, or 20% or more, compared to the control in which only CCL15 is added and no antibody is added, for the above-mentioned reaction accompanying human CCR1 activation by human CCL15. , 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more. The concentration of human CCL15 can be appropriately adjusted to a concentration at which the activity of the above reaction when human CCL15 is added becomes a maximum value depending on the measurement system. For example, when the migration of CCR1-expressing cells is measured by the method described in the Examples of the present application, the concentration of CCL15 is preferably 1 ng / mL. The concentration of the antibody of the present invention can also be adjusted as appropriate by the measurement system. For example, when measuring the migration of CCR1-expressing cells by the method described in this example, the antibody concentration of the present invention is 0.3 μg / mL or more, preferably 1 μg / mL or more, more preferably 3 μg / mL or more, Most preferably, it is 10 μg / mL or more.

　本発明において、ヒトＣＣＬ１５は、ＣＣＲ１を活性化するものであれば、全長ＣＣＬ１５および活性化ヒトＣＣＬ１５のいずれのＣＣＬ１５であってもいい。 In the present invention, human CCL15 may be either full-length CCL15 or activated human CCL15 as long as it activates CCR1.

　ヒトＣＣＲ１発現細胞としては、ヒトＣＣＲ１を発現している細胞であればいずれの細胞でもよく、例えば、ヒト細胞、ヒト細胞株およびヒトＣＣＲ１強制発現株などが挙げられる。 The human CCR1-expressing cell may be any cell as long as it expresses human CCR1, and examples thereof include human cells, human cell lines, and human CCR1 forced expression lines.

　ヒトＣＣＲ１を発現しているヒト細胞としては、例えば、好中球、好酸球、好塩基球、単球、マクロファージ、樹状細胞、ＮＫ細胞、Ｔ細胞、Ｂ細胞、未成熟骨髄球（ｉＭＣ）および骨髄由来免疫抑制性細胞（ＭＤＳＣ）などが挙げられる。 Examples of human cells expressing human CCR1 include neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, NK cells, T cells, B cells, immature myelocytes (iMC). ) And bone marrow-derived immunosuppressive cells (MDSC).

　ヒトＣＣＲ１の細胞外領域としては、ヒトＣＣＲ１のアミノ酸配列のＮ末端から１～３１番目のアミノ酸配列を含むＮ末端領域、９７～１０３番目のアミノ酸配列を含む細胞外ループ１領域、１７２～１９５番目のアミノ酸配列を含む細胞外ループ２領域および２６６～２７８番目のアミノ酸配列を含む細胞外ループ３領域が挙げられる［Cell 72.3(1993):415-425］。 The extracellular region of human CCR1 includes an N-terminal region containing the 1st to 31st amino acid sequence from the N-terminal of the amino acid sequence of human CCR1, an extracellular loop 1 region containing the 97th to 103rd amino acid sequence, and 172nd to 195th And the extracellular loop 2 region containing the amino acid sequence of 266 to 278 [Cell 72.3 (1993): 415-425].

　Ｎ末端領域、細胞外ループ１領域、細胞外ループ２領域および細胞外ループ３領域として、具体的には、それぞれ配列番号２のアミノ酸配列における１～３１番目、９７～１０３番目、１７２～１９５番目および２６６～２７８番目のアミノ酸配列が挙げられる。 As the N-terminal region, extracellular loop 1 region, extracellular loop 2 region and extracellular loop 3 region, specifically, the 1st to 31st positions, the 97th to 103rd positions, and the 172th to 195th positions in the amino acid sequence of SEQ ID NO: 2, respectively And amino acid sequences from 266 to 278.

　本発明の抗体としては、上記のヒトＣＣＲ１の細胞外領域に結合する抗体であればいかなるものでもよいが、ヒトＣＣＲ１の細胞外ループ２領域のアミノ酸配列のうち、少なくとも１つのアミノ酸残基に結合する抗体であることが好ましい。かかる抗体としては、配列番号２のアミノ酸配列の１７２～１９５番目のアミノ酸配列のうち、少なくとも１つのアミノ酸残基に結合する抗体などが挙げられる。 The antibody of the present invention may be any antibody that binds to the extracellular region of human CCR1 described above, but binds to at least one amino acid residue in the amino acid sequence of the extracellular loop 2 region of human CCR1. It is preferable that it is an antibody. Examples of such an antibody include an antibody that binds to at least one amino acid residue in the amino acid sequence of positions 172 to 195 of the amino acid sequence of SEQ ID NO: 2.

　また、本発明の抗体としてより具体的には、下記（ａ）～（ｌ）から選ばれるいずれか一つの抗体が挙げられる。
（ａ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号６９、７０および７１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７２、７３および７４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｂ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７５、７６および７７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７８、７９および８０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｃ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８１、８２および８３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８４、８５および８６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｄ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８７、８８および８９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９０、９１および９２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｅ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９３、９４および９５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９６、９７および９８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｆ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９９、１００および１０１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０２、１０３および１０４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｇ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０５、１０６および１０７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０８、１０９および１１０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｈ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１１、１１２および１１３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１４、１１５および１１６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｉ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１７、１１８および１１９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１２０、１２１および１２２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｊ）前記（ａ）～（ｉ）に記載の少なくとも一つの抗体と、ヒトＣＣＲ１への結合について競合する抗体。
（ｋ）前記（ａ）～（ｉ）に記載のいずれか一つの抗体が結合するエピトープを含むエピトープに結合する抗体。
（ｌ）前記（ａ）～（ｉ）に記載のいずれか一つの抗体が結合するエピトープと同じエピトープに結合する抗体。 More specifically, the antibody of the present invention includes any one antibody selected from the following (a) to (l).
(A) The amino acid sequences of CDRs 1 to 3 of VH include the amino acid sequences described in SEQ ID NOs: 69, 70 and 71, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 72 The antibody which is an amino acid sequence containing the amino acid sequence described in 73 and 74.
(B) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 75, 76, and 77, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 78, respectively. 79. An antibody having an amino acid sequence comprising the amino acid sequence described in 79 and 80.
(C) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 81, 82 and 83, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 84 , 85 and 86, an antibody having an amino acid sequence comprising the amino acid sequence described in 85 and 86.
(D) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 87, 88, and 89, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 90 , 91 and 92, an antibody having an amino acid sequence comprising the amino acid sequence described in 91 and 92.
(E) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 93, 94 and 95, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 96, respectively. The antibody which is an amino acid sequence containing the amino acid sequence described in 97 and 98.
(F) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 99, 100 and 101, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 102. , 103 and 104. An antibody having an amino acid sequence comprising the amino acid sequence described in 103 and 104.
(G) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 105, 106 and 107, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 108 , 109 and 110, an antibody having an amino acid sequence comprising the amino acid sequence described in 109 and 110.
(H) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 111, 112, and 113, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 114, respectively. , 115 and 116, an antibody having an amino acid sequence comprising the amino acid sequence described in 115 and 116.
(I) The amino acid sequences of CDRs 1 to 3 of VH are amino acid sequences including the amino acid sequences described in SEQ ID NOs: 117, 118, and 119, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 120, respectively. , 121 and 122, which is an amino acid sequence comprising the amino acid sequence described in 121 and 122.
(J) An antibody that competes with at least one antibody described in (a) to (i) for binding to human CCR1.
(K) An antibody that binds to an epitope including the epitope to which any one of the antibodies (a) to (i) binds.
(L) An antibody that binds to the same epitope as that to which any one of the antibodies (a) to (i) binds.

　本発明の抗体としては、上記（ａ）～（ｉ）に記載されるいずれか一つの抗体のＶＨのＣＤＲ１～３およびＶＬのＣＤＲ１～３のアミノ酸配列と、それぞれ９０％以上の相同性を示す抗体のＶＨのＣＤＲ１～３およびＶＬのＣＤＲ１～３のアミノ酸配列を有する抗体を含む。９０％以上の相同性とは、具体的には９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％および９９％の相同性などが挙げられる。 The antibody of the present invention shows 90% or more homology with the VH CDR1 to 3 and VL CDR1 to 3 amino acid sequences of any one of the antibodies described in (a) to (i) above. It includes an antibody having the amino acid sequences of CDR1-3 of VH and CDR1-3 of VL. The 90% or higher homology specifically includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology.

　本発明において、上記（ａ）～（ｉ）に記載される抗体の一態様としては、それぞれマウス抗ヒトＣＣＲ１モノクローナル抗体ＫＭ５９０７抗体、ＫＭ５９０８抗体、ＫＭ５９０９抗体、ＫＭ５９１１抗体、ＫＭ５９１５抗体、ＫＭ５９１６抗体、ＫＭ５９５４抗体、ＫＭ５９５５抗体およびＫＭ５９５６抗体が挙げられる。また、上記（ａ）～（ｉ）に記載される抗体の一態様として、それぞれ抗ヒトＣＣＲ１キメラ抗体ｃｈＫＭ５９０７抗体、ｃｈＫＭ５９０８抗体、ｃｈＫＭ５９０９抗体、ｃｈＫＭ５９１１抗体、ｃｈＫＭ５９１５抗体、ｃｈＫＭ５９１６抗体、ｃｈＫＭ５９５４抗体、ｃｈＫＭ５９５５抗体およびｃｈＫＭ５９５６抗体が挙げられる。上記（ａ）～（ｉ）に記載される抗体の一態様として、他には上記（ａ）～（ｉ）に記載されるいずれか一つの抗体のＶＨのＣＤＲ１～３およびＶＬのＣＤＲ１～３のアミノ酸配列を有するヒト化抗体およびヒト抗体などが挙げられる。 In the present invention, one embodiment of the antibodies described in the above (a) to (i) includes mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, and KM5954 antibody, respectively. KM5955 antibody and KM5956 antibody. Further, as one aspect of the antibodies described in (a) to (i) above, the anti-human CCR1 chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and An example is the chKM5956 antibody. As an embodiment of the antibody described in (a) to (i) above, the CDR1 to V3 of VH and the CDR1 to 3 of VL of any one of the antibodies described in (a) to (i) above. And humanized antibodies having the amino acid sequence of:

　本発明の上記（ｊ）の抗体とは、上記（ａ）～（ｉ）に記載の抗体を第１抗体とした時に、該第１抗体とヒトＣＣＲ１との結合を阻害する第２抗体をいう。本発明の上記（ｋ）の抗体とは、上記（ａ）～（ｉ）に記載の抗体を第１抗体、及び第１抗体が結合するエピトープを第１エピトープとした場合、当該第１エピトープを含む、第２エピトープに結合する第２抗体をいう。また、本発明の上記（ｌ）の抗体とは、上記（ａ）～（ｉ）に記載の抗体を第１抗体、及び第１抗体が結合するエピトープを第１エピトープとした場合に、当該第１エピトープに結合する第２抗体をいう。 The antibody (j) of the present invention refers to a second antibody that inhibits the binding between the first antibody and human CCR1 when the antibodies described in (a) to (i) above are used as the first antibody. . The antibody of (k) of the present invention is the antibody described in (a) to (i) above when the first antibody is used, and the epitope to which the first antibody binds is the first epitope. A second antibody that binds to a second epitope. In addition, the antibody of (1) of the present invention refers to the antibody described in (a) to (i) above when the first antibody is used, and the epitope to which the first antibody binds is used as the first epitope. A second antibody that binds to one epitope.

　また、本発明の抗体として、具体的には、下記（ａ）～（ｉ）から選ばれるいずれか一つの抗体も挙げられる。
（ａ）ＶＨのアミノ酸配列が配列番号５１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｂ）ＶＨのアミノ酸配列が配列番号５３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｃ）ＶＨのアミノ酸配列が配列番号５５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｄ）ＶＨのアミノ酸配列が配列番号５７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｅ）ＶＨのアミノ酸配列が配列番号５９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｆ）ＶＨのアミノ酸配列が配列番号６１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｇ）ＶＨのアミノ酸配列が配列番号６３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｈ）ＶＨのアミノ酸配列が配列番号６５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｉ）ＶＨのアミノ酸配列が配列番号６７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。 The antibody of the present invention also specifically includes any one antibody selected from the following (a) to (i).
(A) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 51, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 52.
(B) An antibody wherein the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 53, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 54.
(C) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 55, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 56.
(D) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 57, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 58.
(E) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 59, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 60.
(F) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 61, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 62.
(G) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 63, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 64.
(H) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 65, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 66.
(I) An antibody wherein the amino acid sequence of VH is an amino acid sequence containing the amino acid sequence set forth in SEQ ID NO: 67, and the amino acid sequence of VL is an amino acid sequence containing the amino acid sequence set forth in SEQ ID NO: 68.

　本発明の抗体としては、上記（ａ）～（ｉ）に記載されるいずれか一つの抗体のＶＨおよびＶＬのアミノ酸配列と、それぞれ９０％以上の相同性を示す抗体のＶＨおよびＶＬのアミノ酸配列を有する抗体を含む。９０％以上の相同性とは、具体的には９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％および９９％の相同性などが挙げられる。 The antibody of the present invention includes the VH and VL amino acid sequences of any one of the antibodies described in (a) to (i) above, and the VH and VL amino acid sequences of antibodies that exhibit 90% or more homology, respectively. An antibody having The 90% or higher homology specifically includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology.

　本発明において、上記（ａ）～（ｉ）に記載される抗体の一態様としては、それぞれマウス抗ヒトＣＣＲ１モノクローナル抗体ＫＭ５９０７抗体、ＫＭ５９０８抗体、ＫＭ５９０９抗体、ＫＭ５９１１抗体、ＫＭ５９１５抗体、ＫＭ５９１６抗体、ＫＭ５９５４抗体、ＫＭ５９５５抗体およびＫＭ５９５６抗体が挙げられる。 In the present invention, one embodiment of the antibodies described in the above (a) to (i) includes mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, and KM5954 antibody, respectively. KM5955 antibody and KM5956 antibody.

　また、上記（ａ）～（ｉ）に記載される抗体の一態様として、それぞれ抗ヒトＣＣＲ１キメラ抗体ｃｈＫＭ５９０７抗体、ｃｈＫＭ５９０８抗体、ｃｈＫＭ５９０９抗体、ｃｈＫＭ５９１１抗体、ｃｈＫＭ５９１５抗体、ｃｈＫＭ５９１６抗体、ｃｈＫＭ５９５４抗体、ｃｈＫＭ５９５５抗体およびｃｈＫＭ５９５６抗体が挙げられる。 Further, as one aspect of the antibodies described in (a) to (i) above, the anti-human CCR1 chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and An example is the chKM5956 antibody.

　本発明においてヒトＣＣＲ１としては、配列番号２に記載のアミノ酸配列若しくはＮＣＢＩアクセッション番号ＮＰ＿００１２８６のアミノ酸配列を含むポリペプチド、配列番号２に記載のアミノ酸配列若しくはＮＣＢＩアクセッション番号ＮＰ＿００１２８６のアミノ酸配列において１つ以上のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつヒトＣＣＲ１の機能を有するポリペプチド、あるいは配列番号２に記載のアミノ酸配列若しくはＮＣＢＩアクセッション番号ＮＰ＿００１２８６のアミノ酸配列と６０％以上、好ましくは８０％以上、さらに好ましくは９０％以上、最も好ましくは９５％以上の相同性を有するアミノ酸配列から成り、かつヒトＣＣＲ１の機能を有するポリペプチドなどが挙げられる。 In the present invention, human CCR1 is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286, one in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286. A polypeptide having the amino acid sequence in which the above amino acids are deleted, substituted or added and having the function of human CCR1, or the amino acid sequence described in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286, and 60% or more, Preferred examples include a polypeptide having an amino acid sequence having a homology of 80% or more, more preferably 90% or more, and most preferably 95% or more, and having the function of human CCR1.

　配列番号２に記載のアミノ酸配列またはＮＣＢＩアクセッション番号ＮＰ＿００１２８６で示されるアミノ酸配列において１つ以上のアミノ酸が欠失、置換または付加されたアミノ酸配列を有するポリペプチドは、部位特異的変異導入法［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)］などを用いて、例えば配列番号２のアミノ酸配列を含むポリペプチドをコードするＤＮＡに、部位特異的変異を導入することにより得ることができる。 A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown by NCBI accession number NP — 001286 is a site-directed mutagenesis method [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 (tl), Proc. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)] For example, by introducing a site-specific mutation into DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

　欠失、置換または付加されるアミノ酸の数は特に限定されないが、好ましくは１個～数十個、例えば、１～２０個、より好ましくは１個～数個、例えば、１～５個のアミノ酸である。 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.

　ヒトＣＣＲ１をコードする遺伝子としては、配列番号１に記載の塩基配列、およびＮＣＢＩアクセッション番号ＮＭ＿００１２９５の塩基配列が挙げられる。配列番号１に記載の塩基配列若しくはＮＭ＿００１２９５の塩基配列において、１以上の塩基が欠失、置換または付加された塩基配列から成り、かつヒトＣＣＲ１の機能を有するポリペプチドをコードするＤＮＡを含む遺伝子、配列番号１に記載の塩基配列若しくはＮＭ＿００１２９５の塩基配列と少なくとも６０％以上の相同性を有する塩基配列、好ましくは８０％以上の相同性を有する塩基配列、さらに好ましくは９５％以上の相同性を有する塩基配列から成り、かつヒトＣＣＲ１の機能を有するポリペプチドをコードするＤＮＡを含む遺伝子または配列番号１に記載の塩基配列、若しくはＮＭ＿００１２９５の塩基配列を含むＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡから成り、かつヒトＣＣＲ１の機能を有するポリペプチドをコードする遺伝子なども本発明のヒトＣＣＲ１をコードする遺伝子に含有される。 Examples of the gene encoding human CCR1 include the nucleotide sequence set forth in SEQ ID NO: 1 and the nucleotide sequence of NCBI accession number NM_001295. A gene comprising a DNA encoding a polypeptide consisting of a base sequence described in SEQ ID NO: 1 or a base sequence in which one or more bases are deleted, substituted or added in the base sequence of NM_001295 and having the function of human CCR1; A nucleotide sequence having at least 60% homology with the nucleotide sequence set forth in SEQ ID NO: 1 or NM_001295, preferably having a homology of 80% or more, more preferably having a homology of 95% or more DNA that hybridizes under stringent conditions with a gene comprising a nucleotide sequence and comprising a DNA encoding a polypeptide having the function of human CCR1, or the nucleotide sequence set forth in SEQ ID NO: 1, or a DNA comprising the nucleotide sequence of NM_001295 And has the function of human CCR1 Such as a gene encoding Ripepuchido also contained in the gene coding for the human CCR1 of the present invention.

　ストリンジェントな条件下でハイブリダイズするＤＮＡとしては、配列番号１に記載の塩基配列またはＮＭ＿００１２９５の塩基配列を含むＤＮＡをプローブに用いた、コロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法、サザンブロット・ハイブリダイゼーション法またはＤＮＡマイクロアレイ法などにより得られるハイブリダイズ可能なＤＮＡのことをいう。 As DNA that hybridizes under stringent conditions, a colony hybridization method, a plaque hybridization method, a Southern blotting method using a DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence of NM_001295 as a probe, This refers to hybridizable DNA obtained by a hybridization method or a DNA microarray method.

　具体的には、ハイブリダイズしたコロニーあるいはプラーク由来のＤＮＡ、または該配列を有するＰＣＲ産物若しくはオリゴＤＮＡを固定化したフィルター若しくはスライドガラスを用いて、０．７～１．０ｍｏｌ／Ｌの塩化ナトリウム存在下、６５℃でハイブリダイゼーション法［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, Oxford University, (1995)］を行った後、０．１～２倍濃度のＳＳＣ溶液（１倍濃度のＳＳＣ溶液の組成は、１５０ｍｍｏｌ／Ｌ塩化ナトリウム、１５ｍｍｏｌ／Ｌクエン酸ナトリウムよりなる）を用い、６５℃条件下でフィルターまたはスライドガラスを洗浄することにより同定できるＤＮＡを挙げることができる。 Specifically, 0.7 to 1.0 mol / L of sodium chloride is present using a hybridized colony or plaque-derived DNA or a filter or slide glass on which a PCR product or oligo DNA having the sequence is immobilized. Below, hybridization method at 65 ° C [Molecular lon Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), DNA Cloningique 1: , A Practical Approach, Second Edition, Oxford University, (1995)], 0.1 to 2 times concentrated SSC solution (the composition of 1 times concentrated SSC solution is 150 mmol / L sodium chloride, 15 mmol / L Wash the filter or glass slide at 65 ° C using sodium citrate) The DNA which can be identified by doing is mentioned.

　ハイブリダイズ可能なＤＮＡとしては配列番号１に記載の塩基配列、またはＮＭ＿００１２９５の塩基配列と少なくとも６０％以上の相同性を有するＤＮＡ、好ましくは８０％以上の相同性を有するＤＮＡ、さらに好ましくは９５％以上の相同性を有するＤＮＡを挙げることができる The hybridizable DNA is DNA having at least 60% homology with the nucleotide sequence of SEQ ID NO: 1 or NM_001295, preferably DNA having 80% or more homology, more preferably 95% DNA having the above homology can be mentioned

　真核生物のタンパク質をコードする遺伝子の塩基配列には、しばしば遺伝子の多型が認められる。本発明において用いられる遺伝子に、このような多型によって塩基配列に小規模な変異を生じた遺伝子も本発明のヒトＣＣＲ１をコードする遺伝子に含有される。 In the base sequence of a gene encoding a eukaryotic protein, a polymorphism of the gene is often observed. 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 included in the gene encoding human CCR1 of the present invention.

　本発明における相同性の数値は、特に明示した場合を除き、当業者に公知の相同性検索プログラムを用いて算出される数値であってよいが、塩基配列については、ＢＬＡＳＴ［J. Mol. Biol., 215, 403 (1990)］においてデフォルトのパラメータを用いて算出される数値など、アミノ酸配列については、ＢＬＡＳＴ２［Nucleic Acids Res.,25, 3389 (1997)、Genome Res., 7, 649 (1997)、http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.htmL］においてデフォルトのパラメータを用いて算出される数値などが挙げられる。 Unless otherwise specified, 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, but for the base sequence, 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.htmL], numerical values calculated using default parameters.

　デフォルトのパラメータとしては、Ｇ（Ｃｏｓｔ　ｔｏ　ｏｐｅｎ　ｇａｐ）が塩基配列の場合は５、アミノ酸配列の場合は１１、－Ｅ（Ｃｏｓｔ　ｔｏ　ｅｘｔｅｎｄ　ｇａｐ）が塩基配列の場合は２、アミノ酸配列の場合は１、－ｑ（Ｐｅｎａｌｔｙ　ｆｏｒ　ｎｕｃｌｅｏｔｉｄｅ　ｍｉｓｍａｔｃｈ）が－３、－ｒ（ｒｅｗａｒｄ　ｆｏｒ　ｎｕｃｌｅｏｔｉｄｅ　ｍａｔｃｈ）が１、－ｅ（ｅｘｐｅｃｔ　ｖａｌｕｅ）が１０、－Ｗ（ｗｏｒｄｓｉｚｅ）が塩基配列の場合は１１残基、アミノ酸配列の場合は３残基、－ｙ［Ｄｒｏｐｏｆｆ（Ｘ）ｆｏｒ　ｂｌａｓｔ　ｅｘｔｅｎｓｉｏｎｓ　ｉｎ　ｂｉｔｓ］がｂｌａｓｔｎの場合は２０、ｂｌａｓｔｎ以外のプログラムでは７、－Ｘ（Ｘ　ｄｒｏｐｏｆｆ　ｖａｌｕｅ　ｆｏｒ　ｇａｐｐｅｄ　ａｌｉｇｎｍｅｎｔ　ｉｎ　ｂｉｔｓ）が１５および－Ｚ（ｆｉｎａｌ　Ｘ　ｄｒｏｐｏｆｆ　ｖａｌｕｅ　ｆｏｒ　ｇａｐｐｅｄ　ａｌｉｇｎｍｅｎｔ　ｉｎ　ｂｉｔｓ）がｂｌａｓｔｎの場合は５０、ｂｌａｓｔｎ以外のプログラムでは２５である（http://www.ncbi.nlm.nih.gov/blast/htmL/blastcgihelp.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, amino acid sequence 3 residues, -y [Dropoff (X) for blast extensions in bits] is 20 when blastn, 7 for programs other than blastn, -X (X dropoff value for If the grouped alignment in bits is 15 and -Z (final X dropoff value for grouped alignment in bits) is blastn, it is 50, and 25 for programs other than blastn (http: //www.ncbi.nlmnih.vlm. /blast/htmL/blastcgihelp.htmL).

　配列番号２に記載のアミノ酸配列またはＮＣＢＩアクセッション番号ＮＰ＿００１２８６のアミノ酸配列の部分配列を含むポリペプチドは、当業者に公知の方法によって作製することができる。具体的には、配列番号２のアミノ酸配列をコードするＤＮＡの一部を欠失させ、これを含む発現ベクターを導入した形質転換体を培養することにより作製することができる。また、上記と同様の方法により、配列番号２に記載のアミノ酸配列またはＮＣＢＩアクセッション番号ＮＰ＿００１２８６のアミノ酸配列において１以上のアミノ酸が欠失、置換または付加されたアミノ酸配列を有するポリペプチドを得ることができる。さらに、配列番号２に記載のアミノ酸配列若しくはＮＣＢＩアクセッション番号ＮＰ＿００１２８６のアミノ酸配列からなるポリペプチド、または配列番号２に記載のアミノ酸配列若しくはＮＣＢＩアクセッション番号ＮＰ＿００１２８６のアミノ酸配列において１以上のアミノ酸が欠失、置換あるいは付加されたアミノ酸配列を有するポリペプチドは、フルオレニルメチルオキシカルボニル（Ｆｍｏｃ）法、ｔ－ブチルオキシカルボニル（ｔＢｏｃ）法などの化学合成法によって製造することもできる。 A polypeptide containing a partial sequence of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286 can be prepared by methods known to those skilled in the art. Specifically, it can be prepared by deleting a part of DNA encoding the amino acid sequence of SEQ ID NO: 2 and culturing a transformant into which an expression vector containing the DNA is introduced. In addition, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286 can be obtained by the same method as described above. it can. Furthermore, one or more amino acids are deleted in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286, or the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_001286. A polypeptide having a substituted or added amino acid sequence can also be produced by a chemical synthesis method such as the fluorenylmethyloxycarbonyl (Fmoc) method or the t-butyloxycarbonyl (tBoc) method.

　本発明の抗体としては、ポリクローナル抗体、モノクローナル抗体およびオリゴクローナル抗体のいずれの抗体をも包含する。ポリクローナル抗体とは、異なるクローンの抗体産生細胞が分泌する抗体分子の集団をいう。モノクローナル抗体とは、単一クローンの抗体産生細胞が分泌する抗体であり、ただ一つのエピトープ（抗原決定基ともいう）を認識し、モノクローナル抗体を構成するアミノ酸配列（一次配列）が均一である抗体をいう。オリゴクローナル抗体とは、複数の異なるモノクローナル抗体を混合した抗体分子の集団をいう。 The antibody of the present invention includes any of polyclonal antibodies, monoclonal antibodies and oligoclonal antibodies. Polyclonal antibody refers to a population of antibody molecules secreted by antibody-producing cells of different clones. A monoclonal antibody is an antibody that is secreted by an antibody-producing cell of a single clone, recognizes only one epitope (also called an antigenic determinant), and has a uniform amino acid sequence (primary sequence) that constitutes a monoclonal antibody. Say. An oligoclonal antibody refers to a population of antibody molecules in which a plurality of different monoclonal antibodies are mixed.

　本発明におけるモノクロ－ナル抗体としては、ハイブリドーマにより産生される抗体、または抗体遺伝子を含む発現ベクターで形質転換した形質転換体により産生される遺伝子組換え抗体をあげることができる。 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.

　エピトープとは、モノクローナル抗体が認識し、結合する単一のアミノ酸配列、アミノ酸配列からなる立体構造、翻訳後修飾により修飾されたアミノ酸配列および該アミノ酸配列からなる立体構造などが挙げられる。 Examples of 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.

　翻訳後修飾により修飾されたアミノ酸配列としては、糖鎖がＯＨ置換基を有するＴｙｒおよびＳｅｒに結合したＯ結合型糖鎖、ＮＨ２置換基を有するＧｌｎおよびＡｓｎに結合したＮ結合型糖鎖ならびに硫酸分子がＯＨ置換基を有するＴｙｒに結合した硫酸基などが結合したアミノ酸配列が挙げられる。 The amino acid sequence modified by post-translational modification includes an O-linked sugar chain bonded to Tyr and Ser having an OH substituent, an N-linked sugar chain bonded to Gln and Asn having an NH 2 substituent, and Examples thereof include an amino acid sequence in which a sulfate group or the like in which a sulfuric acid molecule is bonded to Tyr having an OH substituent.

　本発明の抗体がヒトＣＣＲ１の細胞外領域に結合することは、ヒトＣＣＲ１発現細胞に対する本発明の抗体の結合性を、ＥＬＩＳＡ、フローサイトメトリーおよび表面プラズモン共鳴法などを用いて測定することにより確認することができる。また、公知の免疫学的検出法［Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996)、Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)、単クローン抗体実験マニュアル、講談社サイエンティフィック（１９８７）］などを組み合わせて確認することもできる。 The binding of the antibody of the present invention to the extracellular region of human CCR1 was confirmed by measuring the binding of the antibody of the present invention to human CCR1-expressing cells using ELISA, flow cytometry, surface plasmon resonance, etc. can do. Also known immunological detection methods [Monoclonal-Antibodies-Principles-and-practice, Third-edition, Academic-Press- (1996), Antibodies-A-Laboratory-Manual, Cold-Spring-Harbor-Laboratory- (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] etc. can also be confirmed.

　本発明の抗体が結合するヒトＣＣＲ１のアミノ酸残基またはエピトープは、ヒトＣＣＲ１の一部のドメインを欠失させた欠損体、他のタンパク質由来のドメインと置換させた変異体およびヒトＣＣＲ１の部分ペプチド断片等を用いて抗体の結合実験を行うことにより決定することができる。また、上記欠損体または変異体の発現細胞を用いて抗体の結合実験を行うこともできる。 The amino acid residue or epitope of human CCR1 to which the antibody of the present invention binds is a deletion in which a partial domain of human CCR1 is deleted, a mutant in which a domain derived from another protein is substituted, and a partial peptide of human CCR1 It can be determined by conducting antibody binding experiments using fragments or the like. Antibody binding experiments can also be performed using cells expressing the above-mentioned mutants or mutants.

　または、本発明の抗体が結合するヒトＣＣＲ１のアミノ酸残基またはエピトープは、タンパク質分解酵素にて消化したヒトＣＣＲ１のペプチド断片に本発明の抗体を添加し、既知の質量分析法を用いてエピトープマッピングを行うことによっても決定することができる。 Alternatively, the amino acid residue or epitope of human CCR1 to which the antibody of the present invention binds can be epitope mapped using a known mass spectrometry method by adding the antibody of the present invention to a peptide fragment of human CCR1 digested with a proteolytic enzyme. It can also be determined by performing.

　本発明の抗体がヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害することは、例えば、ヒトＣＣＲ１発現細胞内でのＣＣＲ１依存的なシグナル伝達、ＰＬＣの活性化、細胞内のカルシウムイオン濃度上昇、ＮＦ－κＢ活性化、またはヒトＣＣＲ１発現細胞の遊走を指標として確認することができる。 That the antibody of the present invention inhibits the activation of human CCR1 by human CCL15 is, for example, CCR1-dependent signaling in human CCR1-expressing cells, activation of PLC, increase in intracellular calcium ion concentration, NF- κB activation or migration of human CCR1-expressing cells can be confirmed as an index.

　細胞の遊走は、以下に記載するケモタキシスアッセイを用いて測定することができる。例えば、ケモタキシスアッセイチャンバーの上部にヒトＣＣＲ１発現細胞を、該チャンバーの下部に１）培地またはＤＰＢＳなどのネガティブコントロール、２）ヒトＣＣＬ１５および３）ヒトＣＣＬ１５と本発明の抗体をそれぞれ添加し、一定時間培養した後に、該チャンバー下部に存在するヒトＣＣＲ１発現細胞数を適当な方法で測定する。得られた結果について、ヒトＣＣＬ１５を添加した時の細胞数が、培地を添加した時の細胞数よりも増加する条件下で、ヒトＣＣＬ１５と本発明の抗体を添加した時の細胞数が、ヒトＣＣＬ１５を添加した時の細胞数よりも減少していれば、本発明の抗体は、ヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害すると判定することができる。 Cell migration can be measured using the chemotaxis assay described below. For example, human CCR1-expressing cells are added to the upper part of the chemotaxis assay chamber, 1) a negative control such as a medium or DPBS, 2) human CCL15 and 3) human CCL15 and the antibody of the present invention are added to the lower part of the chamber, After culturing for a certain time, the number of human CCR1-expressing cells present in the lower part of the chamber is measured by an appropriate method. Regarding the obtained results, the number of cells when the human CCL15 and the antibody of the present invention were added under the condition that the number of cells when the human CCL15 was added was larger than the number of cells when the medium was added. If the number of cells is smaller than that when CCL15 is added, it can be determined that the antibody of the present invention inhibits the activation of human CCR1 by human CCL15.

　また、本発明の抗体が、ヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害することは、ヒトＣＣＲ１発現細胞内でのカルシウムイオン濃度の変化を指標として確認することができる。細胞内のカルシウム濃度の変化は、公知の方法で測定することができ、例えば、細胞内Ｃａ測定キット（Ｗａｋｏ社製）などを使用し、添付のプロトコールに従い測定することができる。 In addition, the inhibition of the activation of human CCR1 by human CCL15 by the antibody of the present invention can be confirmed by using a change in the calcium ion concentration in human CCR1-expressing cells as an index. The change in intracellular calcium concentration can be measured by a known method, for example, using an intracellular Ca measurement kit (manufactured by Wako) and the like, and can be measured according to the attached protocol.

　確認方法としては、例えばヒトＣＣＲ１発現細胞に、１）培地またはＤＰＢＳなどのネガティブコントロール、２）ヒトＣＣＬ１５および３）ヒトＣＣＬ１５と本発明の抗体をそれぞれ添加した時の細胞内カルシウム濃度の変化を、上記の方法に従い測定する。ヒトＣＣＬ１５を添加した時の細胞内カルシウムイオン濃度が、培地を添加した時の細胞内カルシウムイオン濃度よりも増加する条件下で、ヒトＣＣＬ１５と本発明の抗体を添加した時の細胞内カルシウムイオン濃度が、ヒトＣＣＬ１５を添加した時の細胞内カルシウムイオン濃度よりも減少していれば、本発明の抗体は、ヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害すると判定することができる。 As a confirmation method, for example, changes in intracellular calcium concentration when human CCR1-expressing cells were added with 1) a negative control such as medium or DPBS, 2) human CCL15 and 3) human CCL15 and the antibody of the present invention, respectively. Measure according to the above method. Intracellular calcium ion concentration when human CCL15 and the antibody of the present invention are added under conditions where the intracellular calcium ion concentration when human CCL15 is added is higher than the intracellular calcium ion concentration when medium is added However, if the intracellular calcium ion concentration when human CCL15 is added is decreased, the antibody of the present invention can be determined to inhibit the activation of human CCR1 by human CCL15.

　抗体分子はイムノグロブリン（以下、Ｉｇと表記する）とも称され、ヒト抗体は、分子構造の違いに応じて、ＩｇＡ１、ＩｇＡ２、ＩｇＤ、ＩｇＥ、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４およびＩｇＭのアイソタイプに分類される。アミノ酸配列の相同性が比較的高いＩｇＧ１、ＩｇＧ２、ＩｇＧ３およびＩｇＧ４を総称してＩｇＧともいう。 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.

　抗体分子は重鎖（Ｈｅａｖｙ　ｃｈａｉｎ、以下Ｈ鎖と記す）および軽鎖（Ｌｉｇｈｔ　ｃｈａｉｎ、以下Ｌ鎖と記す）と呼ばれるポリペプチドより構成される。また、Ｈ鎖はＮ末端側よりＶＨ、Ｈ鎖定常領域（ＣＨとも表記される）、Ｌ鎖はＮ末端側よりＶＬ、Ｌ鎖定常領域（ＣＬとも表記される）の各領域により、それぞれ構成される。ＣＨは各サブクラスごとに、α、δ、ε、γおよびμ鎖がそれぞれ知られている。ＣＨはさらに、Ｎ末端側よりＣＨ１ドメイン、ヒンジドメイン、ＣＨ２ドメイン、ＣＨ３ドメインの各ドメインにより構成される。ドメインとは、抗体分子の各ポリペプチドを構成する機能的な構造単位をいう。また、ＣＨ２ドメインとＣＨ３ドメインを併せてＦｃ領域または単にＦｃという。ＣＬは、Ｃλ鎖およびＣκ鎖が知られている。 Antibody molecules are composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (light chains). The H chain is composed of VH and H chain constant regions (also referred to as CH) from the N-terminal side, and the L chain is composed of VL and L chain constant regions (also referred to as CL) from the N-terminal side. Is done. As for CH, α, δ, ε, γ, and μ chains are known 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. As for CL, C λ chain and C κ chain are known.

　本発明におけるＣＨ１ドメイン、ヒンジドメイン、ＣＨ２ドメイン、ＣＨ３ドメインおよびＦｃ領域は、ＥＵインデックス［Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)］により、Ｎ末端からのアミノ酸残基の番号で特定することができる。具体的には、ＣＨ１はＥＵインデックス１１８～２１５番のアミノ酸配列、ヒンジはＥＵインデックス２１６～２３０番のアミノ酸配列、ＣＨ２はＥＵインデックス２３１～３４０番のアミノ酸配列、ＣＨ３はＥＵインデックス３４１～４４７番のアミノ酸配列とそれぞれ特定される。 The CH1 domain, hinge domain, CH2 domain, CH3 domain and Fc region in the present invention are identified from the N-terminus by the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. It can be specified by the amino acid residue number. Specifically, 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, and CH3 is an EU index 341 to 447. Each amino acid sequence is identified.

　本発明の抗体としては、特に遺伝子工学的に作製された組換えマウス抗体、組換えラット抗体、組換えラビット抗体、ヒト型キメラ抗体（以下、単にキメラ抗体とも略記する）、ヒト化抗体（ヒト型相補性決定領域ＣＤＲ移植抗体ともいう）およびヒト抗体などの遺伝子組換え抗体も含まれる。 Examples of the antibody of the present invention include a recombinant mouse antibody, a recombinant rat antibody, a recombinant rabbit antibody, a human chimeric antibody (hereinafter simply abbreviated as a chimeric antibody), a humanized antibody (human) prepared in particular by genetic engineering. Type complementarity determining region CDR-grafted antibody) and recombinant antibodies such as human antibodies are also included.

　キメラ抗体とは、ヒト以外の動物（非ヒト動物）の抗体のＶＨおよびＶＬと、ヒト抗体のＣＨおよびＣＬからなる抗体を意味する。非ヒト動物としては、マウス、ラット、ハムスター、ラビット等、ハイブリドーマを作製することが可能であれば、いかなるものも用いることができる。 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. As the non-human animal, any mouse, rat, hamster, rabbit or the like can be used as long as it can produce a hybridoma.

　ハイブリドーマとは、非ヒト動物に抗原を免疫して取得されたＢ細胞と、マウスなどに由来するミエローマ細胞とを細胞融合させて得られる、所望の抗原特異性を有したモノクローナル抗体を産生する細胞をいう。したがって、ハイブリドーマが産生する抗体を構成する可変領域は、非ヒト動物抗体のアミノ酸配列からなる。 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. Say. Therefore, the variable region constituting the antibody produced by the hybridoma consists of the amino acid sequence of a non-human animal antibody.

　ヒト型キメラ抗体は、モノクローナル抗体を生産する非ヒト動物細胞由来のハイブリドーマより、該モノクローナル抗体のＶＨおよびＶＬをコードするｃＤＮＡを取得し、ヒト抗体のＣＨおよびＣＬをコードするＤＮＡを有する動物細胞用発現ベクターにそれぞれ挿入してヒト型キメラ抗体発現ベクターを構築し、動物細胞へ導入することにより発現させ、製造することができる。 A human chimeric antibody is obtained from a hybridoma derived from a non-human animal cell that produces a monoclonal antibody, and obtains cDNA encoding the VH and VL of the monoclonal antibody, and for animal cells having DNA encoding the human antibody CH and CL. A human chimeric antibody expression vector can be constructed by inserting each into an expression vector and introduced into an animal cell for expression and production.

　ヒト化抗体とは、非ヒト動物抗体のＶＨおよびＶＬのＣＤＲのアミノ酸配列をヒト抗体のＶＨおよびＶＬの対応するＣＤＲに移植した抗体をいう。ＶＨおよびＶＬのＣＤＲ以外の領域はフレームワーク領域（以下、ＦＲと表記する）と称される。 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).

　ヒト化抗体は、非ヒト動物抗体のＶＨのＣＤＲのアミノ酸配列と任意のヒト抗体のＶＨのＦＲのアミノ酸配列からなるＶＨのアミノ酸配列をコードするｃＤＮＡと、非ヒト動物抗体のＶＬのＣＤＲのアミノ酸配列と任意のヒト抗体のＶＬのＦＲのアミノ酸配列からなるＶＬのアミノ酸配列をコードするｃＤＮＡを構築し、ヒト抗体のＣＨおよびＣＬをコードするＤＮＡを有する動物細胞用発現ベクターにそれぞれ挿入してヒト化抗体発現ベクターを構築し、動物細胞へ導入することにより発現させ、製造することができる。 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.

　ヒト抗体は、元来、ヒト体内に天然に存在する抗体をいうが、最近の遺伝子工学的、細胞工学的、発生工学的な技術の進歩により作製されたヒト抗体ファージライブラリーおよびヒト抗体産生トランスジェニック動物から得られる抗体等も含まれる。 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.

　ヒト抗体は、ヒトイムノグロブリン遺伝子を保持するマウス（Tomizuka K. et. al., Proc Natl Acad Sci U S A.  97, 722-7, 2000）に所望の抗原を免疫することにより、取得することが出来る。また、ヒト由来のＢ細胞から抗体遺伝子を増幅したｐｈａｇｅ　ｄｉｓｐｌａｙライブラリーを用いることにより、所望の結合活性を有するヒト抗体を選択することで、免疫を行わずにヒト抗体を取得することができる（Winter G. et. al., Annu Rev Immunol.12:433-55. 1994)。さらに、ＥＢウイルスを用いてヒトＢ細胞を不死化することにより、所望の結合活性を有するヒト抗体を生産する細胞を作製し、ヒト抗体を取得することができる（Rosen A. et. al., Nature 267, 52-54.1977）。 A human antibody is obtained by immunizing a mouse carrying a human immunoglobulin gene (Tomizuka K. et. Al., Proc Natl Acad Sci U S A. 97, 722-7, 2000). 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).

　ヒト体内に存在する抗体は、例えば、ヒト末梢血から単離したリンパ球を、ＥＢウイルス等を感染させることによって不死化した後、クローニングすることにより、該抗体を産生するリンパ球を得ることができ、該リンパ球を培養した培養物中より該抗体を精製することができる。 The antibody present in the human body can be obtained by, for example, immortalizing lymphocytes isolated from human peripheral blood by infecting EB virus or the like and then cloning to obtain lymphocytes that produce the antibody. The antibody can be purified from the culture in which the lymphocytes are cultured.

　ヒト抗体ファージライブラリーは、ヒトＢ細胞から調製した抗体遺伝子をファージ遺伝子に挿入することによりＦａｂ、ｓｃＦｖ等の抗体断片を表面に発現させたファージのライブラリーである。該ライブラリーより、抗原を固定化した基質に対する結合活性を指標として所望の抗原結合活性を有する抗体断片を発現しているファージを回収することができる。該抗体断片は、更に遺伝子工学的手法により、２本の完全なＨ鎖および２本の完全なＬ鎖からなるヒト抗体分子へも変換することができる。 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.

　ヒト抗体産生トランスジェニック動物は、ヒト抗体遺伝子が宿主動物の染色体内に組込まれた動物をいう。具体的には、マウスＥＳ細胞へヒト抗体遺伝子を導入し、該ＥＳ細胞を他のマウスの初期胚へ移植後、発生させることによりヒト抗体産生トランスジェニック動物を作製することができる。ヒト抗体産生トランスジェニック動物からのヒト抗体の作製方法は、通常のヒト以外の哺乳動物で行われているハイブリドーマ作製方法によりヒト抗体産生ハイブリドーマを取得し、培養することで培養物中にヒト抗体を産生蓄積させることができる。 A human antibody-producing transgenic animal is an animal in which a human antibody gene is integrated into the chromosome of a host animal. Specifically, 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.

　本発明の抗体のＶＨおよびＶＬのアミノ酸配列としては、ヒト抗体のＶＨおよびＶＬのアミノ酸配列、非ヒト動物抗体のＶＨおよびＶＬのアミノ酸配列または非ヒト動物抗体のＣＤＲを、任意のヒト抗体のフレームワークに移植したヒト化抗体のＶＨおよびＶＬのアミノ酸配列のいずれでもよい。 As the VH and VL amino acid sequences of the antibody of the present invention, the VH and VL amino acid sequences of a human antibody, the VH and VL amino acid sequences of a non-human animal antibody, or the CDR of a non-human animal antibody are used. Any of the VH and VL amino acid sequences of the humanized antibody transplanted to the workpiece may be used.

　本発明の抗体におけるＣＬのアミノ酸配列としては、ヒト抗体のアミノ酸配列または非ヒト動物抗体のアミノ酸配列のいずれでもよいが、ヒト抗体のアミノ酸配列のＣκまたはＣλが好ましい。 The amino acid sequence of CL in the antibody of the present invention, may be any amino acid sequence or non-human animal antibody amino acid sequences of a human antibody, preferably C kappa or C lambda amino acid sequences of a human antibody.

　本発明の抗体のＣＨとしては、イムノグロブリンに属すればいかなるものでもよいが、好ましくはＩｇＧクラスに属するサブクラス、γ１（ＩｇＧ１）、γ２（ＩｇＧ２）、γ３（ＩｇＧ３）およびγ４（ＩｇＧ４）のいずれも用いることができる。 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, γ1 (IgG1), γ2 (IgG2), γ3 (IgG3) and γ4 (IgG4) Can also be used.

　本発明の抗体としては、Ｆｃと抗体断片とが結合したＦｃ融合タンパク質、Ｆｃと天然に存在するリガンドまたは受容体とが結合したＦｃ融合タンパク質（イムノアドヘシンともいう）、複数のＦｃ領域を融合させたＦｃ融合タンパク質等も本発明に包含される。また、抗体を安定化させるためおよび血中半減期を制御するために、アミノ酸残基を改変したＦｃ領域なども本発明の抗体に用いることができる。 The antibodies of the present invention include 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. Fc fusion proteins and the like that have been made are also encompassed by the present invention. In addition, in order to stabilize the antibody and to control the blood half-life, an Fc region with a modified amino acid residue can also be used in the antibody of the present invention.

　本発明の抗体又は該抗体断片は、翻訳後修飾されたいかなるアミノ酸残基を含む抗体をも包含する。翻訳後修飾としては、例えば、Ｈ鎖のＣ末端におけるリジン残基の欠失［リジン・クリッピング（ｌｙｓｉｎｅ　ｃｌｉｐｐｉｎｇ）］またはポリペプチドのＮ末端におけるグルタミン残基のピログルタミン（ｐｙｒｏＧｌｕ）への変換などが挙げられる［Beck et al, Analytical Chemistry, 85, 715-736（2013）］。 The antibody of the present invention or the antibody fragment includes an antibody containing any amino acid residue modified after translation. Post-translational modifications include, for example, deletion of a lysine residue at the C-terminus of the H chain [lysine clipping] or conversion of a glutamine residue at the N-terminus of a polypeptide to pyroglutin (pyroGlu). [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].

　本発明において、抗体断片とは、ヒトＣＣＲ１の細胞外領域に結合し、ヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害する、抗原結合活性を有する抗体断片である。本発明において抗体断片としては、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、ｓｃＦｖ、ｄｉａｂｏｄｙ、ｄｓＦｖまたは複数のＣＤＲを含むペプチドなどが挙げられる。Ｆａｂは、ＩｇＧ抗体をタンパク質分解酵素パパインで処理して得られる断片のうち（Ｈ鎖の２２４番目のアミノ酸残基で切断される）、Ｈ鎖のＮ末端側約半分とＬ鎖全体がジスルフィド結合（Ｓ－Ｓ結合）で結合した、分子量約５万の抗原結合活性を有する抗体断片である。 In the present invention, an antibody fragment is an antibody fragment having an antigen-binding activity that binds to the extracellular region of human CCR1 and inhibits activation of human CCR1 by human CCL15. Examples of the antibody fragment in the present invention include Fab, Fab ′, F (ab ′) 2 , scFv, diabody, dsFv, or 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 having a molecular weight of about 50,000, bound by (SS bond).

　Ｆ（ａｂ’）２は、ＩｇＧをタンパク質分解酵素ペプシンで処理して得られる断片のうち（Ｈ鎖の２３４番目のアミノ酸残基で切断される）、Ｆａｂがヒンジ領域のＳ－Ｓ結合を介して結合されたものよりやや大きい、分子量約１０万の抗原結合活性を有する抗体断片である。Ｆａｂ’は、上記Ｆ（ａｂ’）２のヒンジ領域のＳ－Ｓ結合を切断した分子量約５万の抗原結合活性を有する抗体断片である。 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は、１本のＶＨと１本のＶＬとを４個のＧｌｙおよび１個のＳｅｒ残基からなるリンカー（Ｇ４Ｓ）を任意の個数つなげたリンカーペプチドなどの適当なペプチドリンカー（Ｐ）を用いて連結した、ＶＨ－Ｐ－ＶＬないしはＶＬ－Ｐ－ＶＨポリペプチドで、抗原結合活性を有する抗体断片である。 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. VH-P-VL or VL-P-VH polypeptide, which is an antibody fragment having antigen-binding activity.

　Ｄｉａｂｏｄｙは、抗原結合特異性の同じまたは異なるｓｃＦｖが２量体を形成した抗体断片で、同じ抗原に対する２価の抗原結合活性または異なる抗原に対する特異的な抗原結合活性を有する抗体断片である。 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 specific antigen-binding activity for different antigens.

　ｄｓＦｖは、ＶＨおよびＶＬ中のそれぞれ１アミノ酸残基をシステイン残基に置換したポリペプチドを該システイン残基間のＳ－Ｓ結合を介して結合させたものをいう。 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.

　ＣＤＲを含むペプチドは、ＶＨまたはＶＬのＣＤＲの少なくとも１領域以上を含んで構成される。複数のＣＤＲを含むペプチドは、ＣＤＲ同士を直接または適当なペプチドリンカーを介して結合させることができる。本発明の改変抗体のＶＨおよびＶＬのＣＤＲをコードするＤＮＡを構築し、該ＤＮＡを原核生物用発現ベクターまたは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物または真核生物へ導入することにより発現させ、製造することができる。また、ＣＤＲを含むペプチドは、Ｆｍｏｃ法またはｔＢｏｃ法などの化学合成法によって製造することもできる。 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. Moreover, the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc method.

　本発明のモノクローナル抗体には、本発明のヒトＣＣＲ１に結合するモノクローナル抗体またはその抗体断片に放射性同位元素、低分子の薬剤、高分子の薬剤、タンパク質または抗体医薬などを化学的または遺伝子工学的に結合させた抗体の誘導体を包含する。 The monoclonal antibody of the present invention includes a radioisotope, a low molecular drug, a high molecular drug, a protein or an antibody drug chemically or genetically engineered to the monoclonal antibody or antibody fragment thereof that binds to human CCR1 of the present invention. Includes derivatives of conjugated antibodies.

　抗体の誘導体は、本発明のヒトＣＣＲ１に結合するモノクローナル抗体またはその抗体断片のＨ鎖若しくはＬ鎖のＮ末端側、Ｃ末端側、抗体分子中の適当な置換基または側鎖あるいは糖鎖などに、放射性同位元素、低分子の薬剤、高分子の薬剤、免疫賦活剤、タンパク質、抗体医薬または核酸医薬などを化学的手法［抗体工学入門、地人書館（１９９４）］により結合させることにより製造することができる。 The derivative of the antibody is added to the N-terminal side or C-terminal side of the H chain or L chain of the monoclonal antibody or antibody fragment thereof binding to human CCR1 of the present invention, an appropriate substituent in the antibody molecule, the side chain or sugar chain, etc. , Radioisotopes, low molecular weight drugs, high molecular weight drugs, immunostimulants, proteins, antibody drugs, or nucleic acid drugs, etc., are combined by chemical techniques [Introduction to Antibody Engineering, Jinshokan (1994)]. be able to.

　また、本発明のヒトＣＣＲ１に結合するモノクローナル抗体またはその抗体断片をコードするＤＮＡと、結合させたいタンパク質または抗体医薬をコードするＤＮＡを連結させて発現ベクターに挿入し、該発現ベクターを適当な宿主細胞へ導入し、発現させる遺伝子工学的手法より製造することができる。 Also, the DNA encoding the monoclonal antibody or antibody fragment thereof that binds to human CCR1 of the present invention and the DNA encoding the protein or antibody drug to be bound are linked and inserted into an expression vector, and the expression vector is inserted into a suitable host. It can be produced by genetic engineering techniques that are introduced into cells and expressed.

　放射性同位元素としては、例えば、１１１Ｉｎ、１３１Ｉ、１２５Ｉ、９０Ｙ、６４Ｃｕ、９９Ｔｃ、７７Ｌｕまたは２１１Ａｔなどが挙げられる。放射性同位元素は、クロラミンＴ法などによって抗体に直接結合させることができる。また、放射性同位元素をキレートする物質を抗体に結合させてもよい。キレート剤としては、例えば、１－イソチオシアネートベンジル－３－メチルジエチレントリアミンペンタ酢酸（ＭＸ－ＤＴＰＡ）などが挙げられる。 Examples of the radioisotope 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. Examples of the chelating agent include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).

　低分子の薬剤としては、例えば、アルキル化剤、ニトロソウレア剤、代謝拮抗剤、抗生物質、植物アルカロイド、トポイソメラーゼ阻害剤、ホルモン療法剤、ホルモン拮抗剤、アロマターゼ阻害剤、Ｐ糖蛋白阻害剤、白金錯体誘導体、Ｍ期阻害剤若しくはキナーゼ阻害剤などの抗癌剤［臨床腫瘍学、癌と化学療法社（１９９６）］、ハイドロコーチゾン若しくはプレドニゾンなどのステロイド剤、アスピリン若しくはインドメタシンなどの非ステロイド剤、金チオマレート若しくはペニシラミンなどの免疫調節剤、サイクロフォスファミド若しくはアザチオプリンなどの免疫抑制剤またはマレイン酸クロルフェニラミン若しくはクレマシチンのような抗ヒスタミン剤などの抗炎症剤［炎症と抗炎症療法、医歯薬出版株式会社（１９８２）］などが挙げられる。 Examples of low molecular weight drugs include alkylating agents, nitrosourea agents, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P glycoprotein inhibitors, platinum Anticancer agents such as complex derivatives, M-phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroidal agents such as hydrocortisone or prednisone, non-steroidal agents such as aspirin or indomethacin, gold thiomalate or Anti-inflammatory agents such as immunomodulators such as penicillamine, immunosuppressive agents such as cyclophosphamide or azathioprine, or antihistamines such as chlorpheniramine maleate or clemacytin [Inflammation and anti-inflammatory therapy, Ishiyaku Shuppan Co., Ltd. (1982) ]] Etc., and the like.

　抗癌剤としては、例えば、アミフォスチン（エチオール）、シスプラチン、ダカルバジン（ＤＴＩＣ）、ダクチノマイシン、メクロレタミン（ナイトロジェンマスタード）、ストレプトゾシン、シクロフォスファミド、イホスファミド、カルムスチン（ＢＣＮＵ）、ロムスチン（ＣＣＮＵ）、ドキソルビシン（アドリアマイシン）、エピルビシン、ゲムシタビン（ゲムザール）、ダウノルビシン、プロカルバジン、マイトマイシン、シタラビン、エトポシド、メトトレキセート、５－フルオロウラシル、フルオロウラシル、ビンブラスチン、ビンクリスチン、ブレオマイシン、ダウノマイシン、ペプロマイシン、エストラムスチン、パクリタキセル（タキソール）、ドセタキセル（タキソテア）、アルデスロイキン、アスパラギナーゼ、ブスルファン、カルボプラチン、オキサリプラチン、ネダプラチン、クラドリビン、カンプトテシン、１０－ヒドロキシ－７－エチル－カンプトテシン（ＳＮ３８）、フロクスウリジン、フルダラビン、ヒドロキシウレア、イダルビシン、メスナ、イリノテカン（ＣＰＴ－１１）、ノギテカン、ミトキサントロン、トポテカン、ロイプロリド、メゲストロール、メルファラン、メルカプトプリン、ヒドロキシカルバミド、プリカマイシン、ミトタン、ペガスパラガーゼ、ペントスタチン、ピポブロマン、タモキシフェン、ゴセレリン、リュープロレニン、フルタミド、テニポシド、テストラクトン、チオグアニン、チオテパ、ウラシルマスタード、ビノレルビン、クロラムブシル、ハイドロコーチゾン、プレドニゾロン、メチルプレドニゾロン、ビンデシン、ニムスチン、セムスチン、カペシタビン、トムデックス、アザシチジン、ＵＦＴ、オキザロプラチン、ゲフィチニブ（イレッサ）、イマチニブ（ＳＴＩ５７１）、エルロチニブ、ＦＭＳ－ｌｉｋｅ　ｔｙｒｏｓｉｎｅ　ｋｉｎａｓｅ　３（Ｆｌｔ３）阻害剤、ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｇｒｏｗｔｈ　ｆａｃｏｔｒ　ｒｅｃｅｐｔｏｒ（ＶＥＧＦＲ）阻害剤、ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ（ＦＧＦＲ）阻害剤、イレッサ若しくはタルセバなどのｅｐｉｄｅｒｍａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ（ＥＧＦＲ）阻害剤、ラディシコール、１７－アリルアミノ－１７－デメトキシゲルダナマイシン、ラパマイシン、アムサクリン、オール－トランスレチノイン酸、サリドマイド、レナリドマイド、アナストロゾール、ファドロゾール、レトロゾール、エキセメスタン、金チオマレート、Ｄ－ペニシラミン、ブシラミン、アザチオプリン、ミゾリビン、シクロスポリン、ラパマイシン、ヒドロコルチゾン、ベキサロテン（ターグレチン）、タモキシフェン、デキサメタゾン、プロゲスチン類、エストロゲン類、アナストロゾール（アリミデックス）、ロイプリン、アスピリン、インドメタシン、セレコキシブ、ペニシラミン、金チオマレート、マレイン酸クロルフェニラミン、クロロフェニラミン、クレマシチン、トレチノイン、ベキサロテン、砒素、ボルテゾミブ、アロプリノール、カリケアマイシン、イブリツモマブチウキセタン、タルグレチン、オゾガミン、クラリスロマシン、ロイコボリン、ケトコナゾール、アミノグルテチミド、スラミンまたはメイタンシノイドあるいはその誘導体などが挙げられる。 Examples of 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, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea, idarubicin, mesna, irinotecan (CPT-11), nogitecan, mitoxan Thorone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, hydroxycarbamide, pricamycin, mitotan, pegaseparagase, pentostatin, pipobroman, tamoxifen, goserelin, leuprorenin, flutamide, teniposide, test lactone, thioguanine, thiotepa , Uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone, methylprednisolone Vindesine, Nimustine, Semustine, Capecitabine, Tomdex, Azacitidine, UFT, Oxaroplatin, Gefitinib (Iressa), Imatinib (STI571), Erlotinib, FMS-like tyrosine Kinase3 (Flt3) Inhibitors, fibroblast growth factor receptor (FGFR) inhibitors, epidermal growth receptor (EGFR) inhibitors such as Iressa or Tarceva, radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, rapamycin Retinoic acid, thalidomide, lenalidomide, anastrozole, fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (tergretin), tamoxifen, dexamethasone , Anastrozole (arimidex), leuprin, aspirin, indomethacin, celecoxib, penicillamine, gold thiomalate, chlorpheniramine maleate, chloropheniramine, clemacytine, tretinoin, bexarotene, arsenic, bortezomib, allopurinol, calicheamicin, ibritummo Mabuchiuxetane, Targretin, Ozogamin, Clarislo Machine, Roy Bolin, ketoconazole, aminoglutethimide, etc. suramin or maytansinoid or derivatives thereof.

　低分子の薬剤と抗体とを結合させる方法としては、例えば、グルタールアルデヒドを介して薬剤と抗体のアミノ基間を結合させる方法または水溶性カルボジイミドを介して薬剤のアミノ基と抗体のカルボキシル基を結合させる方法などが挙げられる。 Examples of a method for binding a low molecular weight drug and an antibody include, for example, a method in which a drug and an amino group of an antibody are bonded via glutaraldehyde, or an amino group of a drug and a carboxyl group of an antibody via a water-soluble carbodiimide. The method of combining is mentioned.

　高分子の薬剤としては、例えば、ポリエチレングリコール（以下、ＰＥＧと表記する）、アルブミン、デキストラン、ポリオキシエチレン、スチレンマレイン酸コポリマー、ポリビニルピロリドン、ピランコポリマー、またはヒドロキシプロピルメタクリルアミドなどが挙げられる。これらの高分子化合物を抗体またはその抗体断片に結合させることにより、（１）化学的、物理的若しくは生物的な種々の因子に対する安定性の向上、（２）血中半減期の顕著な延長、または（３）免疫原性の消失若しくは抗体産生の抑制、などの効果が期待される［バイオコンジュゲート医薬品、廣川書店（１９９３）］。 Examples of the polymer drug include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer, and hydroxypropyl methacrylamide. By linking these macromolecular compounds to antibodies or antibody fragments thereof, (1) improved stability to various chemical, physical or biological factors, (2) significant increase in blood half-life, Alternatively, (3) an effect such as loss of immunogenicity or suppression of antibody production is expected [Bioconjugate Pharmaceutical, Yodogawa Shoten (1993)].

　例えば、ＰＥＧと抗体を結合させる方法としては、ＰＥＧ化修飾試薬と反応させる方法などが挙げられる［バイオコンジュゲート医薬品、廣川書店（１９９３）］。ＰＥＧ化修飾試薬としては、リジンのε－アミノ基への修飾剤（日本国特開昭６１－１７８９２６号公報）、アスパラギン酸およびグルタミン酸のカルボキシル基への修飾剤（日本国特開昭５６－２３５８７号公報）、またはアルギニンのグアニジノ基への修飾剤（日本国特開平２－１１７９２０号公報）などが挙げられる。 For example, as a method for linking PEG and an antibody, a method of reacting with a PEGylation modifying reagent and the like can be mentioned [Bioconjugate pharmaceutical, Yodogawa Shoten (1993)]. Examples of the PEGylation modifying reagent include a modifier for the ε-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).

　免疫賦活剤としては、イムノアジュバントとして知られている天然物でもよく、具体例としては、免疫を亢進する薬剤が、β（１→３）グルカン（例えば、レンチナンまたはシゾフィラン）またはαガラクトシルセラミド（ＫＲＮ７０００）などが挙げられる。 The immunostimulant may be a natural product known as an immunoadjuvant, and specific examples include β (1 → 3) glucan (eg, lentinan or schizophyllan) or α-galactosylceramide (KRN7000). ) And the like.

　タンパク質としては、例えば、ＮＫ細胞、マクロファージまたは好中球などの免疫担当細胞を活性化するサイトカイン若しくは増殖因子または毒素タンパク質などが挙げられる。 Examples of proteins include cytokines, growth factors or toxin proteins that activate immunocompetent cells such as NK cells, macrophages or neutrophils.

　サイトカインまたは増殖因子としては、例えば、インターフェロン（以下、ＩＦＮと記す）－α、ＩＦＮ－β、ＩＦＮ－γ、インターロイキン（以下、ＩＬと記す）－２、ＩＬ－１２、ＩＬ－１５、ＩＬ－１８、ＩＬ－２１、ＩＬ－２３、顆粒球コロニー刺激因子（Ｇ－ＣＳＦ）、顆粒球／マクロファージコロニー刺激因子（ＧＭ－ＣＳＦ）またはマクロファージコロニー刺激因子（Ｍ－ＣＳＦ）などが挙げられる。毒素タンパク質としては、例えば、リシン、ジフテリアトキシンまたはＯＮＴＡＫなどが挙げられ、毒性を調節するためにタンパク質に変異を導入したタンパク毒素も含まれる。 Examples of cytokines or growth factors include interferon (hereinafter referred to as IFN) -α, IFN-β, IFN-γ, 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) or macrophage colony stimulating factor (M-CSF). Examples of the toxin protein include ricin, diphtheria toxin, ONTAK, and the like, and also include protein toxins in which mutations are introduced into the protein to regulate toxicity.

　抗体医薬としては、例えば、抗体の結合によりアポトーシスが誘導される抗原、腫瘍の病態形成に関わる抗原、免疫機能を調節する抗原または病変部位の血管新生に関与する抗原に対する抗体が挙げられる。 Examples of the antibody drug include an antigen against which apoptosis is induced by antibody binding, an antigen associated with tumor pathogenesis, an antigen that regulates immune function, or an antigen associated with angiogenesis of a lesion site.

　抗体の結合によりアポトーシスが誘導される抗原としては、例えば、ｃｌｕｓｔｅｒ　ｏｆ　ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ（以下、ＣＤと記載する）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またはＥｐｉｄｅｒｍａｌ　Ｇｒｏｗｔｈ　Ｆａｃｔｏｒ　Ｒｅｃｅｐｔｏｒ（ＥＧＦＲ）などが挙げられる。 Examples of 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 or Epidemial GrowthFactor ) And the like.

　腫瘍の病態形成に関わる抗原または免疫機能を調節する抗体の抗原としては、例えば、ＣＤ４、ＣＤ４０、ＣＤ４０リガンド、Ｂ７ファミリー分子（例えば、ＣＤ８０、ＣＤ８６、ＣＤ２７４、Ｂ７－ＤＣ、Ｂ７－Ｈ２、Ｂ７－Ｈ３またはＢ７－Ｈ４）、Ｂ７ファミリー分子のリガンド（例えば、ＣＤ２８、ＣＴＬＡ－４、ＩＣＯＳ、ＰＤ－１またはＢＴＬＡ）、ＯＸ－４０、ＯＸ－４０リガンド、ＣＤ１３７、ｔｕｍｏｒ　ｎｅｃｒｏｓｉｓ　ｆａｃｔｏｒ（ＴＮＦ）受容体ファミリー分子（例えば、ＤＲ４、ＤＲ５、ＴＮＦＲ１またはＴＮＦＲ２）、ＴＮＦ－ｒｅｌａｔｅｄ　ａｐｏｐｔｏｓｉｓ－ｉｎｄｕｃｉｎｇ　ｌｉｇａｎｄ　ｒｅｃｅｐｔｏｒ（ＴＲＡＩＬ）ファミリー分子、ＴＲＡＩＬファミリー分子の受容体ファミリー（例えば、ＴＲＡＩＬ－Ｒ１、ＴＲＡＩＬ－Ｒ２、ＴＲＡＩＬ－Ｒ３またはＴＲＡＩＬ－Ｒ４）、ｒｅｃｅｐｔｏｒ　ａｃｔｉｖａｔｏｒ　ｏｆ　ｎｕｃｌｅａｒ　ｆａｃｔｏｒ　ｋａｐｐａ　Ｂ　ｌｉｇａｎｄ（ＲＡＮＫ）、ＲＡＮＫリガンド、ＣＤ２５、葉酸受容体、サイトカイン［例えば、ＩＬ－１α、ＩＬ－１β、ＩＬ－４、ＩＬ－５、ＩＬ－６、ＩＬ－１０、ＩＬ－１３、ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＴＧＦ）βまたはＴＮＦαなど］若しくはこれらのサイトカインの受容体、またはケモカイン（例えば、ＳＬＣ、ＥＬＣ、Ｉ－３０９、ＴＡＲＣ、ＭＤＣまたはＣＴＡＣＫなど）若しくはこれらのケモカインの受容体が挙げられる。 Examples of 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 ( For example, TRAIL-R1, TRAIL-R2, TRAIL-R3 or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor, cytokine [eg IL-1αIL- 1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF) β or TNFα, etc.] or receptors for these cytokines, or chemokines (eg, SLC, ELC, I-309, TARC, MDC or CTACK) or a receptor for these chemokines.

　病変部位の血管新生を阻害する抗体の抗原としては、例えば、ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＶＥＧＦ）、ａｎｇｉｏｐｏｉｅｔｉｎ、ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＦＧＦ）、ＥＧＦ、ｈｅｐａｔｏｃｙｔｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＨＧＦ）、ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＰＤＧＦ）、ｉｎｓｕｌｉｎ－ｌｉｋｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（ＩＧＦ）、ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ（ＥＰＯ）、ＴＧＦβ、ＩＬ－８、ｅｐｈｒｉｎまたはＳＤＦ－１若しくはこれらの受容体などが挙げられる。 Examples of 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), TGFβ, IL-8, ephrin, SDF-1, or their receptors.

　タンパク質または抗体医薬との融合抗体は、モノクローナル抗体または抗体断片をコードするｃＤＮＡにタンパク質または抗体医薬に含まれる抗体をコードするｃＤＮＡを連結させ、融合抗体をコードするＤＮＡを構築し、該ＤＮＡを原核生物または真核生物用発現ベクターに挿入し、該発現ベクターを原核生物または真核生物へ導入することにより発現させ、融合抗体を製造することができる。 A fusion antibody with a protein or antibody drug is obtained by linking a cDNA encoding an antibody contained in a protein or antibody drug to a cDNA encoding a monoclonal antibody or antibody fragment, constructing a DNA encoding the fusion antibody, and A fusion antibody can be produced by inserting into an expression vector for organisms or eukaryotes and expressing the expression vector by introducing the vector into prokaryotes or eukaryotes.

　核酸医薬としては、例えば、遺伝子の機能を制御することによって生体に作用するｓｍａｌｌ　ｉｎｔｅｒｆｅｒｅｎｃｅ　ｒｉｂｏｎｕｃｌｅｉｃ　ａｃｉｄ（ｓｉＲＮＡ）またはｍｉｃｒｏＲＮＡなどの核酸を含む医薬品が挙げられる。例えば、Ｔｈ１７細胞のマスター転写因子ＲＯＲγｔを抑制する核酸医薬とのコンジュゲートが考えられる。 Examples of nucleic acid drugs include pharmaceuticals containing nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA that act on a living body by controlling the function of the gene. For example, a conjugate with a nucleic acid drug that suppresses the master transcription factor RORγt of Th17 cells is conceivable.

　本発明の抗体の誘導体をヒトＣＣＲ１の検出および測定ならびにヒトＣＣＲ１関連疾患の診断に使用する場合に、当該抗体に結合する薬剤としては、通常の免疫学的検出または測定法で用いられる標識体が挙げられる。標識体としては、例えば、アルカリフォスファターゼ、ペルオキシダーゼ若しくはルシフェラーゼなどの酵素、アクリジニウムエステル若しくはロフィンなどの発光物質、またはフルオレセインイソチオシアネート（ＦＩＴＣ）若しくはテトラメチルローダミンイソチオシアネート（ＲＩＴＣ）などの蛍光物質などが挙げられる。 When the derivative of the antibody of the present invention is used for detection and measurement of human CCR1 and diagnosis of a human CCR1-related disease, the drug that binds to the antibody includes a label used in usual immunological detection or measurement methods. Can be mentioned. Examples of the label include an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC). Can be mentioned.

　また、本発明は、ヒトＣＣＲ１に結合するモノクローナル抗体またはその抗体断片を有効成分として含有する組成物を含む。 The present invention also includes a composition containing a monoclonal antibody that binds to human CCR1 or an antibody fragment thereof as an active ingredient.

　また、本発明は、ヒトＣＣＲ１に結合するモノクローナル抗体またはその抗体断片を有効成分として含有する、ヒトＣＣＲ１関連疾患の治療薬に関する。また、本発明は、ヒトＣＣＲ１に結合するモノクローナル抗体またはその抗体断片を投与することを含む、ヒトＣＣＲ１関連疾患の治療方法に関する。 The present invention also relates to a therapeutic agent for human CCR1-related diseases, which contains a monoclonal antibody that binds to human CCR1 or an antibody fragment thereof as an active ingredient. The present invention also relates to a method for treating human CCR1-related diseases, comprising administering a monoclonal antibody that binds to human CCR1 or an antibody fragment thereof.

　ヒトＣＣＲ１関連疾患としては、ヒトＣＣＲ１又はヒトＣＣＲ１のリガンドが関与する疾患であればいかなるものでもよく、例えば、癌、自己免疫疾患および炎症性疾患が挙げられる。癌疾患としては、例えば、びまん性大細胞型Ｂ細胞性リンパ腫、濾胞性リンパ腫、Ｂ細胞リンパ腫、Ｔ細胞リンパ腫、形質細胞性骨髄腫、急性骨髄性白血病、ホジキンリンパ腫、慢性リンパ性白血病、有毛細胞白血病、マントル細胞リンパ腫、濾胞辺縁帯リンパ腫、小リンパ球性リンパ腫、多発性骨髄腫、肝細胞癌、結腸直腸癌、非小細胞肺癌、口腔扁平上皮癌、卵巣癌、前立腺癌、乳癌、神経膠腫または骨肉腫などが挙げられる。自己免疫疾患または炎症性疾患としては、例えば、関節リウマチ、多発性硬化症、慢性閉塞肺疾患、全身性エリテマトーデス、ループス腎炎、喘息、アトピー性皮膚炎、炎症性大腸炎、クローン病またはベーチェット病などが挙げられる。 The human CCR1-related disease may be any disease involving human CCR1 or a human CCR1 ligand, and examples thereof include cancer, autoimmune diseases and inflammatory diseases. Examples of cancer diseases include diffuse large B cell lymphoma, follicular lymphoma, B cell lymphoma, T cell lymphoma, plasma cell myeloma, acute myeloid leukemia, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy Cell leukemia, mantle cell lymphoma, follicular marginal zone lymphoma, small lymphocytic lymphoma, multiple myeloma, hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, oral squamous cell carcinoma, ovarian cancer, prostate cancer, breast cancer, Examples include glioma or osteosarcoma. Examples of autoimmune diseases or inflammatory diseases include rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, lupus nephritis, asthma, atopic dermatitis, inflammatory bowel disease, Crohn's disease or Behcet's disease Is mentioned.

　本発明の抗体または該抗体断片を含有する治療剤は、有効成分としての該抗体または該抗体断片のみを含むものであってもよいが、通常は薬理学的に許容される１以上の担体と一緒に混合し、製剤学の技術分野において公知の任意の方法により製造した医薬製剤として提供するのが好ましい。 The therapeutic agent containing the antibody or the antibody fragment of the present invention may contain only the antibody or the antibody fragment as an active ingredient, but usually one or more pharmacologically acceptable carriers and They are preferably mixed together and provided as pharmaceutical preparations produced by any method known in the pharmaceutical arts.

　投与経路は、治療に際して最も効果的なものを使用するのが好ましく、経口投与、または口腔内、気道内、直腸内、皮下、筋肉内若しくは静脈内などの非経口投与が挙げられ、好ましくは静脈内投与を挙げられる。投与形態としては、例えば、噴霧剤、カプセル剤、錠剤、散剤、顆粒剤、シロップ剤、乳剤、座剤、注射剤、軟膏またはテープ剤などが挙げられる。 The administration route is preferably the most effective for treatment, and includes oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular or intravenous, preferably intravenous. Internal administration can be mentioned. Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or tapes.

　投与量または投与回数は、目的とする治療効果、投与方法、治療期間、年齢および体重などにより異なるが、通常成人１日当たり１０μｇ／ｋｇ～１０ｍｇ／ｋｇである。 The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age and weight, but is usually 10 μg / kg to 10 mg / kg per day for an adult.

　本発明はヒトＣＣＲ１に結合するモノクローナル抗体または該抗体断片を含有する、ＣＣＲ１の検出若しくは測定用試薬、またはヒトＣＣＲ１に結合するモノクローナル抗体または該抗体断片を用いたＣＣＲ１の検出若しくは測定方法に関する。本発明においてヒトＣＣＲ１を検出または測定する方法としては、任意の公知の方法が挙げられる。例えば、免疫学的検出または測定方法などが挙げられる。 The present invention relates to a reagent for detecting or measuring CCR1 containing a monoclonal antibody that binds to human CCR1 or the antibody fragment, or a method for detecting or measuring CCR1 using the monoclonal antibody that binds to human CCR1 or the antibody fragment. In the present invention, any known method may be used as a method for detecting or measuring human CCR1. Examples include immunological detection or measurement methods.

　免疫学的検出または測定方法とは、標識を施した抗原または抗体を用いて、抗体量または抗原量を検出または測定する方法である。免疫学的検出または測定方法としては、例えば、放射性物質標識免疫抗体法（ＲＩＡ）、酵素免疫測定法（ＥＩＡまたはＥＬＩＳＡ）、蛍光免疫測定法（ＦＩＡ）、発光免疫測定法（ｌｕｍｉｎｅｓｃｅｎｔ　ｉｍｍｕｎｏａｓｓａｙ）、ウエスタンブロット法または物理化学的手法などが挙げられる。 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. Examples of the immunological detection or measurement method include radioactive substance-labeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (Western immunoassay), Western Examples include blotting or physicochemical techniques.

　本発明はヒトＣＣＲ１に結合するモノクローナル抗体又は該抗体断片を含む、ＣＣＲ１関連疾患の診断薬、またはヒトＣＣＲ１に結合するモノクローナル抗体又は該抗体断片を用いてＣＣＲ１の検出または測定をすることを含む、ＣＣＲ１関連疾患の診断方法に関する。本発明のモノクローナル抗体または該抗体断片を用いて、上記の方法に従いヒトＣＣＲ１が発現した細胞を検出または測定することにより、ヒトＣＣＲ１が関連する疾患を診断することができる。 The present invention includes a diagnostic agent for a CCR1-related disease, comprising a monoclonal antibody that binds to human CCR1 or the antibody fragment, or detection or measurement of CCR1 using a monoclonal antibody that binds to human CCR1 or the antibody fragment. The present invention relates to a method for diagnosing CCR1-related diseases. By using the monoclonal antibody or the antibody fragment of the present invention to detect or measure a cell in which human CCR1 is expressed according to the above method, a disease associated with human CCR1 can be diagnosed.

　本発明においてヒトＣＣＲ１を検出または測定する対象となる生体試料としては、例えば、組織、細胞、血液、血漿、血清、膵液、尿、糞便、組織液または培養液など、ヒトＣＣＲ１又はヒトＣＣＲ１が発現している細胞を含む可能性のあるものであれば特に限定されない。 Examples of biological samples to be detected or measured for human CCR1 in the present invention include human CCR1 or human CCR1 expressed in tissues, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid. The cell is not particularly limited as long as it may contain cells.

　本発明のモノクローナル抗体またはその抗体断片を含有する診断薬は、目的の診断法に応じて、抗原抗体反応を行なうための試薬、該反応の検出用試薬を含んでもよい。抗原抗体反応を行なうための試薬としては、緩衝剤、塩などが挙げられる。検出用試薬としては、該モノクローナル抗体若しくはその抗体断片を認識する標識された二次抗体、または標識に対応した基質などの通常の免疫学的検出または測定法に用いられる試薬が挙げられる。 The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present invention may contain a reagent for conducting an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method. Examples of the reagent for performing the antigen-antibody reaction include a buffer and a salt. Examples of the detection reagent include a reagent used in a usual immunological detection or measurement method such as a labeled secondary antibody that recognizes the monoclonal antibody or an antibody fragment thereof, or a substrate corresponding to the label.

　また、本発明はＣＣＲ１関連疾患の治療薬若しくは診断薬の製造のための、抗ヒトＣＣＲ１モノクローナル抗体または該抗体断片の使用に関する。 The present invention also relates to the use of an anti-human CCR1 monoclonal antibody or the antibody fragment for the manufacture of a therapeutic or diagnostic agent for CCR1-related diseases.

　以下に、本発明の抗体の製造方法、疾患の治療方法、および疾患の診断方法について、具体的に説明する。 The antibody production method, disease treatment method, and disease diagnosis method of the present invention will be specifically described below.

１．抗体の製造方法
（１）抗原の調製
　抗原となるヒトＣＣＲ１またはヒトＣＣＲ１発現細胞は、ヒトＣＣＲ１全長またはその部分長をコードするｃＤＮＡを含む発現ベクターを、大腸菌、酵母、昆虫細胞または動物細胞などに導入することで得ることができる。また、ヒトＣＣＲ１は、ヒトＣＣＲ１を多量に発現している各種ヒト細胞株、ヒト細胞およびヒト組織などからヒトＣＣＲ１を精製することによっても得ることができる。また、これらヒト細胞株、ヒト細胞およびヒト組織などをそのまま抗原として使用することもできる。さらに、Ｆｍｏｃ法またはｔＢｏｃ法などの化学合成法によりヒトＣＣＲ１の部分配列を有する合成ペプチドを調製し、抗原に用いることもできる。ヒトＣＣＲ１またはヒトＣＣＲ１の部分配列を有する合成ペプチドには、Ｃ末端またはＮ末端にＦＬＡＧまたはＨｉｓなどの公知のタグが付加されていてもよい。 1. Antibody production method (1) Preparation of antigen Human CCR1 or human CCR1-expressing cells to be used as an antigen are expressed in Escherichia coli, yeast, insect cells, animal cells, or the like using an expression vector containing cDNA encoding the full length of human CCR1 or a partial length thereof. It can be obtained by introduction. Human CCR1 can also be obtained by purifying human CCR1 from various human cell lines, human cells, human tissues, etc. that express human CCR1 in large amounts. In addition, these human cell lines, human cells, human tissues, and the like can be used as antigens as they are. Furthermore, a synthetic peptide having a partial sequence of human CCR1 can be prepared by a chemical synthesis method such as Fmoc method or tBoc method and used as an antigen. A known peptide such as FLAG or His may be added to the C-terminus or N-terminus of the synthetic peptide having human CCR1 or a partial sequence of human CCR1.

　本発明で用いられるヒトＣＣＲ１は、Molecular Cloning,A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)やCurrent Protocols In Molecular Biology, John Wiley & Sons (1987-1997)などに記載された方法などを用い、例えば以下の方法により、該ヒトＣＣＲ１をコードするＤＮＡを宿主細胞中で発現させて、製造することができる。 Human CCR1 used in the present invention is a method described in MolecularMCloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), etc. Can be produced by expressing the DNA encoding human CCR1 in a host cell by the following method, for example.

　まず、ヒトＣＣＲ１をコードする部分を含む完全長ｃＤＮＡを適当な発現ベクターのプロモーターの下流に挿入することにより、組換えベクターを作製する。上記完全長ｃＤＮＡの代わりに、完全長ｃＤＮＡをもとにして調製された、ポリペプチドをコードする部分を含む適当な長さのＤＮＡ断片を用いてもよい。次に、得られた該組換えベクターを、該発現ベクターに適合した宿主細胞に導入することにより、ポリペプチドを生産する形質転換体を得ることができる。 First, a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding human CCR1 downstream of a promoter of an appropriate expression vector. Instead of the full-length cDNA, a DNA fragment of an appropriate length containing a polypeptide-encoding portion prepared based on the full-length cDNA may be used. Next, a transformant producing the polypeptide can be obtained by introducing the obtained recombinant vector into a host cell suitable for the expression vector.

　発現ベクターとしては、使用する宿主細胞における自律複製または染色体中への組込みが可能で、ポリペプチドをコードするＤＮＡを転写できる位置に、適当なプロモーターを含有しているものであればいずれも用いることができる。宿主細胞としては、大腸菌などのエシェリヒア属などに属する微生物、酵母、昆虫細胞または動物細胞など、目的とする遺伝子を発現できるものであればいずれも用いることができる。 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. As the host cell, any microorganisms belonging to the genus Escherichia such as Escherichia coli, yeast, insect cells or animal cells can be used so long as they can express the target gene.

　大腸菌などの原核生物を宿主細胞として用いる場合、組換えベクターは、原核生物中で自律複製が可能であると同時に、プロモーター、リボソーム結合配列、ヒトＣＣＲ１をコードする部分を含むＤＮＡ、および転写終結配列を含むベクターであることが好ましい。また、該組換えベクターには、転写終結配列は必ずしも必要ではないが、構造遺伝子の直下に転写終結配列を配置することが好ましい。さらに、該組換えベクターには、プロモーターを制御する遺伝子を含んでいてもよい。 When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in the prokaryote, and at the same time a promoter, a ribosome binding sequence, DNA containing a portion encoding human CCR1, 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.

　該組換えベクターとしては、リボソーム結合配列であるシャイン・ダルガルノ配列（ＳＤ配列ともいう）と開始コドンとの間を適当な距離（例えば６～１８塩基）に調節したプラスミドを用いることが好ましい。 As 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).

　また、該ヒトＣＣＲ１をコードするＤＮＡの塩基配列としては、宿主内での発現に最適なコドンとなるように塩基を置換することができ、これにより目的とするヒトＣＣＲ１の生産率を向上させることができる。 In addition, the base sequence of DNA encoding the human CCR1 can be substituted so that the codon is optimal for expression in the host, thereby improving the production rate of the target human CCR1. Can do.

　発現ベクターとしては、使用する宿主細胞中で機能を発揮できるものであればいずれも用いることができ、例えば、ｐＢＴｒｐ２、ｐＢＴａｃ１、ｐＢＴａｃ２（以上、ロシュ・ダイアグノスティックス社製）、ｐＫＫ２３３―２（ファルマシア社製）、ｐＳＥ２８０（インビトロジェン社製）、ｐＧＥＭＥＸ－１（プロメガ社製）、ｐＱＥ－８（キアゲン社製）、ｐＫＹＰ１０（日本国特開昭５８－１１０６００号公報）、ｐＫＹＰ２００［Agricultural Biological Chemistry, 48, 669 (1984)］、ｐＬＳＡ１［Agric. Biol. Chem., 53, 277 (1989)］、ｐＧＥＬ１［Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)］、ｐＢｌｕｅｓｃｒｉｐｔ　ＩＩ　ＳＫ（－）（ストラタジーン社製）、ｐＴｒｓ３０［大腸菌ＪＭ１０９／ｐＴｒＳ３０（ＦＥＲＭ　ＢＰ－５４０７）より調製］、ｐＴｒｓ３２［大腸菌ＪＭ１０９／ｐＴｒＳ３２（ＦＥＲＭ　ＢＰ－５４０８）より調製］、ｐＧＨＡ２［大腸菌ＩＧＨＡ２（ＦＥＲＭ　ＢＰ－４００）より調製、日本国特開昭６０－２２１０９１号公報］、ｐＧＫＡ２［大腸菌ＩＧＫＡ２（ＦＥＲＭ　ＢＰ－６７９８）より調製、日本国特開昭６０－２２１０９１号公報］、ｐＴｅｒｍ２（米国特許第４，６８６，１９１号明細書、米国特許第４，９３９，０９４号明細書、米国特許第１６０，７３５号明細書）、ｐＳｕｐｅｘ、ｐＵＢ１１０、ｐＴＰ５、ｐＣ１９４、ｐＥＧ４００［J. Bacteriol., 172, 2392 (1990)］、ｐＧＥＸ（ファルマシア社製）、ｐＥＴシステム（ノバジェン社製）またはｐＭＥ１８ＳＦＬ３などが挙げられる。 Any expression vector can be used as long as it can function in the host cell to be used. For example, pBTrp2, pBTac1, pBTac2 (hereinafter, Roche Diagnostics), pKK233-2 ( Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Patent Laid-Open Publication No. 58-110600), pKYP200 (Agricultural Biobiological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (- ) (Manufactured by Stratagene), pTrs30 [E. coli JM109 / pTrS30 (FERM BP-5407) Prepared], pTrs32 [prepared from E. coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from E. coli IGHA2 (FERM BP-400), Japanese Unexamined Patent Publication No. 60-221091], pGKA2 [E. coli IGKA2 ( FERM BP-6798), Japanese Patent Application Laid-Open No. 60-221091], pTerm2 (US Pat. No. 4,686,191, US Pat. No. 4,939,094, US Pat. No. 160) , 735 specification), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392Ｇ (1990)], pGEX (Pharmacia), pET system (Novagen), or pME18SFL3. .

　プロモーターとしては、使用する宿主細胞中で機能を発揮できるものであればいかなるものでもよい。例えば、ｔｒｐプロモーター（Ｐｔｒｐ）、ｌａｃプロモーター、ＰＬプロモーター、ＰＲプロモーターまたはＴ７プロモーターなどの、大腸菌またはファージなどに由来するプロモーターが挙げられる。また、例えば、Ｐｔｒｐを２つ直列させたタンデムプロモーター、ｔａｃプロモーター、ｌａｃＴ７プロモーターまたはｌｅｔ　Ｉプロモーターなどの人為的に設計改変されたプロモーターなどが挙げられる。 As the promoter, any promoter can be used as long as it can function in the host cell to be used. Examples thereof include promoters derived from Escherichia coli or phage, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter or T7 promoter. Further, for example, 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 can be mentioned.

　宿主細胞としては、例えば、大腸菌ＸＬ１－Ｂｌｕｅ、大腸菌ＸＬ２－Ｂｌｕｅ、大腸菌ＤＨ１、大腸菌ＭＣ１０００、大腸菌ＫＹ３２７６、大腸菌Ｗ１４８５、大腸菌ＪＭ１０９、大腸菌ＨＢ１０１、大腸菌Ｎｏ．４９、大腸菌Ｗ３１１０、大腸菌ＮＹ４９または大腸菌ＤＨ５αなどが挙げられる。 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 or E. coli DH5α.

　宿主細胞への組換えベクターの導入方法としては、使用する宿主細胞へＤＮＡを導入する方法であればいずれも用いることができ、例えば、カルシウムイオンを用いる方法［Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)、Gene, 17, 107 (1982)、Molecular & General Genetics, 168, 111 (1979)］が挙げられる。 As a method for introducing a recombinant vector into a host cell, any method for introducing DNA into a host cell to be used can be used. For example, a method using calcium ion [Proc. Natl. Acad. Sci. USA] 69, 2110 (1972), Gene 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

　動物細胞を宿主として用いる場合、発現ベクターとしては、動物細胞中で機能を発揮できるものであればいずれも用いることができ、例えば、ｐｃＤＮＡＩ、ｐＣＤＭ８（フナコシ社製）、ｐＡＧＥ１０７［日本国特開平３－２２９７９号公報；Cytotechnology, 3, 133 (1990)］、ｐＡＳ３－３（日本国特開平２－２２７０７５号公報）、ｐＣＤＭ８［Nature, 329, 840 (1987)］、ｐｃＤＮＡＩ／Ａｍｐ（インビトロジェン社製）、ｐｃＤＮＡ３．１（インビトロジェン社製）、ｐＲＥＰ４（インビトロジェン社製）、ｐＡＧＥ１０３［J. Biochemistry, 101, 1307 (1987)］、ｐＡＧＥ２１０、ｐＭＥ１８ＳＦＬ３、ｐＫＡＮＴＥＸ９３（国際公開第９７／１０３５４号）、Ｎ５ＫＧ１ｖａｌ（米国特許第６，００１，３５８号明細書）、ＩＮＰＥＰ４（Ｂｉｏｇｅｎ－ＩＤＥＣ社製）およびトランスポゾンベクター（国際公開第２０１０／１４３６９８号）などが挙げられる。 When animal cells are used as hosts, any expression vector can be used as long as it can function in animal cells. For example, pcDNAI, pCDM8 (manufactured by Funakoshi), pAGE107 [Japanese Unexamined Patent Publication No. 3]. No. 22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen) , PcDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pKANTEX93 (International Publication No. 97/10354), N5KG1val (US Patent) No. 6,001,358), INPEP4 (Bi gen-IDEC, Inc.) and transposon vector (WO 2010/143698), and the like.

　プロモーターとしては、動物細胞中で機能を発揮できるものであればいずれも用いることができ、例えば、サイトメガロウイルス（ＣＭＶ）のｉｍｍｅｄｉａｔｅ　ｅａｒｌｙ（ＩＥ）遺伝子のプロモーター、ＳＶ４０の初期プロモーター、レトロウイルスのプロモーター、メタロチオネインプロモーター、ヒートショックプロモーター、ＳＲαプロモーターまたはモロニーマウス白血病ウイルスのプロモーター若しくはエンハンサーが挙げられる。また、ヒトＣＭＶのＩＥ遺伝子のエンハンサーをプロモーターと共に用いてもよい。 Any promoter can be used as long as it can function in animal cells. For example, a cytomegalovirus (CMV) immediate early (IE) gene promoter, an SV40 early promoter, a retroviral promoter. , Metallothionein promoter, heat shock promoter, SRα promoter or Moloney murine leukemia virus promoter or enhancer. In addition, an enhancer of human CMV IE gene may be used together with a promoter.

　宿主細胞としては、例えば、ヒト白血病細胞Ｎａｍａｌｗａ細胞、サル細胞ＣＯＳ細胞、チャイニーズ・ハムスター卵巣細胞ＣＨＯ細胞［Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60 , 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 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. 883-900)］；ジヒドロ葉酸還元酵素遺伝子（以下、ｄｈｆｒと表記する）が欠損したＣＨＯ細胞（ＣＨＯ／ＤＧ４４細胞）［Proc.Natl.Acad.Sci.USA,77,4216(1980)］、ＣＨＯ－Ｋ１（ＡＴＣＣ　ＣＣＬ－６１）、ＤＵｋＸＢ１１（ＡＴＣＣ　ＣＣＬ－９０９６）、Ｐｒｏ－５（ＡＴＣＣ　ＣＣＬ－１７８１）、ＣＨＯ－Ｓ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ、Ｃａｔ＃１１６１９）、Ｐｒｏ－３、ラットミエローマ細胞ＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０（またはＹＢ２／０ともいう）、マウスミエローマ細胞ＮＳＯ、マウスミエローマ細胞ＳＰ２／０－Ａｇ１４、シリアンハムスター細胞ＢＨＫまたはＨＢＴ５６３７（日本国特開昭６３－０００２９９号公報）などが挙げられる。 Examples of host cells 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); Chromosoma, 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, sAppendix I, II (pp. 883- 900)]; CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) (CHO / DG44 cells) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC) CCL-1781), CHO-S (Life Technologies, Cat # 11619), Pro-3, rat myeloma cells YB2 / 3HL. P2. G11.16 Ag. 20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14, Syrian hamster cell BHK or HBT5637 (Japanese Unexamined Patent Publication No. 63-000299).

　宿主細胞への組換えベクターの導入方法としては、動物細胞にＤＮＡを導入する方法であればいずれも用いることができ、例えば、エレクトロポレーション法［Cytotechnology, 3, 133 (1990)］、リン酸カルシウム法（日本国特開平２－２２７０７５号公報）またはリポフェクション法［Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)］、などが挙げられる。 As a method for introducing a recombinant vector into a host cell, any method can be used as long as it introduces DNA into animal cells. For example, electroporation [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

　以上のようにして得られるヒトＣＣＲ１をコードするＤＮＡを組み込んだ組換えベクターを保有する微生物または動物細胞などの由来の形質転換体を培地中で培養し、培養液中に該ヒトＣＣＲ１を生成蓄積させ、該培養液から採取することにより、ヒトＣＣＲ１を製造することができる。該形質転換体を培地中で培養する方法は、宿主の培養に用いられる通常の方法に従って行うことができる。 A transformant derived from a microorganism or animal cell having a recombinant vector incorporating a DNA encoding human CCR1 obtained as described above is cultured in a medium, and the human CCR1 is produced and accumulated in the culture solution. Human CCR1 can be produced by collecting from the culture medium. The method of culturing the transformant in a medium can be carried out according to a usual method used for culturing a host.

　真核生物由来の細胞で発現させた場合には、糖または糖鎖が付加されたヒトＣＣＲ１を得ることができる。 When expressed in cells derived from eukaryotes, human CCR1 with an added sugar or sugar chain can be obtained.

　誘導性のプロモーターを用いた組換えベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、ｌａｃプロモーターを用いた組換えベクターで形質転換した微生物を培養する場合にはイソプロピル－β－Ｄ－チオガラクトピラノシドなどを、ｔｒｐプロモーターを用いた組換えベクターで形質転換した微生物を培養する場合にはインドールアクリル酸などを培地に添加してもよい。 When culturing a microorganism transformed with a recombinant vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, cultivate a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like using a recombinant vector using the trp promoter. In this case, indole acrylic acid or the like may be added to the medium.

　動物細胞を宿主として得られた形質転換体を培養する培地としては、例えば、一般に使用されているＲＰＭＩ１６４０培地［The Journal of the American Medical Association, 199, 519 (1967)］、ＥａｇｌｅのＭＥＭ培地［Science, 122, 501 (1952)］、ダルベッコ改変ＭＥＭ培地［Virology, 8, 396 (1959)］、199培地[Proc. Soc. Exp. Biol. Med., 73, 1 (1950)］若しくはＩｓｃｏｖｅ’ｓ　Ｍｏｄｉｆｉｅｄ　Ｄｕｌｂｅｃｃｏ’ｓ　Ｍｅｄｉｕｍ（ＩＭＤＭ）培地またはこれら培地に牛胎児血清（ＦＢＳ）などを添加した培地などが挙げられる。培養は、通常ｐＨ６～８、３０～４０℃、５％ＣＯ２存在下などの条件下で１～７日間行う。また、培養中に必要に応じて、カナマイシンまたはペニシリンなどの抗生物質を培地に添加してもよい。 As a medium for culturing a transformant obtained using animal cells as a host, for example, 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)] or Iscove's Modified Examples include a Dulbecco's Medium (IMDM) medium or a medium obtained by adding fetal bovine serum (FBS) or the like to these mediums. The culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO 2 . Moreover, you may add antibiotics, such as kanamycin or penicillin, to a culture medium during culture | cultivation as needed.

　ヒトＣＣＲ１をコードする遺伝子の発現方法としては、例えば、直接発現以外に、分泌生産または融合タンパク質発現などの方法［Molecular Cloning, A Laboratory Manual,Second Edition,Cold Spring Harbor Laboratory Press (1989)］が挙げられる。 As an expression method of the gene encoding human CCR1, for example, in addition to direct expression, methods such as secretory production or fusion protein expression [Molecular® Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] can be mentioned. It is done.

　ヒトＣＣＲ１の生産方法としては、例えば、宿主細胞内に生産させる方法、宿主細胞外に分泌させる方法、または宿主細胞外膜上に生産させる方法が挙げられ、使用する宿主細胞または生産させるヒトＣＣＲ１の構造を変えることにより、適切な方法を選択することができる。 Examples of the method for producing human CCR1 include a method for producing it in a host cell, a method for producing it secreted outside the host cell, and a method for producing it on the host cell outer membrane. By changing the structure, an appropriate method can be selected.

　ヒトＣＣＲ１が宿主細胞内又は宿主細胞外膜上に生産される場合、ポールソンらの方法［J. Biol. Chem., 264, 17619 (1989)］、ロウらの方法［Proc. Natl. Acad. Sci., USA, 86, 8227 (1989)、Genes Develop., 4, 1288 (1990)］、日本国特開平０５－３３６９６３号公報または国際公開第９４／２３０２１号などに記載の方法を用いることにより、ヒトＣＣＲ１を宿主細胞外に積極的に分泌させることができる。また、ジヒドロ葉酸還元酵素遺伝子などを用いた遺伝子増幅系（日本国特開平２－２２７０７５号公報）を利用してヒトＣＣＲ１の生産量を上昇させることもできる。 When human CCR1 is produced in or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 1989 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci , USA, 86, 19898227 (1989), Genes Develop., 4, 1288 (1990)], Japanese Patent Application Laid-Open No. 05-336963, International Publication No. 94/23021, etc. Human CCR1 can be actively secreted outside the host cell. In addition, the production amount of human CCR1 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Application Laid-Open No. 2-227075).

　得られたヒトＣＣＲ１は、例えば、以下のようにして単離、精製することができる。ヒトＣＣＲ１が細胞内に溶解状態で発現した場合には、培養終了後に細胞を遠心分離により回収し、水系緩衝液に懸濁後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザーまたはダイノミルなどを用いて細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、通常のタンパク質の単離精製法、即ち、溶媒抽出法、硫安などによる塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（ＤＥＡＥ）－セファロース、ＤＩＡＩＯＮ　ＨＰＡ－７５（三菱化学社製）などのレジンを用いた陰イオン交換クロマトグラフィー法、Ｓ－Ｓｅｐｈａｒｏｓｅ　ＦＦ（ファルマシア社製）などのレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロースなどのレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、または等電点電気泳動などの電気泳動法などの手法を単独または組み合わせて用い、精製標品を得ることができる。 The obtained human CCR1 can be isolated and purified as follows, for example. When human CCR1 is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, and then used with an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, or a dynomill. The cells are disrupted to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary 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 hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing To obtain purified preparations It is possible.

　ヒトＣＣＲ１が細胞内に不溶体を形成して発現した場合は、上記と同様に細胞を回収後破砕し、遠心分離を行うことにより、沈殿画分として該ヒトＣＣＲ１の不溶体を回収する。回収した該ヒトＣＣＲ１の不溶体をタンパク質変性剤で可溶化する。該可溶化液を希釈または透析することにより、該ヒトＣＣＲ１を正常な立体構造に戻した後、上記と同様の単離精製法によりポリペプチドの精製標品を得ることができる。 When human CCR1 is expressed in the form of an insoluble substance in the cell, the cell is collected and disrupted in the same manner as described above, and centrifuged to collect the insoluble substance of human CCR1 as a precipitate fraction. The recovered insoluble body of human CCR1 is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to return the human CCR1 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.

　ヒトＣＣＲ１またはその糖修飾体などの誘導体が細胞外に分泌された場合には、培養上清において該ヒトＣＣＲ１またはその糖修飾体などの誘導体を回収することができる。該培養物を上記と同様に遠心分離などの手法により処理することにより可溶性画分を取得し、該可溶性画分から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。 When a derivative such as human CCR1 or a modified sugar thereof is secreted extracellularly, the derivative such as human CCR1 or a modified sugar 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.

　また、本発明において用いられるヒトＣＣＲ１は、Ｆｍｏｃ法またはｔＢｏｃ法などの化学合成法によっても製造することができる。また、アドバンストケムテック社製、パーキン・エルマー社製、ファルマシア社製、プロテインテクノロジインストルメント社製、シンセセル－ベガ社製、パーセプチブ社製または島津製作所社製などのペプチド合成機を利用して化学合成することもできる。 In addition, human CCR1 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

（２）動物の免疫と融合用抗体産生細胞の調製
　３～２０週令のマウス、ラットまたはハムスターなどの動物に、（１）で得られる抗原を免疫して、その動物の脾、リンパ節、末梢血中の抗体産生細胞を採取する。また、マウスＣＣＲ１ノックアウトマウスを被免疫動物として用いることもできる。 (2) Immunization of animals and preparation of antibody-producing cells for fusion 3 to 20-week-old animals such as mice, rats or hamsters are immunized with the antigen obtained in (1), and the spleen, lymph nodes, Collect antibody-producing cells in peripheral blood. Mouse CCR1 knockout mice can also be used as immunized animals.

　免疫は、動物の皮下、静脈内または腹腔内に、例えば、フロインドの完全アジュバント、または水酸化アルミニウムゲルと百日咳菌ワクチンなどの適当なアジュバントとともに抗原を投与することにより行う。抗原が部分ペプチドである場合には、ＢＳＡ（ウシ血清アルブミン）またはＫＬＨ（Ｋｅｙｈｏｌｅ　Ｌｉｍｐｅｔ　ｈｅｍｏｃｙａｎｉｎ）などのキャリアタンパク質とコンジュゲートを作製し、これを免疫原として用いる。 Immunization is performed by administering the antigen subcutaneously, intravenously or intraperitoneally to the animal together with an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine. When 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.

　抗原の投与は、１回目の投与の後、１～２週間おきに５～１０回行う。各投与後３～７日目に眼底静脈叢より採血し、その血清の抗体価を酵素免疫測定法［Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)］などを用いて測定する。免疫に用いた抗原に対し、その血清が十分な抗体価を示した動物を融合用抗体産生細胞の供給源とする。 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.

　抗原の最終投与後３～７日目に、免疫した動物より脾臓などの抗体産生細胞を含む組織を摘出し、抗体産生細胞を採取する。脾臓細胞を用いる場合には、脾臓を細断、ほぐした後、遠心分離し、さらに赤血球を除去して融合用抗体産生細胞を取得する。 3 to 7 days after the final administration of the antigen, tissues containing antibody-producing cells such as the spleen are removed from the immunized animal, and antibody-producing cells are collected. When spleen cells are used, the spleen is shredded and loosened, and then centrifuged, and the erythrocytes are removed to obtain antibody producing cells for fusion.

（３）骨髄腫細胞の調製
　骨髄腫細胞としては、マウスから得られた株化細胞を用い、例えば、８－アザグアニン耐性マウス（ＢＡＬＢ／ｃ由来）骨髄腫細胞株Ｐ３－Ｘ６３Ａｇ８－Ｕ１（Ｐ３－Ｕ１）［Current Topics in Microbiology and Immunology, 18, 1 (1978)］、Ｐ３－ＮＳ１／１－Ａｇ４１（ＮＳ－１）［European J. Immunology, 6, 511 (1976)］、ＳＰ２／０－Ａｇ１４（ＳＰ－２）［Nature, 276, 269 (1978)]、Ｐ３－Ｘ６３－Ａｇ８６５３（６５３）［J. Immunology, 123, 1548 (1979)］またはＰ３－Ｘ６３－Ａｇ８（Ｘ６３）［Nature, 256, 495 (1975)］などが用いられる。 (3) Preparation of myeloma cells As a myeloma cell, a cell line obtained from a mouse is used. For example, an 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (P3- U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1 / 1-Ag41 (NS-1) [European J. Immunology, 6, 511 (1976)], SP2 / 0-Ag14 ( SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)] or P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] is used.

　該骨髄腫細胞は、正常培地［グルタミン、２－メルカプトエタノール、ジェンタマイシン、ＦＢＳ、および８－アザグアニンを加えたＲＰＭＩ１６４０培地］で継代し、細胞融合の３～４日前に正常培地に継代し、融合当日２×１０７個以上の細胞数を確保する。 The myeloma cells are passaged in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine] and passaged to normal medium 3-4 days prior to cell fusion. On the day of fusion, secure a cell count of 2 × 10 7 or more.

（４）細胞融合とモノクローナル抗体産生ハイブリドーマの調製
　（２）で得られる融合用抗体産生細胞と（３）で得られる骨髄腫細胞をＭｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ（ＭＥＭ）培地またはＰＢＳ（リン酸二ナトリウム１．８３ｇ、リン酸一カリウム０．２１ｇ、食塩７．６５ｇ、蒸留水１リットル、ｐＨ７．２）でよく洗浄し、細胞数が、融合用抗体産生細胞：骨髄腫細胞＝５～１０：１になるよう混合し、遠心分離した後、上清を除く。沈澱した細胞群をよくほぐした後、ポリエチレングリコール－１０００（ＰＥＧ－１０００）、ＭＥＭ培地およびジメチルスルホキシドの混液を３７℃で、攪拌しながら加える。さらに１～２分間毎にＭＥＭ培地１～２ｍＬを数回加えた後、ＭＥＭ培地を加えて全量が５０ｍＬになるようにする。遠心分離後、上清を除く。沈澱した細胞群をゆるやかにほぐした後、融合用抗体産生細胞にＨＡＴ培地［ヒポキサンチン、チミジン、およびアミノプテリンを加えた正常培地］中にゆるやかに細胞を懸濁する。この懸濁液を５％ＣＯ２インキュベーター中、３７℃にて７～１４日間培養する。 (4) Cell Fusion and Preparation of Monoclonal Antibody-Producing Hybridoma The antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) are treated with Minimum Essential Medium (MEM) medium or PBS (disodium phosphate 1. 83 g, monopotassium phosphate 0.21 g, sodium chloride 7.65 g, distilled water 1 liter, pH 7.2), and the number of cells becomes fusion antibody-producing cells: myeloma cells = 5 to 10: 1 After mixing and centrifuging, remove the supernatant. After loosening the precipitated cells, a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethyl sulfoxide is added at 37 ° C. with stirring. Add 1-2 mL of MEM medium several times every 1-2 minutes, and then add MEM medium to a total volume of 50 mL. After centrifugation, remove the supernatant. After loosening the precipitated cells, 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.

　培養後、培養上清の一部を抜き取り、後述のバインディングアッセイなどのハイブリドーマの選択方法により、ヒトＣＣＲ１を含む抗原に反応し、ヒトＣＣＲ１を含まない抗原に反応しない細胞群を選択する。次に、限界希釈法によりクローニングを行い、安定して強い抗体価の認められたものをモノクローナル抗体産生ハイブリドーマとして選択する。 After culturing, a part of the culture supernatant is extracted, and a cell group that reacts with an antigen containing human CCR1 and does not react with an antigen not containing human CCR1 is selected by a hybridoma selection method such as a binding assay described later. Next, cloning is performed by a limiting dilution method, and a stable and strong antibody titer recognized is selected as a monoclonal antibody-producing hybridoma.

（５）精製モノクローナル抗体の調製
　プリスタン処理［２，６，１０，１４－テトラメチルペンタデカン（Ｐｒｉｓｔａｎｅ）０．５ｍＬを腹腔内投与し、２週間飼育する］した８～１０週令のマウスまたはヌードマウスに、（４）で得られるモノクローナル抗体産生ハイブリドーマを腹腔内に注射する。１０～２１日でハイブリドーマは腹水癌化する。このマウスから腹水を採取し、遠心分離して固形分を除去後、４０～５０％硫酸アンモニウムで塩析し、カプリル酸沈殿法、ＤＥＡＥ－セファロースカラム、プロテインＡ－カラムまたはゲル濾過カラムによる精製を行ない、ＩｇＧまたはＩｇＭ画分を集め、精製モノクローナル抗体とする。 (5) Preparation of Purified Monoclonal Antibody 8- to 10-week-old mice or nude mice treated with pristane [2,6,10,14-tetramethylpentadecane (Pristane) 0.5 mL intraperitoneally and bred for 2 weeks] Then, the monoclonal antibody-producing hybridoma obtained in (4) is injected intraperitoneally. In 10 to 21 days, the hybridoma becomes ascites tumor. Ascites was collected from this mouse, centrifuged to remove solids, salted out with 40-50% ammonium sulfate, and purified by caprylic acid precipitation, DEAE-Sepharose column, Protein A-column or gel filtration column. The IgG or IgM fraction is collected and used as a purified monoclonal antibody.

　また、（４）で得られるモノクローナル抗体産生ハイブリドーマを、１０％ＦＢＳを添加したＲＰＭＩ１６４０培地などで培養した後、遠心分離により上清を除き、Ｈｙｂｒｉｄｏｍａ　ＳＦＭ培地に懸濁し、３～７日間培養する。得られた細胞懸濁液を遠心分離し、得られた上清よりプロテインＡ－カラムまたはプロテインＧ－カラムによる精製を行ない、ＩｇＧ画分を集め、精製モノクローナル抗体を得ることもできる。なお、Ｈｙｂｒｉｄｏｍａ　ＳＦＭ培地には５％ダイゴＧＦ２１を添加することもできる。 Further, after culturing the monoclonal antibody-producing hybridoma obtained in (4) in an RPMI1640 medium supplemented with 10% FBS, the supernatant is removed by centrifugation, 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. In addition, 5% Digo GF21 can also be added to the Hybridoma SFM medium.

　抗体のサブクラスの決定は、サブクラスタイピングキットを用いて酵素免疫測定法により行う。タンパク質量の定量は、ローリー法または２８０ｎｍでの吸光度より算出する。 The antibody subclass is determined by enzyme immunoassay using a sub-clustering kit. The amount of protein is calculated from the Raleigh method or absorbance at 280 nm.

（６）モノクローナル抗体の選択
　モノクローナル抗体の選択は以下に示すように、フローサイトメトリーを用いて、ヒトＣＣＲ１発現細胞への抗体の結合性を測定することなどにより行う。ヒトＣＣＲ１発現細胞は、細胞表面上にヒトＣＣＲ１が発現していればいずれの細胞でもよく、例えば、ヒト細胞、ヒト細胞株および（１）で得られるヒトＣＣＲ１強制発現細胞株などが挙げられる。 (6) Selection of monoclonal antibody The monoclonal antibody is selected by measuring the binding of the antibody to human CCR1-expressing cells using flow cytometry, as shown below. The human CCR1-expressing cells may be any cells as long as human CCR1 is expressed on the cell surface, and examples thereof include human cells, human cell lines, and human CCR1 forced expression cell lines obtained in (1).

　ヒトＣＣＲ１発現細胞を９６ウェルプレートなどのプレートに分注した後、第１抗体として血清、ハイブリドーマの培養上清又は精製モノクローナル抗体などの被験物質を分注し、反応させる。反応後の細胞を１～１０％ｂｏｖｉｎｅ　ｓｅｒｕｍ　ａｌｂｕｍｉｎ（ＢＳＡ）を含むＰＢＳ（以下、ＢＳＡ－ＰＢＳと記す）などで、よく洗浄した後、第２抗体として蛍光試薬などで標識した抗イムノグロブリン抗体を分注して反応させる。ＢＳＡ－ＰＢＳなどでよく洗浄した後、フローサイトメーターを用いて標識化抗体の蛍光量を測定することにより、ヒトＣＣＲ１発現細胞に対して特異的に反応するモノクローナル抗体を選択する。 After dispensing human CCR1-expressing cells into a plate such as a 96-well plate, a test substance such as serum, hybridoma culture supernatant or purified monoclonal antibody is dispensed as a first antibody and allowed to react. The cells after the reaction are thoroughly washed with PBS containing 1 to 10% bovine serum albumin (BSA) (hereinafter referred to as BSA-PBS), and then anti-immunoglobulin antibody labeled with a fluorescent reagent or the like as a second antibody. Dispense and react. After thoroughly washing with BSA-PBS or the like, a monoclonal antibody that specifically reacts with human CCR1-expressing cells is selected by measuring the amount of fluorescence of the labeled antibody using a flow cytometer.

　また、本発明の抗体と競合してヒトＣＣＲ１に結合する抗体は、上述のフローサイトメトリーを用いた測定系に、被験抗体を添加して反応させることで取得できる。すなわち、被験抗体を加えた時に本発明の抗体とヒトＣＣＲ１との結合が阻害される抗体をスクリーニングすることにより、ヒトＣＣＲ１のアミノ酸配列、又はその立体構造への結合について、本発明の抗体と競合するモノクローナル抗体を取得することができる。　 In addition, an antibody that competes with the antibody of the present invention and binds to human CCR1 can be obtained by adding a test antibody to the above-described measurement system using flow cytometry and reacting. That is, by screening for an antibody that inhibits the binding between the antibody of the present invention and human CCR1 when a test antibody is added, it competes with the antibody of the present invention for binding to the amino acid sequence of human CCR1 or its three-dimensional structure. Monoclonal antibodies can be obtained. *

　また、本発明のヒトＣＣＲ１に結合するモノクローナル抗体が結合するエピトープを含むエピトープに結合する抗体は、上述のスクリーニング方法で取得された抗体のエピトープを公知の方法で同定し、同定したエピトープを含む合成ペプチド、またはエピトープの立体構造に擬態させた合成ペプチド等を作製し、免疫することで取得することができる。 In addition, an antibody that binds to an epitope containing an epitope to which a monoclonal antibody that binds to human CCR1 of the present invention binds is identified by a known method in which the epitope of the antibody obtained by the screening method described above is synthesized. A peptide or a synthetic peptide that mimics the three-dimensional structure of an epitope can be prepared and obtained by immunization.

　更に、本発明のヒトＣＣＲ１に結合するモノクローナル抗体が結合するエピトープと、同じエピトープに結合する抗体は、上述のスクリーニング方法で取得された抗体のエピトープを同定し、同定したエピトープの部分的な合成ペプチド、またはエピトープの立体構造に擬態させた合成ペプチド等を作製し、免疫することで、取得することができる。 Furthermore, the epitope that the monoclonal antibody that binds to human CCR1 of the present invention binds to the same epitope is identified by identifying the epitope of the antibody obtained by the screening method described above, and a partial synthetic peptide of the identified epitope Alternatively, a synthetic peptide or the like that mimics the three-dimensional structure of an epitope is prepared and immunized.

２．遺伝子組換え抗体の作製
　遺伝子組換え抗体の作製例として、以下にヒト型キメラ抗体およびヒト化抗体の作製方法を示す。遺伝子組換えのマウス抗体、ラット抗体およびラビット抗体なども同様の方法で作製することができる。 2. Production of Recombinant Antibody As a production example of a recombinant antibody, a method for producing a human chimeric antibody and a humanized antibody is shown below. Recombinant mouse antibodies, rat antibodies, rabbit antibodies, and the like can be prepared by the same method.

（１）遺伝子組換え抗体発現用ベクターの構築
　遺伝子組換え抗体発現用ベクターは、ヒト抗体のＣＨおよびＣＬをコードするＤＮＡが組み込まれた動物細胞用発現ベクターであり、動物細胞用発現ベクターにヒト抗体のＣＨおよびＣＬをコードするＤＮＡをそれぞれクローニングすることにより構築することができる。 (1) Construction of Recombinant Antibody Expression Vector 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.

　ヒト抗体のＣ領域は任意のヒト抗体のＣＨおよびＣＬを用いることができる。例えば、ヒト抗体のγ１サブクラスのＣＨおよびκクラスのＣＬなどを用いる。ヒト抗体のＣＨおよびＣＬをコードするＤＮＡには、ｃＤＮＡを用いるが、エキソンとイントロンからなる染色体ＤＮＡを用いることもできる。動物細胞用発現ベクターには、ヒト抗体のＣ領域をコードする遺伝子を組込み発現できるものであればいかなるものでも用いることができる。例えば、ｐＡＧＥ１０７［Cytotechnol., 3, 133 (1990)］、ｐＡＧＥ１０３［J. Biochem., 101, 1307 (1987)］、ｐＨＳＧ２７４［Gene, 27, 223 (1984)］、ｐＫＣＲ［Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)］、ｐＳＧ１ｂｄ２－４［Cytotechnol., 4, 173 (1990)］またはｐＳＥ１ＵＫ１Ｓｅｄ１－３［Cytotechnol., 13, 79 (1993)］などを用いる。動物細胞用発現ベクターのうちプロモーターとエンハンサーには、ＳＶ４０の初期プロモーター［J. Biochem., 101, 1307 (1987)］、モロニーマウス白血病ウイルスＬＴＲ［Biochem. Biophys. Res. Commun., 149, 960 (1987)］または免疫グロブリンＨ鎖のプロモーター［Cell, 41, 479 (1985)］とエンハンサー［Cell, 33, 717 (1983)］などが挙げられる。 Any human antibody CH and CL can be used for the C region of a human antibody. For example, γ1 subclass CH and κ class CL of human antibodies are used. Although 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. For example, pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)] or pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)]. Among the expression vectors for animal cells, 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)].

　遺伝子組換え抗体発現用ベクターには、遺伝子組換え抗体発現ベクターの構築の容易さ、動物細胞への導入の容易さ、動物細胞内での抗体Ｈ鎖およびＬ鎖の発現量のバランスが均衡するなどの点から、抗体Ｈ鎖およびＬ鎖が同一のベクター上に存在するタイプ（タンデム型）の遺伝子組換え抗体発現用ベクター［J．Immunol． Methods， 167， 271（1994）］を用いるが、抗体H鎖およびL鎖が別々のベクター上に存在するタイプを用いることもできる。タンデム型の遺伝子組換え抗体発現用ベクターには、ｐＫＡＮＴＥＸ９３（国際公開第９７／１０３５４号）、ｐＥＥ１８［Hybridoma, 17, 559 (1998)］などを用いる。 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. In view of the above, 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)], but a type in which the antibody H chain and L chain are present on separate vectors can also be used. As a tandem recombinant antibody expression vector, pKANTEX93 (International Publication No. 97/10354), pEE18 [Hybridoma, 17, 559 (1998)] or the like is used.

（２）ヒト以外の動物由来の抗体のＶ領域をコードするｃＤＮＡの取得およびアミノ酸配列の解析
　非ヒト抗体のＶＨ及びＶＬをコードするｃＤＮＡの取得およびアミノ酸配列の解析は以下のようにして行うことができる。 (2) Obtaining cDNA encoding the V region of an antibody derived from a non-human animal and analyzing the amino acid sequence Obtaining cDNA encoding the VH and VL of the non-human antibody and analyzing the amino acid sequence should be performed as follows. Can do.

　非ヒト抗体を産生するハイブリドーマ細胞よりｍＲＮＡを抽出し、ｃＤＮＡを合成する。合成したｃＤＮＡをファージまたはプラスミドなどのベクターにクローニングしてｃＤＮＡライブラリーを作製する。該ライブラリーより、マウス抗体のＣ領域部分またはＶ領域部分をコードするＤＮＡをプローブとして用い、ＶＨ若しくはＶＬをコードするｃＤＮＡを有する組換えファージまたは組換えプラスミドをそれぞれ単離する。組換えファージまたは組換えプラスミド上の目的とするマウス抗体のＶＨまたはＶＬの全塩基配列をそれぞれ決定し、塩基配列よりＶＨまたはＶＬの全アミノ酸配列をそれぞれ推定する。 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.

　非ヒト抗体を産生するハイブリドーマ細胞を作製するヒト以外の動物には、マウス、ラット、ハムスターまたはラビットなどを用いるが、ハイブリドーマ細胞を作製することが可能であれば、いかなる動物も用いることができる。 As a non-human animal for producing a hybridoma cell producing a non-human antibody, mouse, rat, hamster, rabbit or the like is used, but any animal can be used as long as it can produce a hybridoma cell.

　ハイブリドーマ細胞からの全ＲＮＡの調製には、チオシアン酸グアニジン－トリフルオロ酢酸セシウム法［Methods in Enzymol., 154, 3 (1987)］、またはＲＮＡ　ｅａｓｙ　ｋｉｔ（キアゲン社製）などのキットなどを用いる。 For preparation of total RNA from hybridoma cells, a kit such as the guanidine thiocyanate-cesium acetate trifluoroacetate method [Methods in Enzymol., 154, 31987 (1987)] or RNA easy kit (manufactured by Qiagen) is used.

　全ＲＮＡからのｍＲＮＡの調製には、オリゴ（ｄＴ）固定化セルロースカラム法［Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)］、またはＯｌｉｇｏ－ｄＴ３０＜Ｓｕｐｅｒ＞ｍＲＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ（登録商標）Ｋｉｔ（タカラバイオ社製）などのキットなどを用いる。また、Ｆａｓｔ　Ｔｒａｃｋ　ｍＲＮＡ　Ｉｓｏｌａｔｉｏｎ（登録商標）Ｋｉｔ（インビトロジェン社製）、またはＱｕｉｃｋＰｒｅｐ　ｍＲＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ（登録商標）Ｋｉｔ（ファルマシア社製）などのキットを用いてハイブリドーマ細胞からｍＲＮＡを調製することもできる。 For preparation of mRNA from total RNA, oligo (dT) immobilized cellulose column method [MolecularMCloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], or Oligo-dT30 <Super> mRNA Purification ( A kit such as a registered trademark Kit (manufactured by Takara Bio Inc.) is used. Alternatively, 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).

　ｃＤＮＡの合成およびｃＤＮＡライブラリーの作製には、公知の方法［Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)、Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)］、またはＳｕｐｅｒＳｃｒｉｐｔ　Ｐｌａｓｍｉｄ　Ｓｙｓｔｅｍ　ｆｏｒ　ｃＤＮＡ　Ｓｙｎｔｈｅｓｉｓ　ａｎｄ　Ｐｌａｓｍｉｄ　Ｃｌｏｎｉｎｇ（インビトロジェン社製）若しくはＺＡＰ－ｃＤＮＡ　Ｓｙｎｔｈｅｓｉｓ（登録商標）Ｋｉｔ（ストラタジーン社製）などのキットなどを用いる。 For synthesis of cDNA and preparation of cDNA library, known methods [Molecular Cloning, A Laboratory Manual, C Second Spring, Harbor Spring Laboratory Press (1989), Current Protocols in Molecular Molecular Biology, Supplement John 1, Wilson & Sons (1987 -1997)], or a kit such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Invitrogen) or ZAP-cDNA Synthesis (registered trademark) Kit (manufactured by Stratagene).

　ｃＤＮＡライブラリーの作製の際、ハイブリドーマ細胞から抽出したｍＲＮＡを鋳型として合成したｃＤＮＡを組み込むベクターには、該ｃＤＮＡを組み込めるベクターであればいかなるものでも用いることができる。例えば、ＺＡＰ　Ｅｘｐｒｅｓｓ［Strategies, 5, 58 (1992)］、ｐＢｌｕｅｓｃｒｉｐｔ　ＩＩ　ＳＫ（＋）［Nucleic Acids Research, 17, 9494 (1989)］、λＺＡＰＩＩ（Ｓｔｒａｔａｇｅｎｅ社製）、λｇｔ１０、λｇｔ１１［DNA Cloning:A Practical Approach, I, 49 (1985)］、Ｌａｍｂｄａ　ＢｌｕｅＭｉｄ（クローンテック社製）、λＥｘＣｅｌｌ、ｐＴ７Ｔ３－１８Ｕ（ファルマシア社製）、ｐＣＤ２［Mol. Cell. Biol., 3, 280 (1983)］またはｐＵＣ１８［Gene, 33, 103 (1985)］などを用いる。 When preparing a cDNA library, any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma cell as a template is incorporated. For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPIII (manufactured by Stratagene), λgt10, λgt11: DNA C11 Approach, I, 49 (1985)], Lambda BlueMid (manufactured by Clontech), λExCell, pT7T3-18U (manufactured by Pharmacia), pCD2 [Mol. Cell. Biol., 3, 280 (1983)] or pUC18 [Gene , 33, 103 (1985)].

　ファージまたはプラスミドベクターにより構築されるｃＤＮＡライブラリーを導入する大腸菌には、該ｃＤＮＡライブラリーを導入、発現および維持できるものであればいかなるものでも用いることができる。例えば、ＸＬ１－Ｂｌｕｅ　ＭＲＦ’［Strategies, 5, 81 (1992)］、Ｃ６００［Genetics, 39, 440 (1954)］、Ｙ１０８８、Ｙ１０９０［Science, 222, 778 (1983)］、ＮＭ５２２［J. Mol. Biol., 166, 1 (1983)］、Ｋ８０２［J. Mol. Biol., 16, 118 (1966)］またはＪＭ１０５［Gene, 38, 275 (1985)］などを用いる。 Any 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. For example, XL1-Blue MRF ′ [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)] or JM105 [Gene, 38, 275 (1985)] or the like is used.

　ｃＤＮＡライブラリーからの非ヒト抗体のＶＨまたはＶＬをコードするｃＤＮＡクローンの選択には、アイソトープ若しくは蛍光標識したプローブを用いたコロニー・ハイブリダイゼーション法、またはプラーク・ハイブリダイゼーション法［Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)］などを用いる。 For selection of cDNA clones encoding non-human antibody VH or VL from a cDNA library, a colony hybridization method using an isotope or fluorescently labeled probe, or a plaque hybridization method [Molecular Cloning, A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press (1989)].

　また、プライマーを調製し、ｍＲＮＡから合成したｃＤＮＡまたはｃＤＮＡライブラリーを鋳型として、Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ法［以下、ＰＣＲ法と表記する、Molecular Cloning, A Laboratory Manual, Second Edition , Cold Spring Harbor Laboratory Press (1989)、Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)］を行うことよりＶＨまたはＶＬをコードするｃＤＮＡを調製することもできる。 In addition, primers were prepared, and cDNA or cDNA library synthesized from mRNA was used as a template. Polymerase Chain Reaction method [hereinafter referred to as PCR method, Molecular® Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989 ), Current Protocols in Molecular Molecular Biology, Supplements 1, John Wiley & Sons (1987-1997)], cDNA encoding VH or VL can also be prepared.

　選択されたｃＤＮＡを、適当な制限酵素などで切断後、ｐＢｌｕｅｓｃｒｉｐｔ　ＳＫ（－）（ストラタジーン社製）などのプラスミドにクローニングし、通常用いられる塩基配列解析方法などにより該ｃＤＮＡの塩基配列を決定する。塩基配列解析方法には、例えば、ジデオキシ法［Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)］などの反応を行った後、ＡＢＩ　ＰＲＩＳＭ３７００（ＰＥバイオシステムズ社製）またはＡ．Ｌ．Ｆ．ＤＮＡシークエンサー（ファルマシア社製）などの塩基配列自動分析装置などを用いる。 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. . The base sequence analysis method includes, for example, a dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], ABI PRISM 3700 (PE Biosystems) or A.I. L. F. An automatic base sequence analyzer such as a DNA sequencer (Pharmacia) is used.

　決定した塩基配列からＶＨおよびＶＬの全アミノ酸配列をそれぞれ推定し、既知の抗体のＶＨおよびＶＬの全アミノ酸配列［Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)］と比較することにより、取得したｃＤＮＡが分泌シグナル配列を含む抗体のＶＨおよびＶＬの完全なアミノ酸配列をコードしているかをそれぞれ確認する。分泌シグナル配列を含む抗体のＶＨおよびＶＬの完全なアミノ酸配列に関しては、既知の抗体のＶＨおよびＶＬの全アミノ酸配列［Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)］と比較することにより、分泌シグナル配列の長さおよびＮ末端アミノ酸配列を推定でき、更にはそれらが属するサブグループを知ることができる。また、ＶＨおよびＶＬの各ＣＤＲのアミノ酸配列についても、既知の抗体のＶＨおよびＶＬのアミノ酸配列［Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)］と比較することによって見出すことができる。 The total amino acid sequences of VH and VL are estimated from the determined nucleotide sequences, respectively, and compared with the entire amino acid sequences of known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] Thus, it is confirmed whether the obtained cDNA codes for the complete amino acid sequence of VH and VL of the antibody including the secretory signal sequence. Compare the complete amino acid sequence of the VH and VL of the antibody containing the secretory signal sequence with the entire amino acid sequence of the known antibody VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] By doing so, the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and further, the subgroup to which they belong can be known. In addition, the amino acid sequences of CDRs of VH and VL are also found by comparing with the VH and VL amino acid sequences of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Can do.

　また、得られたＶＨおよびＶＬの完全なアミノ酸配列を用いて、例えば、ＳＷＩＳＳ－ＰＲＯＴまたはＰＩＲ－Ｐｒｏｔｅｉｎなどの任意のデータベースに対してＢＬＡＳＴ法［J. Mol. Biol., 215, 403 (1990)］などの相同性検索を行い、ＶＨおよびＶＬの完全なアミノ酸配列が新規なものかを確認できる。 Further, using the obtained complete amino acid sequences of VH and VL, for example, the BLAST method [J. Mol. Biol., 215, 403 (1990) against any database such as SWISS-PROT or PIR-Protein] ] Or the like, and the complete amino acid sequences of VH and VL can be confirmed.

（３）ヒト型キメラ抗体発現ベクターの構築
　（１）で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流に、それぞれ非ヒト抗体のＶＨまたはＶＬをコードするｃＤＮＡをそれぞれクローニングすることで、ヒト型キメラ抗体発現ベクターを構築することができる。 (3) Construction of human chimeric antibody expression vector The non-human antibody VH or VL is upstream of each gene encoding CH or CL of the human antibody of the recombinant antibody expression vector obtained in (1), respectively. A human chimeric antibody expression vector can be constructed by cloning each of the encoded cDNAs.

　非ヒト抗体のＶＨまたはＶＬをコードするｃＤＮＡの３’末端側と、ヒト抗体のＣＨまたはＣＬの５’末端側とを連結するために、連結部分の塩基配列が適切なアミノ酸をコードし、かつ適当な制限酵素認識配列になるように設計したＶＨおよびＶＬのｃＤＮＡを作製する。作製されたＶＨおよびＶＬのｃＤＮＡを、（１）で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流にそれらが適切な形で発現する様にそれぞれクローニングし、ヒト型キメラ抗体発現ベクターを構築する。 In order to link the 3 ′ end of cDNA encoding VH or VL of a non-human antibody and the 5 ′ end of CH or CL of a human antibody, 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 respectively expressed so that they are expressed in an appropriate form upstream of each gene encoding the human antibody CH or CL of the recombinant antibody expression vector obtained in (1). Cloning to construct a human chimeric antibody expression vector.

　また、非ヒト抗体ＶＨまたはＶＬをコードするｃＤＮＡを、適当な制限酵素の認識配列を両端に有する合成ＤＮＡを用いてＰＣＲ法によりそれぞれ増幅し、（１）で得られる遺伝子組換え抗体発現用ベクターにクローニングすることもできる。 Further, 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.

（４）ヒト化抗体のＶ領域をコードするｃＤＮＡの構築
　ヒト化抗体のＶＨまたはＶＬをコードするｃＤＮＡは、以下のようにして構築することができる。 (4) Construction of cDNA Encoding V Region of Humanized Antibody cDNA encoding VH or VL of humanized antibody can be constructed as follows.

　非ヒト抗体のＶＨまたはＶＬのＣＤＲのアミノ酸配列を移植するための、ヒト抗体のＶＨまたはＶＬのＦＲのアミノ酸配列をそれぞれ選択する。選択するＦＲのアミノ酸配列には、ヒト抗体由来のものであれば、いずれのものでも用いることができる。例えば、Ｐｒｏｔｅｉｎ　Ｄａｔａ　Ｂａｎｋなどのデータベースに登録されているヒト抗体のＦＲのアミノ酸配列、またはヒト抗体のＦＲの各サブグループの共通アミノ酸配列［Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)］などを用いる。抗体の結合活性の低下を抑えるため、元の抗体のＶＨまたはＶＬのＦＲのアミノ酸配列とできるだけ高い相同性（少なくとも６０％以上）のＦＲのアミノ酸配列を選択する。 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. For example, human antibody FR amino acid sequences registered in databases such as Protein Data Bank, or common amino acid sequences of each subgroup of human antibody FRs [Sequencesenceof Proteins of Immunological Interest, estUS Dept.eptHealth and Human Services ( 1991)]. In order to suppress a decrease in the binding activity of the antibody, 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.

　次に、選択したヒト抗体のＶＨまたはＶＬのＦＲのアミノ酸配列に、もとの抗体のＣＤＲのアミノ酸配列をそれぞれ移植し、ヒト化抗体のＶＨまたはＶＬのアミノ酸配列をそれぞれ設計する。設計したアミノ酸配列を抗体の遺伝子の塩基配列に見られるコドンの使用頻度［Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)］を考慮してＤＮＡ配列に変換し、ヒト化抗体のＶＨまたはＶＬのアミノ酸配列をコードするＤＮＡ配列をそれぞれ設計する。 Next, the amino acid sequence of CDR of the original antibody is transplanted to the amino acid sequence of FR of VH or VL of the selected human antibody, respectively, and the amino acid sequence of VH or VL of the humanized antibody is designed respectively. The designed amino acid sequence is converted into a DNA sequence in consideration of the frequency of codon usage [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. A DNA sequence encoding the amino acid sequence of VH or VL is designed.

　設計したＤＮＡ配列に基づき、１００塩基前後の長さからなる数本の合成ＤＮＡを合成し、それらを用いてＰＣＲ反応を行う。この場合、ＰＣＲ反応での反応効率及び合成可能なＤＮＡの長さから、好ましくはＶＨ、ＶＬ各々について各６本の合成ＤＮＡを設計する。さらに、両端に位置する合成ＤＮＡの５’または３’末端に適当な制限酵素の認識配列を導入することで、ヒト化抗体のＶＨまたはＶＬをコードするｃＤＮＡを、（１）で得られる遺伝子組換え抗体発現用ベクターへ容易にクローニングすることができる。 Based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR reaction is performed using them. In this case, from the reaction efficiency in the PCR reaction and the length of DNA that can be synthesized, preferably 6 synthetic DNAs are designed for each of VH and VL. Further, by introducing an appropriate restriction enzyme recognition sequence into the 5 ′ or 3 ′ end of the synthetic DNA located at both ends, a cDNA encoding the humanized antibody VH or VL can be obtained in the gene set obtained in (1). It can be easily cloned into a vector for expression of the replacement antibody.

　ＰＣＲ反応後、増幅産物をｐＢｌｕｅｓｃｒｉｐｔ　ＳＫ（－）（ストラタジーン社製）などのプラスミドにそれぞれクローニングし、（２）に記載の方法と同様の方法により、塩基配列を決定し、所望のヒト化抗体のＶＨまたはＶＬのアミノ酸配列をコードするＤＮＡ配列を有するプラスミドを取得する。 After the PCR reaction, the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene), the base sequence is determined by the same method as described in (2), and the desired humanized antibody is obtained. A plasmid having a DNA sequence encoding the amino acid sequence of VH or VL is obtained.

　または、設計したＤＮＡ配列に基づき、ＶＨ全長およびＶＬ全長を各々１本の長鎖ＤＮＡとして合成したものを、上記ＰＣＲ増幅産物に代えて用いることもできる。さらに、合成長鎖ＤＮＡの両端に適当な制限酵素の認識配列を導入することで、ヒト化抗体のＶＨまたはＶＬをコードするｃＤＮＡを、（１）で得られる遺伝子組換え抗体発現用ベクターへ容易にクローニングすることができる。 Alternatively, 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. Furthermore, by introducing an appropriate restriction enzyme recognition sequence at both ends of the synthetic long-chain DNA, the cDNA encoding the humanized antibody VH or VL can be easily converted into the recombinant antibody expression vector obtained in (1). Can be cloned into.

（５）ヒト化抗体のＶ領域のアミノ酸配列の改変
　ヒト化抗体は、非ヒト抗体のＶＨおよびＶＬのＣＤＲのみをヒト抗体のＶＨおよびＶＬのＦＲに移植しただけでは、その抗原結合活性は元の非ヒト抗体に比べて低下する［BIO/TECHNOLOGY, 9, 266 (1991)］。ヒト化抗体では、ヒト抗体のＶＨおよびＶＬのＦＲのアミノ酸配列の中で、直接抗原との結合に関与しているアミノ酸残基、ＣＤＲのアミノ酸残基と相互作用するアミノ酸残基、および抗体の立体構造を維持し、間接的に抗原との結合に関与しているアミノ酸残基を同定し、それらのアミノ酸残基を元の非ヒト抗体のアミノ酸残基に置換することにより、低下した抗原結合活性を上昇させることができる。 (5) Modification of the amino acid sequence of the V region of a humanized antibody A humanized antibody can only have its antigen-binding activity by transplanting only the VH and VL CDRs of a non-human antibody into the VH and VL FRs of a human antibody. [BIO / TECHNOLOGY, 9, 266 (1991)]. In humanized antibodies, among the amino acid sequences of human antibody VH and VL FRs, the amino acid residues that are directly involved in binding to the antigen, the amino acid residues that interact with the CDR amino acid residues, and the antibody Reduced antigen binding by identifying amino acid residues that maintain conformation and indirectly participate in antigen binding and replace those amino acid residues with the amino acid residues of the original non-human antibody The activity can be increased.

　抗原結合活性に関わるＦＲのアミノ酸残基を同定するために、Ｘ線結晶解析［J. Mol. Biol., 112, 535 (1977)］またはコンピューターモデリング［Protein Engineering, 7, 1501 (1994）］などを用いることにより、抗体の立体構造の構築および解析を行うことができる。また、それぞれの抗体について数種の改変体を作製し、それぞれの抗原結合活性との相関を検討することを繰り返し、試行錯誤することで必要な抗原結合活性を有するヒト化抗体を取得できる。 In order to identify the amino acid residues of FRs involved in antigen binding activity, X-ray crystallography [J. Mol. Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)] etc. Can be used to construct and analyze the three-dimensional structure of an antibody. In addition, it is possible to obtain humanized antibodies having necessary antigen-binding activity by preparing several types of variants for each antibody, repeatedly examining the correlation with each antigen-binding activity, and by trial and error.

　ヒト抗体のＶＨ及びＶＬのＦＲのアミノ酸残基は、改変用合成ＤＮＡを用いて（４）に記載のＰＣＲ反応を行うことにより、改変させることができる。ＰＣＲ反応後の増幅産物について（２）に記載の方法により、塩基配列を決定し、目的の改変が施されたことを確認する。 The amino acid residues of FR of human antibody VH and VL can be modified by performing the PCR reaction described in (4) using the synthetic DNA for modification. For the amplified product after the PCR reaction, the base sequence is determined by the method described in (2) and it is confirmed that the target modification has been performed.

（６）ヒト化抗体発現ベクターの構築
　（１）で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流に、構築した遺伝子組換え抗体のＶＨまたはＶＬをコードするｃＤＮＡをそれぞれクローニングし、ヒト化抗体発現ベクターを構築することができる。 (6) Construction of humanized antibody expression vector VH or VL of the constructed recombinant antibody upstream of each gene encoding CH or CL of the human antibody of the recombinant antibody expression vector obtained in (1) Each of the cDNAs encoding can be cloned to construct a humanized antibody expression vector.

　例えば、（４）および（５）で得られるヒト化抗体のＶＨまたはＶＬを構築する際に用いる合成ＤＮＡのうち、両端に位置する合成ＤＮＡの５’または３’末端に適当な制限酵素の認識配列を導入することで、（１）で得られる遺伝子組換え抗体発現用ベクターのヒト抗体のＣＨまたはＣＬをコードするそれぞれの遺伝子の上流にそれらが適切な形で発現するようにそれぞれクローニングする。 For example, among the synthetic DNAs used in constructing the humanized antibody VH or VL obtained in (4) and (5), recognition of appropriate restriction enzymes at the 5 ′ or 3 ′ ends of the synthetic DNA located at both ends By introducing the sequence, the gene is cloned so that it is expressed in an appropriate form upstream of each gene encoding CH or CL of the human antibody of the recombinant antibody expression vector obtained in (1).

（７）遺伝子組換え抗体の一過性発現
　（３）および（６）で得られる遺伝子組換え抗体発現ベクター、またはそれらを改変した発現ベクターを用いて遺伝子組換え抗体の一過性発現を行い、作製した多種類のヒト型キメラ抗体、ヒト化抗体の抗原結合活性を効率的に評価することができる。 (7) 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 Thus, the antigen-binding activity of the various types of human chimeric antibodies and humanized antibodies can be efficiently evaluated.

　発現ベクターを導入する宿主細胞には、遺伝子組換え抗体を発現できる宿主細胞であれば、いかなる細胞でも用いることができるが、例えばＣＯＳ－７細胞［American Type Culture Collection(ATCC)番号:CRL1651］を用いる［Methods in Nucleic Acids Res., CRC press, 283 (1991)］。 Any host cell capable of expressing a recombinant antibody can be used as a host cell into which an expression vector is introduced. For example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651] are used. [Methods in Nucleic Acids Res., CRC press, 283 (1991)].

　ＣＯＳ－７細胞への発現ベクターの導入には、ＤＥＡＥ－デキストラン法［Methods in Nucleic Acids Res., CRC press (1991)］、またはリポフェクション法［Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)］などを用いる。 The expression vector is introduced into COS-7 cells by DEAE-dextran method [Methods in Nucleic Acids Res., CRC press (1991)] or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 ( 1987)].

　発現ベクターの導入後、培養上清中の遺伝子組換え抗体の発現量および抗原結合活性は酵素免疫抗体法［Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996)、Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)、単クローン抗体実験マニュアル、講談社サイエンティフィック（１９８７）］などを用いて測定する。 After the introduction of the expression vector, the expression level and antigen binding activity of the recombinant antibody in the culture supernatant are measured by enzyme immunoassay [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like.

（８）遺伝子組換え抗体を安定に発現する形質転換株の取得と遺伝子組換え抗体の調製
（３）および（６）で得られた遺伝子組換え抗体発現ベクターを適当な宿主細胞に導入することにより遺伝子組換え抗体を安定に発現する形質転換株を得ることができる。
　宿主細胞への発現ベクターの導入には、エレクトロポレーション法［日本国特開平２－２５７８９１号公報、Cytotechnology, 3, 133 (1990)］などを用いる。 (8) Obtaining a transformant that stably expresses the recombinant antibody and preparation of the recombinant antibody Introducing the recombinant antibody expression vector obtained in (3) and (6) into an appropriate host cell Thus, a transformed strain that stably expresses the recombinant antibody can be obtained.
For introducing the expression vector into the host cell, an electroporation method [Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)] or the like is used.

　遺伝子組換え抗体発現ベクターを導入する宿主細胞には、遺伝子組換え抗体を発現させることができる宿主細胞であれば、いかなる細胞でも用いることができる。例えば、ＣＨＯ－Ｋ１（ＡＴＣＣ　ＣＣＬ－６１）、ＤＵＫＸＢ１１（ＡＴＣＣ　ＣＣＬ－９０９６）、Ｐｒｏ－５（ＡＴＣＣ　ＣＣＬ－１７８１）、ＣＨＯ－Ｓ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ、Ｃａｔ＃１１６１９）、ラットミエローマ細胞ＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０（ＡＴＣＣ番号：ＣＲＬ１６６２、またはＹＢ２／０ともいう）、マウスミエローマ細胞ＮＳ０、マウスミエローマ細胞ＳＰ２／０－Ａｇ１４（ＡＴＣＣ番号：ＣＲＬ１５８１）、マウスＰ３Ｘ６３－Ａｇ８．６５３細胞（ＡＴＣＣ番号：ＣＲＬ１５８０）、ジヒドロ葉酸還元酵素遺伝子（Ｄｉｈｙｄｒｏｆｏｒａｔｅ　Ｒｅｄｕｃｔａｓｅ、以下、ｄｈｆｒと表記する）が欠損したＣＨＯ細胞（ＣＨＯ／ＤＧ４４細胞）［Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)］などを用いる。 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. For example, CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat # 11619), rat myeloma cell YB2 / 3HL. P2. G11.16 Ag. 20 (ATCC number: also referred to as CRL1662 or YB2 / 0), mouse myeloma cell NS0, mouse myeloma cell SP2 / 0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cells (ATCC number: CRL1580), dihydro CHO cells (CHO / DG44 cells) lacking a folate reductase gene (Dihydroformate Reductase, hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] are used.

　また、細胞内糖ヌクレオチドＧＤＰ－フコースの合成に関与する酵素などのタンパク質、Ｎ－グリコシド結合複合型糖鎖の還元末端のＮ－アセチルグルコサミンの６位にフコースの１位がα結合する糖鎖修飾に関与する酵素などのタンパク質若しくは細胞内糖ヌクレオチドＧＤＰ－フコースのゴルジ体への輸送に関与するタンパク質などの活性が低下または欠失した宿主細胞、例えばα１，６－フコース転移酵素遺伝子が欠損したＣＨＯ細胞（国際公開第２００５／０３５５８６号、国際公開第０２／３１１４０号）、レクチン耐性を獲得したＬｅｃ１３［Somatic Cell and Molecular genetics, 12, 55 (1986)］などを用いることもできる。 In addition, a protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or a sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex sugar chain A host cell in which the activity of a protein such as an enzyme involved in the protein or a protein involved in the transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus is reduced or deleted, such as a CHO deficient in the α1,6-fucose transferase gene 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 can also be used.

　発現ベクターの導入後、遺伝子組換え抗体を安定に発現する形質転換株は、Ｇ４１８硫酸塩（以下、Ｇ４１８と表記する）などの薬剤を含む動物細胞培養用培地で培養することにより選択する（日本国特開平２－２５７８９１号公報）。 After the introduction of the expression vector, 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). (Japanese Patent Laid-Open No. 2-257891).

　動物細胞培養用培地には、ＲＰＭＩ１６４０培地（インビトロジェン社製）、ＧＩＴ培地（日本製薬社製）、ＥＸ－ＣＥＬＬ３０１培地（ジェイアールエイチ社製）、ＩＭＤＭ培地（インビトロジェン社製）若しくはＨｙｂｒｉｄｏｍａ－ＳＦＭ培地（インビトロジェン社製）、またはこれら培地にＦＢＳなどの各種添加物を添加した培地などを用いる。得られた形質転換株を培地中で培養することで培養上清中に遺伝子組換え抗体を発現蓄積させる。培養上清中の遺伝子組換え抗体の発現量および抗原結合活性はＥＬＩＳＡ法などにより測定できる。また、ｄｈｆｒ遺伝子増幅系（日本国特開平２－２５７８９１号公報）などを利用して、形質転換株の産生する遺伝子組換え抗体の発現量を向上させることができる。 RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), or Hybridoma-SFM medium (manufactured by Invitrogen) Invitrogen) or a medium obtained by adding various additives such as FBS to these mediums. By culturing the obtained transformant in a medium, the recombinant antibody is expressed and accumulated in the culture supernatant. The expression level and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA method or the like. In addition, the expression level of the recombinant antibody produced by the transformed strain can be improved using a dhfr gene amplification system (Japanese Patent Laid-Open No. 2-257891).

　遺伝子組換え抗体は、形質転換株の培養上清よりプロテインＡ－カラムを用いて精製する［Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996)、Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)］。また、ゲル濾過、イオン交換クロマトグラフィーおよび限外濾過などのタンパク質の精製で用いられる方法を組み合わすこともできる。 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 Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. It is also possible to combine methods used in protein purification such as gel filtration, ion exchange chromatography and ultrafiltration.

　精製した遺伝子組換え抗体のＨ鎖、Ｌ鎖或いは抗体分子全体の分子量は、ポリアクリルアミドゲル電気泳動法［Nature, 227, 680 (1970)］、またはウェスタンブロッティング法［Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996)、Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory(1988)］など用いて測定することができる。 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 practice, Third edition, “Academic” Press (1996), Antibodies—A Laboratory, Manual, Cold Spring, Laboratory (1988)].

３．精製モノクローナル抗体またはその抗体断片の活性評価
　精製した本発明のモノクローナル抗体またはその抗体断片の活性評価は、以下のように行うことができる。 3. 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.

　本発明の抗体または該抗体断片のヒトＣＣＲ１に対する結合活性は、前述の１－（６）記載のフローサイトメトリーを用いて測定する。また、蛍光抗体法［Cancer Immunol. Immunother., 36, 373 (1993)］などを用いて測定できる。 The binding activity of the antibody of the present invention or the antibody fragment to human CCR1 is measured using the flow cytometry described in 1- (6) above. Further, it can be measured using a fluorescent antibody method [Cancer Immunol. Immunolother, 36, 373 (1993)].

　本発明の抗体または該抗体断片のヒトＣＣＬ１５によるヒトＣＣＲ１発現細胞の遊走を阻害する活性は、上述のケモタキシスアッセイを用いて測定することができる。 The activity of inhibiting the migration of human CCR1-expressing cells by human CCL15 of the antibody of the present invention or the antibody fragment thereof can be measured using the chemotaxis assay described above.

　ヒトＣＣＲ１発現細胞に対するＣＤＣ活性、又はＡＤＣＣ活性は公知の測定方法［Cancer Immunol. Immunother., 36, 373(1993); Current protocols in Immunology, Chapter7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons,Inc.,(1993)］により測定することができる。 CDC activity or ADCC activity against human CCR1-expressing cells can be measured by a known assay method [Cancer Immunol. Immunother., 36, 373 (1993); Current protocols in Immunology, Chapter 7 Immunologic studies in humans, Editor, John ., John Wiley & Sons, Inc., (1993)].

4. 抗体のエフェクター活性を制御する方法
　本発明のモノクローナル抗体のエフェクター活性を制御する方法としては、抗体のＦｃ領域の２９７番目のアスパラギン（Ａｓｎ）に結合するＮ結合複合型糖鎖の還元末端に存在するＮ－アセチルグルコサミン（ＧｌｃＮＡｃ）にα１，６結合するフコース（コアフコースともいう）の量を制御する方法（国際公開第２００５／０３５５８６号、国際公開第２００２／３１１４０号、国際公開第００／６１７３９号）、又は抗体のＦｃ領域のアミノ酸残基を改変することで制御する方法などが知られている。本発明のモノクローナル抗体にはいずれの方法を用いても、エフェクター活性を制御することができる。　 4. Method for controlling the effector activity of the antibody As a method for controlling the effector activity of the monoclonal antibody of the present invention, the N-linked complex type sugar chain binding to the 297th asparagine (Asn) of the Fc region of the antibody is used. Methods for controlling the amount of fucose (also referred to as core fucose) that binds α1,6 to N-acetylglucosamine (GlcNAc) present (International Publication No. 2005/035586, International Publication No. 2002/31140, International Publication No. 00/61739) No.), or 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 monoclonal antibody of the present invention.

　エフェクター活性とは、抗体のＦｃ領域を介して引き起こされる抗体依存性の活性をいい、ＡＤＣＣ活性、ＣＤＣ活性、又はマクロファージ若しくは樹状細胞などの食細胞による抗体依存性ファゴサイトーシス（Ａｎｔｉｂｏｄｙ－ｄｅｐｅｎｄｅｎｔ　ｐｈａｇｏｃｙｔｏｓｉｓ、ＡＤＰ活性）などが知られている。　 The effector activity refers to antibody-dependent activity caused through the Fc region of an antibody, and includes ADCC activity, CDC activity, or antibody-dependent phagocytosis by phagocytic cells such as macrophages or dendritic cells (Antibody-dependent phagocytosis). , ADP activity) and the like are known. *

　エフェクター活性の測定法として、例えば、標的として炎症性細胞、エフェクターとしてヒト末梢血単核球（ＰＢＭＣ）、そして炎症性細胞特異的な抗体を混合し、４時間程度インキュベーションした後、細胞傷害の指標として遊離してきた乳酸脱水素酵素（ＬＤＨ）を測定することができる。若しくは、ヒト全血に、例えばＣＤ２０の様な血液細胞特異的な抗原を認識する抗体を添加し、インキュベーションした後、標的となる血液細胞数の減少をエフェクター活性として測定することができる。または、例えば、ヒト全血に別の標的細胞を混合し、さらに標的細胞に特異的な抗体を添加しインキュベーションした後、標的細胞数の減少をエフェクター活性として測定することができる。いずれの場合においてもエフェクター活性は、遊離ＬＤＨ法、遊離５１Ｃｒ法またはフローサイトメトリー法などによって測定することができる。 As a method for measuring effector activity, for example, inflammatory cells as targets, human peripheral blood mononuclear cells (PBMC) as effectors, and inflammatory cell-specific antibodies are mixed and incubated for about 4 hours, and then an indication of cytotoxicity Lactate dehydrogenase (LDH) released as can be measured. Alternatively, an antibody that recognizes a blood cell-specific antigen such as CD20 is added to human whole blood, and after incubation, a decrease in the number of blood cells to be targeted can be measured as an effector activity. Alternatively, for example, after mixing another target cell with human whole blood, adding an antibody specific to the target cell and incubating, the decrease in the number of target cells can be measured as the effector activity. In any case, the effector activity can be measured by a free LDH method, a free 51 Cr method, a flow cytometry method, or the like.

　抗体のＦｃのＮ結合複合型糖鎖のコアフコースの含量を制御することで、抗体のエフェクター活性を増加又は低下させることができる。抗体のＦｃに結合しているＮ結合複合型糖鎖に結合するフコースの含量を低下させる方法としては、α１，６－フコース転移酵素遺伝子が欠損したＣＨＯ細胞を用いて抗体を発現することで、フコースが結合していない抗体を取得することができる。フコースが結合していない抗体は高いＡＤＣＣ活性を有する。　 By controlling the content of core fucose in the N-linked complex sugar chain of the Fc of the antibody, the effector activity of the antibody can be increased or decreased. As a method for reducing the content of fucose bound to the N-linked complex type sugar chain bound to the Fc of the antibody, expressing the antibody using CHO cells deficient in the α1,6-fucose transferase gene, An antibody to which fucose is not bound can be obtained. Antibodies without fucose binding have high ADCC activity. *

　一方、抗体のＦｃに結合しているＮ結合複合型糖鎖に結合するフコースの含量を増加させる方法としては、α１，６－フコース転移酵素遺伝子を導入した宿主細胞を用いて抗体を発現させることで、フコースが結合している抗体を取得できる。フコースが結合している抗体は、フコースが結合していない抗体よりも低いＡＤＣＣ活性を有する。　 On the other hand, as a method for increasing the content of fucose bound to the N-linked complex type sugar chain bound to the Fc of the antibody, the antibody is expressed using a host cell into which an α1,6-fucose transferase gene has been introduced. Thus, 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. *

　また、抗体のＦｃ領域のアミノ酸残基を改変することでＡＤＣＣ活性又はＣＤＣ活性を増加又は低下させることができる。例えば、米国特許出願公開第２００７／０１４８１６５号明細書に記載のＦｃ領域のアミノ酸配列を用いることで、抗体のＣＤＣ活性を増加させることができる。　 Furthermore, ADCC activity or CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of the antibody. For example, 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. *

　また、米国特許第６，７３７，０５６号明細書、米国特許第７，２９７，７７５号明細書又は米国特許第７，３１７，０９１号明細書に記載のアミノ酸改変を行うことで、ＡＤＣＣ活性又はＣＤＣ活性を、増加させることも低下させることもできる。また本発明の抗体には、上述の抗体定常領域におけるアミノ酸改変または糖鎖改変に合わせて、例えば、日本国特開第２０１３－１６５７１６号公報または日本国特開第２０１２－０２１００４号公報などに記載のアミノ酸改変を行うことにより、Ｆｃ受容体への反応性を制御することで血中半減期を制御した抗体も含まれる。 Further, by performing amino acid modification described in US Pat. No. 6,737,056, US Pat. No. 7,297,775 or US Pat. No. 7,317,091, ADCC activity or CDC activity can be increased or decreased. The antibody of the present invention is described in, for example, Japanese Patent Application Publication No. 2013-165716 or Japanese Patent Application Publication No. 2012-021004 according to amino acid modification or sugar chain modification in the antibody constant region described above. Also included are antibodies whose half-life in blood is controlled by controlling the reactivity to Fc receptors by performing the amino acid modification of.

　さらに、上述の方法を組み合わせて、一つの抗体に使用することにより、抗体のエフェクター活性や血中半減期が制御された抗体を取得することができる。 Furthermore, by using the above-mentioned methods in combination with one antibody, it is possible to obtain an antibody in which the effector activity or blood half-life of the antibody is controlled.

５．本発明の抗ヒトＣＣＲ１モノクローナル抗体またはその抗体断片を用いた疾患の治療方法
　本発明のモノクローナル抗体またはその抗体断片は、ヒトＣＣＲ１依存的な細胞遊走、病変などＣＣＲ１が関連する疾患であれば、いずれのヒトＣＣＲ１関連疾患の治療に用いることができる。 5). Method for treating diseases using anti-human CCR1 monoclonal antibody or antibody fragment thereof of the present invention The monoclonal antibody or antibody fragment of the present invention can be any one of CCR1-related diseases such as human CCR1-dependent cell migration and lesions. Can be used to treat human CCR1-related diseases.

　本発明のモノクローナル抗体またはその抗体断片を含有する治療剤は、有効成分としての該抗体または該抗体断片のみを含むものであってもよいが、通常は薬理学的に許容される１以上の担体と一緒に混合し、製剤学の技術分野において公知の方法により製造した医薬製剤として提供される。 The therapeutic agent containing the monoclonal antibody or antibody fragment thereof of the present invention may contain only the antibody or the antibody fragment as an active ingredient, but usually one or more pharmacologically acceptable carriers And a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics.

　投与経路としては、例えば、経口投与、または口腔内、気道内、直腸内、皮下、筋肉内若しくは静脈内などの非経口投与が挙げられる。投与形態としては、例えば、噴霧剤、カプセル剤、錠剤、散剤、顆粒剤、シロップ剤、乳剤、座剤、注射剤、軟膏またはテープ剤などが挙げられる。 Examples of the administration route include oral administration and parenteral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular or intravenous. Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments or tapes.

　経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤または顆粒剤などが挙げられる。 Preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders or granules.

　乳剤またはシロップ剤のような液体調製物は、水、ショ糖、ソルビトール若しくは果糖などの糖類、ポリエチレングリコール若しくはプロピレングリコールなどのグリコール類、ごま油、オリーブ油若しくは大豆油などの油類、ｐ－ヒドロキシ安息香酸エステル類などの防腐剤またはストロベリーフレーバー若しくはペパーミントなどのフレーバー類などを添加剤として用いて製造する。 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 Manufactured using preservatives such as esters or flavors such as strawberry flavor or peppermint as additives.

　カプセル剤、錠剤、散剤または顆粒剤などは、乳糖、ブドウ糖、ショ糖若しくはマンニトールなどの賦形剤、デンプン若しくはアルギン酸ナトリウムなどの崩壊剤、ステアリン酸マグネシウム若しくはタルクなどの滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース若しくはゼラチンなどの結合剤、脂肪酸エステルなどの界面活性剤またはグリセリンなどの可塑剤などを添加剤として用いて製造する。 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.

　非経口投与に適当な製剤としては、注射剤、座剤または噴霧剤などである。注射剤は、塩溶液若しくはブドウ糖溶液、またはその両者の混合物からなる担体などを用いて製造する。座剤はカカオ脂、水素化脂肪またはカルボン酸などの担体を用いて製造する。 製 剤 Suitable formulations for parenteral administration include injections, suppositories or sprays. An injection is produced using a carrier comprising a salt solution or a glucose solution, or a mixture of both. Suppositories are produced using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.

　噴霧剤は受容者の口腔および気道粘膜を刺激せず、かつ本発明のモノクローナル抗体またはその抗体断片を微細な粒子として分散させ、吸収を容易にさせる担体などを用いて製造する。担体としては、例えば乳糖またはグリセリンなどを用いる。また、エアロゾルまたはドライパウダーとして製造することもできる。さらに、上記非経口剤においても、経口投与に適当な製剤で添加剤として例示した成分を添加することもできる。 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. As the carrier, for example, lactose or glycerin is used. It can also be produced as an aerosol or dry powder. Furthermore, in the above parenteral preparations, the components exemplified as additives in preparations suitable for oral administration can also be added.

６．本発明の抗ヒトＣＣＲ１モノクローナル抗体またはその抗体断片を用いた疾患の診断方法
　本発明のモノクローナル抗体または該抗体断片を用いて、ヒトＣＣＲ１またはヒトＣＣＲ１が発現した細胞を検出または測定することにより、ヒトＣＣＲ１関連疾患を診断することができる。 6). Method for diagnosing disease using anti-human CCR1 monoclonal antibody or antibody fragment thereof of the present invention By detecting or measuring human CCR1 or cells expressing human CCR1 using the monoclonal antibody or the antibody fragment of the present invention, CCR1-related diseases can be diagnosed.

　ヒトＣＣＲ１関連疾患である癌疾患、自己免疫性疾患および炎症性疾患の診断は、例えば患者体内に存在するヒトＣＣＲ１を免疫学的手法により検出または測定して行うことができる。また、患者体内の細胞に発現しているヒトＣＣＲ１をフローサイトメトリーなどの免疫学的手法を用いて検出することにより診断を行うことができる。 The diagnosis of cancer diseases, autoimmune diseases, and inflammatory diseases, which are human CCR1-related diseases, can be performed, for example, by detecting or measuring human CCR1 present in a patient by an immunological technique. In addition, diagnosis can be performed by detecting human CCR1 expressed in cells in a patient using an immunological technique such as flow cytometry.

　免疫学的手法とは、標識を施した抗原または抗体を用いて、抗体量または抗原量を検出または測定する方法である。例えば、放射性物質標識免疫抗体法、酵素免疫測定法、蛍光免疫測定法、発光免疫測定法、ウエスタンブロット法または物理化学的手法などを用いる。 An immunological technique is a method for detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody. For example, a radioactive substance-labeled immunoantibody method, an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blot method or a physicochemical method is used.

　放射性物質標識免疫抗体法は、例えば、抗原または抗原を発現した細胞などに、本発明の抗体または該抗体断片を反応させ、さらに放射線標識を施した抗イムノグロブリン抗体または該抗体断片を反応させた後、シンチレーションカウンターなどで測定する。 In the radiolabeled immunoantibody method, for example, the antibody of the present invention or the antibody fragment thereof is reacted with an antigen or an antigen-expressing cell, and the anti-immunoglobulin antibody or the antibody fragment with a radiolabel is further reacted. Then, measure with a scintillation counter.

　酵素免疫測定法は、例えば、抗原または抗原を発現した細胞などに、本発明の抗体または該抗体断片を反応させ、さらに酵素などで標識を施した抗イムノグロブリン抗体または結合断片を反応させた後、基質を添加して反応液の吸光度を吸光光度計で測定する。例えばサンドイッチＥＬＩＳＡ法などを用いる。酵素免疫測定法で用いる標識体としては、公知［酵素免疫測定法、医学書院（１９８７）］の酵素標識を用いることができる。 In the enzyme immunoassay, for example, an antigen or a cell expressing the antigen is reacted with the antibody of the present invention or the antibody fragment, and further reacted with an anti-immunoglobulin antibody or binding fragment labeled with an enzyme or the like. The substrate is added and the absorbance of the reaction solution is measured with an absorptiometer. For example, a sandwich ELISA method is used. As a label used in the enzyme immunoassay, an enzyme label known in the art [Enzyme immunoassay, Medical School (1987)] can be used.

　例えば、アルカリフォスファターゼ標識、ペルオキシダーゼ標識、ルシフェラーゼ標識またはビオチン標識などを用いる。サンドイッチＥＬＩＳＡ法は、固相に抗体を結合させた後、検出または測定対象である抗原をトラップさせ、トラップされた抗原に第２の抗体を反応させる方法である。該ＥＬＩＳＡ法では、検出または測定したい抗原を認識する抗体または抗体断片であって、抗原認識部位の異なる２種類の抗体を準備し、そのうち、第１の抗体または抗体断片を予めプレート（例えば、９６ウェルプレート）に吸着させ、次に第２の抗体または抗体断片をＦＩＴＣなどの蛍光物質、ペルオキシダーゼなどの酵素またはビオチンなどで標識しておく。上記の抗体が吸着したプレートに、生体内から分離された、細胞またはその破砕液、組織またはその破砕液、細胞培養上清、血清、胸水、腹水または眼液などを反応させた後、標識したモノクローナル抗体または抗体断片を反応させ、標識物質に応じた検出反応を行う。濃度既知の抗原を段階的に希釈して作成した検量線より、被験サンプル中の抗原濃度を算出する。サンドイッチＥＬＩＳＡ法に用いる抗体としては、ポリクローナル抗体またはモノクローナル抗体のいずれを用いてもよく、Ｆａｂ、Ｆａｂ’またはＦ（ａｂ）２などの抗体フラグメントを用いてもよい。サンドイッチＥＬＩＳＡ法で用いる２種類の抗体の組み合わせとしては、異なるエピトープを認識するモノクローナル抗体または抗体断片の組み合わせでもよいし、ポリクローナル抗体とモノクローナル抗体または抗体断片との組み合わせでもよい。 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. In the ELISA method, two types of antibodies or antibody fragments that recognize an antigen to be detected or measured and that have different antigen recognition sites are prepared, of which the first antibody or antibody fragment is pre-plated (for example, 96 Next, the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, biotin, or the like. The above-mentioned antibody-adsorbed plate was labeled after reacting cells or its lysate, tissue or its lysate, cell culture supernatant, serum, pleural effusion, ascites or ocular fluid, etc., separated from the living body. A 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. As an antibody used for 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.

　蛍光免疫測定法は、文献［Monoclonal Antibodies-Principles and practice, Third edition,Academic Press (1996)、単クローン抗体実験マニュアル、講談社サイエンティフィック (1987)］などに記載された方法で測定する。蛍光免疫測定法で用いる標識体としては、公知［蛍光抗体法、ソフトサイエンス社（１９８３）］の蛍光標識を用いることができる。例えば、ＦＩＴＣまたはＲＩＴＣなどを用いる。 Fluorescence immunoassay is measured by the method described in the literature [Monoclonal Antibodies-Principles and Practice, A Third Edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)]. As a label used in the fluorescence immunoassay, a fluorescent label known in the art [fluorescent antibody method, Soft Science (1983)] can be used. For example, FITC or RITC is used.

　発光免疫測定法は文献［生物発光と化学発光　臨床検査４２、廣川書店（１９９８）］などに記載された方法で測定する。発光免疫測定法で用いる標識体としては、公知の発光体標識が挙げられ、アクリジニウムエステルまたはロフィンなどを用いる。 Luminescent immunoassay is performed 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 or lophine is used.

　ウエスタンブロット法は、抗原または抗原を発現した細胞などをＳＤＳ（ドデシル硫酸ナトリウム）－ＰＡＧＥ（ポリアクリルアミドゲル）［Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)］で分画した後、該ゲルをポリフッ化ビニリデン（ＰＶＤＦ）膜またはニトロセルロース膜にブロッティングし、該膜に抗原を認識する抗体または抗体断片を反応させ、さらにＦＩＴＣなどの蛍光物質、ペルオキシダーゼなどの酵素標識またはビオチン標識などを施した抗マウスＩｇＧ抗体または結合断片を反応させた後、該標識を可視化することによって測定する。 Western blotting involves fractionating the antigen or antigen-expressing cells with SDS (sodium dodecyl sulfate) -PAGE (polyacrylamide gel) [Antibodies-A Laboratory Manual Cold Spring Harbor Laboratory (1988)], Blotting on a polyvinylidene fluoride (PVDF) membrane or nitrocellulose membrane, causing the membrane to react with an antibody or antibody fragment that recognizes an antigen, and further applying a fluorescent substance such as FITC, an enzyme label such as peroxidase, or a biotin label It is measured by visualizing the label after reacting with mouse IgG antibody or binding fragment.

　一例を以下に示す。配列番号２のアミノ酸配列を有するポリペプチドを発現している細胞や組織を溶解し、還元条件下でレーンあたりのタンパク量として０．１～３０μｇをＳＤＳ－ＰＡＧＥ法により泳動する。泳動されたタンパク質をＰＶＤＦ膜にトランスファーし１～１０％ＢＳＡを含むＰＢＳ（以下、ＢＳＡ－ＰＢＳと表記する）に室温で３０分間反応させブロッキング操作を行う。ここで本発明のモノクローナル抗体を反応させ、０．０５～０．１％のＴｗｅｅｎ－２０を含むＰＢＳ（以下、Ｔｗｅｅｎ－ＰＢＳと表記する）で洗浄し、ペルオキシダーゼ標識したヤギ抗マウスＩｇＧを室温で２時間反応させる。Ｔｗｅｅｎ－ＰＢＳで洗浄し、ＥＣＬ　Ｗｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇ　Ｄｅｔｅｃｔｉｏｎ　Ｒｅａｇｅｎｔｓ（アマシャム社製）などを用いてモノクローナル抗体が結合したバンドを検出することにより、配列番号２のアミノ酸配列を有するポリペプチドを検出する。ウェスタンブロッティングでの検出に用いられる抗体としては、天然型の立体構造を保持していないポリペプチドに結合できる抗体が用いられる。 An example is shown below. Cells and tissues expressing a polypeptide having the amino acid sequence of SEQ ID NO: 2 are lysed, and 0.1-30 μg of protein per lane is electrophoresed by SDS-PAGE under reducing conditions. The migrated protein is transferred to a PVDF membrane and reacted with PBS containing 1 to 10% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes to perform a blocking operation. Here, 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 having the amino acid sequence of 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). As 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.

　物理化学的手法は、例えば、抗原であるヒトＣＣＲ１と本発明のモノクローナル抗体またはその抗体断片とを結合させることにより凝集体を形成させて、この凝集体を検出することにより行う。この他に物理化学的手法として、毛細管法、一次元免疫拡散法、免疫比濁法またはラテックス免疫比濁法［臨床検査法提要、金原出版（１９９８）］などを用いることもできる。ラテックス免疫比濁法は、抗体または抗原を感作させた粒径０．１～１μｍ程度のポリスチレンラテックスなどの担体を用い、対応する抗原または抗体により抗原抗体反応を起こさせると、反応液中の散乱光は増加し、透過光は減少する。この変化を吸光度または積分球濁度として検出することにより被験サンプル中の抗原濃度などを測定する。 The physicochemical method is performed by, for example, forming an aggregate by binding human CCR1 as an antigen and the monoclonal antibody of the present invention or an antibody fragment thereof, and detecting the aggregate. In addition, as 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 μm 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.

　ヒトＣＣＲ１が発現している細胞の検出または測定は、公知の免疫学的検出法を用いることができるが、中でも、免疫沈降法、免疫細胞染色法、免疫組織染色法または蛍光抗体染色法などを用いることが好ましい。 For detection or measurement of cells expressing human CCR1, known immunological detection methods can be used. Among them, immunoprecipitation, immunocytostaining, immunohistochemical staining, fluorescent antibody staining, etc. can be used. It is preferable to use it.

　免疫沈降法は、ヒトＣＣＲ１を発現した細胞などを本発明のモノクローナル抗体またはその抗体断片と反応させた後、プロテインＧ－セファロースなどのイムノグロブリンに特異的な結合能を有する担体を加えて抗原抗体複合体を沈降させる。または以下のような方法によっても行うことができる。ＥＬＩＳＡ用９６ウェルプレートに上述した本発明のモノクローナル抗体またはその抗体断片を固相化した後、ＢＳＡ－ＰＢＳによりブロッキングする。抗体が、例えばハイブリドーマ培養上清などの精製されていない状態である場合には、抗マウスイムノグロブリン、抗ラットイムノグロブリン、プロテイン－Ａまたはプロテイン－ＧなどをあらかじめＥＬＩＳＡ用９６ウェルプレートに固相化し、ＢＳＡ－ＰＢＳでブロッキングした後、ハイブリドーマ培養上清を分注して結合させる。次に、ＢＳＡ－ＰＢＳを捨てＰＢＳでよく洗浄した後、ヒトＣＣＲ１を発現している細胞や組織の溶解液を反応させる。よく洗浄した後のプレートより免疫沈降物をＳＤＳ－ＰＡＧＥ用サンプルバッファーで抽出し、上記のウェスタンブロッティングにより検出する。 In the immunoprecipitation method, cells expressing human CCR1 are reacted with the monoclonal antibody of the present invention or an antibody fragment thereof, and then a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added. Allow the complex to settle. Alternatively, 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. 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. Next, after discarding BSA-PBS and thoroughly washing with PBS, a lysate of cells or tissues expressing human CCR1 is reacted. Immunoprecipitates are extracted from the well-washed plate with SDS-PAGE sample buffer and detected by Western blotting as described above.

　免疫細胞染色法または免疫組織染色法は、抗原を発現した細胞または組織などを、場合によっては抗体の通過性を良くするため界面活性剤やメタノールなどで処理した後、本発明のモノクローナル抗体と反応させ、さらにＦＩＴＣなどの蛍光標識、ペルオキシダーゼなどの酵素標識またはビオチン標識などを施した抗イムノグロブリン抗体またはその結合断片と反応させた後、該標識を可視化し、顕微鏡にて顕鏡する方法である。また、蛍光標識の抗体と細胞を反応させ、フロ－サイトメーターにて解析する蛍光抗体染色法［Monoclonal Antibodies-Principles and practice, Third edition,Academic Press (1996)、単クローン抗体実験マニュアル、講談社サイエンティフィック (1987)］により検出を行うことができる。特に、ヒトＣＣＲ１に結合する、本発明のモノクローナル抗体またはその抗体断片は、蛍光抗体染色法により天然型の立体構造を保持して発現している細胞の検出ができる。 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 . In addition, fluorescent antibody staining methods that react with fluorescently labeled antibodies and cells and analyze with a flow cytometer [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific Fick (1987)] can be used for detection. In particular, the monoclonal antibody of the present invention or an antibody fragment thereof that binds to human CCR1 can detect cells expressing and retaining a natural three-dimensional structure by fluorescent antibody staining.

　また、蛍光抗体染色法のうち、ＦＭＡＴ８１００ＨＴＳシステム（アプライドバイオシステム社製）などを用いた場合には、形成された抗体－抗原複合体と、抗体－抗原複合体の形成に関与していない遊離の抗体または抗原とを分離することなく、抗原量または抗体量を測定できる。 In addition, among the fluorescent antibody staining methods, when the FMAT8100HTS system (Applied Biosystems) or the like is used, the formed antibody-antigen complex and the free that is not involved in the formation of the antibody-antigen complex The amount of antigen or antibody can be measured without separating the antibody or antigen.

　以下、本発明を実施例により具体的に説明するが、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

［実施例１］ヒト、マウスＣＣＲ１の発現ベクターの作製
（１）各ＣＣＲ１遺伝子の作製
　下記、１～７のヒトまたはマウスのＣＣＲ１若しくはＣＣＲ１－ＣＣＲ３キメラ受容体をコードするＤＮＡを合成した（ジェンスクリプトジャパン社）。合成の際には各ベクターに組み込むための制限酵素サイト（ＢａｍＨＩおよびＮｏｔＩ）と、Ｋｏｚａｋ配列を付加した。
１．ヒトＣＣＲ１（以下、ｈＣＣＲ１）をコードするｃＤＮＡ配列（配列番号１）
２．マウスＣＣＲ１（以下、ｍＣＣＲ１）をコードするｃＤＮＡ配列（配列番号３）
３．ヒトＣＣＲ３（以下、ｈＣＣＲ３）をコードするｃＤＮＡ配列（配列番号５）
４．ヒトＣＣＲ１の１番目から３１番目のアミノ酸配列をヒトＣＣＲ３の対応するＮ末端のアミノ酸配列に置換したキメラ受容体（以下、ＮＣ３－ｈＣＣＲ１）をコードするｃＤＮＡ配列（配列番号６）
５．マウスＣＣＲ１の１番目から３１番目のアミノ酸配列をヒトＣＣＲ３の対応するＮ末端のアミノ酸配列に置換したキメラ受容体（以下、ＮＣ３－ｍＣＣＲ１）をコードするｃＤＮＡ配列（配列番号７）
６．ヒトＣＣＲ３の１７１番目から１９４番目のアミノ酸配列をヒトＣＣＲ１の１７１番目から１９４番目のアミノ酸配列に置換したキメラ受容体（以下、ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１）をコードするｃＤＮＡ配列（配列番号８）
７．ヒトＣＣＲ３の１７１番目から１９４番目のアミノ酸配列をマウスＣＣＲ１の１７１番目から１９４番目のアミノ酸配列に置換したキメラ受容体（以下、ｈＣＣＲ３＿ＥＬ２ｍＣＣＲ１）をコードするｃＤＮＡ配列（配列番号９） [Example 1] Preparation of expression vectors for human and mouse CCR1 (1) Preparation of each CCR1 gene DNA encoding human or mouse CCR1 or CCR1-CCR3 chimeric receptors 1 to 7 below was synthesized (Genscript) Japan). In the synthesis, restriction enzyme sites (BamHI and NotI) for incorporation into each vector and a Kozak sequence were added.
1. CDNA sequence encoding human CCR1 (hereinafter, hCCR1) (SEQ ID NO: 1)
2. CDNA sequence encoding mouse CCR1 (hereinafter, mCCR1) (SEQ ID NO: 3)
3. CDNA sequence encoding human CCR3 (hereinafter hCCR3) (SEQ ID NO: 5)
4). CDNA sequence (SEQ ID NO: 6) encoding a chimeric receptor (hereinafter referred to as NC3-hCCR1) in which the amino acid sequence from human 31 to human CCR1 is substituted with the corresponding N-terminal amino acid sequence of human CCR3
5). CDNA sequence (SEQ ID NO: 7) encoding a chimeric receptor (hereinafter, NC3-mCCR1) in which the first to 31st amino acid sequences of mouse CCR1 are replaced with the corresponding N-terminal amino acid sequences of human CCR3
6). A cDNA sequence (SEQ ID NO: 8) encoding a chimeric receptor (hereinafter, hCCR3_EL2hCCR1) in which the 171st to 194th amino acid sequences of human CCR3 are replaced with the 171st to 194th amino acid sequences of human CCR1
7). CDNA sequence (SEQ ID NO: 9) encoding a chimeric receptor (hereinafter, hCCR3_EL2mCCR1) in which the 171st to 194th amino acid sequences of human CCR3 are replaced with the 171st to 194th amino acid sequences of mouse CCR1

（２）ヒトＣＣＲ１発現ベクターの作製
　上記（１）－１で合成したｈＣＣＲ１をコードするＤＮＡを制限酵素ＢａｍＨＩおよびＮｏｔＩ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂ社）で処理し、ＤＮＡ断片を精製した。Ｔｏｌ２トランスポゾンベクター（国際公開第２０１０／１４３６９８号）（以下、Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏと記載する）を同制限酵素で処理し、ＣＣＲ１をコードするＤＮＡ断片と混ぜたうえで、ＤＮＡ　ｌｉｇａｓｅ（タカラバイオ社）処理によって連結した。ライゲーション後のＤＮＡを大腸菌コンピテントセル（タカラバイオ社）に導入し、薬剤耐性を獲得したコロニーの中から目的のプラスミドＤＮＡを持つ大腸菌株を選択した。この大腸菌株を再度培養し、培養液からトランスフェクション用のＤＮＡを精製した（以下、このように作製したプラスミドをｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏと表わす。）。 (2) Preparation of human CCR1 expression vector The DNA encoding hCCR1 synthesized in (1) -1 was treated with restriction enzymes BamHI and NotI (New England Biolab) to purify the DNA fragment. Tol2 transposon vector (International Publication No. 2010/143698) (hereinafter referred to as Tn-pMug-Hygro) was treated with the same restriction enzyme, mixed with a DNA fragment encoding CCR1, and then DNA ligase (Takara Bio Inc.). ) Linked by processing. The ligated DNA was introduced into an E. coli competent cell (Takara Bio), and an E. coli strain having the desired plasmid DNA was selected from colonies that had acquired drug resistance. This Escherichia coli strain was cultured again, and DNA for transfection was purified from the culture solution (hereinafter, the plasmid thus prepared is referred to as hCCR1 / Tn-pMug-Hygro).

（３）各種ＣＣＲ発現ベクターの作製
　上記（２）と同様の方法で、上記（１）で合成したｍＣＣＲ１、ｈＣＣＲ３、ＮＣ３－ｈＣＣＲ１、ＮＣ３－ｍＣＣＲ１、ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１、ｈＣＣＲ３＿ＥＬ２ｍＣＣＲ１をＴｎ－ｐＭｕｇ－Ｈｙｇｒｏに連結させ、発現ベクターを構築した（以下、それぞれｍＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ｈＣＣＲ３／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ＮＣ３－ｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ＮＣ３－ｍＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ｈＣＣＲ３＿ＥＬ２ｍＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏと表わす。）。 (3) Preparation of various CCR expression vectors The mCCR1, hCCR3, NC3-hCCR1, NC3-mCCR1, hCCR3_EL2hCCR1, and hCCR3_EL2mCCR1 synthesized in (1) above were ligated to Tn-pMug-Hygro in the same manner as in (2) above. In the following, expression vectors were constructed (hereinafter, mCCR1 / Tn-pMug-Hygro, hCCR3 / Tn-pMug-Hygro, NC3-hCCR1 / Tn-pMug-Hygro, NC3-mCCR1 / Tn-pMug-Hygro, hCCR3_EL2hCCR1 / Tn-T pMug-Hygro, represented as hCCR3_EL2mCCR1 / Tn-pMug-Hygro).

（４）ｍＣＣＲ１発現ベクターの作製
　上記（２）と同様の方法で、上記（１）で合成したｍＣＣＲ１をコードするＤＮＡを、ｐＣＡＧＧＳ［Gene. 1991 Dec 15;108(2):193-9.］にｉｎｔｅｒｎａｌ　ｒｉｂｏｓｏｍａｌ　ｅｎｔｒｙ　ｓｉｔｅ（ＩＲＥＳ）とネオマイシン耐性遺伝子が付加されたベクターであるｐＣＡＧ－ＩＲＥＳ－ｎｅｏに連結させ、発現ベクターを構築した（以下、ｍＣＣＲ１／ｐＣＡＧ－ＩＲＥＳ－ｎｅｏと表わす。）。 (4) Preparation of mCCR1 expression vector DNA encoding mCCR1 synthesized in (1) above was transformed into pCAGGS [Gene. 1991 Dec 15; 108 (2): 193-9.] In the same manner as in (2) above. Was ligated to pCAG-IRES-neo, which is a vector to which an internal ribosomal entry site (IRES) and a neomycin resistance gene were added, to construct an expression vector (hereinafter referred to as mCCR1 / pCAG-IRES-neo).

［実施例２］ＣＣＲ１発現細胞株の作製
（１）ｈＣＣＲ１発現細胞の作製
　実施例１で作製したプラスミドＤＮＡであるｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－ＨｙｇｒｏとＴｏｌ２　ｔｒａｎｓｐｏｓａｓｅ発現ベクターＴＰＥＸ＿ｐＭｕｇ（国際公開第２０１３／００５６４９号）をＣＨＯ－Ｓ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）に共導入し、発現細胞株を作製した。遺伝子導入はＦｕｇｅｎｅ　ＨＤ（プロメガ社）を用いて下記のように行った。１×１０５細胞／ｍＬに調製した細胞を６ウェルプレートに２．５ｍＬずつ播種し、２４時間後に、ｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ＴＰＥＸ＿ｐＭｕｇ、Ｆｕｇｅｎｅ　ＨＤの混合物を培養液に添加した。添加後７２時間後に１ｍｇ／ｍＬのハイグロマイシン（Ｉｎｖｉｔｒｏｇｅｎ社）を添加し、約２週間の薬剤選択を行った。薬剤耐性を獲得した細胞を回収し、フローサイトメトリー（ＦＡＣＳ　Ｃａｌｉｂｕｒ、ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）による発現解析を行ったところ、導入したｈＣＣＲ１の発現が確認された。この細胞株をＣＨＯ－Ｓ－ｈＣＣＲ１と表わす。 [Example 2] Preparation of CCR1-expressing cell line (1) Preparation of hCCR1-expressing cell hCCR1 / Tn-pMug-Hygro and Tol2 transposase expression vector TPEX_pMug (International Publication No. 2013/005649) prepared in Example 1 ) Was co-introduced into CHO-S (Thermo Fisher Scientific) to produce an expression cell line. Gene transfer was performed using Fugene HD (Promega) as follows. Cells prepared at 1 × 10 5 cells / mL were seeded at 2.5 mL each in a 6-well plate, and 24 hours later, a mixture of hCCR1 / Tn-pMug-Hygro, TPEX_pMug, and Fugene HD was added to the culture medium. 72 hours after the addition, 1 mg / mL hygromycin (Invitrogen) was added, and drug selection was performed for about 2 weeks. Cells that acquired drug resistance were collected and analyzed for expression by flow cytometry (FACS Calibur, BD Biosciences). As a result, the expression of the introduced hCCR1 was confirmed. This cell line is designated CHO-S-hCCR1.

（２）各種ＣＣＲ発現細胞の作製
　実施例１で作製したｍＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ｈＣＣＲ３／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ＮＣ３－ｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ＮＣ３－ｍＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏ、ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－ＨｙｇｒｏおよびｈＣＣＲ３＿ＥＬ２ｍＣＣＲ１／Ｔｎ－ｐＭｕｇ－Ｈｙｇｒｏについて、上記（１）と同様の方法でＣＨＯ－Ｓ細胞に導入し、発現細胞株を作製した。以下、これらの細胞株をそれぞれＣＨＯ－Ｓ－ｍＣＣＲ１、ＣＨＯ－Ｓ－ｈＣＣＲ３、ＣＨＯ－Ｓ－ＮＣ３－ｈＣＣＲ１、ＣＨＯ－Ｓ－ＮＣ３－ｍＣＣＲ１、ＣＨＯ－Ｓ－ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１およびＣＨＯ－Ｓ－ｈＣＣＲ３＿ＥＬ２ｍＣＣＲ１と表わす。 (2) Production of various CCR-expressing cells mCCR1 / Tn-pMug-Hygro, hCCR3 / Tn-pMug-Hygro, NC3-hCCR1 / Tn-pMug-Hygro, NC3-mCCR1 / Tn-pMug-Hygro prepared in Example 1 HCCR3_EL2hCCR1 / Tn-pMug-Hygro and hCCR3_EL2mCCR1 / Tn-pMug-Hygro were introduced into CHO-S cells in the same manner as in (1) above to produce expression cell lines. Hereinafter, these cell lines are referred to as CHO-S-mCCR1, CHO-S-hCCR3, CHO-S-NC3-hCCR1, CHO-S-NC3-mCCR1, CHO-S-hCCR3_EL2hCCR1 and CHO-S-hCCR3_EL2mCCR1, respectively.

（３）ＲＬ３３－ｈＣＣＲ１細胞の作製
　実施例１で作製したｈＣＣＲ１／Ｔｎ－ｐＭｕｇ－ＨｙｇｒｏとＴｏｌ２　ｔｒａｎｓｐｏｓａｓｅ発現ベクターＴＰＥＸ＿ｐＭｕｇ（国際公開第２０１３／００５６４９号）をウサギ細胞株ＲＬ－３３［Yoshii et al., Jpn J Med Sci Biol. 1977 Jun;30(3):149-57］に共導入し、ｈＣＣＲ１発現細胞株を作製した。遺伝子導入はＬｉｐｏｆｅｃｔａｍｉｎｅ　ＬＴＸ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を使用し、以下のように行った。１×１０５細胞／ｍＬに調製した細胞を６ウェルプレートに２ｍＬずつ播種し、２．５μｇのプラスミドＤＮＡ、５μＬのＬｉｐｏｆｅｃｔａｍｉｎｅ　ＬＴＸの混合物を培地中に添加した。添加後７２時間後に１ｍｇ／ｍＬのハイグロマイシンを添加し、約２週間の薬剤選択を行った。薬剤耐性を獲得した細胞を回収し、フローサイトメトリーによる発現解析を行ったところ、導入したｈＣＣＲ１の発現が確認された。以下、この細胞株をＲＬ３３－ｈＣＣＲ１と表わす。 (3) Preparation of RL33-hCCR1 cells The hCCR1 / Tn-pMug-Hygro and Tol2 transposase expression vector TPEX_pMug (International Publication No. 2013/005649) prepared in Example 1 was used as the rabbit cell line RL-33 [Yoshii et al., Jpn J Med Sci Biol. 1977 Jun; 30 (3): 149-57] to prepare an hCCR1-expressing cell line. The gene transfer was performed as follows using Lipofectamine LTX (Thermo Fisher Scientific). Cells prepared to 1 × 10 5 cells / mL were seeded in 2 mL each in a 6-well plate, and a mixture of 2.5 μg of plasmid DNA and 5 μL of Lipofectamine LTX was added to the medium. 72 hours after the addition, 1 mg / mL hygromycin was added, and drug selection was performed for about 2 weeks. Cells that acquired drug resistance were collected and analyzed for expression by flow cytometry. As a result, the expression of the introduced hCCR1 was confirmed. Hereinafter, this cell line is designated as RL33-hCCR1.

（４）ＲＬ３３－ｍＣＣＲ１細胞の作製
　実施例１（４）で作製したｍＣＣＲ１／ｐＣＡＧ－ＩＲＥＳ－ｎｅｏを上記（３）と同様の方法でＲＬ－３３に導入し、発現細胞株を作製した。薬剤選択は０．５ｍｇ／ｍＬのＧ４１８で行った。以下、この細胞株をＲＬ３３－ｍＣＣＲ１と表わす。 (4) Production of RL33-mCCR1 cells The mCCR1 / pCAG-IRES-neo produced in Example 1 (4) was introduced into RL-33 in the same manner as in (3) above to produce an expression cell line. Drug selection was performed with 0.5 mg / mL G418. Hereinafter, this cell line is designated as RL33-mCCR1.

［実施例３］抗ＣＣＲ１ウサギポリクローナル抗体の作製
　以下の方法で抗ＣＣＲ１ウサギポリクローナル抗体を作製した。ヒトＣＣＲ１のＮ末端ペプチド（配列番号１０）を合成し、２匹のウサギ（Ｎｅｗ　Ｚｅａｌａｎｄ　Ｗｈｉｔｅ）を２週おきに５回免疫した。免疫は、初回のみＣｏｍｐｌｅｔｅ　Ｆｒｅｕｎｄ’ｓ　Ａｄｊｕｖａｎｔ（ＣＦＡ）を用い、二回目以降はＩｎｃｏｍｐｌｅｔｅ　Ｆｒｅｕｎｄ’ｓ　Ａｄｊｕｖａｎｔ（ＩＦＡ）を使用し、背部の複数個所に皮下注入で行った。免疫後に抗体価の上昇した個体から血清を採取し、Ｐｒｏｔｅｉｎ　Ａカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社）によるアフィニティ精製でＩｇＧを精製した。このようにして作製した抗ＣＣＲ１ウサギポリクローナル抗体はＥ５９７１と表わす。 [Example 3] Preparation of anti-CCR1 rabbit polyclonal antibody An anti-CCR1 rabbit polyclonal antibody was prepared by the following method. Human CCR1 N-terminal peptide (SEQ ID NO: 10) was synthesized, and two rabbits (New Zealand White) were immunized 5 times every 2 weeks. Immunization was carried out by subcutaneous injection at multiple locations on the back using Complete Freund's Adjuvant (CFA) only for the first time and Incomplete Freund's Adjuvant (IFA) for the second and subsequent times. Sera were collected from individuals whose antibody titers increased after immunization, and IgG was purified by affinity purification using a Protein A column (GE Healthcare). The anti-CCR1 rabbit polyclonal antibody thus prepared is designated E5971.

［実施例４］フローサイトメトリーによる発現解析
（１）ＣＣＲ１発現の確認
　実施例２で作製したＣＣＲ１発現細胞株について、実施例３で作製した抗ＣＣＲ１ウサギポリクローナル抗体Ｅ５９７１を用いて染色し、フローサイトメトリー（ＦＣＭ）でＣＣＲ１の発現を確認した。ＦＣＭ解析は以下のように行った。細胞を９６ウェルプレートに２×１０５細胞／ウェルで播種し、染色バッファー［３％ＦＢＳ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）／ＤＰＢＳ（ナカライテスク社）／０．１％アジ化ナトリウム（ナカライテスク社）］で洗浄した。該細胞を１０μｇ／ｍＬのＥ５９７１によって１時間氷上で処理し、染色バッファーによる洗浄後、二次抗体Ａｌｅｘａ　Ｆｌｕｏｒ　６４７　ｇｏａｔ　Ａｎｔｉ－Ｒａｂｂｉｔ　ＩｇＧ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を終濃度１μｇ／ｍＬで添加し、室温にて３０分間処理した。該細胞について、再度染色バッファーによる洗浄を行った後に、染色バッファーにて懸濁し、ＢＤ　ＦＡＣＳＣａｌｉｂｕｒ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）を用いて解析を行った。これにより、作製したＣＣＲ１発現細胞株について、導入したＣＣＲ１が発現していることを確認できた。 [Example 4] Expression analysis by flow cytometry (1) Confirmation of CCR1 expression The CCR1-expressing cell line prepared in Example 2 was stained with the anti-CCR1 rabbit polyclonal antibody E5971 prepared in Example 3, and the flow site CCR1 expression was confirmed by measurement (FCM). FCM analysis was performed as follows. Cells were seeded in 96-well plates at 2 × 10 5 cells / well and stained buffer [3% FBS (Thermo Fisher Scientific) / DPBS (Nacalai Tesque) /0.1% sodium azide (Nacalai Tesque)] Washed with. The cells were treated with 10 μg / mL E5971 for 1 hour on ice, washed with staining buffer, and then added with secondary antibody Alexa Fluor 647 goat Anti-Rabbit IgG (Thermo Fisher Scientific) at a final concentration of 1 μg / mL. Treated for 30 minutes at room temperature. The cells were washed again with a staining buffer, suspended in the staining buffer, and analyzed using BD FACSCalibur (BD Biosciences). This confirmed that the introduced CCR1 was expressed in the produced CCR1-expressing cell line.

（２）ＣＣＲ３発現の確認
　実施例２で作製したＣＨＯ－Ｓ－ｈＣＣＲ３についても、上記（１）と同様の方法でＣＣＲ３の発現を確認した。一次抗体には、市販の抗ＣＣＲ３抗体である４４４－１１抗体（ＭＢＬ社）を、二次抗体にはＡｌｅｘａ　Ｆｌｕｏｒ　６４７　ｇｏａｔ　Ａｎｔｉ－ｍｏｕｓｅＩｇＧ（Ｈ＋Ｌ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を使用した。これにより、ＣＨＯ－Ｓ－ｈＣＣＲ３で導入したＣＣＲ３が発現していることを確認できた。 (2) Confirmation of CCR3 Expression Regarding CHO-S-hCCR3 prepared in Example 2, the expression of CCR3 was confirmed by the same method as in (1) above. A commercially available anti-CCR3 antibody, 444-11 antibody (MBL) was used as the primary antibody, and Alexa Fluor 647 goat Anti-mouse IgG (H + L) (Thermo Fisher Scientific) was used as the secondary antibody. This confirmed that CCR3 introduced by CHO-S-hCCR3 was expressed.

［実施例５］ＣＣＲ１ノックアウトマウスを用いたモノクローナル抗体の作製
　マウス交差性抗体を得るため、市販のＣＣＲ１ノックアウト（ＫＯ）マウス（Ｂ６．１２９Ｓ４－Ｃｃｒ１ｔｍ１Ｇａｏ　Ｎ１０＋Ｎ５）（非特許文献１７）（Ｔａｃｏｎｉｃ社）を用いてモノクローナル抗体を作製した。抗体作製は以下の手順で行った。 [Example 5] Production of monoclonal antibody using CCR1 knockout mouse To obtain a mouse cross-linking antibody, commercially available CCR1 knockout (KO) mouse (B6.129S4-Ccr1 tm1Gao N10 + N5) (Non-patent Document 17) (Taconic) Were used to prepare monoclonal antibodies. Antibody production was performed according to the following procedure.

（１）免疫感作
　免疫原として、実施例２で作製したＣＨＯ－Ｓ－ｈＣＣＲ１、ＣＨＯ－Ｓ－ｍＣＣＲ１、ＲＬ３３－ｈＣＣＲ１、およびＲＬ３３－ｍＣＣＲ１を用いた。一回の免疫あたり１×１０７細胞／匹を用いた。５～９週齢のＣＣＲ１　ＫＯ　マウスに、初回免疫時のみアジュバントとしてＡｌｕｍゲル（エル・エス・エル社）（８０μＬ／匹）及び百日咳ワクチン（ナカライテスク社）（１×１０７細胞／匹）を加え腹腔内投与により免疫感作を行った。いずれの免疫も投与量が５００μＬ／匹になるようにＰＢＳで調製した。初回免疫２週間後に２回目、さらに１週間後に３回目の免疫を行い、３日後に部分採血を行った。 (1) Immunization As the immunogen, CHO-S-hCCR1, CHO-S-mCCR1, RL33-hCCR1, and RL33-mCCR1 prepared in Example 2 were used. 1 × 10 7 cells / mouse were used per immunization. 5-9 week old CCR1 KO mice were treated with Alum gel (ELS) (80 μL / mouse) and pertussis vaccine (Nacalai Tesque) (1 × 10 7 cells / mouse) as adjuvants only at the time of the first immunization. In addition, immunization was performed by intraperitoneal administration. All immunizations were prepared with PBS so that the dose was 500 μL / animal. The second immunization was performed 2 weeks after the first immunization, and the third immunization was further performed 1 week later, and partial blood collection was performed 3 days later.

（２）抗血清評価（ＦＣＭ）
　実施例２で作製した各種ＣＣＲ１発現細胞を用いて、血清中の特異的抗体価をＦＣＭで測定した。測定は以下の手順で行った。細胞をそれぞれ１×１０５細胞／ウェルになるように、１％ＢＳＡ（ナカライテスク社）－ＰＢＳ（ナカライテスク社）［０．０２％ＥＤＴＡ（ナカライテスク社）、０．０５％ＮａＮ３（ナカライテスク社）を含む］で調製し、９６ウェル細胞培養用プレートＵ底に５０μＬ／ウェルで分注した。そこへ被験サンプルとして被免疫動物から採取した血清を、終濃度２００倍希釈、１０００倍希釈、及び５０００倍希釈になるように１％ＢＳＡ－ＰＢＳ（０．０２％ＥＤＴＡ、０．０５％ＮａＮ３）で調製し５０μＬ／ウェルで分注し、４℃で３０分間放置した。遠心分離（２０００ｒｐｍ、２分間）した後に上清を吸引し、プレートシェーカーで細胞のペレットを解した。１％ＢＳＡ－ＰＢＳ（０．０２％ＥＤＴＡ、０．０５％ＮａＮ３）を２００μＬ／ウェルで分注し、再度遠心分離（２０００ｒｐｍ、２分間）した後に上清を吸引し、プレートシェーカーで細胞のペレットを解した。そこへＡｌｅｘａ　Ｆｌｕｏｒ６４７　ｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ（Ｈ＋Ｌ）、若しくはＡｌｅｘａ　Ｆｌｕｏｒ４８８　ｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ（Ｈ＋Ｌ）を最終的に３００倍希釈になるように１％ＢＳＡ－ＰＢＳ（０．０２％ＥＤＴＡ、０．０５％ＮａＮ３）で調製し、５０μＬ／ウェルで分注し遮光下４℃で３０分間放置した。遠心分離（２０００ｒｐｍ、２分間）した後に上清を吸引し、プレートシェーカーで細胞のペレットを解した。１％ＢＳＡ－ＰＢＳ（０．０２％ＥＤＴＡ、０．０５％ＮａＮ３）を２００μＬ／ウェルで分注し、再度遠心分離（２０００ｒｐｍ、２分間）した後に上清を吸引し、プレートシェーカーで細胞のペレットを解した。そこへ１％ＢＳＡ－ＰＢＳ（０．０２％ＥＤＴＡ、０．０５％ＮａＮ３）を５０μＬ／ウェルで分注し、フローサイトメーター［ＦＡＣＳＣａｎｔｏ（商標）ＩＩ／ＢＤ］で蛍光強度を測定した。これにより、抗体化の上昇が確認された個体を選択し、脾臓を摘出した。 (2) Antiserum evaluation (FCM)
Using various CCR1-expressing cells prepared in Example 2, the specific antibody titer in serum was measured by FCM. The measurement was performed according to the following procedure. 1% BSA (Nacalai Tesque) -PBS (Nacalai Tesque) [0.02% EDTA (Nacalai Tesque), 0.05% NaN 3 (Nacalai Tesque) so that each cell is 1 × 10 5 cells / well. And dispensed to the bottom of a 96-well cell culture plate U at 50 μL / well. Serum collected from the immunized animal as a test sample was diluted with 1% BSA-PBS (0.02% EDTA, 0.05% NaN 3) so that the final concentrations were 200-fold dilution, 1000-fold dilution, and 5000-fold dilution. ) And dispensed at 50 μL / well and left at 4 ° C. for 30 minutes. After centrifugation (2000 rpm, 2 minutes), the supernatant was aspirated, and the cell pellet was broken with a plate shaker. Dispense 1% BSA-PBS (0.02% EDTA, 0.05% NaN 3 ) at 200 μL / well, re-centrifuge (2000 rpm, 2 minutes), aspirate the supernatant, and remove the cells on a plate shaker. The pellet was unwound. Alexa Fluor 647 goat anti-mouse IgG (H + L) or Alexa Fluor 488 goat anti-mouse IgG (H + L) was finally diluted 300-fold with 1% BSA-PBS (0.02% EDTA, 0.05 % NaN 3 ), dispensed at 50 μL / well, and allowed to stand at 4 ° C. for 30 minutes in the dark. After centrifugation (2000 rpm, 2 minutes), the supernatant was aspirated, and the cell pellet was broken with a plate shaker. Dispense 1% BSA-PBS (0.02% EDTA, 0.05% NaN 3 ) at 200 μL / well, re-centrifuge (2000 rpm, 2 minutes), aspirate the supernatant, and remove the cells on a plate shaker. The pellet was unwound. 1% BSA-PBS (0.02% EDTA, 0.05% NaN 3 ) was dispensed thereto at 50 μL / well, and the fluorescence intensity was measured with a flow cytometer [FACSCanto ™ II / BD]. Thereby, an individual in which an increase in antibody was confirmed was selected, and the spleen was removed.

（３）細胞融合によるハイブリドーマ作製
　マウスミエローマ細胞株　Ｐ３－Ｕ１（Ｐ３Ｘ６３Ａｇ８Ｕ．１、ＡＴＣＣ　ＣＲＬ－１５９７）をエスクロンクローニングメディウム（エーディア社）で培養して無血清馴化したのち、細胞融合の親株として用いた。被免疫動物の脾臓を無菌的に採取しＲＥＤ　ＢＬＯＯＤ　ＣＥＬＬ　ＬＹＳＩＮＧ　ＢＵＦＦＥＲ（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）で溶血後、ＰＢＳで２回洗浄し、脾細胞とＰ３－Ｕ１の細胞数が、脾臓細胞：Ｐ３－Ｕ１＝８：１になるよう混合し、遠心分離（１２００ｒｐｍ、５分間）した。得られた沈澱画分の細胞群をよくほぐした後、攪拌しながら３７℃にて１ｇのポリエチレングリコール－１０００（ＰＥＧ－１０００、純正化学社）、１ｍＬのＭＥＭ培地（ナカライテスク社）および０．３５ｍＬのジメチルスルホキシド（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）の混液を０．５ｍＬ加え、そこに１分間毎に１ｍＬのＭＥＭ培地を５回加えた後、ＭＥＭ培地を加えて全量が５０ｍＬになるようにした。細胞懸濁液を遠心分離（９００ｒｐｍ、５分間）し、得られた沈澱画分の細胞をゆるやかにほぐした後、ＨＡＴ　ＳＵＰＰＬＥＭＥＮＴ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を添加したエスクロンクローニングメディウムで脾臓細胞１．５×１０７細胞／９ｍＬの細胞濃度に懸濁した。事前に９６ウェル培養用プレートにはＨＡＴ添加クローニングメディウムを１００μＬ／ウェルで分注しておき、そこへ細胞懸濁液を１００μＬ／ウェルずつ分注し、ＣＯ２インキュベーター（５％ＣＯ２、３７℃）で８～１０日間培養した。 (3) Hybridoma preparation by cell fusion Mouse myeloma cell line P3-U1 (P3X63Ag8U.1, ATCC CRL-1597) was cultured in Escron Cloning Medium (Aedia) and used as a parent strain for cell fusion It was. The spleen of the immunized animal was aseptically collected, hemolyzed with RED BLOOD CELL LYSING BUFFER (Sigma-Aldrich), washed twice with PBS, and the number of spleen cells and P3-U1 was determined as spleen cells: P3-U1 = 8: 1 and mixed (1200 rpm, 5 minutes). After the cells of the resulting precipitate fraction are thoroughly loosened, 1 g of polyethylene glycol-1000 (PEG-1000, Junsei Chemical), 1 mL of MEM medium (Nacalai Tesque) and 0. 0.5 mL of a mixed solution of 35 mL of dimethyl sulfoxide (Sigma-Aldrich) was added, and then 1 mL of MEM medium was added 5 times every minute, and then the MEM medium was added so that the total volume became 50 mL. After centrifuging the cell suspension (900 rpm, 5 minutes) and gently loosening the cells of the obtained precipitate fraction, the spleen cells 1. were added with an escron cloning medium supplemented with HAT SUPPLEMENT (Thermo Fisher Scientific). Suspended to a cell concentration of 5 × 10 7 cells / 9 mL. A 96-well culture plate was previously dispensed with HAT-added cloning medium at 100 μL / well, and the cell suspension was dispensed at 100 μL / well into a CO 2 incubator (5% CO 2 , 37 ° C.). ) For 8-10 days.

（４）ハイブリドーマのスクリーニング
　ハイブリドーマ培養上清に含まれる抗体のＣＣＲ１への結合活性をＦＣＭで評価した。被験サンプルにはハイブリドーマ培養上清を使用し、染色と測定は上記（２）と同様の手順で実施した。 (4) Screening of hybridoma The binding activity of the antibody contained in the hybridoma culture supernatant to CCR1 was evaluated by FCM. Hybridoma culture supernatant was used as a test sample, and staining and measurement were performed in the same procedure as in (2) above.

（５）ハイブリドーマのサブクローニング
　スクリーニングで陽性であったウェルの細胞についてサブクローニングを行い、クローニングメディウム中で７～１０日ほど培養した。 (5) Subcloning of hybridoma Subcloning was performed on cells in the wells that were positive in the screening, and the cells were cultured in a cloning medium for about 7 to 10 days.

（６）抗体サブクラスの決定
　サブクラス特異的な二次抗体を用いたＦＣＭで各抗体のサブクラスを決定した。染色、測定の手順は上記（２）と同様に行った。被験サンプルにはハイブリドーマ培養上清を使用した。検出抗体にはＡｌｅｘａ　Ｆｌｕｏｒ４８８　ｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ（Ｈ＋Ｌ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）及び、各サブクラス特異的な抗体（Ａｌｅｘａ　Ｆｌｕｏｒ４８８　ｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ１（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ａｌｅｘａ　Ｆｌｕｏｒ４８８　ｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ２ａ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、　Ａｌｅｘａ　Ｆｌｕｏｒ４８８　ｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ２ｂ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、Ａｌｅｘａ　Ｆｌｕｏｒ４８８　ｇｏａｔ　ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ３）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を使用した。 (6) Determination of antibody subclass The subclass of each antibody was determined by FCM using a subclass-specific secondary antibody. The procedure for staining and measurement was the same as (2) above. Hybridoma culture supernatant was used as a test sample. The detection antibodies include Alexa Fluor 488 goat anti-mouse IgG (H + L) (Thermo Fisher Scientific), and each subclass-specific antibody (Alexa Fluor 488 goat anti-mouse IgG 8 (Thermo Fisher 8). (Thermo Fisher Scientific), Alexa Fluor 488 goat anti-mouse IgG2b (Thermo Fluor 488 goat anti-mouse IgG used)

（７）ハイブリドーマ培養上清からの抗体精製
　上記のようにクローニングしたハイブリドーマの培養上清から抗体を精製した。精製はＰｒｏｔｅｉｎ　Ｇ　Ｓｅｐｈａｒｏｓｅ　４Ｆａｓｔ　Ｆｌｏｗ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社）を使用した。培養上清を遠心分離して沈殿物を除き、フィルターでろ過した。カラムに４００μＬの担体を充填し、ＤＰＢＳでバッファーを置換した。培養上清を添加し、単体に抗体を吸着させた後に、１０ｍＬのＤＰＢＳで２回洗浄した。０．４ｍＬのＩｇＧ　Ｅｌｕｔｉｏｎ　Ｂｕｆｆｅｒ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を添加して溶出し、直後に０．１ｍＬの１Ｍ　Ｔｒｉｓ－ＨＣｌ（ニッポンジーン社）ｐＨ８．６にて中和した。ＮＡＰカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社）を用いて脱塩とＤＰＢＳへのバッファー置換を行い、以降の解析に使用した。作製された抗体のクローン名、由来、およびサブクラスを表１に示す。 (7) Antibody Purification from Hybridoma Culture Supernatant Antibodies were purified from the hybridoma culture supernatants cloned as described above. For purification, Protein G Sepharose 4Fast Flow (GE Healthcare) was used. The culture supernatant was centrifuged to remove the precipitate and filtered through a filter. The column was packed with 400 μL of carrier and the buffer was replaced with DPBS. The culture supernatant was added, and the antibody was adsorbed to the simple substance, followed by washing twice with 10 mL of DPBS. 0.4 mL of IgG Elution Buffer (Thermo Fisher Scientific) was added and eluted, and immediately neutralized with 0.1 mL of 1 M Tris-HCl (Nippon Gene) pH 8.6. Desalting and buffer replacement with DPBS were performed using a NAP column (GE Healthcare) and used for the subsequent analysis. Table 1 shows the clone name, origin, and subclass of the prepared antibody.

Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001

［実施例６］ＴＨＰ－１遊走（ケモタキシス）アッセイ
　ＣＣＲ１を発現するヒト細胞株としてはヒト単球性白血病細胞株ＴＨＰ－１が知られている。本細胞はＣＣＬ３、ＣＣＬ５、ＣＣＬ１５またはＣＣＬ２３等のＣＣＲ１リガンドの濃度勾配に対して走化性を示すことが知られており、ＴＨＰ－１を用いた遊走アッセイはＣＣＲ１阻害剤の評価系として汎用される系である。よって、実施例５で得られた抗ヒトＣＣＲ１抗体についても、この実験系を用いてヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害するか否かを評価した。 [Example 6] THP-1 migration (chemotaxis) assay The human monocytic leukemia cell line THP-1 is known as a human cell line expressing CCR1. This cell is known to exhibit chemotaxis to a concentration gradient of CCR1 ligand such as CCL3, CCL5, CCL15 or CCL23, and a migration assay using THP-1 is widely used as an evaluation system for CCR1 inhibitors. System. Therefore, the anti-human CCR1 antibody obtained in Example 5 was also evaluated using this experimental system to inhibit the activation of human CCR1 by human CCL15.

　遊走アッセイの方法を以下に記す。ＴＨＰ－１細胞はＡＴＣＣより入手した。ＴＨＰ－１細胞を５μＭ　Ａｌｌ－ｔｒａｎｓ－ｒｅｔｉｎｏｉｃ　ａｃｉｄ（ＡＴＲＡ）（和光純薬工業社）存在下で３日間培養し分化誘導した上で回収し、３７℃で加温したアッセイ培地［１％ＦＢＳ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）／ＲＰＭＩ１６４０（ナカライテスク社）］にて洗浄後、同培地に再懸濁した。１×１０６細胞／ｍＬに調製し、ポアサイズ５μｍのＴｒａｎｓｗｅｌｌ（Ｃｏｒｎｉｎｇ社、＃３４２１）の上層に１００μＬ／ウェルで細胞を分注した。下層にはケモアトラクタントとして、１ｎｇ／ｍＬの組換えヒトＣＣＬ１５（６８ａａ）（Ｒ＆Ｄ　ｔｅｃｈｎｏｌｏｇｉｅｓ社、＃６２８－ＬＫ）を添加したアッセイ培地を入れ、３７℃にて５％ＣＯ２インキュベーター内で４－６時間培養した後に、下層に移動した細胞数をＣｅｌｌｔｉｔｅｒ－Ｇｌｏ（プロメガ社）で定量した。 The method of migration assay is described below. THP-1 cells were obtained from ATCC. THP-1 cells were cultured for 3 days in the presence of 5 μM All-trans-retinoic acid (ATRA) (Wako Pure Chemical Industries, Ltd.), induced to differentiate, recovered, and incubated at 37 ° C. [1% FBS ( (Thermo Fisher Scientific) / RPMI1640 (Nacalai Tesque)] and then resuspended in the same medium. 1 × 10 6 cells / mL were prepared, and cells were dispensed at 100 μL / well on the upper layer of Transwell (Corning, # 3421) having a pore size of 5 μm. The lower layer is filled with assay medium supplemented with 1 ng / mL recombinant human CCL15 (68aa) (R & D technologies, # 628-LK) as a chemoattractant, and 4-fold in a 5% CO 2 incubator at 37 ° C. After culturing for 6 hours, the number of cells migrated to the lower layer was quantified with Celltiter-Glo (Promega).

　本測定系を用いて精製抗体の細胞遊走を評価する際には、予め１．５ｍＬチューブ内で９０μＬの細胞懸濁液と１０μＬの精製抗体溶液を混合し、３７℃にて１時間インキュベートした上で、細胞をＴｒａｓｎｗｅｌｌの上層に分注した。抗体は、終濃度を０．３、１、３および１０μｇ／ｍＬに調整し、測定に使用した。 When evaluating the cell migration of the purified antibody using this measurement system, 90 μL of the cell suspension and 10 μL of the purified antibody solution were mixed in advance in a 1.5 mL tube and incubated at 37 ° C. for 1 hour. The cells were then dispensed into the upper layer of Trasnwell. The antibody was used for the measurement after adjusting the final concentration to 0.3, 1, 3, and 10 μg / mL.

　得られた結果を図１（ａ）および図１（ｂ）に示す。図１（ａ）および図１（ｂ）に示すように、実施例５で得られたマウス抗ヒトＣＣＲ１モノクローナル抗体ＫＭ５９０７抗体、ＫＭ５９０８抗体、ＫＭ５９０９抗体、ＫＭ５９１１抗体、ＫＭ５９１５抗体、ＫＭ５９１６抗体、ＫＭ５９５４抗体、ＫＭ５９５５抗体およびＫＭ５９５６抗体は、いずれも活性化ＣＣＬ１５により誘導されるＴＨＰ－１の遊走を濃度依存的に阻害した。 The obtained results are shown in FIG. 1 (a) and FIG. 1 (b). As shown in FIG. 1 (a) and FIG. 1 (b), the mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody obtained in Example 5 Both KM5955 antibody and KM5956 antibody inhibited THP-1 migration induced by activated CCL15 in a concentration-dependent manner.

　以上より、本発明のマウス抗ヒトＣＣＲ１モノクローナル抗体は、いずれもヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害する抗体であることが明らかになった。 From the above, it was revealed that the mouse anti-human CCR1 monoclonal antibody of the present invention is an antibody that inhibits the activation of human CCR1 by human CCL15.

［実施例７］抗ヒトＣＣＲ１抗体のヒトＣＣＲ１結合領域の決定
　実施例５で得られたマウス抗ヒトＣＣＲ１モノクローナル抗体のヒトＣＣＲ１の結合領域を、ＣＣＲ１－ＣＣＲ３キメラ受容体発現細胞を用いてＦＣＭで調べた。測定は実施例４と同様の方法で行った。 [Example 7] Determination of human CCR1 binding region of anti-human CCR1 antibody The human CCR1 binding region of the mouse anti-human CCR1 monoclonal antibody obtained in Example 5 was subjected to FCM using cells expressing CCR1-CCR3 chimeric receptor. Examined. The measurement was performed in the same manner as in Example 4.

　ＣＣＲ１－ＣＣＲ３キメラ受容体発現細胞として、実施例２で作製したＣＨＯ－Ｓ－ｈＣＣＲ３、ＣＨＯ－Ｓ－ＮＣ３－ｈＣＣＲ１、ＣＨＯ－Ｓ－ＮＣ３－ｍＣＣＲ１、およびＣＨＯ－Ｓ－ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１を使用した。また、ネガティブコントロールとしてＣＨＯ－Ｓを使用した。 CHO-S-hCCR3, CHO-S-NC3-hCCR1, CHO-S-NC3-mCCR1, and CHO-S-hCCR3_EL2hCCR1 prepared in Example 2 were used as CCR1-CCR3 chimeric receptor-expressing cells. Moreover, CHO-S was used as a negative control.

　被験抗体としては、１０倍希釈した各ハイブリドーマ培養上清、既存のマウス抗ヒトＣＣＲ１モノクローナル抗体　５３５０４抗体（Ｒ＆Ｄ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）およびマウス抗ヒトＣＣＲ３モノクローナル抗体　４４４－１１抗体（ＭＢＬ社）を使用した。 As test antibodies, each hybridoma culture supernatant diluted 10-fold, existing mouse anti-human CCR1 monoclonal antibody 53504 antibody (R & D Technologies) and mouse anti-human CCR3 monoclonal antibody 444-11 antibody (MBL) were used.

　測定結果について、ある細胞を、ある被験抗体（各ハイブリドーマ培養上清、５３５０４抗体または４４４－１１抗体）および二次抗体で染色した時の蛍光強度を、該細胞を二次抗体のみで染色した時の蛍光強度で除した。得られた数値が１０以上の時は、該被験抗体は該細胞に結合すると判定し、１０未満の時は、該被験抗体は該細胞に結合しないと判定し、表２中にそれぞれ○、×で示した。 Regarding the measurement results, the fluorescence intensity when a certain cell was stained with a certain test antibody (each hybridoma culture supernatant, 53504 antibody or 444-11 antibody) and a secondary antibody was measured when the cell was stained only with the secondary antibody. Was divided by the fluorescence intensity. When the obtained numerical value is 10 or more, it is determined that the test antibody binds to the cell, and when it is less than 10, it is determined that the test antibody does not bind to the cell. It showed in.

Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

　表２より、マウス抗ヒトＣＣＲ１モノクローナル抗体ＫＭ５９０７抗体、ＫＭ５９０８抗体、ＫＭ５９０９抗体、ＫＭ５９１１抗体、ＫＭ５９１５抗体、ＫＭ５９１６抗体、ＫＭ５９５４抗体、ＫＭ５９５５抗体およびＫＭ５９５６抗体は、いずれもＣＨＯ－Ｓ－ｈＣＣＲ３には結合せず、ＣＨＯ－Ｓ－ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１に結合した。したがって本発明のマウス抗ヒトＣＣＲ１モノクローナル抗体はいずれもヒトＣＣＲ１の細胞外ループ２に結合することが明らかとなった。 From Table 2, mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody do not bind to CHO-S-hCCR3. , CHO-S-hCCR3_EL2hCCR1. Therefore, it was revealed that any of the mouse anti-human CCR1 monoclonal antibodies of the present invention binds to the extracellular loop 2 of human CCR1.

［実施例８］既存の抗ヒトＣＣＲ１抗体と抗ヒトＣＣＲ１抗体を用いたケモタキシスアッセイ
（１）既存のマウス抗ヒトＣＣＲ１モノクローナル抗体２Ｄ４抗体の調製
　既存の抗ヒトＣＣＲ１抗体である２Ｄ４抗体（米国特許第６，７５６，０３５号明細書）を産生するハイブリドーマ（ＬＳ－１２５－２Ｄ４－１１－１０－１）をＡＴＣＣより入手した。本ハイブリドーマをＨｙｂｒｉｄｏｍａ－ＳＦＭ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を用いて培養し、培養上清から抗体を精製した。精製はＰｒｏｔｅｉｎ　Ｇ　Ｓｅｐｈａｒｏｓｅ　４Ｆａｓｔ　Ｆｌｏｗ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社）を使用した。培養上清を遠心分離して、得られた培養上清をフィルターでろ過した。カラムに４００μＬの担体を充填し、ＤＰＢＳでバッファーを置換した。該カラムに該培養上清を添加し、担体に抗体を吸着させた後に、１０ｍＬのＤＰＢＳで２回洗浄した。該カラムに０．４ｍＬのＩｇＧ　Ｅｌｕｔｉｏｎ　Ｂｕｆｆｅｒ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ社）を添加して抗体を溶出させ、すぐに０．１ｍＬの１Ｍ　Ｔｒｉｓ－Ｃｌ（ナカライテスク社）ｐＨ８．６にて抗体溶液を中和した。ＮＡＰカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社）を用いて該抗体溶液の脱塩とＤＰＢＳへのバッファー置換を行い、以降の解析に使用した。 [Example 8] Chemotaxis assay using existing anti-human CCR1 antibody and anti-human CCR1 antibody (1) Preparation of existing mouse anti-human CCR1 monoclonal antibody 2D4 antibody 2D4 antibody, an existing anti-human CCR1 antibody (USA) A hybridoma (LS-125-2D4-11-10-1) producing Patent No. 6,756,035) was obtained from ATCC. This hybridoma was cultured using Hybridoma-SFM (Thermo Fisher Scientific), and the antibody was purified from the culture supernatant. For purification, Protein G Sepharose 4Fast Flow (GE Healthcare) was used. The culture supernatant was centrifuged, and the obtained culture supernatant was filtered with a filter. The column was packed with 400 μL of carrier and the buffer was replaced with DPBS. The culture supernatant was added to the column, and the antibody was adsorbed onto the carrier, followed by washing twice with 10 mL of DPBS. 0.4 mL of IgG Elution Buffer (Thermo Scientific) was added to the column to elute the antibody, and the antibody solution was immediately neutralized with 0.1 mL of 1 M Tris-Cl (Nacalai Tesque) pH 8.6. . The antibody solution was desalted and buffered with DPBS using a NAP column (GE Healthcare) and used for the subsequent analysis.

　精製した２Ｄ４抗体について、還元条件でのＳＤＳ－ＰＡＧＥを通常の方法で行い、抗体が精製されていることを確認した。 The purified 2D4 antibody was subjected to SDS-PAGE under reducing conditions by a conventional method, and it was confirmed that the antibody was purified.

　また、２Ｄ４抗体のヒトＣＣＲ１への結合活性を、実施例４に記載する方法に準じてＦＣＭで確認した。２Ｄ４抗体は０．１、１μｇ／ｍＬで反応させ、細胞は、ヒトＣＣＲ１発現細胞としてＣＨＯ－Ｓ－ｈＣＣＲ１を、ネガティブコントロールとしてＣＨＯ－Ｓを使用した。その結果、２Ｄ４抗体は、ＣＨＯ－Ｓには結合せず、ＣＨＯ－Ｓ－ｈＣＣＲ１には濃度依存的に結合した。よって、精製した２Ｄ４抗体は、市販されている２Ｄ４抗体と同様に、ヒトＣＣＲ１への結合性を有していることが確認できた。 Also, the binding activity of the 2D4 antibody to human CCR1 was confirmed by FCM according to the method described in Example 4. The 2D4 antibody was reacted at 0.1 and 1 μg / mL, and the cells used were CHO-S-hCCR1 as human CCR1-expressing cells and CHO-S as a negative control. As a result, the 2D4 antibody did not bind to CHO-S and bound to CHO-S-hCCR1 in a concentration-dependent manner. Therefore, it was confirmed that the purified 2D4 antibody has a binding property to human CCR1 similarly to the commercially available 2D4 antibody.

（２）ケモタキシスアッセイ
　既存の抗ヒトＣＣＲ１抗体と、実施例５で得られたマウス抗ヒトＣＣＲ１抗体モノクローナル抗体ＫＭ５９０８抗体およびＫＭ５９１６抗体について、実施例６に記載した方法に準じてＣＣＲ１の活性化を阻害する活性を測定し、得られた結果を抗体毎に比較した。 (2) Chemotaxis assay For the existing anti-human CCR1 antibody and the mouse anti-human CCR1 antibody monoclonal antibody KM5908 antibody and KM5916 antibody obtained in Example 5, activation of CCR1 according to the method described in Example 6 The activity which inhibits was measured, and the obtained results were compared for each antibody.

　既存の抗ヒトＣＣＲ１抗体としては、（１）で作製した２Ｄ４抗体、１４１－２抗体（ＭＢＬ社）および５３５０４抗体（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）を使用した。 As the existing anti-human CCR1 antibody, 2D4 antibody, 141-2 antibody (MBL) and 53504 antibody (R & D Systems) prepared in (1) were used.

　得られた結果を図２に示す。図２に示すように、既存の抗ヒトＣＣＲ１抗体である２Ｄ４抗体、１４１－２抗体、および５３５０４抗体は、いずれも活性化ＣＣＬ１５により誘導されるＴＨＰ－１細胞の遊走を阻害しない一方で、マウス抗ヒトＣＣＲ１モノクローナル抗体ＫＭ５９０８抗体およびＫＭ５９１６抗体は、濃度依存的に上記細胞の遊走を阻害した。 The obtained results are shown in FIG. As shown in FIG. 2, the existing anti-human CCR1 antibodies 2D4 antibody, 141-2 antibody, and 53504 antibody did not inhibit THP-1 cell migration induced by activated CCL15, while mice Anti-human CCR1 monoclonal antibody KM5908 antibody and KM5916 antibody inhibited the migration of the cells in a concentration-dependent manner.

　実施例６にて記載したように、実施例５で取得した抗ヒトＣＣＲ１抗体は、いずれも本実施例と同じ実験条件で、抗体濃度依存的に活性化ＣＣＬ１５により誘導されるＴＨＰ－１細胞の遊走を阻害した［図１（ａ）および図１（ｂ）］。 As described in Example 6, all of the anti-human CCR1 antibodies obtained in Example 5 were THP-1 cells induced by activated CCL15 in an antibody concentration-dependent manner under the same experimental conditions as in this Example. Migration was inhibited [FIGS. 1 (a) and 1 (b)].

　したがって、既存の抗ヒトＣＣＲ１抗体は、ヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害しない一方で、実施例５で得られたマウス抗ヒトＣＣＲ１モノクローナル抗体ＫＭ５９０７抗体、ＫＭ５９０８抗体、ＫＭ５９０９抗体、ＫＭ５９１１抗体、ＫＭ５９１５抗体、ＫＭ５９１６抗体、ＫＭ５９５４抗体、ＫＭ５９５５抗体およびＫＭ５９５６抗体は、いずれもヒトＣＣＬ１５によるヒトＣＣＲ１の活性化を阻害する抗体であることが示された。 Therefore, the existing anti-human CCR1 antibody does not inhibit the activation of human CCR1 by human CCL15, while the mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 obtained in Example 5 Antibodies, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody were all shown to be antibodies that inhibit activation of human CCR1 by human CCL15.

［実施例９］遺伝子組換え抗体の作製
（１）抗体可変領域遺伝子のクローニングと配列決定
　実施例５でクローニングしたハイブリドーマからＴｒｉｚｏｌ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を用いてトータルＲＮＡを抽出し、５’－ＲＡＣＥ法により抗体遺伝子を増幅させた。ＲＡＣＥ用ｃＤＮＡの合成にはＳＭＡＲＴｅｒ　ＲＡＣＥ　Ｋｉｔ（Ｃｌｏｎｔｅｃｈ社）を用いた。ＲＡＣＥ用ｃＤＮＡ合成過程で付加される配列に特異的なプライマーと、マウスＩｇ　ｇａｍｍａ鎖またはｋａｐｐａ鎖増幅用プライマー（配列番号１１－１４）を用いたＰＣＲによって抗体可変領域断片を増幅させ、クローニングして該ＤＮＡ断片の塩基配列を確認した。 [Example 9] Production of recombinant antibody (1) Cloning and sequencing of antibody variable region gene Total RNA was extracted from the hybridoma cloned in Example 5 using Trizol (Life Technologies), and 5'-RACE. Antibody genes were amplified by the method. A SMARTER RACE Kit (Clontech) was used for the synthesis of RACE cDNA. Antibody variable region fragments are amplified by PCR using primers specific to the sequence added in the RACE cDNA synthesis process and a mouse Ig gamma chain or kappa chain amplification primer (SEQ ID NO: 11-14), and cloned. The base sequence of the DNA fragment was confirmed.

　実施例５で得られたの各抗ヒトＣＣＲ１抗体について、重鎖および軽鎖の可変領域のアミノ酸配列をコードする塩基配列、該塩基配列から推定されるアミノ酸配列および該アミノ酸配列からシグナル配列を除いたアミノ酸配列を表す配列番号を、それぞれ表３に示す。さらに、本発明の各抗体のＣＤＲのアミノ酸配列を表す配列番号を、それぞれ表４に示す。 For each anti-human CCR1 antibody obtained in Example 5, the base sequence encoding the amino acid sequence of the variable region of the heavy chain and the light chain, the amino acid sequence deduced from the base sequence, and the signal sequence were removed from the amino acid sequence Table 3 shows SEQ ID NOs representing the amino acid sequences. Further, Table 4 shows SEQ ID NOs representing the amino acid sequences of CDRs of the respective antibodies of the present invention.

Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003

Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004

（２）キメラ抗体の発現ベクターの作製
　実施例５で作製した各抗ヒトＣＣＲ１抗体について、定常領域をＳ２２８Ｐ、Ｌ２３５ＥおよびＲ４０９Ｋのアミノ酸改変を含むヒトＩｇＧ４定常領域（ヒトＩｇＧ４ＰＥ＿Ｒ４０９Ｋ）に置換したキメラ抗体を以下に記載する方法で作製した。（１）で作製した各抗体の可変領域のアミノ酸配列をコードする塩基配列がクローニングされたプラスミドＤＮＡを鋳型として、相同組換え用の塩基配列を付加したプライマーを用いたＰＣＲで各抗体の可変領域のアミノ酸配列をコードする塩基配列を増幅させた。Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（Ｃｌｏｎｔｅｃｈ社）を使用して、該塩基配列を、Ｎ５ＫＧ４ＰＥ　Ｒ４０９Ｋベクター［Ｎ５ＫＧ１ベクター（米国特許第６，００１，３５８号明細書）中のヒトＩｇＧ１の定常領域をコードする塩基配列を、上記のアミノ酸改変を含む変異ヒトＩｇＧ４の定常領域をコードする塩基配列に置換したベクター（以下Ｎ５ＫＧ４ＰＥ　Ｒ４０９Ｋベクターと記載する）］に連結させ、キメラ抗体の発現ベクターを作製した。実験の手順はキットに付属のマニュアルに従った。 (2) Preparation of expression vector of chimeric antibody For each anti-human CCR1 antibody prepared in Example 5, a chimeric antibody in which the constant region was replaced with human IgG4 constant region (human IgG4PE_R409K) containing amino acid modifications of S228P, L235E and R409K It was produced by the method described below. The variable region of each antibody is obtained by PCR using a primer with a base sequence for homologous recombination, using as a template the plasmid DNA into which the nucleotide sequence encoding the amino acid sequence of the variable region of each antibody prepared in (1) has been cloned. The base sequence encoding the amino acid sequence of was amplified. Using In-Fusion HD Cloning Kit (Clontech), the nucleotide sequence is converted into the base sequence encoding the constant region of human IgG1 in N5KG4PE R409K vector [N5KG1 vector (US Pat. No. 6,001,358)]. The sequence was ligated to a vector (hereinafter referred to as N5KG4PE R409K vector) in which the base sequence encoding the constant region of mutant human IgG4 containing the above-mentioned amino acid modification was substituted to prepare an expression vector for the chimeric antibody. The experimental procedure followed the manual that came with the kit.

（３）キメラ抗体の産生と精製
　（２）で作製した発現ベクターおよびＥｘｐｉ２９３　Ｅｘｐｒｅｓｓｉｏｎ　Ｓｙｓｔｅｍ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を使用して、キメラ抗体を産生した。手順は付属マニュアルに従い以下のように行った。Ｅｘｐｉ２９３Ｆ細胞（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を２×１０６細胞／ｍＬの密度で３７℃、２４時間培養し、その後、１反応あたり１．２５×１０８細胞を、４２．５ｍＬのＥｘｐｉ２９３　Ｅｘｐｒｅｓｓｉｏｎ　Ｍｅｄｉｕｍ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）に加えた。Ｏｐｔｉ－ＭＥＭ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）に５０μｇのプラスミドＤＮＡとＥｘｐｉＦｅｃｔａｍｉｎ　２９３　Ｒｅａｇｅｎｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を添加し、３０分間静置した後に、該プラスミド溶液を上記の細胞液に添加した。さらに一晩の培養後に、該細胞液にＥｘｐｉＦｅｃｔａｍｉｎ　２９３　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｅｎｈａｎｃｅｒを添加した（培養ボリュームはトータルで５０ｍＬ）。該細胞液を７～１０日間培養した後に、培養上清を回収した。 (3) Production and Purification of Chimeric Antibody Chimeric antibody was produced using the expression vector prepared in (2) and Expi293 Expression System (Life Technologies). The procedure was performed as follows according to the attached manual. Expi293F cells (Thermo Fisher Scientific) were cultured at a density of 2 × 10 6 cells / mL at 37 ° C. for 24 hours, and then 1.25 × 10 8 cells per reaction were expanded to 42.5 mL of Expi293 Expression Medium (Thermo). Fisher Scientific). 50 μg of plasmid DNA and Expifectamine 293 Reagent (Thermo Fisher Scientific) were added to Opti-MEM (Thermo Fisher Scientific), and allowed to stand for 30 minutes, and then the plasmid solution was added to the above cell solution. Further, after overnight culture, Expifectamine 293 Transfection Enhancer was added to the cell solution (total culture volume was 50 mL). After culturing the cell solution for 7 to 10 days, the culture supernatant was recovered.

　抗体の精製には、Ｐｒｏｔｅｉｎ　Ｇ　Ｓｅｐｈａｒｏｓｅ　４Ｆａｓｔ　Ｆｌｏｗ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社）を使用した。回収した培養上清を遠心分離し、得られた培養上清をフィルターでろ過した。カラムに４００μＬの担体を充填し、ＤＰＢＳでバッファーを置換した。該カラムに培養上清を添加し、単体に抗体を吸着させた後に、該カラムを１０ｍＬのＤＰＢＳで２回洗浄した。該カラムに０．４ｍＬのＩｇＧ　Ｅｌｕｔｉｏｎ　Ｂｕｆｆｅｒ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ社）を添加して抗体を溶出させ、ただちに該抗体溶液に０．１ｍＬの１Ｍ　Ｔｒｉｓ－Ｃｌ　ｐＨ８．６を加えて中和した。ＮＡＰカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社）を用いて該抗体溶液を脱塩し、以降の解析に使用した。 Protein G Sepharose 4Fast Flow (GE Healthcare) was used for antibody purification. The collected culture supernatant was centrifuged, and the obtained culture supernatant was filtered with a filter. The column was packed with 400 μL of carrier and the buffer was replaced with DPBS. The culture supernatant was added to the column, and the antibody was adsorbed to the single body, and then the column was washed twice with 10 mL of DPBS. The antibody was eluted by adding 0.4 mL of IgG Elution Buffer (Thermo Scientific) to the column, and immediately 0.1 mL of 1 M Tris-Cl pH 8.6 was added to the antibody solution for neutralization. The antibody solution was desalted using a NAP column (GE Healthcare) and used for the subsequent analysis.

　こうして得られたマウス抗ヒトＣＣＲ１モノクローナル抗体ＫＭ５９０７抗体、ＫＭ５９０８抗体、ＫＭ５９０９抗体、ＫＭ５９１１抗体、ＫＭ５９１５抗体、ＫＭ５９１６抗体、ＫＭ５９５４抗体、ＫＭ５９５５抗体およびＫＭ５９５６抗体のキメラ抗体を、それぞれｃｈＫＭ５９０７抗体、ｃｈＫＭ５９０８抗体、ｃｈＫＭ５９０９抗体、ｃｈＫＭ５９１１抗体、ｃｈＫＭ５９１５抗体、ｃｈＫＭ５９１６抗体、ｃｈＫＭ５９５４抗体、ｃｈＫＭ５９５５抗体およびｃｈＫＭ５９５６抗体と記載する。 The thus obtained mouse anti-human CCR1 monoclonal antibody KM5907 antibody, KM5908 antibody, KM5909 antibody, KM5911 antibody, KM5915 antibody, KM5916 antibody, KM5954 antibody, KM5955 antibody and KM5956 antibody chimeric antibody were chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, respectively. ChKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and chKM5956 antibody.

［実施例１０］キメラ抗体の結合性の評価
　実施例９で作製したキメラ抗体ｃｈＫＭ５９０７抗体、ｃｈＫＭ５９０８抗体、ｃｈＫＭ５９０９抗体、ｃｈＫＭ５９１１抗体、ｃｈＫＭ５９１５抗体、ｃｈＫＭ５９１６抗体、ｃｈＫＭ５９５４抗体、ｃｈＫＭ５９５５抗体およびｃｈＫＭ５９５６抗体について、ヒトおよびマウスＣＣＲ１への結合性を実施例４に記載する方法に準じてＦＣＭで測定した。ヒトＣＣＲ１発現細胞およびマウスＣＣＲ１発現細胞としては、それぞれ実施例２で作製したＣＨＯ－Ｓ－ｈＣＣＲ１、ＣＨＯ－Ｓ－ｍＣＣＲ１を使用した。その結果、ｃｈＫＭ５９５５抗体は、ヒトＣＣＲ１に結合することが示された。その他のキメラ抗体は、ヒトおよびマウスＣＣＲ１に結合することが示された。 Example 10 Evaluation of Chimeric Antibody Binding Chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and chKM5956 antibody prepared in Example 9 Binding to mouse CCR1 was measured by FCM according to the method described in Example 4. As human CCR1-expressing cells and mouse CCR1-expressing cells, CHO-S-hCCR1 and CHO-S-mCCR1 prepared in Example 2 were used, respectively. As a result, the chKM5955 antibody was shown to bind to human CCR1. Other chimeric antibodies have been shown to bind to human and mouse CCR1.

［実施例１１］キメラ抗体を用いたケモタキシスアッセイ
　実施例９で作製したキメラ抗体ｃｈＫＭ５９０７抗体、ｃｈＫＭ５９０８抗体、ｃｈＫＭ５９０９抗体、ｃｈＫＭ５９１１抗体、ｃｈＫＭ５９１５抗体、ｃｈＫＭ５９１６抗体、ｃｈＫＭ５９５４抗体、ｃｈＫＭ５９５５抗体およびｃｈＫＭ５９５６抗体について、実施例６に記載する方法に準じて、ヒトＣＣＲ１活性化を阻害する活性測定した。その結果、いずれのキメラ抗体も、活性化ヒトＣＣＬ１５によるＴＨＰ－１の遊走を阻害することが示された。 [Example 11] Chemotaxis assay using chimeric antibody The chimeric antibody chKM5907 antibody, chKM5908 antibody, chKM5909 antibody, chKM5911 antibody, chKM5915 antibody, chKM5916 antibody, chKM5954 antibody, chKM5955 antibody and chKM5956 antibody prepared in Example 9 According to the method described in Example 6, the activity of inhibiting human CCR1 activation was measured. As a result, all of the chimeric antibodies were shown to inhibit THP-1 migration by activated human CCL15.

　本発明を特定の態様を用いて詳細に説明したが、本発明の意図と範囲を離れることなく様々な変更および変形が可能であることは、当業者にとって明らかである。なお、本出願は、２０１６年１月２０日付けで出願された日本特許出願（特願２０１６－８９８３号）に基づいており、その全体が引用により援用される。 Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. The present application is based on a Japanese patent application (Japanese Patent Application No. 2016-8983) filed on January 20, 2016, and is incorporated by reference in its entirety.

配列番号６－人工配列の説明：ＮＣ３－ｈＣＣＲ１の塩基配列
配列番号７－人工配列の説明：ＮＣ３－ｍＣＣＲ１の塩基配列
配列番号８－人工配列の説明：ｈＣＣＲ３＿ＥＬ２ｈＣＣＲ１の塩基配列
配列番号９－人工配列の説明：ｈＣＣＲ３＿ＥＬ２ｍＣＣＲ１の塩基配列
配列番号１０－人工配列の説明：Ｎ末端ｈＣＣＲ１　ｐｅｐｔｉｄｅのアミノ酸配列
配列番号１１－人工配列の説明：プライマー＿ｍｏｕｓｅ＿ｇａｍｍａ＿ｒ１の塩基配列
配列番号１２－人工配列の説明：プライマー＿ｍｏｕｓｅ＿ｇａｍｍａ＿ｒ２の塩基配列
配列番号１３－人工配列の説明：プライマー＿ｍｏｕｓｅ＿ｋａｐｐａ＿ｒ１の塩基配列
配列番号１４－人工配列の説明：プライマー＿ｍｏｕｓｅ＿ｋａｐｐａ＿ｒ２の塩基配列
配列番号５１－人工配列の説明：シグナル配列を除いたＫＭ５９０７　ＶＨのアミノ酸配列
配列番号５２－人工配列の説明：シグナル配列を除いたＫＭ５９０７　ＶＬのアミノ酸配列
配列番号５３－人工配列の説明：シグナル配列を除いたＫＭ５９０８　ＶＨのアミノ酸配列
配列番号５４－人工配列の説明：シグナル配列を除いたＫＭ５９０８　ＶＬのアミノ酸配列
配列番号５５－人工配列の説明：シグナル配列を除いたＫＭ５９０９　ＶＨのアミノ酸配列
配列番号５６－人工配列の説明：シグナル配列を除いたＫＭ５９０９　ＶＬのアミノ酸配列
配列番号５７－人工配列の説明：シグナル配列を除いたＫＭ５９１１　ＶＨのアミノ酸配列
配列番号５８－人工配列の説明：シグナル配列を除いたＫＭ５９１１　ＶＬのアミノ酸配列
配列番号５９－人工配列の説明：シグナル配列を除いたＫＭ５９１５　ＶＨのアミノ酸配列
配列番号６０－人工配列の説明：シグナル配列を除いたＫＭ５９１５　ＶＬのアミノ酸配列
配列番号６１－人工配列の説明：シグナル配列を除いたＫＭ５９１６　ＶＨのアミノ酸配列
配列番号６２－人工配列の説明：シグナル配列を除いたＫＭ５９１６　ＶＬのアミノ酸配列
配列番号６３－人工配列の説明：シグナル配列を除いたＫＭ５９５４　ＶＨのアミノ酸配列
配列番号６４－人工配列の説明：シグナル配列を除いたＫＭ５９５４　ＶＬのアミノ酸配列
配列番号６５－人工配列の説明：シグナル配列を除いたＫＭ５９５５　ＶＨのアミノ酸配列
配列番号６６－人工配列の説明：シグナル配列を除いたＫＭ５９５５　ＶＬのアミノ酸配列
配列番号６７－人工配列の説明：シグナル配列を除いたＫＭ５９５６　ＶＨのアミノ酸配列
配列番号６８－人工配列の説明：シグナル配列を除いたＫＭ５９５６　ＶＬのアミノ酸配列
配列番号６９－人工配列の説明：ＫＭ５９０７　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号７０－人工配列の説明：ＫＭ５９０７　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号７１－人工配列の説明：ＫＭ５９０７　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号７２－人工配列の説明：ＫＭ５９０７　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号７３－人工配列の説明：ＫＭ５９０７　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号７４－人工配列の説明：ＫＭ５９０７　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号７５－人工配列の説明：ＫＭ５９０８　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号７６－人工配列の説明：ＫＭ５９０８　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号７７－人工配列の説明：ＫＭ５９０８　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号７８－人工配列の説明：ＫＭ５９０８　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号７９－人工配列の説明：ＫＭ５９０８　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号８０－人工配列の説明：ＫＭ５９０８　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号８１－人工配列の説明：ＫＭ５９０９　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号８２－人工配列の説明：ＫＭ５９０９　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号８３－人工配列の説明：ＫＭ５９０９　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号８４－人工配列の説明：ＫＭ５９０９　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号８５－人工配列の説明：ＫＭ５９０９　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号８６－人工配列の説明：ＫＭ５９０９　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号８７－人工配列の説明：ＫＭ５９１１　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号８８－人工配列の説明：ＫＭ５９１１　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号８９－人工配列の説明：ＫＭ５９１１　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号９０－人工配列の説明：ＫＭ５９１１　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号９１－人工配列の説明：ＫＭ５９１１　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号９２－人工配列の説明：ＫＭ５９１１　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号９３－人工配列の説明：ＫＭ５９１５　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号９４－人工配列の説明：ＫＭ５９１５　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号９５－人工配列の説明：ＫＭ５９１５　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号９６－人工配列の説明：ＫＭ５９１５　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号９７－人工配列の説明：ＫＭ５９１５　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号９８－人工配列の説明：ＫＭ５９１５　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号９９－人工配列の説明：ＫＭ５９１６　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号１００－人工配列の説明：ＫＭ５９１６　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号１０１－人工配列の説明：ＫＭ５９１６　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号１０２－人工配列の説明：ＫＭ５９１６　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号１０３－人工配列の説明：ＫＭ５９１６　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号１０４－人工配列の説明：ＫＭ５９１６　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号１０５－人工配列の説明：ＫＭ５９５４　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号１０６－人工配列の説明：ＫＭ５９５４　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号１０７－人工配列の説明：ＫＭ５９５４　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号１０８－人工配列の説明：ＫＭ５９５４　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号１０９－人工配列の説明：ＫＭ５９５４　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号１１０－人工配列の説明：ＫＭ５９５４　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号１１１－人工配列の説明：ＫＭ５９５５　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号１１２－人工配列の説明：ＫＭ５９５５　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号１１３－人工配列の説明：ＫＭ５９５５　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号１１４－人工配列の説明：ＫＭ５９５５　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号１１５－人工配列の説明：ＫＭ５９５５　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号１１６－人工配列の説明：ＫＭ５９５５　ＶＬ　ＣＤＲ３のアミノ酸配列
配列番号１１７－人工配列の説明：ＫＭ５９５６　ＶＨ　ＣＤＲ１のアミノ酸配列
配列番号１１８－人工配列の説明：ＫＭ５９５６　ＶＨ　ＣＤＲ２のアミノ酸配列
配列番号１１９－人工配列の説明：ＫＭ５９５６　ＶＨ　ＣＤＲ３のアミノ酸配列
配列番号１２０－人工配列の説明：ＫＭ５９５６　ＶＬ　ＣＤＲ１のアミノ酸配列
配列番号１２１－人工配列の説明：ＫＭ５９５６　ＶＬ　ＣＤＲ２のアミノ酸配列
配列番号１２２－人工配列の説明：ＫＭ５９５６　ＶＬ　ＣＤＲ３のアミノ酸配列 SEQ ID NO: 6-description of artificial sequence: NC3-hCCR1 base sequence SEQ ID NO: 7-description of artificial sequence: NC3-mCCR1 base sequence SEQ ID NO: 8-description of artificial sequence: hCCR3_EL2 hCCR1 base sequence SEQ ID NO: 9- Description: nucleotide sequence of hCCR3_EL2mCCR1 SEQ ID NO: 10-description of artificial sequence: amino acid sequence of N-terminal hCCR1 peptide SEQ ID NO: 11-description of artificial sequence: primer_mouse_gamma_r1 nucleotide sequence SEQ ID NO: 12-description of artificial sequence: primer_mouse_gamma_r2 nucleotide sequence SEQ ID NO: 13—Description of artificial sequence: base sequence of primer_mouse_kappa_r1 SEQ ID NO: 14—Description of artificial sequence: base sequence of primer_mouse_kappa_r2 SEQ ID NO: 51—Description of artificial sequence : KM5907 VH amino acid sequence excluding signal sequence SEQ ID NO: 52-description of artificial sequence: KM5907 VL amino acid sequence excluding signal sequence SEQ ID NO: 53-description of artificial sequence: amino acid sequence sequence of KM5908 VH excluding signal sequence No. 54-description of artificial sequence: amino acid sequence of KM5908 VL excluding signal sequence SEQ ID NO: 55-description of artificial sequence: amino acid sequence of KM5909 VH excluding signal sequence SEQ ID NO: 56-description of artificial sequence: excluding signal sequence KM5909 VL amino acid sequence SEQ ID NO: 57-description of artificial sequence: KM5911 VH amino acid sequence number 58 excluding signal sequence-description of artificial sequence: KM5911 VL amino acid sequence excluding signal sequence SEQ ID NO: 59-artificial sequence Description: Excluding signal sequence KM5915 VH amino acid sequence SEQ ID NO: 60-description of artificial sequence: KM5915 VL amino acid sequence SEQ ID NO: 61 excluding signal sequence-description of artificial sequence: KM5916 VH amino acid sequence excluding signal sequence SEQ ID NO: 62-of artificial sequence Description: KM5916 VL amino acid sequence excluding signal sequence SEQ ID NO: 63-description of artificial sequence: KM5954 VH amino acid sequence excluding signal sequence SEQ ID NO: 64-description of artificial sequence: KM5954 VL amino acid sequence excluding signal sequence SEQ ID NO: 65—Description of artificial sequence: amino acid sequence of KM5955 VH excluding signal sequence SEQ ID NO: 66—Description of artificial sequence: amino acid sequence of KM5955 VL excluding signal sequence SEQ ID NO: 67—Description of artificial sequence: Signal sequence Excluded KM5956 VH Mino acid sequence SEQ ID NO: 68-description of artificial sequence: KM5956 VL amino acid sequence excluding signal sequence SEQ ID NO: 69-description of artificial sequence: KM5907 VH CDR1 amino acid sequence SEQ ID NO: 70-description of artificial sequence: KM5907 VH CDR2 Amino acid sequence SEQ ID NO: 71-description of artificial sequence: KM5907 VH CDR3 amino acid sequence SEQ ID NO: 72-description of artificial sequence: KM5907 VL CDR1 amino acid sequence SEQ ID NO: 73-description of artificial sequence: KM5907 VL CDR2 amino acid sequence SEQ ID NO: 74 -Description of artificial sequence: KM5907 VL CDR3 amino acid sequence SEQ ID NO: 75-Description of artificial sequence: KM5908 VH CDR1 amino acid sequence SEQ ID NO: 76-Description of artificial sequence: KM5908 VH CDR2 amino acid sequence SEQ ID NO: 77-Artificial sequence Description: KM5908 VH CDR3 amino acid sequence SEQ ID NO: 78-description of artificial sequence: KM5908 VL CDR1 amino acid sequence SEQ ID NO: 79-description of artificial sequence: KM5908 VL CDR2 amino acid sequence SEQ ID NO: 80-description of artificial sequence: KM5908 VL CDR3 amino acid sequence SEQ ID NO: 81-description of artificial sequence: KM5909 VH CDR1 amino acid sequence SEQ ID NO: 82-description of artificial sequence: KM5909 VH CDR2 amino acid sequence SEQ ID NO: 83-description of artificial sequence: KM5909 VH CDR3 amino acid sequence No. 84-description of artificial sequence: amino acid sequence of KM5909 VL CDR1 SEQ ID NO: 85-description of artificial sequence: amino acid sequence of KM5909 VL CDR2 SEQ ID NO: 86-description of artificial sequence: amino of KM5909 VL CDR3 SEQ ID NO: 87-description of artificial sequence: amino acid sequence of KM5911 VH CDR1 SEQ ID NO: 88-description of artificial sequence: amino acid sequence of KM5911 VH CDR2 SEQ ID NO: 89-description of artificial sequence: amino acid sequence of KM5911 VH CDR3 SEQ ID NO: 90- Description of Artificial Sequence: KM5911 VL CDR1 Amino Acid Sequence SEQ ID NO: 91—Description of Artificial Sequence: KM5911 VL CDR2 Amino Acid Sequence SEQ ID NO: 92—Description of Artificial Sequence: KM5911 VL CDR3 Amino Acid Sequence SEQ ID NO: 93—Description of Artificial Sequence: KM5915 VH CDR1 amino acid sequence SEQ ID NO: 94-description of artificial sequence: KM5915 VH CDR2 amino acid sequence SEQ ID NO: 95-description of artificial sequence: KM5915 VH CDR3 amino acid sequence SEQ ID NO: 96-description of artificial sequence: KM5 15 VL CDR1 amino acid sequence SEQ ID NO: 97-description of artificial sequence: KM5915 VL CDR2 amino acid sequence SEQ ID NO: 98-description of artificial sequence: KM5915 VL CDR3 amino acid sequence SEQ ID NO: 99-description of artificial sequence: KM5916 VH CDR1 amino acid SEQ ID NO: 100—Description of artificial sequence: amino acid sequence of KM5916 VH CDR2 SEQ ID NO: 101—Description of artificial sequence: amino acid sequence of KM5916 VH CDR3 SEQ ID NO: 102—Description of artificial sequence: amino acid sequence of KM5916 VL CDR1 SEQ ID NO: 103— Description of artificial sequence: amino acid sequence of KM5916 VL CDR2 SEQ ID NO: 104-Description of artificial sequence: amino acid sequence of KM5916 VL CDR3 SEQ ID NO: 105- Description of artificial sequence: amino acid sequence of KM5954 VH CDR1 SEQ ID NO: 106-description of artificial sequence: amino acid sequence of KM5954 VH CDR2 SEQ ID NO: 107-description of artificial sequence: amino acid sequence of KM5954 VH CDR3 SEQ ID NO: 108-description of artificial sequence: amino acid sequence of KM5954 VL CDR1 SEQ ID NO: 109-artificial Sequence Description: KM5954 VL CDR2 amino acid sequence SEQ ID NO: 110-Artificial Sequence Description: KM5954 VL CDR3 amino acid sequence SEQ ID NO: 111-Artificial Sequence Description: KM5955 VH CDR1 amino acid sequence SEQ ID NO: 112-Artificial Sequence Description: KM5955 VH CDR2 amino acid sequence SEQ ID NO: 113-description of artificial sequence: KM 5955 VH CDR3 amino acid sequence SEQ ID NO: 114-description of artificial sequence: KM 5955 VL CDR1 amino acid sequence SEQ ID NO: 115-artificial sequence Description: KM5955 VL CDR2 amino acid sequence SEQ ID NO: 116-description of artificial sequence: KM5955 VL CDR3 amino acid sequence SEQ ID NO: 117-description of artificial sequence: KM5956 VH CDR1 amino acid sequence SEQ ID NO: 118-description of artificial sequence: KM5956 VH CDR2 amino acid sequence SEQ ID NO: 119-description of artificial sequence: KM5956 VH CDR3 amino acid sequence SEQ ID NO: 120-description of artificial sequence: KM5956 VL CDR1 amino acid sequence SEQ ID NO: 121-description of artificial sequence: KM5956 VL CDR2 amino acid sequence No. 122—Description of Artificial Sequence: Amino Acid Sequence of KM5956 VL CDR3

Claims (21)

　ヒトＣＣケモカイン受容体１（ＣＣ　ｃｈｅｍｏｋｉｎｅ　ｒｅｃｅｐｔｏｒ　１；以下ＣＣＲ１と略記する）の細胞外領域に結合し、ヒトＣＣ　ｃｈｅｍｏｋｉｎｅ　ｌｉｇａｎｄ（以下、ＣＣＬと略記する）１５によるヒトＣＣＲ１の活性化を阻害するモノクローナル抗体または該抗体断片。 Monoclonal antibody that binds to the extracellular region of human CC chemokine receptor 1 (CC chemokin receptor 1; hereinafter abbreviated as CCR1) and inhibits activation of human CCR1 by human CC chemokin ligand (hereinafter abbreviated as CCL) 15 Or the antibody fragment. 　ヒトＣＣＬ１５により誘導されるヒトＣＣＲ１発現細胞の遊走を阻害する請求項１に記載のモノクローナル抗体または該抗体断片。 The monoclonal antibody or the antibody fragment thereof according to claim 1, which inhibits migration of human CCR1-expressing cells induced by human CCL15. 　ヒトＣＣＲ１の細胞外ループ２領域のアミノ酸配列のうち、少なくとも１つのアミノ酸残基に結合する、請求項１または２に記載のモノクローナル抗体または該抗体断片。 The monoclonal antibody or the antibody fragment according to claim 1 or 2, which binds to at least one amino acid residue in the amino acid sequence of the extracellular loop 2 region of human CCR1. 　モノクローナル抗体が、下記（ａ）～（ｌ）から選ばれるいずれか一つの抗体である、請求項１～３のいずれか１項に記載のモノクローナル抗体または該抗体断片。
（ａ）重鎖可変領域（ｈｅａｖｙ　ｃｈａｉｎ　ｖａｒｉａｂｌｅ　ｒｅｇｉｏｎ；以下、ＶＨと略記する）の相補性決定領域（ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ；以下ＣＤＲと略記する）１～３のアミノ酸配列が、それぞれ配列番号６９、７０および７１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつ軽鎖可変領域（ｌｉｇｈｔ　ｃｈａｉｎ　ｖａｒｉａｂｌｅ　ｒｅｇｉｏｎ；以下、ＶＬと略記する）のＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７２、７３および７４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｂ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７５、７６および７７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号７８、７９および８０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｃ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８１、８２および８３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８４、８５および８６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｄ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号８７、８８および８９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９０、９１および９２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｅ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９３、９４および９５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９６、９７および９８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｆ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号９９、１００および１０１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０２、１０３および１０４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｇ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０５、１０６および１０７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１０８、１０９および１１０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｈ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１１、１１２および１１３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１４、１１５および１１６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｉ）ＶＨのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１１７、１１８および１１９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのＣＤＲ１～３のアミノ酸配列が、それぞれ配列番号１２０、１２１および１２２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｊ）前記（ａ）～（ｉ）に記載の少なくとも一つの抗体と、ヒトＣＣＲ１への結合について競合する抗体。
（ｋ）前記（ａ）～（ｉ）に記載のいずれか一つの抗体が結合するエピトープを含むエピトープに結合する抗体。
（ｌ）前記（ａ）～（ｉ）に記載のいずれか一つの抗体が結合するエピトープと同じエピトープに結合する抗体。 The monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 3, wherein the monoclonal antibody is any one antibody selected from the following (a) to (l):
(A) The amino acid sequences of complementarity determining regions (complementary determining regions; hereinafter abbreviated as CDR) 1 to 3 of heavy chain variable regions (hereinafter abbreviated as VH) are SEQ ID NOs: 69 and 70, respectively. And 71, and the amino acid sequences of CDR1 to CDR3 of the light chain variable region (hereinafter abbreviated as VL) are SEQ ID NOs: 72, 73 and 73, respectively. 74. An antibody having an amino acid sequence comprising the amino acid sequence described in 74.
(B) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 75, 76, and 77, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 78, respectively. 79. An antibody having an amino acid sequence comprising the amino acid sequence described in 79 and 80.
(C) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 81, 82 and 83, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 84 , 85 and 86, an antibody having an amino acid sequence comprising the amino acid sequence described in 85 and 86.
(D) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 87, 88, and 89, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 90 , 91 and 92, an antibody having an amino acid sequence comprising the amino acid sequence described in 91 and 92.
(E) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 93, 94 and 95, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 96, respectively. The antibody which is an amino acid sequence containing the amino acid sequence described in 97 and 98.
(F) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 99, 100 and 101, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 102. , 103 and 104. An antibody having an amino acid sequence comprising the amino acid sequence described in 103 and 104.
(G) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 105, 106 and 107, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are respectively SEQ ID NO: 108 , 109 and 110, an antibody having an amino acid sequence comprising the amino acid sequence described in 109 and 110.
(H) The amino acid sequences of CDR1 to V3 of VH include the amino acid sequences described in SEQ ID NOs: 111, 112, and 113, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 114, respectively. , 115 and 116, an antibody having an amino acid sequence comprising the amino acid sequence described in 115 and 116.
(I) The amino acid sequences of CDRs 1 to 3 of VH are amino acid sequences including the amino acid sequences described in SEQ ID NOs: 117, 118, and 119, respectively, and the amino acid sequences of CDRs 1 to 3 of VL are SEQ ID NO: 120, respectively. , 121 and 122, which is an amino acid sequence comprising the amino acid sequence described in 121 and 122.
(J) An antibody that competes with at least one antibody described in (a) to (i) for binding to human CCR1.
(K) An antibody that binds to an epitope including the epitope to which any one of the antibodies (a) to (i) binds.
(L) An antibody that binds to the same epitope as that to which any one of the antibodies (a) to (i) binds. 　モノクローナル抗体が、下記（ａ）～（ｉ）から選ばれるいずれか一つの抗体である、請求項１～４のいずれか１項に記載のモノクローナル抗体または該抗体断片。
（ａ）ＶＨのアミノ酸配列が配列番号５１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｂ）ＶＨのアミノ酸配列が配列番号５３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｃ）ＶＨのアミノ酸配列が配列番号５５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｄ）ＶＨのアミノ酸配列が配列番号５７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号５８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｅ）ＶＨのアミノ酸配列が配列番号５９に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６０に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｆ）ＶＨのアミノ酸配列が配列番号６１に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６２に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｇ）ＶＨのアミノ酸配列が配列番号６３に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６４に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｈ）ＶＨのアミノ酸配列が配列番号６５に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６６に記載されるアミノ酸配列を含むアミノ酸配列である抗体。
（ｉ）ＶＨのアミノ酸配列が配列番号６７に記載されるアミノ酸配列を含むアミノ酸配列であって、かつＶＬのアミノ酸配列が配列番号６８に記載されるアミノ酸配列を含むアミノ酸配列である抗体。 The monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 4, wherein the monoclonal antibody is any one antibody selected from the following (a) to (i):
(A) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 51, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 52.
(B) An antibody wherein the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 53, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 54.
(C) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 55, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 56.
(D) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 57, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 58.
(E) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 59, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 60.
(F) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 61, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 62.
(G) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 63, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 64.
(H) An antibody in which the amino acid sequence of VH is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 65, and the amino acid sequence of VL is an amino acid sequence including the amino acid sequence described in SEQ ID NO: 66.
(I) An antibody wherein the amino acid sequence of VH is an amino acid sequence containing the amino acid sequence set forth in SEQ ID NO: 67, and the amino acid sequence of VL is an amino acid sequence containing the amino acid sequence set forth in SEQ ID NO: 68. 　モノクローナル抗体が遺伝子組換え抗体である請求項１～５のいずれか１項に記載のモノクローナル抗体または該抗体断片。 The monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 5, wherein the monoclonal antibody is a gene recombinant antibody. 　遺伝子組換え抗体が、ヒト型キメラ抗体、ヒト化抗体およびヒト抗体から選ばれる１の遺伝子組換え抗体である、請求項６に記載のモノクローナル抗体または該抗体断片。 The monoclonal antibody or the antibody fragment according to claim 6, wherein the recombinant antibody is one recombinant antibody selected from a human chimeric antibody, a humanized antibody and a human antibody. 　Ｆａｂ、Ｆａｂ’、（Ｆａｂ’）２、一本鎖抗体（ｓｃＦｖ）、二量体化Ｖ領域（ｄｉａｂｏｄｙ）、ジスルフィド安定化Ｖ領域（ｄｓＦｖ）およびＣＤＲを含むペプチドから選ばれる１の抗体断片である、請求項１～７のいずれか１項に記載の抗体断片。 1 antibody fragment selected from peptides including 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 claims 1 to 7, wherein 　請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片を産生するハイブリドーマ。 A hybridoma producing the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8. 　請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片をコードする塩基配列を有する核酸。 A nucleic acid having a base sequence encoding the monoclonal antibody or the antibody fragment according to any one of claims 1 to 8. 　請求項１０に記載の核酸を含むベクターを含む形質転換細胞。 A transformed cell comprising a vector comprising the nucleic acid according to claim 10. 　請求項９に記載のハイブリドーマまたは請求項１１に記載の形質転換細胞を培養し、培養液から請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片を採取することを含む、請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片の製造方法。 Culturing the hybridoma according to claim 9 or the transformed cell according to claim 11, and collecting the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8 from the culture solution, The method for producing the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8. 　請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片を含む、ヒトＣＣＲ１の検出または測定用試薬。 A reagent for detecting or measuring human CCR1, comprising the monoclonal antibody or the antibody fragment according to any one of claims 1 to 8. 　請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片を含む、ヒトＣＣＲ１関連疾患の診断薬。 A diagnostic agent for a human CCR1-related disease comprising the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8. 　ヒトＣＣＲ１関連疾患が、癌、自己免疫疾患または炎症性疾患である、請求項１４に記載の診断薬。 The diagnostic agent according to claim 14, wherein the human CCR1-related disease is cancer, autoimmune disease or inflammatory disease. 　請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片を有効成分として含有する、ヒトＣＣＲ１関連疾患の治療薬。 A therapeutic agent for a human CCR1-related disease comprising the monoclonal antibody or the antibody fragment according to any one of claims 1 to 8 as an active ingredient. 　ヒトＣＣＲ１関連疾患が、癌、自己免疫疾患または炎症性疾患である、請求項１６に記載の治療薬。 The therapeutic agent according to claim 16, wherein the human CCR1-related disease is cancer, autoimmune disease or inflammatory disease. 　請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片を用いたヒトＣＣＲ１関連疾患の診断方法。 A method for diagnosing a human CCR1-related disease using the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8. 　請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片を用いたヒトＣＣＲ１関連疾患の治療方法。 A method for treating a human CCR1-related disease using the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8. 　ヒトＣＣＲ１関連疾患の診断薬を製造するための、請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片の使用。 Use of the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8, for producing a diagnostic agent for human CCR1-related diseases. 　ヒトＣＣＲ１関連疾患の治療薬を製造するための、請求項１～８のいずれか１項に記載のモノクローナル抗体または該抗体断片の使用。 Use of the monoclonal antibody or the antibody fragment thereof according to any one of claims 1 to 8, for producing a therapeutic drug for human CCR1-related diseases.
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