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WO2004018679A1 - Methode et trousse de diagnostic du cancer - Google Patents

Methode et trousse de diagnostic du cancer Download PDF

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Publication number
WO2004018679A1
WO2004018679A1 PCT/JP2003/010676 JP0310676W WO2004018679A1 WO 2004018679 A1 WO2004018679 A1 WO 2004018679A1 JP 0310676 W JP0310676 W JP 0310676W WO 2004018679 A1 WO2004018679 A1 WO 2004018679A1
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WIPO (PCT)
Prior art keywords
cancer
polypeptide
nucleic acid
subject
factor
Prior art date
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PCT/JP2003/010676
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English (en)
Japanese (ja)
Inventor
Hideaki Shimada
Takeshi Tomonaga
Kazuyuki Matsushita
Takenori Ochiai
Fumio Nomura
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Japan Science and Technology Agency
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Japan Science and Technology Agency
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Priority to JP2004530611A priority Critical patent/JPWO2004018679A1/ja
Publication of WO2004018679A1 publication Critical patent/WO2004018679A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G01N33/5758—

Definitions

  • the invention of the present application relates to the field of cancer. More particularly, the present invention relates to methods and kits for diagnosing cancer. More specifically, the present invention relates to an antigenic peptide for human colon cancer and a method for diagnosing colorectal cancer, comprising two novel antigenic peptides expressed in human colon cancer cells, and colon cancer using one or more of these peptides. It relates to a method of diagnosing with high accuracy. Background art
  • colorectal cancer rectal cancer and colon cancer
  • rectal cancer and colon cancer which is a typical example of cancer
  • Metastases such as lymphatic, hematogenous, and peritoneal dissemination occur, and particularly hematogenous metastases often progress to liver cancer.
  • colorectal cancer As with other malignancies, early detection of carcinoma is the most important issue in combating colorectal cancer. In particular, cancers arising in the upper part of the large intestine have poor subjective symptoms, and there is a high risk that the disease is progressing when they are discovered. Conventionally, colorectal cancer has been mainly screened for fecal occult blood reaction, diagnosed with serum markers such as CEA or CA19-9, and diagnosed with the course of treatment. However, these methods have a high positive rate for advanced cancers, but extremely low for early cancers, making accurate diagnosis difficult. As a method that enables simple and reliable early diagnosis of malignant tumors, a molecular biological diagnosis method using a cancer tissue-specific protein marker has been proposed.
  • Japanese Patent Publication No. 7-51065 discloses the use of glycoprotein 39 as a tumor marker.
  • the glycoprotein described in Japanese Patent Application Laid-Open No. 7-51065 is actually used as a tumor marker, and therefore it cannot be said that its accuracy is high. It also does not state that it can be used for definitive diagnosis.
  • the invention of the present application has been made in view of the above circumstances, and has an object to provide a novel antigenic peptide that is effective for diagnosing cancer (for example, colon cancer).
  • Another object of the present invention is to provide an antibody against the above-mentioned antigenic peptide and a method for diagnosing cancer using the same. Summary of the invention
  • the present invention has been accomplished by discovering that FIR and CENPA can be used unexpectedly as indicators of cancer. Further, the present invention achieves a more excellent effect by finding that a normal type and several variants thereof are present in the FIR, and it is found that a definitive diagnosis of cancer can be made by their identification. Therefore, the present invention provides the following. 1. (a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, its complement, or its fragment sequence;
  • one or more amino acids is a modified polypeptide having at least one mutation selected from the group consisting of substitution, addition, and deletion, A polynucleotide encoding a variant polypeptide having biological activity;
  • polynucleotide consisting of a polynucleotide having at least 70% identity to the polynucleotide of any one of (a) to (c) or a complementary sequence thereof, and having a biological activity.
  • a nucleic acid molecule A nucleic acid molecule.
  • nucleic acid molecule of claim 1 having a length of at least 8 contiguous nucleotides.
  • nucleic acid molecule of claim 1 wherein said biological activity is the ability to bind FBP.
  • nucleic acid molecule according to claim 3 wherein the ability to bind to FBP is defined by the sequence set forth in positions 372 to 442 in SEQ ID NO: 2.
  • the factor according to claim 5 wherein the factor is a nucleic acid molecule and is used as a primer.
  • the label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen antibody reaction.
  • nucleic acid molecule comprising the sequence shown in SEQ ID NO: 1 or a complement thereof.
  • nucleic acid molecule comprising:
  • said alteration is a substitution; an addition between positions 369 and 370 of SEQ ID NO: 1 or between positions 176 and 177 of SEQ ID NO: 15; or up to position 369 of SEQ ID NO: 1 or SEQ ID NO: 14.
  • nucleic acid molecule of claim 13 wherein said modification is in the range of about 400 bases from the 5 'end of SEQ ID NO: 1.
  • the nucleic acid molecule of claim 13, wherein the modification is in the range of about position 150 to about position 360 in SEQ ID NO: 1. 17.
  • the nucleic acid molecule of claim 13, wherein the modification comprises a non-conservative substitution of an encoded amino acid in SEQ ID NO: 1.
  • nucleic acid molecule according to claim 13 comprising a sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, 17, 19 and 21.
  • the agent of claim 25, wherein the label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen antibody reaction.
  • the factor of claim 27, wherein said factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof.
  • agent of claim 27 wherein said agent is a nucleic acid molecule having a length of at least 8 contiguous nucleotides.
  • said factor is a nucleic acid molecule and is used as a primer.
  • a cancer diagnostic kit comprising:
  • a cancer diagnostic kit comprising:
  • kits of claim 39 wherein the cancer comprises a c-myc overexpressing cancer.
  • the cancer comprises rectal and colon cancer.
  • the detection of the nucleic acid molecule in a subject portion of the subject of the subject comprises expression of the nucleic acid molecule in a subject not afflicted with cancer or a portion not afflicted with cancer.
  • kit according to claim 43 wherein the cancer comprises c-myc overexpressing cancer.
  • the abundance of the third nucleic acid molecule is compared with the abundance of the same nucleic acid molecule in a sample from a subject not having cancer or a portion not having cancer. 48.
  • the method of claim 47 wherein if the amount of the nucleic acid molecule in a sample from a subject portion of the subject is increasing, the subject of the subject is determined to be a cancer patient. .
  • C) means for identifiably detecting a signal caused by the second factor and the third factor and measuring the expression levels of the second nucleic acid molecule and the third nucleic acid molecule,
  • the expression of the second nucleic acid molecule in a target portion of the subject is higher than the expression of the second nucleic acid molecule in a subject not having cancer or a portion not having cancer, and If the expression of the third nucleic acid molecule in the subject portion of the subject is higher than the expression of the third nucleic acid molecule in a subject not afflicted with cancer or a portion not afflicted with cancer, Determining that the subject is a cancer patient, means,
  • a cancer diagnostic kit comprising: 50.
  • kit of claim 49 wherein the cancer comprises c-myc overexpressing cancer.
  • the second factor does not specifically react with the nucleic acid molecule according to claim 13, or the third factor does not specifically react with the nucleic acid molecule according to claim 12,
  • kit of claim 49 wherein the second factor and the third factor are or are identifiably labeled.
  • kit according to claim 49 further comprising a factor that distinguishably labels the second factor and the third factor.
  • the ratio of the expression amount of the third nucleic acid molecule to the expression amount of the first nucleic acid molecule in a subject not afflicted with cancer or a portion not afflicted with cancer is the ratio of the expression amount of the third nucleic acid molecule in the target portion of the subject.
  • the ratio of the expression amount of the third nucleic acid molecule to the expression amount of the first nucleic acid molecule is higher than the ratio, the subject of the subject is determined to be a cancer patient.
  • a cancer diagnostic kit comprising:
  • kits according to claim 57 wherein said C) means includes means for comparing a relative abundance ratio or an absolute abundance with said third nucleic acid molecule.
  • kits of claim 57, wherein the cancer comprises a c-myc overexpressing cancer.
  • kit of claim 57 wherein the cancer comprises rectal and colon cancer.
  • the first factor does not specifically react with the nucleic acid molecule according to claim 13 or the first factor is the nucleic acid molecule according to claim 13 and the nucleic acid molecule according to claim 12.
  • the kit according to claim 57 which specifically reacts with both of the nucleic acid molecules.
  • kit of claim 57 wherein the first factor and the third factor are or are identifiably labeled.
  • kit according to claim 57 further comprising a factor that distinguishably labels the first factor and the third factor.
  • the ratio of the expression amount of the third nucleic acid molecule in the amount is Determining that the subject of the subject is a cancer patient if the ratio of the expression amount of the third nucleic acid molecule to the expression amount of the first nucleic acid molecule in the target portion of the test subject is higher than , Method.
  • polypeptide comprising a polynucleotide having at least 70% identity to the polynucleotide of any one of (a) to (c) or a complementary sequence thereof, and having a biological activity.
  • nucleic acid molecule for the manufacture of a diagnostic agent for cancer.
  • polypeptide which has at least one mutation selected from the group consisting of substitution, addition, and deletion in the amino acid sequence of SEQ ID NO: 2 and has biological activity, ;
  • polypeptide having an amino acid sequence having at least 70% identity to the polypeptide of any one of (a) to (d) and having biological activity
  • polypeptide of claim 66 having at least three consecutive amino acids in length.
  • polypeptide of claim 66 wherein said biological activity is the ability to bind FBP.
  • polypeptide of claim 68, wherein said ability to bind to FBP is defined by the sequence set forth in positions 372-442 in SEQ ID NO: 2.
  • polypeptide of claim 66 wherein the polypeptide is or can be labeled.
  • the factor of claim 71, wherein the factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof.
  • the factor of claim 71 wherein said factor is an antibody or derivative thereof. 74. The agent of claim 71, wherein said agent is used as a probe. 75. The agent of claim 71, wherein the agent is or can be labeled.
  • the agent of claim 75, wherein the label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen antibody reaction.
  • polypeptide of claim 66 comprising the sequence set forth in SEQ ID NO: 2.
  • amino acid sequence of SEQ ID NO: 2 comprising at least one modification selected from the group consisting of substitution, addition and deletion;
  • polypeptide of claim 66 comprising:
  • the alteration is a substitution; an addition between positions 102 and 103 of SEQ ID NO: 2 or between positions 59 and 60 of SEQ ID NO: 16; or position 102 of SEQ ID NO: 2 or SEQ ID NO: 12.
  • polypeptide of claim 78 wherein the modification is in the range from the N-terminus of SEQ ID NO: 2 to about 135 amino acids.
  • polypeptide of claim 78 wherein the modification is a modification in the range of about position 50 to about position 120 in SEQ ID NO: 2.
  • polypeptide of claim 78 wherein said alteration comprises a non-conservative substitution in SEQ ID NO: 2.
  • polypeptide of claim 78 comprising a sequence selected from the group consisting of SEQ ID NOs: 12, 14, 16, 18, 20, and 22.
  • the factor of claim 84 wherein said factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof.
  • the factor of claim 84 wherein said factor is an antibody or derivative thereof.
  • said agent is used as a probe.
  • the agent is labeled or can be labeled.
  • agent of claim 89 wherein said label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen antibody reaction.
  • the polypeptide does not specifically react with the polypeptide of claim 77.
  • the factor of claim 91 wherein the factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof.
  • the factor of claim 91 wherein said factor is an antibody or derivative thereof. 95. The factor of claim 91, wherein said factor is used as a probe. 96. The agent of claim 91, wherein the agent is or can be labeled.
  • a cancer diagnostic kit comprising:
  • kits of claim 98, wherein the cancer comprises a c-myc overexpressing cancer. 100.
  • said cancer comprises rectal and colon cancer. 1 0 1.
  • a method for diagnosing cancer
  • the expression level of the B polypeptide is different from the expression level of the B polypeptide in the target portion of the subject, diagnosing that the subject is a high-risk cancer patient;
  • a cancer diagnostic kit comprising:
  • the kit of claim 102 wherein the cancer comprises a c-myc overexpressing cancer.
  • the cancer comprises rectal and colon cancer. 1 0 5.
  • a cancer diagnostic kit comprising: means for determining that the subject is a cancer patient.
  • the detection of the C polypeptide in the target portion of the subject is performed by comparing the expression level of the polypeptide in the subject not afflicted with the cancer or the portion not afflicted with the cancer.
  • the kit according to claim 106 wherein the kit detects high expression of said polypeptide in a target portion of said subject.
  • the kit of claim 106 wherein the cancer comprises c-myc overexpressing cancer.
  • the cancer comprises rectal and colon cancer.
  • the abundance of the C polypeptide is compared with the abundance of the same nucleic acid molecule in a sample from a subject not having cancer or a portion not having cancer.
  • the method of claim 110 wherein if the amount of tide is increasing, the subject of the subject is to be determined to be a cancer patient.
  • the C) means for discriminatively detecting the signal caused by the factor B and the factor C, and measuring the expression levels of the B polypeptide and the C polypeptide, respectively.
  • the expression of the B polypeptide is higher in the target portion of the subject compared to the expression of the B polypeptide in the non-infected subject or the portion not afflicted with cancer, and the subject has cancer.
  • a subject is a cancer patient if the expression of the C polypeptide in the subject portion of the subject is high relative to the expression of the C polypeptide in a subject without or in a portion not afflicted with cancer. Means to determine that there is,
  • a cancer diagnostic kit comprising:
  • kit according to claim 11 wherein said C) means includes means for comparing the relative abundance ratio of said B polypeptide and said C polypeptide.
  • kit of claim 112 wherein the cancer comprises rectal and colon cancer.
  • kits according to claim 11 wherein the cancer comprises a c-myc overexpressing cancer.
  • the factor B does not specifically react with the nucleic acid molecule according to claim 78
  • the factor C does not specifically react with the nucleic acid molecule according to claim 77, or both.
  • kit according to claim 11 further comprising a factor for distinguishably labeling the factor B and the factor C.
  • the C) means for discriminatively detecting the signal caused by the factor A and the factor C, and measuring the expression levels of the A polypeptide and the C polypeptide, respectively.
  • the ratio of the expression level of the C polypeptide to the expression level of the A polypeptide in a non-infected subject or a portion not affected by cancer is the expression of the A polypeptide in the target portion of the subject. Determining that the subject of the subject is a cancer patient if the ratio is higher than the ratio of the expression level of the C polypeptide in the amount.
  • a cancer diagnostic kit comprising:
  • kit according to claim 120 wherein said C) means includes means for comparing the relative abundance or absolute abundance of said A polypeptide and said C polypeptide.
  • the kit of claim 120 wherein the cancer comprises a c-myc overexpressing cancer.
  • the cancer comprises rectal and colon cancer. 1 2 4.
  • the factor A does not specifically react with the polypeptide according to claim 78, or is specific to the polypeptide according to claim 77 and the polypeptide according to claim 78.
  • the kit according to claim 120 which reacts with:
  • kit of claim 120 wherein said factor A and said factor C are or are identifiably labeled.
  • kit according to claim 120 further comprising a factor for distinguishably labeling said factor A and said factor C.
  • C) a step of measuring the expression level of each of the A polypeptide and the C polypeptide, wherein the A polypeptide is in a subject not having cancer or a portion not having cancer.
  • polypeptide which has at least one mutation selected from the group consisting of substitution, addition, and deletion in the amino acid sequence of SEQ ID NO: 2 and has biological activity ;
  • polypeptide having an amino acid sequence having at least 70% identity to the polypeptide of any one of (a) to (d) and having biological activity
  • polypeptide for the manufacture of a diagnostic agent for cancer.
  • polynucleotide consisting of a polynucleotide having at least 70% identity to the polynucleotide of any one of (a) to (c) or a complementary sequence thereof, and having a biological activity.
  • a nucleic acid molecule A nucleic acid molecule.
  • nucleic acid molecule of claim 129 wherein the nucleic acid molecule has a length of at least 8 contiguous nucleotides.
  • nucleic acid molecule of claim 129 wherein said biological activity is binding to centromere chromatin.
  • nucleic acid molecule of claim 129 An agent specific for the nucleic acid molecule of claim 129.
  • 133. The factor of claim 132, wherein said factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof.
  • said factor is a nucleic acid molecule having a length of at least 8 contiguous nucleotides.
  • the factor of claim 132 wherein the factor is a nucleic acid molecule and is used as a primer.
  • the factor of claim 132 wherein said factor is used as a probe.
  • 133. The factor of claim 132, wherein said factor is or can be labeled.
  • agent of claim 137 wherein said label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reactions and antigen-antibody reactions.
  • a cancer diagnostic kit comprising:
  • the kit of claim 1339, wherein the cancer comprises rectal and colon cancer.
  • (e) encodes a polynucleotide having at least 70% identity to the polynucleotide of any one of (a) to (c) or a complementary sequence thereof and having biological activity.
  • nucleic acid molecule for the manufacture of a diagnostic agent for cancer.
  • polypeptide which has at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 4 and has biological activity, ;
  • polypeptide having an amino acid sequence having at least 70% identity to the polypeptide of any one of (a) to (d) and having biological activity
  • polypeptide of claim 142 having at least 3 contiguous amino acids in length.
  • polypeptide of claim 142 wherein said biological activity is binding to centromere chromatin.
  • polypeptide of claim 142 wherein said polypeptide is or can be labeled.
  • the factor of claim 147 wherein the factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof.
  • agent of claim 147 wherein said agent is used as a probe.
  • agent is labeled or can be labeled.
  • agent of claim 151 wherein said label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen-antibody reaction.
  • a cancer diagnostic kit comprising:
  • the kit of claim 153, wherein the cancer comprises rectal and colon cancer. 1 5 5.
  • a method for diagnosing cancer
  • polypeptide which has at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 4, and has biological activity ;
  • polypeptide which is a species homolog of the amino acid sequence of SEQ ID NO: 4, or (e) a polypeptide having an amino acid sequence having at least 70% identity to the polypeptide of any one of (a) to (d), and having biological activity;
  • the present application also provides the following inventions (S1) to (S26) to solve the above problems.
  • a method for diagnosing human colorectal cancer comprising determining the body as a colorectal cancer patient or a colorectal cancer high-risk person.
  • An antigen peptide that binds to the antibody of the above-mentioned invention (S11) or (S12) or the labeled antibody of the above-mentioned invention (S13) and Z or (S14) is present in the biological sample of the subject.
  • a method for diagnosing human colorectal cancer comprising testing whether or not the antigen peptide is present in a sample as a colorectal cancer patient or a person at risk of colorectal cancer.
  • the biological sample of the subject is tested for the presence of the mRNAs of the invention (S3) and (S4) or (S4), and the subject whose mRNA is present in the sample is referred to as a colorectal cancer patient.
  • a method for diagnosing human colorectal cancer wherein the method is determined to be a person at high risk for colorectal cancer.
  • S20 At least the following elements: (a) an antigenic peptide of the above-mentioned invention (S1) and Z or (S2); and (b) a colorectal cancer comprising: a labeled antibody that specifically binds to an antibody in serum. Diagnostic kit.
  • (521) At least the following elements: (a) a plate on which the antigen peptide of the invention (S1) and Z or (S2) is immobilized; and (b) a labeled antibody that specifically binds to a serum antibody.
  • a diagnostic kit for colorectal cancer comprising at least the labeled antibody of the invention (S13) and Z or (S14).
  • (523) At least the following elements: (a) the antibody of the above-mentioned invention (S11) and Z or (S12); and (W the above-mentioned invention (S13) and Z which bind to the same antigen peptide as the antibody of the above (a) Or a colon cancer diagnostic kit comprising the labeled antibody of (S14).
  • (S24) At least the following elements: (a) a plate on which the antibody of the invention (S11) and Z or (S12) is immobilized; and (b) the same antigen as the antibody immobilized on the plate of (a) above A colorectal cancer diagnostic kit comprising the labeled antibody of the invention (S13) and / or (S14) that binds to a peptide.
  • the present inventors have obtained the informed consent from the normal and cancerous parts of a surgical specimen of a colorectal cancer patient, extracted proteins and mRNA, and analyzed them to identify proteins and mRNAs in colorectal cancer cells. Two types of antigenic peptides that were expressed in a specific manner and whose functions as tumor markers were not known before were found.
  • the invention of the present application provides novel antigenic peptides as described above (SEQ ID NOS: 2 and 4, etc.), mRNAs transcribed from the gene DM encoding them, and DNA fragments synthesized from these mRNAs (cDNA: SEQ ID NO: 1, 3).
  • the base sequence and the amino acid sequence shown in SEQ ID NOs: 14 and 14 are each based on addition or deletion of one or more bases, substitution with another base, or mutation based on these bases. It also includes addition and deletion of the above amino acid residues and substitution with other amino acids.
  • the “serum antibody” in certain embodiments refers to an antibody IgG present in the serum of a cancer patient (for example, a colorectal cancer patient) and binding to the antigenic peptide of the inventions (S1) and (S2). I do.
  • the “antibody” of the invention (S11) (S12) and the “labeled antibody” of the invention (S13) (S14) are a polyclonal antibody prepared using the antigen peptide of the invention (S1) (S2) as an immunogen. Monoclonal antibody is meant.
  • FIG. 1 shows the results of Western blot analysis of the expression of the antigenic peptide of the present invention (SEQ ID NO: 2) in cancer tissues and normal tissues.
  • FIG. 2 shows the results of RT-PCR in which the expression of the gene encoding the antigenic peptide of the present invention in cancer tissues and normal tissues was analyzed.
  • FIG. 3 shows the results of Western blot analysis of the expression of the antigenic peptide of the present invention (SEQ ID NO: 4) in cancer tissues and normal tissues.
  • CENP-A protein was increased in primary colorectal cancer. Total protein lysates were prepared from corresponding samples of tumor tissue (T) and nearby normal tissue (N). Equal amounts of protein from each pair were separated on a 7.5% to 15% gradient of polyacrylamide gel and immunoplotted with several antibodies. A. The CENP-A protein is indicated by an arrow. Immunoblotting was also performed using the] 3-actin antibody as a loading control. The intensity of each band was measured by NIH imaging, and the relative average CENP-A protein level between tumor and normal tissue normalized using i3-actin was calculated from at least three experiments.
  • CENP-A protein levels were detected using anti-CENP-A antibody instead of ANA serum.
  • FIG. 4 shows the results of RT-PCR analysis of the expression of the mRNA encoding the antigen peptide of the present invention in cancer tissues and normal tissues.
  • the CENP-A gene is overexpressed in colorectal cancer but is not amplified.
  • A. Total RNA was prepared from corresponding samples of tumor tissue (T) and nearby normal tissue (N), and RT-PCR was performed. The intensity of each band was measured by NIH image, and GAPDH m The relative mean C ENP-A mRNA levels between tumor and normal tissues, normalized using RNA levels, were calculated from at least three experiments. In most cases, CENP-A mRNA in tumors was elevated compared to normal tissue.
  • B Real-time quantitative RT-PCR comparison of CENP-A mRNA levels between tumor tissue and nearby normal tissue by PCR.
  • C. Quantification of the number of copies of the CENP-A gene in tumor and normal tissues by real-time quantitative PCR.
  • FIG. 5 shows immunostaining of human colon tissue with anti-CENP-A antibody.
  • Normal colon epithelium (A and B) and colon cancer tissue (C and D) were fixed with 4% paraformaldehyde and stained with H & E (A and C) or anti-CENP-A antibodies (B and D).
  • CENP-A staining was increased in cancer cells when compared to normal epithelial cells.
  • the initial magnification is 40x. Higher magnification (63x) is shown in B and D inset.
  • FIG. 6 shows simultaneous immunostaining of human colon tissue with anti-CENP-A and anti-CENP-B antibodies.
  • A Normal colon epithelial tissue and colon cancer tissue were fixed with acetone and stained with anti-CENP-A and anti-CENP-B antibodies. Arrowheads indicate CENP-A that does not coexist with C ENP-B. The arrow indicates CENP-B that does not coexist with CENP-A. CENP-A and CENP-B all co-existed in normal cells, whereas some signals of C ENP-A and CENP-B did not co-exist in tumor cells.
  • B Number of CENP-A and CENP-B dots with or without coexistence in normal and tumor cells
  • FIG. 7 shows that the amino terminus of the FIR is important in repressing transcription from the c-my c promoter overnight.
  • HA-FIR and HA-FIR lmN77 were cotransfected with the CAT repo overnight plasmid 75 Ong driven by the c-myc promoter in HeLa cells, and the CAT activity was measured.
  • Each lane was composed of 36 fmo1 HA vector alone (lane 1), 18 fmo1 and 36 fmo1 HA-FIR (lanes 2 and 3), 18 fmo1 and 36 fmo1, respectively.
  • HA—FI RAN 77 (Lanes 4, 5).
  • the lower panel shows a histogram of the average CAT activity in multiple experiments.
  • Lane 2 (18 mol HA-FIR), 3 (36 f mo 1 HA-FIR), 4 (18 f mo 1 HA-FI RAN 77), and 5 (36 f mo 1 HA-FIR)
  • the relative CAT activity of FIR mu N77) is 0.25, 0.16, 0.52, and 0.43, respectively, as compared to lane 1 (36 ⁇ mo1 HA vector).
  • FIG. 8A shows that FIR represses c-Myc, and that its amino terminus is required for repression.
  • 100 fmo1 of HA-FIR or HA-FIRM N77 was transfected into HeLa cells in a 6-well plate. Cells were immunostained for c-Myc (left, green) or HA (middle, red) antibodies. Arrowheads and arrows indicate cells in which HA-FIR and HA-FIRmN77 were expressed, respectively. c-Myc expression was significantly reduced in most HA-FIR expressing cells (arrowheads) when compared to HA-FIRAN77 expressing cells (arrows).
  • FIG. 8B shows that FIR represses c-Myc, and that its amino terminus is required for repression.
  • C-Myc inhibition by HA-FIR or HA-FI RAN77 transfection was quantified by flow cytometric analysis.
  • Transfected cells were identified as PE positive cells shown along the X-axis.
  • FITC positive cells along the y-axis indicate c-Myc expression.
  • HA-FIR suppresses c-Myc expression (left), but HA vector alone does not suppress c-Myc expression (right).
  • the lower panel shows a histogram of c-Myc expression in the boxed area in the upper panel. In this enclosed area 0676
  • FIG. 9A shows that FIR induced apoptosis, but FIR in which the amino terminus was deleted abolished its apoptotic activity.
  • FIG. 9A shows a test of apoptotic cells by TUNEL assay. 150 fmol of HA-FIR, HA-FIRAN77, and empty vector plasmids were transfected into HeLa cells in 6-well plates, and TUNEL assays were performed 24 hours later. The upper panel shows apoptotic cells (arrows) after HA-FIR transfection into HeLa cells. The middle and lower panels show cells transfected with HA-FIRAN77 and HA vector, respectively.
  • FIG. 9B shows that FIR induced apoptosis, but FIR in which the amino terminus was deleted abolished its apoptosis activity.
  • FIG. 9B shows quantification of apoptotic cells by two-color analysis. Cells were identified as PI positive cells shown along the X axis and FITC positive cells shown along the y axis. Apoptosis cells are shown in the upper boxed area in each panel shown in the figure. The percentage of apoptotic cells per 10,000 events in HA-FIR transfection was 16.5%, while HA-FIRAN77, HA empty vector, and DNase I-treated cells (positive control, 6.6%, 2.0%, and 75.6%, respectively.
  • FIG. 1 OA shows that FIR is overexpressed in colorectal cancer tissue.
  • Total protein lysates were prepared from corresponding tumor samples (T) and nearby normal epithelial tissue (N). Equal amounts of proteins from each pair were separated on an 8% polyacrylamide gel and immunoblotted with anti-FIR antibody. Immunlotting was also performed using the i3-actin antibody as a reading control. The intensity of each band was measured by NIH imaging, and the relative average of FIR protein levels between tumor and normal epithelial tissue normalized with / 3-actin was calculated below the figure. FIR expression is much more activated in tumor tissue than in the corresponding non-tumor epithelium. The stages of Duke's classification are also listed at the top of the figure.
  • FIG. 10B shows that FIR is overexpressed in colorectal cancer tissue. Total RNA was prepared from the corresponding samples of (T) and (N), and RT-FCR was performed. (T) FIR mRNA levels in
  • FIG. 10C shows that FIR is overexpressed in colorectal cancer tissue.
  • FIG. 10C is a histogram of FIR mRNA expression in (T) and (N) detected by quantitative real-time PCR. FIR mRNA expression in (T) is significantly higher than FIR mRNA expression in (N) (p ⁇ 0.0056 for t-test; ⁇ ⁇ 0.008 for Wi1 coXon test). .
  • FIG. 10D shows that FIR is overexpressed in colorectal cancer tissue.
  • the expression ratio of (T) / (N) of FIR mRNA and c-myc mRNA in each colorectal cancer tissue was significantly correlated.
  • FIG. 11 is a comparative schematic diagram of four types of variants of the FIR of the present invention.
  • SEQ ID NO: 1 Nucleic acid sequence encoding FI R542.
  • SEQ ID NO: 2 Amino acid sequence of FI R542.
  • SEQ ID NO: 3 Nucleic acid sequence encoding CENP-A.
  • SEQ ID NO: 4 Amino acid sequence of CENP-A.
  • SEQ ID NO: 5 Specific antigenic peptide of FIR amino acid sequence (31-45 amino acid sequence of SEQ ID NO: 2).
  • SEQ ID NO: 6 Another specific antigenic peptide of the FIR amino acid sequence (528-542 amino acid sequence of SEQ ID NO: 2).
  • SEQ ID NO: 9 Example of PCR forward primer for identifying CENP-A SEQ ID NO: 10
  • SEQ ID NO: 13 nucleic acid sequence encoding FIR516.
  • SEQ ID NO: 15 Nucleic acid sequence encoding FFI R499.
  • SEQ ID NO: 17 Nucleic acid sequence of a mutant example of FIR542 (118K).
  • SEQ ID NO: 26 Control sequence reverse primer 2
  • SEQ ID NO: 27 Control sequence forward primer 3
  • SEQ ID NO: 28 Control sequence reverse primer 1
  • SEQ ID NO: 29 FIR genome DNA Forward primer
  • SEQ ID NO: 30 FIR genome DNA reverse primer
  • SEQ ID NO: 31 c-myc DNA forward primer
  • SEQ ID NO: 32 c-myc DNA reverse primer
  • SEQ ID NO: 33 ⁇ -actin DNA forward primer
  • SEQ ID NO: 34 ⁇ -actin DNA reverse primer
  • FIR refers to an FBP-interacting librarian, typically the factor specified by SEQ ID NOS: 1 and 2.
  • the FIR is: It was found with such a style. The specific sequence and other forms of the FIR are described in detail herein below.
  • c—Myc plays an important role in cell proliferation and tumorigenesis.
  • the c-myc proto-oncogene is activated in a variety of tumors and its ectopic expression induces cell growth, proliferation and transformation.
  • both under- and over-expression of c-Myc can lead to apoptotic cell death (FIR ref. 1).
  • Down-regulation of c-Myc is essential for dalcocorticoid-induced apoptosis in human leukemia cells (FIR refs. 2-5), and reduction of c-myc in B cells is apoptotic. Double the action of cis-inducing agents (FIR refs. 6-9).
  • c-myc Treatment of c-myc with antisense oligonucleotides enhances apoptosis in various cells (FIR ref. 1).
  • an increase in c-myc expression may also prove to be pro-apo 1 ⁇ -cis (pr-apoptotic; a pre-stage of apoptosis) under certain circumstances.
  • Forced expression of c-myc in IL-13-dependent myeloid cells promotes cell death due to IL-13 deficiency (FIR reference 10).
  • Serum-deficient Rat1 fibroblasts that overexpress c-Myc can also cause apoptosis (FIR ref. 11).
  • c-Myc expression is tightly regulated, and in turn c-Myc alters the expression of a broad and diverse set of target genes.
  • the molecules and mechanisms that regulate c-myc expression are not fully enumerated, elucidated, or understood.
  • Far Ustream am Element is a sequence required for accurate expression of the human c-myc gene (FIR reference 12).
  • FUSE, c-1 my c promoter P1 1. Located 5 kb upstream, it binds to FUSE binding protein (FBP), a transcription factor that stimulates c-myc expression in a FUSE-dependent manner (FIR references 13 to 15).
  • FBP FUSE binding protein
  • FIR FBP-interacting liberator
  • c-Myc is a transcription factor that regulates cell growth, differentiation and apoptosis. Both up-regulation and down-regulation of c-Myc expression can induce apoptosis under certain circumstances.
  • FIR FBP-interacting Libessa-1
  • a repressor of the c-myc gene induced apoptosis in HeLa cells. Deletion of the amino-terminal repressor domain of the FIR abolished FIR-driven apoptosis, which indicates that cell death is a target of the FIR transcriptional target (eg, cm yc).
  • the present invention has revealed that there are at least four types of variants in the FIR.
  • the nucleic acid sequence is shown in SEQ ID NOs: 1, 11, 13, and 15, and the amino acid sequence is shown in SEQ ID NOs: 2, 12, 14, and 16.
  • the FIRs specified in SEQ ID NOs: 1 and 2 are also referred to herein as FIR542 and are normal sequence.
  • the function of FIR is reported to be c-myc expression suppression (FIR reference 16), and FIR542 is believed to play a role.
  • FIR 559 The FIRs specified in SEQ ID NOs: 11 and 12 are referred to as FIR 559 or PUF 60 due to the length of the 559 amino acid sequence.
  • PUF 60 is reported in RNA Vol. 5 (12), 1548-1560 (1999), and has been implicated in RNA splicing. This FIR 559 has 17 amino acids inserted between positions 102 and 103 in SEQ ID NO: 1.
  • FIR516 The FIRs specified in SEQ ID NOs: 13 and 14 are called FIR516 because they have a length of 516 amino acid sequences.
  • FI R516 has a form deleted from FI R559.
  • the FIRs specified in SEQ ID NOs: 15 and 16 are called FIR499 because they have a length of 499 amino acid sequences.
  • FI R499 has 43 amino acids deleted from the N-terminus from FI R542.
  • variants having substitutions in the amino acid sequence have also been found by the present invention. This substitution is frequently found in the region of about 135 amino acids present at the N-terminal 400 base site.
  • FIR 542 preferably lacks at least one of the normal functions of FIR.
  • an FIR having a normal function has a therapeutic effect
  • a variant of the FIR having a normal function should be used for treatment. Can be.
  • CENP_A refers to a type of protein identified as a centromere-related protein, and typically refers to the factor specified by SEQ ID NOs: 3 and 4. CENP-A was discovered as follows. The specific sequence and other forms of CENP-A are described in detail herein below.
  • CENP—A reference Gene instability in human cancers seems to be widely recognized. In most cancers, this instability is observed primarily at the chromosomal level as gain or loss of whole chromosomes, also known as aneuploidy (CENP—A reference). Dedication 1). Aneuploidy has been found in almost all tumor types and occurs early in tumorigenesis, as seen in colon, cervical, and esophageal carcinoma in situ or precancerous lesions (CENP-A reference 2 and CENP-A reference 3). In addition, in vivo studies with human and rodent cells have shown that aneuploidy is required for neoplastic transformation (CENP-A ref. 4, CENP-A ref. Reference 5). These results suggest that aneuploidy plays an important role in the development and progression of cancer.
  • aneuploidy represents an abnormal number of chromosomes, it can be due to chromosomal mis-segregation during cell division. Potential obstacles in many mitotic processes can lead to uneven separation of chromosomes.
  • the mitotic process involves chromosome condensation, sister chromatid attachment, centromere assembly, centrosome replication, microtubule dynamics, and checkpoints for proper progression through the cell cycle.
  • regulatory genes for these mitotic targets have been shown to be involved in aneuploidy and carcinogenesis induction. For example, STK15 / BTAK / aurora 2 and the vertebrate sekiyurin were overexpressed in human tumors and exhibited transformant activity at the in vivo mouth (CENP-A references 6-8).
  • checkpoint genes hBUB1 and hBUBR1 have been found to be mutated in a small fraction of human colorectal cancer, and the exogenous expression of the mutated BUB1 is associated with abnormal spindle Give a checkpoint (CENP-A reference 9).
  • centromere is a large multiprotein complex built in the centromere DNA, which functions as an attachment site for spindle microtubules, essential for proper chromosome segregation during mitosis. Its dysfunction can be a major factor in aneuploidy, but the direct relationship between centromere and tumorigenesis is unknown.
  • CENP- A 3 is one of first identified centromere component Te human smell (CENP- A reference 10).
  • CENP- A 3 are found only in active Sentoromea, same Sentoromea A unique histone H3-like protein that is thought to be a central element in epigenetic maintenance of identity (see CENP-A ref. 11 and CENP-A ref. 12) thing) .
  • CENP-A is well conserved among eukaryotes (CENP-A references 13-15), and mutations or knockouts of C ENP-A cause chromosomal missegregation (CE NP-A references 16)
  • CENP-A protein levels in colorectal cancer The present inventors have found that CENP-A is highly overexpressed in most cancer tissues as compared to nearby normal mucosa. In addition, CENP-A did not co-exist in part with tumor cells with the centromere-associated DNA binding protein CENP-B. Our results suggest that high-level expression of CENP-A in colorectal cancer causes binding of chromatin to non-centromeric regions and partial rupture of the centromere complex.
  • Centromere protein (CENP) -A is a centromere-specific histone H3-like variant essential for centromere structure and function. This variant plays a central role in the formation of a protein complex called centromere, which is essential for chromosome apposition.
  • centromere protein CENP-A was over-expressed in all 11 primary human colorectal cancer tissues.
  • CENP-A mRNA is also up-regulated, indicating that overexpression of CENP-A occurs at the transcriptional level. Immunostaining with an anti-CENP-A antibody indicated that CE NP-A signals were increased in these tumor cells. In addition, co-immunostaining of CENP-A and C ENP-B (Centromere-associated DNA binding protein) shows that CENP-A is mistargeted to non-centromeric chromatin in these tumor cells. Was done. These results suggest that overexpression of CENP-A may play an important role in aneuploidy in colorectal cancer.
  • CENP-A reference 10 Palmer, D.. Et al., Proc. Atl.Acad.Sci. USA, 88: 3734-3738, 1991 (CENP-A reference 11).
  • protein protein
  • polypeptide oligopeptide
  • peptide refers to a polymer of amino acids of any length.
  • the polymer may be linear, branched, or cyclic.
  • Amino acids may be natural or non-natural, and may be modified amino acids.
  • the term may also include those assembled into a complex of multiple polypeptide chains.
  • the term also embraces naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (eg, Conjugate with a labeling component).
  • This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other Are included.
  • Gene products such as FIR and CENP-A usually take the form of a polypeptide.
  • the polypeptide of the present invention usually has a specific sequence (SEQ ID NO: 2, 4, etc., or a variant thereof).
  • Gene products of genes such as FIR and CENP-A usually take a polypeptide form having such a sequence.
  • the modified sequence can be used for diagnostic purposes in the present invention.
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. The term “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual linkage between nucleotides, and is used interchangeably.
  • oligonucleotides include 2′-0-methyl-liponucleotides, derivative oligonucleotides in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and oligonucleotides in an oligonucleotide.
  • Derivative oligonucleotide in which report in oligonucleotide and phosphodiester bond are converted to peptide nucleic acid bond A derivative oligonucleotide in which peracyl in the oligonucleotide is substituted with C-15 propynyl peracyl, a derivative in which peracyl in the oligonucleotide is substituted with C-15 thiazole peracyl, and a cytosine in oligo nucleotide is C One 5-propier cytosine Substituted derivatives O oligo nucleotides, Derivative oligonucleotides in which the amino acid is substituted with phenoxazine-modified cytosine, the derivative in the DNA is 2 '— Derivatives in which the nucleotide in the oligonucleotide is substituted with 0'-propyl report, and the report in the oligonucleotide is Derivative oli
  • a particular nucleic acid sequence may also include conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Is contemplated. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons has been replaced with a mixed base and a Z or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al.> Mol Cell. Probes 8: 91-98 (1994)). Genes such as FIR and C ENP-A usually take this polynucleotide form.
  • nucleic acid molecule as used herein is also used interchangeably herein with nucleic acids, oligonucleotides, and polynucleotides, and includes cDNA, mRNA, genomic DNA, and the like.
  • nucleic acids and nucleic acid molecules may be included in the term "gene”.
  • a nucleic acid molecule encoding a gene sequence also includes "splice variants (variants, variants).”
  • a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of the nucleic acid.
  • splice variants are the products of alternative splicing of a gene.
  • the initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • different (alternate) nucleic acid splice products encode different polypeptides.
  • other polypeptides derived from the same nucleic acid by c- read-through transcription including alternative exonic splicing, are also included in this definition. Included. Any product of a splicing reaction, including recombinant forms of the splice product, is included in this definition.
  • a gene such as FIR, CENP-A may also include splice variants such as FIR, CENP-A. Such variants are useful in the diagnosis and treatment of the present invention.
  • gene refers to a factor that defines a genetic trait. Those that define the primary structure of a protein are called structural genes, and those that control its expression are called regulatory genes (for example, Promoichi). As used herein, a gene includes a structural gene and a regulatory gene unless otherwise specified. Therefore, reference to a gene such as FIR, CENP-A usually includes both a structural gene such as FIRR and CENP-A and a transcriptional or translational regulatory sequence such as a promoter such as FIR and CENP-A. In the present invention, these regulatory sequences can also be used for diagnosis.
  • gene may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and Z or “protein”, “polypeptide”, “oligopeptide” and “peptide”.
  • gene product also refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” as well as “protein”, “polypeptide”, “oligopeptide” and “polynucleotide” expressed by a gene. Peptide ". Those skilled in the art can understand what a gene product is, depending on the situation.
  • the term “homology” of a gene refers to the degree of identity between two or more gene sequences.
  • the higher the homology between two genes the higher the identity or similarity between their sequences.
  • Whether the two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, by a hybridization method under stringent conditions.
  • D If the NA sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 9% If they are 7%, 98% or 99% identical, the genes are homologous.
  • similarity of a gene refers to the homology of two or more gene sequences when conservative substitutions are regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, if there are conservative substitutions, identity and similarity will differ depending on the presence of the conservative substitution. When there is no conservative substitution, identity and similarity show the same numerical value.
  • the comparison of the similarity, identity and homology between the amino acid sequence and the base sequence is calculated using FASTA, a sequence analysis tool, using default parameters.
  • amino acid may be natural or non-natural as long as the object of the present invention is satisfied.
  • derivative amino acids or “amino acid analogs” refer to amino acids that differ from naturally occurring amino acids but have the same function as the original amino acids. Such derivative amino acids and amino acid analogs are well known in the art.
  • natural amino acid means the L-isomer of a natural amino acid.
  • Natural amino acids include glycine, alanine, palin, leucine, isoleucine, serine, methionine, threonine, fenylalanine, tyrosine, tributphan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, glutamine, glutamine Rupoxyglutamic acid, arginine, orditin, and lysine. Unless otherwise indicated, all amino acids in the present specification are L-forms, but forms using D-form amino acids are also within the scope of the present invention.
  • unnatural amino acid refers to an amino acid that is not normally found in a protein.
  • unnatural amino acids include norleucine, paranitrophenylalanine, homophenylalanine, parafluorophenylalanine, 3-amino-2-benzylpropionic acid, D-form or L-form of homoarginine, and D-phenylalanine.
  • amino acid analog refers to a molecule that is not an amino acid, but that is similar to the physical properties and Z or function of an amino acid.
  • amino acid analog include, but are not limited to, etyonin, canavanine, 2-methylglutamine, and the like.
  • Amino acid mimetics as examples of amino acid analogs, are compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • nucleotide may be natural or non-natural.
  • “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that differs from a naturally occurring nucleotide but has a function similar to that of the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include phosphorothioate, phosphoramidate, methyl phosphonate, chiral methyl phosphonate, 2-O-methyl liponucleotide. Peptide-nucleic acid (PNA). However, it is not limited to these.
  • Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC—IUB Biochemica Nomenclature Commission. Nucleotides may also be referred to by the generally recognized one-letter code.
  • the term "corresponding" amino acid and nucleic acid refer to a polypeptide or nucleic acid molecule having the same action as or having the same action as a predetermined amino acid or nucleic acid in a polypeptide or nucleic acid molecule as a reference for comparison.
  • Amino acids and nucleic acids that are predicted to be active refer to amino acids that exist at similar positions in the active site and make similar contributions to catalytic activity and nucleic acids that encode them.
  • it may be a similar part in the ortholog corresponding to a specific part of the antisense molecule.
  • the corresponding gene for a gene may be an ortholog of that gene.
  • genes corresponding to genes such as human FIR and CENPA-A can be found in other animals (mouse, rat, bush, pest, etc.). Such corresponding genes can be identified using techniques well known in the art. Therefore, for example, the corresponding gene in an animal can be determined by using the sequence of the reference gene of the corresponding gene (eg, a gene such as human FIR or CENP-A) as a query sequence in the animal (eg, mouse, rat). By searching a sequence database.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (having a length of n).
  • the length of the fragment can be appropriately changed depending on the purpose.
  • the lower limit of the length is 3, 4, 5, 6, 7, 8, 9 for polypeptides. , 10, 15, 20, 25, 30, 40, 50 and more amino acids, and the lengths represented by integers not specifically listed here (eg, 11) are also included. May be appropriate as a lower limit.
  • 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, and more nucleotides are listed, and specific listings here are provided.
  • Length as an integer without a number for example, 1 1 Etc.
  • the lengths of the polypeptide and the polynucleotide can be represented by the number of amino acids or nucleic acids, respectively, as described above.
  • the above-mentioned number as an addition or subtraction is intended to include a few above and below (or, for example, 10% above and below) the number.
  • "about” may be used before the number. It should be understood, however, that the presence or absence of “about” does not affect the interpretation of the numerical value herein.
  • the term “specifically interacting factor” or “specific factor” for a biological agent is used interchangeably and refers to Affinities for other irrelevant agents (eg, less than 30% identity, or, in certain cases, less than 99% identity) for a specific agent (eg, a polynucleotide or polypeptide).
  • the affinity for a biological agent is typically greater than or equal to, and preferably less than, the affinity for a biological agent (eg, a polynucleotide or polypeptide). Means significantly higher.
  • the factor specific for the polypeptide includes a specific antibody, and in certain embodiments, the specific factor of the invention includes a specific antibody C It is understood that factors specific for such specific antibodies include the polypeptide of interest itself.
  • any substance or element may be any £ such materials so long as it can achieve the intended purpose, for example, proteins (for example, including antibodies), polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides Nucleotides, nucleotides, nucleic acids (including cDNA, genomic DNA such as DNA, RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (eg, hormones, ligands, information Transmitters, small organic molecules, molecules synthesized by compinatrial chemistry, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.), and their complex molecules (eg, glycoproteins, glycolipids, etc.) ), But are not limited thereto.
  • proteins for example, including antibodies
  • a polynucleotide having a certain degree of sequence homology to the sequence of the polynucleotide typically, a polynucleotide having a certain degree of sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and having complementarity.
  • examples include, but are not limited to, nucleotides and polypeptides such as transcription factors that bind to the promoter region.
  • transcription factors include, but are not limited to, for example, antisense, RNAi, and the like.
  • an antibody specifically directed to the polypeptide, a derivative thereof or an analog thereof eg, a single-chain antibody
  • the polypeptide is a receptor
  • specific ligands or receptors in the case of ligands, and substrates in the case of polypeptides are enzymes, include, but are not limited to, enzymes.
  • small organic molecule refers to an organic molecule having a relatively small molecular weight.
  • the organic low molecular weight refers to those having a molecular weight of about 1000 or less, but may have a higher molecular weight.
  • Small organic molecules can usually be synthesized using methods known in the art or by combining them. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (eg, small molecule ligands). It is not limited to.
  • antibody refers to a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a multispecific antibody, a chimeric antibody, and an anti-idy antibody.
  • monoclonal antibody j refers to an antibody composition having a homogeneous antibody population. The term is not limited in the manner in which it is made. Includes globulin molecules and Fab molecules, F (ab ') 2 fragments, Fv fragments, and other molecules that exhibit immunological binding properties of the original monoclonal antibody molecule. Methods for making are known in the art and are described more fully below.
  • Monoclonal antibodies can be obtained using standard techniques well known in the art (eg, Kohler and M1stein, Nature (1975) 256: 495) or modifications thereof (eg, Buck et al. (1982) In V It is prepared using itro 18: 3 77).
  • a mouse or rat is immunized with a protein conjugated to a protein carrier, boosted, and the spleen (and several large lymph nodes, if necessary) is removed, and the single cell is harvested. Dissociate.
  • the spleen cells can be screened after removal of non-specific adherent cells by applying a cell suspension to a plate or well coated with antigen.
  • B cells expressing immunoglobulin specific for the antigen bind to the plate and are not rinsed off of the suspension residue.
  • the obtained B cells ie, all detached spleen cells
  • myeloma cells are fused with myeloma cells to obtain a hybridoma, and the monoclonal antibody is produced using the hybridoma.
  • antigen refers to any substrate that can be specifically bound by an antibody molecule.
  • antigen Nogen refers to an antigen that can initiate lymphocyte activation that produces an antigen-specific immune response.
  • single-chain antibody refers to an antibody formed by linking the heavy chain fragment and the light chain fragment of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide.
  • complex molecule refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, fats, sugars, and small molecules.
  • complex molecules include, but are not limited to, glycolipids, glycopeptides, and the like.
  • a gene such as FIR or CENP-A or a product thereof or a factor thereof of the present invention is referred to as a gene or product of FIR or CENP-A or a factor of the present invention, respectively, as long as it has a function similar to that of the gene of the present invention.
  • Complex molecules can also be used.
  • an “isolated” substance eg, a biological factor such as a nucleic acid or protein
  • a biological factor for example, in the case of a nucleic acid, a factor other than the nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; and in the case of a protein, a factor other than the protein and the target protein Etc.
  • isolated nucleic acids and proteins include nucleic acids and proteins that have been purified by standard purification methods.
  • isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.
  • a “purified” substance eg, a biological factor such as a nucleic acid or protein
  • a purified substance refers to a substance from which at least a part of a factor naturally associated with the substance has been removed. Therefore, the purity of the substance in the purified substance is usually higher (ie, more concentrated) than in the state in which the substance is normally present. 6
  • purified and isolated preferably refer to at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and Most preferably, it means that at least 98% by weight of the same type of substance is present.
  • the term “expression” of a gene product such as a gene, a polynucleotide, or a polypeptide means that the gene (usually, a DNA form) or the like undergoes a certain action in vivo and changes to another form. .
  • it means that a gene, polynucleotide, or the like is transcribed and translated to form a polypeptide, but transcription and production of mRNA may also be a form of expression.
  • such forms of the polypeptide may have undergone post-translational processing.
  • the term “reduction” in “expression” of a gene, polynucleotide, polypeptide, etc. means that when the factor of the present invention is applied, the amount of expression is significantly reduced as compared to when the factor is not applied.
  • the decrease in expression includes a decrease in the expression level of the polypeptide.
  • the term “increase” in “expression” of a gene, a polynucleotide, a polypeptide, or the like means that the amount of expression is significantly increased when the factor of the present invention is acted on as compared to when it is not acted on.
  • the increase in expression includes an increase in the expression level of the polypeptide.
  • induction of “expression” of a gene means that a certain factor acts on a certain cell to increase the expression level of that gene. Therefore, the induction of expression should be such that the gene is expressed when the expression of the gene is not observed at all, and the expression of the gene is detected when the expression of the gene is already observed. Includes increasing.
  • telomere As used herein, "specifically express" a gene means that the gene is expressed at a particular site or stage of the plant at a different (preferably higher) level than at other sites or stages. Say. To express specifically, a certain site (Eg, a specific site such as a cancer-affected site), or may be expressed at other sites. Preferably, specific expression means expression at only a certain site.
  • biological activity refers to an activity that a certain factor (eg, a polypeptide or a protein) may have in a living body, and exerts various functions (eg, a transcription promoting activity). Activity is included. For example, if two factors interact (eg, bind FIR, CENP-A, etc. to its receptor), then the biological activity will be between the FIR, CENP-A, etc. and its receptor And biological changes resulting therefrom (eg, apoptosis) and the like. FIR includes binding to FBP.
  • CENP-A includes, but is not limited to, binding to centromere chromatin.
  • the biological activity includes the enzymatic activity.
  • an agent is a ligand
  • the ligand involves binding to the corresponding receptor.
  • a biological activity can be measured by techniques well known in the art.
  • the case where the variant molecule has the same activity as that of the variant molecule in the living body can also be included in the definition of having the biological activity.
  • antisense refers to an activity capable of specifically suppressing or reducing the expression of a target gene. Antisense activity is usually achieved by a nucleic acid sequence of at least 8 contiguous nucleotides that is complementary to the nucleic acid sequence of the gene of interest (eg, FIR, CENP-A, etc.). A nucleic acid molecule having such an activity is called an antisense molecule. Such nucleic acid sequences are preferably at least 9 contiguous nucleotides in length, more preferably 10 contiguous nucleotides in length, even more preferably 11 contiguous nucleotides in length, 12 contiguous nucleotides in length.
  • nucleotide length of 13 consecutive nucleotides in length, of 14 consecutive nucleotides in length, of 15 consecutive nucleotides in length, It can be a nucleic acid sequence of 20 contiguous nucleotides in length, 25 contiguous nucleotides in length, 30 contiguous nucleotides in length, 40 contiguous nucleotides in length, 50 contiguous nucleotides in length.
  • Such nucleic acid sequences may include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, and most preferably 95% homologous to the above-described sequences. included.
  • antisense activity is preferably complementary to the sequence at the 5 'end of the nucleic acid sequence of the gene of interest.
  • antisense nucleic acid sequences also include those with one or more or one or more nucleotide substitutions, additions and Zs or deletions with respect to the sequences described above.
  • RNA i is an abbreviation for RNA interference, and when a factor that causes RNA i, such as double-stranded RNA (also referred to as ds RNA), is introduced into cells, homologous mRNA is specifically identified. Refers to the phenomenon in which biosynthesis is degraded and the synthesis of gene products is suppressed, and the technology used for it. As used herein, RNAi may also be used synonymously with factors that cause RNAi in some cases.
  • factor that causes RNAi refers to any factor that can cause RNAi.
  • factor causing RNAi refers to causing RNAi relating to the gene and achieving the effect of RNAi (for example, suppressing the expression of the gene).
  • Factors that cause such RNAi include, for example, sequences that have at least about 70% homology to a portion of the nucleic acid sequence of the target gene or that include sequences that hybridize under stringent conditions. Examples include, but are not limited to, RNAs or variants thereof containing a double-stranded portion both of which are 10 nucleotides in length.
  • the factor is preferably a 3 'overhang More preferably, the 3 ′ overhanging end may be a DNA having a length of 2 nucleotides or more (eg, a DNA having a length of 2 to 4 nucleotides).
  • RNAi RNAi
  • D icer an RN asell I-like nuclease having a helicase domain excises the molecule from the 3 'end by about 20 base pairs in the presence of ATP, and the short ds RNA (also called siRNA) ).
  • s ho rtinterferi ng is an abbreviation of RNA, artificially or not been or biochemically synthesized chemically synthesized, or even synthesized in an organism Or short-chain double-stranded RNA of 10 base pairs or more, which is the result of the degradation of double-stranded RNA of about 40 bases or more in the body, and usually has the structure of 5'-phosphate and 3'-OH And the 3 'end is protruding about 2 bases.
  • a specific protein binds to this siRNA to form RISC (RNA-induced-si1encing-comp1ex).
  • This complex recognizes and binds mRNA having the same sequence as the siRNA, and cleaves the mRNA at the center of the siRNA by RNase III-like enzyme activity.
  • the relationship between the sequence of the siRNA and the sequence of the mRNA to be cleaved as a target is preferably 100%.
  • mutation of a nucleotide at a position outside the center of siRNA does not completely eliminate RNAi cleavage activity, but retains partial activity.
  • mutation of the base at the center of the siRNA has a large effect, and the activity of cleaving mRNA by RNAi is extremely reduced.
  • siRNA for mRNAs with mutations, it is possible to synthesize siRNA with the mutation in the center and introduce it into cells to specifically degrade only the mRNA containing the mutation. it can. Therefore, in the present invention, the siRNA itself can be used as a factor that causes RNAi, and a factor that produces siRNA (eg, For example, ds RNA (typically about 40 bases or more) can be used as such a factor.
  • ds RNA typically about 40 bases or more
  • siRNAs bind the antisense strand of the siRNA to the mRNA and act as primers for RNA-dependent RNA polymerase (RdRP), d
  • RdRP RNA-dependent RNA polymerase
  • sRNA will be synthesized, and this dsRNA will again become a substrate for Dicer, generating new siRNA and amplifying the effect. Therefore, in the present invention, the si RNA itself and the factor that produces the si RNA are also useful. In fact, in insects and the like, for example, 35 dsRNA molecules almost completely degrade mRNA in more than 1,000 copies of cells, so that there are factors that generate siRNA itself and siRNA. It will be appreciated that it is useful.
  • siRNA double-stranded RNA having a length of about 20 bases (for example, typically about 2;! To 23 bases in length) or less, which is called si RNA, can be used.
  • siRNA suppresses gene expression by expressing it in cells and suppresses expression of pathogenic genes targeted by the siRNA, so that it can be used for treatment, prevention, prognosis, etc. of disease .
  • the siRNA used in the present invention may take any form as long as it can cause RNAi.
  • the factor that causes RNAi of the present invention may be a short hairpin structure (shRNA; shorthairpin RNA) having an overhang at the 3 ′ end.
  • shRNA refers to a single-stranded RNA that contains a partially palindromic base sequence, takes a double-stranded structure in the molecule, and forms a structure like a hairpin. Refers to molecules of base pairs or more.
  • shRNAs are artificially chemically synthesized.
  • such shRNAs can be prepared by synthesizing DNA at the mouth of T7 RNA polymerase using a hairpin DNA with the sense and antisense strand DNA sequences ligated in opposite directions. Therefore, it can be generated.
  • shRNAs can be as long as about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in a cell after introduction into the cell. It should be understood that they are degraded and cause RNAi in the same manner as siRNA, and have the therapeutic effect of the present invention. It is to be understood that such effects are exerted on a wide variety of organisms, including insects, plants, animals (including mammals). As described above, shRNA causes RNAi similarly to siRNA, and thus can be used as an active ingredient of the present invention. The shRNA may also preferably have a 3 'overhang.
  • the length of the double-stranded portion is not particularly limited, but may be preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more.
  • the 3 ′ protruding end may be preferably DNA, more preferably DNA having at least 2 nucleotides or more, and further preferably DNA having 2 to 4 nucleotides in length.
  • RNAi used in the present invention can be either artificially synthesized (for example, chemically or biochemically) or a naturally occurring factor. There is no essential difference. Chemically synthesized products are preferably purified by liquid chromatography or the like.
  • RNAi used in the present invention can also be synthesized in vitro.
  • antisense and sense RNA are synthesized from type I DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is induced through the mechanism described above, and the effects of the present invention are achieved.
  • RNAi can be introduced into cells by the calcium phosphate method.
  • Factors that cause NAi also include factors such as single strands, or all similar analogs of nucleic acids, that can hybridize to mRNA. Such factors are also useful in the treatment methods and compositions of the present invention.
  • polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a polynucleotide selected from the polynucleotides of the present invention as a probe by using a colony hybridization method, a plaque hybridization method, a Southern plot hybridization method, or the like. A polynucleotide can be obtained. Specifically, polynucleotides that hybridize under stringent end conditions can be obtained at 65 ° C in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques is immobilized.
  • SSC saline-sodium citrate
  • the composition of 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate.
  • SSC saline-sodium citrate
  • Hybridization has been published in laboratory books such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). This can be done according to the method described.
  • sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions.
  • Hybridizable polynucleotide refers to a polynucleotide that can hybridize to another polynucleotide under the above eight hybridization conditions. Specific examples of the hybridizable polynucleotide include salts of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention.
  • a polynucleotide having at least 60% or more homology with the base sequence preferably a polynucleotide having 80% or more homology, more preferably a polynucleotide having 95% or more homology.
  • probe refers to a substance to be searched for in a biological experiment such as in vitro and / or in vivo screening, and is, for example, a nucleic acid molecule containing a specific base sequence or a specific substance. But not limited thereto.
  • Nucleic acid molecules commonly used as probes include those having a nucleic acid sequence of at least 8 contiguous nucleotides that is homologous or complementary to the nucleic acid sequence of the gene of interest. Such a nucleic acid sequence is preferably at least 9 contiguous nucleotides in length, more preferably 10 contiguous nucleotides in length, even more preferably 11 contiguous nucleotides in length, 12 contiguous nucleotides in length.
  • nucleic acid sequence may be 40 contiguous nucleotides in length, 50 contiguous nucleotides in length.
  • Nucleic acid sequences used as probes include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, and 95% homologous to the above-described sequences. It is.
  • search refers to finding a nucleobase sequence having a specific function and / or property by using one nucleobase sequence electronically, biologically, or by another method. That means.
  • BLAST Altschul et al., J. Mol, Biol. 215: 403-410 (1990)
  • FASTA Pearson S Liman, Pro Natl. Acad. Sci., USA 85: 2444- 2448 (1988)
  • Smith and Waterman method Smith and Waterman, J. Mol. Biol. 17: 195-197 (1981)
  • Need 1 emaand Wunsch method Needleman and Wunsch, J. ol. Biol.
  • Bio searches include stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, microarrays (microarray assays) attached to glass plates, PCR and insit11 hybrids. Examples include, but are not limited to. In the present specification, it is intended that FIR, CENP-A, etc. should also include the corresponding gene identified by such electronic search and biological search.
  • the term “primer” refers to a substance necessary for initiating the reaction of a synthesized polymer compound in a polymer synthase reaction.
  • a nucleic acid molecule synthesis reaction a nucleic acid molecule complementary to a partial sequence of a polymer compound to be synthesized (for example,
  • DNA or RNA can be used.
  • Nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 contiguous nucleotides that is complementary to the nucleic acid sequence of the gene of interest. Such nucleic acid sequences are preferably at least 9 contiguous nucleotides in length, more preferably 10 contiguous nucleotides in length, and even more preferably 12 contiguous nucleotides in length, 12 contiguous nucleotides in length.
  • nucleotide length 13 contiguous nucleotides, 14 contiguous nucleotides, 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 25 contiguous nucleotides, 30 contiguous nucleotides, 40 contiguous nucleotides
  • the nucleic acid sequence may be 50 consecutive nucleotides in length, 50 consecutive nucleotides in length.
  • the nucleic acid sequence used as a probe has at least 70% homology to the above-mentioned sequence, more preferably at least 80% homology, still more preferably 90% homology, and 95% homology. Homologous nucleic acid sequences are included. Sequences suitable as primers are synthesized (enhanced) Can vary depending on the nature of the intended sequence, but those skilled in the art will be able to design primers as appropriate for the intended sequence. The design of such primers is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, Primer Election, DNA Star).
  • epitope refers to a group constituting a structure that determines an antigen.
  • the epitope may be a set of amino acid residues involved in recognition by a particular immunoglobulin, or, in the case of T cells, the T cell receptor protein and Z or major histocompatibility complex (MHC) receptor.
  • MHC major histocompatibility complex
  • epitopes are molecular features (eg, primary, secondary or tertiary peptide structures and charges) and are recognized by immunoglobulins, T cell receptors or HLA molecules Form the site.
  • Epitopes containing peptides may contain more than two amino acids in a spatial conformation unique to the epitope.
  • an epitope consists of at least five such amino acids, and typically consists of at least six, seven, eight, nine, or ten such amino acids. Longer peptides are generally preferred because they resemble the antigenicity of the original peptide, but may not always be so in view of conformation.
  • a sequence of at least 3 amino acids in length is required, preferably this sequence is at least 4 amino acids, more preferably 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids.
  • a sequence of amino acids, 9, 10, 15, 20, 25 amino acids in length may be required.
  • the FIR and CENP-A of the present invention can be used after being artificially modified.
  • certain amino acids in the sequence are replaced by other amino acids in the protein structure, such as, for example, in the cationic domain or the binding site of a substrate molecule, without any apparent loss or loss of interaction binding capacity. obtain. It is the protein's ability to interact and its properties that define the biological function of a protein.
  • certain amino acid substitutions can be made in the amino acid sequence, or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after the substitution. Accordingly, various modifications may be made in the peptide disclosed herein or the corresponding nucleic acid molecule encoding this peptide without appreciable loss of biological utility.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyte. J and Doo little, RFJ Mo 1. Biol. 157 (1): 105-132, 1982).
  • the hydrophobic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of that protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.) .
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); feniralanine (+2.8); cysteine Z cystine (+2.5); methionine.
  • one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still yield a protein having a similar biological function (eg, a protein equivalent in enzymatic activity). It is.
  • the hydrophobicity index is preferably within 2 or less, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.
  • Polypeptides can also be modified to take into account the hydrophilicity index. As described in U.S. Patent No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); Formic acid (+ 3.0 ⁇ 1); glutamic acid (+ 3.0 ⁇ 1); serine (+0.
  • Threonine (10.4); Proline (10.5 ⁇ 1); Alanine (10.5); Histidine (10.5); Cystine (11.0); Methionine (-1 3); Valine (1-1.5); Leucine (1-1.8); Isoloicin
  • the hydrophilicity index is preferably within 2 soils, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • conservative substitution refers to an amino acid substitution in which the original amino acid and the amino acid to be substituted have similar hydrophilicity indexes or Z and hydrophobicity indexes as described above.
  • conservative substitution include, for example, those having a hydrophilicity index or a hydrophobicity index within ⁇ 2, preferably within ⁇ 1, more preferably within ⁇ 0.5. But not limited to them.
  • conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; Leucine, and isoleucine, but are not limited thereto.
  • variant refers to a substance in which a substance such as an original polypeptide or a polynucleotide is partially modified.
  • variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like.
  • variants include one or several substitutions or additions to a reference nucleic acid molecule or polypeptide. 6
  • Alleles refer to genetic variants that belong to the same locus and are distinct from each other. Therefore, “allelic variant” refers to a variant that has an allelic relationship to a certain gene. Such allelic variants usually have sequences that are identical or very similar to their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have activity. "Species homolog or homolog” refers to homology (preferably 60% or more homology, more preferably 60% or more, with a certain gene at the amino acid or nucleotide level) in a certain species. , 80% or more, 85% or more, 90% or more, 95% or more homology).
  • ortholog is also known as an orthologous gene, and refers to a gene derived from speciation from a common ancestor with two genes.
  • the human and mouse hemoglobin genes are orthologs, while the human alpha hemoglobin gene and / 3 hemoglobin gene are paralogs (genes generated by gene duplication). ).
  • Orthologs are useful for estimating molecular phylogenetic trees.
  • An ortholog of the present invention can also be useful in the present invention, since an ortholog can usually perform the same function as the original species in another species.
  • conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence; if the nucleic acid does not encode an amino acid sequence, it is essentially the same.
  • An array Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU All encode the amino acid alanine.
  • nucleic acid variation is a "silent modification (mutation)," which is one type of conservatively modified mutation.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. In that field, each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tributophan), produces functionally identical molecules. It will be appreciated that the Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • Silent mutations may also be indicative of the diagnosis of the present invention.
  • modifications can be made to avoid substitution of cysteine, an amino acid that has a significant effect on the conformation of the polypeptide.
  • Such modifications of the base sequence include cleavage with a restriction enzyme or the like, ligation by a treatment with a DNA polymerase, a K1 enow fragment, DNA ligase, etc.
  • Substitution method (specific site-directed mutagenesis method; Mark Zoller and Michae 1 Smith, Methodsin Enzymology, 100, 468-500 (1983)). Modifications can also be made by methods used in the field of biology.
  • amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides.
  • Amino acid substitution refers to substitution of one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3, amino acids of the original peptide.
  • the addition of amino acids refers to adding one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids to the original peptide chain.
  • Amino acid deletion refers to deletion of one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3, amino acids from the original peptide.
  • Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like.
  • the amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.
  • peptide analog refers to a compound that is different from a peptide, but that has at least one chemical or biological function equivalent to the peptide.
  • peptide analogs include those in which one or more amino acid analogs or amino acid derivatives have been added or substituted with the original peptide.
  • Peptide analogs have a function similar to that of the original peptide (e.g., similar pKa values, similar functional groups, similar binding modes to other molecules). Such additions or substitutions are made in substantially the same manner as described above.
  • Such peptide analogs can be made using techniques well known in the art.
  • a peptide analog can be a polymer comprising an amino acid analog.
  • polynucleotide analog and “nucleic acid analog” refer to a compound that is different from a polynucleotide or nucleic acid, but has at least one chemical or biological function equivalent to the polynucleotide or nucleic acid.
  • polynucleotide or nucleic acid analogs include those in which one or more nucleotide analogs or nucleotide derivatives have been added or substituted with respect to the original peptide.
  • a nucleic acid molecule is a nucleic acid molecule as described above, as long as the expressed polypeptide has substantially the same activity as the native polypeptide. A part of the sequence may be deleted or replaced by another base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and Z or 3,3 ends. Further, a nucleic acid molecule which encodes a polypeptide having substantially the same function as that of a polypeptide obtained by hybridizing a gene encoding the polypeptide under stringent conditions may be used. Such genes are known in the art and can be used in the present invention.
  • Such a nucleic acid can be obtained by the well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method, a hybridization method, and the like.
  • substitution As used herein, “substitution”, “addition” and “deletion” of a polypeptide or a polynucleotide refer to an amino acid or a substitute thereof, or a nucleotide or a Substitutes are replaced, added, and removed. Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques.
  • the number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number may be a function of interest in a variant having the substitutions, additions or deletions (for example, a binding function to FBP). As long as the function of binding to the centromere, the function of transmitting information on hormones and site forces, etc. is maintained. For example, such a number may be one or several, and preferably is within 20%, within 10%, or below 100, below 50, below 25 of the total length It may be as follows.
  • “detection” or “quantification” of gene expression can be achieved using appropriate methods, including, for example, mRNA measurement and immunological measurement methods.
  • molecular biological measurement methods include Northern blotting, dot plotting, and PCR.
  • immunological measurement method include, for example, an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a western plot method, and an immunohistochemical staining method.
  • Examples of the quantification method include the ELISA method and the RIA method. Genetic analysis using an array (for example, a DNA array or a tin array) can also be performed.
  • the DNA array is widely reviewed in (Shujunsha, edited by Cell Engineering, “DNA microarray and the latest PCR method”).
  • the protein array is described in detail in Nat Genet. 2002 Dec; 32 Suppl: 526-32.
  • Methods for analyzing gene expression include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid system, in vitro translation, and the like, in addition to those described above.
  • Such further analysis methods are described, for example, in the Genome Analysis Experiment Method 'Yusuke Nakamura Lab ⁇ Manual, Editing ⁇ Yusuke Nakamura Yodosha (2002), etc. Incorporated.
  • the term “expression level” refers to the level at which a polypeptide or mRNA is expressed in a target cell or the like. Such an expression level can be determined using an antibody of the present invention by any suitable method including immunological measurement methods such as ELISA, RIA, fluorescent antibody, Western blotting, and immunohistological staining. Expression level at the protein level of the polypeptide of the present invention, as assessed by The expression level of the polypeptide of the present invention at the mRNA level, which is evaluated by any appropriate method including a molecular biological measurement method such as a plotting method, a dot plotting method, and a PCR method, may be mentioned. “Change in expression level” refers to the expression level of the polypeptide of the present invention at the protein level or mRNA level, which is evaluated by any appropriate method including the above-described immunological measurement method or molecular biological measurement method. Means increasing or decreasing.
  • a transformant derived from a microorganism, animal cell, or the like having a recombinant vector into which a DNA encoding the polypeptide of the present invention has been incorporated is cultured according to a conventional culture method, and the polypeptide of the present invention is purified.
  • the polypeptide of the present invention can be produced by producing, accumulating, and collecting the polypeptide of the present invention from the culture of the present invention.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the organism of the present invention. Either a natural medium or a synthetic medium can be used as long as the transformant can be efficiently cultured.
  • the carbon source may be any one that can be assimilated by each microorganism, such as dalcose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Alcohols such as ethanol, ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., various inorganic or organic acid ammonium salts, other nitrogen-containing substances, and peptone, meat extract, yeast extract, coa Steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • potassium (II) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • the culture is performed under aerobic conditions such as shaking culture or submerged aeration stirring culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 5 hours to 7 days.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted by using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • indole acrylic acid or the like may be added to the medium.
  • the cells or organs of the plant into which the gene has been introduced can be cultured in large quantities using a jar armmenter.
  • the medium to be cultured may be a commonly used Murashige 'and' Skoog (MS) medium, White medium, or a medium supplemented with plant hormones such as auxin or cytokinin. it can.
  • the medium for culturing the cells of the present invention may be a commonly used RPMI 1640 medium [The Journal of Medicine, Medical As sociati on, 199, 519 (1967)]. ], Eagle's MEM medium [Science, 122, 50 1 (1952)], DMEM medium [Viril ogy, 8, 396 (195 9)], 199 medium [Proceedingsoft he Societyfort he Biological medicine, 73, 1 (1950)] (4) A medium containing fetal serum or the like is used.
  • Cultivation is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C, and the presence of 5% CO 2. If necessary, antibiotics such as kanamycin, penicillin, and streptomycin may be added to the medium during the culturing.
  • a conventional enzyme which is well-known and commonly used in the art, may be used.
  • a purification method can be used. For example, when the polypeptide of the present invention is secreted extracellularly from the transformant for producing the polypeptide of the present invention, the culture is treated by a method such as centrifugation. Obtain the soluble fraction.
  • the cells in the culture are collected by centrifuging the culture to obtain the cells. After washing the cells, the cells are crushed with an ultrasonic crusher, French press, Mantongaulin homogenizer, Dynomill, etc., and cell-free extraction Obtain a liquid.
  • a purified sample can be obtained.
  • the polypeptide of the present invention is expressed by forming an insoluble form in the cells, the cells of the present invention are similarly recovered, crushed, and centrifuged. After recovering the polypeptide, the insoluble form of the polypeptide is solubilized with a polypeptide denaturing agent. The solubilized solution is diluted or dialyzed into a solution containing no polypeptide denaturing agent or diluted so that the concentration of the polypeptide denaturing agent does not denature the polypeptide, and the polypeptide of the present invention is restored to a normal three-dimensional structure. After the composition, a purified sample can be obtained by the same isolation and purification method as described above.
  • the protein can be purified according to a general protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458].
  • the polypeptide of the present invention can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having an affinity for the fused protein [Akio Yamakawa, Experiment Medical (Experimental Medicine), 13, 469-474 (1995)]
  • affinity a substance having an affinity for the fused protein [Akio Yamakawa, Experiment Medical (Experimental Medicine), 13, 469-474 (1995)]
  • the method of Lowe et al. Proc. Natl. Acad.
  • the polypeptide of the present invention can be produced as a fusion protein with protein A, and purified by affinity chromatography using immunoglobulin G.
  • the polypeptide of the present invention can be produced as a fusion protein with a FLAG peptide, and purified by affinity chromatography using an anti-FLAG antibody [Pro atl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)].
  • polypeptide of the present invention can be prepared in invi 1 according to a known method [J. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)]. : 1 "0 transcripts" can be produced using a translation system.
  • the polypeptide obtained above can also be obtained by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • the polypeptide of the present invention can be produced.
  • Advanced ChemTech, Applied Biosyst ems, Pharmacia Biote ch, Protein Techno 1 ogy Instrumen t :, Synthecel 1 -Vega, Per Septive, Shimadzu Chemical synthesis can also be performed using a peptide synthesizer at a manufacturing facility.
  • Structural analysis of the purified polypeptide of the present invention can be performed by a method generally used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hisashi Hirano, Tokyo Chemical Dojin, 1993). It is.
  • the physiological activity of the novel Ps20-like polypeptide of the present invention can be determined by known methods [Cell, 75, 1389 (1993), J. Cell Bio. 1146, 233 (1999), Cancer Res. 58, 1238 (1998). , Neuron 17, 1157 (1996), Science 289, 1197 (2000)].
  • a polyclonal antibody is prepared by administering a purified full-length or partial fragment of the obtained polypeptide or a peptide having a partial amino acid sequence of the protein of the present invention to an animal as an antigen. Can be done.
  • animals to be administered include egrets, goats, rats, mice, hamsters and the like.
  • the dose of the antigen is preferably 50 to 100 g per animal.
  • the antigen be a covalently bound peptide to a carrier protein such as keyhole limpet haemocyanin or thyroglobulin.
  • the peptide serving as the antigen can be synthesized by a peptide synthesizer.
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. On days 3 to 7 after each administration, blood is collected from the fundus venous plexus to prevent the serum from reacting with the antigen used for immunization.
  • Epidemiological assay [Enzyme immunoassay (ELISA): published by Medical Shoin, 1976, Ant ibodies-A Laboratory Manual, Cold Spring Harbor Lavoratory (1988)].
  • Serum can be obtained from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and the polyclonal antibody can be separated and purified from the serum using well-known techniques. Production of monoclonal antibodies is also well known in the art. In order to prepare antibody-producing cells, first, a rat whose serum shows a sufficient antibody titer against the polypeptide fragment fragment of the polypeptide of the present invention used for immunization was used as a source of antibody-producing cells. To produce hybridomas by fusion with myeloma cells. Thereafter, a hybridoma that specifically reacts with the polypeptide partial fragment polypeptide of the present invention is selected by enzyme immunoassay or the like. The monoclonal antibody produced from the hybridoma thus obtained can be used for various purposes.
  • the antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, by chemical synthesis, or preferably, by recombinant expression techniques.
  • Recombinant expression of an antibody of the present invention, or a fragment, derivative or analog thereof comprises a polynucleotide encoding the antibody. It requires the construction of an expression vector that can be used. Once a polynucleotide encoding the antibody molecule or antibody heavy or light chain or portions thereof (preferably containing the heavy or light chain variable domain) of the invention is obtained, the antibody Vectors for the production of the molecule can be produced by recombinant DNA technology using techniques well known in the art.
  • the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the present invention, or a heavy or light chain thereof, or a variable domain of a heavy or light chain, operably linked to a promoter. I will provide a.
  • variable domain of the antibody can include the nucleotide sequence and can be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce an antibody of the present invention.
  • the present invention includes host cells comprising a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains can be co-expressed in a host cell for expression of the entire immunoglobulin molecule.
  • Such an antibody can be used, for example, in a method for immunologically detecting the polypeptide of the present invention.
  • a method for immunologically detecting the polypeptide of the present invention using the antibody of the present invention microtiter ELISA using plate • Fluorescent antibody method, Western blot method, immunohistological staining method, etc.
  • polypeptides are sub sandwiches ELISA method using Epito one flop two different monoclonal antibodies of the antibody reaction with polypeptides of the present invention in the liquid phase, 1 2 6 I
  • the tamper of the present invention labeled with a radioisotope such as Radioimmunoassay method using a protein and an antibody that recognizes the protein of the present invention.
  • Methods for quantifying mRNA of the polypeptide of the present invention are also well known in the art.
  • the expression level of the DNA encoding the polypeptide of the present invention at the mRNA level is determined by the Northern hybridization method or the PCR method. It can be quantified.
  • Such techniques are well-known in the art and are also described in the references listed herein.
  • polynucleotides may be obtained by any method known in the art, and the nucleotide sequence of these polynucleotides may be determined.
  • the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (see, for example, Kutmeier et al., BioTechniques 17: 242 (1994)). This, as described, briefly, involves the synthesis of overlapping nucleotides containing portions of the antibody-encoding sequence, annealing and ligation of those oligonucleotides, and then this ligation by PCR. Includes amplified oligonucleotides.
  • Polynucleotides encoding an antibody can be made from nucleic acid from a suitable source. If a clone containing the nucleic acid encoding the antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or can be obtained from a suitable source (eg, An antibody cDNA library or a cDNA library generated from, or isolated from, any tissue or cell that expresses an antibody (eg, a hybridoma cell selected for expression of an antibody of the invention).
  • a suitable source eg, An antibody cDNA library or a cDNA library generated from, or isolated from, any tissue or cell that expresses an antibody (eg, a hybridoma cell selected for expression of an antibody of the invention).
  • the 3 ′ and 5 ′ ends of the sequence can be hybridized to It can be obtained by PCR amplification using a functional synthetic primer or by cloning using an oligonucleotide probe specific for that particular gene sequence.
  • the amplified nucleic acid generated by PCR can be cloned into a replicable cloning vector using any method known in the art.
  • the nucleotide sequence of the antibody can be determined by methods well known in the art for manipulating nucleotide sequences (eg, recombinant DNA technology, site-directed mutagenesis, PCR). (See, for example, the techniques described in Sambrook et al., Supra, and eds. And Ausubel et al., Eds., Both of which are incorporated herein by reference in their entirety.)). Engineered with, for example, antibodies that have different amino acid sequences to generate amino acid substitutions, deletions, and / or insertions.
  • the amino acid sequence of the heavy and / or light chain variable domains is determined by methods well known in the art for identification of the sequence of the complementarity determining regions (CDRs), eg, (By comparing with the known amino acid sequences of other heavy and light chain variable regions) to determine the variable region.
  • CDRs complementarity determining regions
  • one or more CDRs are placed in a framework region as described above (eg, in a human framework region to humanize a non-human antibody). Can be entered.
  • This framework region can be naturally occurring or can be a consensus framework region, and preferably can be a human framework region (eg, for the listed human framework regions, see Chothia et al .; Biol. 278: 457-479 (1998)).
  • the polynucleotide generated by the combination of the framework regions and the CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions can be made within a framework region, and Preferably, the amino acid substitution improves binding of the antibody to its antigen.
  • such methods may involve amino acid substitution of cysteine residues in one or more of the variable regions involved in intrachain disulfide bonds so as to generate an antibody molecule lacking one or more intrachain disulfide bonds. Or it can be used to create deletions.
  • Other changes to polynucleotides are encompassed by the present invention and in the art.
  • a technique developed for the production of “chimeric antibodies” by splicing genes from mouse antibody molecules of appropriate antigen specificity with genes from human antibody molecules of appropriate biological activity (Morrison Natl. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985). ) Can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, and such molecules have variable regions derived from the constant regions of mouse mAbs and human immunoglobulins (eg, Antibodies).
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E.coli may also be used (Skerra et al., Science 242: 1038-1041 (1988)).
  • screening refers to a target, such as an organism or a substance having a certain specific property of interest, in a population that includes a large number of targets by a specific operation Z evaluation method. Means to select from.
  • agents eg, antibodies
  • polypeptides or nucleic acid molecules of the invention can be used.
  • a system using real substances such as in vitro and in vivo may be used, or a library generated using an in silico (computer-based) system may be used.
  • in silico computer-based
  • diagnosis refers to identifying various parameters related to the type of disease, the type and degree of a disorder, a physical condition, and the like in a subject, and the current state of such a disease, disorder, condition, and drug. This refers to predicting reactivity, predicting changes in disease state, or determining the cause.
  • a disease can be analyzed and correlated with a disease state, and such information can be used to control the disease, disorder, condition, or administration in a subject.
  • Various parameters can be selected, such as formulation or method for treatment or prevention, such as type and amount of drug.
  • the diagnostic method of the present invention is industrially useful because, in principle, it can be used from the body, and can be carried out without the hands of medical staff such as doctors.
  • high risk when used for a disease means that the subject is likely to have the disease (eg, about 25%, about 50%, about 7%). 5%). Subjects who have a high-risk diagnosis can make a definitive diagnosis by another diagnostic method.
  • clusive diagnosis when used for a certain disease, refers to definitively diagnosing a subject as suffering from the disease.
  • Factors, polypeptides, nucleic acid molecules, methods and kits for cancer are included in the present invention.
  • a nucleic acid comprising a nucleic acid sequence of a normal gene of the invention, a nucleic acid comprising a sequence encoding an antibody or a functional derivative thereof is a disease or disease associated with abnormal expression and Z or activity of a polypeptide of the invention.
  • Gene therapy refers to therapy performed by the administration of an expressed or expressible nucleic acid to a subject.
  • the nucleic acids produce their encoded protein, which protein mediates a therapeutic effect.
  • in vitro assays for demonstrating the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on a cell line or patient tissue sample.
  • the effect of a compound or composition on a cell line and / or tissue sample can be determined using techniques known to one of skill in the art, including, but not limited to, cell lysis assays.
  • in vitro assays that can be used to determine whether administration of a particular compound is indicated include in vitro cell culture assays in which a patient tissue sample is grown in culture. And then exposed to or otherwise administered the compound, and the effect of such compound on the tissue sample is observed.
  • prevention refers to treating a disease or disorder before such condition is caused so that the condition does not occur.
  • the term “treatment” refers to preventing the deterioration of a disease or disorder when such a condition occurs, preferably maintaining the status quo, more preferably reducing the disease, It preferably refers to fluctuating.
  • cancer or “cancer” are used interchangeably, are atypical, proliferate faster than normal cells, and are capable of destructively invading surrounding tissues or causing malignancy or metastasis thereof. It refers to a state in which such a malignant tumor exists.
  • cancers include, but are not limited to, solid cancers and hematopoietic tumors.
  • solid cancer refers to a cancer having a solid form, and is a concept that is opposed to hematopoietic tumors such as leukemia.
  • solid cancers include, for example, breast, liver, stomach, lung, head and neck, cervix, prostate, retinoblastoma, malignant lymphoma, esophagus, brain and bone tumors. But not limited to them.
  • subject refers to an organism to which the treatment of the present invention is applied, and is also referred to as “patient”. The patient or subject can preferably be a human.
  • the present invention provides methods of treatment, inhibition and prevention by administering to a subject an effective amount of a compound or pharmaceutical composition of the present invention.
  • the compound can be substantially purified (eg, in a state where there is substantially no substance that limits its effect or produces undesired side effects).
  • the animal targeted by the present invention may be any organism (eg, an animal (eg, a vertebrate, an invertebrate)) having a nervous system or a similar system.
  • a vertebrate for example, black eel, smelt, chondrichthyes, teleost, amphibians, reptiles, birds, mammals, etc.
  • mammals for example, monops, marsupials
  • Illustrative subjects include, but are not limited to, animals such as, for example, ass, bush, puma, chicken, cat, dog, and the like.
  • such a composition may further include a pharmaceutically acceptable carrier and the like.
  • Pharmaceutically acceptable carriers included in the medicament of the present invention include any substance known in the art.
  • suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers Agents, delivery vehicles, diluents, excipients and Z or pharmaceutical adjuvants.
  • the medicament is administered in the form of a composition comprising the active ingredient together with one or more physiologically acceptable carriers, excipients or diluents.
  • suitable vehicles may be water for injection, physiological solutions, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.
  • an acceptable carrier, excipient, or stabilizer is non-toxic to the recipient, and is preferably inert at the dosages and concentrations employed, e.g., Phosphate, citrate or other organic acids; ascorbic acid, human tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymers (eg, polyvinylpyrrolidone) Amino acids (eg, glycine, dal Yumin, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, , Mannitol or sorbitol ); Salt-forming counterions (eg, sodium); and Z or non-ionic surface activators (eg, Tween, p1 uronic, or polyethylene glycol (PEG)).
  • Exemplary suitable carriers include neutral buffered saline, or saline mixed with serum albumin.
  • the product is formulated as a lyophilizate using appropriate excipients (eg, sucrose).
  • excipients eg, sucrose
  • Other standard carriers, diluents and excipients may be included as desired.
  • Other exemplary compositions include a Tris buffer at pH 7.0-8.5 or pH 4.0-5.
  • acetate buffers which may further include sorbitol or a suitable substitute thereof.
  • the medicament of the present invention can be administered orally or parenterally.
  • the medicament of the present invention can be administered intravenously or subcutaneously.
  • the medicament used in this may be in the form of a pyrogen-free, pharmaceutically acceptable aqueous solution. Preparation of such a pharmaceutically acceptable composition can be easily performed by those skilled in the art by considering PH, isotonicity, stability, and the like.
  • the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, topical administration to the affected area, Skin administration).
  • Formulations for such administration may be provided in any formulation.
  • Such preparation forms include, for example, liquid preparations, injections, and sustained-release preparations.
  • the medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizer (Japanese Pharmacopoeia 14th edition or its latest edition (including supplements), Remington's Pharmaceutical Sc iences, 18th Edition, AR Gennaro, ed., Mack Publishing Co immediately any, 1990, etc.) and a sugar chain composition having a desired degree of purity. It can be prepared and stored in the form of a cake or an aqueous solution.
  • the therapeutically effective amount (ie, effective amount) of the protein or polypeptide is in the range of about 0.001 to 30 mg / lig body weight, preferably about 0.01 to 25 mg / kg body weight, more preferably about 0.1. -20 mg / kg body weight, even more preferably in the range of about l-10 mg / kg, 2-9 mg / kg, 3-8 mg / kg, 4-7 mg / kg or 5-6 mg / kg body weight.
  • a polynucleotide encoding a protein is introduced by a method such as gene therapy, a polynucleotide capable of expressing the protein in the above-mentioned range may be administered.
  • the amount of the sugar chain composition used in the treatment method of the present invention depends on the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. In consideration of the above, a person skilled in the art can easily determine.
  • the frequency of applying the treatment method of the present invention to a subject (or patient) also depends on the purpose of use, the target disease, and the like.
  • Frequency may include, for example, daily—once every few months (eg, once a week—once a month). It is preferable to administer once a week and once a month while observing the progress.
  • the “instruction” describes a method of administering or diagnosing the medicament or the like of the present invention to a doctor, a patient or the like who administers or diagnoses (possibly a patient). Things. This instruction describes a word indicating the procedure for administering the diagnostic agent, medicament, etc. of the present invention.
  • the instruction sheet is a so-called package insert, which is usually provided in a paper medium, but is not limited thereto.
  • an electronic medium eg, a homepage (website) provided on the Internet, an e-mail
  • SMS SMS
  • PDF documents etc.
  • the polypeptide having the amino acid sequence of SEQ ID NO: 2 is a transcription factor FBP (Far Ups stream Binding Protein: EMBO J. 19 (5): 1034-1044, which binds 1.5 kb upstream of the c-myc gene promoter. 2000) interacts with the transcription factor FIR (FBP W 200
  • Interacting Receptor A protein known as Cell 104 (3): 343-363, 2001), but its association with cancer was not known at all.
  • the polypeptide having the amino acid sequence of SEQ ID NO: 4 is a type of centromere-specific protein (CENP-A) (Proc. Natl. Acad. Sci. USA 88 (9) : 3734-3738, 1991), but this CEMP-1 was also not known to have any association with cancer.
  • CENP-A centromere-specific protein
  • polypeptides having the amino acid sequence of SEQ ID NO: 2 and variants thereof for example, SEQ ID NOs: 12, 14, 16, 18, 18, 20, 22, etc.
  • polypeptides having the amino acid sequence of SEQ ID NO: 4 The peptide was tested for the presence or absence of an antibody that binds to the antigenic peptide of the present invention in the serum of the subject, and the subject in which the antibody was present in the serum was compared with a colorectal cancer patient or a person with colon cancer risk.
  • a method for diagnosing colorectal cancer associated with a method for diagnosing human colorectal cancer preferably, testing the binding between the antibody of the subject's serum and the antigenic peptide on a plate on which the antigenic peptide is immobilized). be able to.
  • antigenic peptides are synthesized, for example, as a type III mRNA transcribed from a gene DNA encoding a peptide having the amino acid sequence of SEQ ID NO: 2 or 4, and have a nucleotide sequence of SEQ ID NO: 1 or 3 or a partial sequence thereof.
  • a peptide can be expressed in vitro by preparing RNA by in vitro transcription from a recombinant expression vector having a DNA fragment consisting of a sequence (these are also included in the present invention) and performing in vitro translation using this as a type III.
  • Transformed cells are prepared by introducing the recombinant expression vector into prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, and mammalian cells. Then, the peptide encoded by the DNA fragment can be expressed from the transformed cells.
  • prokaryotic cells such as Escherichia coli and Bacillus subtilis
  • eukaryotic cells such as yeast, insect cells, and mammalian cells.
  • the above DNA fragment is inserted into a vector having an RNA polymerase promoter to prepare a recombinant expression vector, and this vector is used to prepare an RM polymerase corresponding to the promoter.
  • RNA polymerase promoter include T7, T3, SP6 and the like.
  • vectors containing these RNA polymerase promoters all over the world include pKAl, pCDM8, ⁇ 3 / ⁇ 718, ⁇ 7 / 319, pBluescript II and the like.
  • the expression vector containing the origin, promoter, ribosome binding site, DNA cloning site, and one minute and one minute that can be replicated in the microorganism is used as described above.
  • An expression vector is prepared by recombining the DNA fragment of the above, a host cell is transformed with the expression vector, and the resulting transformant is cultured, whereby the antigen peptide encoded by the DNA fragment can be obtained from the microorganism. Can be expressed. At this time, it can be expressed as a fusion protein with another protein.
  • Examples of expression vectors for Escherichia coli include a PUC system, pBluescript II, a pET expression system, a pGEX expression system, and the like.
  • the above DNA fragment is inserted into an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, etc.
  • an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, etc.
  • the antigen peptide can be expressed in transformed eukaryotic cells.
  • expression vectors include pKAK pC fraction, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2 and the like.
  • pIND / V5-His, pFLAG-CMV-2, pEGFP-NL pEGFP-CI, etc. are used as expression vectors, fusion with various tags such as His tag, FLAG tag, iyc tag, HA tag, GFP etc.
  • Antigen peptides can also be expressed as proteins.
  • mammalian cultured cells such as monkey kidney cell C0S7, Chinese eight-mester ovary cell CH0, budding yeast, fission yeast, silkworm cells, African egg cells and the like are generally used. Any eukaryotic cell that can express the antigenic peptide may be used.
  • the target peptide can be isolated and purified from the culture by a combination of known separation procedures. For example, treatment with denaturing agents such as urea or surfactants, ultrasonic treatment, enzyme elimination, salting out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, etc. Electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, and the like.
  • the recombinant antigen peptide obtained by the above method includes a fusion protein with any other protein.
  • a fusion protein with dalzytin-S-transferase (GST) or green fluorescent protein (GFP) can be exemplified.
  • GST dalzytin-S-transferase
  • GFP green fluorescent protein
  • a peptide expressed in a transformed cell may undergo various modifications in the cell after being translated. Therefore, modified peptides are also included in the scope of the antigenic peptides of the present invention. Examples of such post-translational modifications include N-terminal methionine elimination, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation and the like.
  • the fflRNA of the present invention is an mRNA transcribed from a gene DNA encoding a colon cancer antigen peptide. These mRNAs can be isolated by known methods, for example, from RNA isolated from colon cancer cells. These mRNAs are tested for the presence of the mRNA of the present invention in a biological sample of a subject, and the subject in which the mRNA is present in the sample is determined as a colorectal cancer patient or a person at high risk for colorectal cancer. And a method for diagnosing human colorectal cancer characterized by:
  • a DNA fragment synthesized from the mRNA of the present invention as type III and consisting of the nucleotide sequence of SEQ ID NO: 1 or a partial continuous sequence thereof is synthesized from the mRNA of the present invention and has the nucleotide sequence of SEQ ID NOS: 1 and 3, or a fragment thereof.
  • the “partially contiguous sequence” is, for example, a nucleotide sequence covering a region (0RF or CDS region) encoding an antigen peptide in each nucleotide sequence.
  • the nucleotide sequences of SEQ ID NOS: 1 and 3 can be obtained by, for example, a known method (Mol. Cell Biol. 2, 161-170, 1982; J. Gene 25, 263-269, 1983; Gene, 150, 243-250, 1994).
  • the cDNA can be obtained by a method in which each cDNA is synthesized using a probe DNA prepared based on the nucleotide sequence of SEQ ID NO: 1 or 3 and the respective cDNAs are isolated.
  • each cDNA can be obtained by RT-PCR using a primer set for PCR-amplifying the mRNA of the present invention and using the mRNA isolated from human cells as type III.
  • the obtained cDNA can be used for a gene amplification method such as PCR (Polymerase Chain Reaction), ASBN (Nucleic acid sequence based amplification), TA (Transcription-mediated amplification) and SDA (Strand Displacement Amplification). Can be amplified.
  • PCR Polymerase Chain Reaction
  • ASBN Nucleic acid sequence based amplification
  • TA Transcription-mediated amplification
  • SDA String Displacement Amplification
  • the antibody that binds to the peptide of the present invention can be a polyclonal antibody or a monoclonal antibody, and a whole molecule capable of binding to the epitope of the antigenic peptide of the present invention, and Fab, F (ab ′) 2 , and Fv fragments, respectively. Etc. are all included.
  • a polyclonal antibody such an antibody can be obtained from serum after immunizing an animal using an antigen peptide or a partial fragment thereof as an immunogen.
  • it can be prepared by introducing the above expression vector for eukaryotic cells into the muscle or skin of an animal by injection or gene gun, and then collecting serum.
  • animals mice, rats, egrets, goats, and chickens are used.
  • Monoclonal antibodies can be prepared using known monoclonal antibody preparation methods ("Monoclonal Antibody", James W. Goding, third edition, Academic Press, 1990); 1996), for example, by the following procedure.
  • 1 Preparation of hybridoma cell group An immunogen containing each of the antigenic peptides of the present invention or a partial fragment thereof is prepared. To immunize mammals, and boost as appropriate to boost the animals. Next, antibody-producing cells (lymphocytes or spleen cells) are excised from the animal, and fused cells of this with a myeloma (myeloma) cell line are obtained.
  • myeloma myeloma
  • a hybridoma cell group can be obtained by selecting and culturing a plurality of cells each producing a desired monoclonal antibody from these fusion cell lines.
  • each step will be described in detail.
  • a) Preparation of immunogen For example, an antigen peptide expressed by in vitro transcription from a recombinant expression vector of the present invention or an antigen peptide expressed from a transformed cell of the present invention can be used as an immunogen. Wear.
  • a partial fragment (10-20 amino acids) it may be a synthetic peptide prepared based on the amino acid sequence of SEQ ID NO: 2 or 4.
  • immunogens can also be used in the form of fusion proteins with other proteins (for example, Daryuthion-S-transferase: GST).
  • fusion proteins with other proteins (for example, Daryuthion-S-transferase: GST).
  • GST Daryuthion-S-transferase
  • the use of such a fusion protein is particularly preferred in that the isolation of the target protein from the expression product of the host-vector system and the screening step of the hybridoma cells described later are easy and reliable.
  • b) Immunization of animals As animals to be immunized, mammals used for known methods for producing hybridomas can be used. Specifically, for example, a mouse, a rat, a goat, a hidge, a horse, a horse, and the like.
  • mice or rats as immunized animals.
  • the strains of mice and rats actually used are not particularly limited. In the case of mice, for example, each strain A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C57BR, C57L, FL, HTH, HT1, LP, rat NZW RF, RI II, SJL, SWR, WB, 129 etc.
  • rats for example, Low, Lewis, Spraque, Daweley ACI, BN, Fischer Etc. can be used.
  • the BALB / c strain in mice and the low strain in rats Is particularly preferred.
  • the age of these mice or rats at the time of immunization is preferably 5 to 12 weeks.
  • Immunization of animals can be performed by administering a solution of the immunogen intradermally or intraperitoneally to the animal.
  • the administration schedule of the immunogen varies depending on the type of the animal to be immunized, individual differences, and the like.
  • the dose of the antigen varies depending on the type of animal, individual differences, etc., but is generally about -100 xg /.
  • Cell fusion Spleen cells or lymph cells containing antibody-producing cells are aseptically removed from the immunized animal 1 to 5 days after the last immunization on the above administration schedule.
  • the antibody-producing cells can be separated from these spleen cells or lymph cells according to a known method.
  • the antibody-producing cells and the myeloma cells are fused.
  • the myeoma cells can be appropriately selected from known cell lines and used.
  • HGPRT Hydroxan thine-guanine phosphoribosyl transferase
  • the fusion of the antibody-producing cell and the myeloma cell can be appropriately performed according to a known method under conditions that do not extremely reduce the cell viability.
  • a chemical method of mixing antibody-producing cells and myeloma cells in a high-concentration polymer solution such as polyethylene glycol or a physical method using electrical stimulation can be used.
  • the selection of fused cells and non-fused cells is preferably performed by, for example, a known HAT (hypoxanthine / aminopterin / thymidine) selection method. This method is effective when obtaining fusion cells using myeloma cells of an HGPRT-deficient strain that cannot survive in the presence of aminopterin.
  • HAT hyperxanthine / aminopterin / thymidine
  • hybridoma cells producing the desired monoclonal antibody can be performed by known enzyme immunoassay (EIA: Enzyme Immunoassay), radioimmunoassay (RIA: Radio Iimunoassay), fluorescent antibody method, etc. Can be performed.
  • EIA Enzyme Immunoassay
  • RIA Radio Iimunoassay
  • fluorescent antibody method etc.
  • EIA Enzyme Immunoassay
  • hybridoma cells can be more reliably screened by carrying out the above-mentioned screening methods together for a fusion partner protein.
  • the hybridoma cells after screening are cloned by a known method such as a methylcellulose method, a soft agarose method, or a limiting dilution method, and used for antibody production.
  • the hybridoma cells obtained by the above method can be stored in a frozen state in liquid nitrogen or in a freezer at -80 ° C or lower.
  • 2 Acquisition and Purification of Monoclonal Antibody
  • the culture may be performed, for example, in a medium of the same composition used in the cloning method described above, or for production of a large amount of monoclonal antibody, a mouse.
  • Hybridoma cells may be injected intraperitoneally and monoclonal antibodies may be collected from the ascites fluid.
  • the monoclonal antibody thus obtained can be purified by, for example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, or the like.
  • the labeled antibody of the present invention is the same polyclonal antibody or monoclonal antibody as the antibody of the present invention, and is an antibody labeled with a labeling substance.
  • a labeling substance an enzyme, a radioisotope or a fluorescent dye can be used. There are no particular restrictions on the enzyme as long as it meets the conditions such as a large turnover number, stability even when bound to the antibody, and specific coloring of the substrate, and it is used for normal EIA.
  • Enzymes for example, peroxidase,
  • enzyme inhibitors, coenzymes, and the like can also be used.
  • the binding between the enzyme and the antibody can be performed by a known method using a crosslinking agent such as a maleimide compound.
  • a known substance can be used depending on the type of the enzyme to be used.
  • 3,3 ′, 5,5′-tetramethylbendicine can be used when peroxidase is used as an enzyme, and paranitrophenol can be used when alkaline phosphatase is used as an enzyme.
  • Radioisotopes can be used for even is commonly used in RIA such as I or 3 H.
  • the fluorescent dye those used in a usual fluorescent antibody method such as fluorescence isothiosineate (FITC) and tetramethylrhodamine isothiosinate (TRITC) can be used.
  • the method for diagnosing cancer of the present invention tests whether or not an antibody that binds to the antigenic peptide of the present invention is present in the serum of the subject, and the subject in which the antibody is present in the serum is treated as a cancer patient or The patient is determined to be at high risk for cancer. That is, since the antigenic peptide of the present invention is a peptide that binds to an antibody (IgG) in the serum of a patient, it is reacted with the serum of a subject, and the serum containing the antibody that binds to these antigenic peptides is used as a cancer patient. Alternatively, it can be determined as the serum of a patient with the eight risks. In this case, it is preferable to determine the binding of the two antigen peptides to the serum antibody.
  • IgG an antibody
  • a subject serum is brought into contact with an antigen peptide, and the antigen peptide is reacted with an IgG antibody in the subject serum in a liquid phase.
  • a signal of the labeled IgG antibody may be detected by reacting a labeled IgG antibody that specifically binds to the IgG antibody in the serum.
  • an enzyme, a radioisotope or a fluorescent dye as exemplified in the above-mentioned labeled antibody can be used.
  • an enzyme When an enzyme is used, a substrate that decomposes by the action of an enzyme and develops a color is added, and the enzyme activity is determined by optically measuring the amount of decomposition of the substrate, converted to the amount of bound antibody, and compared with a standard value. Is used to calculate the amount of antibody.
  • radioactive isotopes measure the radiation dose emitted by the radioisotope using a scintillation counter or the like.
  • the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope.
  • Western blot analysis as shown in the Examples can be employed.
  • the conjugate of the antigen peptide + serum antibody + labeled IgG antibody is separated by known separation means (chromatography, salting out, alcohol precipitation, enzymatic method, solid phase method, etc.), and the signal of the labeled IgG antibody is separated. May be detected.
  • a diagnostic kit of the present invention is provided as one that enables simple and wide-ranging implementation of such a diagnostic method.
  • the diagnostic method of the present invention also includes a method of immobilizing one or more antigen peptides on a plate and testing the binding of the serum of the subject to the antibody on the substrate (this is also included in the present invention). ). By immobilizing the antigen peptide on the substrate, unbound labeled binding molecules can be easily removed. Further, a diagnostic kit of the present invention is provided, which enables simple and wide-ranging implementation of such a diagnostic method.
  • the diagnostic method of the present invention tests whether or not an antigen of the present invention or an antigen peptide that binds to the labeled antibody of the present invention is present in a biological sample of a subject, and the antigen peptide is contained in the sample.
  • the subject in which is present is determined to be a colorectal cancer patient or a high-risk subject thereof. That is, since the antibody or labeled antibody used here is an antibody that specifically binds to an antigen peptide expressed in colorectal cancer cells, a biological sample containing an antigen peptide that binds to this antibody can be used for colorectal cancer patients or It can be determined as a sample for patients at high risk for colorectal cancer. In this case, it is preferable to determine the binding of the two antibodies to the antigen peptide in the sample.
  • blood and blood cells eg, mononuclear cells
  • One embodiment of the diagnostic method of the present invention is a method of binding an antibody and an antigen peptide in a liquid phase system.
  • the labeled antibody of the present invention is brought into contact with a biological sample to bind the labeled antibody and the antigen peptide, the conjugate is separated by the method described herein, and the labeled signal is obtained by the same method.
  • the diagnostic kit of the present invention is provided as a method that enables such a diagnostic method to be performed simply and widely.
  • Another method of diagnosis in a liquid phase system is as follows: the antibody (primary antibody) of the present invention is brought into contact with a biological sample to bind the primary antibody and the antigen peptide; And the labeled signal in the conjugate is detected.
  • an unlabeled secondary antibody is first bound to the antibody + antigen peptide conjugate, and the labeling substance is bound to this secondary antibody. You may do it.
  • binding of the labeling substance to the secondary antibody can be carried out, for example, by biotinylating the secondary antibody and avidinizing the labeling substance.
  • an antibody (tertiary antibody) recognizing a partial region (for example, Fc region) of the secondary antibody may be labeled, and the tertiary antibody may be bound to the secondary antibody.
  • the primary antibody and the secondary antibody both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be performed as described herein.
  • a diagnostic kit of the present invention is provided as one that enables simple and wide-ranging implementation of such a diagnostic method.
  • Another aspect of the diagnostic method of the present invention is a method for testing the binding between an antibody and an antigen peptide in a solid phase system.
  • This method using a solid phase system is a preferable method for detecting a trace amount of antigen peptide and simplifying the operation. That is, in this solid phase method, the antibody (primary antibody) of the present invention is immobilized on a resin plate or the like, the antigen peptide is bound to the immobilized antibody, and the unbound peptide is washed off, and then left on the plate. In this method, a labeled antibody (secondary antibody) is bound to the antibody + antigen peptide conjugate, and the signal of this secondary antibody is detected. This method is a so-called “sandwich method”. When an enzyme is used as a marker,
  • ELISA enzyme linked immunosorbent assay
  • the diagnostic kit of the present invention is a reagent kit for performing the diagnostic method of the present invention. Various such kits are commercially available depending on the type of the test component.
  • the diagnostic kit of the present invention also includes the antigenic peptide, antibody, Except for using a labeled antibody and / or a labeled antibody, it can be constituted by each element used in a known kit.
  • the diagnostic method of the present invention tests whether or not the mRNA of the present invention is present in a biological sample of a subject, and determines a subject having the mRNA in the sample as a colorectal cancer patient or a high-risk subject thereof I do.
  • the biological sample may be stool, blood, or blood cells (eg, mononuclear cells).
  • Detection and measurement of mRNA can be performed by a known RT-PCR method or quantitative RT-PCT method, and in that case, the primer set of the present invention can be used for PCR.
  • These primer sets can be designed based on the nucleotide sequence of SEQ ID NO: 1 or 3, and can be prepared through each step of synthesis and purification.
  • the size (number of bases) of the primer is 15 to 40 bases, preferably 15 to 30 bases in consideration of satisfying specific annealing with type I DNA. However, when performing LA (long accurate) PCR, at least 30 bases are effective. Avoid the complementary sequence between both primers so that one or two pairs of primers consisting of the sense strand (5 'end) and the antisense strand (3' end) do not anneal to each other. Avoid self-complementary sequences to prevent the formation of intra-hairpin structures.
  • the GC content should be about 50% to ensure stable binding to type I DNA, so that GC-rich or AT-rich is not unevenly distributed in the primers.
  • the annealing temperature depends on the Tm temperature
  • select primers that have a Tm value of 55-65 ° C and are close to each other in order to obtain a highly specific PCR product. It is also necessary to pay attention to the final concentration of one primer used in PCR to be about 0.1 to about 1 M.
  • commercially available software for primer design for example, Oligo TM [manufactured by National Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Inc. (Japan)], and the like can also be used.
  • the diagnostic method of the present invention can also be carried out by a microarray provided with the polynucleotide or the oligonucleotide provided by the present invention.
  • a method for producing a microarray a method of directly synthesizing an oligonucleotide on the surface of a solid support (on-chip method) and a method of immobilizing an oligonucleotide prepared in advance on the surface of a solid support are known.
  • the microarray used in the present invention can be manufactured by any of these methods.
  • the on-chip method combines the use of protective groups that are selectively removed by light irradiation with the photolithography and solid-phase synthesis technologies used in semiconductor manufacturing to achieve the required It can be performed by a method of performing selective synthesis in a region (masking technique: for example, Fodor, S. et al. Science 251: 767, 1991).
  • oligonucleotide prepared in advance is immobilized on the surface of a solid support
  • an oligonucleotide into which a functional group is introduced is synthesized, and oligonucleotides are spotted on the surface of the surface-treated solid support and shared.
  • Oligonucleotides are generally covalently bonded to a surface-treated solid support via a spacer or a crosslinker.
  • a method is also known in which a small piece of polyacrylamide gel is aligned on a glass surface and a synthetic oligonucleotide is covalently bonded thereto (Yershov, G, et al. Proc. Natl. Acad. Sci. USA 94: 4913, 1996). ). Also, an array of microelectrodes was prepared on a silica microarray, and an agarose permeable layer containing streptapidine was provided on the electrodes to serve as a reaction site, and this site was positively charged to convert the biotin into a biotin.
  • a method is also known that enables fast and strict hybridization by immobilizing oligonucleotides and controlling the charge at the site (Sosno ski, R, G. et al. Proc. Natl. Acad. Sci. USA 94: 1119-1123, 1997).
  • cDNA is synthesized using the mRNA isolated from the subject's cells as type III, and PCR is performed. Width. At this time, labeled dNTPs are incorporated into the labeled cDNA.
  • the labeled cDNA is brought into contact with the macroarray, and the cDNA hybridized to the microarray capture 5-probe (oligonucleotide or polynucleotide) is detected.
  • Hybridization can be carried out by dispensing a 96-well or 384-well plastic plate and dropping the labeled cDNA aqueous solution on a microarray.
  • the amount of spotting can be about l to 0 nl.
  • the hybridization is preferably carried out at a temperature in the range of room temperature to 70 ° C. and in the range of 6 to time. After completion of the hybridization, wash with a mixed solution of surfactant and buffer to remove unreacted labeled cDNA.
  • the present invention can make a diagnosis using an array.
  • DNA arrays are widely reviewed in (Shujunsha eds., Cell Engineering Separate Volume “DNA Microarrays and the Latest PCR Method”). The protein array is described in detail in Nat Genet. 2002 Dec; 32 Supp 1: 526-32.
  • Methods for analyzing gene expression include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like, in addition to those described above.
  • Such further analysis methods are described in, for example, Genome Analysis Experimental Method ⁇ Yusuke Nakamura Lab ⁇ Manual and Editing ⁇ Yusuke Nakamura Yodosha (2002), and all the descriptions in this specification are incorporated by reference. Is done. Hereinafter, the present invention will be described in more detail.
  • the present invention relates to a cancer (for example, a cancer that overexpresses c-myc, including rectal cancer or colon cancer (collectively referred to as colorectal cancer)) comprising a nucleic acid molecule encoding FIR.
  • a cancer for example, a cancer that overexpresses c-myc, including rectal cancer or colon cancer (collectively referred to as colorectal cancer)
  • colorectal cancer a cancer comprising a nucleic acid molecule encoding FIR.
  • the amount effective for diagnosis, prevention, treatment or prognosis can be determined by a person skilled in the art using techniques known in the art while considering various parameters, and such an amount is determined. For this purpose, those skilled in the art can easily determine the purpose of use, target disease (type, severity, etc.), patient's age, weight, sex, medical history, cell morphology or type, etc.
  • the present invention has revealed that onset of cancer is unexpectedly associated with abnormal expression of FIR, and that administration of normal FIR can cure cancer. Such a relationship and the diagnostic and therapeutic effects using the same have not been known so far. Therefore, the nucleic acid form of the FIR of the present invention and the invention related thereto show more excellent effects than the prior art.
  • the present invention provides: (a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 (encoding FIR542) or a complement thereof or a fragment sequence thereof; A polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (c) in the amino acid sequence of SEQ ID NO: 2, one or more amino acids are selected from the group consisting of substitution, addition, and deletion A variant polypeptide having at least one mutation, wherein the polynucleotide encodes a variant polypeptide having biological activity; and (d) any one of (a) to (c).
  • a nucleic acid molecule comprising: a polynucleotide having a base sequence of 0% and encoding a polypeptide having biological activity.
  • the number of substitutions, additions and deletions in (c) above is limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less , 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less. Lesser numbers of substitutions, additions and deletions are preferred, but retain biological activity (preferably, have activity similar or substantially identical to FIR, or aberrant activity of FIR (eg, , Poor connection with FBP, etc.)
  • the biological activity of the polypeptide is, for example, the interaction with an antibody specific to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.
  • FBP fluorescence-activated protein
  • the ability to bind to FBP is defined by the sequence described at positions 372 to 442 in SEQ ID NO: 2, and can be measured using FBP.
  • the allelic variant according to (d) advantageously has at least 99% homology with the nucleic acid sequence shown in SEQ ID NO: 1.
  • the above three homologues are identified by searching the FIR (or more preferably, FIR542) of the present invention as a query sequence against the database, if a gene sequence database of the species exists. can do. Alternatively, it can be identified by screening a gene library of that type using all or a part of the FIR (or more preferably, FIR542) of the present invention as a probe or primer. Such identification methods are well known in the art and have been described in the literature described herein. Species homologs are, for example, at least about 30% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1.
  • the species homolog is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% Can be at least about 98% homologous.
  • the identity to the polynucleotide of any one of (a) to (e) above or a complementary sequence thereof may be at least about 80%, more preferably at least about 90%, and even more preferably Can be at least about 98%, most preferably at least about 99%.
  • the FIR of the present invention is FIR542.
  • the FIR-encoding nucleic acid molecules of the invention or fragments and variants thereof can be at least 8 contiguous nucleotides in length.
  • the appropriate nucleotide length of the nucleic acid molecule of the present invention may vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides in length, even more preferably at least 15 contiguous nucleotides in length, and even more preferably at least 20 contiguous nucleotides in length. .
  • the lower limit of these nucleotide lengths is not limited to the specific number, but also the number between them (for example, 9, 11, 12, 13, 14, 16, etc.) or more (for example, For example, 21, 22,... 30, etc.).
  • the nucleic acid molecule of the present invention has an upper limit as long as it can be used for the intended use (for example, it can interact with antisense, RNAi, a marker, a primer, a probe, or a predetermined factor).
  • the length may be the entire length of the sequence shown in SEQ ID NO: 1, or may be longer.
  • when used as a primer it can usually be at least about 8 nucleotides in length, and preferably about 10 nucleotides in length.
  • When used as a probe it can usually be at least about 15 nucleotides in length, and preferably about 17 nucleotides in length.
  • the FIR encoding nucleic acid molecule comprises the entire nucleic acid sequence of SEQ ID NO: 1. More preferably, the nucleic acid molecule encoding FIR consists of the entire nucleic acid sequence of SEQ ID NO: 1. (Specific FIR nucleic acid forms)
  • the present invention provides a nucleic acid molecule comprising the sequence shown in SEQ ID NO: 1 or its complement.
  • a sequence is also expressed in normal tissues and can be used as an indicator of normality.
  • the expression level is abnormal, it can be determined that there is a risk of developing cancer.
  • Such a normal FIR nucleic acid form can cause apoptosis of c-myc-expressing cells by external administration, and thus can be used as an anticancer agent.
  • the present invention provides a method comprising: (a) a sequence comprising at least one modification selected from the group consisting of substitution, addition and deletion in SEQ ID NO: 1 or its complement; or (b) a sequence comprising No. 11, at least one sequence selected from the group consisting of 13, 13 and 15 or its complement, or selected from the group consisting of substitution, addition and deletion in said at least one sequence or its complement
  • a nucleic acid molecule comprising a sequence comprising at least one modification.
  • Such a sequence is not expressed or rarely expressed in normal tissues, and is specifically expressed in cancer tissues. Therefore, the expression itself is an index for cancer diagnosis. Can be used. Since all of these nucleic acid molecules are specific to cancer tissues, they can also be used as definitive diagnosis of cancer.
  • a definitive diagnosis of cancer can be made by comparing their expression with expression in tissues not affected by cancer.
  • a cancer is preferably a c-myc overexpressing cancer (including, for example, rectal cancer and Z or colon cancer).
  • Factors that reduce such nucleic acid molecules include: May be used as a therapeutic. Therefore, it is understood that such factors are also within the scope of the present invention.
  • said alteration in said nucleic acid molecule of the invention is a nucleic acid substitution; between positions 369 and 370 of SEQ ID NO: 1 or 176 and 1 of SEQ ID NO: 15. Addition of nucleic acid between positions 7 and 7; or up to position 369 of SEQ ID NO: 1 or up to position 30 of SEQ ID NO: 11, or up to position 176 of SEQ ID NO: 13 or SEQ ID NO: 15 Or a combination thereof. While not wishing to be bound by theory, such specific modifications are causes or consequences that cause the abnormal occurrence of cancer cells by altering, and preferably exacerbating, the normal function of the FIR of the present invention. obtain. Therefore, by detecting a nucleic acid molecule having or containing such a specific sequence, it has utility that it can be used for cancer diagnosis.
  • preferred modifications in the FIR nucleic acid molecules of the present invention are in the range from the 5 'end of SEQ ID NO: 1 to about 400 bases. This is because modification of this range is considered to have a deep relationship with cancer, but is not limited thereto. More preferably, the modification may be in the range of about 150 to about 360 in SEQ ID NO: 1. This is because a case in which the modification in this range is considered to be more likely to be associated with cancer was found, but it is understood that the present invention is not limited thereto. Therefore, it is understood that any modification that causes abnormal expression or dysfunction of FIR can be used for the diagnosis of the present invention.
  • the modification in the FIR nucleic acid molecule of the present invention comprises a non-conservative substitution of the encoding amino acid in SEQ ID NO: 1.
  • Such non-conservative substitution may cause an abnormality in the function of the polypeptide borne by the encoded amino acid sequence.
  • One of the normal functions of FIR is to induce apoptosis in c-myc-expressing cells. It is thought that abnormal expression or presence of a molecule that interferes with normal FIR function is closely related to the development of cancer.
  • the FIR nucleic acid molecule of the present invention comprising a modification is selected from the group consisting of SEQ ID NOS: 11, 13, 15, 15, 17, 19 and 21 Contains an array. Since these specific sequences are not expressed in normal tissues and are specifically expressed in cancer tissues, they can be exemplified as specific sequences of the modified FIR of the present invention. Not limited to them. (Factor specific to FIR nucleic acid form)
  • the invention includes cancers (eg, rectal or colon cancer (collectively referred to as colorectal cancer)) that include an agent that specifically interacts with a nucleic acid molecule encoding an FIR. c) a composition for the treatment, prevention, diagnosis, or prognosis of myc high-expressing cancer).
  • cancers eg, rectal or colon cancer (collectively referred to as colorectal cancer)
  • an agent that specifically interacts with a nucleic acid molecule encoding an FIR.
  • a composition for the treatment, prevention, diagnosis, or prognosis of myc high-expressing cancer e.g, the present invention provides agents specific for nucleic acid molecules encoding any of the FIRs described herein, or variants or fragments thereof.
  • the amount effective for diagnosis, prevention, treatment, or prognosis can be determined by those skilled in the art using techniques well known in the art while considering various parameters, and such an amount is determined.
  • the target disease type, severity, etc.
  • the patient's age, weight, sex, medical history, cell morphology or type, etc. See, eg, Cancer Clinic Resident Manual, Second Edition, Editing: Internal Resident, National Cancer Center-Central Hospital, Medical School, 2000.
  • the present invention has revealed that the onset of cancer is unexpectedly associated with abnormal expression of FIR, and that administration of normal FIR can cure cancer.
  • Such an association, and the diagnostic and therapeutic effects utilizing it, have not been known in the past, and thus the factors of the present invention exhibit superior effects to those provided by the prior art.
  • the agent of the present invention can be an agent selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof. It can be understood that such factors may be any as long as they specifically bind to the nucleic acid molecule of the present invention.
  • the agent of the invention is a nucleic acid molecule.
  • a nucleic acid molecule can be at least 8 consecutive nucleotides in length, and is preferably specific for a nucleic acid sequence of an FIR (eg, SEQ ID NO: 1). Can be combined.
  • the appropriate nucleotide length of the nucleic acid molecule of the present invention may vary depending on the purpose of use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides in length, even more preferably at least 15 contiguous nucleotides in length, and even more preferably at least 20 contiguous nucleotides in length.
  • nucleotide lengths are not only the numbers specifically listed, but also the numbers between them (eg, 9, 11, 11, 12, 13, 14, 16, etc.) or more. It may be a number (eg, 21, 22, 22, 30 etc.).
  • the nucleic acid molecules of the present invention can be used as intended applications (eg, antisense, RNAi, markers, primers, probes) or as long as they can interact with a given factor.
  • the upper limit length may be the full length of the sequence shown in SEQ ID NO: 1, 11 or the complement thereof, or may be longer.
  • primer when used as a primer, it can usually be at least about 8 nucleotides in length, and preferably about 10 nucleotides in length.
  • the agent of the present invention comprises a sequence complementary to the nucleic acid sequence of the FIR polynucleotide of any of (a) to (e) or at least 70% thereof.
  • the nucleic acid molecule may have a sequence having the following identity:
  • the agent of the present invention comprises: (a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or a complement thereof or a fragment thereof; (b) the amino acid of SEQ ID NO: 2 A polynucleotide encoding the sequence or a fragment thereof; (c) in the amino acid sequence of SEQ ID NO: 2, at least one mutation in which one or more amino acids is selected from the group consisting of substitution, addition and deletion.
  • a polynucleotide that hybridizes and encodes a polypeptide having biological activity or (e) (a) to (c).
  • the factor of the present invention is preferably a factor specific to a nucleic acid molecule containing the sequence shown in SEQ ID NO: 1 or its complement. Such a sequence is useful for examining the degree of canceration of a certain tissue or individual, since the form of FIR present in normal tissues can be identified.
  • the factor specific to the nucleic acid molecule comprising the sequence shown in SEQ ID NO: 1 or its complement is selected from the group consisting of: (a) substitution, addition and deletion in SEQ ID NO: 1 or its complement Or (b) at least one sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, or a complement thereof, or the at least one sequence Or at least one selected from the group consisting of substitution, addition and deletion in the complement thereof.
  • it does not specifically react with nucleic acid molecules containing a sequence containing one modification.
  • a variant FIR can be discriminated from a normal FIR and detected.
  • Such detection can provide an accurate and previously impossible diagnosis of cancer, especially c-myc overexpressing cancers, including (rectal or colon (collectively referred to as colorectal) cancers). Achieve the superior effect and usefulness of being able to.
  • the agent of the present invention comprises: (a) a sequence comprising at least one modification selected from the group consisting of substitutions, additions and deletions in SEQ ID NO: 1 or its complement; or (b) ) At least one sequence selected from the group consisting of SEQ ID NOs: 11, 13 and 15 or its complement, or a group consisting of substitution, addition and deletion in said at least one sequence or its complement It is preferable to specifically react with a nucleic acid molecule containing a sequence containing at least one modification selected from the group consisting of: The use of an agent specific for such a variant provides the utility of facilitating the detection of an individual or tissue portion affected by cancer.
  • this factor preferably does not specifically react with nucleic acid molecules comprising the sequence shown in SEQ ID NO: 1 or its complement.
  • a variant of FIR can be discriminated from normal FIR and detected.
  • detection will provide an accurate diagnosis of cancer (particularly c-myc overexpressing cancer, including colorectal or colon cancer (collectively referred to as colorectal cancer)). To achieve an excellent effect and usefulness.
  • the agent of the invention is an antisense or RNAi of a nucleic acid molecule of a FIR.
  • RNA i may be siRNA or sh RNA, and such an example is, for example, about 20 bases (eg, typically about 21 to 23 bases in length) Or a shorter double-stranded RNA, preferably having a 5′-phosphate, 3′-OH structure, and a 3′-terminal It protrudes about 2 bases.
  • the shRNA may also preferably have a 3, overhanging end.
  • the length of the double-stranded portion is not particularly limited, but may be preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more.
  • the 3 ′ protruding end may be preferably DNA, more preferably DNA having at least 2 nucleotides or more, and further preferably DNA having 2 to 4 nucleotides in length.
  • the agent of the present invention is a nucleic acid molecule, which can be used as a primer in a nucleic acid amplification reaction such as PCR or the like, or can be used as a probe such as a Southern blot.
  • the agent of the invention may be labeled or capable of binding to a label.
  • a label can be any label as long as it is identifiably labeled, including techniques such as fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme-substrate reactions, and antigen-antibody reactions. Not limited.
  • an immune reaction such as an antibody
  • a system often used in an immune reaction such as biotin-streptavidin may be used. (Diagnostic kit and method using FIR nucleic acid form)
  • the present invention provides: A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 (encoding FIR542) or a complement thereof or a fragment thereof; A polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (c) in the amino acid sequence of SEQ ID NO: 2, one or more amino acids are selected from the group consisting of substitution, addition, and deletion A variant polypeptide having at least one mutation, A polynucleotide encoding a variant polypeptide having biological activity;
  • a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides of (a) to (c) and encodes a polypeptide having biological activity; or (e) (a) ) A polynucleotide comprising a base sequence having at least 70% identity to the polynucleotide of any one of (c) to (c) or a complementary sequence thereof, and a polynucleotide encoding a polypeptide having biological activity.
  • the method comprising the steps of: If the expression of the nucleic acid molecule in the target portion of the subject is higher than the expression level of the nucleic acid molecule in a non-affected subject or a portion not affected by cancer, the subject of the subject Diagnosed as being at high risk of cancer, means, comprises, providing a cancer diagnostic kit.
  • the cancer to be diagnosed is preferably a cancer with high expression of c-myc, but is not limited thereto.
  • Such high c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • an FIR-specific factor any factor as described herein can be used.
  • the first factor may specifically react with both the normal form and the variant of the FIR, or may react specifically with either the normal form or the variant of the FIR. .
  • any factor having any specificity can be used in the present invention. Therefore, such a factor and a kit containing the same can be used in the present invention. It is understood that it is in the range of.
  • the common sequence between the normal type and the variant for example, the sequence at the 3 ′ terminal side of SEQ ID NO: 1, 11, 13, 15, 15, 17, 19, 21 etc.
  • a factor specific to for example, a sequence having a length of 100 nucleotides
  • the measurement of the expression level of the first nucleic acid molecule can be performed using any signal caused by the first factor.
  • signals include, for example, physical signals (eg, fluorescence, luminescence, etc.), chemical signals (eg, staining, etc.), biochemical signals (eg, enzyme reactions), biological signals (eg, microorganisms) Determination of survival) and the like, but are not limited thereto. Any such signal may be used as long as it can be determined whether or not the mRNA of the present invention is directly or indirectly tested in the biological sample of the subject. can do. In this case, when such a signal is detected, the subject in which the abundance of the iRNA is present in the sample can be determined as a cancer patient or a high-risk subject.
  • the present invention provides: A) a second factor specific for a nucleic acid molecule (second nucleic acid molecule) comprising the sequence set forth in SEQ ID NO: 1 or a complement thereof; Means for measuring the expression level of the nucleic acid molecule using a signal caused by an agent, the expression level of the nucleic acid molecule in a subject not having cancer or a portion not having cancer, When the expression level of the nucleic acid molecule in the target portion of the subject is different, the means for diagnosing that the subject is a dirisk of cancer is provided.
  • a FIR-specific factor may be any second factor as described herein. Any second factor can be used as long as it specifically reacts with normal FIR.
  • the second factor does not specifically react with the variant.
  • any second factor is provided with an understanding of even its reaction specificity, and any factor having any specificity can be used in the present invention. It is understood that such factors and kits containing them are within the scope of the present invention.
  • the modified sequence of the normal type and the variant for example, a sequence at the 5 ′ terminal side, for example, about 400 nucleotides
  • Factors such as, but not limited to, any sequence in length).
  • the measurement of the expression level of the second nucleic acid molecule can be performed using any signal caused by the second factor.
  • signals include, for example, physical signals (eg, fluorescence, luminescence, etc.), chemical signals (eg, staining, etc.), biochemical signals (eg, enzyme reactions), biological signals (eg, microorganisms). Determination of survival) and the like, but are not limited thereto. Any such signal can be used as long as it can be determined directly or indirectly whether or not the mRNA of the present invention is present in a biological sample of a subject. can do. In this case, when such a signal is detected, a subject having an abnormality in the expression level of its mRNA in the sample can be determined to be a cancer patient or a high-risk subject thereof.
  • the present invention provides a method comprising: (A) a sequence comprising at least one alteration selected from the group consisting of: substitutions, additions and deletions in (a) SEQ ID NO: 1 or a complement thereof; or (b) SEQ ID NO: 11.At least one sequence selected from the group consisting of 1, 13 and 15 or a complement thereof, or at least one selected from the group consisting of substitution, addition and deletion in the at least one sequence or the complement thereof A third factor specific to a third nucleic acid molecule comprising a sequence containing one modification; and B) measuring the expression level of the third nucleic acid molecule using a signal caused by the third factor.
  • the third factor can take any form as described in the above section “FIR nucleic acid form-specific factor”.
  • the third factor reacts specifically with a variant of the FIR and may or may not specifically react with a normal form of the FIR.
  • the second factor does not react with the normal form.
  • any factor having any specificity can be used in the present invention as long as its reaction specificity is understood and provided.
  • the variant-specific portion for example, the sequence at the 5 ′ end of SEQ ID NO: 11, 13, 15, 15, 17, 19, 21; For example, a sequence having a length of 100 nucleotides).
  • the measurement of the expression level of the third nucleic acid molecule can be performed using any signal caused by the third factor.
  • signals include, for example, physical signals (eg, fluorescence, luminescence, etc.), chemical signals (eg, staining, etc.), biochemical signals (eg, enzyme reactions), biological signals (eg, microorganisms). Determination of survival) and the like, but are not limited thereto.
  • a signal can be determined as long as it can be determined directly or indirectly whether or not the mRNA corresponding to the third nucleic acid molecule of the present invention is present in the biological sample of the subject. Anything can be used. In such a case, if such a signal is detected, the subject having the expression of the mRNA in the sample can be determined to be a cancer patient or a high-risk person thereof.
  • the detection of the third nucleic acid molecule in a target portion of the subject is performed by detecting the third nucleic acid molecule in a subject not having cancer or a portion not having cancer. It may be to detect that the expression of the S-th nucleic acid molecule is higher in the target portion of the subject compared to the expression amount of the nucleic acid molecule.
  • the present invention provides a method comprising: A) a second agent specific for a nucleic acid molecule (second nucleic acid molecule) comprising the sequence set forth in SEQ ID NO: 1 or a complement thereof. B) (a) a sequence comprising at least one modification selected from the group consisting of substitutions, additions and deletions in SEQ ID NO: 1 or its complement; or (b) SEQ ID NOs: 11, 13, and 1. At least one sequence selected from the group consisting of 5 or a complement thereof, or at least one modification selected from the group consisting of substitution, addition and deletion in the at least one sequence or the complement thereof.
  • a third factor specific to a nucleic acid molecule comprising: C) a signal originating from said second factor and said third factor in a distinguishable manner;
  • the expression of the second nucleic acid molecule in the target portion is high, and compared to the expression of the third nucleic acid molecule in the subject without cancer or the portion without cancer, in the target portion of the subject subject.
  • Means for determining that the subject of the subject is a cancer patient when the expression of the third nucleic acid molecule is high comprising:
  • the means C) includes means for comparing the relative abundance ratio between the second nucleic acid molecule and the third nucleic acid molecule.
  • the second agent does not specifically react with the tertiary nucleic acid molecule, or that the third agent does not specifically react with the second nucleic acid molecule. This is because both nucleic acid molecules can be detected in a distinguishable manner.
  • both the second agent does not specifically react with the third nucleic acid molecule and the third agent does not specifically react with the second nucleic acid molecule. is there. This is because the signal intensity detected by these factors is accurately reflected by the amount of the target nucleic acid molecule.
  • these second and third factors are or are identifiably labeled.
  • a factor that is identifiably labeled means that the factor already has an identifiable label, and that two or more factors can be identifiably labeled.
  • the kit of the present invention further includes a factor that discriminately labels the second factor and the third factor. Identification of such identifiable labels can be physical (eg, fluorescence, luminescence, etc.), chemical (eg, specific chemical cleavage, staining, etc.), biochemical (eg, enzymatic reactions), It can be performed by a biological method (for example, determination of the survival of a microorganism).
  • the present invention provides: A) (a) a polynucleotide having a base sequence set forth in SEQ ID NO: 1 or a complement thereof or a fragment thereof; (b) an amino acid set forth in SEQ ID NO: 2 A polynucleotide encoding the sequence or a fragment thereof; (c) at least one amino acid selected from the group consisting of substitutions, additions and deletions in the amino acid sequence of SEQ ID NO: 2; (D) a variant polypeptide having three mutations, wherein the polynucleotide encodes a variant polypeptide having biological activity; and (d) any one of the polynucleotides of (a) to (c), A polynucleotide that hybridizes under gentle conditions and encodes a polypeptide having biological activity; or any of (e), (a) to (c) A nucleic acid molecule (a first nucleic acid) comprising: a polynucleotide comprising a nucleotide sequence
  • the FIR of the present invention has a high relationship with c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • the detection of the nucleic acid molecule can be performed using an agent specific to such a nucleic acid molecule.
  • any factors specific for the nucleic acid forms of the FIR described herein can be used.
  • the first factor is specific to the first nucleic acid molecule
  • the third factor is specific to the tertiary nucleic acid molecule. Since the first nucleic acid molecule can include both normal forms of FIR and variants thereof, the total amount of FIR-related molecules can be detected.
  • the first factor is advantageously capable of reacting specifically to both normal and variant forms of FIR.
  • the third nucleic acid molecule is a variant of FIR and can be used as an indicator of cancer because it is specifically present in cancer tissues.
  • the means C) preferably includes means for comparing the relative abundance or absolute abundance with the third nucleic acid molecule. The relative abundance can be calculated by directly comparing the signal (eg, fluorescence intensity) due to the factor. Therefore, it is preferable in such a situation because it is not necessary to calculate the absolute amount.
  • the first factor used in the diagnostic kit of the invention does not specifically react with the third nucleic acid molecule, or the first factor is a tertiary nucleic acid molecule and a second nucleic acid molecule. It may be advantageous to react specifically to both. If it does not specifically react with the third nucleic acid molecule, the first and third factors can be used to detect the abundance ratio between a specific FIR variant and another FIR molecule. .
  • the kit of the present invention further includes an agent that discriminately labels the first factor and the third factor.
  • the identification of such identifiable labels can be in physical form (eg, fluorescence, luminescence, etc.), chemical form (eg, specific chemical cleavage, staining, etc.), biochemical form (eg, enzymatic reaction).
  • the biological mode eg, determination of the survival of microorganisms.
  • biological samples include stool, urine, sweat, biopsy tissue, saliva, blood (for example, whole blood, serum, plasma, etc.), blood cells (mononuclear cells, etc.), and others.
  • a body fluid of an affected part, a cell in an affected part, and the like can be targeted.
  • Detection and measurement of mRNA can be performed by a known RT-PCR method or quantitative RT-PCT method, and in such a case, the primer set of the present invention can be used for PCR.
  • These primer sets can be designed based on the nucleotide sequences of SEQ ID NOS: 1, 11, 13, 15, 15, 17, and 19 or modified versions thereof, and can be prepared through each step of synthesis and purification.
  • the size (number of bases) of the primer is 15-40 bases, preferably 15-30 bases, in order to satisfy specific annealing with type I DNA. However, when performing LA (long accurate) PCR, at least 30 bases are effective.
  • designing- use both primers so that one or one pair (two) of the sense strand (5 'end) and the antisense strand (3' end) do not anneal to each other. Avoid inter-complementary sequences and avoid self-complementary sequences to prevent the formation of adenosine structures in the primers.
  • the measurement target is an addition type or a deletion type (for example, SEQ ID NO: 1 and SEQ ID NO: 11 (this corresponds to the amino acid sequence between positions 102 and 103 in the sequence shown in SEQ ID NO: 1). Amino acid residue inserted
  • SEQ ID NO: 1 and SEQ ID NO: 17 the nucleic acid encoding 43 amino acids is deleted from the sequence shown in SEQ ID NO: 1. It is preferable to use a sequence that is present in one of the primers and a sequence that is not present in the other (for example, a sequence of a deleted or added portion) as at least one of the primers, and more preferably as both of the primer pairs.
  • the GC content should be about 50% to ensure stable binding to type I DNA, so that GC-rich or AT-rich is not unevenly distributed in the primer.
  • the annealing temperature depends on the Tm (melting time immediately)
  • primers having a Tm value of 55-65 ° C and close to each other are selected. It is also necessary to take care to adjust the final concentration of the primer used in PCR to be about 0.1 to about 1 M.
  • commercially available software for primer design for example, Oligo TM [manufactured by National Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)] and the like can be used.
  • kit or diagnostic method of the invention can be performed using an array.
  • the method of executing the array is as described above.
  • the present invention provides a method for diagnosing cancer. This cancer diagnosis method
  • Samples used in this cancer diagnostic method include stool, urine, sweat, biopsy tissue, saliva, blood (eg, whole blood, serum, plasma, etc.), blood cells (monocytes, etc.), other body fluids
  • the diseased part can be targeted to cells and the like.
  • Samples from known unaffected sites or from other unaffected subjects can be used for comparison purposes.
  • such a sample can be a sample derived from a site similar to the target site of the subject in question. .
  • the cancer to be diagnosed is c—mv It is preferably, but not limited to, a high C-expressing cancer.
  • the FIR of the present invention has a high relationship with c-my c.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • Detection of the nucleic acid molecule can be performed using a factor specific to such a nucleic acid molecule. As such factors, any factors specific for the nucleic acid form of the FIR described herein can be used.
  • the present invention provides a method comprising: A) providing a sample from a subject portion of a subject of interest; B) providing a sample comprising the sequence set forth in SEQ ID NO: 1 or a complement thereof in the sample. Measuring the abundance of the nucleic acid molecule; C) determining the abundance of the second nucleic acid molecule and the abundance of the same nucleic acid molecule in a sample from a subject not having cancer or a portion not having cancer. Comparing, and if they are different, diagnosing the subject as being at high risk for cancer.
  • the second nucleic acid molecule containing the sequence represented by SEQ ID NO: 1 or its complement is normally expressed in normal tissues, and is known to cause apoptosis in cells that highly express c-myc.
  • the mutation eg, substitution, loss, addition, etc.
  • Such mutations may be a cause and indicator of apoptotic abnormalities (eg, cancer, etc.).
  • abnormalities in their normal expression levels may also be causes and indicators of abnormal apoptosis (eg, cancer). Therefore, detection of such an abnormal type and detection of an abnormal expression level of a normal type are useful as diagnostic indices of apoptosis abnormality.
  • the second factor can take any form as described herein above in the "FIR Nucleic Acid Form Specific Factors" section.
  • the second factor reacts specifically with the normal form of FIR and may or may not specifically react with its variant. Preferably, the second factor does not react with the variant.
  • the reaction specificity is understood and provided, It is understood that factors having specificity can also be used in the present invention, and thus such factors and kits containing them are within the scope of the present invention.
  • a factor specific to a portion specific to a normal form for example, a sequence at the 5 'end of SEQ ID NO: 1, for example, a sequence having a length of 100 nucleotides
  • the present invention provides a method comprising: A) providing a sample from a subject portion of a subject of interest; B) in said sample: (a) substituting, adding and substituting SEQ ID NO: 1 or its complement. A sequence containing at least one modification selected from the group consisting of deletions; or (b) at least one sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, or a complement thereof, or Detecting a nucleic acid molecule comprising at least one sequence selected from the group consisting of substitution, addition and deletion in one sequence or its complement; C) when the nucleic acid molecule is detected, Determining that the subject of the subject is a cancer patient.
  • Samples used in this cancer diagnostic method include stool, urine, sweat, biopsy tissue, saliva, blood (eg, whole blood, serum, plasma, etc.), blood cells (monocytes, etc.), other body fluids
  • cells in an affected part can be targeted.
  • Samples from known unaffected sites or from other unaffected subjects can be used for comparison purposes.
  • a sample from another unaffected subject preferably, such a sample can be a sample derived from a site similar to the target site of the subject in question.
  • the cancer to be diagnosed is preferably a c-myc high expression cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-my c.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon II.
  • the detection of the nucleic acid molecule can be performed using an agent specific to such a nucleic acid molecule.
  • factors include the book Any factor specific for the nucleic acid form of the FIR described herein can be used.
  • the abundance of the nucleic acid molecule is compared with the abundance of the same nucleic acid molecule in a sample from a subject not having cancer or a portion not having cancer. However, if the amount of the nucleic acid molecule in the sample from the target portion of the subject is increasing, it can be determined that the subject is a cancer patient.
  • the present invention provides a method comprising: A) providing a sample from a target portion of a subject of interest; B) determining an abundance of a second nucleic acid molecule and a third nucleic acid molecule in said sample.
  • the cancer to be diagnosed is preferably a c-myc high expression cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-myc.
  • examples of such a c-myc overexpressing cancer include, but are not limited to, rectal cancer and colon cancer.
  • the detection of the nucleic acid molecule can be performed using an agent specific to such a nucleic acid molecule. As such factors, any factors specific for the nucleic acid forms of the FIR described herein can be used.
  • the present invention provides a method comprising: A) providing a sample from a subject portion of a subject of interest; B) wherein said sample comprises: (a) a base sequence as set forth in SEQ ID NO: 1 or a sequence thereof. Polynucleotide having the complement or its fragment sequence (B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (c) one or more amino acids in the amino acid sequence of SEQ ID NO: 2 which are substituted, added and deleted.
  • (D) (a) a variant polypeptide having at least one mutation selected from the group consisting of: a polypeptide encoding a variant polypeptide having biological activity; A) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides of (c) to (c) and encodes a polypeptide having biological activity; or (e) a polynucleotide of (a) to (c).
  • a polypeptide consisting of a base sequence having at least 70% identity to any one of the polynucleotides or its complementary sequence, and having a biologically active polypeptide.
  • a nucleic acid molecule comprising: (a) at least one modification selected from the group consisting of substitution, addition and deletion in SEQ ID NO: 1 or its complement Or (b) at least one sequence selected from the group consisting of SEQ ID NOs: 11, 13 and 15 or its complement, or substitution, addition and deletion in the at least one sequence or its complement.
  • the step of measuring, wherein the ratio of the expression amount of the third nucleic acid molecule to the expression amount of the first nucleic acid molecule in a subject not having cancer or a portion not having cancer is represented by Determining that the subject is a cancer patient if the ratio of the expression amount of the third nucleic acid molecule to the expression amount of the first nucleic acid molecule in the target portion of the elephant subject is higher than the ratio of the expression amount of the third nucleic acid molecule.
  • a method for diagnosing cancer is provided.
  • the cancer to be diagnosed is preferably a c_myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention is highly related to c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • the above nucleic acid molecule Can be detected using factors specific for such nucleic acid molecules. As such factors, any factors specific for the nucleic acid forms of the FIR described herein can be used.
  • the present invention provides: (a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or its complement or a fragment thereof; (b) encoding the amino acid sequence of SEQ ID NO: 2 or a fragment thereof (C) a variant polypeptide having at least one mutation selected from the group consisting of substitution, addition, and deletion in the amino acid sequence of SEQ ID NO: 2, wherein one or more amino acids are substituted; A polynucleotide encoding a variant polypeptide having biological activity; hybridizing to any one of the polynucleotides (d), (a) to (c) under stringent conditions; A polynucleotide encoding a polypeptide having biological activity; or a polynucleotide of any one of (e), (a) to (c) A polynucleotide comprising a nucleotide sequence having at least 70% identity to leotide or its complementary sequence, and a polynucleotide
  • the cancer to be diagnosed is preferably a cancer with high expression of c-myc, but is not limited thereto.
  • the FIR of the present invention is highly related to c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • the detection of the nucleic acid molecule can be performed using a factor specific to such a nucleic acid molecule. As such factors, any factors specific for the nucleic acid form of the FIR described herein can be used.
  • the present invention provides: A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 (encoding FIR 542) or its complement or a fragment thereof; (b) SEQ ID NO: 2 A polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (c) in the amino acid sequence of SEQ ID NO: 2, wherein at least one amino acid is selected from the group consisting of substitution, addition, and deletion (D) a variant polypeptide having three mutations, wherein the polynucleotide encodes a variant polypeptide having biological activity; (d) any one of (a) to (c) being a stringent polynucleotide A polynucleotide that hybridizes under a variety of conditions and encodes a polypeptide having biological activity; or (e) any of (a) to (c) A cancer therapeutic agent comprising a nucleotide sequence of SEQ ID NO: 1 (encoding FIR 542) or its
  • the cancer to be treated is preferably c-myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-myc.
  • examples of such cancers with high expression of c-myc include, but are not limited to, rectal cancer and colon cancer.
  • the nucleic acid form of such a FIR any nucleic acid molecule as described herein can be used.
  • a nucleic acid molecule capable of expressing a normal form of FIR is used.
  • the nucleic acid molecule contained in the therapeutic agent of the present invention includes a nucleic acid molecule encoding a polypeptide (B polypeptide) comprising the sequence shown in SEQ ID NO: 2.
  • the present invention provides a cancer treatment method using the above-mentioned cancer treatment agent. This treatment can use any of the treatment techniques described herein. (Polypeptide form of FIR)
  • the present invention provides a method for expressing c-myc, such as cancer (eg, rectal cancer or colon cancer (collectively referred to as colorectal cancer)), which comprises a polypeptide form of FIR.
  • Composition for the treatment, prevention, diagnosis or prognosis of cancer can be determined by a person skilled in the art using techniques well known in the art while considering various parameters, and such an amount is determined.
  • the person skilled in the art can easily determine the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc.
  • the present invention provides: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (b) an amino acid sequence of SEQ ID NO: 2; A polypeptide wherein the amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion, and has biological activity; (C) a splice variant of the nucleotide sequence of SEQ ID NO: 1. Or a polypeptide encoded by an allelic variant; A polypeptide which is a species homolog of the amino acid sequence described in SEQ ID NO: 2; or (e) has an amino acid sequence having at least 70% identity to any one of the polypeptides (a) to (d). And a polypeptide having biological activity.
  • the number of substitutions, additions and deletions in (b) above may be limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less. Hereafter, it is preferably 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • a smaller number of substitutions, additions and deletions is preferred, but retains biological activity (preferably similar or substantially identical to a normal FIR consisting of the sequence shown in SEQ ID NO: 2. As long as it has an activity or is an abnormal type of FIR activity (eg, poor binding to FBP), the number may be as large as possible.
  • the splice variant or allelic variant in (c) above preferably has at least 99% homology with the amino acid sequence shown in SEQ ID NO: 2.
  • the species homolog can be identified as described herein above, and preferably has at least about 30% homology with the amino acid sequence set forth in SEQ ID NO: 2.
  • the species homolog is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95% 98% may be homologous.
  • the species homolog can be identified by searching the database of the gene sequence of the species, if available, using the FIR of the present invention (or more preferably, FIR 542) as a query sequence. it can.
  • the gene library of the species is screened using all or a part of the FIR (or more preferably, FIR 542) of the present invention as a probe or primer.
  • the species homolog preferably has, for example, at least about 30% homology with the nucleic acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2.
  • the species homolog is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 90%, About 95%, at least about 98%, can be homologous.
  • the biological activity of the variant polypeptide in the above (e) is, for example, a specific activity for a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.
  • the ability to bind to FBP is defined by the sequence described at positions 372-442 in SEQ ID NO: 2, and can be measured using FBP.
  • the homology to the polypeptide of any one of (a) to (d) above may be at least about 80%, more preferably at least about 90%, Preferably it can be at least about 98%, and more preferably at least about 99%.
  • the FIR polypeptide of the invention is FIR542.
  • the polypeptides of the present invention usually have at least three consecutive amino acid sequences.
  • the amino acid length of the polypeptide of the present invention may be any length as long as it is suitable for the intended use, but preferably, a longer sequence may be used. Thus, it may be preferably at least 4 amino acids long, more preferably at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long. More preferably it can be at least 15 amino acids long, and still more preferably Can be at least 20 amino acids in length.
  • the lower limit of these amino acid lengths is not limited to the specific numbers, but also numbers between them (for example, 11, 12, 13, 13, 14, 16, etc.) or larger numbers (for example, , 21, 22,... 30, etc.).
  • its upper limit may be the same as the full length of the sequence shown in SEQ ID NO: 2, or may be longer than that. Good.
  • the FIR in polypeptide form of the invention is labeled or can be labeled. This is because the amount of an antibody against FIR in the body can be measured by using such a labeled or capable of being labeled FIR, whereby the expression level of FIR can be measured indirectly.
  • the present invention provides a polypeptide comprising the sequence shown in SEQ ID NO: 2.
  • a sequence is also expressed in normal tissues and can be used as an indicator of normality.
  • the expression level is abnormal, it can be determined that there is a risk of developing cancer.
  • Such a normal FIR polypeptide form can cause apoptosis to c-myc-expressing cells by external administration, and thus can be used as an anticancer agent.
  • the present invention provides an amino acid sequence comprising: (a) an amino acid sequence comprising at least one modification selected from the group consisting of substitution, addition and deletion in SEQ ID NO: 2; (b) SEQ ID NO: 1 At least one sequence selected from the group consisting of 2, 14 and 16 or a sequence comprising at least one modification selected from the group consisting of substitution, addition and deletion in the at least one sequence.
  • SEQ ID NO: 1 At least one sequence selected from the group consisting of 2, 14 and 16 or a sequence comprising at least one modification selected from the group consisting of substitution, addition and deletion in the at least one sequence.
  • a repeptide Is such a sequence not or rarely expressed in normal tissues and specifically expressed in cancer tissues? Thus, the expression itself can be used as an index for cancer diagnosis. Since all of these polypeptides are specific to cancer tissues, they can be used as a definitive diagnosis of cancer.
  • a definitive diagnosis of cancer can be made by comparing their expression with expression in tissues not affected by cancer.
  • a cancer is preferably a c-myc overexpressing cancer (including, for example, rectal cancer and / or colon cancer).
  • Factors that reduce such polypeptides could potentially be used as therapeutics. Therefore, it is understood that such factors are also within the scope of the present invention.
  • the alteration in the polypeptide of the invention is a substitution; between positions 102 and 103 of SEQ ID NO: 2 or positions 59 and 60 of SEQ ID NO: 16. Or a deletion up to position 102 of SEQ ID NO: 2 or SEQ ID NO: 12 or a position up to position 59 of SEQ ID NO: 14 or SEQ ID NO: 16; or a combination thereof. . While not wishing to be bound by theory, it is believed that such specific modifications alter or preferably exacerbate the normal function of the FIR of the present invention, thereby causing or causing cancer cells to become abnormal. possible. Therefore, by detecting a polypeptide having or containing such a specific sequence, it has utility that it can be used for cancer diagnosis.
  • preferred modifications in the FIR polypeptides of the invention are in the range from the N-terminus of SEQ ID NO: 2 to about 135 amino acids (this is also referred to as the TFIIH binding site. M olecular Ce 11 Vol. 5, 3 3 1-3 4 1. This is because modification of this range is considered to have a deep relationship with cancer, but is not limited thereto. More preferably, the modification may be in the range of about 50 to about 120 in SEQ ID NO: 2. Cases in which alterations in this range are considered to be more relevant to cancer However, it is understood that the present invention is not limited thereto. Therefore, it is understood that any modification that causes abnormal expression or dysfunction of FIR can be used in the diagnosis of the present invention.
  • the modification in the FIR polypeptide of the invention comprises a non-conservative substitution in SEQ ID NO: 2. This is because including such a non-conservative substitution may cause an abnormality in the function of the FIR polypeptide.
  • One of the normal functions of FIR is to induce apoptosis in c-myc expressing cells, and that its function is abnormal or that there are molecules that inhibit normal FIR function. It is thought to be closely related to the onset of cancer.
  • the FIR polypeptide of the present invention comprising a modification is selected from the group consisting of SEQ ID NOS: 12, 14, 16, 18, 20, and 22. Sequence. Since these specific sequences are not expressed in normal tissues but are specifically expressed in cancer tissues, they can be exemplified as specific sequences of the modified FIR of the present invention. Is not limited to them.
  • the invention includes cancers (eg, rectal or colon cancer (collectively referred to as colorectal cancer)) that include factors specific for the polypeptide form of the FIR.
  • cancers eg, rectal or colon cancer (collectively referred to as colorectal cancer)
  • the present invention provides agents specific for any of the FIR polypeptides described herein or variants or fragments thereof.
  • the amount effective for diagnosis, prevention, treatment or prognosis can be determined by a person skilled in the art using techniques well known in the relevant field while taking into consideration various parameters. For example, the purpose of use, target disease (type, severity, etc.), patient's age, weight, gender, medical history, cell morphology, etc.
  • the present invention has revealed that the onset of cancer is unexpectedly associated with abnormal expression of FIR, and that administration of normal FIR can cure cancer. Such an association, and the diagnostic and therapeutic effects utilizing it, have not been known in the art, and thus the factors of the present invention show superior effects to those provided by the prior art.
  • the agent of the present invention may be an agent selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof. It can be understood that such factors may be any that specifically bind to the polypeptide of the present invention. More preferably, the agent of the present invention is an antibody or a derivative thereof (eg, a single-chain antibody). Accordingly, the agents of the present invention can be used as probes and Z or inhibitors.
  • the agent of the invention may be labeled or capable of binding to a label.
  • a label can be any label as long as it is identifiably labeled, including techniques such as fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme-substrate reactions, and antigen-antibody reactions. Not limited.
  • an immune reaction such as an antibody
  • a system often used in an immune reaction such as biotin-streptavidin may be used.
  • the agent of the present invention comprises: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (b) one or more of the amino acid sequences of SEQ ID NO: 2, Amino acids have substitutions, additions and A polypeptide having at least one mutation selected from the group consisting of deletion and having biological activity; (c) a splice variant or allelic variant of the nucleotide sequence of SEQ ID NO: 1 Encoded by a polypeptide;
  • polypeptide (d) a polypeptide which is a species homolog of the amino acid sequence of SEQ ID NO: 2; or (e) has at least 70% identity to the polypeptide of any one of (a) to (d).
  • the polypeptide may be a factor (for example, an antibody or a derivative thereof) having an amino acid sequence of 1% and having biological activity, including a polypeptide.
  • the factor of the present invention is preferably a factor specific to the polypeptide represented by SEQ ID NO: 2.
  • SEQ ID NO: 2 Such a sequence is useful for examining the degree of canceration of a certain tissue or individual, since the form of FIR existing in normal tissues can be identified.
  • the factor specific to the polypeptide comprising the sequence represented by SEQ ID NO: 2 is (a) an amino acid comprising at least one modification selected from the group consisting of substitution, addition and deletion in SEQ ID NO: 2 (B) at least one sequence selected from the group consisting of SEQ ID NOs: 12, 14, and 16, or at least one selected from the group consisting of substitution, addition and deletion in at least one of the sequences;
  • it does not react specifically to polypeptides containing sequences containing one modification.
  • a variant of FIR and normal FIR can be discriminated and detected.
  • Such detection will provide an accurate diagnosis of cancer (particularly c-myc overexpressing cancer, including (rectal or colon cancer, collectively referred to as colorectal cancer)) that was previously impossible. To achieve excellent effects and usefulness.
  • the agent of the present invention comprises: (a) an amino acid sequence comprising at least one modification selected from the group consisting of substitution, addition and deletion in SEQ ID NO: 2; (b) SEQ ID NO: 1 Select from the group consisting of 2, 14 and 16 It is preferable to specifically react with a polypeptide comprising at least one sequence to be obtained or a sequence containing at least one modification selected from the group consisting of substitution, addition and deletion in the at least one sequence.
  • SEQ ID NO: 1 Select from the group consisting of 2, 14 and 16 It is preferable to specifically react with a polypeptide comprising at least one sequence to be obtained or a sequence containing at least one modification selected from the group consisting of substitution, addition and deletion in the at least one sequence.
  • the use of an agent specific for such a variant provides the utility of facilitating the detection of an individual or tissue portion affected by cancer. This factor advantageously does not specifically react with a polypeptide comprising the sequence shown in SEQ ID NO: 2.
  • a variant FIR By reacting in a discriminable manner in this manner, a variant FIR can be discriminated from a normal FIR and detected. Such detection may provide an accurate diagnosis of cancer (particularly cancers that overexpress c-myc, including colorectal or colon cancer (collectively referred to as colorectal cancer)), which was previously impossible. Achieve the superior effect and usefulness of being able to do.
  • the present invention provides: A) (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (b) one or more amino acids in the amino acid sequence of SEQ ID NO: 2 A polypeptide having at least one mutation selected from the group consisting of addition and deletion, and having biological activity; (c) a splice variant or allele of the nucleotide sequence of SEQ ID NO: 1.
  • a polypeptide encoded by the mutant comprising a polypeptide (A polypeptide) An abnormal factor A; and B) a means for measuring the expression level of the polypeptide using a signal caused by the factor A, wherein the subject has no cancer or has cancer.
  • Means for diagnosing the subject as being at high risk for cancer when the expression of the repeptide is high comprising:
  • the cancer to be diagnosed is preferably, but is not limited to, c-myc highly expressing cancer.
  • the FIR of the present invention has a high relationship with c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • any factor as described herein can be used. Factor A may specifically react with either the normal type or variant of FIR, or may specifically react with either the normal type or variant of FIR.
  • any factor having any specificity can be used in the present invention. It is understood that it is within the scope of the present invention.
  • a common sequence between the normal type and the variant for example, a C-terminal sequence such as SEQ ID NO: 2, 12, 14, 16, 16, 18, 20, 22
  • a sequence having a length of 30 amino acids for example, a sequence having a length of 30 amino acids.
  • the A polypeptide itself can be used as the A factor.
  • the A polypeptide since the antibody specific to the A polypeptide can be recognized, the amount of the antibody specific to the A polypeptide in the living body is calculated, and thereby, the amount of the A polypeptide in the body of the A polypeptide is determined. This is because it is possible to identify the presence of the compound.
  • the measurement of the expression level of the polypeptide can be performed using any signal caused by factor A.
  • the present invention provides: A) a factor B specific for a polypeptide comprising the sequence set forth in SEQ ID NO: 2 (B polypeptide); and B) a signal resulting from said factor B Means for measuring the expression level of said polypeptide by using a subject not having cancer or a portion not having cancer.
  • a cancer diagnostic kit for diagnosing that the subject is a high-risk person for cancer.
  • the cancer to be diagnosed is preferably c-myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-my c.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • any factor as described herein can be used. Any factor B may be used as long as it specifically reacts with normal FIR. Preferably, it may be advantageous that factor B does not react specifically with the variant. As long as it has such properties, any factor B can be provided by understanding its reaction specificity, and any factor having any specificity can be used in the present invention. It is understood that such factors and kits containing them are within the scope of the present invention.
  • the sequence is specific for a modified sequence between a normal type and a variant (for example, an N-terminal sequence of SEQ ID NO: 2, for example, any sequence having a length of about 135 amino acids).
  • Factors but are not limited thereto.
  • the B polypeptide itself can be used as the factor B.
  • the antibody specific to B polypeptide can be recognized, the amount of antibody specific to B polypeptide in the living body is calculated. This is because it is possible to identify the presence in the body.
  • the measurement of the expression level of the polypeptide can be performed using any signal caused by factor B.
  • the present invention provides: A) a factor C specific for a polypeptide comprising the sequence set forth in SEQ ID NO: 2 (C polypeptide); and B) a signal resulting from said factor C
  • a cancer diagnostic kit comprising: means for determining that the subject is a cancer patient if the polypeptide is detected in a target portion of the subject.
  • the detection of the polypeptide in the target portion of the subject is performed by comparing the expression level of the polypeptide in the subject not afflicted with the cancer or the portion not afflicted with the cancer.
  • detecting high expression of the polypeptide in the subject portion of the subject is performed by comparing the expression level of the polypeptide in the subject not afflicted with the cancer or the portion not afflicted with the cancer.
  • the cancer to be diagnosed is preferably c-myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention is highly related to c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • any factor as described herein can be used.
  • Any factor C may be used as long as it specifically reacts with the variant of FIR.
  • it may be advantageous that factor C does not react specifically with the FIR normal form.
  • any factor C can be provided by understanding its reaction specificity, and any factor having any specificity can be used in the present invention.
  • the modified sequence between the normal type and the variant is specific.
  • Factors include, but are not limited to.
  • the C polypeptide itself can be used as the C factor.
  • the amount of the antibody specific to the C polypeptide in the living body is calculated. It is because it is possible to identify the existence.
  • the measurement of the expression level of the polypeptide can be performed using any signal derived from factor C.
  • the invention relates to a factor B specific for a polypeptide comprising the sequence set forth in SEQ ID NO: 2 (B polypeptide); B) (a) in SEQ ID NO: 2 An amino acid sequence containing at least one modification selected from the group consisting of additions and deletions; (b) at least one sequence selected from the group consisting of SEQ ID NOs: 12, 14, and 16 or a complement thereof, or A C factor specific for a polypeptide comprising a sequence comprising at least one modification selected from the group consisting of substitutions, additions and deletions in said at least one sequence (C polypeptide); and C)
  • This is a means for discriminatively detecting a signal caused by the factor B and the factor C and measuring the expression levels of the B polypeptide and the C polypeptide, respectively.
  • Expression of the B polypeptide in the target portion of the subject compared to the expression of the B polypeptide in the subject or in a portion not having the cancer, and in some subjects not having the cancer Indicates that the expression of the C polypeptide in the target portion of the subject is higher than the expression of the C polypeptide in the portion not afflicted with cancer.
  • the present invention provides a diagnostic kit for cancer (for example, rectal cancer or colon cancer (collectively referred to as colorectal cancer)), which comprises means for determining that the body is a cancer patient.
  • a diagnostic kit for cancer for example, rectal cancer or colon cancer (collectively referred to as colorectal cancer)
  • colorectal cancer for example, rectal cancer or colon cancer (collectively referred to as colorectal cancer)
  • the C) means includes a means for comparing the relative abundance ratio between the B polypeptide and the C polypeptide.
  • factor B does not specifically react to the C polypeptide, or that factor C does not specifically react to the B polypeptide. This is because both polypeptides can be detected in a distinguishable manner.
  • both factor B does not specifically react with the C polypeptide and that factor C does not specifically react with the B polypeptide. This is because the signal intensity detected by these factors is accurately reflected by the amount of the target polypeptide.
  • these factor B and factor C are or are capable of being distinguishably labeled.
  • a factor that is identifiably labeled means that the factor already has an identifiable label, and that two or more factors can be identifiably labeled. Performing the above processing on the factor means that two or more factors can be identified.
  • the kit of the present invention further includes a factor that discriminately labels factor B and factor C.
  • the identification of such identifiable labels can be in physical form (eg, fluorescence, luminescence, etc.), chemical form (eg, specific chemical cleavage, staining, etc.), biochemical form (eg, enzymatic reaction).
  • the biological mode eg, determination of the survival of microorganisms).
  • the present invention provides: A) a polypeptide consisting of (a) the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (b) the amino acid sequence of SEQ ID NO: 2 A polypeptide, wherein the above amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion, and has biological activity; and (C) a splice of the base sequence of SEQ ID NO: 1.
  • a polypeptide B (a) an amino acid sequence comprising at least one modification selected from the group consisting of substitution, addition and deletion in SEQ ID NO: 2; (b) SEQ ID NO: 12, 1 And at least one sequence selected from the group consisting of 4 and 16 or a sequence comprising at
  • C polypeptide Factor C specific to peptide
  • C Distinguishes the factor A and the signal caused by the factor C Means for measuring the expression level of each of the A polypeptide and the B polypeptide, wherein the A polypeptide in a subject not having cancer or a portion not having cancer is detected. If the ratio of the expression level of the C polypeptide to the expression level of the C polypeptide is higher than the ratio of the expression level of the C polypeptide to the expression level of the A polypeptide in the target portion of the subject, Provided is a cancer diagnostic kit comprising: means for confirming that the patient is a patient.
  • the cancer to be diagnosed is preferably c-myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-myc.
  • Such high c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • factor A is specific for the A polypeptide and factor C is specific for the C polypeptide. Since the A polypeptide can include both normal forms of the FIR and variants thereof, the total amount of FIR-related molecules can be detected. Thus, preferably, factor A is advantageously capable of specifically reacting with both normal and variant forms of FIR.
  • the C polypeptide is a variant of FIR and can be used as an indicator of cancer because it is specifically present in cancer tissues.
  • the above-mentioned means C) preferably includes means for comparing the relative abundance or absolute abundance with the C polypeptide.
  • the relative abundance ratio can be calculated by directly comparing the signal (eg, fluorescence intensity) due to the factor. Therefore, it is preferable in such a situation because it is not necessary to calculate the absolute amount.
  • a calibration curve can be created using each factor based on a known amount of a standard sample, and the absolute amount can be calculated accordingly. Such methods are well-known in the art.
  • factor A does not specifically react with the C polypeptide, or factor A reacts specifically with the B or C polypeptide. It can be advantageous. When the factor A does not specifically react with the C polypeptide, the factor A and the factor C can be used to detect the abundance ratio between a specific FIR variant and a non-specific FIR molecule. Alternatively, when factor A specifically reacts with both C polypeptide and B polypeptide, the total amount of FIR expression and the expression amount of variant can be compared.
  • Factor A and Factor C are or are differentially labeled. In such a case, the kit of the present invention further includes a factor that discriminately labels factor A and factor C.
  • Identification of such identifiable labels can be in a physical format (eg, fluorescence, luminescence, etc.), a chemical format (eg, specific chemical cleavage, staining, etc.), a biochemical format (eg, enzyme reaction). ), Biological modes (eg, determination of the survival of microorganisms), and the like.
  • a physical format eg, fluorescence, luminescence, etc.
  • a chemical format eg, specific chemical cleavage, staining, etc.
  • a biochemical format eg, enzyme reaction
  • Biological modes eg, determination of the survival of microorganisms
  • the present invention provides: A) providing a sample from a target portion of a subject; B) comprising in the sample: (a) an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof; (B) in the amino acid sequence of SEQ ID NO: 2, one or more amino acids have at least one mutation selected from the group consisting of substitution, addition and deletion, and have a biological activity A polypeptide; (c) a polypeptide encoded by a splice variant or an allelic variant of the nucleotide sequence of SEQ ID NO: 1; (d) a species homolog of the amino acid sequence of SEQ ID NO: 2; A polypeptide; or (e) having an amino acid sequence having at least 70% identity to the polypeptide of any one of (a) to (d), and having a biological activity.
  • Measuring the abundance of the polypeptide comprising: C) the abundance of the polypeptide and a subject not having cancer or a portion not having cancer. Comparing the abundance of the same nucleic acid molecule in the sample from the subject, and if the amount of the polypeptide in the sample from the target portion of the subject is increased, the subject in the subject is at high risk of cancer Providing a method for diagnosing cancer, comprising: Offer.
  • Samples used in this cancer diagnosis method include stool, urine, sweat, biopsy tissue, saliva, blood (eg, whole blood, serum, plasma, etc.), blood cells (eg, mononuclear cells), other body fluids, Cells in the affected part can be targeted.
  • Samples from apparently unaffected sites or from other unaffected subjects can be used for comparison purposes.
  • a sample from another unaffected subject preferably, such a sample can be a sample derived from a site similar to the target site of the subject in question.
  • the cancer to be diagnosed is preferably c-myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-my c.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • Detection of the polypeptide can be performed using a factor (for example, factor A) specific to such a polypeptide (A polypeptide).
  • any factors specific for the polypeptide form of the FIR described herein can be used. Any factor A may be used as long as it specifically reacts with FIR. Preferably, factor A may advantageously respond specifically to both the normal FIR and its variants. Alternatively, factor A may be specific for the variant alone or only for the normal form. As long as it has such properties, any factor A can be provided with understanding of its reaction specificity, and any specific factor can be used in the present invention. It is understood that such factors and kits containing them are within the scope of the present invention.
  • the sequence is specific for a modified sequence between a normal type and a variant (for example, a sequence on the N-terminal side of SEQ ID NO: 2, for example, any sequence of about 135 amino acids in length).
  • Factors include, but are not limited to.
  • the invention provides a method comprising: A) providing a sample from a subject portion of a subject of interest; B) a polypeptide comprising the sequence set forth in SEQ ID NO: 2 (B polypeptide) in said sample. Measuring the abundance of the peptide); C) comparing the abundance of the polypeptide with the abundance of the same nucleic acid molecule in a sample from a subject without cancer or a part without cancer.
  • the polypeptide containing the sequence represented by SEQ ID NO: 2 is normally expressed in normal tissues, and is known to cause apoptosis in cells that highly express c-myc. , Substitution, loss, addition, etc.) can impair normal functioning.
  • mutations may be a cause and indicator of apoptotic abnormalities (eg, cancer, etc.).
  • the abnormal expression level of the normal form may also be a cause and an indicator of apoptosis abnormality (for example, cancer).
  • any factor eg, factor B
  • Factor B can be in any form as described herein above in the section “FIR Polypeptide Form-Specific Factors” section.
  • Factor B reacts specifically with normal FIR and may or may not react specifically with its variant.
  • the second factor does not react with the variant.
  • any factor having any specificity can be used in the present invention as long as the reaction specificity is understood and provided, and therefore, such a factor and a kit containing the same can be used. It is understood that it is within the scope of the present invention.
  • a portion specific to a normal type for example, a sequence at the 5 ′ terminal side of SEQ ID NO: 1; (Such as a sequence of a nucleotide length), but not limited thereto.
  • the invention provides a method comprising: A) providing a sample from a subject portion of a subject of interest; B) in said sample: (a) substitutions, additions and deletions at An amino acid sequence comprising at least one modification selected from the group consisting of: (b) at least one sequence selected from the group consisting of SEQ ID NOs: 12, 14, and 16, or a substitution in the at least one sequence; Detecting a polypeptide (C polypeptide) containing a sequence containing at least one modification selected from the group consisting of additions and deletions; C) if the polypeptide is detected, the subject is cancer
  • a method for diagnosing cancer comprising: determining a patient.
  • Samples used in this cancer diagnostic method include stool, urine, sweat, biopsy tissue, saliva, blood (eg, whole blood, serum, plasma, etc.), blood cells (monocytes, etc.), other body fluids, Cells in the affected part can be targeted. Samples from sites known to be unaffected or from other unaffected subjects can be used for comparison purposes. When a sample from another unaffected subject is used, preferably, such a sample can be a sample derived from a site similar to the target site of the subject.
  • the cancer to be diagnosed is preferably a cancer with high expression of c-myc, but is not limited thereto.
  • the FIR of the present invention is highly related to c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • Detection of the polypeptide can be performed using factors specific for such polypeptide.
  • any factor eg, factor C
  • the subject can be to be determined to be a cancer patient.
  • the present invention provides a method comprising: A) providing a sample from a subject portion of a subject of interest; B) measuring the abundance of B and C polypeptides in said sample. C) comparing the abundance of the B polypeptide with the abundance of the same polypeptide in a sample from a subject not afflicted with cancer or a portion not afflicted with cancer; Increased expression of the B polypeptide in samples from, and the presence of C polypeptide in samples from non-cancer-free subjects or cancer-free parts Comparing the abundance of the same polypeptide, if the expression level of C polypeptide is increased in the sample from the target portion of the subject, it is determined that the subject is a cancer patient Do, process, cancer (for example, the present invention provides a method for diagnosing rectal cancer or colon cancer (collectively referred to as colorectal cancer), wherein the cancer to be diagnosed is preferably c-myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-myc.
  • examples include, but are not limited to, rectal cancer and colon cancer
  • the detection of the polypeptide can be performed using factors specific for such polypeptides. Any factor specific for the polypeptide form of the FIR described herein can be used.
  • the present invention provides: A) a step of providing a sample from a target portion of a subject; B) in the sample, (a) comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (B) a group consisting of substitution, addition and deletion of one or more amino acids in the amino acid sequence of SEQ ID NO: 2; A polypeptide having at least one mutation selected from the group consisting of: a polypeptide having biological activity; and (c) a polypeptide encoded by a splice variant or allelic variant having the nucleotide sequence of SEQ ID NO: 1.
  • C) A polypeptide and C polypeptide A step of measuring the expression level of each of the polypeptides, wherein the ratio of the expression level of the C polypeptide to the expression level of the A polypeptide in a subject not having cancer or a portion not having cancer Determining that the subject in the subject is a cancer patient if the ratio is higher than the ratio of the expression level of the C polypeptide to the expression level of the A polypeptide in the subject portion of the subject.
  • the cancer to be diagnosed is preferably c-myc high-expressing cancer, but is not limited thereto.
  • the FIR of the present invention has a high relationship with c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer.
  • Detection of the polypeptide can be performed using factors specific for such polypeptides. As such factors, any factors specific for the polypeptide form of the FIR described herein can be used.
  • the invention provides a polypeptide comprising: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (b) one or more amino acids of the amino acid sequence of SEQ ID NO: 2 A polypeptide having at least one mutation selected from the group consisting of substitution, addition and deletion and having biological activity; (c) a splice variant of the nucleotide sequence of SEQ ID NO: 1.
  • a polypeptide encoded by an allelic variant comprising: Provides use for build-in.
  • the cancer to be diagnosed is preferably, but not limited to, a cancer with high expression of c-myc.
  • the FIR of the present invention is highly related to c-myc.
  • Such c-myc overexpressing cancers include, but are not limited to, for example, rectal cancer and colon cancer. Detection of the polypeptide can be performed using factors specific for such polypeptide. As such factors, any factors specific for the polypeptide form of the FIR described herein can be used.
  • the antigenic peptide of the present invention is a peptide that binds to an antibody (IgG) in the serum of a patient, it is reacted with the serum of the subject, and the serum containing the antibody that binds to these antigenic peptides is used as a cancer. It can be determined as the serum of the patient or his high-risk patient. In this case, it is preferable to determine the binding of the two types of antigen peptides to antibodies in serum.
  • the serum of the subject is brought into contact with the antigen peptide, and the antigen peptide is reacted with the IgG antibody in the serum of the subject in a liquid phase.
  • a signal of the labeled IgG antibody may be detected by reacting a labeled IgG antibody that specifically binds to the IgG antibody in the serum.
  • a labeling substance of the labeled IgG antibody an enzyme, a radioisotope or a fluorescent dye as exemplified in the above-mentioned labeled antibody can be used.
  • the enzyme activity is determined by optically measuring the amount of decomposition of the substrate, and this is converted to the amount of bound antibody, and compared with the standard value. Is used to calculate the amount of antibody.
  • radioactive isotopes measure the radiation dose emitted by the radioisotope using a scintillation counter or the like.
  • the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope.
  • Western blot analysis as described in the Examples can be employed.
  • the conjugate of the antigen peptide + serum antibody + labeled IgG antibody is separated by known separation means (chromatography, salting out, alcohol precipitation, enzymatic method, solid phase method, etc.), and the signal of the labeled IgG antibody is separated. May be detected.
  • the diagnostic kit of the present invention is provided as a method that enables simple and wide-ranging implementation of such a diagnostic method.
  • the diagnostic method of the present invention also includes a method of immobilizing one or more antigen peptides on a plate and testing the binding of the test subject's serum to the antibody on the substrate (this is also included in the present invention). ). By immobilizing the antigen peptide on the substrate, unbound labeled binding molecules can be easily removed.
  • an embodiment of the diagnostic kit of the present invention is provided as enabling such a diagnostic method to be performed simply and widely.
  • the diagnostic method of the present invention tests whether a biological sample of a subject contains an antibody of the present invention or an antigen peptide that binds to the labeled antibody of the present invention, A subject whose antigen peptide is present in the sample is determined to be a cancer patient or a high-risk subject thereof. That is, since the antibody or labeled antibody used here is an antibody that specifically binds to an antigen peptide expressed in colorectal cancer cells, a biological sample containing the antigen peptide that binds to this antibody is used for colorectal cancer patients. Alternatively, it can be determined as a sample of a colorectal cancer high-risk patient.
  • the diagnostic method of the present invention is a method of binding an antibody and an antigen peptide in a liquid phase system.
  • the labeled antibody of the present invention is brought into contact with a biological sample to bind the labeled antibody and the antigen peptide, the conjugate is separated by the method described herein, and the labeled signal is obtained by the same method.
  • the diagnostic kit of the present invention is provided as one that enables simple and wide-ranging implementation of such a diagnostic method.
  • Another method of diagnosis in a liquid phase system is to contact the antibody (primary antibody) of the present invention (primary antibody) with a biological sample to bind the primary antibody and the antigen peptide, and to the conjugate, the labeled antibody (secondary antibody) of the present invention (secondary antibody). Antibody), and the labeled signal of the conjugate is detected.
  • an unlabeled secondary antibody may first be bound to the antibody + antigen peptide conjugate, and a labeling substance may be bound to this secondary antibody.
  • binding of the labeling substance to the secondary antibody can be carried out, for example, by biotinylating the secondary antibody and avidinizing the labeling substance.
  • an antibody (tertiary antibody) recognizing a partial region (for example, Fc region) of the secondary antibody may be labeled, and the tertiary antibody may be bound to the secondary antibody.
  • the primary antibody and the secondary antibody both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be performed as described herein.
  • a diagnostic method is simple and broad.
  • One embodiment of the diagnostic kit of the present invention is provided to enable a wide range of implementations.
  • Another aspect of the diagnostic method of the present invention is a method for testing the binding between an antibody and an antigen peptide in a solid phase system.
  • This method using a solid phase system is a preferable method for detecting a trace amount of antigen peptide and simplifying the operation. That is, in this solid phase method, the antibody (primary antibody) of the present invention is immobilized on a resin plate or the like, the antigen peptide is bound to the immobilized antibody, and the unbound peptide is washed off, and then left on the plate. In this method, a labeled antibody (secondary antibody) is bound to the antibody + antigen peptide conjugate, and the signal of this secondary antibody is detected. This method is a so-called “sandwich method”.
  • the method is widely used as an enzyme-linked immunosorbent assay (I ELISA).
  • I ELISA enzyme-linked immunosorbent assay
  • the primary antibody and the secondary antibody both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Detection of the signal can be performed as described herein. Further, an embodiment of the diagnostic kit of the present invention is provided as enabling simple and wide-ranging implementation of such a diagnostic method.
  • the diagnostic kit of the present invention can be a reagent kit for performing the diagnostic method of the present invention. Various such kits are commercially available depending on the type of the test component.
  • the diagnostic kit of the present invention also uses the antigenic peptide, antibody and Z or labeled antibody provided by the present invention. Except for the above, it can be constituted by each element used in a kit known in the art.
  • kits or diagnostic method of the invention can be performed using an array.
  • the method of executing the diagnosis using the array is as described above.
  • the present invention provides: A) a polypeptide consisting of (a) the amino acid sequence of SEQ ID NO: 2 or a fragment thereof; (b) one or more amino acids in the amino acid sequence of SEQ ID NO: 2 A polypeptide having at least one mutation selected from the group consisting of addition and deletion, and having biological activity; (c) a splice variant or allele of the nucleotide sequence of SEQ ID NO: 1.
  • a polypeptide encoded by the variant (d) a polypeptide that is a species homolog of the amino acid sequence set forth in SEQ ID NO: 2, or (e) a polypeptide of any one of (a) to (d).
  • a polypeptide having an amino acid sequence of at least 70% identity and having biological activity including a polypeptide, a polypeptide (A polypeptide), a cancer therapeutic agent
  • the cancer to be treated is preferably c-myc high-expressing cancer, but is not limited thereto.
  • Such cancers with high expression of c-myc include, for example, rectal cancer and colon cancer, which are not determined.
  • an FIR-specific factor any factor as described herein can be used.
  • the A polypeptide has at least one of the FIR normal functions, and preferably has the ability to bind to FBP and the activity of inhibiting C or myc.
  • a molecule represented by SEQ ID NO: 2, 12, 14, 16, 18, 20, 22, or a variant thereof, which has a function equivalent to SEQ ID NO: 2 Can be.
  • the polypeptide contained in the therapeutic agent of the present invention includes a polypeptide (B polypeptide) containing the sequence shown in SEQ ID NO: 2.
  • the present invention provides a cancer treatment method using the above-mentioned cancer treatment agent.
  • This treatment can use any of the treatment techniques described herein. (CENP-A nucleic acid form)
  • the present invention relates to c-myc overexpressing cancers, including cancers (eg, rectal cancer or colon cancer (collectively referred to as colorectal cancers)) comprising a nucleic acid molecule encoding CENP-A.
  • a diagnostically, prophylactically, therapeutically or prognostically effective amount can be determined by those skilled in the art using techniques well known in the art while considering various parameters. Can be easily determined by those skilled in the art, taking into account, for example, the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, etc.
  • the present invention has revealed that the onset of cancer is unexpectedly associated with abnormal expression of CENP-A. Such a relationship, as well as the diagnostic and therapeutic effects utilizing it, have not been known so far. Therefore, the nucleic acid form of CENP-A of the present invention and the invention related thereto show superior effects over the prior art.
  • the present invention provides: a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 or its complement or a fragment thereof; (C) in the amino acid sequence of SEQ ID NO: 4, wherein one or more amino acids has at least one mutation selected from the group consisting of substitution, addition, and deletion. (D) a polynucleotide that encodes a biologically active variant polypeptide; and (d) hybridizes under stringent conditions to any one of the polynucleotides (a) to (c). And (e) a polynucleotide encoding a biologically active polypeptide; or (e) any one of (a) to (c). A nucleotide sequence having at least 70% identity to a nucleotide or its complementary sequence, and A polynucleotide encoding a polypeptide having biological activity.
  • the number of substitutions, additions and deletions in (c) above is limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less , 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less. Less number of substitutions, additions and deletions are preferred, but retains biological activity (preferably, has activity similar or substantially identical to CENP-A, or an aberrant form thereof) (For example, binding to non-centromeric chromatin).
  • the biological activity of the polypeptide is, for example, an interaction with an antibody specific to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof. And interaction with centromere chromatin, but are not limited thereto. These can be measured, for example, by an immunological assay, a labeling assay, or the like.
  • the allelic variant according to (d) advantageously has at least 99% homology with the nucleic acid sequence shown in SEQ ID NO: 1.
  • the above-described species homolog can be identified by searching CENP-A of the present invention as a query sequence for the database, if a gene sequence database of the species exists.
  • all or part of CENP-A of the present invention can be identified as a probe or primer by screening such a gene library. Such identification methods are well known in the art and are also described in the literature described herein.
  • the species homolog has, for example, at least about 30% homology with the nucleic acid sequence shown in SEQ ID NO: 3.
  • the species homolog is at least about 40%, at least about 50%, at least about 60%, At least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, may be homologous.
  • the identity to the polynucleotide of any one of (a) to (e) above or a complementary sequence thereof may be at least about 80%, more preferably at least about 90%, and even more preferably Can be at least about 98%, most preferably at least about 99%.
  • CENP-A of the present invention has a sequence 100% identical to the sequence set forth in SEQ ID NO: 3.
  • the nucleic acid molecule encoding CENP-A of the present invention, or fragments and variants thereof can be at least 8 contiguous nucleotides in length.
  • the appropriate nucleotide length of the nucleic acid molecule of the present invention may vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides in length, even more preferably at least 15 contiguous nucleotides in length, and even more preferably at least 20 contiguous nucleotides in length. possible.
  • the lower limit of these nucleotide lengths is not limited to the specific numbers, but also numbers between them (eg, 9, 11, 12, 13, 14, 16, etc.) or larger numbers (eg, 21 , 22,... 30, etc.).
  • the nucleic acid molecule of the present invention has an upper limit as long as it can be used for the intended use (for example, it can interact with antisense, RNAi, a marker, a primer, a probe, and a predetermined factor).
  • the length may be the entire length of the sequence shown in SEQ ID NO: 1, or may be longer.
  • when used as a primer it can usually be at least about 8 nucleotides in length, and preferably about 10 nucleotides in length.
  • the nucleic acid molecule encoding CENP-A When used as a probe, it can usually be at least about 15 nucleotides in length, and preferably about 17 nucleotides in length.
  • the nucleic acid molecule encoding CENP-A includes the entire nucleic acid sequence of SEQ ID NO: 3. More preferably, the nucleic acid molecule encoding CENP-A consists of the entire range of the nucleic acid sequence of SEQ ID NO: 3. (CENP—specific factor for A)
  • the present invention provides a cancer (eg, a rectal cancer or a colon cancer (collectively referred to as a colon cancer), comprising an agent that specifically interacts with a nucleic acid molecule encoding CENP-A. )
  • a cancer eg, a rectal cancer or a colon cancer (collectively referred to as a colon cancer)
  • the present invention provides an agent specific for any of the nucleic acid molecules encoding CENP-A or variants or fragments thereof described herein.
  • the amount effective for diagnosis, prevention, treatment or prognosis can be determined by those skilled in the art using techniques well known in the art while considering various parameters, and such an amount is determined.
  • the agent of the present invention can be an agent selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof. It can be understood that such factors may be any as long as they specifically bind to the nucleic acid molecule of the present invention.
  • the agent of the invention is a nucleic acid molecule.
  • such a nucleic acid molecule can be at least 8 contiguous nucleotides in length, and is preferably specific for a CENP-A nucleic acid sequence (eg, SEQ ID NO: 3). Can be combined.
  • nucleic acid molecule of the present invention may vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides in length, even more preferably at least 15 contiguous nucleotides in length, and still more preferably at least 20 contiguous nucleotides in length. It can be of consecutive nucleotide length.
  • the lower limit for these nucleotide lengths is not only the numbers specifically listed, but also the numbers between them (eg, 9, 11, 12, 13, 13, 14, 16, etc.) or larger numbers. (For example, 21, 22, 2, 30, etc.).
  • the nucleic acid molecules of the present invention can be used as intended applications (eg, antisense, RNAi, markers, primers, probes) or can interact with predetermined factors.
  • the upper limit length may be the full length of the sequence shown in SEQ ID NO: 3 or the complement thereof, or may be longer.
  • when used as a primer it can usually be at least about 8 nucleotides in length, and preferably about 10 nucleotides in length.
  • When used as a probe it can usually be at least about 15 nucleotides in length, and preferably about 17 nucleotides in length.
  • the agent of the invention comprises a sequence complementary to the nucleic acid sequence of any of the above CENP-A polynucleotides (a)-(e) or at least 7 It may be a nucleic acid molecule having a sequence with 0% identity.
  • the agent of the present invention is an antisense or RNAi of a CENP-A nucleic acid molecule.
  • RNA i may be either si RNA or sh RNA, such as about 20 bases (eg, For example, typically about 21 to 23 bases in length) or less, and preferably has a 5′-phosphate, 3′-OH structure, The 3 'end protrudes about 2 bases.
  • the shRNA may also preferably have a 3 'overhang.
  • the length of the double-stranded portion is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more.
  • the 3 'protruding end may be preferably DNA, more preferably DNA having at least 2 nucleotides or more, and further preferably DNA having 2 to 4 nucleotides in length.
  • the agent of the present invention is a nucleic acid molecule, and can be used as a primer in a nucleic acid amplification reaction such as PCR, or can be used as a probe in a Southern blot or the like.
  • the agent of the invention may be labeled or capable of binding to a label.
  • a label can be any label as long as it is identifiably labeled, including techniques such as fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme-substrate reactions, and antigen-antibody reactions. Not limited.
  • an immune reaction such as an antibody
  • a system often used in an immune reaction such as biotin-streptavidin may be used.
  • the factor specific to CENP-A of the present invention is specific to a polynucleotide consisting of the sequence shown in SEQ ID NO: 3 (or a variant present in a normal subject).
  • it is a factor.
  • Such a factor can detect a normal form of CENP-A.
  • the factor specific to C ENP-A of the present invention is CENP-A other than a polynucleotide having the sequence shown in SEQ ID NO: 3 (or a variant present in a normal subject).
  • it is a factor specific for the variant.
  • Such factors can detect variants of C ENP-A.
  • the present invention provides: A) a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 or a complement or fragment thereof;
  • a cancer diagnostic kit comprising: means for diagnosing a person at risk.
  • cancers to be diagnosed include, but are not limited to, rectal cancer and colon cancer.
  • any factor as described herein can be used.
  • any factor having any specificity can be used in the present invention, as long as its reaction specificity is understood and provided. It is understood that and kits containing them are within the scope of the present invention.
  • One embodiment includes, but is not limited to, factors specific to the sequence comprising any 20 nucleotides in SEQ ID NO: 3.
  • the measurement of the expression level of the nucleic acid molecule can be performed using any signal caused by a factor specific to the nucleic acid form of CENP-A.
  • signals include, for example, physical signals (eg, fluorescence, luminescence, etc.), chemical signals (eg, staining, etc.), biochemical signals (eg, enzyme reactions), biological signals (eg, , Determination of the survival of microorganisms) and the like, but are not limited thereto.
  • a signal may be any signal that can be determined in a biological sample of a subject, for example, by directly or indirectly testing whether or not the mRNA of the present invention is present. Anything can be used. In this case, if such a signal is detected, the subject in which the mRNA is present in the sample can be determined to be a cancer patient or a high-risk subject.
  • RNA samples include stool, urine, sweat, biopsy tissue, saliva, blood (for example, whole blood, serum, plasma, etc.), blood cells (mononuclear cells, etc.).
  • blood for example, whole blood, serum, plasma, etc.
  • blood cells mononuclear cells, etc.
  • Detection and measurement of mRNA can be performed by a known RT-PCR method or a quantitative RT-PCT method, and in that case, a set of primers of the present invention can be used for PCR.
  • These primer sets can be designed based on the nucleotide sequence of SEQ ID NO: 3 or a variant thereof, and can be prepared through each step of synthesis and purification.
  • the size (number of bases) of the primer is 15 to 40 bases, preferably 15 to 30 bases in consideration of satisfying specific annealing with type I DNA. However, when performing LA (long accurate) PCR, at least 30 bases are effective.
  • one pair or two pairs of primers consisting of the sense strand (5 'end) and the antisense strand (3' end) should not anneal to each other. Avoid the complementary sequence between both primers, and also avoid the self-complementary sequence to prevent the formation of the avian structure within the primer.
  • a sequence that is present on one side and not on the other side for example, a sequence of a deleted or added portion
  • the GC content should be about 50% to ensure stable binding to type I DNA, so that GC-rich or AT-rich is not unevenly distributed in the primer. Since the annealing temperature depends on the melting temperature (Tm), primers with a Tm value of 55-65 ° C and close to each other should be selected to obtain highly specific PCR products. It is also necessary to take care to adjust the final concentration of the primer used in PCR to be about 0.1 to about 1 M.
  • commercially available software for primer design for example, Olio TM [manufactured by Natal Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Inc. (Japan)], and the like can also be used.
  • kits or diagnostic method of the invention can be performed using an array.
  • the method of executing the array is as described above.
  • the present invention provides: A) a step of providing a sample from a target portion of a subject; B) in the sample: a) a base sequence of SEQ ID NO: 3 or a complement thereof or A polynucleotide having the fragment sequence;
  • Samples used in this cancer diagnostic method include stool, urine, sweat, biopsy tissue, saliva, blood (eg, whole blood, serum, plasma, etc.), blood cells (monocytes, etc.), other body fluids
  • cells in an affected part can be targeted.
  • Samples from known unaffected sites or from other unaffected subjects can be used for comparison purposes.
  • a sample from another unaffected subject is used, preferably, such a sample can be a sample derived from a site similar to the target site of the subject in question.
  • cancers to be diagnosed include, but are not limited to, rectal cancer and colon cancer.
  • the detection of the nucleic acid molecule can be performed using an agent specific to such a nucleic acid molecule.
  • the present invention provides: (a) a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 or its complement or a fragment thereof; (b) encoding the amino acid sequence of SEQ ID NO: 4 or a fragment thereof (C) an amino acid sequence represented by SEQ ID NO: 4; A polynucleotide, wherein the amino acid is a variant polypeptide having at least one mutation selected from the group consisting of substitution, addition, and deletion, which encodes a variant polypeptide having biological activity; d) a polynucleotide encoding a polypeptide having biological activity of any one of (a) to (c); or (e) a polynucleotide encoding any one of (a) to (c) or a complementary sequence thereof.
  • a polynucleotide comprising a nucleotide sequence having at least 70% identity and encoding a polypeptide having biological activity comprising: a nucleic acid molecule for use in the manufacture of a diagnostic agent for cancer.
  • cancers to be diagnosed include, but are not limited to, rectal cancer and colon cancer.
  • the detection of the nucleic acid molecule can be performed using a factor specific to such a nucleic acid molecule.
  • any factors specific for the nucleic acid form of CENP-A described herein can be used. (Therapeutic method and therapeutic agent using nucleic acid form of CENP-A)
  • the present invention provides: A) a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 or a complement or fragment thereof; (b) an amino acid sequence of SEQ ID NO: 4 Or a polynucleotide encoding the fragment thereof; (c) a variant having at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 4, wherein one or more amino acids are substituted; (D) a polynucleotide that encodes a biologically active variant polypeptide; (d) hybridizes to any one of the polynucleotides under stringent conditions; A polynucleotide encoding a soybean and biologically active polypeptide; or (e) a polynucleotide of any one of (a) to (c) De or identity to the complement sequence is the nucleotide sequence which is at least 70%, and biology A polynucleotide encoding a polypeptide having the amino acid sequence
  • examples of the cancer to be treated include, but are not limited to, rectal cancer and colon cancer.
  • any nucleic acid molecule as described herein can be used.
  • a nucleic acid molecule capable of expressing a normal form of CENP-A is used.
  • a molecule represented by SEQ ID NO: 3 or a variant thereof, which expresses a molecule having a function equivalent to SEQ ID NO: 3 or a molecule having a function equivalent to SEQ ID NO: 2 Can be used.
  • the nucleic acid molecule contained in the therapeutic agent of the present invention includes a nucleic acid molecule encoding a polypeptide comprising the sequence shown in SEQ ID NO: 3.
  • the present invention provides a cancer treatment method using the above-mentioned cancer treatment agent.
  • This treatment may use any of the treatment techniques described herein. (Polypeptide form of CENP—A)
  • the present invention provides a method for treating a condition of cancer (eg, rectal or colon cancer (collectively referred to as colorectal cancer)) comprising CENP-A in a polypeptide form.
  • a condition of cancer eg, rectal or colon cancer (collectively referred to as colorectal cancer)
  • CENP-A in a polypeptide form.
  • Prophylactic, diagnostic or prognostic compositions the amount effective for diagnosis, prevention, treatment or prognosis can be determined by a person skilled in the art using techniques well known in the art while considering various parameters, and such an amount is determined. For example, a person skilled in the art can easily determine the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc.
  • the present invention provides: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof; (b) an amino acid sequence of SEQ ID NO: 4; A polypeptide, wherein the amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion, and has biological activity; (c) a splice variant of the base sequence of SEQ ID NO: 3 Or a polypeptide encoded by an allelic variant; (d) a species homolog of the amino acid sequence of SEQ ID NO: 4, a polypeptide; or (e) any one of (a) to (d) Polypeptides having an amino acid sequence that is at least 70% identical to two polypeptides and having biological activity.
  • the number of substitutions, additions and deletions in (b) above may be limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less. Hereafter, it is preferably 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • a smaller number of substitutions, additions and deletions is preferred, but retains biological activity (preferably similar or substantially similar to normal CENP-A consisting of the sequence shown in SEQ ID NO: 4. A larger number may be used as long as they have the same activity or have an abnormal activity of CENP-A (eg, binding to non-centromeric chromatin).
  • the splice variant or allelic variant in (c) above preferably has at least 99% homology with the amino acid sequence shown in SEQ ID NO: 4.
  • the species homolog can be identified as described herein above and comprises at least about 30% of the amino acid sequence set forth in SEQ ID NO: 4. Preferably, they have homology. Preferably, the species homolog is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95% 98% may be homologous.
  • the above-mentioned species homolog can be identified by searching the database of the gene sequence of the species, if any, for CENP-A of the present invention as a query sequence. Alternatively, all or part of the CENP-A of the present invention can be identified as a probe or primer by screening such a gene library. Such identification methods are well known in the art and are also described in the literature described herein.
  • the species homolog preferably has, for example, at least about 30% homology with the nucleic acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 4.
  • the species homolog is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% %, At least about 98%, can be homologous.
  • the biological activity of the variant polypeptide in the above (e) is, for example, specific to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 or a fragment thereof. Interaction with a specific antibody, interaction with centromere chromatin, and the like, but are not limited thereto. These can be measured by, for example, an immunological assay, a fluorescent assay, or the like.
  • the homology to the polypeptide of any one of (a) to (d) above may be at least about 80%, more preferably at least about 90%, and more preferably Can be at least about 98% and have And preferably at least about 99%.
  • the CENP-A polypeptide of the invention has a sequence consisting of SEQ ID NO: 4.
  • the polypeptides of the present invention usually have at least three consecutive amino acid sequences.
  • the amino acid length of the polypeptide of the present invention may be any length as long as it matches the intended use, but preferably, a longer sequence may be used. Thus, it may be preferably at least 4 amino acids long, more preferably at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long. More preferably it can be at least 15 amino acids long, and still more preferably at least 20 amino acids long.
  • the lower limit of these amino acid lengths is not limited to the specific numbers, but also numbers between them (for example, 11, 12, 13, 14, 14, 16, etc.) or larger numbers (for example, 2 1, 22,... 30, etc.).
  • the upper limit length may be the same as or greater than the entire length of the sequence shown in SEQ ID NO: 4. Good.
  • the polypeptide form of CENP-A of the present invention is labeled or can be labeled.
  • the amount of antibody against CENP-A in the body can be measured, thereby indirectly measuring the expression level of CENP-A. Because you can do it.
  • the present invention relates to a cancer (eg, rectal or colon cancer (collectively referred to as colorectal cancer)) comprising an agent specific for the polypeptide form of CENP-A, Compositions for treatment, prevention, diagnosis or prognosis are provided. Accordingly, the present invention relates to any of the CENP-A polypeptides described herein. An agent specific for the tide or a variant or fragment thereof is provided.
  • the amount effective for diagnosis, prevention, treatment or prognosis can be determined by a person skilled in the art using techniques well known in the art while considering various parameters, and such an amount is determined.
  • a person skilled in the art can easily determine the purpose of use, target disease (type, severity, etc.), patient's age, weight, sex, medical history, cell morphology or type, etc. (See, eg, Cancer Resident Resident Manual Second Edition, edited by the Internal Resident of the National Cancer Center Hospital, Medical College, 2000).
  • target disease type, severity, etc.
  • patient's age, weight, sex medical history, cell morphology or type, etc.
  • the agent of the present invention may be an agent selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof. It can be understood that such factors may be any that specifically bind to the polypeptide of the present invention. More preferably, the agent of the present invention is an antibody or a derivative thereof (eg, a single-chain antibody). Thus, the agents of the present invention can be used as probes and probes or inhibitors.
  • the agent of the invention may be advantageously labeled or capable of binding to a label.
  • a label can be any label as long as it is identifiably labeled, including techniques such as fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme-substrate reactions, and antigen-antibody reactions. Not limited.
  • an immune reaction such as an antibody, 3010676
  • a system often used in an immune reaction such as biotin-streptavidin may be used.
  • the factor specific to CENP-A of the present invention is a polypeptide specific to the polypeptide consisting of the sequence shown in SEQ ID NO: 4 (or a variant present in a normal subject).
  • it is a factor.
  • Such factors can detect the normal form of CENP-A.
  • the factor specific to CENP-A of the present invention is a variant of CENP-A other than a polypeptide consisting of the sequence shown in SEQ ID NO: 4 (or a variant present in a normal subject).
  • it is a specific factor.
  • Such factors can detect variants of CENP-A.
  • the present invention provides: A) a polypeptide consisting of (a) the amino acid sequence of SEQ ID NO: 4 or a fragment thereof; (b) one or more amino acids in the amino acid sequence of SEQ ID NO: 4 A polypeptide having at least one mutation selected from the group consisting of addition and deletion, and having biological activity; (c) a splice variant or allele of the nucleotide sequence of SEQ ID NO: 3.
  • a polypeptide encoded by the mutant comprising: a polypeptide; a factor specific to the polypeptide; and B) the factor Means for measuring the expression level of the polypeptide using a signal resulting from the method, wherein the expression level is compared with the expression level of the polypeptide in a subject not afflicted with cancer or a portion not afflicted with cancer.
  • cancer diagnostic kit Diagnosing the subject as being at high risk for cancer if the expression of the polypeptide is high in the subject portion of the subject.
  • cancer to be diagnosed include, but are not limited to, rectal cancer and colon cancer.
  • any factor as described herein can be used. Such factors may specifically react with either the normal or modified form of CENP-A, or may specifically react with either the normal or modified form of FIR. As long as the reaction specificity is understood and provided, any factor having any specificity can be used in the present invention. Is understood to be within the range.
  • examples include, but are not limited to, factors specific to a common sequence between the normal form and the variant (eg, a 30 amino acid long sequence in SEQ ID NO: 4).
  • the CENP-A polypeptide itself can be used as this factor.
  • B polypeptide since the antibody specific to CENP_A polypeptide can be recognized, the amount of antibody specific to CENP-A polypeptide in the living body is calculated. This is because it is possible to identify the presence of the CENP-A polypeptide in the body.
  • the measurement of the expression level of the CENP-A polypeptide can be performed using any signal derived from a factor specific to the CENP_A polypeptide form of the present invention.
  • the method for diagnosing cancer of the present invention tests whether or not an antibody that binds to the antigenic peptide of the present invention is present in the serum of the subject, and subjects the subject having the antibody in the serum to cancer. It can also be determined as a patient or a person at high risk for cancer. That is, since the antigenic peptide of the present invention is a peptide that binds to an antibody (IgG) in the serum of a patient, it is reacted with the serum of a subject, and the serum containing the antibody that binds to these antigenic peptides is used for cancer. Can be determined as the serum of the patient or its high-risk patients You. At this time, it is preferable to determine the binding of the two types of antigen peptides to the antibody in serum.
  • IgG an antibody
  • a subject serum is brought into contact with an antigen peptide, and the antigen peptide is reacted with an IgG antibody in the subject serum in a liquid phase.
  • a signal of the labeled IgG antibody may be detected by reacting a labeled IgG antibody that specifically binds to the IgG antibody in the serum.
  • an enzyme, a radioisotope or a fluorescent dye as exemplified in the above-mentioned labeled antibody can be used.
  • an enzyme When an enzyme is used, a substrate that decomposes by the action of an enzyme and develops a color is added, and the enzyme activity is determined by optically measuring the amount of decomposition of the substrate, converted to the amount of bound antibody, and compared with a standard value. Is used to calculate the amount of antibody.
  • radioactive isotopes measure the radiation dose emitted by the radioisotope using a scintillation counter or the like.
  • a fluorescent dye When a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescent microscope.
  • Western blot analysis as shown in the Examples can be employed.
  • the conjugate of the antigen peptide + serum antibody + labeled IgG antibody is separated by known separation means (chromatography, salting out, alcohol precipitation, enzymatic method, solid phase method, etc.), and the signal of the labeled IgG antibody is separated. May be detected.
  • the diagnostic kit of the present invention is provided as one that enables simple and wide-ranging implementation of such a diagnostic method.
  • the diagnostic method of the present invention also includes a method of immobilizing one or more antigen peptides on a plate and testing the binding of the test subject's serum to the antibody on the substrate (also encompassed by the present invention). It can also be implemented as By immobilizing the antigen peptide on the substrate, unbound labeled binding molecules can be easily removed.
  • an embodiment of the diagnostic kit of the present invention is provided as enabling such a diagnostic method to be performed simply and widely.
  • the diagnostic method of the present invention tests whether or not an antibody of the present invention or an antigen peptide that binds to the labeled antibody of the present invention is present in a biological sample of a subject.
  • a subject in which the antigen peptide is present is determined to be a cancer patient or a high-risk subject thereof. That is, since the antibody or labeled antibody used here is an antibody that specifically binds to an antigen peptide expressed in colorectal cancer cells, a biological sample containing an antigen peptide that binds to this antibody can be used for colorectal cancer. It can be determined as a sample of a patient or a patient at high risk for colorectal cancer. In this case, it is preferable to determine the binding of the two antibodies to the antigen peptide in the sample. In addition, blood and blood cells (eg, mononuclear cells) can be used as a biological sample.
  • blood and blood cells eg, mononuclear cells
  • One embodiment of the diagnostic method of the present invention is a method of binding an antibody and an antigen peptide in a liquid phase system.
  • the labeled antibody of the present invention is brought into contact with a biological sample to bind the labeled antibody and the antigen peptide, the conjugate is separated by the method described herein, and the labeled signal is obtained by the same method.
  • the diagnostic kit of the present invention is provided as a method which enables such a diagnostic method to be carried out simply and widely.
  • Another method of diagnosis in a liquid phase system is to contact the antibody (primary antibody) of the present invention (primary antibody) with a biological sample to bind the primary antibody and the antigen peptide, and to the conjugate, the labeled antibody (secondary antibody) of the present invention (secondary antibody). Antibody), and the labeled signal of the conjugate is detected.
  • an unlabeled secondary antibody may first be bound to the antibody + antigen peptide conjugate, and a labeling substance may be bound to this secondary antibody.
  • binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylating the secondary antibody and avidinizing the labeling substance.
  • an antibody (tertiary antibody) recognizing a partial region (for example, Fc region) of the secondary antibody may be labeled, and the tertiary antibody may be bound to the secondary antibody.
  • the primary antibody and the secondary antibody both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Can also be. Separation of the conjugate from the liquid phase and detection of the signal can be performed as described herein.
  • an embodiment of the diagnostic kit of the present invention is provided as enabling simple and wide-ranging implementation of such a diagnostic method.
  • Another aspect of the diagnostic method of the present invention is a method for testing the binding between an antibody and an antigen peptide in a solid phase system.
  • This method using a solid phase system is a preferable method for detecting a trace amount of antigen peptide and simplifying the operation. That is, in this solid phase method, the antibody (primary antibody) of the present invention is immobilized on a resin plate or the like, the antigen peptide is bound to the immobilized antibody, and the unbound peptide is washed off, and then left on the plate. In this method, a labeled antibody (secondary antibody) is bound to the antibody + antigen peptide conjugate, and the signal of this secondary antibody is detected. This method is a so-called “sandwich method”.
  • an enzyme When an enzyme is used as a marker, it is used as a factory ELISA (enzyme 1 inked immunosorbent assay).
  • ELISA enzyme 1 inked immunosorbent assay
  • the primary antibody and the secondary antibody both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Detection of the signal can be performed as described herein. Further, an embodiment of the diagnostic kit of the present invention is provided as enabling simple and wide-ranging implementation of such a diagnostic method.
  • the diagnostic kit of the present invention can be a reagent kit for performing the diagnostic method of the present invention. Various such kits are commercially available depending on the type of the test component.
  • the diagnostic kit of the present invention also uses the antigenic peptide, antibody and Z or labeled antibody provided by the present invention. Except for the above, it can be constituted by each element used in a kit known in the art.
  • kits or diagnostic method of the invention can be performed using an array.
  • the method of executing the array is as described above.
  • the present invention provides a method comprising: A) providing a sample from a target portion of a subject; B) measuring the abundance of the polypeptide according to claim 142 in the sample. C) comparing the abundance of the polypeptide with the abundance of the same nucleic acid molecule in a sample not affected by cancer or a sample from a portion not affected by cancer; Diagnosing that the subject of the subject is at high risk for cancer when the amount of the polypeptide in the sample from the subject portion is increased.
  • Samples used in this cancer diagnostic method include stool, urine, sweat, biopsy tissue, saliva, blood (eg, whole blood, serum, plasma, etc.), blood cells (monocytes, etc.), other body fluids
  • cells in the affected part can be targeted.
  • Samples from apparently unaffected sites or from other unaffected subjects may be used for comparison purposes.
  • a sample from another unaffected subject is used, preferably, such a sample can be a sample derived from a site similar to the target site of the subject.
  • examples of the cancer to be diagnosed include, but are not limited to, rectal cancer and colon cancer. Detection of the above polypeptide can be performed using a factor (for example, an antibody) specific to such a polypeptide (CENP-A polypeptide).
  • any factors specific for the polypeptide form of CENP-A described herein can be used.
  • such an agent may advantageously respond specifically to both normal CENP-A having the sequence shown in SEQ ID NO: 4 and variants thereof.
  • the factor may be specific for the variant alone or only for the normal form.
  • any factor can be provided with an understanding of even its reaction specificity, and any factor having any specificity can be used in the present invention. It is understood that the factors and kits containing them are within the scope of the present invention Is done.
  • factors include, but are not limited to, those sequence portions that differ between the normal and the variant.
  • the present invention provides: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof; (b) in the amino acid sequence of SEQ ID NO: 4, wherein one or more amino acids are A polypeptide having at least one mutation selected from the group consisting of substitution, addition and deletion, and having biological activity; (c) a splice variant or allele of the nucleotide sequence of SEQ ID NO: 3.
  • a polypeptide encoded by a gene variant comprising: (d) a polypeptide which is a species homolog of the amino acid sequence set forth in SEQ ID NO: 4; or (e) a polypeptide of any one of (a) to (d) A polypeptide having an amino acid sequence having an identity of at least 70% with respect to and having biological activity, for the manufacture of a diagnostic agent for cancer, comprising: It provides for the use.
  • examples of the cancer to be diagnosed include, but are not limited to, rectal cancer and colon cancer. Detection of the above polypeptides can be performed using factors specific to such polypeptides. As such factors, any factors specific for the polypeptide form of CENP-A described herein can be used. (Therapeutic method and therapeutic agent using polypeptide form of FIR)
  • the present invention provides: A) (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof; (b) one or more amino acids in the amino acid sequence of SEQ ID NO: 4 A polypeptide having at least one mutation selected from the group consisting of, addition and deletion, and having biological activity; (c) a splice variant or allele of the nucleotide sequence of SEQ ID NO: 3.
  • a polypeptide encoded by the variant (d) SEQ ID NO: 4 A polypeptide which is a species homologue of the amino acid sequence described above; or (e) an amino acid sequence having at least 70% identity to the polypeptide of any one of (a) to (d), And a polypeptide having biological activity, comprising: a polypeptide; and a cancer therapeutic agent comprising the polypeptide.
  • examples of the cancer to be treated include, but are not limited to, rectal cancer and colon cancer.
  • any factor as described herein can be used.
  • the polypeptide has at least one of the normal functions of CENP-A, and preferably has the ability to bind to centromere chromatin.
  • a molecule having a function equivalent to that of SEQ ID NO: 4, which is a sequence represented by SEQ ID NO: 4 or a variant thereof, can be used.
  • the polypeptide contained in the therapeutic agent of the present invention comprises a polypeptide comprising the sequence shown in SEQ ID NO: 4.
  • the present invention provides a cancer treatment method using the above-mentioned cancer treatment agent.
  • This treatment can use any of the treatment techniques described herein.
  • the underlines correspond to the 31-45 amino acid sequence and the 528-542 amino acid sequence of SEQ ID NO: 2, respectively.
  • the synthesized peptides were analyzed by HPLC to ensure purity, and the molecular weight was identified by mass spectrometry.
  • This synthetic peptide was used to produce aseptic Caesarean sections from New Zealand.
  • the S.P.F. standard (pathogenic bacteria) magpie was immunized by a conventional method, the serum was extracted, and an affinity-purified anti-FIR antibody was prepared.
  • frozen specimens were collected from each of the cancerous and non-cancerous tissues immediately after removal of colorectal cancer and stored at -80 ° C.
  • An appropriate amount of the frozen sample is homogenized in a 9.5 M Uera, 2% CHAPS, 1% DTT solution, and then centrifuged at 15,000 g in a desktop high-speed centrifuge (Beckman), and the supernatant (protein solution) is added to the supernatant for one week. Then, the protein concentration was identified by the absorbance.
  • the SO g of each protein obtained from the cancerous and non-cancerous tissues was electrophoresed on 8% Tris / Glycine SDS-Polyacrylamide gel.
  • the electrophoresed protein was transferred to a nitrocellulose membrane, and eastern blot was performed using the antibody (1-2).
  • the transcription of the transcript (mRNA) of the gene encoding the antigenic peptide FIR of the present invention in cancer tissues and normal tissues was analyzed by RT-PCR.
  • MRNA was extracted from frozen specimens collected from cancerous and non-cancerous tissues immediately after removal of colorectal cancer using the RNeasy Mini Kit (Qiagen). This mRNA (5 g / 201) was converted into cDNA using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV, cat no. 1 483 188, Roche) and amplified by PCR.
  • the primer sets used for PCR are as follows.
  • Reverse primer 5 '-ggggctgggccagggtcag-3' (SEQ ID NO: 8)
  • the PCR conditions are as follows.
  • Example 3 The expression of CENP-A (SEQ ID NO: 4) of the present invention in cancer tissues and normal tissues was analyzed by Western blotting.
  • lysis buffer 7M urea, 2M thiourea, 2% 3-[(3-cholamide propyl) dimethyl ammonium]] In 1-propanesulfonate, 0.1 M DTT, 2% IPG buffer (Amersh am Pharmamacia Biotech, Buc nghams hire, United Kingdom), and 4 OmM Tris).
  • the lysate was dissolved using a Po ytron homogenizer (Kine matica, Littau-Luzern, Switzerland).
  • the protein in the supernatant was separated by electrophoresis on a 7.5 to 15% gradient gel (Nikkyo Technology DRC, Tokyo, Japan).
  • the protein is transferred to a polyvinylidene fluoride membrane (Mi 11 ipore, Bedford, MA) in a tank transfer device (Bio-Rad, Hercu 1es, CA), and the membrane is 5% skimmed. Blocked with PBS containing milk. Nuclear centromere autoantibody positive control (1: 10000 diluted in blocking buffer) (ANA serum; TeBinding Site, Birmingham, UnitedKingdom), 1 : 500-diluted anti-human CENP-A monoclonal antibody (Ando, S. eta 1., Mo 1. Cell.
  • Tissues were fixed on glass slides using PBS containing 4% paraformaldehyde for 5 minutes at room temperature. After washing three times with PBS, samples were permeabilized with PBS containing 0.5% Triton X-100 for 5 minutes and re-fixed with 4% paraformaldehyde ZPBS for 5 minutes. In some experiments, tissues were fixed with acetone at 4t: for 10 minutes. Nonspecific binding of antibodies was blocked with blocking buffer (1% BSA or 10% fetal calf serum / PBS) for 1 hour. Samples were prepared using anti-human CENP-A monoclonal antibody diluted 1: 500 in blocking buffer and goat anti-human CENP-B (Y-17) antibody diluted in Z or 1:25 (Santa Cruz).
  • RNA and genomic DNA were extracted from tumor and non-tumor tissues using the RNeasy Minikit and Dneasy T issuekit (Qiagen).
  • str and cDNA Syn tesiskit for PT-PCR (Ro che, Mannhe im, CDNA was synthesized from total RNA using Germany).
  • cDNA was amplified using the following primers (forward: 5 '-TAGGCGCTTCCTCCCATCAA-3' (SEQ ID NO: 9), reverse: 5 '-GCCGAGTCCCTCCTCAAG- 3 '(SEQ ID NO: 10)).
  • GAPDH cDNA was amplified for control. Serial dilutions of the template cDNA were made for the PCR reaction and the PCR product was optimized within the linear range.
  • Real-time quantitative PCR of CENP-A cDNA using the Light Cy1 er instrument (Ro che) was performed using the Light Cy1 er DNA Master SYBR Green I (Fast Start T aq DN- A polymerase, deoxynucleotide triphosphate, buffer, SYBR Green I), 3.
  • Chromosome 7 (CEP 7 Spectrum rum range), Chromosome 8 (CEP 8 Spectrum O range), Chromosome 12 (CEP 12 Spectrum ⁇ range), and Chromosome 15 (CEP 15 Sp ectr um Gr e en; Vy sis, Downer s Gr ove,
  • CENP-A a protein essential for kinetochore assembly
  • ANA serum an anti-centromere antibody
  • FIG. 3B The overexpression of CENP-A in the colorectal tumors shown above was also confirmed by Western plot using an anti-human CENP-A monoclonal antibody (FIG. 3B). Since CENP-A is a component of the human centromere complex, other centromere proteins may also be overexpressed.
  • ANA serum contains antibodies to CENP-B and CENP-A in addition to CENP-A. Therefore, the expression of these proteins was also tested. As shown in FIG. 3C, expression levels of CENP-B protein were similar in tumor tissue and nearby normal tissue. The band corresponding to CENP-C was not detectable by the ANA serum (data not shown). This overexpression of CENP-A in colorectal cancer is not due to the high rate of tumor cell growth, and the relative expression levels of the PCNA protein in most cases are high in tumor and normal tissues. This is due to the similarity between the two (Fig. 3D).
  • CENP-A mRNA levels in colorectal cancer cells and normal colon epithelium were tested by RT-PCR (Figure 4A). . All cases except case 32 showed higher CENP-A mRNA levels in tumor cells than in normal cells. Also, the relative mRNA levels in the tumor cells correlated well with the relative protein levels shown in Figure 3A. Increased CE NP-A mRNA levels in colorectal cancer cells were further confirmed by real-time quantitative RT-PCR (FIG. 4B). CENP-A gene amplification was also used in the above experiments The same corresponding tumor Z was tested by real-time quantitative PCR using genomic DNA from normal samples. As shown in FIG. 4C, no amplification was observed in the tumor. These results indicate that overexpression of CENP-A occurred at the transcript level.
  • Chromosomes have been shown to be reduced or increased in most cancers, and some colorectal cancer cell lines are often polysomal (Lengauer, C. et al. , Nature, 396: 643-64 9, 1998). These observations raise the question whether overexpression of CENP-A could be a consequence of ploidy. To address this question, FISH analysis of normal and tumor cells was performed using several centromeric probes. Three preparations of colorectal cancer overexpressing CENP-A were obtained and the number of centromere signals for chromosomes 7, 8, 12, and 15 was counted (Table 2).
  • Tumor 2 Tumor 3 Chromosome number (%) ⁇ Chromosome number (%) Chromosome number (%) Chromosome number (%) Chromosome number (%)
  • tissue sections of colorectal cancer tissue and nearby normal tissue were analyzed using anti-human CENP-A Staining was performed using a monoclonal antibody (Ando, S. eta 1., Mo 1. Cell. Biol., 22: 222 9-2241, 202).
  • CENP-A was present as a dispersed dot in the nucleus. This is typically seen for centoral centromere staining in both normal and tumor cells. Examination of some tissue sections under a microscope shows that CENP-A staining is increased in tumor cells as compared to normal epithelial cells. This indicates that CENP-A is actually increased in tumor cells (FIGS. 5B and 5D).
  • CENP-A is known as a central component of the centromere and recruits a subset of other centromeric proteins ⁇ Thus, mislocalization of CENP-A (mis10ca1ization) Can cause ectopic centromere formation and cause chromosome missegregation.
  • tissue sections were co-stained with anti-CENP-A and anti-CENP-B antibodies, and individual cells were tested at high magnification (Figure 6A). Considering binding to ⁇ satellite DN ⁇ , CENP-B was used as a centromere.
  • CENP-A SEQ ID NO: 4
  • T cancer tissue
  • N normal tissue
  • FIG. 4 shows a summary of the results obtained by analyzing the transcription of the transcript (mRNA) of the gene encoding CENP-A of the present invention in cancer tissues and normal tissues by RT-PCR. From cancer tissue (T) and normal tissue (N) of 11 colorectal cancer patients
  • CENP-A mRNA When the transcription of CENP-A mRNA was compared, specific expression of CENP-A mRNA was confirmed in cancer tissue cells.
  • CENP-A is an essential protein for centromere function.
  • CENP-A distinguishes centromeric chromatin from other chromatins by replacing histone H3 in centromeric-specific nucleosomes (Yoda, K. eta 1. Proc. Natl. Actl. Ac ad. Sci. USA, 97: 7266-7271, 2000).
  • CENP-A is required for recruitment of other centromeric proteins to the centromere (Howman, EV eta 1., Proc. Nat. 1. Ac ad. Sci. USA, 97: 1148 — 1153, 2000).
  • inappropriate expression of CENP-A can induce abnormal centromere function and chromosomal mis-segregation.
  • CENP-A The deletion of CENP-A from certain centromeres is somewhat unexpected. Although the exact mechanism is unclear, overexpression of CENP-A can destroy other centromeric kinetochore components and disrupt kinetochore complexes. Another possibility is that mistargeting of CENP-A to the non-centromeric region of chromatin alters the chromosomal conformation, which may prevent normal centromere assembly. In fact, CENP-A, which was overexpressed in HeLa cells, was found prominently on the chromosomal orthochromatic arm, reducing CENP-A levels in heterochromatin near the center of certain chromosomes. (Van Hooser, AA et al., J. Cell. Sci., 114: 3529-3542, 2001). Further investigation is needed to clarify the mechanism of mistargeting of CENP-A in colorectal cancer and how it contributes to chromosomal instability.
  • CENP-A Overexpressed in Colorectal Cancer The present inventors have demonstrated that CENP-A mRNA is also increased. This indicates that overexpression of CENP-A occurs at the transcriptional level. Thus, although some enhancer proteins may be active, no such transcription factor or enhancer element is known. Research on the regulation of CEN P-A transcription is needed.
  • CENP-A expression is also known to be regulated in the cell cycle.
  • CENP- A is synthesized in G 2 phase after DNA synthesis is performed, may be important in Centro Mea nucleocapsid Seo Ichimu set (She l by, RD eta 1. , J. Ce ll. B io 1 , 151: 11 13—1118, 2000).
  • CENP-A expression and centromere assembly are tightly regulated during the cell cycle, and this dysregulation can induce aneuploidy. It is important to test the expression level of CE NP-A throughout the cell cycle in colorectal cancer cell lines.
  • CENP-A has a CO OH-terminal histone fold domain similar to histone H 3 and highly variable NH 2 terminal domain. This unique NH 2 -terminal domain may be a useful target for designing anti-cancer drugs.
  • Example 4 FIR analysis using further cases
  • FIR was identified using samples from three additional colorectal cancer patients (118K, 1 ⁇ , 28 ⁇ ), and sequence analysis was performed on the identified FIR. Sequencing was performed using a sequencer commercially available from ⁇ I. As a result, the sequence identified by SEQ ID NOs: 17 and 18 from case 118K, the sequence identified by SEQ ID NOs: 19 and 20 from case 1K, and the sequence identified by case 28K from SEQ ID NO: The sequences specified by 21 and 22 were found.
  • a reporter plasmid containing the upstream myc-myc promoter of the chloramphenicylacetyltransferase (CAT) gene was used for CAT access (Avigan, M., I., Strober, B., and Levens , D., J. Biol. ⁇ Chem., 265: 18538-18545, 1990).
  • CAT chloramphenicylacetyltransferase
  • HA-FIR full-length FIR cDNA
  • HA-FIRAN 77 FIR mutant with the first 77 amino acids deleted
  • pCGNM2 vector-plasmid Liu, L, Akoulitchev, S., Weber, A., Ge, H., Cuikov, S., Libutti, D., Wang, XW, Conaway, JW, Harris, CC, Conaway, RC, Reinberg, D., and Levens, D., Cell. , 104: 353-363, 2001
  • All plasmids were prepared by CsC1 ultracentrifugation and sequence verified.
  • HeLa cells were cultured in DuIbecco's ModifiedEalle's Medium (DMEM, Gibco-BRL) containing 10% fetal calf serum. Cells were transfected by electroporation, harvested 48 hours after transfection, and assayed for CAT activity as previously described (Tomonaga, T., and Levens, D., J. Biol. C Em., 270: 4875-4881, 1995).
  • DMEM DuIbecco's ModifiedEalle's Medium
  • HeLa cells were grown on coverslips and then transfected with plasmid using Lipofect amine P1us reagent (Gibco BRL). Eighteen hours after plasmid transfection, cells were processed for immunocytochemistry as previously described (He, L., Liu, J., Collins, I., Sanford, S., 0 Connell , B., Benham, CJ, and Levens, D., EMB0 J., 19, 1034-44, 2000). A mouse monoclonal primary antibody against HA (Santa CruZ Biotechnology, CA) and a primary heron polyclonal antibody against c-Myc (Upstate Biotecnology, NY) were used in blocking buffer.
  • DAP I diamidinophenylindole
  • Leica QF IS H Leica Microsystems ems, Tokyo, Japan

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Abstract

La présente invention concerne de nouveaux peptides antigéniques qui sont efficaces dans le diagnostic du cancer, des anticorps dirigés contre ces peptides antigéniques ainsi qu'une méthode de diagnostic du cancer (par exemple le cancer du rectum ou le cancer du côlon) dans laquelle on utilise ces derniers. De manière plus spécifique il s'agit d'un peptide antigénique du cancer humain comportant une séquence d'acides aminés représentée par SEQ ID NO:2, 4 ou autre, et une méthode de diagnostic du cancer qui consiste à examiner s'il existe ou non, dans un échantillon de sérum du patient, un anticorps se liant audit peptide antigénique. La présente invention concerne ainsi un anticorps se liant au peptide antigénique, une méthode de diagnostic du cancer qui consiste à examiner s'il existe ou non, dans un prélèvement biologique du patient, un peptide antigénique se liant audit anticorps et des méthodes correspondantes.
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WO2004093900A1 (fr) * 2003-04-21 2004-11-04 Japan Science And Technology Agency Agent induisant une apoptose et procede pour induire une apoptose
WO2006080192A1 (fr) 2005-01-05 2006-08-03 National University Corporation Chiba University Gene specifique du cancer et kit de diagnostic utilisant ce dernier
WO2006080193A1 (fr) * 2005-01-06 2006-08-03 National University Corporation Chiba University Oncogene et kit de diagnostic utilisant ce dernier
WO2007086342A1 (fr) * 2006-01-27 2007-08-02 National University Corporation Chiba University Procédé pour détecter le cancer au moyen d'une variante d'épissage du régulateur fir de transcription du gène c-myc ou d'une séquence de répétition à quatre bases dans l'intron 2

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004093900A1 (fr) * 2003-04-21 2004-11-04 Japan Science And Technology Agency Agent induisant une apoptose et procede pour induire une apoptose
WO2006080192A1 (fr) 2005-01-05 2006-08-03 National University Corporation Chiba University Gene specifique du cancer et kit de diagnostic utilisant ce dernier
JPWO2006080192A1 (ja) * 2005-01-05 2008-06-19 国立大学法人 千葉大学 癌に特異的な遺伝子及びそれを用いた診断キット
JP4677565B2 (ja) * 2005-01-05 2011-04-27 国立大学法人 千葉大学 癌に特異的な遺伝子及びそれを用いた診断キット
US7998693B2 (en) 2005-01-05 2011-08-16 National University Corporation Chiba University Gene specific to cancer and diagnosis kit using the same
WO2006080193A1 (fr) * 2005-01-06 2006-08-03 National University Corporation Chiba University Oncogene et kit de diagnostic utilisant ce dernier
JPWO2006080193A1 (ja) * 2005-01-06 2008-06-19 国立大学法人 千葉大学 癌遺伝子及びそれを利用した診断キット
JP4677566B2 (ja) * 2005-01-06 2011-04-27 国立大学法人 千葉大学 癌遺伝子及びそれを利用した診断キット
WO2007086342A1 (fr) * 2006-01-27 2007-08-02 National University Corporation Chiba University Procédé pour détecter le cancer au moyen d'une variante d'épissage du régulateur fir de transcription du gène c-myc ou d'une séquence de répétition à quatre bases dans l'intron 2
JPWO2007086342A1 (ja) * 2006-01-27 2009-06-18 国立大学法人 千葉大学 ｃ−ｍｙｃ遺伝子転写抑制因子ＦＩＲのスプライシングバリアント又はイントロン２内の４塩基繰り返し配列による癌検出方法
JP4806776B2 (ja) * 2006-01-27 2011-11-02 国立大学法人 千葉大学 ｃ−ｍｙｃ遺伝子転写抑制因子ＦＩＲのスプライシングバリアント又はイントロン２内の４塩基繰り返し配列による癌検出方法

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