Coatomer protein complex subunit beta-2 as biomarker for cancer diagnosis

Abstract

The present invention relates to a biomarker for cancer diagnosis in a blood sample, wherein said biomarker contains a coatomer protein complex subunit beta-2 (COPB2).

Description

Coatomer protein complex subunit beta 2 as a biomarker for cancer diagnosis

[0001] The present invention relates to a biomarker for cancer diagnosis.

[0002] Cancer is a disease with a high mortality rate in Japan, and its treatment remains difficult. While early detection and treatment are effective for cancer treatment, the high false positive rate in imaging diagnosis such as X-ray examination and low-dose CT screening is a concern. Therefore, a two-stage screening that performs imaging diagnosis after narrowing down the high-risk group using biomarkers is an important strategy for avoiding false positives and achieving early detection of cancer.

[0003] As a biomarker for cancer, research is underway to utilize biomolecules contained in tumor cells, such as driver genes possessed by tumor cells and proteins expressed thereby, as biomarkers.

[0004] Coatomer protein complex subunit beta 2 (COPB2) is known to be related to prostate cancer, breast cancer, etc. (Non-Patent Documents 1 to 7). Patent Document 1 also discloses an antibody against coatomer protein complex subunit beta 2.

[0005] International Publication No. 2024 / 101357

[0006] Arch Med Res. 2016 Aug;47(6):411-418.J Cell Mol Med. 2019 Aug;23(8):5235-5245.Exp Ther Med. 2021 Aug;22(2):792.CNS Neurosci Ther. 2020 Mar; 26(3): 309-318.Biomed Res Int. 2021 March 20;2021:6648078Squamous Cell Carcinoma Pathogenesis Cancers (Basel). 2022 Apr 18;14(8):2038.BMC Cancer. 2022 Jun 17;22(1):667

[0007] However, using biomolecules contained in tumor cells as biomarkers requires direct access to the affected area for sample collection. Therefore, the present invention aims to provide a novel cancer diagnostic biomarker present in the blood.

[0008] As a result of diligent research by the inventors, we discovered that COPB2 can be used as a biomarker in blood samples for the diagnosis of cancer, and thus completed the present invention.

[0009] In other words, the present invention is as follows: [1] A biomarker for cancer diagnosis in a blood sample comprising cortomer protein complex subunit beta 2 (COPB2). [2] The cancer diagnostic biomarker according to [1], wherein the COPB2 is derived from extracellular vesicles (EVs) in the blood. [3] The cancer diagnostic biomarker according to [1] or [2], wherein the cancer is one or more selected from the group consisting of lung cancer, breast cancer, glioma, gastrointestinal cancer, hepatocellular carcinoma, liver cancer, prostate cancer, and pancreatic cancer. [4] A method for obtaining data for diagnosing whether a subject has cancer, comprising the step of measuring the level of cortomer protein complex subunit beta 2 (COPB2) in a blood sample derived from the subject. [5] The method according to [4], wherein the COPB2 is derived from extracellular vesicles (EVs) in the blood. [6] The method according to [4] or [5], wherein the cancer is one or more selected from the group consisting of lung cancer, breast cancer, glioma, gastrointestinal cancer, hepatocellular carcinoma, liver cancer, prostate cancer, and pancreatic cancer. [7] A kit for diagnosing cancer in a blood sample, comprising a substance capable of binding to COPB2. [8] The cancer diagnostic kit according to [7], wherein the substance capable of binding to COPB2 is an anti-COPB2 antibody or an antigen-binding fragment thereof.

[0010] According to the present invention, a biomarker for cancer diagnosis can be provided.

[0011] The full amino acid sequence of human COPB2 is shown. The fragment amino acid sequence of human COPB2 is shown. The particle profile of the EV fraction obtained by NTA (Nanoparticle Tracking Analysis) is shown. The results of Western blotting are shown. Cell lysate: Indicates that the sample originates from cell lysate. Small EV: Indicates that the sample originates from the EV fraction. BEAS-2B: Indicates that the sample originates from non-lung cancer cells. NCI-H1975: Indicates that the sample originates from lung cancer cells. COPB2: Shows the band of the coatmer protein complex subunit beta 2. CD9: EV marker. GM130: Proof that no cellular components are present. β-actin: Internal cell standard. A comparison of the relative values ​​of the COPB2 band / CD9 in BEAS-2B and NCI-H1975 is shown. (Left) Box plots of COPB2 levels in the blood of cancer patients and non-cancer patients are shown. (Right) ROC diagram created from a comparison of COPB2 levels in the blood of cancer patients and non-cancer patients is shown. Comparison results of COPB2 levels in the blood of non-smokers and smokers are shown. The amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone A2-7C6 (Figure 8A) and the amino acid sequences of the heavy chain variable region and light chain variable region (Figure 8B) are shown. The anoic acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone A2-7D11 (Figure 9A) and the amino acid sequences of the heavy chain variable region and light chain variable region (Figure 9B) are shown. The amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone A3-8C9 (Figure 10A) and the amino acid sequences of the heavy chain variable region and light chain variable region (Figure 10B) are shown. Figure 11A shows the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone TA201C003a (identification number: #003), as well as the amino acid sequences of the heavy chain variable region and the light chain variable region (Figure 11B). Figure 12A shows the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of clone TA201B010a (identification number: #010), as well as the amino acid sequences of the heavy chain variable region and the light chain variable region (Figure 12B).

[0012] The embodiments of the present invention will be described below, but the present invention is not limited by the following examples.

[0013] This invention relates to a biomarker for cancer diagnosis in blood samples, comprising coated protein complex subunit beta-2 (COPB2).

[0014] (Coated Protein Complex Subunit Beta-2) In this specification, the term "coated protein complex subunit beta-2" is also referred to as COPB2. COPB2 is one of the non-clathrin-coated endoplasmic reticulum subunits that form a coated protein involved in membrane transport between the endoplasmic reticulum and the Golgi apparatus within a cell, and is a coated protein involved in membrane transport between the endoplasmic reticulum and the Golgi apparatus. In this embodiment, the COPB2 that serves as a biomarker for cancer diagnosis is not particularly limited as long as it is a protein classified as COPB2, but it is preferably human COPB2. It is preferable that the COPB2 has the full-length amino acid sequence of human COPB2 (SEQ ID NO: 31). As a COPB2 for cancer diagnosis, any COPB2 protein present in the body is acceptable, and it is preferable that the COPB2 has the amino acid sequence represented by SEQ ID NO: 31, and as long as it is present in the body, it may also be a COPB2 having an amino acid sequence with a mutation in SEQ ID NO: 31. As long as it is present in the body, the COPB2 for cancer diagnosis may also be a protein having a partial amino acid sequence of COPB2. COPB2 may be a protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 31, as long as it has the same function as COPB2, and may also be a protein containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 31.

[0015] (Biomarkers for cancer diagnosis) In this specification, the term "biomarker for cancer diagnosis" refers to a "biomarker" that can serve as an indicator for cancer diagnosis. In this specification, the term "biomarker" refers to a substance that can diagnose a specific disease in a subject through testing. Specifically, the term "biomarker" refers to a substance that can serve as a criterion for determining whether or not a subject is suffering from a specific disease by detecting the concentration or mutation status of the biomarker in the subject. In this embodiment, the biomarker for cancer diagnosis is present in a blood sample. In this embodiment, the term "biomarker for cancer diagnosis" may also be used as "tumor biomarker," "diagnostic marker," "predictive marker," "monitoring marker," "marker," etc., as long as it is for cancer diagnosis.

[0016] While not particularly limited, the inventors speculate that the following is the reason why COPB2 is effective as a biomarker for diagnosing cancer. Generally, cancer cells increase the production of proteins containing amino acid sequence mutations due to gene mutations. Such mutated proteins can sometimes undergo misfolding, where they cannot assume a normal folded structure. The endoplasmic reticulum is an organelle that performs protein synthesis and folding within the cell, and when misfolded proteins accumulate excessively, so-called endoplasmic reticulum stress is caused. Endoplasmic reticulum stress is a state in which the endoplasmic reticulum is subjected to a load that exceeds its protein folding capacity, and cells have various mechanisms to relieve endoplasmic reticulum stress. One of these is a mechanism that expels misfolded proteins by endocytosis. Therefore, in cancer cells, it is speculated that an extracellular vesicle containing COPB2 takes up misfolded proteins and expels the extracellular vesicle along with the proteins, thereby maintaining intracellular order. In other words, it is speculated that the secretion of extracellular vesicles containing COPB2 is activated from cancer cells and surrounding cells, and as a result, the amount of COPB2 in the blood of patients with cancer increases. Therefore, in this embodiment, COPB2 in the blood can be used as a biomarker for cancer diagnosis, and it is preferable to use COPB2 derived from extracellular vesicles in the blood as a biomarker for cancer diagnosis.

[0017] The present invention relates to a method for acquiring data for diagnosing whether a subject has cancer, the method comprising the step of measuring the level of coated protein complex subunit beta 2 (COPB2) in a blood sample derived from the subject. In this embodiment, data for diagnosing whether a subject has cancer is acquired. This data is obtained by measuring the presence of coated protein complex subunit beta 2 (COPB2) in a blood sample derived from the subject.

[0018] (Diagnosis) In this specification, the term "diagnosis" refers to determining the presence or likelihood of a disease, or the state of the disease. Normally, the act of diagnosis is the exclusive domain of physicians, veterinarians, or dentists, but in this specification, diagnosis includes auxiliary actions that assist in the diagnosis by physicians, etc., without the involvement of physicians, etc., such as the act of obtaining data for diagnosis. "Differentiation of disease" refers to determining whether or not a person has a particular disease. This also includes distinguishing between other diseases with similar lesions or symptoms. In this specification, the term "likelihood of disease" refers to the probability of developing a particular disease in the future. In this specification, if the specific disease is cancer, this also includes the possibility of progressing from a non-cancerous state to cancer. Furthermore, in this specification, the term "state of the disease" refers to the condition, stage, phase, and severity of a particular disease. For example, the severity of the condition may be mild, moderate, and severe, and the stage of the disease may be early, middle, and late.

[0019] "Pathological condition" includes not only the state of the patient's illness at a specific point in time, but also the progression of the illness from one point in time to another. For example, it may include the progression and degree of cancer, where the cancer worsens over time, or conversely, the degree of cancer remission, where it improves over time. Therefore, in this specification, "determination of pathological condition" includes the determination of the state and degree at a specific point in time, and the determination of the progression of the state from one point in time to another (for example, whether the cancer is progressing or improving).

[0020] In this embodiment, data for diagnosing whether a subject has cancer is obtained by measuring the level of cancer diagnostic biomarkers in the subject. In this embodiment, since COPB2 is used as the cancer diagnostic biomarker, the level of COPB2 in a blood sample derived from the subject is measured as data for diagnosing whether the subject has cancer.

[0021] In this specification, the term "level" means an index relating to a quantified abundance, and includes, for example, concentration, quantity, or an index that can be used instead (preferably a numerical index). Thus, in this specification, the term "level of biomarker" may be, for example, the measured value of the fluorescence intensity of the band itself, or, for example, a value converted to the number of extracellular vesicles and / or COPB2s in a given sample. Furthermore, the level may be an absolute numerical value (abundance, abundance per unit area, etc.) or a relative numerical value compared to a set control as necessary. Methods for measuring the level of a cancer diagnostic biomarker in a subject are not particularly limited, but include general methods known to those skilled in the art. Among these, examples include immunological assays (e.g., dot blot assay, Western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, immunoelectrophoresis, single-component radioimmunodiffusion assay (SRID), radioimmunoassay (RIA), enzyme-linked immunoassay (EIA), latex immunoassay (LIA), and fluorescence immunoassay (FIA)), and affinity assays (e.g., intermolecular interaction analysis, surface plasmon resonance (SPR), probe microscopy (SPM)). The above measurements may also be performed using commercially available measurement kits.

[0022] In this embodiment, after measuring the level, a comparison step may be included in which the level is compared with some index. If the measured level of the cancer diagnostic biomarker in the blood sample of the subject is at a level sufficient to diagnose cancer, the subject may be diagnosed with cancer by comparing it with the index. Alternatively, a subject without cancer may be used as a control, and the level of the cancer diagnostic biomarker in the blood sample of the control may be compared with the index to determine if the subject has cancer. A subject without cancer may be a healthy person who is completely free from cancer, or a person who does not have the cancer suspected of being present in the subject to whom the method of this embodiment is applied. In this case, for example, if the level of the cancer diagnostic biomarker in the subject is higher than that of a healthy person, the subject may be identified as having a high probability of having cancer, or if it is lower than or equal to that of a healthy person, the subject may be identified as having a low probability of having cancer.

[0023] In one embodiment of this embodiment, a method for acquiring data to diagnose whether a subject has cancer may include the steps of: mixing a blood sample obtained from the subject with a substance that can bind to a cancer diagnostic biomarker; detecting the cancer diagnostic biomarker; and measuring the level of the cancer diagnostic biomarker. The marker detection step and the level measurement step may be performed simultaneously. In one embodiment, the measurement may be performed using the cancer diagnostic kit of the present invention and / or a general method known to those skilled in the art. In one embodiment of this embodiment, it may also be a method for diagnosing whether a subject has cancer. In this embodiment, if it is a diagnostic method, the description herein of a method for acquiring data to diagnose whether a subject has cancer may apply to the diagnostic method.

[0024] In this specification, the term "mixing" means that a blood sample may be mixed with a substance that can bind to a cancer diagnostic biomarker, or a blood sample may be mixed with a substance that can bind to a cancer diagnostic biomarker. The method of mixing is not particularly limited. By mixing a blood sample with a substance that can bind to a cancer diagnostic biomarker, if the cancer diagnostic biomarker COPB2 is present in the blood sample, the cancer diagnostic biomarker and the substance that can bind to the cancer diagnostic biomarker will come into contact. In this specification, the term "contact" means bringing the cancer diagnostic biomarker and the substance that can bind to the cancer diagnostic biomarker close enough to interact. Such interactions are not particularly limited, but include chemical bonds such as covalent bonds, coordination bonds, metal-metal bonds, ionic bonds, metallic bonds, hydrogen bonds, and van der Waals bonds, intermolecular forces and interatomic forces such as Coulomb forces, interionic interactions, hydrogen bonds, dipole interactions, and van der Waals forces, as well as other interactions, charge transfer interactions, transcyclic interactions, hydrophobic interactions, and associations between peptides and biomolecules. By mixing a blood sample obtained from a subject with a substance capable of binding to a cancer diagnostic biomarker, if the blood sample contains COPB2, which is a cancer diagnostic biomarker, then COPB2 and the substance capable of binding to the cancer diagnostic biomarker come into contact. If COPB2 is not present in the blood sample, then COPB2 and the substance capable of binding to the cancer diagnostic biomarker do not come into contact. However, even if COPB2 is not present in the blood sample, in this specification, this means that the process involves bringing the cancer diagnostic biomarker into contact with the substance capable of binding to the said cancer diagnostic biomarker.The contact process is not particularly limited, but for example, it may be a process of dropping a blood sample containing or not containing a cancer diagnostic biomarker onto a solid-phase substance capable of binding to a cancer diagnostic biomarker and bringing them into contact, or it may be a process of dropping a substance capable of binding to a cancer diagnostic biomarker onto a substrate prepared by solidifying a blood sample containing or not containing a cancer diagnostic biomarker and bringing it into contact. The contact conditions in the contact process can be set, for example, as general conditions under which binding between an antibody and an antigen occurs.

[0025] In this specification, the term "detection" means detecting the interaction between a cancer diagnostic biomarker and a substance that can bind to the cancer diagnostic biomarker, and confirming the presence or absence of the cancer diagnostic biomarker. The detection of the degree of such interaction may be by measuring the level of the biomarker. In one embodiment of this specification, the substance that can bind to the cancer diagnostic biomarker may be treated with fluorescent labeling, colorimetric labeling, high electron density labeling, chemiluminescence labeling, and radioactive labeling for use in the detection step.

[0026] (Blood Sample) In this specification, the term "blood sample" refers to blood collected from a specimen and samples prepared from blood. As a blood sample, whole blood may be used as is, but examples of blood samples include plasma, serum, hemolysis, and other fractions or processed products of blood. Blood samples may also be diluents or concentrates. Whole blood may be obtained from the subject by conventionally known methods, and fractions or processed products of blood may also be obtained by known methods. Dilution and concentration can also be carried out by appropriately known methods. In this embodiment, the blood sample may be a fraction of extracellular vesicles (EVs) in the blood.

[0027] (Extracellular Vesicles) In this specification, the term "extracellular vesicle" refers to fine particles released from cells that consist of a lipid membrane and contain biomolecules such as nucleic acids, proteins, peptides, and hormones. Extracellular vesicles are also called EVs, from the initials of Extracellular Vesicles. Extracellular vesicles are referred to as large EVs if their particle diameter is greater than 200 nm, and small EVs if it is smaller than 200 nm. The particle diameter of extracellular vesicles is not particularly limited, but is generally between 30 and 1000 nm. The particle diameter of extracellular vesicles may be 30-400 nm, 50-400 nm, or 50-300 nm. Extracellular vesicles are classified into exosomes, microvesicles, and apoptotic vesicles, etc., based on differences in their in vivo production mechanisms. In this specification, any fine particles released from cells that consist of a lipid membrane encapsulating biomolecules such as nucleic acids, proteins, peptides, and hormones may be used, including exosomes, microvesicles, or apoptotic vesicles.

[0028] (Extracellular Vesicle Fraction) In this specification, the term "extracellular vesicle fraction" refers to a portion of a blood sample that contains at least extracellular vesicles. The portion of a blood sample containing extracellular vesicles can be separated and obtained by methods known to those skilled in the art. The portion of a blood sample containing extracellular vesicles may be a fraction collected from a blood sample, preferably from serum, by ultracentrifugation, as described in the examples below.

[0029] Methods for separating extracellular vesicle fractions from blood samples include ultracentrifugation (e.g., Therry C., Curr. Protoc. Cell Biol. (2006) Chapter 3: Unit 3.22.), polymer precipitation, immunoprecipitation, FACS, ultrafiltration, gel filtration, HPLC, sorting using microfluidic devices, and separation by adsorption onto carriers such as beads using antibodies or lectins. Alternatively, the extracellular vesicle fraction may be recovered using commercially available exosome recovery kits.

[0030] The method for separating the extracellular vesicle fraction from a blood sample by adsorption onto a carrier may use a predetermined buffer. The buffer is not particularly limited, but examples include MES (2-(N-morpholino)ethanesulfonic acid), ADA (N-(2-acetamide)iminodiacetic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)sesquisodium salt monohydrate), ACES (N-(2-acetamide)-2-aminoethanesulfonic acid), Bis-Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-morpholinopropanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), H Examples include EPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), POPSO (piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate), HEPPSO (2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid) monohydrate), EPPS (3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid), Tricinene (N-[tris(hydroxymethyl)methyl]glycine), Bicine (N,N-bis(2-hydroxyethyl)glycine), TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), CHES (N-cyclohexyl-2-aminoethanesulfonic acid), etc.

[0031] The separation of the extracellular vesicle fraction from a blood sample may include a step of dissociation to recover EVs from the carrier. The dissociation step may use a predetermined dissociation buffer. The dissociation buffer may be a buffer containing a substance that neutralizes the interaction between EVs and the carrier to cause dissociation, or a substance that causes EVs to dissociate from the carrier through a stronger interaction. The substance to be dissociated is not particularly limited, but examples include metal cations and surfactants. The metal cations are not particularly limited, but examples include monovalent metal cations such as sodium ions, potassium ions, lithium ions, silver ions, and copper(I) ions; divalent metal cations such as magnesium ions, calcium ions, zinc ions, nickel ions, barium ions, copper(II) ions, iron(II) ions, tin(II) ions, cobalt(II) ions, and lead(II) ions; and trivalent metal cations such as aluminum ions and iron(III) ions. The surfactant may be either an ionic surfactant or a nonionic surfactant, and is not particularly limited, but examples include Tween-20, Nonidet P-40, Triton X-100, Brig 35, SDS, CHAPS, sodium cholate, sodium deoxycholate, Deoxy-BIGCHAP, BIGCHAP, NIKKOLBL-9EX, MEGA-8, MEGA-9, MEGA-10, Triton X-114, Tween-40, Brig 58, NP-40, DDM, Tween-80, sarcosyl, urea, and Polyo. xyethylene (20) Sorbitan Trioleate, IGEPALCA-630, Digitonin, saponin, n -Heptyl-β-D-thioglucopyranoside, n-Dodecyl-β-D-maltopyranoside, n-Octyl-β-D-thioglucopyranoside, n-Nonyl-β-D-thiomaltoside, Oct Examples include yl-β-D-glucoside, Zwittergent3-12, Dodecyl-β-D-maltoside, and the like. The step of dissociating EVs to recover them from the carrier may be a step of dissociation by physical stimulation (heating, freeze-crushing, ultrasonic treatment, etc.).

[0032] (Kit) In this specification, the term "kit" may mean a combination of articles that perform a process relating to the diagnosis of cancer, such as collecting, storing, separating, measuring, diagnosing, and labeling a blood sample.

[0033] The kit may include chemical reagents (e.g., biomolecules such as antibodies, polypeptides, and polynucleotides), as well as other components. Specific examples of other components, though not limited to those specified, include: apparatus and reagents for blood sample collection and / or purification; apparatus and reagents for stable storage and / or transport of blood samples; apparatus and reagents for separation and / or purification of target fractions; apparatus and reagents for biomarker measurement; apparatus and reagents for displaying diagnostic results; suitable blood samples for standardization, normalization, and / or control; sample tubes; holders; trays; racks; dishes; plates; user instructions; solutions; buffer solutions; or other chemical reagents. The kit may also be packaged in a box with a lid for convenient storage and safe shipping.

[0034] In this embodiment, the kit may be a diagnostic kit for cancer. The diagnostic kit may be a simple diagnostic kit that provides data for diagnosing whether a subject has cancer, preferably using an extracellular vesicle fraction prepared from a blood sample.

[0035] (Cancer) In this specification, the term "cancer" refers to a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. The term "cancer" is also used in other terms such as "carcinoma," "tumor," and "neoplasm."

[0036] Cancers are not particularly limited, but examples include primary tumors, advanced cancers, recurrent cancers, metastatic cancers, and combinations thereof.

[0037] In this specification, the term "recurrence" refers to the reappearance of cancer after a period of time when cancer was not detected, the re-growth of cancer that had shrunk due to treatment, or the emergence of the same cancer in a different location. In this specification, the term "metastasis" refers to the movement of cancer cells from their original site of origin to another organ or tissue via blood vessels or lymphatic system, where they proliferate.

[0038] The cancer may originate in any organ or tissue, but it is preferable that it originates in an organ or tissue where COPB2 is distributed. The cancer is not particularly limited, but examples include cancers that originate in the brain, eyes, endocrine tissue, respiratory system, digestive tract, liver, gallbladder, pancreas, kidneys, bladder, reproductive tissue, muscle tissue, connective tissue, soft tissue, skin, bone marrow, lymphoid tissue, etc. Furthermore, it may be either an epithelial malignant tumor or a non-epithelial malignant tumor.

[0039] Specific cancers include, but are not limited to, thyroid cancer, adrenocortical carcinoma (ACC), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma (LUAD), squamous cell carcinoma of the lung, etc.), gastrointestinal cancer (e.g., esophageal cancer, stomach cancer, colorectal cancer, rectal adenocarcinoma (READ), etc.), bile duct cancer (CHOL), liver cancer, hepatocellular carcinoma, hepatoma, pancreatic cancer, kidney cancer (e.g., renal pigment atrophy (KICH), renal papillary cell carcinoma (KIRP), etc.), bladder cancer, peritoneal cancer, salivary gland cancer, skin cancer (e.g., cutaneous melanoma (SKCM), etc.), squamous cell carcinoma, multiple myeloma, glioma, brain tumor (e.g., low-grade glioma (LGG), glioblastoma multiforme (GBM), etc.), pheochromocytoma, paracancerous glioma (PCPG), uterine cancer ( Examples include endometrial cancer, uterine cancer, cervical cancer, uterine carcinosarcoma (UCS), ovarian cancer (e.g., serous cystadenocarcinoma of the ovary (OV)), breast cancer (e.g., mammary gland cancer, invasive mammary gland cancer (BRCA)), prostate cancer, testicular cancer (e.g., testicular germ cell tumor (TGCT)), vulvar cancer, thymoma (THYM), leukemia (e.g., AML (acute myeloid leukemia), CML (chronic myeloid leukemia)), malignant lymphoma, ALL (acute lymphoblastic leukemia), CLL (chronic lymphocytic leukemia), Hodgkin lymphoma, non-Hodgkin lymphoma, lymphoid neoplasms such as diffuse large B-cell lymphoma (DLBC), and other lymphocyte proliferative disorders.

[0040] Among these, it is preferable that the cancer be one or more selected from the group consisting of lung cancer, breast cancer, glioma, gastrointestinal cancer, hepatocellular carcinoma, liver cancer, prostate cancer, and pancreatic cancer, and preferably it may be used for the diagnosis of lung cancer.

[0041] (Anti-COPB2 antibody or its antigen-binding fragment) In this specification, the term "anti-COPB2 antibody" refers to an antibody against COPB2. The description of anti-COPB2 antibody also applies to the antigen-binding fragment of anti-COPB2 antibody. Therefore, in this specification, even if there is no mention of the antigen-binding fragment of anti-COPB2 antibody, the description of anti-COPB2 antibody shall be understood as a description of the antigen-binding fragment of anti-COPB2 antibody.

[0042] In one aspect, the anti-COPB2 antibody is an antibody that specifically binds to COPB2, and the antigen-binding fragment of the anti-COPB2 antibody can also be an antigen-binding fragment that specifically binds to COPB2.

[0043] The anti-COPB2 antibody may be any antibody that specifically binds to COPB2 and can be used for the detection of a biomarker for cancer diagnosis. Although not particularly limited, for example, the use of an antibody that binds to human COPB2 so that human COPB2 cannot bind to the human COPB2 receptor can be mentioned.

[0044] The binding site of COPB2 to which the anti-COPB2 antibody binds is not particularly limited, but for example, it preferably binds to one or more peptides having the amino acid sequence of human COPB2.

[0045] The anti-COPB2 antibody may have a structure known as an antibody and may be of any isotype of IgG, IgM, IgA, IgD, or 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.

[0046] The anti-COPB2 antibody may be a chimeric antibody, a humanized antibody, a human antibody, etc. The antigen-binding fragment of the anti-COPB2 antibody may be an antigen-binding fragment of a chimeric antibody, a humanized antibody, a human antibody, etc. A chimeric antibody is an antibody in which fragments of antibodies derived from different species are linked. A humanized antibody is an antibody in which non-human CDR amino acid sequences are transplanted into a human antibody. Although not particularly limited, examples of humanized antibodies include antibodies having heavy chain CDR1-3 and light chain CDR1-3 of an antibody produced by immunizing a mouse or a rat, and all other regions being derived from a human antibody. In the case of a humanized antibody, it may be a human chimeric antibody, a mutant human antibody, or a human-type antibody.

[0047] In one embodiment of this invention, the anti-COPB2 antibody or its antigen-binding fragment may include any of the following sets of CDRs (a) to (e). (a) Heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 Heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2 Heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 Light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4 Light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 (b) Heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 7 Heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8 Heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 9 Light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10 Light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 12 (c) Heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 13 Heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 14 Heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 15 Light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 16 (d) Light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 17, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 18 Heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 32 Heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 33 Heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 34 Light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 35 Light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 36, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 37 (e) Heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 38 Heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 39 Heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 40 Light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 41 Light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 42, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 43

[0048] The anti-COPB2 antibody or its antigen-binding fragment can specifically bind to COPB2 by having any of the sets of CDRs of (a) to (e) above.

[0049] In one embodiment of this invention, the anti-COPB2 antibody or its antigen-binding fragment may include any of the following sets of CDRs (a1) to (e1). (a1) Heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 1 Heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 2 Heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 3 Light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 4 Light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 5, and Light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 6 (b1) Heavy chain CDR1 consists of an amino acid sequence represented by SEQ ID NO: 7, or an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 7. Heavy chain CDR2 consists of an amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 8. Heavy chain CDR3 consists of an amino acid sequence represented by SEQ ID NO: 9, or an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 9. Light chain CDR1 consists of an amino acid sequence represented by SEQ ID NO: 10, or an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 10.Light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 11, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 11, and Light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 12, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 12 (c1) Heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 13, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 13 Heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 14, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 14 Heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 15, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 15 Light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 16, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 16; Light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 17; and Light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 18 (d1) Heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 32, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 13; Heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 33, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 14 A heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 34, or an amino acid sequence in which one to several amino acids are added, substituted, or deleted from the amino acid sequence represented by SEQ ID NO: 15.Light chain CDR1 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 35 or the amino acid sequence represented by SEQ ID NO: 16, with the addition, substitution, or deletion of one to several amino acids. Light chain CDR2 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 36 or the amino acid sequence represented by SEQ ID NO: 17, with the addition, substitution, or deletion of one to several amino acids, and light chain CDR3 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 37 or the amino acid sequence represented by SEQ ID NO: 18, with the addition, substitution, or deletion of one to several amino acids. (e1) Heavy chain CDR1 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 38 or the amino acid sequence represented by SEQ ID NO: 13, with the addition, substitution, or deletion of one to several amino acids. Heavy chain CDR2 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 39 or the amino acid sequence represented by SEQ ID NO: 14, with the addition, substitution, or deletion of one to several amino acids. Heavy chain CDR3 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 40 or the amino acid sequence represented by SEQ ID NO: 15, with the addition, substitution, or deletion of one to several amino acids. Light chain CDR1 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 41 or the amino acid sequence represented by SEQ ID NO: 16, with the addition, substitution, or deletion of one to several amino acids. Light chain CDR2 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 42 or the amino acid sequence represented by SEQ ID NO: 17, with the addition, substitution, or deletion of one to several amino acids, and light chain CDR3 consisting of an amino acid sequence that is either the amino acid sequence represented by SEQ ID NO: 43 or the amino acid sequence represented by SEQ ID NO: 18, with the addition, substitution, or deletion of one to several amino acids.

[0050] The anti-COPB2 antibody is preferably an antibody containing any of the CDR sets (a) to (e) above, but may also be an antibody containing any of the CDR sets (a1) to (e1) above. In other words, the above description is understood to mean that the antigen-binding fragment of the anti-COPB2 antibody is preferably an antigen-binding fragment containing any of the CDR sets (a) to (e) above, but may also be an antigen-binding fragment containing any of the CDR sets (a1) to (e1) above. In the CDR sets (a1) to (e1) above, the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 may be any of the CDR sets (a) to (e) above. In the set of CDRs described in (a1) to (e1) above, the amino acid sequence of any of the CDRs may be an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by sequence numbers 1 to 6, and there may be one to six CDRs that are amino acid sequences containing the addition, substitution, or deletion of one to several amino acids.

[0051] If an anti-COPB2 antibody or its antigen-binding fragment has any of the CDR sets described in (a1) to (e1) above, then any of the CDR sets is a set that has or retains the ability to bind to COPB2.

[0052] In this specification, when we say "including the addition, substitution, or deletion of one to several amino acids," the number of amino acids to be added, substituted, or deleted may be, for example, one, two, three, four, five, six, seven, eight, nine, or ten.

[0053] When the amino acid sequence of the CDR is described as "containing the addition, substitution, or deletion of one to several amino acids," the number of amino acids added, substituted, or deleted is not particularly limited as long as the anti-COPB2 antibody has binding affinity to COPB2, but may be, for example, one, two, three, or four. It may contain "the addition, substitution, or deletion of one amino acid," "the addition, substitution, or deletion of one to two amino acids," "the addition or deletion of one to three amino acids," or "the addition, substitution, or deletion of one to four amino acids." In the amino acid sequence of the CDR, "the addition, substitution, or deletion of one to two amino acids" is preferred, and "the addition, substitution, or deletion of one amino acid" is preferred.

[0054] The location of the amino acid addition, substitution, or deletion may be any position in the CDR amino acid sequence, as long as binding to COPB2 is maintained. The heavy chain variable region and light chain variable region of the antibody include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus, with the heavy chain CDR3 being a particularly important part that determines the antigen specificity of the antibody. If an anti-COPB2 antibody has a mutation in the CDR, it may have one to several amino acid additions, substitutions, or deletions in CDR1 and / or CDR2 other than CDR3, or even just one amino acid addition, substitution, or deletion, as long as antigen specificity is maintained.

[0055] In one embodiment of this invention, the anti-COPB2 antibody or its antigen-binding fragment may include any of the following sets of CDRs (a2) to (e2). (a2) Heavy chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 Heavy chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 Heavy chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3 Light chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 4 Light chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 5, and light chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 6 (b2) Heavy chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 7 Heavy chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 8 Heavy chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 9 Light chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 10 Light chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 11, and light chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 12 (c2) Heavy chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13 Heavy chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14 Heavy chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15 Light chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 16 Light chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 17, and light chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 18 (d2)Heavy chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 32 Heavy chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 33 Heavy chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 34 Light chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 35 Light chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 36, and light chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 37 (e2) Heavy chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 38 Heavy chain CDR2 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 39 Heavy chain CDR3 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 40 Light chain CDR1 consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 41 Light chain CDR2 consists of an amino acid sequence that is 80% or more identical to the amino acid sequence represented by SEQ ID NO: 42, and light chain CDR3 consists of an amino acid sequence that is 80% or more identical to the amino acid sequence represented by SEQ ID NO: 43.

[0056] The anti-COPB2 antibody is preferably an antibody containing any of the CDR sets (a) to (e) above, but may also be an antibody containing any of the CDR sets (a2) to (e2) above. In other words, the above description is understood to mean that the antigen-binding fragment of the anti-COPB2 antibody is preferably an antigen-binding fragment containing any of the CDR sets (a) to (e) above, but may also be an antigen-binding fragment containing any of the CDR sets (a2) to (e2) above. In the CDR sets (a2) to (e2) above, the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 may be any of the CDR sets (a) to (e) above. In the sets of CDRs described in (a2) to (e2) above, if the set is not one of the sets of CDRs described in (a) to (e), the amino acid sequence of any of the CDRs may be an amino acid sequence that has 80% or more identity with the amino acid sequence represented by sequence numbers 1 to 6, and there may be 1 to 6 CDRs that have 80% or more identity with the amino acid sequence.

[0057] If an anti-COPB2 antibody or its antigen-binding fragment has any of the CDR sets described in (a2) to (e2) above, then any of the CDR sets is a set that has or retains the ability to bind to COPB2.

[0058] In this specification, "having 80% or more identity" means that when the amino acid sequence before mutation and the amino acid sequence mutated from that amino acid sequence are aligned to maximize the match, the number of common amino acid residues is 80% or more of the number of amino acids in the amino acid sequence before mutation. The identity may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The identity may also be 100%. A set of heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 consisting only of amino acid sequences having 100% identity is any of the CDR sets described in (a) to (e) above.

[0059] In one embodiment of this invention, the anti-COPB2 antibody or its antigen-binding fragment may be an anti-COPB2 antibody or its antigen-binding fragment that includes the following heavy chain variable regions and / or light chain variable regions (f) to (j). The following heavy chain variable regions and / or light chain variable regions (f) to (j) preferably have any of the above CDR sets, and more preferably have any of the above CDR sets (a) to (e). Taking the case of (f) as an example (the same applies to (g) to (j)), the heavy chain variable region and / or light chain variable region of (f) is any of the heavy chain variable regions described in (f), any of the light chain variable regions described in (f), any of the heavy chain variable regions described in (f), and any of the light chain variable regions described in (f). Furthermore, the anti-COPB2 antibody preferably contains the following heavy chain variable regions and light chain variable regions (f) to (j). In this case, for example, in the case of (f), it is more preferable to contain either of the heavy chain variable regions of (f) and either of the light chain variable regions of (f). (f) A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 19; a heavy chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 19; or a heavy chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 19; a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 20; a light chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 20; or a light chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 20; (g) A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 21; a heavy chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 21; or a heavy chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 21; a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 22;(h) A light chain variable region containing an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 22; or a light chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 22. (h) A heavy chain variable region containing an amino acid sequence represented by SEQ ID NO: 23; a heavy chain variable region containing an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 23; or a heavy chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 23. A light chain variable region containing an amino acid sequence represented by SEQ ID NO: 24; a light chain variable region containing an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 24; or a light chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 24. (i) A heavy chain variable region containing an amino acid sequence represented by SEQ ID NO: 44; (j) A heavy chain variable region containing an amino acid sequence in the amino acid sequence represented by SEQ ID NO: 44 that includes the addition, substitution, or deletion of one to several amino acids; or a heavy chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 44; A light chain variable region containing an amino acid sequence in the amino acid sequence represented by SEQ ID NO: 45; A light chain variable region containing an amino acid sequence in the amino acid sequence represented by SEQ ID NO: 45 that includes the addition, substitution, or deletion of one to several amino acids; or a light chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 45; (j) A heavy chain variable region containing an amino acid sequence represented by SEQ ID NO: 46; A heavy chain variable region containing an amino acid sequence in the amino acid sequence represented by SEQ ID NO: 46 that includes the addition, substitution, or deletion of one to several amino acids; or a heavy chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 46; A light chain variable region containing an amino acid sequence in the amino acid sequence represented by SEQ ID NO: 47 that includes the addition, substitution, or deletion of one to several amino acids;Or a light chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by Sequence ID No. 47;

[0060] The heavy chain variable region and the light chain variable region have a framework region (FR) in addition to the CDR. The amino acid sequence of the FR may be the same amino acid sequence derived from the FR of each variable region of each immunoglobulin, or it may be a sequence with mutations in the amino acid sequence, or it may be a partially modified version of the amino acid sequence derived from the FR, such as by introducing a restriction enzyme recognition site into a part of it. In the amino acid sequence of the heavy chain variable region and / or the light chain variable region, amino acid mutations may be in regions other than the CDR, i.e., within the FR, from the viewpoint of having little effect on reactivity with the antigen.

[0061] The anti-COPB2 antibody comprises the following set of CDRs: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3, light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6, and may also include any of the following heavy chain variable regions and / or any of the following light chain variable regions. A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 19; a heavy chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 19; or a heavy chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 19; a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 20; a light chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 20; or a light chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 20

[0062] The anti-COPB2 antibody comprises the following set of CDRs: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 7, heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8, heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 9, light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10, light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 12, and may also include any of the following heavy chain variable regions and / or any of the following light chain variable regions. A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 21; a heavy chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 21; or a heavy chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 21; a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 22; a light chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 22; or a light chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 22

[0063] The anti-COPB2 antibody comprises the following set of CDRs: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 13, heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 14, heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 15, light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 16, light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 17, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 18, and may also include any of the following heavy chain variable regions and / or any of the following light chain variable regions. A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 23; a heavy chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 23; or a heavy chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 23; a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 24; a light chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 24; or a light chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 24

[0064] The anti-COPB2 antibody comprises the following set of CDRs: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 32, heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 33, heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 34, light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 35, light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 36, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 37, and may also include any of the following heavy chain variable regions and / or any of the following light chain variable regions. A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 44; a heavy chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 44; or a heavy chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 44; a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 45; a light chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 45; or a light chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 45

[0065] The anti-COPB2 antibody comprises the following set of CDRs: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 38, heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 39, heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 40, light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 41, light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 42, and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 43, and may also include any of the following heavy chain variable regions and / or any of the following light chain variable regions. A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 46; a heavy chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 46; or a heavy chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 46; a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 47; a light chain variable region containing the amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 47; or a light chain variable region containing the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 47

[0066] In anti-COPB2 antibodies, regions other than the CDR when a CDR is specified, and regions other than the variable region when a heavy chain variable region and / or light chain variable region are specified, may be selected from regions that are normally present in antibodies, as long as the anti-COPB2 antibody has the ability to bind to COPB2. Furthermore, the heavy chain variable region and light chain variable region in anti-COPB2 antibodies may be amino acid sequences in which amino acids constituting the signal sequence are deleted from the amino acid sequences represented by SEQ ID NOs: 19-24 and 44-47. That is, anti-COPB2 antibodies may include a heavy chain variable region containing an amino acid sequence in which amino acids constituting the signal sequence are deleted from the amino acid sequence represented by any of SEQ ID NOs: 19, 21, 23, 44, or 46, and / or a light chain variable region containing an amino acid sequence in which amino acids constituting the signal sequence are deleted from the amino acid sequence represented by any of SEQ ID NOs: 20, 22, 24, 45, or 47. The heavy chain variable region or light chain variable region may include an amino acid sequence containing the addition, substitution, or deletion of one to several amino acids, or it may be an amino acid sequence in which an amino acid constituting the signal sequence is deleted, as represented by any of the amino acid sequences represented by SEQ ID NOs: 19-24 and 44-47. Furthermore, the heavy chain variable region or light chain variable region may include an amino acid sequence having 80% or more identity with an amino acid sequence in which an amino acid constituting the signal sequence is deleted, as represented by any of the amino acid sequences represented by SEQ ID NOs: 19-24 and 44-47. The signal sequence corresponds to approximately 20 amino acids from the N-terminal amino acid in the heavy chain variable region and light chain variable region.

[0067] The amino acid sequences corresponding to the signal sequences are as follows: SEQ ID NO: 19 (positions 1-28) SEQ ID NO: 20 (positions 1-36) SEQ ID NO: 21 (positions 1-28) SEQ ID NO: 22 (positions 1-22) SEQ ID NO: 23 (positions 1-19) SEQ ID NO: 24 (positions 1-19) SEQ ID NO: 44 (positions 1-19) SEQ ID NO: 45 (positions 1-20) SEQ ID NO: 46 (positions 1-19) SEQ ID NO: 47 (positions 1-22) Therefore, for an anti-COPB2 antibody or its antigen-binding fragment containing the heavy chain variable regions and / or light chain variable regions of (f) to (j) above, an antibody or its antigen-binding fragment that does not contain the signal sequence may also contain the following heavy chain variable regions and / or light chain variable regions of (f1) to (j1) below. (f1) Heavy chain variable region containing the amino acid sequence represented by position 29-145 of SEQ ID NO: 19 (SEQ ID NO: 25); Heavy chain variable region containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by position 29-145 of SEQ ID NO: 19; or Heavy chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by position 29-145 of SEQ ID NO: 19; Light chain variable region containing the amino acid sequence represented by position 37-143 of SEQ ID NO: 20 (SEQ ID NO: 26); Light chain variable region containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by position 37-143 of SEQ ID NO: 20; or Light chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by position 37-143 of SEQ ID NO: 20; (g1) Heavy chain variable region containing the amino acid sequence represented by position 29-145 of SEQ ID NO: 21 (SEQ ID NO: 27); A heavy chain variable region containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by position 29 to 145 of SEQ ID NO: 21; or a heavy chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by position 29 to 145 of SEQ ID NO: 21; a light chain variable region containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by position 23 to 129 of SEQ ID NO: 22;Or a light chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by position 23 to 129 of SEQ ID NO: 22 (h1) A heavy chain variable region containing an amino acid sequence represented by position 20 to 134 of SEQ ID NO: 23 (SEQ ID NO: 29); A heavy chain variable region containing an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by position 20 to 134 of SEQ ID NO: 23; Or a heavy chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by position 20 to 134 of SEQ ID NO: 23; A light chain variable region containing an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by position 20 to 131 of SEQ ID NO: 24; Or a light chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by position 20 to 131 of SEQ ID NO: 24 (i1) A heavy chain variable region containing the amino acid sequence represented by position 20-146 of SEQ ID NO: 44 (SEQ ID NO: 48); a heavy chain variable region containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by position 20-146 of SEQ ID NO: 44; or a heavy chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by position 20-146 of SEQ ID NO: 44; a light chain variable region containing the amino acid sequence represented by position 21-132 of SEQ ID NO: 45 (SEQ ID NO: 49); a light chain variable region containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by position 21-132 of SEQ ID NO: 45; or a light chain variable region containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by position 21-132 of SEQ ID NO: 45; (j1) A heavy chain variable region containing the amino acid sequence represented by position 20-144 of SEQ ID NO: 46 (SEQ ID NO: 50); A heavy chain variable region containing an amino acid sequence that includes the addition, substitution, or deletion of one to several amino acids in the amino acid sequence represented by positions 20 to 144 of Sequence ID No. 46;Or a heavy chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by position 20 to 144 of SEQ ID NO: 46; a light chain variable region containing an amino acid sequence represented by position 23 to 129 of SEQ ID NO: 47 (SEQ ID NO: 51); a light chain variable region containing an amino acid sequence in which one to several amino acids are added, substituted, or deleted in the amino acid sequence represented by position 23 to 129 of SEQ ID NO: 47; or a light chain variable region containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by position 23 to 129 of SEQ ID NO: 47;

[0068] The anti-COPB2 antibody or its antigen-binding fragment, which includes the heavy chain variable region and / or light chain variable region described above (f1) to (j1), is preferably an antibody or its antigen-binding fragment that includes any of the CDR sets described above (a) to (e), (a1) to (e1), or (a2) to (e2). In other words, an anti-COPB2 antibody or its antigen-binding fragment containing the heavy chain variable region and / or light chain variable region of (f1) to (j1) may, in the case of an anti-COPB2 antibody or its antigen-binding fragment containing the heavy chain variable region and / or light chain variable region of (f1), preferably contain any of the CDR sets of (a), (a1), or (a2), and more preferably contain the CDR set of (a); in the case of an anti-COPB2 antibody or its antigen-binding fragment containing the heavy chain variable region and / or light chain variable region of (g1), preferably contain any of the CDR sets of (b), (b1), or (b2), and more preferably contain the CDR set of (b); in the case of an anti-COPB2 antibody or its antigen-binding fragment containing the heavy chain variable region and / or light chain variable region of (h1), preferably contain any of the CDR sets of (c), (c1), or (c2), and more preferably contain the CDR set of (c); In the case of an anti-COPB2 antibody or its antigen-binding fragment containing the heavy chain variable region and / or light chain variable region of (i1), it is preferable to include any of the CDR sets of (d), (d1), or (d2), and more preferably the set of CDRs of (d). In the case of an anti-COPB2 antibody or its antigen-binding fragment containing the heavy chain variable region and / or light chain variable region of (j1), it is preferable to include any of the CDR sets of (e), (e1), or (e2), and more preferably the set of CDRs of (e).

[0069] An anti-COPB2 antibody or its antigen-binding fragment may include any of the sets of CDRs (a) to (e) that include the heavy chain variable regions and / or light chain variable regions described above as (f1) to (j1). The descriptions of the heavy chain variable regions and / or light chain variable regions (f) to (j) shall be understood as descriptions of the heavy chain variable regions and / or light chain variable regions (f1) to (j1). Similarly, in the descriptions of biomarkers, methods, kits, etc. in this specification, the descriptions of the heavy chain variable regions and / or light chain variable regions (f1) to (j) shall be understood as descriptions of the heavy chain variable regions and / or light chain variable regions (f1) to (j1).

[0070] In one embodiment of this invention, the anti-COPB2 antibody or its antigen-binding fragment may be a commercially available anti-COPB2 antibody or its antigen-binding fragment. The anti-COPB2 antibody is not particularly limited as long as it is generally available, but examples include the anti-COPB2 monoclonal antibody from Novus Biologicals (product name: COPB2 Antibody (M3A5), product code: NB600-102, etc.) and the anti-COPB2 polyclonal antibody from Bethyl Laboratories (product name: COPB2 Polyclonal Antibody) y, Catalog numbers: Cat#A304-522A, Cat#A304-523A, Cat#A304-522A-M, Cat#A304-523A-M, etc.), anti-COPB2 polyclonal antibodies manufactured by Invitrogen (product name: COPB2PolyclonalAntibody, catalog numbers: Catalogue#PA5-77106, #PA5-96557, #PA5-49113, #PA5 Anti-COPB2 antibodies from AssayGenie (product name: Anti-COPB2 Antibody, catalog number: PACO44494, CAB7036, etc.), anti-COPB2 polyclonal antibodies from Solarbio (product name: Anti-COPB2 Polyclonal Antibody, catalog number: K110122P-SAlexaFluor) Examples include anti-COPB2 polyclonal antibodies from Abcam (product name: Anti-COPB2 antibody, product codes: ab229639, ab2915, ab192924, etc.) and anti-COPB2 antibodies from Atlas Antibodies (product name: Anti-COPB2antibodyproducedinrabbit, product numbers: HPA036867, HPA058180, etc.). In addition, the anti-COPB2 antibody or its antigen-binding fragment may contain an amino acid sequence that has 80% or more identity with the amino acid sequence of a commercially available anti-COPB2 antibody.

[0071] In one aspect of this embodiment, the anti-COPB2 antibody or its antigen-binding fragment may be an anti-COPB2 antibody or its antigen-binding fragment that binds to the following antigens, and the antigen is not particularly limited as long as it is derived from the amino acid sequence of COPB2. The amino acid sequence of the antigen is not particularly limited, but for example it is as follows: Full-length amino acid sequence of human COPB2 (SEQ ID NO: 31) Amino acid sequence corresponding to positions 286 to 380 of the amino acid sequence of human COPB2 Amino acid sequence corresponding to positions 494 to 511 of the amino acid sequence of human COPB2 Amino acid sequence corresponding to positions 650 to 906 of the amino acid sequence of human COPB2 Amino acid sequence corresponding to positions 657 to 906 of the amino acid sequence of human COPB2 Amino acid sequence corresponding to positions 707 to 786 of the amino acid sequence of human COPB2 Amino acid sequence corresponding to positions 800 to 850 of the amino acid sequence of human COPB2 Among the amino acid sequences of B2, the amino acid sequence corresponding to positions 814 to 843; among the amino acid sequences of human COPB2, the amino acid sequence corresponding to positions 850 to 906; among the amino acid sequences of human COPB2, the amino acid sequence corresponding to positions 856 to 906; among these, the amino acid sequence of the antigen is not particularly limited, but it is preferably an amino acid sequence consisting of 25 to 257 consecutive amino acid residues selected from the amino acid sequence corresponding to positions 650 to 906 of the human COPB2 amino acid sequence, and more preferably the following amino acid sequences: among the amino acid sequences of human COPB2, the amino acid sequence corresponding to positions 650 to 906; among the amino acid sequences of human COPB2, the amino acid sequence corresponding to positions 850 to 906; and among the amino acid sequences of human COPB2, the amino acid sequence corresponding to positions 856 to 906.

[0072] The anti-COPB2 antibody or its antigen-binding fragment may be an antibody against an antigen having the amino acid sequence represented by SEQ ID NO: 31, or an antigen-binding fragment thereof. Furthermore, the anti-COPB2 antibody or its antigen-binding fragment may be an antibody against an antigen having the amino acid sequence from position 286 to 380, from position 494 to 511, from position 650 to 906, from position 657 to 906, from position 707 to 786, from position 800 to 850, from position 814 to 843, from position 850 to 906, or from position 856 to 906 in the amino acid sequence represented by SEQ ID NO: 31, or an antigen-binding antibody thereof. The sequence may be an antibody against an antigen having the amino acid sequence from position 650 to 906, from position 657 to 906, from position 707 to 786, from position 800 to 850, from position 814 to 843, from position 850 to 906, or from position 856 to 906, or an antigen-binding antibody thereof. The amino acid sequence represented by Sequence ID No. 31 may be an antibody against an antigen having the amino acid sequence from position 650 to 906, from position 850 to 843, or from position 856 to 906, or an antigen-binding fragment thereof.

[0073] In this embodiment, the acquisition of data for diagnosing whether or not a person has cancer may be combined with other methods for diagnosing cancer. While not particularly limited, the present invention may be used as one of two-stage or multi-stage screenings, such as X-ray examination, CT examination, low-dose CT screening, sputum cytology, exfoliation cytology, and fine-needle aspiration cytology, for the purpose of identifying high-risk populations.

[0074] Some embodiments of the above-described embodiments of the present invention are described below. The present invention will be specifically explained below with reference to these embodiments, but the present invention is not limited to these embodiments.

[0075] (1. Increase in COPB2 levels in EVs derived from lung cancer cells) To verify that EVs released by cancer cells contain COPB2, we compared the levels of COPB2 in EVs derived from non-cancer cells and EVs derived from cancer cells.

[0076] (1-1. Recovery of EVs fraction) 60 mL of culture supernatant recovered from non-lung cancer airway epithelial cells (BEAS-2B) and lung cancer cells (NCI-H1975) was centrifuged at 2,000 × g at 4°C for 10 minutes to settle the suspended matter. The supernatant was collected and impurities were removed using a 0.22 μm filter (MERCK, catalog number #SLGVR33RS). Ultracentrifugation was performed using an ultracentrifuge (himac, catalog number #CP100NX) at 210,000 × g at 4°C for 70 minutes. After removing the supernatant, PBS(-) passed through a 0.22 μm filter was added to resuspend the precipitate, and ultracentrifugation was performed again at 210,000 × g at 4°C for 70 minutes to wash the EVs. After washing, the supernatant was removed again, and the precipitated EVs were suspended in 100 μL of PBS(-), dispensed into Protein LoBind® Tubes (Eppendorf), and stored at 4°C.

[0077] (1-2. Western Blotting) Using NanoSight (Malvern Panalogical, catalog number #NS300), it was confirmed that a suitable group of particles as EVs had been recovered in the sample (cell lysate or EV suspension) (Figure 3). Subsequently, the sample concentration was adjusted, 4X SDS Sample Buffer (Novagen, catalog number #70607-3) was added, and 2-Mercaptethanol (Sigma, catalog number #M-6250) was added to the cell lysate, and the volume was adjusted to 10-15 μL. For electrophoresis, Mini-PEOTEAN TGX Gels (Bio-Rad, catalog number #4561096) and Running Buffer Solution (10X) for SDS-PAGE (Nacalai Tesque, catalog number #30329-61) were used. The gels were placed in the electrophoresis tank, filled with Running Buffer diluted 10-fold with Milli-Q water, and then the prepared samples were applied. Precision Plus Protein (registered trademark) was applied in parallel to determine the band size. Dual-color standards (Bio-Rad, catalog number #1610374) were applied simultaneously, and electrophoresis was performed at a constant voltage of 150V for 45 minutes. The gel after electrophoresis was attached to an Immobilon-P PVDF 0.45um 7x8.4cm sheet (Millipore, catalog number #IPVH0785), sandwiched between Mini Trans-Blot® Filter paper (Bio-Rad, catalog number #1703932), immersed in Transfer Buffer, and transferred at a constant voltage of 100 V for 60 minutes. The composition of the Transfer Buffer is as follows.

[0078]

[0079] After transfer, the membrane was blocked for 1 hour, then immersed in a diluted primary antibody solution and shaken for 1 hour. After shaking, it was washed three times for 5 minutes with TBS-T, exposed to diluted secondary antibody, and shaken for 60 minutes. Then it was washed three times for 10 minutes with TBS-T, and imaging was performed using an ECL select Western Blotting Detection Reagent (Cytiva, catalog number #RPN2235) with ChemiDoc® XRS+ with Image Lab® Software (Bio-Rad). ImageJ (U.S. National Institutes of Health) was used for band quantification. Furthermore, the primary antibodies used were Purified anti-human CD9 Antibody (BioLedgend, catalog number #312102), COPB2 (A3-8C9), GOLGA2 / GM130 Polychloronal Antibody (proteintech, catalog number #11308-1-AP), and Anti-β-Actin (ACTB) Antibody (sigma-aldrich, catalog number #A2228). The secondary antibodies used were Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (cytiva, catalog number #NA931) and Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (cytiva, part number #NA934) was used.

[0080] (1-3. Results) EVs were recovered from BEAS-2B and NCI-H1975 by ultracentrifugation and Western blotting was performed. More COPB2 was detected in EVs derived from NCI-H1975 than in EVs derived from BEAS-2B (Figures 4 and 5). This suggests that cancer cells secrete EVs containing more COPB2 into the extracellular space than healthy cells. The expression level of the protein CD9, which is known to be abundant in EVs, confirmed that the recovery of EVs by ultracentrifugation was performed correctly. Furthermore, the absence of GM130 in the EVs confirmed that there was no contamination with cellular components and that the EVs were properly purified (Figure 4). The expression level of β-actin was confirmed as a loading control of cells, and it was confirmed that β-actin was present in EVs derived from NCI-H1975 (Figure 4). This suggests that extracellular proteins (EVs) containing COPB2 derived from cancer cells are secreted via a different production pathway than EVs derived from normal cells. Furthermore, β-actin contained in EVs may serve as a cancer diagnostic biomarker linked to increased COPB2 protein production in EVs derived from cancer cells.

[0081] (2. Differentiation of lung cancer / non-lung cancer patients using COPB2 as a cancer diagnostic biomarker) To verify the effectiveness of COPB2 as a cancer diagnostic biomarker, the amount of COPB2 contained in the blood of cancer patients and non-cancer patients was compared.

[0082] (2-1. Selection of specimens) Serum samples were collected from 10 lung cancer patients and 10 non-lung cancer patients who visited the Department of Surgery (Respiratory Medicine) at Keio University School of Medicine, with written consent obtained.

[0083] (2-2. Purification of serum EVs) Blood collected from patients was transferred to serum centrifuge tubes and allowed to stand at room temperature for more than 30 minutes but less than 1 hour. Then, it was centrifuged in a swing centrifuge at 1500 x g and 25°C for 10 minutes. After centrifugation was complete, 1.1 mL was dispensed into Protein LoBind Tubes and immediately stored at -80°C. Then, the serum was thawed on ice and centrifuged at 10,000 x g and 4°C for 10 minutes to precipitate impurities, and the supernatant was further ultracentrifuged at 210,000 x g and 4°C for 25 minutes to precipitate the EVs fraction. After removing the supernatant by decanting, PBS(-) filtered through a 0.22 μm was added and the mixture was resuspended. The EVs were then washed by centrifugation again at 210,000 × g at 4°C for 10 minutes. The supernatant was removed by decanting, and the precipitated EVs were suspended in approximately 30-50 μL of remaining PBS(-) by vortexing. The mixture was then transferred to Protein LoBind® Tubes (Eppendorf) and stored at 4°C.

[0084] (2-3. Western Blot) The EV concentration of each sample was measured by Nanoparticle Tracking Analysis, and 4X SDS Sample Buffer was added to EV suspensions adjusted to the same particle number. Subsequent processing was carried out according to the method described in (1-2. Western Blot).

[0085] (2-4. Statistical Analysis) The comparison of the means between the two groups was evaluated using Welch's t-test. The discriminant performance between the two groups was evaluated using ROC analysis.

[0086] (2-5. Results) Western blotting was performed on serum EVs purified from 10 lung cancer patients and 10 non-lung cancer patients. Band intensity was quantified using ImageJ, and the results were summarized in an ROC curve. The experiment was conducted without the researchers knowing which samples originated from lung cancer patients. The analysis confirmed that the amount of COPB2 protein per unit number was significantly higher in lung cancer patients (Figure 6). This indicates that EVs rich in COPB2 secreted by cancer cells are released into the circulatory system and can be measured as COPB2 in the blood. Furthermore, no association was found between the presence or absence of smoking, a risk factor for lung cancer, and the amount of COPB2 per unit number (Figure 7). This suggests that the COPB2 detected in the blood originates from the cancer cells themselves, rather than being a risk factor for cancer. In summary, COPB2 is shown to be effective as a biomarker for cancer diagnosis.

[0087] The anti-COPB2 antibody available in this embodiment can be manufactured by the method disclosed in International Publication No. 2024 / 101357, or by a method similar to that disclosed. It may also be manufactured by the following method.

[0088] Five TC-mAb mice (Trans Chromosomics Co., Ltd.) (individual numbers: 7337, 7366, 7397, 7412, 7484) were immunized with purified human COPB2 (650-906) protein. Immune sites were multiple on the mice, and administration was performed at least twice over a three-week period. After three weeks, blood was collected, and serum antibody titers were confirmed by ELISA.

[0089] Similar evaluations were performed on the plasma of pancreatic cancer patients and liver cancer patients, confirming that COPB2 is effective as a biomarker for cancer diagnosis.

Claims

1. A biomarker for cancer diagnosis in blood samples, containing the coater protein complex subunit beta-2 (COPB2).

2. The cancer diagnostic biomarker according to claim 1, wherein the COPB2 is derived from extracellular vesicles (EVs) in the blood.

3. The cancer diagnostic biomarker according to claim 1, wherein the cancer is one or more selected from the group consisting of lung cancer, breast cancer, glioma, gastrointestinal cancer, hepatocellular carcinoma, liver cancer, prostate cancer, and pancreatic cancer.

4. A method for obtaining data to diagnose whether a subject has cancer, comprising the step of measuring the level of coatmer protein complex subunit beta 2 (COPB2) in a blood sample derived from the subject.

5. The method according to claim 4, wherein the COPB2 is derived from extracellular vesicles (EVs) in the blood.

6. The method according to claim 4, wherein the cancer is one or more selected from the group consisting of lung cancer, breast cancer, glioma, gastrointestinal cancer, hepatocellular carcinoma, liver cancer, prostate cancer, and pancreatic cancer.

7. A kit for diagnosing cancer in blood samples, containing a substance that can bind to COPB2.

8. The cancer diagnostic kit according to claim 7, wherein the substance capable of binding to COPB2 is an anti-COPB2 antibody or an antigen-binding fragment thereof.

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