WO2019050273A1 - Méthode de diagnostic du cancer du pancréas à l'aide de méthionyl-arnt synthétase et marqueur spécifique aux cellules acineuses - Google Patents

Méthode de diagnostic du cancer du pancréas à l'aide de méthionyl-arnt synthétase et marqueur spécifique aux cellules acineuses Download PDF

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WO2019050273A1
WO2019050273A1 PCT/KR2018/010366 KR2018010366W WO2019050273A1 WO 2019050273 A1 WO2019050273 A1 WO 2019050273A1 KR 2018010366 W KR2018010366 W KR 2018010366W WO 2019050273 A1 WO2019050273 A1 WO 2019050273A1
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cell
mrs
protein
pancreatic cancer
cells
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Korean (ko)
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김성훈
권남훈
이동기
임범진
장성일
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(주)온코태그디아그노스틱
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Publication of WO2019050273A1 publication Critical patent/WO2019050273A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/0101—Methionine-tRNA ligase (6.1.1.10)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/976—Trypsin; Chymotrypsin

Definitions

  • the present invention relates to a method for diagnosing pancreatic cancer using methionyl-tRNA synthetase (MRS) and a precursor cell-specific marker, and more particularly, to a method for diagnosing pancreatic cancer using a methionyl-tRNA synthetase And a preparation for measuring the expression level of a precursor cell specific marker protein, a kit comprising the same, and a method for enhancing the accuracy of diagnosis of pancreatic cancer using the two proteins as a double marker.
  • MRS methionyl-tRNA synthetase
  • a preparation for measuring the expression level of a precursor cell specific marker protein a kit comprising the same, and a method for enhancing the accuracy of diagnosis of pancreatic cancer using the two proteins as a double marker.
  • Cancer is a group of diseases that can start from the growth of uncontrolled cells, infiltrate into the surrounding normal tissues or organs, destroy them, create new growth places, and take away the life of the individual. Despite having made remarkable progress in the search for new targets, including regulation of cell cycle or apoptosis, and cancer genes or cancer-suppressing genes, to conquer cancer over the last decade, It is increasing with development.
  • pancreatic cancer is the fatal cancer with a 5-year survival rate of 1-4% and a median survival time of 5 months. It has the worst prognosis of cancer of the human body. In the 80-90% of patients, the diagnosis is made in a state in which a radical resection is not possible, which is expected to be cured. Therefore, the prognosis is poor and the treatment depends on chemotherapy. Until now, it has been reported that 5- The therapeutic efficacy of several anticancer drugs including fluoro uracil, gemcitabine, and tarceva is extremely disappointing and the response rate to chemotherapy is only about 15%, which improves the prognosis of patients with pancreatic cancer This suggests that a more accurate and faster method of diagnosis is required for the diagnosis.
  • pathological examination is a test to identify the origin of disease mainly from morphological standpoint using extracted cells, tissues or organs, and it is a method to grasp gross finding, optical, electron microscope search It is an important test that is used for the diagnosis of disease.
  • pathological tests include histopathology and cytopathology.
  • histologic examination and cytodiagnosis test there are many differences between histologic examination and cytodiagnosis test, and it is known that there is a large difference between the histopathologic and cytological test results in the accuracy of diagnosis, sensitivity and specificity in well-known cancer markers . Therefore, even if it is a known cancer marker, it is considered as a separate problem whether the diagnostic efficacy can be practically achieved according to a specific specimen (tissue or cell).
  • TBS bethesda system
  • pancreatic cancer In the case of pancreatic cancer that can be operated through imaging studies, definitive diagnosis of pancreatic cancer is necessary through biopsy or endoscopic ultrasound cytology before the operation. Even if surgery is not possible, biopsy or cytology is necessary for histologic diagnosis for chemotherapy or radiotherapy.
  • Tumor markers for diagnosis of pancreatic cancer are CEA (reference value: 5.0 ng / mL) and CA 19-9 (reference value: 37 U / mL).
  • CA19-9 there is a problem that the specificity is low as a marker for diagnosing pancreatic cancer. According to one report, CA19-9 was elevated in about 1% of those who underwent physical examinations, and only 2/4 of cancer-free patients with elevated CA19-9 levels without symptoms were reported. According to the American Cancer Society guidelines, CA19-9 has been shown to have reduced sensitivity and specificity in pancreatic cancer screening.
  • Korean Patent No. 10-0819122 discloses a biomarker for diagnosing pancreatic cancer, including matrilin, transthyretin and stratin in, Has been disclosed as a pancreatic cancer marker
  • Korean Patent Application Publication No. 2008-0082372 discloses a technique using various pancreatic cancer markers.
  • Korean Patent Laid-Open Publication No. 2009-0003308 discloses a method for diagnosing pancreatic cancer by detecting the expression level of REG4 protein in a blood sample of an individual.
  • Korean Published Application No. 2012-0009781 discloses a method for diagnosing pancreatic cancer Korean Patent Application Laid-open No.
  • 2007-0119250 discloses an assay method for measuring the expression level of XIST RNA in cancer tissues isolated from an individual in order to provide a new gene LBFL313 differentially expressed in human pancreatic cancer tissue
  • U.S. Patent Application Publication No. 2011/0294136 discloses a method for diagnosing pancreatic cancer using biomarkers such as keratin 8 protein.
  • biomarkers such as keratin 8 protein.
  • the above-mentioned markers have a limitation in that they show a great difference in the diagnostic efficiency and accuracy of each marker, and in particular, they do not depend on the cytology analysis method and are clinically diagnosed as tumor cells or other disease states
  • there is no definite marker for determining whether a clear diagnosis of pancreatic cancer is more important In other words, in the case of the previously reported pancreatic cancer diagnostic markers, in the application of the cytological examination, Level diagnosis, the sensitivity and specificity are not good enough to be effective.
  • pancreatic cancer in order to diagnose pancreatic cancer, expensive examination such as CT, endoscopic retrograde cholangiopancreatography (ERCP), ultrasound endoscopy (EUS), and angiography is demanded in addition to the above tumor markers , It is very difficult to diagnose pancreatic cancer accurately.
  • the diagnosis of pancreatic cancer is made through endoscopic ultrasonic micro needle aspiration test in a patient who can not undergo surgery because the pancreatic cancer is a progressive cancer that is not easily resectable at the time of diagnosis and 10-15% .
  • endoscopic ultrasound microscopic needle aspiration cytology is difficult to compare with surrounding tissues or cells compared with pancreatic cancer tissues.
  • pancreatic cancer Because of this, we are still relying on pathological diagnosis based on general staining, such as H & E staining or pap staining, to differentiate cells from pancreatic cancer cells and other diseases (eg pancreatitis).
  • diagnosis of pancreatic cancer by the above-mentioned conventional staining method may be different according to the experience and the interpretation technique of the medical staff.
  • H & E staining or pap staining It is very difficult to distinguish between benign disease and other diseases.
  • it is difficult to prevent unnecessary surgery because the diagnosis of the cell can not be performed properly for the pancreatic cancer patient who can undergo surgery because the diagnosis is not accurate. Therefore, accurate diagnosis of cytology is needed to increase the therapeutic effect of pancreatic cancer clinically.
  • pancreatic cell sample High expression of MRS (methionyl tRNA synthetase) can be clearly distinguished from malignant tumor cells.
  • MRS methionyl tRNA synthetase
  • this classification is judged as atypical cells by conventional cytopathologic examination using H & E staining or pap staining (MRS) expression and the use of double-specific marker (s), such as chymotrypsin, as a dual marker, may be useful in the diagnosis of pancreatic cancer.
  • s double-specific marker
  • an object of the present invention is to provide a method for detecting a methionyl tRNA synthetase (MRS) protein from a pancreas sample collected from a potential patient to provide information necessary for diagnosis of pancreatic cancer. It is an object of the present invention to provide a method for detecting the expression of a precursor cell-specific marker protein and an MRS protein in a pancreatic sample collected from a latent patient, And
  • test sample of step (a) is a pancreatic cancer cell when the precursor cell-specific marker protein is not expressed and the MRS protein is expressed.
  • the cell is a pancreatic cancer cell.
  • pancreatic cancer (a) measuring the level of expression of the precursor cell-specific marker protein and the MRS protein in the pancreas sample collected from a potential patient; And (b) determining that the cell is a pancreatic cancer cell if the expression of the MRS protein is not expressed in the step (a) but the precursor cell specific marker protein is not expressed, and (Diagnosis of pancreatic cancer) that provides information necessary for diagnosis of pancreatic cancer.
  • the present invention provides an agent for measuring the expression level of a methionyl-tRNA synthetase (MRS) protein and an agent for measuring an expression level of an acinar cell specific
  • MRS methionyl-tRNA synthetase
  • the present invention provides a composition for diagnosing pancreatic cancer comprising an agent for measuring the expression level of a marker protein and a kit for diagnosing pancreatic cancer comprising the same.
  • the present invention provides a method for diagnosing pancreatic cancer, And a method for qualitative or quantitative analysis of the expression level of the MRS protein and the secretory cell-specific marker protein from the collected pancreas sample.
  • A measuring the expression level of a secretory cell-specific marker protein and MRS protein in a pancreas sample collected from a latent patient;
  • test sample of step (a) is a pancreatic cancer cell when the precursor cell-specific marker protein is not expressed and the MRS protein is expressed.
  • the present invention provides a method for improving sensitivity or specificity, comprising the steps of:
  • step (b) when the precursor cell-specific marker protein is not expressed in step (a) and MRS If the expression of the protein is increased, it is determined that the cell is a pancreatic cancer cell.
  • the present invention provides a method for the cytodiagnosis or histology of pancreatic cancer
  • the pancreatic cancer cell is further characterized in that it is a pancreatic cancer cell (Diagnosis of pancreatic cancer) that provides information necessary for diagnosis of pancreatic cancer.
  • a description in the range of 1 to 5 may be applied to individual values within the range, such as 1, 2, 2.7, 3, 3.5, 4.3 and 5, as well as from 1 to 3, 1 to 4, 5, 2 to 3, 2 to 4, 3 to 4, and the like. This applies irrespective of the width of the range.
  • &quot comprising " of the present invention is used synonymously with " containing " or " characterized ", and does not exclude additional component elements or method steps not mentioned in the composition or method .
  • 'consisting of' is used in the same way as 'consisting of' Quot; means excluding any additional elements, steps or components not listed.
  • Essential consistency of &quot means, in the context of a composition or method, to include a constituent element or step as well as constituent elements or steps which do not materially affect its essential properties, do.
  • pancreatic cancer refers to malignant tumor or cancer occurring in the pancreas. It refers to a malignant tumor or cancer which has a rapid growth rate and has a characteristic of penetrating into surrounding tissue and metastasizing to other organs )
  • the neo-bi means to come.
  • the malignant tumor or cancer is distinguished from a benign tumor, which has a slow growth rate and does not metastasize. More than 90% of cancers in the pancreas occur in pancreatic duct cells, and pancreatic cancer usually refers to pancreatic cancer or pancreatic ductal cancer. Therefore, preferably, the pancreatic cancer in the present invention may be a pancreatic duct (adeno) carcinoma.
  • the cause of the pancreatic cancer for the purpose of diagnosis in the present invention is not particularly limited as long as it is cancer of the pancreas due to metastasis or primary cancer. Preferably, it may be targeted to primary cancer.
  • " normal " in the present invention means a malignant tumor or a non-cancerous state (negative malignancy, negative malignancy), and includes a complete steady state without any disease, pancreatitis other than malignant tumor Other disease states, and / or " benign ".
  • the positive mark described as 'benign' in the clinical (final) disease state determination is distinguished from the positive mark on the corresponding test indicated by 'posit ive', and the positive mark as 'posit ive' It means that there is a reaction in the test method and the result of the test means the possibility of cancer.
  • pancreatic cancer is diagnosed by computed tomography (CT) or magnetic resonance imaging (MRI), and imaging or CT or MRI is used.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • pancreatic cancer is made indirectly through histological examination or cytology.
  • the histological examination can confirm that cancer is present in a specific area through comparison with surrounding structures or cells.
  • the cytological examination since the individual cells are extracted and stained, the relationship with the surrounding tissues can not be proved. And cytologic examination are fundamentally different.
  • pancreatic cancer protein analysis in body fluids such as plasma is measured at the same time to determine the presence or the possibility of pancreatic cancer.
  • serologic methods have only a meaningful role as a reference in the diagnosis of pancreatic cancer, and do not provide crucial information for the diagnosis of pancreatic cancer.
  • the most commonly used tumor markers associated with pancreatic cancer are carbohydrate antigene 19-9 (CA19-9) or carcinoembryogenic antigen (CEA).
  • CA19-9 is known to be inadequate for the diagnosis of pancreatic cancer because its serum concentration is elevated even in non-cancerous diseases such as hepatitis, cirrhosis and pancreatitis.
  • CA19-9 is not detectable in pancreatic cell itself, , It can not be said to be pancreatic cancer-specific. Therefore, CA19-9 serum levels are monitored after surgery to determine the prognosis of patients.
  • CEA does not exhibit sufficient sensitivity, specificity, positive predictive value, and / or negative predictive value for pancreatic cancer, which is a limitation in accurately diagnosing pancreatic cancer.
  • MRS was expressed in (high) pancreatic cancer, and found that when using MRS as a pancreatic cancer marker, diagnosis results can be obtained not only in histology but also in cytology. That is, MRS is highly expressed specifically in pancreatic cancer as compared to normal pancreatic cells (ie, non-tumorous pancreatic cells), and MRS is more sensitive to pancreatic cell samples than CEA, which is conventionally used as a pancreatic cancer marker Pancreatic cancer can be diagnosed, especially in the cytogenetic diagnosis Has demonstrated a remarkable effect of allowing highly precise identification of pancreatic cancer cells for atypical cells which are difficult to diagnose accurately by conventional cytopathologic examination methods (for example, H & E staining or pap staining). Especially, the diagnosis accuracy of pancreatic cancer was remarkably elevated when it was diagnosed using double marker as an additional marker specific protein such as chymotrypsin.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a preparation for measuring the expression level of a methionyl-tRNA synthetase (MRS) protein and a preparation for measuring the expression level of an acinar cell specific marker protein.
  • a diagnostic kit for pancreatic cancer comprising the same.
  • a composition for diagnosing pancreatic cancer which comprises an agent for measuring the expression level of the MRS protein and an agent for measuring the expression level of the secretory cell specific marker protein, and a kit for diagnosing pancreatic cancer comprising the same.
  • compositions for measuring the expression level of MRS protein and a composition for diagnosing pancreatic cancer which is essentially constituted as an agent for measuring the expression level of a precursor cell specific marker protein, and a kit for diagnosing pancreatic cancer comprising the same.
  • the present invention also provides the use of a system for measuring the expression levels of MRS protein and secretory cell-specific marker protein for preparing a diagnostic agent for pancreatic cancer, particularly a measurement agent for each of MRS or precursor cell marker proteins.
  • the term " diagnosis " means identifying (identifying) the presence or characteristic of a pathological condition.
  • the diagnosis may be performed by measuring the expression level or expression level of the MRS protein to confirm the presence or absence of pancreatic cancer.
  • 'MRS' means a methionyl-tRNA synthetase
  • the MRS is an enzyme that mediates aminoacylate ion reaction between methionine and tRNA.
  • the MRS protein of the present invention can be any of the specific sequences of MRS amino acid sequences known in the art And its biological origin are not particularly limited. For example, in humans, it is encoded in MARS gene, and the sequence information of MRS is ⁇ 004990 (111 ⁇ ), NP_004981.2, P56192. 2 protein) is known as the Genbank (NCBI) accession number.
  • the MRS of the present invention may include a human MRS protein amino acid sequence represented by SEQ ID NO: 1.
  • the MRS protein of the present invention may comprise the amino acid sequence of SEQ ID NO: 1.
  • MRS cyto lasmic form
  • mi tochondrial form mi tochondrhal methionyl-tRNA synthetase
  • the MRS in the present invention may preferably be a cytoplasmic form.
  • &quot express ion " means that a protein or nucleic acid is produced in a cell.
  • " protein " in the present invention is used interchangeably with a 'polypeptide ide' or a 'pept ide', for example, a polymer of amino acid residues as commonly found in natural state proteins.
  • the agent for measuring the expression level of the MRS protein may be an antibody or an aptamer that specifically binds to the MRS protein, although the type thereof is not particularly limited as long as it is known to be usable for measuring the expression level of the protein in the sugar chain .
  • " ant ibody " in the present invention means an immunoglobulin that specifically binds to an antigenic site. More specifically, it refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected to each other by a disulfide bond.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CHI, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)) in which more conserved regions, referred to as framework regions (FR), are scattered.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each of VH and VL consists of three CDRs and four FRs arranged in amino-terminal to carboxy-terminal to FRl, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • the constant region of an antibody can mediate the binding of immunoglobulins to the host tissue or factor, including the various components of the immune system (e. G., Effector cells) and the first component of the traditional complement system (Clq).
  • the anti-MRS antibody is an antibody that specifically binds only to the MRS protein without attacking other proteins including other aminoacyl thiourea synthetases in addition to MRS.
  • the antibody specifically binding to the MRS protein in the present invention may preferably be an antibody that specifically binds to a protein (MRS) comprising the amino acid sequence represented by SEQ ID NO: 1.
  • the anti-MRS antibody may be produced by cloning the MRS gene into an expression vector to obtain a protein encoded by the gene and obtaining an antibody produced by injecting the obtained protein into an animal, can do.
  • the MRS antibody may be prepared through an MRS full length sequence protein, or may be prepared by using a fragment of an MRS protein containing an MRS antigenic site to produce an MRS protein specific antibody.
  • the specific sequence and form of the antibody of the present invention are not particularly limited and include polyclonal antibody or monoclonal antibody.
  • the type of the immunoglobulin to be provided is not particularly limited, and may be selected from the group consisting of IgG, IgA, IgM, IgE and IgD, preferably an IgG antibody.
  • the antibody of the present invention includes a special antibody such as a humanized antibody, a chimeric antibody and a recombinant antibody as long as it can specifically bind to the MRS protein.
  • Some of the whole antibodies are also included in the antibody of the present invention as long as they have antigen-antibody binding (anti-human), and include all kinds of immunoglobulin antibodies that specifically bind to MRS.
  • Fab fragment antigene- binding
  • F (ab ') 2 is a fragment produced by hydrolyzing an antibody to pepsin, and two Fabs are linked from a medium chain hinge to a disulfide bond.
  • F (ab ') is the F (ab') 2 fragment Is a monomeric antibody fragment in which a heavy chain hinge is added to a Fab separated by reducing disulfide bonds.
  • Fv (variable fragment) is an antibody fragment consisting of only variable regions of heavy and light chains, respectively.
  • the single chain variable fragment is a recombinant antibody fragment in which the heavy chain variable region (VH) and the light chain variable region (VU) are linked by a flexible peptide linker.
  • the diabody is a linker with very short VH and VL of scFv Refers to fragments of the same type that bind to VL and VH of other scFvs and bind to each other to form a dimer.
  • fragments of the antibody are characterized by binding specificity to human-derived MRS protein It is not limited by structure or form.
  • the site where the antibody interacts with (i.e., binds to) MRS is not particularly limited, Or an antibody or a functional fragment thereof, which specifically binds to an epitope of a region including the amino acid sequence represented by SEQ ID NO: 2 in the MRS. More preferably an antibody or a functional fragment thereof that specifically binds to an epitope comprising the 861th to 900th amino acid regions of the MRS (methionyl-tRNA synthetase) protein represented by SEQ ID NO: 1 have.
  • the present inventors obtained an antibody that epitopes the amino acid sequence region represented by SEQ ID NO: 2 in MRS, It has been confirmed that it is possible to provide a high-sensitivity detection capability.
  • the antibody specifically binding to the epitope of the region including the amino acid sequence represented by SEQ ID NO: 2 is not particularly limited as long as it has the desired specific binding ability,
  • a light chain complementarity determining region KCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16
  • Light chain complementarity determining region 2 comprising the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 18
  • a light chain variable region (VU) comprising a light chain complementarity determining region 3 (CDR3) comprising an amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 20
  • a heavy chain complementarity determining region 1 (CDR1) comprising an amino acid sequence represented by SEQ ID NO: 10 or SEQ ID NO: 22
  • a heavy chain complementarity determining region 2 comprising an amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 24
  • a light chain variable region (VH) comprising a heavy chain complementarity determining region 3 (CDR3) comprising an amino acid sequence
  • the antibody (including its functional fragment) of the present invention may have a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 28 and a heavy chain variable region including the amino acid sequence represented by SEQ ID NO: 30 . ≪ / RTI >
  • the antibody (including functional fragments thereof) of the present invention may comprise a light chain variable region having an amino acid sequence represented by SEQ ID NO: 32 and a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: And may include amino acid sequences.
  • the present invention provides an antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 36 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 37.
  • the present invention provides an antibody comprising a light chain consisting of the amino acid sequence of SEQ ID NO: 38 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 39.
  • luciferin 2, 3-dihydropthalazine dione, maleate dihydrogenase, urase, Glucosamidase, lysozyme, saccharide oxidase (for example, glucose oxidase, galactose oxidase, and glucose-6) such as horseradish peroxidase (HRPO), alkaline phosphatase,? -Galactosidase, glucoamylase, -Phosphate dehydrogenase), heterocyclic oxidases (e. G., Free radicals and xanthine oxidase), lactoperoxidases, microperoxidases, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • glucoamylase glucoamylase
  • -Phosphate dehydrogenase heterocyclic oxidases
  • Biotin selectively binds to avidin, and thus this label can be conjugated to the antibody in this indirect manner.
  • the antibody may be conjugated to a small hapten (e. G., Digoxin) and one of the different types of labels mentioned above may be conjugated to an anti- Hapten antibody (e. G., An anti-diphoshin antibody).
  • a small hapten e. G., Digoxin
  • an anti- Hapten antibody e. G., An anti-diphoshin antibody
  • 'timer' refers to a single-stranded nucleic acid (DNA, RA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding with an analyte to be detected in a sample, The presence of the target protein in the sample can be confirmed.
  • Aptamer can be produced by selecting a target protein to be identified and having a high binding strength according to a general method of preparing an aptamer, determining the sequence of the nucleotide and synthesizing the oligonucleotide. The 5' end or the 3 ' But not limited to, a moiety capable of binding to the functional group of the aptamer chip, e.g., -SH, -OM, -OH or NH2.
  • the term 'acinar cell specific marker' means a marker showing a significant difference between a secretory cell and a non-secretory cell. Specifically, the presence (expression) or abundance (expression amount) of the gene is detected by detecting the presence (expression) or presence (expression) Objective measurement that can be distinguished Possible markers.
  • a marker proteins, DNA, RNA, metabolites and the like are generally used.
  • chymotrypsin phospholipase A2 group IB (PLA2G1B, phospholipase A2 group IB), and amylase A2 (SEQ ID NO: 2) are known to be known as specific cell-specific marker proteins in the art, amylase 2A) may be used.
  • the above-mentioned 'chymotrypsin' is not particularly limited as long as it is known in the art as chymotrypsin (particularly, human), and its specific amino acid sequence is not particularly limited.
  • double-staining was performed with an MRS antibody using an antibody having a marking of a chymotrypsin protein represented by NP_009203.2 (see SEQ ID NO: 3).
  • PLA2G1B Phospho 1 ipase A2 group IB
  • PLA2G1B protein in particular, human
  • NCBI NCBI accession numbers AAI06727.1, AAI06726.1, AAH05386.1, NP_000919.1, and so on.
  • amylase A2 (amylase 2 ⁇ ) is not particularly limited as long as it is known in the art as amylase A2 (particularly human).
  • Genbank NCBI accession number AAI46998.1 , AAH07060.1, BAD97183.1, AAA51723.1, and the like.
  • the expression level of the precursor cell-specific marker protein may be measured in a manner known to those skilled in the art.
  • the type of the marker protein may be specifically Binding antibody or aptamer, and a specific description thereof can be given by the above-mentioned anti-MRS Antibody and ⁇ Tumor.
  • the kit for diagnosing pancreatic cancer according to the present invention may further comprise one or more antibodies or uptamers which specifically recognize the proteins individually, Further, other component compositions, solutions or devices may be included.
  • the kit Western blot, ELISA, radioimmunoassay analysis, "radiation immunodiffusion, OY greater interrogating you immunodiffusion, rocket immunoelectrophoresis, immunological staining, immunoprecipitation assay, complement fixation assay, FACS, SPR or how a protein chip
  • the kit comprises an antibody specific for the MRS protein.
  • the antibody is a monoclonal antibody, a polyclonal antibody or a recombinant antibody, which has high specificity and affinity for a target marker protein and has substantially no cross reactivity to other proteins.
  • the kit may further comprise an antibody specific for any control protein.
  • the antibody provided in the kit may itself be labeled with a detectable moiety, as described above.
  • the kit may further comprise a separate reagent capable of detecting the bound antibody, for example, a labeled secondary antibody, chromophores, an enzyme (in conjugated form with the antibody) and its substrate, or an antibody And other materials that can be used.
  • the kit of the present invention may include a washing solution or an eluting solution capable of removing surplus chromogenic substrate and unbound protein and retaining only the protein marker bound to the antibody.
  • the agent that measures the level of expression of the MRS protein may also include an agent that detects the level of expression of the MRS gene (MARS).
  • MRS MRS gene
  • MRNA messenger RNA
  • the agent for detecting the MRS mRNA is not particularly limited as long as it is a ligand that specifically binds or hybridises to MRS mRNA, For example, a primer (pair) or a probe.
  • the agent for measuring the expression level of acinar cell specific marker protein includes an agent for detecting the expression level of the gene encoding the secretory cell specific marker protein.
  • an agent for detecting the expression level of the gene encoding the secretory cell specific marker protein for example, a preparation for detecting mRNA coding for a precursor cell-specific marker protein can be used.
  • the ligand is a ligand that specifically binds or reacts with the mRNA (11) ( ⁇ 23 011), the kind thereof is not particularly limited , For example a primer (pair) or a probe.
  • the 'primer' is a nucleic acid sequence having a short free 3 'hydroxyl group and can form a base pair with a complementary template and a short nucleic acid sequence serving as a starting point for template strand copying It says.
  • the primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at the appropriate complete solution and temperature.
  • reagents for polymerization i. E., DNA polymerase or reverse transcriptase
  • the PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.
  • the sequence of the primer does not need to have a sequence completely complementary to a partial nucleotide sequence of the template, but is stratified if it has a superficial complementarity within a range that can be reacted with the template and has a primer-specific action. Therefore, in the present invention, the primer for measuring the expression level of the target mRNA does not need to have a sequence completely complementary to the coding gene sequence of the protein of interest, amplifies a specific region of mRNA or cDNA through DNA synthesis, The amount of which is complementary to the length for which the purpose is to be measured.
  • the primer for the amplification reaction consists of a pair (pair) complementarily binding to a template (or sense) at opposite ends of a specific region of an mRNA to be amplified and an opposite region (antisense), respectively.
  • Primers can be readily designed by those skilled in the art with reference to mRNA or cDNA sequences encoding the protein.
  • Probe '. MRNA or cDNA (complementary DNA) of a specific gene refers to a fragment of a polynucleotide such as RNA or DNA having a length of several hundreds to several hundreds of bases that can be specifically bound and is labeled so that the presence or absence of mRNA or cDNA to be bound Presence, and expression of the cells.
  • a probe complementary to a target mRNA can be used for diagnosis by measuring the expression amount of a target mRNA by performing a reaction (11 13 ( ⁇ 2 1011) with a sample of a test sample.
  • the sanitizing conditions may be appropriately selected according to techniques known in the art.
  • the primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods.
  • the primer or the probe may be variously modified according to methods known in the art, so long as the primer or the probe does not interfere with the PCR with MRS mRNA.
  • modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) ) Or charged conjugate (eg, phosphorothioate, phosphorodithioate, etc.), and the labeling of the labeling material with fluorescence or enzymes.
  • uncharged linkers e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.
  • charged conjugate eg, phosphorothioate, phosphorodithioate, etc.
  • the diagnostic kit of the present invention may further comprise one or more other component compositions suitable for the assay, as well as primers or probes for recognizing mRNA for each of them in order to measure the expression level of MRS protein and / or secretory cell specific marker protein, Solutions or devices may optionally be included.
  • the kit is not particularly limited as long as it is known as an assay kit that provides a primer (primer pair) or a probe as a component in the art.
  • the kit includes PCR (polymerase chain reaction), RNase protection assay , Northern blotting, Southern blotting or kits for DNA microarray chips, and the like.
  • the diagnostic kit may be a diagnostic kit characterized by comprising essential elements necessary for performing the polymerase antagonism.
  • the Polymerase Enzyme Kit contains a respective pair of primers specific for the marker gene (mRNA).
  • a primer is a nucleotide having a sequence specific to the nucleotide sequence of each marker gene (mRNA), and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also contain a primer specific for the nucleic acid sequence of the control gene.
  • the polymerase kit can be used in a test tube or other suitable container, a reaction complete layer (with varying pH and magnet concentrations), deoxynucleotides (dNTPs), a DNA polymerase (such as Taq polymerase) DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.
  • a reaction complete layer with varying pH and magnet concentrations
  • dNTPs deoxynucleotides
  • a DNA polymerase such as Taq polymerase
  • DNAse DNAse
  • RNAse inhibitor DEPC-water sterile water, and the like.
  • the present invention also provides a method for qualitatively or quantitatively analyzing the expression level of MRS protein and secretory cell-specific marker protein from a pancreas sample collected from a potential patient to provide information necessary for diagnosing pancreatic cancer. That is, the present invention provides a method for diagnosing pancreatic cancer, which comprises measuring the expression level of MRS protein and secretory cell-specific marker protein in a sample of a subject. Specifically,
  • step (b) determining that the test sample of step (a) is a pancreatic cancer cell when the MRS protein is expressed without expressing the precursor cell-specific marker protein.
  • the term 'analysis' may mean 'measurement', and the qualitative analysis may be a measurement and confirmation of the presence or absence of a target substance, It may be meant to measure and confirm changes in the level of expression (level of expression) or amount.
  • the analysis or measurement can be performed without limitation, including both qualitative and quantitative methods. Therefore, detection of MRS protein includes detection of the presence of MRS protein, or confirmation of an increase (up-regulation) of the amount of protein expression.
  • the step (a) comprises providing a pancreatic sample collected from a latent patient and measuring the expression level of a secretory cell-specific marker protein and MRS protein in the sample .
  • the subject of the present invention may be an animal, preferably an animal including a mammal, particularly a human, and includes the animal-derived cells, tissues, organs and the like. More preferably, it may be a human or a patient who needs a diagnosis.
  • it is possible to perform a step of providing a sample from a subject or a potential patient before the step (a).
  • the term " potential patient " in the present invention means a patient suspected of having pancreatic cancer, and means a patient suspected of having pancreatic cancer through various tests such as clinical symptoms, hematological examination or imaging test.
  • the latent patient includes a patient who can be diagnosed as a pancreatic cancer based on an imaging test, and a patient who can not be diagnosed, and means a patient suspected of having a pancreatic cancer in a clinical symptom or hematological test even if the pancreatic cancer diagnosis is impossible.
  • Clinical manifestations of pancreatic cancer include abdominal pain, jaundice, weight loss digestive disorder, and diabetes, but these symptoms are not specific to pancreatic cancer. Hematologic tests may also increase the number of tumor markers such as jaundice, diabetes mellitus, CEA, and CA19-9. Abdominal ultrasonography, abdominal CT, abdominal MRI, and PET-CT can be used for imaging. Pancreatic cancer is suspected when there is a pancreatic mass.
  • pancreatic cancer may be suspicious for pancreatic cancer but not for pancreatic cancer.
  • the final diagnosis of pancreatic cancer is pathologic, and in patients who can undergo surgery, the disease is confirmed by the tissue obtained after surgery, and in patients whose operation is not possible, it is confirmed by cytology.
  • the latent patient (suspected patient of pancreatic cancer) of the present invention has general symptoms such as abdominal pain, jaundice, diminution of body weight, digestive disorder, diabetes and the like which are generally observed in pancreatic cancer, and diagnostic equipment such as CT, ultrasound, MRI The patient may not be able to confirm with pancreatic cancer.
  • the latent patient has a need for definitive diagnosis of pancreatic cancer dependent on cytology (cytogenetic) as it is impossible to perform a wide area invasive biopsy or unnecessary patient (i.e., because of the impossibility or unnecessary inspection of pancreatic tissue by surgery) . That is, it may be, but is not limited to, a patient in need of definitive diagnosis of pancreatic cancer by cellular level analysis.
  • the sample is not particularly limited as long as it is collected from an individual (patient) or a subject to be diagnosed for the presence or absence of pancreatic cancer, but may be preferably a pancreatic tissue or a pancreatic cell.
  • the pancreatic tissue can be obtained from all parts of the pancreas including the pancreatic duct, in particular, lesion suspected areas.
  • the pancreatic tissue may be one usually obtained by biopsy or surgery in the pancreas.
  • the method for isolating the pancreatic cells is not particularly limited and is understood to include not only the method currently used in the art for separating cells of a human tissue but also a new method to be developed for the same purpose in the future.
  • it may be brushing cytology or fine needle aspiration (FNA) or fine needle aspiration, and most preferably endoscopic ultrasonic microneedle inhalation (EUS-FNA).
  • FNA fine needle aspiration
  • EUS-FNA endoscopic ultrasonic microneedle inhalation
  • the term 'brush cytology' refers to a method of collecting cells by rubbing the pancreas, especially the surface of the pancreatic duct (particularly, the suspicious part of the lesion) using a conventional cytology brush.
  • the term 'separated by the fine needle aspiration method' in the present invention means a collection method in which cells of a lesion (suspected part of a pancreatic cancer) are aspirated and extracted using a thin needle commonly used for cytodiagnosis .
  • the pancreatic tissue or pancreatic cell sample essentially comprises pancreatic duct cells.
  • the pancreatic duct is distinct from the pancreatic parenchyma.
  • pancreatic cells or tissues may be pretreated according to conventional sample preparation methods (for example, fixed, centrifugal, slide-to-slide) methods known in the art.
  • the pancreatic cell or tissue sample may be pretreated by conventional paraffin block or paraffin section preparation methods and provided on a slide (slide) have.
  • the pancreatic cell or tissue sample may be prepared by a conventional method of preparing a liquid monolayer cell slide (slide production method for liquid cell examination), and may be prepared by, for example, ThinPrep, SurePath, of (Sl ide) by means of a liquid-based monolayer attachment method.
  • the method for diagnosing pancreatic cancer of the present invention may be preferably for analyzing pancreatic cells.
  • Cytology analysis methods for directly analyzing pancreatic cells are very different from those of the pancreatic cancer screening method, and therefore, there are many differences from the pancreatic cancer diagnostic methods reported in the above-mentioned prior art documents.
  • the pancreas cells themselves are used, there is no room for controversy with tumors of other organs.
  • histological examination was performed by endoscopically observing the target site or collecting tissues of a certain area ranging from 1 to 10 9 cel ls from the tissue suspected of being transformed into cancer, and then diagnosing cancer through a biochemical method such as dyeing .
  • Measuring the expression level of the protein means measuring the expression level (i.e., measuring the presence or absence of expression), or measuring the level of qualitative and quantitative change of the protein.
  • the measurement can be performed without limitation, including both qualitative (analysis) and quantitative methods.
  • the types of qualitative and quantitative methods for measuring protein levels are well known in the art and include the experimental methods described herein. Methods for comparing specific protein levels for each method are well known in the art.
  • the term 'detection' refers to measuring and confirming the presence (expression) of a desired substance (marker protein, MRS and / or secretory cell-specific marker in the present invention) Expression level) of the test compound.
  • the term " protein detection " in the present invention is meant to include detection of the presence of a target protein or confirmation of an increase (up-regulation) of the expression amount of the protein.
  • the term " increased expression (or high expression) " of a protein means that an expression is expressed (i.e., an undetectable one is detected) or a relatively overexpressed (i.e., ). This may involve performing a process involving comparison or contrast with a negative control.
  • the meaning of the opposite term thereof can be understood by one of ordinary skill in the art to have the opposite meaning according to the above definition.
  • the method is not particularly limited. For example, detection using an antibody specifically binding to the protein Or measured.
  • the antibody specifically binding to the target protein in the present invention is as described above.
  • the way how to measure the protein expression levels are known in the art and not particularly limited, for example, Western blotting, ELISA (enzyme-l inked i ⁇ unospeci 'f ic assay, ELISA), radioimmunoassay analysis, Immunoprecipitation, Immunoprecipitation, Immunoprecipitation, Immunostaining, FACS (Immunohistochemical Staining, Immunohistochemical staining, Immunofluorescence staining), Immunoprecipitation, Immunoprecipitation, cel l sorter), surface plasmon resonance (SPR), or protein chip method.
  • the measurement method is understood to be the method of measuring the MRS protein and the precursor cell specific marker protein expression level provided by the present invention, and the measurement method thereof in accordance with the description of the kit containing the same.
  • pancreatic cancer pancreatic cancer
  • abnormal cells atypical cells
  • pancreatic cancer markers have not been effective in cytologic examination due to poor sensitivity and specificity in diagnosis of cell - level, unlike histologic examination. This is well illustrated in one embodiment of the specification of the present invention.
  • CEA one of the tumor markers most commonly used for the diagnosis of pancreatic cancer, was not detected in pancreatic tumor cells, but was weakly expressed, and H & E staining revealed it to be an atypical cell It is also evident from the fact that no CEA was detected in the pancreatic cytosolic samples of patients who were finally confirmed as pancreatic cancer. That is, in the case of the previously reported pancreatic cancer markers, it is impossible to definitively diagnose pancreatic cancer or non-coexisting abnormal cells in the atypical cells as a pathological cell diagnostic method using conventional H & E staining, In the case of the MRS according to the present invention, it is possible to clearly determine whether the tumor is a pancreatic cell, which is determined to be atypical.
  • the MRS exhibits a remarkable effect of diagnosing pancreatic cancer more accurately. That is, the accuracy of the MRS according to the present invention is very high even when applied to the cytopathological examination. Especially, when the MRS according to the present invention is combined with the precursor cell-specific marker, the accuracy of false-positive judgment by the precursor cells is remarkably reduced, It has a rising effect.
  • the present invention further provides the following steps (i) and (ii) before, simultaneously or after the measurement of the expression level of the precursor cell-specific marker protein and the MRS protein (for example, Thereby further enhancing the diagnostic effect:
  • DAPH4 6-diamidino-2-phenyl indole, which stains pancreatic cells from potential patients, methylene blue, acetocarmine, toluidine blue, At least one staining solution selected from the group consisting of hematoxylin and Hoechst and a group consisting of eosin, crystal violet and orange G staining cytoplasm Staining the cells with the selected one or more staining solutions;
  • pancreatic cells (ii) judging the pancreatic cells as malignant tumor cells, atypical cells or normal cells by the cell staining.
  • the atypical cells identified in the step (ii) are understood to be undefined and undefined cells, but are not limited thereto. More specifically, the morphological diagnostic pathological test may also include the determination of Suspicious of malignancy (malignant tumor cell suspicion) May be inclusive.
  • the above steps (i) and (ii) are cytodiagnosis methods based on a conventional pathological examination of a morphological diagnosis method, which are based on H & E or pap staining used in an embodiment of the present invention.
  • the term " morphological diagnostic pathology " or " morphological examination &quot refers to examining abnormal morphological changes when normal cells are transformed into cancers.
  • the specific content is not specifically limited, preferably the cell gunjipseong; Nuclear / cytoplasmic ratio (N / C rat io); Shape of nuclear membrane (nuclear membrane irregularity); Aggregation of chromatin; Appearance of nuclear bodies in the nucleus; And the appearance of mitosis.
  • the morphological examination can be performed simultaneously or simultaneously with a method of providing information necessary for diagnosing pancreatic cancer by qualitative or quantitative analysis of expression levels of MRS protein and secretory cell-specific marker protein provided by the present invention (pancreatic cancer diagnosis method) Can be performed separately or sequentially.
  • the pancreatic cell sample is identified as a malignant tumor cell, an atypical cell or a normal cell.
  • the abnormal cell morphological change when the normal cell is transformed into cancer And the specific discrimination criteria are well known in the art.
  • the morphological change of the atypical cell means a malignant tumor cell (cancer cell) or a cell which can not be clearly determined as a normal cell.
  • the determination of the pancreatic cell sample as a malignant tumor cell, an atypical cell or a normal cell from the cell staining result of the step (i) in the step (ii) may be performed: the cell is plastically trichotomized; High nuclear / cytoplasmic ratio (N / c rat io, nuclear to cytoplasmic rat io); Appearance of chromatin aggregation; A rough nuclear membrane (the degree of irregularity of the nuclear membrane becomes larger); Emergence of nuclear bodies; And the appearance of mitosis, the tumor is judged to be a malignant tumor cell.
  • the cells are laminated in one layer, and the nucleus / cytoplasmic ratio (N / C rat io) is small and the nuclear membrane In the case of a smooth shape, it is judged as a normal cell, If the change in the cell does not reach the malignant cells but can not be determined as normal (including benign), it is judged to be an atypical cell (aypical cell).
  • the cell level test (ie, cytology test) It is characterized by what can be obtained.
  • the cell level test in the case of performing the steps (i) and (ii) before the MRS detection, in the case of pancreatic cells judged to be malignant tumor cells or normal cells through cell staining, By further re-analyzing the expression level (or absence) of the MRS and the precursor cell-specific marker protein, it is possible to more accurately determine whether the cancer is pancreatic cancer or not, thereby making it possible to significantly reduce the diagnosis error. , It is possible to make a clear judgment as to whether the tumor is a tumor by analyzing the secretory cell specific marker protein and the MRS expression level (or not) in the subsequent step (a).
  • the present invention is characterized by the fact that a double-staining method of MRS and a precursor cell-specific marker protein enables accurate diagnosis with high accuracy in examination not only of tissue but also of cell level.
  • a double-staining method of MRS and a precursor cell-specific marker protein enables accurate diagnosis with high accuracy in examination not only of tissue but also of cell level.
  • the present invention which provides accurate diagnosis at the cell level has a great advantage.
  • step (b) when the MRS protein is expressed (increased expression) in the measurement sample of step (a) without expression of the secretory cell-specific marker protein, it is determined to be a pancreatic cancer cell.
  • MRS was not detected (increased expression) in pancreatic cells determined to be normal cells through H & E staining, but MRS It was confirmed to be strongly expressed. That is, it is confirmed that MRS can be used as a pancreatic cancer diagnostic marker.
  • MRS was detected in the pancreatic cells of patients whose diagnosis was finally confirmed as pancreatic cancer after H & E staining was confirmed to be atypical.
  • MRS is not expressed in most of the normal cells, but in a few normal samples, MRS is expressed, resulting in false positive. This false positive result is confirmed by MRS in acinar cell Which is caused by the phenomenon being expressed.
  • the step (b) is advantageous in that pancreatic cancer (particularly pancreatic cancer or pancreatic ductal adenocarcinoma) can be detected directly through detection of (high) expression of MRS without comparison with another control group (sample).
  • the level of MRS detection (the level of MRS expression, particularly the level of increase), which is a standard for diagnosis of pancreatic cancer, can be determined by dividing the degree of detection (expression) or the degree of detection (expression) according to the measurement method selected by a person skilled in the art. For example, by measuring the levels of MRS expression in a number of normal subjects and patients, data can be accumulated and analyzed to determine appropriate criteria for diagnosis, such as normal category and pancreatic cancer occurrence category according to the level of MRS detection (expression) .
  • the step (b) may be performed relatively to the negative control sample (in particular, the normal control sample).
  • the method may further comprise comparing the MRS protein level detected from the pancreatic sample collected from the potential patient with the negative control sample in step (b) or after step (b).
  • the term normal control group refers to a pancreatic sample collected from a normal human part in a pancreas of a potential patient to be examined (i.e., the same individual as a patient to be examined in the step (a)) or other normal individuals It is meant to include all samples taken from the pancreas.
  • the level of MRS protein detected in the pancreas samples taken from the patient is higher than the negative control (especially normal control) level, it can be judged to be a pancreatic cancer patient.
  • Information on such a control group for example, detection intensity or expression intensity
  • MRS expression level measurement preparation provided in the present invention described below and a kit containing the same, .
  • the control group it is interesting to judge that the level of MRS expression level in the test sample is a pancreatic cancer patient.
  • test sample of step (a) is a pancreatic cancer cell when the precursor cell-specific marker protein is not expressed and the MRS protein is expressed.
  • step (b) comparing the level of methionyl thi ene synthase measured in the step (a) with that of the negative control, the expression level of methionyl thiourea synthetase was increased and the secretory cell specific marker protein was increased And determining that the cell is a pancreatic cancer cell if the cell is not expressed.
  • " normal pancreatic cell " or " normal control group " in the present invention means a non-tumor (cancer) pancreatic cell. It means a pancreatic cell in a state other than a cancer (cancer) , Benign (benign), and fully healthy pancreatic cells (atherosclerotic pancreatic cells).
  • the present invention also provides a method for distinguishing atypical cells from cancer cells (malignant tumor cells) and normal cells (non-cancer cells), including steps (a) and (b) described above.
  • pancreatic cancer In order to diagnose pancreatic cancer, it is necessary to perform expensive and thorough examination such as computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP), ultrasound endoscopy (EUS) and angiography. It is very difficult to diagnose correctly. Ultimately, pathologic methods such as biopsy or cytology should be performed to confirm pancreatic cancer. On the other hand, pancreatic cancer is a case of progressive cancer that is not easily resectable at the time of diagnosis, and in cases where surgery is impossible, only 10-15% of patients can not undergo surgery. Diagnosis of pancreatic cancer is performed through endoscopic ultrasonic micro needle aspiration test .
  • pancreatic cancer there is a limitation in confirming the diagnosis of the cell diagnosis by the endoscopic ultrasonic microneedle suction test because it is difficult to compare with the surrounding structure or cells compared with the postoperative pancreatic cancer tissue.
  • Existing cytopathologic diagnosis depends on general staining such as H & E staining or pap staining to differentiate cells of pancreatic cancer cells and other diseases (for example, pancreatitis).
  • the diagnosis of pancreatic cancer by conventional cytopathologic diagnosis based on conventional conventional staining is different according to the experience and interpretation technique of the medical staff.
  • pancreatic cancer or normal cells non-pancreatic cancer cells (Eg, pancreatic cancer cells, etc.)
  • pancreatic cancer cells Eg, pancreatic cancer cells, etc.
  • it is often an atypical cell and it is often necessary to perform additional and frequent repeated examinations .
  • it is judged as atypical cell in pancreatic cell observation through H & E staining or pap staining it is very difficult to distinguish whether it is pancreatic cancer or other benign disease.
  • the accurate diagnosis of the cell division is delayed, it is impossible to appropriately treat a pancreatic cancer patient who can undergo surgery, and conversely, it is difficult to prevent unnecessary surgery. Therefore, accurate diagnosis of cytology is needed to increase the therapeutic effect of pancreatic cancer clinically.
  • H & E staining and pap staining which are commonly used to diagnose cancer, are atypical cells that are difficult to distinguish between tumor or non-tumor.
  • the diagnosis of tumor is very important clinically, and it can be said that it is very meaningful in that the expression of MRS (high) in these atypical cells is confirmed (except for the precursor cells), so it can be determined as a tumor cell.
  • MRS high
  • the physical burden on the patient is increased in the acquisition of the sample rather than in the cytological examination.
  • the operation can not be performed according to the progress of the cancer,
  • the diagnostic method of the present invention which provides an accurate diagnosis even at the cellular level, has a great advantage.
  • the sensitivity and specificity In the diagnosis of pancreatic cancer, when employing the method of the present invention in which the MRS protein and the secretory cell-specific marker protein are simultaneously used at the same time and their detection patterns are analyzed, the sensitivity and specificity , The positive predictive value and / or the negative predictive value are almost 100%.
  • the present invention provides a method for improving sensitivity or specificity in cytodiagnosis or histology of pancreatic cancer comprising the steps of:
  • the cell is a pancreatic cancer cell.
  • the term 'sensitivity' refers to the rate at which a final clinical pathological diagnosis is made for a pancreatic cancer sample or a patient through a subject test method (eg, the test method of the present invention).
  • the term 'specificity' refers to the rate at which a normal determination is made through a subject test method (eg, the test method of the present invention) for a sample or patient whose final clinical pathological diagnosis is normal.
  • a subject test method eg, the test method of the present invention
  • at least one selected from the group consisting of sensitivity, specificity, positive predictive value, and negative predictive value is 80% to 100%>, preferably 85% to 99%, more preferably 90 to 98% Level). Specific figures for this level are 80, 81, 82, and 83%.
  • a boundary value of 80% and 95% in the numerical range may be selected, so that all values in the range of 80% to 95.4% It will be apparent to those skilled in the art.
  • the sensitivity, specificity, positive predictive value and / or negative predictive value are preferably at least 85% (85% to 100%, preferably 87% to 99%, more preferably, 90 to 98%), but is not limited thereto.
  • the method of the present invention can be understood as a method of improving the accuracy, and it is preferable that the accuracy is in the range of 90% to 100%, and more preferably the accuracy is in the range of 90% to 99%.
  • the specific values of the above levels were 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% , 97.5%, 98%, 98.5%, 99%, 99.5%, and 100%.
  • a boundary value of 92.5% and 99.5% in the numerical range may be selected, so that all values in the range of 93.5% to 99.5% It will be apparent to those skilled in the art. In yet another embodiment, it is more preferred that it exhibits a level of from 93% to 98%, most preferably from 95% to 98%, but is not limited thereto.
  • the present invention also relates to a method of screening for cytodiagnosis or histologic examination of pancreatic cancer
  • step (b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and the expression of the MRS protein is increased to be a pancreatic cancer cell
  • the morphological examination includes all of the other morphological examination methods according to this method, including the tests performed including the steps (i) and (ii) described above as a preferable example. A description thereof will be made by those skilled in the art with reference to the above description. .
  • steps (a) and (b) A detailed description of the steps (a) and (b) is as described above, and when this step is performed as an adjuvant (i.e., as an adjuvant therapy), the morphological examination is simulataneous, ) Or sequentially (sequential).
  • the determination method including steps (a) and (b) above can be performed before, simultaneously, or after the morphological inspection.
  • MRS is more accurate than conventional pancreatic cancer markers such as CEA. Especially, when double-marker markers are used as markers of chimotrypsin, Lt; / RTI >
  • FIG. 1 is a graph showing the MRS binding strength and specificity of anti-i-MRS antibodies (1E8, 8A12), using cell extracts of H460 cells treated with si-MRS,
  • FIG. 2 shows the MRS binding strength and specificity of the ant i-MRS antibodies (1E8, 8A12) of the present invention using the cell extracts of the PANC-1 cell line (pancreatic cancer cell line) and the SCK cell line (non-pancreatic cancer cell line) Western blot compared to antibody (Abl 37105).
  • Figure 3 shows the effect of different ARS (aminoacyl-tRNA synthetase) of 1E8 antibody on AIMP protein The results of ELISA for confirming the cross-reactivity of the compounds are shown in the graph.
  • ARS aminoacyl-tRNA synthetase
  • FIG. 4 is a graph showing the results of ELISA performed to confirm the cross-reactivity of the 8A12 antibody against other ARS (amino acid synthetase), AIMP protein.
  • FIG. 5 shows SPR (surface plasmon resonance) test results for confirming antibody affinity of 1E8 antibody against MRS + AIMP3 protein.
  • Fig. 6 shows the result of SPR (Surface lasmon resonance) test in which 1E8 antibody was confirmed to have no anti-maleicity against AIMP3.
  • FIG. 7 shows SPR (surface plasmon resonance) test results performed to confirm antibody affinity of 8A12 antibody against MRS + AIMP3 protein.
  • Fig. 8 shows the result of SPR (surface lasmon resonance) test in which 8A12 antibody was confirmed to be non-reactive for AIMP3.
  • Fig. 10 shows the results of immunohistochemical staining of Thinprep slides similar to clinical conditions with PANC-1 cell line using Thinprep equipment (Hologi c. Inc.) Used for patient cell sample processing at clinical sites and immunization of 1E8 antibody and 8A12 antibody of the present invention Fluorescence staining was performed.
  • FIG. 11 shows the results of H & E staining of pancreatic cells isolated from normal patients, showing the expression of MRS Observation results and observations of the expression of CEA are shown (each patient's code number).
  • FIG. 12 shows the results of H & E staining, MRS expression, and CEA expression in pancreatic cancer cells isolated from patients with pancreatic cancer.
  • FIG. 13 shows the result of observation of the expression of MRS and the observation of the expression of CEA in the pancreatic cytosine sample of a patient confirmed to be atypical as a result of H & E staining.
  • FIG. 14 shows the results of observing MRS expression intensity in normal donor cells (acinar cells) in normal pancreatic tissues.
  • FIG. 15 shows the results of H & E for acinar cells in normal pancreatic tissues
  • FIG. 16 shows the result of double detection of MRS and chymotrypsin protein for acinar cells in normal pancreatic tissues.
  • FIG. 17 shows a pattern pattern in which a representative staining pattern of two cells of the pancreatic precursor cell cytosine, when the double staining method of the present invention is applied, is very weakly detected and relatively strong detection of chymotrypsin is observed .
  • FIG. 18 shows that, when the double staining method of the present invention was applied to the pancreatic precursor cell cytology sample, the MRS detection was significant as well as the chymotrypsin among the representative staining patterns represented by the precursor cells, but the pattern pattern in which the detection of the chymotrypsin was relatively strong Lt; / RTI >
  • FIG. 19 shows the results of performing the double staining method of the present invention in a pancreatic cell sample of a pancreas cancer confirmed by papain staining and finally a confirmed pancreatic cancer patient.
  • Example 1 Preparation of a useful antibody for the pancreatic cancer test method of the present invention (Obtaining an antibody having high specificity for MRS) MRS (methyonyl-tRNA synthetase) in vivo is an AMP3 (Aminoacyl tRNA synthetase com lex- protein 3), and it is known that such binding state is separated by UV irradiation or the like. Therefore, in order to accurately detect MRS, it is necessary to specifically detect MRS only in a state where MRS binds to AIMP3. However, since there are many similarities in protein structure between AIMP and ARS species, AIMP and ARS species. Thus, for the diagnostic accuracy of the pancreatic cancer assay of the present invention, the present inventors produced MRS antibodies with high sensitivity without cross-reactivity to other proteins by the following method.
  • MRS methyonyl-tRNA synthetase
  • AMP3 Aminacyl tRNA synthetase com lex- protein 3
  • MRS-AIMP3 protein The MRS-AIMP3 co-purif ied protein was expressed on E. coli
  • the specific experimental method is as follows. MRS (SEQ ID NO: 1) and AIMP3 SEQ ID NO: 40 using the BL21DE3 strain, NCBI ref. After culturing in LB medium, single colonies were cultured in 5 ml LB liquid medium containing ampicillin so that the 0D600 value was 0.6-0.8. Then, ImM IPTG was added, followed by culturing at 37 ° C for 3 hours, followed by centrifugation for 10 minutes to obtain only cells. SDS-PAGE was performed with the cell solution, and expression of the proteins was confirmed using a coomassie stain.
  • the cell solution which induced the overexpression by IPTG was collected and centrifuged to obtain cells.
  • the cells were lysed with imi DPBS and then lysed with an ultrasonic mill, and then centrifuged with the lysed cells to isolate the MRS-AIMP3 co-puried protein.
  • mice were firstly injected intraperitoneally. 10-week old BALB / c mice weighing 25-30 g were purchased from Orient Bio Co. (Sungnam, KyungKiDo, Republ ic of Korea), and animals were layered under constant conditions (temperature: 20 ⁇ 2 ° C, humidity: 40-60%, darkness: 12 hour light / dark cycle) We used this study. Animal experiments were conducted in accordance with the guidelines of the University Animal Care and Use Committee at Seoul National University.
  • MRS-AIMP3 co-puri fi ed proteins of the same dose were injected into the abdominal cavity two weeks later.
  • MRS-AIMP3 co-puried protein was injected into the tail vein of mice three days before the cell fusion experiment.
  • the immunized mice were anesthetized with ether, and blood was collected from the heart with a heparinized syringe. The blood was allowed to stand overnight at 4 ° C and centrifuged to separate the serum. Separated serum was appropriately divided and stored at -80 ° C.
  • Myeloma cells were prepared for cell fusion. Myeloma cells were cultured and the cell density was 2.5 to 5> 10 ⁇ 11 > / 111 lbs. Myeloma cells were prepared by concentrating 1/3 of the cells 24 hours before cell fusion. The mice immunized in the above Example 1-2 The cells were anesthetized with ether and spleens were harvested to separate B cells, followed by washing with SF-DMEM2 (DMEM + 2XAA) and eluting the cells. The cell suspension was collected, placed in a tube, allowed to settle, and the supernatant was transferred to a new tube and centrifuged at 1500 rpm for 5 minutes.
  • SF-DMEM2 DMEM + 2XAA
  • the supernatant of the centrifuged splenocytes was removed and tapping followed by filling of SF-DMEM2.
  • B cells and myeloma cells were each centrifuged and washed, and the washing procedure was repeated one more time.
  • the upper layer of the washed myeloma cells was removed, tapped and filled with SF-DMEM2.
  • the cells were wrapped, and then red blood cells (RBCs) were added to the LBClysis buffer ⁇ , followed by filling with SF-DMEM2.
  • B cells and myeloma cells were centrifuged, centrifuged B cells and myeloma cell supernatants were removed, and tapping and SF-DMEM2 10 ⁇ was filled.
  • the B cells with myeloma cells was determined for each concentration of 100 fold dilution and counting in a tube e- [concentration of the B-cell levels (1X10 8, 8X10 7, 5X10 7), myeloma cells (1X10 7, 8X10 6, 5 X10 6 ).
  • B cells and myeloma cells were determined at a ratio of 10: 1. The determined concentration of B cells and myeloma cells were put into tubes and centrifuged.
  • the supernatant was spun down on the alcohol solution, semi-dried and tapped for 30 seconds to 1 minute. PEG (2) was slowly added for 1 minute while pipetting and reacted. The tube was shaken while inserting SF-DMEM2, followed by centrifugation. After centrifugation, the supernatant was removed and the HT medium (HT50X (HT (sigma) 1 vial + SF-DMEM1OmI) li, FBS lOm ⁇ , SF-DMEMKDMEM + lxAA) 30] , And gradually increased to 50 at the same time. The suspension was again incubated at 37 ° C in a 5% CO 2 incubator for 3 hours.
  • HT50X HT (sigma) 1 vial + SF-DMEM1OmI) li, FBS lOm ⁇ , SF-DMEMKDMEM + lxAA
  • Example 1-4 Selection of Hybridoma Cells Producing MRS-Specific Monoclonal Antibodies Cells that did not recognize AIMP3 were selected while confirming MRS among the fusion cell groups prepared in Example 1-3, The experiment was carried out as follows. First, the medium was changed from 8 to 9 days after cell fusion, and cultured in 96 wells and 24 wells were cultured in cDMEM2 until well grown. After the medium was changed, the supernatant of the color-changed wells was collected on day 5-7 and filled with CDMEM2. ELISA test for the binding of MRS and AIMP3 to the antibody produced in each fusion cell was performed. After the ELISA test, wells were selected and cultured in 24 wells.
  • Fusion cells selected on the basis of the primary screening were transferred to 24 wells, cultured and centrifuged, and the supernatant was collected and subjected to secondary screening by ELISA and Western blotting.
  • the absorbance (0.D value) of the fused cells grown in 24 wells was confirmed by ELISA. Only the fusion cells with the absorbance exceeding 1.0 were selected and transferred to a 25T / C culture flask. The cells were cultured and centrifuged, and the supernatant was collected and subjected to ELISA Screening was carried out.
  • the fusion cells selected on the basis of the tertiary screening were transferred to a 75T / C culture flask, cultured, and the absorbance was confirmed by ELISA to select cells that did not recognize AIMP3 while recognizing MRS. Finally, “1E8" and “8A12" clones Respectively.
  • Monoclonal antibodies to MRS can be obtained from the final fusion cells (hybridoma cells 1E8 or 8A12) selected in Examples 1-4, respectively, by the following two methods.
  • the ascites was left overnight at 4 ° C, and centrifuged the next day to remove lumpy material including the yellow fat layer and separate only the supernatant.
  • the supernatant was separated and stored at -2CTC.
  • the column was filled with Protein A, which was stored in a stock solution (20% ethanol) for antibody purification, and 20% ethanol was added to the column.
  • the aliquots were dialyzed with an appropriate amount of phosphate buffer solution and loaded onto a Protein A column. After binding with 3 bed volume binding buffer (20 mM sodium phosphate, pH 7.0), 3 bed (0.5 M glycine buffer, H 3.0 2.5).
  • Each fraction was neutralized with the neutralization buffer (1 M Tris-HCl, pH 9.0). Through SDS-PAGE, the purity of the fractions was determined and desalted with an Ammersharm GE column.
  • Hybridoma cells obtained in the above Example 1-4 were acclimated in a serum-free medium (Thermo) supplemented with GlutaMAX (Gibco) (final 5 mM) and lx Cholesterol I ipid concentrate (Gibco) Hybridoma cells are also named 34-8F2). The cells were then cultured in Cel lstack-5 (Corning, Corning, NY) at a maximum culture volume of 860 mL.
  • GlutaMAX (Gibco) (final 5 mM) and lx Cholesterol l ipid concentrate (Gibco) were added to serum-free medium (Thermo) and the initial cell concentration was inoculated at 1.4-2.0 x 10 5 cel / mL. After 4 to 5 days of inoculation, the cells were centrifuged at 2000 rpm for 10 minutes, and the supernatant was recovered. After confirming the pH of the supernatant, the pH was adjusted to 7.6 using a 20X binding solution (1M Potassium phosphate dibasic) (pH 9.0). And then filtered using a 0.22um filter to obtain a neutralized antibody culture. The obtained antibody culture was purified through a protein A column.
  • the GE PD-10 column was equilibrated with 25 ml of physiological saline and then centrifuged (1000 g , 2 minutes). Then, 2.5 ml of the antibody eluant obtained from the protein A column was added to the above GE PD-10 column, centrifuged (lOOOg, 2 minutes), and the antibody-exchanged antibody was collected with physiological saline. The antibody concentration was determined by measuring absorbance at 280 nm, divided and stored at -80 ° C.
  • the results of identification of the antigen binding site of the 1E8 antibody are shown in Table 1, and the results of confirming the sequence of the antigen binding site of the 8A12 antibody are shown in Table 2.
  • Fab was synthesized with the confirmed sequence and confirmed by ELISA that MRS shows high binding ability. Also, it was confirmed that the sequence identified above was consistent with the result of protein sequence analysis (mass spectrometry result) of the antibody obtained through multiple purification after the hybridoma cells were injected into mouse abdominal cavity in Example 1-5.
  • the resulting 1E8 Fab or 8A12 Fab sequences were cloned into mouse IgG heavy chain (pFUSE-mIgG2a-Fc, InvivoGen) and mouse lute chain sequencing vectors (pFUSE2-CLIg-mK, InvivoGen).
  • the vector was then co-transformed into freestyle 293F cells using PEKPolysciences, 23966-2) so that the light and heavy chains of the antibody were coexpressed simultaneously in the cells.
  • Transformed 293F cells were cultured for 7 days at 37 ° C and 8% CO 2 . Cells were then harvested and centrifuged to obtain supernatants.
  • the pH of the supernatant was adjusted to 7.6 using the prepared 20X binding solution (1M Potassium phosphate dibasic) (pH .9.0). The supernatant was then filtered with a 0.22 [mu] pi filter to obtain a neutralized antibody culture.
  • Antibodies are obtained from the antibody culture by the method described in 2) of Example 1-5.
  • the total antibody of 1E8 IgG thus obtained was confirmed to consist of a light chain consisting of the amino acid sequence of SEQ ID NO: 36 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 37. It was also confirmed that the whole antibody of 8A12 IgG was composed of the light chain consisting of the amino acid sequence of SEQ ID NO: 38 and the heavy chain consisting of the amino acid sequence of SEQ ID NO: 39.
  • H460 cells were cultured in DMEM (Hyclone, GE li fesciences) medium containing 10% FBSCFetal bovine serum, Hyclone, GE-1 i fesciences and 1% penicillin (Hyclone, GE li fesciences). Each cell was cultured under the conditions of 5% CO 2 and 37 ° C. The cultured H460 cells were treated with si-MRS for 72 hours.
  • the H460 cells were then harvested, lysed and then subjected to western blotting with H460 cell lysate. The experiment was repeated twice.
  • 1E8 antibody or 8A12 antibody was used at 1: 5000 (0.2 yg / ml), and MRS antibody (Abeam, Ab50793) distributed in the market was used in the same manner for comparison of binding ability. (Tublin) was used.
  • Experimental Results As shown in Fig. 1, conventional MRS antibodies circulating in the market In the si-MRS treated group, no MRS was detected. In the si-MRS-exposed group, the detection ability (binding ability to MRS) was significantly lower than that of the 8A12 antibody and 1E8 antibody of the present invention.
  • the 1E8 antibody and the 8A12 antibody of the present invention were remarkably superior to the conventional MRS antibody in MRS specific binding ability and sensitivity, and the 8A12 antibody showed excellent binding ability and sensitivity.
  • PANC-1 pancreatic cancer cell line and SCK (non-pancreatic cancer cell line) cell line were used to further confirm the MRS binding ability of the 1E8 antibody and the 8A12 antibody.
  • a commercially available MRS antibody (Abeam, Abl 37105) was used (antibodies were used at a ratio of 1: 1000, 0.137 / / ⁇ 1) Western blot experiment.
  • the 1E8 antibody and the 8A12 antibody of the present invention specifically detected the MRS, but the existing commercial antibody Abl37105 antibody exhibited a large number of nonspecific bands under the same conditions, indicating that the selective detection ability was very poor.
  • MRS was not detected in SCK cell line (non-pancreatic cancer cell line) under this experimental condition.
  • MRS protein His-MRS, MRS ful1
  • other ARS proteins DX2 tag free, 34S-DX2, 34S-AIMP2, His
  • 1E8 antibody or 8A12 antibody was added to a 96-well plate coated with each of the ARS proteins at a concentration of 500 ng / ml and reacted for 1 hour.
  • HRP-conjugated anti-mouse IgG secondary antibody was added thereto for 1 hour And subjected to ELISA at 450 nm. Absorbance was measured.
  • the substrates include, but are not limited to, ⁇ (3,3 ', 5,5'
  • Tetramethylbenzidine Tetramethylbenzidine
  • the 1E8 antibody reacts only with MRS, But not to other ARS and AIMP proteins.
  • the 8A12 antibody was found to bind only to MRS and to counteract other ARS and AIMP proteins. As a result, it was confirmed that the 1E8 antibody and the 8A12 antibody had no cross-reactivity to other ARS protein and AIMP protein and specifically detected only MRS.
  • MRS + AIMP3 protein MRS-AIMP3 co-puried protein
  • AIMP3 protein MRS-AIMP3 co-puried protein
  • MRS + AIMP3 or AIMP3 protein was coated on a CM5 chip and the degree of binding reaction with protein was measured while flowing 1E8 antibody or 8A12 antibody at various concentrations.
  • the analytes and buffers were injected at a flow rate of 30 l / min for 8 minutes and washed for 20 minutes. As a result, as shown in FIG. 5 and FIG.
  • the positions of the respective MRS fragments were 1 to 266aa fragment, 267 to 597aa fragment, 1 to 598aa fragment, 598 to 900aa fragment, 660 to 860aa fragment, 660-900 fragment, and 730-900 fragment in the entire amino acid sequence of MRS in SEQ ID NO: And other sub-areas.
  • the Myc protein was bound to the N-terminal of each peptide, Myc protein was used as a control.
  • H460 cells were transfected (transfect ion) using Turbofect (Thermo) along a vector DNA Cloning 2 U g to the manufacturer's instructions. After 24 hours, cells were obtained and subjected to Western blotting.
  • 1E8 antibody and 8A12 antibody were used as a primary antibody by 1: 5000 (0.2 U g / mL). Through these experiments, it was found that the 1E8 antibody and the 8A12 antibody have at least 598-900 aa region epeptide in the MRS protein of SEQ ID NO: 1.
  • 1E8 antibody or 8A12 antibody was used (1x PBST-Tween 0.05%) at a concentration of ⁇ O ⁇ M
  • HRP conjugated Goat anti-mouse IgG was diluted 1: 10000 with a secondary antibody (lxPBST-TweenO .05%), and absorbance was measured at 450 nm.
  • PANC-1 pancreatic cancer cell line and SCK (non-pancreatic cancer cell line) cell line PANC-1 pancreatic cancer cell line and SCK (non-pancreatic cancer cell line) cell line.
  • MRS fluorescence staining was performed using 1E8 antibody or 8A12 antibody, respectively.
  • a commercially available MRS antibody (Abeam, Abl 37105) was used as a control.
  • dyeing was carried out by the following procedure. To each of the target cells prepared on the slide, 0.2% tween 20 was treated with PBS to increase permeability and then blocked with 2% goat serum for 1 hour.
  • the antibodies (1E & antibody or 8A12 antibody, manufactured by Oncotag) or Abl37105 antibody (Abeam) were treated with lyg / ml 37 ° C for 1 hour and washed three times with 0.05% TBSTC . Then, as a secondary ant ibody with a fluorescent substance bound thereto, Al exa-488-conjugated secondary .
  • Ant i id ies purchased from Molecular Probes, Cat. No. A11001
  • the mounting solution with DAPI Pro mg Gold ant i fade regent with DAPI / Molecular probes, Cat. No. P36931
  • DAPI Pro mg Gold ant i fade regent with DAPI / Molecular probes, Cat. No. P36931
  • 20 ⁇ l of tissue on the slides, covered with coverslips, And fluorescence microscopy As shown in FIG. 9, the commercially available Abl37105 antibody, which was confirmed to have low MRS specificity in Example 1-7, nonspecifically stained both PANC-1 pancreatic cancer cells and SCK cells (non-pancreatic cancer cells) It was confirmed that the antibody and 8A12 antibody can specifically stain only MRS.
  • Thinprep slides were prepared by using PANC-1 cell line similar to the clinical conditions (see Example 2 below), using the Thinprep equipment (Hologic. Inc.) Used for patient cell sample processing at the clinical site, and the 1E8 antibody And the 8A12 antibody. As a result, as shown in FIG. 10, it was confirmed that the 1E8 antibody and 8A12 antibody of the present invention can be used together with a sample providing method (Thinprep slide, etc.) commonly used in a clinical field.
  • a sample providing method Thinprep slide, etc.
  • Example 2 Identification and effect of pancreatic cancer cell-specific MRS expression detection (staining method) in cytodiagnosis assay
  • Pancreas cells as a sample were obtained according to a conventional endoscopic ultrasonic microhip lips (EUS-FNA).
  • EUS-FNA endoscopic ultrasonic microhip lips
  • the endoscopic ultrasonic instrument : the (a l inear array echoendoscope EUS, product name GF-UCT140 or GF-UCT180, Ltd. Olympus, Japan) as above or the duodenum
  • the pancreatic mass was confirmed as an ultrasound image by using an ultrasound device installed at the end of the endoscope.
  • Pancreatic cells were obtained by introducing a needle for fine needle aspiration (product name: 22G Echo-ultraTM, company: Cook Medical, Cork, Ireland) into an ultrasonically collimated mass.
  • pancreatic cells were collected by a conventional method using a Cellular Automated Cell Block System (Hologic) (Antonio Ieni et al., Cell-block procedure in endoscopic ultrasound guided fine needle aspiration of gastrointestinal sol id neoplastic lesions , World J Gastrointest Endosc 2015 August 25; 7 (11): 1014-1022) as cell-based paraffin sections.
  • Hologic Cellular Automated Cell Block System
  • the pancreatic cell sample can also be provided by thinning or direct smearing on a ThinPrep slide in a conventional manner using ThinPrep (Hologic, Inc.) (de Luna R et al., Comparison of ThinPrep and conventional preparations in pancreatic fine-needle aspiration biopsy, Diagnostic Cytopathology, 2004 Feb; 30 (2): 71-6.). The results of these cell samples were compared by the following test methods.
  • H & E staining was carried out using hematoxylin and eosin according to conventional protocols (see detailed protocol in Example 3 below). It can also be reduced by staining with Pap staining method.
  • the Pap staining was performed using hematoxylin, 0G-6 (Orange G-6), and eosin azure according to conventional protocols (see detailed protocol in Example 3 below).
  • a paraffin slice sample is used, paraffin removal and hydration are performed by a conventional method, and the dye materials are treated. Cells are stained with a single layer on the slide.
  • N / C ratio When the nucleus / cytoplasm ratio (N / C ratio) is small and the nuclear membrane is smooth, the cell is judged as benign (normal) The ratio is high, chromatin aggregation is seen, the nuclear membrane is coarse, and the nucleolus and mitosis appear, and it is judged to be a malignant tumor cell. If the cell change does not reach the malignant cell but it can not be judged as benign, (atypical cell). 3) Clinical end result was determined by imaging (abdominal ultrasonography, abdominal computed tomography, abdominal magnetic resonance imaging, endoscopic retrograde ganglionectomy, positron emission tomography) and pathological examination (cytology, biopsy) Based on the results, the doctor made a final judgment.
  • Anti-i-MRS antibody (representative of using 8A12 antibody of the present invention, manufactured by Oncotag) was diluted 1: 300, treated overnight at 4 ° C, washed three times with PBS for 5 minutes
  • Alexa-488-conjugated secondary antibody (Molecular probes, Cat. No. A11001) was used as a secondary antibody with a fluorescent substance attached. It was diluted 1: 200 ⁇ 1: Treated for 1 hour, washed three times with PBS for 5 minutes each
  • PPV positive predictive value
  • NPV negative predictive value
  • PPV positive predictive value
  • NPV negative predictive value
  • Example 3 Comparison of pancreatic cancer discriminating ability between CEA of the present invention and MRS of the present invention at the cellular level
  • pancreatic cell specimens were collected and obtained by endoscopic ultrasound-assisted cell resection (fine needle aspiration). The study was approved by the Research Ethics Committee of the Gangnam Severance Hospital. Pancreatic cell samples from the above patients were obtained through endoscopic ultrasonic fine-needle aspiration (EUS-FNA) in the same manner as in Example 2 above. The pancreatic cells thus obtained were prepared by a conventional method using Cellular Automated Cell Block System Otologic (see Example 2) in the form of paraffin sections (Cellent paraffin sections) or Thinprep method.
  • EUS-FNA endoscopic ultrasonic fine-needle aspiration
  • pancreatic cancer Twenty - six patients with suspected pancreatic cancer were followed - up for pancreatic cancer (13 cases) and normal pancreas (13 cases). Of the 13 patients who were diagnosed as pancreatic cancer, 7 were classified as pancreatic cancer cells by histologic examination by pathologist. The remaining 6 patients were classified as atypical cells by histologic examination. And finally diagnosed as pancreatic cancer. At the time of pancreatic cell harvesting, the patient was informed by papers, and pancreatic cancer cells, atypical cells, and normal cells were histologically confirmed by a pathologist (see judgment criteria of Example 2). Representative diagnostic examples of each type of normal cells, tumor cells, and atypical cells in the 26 samples obtained are shown in the drawings of each experiment. 2) Conventional cytogenetic method
  • H & E staining Paraffin slices were treated with Xylene three times for 5 min, 100% ethanol for 2 min, 95% ethane for 2 min, 90% ethanol for 2 min, 70% ethanol for 2 min, For 10 min, respectively. Paraffin removal and hydration were performed. The hematoxylin was counterstained for 30 seconds at room temperature and washed with water for 10 minutes. At room temperature eos i n ol 1 min and the reaction can and washed for 10 minutes by deutmul. Dehydration and clearing were carried out for 1 min in 70% ethanol, 1 min in 90% ethane, 1 min in 95% ethane, 1 min in 100% ethane, and 3 x 5 min in xylene. The mounting solution was dropped onto the slide tissue, the cells were covered with a coverslide, and the sample was observed under an optical microscope.
  • Papanicolaou stain was performed using a Varistain 24-4 stainer from Thermo Scientific according to the instrument's built-in protocol. The protocol is shown in Table 6 below.
  • Atypia is defined as the nucleus of the cell is enlarged and the cytoplasm is decreased, and the nucleus to cytoplasmic ratio (N / C rat io) increases; Chromatography is not uniformly distributed in the nucleus, but clumping occurs partially, nucleolus appears in the nucleus, and mitosis appears. If all these atypical features are present and the severity is significant, it is possible to diagnose malignant tumors by cytologic examination. However, if these findings are partial, or if they are weak, they should be classified as atypical cells. These lesions may be partially seen in benign lesions such as severe inflammation. Conversely, if none of these findings are visible, normal cells can be determined. Of course, It is presupposed that it is a cell that can be observed at the site.
  • Example 2 Immunostaining for MRS was carried out in the same manner as described in Example 2.
  • CEA Carcinoembryonic Antigen
  • ant ibody purchased from Dako, Cat. No. M7072
  • the same procedure as in Example 2 was carried out except that the optimum treatment conditions were used.
  • pancreatic cells obtained from four patients did not show any tumor findings in H & E staining and were judged to be normal cells. In the normal pancreas cells, it was confirmed that neither MRS nor CEA stained .
  • pancreatic cells obtained from four patients were judged to be tumor cells in H & E staining.
  • MRS or CEA staining was performed using pancreatic cells of a patient who was finally identified as a tumor by following up the prognosis of the patient, which is an atypical cell which is difficult to clearly determine whether it is a tumor cell alone with H & E staining.
  • the results are shown in Fig.
  • pancreatic cells classified into atypical cells As shown in Fig. 13, it was found that the division of pancreatic cells classified into atypical cells through H & E staining was not clear whether the tumor was a tumor or a normal cell.
  • the patients included in the atypical cell group are all those who have been diagnosed as pancreatic cancer in the future.
  • CEA staining was not observed in all four atypical pancreatic cells.
  • pancreatic cancer cells diagnosed with pancreatic cancer can be diagnosed with higher accuracy than other tumor markers by performing H & E staining and MRS staining together with pancreatic cancer cells isolated from suspected pancreatic cancer patients.
  • conventional tumor markers eg, CEA
  • Example 4-1 Investigation of Causes of False Positive Results
  • MRS was proved to be able to diagnose pancreatic cancer with higher accuracy than existing pancreatic cancer markers such as CEA.
  • the experiment in the above- It is necessary to confirm the degree of accuracy and diagnosis of blind (unknown) status after tissue collection from patients suspected of having pancreatic cancer.
  • MRS showed a very good sensitivity in the diagnosis of pancreatic cancer, but the specificity of MRS alone was slightly lower than the sensitivity in the case of pancreatic cancer.
  • the cause of the false positive judgment was determined by performing MRS staining after discriminating cancer tissues and normal tissues with H & E stain for all the specimens.
  • MRS staining As a result, as shown in FIG. 14, acinar cel l, pancreatic parenchymal cells) showed high expression of MRS.
  • MRS Diagnosis Accuracy Improvement Strategy Establishment Example 4-1 As shown in the results of the item, MRS is expressed at a high level in normal recipient cells (acinar cel 1), so that it contributes to the false positive rate of pancreatic cancer judgment alone (that is, And to reduce the false positive rate, the double staining method was devised using MRS and acinar cell specific marker proteins.
  • the present inventors Of the acinar cell specific marker candidates for example, chymotrypsin was used to confirm the staining pattern in the secretory cells.
  • ICC Immunohistochemistry
  • the tissue of FIG. 16 is a normal tissue as shown in the H & E staining image of FIG. FIG. 16 shows an enlarged view of the acinar cell and the surrounding cells when the double staining of the present invention is performed on this normal tissue.
  • MRS and chymotrypsin were both strongly expressed in acinar cells, and the color was strongly overlapped, and MRS was not detected in other parts of normal tissues (FIG. 16). Based on this staining pattern, cell and tissue regions in which a precursor cell marker (typically chymotrypsin) is detected at high intensity can be excluded from the process of pancreatic cancer discrimination.
  • pancreatic cancer is classified as Posit ive, and in the remaining cases, it is classified as Negat ive.
  • chimotrypsin was strongly detected (expressed) in red cells, and the red color was very strong. Specifically, as shown in FIG. 17, MRS was detected very weakly and relatively strongly detected chymotrypsin As shown in Fig. 18, there was a pattern in which the MRS detection was significant with chymotrypsin. However, even in the case of the staining pattern shown in Fig. 18, the red color representing the chymotrypsin appears to have a high intensity in the merge image.
  • pancreatic cancer confirmed by conventional cytopathologic examination and ultimately confirmed by pancreatic cancer
  • the cells isolated from the pancreas stained with pancreatic cancer were analyzed by pap staging, and the staining pattern of the cell sample judged to be tumor cells was determined by the double staining method Respectively.
  • the cytopenic samples used in this experiment show little red color and very strong green color, that is, chymotrypsin is almost And the expression of MRS was very strongly expressed. Thus, it was confirmed that it can be judged as pancreatic cancer.
  • pancytopenia in patients with confirmed pancreatic carcinoma was confirmed by conventional cytopathologic examination. However, in the case of confirmed pancreatic cancer patients, pancytopenia (atypical eel Is) The cytological (cell sample) staining pattern was evaluated by the double staining method of the present invention.
  • the expression intensity of MRS was very high, and the detection strength of chymotrypsin was relatively low, confirming that it was a cancer cell (see merge image).
  • the double staining method of the present invention confirmed that cancer can be judged also on non-specific cells.
  • Results A total of 26 cell specimens were subjected to the double staining method of the present invention The results were compared with the final clinical diagnosis, and the results are shown in Table 8 below. As a result of the double staining of the present invention, it is classified as pancreatic cancer Posit ive in the case of MRS (+) and osteosarcoma marker (-) and in the other cases (that is, as MRS (+ (-) and prefrontal cell markers (-), or MRS (-) and prefrontal cell markers (+)) are classified as Negat ive.
  • pancreatic cancer by the MRS and pre-oocyte markers (representatively, chymotrypsin) double staining method of the present invention is sensitive to the sensitivity, the specificity, and the specificity (100%), positive predictive value (PPV) 100% and negative predictive value (NPV) 100%.
  • the double staining method of the present invention can not be confirmed or undetected by conventional cytopathological judgment methods (pap-staining or H & E stainig based morphological judgment method) including atypical cells It was confirmed that the pancreatic cell (particularly, Atypia) can be clearly distinguished from malignant tumor cells, and the diagnosis efficiency in the cytology can be remarkably increased.
  • conventional cytopathological judgment methods pap-staining or H & E stainig based morphological judgment method
  • the present invention relates to a method for diagnosing pancreatic cancer using a methionyl-thi ene synthase and a secretory cell-specific marker.
  • the MRS The diagnostic accuracy of pancreatic cancer markers is higher than that of pancreatic cancer markers such as CEA.
  • additional marker markers such as chymotrypsin, as an additional dual marker, in addition to the expression of MRS, , In vitro diagnostic industry, and the like.

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Abstract

La présente invention concerne une méthode de diagnostic du cancer du pancréas à l'aide de méthionyl-ARNt synthétase et un marqueur spécifique aux cellules acineuses. La MRS a un niveau plus élevé de précision de diagnostic que les marqueurs du cancer du pancréas classiques tels que le CEA, permet en particulier une augmentation significative de la précision de diagnostic du cancer du pancréas lorsque, conjointement avec l'expression de la MRS, une protéine marqueur spécifique aux cellules acineuses, telle que la chymotrypsine, est utilisée en outre en tant que marqueur double, et présente ainsi une applicabilité industrielle nettement supérieure dans des domaines tels que l'industrie de diagnostic in vitro.
PCT/KR2018/010366 2017-09-05 2018-09-05 Méthode de diagnostic du cancer du pancréas à l'aide de méthionyl-arnt synthétase et marqueur spécifique aux cellules acineuses WO2019050273A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4067376A1 (fr) * 2021-03-30 2022-10-05 Diaccurate Anticorps monoclonaux anti-pla2g1b et leurs utilisations

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011072265A1 (fr) * 2009-12-11 2011-06-16 Atyr Pharma, Inc. AMINOACYL-ARNt SYNTHÉTASES DESTINÉES À MODULER UNE INFLAMMATION
US8981045B2 (en) * 2010-05-03 2015-03-17 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-tRNA synthetases
KR101771070B1 (ko) * 2015-11-13 2017-08-24 재단법인 의약바이오컨버젼스연구단 Mrs와 cdk4의 결합을 저해하는 항암제 스크리닝 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011072265A1 (fr) * 2009-12-11 2011-06-16 Atyr Pharma, Inc. AMINOACYL-ARNt SYNTHÉTASES DESTINÉES À MODULER UNE INFLAMMATION
US8981045B2 (en) * 2010-05-03 2015-03-17 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-tRNA synthetases
KR101771070B1 (ko) * 2015-11-13 2017-08-24 재단법인 의약바이오컨버젼스연구단 Mrs와 cdk4의 결합을 저해하는 항암제 스크리닝 방법

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KAYED, H. ET AL.: "BGLAP Is Expressed in Pancreatic Cancer Cells and Increases Their Growth and Invasion", MOLECULAR CANCER, vol. 6, 28 December 2007 (2007-12-28), pages 1 - 9, XP021036927 *
KIM, S. ET AL.: "Aminoacyl-tRNA Synthetases and Tumorigenesis: More than Housekeeping", NATURE REVIEWS CANCER, vol. 1, no. 1, October 2011 (2011-10-01), pages 708 - 718, XP055580901 *
KWON, N. H. ET AL.: "Dual Role of Methionyl-tRNA Synthetase in the Regulation of Translation and Tumor Suppressor Activity of Aminoacyl-tRNA Synthetase-interacting Multifunctional Protein-3", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 108, no. 49, 6 December 2011 (2011-12-06), pages 19635 - 19640, XP055580911 *
LA ROSA, S. ET AL.: "Acinar Cell Carcinoma of the Pancreas: Overview of Clinicopathologic Features and Insights into the Molecular Pathology", FRONTIERS IN MEDICINE, vol. 2, 2015, pages 1 - 13, XP055580906 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4067376A1 (fr) * 2021-03-30 2022-10-05 Diaccurate Anticorps monoclonaux anti-pla2g1b et leurs utilisations
WO2022207674A1 (fr) * 2021-03-30 2022-10-06 Diaccurate Anticorps monoclonaux anti-pla2g1b et leurs utilisations

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