Antibodies against substant protein complex subset

Abstract

An antibody or an antigen-binding fragment thereof against the cocoat protein complex subunit [beta] 2 (COPB2), comprising any one of the CDR groups of (a) to (c) described below, (a) 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; (b) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (c) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18.

Description

Technical Field

[0001] The present invention relates to an antibody against coatomer protein complex subunit beta-2 (COPB2) capable of detecting COPB2, and the like. Background Art

[0002] In recent years, the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (COVID-19) has raged around the world.

[0003] Infected persons with COVID-19 show early symptoms such as fever and cough similar to those of a cold after a relatively long incubation period of 1 to 14 days. However, most infected persons are mild cases or asymptomatic infections. What is problematic in COVID-19 is that the remaining part of the infected persons develop severe respiratory disorders such as acute respiratory distress syndrome (ARDS), and then develop life-threatening complications such as myocarditis, vasculitis, and encephalomeningitis, that is, the problem of severe aggravation.

[0004] SARS-CoV-2, the causative virus of COVID-19, is a virus belonging to the genus Coronavirus, and PCR etc. are used for its detection. However, these detection methods can only determine the presence or absence of infection, and there is a problem that the subsequent severe aggravation cannot be predicted.

[0005] Patent Document 1 discloses a method for determining the possibility of severe aggravation in COVID-19 patients, which includes measuring the levels of one or more marker proteins present in exosomes derived from the patients, and COPB2 is one of the marker proteins.

[0006] COPB2 is one of the non-clathrin-coated endoplasmic reticulum subunits that form coatomers involved in membrane trafficking between the endoplasmic reticulum and the Golgi apparatus, and is a coatomer protein involved in membrane trafficking between the endoplasmic reticulum and the Golgi apparatus. As a coatomer protein, COPB2 plays an important role in embryogenesis and tumor progression, is involved in various conditions, and is also involved in controlling the proliferation and metastasis of cancer cells, etc., and thus has been studied as an important factor in many cancers (Non-Patent Document 1).

[0007] Prior Art Documents

[0008] Patent Documents

[0009] Patent Document 1: International Publication No. 2022 / 071601

[0010] Non-Patent Documents

[0011] Non - Patent Document 1: Clin Transl Oncol, 2021(11):2195 - 2205 Summary of the Invention

[0012] Problems to be Solved by the Invention

[0013] An object of the present invention is to provide a novel antibody capable of detecting COPB2 protein, etc.

[0014] Means for Solving the Problems

[0015] The inventors of the present application conducted in - depth research and as a result, discovered a novel antibody having CDRs containing specific sequences.

[0016] That is, the present invention is as described below. (1)

[0018] An antibody or an antigen - binding fragment thereof against coatomer protein complex subunit beta - 2 (COPB2), which comprises any one of the following CDR groups (a) to (c),

[0019] (a)

[0020] Heavy - chain CDR1 comprising the amino - acid sequence represented by SEQ ID NO: 1

[0021] Heavy - chain CDR2 comprising the amino - acid sequence represented by SEQ ID NO: 2

[0022] Heavy - chain CDR3 comprising the amino - acid sequence represented by SEQ ID NO: 3

[0023] Light - chain CDR1 comprising the amino - acid sequence represented by SEQ ID NO: 4

[0024] Light - chain CDR2 comprising the amino - acid sequence represented by SEQ ID NO: 5, and

[0025] Light - chain CDR3 comprising the amino - acid sequence represented by SEQ ID NO: 6

[0026] (b)

[0027] Heavy - chain CDR1 comprising the amino - acid sequence represented by SEQ ID NO: 7

[0028] Heavy - chain CDR2 comprising the amino - acid sequence represented by SEQ ID NO: 8

[0029] Heavy - chain CDR3 comprising the amino - acid sequence represented by SEQ ID NO: 9

[0030] Light - chain CDR1 comprising the amino - acid sequence represented by SEQ ID NO: 10

[0031] Light - chain CDR2 comprising the amino - acid sequence represented by SEQ ID NO: 11, and

[0032] The light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 12

[0033] (c)

[0034] The heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 13

[0035] The heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 14

[0036] The heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 15

[0037] The light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 16

[0038] The light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 17, and

[0039] The light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 18.

[0040] (1-1)

[0041] An antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), which comprises any one of the CDR sets of (a1)-(c1) below,

[0042] (a1)

[0043] The heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 1

[0044] The heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 2

[0045] The heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 3

[0046] The light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 4

[0047] The light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 5, and

[0048] The light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 6

[0049] (b1)

[0050] The heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 7

[0051] The heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 8

[0052] The heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 9

[0053] The light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 10

[0054] The light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 11, and

[0055] The light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 12

[0056] (c1)

[0057] The heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 13

[0058] The heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 14

[0059] The heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or an amino acid sequence having addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 15

[0060] The light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, or an amino acid sequence having 1 to several amino acids added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 16

[0061] The light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having 1 to several amino acids added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 17, and

[0062] The light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence having 1 to several amino acids added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 18.

[0063] (1-2)

[0064] An antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), which comprises any one of the following CDR sets of (a2)-(c2),

[0065] (a2)

[0066] The heavy chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1

[0067] The heavy chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2

[0068] The heavy chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3

[0069] The light chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 4

[0070] The light chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 5, and

[0071] The light chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 6

[0072] (b2)

[0073] The heavy chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 7

[0074] The heavy chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 8

[0075] The heavy-chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 9

[0076] The light-chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 10

[0077] The light-chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 11, and

[0078] The light-chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 12

[0079] (c2)

[0080] The heavy-chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13

[0081] The heavy-chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14

[0082] The heavy-chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15

[0083] The light-chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 16

[0084] The light-chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 17, and

[0085] The light-chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 18. (2)

[0087] An antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), which comprises the following heavy-chain variable region and / or light-chain variable region of (d) to (f),

[0088] (d)

[0089] The heavy-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19;

[0090] The heavy-chain variable region comprising an amino acid sequence having 1 to several amino acids added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 19; or

[0091] The heavy-chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 19,

[0092] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 20;

[0093] A light chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 20; or

[0094] A light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 20

[0095] (e)

[0096] A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 21;

[0097] A heavy chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 21; or

[0098] A heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 21,

[0099] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 22;

[0100] A light chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 22; or

[0101] A light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 22

[0102] (f)

[0103] A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 23;

[0104] A heavy chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 23; or

[0105] A heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 23,

[0106] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 24;

[0107] A light chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 24; or

[0108] A light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 24.

[0109] An antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or light chain variable region of (d) to (f) above is preferably the antibody or antigen-binding fragment thereof described in any one of (1) to (1-2) above.

[0110] That is, with respect to an antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or light chain variable region of (d) to (f) above, in the case of an antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or light chain variable region of (d), any one of the CDR sets of (a), (a1), or (a2) is preferably included, and more preferably the CDR set of (a); in the case of an antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or light chain variable region of (e), any one of the CDR sets of (b), (b1), or (b2) is preferably included, and more preferably the CDR set of (b); in the case of an antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or light chain variable region of (f), any one of the CDR sets of (c), (c1), or (c2) is preferably included, and more preferably the CDR set of (c).

[0111] It may also be an antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) as described in (1) above, comprising the heavy chain variable region and / or light chain variable region of (d) to (f) above. (3)

[0113] A nucleic acid encoding the antibody or antigen-binding fragment thereof described in any one of (1) to (2) above.

[0114] In this paragraph, (1) to (2) refer to (1), (1-1), (1-2), and (2). (4)

[0116] An expression vector comprising the nucleic acid described in (3) above. (5)

[0118] A transformant comprising the expression vector described in (4) above. (6)

[0120] A kit for detecting COPB2 protein in a sample, comprising the antibody or antigen-binding fragment thereof described in (1) to (2) above. (7)

[0122] The kit as described in (6) above, wherein the detection is carried out by immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, enzyme-linked immunosorbent assay (ELISA), or immunoblotting.

[0123] Advantages of the Invention

[0124] According to the present invention, novel antibodies capable of detecting the COPB2 protein can be provided. Brief Description of the Drawings

[0125] Figure 1 shows the principle of measuring the antibody titer after immunization with purified human COPB2 (650-906) protein in mice.

[0126] Figure 2 shows the antibody titer after two immunizations with purified human COPB2 protein in mice. High antibody titers of more than 800,000 counts were confirmed in A / Jackson mice, and antibody titers of less than 200,000 counts were confirmed in Balb / c mice.

[0127] Figure 3 ​​​Study showing the combination of solid-phase antibody and labeled antibody in sandwich ELISA. Shown by 4 panels according to the detection antibody. The 4 line graphs within the panel represent the absorbance (OD450) obtained for each solid-phase antibody. In the figure showing the results when the detection antibody is clone #A3-3G10, regarding the values of OD450 (vertical axis), from above (in the order of decreasing values), the results of clone #A2-7D11, clone #A2-7C6, clone #A3-8C9, and clone #A3-3G10 are shown. The results of clone #A2-7D11 and clone #A2-7C6 are close, and the results of clone #A3-8C9 and clone #A3-3G10 are close. In the figure showing the results when the detection antibody is clone #A3-8C9, the results are the same as when the detection antibody is clone #A3-3G10. In the figure showing the results when the detection antibody is clone #A2-7C6, regarding the values of OD450 (vertical axis), from above (in the order of decreasing values), the results of clone #A2-7D11, clone #A3-8C9, clone #A3-3G10, and clone #A2-7C6 are shown. In the figure showing the results when the detection antibody is clone #A2-7D11, regarding the values of OD450 (vertical axis), from above (in the order of decreasing values), the results of clone #A2-7C6, clone #A2-7D11, clone #A3-8C9, and clone #A3-3G10 are shown. Regardless of which clone the detection antibody is, the relative relationship of clone #A2-7D11, clone #A3-8C9, and clone #A3-3G10 remains unchanged from above (in the order of decreasing values) for the values of OD450 (vertical axis), while the order of clone #A2-7C6 is relatively different. It was confirmed that when clone #A2-7D11 was used as the solid-phase antibody and clone #A2-7C6 was used as the labeled antibody with reference to the results when the detection antibody was clone #A2-7C6, the strongest signal was detected.

[0128] Figure 4 Study showing whether the COPB2 protein can be detected in human cells and mouse cells by Western blotting. Lane 1: 10 μg of HEK293 cell extract, Lane 2: 5 μg of mouse liver extract, Lane 3: 10 μg of mouse liver extract.

[0129] [Figure 5] shows the results of immunostaining using BEAS-2B respiratory epithelial cells. The scale bar represents 50 μm. Only the antibody ( Figure 5A ). The antibody and nuclear staining ( Figure 5B ). For any antibody, there is an accumulation of stained areas in the region around the nucleus, which is considered to be near the Golgi apparatus morphologically. For A2-7C6, only the Golgi apparatus is stained most clearly.

[0130] ​[Figure 6] shows the results of immunostaining using the human lung cancer cell line NCl-H1975. The scale bar indicates 50 μm. Only the antibody ( Figure 6A ). The antibody and nuclear staining ( Figure 6B ). Some differences in the local presence from BEAS-2B were confirmed, and it was present in a punctate and dispersed manner.

[0131] [Figure 7] shows the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone #A2-7C6 ( Figure 7A ), as well as the amino acid sequences of the heavy chain variable region and light chain variable region ( Figure 7B ).

[0132] [Figure 8] shows the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone #A2-7D11 ( Figure 8A ), as well as the amino acid sequences of the heavy chain variable region and light chain variable region ( Figure 8B ).

[0133] [Figure 9] shows the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone #A3-8C9 ( Figure 9A ), as well as the amino acid sequences of the heavy chain variable region and light chain variable region ( Figure 9B ). Detailed Embodiments

[0134] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following examples.

[0135] One embodiment of the present embodiment is an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), which comprises any one of the following CDR groups (a)-(c),

[0136] (a)

[0137] Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1

[0138] Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2

[0139] Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3

[0140] Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4

[0141] Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and

[0142] Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6

[0143] (b)

[0144] Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7

[0145] Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8

[0146] Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9

[0147] Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10

[0148] Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and

[0149] Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12

[0150] (c)

[0151] Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13

[0152] Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14

[0153] Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15

[0154] Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16

[0155] Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and

[0156] Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18.

[0157] By making an antibody against coatomer protein complex subunit beta-2 (COPB2) or an antigen-binding fragment thereof have any one of the CDR sets of (a) to (c) above, it is possible to detect COPB2 protein by specifically binding to COPB2.

[0158] In the present specification, an antibody against coatomer protein complex subunit beta-2 (COPB2) is also referred to as an anti-COPB2 antibody. The description of the anti-COPB2 antibody also applies to the antigen-binding fragment of the anti-COPB2 antibody. Therefore, even if the antigen-binding fragment of the anti-COPB2 antibody is not mentioned in the present specification, the description of the anti-COPB2 antibody can be directly understood as the description of the antigen-binding fragment of the anti-COPB2 antibody.

[0159] The anti-COPB2 antibody is preferably an antibody comprising any one of the CDR groups of (a) to (c) above, but may also be an antibody comprising any one of the CDR groups of (a1) to (c1) above, or may also be an antibody comprising any one of the CDR groups of (a2) to (c2) above. That is, the above description can be directly understood as: the antigen-binding fragment of the anti-COPB2 antibody is preferably an antigen-binding fragment comprising any one of the CDR groups of (a) to (c) above, but may also be an antigen-binding fragment comprising any one of the CDR groups of (a1) to (c1) above, or may also be an antigen-binding fragment comprising any one of the CDR groups of (a2) to (c2) above.

[0160] COPB2 is a protein that is widely conserved from yeast to humans. In one aspect, the anti-COPB2 antibody is an antibody that specifically binds to human COPB2, and the antigen-binding fragment of the anti-COPB2 antibody may also be an antigen-binding fragment that specifically binds to human COPB2.

[0161] In the present specification, the term "antibody" refers to a molecule having a structure in which two heavy chains (H chains) and two light chains (L chains) associated with each other are stabilized by a pair of disulfide bonds.

[0162] The two heavy chains are respectively formed by a heavy-chain variable region VH, a heavy-chain constant region CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain is formed by a light-chain variable region VL and a light-chain constant region CL. In an antibody, the variable-region fragment (Fv) formed by VH and VL is the region directly involved in binding to an antigen. In addition, the antigen-binding region formed by VL, CL, VH, and CH1 is called the Fab region, and the region formed by the hinge region, CH2, and CH3 is called the Fc region.

[0163] Among the variable regions, the region that directly contacts the antigen varies particularly greatly and is called the complementarity-determining region (CDR). The part with fewer mutations outside the CDR is called the framework region (FR). There are 3 CDRs (heavy-chain CDR1 to 3 and light-chain CDR1 to 3) respectively in the variable regions of the light chain and the heavy chain.

[0164] The antibody is not particularly limited. For example, it may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred.

[0165] In this specification, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies. The monoclonal antibody is not particularly limited and can be produced, for example, by the hybridoma method, the recombinant DNA method, the phage display method, or a method using a genetically engineered animal containing all or part of the human immunoglobulin locus, or a combination of these methods.

[0166] In this specification, the term "polyclonal antibody" refers to a mixture of different antibodies that recognize multiple epitopes on one antigen. The polyclonal antibody is not particularly limited and can be produced, for example, by administering an immunogen containing the target antigen to a mammal (e.g., rat, mouse, rabbit, cattle, monkey, etc.) or a bird (e.g., chicken, etc.).

[0167] The anti-COPB2 antibody can be labeled or can be a conjugate with other agents, i.e., an antibody-drug conjugate (ADC).

[0168] In this specification, the term "antigen-binding fragment" refers to a molecule that contains a part of an antibody and can bind to the same antigen as the antibody. The antigen-binding fragment is not particularly limited, and examples include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv), etc.

[0169] The anti-COPB2 antibody can have a structure known for an antibody and can be any isotype among IgG, IgM, IgA, IgD, and IgE. IgG is an immunoglobulin having a γ heavy chain and is produced as part of the secondary immune response to an antigen. IgM is an immunoglobulin having a μ heavy chain and exists as a pentamer in mammals. IgA is an immunoglobulin having an α heavy chain, IgD is an immunoglobulin having an ε heavy chain, and IgE is an immunoglobulin having a δ heavy chain.

[0170] The anti-COPB2 antibody can be a chimeric antibody, a humanized antibody, etc. The antigen-binding fragment of the anti-COPB2 antibody can be an antigen-binding fragment of a chimeric antibody, an antigen-binding fragment of a humanized antibody, etc. A chimeric antibody is an antibody formed by linking antibody fragments from different species. A humanized antibody is an antibody in which the CDR amino acid sequences from non-human sources are transplanted into a human antibody. The humanized antibody is not particularly limited, and examples include an antibody having the heavy chain CDR1-3 and light chain CDR1-3 of an antibody produced by immunizing a mouse or a rat and the other regions derived from a human antibody. When referring to a humanized antibody, it sometimes also refers to a human chimeric antibody.

[0171] One aspect of this embodiment is an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), which comprises any one of the following CDR groups (a1) to (c1).

[0172] (a1)

[0173] Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 1

[0174] Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 2

[0175] Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 3

[0176] Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 4

[0177] Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 5, and

[0178] Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 6

[0179] (b1)

[0180] Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 7

[0181] Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 8

[0182] Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9, or an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 9

[0183] The light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 10

[0184] The light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 11, and

[0185] The light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 12

[0186] (c1)

[0187] The heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 13

[0188] The heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 14

[0189] The heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 15

[0190] The light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 16

[0191] The light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 17, and

[0192] The light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 18.

[0193] In the CDR groups of (a1) to (c1) above, the heavy-chain CDR1 to CDR3 and the light-chain CDR1 to CDR3 can be the CDR groups of any one of (a) to (c) above. In the CDR groups of (a1) to (c1) above, the amino acid sequences of the CDRs of any one of (a1) to (c1) can be amino acid sequences containing addition, substitution, or deletion of 1 to several amino acids in the amino acid sequences represented by SEQ ID NOs: 1 to 6, and the CDRs as amino acid sequences containing addition, substitution, or deletion of 1 to several amino acids can be 1 to 6.

[0194] In one aspect of the present embodiment, when the antibody or its antigen-binding fragment against coatomer protein complex subunit beta-2 (COPB2) contains any one of the CDR groups of (a1) to (c1) above, any CDR group is a group having or maintaining the binding ability to coatomer protein complex subunit beta-2 (COPB2).

[0195] In this specification, in the case of "containing addition, substitution, or deletion of 1 to several amino acids", the number of added, substituted, or deleted amino acids can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0196] In the case of "containing addition, substitution, or deletion of 1 to several amino acids" in the amino acid sequence of the CDR, as long as the anti-COPB2 antibody has binding affinity to COPB2, the number of added, substituted, or deleted amino acids is not particularly limited, and can be, for example, 1, 2, 3, or 4. It can include "addition, substitution, or deletion of 1 amino acid", can include "addition, substitution, or deletion of 1 to 2 amino acids", can include "addition or deletion of 1 to 3 amino acids", can include "addition, substitution, or deletion of 1 to 4 amino acids". In the amino acid sequence of the CDR, "addition, substitution, or deletion of 1 to 2 amino acids" is preferred, and "addition, substitution, or deletion of 1 amino acid" is preferred.

[0197] As long as the binding affinity to COPB2 is maintained, the positions of amino acid addition, substitution, or deletion can be any position in the amino acid sequence of the CDR. The heavy-chain variable region and the light-chain variable region of the antibody contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. In particular, the heavy-chain CDR3 is an important part determining the antigen specificity of the antibody. For the anti-COPB2 antibody, when there are mutations in the CDR, as long as the antigen specificity is maintained, 1 to several amino acids can be added, substituted, or deleted in CDR1 and / or CDR2 other than CDR3, and it can also be addition, substitution, or deletion of 1 amino acid.

[0198] One aspect of the present embodiment is an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), which comprises any one of the following CDR groups (a2) to (c2),

[0199] (a2)

[0200] Heavy chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1

[0201] Heavy chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2

[0202] Heavy chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3

[0203] Light chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 4

[0204] Light chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 5, and

[0205] Light chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 6

[0206] (b2)

[0207] Heavy chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 7

[0208] Heavy chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 8

[0209] Heavy chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 9

[0210] Light chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 10

[0211] Light chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 11, and

[0212] Light chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 12

[0213] (c2)

[0214] The heavy-chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13

[0215] The heavy-chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14

[0216] The heavy-chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15

[0217] The light-chain CDR1 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 16

[0218] The light-chain CDR2 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 17, and

[0219] The light-chain CDR3 comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 18.

[0220] In the CDR groups of (a2) to (c2) above, the heavy-chain CDR1 to CDR3 and the light-chain CDR1 to CDR3 may be the CDR groups of any one of (a) to (c) above. In the CDR groups of (a2) to (c2) above, when they are not the CDR groups of any one of (a) to (c) above, the amino acid sequences of the CDRs of any one of (a) to (c) above may be amino acid sequences having 80% or more identity with the amino acid sequences represented by SEQ ID NOs: 1 to 6, and the CDRs having amino acid sequences with 80% or more identity may be 1 to 6.

[0221] In one aspect of the present embodiment, when an antibody against coatomer protein complex subunit beta-2 (COPB2) or an antigen-binding fragment thereof comprises any one of the CDR groups of (a2) to (c2) above, any CDR group is a group having or retaining the binding ability to coatomer protein complex subunit beta-2 (COPB2).

[0222] In the present specification, "having 80% or more identity" means that when the alignment is performed in such a way that the coincidence rate of the amino acid sequence before mutation and the amino acid sequence mutated from the amino acid sequence becomes the maximum, the number of common amino acid residues is 80% or more of the number of amino acids in the amino acid sequence before mutation.

[0223] The identity may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. The identity may also be 100%.

[0224] A group consisting only of heavy-chain CDR1 to CDR3 and light-chain CDR1 to CDR3 having 100% identity in amino acid sequence is a CDR group of any one of the above (a) to (c).

[0225] One aspect of the present embodiment is an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), which comprises the following heavy-chain variable regions and / or light-chain variable regions of (d) to (f).

[0226] Among the following heavy-chain variable regions and / or light-chain variable regions of (d) to (f), a CDR group of any one of the above is preferably included, and more preferably a CDR group of any one of the above (a) to (c) is included.

[0227] When taking the case of (d) as an example for description (the same applies to (e) and (f)), the heavy-chain variable region and / or light-chain variable region of (d) means: any one of the heavy-chain variable regions described in (d), or any one of the light-chain variable regions described in (d); or any one of the heavy-chain variable regions described in (d) and any one of the light-chain variable regions described in (d).

[0228] It should be noted that the anti-COPB2 antibody preferably comprises the following heavy-chain variable regions and light-chain variable regions. In this case, for example, in the case of (d), it more preferably comprises any one of the heavy-chain variable regions in (d) and any one of the light-chain variable regions in (d).

[0229] (d)

[0230] A heavy-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19;

[0231] A heavy-chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 19; or

[0232] A heavy-chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 19,

[0233] A light-chain variable region comprising the amino acid sequence represented by SEQ ID NO: 20;

[0234] A light-chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 20; or

[0235] A light-chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 20

[0236] (e)

[0237] The heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 21;

[0238] The heavy chain variable region comprising an amino acid sequence having one to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 21; or

[0239] The heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 21,

[0240] The light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 22;

[0241] The light chain variable region comprising an amino acid sequence having one to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 22; or

[0242] The light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 22

[0243] (f)

[0244] The heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 23;

[0245] The heavy chain variable region comprising an amino acid sequence having one to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 23; or

[0246] The heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 23,

[0247] The light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 24;

[0248] The light chain variable region comprising an amino acid sequence having one to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 24; or

[0249] The light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 24.

[0250] In addition to the CDRs, the heavy chain variable region and the light chain variable region also have framework regions (FRs). The amino acid sequences of the FRs can be the amino acid sequences of the FRs of the respective variable regions of each immunoglobulin themselves, can be sequences having mutations in the amino acid sequences, or can be sequences that have been partially modified by introducing restriction enzyme recognition sites, etc. into a part of the amino acid sequences derived from the FRs. In the amino acid sequences of the heavy chain variable region and / or the light chain variable region, from the viewpoint of having little influence on the reactivity with an antigen, the amino acid mutations can be in regions other than the CDRs, that is, in the FRs.

[0251] The anti-COPB2 antibody comprises the following CDR groups,

[0252] Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1

[0253] Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2

[0254] Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3

[0255] Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4

[0256] Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and

[0257] Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6,

[0258] and may comprise a heavy chain variable region of any one of the following and / or a light chain variable region of any one of the following,

[0259] A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19;

[0260] A heavy chain variable region comprising an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 19; or

[0261] A heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 19,

[0262] A light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 20;

[0263] A light chain variable region comprising an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 20; or

[0264] A light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 20.

[0265] The anti-COPB2 antibody comprises the following CDR groups,

[0266] Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7

[0267] Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8

[0268] Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9

[0269] Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10

[0270] The light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and

[0271] the light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12,

[0272] and may comprise a heavy chain variable region of any one of the following and / or a light chain variable region of any one of the following,

[0273] a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 21;

[0274] a heavy chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 21; or

[0275] a heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 21,

[0276] a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 22;

[0277] a light chain variable region comprising an amino acid sequence having an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 22; or

[0278] a light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 22.

[0279] The anti-COPB2 antibody comprises the following CDR set,

[0280] the heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13

[0281] the heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14

[0282] the heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15

[0283] the light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16

[0284] the light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and

[0285] the light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18,

[0286] and may comprise a heavy chain variable region of any one of the following and / or a light chain variable region of any one of the following,

[0287] a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 23;

[0288] The heavy chain variable region of an amino acid sequence containing an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 23; or

[0289] The heavy chain variable region of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 23,

[0290] The light chain variable region of an amino acid sequence represented by SEQ ID NO: 24;

[0291] The light chain variable region of an amino acid sequence containing an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 24; or

[0292] The light chain variable region of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 24.

[0293] In the anti-COPB2 antibody, for the region other than the CDR in the case where the CDR is defined, and the region other than the variable region in the case where the heavy chain variable region and / or the light chain variable region is defined, as long as the anti-COPB2 antibody has the binding ability to COPB2, it can be selected from the regions possessed by a normal antibody.

[0294] In addition, for the heavy chain variable region and the light chain variable region in the anti-COPB2 antibody, the amino acid sequence obtained by deleting the amino acids constituting the signal sequence from the amino acid sequences represented by SEQ ID NOs: 19 to 24 can be used.

[0295] That is, the anti-COPB2 antibody may contain:

[0296] The heavy chain variable region of an amino acid sequence obtained by deleting the amino acids constituting the signal sequence from any one of the amino acid sequences represented by SEQ ID NOs: 19, 21, and 23, and / or

[0297] The light chain variable region of an amino acid sequence obtained by deleting the amino acids constituting the signal sequence from any one of the amino acid sequences represented by SEQ ID NOs: 20, 22, and 24.

[0298] As the heavy chain variable region or the light chain variable region containing an amino acid sequence with an addition, substitution, or deletion of 1 to several amino acids, it can also be the heavy chain variable region or the light chain variable region containing an amino acid sequence with an addition, substitution, or deletion of 1 to several amino acids in the amino acid sequence obtained by deleting the amino acids constituting the signal sequence from any one of the amino acid sequences represented by SEQ ID NOs: 19 to 24.

[0299] Alternatively, as the heavy chain variable region or the light chain variable region containing an amino acid sequence having 80% or more identity, it may also be a heavy chain variable region or a light chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence obtained by deleting the amino acids constituting the signal sequence from the amino acid sequences represented by any of SEQ ID NOs: 19 to 24.

[0300] The signal sequence corresponds to approximately 20 amino acids from the N-terminal amino acid in the heavy chain variable region and the light chain variable region.

[0301] The amino acid sequence corresponding to the signal sequence is as follows.

[0302] SEQ ID NO: 19, positions 1 to 28

[0303] SEQ ID NO: 20, positions 1 to 36

[0304] SEQ ID NO: 21, positions 1 to 28

[0305] SEQ ID NO: 22, positions 1 to 22

[0306] SEQ ID NO: 23, positions 1 to 19

[0307] SEQ ID NO: 24, positions 1 to 19

[0308] Therefore, with respect to the antibody or its antigen-binding fragment against coatomer protein complex subunit beta-2 (COPB2) containing the heavy chain variable region and / or the light chain variable region of (d) to (f) above, as the antibody or its antigen-binding fragment not containing the signal sequence, it may be the antibody or its antigen-binding fragment against coatomer protein complex subunit beta-2 (COPB2) containing the heavy chain variable region and / or the light chain variable region of the following (d1) to (f1).

[0309] (d1)

[0310] A heavy chain variable region containing the amino acid sequence represented by positions 29 to 145 of SEQ ID NO: 19;

[0311] A heavy chain variable region containing an amino acid sequence having 1 to several amino acid additions, substitutions, or deletions in the amino acid sequence represented by positions 29 to 145 of SEQ ID NO: 19; or

[0312] A heavy chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by positions 29 to 145 of SEQ ID NO: 19,

[0313] A light chain variable region containing the amino acid sequence represented by positions 37 to 143 of SEQ ID NO: 20;

[0314] The light chain variable region having an amino acid sequence in which 1 to several amino acids are added, substituted, or deleted in the amino acid sequence represented by positions 37 to 143 of SEQ ID NO: 20; or

[0315] The light chain variable region having an amino acid sequence with 80% or more identity to the amino acid sequence represented by positions 37 to 143 of SEQ ID NO: 20

[0316] (e1)

[0317] The heavy chain variable region containing the amino acid sequence represented by positions 29 to 145 of SEQ ID NO: 21;

[0318] The heavy chain variable region having an amino acid sequence in which 1 to several amino acids are added, substituted, or deleted in the amino acid sequence represented by positions 29 to 145 of SEQ ID NO: 21; or

[0319] The heavy chain variable region having an amino acid sequence with 80% or more identity to the amino acid sequence represented by positions 29 to 145 of SEQ ID NO: 21,

[0320] The light chain variable region containing the amino acid sequence represented by positions 23 to 129 of SEQ ID NO: 22;

[0321] The light chain variable region having an amino acid sequence in which 1 to several amino acids are added, substituted, or deleted in the amino acid sequence represented by positions 23 to 129 of SEQ ID NO: 22; or

[0322] The light chain variable region having an amino acid sequence with 80% or more identity to the amino acid sequence represented by positions 23 to 129 of SEQ ID NO: 22

[0323] (f1)

[0324] The heavy chain variable region containing the amino acid sequence represented by positions 20 to 134 of SEQ ID NO: 23;

[0325] The heavy chain variable region having an amino acid sequence in which 1 to several amino acids are added, substituted, or deleted in the amino acid sequence represented by positions 20 to 134 of SEQ ID NO: 23; or

[0326] The heavy chain variable region having an amino acid sequence with 80% or more identity to the amino acid sequence represented by positions 20 to 134 of SEQ ID NO: 23,

[0327] The light chain variable region containing the amino acid sequence represented by positions 20 to 131 of SEQ ID NO: 24;

[0328] The light chain variable region having an amino acid sequence in which 1 to several amino acids are added, substituted, or deleted in the amino acid sequence represented by positions 20 to 131 of SEQ ID NO: 24; or

[0329] A light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by positions 20 to 131 of SEQ ID NO: 24

[0330] An antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or the light chain variable region of (d1)-(f1) above is preferably the antibody or the antigen-binding fragment thereof described in any one of (1)-(1-2) above.

[0331] That is, with respect to an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or the light chain variable region of (d1)-(f1) above, in the case of an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or the light chain variable region of (d1), any one of the CDR groups of (a), (a1) or (a2) is preferably included, more preferably the CDR group of (a); in the case of an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or the light chain variable region of (e1), any one of the CDR groups of (b), (b1) or (b2) is preferably included, more preferably the CDR group of (b); in the case of an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) comprising the heavy chain variable region and / or the light chain variable region of (f1), any one of the CDR groups of (c), (c1) or (c2) is preferably included, more preferably the CDR group of (c).

[0332] It may also be an antibody or an antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) as described in (1) above, comprising the heavy chain variable region and / or the light chain variable region of (d1)-(f1) above.

[0333] The description of the heavy chain variable region and / or the light chain variable region of (d)-(f) can be understood as the description of the heavy chain variable region and / or the light chain variable region of (d1)-(f1). The following descriptions in this specification regarding nucleic acids, expression vectors, transformants, production methods, kits, etc. are also the same as the description of the heavy chain variable region and / or the light chain variable region of (d)-(f), and can be understood as the description of the heavy chain variable region and / or the light chain variable region of (d1)-(f1).

[0334] (Nucleic acid)

[0335] One aspect of this embodiment is a nucleic acid encoding an antibody or an antigen-binding fragment thereof against COPB2.

[0336] The nucleic acid is not particularly limited and can be a natural nucleic acid or an artificial nucleic acid. For example, DNA, RNA, and chimeric nucleic acids of DNA and RNA can be cited. The base sequence of the nucleic acid encoding the anti-COPB2 antibody or its antigen-binding fragment is determined based on the amino acid sequence, and the nucleic acid can be prepared by methods known to those skilled in the art or methods based thereon.

[0337] The nucleic acid can be present in a single vector or a separate vector, or can be present at one or more sites within the host cell. The nucleic acid encoding the anti-COPB2 antibody or its antigen-binding fragment can be one or more nucleic acid molecules encoding the heavy and light chains of the antibody or their antigen-binding fragments.

[0338] (Expression vector)

[0339] One embodiment of the present embodiment is an expression vector containing a nucleic acid.

[0340] The expression vector is not particularly limited and can be appropriately selected according to the host cell to be used. For example, plasmids, retroviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, plant virus vectors (e.g., cauliflower mosaic virus vectors, tobacco mosaic virus vectors, etc.), cosmids, YACs, EBV-derived episomes, etc. can be cited. The nucleic acid can be inserted into these expression vectors by known methods (such as methods using restriction endonucleases) or methods based thereon to obtain an expression vector containing the nucleic acid.

[0341] In addition to the nucleic acid encoding the antibody against COPB2 or its antigen-binding fragment, the expression vector can also contain a promoter, an origin of replication, a selectable marker gene, etc. that regulate the expression of the nucleic acid gene encoding the antibody against COPB2 or its antigen-binding fragment. The promoter and the origin of replication can be appropriately selected according to the type of the host cell and the vector.

[0342] (Transformant)

[0343] One embodiment of the present embodiment is a transformant containing a vector.

[0344] The transformant is not particularly limited and can be obtained by transfecting an appropriate host cell with the expression vector. That is, the transformant can be a cell (host cell) containing the expression vector. The host cell is not particularly limited. For example, eukaryotic cells such as mammalian cells (CHO cells, COS cells, myeloma cells, HeLa cells, Vero cells, etc.), insect cells, plant cells, fungal cells (yeast genus, Aspergillus genus, etc.), and prokaryotic cells such as Escherichia coli (E.Coli), Bacillus subtilis, etc. can be cited.

[0345] (Method for producing an antibody against COPB2 or its antigen-binding fragment)

[0346] The method for producing an anti-COPB2 antibody or its antigen-binding fragment is not particularly limited. For example, in the case of a monoclonal antibody, it can be the following method: isolating antibody-producing cells from a non-human mammal immunized with COPB2 or its fragment, fusing them with myeloma cells, etc. to produce a hybridoma, and purifying the antibody produced by the hybridoma. In addition, a polyclonal antibody can be obtained from the serum of an animal immunized with COPB2 or its fragment.

[0347] In the case of producing an anti-COPB2 antibody or its antigen-binding fragment by genetic recombination methods, for example, an appropriate host cell can be transformed with an expression vector containing a nucleic acid encoding the anti-COPB2 antibody or its antigen-binding fragment, and the obtained transformant can be cultured under appropriate conditions to express the antibody or antigen-binding fragment. Then, it can be isolated and purified by known methods or methods based thereon.

[0348] The method for separating and purifying the antibody or antigen-binding fragment is not particularly limited. For example, affinity columns using Protein A, etc., column purification using other chromatographic columns, filter filtration, ultrafiltration, salting out, dialysis, etc. can be cited, and they can also be appropriately combined.

[0349] In the case where the anti-COPB2 antibody has a CDR having an amino acid sequence obtained by adding, substituting, or deleting amino acids in the amino acid sequences of heavy-chain CDR1-3 and light-chain CDR1-3, or in the case where the anti-COPB2 antibody has a CDR having an amino acid sequence having 80% or more identity with the amino acid sequences of heavy-chain CDR1-3 and light-chain CDR1-3, the anti-COPB2 antibody can be produced by known methods such as site-directed mutagenesis, random mutagenesis introduction method, chain replacement method, CDR walking method, etc.

[0350] Those skilled in the art are familiar that by phage display method, antibodies or their antigen-binding fragments with various mutations in the CDR are presented on the phage surface and screened using an antigen, so that antibodies or their antigen-binding fragments containing a more affinity-matured CDR group can be obtained (for example, Wu et al., PNAS, 95:6037-6042 (1998); Schier, R. et al., J. Mol. Bio. 263:551-567 (1996); Schier, R. et al., J. Mol. Biol. 255:28-43 (1996); Yang, W.P. et al., J. Mol. Biol., 254:392-403 (1995)). Therefore, an anti-COPB2 antibody can be produced by this method.

[0351] Chimeric antibodies and humanized antibodies can be produced by known methods or methods based thereon.

[0352] It can be prepared using the following gene: The gene is obtained by cloning an antibody gene from the mRNA of a hybridoma that produces antibodies of animals other than humans and ligating it to a part of the human antibody gene through genetic recombination technology.

[0353] As the gene for the anti-COPB2 antibody, it can also be determined based on the amino acid sequences of the CDRs and the amino acid sequence of the variable region described in the above (a) to (f).

[0354] In the production of chimeric antibodies, there is no particular limitation. For example, it can be produced using a nucleic acid obtained by genetically recombining and replacing the heavy chain variable region (VH) and the light chain variable region (LH) of the above (d) to (f) with sequences derived from human antibodies.

[0355] In addition, the constant regions of the heavy chain and light chain of human IgG1 are cloned. For the heavy chain and light chain, for example, the heavy chain variable region (VH) and the light chain variable region (LH) of the above (d) to (f) can be ligated to the constant region derived from human antibodies by the PCR-based Overlapping Hanging method. The amplified DNA is inserted into an appropriate vector and transformed to obtain a chimeric antibody.

[0356] In the production of humanized antibodies, the variable part of the human antibody is cloned and modified by site-selective mutagenesis using the Megaprimer method to the base sequence of the CDR of the anti-COPB2 antibody. When the amino acid sequence of the framework region cannot specifically bind to the antigen after humanizing, a part of the amino acids in the framework region can be converted from the human type to the rat type.

[0357] CDRs containing amino acid sequences with one or more amino acid deletions, substitutions, or additions in the amino acid sequence of the original CDR, and CDRs containing amino acid sequences with 80% or more identity to the original sequence can be prepared using known methods such as site-directed mutagenesis, random mutagenesis, chain replacement method, and CDR stepping method.

[0358] The antigen-binding fragment of the anti-COPB2 antibody can be expressed using the nucleic acid encoding the fragment by the above method. In addition, it can also be fragmented by treatment with enzymes such as papain and pepsin after obtaining the full-length antibody.

[0359] By using an anti-COPB2 antibody, the amount of COPB2 protein in COVID-19 patients can be measured. For example, for a biological sample collected from a COVID-19 patient, its presence amount is determined by a method of detecting the presence amount of COPB2 protein, and by comparing it with the biological sample of a patient without COVID-19, the trend of COVID-19 exacerbation can be predicted.

[0360] (Kit)

[0361] One aspect of the present embodiment is a kit for detecting COPB2 protein in a sample,

[0362] The kit contains an antibody against COPB2 or an antigen-binding fragment thereof.

[0363] The antibody against COPB2 or an antigen-binding fragment thereof contained in the kit may be the anti-COPB2 antibody or an antigen-binding fragment thereof described in this specification.

[0364] The kit may be accompanied by instructions for use of the kit.

[0365] In addition to the anti-COPB2 antibody or an antigen-binding fragment thereof, the kit may also contain reagents required for detecting COPB2 protein in a sample, etc.

[0366] The reagent is not particularly limited. For example, antigen retrieval agent, enzymatic antigen retrieval agent, blocking agent, washing solution, fluorescent dye, labeled enzyme, labeled secondary antibody, stain, chromogenic agent, fixative for cells, etc., membrane permeabilization agent, dehydrating agent, mounting agent, detection agent, reaction termination solution, buffer solution, cell lysis buffer solution, loading buffer solution, electrophoresis buffer solution, transfer buffer solution, etc. The kit may contain at least one of these reagents.

[0367] The sample to be detected for COPB2 protein using the anti-COPB2 antibody or an antigen-binding fragment thereof is not particularly limited. For example, it may be a biological sample isolated from an animal such as a human. As a mammal other than a human, there is no particular limitation, and it may be a rodent such as a mouse or a rat, or it may be a dog, a cow, a horse, a monkey, a rabbit, a pig, etc.

[0368] The biological sample is not particularly limited. For example, tracheal swab, oral swab, nasal swab fluid, pharyngeal swab fluid, nasal wash fluid, oral wash fluid, nasal aspiration fluid, saliva, sputum, blood, serum, urine, tissue, cell, crushed product of tissue or cell, etc. Among them, blood or serum is preferred.

[0369] Since COPB2 is known to be present in exosomes, the COPB2 protein in exosomes can also be used as a detection target. In addition, an anti-COPB2 antibody or an antigen-binding fragment thereof can also be used to detect exosomes in a sample by detecting the COPB2 protein. Exosomes are secreted by cells and circulate in the body, existing not only between cells but also in body fluids (such as blood, cerebrospinal fluid, urine, etc.). There is no particular limitation on the biological sample used for the detection of exosomes or the detection of the COPB2 protein in exosomes. For example, body fluids can be cited.

[0370] When using blood as a biological sample, there is no particular limitation on the sample. For example, whole blood, plasma, serum, etc. can be directly used, or it can be a processed product obtained by treating blood, for example, for the purpose of hemolysis treatment or obtaining specific components.

[0371] Regarding the sample, the biological sample can be appropriately diluted or concentrated. In addition, regarding the sample, in order to stably exist the COPB2 protein, there is no particular limitation. For example, it can contain a pH regulator, glycine, polysaccharide, sodium azide, thimerosal, EDTA, DTT, 2-mercaptoethanol, bovine serum albumin, glycerol, ethylene glycol, etc.

[0372] There is no particular limitation on the detection of the COPB2 protein. For example, it can be carried out on the COPB2 protein in the sample by an immunological assay method using an anti-COPB2 antibody or an antigen-binding fragment thereof.

[0373] Immunoassay for detecting a target protein by immunological assay is well-known in the art.

[0374] The reagent classes required in each immunoassay are also well-known. An anti-COPB2 antibody or an antigen-binding fragment thereof can be used as the antibody used in the immunoassay, and the immunological detection of the COPB2 protein can be carried out using a usual immunoassay kit.

[0375] Immunoassay is a method for qualitative and quantitative analysis of trace components by utilizing the specific reaction between an antigen and an antibody. Since the antigen-antibody reaction has high sensitivity and selectivity of the reaction, it is widely used, and there are various assay methods according to the difference in its assay principle.

[0376] There is no particular limitation on the immunoassay, and the methods shown in the following table can be cited.

[0377] [Table 1]

[0378]

[0379] As for the immunoassay, there is no particular limitation. For example, immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunoblotting, etc. are preferred.

[0380] Diseases (such as cancer or infectious diseases) can be diagnosed by detecting the COPB2 protein. A kit containing an anti-COPB2 antibody can be used as a tool for performing related diagnoses.

[0381] For example, an anti-COPB2 antibody or its antigen-binding fragment can be used to predict the occurrence or progression of cancer, to predict the possibility of cancer metastasis, or to predict the diagnosis and recurrence possibility of cancer prognosis.

[0382] The disclosures of all patent documents and non-patent documents cited in this specification are incorporated herein by reference in their entirety.

[0383] Examples

[0384] Some examples of the above-described embodiments of the present application are described below.

[0385] Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

[0386] 1. Preparation of antigen protein

[0387] cDNA encoding 255 amino acid residues from glutamine at position 650 to aspartic acid at position 906 in the amino acid sequence corresponding to human COPB2 was synthesized, and cDNA in the form of a 6xHis tag was ligated downstream. It was cloned into the expression vector pcDNA5 / TO (ThermoFisher Scientific). This plasmid was transfected into Expi293 cells (ThermoFisher Scientific) to express the human COPB2 antigen protein. The transfection was carried out using Expi293fectamine (ThermoFisher Scientific) according to the method described in the instruction manual. The culture medium and cells were recovered separately 6 days after transfection, and a cell extract was prepared from the cells by RIPA (Invitrogen). 10 μL of the culture medium and 10 μg of the cell extract were subjected to SDS-PAGE under reducing conditions, and the expression of COPB2 (650-906) was confirmed by Western blotting based on an anti-His tag antibody (proteintech).

[0388] As a result, the expression of COPB2 (650-906) was confirmed both in the culture medium and in the cells, and more expression and accumulation of COPB2 (650-906) were confirmed in the cells.

[0389] The culture medium of Expi293 cells after transfection and the cells were used to purify COPB2 (650 - 906) protein with TALON resin (Clontech). Through two transfection experiments, a total of 524 μg of purified COPB2 (650 - 906) protein was obtained from the culture medium and the cells.

[0390] 2. Preparation of monoclonal antibodies

[0391] Three A / Jackson mice and three Balb / c mice were immunized with purified human COPB2 (650 - 906) protein. That is, 50 μg of purified human COPB2 (650 - 906) protein was administered intraperitoneally to each mouse and 50 μg of purified human COPB2 (650 - 906) protein was administered subcutaneously to the neck of each mouse every time, and this was carried out twice every three weeks. Blood was collected after two immunizations to measure the antibody titer against the antigen protein. The principle of the measurement method is shown in Figure 1 .

[0392] As a result, it was confirmed that all A / Jackson mice immunized with human COPB2 protein had a high antibody titer of 800,000 counts or more. On the other hand, in Balb / c mice, an antibody titer of 200,000 counts or less was confirmed ( Figure 2 ).

[0393] One A / Jackson mouse (A / J#3 mouse) that showed a high antibody titer after being immunized twice with purified human COPB2 protein was given the antigen protein as the final booster. Four days after administration, the spleen was removed and splenocytes were prepared, and then cell fusion of splenocytes and myeloma cells (P3U1) was carried out. Hybridomas were inoculated into 10 96-well plates, and the antibody titer against human COPB2 protein as the immunogen was measured in the culture supernatants of a total of 928 wells. Five positive samples were confirmed. The cells contained in the wells of these five positive samples were further monoclonalized by two rounds of limiting dilution method. As a result, two clones of hybridomas were established as cell lines (#A3 - 3G10, #A3 - 8C9) that produce antibodies showing high reactivity with COPB2 protein.

[0394] Furthermore, splenocytes were also prepared from a mouse (A / J#2 mouse) that showed a high antibody titer after the third immunization, and cell fusion was carried out in the same way, and screening of 768 samples of hybridomas was carried out. As a result, two positive samples were detected and further monoclonalized by two rounds of limiting dilution method respectively (#A2 - 7C6, #A2 - 7D11).

[0395] As described above, a total of four clones of anti-COPB2 monoclonal antibodies were prepared.

[0396] 3. Preparation of sandwich ELISA

[0397] For the sandwich ELISA of all combinations using the four obtained clones (clone #A3-3G10, #A3-8C9, #A2-7C6, #A2-7D11) as the solid-phase antibody and the labeled antibody, their performance was evaluated. That is, each antibody solution prepared at 5 μg / mL with phosphate-buffered saline (PBS) was added at 100 μL / well to a 96-well microplate and allowed to react overnight. Then, the reaction solution was removed, and after washing once with a washing solution (PBS containing 0.01% Tween 20 and 0.1% ProClin 150), PBS containing 2% Block ACE (Dainippon Sumitomo Pharma Co., Ltd.) and 10% sucrose (FUJIFILM Wako Pure Chemical Corporation) was added at 200 μL / well each, and incubated at 37 °C for 2 hours for blocking, thereby preparing an antibody solid-phase plate.

[0398] On the other hand, for all the antibodies, they were reacted with 20-fold molar amount of Sulfo-NHS-LC-Biotin (ThermoFisher Scientific) at room temperature for 2 hours and subjected to gel filtration using a Zeba desalting column (ThermoFisher Scientific), thereby preparing the labeled antibody.

[0399] Purified COPB2 protein was added to the antibody-solidified plate at concentrations of 0, 0.1, 1, 10 ng / mL, stirred, and then reacted at room temperature for 2 hours. Then, the reaction solution was removed, and after washing 3 times with the above washing solution, 100 μL of the above-prepared labeled antibody at 1 μg / mL was added, stirred, and reacted at room temperature for 2 hours. After washing 3 times, 100 μL of HRP (horseradish peroxidase)-labeled streptavidin (R&D) was added and reacted at room temperature for 20 minutes. Similarly, after washing 3 times, 100 μL of TMB solution (Colorburst Blue, MOSS) was added and reacted at room temperature for 20 minutes. 50 μL of 2N sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm of each well was measured ( Figure 3 ). As a result, it was found that when clone #A2-7D11 among the four anti-COPB2 antibodies was used as the solid-phase antibody and clone #A2-7C6 was used as the labeled antibody, the strongest signal was detected.

[0400] 4. Western blot study

[0401] For four cloned antibodies, a study was conducted on whether the COPB2 protein in human cells and mouse cells could be detected by Western blotting. That is, 10 μg of cell extract prepared from human HEK293 cells using RIPA lysis buffer (manufactured by Santa Cruz Biotechnology, Inc.) and 5 or 10 μg of tissue extract prepared from mouse liver using T-PER lysis buffer (manufactured by ThermoFisher Scientific) were subjected to SDS-PAGE under reducing conditions, transferred to an Immobilon-P membrane (Merck Millipore), blocked with TBS-T (0.05 M Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, pH 7.6) containing 3% skim milk at room temperature for 1 hour, and reacted overnight at 4°C with a solution prepared by dissolving each antibody at a concentration of 1 μg / mL in TBS-T containing 3% skim milk. After the reaction, each membrane was washed 3 times with TBS-T for 10 minutes, and then reacted with a solution prepared by diluting an HRP-labeled rabbit anti-mouse IgG polyclonal antibody (manufactured by Dako) 1000-fold with TBS-T containing 3% skim milk at 4°C for 2 hours, followed by washing 3 times with TBS-T for 10 minutes. Then, the antibody was detected by Chemi-Lumi One Super (manufactured by Nacalai Tesque) ( Figure 4 ). The results showed that all antibodies reacted strongly with human COPB2, and clones #A2-7C6 and #A2-7D11 also cross-reacted with mouse COPB2. In addition, clone #A3-3G10 showed weak cross-reactivity with mouse COPB2, and clone #A3-8C9 did not react with mouse COPB2 at all.

[0402] 5. Fluorescent immunohistochemical staining

[0403] a) Preparation

[0404] 1) To improve the hydrophilicity of the coverslip, polyethyleneimine or poly-L-lysine was coated at room temperature for 1 hour.

[0405] 2) The coverslip was rinsed with sterile water (3 times for 1 hour).

[0406] 3) After the coverslip was completely dry, it was sterilized under UV light for at least 4 hours.

[0407] 4) Cells were cultured on the coverslip. Alternatively, samples were prepared by smearing cells or fixation based on Cytospin (trademark).

[0408] 5) Wash gently with PBS or PBS containing 0.1% Tween 20.

[0409] b) Fixation

[0410] 1) Incubate for 5 minutes at room temperature in 100% methanol cooled to -20°C.

[0411] 2) Incubate for 10 minutes at room temperature in PBS (pH 7.4) containing 4% paraformaldehyde.

[0412] 3) Wash the cells 3 times with ice-cold PBS.

[0413] c) Antigen retrieval (optional)

[0414] 1) Preheat the antigen retrieval solution (100 mM Tris, 5% [w / v] urea, pH 9.5) to 95°C. Fill the staining jar with the heated solution and immerse it entirely in a 95°C water bath.

[0415] 2) Carefully place the coverslip in the antigen retrieval solution in the staining jar using forceps with a flat tip.

[0416] 3) Incubate at 95°C for 10 minutes.

[0417] 4) Remove the coverslip from the antigen retrieval solution and immerse the coverslip with the cell side up in a 6-well tissue culture plate pre-filled with PBS.

[0418] 5) Wash the sample 3 times with PBS for 5 minutes.

[0419] d) Permeabilization

[0420] 1) Incubate the sample with PBS containing 0.1 - 0.25% Triton X-100 (or 100 μM digitonin, 0.5% saponin, etc.) for 10 minutes.

[0421] 2) Triton X-100 is the most widely used surfactant in permeabilization, but it causes more damage to the cell membrane compared to digitonin and saponin. Therefore, caution is required when staining targets present on the cell membrane simultaneously. In such cases, it is advisable to study the concentration of Triton X-100 for each experiment.

[0422] 3) After incubation, wash the cells 3 times with PBS for 5 minutes.

[0423] e) Blocking and immunostaining

[0424] 1) To block non-specific binding of antibodies, the sample was incubated with a blocking solution (PBST (PBS + 0.1% Tween 20) containing 1% BSA and 22.52 mg / mL glycine) at room temperature for 30 minutes. It is also possible to use 1% gelatin, 10% serum (from the same animal species as the host of the secondary antibody), etc. to replace the above solution.

[0425] 2) In a humidified chamber, the sample was incubated with a primary antibody diluted with PBST containing 1% BSA at room temperature for 1 hour or at 4 °C overnight.

[0426] 3) The antibody solution was removed from the sample, and the sample was washed 3 times with PBS for 5 minutes each.

[0427] 4) The sample was incubated with a fluorescently labeled secondary antibody (Thermo, A28175, goat anti-mouse IgG (H + L), Superclonal™ recombinant secondary antibody, AlexaFluor™ 488) diluted with PBST containing 1% BSA in the dark at room temperature for 1 hour.

[0428] 5) The secondary antibody solution was removed from the sample, and the sample was washed 3 times with PBS for 5 minutes each as much as possible in the dark.

[0429] f) Nuclear counterstaining

[0430] 1) 0.1 - 1 μg / mL of Hoechst 33342 nuclear stain (Thermo, H3570) or DAPI was added to the sample and incubated for 1 minute.

[0431] 2) The sample was washed with PBS.

[0432] g) Mounting

[0433] 1) One drop of mounting medium was added to the sample, and the sample was mounted with a coverslip.

[0434] 2) To prevent the sample from moving, the coverslip was sealed with clear nail polish.

[0435] 3) The sample was stored at 4 °C or -20 °C in the dark.

[0436] h) Microscopic observation

[0437] Observation was carried out using a fluorescence microscope (Keyence BZ-X810), and images were recorded (Figures 5 and 6).

[0438] 6. Structural analysis of anti-COPB2 antibody

[0439] The structural analysis of the heavy chain variable region and the light chain variable region of the anti-COPB2 monoclonal antibodies A2-7C6, A2-7D11, and A3-8C9 was performed using the bioinformatics analysis system of Repertoire Genesis, Inc.

[0440] The amino acid sequences of the heavy chain CDR1-3 and the light chain CDR1-3, as well as the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-COPB2 monoclonal antibodies A2-7C6, A2-7D11, and A3-8C9, are shown in Figures 7-9.

Claims

1. An antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2), comprising any one of the CDR sets of (a) to (c) below, (a) Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1 Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2 Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3 Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4 Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6 (b) Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7 Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8 Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9 Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10 Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12 (c) Heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13 Heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14 Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15 Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16 Light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and Light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

18.

2. The antibody or antigen-binding fragment thereof against coatomer protein complex subunit beta-2 (COPB2) according to claim 1, comprising the following heavy chain variable region and / or light chain variable region of (d) to (f), (d) Heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 19; Heavy chain variable region comprising an amino acid sequence containing one or several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 19; or Heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 19, Light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 20; Light chain variable region comprising an amino acid sequence containing one or several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 20; or Light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 20 (e) Heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 21; Heavy chain variable region comprising an amino acid sequence containing one or several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 21; or Heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 21, Light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 22; Light chain variable region comprising an amino acid sequence containing one or several amino acid additions, substitutions, or deletions in the amino acid sequence represented by SEQ ID NO: 22; or Light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 22 (f) Heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 23; The heavy chain variable region comprising an amino acid sequence in which 1 to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 23; or The heavy chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 23, The light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 24; The light chain variable region comprising an amino acid sequence in which 1 to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 24; or The light chain variable region comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO:

24.

3. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. An expression vector comprising the nucleic acid according to claim 3.

5. A transformant comprising the expression vector according to claim 4.

6. A kit for detecting COPB2 protein in a sample, comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. The kit according to claim 6, wherein, Detection is performed by immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, enzyme-linked immunosorbent assay (ELISA), or immunoblotting.

Images