JP2005089354A - Antibody selectively recognizing phosphorylated beta-catenin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnostic agent for diagnosing the development, metastasis or infiltration of cancer, and a treating agent for inhibiting the development, metastasis, growth or infiltration of cancer. <P>SOLUTION: An antibody which selectively recognizes β-catenin whose tyrosine residue at the 86th-position or the 254th-position of a specific amino acid sequence is phosphorylated and its immunoreactive segment are disclosed. The diagnostic agent for diagnosing the development, metastasis or infiltration of cancer has them as active ingredients, and the treating agent for inhibiting the development, metastasis, infiltration or growth of cancer has them as active ingredients. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an antibody that selectively recognizes phosphorylated β-catenin at the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1, and the 254th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1. The present invention relates to antibodies that selectively recognize phosphorylated β-catenin and methods for using them.

  Cell adhesion between epithelial cells is maintained by homophilic binding of E-cadherin, a calcium-dependent cell adhesion factor that constitutes an adherence junction. β-catenin was found as one of the proteins that bind to the intracellular domain of E-cadherin. In addition, β-catenin bound to E-cadherin binds to the actin cytoskeleton via α-catenin. That is, α-catenin and β-catenin mediate the binding between E-cadherin and the actin cytoskeleton. In addition, since E-cadherin lacking the intracellular domain loses the ability to adhere between cells, the binding between E-cadherin and actin cytoskeleton via α-catenin and β-catenin is essential for maintaining cell-cell adhesion. (Non-patent Document 1).

  The first stage of cancer invasion and cancer metastasis is the migration of cancer cells from the cancer foci. The E-cadherin intercellular adhesion mechanism is thought to be involved in the migration of cancer cells and to have a function of suppressing cancer invasion and cancer metastasis. In fact, it is known that the E-cadherin intercellular adhesion mechanism is inactivated by various factors in cancer cells. For example, E cadherin or catenin gene deletion or mutation introduction may be mentioned. Or, even if the coding region of the gene is normal, there is a decrease in the expression of a protein involved in the cell-cell adhesion mechanism caused by CpG methylation in the promoter region.

  Further, in poorly differentiated adenocarcinoma showing a diffuse infiltrated histology with poor adhesion between cells, gene abnormalities or abnormal expression of E-cadherin and β-catenin are frequently observed. Such inactivation of the cell-cell adhesion mechanism due to abnormal gene expression of E-cadherin and β-catenin is one of the triggers of cancer invasion and metastasis.

  On the other hand, in well-differentiated adenocarcinoma showing a histological image that maintains cell-cell adhesion, the frequency of E-cadherin and β-catenin gene abnormality or expression abnormality is low. In well-differentiated adenocarcinoma, adhesion between cells is lost only at the advanced part of the tumor, and the cancer cells have a histopathological finding that infiltrates with stromal hyperplasia. Moreover, the adhesion ability between cells has recovered in the liver metastasis of the same case, and the adhesion mechanism between E cadherin cells is reversibly controlled in well-differentiated adenocarcinoma.

In an in vitro using cell lines experiments, the enhanced phosphorylation of intracellular, it was confirmed that cell-cell adhesion are dissociated. Further, when stimulated by growth factors such as epidermal growth factor (hereinafter referred to as EGF) and hepatocyte growth factor (hereinafter referred to as HGF), cell-cell adhesion is dissociated. In addition, it was found that the phosphorylation catalytic domain of c-erbB-2, an EGF receptor, is associated with β-catenin in the cell. Currently, phosphorylation of β-catenin by tyrosine phosphorylation of various growth factor receptors is thought to contribute to inactivation of the E-cadherin intercellular adhesion mechanism (non-patented). Reference 2).

  Correlation between intracellular phosphotyrosine level and expression level of keratinocyte growth factor (KGF) receptor with phosphorylation catalytic domain in immunohistochemical staining of undifferentiated adenocarcinoma of the stomach (P <0.001) It is reported that the amount of intracellular phosphorylated tyrosine and the amount of E-cadherin and β-catenin expressed in the cytoplasm are correlated (P <0.05) (Non-patent Document 3). When an extract of a cancer site of a clinical specimen was immunoprecipitated with an anti-β-catenin antibody and then detected with an anti-phosphotyrosine antibody, phosphorylated β-catenin was detected. That is, a significant amount of phosphorylated β-catenin is present in the cancer tissue. On the other hand, phosphorylated β-catenin was not detected at the non-cancer site adjacent to the cancer site. Therefore, also in clinical practice, inactivation of the cell-cell adhesion mechanism accompanying phosphorylation of β-catenin is considered to be involved in cancer invasion and cancer metastasis.

  A transformant of human gastric cancer cell line TMK-1 cell introduced with a gene encoding an N-terminal deletion of β-catenin that can bind to erbB-2 receptor having phosphorylation catalytic domain but not E cadherin They were transplanted into the abdominal cavity of nude mice and examined for metastasis to the peritoneum. When compared with TMK-1 cells into which no gene had been introduced, the transformant of the β-catenin N-terminal deletion was found to have an effect of suppressing metastasis to the peritoneum (Non-patent Document 4). That is, in the transformant of β-catenin N-terminal deletion, E-cadherin is obtained by competitively inhibiting tyrosine phosphorylation of wild-type β-catenin by the receptor erbB-2. It was thought that the mechanism of cell-cell adhesion was normalized, and metastasis of cancer cells was suppressed.

  In addition, there is a report that β-catenin functions as a transcription activator in the nucleus by transmitting a wnt signal (Non-patent Document 5). β-catenin is degraded by phosphorylation by glycogen synthase kinase-3β (hereinafter referred to as GSK-3β), binding to APC gene products and other factors that are tumor suppressor genes, and ubiquitination. The abundance in the cytoplasm is kept low. The binding of wnt and its receptor Frizzled inactivates GSK-3β, and β-catenin is not phosphorylated and accumulates in the cytoplasm free from the degradation mechanism. Accumulated β-catenin is a transcription factor lymphocyte enhancer binding factor-1 (hereinafter referred to as LEF1) or T cell factor-4 (hereinafter referred to as TCF4). It is thought that it translocates into the nucleus and activates transcription of cancer-related genes such as c-myc, Cyclin D, and PPARδ (peroxisome proliferator activated receptor δ).

  In addition, in many colorectal cancers, deletion or mutation of the APC gene, which is thought to be involved in β-catenin degradation, has been found. Similarly, in many cancers, β-catenin is phosphorylated by GSK-3β. Mutation is found in the receiving site. That is, it is considered that the accumulation of β-catenin in the cytoplasm is deeply related to the occurrence and growth of cancer.

  In addition, geldanamycin, an agent that induces the degradation of EGF receptor erbB-2, is allowed to act on melanoma cell lines in which the degradation system of β-catenin is incomplete due to mutations in the APC gene or β-catenin gene Phosphorylation of β-catenin tyrosine residues is suppressed, and the binding of β-catenin and E-cadherin is enhanced. At the same time, the amount of nuclear β-catenin and the activity of β-catenin-dependent transcriptional activators are reduced. Recognized (Non-Patent Document 6). That is, in cancer cells in which the β-catenin degradation system is defective, tyrosine phosphorylation of β-catenin by the phosphorylation catalytic domain of the growth factor receptor inactivates the intercellular adhesion mechanism of E-cadherin and It was suggested that it may contribute to cancer development and cancer growth through positive control of the wnt signaling system as well as causing cancer metastasis.

Although the molecular mechanism of inactivation of the E-cadherin intercellular adhesion mechanism by phosphorylation of β-catenin has not yet been clarified, several hypotheses have been proposed. Phosphorylated β-catenin has a reduced affinity for E-cadherin, and the bond with E-cadherin is dissociated. In an experiment using a recombinant protein in vitro , the phosphorylation site of β-catenin by the product of c-Src gene, a proto-oncogene, is 86-tyrosine or 654-tyrosine, and a mutation in which tyrosine is substituted with phenylalanine From the examination using the body, it became clear that phosphorylation of position 654 tyrosine is involved in the decrease in the affinity between β-catenin and E-cadherin (Non-patent Document 7).

  When a tyrosine phosphatase inhibitor is allowed to act on a transformant in which an E-cadherin gene has been introduced into the lymphoma cell line K562 cell and an intercellular adhesion mechanism has been introduced, the intracellular phosphorylation is enhanced. To do. At that time, the bond between E-cadherin and β-catenin is not dissociated, and the bond between β-catenin and α-catenin is dissociated. Further, in the transformant in which the chimeric gene of E-cadherin and α-catenin was introduced into K562 cells, dissociation of cell aggregates was not observed by the tyrosine phosphatase inhibitor. Therefore, it was suggested that the release of α-catenin from the complex of E-cadherin and α-catenin and β-catenin is the cause of inactivation of the cell-cell adhesion mechanism. (Non-patent document 8).

  Thus, phosphorylation of β-catenin is thought to be deeply involved in cancer development, invasion, metastasis, and proliferation. However, an important tyrosine phosphorylation site of β-catenin has been identified so far. Not.

  When such a phosphorylated amino acid residue is specifically detected, an antibody that specifically reacts with a peptide containing the phosphorylated amino acid residue is generally used. To date, rabbit anti-phosphorylated β-catenin polyclonal antibodies are commercially available as antibodies that specifically react with phosphorylated β-catenin. The antibody recognizes β-catenin in which 41-position tyrosine and 45-position serine are phosphorylated, or 33-, 37-position serine and 41-position tyrosine phosphorylated β-catenin. However, there are no reports of anti-β-catenin monoclonal antibodies that can recognize specific phosphorylation sites. Furthermore, no anti-phosphorylated β-catenin monoclonal antibody that recognizes only one phosphorylation site is known, and in particular, an anti-phosphorylated β-catenin antibody that can selectively recognize phosphorylated tyrosine at position 86 or phosphorylated tyrosine at position 254 is unknown.

EMBO Journal, 7, 3679, 1988 Japanese Journal of Cancer and Chemotherapy, 26, 565, 1999 Japanese Journal of Cancer Research, 89, 829, 1998 Oncogene, 13, 883, 1996 Cell engineering, 16, 793, 1997 Cancer Research, 61, 1671, 2001 Journal of Biological Chemistry, 274, 36734, 1999 The Journal of Cell Biology, 127, 1375, 1994

  An object of the present invention is to provide a diagnostic agent for diagnosing cancer, a therapeutic agent for cancer, and a method for screening for a therapeutic agent for cancer.

The present invention relates to the following (1) to (16).
(1) An antibody that selectively recognizes β-catenin phosphorylated at the 86th or 254th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1.
(2) The antibody according to (1), wherein the antibody is a monoclonal antibody.
(3) The monoclonal antibody according to (2), wherein the antibody is produced from hybridoma KM2853 or KM2891.
(4) The antibody according to any one of (1) to (3), which is an IgG class antibody.
(5) An immunoreactive fragment of the antibody according to any one of (1) to (4).
(6) The derivative of the antibody according to any one of (1) to (4).
(7) A hybridoma that produces a monoclonal antibody that selectively recognizes β-catenin in which the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.
(8) A hybridoma that produces a monoclonal antibody that selectively recognizes β-catenin in which the tyrosine residue at position 254 in the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.
(9) The hybridoma according to (7), wherein the hybridoma is identified by an accession number FERM BP-8262.
(10) The hybridoma according to (8), wherein the hybridoma is identified by an accession number FERM BP-8261.
(11) A therapeutic agent for cancer comprising the antibody according to any one of (1) to (4), an immunoreactive fragment or a derivative thereof.
(12) A diagnostic agent for cancer comprising the antibody according to any one of (1) to (4), an immunoreactive fragment or a derivative thereof.
(13) A method for detecting tyrosine phosphorylation of β-catenin by an immunoassay using the antibody, immunoreactive fragment or derivative thereof according to any one of (1) to (4).
(14) A method for discriminating cancer using the antibody, immunoreactive fragment or derivative thereof according to any one of (1) to (4).
(15) Using the antibody, immunoreactive fragment or derivative thereof according to any one of (1) to (4), a tyrosine residue at position 86 or 254 of β-catenin represented by the amino acid sequence of SEQ ID NO: 1 A method of screening for an agent that inhibits phosphorylation of a group.
(16) A method for screening a therapeutic agent for cancer using the antibody, immunoreactive fragment or derivative thereof according to any one of (1) to (4).

  The present invention relates to β-catenin phosphorylated at the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 or βcatenin phosphorylated at the 254th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1. Provided are antibodies that selectively recognize and uses thereof. In addition, since the antibody of the present invention can specifically recognize tyrosine phosphorylation at position 86 or 254 of β-catenin represented by the amino acid sequence of SEQ ID NO: 1, it is represented by the amino acid sequence of SEQ ID NO: 1. It can also be used to screen for substances that inhibit tyrosine phosphorylation at position 86 or 254 of β-catenin.

Antibody of the Present Invention An antibody that selectively recognizes β-catenin phosphorylated at the 86th or 254th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 of the present invention is represented by SEQ ID NO: 1. An antibody that selectively recognizes β-catenin phosphorylated at tyrosine 86 at the amino acid sequence of β-catenin (hereinafter referred to as “86-tyrosine phosphorylation selective anti-β-catenin antibody”), and tyrosine 254 Antibodies that selectively recognize phosphorylated β-catenin (hereinafter referred to as “254th tyrosine phosphorylation selective anti-β-catenin antibody”) are included.

  The 86th tyrosine phosphorylation selective anti-β-catenin antibody refers to an antibody that can specifically bind to β-catenin phosphorylated at the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1. The anti-β-catenin antibody selective for tyrosine phosphorylation at position 254 refers to an antibody that can specifically bind to β-catenin phosphorylated at the tyrosine residue at position 254 in the amino acid sequence represented by SEQ ID NO: 1.

  Here, an antibody capable of specifically binding to β-catenin phosphorylated at the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 means that the 86th tyrosine residue in the amino acid sequence of βcatenin is phosphorylated. It refers to an antibody that specifically binds with higher affinity to β-catenin in which the 86th tyrosine residue is phosphorylated than β-catenin that is not.

  The antibody of the present invention includes both polyclonal antibodies and monoclonal antibodies, and monoclonal antibodies are preferred.

  Examples of polyclonal antibodies include antisera from immunized animals and polyclonal antibodies purified from the antisera.

  Examples of the monoclonal antibody include an antibody produced by a hybridoma and a gene recombinant antibody produced by a transformant transformed with an expression vector containing the antibody gene.

  Recombinant antibodies include antibodies produced by genetic recombination, such as humanized antibodies and human antibodies. As a genetically modified antibody, one having properties equivalent to those of a monoclonal antibody, having reduced antigenicity in the human body, and having an extended blood half-life is preferably used. Properties equivalent to those of a monoclonal antibody include binding activity and neutralization activity.

  Humanized antibodies include human chimeric antibodies and human complementarity determining regions (hereinafter referred to as CDR) grafted antibodies.

  A human chimeric antibody is a non-human animal antibody heavy chain (hereinafter referred to as H chain) V region (hereinafter referred to as antibody heavy chain variable region as VH) and light chain (hereinafter referred to as L chain). ) V region (hereinafter referred to as antibody L chain variable region as VL), human antibody heavy chain constant region (hereinafter referred to as CH) and human antibody light chain constant region (hereinafter referred to as CL). ).

  In the present invention, the human chimeric antibody produces a mouse monoclonal antibody that specifically binds to β-catenin phosphorylated at the 86th tyrosine residue or the 254th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1. From the hybridoma, cDNA encoding VH and VL is obtained and inserted into an expression vector for animal cells having genes encoding human antibody CH and human antibody CL to construct a human chimeric antibody expression vector. It can be produced by introducing into and expressing.

  The structure of the C region of the human chimeric antibody of the present invention may belong to any immunoglobulin (Ig) class, but may be an IgG type, or an IgG1, IgG2, IgG3, IgG4, or other immunoglobulin belonging to the IgG type. The C region is preferred.

  The human CDR-grafted antibody is constructed by constructing a gene encoding a V region obtained by substituting the VH and VL sequences of any human antibody with the CDR sequences of the VH and VL of an antibody of a non-human animal, respectively, It can be produced by introducing it into an expression vector for animal cells having a gene encoding CL of human antibody and expressing it.

  The structure of the C region of the human CDR-grafted antibody of the present invention may belong to any immunoglobulin (Ig) class, but may be an IgG type, or an IgG1, IgG2, IgG3, IgG4, or other immunogen belonging to the IgG type. The C region of globulin is preferred.

  A human antibody originally means an antibody that naturally exists in the human body, but a human antibody phage library created by recent advances in genetic engineering, cell engineering, and developmental technology, and human antibodies It also includes antibodies obtained from production transgenic animals.

  The antibody present in the human body is obtained by, for example, isolating human peripheral blood lymphocytes that produce human antibodies, immortalizing them by infecting EB virus, etc. It can be produced by culturing and purifying the antibody from the culture.

  A human antibody phage library refers to a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting antibody cDNA prepared from human antibody-producing cells such as human B cells into a phage vector. Say. From the library, phages expressing antibody fragments having a desired antigen-binding activity can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule consisting of two complete heavy chains and two complete light chains using genetic engineering techniques.

  A human antibody-producing transgenic animal refers to an animal in which a human antibody gene is integrated into the genome. Specifically, a human antibody-producing transgenic animal can be created by introducing a human antibody gene into an ES cell such as a mouse, transplanting the ES cell into another mouse early embryo, and generating it. Human antibodies are produced from human antibody-producing transgenic animals by obtaining human antibody-producing hybridomas by a conventional method for producing hybridomas in mammals other than humans, and culturing them to obtain the antibodies in the culture. It can be carried out by purification.

Immunoreactive fragment of antibody The immunoreactive fragment of the antibody of the present invention is a fragment of the antibody of the present invention as described above, wherein the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated. Β-catenin or an antibody fragment that can specifically bind to β-catenin phosphorylated at the 254th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1. Such immunoreactive fragments include antibody fragments obtained by digesting the antibody of the present invention with an appropriate peptidase, and antibody fragments produced using recombinant DNA technology. The immunoreactive fragment of the present invention includes Fab (Fragment of antigen binding), F (ab ′) ₂ , Fab ′, single chain antibody (Single chain Fv; hereinafter referred to as scFv) of the antibody of the present invention. [Science, 242, 423, 1988] Dimerization variable region (hereinafter referred to as Diabody) [Nature Biotechnol., 15, 629, 1997], and disulfide stabilized Fv fragment (hereinafter referred to as dsFv) [Molecular Immunol., 32, 249, 1995]. Further, the antibody fragment of the present invention includes a peptide containing the CDR amino acid sequences of the antibody heavy chain V region (VH) and antibody light chain V region (VL) (hereinafter referred to as a peptide containing CDR) [J. Biol. Chem., 271, 2966, 1996] are also included.

  Fab is composed of the N-terminal half of the H chain obtained by cleaving the upper peptide part of the two disulfide bonds that crosslink the two H chains at the hinge region of IgG with the enzyme papain, and the entire L chain. It is an antibody fragment having an antigen binding activity of about 30,000 molecular weight.

  The Fab of the present invention can be obtained by treating the 86-position tyrosine phosphorylation selective anti-β-catenin antibody or the 254-position tyrosine phosphorylation-selective anti-β-catenin antibody with the enzyme papain. Alternatively, a Fab can be produced by inserting a DNA encoding the Fab fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic or eukaryotic vector. .

F (ab ') ₂ can be obtained by treating the lower part of two pairs of disulfide bonds in the hinge region of IgG with the enzyme trypsin. Antigen binding activity with a molecular weight of about 100,000, in which two Fab regions are bound at the hinge portion. It is an antibody fragment having

The F (ab ′) ₂ of the present invention can be obtained by treating the 86-position tyrosine phosphorylation selective anti-β-catenin antibody or the 254-position tyrosine phosphorylation-selective anti-β-catenin antibody with the enzyme pepsin. Alternatively, DNA encoding the F (ab ′) ₂ fragment of the antibody is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the vector is introduced into a prokaryotic or eukaryotic expression, and expressed, and F ( ab ') ₂ can be produced.

Fab ′ is an antibody fragment having an antigen binding activity of about 50,000 molecular weight obtained by cleaving the disulfide bond between the hinges of F (ab ′) _{2 described} below.

The Fab ′ of the present invention can be obtained by treating F (ab ′) ₂ of the 86th tyrosine phosphorylation selective anti-β-catenin antibody or the 254th tyrosine phosphorylation-selective anti-βcatenin antibody with the reducing agent dithiothreitol. it can. Alternatively, Fab ′ is produced by inserting a DNA encoding the Fab ′ fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and expressing the vector by introducing the vector into a prokaryotic or eukaryotic organism. Can do.

  scFv is a fragment represented as VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P). Say. As VH and VL contained in scFv used in the present invention, any of the 86th tyrosine phosphorylation selective anti-β-catenin antibody or the 254th tyrosine phosphorylation selective anti-βcatenin antibody of the present invention may be used. it can.

  The scFv of the present invention obtains VH and VL cDNAs from the 86-position tyrosine phosphorylation selective anti-β-catenin antibody or the 254-position tyrosine phosphorylation-selective anti-β-catenin antibody, and constructs a DNA encoding the scFv fragment, It can be produced by inserting into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism for expression.

  Diabody is an antibody fragment in which scFv having the same or different antigen binding specificity forms a dimer, and is an antibody fragment having a bivalent antigen-binding activity for the same antigen or a bispecific antigen-binding activity for different antigens. .

  The Diabody of the present invention obtains VH and VL cDNAs from the 86th tyrosine phosphorylation selective anti-β-catenin antibody or the 254th tyrosine phosphorylation selective anti-βcatenin antibody, and constructs a DNA encoding the Diabody fragment, It can be produced by inserting into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism for expression.

  dsFv is a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are linked by a disulfide bond. The amino acid residue to be substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. [Protein Engineering, 7, 697, 1994].

  As the VH and VL contained in the dsFv of the present invention, any of the 86th tyrosine phosphorylation selective anti-β-catenin antibody or the 254th tyrosine phosphorylation selective anti-βcatenin antibody can be used.

  The dsFv of the present invention obtains VH and VL cDNAs from the 86th tyrosine phosphorylation selective anti-β-catenin antibody or the 254th tyrosine phosphorylation-selective anti-βcatenin antibody, and constructs a DNA encoding the dsFv fragment, It can be produced by inserting into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism for expression.

  The peptide containing CDR is composed of at least one region of CDR of VH or VL. A peptide containing a plurality of CDRs can be produced by binding directly or via an appropriate peptide linker.

  The CDR-containing peptide of the present invention encodes a peptide containing CDR by obtaining VH and VL cDNAs from the 86th tyrosine phosphorylation-selective anti-β-catenin antibody or the 254th tyrosine phosphorylation-selective anti-β-catenin antibody. It can be produced by constructing DNA, inserting it into a prokaryotic expression vector or eukaryotic expression vector, introducing the vector into a prokaryotic or eukaryotic organism, and expressing it. A peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluormethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method).

Antibody Derivative The derivative of the antibody of the present invention includes a radioactive tyrosine phosphorylation selective anti-β-catenin antibody or an immunoreactive fragment thereof, or a tyrosine phosphorylation selective anti-β-catenin antibody or an immunoreactive fragment thereof at position 254. Examples thereof include derivatives of antibodies in which isotopes, proteins or drugs are chemically or genetically bound.

  The derivative of the antibody of the present invention is an H chain of the 86th tyrosine phosphorylation selective anti-β-catenin antibody or immunoreactive fragment thereof, or the 254th tyrosine phosphorylation-selective anti-βcatenin antibody or immunoreactive fragment thereof of the present invention. Alternatively, a radioisotope, protein or drug (preferably a low isotope) is added to the N- or C-terminal side of the L chain, an appropriate substituent or side chain in the antibody or antibody fragment, or a sugar chain in the antibody or antibody fragment. Molecular drugs) and the like can be produced using chemical methods [Introduction to Antibody Engineering, Osamu Kanmitsu, Jinjinshokan Co., Ltd., 1994].

  Alternatively, the DNA encoding the 86-position tyrosine phosphorylation-selective anti-β-catenin antibody or the 254-position tyrosine phosphorylation-selective anti-β-catenin antibody or an immunoreactive fragment thereof was linked to the DNA encoding the protein to be bound. DNA can be produced by inserting into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism.

Examples of the radioisotope include ¹³¹ I and ¹²⁵ I, and can be bound to an antibody by, for example, the chloramine T method.

  As drugs, alkylating agents such as nitrogen mustard and cyclophosphamide, antimetabolites such as 5-fluorouracil and methotrexate, antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin and doxorubicin, vincristine, vinblastine, vindesine Anticancer agents such as hormonal agents such as plant alkaloids, tamoxifen, dexamethasone [Clinical Oncology, edited by Japan Clinical Oncology Society, Cancer and Chemotherapy Co., 1996], or steroids such as hydrocortisone, prednisone, aspirin, indomethacin Non-steroids such as gold thiomalate, immunomodulators such as penicillamine, immunosuppressants such as cyclophosphamide, azathioprine, chlorpheniramine maleate, clemastine Anti-inflammatory agents such as antihistamines [Inflammation and anti-inflammatory therapy, Ishiyaku Shuppan Co., Ltd., 1982]. For example, as a method of binding daunomycin and antibody, there are a method of binding between daunomycin and the amino group of the antibody via glutaraldehyde, a method of binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carbodiimide, etc. Can be mentioned.

  Examples of proteins include cytokines that activate immunocompetent cells, such as human interleukin 2, human granulocyte macrophage colony stimulating factor, human macrophage colony stimulating factor, and human interleukin 12. In addition, toxins such as ricin and diphtheria toxin having an activity of directly damaging cancer cells can be used. For example, for a fusion antibody in which a protein is bound to an antibody or an immunoreactive antibody fragment thereof, a cDNA encoding the protein is linked to a cDNA encoding the antibody or antibody fragment, and a DNA encoding the fusion antibody is constructed, A fusion antibody can be produced by inserting DNA into a prokaryotic or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.

Β-catenin phosphorylated at the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 of the antibody of the present invention or an immunoreactive fragment thereof, or tyrosine at the 254th position in the amino acid sequence represented by SEQ ID NO: 1 The binding activity to β-catenin whose residue is phosphorylated can be measured by a commonly used immunoassay. Examples of immunoassay include ELISA [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988] and Western blotting [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988]. By these methods, it can be measured in vitro .

  Hereinafter, methods for producing 86-position tyrosine phosphorylation selective anti-β-catenin antibody or 254-position tyrosine phosphorylation-selective anti-β-catenin antibody, immunoreactive fragments and derivatives thereof constituting the present invention, uses thereof, and The use of the contained drug will be described.

1. Method for producing anti-β-catenin antibody selective for tyrosine phosphorylation or anti-β-catenin antibody selective for tyrosine phosphorylation at position 254
(1) Preparation of Peptide as Antigen 86-Tyrosine Phosphorylation Selective β-Catenin Antibody in the Amino Acid Sequence Represented by SEQ ID NO: 1 or 254 Tyrosine Phosphorylation-Selective β-Catenin in the Amino Acid Sequence Represented by SEQ ID NO: 1 The antibody comprises 5 or more consecutive residues, specifically 6 to 30 residues, including tyrosine at position 86 or 254 in the amino acid sequence represented by SEQ ID NO: 1, respectively, among the amino acid sequence of β-catenin, preferably Consists of an amino acid sequence of 10 to 25 residues and chemically synthesizes a peptide in which the tyrosine at position 86 or 254 in the amino acid sequence represented by SEQ ID NO: 1 is replaced with phosphorylated tyrosine, and immunizes animals using the peptide as an antigen By doing so, it can be produced respectively. Specific examples of the antigen peptide for producing the 86-position tyrosine phosphorylation selective β-catenin antibody in the amino acid sequence represented by SEQ ID NO: 1 include a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 and the like. Specific examples of the antigen peptide for producing the 86th tyrosine phosphorylation selective β-catenin antibody in the amino acid sequence represented by SEQ ID NO: 1 include a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5 and the like. That is, the 86th tyrosine phosphorylation selective anti-β-catenin antibody of the present invention specifically binds to the phosphorylated peptide portion using the phosphorylated peptide portion in the 86th tyrosine phosphorylated β-catenin as an epitope. Similarly, the tyrosine phosphorylation-selective anti-β-catenin antibody also specifically binds to the phosphorylated peptide portion using the above-mentioned phosphorylated peptide portion in the tyrosine phosphorylated β-catenin as the epitope.

  If necessary, a cysteine residue for use in binding to a carrier protein described later is added to the N-terminus or C-terminus of the peptide. Peptides phosphorylated on tyrosine were prepared according to literature methods [Helv. Chim. Acta, 74, 1314, 1991, Helv. Chem. Acta, 75, 707, 1992, Tetrahedron Lett., 30, 6229, 1989, Tetrahedron Lett. , 29, 3591, 1988] can be chemically synthesized by solid phase synthesis using a peptide synthesizer or the like.

(2) Preparation of polyclonal antibodies Antisera containing polyclonal antibodies against the above peptides are described in the literature [Antibodies: A Laboratory Manual, Cold Spring Harbor Press. 1988] (hereinafter abbreviated as "Antibodies: A Laboratory Manual"). By general methods, immunization is carried out by administering the antigens several times every 10 to 4 weeks together with an appropriate adjuvant, subcutaneously, intravenously or intraperitoneally in animals such as mice, rats, hamsters, rabbits, etc. Can be prepared. Immunization may be performed with one of the peptide antigen containing 86 phosphorylated tyrosine and the peptide antigen containing 254 phosphorylated tyrosine, or with a mixture containing both. When immunization is performed using the mixture, a polyclonal antibody containing an 86-position tyrosine phosphorylation-selective anti-β-catenin antibody and an 254-position tyrosine phosphorylation-selective anti-β-catenin antibody is obtained.

  If only the peptide prepared in (1) is weak in raising an antibody as an antigen, the peptide is keyhole limpet hemocyanine (hereinafter referred to as KLH), bovine serum albumin, ovalbumin, tetanus A substance bound to a carrier protein such as a toxin is used as an antigen. For example, 200 to 1000 μg for rabbits and 10 to 100 μg for mice are administered at a time. The peptide and carrier protein can be linked using m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) or glutaraldehyde. Examples of adjuvants include Complete Freund's Adjuvant or aluminum hydroxide gel and pertussis vaccine. Three to ten days after each administration, blood is collected from the fundus venous plexus or tail vein of the immunized animal, and the reactivity against the phosphorylated peptide used for the antigen is confirmed by enzyme immunoassay. Serum collected from the indicated animals is antiserum. In the enzyme immunoassay method, the phosphorylated peptide used for the antigen is coated on a plate, the sample serum is reacted as the first antibody, and further labeled with biotin, enzyme, chemiluminescent substance or radioisotope as the second antibody. This is a method of detecting and quantifying the antibody that recognizes and binds to the antigenic peptide by reacting with the anti-immunoglobulin antibody (antibody against the immunoglobulin of the animal used for immunization) and then reacting according to the labeling substance [enzyme Immunoassay (ELISA method): published by Medical School, 1976].

  The peptide for antigen prepared in (1) is, for example, Sepharose-4B (manufactured by Amersham Pharmacia Biotech) activated with N-hydroxysuccinimide (NHS) or Hitrap NHS- A polyclonal antibody that binds to the antigenic peptide by affinity chromatography on the antiserum using an affinity column prepared by immobilization on an activation column (HiTrap NHS-activated column, manufactured by Amersham Pharmacia Biotech). The antibody can be purified. Peptide immobilization and affinity chromatography can be performed according to the manufacturer's attachment.

  The thus purified polyclonal antibody has a peptide other than the phosphorylated tyrosine in the amino acid sequence of the peptide used as the antigen as an epitope, or 86 in the amino acid sequence represented by SEQ ID NO: 1. Antibodies capable of binding β-catenin not phosphorylated at position 86 or 254 other than β-catenin phosphorylated at the tyrosine residue at position 254 or 254 are also included. Therefore, the polyclonal antibody purified above is passed through an affinity column in which a peptide in which the tyrosine is not phosphorylated in the same amino acid sequence as the antigen peptide prepared in (1) is immobilized using the above method. In addition, the 86th tyrosine phosphorylation selective anti-βcatenin polyclonal antibody is removed by binding an antibody that recognizes β-catenin in which the 86th or 254th position in the amino acid sequence represented by SEQ ID NO: 1 is not phosphorylated. Antibodies or 254 tyrosine phosphorylation selective anti-β-catenin polyclonal antibodies can be purified.

(3) Production of monoclonal antibodies
In the same manner as in (2), mice or rats aged 3 to 20 weeks are immunized, and antibody-producing cells are collected from the spleen, lymph nodes, and peripheral blood of animals that show a sufficient antibody titer in the serum. For example, the spleen is removed and spleen cells are collected.

In order to obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. Generally available cell lines can be used as myeloma cells to be fused with antibody-producing cells. As the myeloma cell, for example, 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (P3-U1) which is a cell line obtained from a mouse [Kohler G. et al. , Europ. J. Immunol., 6, 511, 1976], SP2 / 0-Ag14 (SP-2 / 0) [Shulmanet M. et al., Nature, 276, 269, 1978], P3-X63Ag8653 (653) [Kearney JF et al., J. Immunol., 123, 1548, 1979], P3-X63Ag8 (X63) [Kohler G. et al., Nature, 256, 495, 1975], etc., in vitro Any myeloma cells that can be grown in Regarding the culture and passage of these cell lines, according to a known method [Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988], a cell number of 2 × 10 ⁷ or more is secured by the time of cell fusion.

The antibody-producing cells and myeloma cells are washed with minimal essential medium (hereinafter referred to as MEM) or phosphate buffered saline (hereinafter referred to as PBS), and then polyethylene glycol. Add cell aggregating media such as -1000 to allow cell fusion. The fused cells were added to HAT medium [normal medium (RPMI-1640 medium with 1.5 mmol / L glutamine, 5 × 10 ⁻⁵ mol / L 2-mercaptoethanol, 10 μg / mL gentamicin and 10% fetal calf serum (FCS) Medium)), suspended in ^10-4 mol / L hypoxanthine, ^1.5x10-5 mol / L thymidine and ^4x10-7 mol / L aminopterin], and dispensed into a 96-well plate. Incubate.

  After culturing, a portion of the culture supernatant of each well is taken and enzyme immunoassay is used to increase the reactivity of the phosphorylated peptide as an antigen compared to a non-phosphorylated peptide having the same sequence as the antigen peptide. Select what to show. Cloning was repeated twice from the cells in the selected well by the limiting dilution method [the first time, HT medium (medium obtained by removing aminopterin from HAT medium), the second time using normal medium], stably Those having a strong antibody titer are selected as the 86th tyrosine phosphorylation selective anti-βcatenin antibody or the 254th tyrosine phosphorylation selective anti-βcatenin antibody-producing hybridoma strain.

  The subclass of the purified monoclonal antibody can be determined using a monoclonal antibody typing kit or the like. The protein mass can be calculated by the Raleigh method or the absorbance at a wavelength of 280 nm.

As a method for obtaining a purified monoclonal antibody, 0.5 ml of pristane (Pristane, 2, 6, 10, 14-tetramethylpentadecane) was intraperitoneally administered, and the above was applied to 8-10 week old mice or nude mice raised for 2 weeks. injected 86 of tyrosine phosphorylation selective anti β-catenin antibody-producing hybridoma cells or 254 of tyrosine phosphorylation selective anti β-catenin antibody-producing hybridoma cells 5 × 10 ⁶ ~2 × 10 ⁷ cells / animal, obtained by intraperitoneally To do. The hybridoma becomes ascites tumor in 10-21 days. Ascites is collected from the mouse or nude mouse, centrifuged, salted out with 40-50% saturated ammonium sulfate, caprylic acid precipitation method, DEAE-Sepharose column, Protein A column or Cellulofine GSL2000 (Seikagaku Corporation) column, etc. Use to collect IgG or IgM fractions to obtain purified monoclonal antibodies.

  Specific examples of the hybridoma of the present invention include the 86-tyrosine phosphorylation selective anti-β-catenin antibody-producing hybridoma KM2891, the 254-position tyrosine phosphorylation-selective anti-β-catenin antibody-producing hybridoma KM2853, and the like. Hybridoma KM2853 has been deposited as FERM BP-8261 on December 19, 2002 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, Tsukuba City, Ibaraki). Hybridoma KM2891 was deposited as FERM BP-8262 on December 19, 2002 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1st Street, Tsukuba City, Ibaraki). Yes.

2. Use of 86-tyrosine phosphorylation-selective anti -β-catenin antibody, or 254-position tyrosine-phosphorylation-selective anti-β-catenin antibody, immunoreactive fragment and derivative thereof 86-position tyrosine phosphorylation-selective anti-β-catenin antibody of the present invention Alternatively, the anti-β-catenin antibody selective for tyrosine phosphorylation at position 254 can specifically bind to phosphorylation of β-catenin at tyrosine at position 86 or tyrosine at position 254, respectively. The antibody of the present invention having such properties can be used for the following uses.

(1) A method for detecting phosphorylation of β-catenin using the antibody of the present invention, a diagnostic agent for cancer and a method for discriminating cancer Cellular phosphorylation of β-catenin is carried out using the antibody obtained in 1 above, It can be detected by immunological methods.

  In addition, since the increase in β-catenin in cells is considered to be involved in cancer, the occurrence of cancer, cancer growth, cancer invasion and cancer can be determined by quantifying β-catenin using the immunological method described above. The state of metastasis can be determined. Therefore, the antibody of the present invention can be used as a diagnostic agent for cancer.

  Specifically, the antibody of the present invention is brought into contact with a cell extract or a test sample, and the binding between the antibody of the present invention and β-catenin is measured qualitatively or quantitatively. As described above, the occurrence of cancer, cancer growth, cancer invasion and cancer metastasis can be predicted by quantifying phosphorylation of β-catenin in the cell extract or test sample.

  The test sample is not particularly limited, and examples thereof include biological samples, specifically extracts thereof such as a diseased tissue suspected of being cancerous, and biopsy samples, surgically removed tissues, and the like are used. preferable.

  Examples of the method for examining the degree of phosphorylation of β-catenin according to the present invention include immunological measurement methods. Immunoassay methods include immunoassay methods, immunoblotting methods, agglutination reactions, complement binding reactions, hemolysis reactions, sedimentation reactions, gold colloid methods, chromatographic methods, immunostaining methods, etc. Any method is included, but preferably, an enzyme immunoassay (ELISA method), an immunoblotting method, an immunohistochemical staining method or the like is used.

  Examples of the ELISA method include a sandwich ELISA and a competitive ELISA, and a sandwich ELISA is preferable. As an antibody used in the sandwich ELISA method, any of antibodies such as monoclonal antibodies and polyclonal antibodies and immunoreactive fragments thereof may be used. As the combination of the two types of antibodies used in the ELISA method, any of monoclonal antibodies and polyclonal antibodies that recognize different epitopes and their immunoreactive fragments may be used, but a highly sensitive sandwich ELISA is performed. Therefore, a combination of two kinds of monoclonal antibodies having different antigen binding activities is preferable.

  In the case of specifically detecting phosphorylated β-catenin, one antibody is a β-catenin-specific antibody, and one antibody is the 86th tyrosine phosphorylation-selective anti-β-catenin antibody of the present invention or the 254th tyrosine. It is preferred to use a phosphorylation selective anti-β-catenin antibody.

  When non-phosphorylated β-catenin is specifically detected, any β-catenin binding can be used as long as the two types of antibodies used do not recognize the same epitope and do not inhibit binding to β-catenin. Antibodies can also be used.

  The antibody to be bound to the solid phase and the antibody to be labeled may be any of the above-described antibodies. Examples of the labeling method of the antibody include a method of labeling with a radioisotope, enzyme, fluorescence, luminescence, etc., but labeling with an enzyme is preferable.

  Examples of the method for examining the degree of phosphorylation of β-catenin using the ELISA method include the following methods.

  First, in the sandwich ELISA described above, an antibody that binds to non-phosphorylated β-catenin is used as an antibody that binds to the solid phase and a labeled antibody, and β-catenin that is not phosphorylated contained in the test sample is used. To detect.

  Second, using either the 86-tyrosine phosphorylation selective anti-β-catenin antibody or the 254-tyrosine phosphorylation-selective anti-β-catenin antibody of the present invention for either the antibody to be bound to the solid phase or the labeled antibody, Only β-catenin in which the 86th or 254th tyrosine in the amino acid sequence represented by SEQ ID NO: 1 contained in the test sample is phosphorylated is specifically detected.

  By comparing the amount of non-phosphorylated β-catenin and the amount of phosphorylated β-catenin detected in this manner, the amount of β-catenin and the degree of phosphorylation of β-catenin in the test sample can be measured.

  As a method for examining the degree of phosphorylation of β-catenin using the immunoblotting method, specifically, the cell extract or test substance immobilized on the membrane was reacted with the first antibody and then labeled. A method of reacting the second antibody can be used.

As an antibody used in the immunoblotting method, either a polyclonal antibody or a monoclonal antibody may be used, and an immunoreactive fragment such as Fab, Fab ′, F (ab ′) ₂ described above may be used.

  Examples of the first antibody include the 86th tyrosine phosphorylation-selective anti-β-catenin monoclonal antibody or the 254th tyrosine phosphorylation-selective anti-β-catenin monoclonal antibody of the present invention, an immunoreactive fragment thereof, or non-phosphorylated β A catenin monoclonal antibody or an immunoreactive fragment thereof can be used.

  As the second antibody, any antibody may be used as long as it can react with the first antibody. For example, an anti-mouse immunoglobulin antibody is used.

  Examples of the labeling method include labeling with a radioisotope, enzyme, fluorescence, luminescence, etc., but labeling with an enzyme is preferable.

  A method for examining the degree of β-catenin phosphorylation using an immunohistological staining method can be performed in the same manner as the immunoblotting method. Specifically, after the clinical tissue material fixed on the preparation and the first antibody are reacted, the labeled second antibody is reacted. Examples of clinical tissues include tissues suspected of becoming cancerous and extracts thereof, and it is preferable to use biopsy samples and surgically removed tissues.

  The site | part of the structure | tissue dye | stained in the immunohistochemical staining method shows that 86th or 254th tyrosine of β-catenin represented by the amino acid sequence of SEQ ID NO: 1 is phosphorylated. A patient having a tissue with a high degree of phosphorylation can be identified as a cancer patient.

  In addition, the tumor tissue of cancer patients is stained over time by the same method as described above to measure the degree of phosphorylation of β-catenin at 86th or 254th tyrosine to discriminate cancer growth or invasion. Can do.

  Furthermore, the tumor tissue of a cancer patient and the tissue in the vicinity of the tumor tissue are stained by the same method as described above, and the metastasis of cancer is discriminated by comparing the degree of phosphorylation of β-catenin at 86th or 254th tyrosine. Can do.

  The immunoblotting method and immunohistochemical staining can be performed by the methods described in experimental documents such as Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.

(2) Cancer therapeutic agent using the antibody of the present invention, immunoreactive fragment or derivative thereof The antibody of the present invention, immunoreactive fragment or derivative thereof is the tyrosine at position 86 in the amino acid sequence represented by SEQ ID NO: 1. Alternatively, β-catenin in which tyrosine at position 254 is phosphorylated can be selectively recognized and specifically bound to phosphorylated tyrosine at position 86 or 254 of phosphorylated β-catenin. Therefore, it can be used as a therapeutic agent for cancer by suppressing transcriptional activation of cancer-related genes related to phosphorylated β-catenin.

  The aforementioned drugs can be administered alone, but are usually mixed together with one or more pharmacologically acceptable carriers and produced by any method well known in the pharmaceutical arts. It is desirable to provide it as a pharmaceutical preparation.

  It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous can be used. In the case of a preparation, preferably, intravenous administration can be mentioned.

  Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

  Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.

  Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.

  Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin and the like can be used as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
The injection is prepared using a carrier composed of a salt solution, a glucose solution, or a mixture of both.
Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.

  The spray is prepared using a carrier that does not irritate the compound or antibody itself or the recipient's oral cavity and airway mucosa, and that disperses the compound or antibody as fine particles to facilitate absorption.

  Specific examples of the carrier include lactose and glycerin. Depending on the properties of the compound or antibody and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

  The dosage of the present pharmaceutical composition varies depending on the age, symptoms, etc. of the patient, but is administered to mammals including humans as 0.1-20 mg / kg / day as each compound or antibody. When administering each compound or antibody at the same time, administer once a day (single dose or daily dose) or intermittently 1 to 3 times a week, once every 2 or 3 weeks, separately In some cases, each compound or antibody is injected intravenously once a day (single dose or daily dose) or intermittently once to 3 times a week, once every 2 or 3 weeks, at appropriate times. Do.

(3) Method for screening a substance that specifically inhibits phosphorylation of tyrosine 86 or 254 tyrosine of β-catenin Using the antibody obtained in 1 above, an immunoreactive fragment or derivative thereof, and a cell expressing β-catenin Thus, a substance that inhibits phosphorylation of tyrosine at position 86 or 254 of β-catenin represented by the amino acid sequence of SEQ ID NO: 1 can be screened.

  Specifically, after contacting a test sample with a cell suspected of expressing β-catenin or an extract of the cell and a substance capable of phosphorylating β-catenin, the β described in (1) above. Using the method for measuring the phosphorylation amount of catenin, the phosphorylation amount of β-catenin is measured, and the phosphorylation of tyrosine at position 86 or 254 of β-catenin represented by the amino acid sequence of SEQ ID NO: 1 contained in the test sample Substances that inhibit oxidation can be selected.

  The test substance is not particularly limited as long as it can be mixed with β-catenin in a solution state, and examples thereof include a low molecular compound, a high molecular compound, an organic compound, an inorganic compound, a protein, a gene, a virus, and a cell. The test substance excluding the gene may be added directly to the reaction solution.

  As a method for efficiently introducing a gene into a culture system, a method of adding the gene to a virus vector such as a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, lentivirus, or the like, or an artificial method such as a liposome. For example, a method of enclosing in a panicle structure and adding to a culture system can be mentioned. Specific examples include reports on gene analysis using recombinant viral vectors [Proc. Natl. Acad. Sci. USA, 92, 6733, 1995, Nucleic Acids Res., 18, 3587, 1990, Nucleic Acids Res., 23, 3816, 1995].

  The screening method of the present invention includes a screening method using an immunological assay. Examples of the immunological measurement method include any known immunological measurement method. Examples of the immunoassay include a competitive method and a sandwich method [Immunology Illustrated 5th Edition (Nanedo)], with the sandwich method being preferred.

  Specifically, the screening method using the sandwich method involves binding the first antibody to the solid phase, then containing the test sample and growth factors such as EGF (epidermal growth factor) and HGF (hepatocyte growth factor). This is a method of reacting a cell or an extract of a cell that is considered to express β-catenin cultured in a medium and then reacting with a labeled second antibody.

As an antibody used in the sandwich method, any of polyclonal antibody monoclonal antibodies may be used, and immunoreactive fragments such as Fab, Fab ′, F (ab ′) ₂ described above may be used.

  As a combination of two types of antibodies used in the sandwich method, any of monoclonal antibodies, polyclonal antibodies, and other immunoreactive fragments that recognize different epitopes may be used, but a highly sensitive sandwich ELISA is performed. Therefore, a combination of two kinds of monoclonal antibodies having different antigen binding activities is preferable.

  For example, an anti-β-catenin antibody or an antibody fragment that recognizes an epitope different from the 86-position tyrosine phosphorylation-selective anti-β-catenin antibody or the 254-position tyrosine phosphorylation-selective anti-β-catenin antibody, and the 86-position tyrosine phosphorylation-selective A combination with an anti-β-catenin antibody or a tyrosine phosphorylation-selective anti-β-catenin antibody or an immunoreactive fragment thereof is mentioned. The antibody to be bound to the solid phase and the antibody to be labeled may be either of the two antibodies.

  Examples of the labeling method include labeling with a radioisotope, enzyme, fluorescence, luminescence, etc., but labeling with an enzyme is preferable.

Hereinafter, one embodiment of the screening method of the present invention will be specifically described.
Dispense the 86th tyrosine phosphorylation-selective anti-β-catenin antibody or the 254th tyrosine phosphorylation-selective anti-β-catenin antibody into a 96-well plate, and allow it to stand overnight at 4 ° C for adsorption. After washing, PBS containing 1% bovine serum albumin is added and left at room temperature for 1 hour to block non-specific adsorption. After washing with PBS, after dispensing cell extracts of cells expressing β-catenin cultured in a medium that does not contain growth factors such as EGF (epidermal growth factor) and HGF (hepatocyte growth factor), EGF and HGF A growth factor such as the above and a test substance are added to activate and react β-catenin. After washing, the amount of β-catenin bound to the 86-position tyrosine phosphorylation selective anti-β-catenin antibody or the 254-position tyrosine phosphorylation-selective anti-β-catenin antibody, Measurement is performed by an enzyme immunoassay using an anti-β-catenin antibody that recognizes an epitope different from the oxidation-selective anti-β-catenin antibody.

  Alternatively, the amount of the adapter molecule that can bind to β-catenin bound to the well is measured by an enzyme immunoassay using a label of the adapter molecule or an antibody against the adapter molecule. By comparing the binding amount of β-catenin when no test substance is added or the binding amount of adapter molecules bound to β-catenin and the binding amount when the test substance is added, the amino acid sequence of SEQ ID NO: 1 is represented. A substance that inhibits phosphorylation of tyrosine at position 86 or 254 of β-catenin, or β-catenin phosphorylated at tyrosine at position 86 or 254 in the amino acid sequence represented by SEQ ID NO: 1 and an adapter molecule Inhibiting substances can be screened.

  Examples of adapter molecules include E-cadherin, α-catenin, transcription factor LEF1, or TCF-4.

(4) Method of obtaining a substance that specifically inhibits phosphorylation of 86th or 254th tyrosine phosphorylation of β-catenin by drug design X-ray crystallography or NMR analysis of β-catenin intracellular region and tyrosine kinase domain structure Based on the numerical values obtained from the structural analysis of the above, a computer simulation is performed to estimate the binding region between β-catenin and signaling molecules, and existing compounds that can inhibit the binding between these molecules Inhibitors of phosphorylation of β-catenin can be obtained by synthesizing the compound by designing using the above database or computer software.

  The tyrosine kinase domain may be any enzyme protein that can interact with β-catenin and phosphorylate β-catenin. For example, a tyrosine kinase of a growth factor receptor such as an EGF receptor or an HGF receptor. Domain, or the tyrosine kinase domain of GSK-3β that transmits the wnt signal.

  A substance that specifically inhibits phosphorylation of tyrosine at position 86 or 254 of β-catenin has a structure of a tyrosine kinase domain that phosphorylates at position 86 or 254 in the amino acid sequence represented by SEQ ID NO: 1. By computer simulation based on numerical values obtained by structural analysis such as line crystal analysis or NMR analysis, select new compounds from existing databases or design using computer software, A substance that specifically inhibits phosphorylation of β-catenin can be designed.

  As the computer software, any software can be used as long as it is computer software generally used for drug design from the prediction of the three-dimensional structure of a protein. Specifically, (1) Insight II / Modeler (Accelrys) is used, for example, when the three-dimensional structure of a protein with an unknown structure is automatically constructed from the three-dimensional structure of a similar protein with a known structure. (2) When predicting a ligand binding site from the three-dimensional structure of a protein, for example, Cerius2 / LigandFit (Accelrys) is used. (3) When the three-dimensional structure of a protein is known and the ligand binding site is known (as a region), when the ligand binding mode is predicted, or whether any compound binds (inhibits) In this case, for example, Insight II / Affinity (Accelrys), GOLD (CCDC), FlexX (Tripos), and DOCK (Prof. Kuntz) are used. (4) When designing a compound that is expected to bind (inhibit) from the ligand binding site, for example, Insight II / Ludi (Accelrys) is used.

Whether or not the compound has the inhibitory activity can be confirmed by producing the compound designed above and subjecting it to the assay method shown in the screening method of (2) above.
Examples of the present invention are shown below, but the present invention is not limited thereto.

Abbreviations Abbreviations for amino acids and their protecting groups used in the present invention are the recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature [European Journal of Biochemistry, 138, 9, 1984]. I followed.

The following abbreviations represent the corresponding amino acids below unless otherwise specified.
Ala: L-alanine
Asp: L-aspartic acid
Asx: L-Aspartic acid or L-Asparagine
Arg: L-Arginine
Cys: L-cysteine
Gln: L-glutamine
Glx: L-glutamic acid or L-glutamine
Gly: Glycine
Ile: L-isoleucine
Leu: L-leucine
Met: L-methionine
Phe: L-Phenylalanine
Ser: L-serine
Thr: L-threonine
Tyr: L-tyrosine
pTyr: L-phosphotyrosine
Met: L-methionine
Val: L-Valine

The following abbreviations represent the following amino acid protecting groups and side chain protected amino acids.
Fmoc: 9-fluorenylmethyloxycarbonyl
tBu: t-butyl
Bzl: Benzyl
Trt: Trityl
Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl
Fmoc-Arg (Pmc) -OH: N ^α -9-fluorenylmethyloxycarbonyl-N ^g -2,2,5,7,8-pentamethylchroman-6-sulfonyl-L-arginine
Fmoc-Asp (OtBu) -OH: N ^α -9-fluorenylmethyloxycarbonyl-L-aspartic acid-β-t-butyl ester
Fmoc-Cys (Trt) -OH: N ^α -9-fluorenylmethyloxycarbonyl-S-trityl-L-cysteine
Fmoc-Gln (Trt) -OH: N ^α -9-fluorenylmethyloxycarbonyl-N ^ε -trityl-L-glutamine
Fmoc-Ser (tBu) -OH: N ^α -9-fluorenylmethyloxycarbonyl-Ot-butyl-L-serine
Fmoc-Thr (tBu) -OH: N ^α -9-fluorenylmethyloxycarbonyl-Ot-butyl-L-threonine
Fmoc-Tyr (tBu) -OH: N ^α -9-fluorenylmethyloxycarbonyl-Ot-butyl-L-tyrosine
Fmoc-Tyr (PO (OBzl) OH) -OH: N ^α -9-fluorenylmethyloxycarbonyl-O-benzyl-L-phosphotyrosine

The following abbreviations represent the following reaction solvents, reaction reagents, and the like.
HBTU: 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
HOBt: N-hydroxybenzotriazole
DIEA: Diisopropylethylamine
DMF: N, N-dimethylformamide
TFA: trifluoroacetic acid

In the following examples, the physicochemical properties of the compounds were measured by the following methods.
Mass spectrometry was performed by the FAB-MS method using JEOL JMS-HX110A, or by the MALDI-TOFMS method using the Bruker mass spectrometer REFLEX. Amino acid analysis was performed by the method of Cohen et al. [Analytical Biochemistry, 222, 19, 1994]. Hydrolysis was carried out at 110 ° C. for 20 hours in hydrochloric acid vapor, and the amino acid composition of the hydrolyzate was analyzed using a Waters AccQ-Tag amino acid analyzer (manufactured by Waters).

Example 1. Production of tyrosine phosphorylation selective anti-β-catenin antibody at position 1.86 or 254
(1) Preparation of antigenic peptide The amino acid sequence of the β-catenin protein shown in SEQ ID NO: 1 was analyzed, and the highly hydrophilic part, N-terminal, C-terminal, secondary structure turn part, and part with random coil structure From these, two partial amino acid sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4 considered to be suitable as antigens were selected, and peptide compounds were prepared based on the amino acid sequences (compounds 1 and 3). Furthermore, a tyrosine residue which is considered to be a phosphorylation site in each partial sequence is specified, and a peptide in which tyrosine residues are phosphorylated in two partial sequences shown in SEQ ID NO: 3 and SEQ ID NO: 5 and phosphorylation Untreated peptides (compounds 2 and 4) were made as follows.

(1) -1 Compound 1 (bCAT-86Y) (SEQ ID NO: 2)
Synthesis of (H-Cys-Asp-Ile-Asp-Gly-Gln-Tyr-Ala-Met-Thr-Arg-Ala-NH ₂ )
Fmoc-NH, 27.5μmol-bound carrier resin (Rink amide MBHA resin resin, manufactured by Novabiochem) 50mg is placed in a reaction vessel of an automatic synthesizer (Shimadzu Corporation), 600μl of DMF is added, and the solution is stirred for 3 minutes. After discharging, the following operation was performed according to the synthesis program of Shimadzu Corporation.
(a) 500 μl of 30% piperidine-DMF solution was added, the mixture was stirred for 4 minutes, the solution was drained, and this operation was repeated once more.
(b) The carrier resin was washed with 600 μl of DMF for 1 minute, the solution was discharged, and this operation was repeated 5 times.
(c) Fmoc-Ala-OH (275 μmol), HBTU (275 μmol), HOBt monohydrate (275 μmol) and DIEA (550 μmol) were stirred in DMF (1.10 ml) for 3 minutes. In addition, the mixture was stirred for 60 minutes and the solution was drained.

  After washing the carrier resin with 600 μl of DMF for 1 minute, the solution was discharged, and this was repeated 5 times. In this way, Fmoc-Ala-NH was synthesized on the support.

Next, after the steps (a) and (b), a condensation reaction is performed using Fmoc-Arg (Pmc) -OH in the step (c), and after the washing step (d), Fmoc-Arg (Pmc ) -Ala-NH was synthesized on the support.
Hereinafter, in step (c), Fmoc-Thr (tBu) -OH, Fmoc-Met-OH, Fmoc-Ala-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly (A) to (d) were repeated using -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ile-OH, Fmoc-Asp (OtBu) -OH, and Fmoc-Cys (Trt) -OH in this order. Thereafter, after deprotection and washing steps of (a) and (b), it was washed successively with methanol and butyl ether and dried under reduced pressure for 12 hours to obtain a carrier resin to which a side chain protected peptide was bound. This is a mixture of TFA (82.5%), thioanisole (5%), water (5%), ethyl methyl sulfide (3%), 1,2-ethanedithiol (2.5%) and thiophenol (2%) 1 ml of the solution was added and allowed to stand at room temperature for 8 hours to remove the side chain protecting group and excise the peptide from the resin. After filtration of the resin, about 10 ml of ether was added to the resulting solution, and the resulting precipitate was collected by centrifugation and decantation to obtain 41.5 mg as a crude peptide. The entire amount of the crude product was dissolved in an acetic acid aqueous solution, and then purified by HPLC using a reverse phase column (manufactured by Shiseido, CAPCELL PAK C18 30 mm I.D. X 250 mm). Elution was performed by a linear concentration gradient method in which 90% acetonitrile aqueous solution containing 0.1% TFA was added to 0.1% TFA aqueous solution, and detection was performed at 220 nm to obtain a fraction containing Compound 1. This fraction was lyophilized to give 11.4 mg of compound 1 having the following physicochemical properties:
Mass spectrometry [TOFMS]; m / z = 1342.5 (M + H ⁺ )
Amino acid analysis; Asx 2.1 (2), Glx 1.1 (1), Gly 1.1 (1), Arg 1.1 (1), Thr 1.0 (1), Ala 2.1 (2), Tyr 0.9 (1), Met 0.6 (1) , Ile 1.0 (1), Cys 1.4 (1)

(1) -2 Compound 2 (bCAT-86pY) (SEQ ID NO: 3)
Synthesis of (H-Cys-Asp-Ile-Asp-Gly-Gln-pTyr-Ala-Met-Thr-Arg-Ala-NH ₂ )
Fmoc-Ala-OH, Fmoc-Arg (Pmc), starting from 50mg of Fmoc-NH, 27.5μmol-bound carrier resin (Rink amide MBHA resin resin, Novaviokem) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Met-OH, Fmoc-Ala-OH, Fmoc-Tyr (PO (OBzl) OH) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly- OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ile-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Cys (Trt) -OH are condensed sequentially, and after Fmoc group removal, washing and drying, A carrier resin having a side chain-protected peptide bound thereto was obtained. This is a mixture of TFA (82.5%), thioanisole (5%), water (5%), ethyl methyl sulfide (3%), 1,2-ethanedithiol (2.5%) and thiophenol (2%) 1 ml of the solution was added and allowed to stand at room temperature for 8 hours to remove the side chain protecting group and excise the peptide from the resin. After filtration of the resin, about 10 ml of ether was added to the resulting solution, and the resulting precipitate was collected by centrifugation and decantation to obtain 38.7 mg as a crude peptide. The entire amount of the crude product was dissolved in an acetic acid aqueous solution, and then purified by HPLC using a reverse phase column (manufactured by Shiseido, CAPCELL PAK C18 30 mm I.D. X 250 mm). Elution was performed by a linear concentration gradient method in which a 90% acetonitrile aqueous solution containing 0.1% TFA was added to a 0.1% TFA aqueous solution, and detection was performed at 220 nm to obtain a fraction containing Compound 2. This fraction was lyophilized to give 14.1 mg of compound 2 having the following physicochemical properties:
Mass spectrometry [TOFMS]; m / z = 1422.6 (M + H ⁺ )
Amino acid analysis; Asx 2.1 (2), Glx 1.1 (1), Gly 1.0 (1), Arg 1.0 (1), Thr 1.0 (1), Ala 2.0 (2), Tyr 0.9 (1), Met 1.0 (1) , Ile 0.9 (1), Cys 1.4 (1)
However, phosphotyrosine was detected as tyrosine by hydrolysis in amino acid analysis.

(1) -3 Compound 3 (bCAT-254Y) (SEQ ID NO: 4)
Synthesis of (H-Cys-Asp-Ser-Val-Leu-Phe-Tyr-Ala-Ile-Thr-Thr-Leu-NH ₂ )
Fmoc-NH, 27.5 μmol-bound carrier resin (Rink Amide MBHA resin resin, manufactured by Novabiochem) 50 mg as a starting material, as in (1) -1 above, Fmoc-Leu-OH, Fmoc-Thr (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Val-OH , Fmoc-Ser (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Cys (Trt) -OH were sequentially condensed, and then Fmoc group removal, washing, and drying were followed by binding of the side chain protective peptide. A carrier resin was obtained. To this was added 1 ml of a mixed solution consisting of TFA (90%), thioanisole (5%) and 1,2-ethanedithiol (5%) and left at room temperature for 2 hours to remove side chain protecting groups and resin The peptide was cut out. After the resin was filtered off, about 10 ml of ether was added to the resulting solution, and the resulting precipitate was collected by centrifugation and decantation to obtain 30.4 mg as a crude peptide. The total amount of this crude product was dissolved in a mixed solution consisting of dithiothreitol and DMF, and purified by HPLC using a reverse phase column (manufactured by Shiseido, CAPCELL PAK C18 30 mm I.D. X 250 mm). Elution was performed by a linear concentration gradient method in which 90% acetonitrile aqueous solution containing 0.1% TFA was added to 0.1% TFA aqueous solution, and detection at 220 nm gave a fraction containing compound 3. This fraction was lyophilized to give 2.9 mg of compound 3 having the following physicochemical properties:
Mass spectrometry [TOFMS]; m / z = 1936.0 (M + H ⁺ )
Amino acid analysis; Asx 1.1 (1), Gly 0.9 (1), Thr 2.1 (2), Ala 1.2 (1), Tyr 1.0 (1), Val 0.7 (1), Ile 1.1 (1), Leu 1.9 (2) , Phe 1.0 (1), Cys 1.3 (1)

(1) -4 Compound 4 (bCAT-254pY) (SEQ ID NO: 5)
Synthesis of (H-Cys-Asp-Ser-Val-Leu-Phe-pTyr-Ala-Ile-Thr-Thr-Leu-NH ₂ )
Fmoc-NH, 27.5 μmol-bound carrier resin (NovaSyn TGR resin resin, manufactured by Novabiochem) 50 mg as the starting material, Fmoc-Leu-OH, Fmoc-Thr (tBu)- OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Tyr (PO (OBzl) OH) -OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc- After condensing Val-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asp (OtBu) -OH, and Fmoc-Cys (Trt) -OH sequentially, the Fmoc group is removed, washed, and dried, and then side chain protected peptide To obtain a bound carrier resin. To this was added 1 ml of a mixed solution consisting of TFA (90%), thioanisole (5%) and 1,2-ethanedithiol (2.5%) and left at room temperature for 2 hours to remove side chain protecting groups and resin The peptide was cut out. After filtration of the resin, about 10 ml of ether was added to the resulting solution, and the resulting precipitate was collected by centrifugation and decantation to obtain 34.1 mg as a crude peptide. The total amount of this crude product was dissolved in a mixed solution consisting of dithiothreitol and dimethyl sulfoxide, and purified by HPLC using a reverse phase column (CAPE CELL PAK C18 30 mm I.D. X 250 mm, manufactured by Shiseido). Elution was performed by a linear concentration gradient method in which 90% acetonitrile aqueous solution containing 0.1% TFA was added to 0.1% TFA aqueous solution, and detection was performed at 220 nm to obtain a fraction containing compound 4. This fraction was lyophilized to give 2.9 mg of compound 4 having the following physicochemical properties:
Mass spectrometry [TOFMS]; m / z = 1424.6 (M + H ⁺ )
Amino acid analysis; Asx 1.1 (1), Gly 1.0 (1), Thr 2.0 (2), Ala 1.0 (1), Tyr 0.9 (1), Val 0.9 (1), Ile 1.0 (1), Leu 2.0 (2) , Phe 1.0 (1), Cys 1.3 (1)
However, phosphotyrosine was detected as tyrosine by hydrolysis in amino acid analysis.

(2) Preparation of immunogen Of the peptides represented by compounds 1 to 4 obtained in Example 1 (1), compound 2 (bCAT-86pY) and compound 4 (bCAT-254pY) enhance immunogenicity. For the purpose, a conjugate with KLH (manufactured by Nacalai Tesque) was prepared by the following method and used as an immunogen. That is, KLH was dissolved in PBS to prepare 10 m / mL, and 1/10 volume of 25 m / mL MBS [N- (m-Maleimidobenzoyloxy) succinimide; manufactured by Nacalai Tesque] was added dropwise and reacted for 30 minutes with stirring. . Peptide 1 mg obtained by dissolving 2.5 mg of KLH-MBS obtained by removing unreacted MBS in a gel filtration column such as Sephadex G-25 column previously equilibrated with PBS in 0.1 M sodium phosphate buffer (pH 7.0) And stirred for 3 hours at room temperature. After the reaction, dialyzed with PBS was used as an immunogen.

(3) Immunization of animals and preparation of antibody-producing cells 100 μg of the KLH conjugates of Compound 2 and Compound 4 prepared in Example 1 (2) were each added with an aluminum hydroxide adjuvant [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 99 , 1988] 2 mg and pertussis vaccine (Chiba Prefectural Serum Research Institute) 1 × 10 ⁹ cells were administered to each of three 6-week-old female BDF1 mice. From 2 weeks after administration, 100 μg of each KLH conjugate was administered once a week for a total of 4 times. Blood was collected from the fundus venous plexus of the mouse, the serum antibody titer thereof was examined by the enzyme immunoassay shown below, and the spleen was removed 3 days after the final immunization from the mouse exhibiting a sufficient antibody titer.

  The spleen was shredded in MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical), loosened with tweezers, and centrifuged (280 × g, 5 minutes). Tris-ammonium chloride buffer (pH 7.6) was added to the obtained precipitate fraction, and erythrocytes were removed by treatment for 1 to 2 minutes. The obtained precipitate fraction (cell fraction) was washed 3 times with MEM medium and used for cell fusion.

(4) Preparation of mouse myeloma cells
8-Azaguanine-resistant mouse myeloma cell line P3-X63Ag8-U1 (P3-U1: purchased from ATCC) was cultured in normal medium (RPMI medium supplemented with 10% fetal bovine serum), and 2 × 10 ⁷ or more cells were fused during cell fusion. Cells were secured and served as parental strain for cell fusion.

(5) Preparation of hybridoma The mouse spleen cells obtained in Example 1 (3) and the myeloma cells obtained in Example 1 (4) were mixed at a ratio of 10: 1 and centrifuged (280 × g 5 minutes). 10 ⁸ mice with a mixture of polyethylene glycol-1000 (PEG-1000) 2 g, MEM medium 2 mL and dimethyl sulfoxide 0.7 mL at 37 ° C. while thoroughly aggregating the cell group of the obtained precipitate fraction 0.5 mL per splenocyte was added, and 1 mL of MEM medium was added several times to the suspension every 1-2 minutes, and then the MEM medium was added so that the total volume became 50 mL.

The suspension was centrifuged (170 × g, 5 minutes), and the cells of the obtained precipitate fraction were loosened gently. Suspended in 100 mL of a medium in which a HAT medium additive (GIBCO BRL) was added to an RPMI medium supplemented with fetal serum]. The suspension was dispensed into a 96-well culture plate at 200 μL / well and cultured at 37 ° C. for 10-14 days in a 5% CO ₂ incubator.

  After culture, the culture supernatant was subjected to the enzyme immunoassay described in Example 1 (6), and the control was the same amino acid sequence but not phosphorylated with respect to compound 2 and compound 4 used as antigenic peptides. Compared with compounds 1 and 3 used as peptides, wells showing strong reactivity were selected, and cloning by limiting dilution was repeated twice from the cells contained therein, and the 86th tyrosine phosphorylation selective anti-β-catenin monoclonal was selected. An antibody-producing hybridoma or a hybridoma producing an anti-β-catenin monoclonal antibody selective for tyrosine phosphorylation at position 254 was established. A total of 14 clones of KM2890 and KM2891 were obtained using compound 2 as an antigen peptide, and KM2850 to KM2858 and KM2972 to KM2974 were obtained using compound 4 as an antigen peptide.

(6) Enzyme immunoassay (binding ELISA)
As an antigen for assay, a compound obtained by conjugating compounds 1 to 4 obtained in Example 1 (1) with thyroglobulin (hereinafter referred to as THY) was used. The preparation method is as described in Example 1 (2), but SMCC [4- (N-Maleimidomethyl) -cyclohexane-1-carboxylic acid N-hydroxysuccinimido ester; manufactured by Sigma Co.] is used instead of MBS as the crosslinking agent. Was used. For compound 2, compound 1 (bCAT-86Y), which is a non-phosphorylated peptide having the same sequence as compound 2, and for compound 4, compound 3 (bCAT-254Y) is also used as each negative control peptide. And used in a conjugate. The conjugate prepared as described above was dispensed into a 96-well ELISA plate (manufactured by Greiner) at 5 μg / mL at 50 μL / well and allowed to stand overnight at 4 ° C. for adsorption. After washing the plate, 100 μL / well of Dulbecco's phosphate buffer (PBS) containing 1% bovine serum albumin (hereinafter referred to as BSA) (hereinafter referred to as 1% BSA / PBS) was added, The remaining active groups were blocked by standing at room temperature for 1 hour. 1% BSA / PBS was discarded, and immunized mouse antiserum, hybridoma culture supernatant or purified monoclonal antibody was dispensed at 50 μL / well as a primary antibody to the plate and left for 2 hours. The plate was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate [(ICI trademark Tween 20 equivalent: Wako Pure Chemical Industries)] / PBS (hereinafter referred to as Tween-PBS), and then secondary antibody Peroxidase-labeled rabbit anti-mouse immunoglobulin (Dako) was added at 50 μL / well and allowed to stand at room temperature for 1 hour. After the plate was washed with Tween-PBS, ABTS substrate solution [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid), 1 mmol / L / 0.1 mol / L citrate buffer (pH 4.2)] was added at 50 μL / Color was developed by adding in wells, and the absorbance at OD 415 nm was measured using a plate reader (Emax; manufactured by Molecular Devices).

(7) Purification of monoclonal antibody The hybridoma strain obtained in Example 1 (5) was intraperitoneally injected into pristane-treated 8-week-old nude female mice (BALB / c) at 5 × 10 ⁶ to 2 × 10 ⁷ cells / mouse. Injected. After 10 to 21 days, ascites was collected from the mice in which ascites accumulated due to the hybridoma becoming ascites cancer.

  The ascites was centrifuged (1200 × g, 5 minutes) to remove solids. The purified IgG monoclonal antibody was obtained by purification by the caprylic acid precipitation method [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988]. The subclasses of the monoclonal antibodies produced by the hybridomas KM2850 to KM2858, KM2890, KM2891 and KM2972 to KM2974 were determined as shown in Table 1 by ELISA using a subclustering kit.

Example 2 Examination of reactivity of monoclonal antibody
(1) Reactivity with antigen peptide in antigen solid phase system (binding ELISA)
This was carried out according to the method shown in Example 1 (6). As the primary antibody, the hybridoma culture supernatant obtained in Example 1 (5) was diluted 6-fold with 5-fold dilution. The results are shown in FIG. All monoclonal antibodies showed strong reactivity to the phosphorylated antigenic peptide (compound 2 or 4) compared to the negative control non-phosphorylated peptide (compound 1 or 3).

(2) Reactivity to antigenic peptides in the antigen liquid phase system (Inhibition ELISA)
The reactivity of the 86-position tyrosine phosphorylation-selective anti-β-catenin monoclonal antibody prepared by using compound 2 as an antigen peptide in Example 1 (6) with respect to various peptides shown in compounds 1 to 4 was determined by an inhibition ELISA. I examined it. Prepare an antigen-immobilized plate in the same manner as in Example 1 (6), dispense compounds diluted 10-fold from 10 μg / mL in 10-fold dilutions at 50 μL / well, and then incubate the hybridoma KM2890 or KM2891. The supernatant was diluted 2000 times, dispensed at 50 μL / well, mixed in the well, and allowed to react at room temperature for 2 hours. The wells were washed with Tween-PBS, diluted peroxidase-labeled rabbit anti-mouse immunoglobulin (Dako) was added at 50 μL / well, reacted at room temperature for 1 hour, washed with Tween-PBS, and then ABTS substrate solution [2.2-Adinobis (3-Ethylbenzothiazole-6-sulfonic acid) ammonium] was added at 50 μL / well for color development, and the absorbance at OD 415 nm was measured with a plate reader (Emax; manufactured by Wako Pure Chemical Industries, Ltd.).

  As shown in FIG. 2, the 86-position tyrosine phosphorylation selective anti-β-catenin monoclonal antibodies (KM2890-derived (1) and KM2891-derived (2)) both reacted with compounds 3 and 4 having different amino acid sequences in the liquid phase system. I did not. Moreover, it reacted 10 to 100 times more strongly with the compound 2 which is a phosphorylated peptide compared with the compound 1 which is the negative control which is the same amino acid sequence but is not phosphorylated.

(3) Western blotting Human squamous cell carcinoma cell line A431 (ATCC No. CRL-1555) and human gastric cancer cell line TMK-1 [Jpn. J. Cancer Rec., 76, 1064-1071, 1985] After culturing in RPMI medium for 48 hours, epidermal growth factor EGF was added to 50 ng / mL, and after 0 hours and 0.5 hours, it was collected using EDTA solution to prepare a cell lysate as follows. As a comparative control, a cell lysate was also prepared from cells cultured in serum-free culture and RPMI medium supplemented with 10% FCS without treatment with epidermal growth factor EGF.

The collected cells were washed with PBS, and then a cell lysis buffer (50 mM Tris-HCl pH 7.2, 1% TritonX, 50 mM NaCl, 2 mM MgCl ₂ , 2 mM CaCl ₂ , 0.1% NaN ₃ , 50 mM iodoacetamide, 50 mM N -Ethylmaleimide, 1 mg / ml leupeptin, 0.1 mM dithiothreitol) was added to 5 × 10 ⁷ cells, left at 4 ° C. for 0.5 hours, and centrifuged to obtain the supernatant fraction. Using. SDS-PAGE sample buffer was added to the resulting cell lysate, heated at 100 ° C for 5 minutes, and SDS-polyacrylamide electrophoresis (5% concentrated gel, 7.5% separation gel, 200 V at 1 x 10 ⁵ cells / lane) 45 minutes) After fractionation with [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988], blotting was performed on a PVDF membrane (25 V, 30 minutes or 60 minutes) with a semi-dry blotting apparatus. After blocking with 5% Skimmilk, 1% BSA, and 0.1% Tween-PBS for 2 hours, the 86th tyrosine phosphorylation selective anti-βcatenin monoclonal antibody hybridoma (KM2891) or the 254th tyrosine phosphorylation selective anti-βcatenin monoclonal antibody hybridoma ( The culture supernatant of KM2853) was reacted overnight at 4 ° C. After thoroughly washing with 0.1% Tween-20, 1 mM Orthovanadate-PBS, a peroxidase-labeled rabbit anti-mouse immunoglobulin antibody (Zymet) was reacted for 1 hour as the second antibody. After thoroughly washing with 0.1% Tween, 1 mM Orthovanadate-PBS, detection was performed using an ECL detection kit (Amersham-Pharmacia) and exposed on an X-ray film.

  FIG. 3 shows the results of Western blotting with the 86-position tyrosine phosphorylation selective anti-β-catenin monoclonal antibody (derived from KM2891). TMK-1 cells 0.5 hours after addition of epidermal growth factor EGF after serum-free culture were compared to TMK-1 cells immediately after addition of epidermal growth factor EGF. The signal of the band detected with the antibody (from KM2891) was clearly enhanced. Therefore, the 86th tyrosine phosphorylation selective anti-β-catenin monoclonal antibody (derived from KM2891) is converted to β-catenin phosphorylated at the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 by stimulation with epidermal growth factor EGF. It was shown that β-catenin, which binds more strongly than non-phosphorylated β-catenin and phosphorylates the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 by Western blotting, can be detected.

  FIG. 4 shows the results of Western blotting using an anti-β-catenin monoclonal antibody selective for tyrosine phosphorylation at position 254 (derived from KM2853). After serum-free culture, A431 cells 0.5 hours after addition of epidermal growth factor EGF were compared with A431 cells immediately after addition of epidermal growth factor EGF, and 254 tyrosine phosphorylation selective anti-β-catenin monoclonal antibody (derived from KM2853) The signal of the band detected in) was clearly enhanced. Therefore, the anti-β-catenin monoclonal antibody selective for tyrosine phosphorylation at position 254 (derived from KM2853) does not react with β-catenin in which the tyrosine residue at position 254 in the amino acid sequence represented by SEQ ID NO: 1 by phosphorylation of epidermal growth factor EGF is phosphorylated. It was shown that β-catenin, which binds stronger than phosphorylated β-catenin and phosphorylates the tyrosine residue at position 254 in the amino acid sequence represented by SEQ ID NO: 1 by Western blotting, can be detected.

(4) Immunohistochemical staining (ABC method)
As a specimen, a section of human tongue cancer (squamous cell carcinoma) subjected to paraffin embedding treatment after formalin fixation was used. The sections were deparaffinized and hydrophilized with a xylene-ethanol series, washed with running water, and endogenous peroxidase was inactivated with a 0.3% hydrogen peroxide-methanol solution. As an antigen activation treatment, a microwave treatment (95 ° C., 20 minutes) was performed in 1 mM Orthovanadate-0.01M citrate buffer (PH6.0). After washing with running water and 0.1% Tween-PBS, blocking was performed with 5% Skimmilk-1% BSA-1 mM Orthovanadate-0.1% Tween-PBS for 40 minutes. After treatment with 2% NSS-1 mM Orthovanadate-PBS (3 minutes), the hybridoma culture supernatant of 86- or 254-position tyrosine phosphorylation-selective anti-β-catenin monoclonal antibody as a primary antibody was reacted overnight at 4 ° C. . After washing with 0.1% Tween-PBS and treatment with 2% NSS-1 mM Orthovanadate-PBS (3 minutes), a biotinylated anti-mouse immunoglobulin (manufactured by VECTOR) was reacted as a secondary antibody for 45 minutes at room temperature. . After washing with 0.1% Tween-PBS and treatment with 2% NSS-1 mM Orthovanadate-PBS (3 minutes), sABC reagent (manufactured by Dako) was reacted at room temperature for 40 minutes. After washing with 0.1% Tween-PBS, the diaminobenzidine aqueous solution was reacted for 3 to 20 minutes to develop color. After washing with running water, the cells were stained with hematoxylin, washed with running water, dehydrated with ethanol-xylene series, passed through, sealed and examined.

  As a result of staining with 86-position tyrosine phosphorylation-selective anti-β-catenin monoclonal antibody (from KM2891) or 254-position tyrosine phosphorylation-selective anti-β-catenin monoclonal antibody (from KM2853) as a primary antibody, the cell membrane part of cancer cells is specific Stained. Therefore, it was revealed that these antibodies can detect phosphorylation of 86th or 254th tyrosine phosphorylation of β-catenin represented by the amino acid sequence of SEQ ID NO: 1 in cancer tissue by immunohistochemical staining.

The antigen peptide selective reactivity in monoclonal antibody binding ELISA is shown. The horizontal axis represents the dilution factor of the culture supernatant of each antibody-producing hybridoma used in ELISA, and the vertical axis represents the absorbance. The antigenic peptide selective reactivity of the monoclonal antibody in the inhibition ELISA is shown. The horizontal axis represents the concentration of compounds 1 to 4 which are peptides added to the reaction system, and the vertical axis represents absorbance. The reactivity in Western blotting of 86th tyrosine phosphorylation selective anti-β-catenin monoclonal antibody (derived from KM2891) is shown. In the figure, SFM indicates serum-free culture, and FBS M indicates serum-added culture. The lane shows cells from the left for each cell used, immediately after addition of EGF after serum-free culture, 0.5 hour later, and further during serum-added culture. The arrow indicates the β-catenin band. The reactivity in the Western blotting of a tyrosine phosphorylation selective anti-β-catenin monoclonal antibody (derived from KM2853) is shown. Each lane using A431 cells shows cells from the left for each cell used, immediately after addition of EGF after serum-free culture, 0.5 hour later, and further during serum-added culture.

Claims (16)

  An antibody that selectively recognizes β-catenin in which the tyrosine residue at position 86 or 254 in the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.   The antibody according to claim 1, wherein the antibody is a monoclonal antibody.   The monoclonal antibody according to claim 2, wherein the antibody is produced from hybridoma KM2853 or KM2891.   The antibody according to any one of claims 1 to 3, which is an IgG class antibody.   An immunoreactive fragment of the antibody according to any one of claims 1 to 4.   The derivative | guide_body of the antibody of any one of Claims 1-4.   A hybridoma that produces a monoclonal antibody that selectively recognizes β-catenin in which the 86th tyrosine residue in the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.   A hybridoma that produces a monoclonal antibody that selectively recognizes β-catenin in which the tyrosine residue at position 254 in the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.   The hybridoma according to claim 7, which is specified by the accession number FERM BP-8262.   The hybridoma according to claim 8, which is specified by the accession number FERM BP-8261.   The therapeutic agent of cancer containing the antibody of any one of Claims 1-4, its immunoreactive fragment, or a derivative.   A diagnostic agent for cancer comprising the antibody according to any one of claims 1 to 4, an immunoreactive fragment or derivative thereof.   A method for detecting tyrosine phosphorylation of β-catenin by an immunological assay using the antibody, immunoreactive fragment or derivative thereof according to any one of claims 1 to 4.   A method for discriminating cancer using the antibody, immunoreactive fragment or derivative thereof according to any one of claims 1 to 4.   A phosphorylation of a tyrosine residue at position 86 or 254 of β-catenin represented by the amino acid sequence of SEQ ID NO: 1, using the antibody, immunoreactive fragment or derivative thereof according to any one of claims 1 to 4. A method of screening for an agent that inhibits oxidation.   A method for screening for a therapeutic agent for cancer using the antibody, immunoreactive fragment or derivative thereof according to any one of claims 1 to 4.

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