JP2012240942A - Anti-atbf1 antibody and use thereof - Google Patents

Abstract

The present invention provides a novel antibody extremely useful as a means for determining the malignancy of cancer and its use.
A rabbit-derived polyclonal antibody that recognizes a specific amino acid sequence of AT motif binding factor 1 (ATBF1). And a cancer cell malignancy determination reagent and a malignancy determination method using the antibody. If ATBF1 exists in the nucleus, it is determined that the malignancy is low.
[Selection figure] None

Description

  The present invention relates to an anti-ATBF1 (AT motif binding factor 1) antibody and use thereof. More specifically, the present invention relates to an anti-ATBF1 antibody useful for determining the malignancy of cancer cells and uses thereof (cancer malignancy determination reagents and kits, cancer malignancy determination methods, etc.).

  ATBF1 was cloned in 1991 as a DNA binding protein (AT-rich binding factor 1) that binds to A (adenine) and T (thymine) -rich (AT-rich) DNA sequences in the expression control region upstream of the AFP gene promoter. (Non-Patent Document 1). First, 9 kb of ATBF1 mRNA was cloned, and in 1995, the 12 kb full length ATBF1 mRNA was named ATBF1-A as a major gene transcript. For ATBF1-A, functions related to neuronal differentiation have been clarified (Non-patent Documents 2 and 3). Most recently, the name of ATBF1 was renamed ZFHX3 (zinc finger homeobox 3) by the Human Genome Organization.

  The presence of ATBF1 gene mutation has been reported in prostate cancer, gastric cancer, and liver cancer (Non-Patent Documents 4 to 10), and its relationship with cancer malignancy has been pointed out. Detection of gene mutations requires DNA sequencing of the full length of ATBF1, and there are technical limitations in clinical application at general facilities. Our research group devised a method that can determine the malignancy of cancer by using anti-ATBF1 antibody instead of mutation detection. It is an attempt to predict the malignancy of cancer by paying attention to the phenomenon that ATBF1 protein, which may have been mutated, accumulates in the cytoplasm instead of the cell nucleus (Patent Document 1). In addition, for the practical application as a pathological diagnosis reagent, an attempt was made to monoclonalize the antibody (Patent Document 2).

  By the way, cancer, which refers to all malignant tumors, exhibits various pathologies depending on the tissue in which it forms, the genetic predisposition of the patient, environmental factors, and the like. In the treatment of cancer, in general, a therapy considered to be the most effective is preferentially selected from among chemotherapy, radiation therapy, and surgical operation. It is extremely important to accurately determine the malignancy of cancer cells when deciding on a cancer treatment policy. The grade of cancer is generally determined by the ability of cancer cells to grow and the effects of chemotherapy or radiation therapy. A cancer with low malignancy has a low proliferative ability and is easy to be surgically removed, or chemotherapy or radiation therapy is effective, and the prognosis is good. Cancers with high malignancy have high proliferative capacity and are difficult to remove surgically, or chemotherapy and radiation therapy are ineffective, resulting in poor prognosis. For cancers with particularly high malignancy, rapid and accurate surgical removal is desired, and accurate adjuvant treatment (radiotherapy or chemotherapy) is essential. If the grade of malignancy is mistaken, the expected therapeutic effect cannot be obtained, leading to worsening of the disease state, occurrence of serious side effects, or recurrence. In fact, because there is no effective means of determining the characteristics of cancer, especially the grade of malignancy, many cases have been treated under the wrong treatment policy, resulting in unfortunate results without effective treatment effects. . The reports related to the present invention are listed below.

International Publication No. 2006/011587 Pamphlet JP 2010-241783 A

Mol. Cell. Biol. 11: 6041-6049, 1991 J. Biol. Chem. 270: 26840-26848, 1995 Development 132: 5137-5145, 2005 Nat. Genet 37: 407-412, 2005 Prostate 66: 1082-1085, 2006 Oncogene 20: 869-873, 2001 Clic. Cancer Res. 13: 4355-4359, 2007 Histopathology 52: 552-559, 2008 Clin. Cancer Res. 11: 193-198, 2005 BMC Cancer 8: 262, 2008

  An object of the present invention is to provide a novel antibody extremely useful as a means for determining the malignancy of cancer and its use.

  In the past few years, attempts to put it into practical use have not progressed easily. However, the analysis results of ATBF1 molecules were accumulated during the research and development process, and the phenomenon that ATBF1, which was previously thought to be a single large protein, was processed at the protein level, yielded significant research results related to disease pathology. The present inventors have taken a policy of further clarifying the intracellular localization mode of the ATBF1 protein in malignant cells by making use of these research results and increasing the types of anti-ATBF1 antibodies to be used. Specifically, more than 20 types of anti-ATBF1 antibodies (each recognizing different antigenic peptides) are prepared to cover the full length of anti-ATBF1, and their characteristics (specificity and possibility of application to pathological diagnostics, etc.) ) Was examined in detail. As a result, there was a correlation between the evaluation of immunostaining using an antibody prepared by using amino acids 1504 to 1520 of the ATBF1 protein as an antigen peptide (hereinafter referred to as “MB0039 antibody”) and the survival rate of bladder cancer. recognized. In other words, it became clear that the staining with the antibody becomes a prognostic factor (biomarker for prognosis estimation) of bladder cancer.

  Here, in order to diagnose the malignancy of cancer, a histopathological diagnosis method using hematoxylin and eosin staining is effective. In bladder cancer, first, the depth of penetration is classified into intramucosal (pTa, pTis) and below (pT1, pT2, pT3, pT4). A fairly clear prognosis can be achieved by classifying WHO into low grade and high grade. However, high-grade cancer cases that cannot be selected by the WHO classification were overlooked in pTa, low grade, and it was pointed out that the appropriate treatment time was missed early. If the morphological abnormality of cancer cells progresses and the cancer progresses and the grade of malignancy becomes clear, the diagnosis of the grade of malignancy is easy, but it seems to be low-grade morphologically very early. There was no suitable biomarker to find high-grade cancer cases. The method for evaluating nuclear staining with the MB0039 antibody, which has been successfully obtained by the present inventors, can provide a solution to this problem, and its value is extremely high.

  On the other hand, it can be said that the MB0039 antibody can also be applied to malignancy determination of neuroblastoma and the like by summarizing large-scale epidemiological survey reports of gene mutations (Nature 455: 971-974, 2008, etc.).

The present invention described below is based on the above results or considerations.
[1] An anti-ATBF1 (AT motif binding factor 1) antibody that recognizes the antigen peptide SPTGSDSGSVQEDSGSEC (SEQ ID NO: 1).
[2] The anti-ATBF1 antibody according to [1], which is a rabbit-derived antibody.
[3] The anti-ATBF1 antibody according to [1] or [2], which is a polyclonal antibody.
[4] A reagent for determining the malignancy of a test cancer cell, comprising the anti-ATBF1 antibody according to any one of [1] to [3].
[5] The reagent for determining malignancy according to [4], which is used for estimating a prognosis of cancer.
[6] The reagent for determining malignancy according to [5], wherein the cancer is bladder cancer.
[7] A kit for determining the malignancy of a test cancer cell, comprising the reagent according to any one of [4] to [6].
[8] (1) The anti-ATBF1 antibody according to any one of [1] to [3], the reagent according to any one of [4] to [6], or the kit according to [7] Detecting the amount of ATBF1 in a test cancer cell separated from a living body,
(2) determining the malignancy of the test cell based on the detection result;
A method for determining the malignancy of a test cancer cell, comprising:
[9] The malignancy determination method according to [8], wherein the determination of step (2) is performed according to a determination criterion that the malignancy is low if ATBF1 is present in the nucleus.
[10] The malignancy determination method according to [8] or [9], further including a step of estimating a prognosis of cancer based on the determination result of step (2).
[11] The malignancy determination method according to [10], wherein the cancer is bladder cancer.

List of antigen peptides used for peptide antibody production. The position and sequence of amino acids in ATBF1 protein are shown. List of anti-ATBF1 antibodies against ATBF1 protein with a total length of 3703 amino acids. The name of the antibody and the amino acid number of the antigen peptide used are shown. ● NLS with a mark represents Nuclear Localization Signal (amino acid sequence of nuclear localization signal). D1-120 and AT-6 are existing antibodies. Reactivity of MB0039 antibody against bladder cancer cell line. A: Results of immunostaining with MB0039 antibody. B: Western blot (left) using whole cell extracted protein as a sample and Western blot (right) using nuclear fraction (lanes 7-9) and cytoplasmic fraction (10-12) as samples. Reactivity of various antibodies against clinical specimens of bladder cancer. A: HE (hematoxylin and eosin) -stained images of low-grade (left) and high-grade (right) samples. B: Immunostained images of a low-grade sample (top) and a high-grade sample (bottom). MB00033 antibody (No.33), MB0034 antibody (No.34), MB0039 antibody (No.39), D1-120 antibody, MB0043 antibody (No.43), MB0044 antibody (No.43) against clinical specimens (No.1-21) No. 44), MB0047 antibody (No. 47), MB0049 antibody (No. 49) reactivity. Solid line: Nucleus-based staining (nucleus> cytoplasm). Dotted line: cytoplasmic staining (nucleus <cytoplasm). Results of immunostaining using tissues collected by the first transurethral bladder tumor resection (TUR). A: Stained using MB0039 antibody and observed under a microscope. If the nuclear staining is significantly stronger than the cytoplasmic staining, it is judged as nuclear staining (positive), and the survival rate is divided into two groups: nuclear staining (positive) and nuclear staining (negative). -Used for Mayer analysis. B: Results of Kaplan-Mayer analysis of survival rate divided into two groups depending on the presence or absence of overexpression of p53. C: Results of Kaplan-Mayer analysis of survival rate divided into two groups depending on the presence or absence of p21 expression.

(the term)
In the present invention, the “test cancer cell” is a cell to be determined for malignancy in the method of the present invention. The test cancer cell is separated from the living body. That is, the present invention is applied to a test cancer cell in a state separated from a living body. “Separated from the living body” refers to a state in which the test cancer cell is completely isolated from the living body derived from the part of the living tissue in which the test cancer cell exists. The test cancer cells are usually prepared in a state existing in the living body, that is, in a state of being bound to surrounding cells, and used for the method of the present invention. The test cancer cells may be used in the method of the present invention after being separated (isolated) from surrounding cells.

  The test cancer cells in the present invention include cells that are determined to be cancer by other diagnostic methods, cells that are determined to have a high probability of being cancer, and cells that are likely to be cancer. Preferably, cells that are determined to be cancer by other diagnostic methods or cells that are determined to have a high probability of being cancer are used. Examples of other diagnostic methods here include X-ray contrast examination, endoscopy, ultrasonic examination, CT examination, MRI examination, PET examination, and a diagnostic method using a tumor marker. Usually, test cancer cells are collected from a tissue suspected of having cancer due to one or more of these.

  In the present invention, “cancer” is to be interpreted broadly and includes carcinoma and sarcoma. In the present invention, the term “cancer” is used interchangeably with “tumor”. It may also include benign tumors, benign malignant border lesions, and malignant tumors before the pathological diagnosis is confirmed, that is, before any benign or malignant tumor is confirmed. possible.

  “ATBF1” refers to AT motif binding factor 1 (AT motif binding factor 1). ATBF1 is known to be a transcription factor that binds to the AT-rich domain of an AFP (alphafetoprotein) regulator and down-regulates the expression of the AFP gene (see Non-Patent Document 2). As for ATBF1, ATBF1-A (404 kDa, amino acid sequence is shown in SEQ ID NO: 2), which are two isoforms having different molecular weights, formed by using different promoters and alternative splicing. The base sequence (GenBank accession number: see L32832) is shown in SEQ ID NO: 3) and ATBF1-B (306 kDa, the amino acid sequence is shown in SEQ ID NO: 4. In addition, the base sequence encoding ATBF1-B (GenBank accession number : See L32833) is shown in SEQ ID NO: 5.) is known (see Non-Patent Document 2). ATBF1-A has a structure in which the N-terminal side of the protein is 920 amino acids longer than ATBF1-B. The term “ATBF1” is used herein as an expression encompassing these two isoforms. Therefore, unless otherwise specified, “ATBF1 amount” means the sum of the abundance of each isoform. In the method of the present invention, in principle, the sum is used as a detection target. However, this does not preclude the detection of only the amount of any isoform. It should be noted that when “ATBF1” is simply described, it means ATBF1 protein except when it is clear that it has other meanings.

  The “ATBF1 amount in the test cancer cell” refers to the total amount of ATBF1 present in the nucleus and cytoplasm of the test cancer cell. In the present specification, “the amount of ATBF1 in the test cancer cell” is also referred to as “the amount of ATBF1 in the whole cell of the test cancer cell”. On the other hand, “the amount of ATBF1 in the nucleus of the test cancer cell” refers to the amount of ATBF1 present in the nucleus of the test cancer cell. Similarly, “the amount of ATBF1 in the cytoplasm of the test cancer cell” refers to the amount of ATBF1 present in the cytoplasm of the test cancer cell.

  “Detecting the amount of ATBF1” means grasping the amount of ATBF1 as an absolute amount or a relative amount. The reference of the relative amount here can be, for example, the ATBF1 amount of a standard sample prepared according to the malignancy. Alternatively, when the amount of ATBF1 in the nucleus is a detection target, the amount of ATBF1 in the cytoplasm can be used as a reference. Similarly, when the amount of ATBF1 in the cytoplasm is a detection target, the amount of ATBF1 in the nucleus can be used as a reference. Note that “detecting the amount of ATBF1” includes checking whether ATBF1 is present. Usually, the presence or absence of ATBF1 and the amount thereof will be examined. It is not essential to quantitate the amount of ATBF1 strictly.For example, by comparing with the amount of ATBF1 of a control that is an index of malignancy, the amount of ATBF1 is determined to such an extent that the malignancy of a test cancer cell can be determined. It only needs to be able to measure.

1. Anti-ATBF1 Antibody and Preparation Method Thereof The first aspect of the present invention relates to an anti-ATBF1 antibody (hereinafter abbreviated as “the antibody of the present invention”). The antibody of the present invention recognizes the antigen peptide SPTGSDSGSVQEDSGSEC (SEQ ID NO: 1) consisting of amino acids 1504 to 1520 of the ATBF1 protein. In other words, the epitope of the antibody of the present invention exists in the peptide portion.

  The method for preparing the antibody of the present invention is not particularly limited. The antibody of the present invention can be prepared using an immunological technique, a phage display method, a ribosome display method, or the like. Specific examples of the method for preparing the antibody of the present invention are shown in the Examples section described later.

  In one embodiment, the antibody of the present invention is prepared by an immunological technique using rabbits as immunized animals. In the present specification, the antibody is referred to as “rabbit-derived antibody”.

  The antibody of the present invention is provided as a polyclonal antibody, oligoclonal antibody or monoclonal antibody.

  The antibody of the present invention can be prepared by the following procedure using an immunological technique. First, the antigen peptide is prepared, and an animal (rabbit, goat, chicken, mouse, rat, etc.) is immunized using the antigen peptide.

  It is possible to use chemically synthesized antigens or recombinant antigens. An antigen to which a carrier protein is bound may be used in order to enhance the immunity-inducing action. As the carrier protein, KLM (Keyhole Light Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen obtained by expressing the above antigen peptide as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

  Immunization is repeated as necessary, and blood is collected when the antibody titer sufficiently increases, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.

  On the other hand, in order to obtain a monoclonal antibody, immunization is carried out in the same procedure as described above, and antibody-producing cells are extracted from the immunized animal when the antibody titer has sufficiently increased. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone producing an antibody having high specificity for the antigen peptide is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

  The antibody obtained on the condition that it retains the characteristics required for the antibody of the present invention, that is, the characteristics of specifically recognizing the ATBF1 protein site (amino acids 1504 to 1520) corresponding to the antigenic peptide Various modifications can be made to. Such modified antibodies are also one aspect of the present invention.

A fusion antibody or labeled antibody in which a low molecular weight compound, protein, labeling substance or the like is fused or bound can be constituted. Such modified antibodies are also one aspect of the present invention. Examples of the labeling substance include fluorescent dyes such as fluorescein, rhodamine, Texas red, oregon green, enzymes such as horseradish peroxidase, microperoxidase, alkaline phosphatase, β-D-galactosidase, chemical or bioluminescent compounds such as luminol, acridine dye, etc. , ³² P, ¹³¹ I, ¹²⁵ I and the like, and biotin.

2. Reagent and kit for determining malignancy of cancer cell The second aspect of the present invention relates to the use of the antibody of the present invention. As reported in the previous patent application (WO 2006/011587 pamphlet), the anti-ATBF1 antibody is extremely useful for determining the malignancy of cancer cells. Based on this finding, in this aspect, a reagent for determining the malignancy of a test cancer cell comprising the antibody of the present invention is provided. A kit for determining the malignancy of a test cancer cell comprising the reagent as a constituent element is also provided.

  The reagents and kits of the present invention can be applied to various cancer cells. That is, the cancer type whose malignancy is determined by the reagent and kit for use in the present invention is not particularly limited. However, in view of the fact that the immunostaining evaluation using the MB0039 antibody has been correlated with the survival rate of bladder cancer, the reagent and kit of the present invention are particularly useful for determining the malignancy of cells of bladder cancer. Useful. On the other hand, it can be said that the MB0039 antibody can be applied to the determination of the malignancy of neuroblastoma, etc. by summarizing large-scale epidemiological studies on gene mutations (Nature 455: 971-974, 2008, etc.).

The reagent of the present invention may be constituted using antibody fragments such as Fab, Fab ′, F (ab ′) ₂ , scFv, and dsFv antibodies.

  The kit of the present invention contains the reagent of the present invention as a main component. By using the kit of the present invention, it is possible to carry out the determination method described later (that is, the malignancy determination method of test cancer cells) more easily and in a shorter time. In the case of a kit for a method for directly detecting the binding amount of an anti-ATBF1 antibody, a reagent comprising a labeled anti-ATBF1 antibody is used. On the other hand, in the case of a kit for an indirect detection method, a reagent comprising an unlabeled anti-ATBF1 antibody is used. In this case, a secondary antibody labeled with a labeling substance (labeled secondary antibody) may be included in the kit. When a kit for a detection method using a polymer in which a secondary antibody and a labeling substance are bound to each other, the polymer may be included in the kit.

  On the other hand, an antigen peptide (typically SPTGSDSGSVQEDSGSEC), human ATBF1, etc. may be further included. The human ATBF1 here may be full length or a fragment. The origin and preparation method of the antigenic peptide and human ATBF1 are not particularly limited. For example, a recombinant antigen peptide prepared by recombinant DNA technology can be used. Antigenic peptides are typically used to confirm that the staining obtained using the kit is based on specific binding between the anti-ATBF1 antibody and its epitope. Specifically, first, an anti-ATBF1 antibody is treated with an antigen peptide. Immunostaining is performed using the anti-ATBF1 antibody after treatment. The obtained stained image is compared with the stained image obtained using the untreated anti-ATBF1 antibody. If the latter staining image shows a strong staining property, it can be confirmed that the staining property is based on the specific binding between the anti-ATBF1 antibody and its epitope.

  On the other hand, when an anti-ATBF1 antibody prepared using a fusion protein with a tag or carrier protein (hereinafter referred to as a tag or the like) as an antigen is used in the kit, the used tag or the like may be further included in the kit. The anti-ATBF1 antibody constituting the kit is required when there is a possibility that a reactive antibody is mixed with the tag used in the preparation process. If the tag or the like is used as described below, it can be confirmed that the staining property obtained using the kit is based on the specific binding between the anti-ATBF1 antibody and the ATBF1 antibody. First, an anti-ATBF1 antibody is treated with this tag or the like. The specimen is immunostained with the anti-ATBF1 antibody after treatment. The obtained stained image is compared with the stained image obtained using the untreated anti-ATBF1 antibody. If there is no difference in staining between the two, it can be confirmed that the staining in the latter stained image is based on specific binding between the anti-ATBF1 antibody and ATBF1.

  One or more reagents necessary for carrying out immunostaining such as antigen-antibody reaction and staining (for example, formalin and paraffin for tissue fixation / embedding, BSA for inhibiting non-specific binding) , A coloring reagent such as DAB, a hematoxylin solution for nuclear staining, etc.) and instruments may be further included. Usually, an instruction manual is attached to the kit of the present invention.

3. A further aspect of the present invention relates to a method (malignancy determination method) for determining the malignancy of a test cancer cell. In the present invention, the “malignancy determination method” refers to a method of determining the malignancy of a test cancer cell. The grade of malignancy of cancer is generally classified (determined) based on cell atypia, cell, tissue structure atypia, proliferation, invasion, metastasis, and the like. In general, low-grade cancer has a slow cell growth rate and is less likely to cause invasion and metastasis, so that surgical resection is easy, and chemotherapy and radiation therapy are effective, and the tumor can be easily reduced These can be combined to improve the prognosis. On the other hand, cancer with high malignancy has a fast cell growth rate and is prone to infiltrate and metastasis, and surgical resection is difficult, and because chemotherapy and radiation therapy are ineffective, the tumor shrinks. Is difficult, and the combination of these results in poor prognosis. For cancers with particularly high malignancy, rapid and accurate surgical removal is desired, and multidisciplinary treatment including accurate adjuvant treatment (radiotherapy or chemotherapy) is essential.

  In the present invention, (1) a step of detecting the amount of ATBF1 in a test cancer cell separated from a living body, and (2) a step of determining the malignancy of the test cell based on the detection result are performed. In step (1), the antibody of the present invention or the reagent of the present invention (including the reagent constituting the kit of the present invention) is used.

  The test cancer cell can be collected from the suspected cancer tissue. Specifically, a part of the suspicious cancer tissue can be collected by biopsy (biopsy), and used as a sample containing test cancer cells for the method of the present invention.

  In the present invention, the amount of ATBF1 is not limited to this, but is preferably performed using an immunohistochemical staining method. According to the immunohistochemical staining method, the amount of ATBF1 can be detected quickly and with high sensitivity. Also, the operation is simple. Therefore, the burden on the subject (patient) accompanying the detection of the amount of ATBF1 and the like is reduced. In the immunohistochemical staining method, usually, a test cancer cell is first contacted with an antibody (for example, an anti-ATBF1 antibody) that is specific to the detection target. Thereafter, the amount of the antibody bound to the whole cell, nucleus, and / or cytoplasm is measured. From the measurement result, the abundance of the detection target in the whole cell, the nucleus, and / or the cytoplasm of the test cancer cell is calculated. Specifically, the method of the present invention can be carried out according to the immunohistochemical staining method shown below.

In general, immunohistochemical staining of living tissue is performed by the following procedures (1) to (9). Various documents and books can be referred to for immunohistochemical staining of biological tissues (for example, “Enzyme Antibody Method, Revised 3rd Edition”, edited by Keiichi Watanabe and Kazuho Nakane, interdisciplinary project).
(1) Fixing and embedding in paraffin Fix tissue surgically collected from a living body with formalin, paraformaldehyde, anhydrous ethyl alcohol, or the like. Then embedded in paraffin. Generally dehydrated with alcohol, treated with xylene, and finally embedded in paraffin. A specimen embedded in paraffin is sliced into a desired thickness (for example, 3 to 5 μm thick) and spread on a glass slide. In place of the paraffin-embedded specimen, an alcohol-fixed specimen, a dry-sealed specimen, a frozen specimen, or the like may be used.
(2) Deparaffinization Generally, treat with xylene, alcohol, and purified water in order.
(3) Pretreatment (antigen activation)
If necessary, enzyme treatment, heat treatment and / or pressure treatment are performed for antigen activation. In the case of MB0039 successfully obtained by the present inventors, this antigen activation process can be omitted to simplify the operation. In addition, it can be said that omitting the pretreatment is preferable from the viewpoint of improving reliability and sensitivity. That is, if the treatment is omitted, the possibility that the cell membrane is disrupted and ATBF1 leaks is reduced, and ATBF1 can be detected in its original state.
(4) Endogenous peroxidase removal When peroxidase is used as a labeling substance in staining, endogenous peroxidase activity is removed by treatment with hydrogen peroxide.
(5) Inhibition of non-specific reaction The section is treated with bovine serum albumin solution (for example, 1% solution) for several minutes to several tens of minutes to inhibit non-specific reaction. Note that this step may be omitted by carrying out the next primary antibody reaction using an antibody solution containing bovine serum albumin.
(5) Primary antibody reaction An antibody diluted to an appropriate concentration is dropped onto a section on a slide glass and then allowed to react for several tens of minutes to several hours. After completion of the reaction, wash with an appropriate buffer such as phosphate buffer.
(6) Addition of labeling reagent Peroxidase is frequently used as a labeling substance. A secondary antibody to which peroxidase is bound is dropped onto a section on a slide glass, and then allowed to react for several tens of minutes to several hours. After completion of the reaction, wash with an appropriate buffer such as phosphate buffer.
(7) Color reaction DAB (3,3'-diaminobenzidine) is dissolved in Tris buffer. Subsequently, hydrogen peroxide is added. The coloring solution thus prepared is allowed to infiltrate into the section for several minutes (for example, 5 minutes) to develop a color. After color development, the section is thoroughly washed with tap water to remove DAB.
(8) Nuclear staining Nuclear reaction is performed by reacting Meyer's hematoxylin for several seconds to several tens of seconds. Wash with running water and color (usually for a few minutes).
(9) Dehydration, clearing, sealing After dehydrating with alcohol, clearing with xylene, and finally sealing with synthetic resin, glycerin, rubber syrup, etc.

  In step (2) in the present invention, the malignancy is determined based on the detection result in step (1). In this case, typically, a criterion of “malignancy is low if ATBF1 exists mainly in the nucleus” is used. “ATBF1 exists mainly in the nucleus” means that the amount of ATBF1 in the nucleus is greater than the amount of ATBF1 in the cytoplasm. Therefore, in the case where the immunostaining method is used for the detection in step (1), it is determined that the degree of malignancy is low if the staining mainly of the nucleus is observed. As a matter of course, a criterion that is inextricably linked to the criterion of “low malignancy if ATBF1 exists mainly in the nucleus”, that is, “malignancy if ATBF1 exists mainly in the cytoplasm” The criterion “high” may be adopted. Note that the evaluation / determination here can be automatically / mechanically performed irrespective of the judgment of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the judgment criteria.

  On the other hand, if there is no difference between the amount of ATBF1 in the nucleus and the amount of ATBF1 in the cytoplasm, it is usually judged that the malignancy is high. In the case where there is no difference between the amount of ATBF1 in the nucleus and the amount of ATBF1 in the cytoplasm, in addition to the case where the amount of ATBF1 in the cytoplasm is similar to that in the nucleus, the ATBF1 can be detected both in the nucleus and in the cytoplasm. The case where is not detected is included.

  When a clinical sample is used, the determination result according to the present invention can be used for, for example, determination of a treatment policy (selection of an effective treatment method, etc.). By using the determination result, improvement of treatment results, improvement of prognosis, improvement of patient's quality of life (QOL) and the like are brought about. Thus, the malignancy determination method of the present invention provides information extremely useful for cancer treatment.

  In one embodiment of the present invention, the determination result is used for cancer prognosis estimation. In this aspect, the prognosis of cancer is estimated based on the determination result of step (2). Typically, it is estimated that the prognosis is bad (poor prognosis) in the case of a determination result that the malignancy is high, and the prognosis is good (good prognosis) in the case of the determination result that the malignancy is low. The prognostic level is set in advance corresponding to the malignancy level (for example, 1: very good, 2: good, 3: relatively good, 4: relatively bad, 5: bad, 6: very good. It is also possible to determine which level corresponds to a plurality of levels.

  The type of cancer targeted by the present invention is not particularly limited. For example, the present invention can be applied to determine the malignancy of various tumors including bladder cancer, stomach cancer, skin tumor, tongue cancer and the like. However, the present invention is particularly useful for the purpose of determining the malignancy of bladder cancer.

1. Production of novel antibodies that recognize human ATBF1
After predicting antigenicity from the ATBF1 sequence, about 20 types of antigen peptides were set to cover the entire length of ATBF1 (FIG. 1). Using these antigenic peptides, we attempted to prepare polyclonal antibodies that recognize human ATBF1 by immunological techniques. As an example, a protocol when an antigen peptide (SPTGSDSGSVQEDSGSEC: SEQ ID NO: 1) is used as an immunogen is shown below. This also applies to the protocol when other antigens are used.

(1) Antigen preparation “Amino acid sequence SPTGSDSGSVQEDSGSEC (SEQ ID NO: 1) corresponding to amino acids 1504 to 1520 of ATBF1 was synthesized by a solid phase peptide synthesizer (AAPPTec / Apex 396-DC). The crude peptide fraction was recovered by ether precipitation, and the collected crude peptide fraction was purified by HPLC, collected after confirmation of molecular weight by MALDI TOF-Mass (PerSeptive Biosystems / Voyager-DE STR), and lyophilized. By a similar method, the carrier protein KLH was bound via a cysteine at the terminal of the synthesized peptide to obtain an immunogen.

(2) Immunization Rabbits (New Zealand White Rabbit) were used as immunized animals. The immunogen (KLH-binding synthetic peptide) was adjusted to 0.5 mg / mL with 1% DMSO-PBS. First, 400 μL of immunogen and 400 μL of adjuvant (complete adjuvant (FREUND) (Sigma) for the first time only, 2 to 6 times Incomplete adjuvant (Sigma)) were mixed to form an emulsion, and immunization was performed in the rabbit's back skin. Blood was collected from the ear vein and heart after the second immunization 14 days later and the third to sixth immunizations (final immunization) every 7 days. The serum was separated and collected, and then purified by affinity chromatography with the antigen peptide immobilized thereon to obtain a polyclonal antibody MB0039 (No. 39).

  A list of successfully acquired antibodies is shown in FIG. Existing antibodies (D1-120 and AT-6) are also shown.

2. Preparation of existing anti-ATBF1 antibody (1) D1-120 antibody Mouse ATBF1 (Ido et al., (1996). Gene, 168, 227-231) 41 amino acid residues (2114-2154: LQTLPAQLPPQLGPVEPLPADLAQLYQHQLNPTLLQQQNKR: SEQ ID NO: 6) A recombinant peptide fused with glutathione S-transferase (GST) is used as an antigen. The 41 amino acid residues described above completely match the amino acid residues (2170-2147) of human ATBF1.

  Specifically, the antigen is prepared by the following procedure. Details of antigen preparation and antibody production are described in a previous report (J. Compartive Neurology (2003) 465: 57-71).

First, as described above, the target amino acid portion is excised from mouse cDNA and recombined (subcloned) into a vector pGEX-K for preparing a GST fusion protein. E. coli AD202 is introduced into the gene, and the protein expressed in AD202 is purified by a conventional method using Sepharose-glutathione beaded agarose (Sigma) (for example, “First Recombinant Protein Purification Handbook” published in 1999, See Amersham Pharmacia Biotech, Inc.). Using the antigen (ATBF1-GST fusion protein) prepared as described above, an anti-ATBF1 antibody (D1-120) is obtained by the following procedures (a) to (d).
(a) An antigen (1 mg / ml) melted in PBS (pH 7.5) is mixed with an equal amount of Titer Max Gold (CytRx) to prepare an emulsion for immunization.
(b) Immunization (over 5 degrees on days 0, 14, 28, 49, 70) is performed by subcutaneous injection of 2 ml of emulsion into the back of the rabbit.
(c) When 91 days have passed, the blood is sacrificed and the serum is separated.
(d) Prepare an antigen column with the antigen used for immunization, and obtain the anti-ATBF1 antibody after purifying the antigen-antibody column from serum (for example, see “Amersham Pharmacia Biotech Co., Ltd.” published in 2000, “First Antibody Purification Handbook”) ).

(2) AT-6 antibody (polyclonal antibody)
Amino acid residues common to human ATBF1-A and B (3405-3549: PGAPSPDKDPAKESPKPEEQKNTPREVSPLLPKLPEEPEAESKSADSLYDPFIVPKVQYKLVCRKCQAGFSDEEAARSHLKSLCFFGQSVVNLQEMVLHVPTGGGGGGSGGGGGGGGGGGGGSGGGGGGGGGGGGGGSYHCLACES An antibody (polyclonal) is prepared and purified using the prepared antigen (for example, “Enzyme Antibody Method, Revised 3rd Edition”, edited by Keiichi Watanabe and Kazuho Nakane, interdisciplinary project).

3. Evaluation using bladder cancer cell lines Cell lines derived from human bladder cancer T24 (with p53 mutation, high grade), HT1376 (with p21 mutation, intermediate grade) and RT4 (p53, p21 mutation free, low malignancy) The degree of staining of each antibody was examined by the following procedure. The cell line is cultured in an RMI1640 culture medium containing 10% serum, released from the culture dish by trypsin treatment, and collected by attaching cells to a slide glass by cytospin, followed by 4% paraformaldehyde fixation, primary antibody MB0039 antibody DAB staining was performed at 1/1000 dilution for 1 hour at room temperature. (Please write and correct here.)

  The results of immunostaining with the MB0039 antibody are shown in FIG. 3A. ATBF1 staining for both T24, HT1376, and RT4 stains both in the nucleus and cytoplasm, but T24 stains more strongly in the cytoplasm than nuclei, and HT1376 stains the nucleus and cytoplasm almost the same The RT was stained more strongly in the nucleus than in the cytoplasm.

  On the other hand, protein was extracted from each cell, and the reactivity with each antibody was examined by Western blot. The results detected with the MB0039 antibody are shown in FIG. 3B (left: when whole cell extract protein is used as a sample, right: when nuclear fraction (lanes 7-9) and cytoplasmic fraction (10-12) are used as samples). . Multiple bands are observed (FIG. 3B left), indicating that antibody MB0039 can recognize both full-length ATBF1 and fragmented ATBF1. Comparing the detection results of whole cell extract protein (Fig. 3B left) with detection results of nuclear fraction and cytoplasmic fraction (Fig. 3B right), the full-length ATBF1 400 kDa and the high molecular weight fragment were 230 kDa 160 in the nuclear fraction. It can be seen that ATBF1 (55 kDa, 40 kDa, 15 kDa) detected at the kDa position and fragmented to a lower molecular weight is mainly present in the cytoplasm.

4). Evaluation using clinical specimens of bladder cancer In order to examine the possibility of applying the novel antibody to pathological diagnosis, the reactivity of each antibody against the clinical specimen of bladder cancer was examined by the following method. For bladder cancer, transurethral bladder tumor resection (TUR) is performed for diagnostic and therapeutic purposes. Immunostaining using 8 types of anti-ATBF1 antibodies including MB0039 antibody was performed on the first TUR specimen in each case, and the staining property for each case was evaluated.

  The detection result (stained image) is shown in FIG. FIG. 4A shows the result of HE staining. Representative stained images were shown for low-grade (left) and high-grade (right) specimens. FIG. 4B shows the results of immunostaining with each antibody. The staining (lower) for low-grade specimens was compared with the staining (higher) for high-grade specimens. When the MB0039 antibody (No. 39) is used, the staining property is clearly and decisively different between the low grade and the high grade. Specifically, it shows a nucleus-based staining property at a low grade, while a cytoplasm-based staining property is shown at a high grade. Some of the other antibodies showed similar staining properties, but the border between the nucleus and the cytoplasm was unclear compared to when the MB0039 antibody was used, and the color development was not sufficient. The staining is much worse than when the MB0039 antibody is used. The staining ability of the existing antibody (D1-120) is not far from that of MB0039 antibody.

  The reactivity of each antibody against clinical specimens of various grades is summarized in FIG. In FIG. 5, the solid line indicates that the staining property is mainly of the nucleus (nucleus> cytoplasm), and the dotted line indicates that the staining property is mainly of the cytoplasm (nucleus <cytoplasm). When the MB0039 antibody is used, the low-malignant specimen (1 to 18) has a nuclear-dominant staining property, and the high-grade specimen (19 to 21) has a nuclear-dominant staining property. That is, according to the MB0039 antibody, it was shown that low malignancy and high malignancy can be discriminated by the difference in staining (nucleus or cytoplasm). Such discrimination is not possible with antibodies other than the MB0039 antibody.

  On the other hand, using the tissue collected by the first transurethral bladder tumor resection (TUR) as a sample, immunostaining was evaluated using the MB0039 antibody. If the nuclear staining is significantly stronger than the cytoplasmic staining, it is judged as nuclear staining (positive), and the survival rate is divided into two groups: nuclear staining (positive) and nuclear staining (negative). -Used for Mayer analysis. As shown in FIG. 6A, the survival rate of the group positive for nuclear staining was high, and the prognosis was significantly good.

  The above tissue samples were divided into two groups according to the presence or absence of overexpression of p53, and the survival rate was analyzed by Kaplan-Mayer analysis, and no significant difference was observed (FIG. 6B). Further, no significant difference was observed when Kaplan-Mayer analysis was performed on the survival rate divided into two groups depending on the presence or absence of p21 expression (FIG. 6C).

5. Summary (1) The MB0039 antibody gives a clear histopathologically stained image. This means that the use of MB0039 antibody enables pathological diagnosis with high accuracy.
(2) According to the MB0039 antibody, the malignancy of cancer can be easily and accurately determined using the difference in staining properties as an index. In addition, prognosis can be estimated. Judgment / estimation using staining with MB0039 antibody is particularly effective when it is difficult to determine the malignancy of cancer from a morphological viewpoint. As described above, it was pointed out that a cancer case with high malignancy that cannot be selected by WHO classification was overlooked in grade I of bladder cancer, and an appropriate treatment time was missed early. The staining with the MB0039 antibody can provide a useful judgment material for the diagnosis of such cases.

  The anti-ATBF1 antibody of the present invention is useful as a tool for determining the malignancy of cancer cells. According to immunohistochemistry using the anti-ATBF1 antibody of the present invention, useful information for pathological diagnosis can be obtained. In particular, application to pathological diagnosis of bladder cancer (judgment of malignancy and estimation of prognosis) is assumed.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (11)

  An anti-ATBF1 (AT motif binding factor 1) antibody that recognizes the antigen peptide SPTGSDSGSVQEDSGSEC (SEQ ID NO: 1).   The anti-ATBF1 antibody according to claim 1, which is a rabbit-derived antibody.   The anti-ATBF1 antibody according to claim 1 or 2, which is a polyclonal antibody.   A reagent for determining the malignancy of a test cancer cell, comprising the anti-ATBF1 antibody according to any one of claims 1 to 3.   The reagent for judging malignancy according to claim 4, which is used for cancer prognosis estimation.   The reagent for determining malignancy according to claim 5, wherein the cancer is bladder cancer.   A kit for determining the malignancy of a test cancer cell, comprising the reagent according to any one of claims 4 to 6. (1) Using the anti-ATBF1 antibody according to any one of claims 1 to 3, the reagent according to any one of claims 4 to 6, or the kit according to claim 7, it is separated from a living body. Detecting the amount of ATBF1 in the test cancer cells,
(2) determining the malignancy of the test cell based on the detection result;
A method for determining the malignancy of a test cancer cell, comprising:   The malignancy determination method according to claim 8, wherein the determination of step (2) is performed according to a determination criterion that the malignancy is low if ATBF1 is present in the nucleus.   The malignancy determination method according to claim 8 or 9, further comprising a step of estimating a prognosis of cancer based on the determination result of step (2).   The malignancy evaluation method according to claim 10, wherein the cancer is bladder cancer.

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