Antibody, nucleic acid, cell, and medicament

Abstract

To provide a new anti-human CXCR3 antibody useful for an antibody drug.SOLUTION: Provided is an antibody that specifically binds to human CXCR3, wherein the antibody specifically binds to an extracellular domain of human CXCR3, has an activity of blocking a CXCR3-dependent cellular function, and does not specifically bind to human vascular endothelial cells. The antibodies have heavy chain CDRs1-3 and light chain CDRs1-3 comprising specific amino acid sequences. The antibody is preferably a humanized antibody or a chimeric antibody.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[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. [Background technology]

[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 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 interact redundantly, synergistically, or antagonistically in vivo (Non-Patent Documents 2 and 3). Activation of CXCR3A by CXCL9, CXCL10, and CXCL11 leads to activation of Gαq and Gαi, inducing cell chemotaxis, cell proliferation, cell survival, and tumor cell metastasis.

[0005] CXCR3B has an N-terminal domain that is 47 amino acids longer than CXCR3A due to alternative splicing. CXCR3B expression has been confirmed in tissues such as the heart, skeletal muscle, liver, and kidney, as well as in vascular endothelial cells. The primary ligand for CXCR3B is CXCL4, and in vitro, CXCL9, CXCL10, and CXCL11 also bind to CXCR3B. 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 have opposing effects. 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 contribute to various inflammatory diseases. Examples include Th1-related diseases such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and acute cardiac allograft rejection (Non-Patent Document 3). More recent studies have also suggested a link with a wider range of diseases, including viral infections such as influenza and COVID-19, and tumors (Non-Patent Documents 3 and 4). Therefore, there is a need for substances (e.g., antibodies) and methods that target CXCR3 signaling to mitigate the progression of disorders associated with CXCR3 signaling.

[0007] Substantial 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 multiple myeloma), and the anti-CCR4 monoclonal antibody mogamuzulimab (a drug for 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 progressed. However, only two small molecule CXCR3 antagonists have progressed to clinical trials: AMG487 and ACT-777991. AMG487 progressed to a Phase IIa trial for psoriasis, but efficacy was not demonstrated. ACT-777991, which is being developed as a treatment for type 1 diabetes, has completed a Phase I trial and demonstrated good tolerability, kinetics, and safety in healthy subjects, suggesting further clinical development is anticipated (Non-Patent Document 6). Meanwhile, a Phase II clinical trial of the human monoclonal antibody eldelumab (BMS-936557), targeting CXCL10, for the treatment of ulcerative colitis (UC) and Crohn's disease suggested that it may be an effective treatment for moderate to severe active UC (Non-Patent Document 7). These results suggest the clinical value of therapeutics targeting the CXCL10-CXCR3 cascade for Th1-related diseases.

[0009] Patent Document 1 discloses six 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 monoclonal antibodies that specifically bind to human CXCR3 and their humanized antibodies. The amino acid sequences of the complementarity-determining regions, heavy chain variable regions, and light chain variable regions of each monoclonal antibody are also disclosed. 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. It also discloses the amino acid sequences of the complementarity-determining regions, heavy chain variable region, and light chain variable region of this monoclonal antibody. It has been shown that depletion of CXCR3 with this antibody is 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 alternative or improved anti-human CXCR3 antibodies with superior properties as pharmaceutical raw materials. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. 2008 / 094942 [Patent Document 2] International Publication No. 2013 / 109974 [Patent Document 3] International Publication No. 2018 / 119288 [Non-patent literature]

[0014] [Non-Patent Document 1] Catherine E. Hughes and Robert JB, "A guide to chemokines and their receptors." FEBS J. 2018 Aug; 285(16): 2944-2971. [Non-patent document 2] 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. [Non-patent document 3] Joanna R Groom and Andrew D Luster, "CXCR3 ligands: redundant, collaborative and antagonistic functions" Immunol Cell Biol. 2011 Feb; 89(2),207-215.

Outdoor Tools 4

Direct Environment 5

Outdoor Configuration6

[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. [Means for solving the problem]

[0016] 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 to block CXCR3A-dependent cellular functions, does not specifically bind to human vascular endothelial cells, and is selected from the group consisting of the following antibodies (AB1) to (AB9) and (AB12) to (AB27): (AB26) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 251, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 252, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 253, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 254, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 255, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 256. (AB23) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 221, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 222, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 223, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 224, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 225, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 226. (AB24) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 231, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 232, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 233, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 234, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 235, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 236. (AB25) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 242, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 245, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 246. (AB27) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 261, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 262, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 263, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 264, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 265, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 266. (AB1) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, (AB2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16. (AB3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26. (AB5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46. (AB8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76. (AB16) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 151, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 152, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 153, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 154, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 155, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 156. (AB17) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 161, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 162, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 163, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 164, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 165, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 166. (AB18) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 171, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 172, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 173, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 174, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 175, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 176. (AB20) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 191, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 192, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 193, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 194, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 195, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 196. (AB21) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 201, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 202, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 203, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 204, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 205, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 206. (AB22) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 211, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 212, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 213, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 214, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 215, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 216. (AB4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36. (AB6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56. (AB7) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66. (AB9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86. (AB12) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116. (AB13) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 126. (AB14) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 132, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 133, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 135, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 136. (AB15) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 141, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 142, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 143, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 144, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 145, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 146. (AB19) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 181, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 182, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 183, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 184, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 185, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 186. It is an antibody that satisfies any one of the following conditions.

[0017] One embodiment of the present disclosure provides an 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 is selected from the group consisting of the following (C1) to (C9) and (C12) to (C27): (C26) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 257 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 259, (C23) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 227 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 229, (C24) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 237, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 239, (C25) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 247, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 249, (C27) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 267 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 269, (C1) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9, (C2) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19. (C3) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 29, (C5) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 49; (C8) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 79; (C16) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 157 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 159, (C17) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 167 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 169, (C18) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 177 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 179, (C20) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 197 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 199; (C21) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 207 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 209, (C22) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 217 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 219, (C4) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 39, (C6) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 59, (C7) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 67 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 69, (C9) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89; (C12) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 117 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 119, (C13) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 127 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 129, (C14) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139, (C15) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 147 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 149, (C19) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 187 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 189, It is an antibody that satisfies any one of the following conditions.

[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 treating 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. [Effects of the Invention]

[0030] According to the present disclosure, it is possible to provide a novel anti-human CXCR3 antibody that has superior properties as a pharmaceutical raw material. [Brief explanation of the drawings]

[0031] [Figure 1] 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. [Figure 2] 1 is a histogram showing the results of flow cytometry performed in Example 7, illustrating the binding properties of the antibody (13B4, 201009-2-C) to CHO cells stably expressing human CXCR3A and vascular endothelial cells. [Figure 3A] 1 is a graph showing the results of assessing the binding ability of an antibody (13B4) to CHO cells stably expressing human CXCR3A. [Figure 3B] Fig. 1 is a graph showing the results of assessing the binding ability of antibody (11F11) to CHO cells stably expressing human CXCR3A. [Figure 3C]Fig. 10 is a graph showing the results of assessing the binding ability of an antibody (201006-3-H) to CHO cells stably expressing human CXCR3A. [Figure 3D] Fig. 10 is a graph showing the results of assessing the binding ability of an antibody (201006-5-F) to CHO cells stably expressing human CXCR3A. [Figure 3E] Fig. 10 is a graph showing the results of assessing the binding ability of antibody (201006-7-G) to CHO cells stably expressing human CXCR3A. [Figure 3F] Fig. 10 is a graph showing the results of assessing the binding ability of an antibody (201009-2-C) to CHO cells stably expressing human CXCR3A. [Figure 3G] Fig. 10 is a graph showing the results of assessing the binding ability of an antibody (201009-3-H) to CHO cells stably expressing human CXCR3A. [Figure 3H] Fig. 10 is a graph showing the results of assessing the binding ability of an antibody (201009-1-C) to CHO cells stably expressing human CXCR3A. [Figure 4A] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (13B4) against human CXCR3A. [Figure 4B] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (11F11) against human CXCR3A. [Figure 4C] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-3-H) against human CXCR3A. [Figure 4D] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-5-F) against human CXCR3A. [Figure 4E] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201006-7-G) against human CXCR3A. [Figure 4F] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201009-2-C) against human CXCR3A. [Figure 4G] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201009-3-H) against human CXCR3A. [Figure 4H]1 is a graph showing the dose dependency of the inhibitory activity of an antibody (201009-1-C) against human CXCR3A. [Figure 5] 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. [Figure 6A] 1 is a graph showing the results of evaluating the binding ability of an antibody (13B4) to human peripheral blood mononuclear cell-derived T cells. [Figure 6B] 1 is a graph showing the results of assessing the binding ability of an antibody (11F11) to human peripheral blood mononuclear cell-derived T cells. [Figure 6C] 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-3-H) to human peripheral blood mononuclear cell-derived T cells. [Figure 6D] 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-5-F) to human peripheral blood mononuclear cell-derived T cells. [Figure 6E] 1 is a graph showing the results of evaluating the binding ability of an antibody (201006-7-G) to human peripheral blood mononuclear cell-derived T cells. [Figure 6F] 1 is a graph showing the results of evaluating the binding ability of an antibody (201009-2-C) to human peripheral blood mononuclear cell-derived T cells. [Figure 7] 1 is a graph showing the dose dependency of the inhibitory activity of antibody (201009-2-C) on the migration of human peripheral blood mononuclear cell-derived T cells. [Figure 8] 1 is a graph showing the results of flow cytometry performed in Example 14, demonstrating that antibodies are internalized together with receptors in human peripheral blood mononuclear cell-derived T cells. [Figure 9A] FIG. 1 is an explanatory diagram showing the alignment of the heavy chain variable regions of humanized anti-CXCR3 antibodies. [Figure 9B] FIG. 1 is an explanatory diagram showing the alignment of the light chain variable regions of humanized anti-CXCR3 antibodies. [Figure 10A] 1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL1) to human peripheral blood mononuclear cell-derived T cells. [Figure 10B]1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL2) to human peripheral blood mononuclear cell-derived T cells. [Figure 10C] 1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL3) to human peripheral blood mononuclear cell-derived T cells. [Figure 10D] 1 is a graph showing the results of evaluating the binding of antibodies (VH1+VL4) to human peripheral blood mononuclear cell-derived T cells. [Figure 10E] 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL1) to human peripheral blood mononuclear cell-derived T cells. [Figure 10F] 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL2) to human peripheral blood mononuclear cell-derived T cells. [Figure 10G] 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL3) to human peripheral blood mononuclear cell-derived T cells. [Figure 10H] 1 is a graph showing the results of evaluating the binding of antibodies (VH2+VL4) to human peripheral blood mononuclear cell-derived T cells. [Figure 11A] 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH1+VL1) on the migration of human peripheral blood mononuclear cell-derived T cells. [Figure 11B] 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH1+VL4) on the migration of human peripheral blood mononuclear cell-derived T cells. [Figure 11C] 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH2+VL1) on the migration of human peripheral blood mononuclear cell-derived T cells. [Figure 11D] 1 is a graph showing the dose dependency of the inhibitory activity of antibodies (VH2+VL4) on the migration of human peripheral blood mononuclear cell-derived T cells. DETAILED DESCRIPTION OF THE INVENTION

[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) that spans the cell membrane seven times, with its N-terminus facing extracellularly and its C-terminus facing intracellularly. Human CXCR3 exists in three isoforms: CXCR3A, CXCR3B, and CXCR3Alt. The gene (cDNA) encoding human CXCR3 has already been isolated, and the amino acid sequence of human CXCR3 is also known. This 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 indicates the amino acid number, and the right side indicates each domain. Note that the boundaries between each domain may vary slightly. 1-53: N-terminal domain 81-89: Intracellular first loop domain 111-125: extracellular first loop domain 148-169: Intracellular second loop domain 190-212: extracellular second loop domain 234-255: Intracellular third loop domain 278-298: Extracellular third loop domain 322-368: C-terminal domain

[0035] <Anti-human CXCR3 antibody> The antibody disclosed in the present specification is an antibody (anti-human CXCR3 antibody) that specifically binds to human CXCR3, 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] The antibody according to one embodiment comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, or 271; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, or 272; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263, or 273; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, or 274; 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 It has 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] The antibody according to one embodiment comprises: Below (A1)~(A28): (A1) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3. (A2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13. (A3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23. (A4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33. (A5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43. (A6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53. (A7) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63. (A8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73. (A9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83. (A10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93. (A11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103. (A12) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113. (A13) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123. (A14) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 132, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 133. (A15) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 141, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 142, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 143. (A16) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 151, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 152, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 153. (A17) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 161, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 162, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 163. (A18) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 171, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 172, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 173. (A19) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 181, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 182, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 183. (A20) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 191, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 192, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 193. (A21) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 201, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 202, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 203. (A22) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 211, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 212, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 213. (A23) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 221, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 222, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 223. (A24) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 231, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 232, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 233. (A25) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 242, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 243. (A26) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 251, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 252, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 253. (A27) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 261, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 262, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 263. (A28) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 271, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 272, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 273. Satisfy one of the following.

[0038] The antibody according to one embodiment comprises: Below (B1)~(B28): (B1) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, (B2) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16. (B3) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26. (B4) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36. (B5) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46. (B6) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 56. (B7) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66. (B8) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 76. (B9) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 86. (B10) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96. (B11) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106. (B12) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116. (B13) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 126. (B14) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 135, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 136. (B15) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 144, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 145, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 146. (B16) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 154, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 155, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 156. (B17) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 164, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 165, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 166. (B18) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 174, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 175, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 176. (B19) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 184, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 185, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 186. (B20) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 194, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 195, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 196. (B21) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 204, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 205, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 206. (B22) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 214, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 215, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 216. (B23) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 224, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 225, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 226. (B24) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 234, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 235, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 236. (B25) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 245, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 246, (B26) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 254, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 255, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 256. (B27) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 264, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 265, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 266. (B28) A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 274, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 275, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 276. Satisfy one of the following.

[0039] In one embodiment, the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 of the antibody satisfy any one of (AB1) to (AB9) and (AB12) to (AB27) above.

[0040] In one embodiment, the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 of the antibody are (AB10), (AB11), or (AB28) shown below: (AB10) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 92, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 93, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 96. (AB11) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 103, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 106. (AB28) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 271, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 272, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 273, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 274, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 275, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 276. Satisfy one of the following.

[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 selected from the group consisting of (C10), (C11), and (C28) below: (C10) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 97 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 99, (C11) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 107 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 109, (C28) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 277 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 279; Satisfy one of the following.

[0043] The heavy chain variable region specified by (C1) (SEQ ID NO: 7) contains heavy chain CDRs 1 to 3 (SEQ ID NOs: 1 to 3) specified by (A1) or (AB1). The light chain variable region specified by (C1) (SEQ ID NO: 9) 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 specified by (C2) (SEQ ID NO: 17) contains heavy chain CDRs 1 to 3 specified by (A2) or (AB2) (SEQ ID NO: 11 to 13). The light chain variable region specified by (C2) (SEQ ID NO: 19) contains light chain CDRs 1 to 3 specified by (B2) or (AB2) (SEQ ID NO: 14 to 16). 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 specified by (C3) (SEQ ID NO: 27) contains heavy chain CDRs 1 to 3 specified by (A3) or (AB3) (SEQ ID NO: 21 to 23). The light chain variable region specified by (C3) (SEQ ID NO: 29) contains light chain CDRs 1 to 3 specified by (B3) or (AB3) (SEQ ID NO: 24 to 26). 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 CDRs 1 to 3 specified by (A4) or (AB4) (SEQ ID NO: 31 to 33). The light chain variable region specified by (C4) (SEQ ID NO: 39) contains light chain CDRs 1 to 3 specified by (B4) or (AB4) (SEQ ID NO: 34 to 36). 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 specified by (C5) (SEQ ID NO: 47) contains heavy chain CDRs 1 to 3 specified by (A5) or (AB5) (SEQ ID NO: 41 to 43). The light chain variable region specified by (C5) (SEQ ID NO: 49) contains light chain CDRs 1 to 3 specified by (B5) or (AB5) (SEQ ID NO: 44 to 46). 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 CDRs 1 to 3 specified by (A7) or (AB7) (SEQ ID NO: 61 to 63). The light chain variable region specified by (C7) (SEQ ID NO: 69) contains light chain CDRs 1 to 3 specified by (B7) or (AB7) (SEQ ID NO: 64 to 66). 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 CDRs 1 to 3 specified by (A9) or (AB9) (SEQ ID NO: 81 to 83). The light chain variable region specified by (C9) (SEQ ID NO: 89) contains light chain CDRs 1 to 3 specified by (B9) or (AB9) (SEQ ID NO: 84 to 86). An example of an antibody that satisfies (A9), (B9), (AB9), or (C9) is "201112-1-C," which is described in the Examples below.

[0052] The heavy chain variable region specified by (C10) (SEQ ID NO: 97) contains 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) contains 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 CDRs 1 to 3 specified by (A12) or (AB12) (SEQ ID NO: 111 to 113). The light chain variable region specified by (C12) (SEQ ID NO: 119) contains light chain CDRs 1 to 3 specified by (B12) or (AB12) (SEQ ID NO: 114 to 116). An example of an antibody that satisfies (A12), (B12), (AB12), or (C12) is "201006-4-H," which is 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," which is 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 specified by (C15) (SEQ ID NO: 147) contains heavy chain CDRs 1 to 3 specified by (A15) or (AB15) (SEQ ID NO: 141 to 143). The light chain variable region specified by (C15) (SEQ ID NO: 149) contains light chain CDRs 1 to 3 specified by (B15) or (AB15) (SEQ ID NO: 144 to 146). An example of an antibody that satisfies (A15), (B15), (AB15), or (C15) is "201006-4-A," which is 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," which is 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," which is 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 that satisfies (A19), (B19), (AB19), or (C19) is "201006-4-G," which is 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 that satisfies (A21), (B21), (AB21), or (C21) is "201006-2-D," which is described in the Examples below.

[0064] The heavy chain variable region specified by (C22) (SEQ ID NO: 217) contains heavy chain CDRs 1 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 CDRs 1 to 3 specified by (B22) or (AB22) (SEQ ID NO: 214 to 216). An example of an antibody that satisfies (A22), (B22), (AB22), or (C22) is "201006-2-C," which is described in the Examples below.

[0065] The heavy chain variable region specified by (C23) (SEQ ID NO: 227) contains heavy chain CDRs 1 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 CDRs 1 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," which is described in the Examples below.

[0066] The heavy chain variable region specified by (C24) (SEQ ID NO: 237) contains heavy chain CDRs 1 to 3 specified by (A24) or (AB24) (SEQ ID NO: 231 to 233). The light chain variable region specified by (C24) (SEQ ID NO: 239) contains light chain CDRs 1 to 3 specified by (B24) or (AB24) (SEQ ID NO: 234 to 236). An example of an antibody that satisfies (A24), (B24), (AB24), or (C24) is "201006-5-F," which is described in the Examples below.

[0067] The heavy chain variable region specified by (C25) (SEQ ID NO: 247) contains heavy chain CDRs 1 to 3 specified by (A25) or (AB25) (SEQ ID NO: 241 to 243). The light chain variable region specified by (C25) (SEQ ID NO: 249) contains light chain CDRs 1 to 3 specified by (B25) or (AB25) (SEQ ID NO: 244 to 246). An example of an antibody that satisfies (A25), (B25), (AB25), or (C25) is "201006-7-G," which is described in the Examples below.

[0068] The heavy chain variable region specified by (C26) (SEQ ID NO: 257) contains heavy chain CDRs 1 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 CDRs 1 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," which is described in the Examples below.

[0069] The heavy chain variable region specified by (C27) (SEQ ID NO: 267) contains heavy chain CDRs 1 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 CDRs 1 to 3 specified by (B27) or (AB27) (SEQ ID NO: 264 to 266). An example of an antibody that satisfies (A27), (B27), (AB27), or (C27) is "201009-3-H," which is 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," which is 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 NO:44 and SEQ ID NO:54 are the same. The amino acid sequences of SEQ ID NO:64 and SEQ ID NO:124 are the same. The amino acid sequences of SEQ ID NO:74, SEQ ID NO:144, SEQ ID NO:224, SEQ ID NO:254, and SEQ ID NO:264 are the same. The amino acid sequences of SEQ ID NO:84, SEQ ID NO:94, and SEQ ID NO:104 are the same. The amino acid sequences of SEQ ID NO:154 and SEQ ID NO:164 are the same. The amino acid sequences of SEQ ID NO:174, SEQ ID NO:184, SEQ ID NO:204, and SEQ ID NO:214 are the same.

[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 above-mentioned antibody may be a functional fragment of an antibody. Here, "functional fragment of an antibody" refers to a partial fragment of an antibody (i.e., an immunoglobulin) that retains at least one action against an antigen. Examples of the partial fragment include F(ab')2, Fab, Fv, disulfide-linked Fv, single-chain antibodies (scFv, VH-VL), VH, polymers thereof, and fusions of these with a heavy chain CH3 region. Further examples include each CDR, 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 the antibodies described above. A partial fragment of an antibody may also be referred to as an "antibody fragment."

[0078] The above-mentioned antibody may be a multispecific antibody. The multispecific antibody of this embodiment specifically binds to the extracellular domain of human CXCR3A, has the activity of blocking CXCR3A-dependent cellular functions, and has at least a first specific binding property that does not specifically bind to human vascular endothelial cells, and a second specific binding property 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 embodiment, 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 comprise 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 the activity of blocking CXCR3A-dependent cellular functions. CXCR3A-dependent cellular functions are induced by the activation of proteins, such as trimeric G proteins and β-arrestins, that conjugate with the intracellular portion of CXCR3 and are involved in intracellular signal transduction. 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 share the same epitope as the above-described antibodies. For example, the epitopes of 28 anti-human CXCR3 antibodies specifically described 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 amino acid sequence having 1 to 10 amino acid substitutions, additions, or deletions in the amino acid sequence of the SEQ ID NOs. defined in (C1) to (C28) above, or an antibody having 90% or greater identity to the amino acid sequence of the SEQ ID NOs. defined in (C1) to (C28) above. The number of substituted, added, or deleted amino acids is preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3. The identity is preferably 92% or greater, more preferably 95% or greater, and particularly preferably 97% or greater.

[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 a first antibody, to the receptor is competitively inhibited by a second antibody to be 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 the activity of blocking 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 antibody> 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 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 antibodies, including, for example, a first nucleic acid encoding a heavy chain variable region and / or a second nucleic acid encoding a light chain variable region.

[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 3 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] <Antibody manufacturing method> The antibody can be produced using genetic recombination techniques, i.e., by constructing recombinant cells that express the nucleic acid, the antibody can be obtained from a culture of the cells.

[0094] <Construction and preparation of humanized antibodies> Methods for constructing and producing humanized antibodies are described below. As an example, a method for constructing and producing a humanized antibody having heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 as specified in (AB1) above is 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 region of VH of any human antibody. Similarly, DNAs encoding variable regions in which light chain CDRs 1 to 3 are grafted onto the framework region of VL of any human antibody are prepared. Each of the prepared DNAs is inserted into a vector containing a sequence encoding the CH or CL of a human antibody to construct a humanized antibody expression vector. The constructed expression vector is introduced into host cells to obtain recombinant cells that express the humanized antibody. The recombinant cells are then cultured, and the desired humanized antibody is obtained from the culture.

[0096] Humanized antibodies having heavy chain CDR1 to 3 and light chain CDR1 to 3 specified above in (AB2) to (AB28) can also be constructed and produced in a similar manner.

[0097] Methods for constructing and producing chimeric antibodies are described below. 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 is described. First, a DNA encoding the amino acid sequence shown in SEQ ID NO: 7 is prepared as the DNA encoding VH. Furthermore, a DNA encoding the amino acid sequence shown in SEQ ID NO: 9 is prepared as the DNA encoding VL. 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 carrying a sequence encoding the CH or CL of a human antibody, respectively, to construct a chimeric antibody expression vector. Vectors carrying a sequence encoding the CH or CL of a human antibody are commercially available. The constructed expression vector is introduced into host cells to obtain recombinant cells that express the chimeric antibody. These recombinant cells are 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 an anti-human CXCR3 antibody or a fragment thereof of the present disclosure to another antibody or a fragment thereof. Another example is co-expressing an anti-human CXCR3 antibody or a fragment thereof of the present disclosure with another antibody or a fragment thereof in a host cell. Other linking methods include chemical coupling, gene fusion, non-covalent association, etc.

[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 antibodies 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 cells can be collected and 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 antibody> The antibody of the present disclosure may be a modified antibody to which another molecule is bound. Examples of the other molecule include peptides, proteins, small molecules, radioisotopes, light absorbers, etc. Methods for binding to the other molecule include chemical coupling, gene fusion, non-covalent association, etc. Note that when the other molecule to be bound is an antibody, the modified antibody may be a multispecific antibody. In this respect, a multispecific antibody can be considered a type of modified antibody.

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

[0106] Antibodies can block CXCR3A-dependent cellular functions in various ways, including by binding to the ligand-binding site on the cell surface and competitively or noncompetitively inhibiting ligand binding to the receptor; by binding to a site other than the ligand-binding site and altering receptor structure to inhibit activation; and by binding to the receptor and inducing receptor internalization, thereby reducing receptor expression levels on the cell surface and thereby reducing cellular ligand responsiveness. 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 to treat a disease, disorder, or condition caused by impaired CXCR3-dependent cellular function, including Th1-related diseases such as atherosclerosis, myocarditis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, type 1 diabetes, psoriasis, rheumatism, inflammatory bowel disease, systemic lupus erythematosus, and acute cardiac allograft rejection, as well as infectious diseases and tumors such as influenza and COVID-19.

[0108] In particular, drugs that selectively inhibit CXCR3A are thought to be highly useful in therapies aimed at suppressing the infiltration, proliferation, and activation of inflammatory cells. Increased CXCR3A ligands 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] In addition, drugs that selectively inhibit CXCR3A may 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 interactions promote tumor growth and metastasis, while CXCR3B-ligand interactions promote 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 to be an inhibitor that preferentially inhibits CXCR3A over CXCR3B, but no CXCR3A-specific inhibitors have been reported (K. Boye et al., Sci Rep. 2017 Sep 6;7(1):10703.).

[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 administration, pulmonary administration, and transdermal administration. Injections can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. The administration method can be selected appropriately depending on the age and symptoms of the patient. The dosage of the above-mentioned antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg of antibody per patient. However, the dosage of the above-mentioned antibody is not limited to these ranges.

[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, etc., 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.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. If necessary, the antibody can also be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.) or made into colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) (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 nucleic acids can also be incorporated into gene therapy vectors or mRNA that can be translated into proteins in vivo to produce gene therapy drugs. Methods for administering such gene therapy drugs (recombinant vectors) include direct administration using naked plasmids, administration via packaging in liposomes, administration via incorporation into various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, and HVJ vectors (see Adolph, "Viral Genome Methods," CRC Press, Florid (1996)), and administration via coating on bead carriers 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. Pat. No. 4,945,050), or viral infection, and then the cells may be reintroduced into an animal.

[0117] Furthermore, the present disclosure also includes methods and therapeutic agents for treating a disease or disorder caused by abnormally enhanced CXCR3-dependent cell function in a mammal suffering from the disease. Here, the term "treatment" means preventing or alleviating the progression and worsening of the pathological condition of a disease in a mammal that is at risk of contracting or is contracting the disease, and is used to mean a therapeutic procedure aimed at preventing or alleviating the progression and worsening of the symptoms of the disease. Furthermore, the term "disease" refers to any disease that develops due to abnormally enhanced CXCR3-dependent cell function, and is not particularly limited to, but includes, for example, 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, tumors, and other Th1-related immune disorders. It also includes cardiovascular disorders, nervous system disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, infectious diseases, blood cancers, and solid cancers. The term "mammal" to be treated refers to any animal classified as a mammal, and includes, but is not limited to, humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is a human.

[0118] <Antibody immobilization 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 manufacturing a medicament for treating a disease, disorder, or condition involving impaired CXCR3-dependent cellular function. The disease, disorder, or condition includes at least cardiovascular disorders, nervous system disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, infectious diseases, blood cancers, and solid cancers. [Example]

[0123] [Example 1] Immunization of animals to produce anti-CXCR3 antibodies (1) Preparation of plasmid for DNA immunization The template plasmid vector pCI-hEP4-GroEL was constructed as follows. Based on the sequence information of human EP4 registered in the NCBI Gene Bank (NM_000958, SEQ ID NO: 293), an artificial gene was synthesized in which a signal sequence (SEQ ID NO: 294) was added to the 5' end of the human EP4 gene. Using this as a template, a human EP4 gene fragment (hereinafter referred to as "DNA fragment A") was prepared in which the GCTAGC sequence was added to the 5' end and the GTCGAC sequence to the 3' end by PCR.

[0124] Genomic DNA was extracted and purified from E. coli strain HMS174(DE3) (Novagen). Next, PCR was performed using the purified genomic DNA as a template and the primer pair shown in SEQ ID NOs: 295 and 296 to amplify a DNA fragment containing the GroEL subunit gene (SEQ ID NO: 297; hereafter referred to as "DNA fragment B"). DNA fragment B contained a SalI site at the 5' end and a sequence encoding two stop codons (TAATAG) and a NotI site at the 3' end, derived from the primers.

[0125] The mammalian expression vector pCI-Mammalian Expression Vector (Promega) was digested with the restriction enzymes NheI and SalI, and the termini were dephosphorylated with bacterial alkaline phosphatase (BAP), followed by insertion of DNA fragment A. This expression vector was further digested with SalI and NotI, and the termini were dephosphorylated with BAP, followed by insertion of DNA fragment B, 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 GCTAGC sequence to the 5' end and the GTCGAC sequence 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 to construct the immunization vector pCI-hCXCR3A-GroEL. This vector expresses a fusion protein of human CXCR3A and GroEL.

[0127] (2) DNA immunity The vector pCI-hCXCR3A-GroEL was dissolved in PBS to a concentration of 2 mg / mL to prepare an immunization composition. Eight-week-old mice were immunized by injecting 25 μL of this immunization composition into the thigh muscles of both legs (day 0). This resulted in 50 μg of pCI-hCXCR3A-GroEL being administered to each leg, i.e., 100 μg per mouse per injection. Subsequently, the mice were immunized repeatedly 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 each leg, i.e., 160 μg per rat per injection. Subsequently, the rats were immunized repeatedly 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) Generation of cells stably expressing human CXCR3A An artificial gene was synthesized based on the human CXCR3A sequence (AAO92295, SEQ ID NO: 281) registered with the NCBI Gene Bank and introduced into pEF-FRT (Thermo Fisher Scientific) to construct pEF-FRT-hCXCR3A. pEF-FRT-hCXCR3A and the pOG44 plasmid (Thermo Fisher Scientific) were co-transfected into CHO-FlpIn cells (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific). Cells resistant to hygromycin and stably expressing human CXCR3A were cloned. These human CXCR3A-expressing cells are hereafter referred to as "human CXCR3A stably expressing CHO cells."

[0130] (4) Flow cytometry evaluation of serum antibodies for human CXCR3A Human CXCR3A stably expressing CHO cells and CHO-FlpIn cells were washed with FACS buffer (PBS containing 1% FBS) to a cell concentration of 1 × 10 7 The cells were suspended in FACS buffer at a concentration of 100 cells / mL. Fc Block (Tonbo Biosciences) was added at a volume of 1 / 500 of the cell suspension, and blocking was performed at 4°C. After blocking, 1 to 2 × 10 5 Cells were suspended at a concentration of 50 μL per cell. This cell suspension was mixed with the immunized animal serum, which served 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 for 1 hour at 4°C. After washing twice with 100 μL of FACS buffer, the cells were suspended in 80 μL of FACS buffer and the cell surface fluorescence intensity was measured using an iQue flow cytometer (Sartorius) to evaluate antibody binding. The immunized animal serum specifically reacted 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 with transiently expressing cells and harvesting 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 mice or rats (2). Three days later, the spleen, inguinal lymph nodes, and iliac lymph nodes were harvested. Splenocytes and lymph node-derived cells were isolated from each tissue, 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 5 A cell suspension of 500 μL was prepared. This cell suspension was loaded into a microchamber for the cell picking system. The microchamber was centrifuged five times at 300 rpm for 1 minute to allow one or two CHO cells stably expressing human CXCR3A to be placed in each microwell. The microchamber was washed with F-12 medium and then 500 μL of F-12 medium was added. The microchamber was incubated at 37°C for 1 hour in a CO2 incubator to allow the 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 the cells. After washing three times with F-12 medium to remove excess CytoRed, 1 mL of F-12 medium was added to the microchamber.

[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 to allow 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 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 then placed in a cell picking system (AS ONE Corporation) to acquire transmitted light images and two types of fluorescent images of all microwells. CytoRed fluorescence detection was performed at an excitation wavelength of 543 nm and an emission wavelength of 593 nm. Alexa Fluor 488 fluorescence detection was performed at an excitation wavelength of 482 nm and an emission wavelength of 536 nm. From microwells where it was determined based on the scanned images of transmitted light, CytoRed, and Alexa Fluor 488 that antibodies binding to CHO cells stably expressing human CXCR3A were present, antibody-producing cells were collected into a cell lysate using capillaries (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 isolated 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, cDNA synthesis was performed by reverse transcription after washing the oligo-dT magnet with washing solution. After further washing the magnet, 5'-terminal translational reaction was performed. Using the synthesized cDNA, the antibody heavy chain variable region (VH region) gene and antibody light chain variable region (VL region) gene were isolated and amplified by 5'-race PCR.

[0135] To enhance the specificity of the amplified product, PCR was performed twice. In the first PCR for the mouse antibody, a mixture of a first forward primer (SEQ ID NO: 282) that amplifies both the VH and VL regions, 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 was used. In the second PCR, the amplified product from the first PCR was used as a template. For amplification of the VH region, a second forward primer (SEQ ID NO: 285) and a mouse VH second reverse primer (SEQ ID NO: 286) that specifically amplifies the VH region were used, and for amplification of the VL region, a second forward primer (SEQ ID NO: 285) and a mouse VL second reverse primer (SEQ ID NO: 287) that specifically amplifies the VL region were used.

[0136] In addition, in the first PCR for antibodies obtained from rats, 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. For amplification of the VH region, a 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, and for amplification of the VL region, a 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.

[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 units 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 by PCR to construct an antibody expression unit expressing 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 by PCR to construct an antibody expression unit expressing a full-length antibody light chain.

[0139] (4) Introducing an antibody expression unit into mammalian cells Expi293F cells (Thermo Fisher Scientific) were cultured at 7.5 × 10 in a 6-well cell culture plate. 6 Cells were seeded at 3 mL / well. Expifectamine 293 Reagent (Thermo Fisher Scientific) was used to co-transfect the heavy and light chain antibody expression units constructed in (3) into Expi293F cells. Cell supernatants were collected on day 5 after transfection and used to evaluate the binding activity of the expressed antibodies in the same manner as in Example 1 (4). Antibody units that bound to CHO cells stably expressing human CXCR3A but did not bind to CHO-FlpIn cells were designated as primary hit antibodies.

[0140] [Example 3] Preparation of antibodies <2> (1) Hybridoma production and screening for binding clones The cells collected in Example 1(5) and myeloma cells SP2 / 0 were fused using the cell fusion device NEPAGENE ECFG21 (Nepper Gene). The fused cells (hybridomas) were suspended in RPMI1640 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 CHO cells stably expressing human CXCR3A were screened using the same method as in Example 1(4).

[0141] (2) Hybridoma cloning Hybridomas confirmed to produce antibodies in (1) were cloned by limiting dilution. Specifically, the hybridomas were seeded into 96-well plates at a density of 1 cell or less per well and cultured. After two 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 5 The cells were then 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 procedure as in Example 2(2) was carried out.

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

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

[0146] [Example 4] Determination of antibody heavy chain variable region and CDR sequence of antibody heavy chain variable region 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 region comprehensively including the regions determined by both numbering methods was determined as the CDR.

[0147] Based on the determined sequence information for the antibody heavy chain variable region and antibody light chain variable region, 28 types of antibodies were selected that were confirmed to have independent antibody heavy chain variable region and 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 derived from rats, and the others are derived from mice.

[0148] 201001-1-F (Table 1-1; SEQ ID NOs: 1-10) 201001-5-B (Tables 1-2; SEQ ID NOs: 11-20) 201001-2-C (Tables 1-3; SEQ ID NOs: 21-30) 201001-4-E (Tables 1-4; SEQ ID NOs: 31-40) 201001-1-C (Tables 1-5; SEQ ID NOs: 41-50) 201001-5-A (Tables 1-6; SEQ ID NOs: 51-60) 201009-1-D (Tables 1-7; SEQ ID NOs: 61-70) 201009-5-F (Tables 1-8; SEQ ID NOs: 71-80) 201112-1-C (Tables 1-9; SEQ ID NOs: 81-90) 13B4 (Tables 1-10; SEQ ID NOs: 91-100) 11F11 (Tables 1-11; SEQ ID NOs: 101-110) 201006-4-H (Tables 1-12; SEQ ID NOs: 111-120) 201006-4-F (Tables 1-13; SEQ ID NOs: 121-130) 201006-5-A (Tables 1-14; SEQ ID NOs: 131-140) 201006-4-A (Tables 1-15; SEQ ID NOs: 141-150) 201006-1-H (Tables 1-16; SEQ ID NOs: 151-160) 201006-3-D (Tables 1-17; SEQ ID NOs: 161-170) 201006-1-F (Tables 1-18; SEQ ID NOs: 171-180) 201006-4-G (Tables 1-19; SEQ ID NOs: 181-190) 201006-7-A (Tables 1-20; SEQ ID NOs: 191-200) 201006-2-D (Tables 1-21; SEQ ID NOs: 201-210) 201006-2-C (Tables 1-22; SEQ ID NOs: 211-220) 201006-3-H (Table 1-23; SEQ ID NOs: 221-230) 201006-5-F (Table 1-24; SEQ ID NOs: 231-240) 201006-7-G (Table 1-25; SEQ ID NOs: 241-250) 201009-2-C (Table 1-26; SEQ ID NOs: 251-260) 201009-3-H (Table 1-27; SEQ ID NOs: 261-270) 201009-1-C (Table 1-28; SEQ ID NOs: 271-280)

[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] [Table 1-1]

[0151] [Table 1-2]

[0152] [Table 1-3]

[0153] [Table 1-4]

[0154] [Table 1-5]

[0155] [Table 1-6]

[0156] [Table 1-7]

[0157] [Table 1-8]

[0158] [Table 1-9]

[0159] Table 1-10

[0160] Table 1-11

[0161] Table 1-12

[0162] Table 1-13

[0163] Table 1-14

[0164] Table 1-15

[0165] Table 1-16

[0166] Table 1-17

[0167] Table 1-18

[0168] Table 1-19

[0169] [Table 1-20]

[0170] [Table 1-21]

[0171] [Table 1-22]

[0172] [Table 1-23]

[0173] [Table 1-24]

[0174] [Table 1-25]

[0175] [Table 1-26]

[0176] [Table 1-27]

[0177] [Table 1-28]

[0178] [Example 5] Intracellular Ca 2+ Antibody screening by signal transduction inhibitory activity assessment The culture supernatant of the clone selected as the primary hit antibody in Example 2(4) or Example 3(5) was used to obtain a purified antibody by Protein A affinity purification. The purified primary hit antibody was 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 2 days. After 2 days, the culture medium was replaced with a solution containing 3 μM Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Thermo Fisher Scientific). After 1 hour, primary hit antibodies or control antibodies diluted within the 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 Corporation) or rat isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) were used as control antibodies. After incubation, the assay plate was placed in Ca 2+ The signal was analyzed using the FDSSμCELL signal analyzer (Hamamatsu Photonics), and the intracellular Ca2+ levels were 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 normalized by setting the inhibition rate of "no antibody, with IP-10" at 0% and the inhibition rate of "no antibody, without IP-10" at 100%, and calculated as a relative value. 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 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 to determine whether they could induce stable intracellular Ca2+ binding. 2+ The antibody was selected as one that exhibits signal transduction inhibitory activity.

[0181] [Table 2]

[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 the RNeasy Mini Kit (QIAGEN) according to the kit's protocol. Genomic DNA was removed from the RNA using 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 alternative 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 tests.

[0186] CHO cells stably expressing human CXCR3A, 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 at a concentration of 1 × 10 cells / mL. Fc Block (Tonbo Biosciences) was added at a volume of 1 / 500 of the cell suspension, or goat serum diluted 1:1, and blocking was performed at 4°C for 30 minutes. After blocking, 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 mouse isotype control antibody (Fujifilm Wako Pure Chemical Corporation), rat isotype control antibody (Fujifilm Wako Pure Chemical Corporation), or 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, 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, Figure 2 shows histograms comparing 13B4, 201009-2-C, a CXCR3B-specific antibody, or a prior art antibody (all of which are anti-chemokine antibodies) with a control antibody (isotype antibody). In Figure 2, solid black indicates an isotype antibody, and open white indicates an anti-chemokine antibody. A rightward shift in the histogram was determined to indicate "binding." The CXCR3B-specific antibody was confirmed to bind to two types of human vascular endothelial cells but not to CHO cells stably expressing human CXCR3A. 13B4 and the prior art antibody were confirmed to bind to CHO cells stably expressing human CXCR3A and the 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. A rightward shift in the histogram is indicated by "+" and no shift is indicated by "-." Sixteen antibodies, including 201009-2-C, were confirmed to specifically bind to human CXCR3A and not to human vascular endothelial cells, which predominantly express human CXCR3B.

[0189] [Table 3]

[0190] [Example 8] Preparation 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) Introducing antibody expression plasmids into mammalian cells In a 125 mL Erlenmeyer flask (vent cap) for cell culture, 1.8 × 10 ExpiCHO cells (Thermo Fisher Scientific) were cultured. 8 The cells were seeded at 30 mL per flask. The Expifectamine CHO Tranfection Kit (Thermo Fisher Scientific) was used to co-transfect the heavy and light chain antibody expression plasmids constructed in (1) into ExpiCHO cells. On day 5 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 types of antibodies obtained in Example 8, namely, 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 7The cells were suspended in FACS buffer at a concentration of 1 × 10 cells / mL. Fc Block (Tonbo Biosciences) was added at a volume 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 volume 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. After washing twice with 100 μL of FACS buffer, cells were suspended in 50 μL of FACS buffer and the cell surface fluorescence intensity was measured using a Novocyte flow cytometer (Agilent) to evaluate antibody binding.

[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] [Table 4]

[0196] Example 10: Intracellular Ca 2+ Signal transduction inhibition assessment The eight types of antibodies obtained in Example 8, namely, 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. 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, eight antibodies diluted to concentrations ranging from 200 nM to 0.8 nM or a control antibody were added to each well and incubated for 15 minutes. Mouse isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) or rat isotype control antibodies (Fujifilm Wako Pure Chemical Corporation) were used as control antibodies. After incubation, the assay plate was resuspended in Ca 2+ The signal was analyzed using the FDSSμCELL signal analyzer (Hamamatsu Photonics), and the intracellular Ca2+ levels were measured when each cell was stimulated with 100 nM IP-10 (PEPROTECH). 2+ The inhibitory activity against a transient increase in the concentration of the antibody was measured. The inhibitory activity was normalized to a 0% inhibition rate for "no antibody, with IP-10" and a 100% inhibition rate for "no antibody, without IP-10," and calculated as a relative value. 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 Ca2+ expression in CHO cells stably expressing human CXCR3A. 2+ It was confirmed that the antibodies inhibited signal transduction in a dose-dependent manner, demonstrating their ability to block 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] [Table 5]

[0200] [Example 11] Evaluation of antibody binding specificity To evaluate the binding specificity of antibodies to CXCR3A, 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.

[0201] Human CXCR3A stably expressed CHO cells and other chemokine receptor expressing cells, human CCR7 stably expressed CHO cells (manufactured in-house) and human CCR6 stably expressed 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 at a concentration of 1 × 10 cells / mL. Fc Block (Tonbo Biosciences) was added at a volume 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 volume of 50 μL per cell. This cell suspension was mixed with 50 μL of diluted six anti-CXCR3 antibodies, anti-CCR7 antibodies (in-house manufactured), anti-CCR6 antibodies (in-house manufactured), or a control antibody, 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 diluted secondary antibodies (PE-labeled anti-mouse IgG antibody (Southern Biotech), APC-labeled anti-rat IgG antibody (Abcam), or APC-labeled anti-human IgG antibody (Invitrogen)) were added 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, and human CCR6 antibody (anti-chemokine antibodies) with control antibodies (isotype antibodies) are shown in Figure 5. In Figure 5, solid lines represent isotype antibodies, and open lines represent anti-chemokine antibodies. A rightward shift in the histogram was considered "bound." Binding of 201006-7-G and 201009-2-C was observed only with CHO cells stably expressing human CXCR3A. These results confirmed that 201006-7-G and 201009-2-C specifically bind to human CXCR3A but do not bind to other chemokine receptors. Similar results to Figure 5 were obtained for 13B4, 11F11, 201006-3-H, and 201006-5-F. The binding specificities of six types of 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] [Table 6]

[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 tests.

[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 in FACS buffer (PBS, containing 1% FBS) with goat serum (Thermo Fisher Scientific) at a concentration of 1 x 10 7 The cells were suspended at 4 × 10 cells / mL and blocked for 30 minutes at 4 °C. 6 Cells 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. 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 as the secondary antibody 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 the 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] [Table 7]

[0208] [Example 13] Evaluation of antibody activity to inhibit migration of human peripheral blood-derived T cells 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 tests.

[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 to a cell density of 2.0 to 2.4 × 10. 6The cells were incubated at 37°C for 60 minutes at 2.4 nM IP-10 (PEPROTECH) in the wells of a 96-well Transwell receiver plate with an integrated insert 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 to 0% inhibition for "no antibody, with IP-10" and 100% inhibition for "no antibody, without IP-10." Figure 7 shows the inhibitory effect of 201009-2-C on IP-10-dependent T cell migration. The horizontal axis represents antibody concentration, while the vertical axis represents % inhibition. The T cell migration inhibition rate of each antibody is summarized in Table 8.

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

[0211] [Table 8]

[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 tests.

[0213] 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 diluted 1:1 with FACS buffer (PBS containing 1% FBS) and diluted to 1 × 10 with goat serum (Thermo Fisher Scientific). 7The cells were suspended at 1 × 10 cells / mL and blocked for 30 minutes at 4°C. 5 Cells 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, the cells were washed twice with cold FACS buffer. To promote antibody internalization, a portion of the washed cells was incubated at 37°C for 30 minutes. The remaining cells were 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 flow cytometer Novocyte (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 incubated with antibody at 4°C was normalized to 100%, and the median fluorescence intensity of cells incubated 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 incubated 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] Preparation of humanized anti-CXCR3 antibody The case of 201009-2-C is taken as an example. To create a humanized antibody with grafted CDRs from 201009-2-C, human frameworks were selected based on the homology between 201009-2-C and human germline VH and VL genes. Based on computer modeling, several antibodies were designed by adding mutations to the selected human germline VH and VL sequences to support the predicted antibody conformation of 201009-2-C: "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 transfected into CHO cells, and purified antibodies were produced from the culture medium by affinity chromatography and subjected to the following tests. The purity of all purified antibodies was confirmed to be 90% or higher when tested by SDS-PAGE.

[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 inhibitory 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 nearly equivalent to those of 201009-2-C, confirming that conversion to the constant surface region of a human antibody does not affect binding activity or 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] [Table 9]

[0219] [Table 10]

[0220] [Table 11]

Claims

1. An antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A; It has the activity of blocking CXCR3A-dependent cell functions, It does not specifically bind to human vascular endothelial cells, The following (AB1) to (AB9), (AB12) to (AB27): (AB26) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 251, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 252, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 253, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 254, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 255, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

256. (AB23) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 221, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 222, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 223, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 224, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 225, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 226, (AB24) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 231, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 232, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 233, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 234, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 235, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

236. (AB25) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 241, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 242, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 243, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 244, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 245, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 246, (AB27) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 261, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 262, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 263, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 264, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 265, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

266. (AB1) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, (AB2) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16, (AB3) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26, (AB5) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 46, (AB8) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 72, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 73, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

76. (AB16) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 151, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 152, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 153, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 154, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 155, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

156. (AB17) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 161, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 162, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 163, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 164, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 165, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

166. (AB18) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 171, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 172, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 173, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 174, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 175, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

176. (AB20) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 191, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 192, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 193, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 194, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 195, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

196. (AB21) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 201, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 202, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 203, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 204, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 205, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 206, (AB22) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 211, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 212, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 213, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 214, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 215, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

216. (AB4) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

36. (AB6) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 52, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 53, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

56. (AB7) A antibody having a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 63, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 66, (AB9) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

86. (AB12) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 111, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 112, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 113, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 114, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 115, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

116. (AB13) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 121, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 122, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 123, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 124, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 125, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

126. (AB14) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 131, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 132, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 133, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 134, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 135, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

136. (AB15) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 141, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 142, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 143, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 144, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 145, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

146. (AB19) A heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 181, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 182, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 183, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 184, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 185, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

186. Antibodies that meet any of the above criteria.

2. An antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A; It has the activity of blocking CXCR3A-dependent cell functions, It does not specifically bind to human vascular endothelial cells, The following (C1) to (C9), (C12) to (C27): (C26) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 257, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 259; (C23) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 227, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 229; (C24) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 237, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 239; (C25) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 247, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 249; (C27) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 267, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 269; (C1) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 9; (C2) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

19. (C3) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

29. (C5) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 49; (C8) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 77, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 79; (C16) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 157, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

159. (C17) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 167, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 169; (C18) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 177, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 179; (C20) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 197, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 199; (C21) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 207, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 209; (C22) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 217, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 219, (C4) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 39, (C6) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

59. (C7) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 69; (C9) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

89. (C12) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 117, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

119. (C13) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 127, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

129. (C14) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:

139. (C15) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 147, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 149; (C19) A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 187, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 189; Antibodies that meet any of the above criteria.

3. An antibody that specifically binds to human CXCR3, specifically binds to the extracellular domain of human CXCR3A; It has the activity of blocking CXCR3A-dependent cell functions, It does not specifically bind to human vascular endothelial cells, A second antibody that competitively inhibits the binding of the first antibody, which is the antibody of claim 2, to a receptor.

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 3, which is a multispecific antibody.

6. The antibody according to any one of claims 1 to 3, which has internalization activity.

7. The antibody according to any one of claims 1 to 3, 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 3.

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

11. A pharmaceutical comprising the antibody according to any one of claims 1 to 3 as an active ingredient.

12. The pharmaceutical agent according to claim 11, which is used to treat a disease, disorder, or condition associated with impairment of 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.

15. A nucleic acid encoding the antibody of claim 4.

16. A cell comprising the nucleic acid of claim 15.

17. A pharmaceutical comprising the antibody according to claim 4 as an active ingredient.

Images