Anti-CK2α antibody or fragment thereof

Abstract

The present invention addresses the problem of providing a novel anti-CK2α antibody having improved specificity and sensitivity.　Provided is an anti-CK2α antibody or a fragment thereof comprising: (1) a heavy-chain variable region including CDR1 containing the amino acid sequence represented by SEQ ID NO: 5, CDR2 containing the amino acid sequence represented by SEQ ID NO: 6, and CDR3 containing the amino acid sequence FV, and a light-chain variable region including CDR1 containing the amino acid sequence represented by SEQ ID NO: 8, CDR2 containing the amino acid sequence represented by SEQ ID NO: 9, and CDR3 containing the amino acid sequence represented by SEQ ID NO: 10; and (2) a heavy-chain variable region including CDR1 containing the amino acid sequence represented by SEQ ID NO: 17, CDR2 containing the amino acid sequence represented by SEQ ID NO: 18, and CDR3 containing the amino acid sequence FV, and a light-chain variable region including CDR1 containing the amino acid sequence represented by SEQ ID NO: 20, CDR2 containing the amino acid sequence represented by SEQ ID NO: 21, and CDR3 containing the amino acid sequence represented by SEQ: 22.

Description

Anti-CK2α antibody or fragment thereof

[0001] The present invention relates to an anti-CK2α antibody or a fragment thereof, a kit for predicting the prognosis of a cancer patient, a method for predicting the prognosis of a cancer patient, and the like.

[0002] In Japan, cancer is the leading cause of death, accounting for approximately 30% of all deaths. For example, breast cancer is the leading cause of death among women aged 30 to 64, with approximately 14,000 deaths from breast cancer in 2018. While advances in cancer detection and / or treatment have improved survival rates for cancer patients, there are still patients with poor prognoses who are at high risk for recurrence, metastasis, or death. Therefore, in order to improve the quality of cancer treatment, including breast cancer, it is extremely important to predict the prognosis of cancer patients and provide individualized management based on the results.

[0003] The present inventors have discovered CK2α (Casein kinase 2α) protein as a new biomarker capable of predicting the prognosis of cancer patients, including breast cancer patients, with high accuracy (Patent Document 1). CK2α protein is present throughout the cell in normal cells, but is highly expressed in the nucleus of cancer cells and localized in the nucleolus, which correlates with poor prognosis. The present inventors have demonstrated that the nuclear expression level and nucleolar staining level of CK2α protein are strongly correlated with poor prognosis in cancer patients and are also strongly associated with the risk of recurrence (Patent Document 1). The method of using CK2α protein in the nucleolus as a biomarker is a groundbreaking technology that enables highly accurate prediction of the prognosis of cancer patients. However, existing anti-CK2α antibodies used for prognosis prediction lack sufficient specificity and sensitivity for CK2α protein.

[0004] Therefore, there is a need for new anti-CK2α antibodies that have improved specificity and sensitivity compared to conventional anti-CK2α antibodies.

[0005] International Publication No. 2021 / 132544

[0006] An object of the present invention is to provide new anti-CK2α antibodies with improved specificity and sensitivity.

[0007] The CK2α protein, together with the CK2α' and CK2β proteins, constitutes the serine-threonine kinase CK2 (Casein kinase 2). The CK2α protein is structurally similar to the CK2α' protein, and conventional anti-CK2α antibodies could not distinguish between the two.

[0008] To solve the above-mentioned problems, the present inventors developed mAb (6A3), a new antibody that exhibits extremely high specificity for CK2α protein. Many anti-CK2α antibodies produced in mice immunized with human CK2α protein exhibit cross-reactivity with CK2α' protein. By applying the criterion that mAb (6A3) binds only to CK2α protein and not to CK2α' protein, the present inventors succeeded in developing mAb (6A3) with extremely high specificity for CK2α protein. In Western blotting and immunoprecipitation of CK2α protein, mAb (6A3) demonstrated significantly higher specificity and sensitivity than conventional antibodies. Furthermore, mAb (6A3) detects CK2α protein localized in nucleoli with extremely high specificity and sensitivity in cancer tissues derived from cancer patients with poor prognosis, providing an extremely useful tool for biomarker detection. Based on the above findings, the present invention provides the following:

[0009] (1) An anti-CK2α antibody or a fragment thereof, comprising: (i) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence FV; and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 8, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10; (ii) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 17, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 18, and CDR3 consisting of the amino acid sequence FV; and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 21, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22; (iii) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 25, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 26, and CDR3 consisting of the amino acid sequence FV; (iv) a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 28, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 29, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 30; (iv) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 33, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and CDR3 consisting of the amino acid sequence FV, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 36, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 38; (v) a heavy chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 41, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 42, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 43, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 44, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 46; or (vi) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 49;The anti-CK2α antibody or a fragment thereof comprises a heavy chain variable region comprising CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 50 and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 51, and a light chain variable region comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 52, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 54. (2) The anti-CK2α antibody or a fragment thereof according to (1), comprising: (a) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12; (b) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24; (d) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 32; (e) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40; or (f) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 47, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 48. (3) A kit for predicting the prognosis of a cancer patient, comprising the anti-CK2α antibody or a fragment thereof according to (1) or (2). (4) The kit according to (3), wherein the prognosis includes a risk of recurrence. (5) The kit according to (3) or (4), wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma. (6) A method for predicting the prognosis of a cancer patient, comprising the steps of detecting CK2α protein or a fragment thereof in nucleoli in cancer cells or tissues obtained from the cancer patient, and predicting a poor prognosis when the CK2α protein or a fragment thereof is detected in the nucleoli at a higher level than in other cell fractions, and / or predicting a good prognosis when the CK2α protein or a fragment thereof is not detected in the nucleoli at a higher level than in other cell fractions.The method according to (6), wherein the CK2α protein or a fragment thereof is detected using the anti-CK2α antibody or a fragment thereof according to (1) or (2). (7) The method according to (6), wherein the prognosis includes a risk of recurrence. (8) The method according to (6) or (7), wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, renal cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma. (9) The method according to (8), wherein the cancer is breast cancer, and the presence or absence of detection of the CK2α protein or a fragment thereof is combined with at least one of classification by stage, classification by hormone receptor expression status, and classification by HER2 gene and / or protein expression status to predict the prognosis of the breast cancer patient. This specification incorporates the disclosures of Japanese Patent Application No. 2022-055606, from which the present application claims priority.

[0010] The present invention provides a new anti-CK2α antibody with improved specificity and sensitivity.

[0011] Figure 1 shows the results of Western blot analysis using mouse antisera and hybridoma culture supernatants. Figure 1A shows the results of Western blot analysis of recombinant CK2α protein (α) and recombinant CK2α' protein (α') using antisera from mice immunized with the antigen polypeptide. Figure 1B shows the results of Western blot analysis of recombinant CK2α protein (α) and recombinant CK2α' protein (α') using culture supernatants from clones 6A, 6B, 6C, and 7A. Figure 2 shows the results of Western blot analysis of CK2α protein in cytoplasmic lysates of HEK293 cells using culture supernatants from clones 6A1, 6A2, and 6A3 and a commercially available mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK; indicated as "control antibody" in the figure). A nonspecific band, indicated by an arrow, was detected with the control antibody. Figure 3 shows the results of Western blotting using mAb (6A3) and a control antibody to detect intracellular CK2α protein in HEK293 cells. Figure 3A shows the results of Western blotting. Figure 3B shows the results of quantification of the staining intensity of the band corresponding to CK2α protein. The figure shows the mean of three results for each condition. Error bars indicate SEM. Figure 4 shows the results of Western blotting using mAb (6A3) and a control antibody to detect Flag-CK2α protein and endogenous CK2α protein in cytoplasmic lysates from HEK293 cells expressing Flag-CK2α protein (Flag-CK2α(+) lane) and HEK293 cells not expressing Flag-CK2α protein (Flag-CK2α(-) lane). A nonspecific band was detected with the control antibody (right panel, arrow), whereas it was not detected with mAb (6A3) (left panel, arrow). Figure 5 shows the results of Western blotting of immunoprecipitates. The results are shown in Table 1. Lysates prepared from the cytoplasm (C) and nucleus (N) of HEK293 cells were immunoprecipitated with mAb (6A3) or a control antibody, and the immunoprecipitates were subjected to Western blotting with mAb (6A3) or a control antibody."No immunoprecipitation" indicates samples loaded with cytoplasmic (C) and nuclear (N) lysates without immunoprecipitation. Figure 6 shows the results of Western blotting of immunoprecipitates. The staining intensity of the band corresponding to CK2α was quantified in the left and right membranes in Figure 5. The figure shows the mean value of three results, and error bars indicate SEM. Figure 7 shows the results of Western blotting of immunoprecipitates. The immunoprecipitates obtained by immunoprecipitation of Flag-CK2α-expressing RPE cell lysates using mAb (6A3) or a control antibody were subjected to Western blotting using mAb (6A3) or an anti-Flag antibody. Figure 8 shows the results of Western blotting of immunoprecipitates. The staining intensity of the bands (two bands indicated by arrows in Figure 7) was quantified when mAb (6A3) or a control antibody was used for immunoprecipitation in Figure 7. The figure shows the mean value of three results, and error bars indicate SEM. Figure 9 shows representative images of immunohistochemical staining of the cancer infiltrate (lesion) of invasive ductal carcinoma. Figure 9A shows an image (magnification ×400) of the cancer infiltrate (lesion) stained with 0.1 μg / mL of mAb (6A3). A magnified image of the area marked with a black box is shown in the lower left. Figure 9B shows an image (magnification ×400) of the cancer infiltrate (lesion) stained with 2 μg / mL of a control antibody. A magnified image of the area marked with a black box is shown in the lower left. Figure 10 shows the results of Western blot analysis of immunoprecipitates from either MCF-7 cell line or HEK293 cells expressing Flag-CK2α protein, using IgG purified from 6A3 and several established CK2 antibody clones. The upper panel shows the results of detecting endogenous CK2α protein in MCF-7 cell line. The lower panel shows the results of detecting endogenous CK2α protein and Flag-CK2α protein in HEK293 cells expressing Flag-CK2α. Figure 11 shows the results of ChIP-qPCR targeting the HMGB2 locus performed on fractions obtained by chromatin immunoprecipitation with IgG purified from several CK2 antibody clones established in addition to 6A3.The "Control" lane indicates a sample loaded without IgG addition to an equivalently fractionated chromatin fraction. Figure 12 shows the results of Western blot analysis of purified recombinant CK2α protein. Figure 13 shows representative images of immunohistochemical staining of the cancer infiltrate (lesion) of invasive ductal carcinoma. Figure 13A shows an image (magnification ×400) of the cancer infiltrate (lesion) stained with 0.1 μg / mL of mAb (21B1). Figure 13B shows an image (magnification ×400) of the cancer infiltrate (lesion) stained with 2 μg / mL of the control antibody. Figure 14 shows the recurrence-free survival rates of the CK2α nucleolus-positive group ("CK2-NO(+)") and the CK2α nucleolus-negative group ("CK2-NO(-)") based on the results of histochemical staining using the 6A3 clone in FFPE samples from patients with primary lung adenocarcinoma who underwent resection. Figure 15 shows the results of univariate and multivariate analyses based on the results of histochemical staining using the 6A3 clone for FFPE samples from primary lung adenocarcinoma patients. Figure 15A shows the results of univariate analysis. Figure 15B shows the results of multivariate analysis. Figure 15C shows the results of analyzing the effects of factors determining recurrence-free survival in multivariate analysis. Figure 16 shows the results of analyzing variables determining the time to recurrence using two recurrence prediction models. Figure 16A shows the results of recurrence prediction model 1 based on six variables. Figure 16B shows the results of recurrence prediction model 2 based on seven variables, including age. Figure 17 shows the classification of stage I to III primary lung adenocarcinoma patients based on stage and CK2α staining assessment, distinguishing between those with and without recurrence.

[0012] (Definition of Terms) As used herein, "CK2α protein" refers to the α subunit of casein kinase 2 (CK2). The CK2α protein is also called casein kinase 2 alpha 1 or casein kinase II subunit alpha, CK2α1 protein, or CSNK2A1 protein. The CK2α protein, together with the CK2α' protein and the CK2β protein, constitutes the tetramer CK2.

[0013] Casein kinase 2 (KK2) is a serine-threonine kinase known to be involved in the pro-survival pathway. It functions as a tetramer consisting of an α subunit (CK2α protein), an α' subunit (CK2α' protein), and two β subunits (CK2β proteins). The α subunit (CK2α protein) and the α' subunit (CK2α' protein) function as catalytic subunits of KK2.

[0014] Herein, the CK2α protein or a fragment thereof can be a biomarker for predicting the prognosis of a cancer patient. For example, if the patient is human, the human CK2α protein or a fragment thereof can be a biomarker.

[0015] International Publication No. 2021 / 132544 discloses that CK2α protein in nucleoli can be a biomarker for predicting the prognosis of cancer patients, such as breast cancer patients. Specifically, immunohistochemical staining of CK2α protein was performed on formalin-fixed, paraffin-embedded breast cancer tissue specimens resected from primary breast cancer patients who underwent curative resection. Results revealed that while CK2α protein is present throughout the cell in normal cells, it is frequently highly expressed in the nucleus of breast cancer cells. Furthermore, it was found that in a proportion of breast cancer patients (slightly less than 40%), CK2α protein is localized in the nucleolus. Furthermore, higher levels of nuclear expression and nucleolar staining of CK2α protein are associated with a higher proportion of cases of higher stage breast cancer. Furthermore, evaluation of the prognosis of primary breast cancer patients revealed that nucleolar localization of CK2α protein is associated with a higher relative risk for both recurrence and survival, and that the presence or absence of nucleolar localization of CK2α protein is a strong predictor of recurrence. In addition, we investigated the localization of CK2α protein in various cancer tissues other than breast cancer and found that CK2α protein can be localized in the nucleoli in cancers in general, including breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer (rectal cancer and colon cancer), bladder cancer, lung cancer (pulmonary adenocarcinoma and lung squamous cell carcinoma), thyroid cancer, and glioma.

[0016] Unless otherwise specified, the term "marker" as used herein refers to a biomarker consisting of the CK2α protein or a fragment thereof, or a biomarker in the nucleolus consisting of the CK2α protein or a fragment thereof.

[0017] In the present specification, the type of "cancer" is not limited, and examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, etc. Specific examples of the type of cancer include malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder cancer and bile duct cancer), brain tumor, head and neck cancer, mesothelioma, osteosarcoma, glioma, pediatric tumors such as neuroblastoma, leukemia, lymphoma, etc. The cancer is preferably breast cancer, uterine cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, biliary tract cancer (e.g., gallbladder cancer or bile duct cancer), kidney cancer, colorectal cancer (e.g., rectal cancer or colon cancer), bladder cancer, lung cancer (e.g., lung adenocarcinoma or lung squamous cell carcinoma), thyroid cancer, or glioma (e.g., astrocytoma), more preferably breast cancer.

[0018] As used herein, the type of "breast cancer" is not limited, and examples include ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ, and special types of cancer such as medullary carcinoma, mucinous carcinoma, and tubular carcinoma.

[0019] As used herein, "prognosis" refers to the predicted course of a cancer patient (e.g., whether or not there is a recurrence, or whether the patient will survive or die). "Prognosis prediction" may be prediction of recurrence risk (e.g., recurrence-free survival rate), survival time, or survival rate at a certain time after surgery (e.g., 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years or more), relapse-free survival rate (RFS), or disease-specific survival rate (DFS). In one embodiment, prognosis prediction includes prediction of recurrence risk (e.g., relapse-free survival rate). Note that, as used herein, relapse-free survival rate refers to the proportion of patients who do not develop recurrent cancer, such as cancer associated with the primary cancer, and disease-specific survival rate refers to the proportion of patients who do not die from a disease associated with the primary cancer. Prognosis prediction can also be referred to as prognosis determination, evaluation, or diagnosis, or assistance thereto.

[0020] A specific example of the CK2α protein is a human-derived CK2α (human CK2α) protein that comprises or consists of the amino acid sequence shown in SEQ ID NO:2.

[0021] The CK2α protein also includes CK2α variants and CK2α orthologs of other organisms that have functionally equivalent activity to the CK2α protein shown in SEQ ID NO: 2. Specifically, the CK2α protein includes an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2, or a CK2α protein that has 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 2.

[0022] As used herein, "several" refers to, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 amino acids. Furthermore, conservative amino acid substitutions are desirable for amino acid substitutions. "Conservative amino acid substitutions" refer to substitutions between amino acids with similar properties, such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties can be classified into, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acids (leucine, valine, isoleucine), and aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine).

[0023] As used herein, "amino acid identity" refers to the percentage (%) of identical amino acid residues between two amino acid sequences relative to the total amino acid residues of the CK2α protein containing the amino acid sequence set forth in SEQ ID NO: 2, when the two amino acid sequences are aligned, with gaps introduced as necessary, to maximize the degree of amino acid identity between the two sequences. Amino acid identity can be calculated using a protein search system such as BLAST or FASTA. For details on how to determine identity, see, for example, Altschul et al., Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990.

[0024] The CK2α protein is encoded by the CK2α gene. A specific example of the CK2α gene is the human CK2α gene that encodes the human CK2α protein comprising the amino acid sequence shown in SEQ ID NO: 2. More specifically, the CK2α gene includes a gene comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 1.

[0025] The CK2α gene also encompasses CK2α variants having activity functionally equivalent to that of the CK2α protein encoded by the CK2α gene shown in SEQ ID NO: 1, and CK2α genes encoding CK2α orthologs from other organisms. Specifically, the CK2α gene encompasses a nucleotide sequence in which one or more nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 1, or a CK2α gene having 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more nucleotide identity to the nucleotide sequence shown in SEQ ID NO: 1. Furthermore, the CK2α gene encompasses a gene that contains a nucleotide sequence that hybridizes under highly stringent conditions to a nucleic acid fragment containing a portion of the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, and encodes a protein having activity functionally equivalent to that of the CK2α protein.

[0026] As used herein, "base identity" refers to the percentage (%) of identical bases between two base sequences relative to the total bases of the CK2α gene, including the base sequence shown in SEQ ID NO: 2, when the two base sequences are aligned and gaps are introduced, if necessary, to maximize the degree of base identity between the two.

[0027] As used herein, "hybridizing under highly stringent conditions" refers to hybridization and washing under conditions of low salt concentration and / or high temperature. For example, incubation with a probe in 6xSSC, 5xDenhardt's reagent, 0.5% SDS, and 100 μg / mL denatured, fragmented salmon sperm DNA at 65°C to 68°C can be exemplified, followed by washing in a 2xSSC, 0.1% SDS wash solution starting at room temperature, lowering the salt concentration in the wash solution to 0.1xSSC, and raising the temperature to 68°C until no background signal is detected. Highly stringent hybridization conditions are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and may be used for reference.

[0028] Such nucleotide sequence information of the CK2α gene can be searched for in public databases (GenBank, EMBL, DDBJ). For example, based on the known nucleotide sequence information of the CK2α gene shown in SEQ ID NO: 1, genes with high nucleotide identity can be searched for and obtained from the database.

[0029] As used herein, a "fragment" of a CK2α protein refers to a peptide fragment that contains or consists of a portion of the amino acid sequence constituting the CK2α protein and can be identified as a fragment of the CK2α protein from the amino acid sequence constituting the fragment. For example, a "fragment" may be 5 or more, 8 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid residues of the full-length amino acid sequence of the CK2α protein, or may be a peptide consisting of 200 or less, 150 or less, 120 or less, 100 or less, or 80 or less consecutive amino acid residues. For example, a "fragment" may be a peptide consisting of 5 to 200, 10 to 120, or 50 to 80 consecutive amino acid residues.

[0030] As used herein, the term "nucleolus" refers to a region in the nucleus of a eukaryotic cell that is densely packed with molecules and where rRNA transcription and ribosome production occur. Nucleoli can generally be observed with an optical microscope. Usually, one nucleolus is observed within a nucleus, but multiple nucleoli may also be observed.

[0031] (Anti-CK2α antibody or fragment thereof) In one aspect, the present invention relates to an anti-CK2α antibody or a fragment thereof. The anti-CK2α antibody or fragment thereof of the present invention can predict the prognosis of a cancer patient by detecting the CK2α protein or a peptide fragment thereof, which can be localized in the nucleolus in highly malignant cancers.

[0032] (1) Anti-CK2α Antibody As used herein, the term "anti-CK2α antibody" refers to an antibody that exhibits immunoreactivity to the CK2α protein or a peptide fragment thereof. The species from which the anti-CK2α antibody of the present invention is derived is not particularly limited. Antibodies derived from birds and mammals are preferred. Examples include chicken, ostrich, mouse, rat, guinea pig, rabbit, goat, donkey, sheep, camel, horse, and human.

[0033] The anti-CK2α antibody of the present invention is a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to a single type of immunoglobulin, or at least one set of light chain variable regions (V) contained in an immunoglobulin, that contains a framework region (hereinafter referred to as "FR") and a complementarity determining region (hereinafter referred to as "CDR") and is capable of specifically binding to and recognizing an antigen. L region) and heavy chain variable region (V H A recombinant or synthetic antibody refers to an antibody that contains a specific region.

[0034] When an anti-CK2α antibody is composed of an immunoglobulin molecule, the immunoglobulin can be of any class (e.g., IgG, IgE, IgM, IgA, IgD, and IgY) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The epitope of the CK2α protein or a peptide fragment thereof recognized by the anti-CK2α antibody of the present invention is an epitope specifically contained in the CK2α protein. Preferably, this epitope is not contained in the CK2α' protein.

[0035] A specific example of an anti-CK2α antibody that recognizes the above epitope is the murine anti-CK2α monoclonal antibody clone 6A3 (herein referred to as "mAb (6A3)") described in the Examples below. This mAb (6A3) has a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12. According to Kabat's rules (Kabat EA, et al., 1991, Sequences of proteins of immunological interest, Vol. 1, eds. 5, NIH publication), in the heavy chain variable region of mAb (6A3), CDR1 (HCDR1) consists of the amino acid sequence shown in SEQ ID NO: 5, CDR2 (HCDR2) consists of the amino acid sequence shown in SEQ ID NO: 6, and CDR3 (HCDR3) consists of the amino acid sequence shown in SEQ ID NO: 7 (FV). In the light chain variable region of mAb (6A3), CDR1 (LCDR1) consists of the amino acid sequence shown in SEQ ID NO: 8, CDR2 (LCDR2) consists of the amino acid sequence shown in SEQ ID NO: 9, and CDR3 (LCDR3) consists of the amino acid sequence shown in SEQ ID NO: 10. The amino acid sequences of SEQ ID NOs: 5 to 12 are shown in Table 1 below.

[0036]

[0037] Further specific examples of anti-CK2α antibodies that recognize the above-mentioned epitopes include the mouse anti-CK2α monoclonal antibody clones 10B2 (herein referred to as "mAb (10B2)"), 15C1 (herein referred to as "mAb (15C1)"), 16C2 (herein referred to as "mAb (16C2)"), 19C2 (herein referred to as "mAb (19C2)"), and 21B1 (herein referred to as "mAb (21B1)") described in the Examples below.

[0038] The heavy chain variable region of mAb (10B2) consists of the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 16. According to Kabat's rules, in the heavy chain variable region of mAb (10B2), CDR1 (HCDR1) consists of the amino acid sequence shown in SEQ ID NO: 17, CDR2 (HCDR2) consists of the amino acid sequence shown in SEQ ID NO: 18, and CDR3 (HCDR3) consists of the amino acid sequence shown in SEQ ID NO: 19. In addition, in the light chain variable region of mAb (10B2), CDR1 (LCDR1) consists of the amino acid sequence shown in SEQ ID NO: 20, CDR2 (LCDR2) consists of the amino acid sequence shown in SEQ ID NO: 21, and CDR3 (LCDR3) consists of the amino acid sequence shown in SEQ ID NO: 22.

[0039] The heavy chain variable region of mAb (15C1) consists of the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 24. According to Kabat's rules, in the heavy chain variable region of mAb (15C1), CDR1 (HCDR1) consists of the amino acid sequence shown in SEQ ID NO: 25, CDR2 (HCDR2) consists of the amino acid sequence shown in SEQ ID NO: 26, and CDR3 (HCDR3) consists of the amino acid sequence shown in SEQ ID NO: 27. In addition, in the light chain variable region of mAb (15C1), CDR1 (LCDR1) consists of the amino acid sequence shown in SEQ ID NO: 28, CDR2 (LCDR2) consists of the amino acid sequence shown in SEQ ID NO: 29, and CDR3 (LCDR3) consists of the amino acid sequence shown in SEQ ID NO: 30.

[0040] The heavy chain variable region of mAb (16C2) consists of the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 32. According to Kabat's rules, in the heavy chain variable region of mAb (16C2), CDR1 (HCDR1) consists of the amino acid sequence shown in SEQ ID NO: 33, CDR2 (HCDR2) consists of the amino acid sequence shown in SEQ ID NO: 34, and CDR3 (HCDR3) consists of the amino acid sequence shown in SEQ ID NO: 35. In addition, in the light chain variable region of mAb (16C2), CDR1 (LCDR1) consists of the amino acid sequence shown in SEQ ID NO: 36, CDR2 (LCDR2) consists of the amino acid sequence shown in SEQ ID NO: 37, and CDR3 (LCDR3) consists of the amino acid sequence shown in SEQ ID NO: 38.

[0041] The heavy chain variable region of mAb (19C2) consists of the amino acid sequence shown in SEQ ID NO: 39, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 40. According to Kabat's rules, in the heavy chain variable region of mAb (19C2), CDR1 (HCDR1) consists of the amino acid sequence shown in SEQ ID NO: 41, CDR2 (HCDR2) consists of the amino acid sequence shown in SEQ ID NO: 42, and CDR3 (HCDR3) consists of the amino acid sequence shown in SEQ ID NO: 43. In addition, in the light chain variable region of mAb (19C2), CDR1 (LCDR1) consists of the amino acid sequence shown in SEQ ID NO: 44, CDR2 (LCDR2) consists of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 (LCDR3) consists of the amino acid sequence shown in SEQ ID NO: 46.

[0042] The heavy chain variable region of mAb (21B1) consists of the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 48. According to Kabat's rules, in the heavy chain variable region of mAb (21B1), CDR1 (HCDR1) consists of the amino acid sequence shown in SEQ ID NO: 49, CDR2 (HCDR2) consists of the amino acid sequence shown in SEQ ID NO: 50, and CDR3 (HCDR3) consists of the amino acid sequence shown in SEQ ID NO: 51. In addition, in the light chain variable region of mAb (21B1), CDR1 (LCDR1) consists of the amino acid sequence shown in SEQ ID NO: 52, CDR2 (LCDR2) consists of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 (LCDR3) consists of the amino acid sequence shown in SEQ ID NO: 54.

[0043] An example of a nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 11, which corresponds to the heavy chain variable region of mAb (6A3), is a nucleic acid consisting of the base sequence shown in SEQ ID NO: 13. An example of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 12, which corresponds to the light chain variable region of mAb (6A3), is a nucleic acid consisting of the base sequence shown in SEQ ID NO: 14.

[0044] An example of a nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 15, which corresponds to the heavy chain variable region of mAb (10B2), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 55. Furthermore, an example of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 16, which corresponds to the light chain variable region of mAb (10B2), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 56.

[0045] An example of a nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 23, which corresponds to the heavy chain variable region of mAb (15C1), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 57. Furthermore, an example of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 24, which corresponds to the light chain variable region of mAb (15C1), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 58.

[0046] An example of a nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 31, which corresponds to the heavy chain variable region of mAb (16C2), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 59. Furthermore, an example of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 32, which corresponds to the light chain variable region of mAb (16C2), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 60.

[0047] An example of a nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 39, which corresponds to the heavy chain variable region of mAb (19C2), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 61. Furthermore, an example of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 40, which corresponds to the light chain variable region of mAb (19C2), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 62.

[0048] An example of a nucleic acid (nucleotide) encoding the amino acid sequence shown in SEQ ID NO: 47, which corresponds to the heavy chain variable region of mAb (21B1), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 63. Furthermore, an example of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 48, which corresponds to the light chain variable region of mAb (21B1), is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 64.

[0049] "Recombinant antibody" refers to a chimeric antibody or a humanized antibody. A "chimeric antibody" is an antibody created by combining the amino acid sequences of antibodies from different animals, in which the constant region (C region) of one antibody is replaced with the C region of another antibody. For example, an antibody in which the C region of a mouse monoclonal antibody is replaced with the C region of a human antibody falls into this category. A specific example is an antibody in which the heavy chain variable region of a human antibody against a given antigen is replaced with the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11 of the aforementioned mAb (6A3), and the light chain variable region of the human antibody is replaced with the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12. This can reduce the immune response to the antibody in the human body. A "humanized antibody" is a mosaic antibody in which the CDRs of a human antibody are replaced with the CDRs of an antibody derived from a non-human mammal. The variable region (V region) of an immunoglobulin molecule is composed of four FRs (FR1, FR2, FR3, and FR4) and three CDRs (CDR1, CDR2, and CDR3) linked in the following order from the N-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The FRs are relatively conserved regions that form the framework of the variable region, while the CDRs directly contribute to the antigen-binding specificity of the antibody. Humanized antibodies can be constructed, for example, by replacing a set of CDR1, CDR2, and CDR3 in the light or heavy chain of mouse-derived mAb (6A3) with a set of CDR1, CDR2, and CDR3 in the light or heavy chain of a human antibody specific for any antigen, thereby resulting in a human antibody that inherits the antigen-binding specificity of the mouse antibody mAb (6A3). Specific examples include antibodies in which CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 7, which are derived from the heavy chain of the aforementioned mAb (6A3), are substituted with the heavy chain CDR1, CDR2, and CDR3 of a human antibody, respectively, and in which CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 8, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 9, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10, which are derived from the light chain of the aforementioned mAb (6A3), are substituted with the light chain CDR1, CDR2, and CDR3 of a human antibody, respectively.Such humanized antibodies are derived from human antibodies except for the CDRs, and therefore can reduce immune responses against the antibodies in the human body more than chimeric antibodies.

[0050] "Synthetic antibody" refers to an antibody synthesized chemically or by using recombinant DNA technology. For example, it includes antibodies newly synthesized using recombinant DNA technology. Specific examples include scFv (single chain fragment of variable region), diabody, triabody, tetrabody, etc. In an immunoglobulin molecule, a set of variable regions (light chain variable region V) that form a functional antigen-binding site are included. L and heavy chain variable region V H ) are located on separate polypeptide chains, known as light and heavy chains. scFvs are fragments of an immunoglobulin molecule that are separated by a V L and V H A synthetic antibody with a molecular weight of approximately 35 kDa or less has a structure in which two variable regions are linked by a flexible linker of sufficient length and incorporated into a single polypeptide chain. Within an scFv, a pair of variable regions can self-assemble to form a functional antigen-binding site. An scFv can be obtained by incorporating recombinant DNA encoding it into a vector using known techniques and expressing it. A diabody is a molecule based on the dimeric structure of scFvs (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). For example, if the linker length is shorter than approximately 12 amino acid residues, the two variable regions within the scFv cannot self-assemble, but by allowing two scFvs to interact to form a diabody, the V of one scFv can be disassembled. L is the V of the other scFv HThe scFv fragments can aggregate with each other to form two functional antigen-binding sites. Furthermore, adding a cysteine ​​residue to the C-terminus of an scFv allows disulfide bonding between two scFvs, resulting in the formation of a stable diabody. Thus, diabodies are bivalent antibody fragments. Triabodies and tetrabodies, like diabodies, are trivalent and tetravalent antibodies based on the scFv structure, possessing trimer and tetramer structures, respectively. Diabodies, triabodies, and tetrabodies may also be multispecific antibodies. A "multispecific antibody" refers to a multivalent antibody, i.e., an antibody that has multiple antigen-binding sites within a single molecule, each of which binds to a different epitope. For example, a diabody may be a bispecific antibody, in which each antigen-binding site binds to a different epitope. Specifically, for example, the anti-CK2α antibody of the present invention corresponds to a diabody in which one antigen-binding site comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12, and the other antigen-binding site binds to a different epitope.

[0051] The anti-CK2α antibodies of the present invention can also be modified. As used herein, "modification" includes functional modifications necessary for antigen-specific binding activity, such as glycosylation, and labeling modifications necessary for antibody detection.

[0052] Glycosylation modifications on anti-CK2α antibodies are performed to adjust the affinity of the anti-CK2α antibody for the target CK2α protein or a peptide fragment thereof. Specific examples include modifications such as introducing substitutions into the amino acid residues that make up the glycosylation in the FR of the anti-CK2α antibody to remove the glycosylation site, thereby eliminating the glycosylation at that site.

[0053] The anti-CK2α antibody can be labeled with, for example, fluorescent dyes (FITC, rhodamine, Texas Red, Cy3, Cy5), fluorescent proteins (e.g., PE, APC, GFP), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase), radioisotopes (e.g., 3H, 14 C. 35 Examples of such labels include those labeled with ATP (S) or biotin or (strept)avidin.

[0054] The anti-CK2α antibody of the present invention has a dissociation constant with CK2α protein of 10 -7 It is preferable that the value is equal to or less than M, for example, 10 -8 It is preferable that the affinity is as high as 10 M or less, and more preferably 10 -9 M or less, particularly preferably 10 -10 The dissociation constant is equal to or less than M. The dissociation constant can be measured using techniques known in the art. For example, it may be measured using a Biacore system (GE Healthcare) with rate evaluation kit software.

[0055] (2) Fragments thereof As used herein, the term "fragment thereof" refers to an antibody fragment that comprises a portion of an anti-CK2α antibody and exhibits immunoreactivity to the CK2α protein or a fragment thereof, similar to the anti-CK2α antibody. Examples of such fragments include Fab, F(ab')2, Fab', Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), dimeric scFv (bivalent diabodies), multispecific antibodies, heavy-chain antibodies such as camelized single-domain antibodies (camelized antibodies; VHH antibodies), nanobodies, domain antibodies, and bivalent domain antibodies.

[0056] Fab is an antibody fragment generated by cleavage of an IgG molecule with papain at the N-terminal side of the hinge disulfide bond, and contains the heavy chain constant region (heavy chain constant region: hereinafter referred to as C H The three domains that make up the H 1. C H 2. C H 3) V H C adjacent to H 1 and V H , and a full-length L chain.

[0057] F(ab')2 is a dimer of Fab' produced by pepsin cleavage of an IgG molecule at the C-terminal side of the disulfide bond in the hinge region. Fab' has a slightly longer heavy chain than Fab due to the inclusion of the hinge region, but has essentially the same structure as Fab. Fab' can be obtained by reducing F(ab')2 under mild conditions to cleave the disulfide bond in the hinge region. Since all of these antibody fragments contain an antigen-binding site, they have the ability to specifically bind to an antigen epitope.

[0058] (3) Preparation of Anti-CK2α Antibody The anti-CK2α antibody of the present invention can be obtained by conventional methods in the art. Furthermore, if the amino acid sequence of a monoclonal antibody is known, it can also be prepared by chemical synthesis or recombinant DNA technology based on the amino acid sequence. Furthermore, a monoclonal antibody can also be obtained from a hybridoma that produces the antibody.

[0059] The antigenic polypeptide or antigenic peptide that can be used as an immunogen for the anti-CK2α antibody of the present invention is any part or the full-length of the CK2α protein (hereinafter referred to as a "CK2α antigenic polypeptide"). For example, an example of an antigenic polypeptide that can be used as an immunogen for the anti-CK2α antibody of the present invention is the full-length human CK2α protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. The CK2α antigenic peptide can be prepared, for example, by chemical synthesis or DNA recombinant technology.

[0060] In one embodiment, the present invention provides an anti-CK2α antibody or a fragment thereof for predicting the prognosis of a cancer patient.

[0061] The anti-CK2α antibody or a fragment thereof of the present invention can specifically detect CK2α protein. For example, highly sensitive and specific detection is possible by using the anti-CK2α antibody or a fragment thereof of the present invention in immunohistochemical staining, enzyme-linked immunosorbent assay (including ELISA and EIA), Western blotting, radioimmunoassay (RIA), immunoprecipitation, chromatin immunoprecipitation (CHIP), or flow cytometry.

[0062] (Kit for predicting the prognosis of cancer patients) In one aspect, the present invention relates to a kit for predicting the prognosis of cancer patients. The kit for predicting the prognosis of cancer patients of the present invention comprises the above-mentioned anti-CK2α antibody or an immunoreactive fragment thereof as an essential component, and is capable of detecting a biomarker for predicting the prognosis of cancer patients, which biomarker consists of CK2α protein. The kit for predicting the prognosis of cancer patients of the present invention comprises, as a selected component, an antibody (hereinafter referred to as "other prognosis prediction antibody") against a biomarker for predicting the prognosis of cancer patients other than CK2α protein or a peptide fragment thereof (hereinafter referred to as "other prognosis prediction biomarker").

[0063] (1) Essential Components The kit for predicting the prognosis of a cancer patient of the present invention contains the above-mentioned anti-CK2α antibody or a fragment thereof as an essential component. The anti-CK2α antibody contained in the kit for predicting the prognosis of a cancer patient of the present invention may be a single type or multiple types.

[0064] (2) Selected Components The kit for predicting the prognosis of a cancer patient of the present invention may further include one or more other prognostic antibodies or immunoreactive fragments thereof as selected components. The other prognostic antibodies may be any antibodies that can improve the accuracy of prognosis prediction for a cancer patient when used in combination with the anti-CK2α antibody. Antibodies against any cancer prognostic biomarkers can be used. Such biomarkers can be selected from known cancer markers.

[0065] In addition to the above-mentioned essential components, the kit for predicting the prognosis of cancer patients of the present invention may also include other reagents necessary for predicting the prognosis of cancer patients, such as known reagents for immunohistochemical staining, ELISA, Western blotting, etc., such as labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, instruments and control buffers necessary for the test, and instructions for use in detection and interpretation of the results.

[0066] (Method for predicting the prognosis of a cancer patient) In one aspect, the present invention relates to a method for predicting the prognosis of a cancer patient.

[0067] In one embodiment, the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, biliary tract cancer, kidney cancer, colon cancer, bladder cancer, lung cancer, thyroid cancer, and glioma.

[0068] In one embodiment, the present invention predicts the prognosis of cancer patients by combining a marker consisting of CK2α protein or a fragment thereof with factors such as stage classification, tumor size, the presence or absence of lymph node metastasis, and histological grade.

[0069] In one embodiment, the cancer is breast cancer, and the marker is combined with at least one, for example, two, and preferably all three, of classifications based on stage, hormone receptor expression status, and HER2 gene and / or protein expression status to predict the prognosis of breast cancer patients. In one embodiment, the marker is combined with other factors such as tumor size, presence or absence of lymph node metastasis, and histological grade, in addition to or separately from the above classifications, to predict the prognosis of breast cancer patients. Combining the marker with other classifications or factors can have the effect of enabling better prognosis prediction.

[0070] As used herein, stage classification refers to a stage classification based on the TNM classification of the Union for International Cancer Control (UICC) (UICC International Convention, LH Sobin, MK Gospodarowicz and Ch. Wittekind, TNM Classification of Malignant Tumors, 7th edition). The above-mentioned TNM classification of the Union for International Cancer Control (UICC) is referred to herein as the UICC-TNM classification. In the UICC-TNM classification, breast cancer is classified into stages 0, I, II, III, and IV, in descending order of progression. The UICC-TNM classification classifies the progression of cancerous lesions based on three factors: the size of the lump and its spread within the breast (T classification), lymph node metastasis (N classification), and distant metastasis (M classification). Determining the stage of disease based on the UICC-TNM classification can be done according to the common knowledge of those skilled in the art.

[0071] Specifically, stage 0 is when breast cancer is confined to the milk ducts, stage I is when the breast cancer tumor is 2cm or less in diameter and has no axillary lymph node metastasis or has micrometastasis of 0.2mm or less, stage II is when the tumor is more than 2cm in diameter and has no axillary lymph node metastasis or is less than 5cm in diameter and has three or fewer axillary lymph node metastases, stage III is when the tumor has 4-9 axillary lymph node metastasis regardless of size (including cases where there is no axillary lymph node metastasis but clinically clear parasternal lymph node metastasis), or when the tumor is more than 5cm in diameter and has nine or fewer axillary lymph node metastasis, or when the tumor has invaded the chest wall, or when there are skin ulcers, satellite nodules, or skin edema, regardless of tumor size, or in the case of inflammatory breast cancer, regardless of lymph node metastasis. If there are 10 or more axillary lymph node metastases, or if there are axillary and parasternal lymph node metastases, or ipsilateral supraclavicular lymph node metastases, the tumor is classified as stage III, regardless of its condition. If there is distant metastasis, the tumor is classified as stage IV. In the examples described below, all stages are shown as stages after postoperative pathological diagnosis (p stage).

[0072] Classification based on hormone receptor expression status refers to classification based on the expression status of estrogen receptor (ER) and / or progesterone receptor (PgR), for example, the presence or absence of expression (positive or negative) or the level of expression. The expression status of ER and PgR may refer to the expression status of genes encoding these proteins, but is preferably the expression status of these proteins. Classification based on the expression status of HER2 gene and / or protein may refer to the presence or absence of expression (positive or negative) or the level of expression of HER2 gene and / or protein. Methods for measuring the expression status of ER, PgR, and HER2 are known to those skilled in the art and include, but are not limited to, immunological detection methods such as immunohistochemical staining for protein detection, and nucleic acid detection methods such as nucleic acid amplification methods using primers or hybridization methods using probes (e.g., FISH (Fluorescence In Situ Hybridization)).

[0073] When classifying tumors by combining ER, PgR, and HER2, they can be classified into the following three groups: (1) hormone receptor positive / HER2 negative, in which ER and / or PgR are expressed but HER2 is not; (2) HER2 positive, in which HER2 is expressed regardless of whether ER and PgR are expressed; and (3) triple negative, in which none of ER, PgR, or HER2 is expressed.

[0074] In one embodiment, the presence or absence or level of expression of CK2α protein or a fragment thereof in the nucleolus is used as a marker for predicting the prognosis of a cancer patient. The presence or absence or level of expression is described in detail below.

[0075] In one aspect, the present invention relates to a method for predicting the prognosis of a cancer patient. The method includes the steps of detecting CK2α protein or a fragment thereof in nucleoli in cancer cells or tissues obtained from the cancer patient, and predicting a poor prognosis if CK2α protein or a fragment thereof is detected, and / or predicting a good prognosis if CK2α protein or a fragment thereof is not detected. The detection step can be performed in vitro. Furthermore, the detection of CK2α protein or a fragment thereof in nucleoli can be performed using the above-mentioned anti-CK2α antibody or an immunoreactive fragment thereof.

[0076] In one aspect, the present invention relates to a method for predicting the prognosis of a cancer patient. The method includes the steps of detecting CK2α protein or a fragment thereof in nucleoli in cancer cells or tissues obtained from the cancer patient, and predicting a poor prognosis if CK2α protein or a fragment thereof is detected in nucleoli at a higher level than in other cellular fractions, and / or predicting a good prognosis if CK2α protein or a fragment thereof is not detected in nucleoli at a higher level than in other cellular fractions. Here, "other cellular fractions" are not limited to cellular fractions other than nucleoli, and may include, for example, cytoplasm or nucleoplasm (nuclear fluid). Furthermore, "when CK2α protein or a fragment thereof is not detected in nucleoli at a higher level than in other cellular fractions" includes cases where CK2α protein or a fragment thereof is detected in nucleoli at a similar level to other cellular fractions (including cases where CK2α protein or a fragment thereof is detected uniformly throughout the cell) and cases where CK2α protein or a fragment thereof is detected in other cellular fractions at a higher level than in nucleoli. The detection step can be performed in vitro. Furthermore, the CK2α protein or a fragment thereof in the nucleolus can be detected using the above-mentioned anti-CK2α antibody or a fragment thereof having immunoreactivity.

[0077] Each step will be specifically described below. (1) Detection Step The stage of cancer suffered by patients who are the subject of the present invention is not limited. For example, in the case of breast cancer, the breast cancer suffered by patients who are the subject of the present invention may be stage I to IV, such as stage I to III or stage III. Cancer patients in the present invention are, for example, mammals, preferably primates, and more preferably humans.

[0078] The cancer cells or tissues used in the present invention are not particularly limited and can be obtained from cancer patients by, for example, biopsy or surgical resection. The cells or tissues may be used directly for marker detection, or may be appropriately pretreated for measurement. For example, when detecting markers by immunohistochemical staining, paraffin-embedded sections may be prepared from patient-derived samples. Furthermore, when detecting biomarkers by Western blotting, for example, nuclei or nucleoli may be isolated from patient-derived samples to prepare protein extracts.

[0079] The marker detected by this method may be either the CK2α protein or a fragment thereof. Detection includes measuring the presence or absence of expression, the amount of expression, or the level of expression concentration. As used herein, the term "detection" includes measurement, qualitative, quantitative, and semi-quantitative.

[0080] The method for detecting the CK2α protein or a fragment thereof may be any known protein detection method, and is not particularly limited, but examples thereof include immunological detection methods.

[0081] The "immunological detection method" is a method for measuring the amount of a target molecule (antigen) using an antibody or antibody fragment that specifically binds to the target molecule. Specifically, the above-mentioned CK2α protein or a fragment thereof can be used in this step.

[0082] Immunological detection methods include, for example, immunohistochemical staining, enzyme-linked immunosorbent assay (including ELISA and EIA), Western blotting, radioimmunoassay (RIA), immunoprecipitation, chromatin immunoprecipitation (CHIP), or flow cytometry.

[0083] Known methods can be used for the "immunohistochemical staining method." For example, a patient-derived sample may be fixed in formalin, embedded in paraffin, sliced ​​into tissue pieces, and attached to a glass slide to be used as a section sample. Immunohistochemical staining may be performed on the section sample, optionally after activating the antigen by heat treatment, using a primary antibody that recognizes the CK2α protein or a fragment thereof (specifically, the above-mentioned CK2α protein or a fragment thereof) and a labeled secondary antibody that recognizes the primary antibody.

[0084] Furthermore, for methods that cannot confirm the expression site, such as Western blotting, the expression of CK2α protein or a fragment thereof in the nucleolus can be confirmed by performing the method on a sample from which nucleoli have been isolated in advance.

[0085] The above-mentioned measurement methods are all well known in the art. Therefore, specific measurement methods may be performed in accordance with known methods. For example, the method described in Green, MR and Sambrook, J., 2012 (mentioned above) can be used as a reference.

[0086] (2) Prediction Step In this step, the prognosis of the cancer patient is predicted based on the measurement results obtained in the measurement step. In one embodiment, this step includes determining whether the cancer cells or tissue are positive or negative for the marker from the results obtained in the detection step. If the cancer cells or tissue are negative for the marker, the prognosis of the cancer patient can be predicted to be good. On the other hand, if the cancer cells or tissue are positive for the marker, the prognosis of the cancer patient can be predicted to be poor.

[0087] When immunohistochemical staining is used, for example, staining of one or more cells or cell clusters can be considered positive, while the absence of stained tumor cells can be considered negative. Alternatively, a positive result can be determined when the number of stained tumor cells exceeds a certain percentage (e.g., 10%, 15%, or 20%) of the total number of tumor cells, and a negative result can be determined when the number of stained tumor cells is equal to or less than this certain percentage. In immunohistochemical staining, sections can be classified into the following five categories: I, II, III, IV, and V. I: Staining of the entire cell, but no clear nuclear staining. II: Nuclear staining (+), nuclear staining more clearly than cytoplasm. III: Nuclear staining (++), nuclear staining at a higher level than II. IV: Nuclear staining (+, ++), and nucleolar staining (+). V: Nuclear staining (-), nucleolar staining (+). In the above classification, IV and V can be considered to indicate positive CK2α protein or its fragments in the nucleolus.

[0088] In one embodiment, the prediction step comprises determining whether the expression level of a marker in the cancer cells or tissue obtained in the detection step is higher or lower (e.g., than a predetermined threshold). If the expression level of a marker in the cancer cells or tissue is lower (e.g., statistically significantly lower) than a predetermined threshold, the prognosis of the cancer patient may be predicted to be good (e.g., relative to a population having an expression level higher than the predetermined threshold). On the other hand, if the expression level of a biomarker in the cancer cells or tissue is higher (e.g., statistically significantly higher) than a predetermined threshold, the prognosis of the cancer patient may be predicted to be poor (e.g., relative to a population having an expression level lower than the predetermined threshold).

[0089] The predetermined threshold may be a control amount measured in a control sample (control cells or tissue, e.g., control breast cells or breast tissue). The control sample may be derived from a healthy individual (e.g., a healthy person), a benign breast tumor, or a breast cancer patient (e.g., a stage II breast cancer patient). In the present invention, a "healthy individual" refers to a healthy individual of the same species as the test individual who is not affected with cancer.

[0090] For example, the expression level in these individuals, or the median, average, upper limit, lower limit, or range of the expression level in multiple individuals can be used as the predetermined threshold. The threshold can be set appropriately depending on the accuracy of prediction, and can be determined, for example, by ROC (receiver operating characteristic curve) analysis.

[0091] As used herein, "statistically significant" refers to a case where the risk rate (significance level) of the obtained value is small, specifically, p<0.05 (less than 5%), p<0.01 (less than 1%), or p<0.001 (less than 0.1%). The statistical test method is not particularly limited and any known test method capable of determining the presence or absence of significance may be used as appropriate. For example, the Student's t-test, multiple comparison test, or log-rank test may be used.

[0092] As used herein, "poor prognosis" refers to poor clinical outcomes (e.g., after surgical resection) (e.g., a high risk or rate of recurrence of cancer such as breast cancer, a low recurrence-free survival rate, a low disease (cancer)-specific survival rate, or a low overall survival rate). In the case of a poor prognosis, the 5-year recurrence-free survival rate or disease-specific survival rate may be 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less. In the present invention, survival rate refers to cumulative survival rate.

[0093] As used herein, "good prognosis" refers to a good clinical outcome. A good prognosis may be a recurrence-free survival rate or survival rate 5 years after cancer resection surgery of 90% or more, 95% or more, or 100%.

[0094] According to the present invention, it is possible to predict the prognosis of a cancer patient, and based on the results, it is possible to determine a treatment plan (e.g., type of anticancer drug, dosage, administration interval, etc.) or determine the intervals between tests for cancer recurrence and metastasis.

[0095] When a cancer patient is predicted to have a poor prognosis according to the present invention, the patient may be administered chemotherapy and / or radiation therapy to prevent cancer recurrence, improve the prognosis, or improve the survival rate. Therefore, the present invention also provides a method for preventing cancer recurrence, improve the prognosis, or improve the survival rate, comprising administering at least one of chemotherapy and radiation therapy to a cancer patient predicted to have a poor prognosis according to the method of the present invention. Furthermore, when a cancer patient is predicted to have a poor prognosis, the frequency of examinations can be increased to detect cancer recurrence early.

[0096] Drugs include, but are not limited to, anticancer drugs such as doxorubicin, cyclophosphamide, 5-fluorouracil (5-FU), capecitabine, oxaliplatin, and irinotecan; hormone therapy drugs such as antiestrogens (e.g., tamoxifen), LH-RH agonist preparations (e.g., leuprorelin), aromatase inhibitors (e.g., anastrozole), and progesterone preparations; and antibody drugs such as HER2 antibodies (e.g., trastuzumab). Drugs can be used alone or in combination. Drugs can be administered by injection, intravenous administration, oral administration, or other routes.

[0097] In one embodiment, the methods described herein predict the prognosis of a cancer patient by combining the presence or absence of detection of CK2α protein or a fragment thereof with factors such as stage classification, tumor size, the presence or absence of lymph node metastasis, and histological grade.

[0098] In one embodiment, in the method described herein, the cancer is breast cancer, and the prognosis of a breast cancer patient is predicted based on the presence or absence of detection of CK2α protein or a fragment thereof in combination with at least one of classification by stage, classification by hormone receptor expression status, and classification by HER2 gene and / or protein expression status. Classification by stage, classification by hormone receptor expression status, and HER2 gene and / or protein expression status are as described in the section (Use as a marker for prognosis prediction). In one embodiment, the method described herein predicts the prognosis of a breast cancer patient by combining the presence or absence of detection of CK2α protein or a fragment thereof with other factors, such as tumor size, presence or absence of lymph node metastasis, and histological grade, in addition to or separately from the above classification. Combining this with other classifications or factors can have the effect of enabling more accurate prognosis prediction.

[0099] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0100] Example 1: Preparation of anti-CK2α monoclonal antibody (Objective) To develop a monoclonal antibody (anti-CK2α monoclonal antibody) that can specifically and sensitively detect CK2α protein.

[0101] (Methods and Results) (1) Immunization with Antigen Polypeptide The production of anti-CK2α monoclonal antibodies was partially outsourced to Immuno-Biological Laboratories Co., Ltd. Specifically, the human CK2α protein shown in SEQ ID NO: 2 to which a GST tag was attached was expressed in Escherichia coli (DE3), and the GST tag was then cleaved by thrombin treatment, resulting in the purified polypeptide, which was then produced as an antigen. Mice were then immunized by Immuno-Biological Laboratories Co., Ltd. After immunization, test blood samples were taken and antibody titers were confirmed by ELISA.

[0102] Figure 1A shows the results of Western blot analysis of antisera obtained from immunized mice against recombinant CK2α protein and recombinant CK2α' protein. Western blot analysis was performed using anti-mouse IgG-HRP antibody (Abcam, #6789) as the secondary antibody and chemiluminescent detection reagent (Thermo Scientific, #32209). Antisera obtained from mice showed reactivity to both CK2α protein and the structurally similar CK2α' protein. This suggests that many of the anti-CK2α antibodies produced in mice immunized with human CK2α protein exhibit cross-reactivity to CK2α' protein.

[0103] (2) Cell fusion and screening. Lymphocytes were isolated from immunized mice and fused with myeloma cells. The culture supernatants were screened for antibody production using ELISA. Hybridomas were then cloned by limiting dilution using good wells and screened by ELISA. Four hybridoma clones, 6A, 6B, 6C, and 7A, were obtained that selectively reacted with CK2α protein but not with CK2α' protein.

[0104] (3) Analysis of Selected Clones Figure 1B shows the results of Western blot analysis of the culture supernatants of clones 6A, 6B, 6C, and 7A against recombinant CK2α protein and recombinant CK2α' protein. Clones 6A, 6B, 6C, and 7A all selectively reacted with CK2α protein but showed no reactivity with CK2α' protein.

[0105] (4) Further Cloning of Clone 6A Clones 6A1, 6A2, and 6A3 were obtained by further limiting dilution cloning of clone 6A. Figure 2 shows the results of detecting CK2α protein in cytoplasmic lysates of Flag-CK2α-expressing HEK293 cells using the culture supernatants of clones 6A1, 6A2, and 6A3. Figure 2 also shows the results of detection using a commercially available mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK) as a control (Figure 2, "Control antibody"). In addition to the CK2α protein band, the control antibody detected a nonspecific band at the higher molecular weight. Clones 6A1, 6A2, and 6A3 did not detect such a band. These clones 6A1, 6A2, and 6A3 demonstrated highly specific antibodies.

[0106] (5) Purification of IgG from Clone 6A3 Clone 6A3 was grown in serum-supplemented culture and further cultured in serum-free medium, and the culture supernatant was collected. IgG was purified from the collected culture supernatant using a protein A column. Hereinafter, the purified monoclonal antibody is referred to as "mAb (6A3)."

[0107] Example 2: Western blotting and immunoprecipitation using anti-CK2α monoclonal antibody mAb (6A3) (Objective) Western blotting and immunoprecipitation were performed using the anti-CK2α monoclonal antibody mAb (6A3) prepared in Example 1. The performance of mAb (6A3) was compared with that of a commercially available mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK; hereafter referred to as the "control antibody").

[0108] (Methods and Results) (1) Western Blot for Intracellular CK2α Protein. Endogenous CK2α protein was detected by Western blot analysis of cytoplasmic lysates of HEK293 cells using mAb (6A3) at 0.1 μg / mL or 0.5 μg / mL. The results are shown in Figure 3. Figure 3 also shows the detection results using a mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK) at 0.5 μg / mL as a control (Figure 3, "Control antibody"). As shown in Figure 3B, mAb (6A3) at 0.5 μg / mL was able to detect endogenous CK2α protein with over 3.5-fold higher sensitivity than the control antibody at the same concentration. Furthermore, mAb (6A3) at 0.1 μg / mL was able to detect endogenous CK2α protein with over 2-fold higher sensitivity than the control antibody at 0.5 μg / mL. These results demonstrated that mAb (6A3) can detect endogenous CK2α protein with significantly higher sensitivity than conventional antibodies.

[0109] (2) Western blot analysis of Flag-CK2α protein. Western blot analysis of cytoplasmic lysates from HEK293 cells expressing Flag-CK2α protein was performed using 0.1 μg / mL of mAb (6A3) and 0.5 μg / mL of the control antibody. The results are shown in Figure 4. The control antibody detected nonspecific bands at the same position as Flag-CK2α protein even in cells that did not express Flag-CK2α protein (Flag-CK2α(-) lane) (Figure 4, right, two bands indicated by arrows). In contrast, detection with mAb (6A3) detected Flag-CK2α protein bands only in cells that expressed Flag-CK2α protein (Flag-CK2α(+) lane), but no nonspecific bands were detected at the same position as Flag-CK2α protein in cells that did not express Flag-CK2α protein (Flag-CK2α(-) lane) (Figure 4, left, arrows).

[0110] These results demonstrated that mAb (6A3) can detect CK2α protein with higher specificity than conventional antibodies, and that mAb (6A3) can specifically detect both endogenous CK2α and exogenously expressed CK2α in cell lysates.

[0111] (3) Western Blotting of Immunoprecipitates. Lysates were prepared from the cytoplasm and nuclei of HEK293 cells. Immunoprecipitation was performed using 1 μg of mAb (6A3) or mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK) from each lysate. Specifically, cultured HEK293 cells were washed with PBS and harvested from the plate. The cytoplasmic and nuclear fractions were partially purified according to standard procedures. CK2α immune complexes were formed by adding anti-CK2α antibody and gently mixing at 4°C for 2 hours. The immunoprecipitates obtained by centrifugation were then treated with SDS and electrophoresed. Western blotting was performed using 4 μg / mL of mAb (6A3) or mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK).

[0112] The results of Western blotting are shown in Figure 5. The staining intensities of each CK2α band in the gel shown on the left side of Figure 5 and each CK2α band in the gel shown on the right side of Figure 5 were quantified and summed, and the results are shown in Figure 6. When mAb (6A3) or the control antibody was used as the immunoprecipitation antibody, no significant difference was detected in the amount of endogenous CK2α protein immunoprecipitated. On the other hand, when mAb (6A3) or the control antibody was used as the Western blotting antibody, endogenous CK2α protein could be detected with high sensitivity by mAb (6A3).

[0113] (4) Further Western Blotting of Immunoprecipitates Cytoplasmic lysates were prepared from Flag-CK2α-expressing RPE cells. Immunoprecipitation was performed from these lysates using 1 μg of mAb (6A3) or mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK). Western blot analysis of the resulting immunoprecipitates was performed using 0.1 μg / mL of mAb (6A3) or 2 μg / mL of anti-Flag antibody (Sigma, #1805).

[0114] The results of Western blotting are shown in Figure 7. The staining intensities of the two bands indicated by arrows in Figure 7 were quantified and are shown in Figure 8. When mAb (6A3) or the control antibody was used as the immunoprecipitation antibody, the amount of Flag-CK2α protein immunoprecipitated was greater with mAb (6A3) (compare the two bands indicated by arrows in Figure 7).

[0115] Example 3: Immunohistochemistry of breast cancer tissue using anti-CK2α monoclonal antibody mAb (6A3) (Objective) Breast cancer tissue was immunostained using anti-CK2α monoclonal antibody mAb (6A3). CK2α protein localized in the nucleoli in cancer cells was detected using mAb (6A3) and a commercially available mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK; hereafter referred to as the "control antibody"), and the performance of the two antibodies was compared.

[0116] (Methods and Results) Formalin-fixed, paraffin-embedded specimens of the invasive cancer (lesion) in breast cancer tissues resected from patients with primary breast cancer (invasive ductal carcinoma) who underwent primary mastectomy at Hoshi General Hospital between 2007 and 2014 were used. The tumor stage was p Stage IIB according to the TNM classification of malignant tumors (UICC International Code, LH Sobin, MK Gospodarowicz and Ch. Wittekind, TNM Classification of Malignant Tumors, 7th edition). This study was approved by the review boards of Hoshi General Hospital and Fukushima Medical University.

[0117] Formalin blocks were cut into 4-μm-thick sections and mounted on glass plates. They were deparaffinized and rehydrated using a Tissue Tech Prisma 6120 (Sakura Finetech Japan Co., Ltd.) according to standard procedures. Antigen retrieval was performed by autoclaving in 10 mM sodium bicarbonate buffer (pH 8.0) at 105°C for 10 minutes. The sections were blocked with goat serum diluted 200-fold in 10 mM phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 30 minutes at room temperature. After washing with PBS, the sections were incubated overnight at 4°C with anti-CK2α monoclonal antibody mAb (6A3) or mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK) in PBS containing BSA and 0.05% Tween® 20. Sixteen hours later, the cells were incubated with biotin-conjugated anti-mouse IgG (BA-9200, Vector Laboratories, US) for 30 minutes at room temperature, followed by a 30-minute incubation with avidin-horseradish peroxidase complex using the Vectastain™ Elite ABC HRP kit (PK-6102, Vector Laboratories, US). The anti-CK2α antibody was then visualized with diaminobenzidine (DOJINDO, Japan) under acidic conditions.

[0118] The anti-CK2α monoclonal antibody mAb (6A3) was used at a 1,000-fold dilution (0.1 μg / mL), and the mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK) was used at a 1,000-fold dilution (2 μg / mL).

[0119] Figure 9 shows the staining results for the infiltrated area (lesion) of invasive ductal carcinoma. Staining with 0.1 μg / mL of mAb (6A3) strongly stained the nucleoli, which are intranuclear structures, while background staining was weak (Figure 9A). As a result, nucleoli, where CK2α protein is localized, were clearly identified in the cancer cells. On the other hand, staining with 2 μg / mL of the control antibody weakly stained the nucleoli, and the difference from the background was not clear (Figure 9B).

[0120] Example 4: Determination of CDR sequences of anti-CK2α monoclonal antibody mAb (6A3) (Objective) To determine the variable region sequences of the heavy and light chains and the CDR sequences of anti-CK2α monoclonal antibody mAb (6A3).

[0121] (Methods and Results) We commissioned FASMAC Co., Ltd. to determine the antibody amino acid sequence of clone 6A3. The results of determining the sequences of the heavy and light chain variable regions and CDRs are shown below. CDRs were identified according to the Kabat antibody numbering system. It was also determined that mAb (6A3) is an IgG2b.

[0122]

[0123] Example 5: Production of further anti-CK2α monoclonal antibodies (Objective) To further develop monoclonal antibodies (anti-CK2α monoclonal antibodies) that can specifically and sensitively detect CK2α protein, and to determine the heavy and light chain variable region sequences and CDR sequences for each clone.

[0124] (Methods and Results) Mice were immunized with human CK2α protein as an antigen according to the method described in "(1) Immunization with Antigen Polypeptide" in Example 1. Lymphocytes were isolated from immunized mice according to the methods described in "(2) Cell Fusion and Screening," "(3) Analysis of Selected Clones," and "(4) Further Cloning of Clone 6A" in Example 1. Lymphocytes were then fused with myeloma cells to generate antibody-producing hybridomas, yielding five clones: clones 10B2, 15C1, 16C2, 19C2, and 21B1. Furthermore, the culture supernatants of each clone were purified according to the method described in "(5) Purification of IgG from Clone 6A3" in Example 1. The purified monoclonal antibodies "mAb (10B2)," "mAb (15C1)," "mAb (16C2)," "mAb (19C2)," and "mAb (21B1)" were obtained. Next, the heavy and light chain variable region sequences and CDR sequences were determined for each of the obtained anti-CK2α monoclonal antibodies using the same method as in Example 4. The results of determining the sequences of the heavy and light chain variable regions and CDRs are shown below. CDRs were identified according to the Kabat antibody numbering system. Furthermore, it was revealed that the obtained anti-CK2α monoclonal antibodies, "mAb (6A3)," "mAb (10B2)," "mAb (15C1)," and "mAb (16C2)," were all IgG2b, while "mAb (19C2)" and "mAb (21B1)" were IgG1.

[0125]

[0126] Example 6: Western blot and chromatin immunoprecipitation using anti-CK2α monoclonal antibodies (Purpose) Western blot and immunoprecipitation were performed to compare the performance of the anti-CK2α monoclonal antibody mAb (6A3) prepared in Example 1 and the anti-CK2α monoclonal antibodies mAb (10B2), mAb (15C1), mAb (16C2), and mAb (19C2) prepared in Example 5 with a commercially available mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK; referred to as the "control antibody").

[0127] (Methods and Results) (1) Western Blot of Immunoprecipitates from Cell Lysates. Lysates were prepared from the breast cancer cell line MCF-7 (JCRB Cell Bank, #JCRB0134) and HEK293 cells expressing Flag-CK2α protein. Cultured MCF-7 cells or HEK293 cells expressing Flag-CK2α protein were washed with PBS and harvested from the plate. Soluble fractions were partially purified from the cell lysates according to standard methods. Anti-CK2α antibodies (1 μg of six antibodies of the present invention or control antibodies) were added and gently mixed at 4°C for 2 hours. Protein G-agarose beads were then added and centrifuged to form CK2α immune complexes. The immunoprecipitates obtained after centrifugation were then treated with SDS and electrophoresed. Western blotting was performed using 0.1 μg / mL of the antibodies of the present invention or control antibodies.

[0128] The results of Western blot analysis after immunoprecipitation of MCF-7 cell line or HEK293 cells expressing Flag-CK2α protein are shown in Figure 10. In Western blot analysis using the control antibody, in addition to the band for endogenous CK2α, an equally or more intense nonspecific band was detected in the MCF-7 cell line. In HEK293 cells expressing Flag-CK2α protein, in addition to the bands for endogenous CK2α and Flag-CK2α, an equally or more intense nonspecific band was detected. In contrast, this nonspecific band was not detected with mAb (6A3), mAb (10B2), mAb (15C1), mAb (16C2), and mAb (19C2) of the present invention, demonstrating their high specificity for CK2α.

[0129] (2) Chromatin Immunoprecipitation. CK2α protein is recruited to a wide range of sites in the human genome, including the HMGB2 (High Mobility Group Box 2) locus, in the nucleus (unpublished work by the inventors). Based on this, we performed chromatin immunoprecipitation targeting the HMGB2 locus to verify the performance of the antibodies of the present invention. Specifically, lysates were prepared from human retinal pigment epithelial cells (human RPE cells) and human RPE cells in which the CK2 gene had been knocked out using CRISPR-Cas9 (RPE-ko) according to standard chromatin immunoprecipitation methods, and chromatin fractions were prepared. Anti-CK2α antibodies (1 μg of mAb (6A3), mAb (10B2), mAb (15C1), or mAb (16C2)) were added to the chromatin fraction and gently mixed overnight at 4°C using a rotator. Protein G-agarose beads were then added and the mixture was centrifuged to obtain chromatin immune complex fractions. Using nucleic acids contained in the obtained fraction as a template, the nucleotide sequence of the human HMGB2 gene locus was amplified by PCR using the primer pair shown in SEQ ID NOs: 65 and 66. The PCR amplification conditions (temperature and time) were as follows: 95°C for 3 seconds, 60°C for 20 seconds (1 cycle), repeated 40 times. The amount of DNA amplified corresponding to HMGB2 was measured using the KAPA SYBR Fast qPCR kit (KAPA Biosystems) with Applied Biosystems StepOne (Life Technologies).

[0130] The results of chromatin immunoprecipitation are shown in Figure 11. In fractions immunoprecipitated with any of mAb (6A3), mAb (10B2), mAb (15C1), and mAb (16C2), the amount of amplification of the target sequence corresponding to the HMGB2 locus was increased compared to samples without specific antibodies ("Control" in Figure 11). These results demonstrated that chromatin corresponding to the HMGB2 locus was effectively enriched in fractions immunoprecipitated with these antibodies.

[0131] (3) Western Blot of Recombinant CK2α Purified recombinant CK2α protein was detected by Western blot using mAb (6A3), mAb (10B2), mAb (15C1), mAb (16C2), mAb (19C2), and mAb (21B1) of the present invention. The recombinant CK2α protein was purified from human CK2α protein expressed in Escherichia coli by introducing the pGEX2T-CK2α gene. 0.05 μg, 0.1 μg, or 0.2 μg of recombinant CK2α protein was treated with SDS and electrophoresed, followed by detection using 0.1 μg / mL of the six antibodies of the present invention.

[0132] The results of Western blot are shown in Figure 12. It was demonstrated that mAb (6A3), mAb (10B2), mAb (15C1), mAb (16C2), mAb (19C2), and mAb (21B1) were all capable of detecting recombinant CK2α protein.

[0133] Example 7: Immunohistochemistry of breast cancer tissue using anti-CK2α monoclonal antibody mAb (21B1) (Purpose) Using the anti-CK2α monoclonal antibody mAb (21B1) prepared in Example 5 and a control antibody, breast cancer tissue was immunostained to detect CK2α protein localized in the nucleoli in cancer cells.

[0134] (Methods and Results) Immunostaining using anti-CK2α monoclonal antibody mAb (21B1) was performed on formalin-fixed, paraffin-embedded specimens of cancer infiltrates (lesions) in breast cancer tissues according to the method described in Example 3. The anti-CK2α monoclonal antibody mAb (21B1) was used at a 1,000-fold dilution (0.1 μg / mL). The mouse anti-CK2α monoclonal antibody (ab70774, Abcam, UK) was used at a 1,000-fold dilution (2 μg / mL).

[0135] Figure 9 shows the staining results for the infiltrated area (lesion) of invasive ductal carcinoma. Staining with 0.1 μg / mL of mAb (21B1) strongly stained the nucleoli, which are intranuclear structures, while background staining was weak (Figure 13A). As a result, nucleoli, where CK2α protein is localized, were clearly identified in the cancer cells. On the other hand, staining with 2 μg / mL of the control antibody weakly stained the nucleoli, and the difference from the background was not clear (Figure 13B).

[0136] Example 8: Prognosis Evaluation of Patients with Lung Adenocarcinoma After Resection Surgery (Objective) To evaluate the expression and localization of CK2α protein in lung adenocarcinoma tissues resected from patients with lung adenocarcinoma using the monoclonal anti-CK2α antibody mAb (6A3) of the present invention, and to evaluate the prognosis of patients after resection. (Method) To use formalin-fixed, paraffin-embedded lung adenocarcinoma tissue specimens resected from 120 patients with primary lung adenocarcinoma who underwent curative resection at the Department of Thoracic Surgery, Fukushima Medical University, between 2007 and 2014. Tumor stage was determined according to the TNM Classification of Malignant Tumors (UICC International Code, LH Sobin, MK Gospodarowicz, and Ch. Wittekind, TNM Classification of Malignant Tumors, 8th edition). This study was approved by the Fukushima Medical University Review Board.

[0137] Formalin blocks were cut into 4 μm-thick sections and mounted on glass plates. They were deparaffinized and rehydrated using a Tissue Tech Prisma 6120 (Sakura Finetech Japan Co., Ltd.) according to standard procedures. Antigen retrieval was performed by autoclaving in 10 mM sodium bicarbonate buffer (pH 8.0) at 105°C for 10 minutes. The sections were blocked with goat serum diluted 200-fold in 10 mM phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 30 minutes at room temperature. After washing with PBS, the sections were incubated overnight at 4°C with monoclonal anti-CK2α antibody mAb (6A3, 0.1 μg / mL) diluted 1,000-fold in PBS containing BSA and 0.05% Tween® 20. Sixteen hours later, the sections were incubated with biotin-conjugated anti-mouse IgG (BA-9200, Vector Laboratories, US) for 30 minutes at room temperature, followed by a 30-minute incubation with avidin-horseradish peroxidase complex using the Vectastain™ Elite ABC HRP kit (PK-6102, Vector Laboratories, US). Anti-CK2α antibodies were then visualized using diaminobenzidine (DOJINDO, Japan) under acidic conditions. Serial sections were counterstained with hematoxylin.

[0138] Immunohistochemical slides using the CK2α antibody were graded by two independent pathologists blinded to patient information on a five-point scale: I, II, III, IV, and V. I: Staining throughout the cell, but no clear nuclear staining. II: Nuclear staining (+), nuclear staining more clearly than cytoplasm. III: Nuclear staining (++), nuclear staining at a higher level than II. IV: Nuclear staining (+, ++), and nucleolar staining (+). V: Nuclear staining (-), nucleolar staining (+).

[0139] In 118 patients with primary lung adenocarcinoma (stages I to III, excluding stage IV), survival curves were analyzed using the Kaplan-Meier method for the CK2α nucleolar staining-positive (IV+V) group and the CK2α nucleolar staining-negative (I+II+III) group. Specifically, relapse-free survival was analyzed. The significance of the difference between the survival curves for the CK2α nucleolar staining-positive (IV+V) and negative (I+II+III) groups was tested using the log-rank test, and the hazard ratio and its 95% confidence interval were calculated. All statistical analyses were performed using JMP Pro version 14.2.0.

[0140] Furthermore, we evaluated the prognosis of 118 patients with primary lung adenocarcinoma (stages I-III, excluding stage IV). Clinical information was obtained retrospectively by reviewing medical records. Prognostic events were defined as recurrence, death from lung adenocarcinoma, and all-cause mortality, and their association with CK2α staining was examined.

[0141] (Results) The recurrence-free survival rate results are shown in Figure 14. Lung adenocarcinoma patients with CK2α nucleolus staining-positive showed a significantly lower recurrence-free survival rate than patients with nucleolus staining-negative. This indicates that the risk of recurrence in CK2α nucleolus staining-positive cases was significantly higher than that in CK2α nucleolus staining-negative cases (N = 118, P = 0.0031, log-rank test).

[0142] Univariate and multivariate analyses were also performed on 120 patients with primary lung adenocarcinoma. In the univariate analysis, we focused on each variable and evaluated its correlation and strength of influence on the objective variable, postoperative recurrence-free survival (FFS) of newly diagnosed lung adenocarcinoma. In the multivariate analysis, we selected four variables with a P value of less than 0.05 in the univariate analysis and analyzed whether they mutually influenced FFS or whether they defined the objective variable as an independent variable. Figures 15A and 15B show the hazard ratios (HRs) and their 95% confidence intervals calculated in the univariate and multivariate analyses. Furthermore, to analyze the effect of determining FFS in the multivariate analysis, we used a Partition Tree in JMP Pro version 14 to compare the contribution of each variable to the objective variable. Figure 15C shows the results. These results indicated that CK2α nucleolar staining positivity was strongly associated with future recurrence as an independent variable. Furthermore, in multivariate analysis, the indicator of positive CK2α nucleolar staining had the highest Wald value, a test statistic indicating statistical significance.

[0143] FIG. 16 shows the results of an analysis of variables that determine the time to recurrence using two recurrence prediction models.

[0144] Figure 16A shows the results of recurrence prediction model 1 based on six variables. The median time to recurrence was 1397 days, with the top variable being 717 days for cases with peripheral lymph node metastasis at the time of surgery, compared with 1496 days for cases without peripheral lymph node metastasis. On the other hand, the time to recurrence was 1326 days for cases with positive CK2α nucleolus staining, but 745 days for cases with (positive CK2α nucleolus staining and) lymphatic invasion.

[0145] Figure 16B shows the results of recurrence prediction model 2, which is based on seven variables, adding age to the six variables in model 1. The top variable was the presence of peripheral lymph node metastasis at the time of surgery (717 days compared with a median time to recurrence of 1397 days). For cases without peripheral lymph node metastasis (1496 days), the time to recurrence was 845 days for patients aged 80 years or older, while for patients under 80 years old (1566 days), the time to recurrence was 1370 days for cases with CK2α nucleolus staining positivity, of which the time to recurrence was 745 days for cases with lymphatic invasion (CK2α nucleolus staining positivity).

[0146] Figure 17 shows patients with primary lung adenocarcinoma classified into stages I to III based on stage and CK2α staining assessment, and distinguishes between those with and without recurrence. In particular, even in cases classified as early stage due to the size of the primary tumor and small or no metastasis to peripheral lymph nodes, statistical evidence suggests that positive CK2α nucleolar staining is a useful indicator of the possibility of future recurrence (Figure 17).

[0147] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An anti-CK2α antibody or a fragment thereof, (1) CDR1 consisting of the amino acid sequence shown in Sequence ID No. 5, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 6, and CDR3 consisting of amino acid sequence FV Heavy chain variable region including, CDR1, consisting of the amino acid sequence shown in Sequence ID No. 8, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 9, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 10 Light chain variable region including, (2) CDR1 consisting of the amino acid sequence shown in Sequence ID No. 17, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 18, and CDR3 consisting of amino acid sequence FV Heavy chain variable region including, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 21, and CDR3 consisting of the amino acid sequence shown in Sequence ID No. 22 Light chain variable region including, (3) CDR1 consisting of the amino acid sequence shown in Sequence ID No. 25, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 26, and CDR3 consisting of amino acid sequence FV Heavy chain variable region including, CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 28, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 29, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 30 Light chain variable region including, (4) CDR1 consisting of the amino acid sequence shown in Sequence ID No. 33, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and CDR3 consisting of amino acid sequence FV Heavy chain variable region including, CDR1 consisting of the amino acid sequence shown in Sequence ID No. 36, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 37, and CDR3 consisting of the amino acid sequence shown in Sequence ID No. 38 Light chain variable region including, (5) CDR1 consisting of the amino acid sequence shown in Sequence ID No. 41, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 42, and CDR3 consists of the amino acid sequence shown in SEQ ID NO:

43. Heavy chain variable region including, CDR1, consisting of the amino acid sequence shown in SEQ ID NO: 44, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 45, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 46 Light chain variable region including, or (6) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 49, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 50, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 51 Heavy chain variable region including, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 52, CDR2 consisting of the amino acid sequence shown in Sequence ID No. 53, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 54 Light chain variable region including The anti-CK2α antibody or a fragment thereof, comprising the above.

2. The anti-CK2α antibody or a fragment thereof (a) A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 11, and The light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 12, (b) A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 15, and Light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 16 (c) A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 23, and Light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 24 (d) A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, and Light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 32 (e) A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 39, and A light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 40, or (f) A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 47, and Light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 48 The anti-CK2α antibody or fragment thereof according to claim 1, comprising:

3. A kit for predicting the prognosis of cancer patients, comprising an anti-CK2α antibody or a fragment thereof according to claim 1 or 2.

4. The kit according to claim 3, wherein the prognosis includes the risk of recurrence.

5. The kit according to claim 3, wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma.

6. A method for predicting the prognosis of cancer patients, A step of detecting CK2α protein or a fragment thereof in the nucleolus of cancer cells or tissue obtained from a cancer patient, and A process in which a poor prognosis is predicted if CK2α protein or its fragments are detected in high concentrations in the nucleolus compared to other cell fractions, and / or a good prognosis is predicted if CK2α protein or its fragments are not detected in high concentrations in the nucleolus compared to other cell fractions. The method comprising detecting the CK2α protein or a fragment thereof using the anti-CK2α antibody or fragment thereof according to claim 1 or 2.

7. The method according to claim 6, wherein the prognosis includes the risk of recurrence.

8. The method according to claim 6, wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, kidney cancer, colorectal cancer, bladder cancer, lung cancer, thyroid cancer, and glioma.

9. The method according to claim 8, wherein the cancer is breast cancer, and the presence or absence of detection of CK2α protein or a fragment thereof is combined with at least one of the following classifications: classification by stage, classification by hormone receptor expression status, and classification by HER2 gene and / or protein expression status to predict the prognosis of a breast cancer patient.