Antibody that inhibits chloride ion excretion and method for inhibiting cancer invasion using the same

Abstract

To provide other means for exerting the effect of chloride ion excretion inhibition, particularly means capable of inhibiting CLIC1 channel activity even when cells are severely deformed, specifically an antibody that inhibits chloride ion excretion, and to provide a method for inhibiting cancer invasion using the means, specifically a method for inhibiting cancer invasion using the antibody.SOLUTION: Provided is an anti-CLIC1 (chloride intracellular channel protein 1) antibody or an antigen-binding fragment thereof that specifically bind to an extracellular region of CLIC1 and inhibit chloride ion excretion from CLIC1-expressing cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an antibody that inhibits chloride ion excretion and a method for suppressing cancer invasion using the same. [Background technology]

[0002] The chloride ion channel protein CLIC1 (Chloride intracellular channel protein 1) is a protein involved in the cell volume regulation mechanism. It has been known that CLIC1 is associated with poor prognosis in various cancers and is involved in the invasion of cancer cells (Non-Patent Document 1). It has also been shown that knocking out CLIC1 reduces the invasiveness of cancer cells. It has been reported that (Non-Patent Document 2).

[0003] IAA-94 (indanyloxyacetic acid-94; Methylindazone) has been reported as a drug that inhibits CLIC1 activity. When added to gastric cancer cells, it suppresses the production of reactive oxygen species and improves migration and It has been reported that IAA-94 reduced the invasive ability of CLIC1 (Non-Patent Document 3). Although IAA-94 cannot be used in vivo due to its side effects, a monoclonal antibody that recognizes the CLIC1 extracellular domain has been reported, and it has been reported that it has a 90% inhibitory effect compared to IAA-94 (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2022 / 112486 [Patent Document 2] Patent Publication No. 2021-191238 [Non-patent literature]

[0005] [Non-Patent Document 1] Cellular Physiology and Biochemistry, 2018;46:907-924 [Non-Patent Document 2] Clinical and Translational Oncology, 2021;23:514-525 [Non-Patent Document 3] Molecular Medicine Reports, 2015;12:8041-8047 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, existing drugs that inhibit CLIC1 activity include IAA-94 and monoclonal antibodies. It is known that the inhibitory effect on cancer cell invasion has been verified by cell and animal experiments, and its pharmacological effect is being proven. The inventors have demonstrated that applying a mechanical external force to cancer cells inhibits the release of CLIC1 from We have developed a method to forcibly induce chloride ion excretion (Patent Document 2). We have also developed a method to directly detect chloride ions excreted outside the cell using an FET sensor at the same time as applying an external force. (Patent application 2023-093435) and demonstrated that this method can induce the discharge of chloride ions. Furthermore, they found that the inhibitory effect of IAA-94 on chloride ion efflux decreased depending on the magnitude of the applied external force. This indicates that CLIC1 inhibits chloride ion efflux by applying external force. It is thought that pores are opened by cell membrane stretching induced by IAA-94, suggesting that IAA-94 may dissociate from CLIC1 when cancer cells undergo significant deformation during cancer cell invasion. Therefore, IAA-94 or drugs with equivalent inhibitory effects can easily bind CLIC1 to the membrane surface of cancer cells. On the other hand, it is thought that the stable binding of CLIC1 causes the dissociation of the peptide and prevents it from exerting its full pharmacological effect. It is expected that cancer invasion can be inhibited more effectively if the channel activity can be inhibited. Therefore, the present invention relates to another means for exerting a chloride ion excretion inhibitory effect, in particular, a means for stably inhibiting CLIC1 channel activity even when a high external force is applied, and a method for inhibiting cancer invasion using the means. Specifically, the objective of the present invention is to provide an antibody that inhibits chloride ion excretion and a method for suppressing cancer invasion using the antibody. [Means for solving the problem]

[0007] As a result of extensive investigations aimed at solving the above problems, the present inventors have discovered that the extracellular domain of CLIC1 specifically binds to the CLIC1 protein. The antibody binds to CLIC1-expressing cells under conditions where IAA-94 cannot exert its inhibitory effect. The inventors have found that the antibody inhibits chloride ion excretion from cells and that it is possible to effectively suppress cancer invasion by inhibiting volume regulation during cancer invasion using the antibody.

[0008] That is, the present invention is as follows. [1] Binds specifically to the extracellular domain of CLIC1 (chloride intracellular channel protein 1) and an anti-CLIC1 antibody or an antigen-binding fragment thereof that inhibits chloride ion efflux from CLIC1-expressing cells, and that exhibits a higher chloride ion efflux inhibitory effect than IAA-94. Antigen-binding fragment. [2] The extracellular domain of CLIC1 is represented by amino acid numbers 1 to 24 in SEQ ID NO: 136. The anti-CLIC1 antibody or antigen-binding fragment thereof according to [1], Piece. [3] The anti-CLIC1 antibody or antigen-binding fragment thereof according to [1] or [2], having a heavy chain variable region and a light chain variable region represented by any one of the following: (1) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 11; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:13. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:14; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 16; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 17; (2) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 17; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:19. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:20; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 23; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 22; (3) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 23; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:25. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:26; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:28; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:29; (4) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 29; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:31. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:32; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 34; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 34; (5) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 35; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:37. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:38; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 40; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 40; (6) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 41; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:43. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:44; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:46; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:47; (7) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 47; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:49. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:50; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:52; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:53; (8) heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 53; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:55. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:56; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:58; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:59; (9) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 59; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:61. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:62; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 64; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 64; (10) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 65; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:67. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:68; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 70; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 70; (11) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 71; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:73. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 74; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 76; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 77; (12) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 77; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:79. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 80; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 82; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 83; (13) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 83; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 85. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 86; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 88; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 88; (14) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 89; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:91. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 92; and a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:94. a light chain variable region comprising: (15) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 95; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:97. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 98; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 100; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 100; (16) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 101; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 103. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 104; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 105; (17) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 106; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 108. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 109; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 110; (18) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 111; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:113. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 114; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 115; (19) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 116; and a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO:118. a heavy chain variable region comprising a heavy chain CDR3 having the formula: A light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 119; a light chain CDR2 having the amino acid sequence represented by TAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 120; or (20) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 121; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 123. a heavy chain variable region comprising a heavy chain CDR3 having the formula: A light chain variable region comprising: a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 124; a light chain CDR2 having the amino acid sequence represented by SAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 125. [4] A heavy chain variable region and a light chain variable region represented by any of the following: The anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of [1] to [3], which has an activity: (1) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:1, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:1; and a light chain variable region having the amino acid sequence represented by SEQ ID NO:2, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:2; (2) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:3, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:3; and a light chain variable region having the amino acid sequence represented by SEQ ID NO:4, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:4; (3) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:5, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:5; and A light chain variable region having the amino acid sequence represented by SEQ ID NO:6, or a light chain variable region having an amino acid sequence that is 90% or more identical to an amino acid sequence of (4) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 7, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 7; and A light chain variable region having the amino acid sequence represented by SEQ ID NO:8, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:8; or (5) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:9, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:9; and A light chain variable region having the amino acid sequence represented by SEQ ID NO:10, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:10. [5] The anti-CLIC1 antibody or its antigen according to any one of [1] to [4], wherein the antibody is derived from a mouse antibody, a rat antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody. Combined fragment. [6] The anti-CLIC1 antibody or antigen-binding fragment thereof is a complete antibody, F(ab')2, Fab' The anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of [1] to [5], which is a Fab, Fv, scFv, nanobody, VHH, minibody, diabody, or triabody. [7] The anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of [1] to [6], and a pharmaceutical A pharmaceutical composition comprising a carrier acceptable for [8] The anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of [1] to [6], or 7] A chloride ion efflux inhibitor comprising the pharmaceutical composition described in . [9] The anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of [1] to [6], or 7] An agent for suppressing cancer invasion and / or metastasis, comprising the pharmaceutical composition described in 7).

[10] The anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of [1] to [6], or A cancer therapeutic agent comprising the pharmaceutical composition described in [7].

[11] The cancer therapeutic drug described in

[10] , wherein the cancer is a brain tumor, breast cancer, stomach cancer, lung cancer, pancreatic cancer, liver cancer, colon cancer, prostate cancer, or cervical cancer.

[12] A vector encoding the anti-CLIC1 antibody or an antigen-binding fragment thereof according to any one of [1] to [6]. Isolated DNA. Effect of the Invention

[0009] INDUSTRIAL APPLICABILITY The present invention makes it possible to provide an antibody that inhibits chloride ion excretion and a method for suppressing cancer invasion using the same. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows the chloride ion efflux inhibitory effect using a CLIC1 extracellular domain-recognizing polyclonal antibody. (A) A shows a schematic diagram of the recognition site of the anti-CLIC1 antibody. (B) Relative fluorescence intensity upon application of external force in MQAE-introduced cells. [Diagram 2] FIG. 13 shows the intracellular MQAE relative fluorescence intensity when an external force was applied to MDA-MB-231 cells to which a monoclonal antibody recognizing the CLIC1 extracellular domain had been added. [Diagram 3] Figure 1 shows the relative fluorescence intensity of MQAE in MDA-MB-231 cells 5 minutes after application of external force. To evaluate the statistical significance between the results of cells without antibody and the results of cells with each antibody added, t-tests were performed and p-values ​​are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] <Anti-CLIC1 antibody or antigen-binding fragment thereof> One embodiment of the present invention is a method for detecting extracellular expression of CLIC1 (chloride intracellular channel protein 1). The antibody or antigen-binding fragment thereof of the present invention specifically binds to the CLIC1 region and inhibits chloride ion efflux from CLIC1-expressing cells. The antibody or antigen-binding fragment thereof exhibits a higher chloride ion efflux inhibitory effect than IAA-94.

[0012] The inhibitory effect on chloride ion excretion can be evaluated using any method known to those skilled in the art. For example, the method described in Patent Document 2 or Patent Application No. 2023-093435 can be used to apply a mechanical force to cells. By applying a voltage to the nucleus, we forcibly induced chloride ion efflux from CLIC1, resulting in the efflux of It can be evaluated based on the amount of chloride ions released.

[0013] For example, an external mechanical force can be applied to CLIC1-expressing cells by the following method. The external force load is applied to the contact area of ​​the cell, which is equal to or 1 / 10 of the cell's surface area (e.g., a contact area of ​​30 to 300 μm 2 ). Such a member may be, for example, a member having a cell presser that contacts the cell at the tip and applies pressure, or may be a member having a flat tip. The shape of the cell presser is not limited as long as it has the above-mentioned contact surface, and may be pyramidal (conical or pyramidal with an apex angle that does not destroy the cell membrane even when a desired pressure is applied to the cell membrane), cylindrical, prism-shaped, truncated cone-shaped, truncated pyramidal, spherical (including approximately spherical), or hemispherical (including approximately hemispherical). The material of the cell presser is not particularly limited as long as it has the above-mentioned contact surface, and examples of the material include organic polymers and metals. The size of the cell presser is not limited as long as it has the above-mentioned contact surface, and for example, in the case of pyramidal, cylindrical, prism-shaped, truncated cone-shaped, or truncated pyramidal, the diameter at the base is 5 to 20 μm. In the case of a spherical or semispherical shape, the particle size may be 5 to 20 μm. One example is a cantilever used in a scanning probe microscope (SPM) or atomic force microscope (AFM), or a tipless cantilever with a cell pressure sensor attached to the tip thereof. The material of the cell presser is not particularly limited, but examples include silicon (such as single crystal silicon), silicon nitride, gold, chromium, indium arsenide, silica, polyethylene terephthalate, polyethylene, polypropylene, polystyrene, polycarbonate, and acrylic resin. The strength of the applied external force should be such that the excretion of chloride ions through the cell membrane is observed, and is preferably a strength that can stretch the cell membrane laterally, for example, 1 to 100 nN, preferably 3 to 50 nN, and more preferably 10 to 30 nN. The duration of application of the external force may be any duration sufficient for observing the discharge of chloride ions due to the application of the external force, and may be, for example, 1 second to 15 minutes, or 10 seconds to 5 minutes. Thus, applying mechanical force to cells inhibits chloride ion expulsion from CLIC1. It is possible to forcibly induce the exit of the

[0014] The amount of excreted chloride ions can be measured by any method. For example, a cell membrane-permeable fluorescent dye (e.g., N-Ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE)) whose fluorescence intensity decreases when bound to chloride ions can be used to measure the amount of excreted chloride ions caused by fluorescent dyes such as MQAE. The amount of chloride ions discharged from the cell can be measured by directly detecting the chloride ions discharged from the cell using a FET sensor at the same time as the application of an external force (Patent Document 2). This is also possible (Patent application no. 2023-093435).

[0015] The antibody or antigen-binding fragment thereof of the present invention has a stronger inhibitory effect on chloride ion efflux than IAA-94 (e.g., 100 μM). In the present invention, when chloride ion efflux from CLIC1 is forcibly induced by applying an external mechanical force, the cells to which IAA-94 was added exhibited a higher inhibitory effect on chloride ion efflux. It can be determined that chloride ion excretion is inhibited by the fact that the amount of chloride ion excreted in the cells to which the antibody of the present invention or an antigen-binding fragment thereof has been added is smaller than that in the cells to which the antibody of the present invention or an antigen-binding fragment thereof has been added. In this case, the amount of IAA-94 added is not particularly limited as long as it is a commonly used concentration, and may be, for example, 50 μM to 100 μM.

[0016] Although there are existing monoclonal antibodies that target CLIC1, most of these antibodies is intended for use in cell staining and other applications, and recognizes the intracellular domain of CLIC1. However, when trying to control the function of cancer cells from outside the cells, it is difficult to use antibodies that target such intracellular regions. An antibody that binds to the extracellular domain of CLIC1 has been disclosed, but its inhibitory effect on chloride ion efflux was less than that of IAA-94 and was insufficient. The antibody of the present invention targets the extracellular domain of CLIC1 and is more effective than IAA-94. This has a strong inhibitory effect on chloride ion excretion. , which can exert a strong chloride ion efflux inhibitory effect, can efficiently inhibit chloride ion efflux from CLIC1 in proliferating and invading cancer cells.

[0017] An example of the extracellular domain of CLIC1 is the amino acid sequence represented by amino acid numbers 1 to 24 in SEQ ID NO: 136, which is the amino acid sequence of human CLIC1. The antibody or antigen-binding fragment thereof of the present invention may recognize all of these amino acid sequences represented by amino acid numbers 1 to 24, or may recognize only a part of these amino acid sequences. Furthermore, it may also recognize sequences other than the amino acid sequences represented by amino acid numbers 1 to 24 in addition to these. The amino acid number refers to the number of amino acid residues counted from the amino terminal amino acid residue of the amino acid sequence.

[0018] The antibody or antigen-binding fragment thereof of the present invention may have, for example, a heavy chain variable region and a light chain variable region represented by any of the following: (1) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 11; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:13. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:14; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 16; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 17; (2) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 17; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:19. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:20; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 23; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 22; (3) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 23; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:25. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:26; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:28; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:29; (4) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 29; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:31. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:32; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 34; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 34; (5) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 35; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:37. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:38; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 40; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 40; (6) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 41; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:43. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:44; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:46; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:47; (7) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 47; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:49. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:50; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:52; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:53; (8) heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 53; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:55. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:56; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:58; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:59; (9) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 59; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:61. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:62; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 64; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 64; (10) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 65; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:67. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:68; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 70; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 70; (11) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 71; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:73. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 74; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 76; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 77; (12) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 77; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:79. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 80; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 82; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 83; (13) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 83; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 85. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 86; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 88; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 88; (14) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 89; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:91. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 92; and a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:94. a light chain variable region comprising: (15) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 95; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:97. a heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 98; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 100; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 100; (16) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 101; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 103. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 104; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 105; (17) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 106; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 108. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 109; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 110; (18) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 111; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:113. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 114; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 115; (19) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 116; and a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO:118. a heavy chain variable region comprising a heavy chain CDR3 having the formula: A light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 119; a light chain CDR2 having the amino acid sequence represented by TAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 120; or (20) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 121; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO: 123. a heavy chain variable region comprising a heavy chain CDR3 having the formula: A light chain variable region comprising: a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 124; a light chain CDR2 having the amino acid sequence represented by SAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 125. The CDR sequences in (1) to (5) above are identified by Kabat numbering. Each CDR sequence in (6) to (10) above is identified by Chothia numbering. The CDR sequences in (11) to (15) above are numbered according to Contact numbering. Each CDR sequence in (16) to (20) above is specified by the IMGT numbering.

[0019] The positions of the CDRs in the heavy chain variable region and the light chain variable region are numbered according to the known numbering system. The numbering method can be used to determine the number of the chromosome. For example, the numbering method may be the Kabat numbering, Chothia numbering, Contact numbering, or IMGT numbering. It is possible.

[0020] The antibody or antigen-binding fragment thereof of the present invention may have, for example, any of the heavy chain variable regions and light chain variable regions shown below, and have binding activity to CLIC1: (1) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:1, or a heavy chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO:1; and a light chain variable region having the amino acid sequence represented by SEQ ID NO:2, or a light chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO:2; (2) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 3, or a heavy chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region having the amino acid sequence represented by SEQ ID NO:4, or a light chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO:4; (3) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:5, or a heavy chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO:5; and a light chain variable region having the amino acid sequence represented by SEQ ID NO:6, or a light chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO:6; (4) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 7, or a heavy chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO: 7; and A light chain variable region having an amino acid sequence represented by SEQ ID NO:8, or a light chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO:8; or (5) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 9, or a heavy chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO: 9; and A light chain variable region having an amino acid sequence represented by SEQ ID NO:10, or a light chain variable region having an amino acid sequence that is 90% or more identical, preferably 95% or more identical, and more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO:10.

[0021] The term "90% or more, 95% or more, or 98% or more identical to the amino acid sequences represented by SEQ ID NOs: 1 to 10" is not particularly limited as long as it has binding activity to CLIC1. Any amino acid may be substituted, deleted or added in the CDR region of the main or light chain variable region. Any amino acid may be substituted, deleted, or added outside the CDR region. However, from the viewpoint of binding activity, if there are amino acid substitutions, deletions, or additions, the CDR It is preferable that the amino acid substitution is located outside the CLIC1 binding region. There is no particular limitation as long as the substitution has the desired functionality, but conservative substitutions are preferred.

[0022] The origin of the antibody of the present invention is not particularly limited, and may be, for example, a mouse antibody, a rat antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0023] The antibody of the present invention specifically binds to the extracellular domain of CLIC1, which includes the heavy chain variable region and the light chain variable region. The antibody may be a binding antibody and may include a heavy chain variable region and a heavy chain constant region, and a light chain variable region and a light chain constant region. For example, when the antibody is a complete antibody, it may be IgG (IgG1, IgG2, IgG3 or IgG4), IgA, IgE, IgM or IgD. The antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody from the standpoint of stabilizing the quality and binding ability.

[0024] The antigen-binding fragment of the antibody of the present invention is not particularly limited as long as it has binding activity to CLIC1. , for example, F(ab')2, Fab', Fab, Fv, scFv, nanobody, VHH, minibody, diabody, or triabody.

[0025] The antibody of the present invention can be obtained using techniques commonly used in the art, such as chemical synthesis and genetic recombination, and can be obtained, for example, by administering an antigen to an animal that produces an antibody against a specific antigen, such as a mouse, rat, or rabbit, and immunizing the animal.

[0026] The method for producing a monoclonal antibody is not particularly limited, and can be performed by a method known to those skilled in the art. For example, a monoclonal antibody can also be produced by (i) recovering antibody-producing cells from the blood of the animal after the immunization, (ii) screening the antibody-producing cells that produce the desired antibody, (iii) amplifying the target DNA from the antibody gene extracted from the antibody-producing cells by RT-PCR, (iv) inserting the amplified DNA into an expression vector, transforming a host cell with the vector, and culturing the host cell.

[0027] As another method for producing monoclonal antibodies, for example, hybridoma cells can be produced by combining antibody-producing cells collected from the blood of an animal after immunization with myeloma cells, and the hybridoma cells which produce the desired antibody can be screened and cultured to obtain monoclonal antibodies.

[0028] Alternatively, monoclonal antibodies can be obtained based on sequence information using techniques commonly used in the art, such as chemical synthesis and genetic recombination.

[0029] Chimeric antibodies, humanized antibodies, human antibodies, and the like can be obtained using techniques commonly used in the art, such as chemical synthesis and genetic recombination.

[0030] Polyclonal antibodies can be obtained by, for example, collecting serum from the animal after the immunization.

[0031] The antibody of the present invention preferably has the above-mentioned specific combination of heavy chain CDR1 to 3 and light chain CDR1 to 3, but may have any combination thereof as long as it has the ability to bind to CLIC1. That's fine.

[0032] <Pharmaceutical Composition> Another embodiment of the invention is an anti-CLIC1 antibody or antigen-binding fragment thereof, and a pharma- ceutically acceptable The pharmaceutical composition comprises a carrier.

[0033] The pharmaceutical composition of this embodiment contains only one type of the anti-CLIC1 antibody or antigen-binding fragment thereof. It may contain two or more types.

[0034] The pharmaceutical composition of the present embodiment is not particularly limited as long as it can exert the desired pharmaceutical effect, and may optionally contain other ingredients.

[0035] The pharma- ceutically acceptable carrier refers to an ingredient other than the active ingredient contained in the pharmaceutical composition. There is no particular limitation as long as it is one that is usually used in medicines. For example, a buffer solution such as PBS; Examples of the additives include diluents such as saline solution; excipients such as lactose and magnesium stearate; antioxidants such as ascorbic acid; and preservatives.

[0036] The pharmaceutical composition of the present embodiment can be administered in various forms, including parenteral administration using injections, drips, suppositories, etc., and oral administration using tablets, capsules, granules, powders, syrups, etc. Parenteral administration can be intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0037] The dosage can be adjusted depending on symptoms, age, body weight, dosage form, antibody binding titer, and purpose. For example, in parenteral administration, about 0.01 mg to 1000 mg per day is administered by injection to an adult. For oral administration, the daily dose for adults is about 0.01 mg to 1000 mg. If necessary, these may be administered in one or multiple doses. It is possible.

[0038] <Chloride ion excretion inhibitor> Another embodiment of the present invention is the anti-CLIC1 antibody or antigen-binding fragment thereof, or the pharmaceutical The composition is a chloride ion efflux inhibitor.

[0039] The chloride ion efflux inhibitor of this embodiment is an anti-CLIC1 antibody or an antigen-binding fragment thereof that can inhibit chloride ion efflux by CLIC1 by binding to the extracellular domain of CLIC1 present on the surface of cells expressing CLIC1.

[0040] The chloride ion efflux inhibitor of this embodiment comprises the anti-CLIC1 antibody or an antigen-binding fragment thereof. It may contain only one type, or two or more types.

[0041] The chloride ion efflux inhibitor of this embodiment may contain other components as desired, so long as the other components do not impede the chloride ion efflux inhibitory effect.

[0042] Chloride ions can be measured by any method known to those skilled in the art. For example, they can be measured by the method described in Patent Document 1, that is, using a cell membrane permeable fluorescent dye (e.g., N-Ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE)) whose fluorescence intensity decreases when bound to chloride ions, the fluorescence caused by the fluorescent dye such as MQAE is used as an indicator. For example, when cells to which the chloride ion efflux inhibitor of this embodiment has not been added are used as a control, and the cells are stimulated, the amount of chloride ion excretion in the cells to which the chloride ion efflux inhibitor of this embodiment has been added is reduced compared to the control, whereby it can be determined that chloride ion excretion has been inhibited.

[0043] The chloride ion efflux inhibitor of this embodiment can be used for various purposes as needed, and can be used, for example, as a reagent or medicine for inhibiting chloride ion efflux from cells.

[0044] When the chloride ion efflux inhibitor of this embodiment is used as a reagent, the manufacture, use, etc. of the reagent can be performed based on a conventional method in the field of molecular biology. When the chloride ion efflux inhibitor of this embodiment is used as a medicine, the description in the above section <Pharmaceutical composition> can be used for the ingredients, dosage form, dosage, etc. The description in the above section <Anti-CLIC1 antibody or antigen-binding fragment thereof> can be used for the antibody of this embodiment.

[0045] <Cancer invasion and / or metastasis inhibitors> Another embodiment of the present invention is the anti-CLIC1 antibody or antigen-binding fragment thereof, or the pharmaceutical The composition is an agent for suppressing cancer invasion and / or metastasis.

[0046] The cancer invasion and / or metastasis inhibitor of this embodiment is an anti-CLIC1 antibody or an antigen-binding fragment thereof. inhibits cancer invasion and / or metastasis by binding to the extracellular domain of CLIC1 present on the surface of CLIC1-expressing cancer cells and inhibiting chloride ion export by CLIC1 Cancer cells lose cell volume due to the inhibition of chloride ion excretion. As a result, cancer cells cannot pass through the narrow gaps between surrounding cells and cannot spread to surrounding tissue.

[0047] The cancer invasion and / or metastasis inhibitor of this embodiment comprises the anti-CLIC1 antibody or an antigen-binding domain thereof. It may contain only one type of fragment, or two or more types of fragments.

[0048] The cancer invasion and / or metastasis inhibitor of this embodiment may contain other ingredients as long as they do not impede its effect of inhibiting cancer invasion and / or metastasis.

[0049] Cancer invasiveness can be measured by any method known to one of skill in the art, such as a cell invasion assay using a membrane with pores of a desired size.

[0050] The cancer invasion and / or metastasis inhibitor of this embodiment can be used for various purposes as needed, for example, as a reagent or medicine for inhibiting chloride ion excretion from cells.

[0051] When the cancer invasion and / or metastasis inhibitor of this embodiment is used as a reagent, the manufacture, use, etc. of the reagent can be performed based on standard methods in the field of molecular biology. When the cancer invasion and / or metastasis inhibitor of this embodiment is used as a medicine, the description in the above section <Pharmaceutical composition> can be used for the ingredients, dosage form, dosage, etc. The description regarding the antibody of this embodiment can be found in the above section <Anti-CLIC1 antibody or antigen-binding fragment thereof>. The reference can be used as a reference.

[0052] <Cancer drugs> Another embodiment of the present invention is the anti-CLIC1 antibody or antigen-binding fragment thereof, or the pharmaceutical The composition is a cancer treatment agent.

[0053] The cancer therapeutic drug of this embodiment may contain additional active ingredients as long as the additional active ingredients do not impede the effect of cancer treatment. For example, the cancer treatment drug is a compound comprising the anti-CLIC1 antibody or its antigen-binding fragment and a drug such as an anticancer drug. The anti-CLIC1 antibody or the antigen-binding fragment thereof may be an antibody-drug conjugate (ADC) having an attached substance. This allows the anti-CLIC1 antibody or the antigen-binding fragment thereof to bind to cancer cells and inhibit their invasion. While suppressing this, the attached anticancer drug or other drug can be delivered directly to the cancer cells, thereby exerting an antitumor effect.

[0054] The cancer therapeutic drug of the present embodiment inhibits the infiltration of cancer cells from the primary cancer focus and from sites where cancer has already proliferated, thereby inhibiting cancer metastasis, etc. Therefore, it can be suitably used for cancers with high invasiveness in particular. The specific type of cancer is not particularly limited, and examples include brain tumors, breast cancer, stomach cancer, lung cancer, pancreatic cancer, liver cancer, colon cancer, prostate cancer, and cervical cancer.

[0055] In this embodiment, cancer treatment is not particularly limited, but includes, for example, inhibition of cancer cell infiltration, inhibition of cancer cell metastasis, inhibition of cancer cell proliferation, and the like.

[0056] The subject of treatment in the present invention may be any animal that develops cancer, and is preferably a mammal such as a human, mouse, or rat, and more preferably a human.

[0057] The components, dosage form, dosage amount, etc. of this embodiment can be referenced from the description in the above <Pharmaceutical Composition> section. The descriptions in the section entitled "CLIC1 antibody or antigen-binding fragment thereof" can be cited.

[0058] <Isolated DNA> Another embodiment of the present invention relates to an isolated nucleic acid encoding the anti-CLIC1 antibody or antigen-binding fragment thereof. It is DNA that has been

[0059] The anti-CLIC1 antibody or antigen-binding fragment thereof can be produced by any method based on DNA encoding the antibody. For example, the antibody-encoding DNA can be inserted into an expression vector, A monoclonal antibody can be produced by transforming a host cell with the vector and culturing the host cell. The DNA may be inserted into the same expression vector and the vector used to transform a host cell. Alternatively, the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into separate vectors, and the two vectors may be used to transform host cells. EXAMPLES

[0060] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0061] <Example 1> Preparation of antibodies recognizing the extracellular domain of CLIC1 The production of the antibody was outsourced to Cosmobio. A peptide of 24 residues from the N-terminus of the extracellular domain of CLIC1 (sequence number 136) (Figure 1A) was used as an antigen on days 0, 14, 28, and 42 in rabbits (strain name: On the 49th day, whole blood was collected and purified to generate rabbit anti-CLIC1 polyclonal antibody. A local antibody was obtained (Antibody A). Furthermore, lymphocytes were prepared from 10 mL of blood and fluorescently labeled for the production of monoclonal antibodies. Cells with high reactivity to the antigen peptide were cultured as single cells in a 96-well plate by FACS. The antibody genes were amplified from the single cells obtained by RT-PCR, and the obtained antibody genes were used to express Fab antibody proteins by cell-free transcription-translation reaction (PUREfrex 2.0 (Gene Frontier), DsbC Set (Gene Frontier)). The antigen peptide was immobilized. Using the plate, an ELISA test was performed to confirm the binding ability with the antigen peptide. Eleven clones with high peptide binding ability were selected. The DNA sequences of the heavy and light chain variable regions of each clone were then inserted into a cloning vector to prepare plasmids, and the plasmids were then synthesized. The antibody gene sequence was determined by sequence analysis. The DNA sequence of the region was chemically synthesized and expressed in HEK293 cells (Thermo Fisher). After quality confirmation by SDS-PAGE and HPLC, the IgG antibody was prepared as an anti-CLIC1 monoclonal antibody.

[0062] Example 2 Chloride ion excretion from cancer cells using monoclonal antibodies that recognize the CLIC1 extracellular domain Outer Inhibition> The chloride ion excretion inhibitory effect of the above 11 anti-CLIC1 monoclonal antibodies was tested. As a negative control, a commercially available monoclonal antibody that recognizes the intracellular domain of CLIC1 was used. A local antibody was used (Antibody B: Santa Cruz Biotechnology, SC-81873). The five clones that were detected and one clone for which no inhibitory effect was observed are summarized and listed in Table 1.

[0063] [Table 1] Human breast cancer cell line MDA-MB-231 (ATCC, HTB-26) was seeded on a glass base dish and cultured overnight. MQAE (Dojindo, The cells were incubated for 1 hour in a medium containing 5 mM erythrocyte sedimentation buffer (final concentration 5 mM) and an antibody recognizing the extracellular domain of CLIC1 (final concentration 30 μg / mL). After washing with PBS, the cells in PBS were observed using an atomic force microscope (AFM) and Mechanical stimulation was applied for 10 min at a set point of 3 nN using a flat-tip cantilever. Fluorescence images of intracellular MQAE were obtained using a fluorescence microscope, and the fluorescence intensity was calculated using ImageJ. When an external force of 3 nN was applied, the cells treated with the anti-CLIC1 polyclonal antibody (Antibody A) that recognizes the extracellular domain or the chloride ion channel inhibitor IAA-94 (Sigma-Aldrich) showed no significant change. The relative fluorescence intensity after 5 min of application was significantly lower than that of the cells in The CLIC1 intracellular domain used as a control was No inhibitory effect was observed in cells treated with the region-recognizing monoclonal antibody (Antibody B). . In addition, when an external force of 15 nN was applied, the ion concentration in the IAA-94-added cells was significantly higher than that in the IAA-94-added cells when an external force of 3 nN was applied. The inhibition of efflux was attenuated, suggesting that IAA-94 may dissociate from the binding site due to a conformational change in the channel caused by cell membrane expansion. In cells treated with the null antibody, chloride ion excretion was observed at 15 nN as well as at 3 nN. was hindered. These results suggest that the CLIC1 extracellular domain-recognizing polyclonal antibody inhibits the expression of the CLIC1 extracellular domain when the cell membrane is greatly expanded. It was also shown that the excretion of chloride ions by the application of external force was inhibited in the case of

[0064] Next, 11 monoclonal antibody clones that recognize the CLIC1 extracellular domain were tested. We applied external force to cells to which monoclonal antibodies had been added, and evaluated the change in MQAE relative fluorescence intensity over time. As a result, clones were confirmed that showed an inhibitory effect equal to or greater than that of the prepared polyclonal antibodies (Figure 2). In comparison with the level of the 5 clones, chloride ion excretion was significantly inhibited. The heavy chain variable region (SEQ ID NO: 1) of each clone in which the ion excretion inhibitory effect was confirmed was 26 (AT-10), SEQ ID NO: 128 (AT-15), SEQ ID NO: 130 (AT-17), SEQ ID NO: 132 (AT-22), SEQ ID NO: 134 (AT-44), and the light chain variable region (SEQ ID NO: 127 (AT-10), SEQ ID NO: 129 (AT-15), SEQ ID NO: 131 (AT-17), SEQ ID NO: 133 (AT-22), The DNA sequence of No. 135 (AT-44) is shown in the sequence listing. CLIC1 is thought to function as a channel by forming tetramers or higher oligomers on the cell membrane. However, some clones did not inhibit chloride ion excretion. This result suggests that there are clones that have inhibitory activity depending on the binding mode of the monoclonal antibodies that recognize the extracellular domain. These results suggest that there exist clones that do not possess the CLIC1 inhibitory activity. The clones that possess the inhibitory activity may sterically block the channel pore and prevent ions from passing through, or the antibodies may crosslink CLIC1 monomers. It is presumed that this mechanically blocks pore opening and inhibits ion excretion.

[0065] Among the prepared anti-CLIC1 monoclonal antibodies, the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the anti-CLIC1 monoclonal antibodies with high chloride ion efflux inhibitory activity are shown below. In Tables 2 to 5, the CDR sequences are underlined. Each sequence is represented by Kabat numbering. (Table 2), Chothia numbering (Table 3), Contact numbering (Table 4), IMGT numbering The CDR positions were determined by baling (Table 5). In each table, the underlines indicate CDR1, CDR2, and so on. , CDR3 is indicated.

[0066] [Table 2]

[0067] [Table 3]

[0068] [Table 4]

[0069] [Table 5]

[0070] Among the anti-CLIC1 monoclonal antibodies produced, the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 of five clones with high chloride ion efflux inhibitory activity are shown in the following tables. The numbering systems are Kabat numbering (Table 6), Chothia numbering (Table 7), Contact numbering (Table 8), and IMGT numbering (Table 9), respectively.

[0071] [Table 6]

[0072] [Table 7]

[0073] [Table 8]

[0074] [Table 9]

Claims

1. An anti-CLIC1 antibody or its antigen-binding fragment that specifically binds to the extracellular domain of CLIC1 (chloride intracellular channel protein 1) and inhibits chloride ion excretion from CLIC1-expressing cells. There was, Anti-CLIC1 antibody or its antigen-binding fragment exhibiting a higher chloride ion excretion inhibitory effect than IAA-94 Piece.

2. The extracellular domain of CLIC1 is represented by amino acid numbers 1 to 24 in SEQ ID NO:

136. The anti-CLIC1 antibody or antigen-binding fragment thereof according to claim 1, which has the amino acid sequence 。

3. The anti-CLIC1 antibody or antigen-binding fragment thereof of claim 1, having a heavy chain variable region and a light chain variable region represented by any one of the following: (1) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 11; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

13. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:14; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 16; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 17; (2) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 17; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

19. A heavy chain variable region comprising a heavy chain CDR3; and A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:20; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:22; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:23; (3) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 23; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

25. A heavy chain variable region comprising a heavy chain CDR3; and A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:26; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:28; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:29; (4) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 29; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

31. A heavy chain variable region comprising a heavy chain CDR3; and A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:32; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 34; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 35; (5) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 35; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

37. A heavy chain variable region comprising a heavy chain CDR3; and A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:38; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 40; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 40; (6) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 41; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

43. A heavy chain variable region comprising a heavy chain CDR3; and A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:44; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:46; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:47; (7) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 47; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

49. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:50; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:52; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:53; (8) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO:53; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

55. A heavy chain variable region comprising a heavy chain CDR3; and Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:56; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:58; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:59; (9) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO:59; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

61. A heavy chain variable region comprising a heavy chain CDR3; and A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:62; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:64; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:64; (10) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 65; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

67. a heavy chain variable region comprising a heavy chain CDR3 comprising: Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:68; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 70; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 70; (11) A heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 71; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

73. a heavy chain variable region comprising a heavy chain CDR3 comprising: A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:74; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:76; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:77; (12) A heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 77; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

79. a heavy chain variable region comprising a heavy chain CDR3 comprising: Light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 80; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 82; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 83; (13) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 83; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

85. a heavy chain variable region comprising a heavy chain CDR3 comprising: A light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 86; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 87; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 88; (14) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 89; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

91. a heavy chain variable region comprising a heavy chain CDR3 comprising: A light chain CDR1 having the amino acid sequence represented by SEQ ID NO:92; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:94; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:95; (15) Heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 95; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

97. a heavy chain variable region comprising a heavy chain CDR3 comprising: Light chain CDR1 having the amino acid sequence represented by SEQ ID NO:98; a light chain variable region comprising a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 100; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 100; (16) A heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 101; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

103. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 104; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 105; (17) A heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 106; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

108. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 109; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 110; (18) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 111; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

113. a heavy chain variable region comprising a heavy chain CDR3 having the formula: a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 114; a light chain CDR2 having the amino acid sequence represented by DAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 115; (19) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 116; and a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO:

118. a heavy chain variable region comprising a heavy chain CDR3 having the formula: A light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 119; a light chain CDR2 having the amino acid sequence represented by TAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 120; or (20) a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 121; and a heavy chain CDR2 having an amino acid sequence represented by SEQ ID NO:

123. a heavy chain variable region comprising a heavy chain CDR3 having the formula: A light chain variable region comprising: a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 124; a light chain CDR2 having the amino acid sequence represented by SAS; and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:

125.

4. The heavy chain variable region and the light chain variable region have any of the following binding activity to CLIC1: The anti-CLIC1 antibody or antigen-binding fragment thereof of claim 1, comprising: (1) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:1, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:1; and A light chain variable region having the amino acid sequence represented by SEQ ID NO: 2, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 2; (2) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 3, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 3; and A light chain variable region having the amino acid sequence represented by SEQ ID NO: 4, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 4; (3) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO:5, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:5; and A light chain variable region having the amino acid sequence represented by SEQ ID NO:6, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:6; (4) A heavy chain variable region having the amino acid sequence represented by SEQ ID NO: 7, or a heavy chain variable region having the amino acid sequence represented by SEQ ID NO:

7. A heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in A light chain variable region having the amino acid sequence represented by SEQ ID NO:8, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:8; or (5) a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 9, or a heavy chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 9; and A light chain variable region having the amino acid sequence represented by SEQ ID NO:10, or a light chain variable region having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO:

10.

5. The anti-CLIC1 antibody or its antigen-binding fragment according to claim 1 , wherein the antibody is derived from a mouse antibody, a rat antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody.

6. The anti-CLIC1 antibody or antigen-binding fragment thereof may be a complete antibody, an F(ab') ２ , Fab', F The anti-CLIC1 antibody or antigen-binding fragment thereof of claim 1, which is an ab, Fv, scFv, nanobody, VHH, minibody, diabody, or triabody.

7. The anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, A pharmaceutical composition comprising an acceptable carrier.

8. An anti-CLIC1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, or A pharmaceutical composition comprising the anti-CLIC1 antibody or an antigen-binding fragment thereof and a pharma- ceutical acceptable carrier. A chloride ion efflux inhibitor comprising:

9. An anti-CLIC1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, or A pharmaceutical composition comprising the anti-CLIC1 antibody or an antigen-binding fragment thereof and a pharma- ceutical acceptable carrier.

2. An agent for suppressing cancer invasion and / or metastasis, comprising the composition.

10. An anti-CLIC1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, or A pharmaceutical composition comprising the anti-CLIC1 antibody or an antigen-binding fragment thereof and a pharma- ceutical acceptable carrier. A cancer treatment drug, comprising:

11. The cancer therapeutic agent according to claim 10, wherein the cancer is brain cancer, breast cancer, stomach cancer, lung cancer, pancreatic cancer, liver cancer, colon cancer, prostate cancer, or cervical cancer.

12. A gene encoding the anti-CLIC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. Isolated DNA.

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