[go: up one dir, main page]

WO2025164598A1 - Antibody, nucleic acid, cell, and medicine - Google Patents

Antibody, nucleic acid, cell, and medicine

Info

Publication number
WO2025164598A1
WO2025164598A1 PCT/JP2025/002546 JP2025002546W WO2025164598A1 WO 2025164598 A1 WO2025164598 A1 WO 2025164598A1 JP 2025002546 W JP2025002546 W JP 2025002546W WO 2025164598 A1 WO2025164598 A1 WO 2025164598A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
sequence represented
heavy chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/002546
Other languages
French (fr)
Japanese (ja)
Inventor
仁己 芦田
美樹 中川
雅彦 品川
龍馬 田中
ゆき 林
曜子 鎌田
沙弥 栗林
宏 喜田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NB Health Labs Co Ltd
Original Assignee
NB Health Labs Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by NB Health Labs Co Ltd filed Critical NB Health Labs Co Ltd
Priority to JP2025546599A priority Critical patent/JP7784193B1/en
Publication of WO2025164598A1 publication Critical patent/WO2025164598A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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

Definitions

  • the present disclosure relates to an antibody that specifically binds to the extracellular domain of human CXCR3, a nucleic acid encoding the antibody, a cell containing the nucleic acid, and a pharmaceutical containing the antibody as an active ingredient.
  • Chemokines are chemotactic cytokines secreted by various cells. Chemokines are classified into four groups based on their amino acid sequence and structure: CXC, CC, C, and CX3C. Chemokines play an important role in immune cell differentiation and homeostasis, primary and secondary immune responses, and immune cell migration in various diseases. Nearly 50 types of chemokines have been reported in humans and mice (Non-Patent Document 1). Chemokine action is expressed through binding to classical chemokine receptors, which are seven-transmembrane G protein-coupled receptors (GPCRs).
  • GPCRs seven-transmembrane G protein-coupled receptors
  • Non-Patent Document 1 Atypical chemokine receptors, which bind to chemokines but are not coupled to G proteins, also exist and are responsible for the formation of chemokine concentration gradients. Approximately 20 classical chemokine receptors and four atypical chemokine receptors have been identified in mammals (Non-Patent Document 1).
  • CXCR3 also known as G protein-coupled receptor 9 (GPR9) or CD183, is a chemokine receptor expressed primarily on T cells.
  • CXCR3 is expressed in a variety of organisms, including humans, mice, rats, cattle, chimpanzees, macaques, dogs, frogs, platypus, pigs, and zebrafish. In humans, there are three isoforms: CXCR3A, CXCR3B, and CXCR3Alt.
  • CXCR3A is the most predominant isoform in vivo and is highly expressed in activated CD8+ T cells, memory CD4+ T cells, memory CD8+ T cells, and NK cells. It plays an important role in immunophysiological processes, such as effector cell homing to inflammatory sites and pathogen clearance. It is also expressed in immune cells such as subsets of dendritic cells, B cell subsets, macrophages, Th17 cells, regulatory T cells, and neutrophils (Non-Patent Documents 1 and 2).
  • the functional ligands for CXCR3A are the interferon- ⁇ (IFN- ⁇ )-inducible chemokines CXCL9 (MIG-9), CXCL10 (IP-10), and CXCL11 (I-TAC).
  • Non-Patent Documents 2 and 3 Activation of CXCR3A by CXCL9, CXCL10, and CXCL11 leads to activation of G ⁇ q and G ⁇ i, inducing cell chemotaxis, cell proliferation, cell survival, tumor cell metastasis, and more.
  • CXCR3B has an N-terminal domain that is 47 amino acids longer than CXCR3A due to alternative splicing.
  • CXCR3B expression has been confirmed in tissues such as the heart, skeletal muscle, liver, and kidney, as well as in vascular endothelial cells.
  • the primary ligand for CXCR3B is CXCL4, and in vitro, CXCL9, CXCL10, and CXCL11 also bind to CXCR3B.
  • Ligand binding to CXCR3B activates Gas, inhibiting the growth and migration of endothelial cells and tumor cells and promoting apoptosis. In other words, CXCR3A and CXCR3B are thought to have opposing effects.
  • CXCR3Alt is co-expressed in small amounts in CXCR3A-expressing cells and plays a role in chemotaxis toward CXCL11 (Non-Patent Document 2).
  • CXCL9, CXCL10, and CXCL11 are produced in large quantities at sites of inflammation, such as autoimmune diseases, transplants, infections, and tumors. They are thought to be involved in the recruitment of immune cells to inflammatory sites and are involved in various inflammatory diseases.
  • Th1-related diseases such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and acute cardiac allograft rejection (Non-Patent Document 3).
  • Th1-related diseases such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and acute cardiac allograft rejection.
  • Non-Patent Document 3 Furthermore, more recent studies have suggested an association with a wider range of diseases, including influenza, viral infections such as COVID-19, and tumors (Non-Patent Documents 3, 4). Therefore, there is
  • CXCR3 has also been the target of many drug discovery programs, and the development of small molecule antagonists and antibodies has progressed.
  • AMG487 progressed to a Phase IIa trial for psoriasis, but efficacy was not demonstrated.
  • ACT-777991 which is being developed as a treatment for type 1 diabetes, has completed a Phase I trial, demonstrating good tolerability, kinetics, and safety in healthy subjects, and is expected to progress to further clinical development (Non-Patent Document 6).
  • Non-Patent Document 7 a Phase II clinical trial of the human monoclonal antibody eldelumab (BMS-936557), which targets CXCL10, for the treatment of ulcerative colitis (UC) and Crohn's disease suggested that it may be an effective treatment for moderate to severe active UC (Non-Patent Document 7).
  • Patent Document 1 discloses six humanized monoclonal antibodies that specifically bind to human CXCR3. It also discloses the amino acid sequences of the complementarity-determining regions, heavy chain variable regions, and light chain variable regions of each monoclonal antibody.
  • Patent Document 2 discloses four types of monoclonal antibodies that specifically bind to human CXCR3, as well as humanized antibodies thereof. It also discloses the amino acid sequences of the complementarity-determining regions, heavy chain variable regions, and light chain variable regions of each monoclonal antibody. These anti-human CXCR3 antibodies have been shown to be particularly effective in treating type 1 diabetes.
  • Patent Document 3 discloses a humanized version of a monoclonal antibody that specifically binds to human CXCR3. It also discloses the amino acid sequences of the complementarity-determining region, heavy chain variable region, and light chain variable region of this monoclonal antibody. CXCR3 depletion using this antibody has been shown to be effective in treating vitiligo vulgaris.
  • Patent Documents 1 to 3 have not yet been put to practical use as therapeutic agents. There is a need in the art for additional or improved anti-human CXCR3 antibodies with superior properties as pharmaceutical raw materials.
  • the purpose of this disclosure is to provide a novel anti-human CXCR3 antibody with superior properties as a pharmaceutical ingredient.
  • One aspect of the present disclosure is an antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A, has activity of blocking CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and is selected from the group consisting of the following (AB1) to (AB9), (AB12) to (AB27): (AB26)
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 251, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 252, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 253, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 254, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 255, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 256.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 151, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 152, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 153, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 154, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 155, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 156.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 161, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 162, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 163, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 164, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 165, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 166.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 171, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 172, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 173, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 174, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 175, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 176.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 191, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 192, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 193, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 194, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 195, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 196.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 126.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 132, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 133, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 135, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 136.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 141, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 142, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 143, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 144, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 145, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 146.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 181, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 182, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 183, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 184, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 185, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 186. It is an antibody that satisfies any of the following criteria.
  • One aspect of the present disclosure is an antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A, has activity of blocking CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and is selected from the group consisting of the following (C1) to (C9), (C12) to (C27): (C26) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 257, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 259; (C23) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 227, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 229; (C24) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 237, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 239; (C25) A heavy chain variable region comprising the amino
  • C3 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29.
  • C5 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 49.
  • (C8) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 77, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 79;
  • (C16) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 157, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 159;
  • (C17) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 167, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 169;
  • (C18) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 177, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 179;
  • (C20) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 197, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 199;
  • (C21) A
  • C7 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 69;
  • C9 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89.
  • C12 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 117, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 119.
  • C13 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 127, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 129.
  • C14 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139.
  • C15 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 147, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 149;
  • C19 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 187, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 189; It is an antibody that satisfies any one of the following conditions.
  • One aspect of the present disclosure is a second antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A, has activity to block CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and competitively inhibits the binding of the first antibody described above to the receptor.
  • the antibody is a humanized antibody or a chimeric antibody.
  • the antibody is a multispecific antibody.
  • the antibody has internalization activity.
  • the antibody is a modified antibody to which another molecule is bound.
  • the modified antibody is an antibody-drug conjugate.
  • One aspect of the present disclosure is a nucleic acid encoding the above-described antibody.
  • One aspect of the present disclosure is a cell containing the above-mentioned nucleic acid.
  • One aspect of the present disclosure is a pharmaceutical containing the above-described antibody as an active ingredient.
  • the pharmaceutical is used to treat a disease, disorder, or condition involving impairment of CXCR3-dependent cellular function.
  • the disease, disorder, or condition is a Th1 immune disorder.
  • the Th1 immune disorder is a cardiovascular disorder, a nervous system disorder, an inflammatory disease, an autoimmune disease, a metabolic disease, an infectious disease, a blood cancer, or a solid cancer.
  • This disclosure makes it possible to provide a novel anti-human CXCR3 antibody with superior properties as a pharmaceutical ingredient.
  • 1 is a graph showing the results of quantifying gene expression of CXCR3A and CXCR3B performed in Example 6, showing that human vascular endothelial cells predominantly express CXCR3B.
  • 1 is a histogram showing the results of flow cytometry performed in Example 7, illustrating the binding properties of the antibody (13B4, 201009-2-C) to CHO cells stably expressing human CXCR3A and vascular endothelial cells.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (13B4) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (11F11) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-3-H) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-5-F) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-7-G) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201009-2-C) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201009-3-H) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201009-1-C) to CHO cells stably expressing human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibody (13B4) against human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibody (11F11) against human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-3-H) against human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-5-F) against human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-7-G) against human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (201009-2-C) against human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201009-3-H) against human CXCR3A.
  • 1 is a graph showing the dose dependency of the inhibitory activity of an antibody (201009-1-C) against human CXCR3A.
  • 1 is a histogram showing the results of flow cytometry performed in Example 11, showing the specific binding of antibodies (201006-7-G, 201009-2-C) to human CXCR3A.
  • 1 is a graph showing the results of evaluating the binding ability of antibody (13B4) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (11F11) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-3-H) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-5-F) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-7-G) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding ability of an antibody (201009-2-C) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201009-2-C) on the migration of human peripheral blood mononuclear cell-derived T cells.
  • FIG. 1 is a graph showing the results of flow cytometry performed in Example 14, demonstrating that antibodies are internalized together with receptors in human peripheral blood mononuclear cell-derived T cells.
  • FIG. 1 is an explanatory diagram showing the alignment of the heavy chain variable regions of humanized anti-CXCR3 antibodies.
  • FIG. 1 is an explanatory diagram showing the alignment of the light chain variable region of a humanized anti-CXCR3 antibody.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL1) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL2) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL3) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL4) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL1) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL2) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL3) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL4) to human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH1+VL1) on the migration of human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH1+VL4) on the migration of human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH2+VL1) on the migration of human peripheral blood mononuclear cell-derived T cells.
  • 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH2+VL4) on the migration of human peripheral blood mononuclear cell-derived T cells.
  • complementarity determining region is abbreviated as CDR.
  • heavy chain variable region may be abbreviated as VH, heavy chain constant region as CH, light chain variable region as VL, and light chain constant region as CL.
  • antibody may be replaced with “immunoglobulin.”
  • nucleic acid may be replaced with "DNA” or "gene.”
  • the human CXCR3 receptor is a type of G protein-coupled receptor (GPCR), which penetrates the cell membrane seven times and is present with its N-terminus facing extracellularly and its C-terminus facing intracellularly.
  • Human CXCR3 exists in three isoforms: CXCR3A, CXCR3B, and CXCR3Alt.
  • the gene (cDNA) encoding human CXCR3 has already been isolated, and the amino acid sequence of human CXCR3 is also known.
  • the sequence information can be obtained from a gene database (e.g., NCBI Reference Sequence: AAO92295).
  • the amino acid sequence of human CXCR3A is shown in SEQ ID NO: 281, and the amino acid sequence of human CXCR3B is shown in SEQ ID NO: 292.
  • Each domain of human CXCR3A is thought to correspond to the following portion of the amino acid sequence shown in SEQ ID NO: 281.
  • the left side indicates the amino acid number, and the right side indicates each domain. Note that the boundaries between each domain may vary slightly.
  • the antibody disclosed in the present specification is an antibody that specifically binds to human CXCR3 (anti-human CXCR3 antibody), which specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, and does not specifically bind to human vascular endothelial cells.
  • the antibody comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, or 271; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, or 272; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213,
  • the antibody comprises: The following (A1) to (A28): (A1) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; (A2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13; (A3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; (A4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103;
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113;
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123;
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid
  • the antibody comprises: The following (B1) to (B28): (B1) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6; (B2) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16; (B3) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26; (B4) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented
  • a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106;
  • a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116;
  • a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 126;
  • a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the
  • the heavy chain CDR1-3 and light chain CDR1-3 of the antibody satisfy any of the above (AB1)-(AB9) and (AB12)-(AB27).
  • the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 of the antibody are selected from the group consisting of (AB10), (AB11), and (AB28): (AB10)
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 271, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 272, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 273, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 274, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 275, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 276. Satisfy one of the following.
  • the heavy chain variable region and light chain variable region of the antibody satisfy any of the above (C1) to (C9) and (C12) to (C27).
  • the antibody has a heavy chain variable region and a light chain variable region selected from the group consisting of (C10), (C11), and (C28) below:
  • (C10) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 99.
  • (C11) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 107, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 109.
  • C28 A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 277, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 279; Satisfy one of the following.
  • the heavy chain variable region (SEQ ID NO: 7) specified by (C1) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 1 to 3) specified by (A1) or (AB1).
  • the light chain variable region (SEQ ID NO: 9) specified by (C1) contains light chain CDRs 1 to 3 (SEQ ID NOs: 4 to 6) specified by (B1) or (AB1).
  • An example of an antibody that satisfies (A1), (B1), (AB1), or (C1) is "201001-1-F," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 17) specified by (C2) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 11 to 13) specified by (A2) or (AB2).
  • the light chain variable region (SEQ ID NO: 19) specified by (C2) contains light chain CDRs 1 to 3 (SEQ ID NOs: 14 to 16) specified by (B2) or (AB2).
  • An example of an antibody that satisfies (A2), (B2), (AB2), or (C2) is "201001-5-B," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 27) specified by (C3) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 21 to 23) specified by (A3) or (AB3).
  • the light chain variable region (SEQ ID NO: 29) specified by (C3) contains light chain CDRs 1 to 3 (SEQ ID NOs: 24 to 26) specified by (B3) or (AB3).
  • An example of an antibody that satisfies (A3), (B3), (AB3), or (C3) is "201001-2-C," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 37) specified by (C4) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 31 to 33) specified by (A4) or (AB4).
  • the light chain variable region (SEQ ID NO: 39) specified by (C4) contains light chain CDRs 1 to 3 (SEQ ID NOs: 34 to 36) specified by (B4) or (AB4).
  • An example of an antibody that satisfies (A4), (B4), (AB4), or (C4) is "201001-4-E," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 47) specified by (C5) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 41 to 43) specified by (A5) or (AB5).
  • the light chain variable region (SEQ ID NO: 49) specified by (C5) contains light chain CDRs 1 to 3 (SEQ ID NOs: 44 to 46) specified by (B5) or (AB5).
  • An example of an antibody that satisfies (A5), (B5), (AB5), or (C5) is "201001-1-C," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 57) specified by (C6) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 51 to 53) specified by (A6) or (AB6).
  • the light chain variable region (SEQ ID NO: 59) specified by (C6) contains light chain CDRs 1 to 3 (SEQ ID NOs: 54 to 56) specified by (B6) or (AB6).
  • An example of an antibody that satisfies (A6), (B6), (AB6), or (C6) is "201001-5-A," which is described in the Examples below.
  • the heavy chain variable region identified by (C7) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 61 to 63) identified by (A7) or (AB7).
  • the light chain variable region identified by (C7) (SEQ ID NO: 69) contains light chain CDRs 1 to 3 (SEQ ID NOs: 64 to 66) identified by (B7) or (AB7).
  • An example of an antibody that satisfies (A7), (B7), (AB7), or (C7) is "201009-1-D," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 77) identified by (C8) contains heavy chain CDRs 1-3 (SEQ ID NOs: 71-73) identified by (A8) or (AB8).
  • the light chain variable region (SEQ ID NO: 79) identified by (C8) contains light chain CDRs 1-3 (SEQ ID NOs: 74-76) identified by (B8) or (AB8).
  • An example of an antibody that satisfies (A8), (B8), (AB8), or (C8) is "201009-5-F," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 87) identified by (C9) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 81 to 83) identified by (A9) or (AB9).
  • the light chain variable region (SEQ ID NO: 89) identified by (C9) contains light chain CDRs 1 to 3 (SEQ ID NOs: 84 to 86) identified by (B9) or (AB9).
  • An example of an antibody that satisfies (A9), (B9), (AB9), or (C9) is "201112-1-C," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 97) specified by (C10) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 91 to 93) specified by (A10) or (AB10).
  • the light chain variable region (SEQ ID NO: 99) specified by (C10) contains light chain CDRs 1 to 3 (SEQ ID NOs: 94 to 96) specified by (B10) or (AB10).
  • An example of an antibody that satisfies (A10), (B10), (AB10), or (C10) is "13B4," which is described in the Examples below.
  • the heavy chain variable region specified by (C11) contains heavy chain CDRs 1 to 3 specified by (A11) or (AB11) (SEQ ID NO: 101 to 103).
  • the light chain variable region specified by (C11) contains light chain CDRs 1 to 3 specified by (B11) or (AB11) (SEQ ID NO: 104 to 106).
  • An example of an antibody that satisfies (A11), (B11), (AB11), or (C11) is "11F11," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 117) specified by (C12) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 111 to 113) specified by (A12) or (AB12).
  • the light chain variable region (SEQ ID NO: 119) specified by (C12) contains light chain CDRs 1 to 3 (SEQ ID NOs: 114 to 116) specified by (B12) or (AB12).
  • An example of an antibody that satisfies (A12), (B12), (AB12), or (C12) is "201006-4-H," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 127) specified by (C13) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 121 to 123) specified by (A13) or (AB13).
  • the light chain variable region (SEQ ID NO: 129) specified by (C13) contains light chain CDRs 1 to 3 (SEQ ID NOs: 124 to 126) specified by (B13) or (AB13).
  • An example of an antibody that satisfies (A13), (B13), (AB13), or (C13) is "201006-4-F," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 137) specified by (C14) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 131 to 133) specified by (A14) or (AB14).
  • the light chain variable region (SEQ ID NO: 139) specified by (C14) contains light chain CDRs 1 to 3 (SEQ ID NOs: 134 to 136) specified by (B14) or (AB14).
  • An example of an antibody that satisfies (A14), (B14), (AB14), or (C14) is "201006-5-A," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 147) identified by (C15) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 141 to 143) identified by (A15) or (AB15).
  • the light chain variable region (SEQ ID NO: 149) identified by (C15) contains light chain CDRs 1 to 3 (SEQ ID NOs: 144 to 146) identified by (B15) or (AB15).
  • An example of an antibody that satisfies (A15), (B15), (AB15), or (C15) is "201006-4-A," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 157) identified by (C16) contains heavy chain CDRs 1-3 (SEQ ID NOs: 151-153) identified by (A16) or (AB16).
  • the light chain variable region (SEQ ID NO: 159) identified by (C16) contains light chain CDRs 1-3 (SEQ ID NOs: 154-156) identified by (B16) or (AB16).
  • An example of an antibody that satisfies (A16), (B16), (AB16), or (C16) is "201006-1-H," which is described in the Examples below.
  • the heavy chain variable region identified by (C17) contains heavy chain CDRs 1-3 (SEQ ID NOs: 161-163) identified by (A17) or (AB17).
  • the light chain variable region identified by (C17) contains light chain CDRs 1-3 (SEQ ID NOs: 164-166) identified by (B17) or (AB17).
  • An example of an antibody that satisfies (A17), (B17), (AB17), or (C17) is "201006-3-D," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 177) identified by (C18) contains heavy chain CDRs 1-3 (SEQ ID NOs: 171-173) identified by (A18) or (AB18).
  • the light chain variable region (SEQ ID NO: 179) identified by (C18) contains light chain CDRs 1-3 (SEQ ID NOs: 174-176) identified by (B18) or (AB18).
  • An example of an antibody that satisfies (A18), (B18), (AB18), or (C18) is "201006-1-F," which is described in the Examples below.
  • the heavy chain variable region identified by (C19) contains heavy chain CDRs 1-3 (SEQ ID NOs: 181-183) identified by (A19) or (AB19).
  • the light chain variable region identified by (C19) contains light chain CDRs 1-3 (SEQ ID NOs: 184-186) identified by (B19) or (AB19).
  • An example of an antibody that satisfies (A19), (B19), (AB19), or (C19) is "201006-4-G,” which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 197) identified by (C20) contains heavy chain CDRs 1-3 (SEQ ID NOs: 191-193) identified by (A20) or (AB20).
  • the light chain variable region (SEQ ID NO: 199) identified by (C20) contains light chain CDRs 1-3 (SEQ ID NOs: 194-196) identified by (B20) or (AB20).
  • An example of an antibody that satisfies (A20), (B20), (AB20), or (C20) is "201006-7-A," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 207) identified by (C21) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 201 to 203) identified by (A21) or (AB21).
  • the light chain variable region (SEQ ID NO: 209) identified by (C21) contains light chain CDRs 1 to 3 (SEQ ID NOs: 204 to 206) identified by (B21) or (AB21).
  • An example of an antibody that satisfies (A21), (B21), (AB21), or (C21) is "201006-2-D," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 217) identified by (C22) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 211 to 213) identified by (A22) or (AB22).
  • the light chain variable region (SEQ ID NO: 219) identified by (C22) contains light chain CDRs 1 to 3 (SEQ ID NOs: 214 to 216) identified by (B22) or (AB22).
  • An example of an antibody that satisfies (A22), (B22), (AB22), or (C22) is "201006-2-C," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 227) identified by (C23) contains heavy chain CDRs 1-3 (SEQ ID NOs: 221-223) identified by (A23) or (AB23).
  • the light chain variable region (SEQ ID NO: 229) identified by (C23) contains light chain CDRs 1-3 (SEQ ID NOs: 224-226) identified by (B23) or (AB23).
  • An example of an antibody that satisfies (A23), (B23), (AB23), or (C23) is "201006-3-H," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 237) identified by (C24) contains heavy chain CDRs 1-3 (SEQ ID NOs: 231-233) identified by (A24) or (AB24).
  • the light chain variable region (SEQ ID NO: 239) identified by (C24) contains light chain CDRs 1-3 (SEQ ID NOs: 234-236) identified by (B24) or (AB24).
  • An example of an antibody that satisfies (A24), (B24), (AB24), or (C24) is "201006-5-F," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 247) identified by (C25) contains heavy chain CDRs 1-3 (SEQ ID NOs: 241-243) identified by (A25) or (AB25).
  • the light chain variable region (SEQ ID NO: 249) identified by (C25) contains light chain CDRs 1-3 (SEQ ID NOs: 244-246) identified by (B25) or (AB25).
  • An example of an antibody that satisfies (A25), (B25), (AB25), or (C25) is "201006-7-G," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 257) identified by (C26) contains heavy chain CDRs 1-3 (SEQ ID NOs: 251-253) identified by (A26) or (AB26).
  • the light chain variable region (SEQ ID NO: 259) identified by (C26) contains light chain CDRs 1-3 (SEQ ID NOs: 254-256) identified by (B26) or (AB26).
  • An example of an antibody that satisfies (A26), (B26), (AB26), or (C26) is "201009-2-C," which is described in the Examples below.
  • the heavy chain variable region identified by (C27) contains heavy chain CDRs 1-3 (SEQ ID NOs: 261-263) identified by (A27) or (AB27).
  • the light chain variable region identified by (C27) (SEQ ID NO: 269) contains light chain CDRs 1-3 (SEQ ID NOs: 264-266) identified by (B27) or (AB27).
  • An example of an antibody that satisfies (A27), (B27), (AB27), or (C27) is "201009-3-H," which is described in the Examples below.
  • the heavy chain variable region (SEQ ID NO: 277) identified by (C28) contains heavy chain CDRs 1-3 (SEQ ID NOs: 271-273) identified by (A28) or (AB28).
  • the light chain variable region (SEQ ID NO: 279) identified by (C28) contains light chain CDRs 1-3 (SEQ ID NOs: 274-276) identified by (B28) or (AB28).
  • An example of an antibody that satisfies (A28), (B28), (AB28), or (C28) is "201009-1-C," which is described in the Examples below.
  • amino acid sequences of SEQ ID NO:1, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:111, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO:161, and SEQ ID NO:251 are the same.
  • the amino acid sequences of SEQ ID NO:81 and SEQ ID NO:101 are the same.
  • the amino acid sequences of SEQ ID NO:171 and SEQ ID NO:181 are the same.
  • the amino acid sequences of SEQ ID NO:191, SEQ ID NO:201, and SEQ ID NO:211 are the same.
  • the amino acid sequences of SEQ ID NO:221, SEQ ID NO:231, SEQ ID NO:241, and SEQ ID NO:261 are the same.
  • amino acid sequences of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:72, SEQ ID NO:132, SEQ ID NO:142, SEQ ID NO:162, SEQ ID NO:182, SEQ ID NO:192, and SEQ ID NO:222 are the same.
  • the amino acid sequences of SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:62, and SEQ ID NO:202 are the same.
  • the amino acid sequences of SEQ ID NO:42 and SEQ ID NO:52 are the same.
  • the amino acid sequences of SEQ ID NO:152 and SEQ ID NO:212 are the same.
  • the amino acid sequences of SEQ ID NO:172, SEQ ID NO:232, and SEQ ID NO:262 are the same.
  • amino acid sequences of SEQ ID NO:3 and SEQ ID NO:13 are the same.
  • the amino acid sequences of SEQ ID NO:63, SEQ ID NO:123, SEQ ID NO:153, SEQ ID NO:163, SEQ ID NO:173, SEQ ID NO:183, SEQ ID NO:193, SEQ ID NO:203, SEQ ID NO:213, and SEQ ID NO:243 are the same.
  • the amino acid sequences of SEQ ID NO:83 and SEQ ID NO:93 are the same.
  • the amino acid sequences of SEQ ID NO:113, SEQ ID NO:133, SEQ ID NO:143, SEQ ID NO:223, SEQ ID NO:233, SEQ ID NO:253, and SEQ ID NO:263 are the same.
  • the amino acid sequences of SEQ ID NO:44 and SEQ ID NO:54 are the same.
  • the amino acid sequences of SEQ ID NO:64 and SEQ ID NO:124 are the same.
  • the amino acid sequences of SEQ ID NO:74, SEQ ID NO:144, SEQ ID NO:224, SEQ ID NO:254 and SEQ ID NO:264 are the same.
  • the amino acid sequences of SEQ ID NO:84, SEQ ID NO:94 and SEQ ID NO:104 are the same.
  • the amino acid sequences of SEQ ID NO:154 and SEQ ID NO:164 are the same.
  • the amino acid sequences of SEQ ID NO:174, SEQ ID NO:184, SEQ ID NO:204 and SEQ ID NO:214 are the same.
  • the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:15 are the same.
  • the amino acid sequences of SEQ ID NO:25, SEQ ID NO:145, SEQ ID NO:175, SEQ ID NO:185, and SEQ ID NO:265 are the same.
  • the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:55 are the same.
  • the amino acid sequences of SEQ ID NO:65, SEQ ID NO:125, and SEQ ID NO:135 are the same.
  • the amino acid sequences of SEQ ID NO:75, SEQ ID NO:225, and SEQ ID NO:255 are the same.
  • the amino acid sequences of SEQ ID NO:85, SEQ ID NO:95, and SEQ ID NO:105 are the same.
  • amino acid sequences of SEQ ID NO:155, SEQ ID NO:165, and SEQ ID NO:245 are the same.
  • amino acid sequences of SEQ ID NO:195, SEQ ID NO:205, and SEQ ID NO:215 are the same.
  • amino acid sequences of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:46, and SEQ ID NO:56 are the same.
  • the amino acid sequences of SEQ ID NO:66, SEQ ID NO:76, SEQ ID NO:136, SEQ ID NO:196, SEQ ID NO:206, SEQ ID NO:216, SEQ ID NO:226, SEQ ID NO:246, and SEQ ID NO:266 are the same.
  • the amino acid sequences of SEQ ID NO:96 and SEQ ID NO:106 are the same.
  • the amino acid sequences of SEQ ID NO:126, SEQ ID NO:176, and SEQ ID NO:186 are the same.
  • the amino acid sequences of SEQ ID NO:146, SEQ ID NO:236, and SEQ ID NO:256 are the same.
  • the amino acid sequences of SEQ ID NO:156 and SEQ ID NO:166 are the same.
  • the above-mentioned antibody may be a functional fragment of an antibody.
  • “functional fragment of an antibody” refers to a partial fragment of an antibody (i.e., an immunoglobulin) that retains at least one action against an antigen.
  • partial fragments include F(ab')2, Fab, Fv, disulfide-linked Fv, single-chain antibodies (scFv, VH-VL), VH, and polymers thereof, as well as fusions of these with heavy chain CH3 regions.
  • CDRs such as CDR1, CDR2, and CDR3, conjugates of these CDRs, and fusions of these CDRs or CDR conjugates with heavy chain CH3 regions.
  • the antibodies of the present disclosure also include partial fragments of antibodies such as those described above. Partial fragments of antibodies are sometimes referred to as "antibody fragments.”
  • the above-mentioned antibody may be a multispecific antibody.
  • the multispecific antibody of this embodiment specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, and has at least a first specific binding property that does not specifically bind to human vascular endothelial cells, and a second specific binding property that is different from the first specific binding property.
  • the second specific binding property may be for human CXCR3, or may be for another target molecule.
  • An example of a multispecific antibody is a diabody, which is a type of bispecific antibody.
  • the multispecific antibody comprises two or more (e.g., 2, 3, 4, 5, or more) antigen-binding domains.
  • the multispecific antibody can bind to two or more CXCR3 molecules, for example, at the same or different epitopes.
  • the multispecific antibody can bind to CXCR3 and at least one other antigen with high affinity, for example.
  • the antigen-binding portion of the antibody can comprise one or more fragments of the antibody that retain the ability to specifically bind to the antigen. These fragments can comprise heavy and/or light chain variable regions from a parent antibody or a variant of the parent antibody.
  • the class (isotype) of the antibody is not particularly limited.
  • it may be any class, such as IgG, IgM, IgA, IgD, or IgE.
  • the subclass of the antibody is not particularly limited.
  • it may be any subclass, such as IgG1, IgG2, IgG3, or IgG4.
  • the antibody has the activity of blocking CXCR3A-dependent cellular functions.
  • CXCR3A-dependent cellular functions are induced by the activation of proteins that couple to the intracellular portion of CXCR3 and are responsible for intracellular signal transduction, such as trimeric G proteins and ⁇ -arrestins.
  • Activation of CXCR3A-dependent cellular functions is induced by stimulation with a CXCR3 ligand. Examples include fluctuations in intracellular calcium ions, fluctuations in intracellular cyclic adenosine monophosphate (cAMP), GTP binding to the small G protein Rho, cell chemotaxis, and receptor internalization.
  • cAMP cyclic adenosine monophosphate
  • the antibody does not specifically bind to human vascular endothelial cells.
  • human vascular endothelial cells predominantly express CXCR3B.
  • the extracellular domain of CXCR3A to which the above-mentioned antibody specifically binds may be any of the N-terminal domain, the extracellular first loop domain, the extracellular second loop domain, and the extracellular third loop domain. Furthermore, the above-mentioned antibody may bind to only one of these extracellular domains, or to two or more of them.
  • the present disclosure includes antibodies that are "functionally equivalent" to the above-mentioned anti-human CXCR3 antibodies.
  • Functionally equivalent antibodies include antibodies that have the same epitope as the above-mentioned antibodies.
  • the epitopes of 28 anti-human CXCR3 antibodies specifically shown in the Examples below are analyzed by epitope mapping using partial peptides of human CXCR3, etc.
  • synthetic peptides containing the identified epitopes can be used as antigens to obtain anti-human CXCR3 antibodies that bind to the same epitopes as the above-mentioned 28 anti-human CXCR3 antibodies.
  • the amino acid sequences of the heavy chain variable region and light chain variable region of the obtained anti-human CXCR3 antibody can be determined, and the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 can be identified.
  • the present disclosure provides an antibody that specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, and does not specifically bind to human vascular endothelial cells.
  • the antibody includes an amino acid sequence having 1 to 10 amino acid substitutions, additions, or deletions in the amino acid sequence of the SEQ ID NOs defined in (C1) to (C28) above, or an antibody having 90% or greater identity to the amino acid sequence of the SEQ ID NOs defined in (C1) to (C28) above.
  • the number of substituted, added, or deleted amino acids is preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3.
  • the identity is preferably 92% or greater, more preferably 95% or greater, and particularly preferably 97% or greater.
  • a competitive experiment can be used to determine whether two antibodies have the same epitope. For example, if the binding of the 28 anti-human CXCR3 antibodies or functional fragments thereof, which serve as first antibodies, to the receptor is competitively inhibited by the second antibody being tested, then the second antibody can be said to bind to the same epitope as the first antibody.
  • the present disclosure includes an antibody (second antibody) that specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and competitively inhibits the binding of the above antibody (first antibody) to the receptor.
  • the CDRs and other regions can each be selected independently and combined in various combinations with other CDRs or framework regions of a particular antibody.
  • the VH and/or VL, CDR and framework sequences can be present in any combination in an antibody that specifically binds to the extracellular domain of human CXCR3A, has activity to block CXCR3A-dependent cellular functions, and does not specifically bind to human vascular endothelial cells.
  • a humanized antibody is an antibody in which the CDRs are derived from a non-human animal and the other regions (such as framework and constant regions) are derived from humans. Methods for constructing and producing humanized antibodies are described below.
  • the present disclosure includes nucleic acids (DNA) encoding the antibodies, including, for example, a first nucleic acid encoding a heavy chain variable region and/or a second nucleic acid encoding a light chain variable region.
  • DNA nucleic acids
  • the heavy chain variable region encoded by the first nucleic acid may, for example, include a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, or 271; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263, or 273.
  • a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 16
  • the heavy chain variable region encoded by the first nucleic acid comprises, for example, heavy chain CDR1 to CDR3 specified by any of (A1) to (A28) above.
  • the heavy chain variable region encoded by the first nucleic acid is, for example, one specified by any of (C1) to (C28) above.
  • the light chain variable region encoded by the second nucleic acid may, for example, include a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, or 274; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, or 276.
  • a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124
  • the light chain variable region encoded by the second nucleic acid comprises, for example, heavy chain CDR1 to CDR3 specified in any of (B1) to (B28) above.
  • the light chain variable region encoded by the second nucleic acid is, for example, one specified in any of (C1) to (C28) above.
  • the above nucleic acid may be incorporated into a vector.
  • the vector is selected appropriately depending on the type of host cell into which it will be introduced.
  • Vectors include vectors for gene therapy. In this case, the vector itself can be administered directly into the body.
  • the antibody can be produced using genetic recombination techniques, i.e., by constructing recombinant cells that express the nucleic acid, the antibody can be obtained from a culture of the cells.
  • DNAs encoding the amino acid sequences shown in SEQ ID NOS: 1 to 6 are prepared as DNAs encoding each CDR.
  • DNAs can be prepared using known methods such as PCR.
  • the DNAs can also be prepared by chemical synthesis.
  • DNAs encoding variable regions in which heavy chain CDRs 1 to 3 are grafted onto the framework region of VH of any human antibody are prepared.
  • DNAs encoding variable regions in which light chain CDRs 1 to 3 are grafted onto the framework region of VL of any human antibody are prepared.
  • Each of the prepared DNAs is inserted into a vector containing a sequence encoding the CH or CL of a human antibody to construct a humanized antibody expression vector.
  • the constructed expression vector is introduced into host cells to obtain recombinant cells that express the humanized antibody. These recombinant cells are then cultured, and the desired humanized antibody is obtained from the culture.
  • Humanized antibodies having heavy chain CDR1-3 and light chain CDR1-3 specified above in (AB2) to (AB28) can also be constructed and produced in a similar manner.
  • a method for constructing and producing a chimeric antibody will be described.
  • a method for constructing and producing a chimeric antibody having a heavy chain variable region (VH) and a light chain variable region (VL) as specified in (C1) above will be described.
  • DNA encoding the amino acid sequence shown in SEQ ID NO: 7 is prepared as DNA encoding VH.
  • DNA encoding the amino acid sequence shown in SEQ ID NO: 9 is prepared as DNA encoding VL.
  • DNA can be prepared using known methods such as PCR.
  • the DNA can also be prepared by chemical synthesis.
  • the resulting DNA encoding the VH or VL is inserted into a vector containing a sequence encoding the CH or CL of a human antibody, respectively, to construct a chimeric antibody expression vector.
  • vectors containing a sequence encoding the CH or CL of a human antibody are commercially available.
  • Chimeric antibodies having the VH and VL specified above in (C2) to (C28) can also be constructed and produced in a similar manner.
  • Methods for producing multispecific antibodies include, for example, linking an anti-human CXCR3 antibody or a fragment thereof of the present disclosure to another antibody or a fragment thereof. Another example includes co-expressing an anti-human CXCR3 antibody or a fragment thereof of the present disclosure with another antibody or a fragment thereof in a host cell. Other linking methods include chemical coupling, gene fusion, non-covalent association, etc.
  • bispecific antibody An example of a multispecific antibody (bispecific antibody) that has been put into practical use is Blincyto (registered trademark), which binds to both CD3 and CD19. Another example is Hemlibra (registered trademark), which binds to both coagulation factor IXa and coagulation factor X.
  • Techniques for producing multispecific antibodies are known in the art, and these known production techniques can also be applied to the anti-human CXCR3 antibody of the present disclosure.
  • the method for purifying the antibody is not particularly limited, and known methods can be used.
  • the culture supernatant of the recombinant cell can be collected and purified by combining known methods such as various types of chromatography, salting out, dialysis, and membrane separation.
  • the antibody isotype is IgG, it can be easily purified by affinity chromatography using protein A.
  • the antibody of the present disclosure may be a modified antibody to which another molecule is bound.
  • the other molecule include peptides, proteins, small molecules, radioisotopes, light absorbers, etc.
  • Methods for binding to the other molecule include chemical coupling, gene fusion, non-covalent association, etc.
  • the modified antibody may be a multispecific antibody. In this respect, a multispecific antibody can be considered a type of modified antibody.
  • the modified antibody is an antibody-drug conjugate (ADC).
  • Drugs to be conjugated include antiviral drugs; immunosuppressants; immunomodulators; proteins or polypeptides with biological activity such as cytokines; radioisotopes; and light absorbers.
  • Antibody-drug conjugates can be produced by conjugating these drugs directly or indirectly via a linker or chelator.
  • the present disclosure includes a pharmaceutical comprising the antibody as an active ingredient.
  • the pharmaceutical may be a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.
  • the pharmaceutical blocks CXCR3A-dependent cellular functions.
  • the modes by which antibodies block CXCR3A-dependent cellular functions include antibodies that bind to the ligand-binding site on the cell surface and competitively or non-competitively inhibit ligand binding to the receptor, antibodies that bind to a site other than the ligand-binding site and inhibit activation by altering the receptor structure, and antibodies that induce receptor internalization through binding to the receptor, thereby reducing the amount of receptor expression on the cell surface and thereby reducing the ligand responsiveness of the cell.
  • Antibodies that induce receptor internalization are also used in ADCs.
  • the above-mentioned antibodies have internalization activity. In other words, they induce receptor internalization.
  • the above-mentioned pharmaceutical is used to treat a disease, disorder, or condition caused by impaired CXCR3-dependent cellular function.
  • diseases, disorders, or conditions caused by impaired CXCR3-dependent cellular function include atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatism, inflammatory bowel disease, systemic lupus erythematosus, Th1-related diseases such as acute cardiac allograft rejection, influenza, infectious diseases such as COVID-19, and tumors.
  • drugs that selectively inhibit CXCR3A are thought to be highly useful in treatments aimed at suppressing the infiltration, proliferation, and activation of inflammatory cells.
  • Increases in CXCR3A ligands in patients can be disease-specific.
  • increases in CXCL10 have been reported in autoimmune colitis, type 1 diabetes, and acute lung injury; CXCL9 and CXCL10 in multiple sclerosis; and CXCL9, CXCL10, and CXCL11 in atherosclerosis (Meri K. Tulic et al., Nat Commun. 2019 May 16;10(1):2178).
  • Knowledge about the role of CXCR3B-ligand interactions in inflammatory diseases is limited, and drugs that inhibit both CXCR3A and CXCR3B may cause unexpected side effects due to CXCR3B inhibition.
  • CXCR3A-ligand interaction promotes tumor growth and metastasis
  • CXCR3B-ligand interaction promotes anti-tumor growth, angiogenesis inhibition, and apoptosis.
  • CXCR3A-specific CXCR3 inhibitors may have a more potent anti-tumor effect, suppressing tumor growth and metastasis while maintaining the anti-tumor effects of CXCR3B.
  • SCH546738 is known to be an inhibitor that preferentially inhibits CXCR3A over CXCR3B, but no CXCR3A-specific inhibitors have been reported (K. Boye et al., Sci Rep. 2017 Sep 6;7(1):10703).
  • vitiligo drugs that inhibit both CXCR3A and CXCR3B may have a greater therapeutic effect.
  • migration of pathogenic T cells is stimulated via CXCR3A, and apoptosis of melanocytes is promoted via CXCR3B (Meri K. Tulic et al., Nat Commun. 2019 May 16;10(1):2178.).
  • the above-mentioned pharmaceuticals can be administered orally or parenterally, systemically or locally.
  • administration forms include injections, intranasal administrations, pulmonary administrations, and transdermal administrations.
  • Injections can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.
  • the administration method can be selected appropriately depending on the age and symptoms of the patient.
  • the dosage of the above-mentioned antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg of antibody per patient. However, the dosage of the above-mentioned antibody is not limited to these ranges.
  • the above-mentioned pharmaceuticals can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA).
  • the above-mentioned pharmaceuticals can contain pharmaceutically acceptable carriers and additives.
  • the carriers and additives include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphate, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents, but are not limited to these.
  • composition may contain other low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, immunoglobulins; and amino acids such as glycine, glutamine, asparagine, arginine, lysine, etc.
  • examples include isotonic solutions containing physiological saline, glucose, or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, or sodium chloride. These may be used in combination with appropriate solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), or nonionic surfactants (polysorbate 80, HCO-50).
  • solubilizers such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), or nonionic surfactants (polysorbate 80, HCO-50).
  • the antibody can also be encapsulated in microcapsules (such as hydroxymethylcellulose, gelatin, or poly(methyl methacrylate)), or in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remingto's Pharmaceutical Science 16th Edition,” Oslo, Ed. 1980).
  • microcapsules such as hydroxymethylcellulose, gelatin, or poly(methyl methacrylate)
  • colloidal drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules
  • the nucleic acid may be incorporated into a gene therapy vector or mRNA that can be translated into a protein in vivo to produce a gene therapy drug.
  • Methods for administering the gene therapy drug include direct administration using a naked plasmid, administration by packaging the nucleic acid in a liposome or the like, administration by incorporating the nucleic acid into various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, and HVJ vectors (see Adolph, "Viral Genome Methods," CRC Press, Florid (1996)), and administration by coating the nucleic acid on a bead carrier such as colloidal gold particles (e.g., WO 93/17706).
  • the gene therapy drug may be administered by any method as long as the antibody is expressed in vivo and can exert its effect.
  • a sufficient amount is administered via an appropriate parenteral route, such as intravenous, intraperitoneal, subcutaneous, intradermal, intraadipose tissue, intramammary tissue, inhalation, or intramuscular route, by injection, infusion, gas-induced particle bombardment (using an electron gun, etc.), or via a mucosal route such as a nasal spray.
  • the gene therapy drug may be administered ex vivo to cells using liposome transfection, particle bombardment (U.S. Patent No. 4,945,050), or viral infection, and then the cells may be reintroduced into an animal.
  • the present disclosure also includes methods and therapeutic agents for treating a disease or disorder caused by abnormally enhanced CXCR3-dependent cell function in a mammal suffering from the disease.
  • treatment means preventing or alleviating the progression and worsening of the pathological condition of a disease in a mammal that is at risk of contracting or is contracting the disease, and is used to mean a therapeutic procedure aimed at preventing or alleviating the progression and worsening of the symptoms of the disease.
  • the term “disease” refers to any disease that develops due to the abnormal enhancement of CXCR3-dependent cellular functions, and is not particularly limited thereto.
  • Th1-related immune disorders such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatism, inflammatory bowel disease, systemic lupus erythematosus, acute cardiac allograft rejection, influenza, COVID-19, and tumors.
  • Th1-related immune disorders such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatism, inflammatory bowel disease, systemic lupus erythematosus, acute cardiac allograft rejection, influenza, COVID-19, and tumors.
  • the term also encompasses cardiovascular disorders, nervous system disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, infectious diseases, blood cancers, and solid cancers.
  • mammal to be treated refers to any animal classified as a mammal, and includes, but is not limited to, humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses.
  • a particularly preferred "mammal” is a human.
  • the present disclosure includes a support on which the above-mentioned antibody is immobilized (antibody-immobilized support).
  • the antibody-immobilized support is used to remove CXCR3-expressing cells from a body fluid containing CXCR3-expressing cells by contacting the support with the antibody-immobilized support.
  • An example of the body fluid is blood.
  • the above-mentioned antibody immobilized on the support may be one type only or two or more types.
  • the antibody-immobilized carrier of the present disclosure include a carrier in which the antibody is immobilized on a water-insoluble carrier and packed in a container. While any material can be used as the water-insoluble carrier, preferred materials in terms of moldability, sterilization, and low cytotoxicity include synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, and polyurethane; natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alginate; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and metal materials such as stainless steel and titanium.
  • synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, and polyurethane
  • natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alg
  • the carrier may be in the form of granules, cotton, woven fabric, nonwoven fabric, porous sponge, or flat plate, but granules, cotton, woven fabric, nonwoven fabric, or porous sponge are preferred due to their large surface area per volume.
  • a kit for removing CXCR3-expressing cells can be produced by combining the antibody-immobilized carrier of the present disclosure with other components.
  • Such other components include an anticoagulant, an extracorporeal circulation circuit, etc.
  • the present disclosure includes a method for treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function, comprising administering an effective amount of the antibody to a patient suffering from the disease, disorder, or condition.
  • the present disclosure includes the antibody for use in treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function.
  • the present disclosure includes use of the antibody for manufacturing a medicament for treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function.
  • the diseases, disorders, or conditions include at least cardiovascular disorders, nervous system disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, infectious diseases, blood cancers, and solid cancers.
  • Example 1 Immunization of Animals to Produce Anti-CXCR3 Antibodies
  • the template plasmid vector pCI-hEP4-GroEL was constructed as follows. Based on the sequence information of human EP4 registered in the NCBI Gene Bank (NM_000958, SEQ ID NO: 293), an artificial gene was synthesized in which a signal sequence (SEQ ID NO: 294) was added to the 5' end of the human EP4 gene. Using this as a template, a human EP4 gene fragment (hereinafter referred to as "DNA fragment A”) was prepared in which the sequence GCTAGC was added to the 5' end and the sequence GTCGAC was added to the 3' end by PCR.
  • DNA fragment A human EP4 gene fragment
  • Genomic DNA was extracted and purified from E. coli HMS174(DE3) strain (Novagen). Next, PCR was performed using the purified genomic DNA as a template and the primer pair shown in SEQ ID NOs: 295 and 296 to amplify a DNA fragment containing the GroEL subunit gene (SEQ ID NO: 297; hereafter referred to as "DNA fragment B").
  • DNA fragment B contained a SalI site at the 5' end and a sequence encoding two stop codons (TAATAG) and a NotI site at the 3' end, derived from the primers.
  • the mammalian expression vector pCI-Mammalian Expression Vector (Promega) was digested with the restriction enzymes NheI and SalI, the ends were dephosphorylated with bacterial alkaline phosphatase (BAP), and DNA fragment A was inserted.
  • This expression vector was further digested with SalI and NotI, the ends were dephosphorylated with BAP, and DNA fragment B was inserted to construct the template plasmid vector pCI-hEP4-GroEL.
  • DNA fragment C Based on the sequence information for human CXCR3A registered in the NCBI Gene Bank (AAO92295, SEQ ID NO: 281), an artificial gene was synthesized by adding the GCTAGC sequence to the 5' end of the human CXCR3A gene and the GTCGAC sequence to the 3' end. This artificial gene was then introduced into a cloning vector, and the resulting vector was cleaved at the NheI and SalI sites to prepare a DNA fragment (hereinafter referred to as "DNA fragment C").
  • the template plasmid vector pCI-hEP4-GroEL was digested with the restriction enzymes NheI and SalI, and DNA fragment C was inserted to construct the immunization vector pCI-hCXCR3A-GroEL.
  • This vector expresses a fusion protein of human CXCR3A and GroEL.
  • Eight-week-old rats were immunized by injecting 40 ⁇ L of the above immunization composition into the thigh muscles of both legs (day 0). This resulted in 80 ⁇ g of pCI-hCXCR3A-GroEL being administered to both legs, i.e., 160 ⁇ g per rat per injection. Subsequently, the animals were immunized in the same manner on days 14 and 28. On days 21 and 35 after the initial immunization, blood was collected from the immunized animals, and the serum was separated, collected, and stored at -30°C.
  • human CXCR3A stably expressing CHO cells The cells were cultured in a medium containing hygromycin (Thermo Fisher Scientific), and hygromycin-resistant cells stably expressing human CXCR3A were cloned.
  • hygromycin Thermo Fisher Scientific
  • hygromycin-resistant cells stably expressing human CXCR3A were cloned.
  • human CXCR3A-expressing cells are referred to as "human CXCR3A stably expressing CHO cells.”
  • the human CXCR3A gene was introduced into the mammalian expression vector pCI Mammalian Expression Vector (Promega) to construct pCI-hCXCR3A.
  • pCI-hCXCR3A was transfected into HEK293FT cells using Lipofectamine 2000 to transiently express human CXCR3A. These cells were administered to the spleen of the individual (mouse or rat) described in (2), and three days later, the spleen, inguinal lymph nodes, and iliac lymph nodes were sampled. Splenocytes and lymph node-derived cells were isolated from each sample, aliquoted, and stored at -80°C until screening.
  • Example 2 Preparation of antibodies ⁇ 1> (1) Selection of specific antibody-producing lymphocytes using microwells. CHO cells stably expressing human CXCR3A were suspended in F-12 medium (containing 10% FBS, penicillin/streptomycin) to prepare a cell suspension of 3.5 x 10 cells/500 ⁇ L. This cell suspension was loaded into a microchamber for a cell picking system. The microchamber was centrifuged five times at 300 rpm for 1 minute, and one or two CHO cells stably expressing human CXCR3A were placed in each microwell. The microchamber was washed with F-12 medium, and then 500 ⁇ L of F-12 medium was added.
  • F-12 medium containing 10% FBS, penicillin/streptomycin
  • the mixture was incubated at 37°C in a CO2 incubator for 1 hour, allowing the CHO cells stably expressing human CXCR3A to adhere to the bottom of the microwell.
  • a CytoRed solution (Dojindo Laboratories) adjusted to a concentration of 10 nM in F-12 medium was added, and the cells were further incubated for 1 hour at 37°C to stain the cells. After washing three times with F-12 medium to remove excess CytoRed, the microchamber was filled with 1 mL of F-12 medium.
  • Antibody-producing cells were enriched from the cells collected in Example 1(5) after DNA immunization using EasySep Mouse Biotin Positive Selection Kit (STEMCELL Technologies). 5 x 104 to 2 x 105 antibody-producing cells were suspended in 500 ⁇ L of F-12 medium and loaded into the microchamber. The microchamber was centrifuged five times at 300 rpm for 1 minute, and the microchamber was adjusted so that one or two antibody-producing cells were contained in each microwell. After washing the microchamber with medium, an appropriate amount of medium was added and the mixture was incubated at 37°C for 30 minutes to allow the antibody to be secreted from the antibody-producing cells.
  • Alexa Fluor 488-labeled anti-mouse IgG antibody (Abcam) or Alexa Fluor 488-labeled anti-rat IgG antibody (Abcam) diluted 500-fold in RPMI 1640 (containing 10% FBS) was added and incubated at 37°C for 30 minutes. After washing three times with FACS buffer (PBS containing 1% FBS), 1 mL of FACS buffer was added. The microchamber was placed in a cell picking system (AS ONE) to acquire transmitted light images and two types of fluorescent image information for all microwells. CytoRed fluorescence detection was performed at an excitation wavelength of 543 nm and an emission wavelength of 593 nm.
  • Alexa Fluor 488 fluorescence detection was performed at an excitation wavelength of 482 nm and an emission wavelength of 536 nm. From microwells where it was determined based on the scanned images of transmitted light, CytoRed, and Alexa Fluor 488 that antibodies binding to CHO cells stably expressing human CXCR3A were present, antibody-producing cells were recovered into a cell lysate using a capillary tube (As One Corporation) with a diameter of several to several tens of micrometers.
  • Antibody genes were obtained from antibody-producing cells using the MAGrahd method (Kurosawa N, Yoshioka M, Fujimoto R, Yamagishi F, Isobe M. "Rapid production of antigen-specific monoclonal antibodies from a variety of animals.” BMC Biol. 2012; 10: 80). Specifically, 5 ⁇ L of the cell lysate obtained in (1) was mixed with 5 ⁇ g of oligo-dT magnet, and cell-derived mRNA was captured on the oligo-dT magnet.
  • the oligo-dT magnet was washed with a washing solution, and then cDNA synthesis was performed by reverse transcription. After further washing the magnet, a 5'-terminal translational reaction was performed.
  • the synthesized cDNA the antibody heavy chain variable region (VH region) gene and the antibody light chain variable region (VL region) gene were isolated and amplified by 5'-race PCR.
  • PCR was performed twice.
  • a mixture of the first forward primer (SEQ ID NO: 282), which amplifies both the VH and VL regions, the mouse VH first reverse primer (SEQ ID NO: 283), which specifically amplifies the VH region, and the mouse VL first reverse primer (SEQ ID NO: 284), which specifically amplifies the VL region was used.
  • the amplified product from the first PCR was used as a template, and the second forward primer (SEQ ID NO: 285) and the mouse VH second reverse primer (SEQ ID NO: 286), which specifically amplifies the VH region, were used as primers for amplifying the VH region, and the second forward primer (SEQ ID NO: 285) and the mouse VL second reverse primer (SEQ ID NO: 287), which specifically amplifies the VL region, were used as primers for amplifying the VL region.
  • a mixture of a first forward primer (SEQ ID NO: 282) that amplifies both the VH and VL regions, a rat VH first reverse primer (SEQ ID NO: 288) that specifically amplifies the VH region, and a rat VL first reverse primer (SEQ ID NO: 289) that specifically amplifies the VL region was used.
  • the first amplification product was used as a template, and the second forward primer (SEQ ID NO: 285) and a rat second reverse primer (SEQ ID NO: 290) that specifically amplifies the VH region were used to amplify the VH region, and the second forward primer (SEQ ID NO: 285) and a rat VL second reverse primer (SEQ ID NO: 291) that specifically amplifies the VL region were used to amplify the VL region.
  • Example 3 Preparation of antibody ⁇ 2> (1) Preparation of Hybridomas and Screening of Binding Clones The cells collected in Example 1(5) and myeloma cells SP2/0 were fused using a cell fusion device NEPAGENE ECFG21 (Nepper Gene). The fused cells (hybridomas) were suspended in RPMI 1640 medium containing HAT Supplement (Invitrogen) and seeded onto a 96-well plate. After culturing for approximately 8 days, a portion of the culture supernatant was collected. Hybridomas producing antibodies that specifically bind to human CXCR3A stably expressing CHO cells were screened using the same method as in Example 1(4).
  • Hybridomas confirmed to produce antibodies in (1) were cloned by limiting dilution. Specifically, the hybridomas were seeded into a 96-well plate at 1 cell or less per well and cultured. After 2 weeks, antibodies that specifically bind to CHO cells stably expressing human CXCR3A were screened using the same method as in Example 1(4). Antibodies that bound to CHO cells stably expressing human CXCR3A but did not bind to CHO-FlpIn cells were determined as primary hit antibodies.
  • Hybridomas producing the primary hit antibodies were expanded and then replaced with CD Hybridoma medium (Gibco) at a concentration of 5 x 10 cells/mL and cultured for 7 days. The culture supernatant was collected and centrifuged to precipitate cell debris. The supernatant was passed through a 0.45 ⁇ m filter (Sartorius) and stored at 4°C until purification.
  • CD Hybridoma medium Gibco
  • Example 4 Determination of CDR sequences of antibody heavy chain variable regions and antibody heavy chain variable regions
  • the second PCR amplification products containing the antibody heavy chain variable region and antibody light chain variable region obtained in Example 2(2) or Example 3(3) were purified using a PCR product purification kit (FastGene).
  • the sequences of the antibody heavy chain variable region and antibody light chain variable region were determined by direct sequencing using a mouse VH second reverse primer (SEQ ID NO: 286) as the sequence primer for the antibody heavy chain variable region and a mouse VL second reverse primer (SEQ ID NO: 287) as the sequence primer for the antibody light chain variable region, respectively.
  • the sequences of the antibody heavy chain variable region and antibody light chain variable region were determined by direct sequencing using a rat VH second reverse primer (SEQ ID NO: 290) as the sequence primer for the antibody heavy chain variable region and a rat VL second reverse primer (SEQ ID NO: 291) as the sequence primer for the antibody light chain variable region, respectively.
  • the CDRs were determined by combining the Kabat numbering method and the IMGT numbering method, and the regions comprehensively including the regions determined by both numbering methods were determined as CDRs.
  • 201001-1-F (Table 1-1; SEQ ID NOs: 1-10) 201001-5-B (Table 1-2; SEQ ID NOs: 11-20) 201001-2-C (Tables 1-3; SEQ ID NOs: 21-30) 201001-4-E (Tables 1-4; SEQ ID NOs: 31-40) 201001-1-C (Tables 1-5; SEQ ID NOs: 41-50) 201001-5-A (Tables 1-6; SEQ ID NOs: 51-60) 201009-1-D (Tables 1-7; SEQ ID NOs: 61-70) 201009-5-F (Tables 1-8; SEQ ID NOs: 71-80) 201112-1-C (Tables 1-9; SEQ ID NOs: 81-90) 13B4 (Tables 1-10; SEQ ID NOs: 91-100) 11F11 (Tables 1-11; SEQ ID NOs: 101-110) 201006-4-H (Tables 1-12; SEQ ID NOs: 111-120) 201006-4-F (Tables 1-13;
  • Example 5 Antibody screening by evaluating the activity of inhibiting intracellular Ca2 + signaling
  • Purified antibodies were obtained by Protein A affinity purification using the culture supernatants of the clones selected as primary hit antibodies in Example 2 (4) or Example 3 (5).
  • the purified primary hit antibodies were subjected to the following tests.
  • Human CXCR3A-stably expressing CHO cells were seeded into a 96-well microplate at an initial cell concentration of 2 x 104 cells/100 ⁇ L per well and cultured for 2 days. After 2 days, the culture medium was replaced with a solution containing 3 ⁇ M Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Thermo Fisher Scientific). After 1 hour, primary hit antibodies or control antibodies diluted within the 100 nM to 10 nM concentration range were added to each well and incubated for 15 minutes. Mouse isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) were used as control antibodies.
  • the assay plate was placed in a Ca2 + signal analyzer FDSS ⁇ CELL (Hamamatsu Photonics), and the inhibitory activity against the transient increase in intracellular Ca2 + concentration when each cell type was stimulated with 100 nM IP-10 (PEPROTECH) was measured.
  • the inhibitory activity was calculated as a relative value by standardizing the case of "no antibody, with IP-10" as an inhibition rate of 0% and the case of "no antibody, without IP-10" as an inhibition rate of 100%.
  • the inhibitory activity of 28 types of antibodies with different CDRs is shown in Table 2. N/A in the table indicates that the measurement was not performed.
  • RNA was also obtained from the human renal adenocarcinoma cell line ACHN, which is known to express both CXCR3A and CXCR3B, by similar treatment (Takanobu Utsumi, Takahito Suyama, Yusuke Imamura, Miki Fuse, Shinichi Sakamoto, Naoki Nihei, Takeshi Ueda, Hiroyoshi Suzuki, Naohiko Seki, Tomohiko Ichikawa, "The association of CXCR3 and renal cell carcinoma metastasis," J. Urol. 2014 Aug;192(2):567-74).
  • cDNA was synthesized from RNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems). CXCR3A and CXCR3B genes contained in 100 ng of cDNA were detected by quantitative real-time PCR. 18S was used as an internal standard gene, and TaqMan Gene Expression Assays (Applied Biosystems) were used as 18S detection primers.
  • Ct values were analyzed using the ⁇ Ct method, and the relative expression levels of CXCR3A and CXCR3B in each cell were calculated, with the CXCR3B expression level in ACHN set at 1. As shown in Figure 1, it was confirmed that CXCR3B is expressed more predominantly than CXCR3A in human umbilical vascular endothelial cells and human microvascular endothelial cells.
  • Example 7 Evaluation of binding to vascular endothelial cells by flow cytometry Of the 28 anti-CXCR3 antibodies shown in Table 2, 19 antibodies were subjected to the following test.
  • CHO cells stably expressing human CXCR3A, human umbilical vascular endothelial cells (Lonza) confirmed in Example 6 to predominantly express CXCR3B, and human microvascular endothelial cells (Cell Systems) were washed with FACS buffer (PBS containing 1% FBS) and suspended in FACS buffer to a cell concentration of 1 x 10 cells/mL.
  • Fc Block (Tonbo Biosciences) was added at a volume of 1/500 of the cell suspension, or goat serum diluted 1:1, and blocking was performed at 4°C for 30 minutes. After blocking, the cells were suspended at a concentration of 1 x 10 cells/50 ⁇ L.
  • This cell suspension was mixed with 50 ⁇ L of each diluted antibody and incubated at 4°C for 1 hour.
  • the antibodies used were 19 types of anti-CXCR3 antibodies, a CXCR3B-specific antibody (Proteintech), a prior art antibody, or a control antibody.
  • Prior art antibodies used included anti-CXCR3 antibody #49801 (R&D) and anti-CXCR3 antibody 4Hu3 (described in Patent Document 2).
  • Control antibodies included a mouse isotype control antibody (Fujifilm Wako Pure Chemical Industries, Ltd.), a rat isotype control antibody (Fujifilm Wako Pure Chemical Industries, Ltd.), or a human isotype control antibody (GeneScript).
  • the cells were washed twice with 100 ⁇ L of FACS buffer. 50 ⁇ L of a diluted solution of a secondary antibody, such as PE-labeled anti-mouse IgG antibody (Southern Biotech), Alexa Flour 488-labeled anti-rat IgG antibody (Abcam), APC-labeled anti-rat IgG antibody (Abcam), or APC-labeled anti-human IgG antibody (Invitrogen), was added to each well and incubated for 1 hour at 4°C. After washing twice with 100 ⁇ L of FACS buffer, the cells were suspended in 50 ⁇ L of FACS buffer and the fluorescence intensity on the cell surface was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.
  • a secondary antibody such as PE-labeled anti-mouse IgG antibody (Southern Biotech), Alexa Flour 488-labeled anti-rat IgG antibody (Abcam), APC-labeled anti-rat IgG antibody (Ab
  • Figure 2 shows histograms comparing 13B4, 201009-2-C, a CXCR3B-specific antibody, or a prior art antibody (all of which are anti-chemokine antibodies) with a control antibody (isotype antibody).
  • solid black indicates an isotype antibody
  • open white indicates an anti-chemokine antibody.
  • a rightward shift in the histogram was determined to indicate "binding.”
  • the CXCR3B-specific antibody was confirmed to bind to two types of human vascular endothelial cells, but not to CHO cells stably expressing human CXCR3A.
  • 13B4 and the prior art antibody were confirmed to bind to CHO cells stably expressing human CXCR3A and two types of human vascular endothelial cells.
  • 201009-2-C bound only to CHO cells stably expressing human CXCR3A, and did not bind to human vascular endothelial cells.
  • the cell binding specificity of the antibodies is shown in Table 3. A rightward shift in the histogram is indicated by a "+”, and no rightward shift is indicated by a "-”. It was confirmed that 16 antibodies, including 201009-2-C, specifically bind to human CXCR3A and do not bind to human vascular endothelial cells, which predominantly express human CXCR3B.
  • Example 8 Production of antibodies (1) cDNA cloning Using the eight antibody genes selected in Example 5, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, 201009-2-C, 201009-3-H, and 201009-1-C, heavy and light chains were cloned into pET vectors by homologous sequence-selective recombination cloning (Kurosawa N, Yoshioka M, Isobe M. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells. BMC Biotechnol. 2011 Apr 13;11:39.).
  • Example 9 Evaluation of binding to CXCR3A by flow cytometry The eight antibodies obtained in Example 8, i.e., 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, 201009-2-C, 201009-3-H, and 201009-1-C, were subjected to the following tests.
  • CHO cells stably expressing human CXCR3A were washed with FACS buffer (PBS containing 1% FBS) and suspended in FACS buffer to a cell concentration of 1 x 10 cells/mL.
  • Fc Block (Tonbo Biosciences) was added at 1/500 the volume of the cell suspension, and blocking was performed at 4°C for 30 minutes. After blocking, the cells were suspended at 1 x 10 cells/50 ⁇ L. This cell suspension was mixed with 50 ⁇ L each of eight diluted anti-CXCR3 antibodies or a control antibody, and incubated at 4°C for 1 hour.
  • Mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation) was used as the control antibody.
  • the cells were washed twice with 100 ⁇ L of FACS buffer.
  • 50 ⁇ L of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech) or Alexa Flour 488-labeled anti-rat IgG antibody (Abcam) as a secondary antibody was added to each well and incubated for 1 hour at 4°C.
  • After washing the cells twice with 100 ⁇ L of FACS buffer they were suspended in 50 ⁇ L of FACS buffer and the fluorescence intensity on the cell surface was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.
  • a graph was created in which the horizontal axis represents antibody concentration and the vertical axis represents the median fluorescence intensity of the flow cytometer histogram. All eight antibodies were confirmed to bind to CHO cells stably expressing human CXCR3A in a dose-dependent manner. The results of binding of each antibody to CHO cells stably expressing human CXCR3A are shown in Figures 3A to 3H. The 50% binding concentration (EC 50 ) and 80% binding concentration (EC 80 ) were calculated and are summarized in Table 4.
  • Example 10 Evaluation of inhibition of intracellular Ca2 + signaling The eight antibodies obtained in Example 8, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, 201009-2-C, 201009-3-H, and 201009-1-C, were subjected to the following tests.
  • Human CXCR3A-stably expressing CHO cells were seeded into a 96-well microplate at an initial cell concentration of 2 x 104 cells/100 ⁇ L per well and cultured for 2 days. After 2 days, the culture medium was replaced with a solution containing 3 ⁇ M Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Thermo Fisher Scientific). After 1 hour, eight antibodies diluted to concentrations ranging from 200 nM to 0.8 nM or a control antibody were added to each well and incubated for 15 minutes.
  • Example 11 Evaluation of antibody binding specificity To evaluate the binding specificity of antibodies to CXCR3A, the six antibodies selected in Example 10, i.e., 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, and 201009-2-C, were subjected to the following tests.
  • Human CXCR3A-stably expressing CHO cells and other chemokine receptor-expressing cells including human CCR7-stably expressing CHO cells (manufactured in-house) and human CCR6-stably expressing CHO cells (manufactured in-house), were washed with FACS buffer (PBS containing 1% FBS) and suspended in FACS buffer to a cell concentration of 1 x 10 cells/mL.
  • Fc Block Teonbo Biosciences
  • This cell suspension was mixed with 50 ⁇ L each of six diluted anti-CXCR3 antibodies, anti-CCR7 antibodies (manufactured in-house), anti-CCR6 antibodies (manufactured in-house), or a control antibody, and incubated at 4°C for 1 hour.
  • Control antibodies included mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation), rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation), and human isotype control antibody (GeneScript). After incubation, cells were washed twice with 100 ⁇ L of FACS buffer.
  • Figure 5 shows histograms comparing 201006-7-G, 201009-2-C, human CCR7 antibody, or human CCR6 antibody (all anti-chemokine antibodies) with a control antibody (isotype antibody).
  • the solid black indicates the isotype antibody
  • the open white indicates the anti-chemokine antibody.
  • a rightward shift in the histogram was considered to indicate "binding.” Binding of 201006-7-G and 201009-2-C was observed only in CHO cells stably expressing human CXCR3A. These results confirmed that 201006-7-G and 201009-2-C do not bind to other chemokine receptors, but specifically bind to human CXCR3A.
  • Example 12 Evaluation of binding to human peripheral blood-derived T cells by flow cytometry The six antibodies selected in Example 10, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, and 201009-2-C, were subjected to the following test.
  • Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were thawed and activated by culturing in TexMACS medium (Miltenyi Biotech) containing 1% T cell TransAct, human, and 10 ng/mL IL-2 for 3 days. The medium was replaced every 2–3 days (TexMACS medium containing 10 ng/mL IL-2) for expansion. T cells were used for testing after 10 days of expansion. Human T cells were suspended at 1 ⁇ 10 cells/mL in goat serum (Thermo Fisher Scientific) diluted 1:1 with FACS buffer (PBS containing 1% FBS) and blocked for 30 minutes at 4°C. After blocking, the cells were suspended at 4 ⁇ 10 cells/mL.
  • FACS buffer PBS containing 1% FBS
  • This cell suspension was mixed with 25 ⁇ L of each of the six diluted antibodies or a control antibody and incubated at 4°C for 1 hour.
  • Mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation) was used as a control antibody.
  • cells were washed twice with 120 ⁇ L of FACS buffer.
  • 25 ⁇ L of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech) or Alexa Flour 488-labeled anti-rat IgG antibody (Abcam) was added to each well as a secondary antibody and incubated at 4°C for 1 hour.
  • After washing twice with FACS buffer cells were suspended in 50 ⁇ L of FACS buffer and cell surface fluorescence intensity was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.
  • Example 13 Evaluation of antibody activity to inhibit migration of human peripheral blood-derived T cells
  • Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were cultured as described in Example 12, and T cells from 10 days of expansion culture were used for the study.
  • Human T cells were washed with assay buffer (RPMI-1640 + 0.1% BSA).
  • Assay buffer RPMI-1640 + 0.1% BSA
  • Serially diluted antibodies were added to the cells and incubated at 37°C for 60 minutes at a cell density of 2.0-2.4 x 10 /mL.
  • 2.4 nM IP-10 (PEPROTECH) was added to the wells of a receiver plate equipped with an insert-integrated 96-well Transwell with a 3.0 ⁇ m polycarbonate membrane (Corning).
  • the inhibitory activity was calculated as a relative value by standardizing the inhibition rate of "no antibody, with IP-10" to 0% and the inhibition rate of "no antibody, without IP-10" to 100%.
  • the inhibitory effect of 201009-2-C on IP-10-dependent T cell migration is shown in Figure 7.
  • the horizontal axis represents the antibody concentration, and the vertical axis represents the inhibition rate (%).
  • the T cell migration inhibition rate of each antibody is summarized in Table 8.
  • Example 14 Evaluation of antibody internalization by flow cytometry Five types of antibodies, 13B4, 11F11, 201006-5-F, 201006-7-G, and 201009-2-C, were subjected to the following test.
  • Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were cultured as described in Example 12, and T cells from 10 days of expansion were used for the study.
  • Human T cells were suspended at 1 x 10 cells/mL in goat serum (Thermo Fisher Scientific) diluted 1:1 with FACS buffer (PBS containing 1% FBS) and blocked at 4°C for 30 minutes. After blocking, the cells were suspended at 1 x 10 cells/50 ⁇ L. This cell suspension was mixed with five antibodies or a control antibody at an antibody concentration of 1 nM and incubated at 4°C for 30 minutes.
  • Mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation) was used as the control antibody.
  • the cells were washed twice with cold FACS buffer. To promote antibody internalization, a portion of the washed cells was incubated at 37°C for 30 minutes. The remaining cells were allowed to stand at 4°C for 30 minutes. 50 ⁇ L of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech) or Alexa Flour 488-labeled anti-rat IgG antibody (Abcam) was added to each well as a secondary antibody, and the cells were incubated at 4°C for 15 minutes. After washing the cells twice with cold FACS buffer, they were suspended in 50 ⁇ L of cold FACS buffer, and the fluorescence intensity of the cell surface was measured using a Novocyte flow cytometer (Agilent).
  • Antibody internalization was indirectly evaluated based on the expression level of the receptor on the cell surface. That is, the median fluorescence intensity of cells reacted with antibody at 4°C was normalized to 100%, and the median fluorescence intensity of cells reacted with antibody at 37°C was shown as a relative value.
  • Figure 8 shows the median fluorescence intensity % under each condition. As shown by the decrease in median fluorescence intensity % in cells reacted at 37°C compared to 4°C, it was confirmed that the five antibodies were internalized into the cells along with the receptor.
  • Example 15 Production of humanized anti-CXCR3 antibodies The case of 201009-2-C will be exemplified. To produce a humanized antibody into which the CDRs of 201009-2-C were grafted, human frameworks were selected based on the homology between 201009-2-C and human germline VH and VL genes.
  • VL1 SEQ ID NO:300
  • VL2 SEQ ID NO:301
  • VL3 SEQ ID NO:302
  • VL4 SEQ ID NO:303
  • VL region of 201009-2-C SEQ ID NO:259
  • binding activity to human T cells was measured according to the method of Example 12.
  • Graphs were created by plotting antibody concentration on the horizontal axis and median fluorescence intensity of flow cytometer histograms on the vertical axis.
  • the results of binding of each antibody to human T cells are shown in Figures 10A to 10H, and the calculated 50% binding concentrations (EC 50 ) are summarized in Table 9.
  • the human T cell migration inhibitory activity of each antibody was evaluated according to the method of Example 13.
  • the dose dependency of the T cell migration inhibition rate for each antibody is summarized in Table 10.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Cardiology (AREA)
  • Oncology (AREA)
  • Cell Biology (AREA)
  • Rheumatology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Communicable Diseases (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Diabetes (AREA)

Abstract

Provided is a novel anti-human CXCR3 antibody useful for an antibody medicine.　Provided is an antibody which specifically binds to human CXCR3, specifically binds to an extracellular domain of human CXCR3A, has activity of blocking CXCR3A-dependent cellular functions, and does not specifically bind to human vascular endothelial cells. Said antibody has heavy chains CDR1-3 and light chains CDR1-3 which contain a specific amino acid sequence. Said antibody is preferably a humanized antibody or a chimeric antibody.

Description

抗体、核酸、細胞、及び医薬Antibodies, nucleic acids, cells, and drugs

　本開示は、ヒトＣＸＣＲ３の細胞外ドメインに特異的に結合する抗体、当該抗体をコードする核酸、当該核酸を含む細胞、及び当該抗体を有効成分として含有する医薬に関する。 The present disclosure relates to an antibody that specifically binds to the extracellular domain of human CXCR3, a nucleic acid encoding the antibody, a cell containing the nucleic acid, and a pharmaceutical containing the antibody as an active ingredient.

　ケモカインは、種々の細胞から分泌される走化性サイトカインである。ケモカインは、アミノ酸配列と構造の違いにより、ＣＸＣ、ＣＣ、Ｃ、ＣＸ３Ｃの４グループに分類される。ケモカインの機能は、免疫細胞の分化と恒常性、一次免疫応答、二次免疫応答の誘導、種々の疾患における免疫細胞の遊走に重要である。ヒトおよびマウスでは５０種類近くのケモカインが報告されている（非特許文献１）。ケモカインの作用は、７回膜貫通型Ｇタンパク質共役受容体（ＧＰＣＲ）である古典的ケモカイン受容体への結合により発現する。ケモカインと結合するがＧタンパク質と共役しない非定型ケモカイン受容体も存在し、ケモカインの濃度勾配の形成を担っている。哺乳動物においては、約２０個の古典的ケモカイン受容体と４個の非定型ケモカイン受容体が見つかっている（非特許文献１）。 Chemokines are chemotactic cytokines secreted by various cells. Chemokines are classified into four groups based on their amino acid sequence and structure: CXC, CC, C, and CX3C. Chemokines play an important role in immune cell differentiation and homeostasis, primary and secondary immune responses, and immune cell migration in various diseases. Nearly 50 types of chemokines have been reported in humans and mice (Non-Patent Document 1). Chemokine action is expressed through binding to classical chemokine receptors, which are seven-transmembrane G protein-coupled receptors (GPCRs). Atypical chemokine receptors, which bind to chemokines but are not coupled to G proteins, also exist and are responsible for the formation of chemokine concentration gradients. Approximately 20 classical chemokine receptors and four atypical chemokine receptors have been identified in mammals (Non-Patent Document 1).

　ＣＸＣＲ３は、Ｇタンパク質共役受容体９（ＧＰＲ９）、ＣＤ１８３としても知られている、主にＴ細胞に発現するケモカイン受容体である。ＣＸＣＲ３は、例えば、ヒト、マウス、ラット、ウシ、チンパンジー、マカク、イヌ、カエル、カモノハシ、ブタ、ゼブラフィッシュなど、さまざまな生物で発現している。ヒトでは、ＣＸＣＲ３Ａ、ＣＸＣＲ３Ｂ、ＣＸＣＲ３Ａｌｔの３種類のアイソフォームが存在する。 CXCR3, also known as G protein-coupled receptor 9 (GPR9) or CD183, is a chemokine receptor expressed primarily on T cells. CXCR3 is expressed in a variety of organisms, including humans, mice, rats, cattle, chimpanzees, macaques, dogs, frogs, platypus, pigs, and zebrafish. In humans, there are three isoforms: CXCR3A, CXCR3B, and CXCR3Alt.

　ＣＸＣＲ３Ａは、生体内において最も主要なアイソフォームであり、活性化ＣＤ８陽性Ｔ細胞、メモリーＣＤ４陽性Ｔ細胞、メモリーＣＤ８陽性Ｔ細胞、ＮＫ細胞において高度に発現し、炎症部位へのエフェクター細胞のホーミング、病原体のクリアランスなど、免疫生理学プロセスにおいて重要な役割を果たしている。その他、樹状細胞のサブセット、Ｂ細胞のサブセット、マクロファージ、Ｔｈ１７細胞、制御性Ｔ細胞、好中球などの免疫細胞においても発現が認められている（非特許文献１、２）。ＣＸＣＲ３Ａの機能的リガンドは、インターフェロン－γ（ＩＦＮ－γ）誘導性のケモカインＣＸＣＬ９（ＭＩＧ－９）、ＣＸＣＬ１０（ＩＰ－１０）、ＣＸＣＬ１１（Ｉ－ＴＡＣ）である。これら３つのリガンドは、生体内で重複して、相乗的に、もしくは拮抗的に、作用することが示されている（非特許文献２、３）。ＣＸＣＬ９、ＣＸＣＬ１０、ＣＸＣＬ１１によるＣＸＣＲ３Ａの活性化は、Ｇαｑ、Ｇαｉの活性化をもたらし、細胞走化性、細胞増殖、細胞生存、腫瘍細胞の転移などを誘発する。 CXCR3A is the most predominant isoform in vivo and is highly expressed in activated CD8+ T cells, memory CD4+ T cells, memory CD8+ T cells, and NK cells. It plays an important role in immunophysiological processes, such as effector cell homing to inflammatory sites and pathogen clearance. It is also expressed in immune cells such as subsets of dendritic cells, B cell subsets, macrophages, Th17 cells, regulatory T cells, and neutrophils (Non-Patent Documents 1 and 2). The functional ligands for CXCR3A are the interferon-γ (IFN-γ)-inducible chemokines CXCL9 (MIG-9), CXCL10 (IP-10), and CXCL11 (I-TAC). These three ligands have been shown to act redundantly, synergistically, or antagonistically in vivo (Non-Patent Documents 2 and 3). Activation of CXCR3A by CXCL9, CXCL10, and CXCL11 leads to activation of Gαq and Gαi, inducing cell chemotaxis, cell proliferation, cell survival, tumor cell metastasis, and more.

　ＣＸＣＲ３Ｂは、選択的スプライシングによってＣＸＣＲ３Ａよりも４７アミノ酸長いＮ末端ドメインを持つ。ＣＸＣＲ３Ｂの発現は、心臓、骨格筋、肝臓、腎臓などの組織、血管内皮細胞において確認されている。ＣＸＣＲ３Ｂの主要なリガンドはＣＸＣＬ４であり、Ｉｎ　ｖｉｔｒｏにおいては、ＣＸＣＬ９、ＣＸＣＬ１０、ＣＸＣＬ１１もＣＸＣＲ３Ｂに結合する。リガンドのＣＸＣＲ３Ｂへの結合は、Ｇαｓを活性化し、内皮細胞、腫瘍細胞の増殖阻害、遊走阻害、アポトーシス促進作用を示す。すなわち、ＣＸＣＲ３ＡとＣＸＣＲ３Ｂは、互いに相反する作用を担っていると考えられる。ＣＸＣＲ３Ａｌｔは、ＣＸＣＲ３Ａ発現細胞にわずかに共発現し、ＣＸＣＬ１１に対する走化性において役割を持つ（非特許文献２）。 CXCR3B has an N-terminal domain that is 47 amino acids longer than CXCR3A due to alternative splicing. CXCR3B expression has been confirmed in tissues such as the heart, skeletal muscle, liver, and kidney, as well as in vascular endothelial cells. The primary ligand for CXCR3B is CXCL4, and in vitro, CXCL9, CXCL10, and CXCL11 also bind to CXCR3B. Ligand binding to CXCR3B activates Gas, inhibiting the growth and migration of endothelial cells and tumor cells and promoting apoptosis. In other words, CXCR3A and CXCR3B are thought to have opposing effects. CXCR3Alt is co-expressed in small amounts in CXCR3A-expressing cells and plays a role in chemotaxis toward CXCL11 (Non-Patent Document 2).

　ＣＸＣＲ３へのリガンドの結合によるカスケードの活性化は、主に、組織への免疫細胞の遊走、インテグリン活性化および細胞浸潤を誘導する。ＣＸＣＬ９、ＣＸＣＬ１０、ＣＸＣＬ１１は、自己免疫疾患、移植、感染、腫瘍などの炎症局所において大量に産生され、炎症部位への免疫細胞の動員に関与し、様々な炎症性疾患に関与すると考えられている。例としては、アテローム性動脈硬化症、心筋炎、多発性硬化症、喘息、慢性閉塞性肺疾患、肺線維症、１型糖尿病、乾癬、リウマチ、炎症性腸炎、全身性エリテマトーデス、急性心臓同種移植片拒絶反応などのＴｈ１関連疾患がある（非特許文献３）。また、より最近の研究では、インフルエンザ、新型コロナウイルス感染症（ＣＯＶＩＤ－１９）などのウイルス感染症や腫瘍など、より広範囲の疾患との関連が示唆されている（非特許文献３、４）。したがって、ＣＸＣＲ３シグナル伝達に関連する障害の進行を軽減するために、ＣＸＣＲ３シグナル伝達を標的とする物質（例えば、抗体）および方法が必要とされている。 Activation of the cascade by ligand binding to CXCR3 primarily induces immune cell migration into tissues, integrin activation, and cell infiltration. CXCL9, CXCL10, and CXCL11 are produced in large quantities at sites of inflammation, such as autoimmune diseases, transplants, infections, and tumors. They are thought to be involved in the recruitment of immune cells to inflammatory sites and are involved in various inflammatory diseases. Examples include Th1-related diseases such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and acute cardiac allograft rejection (Non-Patent Document 3). Furthermore, more recent studies have suggested an association with a wider range of diseases, including influenza, viral infections such as COVID-19, and tumors (Non-Patent Documents 3, 4). Therefore, there is a need for agents (e.g., antibodies) and methods that target CXCR3 signaling to reduce the progression of disorders associated with CXCR3 signaling.

　ケモカイン受容体を特異的に標的とする薬剤の開発に対して、多大な努力が重ねられてきた。しかし、米国食品医薬品局（Food and Drug Administration; FDA）によって承認されている薬剤は、ＣＣＲ５インバースアゴニストであるマラビロク（抗レトロウイルス薬）、ＣＸＣＲ４アンタゴニストであるプレリキサフォル（多発性骨髄腫治療薬）、抗ＣＣＲ４モノクローナル抗体モガムズリマブ（Ｔ細胞白血病治療薬）の３つのみである。受容体とリガンドの結合の重複性、生体における役割の病的および生理学的多様性、受容体構造の類似性による特異的低分子開発の難しさなどが、薬剤開発を困難にしていると考えられる（非特許文献５）。 Enormous efforts have been made to develop drugs that specifically target chemokine receptors. However, only three drugs have been approved by the U.S. Food and Drug Administration (FDA): the CCR5 inverse agonist maraviroc (an antiretroviral drug), the CXCR4 antagonist plerixafor (a drug for treating multiple myeloma), and the anti-CCR4 monoclonal antibody mogamuzulimab (a drug for treating T-cell leukemia). Redundancy in receptor-ligand binding, the pathological and physiological diversity of roles in the body, and the difficulty of developing specific small molecules due to similarity in receptor structure are thought to complicate drug development (Non-Patent Document 5).

　ＣＸＣＲ３に関しても、多くの創薬プログラムのターゲットとなっており、低分子アンタゴニスト、抗体の開発が進められてきた。しかし、臨床試験に進んだ低分子ＣＸＣＲ３アンタゴニストは、ＡＭＧ４８７、ＡＣＴ－７７７９９１の２つのみである。ＡＭＧ４８７については乾癬を対象とする第ＩＩａ相試験まで進んだが、有効性は示されなかった。１型糖尿病治療薬として開発が進んでいるＡＣＴ－７７７９９１については、第Ｉ相試験が完了し、健常人において良好な忍容性、動態、安全性が確認され、さらなる臨床開発段階へ進展することが期待される（非特許文献６）。一方で、ＣＸＣＬ１０を標的とするヒトモノクローナル抗体エルデルマブ（ＢＭＳ－９３６５５７）は、潰瘍性大腸炎（ＵＣ）とクローン病の治療に関する第ＩＩ相臨床試験において、中等度から重度の活動性ＵＣに対して効果的な治療法である可能性が示唆された（非特許文献７）。この結果は、Ｔｈ１関連疾患に対するＣＸＣＬ１０－ＣＸＣＲ３カスケードを標的とする治療の臨床的価値を示唆している。 CXCR3 has also been the target of many drug discovery programs, and the development of small molecule antagonists and antibodies has progressed. However, only two small molecule CXCR3 antagonists have progressed to clinical trials: AMG487 and ACT-777991. AMG487 progressed to a Phase IIa trial for psoriasis, but efficacy was not demonstrated. ACT-777991, which is being developed as a treatment for type 1 diabetes, has completed a Phase I trial, demonstrating good tolerability, kinetics, and safety in healthy subjects, and is expected to progress to further clinical development (Non-Patent Document 6). Meanwhile, a Phase II clinical trial of the human monoclonal antibody eldelumab (BMS-936557), which targets CXCL10, for the treatment of ulcerative colitis (UC) and Crohn's disease suggested that it may be an effective treatment for moderate to severe active UC (Non-Patent Document 7). These results suggest the clinical value of treatments targeting the CXCL10-CXCR3 cascade for Th1-related diseases.

　特許文献１には、ヒトＣＸＣＲ３に特異的に結合する６種類のモノクローナル抗体のヒト化抗体が開示されている。そして、各モノクローナル抗体の相補性決定領域、重鎖可変領域、軽鎖可変領域のアミノ酸配列が開示されている。 Patent Document 1 discloses six humanized monoclonal antibodies that specifically bind to human CXCR3. It also discloses the amino acid sequences of the complementarity-determining regions, heavy chain variable regions, and light chain variable regions of each monoclonal antibody.

　特許文献２には、ヒトＣＸＣＲ３に特異的に結合する４種のモノクローナル抗体とそのヒト化抗体が開示されている。そして、各モノクローナル抗体の相補性決定領域、重鎖可変領域、軽鎖可変領域のアミノ酸配列が開示されている。これらの抗ヒトＣＸＣＲ３抗体は、特に１型糖尿病の治療に有効であることが示されている。 Patent Document 2 discloses four types of monoclonal antibodies that specifically bind to human CXCR3, as well as humanized antibodies thereof. It also discloses the amino acid sequences of the complementarity-determining regions, heavy chain variable regions, and light chain variable regions of each monoclonal antibody. These anti-human CXCR3 antibodies have been shown to be particularly effective in treating type 1 diabetes.

　特許文献３には、ヒトＣＸＣＲ３に特異的に結合する１種のモノクローナル抗体のヒト化抗体が開示されている。そして、このモノクローナル抗体の相補性決定領域、重鎖可変領域、軽鎖可変領域のアミノ酸配列が開示されている。この抗体によるＣＸＣＲ３の枯渇は、尋常性白斑の治療に有効であることが示されている。 Patent Document 3 discloses a humanized version of a monoclonal antibody that specifically binds to human CXCR3. It also discloses the amino acid sequences of the complementarity-determining region, heavy chain variable region, and light chain variable region of this monoclonal antibody. CXCR3 depletion using this antibody has been shown to be effective in treating vitiligo vulgaris.

　しかし、特許文献１～３に記載されている抗体は、治療薬としての実用化には至っていない。当技術分野において、医薬品原料としてより優れた特性を有するさらに別の又は改良された抗ヒトＣＸＣＲ３抗体が求められている。 However, the antibodies described in Patent Documents 1 to 3 have not yet been put to practical use as therapeutic agents. There is a need in the art for additional or improved anti-human CXCR3 antibodies with superior properties as pharmaceutical raw materials.

国際公開第２００８／０９４９４２号WO 2008/094942 国際公開第２０１３／１０９９７４号International Publication No. 2013/109974 国際公開第２０１８／１１９２８８号International Publication No. 2018/119288

Catherine E. Hughes and Robert J. B., "A guide to chemokines and their receptors." FEBS J. 2018 Aug; 285(16): 2944-2971.Catherine E. Hughes and Robert J. B., "A guide to chemokines and their receptors." FEBS J. 2018 Aug; 285(16): 2944-2971. Devi Satarkar, Chinmoy Patra, "Evolution, Expression and Functional Analysis of CXCR3 in Neuronal and Cardiovascular Diseases: A Narrative Review", Front Cell Dev Biol. 2022 Jun 20:10:882017.Devi Satarkar, Chinmoy Patra, "Evolution, Expression and Functional Analysis of CXCR3 in Neuronal and Cardiovascular Diseases: A Narrative Review", Front Cell Dev Biol. 2022 Jun 20:10:882017. Joanna R Groom and Andrew D Luster, "CXCR3 ligands: redundant, collaborative and antagonistic functions" Immunol Cell Biol. 2011 Feb; 89(2),207-215.Joanna R Groom and Andrew D Luster, “CXCR3 ligands: redundant, collaborative and antagonistic functions” Immunol Cell Biol. 2011 Feb; 89(2),207-215. Daniel Reynolds, Cristina Vazquez Guillamet, Aaron Day, Nicholas Borcherding, Rodrigo Vazquez Guillamet, Jose Alberto Choreno-Parra, Stacey L. House, Jane A. O'Halloran, Joaquin Zuniga, Ali H. Ellebedy, Derek E. Byers, and Philip A. Mudd, "Comprehensive Immunologic Evaluation of Bronchoalveolar Lavage Samples from Human Patients with Moderate and Severe Seasonal Influenza and Severe COVID-19", J Immunol. 2021 Sep 1;207(5):1229-1238.Daniel Reynolds, Cristina Vazquez Guillamet, Aaron Day, Nicholas Borcherding, Rodrigo Vazquez Guillamet, Jose Alberto Choreno-Parra, Stacey L. House, Jane A. O'Halloran, Joaquin Zuniga, Ali H. Ellebedy, Derek E. Byers, and Philip A. Mudd, “Comprehensive Immunologic Evaluation of Bronchoalveolar Lavage Samples from Human Patie nts with Moderate and Severe Seasonal Influenza and Severe COVID-19", J Immunol. 2021 Sep 1;207(5):1229-1238. Wing Yee Lai and Anja MuellerLatest, "Latest update on chemokine receptors as therapeutic targets", Biochem Soc Trans. 2021 Jun 30; 49(3): 1385-1395.Wing Yee Lai and Anja MuellerLatest, “Latest update on chemokine receptors as therapeutic targets”, Biochem Soc Trans. 2021 Jun 30; 49(3): 1385-1395. Marie-Laure Boof, Martine Gehin, Christine Voors-Pette, Chih-Hsuan Hsin, Virginie Sippel, Daniel S Strasser, Jasper Dingemanse, "Pharmacokinetics, pharmacodynamics and safety of the novel C-X-C chemokine receptor 3 antagonist ACT-777991: Results from the first-in-human study in healthy adults", Br J Clin Pharmacol. 2024 Feb;90(2):588-599.Marie-Laure Boof, Martine Gehin, Christine Voors-Pette, Chih-Hsuan Hsin, Virginie Sippel, Daniel S Strasser, Jasper Dingemanse, "Pharmacokinetics, pharmacodynamics and sa fety of the novel C-X-C chemokine receptor 3 antagonist ACT-777991: Results from the first-in-human study in healthy adults", Br J Clin Pharmacol. 2024 Feb;90(2):588-599. Lloyd Mayer, William J Sandborn, Yuriy Stepanov, Karel Geboes, Robert Hardi, Michael Yellin, Xiaolu Tao, Li An Xu, Luisa Salter-Cid, Sheila Gujrathi, Richard Aranda, Allison Y Luo, "Anti-IP-10 antibody (BMS-936557) for ulcerative colitis: a phase II randomised study", Gut. 2014 Mar;63(3):442-50.Lloyd Mayer, William J Sandborn, Yuriy Stepanov, Karel Geboes, Robert Hardi, Michael Yellin, Xiaolu Tao, Li An Xu, Luisa Salter-Cid, Sheila Gujrathi , Richard Aranda, Allison Y Luo, “Anti-IP-10 antibody (BMS-936557) for ulcerative colitis: a phase II randomised study”, Gut. 2014 Mar;63(3):442-50.

　上記したように、ＣＸＣＲ３の機能を阻害する抗体は、抗体医薬としての有用性が期待されているが、未だ治療薬として実用化に至ったものがない。そこで本開示は、医薬品原料としてより優れた特性を有する新規の抗ヒトＣＸＣＲ３抗体を提供することを目的とする。 As mentioned above, antibodies that inhibit the function of CXCR3 are expected to be useful as antibody drugs, but none have yet been put into practical use as therapeutic agents. Therefore, the purpose of this disclosure is to provide a novel anti-human CXCR3 antibody with superior properties as a pharmaceutical ingredient.

　本開示の一態様は、ヒトＣＸＣＲ３に特異的に結合する抗体であって、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合せず、下記（ＡＢ１）～（ＡＢ９）、（ＡＢ１２）～（ＡＢ２７）：
（ＡＢ２６）配列番号２５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２３）配列番号２２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２４）配列番号２３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２５）配列番号２４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２７）配列番号２６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１）配列番号１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２）配列番号１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ３）配列番号２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ５）配列番号４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ８）配列番号７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１６）配列番号１５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１７）配列番号１６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１８）配列番号１７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２０）配列番号１９１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１９２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１９３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１９４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１９５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１９６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２１）配列番号２０１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２０２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２０３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２０４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２０５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２０６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２２）配列番号２１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ４）配列番号３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ６）配列番号５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ７）配列番号６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ９）配列番号８１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号８２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号８３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号８４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号８５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号８６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１２）配列番号１１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１３）配列番号１２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１４）配列番号１３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１５）配列番号１４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１９）配列番号１８１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１８２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１８３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１８４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１８５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１８６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
のいずれかを満たす、抗体である。 One aspect of the present disclosure is an antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A, has activity of blocking CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and is selected from the group consisting of the following (AB1) to (AB9), (AB12) to (AB27):
(AB26) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 251, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 252, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 253, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 254, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 255, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 256.
(AB23) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 221, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 222, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 223, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 224, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 225, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 226,
(AB24) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 231, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 232, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 233, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 234, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 235, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 236.
(AB25) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 242, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 245, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 246,
(AB27) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 261, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 262, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 263, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 264, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 265, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 266.
(AB1) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6,
(AB2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16,
(AB3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26,
(AB5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46,
(AB8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76.
(AB16) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 151, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 152, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 153, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 154, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 155, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 156.
(AB17) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 161, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 162, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 163, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 164, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 165, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 166.
(AB18) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 171, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 172, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 173, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 174, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 175, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 176.
(AB20) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 191, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 192, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 193, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 194, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 195, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 196.
(AB21) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 201, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 202, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 203, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 204, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 205, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 206,
(AB22) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 211, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 212, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 213, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 214, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 215, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 216.
(AB4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.
(AB6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56.
(AB7) A antibody having a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66,
(AB9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86.
(AB12) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116.
(AB13) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 126.
(AB14) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 132, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 133, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 135, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 136.
(AB15) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 141, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 142, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 143, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 144, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 145, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 146.
(AB19) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 181, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 182, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 183, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 184, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 185, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 186.
It is an antibody that satisfies any of the following criteria.

　本開示の一態様は、ヒトＣＸＣＲ３に特異的に結合する抗体であって、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合せず、下記（Ｃ１）～（Ｃ９）、（Ｃ１２）～（Ｃ２７）：
（Ｃ２６）配列番号２５７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２５９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２３）配列番号２２７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２２９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２４）配列番号２３７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２３９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２５）配列番号２４７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２４９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２７）配列番号２６７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２６９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１）配列番号７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２）配列番号１７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ３）配列番号２７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ５）配列番号４７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号４９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ８）配列番号７７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号７９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１６）配列番号１５７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１５９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１７）配列番号１６７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１６９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１８）配列番号１７７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１７９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２０）配列番号１９７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１９９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２１）配列番号２０７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２０９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２２）配列番号２１７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２１９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ４）配列番号３７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号３９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ６）配列番号５７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号５９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ７）配列番号６７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号６９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ９）配列番号８７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号８９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１２）配列番号１１７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１１９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１３）配列番号１２７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１２９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１４）配列番号１３７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１３９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１５）配列番号１４７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１４９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１９）配列番号１８７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１８９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
のいずれかを満たす、抗体である。 One aspect of the present disclosure is an antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A, has activity of blocking CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and is selected from the group consisting of the following (C1) to (C9), (C12) to (C27):
(C26) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 257, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 259;
(C23) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 227, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 229;
(C24) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 237, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 239;
(C25) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 247, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 249;
(C27) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 267, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 269;
(C1) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9;
(C2) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19.
(C3) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29.
(C5) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 49.
(C8) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 77, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 79;
(C16) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 157, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 159;
(C17) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 167, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 169;
(C18) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 177, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 179;
(C20) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 197, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 199;
(C21) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 207, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 209;
(C22) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 217, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 219,
(C4) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 39,
(C6) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 59.
(C7) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 69;
(C9) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89.
(C12) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 117, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 119.
(C13) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 127, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 129.
(C14) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139.
(C15) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 147, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 149;
(C19) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 187, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 189;
It is an antibody that satisfies any one of the following conditions.

　本開示の一態様は、ヒトＣＸＣＲ３に特異的に結合する抗体であって、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合せず、上記の抗体である第一抗体と受容体との結合を競合阻害する、第二抗体である。 One aspect of the present disclosure is a second antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A, has activity to block CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and competitively inhibits the binding of the first antibody described above to the receptor.

　好ましくは、前記抗体は、ヒト化抗体又はキメラ型抗体である。 Preferably, the antibody is a humanized antibody or a chimeric antibody.

　好ましくは、前記抗体は、多重特異性抗体である。 Preferably, the antibody is a multispecific antibody.

　好ましくは、前記抗体は、内在化活性を有する。 Preferably, the antibody has internalization activity.

　好ましくは、前記抗体は、他の分子が結合した修飾抗体である。 Preferably, the antibody is a modified antibody to which another molecule is bound.

　好ましくは、前記修飾抗体が、抗体薬物複合体である。 Preferably, the modified antibody is an antibody-drug conjugate.

　本開示の一態様は、上記の抗体をコードする、核酸である。 One aspect of the present disclosure is a nucleic acid encoding the above-described antibody.

　本開示の一態様は、上記の核酸を含む、細胞である。 One aspect of the present disclosure is a cell containing the above-mentioned nucleic acid.

　本開示の一態様は、上記の抗体を有効成分として含有する、医薬である。 One aspect of the present disclosure is a pharmaceutical containing the above-described antibody as an active ingredient.

　好ましくは、前記医薬は、ＣＸＣＲ３依存的な細胞機能の障害が関与している疾患、障害、又は病状の治療に用いられる。 Preferably, the pharmaceutical is used to treat a disease, disorder, or condition involving impairment of CXCR3-dependent cellular function.

　好ましくは、前記疾患、障害、又は病状が、Ｔｈ１免疫異常疾患である。 Preferably, the disease, disorder, or condition is a Th1 immune disorder.

　好ましくは、前記Ｔｈ１免疫異常疾患が、心血管障害、神経系障害、炎症性疾患、自己免疫疾患、代謝性疾患、感染症、血液癌、又は固形癌である。 Preferably, the Th1 immune disorder is a cardiovascular disorder, a nervous system disorder, an inflammatory disease, an autoimmune disease, a metabolic disease, an infectious disease, a blood cancer, or a solid cancer.

　本開示によれば、医薬品原料としてより優れた特性を有する新規の抗ヒトＣＸＣＲ３抗体を提供することができる。 This disclosure makes it possible to provide a novel anti-human CXCR3 antibody with superior properties as a pharmaceutical ingredient.

実施例６で行ったＣＸＣＲ３Ａ、ＣＸＣＲ３Ｂの遺伝子発現を定量化した結果を表すグラフであり、ヒト血管内皮細胞がＣＸＣＲ３Ｂを優位に発現することを示している。1 is a graph showing the results of quantifying gene expression of CXCR3A and CXCR3B performed in Example 6, showing that human vascular endothelial cells predominantly express CXCR3B. 実施例７で行ったフローサイトメトリーの結果を表すヒストグラムであり、抗体（１３Ｂ４、２０１００９－２－Ｃ）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞、血管内皮細胞への結合特性を示している。1 is a histogram showing the results of flow cytometry performed in Example 7, illustrating the binding properties of the antibody (13B4, 201009-2-C) to CHO cells stably expressing human CXCR3A and vascular endothelial cells. 抗体（１３Ｂ４）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (13B4) to CHO cells stably expressing human CXCR3A. 抗体（１１Ｆ１１）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (11F11) to CHO cells stably expressing human CXCR3A. 抗体（２０１００６－３－Ｈ）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201006-3-H) to CHO cells stably expressing human CXCR3A. 抗体（２０１００６－５－Ｆ）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201006-5-F) to CHO cells stably expressing human CXCR3A. 抗体（２０１００６－７－Ｇ）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201006-7-G) to CHO cells stably expressing human CXCR3A. 抗体（２０１００９－２－Ｃ）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201009-2-C) to CHO cells stably expressing human CXCR3A. 抗体（２０１００９－３－Ｈ）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201009-3-H) to CHO cells stably expressing human CXCR3A. 抗体（２０１００９－１－Ｃ）のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201009-1-C) to CHO cells stably expressing human CXCR3A. 抗体（１３Ｂ４）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibody (13B4) against human CXCR3A. 抗体（１１Ｆ１１）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibody (11F11) against human CXCR3A. 抗体（２０１００６－３－Ｈ）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-3-H) against human CXCR3A. 抗体（２０１００６－５－Ｆ）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-5-F) against human CXCR3A. 抗体（２０１００６－７－Ｇ）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-7-G) against human CXCR3A. 抗体（２０１００９－２－Ｃ）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of an antibody (201009-2-C) against human CXCR3A. 抗体（２０１００９－３－Ｈ）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibody (201009-3-H) against human CXCR3A. 抗体（２０１００９－１－Ｃ）のヒトＣＸＣＲ３Ａに対する阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of an antibody (201009-1-C) against human CXCR3A. 実施例１１で行ったフローサイトメトリーの結果を表すヒストグラムであり、抗体（２０１００６－７－Ｇ、２０１００９－２－Ｃ）とヒトＣＸＣＲ３Ａの特異的結合を示している。1 is a histogram showing the results of flow cytometry performed in Example 11, showing the specific binding of antibodies (201006-7-G, 201009-2-C) to human CXCR3A. 抗体（１３Ｂ４）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of antibody (13B4) to human peripheral blood mononuclear cell-derived T cells. 抗体（１１Ｆ１１）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (11F11) to human peripheral blood mononuclear cell-derived T cells. 抗体（２０１００６－３－Ｈ）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201006-3-H) to human peripheral blood mononuclear cell-derived T cells. 抗体（２０１００６－５－Ｆ）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201006-5-F) to human peripheral blood mononuclear cell-derived T cells. 抗体（２０１００６－７－Ｇ）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201006-7-G) to human peripheral blood mononuclear cell-derived T cells. 抗体（２０１００９－２－Ｃ）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding ability of an antibody (201009-2-C) to human peripheral blood mononuclear cell-derived T cells. 抗体（２０１００９－２－Ｃ）のヒト末梢血単核球由来Ｔ細胞の遊走阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibody (201009-2-C) on the migration of human peripheral blood mononuclear cell-derived T cells. 実施例１４で行ったフローサイトメトリーの結果を示すグラフであり、ヒト末梢血単核球由来Ｔ細胞において抗体が受容体とともに内在化することを示している。1 is a graph showing the results of flow cytometry performed in Example 14, demonstrating that antibodies are internalized together with receptors in human peripheral blood mononuclear cell-derived T cells. ヒト化抗ＣＸＣＲ３抗体の重鎖可変領域のアライメントを表す説明図である。FIG. 1 is an explanatory diagram showing the alignment of the heavy chain variable regions of humanized anti-CXCR3 antibodies. ヒト化抗ＣＸＣＲ３抗体の軽鎖可変領域のアライメントを表す説明図である。FIG. 1 is an explanatory diagram showing the alignment of the light chain variable region of a humanized anti-CXCR3 antibody. 抗体（ＶＨ１＋ＶＬ１）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL1) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ１＋ＶＬ２）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL2) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ１＋ＶＬ３）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL3) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ１＋ＶＬ４）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL4) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ２＋ＶＬ１）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL1) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ２＋ＶＬ２）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL2) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ２＋ＶＬ３）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL3) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ２＋ＶＬ４）のヒト末梢血単核球由来Ｔ細胞への結合性評価の結果を表すグラフである。1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL4) to human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ１＋ＶＬ１）のヒト末梢血単核球由来Ｔ細胞の遊走阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH1+VL1) on the migration of human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ１＋ＶＬ４）のヒト末梢血単核球由来Ｔ細胞の遊走阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH1+VL4) on the migration of human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ２＋ＶＬ１）のヒト末梢血単核球由来Ｔ細胞の遊走阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH2+VL1) on the migration of human peripheral blood mononuclear cell-derived T cells. 抗体（ＶＨ２＋ＶＬ４）のヒト末梢血単核球由来Ｔ細胞の遊走阻害活性の用量依存性を表すグラフである。1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH2+VL4) on the migration of human peripheral blood mononuclear cell-derived T cells.

　本開示において、相補性決定領域（ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）をＣＤＲと略記する。本開示において、重鎖可変領域をＶＨ、重鎖定常領域をＣＨ、軽鎖可変領域をＶＬ、軽鎖定常領域をＣＬと、それぞれ略記することがある。本開示において、「抗体」という文言は「免疫グロブリン」に置き換えることができる。本開示において、「核酸」という文言は「ＤＮＡ」又は「遺伝子」に置き換えることができる。 In this disclosure, complementarity determining region is abbreviated as CDR. In this disclosure, heavy chain variable region may be abbreviated as VH, heavy chain constant region as CH, light chain variable region as VL, and light chain constant region as CL. In this disclosure, the term "antibody" may be replaced with "immunoglobulin." In this disclosure, the term "nucleic acid" may be replaced with "DNA" or "gene."

＜ヒトＣＸＣＲ３＞
　ヒトＣＸＣＲ３受容体はＧタンパク質共役型受容体（ＧＰＣＲ）の一種であり、細胞膜を７回貫通し、そのＮ末端を細胞外に、Ｃ末端を細胞内に向けて存在している。ヒトＣＸＣＲ３には、ＣＸＣＲ３Ａ、ＣＸＣＲ３Ｂ、ＣＸＣＲ３Ａｌｔの３種類のアイソフォームが存在する。ヒトＣＸＣＲ３をコードする遺伝子（ｃＤＮＡ）はすでに単離されており、ヒトＣＸＣＲ３のアミノ酸配列も知られている。当該配列情報は、遺伝子のデータベースから得ることができる（例えば、NCBI Reference Sequence：ＡＡＯ９２２９５）。その一例として、ヒトＣＸＣＲ３Ａのアミノ酸配列を配列番号２８１に、ヒトＣＸＣＲ３Ｂのアミノ酸配列を配列番号２９２にそれぞれ示す。 <Human CXCR3>
The human CXCR3 receptor is a type of G protein-coupled receptor (GPCR), which penetrates the cell membrane seven times and is present with its N-terminus facing extracellularly and its C-terminus facing intracellularly. Human CXCR3 exists in three isoforms: CXCR3A, CXCR3B, and CXCR3Alt. The gene (cDNA) encoding human CXCR3 has already been isolated, and the amino acid sequence of human CXCR3 is also known. The sequence information can be obtained from a gene database (e.g., NCBI Reference Sequence: AAO92295). As an example, the amino acid sequence of human CXCR3A is shown in SEQ ID NO: 281, and the amino acid sequence of human CXCR3B is shown in SEQ ID NO: 292.

　ヒトＣＸＣＲ３Ａの各ドメインは、配列番号２８１に示すアミノ酸配列における以下の部分に相当すると考えられている。左側がアミノ酸番号、右側が各ドメインである。なお各ドメイン間の境界については、多少の前後が生じ得る。
　　　１～　５３：Ｎ末端ドメイン
　　８１～　８９：細胞内第１ループドメイン
　１１１～１２５：細胞外第１ループドメイン
　１４８～１６９：細胞内第２ループドメイン
　１９０～２１２：細胞外第２ループドメイン
　２３４～２５５：細胞内第３ループドメイン
　２７８～２９８：細胞外第３ループドメイン
　３２２～３６８：Ｃ末端ドメイン Each domain of human CXCR3A is thought to correspond to the following portion of the amino acid sequence shown in SEQ ID NO: 281. The left side indicates the amino acid number, and the right side indicates each domain. Note that the boundaries between each domain may vary slightly.
1-53: N-terminal domain 81-89: Intracellular loop 1 domain 111-125: Extracellular loop 1 domain 148-169: Intracellular loop 2 domain 190-212: Extracellular loop 2 domain 234-255: Intracellular loop 3 domain 278-298: Extracellular loop 3 domain 322-368: C-terminal domain

＜抗ヒトＣＸＣＲ３抗体＞
　本明細書で開示される抗体は、ヒトＣＸＣＲ３に特異的に結合する抗体（抗ヒトＣＸＣＲ３抗体）であって、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合しないものである。 <Anti-human CXCR3 antibody>
The antibody disclosed in the present specification is an antibody that specifically binds to human CXCR3 (anti-human CXCR3 antibody), which specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, and does not specifically bind to human vascular endothelial cells.

　一態様に係る抗体は、
　配列番号１、１１、２１、３１、４１、５１、６１、７１、８１、９１、１０１、１１１、１２１、１３１、１４１、１５１、１６１、１７１、１８１、１９１、２０１、２１１、２２１、２３１、２４１、２５１、２６１、又は２７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、
　配列番号２、１２、２２、３２、４２、５２、６２、７２、８２、９２、１０２、１１２、１２２、１３２、１４２、１５２、１６２、１７２、１８２、１９２、２０２、２１２、２２２、２３２、２４２、２５２、２６２、又は２７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、
　配列番号３、１３、２３、３３、４３、５３、６３、７３、８３、９３、１０３、１１３、１２３、１３３、１４３、１５３、１６３、１７３、１８３、１９３、２０３、２１３、２２３、２３３、２４３、２５３、２６３、又は２７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、
　配列番号４、１４、２４、３４、４４、５４、６４、７４、８４、９４、１０４、１１４、１２４、１３４、１４４、１５４、１６４、１７４、１８４、１９４、２０４、２１４、２２４、２３４、２４４、２５４、２６４、又は２７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、
　配列番号５、１５、２５、３５、４５、５５、６５、７５、８５、９５、１０５、１１５、１２５、１３５、１４５、１５５、１６５、１７５、１８５、１９５、２０５、２１５、２２５、２３５、２４５、２５５、２６５、又は２７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び
　配列番号６、１６、２６、３６、４６、５６、６６、７６、８６、９６、１０６、１１６、１２６、１３６、１４６、１５６、１６６、１７６、１８６、１９６、２０６、２１６、２２６、２３６、２４６、２５６、２６６、又は２７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する。 The antibody according to one embodiment comprises:
a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, or 271;
a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, or 272;
a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263, or 273;
a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, or 274;
and a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, or 275; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, or 276.

　一態様に係る抗体は、
　下記（Ａ１）～（Ａ２８）：
（Ａ１）配列番号１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２）配列番号１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ３）配列番号２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ４）配列番号３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ５）配列番号４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ６）配列番号５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ７）配列番号６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ８）配列番号７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ９）配列番号８１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号８２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号８３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１０）配列番号９１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号９２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号９３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１１）配列番号１０１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１０２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１０３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１２）配列番号１１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１３）配列番号１２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１４）配列番号１３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１５）配列番号１４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１６）配列番号１５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１７）配列番号１６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１８）配列番号１７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ１９）配列番号１８１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１８２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１８３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２０）配列番号１９１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１９２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号１９３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２１）配列番号２０１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２０２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２０３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２２）配列番号２１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２３）配列番号２２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２４）配列番号２３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２５）配列番号２４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２６）配列番号２５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２７）配列番号２６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
（Ａ２８）配列番号２７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、及び配列番号２７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３を有する、
のいずれかを満たす。 The antibody according to one embodiment comprises:
The following (A1) to (A28):
(A1) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3;
(A2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13;
(A3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23;
(A4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33;
(A5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43;
(A6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53;
(A7) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63;
(A8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73;
(A9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83;
(A10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93.
(A11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103;
(A12) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113;
(A13) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123;
(A14) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 132, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 133;
(A15) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 141, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 142, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 143;
(A16) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 151, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 152, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 153;
(A17) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 161, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 162, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 163;
(A18) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 171, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 172, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 173;
(A19) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 181, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 182, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 183;
(A20) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 191, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 192, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 193;
(A21) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 201, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 202, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 203;
(A22) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 211, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 212, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 213;
(A23) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 221, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 222, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 223;
(A24) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 231, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 232, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 233;
(A25) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 242, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 243;
(A26) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 251, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 252, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 253;
(A27) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 261, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 262, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 263;
(A28) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 271, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 272, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 273;
Satisfy one of the following.

　一態様に係る抗体は、
　下記（Ｂ１）～（Ｂ２８）：
（Ｂ１）配列番号４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２）配列番号１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ３）配列番号２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ４）配列番号３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ５）配列番号４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ６）配列番号５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ７）配列番号６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ８）配列番号７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ９）配列番号８４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号８５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号８６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１０）配列番号９４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号９５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号９６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１１）配列番号１０４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１０５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１０６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１２）配列番号１１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１３）配列番号１２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１４）配列番号１３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１５）配列番号１４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１６）配列番号１５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１７）配列番号１６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１８）配列番号１７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ１９）配列番号１８４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１８５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１８６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２０）配列番号１９４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１９５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１９６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２１）配列番号２０４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２０５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２０６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２２）配列番号２１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２３）配列番号２２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２４）配列番号２３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２５）配列番号２４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２６）配列番号２５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２７）配列番号２６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（Ｂ２８）配列番号２７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
のいずれかを満たす。 The antibody according to one embodiment comprises:
The following (B1) to (B28):
(B1) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6;
(B2) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16;
(B3) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26;
(B4) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36;
(B5) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46;
(B6) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56;
(B7) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66;
(B8) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76;
(B9) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86;
(B10) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96.
(B11) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106;
(B12) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116;
(B13) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 126;
(B14) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 135, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 136;
(B15) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 144, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 145, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 146;
(B16) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 154, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 155, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 156;
(B17) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 164, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 165, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 166;
(B18) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 174, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 175, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 176;
(B19) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 184, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 185, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 186;
(B20) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 194, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 195, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 196;
(B21) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 204, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 205, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 206;
(B22) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 214, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 215, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 216;
(B23) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 224, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 225, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 226;
(B24) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 234, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 235, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 236;
(B25) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 245, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 246;
(B26) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 254, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 255, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 256;
(B27) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 264, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 265, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 266;
(B28) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 274, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 275, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 276;
Satisfy one of the following.

　一態様に係る抗体は、重鎖ＣＤＲ１～３と軽鎖ＣＤＲ１～３が、上記（ＡＢ１）～（ＡＢ９）、（ＡＢ１２）～（ＡＢ２７）のいずれかを満たす。 In one embodiment, the heavy chain CDR1-3 and light chain CDR1-3 of the antibody satisfy any of the above (AB1)-(AB9) and (AB12)-(AB27).

　一態様に係る抗体は、重鎖ＣＤＲ１～３と軽鎖ＣＤＲ１～３が、下記（ＡＢ１０）、（ＡＢ１１）、（ＡＢ２８）：
（ＡＢ１０）配列番号９１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号９２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号９３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号９４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号９５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号９６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１１）配列番号１０１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１０２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１０３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１０４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１０５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１０６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２８）配列番号２７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
のいずれかを満たす。 In one embodiment, the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 of the antibody are selected from the group consisting of (AB10), (AB11), and (AB28):
(AB10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96.
(AB11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106.
(AB28) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 271, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 272, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 273, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 274, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 275, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 276.
Satisfy one of the following.

　一態様に係る抗体は、重鎖可変領域と軽鎖可変領域が、上記（Ｃ１）～（Ｃ９）、（Ｃ１２）～（Ｃ２７）のいずれかを満たす。 In one embodiment, the heavy chain variable region and light chain variable region of the antibody satisfy any of the above (C1) to (C9) and (C12) to (C27).

　一態様に係る抗体は、重鎖可変領域と軽鎖可変領域が、下記（Ｃ１０）、（Ｃ１１）、（Ｃ２８）：
（Ｃ１０）配列番号９７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号９９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１１）配列番号１０７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１０９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２８）配列番号２７７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２７９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
のいずれかを満たす。 In one embodiment, the antibody has a heavy chain variable region and a light chain variable region selected from the group consisting of (C10), (C11), and (C28) below:
(C10) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 99.
(C11) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 107, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 109.
(C28) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 277, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 279;
Satisfy one of the following.

　（Ｃ１）で特定される重鎖可変領域（配列番号７）は、（Ａ１）又は（ＡＢ１）で特定される重鎖ＣＤＲ１～３（配列番号１～３）を含む。（Ｃ１）で特定される軽鎖可変領域（配列番号９）は、（Ｂ１）又は（ＡＢ１）で特定される軽鎖ＣＤＲ１～３（配列番号４～６）を含む。（Ａ１）、（Ｂ１）、（ＡＢ１）、又は（Ｃ１）を満たす抗体の例としては、後述の実施例に記載された「２０１００１－１－Ｆ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 7) specified by (C1) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 1 to 3) specified by (A1) or (AB1). The light chain variable region (SEQ ID NO: 9) specified by (C1) contains light chain CDRs 1 to 3 (SEQ ID NOs: 4 to 6) specified by (B1) or (AB1). An example of an antibody that satisfies (A1), (B1), (AB1), or (C1) is "201001-1-F," which is described in the Examples below.

　（Ｃ２）で特定される重鎖可変領域（配列番号１７）は、（Ａ２）又は（ＡＢ２）で特定される重鎖ＣＤＲ１～３（配列番号１１～１３）を含む。（Ｃ２）で特定される軽鎖可変領域（配列番号１９）は、（Ｂ２）又は（ＡＢ２）で特定される軽鎖ＣＤＲ１～３（配列番号１４～１６）を含む。（Ａ２）、（Ｂ２）、（ＡＢ２）、又は（Ｃ２）を満たす抗体の例としては、後述の実施例に記載された「２０１００１－５－Ｂ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 17) specified by (C2) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 11 to 13) specified by (A2) or (AB2). The light chain variable region (SEQ ID NO: 19) specified by (C2) contains light chain CDRs 1 to 3 (SEQ ID NOs: 14 to 16) specified by (B2) or (AB2). An example of an antibody that satisfies (A2), (B2), (AB2), or (C2) is "201001-5-B," which is described in the Examples below.

　（Ｃ３）で特定される重鎖可変領域（配列番号２７）は、（Ａ３）又は（ＡＢ３）で特定される重鎖ＣＤＲ１～３（配列番号２１～２３）を含む。（Ｃ３）で特定される軽鎖可変領域（配列番号２９）は、（Ｂ３）又は（ＡＢ３）で特定される軽鎖ＣＤＲ１～３（配列番号２４～２６）を含む。（Ａ３）、（Ｂ３）、（ＡＢ３）、又は（Ｃ３）を満たす抗体の例としては、後述の実施例に記載された「２０１００１－２－Ｃ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 27) specified by (C3) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 21 to 23) specified by (A3) or (AB3). The light chain variable region (SEQ ID NO: 29) specified by (C3) contains light chain CDRs 1 to 3 (SEQ ID NOs: 24 to 26) specified by (B3) or (AB3). An example of an antibody that satisfies (A3), (B3), (AB3), or (C3) is "201001-2-C," which is described in the Examples below.

　（Ｃ４）で特定される重鎖可変領域（配列番号３７）は、（Ａ４）又は（ＡＢ４）で特定される重鎖ＣＤＲ１～３（配列番号３１～３３）を含む。（Ｃ４）で特定される軽鎖可変領域（配列番号３９）は、（Ｂ４）又は（ＡＢ４）で特定される軽鎖ＣＤＲ１～３（配列番号３４～３６）を含む。（Ａ４）、（Ｂ４）、（ＡＢ４）、又は（Ｃ４）を満たす抗体の例としては、後述の実施例に記載された「２０１００１－４－Ｅ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 37) specified by (C4) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 31 to 33) specified by (A4) or (AB4). The light chain variable region (SEQ ID NO: 39) specified by (C4) contains light chain CDRs 1 to 3 (SEQ ID NOs: 34 to 36) specified by (B4) or (AB4). An example of an antibody that satisfies (A4), (B4), (AB4), or (C4) is "201001-4-E," which is described in the Examples below.

　（Ｃ５）で特定される重鎖可変領域（配列番号４７）は、（Ａ５）又は（ＡＢ５）で特定される重鎖ＣＤＲ１～３（配列番号４１～４３）を含む。（Ｃ５）で特定される軽鎖可変領域（配列番号４９）は、（Ｂ５）又は（ＡＢ５）で特定される軽鎖ＣＤＲ１～３（配列番号４４～４６）を含む。（Ａ５）、（Ｂ５）、（ＡＢ５）、又は（Ｃ５）を満たす抗体の例としては、後述の実施例に記載された「２０１００１－１－Ｃ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 47) specified by (C5) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 41 to 43) specified by (A5) or (AB5). The light chain variable region (SEQ ID NO: 49) specified by (C5) contains light chain CDRs 1 to 3 (SEQ ID NOs: 44 to 46) specified by (B5) or (AB5). An example of an antibody that satisfies (A5), (B5), (AB5), or (C5) is "201001-1-C," which is described in the Examples below.

　（Ｃ６）で特定される重鎖可変領域（配列番号５７）は、（Ａ６）又は（ＡＢ６）で特定される重鎖ＣＤＲ１～３（配列番号５１～５３）を含む。（Ｃ６）で特定される軽鎖可変領域（配列番号５９）は、（Ｂ６）又は（ＡＢ６）で特定される軽鎖ＣＤＲ１～３（配列番号５４～５６）を含む。（Ａ６）、（Ｂ６）、（ＡＢ６）、又は（Ｃ６）を満たす抗体の例としては、後述の実施例に記載された「２０１００１－５－Ａ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 57) specified by (C6) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 51 to 53) specified by (A6) or (AB6). The light chain variable region (SEQ ID NO: 59) specified by (C6) contains light chain CDRs 1 to 3 (SEQ ID NOs: 54 to 56) specified by (B6) or (AB6). An example of an antibody that satisfies (A6), (B6), (AB6), or (C6) is "201001-5-A," which is described in the Examples below.

　（Ｃ７）で特定される重鎖可変領域（配列番号６７）は、（Ａ７）又は（ＡＢ７）で特定される重鎖ＣＤＲ１～３（配列番号６１～６３）を含む。（Ｃ７）で特定される軽鎖可変領域（配列番号６９）は、（Ｂ７）又は（ＡＢ７）で特定される軽鎖ＣＤＲ１～３（配列番号６４～６６）を含む。（Ａ７）、（Ｂ７）、（ＡＢ７）、又は（Ｃ７）を満たす抗体の例としては、後述の実施例に記載された「２０１００９－１－Ｄ」が挙げられる。 The heavy chain variable region identified by (C7) (SEQ ID NO: 67) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 61 to 63) identified by (A7) or (AB7). The light chain variable region identified by (C7) (SEQ ID NO: 69) contains light chain CDRs 1 to 3 (SEQ ID NOs: 64 to 66) identified by (B7) or (AB7). An example of an antibody that satisfies (A7), (B7), (AB7), or (C7) is "201009-1-D," which is described in the Examples below.

　（Ｃ８）で特定される重鎖可変領域（配列番号７７）は、（Ａ８）又は（ＡＢ８）で特定される重鎖ＣＤＲ１～３（配列番号７１～７３）を含む。（Ｃ８）で特定される軽鎖可変領域（配列番号７９）は、（Ｂ８）又は（ＡＢ８）で特定される軽鎖ＣＤＲ１～３（配列番号７４～７６）を含む。（Ａ８）、（Ｂ８）、（ＡＢ８）、又は（Ｃ８）を満たす抗体の例としては、後述の実施例に記載された「２０１００９－５－Ｆ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 77) identified by (C8) contains heavy chain CDRs 1-3 (SEQ ID NOs: 71-73) identified by (A8) or (AB8). The light chain variable region (SEQ ID NO: 79) identified by (C8) contains light chain CDRs 1-3 (SEQ ID NOs: 74-76) identified by (B8) or (AB8). An example of an antibody that satisfies (A8), (B8), (AB8), or (C8) is "201009-5-F," which is described in the Examples below.

　（Ｃ９）で特定される重鎖可変領域（配列番号８７）は、（Ａ９）又は（ＡＢ９）で特定される重鎖ＣＤＲ１～３（配列番号８１～８３）を含む。（Ｃ９）で特定される軽鎖可変領域（配列番号８９）は、（Ｂ９）又は（ＡＢ９）で特定される軽鎖ＣＤＲ１～３（配列番号８４～８６）を含む。（Ａ９）、（Ｂ９）、（ＡＢ９）、又は（Ｃ９）を満たす抗体の例としては、後述の実施例に記載された「２０１１１２－１－Ｃ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 87) identified by (C9) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 81 to 83) identified by (A9) or (AB9). The light chain variable region (SEQ ID NO: 89) identified by (C9) contains light chain CDRs 1 to 3 (SEQ ID NOs: 84 to 86) identified by (B9) or (AB9). An example of an antibody that satisfies (A9), (B9), (AB9), or (C9) is "201112-1-C," which is described in the Examples below.

　（Ｃ１０）で特定される重鎖可変領域（配列番号９７）は、（Ａ１０）又は（ＡＢ１０）で特定される重鎖ＣＤＲ１～３（配列番号９１～９３）を含む。（Ｃ１０）で特定される軽鎖可変領域（配列番号９９）は、（Ｂ１０）又は（ＡＢ１０）で特定される軽鎖ＣＤＲ１～３（配列番号９４～９６）を含む。（Ａ１０）、（Ｂ１０）、（ＡＢ１０）、又は（Ｃ１０）を満たす抗体の例としては、後述の実施例に記載された「１３Ｂ４」が挙げられる。 The heavy chain variable region (SEQ ID NO: 97) specified by (C10) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 91 to 93) specified by (A10) or (AB10). The light chain variable region (SEQ ID NO: 99) specified by (C10) contains light chain CDRs 1 to 3 (SEQ ID NOs: 94 to 96) specified by (B10) or (AB10). An example of an antibody that satisfies (A10), (B10), (AB10), or (C10) is "13B4," which is described in the Examples below.

　（Ｃ１１）で特定される重鎖可変領域（配列番号１０７）は、（Ａ１１）又は（ＡＢ１１）で特定される重鎖ＣＤＲ１～３（配列番号１０１～１０３）を含む。（Ｃ１１）で特定される軽鎖可変領域（配列番号１０９）は、（Ｂ１１）又は（ＡＢ１１）で特定される軽鎖ＣＤＲ１～３（配列番号１０４～１０６）を含む。（Ａ１１）、（Ｂ１１）、（ＡＢ１１）、又は（Ｃ１１）を満たす抗体の例としては、後述の実施例に記載された「１１Ｆ１１」が挙げられる。 The heavy chain variable region specified by (C11) (SEQ ID NO: 107) contains heavy chain CDRs 1 to 3 specified by (A11) or (AB11) (SEQ ID NO: 101 to 103). The light chain variable region specified by (C11) (SEQ ID NO: 109) contains light chain CDRs 1 to 3 specified by (B11) or (AB11) (SEQ ID NO: 104 to 106). An example of an antibody that satisfies (A11), (B11), (AB11), or (C11) is "11F11," which is described in the Examples below.

　（Ｃ１２）で特定される重鎖可変領域（配列番号１１７）は、（Ａ１２）又は（ＡＢ１２）で特定される重鎖ＣＤＲ１～３（配列番号１１１～１１３）を含む。（Ｃ１２）で特定される軽鎖可変領域（配列番号１１９）は、（Ｂ１２）又は（ＡＢ１２）で特定される軽鎖ＣＤＲ１～３（配列番号１１４～１１６）を含む。（Ａ１２）、（Ｂ１２）、（ＡＢ１２）、又は（Ｃ１２）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－４－Ｈ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 117) specified by (C12) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 111 to 113) specified by (A12) or (AB12). The light chain variable region (SEQ ID NO: 119) specified by (C12) contains light chain CDRs 1 to 3 (SEQ ID NOs: 114 to 116) specified by (B12) or (AB12). An example of an antibody that satisfies (A12), (B12), (AB12), or (C12) is "201006-4-H," which is described in the Examples below.

　（Ｃ１３）で特定される重鎖可変領域（配列番号１２７）は、（Ａ１３）又は（ＡＢ１３）で特定される重鎖ＣＤＲ１～３（配列番号１２１～１２３）を含む。（Ｃ１３）で特定される軽鎖可変領域（配列番号１２９）は、（Ｂ１３）又は（ＡＢ１３）で特定される軽鎖ＣＤＲ１～３（配列番号１２４～１２６）を含む。（Ａ１３）、（Ｂ１３）、（ＡＢ１３）、又は（Ｃ１３）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－４－Ｆ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 127) specified by (C13) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 121 to 123) specified by (A13) or (AB13). The light chain variable region (SEQ ID NO: 129) specified by (C13) contains light chain CDRs 1 to 3 (SEQ ID NOs: 124 to 126) specified by (B13) or (AB13). An example of an antibody that satisfies (A13), (B13), (AB13), or (C13) is "201006-4-F," which is described in the Examples below.

　（Ｃ１４）で特定される重鎖可変領域（配列番号１３７）は、（Ａ１４）又は（ＡＢ１４）で特定される重鎖ＣＤＲ１～３（配列番号１３１～１３３）を含む。（Ｃ１４）で特定される軽鎖可変領域（配列番号１３９）は、（Ｂ１４）又は（ＡＢ１４）で特定される軽鎖ＣＤＲ１～３（配列番号１３４～１３６）を含む。（Ａ１４）、（Ｂ１４）、（ＡＢ１４）、又は（Ｃ１４）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－５－Ａ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 137) specified by (C14) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 131 to 133) specified by (A14) or (AB14). The light chain variable region (SEQ ID NO: 139) specified by (C14) contains light chain CDRs 1 to 3 (SEQ ID NOs: 134 to 136) specified by (B14) or (AB14). An example of an antibody that satisfies (A14), (B14), (AB14), or (C14) is "201006-5-A," which is described in the Examples below.

　（Ｃ１５）で特定される重鎖可変領域（配列番号１４７）は、（Ａ１５）又は（ＡＢ１５）で特定される重鎖ＣＤＲ１～３（配列番号１４１～１４３）を含む。（Ｃ１５）で特定される軽鎖可変領域（配列番号１４９）は、（Ｂ１５）又は（ＡＢ１５）で特定される軽鎖ＣＤＲ１～３（配列番号１４４～１４６）を含む。（Ａ１５）、（Ｂ１５）、（ＡＢ１５）、又は（Ｃ１５）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－４－Ａ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 147) identified by (C15) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 141 to 143) identified by (A15) or (AB15). The light chain variable region (SEQ ID NO: 149) identified by (C15) contains light chain CDRs 1 to 3 (SEQ ID NOs: 144 to 146) identified by (B15) or (AB15). An example of an antibody that satisfies (A15), (B15), (AB15), or (C15) is "201006-4-A," which is described in the Examples below.

　（Ｃ１６）で特定される重鎖可変領域（配列番号１５７）は、（Ａ１６）又は（ＡＢ１６）で特定される重鎖ＣＤＲ１～３（配列番号１５１～１５３）を含む。（Ｃ１６）で特定される軽鎖可変領域（配列番号１５９）は、（Ｂ１６）又は（ＡＢ１６）で特定される軽鎖ＣＤＲ１～３（配列番号１５４～１５６）を含む。（Ａ１６）、（Ｂ１６）、（ＡＢ１６）、又は（Ｃ１６）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－１－Ｈ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 157) identified by (C16) contains heavy chain CDRs 1-3 (SEQ ID NOs: 151-153) identified by (A16) or (AB16). The light chain variable region (SEQ ID NO: 159) identified by (C16) contains light chain CDRs 1-3 (SEQ ID NOs: 154-156) identified by (B16) or (AB16). An example of an antibody that satisfies (A16), (B16), (AB16), or (C16) is "201006-1-H," which is described in the Examples below.

　（Ｃ１７）で特定される重鎖可変領域（配列番号１６７）は、（Ａ１７）又は（ＡＢ１７）で特定される重鎖ＣＤＲ１～３（配列番号１６１～１６３）を含む。（Ｃ１７）で特定される軽鎖可変領域（配列番号１６９）は、（Ｂ１７）又は（ＡＢ１７）で特定される軽鎖ＣＤＲ１～３（配列番号１６４～１６６）を含む。（Ａ１７）、（Ｂ１７）、（ＡＢ１７）、又は（Ｃ１７）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－３－Ｄ」が挙げられる。 The heavy chain variable region identified by (C17) (SEQ ID NO: 167) contains heavy chain CDRs 1-3 (SEQ ID NOs: 161-163) identified by (A17) or (AB17). The light chain variable region identified by (C17) (SEQ ID NO: 169) contains light chain CDRs 1-3 (SEQ ID NOs: 164-166) identified by (B17) or (AB17). An example of an antibody that satisfies (A17), (B17), (AB17), or (C17) is "201006-3-D," which is described in the Examples below.

　（Ｃ１８）で特定される重鎖可変領域（配列番号１７７）は、（Ａ１８）又は（ＡＢ１８）で特定される重鎖ＣＤＲ１～３（配列番号１７１～１７３）を含む。（Ｃ１８）で特定される軽鎖可変領域（配列番号１７９）は、（Ｂ１８）又は（ＡＢ１８）で特定される軽鎖ＣＤＲ１～３（配列番号１７４～１７６）を含む。（Ａ１８）、（Ｂ１８）、（ＡＢ１８）、又は（Ｃ１８）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－１－Ｆ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 177) identified by (C18) contains heavy chain CDRs 1-3 (SEQ ID NOs: 171-173) identified by (A18) or (AB18). The light chain variable region (SEQ ID NO: 179) identified by (C18) contains light chain CDRs 1-3 (SEQ ID NOs: 174-176) identified by (B18) or (AB18). An example of an antibody that satisfies (A18), (B18), (AB18), or (C18) is "201006-1-F," which is described in the Examples below.

　（Ｃ１９）で特定される重鎖可変領域（配列番号１８７）は、（Ａ１９）又は（ＡＢ１９）で特定される重鎖ＣＤＲ１～３（配列番号１８１～１８３）を含む。（Ｃ１９）で特定される軽鎖可変領域（配列番号１８９）は、（Ｂ１９）又は（ＡＢ１９）で特定される軽鎖ＣＤＲ１～３（配列番号１８４～１８６）を含む。（Ａ１９）、（Ｂ１９）、（ＡＢ１９）、又は（Ｃ１９）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－４－Ｇ」が挙げられる。 The heavy chain variable region identified by (C19) (SEQ ID NO: 187) contains heavy chain CDRs 1-3 (SEQ ID NOs: 181-183) identified by (A19) or (AB19). The light chain variable region identified by (C19) (SEQ ID NO: 189) contains light chain CDRs 1-3 (SEQ ID NOs: 184-186) identified by (B19) or (AB19). An example of an antibody that satisfies (A19), (B19), (AB19), or (C19) is "201006-4-G," which is described in the Examples below.

　（Ｃ２０）で特定される重鎖可変領域（配列番号１９７）は、（Ａ２０）又は（ＡＢ２０）で特定される重鎖ＣＤＲ１～３（配列番号１９１～１９３）を含む。（Ｃ２０）で特定される軽鎖可変領域（配列番号１９９）は、（Ｂ２０）又は（ＡＢ２０）で特定される軽鎖ＣＤＲ１～３（配列番号１９４～１９６）を含む。（Ａ２０）、（Ｂ２０）、（ＡＢ２０）、又は（Ｃ２０）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－７－Ａ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 197) identified by (C20) contains heavy chain CDRs 1-3 (SEQ ID NOs: 191-193) identified by (A20) or (AB20). The light chain variable region (SEQ ID NO: 199) identified by (C20) contains light chain CDRs 1-3 (SEQ ID NOs: 194-196) identified by (B20) or (AB20). An example of an antibody that satisfies (A20), (B20), (AB20), or (C20) is "201006-7-A," which is described in the Examples below.

　（Ｃ２１）で特定される重鎖可変領域（配列番号２０７）は、（Ａ２１）又は（ＡＢ２１）で特定される重鎖ＣＤＲ１～３（配列番号２０１～２０３）を含む。（Ｃ２１）で特定される軽鎖可変領域（配列番号２０９）は、（Ｂ２１）又は（ＡＢ２１）で特定される軽鎖ＣＤＲ１～３（配列番号２０４～２０６）を含む。（Ａ２１）、（Ｂ２１）、（ＡＢ２１）、又は（Ｃ２１）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－２－Ｄ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 207) identified by (C21) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 201 to 203) identified by (A21) or (AB21). The light chain variable region (SEQ ID NO: 209) identified by (C21) contains light chain CDRs 1 to 3 (SEQ ID NOs: 204 to 206) identified by (B21) or (AB21). An example of an antibody that satisfies (A21), (B21), (AB21), or (C21) is "201006-2-D," which is described in the Examples below.

　（Ｃ２２）で特定される重鎖可変領域（配列番号２１７）は、（Ａ２２）又は（ＡＢ２２）で特定される重鎖ＣＤＲ１～３（配列番号２１１～２１３）を含む。（Ｃ２２）で特定される軽鎖可変領域（配列番号２１９）は、（Ｂ２２）又は（ＡＢ２２）で特定される軽鎖ＣＤＲ１～３（配列番号２１４～２１６）を含む。（Ａ２２）、（Ｂ２２）、（ＡＢ２２）、又は（Ｃ２２）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－２－Ｃ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 217) identified by (C22) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 211 to 213) identified by (A22) or (AB22). The light chain variable region (SEQ ID NO: 219) identified by (C22) contains light chain CDRs 1 to 3 (SEQ ID NOs: 214 to 216) identified by (B22) or (AB22). An example of an antibody that satisfies (A22), (B22), (AB22), or (C22) is "201006-2-C," which is described in the Examples below.

　（Ｃ２３）で特定される重鎖可変領域（配列番号２２７）は、（Ａ２３）又は（ＡＢ２３）で特定される重鎖ＣＤＲ１～３（配列番号２２１～２２３）を含む。（Ｃ２３）で特定される軽鎖可変領域（配列番号２２９）は、（Ｂ２３）又は（ＡＢ２３）で特定される軽鎖ＣＤＲ１～３（配列番号２２４～２２６）を含む。（Ａ２３）、（Ｂ２３）、（ＡＢ２３）、又は（Ｃ２３）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－３－Ｈ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 227) identified by (C23) contains heavy chain CDRs 1-3 (SEQ ID NOs: 221-223) identified by (A23) or (AB23). The light chain variable region (SEQ ID NO: 229) identified by (C23) contains light chain CDRs 1-3 (SEQ ID NOs: 224-226) identified by (B23) or (AB23). An example of an antibody that satisfies (A23), (B23), (AB23), or (C23) is "201006-3-H," which is described in the Examples below.

　（Ｃ２４）で特定される重鎖可変領域（配列番号２３７）は、（Ａ２４）又は（ＡＢ２４）で特定される重鎖ＣＤＲ１～３（配列番号２３１～２３３）を含む。（Ｃ２４）で特定される軽鎖可変領域（配列番号２３９）は、（Ｂ２４）又は（ＡＢ２４）で特定される軽鎖ＣＤＲ１～３（配列番号２３４～２３６）を含む。（Ａ２４）、（Ｂ２４）、（ＡＢ２４）、又は（Ｃ２４）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－５－Ｆ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 237) identified by (C24) contains heavy chain CDRs 1-3 (SEQ ID NOs: 231-233) identified by (A24) or (AB24). The light chain variable region (SEQ ID NO: 239) identified by (C24) contains light chain CDRs 1-3 (SEQ ID NOs: 234-236) identified by (B24) or (AB24). An example of an antibody that satisfies (A24), (B24), (AB24), or (C24) is "201006-5-F," which is described in the Examples below.

　（Ｃ２５）で特定される重鎖可変領域（配列番号２４７）は、（Ａ２５）又は（ＡＢ２５）で特定される重鎖ＣＤＲ１～３（配列番号２４１～２４３）を含む。（Ｃ２５）で特定される軽鎖可変領域（配列番号２４９）は、（Ｂ２５）又は（ＡＢ２５）で特定される軽鎖ＣＤＲ１～３（配列番号２４４～２４６）を含む。（Ａ２５）、（Ｂ２５）、（ＡＢ２５）、又は（Ｃ２５）を満たす抗体の例としては、後述の実施例に記載された「２０１００６－７－Ｇ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 247) identified by (C25) contains heavy chain CDRs 1-3 (SEQ ID NOs: 241-243) identified by (A25) or (AB25). The light chain variable region (SEQ ID NO: 249) identified by (C25) contains light chain CDRs 1-3 (SEQ ID NOs: 244-246) identified by (B25) or (AB25). An example of an antibody that satisfies (A25), (B25), (AB25), or (C25) is "201006-7-G," which is described in the Examples below.

　（Ｃ２６）で特定される重鎖可変領域（配列番号２５７）は、（Ａ２６）又は（ＡＢ２６）で特定される重鎖ＣＤＲ１～３（配列番号２５１～２５３）を含む。（Ｃ２６）で特定される軽鎖可変領域（配列番号２５９）は、（Ｂ２６）又は（ＡＢ２６）で特定される軽鎖ＣＤＲ１～３（配列番号２５４～２５６）を含む。（Ａ２６）、（Ｂ２６）、（ＡＢ２６）、又は（Ｃ２６）を満たす抗体の例としては、後述の実施例に記載された「２０１００９－２－Ｃ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 257) identified by (C26) contains heavy chain CDRs 1-3 (SEQ ID NOs: 251-253) identified by (A26) or (AB26). The light chain variable region (SEQ ID NO: 259) identified by (C26) contains light chain CDRs 1-3 (SEQ ID NOs: 254-256) identified by (B26) or (AB26). An example of an antibody that satisfies (A26), (B26), (AB26), or (C26) is "201009-2-C," which is described in the Examples below.

　（Ｃ２７）で特定される重鎖可変領域（配列番号２６７）は、（Ａ２７）又は（ＡＢ２７）で特定される重鎖ＣＤＲ１～３（配列番号２６１～２６３）を含む。（Ｃ２７）で特定される軽鎖可変領域（配列番号２６９）は、（Ｂ２７）又は（ＡＢ２７）で特定される軽鎖ＣＤＲ１～３（配列番号２６４～２６６）を含む。（Ａ２７）、（Ｂ２７）、（ＡＢ２７）、又は（Ｃ２７）を満たす抗体の例としては、後述の実施例に記載された「２０１００９－３－Ｈ」が挙げられる。 The heavy chain variable region identified by (C27) (SEQ ID NO: 267) contains heavy chain CDRs 1-3 (SEQ ID NOs: 261-263) identified by (A27) or (AB27). The light chain variable region identified by (C27) (SEQ ID NO: 269) contains light chain CDRs 1-3 (SEQ ID NOs: 264-266) identified by (B27) or (AB27). An example of an antibody that satisfies (A27), (B27), (AB27), or (C27) is "201009-3-H," which is described in the Examples below.

　（Ｃ２８）で特定される重鎖可変領域（配列番号２７７）は、（Ａ２８）又は（ＡＢ２８）で特定される重鎖ＣＤＲ１～３（配列番号２７１～２７３）を含む。（Ｃ２８）で特定される軽鎖可変領域（配列番号２７９）は、（Ｂ２８）又は（ＡＢ２８）で特定される軽鎖ＣＤＲ１～３（配列番号２７４～２７６）を含む。（Ａ２８）、（Ｂ２８）、（ＡＢ２８）、又は（Ｃ２８）を満たす抗体の例としては、後述の実施例に記載された「２０１００９－１－Ｃ」が挙げられる。 The heavy chain variable region (SEQ ID NO: 277) identified by (C28) contains heavy chain CDRs 1-3 (SEQ ID NOs: 271-273) identified by (A28) or (AB28). The light chain variable region (SEQ ID NO: 279) identified by (C28) contains light chain CDRs 1-3 (SEQ ID NOs: 274-276) identified by (B28) or (AB28). An example of an antibody that satisfies (A28), (B28), (AB28), or (C28) is "201009-1-C," which is described in the Examples below.

　配列番号１、配列番号３１、配列番号４１、配列番号５１、配列番号６１、配列番号７１、配列番号１１１、配列番号１２１、配列番号１３１、配列番号１６１、配列番号２５１のアミノ酸配列は同じである。配列番号８１、配列番号１０１のアミノ酸配列は同じである。配列番号１７１、配列番号１８１のアミノ酸配列は同じである。配列番号１９１、配列番号２０１、配列番号２１１のアミノ酸配列は同じである。配列番号２２１、配列番号２３１、配列番号２４１、配列番号２６１のアミノ酸配列は同じである。 The amino acid sequences of SEQ ID NO:1, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:111, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO:161, and SEQ ID NO:251 are the same. The amino acid sequences of SEQ ID NO:81 and SEQ ID NO:101 are the same. The amino acid sequences of SEQ ID NO:171 and SEQ ID NO:181 are the same. The amino acid sequences of SEQ ID NO:191, SEQ ID NO:201, and SEQ ID NO:211 are the same. The amino acid sequences of SEQ ID NO:221, SEQ ID NO:231, SEQ ID NO:241, and SEQ ID NO:261 are the same.

　配列番号２、配列番号１２、配列番号７２、配列番号１３２、配列番号１４２、配列番号１６２、配列番号１８２、配列番号１９２、配列番号２２２のアミノ酸配列は同じである。配列番号２２、配列番号３２、配列番号６２、配列番号２０２のアミノ酸配列は同じである。配列番号４２、配列番号５２のアミノ酸配列は同じである。配列番号１５２、配列番号２１２のアミノ酸配列は同じである。配列番号１７２、配列番号２３２、配列番号２６２のアミノ酸配列は同じである。 The amino acid sequences of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:72, SEQ ID NO:132, SEQ ID NO:142, SEQ ID NO:162, SEQ ID NO:182, SEQ ID NO:192, and SEQ ID NO:222 are the same. The amino acid sequences of SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:62, and SEQ ID NO:202 are the same. The amino acid sequences of SEQ ID NO:42 and SEQ ID NO:52 are the same. The amino acid sequences of SEQ ID NO:152 and SEQ ID NO:212 are the same. The amino acid sequences of SEQ ID NO:172, SEQ ID NO:232, and SEQ ID NO:262 are the same.

　配列番号３、配列番号１３のアミノ酸配列は同じである。配列番号６３、配列番号１２３、配列番号１５３、配列番号１６３、配列番号１７３、配列番号１８３、配列番号１９３、配列番号２０３、配列番号２１３、配列番号２４３のアミノ酸配列は同じである。配列番号８３、配列番号９３のアミノ酸配列は同じである。配列番号１１３、配列番号１３３、配列番号１４３、配列番号２２３、配列番号２３３、配列番号２５３、配列番号２６３のアミノ酸配列は同じである。 The amino acid sequences of SEQ ID NO:3 and SEQ ID NO:13 are the same. The amino acid sequences of SEQ ID NO:63, SEQ ID NO:123, SEQ ID NO:153, SEQ ID NO:163, SEQ ID NO:173, SEQ ID NO:183, SEQ ID NO:193, SEQ ID NO:203, SEQ ID NO:213, and SEQ ID NO:243 are the same. The amino acid sequences of SEQ ID NO:83 and SEQ ID NO:93 are the same. The amino acid sequences of SEQ ID NO:113, SEQ ID NO:133, SEQ ID NO:143, SEQ ID NO:223, SEQ ID NO:233, SEQ ID NO:253, and SEQ ID NO:263 are the same.

　配列番号４４、配列番号５４のアミノ酸配列は同じである。配列番号６４、配列番号１２４のアミノ酸配列は同じである。配列番号７４、配列番号１４４、配列番号２２４、配列番号２５４、配列番号２６４のアミノ酸配列は同じである。配列番号８４、配列番号９４、配列番号１０４のアミノ酸配列は同じである。配列番号１５４、配列番号１６４のアミノ酸配列は同じである。配列番号１７４、配列番号１８４、配列番号２０４、配列番号２１４のアミノ酸配列は同じである。 The amino acid sequences of SEQ ID NO:44 and SEQ ID NO:54 are the same. The amino acid sequences of SEQ ID NO:64 and SEQ ID NO:124 are the same. The amino acid sequences of SEQ ID NO:74, SEQ ID NO:144, SEQ ID NO:224, SEQ ID NO:254 and SEQ ID NO:264 are the same. The amino acid sequences of SEQ ID NO:84, SEQ ID NO:94 and SEQ ID NO:104 are the same. The amino acid sequences of SEQ ID NO:154 and SEQ ID NO:164 are the same. The amino acid sequences of SEQ ID NO:174, SEQ ID NO:184, SEQ ID NO:204 and SEQ ID NO:214 are the same.

　配列番号５、配列番号１５のアミノ酸配列は同じである。配列番号２５、配列番号１４５、配列番号１７５、配列番号１８５、配列番号２６５のアミノ酸配列は同じである。配列番号４５、配列番号５５のアミノ酸配列は同じである。配列番号６５、配列番号１２５、配列番号１３５のアミノ酸配列は同じである。配列番号７５、配列番号２２５、配列番号２５５のアミノ酸配列は同じである。配列番号８５、配列番号９５、配列番号１０５のアミノ酸配列は同じである。配列番号１５５、配列番号１６５、配列番号２４５のアミノ酸配列は同じである。配列番号１９５、配列番号２０５、配列番号２１５のアミノ酸配列は同じである。 The amino acid sequences of SEQ ID NO:5 and SEQ ID NO:15 are the same. The amino acid sequences of SEQ ID NO:25, SEQ ID NO:145, SEQ ID NO:175, SEQ ID NO:185, and SEQ ID NO:265 are the same. The amino acid sequences of SEQ ID NO:45 and SEQ ID NO:55 are the same. The amino acid sequences of SEQ ID NO:65, SEQ ID NO:125, and SEQ ID NO:135 are the same. The amino acid sequences of SEQ ID NO:75, SEQ ID NO:225, and SEQ ID NO:255 are the same. The amino acid sequences of SEQ ID NO:85, SEQ ID NO:95, and SEQ ID NO:105 are the same. The amino acid sequences of SEQ ID NO:155, SEQ ID NO:165, and SEQ ID NO:245 are the same. The amino acid sequences of SEQ ID NO:195, SEQ ID NO:205, and SEQ ID NO:215 are the same.

　配列番号６、配列番号１６、配列番号４６、配列番号５６のアミノ酸配列は同じである。配列番号６６、配列番号７６，配列番号１３６、配列番号１９６、配列番号２０６、配列番号２１６、配列番号２２６、配列番号２４６、配列番号２６６のアミノ酸配列は同じである。配列番号９６、配列番号１０６のアミノ酸配列は同じである。配列番号１２６、配列番号１７６、配列番号１８６のアミノ酸配列は同じである。配列番号１４６、配列番号２３６、配列番号２５６のアミノ酸配列は同じである。配列番号１５６、配列番号１６６のアミノ酸配列は同じである。 The amino acid sequences of SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:46, and SEQ ID NO:56 are the same. The amino acid sequences of SEQ ID NO:66, SEQ ID NO:76, SEQ ID NO:136, SEQ ID NO:196, SEQ ID NO:206, SEQ ID NO:216, SEQ ID NO:226, SEQ ID NO:246, and SEQ ID NO:266 are the same. The amino acid sequences of SEQ ID NO:96 and SEQ ID NO:106 are the same. The amino acid sequences of SEQ ID NO:126, SEQ ID NO:176, and SEQ ID NO:186 are the same. The amino acid sequences of SEQ ID NO:146, SEQ ID NO:236, and SEQ ID NO:256 are the same. The amino acid sequences of SEQ ID NO:156 and SEQ ID NO:166 are the same.

　上記抗体は、抗体の機能性断片であってもよい。ここで「抗体の機能的断片」とは、抗体（すなわち免疫グロブリン）の部分断片であって、抗原に対する作用を少なくとも１つ保持するものを指す。前記部分断片の例としては、Ｆ（ａｂ’）２、Ｆａｂ、Ｆｖ、ジスルフィド結合Ｆｖ、一本鎖抗体（ｓｃＦｖ、ＶＨ－ＶＬ）、ＶＨ、及びこれらの重合体、並びに、これらと重鎖ＣＨ３領域との融合体が挙げられる。また、ＣＤＲ１、ＣＤＲ２、ＣＤＲ３等の各ＣＤＲ、及びこれらＣＤＲの連結体、並びに、これらＣＤＲ又はＣＤＲ連結体と重鎖ＣＨ３領域との融合体が挙げられる。すなわち本開示の抗体には、前記したような抗体の部分断片も含まれる。抗体の部分断片を「抗体断片」と称することもある。 The above-mentioned antibody may be a functional fragment of an antibody. Here, "functional fragment of an antibody" refers to a partial fragment of an antibody (i.e., an immunoglobulin) that retains at least one action against an antigen. Examples of such partial fragments include F(ab')2, Fab, Fv, disulfide-linked Fv, single-chain antibodies (scFv, VH-VL), VH, and polymers thereof, as well as fusions of these with heavy chain CH3 regions. Also included are CDRs such as CDR1, CDR2, and CDR3, conjugates of these CDRs, and fusions of these CDRs or CDR conjugates with heavy chain CH3 regions. In other words, the antibodies of the present disclosure also include partial fragments of antibodies such as those described above. Partial fragments of antibodies are sometimes referred to as "antibody fragments."

　上記抗体は、多重特異性抗体であってもよい。本態様の多重特異性抗体は、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合しないという第一の特異的結合性と、第一の特異的結合性とは異なる第二の特異的結合性を、少なくとも有する。第二の特異的結合性は、ヒトＣＸＣＲ３に対するものであってもよいし、他の標的分子に対するものであってもよい。多重特異性抗体の例としては、二重特異性抗体の一種であるダイアボディが挙げられる。 The above-mentioned antibody may be a multispecific antibody. The multispecific antibody of this embodiment specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, and has at least a first specific binding property that does not specifically bind to human vascular endothelial cells, and a second specific binding property that is different from the first specific binding property. The second specific binding property may be for human CXCR3, or may be for another target molecule. An example of a multispecific antibody is a diabody, which is a type of bispecific antibody.

　一態様では、上記多重特異性抗体は、２つ以上（例えば、２、３、４、５、またはそれ以上）の抗原結合ドメインを含む。この多重特異性抗体は、例えば、同一又は異なるエピトープで、２つ以上のＣＸＣＲ３分子に結合することができる。この多重特異性抗体は、例えば、ＣＸＣＲ３および少なくとも１つの他の抗原に、高い親和性で結合することができる。抗体の抗原結合部分は、抗原に特異的に結合する能力を保持する抗体の１つ以上のフラグメントを含むことができる。これらのフラグメントは、親抗体または親抗体の変異体からの重鎖および／または軽鎖可変領域を含むことができる。 In one aspect, the multispecific antibody comprises two or more (e.g., 2, 3, 4, 5, or more) antigen-binding domains. The multispecific antibody can bind to two or more CXCR3 molecules, for example, at the same or different epitopes. The multispecific antibody can bind to CXCR3 and at least one other antigen with high affinity, for example. The antigen-binding portion of the antibody can comprise one or more fragments of the antibody that retain the ability to specifically bind to the antigen. These fragments can comprise heavy and/or light chain variable regions from a parent antibody or a variant of the parent antibody.

　上記抗体のクラス（アイソタイプ）は特に限定されない。例えば、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ等、いずれのクラスであってもよい。さらに、上記抗体のサブクラスについても特に限定はなく、例えば、ＩｇＧであれば、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４等のいずれのサブクラスであってもよい。 The class (isotype) of the antibody is not particularly limited. For example, it may be any class, such as IgG, IgM, IgA, IgD, or IgE. Furthermore, the subclass of the antibody is not particularly limited. For example, if it is IgG, it may be any subclass, such as IgG1, IgG2, IgG3, or IgG4.

　一態様では、上記抗体は、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有する。ＣＸＣＲ３Ａ依存的な細胞機能は、３量体Ｇタンパク質やβアレスチン等の、ＣＸＣＲ３の細胞内部分に共役して細胞内情報伝達を担うタンパク質が活性化することによって誘発される機能である。ＣＸＣＲ３Ａ依存的な細胞機能の活性化は、ＣＸＣＲ３リガンドの刺激によって誘発される。例えば、細胞内カルシウムイオンの変動、細胞内Ｃｙｃｌｉｃ　ａｄｅｎｏｓｉｎｅ　ｍｏｎｏｐｈｏｓｐｈａｔｅ（ｃＡＭＰ）の変動、低分子量Ｇタンパク質ＲｈｏへのＧＴＰ結合、細胞走化性、受容体の内在化などが挙げられる。 In one aspect, the antibody has the activity of blocking CXCR3A-dependent cellular functions. CXCR3A-dependent cellular functions are induced by the activation of proteins that couple to the intracellular portion of CXCR3 and are responsible for intracellular signal transduction, such as trimeric G proteins and β-arrestins. Activation of CXCR3A-dependent cellular functions is induced by stimulation with a CXCR3 ligand. Examples include fluctuations in intracellular calcium ions, fluctuations in intracellular cyclic adenosine monophosphate (cAMP), GTP binding to the small G protein Rho, cell chemotaxis, and receptor internalization.

　一態様では、上記抗体は、ヒト血管内皮細胞に特異的に結合しない。後述の実施例で示すように、ヒト血管内皮細胞はＣＸＣＲ３Ｂを優位に発現する。 In one embodiment, the antibody does not specifically bind to human vascular endothelial cells. As shown in the examples below, human vascular endothelial cells predominantly express CXCR3B.

　上記抗体が特異的に結合するＣＸＣＲ３Ａの細胞外ドメインは、Ｎ末端ドメイン、細胞外第１ループドメイン、細胞外第２ループドメイン、細胞外第３ループドメインのいずれでもよい。また上記抗体は、これらの細胞外ドメインのいずれか１つのみに結合するものでもよいし、２つ以上に結合するものでもよい。 The extracellular domain of CXCR3A to which the above-mentioned antibody specifically binds may be any of the N-terminal domain, the extracellular first loop domain, the extracellular second loop domain, and the extracellular third loop domain. Furthermore, the above-mentioned antibody may bind to only one of these extracellular domains, or to two or more of them.

　本開示は、上記した抗ヒトＣＸＣＲ３抗体と「機能的に同等」な抗体を含む。機能的に同等な抗体には、上記抗体とエピトープが同一の抗体が含まれる。例えば、後述の実施例で具体的に示す２８種の抗ヒトＣＸＣＲ３抗体のエピトープを、ヒトＣＸＣＲ３の部分ペプチド等を用いたエピトープマッピング法により解析する。そして、同定されたエピトープを含む合成ペプチドを抗原として用い、上記２８種の抗ヒトＣＸＣＲ３抗体と同一のエピトープに結合する抗ヒトＣＸＣＲ３抗体を得ることができる。さらに、得られた抗ヒトＣＸＣＲ３抗体の重鎖可変領域と軽鎖可変領域のアミノ酸配列を決定し、重鎖ＣＤＲ１～３と軽鎖ＣＤＲ１～３のアミノ酸配列を特定することができる。 The present disclosure includes antibodies that are "functionally equivalent" to the above-mentioned anti-human CXCR3 antibodies. Functionally equivalent antibodies include antibodies that have the same epitope as the above-mentioned antibodies. For example, the epitopes of 28 anti-human CXCR3 antibodies specifically shown in the Examples below are analyzed by epitope mapping using partial peptides of human CXCR3, etc. Then, synthetic peptides containing the identified epitopes can be used as antigens to obtain anti-human CXCR3 antibodies that bind to the same epitopes as the above-mentioned 28 anti-human CXCR3 antibodies. Furthermore, the amino acid sequences of the heavy chain variable region and light chain variable region of the obtained anti-human CXCR3 antibody can be determined, and the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 can be identified.

　上記抗体と機能的に同等な抗体の一例として、本開示は、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合しない抗体であって、上記した（Ｃ１）～（Ｃ２８）で規定された配列番号のアミノ酸配列において、１～１０個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を有するもの、或いは、上記した（Ｃ１）～（Ｃ２８）で規定された配列番号のアミノ酸配列と９０％以上の同一性を有するもの、を含む。置換、付加、若しくは欠失した前記アミノ酸の数は、好ましくは１～８個、より好ましくは１～５個、特に好ましくは１～３個である。前記同一性は、好ましくは９２％以上、より好ましくは９５％以上、特に好ましくは９７％以上である。 As an example of an antibody functionally equivalent to the above antibody, the present disclosure provides an antibody that specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, and does not specifically bind to human vascular endothelial cells. The antibody includes an amino acid sequence having 1 to 10 amino acid substitutions, additions, or deletions in the amino acid sequence of the SEQ ID NOs defined in (C1) to (C28) above, or an antibody having 90% or greater identity to the amino acid sequence of the SEQ ID NOs defined in (C1) to (C28) above. The number of substituted, added, or deleted amino acids is preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3. The identity is preferably 92% or greater, more preferably 95% or greater, and particularly preferably 97% or greater.

　２つの抗体についてエピトープが同一か否かを調べる方法としては、競合実験による方法が挙げられる。例えば、第一抗体たる前記２８種の抗ヒトＣＸＣＲ３抗体又はその機能的断片と受容体との結合が、試験対象である第二抗体によって競合阻害を受ける場合には、当該第二抗体は、前記第一抗体と同じエピトープに結合するものであるといえる。そして、上記抗体と機能的に同等な抗体の一例として、本開示は、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合しない抗体（第二抗体）であって、上記抗体（第一抗体）と受容体との結合を競合阻害する抗体を含む。 A competitive experiment can be used to determine whether two antibodies have the same epitope. For example, if the binding of the 28 anti-human CXCR3 antibodies or functional fragments thereof, which serve as first antibodies, to the receptor is competitively inhibited by the second antibody being tested, then the second antibody can be said to bind to the same epitope as the first antibody. As an example of an antibody functionally equivalent to the above antibody, the present disclosure includes an antibody (second antibody) that specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and competitively inhibits the binding of the above antibody (first antibody) to the receptor.

　本明細書で開示されたＣＤＲ及びその他の領域（フレームワーク領域や定常領域など）は、それぞれを独立して選択し、特定の抗体の他のＣＤＲ又はフレームワーク領域とさまざまな組み合わせで組み合わせることができる。特定の実施形態では、ＶＨ及び/又はＶＬ、ＣＤＲ及びフレームワーク配列は、ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、ヒト血管内皮細胞に特異的に結合しない抗体において任意の組み合わせで存在することができる。 The CDRs and other regions (e.g., framework regions and constant regions) disclosed herein can each be selected independently and combined in various combinations with other CDRs or framework regions of a particular antibody. In certain embodiments, the VH and/or VL, CDR and framework sequences can be present in any combination in an antibody that specifically binds to the extracellular domain of human CXCR3A, has activity to block CXCR3A-dependent cellular functions, and does not specifically bind to human vascular endothelial cells.

＜ヒト化抗体＞
　本開示は、ヒト化抗体である上記抗体を含む。ヒト化抗体とは、ＣＤＲがヒト以外の動物由来のもので、その他の領域（フレームワーク領域や定常領域など）がヒト由来のものである抗体を指す。ヒト化抗体を構築及び生産する方法については後述する。 <Humanized antibody>
The present disclosure includes the above antibodies that are humanized. A humanized antibody is an antibody in which the CDRs are derived from a non-human animal and the other regions (such as framework and constant regions) are derived from humans. Methods for constructing and producing humanized antibodies are described below.

＜核酸＞
　本開示は、上記抗体をコードする核酸（ＤＮＡ）を含む。当該核酸は、例えば、重鎖可変領域をコードする第一核酸、及び／又は、軽鎖可変領域をコードする第二核酸を含む。 <Nucleic acid>
The present disclosure includes nucleic acids (DNA) encoding the antibodies, including, for example, a first nucleic acid encoding a heavy chain variable region and/or a second nucleic acid encoding a light chain variable region.

　第一核酸がコードする重鎖可変領域は、例えば、配列番号１、１１、２１、３１、４１、５１、６１、７１、８１、９１、１０１、１１１、１２１、１３１、１４１、１５１、１６１、１７１、１８１、１９１、２０１、２１１、２２１、２３１、２４１、２５１、２６１又は２７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１；配列番号２、１２、２２、３２、４２、５２、６２、７２、８２、９２、１０２、１１２、１２２、１３２、１４２、１５２、１６２、１７２、１８２、１９２、２０２、２１２、２２２、２３２、２４２、２５２、２６２又は２７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２；及び、配列番号３、１３、２３、３３、４３、５３、６３、７３、８３、９３、１０３、１１３、１２３、１３３、１４３、１５３、１６３、１７３、１８３、１９３、２０３、２１３、２２３、２３３、２４３、２５３、２６３又は２７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、を含む。第一核酸がコードする重鎖可変領域は、例えば、上記（Ａ１）～（Ａ２８）のいずれかで特定される重鎖ＣＤＲ１～３を含む。第一核酸がコードする重鎖可変領域は、例えば、上記（Ｃ１）～（Ｃ２８）のいずれかで特定されるものである。 The heavy chain variable region encoded by the first nucleic acid may, for example, include a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, or 271; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263, or 273. The heavy chain variable region encoded by the first nucleic acid comprises, for example, heavy chain CDR1 to CDR3 specified by any of (A1) to (A28) above. The heavy chain variable region encoded by the first nucleic acid is, for example, one specified by any of (C1) to (C28) above.

　第二核酸がコードする軽鎖可変領域は、例えば、配列番号４、１４、２４、３４、４４、５４、６４、７４、８４、９４、１０４、１１４、１２４、１３４、１４４、１５４、１６４、１７４、１８４、１９４、２０４、２１４、２２４、２３４、２４４、２５４、２６４又は２７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１；配列番号５、１５、２５、３５、４５、５５、６５、７５、８５、９５、１０５、１１５、１２５、１３５、１４５、１５５、１６５、１７５、１８５、１９５、２０５、２１５、２２５、２３５、２４５、２５５、２６５又は２７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２；及び、配列番号６、１６、２６、３６、４６、５６、６６、７６、８６、９６、１０６、１１６、１２６、１３６、１４６、１５６、１６６、１７６、１８６、１９６、２０６、２１６、２２６、２３６、２４６、２５６、２６６又は２７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３、を含む。第二核酸がコードする軽鎖可変領域は、例えば、上記（Ｂ１）～（Ｂ２８）のいずれかで特定される重鎖ＣＤＲ１～３を含む。第二核酸がコードする軽鎖可変領域は、例えば、上記（Ｃ１）～（Ｃ２８）のいずれかで特定されるものである。 The light chain variable region encoded by the second nucleic acid may, for example, include a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, or 274; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, or 276. The light chain variable region encoded by the second nucleic acid comprises, for example, heavy chain CDR1 to CDR3 specified in any of (B1) to (B28) above. The light chain variable region encoded by the second nucleic acid is, for example, one specified in any of (C1) to (C28) above.

　上記核酸は、ベクターに組み込まれていてもよい。ベクターは、それが導入される宿主細胞の種類等によって適宜選択される。ベクターには遺伝子治療用ベクターが含まれる。この場合には、ベクターそのものを生体内に直接投与可能である。 The above nucleic acid may be incorporated into a vector. The vector is selected appropriately depending on the type of host cell into which it will be introduced. Vectors include vectors for gene therapy. In this case, the vector itself can be administered directly into the body.

＜抗体の製造方法＞
　上記抗体は、遺伝子組換えの手法を用いて製造することができる。すなわち、上記核酸を発現する組換え細胞を構築し、当該細胞の培養物から上記抗体を取得することができる。 <Method of producing antibodies>
The antibody can be produced using genetic recombination techniques, i.e., by constructing recombinant cells that express the nucleic acid, the antibody can be obtained from a culture of the cells.

＜ヒト化抗体の構築及び調製＞
　ヒト化抗体を構築及び生産する方法について説明する。一例として、上記（ＡＢ１）で特定される重鎖ＣＤＲ１～３及び軽鎖ＣＤＲ１～３を有するヒト化抗体を構築及び生産する方法について述べる。まず、各ＣＤＲをコードするＤＮＡとして、配列番号１～６に示すアミノ酸配列をコードするＤＮＡを調製する。ＤＮＡの調製は、ＰＣＲ等の公知の方法を用いて行うことができる。化学合成により当該ＤＮＡを調製することもできる。 Construction and preparation of humanized antibodies
Methods for constructing and producing humanized antibodies will be described. As an example, a method for constructing and producing a humanized antibody having heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 as specified in (AB1) above will be described. First, DNAs encoding the amino acid sequences shown in SEQ ID NOS: 1 to 6 are prepared as DNAs encoding each CDR. DNAs can be prepared using known methods such as PCR. The DNAs can also be prepared by chemical synthesis.

　次に、これらのＤＮＡを用いて、任意のヒト抗体におけるＶＨのフレームワーク領域に重鎖ＣＤＲ１～３が移植された可変領域をコードするＤＮＡを作製する。同様に、任意のヒト抗体におけるＶＬのフレームワークに軽鎖ＣＤＲ１～３が移植された可変領域をコードするＤＮＡを作製する。作製した各ＤＮＡを、ヒト抗体のＣＨ又はＣＬをコードする配列を有するベクターに挿入し、ヒト化抗体発現ベクターを構築する。構築した発現ベクターを宿主細胞に導入することにより、ヒト化抗体を発現する組換え細胞を得る。そして、この組換え細胞を培養し、当該培養物から所望のヒト化抗体を取得する。 Next, these DNAs are used to prepare DNAs encoding variable regions in which heavy chain CDRs 1 to 3 are grafted onto the framework region of VH of any human antibody. Similarly, DNAs encoding variable regions in which light chain CDRs 1 to 3 are grafted onto the framework region of VL of any human antibody are prepared. Each of the prepared DNAs is inserted into a vector containing a sequence encoding the CH or CL of a human antibody to construct a humanized antibody expression vector. The constructed expression vector is introduced into host cells to obtain recombinant cells that express the humanized antibody. These recombinant cells are then cultured, and the desired humanized antibody is obtained from the culture.

　上記（ＡＢ２）～（ＡＢ２８）で特定される重鎖ＣＤＲ１～３及び軽鎖ＣＤＲ１～３を有するヒト化抗体についても、同様の方法で構築及び生産することができる。 Humanized antibodies having heavy chain CDR1-3 and light chain CDR1-3 specified above in (AB2) to (AB28) can also be constructed and produced in a similar manner.

　キメラ型抗体を構築及び生産する方法について説明する。一例として、上記（Ｃ１）で特定される重鎖可変領域（ＶＨ）と軽鎖可変領域（ＶＬ）を有するキメラ型抗体を構築及び生産する方法について述べる。まず、ＶＨをコードするＤＮＡとして、配列番号７に示すアミノ酸配列をコードするＤＮＡを調製する。また、ＶＬをコードするＤＮＡとして、配列番号９に示すアミノ酸配列をコードするＤＮＡを調製する。ＤＮＡの調製は、ＰＣＲ等の公知の方法を用いて行うことができる。化学合成により当該ＤＮＡを調製することもできる。 A method for constructing and producing a chimeric antibody will be described. As an example, a method for constructing and producing a chimeric antibody having a heavy chain variable region (VH) and a light chain variable region (VL) as specified in (C1) above will be described. First, DNA encoding the amino acid sequence shown in SEQ ID NO: 7 is prepared as DNA encoding VH. Furthermore, DNA encoding the amino acid sequence shown in SEQ ID NO: 9 is prepared as DNA encoding VL. DNA can be prepared using known methods such as PCR. The DNA can also be prepared by chemical synthesis.

　得られたＶＨ又はＶＬをコードするＤＮＡを、ヒト抗体のＣＨ又はＣＬをコードする配列を有するベクターにそれぞれ挿入し、キメラ型抗体発現ベクターを構築する。なお、ヒト抗体のＣＨ又はＣＬをコードする配列を有するベクターは、市場から入手できる。構築した発現ベクターを宿主細胞に導入することにより、キメラ型抗体を発現する組換え細胞を得る。そして、この組換え細胞を培養し、当該培養物から所望のキメラ型抗体を取得する。 The resulting DNA encoding the VH or VL is inserted into a vector containing a sequence encoding the CH or CL of a human antibody, respectively, to construct a chimeric antibody expression vector. Note that vectors containing a sequence encoding the CH or CL of a human antibody are commercially available. By introducing the constructed expression vector into a host cell, recombinant cells that express the chimeric antibody are obtained. These recombinant cells are then cultured, and the desired chimeric antibody is obtained from the culture.

　上記（Ｃ２）～（Ｃ２８）で特定されるＶＨ及びＶＬを有するキメラ型抗体についても、同様の方法で構築及び生産することができる。 Chimeric antibodies having the VH and VL specified above in (C2) to (C28) can also be constructed and produced in a similar manner.

＜多重特異性抗体の製法＞
　多重特異性抗体を製造する方法としては、例えば、本開示の抗ヒトＣＸＣＲ３抗体又はその断片と、別の抗体又はその断片を、連結させることが挙げられる。他の例として、本開示の抗ヒトＣＸＣＲ３抗体又はその断片と、別の抗体又はその断片を、宿主細胞内で共に同時発現させることが挙げられる。別の連結の方法としては、化学的カップリング、遺伝子融合、非共有結合性会合、等が挙げられる。 <Method for producing multispecific antibodies>
Methods for producing multispecific antibodies include, for example, linking an anti-human CXCR3 antibody or a fragment thereof of the present disclosure to another antibody or a fragment thereof. Another example includes co-expressing an anti-human CXCR3 antibody or a fragment thereof of the present disclosure with another antibody or a fragment thereof in a host cell. Other linking methods include chemical coupling, gene fusion, non-covalent association, etc.

　なお、実用化された多重特異性抗体（二重特異性抗体）の例としては、ＣＤ３とＣＤ１９に結合するビーリンサイト（登録商標）が挙げられる。他の例としては、凝固因子ＩＸａと凝固因子Ｘに結合するヘムライブラ（登録商標）が挙げられる。多重特異性抗体の製造技術は当該技術分野では公知であり、これら公知の製造技術は、本開示の抗ヒトＣＸＣＲ３抗体にも適用できる。 An example of a multispecific antibody (bispecific antibody) that has been put into practical use is Blincyto (registered trademark), which binds to both CD3 and CD19. Another example is Hemlibra (registered trademark), which binds to both coagulation factor IXa and coagulation factor X. Techniques for producing multispecific antibodies are known in the art, and these known production techniques can also be applied to the anti-human CXCR3 antibody of the present disclosure.

＜精製方法＞
　上記抗体の精製方法としては特に限定はなく、公知の手法を採用することができる。例えば、前記組換え細胞の培養上清を回収し、各種クロマトグラフィー、塩析、透析、膜分離等の公知の手法を組み合わせて、上記抗体を精製することができる。例えば、抗体のアイソタイプがＩｇＧである場合には、プロテインＡを用いたアフィニティクロマトグラフィーによって簡便に精製することもできる。 <Purification method>
The method for purifying the antibody is not particularly limited, and known methods can be used. For example, the culture supernatant of the recombinant cell can be collected and purified by combining known methods such as various types of chromatography, salting out, dialysis, and membrane separation. For example, when the antibody isotype is IgG, it can be easily purified by affinity chromatography using protein A.

＜修飾抗体＞
　本開示の抗体は、他の分子が結合した修飾抗体であってもよい。他の分子の例としては、ペプチド、タンパク質、低分子化合物、放射性同位体、光吸収体、などが挙げられる。他の分子と結合させる方法としては、化学的カップリング、遺伝子融合、非共有結合性会合、等が挙げられる。なお、結合させる他の分子が抗体である場合には、その修飾抗体は、多重特異性抗体となり得る。この点において、多重特異性抗体は、修飾抗体の一種と捉えることができる。 <Modified antibody>
The antibody of the present disclosure may be a modified antibody to which another molecule is bound. Examples of the other molecule include peptides, proteins, small molecules, radioisotopes, light absorbers, etc. Methods for binding to the other molecule include chemical coupling, gene fusion, non-covalent association, etc. Note that when the other molecule to be bound is an antibody, the modified antibody may be a multispecific antibody. In this respect, a multispecific antibody can be considered a type of modified antibody.

　好ましい態様では、上記修飾抗体が抗体薬物複合体（Ａｎｔｉｂｏｄｙ　Ｄｒｕｇ　Ｃｏｎｊｕｇａｔｅ；ＡＤＣ）である。コンジュゲートさせる薬物としては、抗ウイルス薬；免疫抑制剤；免疫調節薬；サイトカイン等の生物学的活性を有するタンパク質又はポリペプチド；放射性同位体；光吸収体、などが挙げられる。これらの薬物を直接的に、又はリンカー若しくはキレート剤を介して間接的にコンジュゲートして、抗体薬物複合体を製造することができる。 In a preferred embodiment, the modified antibody is an antibody-drug conjugate (ADC). Drugs to be conjugated include antiviral drugs; immunosuppressants; immunomodulators; proteins or polypeptides with biological activity such as cytokines; radioisotopes; and light absorbers. Antibody-drug conjugates can be produced by conjugating these drugs directly or indirectly via a linker or chelator.

＜医薬＞
　本開示は、上記抗体を有効成分として含有する医薬を包含する。当該医薬は、上記抗体と、薬学的に許容される担体とを含む医薬組成物であり得る。好ましくは、上記医薬は、ＣＸＣＲ３Ａ依存的な細胞機能を遮断するものである。 <Pharmaceuticals>
The present disclosure includes a pharmaceutical comprising the antibody as an active ingredient. The pharmaceutical may be a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical blocks CXCR3A-dependent cellular functions.

　抗体のＣＸＣＲ３Ａ依存的な細胞機能の遮断の様式としては、抗体が細胞表面のリガンド結合部位に結合し、リガンドの受容体結合を競合的または非競合的に阻害するもの、抗体がリガンド結合部位以外の部位に結合し、受容体構造を変化させることにより活性化を阻害するもの、抗体と受容体の結合が受容体の内在化を誘発し、細胞表面における受容体発現量を低下させることにより細胞のリガンド応答性を低減させるもの、などが挙げられる。受容体の内在化を誘発する抗体は、ＡＤＣにおいても活用される。好ましい態様では、上記抗体は、内在化活性を有する。換言すれば、受容体の内在化を誘発する。 The modes by which antibodies block CXCR3A-dependent cellular functions include antibodies that bind to the ligand-binding site on the cell surface and competitively or non-competitively inhibit ligand binding to the receptor, antibodies that bind to a site other than the ligand-binding site and inhibit activation by altering the receptor structure, and antibodies that induce receptor internalization through binding to the receptor, thereby reducing the amount of receptor expression on the cell surface and thereby reducing the ligand responsiveness of the cell. Antibodies that induce receptor internalization are also used in ADCs. In a preferred embodiment, the above-mentioned antibodies have internalization activity. In other words, they induce receptor internalization.

　好ましい態様では、上記医薬は、ＣＸＣＲ３依存的な細胞機能の障害が原因として関与している疾患、障害又は病状の治療に用いられる。ＣＸＣＲ３依存的な細胞機能の障害が原因として関与している疾患、障害又は病状の例としては、アテローム性動脈硬化症、心筋炎、多発性硬化症、喘息、慢性閉塞性肺疾患、肺線維症、１型糖尿病、乾癬、リウマチ、炎症性腸炎、全身性エリテマトーデス、急性心臓同種移植片拒絶反応などのＴｈ１関連疾患、インフルエンザ、ＣＯＶＩＤ－１９などの感染症や腫瘍等がある。 In a preferred embodiment, the above-mentioned pharmaceutical is used to treat a disease, disorder, or condition caused by impaired CXCR3-dependent cellular function. Examples of diseases, disorders, or conditions caused by impaired CXCR3-dependent cellular function include atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatism, inflammatory bowel disease, systemic lupus erythematosus, Th1-related diseases such as acute cardiac allograft rejection, influenza, infectious diseases such as COVID-19, and tumors.

　特に、炎症細胞の浸潤、増殖、活性化を抑制することを目的とする治療においては、ＣＸＣＲ３Ａを選択的に阻害する薬剤の有用性が高いと考えられる。患者におけるＣＸＣＲ３Ａリガンドの増加は疾患によって特異的である場合があり、例えば、自己免疫性大腸炎、１型糖尿病、急性肺障害においてはＣＸＣＬ１０、多発性硬化症においてはＣＸＣＬ９とＣＸＣＬ１０、アテローム動脈硬化症においては、ＣＸＣＬ９、ＣＸＣＬ１０、ＣＸＣＬ１１が増加することが報告されている（Meri K. Tulic et al., Nat Commun. 2019 May 16;10(1):2178.）。炎症性疾患におけるＣＸＣＲ３Ｂ‐リガンド相互作用の役割に関する知見は少なく、ＣＸＣＲ３ＡとＣＸＣＲ３Ｂの両方を阻害する薬剤においては、ＣＸＣＲ３Ｂを阻害することによる予期せぬ副作用を引き起こす可能性を否定できない。 In particular, drugs that selectively inhibit CXCR3A are thought to be highly useful in treatments aimed at suppressing the infiltration, proliferation, and activation of inflammatory cells. Increases in CXCR3A ligands in patients can be disease-specific. For example, increases in CXCL10 have been reported in autoimmune colitis, type 1 diabetes, and acute lung injury; CXCL9 and CXCL10 in multiple sclerosis; and CXCL9, CXCL10, and CXCL11 in atherosclerosis (Meri K. Tulic et al., Nat Commun. 2019 May 16;10(1):2178). Knowledge about the role of CXCR3B-ligand interactions in inflammatory diseases is limited, and drugs that inhibit both CXCR3A and CXCR3B may cause unexpected side effects due to CXCR3B inhibition.

　また、いくつかの腫瘍においては、ＣＸＣＲ３Ａを選択的に阻害する薬剤の有用性が高いと考えられる。大腸癌、胃癌、膵臓癌、乳癌、前立腺癌、腎癌、卵巣癌、メラノーマ、膠芽腫、多発性骨髄腫などは、ＣＸＣＲ３の発現と予後悪化の関連性が示唆されている（Xiaoming Wang et al., Front Oncol. 2022 Nov 21:12:1022688.）。ＣＸＣＲ３Ａ‐リガンド相互作用は腫瘍増殖、転移を促進し、ＣＸＣＲ３Ｂ‐リガンド相互作用は抗腫増殖、血管新生阻害、アポトーシスを促進する。したがって、ＣＸＣＲ３Ａ特異的なＣＸＣＲ３阻害剤は、ＣＸＣＲ３Ｂの抗腫瘍効果を維持しながら腫瘍の成長、転移を抑制し、より強力な抗腫瘍効果を持つ可能性がある。ＣＸＣＲ３ＢよりもＣＸＣＲ３Ａを優先的に阻害する阻害剤として、ＳＣＨ５４６７３８が知られているが、ＣＸＣＲ３Ａ特異的阻害剤の報告はない（K. Boye et al., Sci Rep. 2017 Sep 6;7(1):10703.）。 In addition, drugs that selectively inhibit CXCR3A are thought to be highly effective in some tumors. CXCR3 expression has been suggested to be associated with poor prognosis in colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, renal cancer, ovarian cancer, melanoma, glioblastoma, and multiple myeloma (Xiaoming Wang et al., Front Oncol. 2022 Nov 21:12:1022688). CXCR3A-ligand interaction promotes tumor growth and metastasis, while CXCR3B-ligand interaction promotes anti-tumor growth, angiogenesis inhibition, and apoptosis. Therefore, CXCR3A-specific CXCR3 inhibitors may have a more potent anti-tumor effect, suppressing tumor growth and metastasis while maintaining the anti-tumor effects of CXCR3B. SCH546738 is known to be an inhibitor that preferentially inhibits CXCR3A over CXCR3B, but no CXCR3A-specific inhibitors have been reported (K. Boye et al., Sci Rep. 2017 Sep 6;7(1):10703).

　一方、白斑においては、ＣＸＣＲ３ＡおよびＣＸＣＲ３Ｂの両方を阻害する薬剤が、より高い治療効果を発揮する可能性がある。白斑においては、ＣＸＣＲ３Ａを介して病原性Ｔ細胞の遊走が刺激され、ＣＸＣＲ３Ｂを介してメラノサイトのアポトーシスが促進される（Meri K. Tulic et al., Nat Commun. 2019 May 16;10(1):2178.）。 On the other hand, in vitiligo, drugs that inhibit both CXCR3A and CXCR3B may have a greater therapeutic effect. In vitiligo, migration of pathogenic T cells is stimulated via CXCR3A, and apoptosis of melanocytes is promoted via CXCR3B (Meri K. Tulic et al., Nat Commun. 2019 May 16;10(1):2178.).

＜投与方法＞
　上記医薬は、経口あるいは非経口的に、全身あるいは局部的に投与することができる。投与の形態としては、注射剤型、経鼻投与剤型、経肺投与剤型、経皮投与型などが挙げられる。注射剤型の場合には、例えば、静脈内注射、筋肉内注射、腹腔内注射、皮下注射などにより、全身又は局部的に投与することができる。また、患者の年齢、症状により、適宜、投与方法を選択することができる。上記抗体の投与量としては、例えば、一回につき体重１ｋｇあたり０．０００１ｍｇから１０００ｍｇの範囲で選ぶことができる。あるいは、例えば、患者あたり抗体０．００１～１０００００ｍｇ／ｂｏｄｙの範囲で投与量を選ぶことができる。しかしながら、上記抗体の投与量は、これらの範囲に限定されるものではない。 <Administration method>
The above-mentioned pharmaceuticals can be administered orally or parenterally, systemically or locally. Examples of administration forms include injections, intranasal administrations, pulmonary administrations, and transdermal administrations. Injections can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. The administration method can be selected appropriately depending on the age and symptoms of the patient. The dosage of the above-mentioned antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg of antibody per patient. However, the dosage of the above-mentioned antibody is not limited to these ranges.

　＜製剤化＞
　上記医薬は、常法に従って製剤化することができる（例えば、Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A）。上記医薬には、薬学的に許容される担体や添加物を含めることができる。前記担体あるいは前記添加物の例としては、界面活性剤（ＰＥＧ、Ｔｗｅｅｎ等）、賦形剤、酸化防止剤（アスコルビン酸等）、着色料、着香料、保存料、安定剤、緩衝剤（リン酸、クエン酸、他の有機酸等）、キレート剤（ＥＤＴＡ等）、懸濁剤、等張化剤、結合剤、崩壊剤、滑沢剤、流動性促進剤、矯味剤等が挙げられるが、これらに限定されず、その他常用の担体等を適宜使用することができる。具体的には、軽質無水ケイ酸、乳糖、結晶セルロース、マンニトール、デンプン、カルメロースカルシウム、カルメロースナトリウム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルアセタールジエチルアミノアセテート、ポリビニルピロリドン、ゼラチン、中鎖脂肪酸トリグリセライド、ポリオキシエチレン硬化ヒマシ油６０、白糖、カルボキシメチルセルロース、コーンスターチ、無機塩類等を挙げることができる。また、その他の低分子量のポリペプチド；血清アルブミン、ゼラチン、免疫グロブリン等のタンパク質；グリシン、グルタミン、アスパラギン、アルギニン、リシン等のアミノ酸、を含んでいてもよい。 <Formulation>
The above-mentioned pharmaceuticals can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). The above-mentioned pharmaceuticals can contain pharmaceutically acceptable carriers and additives. Examples of the carriers and additives include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphate, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents, but are not limited to these. Other commonly used carriers and the like can also be used as appropriate. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. Furthermore, the composition may contain other low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, immunoglobulins; and amino acids such as glycine, glutamine, asparagine, arginine, lysine, etc.

　注射用の水溶液とする場合には、例えば生理食塩水、ブドウ糖やその他の補助薬を含む等張液、例えば、Ｄ－ソルビトール、Ｄ－マンノース、Ｄ－マンニトール、塩化ナトリウムが挙げられ、適当な溶解補助剤、例えばアルコール（エタノール等）、ポリアルコール（プロピレングリコール、ＰＥＧ等）、非イオン性界面活性剤（ポリソルベート８０、ＨＣＯ－５０）等と併用してもよい。また必要に応じて、上記抗体をマイクロカプセル（ヒドロキシメチルセルロース、ゼラチン、ポリ［メチルメタクリル酸］等のマイクロカプセル）に封入したり、コロイドドラッグデリバリーシステム（リポソーム、アルブミンミクロスフェア、マイクロエマルジョン、ナノ粒子及びナノカプセル等）とすることもできる（"Remingto's Pharmaceutical Science 16th edition", Oslo Ed. 1980)等参照）。 When preparing an aqueous solution for injection, examples include isotonic solutions containing physiological saline, glucose, or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, or sodium chloride. These may be used in combination with appropriate solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), or nonionic surfactants (polysorbate 80, HCO-50). If necessary, the antibody can also be encapsulated in microcapsules (such as hydroxymethylcellulose, gelatin, or poly(methyl methacrylate)), or in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remingto's Pharmaceutical Science 16th Edition," Oslo, Ed. 1980).

　さらに、薬剤を徐放させる技術が知られており、上記医薬に適用し得る（Langer et al., J. Biomed. Master. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:98-105 (1982)；米国特許第３，７７３，９１９号；欧州特許出願公開第５８，４８１号；Sidman et al., Biopolymers 22: 547-556 (1983)；欧州特許出願公開第１３３，９８８号）。 Furthermore, techniques for sustained drug release are known and may be applied to the above-mentioned pharmaceuticals (Langer et al., J. Biomed. Master. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:98-105 (1982); U.S. Patent No. 3,773,919; European Patent Application Publication No. 58,481; Sidman et al., Biopolymers 22:547-556 (1983); European Patent Application Publication No. 133,988).

＜遺伝子治療への応用＞
　上記核酸を遺伝子治療用ベクターあるいは生体内でタンパク質に翻訳可能なｍＲＮＡに組込み、遺伝子治療薬とすることも考えられる。当該遺伝子治療薬（組換えベクター）の投与方法としては、ｎａｋｅｄプラスミドによる直接投与の他、リポソーム等にパッケージングして投与する方法、レトロウイルスベクター、アデノウイルスベクター、ワクシニアウイルスベクター、ポックスウイルスベクター、アデノ随伴ウイルスベクター、ＨＶＪベクター等の各種ウイルスベクターに組み込んで投与する方法（Adolph『ウイルスゲノム法』，CRC Press, Florid (1996)参照）、コロイド金粒子等のビーズ担体に被覆（国際公開第９３／１７７０６号パンフレット等）して投与する方法、等が挙げられる。 <Application to gene therapy>
The nucleic acid may be incorporated into a gene therapy vector or mRNA that can be translated into a protein in vivo to produce a gene therapy drug. Methods for administering the gene therapy drug (recombinant vector) include direct administration using a naked plasmid, administration by packaging the nucleic acid in a liposome or the like, administration by incorporating the nucleic acid into various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, and HVJ vectors (see Adolph, "Viral Genome Methods," CRC Press, Florid (1996)), and administration by coating the nucleic acid on a bead carrier such as colloidal gold particles (e.g., WO 93/17706).

　前記遺伝子治療薬は、生体内において上記抗体が発現され、その作用を発揮できる限り、いかなる方法により投与してもよい。好ましくは、適当な非経口経路、例えば、静脈内、腹腔内、皮下、皮内、脂肪組織内、乳腺組織内、吸入、筋肉内、等の経路を介して、注射、注入、ガス誘導性粒子衝撃法（電子銃等による）、添鼻薬等粘膜経路を介する方法、等により十分な量が投与される。さらに、前記遺伝子治療薬は、ｅｘ　ｖｉｖｏにおいてリポソームトランスフェクション、粒子衝撃法（米国特許第４，９４５，０５０号）、又はウイルス感染を利用して細胞に投与し、当該細胞を動物に再導入することにより投与してもよい。 The gene therapy drug may be administered by any method as long as the antibody is expressed in vivo and can exert its effect. Preferably, a sufficient amount is administered via an appropriate parenteral route, such as intravenous, intraperitoneal, subcutaneous, intradermal, intraadipose tissue, intramammary tissue, inhalation, or intramuscular route, by injection, infusion, gas-induced particle bombardment (using an electron gun, etc.), or via a mucosal route such as a nasal spray. Furthermore, the gene therapy drug may be administered ex vivo to cells using liposome transfection, particle bombardment (U.S. Patent No. 4,945,050), or viral infection, and then the cells may be reintroduced into an animal.

　さらに、本開示には、ＣＸＣＲ３依存的な細胞機能の異常亢進によって発症する疾患又は疾病に罹患した哺乳動物の当該疾患に関する治療方法と治療薬も含まれる。
　ここで「治療」とは、疾患に罹患するおそれがあるか又は罹患した哺乳動物において、当該疾患の病態の進行及び悪化を阻止又は緩和することを意味し、これによって当該疾患の諸症状等の進行及び悪化を阻止又は緩和することを目的とする治療的処置の意味として使用される。
　また、「疾患」とは、ＣＸＣＲ３依存的な細胞機能の異常亢進に起因して発症する疾患全般のことを意味し、特に限定されるものではなく、例えば、アテローム性動脈硬化症、心筋炎、多発性硬化症、喘息、慢性閉塞性肺疾患、肺線維症、１型糖尿病、乾癬、リウマチ、炎症性腸炎、全身性エリテマトーデス、急性心臓同種移植片拒絶反応、インフルエンザ、ＣＯＶＩＤ－１９、腫瘍など、Ｔｈ１関連免疫異常を含む概念である。また、心血管障害、神経系障害、炎症性疾患、自己免疫疾患、代謝性疾患、感染症、血液癌、固形癌を含む概念である。治療の対象となる「哺乳動物」は、哺乳類に分類される任意の動物を意味し、特に限定はしないが、例えば、ヒトの他、イヌ、ネコ、ウサギなどのペット動物、ウシ、ブタ、ヒツジ、ウマなどの家畜動物などのことである。特に好ましい「哺乳動物」は、ヒトである。 Furthermore, the present disclosure also includes methods and therapeutic agents for treating a disease or disorder caused by abnormally enhanced CXCR3-dependent cell function in a mammal suffering from the disease.
Here, "treatment" means preventing or alleviating the progression and worsening of the pathological condition of a disease in a mammal that is at risk of contracting or is contracting the disease, and is used to mean a therapeutic procedure aimed at preventing or alleviating the progression and worsening of the symptoms of the disease.
Furthermore, the term "disease" refers to any disease that develops due to the abnormal enhancement of CXCR3-dependent cellular functions, and is not particularly limited thereto. The term encompasses Th1-related immune disorders such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatism, inflammatory bowel disease, systemic lupus erythematosus, acute cardiac allograft rejection, influenza, COVID-19, and tumors. The term also encompasses cardiovascular disorders, nervous system disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, infectious diseases, blood cancers, and solid cancers. The term "mammal" to be treated refers to any animal classified as a mammal, and includes, but is not limited to, humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is a human.

＜抗体固定化担体＞
　本開示は、上記抗体が固定化された担体（抗体固定化担体）を含む。好ましい態様では、当該抗体固定化担体は、ＣＸＣＲ３発現細胞を含む体液を接触させて、前記体液からＣＸＣＲ３発現細胞を除去するために用いられる。体液の例としては、血液が挙げられる。担体に固定化される上記抗体は、１種類のみでもよいし、２種以上でもよい。 <Antibody-immobilized carrier>
The present disclosure includes a support on which the above-mentioned antibody is immobilized (antibody-immobilized support). In a preferred embodiment, the antibody-immobilized support is used to remove CXCR3-expressing cells from a body fluid containing CXCR3-expressing cells by contacting the support with the antibody-immobilized support. An example of the body fluid is blood. The above-mentioned antibody immobilized on the support may be one type only or two or more types.

　本開示の抗体固定化担体の具体的形態としては、例えば、上記抗体が水不溶性担体に固定され、容器に充填されたものが挙げられる。ここで、水不溶性担体としてはいかなる材質も使用可能であるが、成型性、滅菌性や、細胞毒性が低いという点で好ましいものを列記すると、ポリエチレン、ポリプロピレン、ポリスチレン、アクリル樹脂、ナイロン、ポリエステル、ポリカーボネート、ポリアクリルアミド、ポリウレタン等の合成高分子、アガロース、セルロース、酢酸セルロース、キチン、キトサン、アルギン酸塩等の天然高分子、ハイドロキシアパタイト、ガラス、アルミナ、チタニア等の無機材料、ステンレス、チタン等の金属材料が挙げられる。 Specific examples of the antibody-immobilized carrier of the present disclosure include a carrier in which the antibody is immobilized on a water-insoluble carrier and packed in a container. While any material can be used as the water-insoluble carrier, preferred materials in terms of moldability, sterilization, and low cytotoxicity include synthetic polymers such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, and polyurethane; natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alginate; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and metal materials such as stainless steel and titanium.

　担体の形状としては、粒状、綿状、織布、不織布、スポンジ状多孔質体、平板状、などが挙げられるが、体積当たりの表面積が大きいという点で粒状、綿状、織布、不織布、スポンジ状多孔質体が好ましい。 The carrier may be in the form of granules, cotton, woven fabric, nonwoven fabric, porous sponge, or flat plate, but granules, cotton, woven fabric, nonwoven fabric, or porous sponge are preferred due to their large surface area per volume.

　本開示の抗体固定化担体と他の構成要素とを組み合わせて、ＣＸＣＲ３発現細胞除去用キットを作製することができる。当該他の構成要素としては、抗凝固剤、体外循環回路などが挙げられる。 A kit for removing CXCR3-expressing cells can be produced by combining the antibody-immobilized carrier of the present disclosure with other components. Such other components include an anticoagulant, an extracorporeal circulation circuit, etc.

　本開示は、上記抗体の有効量を、ＣＸＣＲ３依存的な細胞機能の障害が関与している疾患、障害、又は病状の患者に投与することを含む、当該疾患、障害、又は病状の治療方法、を包含する。本開示は、ＣＸＣＲ３依存的な細胞機能の障害が関与している疾患、障害、又は病状の治療における使用のための上記抗体、を包含する。本開示は、ＣＸＣＲ３依存的な細胞機能の障害が関与している疾患、障害、又は病状の治療に用いられる医薬を製造するための上記抗体の使用、を包含する。前記疾患、障害、又は病状には、少なくとも、心血管障害、神経系障害、炎症性疾患、自己免疫疾患、代謝性疾患、感染症、血液癌、固形癌が含まれる。 The present disclosure includes a method for treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function, comprising administering an effective amount of the antibody to a patient suffering from the disease, disorder, or condition. The present disclosure includes the antibody for use in treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function. The present disclosure includes use of the antibody for manufacturing a medicament for treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function. The diseases, disorders, or conditions include at least cardiovascular disorders, nervous system disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, infectious diseases, blood cancers, and solid cancers.

〔実施例１〕抗ＣＸＣＲ３抗体作製のための動物への免疫
（１）ＤＮＡ免疫用プラスミドの調製
　鋳型プラスミドベクターｐＣＩ－ｈＥＰ４－ＧｒｏＥＬを、下記の様に構築した。ＮＣＢＩジーンバンク登録済みのヒトＥＰ４の配列情報（ＮＭ＿０００９５８、配列番号２９３）を基に、ヒトＥＰ４遺伝子の５’末端にシグナル配列（配列番号２９４）を付加した人工遺伝子を合成した。これを鋳型とし、５’末端にＧＣＴＡＧＣ配列、３’末端にＧＴＣＧＡＣ配列をＰＣＲにて付加したヒトＥＰ４遺伝子断片（以下、「ＤＮＡ断片Ａ」と称する）を調製した。 Example 1 Immunization of Animals to Produce Anti-CXCR3 Antibodies (1) Preparation of Plasmid for DNA Immunization The template plasmid vector pCI-hEP4-GroEL was constructed as follows. Based on the sequence information of human EP4 registered in the NCBI Gene Bank (NM_000958, SEQ ID NO: 293), an artificial gene was synthesized in which a signal sequence (SEQ ID NO: 294) was added to the 5' end of the human EP4 gene. Using this as a template, a human EP4 gene fragment (hereinafter referred to as "DNA fragment A") was prepared in which the sequence GCTAGC was added to the 5' end and the sequence GTCGAC was added to the 3' end by PCR.

　大腸菌ＨＭＳ１７４（ＤＥ３）株（ノバジェン社）からゲノムＤＮＡを抽出・精製した。次に、精製したゲノムＤＮＡを鋳型とし、配列番号２９５及び２９６に示すプライマー対を用いてＰＣＲを行い、ＧｒｏＥＬサブユニット遺伝子を含むＤＮＡ断片（配列番号２９７；以下、「ＤＮＡ断片Ｂ」称する。）を増幅した。ＤＮＡ断片Ｂには、プライマーに由来して、５’末端にＳａｌＩサイト、３’末端に２個の停止コドン（ＴＡＡＴＡＧ）をコードする配列およびＮｏｔＩサイトが導入された。 Genomic DNA was extracted and purified from E. coli HMS174(DE3) strain (Novagen). Next, PCR was performed using the purified genomic DNA as a template and the primer pair shown in SEQ ID NOs: 295 and 296 to amplify a DNA fragment containing the GroEL subunit gene (SEQ ID NO: 297; hereafter referred to as "DNA fragment B"). DNA fragment B contained a SalI site at the 5' end and a sequence encoding two stop codons (TAATAG) and a NotI site at the 3' end, derived from the primers.

　哺乳動物発現ベクターｐＣＩ－Ｍａｍｍａｌｉａｎ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒ（プロメガ）を制限酵素ＮｈｅＩとＳａｌＩで消化し、バクテリア由来アルカリフォスファターゼ（ＢＡＰ）にて末端を脱リン酸化した後、ＤＮＡ断片Ａを挿入した。さらに、この発現ベクターをＳａｌＩおよびＮｏｔＩで消化し、ＢＡＰにて末端を脱リン酸化処理した後、ＤＮＡ断片Ｂを挿入し、鋳型プラスミドベクターｐＣＩ－ｈＥＰ４－ＧｒｏＥＬを構築した。 The mammalian expression vector pCI-Mammalian Expression Vector (Promega) was digested with the restriction enzymes NheI and SalI, the ends were dephosphorylated with bacterial alkaline phosphatase (BAP), and DNA fragment A was inserted. This expression vector was further digested with SalI and NotI, the ends were dephosphorylated with BAP, and DNA fragment B was inserted to construct the template plasmid vector pCI-hEP4-GroEL.

　ＮＣＢＩジーンバンク登録済みのヒトＣＸＣＲ３Ａの配列情報（ＡＡＯ９２２９５、配列番号２８１）を基に、ヒトＣＸＣＲ３Ａ遺伝子の５’末端にＧＣＴＡＧＣ配列、３’末端にＧＴＣＧＡＣ配列を付加した人工遺伝子を合成した。この人工遺伝子をクローニングベクターに導入した後、得られたベクターをＮｈｅＩとＳａｌＩサイトで切断してＤＮＡ断片（以下、「ＤＮＡ断片Ｃ」と称する。）を調製した。鋳型プラスミドベクターｐＣＩ－ｈＥＰ４－ＧｒｏＥＬを制限酵素ＮｈｅＩとＳａｌＩで消化し、ＤＮＡ断片Ｃを挿入し、免疫用ベクターｐＣＩ－ｈＣＸＣＲ３Ａ－ＧｒｏＥＬを構築した。このベクターは、ヒトＣＸＣＲ３ＡとＧｒｏＥＬとの融合タンパク質を発現する。 Based on the sequence information for human CXCR3A registered in the NCBI Gene Bank (AAO92295, SEQ ID NO: 281), an artificial gene was synthesized by adding the GCTAGC sequence to the 5' end of the human CXCR3A gene and the GTCGAC sequence to the 3' end. This artificial gene was then introduced into a cloning vector, and the resulting vector was cleaved at the NheI and SalI sites to prepare a DNA fragment (hereinafter referred to as "DNA fragment C"). The template plasmid vector pCI-hEP4-GroEL was digested with the restriction enzymes NheI and SalI, and DNA fragment C was inserted to construct the immunization vector pCI-hCXCR3A-GroEL. This vector expresses a fusion protein of human CXCR3A and GroEL.

（２）ＤＮＡ免疫
　ＰＢＳにベクターｐＣＩ－ｈＣＸＣＲ３Ａ－ＧｒｏＥＬを２ｍｇ／ｍＬの濃度になるよう溶解し、免疫用組成物を調製した。この免疫用組成物を、８週齢のマウスの両足大腿筋に各２５μＬずつ注射を行い、免疫した（０日目）。これにより、ｐＣＩ－ｈＣＸＣＲ３Ａ－ＧｒｏＥＬを両足に５０μｇずつ、すなわち、１匹につき１回あたり１００μｇ投与した。その後、１４日目、２８日目にも同様に繰り返し免疫した。初回免疫から２１、３５日目に、免疫動物から血液を採取し、血清を分離、回収し、－３０℃にて保存した。 (2) DNA Immunization The vector pCI-hCXCR3A-GroEL was dissolved in PBS to a concentration of 2 mg/mL to prepare an immunization composition. 25 μL of this immunization composition was injected into the thigh muscles of both legs of 8-week-old mice for immunization (day 0). Thus, 50 μg of pCI-hCXCR3A-GroEL was administered to both legs, i.e., 100 μg per mouse per administration. Subsequently, immunization was repeated in the same manner on days 14 and 28. On days 21 and 35 after the initial immunization, blood was collected from the immunized animals, and the serum was separated, collected, and stored at -30°C.

　上記の免疫用組成物を、８週齢のラットの両足大腿筋に各４０μＬずつ注射を行い、免疫した（０日目）。これにより、ｐＣＩ－ｈＣＸＣＲ３Ａ－ＧｒｏＥＬを両足に８０μｇずつ、すなわち、１匹につき１回あたり１６０μｇ投与した。その後、１４日目、２８日目にも同様に繰り返し免疫した。初回免疫から２１、３５日目に、免疫動物から血液を採取し、血清を分離、回収し、－３０℃にて保存した。 Eight-week-old rats were immunized by injecting 40 μL of the above immunization composition into the thigh muscles of both legs (day 0). This resulted in 80 μg of pCI-hCXCR3A-GroEL being administered to both legs, i.e., 160 μg per rat per injection. Subsequently, the animals were immunized in the same manner on days 14 and 28. On days 21 and 35 after the initial immunization, blood was collected from the immunized animals, and the serum was separated, collected, and stored at -30°C.

（３）ヒトＣＸＣＲ３Ａを安定に発現する細胞の作製
　ＮＣＢＩジーンバンク登録済みのヒトＣＸＣＲ３Ａの配列情報（ＡＡＯ９２２９５、配列番号２８１）を基に人工遺伝子を合成してｐＥＦ－ＦＲＴ（Thermo Fisher Scientific社）に導入し、ｐＥＦ－ＦＲＴ－ｈＣＸＣＲ３Ａを構築した。ＣＨＯ－ＦｌｐＩｎ細胞（Thermo Fisher Scientific社）に、リポフェクトアミン２０００（Thermo Fisher Scientific社）を用いて、ｐＥＦ－ＦＲＴ－ｈＣＸＣＲ３ＡとｐＯＧ４４プラスミド（Thermo Fisher Scientific社）を同時に導入した。ハイグロマイシン（Thermo Fisher Scientific社）を含む培地で培養し、ハイグロマイシン耐性でヒトＣＸＣＲ３Ａを安定に発現する細胞をクローニングした。以下、このヒトＣＸＣＲ３Ａ発現細胞を「ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞」と称する。 (3) Preparation of Cells Stably Expressing Human CXCR3A An artificial gene was synthesized based on the sequence information of human CXCR3A registered in the NCBI Gene Bank (AAO92295, SEQ ID NO: 281) and introduced into pEF-FRT (Thermo Fisher Scientific) to construct pEF-FRT-hCXCR3A. pEF-FRT-hCXCR3A and pOG44 plasmid (Thermo Fisher Scientific) were simultaneously introduced into CHO-FlpIn cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific). The cells were cultured in a medium containing hygromycin (Thermo Fisher Scientific), and hygromycin-resistant cells stably expressing human CXCR3A were cloned. Hereinafter, these human CXCR3A-expressing cells are referred to as "human CXCR3A stably expressing CHO cells."

（４）フローサイトメトリーを用いた血清中抗体のヒトＣＸＣＲ３Ａに対する結合性評価
　ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞およびＣＨＯ－ＦｌｐＩｎ細胞を、ＦＡＣＳバッファー（ＰＢＳ，１％　ＦＢＳ含有）で洗浄し、細胞濃度が１×１０７細胞／ｍＬとなるようにＦＡＣＳバッファーで懸濁した。Ｆｃ　Ｂｌｏｃｋ（Tonbo biosciences社）を細胞懸濁液の１／５００量加え、４℃でブロッキングを行った。ブロッキング後、１～２×１０５細胞／５０μＬとなるように細胞を懸濁した。この細胞懸濁液と、１次抗体サンプルとなる免疫動物血清を混合し、４℃でインキュベートした。インキュベート後、１００μＬのＦＡＣＳバッファーで細胞を２回洗浄した。二次抗体としてＰＥ標識抗マウスＩｇＧ抗体（サザンバイオテック社）、またはＡｌｅｘａ　Ｆｌｏｕｒ　４８８標識抗ラットＩｇＧ抗体（Abcam社）の希釈液を各ウェルに５０μＬずつ加え、４℃で１時間インキュベートした。１００μＬのＦＡＣＳバッファーで細胞を２回洗浄した後、８０μＬのＦＡＣＳバッファーに懸濁し、フローサイトメーターｉＱｕｅ（ザルトリウス社）にて細胞表面の蛍光強度を測定し、抗体の結合性を評価した。免疫動物血清は、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に特異的に反応した。これにより、ｐＣＩ－ｈＣＸＣＲ３Ａ－ＧｒｏＥＬによる免疫により、免疫動物においてヒトＣＸＣＲ３Ａを特異的に認識する抗体の産生が誘導されたことを確認できた。 (4) Evaluation of serum antibody binding to human CXCR3A using flow cytometry. Human CXCR3A stably expressing CHO cells and CHO-FlpIn cells were washed with FACS buffer (PBS containing 1% FBS) and suspended in FACS buffer to a cell concentration of 1 x 10 cells/mL. Fc Block (Tonbo Biosciences) was added in an amount of 1/500 of the cell suspension, and blocking was performed at 4°C. After blocking, the cells were suspended to a concentration of 1-2 x 10 cells/50 μL. This cell suspension was mixed with immunized animal serum, which served as a primary antibody sample, and incubated at 4°C. After incubation, the cells were washed twice with 100 μL of FACS buffer. 50 μL of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech) or Alexa Flour 488-labeled anti-rat IgG antibody (Abcam) was added to each well as a secondary antibody, followed by incubation at 4°C for 1 hour. After washing the cells twice with 100 μL of FACS buffer, they were suspended in 80 μL of FACS buffer and the cell surface fluorescence intensity was measured using an iQue flow cytometer (Sartorius) to evaluate antibody binding. Serum from immunized animals reacted specifically with CHO cells stably expressing human CXCR3A. This confirmed that immunization with pCI-hCXCR3A-GroEL induced the production of antibodies that specifically recognize human CXCR3A in immunized animals.

（５）一過性発現細胞を用いたブーストおよび採材
　哺乳動物発現ベクターｐＣＩ　Ｍａｍｍａｌｉａｎ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒ（プロメガ社）にヒトＣＸＣＲ３Ａ遺伝子を導入し、ｐＣＩ－ｈＣＸＣＲ３Ａを構築した。ｐＣＩ－ｈＣＸＣＲ３Ａを、リポフェクタミン２０００を用いてＨＥＫ２９３ＦＴ細胞にトランスフェクションして、ヒトＣＸＣＲ３Ａを一過性に発現させた。この細胞を（２）の個体（マウス又はラット）の脾臓に投与し、３日後に脾臓、鼠径リンパ節および腸骨リンパ節を採材した。採材した各組織から脾細胞およびリンパ節由来細胞を分離し、小分け分注し、スクリーニングまで－８０℃で保存した。 (5) Boosting and Sampling Using Transiently Expressing Cells The human CXCR3A gene was introduced into the mammalian expression vector pCI Mammalian Expression Vector (Promega) to construct pCI-hCXCR3A. pCI-hCXCR3A was transfected into HEK293FT cells using Lipofectamine 2000 to transiently express human CXCR3A. These cells were administered to the spleen of the individual (mouse or rat) described in (2), and three days later, the spleen, inguinal lymph nodes, and iliac lymph nodes were sampled. Splenocytes and lymph node-derived cells were isolated from each sample, aliquoted, and stored at -80°C until screening.

〔実施例２〕抗体の作製＜１＞
（１）マイクロウェルを用いた特異的抗体産生リンパ球の選抜
　ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞をＦ－１２培地（１０％　ＦＢＳ，Ｐｅｎｉｃｉｌｌｉｎ／Ｓｔｒｅｐｔｏｍｙｃｉｎ含有）で懸濁し、３．５×１０５細胞／５００μＬの細胞懸濁液を調製した。この細胞懸濁液をセルピッキングシステム用のマイクロチャンバーに充填した。マイクロチャンバーを３００ｒｐｍで１分間、５回遠心し、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞が各マイクロウェルに１又は２個格納されるように調製した。マイクロチャンバーをＦ－１２培地で洗浄した後、５００μＬのＦ－１２培地を入れた。ＣＯ２インキュベーターにて３７℃で１時間インキュベートし、マイクロウェルの底面にヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞を接着させた。Ｆ－１２培地で１０ｎＭ濃度に調整したＣｙｔｏＲｅｄ溶液（同仁化学研究所社）を加え、さらに３７℃で１時間インキュベートし細胞を染色した。Ｆ－１２培地で３回洗浄して余剰のＣｙｔｏＲｅｄを除去した後、１ｍＬのＦ－１２培地をマイクロチャンバー内に満たした。 [Example 2] Preparation of antibodies <1>
(1) Selection of specific antibody-producing lymphocytes using microwells. CHO cells stably expressing human CXCR3A were suspended in F-12 medium (containing 10% FBS, penicillin/streptomycin) to prepare a cell suspension of 3.5 x 10 cells/500 μL. This cell suspension was loaded into a microchamber for a cell picking system. The microchamber was centrifuged five times at 300 rpm for 1 minute, and one or two CHO cells stably expressing human CXCR3A were placed in each microwell. The microchamber was washed with F-12 medium, and then 500 μL of F-12 medium was added. The mixture was incubated at 37°C in a CO2 incubator for 1 hour, allowing the CHO cells stably expressing human CXCR3A to adhere to the bottom of the microwell. A CytoRed solution (Dojindo Laboratories) adjusted to a concentration of 10 nM in F-12 medium was added, and the cells were further incubated for 1 hour at 37°C to stain the cells. After washing three times with F-12 medium to remove excess CytoRed, the microchamber was filled with 1 mL of F-12 medium.

　ＤＮＡ免疫後に実施例１（５）で回収した細胞から、ＥａｓｙＳｅｐ　Ｍｏｕｓｅ　Ｂｉｏｔｉｎ　Ｐｏｓｉｔｉｖｅ　Ｓｅｌｅｃｔｉｏｎ　Ｋｉｔ（STEMCELL Technologies社）を用いて抗体産生細胞を濃縮した。５×１０４個～２×１０５個の抗体産生細胞を５００μＬのＦ－１２培地で懸濁し、マイクロチャンバーに充填した。マイクロチャンバーを３００ｒｐｍで１分間、５回遠心し、抗体産生細胞が各マイクロウェルに１又は２個格納されるように調製した。マイクロチャンバーを培地で洗浄した後、適量の培地を入れて３７℃で３０分間インキュベートし、抗体産生細胞から抗体を分泌させた。マイクロウェルを洗浄して上清を除去した後、ＲＰＭＩ１６４０（１０％　ＦＢＳ含有）で５００倍希釈したＡｌｅｘａ　Ｆｌｕｏｒ　４８８標識抗マウスＩｇＧ抗体（Abcam社）、またはＡｌｅｘａ　Ｆｌｕｏｒ　４８８標識抗ラットＩｇＧ抗体（Abcam社）を添加し、３７℃で３０分間インキュベートした。ＦＡＣＳバッファー（ＰＢＳ，１％　ＦＢＳ含有）にて３回洗浄した後、ＦＡＣＳバッファーを１ｍＬ入れた。セルピッキングシステム（アズワン社）にマイクロチャンバーをセットし、全てのマイクロウェルの透過光画像および２種類の蛍光画像の情報を取得した。ＣｙｔｏＲｅｄの蛍光検出は、励起波長５４３ｎｍ、蛍光波長５９３ｎｍの条件で行った。Ａｌｅｘａ　Ｆｌｕｏｒ　４８８の蛍光検出は、励起波長４８２ｎｍ、蛍光波長５３６ｎｍの条件で行った。透過光、ＣｙｔｏＲｅｄ、およびＡｌｅｘａ　Ｆｌｕｏｒ　４８８のスキャン画像をもとにヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に結合する抗体が存在すると判断できたマイクロウェルから、直径数μｍ～数十μｍのキャピラリー（アズワン社）を用いて、抗体生産細胞を細胞溶解液の中に回収した。 Antibody-producing cells were enriched from the cells collected in Example 1(5) after DNA immunization using EasySep Mouse Biotin Positive Selection Kit (STEMCELL Technologies). 5 x 104 to 2 x 105 antibody-producing cells were suspended in 500 μL of F-12 medium and loaded into the microchamber. The microchamber was centrifuged five times at 300 rpm for 1 minute, and the microchamber was adjusted so that one or two antibody-producing cells were contained in each microwell. After washing the microchamber with medium, an appropriate amount of medium was added and the mixture was incubated at 37°C for 30 minutes to allow the antibody to be secreted from the antibody-producing cells. After washing the microwells and removing the supernatant, Alexa Fluor 488-labeled anti-mouse IgG antibody (Abcam) or Alexa Fluor 488-labeled anti-rat IgG antibody (Abcam) diluted 500-fold in RPMI 1640 (containing 10% FBS) was added and incubated at 37°C for 30 minutes. After washing three times with FACS buffer (PBS containing 1% FBS), 1 mL of FACS buffer was added. The microchamber was placed in a cell picking system (AS ONE) to acquire transmitted light images and two types of fluorescent image information for all microwells. CytoRed fluorescence detection was performed at an excitation wavelength of 543 nm and an emission wavelength of 593 nm. Alexa Fluor 488 fluorescence detection was performed at an excitation wavelength of 482 nm and an emission wavelength of 536 nm. From microwells where it was determined based on the scanned images of transmitted light, CytoRed, and Alexa Fluor 488 that antibodies binding to CHO cells stably expressing human CXCR3A were present, antibody-producing cells were recovered into a cell lysate using a capillary tube (As One Corporation) with a diameter of several to several tens of micrometers.

（２）回収した抗体産生細胞からの抗体遺伝子単離
　抗体産生細胞から、ＭＡＧｒａｈｄ法（Kurosawa N, Yoshioka M, Fujimoto R, Yamagishi F, Isobe M. "Rapid production of antigen-specific monoclonal antibodies from a variety of animals." BMC Biol. 2012; 10: 80）にて抗体遺伝子を取得した。すなわち、（１）で得た細胞溶解液５μＬとオリゴｄＴマグネット５μｇとを混合し、オリゴｄＴマグネット上に細胞由来のｍＲＮＡを捕捉した。ＭＡＧｒａｈｄリアクタートレーとネオジム磁石を使い、オリゴｄＴマグネットを洗浄溶液で洗浄後、逆転写反応によるｃＤＮＡ合成を行った。さらにマグネットを洗浄後、５’ターミナルトランスレーショナル反応を実施した。合成した上記ｃＤＮＡを用い、５’ｒａｃｅＰＣＲ法にて、抗体重鎖可変領域（ＶＨ領域）の遺伝子と、抗体軽鎖可変領域（ＶＬ領域）の遺伝子を単離増幅した。 (2) Isolation of antibody genes from recovered antibody-producing cells. Antibody genes were obtained from antibody-producing cells using the MAGrahd method (Kurosawa N, Yoshioka M, Fujimoto R, Yamagishi F, Isobe M. "Rapid production of antigen-specific monoclonal antibodies from a variety of animals." BMC Biol. 2012; 10: 80). Specifically, 5 μL of the cell lysate obtained in (1) was mixed with 5 μg of oligo-dT magnet, and cell-derived mRNA was captured on the oligo-dT magnet. Using a MAGrahd reactor tray and a neodymium magnet, the oligo-dT magnet was washed with a washing solution, and then cDNA synthesis was performed by reverse transcription. After further washing the magnet, a 5'-terminal translational reaction was performed. Using the synthesized cDNA, the antibody heavy chain variable region (VH region) gene and the antibody light chain variable region (VL region) gene were isolated and amplified by 5'-race PCR.

　なお、増幅産物の特異性を高めるために、ＰＣＲは２回行った。マウスから取得した抗体に対する１回目のＰＣＲでは、ＶＨ領域とＶＬ領域を共通して増幅させる第一フォワードプライマー（配列番号２８２）と、ＶＨ領域を特異的に増幅させるマウスＶＨ第一リバースプライマー（配列番号２８３）と、ＶＬ領域を特異的に増幅させるマウスＶＬ第一リバースプライマー（配列番号２８４）を混合して使用した。２回目のＰＣＲでは、１回目の増幅産物を鋳型とし、ＶＨ領域の増幅については第二フォワードプライマー（配列番号２８５）とＶＨ領域を特異的に増幅させるマウスＶＨ第二リバースプライマー（配列番号２８６）、ＶＬ領域の増幅については第二フォワードプライマー（配列番号２８５）とＶＬ領域特異的に増幅させるマウスＶＬ第二リバースプライマー（配列番号２８７）をそれぞれプライマーとして使用した。 In order to increase the specificity of the amplified product, PCR was performed twice. In the first PCR for the mouse antibody, a mixture of the first forward primer (SEQ ID NO: 282), which amplifies both the VH and VL regions, the mouse VH first reverse primer (SEQ ID NO: 283), which specifically amplifies the VH region, and the mouse VL first reverse primer (SEQ ID NO: 284), which specifically amplifies the VL region, was used. In the second PCR, the amplified product from the first PCR was used as a template, and the second forward primer (SEQ ID NO: 285) and the mouse VH second reverse primer (SEQ ID NO: 286), which specifically amplifies the VH region, were used as primers for amplifying the VH region, and the second forward primer (SEQ ID NO: 285) and the mouse VL second reverse primer (SEQ ID NO: 287), which specifically amplifies the VL region, were used as primers for amplifying the VL region.

　また、ラットから取得した抗体に対する１回目のＰＣＲでは、ＶＨ領域とＶＬ領域を共通して増幅させる第一フォワードプライマー（配列番号２８２）と、ＶＨ領域を特異的に増幅させるラットＶＨ第一リバースプライマー（配列番号２８８）と、ＶＬ領域を特異的に増幅させるラットＶＬ第一リバースプライマー（配列番号２８９）を混合して使用した。２回目のＰＣＲでは、１回目の増幅産物を鋳型とし、ＶＨ領域の増幅については第二フォワードプライマー（配列番号２８５）とＶＨ領域を特異的に増幅させるラット第二リバースプライマー（配列番号２９０）、ＶＬ領域の増幅については第二フォワードプライマー（配列番号２８５）とＶＬ領域特異的に増幅させるラットＶＬ第二リバースプライマー（配列番号２９１）をそれぞれプライマーとして使用した。 In addition, in the first PCR for the antibody obtained from rat, a mixture of a first forward primer (SEQ ID NO: 282) that amplifies both the VH and VL regions, a rat VH first reverse primer (SEQ ID NO: 288) that specifically amplifies the VH region, and a rat VL first reverse primer (SEQ ID NO: 289) that specifically amplifies the VL region was used. In the second PCR, the first amplification product was used as a template, and the second forward primer (SEQ ID NO: 285) and a rat second reverse primer (SEQ ID NO: 290) that specifically amplifies the VH region were used to amplify the VH region, and the second forward primer (SEQ ID NO: 285) and a rat VL second reverse primer (SEQ ID NO: 291) that specifically amplifies the VL region were used to amplify the VL region.

　２回目のＰＣＲ後のサンプルについてアガロースゲル電気泳動を行ったところ、約７５０ｂｐの位置にＶＨ領域、約５５０ｂｐの位置にＶＬ領域にそれぞれ対応する増幅産物が確認できた。 When the sample after the second PCR was subjected to agarose gel electrophoresis, amplification products corresponding to the VH region at approximately 750 bp and the VL region at approximately 550 bp were confirmed.

（３）抗体発現ユニットの構築
　ＴＳ－ｊＰＣＲ法（Yoshioka M, Kurosawa N, Isobe M. "Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells." BMC Biotechnol. 2011; 11: 75）にて抗体発現ユニットを構築した。すなわち、（２）で増幅したＶＨ領域の遺伝子と、抗体重鎖定常領域の遺伝子と、遺伝子発現に必要なプロモーター領域を含む遺伝子とをＰＣＲを用いて融合し、完全長抗体重鎖を発現する抗体発現ユニットを構築した。同様に、（２）で増幅したＶＬ領域の遺伝子と、抗体軽鎖定常領域の遺伝子と、遺伝子発現に必要なプロモーター領域を含む遺伝子とをＰＣＲを用いて融合し、完全長抗体軽鎖を発現する抗体発現ユニットを構築した。 (3) Construction of antibody expression unit An antibody expression unit was constructed using the TS-jPCR method (Yoshioka M, Kurosawa N, Isobe M. "Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells." BMC Biotechnol. 2011; 11: 75). Specifically, the VH region gene amplified in (2), the antibody heavy chain constant region gene, and a gene containing a promoter region required for gene expression were fused using PCR to construct an antibody expression unit that expresses a full-length antibody heavy chain. Similarly, the VL region gene amplified in (2), the antibody light chain constant region gene, and a gene containing a promoter region required for gene expression were fused using PCR to construct an antibody expression unit that expresses a full-length antibody light chain.

（４）哺乳動物細胞への抗体発現ユニット導入
　細胞培養用６ウェルプレートに、Ｅｘｐｉ２９３Ｆ細胞（Thermo Fisher Scientific社）を７．５×１０６細胞／３ｍＬ／ウェルとなるように播種した。Ｅｘｐｉｆｅｃｔａｍｉｎｅ　２９３　Ｒｅａｇｅｎｔ（Thermo Fisher Scientific社）を用いて、（３）で構築した重鎖および軽鎖の２種の抗体発現ユニットをＥｘｐｉ２９３Ｆ細胞に共導入した。導入５日目に細胞上清を回収して、実施例１（４）と同様の方法で、発現された抗体の結合性評価に用いた。ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に結合し、ＣＨＯ－ＦｌｐＩｎ細胞に結合しなかった抗体ユニットを一次ヒット抗体とした。 (4) Transfection of antibody expression units into mammalian cells Expi293F cells (Thermo Fisher Scientific) were seeded into a 6-well cell culture plate at 7.5 x 10 cells/3 mL/well. The two antibody expression units, heavy and light chains, constructed in (3) were co-transfected into Expi293F cells using Expifectamine 293 Reagent (Thermo Fisher Scientific). Five days after transfection, the cell supernatant was collected and used to evaluate the binding activity of the expressed antibody in the same manner as in Example 1(4). The antibody unit that bound to human CXCR3A stably expressing CHO cells but did not bind to CHO-FlpIn cells was designated as the primary hit antibody.

〔実施例３〕抗体の作製＜２＞
（１）ハイブリドーマの作製と結合性クローンのスクリーニング
　実施例１（５）で回収した細胞とミエローマ細胞であるＳＰ２／０を材料とし、細胞融合装置ＮＥＰＡＧＥＮＥ　ＥＣＦＧ２１（ネッパージーン）を用いて融合させた。融合した細胞（ハイブリドーマ）をＨＡＴ　Ｓｕｐｐｌｅｍｅｎｔ（Invitrogen社）を含むＲＰＭＩ１６４０培地に懸濁し、９６ウェルプレートに播種した。８日間程度の培養を行い、培養上清の一部を回収した。実施例１（４）と同様の方法で、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に特異的に結合する抗体を産生するハイブリドーマをスクリーニングした。 [Example 3] Preparation of antibody <2>
(1) Preparation of Hybridomas and Screening of Binding Clones The cells collected in Example 1(5) and myeloma cells SP2/0 were fused using a cell fusion device NEPAGENE ECFG21 (Nepper Gene). The fused cells (hybridomas) were suspended in RPMI 1640 medium containing HAT Supplement (Invitrogen) and seeded onto a 96-well plate. After culturing for approximately 8 days, a portion of the culture supernatant was collected. Hybridomas producing antibodies that specifically bind to human CXCR3A stably expressing CHO cells were screened using the same method as in Example 1(4).

（２）ハイブリドーマのクローニング
　（１）で抗体産生が確認されたハイブリドーマを、限界希釈法にてクローニングした。すなわち、ハイブリドーマを、９６ウェルプレートに細胞１個以下／１穴になるように播種し、培養した。２週間後、実施例１（４）と同様の方法で、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に特異的に結合する抗体をスクリーニングした。ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に結合し、ＣＨＯ－ＦｌｐＩｎ細胞に結合しなかった抗体を一次ヒット抗体とした。 (2) Cloning of Hybridomas Hybridomas confirmed to produce antibodies in (1) were cloned by limiting dilution. Specifically, the hybridomas were seeded into a 96-well plate at 1 cell or less per well and cultured. After 2 weeks, antibodies that specifically bind to CHO cells stably expressing human CXCR3A were screened using the same method as in Example 1(4). Antibodies that bound to CHO cells stably expressing human CXCR3A but did not bind to CHO-FlpIn cells were determined as primary hit antibodies.

　一次ヒット抗体を産生するハイブリドーマを拡大培養したのち、５×１０５細胞／ｍＬの濃度でＣＤ　Ｈｙｂｒｉｄｏｍａ培地（Gibco社）に置換し、７日間培養した。培養上清を回収したのち遠心して細胞残骸を沈降させた。その上清を０．４５μｍフィルター（SARTORIUS社）に通し、精製するまで４℃で保存した。 Hybridomas producing the primary hit antibodies were expanded and then replaced with CD Hybridoma medium (Gibco) at a concentration of 5 x 10 cells/mL and cultured for 7 days. The culture supernatant was collected and centrifuged to precipitate cell debris. The supernatant was passed through a 0.45 μm filter (Sartorius) and stored at 4°C until purification.

（３）抗体遺伝子の単離
　実施例２（２）と同様の操作を行った。 (3) Isolation of antibody genes The same procedures as in Example 2(2) were carried out.

（４）抗体発現ユニットの構築
　実施例２（３）と同様の操作を行った。 (4) Construction of antibody expression unit The same procedure as in Example 2(3) was carried out.

（５）哺乳動物細胞への抗体発現ユニット導入
　実施例２（４）と同様の操作を行った。 (5) Introduction of antibody expression unit into mammalian cells The same procedure as in Example 2(4) was carried out.

〔実施例４〕抗体重鎖可変領域および抗体重鎖可変領域のＣＤＲ配列の決定
　実施例２（２）又は実施例３（３）で得た抗体重鎖可変領域および抗体軽鎖可変領域を含む２回目のＰＣＲ増幅産物を、ＰＣＲ産物精製キット（FastGene社）を用いて精製した。マウスから取得した抗体の配列決定には、抗体重鎖可変領域のシーケンスプライマーとしてマウスＶＨ第二リバースプライマー（配列番号２８６）を、抗体軽鎖可変領域のシーケンスプライマーとしてマウスＶＬ第二リバースプライマー（配列番号２８７）をそれぞれ用いてダイレクトシーケンス法を実施することで、抗体重鎖可変領域および抗体軽鎖可変領域の配列を決定した。また、ラットから取得した抗体の配列決定には、抗体重鎖可変領域のシーケンスプライマーとしてラットＶＨ第二リバースプライマー（配列番号２９０）を、抗体軽鎖可変領域のシーケンスプライマーとしてラットＶＬ第二リバースプライマー（配列番号２９１）をそれぞれ用いてダイレクトシーケンス法を実施することで、抗体重鎖可変領域および抗体軽鎖可変領域の配列を決定した。ＣＤＲの決定にはｋａｂａｔ　ｎｕｍｂｅｒｉｎｇ法とＩＭＧＴ　ｎｕｍｂｅｒｉｎｇ法を組み合わせて解析し、両方のナンバリング方法によって判定される領域を包括的に含む領域を、ＣＤＲとして決定した。 [Example 4] Determination of CDR sequences of antibody heavy chain variable regions and antibody heavy chain variable regions The second PCR amplification products containing the antibody heavy chain variable region and antibody light chain variable region obtained in Example 2(2) or Example 3(3) were purified using a PCR product purification kit (FastGene). The sequences of the antibody heavy chain variable region and antibody light chain variable region were determined by direct sequencing using a mouse VH second reverse primer (SEQ ID NO: 286) as the sequence primer for the antibody heavy chain variable region and a mouse VL second reverse primer (SEQ ID NO: 287) as the sequence primer for the antibody light chain variable region, respectively. The sequences of the antibody heavy chain variable region and antibody light chain variable region were determined by direct sequencing using a rat VH second reverse primer (SEQ ID NO: 290) as the sequence primer for the antibody heavy chain variable region and a rat VL second reverse primer (SEQ ID NO: 291) as the sequence primer for the antibody light chain variable region, respectively. The CDRs were determined by combining the Kabat numbering method and the IMGT numbering method, and the regions comprehensively including the regions determined by both numbering methods were determined as CDRs.

　決定した抗体重鎖可変領域および抗体軽鎖可変領域の配列情報をもとに、他とは配列が重複せず独立した抗体重鎖可変領域と抗体軽鎖可変領域の配列を有することが確認された２８種類の抗体を選抜した。以下に、各抗体の名称を、後述する表番号と配列番号とともに示す。このうち、２０１１１２－１－Ｃ、１３Ｂ４、１１Ｆ１１はラット由来、その他はマウス由来の抗体である。 Based on the determined sequence information for the antibody heavy chain variable region and antibody light chain variable region, 28 types of antibodies were selected that were confirmed to have independent antibody heavy chain variable region and light chain variable region sequences that do not overlap with other sequences. The name of each antibody is shown below along with the table number and sequence number described below. Of these, 201112-1-C, 13B4, and 11F11 are derived from rats, while the others are derived from mice.

　２０１００１－１－Ｆ（表１－１；配列番号１－１０）
　２０１００１－５－Ｂ（表１－２；配列番号１１－２０）
　２０１００１－２－Ｃ（表１－３；配列番号２１－３０）
　２０１００１－４－Ｅ（表１－４；配列番号３１－４０）
　２０１００１－１－Ｃ（表１－５；配列番号４１－５０）
　２０１００１－５－Ａ（表１－６；配列番号５１－６０）
　２０１００９－１－Ｄ（表１－７；配列番号６１－７０）
　２０１００９－５－Ｆ（表１－８；配列番号７１－８０）
　２０１１１２－１－Ｃ（表１－９；配列番号８１－９０）
　１３Ｂ４（表１－１０；配列番号９１－１００）
　１１Ｆ１１（表１－１１；配列番号１０１－１１０）
　２０１００６－４－Ｈ（表１－１２；配列番号１１１－１２０）
　２０１００６－４－Ｆ（表１－１３；配列番号１２１－１３０）
　２０１００６－５－Ａ（表１－１４；配列番号１３１－１４０）
　２０１００６－４－Ａ（表１－１５；配列番号１４１－１５０）
　２０１００６－１－Ｈ（表１－１６；配列番号１５１－１６０）
　２０１００６－３－Ｄ（表１－１７；配列番号１６１－１７０）
　２０１００６－１－Ｆ（表１－１８；配列番号１７１－１８０）
　２０１００６－４－Ｇ（表１－１９；配列番号１８１－１９０）
　２０１００６－７－Ａ（表１－２０；配列番号１９１－２００）
　２０１００６－２－Ｄ（表１－２１；配列番号２０１－２１０）
　２０１００６－２－Ｃ（表１－２２；配列番号２１１－２２０）
　２０１００６－３－Ｈ（表１－２３；配列番号２２１－２３０）
　２０１００６－５－Ｆ（表１－２４；配列番号２３１－２４０）
　２０１００６－７－Ｇ（表１－２５；配列番号２４１－２５０）
　２０１００９－２－Ｃ（表１－２６；配列番号２５１－２６０）
　２０１００９－３－Ｈ（表１－２７；配列番号２６１－２７０）
　２０１００９－１－Ｃ（表１－２８；配列番号２７１－２８０） 201001-1-F (Table 1-1; SEQ ID NOs: 1-10)
201001-5-B (Table 1-2; SEQ ID NOs: 11-20)
201001-2-C (Tables 1-3; SEQ ID NOs: 21-30)
201001-4-E (Tables 1-4; SEQ ID NOs: 31-40)
201001-1-C (Tables 1-5; SEQ ID NOs: 41-50)
201001-5-A (Tables 1-6; SEQ ID NOs: 51-60)
201009-1-D (Tables 1-7; SEQ ID NOs: 61-70)
201009-5-F (Tables 1-8; SEQ ID NOs: 71-80)
201112-1-C (Tables 1-9; SEQ ID NOs: 81-90)
13B4 (Tables 1-10; SEQ ID NOs: 91-100)
11F11 (Tables 1-11; SEQ ID NOs: 101-110)
201006-4-H (Tables 1-12; SEQ ID NOs: 111-120)
201006-4-F (Tables 1-13; SEQ ID NOs: 121-130)
201006-5-A (Tables 1-14; SEQ ID NOs: 131-140)
201006-4-A (Tables 1-15; SEQ ID NOs: 141-150)
201006-1-H (Tables 1-16; SEQ ID NOs: 151-160)
201006-3-D (Tables 1-17; SEQ ID NOs: 161-170)
201006-1-F (Table 1-18; SEQ ID NOs: 171-180)
201006-4-G (Tables 1-19; SEQ ID NOs: 181-190)
201006-7-A (Tables 1-20; SEQ ID NOs: 191-200)
201006-2-D (Tables 1-21; SEQ ID NOs: 201-210)
201006-2-C (Tables 1-22; SEQ ID NOs: 211-220)
201006-3-H (Table 1-23; SEQ ID NOs: 221-230)
201006-5-F (Table 1-24; SEQ ID NOs: 231-240)
201006-7-G (Table 1-25; SEQ ID NOs: 241-250)
201009-2-C (Table 1-26; SEQ ID NOs: 251-260)
201009-3-H (Table 1-27; SEQ ID NOs: 261-270)
201009-1-C (Table 1-28; SEQ ID NOs: 271-280)

　各抗体のＣＤＲと可変領域のアミノ酸配列、並びに、当該可変領域をコードするＤＮＡのヌクレオチド配列を、配列番号とともに表１－１から表１－２８にまとめた。表中、各可変領域の配列の下線部分はＣＤＲを示す。 The amino acid sequences of the CDRs and variable regions of each antibody, as well as the nucleotide sequences of the DNA encoding the variable regions, are summarized in Tables 1-1 to 1-28, along with the sequence numbers. In the tables, the underlined portions of each variable region sequence indicate the CDRs.

〔実施例５〕細胞内Ｃａ２＋シグナル伝達阻害活性評価による抗体スクリーニング
　実施例２（４）又は実施例３（５）にて一次ヒット抗体として選抜されたクローンの培養上清を用い、Ｐｒｏｔｅｉｎ　Ａアフィニティー精製によって精製抗体を得た。精製一次ヒット抗体を、以下の試験に供した。 [Example 5] Antibody screening by evaluating the activity of inhibiting intracellular Ca2 + signaling Purified antibodies were obtained by Protein A affinity purification using the culture supernatants of the clones selected as primary hit antibodies in Example 2 (4) or Example 3 (5). The purified primary hit antibodies were subjected to the following tests.

　ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞を、９６ウェルマイクロプレートに、各ウェル当たり２×１０４細胞／１００μＬの初期細胞濃度で播種し、２日間培養した。２日後に、培養培地を３μＭ　Ｃａｌ－５２０（AAT Bioquest社）、０．０５％　Ｐｌｕｒｏｎｉｃ－Ｆ１２７、及び２．５ｍＭプロベネシド（Thermo Fisher Scientific社）を含む溶液に交換した。１時間後、各ウェルに、１００ｎＭ～１０ｎＭの濃度範囲内に希釈した一次ヒット抗体、または対照の抗体を添加し、１５分間インキュベートした。対照の抗体として、マウスアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、またはラットアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）を用いた。インキュベート後、アッセイプレートをＣａ２＋シグナル解析装置ＦＤＳＳμＣＥＬＬ（浜松ホトニクス社）に設置し、１００ｎＭのＩＰ－１０（PEPROTECH社）によって各細胞を刺激した際の細胞内Ｃａ２＋濃度の一過的上昇に対する阻害活性を測定した。阻害活性は、「抗体なし、ＩＰ－１０あり」の場合を阻害率０％、「抗体なし、ＩＰ－１０なし」の場合を阻害率１００％として標準化を行い、相対的な値として算出した。代表例として、ＣＤＲの異なる２８種類の抗体の阻害活性を表２に示した。表中のＮ／Ａは、測定未実施を示す。 Human CXCR3A-stably expressing CHO cells were seeded into a 96-well microplate at an initial cell concentration of 2 x 104 cells/100 μL per well and cultured for 2 days. After 2 days, the culture medium was replaced with a solution containing 3 μM Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Thermo Fisher Scientific). After 1 hour, primary hit antibodies or control antibodies diluted within the 100 nM to 10 nM concentration range were added to each well and incubated for 15 minutes. Mouse isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) were used as control antibodies. After incubation, the assay plate was placed in a Ca2 + signal analyzer FDSSμCELL (Hamamatsu Photonics), and the inhibitory activity against the transient increase in intracellular Ca2 + concentration when each cell type was stimulated with 100 nM IP-10 (PEPROTECH) was measured. The inhibitory activity was calculated as a relative value by standardizing the case of "no antibody, with IP-10" as an inhibition rate of 0% and the case of "no antibody, without IP-10" as an inhibition rate of 100%. As representative examples, the inhibitory activity of 28 types of antibodies with different CDRs is shown in Table 2. N/A in the table indicates that the measurement was not performed.

　同様の試験を繰り返し行い、ラットから取得した１３Ｂ４、１１Ｆ１１、マウスから取得した２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃ、２０１００９－３－Ｈおよび２０１００９－１－Ｃの８種類を、安定的な細胞内Ｃａ２＋シグナル伝達阻害活性を示す抗体として選択した。 Similar tests were repeated, and eight antibodies, 13B4 and 11F11 obtained from rats, and 201006-3-H, 201006-5-F, 201006-7-G, 201009-2-C, 201009-3-H, and 201009-1-C obtained from mice, were selected as antibodies that exhibit stable intracellular Ca 2+ signaling inhibitory activity.

〔実施例６〕血管内皮細胞におけるＣＸＣＲ３ＡおよびＣＸＣＲ３Ｂ遺伝子発現定量
　ヒト臍帯血管内皮細胞（ロンザ社）およびヒト微小血管内皮細胞（Cell systems社）のペレットから、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（QIAGEN社）を用いてキットに添付されるプロトコルに従ってＲＮＡを抽出し、ＤＮａｓｅＩ（QIAGEN社）にてＲＮＡに含まれるゲノムＤＮＡを除去した。ＣＸＣＲ３ＡおよびＣＸＣＲ３Ｂの両方を発現することが知られているヒト腎腺癌細胞株ＡＣＨＮについても、同様に処理したＲＮＡを得た（Takanobu Utsumi, Takahito Suyama, Yusuke Imamura, Miki Fuse, Shinichi Sakamoto, Naoki Nihei, Takeshi Ueda, Hiroyoshi Suzuki, Naohiko Seki, Tomohiko Ichikawa, "The association of CXCR3 and renal cell carcinoma metastasis" J Urol. 2014 Aug;192(2):567-74.）。 Example 6: Quantification of CXCR3A and CXCR3B gene expression in vascular endothelial cells. RNA was extracted from pellets of human umbilical vascular endothelial cells (Lonza) and human microvascular endothelial cells (Cell Systems) using an RNeasy Mini Kit (QIAGEN) according to the protocol provided with the kit. Genomic DNA contained in the RNA was removed with DNase I (QIAGEN). RNA was also obtained from the human renal adenocarcinoma cell line ACHN, which is known to express both CXCR3A and CXCR3B, by similar treatment (Takanobu Utsumi, Takahito Suyama, Yusuke Imamura, Miki Fuse, Shinichi Sakamoto, Naoki Nihei, Takeshi Ueda, Hiroyoshi Suzuki, Naohiko Seki, Tomohiko Ichikawa, "The association of CXCR3 and renal cell carcinoma metastasis," J. Urol. 2014 Aug;192(2):567-74).

　Ｈｉｇｈ　ｃａｐａｃｉｔｙ　ＲＮＡ－ｔｏ－ｃＤＮＡ　ｋｉｔ（Applied Biosystems社）を用いて、ＲＮＡからｃＤＮＡを合成した。定量リアルタイムＰＣＲにより、１００ｎｇのｃＤＮＡに含まれるＣＸＣＲ３ＡおよびＣＸＣＲ３Ｂ遺伝子を検出した。１８Ｓを内部標準遺伝子とし、１８Ｓ検出プライマーとしてＴａｑＭａｎ　Ｇｅｎｅ　Ｅｘｐｒｅｓｓｉｏｎ　Ａｓｓａｙｓ（Applied Biosystems社）を用いた。ＣＸＣＲ３ＡおよびＣＸＣＲ３Ｂの検出プライマーとして、論文に記載のＴａｑＭａｎ　Ａｓｓａｙを用いた（Laura Lasagni, Michela Francalanci, Francesco Annunziato, Elena Lazzeri, Stefano Giannini, Lorenzo Cosmi, Costanza Sagrinati, Benedetta Mazzinghi, Claudio Orlando, Enrico Maggi, Fabio Marra, Sergio Romagnani, Mario Serio, Paola Romagnani, "An Alternatively Spliced Variant of CXCR3 Mediates the Inhibition of Endothelial Cell Growth Induced by IP-10, Mig, and I-TAC, and Acts as Functional Receptor for Platelet Factor 4." J Exp Med. 2003 Jun 2;197(11):1537-49.）。 cDNA was synthesized from RNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems). CXCR3A and CXCR3B genes contained in 100 ng of cDNA were detected by quantitative real-time PCR. 18S was used as an internal standard gene, and TaqMan Gene Expression Assays (Applied Biosystems) were used as 18S detection primers. The TaqMan Assay described in the paper was used as the detection primers for CXCR3A and CXCR3B (Laura Lasagni, Michela Francalanci, Francesco Annunziato, Elena Lazzeri, Stefano Giannini, Lorenzo Cosmi, Costanza Sagrinati, Benedetta Mazzinghi, Claudio Orlando, Enrico Maggi, Fabio Marra, Sergio Romagnan). i, Mario Serio, Paola Romagnani, "An Alternative Spliced Variant of CXCR3 Mediates the Inhibition of Endothelial Cell Growth I neduced by IP-10, Mig, and I-TAC, and Acts as Functional Receptor for Platelet Factor 4." J Exp Med. 2003 Jun 2;197(11):1537-49.).

　得られたＣｔ値をΔΔＣｔ法により解析し、ＡＣＨＮのＣＸＣＲ３Ｂ発現量を１としたときの各細胞におけるＣＸＣＲ３Ａ、ＣＸＣＲ３Ｂの相対的な発現量を算出した。図１に示すとおり、ヒト臍帯血管内皮細胞およびヒト微小血管内皮細胞においては、ＣＸＣＲ３ＡよりもＣＸＣＲ３Ｂが優位に発現していることが確認された。 The obtained Ct values were analyzed using the ΔΔCt method, and the relative expression levels of CXCR3A and CXCR3B in each cell were calculated, with the CXCR3B expression level in ACHN set at 1. As shown in Figure 1, it was confirmed that CXCR3B is expressed more predominantly than CXCR3A in human umbilical vascular endothelial cells and human microvascular endothelial cells.

〔実施例７〕フローサイトメトリーによる血管内皮細胞への結合評価
　表２に示した２８種類の抗ＣＸＣＲ３抗体のうち、１９種類の抗体を以下の試験に供した。 [Example 7] Evaluation of binding to vascular endothelial cells by flow cytometry Of the 28 anti-CXCR3 antibodies shown in Table 2, 19 antibodies were subjected to the following test.

　ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞、実施例６にてＣＸＣＲ３Ｂを優位に発現することが確認されたヒト臍帯血管内皮細胞（ロンザ社）、およびヒト微小血管内皮細胞（Cell systems社）をＦＡＣＳバッファー（ＰＢＳ，１％　ＦＢＳ含有）で洗浄し、細胞濃度が１×１０７細胞／ｍＬとなるようにＦＡＣＳバッファーで懸濁した。Ｆｃ　Ｂｌｏｃｋ（Tonbo biosciences社）を細胞懸濁液の１／５００量、または１：１希釈したヤギ血清を加え、４℃で３０分間ブロッキングを行った。ブロッキング後、１×１０５細胞／５０μＬとなるように細胞を懸濁した。この細胞懸濁液と、希釈した各種抗体を５０μＬずつ混合し、４℃で１時間インキュベートした。前記抗体として、１９種類の抗ＣＸＣＲ３抗体、ＣＸＣＲ３Ｂ－ｓｐｅｃｉｆｉｃ　ａｎｔｉｂｏｄｙ（Proteintech社）、先行技術抗体、または対照の抗体を用いた。先行技術抗体として、抗ＣＸＣＲ３抗体＃４９８０１（R＆D社）、または抗ＣＸＣＲ３抗体４Ｈｕ３（特許文献２に記載）を用いた。対照の抗体として、マウスアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、ラットアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、またはヒトアイソタイプコントロール抗体（GeneScript社）を用いた。インキュベート後、１００μＬのＦＡＣＳバッファーで、細胞を２回洗浄した。二次抗体としてＰＥ標識抗マウスＩｇＧ抗体（サザンバイオテック社）、Ａｌｅｘａ　Ｆｌｏｕｒ　４８８標識抗ラットＩｇＧ抗体（Abcam社）、ＡＰＣ標識抗ラットＩｇＧ抗体（Abcam社）、またはＡＰＣ標識抗ヒトＩgＧ抗体（Invitrogen社）の希釈液を各ウェルに５０μＬずつ加え、４℃で１時間インキュベートした。１００μＬのＦＡＣＳバッファーで細胞を２回洗浄した後、５０μＬのＦＡＣＳバッファーに懸濁し、フローサイトメーターＮｏｖｏｃｙｔｅ（Agilent社）にて細胞表面の蛍光強度を測定し、抗体の結合性を評価した。 CHO cells stably expressing human CXCR3A, human umbilical vascular endothelial cells (Lonza) confirmed in Example 6 to predominantly express CXCR3B, and human microvascular endothelial cells (Cell Systems) were washed with FACS buffer (PBS containing 1% FBS) and suspended in FACS buffer to a cell concentration of 1 x 10 cells/mL. Fc Block (Tonbo Biosciences) was added at a volume of 1/500 of the cell suspension, or goat serum diluted 1:1, and blocking was performed at 4°C for 30 minutes. After blocking, the cells were suspended at a concentration of 1 x 10 cells/50 μL. This cell suspension was mixed with 50 μL of each diluted antibody and incubated at 4°C for 1 hour. The antibodies used were 19 types of anti-CXCR3 antibodies, a CXCR3B-specific antibody (Proteintech), a prior art antibody, or a control antibody. Prior art antibodies used included anti-CXCR3 antibody #49801 (R&D) and anti-CXCR3 antibody 4Hu3 (described in Patent Document 2). Control antibodies included a mouse isotype control antibody (Fujifilm Wako Pure Chemical Industries, Ltd.), a rat isotype control antibody (Fujifilm Wako Pure Chemical Industries, Ltd.), or a human isotype control antibody (GeneScript). After incubation, the cells were washed twice with 100 μL of FACS buffer. 50 μL of a diluted solution of a secondary antibody, such as PE-labeled anti-mouse IgG antibody (Southern Biotech), Alexa Flour 488-labeled anti-rat IgG antibody (Abcam), APC-labeled anti-rat IgG antibody (Abcam), or APC-labeled anti-human IgG antibody (Invitrogen), was added to each well and incubated for 1 hour at 4°C. After washing twice with 100 μL of FACS buffer, the cells were suspended in 50 μL of FACS buffer and the fluorescence intensity on the cell surface was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.

　代表例として、１３Ｂ４、２０１００９－２－Ｃ、ＣＸＣＲ３Ｂ－ｓｐｅｃｉｆｉｃ　ａｎｔｉｂｏｄｙ、または先行技術抗体（以上、抗ケモカイン抗体）と、対照の抗体（アイソタイプ抗体）とを比較したヒストグラムを図２に示す。図２において、黒塗りはアイソタイプ抗体、白抜きは抗ケモカイン抗体をそれぞれ示す。ヒストグラムが右方シフトしたものを「結合した」と判断した。ＣＸＣＲ３Ｂ－ｓｐｅｃｉｆｉｃ　ａｎｔｉｂｏｄｙは、２種のヒト血管内皮細胞に結合し、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に結合しないことが確認された。１３Ｂ４および先行技術抗体は、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞と２種のヒト血管内皮細胞に結合することが確認された。２０１００９－２－Ｃは、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞のみに結合し、ヒト血管内皮細胞に結合しなかった。 As representative examples, Figure 2 shows histograms comparing 13B4, 201009-2-C, a CXCR3B-specific antibody, or a prior art antibody (all of which are anti-chemokine antibodies) with a control antibody (isotype antibody). In Figure 2, solid black indicates an isotype antibody, and open white indicates an anti-chemokine antibody. A rightward shift in the histogram was determined to indicate "binding." The CXCR3B-specific antibody was confirmed to bind to two types of human vascular endothelial cells, but not to CHO cells stably expressing human CXCR3A. 13B4 and the prior art antibody were confirmed to bind to CHO cells stably expressing human CXCR3A and two types of human vascular endothelial cells. 201009-2-C bound only to CHO cells stably expressing human CXCR3A, and did not bind to human vascular endothelial cells.

　抗体の細胞への結合特異性を表３に示す。ヒストグラムの右方シフトが観察されたものを「＋」、観察されなかったものを「－」と表示した。２０１００９－２－Ｃを含む１６種類の抗体は、ヒトＣＸＣＲ３Ａに特異的に結合し、ヒトＣＸＣＲ３Ｂを優勢に発現するヒト血管内皮細胞に結合しないことが確認された。 The cell binding specificity of the antibodies is shown in Table 3. A rightward shift in the histogram is indicated by a "+", and no rightward shift is indicated by a "-". It was confirmed that 16 antibodies, including 201009-2-C, specifically bind to human CXCR3A and do not bind to human vascular endothelial cells, which predominantly express human CXCR3B.

〔実施例８〕抗体の作製
（１)ｃＤＮＡクローニング
　実施例５で選択した１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃ、２０１００９－３－Ｈおよび２０１００９－１－Ｃの８種類の抗体遺伝子を用い、相同性配列選択的組み換えクローニング（Kurosawa N, Yoshioka M, Isobe M. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells. BMC Biotechnol. 2011 Apr 13;11:39.）によってｐＥＴ　ｖｅｃｔｏｒへの重鎖および軽鎖のクローニングを行った。 [Example 8] Production of antibodies (1) cDNA cloning Using the eight antibody genes selected in Example 5, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, 201009-2-C, 201009-3-H, and 201009-1-C, heavy and light chains were cloned into pET vectors by homologous sequence-selective recombination cloning (Kurosawa N, Yoshioka M, Isobe M. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells. BMC Biotechnol. 2011 Apr 13;11:39.).

（２）哺乳動物細胞への抗体発現プラスミド導入
　細胞培養用１２５ｍＬ三角フラスコ（ベントキャップ）に、ＥｘｐｉＣＨＯ細胞（Thermo Fisher Scientific社）を１．８×１０８細胞／３０ｍＬ／フラスコとなるように播種した。Ｅｘｐｉｆｅｃｔａｍｉｎｅ　ＣＨＯ　Ｔｒａｎｆｅｃｔｉｏｎ　Ｋｉｔ（Thermo Fisher Scientific社）を用いて、（１）で構築した重鎖および軽鎖の２種の抗体発現プラスミドをＥｘｐｉＣＨＯ細胞に共導入した。導入５日目に細胞上清を回収し、Ｐｒｏｔｅｉｎ　Ａアフィニティー精製によって精製抗体を得た。 (2) Introduction of antibody expression plasmids into mammalian cells. ExpiCHO cells (Thermo Fisher Scientific) were seeded into 125 mL cell culture Erlenmeyer flasks (vented cap) at 1.8 x 10 cells/30 mL/flask. Using an Expifectamine CHO Transfection Kit (Thermo Fisher Scientific), the two antibody expression plasmids constructed in (1), one for the heavy chain and one for the light chain, were co-transfected into ExpiCHO cells. Five days after transfection, the cell supernatant was collected, and purified antibodies were obtained by Protein A affinity purification.

〔実施例９〕フローサイトメトリーによるＣＸＣＲ３Ａへの結合評価
　実施例８にて得た１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃ、２０１００９－３－Ｈ、２０１００９－１－Ｃの８種類の抗体を、以下の試験に供した。 [Example 9] Evaluation of binding to CXCR3A by flow cytometry The eight antibodies obtained in Example 8, i.e., 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, 201009-2-C, 201009-3-H, and 201009-1-C, were subjected to the following tests.

　ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞をＦＡＣＳバッファー（ＰＢＳ，１％　ＦＢＳ含有）で洗浄し、細胞濃度が１×１０７細胞／ｍＬとなるようにＦＡＣＳバッファーで懸濁した。Ｆｃ　Ｂｌｏｃｋ（Tonbo biosciences社）を細胞懸濁液の１／５００量加え、４℃で３０分間ブロッキングを行った。ブロッキング後、１×１０５細胞／５０μＬとなるように細胞を懸濁した。この細胞懸濁液と、希釈した８種の抗ＣＸＣＲ３抗体、または対照の抗体を５０μＬずつ混合し、４℃で１時間インキュベートした。対照の抗体として、マウスアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、またはラットアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）を用いた。インキュベート後、１００μＬのＦＡＣＳバッファーで、細胞を２回洗浄した。二次抗体としてＰＥ標識抗マウスＩｇＧ抗体（サザンバイオテック社）、またはＡｌｅｘａ　Ｆｌｏｕｒ　４８８標識抗ラットＩｇＧ抗体（Abcam社）の希釈液を各ウェルに５０μＬずつ加え、４℃で１時間インキュベートした。１００μＬのＦＡＣＳバッファーで細胞を２回洗浄した後、５０μＬのＦＡＣＳバッファーに懸濁し、フローサイトメーターＮｏｖｏｃｙｔｅ（Agilent社）にて細胞表面の蛍光強度を測定し、抗体の結合性を評価した。 CHO cells stably expressing human CXCR3A were washed with FACS buffer (PBS containing 1% FBS) and suspended in FACS buffer to a cell concentration of 1 x 10 cells/mL. Fc Block (Tonbo Biosciences) was added at 1/500 the volume of the cell suspension, and blocking was performed at 4°C for 30 minutes. After blocking, the cells were suspended at 1 x 10 cells/50 μL. This cell suspension was mixed with 50 μL each of eight diluted anti-CXCR3 antibodies or a control antibody, and incubated at 4°C for 1 hour. Mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation) was used as the control antibody. After incubation, the cells were washed twice with 100 μL of FACS buffer. 50 μL of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech) or Alexa Flour 488-labeled anti-rat IgG antibody (Abcam) as a secondary antibody was added to each well and incubated for 1 hour at 4°C. After washing the cells twice with 100 μL of FACS buffer, they were suspended in 50 μL of FACS buffer and the fluorescence intensity on the cell surface was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.

　横軸に抗体の濃度、縦軸にフローサイトメーターのヒストグラムの蛍光強度の中央値をプロットしたグラフを作成した。８種類の抗体はいずれもヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞に用量依存的に結合することが確認された。各抗体のヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞への結合の結果を図３Ａ～図３Ｈに示した。５０％結合濃度（ＥＣ５０）、８０％結合濃度（ＥＣ８０）を算出し、表４にまとめた。 A graph was created in which the horizontal axis represents antibody concentration and the vertical axis represents the median fluorescence intensity of the flow cytometer histogram. All eight antibodies were confirmed to bind to CHO cells stably expressing human CXCR3A in a dose-dependent manner. The results of binding of each antibody to CHO cells stably expressing human CXCR3A are shown in Figures 3A to 3H. The 50% binding concentration (EC 50 ) and 80% binding concentration (EC 80 ) were calculated and are summarized in Table 4.

〔実施例１０〕細胞内Ｃａ２＋シグナル伝達の阻害評価
　実施例８にて得た１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃ、２０１００９－３－Ｈ、２０１００９－１－Ｃの８種類の抗体を、以下の試験に供した。 [Example 10] Evaluation of inhibition of intracellular Ca2 + signaling The eight antibodies obtained in Example 8, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, 201009-2-C, 201009-3-H, and 201009-1-C, were subjected to the following tests.

　ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞を、９６ウェルマイクロプレートに、各ウェル当たり２×１０４細胞／１００μＬの初期細胞濃度で播種し、２日間培養した。２日後に、培養培地を３μＭ　Ｃａｌ－５２０（AAT Bioquest社）、０．０５％　Ｐｌｕｒｏｎｉｃ－Ｆ１２７、及び２．５ｍＭプロベネシド（Thermo Fisher Scientific社）を含む溶液に交換した。１時間後、各ウェルに２００ｎＭ～０．８ｎＭの濃度範囲内に希釈した８種類の抗体、または対照の抗体を添加し、１５分間インキュベートした。対照の抗体として、マウスアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、またはラットアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）を用いた。インキュベート後、アッセイプレートをＣａ２＋シグナル解析装置ＦＤＳＳμＣＥＬＬ（浜松ホトニクス社）に設置し、１００ｎＭのＩＰ－１０（PEPROTECH社）によって各細胞を刺激した際の細胞内Ｃａ２＋濃度の一過的上昇に対する阻害活性を測定した。阻害活性は、「抗体なし、ＩＰ－１０あり」の場合を阻害率０％、「抗体なし、ＩＰ－１０なし」の場合を阻害率１００％として標準化を行い、相対的な値として算出した。横軸に各抗体の濃度、縦軸に阻害率（％）としてプロットしたグラフを図４Ａ～図４Ｈに示した。 Human CXCR3A-stably expressing CHO cells were seeded into a 96-well microplate at an initial cell concentration of 2 x 104 cells/100 μL per well and cultured for 2 days. After 2 days, the culture medium was replaced with a solution containing 3 μM Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Thermo Fisher Scientific). After 1 hour, eight antibodies diluted to concentrations ranging from 200 nM to 0.8 nM or a control antibody were added to each well and incubated for 15 minutes. Mouse isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) were used as control antibodies. After incubation, the assay plate was placed in a Ca2 + signal analyzer FDSSμCELL (Hamamatsu Photonics), and the inhibitory activity against the transient increase in intracellular Ca2 + concentration when each cell type was stimulated with 100 nM IP-10 (PEPROTECH) was measured. The inhibitory activity was calculated as a relative value by standardizing the inhibition rate of "no antibody, with IP-10" to 0% and the inhibition rate of "no antibody, without IP-10" to 100%. Graphs plotting the concentration of each antibody on the horizontal axis and the inhibition rate (%) on the vertical axis are shown in Figures 4A to 4H.

　８種類の抗体は、いずれもヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞におけるＩＰ－１０誘発の細胞内Ｃａ２＋シグナル伝達を用量依存的に阻害することが確認され、ＣＸＣＲ３依存的な細胞機能を遮断する活性を有するものであることが示された。各抗体の５０％阻害濃度（ＩＣ５０）は表５に示した。
　８種類の抗体のうち、実施例９、実施例１０において優れた活性を示した１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃの６種類の抗体を選択し、以降の解析に使用した。 All eight antibodies were confirmed to dose-dependently inhibit IP-10-induced intracellular Ca 2+ signaling in CHO cells stably expressing human CXCR3A, demonstrating that they have the activity of blocking CXCR3-dependent cellular functions. The 50% inhibitory concentration (IC 50 ) of each antibody is shown in Table 5.
Of the eight antibodies, six antibodies, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, and 201009-2-C, which showed excellent activity in Examples 9 and 10, were selected and used in the subsequent analysis.

〔実施例１１〕抗体の結合特異性評価
　ＣＸＣＲ３Ａに対する抗体の結合特異性を評価するため、実施例１０にて選抜した１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃの６種類の抗体を、以下の試験に供した。 [Example 11] Evaluation of antibody binding specificity To evaluate the binding specificity of antibodies to CXCR3A, the six antibodies selected in Example 10, i.e., 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, and 201009-2-C, were subjected to the following tests.

　ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞および他のケモカイン受容体発現細胞としてヒトＣＣＲ７安定発現ＣＨＯ細胞（社内製造）、ヒトＣＣＲ６安定発現ＣＨＯ細胞（社内製造）をＦＡＣＳバッファー（ＰＢＳ，１％　ＦＢＳ含有）で洗浄し、細胞濃度が１×１０７細胞／ｍＬとなるようにＦＡＣＳバッファーで懸濁した。Ｆｃ　Ｂｌｏｃｋ（Tonbo biosciences社）を細胞懸濁液の１／５００量加え、４℃で３０分間ブロッキングを行った。ブロッキング後、１×１０５細胞／５０μＬとなるように細胞を懸濁した。この細胞懸濁液と、希釈した６種類の抗ＣＸＣＲ３抗体、抗ＣＣＲ７抗体（社内製造）、抗ＣＣＲ６抗体（社内製造）、または対照の抗体を５０μＬずつ混合し、４℃で１時間インキュベートした。対照の抗体として、マウスアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、ラットアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、またはヒトアイソタイプコントロール抗体（GeneScript社）を用いた。インキュベート後、１００μＬのＦＡＣＳバッファーで２回、細胞を洗浄した。二次抗体として、ＰＥ標識抗マウスＩｇＧ抗体（サザンバイオテック社）、ＡＰＣ標識抗ラットＩｇＧ抗体（Abcam社）、またはＡＰＣ標識抗ヒトＩｇＧ抗体（Invitrogen社）の希釈液を各ウェルに５０μＬずつ加え、４℃で１時間インキュベートした。１００μＬのＦＡＣＳバッファーで細胞を２回洗浄した後、５０μＬのＦＡＣＳバッファーに懸濁し、フローサイトメーターＮｏｖｏｃｙｔｅ（Agilent社）にて細胞表面の蛍光強度を測定し抗体の結合性を評価した。 Human CXCR3A-stably expressing CHO cells and other chemokine receptor-expressing cells, including human CCR7-stably expressing CHO cells (manufactured in-house) and human CCR6-stably expressing CHO cells (manufactured in-house), were washed with FACS buffer (PBS containing 1% FBS) and suspended in FACS buffer to a cell concentration of 1 x 10 cells/mL. Fc Block (Tonbo Biosciences) was added at 1/500 the volume of the cell suspension, and blocking was performed at 4°C for 30 minutes. After blocking, the cells were suspended at a concentration of 1 x 10 cells/50 μL. This cell suspension was mixed with 50 μL each of six diluted anti-CXCR3 antibodies, anti-CCR7 antibodies (manufactured in-house), anti-CCR6 antibodies (manufactured in-house), or a control antibody, and incubated at 4°C for 1 hour. Control antibodies included mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation), rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation), and human isotype control antibody (GeneScript). After incubation, cells were washed twice with 100 μL of FACS buffer. 50 μL of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech), APC-labeled anti-rat IgG antibody (Abcam), or APC-labeled anti-human IgG antibody (Invitrogen) was added to each well and incubated for 1 hour at 4°C. After washing twice with 100 μL of FACS buffer, cells were suspended in 50 μL of FACS buffer and the cell surface fluorescence intensity was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.

　代表例として２０１００６－７－Ｇ、２０１００９－２－Ｃ、ヒトＣＣＲ７抗体、またはヒトＣＣＲ６抗体（以上、抗ケモカイン抗体）と、対照の抗体（アイソタイプ抗体）とを比較したヒストグラムを図５に示す。図５において、黒塗りはアイソタイプ抗体、白抜きは抗ケモカイン抗体をそれぞれ示す。ヒストグラムが右方シフトしたものを「結合した」と判断した。２０１００６－７－Ｇ、２０１００９－２－Ｃの結合は、ヒトＣＸＣＲ３Ａ安定発現ＣＨＯ細胞でのみ観察された。この結果から、２０１００６－７－Ｇ、２０１００９－２－Ｃは他のケモカイン受容体とは結合せず、ヒトＣＸＣＲ３Ａに特異的に結合することが確認された。１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆについても、図５と同様の結果が得られた。６種類のＣＸＣＲ３抗体の結合特異性を表６に示す。ヒストグラムの右方シフトが観察されたものを「＋」、観察されなかったものを「－」と表示した。 As representative examples, Figure 5 shows histograms comparing 201006-7-G, 201009-2-C, human CCR7 antibody, or human CCR6 antibody (all anti-chemokine antibodies) with a control antibody (isotype antibody). In Figure 5, the solid black indicates the isotype antibody, and the open white indicates the anti-chemokine antibody. A rightward shift in the histogram was considered to indicate "binding." Binding of 201006-7-G and 201009-2-C was observed only in CHO cells stably expressing human CXCR3A. These results confirmed that 201006-7-G and 201009-2-C do not bind to other chemokine receptors, but specifically bind to human CXCR3A. Similar results to those shown in Figure 5 were obtained for 13B4, 11F11, 201006-3-H, and 201006-5-F. The binding specificities of the six CXCR3 antibodies are shown in Table 6. Histograms showing a rightward shift are indicated by a "+" and those showing no shift are indicated by a "-".

〔実施例１２〕フローサイトメトリーによるヒト末梢血由来Ｔ細胞への結合評価
　実施例１０にて選抜した１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃの６種類の抗体を、以下の試験に供した。 [Example 12] Evaluation of binding to human peripheral blood-derived T cells by flow cytometry The six antibodies selected in Example 10, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, and 201009-2-C, were subjected to the following test.

　凍結ヒト末梢血単核細胞（Cellular Technology Limited社）を融解し、１％ Ｔ　ｃｅｌｌ　ＴｒａｎｓＡｃｔ，　ｈｕｍａｎと１０ｎｇ／ｍＬ　ＩＬ－２を含むＴｅｘＭＡＣＳ培地（Miltenyi Biotech社）で３日間培養し、活性化した。２～３日毎に培地交換（１０ｎｇ／ｍＬ　ＩＬ－２を含むＴｅｘＭＡＣＳ培地）を行い、拡大培養を行った。拡大培養１０日以降のＴ細胞を試験に使用した。ヒトＴ細胞を、ＦＡＣＳバッファー（ＰＢＳ，１％　ＦＢＳ含有）にて１：１希釈したヤギ血清（Thermo Fisher Scientific社）で１×１０７細胞／ｍＬとなるように懸濁し、４℃で３０分間ブロッキングを行った。ブロッキング後、４×１０６細胞／ｍＬになるように細胞を懸濁した。この細胞懸濁液と、希釈した６種類の抗体、または対照の抗体を２５μＬずつ混合し、４℃で１時間インキュベートした。対照の抗体として、マウスアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、またはラットアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）を用いた。インキュベート後、１２０μＬのＦＡＣＳバッファーで２回、細胞を洗浄した。二次抗体としてＰＥ標識抗マウスＩｇＧ抗体（サザンバイオテック社）、またはＡｌｅｘａ　Ｆｌｏｕｒ　４８８標識抗ラットＩｇＧ抗体（Abcam社）の希釈液を各ウェルに２５μＬずつ加え、４℃で１時間インキュベートした。ＦＡＣＳバッファーで細胞を２回洗浄した後、５０μＬのＦＡＣＳバッファーに懸濁し、フローサイトメーターＮｏｖｏｃｙｔｅ（Agilent社）にて細胞表面の蛍光強度を測定し、抗体の結合性を評価した。 Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were thawed and activated by culturing in TexMACS medium (Miltenyi Biotech) containing 1% T cell TransAct, human, and 10 ng/mL IL-2 for 3 days. The medium was replaced every 2–3 days (TexMACS medium containing 10 ng/mL IL-2) for expansion. T cells were used for testing after 10 days of expansion. Human T cells were suspended at 1 × 10 cells/mL in goat serum (Thermo Fisher Scientific) diluted 1:1 with FACS buffer (PBS containing 1% FBS) and blocked for 30 minutes at 4°C. After blocking, the cells were suspended at 4 × 10 cells/mL. This cell suspension was mixed with 25 μL of each of the six diluted antibodies or a control antibody and incubated at 4°C for 1 hour. Mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation) was used as a control antibody. After incubation, cells were washed twice with 120 μL of FACS buffer. 25 μL of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech) or Alexa Flour 488-labeled anti-rat IgG antibody (Abcam) was added to each well as a secondary antibody and incubated at 4°C for 1 hour. After washing twice with FACS buffer, cells were suspended in 50 μL of FACS buffer and cell surface fluorescence intensity was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.

　横軸に抗体の濃度、縦軸にフローサイトメーターのヒストグラムの蛍光強度の中央値をプロットしたグラフを作成した。６種類の抗体は、いずれもヒトＴ細胞に用量依存的に結合することが確認された。各抗体のヒトＴ細胞への結合の結果を図６Ａ～図６Ｆに示した。５０％結合濃度（ＥＣ５０）を算出し、表７にまとめた。 A graph was created in which the horizontal axis represents the antibody concentration and the vertical axis represents the median fluorescence intensity of the flow cytometer histogram. All six antibodies were confirmed to bind to human T cells in a dose-dependent manner. The results of binding of each antibody to human T cells are shown in Figures 6A to 6F. The 50% binding concentration (EC 50 ) was calculated and is summarized in Table 7.

〔実施例１３〕抗体のヒト末梢血由来Ｔ細胞遊走阻害活性評価
　実施例１０にて選抜した１３Ｂ４、１１Ｆ１１、２０１００６－３－Ｈ、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃの６種類の抗体を、以下の試験に供した。 [Example 13] Evaluation of antibody activity to inhibit migration of human peripheral blood-derived T cells Six types of antibodies selected in Example 10, namely, 13B4, 11F11, 201006-3-H, 201006-5-F, 201006-7-G, and 201009-2-C, were subjected to the following test.

　凍結ヒト末梢血単核細胞（Cellular Technology Limited社）を、実施例１２と同様に培養し、拡大培養１０日以降のＴ細胞を試験に使用した。ヒトＴ細胞を、アッセイバッファー（ＲＰＭＩ－１６４０＋０．１％　ＢＳＡ）で洗浄した。階段希釈した抗体を細胞に添加し、細胞密度２．０～２．４×１０６／ｍＬにて３７℃で６０分間静置した。インサート一体型９６ウェルトランズウェル、３．０ μｍポリカーボネート製メンブレン（コーニング社製）のレシーバープレートのウェル内に２．４ｎＭ ＩＰ－１０（PEPROTECH社）を添加した。トランスウェルのインサートをレシーバープレート上に設置後、上記の細胞懸濁液７５μＬをインサート内に播種し、３７℃にて９０分インキュベートした。インキュベート後、インサートを除去し、フローサイトメーターＮｏｖｏｃｙｔｅ（Agilent社）を用いて、遊走した細胞数を測定した。阻害活性は、「抗体なし、ＩＰ－１０あり」の場合を阻害率０％、「抗体なし、ＩＰ－１０なし」の場合を阻害率１００％として標準化を行い、相対的な値として算出した。代表例として、２０１００９－２－ＣのＩＰ－１０依存的Ｔ細胞遊走に対する阻害効果を図７に示す。横軸に抗体の濃度、縦軸に阻害率％をプロットした。各抗体のＴ細胞遊走阻害率を表８にまとめた。 Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were cultured as described in Example 12, and T cells from 10 days of expansion culture were used for the study. Human T cells were washed with assay buffer (RPMI-1640 + 0.1% BSA). Serially diluted antibodies were added to the cells and incubated at 37°C for 60 minutes at a cell density of 2.0-2.4 x 10 /mL. 2.4 nM IP-10 (PEPROTECH) was added to the wells of a receiver plate equipped with an insert-integrated 96-well Transwell with a 3.0 μm polycarbonate membrane (Corning). After placing the Transwell insert on the receiver plate, 75 μL of the above cell suspension was seeded into the insert and incubated at 37°C for 90 minutes. After incubation, the insert was removed, and the number of migrated cells was measured using a Novocyte flow cytometer (Agilent). The inhibitory activity was calculated as a relative value by standardizing the inhibition rate of "no antibody, with IP-10" to 0% and the inhibition rate of "no antibody, without IP-10" to 100%. As a representative example, the inhibitory effect of 201009-2-C on IP-10-dependent T cell migration is shown in Figure 7. The horizontal axis represents the antibody concentration, and the vertical axis represents the inhibition rate (%). The T cell migration inhibition rate of each antibody is summarized in Table 8.

　６種類の抗体は、いずれもヒトＴ細胞におけるＩＰ－１０誘導の細胞遊走を用量依存的に阻害することが確認され、ＣＸＣＲ３依存的な細胞機能を遮断する活性を有するものであることが示された。 All six antibodies were confirmed to dose-dependently inhibit IP-10-induced cell migration in human T cells, demonstrating their ability to block CXCR3-dependent cellular functions.

〔実施例１４〕フローサイトメトリーによる抗体内在化の評価
　１３Ｂ４、１１Ｆ１１、２０１００６－５－Ｆ、２０１００６－７－Ｇ、２０１００９－２－Ｃの５種類の抗体を、以下の試験に供した。 [Example 14] Evaluation of antibody internalization by flow cytometry Five types of antibodies, 13B4, 11F11, 201006-5-F, 201006-7-G, and 201009-2-C, were subjected to the following test.

　凍結ヒト末梢血単核細胞（Cellular Technology Limited社）を、実施例１２と同様に培養し、拡大培養１０日以降のＴ細胞を試験に使用した。ヒトＴ細胞を、ＦＡＣＳバッファー（ＰＢＳ，１％　ＦＢＳ含有）にて１：１希釈したヤギ血清（Thermo Fisher Scientific社）で１×１０７細胞／ｍＬとなるように懸濁し、４℃で３０分間ブロッキングを行った。ブロッキング後、１×１０５細胞／５０μＬになるように細胞を懸濁した。この細胞懸濁液と、５種類の抗体、または対照の抗体を、抗体濃度１ｎＭとなるよう混合し、４℃で３０分間インキュベートした。対照の抗体として、マウスアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）、またはラットアイソタイプコントロール抗体（富士フイルム和光純薬株式会社）を用いた。インキュベート後、冷ＦＡＣＳバッファーで２回、細胞を洗浄した。抗体の内在化を促進するため、洗浄後の細胞の一部を３７℃で３０分間インキュベートした。残りの細胞は４℃で３０分静置した。二次抗体としてＰＥ標識抗マウスＩｇＧ抗体（サザンバイオテック社）、またはＡｌｅｘａ　Ｆｌｏｕｒ　４８８標識抗ラットＩｇＧ抗体（Abcam社）の希釈液を各ウェルに５０μＬずつ加え、４℃で１５分間インキュベートした。冷ＦＡＣＳバッファーで細胞を２回洗浄した後、５０μＬの冷ＦＡＣＳバッファーに懸濁し、フローサイトメーターＮｏｖｏｃｙｔｅ（Agilent社）にて細胞表面の蛍光強度を測定した。 Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were cultured as described in Example 12, and T cells from 10 days of expansion were used for the study. Human T cells were suspended at 1 x 10 cells/mL in goat serum (Thermo Fisher Scientific) diluted 1:1 with FACS buffer (PBS containing 1% FBS) and blocked at 4°C for 30 minutes. After blocking, the cells were suspended at 1 x 10 cells/50 μL. This cell suspension was mixed with five antibodies or a control antibody at an antibody concentration of 1 nM and incubated at 4°C for 30 minutes. Mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation) was used as the control antibody. After incubation, the cells were washed twice with cold FACS buffer. To promote antibody internalization, a portion of the washed cells was incubated at 37°C for 30 minutes. The remaining cells were allowed to stand at 4°C for 30 minutes. 50 μL of a diluted solution of PE-labeled anti-mouse IgG antibody (Southern Biotech) or Alexa Flour 488-labeled anti-rat IgG antibody (Abcam) was added to each well as a secondary antibody, and the cells were incubated at 4°C for 15 minutes. After washing the cells twice with cold FACS buffer, they were suspended in 50 μL of cold FACS buffer, and the fluorescence intensity of the cell surface was measured using a Novocyte flow cytometer (Agilent).

　抗体の内在化は、受容体の細胞表面における発現量から間接的に評価した。すなわち４℃で抗体反応させた細胞の蛍光強度中央値を１００％として標準化し、３７℃で抗体反応させた細胞における蛍光強度中央値を相対的な値として示した。図８に各条件における蛍光強度中央値％を示す。４℃と比べ３７℃で反応させた細胞における蛍光強度中央値％の低下が示す通り、５種の抗体が受容体とともに細胞内に内在化することが確認された。 Antibody internalization was indirectly evaluated based on the expression level of the receptor on the cell surface. That is, the median fluorescence intensity of cells reacted with antibody at 4°C was normalized to 100%, and the median fluorescence intensity of cells reacted with antibody at 37°C was shown as a relative value. Figure 8 shows the median fluorescence intensity % under each condition. As shown by the decrease in median fluorescence intensity % in cells reacted at 37°C compared to 4°C, it was confirmed that the five antibodies were internalized into the cells along with the receptor.

〔実施例１５〕ヒト化抗ＣＸＣＲ３抗体の作製
　２０１００９－２－Ｃの場合を例示する。２０１００９－２－ＣのＣＤＲを移植したヒト化抗体を作製するために、ヒトフレームワークを２０１００９－２－Ｃとヒト生殖系列ＶＨおよびＶＬ遺伝子との相同性に基づいて選択した。コンピュータモデリングに基づいて、２０１００９－２－Ｃの予測される抗体の立体構造を支持することができるように、選択されたヒト生殖系列ＶＨおよびＶＬの配列に変異を加えた複数の抗体、「ＶＨ１＋ＶＬ１」、「ＶＨ１＋ＶＬ２」、「ＶＨ１＋ＶＬ３」、「ＶＨ１＋ＶＬ４」、「ＶＨ２＋ＶＬ１」、「ＶＨ２＋ＶＬ２」、「ＶＨ２＋ＶＬ３」、「ＶＨ２＋ＶＬ４」をデザインした。ＶＨ１（配列番号２９８）、ＶＨ２（配列番号２９９）、および２０１００９－２－ＣのＶＨ領域（配列番号２５７）のアライメントを図９Ａに示す。ＶＬ１（配列番号３００）、ＶＬ２（配列番号３０１）、ＶＬ３（配列番号３０２）、ＶＬ４（配列番号３０３）および２０１００９－２－ＣのＶＬ領域（配列番号２５９）のアライメントを図９Ｂに示す。 Example 15: Production of humanized anti-CXCR3 antibodies The case of 201009-2-C will be exemplified. To produce a humanized antibody into which the CDRs of 201009-2-C were grafted, human frameworks were selected based on the homology between 201009-2-C and human germline VH and VL genes. Based on computer modeling, multiple antibodies were designed by adding mutations to the selected human germline VH and VL sequences so that the predicted antibody conformation of 201009-2-C could be supported: "VH1+VL1,""VH1+VL2,""VH1+VL3,""VH1+VL4,""VH2+VL1,""VH2+VL2,""VH2+VL3," and "VH2+VL4." An alignment of VH1 (SEQ ID NO:298), VH2 (SEQ ID NO:299), and the VH region of 201009-2-C (SEQ ID NO:257) is shown in Figure 9A. An alignment of VL1 (SEQ ID NO:300), VL2 (SEQ ID NO:301), VL3 (SEQ ID NO:302), VL4 (SEQ ID NO:303), and the VL region of 201009-2-C (SEQ ID NO:259) is shown in Figure 9B.

　各抗体のＶＨ領域にヒトＩｇＧ１の重鎖定常領域（配列番号３０４）、ＶＬ領域にκ鎖定常領域（配列番号３０５）を接続した人工遺伝子を合成した。また、比較対象として、２０１００９－２－ＣのＶＨ領域にヒトＩｇＧ１の重鎖定常領域、ＶＬ領域にκ鎖定常領域を接続したキメラ型抗ＣＸＣＲ３抗体「ＶＨ＋ＶＬ－Ｃｈｉｍｅｒａ」の人工遺伝子も合成した。抗体遺伝子はＣＨＯ細胞に導入し、その培養液よりアフィニティクロマトグラフィーにて精製抗体を製造し、以下の試験に供した。いずれの精製抗体もＳＤＳ－ＰＡＧＥ法により純度を検定したところ９０％以上であることを確認した。 Artificial genes were synthesized in which the human IgG1 heavy chain constant region (sequence number 304) was connected to the VH region of each antibody, and the kappa chain constant region (sequence number 305) was connected to the VL region. For comparison, an artificial gene for a chimeric anti-CXCR3 antibody "VH+VL-Chimera" was also synthesized, in which the human IgG1 heavy chain constant region was connected to the VH region of 201009-2-C, and the kappa chain constant region was connected to the VL region. The antibody genes were introduced into CHO cells, and purified antibodies were produced from the culture medium by affinity chromatography and subjected to the following tests. The purity of all purified antibodies was confirmed to be 90% or higher when tested by SDS-PAGE.

　次に、実施例１２の方法に従ってヒトＴ細胞に対する結合活性の測定を行った。横軸に抗体の濃度、縦軸にフローサイトメーターのヒストグラムの蛍光強度の中央値をプロットしたグラフを作成した。各抗体のヒトＴ細胞への結合の結果を図１０Ａ～図１０Ｈに示し、算出された５０％結合濃度（ＥＣ５０）を表９にまとめた。また、実施例１３の方法に従って、各抗体のヒトＴ細胞遊走阻害活性を評価した。各抗体のＴ細胞遊走阻害率の用量依存性を表１０にまとめた。「ＶＨ１＋ＶＬ１」、「ＶＨ１＋ＶＬ４」、「ＶＨ２＋ＶＬ１」、「ＶＨ２＋ＶＬ４」の活性について、横軸に各抗体の濃度、縦軸に阻害率（％）をプロットしたグラフを作成し、図１１Ａ～図１１Ｄに示した。算出された５０％阻害濃度（ＩＣ５０）を表１１にまとめた。ＶＨ＋ＶＬ－Ｃｈｉｍｅｒａの結合活性および阻害活性が、２０１００９－２－Ｃとほぼ同等であることから、ヒト抗体の定常表域への変換が結合活性および阻害活性に影響しないことを確認した。また、ヒト化ＣＸＣＲ３抗体としてデザインした、８種の抗体の結合活性、遊走阻害活性はＶＨ＋ＶＬ－Ｃｈｉｍｅｒａと同等であることから、すべてのデザインにおいてヒト化後に結合活性および阻害活性が維持されていることを確認した。 Next, binding activity to human T cells was measured according to the method of Example 12. Graphs were created by plotting antibody concentration on the horizontal axis and median fluorescence intensity of flow cytometer histograms on the vertical axis. The results of binding of each antibody to human T cells are shown in Figures 10A to 10H, and the calculated 50% binding concentrations (EC 50 ) are summarized in Table 9. Furthermore, the human T cell migration inhibitory activity of each antibody was evaluated according to the method of Example 13. The dose dependency of the T cell migration inhibition rate for each antibody is summarized in Table 10. Graphs were created by plotting the concentration of each antibody on the horizontal axis and the inhibition rate (%) on the vertical axis for the activity of "VH1 + VL1,""VH1 + VL4,""VH2 + VL1," and "VH2 + VL4," and are shown in Figures 11A to 11D. The calculated 50% inhibitory concentrations (IC 50 ) are summarized in Table 11. The binding activity and inhibitory activity of VH+VL-Chimera were almost equivalent to those of 201009-2-C, confirming that conversion to the constant surface region of a human antibody does not affect the binding activity and inhibitory activity. Furthermore, the binding activity and migration inhibitory activity of the eight antibodies designed as humanized CXCR3 antibodies were equivalent to those of VH+VL-Chimera, confirming that the binding activity and inhibitory activity were maintained after humanization in all designs.

Claims (14)

　ヒトＣＸＣＲ３に特異的に結合する抗体であって、
　ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、
　ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、
　ヒト血管内皮細胞に特異的に結合せず、
　下記（ＡＢ１）～（ＡＢ９）、（ＡＢ１２）～（ＡＢ２７）：
（ＡＢ２６）配列番号２５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２３）配列番号２２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２４）配列番号２３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２５）配列番号２４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２７）配列番号２６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１）配列番号１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２）配列番号１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ３）配列番号２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ５）配列番号４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ８）配列番号７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１６）配列番号１５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１７）配列番号１６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１８）配列番号１７１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１７２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１７３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１７４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１７５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１７６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２０）配列番号１９１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１９２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１９３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１９４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１９５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１９６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２１）配列番号２０１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２０２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２０３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２０４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２０５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２０６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ２２）配列番号２１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号２１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号２１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号２１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号２１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号２１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ４）配列番号３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ６）配列番号５１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号５２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号５３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号５４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号５５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号５６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ７）配列番号６１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号６２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号６３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号６４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号６５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号６６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ９）配列番号８１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号８２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号８３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号８４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号８５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号８６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１２）配列番号１１１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１１２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１１３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１１４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１１５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１１６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１３）配列番号１２１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１２２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１２３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１２４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１２５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１２６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１４）配列番号１３１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１３２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１３３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１３４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１３５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１３６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１５）配列番号１４１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１４２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１４３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１４４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１４５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１４６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
（ＡＢ１９）配列番号１８１で表されるアミノ酸配列を含む重鎖ＣＤＲ１、配列番号１８２で表されるアミノ酸配列を含む重鎖ＣＤＲ２、配列番号１８３で表されるアミノ酸配列を含む重鎖ＣＤＲ３、配列番号１８４で表されるアミノ酸配列を含む軽鎖ＣＤＲ１、配列番号１８５で表されるアミノ酸配列を含む軽鎖ＣＤＲ２、及び配列番号１８６で表されるアミノ酸配列を含む軽鎖ＣＤＲ３を有する、
のいずれかを満たす、抗体。 An antibody that specifically binds to human CXCR3,
specifically binds to the extracellular domain of human CXCR3A;
It has the activity of blocking CXCR3A-dependent cell functions,
It does not specifically bind to human vascular endothelial cells,
The following (AB1) to (AB9), (AB12) to (AB27):
(AB26) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 251, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 252, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 253, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 254, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 255, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 256.
(AB23) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 221, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 222, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 223, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 224, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 225, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 226,
(AB24) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 231, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 232, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 233, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 234, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 235, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 236.
(AB25) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 242, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 245, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 246,
(AB27) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 261, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 262, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 263, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 264, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 265, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 266.
(AB1) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6,
(AB2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16,
(AB3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26,
(AB5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46,
(AB8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76.
(AB16) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 151, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 152, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 153, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 154, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 155, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 156.
(AB17) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 161, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 162, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 163, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 164, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 165, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 166.
(AB18) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 171, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 172, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 173, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 174, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 175, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 176.
(AB20) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 191, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 192, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 193, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 194, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 195, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 196.
(AB21) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 201, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 202, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 203, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 204, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 205, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 206,
(AB22) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 211, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 212, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 213, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 214, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 215, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 216.
(AB4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.
(AB6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56.
(AB7) A antibody having a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66,
(AB9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86.
(AB12) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116.
(AB13) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 126.
(AB14) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 132, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 133, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 135, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 136.
(AB15) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 141, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 142, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 143, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 144, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 145, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 146.
(AB19) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 181, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 182, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 183, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 184, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 185, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 186.
Antibodies that meet any of the above criteria. 　ヒトＣＸＣＲ３に特異的に結合する抗体であって、
　ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、
　ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、
　ヒト血管内皮細胞に特異的に結合せず、
　下記（Ｃ１）～（Ｃ９）、（Ｃ１２）～（Ｃ２７）：
（Ｃ２６）配列番号２５７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２５９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２３）配列番号２２７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２２９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２４）配列番号２３７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２３９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２５）配列番号２４７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２４９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２７）配列番号２６７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２６９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１）配列番号７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２）配列番号１７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ３）配列番号２７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ５）配列番号４７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号４９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ８）配列番号７７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号７９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１６）配列番号１５７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１５９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１７）配列番号１６７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１６９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１８）配列番号１７７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１７９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２０）配列番号１９７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１９９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２１）配列番号２０７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２０９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ２２）配列番号２１７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号２１９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ４）配列番号３７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号３９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ６）配列番号５７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号５９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ７）配列番号６７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号６９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ９）配列番号８７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号８９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１２）配列番号１１７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１１９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１３）配列番号１２７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１２９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１４）配列番号１３７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１３９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１５）配列番号１４７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１４９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
（Ｃ１９）配列番号１８７で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号１８９で表されるアミノ酸配列を含む軽鎖可変領域を有する、
のいずれかを満たす、抗体。 An antibody that specifically binds to human CXCR3,
specifically binds to the extracellular domain of human CXCR3A;
It has the activity of blocking CXCR3A-dependent cell functions,
It does not specifically bind to human vascular endothelial cells,
The following (C1) to (C9), (C12) to (C27):
(C26) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 257, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 259;
(C23) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 227, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 229;
(C24) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 237, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 239;
(C25) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 247, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 249;
(C27) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 267, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 269;
(C1) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9;
(C2) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19.
(C3) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29.
(C5) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 49.
(C8) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 77, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 79;
(C16) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 157, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 159.
(C17) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 167, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 169;
(C18) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 177, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 179;
(C20) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 197, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 199;
(C21) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 207, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 209;
(C22) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 217, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 219,
(C4) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 39,
(C6) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 59.
(C7) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 69;
(C9) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89.
(C12) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 117, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 119.
(C13) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 127, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 129.
(C14) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139.
(C15) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 147, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 149;
(C19) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 187, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 189;
Antibodies that meet any of the above criteria. 　ヒトＣＸＣＲ３に特異的に結合する抗体であって、
　ヒトＣＸＣＲ３Ａの細胞外ドメインに特異的に結合し、
　ＣＸＣＲ３Ａ依存的な細胞機能を遮断する活性を有し、
　ヒト血管内皮細胞に特異的に結合せず、
　請求項２に記載の抗体である第一抗体と受容体との結合を競合阻害する、第二抗体。 An antibody that specifically binds to human CXCR3,
specifically binds to the extracellular domain of human CXCR3A;
It has the activity of blocking CXCR3A-dependent cell functions,
It does not specifically bind to human vascular endothelial cells,
A second antibody that competitively inhibits the binding of the first antibody, which is the antibody of claim 2, to a receptor. 　ヒト化抗体又はキメラ型抗体である、請求項１又は３に記載の抗体。 The antibody of claim 1 or 3, which is a humanized antibody or a chimeric antibody. 　多重特異性抗体である、請求項１～４のいずれかに記載の抗体。 The antibody according to any one of claims 1 to 4, which is a multispecific antibody. 　内在化活性を有する、請求項１～５のいずれかに記載の抗体。 An antibody described in any one of claims 1 to 5, which has internalization activity. 　他の分子が結合した修飾抗体である、請求項１～６のいずれかに記載の抗体。 The antibody described in any one of claims 1 to 6, which is a modified antibody to which another molecule is bound. 　前記修飾抗体が、抗体薬物複合体である、請求項７に記載の抗体。 The antibody of claim 7, wherein the modified antibody is an antibody-drug conjugate. 　請求項１～６のいずれかに記載の抗体をコードする、核酸。 A nucleic acid encoding the antibody described in any one of claims 1 to 6. 　請求項９に記載の核酸を含む、細胞。 A cell containing the nucleic acid of claim 9. 　請求項１～８のいずれかに記載の抗体を有効成分として含有する、医薬。 A pharmaceutical comprising the antibody described in any one of claims 1 to 8 as an active ingredient. 　ＣＸＣＲ３依存的な細胞機能の障害が関与している疾患、障害、又は病状の治療に用いられる、請求項１１に記載の医薬。 The pharmaceutical agent described in claim 11, which is used to treat a disease, disorder, or condition involving impairment of CXCR3-dependent cellular function. 　前記疾患、障害、又は病状が、Ｔｈ１免疫異常疾患である、請求項１２に記載の医薬。 The pharmaceutical according to claim 12, wherein the disease, disorder, or condition is a Th1 immune disorder. 　前記Ｔｈ１免疫異常疾患が、心血管障害、神経系障害、炎症性疾患、自己免疫疾患、代謝性疾患、感染症、血液癌、又は固形癌である、請求項１３に記載の医薬。 The pharmaceutical according to claim 13, wherein the Th1 immune dysregulation disease is a cardiovascular disorder, a nervous system disorder, an inflammatory disease, an autoimmune disease, a metabolic disease, an infectious disease, a blood cancer, or a solid cancer.
PCT/JP2025/002546 2024-01-31 2025-01-28 Antibody, nucleic acid, cell, and medicine Pending WO2025164598A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2025546599A JP7784193B1 (en) 2024-01-31 2025-01-28 Antibodies, nucleic acids, cells, and drugs

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463627739P 2024-01-31 2024-01-31
US63/627,739 2024-01-31

Publications (1)

Publication Number Publication Date
WO2025164598A1 true WO2025164598A1 (en) 2025-08-07

Family

ID=96590811

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2025/002546 Pending WO2025164598A1 (en) 2024-01-31 2025-01-28 Antibody, nucleic acid, cell, and medicine

Country Status (2)

Country Link
JP (1) JP7784193B1 (en)
WO (1) WO2025164598A1 (en)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI HAI, RONG SHIKUO, CHEN CHAO, FAN YAYUN, CHEN TUO, WANG YONG, CHEN DONGMEI, YANG CHUN, YANG JIALI: "Disparate roles of CXCR3A and CXCR3B in regulating progressive properties of colorectal cancer cells", MOLECULAR CARCINOGENESIS, JOHN WILEY & SONS, INC., US, vol. 58, no. 2, 1 February 2019 (2019-02-01), US , pages 171 - 184, XP093342075, ISSN: 0899-1987, DOI: 10.1002/mc.22917 *

Also Published As

Publication number Publication date
JP7784193B1 (en) 2025-12-11

Similar Documents

Publication Publication Date Title
JP7284557B2 (en) Pharmaceutical composition
WO2022042690A1 (en) Ccr8 antibody and application thereof
CN105884895B (en) Anti-human CCR7 antibody, hybridoma, nucleic acid, carrier, cell, pharmaceutical composition and antibody immobilized carrier
JP2008044966A (en) Antibody against interleukin-22 and use therefor
IL263611A (en) Pd-l1-specific antibodies and methods of using the same
JP2022101631A (en) Anti-pd-l1 antibody and il-7 fusions
WO2017049452A1 (en) Fully human antibody against human cd137 and use thereof
CN112409483A (en) Anti-PD-L1 Nanobody
US9631024B2 (en) Antibodies that bind LGR4, their use in inhibiting neoplastic cells and in treating tumors
CN116761821B (en) Anti-PAR-2 antibodies and methods of use thereof
WO2011108008A2 (en) Antibody for targeted induction of apoptosis, cdc and adcc mediated killing of cancer cells, tbl-cln1
WO2011038149A2 (en) Methods of treating inflammation
JP7754506B2 (en) Antibodies to the poliovirus receptor (PVR) and uses thereof
JP7784193B1 (en) Antibodies, nucleic acids, cells, and drugs
JP7458049B2 (en) Autoimmune disease treatment agent
EP4549572A1 (en) Fusion protein
CN115181180B (en) Antibodies against human programmed death ligand-1 (PD-L1) and their applications
WO2024098939A1 (en) Bifunctional fusion protein and use thereof
WO2024085081A1 (en) Antibody, nucleic acid, cell, and pharmaceutical
KR20240137027A (en) Antihuman CXCL1 antibody
JP2024506664A (en) Anti-VEGF antibodies and their uses
JP2018505144A (en) Anti-CXCL12 antibody molecules and uses thereof
CN121181698A (en) An anti-LILRB2 antibody or its antigen-binding fragment and its application
EA045980B1 (en) ANTIBODIES AGAINST THE POLIO VIRUS RECEPTOR (PVR) AND THEIR APPLICATION
EA050692B1 (en) MONOCLONAL ANTIBODY AGAINST LAG-3 AND ITS ANTIGEN-BINDING FRAGMENT AND THEIR APPLICATION

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25748180

Country of ref document: EP

Kind code of ref document: A1