Antibodies for determining the presence or absence of phosphorylation-specific reaction of threonine 1010 of NCAPG2 and their use

Abstract

The present invention relates to an antibody for determining the presence or absence of phosphorylation-specific reaction of threonine 1010 in the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) and its use. The antibody of the present invention has high selectivity and binding ability to pT1010 of NCAPG2, and the reactivity of NCAPG2 to pT1010 is suppressed in cancer cells treated with inhibitors of kinases that regulate cell division, such as CDK1, PLK1, Mps1, and Aurora kinase, and is highly detected in tumor tissues compared to non-tumor tissues of cancer patients through immunohistochemical staining. Therefore, it is expected that the antibody of the present invention can be useful in cancer-related research and development fields, such as diagnosing cancer or screening anti-cancer drug candidates, by determining the presence or absence of phosphorylation of threonine 1010 of NCAPG2 using the antibody of the present invention.

Description

[Technical Field]

[0001] The present invention relates to an antibody for confirming the presence or absence of a phosphorylation-specific reaction at threonine 1010 in the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2), and uses thereof.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2021-0066894, filed on May 25, 2021, the entire contents of which are incorporated herein by reference in their entirety in the specification and drawings. [Background technology]

[0003] Cancer is one of the incurable diseases that humanity must solve, and huge amounts of capital are being invested worldwide in developing a cure for it. In Korea, it is the number one cause of death from disease, with more than 100,000 people diagnosed with it each year and more than 60,000 dying from it.

[0004] Most cancer screening methods to date are physical, such as double-contrast, compression, or mucosal X-rays for gastrointestinal examinations. Using an endoscope allows for direct visual inspection of internal organs, making it possible to detect even very small lesions that do not show up on X-rays. It also allows for direct tissue examination of suspected cancer sites, increasing the diagnostic rate. However, this method has drawbacks, such as hygiene issues and the pain that patients endure during the examination process.

[0005] Furthermore, most cancer treatments currently available involve surgical removal of the lesion, which is the only option, especially when a complete cure is being sought. In such surgical resections, the general rule is to remove as much of the tumor as possible, but the extent of the resection may be determined based on the potential for complications from extensive resection. However, even in such cases, if the cancer has metastasized to other organs, curative surgery is impossible. Therefore, in such cases, other options, such as the administration of anticancer drugs, are chosen. However, currently available anticancer drugs only provide temporary relief of symptoms, inhibit post-operative recurrence, and extend survival. They have limitations in terms of fundamental cancer treatment, and can also cause double suffering to patients due to the side effects and financial burden of administering anticancer drugs.

[0006] Therefore, in order to treat cancer, it is of utmost importance to develop a cancer diagnostic method with high sensitivity and specificity at the pre-treatment stage, and such a diagnosis must be able to detect cancer at an early stage. Furthermore, there is a need for a method that can predict the prognosis for specific cancers and provide customized diagnosis and treatment. However, to date, there are very few molecular diagnostic technologies that can specifically detect lesions at an early stage and determine whether or not cancer develops.

[0007] Mitosis, on the other hand, refers to the division of a cell in which all cellular components are separated into two new cells. Mitosis involves chromosome condensation, spindle pole body separation and pole migration, central chromosome alignment, and finally, the separation of all cellular components. As a cell begins to divide, chromosomes must form specific structures for efficient bidirectional segregation. This mitosis-specific chromosome structure is primarily dependent on three multiprotein complexes: two condensin and two cohesin complexes. The cohesin complex binds its sister chromatids, while the condensin complex acts to thicken and shorten the interior of chromosomes. Each condensin complex consists of two ATPase subunit heterodimers, a structural maintenance of chromosomes complex (SMC2 & SMC4), and three non-SMC regulatory subunits. The unique sum of these three regulatory components defines each condensin complex; for example, NCAPD2, NCAPG, and NCAPH are components of condensin complex I, and NCAPD3, NCAPG2, and NCAPH2 are components of condensin complex II. The SMC2 and 4 heterodimers are crosslinkers for mitotic DNA condensation using their ATPase activity. NCAPH and NCAPH2 are kleisin proteins that connect the SMC heterodimer to the other two regulatory subunits, while NCAPG, NCAPG2, NCAPD2, and NCAPD3 are regulatory subunits for each condensin complex that contain HEAT repeat domains corresponding to variable scaffolds. Condensin complex I is located in the cytosol during interphase and is incorporated into chromosomes by aurora kinase B immediately after nuclear envelope breakdown, where it remains attached to chromosome arms until cytokinesis.Condensin complex II, on the other hand, remains in the nucleus during quiescence and contributes to chromosome condensation during cell division. Its intrachromosomal invagination is achieved through a catalytically independent function of protein phosphatase 2A (PP2A). Various other functions, including chromosome decatenation, chromatin remodeling, and complex I condensation, ensure that chromosome condensation is maintained until cytokinesis. Condensin complex I, present in yeast species, is the classical condensin complex for eukaryotic chromosome condensation. Condenser II regulates not only chromosome rigidity but also various cellular processes, such as chromosome segregation, DNA repair, apoptosis, sister chromatid resolution, gene expression regulation, and histone modulation. Interestingly, homozygous mutants of all nematode condensin complex II components exhibit abnormal sizes or uneven nuclear distribution. In human cells, deficiency of any component of condensin complex II causes defects in chromosome alignment or division. Furthermore, NCAPD3 primarily affects centrosome division, while NCAPG2 deficiency frequently results in poor chromosome alignment within the metaphase plate. Regarding chromosome division, recent reports have shown that NCAPD3 contributes to the relocation of PLK1 to chromosome arms.

[0008] However, in the field of cancer-related research and development, such as cancer diagnosis and treatment, antibodies that specifically bind to specific phosphorylated amino acid sequences in the amino acid sequence of NCAPG2 and their uses are not yet well known. Summary of the Invention [Problem to be solved by the invention]

[0009] The inventors confirmed that phosphorylation of threonine at position 1010 in the amino acid sequence of NCAPG2 is associated with cancer, and prepared an antibody specific to pT1010 of NCAPG2 to confirm the presence or absence of phosphorylation of threonine at position 1010 (hereinafter referred to as pT1010) in the amino acid sequence of NCAPG2.They confirmed that the antibody was highly reactive to pT1010 of NCAPG2, and based on this, completed the present invention.

[0010] Therefore, an object of the present invention is to provide an antibody for confirming the presence or absence of phosphorylation of threonine at position 1010 in the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2), and uses thereof.

[0011] However, the technical problems that the present invention aims to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0012] In order to achieve the above object, the present invention provides an antibody for determining whether or not threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) is phosphorylated,

[0013] The antibody is characterized by recognizing the peptide represented by the amino acid sequence of SEQ ID NO: 1 as an antigen and specifically binding to the threonine at position 1010 from the N-terminus of the amino acid sequence of phosphorylated NCAPG2.

[0014] In one embodiment of the present invention, the amino acid sequence of NCAPG2 may be represented by SEQ ID NO:2.

[0015] The present invention also provides a method for producing an antibody for determining whether or not threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is phosphorylated, the method comprising the steps of: isolating the antibody from serum isolated from an individual injected with a peptide represented by the amino acid sequence of SEQ ID NO: 1;

[0016] The antibody has a relatively higher affinity for a peptide in which the 7th amino acid from the N-terminus of the peptide represented by the amino acid sequence of SEQ ID NO: 1 is phosphorylated at threonine, compared to a peptide in which the 7th amino acid from the N-terminus of the peptide represented by the amino acid sequence of SEQ ID NO: 1 is non-phosphorylated at threonine, the 7th amino acid from the N-terminus of the peptide represented by the amino acid sequence of SEQ ID NO: 1.

[0017] The present invention also provides a method for providing information for cancer diagnosis, which includes reacting the antibody with cells expressing NCAPG2 (Non-SMC condensin II complex subunit G2) and confirming the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2.

[0018] In one embodiment of the present invention, the method may further include determining that the cancer is present when threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is phosphorylated.

[0019] In another embodiment of the present invention, the higher the degree of phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2, the lower the degree of differentiation of tumor cells.

[0020] The present invention also provides a method for treating cancer cells with a candidate substance, comprising:

[0021] (b) reacting the antibody with cancer cells treated with the candidate substance to confirm whether or not phosphorylation occurs at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2); and

[0022] (c) A method for screening candidate anticancer drugs is provided, which includes a step of selecting the candidate substance as a candidate anticancer drug if phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is suppressed in step (b).

[0023] In one embodiment of the present invention, the candidate anticancer drug may include at least one selected from the group consisting of a PLK1 (Polo-like kinase 1) inhibitor, an Mps1 (Monopolar spindle 1) inhibitor, an Aurora kinase inhibitor, and a CDK1 (Cyclin-dependent kinase 1) inhibitor.

[0024] In another embodiment of the present invention, the PLK1 inhibitor, Mps1 inhibitor, Aurora kinase inhibitor, or CDK1 inhibitor may be at least one selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, and natural extracts.

[0025] The present invention also relates to the antibody, and Contains candidate anti-cancer drugs, The antibody acts as a prognostic marker (indicator) for confirming the efficacy of the anticancer drug candidate substance, and the kit for confirming the efficacy of the anticancer drug candidate substance is provided.

[0026] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2).

[0027] In one embodiment of the present invention, the substance may be at least one selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, chemical compounds, natural products, and natural extracts.

[0028] The present invention also provides a cancer diagnostic method, which includes the step of reacting the antibody with cells or animals expressing NCAPG2 (Non-SMC condensin II complex subunit G2) and confirming the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2.

[0029] The present invention also provides a method for preventing or treating cancer, which includes administering to an individual in need thereof a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2).

[0030] The present invention also provides a cancer prevention or treatment use of a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2).

[0031] The present invention also provides use of a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) for the production of a drug for preventing or treating cancer. [Effects of the Invention]

[0032] The antibody for determining whether or not there is phosphorylation of threonine at position 1010 in the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) according to the present invention has high selectivity and binding to pT1010 of NCAPG2, and it was confirmed that the reactivity of NCAPG2 to pT1010 was suppressed in cancer cells treated with inhibitors of kinases that regulate cell division, such as CDK1, PLK1, Mps1, and Aurora kinase, and that it was detected at a significantly higher level in tumor tissues than in non-tumor tissues of cancer patients by immunohistochemical staining. Therefore, determining whether or not there is phosphorylation of threonine at position 1010 of NCAPG2 using the antibody according to the present invention is expected to be useful in cancer-related research and development fields, such as diagnosing cancer or screening candidate anti-cancer drugs. [Brief explanation of the drawings]

[0033] [Figure 1a] FIG. 1 shows the reactivity of an antibody against the C-HRGVLS(pT)LIAGPV-amide antigen according to one embodiment of the present invention, compared with the reactivity with a mutant in which threonine at position 1010 of NCAPG2 is substituted with alanine. [Figure 1b] FIG. 1 is a diagram confirming the reactivity of anti-pT1010 antibody to C-HRGVLS(pT)LIAGPV-amide antigen in cells treated with a kinase inhibitor according to an embodiment of the present invention. [Figure 1c] FIG. 1 is a diagram confirming the reactivity of anti-pT1010 antibody to C-HRGVLS(pT)LIAGPV-amide antigen in cells treated with a kinase inhibitor according to an embodiment of the present invention. [Figure 2a] FIG. 1 shows the results of immunocytochemical staining using an anti-NCAPG2 antibody when the expression of NCAPG2 is reduced according to one embodiment of the present invention. [Figure 2b] FIG. 10 shows the results of immunocytochemical staining using anti-pT1010 antibody against CDTPVHRGVLSpTLIA antigen when NCAPG2 expression is reduced according to one embodiment of the present invention. [Figure 2c] FIG. 10 shows the results of immunocytochemical staining using anti-pT1010 antibody against C-HRGVLS(pT)LIAGPV-amide antigen when NCAPG2 expression is reduced according to one embodiment of the present invention. [Figure 3] FIG. 1 shows the results of immunostaining when normal cells and cancer cells are treated with anti-pT1010 antibody against C-HRGVLS(pT)LIAGPV-amide antigen according to one embodiment of the present invention. [Figure 4a] FIG. 1 shows the results of immunohistochemical staining in liver cancer tissue to confirm pT1010 expression of NCAPG2 according to one embodiment of the present invention. [Figure 4b] 1 is a diagram showing the results of quantifying the positive reaction after immunohistochemical staining of liver cancer tissue to confirm pT1010 expression of NCAPG2 according to one embodiment of the present invention using the H-score system. [Figure 4c] 1 shows the results of immunohistochemical staining in bile duct cancer tissues to confirm pT1010 expression of NCAPG2 according to one embodiment of the present invention. [Figure 4d] 1 shows the results of immunohistochemical staining in pancreatic cancer tissues to confirm pT1010 expression of NCAPG2 according to one embodiment of the present invention. [Figure 5a] 1 is a diagram showing the expression of NCAPG2pT1010 according to the degree of tumor differentiation in pancreatic cancer tissues according to an embodiment of the present invention, confirmed by immunohistochemical staining. [Figure 5b] 1 is a diagram showing NCAPG2pT1010 expression according to the degree of tumor differentiation in pancreatic cancer tissues according to an embodiment of the present invention, as determined using a Q-score system. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides an antibody for confirming the presence or absence of phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2),

[0035] The antibody is characterized by recognizing the peptide represented by the amino acid sequence of SEQ ID NO: 1 as an antigen and specifically binding to the threonine at position 1010 from the N-terminus of the amino acid sequence of phosphorylated NCAPG2.

[0036] The NCAPG2 may be derived from humans and may be represented by the amino acid sequence of SEQ ID NO: 2 (NCBI GenBank: AAH43404.1).

[0037] In the present invention, "threonine at position 1010 of NCAPG2" or "threonine at position 1010 from the amino acid sequence of NCAPG2" may mean threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2.

[0038] The amino acid sequence of NCAPG2 represented by SEQ ID NO: 2 contains threonine at the 1010th amino acid from the N-terminus, and in the present invention, "pT1010" means that a phosphate group is bound to threonine, the 1010th amino acid from the N-terminus in the amino acid sequence of NCAPG2.

[0039] The present invention also provides a method for producing an antibody for determining whether or not threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is phosphorylated, the method comprising the steps of: isolating the antibody from serum isolated from an individual injected with a peptide represented by the amino acid sequence of SEQ ID NO: 1;

[0040] The antibody has a relatively high affinity for a peptide in which the 7th amino acid from the N-terminus of the peptide represented by the amino acid sequence of SEQ ID NO: 1 is phosphorylated at threonine, compared to a peptide in which the 7th amino acid from the N-terminus of the peptide represented by the amino acid sequence of SEQ ID NO: 1 is non-phosphorylated at threonine.

[0041] The term "phosphorylation," as used herein, refers to the biochemical reaction of adding a phosphate group to a serine (S), threonine (T), or tyrosine (Y) residue of a specific protein, which is catalyzed by protein kinase enzymes. Phosphorylation typically modifies the function of the target protein to regulate its activity. As part of the homeostatic mechanism of the cell, phosphorylation is merely a transient process that is reversed by other enzymes called phosphatases. Any abnormalities in either aspect of the reaction (phosphorylation vs. dephosphorylation) can disrupt cellular function.

[0042] The term "antibody" as used herein refers to a polypeptide comprising a framework region derived from an immunoglobulin gene or a fragment thereof that specifically binds and recognizes an antigen. Recognized immunoglobulin genes include numerous immunoglobulin variable region genes, including kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, thereby defining the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody is crucial for determining binding specificity and affinity. In the present invention, antibodies or antibody fragments may be derived from different organisms, including, but not limited to, humans, mice, rats, hamsters, camels, and rabbits. In one embodiment of the present invention, antibodies or antibody fragments may be derived from rabbits. The antibodies of the present invention may include antibodies that have been modified or mutated at one or more amino acid positions to improve or adjust a desired function of the antibody (e.g., glycosylation, expression, antigen recognition, effector function, antigen binding, specificity, etc.).

[0043] In the present invention, the antibody may be prepared by injecting the C-HRGVLS(pT)LIAGPV-amide peptide represented by the amino acid sequence of SEQ ID NO: 1 into an individual so that the antibody recognizes it as an antigen, resulting in the highest antibody specificity for pT1010. The peptide may be conjugated with KLH (keyhole limpet hemocyanin) before injection into an individual, but is not limited thereto. Furthermore, the peptide may have modifications at its N- and C-termini to enhance similarity to native proteins and stability. According to one embodiment of the present invention, the peptide represented by the amino acid sequence of SEQ ID NO: 1 may have an acetylated N-terminus (C) and an amidated C-terminus (amidated amide), but is not limited thereto.

[0044] In the present invention, the antibody is not limited in its binding position or type as long as it can confirm the presence or absence of phosphorylation at the threonine site at position 1010 from the N-terminus of the amino acid sequence of NCAPG2. For example, the antibody may specifically bind to a specific amino acid sequence of NCAPG2, or may specifically bind to the phosphate group at the threonine site at position 1010 from the N-terminus of the amino acid sequence of NCAPG2.

[0045] The term "specifically binds" as used herein refers to antibody binding to a predetermined antigen, where the antibody binds to the predetermined antigen with an affinity that is higher, e.g., at least two-fold higher, than the affinity for binding to an unrelated antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein).

[0046] The present invention also provides a method for providing information for cancer diagnosis, which includes reacting the antibody with cells expressing NCAPG2 (Non-SMC condensin II complex subunit G2) and confirming the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2.

[0047] The present invention also provides a cancer diagnostic method, which includes the step of reacting the antibody with cells or animals expressing NCAPG2 (Non-SMC condensin II complex subunit G2) and confirming the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2.

[0048] In the present invention, the method may further include a step of determining that the tumor is cancer if the threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is phosphorylated, and the higher the degree of phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2, the lower the degree of differentiation of the tumor cells.

[0049] The term "diagnosis" as used herein means to confirm the presence or characteristics of a pathological condition. For purposes of the present invention, diagnosis is to confirm the presence or absence of cancer.

[0050] The present invention also provides a method for treating cancer, comprising the steps of:

[0051] (a) reacting the antibody with cells or animals expressing NCAPG2 (Non-SMC condensin II complex subunit G2) and confirming the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2;

[0052] (b) determining that the cancer is present when the threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is phosphorylated; and

[0053] (c) treating the cancer determined in step (b).

[0054] In the present invention, treating cancer at the step (c) may be performed using methods such as chemotherapy, radiotherapy, surgery, or biological therapy.

[0055] In the present invention, the term "chemotherapy" refers to the use of chemical substances to treat a particular disease and the entire range of drugs used in that case.

[0056] In the present invention, the drug may be, for example, paclitaxel, doxorubicin, 5-fluorouracil, cisplatin, imatinib, carboplatin, oxaliplatin, tegafur, irinotecan, docetaxel, cyclophosphamide, cemcitabine, ifosfamide, mitomycin C, or the like. C), vincristine, etoposide, methotrexate, topotecan, tamoxifen, vinorelbine, camptothecin, daunorubicin, chlorambucil, bryostatin-1, calicheamicin, mayatansine, levamisole, DNA recombinant interferon alpha-2a alfa-2a, mitoxantrone, nimustine, interferon alfa-2a, doxifluridine, formestane, leuprolide acetate, megestrol acetateacetate, carmofur, teniposide, bleomycin, carmustine, heptaplatin, exemestane, anastrozole, estramustine, capecitabine, goserelin acetate, potassium polysaccharide, medroxyprogesterone acetate, epirubicin, letrozole, pirarubicin, topotecan, altretamine, toremifene citrate The anticancer agent may be at least one selected from the group consisting of, but not limited to, cyclosporine citrate, BCNU, taxotere, actinomycin D, and synthetic analogs thereof, and modified or modified substances that exhibit the same pharmacological effects.

[0057] In the present invention, radiation therapy refers to the exposure of a patient to high-energy radiation, including, but not limited to, X-rays, gamma rays, and neutrons. Such types of therapy include, but are not limited to, external beam radiation therapy, internal radiation therapy, implantable radiation therapy, brachytherapy, and systemic radiation therapy.

[0058] In the present invention, the term "surgery" includes any therapeutic or diagnostic procedure involving the procedural action of a hand or a hand in conjunction with an instrument on an individual's body to achieve a curative, therapeutic or diagnostic effect.

[0059] In the present invention, the term "biological therapy" refers to a therapy that directly or indirectly utilizes the human immune system using a biological preparation containing a substance derived from an organism or a substance produced using an organism. The biological preparation includes vaccines, allergens, antigens, hormones, cytokines, enzymes, blood and plasma, immune serum, monoclonal antibodies, fermentation products, antitoxins, and laboratory diagnostic agents, the potency and stability of which cannot be evaluated by physical or chemical tests alone.

[0060] In the present invention, the biological agent may be, for example, adalimumab, alemtuzumab, bevacizumab, cetuximab, daratumumab, panitumumab, rituximab, trastuzumab, pertuzumab, ipilimumab, nifedipine ... The antibody may be at least one selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, tocilizumab, sarilumab, satralizumab, and siltuximab, but is not limited thereto.

[0061] In the present invention, the type of the "cell" may be a vertebrate, for example, a mammal including a human (human, monkey, mouse, rat, hamster, cow, etc.), a bird (chicken, ostrich, etc.), an amphibian (frog, etc.), or a fish, or an invertebrate, for example, an insect (silkworm, moth, fruit fly, etc.), a plant, or a microorganism such as yeast, and may preferably be a mammal including a human, but is not limited thereto.

[0062] The term "cancer" as used in the present invention is a general term for diseases caused by cells that have aggressive properties, such as dividing and growing despite normal growth limitations, invasive properties, such as infiltrating surrounding tissues, and metastatic properties, such as spreading to other parts of the body.

[0063] In the present invention, the cancer is not particularly limited in type as long as it is known as a malignant tumor in the art, and examples thereof include breast cancer, colon cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, bile duct cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, trumpet cancer, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, and small intestine cancer. The cancer may be selected from the group consisting of cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or hydroureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, and according to one embodiment of the present invention, may be breast cancer, liver cancer, bile duct cancer, or pancreatic cancer.

[0064] In the present invention, "reacting an antibody with a cell or an animal" means treating the cell or an animal with the antibody or contacting the cell or an animal with the antibody to confirm the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2.

[0065] As used herein, "contacting" refers to the normal meaning of combining two or more preparations (e.g., two polypeptides) or combining a preparation with a cell (e.g., a protein with a cell). Contacting can occur in vitro, such as combining two or more preparations in a test tube or other container, or combining a test preparation with a cell or a cell lysate. Contacting can also occur in cells or in situ, such as contacting two polypeptides from a cell or cell lysate by coexpressing recombinant polynucleotides encoding the two polypeptides in the cell.

[0066] The present invention also provides a method for treating cancer cells with a candidate substance, comprising:

[0067] (b) reacting the antibody with cancer cells treated with the candidate substance to confirm whether or not phosphorylation occurs at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2); and

[0068] (c) A method for screening candidate anticancer drugs is provided, which includes a step of selecting the candidate substance as a candidate anticancer drug if phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is suppressed in step (b).

[0069] The present invention also relates to the antibody, and Contains candidate anti-cancer drugs, The antibody acts as a prognostic marker (indicator) for confirming the efficacy of the anticancer drug candidate substance, and the kit for confirming the efficacy of the anticancer drug candidate substance is provided.

[0070] The term "candidate substance" as used in the present invention refers to an unknown substance used in screening to confirm the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2. According to one embodiment of the present invention, a candidate substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 may be selected as a candidate anticancer drug, and the candidate anticancer drug may be at least one selected from the group consisting of PLK1 (Polo-like kinase 1) inhibitors, Mps1 (Monopolar spindle 1) inhibitors, Aurora kinase inhibitors, and CDK1 (Cyclin-dependent kinase 1) inhibitors. In the present invention, the PLK1 inhibitor, Mps1 inhibitor, Aurora kinase inhibitor, or CDK1 inhibitor may be at least one selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, and natural extracts, but is not limited thereto as long as it is a substance that can inhibit PLK1, Mps1, Aurora kinase, or CDK1.

[0071] In the present invention, a prognostic marker (indicator) can provide some information on prognosis, in addition to tumor size, lymph node status, and histological grade, and can indicate the possibility of response to a particular therapeutic agent, and can be used to select patients for treatment with a particular therapeutic agent. According to one embodiment of the present invention, an NCAPG2 pT1010-specific antibody can act as a prognostic marker to confirm the potential of a PLK1 inhibitor, Mps1 inhibitor, Aurora kinase inhibitor, or CDK1 inhibitor as an anticancer agent by determining whether phosphorylation of NCAPG2 at threonine 1010 is suppressed by treatment with the PLK1 inhibitor, Mps1 inhibitor, Aurora kinase inhibitor, or CDK1 inhibitor.

[0072] In the present invention, the kit can confirm the effectiveness of a candidate anticancer drug substance, and for confirming the effectiveness of such a candidate anticancer drug substance, the kit includes an antibody that specifically binds to threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2, and at least one candidate anticancer drug substance selected from the group consisting of a PLK1 inhibitor, an Mps1 inhibitor, an Aurora kinase inhibitor, and a CDK1 inhibitor, and the antibody and candidate anticancer drug substance can each be applied one or more times without any restriction on the number of times, and there is no restriction on before or after the application of each antibody and candidate anticancer drug substance, and they may be applied simultaneously or separately.

[0073] In the present invention, the kit may include a container, instructions, an antibody that specifically binds to threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2, and at least one candidate anti-cancer drug selected from the group consisting of a PLK1 inhibitor, an Mps1 inhibitor, an Aurora kinase inhibitor, and a CDK1 inhibitor. The container serves to package, store, and fix the antibody that specifically binds to threonine at position 1010 in the amino acid sequence of NCAPG2 and the at least one candidate anti-cancer drug selected from the group consisting of a PLK1 inhibitor, an Mps1 inhibitor, an Aurora kinase inhibitor, and a CDK1 inhibitor. The material of the container may be, for example, but is not limited to, a plastic or glass bottle. Furthermore, the instructions included in the kit for confirming the effectiveness of a candidate anticancer drug substance according to the present invention may contain instructions regarding confirming the effectiveness of the candidate anticancer drug substance, or the instructions may be written on a sheet or booklet separate from the container, and the sheet or booklet may be contained in the container together with an antibody that specifically binds to the threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 or at least one candidate anticancer drug substance selected from the group consisting of a PLK1 inhibitor, an Mps1 inhibitor, an Aurora kinase inhibitor, and a CDK1 inhibitor.

[0074] In the present invention, the method for confirming the presence or absence of phosphorylation at threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 using an antibody specific to pT1010 of NCAPG2 is a conventional method known in the art, such as western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence staining, Ouchterlony, complement fixation assay, protein chip, etc. The measurement may be performed by one or more methods selected from the group consisting of immunoprecipitation, immunocytochemistry, and immunohistochemistry, and according to a specific embodiment of the present invention, the measurement may be performed using, but is not limited to, immunoprecipitation, immunocytochemistry, or immunohistochemistry.

[0075] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2).

[0076] The present invention also provides a method for preventing or treating cancer, which includes administering to an individual in need thereof a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2).

[0077] The present invention also provides a cancer prevention or treatment use of a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2).

[0078] The present invention also provides use of a substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) for the production of a drug for preventing or treating cancer.

[0079] In the present invention, the substance that inhibits the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) may be at least one selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, and natural extracts, but is not limited thereto.

[0080] In the present invention, "prevention" means any action of suppressing cancer or delaying the onset of cancer by administering the composition according to the present invention.

[0081] In the present invention, "treatment" means any action in which the symptoms of cancer are improved or beneficially altered by administering a composition according to the present invention.

[0082] In the present invention, "administration" means providing a given composition of the present invention to an individual by any suitable method.

[0083] In the present invention, the term "individual" refers to a subject in need of treatment for a disease to which the composition of the present invention can be administered, and more specifically refers to mammals such as human or non-human primates, mice, rabbits, dogs, cats, horses, and cows.

[0084] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, such as at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials and controlled-release additives.

[0085] The pharmaceutical composition according to the present invention may be formulated by a conventional method into the form of a powder, granules, sustained-release granules, enteric-coated granules, liquid, eye drops, elucic preparation, emulsion, suspension, spirit preparation, troche, perfume, lemonade, tablet, sustained-release tablet, enteric-coated tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric-coated capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, perfusion solution, plaster, lotion, paste, spray, inhalant, patch, sterile injection, or external preparation such as aerosol, and the external preparation may have the form of a cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, or cataplasm.

[0086] Carriers, excipients and diluents that may be included in pharmaceutical compositions according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0087] When the formulation is made, it is usually prepared by using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc.

[0088] Examples of additives for the tablets, powders, granules, capsules, pills, and lozenges according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC), HPMC1928, HPMC2208, HPMC2906, HPMC2910, propylene glycol, casein, calcium lactate, and Primozel; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, and casein. Binders such as sodium, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. may be used, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resins, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gumgum), baking soda, polyvinylpyrrolidone, calcium phosphate, gelling starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, D-sorbitol liquid, disintegrants such as hard anhydrous silicic acid, calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri Lubricants such as wax, aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard silicic anhydride may also be used.

[0089] Examples of additives that may be used in the liquid preparation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearates, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethyl cellulose.

[0090] The syrup according to the present invention may contain a solution of sucrose, other sugars or sweeteners, and may also contain flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. as needed.

[0091] The emulsion of the present invention may contain purified water, and may contain emulsifiers, preservatives, stabilizers, fragrances, etc., as needed.

[0092] The suspension according to the present invention may contain a suspending agent such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC1828, HPMC2906, or HPMC2910, and may also contain a surfactant, preservative, stabilizer, coloring agent, or fragrance, as needed.

[0093] The injections according to the present invention may contain distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated Ringer's injection, ethanol, propylene glycol, fixed oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, solvents such as ethyl oleate, isopropyl myristate, and benzene benzoate, solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, teufels, nidic acid amide, hexamine, and dimethylacetamide, weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), ), organic compounds, proteins, albumin, peptone, buffers such as gums, isotonicity agents such as sodium chloride, stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid, sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite, soothing agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate, and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0094] Suppositories according to the present invention may contain cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic and oleic acids, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter plus cholesterol, lecithin, lanet wax, glycerol monostearate, Tween or Span, Imhausen, Monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16 (Cebes Pharma). 16), Hexalide Base 95, Cotomar, Hydrocoat SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrocoat 711, Idropostal, Massa Estralium Bases such as estrarium, A, AS, B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospostal-en, Paramound-B, Sposhiro (OSI, OSIX, A, B, C, D, H, L), suppository base IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and Tezestar triglyceride base (TG-95, MA, 57) may also be used.

[0095] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.

[0096] Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents that may be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0097] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level may be determined by factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0098] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that will provide the maximum effect at the minimum dose without causing side effects, and this can be easily determined by a person of ordinary skill in the art to which the present invention pertains.

[0099] The pharmaceutical compositions of the present invention may be administered to an individual by a variety of routes, including, but not limited to, oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like, although all modes of administration are contemplated.

[0100] The pharmaceutical composition of the present invention is determined by the type of drug as the active ingredient, as well as various related factors such as the disease to be treated, the administration route, the patient's age, sex, weight, and severity of the disease.

[0101] In one example of the present invention, an antibody was produced to confirm the presence or absence of phosphorylation of threonine at position 1010 from the N-terminus (pT1010) in the amino acid sequence of NCAPG2 using C-HRGVLS(pT)LIAGPV-amide as an antigen (see Example 1).

[0102] In another embodiment of the present invention, the antibody reactivity of a mutant (TA) in which the threonine (Thr) at position 1010 from the N-terminus in the amino acid sequence of NCAPG2 was replaced with alanine (Ala) was compared with that of an antibody against the CDTPHRGVLSpTLIA antigen, confirming that the antibody prepared above has superior specificity for pT1010 of NCAPG2, and that the pT1010-specific band of NCAPG2 was reduced by immunoprecipitation with treatment with a kinase inhibitor (see Example 2).

[0103] In yet another example of the present invention, the specificity of the prepared antibody was confirmed using immunocytochemistry, and when NCAPG2-specific expression was reduced using siRNA, it was confirmed that the reactivity of the anti-pT1010 antibody against the C-HRGVLS(pT)LIAGPV-amide antigen was sufficiently specific, and it was found that the selectivity and binding strength of the antibody were more suitable than those of the antibody against the CDTPVHRGVLSpTLIA antigen (see Example 3).

[0104] In yet another example of the present invention, a comparison of immunostaining patterns in normal cells and cancer cells revealed that, in normal cells, spindle attachment points (kinetochores) were specifically stained during cell division upon treatment with anti-pT1010 antibody, whereas in cancer cells, the staining intensity was increased and spindle attachment point-specific staining often did not occur during cell division (see Example 4).

[0105] In yet another example of the present invention, the above-prepared antibody was used to confirm the expression of NCAPG2 pT1010 in non-tumor and tumor areas of liver cancer, bile duct cancer, and pancreatic cancer tissues by immunohistochemical staining, and it was confirmed that NCAPG2 pT1010 is mostly expressed in the nuclei of cells and that expression is significantly higher in tumor areas than in non-tumor areas (see Example 5).

[0106] In yet another example of the present invention, immunohistochemical staining for NCAPG2pT1010 was performed in pancreatic cancer tissue, and it was confirmed that the level of NCAPG2pT1010 expression in pancreatic cancer tissue correlates with the degree of tumor differentiation (see Example 6). [Example]

[0107] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention.

[0108] [Example 1. Production of NCAPG2pT1010-specific antibody] To generate antibodies to confirm the presence or absence of phosphorylation of threonine 1010 (pT1010) in the amino acid sequence of NCAPG2, a peptide with a purity of over 90% was synthesized using C-HRGVLS(pT)LIAGPV-amide (SEQ ID NO: 1) as the antigen. This peptide was conjugated with KLH (keyhole limpet hemocyanin) and injected into rabbits. The rabbits were repeatedly injected 4-5 times to increase the production of anti-pT1010 antibodies. Serum titers on days 0, 26, 52, and 72 after immunization were determined by enzyme-linked immunosorbent assay (ELISA). Next, in the antiserum affinity purification and depletion process, a phosphopeptide column was used to remove nonspecific binding proteins and concentrate the target antibodies, thereby isolating antibodies from the isolated serum that have a relative affinity for C-HRGVLS(pT)LIAGPV-amide compared to the non-phosphorylated peptide (C-HRGVLSTLIAGPV-amide). This is a process for preparing phosphospecific antibodies, in which a column with a non-phosphopeptide is used to once again remove phospho-nonspecific antibodies that bind to the non-phosphorylated peptide.

[0109] [Example 2. Confirmation of antibody reactivity] To confirm the reactivity of the antibody prepared in Example 1, the binding of a mutant (TA) in which threonine (Thr) at position 1010 in the amino acid sequence of NCAPG2 was substituted with alanine (Ala) to the protoplast was confirmed by immunoprecipitation.

[0110] Specifically, 3xFLAG-NCAPG2pT1010 wild-type (WT) and pT1010A mutant DNA were overexpressed in HEK293 cells using transfection reagent (LTX / PLUS), and the cells were harvested after 24 hours. After cell lysis, the total protein yield was over 500 μg. NCAPG2 was pulled down using FLAG M2 affinity beads by centrifugation at 20 rpm at 4°C for over 3 hours. To increase purity, an excess of 3xFLAG peptide was added to the beads, and a competition process was performed to release NCAPG2 bound to the FLAG beads from the beads. The reaction was then centrifuged at 20 rpm at 4°C for 30 minutes to 1 hour. The supernatant was separated and used.

[0111] As a result, as shown in Figure 1a, the conventional commercially available NCAPG2 antibody (purchased from SIGMA ATLAS) detected the NCAPG2 mutant (TA) at almost the same level, and C DTPVHRGVLSpTLIA( It was confirmed that antibodies against the antigen (SEQ ID NO: 3) were reduced in the NCAPG2 mutant (TA), but were still detectable. Meanwhile, it was observed that antibodies against the C-HRGVLS(pT)LIAGPV-amide antigen prepared in Example 1 were not detected in the NCAPG2 mutant (TA). This confirmed that the NCAPG2pT1010-specific antibody prepared in the present invention has specific reactivity with the phosphorylation site of threonine at position 1010 in the amino acid sequence of NCAPG2 and has superior specificity for NCAPG2 pT1010 compared to antibodies against the CDTPVHRGVLSpTLIA antigen.

[0112] To identify the kinase responsible for phosphorylation, breast cancer cell line MBAMB231 was treated with the mitotic induction reagent nocodazole and then treated with inhibitors of kinases that regulate mitotic phase for 2 hours, after which the reactivity of NCAPG2 pT1010-specific antibodies was confirmed. The kinase inhibitors used were Aurora kinase B (ZM447439), CDK1 (RO3306), PLK1 (BI2536), Mps1, and Aurora kinase (Reversine). As shown in Figure 1b, inhibition of CDK1, Mps1, and Aurora kinase eliminated the phosphorylation-specific bands. These results were consistent across different inhibitor concentrations, as shown in Figure 1c.

[0113] Example 3: Confirmation of antibody specificity using immunocytochemistry To confirm whether the NCAPG2 pT1010-specific antibody prepared in Example 1 can be used for cell staining, we investigated whether the reactivity of this antibody was sufficiently specific when NCAPG2-specific expression was reduced using siRNA. To this end, siGFP or siNCAPG2 was transfected into MDA-MB-231 (program: X-013) cells using the AMAXA transfection method, and staining was performed 48 hours later.

[0114] For staining, the sections were fixed with 1% PFA and 0.2% Triton X-100, washed with PBS, blocked with 3% BSA / PBS, and then treated with primary antibodies at the following ratios.

[0115] 1) Anti-NCAPG2 1:100 / anti-CenpC 1:5000 / 3% BSA / PBS

[0116] 2) Old anti-pT1010 1:200 / anti-CenpC 1:5000 / 3% BSA / PBS

[0117] 3) New anti-pT1010(1158,2 ndpurify)1:200 / anti-CenpC 1:5000 / 3% BSA / PBS

[0118] Next, after washing with PBS, the sections were treated with secondary antibodies (Alexa 488α-rabbit / Alexa 594α-guineapig) at 1:1000, mounted with VECTASHIELD, and then observed under a confocal microscope.

[0119] Furthermore, after knocking out NCAPG2 using the Cas9 system, the cells were treated with antibodies at the same ratios as above and stained in the same manner.

[0120] After performing immunocytochemistry using the above method, the results for a conventional commercially available NCAPG2-reactive antibody are shown in Figure 2a, the results for the anti-pT1010 antibody of the CDTPVHRGVLSpTLIA antigen are shown in Figure 2b, and the results for the anti-pT1010 antibody of the C-HRGVLS(pT)LIAGPV-amide antigen prepared in Example 1 are shown in Figure 2c. By comparing the positions of the areas stained with anti-CenpC and anti-pT1010 antibodies, it can be seen that when the T1010 moiety of NCAPG2 is phosphorylated, the spindle fiber attachment point (kinetochore) in the chromosome is specifically stained.

[0121] When compared with Figure 1a, it was observed that the anti-pT1010 antibody against the HRGVLSpTLIAGPV-amide antigen was significantly reduced in the TA mutant compared to the previously prepared CDTPVHRGVLSpTLIA pT1010 antibody, indicating that the antibody selectivity and binding strength were more suitable for the HRGVLSpTLIAGPV-amide antigen. Furthermore, when compared with Figures 2a-2c, the results of the cell immunostaining experiment showed that the antibody was boosted and repurified (2 nd When the (purify)HRGVLSpTLIAGPV-amide rabbit antibody was used, it was found that selectivity was increased by showing a clearer staining pattern, and it was confirmed that when NCAPG2 expression was reduced using siRNA that inhibits mRNA or CRISPR-CAS9 that controls DNA, the staining image of the spindle attachment points stained by the antibody of the present invention became weaker.

[0122] This indicates that the selectivity and binding strength of the anti-pT1010 antibody of the present invention to the HRGVLSpTLIAGPV-amide antigen are more suitable than those of the conventionally prepared CDTPVHRGVLSpTLIApT1010 antibody.

[0123] Example 4: Comparison of immunostaining patterns in normal cells and cancer cells Phosphorylation of threonine 1010 of NCAPG2 is an important residue for interaction with PLK1 and normal cell division. Therefore, to investigate whether the phosphorylation pattern of threonine 1010 differs between normal and cancer cells, immunostaining experiments were performed using liver cancer (Hepg2, SNU-449), breast cancer (MDA-MB-468), pancreatic cancer (KP-3, Panc1), lung cancer (A549, H1299), and prostate cancer (LNCaP) cell lines. The antibody was boosted and purified again (2 nd Purify) 1158 rabbit antibody (1158, new anti-pT1010) was used.

[0124] Each cancer cell line was treated with 250 ng / ml nocodazole 4–5 hours before harvesting. Cells were harvested using trypsin / EDTA, washed with PBS, and counted. Subsequently, 5 × 10 5The cells were resuspended in PBS to a concentration of 100 cells / ml. Next, 100 μl of cells were placed in a cuvette and spin-dried at 1500 rpm for 5 minutes using a Cytospin spindle. To fix the cells, they were treated with 1% paraformaldehyde and 0.25% Triton X-100 at room temperature for 10 minutes and then washed with PBS. After blocking (3% BSA / PBS), the cells were incubated with primary antibodies (anti-pT1010 1:100 / anti-CenpC 1:5,000) overnight at 4°C. The next day, the cells were washed with PBS and then incubated with secondary antibodies (Alexa 488, anti-rabbit +594, anti-guinea pig 1:1000) for 2 hours at room temperature. After washing with PBS, the cells were stained with DAPI. After washing again with PBS, the cells were mounted and observed under an LSM780 confocal microscope.

[0125] As a result, as shown in Figure 3, in normal cells (HDFs), when treated with anti-pT1010 antibody, specific staining of CENPC (Centromere protein C), the attachment site between chromosomes and spindle fibers, occurs during cell division. However, in cancer cells, the staining intensity increases, and we confirmed that in many cases, spindle attachment point-specific staining does not occur during cell division due to chromosomal instability.

[0126] [Example 5. Confirmation of immunohistochemistry results in human cancer tissue] Immunohistochemistry is a method that uses antigen-antibody reactions to specifically detect target proteins in tissues, and is a method that visually identifies the location of target proteins that have reacted with antigens through DAB color development.

[0127] Using this immunohistochemical staining method, we confirmed the expression of NCAPG2 pT1010 in non-tumorous and tumorous areas of human liver cancer, cholangiocarcinoma, and pancreatic cancer tissues.

[0128] (5-1. Confirmation of immunohistochemical staining results in liver cancer tissue) Paraffin-embedded slides of human liver cancer tissues prepared through standard tissue processing procedures were subjected to immunohistochemical staining after hydration, antigen retrieval using citrate buffer, blocking of endogenous peroxidase using hydrogen peroxide, blocking of non-specific antibody reactions using normal horse serum, and secondary antibody binding of the NCAPG2 pT1010 antibody to the host to detect the specific antibody reaction against pT1010 of NCAPG2 prepared in Example 1, as well as the antigen-antibody reaction. Next, each patient's tissue was divided into tumor and non-tumor areas, and 3-4 representative cross-sections of each area were photographed at 200x magnification. The H-score system was used to quantify the positive reaction (the H-score is calculated by dividing the degree of positive reaction by 0, 1, 2, or 3, then multiplying the result by the percentage of cells corresponding to each score and adding up all the values; if all cells in the cross-section showed a strong positive reaction, a maximum score of 300 was given).

[0129] Human liver cancer tissue was stained using the above method, and the positive reaction was quantified. As shown in Figures 4a and 4b, the NCAPG2 anti-pT1010 antibody was confirmed to stain at the position of hematoxylin, which is used to stain nuclei and chromosomes in tissue staining methods. This showed that NCAPG2 pT1010 was expressed in the nuclei of most cells, and that its expression was significantly higher in tumor areas than in non-tumor areas in samples from 80 liver cancer patients.

[0130] (5-2. Confirmation of immunohistochemical staining results in bile duct cancer tissue) Immunohistochemical staining for NCAPG2 pT1010 was performed on tissues derived from bile duct cancer patients using the method described in Example 4-1. As a result, as shown in Figure 4c, by confirming the staining location of the antibody for NCAPG2 pT1010, it was found that NCAPG2 pT1010 expression was observed at very high levels in the nuclei of tumor cells in most bile duct cancer tissues, confirming that expression was significantly higher in tumor areas than in non-tumor areas.

[0131] (5-3. Confirmation of immunohistochemical staining results in pancreatic cancer tissue) Using the method of Example 4-1, the non-tumor / tumor regions of tissue samples derived from pancreatic cancer patients were observed under a 200x microscope. As a result, as shown in Figure 4d, by confirming the staining location of the antibody against NCAPG2 pT1010, it was observed that NCAPG2 pT1010 is specifically expressed in the nucleus, and that its expression was significantly higher in pancreatic cancer tissue than in normal pancreatic tissue.

[0132] [Example 6. Confirmation of NCAPG2pT1010 expression pattern depending on the degree of differentiation of pancreatic cancer] Immunohistochemical staining of NCAPG2pT1010 was performed on a total of 126 cases of human pancreatic cancer tissue, and the results confirmed that it was specifically stained in the nucleus, as confirmed in Example 5-3 above. The expression level was categorized into 0 to 3 using the Q-score system, and this expression level was compared with various clinical information such as the degree of tumor differentiation, lymph node metastasis rate, time to recurrence after surgery, and time to death after surgery. As shown in Figures 5a and 5b, the level of NCAPG2pT1010 expression in pancreatic cancer tissue tended to be higher as the tumor became less differentiated.

[0133] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. [Industrial Applicability]

[0134] The antibody for determining whether or not there is phosphorylation of threonine at position 1010 in the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) according to the present invention has high selectivity and binding to pT1010 of NCAPG2, and it was confirmed that the reactivity of NCAPG2 to pT1010 was suppressed in cancer cells treated with inhibitors of kinases that regulate cell division, such as CDK1, PLK1, Mps1, and Aurora kinase, and that it was detected at a significantly higher level in tumor tissues than in non-tumor tissues of cancer patients through immunohistochemical staining. Therefore, determining whether or not there is phosphorylation of threonine at position 1010 of NCAPG2 using the antibody according to the present invention can be useful in cancer-related research and development fields, such as diagnosing cancer or screening anti-cancer drug candidates, and thus has industrial applicability.

Claims

1. A method for producing an antibody for determining whether or not threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is phosphorylated, the method comprising the steps of: isolating the antibody from serum isolated from an individual injected with a peptide represented by the amino acid sequence of SEQ ID NO: 1; The amino acid sequence of NCAPG2 is represented by SEQ ID NO: 2, A method for producing an antibody, characterized in that the antibody has relatively higher affinity for a peptide in which the threonine, the seventh amino acid from the N-terminus of the peptide represented by the amino acid sequence of SEQ ID NO: 1, is phosphorylated compared to a peptide in which the threonine, the seventh amino acid from the N-terminus of the peptide represented by the amino acid sequence of SEQ ID NO: 1, is non-phosphorylated.

2. A method for providing information for cancer diagnosis, comprising: reacting the antibody produced by the method of claim 1 with cells expressing NCAPG2 (Non-SMC condensin II complex subunit G2) and confirming whether or not the threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is phosphorylated; and determining whether the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is an indicator of the presence of cancer cells. Including, The amino acid sequence of NCAPG2 is represented by SEQ ID NO: 2, A method for providing information for cancer diagnosis (excluding medical procedures for humans), wherein the cancer is at least one selected from the group consisting of liver cancer, bile duct cancer, pancreatic cancer, breast cancer, lung cancer, and prostate cancer.

3. The method for providing information for cancer diagnosis according to claim 2, characterized in that the higher the degree of phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2, the lower the degree of differentiation of tumor cells.

4. (a) treating cancer cells with a candidate substance; (b) reacting the cancer cells treated with the candidate substance with the antibody produced by the method of claim 1 to confirm whether or not the threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 (Non-SMC condensin II complex subunit G2) is phosphorylated; (c) selecting the candidate substance as a candidate anticancer drug when the phosphorylation of threonine at position 1010 from the N-terminus of the amino acid sequence of NCAPG2 is suppressed in cancer cells treated with the candidate substance in step (b); The amino acid sequence of NCAPG2 is represented by SEQ ID NO: 2, The method for screening candidate anticancer agents, wherein the cancer is at least one selected from the group consisting of liver cancer, bile duct cancer, pancreatic cancer, breast cancer, lung cancer, and prostate cancer.

5. 5. The method for screening anticancer drug candidate substances according to claim 4, wherein the anticancer drug candidate substances include at least one selected from the group consisting of PLK1 (Polo-like kinase 1) inhibitors, Mps1 (Monopolar spindle 1) inhibitors, Aurora kinase inhibitors, and CDK1 (Cyclin-dependent kinase 1) inhibitors.

6. The method for screening candidate anticancer agents according to claim 5, characterized in that the PLK1 inhibitor, Mps1 inhibitor, Aurora kinase inhibitor, or CDK1 inhibitor is at least one selected from the group consisting of nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, and natural extracts.

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