Antibody, nucleic acid, cell, and medicine

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

Antibodies, nucleic acids, cells, and drugs

[0001] 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 comprising the antibody as an active ingredient.

[0002] 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. Chemokine functions are important in immune cell differentiation and homeostasis, primary and secondary immune responses, and immune cell migration in various diseases. Nearly 50 chemokines have been reported in humans and mice (Non-Patent Document 1). Chemokine activity 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 G protein-coupled, 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).

[0003] CXCR3, also known as G protein-coupled receptor 9 (GPR9) or CD183, is a chemokine receptor primarily expressed 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.

[0004] 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). 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, and induces cell chemotaxis, cell proliferation, cell survival, tumor cell metastasis, and the like.

[0005] CXCR3B has an N-terminal domain 47 amino acids longer than CXCR3A due to alternative splicing. CXCR3B expression has been confirmed in tissues such as 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. Binding of ligands to CXCR3B activates Gas, inhibiting the growth, migration, and promoting apoptosis of endothelial and tumor cells. Thus, CXCR3A and CXCR3B are thought to play opposing roles. CXCR3Alt is weakly co-expressed in CXCR3A-expressing cells and plays a role in chemotaxis toward CXCL11 (Non-Patent Document 2).

[0006] 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 the inflammatory site and are involved in various inflammatory diseases. Examples include Th1-mediated 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). More recent studies have also suggested a link between these CXCLs and a wider range of diseases, including influenza, viral infections such as COVID-19, and tumors (Non-Patent Documents 3 and 4). Thus, there is a need for agents (eg, antibodies) and methods that target CXCR3 signaling to reduce the progression of disorders associated with CXCR3 signaling.

[0007] Significant 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, pathological and physiological diversity in biological roles, and the difficulty of developing specific small molecules due to similar receptor structures are thought to have made drug development difficult (Non-Patent Document 5).

[0008] CXCR3 has also been the target of many drug discovery programs, and the development of small molecule antagonists and antibodies has been progressing. 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, and good tolerability, kinetics, and safety have been confirmed in healthy subjects, suggesting that it may progress to further clinical development stages (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). This result suggests the clinical value of therapies targeting the CXCL10-CXCR3 cascade for Th1-related diseases.

[0009] Patent Document 1 discloses six types of humanized monoclonal antibodies that specifically bind to human CXCR3, and also discloses the amino acid sequences of the complementarity-determining regions, heavy chain variable regions, and light chain variable regions of each monoclonal antibody.

[0010] Patent Document 2 discloses four types of monoclonal antibodies that specifically bind to human CXCR3 and their humanized antibodies. 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.

[0011] Patent Document 3 discloses a humanized version of a monoclonal antibody that specifically binds to human CXCR3. The amino acid sequences of the complementarity-determining regions, heavy chain variable region, and light chain variable region of this monoclonal antibody are also disclosed. CXCR3 depletion using this antibody has been shown to be effective in treating vitiligo vulgaris.

[0012] 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.

[0013] International Publication No. WO 2008 / 094942 International Publication No. WO 2013 / 109974 International Publication No. WO 2018 / 119288

[0014] 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.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.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 CXC 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 randomized study", Gut. 2014 Mar;63(3):442-50.

[0015] As described 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, an object of the present disclosure is to provide a novel anti-human CXCR3 antibody that has superior properties as a pharmaceutical ingredient.

[0016] One aspect of the present disclosure is an antibody that specifically binds to human CXCR3, which specifically binds to the extracellular domain of human CXCR3A and has activity of blocking CXCR3A-dependent cellular functions, but 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) an antibody having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) An antibody that satisfies any one of the following: 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.

[0017] 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 has the following characteristics (C1) to (C9), (C12) to (C27): (C26) has 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) has 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) has 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) has 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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.

[0018] 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, which is the above-mentioned antibody, to the receptor.

[0019] Preferably, the antibody is a humanized antibody or a chimeric antibody.

[0020] Preferably, the antibody is a multispecific antibody.

[0021] Preferably, the antibody has internalizing activity.

[0022] Preferably, the antibody is a modified antibody to which another molecule is attached.

[0023] Preferably, the modified antibody is an antibody-drug conjugate.

[0024] One aspect of the present disclosure is a nucleic acid encoding the above-described antibody.

[0025] One aspect of the present disclosure is a cell comprising the above-described nucleic acid.

[0026] One aspect of the present disclosure is a pharmaceutical comprising the above-described antibody as an active ingredient.

[0027] Preferably, the medicament is used for the treatment of a disease, disorder or condition involving impairment of CXCR3-dependent cellular function.

[0028] Preferably, the disease, disorder or condition is a Th1 immune dysregulation disorder.

[0029] 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.

[0030] According to the present disclosure, a novel anti-human CXCR3 antibody having superior properties as a pharmaceutical raw material can be provided.

[0031]

[0033] Figure 1 is a graph showing the results of quantifying gene expression of CXCR3A and CXCR3B performed in Example 6, demonstrating that human vascular endothelial cells predominantly express CXCR3B.

[0034] Figure 2 is a histogram showing the results of flow cytometry performed in Example 7, illustrating the binding properties of antibodies (13B4, 201009-2-C) to CHO cells stably expressing human CXCR3A and vascular endothelial cells.

[0035] Figure 3 is a graph showing the results of evaluating the binding ability of antibody (13B4) to CHO cells stably expressing human CXCR3A.

[0036] Figure 4 is a graph showing the results of evaluating the binding ability of antibody (11F11) to CHO cells stably expressing human CXCR3A.

[0037] Figure 5 is a graph showing the results of evaluating the binding ability of antibody (201006-3-H) to CHO cells stably expressing human CXCR3A.

[0038] Figure 6 is a graph showing the results of evaluating the binding ability of antibody (201006-5-F) to CHO cells stably expressing human CXCR3A. Graph showing the results of evaluating the binding ability of antibody (201006-7-G) to CHO cells stably expressing human CXCR3A. Graph showing the results of evaluating the binding ability of antibody (201009-2-C) to CHO cells stably expressing human CXCR3A. Graph showing the results of evaluating the binding ability of antibody (201009-3-H) to CHO cells stably expressing human CXCR3A. Graph showing the results of evaluating the binding ability of antibody (201009-1-C) to CHO cells stably expressing human CXCR3A. Graph showing the dose dependency of the inhibitory activity of antibody (13B4) against human CXCR3A. Graph showing the dose dependency of the inhibitory activity of antibody (11F11) against human CXCR3A. Graph showing the dose dependency of the inhibitory activity of antibody (201006-3-H) against human CXCR3A. Graph showing the dose dependency of the inhibitory activity of antibody (201006-5-F) against human CXCR3A. Graph showing the dose dependency of the inhibitory activity of antibody (201006-7-G) against human CXCR3A. Graph showing the dose dependency of the inhibitory activity of antibody (201009-2-C) against human CXCR3A. Graph showing the dose dependency of the inhibitory activity of antibody (201009-3-H) against human CXCR3A. Graph showing the dose dependency of the inhibitory activity of 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. 2 is a graph showing the results of evaluating the binding of antibody (13B4) to human peripheral blood mononuclear cell-derived T cells. 3 is a graph showing the results of evaluating the binding of antibody (11F11) to human peripheral blood mononuclear cell-derived T cells. 4 is a graph showing the results of evaluating the binding of antibody (201006-3-H) to human peripheral blood mononuclear cell-derived T cells. 5 is a graph showing the results of evaluating the binding of antibody (201006-5-F) to human peripheral blood mononuclear cell-derived T cells. 6 is a graph showing the results of evaluating the binding of antibody (201006-7-G) to human peripheral blood mononuclear cell-derived T cells. 1 is a graph showing the results of an evaluation of the binding ability of antibody (201009-2-C) to human peripheral blood mononuclear cell-derived T cells. 2 is a graph showing the dose-dependence of the inhibitory activity of antibody (201009-2-C) on the migration of human peripheral blood mononuclear cell-derived T cells. 3 is a graph showing the results of flow cytometry performed in Example 14, demonstrating that the antibody is internalized together with its receptor in human peripheral blood mononuclear cell-derived T cells. 4 is an explanatory diagram showing the alignment of the heavy chain variable region of a humanized anti-CXCR3 antibody. 5 is an explanatory diagram showing the alignment of the light chain variable region of a humanized anti-CXCR3 antibody. 6 is a graph showing the results of an evaluation of the binding ability of antibody (VH1+VL1) to human peripheral blood mononuclear cell-derived T cells. 7 is a graph showing the results of an evaluation of the binding ability of antibody (VH1+VL2) to human peripheral blood mononuclear cell-derived T cells. 8 is a graph showing the results of an evaluation of the binding ability of antibody (VH1+VL3) to human peripheral blood mononuclear cell-derived T cells. 1 is a graph showing the results of evaluating the binding ability of an antibody (VH1+VL4) to human peripheral blood mononuclear cell-derived T cells. FIG. 2 is a graph showing the results of evaluating the binding ability of an antibody (VH2+VL1) to human peripheral blood mononuclear cell-derived T cells. FIG. 3 is a graph showing the results of evaluating the binding ability of an antibody (VH2+VL2) to human peripheral blood mononuclear cell-derived T cells. FIG. 4 is a graph showing the results of evaluating the binding ability of an antibody (VH2+VL3) to human peripheral blood mononuclear cell-derived T cells. FIG. 5 is a graph showing the results of evaluating the binding ability of an antibody (VH2+VL4) to human peripheral blood mononuclear cell-derived T cells. FIG. 6 is a graph showing the dose dependency of the inhibitory activity of an antibody (VH1+VL1) on the migration of human peripheral blood mononuclear cell-derived T cells.Graphs showing the dose-dependence of the inhibitory activity of antibody (VH1+VL4) on human peripheral blood mononuclear cell-derived T cell migration. Graphs showing the dose-dependence of the inhibitory activity of antibody (VH2+VL1) on human peripheral blood mononuclear cell-derived T cell migration. Graphs showing the dose-dependence of the inhibitory activity of antibody (VH2+VL4) on human peripheral blood mononuclear cell-derived T cell migration.

[0032] In the present disclosure, complementarity determining region is abbreviated as CDR. In the present disclosure, the heavy chain variable region may be abbreviated as VH, the heavy chain constant region as CH, the light chain variable region as VL, and the light chain constant region as CL. In the present disclosure, the term "antibody" may be replaced with "immunoglobulin." In the present disclosure, the term "nucleic acid" may be replaced with "DNA" or "gene."

[0033] <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. A 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.

[0034] 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 is the amino acid number, and the right side is each domain. Note that the boundaries between each domain may vary slightly. 1 to 53: N-terminal domain 81 to 89: Intracellular first loop domain 111 to 125: Extracellular first loop domain 148 to 169: Intracellular second loop domain 190 to 212: Extracellular second loop domain 234 to 255: Intracellular third loop domain 278 to 298: Extracellular third loop domain 322 to 368: C-terminal domain

[0035] <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.

[0036] An 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.

[0037] An antibody according to one embodiment has the following (A1) to (A28): (A1) having 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) having 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) having 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) having 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) having 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) having 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) 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, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63; (A8) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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.

[0038] The antibody according to one embodiment has the following (B1) to (B28): (B1) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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.

[0039] In one embodiment, the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 of the antibody satisfy any one of (AB1) to (AB9) and (AB12) to (AB27) above.

[0040] In one embodiment, the antibody has heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 as follows (AB10), (AB11), (AB28): (AB10) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93, light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96; (AB11) heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103, light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106; (AB28) The antibody satisfies any one of the following: 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.

[0041] In one embodiment, the heavy chain variable region and the light chain variable region of the antibody satisfy any one of (C1) to (C9) and (C12) to (C27) above.

[0042] In one embodiment, the antibody has a heavy chain variable region and a light chain variable region that satisfy any of the following conditions (C10), (C11), and (C28): (C10) having 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) having 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) having 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.

[0043] 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" described in the Examples below.

[0044] 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" described in the Examples below.

[0045] The heavy chain variable region (SEQ ID NO: 27) specified by (C3) contains heavy chain CDR1 to CDR3 (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 CDR1 to CDR3 (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" described in the Examples below.

[0046] The heavy chain variable region specified by (C4) (SEQ ID NO: 37) contains heavy chain CDR1 to CDR3 (SEQ ID NOs: 31 to 33) specified by (A4) or (AB4). The light chain variable region specified by (C4) (SEQ ID NO: 39) contains light chain CDR1 to CDR3 (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" described in the Examples below.

[0047] The heavy chain variable region (SEQ ID NO: 47) specified by (C5) contains heavy chain CDR1 to CDR3 (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 CDR1 to CDR3 (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" described in the Examples below.

[0048] The heavy chain variable region specified by (C6) (SEQ ID NO: 57) contains heavy chain CDRs 1 to 3 specified by (A6) or (AB6) (SEQ ID NO: 51 to 53). The light chain variable region specified by (C6) (SEQ ID NO: 59) contains light chain CDRs 1 to 3 specified by (B6) or (AB6) (SEQ ID NO: 54 to 56). An example of an antibody that satisfies (A6), (B6), (AB6), or (C6) is "201001-5-A," which is described in the Examples below.

[0049] The heavy chain variable region specified by (C7) (SEQ ID NO: 67) contains heavy chain CDR1 to CDR3 (SEQ ID NOs: 61 to 63) specified by (A7) or (AB7). The light chain variable region specified by (C7) (SEQ ID NO: 69) contains light chain CDR1 to CDR3 (SEQ ID NOs: 64 to 66) specified by (B7) or (AB7). An example of an antibody that satisfies (A7), (B7), (AB7), or (C7) is "201009-1-D" described in the Examples below.

[0050] The heavy chain variable region specified by (C8) (SEQ ID NO: 77) contains heavy chain CDRs 1 to 3 specified by (A8) or (AB8) (SEQ ID NO: 71 to 73). The light chain variable region specified by (C8) (SEQ ID NO: 79) contains light chain CDRs 1 to 3 specified by (B8) or (AB8) (SEQ ID NO: 74 to 76). An example of an antibody that satisfies (A8), (B8), (AB8), or (C8) is "201009-5-F" described in the Examples below.

[0051] The heavy chain variable region specified by (C9) (SEQ ID NO: 87) contains heavy chain CDR1 to CDR3 (SEQ ID NOs: 81 to 83) specified by (A9) or (AB9). The light chain variable region specified by (C9) (SEQ ID NO: 89) contains light chain CDR1 to CDR3 (SEQ ID NOs: 84 to 86) specified by (B9) or (AB9). An example of an antibody that satisfies (A9), (B9), (AB9), or (C9) is "201112-1-C" described in the Examples below.

[0052] The heavy chain variable region specified by (C10) (SEQ ID NO: 97) comprises heavy chain CDRs 1 to 3 specified by (A10) or (AB10) (SEQ ID NO: 91 to 93). The light chain variable region specified by (C10) (SEQ ID NO: 99) comprises light chain CDRs 1 to 3 specified by (B10) or (AB10) (SEQ ID NO: 94 to 96). An example of an antibody that satisfies (A10), (B10), (AB10), or (C10) is "13B4," which is described in the Examples below.

[0053] 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.

[0054] The heavy chain variable region specified by (C12) (SEQ ID NO: 117) contains heavy chain CDR1 to CDR3 (SEQ ID NOs: 111 to 113) specified by (A12) or (AB12). The light chain variable region specified by (C12) (SEQ ID NO: 119) contains light chain CDR1 to CDR3 (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" described in the Examples below.

[0055] The heavy chain variable region specified by (C13) (SEQ ID NO: 127) contains heavy chain CDRs 1 to 3 specified by (A13) or (AB13) (SEQ ID NO: 121 to 123). The light chain variable region specified by (C13) (SEQ ID NO: 129) contains light chain CDRs 1 to 3 specified by (B13) or (AB13) (SEQ ID NO: 124 to 126). An example of an antibody that satisfies (A13), (B13), (AB13), or (C13) is "201006-4-F" described in the Examples below.

[0056] The heavy chain variable region specified by (C14) (SEQ ID NO: 137) contains heavy chain CDRs 1 to 3 specified by (A14) or (AB14) (SEQ ID NO: 131 to 133). The light chain variable region specified by (C14) (SEQ ID NO: 139) contains light chain CDRs 1 to 3 specified by (B14) or (AB14) (SEQ ID NO: 134 to 136). An example of an antibody that satisfies (A14), (B14), (AB14), or (C14) is "201006-5-A," which is described in the Examples below.

[0057] The heavy chain variable region (SEQ ID NO: 147) specified by (C15) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 141 to 143) specified by (A15) or (AB15). The light chain variable region (SEQ ID NO: 149) specified by (C15) contains light chain CDRs 1 to 3 (SEQ ID NOs: 144 to 146) specified by (B15) or (AB15). An example of an antibody that satisfies (A15), (B15), (AB15), or (C15) is "201006-4-A" described in the Examples below.

[0058] The heavy chain variable region specified by (C16) (SEQ ID NO: 157) contains heavy chain CDRs 1 to 3 specified by (A16) or (AB16) (SEQ ID NO: 151 to 153). The light chain variable region specified by (C16) (SEQ ID NO: 159) contains light chain CDRs 1 to 3 specified by (B16) or (AB16) (SEQ ID NO: 154 to 156). An example of an antibody that satisfies (A16), (B16), (AB16), or (C16) is "201006-1-H" described in the Examples below.

[0059] The heavy chain variable region specified by (C17) (SEQ ID NO: 167) contains heavy chain CDRs 1 to 3 specified by (A17) or (AB17) (SEQ ID NO: 161 to 163). The light chain variable region specified by (C17) (SEQ ID NO: 169) contains light chain CDRs 1 to 3 specified by (B17) or (AB17) (SEQ ID NO: 164 to 166). An example of an antibody that satisfies (A17), (B17), (AB17), or (C17) is "201006-3-D," which is described in the Examples below.

[0060] The heavy chain variable region specified by (C18) (SEQ ID NO: 177) contains heavy chain CDRs 1 to 3 specified by (A18) or (AB18) (SEQ ID NO: 171 to 173). The light chain variable region specified by (C18) (SEQ ID NO: 179) contains light chain CDRs 1 to 3 specified by (B18) or (AB18) (SEQ ID NO: 174 to 176). An example of an antibody that satisfies (A18), (B18), (AB18), or (C18) is "201006-1-F" described in the Examples below.

[0061] The heavy chain variable region specified by (C19) (SEQ ID NO: 187) contains heavy chain CDRs 1 to 3 specified by (A19) or (AB19) (SEQ ID NO: 181 to 183). The light chain variable region specified by (C19) (SEQ ID NO: 189) contains light chain CDRs 1 to 3 specified by (B19) or (AB19) (SEQ ID NO: 184 to 186). An example of an antibody satisfying (A19), (B19), (AB19), or (C19) is "201006-4-G" described in the Examples below.

[0062] The heavy chain variable region specified by (C20) (SEQ ID NO: 197) contains heavy chain CDRs 1 to 3 specified by (A20) or (AB20) (SEQ ID NO: 191 to 193). The light chain variable region specified by (C20) (SEQ ID NO: 199) contains light chain CDRs 1 to 3 specified by (B20) or (AB20) (SEQ ID NO: 194 to 196). An example of an antibody that satisfies (A20), (B20), (AB20), or (C20) is "201006-7-A," which is described in the Examples below.

[0063] The heavy chain variable region specified by (C21) (SEQ ID NO: 207) contains heavy chain CDRs 1 to 3 specified by (A21) or (AB21) (SEQ ID NO: 201 to 203). The light chain variable region specified by (C21) (SEQ ID NO: 209) contains light chain CDRs 1 to 3 specified by (B21) or (AB21) (SEQ ID NO: 204 to 206). An example of an antibody satisfying (A21), (B21), (AB21), or (C21) is "201006-2-D" described in the Examples below.

[0064] The heavy chain variable region specified by (C22) (SEQ ID NO: 217) contains heavy chain CDR1 to 3 specified by (A22) or (AB22) (SEQ ID NO: 211 to 213). The light chain variable region specified by (C22) (SEQ ID NO: 219) contains light chain CDR1 to 3 specified by (B22) or (AB22) (SEQ ID NO: 214 to 216). An example of an antibody satisfying (A22), (B22), (AB22), or (C22) is "201006-2-C" described in the Examples below.

[0065] The heavy chain variable region specified by (C23) (SEQ ID NO: 227) contains heavy chain CDR1 to 3 specified by (A23) or (AB23) (SEQ ID NO: 221 to 223). The light chain variable region specified by (C23) (SEQ ID NO: 229) contains light chain CDR1 to 3 specified by (B23) or (AB23) (SEQ ID NO: 224 to 226). An example of an antibody that satisfies (A23), (B23), (AB23), or (C23) is "201006-3-H" described in the Examples below.

[0066] The heavy chain variable region specified by (C24) (SEQ ID NO: 237) contains heavy chain CDR1-3 (SEQ ID NOs: 231-233) specified by (A24) or (AB24). The light chain variable region specified by (C24) (SEQ ID NO: 239) contains light chain CDR1-3 (SEQ ID NOs: 234-236) specified by (B24) or (AB24). An example of an antibody satisfying (A24), (B24), (AB24), or (C24) is "201006-5-F" described in the Examples below.

[0067] The heavy chain variable region (SEQ ID NO: 247) specified by (C25) contains heavy chain CDR1-3 (SEQ ID NOs: 241-243) specified by (A25) or (AB25). The light chain variable region (SEQ ID NO: 249) specified by (C25) contains light chain CDR1-3 (SEQ ID NOs: 244-246) specified by (B25) or (AB25). An example of an antibody that satisfies (A25), (B25), (AB25), or (C25) is "201006-7-G" described in the Examples below.

[0068] The heavy chain variable region specified by (C26) (SEQ ID NO: 257) contains heavy chain CDR1 to 3 specified by (A26) or (AB26) (SEQ ID NO: 251 to 253). The light chain variable region specified by (C26) (SEQ ID NO: 259) contains light chain CDR1 to 3 specified by (B26) or (AB26) (SEQ ID NO: 254 to 256). An example of an antibody that satisfies (A26), (B26), (AB26), or (C26) is "201009-2-C" described in the Examples below.

[0069] The heavy chain variable region specified by (C27) (SEQ ID NO: 267) contains heavy chain CDR1 to 3 specified by (A27) or (AB27) (SEQ ID NO: 261 to 263). The light chain variable region specified by (C27) (SEQ ID NO: 269) contains light chain CDR1 to 3 specified by (B27) or (AB27) (SEQ ID NO: 264 to 266). An example of an antibody satisfying (A27), (B27), (AB27), or (C27) is "201009-3-H" described in the Examples below.

[0070] The heavy chain variable region specified by (C28) (SEQ ID NO: 277) contains heavy chain CDRs 1 to 3 specified by (A28) or (AB28) (SEQ ID NO: 271 to 273). The light chain variable region specified by (C28) (SEQ ID NO: 279) contains light chain CDRs 1 to 3 specified by (B28) or (AB28) (SEQ ID NO: 274 to 276). An example of an antibody that satisfies (A28), (B28), (AB28), or (C28) is "201009-1-C" described in the Examples below.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The amino acid sequences of SEQ ID NOs:44 and 54 are the same. The amino acid sequences of SEQ ID NOs:64 and 124 are the same. The amino acid sequences of SEQ ID NOs:74, 144, 224, 254, and 264 are the same. The amino acid sequences of SEQ ID NOs:84, 94, and 104 are the same. The amino acid sequences of SEQ ID NOs:154 and 164 are the same. The amino acid sequences of SEQ ID NOs:174, 184, 204, and 214 are the same.

[0075] 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.

[0076] 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.

[0077] The 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 function 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 a heavy chain CH3 region. Other examples include CDRs such as CDR1, CDR2, and CDR3, conjugates of these CDRs, and fusions of these CDRs or CDR conjugates with a heavy chain CH3 region. In other words, the antibodies of the present disclosure also include partial fragments of antibodies such as those described above. A partial fragment of an antibody may also be referred to as an "antibody fragment."

[0078] The antibody may be a multispecific antibody. The multispecific antibody of this embodiment specifically binds to the extracellular domain of human CXCR3A, has 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 for another target molecule. An example of a multispecific antibody is a diabody, which is a type of bispecific antibody.

[0079] 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, e.g., at the same or different epitopes. The multispecific antibody can, for example, bind to CXCR3 and at least one other antigen with high affinity. 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 include heavy and / or light chain variable regions from a parent antibody or a variant of the parent antibody.

[0080] 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, in the case of IgG, it may be any subclass such as IgG1, IgG2, IgG3, or IgG4.

[0081] In one embodiment, the antibody has activity to block CXCR3A-dependent cellular functions. CXCR3A-dependent cellular functions are functions induced by 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 of such cellular functions 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.

[0082] In one embodiment, the antibody does not specifically bind to human vascular endothelial cells, which predominantly express CXCR3B, as shown in the Examples below.

[0083] The extracellular domain of CXCR3A to which the antibody specifically binds may be any of the N-terminal domain, extracellular loop domain 1, extracellular loop domain 2, and extracellular loop domain 3. The antibody may bind to only one of these extracellular domains, or to two or more of them.

[0084] The present disclosure includes antibodies that are "functionally equivalent" to the above-described anti-human CXCR3 antibodies. Functionally equivalent antibodies include antibodies that have the same epitope as the above-described 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-described 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 the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 can be identified.

[0085] As an example of an antibody functionally equivalent to the above-mentioned 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, and includes an antibody having an amino acid sequence in which 1 to 10 amino acids have been substituted, added, or deleted in the amino acid sequence of the SEQ ID NO: defined in (C1) to (C28) above, or an antibody that has 90% or more identity to the amino acid sequence of the SEQ ID NO: 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 more, more preferably 95% or more, and particularly preferably 97% or more.

[0086] 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, 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-mentioned antibody, the present disclosure includes an antibody (second 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, and competitively inhibits the binding of the above-mentioned antibody (first antibody) to the receptor.

[0087] The CDRs and other regions (e.g., framework regions and constant regions) disclosed herein can each be independently selected 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.

[0088] <Humanized Antibodies> The present disclosure includes the above-described antibodies that are humanized. A humanized antibody refers to an antibody in which the CDRs are derived from a non-human animal and the other regions (such as framework regions and constant regions) are derived from humans. Methods for constructing and producing humanized antibodies are described below.

[0089] <Nucleic Acid> The present disclosure includes nucleic acids (DNA) encoding the above-described antibodies. The nucleic acids include, for example, a first nucleic acid encoding a heavy chain variable region and / or a second nucleic acid encoding a light chain variable region.

[0090] The heavy chain variable region encoded by the first nucleic acid may, for example, be 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.

[0091] The light chain variable region encoded by the second nucleic acid may, for example, be 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.

[0092] The nucleic acid may be incorporated into a vector. The vector is appropriately selected depending on the type of host cell to be introduced, etc. Vectors include vectors for gene therapy. In this case, the vector itself can be directly administered into the body.

[0093] <Method for Producing Antibodies> The antibodies can be produced using genetic recombination techniques. That is, recombinant cells that express the nucleic acids can be constructed, and the antibodies can be obtained from cultures of the cells.

[0094] <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 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.

[0095] Next, these DNAs are used to prepare DNAs encoding variable regions in which heavy chain CDRs 1 to 3 are grafted onto the framework regions of VH of any human antibody. Similarly, DNAs encoding variable regions in which light chain CDRs 1 to 3 are grafted onto the framework regions 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.

[0096] 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.

[0097] 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, a DNA encoding the amino acid sequence shown in SEQ ID NO: 7 is prepared as the DNA encoding the VH. Furthermore, a DNA encoding the amino acid sequence shown in SEQ ID NO: 9 is prepared as the DNA encoding the VL. The DNA can be prepared using known methods such as PCR. The DNA can also be prepared by chemical synthesis.

[0098] The resulting DNA encoding the VH or VL is inserted into a vector having a sequence encoding the CH or CL of a human antibody, respectively, to construct a chimeric antibody expression vector. Vectors having a sequence encoding the CH or CL of a human antibody are commercially available. The constructed expression vector is introduced into a host cell to obtain a recombinant cell that expresses the chimeric antibody. The recombinant cell is then cultured, and the desired chimeric antibody is obtained from the culture.

[0099] Chimeric antibodies having VH and VL specified in (C2) to (C28) above can also be constructed and produced in a similar manner.

[0100] <Method for Producing Multispecific Antibodies> Methods for producing multispecific antibodies include, for example, linking the anti-human CXCR3 antibody or fragment thereof of the present disclosure to another antibody or fragment thereof. Another example includes co-expressing the anti-human CXCR3 antibody or fragment thereof of the present disclosure and the other antibody or fragment thereof in a host cell. Other linking methods include chemical coupling, gene fusion, non-covalent association, etc.

[0101] 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.

[0102] <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 the antibody can be purified by combining known methods such as various types of chromatography, salting out, dialysis, and membrane separation. For example, if the antibody isotype is IgG, it can be easily purified by affinity chromatography using protein A.

[0103] <Modified Antibodies> 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, and light absorbers. Methods for binding to the other molecule include chemical coupling, gene fusion, and non-covalent association. When the other molecule to be bound is an antibody, the modified antibody can be a multispecific antibody. In this respect, a multispecific antibody can be considered a type of modified antibody.

[0104] 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. These drugs can be conjugated directly or indirectly via a linker or chelator to produce an antibody-drug conjugate.

[0105] <Drug> The present disclosure encompasses a drug containing the above-described antibody as an active ingredient. The drug may be a pharmaceutical composition containing the above-described antibody and a pharmaceutically acceptable carrier. Preferably, the drug blocks CXCR3A-dependent cellular functions.

[0106] Examples of the manner in which an antibody blocks 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 changing 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 antibody has internalization activity. In other words, it induces receptor internalization.

[0107] In a preferred embodiment, the pharmaceutical composition is used for treating 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.

[0108] 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. Increased CXCR3A ligand levels in patients can be disease-specific. For example, increased levels of CXCR3A ligands have been reported in autoimmune colitis, type 1 diabetes, and acute lung injury (CXCL10), in multiple sclerosis (CXCL9 and CXCL10), and in atherosclerosis (CXCL9, CXCL10, and CXCL11) (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.

[0109] Furthermore, drugs that selectively inhibit CXCR3A are thought to be highly effective in some tumors. CXCR3 expression has been shown to be associated with poor prognosis in colon 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 antitumor growth, angiogenesis inhibition, and apoptosis. Therefore, CXCR3A-specific CXCR3 inhibitors may have a more potent antitumor effect by suppressing tumor growth and metastasis while maintaining the antitumor effects of CXCR3B. SCH546738 is known as 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).

[0110] On the other hand, drugs that inhibit both CXCR3A and CXCR3B may be more effective in treating vitiligo, where CXCR3A stimulates the migration of pathogenic T cells and CXCR3B promotes melanocyte apoptosis (Meri K. Tulic et al., Nat Commun. 2019 May 16;10(1):2178).

[0111] <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. In the case of injections, they 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.

[0112] <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.

[0113] When preparing an aqueous solution for injection, examples include physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and these may be used in combination with appropriate solubilizing agents, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), and nonionic surfactants (polysorbate 80, HCO-50). If necessary, the antibody can also be encapsulated in microcapsules (such as microcapsules made of hydroxymethylcellulose, gelatin, poly(methyl methacrylate), etc.) 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).

[0114] 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. Pat. 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).

[0115] <Application to Gene Therapy> The above nucleic acids can be incorporated into a gene therapy vector or mRNA that can be translated into a protein in vivo to form 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 retrovirus vectors, adenovirus 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).

[0116] 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, including injection, infusion, gas-induced particle bombardment (using an electron gun, etc.), or mucosal administration such as 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.

[0117] Furthermore, the present disclosure also includes a method and a therapeutic agent for treating a disease or disorder caused by abnormally enhanced CXCR3-dependent cellular function in a mammal suffering from the disease. Here, "treatment" refers to preventing or alleviating the progression or worsening of the pathology of the disease in a mammal at risk of or suffering from the disease, thereby referring to a therapeutic treatment aimed at preventing or alleviating the progression or worsening of symptoms of the disease. Furthermore, the term "disease" refers to any disease caused by abnormally enhanced CXCR3-dependent cellular function, and includes, but is not limited to, 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 concept also includes cardiovascular disorders, nervous system disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, infectious diseases, blood cancers, and solid cancers. The "mammal" to be treated means 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 humans.

[0118] <Antibody-immobilized carrier> The present disclosure includes a carrier on which the above-mentioned antibody is immobilized (antibody-immobilized carrier). In a preferred embodiment, the antibody-immobilized carrier is used to remove CXCR3-expressing cells from a body fluid containing CXCR3-expressing cells by contacting the carrier with the antibody-immobilized carrier. An example of the body fluid is blood. The above-mentioned antibody immobilized on the carrier may be one type only, or two or more types.

[0119] 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, sterilizability, 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.

[0120] The shape of the carrier may be granular, cotton-like, woven fabric, nonwoven fabric, sponge-like porous material, flat plate-like, etc., but granular, cotton-like, woven fabric, nonwoven fabric, and sponge-like porous material are preferred in terms of their large surface area per volume.

[0121] A kit for removing CXCR3-expressing cells can be prepared by combining the antibody-immobilized carrier of the present disclosure with other components, such as an anticoagulant and an extracorporeal circulation circuit.

[0122] 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 the manufacture of a medicament for treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function. The disease, disorder, or condition includes at least a cardiovascular disorder, a nervous system disorder, an inflammatory disease, an autoimmune disease, a metabolic disease, an infectious disease, a blood cancer, and a solid cancer.

[0123] Example 1 Immunization of Animals for Production of 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.

[0124] Genomic DNA was extracted and purified from Escherichia 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; hereinafter 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.

[0125] The mammalian expression vector pCI-Mammalian Expression Vector (Promega) was digested with the restriction enzymes NheI and SalI, and the ends were dephosphorylated with bacterial alkaline phosphatase (BAP), after which DNA fragment A was inserted. This expression vector was further digested with SalI and NotI, and the ends were dephosphorylated with BAP, after which DNA fragment B was inserted to construct the template plasmid vector pCI-hEP4-GroEL.

[0126] Based on the sequence information of human CXCR3A registered in the NCBI Gene Bank (AAO92295, SEQ ID NO: 281), an artificial gene was synthesized by adding the sequence GCTAGC to the 5' end and the sequence GTCGAC to the 3' end of the human CXCR3A gene. This artificial gene was 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 into it to construct the immunization vector pCI-hCXCR3A-GroEL. This vector expresses a fusion protein of human CXCR3A and GroEL.

[0127] (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.

[0128] Eight-week-old rats were immunized by injecting 40 μL of the above-mentioned 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. Thereafter, the rats were repeatedly 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.

[0129] (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."

[0130] (4) Evaluation of Binding of Serum Antibodies 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 incubated at a cell concentration of 1 × 10 ７ The cells were suspended in FACS buffer to a concentration of 100 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, 1 to 2 × 10 ５Cells were suspended at a concentration of 50 μL per cell. This cell suspension was mixed with the immunized animal serum serving as the 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 and incubated at 4°C for 1 hour. After washing twice with 100 μL of FACS buffer, the cells were suspended in 80 μL of FACS buffer. Cell surface fluorescence intensity was measured using a flow cytometer iQue (Sartorius) to evaluate antibody binding. The immunized animal serum 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 the immunized animals.

[0131] (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.

[0132] [Example 2] Preparation of antibodies <1> (1) Selection of specific antibody-producing lymphocytes using microwells Human CXCR3A stably expressing CHO cells were suspended in F-12 medium (containing 10% FBS, penicillin / streptomycin) and incubated at 3.5 × 10 ５A cell suspension of 500 μL of cells was prepared. This cell suspension was filled into a microchamber for a cell picking system. The microchamber was centrifuged at 300 rpm for 1 minute five times, and the cells were prepared so that 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. ２ The cells were incubated in an incubator at 37°C for 1 hour to allow CHO cells stably expressing human CXCR3A to adhere to the bottom of the microwell. CytoRed solution (Dojindo Laboratories) adjusted to a concentration of 10 nM in F-12 medium was added, and the cells were further incubated at 37°C for 1 hour to stain them. After washing three times with F-12 medium to remove excess CytoRed, the microchamber was filled with 1 mL of F-12 medium.

[0133] 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). ４ pieces ~ 2×10 ５Antibody-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, allowing one or two antibody-producing cells to be placed in each microwell. After washing the microchamber with medium, an appropriate amount of medium was added and the microwell was incubated at 37°C for 30 minutes to allow antibody secretion from the antibody-producing cells. After washing the microwell 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 set in a cell picking system (AS ONE Corporation), and transmitted light images and two types of fluorescent image information were acquired for all microwells. Fluorescence detection of CytoRed was performed at an excitation wavelength of 543 nm and an emission wavelength of 593 nm. Fluorescence detection of Alexa Fluor 488 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 transmitted light, CytoRed, and Alexa Fluor 488 scanned images that antibodies binding to human CXCR3A stably expressing CHO cells were present, antibody-producing cells were recovered into a cell lysate using a capillary tube (AS ONE Corporation) with a diameter of several micrometers to several tens of micrometers.

[0134] (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.

[0135] In addition, to increase the specificity of the amplified product, PCR was performed twice. In the first PCR for the antibody obtained from mouse, a first forward primer (SEQ ID NO: 282) that commonly amplifies the VH region and the VL region, a mouse VH first reverse primer (SEQ ID NO: 283) that specifically amplifies the VH region, and a mouse VL first reverse primer (SEQ ID NO: 284) that specifically amplifies the VL region were mixed and used. In the second PCR, the first amplified product 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) that specifically amplifies the VH region were used as primers for amplification of the VH region, and the second forward primer (SEQ ID NO: 285) and the mouse VL second reverse primer (SEQ ID NO: 287) that specifically amplifies the VL region were used as primers for amplification of the VL region.

[0136] In addition, in the first PCR for the antibody obtained from rat, a first forward primer (SEQ ID NO: 282) that commonly amplifies 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 were used in combination. In the second PCR, the amplification product of the first PCR 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 as primers for amplification of 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 as primers for amplification of the VL region.

[0137] When the sample after the second PCR was subjected to agarose gel electrophoresis, amplification products corresponding to the VH region at a position of approximately 750 bp and the VL region at a position of approximately 550 bp were confirmed.

[0138] (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.

[0139] (4) Introducing antibody expression units into mammalian cells. 7.5 × 10 Expi293F cells (Thermo Fisher Scientific) were cultured in a 6-well cell culture plate. ６ The cells were seeded at 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 determined as the primary hit antibody.

[0140] [Example 3] Antibody Production <2> (1) Hybridoma Production and Screening for 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).

[0141] (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.

[0142] Hybridomas producing the primary hit antibodies were expanded and cultured, and then 5 × 10 ５The medium was replaced with CD Hybridoma medium (Gibco) at a concentration of 1000 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.

[0143] (3) Isolation of antibody genes The same procedures as in Example 2(2) were carried out.

[0144] (4) Construction of antibody expression unit The same procedure as in Example 2(3) was carried out.

[0145] (5) Introduction of antibody expression unit into mammalian cells The same procedure as in Example 2(4) was carried out.

[0146] [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.

[0147] Based on the determined sequence information of 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 antibody light chain variable region sequences that did 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 rat-derived antibodies, and the others are mouse-derived antibodies.

[0148] 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 (Table 1-3; SEQ ID NOs: 21-30) 201001-4-E (Table 1-4; SEQ ID NOs: 31-40) 201001-1-C (Table 1-5; SEQ ID NOs: 41-50) 201001-5-A (Table 1-6; SEQ ID NOs: 51-60) 201009-1-D (Table 1-7; SEQ ID NOs: 61-70) 201009-5-F (Table 1-8; SEQ ID NOs: 71-80) 201112-1-C (Table 1-9; SEQ ID NOs: 81-90) 13B4 (Table 1-10; SEQ ID NOs: 91-100) 11F11 (Table 1-11; SEQ ID NOs: 101-110) 201006-4-H (Table 1-12; SEQ ID NOs: 111-120) 201006-4-F (Table 1-13; SEQ ID NOs: 121-130) 201006-5-A (Table 1-14; SEQ ID NOs: 131-140) 201006-4-A (Table 1-15; SEQ ID NOs: 141-150) 201006-1-H (Table 1-16; SEQ ID NOs: 151-160) 201006-3-D (Table 1-17; SEQ ID NOs: 161-170) 201006-1-F (Table 1-18; SEQ ID NOs: 171-180) 201006-4-G (Table 1-19; SEQ ID NOs: 181-190) 201006-7-A (Table 1-20; SEQ ID NOs: 191-200) 201006-2-D (Table 1-21; SEQ ID NOs: 201-210) 201006-2-C (Table 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)

[0149] 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 their SEQ ID NOs. In the tables, the underlined portions of each variable region sequence indicate the CDRs.

[0150]

[0151]

[0152]

[0153]

[0154]

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[0177]

[0178] [Example 5] Intracellular Ca ２＋ Antibody screening by evaluation of signal transduction inhibitory activity 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.

[0179] Human CXCR3A stably expressing CHO cells were placed in a 96-well microplate at 2 × 10 per well. ４ Cells were seeded at an initial cell concentration of 100 μL and cultured for two days. After two 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 one hour, primary hit antibodies or control antibodies diluted within the concentration range of 100 nM to 10 nM were added to each well and incubated for 15 minutes. Mouse isotype control antibodies (Fujifilm Wako Pure Chemical Industries, Ltd.) or rat isotype control antibodies (Fujifilm Wako Pure Chemical Industries, Ltd.) were used as control antibodies. After incubation, the assay plate was placed in Ca ２＋ The signal analyzer FDSSμCELL (Hamamatsu Photonics) was installed, and the intracellular Ca concentration was measured when each cell was stimulated with 100 nM IP-10 (PEPROTECH). ２＋ The inhibitory activity against a transient increase in concentration 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 a representative example, 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.

[0180] Similar tests were repeated, and eight types of 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 tested for their ability to induce stable intracellular Ca2+ binding. ２＋ The antibody was selected as one that exhibits signal transduction inhibitory activity.

[0181]

[0182] 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).

[0183] 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. TaqMan Assays described in the paper were used as 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 Romagnani, Mario Serio, Paola Romagnani, "An 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.)

[0184] The obtained Ct values ​​were analyzed by 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 vein endothelial cells and human microvascular endothelial cells.

[0185] [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.

[0186] Human CXCR3A stably expressing CHO cells, human umbilical vascular endothelial cells (Lonza) confirmed to predominantly express CXCR3B in Example 6, and human microvascular endothelial cells (Cell Systems) were washed with FACS buffer (PBS containing 1% FBS) to a cell concentration of 1 × 10 ７ The cells were suspended in FACS buffer to a concentration of 1 × 10 cells / mL. Fc Block (Tonbo Biosciences) was added in an amount 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, 1 × 10 cells were added. ５ The cells were suspended at a concentration of 50 μL per cell. 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 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 Corporation), a rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation), 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.

[0187] As representative examples, histograms comparing 13B4, 201009-2-C, CXCR3B-specific antibodies, or prior art antibodies (all referred to as anti-chemokine antibodies) with a control antibody (isotype antibody) are shown in Figure 2. In Figure 2, solid black indicates isotype antibodies, and open white indicates anti-chemokine antibodies. 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 but not to human vascular endothelial cells.

[0188] The cell binding specificity of the antibodies is shown in Table 3. Those for which a rightward shift in the histogram was observed are indicated with "+", and those for which no shift was observed are indicated with "-". It was confirmed that 16 types of antibodies, including 201009-2-C, specifically bind to human CXCR3A, but do not bind to human vascular endothelial cells that predominantly express human CXCR3B.

[0189]

[0190] [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.).

[0191] (2) Introduction of antibody expression plasmid into mammalian cells. 1.8 × 10 ExpiCHO cells (Thermo Fisher Scientific) were placed in a 125 mL Erlenmeyer flask (vent cap) for cell culture. ８ The cells were seeded at 30 mL per flask. The heavy and light chain antibody expression plasmids constructed in (1) were co-transfected into ExpiCHO cells using an Expifectamine CHO Transfection Kit (Thermo Fisher Scientific). Five days after transfection, the cell supernatant was collected, and purified antibodies were obtained by Protein A affinity purification.

[0192] [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.

[0193] Human CXCR3A stably expressing CHO cells were washed with FACS buffer (PBS containing 1% FBS) and diluted to a cell concentration of 1 × 10 ７ The cells were suspended in FACS buffer to a concentration of 1 × 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 for 30 minutes. After blocking, 1 × 10 cells were ５Cells were suspended at a concentration of 50 μL per cell. This cell suspension was mixed with 50 μL of each of the 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, 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 as the secondary antibody to each well and incubated at 4°C for 1 hour. The cells were washed twice with 100 μL of FACS buffer and then suspended in 50 μL of FACS buffer. The fluorescence intensity on the cell surface was measured using a Novocyte flow cytometer (Agilent) to evaluate the antibody binding.

[0194] 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 eight antibodies were confirmed to bind to CHO cells stably expressing human CXCR3A in a dose-dependent manner. The results of the binding of each antibody to CHO cells stably expressing human CXCR3A are shown in Figures 3A to 3H. The 50% binding concentration (EC ５０ ), 80% binding concentration (EC ８０ ) were calculated and summarized in Table 4.

[0195]

[0196] [Example 10] Intracellular Ca ２＋ Evaluation of signal transduction inhibition The eight types of 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.

[0197] Human CXCR3A stably expressing CHO cells were placed in a 96-well microplate at 2 × 10 per well. ４Cells were seeded at an initial cell concentration of 100 μL and cultured for two days. After two 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 one hour, eight antibodies diluted to a concentration range of 200 nM to 0.8 nM or a control antibody were added to each well and incubated for 15 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 assay plate was resuspended in Ca ２＋ The signal analyzer FDSSμCELL (Hamamatsu Photonics) was installed, and the intracellular Ca concentration was measured when each cell was stimulated with 100 nM IP-10 (PEPROTECH). ２＋ The inhibitory activity against a transient increase in the concentration of each antibody 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.

[0198] All eight antibodies inhibited IP-10-induced intracellular Ca in CHO cells stably expressing human CXCR3A. ２＋ It was confirmed that the antibodies inhibited signal transduction in a dose-dependent manner, demonstrating that they have the activity of blocking CXCR3-dependent cell functions. ５０ ) are 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.

[0199]

[0200] [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.

[0201] 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) to a cell concentration of 1 × 10 ７ The cells were suspended in FACS buffer to a concentration of 1 × 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 for 30 minutes. After blocking, 1 × 10 cells were ５ Cells were suspended at a concentration of 50 μL per cell. This cell suspension was mixed with 50 μL of diluted six types of anti-CXCR3 antibodies, anti-CCR7 antibodies (manufactured in-house), anti-CCR6 antibodies (manufactured in-house), or control antibodies, and incubated at 4°C for 1 hour. Mouse isotype control antibodies (Fujifilm Wako Pure Chemical Corporation), rat isotype control antibodies (Fujifilm Wako Pure Chemical Corporation), or human isotype control antibodies (GeneScript) were used as control antibodies. 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 as secondary antibodies to each well and incubated at 4°C for 1 hour. The cells were washed twice with 100 μL of FACS buffer, then 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.

[0202] As representative examples, 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) are shown in Figure 5. In Figure 5, solid black indicates isotype antibody, and open white indicates anti-chemokine antibody. A rightward shift in the histogram was determined to be "bound." 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. Those for which a rightward shift in the histogram was observed are indicated by "+", and those for which no shift was observed are indicated by "-".

[0203]

[0204] [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.

[0205] Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were thawed and activated by culturing for 3 days in TexMACS medium (Miltenyi Biotech) containing 1% T cell TransAct, human and 10 ng / mL IL-2. The medium was replaced every 2-3 days with TexMACS medium containing 10 ng / mL IL-2 for expansion. T cells from day 10 onward were used for the study. Human T cells were diluted 1:1 with FACS buffer (PBS, containing 1% FBS) to a concentration of 1 x 10 ７ The cells were suspended at 4 × 10 cells / mL and blocked for 30 minutes at 4°C. ６Cells were suspended at 1000 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 the control antibody. After incubation, cells were washed twice with 120 μL of FACS buffer. As the secondary antibody, 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 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.

[0206] 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 the binding of each antibody to human T cells are shown in Figures 6A to 6F. The 50% binding concentration (EC ５０ ) were calculated and summarized in Table 7.

[0207]

[0208] [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.

[0209] Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were cultured in the same manner as in Example 12, and T cells from 10 days of expansion culture were used for the test. Human T cells were washed with assay buffer (RPMI-1640 + 0.1% BSA). Serially diluted antibodies were added to the cells, and the cells were cultured at a cell density of 2.0 to 2.4 x 10. ６The cells were incubated at 37°C for 60 minutes at 2.4 nM IP-10 (PEPROTECH) in the wells of a receiver plate equipped with an insert-integrated 96-well Transwell and a 3.0 μm polycarbonate membrane (Corning). After placing the Transwell insert on the receiver plate, 75 μL of the 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, normalized by setting "no antibody, with IP-10" as 0% inhibition and "no antibody, without IP-10" as 100% inhibition. 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 antibody concentration, and the vertical axis represents % inhibition. The T cell migration inhibition rate of each antibody is summarized in Table 8.

[0210] All six types of antibodies were confirmed to inhibit IP-10-induced cell migration in human T cells in a dose-dependent manner, demonstrating that they have the activity of blocking CXCR3-dependent cellular functions.

[0211]

[0212] [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.

[0213] Frozen human peripheral blood mononuclear cells (Cellular Technology Limited) were cultured in the same manner as in Example 12, and T cells were used for the test after 10 days of expansion culture. Human T cells were diluted 1:1 with FACS buffer (PBS, containing 1% FBS) containing goat serum (Thermo Fisher Scientific) to a concentration of 1 x 10 ７ The cells were suspended at 1 × 10 cells / mL and blocked at 4°C for 30 minutes. ５Cells were suspended at a concentration of 50 μL per cell. 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, 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 left 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 as the secondary antibody to each well and incubated at 4°C for 15 minutes. The cells were washed twice with cold FACS buffer, suspended in 50 μL of cold FACS buffer, and the fluorescence intensity on the cell surface was measured using a Novocyte flow cytometer (Agilent).

[0214] Antibody internalization was indirectly assessed based on the expression level of the receptor on the cell surface. Specifically, 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 % for 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.

[0215] 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.

[0216] Artificial genes were synthesized in which the human IgG1 heavy chain constant region (SEQ ID NO: 304) was linked to the VH region of each antibody, and the κ chain constant region (SEQ ID NO: 305) was linked 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 linked to the VH region of 201009-2-C, and the κ chain constant region was linked 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 assayed by SDS-PAGE.

[0217] Next, the binding activity to human T cells was measured according to the method of Example 12. Graphs were prepared by plotting the antibody concentration on the horizontal axis and the median fluorescence intensity of the flow cytometer histogram 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 concentration (EC ５０) are summarized in Table 9. Furthermore, the inhibitory activity of each antibody against human T cell migration was evaluated according to the method of Example 13. The dose dependency of the T cell migration inhibition rate of each antibody is summarized in Table 10. For the activity of "VH1+VL1," "VH1+VL4," "VH2+VL1," and "VH2+VL4," graphs were prepared by plotting the concentration of each antibody on the horizontal axis and the inhibition rate (%) on the vertical axis, and these graphs 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.

[0218]

[0219]

[0220]

Claims

1. An antibody that specifically binds to human CXCR3, 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, and is selected from the group consisting of the following (AB1) to (AB9), (AB12) to (AB27): (AB26) an antibody having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) An antibody that satisfies any of the following: 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.

2. An antibody that specifically binds to human CXCR3, 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, and is selected from the group consisting of the following (C1) to (C9), (C12) to (C27): (C26) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) having 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) An antibody that satisfies any of the following: (C13) having 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) having 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) having 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) having 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.

3. 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, which is the antibody described in claim 2, to the receptor.

4. The antibody according to claim 1 or 3, which is a humanized antibody or a chimeric antibody.

5. The antibody according to any one of claims 1 to 4, which is a multispecific antibody.

6. An antibody according to any one of claims 1 to 5, which has internalization activity.

7. The antibody according to any one of claims 1 to 6, which is a modified antibody to which another molecule is bound.

8. The antibody of claim 7, wherein the modified antibody is an antibody-drug conjugate.

9. A nucleic acid encoding the antibody of any one of claims 1 to 6.

10. A cell comprising the nucleic acid of claim 9.

11. A pharmaceutical comprising the antibody according to any one of claims 1 to 8 as an active ingredient.

12. The pharmaceutical agent according to claim 11, which is used to treat a disease, disorder, or condition associated with impaired CXCR3-dependent cellular function.

13. The pharmaceutical according to claim 12, wherein the disease, disorder, or condition is a Th1 immune disorder.

14. The pharmaceutical composition according to claim 13, wherein 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.

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