Method for diagnosing cancer using anti-BAG2 antibodies

Abstract

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

Description

Background of the Invention 1. Technical field:

[0002] The present disclosure relates to a composition for diagnosing cancer, the composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof; and a method for providing information for diagnosing cancer.

[0003] 2. General background and prior art:

[0004] The Bcl-2-associated immortalization gene (BAG) family of co-chaperone proteins mediates a variety of physiological processes, including intracellular protein folding, stress response, neuronal differentiation, apoptosis, and cell proliferation, and functionally binds to various co-chaperone 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. BAG2's primary role in regulating proteins by inhibiting CHIP activity is through stabilizing chaperone-associated proteins such as PINK1 and CFTR, which are associated with neurodegenerative diseases and autosomal recessive genetic diseases. BAG2 has been reported to have pro-apoptotic activity in a manner such that BAG2 expression increases proteasome inhibitor-induced apoptosis, and when thyroid cancer cells are exposed to the proteasome inhibitor MG132, BAG2 knockdown can partially inhibit apoptosis. It has also been reported that in various mutant K-Ras-induced tumors, overexpression of BAG2 promotes the stabilization of the potent oncogene STK33 protein, thereby promoting tumor development. However, despite these findings, the role of BAG2 in cancer progression and metastasis remains unclear.

[0005] Therefore, there is a need to develop compositions and methods for diagnosing cancer using antibodies that specifically bind to BAG2 polypeptides or fragments thereof. Summary of the Invention

[0006] 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.

[0007] One aspect provides a composition for diagnosing cancer, comprising an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof.

[0008] Another aspect provides a kit for diagnosing cancer, the kit comprising the composition.

[0009] Another aspect provides a method of providing information for diagnosing cancer.

[0010] Additional aspects will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the presented embodiments of the disclosure.

[0011] One aspect provides a composition for diagnosing cancer, comprising an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof.

[0012] The BAG2 polypeptide can be derived from a mammal. The mammal can be human (Homo sapiens), mouse (Mus musculus), monkey, cow, or horse. BAG2 can comprise the amino acid sequence of SEQ ID NO:69. The amino acid sequence of SEQ ID NO:69 corresponds to the sequence of NCBI reference SEQ ID NO:NM_004282.4. The BAG2 protein includes variants that have bioequivalent activity to the amino acid sequence of SEQ ID NO:69, even though their amino acid sequences do not match the amino acid sequence of SEQ ID NO:69. The BAG2 polypeptide can comprise an amino acid sequence that has at least 60%, such as 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. The BAG2 protein can be a polypeptide having a sequence identical to 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. Throughout this specification, "polypeptide" and "protein" are used interchangeably.

[0013] Antibodies refer to specific immunoglobulins for antigenic sites. Antibodies refer to polypeptides or polypeptide combinations that specifically bind to BAG2 polypeptides or their fragments. Antibodies include polyclonal antibodies, monoclonal antibodies or recombinant antibodies, such as ScFv fragments, diabodies, single-chain antibodies, etc., and include all immunoglobulin antibodies. Antibodies may include antibodies in the complete form with two full-length light chains and two full-length heavy chains, and may also include functional fragments of their antibody molecules, which retain antigen-binding function due to the inclusion of specific antigen-binding sites (i.e., binding domains), despite the absence of the structure of a complete antibody in the complete form with two light chains and two heavy chains.

[0014] The antigen-binding fragment is a fragment of the entire structure of an immunoglobulin and refers to a portion of the polypeptide that contains a portion capable of binding to an antigen. For example, the antigen-binding fragment can be scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab')2, or a combination thereof.

[0015] There are five heavy chains, γ, δ, α, μ and ε, and the heavy chain can determine the type of antibody. α and γ each include 450 amino acids, and μ and ε each contain 550 amino acids. The heavy chain has two regions, namely the variable region and the constant region.

[0016] There are two types of light chains, kappa and lambda, and can include from about 211 amino acids to about 217 amino acids.A light chain can have a constant region and a variable region.

[0017] The antibody or antigen-binding fragment thereof may comprise: a heavy chain variable region (the heavy chain variable region comprises 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 (the light chain variable region comprises 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);

[0018] a heavy chain variable region (the heavy chain variable region comprises 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 (the light chain variable region comprises 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);

[0019] a heavy chain variable region (the heavy chain variable region comprises 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 (the light chain variable region comprises 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);

[0020] a heavy chain variable region (the heavy chain variable region comprises 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 (the light chain variable region comprises 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);

[0021] a heavy chain variable region (the heavy chain variable region comprises 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 (the light chain variable region comprises 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

[0022] 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.

[0023] The 6th and 7th Xaa in SEQ ID NO:39 can be glycine (Gly) or alanine (Ala). The second Xaa in SEQ ID NO:35 can be tyrosine (Tyr) or histidine (His). The 8th Xaa in SEQ ID NO:41 can be serine (Ser) or threonine (Thr). The 12th Xaa in SEQ ID NO:47 can be tyrosine (Tyr) or histidine (His). The third Xaa in SEQ ID NO:53 can be methionine (Met) or isoleucine (Ile). The second Xaa in SEQ ID NO:59 can be Ala or Ser.

[0024] The antibody or antigen-binding fragment thereof may be 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: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 and in which the 6th and 7th Xaas 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 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; or

[0025] 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: 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.

[0026] The antibody or antigen-binding fragment thereof may be 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: 35, wherein the second Xaa is Tyr, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, wherein the eighth Xaa is Ser, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47, wherein the twelfth Xaa is His; and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53, wherein the third Xaa is Met, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59, wherein the second Xaa is Ala, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 65; 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: 35, wherein the second Xaa is Tyr, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, wherein the eighth Xaa is Ser, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47 NO:37, a VH-CDR1 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.

[0027] The antibody or antigen-binding fragment thereof may be 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: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, wherein 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; 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: 45. 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.

[0028] The antibody or antigen-binding fragment thereof may be 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 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. The VL-CDR1 consists of the amino acid sequence of SEQ ID NO:55, the VL-CDR2 consists of the amino acid sequence of SEQ ID NO:61, and the VL-CDR3 consists of the amino acid sequence of SEQ ID NO:67.

[0029] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs: 21 to 26; a light chain variable region comprising an amino acid sequence selected from any one of SEQ ID NOs: 27 to 32; or the heavy chain variable region and the light chain variable region.

[0030] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 27; a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 28; a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 29; a heavy chain variable region of SEQ ID NO: 24 and a light chain variable region of SEQ ID NO: 30; a heavy chain variable region of SEQ ID NO: 25 and a light chain variable region of SEQ ID NO: 31; or a heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 32; or a combination thereof.

[0031] The 56th and 57th Xaa in SEQ ID NO: 21 may be Gly or Ala. In SEQ ID NO: 23, the 1st Xaa may be glutamine (Gln) or glutamic acid (Glu), the 7th Xaa may be Ser or proline (Pro), the 12th Xaa may be valine (Val) or alanine (Ala), the 27th Xaa may be Tyr or His, the 58th Xaa may be Ser or Thr, the 61st Xaa may be asparagine (Asn) or Ser, the 74th Xaa may be arginine (Arg) or lysine (Lys), the 83rd Xaa may be phenylalanine (Phe) or leucine (Leu), the 92nd Xaa may be Gly or Ala, and the 108th Xaa may be His or Tyr. The 53rd Xaa in SEQ ID NO: 27 may be Ile or Phe. In SEQ ID NO: 29, the 29th Xaa may be Met or Ile, the 51st Xaa may be Ala or Ser, the 79th Xaa may be Glu or aspartic acid (Asp), and the 106th Xaa may be Met or Ile.

[0032] The antibody or antigen-binding fragment thereof may be a monoclonal antibody.

[0033] The antibody or antigen-binding fragment thereof can be labeled with a detectable label or a label capable of emitting a detectable signal. The 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 itself can be detectable and catalyze the chemical modification of a detectable substrate compound or composition. The label can be an immunofluorescent label, a chemiluminescent label, a phosphorescent label, a radioactive label, an epitope tag, avidin / biotin, colloidal gold particles, colored particles, magnetic particles, chromophore labels, ECL labels, enzymes, and the like.

[0034] The antibody or antigen-binding fragment thereof may be produced by a hybridoma cell selected from the group consisting of 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.

[0035] The hybridoma cell refers to a hybrid cell with tumorigenicity produced by the artificial fusion of two cells, and generally speaking, can be used to continuously produce antibodies by fusing B cells and plasmacytoma cells isolated from an immune subject. In this specification, the hybridoma cell may be referred to as a hybridoma cell or a fusion cell.

[0036] The cancer can be a solid cancer or a non-solid cancer. Solid cancer refers to cancerous tumors that occur in organs such as the liver, lungs, breast, and skin. Non-solid cancer is a cancer that occurs in the blood and is also referred to as a blood cancer. The cancer can be a carcinoma, a sarcoma, a cancer derived from hematopoietic cells, a germ cell tumor, or a blastoma. The cancer can 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 tumors, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumors, 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.

[0037] The present inventors have discovered 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 can be identified and its level 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 cell lines is significantly higher than its level in normal cells. Therefore, the cancer diagnosed by using the composition can be selected from breast cancer, pancreatic cancer, glioma, gastric cancer, ovarian cancer, and lymphoma.

[0038] The present inventors have discovered that when using anti-BAG2 antibodies or antigen-binding fragments thereof, and in particular a combination of anti-BAG2 antibodies or antigen-binding fragments thereof, BAG2 is overexpressed in breast cancer patients compared to normal subjects, and when BAG2 levels are high, the breast cancer patients are more likely to have metastatic breast cancer. Therefore, the breast cancer diagnosed using the composition can be metastatic breast cancer.

[0039] Among the molecular subtypes of breast cancer, triple-negative breast cancer (TNBC) is a very aggressive type with a poor prognosis and high mortality despite systemic treatment. TNBC is a heterogeneous type compared to luminal or HER2-enriched types. Although most targeted breast cancer therapies have shown positive results against 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 clearly defined molecular targets, so limited effective treatment options need to be included, such as poly ADP-ribose polymerase (PARP), epidermal growth factor receptor (EGFR), Src tyrosine kinase, etc. Therefore, in order to prevent unnecessary treatment pathways and to select effective treatments for TNBC patients, it is necessary to quickly and accurately distinguish TNBC patients from patients with various types of breast cancer.

[0040] The present inventors have discovered that when using anti-BAG2 antibodies or antigen-binding fragments thereof, and in particular combinations of anti-BAG2 antibodies or antigen-binding fragments thereof, BAG2 is overexpressed in breast cancer patients, and when BAG2 levels are high, the probability of breast cancer patients being diagnosed with TNBC type breast cancer is high. Therefore, the breast cancer that can be diagnosed using the method can be triple-negative breast cancer or TNBC type breast cancer.

[0041] Another aspect provides a kit for diagnosing cancer, the kit comprising the composition.

[0042] The test kit may further include one or more other component compositions, solutions or devices that are suitable for the analytical method used by the test kit, such as Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation, FAGS, protein chip or a combination thereof. For example, in order to detect the immune complex of BAG2 in a sample using an antibody specific for each of them, the test kit may further include a substrate, a suitable buffer, a second antibody labeled with a coloring enzyme or a fluorescent substance, or a coloring substrate. The substrate can be a nitrocellulose membrane, a 96-well plate synthesized with polyethylene resin, a 96-well plate synthesized with polystyrene resin, a glass slide, etc.; the coloring enzyme can be peroxidase, alkaline phosphatase, etc.; the fluorescent substance can be fluorescein isothiocyanate (FITC), rhodamine B-isothiocyanate (RITC), etc.; and the coloring substrate can be 2,2'-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), tetramethylbenzidine (TMB), etc.

[0043] Another aspect provides a method of providing information for diagnosing cancer.

[0044] The method is a method of providing information for diagnosing cancer, wherein the presence of BAG2 in a subject is detected to diagnose the presence of cancer in the subject.

[0045] The method may comprise contacting a sample isolated from a subject with an antibody that specifically binds to a BAG2 polypeptide or a fragment thereof; and measuring the formed complex of the BAG2 polypeptide or fragment thereof and the antibody.

[0046] The method may further comprise determining whether the subject has cancer.The information may be information on whether the BAG2 level measured in the subject is higher, equal to, or lower than the BAG2 level measured in the control group.

[0047] The sample can be a biological sample isolated from a subject to be diagnosed. The biological sample can 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, cerebrospinal fluid, or a combination thereof.

[0048] The sample can be blood, serum, plasma or a combination thereof. The inventors have demonstrated that BAG2 is secreted extracellularly and that BAG2 is actually detected in the serum of breast cancer patients by using anti-BAG2 antibodies or their antigen-binding fragments. Therefore, BAG2 is soluble in blood, serum, plasma or a combination thereof.

[0049] When BAG2 is present in a sample isolated from a subject, an antibody that specifically binds to a BAG2 polypeptide or a fragment thereof can bind to the BAG2 in the sample. The antibody can be labeled with, for example, a fluorophore, a chromophore, or an enzyme that can convert a substrate into a chromophore, for example, to visualize the presence of BAG2 in the sample.

[0050] Due to the binding reaction, BAG2 in the sample forms a complex with the antibody that specifically binds to the BAG2 polypeptide or its fragment, and from the complex, if necessary, the presence or level of BAG2 can be identified or measured using methods known to those skilled in the art. The complex can be measured by Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip or a combination thereof. This measurement can be performed to measure the level of the complex in the sample.

[0051] The control group can be a sample taken 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). Therefore, the BAG2 level of the control group can be a sample taken from a healthy subject, or, if necessary, the mean value of the BAG2 concentration in a sample taken from a patient with breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer or DLBCL.

[0052] The sample used as a control may be of the same type as the sample collected from the same anatomical location for diagnosis. For example, when 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 the control group.

[0053] A healthy subject is a subject who does not suffer from any acute or chronic disease, at least cancer, such as breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer or DLBCL.

[0054] The BAG2 level in the sample collected from healthy subjects may not contain BAG2 substantially. Therefore, when control group is set to have the value obtained from healthy subjects, when finding that experimenter to be diagnosed has BAG2, or when BAG2 level is significantly high, described experimenter may be suspected of having cancer. On the other hand, as shown in the 3rd section of this specification embodiment 2, in the blood of metastatic breast cancer patient, in the blood of the TNBC type patient in breast cancer patient for example, the possibility of identifying the existence of BAG2 is high, and the mean value of BAG2 is significantly high (Fig. 7 and 8). Therefore, when control group is set to the sample separated from breast cancer patient, non-metastatic breast cancer patient or non-TNBC type breast cancer patient, when determining that the level of BAG2 is significantly high in experimenter to be diagnosed, described experimenter may be suspected of having metastatic breast cancer or TNBC type breast cancer.

[0055] Therefore, in one aspect, the present invention relates to a method for detecting the presence of cancer in an individual, the method 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 the complex formed of the BAG2 polypeptide or fragment thereof and the antibody or antigen-binding fragment thereof, wherein the presence of cancer is detected if the BAG2 measured in the sample is higher than the BAG2 level measured in a negative control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] This 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 Office upon request and payment of the necessary fee.

[0057] 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 do not limit the present invention, in which:

[0058] Figure 1 Shown are the results of Western blotting of anti-BAG2 antibodies produced from 10 mouse hybridoma cells;

[0059] Figure 2A and 2B The results of Western blotting of full-length BAG2 polypeptide or fragments thereof of anti-BAG2 antibodies are shown;

[0060] Figure 3 BAG2 domains reactive with corresponding anti-BAG2 antibodies are shown;

[0061] Figure 4 The standard curve of BAG2 protein in 90 possible combinations of anti-BAG2 antibodies is shown;

[0062] Figure 5 A graph showing the specific binding ability of 14 selected antibody combinations to the BAG2 protein;

[0063] FIG6 shows the differences in BAG2 expression patterns in various cancer cell lines observed using the antibody combination of 2A11-3F12 and the antibody combination of 9B12-3G8;

[0064] FIG7 shows the significant difference in BAG2 protein expression in the serum of patients with luminal and TNBC breast cancer observed using the antibody combination of 2A11-3F12 and the antibody combination of 9B12-3G8;

[0065] Figure 8 shows the significant difference in BAG2 protein expression in the serum of patients with luminal and TNBC breast cancer observed using the antibody combination of 8C4-3G8 and the antibody combination of 10H7-3G8; and

[0066] Figures 9A to 9D Receiver operating characteristic curve (ROC) values ​​and area under the curve (AUC) values ​​of the four antibody combinations are shown. DETAILED DESCRIPTION

[0067] The following examples are provided to illustrate the present invention and not by way of limitation.

[0068] Example

[0069] Example 1: Selection, sequencing and antigen-antibody reaction of anti-Bag2 antibodies

[0070] 1. Selection of Monoclonal Antibodies Targeting Bag2 and Their Amino Acid Sequence Analysis

[0071] The present inventors selected antibodies targeting bag2, analyzed their amino acid sequences, and determined the complementarity determining regions (CDRs) of each of the antibodies.

[0072] Specifically, the gene encoding the human BAG2 protein consisting of the nucleotide sequence of SEQ ID NO:70 and the amino acid sequence of SEQ ID NO:69 was cloned into the pCAGGS plasmid and linearized. The linearized construct was then inoculated into the muscles of five six-week-old female BALB / c mice by applying electric shock. The construct consisted of 100 μg of DNA in 100 μl of PBS and was administered intramuscularly three times at three-week intervals. A control group of mice was treated with the same plasmid. To generate therapeutic and diagnostic antibodies, a DNA-based immunization strategy, which is more effective than protein-based antigen injections, was implemented. Blood was collected from the vena cava or tail vein of the mice and tested by enzyme immunoassay, which indicates serum antibody titers. Three days after the last immunization, spleens were harvested from mice that exhibited sufficient antibody titers. B lymphocytes were isolated from the spleens and fused with myeloma cells (the ATCC SP2 / 0-Ag14 cell line) cultured with isolated B lymphocytes to obtain fused cells. After culturing the fused cells in HAT medium containing hypoxanthine, aminopterin, and thymidine, approximately 130 clones were selected to obtain hybridoma cells fused only with myeloma and B lymphocytes. Among the hybridoma cells obtained by immunoblotting selection, 10 hybridoma cells that produced antibodies that specifically bound to human BAG2 protein were obtained.

[0073] From 5x106 total RNA of individual hybridoma cell production anti-BAG2 antibody, and by using SMART RACE cDNA amplification test kit (Clontech) by 100ng total RNA production 5 '-RACE-cDNA according to the specification sheets of manufacturers.By pcr amplification heavy chain variable region (VH) and light chain variable region (VL) coding region, and the gene of amplification is inserted in pGEM-T carrier (Promega, USA), this gene is cloned, and by using automatic gene analyzer (ABI Prism 310, Applied Biosystem Co.) analyze the nucleotide sequence of this gene.By comparing with the nucleotide sequence of previous report, identify the nucleotide sequence of analyzed gene, and the nucleotide sequence identified by artificial translation is for determining the sequence of complementary determining region VH-CDR1 ,-CDR2 and-CDR3 and VL-CDR1 ,-CDR2 and-CDR3.Determining the sequence of complementary determining region is to use the database (http: / / www.bioinf.org.uk / abs / ) of Kabat to perform.

[0074] As a result, 10 anti-BAG2 antibodies that specifically bind to BAG2 were obtained from hybridoma cells. These 10 anti-BAG2 antibodies are 2A11, 4C2, 8C4, 3B5, 9B3, 9B12, 3B10, 10H7, 3GB, and 3F12 antibodies. Furthermore, the amino acid sequences of the heavy and light chain variable regions and their complementarity-determining regions, as shown in Tables 1 to 3, and the nucleotide sequences of the genes encoding these antibodies were determined.

[0075] 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, the 56th and 57th Xaa of SEQ ID NO: 21 are each Gly, and the 53rd Xaa of SEQ ID NO: 27 is Ile. In the 4C2 antibody, the 56th and 57th Xaa of SEQ ID NO: 21 are Gly and Ala, respectively, and the 53rd Xaa of SEQ ID NO: 27 is Phe. In the 8C4 antibody, the 56th and 57th Xaa of SEQ ID NO: 21 are Ala and Gly, respectively, and the 53rd Xaa of SEQ ID NO: 27 is Phe.

[0076] 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. In the 2A11 antibody, the 56th and 57th Xaa residues in SEQ ID NO: 21 are each Gly. Regarding the 4C2 antibody, in SEQ ID NO: 21, the 56th Xaa residue is Gly, and the 57th Xaa residue is Ala. Regarding the 8C4 antibody, in SEQ ID NO: 21, the 56th Xaa residue is Ala, and the 57th Xaa residue is Gly.

[0077] 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. Regarding the 9B3 antibody, in SEQ ID NO: 23, the 1st Xaa is Glu, the 7th Xaa is Ser, the 12th Xaa is Val, the 27th Xaa is Tyr, the 58th Xaa is Ser, the 61st Xaa is Asn, the 74th Xaa is Lys, the 83rd Xaa is Phe, the 92nd Xaa is Ala, and the 108th Xaa is Tyr. Regarding the 9B3 antibody, in SEQ ID NO: 29, the 29th Xaa is Ile, the 51st Xaa is Ala, the 79th Xaa is Glu, and the 106th Xaa is Ile. Regarding the 9B12 antibody, in SEQ ID NO: 23, the 1st Xaa is Gin, the 7th Xaa is Ser, the 12th Xaa is Val, the 27th Xaa is Tyr, the 58th Xaa is Ser, the 61st Xaa is Asn, the 74th Xaa is Arg, the 83rd Xaa is Phe, the 92nd Xaa is Gly, and the 108th Xaa is His. Regarding the 9B12 antibody, in SEQ ID NO: 29, the 29th Xaa is Met, the 51st Xaa is Ala, the 79th Xaa is Glu, and the 106th Xaa is Met. Regarding the 3B10 antibody, in SEQ ID NO: 23, the 1st Xaa is Gln, the 7th Xaa is Pro, the 12th Xaa is Ala, the 27th Xaa is His, the 58th Xaa is Thr, the 61st Xaa is Ser, the 74th Xaa is Arg, the 83rd Xaa is Leu, the 92nd Xaa is Gly, and the 108th Xaa is His. Regarding the 3B10 antibody, in SEQ ID NO: 29, the 29th Xaa is Met, the 51st Xaa is Ser, the 79th Xaa is Asp, and the 106th Xaa is Ile.

[0078] Regarding 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. Regarding the 9B3 antibody, the 2nd Xaa of SEQ ID NO: 35 is Tyr, the 8th Xaa of SEQ ID NO: 41 is Ser, the 12th Xaa of SEQ ID NO: 47 is Tyr, the 3rd Xaa of SEQ ID NO: 53 is Ile, and the 2nd Xaa of SEQ ID NO: 59 is Ala. Regarding the 9B12 antibody, the 2nd Xaa of SEQ ID NO:35 is Tyr, the 8th Xaa of SEQ ID NO:41 is Ser, the 12th Xaa of SEQ ID NO:47 is His, the 3rd Xaa of SEQ ID NO:53 is Met, and the 2nd Xaa of SEQ ID NO:59 is Ala. Regarding the 3B10 antibody, the 2nd Xaa of SEQ ID NO:35 is His, the 8th Xaa of SEQ ID NO:41 is Thr, the 12th Xaa of SEQ ID NO:47 is His, the 3rd Xaa of SEQ ID NO:53 is Met, and the 2nd Xaa of SEQ ID NO:59 is Ser.

[0079] Table 1

[0080]

[0081]

[0082] Table 2

[0083] Antibody name Amino acid sequence of the VH region Amino acid sequence of the VL region 2A11 SEQ ID NO:21 SEQ ID NO:27 4C2 SEQ ID NO:21 SEQ ID NO:27 8C4 SEQ ID NO:21 SEQ ID NO:27 3B5 SEQ ID NO:22 SEQ ID NO:28 9B3 SEQ ID NO:23 SEQ ID NO:29 9B12 SEQ ID NO:23 SEQ ID NO:29 3B10 SEQ ID NO:23 SEQ ID NO:29 10H7 SEQ ID NO:24 SEQ ID NO:30 3G8 SEQ ID NO:25 SEQ ID NO:31 3F12 SEQ ID NO:26 SEQ ID NO:32

[0084] Table 3

[0085]

[0086]

[0087] 2. Identification of Antigen-Antibody Responses of Anti-BAG2 Antibodies in Breast Cancer Cells

[0088] Figure 1The results of immunoblotting of anti-BAG2 antibodies produced from 10 mouse hybridoma cells are shown. Specifically, human breast cancer cell MDA-MB-231 cells are cultured at a temperature of 37°C in DMEM (Welgene) culture medium containing 10% FBS, 100U / ml penicillin and 100μg / ml streptomycin. Cells are harvested from the wells and washed with PBS, and the cells are dissolved in a lysis buffer containing 1% Brij 97, 5mM EDTA, 0.02M HEPES pH 7.3, 0.15M NaCl, 1mM PMSF, 0.5mM NaF, 10μg / rni aprotinin and 0.2mM sodium orthovanadate. After incubation on ice for 15 minutes, the nucleus is removed from the cells by centrifugation and the supernatant is collected. To an appropriate amount of supernatant, a 2X sample buffer consisting of 20% glycerol, 4.6% SOS, 0.125M tris (pH 6.8), 0.1% bromophenol blue is added. Under standard conditions, 10 ug protein sample was carried out to SOS-PAGE analysis on 12% gel by using mini-Protean II system (Bio-Rad Hercules, CA). For immunoblotting, protein was transferred to Millipore (a kind of PVDF membrane). The blocking solution consisting of 0.1% Tween20 and 5% bovine serum albumin (BSA) in TBS was reacted for 1 hour. Subsequently, a 1 / 2000 dilution of the anti-BAG2 antibody extracted from the hybridoma cell culture was used as the primary antibody, and the goat anti-mouse HRP conjugate (Dako) used as the secondary antibody was diluted to 1 / 5000. EGL reagent (Amersham Pharmacia Biotech) was used as substrate to carry out film sensitization in the dark. Photosensitive bands were compared with standard molecular markers to identify the bands corresponding to the size of BAG2.

[0089] Results, such as Figure 1 As shown, antibodies 2A11, 3B5, 3B10, 3F12, 3GB, 4C2, 8C4, 9B3, 9B12, and 10H7 exhibited antigen-antibody reactions targeting BAG2, compared with the commercially available polyclonal anti-BAG2 antibody ab58682 (Abcam) used as a positive control.

[0090] Next, in order to carry out domain mapping to the BAG2 antigen that ten kinds of antibodies identified in Section 1 cells above are directed against, the cell of the GST-empty vector (pcDNA3.1+ / GST vector, NovoProBioscience Inc.China) with a molecular weight of about 26kDa is introduced therein as a negative control. GST-Bag complete vector, GST-Bag F1 vector, GST-Bag F2 vector, GST-Bag F3 vector and GST-Bag F4 vector (each comprising the polynucleotides encoding human BAG2 protein and the polynucleotides encoding the fragment of BAG2 protein) are introduced into cells, and the cells wherein introduced therein with the vector are cultured to express genes, and then cell lysates are obtained. For cell lysates, immunoblotting is performed using each of 10 kinds of antibodies. The polynucleotide encoding human BAG2 protein has the nucleotide sequence of SEQ ID NO:70. GST-Bag F1, -Bag F2, -Bag F3 and -Bag F4 vectors are composed of the nucleotide sequences of SEQ ID NO:71 to 74, respectively.

[0091] Figure 2A and 2B The results of immunoblotting of full-length BAG2 polypeptide or fragments thereof of anti-BAG2 antibodies are shown. In Figure 2, A shows a schematic 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 HEK293T cells by lipofectamine transfection (Thermo Fisher Scientific, Inc., Waltham, MA, 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 37°C for 30 hours, and then the cells were separated. Figure 1 The isolated cells were disrupted in the same manner as described above and analyzed by SOS-PAGE on a 12% gel. For immunoblotting, the cells were isolated by PCR and analyzed by Western blotting. Figure 1 After 1 hour of reaction with the same blocking solution as described above, each of the 10 purified anti-BAG2 antibodies at a concentration of 2 mg / ml was used as the primary antibody at a 1 / 10,000 dilution and allowed to bind to the cells. A goat anti-mouse HRP conjugate was used as the secondary antibody at a 1 / 5000 dilution and film exposure was performed in the dark using EGL reagent (Amersham Pharmacia Biotech) as a substrate. Standard molecular marker size was expressed to confirm the size of BAG2.

[0092] As shown in Figure 2, each anti-BAG2 antibody differentially binds to the full-length BAG2 polypeptide or its fragments. In particular, for each of the 10 anti-BAG2 antibodies, a signal was typically detected at a position of approximately 50 kDa in the cell lysate introduced with the intact GST-Bag vector. This result indicates that all of these antibodies can bind to the full-length BAG2 polypeptide. Finally, the BAG2 domain region with which each anti-BAG2 antibody reacted was identified.

[0093] Figure 3 The BAG2 domains that react with the corresponding anti-BAG2 antibodies are shown. Figure 3 As shown, 9B3, 9B12, 3B10, and 10H7 antibodies bind to the N-terminus of the BAG2 protein, 2A11, 3B5, 4C2, and 8C4 antibodies bind to the middle region of the BAG2 protein, and 3F12 and 3GB antibodies bind to the C-terminus of the BAG2 protein. The N-terminus typically includes a coiled-coil region of 21-60 amino acids, and the middle region binds to a portion of the BNB region of 109-189 amino acids. Therefore, by using a panel of antibodies that bind to different sites, BAG2 protein or fragments thereof present in a sample can be detected with high sensitivity and specificity.

[0094] Example 2: Screening of anti-BAG2 antibody combinations and confirmation of cancer diagnostic efficacy

[0095] 1. Screening for combinations of anti-BAG2 antibodies that can be used for cancer diagnosis

[0096] Figure 4 The standard curve of BAG2 protein in 90 possible combinations of anti-BAG2 antibodies is shown. Figure 4 As shown, among the possible combinations of anti-BAG2 antibodies, 14 antibody combinations showing high slopes of the standard curve were selected, namely 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.

[0097] Figure 5Figures showing the specific binding power of 14 selected antibody combinations for BAG2 protein. In detail, according to the manufacturer's instructions, Myc-tag BAG2 expression vector or Myc-tag empty vector were transduced into human breast cancer cell line MDA-MB-231 using Lipofectamine 2000 (Invitrogen). Using 14 antibody combinations, the relative amount of BAG2 protein secreted from 30ug lysates of cells expressing BAG2 protein, which were introduced into control cells with Myc-tag empty vector and into Myc-tag BAG2 expression vector, was measured. Control cells were labeled with EV, and BAG2 overexpressing cells were labeled with OE. That is, by confirming the relative amount of the secretory BAG2 protein of BAG2 overexpressing cells and control cells, it was possible to determine the specific binding power of the antibody combination for BAG2 protein.

[0098] Results, such as Figure 5 As shown in , four antibody combinations with high specific binding ability to BAG2 protein were selected, namely 2A11-3F12, 9B12-3G8, 8C4-3G8 and 10H7-3F12.

[0099] 2. Identify the efficacy of selected anti-BAG2 antibody combinations in diagnosing various cancers

[0100] To confirm the cancer diagnostic efficacy of the antibody combination selected in Section 1, the antigen-antibody reaction of the antibody combination with BAG2 was confirmed. Specifically, human BAG2 protein with 6 histidine residues attached to the N terminus produced and purified from human 293T cells was obtained as an antigen.

[0101] Sandwich enzyme-linked immunosorbent assays (sandwich ELISAs) were performed using each of the 2A11-3F12 and 9B12-3G8 antibody combinations.

[0102] In detail, sandwich ELISA was performed by the following process. Each of the antibodies 3F12 and 3G8 was reacted with 27ul of 10mM NHS-biotin (succinimidyl biotin, Thermo scientific, catalog number 21435) in an amount of 1mg to prepare a detection antibody bound to biotin. Each of the antibodies 2A11, 9B12, 8C4 and 10H7 as capture antibodies was diluted to a concentration of 3ug / ml using ELISA plate coating buffer (R&D system, DY006) and spread in each well of a 96-well plate in an amount of 100ul, and then coated overnight 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. 1% BSA buffer was used to prepare the human His-tagged BAG2 recombinant protein (Ybiologics, Korea) produced and purified in the cell line HEK293T of human origin at a concentration of 100 ng / ml as BAG2 standard material, which was then diluted 4 times 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 people, 20 patients diagnosed with luminal breast cancer and 38 patients diagnosed with TNBC type breast cancer, then mixed by gentle shaking and allowed to stand at room temperature for 20-30 minutes. The blood was then centrifuged at 2,500 rpm for 10 minutes. The serum stored at below 4 ° C was diluted 1 / 2 times using 1% BSA buffer. 0, 0.02, 0.097, 0.39, 1.56, 6.25, 25 and 100ng / ml of BAG2 standard solution and 100ul of 1 / 2 times diluted patient sample are distributed in each well of the plate having removed the blocking solution, and reacted at room temperature for 1 hour, then washed with wash buffer (R&D system, WA126). 100ul of streptavidin-HRP (Pierce, 21130) diluted by using 1% BSA buffer 1: 50000 is distributed in each well, and reacted at room temperature for 30 minutes, then washed with wash buffer. 100ul of 3,3', 5,5'-tetramethylbenzidine (TMB, R&D system, DY999) is distributed in each well, reacted at room temperature for 15 minutes in cows, and then stop solution is added thereto to terminate the reaction. The concentration of BAG2 protein in patient sample is calculated from the standard curve of the antibody obtained by measuring the absorbance of the reaction solution at 450nm.

[0103] 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 giant B-cell lymphoma observed by using a combination of anti-BAG2 antibodies. Figure 6A The results of sandwich ELISA for the 2A11-3F12 antibody combination are shown, and Figure 6B Shown are the results of sandwich ELISA using the 9B12-3G8 antibody combination.

[0104] like Figure 6A and 6B As shown, 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 giant B-cell lymphoma (DLBCL) cell lines SU-DHL4 and SU-DHL6. In contrast, BAG2 protein was not expressed at all or was expressed very little in normal cell lines T47D, SNU2469, A172, SNU620, OVCAR3, and U2932. In other words, compared with normal cells, it was confirmed by using the 2A11-3F12 and 9B12-3G8 antibody combination that BAG2 protein is significantly highly expressed in cancer cells including breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, and diffuse giant B-cell lymphoma.

[0105] Therefore, when the above-mentioned antibody combination is used, cancer can be effectively diagnosed by identifying whether the expression pattern of the BAG2 protein is similar to the expression pattern of the BAG2 protein shown in cancer cells compared with normal cells.

[0106] 3. Identify the efficacy of selected anti-BAG2 antibody combinations in diagnosing breast cancer

[0107] The difference in the expression pattern of BAG2 protein in serum between patients with luminal and TNBC breast cancer and normal subjects was confirmed by using the same sandwich ELISA method as described in Section 2. In detail, sera were collected from healthy volunteers (N=14), patients with luminal breast cancer (N=38), and patients with TNBC breast cancer (N=38), and the expression pattern of BAG2 protein in the obtained sera was confirmed.

[0108] Figure 7A and 7Band 8A and 8B show significant differences in BAG2 protein expression in the sera of patients with luminal and TNBG breast cancer identified by using a combination of anti-BAG2 antibodies. Figure 7A The case where the 9B12-3G8 antibody combination was used is shown, and Figure 7B The case where the 2A11-3F12 antibody combination was used is shown. Figure 8A The case where the 8G4-3G8 antibody combination was used is shown, and Figure 8B The case where the 10H7-3G8 antibody combination was used is shown.

[0109] like Figure 7A and 7B As shown in Figures 8A and 8B, when the 9B12-3G8, 2A11-3F12, 8G4-3G8, and 1OH7-3F12 antibody combinations were used, the p-values ​​for the corresponding antibody combinations were p<0.0001, p=0.0373, p=0.0009, and p=0.0190, respectively, indicating that the BAG2 protein expression patterns in the serum of patients with luminal and TNBG breast cancers were significantly different from those in normal subjects. Therefore, by observing significant differences in BAG2 protein expression patterns using antibody combinations, breast cancer can be effectively diagnosed.

[0110] 4. Confirm the usefulness of the selected anti-BAG2 antibody combination in breast cancer diagnosis.

[0111] In order to confirm the sensitivity and specificity of cancer diagnosis using each of the four antibody combinations, the results of Section 2 were shown by using ROG curves.

[0112] Figures 9A to 9D Receiver operating characteristic curves (ROG) and areas under the curves (AUG) for the corresponding antibody combinations are shown. Figure 9A The case where the 9B12-3G8 antibody combination was used is shown. Figure 9B The case where the 8G4-3G8 antibody combination was used is shown. Figure 9C The case where the 2A11-3F12 antibody combination was used is shown, and Figure 9D The case where the 10H7-3F12 antibody combination was used is shown.

[0113] like Figures 9A to 9DAs shown in the figure, 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 case of all antibody combinations, the AUG values ​​in the ROG curve were around 0.7, and when the 9B12-3G8, 8G4-3G8 and 2A11-3F12 antibody combinations were used, the AUG values ​​were between 0.8 and 0.9. These results show that each antibody combination can be used to diagnose breast cancer with high sensitivity and specificity. Therefore, this four-antibody combination can be effectively used to diagnose breast cancer.

[0114] The composition for diagnosing cancer comprising an anti-BAG2 antibody or an antigen-binding fragment thereof according to one aspect can provide information for diagnosing cancer.

[0115] According to another aspect of the method for providing information for diagnosing cancer, unlike conventional diagnostic methods that collect tissue, the presence or level of BAG2 polypeptide in blood can be identified or measured from blood, and the diagnostic usefulness is high. Therefore, the method can be used to diagnose cancer in various tissues and institutions.

[0116] [Accession Number]

[0117] Depository: Korea Institute of Bioscience and Biotechnology

[0118] Accession number: KCTC13737BP

[0119] Deposit date: November 28, 2018

[0120] Depository: Korea Institute of Bioscience and Biotechnology

[0121] Accession number: KCTC13738BP

[0122] Deposit date: November 28, 2018

[0123] Depository: Korea Institute of Bioscience and Biotechnology

[0124] Accession number: KCTC13739BP

[0125] Deposit date: November 28, 2018

[0126] Depository: Korea Institute of Bioscience and Biotechnology

[0127] Accession number: KCTC13740BP

[0128] Deposit date: November 28, 2018

[0129] Depository: Korea Institute of Bioscience and Biotechnology

[0130] Accession number: KCTC13741BP

[0131] Deposit date: November 28, 2018

[0132] Depository: Korea Institute of Bioscience and Biotechnology

[0133] Accession number: KCTC13742BP

[0134] Deposit date: November 28, 2018

[0135] Depository: Korea Institute of Bioscience and Biotechnology

[0136] Accession number: KCTC13743BP

[0137] Deposit date: November 28, 2018

[0138] Depository: Korea Institute of Bioscience and Biotechnology

[0139] Accession number: KCTC13744BP

[0140] Deposit date: November 28, 2018

[0141] Depository: Korea Institute of Bioscience and Biotechnology

[0142] Accession number: KCTC13745BP

[0143] Deposit date: November 28, 2018

[0144] Depository: Korea Institute of Bioscience and Biotechnology

[0145] Accession number: KCTC13746BP

[0146] Deposit date: November 28, 2018

[0147] All references cited herein are incorporated by reference in their entirety.

[0148] *****

[0149] 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 described specifically herein. <110> Medpacto Inc. Research and Business Foundation SUNGKYUNKWANUNIVERSITY Industry-Academic Cooperation Foundation, Yonsei University <120> Method for diagnosing cancer using anti-BAG2 antibodies <130> 6987-0201 <160> 74 <170> KoPatentIn 3.0 <210> 1 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> 2A11-VH <400> 1 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgacgctg 60 tcctgcaagg cttcgggcta cacatttact gactatgaaa tgcactgggt gaagcagact 120 cctgtgcatg gcctggaatg gattggagtt attgatcctg aaactggtgc tactgcctac 180 aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag tacagcctac 240 atggagctcc gcagcctgac atctgaggac tctgccgtct attactgtac aagagggaaa 300 ttttattact ccggtcggga ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360 gtctcctca 369 <210> 2 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> 4C2-VH <400> 2 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgacgctg 60 tcctgcaagg cttcgggcta cacatttact gactatgaaa tgcactgggt gaagcagact 120 cctgtgcatg gcctggaatg gattggagtt attgatcctg aaactggtgc tactgcctac 180 aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag tacagcctac 240 atggagctcc gcagcctgac atctgaggac tctgccgtct attactgtac aagagggaaa 300 ttttattact ccggtcggga ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360 gtctcctca 369 <210> 3 <211> 369 <2​​​​​​​​​​​​​cctgtgcatg gcctggaatg gattggagtt attgatcctg aaactgctgg tactgcctac 180 aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag tacagcctac 240 atggagctcc gcagcctgac atctgaggac tctgccgtct attactgtac aagagggaaa 300 ttttattact ccggtcggga ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360 gtctcctca 369 <210> 4 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> 3B5-VH <400> 4 gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagtta 60 tcctgcaagg cttctggtta ctcattcact gactacacct tttactgggt gaggcagagc 120 catggagaga gccttgagtg gattggatat attgatcctt acaatggtgg taatacttat 180 aaccggaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc 240 atgcatctca acagcctgac atctgaagac tctgcagtct attactgtgc gagagggtac 300 tataggtacg gggggggggg ggactttgac tactggggcc aaggcaccac tctcacagtc 360 tcctca 366 <210> 5 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 9B3-VH <400> 5 gaggtccagc tgcaacaatc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tagttattac 180 aatgagaagg tcaagggcaa ggcaacactg accgcagaca aatcctccag cactgcctac 240 atgcagttca gcagcctgac agctgatgac tctgcggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ctattggggc caaggcacca ctctcacagt ctcctca 357 <210> 6 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 9B12-VH <400> 6 caggtccaac tgcagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 caggtccaac tgcagcagcc tggagctgag ctggcaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact aattacatga tagagtggat aaaacagagg 120 tcctgcaagg cttctggcca cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tagttataac 180 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tacttataac 180 aatgagaagg tcaagggcaa ggcaacactg accgcagaca gatcctccag cactgcctac 240 agtgagaagg tcaagggcaa ggcaacactg accgcagaca gatcctccag cactgcctac 240 atgcagttca gcagcctgac agctgatgac tctggggtct atttctgtcg gatctatggt 300 atgcagttca gcagcctgac agctgatgac tctggggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ccattggggc caaggcacca ctctcacagt ctcctca 357 aactacaagg ggtactttga ccattggggc caaggcacca ctctcacagt ctcctca 357 <210> 7<210> 7 <211> 357<211> 357[[ID=十五]] <212> DNA<212> DNA <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <220> <220> <223> 3B10-VH <223> 3B10-VH <400> 7 <400> 7 caggtccaac tgcagcagcc tggagctgag ctggcaaggc ctgggacttc agtgaaggtg 60 caggtccaac tgcagcagcc tggagctgag ctggcaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggcca cgccttcact aattacatga tagagtggat aaaacagagg 120 tcctgcaagg cttctggcca cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tacttataac 180 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tacttataac 180 agtgagaagg tcaagggcaa ggcaacactg accgcagaca gatcctccag cactgcctac 240 agtgagaagg tcaagggcaa ggcaacactg accgcagaca gatcctccag cactgcctac 240 atgcagctca gcagcctgac agctgatgac tctggggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ccattggggc caaggcacca ctctcacagt ctcctca 357 <210> 8 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> 10H7-VH <400> 8 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctggtta taccttcaca gactattcaa ttcactgggt gaggcaggct 120 ccaggaaagg gttttaagtg gatgggctgg ataaacactg agactggtga gccaacatat 180 gcagatgact tcaagggacg gtttgccctc tctttggaaa cctctgccag cactgcctac 240 ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc tagatttgac 300 tacggtacta gttactggta cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360 tca 363 <210> 9 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 3G8-VH <400> 9 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctgggta tagcttcaca aagtatggaa tgaactgggt gaagcaggct 120 ccaggaaagg atatcaagtg gatggggtgg ataaacacca acactggaga ggcaacatat 180 ggtgaagagg tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctat 240 ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagattggga 300 ttgaggtacc ttgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 10 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 3F12-VH <400> 10 caggtgcaac tgcaggagtc aggacctgac ctggtgaaac cttctcagtc actttcactc 60 acctgcactg tcactgggta ctccatcacc agtggttata gctggcactg gatccggcag 120 tttccaggaa acaaattgga atggatgggc tacatatatt atagaggtag cactaactac 180 aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240 ctgctgttga aatctgtgac tactgaggac acagccacat attactgtgc aagagaggct 300 tactggggcc aagggactct ggtcactgtc tcagca 336 <210> 11 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 2A11-VL [[ID=十六]]<400> 11 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acaggttcct 300 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 12 <211> 336 <212> DNA <213> Artificial Sequence <220>[[ID=三十九]] <223> 4C2-VL <400> 12 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgttct acaaagtttc caaccgattt 180 tacctgcaga agccaggcca gtctccaaag ctcctgttct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acaggttcct 300 agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acaggttcct 300 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 13<210> 13 <211> 336<211> 336 <212> DNA<212> DNA <213> Artificial Sequence<213> Artificial Sequence <220> <220> <223> 8C4-VL <223> 8C4-VL <400> 13 <400> 13 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgttct acaaagtttc caaccgattt 180 tacctgcaga agccaggcca gtctccaaag ctcctgttct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 <000agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acaggttcct 300 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 14 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 3B5-VL <400> 14 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatccagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatcc acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggaatt tatttctgct ctcaaaatac acatattcct 300 ccgacgttcg ctggaggcac caagctggaa atcaaa 336 <210> 15 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 9B3-VL <400> 15 gaggtccagc tgcaacaatc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tagttattac 180 aatgagaagg tcaagggcaa ggcaacactg accgcagaca aatcctccag cactgcctac 240 atgcagttca gcagcctgac agctgatgac tctgcggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ctattggggc caaggcacca ctctcacagt ctcctca 357 <210> 16] <211> 321 <212> DNA ]<213> Artificial Sequence <220> <223> 9B12-VL <400> 16 gacattgtga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacatgaat agctatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatatgg gaatttatta ttgtctacag aatgatgagt ttccattcac gttcggctcg 300 gggacaaagc tggaaatgaa g 321 <210> 17 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 3B10-VL <400> 17 gacattgtga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacatgaat agctatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt tcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggactat 240 gaagatatgg gaatttatta ttgtctacag aatgatgagt ttccattcac gttcggctcg 300 gggacaaagc tggaaataaa a 321 <210> 18 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 10H7-VL <400> 18 gatgtccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagccacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaaccc gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct gaagattttg caacttacta ctgtcaacag agttacacta ccccgctcac tttcggtgga ggcaccaagc tggaatcaa acgtggagga gccagcctcg tggaattcaa g <210> 19 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> 3G8-VL <400> 19 60. gacattgtga tgacccagtc tcctgcttcc ttagttgtat ctctggggca gagggccacc atctcatgca gggccagcaa aagtgtcagt acatctgact atagttatat gcactggtac 180. snow snow snow snow snow snow snow snow snow snow snow ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acaataggga gcttcctccc 300 acgttcggtg ctgggaccaa gctggagctg aaa 333 <210> 20 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 3F12-VL <400> 20 gatgttttga tgacccaaac tccactcact ttgtcggtta cctttgggca gccagcctcc 60 atctcttgca ggtcaagtca gagcctctta gatagtgatg gagagacata tttgaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttccg 300 tacacgttcg gaggggggac caagctggaa ataaaa 336 <210> 21 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VH <220> <221> Variant <222> (56)..(57) <223> Xaa is Gly or Ala <400> 21 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asp Pro Glu Thr Xaa Xaa Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Lys Phe Tyr Tyr Ser Gly Arg Asp Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120[[ID=3l]] <210> 22 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VH <400> 22 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Phe Tyr Trp Val Arg Gln Ser His Gly Glu Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Asn Thr Tyr Asn Arg Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Arg Tyr Gly Gly Gly Gly Asp Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 23 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VH <220> <221> Variant <222> (1) <223> Xaa is Glu or Gln <220> <221> Variant <222> (7) <223> Xaa is Ser or Pro <220> <221> Variants <222> (12) <223> Xaa is Val or Ala <220> <221> Variants <222> (27) <223> Xaa is Tyr or His <220> <221> Variants <222> (58) <223> Xaa is Ser or Thr <220> <221> Variants <222> (61) <223> Xaa is Asn or Ser <220> <221> Variants <222> (74) <223> Xaa is Arg or Lys <220> <221> Variants <222> (83) <223> Xaa is Phe or Leu <220> <221> Variants <222> (92) <223> Xaa is Gly or Ala <220> <221> Variants <222> (108) <223> Xaa is His or Tyr <400> twenty three Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Met Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Tyr Tyr Asn Glu Lys Val 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Phe Ser Ser Leu Thr Ala Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Arg Ile Tyr Gly Asn Tyr Lys Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 24 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VH <400> 24 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Phe Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Leu Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Phe Asp Tyr Gly Thr Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 25 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VH <400> 25 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Lys Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Asp Ile Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Ala Thr Tyr Gly Glu Glu Val 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Leu Arg Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VH <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Tyr Tyr Arg Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Leu Leu Lys Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 <210> 27 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VL <220> <221> Variant <222> (53) <223> Xaa is Ile or Phe <400> 27 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Xaa Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly 85 90 95 Ser Gln Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 28 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VL <400> 28 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His Ile Pro Pro Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VL <220> <221> Variants <222> (twenty three) <223> Xaa is Met or Ile <220> <221> Variants <222> (45) <223> Xaa is Ala or Ser <220> <221> Variants <222> (73) <223> Xaa is Glu or Asp <220> <221> Variants <222> (100) <223> Xaa is Met or Ile <400> 29 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Xaa Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Xaa Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Xaa Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Asn Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Xaa Lys 100 105 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> 10H7-VL <400> 30 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 31 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VL <400> 31 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Val Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Asp Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Val Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Asn Arg 85 90 95 Glu Leu Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 32 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VL <400> 32 Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Phe Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Glu Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VHCDR1 <400> 33 Gly Tyr Thr Phe Thr Asp Tyr Glu 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VHCDR1 <400> 34 Gly Tyr Ser Phe Thr Asp Tyr Thr 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VHCDR1 <220> <221> Variants <222> (2) <223> Xaa is Tyr or His <400> 35 Gly Xaa Ala Phe Thr Asn Tyr Met 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VHCDR1 <400> 36 Gly Tyr Thr Phe Thr Asp Tyr Ser 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VHCDR1 <400> 37 Gly Tyr Ser Phe Thr Lys Tyr Gly 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VHCDR1 <400> 38 Gly Tyr Ser Ile Thr Ser Gly Tyr Ser 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VHCDR2 <220> <221> Variants <222> (6)..(7) <223> Xaa is Gly or Ala <400> 39 Ile Asp Pro Glu Thr Xaa Xaa Thr 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VHCDR2 <400> 40 Ile Asp Pro Tyr Asn Gly Gly Asn 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B5-VHCDR2 <220> <221> Variants <222> (8) <223> Xaa is Ser or Thr <400> 41 Ile Asn Pro Gly Ser Gly Gly Xaa 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VHCDR2 <400> 42 Ile Asn Thr Glu Thr Gly Glu Pro 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VHCDR2 <400> 43 Ile Asn Thr Asn Thr Gly Glu Ala 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VHCDR2 <400> 44 Ile Tyr Tyr Arg Gly Ser Thr 1 5 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VHCDR3 <400> 45 Thr Arg Gly Lys Phe Tyr Tyr Ser Gly Arg Asp Tyr Ala Met Asp Tyr 1 5 10 15 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VHCDR3 <400> 46 Ala Arg Gly Tyr Tyr Arg Tyr Gly Gly Gly Gly Asp Phe Asp Tyr 1 5 10 15 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VHCDR3 <220> <221> Variants <222> (12) <223> Xaa is Tyr or His <400> 47 Arg Ile Tyr Gly Asn Tyr Lys Gly Tyr Phe Asp Xaa 1 5 10 <210> 48 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VHCDR3 <400> 48 Ala Arg Phe Asp Tyr Gly Thr Ser Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VHCDR3 <400> 49 Ala Arg Leu Gly Leu Arg Tyr Leu Asp Tyr 1 5 10 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VHCDR3 <400> 50 Ala Arg Glu Ala Tyr 1 5 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VLCDR1 <400> 51 Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VLCDR1 <400> 52 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 53 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VLCDR1 <220> <221> Variants <222> (3) <223> Xaa is Ile or Met <400> 53 Gln Asp Xaa Asn Ser Tyr 1 5 <210> 54 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VLCDR1 <400> 54 Gln Ser Ile Ser Ser Tyr 1 5 <210> 55 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VLCDR1 <400> 55 Lys Ser Val Ser Thr Ser Asp Tyr Ser Tyr 1 5 10 <210> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VLCDR1 <400> 56 Gln Ser Leu Leu Asp Ser Asp Gly Glu Thr Tyr 1 5 10 <210> 57 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VLCDR2 <400> 57 Lys Val Ser 1 <210> 58 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VLCDR2 <400> 58 Lys Val Ser 1 <210> 59 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VLCDR2 <220> <221> Variants <222> (2) <223> Xaa is Ala or Ser <400> 59 Arg Xaa Asn 1 <210> 60 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VLCDR2 <400> 60 Ala Ala Ser 1 <210> 61 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VLCDR2 <400> 61 Leu Ala Ser 1 <210> 62 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VLCDR2 <400> 62 Leu Val Ser 1 <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VLCDR3 <400> 63 Phe Gln Gly Ser Gln Val Pro Pro Thr 1 5 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VLCDR3 <400> 64 Ser Gln Asn Thr His Ile Pro Pro Thr 1 5 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VLCDR3 <400> 65 Leu Gln Asn Asp Glu Phe Pro Phe Thr 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VLCDR3 <400> 66 Gln Gln Ser Tyr Thr Thr Pro Leu Thr 1 5 <210> 67 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VLCDR3 <400> 67 Gln His Asn Arg Glu Leu Pro Pro Thr 1 5 <2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Asn Leu Thr Ala Asn Arg Leu Met Gly Arg Thr Leu Thr Val Glu Val 85 90 95 Ser Val Glu Thr Ile Arg Asn Pro Gln Gln Gln Glu Ser Leu Lys His 100 105 110 Ala Thr Arg Ile Ile Asp Glu Val Val Asn Lys Phe Leu Asp Asp Leu 115 120 125 Gly Asn Ala Lys Ser His Leu Met Ser Leu Tyr Ser Ala Cys Ser Ser 130 135 140 Glu Val Pro His Gly Pro Val Asp Gln Lys Phe Gln Ser Ile Val Ile 145 150 155 160 Gly Cys Ala Leu Glu Asp Gln Lys Lys Ile Lys Arg Arg Leu Glu Thr 165 170 175 Leu Leu Arg Asn Ile Glu Asn Ser Asp Lys Ala Ile Lys Leu Leu Glu 180 185 190 His Ser Lys Gly Ala Gly Ser Lys Thr Leu Gln Gln Asn Ala Glu Ser 195 200 205 Arg Phe Asn 210 <21,0> 70 <211> 636 <212> DNA <213> Homo sapiens <400> 70 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca 300 attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg 360 gtcaataagt ttctggatga tttgggaaat gccaagagtc atttaatgtc gctctacagt 420 gcatgttcat ctgaggtgcc acatgggcca gttgatcaga agtttcaatc catagtaatt 480 ggctgtgctc ttgaagatca gaagaaaatt aagagaagat tagagactct gcttagaaat 540 attgaaaact ctgacaaggc catcaagcta ttagagcatt ctaaaggagc tggttccaaa 600 actctgcaac aaaatgctga aagcagattc aattag 636 <210> 71 <211> 270 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F1 vector <400> 71 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga 270 <210> 72 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F2 vector <400> 72 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca 300 attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg 360 "360" <210> 73 <211> 450 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F3 vector <400> 73 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc "60" atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa "120" gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc "180" cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta "240" aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca "300" attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg "360" gtcaataagt ttctggatga tttgggaaat gccaagagtc atttaatgtc gctctacagt "420" gcatgttcat ctgaggtgcc acatgggcca "450" <210> 74 <211> 540 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F4 Vector <400> 74 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca 300 attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg 360 gtcaataagt ttctggatga tttgggaaat gccaagagtc atttaatgtc gctctacagt 420 gcatgttcat ctgaggtgcc acatgggcca gttgatcaga agtttcaatc catagtaatt 480 ggctgtgctc ttgaagatca gaagaaaatt aagagaagat tagagactct gcttagaaat 540 540

Claims

1. A composition for diagnosing cancer, comprising: an antibody or an antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: 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, wherein the 6th Xaa and the 7th Xaa of SEQ ID NO: 39 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; 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, wherein the 6th Xaa and the 7th Xaa of SEQ ID NO: 39 are Gly and Ala, 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; or 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, wherein the 6th Xaa and the 7th Xaa 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 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.

2. The composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 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: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, wherein the 6th Xaa and the 7th Xaa of SEQ ID NO: 39 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.

3. The composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 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: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, wherein the 6th Xaa and the 7th Xaa of SEQ ID NO: 39 are Gly and Ala, 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.

4. The composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 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: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, wherein the 6th Xaa and the 7th Xaa 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 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.

5. The composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 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: 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 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 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.

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

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

8. The composition of claim 1, wherein the cancer is selected from the group consisting of 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 tumors, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumors, 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.

9. A kit for diagnosing cancer, comprising: The composition of any one of claims 1 to 8.

10. Use of an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof in the preparation of a kit for providing information for diagnosing cancer in a subject, wherein a sample isolated from the subject is contacted with the antibody or antigen-binding fragment thereof; and the formed complex of the BAG2 polypeptide or fragment thereof and the antibody or antigen-binding fragment thereof is measured; wherein the antibody or antigen-binding fragment thereof comprises: 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, wherein the 6th Xaa and the 7th Xaa of SEQ ID NO: 39 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; 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, wherein the 6th Xaa and the 7th Xaa of SEQ ID NO: 39 are Gly and Ala, 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; or 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, wherein the 6th Xaa and the 7th Xaa 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 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.

11. The use according to claim 10, wherein the sample is a biological sample.

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, cerebrospinal fluid, or a combination thereof.

13. The method of claim 10, wherein the cancer is selected from the group consisting of 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 tumors, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumors, 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 use according to claim 10, wherein the information is information about whether the BAG2 level measured in the subject is higher, equal to or lower than the BAG2 level measured in a negative control group.

15. Use of an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof in the preparation of a kit for detecting the presence of cancer in an individual, wherein the antibody or antigen-binding fragment thereof is as defined in any one of claims 1 to 8, and wherein contacting a biological sample isolated from the individual with the antibody or antigen-binding fragment thereof; and The formed complex of the BAG2 polypeptide or fragment thereof and the antibody or antigen-binding fragment thereof is measured, wherein the presence of cancer is detected if the BAG2 measured in the sample is higher than the level of BAG2 measured in the negative control group.

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