Methods for diagnosing cancer using Anti-bag2 antibody

Abstract

To provide a composition for use in diagnosing cancer.SOLUTION: A composition for use in diagnosing cancer comprises: an antibody or an antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] 2. 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. It has been reported that BAG2 has pro-apoptotic activity, such that expression of BAG2 increases proteasome inhibitor-induced apoptosis, and BAG2 knockdown partially inhibits apoptosis when thyroid cancer cells are 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 various mutant K-Ras-induced tumors, thereby promoting tumor development. However, despite these findings, the role of BAG2 in cancer progression and metastasis is not clearly known. Summary of the Invention [Problem to be solved by the invention]

[0003] Thus, there is a need to develop compositions and methods for diagnosing cancer using antibodies that specifically bind to a 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, the 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 of providing information for use in diagnosing 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. The amino acid sequence of SEQ ID NO:69 corresponds to NCBI reference SEQ ID NO:NM_004282.4. The BAG2 protein includes variants having 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 having 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] Antibody refers to a specific immunoglobulin against an antigenic site. Antibody refers to a polypeptide or combination of polypeptides that specifically binds to a BAG2 polypeptide or a fragment thereof. Antibody includes polyclonal, monoclonal, or recombinant antibodies, such as ScFv fragments, diabodies, single-chain antibodies, and the like, and includes all immunoglobulin antibodies. Antibody may include the complete form of an antibody with two full-length light chains and two full-length heavy chains, and may also include functional fragments of an antibody molecule that retain antigen-binding function by including a specific antigen-binding site, i.e., a binding domain, despite the absence of the structure of a complete intact antibody with 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, mu and epsilon each contain 550 amino acids. Heavy chains have two regions, the variable region and the constant region.

[0014] There are two types of light chains, kappa and lambda, which may contain amino acids from about 211 to about 217. 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] 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 (Lie). 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 including a VH-CDR1 having the amino acid sequence of SEQ ID NO:33, a VH-CDR2 having the amino acid sequence of SEQ ID NO:39 in which the 6th and 7th Xaas are Gly, and a VH-CDR3 having the amino acid sequence of SEQ ID NO:45, and a light chain variable region including a VL-CDR1 having the amino acid sequence of SEQ ID NO:51, a VL-CDR2 having the amino acid sequence of SEQ ID NO:57, and a VL-CDR3 having 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 including a VH-CDR1 having the amino acid sequence of SEQ ID NO:38, a VH-CDR2 having the amino acid sequence of SEQ ID NO:44, and a VH-CDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region including a VL-CDR1 having the amino acid sequence of SEQ ID NO:56, a VL-CDR2 having the amino acid sequence of SEQ ID NO:62, and a VL-CDR3 having the amino acid sequence of SEQ ID NO:68.

[0024] The antibody or antigen-binding fragment thereof includes an 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; or a light chain variable region 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: The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region having a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:49, and a light chain variable region having 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 a VH-CDR1 having the amino acid sequence of SEQ ID NO:33, a 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 a VH-CDR3 having the amino acid sequence of SEQ ID NO:45, and a light chain variable region including a VL-CDR1 having the amino acid sequence of SEQ ID NO:51, a VL-CDR2 having the amino acid sequence of SEQ ID NO:57, and a VL-CDR3 having the amino acid sequence of SEQ ID NO:63; or 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 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: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 an 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: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 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 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. 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.

[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 may be glutamine (Gln) or glutamic acid (Glu), Xaa at position 7 may be Ser or proline (Pro), Xaa at position 12 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. In SEQ ID NO:27, Xaa at position 53 may be Ile or Phe. In SEQ ID NO:29, Xaa at position 29 may be Met or Ile, Xaa at position 51 may be Ala or Ser, Xaa at position 79 may be Glu or aspartic acid (Asp), and Xaa at position 106 may be Met or Ile.

[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 substrate compound or composition that is detectable. The label may be an immunofluorescent label, a chemiluminescent label, a phosphorescent label, a radioactive label, an epitope tag, an avidin / biotin, 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 having tumorigenic potential that are the result of 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 immunized subjects. In this specification, hybridoma cells may be referred to as hybridoma cells or fusion cells.

[0034] The cancer may be a solid or non-solid cancer. 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 occurs in the blood, also called blood cancer. The cancer may be a carcinoma, a sarcoma, a cancer derived from hematopoietic cells, a germ cell tumor, or a blastoma. The cancer may be selected from, for example, 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, 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 anti-BAG2 antibodies or antigen-binding fragments thereof, particularly a combination of anti-BAG2 antibodies or antigen-binding fragments thereof selected by BAG2 domain screening, are used, the presence of BAG2 is identified or its level is significantly higher 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. Thus, 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 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 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 by the composition may be metastatic breast cancer.

[0037] Among the molecular subtypes of breast cancer, triple-negative breast cancer (TNBC) is a highly aggressive type, with poor prognosis and high mortality despite systemic treatment. TNBC is a heterogeneous type compared to luminal or HER2-enriched types. Although most breast cancer targeted therapies have shown positive outcomes for hormone receptor and HER2-positive breast cancer, TNBC patients lack three types of target receptors, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2), or other defined molecular targets, resulting in limited options for effective treatment, such as poly ADP-ribose polymerase (PARP), epidermal growth factor receptor (EGFR), Src tyrosine kinase, or similar. Therefore, it is necessary to quickly and accurately distinguish TNBC patients from various types of breast cancer patients in order 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 is diagnosed with TNBC type breast cancer is high.Therefore, the breast cancer that can be diagnosed by using the method can be triple negative breast cancer or TNBC type 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 combinations thereof, etc. For example, for detection of immune complexes of BAG2 in a sample by their respective specific antibodies, the kit may further comprise a substrate, a suitable 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 synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, a glass slide, or the like; the color 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 color 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 of providing information for use in diagnosing cancer.

[0042] The method detects the presence of BAG2 in a subject to diagnose cancer present in the subject, providing 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 a fragment thereof, and measuring a complex formed between the BAG2 polypeptide or a fragment thereof and the antibody.

[0044] The method may further include determining whether the subject has cancer. The information may be about whether the level of BAG2 measured in the subject is higher, 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 is actually detected by using anti-BAG2 antibody or its antigen-binding fragment in the serum of breast cancer patients. 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, for example, to visualize the presence of BAG2 in the sample.

[0048] Due to the binding reaction, BAG2 in the sample forms a complex with the antibody that specifically binds to the BAG2 polypeptide or a fragment thereof, from which the presence or, optionally, the level of BAG2 can be identified or measured by utilizing 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 taken 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 taken from healthy subjects, or, if desired, samples taken 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 taken at the same anatomical location as the diagnostic sample, for example, if the sample is blood collected from the median cubital vein of the subject, the control may be blood collected from the median palatine vein of a control group.

[0051] A healthy subject is a subject who is not affected by 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 the sample collected from a healthy subject may be substantially free of BAG2. Therefore, in the 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 the subject is significantly high, the subject may be suspected to have cancer. On the other hand, as shown in Section 3 of Example 2 herein, there is a high probability of identifying the presence of BAG2 in the blood of metastatic breast cancer patients, such as TNBC type patients among breast cancer patients, and the average value of BAG2 is significantly high (Figures 7 and 8). Therefore, in the 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 to have 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 become 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 description of the drawings]

[0056] [Figure 1] 1 shows the results of Western blotting of anti-BAG2 antibodies produced from 10 mouse hybridoma cells. [Figure 2A] 1 shows the results of Western blotting of anti-BAG2 antibodies or fragments thereof for the full-length BAG2 polypeptide. [Figure 2B] 1 shows the results of Western blotting of anti-BAG2 antibodies or fragments thereof for the full-length BAG2 polypeptide. [Diagram 3] The BAG2 domains that react with each anti-BAG2 antibody are shown. [Figure 4] 1 shows a standard curve of BAG2 protein in 90 possible combinations of anti-BAG2 antibodies. [Diagram 5] FIG. 1 shows a chart depicting the specific binding avidity of 14 selected antibody combinations for the BAG2 protein. [Figure 6] FIG. 1 shows the differences in BAG2 expression patterns in various cancer cell lines observed with the antibody combinations 2A11-3F12 and 9B12-3G8. [Figure 7] 1 shows the significant difference in BAG2 protein expression in serum of luminal and TNBC breast cancer patients observed by using the antibody combination 2A11-3F12 and the antibody combination 9B12-3G8. [Figure 8] 1 shows the significant difference in BAG2 protein expression in serum of luminal and TNBC breast cancer patients observed by using the antibody combination 8C4-3G8 and the antibody combination 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 PREFERRED EMBODIMENTS

[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] Working 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 targeting Bag2, analyzed their amino acid sequences, and determined the complementarity determining regions (CDRs) of each of the antibodies.

[0059] In detail, the 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 pCAGGS plasmid and linearized, and the linearized construct was then inoculated into the muscle of five 6-week-old female BALB / c mice by applying electric shock. The construct was inoculated intramuscularly three times at three-week intervals, consisting of 100 μg of DNA in 100 μl of PBS. At this time, the control plasmid was also subjected to the same procedure. To generate therapeutic and diagnostic antibodies, a DNA vaccine-based immunization strategy was carried out, which is more efficient than protein-based antigen injection. Blood was collected from the vena cava or tail vein of the mice and tested by enzyme immunoassay to show serum antibody titers, and spleens were removed from mice that showed sufficient antibody titers three days after the last immunization. B lymphocytes were isolated from the spleen, and the isolated B lymphocytes were then fused with myeloma cells cultured with ATCC's SP2 / 0-Ag14 cell line, thereby obtaining 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 selection process by immunoblotting, 10 hybridoma cells were obtained that produce antibodies that specifically bind to human BAG2 protein.

[0060] Anti-Bag2 antibody total RNA was added at 5 × 10 65'-RACE-cDNA was generated from 100ng of total RNA by using SMART RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions. The regions encoding the heavy chain variable region (VH) and the light chain variable region (VL) were amplified by PCR, the amplified genes were inserted into pGEM-T vector (Promega, USA), cloned, and their nucleotide sequences were analyzed by using an automatic gene analyzer (ABI Prism 310, Applied Biosystem 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 sequences were determined by using the Kabat database (http: / / www.bioinf.org.uk / abs / ).

[0061] As a result, ten anti-BAG2 antibodies that specifically bind to BAG2 were obtained from the hybridoma cells. The ten anti-BAG2 antibodies were 2A11, 4C2, 8C4, 3B5, 9B3, 9B12, 3B10, 10H7, 3GB, and 3F12 antibodies. In addition, the amino acid sequences of the heavy chain variable region, light chain variable region, and their complementarity determining regions shown in Tables 1 to 3, as well as the nucleotide sequences of the genes encoding the antibodies, 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, the 56th and 57th Xaa in SEQ ID NO:21 are Gly, respectively. For the 4C2 antibody, the 56th Xaa in SEQ ID NO:21 is Gly and the 57th Xaa is Ala. For the 8C4 antibody, the 56th Xaa in SEQ ID NO:21 is Ala and the 57th Xaa 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 29 is Ile, Xaa at position 51 is Ala, Xaa at position 79 is Glu, and Xaa at position 106 is Ile. 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 29 is Met, Xaa at position 51 is Ala, Xaa at position 79 is Glu, and Xaa at position 106 is Met. For the 3B10 antibody, in SEQ ID NO:23, Xaa at position 1 is Gln, Xaa 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. For the 3B10 antibody, in SEQ ID NO:29, Xaa at position 29 is Met, Xaa at position 51 is Ser, Xaa at position 79 is Asp, and Xaa at position 106 is Ile.

[0065] For the 9B3, 9B12 and 3B10 antibodies, the VH-CDR1, -CDR2 and -CDR3 consist of the amino acid sequences of SEQ ID NOS:35, 41 and 47, respectively, and the 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 lie 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 response of anti-BAG2 antibody in breast cancer cells FIG. 1 shows the results of immunoblotting of anti-BAG2 antibodies generated from 10 kinds of mouse hybridoma cells. Specifically, human breast cancer cells, MDA-MB-231 cells, were cultured at a temperature of 37° C. in DMEM (Welgene) medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin. The cells were detached from the wells, washed with PBS, and lysed in a lysis buffer solution 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 / rni aprotinin, and 0.2 mM sodium orthovanadate. After 15 minutes of incubation on ice, the nuclei were removed from the cells by centrifugation, and the supernatant was collected. 2X sample buffer consisting of 20% glycerol, 4.6% SOS, 0.125M Tris pH 6.8, 0.1% bromophenol blue was added to the appropriate amount of supernatant. 10 μg protein samples were subjected to SOS-PAGE analysis on 12% gel under standard conditions by using mini-Protean II system (Bio-Rad, Hercules, CA). For immunoblotting, proteins were transferred onto Millipore PVDF membrane. Blocking solution consisting of 0.1% Tween 20 and 5% bovine serum albumin (BSA) in TBS was reacted for 1 hour. The primary antibody was then 1 / 2000 dilution of anti-BAG2 antibody extracted from hybridoma cell culture, and goat anti-mouse HRP conjugate (Dako) used as secondary antibody was diluted 1 / 5000. 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 a result, 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] Then, for domain mapping of the BAG2 antigen against which the ten antibodies were identified in the previous section 1, cells transfected with a GST-empty vector (pcDNA3.1+ / GST vector, NovoPro Bioscience Inc., China) having a molecular weight of about 26 kDa were used as a negative control. GST-Bag Full vector, GST-Bag F1 vector, GST-Bag F2 vector, GST-Bag F3 vector, and GST-Bag F4 vector, each of which contains a polynucleotide encoding human BAG2 protein and a polynucleotide encoding a fragment of BAG2 protein, were transfected into cells, and the cells transfected with the vectors were cultured to express the genes, and then cell lysates were obtained. For the cell lysates, immunoblotting was performed with each of the ten antibodies. The polynucleotide encoding 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 of anti-BAG2 antibody or its fragments. In Figure 2, A shows the diagram of the vector and BAG2 protein and its fragments, and B shows the results of immunoblotting. In detail, immunoblotting was performed as follows: each of the vectors was introduced into HEK2T cells by Lipofectamine transfection (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA) method, and the resulting transformed cells were cultured in DMEM (Welgene) medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin at a temperature of 37°C for 30 hours, after which the cells were isolated. The isolated cells were disrupted using the same method as described in relation to Figure 1 and subjected to SOS-PAGE analysis on a 12% gel. For immunoblotting, after 1 hour of reaction in the same blocking solution as described in relation to Figure 1, ten purified anti-BAG2 antibodies with a concentration of 2 mg / ml were used as primary antibodies at a dilution of 1 / 10000 to bind to the cells. Goat anti-mouse HRP conjugate was used as secondary antibody at a dilution of 1 / 5000, and film exposure was performed in the dark using EGL reagent (Amersham Pharmacia Biotech) as substrate. Standard molecular marker size was expressed to confirm the size of BAG2.

[0070] As a result, as shown in Figure 2, each anti-BAG2 antibody differentially bound to the full-length BAG2 polypeptide or its fragments. In particular, for each of the 10 anti-BAG2 antibodies, a signal was commonly detected at a position of about 50 kDa in the lysate of 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 reacts was identified.

[0071] FIG. 3 shows the BAG2 domains that react with each anti-BAG2 antibody. As shown in FIG. 3, 9B3, 9B12, 3B10, and 10H7 antibodies were bound to the N-terminus of BAG2 protein, 2A11, 3B5, 4C2, and 8C4 antibodies were bound to the central region of BAG2 protein, and 3F12 and 3GB antibodies were bound to the C-terminus of 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 part 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 may be detected with high sensitivity and specificity.

[0072] Example 2: Screening of combinations of anti-BAG2 antibodies 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 in 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] FIG. 5 shows a chart showing the specific binding ability of the selected 14 antibody combinations for BAG2 protein. In detail, Myc-tagged BAG2 expression vector or Myc-tagged empty vector was transduced into human breast cancer cell line MDA-MB-231 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, respectively. The 14 antibody combinations were used to determine the relative amount of secreted BAG2 protein from 30 μg of lysate of control cells transfected with Myc-tagged empty vector and cells overexpressing BAG2 protein transfected with Myc-tagged BAG2 expression vector. The control cells were labeled with EV, and the BAG2 overexpressing cells were labeled with OE. That is, the specific binding ability of the antibody combination for BAG2 protein can be determined by confirming the relative amount of secreted BAG2 protein of BAG2 overexpressing cells and control cells.

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

[0076] 2. Identification of 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. In detail, human BAG2 protein with six histamine residues attached to the N-terminus, which was produced and purified from human 293T cells, was obtained as an antigen.

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

[0078] In detail, sandwich ELISA was carried out through the following steps. Antibodies 3F12 and 3G8, each in an amount of 1 mg, were reacted with 27 μl of 10 mM NHS-biotin (succinimidyl biotin, Thermo scientific, Cat#21435) to prepare biotin-conjugated detection antibodies. Antibodies 2A11, 9B12, 8C4, and 10H7, each as capture antibodies, were diluted to a concentration of 3 μg / ml by 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, and then coated overnight at a temperature of 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. As a BAG2 standard material, human His-tagged BAG2 recombinant protein (Ybiologics, Korea) produced and purified in the human-derived cell line HEK293T was prepared at a concentration of 100 ng / ml using 1% BSA buffer, and then diluted 4-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 each of 14 healthy subjects, 20 patients diagnosed with luminal breast cancer, and 38 patients diagnosed with TNBC breast cancer, and then mixed by gentle shaking and left at room temperature for 20 to 30 minutes. The blood was then centrifuged at 2,500 rpm for 10 minutes. Serum stored at less than 4°C was diluted 1 / 2-fold using 1% BSA buffer. 100μl of 0, 0.02, 0.097, 0.39, 1.56, 6.25, 25, and 100ng / ml BAG2 standard solution and 1 / 2 diluted patient sample were distributed into each well of the plate from which the blocking solution was removed, reacted at room temperature for 1 hour, and then washed with washing buffer (R&D system, WA126). 100μl of 1:50000 diluted streptavidin-HRP (Pierce, 21130) by using 1% BSA buffer was distributed into each well, reacted at room temperature for 30 minutes, and then washed with washing buffer.100 μl of 3,3',5,5'-tetramethylbenzidine (TMB, R&D system, DY999) was dispensed into each well and reacted in female cow at room temperature for 15 minutes, and then the reaction was stopped by adding stop solution to it. The concentration of BAG2 protein in the patient sample was calculated from the antibody standard curve obtained by measuring the absorbance of the reaction solution at 450 nm.

[0079] Figure 6 shows the difference 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 of the antibody combination 2A11-3F12, and Figure 6B shows the results of sandwich ELISA of the antibody combination 9B12-3G8.

[0080] As shown in Figures 6A and 6B, when the 2A11-3F12 and 9B12-3G8 antibody combination was used, BAG protein was expressed at high levels 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, BAG2 protein was confirmed to be 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 luminal breast cancer patients, TNBC breast cancer patients, and normal subjects was confirmed by using the same sandwich ELISA method described in Section 2. In detail, serum from healthy volunteers (N=4), luminal breast cancer patients (N=4), and TNBC breast cancer patients (N=38) was collected, and the expression pattern of BAG2 protein in the obtained serum was confirmed.

[0083] Figures 7A and 7B and 8A and 8B show the significant difference 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 of using 9B12-3G8 antibody combination, and Figure 7B shows an example of using 2A11-3F12 antibody combination. Figure 8A shows an example of using 8G4-3G8 antibody combination, and Figure 8B shows an example of using 10H7-3G8 antibody combination.

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

[0086] Figures 9A-90 show the receiver operating characteristic curve (ROC) and area under the curve (AUC) of each antibody combination. Figure 9A shows an example of using the 9B12-3G8 antibody combination, Figure 9B shows an example of using the 8G4-3G8 antibody combination, Figure 9G shows an example of using the 2A11-3F12 antibody combination, and Figure 90 shows an example of 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, in the examples of all antibody combinations, the AUG values ​​in the ROG curve 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 indicate that each antibody combination can be used to diagnose breast cancer by utilizing high sensitivity and specificity. Therefore, the 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 is used to diagnose cancer.

[0089] According to the method for providing information used for diagnosing cancer according to another embodiment, unlike the conventional diagnostic method of collecting tissue, 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 use 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 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13738BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13739BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13740BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13741BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13742BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13743BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13744BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13745BP Deposit date: November 28, 2018 Deposit: 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] TIFF2025071090000005.tif499 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. An antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof, The antibody or antigen-binding fragment thereof 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; or a heavy chain variable region including 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 Tyr); 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 Ile), 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). a light chain variable region comprising: a VL-CDR3 consisting of the amino acid sequence of ID NO: 65; Including, An antibody or antigen-binding fragment thereof.

2. A heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 27; or a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 29; The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. An antibody or antigen-binding fragment thereof described in claim 1, which is a monoclonal antibody.

4. An antibody or antigen-binding fragment thereof described in claim 1, which is attached with a detectable label or a label capable of emitting a detectable signal.

5. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is a Fab or scFv.

6. The antibody or antigen-binding fragment thereof described in claim 1, produced by a hybridoma cell selected from the hybridoma cells deposited under accession numbers KCTC 13743BP and KCTC 13744BP.

7. A polynucleotide encoding the antibody or antigen-binding fragment thereof described in claim 1.

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