Methods for diagnosing cancer using anti-BAG2 antibodies

Abstract

The present application discloses a composition for use in diagnosing cancer, the composition comprising an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof.

Description

[Technical Field]

[0001] Background of the Invention 1. Field of the Invention The present disclosure relates to compositions for use in diagnosing cancer, comprising an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof, and methods of providing information used to diagnose cancer. [Background technology]

[0002] 2. General Background and State of the Art The co-chaperone Bcl-2-associated athanogene (BAG) protein family mediates various physiological processes, including intracellular protein folding, stress response, neuronal differentiation, apoptosis, and cell proliferation, and functionally binds to various cooperating proteins. BAG2, a member of the BAG domain family with anti-apoptotic activity, is a negative regulator of the C-terminus of Hsc70-interacting protein (CHIP), a chaperone-associated ubiquitin ligase. The primary role of BAG2 in protein regulation through inhibition of CHIP activity is associated with neurodegenerative and autosomal recessive disorders through stabilization of chaperone-related proteins such as PINK1 and CFTR. BAG2 has been reported to have proapoptotic activity, as its expression increased proteasome inhibitor-induced apoptosis, and BAG2 knockdown partially inhibited apoptosis in thyroid cancer cells exposed to the proteasome inhibitor MG132. It has also been reported that overexpression of BAG2 promotes the stabilization of STK33 protein, a potent oncogene, in tumors induced by various mutant K-Ras, thereby promoting tumor development. However, despite these findings, the role of BAG2 in cancer progression and metastasis remains unclear. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, there is a need to develop compositions and methods for diagnosing cancer using antibodies that specifically bind to the BAG2 polypeptide or a fragment thereof. [Means for solving the problem]

[0004] Summary of the Invention These and other objects of the present invention will be more fully understood from the following description of the invention, its accompanying reference drawings and the appended claims.

[0005] One embodiment provides a composition for use in diagnosing cancer comprising an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof.

[0006] Another aspect provides a kit for use in diagnosing cancer comprising the composition.

[0007] Another aspect provides a method for providing information used to diagnose cancer.

[0008] Additional aspects will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the embodiments presented in this disclosure.

[0009] One embodiment provides a composition for use in diagnosing cancer comprising an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof.

[0010] The BAG2 polypeptide may be derived from a mammal. The mammal may be a human (Homo sapiens), mouse (Mus musculus), monkey, cow, or horse. The BAG2 may comprise the amino acid sequence of SEQ ID NO:69, which corresponds to NCBI reference SEQ ID NO:NM_004282.4. The BAG2 protein includes variants that have biologically equivalent activity to the amino acid sequence of SEQ ID NO:69, but whose amino acid sequences do not match the amino acid sequence of SEQ ID NO:69. The BAG2 polypeptide may comprise an amino acid sequence that has at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:69. A BAG2 protein may be a polypeptide having the same sequence as SEQ ID NO:69 except for at least one amino acid residue, at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, or at least seven amino acid residues. As used herein, "polypeptide" may be used interchangeably with "protein."

[0011] An antibody refers to a specific immunoglobulin directed against an antigenic site. An antibody refers to a polypeptide or combination of polypeptides that specifically binds to a BAG2 polypeptide or a fragment thereof. An antibody includes polyclonal antibodies, monoclonal antibodies, or recombinant antibodies, such as ScFv fragments, diabodies, single-chain antibodies, and the like, and encompasses all immunoglobulin antibodies. An antibody may include the complete form of an antibody having two full-length light chains and two full-length heavy chains, or a functional fragment of an antibody molecule that retains antigen-binding function by including a specific antigen-binding site, i.e., a binding domain, despite not having the structure of a complete, intact antibody having two light chains and two heavy chains.

[0012] An antigen-binding fragment refers to a portion of a polypeptide that is a fragment of the entire structure of an immunoglobulin and contains the portion to which an antigen can bind. For example, the antigen-binding fragment may be an scFv, (scFv)2, Fv, Fab, Fab', FvF(ab')2, or a combination thereof.

[0013] There are five types of heavy chains: gamma, delta, alpha, mu, and epsilon, and the heavy chain may determine the type of antibody. Alpha and gamma each contain 450 amino acids, and mu and epsilon each contain 550 amino acids. Heavy chains have two regions: a variable region and a constant region.

[0014] There are two types of light chains, kappa and lambda, which may contain from about 211 amino acids to about 217 amino acids. The light chain may have a constant region and a variable region.

[0015] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45, and a light chain variable region comprising a complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63;

[0016] a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 52, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 64;

[0017] a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 65;

[0018] a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 60, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 66;

[0019] a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; and

[0020] The antibody may comprise a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO:38, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:44, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:56, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:62, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:68; or a combination thereof.

[0021] The sixth and seventh Xaa in SEQ ID NO:39 may be glycine (Gly) or alanine (Ala). The second Xaa in SEQ ID NO:35 may be tyrosine (Tyr) or histidine (His). The eighth Xaa in SEQ ID NO:41 may be serine (Ser) or threonine (Thr). The twelfth Xaa in SEQ ID NO:47 may be tyrosine (Tyr) or histidine (His). The third Xaa in SEQ ID NO:53 may be methionine (Met) or isoleucine (Ie). The second Xaa in SEQ ID NO:59 may be Ala or Ser.

[0022] the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 in which the 6th and 7th Xaas are Gly, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45, and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; or

[0023] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO:38, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:44, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:56, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:62, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:68.

[0024] The antibody or antigen-binding fragment thereof includes a heavy chain variable region including a VH-CDR1 having the amino acid sequence of SEQ ID NO: 35 in which the second Xaa is Tyr, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 41 in which the eighth Xaa is Ser, and a VH-CDR3 having the amino acid sequence of SEQ ID NO: 47 in which the twelfth Xaa is His, and a light chain variable region including a VL-CDR1 having the amino acid sequence of SEQ ID NO: 53 in which the third Xaa is Met, a VL-CDR2 having the amino acid sequence of SEQ ID NO: 59 in which the second Xaa is Ala, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 65; or an antibody or antigen-binding fragment thereof including a VH-CDR1 having the amino acid sequence of SEQ ID NO: 37, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 43, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 65. The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:67.

[0025] The antibody or antigen-binding fragment thereof includes: an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region including VH-CDR1 having the amino acid sequence of SEQ ID NO: 33, VH-CDR2 having the amino acid sequence of SEQ ID NO: 39 in which the 6th Xaa and the 7th Xaa are Ala and Gly, respectively, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 45; and a light chain variable region including VL-CDR1 having the amino acid sequence of SEQ ID NO: 51, VL-CDR2 having the amino acid sequence of SEQ ID NO: 57, and VL-CDR3 having the amino acid sequence of SEQ ID NO: 63; or a heavy chain variable region including VH-CDR1 having the amino acid sequence of SEQ ID NO: 37, VH-CDR2 having the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 49; and a light chain variable region including VL-CDR1 having the amino acid sequence of SEQ ID NO: 55, VL-CDR2 having the amino acid sequence of SEQ ID NO: 56, and VL-CDR3 having the amino acid sequence of SEQ ID NO: 67. The antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:67.

[0026] The antibody or antigen-binding fragment thereof includes an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 44, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; or a light chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 62. and a light chain variable region comprising VL-CDR3 consisting of the amino acid sequence of NO:67, or an antigen-binding fragment thereof.

[0027] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising any one of amino acid sequences selected from SEQ ID NOs: 21 to 26; a light chain variable region comprising any one of amino acid sequences selected from SEQ ID NOs: 27 to 32; or the heavy chain variable region and the light chain variable region thereof.

[0028] The antibody or antigen-binding fragment thereof may comprise the heavy chain variable region of SEQ ID NO:21 and the light chain variable region of SEQ ID NO:27; the heavy chain variable region of SEQ ID NO:22 and the light chain variable region of SEQ ID NO:28; the heavy chain variable region of SEQ ID NO:23 and the light chain variable region of SEQ ID NO:29; the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:30; the heavy chain variable region of SEQ ID NO:25 and the light chain variable region of SEQ ID NO:31; or the heavy chain variable region of SEQ ID NO:26 and the light chain variable region of SEQ ID NO:32; or a combination thereof.

[0029] Xaa at positions 56 and 57 in SEQ ID NO:21 may be Gly or Ala. In SEQ ID NO:23, Xaa at position 1 is glutamine (G l n) or glutamic acid (Glu), and the seventh Xaa may be Ser or proline. hmm( Xaa at position 53 in SEQ ID NO:27 may be valine (Val) or alanine (Ala), Xaa at position 27 may be Tyr or His, Xaa at position 58 may be Ser or Thr, Xaa at position 61 may be asparagine (Asn) or Ser, Xaa at position 74 may be arginine (Arg) or lysine (Lys), Xaa at position 83 may be phenylalanine (Phe) or leucine (Leu), Xaa at position 92 may be Gly or Ala, and Xaa at position 108 may be His or Tyr. Xaa at position 53 in SEQ ID NO:27 may be Il In SEQ ID NO:29, 2 may be e or Phe. 9 Xaa is Met or Il e, 51 The 7th Xaa may be Ala or Ser, 9 The 10th Xaa may be Glu or aspartic acid (Asp), 6 Xaa is Met orIl It may also be e.

[0030] The antibody or antigen-binding fragment thereof may be a monoclonal antibody.

[0031] The antibody or antigen-binding fragment thereof may be marked with a detectable label or a label capable of emitting a detectable signal. A label refers to a detectable compound or composition that is directly or indirectly conjugated to the antibody to produce a labeled antibody or antigen-binding fragment thereof. The label may be detectable itself or may catalyze the chemical modification of a detectable substrate compound or composition. The label may be an immunofluorescent label, a chemiluminescent label, a phosphorescent label, a radioactive label, an epitope tag, an avidin / biotin label, a colloidal gold particle, a colored particle, a magnetic particle, a chromophore label, an ECL label, an enzyme, or the like.

[0032] The antibody or antigen-binding fragment thereof may be produced by a hybridoma cell selected from the hybridoma cells deposited under accession numbers KCTC 137378P, KCTC 137388P, KCTC 137398P, KCTC 137408P, KCTC 137418P, KCTC 137428P, KCTC 137438P, KCTC 137448P, KCTC 137458P and KCTC 137468P.

[0033] Hybridoma cells refer to hybrid cells capable of tumorigenesis that are formed by artificial fusion of two types of cells, and may generally be used to continuously produce antibodies by fusing B cells with plasmacytoma cells isolated from an immunized subject. Herein, hybridoma cells may be referred to as hybridoma cells or fusion cells.

[0034] Cancer may be solid or non-solid. Solid cancer refers to the development of cancerous tumors in organs such as the liver, lungs, breasts, and skin. Non-solid cancer is cancer that develops in the blood and is also called blood cancer. Cancer may be carcinoma, sarcoma, cancer derived from hematopoietic cells, germ cell tumor, or blastoma. Cancer may be selected from, for example, breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, gastric cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, brain and spinal cord tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndrome (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

[0035] The present inventors have found that when an anti-BAG2 antibody or its antigen-binding fragment, particularly a combination of anti-BAG2 antibodies or their antigen-binding fragments selected by BAG2 domain screening, is used, the presence of BAG2 is identified or its level is significantly elevated in cancer cell lines, including breast cancer cell lines, pancreatic cancer cell lines, glioblastoma multiforme cell lines, gastric cancer cell lines, ovarian cancer cell lines, or diffuse large B-cell lymphoma, unlike in normal cells. Therefore, the cancer diagnosed by using the composition may be selected from breast cancer, pancreatic cancer, glioblastoma, gastric cancer, ovarian cancer, and lymphoma.

[0036] The present inventors have found that when anti-BAG2 antibodies or antigen-binding fragments thereof, particularly a combination of anti-BAG2 antibodies or antigen-binding fragments thereof, are used, BAG2 is overexpressed in breast cancer patients compared to normal subjects, and when the level of BAG2 is high, the breast cancer patients are more likely to have metastatic breast cancer. Therefore, the breast cancer diagnosed with the composition may be metastatic breast cancer.

[0037] Among the molecular subtypes of breast cancer, triple-negative breast cancer (TNBC) is a highly aggressive subtype associated with poor prognosis and high mortality despite systemic treatment. TNBC is a heterogeneous subtype compared to luminal or HER2-enriched subtypes. While most targeted breast cancer therapies have demonstrated positive outcomes in hormone receptor- and HER2-positive breast cancers, TNBC patients lack three target receptors, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2), or other defined molecular targets, limiting effective treatment options, such as poly(ADP-ribose) polymerase (PARP), epidermal growth factor receptor (EGFR), Src tyrosine kinase, or similar. Therefore, rapid and accurate differentiation of TNBC patients from various subtypes of breast cancer is necessary to prevent unnecessary therapeutic approaches and select effective treatments for TNBC patients.

[0038] The present inventors have found that when anti-BAG2 antibody or its antigen-binding fragment, particularly a combination of anti-BAG2 antibody or its antigen-binding fragment, is used, BAG2 is overexpressed in breast cancer patients, and when the level of BAG2 is high, the probability that the breast cancer patient will be diagnosed with TNBC breast cancer is high.Therefore, the breast cancer that can be diagnosed by using the method can be triple-negative breast cancer or TNBC breast cancer.

[0039] Another aspect provides a kit for use in diagnosing cancer comprising the composition.

[0040] The kit may further comprise one or more other component compositions, solutions, or devices suitable for the analytical method used by the kit, such as Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation, FAGS, protein chips, or a combination thereof. For example, for detection of BAG2 immune complexes in a sample with their respective specific antibodies, the kit may further comprise a substrate, an appropriate buffer, a secondary antibody marked with a colored enzyme or fluorescent substance, or a colored substrate. The substrate may be a nitrocellulose membrane, a 96-well plate composed of polyvinyl resin, a 96-well plate composed of polystyrene resin, a glass slide, or the like; the coloring enzyme may be peroxidase, alkaline phosphatase, or the like; the fluorescent substance may be fluorescein isothiocyanate (FITC), rhodamine B-isothiocyanate (RITC), or the like; and the coloring substrate may be 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), tetramethylbenzidine (TMB), or the like.

[0041] Another aspect provides a method for providing information used to diagnose cancer.

[0042] The method detects the presence of BAG2 in a subject to diagnose cancer present in the subject's body and provides information that can be used to diagnose cancer.

[0043] The method may include contacting a sample isolated from a subject with an antibody that specifically binds to a BAG2 polypeptide or fragment thereof, and measuring a complex formed between the BAG2 polypeptide or fragment thereof and the antibody.

[0044] The method may further include determining whether the subject has cancer. The information may be information about whether the level of BAG2 measured in the subject is higher than, the same as, or lower than the level of BAG2 measured in a control group.

[0045] The sample may be a biological sample isolated from a subject to be diagnosed. The biological sample may be a cell, an organ, a cell lysate, whole blood, blood, serum, plasma, lymph, extracellular fluid, body fluid, urine, feces, tissue, bone marrow, saliva, sputum, spinal fluid, or a combination thereof.

[0046] The sample may be blood, serum, plasma, or a combination thereof. The present inventors have demonstrated that BAG2 is secreted from cells and that BAG2 can actually be detected in the serum of breast cancer patients by using anti-BAG2 antibodies or their antigen-binding fragments. Therefore, BAG2 may be soluble in blood, serum, plasma, or a combination thereof.

[0047] If BAG2 is present in a sample isolated from a subject, an antibody that specifically binds to a BAG2 polypeptide or a fragment thereof may bind to BAG2 in the sample. The antibody may be labeled, for example, with a fluorophore, chromophore, or an enzyme capable of converting a substrate to a chromophore, e.g., to visualize the presence of BAG2 in the sample.

[0048] Due to the binding reaction, BAG2 in the sample forms a complex with an antibody that specifically binds to the BAG2 polypeptide or a fragment thereof, and the presence or, if desired, the level of BAG2 can be identified or measured from the complex using methods known to those skilled in the art. The complex can be measured by Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation, FAGS, protein chip, or a combination thereof. Measurements can be performed to measure the level of the complex in the sample.

[0049] The control group may be a sample collected from a healthy subject, or a sample collected from breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, or diffuse large B-cell lymphoma (DLBCL). Thus, the BAG2 level of the control group may be the average concentration of BAG2 in samples collected from healthy subjects, or, if necessary, samples collected from breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, or DLBCL.

[0050] The sample used as the control may be of the same type and collected at the same anatomical location as the diagnostic sample. For example, if the sample is blood collected from the median cubital vein of a subject, the control may be blood collected from the median cubital vein of a control group.

[0051] A healthy subject is a subject who is not suffering from any acute or chronic disease, at least cancer, such as breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, or DLBCL.

[0052] The BAG2 level in a sample collected from a healthy subject may be substantially free of BAG2. Therefore, in an example where the control group is set to have values ​​obtained from healthy subjects, if the subject to be diagnosed is found to have BAG2 or the level of BAG2 in that subject is significantly high, the subject may be suspected of having cancer. On the other hand, as shown in Section 3 of Example 2 herein, the probability of identifying the presence of BAG2 in the blood of metastatic breast cancer patients, for example, TNBC-type breast cancer patients, is high, and the average value of BAG2 is significantly high (Figures 7 and 8). Therefore, in an example where the control group is set to samples isolated from breast cancer patients, non-metastatic breast cancer patients, or non-TNBC-type breast cancer patients, if the level of BAG2 is determined to be significantly high in the subject to be diagnosed, the subject may be suspected of having metastatic breast cancer or TNBC-type breast cancer.

[0053] Thus, in one aspect, the present invention is directed to a method for detecting the presence of cancer in an individual, comprising contacting a biological sample isolated from the individual with an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof, and measuring a complex formed between the BAG2 polypeptide or fragment thereof and the antibody or antigen-binding fragment thereof, wherein the presence of cancer is detected if the level of BAG2 measured in the sample is higher than the level of BAG2 measured in a negative control group.

[0054] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0055] The present invention will be more fully understood from the detailed description given herein below and the accompanying drawings, which are illustrative only and therefore not limiting of the invention. [Brief explanation of the drawings]

[0056] [Figure 1] 1 shows the results of Western blotting of anti-BAG2 antibodies produced from 10 types of mouse hybridoma cells. [Figure 2A] 1 shows the results of Western blotting of the full-length BAG2 polypeptide with anti-BAG2 antibodies or fragments thereof. [Figure 2B] 1 shows the results of Western blotting of the full-length BAG2 polypeptide with anti-BAG2 antibodies or fragments thereof. [Figure 3] The BAG2 domains that react with each anti-BAG2 antibody are shown. [Figure 4] A standard curve of BAG2 protein in 90 possible combinations of anti-BAG2 antibodies is shown. [Figure 5] 1 shows a diagram showing the specific binding avidity of 14 selected antibody combinations for BAG2 protein. [Figure 6] 1 shows the differences in BAG2 expression patterns in various cancer cell lines observed using the antibody combinations 2A11-3F12 and 9B12-3G8. [Figure 7] 1 shows the significant difference in BAG2 protein expression in the serum of luminal and TNBC breast cancer patients observed using the antibody combination 2A11-3F12 and the antibody combination 9B12-3G8. [Figure 8] 1 shows the significant difference in BAG2 protein expression in the serum of luminal and TNBC breast cancer patients observed by using the antibody combinations 8C4-3G8 and 10H7-3G8. [Figure 9A] Receiver operating characteristic curve (ROC) and area under the curve (AUC) values ​​for the four antibody combinations are shown. [Figure 9B] Receiver operating characteristic curve (ROC) and area under the curve (AUC) values ​​for the four antibody combinations are shown. [Figure 9C] Receiver operating characteristic curve (ROC) and area under the curve (AUC) values ​​for the four antibody combinations are shown. [Figure 9D] Receiver operating characteristic curve (ROC) and area under the curve (AUC) values ​​for the four antibody combinations are shown. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description of the Preferred Embodiments The following examples are offered by way of illustration of the present invention, but not by way of limitation.

[0058] Example Example 1: Selection of anti-Bag2 antibodies, sequencing and antigen-antibody reactions 1. Selection of monoclonal antibodies targeting bag2 and analysis of their amino acid sequences The present inventors selected antibodies that target Bag2, analyzed their amino acid sequences, and determined the complementarity-determining regions (CDRs) of each of the antibodies.

[0059] Specifically, a gene consisting of the nucleotide sequence of SEQ ID NO:70, encoding the human BAG2 protein consisting of the amino acid sequence of SEQ ID NO:69, was cloned into the pCAGGS plasmid and linearized. The linearized construct was then inoculated intramuscularly into five 6-week-old female BALB / c mice by electric shock. This construct was administered intramuscularly three times at 3-week intervals, consisting of 100 μg of DNA in 100 μl of PBS. A control plasmid was also administered in the same manner. To generate therapeutic and diagnostic antibodies, a DNA vaccine-based immunization strategy, which is more efficient than protein-based antigen injections, was implemented. Blood was collected from the vena cava or tail vein of the mice and tested by enzyme-linked immunosorbent assay to determine serum antibody titers. Spleens were removed from mice that showed sufficient antibody titers three days after the final immunization. B lymphocytes were isolated from the spleen and subsequently fused with myeloma cells cultured with the ATCC SP2 / 0-Ag14 cell line to obtain fused cells. The fused cells were then cultured in HAT medium containing hypoxanthine, aminopterin, and thymidine, and hybridoma cells fused only with myeloma and B lymphocytes were obtained by selecting approximately 130 clones. From the hybridoma cells obtained through the immunoblotting selection process, 10 hybridoma cells producing antibodies specifically binding to human BAG2 protein were obtained.

[0060] Anti-Bag2 antibody total RNA was added to 5 × 10 65'-RACE cDNA was generated from 100 ng of total RNA from hybridoma cells using the SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions. The regions encoding the heavy chain variable region (VH) and light chain variable region (VL) were amplified by PCR, and the amplified genes were inserted into the pGEM-T vector (Promega, USA) for cloning. The nucleotide sequences were analyzed using an automated genetic analyzer (ABI Prism 310, Applied Biosystems Co.). The nucleotide sequences of the analyzed genes were identified by comparison with previously reported nucleotide sequences, and the identified nucleotide sequences were artificially translated for use in determining the sequences of the complementarity-determining regions VH-CDR1, -CDR2, and -CDR3, and VL-CDR1, -CDR2, and -CDR3. Complementarity determining region sequencing was performed by using the Kabat database (http: / / www.bioinf.org.uk / abs / ).

[0061] As a result, 10 anti-BAG2 antibodies that specifically bind to BAG2 were obtained from the hybridoma cells. The 10 anti-BAG2 antibodies were 2A11, 4C2, 8C4, 3B5, 9B3, 9B12, 3B10, 10H7, 3GB, and 3F12. In addition, the amino acid sequences of the heavy chain variable region, light chain variable region, and their complementarity-determining regions, as well as the nucleotide sequences of the genes encoding the antibodies, shown in Tables 1 to 3, were determined.

[0062] The 2A11, 4C2, and 8C4 antibodies comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:21 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:27. In the 2A11 antibody, Xaa at positions 56 and 57 of SEQ ID NO:21 are Gly, respectively, and Xaa at position 53 of SEQ ID NO:27 is lie. In the 4C2 antibody, Xaa at positions 56 and 57 of SEQ ID NO:21 are Gly and Ala, respectively, and Xaa at position 53 of SEQ ID NO:27 is Phe. In the 8C4 antibody, Xaa at positions 56 and 57 of SEQ ID NO:21 are Ala and Gly, respectively, and Xaa at position 53 of SEQ ID NO:27 is Phe.

[0063] The VH-CDR1, -CDR2, and -CDR3 of the 2A11, 4C2, and 8C4 antibodies consist of the amino acid sequences of SEQ ID NOs: 33, 39, and 45, respectively, and the VL-CDR1, -CDR2, and -CDR3 consist of the amino acid sequences of SEQ ID NOs: 51, 57, and 63, respectively. For the 2A11 antibody, Xaa at positions 56 and 57 of SEQ ID NO: 21 are Gly. For the 4C2 antibody, Xaa at position 56 of SEQ ID NO: 21 is Gly and Xaa at position 57 is Ala. For the 8C4 antibody, Xaa at position 56 of SEQ ID NO: 21 is Ala and Xaa at position 57 is Gly.

[0064] The 9B3, 9B12, and 3B10 antibodies comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:23 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:29. For the 9B3 antibody, in SEQ ID NO:23, Xaa at position 1 is Glu, Xaa at position 7 is Ser, Xaa at position 12 is Val, Xaa at position 27 is Tyr, Xaa at position 58 is Ser, Xaa at position 61 is Asn, Xaa at position 74 is Lys, Xaa at position 83 is Phe, Xaa at position 92 is Ala, and Xaa at position 108 is Tyr. For the 9B3 antibody, in SEQ ID NO:29, Xaa at position 1 is Glu, Xaa at position 7 is Ser, Xaa at position 12 is Val, Xaa at position 27 is Tyr, Xaa at position 58 is Ser, Xaa at position 61 is Asn, Xaa at position 74 is Lys, Xaa at position 83 is Phe, Xaa at position 92 is Ala, and Xaa at position 108 is Tyr. 9 The th Xaa is ll e, 51 The 7th Xaa is Ala, 9 The 10th Xaa is Glu, 6 The th Xaa is ll For the 9B12 antibody, in SEQ ID NO:23, Xaa at position 1 is Gin, Xaa at position 7 is Ser, Xaa at position 12 is Val, Xaa at position 27 is Tyr, Xaa at position 58 is Ser, Xaa at position 61 is Asn, Xaa at position 74 is Arg, Xaa at position 83 is Phe, Xaa at position 92 is Gly, and Xaa at position 108 is His. For the 9B12 antibody, in SEQ ID NO:29, Xaa at position 1 is Gin, Xaa at position 7 is Ser, Xaa at position 12 is Val, Xaa at position 27 is Tyr, Xaa at position 58 is Ser, Xaa at position 61 is Asn, Xaa at position 74 is Arg, Xaa at position 83 is Phe, Xaa at position 92 is Gly, and Xaa at position 108 is His. 9 Xaa is Met, 51 The 7th Xaa is Ala, 9 The 10th Xaa is Glu, 6 For the 3B10 antibody, the first Xaa is Met. lXaa at position 7 is Pro, Xaa at position 12 is Ala, Xaa at position 27 is His, Xaa at position 58 is Thr, Xaa at position 61 is Ser, Xaa at position 74 is Arg, Xaa at position 83 is Leu, Xaa at position 92 is Gly, and Xaa at position 108 is His. 9 In the third, Xaa is Met, 51 The 7th Xaa is Ser, 9 The 10th Xaa is Asp, 6 The th Xaa is ll e.

[0065] For the 9B3, 9B12 and 3B10 antibodies, VH-CDR1, -CDR2 and -CDR3 consist of the amino acid sequences of SEQ ID NOS:35, 41 and 47, respectively, and VL-CDR1, -CDR2 and -CDR3 consist of the amino acid sequences of SEQ ID NOS:53, 59 and 65, respectively. For the 9B3 antibody, the second Xaa of SEQ ID NO:35 is Tyr, the eighth Xaa of SEQ ID NO:41 is Ser, the twelfth Xaa of SEQ ID NO:47 is Tyr, the third Xaa of SEQ ID NO:53 is Ie, and the second Xaa of SEQ ID NO:59 is Ala. For the 9B12 antibody, the second Xaa of SEQ ID NO:35 is Tyr, the eighth Xaa of SEQ ID NO:41 is Ser, the twelfth Xaa of SEQ ID NO:47 is His, the third Xaa of SEQ ID NO:53 is Met, and the second Xaa of SEQ ID NO:59 is Ala. For the 3B10 antibody, the second Xaa of SEQ ID NO:35 is His, the eighth Xaa of SEQ ID NO:41 is Thr, the twelfth Xaa of SEQ ID NO:47 is His, the third Xaa of SEQ ID NO:53 is Met, and the second Xaa of SEQ ID NO:59 is Ser. [Table 1] [Table 2] [Table 3]

[0066] 2. Identification of antigen-antibody responses of anti-BAG2 antibodies in breast cancer cells Figure 1 shows the results of immunoblotting of anti-BAG2 antibodies generated from ten mouse hybridoma cells. Specifically, human breast cancer cells, MDA-MB-231 cells, were cultured at 37°C in DMEM (Welgene) medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells were detached from the wells, washed with PBS, and lysed in a lysis buffer containing 1% Brij97, 5 mM EDTA, 0.02 M HEPES pH 7.3, 0.15 M NaCl, 1 mM PMSF, 0.5 mM NaF, 10 μg / ml aprotinin, and 0.2 mM sodium orthovanadate. After 15 minutes of incubation on ice, nuclei were removed from the cells by centrifugation, and the supernatant was collected. A 2X sample buffer consisting of 20% glycerol, 4.6% SOS, 0.125 M Tris pH 6.8, and 0.1% bromophenol blue was added to an appropriate amount of supernatant. Ten μg of protein samples were subjected to SOS-PAGE analysis on a 12% gel under standard conditions using the mini-Protean II system (Bio-Rad, Hercules, CA). For immunoblotting, proteins were transferred onto Millipore PVDF membranes. A blocking solution consisting of 0.1% Tween 20 and 5% bovine serum albumin (BSA) in TBS was added for 1 hour. The primary antibody was anti-BAG2 antibody extracted from hybridoma cell culture at a 1 / 2000 dilution, and the secondary antibody was a goat anti-mouse HRP conjugate (Dako) at a 1 / 5000 dilution. Film exposure was performed in the dark using EGL reagent (Amersham Pharmacia Biotech) as a substrate. The exposed bands were compared with standard molecular markers to identify the band corresponding to the size of BAG2.

[0067] As shown in Figure 1, antibodies 2A11, 3B5, 3B10, 3F12, 3GB, 4C2, 8C4, 9B3, 9B12, and 10H7 showed antigen-antibody reactions targeting BAG2, compared with ab58682 (Abcam), a commercially available polyclonal anti-BAG2 antibody used as a positive control.

[0068] Next, for domain mapping of the BAG2 antigen, for which the 10 antibodies were identified in Section 1 above, cells transfected with a GST-empty vector (pcDNA3.1+ / GST vector, NovoPro Bioscience Inc., China) having a molecular weight of approximately 26 kDa were used as a negative control. Cells were transfected with the GST-Bag Full vector, GST-Bag F1 vector, GST-Bag F2 vector, GST-Bag F3 vector, and GST-Bag F4 vector, each containing a polynucleotide encoding the human BAG2 protein and a polynucleotide encoding a fragment of the BAG2 protein, respectively. The transfected cells were cultured to express the gene, and then cell lysates were obtained. Immunoblotting of the cell lysates was performed using each of the 10 antibodies. The polynucleotide encoding the human BAG2 protein has the nucleotide sequence of SEQ ID NO:70. The GST-Bag F1, -Bag F2, -Bag F3, and -Bag F4 vectors consist of the nucleotide sequences of SEQ ID NOs: 71 to 74, respectively.

[0069] Figures 2A and 2B show the results of immunoblotting of the full-length BAG2 polypeptide using anti-BAG2 antibodies or fragments thereof. In Figure 2, A shows a diagram of the vector and the BAG2 protein and its fragments, and B shows the results of immunoblotting. Specifically, immunoblotting was performed as follows: each of the vectors was introduced into HEK2T cells by Lipofectamine transfection (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA). The resulting transformed cells were cultured in DMEM (Welgene) medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C for 30 hours, after which the cells were isolated. The isolated cells were disrupted using the same method as described in connection with Figure 1 and subjected to SOS-PAGE analysis on a 12% gel. For immunoblotting, cells were incubated for 1 hour in the same blocking solution as described in Figure 1. Ten purified anti-BAG2 antibodies, each at a concentration of 2 mg / ml, were used as primary antibodies at a 1 / 10,000 dilution and bound to the cells. A goat anti-mouse HRP conjugate was used as the secondary antibody at a 1 / 5,000 dilution, and film exposure was performed in the dark using EGL reagent (Amersham Pharmacia Biotech) as the substrate. Standard molecular marker size expression was performed to confirm the size of BAG2.

[0070] As shown in Figure 2, each anti-BAG2 antibody differentially bound to the full-length BAG2 polypeptide or its fragments. In particular, signals were commonly detected at approximately 50 kDa for each of the 10 anti-BAG2 antibodies in lysates from GST-Bag Full vector-transfected cells. This result indicates that all of these antibodies can bind to the full-length BAG2 antibody. Finally, the domain region of BAG2 to which each BAG2 antibody reacted was identified.

[0071] Figure 3 shows the BAG2 domains that react with each anti-BAG2 antibody. As shown in Figure 3, the 9B3, 9B12, 3B10, and 10H7 antibodies were bound to the N-terminus of the BAG2 protein; the 2A11, 3B5, 4C2, and 8C4 antibodies were bound to the central region of the BAG2 protein; and the 3F12 and 3GB antibodies were bound to the C-terminus of the BAG2 protein. The N-terminus commonly contains a coiled-coil region of 21 to 60 amino acids, and the central region is bound to a portion of the BNB region of 109 to 189 amino acids. Therefore, by using a set of antibodies that bind to different sites, the BAG2 protein or its fragments present in a sample can be detected with high sensitivity and specificity.

[0072] Example 2: Screening of anti-BAG2 antibody combinations and confirmation of their efficacy in cancer diagnosis

[0073] 1. Screening of combinations of anti-BAG2 antibodies useful for cancer diagnosis Figure 4 shows the standard curves of BAG2 protein for 90 possible combinations of anti-BAG2 antibodies. As a result, as shown in Figure 4, 14 antibody combinations showing high-slope standard curves were selected from the possible combinations of anti-BAG2 antibodies: 2A11-3F12, 10H7-3G8, 9B12-3G8, 10H7-3F12, 9B8-3G8, 3G8-3F12, 3B5-3F12, 3G8-4C2, 3F12-3G8, 3B5-3G8, 3B5-4C2, 3G8-8C4, and 9B3-3F12.

[0074] Figure 5 shows a graph depicting the specific binding activity of 14 selected antibody combinations for BAG2 protein. Specifically, a Myc-tagged BAG2 expression vector or a Myc-tagged empty vector was transfected into the human breast cancer cell line MDA-MB-231 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The relative amounts of secreted BAG2 protein from 30 μg of lysates of control cells transfected with the Myc-tagged empty vector and cells overexpressing BAG2 protein transfected with the Myc-tagged BAG2 expression vector were determined using the 14 antibody combinations. Control cells were labeled with EV, and BAG2-overexpressing cells were labeled with OE. Thus, the specific binding activity of the antibody combinations for BAG2 protein can be determined by determining the relative amounts of secreted BAG2 protein from BAG2-overexpressing cells and control cells.

[0075] As a result, as shown in Figure 5, four antibody combinations with high specific binding ability to the BAG2 protein, 2A11-3F12, 9B12-3G8, 8C4-3G8, and 10H7-3F12, were selected.

[0076] 2. Identifying the efficacy of selected anti-BAG2 antibody combinations in the diagnosis of various cancers To confirm the cancer diagnostic efficacy of the antibody combination selected in Section 1, the antigen-antibody reaction between the antibody combination and BAG2 was confirmed. Specifically, human BAG2 protein with six histamine residues attached to the N-terminus, which was produced and purified from human 293T cells, was used as the antigen.

[0077] Sandwich enzyme-linked immunosorbent assays (sandwich ELISA) were performed with each of the 2A11-3F12 and 9B12-3G8 antibody combinations.

[0078] Specifically, sandwich ELISA was performed through the following steps: 1 mg of each of antibodies 3F12 and 3G8 was reacted with 27 μl of 10 mM NHS-biotin (succinimidyl biotin, Thermo Scientific, Cat. #21435) to prepare biotin-conjugated detection antibodies. Each of antibodies 2A11, 9B12, 8C4, and 10H7 was diluted to a concentration of 3 μg / ml using ELISA plate coating buffer (R&D System, DY006) and applied to each well of a 96-well plate in an amount of 100 μl, followed by overnight coating at 4°C. The antibody-coated plate was blocked with 1% bovine serum albumin (BSA) buffer (R&D System, DY995) at room temperature for 1 hour. For the BAG2 standard, human His-tagged BAG2 recombinant protein (Ybiologics, Korea) produced and purified in the human cell line HEK293T was prepared at a concentration of 100 ng / ml using 1% BSA buffer. It was then diluted four-fold to prepare standard solutions of 0.02, 0.097, 0.31, 1.56, 6.25, and 25 ng / ml. For blood samples, 8 ml or more of blood was collected from 14 healthy volunteers, 20 patients diagnosed with luminal breast cancer, and 38 patients diagnosed with TNBC breast cancer. The blood was mixed by gentle shaking and left at room temperature for 20–30 minutes. The blood was then centrifuged at 2,500 rpm for 10 minutes. Serum stored at <4°C was diluted half-fold with 1% BSA buffer. The blocking solution was removed from the plate, and 100 μl of 0, 0.02, 0.097, 0.39, 1.56, 6.25, 25, and 100 ng / ml BAG2 standard solutions and 100 μl of 1 / 2-fold diluted patient samples were dispensed into each well. The wells were incubated at room temperature for 1 hour, followed by washing with washing buffer (R&D system, WA126). 100 μl of 1:50,000 diluted streptavidin-HRP (Pierce, 21130) diluted with 1% BSA buffer was dispensed into each well. The wells were incubated at room temperature for 30 minutes, followed by washing with washing buffer.100 μl of 3,3',5,5'-tetramethylbenzidine (TMB, R&D System, DY999) was dispensed into each well and reacted at room temperature for 15 minutes in a female cow. Then, a stop solution was added to stop the reaction. The concentration of BAG2 protein in the patient samples was calculated from the antibody standard curve obtained by measuring the absorbance of the reaction solution at 450 nm.

[0079] Figure 6 shows the differences in BAG2 expression patterns in cancer cell lines, including breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, and diffuse large B-cell lymphoma, observed by using anti-BAG2 antibody combinations. Figure 6A shows the results of sandwich ELISA for the 2A11-3F12 antibody combination, and Figure 6B shows the results of sandwich ELISA for the 9B12-3G8 antibody combination.

[0080] As shown in Figures 6A and 6B, when the 2A11-3F12 and 9B12-3G8 antibody combination was used, BAG protein was highly expressed in various cancer cell lines, including breast cancer cell lines MDA-MB-231 and Hs578T, pancreatic cancer cell lines SNU2564 and PANC1, glioblastoma multiforme (GBM) cell lines U251MG and T98G, gastric cancer cell lines SNU1 and SNU484, ovarian cancer cell lines SKOV3 and A2780, and diffuse large B-cell lymphoma (DLBCL) cell lines SU-DHL4 and SU-DHL6. In contrast, BAG2 protein was not expressed or was barely expressed in normal cell lines T47D, SNU2469, A172, SNU620, OVCAR3, and U2932. In other words, it was confirmed that BAG2 protein is significantly more highly expressed in cancer cells, including breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, and diffuse large B-cell lymphoma, compared to normal cells, by using the 2A11-3F12 and 9B12-3G8 antibody combination.

[0081] Therefore, when the above antibody combinations are used, cancer can be effectively diagnosed by identifying whether the expression pattern of BAG2 protein is similar to the expression pattern of BAG2 protein exhibited in cancer cells compared to normal cells.

[0082] 3. Identification of the efficacy of selected anti-BAG2 antibody combinations in the diagnosis of breast cancer The difference in the expression pattern of BAG2 protein in serum between patients with luminal breast cancer, patients with TNBC breast cancer, and normal subjects was confirmed using the same sandwich ELISA method described in Section 2. Specifically, serum was collected from healthy volunteers (N = 4), patients with luminal breast cancer (N = 4), and patients with TNBC breast cancer (N = 38), and the expression pattern of BAG2 protein in the obtained serum was confirmed.

[0083] Figures 7A and 7B and 8A and 8B show significant differences in BAG2 protein expression in the serum of luminal and TNBC breast cancer patients identified by using anti-BAG2 antibody combinations. Figure 7A shows an example using the 9B12-3G8 antibody combination, and Figure 7B shows an example using the 2A11-3F12 antibody combination. Figure 8A shows an example using the 8G4-3G8 antibody combination, and Figure 8B shows an example using the 10H7-3G8 antibody combination.

[0084] As shown in Figures 7A and 7B and 8A and 8B, when the 9B12-3G8, 2A11-3F12, 8G4-3G8, and 10H7-3F12 antibody combinations were used, the p-values ​​for each antibody combination were p<0.0001, p=0.0373, p=0.0009, and p=0.0190, respectively, indicating that there were significant differences in the BAG2 protein expression patterns in the serum of luminal and TNBC breast cancer patients compared with normal subjects. Therefore, breast cancer can be effectively diagnosed by observing significant differences in the BAG2 protein expression patterns using antibody combinations.

[0085] 4. Confirmation of the usefulness of selected anti-BAG2 antibody combinations in breast cancer diagnosis To confirm the sensitivity and specificity of cancer diagnosis using each of the four antibody combinations, the results in Section 2 were presented using ROG curves.

[0086] Figures 9A to 90 show the receiver operating characteristic curves (ROC) and area under the curve (AUC) for each antibody combination. Figure 9A shows an example using the 9B12-3G8 antibody combination, Figure 9B shows an example using the 8G4-3G8 antibody combination, Figure 9G shows an example using the 2A11-3F12 antibody combination, and Figure 90 shows an example using the 10H7-3F12 antibody combination.

[0087] As shown in Figures 9A-90, when the 9B12-3G8, 8G4-3G8, 2A11-3F12, and 10H7-3G8 antibody combinations were used, the AUG values ​​were 0.8596, 0.8368, 0.8554, and 0.6736. That is, for all antibody combinations, the AUG values ​​in the ROG curves were approximately 0.7, and when the 9B12-3G8, 8G4-3G8, and 2A11-3F12 antibody combinations were used, the AUG values ​​were measured to be 0.8-0.9. These results demonstrate that each antibody combination can be used to diagnose breast cancer by utilizing high sensitivity and specificity. Therefore, these four antibody combinations can be beneficially used to diagnose breast cancer.

[0088] A composition for use in diagnosing cancer comprising an anti-BAG2 antibody or antigen-binding fragment thereof according to one embodiment may provide information that can be used to diagnose cancer.

[0089] According to another aspect of the method for providing information used to diagnose cancer, unlike conventional diagnostic methods that require tissue collection, the presence or level of BAG2 polypeptide in blood can be identified or measured from blood, and the diagnostic utility is high. Therefore, various tissues can be used in the method for diagnosing cancer.

[0090] The following deposits are international deposits under the Budapest Treaty: [Accession number] Deposited at: Korea Research Institute of Bioscience and Biotechnology Accession number: KCTC13737BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13738BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13739BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13740BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13741BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13742BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13743BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13744BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13745BP Deposit date: November 28, 2018 Deposited at: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13746BP Deposit date: November 28, 2018

[0091] All references cited herein are incorporated by reference in their entirety.

[0092] TIFF0007747268000004.tif487 Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein.

Claims

1. 1. A composition for use in diagnosing cancer comprising a combination of antibodies or antigen-binding fragments thereof that specifically bind to a BAG2 polypeptide or a fragment thereof, The combination is (i) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising a complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas in SEQ ID NO: 39 are Gly), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light-chain variable region comprising a complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; (ii) a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35 (wherein the second Xaa of SEQ ID NO: 35 is Tyr), a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41 (wherein the eighth Xaa of SEQ ID NO: 41 is Ser), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47 (wherein the twelfth Xaa of SEQ ID NO: 47 is His); and a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53 (wherein the third Xaa of SEQ ID NO: 53 is Met), a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59 (wherein the second Xaa of SEQ ID NO: 59 is Ala), and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 59 (wherein the second Xaa of SEQ ID NO: 59 is Ala). and a light chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; (iii) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising a complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas of SEQ ID NO: 39 are Ala and Gly, respectively), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light-chain variable region comprising a complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; or (iv) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 48; and a light-chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 60, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 66; and a light chain variable region comprising a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62 and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; Including, composition.

2. The combination is the antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 in which the 6th Xaa and the 7th Xaa are each Gly, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and the antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 44, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50; and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; The composition of claim 1 comprising:

3. The combination is the antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region including a VH-CDR1 having the amino acid sequence of SEQ ID NO: 35 in which the second Xaa is Tyr, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 41 in which the eighth Xaa is Ser, and a VH-CDR3 having the amino acid sequence of SEQ ID NO: 47 in which the twelfth Xaa is His; and a light chain variable region including a VL-CDR1 having the amino acid sequence of SEQ ID NO: 53 in which the third Xaa is Met, a VL-CDR2 having the amino acid sequence of SEQ ID NO: 59 in which the second Xaa is Ala, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 65; and the antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49; and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; The composition of claim 1 comprising:

4. The combination is the antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 in which the 6th Xaa and the 7th Xaa are Ala and Gly, respectively, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and the antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49; and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; The composition of claim 1 comprising:

5. The composition of claim 1 , wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

6. The composition of claim 1 , wherein the antibody or antigen-binding fragment thereof is labeled with a detectable label or a label capable of emitting a detectable signal.

7. 2. The composition of claim 1, wherein the antibody or antigen-binding fragment thereof is produced by a hybridoma cell selected from the hybridoma cells deposited under accession numbers KCTC 13737BP, KCTC 13740BP, KCTC 13741BP, KCTC 13743BP, KCTC 13745BP, and KCTC 13746BP.

8. 2. The composition of claim 1, wherein the cancer is selected from breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndrome (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

9. A kit for use in diagnosing cancer, comprising the composition of any one of claims 1 to 8.

10. 1. A method for providing information used to diagnose cancer in a subject, comprising contacting a sample isolated from said subject with a combination of antibodies or antigen-binding fragments thereof that specifically bind to a BAG2 polypeptide or fragment thereof, and measuring the complexes formed between said BAG2 polypeptide or fragment thereof and said antibodies or antigen-binding fragments thereof, The combination is (i) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising a complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas in SEQ ID NO: 39 are Gly), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light-chain variable region comprising a complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; (ii) a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35 (wherein the second Xaa of SEQ ID NO: 35 is Tyr), a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41 (wherein the eighth Xaa of SEQ ID NO: 41 is Ser), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47 (wherein the twelfth Xaa of SEQ ID NO: 47 is His); and a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53 (wherein the third Xaa of SEQ ID NO: 53 is Met), a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59 (wherein the second Xaa of SEQ ID NO: 59 is Ala), and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 59 (wherein the second Xaa of SEQ ID NO: 59 is Ala). and a light chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; (iii) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising a complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas of SEQ ID NO: 39 are Ala and Gly, respectively), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light-chain variable region comprising a complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; or (iv) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 48; and a light-chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 60, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 66; and a light chain variable region comprising a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62 and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; Including, method.

11. The method of claim 10, wherein the sample is a biological sample.

12. 12. The method of claim 11, wherein the biological sample is a cell, an organ, a cell lysate, whole blood, blood, serum, plasma, lymph, extracellular fluid, body fluid, urine, feces, tissue, bone marrow, saliva, sputum, spinal fluid, or a combination thereof.

13. 11. The method of claim 10, wherein the cancer is selected from breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndrome, myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

14. The method of claim 10, wherein the information is about whether the level of BAG2 measured in the subject is higher than, the same as, or lower than the level of BAG2 measured in a negative control group.

15. 1. A method for detecting the presence of cancer in an individual, comprising: contacting a biological sample isolated from said individual with a combination of antibodies or antigen-binding fragments thereof that specifically bind to a BAG2 polypeptide or fragment thereof; and measuring the complex formed between the BAG2 polypeptide or fragment thereof and the antibody or antigen-binding fragment thereof, wherein the presence of cancer is detected if the level of BAG2 measured in the sample is higher than the level of BAG2 measured in a negative control group. Including, The combination is (i) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising a complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas in SEQ ID NO: 39 are Gly), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light-chain variable region comprising a complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and the antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; (ii) a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35 (wherein the second Xaa of SEQ ID NO: 35 is Tyr), a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41 (wherein the eighth Xaa of SEQ ID NO: 41 is Ser), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47 (wherein the twelfth Xaa of SEQ ID NO: 47 is His); and a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53 (wherein the third Xaa of SEQ ID NO: 53 is Met), a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59 (wherein the second Xaa of SEQ ID NO: 59 is Ala), and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 59 (wherein the second Xaa of SEQ ID NO: 59 is Ala). and a light chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; (iii) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising a complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas of SEQ ID NO: 39 are Ala and Gly, respectively), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light-chain variable region comprising a complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; and an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; or (iv) the antibody or antigen-binding fragment thereof, comprising: a heavy-chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 48; and a light-chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 60, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 66; and a light chain variable region comprising a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62 and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; Including, method.

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