WO2019050273A1 - Method for diagnosing pancreatic cancer using methionyl-trna synthetase and acinar cell-specific marker - Google Patents

Method for diagnosing pancreatic cancer using methionyl-trna synthetase and acinar cell-specific marker Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
cell
mrs
protein
pancreatic cancer
cells
Prior art date
Application number
PCT/KR2018/010366
Other languages
French (fr)
Korean (ko)
Inventor
김성훈
권남훈
이동기
임범진
장성일
Original Assignee
(주)온코태그디아그노스틱
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by (주)온코태그디아그노스틱 filed Critical (주)온코태그디아그노스틱
Publication of WO2019050273A1 publication Critical patent/WO2019050273A1/en

Links

Classifications

    • 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.

Abstract

The present invention relates to a method for diagnosing pancreatic cancer using methionyl-tRNA synthetase and an acinar cell-specific marker. MRS has a higher level of diagnostic accuracy than conventional pancreatic cancer markers such as CEA, particularly allows a significant increase in the accuracy of diagnosis of pancreatic cancer when, together with the expression of MRS, an acinar cell-specific marker protein such as chymotrypsin is used additionally as a dual marker, and thus has markedly superior industrial applicability in fields such as in the in-vitro diagnostics industry.

Description

【명세서】  【Specification】
【발명의 명칭】 메티오닐 -티알엔에이 합성효소 및 선방세포 특이적 마커를 이용한 췌장암 진단 방법 METHODS FOR DIAGNOSING Pancreatic Cancer Using Methionyl-Thiolated Enzyme and Dendritic Cell-Specific Markers
【기술분야】 본 발명은 메티오닐 -티알엔에이 합성효소 (methionyl-tRNA synthetase , MRS) 및 선방세포 특이적 마커를 이용한 췌장암 진단 방법에 관한 것으로, 보다 상세하게는 MRS 단백질 발현 수준을 측정하는 제제 및 선방세포 특이적 마커 단백질의 발현 수준을 측정하는 제제를 포함하는 췌장암 진단용 조성물， 이를 포함하는 키트 및 상기 두 단백질을 이중 마커로 사용하여 췌장암 진단의 정확도를 높이는 방법에 관한 것이다. TECHNICAL FIELD 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.
【배경기술】 본 출원은 2017년 9월 5일에 출원된 대한민국 특허출원 제 10-2017- 0113372호를 우선권으로 주장하고， 상기 명세서 전체는 본 출원의 참고문헌이다. The present application claims priority from Korean Patent Application No. 10-2017- 0113372, filed on September 5, 2017, the entire contents of which are incorporated herein by reference.
암이란 주로 통제되지 않는 세포의 증식에서 시작되어 주위의 정상조직 또는 기관으로 침윤하여 파괴시키고 새로운 성장 장소를 만들 수 있어 개체의 생명을 빼앗아 갈 수 있는 질환 군을 총칭한다. 지난 10여년 동안 암을 정복하기 위해 세포 주기나 세포사멸 (apoptosis)의 조절과 발암유전자나 암 억제 유전자들을 포함한 새로운 표적을 모색함에 있어서 눈에 띄는 발전을 거듭해 왔음에도 불구하고 암의 발생률은 문명이 발달됨에 따라증가되고 있다. 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.
이 중 췌장암은 5년 생존율이 1—4%, 중앙생존기간 5개월에 이르는 치명적인 암으로 인체의 암 중에서 가장 불량한 예후를 보이고 있다. 80-90% 환자에서 진단시 완치를 기대하는 근치적 절제가 불가능한 상태에서 발견되기 때문에 예후가 불량하고 치료는 주로 항암요법에 의존하고 있으므로， 그 어떤 인체암보다도 조기 진단법 개발이 절실히 요망되고 있다. 현재까지 췌장암에 효과가 있다고 알려진 5- 플루오로유라실， 잼시타민 (gemcitabine) , 타르세바 (tarceva)를 포함한 몇 항암제의 치료 효과는 지극히 실망적이며， 항암치료에 대한 반응율은 15% 내외에 불과하고 이러한 사실은 췌장암 환자의 예후를 향상시키기 위해서는 보다 정확하고, 빠른 진단방법이 필요하다는 것을 시사한다. Among these, 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.
한편， 병리검사 (pathological examinat ion)란 적출한 세포, 조직 또는 장기를 이용해 주로 형태학적 입장에서 질병의 근원을 해명하려 하는 검사를 의미하며, 육안적 소견의 파악， 광학， 전자현미경 검색 등의 방법으로 질병의 진단에 웅용되는 중요한 검사다. 이러한 병리검사에는 조직병리검사와 세포병리검사가 있다. 한편， 조직검사와 세포진 (cytodiagnosi s) 검사는 많은 차이를 지니며, 잘 알려진 암 마커들을 이용한 분석 실험에서 조직검사와 세포진검사 사이에는 진단 민감도， 특이도 등 그 예측 정확도에서 많은 차이를 보이는 것으로 알려졌다. 따라서 기존에 알려진 암 마커라고 하더라도 구체적인 검체 (조직 또는 세포)에 따라서 실질적으로 진단 실효성을 거둘 수 있는지는 별개의 문제로 여겨진다. On the other hand, 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. These pathological tests include histopathology and cytopathology. On the other hand, 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).
체내에서 분리된 세포를 병리학적으로 검사함에 있어서 장애가 되는 것 중에 비정형 세포 (atypical cel l )에 대한 판단의 어려움도 한몫하고 있는 실정이다. 1976년 Melamed 등이 염증성 변화는 아니면서 이형성으로 진단하기에는 미흡한 세포변화를 편평세포 비정형성으로 발표한 후, 비정형적 세포에 대한 진단， 해석 및치료방침결정에 많은 논란이 있어왔다. 따라서 이의 개선을 위하여 The bethesda system(TBS)이 제정되었고， TBS에서는 비정형적 세포 (atypical cel l )라는 용어의 사용을 염증성， 전암성 또는 종양성 세포변화로 진단할 수 없는， 본질을 알수 없는 경우 (undetermined signi f i cance)에만 극히 제한하여 사용하고 있다. 비정형 세포에 대한 치료적 방침과 관련하여 다른 견해가 있을 수 있기 때문에， 문제가 된다. 특히， 실제로 암이 진행되고 있는 상태임에도 불구하고 조직 또는 세포 수준의 검사에서 비정형 세포로 진단되거나 또는 조직 검체에서만 결과가 판정되어 나오는 경우가 상당하다는 데에 문제가 있다. 부정형의 조직 구조나 세포 형태가 염증성 병변인지 신생물인지 구분이 명확하지 않은 경우 atypi sm 또는 cel lular atypia라고 진단하는 경우가 많다. 따라서 다른 검사수단 등에 의하여 여러번 반복적인 재검사의 필요성이 따르며 이에 따른 시간적 경제적 비용이 상당히 소모되고 있는 실정이다. 췌장은 신체 깊숙이 위치하므로， 암이 췌장에 존재하는 경우 이를 검출하기가 매우 어렵다. 영상학적 검사들을 통해 수술이 가능한 췌장암일 경우 수술을 시행 받기 전 종괴에 대한 조직검사나 내시경초음파하 세포진 검사를 통해 췌장암의 확정 진단이 필요하다. 또한 수술올 할 수 없는 경우에도 항암치료나 방사선치료를 위한 조직학적 진단을 위해 조직검사나 세포진 검사가 필요하다. 췌장암을 진단하기 위한 종양 마커는 CEA (참조 값: 5.0 ng/mL) 및 CA 19-9(참조 값: 37 U/mL)가 대표적이다. 그러나, CA19-9의 경우， 췌장암 진단용마커로서는 특이도가 낮다는 문제점이 있다. 한 보고에 의하면 건강검진을 받은 사람 중에서 CA19-9 수치가 증가된 경우가 약 1% 이었는데， 증상 없이 CA19-9 수치가 상승된 사람 중에서 암이 발견된 경우는 실제 2¾>에 불과하였다고 한다. 미국 암학회 가이드라인에 따르면， 췌장암의 선별 검사에서 CA19— 9은 민감도나 특이도가 떨어진다고 판단한 바 있다. The difficulty in judging the atypical cells is one of the obstacles in the pathological examination of cells isolated from the body. In 1976, Melamed et al. Reported that the inflammatory changes were not inflammatory changes, but they were not enough to diagnose dysplasia, and there have been many controversies about the diagnosis, interpretation, and treatment of atypical cells. Therefore, the bethesda system (TBS) has been established for its improvement and the use of the term atypical cell in TBS is not known to be diagnosed as inflammatory, (undetermined signi fi cance). This is problematic because there may be other views regarding the therapeutic strategy for non-malignant cells. Particularly, there is a problem in that it is significant that diagnosis is made of atypical cells in a tissue or cell level test, or the result is judged only in a tissue sample, although the cancer is actually progressing. If atypical SM or celi lular atypia is diagnosed, it is often the case that the indeterminate tissue structure or cell type is not an inflammatory lesion or neoplasm. Therefore, it is necessary to repeatedly repeat the inspection by other inspection means, and accordingly the time and economic costs are considerably consumed. Since the pancreas is deeply in the body, it is very difficult to detect when the cancer is present in the pancreas. 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). However, in the case of 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.
이외에도 췌장암을 진단하기 위한 바이오 마커 개발이 활발하게 이루어지고 있으며， 그 예시로서 대한민국 특허 제 10-0819122호는 마트릴린 (matri l in) , 트랜스티레틴 (transthyret in) 및 스트라티핀 (strat i f in)을 췌장암 마커로 이용한 기술을 개시하고 있으며， 대한민국 출원공개 제 2012-0082372호는 여러 가지 췌장암 마커를 이용한 기술을 개시하고 있다. 또한， 한국 출원공개 제 2009-0003308호는 개체의 혈액 시료에서 REG4 단백질의 발현량을 검출하여 췌장암을 진단하는 방법을 개시하고 있으며， 한국 출원공개 제 2012-0009781호는 개체의 췌장암 진단에 필요한 정보를 제공하기 위하여 개체로부터 분리한 암 조직 중 XIST RNA의 발현량을 측정하는 분석방법을, 한국 출원공개 제 2007-0119250호는 정상 인간의 췌장 조직과 비교하여 인간 췌장암 조직에서 다르게 발현된 신규 유전자 LBFL313 패밀리를 개시하고 있고, 미국 출원공개 제 2011/0294136호는 케라틴 8 단백질 등의 바이오 마커들을 이용한 췌장암 진단방법을 개시하고 있다. 하지만， 상기한 마커들은 마커마다 그 진단 효율 및 정확성에서 큰 차이를 나타낸다는 한계점이 있으며， 특히 세포학적 분석 (Cytologycal analysis)방법에의 한 것이 아닌것으로서, 임상적으로 종양세포인지 또는 기타 다른질환 상태에 있는 세포인지에 대한 구별이 명확하지 않은 비정형 (atypical ) 세포의 경우 췌장암 여부에 대한 명확한 진단의 중요성이 더욱 높다고 할 수 있음에도 불구하고 이를 명확하게 판단해 줄 수 있는 마커가존재하지 않는다. 즉, 종래 보고된 췌장암 진단 마커들의 경우 세포진 검사의 적용에 있어서， 조직 검사에서와는 다르게 세포 수준의 진단에서는 민감도 및 특이도가 좋지 못하여 실효성을 거두고 있지 못하는 실정이다. In addition, biomarkers for diagnosing pancreatic cancer have been actively developed. For example, 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, and 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 And U.S. Patent Application Publication No. 2011/0294136 discloses a method for diagnosing pancreatic cancer using biomarkers such as keratin 8 protein. However, 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 In the case of atypical cells in which the distinction is not clear, 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.
따라서， 췌장암을 진단하기 위해서는 상기 종양마커 이외에도 컴퓨터 단층촬영술 (CT) , 내시경역행 담췌간조영술 (endoscopic retrograde cholangiopancreatography: ERCP) , 초음파내시경검사법 (EUS) , 혈관조영술과 같은 고가의 철저한 검사가 요구되고 있으나, 이를 통해서도 췌장암을 정확하게 진단하는 것이 매우 어려운 실정이다. 또한， 췌장암은 진단 당시 이미 절제가 불가능한 진행암일 경우가 많고， 수술이 가능한 예는 10-15% 밖에 되지 않아 수술을 할 수 없는 환자에서 췌장암의 진단은 내시경 초음파 미세바늘 흡인술 검사를 통해 실시하고 있다. 그러나 내시경초음파 미세바늘 흡인술 검사를 통한 세포진단의 경우 수술 후 췌장암 조직에 비해 주변 구조나 세포와의 비교가 어려워 진단을 확진하는데 한계점이 있다. Therefore, 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% . However, endoscopic ultrasound microscopic needle aspiration cytology is difficult to compare with surrounding tissues or cells compared with pancreatic cancer tissues.
이 때문에 아직까지 췌장암세포와 다른 질환 (예를 들어 췌장염)의 세포를 감별하는데 있어 주로 H&E 염색 또는 pap 염색 등 일반염색을 기반으로 하는 병리학적 진단에 의존하고 하고 있다. 그러나 상기 기존 염색방법에 의해 췌장암을 확진하는 것은 의료진의 경험과 해석기술에 따라 다른 진단이 내려지기도 하며， 특히 H&E염색 또는 pap 염색을 통한 췌장세포 관찰에서 비정형 (atypical ) 세포로 판정된 경우 췌장암인지 다른 양성 (benign)질환인지 여부에 대한 구별이 매우 어려워 환자의 질환에 대한 정확한 진단 및 치료가 빠르게 이루어지지 못하고 있다. 세포진에 대한 진단이 정확하지 않아 수술을 받을 수 있는 췌장암 환자에게 적절한 치료 * 할 수 없으며, 반대로 불필요한 수술을 방지하기도 어려운 문제점이 있다. 따라서 임상적으로 췌장암의 치료 효과를 증대시키기 위해서는 세포진에 대한 정확한 진단법이 필요한실정이다. 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). However, the 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. In particular, when it is judged as atypical cell in the pancreatic cell observation by H & E staining or pap staining, It is very difficult to distinguish between benign disease and other diseases. There is a problem that 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.
【발명의 상세한 설명】 【기술적 과제】 이에 본 발명자들은 췌장암 임상 환자들로부터 얻은 췌장세포 시료를 이용하여 세포 수준에서 보다 정확하게 췌장암을 진단할 수 있는 마커를 찾기 위해 세포학적 분석 (cytological analysis)을 면밀히 수행한 결과, 췌장 세포 시료에서 MRS(methionyl tRNA synthetase)의 (고)발현 검출을 통해 악성종양세포를 명확히 구분할 수 있으며， 특히 이러한 구분은 H&E 염색 또는 pap 염색을 이용하는 기존 세포 병리학적 검사법에 의해 비정형 세포 (atypical cel l )로 판정되어 종양인지 여부에 대한 확진이 불가능하였던 세포 시료에서도 가능하다는 것을 최초로 규명하였으며, MRS 발현과 더불어 키모트립신 (chymotrypsin)과 같은 선방세포 특이적 마커 단백질을 이중 마커 (dual marker )로 사용하면 췌장암 진단의 정확도가 더욱 현저히 상승함을 확인하고 본 발명을 완성하게 되었다. Description of the invention The present inventors have studied cytological analysis of pancreatic cancer samples from pancreatic cancer clinical patients in order to find markers that can diagnose pancreatic cancer more accurately at the cellular level. As a result, in the pancreatic cell sample (High) expression of MRS (methionyl tRNA synthetase) can be clearly distinguished from malignant tumor cells. In particular, 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. And the accuracy is further increased, thereby completing the present invention.
따라서 본 발명의 목적은， 췌장암 진단에 필요한 정보를 제공하기 위하여， 잠재 환자로부터 채취한 췌장 시료로부터 메티오닐 티알엔에이 합성효소 (methionyl tRNA synthetase , MRS) 단백질을 검출하는 방법을 제공하는 것이다. 본 발명의 목적은， (a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 Accordingly, 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
(b) 상기 (a) 단계의 측정 시료에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현되면 췌장암 세포인 것으로 판단하는 단계를 포함하는， 췌장암 진단 방법을 제공하는 것이다. (b) determining that the 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.
본 발명의 또 다른 목적은， 췌장암에 대한 세포진 (cytodiagnosi s) 검사 또는 조직 검사에 있어서， 하기 단계를 포함하는 것을 특징으로 하는 민감도 또는 특이도를 향상시키는 방법을 제공하는 것이다: It is yet another object of the present invention to provide a method for improving sensitivity or specificity in a cytodiagnosis test or biopsy for pancreatic cancer comprising the steps of:
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 증가되었으면 췌장암 세포인 것으로 판단하는 단계. (b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the cell is a pancreatic cancer cell.
본 발명의 또 다른 목적은， 췌장암에 대한 세포진 (cytodiagnosi s) 검사 또는 조직검사에 있어서, 형태학적 검사와 병용하여 It is another object of the present invention to provide a method for screening for cytodiagnosis or histologic examination of pancreatic cancer,
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (b) 상기 (a) 단계에서 선방세포 특어적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 증가되었으면 췌장암 세포인 것으로 판단하는 단계를 포함하는 것을 특징으로 하는 췌장암세포 판별법을 추가로 수행하는 것을 특징으로 하는， 췌장암 진단에 필요한 정보를 제공하는 방법 (췌장암 진단 방법)을 제공하는 것이다. (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.
【기술적 해결방법】 상기와 같은 목적을 달성하기 위하여， 본 발명은 메티오닐 티알엔에이 합성효소 (methionyl-tRNA synthetase , MRS) 단백질의 발현 수준을 측정하는 제제 및 선방세포 (acinar cel l ) 특이적 마커 단백질의 발현 수준을 측정하는 제제를 포함하는 췌장암 진단용 조성물과 이를 포함하는 췌장암 진단용 키트를 제공한다. [Technical Solution] In order to achieve the above object, 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 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.
본 발명의 다른 목적을 달성하기 위하여 본 발명은 췌장암 진단에 필요한 정보를 제공하기 위하여， 잠재 환자로부터. 채취한 췌장 시료로부터 상기 MRS 단백질 및 선방세포 특이적 마커 단백질의 발현 수준을 정성 또는 정량 분석하는 방법을 제공한다. 본 발명은, (a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 In order to achieve the above object, 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; And
(b) 상기 (a) 단계의 측정 시료에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현되면 췌장암 세포인 것으로 판단하는 단계를 포함하는， 췌장암 진단 방법을 제공한다. (b) determining that the 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.
췌장암에 대한 세포진 (cytodi agnosi s) 검사 또는 조직 검사에 있어서， 하기 단계를 포함하는 것을 특징으로 하는 민감도 또는 특이도를 향상시키는 방법을 제공한다: In a cytodiagnosis assay or biopsy for pancreatic cancer, the present invention provides a method for improving sensitivity or specificity, comprising the steps of:
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 증가되었으면 췌장암 세포인 것으로 판단하는 단계 . (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.
본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은 췌장암에 대한 세포진 (cytodiagnosis) 검사 또는 조직검사에 있어서 , 형태학적 검사와 병용하여 In order to accomplish still another object of the present invention, the present invention provides a method for the cytodiagnosis or histology of pancreatic cancer,
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 증가되었으면 췌장암 세포인 것으로 판단하는 단계를 포함하는 것을 특징으로 하는 췌장암세포 판별법을 추가로 수행하는 것을 특징으로 하는， 췌장암 진단에 필요한 정보를 제공하는 방법 (췌장암 진단 방법)을 제공한다. (b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, 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.
이하본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 명세서에 개시된 내용 전반에 걸쳐서， 본 발명과 관련된 다양한 양상 또는 조건들이 범위 형식으로 제안될 수 있다. 본 명세서에서 범위값의 기재는， 별다른 언급이 없는 한 해당 경계값을 포함하는 것으로서 즉， 하한값 이상 내지 상한값 이하의 값을들 모두 포함하는 의미이다. 범위 형식의 서술은 단순히 편의성 및 간략성을 위한 것이며， 본 발명의 범위에 대한 융통성 없는 제한 ( inf lexible l imitat ion)으로서 해석되지 않아야 하는 것으로 이해되어야 한다. 따라서 범위의 서술은 상기 범위 내의 개별적인 수치값들 뿐만 아니라 모든 가능한 하부범위 (subrange)를 구체적으로 개시한 것으로 고려되어야 한다. 예를 들어， 1 내지 5와 같은 범위의 서술은 상기 범위 내의 개별적 수치들， 예를 들어， 1， 2, 2.7， 3, 3.5， 4.3 및 5 뿐만 아니라， 1 내지 3， 1 내지 4, 2 내지 5， 2 내지 3， 2 내지 4， 3 내지 4 등과 같은 하부범위들을 구체적으로 개시한 것으로 간주되어야 한다. 이는 범위의 폭과 무관하게 적용된다. Throughout the disclosure herein, various aspects or conditions relating to the present invention may be suggested in a range format. In this specification, the description of the range value is meant to include the relevant boundary value unless otherwise stated, that is, to include all the values from the lower limit value to the upper limit value. It should be understood that the description of a range format is merely for convenience and simplicity and should not be construed as an inflexible limitation to the scope of the present invention. Thus, the description of a range should be considered as disclosing all possible subranges as well as individual numerical values within the range. For example, 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.
본 발명의 용어 '〜을 포함하는 (comprising)' 이란 '함유하는' 또는 '특징으로 하는' 과 동일하게 사용되며, 조성물 또는 방법에 있어서， 언급되지 않은 추가적인 성분 요소 또는 방법 단계 둥을 배제하지 않는다. 용어 '-로 구성되는 (consist ing of)' 이란 '〜로 이루어지는' 과 동일하게 사용되며， 별도로 기재되지 않은 추가적인 요소， 단계 또는 성분 등을 제외하는 것을 의미한다. 용어The term " 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 . The term 'consisting of' is used in the same way as 'consisting of' Quot; means excluding any additional elements, steps or components not listed. Terms
'필수적으로 구성되는 (essent ial ly consist ing of )' 이란 조성물 또는 방법의 범위에 있어서， 기재된 성분 요소 또는 단계와 더불어 이의 기본적인 특성에 실질적으로 영향을 미치지 않는 성분 요소 또는 단계 등을 포함하는 것을 의미한다. &Quot; Essential consistency of " 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.
본 발명에서 용어 '췌장암 (pancreat ic cancer)' 이란 췌장에 발생한 악성 (mal ignant ) 종양 또는 암을 의미하는 것으로서， 증식속도가 빠르고 주위조직으로 침투 및 다른 기관으로 전이하는 특징을 가진 악성 (mal ignant ) 신생물올 의미한다. 상기 악성 종양 또는 암은, 성장속도가 느리고 전이되지 않는 특성을 지니는 양성 종양 (benign tumor)과 구분된다. 췌장에 생기는 암 중 90% 이상이 췌관세포에 생기며， 췌장암은 보통 췌관암 또는 췌관선암을 의미한다. 따라서 바람직하게, 본 발명에서의 췌장암은 췌관 (선)암종 (pancreat ic ductal (adeno)carcinoma)을 의미하는 것일 수 있다. 본 발명에서 진단의 목적으로 하는 췌장암은， 이것이 원발암이건 전이에 의하여 췌장에 2차 적으로 암이 생긴 것이건 그 발생원인이 특별히 제한되지 않는다. 바람직하게 원발암을 대상으로 하는 것일 수 있다. The term 'pancreatic cancer' as used herein 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.
본 발명에서 용어 '정상' 은 악성 종양 또는 암이 아닌 상태 (Negat ive for mal ignancy, 악성종양세포 음성)를 의미하는 것으로서, 아무런 질환이 없는 완전 정상 상태， 악성 종양 (암)이 아닌 췌장염 등의 다른 질병 상태， 또는 /및 ' Benign (양성)' 에 해당하는 판정 상태를 포함하는 의미이다. 본 명세서에서 임상적인 (최종)질환 상태 판정에 있어서 'Benign' 으로 기재되는 양성 표시는 'posit ive' 로 표시되는 해당 검사법 상에서의 양성 표시와 구분되는 것으로， 상기 'posit ive' 로 표시되는 양성은 해당 검사법에서 반웅이 있는 것으로 나음 또는 해당 검사법에서 암의 가능성을 의미하는 결과가 나음을 의미한다. The term " 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 ". In the present specification, 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.
췌장암 환자 중 약 5-10%는 유전 소인을 가지고 있는데， 췌장암 환자에서 췌장암의 가족력이 있는 경우는 약 7.8% 정도로 일반인에서의 췌장암 발생률 0.6%에 비해 빈도가 높다. 췌장암은 5년 생존율이 5% 이하로 예후가 매우 나쁜 암이다. 그 이유는 대부분 암이 진행된 후에 발견되기 때문에 발견 당시 수술 절제가 가능한 경우가 20% 이내이고， 육안으로 보기에 완전히 절제되었다 하더라도 미세 전이에 의해 생존율 향상이 적으며， 항암제 및 방사선 치료에 대한 반웅이 낮기 때문이다. 현재 췌장암을 진단하는 방법으로는 전산화 단층촬영 (computed tomography ;CT)이나 자기공명 영상장치 (magnet ic resonance imaging; MRI )를 사용하는 조직검사가 있으며， 영상학적인 방법인 CT 또는 MRI가 사용되나， 이는 간접적인 진단 방법이고 최종적으로는 병리학적 방법인 조직 검사나 세포진 검사를 통해 췌장암을 진단한다. 조직검사는 주변의 구조나 세포와 비교를 통해 특정 영역에 암이 있는 것으로 확진가능하지만， 세포진 검사의 경우에는 낱개의 세포를 뽑아내어 도말한 것이므로 주변 조직과의 관계를 증명할 수 없기 때문에， 조직 검사와 세포진 검사는 근본적으로 많은 차이가 있다. About 5% to 10% of pancreatic cancer patients have a genetic predisposition. About 7.8% of pancreatic cancer patients have a family history of pancreatic cancer, compared with 0.6% of pancreatic cancer patients in the general population. Pancreatic cancer is a very poor prognosis with a 5 - year survival rate of less than 5%. The reason is that most of the cancer is found after the progression, , And the survival rate is not improved by micro metastasis even if it is completely resected by the naked eye and the reaction to chemotherapy and radiotherapy is low. Currently, pancreatic cancer is diagnosed by computed tomography (CT) or magnetic resonance imaging (MRI), and imaging or CT or MRI is used. The diagnosis of 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. However, in the case of 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.
최근에는 췌장암을 진단할 수 있는 임상시료로서 혈장과 같은 체액에서의 단백질 분석을 동시에 측정하여 췌장암 존재 또는 발병 가능성 여부를 판단하고 있다. 그러나， 임상적으로 혈청학적 방법은 췌장암 진단에 있어서 참고자료로서의 의미만을 지닐 뿐， 췌장암 진단에 결정적인 정보를 제공하지는 못하고 있다. 현재 췌장암과 관련되어 가장 흔히 쓰이는 종양 표지자는 카보하이드레이트안티젠 19-9 (carbohydrate ant igen 19—9: CA19— 9) 또는 암태아성항원 (carcinoembryogenic ant igen: CEA)을 들 수 있다. 그러나 CA19-9는 간염， 간경변， 췌장염과 같은 비 암성 질환에서도 혈중농도가 상승하기 때문에, 췌장암의 진단으로의 사용은 적절하지 않은 것이 알려져 있고, 췌장세포 자체에서 검출이 가능한 것이 아니라 혈액에서 검출이 되는 마커이기 때문에 췌장암 특이적이라 할 수 없다. 따라서 수술 후 CA19-9 혈청 수준을 감시함으로써 환자의 예후를 판단하기 위해 사용되고 있다. 또한 CEA의 경우에도 췌장암에 대해 충분한 민감도， 특이도, 양성 예측률 및 /또는 음성 예측률을 보이지 못하고 있어 췌장암을 정확하게 진단하는데는 한계점이 있으며， 이는 본 명세서 실시예에 잘 나타나 있다. Recently, as a clinical sample that can diagnose 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. However, clinically, 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). However, 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. It is known that 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. In addition, 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.
이에 반해 본 발명자들은 췌장암에서 MRS가 (고)발현됨을 최초로 규명하였으며, 뿐만 아니라 MRS를 췌장암 마커로 이용할 시에 조직검사뿐만 아니라 세포진에서도 높은 정확도의 진단결과를 수득할 수 있음을 발견하였다. 즉， 정상 췌장 세포 (즉, 비-종양성 췌장 세포)와 대비적으로 MRS가 췌장암에서 특이적으로 고발현되며， MRS가 기존에 췌장암 마커로 많이 사용되고 있는 CEA보다도 높은 정확도로 췌장 세포 시료에 대하여 췌장암 판정이 가능하며， 특히 세포진 진단에서 기존 세포병리학적 검사 방법 (예를 들어 H&E 염색 또는 pap 염색 등)으로 확정진단이 어려운 비정형 (atypical ) 세포에 대해서도 높은 정확도로 췌장암 세포의 구별을 가능하게 한다는 현저한 효과를 밝힌 바 있다. 특히 이러한 췌장암 진단 정확도는， 키모트립신과 같은 선방세포 특이적 마커 단백질을 추가하여 이중 마커로 사용하여 진단하였을 때 현저하게 상승된 것을 확인하였다. On the other hand, the inventors of the present invention firstly confirmed that 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.
따라서 본 발명은 메티오닐 티알엔에이 합성효소 (methionyl-tRNA synthetase, MRS) 단백질의 발현 수준을 측정하는 제제 및 선방세포 (acinar cel l ) 특이적 마커 단백질의 발현 수준을 측정하는 제제를 포함하는 췌장암 진단용 조성물, 및 이를 포함하는 췌장암 진단용 키트를 제공한다. 또한 MRS 단백질의 발현 수준을 측정하는 제제 및 선방세포 특이적 마커 단백질의 발현 수준을 측정하는 제제로 구성되는 췌장암 진단용 조성물， 및 이를 포함하는 췌장암 진단용 키트를 제공한다. 또한 MRS 단백질의 발현 수준을 측정하는 제제 및 선방세포 특이적 마커 단백질의 발현 수준을 측정하는 제제로 필수적으로 구성되는 췌장암 진단용 조성물， 및 이를 포함하는 췌장암 진단용 키트를 제공한다. Therefore, the present invention relates to 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. And a diagnostic kit for pancreatic cancer comprising the same. Also provided is 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. Also disclosed are a composition 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.
또한 본 발명은 췌장암의 진단용 제제를 제조하기 위한 MRS 단백질 및 선방세포 특이적 마커 단백질의 발현 수준올 측정하는 제계 (특히, MRS 또는 선방세포 마커 단백질 각각에 대한 측정 제제)의 용도를 제공한다. 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.
본 명세서에서 용어 '진단' 은 병리 상태의 존재 또는 특징을 확인 (판별)하는 것을 의미한다. 구체적으로， 본 발명에 있어서 상기 진단은 MRS 단백질의 발현 여부 또는 발현 수준을 측정하여 췌장암의 존재 또는 발병 여부를 확인하는 것일 수 있다. As used herein, the term " diagnosis " means identifying (identifying) the presence or characteristic of a pathological condition. Specifically, in the present invention, 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.
본 발명에서 'MRS' 는 메티오닐 티알엔에이 합성효소 (methionyl-tRNA synthetase)를 의미하는 것으로서， 상기 MRS는 아미노산 메티.오닌과 tRNA의 아미노아실레이션 (aminoacylat ion) 반응을 매개하는 효소이다. 본 발명의 MRS 단백질은 당업계에 공지된 MRS 아미노산 서열을 포함하는 것이라면 그 구체적 서열 및 이의 생물 기원이 특별히 제한되지 않는다. 일례로 인간에서는 MARS 유전자에 암호화되어 있으며， MRS의 서열 정보는 匪_004990(111 ^) , NP_004981.2 , P56192. 2 단백질) 등의 Genbank(NCBI ) accession number로 공지되어 있다. 바람직하게 본 발명의 MRS는 서열번호 1로 표시되는 인간의 MRS 단백질 아미노산 서열을 포함하는 것일 수 있다. 더욱 바람직하게 본 발명의 MRS 단백질은 상기 서열번호 1로 표시되는 아미노산 서열로 이루어지는 것일 수 있다. 상기 MRS는 cyto lasmi c form (cyto lasmic methionyl-tRNA synthetase)과 mi tochondrial form(mi tochondr ial methionyl-tRNA ynthetase)의 두 가지 아형 ( i soform)이 있다. 본 발명에서의 MRS는 바람직하게는 cytoplasmic form일 수 있다. In the present invention, 'MRS' means a methionyl-tRNA synthetase, and 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 匪004990 (111 ^), NP_004981.2, P56192. 2 protein) is known as the Genbank (NCBI) accession number. Preferably, the MRS of the present invention may include a human MRS protein amino acid sequence represented by SEQ ID NO: 1. More preferably, the MRS protein of the present invention may comprise the amino acid sequence of SEQ ID NO: 1. There are two subtypes of MRS: cyto lasmic form (cyto lasmic methionyl-tRNA synthetase) and mi tochondrial form (mi tochondrhal methionyl-tRNA synthetase). The MRS in the present invention may preferably be a cytoplasmic form.
본 발명에서 용어 '발현 (express ion) '은 세포에서 단백질 또는 핵산 o 생성되는 것을 의미한다. In the present invention, the term " express ion " means that a protein or nucleic acid is produced in a cell.
본 발명에서 용어 '단백질'은 '폴리펩타이드 (polypept ide) ' 또는 '펩타이드 (pept ide) '와 호환성 있게 사용되며， 예컨대, 자연 상태의 단백질에서 일반적으로 발견되는 바와 같이 아미노산 잔기의 중합체를 말한다. The term " 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.
상기 MRS 단백질의 발현 수준을 측정하는 제제는， 당멉계에 단백질의 발현수준 측정에 사용가능한 것으로 알려진 것이라면 그 종류가 특별히 제한되지 않으나， 바람직하게 MRS 단백질에 특이적으로 결합하는 항체 또는 앱타머일 수 있다. 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 .
본 발명에서 용어 '항체 (ant ibody) '는 항원성 부위에 특이적으로 결합하는 면역글로불린 ( immunoglobul in)을 의미한다. 더욱 구체적으로, 디설파이드 결합에 의해 서로 연결된 적어도 2개의 중 (H) 쇄 및 2개의 경 (L) 쇄를 포함하는 당단백질을 가리킨다. 각각의 중쇄는 중쇄 가변 영역 (이하， HCVR또는 VH로 약기) 및 중쇄불변 영역으로 이루어진다. 중쇄 불변 영역은 3개의 도메인， CHI , CH2 및 CH3으로 이루어진다. 각각의 경쇄는 경쇄 가변 영역 (이하 LCVR 또는 VL로 약기) 및 경쇄 불변 영역으로 이루어진다. 경쇄 불변 영역은 하나의 도메인， CL로 이루어진다. VH및 VL 영역은 프레임워크 영역 (FR)이라 일컬어지는 더욱 보존된 영역이 산재된 초가변성 영역 (상보성 결정 영역 (CDR)이라 일컬어짐)으로 더욱 세분될 수 있다. VH및 VL의 각각은 하기 순서: FRl , CDR1, FR2, CDR2, FR3, CDR3 , FR4로 아미노- 말단으로부터 카르복시-말단으로 배열된 3개의 CDR 및 4개의 FR로 구성된다. 중쇄 및 경쇄의 가변 영역은 항원과 상호작용하는 결합 도메인을 함유한다. 항체의 불변 영역은， 면역 체계의 다양한 세포 (예， 효과기 세포) 및 전통적인 상보 체계의 첫 번째 성분 (Clq)을 포함하여， 숙주 조직 또는 인자에 대한 면역글로불린의 결합을 매개할 수 있다. The term " 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. The 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. 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).
본 발명에서의 항 -MRS 항체는 MRS 외에, 다른 종류의 아미노아실 티알엔에이 합성효소를 포함하는 다른 단백질에는 반웅하지 않고, MRS 단백질에만 특이적으로 결합하는 항체이다. 본 발명에서 MRS 단백질에 특이적으로 결합하는 항체는, 바람직하게는 서열번호 1로 표시되는 아미노산 서열을 포함하는 단백질 (MRS)에 특이적으로 결합하는 항체일 수 있다. 항 -MRS 항체는 MRS유전자를 발현백터에 클로닝하여 상기 유전자에 의해 암호화되는 단백질을 수득하고， 수득한 단백질을 동물에 주입하여 생성되는 항체를 수득하는 등의 당해 기술분야의 통상적인 방법에 따라 제조할 수 있다. 상기 MRS 항체는 MRS 전장서열 단백질을 통해 제작되는 것일 수도 있고， 또는 MRS 항원성 부위를 포함하는 MRS 단백질의 단편올 이용하여 MRS 단백질 특이적인 항체를 제조할 수도 있다. 본 발명의 항체의 구체적 서열과 그 형태는 특별히 체한되지 않으며, 다클론항체 (polyclonal ant ibody) 또는 단일클론항체 (monoclonal ant ibody)를 포함한다. 또한 상기 항체는 제공되는 면역글로불린으로서의 종류가 특별히 제한되지 않으며, 일례로 IgG, IgA, IgM, IgE 및 IgD로 이루어진 군에서 선택되는 것일 수 있으며， 바람직하게는 IgG 항체일 수 있다. 나아가 본 발명의 항체에는 MRS 단백질에 특이적으로 결합할 수 있는 것이라면 인간화 항체, 키메릭 항체 등의 특수 항체와 재조합 항체도 포함된다. 또한 항원 -항체 결합성 (반웅)을 갖는 것이면 전체 항체의 일부도 본 발명의 항체에 포함되며， MRS에 특이적으로 결합하는 모든 종류의 면역글로불린 항체가 포함된다. 예를 들어 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 갖는 완전한 형태의 항체뿐 아니라 항체 분자의 기능적인 단편， 즉 항원 결합 기능을 갖는 Fab, F(ab' ) , F(ab' )2, Fv, 디아바디 (diabody)， scFv등의 형태일 수 있다. In the present invention, 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. In addition, 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. Furthermore, 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. F (ab '), F (ab '), < / RTI > which have antigen binding functions, as well as complete forms of antibodies with two full length light chains and two full length heavy chains, 2, Fv, diabody, scFv, and the like.
Fab( fragment ant i gen— binding )는 항체의 항원 결합 단편으로， 중쇄와 경쇄 각각의 하나의 가변 도메인과 불변 도메인으로 구성되어 있다. F(ab' )2는 항체를 펩신으로 가수분해시켜서 생성되는 단편으로， 두 개의 Fab가 중쇄 경첩 (hinge)에서 이황결합 (disul f ide bond)으로 연결된 형태를 하고 있다. F(ab' )는 F(ab' )2 단편의 이황결합을 환원하여 분리시킨 Fab에 중쇄 경첩이 부가된 형태의 단량체 항체 단편이다. Fv(variable fragment )는 중쇄와 경쇄 각각의 가변영역으로만 구성된 항체 단편이다. scFv(single chain variable fragment )는 중쇄가변영역 (VH)과 경쇄가변 영역 (VU이 유연한 펩티드 링커로 연결되어 있는 재조합 항체 단편이다. 디아바디 (diabody)는 scFv의 VH와 VL가 매우 짧은 링커로 연결되어 서로 결합하지 못하고, 동일한 형태의 다른 scFv의 VL와 VH와 각각 결합하여 이량체를 형성하고 있는 형태의 단편을 의미하며， 본 발명의 목적상 항체의 단편은 인간 유래 MRS 단백질에 대한 결합특이성을 유지하고 있는 것이라면 구조나 형태의 제한을 받지 않는다. Fab (fragment antigene- binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one constant domain of each of the heavy and light chains. 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 ( sc Fv) 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. For the purposes of the present invention, fragments of the antibody are characterized by binding specificity to human-derived MRS protein It is not limited by structure or form.
본원 발명에서 상기 항 -MRS 항체 (이의 기능적 단편 포함)는 MRS 단백질에 특이적으로 결합할 수 있는 것이라면, 상기 항체가 MRS와 상호작용 (즉， 결합)하는 부위 등이 특별히 제한되지 않으나， 바람직하게 MRS에서 서열번호 2로 표시되는 아미노산 서열을 포함하는 영역의 에피토프 (epitope)에 특이적으로 결합하는 것을 특징으로 하는 항체 또는 이의 기능적 단편일 수 있다. 더욱 바람직하게 서열번호 1로 표시되는 MRS(methionyl-tRNA synthetase) 단백질의 861 번째 내지 900번째 아미노산 영역을 포함하는 에피토프 (epitope)에 특이적으로 결합하는 것을 특징으로 하는 항체 또는 이의 기능적 단편이 바람직할수 있다. 본 발명의 일 실시예에서, 본 발명자들은 췌장암 세포에 대한 MRS의 고감도의 검출 (염색)을 위해, MRS에서 서열번호 2로 표시되는 아미노산 서열 영역을 에피토프로 하는 항체를 수득하고 이러한 항체가 MRS에 대한 고감도의 검출능을 제공 가능함을 확인한 바 있다. In the present invention, as long as the anti-MRS antibody (including its functional fragment) is capable of specifically binding to the MRS protein, 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. In one embodiment of the present invention, for the detection (staining) of a high sensitivity of MRS to pancreatic cancer cells, 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.
상기 서열번호 2로 표시되는 아미노산 서열을 포함하는 영역의 에피토프 (epitope)에 특이적으로 결합하는 항체는， 목적하는 특이적 결합능을 가지는 한 그 구체적 서열이 특별히 제한되지 않으나, 바람직하게 서열번호 4 또는 서열번호 16으로 표시되는 아미노산 서열을 포함하는 경쇄 상보성 결정부위 KCDR1) ; 서열번호 6 또는 서열번호 18로 표시되는 아미노산 서열을 포함하는 경쇄 상보성 결정부위 2(CDR2) ; 서열번호 8 또는 서열번호 20으로 표시되는 아미노산 서열을 포함하는 경쇄 상보성 결정부위 3(CDR3)을 포함하는 경쇄가변영역 (VU , 및 서열번호 10 또는 서열번호 22로 표시되는 아미노산 서열을 포함하는 중쇄 상보성 결정부위 l(CDRl) ; 서열번호 12 또는 서열번호 24으로 표시되는 아미노산 서열을 포함하는 중쇄 상보성 결정부위 2(CDR2) ; 서열번호 14또는 서열번호 26으로 표시되는 아미노산 서열을 포함하는 중쇄 상보성 결정부위 3(CDR3)을 포함하는 경쇄가변영역 (VH)을 포함하는 것일 수 있다. 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 (CDR2) 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 (CDR2) 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 represented by SEQ ID NO: 14 or SEQ ID NO: 26.
상기 CDR 구성을 가지는 바람직한 일례로서， 본 발명의 항체 (이의 기능적 단편 포함)는 경쇄가변영역이 서열번호 28로 표시되는 아미노산 서열을 포함할 수 있고， 중쇄 가변영역은 서열번호 30으로 표시되는 아미노산서열을 포함할 수 있다. As a preferred example having the above CDR structure, 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 >
상기 CDR 구성을 가지는 또 다른 바람직한 일례로서， 본 발명의 항체 (이의 기능적 단편 포함)는 경쇄가변영역이 서열번호 32로 표시되는 아미노산 서열을 포함할 수 있고， 중쇄 가변영역은 서열번호 34로 표시되는 아미노산 서열을 포함할 수 있다. In another preferred example of the CDR construct, 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.
가장 바람직한 일례로서， 본원 발명은 서열번호 36의 아미노산 서열로 이루어지는 경쇄 및 서열번호 37의 아미노산 서열로 이루어지는 중쇄로 이루어지는 항체를 제공한다. 또 다른 가장 바람직한 일례로서， 본원 발명은 서열번호 38의 아미노산 서열로 이루어지는 경쇄 및 서열번호 39의 아미노산 서열로 이루어지는 중쇄로 이루어지는 항체를 제공한다. As a most preferred example, 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. As another most preferred example, 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.
본 발명에서 검출 제제들 (대표적으로 항체 및 이의 기능적 단편 등)은 이의 '검출 '을 위하여, 일반적으로 검출가능 모이어티 (moiety)로 표지될 수 있다. 예를 들어， 문헌 [Current Protocol s in Immunology, Volumes 1 and 2 , 1991 , Col igen등 Ed. Wi ley- Interscience , New York, N. Y. , Pubs]에 기술된 기술을 이용하여 , 방사성 동위원소 또는 형광표지로 표지될 수 있다. 또는 다양한 효소 -기질 표지가 이용가능하며, 상기 효소적 표지의 예는 초파리 루시퍼라제 및 세균 루시퍼라제 (미국 특허 제 4，737，456호)와 같은 루시퍼라제， 루시페린 ( luci fer in) , 2，3-다이하이드로프탈라진디오네스， 말레이트 디하이드로게나제, 유라제 (urase) , 호스래디쉬 퍼옥시다제 (HRP0)와 같은 퍼옥시다제， 알칼라인 포스파타제， β - 갈락토시다제, 글루코아밀라제， 라이소자임， 사카라이드 옥시다제 (예를 들어 글루코스옥시다제, 갈락토스 옥시다제， 및 글루코스 -6-포스페이트 디하이드로게나제)， 헤테로사이클릭 옥시다제 (예를 들어 유리카제 및 잔틴 옥시다제)， 락토퍼옥시다제， 마이크로퍼옥시다제 등을 포함한다. 항체에 효소를 접합시키는 기술은 예를 들어, 문헌 [0 ' Sul l ivan 등， 1981， Methods for the Preparat ion of Enzyme—항체 Conjugates for use in Enzyme Immunoassay, in Methods in Enzym . (J . Langone & Η · Van Vunaki s , eds . ) , Academi c press , N . Y . , 73： 147-166]에 기술되어 있다. 표지는 다양한 공지된 기술을 이용하여 항체에 직접 또는 간접적으로 접합될 수 있다. 예를 들어， 항체는 바이오틴 (biot in)에 접합될 수 있고 상기에 언급된 3종의 광범위한 카테고리에 속하는 임의의 표지들이 아비딘과, 또는 그 반대로 접합될 수 있다. 바이오틴은 아비딘 (avidin)에 선택적으로 결합하고， 따라서 이 표지는 이러한 간접적 방식으로 항체에 접합될 수 있다. 또는, 항체에 표지의 간접적 접합을 달성하기 위하여， 항체는 작은 합텐 (hapten) (예를 들에 딕옥신 [digoxin] )과 접합될 수 있고 상기에 언급된 서로 다른 유형의 표지들의 하나가 항 -합텐 항체에 접합될 수 있다 (예컨대， 항-딕옥신 항체) . 따라서， 항체에 대한 표지의 간접적 접합이 달성될 수 있다. Detectives (typically antibodies and functional fragments thereof, etc.) in the present invention may be labeled with a detectable moiety, generally for their detection. See, for example, Current Protocol in Immunology, Volumes 1 and 2, 1991, Coligen et al. Ed. Can be labeled with a radioactive isotope or a fluorescent label using the techniques described in < RTI ID = 0.0 > Wieley-Interscience, New York, NY, Pubs. Examples of such enzymatic labels are luciferase such as Drosophila luciferase and bacterium luciferase (U.S. Patent No. 4,737,456), 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. Techniques for conjugating enzymes to antibodies are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Enzyme Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone and H. Van Vunaki, eds.), Academia c press, N. Y. , ≪ / RTI > 73: 147-166. The label can be conjugated directly or indirectly to the antibody using a variety of known techniques. For example, the antibody can be conjugated to biotin and any of the labels in the three broad categories mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and thus this label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve an indirect conjugation of the label to the antibody, 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). Thus, indirect conjugation of the label to the antibody can be achieved.
본 발명에서 용어 타머' 는 시료 내의 검출하고자 하는 분석물질과 특이적으로 결합할 수 있는 물질로 그 자체로 안정된 삼차 구조를 가지는 단일 가닥 핵산 (DNA , R A, 또는 변형 핵산)을 의미하는 것으로, 특이적으로 시료 내의 표적 단백질의 존재를 확인할 수 있다. '앱타머의 제조는 일반적인 앱타머의 제조 방법에 따라， 확인하고자 하는 표적 단백질에 대해 선택적이고 높은 결합력올 가지는 을리고뉴클레오티드의 서열을 결정하여 합성한 후， 올리고뉴클레오티드의 5 ' 말단이나 3 ' 말단을 앱타머 칩의 관능기에 결합할 수 있도톡， -SH, - OM , -OH또는 NH2로 변형을 시킴으로써 이루어질 수 있으나， 이에 제한되지 않는다. The term 'timer' as used herein 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.
본 발명에서 용어 '선방세포 (ac inar cel l ) 특이적 마커' 란, 선방세포와 선방세포가 아닌 타 세포 간에 유의한 차이를 보이는 표지자를 의미한다. 구체적으로 선방세포에만 특이적으로 존재 (발현)하거나 또는 타 세포에 비해 선방세포에만 존재량 (발현량)이 월등히 높아， 이의 존재 (발현) 여부 또는 존재량 (발현량)을 확인함으로써 선방세포를 구분할 수 있는 객관적으로 측정이 가능한 표지자를 말한다. 이러한 표지자로서는 일반적으로 단백질, DNA, RNA 및 대사물질 등이 이용되며， 본 발명에서는 선방세포 특이적 마커 단백질이 사용되는 것이 바람직할 수 있다. 당업계에 선방세포 특이적 마커 단백질로 알려진 것이라면， 그 종류가 특별히 제한되지 않으나, 예를 들어 키모트립신 (Chymotrypsin) , 포스포리파아제 A2 그룹 IB(PLA2G1B, Phospho l ipase A2 group IB) 및 아밀라아제 A2(amylase 2A)로 이루어지는 군에서 선택되는 하나 이상의 것을사용할 수 있다. In the present invention, 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. As such a marker, proteins, DNA, RNA, metabolites and the like are generally used. In the present invention, it is preferable that a precursor cell-specific marker protein is used. For example, 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.
상기 '키모트립신 (chymotrypsin)' 은 본 발명에서 당업계에 키모트립신 (특히， 인간의 것)으로 알려진 것이라면 그 구체적 아미노산 서열 구성이 특별히 제한되지 않으며， 일례로 Genbank(NCBI ) accession number EAW51726.1, EAW51725.1, CAA74031.1, AAH15118.1, CAA74031.1, NP_009203.2 등으로 공지된 것을 대상으로 할 수 있다. 일례로 본 발명의 일실시예에서는 NP_009203.2(서열번호 3 참조)로 표시되는 키모트립신 단백질을 표지자로 하는 항체를 사용하여 MRS 항체와 함께 이중염색을 실시한 바 있다. 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. For example, Genbank (NCBI) accession number EAW51726.1, EAW51725.1, CAA74031.1, AAH15118.1, CAA74031.1, NP_009203.2, and the like. For example, in one embodiment of the present invention, 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).
상기 '포스포리파아제 A2 그룹 IB(PLA2G1B, Phospho 1 ipase A2 group IB)' 은 본 발명에서 당업계에 PLA2G1B 단백질 (특히， 인간의 것)로 알려진 것이라면 그 구체적 아미노산 서열 구성이 특별히 제한되지 않으며， 일례로 Genbank(NCBI ) accession number AAI06727.1, AAI06726.1 , AAH05386.1 , NP_000919.1 등으로 공지된 것을 대상으로 할수 있다. The specific amino acid sequence constitution of the phospholipase A2 group IB (PLA2G1B, Phospho 1 ipase A2 group IB) is not particularly limited as long as it is known in the art as PLA2G1B protein (in particular, human) (NCBI) accession numbers AAI06727.1, AAI06726.1, AAH05386.1, NP_000919.1, and so on.
상기 '아밀라아제 A2(amylase 2Α)' 은 본 발명에서 당업계에 아밀라아제 Α2(특히， 인간의 것)로 알려진 것이라면 그 구체적 아미노산 서열 구성이 특별히 제한되지 않으며， 일례로 Genbank(NCBI ) accession number AAI46998.1 , AAH07060.1, BAD97183.1, AAA51723.1 등으로 공지된 것을 대상으로 할 수 있다. The amylase A2 (amylase 2α) is not particularly limited as long as it is known in the art as amylase A2 (particularly human). For example, Genbank (NCBI) accession number AAI46998.1 , AAH07060.1, BAD97183.1, AAA51723.1, and the like.
본 발명에서 상기 선방세포 특이적 마커 단백질의 발현수준올 측정하는 제제는， 당업계에 단백질의 발현수준 측정에 사용가능한 것으로 알려진 것이라면 그 종류가 특별히 제한되지 않으나， 바람직하게 상기 마커 단백질에 특이적으로 결합하는 항체 또는 앱타머일 수 있으며， 이에 대한 구체적인 설명은 전술한 항 -MRS 항체 및 ¾타머에 준한다. In the present invention, the expression level of the precursor cell-specific marker protein may be measured in a manner known to those skilled in the art. For example, 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 타 Tumor.
본 발명의 췌장암 진단용 키트에는 MRS 단백질 또는 /및 선방세포 특이적 마커 단백질의 발현 수준을 측정하기 위하여, 선택적으로 상기 단백질을 각각 특이적으로 인식하는 항체 또는 업타머뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물， 용액 또는 장치가포함될 수 있다. 구체적인 양태로서 상기 키트는 웨스턴 블랏， ELISA, 방사선면역분석， ' 방사선 면역 확산법， 오우크테로니 면역 확산법， 로케트 면역전기영동， 면역염색법， 면역침전 분석법, 보체 고정 분석법， FACS, SPR 또는 단백질 칩 방법을 수행하기 위해 필요한 공지의 필수요소 및 부수 요소를 포함하는 것을 특징으로 하는 진단 키트일 수 있으나, 이에 제한되지 않는다. 일례로， 상기 키트는 MRS 단백질에 대한 특이적인 항체를 포함한다. 상기 항체는 목적 마커 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 대한 교차반응성이 거의 없는 (실질적으로 없는) 항체로， 단클론 항체， 다클론 항체 또는 재조합 항체이다. 또한 상기 키트는 추가적으로 임의의 대조군 단백질에 특이적인 항체를 포함할 수 있다. 키트에 제공되는 항체는 그 자체로서 검출 가능한 모이어티로 표지될 수 있으며, 이는 전술한 바와 같다. 그 외 상기 키트는 결합된 항체를 검출할 수 있는 별도의 시약， 예를 들면， 표지된 2차 항체, 발색단 (chromophores) , 효소 (항체와 컨주게이트된 형태로서) 및 그의 기질， 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다. 또한， 본 발명의 키트는 잉여의 발색 기질 및 결합되지 않은 단백질 등은 제거하고 항체와 결합된 단백질 마커만을 보유할수 있는 세척액 또는 용리액을 포함할 수 있다. 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. As a specific embodiment 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 But it should be understood that the invention is not limited thereto. In one embodiment, 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. In addition, 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.
상기 MRS 단백질의 발현 수준을 측정하는 제제는， 또한 MRS 유전자 (MARS)의 발현수준을 검출하는 제제를 포함하는 의미일 수 있다. 단백질 발현 수준의 증가는 상기 단백질을 암호화하는 유전자로부터의 전사물 (예를들어 mRNA)의 증가가 동반되는 것이므로， 당업자라면 전술한 것과 같이 MRS 단백질 자체를 검출하는 수단 뿐만아니라 간접적으로 MRS 단백질 발현과 직접적으로 관계된 전사물들을 검출하는 수단을 사용가능함이 자명하게 이해 가능하다. 상기 MRS mRNA를 검출하는 제제는 MRS mRNA에 특이적으로 부착 또는 흔성화 (hybr idizat ion)하는 리간드라면 그 종류가 특별히 제한되지 않으나， 예를 들어 프라이머 (쌍) 또는 프로브 일 수 있다 . 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). As an increase in the level of protein expression is accompanied by an increase in the transcript (e. G., MRNA) from the gene encoding the protein, those skilled in the art will recognize that the MRS protein itself, It is obviously understandable that means for directly detecting the related transcripts can be used. 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.
또한 선방세포 (acinar cel l ) 특이적 마커 단백질의 발현 수준을 측정하는 제제는， 상기 선방세포 특이적 마커 단백질을 암호화하는 유전자의 발현 수준을 검출하는 제제를 포함한다. 일례로 선방세포 특이적 마커 단백질을 코딩하는 mRNA를 검출하는 제제를 사용가능하며， 상기 mRNA에 특이적으로 부착 또는 흔성화(1 1) (^ 23 011)하는 리간드라면 그 종류가 특별히 제한되지 않으나， 예를 들어 프라이머 (쌍) 또는 프로브 일 수 있다. Also, 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. For example, a preparation for detecting mRNA coding for a precursor cell-specific marker protein can be used. If 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.
상기 '프라이머' 는 짧은 자유 3-말단 수산화기 ( free 3 ' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트 (template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로서 작용하는 짧은 핵산 서열을 말한다. 프라이머는 적절한 완층용액 및 온도에서 중합반응을 위한 시약 (즉， DNA 폴리머라제 또는 역전사효소) 및 상이한 4 가지의 뉴클레오사이드 트리포스페이트의 존재 하에서 DNA 합성을 개시할 수 있다. PCR조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 기술에 따라 적절히 선택될 수 있다. 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. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.
프라이머의 서열은 주형의 일부 염기 서열과 완전하게 상보적인 서열을 가질 필요는 없으며， 주형과 흔성화되어 프라이머 고유의 작용을 할 수 있는 범위 내에서의 층분한 상보성을 가지면 층분하다. 따라서 본 발명에서 목적 mRNA의 발현 수준을 측정하기 위한 프라이머는 대상으로 하는 단백질을 코딩 유전자 서열에 완벽하게 상보적인 서열을 가질 필요는 없으며， DNA 합성을 통해 mRNA 또는 cDNA의 특정 구간을 증폭하여 목적 mRNA의 양을 측정하려는 목적에 맞는 길이와 상보성을 갖는 것이면 층분하다. 상기 증폭 반응을 위한 프라이머는 증폭하고자 하는 mRNA의 특정 구간의 양쪽 끝부분의 주형 (또는 센스， sense)과 반대편 (안티센스， ant isense)에 각각 상보적으로 결합하는 한 세트 (쌍)으로 구성된다. 프라이머는 당업자라면 단백질을 코딩하는 mRNA 또는 cDNA 염기서열을 참조하여 용이하게 디자인할 수 있다. 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.
프로브 (probe) '는. 특정 유전자의 mRNA나 cDNA( complementary DNA)에 특이적으로 결합할 수 있는 짧게는 수개 내지 길게는 수백 개의 염기 (base pai r) 길이의 RNA 또는 DNA 등 폴리뉴클레오티드의 단편을 의미하며, 표지 ( label ing)되어 있어서 결합하는 대상 mRNA나 cDNA의 존재 유무, 발현양 둥을 확인할 수 있다. 본 발명와 목적을 위해서는 목적 mRNA에 상보적인 프로브를 피검체의 시료와 흔성화 반응(11 13 (^ 2 1011)을 수행하여 목적 mRNA의 발현량을 측정함으로써 진단에 이용할 수 있다. 프로브의 선택 및 흔성화 조건은 당업계에 공지된 기술에 따라 적절하게 선택할수 있다. 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. For the purpose of the present invention and its object, 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.
본 발명의 프라이머 또는 프로브는 포스포아미다이트 (phosphoramidi te) 고체 지지체 합성법이나 기타 널리 공지된 방법을 이용하여 화학적으로 합성할 수 있다. 또한 프라이머 또는 프로브는 MRS mRNA와의 흔성화를 방해하지 않는 범위에서 당해 기술분야에 공지된 방법에 따라 다양하게 변형시킬 수 있다. 이러한 변형의 예로는 메틸화， 캡화， 천연 뉴클레오티드 하나 이상의 동족체로의 치환 및 뉴클레오티드 간의 변형， 예를 들면 하전되지 않은 연결체 (예: 메틸 포스포네이트， 포스포트리에스테르， 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체 (예: 포스포로티오에이트， 포스포로디티오에이트 등) , 그리고 형광 또는 효소를 이용한 표지물질 ( label ing mater i al )의 결합 등이 있다. The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, 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. Examples of such 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.
본 발명의 진단용 키트에는 MRS 단백질 또는 /및 선방세포 특이적 마커 단백질의 발현 수준을 측정하기 위하여 각각에 대한 mRNA를 인식하는 프라이머 또는 프로브 뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치가 선택적으로 포함될 수 있다. 상기 키트는 당업계에 프라이머 (프라이머쌍) 또는 프로브를 구성품으로 제공하는 분석 키트로서 알려진 것이라면 그 종류가 특별히 제한되지 않으나, 예를 들어 PCR(polymerase chain react ion, 중합효소연쇄반응)， RNase 보호 분석법， 노던 블랏팅， 서던 블랏팅 또는 DNA마이크로어레이 칩용 키트 등을 포함한다. 일례로, 상기 진단 키트는 중합효소반웅을 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 진단용 키트일 수 있다. 중합효소반웅 키트는 마커 유전자 (mRNA)에 대한 특이적인 각각의 프라이머 쌍을 포함한다. 프라이머는 각 마커 유전자 (mRNA)의 핵산서열에 특이적인 서열을 가지는 뉴클레오타이드로서， 약 7bp 내지 50bp의 길이, 보다 바람직하게는 약 10bp 내지 30bp의 길이이다. 또한 대조군 유전자의 핵산 서열에 특이적인 프라이머를 포함할 수 있다. 그 외 중합효소반웅 키트는 테스트 튜브 또는 다른 적절한 컨테이너， 반응 완층액 (pH 및 마그네슴 농도는 다양)， 데옥시뉴클레오타이드 (dNTPs) , DNA 폴리머라아제 (예를 들어 Taq-폴리머라아제) 및 역전사효소， DNAse , RNAse 억제제 DEPC-수 (DEPC-water )， 멸균수 등을 포함할수 있다. 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. For example, the kit includes PCR (polymerase chain reaction), RNase protection assay , Northern blotting, Southern blotting or kits for DNA microarray chips, and the like. As an example, 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. etc 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.
또한 본 발명은 췌장암 진단에 필요한 정보를 제공하기 위하여， 잠재 환자로부터 채취한 췌장 시료로부터 MRS 단백질 및 선방세포 특이적 마커 단백질의 발현 수준을 정성 또는 정량 분석하는 방법을 제공한다. 즉, 본 발명은 피검체 시료 내의 MRS 단백질 및 선방세포 특이적 마커 단백질의 발현 수준을 측정하는 것을 특징으로 하는 췌장암 진단 방법올 제공한다. 구체적으로 상기 방법은 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,
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계의 측정 시료에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현되면 췌장암 세포인 것으로 판단하는 단계를 포함하는 것일 수 있다. (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.
본 발명에서 용어 '분석' 은 바람직하게 '측정' 을 의미하는 것일 수 있고, 상기 정성분석은 목적하는 물질의 존재 여부를 측정 및 확인하는 것올 의미하는 것일 수 있으며， 상기 정량분석은 목적하는 물질의 존재 수준 (발현 수준) 또는 양의 변화를 측정 및 확인하는 것을 의미하는 것일 수 있다. 본 발명에서 분석 또는 측정은 정성적인 방법과 정량적인 방법을 모두 포함하여 제한 없이 수행될 수 있다. 따라서 MRS 단백질 검출은 MRS 단백질의 존재 여부 검출， 또는 상기 단백질 발현량의 증가 (상향 조절)를 확인하는 것을 포함하는 의미이다. In the present invention, 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. In the present invention, 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.
이하， 상기 방법을 각 단계에 따라 설명한다. Hereinafter, the above method will be described in accordance with each step.
상기 (a) 단계는 잠재 환자로부터 채취한 췌장 시료를 제공하고 상기 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계이다. 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 .
본 발명의 상기 피검체란 동물， 바람직하게는 포유동물, 특히 인간을 포함하는 동물일 수 있으며, 상기 동물 유래 세포, 조직， 기관 등을 포함한다. 더 바람직하게는 진단이 필요한 인간 또는 환자 (pat ient ) 일 수 있다. 본 발명에서는 상기 (a) 단계 이전에 피검체 또는 잠재환자로부터 시료를 제공하는 단계를 수행할 수 있다. 본 발명에서 용어 '잠재 환자' 는 췌장암으로 의심되는 환자를 의미하는 것으로서， 임상 증상， 혈액학적 검사 또는 영상학적 검사상 등 다양한 검사들을 통해 췌장암이 있는 것으로 의심되는 환자를 의미한다. 즉， 상기 잠재 환자는 영상학적 검사상으로 췌장암 판정 가능한 환자 및 판정 불가능 환자를 포함하며， 영상학적 검사상으로 췌장암 판정이 불가능하다고 하더라도 임상 증상, 혈액학적 검사에서 췌장암이 의심되는 환자를 의미한다. 췌장암 환자에서 나타날 수 있는 임상증상으로는 복부 통증， 황달， 체중 감소 소화장애， 당뇨 등이 있으나 이러한 증상들이 췌장암에만 국한된 특이 증상은 아니다. 또한 혈액학적 검사상 황달이나 당뇨 검사 수치， CEA, CA19-9와 같은 종양표지자가 상승될 수 있다. 영상학적 검사는 복부 초음파， 복부 CT, 복부 MRI , PET-CT를 시행할 수 있으며 이러한 영상학적 검사에서 췌장의 종괴가 있을 시 췌장암을 의심하게 된다. 이러한 영상학적 검사들로서는 췌장암을 의심할 수는 있으나 췌장암을 확진할 수는 없다. 췌장암의 최종적인 확진은 병리학적 검사로 진행되며， 수술이 가능한 환자에서는 수술 후 얻어진 조직을 통해 확진되고 수술이 가능하지 않는 환자에서는 세포진 검사를 이용해 확진하게 된다. 바람직하게 본 발명의 상기 잠재 환자 (췌장암 의심환자)는 췌장암에서 일반적으로 관찰되는 증상인 복부통증， 황달， 체증감소， 소화장애, 당뇨 등의 일반증상이 있고， CT, 초음파, MRI 등과 같은 진단 장비를 통해 췌장암으로 확진할 수 없는 환자를 의미하는 것일 수 있다. 더욱 바람직하게, 상기 잠재 환자는 넓은 부위의 침습적 조직 검사가 불가능 또는 불필요한 환자 (즉， 수술에 의한 췌장조직검사가 불가능 또는 불필요하기 때문에)로서 세포검사 (세포진)에 의존적으로 췌장암을 명확히 진단할 필요성이 있는 환자일 수 있다. 즉， 세포 수준의 분석에 의해 췌장암을 명확히 진단할 필요성이 있는 환자일 수 있으나, 이에 제한되는 것은 아니다. 상기 시료는 췌장암의 존재 여부를 진단하고자 하는 개체 (환자) 또는 피검체로부터 채취된 것이라면 특별히 제한되지 않으나, 바람직하게 췌장 조직 또는 췌장 세포일 수 있다. 상기 췌장 조직은 췌관 (pancreat ic duct )을 포함하는 췌장의 모든 부위 (특히， 특히， 병변 의심부위)로부터 수득될 수 있다. 상기 췌장 조직은 일반적으로 췌장에서 생검 (biopsy) 또는 수술에 의하여 수득되는 것일 수 있다. 상기 췌장세포를 분리하는 방법은 특별히 제한되지 않으며， 당업계에서 현재 인체 조직의 세포를 분리하기 위해 사용되고 있는 방법뿐만 아니라， 장래에 동일한 목적으로 개발될 새로운 방법도 포함되는 개념으로 이해된다. 바람직하게는 솔세포진 (brushing cytology) 또는 미세바늘흡입법 (Fine needle aspi rat ion(FNA) , 세침흡인법)일 수 있으며, 가장 바람직하게는 내시경 초음파 미세바늘 흡입법 (EUS- FNA)일 수 있다. 본 발명에서 용어 '솔세포진 (brush cytology) 방법으로 채취 '란 통상의 세포진 솔 (cytology brush)을 이용해 췌장 특히, 췌관 표면 (특히， 환부 의심 부위)올 문질러서 세포를 채취하는 방식을 의미한다. 본 발명에서 용어 '미세바늘흡입법 (세침흡인법 ) 방법으로 분리된' 이란 세포진 (cytodiagnosis)에 통상적으로 사용되는 얇은 바늘을 이용해 병변 (췌장암 의심 부위)의 세포를 흡인하여 뽑아내는 채취 방식을 의미한다. 바람직하게 하나의 실시양태 (embodiment )에서， 상기 췌장 조직 또는 췌장 세포 시료에는 필수적으로 췌관세포가포함된다. 췌관은 췌장실질과 구분된다. 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. In the present invention, 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. That is, 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. These imaging tests 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. Preferably, 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. More preferably, 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. Preferably, it may be brushing cytology or fine needle aspiration (FNA) or fine needle aspiration, and most preferably endoscopic ultrasonic microneedle inhalation (EUS-FNA). In the present invention, 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 . Preferably, in one embodiment, the pancreatic tissue or pancreatic cell sample essentially comprises pancreatic duct cells. The pancreatic duct is distinct from the pancreatic parenchyma.
수득된 췌장 세포 또는 조직은 당업계에 공지된 통상의 시료 전처리 (예를들어 고정， 원심분리， 슬라이드에 도말 둥) 방식에 따라 전처리되어 제공될 수 있다. 바람직한 하나의 실시 양태로서, 상기 췌장 세포 또는 조직 시료는 통상의 파라핀 블톡 (paraf f in block) 또는 파라핀 절편 (paraff in sect ion) 제작법에 의해 전처리되어 실험용 슬라이드 (s l ide) 상에 제공 되는 것일 수 있다. 또한 바람직한 하나의 실시 양태로서， 상기 췌장 세포 또는 조직 시료는 통상의 액상 단층세포 슬라이드 제작법 (액상세포검사용 슬라이드 제작법)에 의해 전처리되어 준비되는 것일 수 있으며， 일례로 ThinPrep , SurePath, Cel l Prep 등을 사용하여 액상 기반 단층 부착 방법에 의해 실험용 슬라이드 (sl ide) 상에 것일 수 있다. The obtained 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. In one preferred embodiment, 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. In one preferred embodiment, 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.
본 발명의 상기 췌장암 진단방법은 바람직하게 췌장 세포를 분석하는 것일 수 있다. 췌장 세포를 직접 분석하는 세포학적 분석 (Cytologycal analysi s)방법은 초직 검사와는 많은 차이가 있고, 때문에 본 명세서에서 전술한 선행기술문헌들에서 보고하고 있는 췌장암 진단방법과는 많은 차이가 있다. 또한 췌장세포 자체를 이용하기 때문에 타 장기의 종양과 흔동될 여지가 없다. 기존에 조직검사는 목적 부위를 내시경적으로 관찰하거나 암으로의 형질전환이 의심되는 조직으로부터 lg 내지 109 cel ls 정도의 일정 영역의 조직을 채취한 후 염색 등과 같은 생화학적 방식을 통하여 암진단을 수행한다. 이러한 조직검사는 주변의 구조나 세포와 비교를 통해 특정 영역에 암이 있는 것으로 확진하기가 비교적 용이한 것으로 알려져 있다. 또한 조직 수준에서의 마커 발현형태는 주변의 정상 조직들과 전체적으로 경향성들의 비교를 통해 확진이 더 용이하다. 하지만 세포진 검사의 경우에는 낱개의 세포를 뽑아내어 도말한 것이므로 주변 조직과의 관계를 증명할 수 없어 진단에 상당한 어려움이 따르기 때문에， 세포 수준에서의 질환 진단이 큰 의미를 지닌다. 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. In addition, because the pancreas cells themselves are used, there is no room for controversy with tumors of other organs. Previously, 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 . It is known that such a biopsy is comparatively easy to confirm that cancer is present in a specific area through comparison with surrounding structures or cells. In addition, the expression pattern of the marker at the tissue level is more easily confirmed by comparing the trends with surrounding normal tissues as a whole. However, in the case of the cytopenia test, the diagnosis of the disease at the cellular level is of great significance because it is difficult to prove the relationship with the surrounding tissues since the individual cells are extracted and smoked.
상기 '단백질의 발현수준을 측정' 하는 것은 발현 여부를 측정하는 것 (즉, 발현 유무를 측정하는 것)， 또는 상기 단백질의 질적， 양적 변화 수준을 측정하는 것을 의미한다. 상기 측정은 정성적인 방법 (분석)과 정량적인 방법을 모두 포함하여 제한 없이 수행될 수 있다. 단백질 수준의 측정에 있어서 정성적 방법과 정량적 방법의 종류는 당업계에 잘 알려져 있으며， 본 명세서에서 기술한 실험법들이 이에 포함된다. 각 방법 별로 구체적 단백질 수준 비교 방식은 당업계에 잘 알려져 있다. 본 발명에서 용어 '검출' 은 목적하는 물질 (본 발명에서의 마커 단백질, MRS 또는 /및 선방세포 특이적 마커)의 존재 (발현) 여부를 측정 및 확인하는 것， 또는 목적하는 물질의 존재 수준 (발현 수준)의 변화를 측정 및 확인하는 것을 모두 포함하는 의미이다. 따라서 본 발명에서 용어 '단백질 검출' 은 목적 단백질의 존재 여부 검출， 또는 상기 단백질 발현량의 증가 (상향 조절)를 확인하는 것을 포함하는 의미이다. 본 발명에서 단백질의 '발현 증가 (또는 고발현)' 라는 의미는 발현되지 않던 것이 발현된 것 (즉, 검출되지 않던 것이 검출된 것) 또는 정상적인 수준보다 상대적으로 과발현된 것 (즉， 검출량이 많아지는 것 )을 의미한다 . 이는 음성대조군과의비교 또는 대조를 포함하는 과정을 수행함을 내포할 수 있다. 이의 반대적 용어의 의미는 당업자라면 상기 정의에 준하여, 반대의미를 가지는 것으로 이해 가능하다. 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. In the present invention, 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. Accordingly, 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. In the present invention, 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.
본 발명에서 MRS 단백질 및 선방세포 특이적 마커 단백질의 검출은 당업계에 공지된 단백질 발현 수준 측정법에 의한 것이라면 그 방법이 특별히 제한되지 않으나， 일례로 상기 단백질에 특이적으로 결합하는 항체를 이용하여 검출하거나 측정할 수 있다. 본 발명에서 목적 단백질에 특이적으로 결합하는 항체에 대해서는 전술한 바와 같다. 단백질 발현 수준을 측정하는 방법은 당업계에서 공지되어있는 방법이라면 특별히 제한되지 않으나， 예를 들어 웨스턴 블랏， ELISA(enzyme-l inked i隱 unospeci'f ic assay, 효소면역분석법)， 방사선면역분석, 방사선 면역 확산법， 오우크테로니 면역 확산법, 로케트 면역전기영동， 면역염색법 (면역조직화학염색， 면역세포화학염색 및 면역형광염색 등 포함) , 면역침전 분석법， 보체 고정 분석법, F ACS (Fluorescence act ivated cel l sorter) , SPR( surface plasmon resonance) 또는 단백질 칩 방법 중 어느 하나를 이용하는 것일 수 있다. 이외에도 상기 측정 방법은, 본원 발명에서 제공하는 MRS 단백질 및 선방세포 특이적 마커 단백질 발현수준 측정 제제 및 이를 포함하는 키트에 대하여 기술된 바에 준하여 그 측정 방법이 이해된다. In the present invention, if the MRS protein and the secretory cell-specific marker protein are detected by a method for assaying protein expression levels known in the art, 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. If 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隱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. In addition, 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.
기존 세포진 검사들의 경우에는 췌장암인지 또는 정상세포 (비췌장암 세포를 포괄적으로 의미하는 것으로 예를 들어 췌장염 세포 등을 포함)인지 여부가 명확하지않은 비정형 세포 (atypical cel l )로서 병리소견을 내는 경우가 많으며， 이러한경우 추가적이고 다수의 반복적인 재검을 필요로 한다. 종래 보고된 췌장암 진단 마커들의 경우 세포진 검사의 적용에 있어서, 조직 검사에서와는 다르게 세포 수준의 진단에서는 민감도 및 특이도가 좋지 못하여 실효성을 거두고 있지 못하는 실정이다. 이는 본 발명의 명세서 일실시예에서 잘 나타나 있다. 기존의 췌장암의 진단에 가장 일반적으로 쓰이고 있는 종양 마커 중 하나인 CEA의 경우， 췌장 종양세포에서 그 발현이 관찰되지 않거나 발현이 되었더라도 그 정도가 매우 약하였으며， H&E 염색결과 비정형 세포로 판단되었고 추후 최종적으로 췌장암으로 확진된 환자의 췌장 세포진 시료에 대하여 CEA가 전혀 검출되지 않았다는 것을 통해서도 나타난다. 즉, 종래 보고된 췌장암 진단 마커들의 경우， 기존 H&E 염색 등을 이용한 병리학적 세포진단 방법으로는 췌장암인지 또는 정상세포인지 여부가 명확하지 않은 비정형 세포에서 일관성 있는 발현이 나타나지 않아 명확한 진단이 불가능하지만， 본 발명에 따른 MRS의 경우에는 비정형으로 판정되는 췌장세포에서도 종양인지 여부에 대한 명확한 판단이 가능하기 때문에 보다 정확하게 췌장암을 진단할 수 있는 현저한 효과를 나타낸다. 즉， 본 발명에 따른 MRS의 경우에는 세포진 검사에 적용하여도 그 정확도가 매우 높으며， 특히 선방세포 특이적 마커와 병용되었올 때 선방세포에 의한 위양성 판단오류를 현저히 감소시키기 때문에 췌장암 진단 정확도가 현저히 상승되는 효과를 가진다. In the case of conventional cytological examinations, atypical cells (pancreatic cancer) or abnormal cells (atypical cells), which are unclear as to whether normal cells (including pancreatic cancer cells, etc., which comprehensively mean non-pancreatic cancer cells) And in these cases additional and multiple repetitive re-examinations are required. Previously reported 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. Thus, 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.
이에 본 발명은， 상기 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하기 (예를 들어 상기 (a) 단계) 이전에, 동시에 또는 이후에 하기 ( i ) 및 ( Π ) 단계를 추가적으로 수행하여 진단 효과를 더욱 높일 수 있다: Accordingly, 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:
( i ) 잠재 환자로부터 채취한 췌장 세포를 세포핵을 염색하는 DAPH4' ,6-diamidino—2-phenyl indole) , 메틸렌블루 (methylene blue) , 아세트산카민 (acetocarmine)， 틀루이딘블루 (toluidine blue) , 헤마특실린 (hematoxyl in) 및 훽스트 (Hoechst )로 이루어진 군에서 선택된 하나 이상의 염색용액， 및 세포질을 염색하는 에오신 (eosin) , 크리스탈바이을렛 (crystal violet ) 및 오렌지 G(orange G)로 이루어진 군에서 선택된 하나 이상의 염색 용액으로 세포 염색하는 단계; 및 (i) 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; And
( i i ) 상기 세포염색에 의해 췌장 세포를 악성종양세포， 비정형세포 (atypical cel l ) 또는 정상세포로 판단하는 단계. (ii) judging the pancreatic cells as malignant tumor cells, atypical cells or normal cells by the cell staining.
상기 ( i i ) 단계에서 판별되는 비정형세포는 미확진 및 확진 불가 세포로 이해되며， 이에 제한되지 않으나, 구체적으로 상기 형태학적 진단 방식의 병리검사에서 Suspicious of mal ignancy (악성 종양세포 의심 ) 판정도 모두 포함하는 의미일 수 있다. 상기 ( i ) 및 ( i i ) 단계는 형태학적 진단 방식의 기존 병리검사에 따른 세포진 (cytodiagnosis) 방법으로서, 본 발명의 일실시예에서 사용하고 있는 H&E 또는 pap 염색에 준하는 것들이다. 본 발명에서， 용어 '형태학적 진단 방식의 병리검사' 또는 '형태학적 검사' 란， 정상 세포가 암으로 변화될 때의 비정상적인 형태학적 변화를 검사하는 것을 의미한다. ' 상기 비정상적인 형태학적 변화에 관한 구체적 검사 항목 또는 기준은， 당업계에 암세포가 가지는 형태학적 변화의' 종류라면 그 구체적 내용이 특별히 제한되지 않으나， 바람직하게 세포 군집성; 세포핵 /세포질 비율 (N/C rat io) ; 핵막의 모양 (핵막 모양의 불규칙성) ; 염색질의 뭉침 현상; 핵 내 핵소체의 출현; 및 유사분열의 출현으로 이루어지는 군에서 선택되는 하나 이상을 검사하는 것일 수 있다. 상기 형태학적 검사는 본 발명에서 제공하는 MRS 단백질 및 선방세포 특이적 마커 단백질의 발현 수준을 정성 또는 정량 분석하는 것에 의해 췌장암 진단에 필요한 정보를 제공하는 방법 (췌장암 진단 방법)과 동시에 (simultaneous) , 별도로 (separate) 또는 순차적 (sequent ial )으로 수행될 수 있다. 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. In the present invention, the term " morphological diagnostic pathology " or " morphological examination " refers to examining abnormal morphological changes when normal cells are transformed into cancers. If the type "specific examination items or criteria related to the abnormal morphological changes, the morphological changes in cancer cells with the art, 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. ≪ RTI ID = 0.0 > [0031] < / RTI > 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.
이에， 상기 ( Π ) 단계에서 상기 ( i ) 단계의 세포 염색결과로부터 췌장 세포 시료를 악성종양세포, 비정형 세포 또는 정상세포로 판별하 ¾ 것은 정상 세포가 암으로 변화될 때의 비정상적인 형태학적 변화에 근거하여 판별되는 것일 수 있으며， 그 구체적 판별 기준은 당업계에 잘 알려져 있다. 이때 비정형 세포란 형태학적 변화로는 악성 종양세포 (암세포) 또는 정상세포로 명확한 판정이 불가한 세포를 의미한다. 본 발명의 바람직한 일 실시양태에서， 상기 ( i i ) 단계에서 상기 ( i ) 단계의 세포 염색결과로부터 췌장 세포 시료를 악성종양세포， 비정형 세포 또는 정상세포로 판별하는 것은 바람직하게 하기와 같은 기준에 의해 수행되는 것일 수 있다: 세포가 3차원으로 도말됨; 세포핵 /세포질 비율 (N/c rat io, Nuclear to cytoplasmic rat io)이 높음; 염색질의 뭉침 현상 출현; 거친 모양의 핵막 (핵막의 불규칙 정도가 커짐) ; 핵소체의 출현; 및 유사분열의 출현으로 이루어지는 군에서선택된 두 가지 이상의 형태학적 이상을 보이는 경우에 악성 종양세포로 판정하며， 세포가 한 겹으로 도말되어 있으며 세포핵 /세포질 비율 (N/C rat io)이 작고 핵막이 매끄러운 모양일 경우에는 정상 세포로 판정하고， 세포의 변화가 악성 세포에는 미치지 못하나 정상 (benign 포함)으로 판정할 수 없는 경우 비정형 세포 (aypical cel l )로 판정한다. From the result of the cell staining in step (i), 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. At this time, 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. In a preferred embodiment of the present invention, 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) It 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).
상기 ( i ) 및 ( Π ) 단계를 MRS 검출 (발현수준 측정) 단계 이전에, 동시에 또는 이후에 병행하여 추가적으로 수행하게 되면， 세포수준의 검사 (즉, 세포진 검사)만으로도 매우 높은 정확도의 진단결과를 얻을 수 있는 것이 특징이다. 일례로 MRS 검출 이전에 ( i ) 및 ( i i ) 단계를 수행하는 경우에 어서, 세포염색을 통해 악성종양세포 또는 정상세포로 판단이 된 췌장 세포의 경우에는 후속적으로 수행되는 (a) 단계에서 MRS 및 선방세포 특이적 마커 단백질의 발현수준 (또는 여부)을 추가적으로 재분석함으로서 보다 확실하게 췌장암인지 정상인지 여부를 판단할 수 있어 진단오류를 현저히 줄일 수 있으며， 상기 세포염색올 통해 비정형 세포로 판단이 된 경우에는 후속적으로 수행되는 (a) 단계에서 선방세포 특이적 마커 단백질과 MRS 발현수준 (여부)을 분석함으로서 종양인지 여부에 대한 명확한 판단이 가능하다. If the above steps (i) and (Π) are performed before, simultaneously or after the MRS detection (expression level measurement) step, the cell level test (ie, cytology test) It is characterized by what can be obtained. For example, 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).
상기 본 발명은 MRS 및 선방세포 특이적 마커 단백질의 이중 염색법을 통해 조직뿐만 아니라 세포수준의 검사에서 정확도 높은 확정 진단이 가능하다는 것이 그 특징이다. 기존에 비정형 세포로 검진결과가 나왔을 때 조직 생검을 다시 수행하여 재진단하여야 하는 번거로움이 있고， 다량의 생검을 필요로 하는 조직검사의 경우 세포진 검사 보다 시료 취득에 있어서 환자에 신체적 부담이 가중되고 뿐만 아니라 췌장암 진행 정도에 따라 다량의 조직 시료를 얻기 어려운 경우가 있는 문제점이 것을 고려하였을 때， 세포 수준에서 정확한 진단을 제공하는 본원 발명은 더욱 큰 장점을 지닌다고 할 수 있다. 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. In the case of non-malignant cells, there is a need to perform a biopsy again, and a biopsy that requires a large amount of biopsy results in a physical burden on the patient In addition, when considering the problem that it is difficult to obtain a large amount of tissue samples depending on the degree of progression of pancreatic cancer, the present invention which provides accurate diagnosis at the cell level has a great advantage.
상기 (b) 단계에서는 상기 (a) 단계의 측정 시료에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 (발현증가)되면 췌장암 세포인 것으로 판단한다. In 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.
본 발명의 일실시예에 따르면， H&E 염색을 통해 정상세포로 판정이 된 췌장 세포에서는 MRS가 전혀 검출 (발현증가)이 되지 않았지만， 종양세포에서는 MRS가 강하게 발현 되는 것으로 확인되었다. 즉, MRS가 췌장암 진단 마커로서 사용될 수 있음을 확인한 것이다. 한편, H&E염색을 통해 비정형 세포 (atypical )로 판정이 되었으나 향후 환자를 추적 관찰하여 본 결과 최종적으로 췌장암으로 진단이 확정된 환자의 췌장세포에서도, MRS가 발현되어 검출되는 것으로 확인되었다. 한편， 췌장암의 진단에 있어서 정상세포에서는 대부분 MRS가 전혀 발현이 되지 않았지만 소수의 정상 시료에서 MRS가 발현되어 위양성올 나타내는 경우가 있는데， 이러한 위양성 판단은 정상 선방세포 (acinar cel l )에서 MRS가 고발현되는 현상에 기인함을 알아내었다. 이에 선방세포 특이적 마커로서 일례로 키모트립신 사용하여 MRS와 이중 마커 (dual marker )로 이용하는 본 발명의 이중 염색법을 개발하였다. 췌장암 세포의 경우 선방세포 마커 (키모트립신)가 발현되지 않고 MRS의 강한 발현 신호가 매우 우세하게 나타나며， 이를 통해 기존 세포진 검사에서 비정형 세포로 분류된 시료를 매우'높은 정확도로 췌장암과 비췌장암 (정상)으로 구분할 수 있다. According to one embodiment of the present invention, 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. In addition, 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. On the other hand, in the diagnosis of pancreatic cancer, 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. For example, a double staining method using MRS and a dual marker using chymotrypsin as a precursor cell specific marker has been developed. In the case of pancreatic cancer cells prior-cell markers (chymotrypsin) is not expressed appears to have strong expression signals of the MRS strongly predominating, through which a very "pancreatic and non-pancreatic cancer with high accuracy to the samples classified as atypical cells in a conventional Pap smear (normal ).
상기 (b) 단계는 다른 대조군 (시료)과 비교 없이도 MRS의 (고)발현 검출을 통해서 바로 췌장암 (특히， 췌관암 또는 췌관선암)의 검출이 가능한 것을 장점으로 한다. 이는 본 발명의 명세서 실시예에 잘 나타나있다. 췌장암 진단의 기준이 되는 MRS 검출 수준 (MRS 발현 수준， 특히 증가 수준)에 대해서는， 당업자가 선택한 측정 방법에 따라 검출 (발현)의 유무로， 혹은 검출 (발현) 정도의 등급을 나누어 결정할 수 있다. 예를 들어, 다수의 정상인과 환자의 시료에서 MRS의 발현 수준을 측정하여 데이터를 축적하고 분석함으로써 MRS 검출 (발현) 수준의 정도에 따라 정상 범주， 췌장암 발병 범주 등으로 구분하여 적절한 진단의 기준을 제공할 수 있다. 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). This is well illustrated in the specification of the present invention. 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) .
또한 상기 (b) 단계는 음성 대조군 (특히， 정상 대조군) 시료와 비교적으로 수행될 수도 있다. 따라서 상기 방법은 (b) 단계에서 또는 (b) 단계 이후에 잠재 환자로부터 채취한 췌장 시료로부터 검출된 MRS 단백질 수준을 음성 대조군 시료와 비교하는 단계를 추가적으로 포함할 수 있다. 본 발명에서 용어 정상 대조군은 검사 대상인 잠재 환자 (즉， 상기 (a) 단계에서 검사 대상이 된 환자와 동일 개체)의 췌장에서 정상인 부위로부터 채취된 췌장 시료 또는 다른 정상 개체 (췌장암이 없는 개체)로부터 채취된 췌장 시료를 모두 포함하는 의미이다. 이때 상기 잠재 환자로부터 채취한 췌장 시료에서 검출된 MRS 단백질 수준이 음성 대조군 (특히， 정상 대조군) 수준보다높으면 췌장암 환자인 것으로 판단할수 있다. 이러한 대조군에 대한 정보 (예를 들어 검출강도 또는 발현강도)는 후술된 본원 발명에서 제공하는 MRS 발현수준 측정 제제 및 이를 포함하는 키트에 명시되는 형태로 제공될 수 있고, 혹은 다른 형태로 부수적으로 제공될 수 있다. 이러한 대조군에 비해 검사 대상 시료에서 MRS 반현수준이 높으면 췌장암 환자인 것으로 판단할수밌다. Also, the step (b) may be performed relatively to the negative control sample (in particular, the normal control sample). Thus, 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). In the present invention, 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. At this time, If 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) may be provided in the form of an MRS expression level measurement preparation provided in the present invention described below and a kit containing the same, . Compared with the control group, it is interesting to judge that the level of MRS expression level in the test sample is a pancreatic cancer patient.
하나의 실시양태 (embodiment )에서 본 발명은 In one embodiment,
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계의 측정 시료에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현되면 췌장암 세포인 것으로 판단하는 단계를 포함하는， 췌장암 진단 방법을 제공한다. (b) determining that the 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.
하나의 실시양태에서， 더욱 바람직하게 본 발명은 In one embodiment, more preferably,
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계에서 측정한 메티오닐 티알엔에이 합성효소 수준을 음성 대조군과 비교하고， 음성 대조군과 비교하여 메티오닐 티알엔에이 합성효소 수준이 발현 증가되었고 선방세포 특이적 마커 단백질이 발현되지 않았으면 췌장암 세포인 것으로 판단하는 단계를 포함하는 췌장암 진단 방법을 제공한다. (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.
본 발명에서 용어 '정상 췌장 세포' 또는 '정상 대조군' 이란 비- 종양 (암)성 췌장 세포를 의미하는 것으로， 종양 (암)이 아닌 다른 질병 (예를 들어， 췌장염 등) 상태에 있는 췌장 세포， Benign (양성) 상태의 세포 및 완전히 건강한 췌장 세포 (무질병 췌장 세포)를 모두 포함하는 의미이다. 또한 본원 발명은 전술한 (a) 및 (b) 단계를 포함하여， 비정형 세포를 암세포 (악성 종양세포)와 정상세포 (비 -암세포)로 구분하는 방법을 제공한다고 할 수 있다. The term " 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.
기존에 췌장암 진단을 위해서 컴퓨터 단층촬영술 (CT) , 내시경역행 담췌간조영술 (endoscopic retrograde cholangiopancreatography: ERCP) , 초음파내시경검사법 (EUS) , 혈관조영술과 같은 고가의 철저한 검사가 요구되고 있으나， 이를 통해서도 췌장암을 정확하게 진단하는 것이 매우 어려운 실정이다. 결국 췌장암의 확진을 위해서는 병리학적인 방법인 조직검사나 세포진 검사를 수행하여야 한다. 한편， 췌장암은 진단 당시 이미 절제가 불가능한 진행암일 경우가 많고， 수술이 가능한 예는 10-15% 밖에 되지 않아 수술을 할 수 없는 환자에서 췌장암의 진단은 내시경 초음파 미세바늘 흡인술 검사를 통해 실시하고 있다. 그런데 이러한 내시경초음파 미세바늘 흡인술 검사를 통한 세포진단의 경우 수술후 췌장암 조직에 비해 주변 구조나 세포와의 비교가 어려워 진단을 확진하는데 한계점이 있다. 기존 세포 병리학적 진단은 췌장암세포와 다른 질환 (예를 들어 췌장염)의 세포를 감별하는데에 주로 H&E 염색 또는 pap 염색 등 일반염색에 의존하고 하고 있다. 그러나 이러한 전통적인 일반 염색에 근거한 기존 세포병리학적 진단 방식에 의해 췌장암을 확진하는 것은 의료진의 경험과 해석기술에 따라 다른 진단이 내려지기도 하며, 또한 이러한 기존 검사들의 경우 췌장암인지 또는 정상세포 (비췌장암 세포를 포괄적으로 의미하는 것으로 예를 들어 췌장염 세포 등을 포함)인지 여부가 명확하지 않은 비정형 세포 (atypical cel l )로서 병리소견을 내는 경우가 많으며， 이러한 경우 추가적이고 다수의 반복적인 재검을 필요로 한다. 특히 H&E염색 또는 pap 염색을 통한 췌장세포 관찰에서 비정형 (atypical ) 세포로 판정된 경우 췌장암인지 다른 양성질환인지 여부에 대한 구별이 매우 어려워 환자의 질환에 대한 정확한 진단 및 치료가 빠르게 이루어지지 못하고 있다. 세포진에 대한 정확한 진단이 늦어짐에 따라 수술을 받을 수 있는 췌장암 환자에게 적절한 치료를 할 수 없으며, 반대로 불필요한 수술을 방지하기도 어려운 문제점이 있다. 따라서 임상적으로 췌장암의 치료 효과를 증대시키기 위해서는 세포진에 대한 정확한 진단법이 필요한 실정이다. 이처럼 암을 진단하기 위해 일반적으로 사용되고 있는 H&E 염색, pap 염색을 통해서는 종양인지 또는 비종양인지 구분이 매우 어려운 비정형 (atypical ) 세포에서 종양여부의 판정이 임상적으로 매우 중요한데, 이러한 비정형 세포에서 MRS의 (고)발현이 확인되면 (선방세포 제외) 종양세포로 판정할 수 있다는 점에서 매우 의미가 있다고 할 수 있다. 또한 상대적으로 많은 양의 생검을 필요로 하는 조직검사의 경우 세포진 검사보다 시료 취득에 있어서 환자에 신체적 부담이 가중되며 어떠한 경우에는 암의 진행 상태에 따라 수술이 불가 및 조직의 채취가 불가한 문제점이 있는 것을 고려하였을 때， 세포 수준에서도 정확한 진단을 제공하는 본원 발명의 진단 방법은 더욱 큰 장점을 지닌다고 할 수 있다. 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 . However, 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). However, 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. In addition, in the case of these conventional tests, pancreatic cancer or normal cells (non-pancreatic cancer cells (Eg, pancreatic cancer cells, etc.), but it is often an atypical cell, and it is often necessary to perform additional and frequent repeated examinations . Especially, when 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. As 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. In the case of biopsy requiring a relatively large amount of biopsy, the physical burden on the patient is increased in the acquisition of the sample rather than in the cytological examination. In some cases, 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.
췌장암 진단에 있어서， MRS 단백질 및 선방세포 특이적 마커 단백질을 동시에 마쩌로 사용하여 이들의 검출 패턴을 분석하는 본원 발명의 방식을 채용하는 경우, 조직 및 세포 수준의 검사에 있어서 진단의 민감도， 특이도， 양성 예측률 및 /또는 음성예측률이 거의 100%수준을 나타내는 것이 특징이다. 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%.
이에， 본원 발명은 췌장암에 대한 세포진 (cytodiagnosis) 검사 또는 조직 검사에 있어서， 하기 단계를 포함하는 것을 특징으로 하는 민감도 또는 특이도를 향상시키는 방법을 제공한다: Accordingly, the present invention provides a method for improving sensitivity or specificity in cytodiagnosis or histology of pancreatic cancer comprising the steps of:
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS단백질의 발현수준을 측정하는 단계 ; 및 (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) 상기 (a) 단계에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 증가되었으면 췌장암 세포인 것으로 판단하는 단계. (b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the cell is a pancreatic cancer cell.
본 발명에서 용어 '민감도' 란 최종 임상병리학적 진단이 췌장암인 시료 또는 환자에 대하여, 대상 검사법 (ex. 본원 발명의 검사법)을 통해 췌장암 판정이 내려진 비율을 의미한다. In the present invention, 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).
본 발명에서 용어 '특이도' 란 최종 임상병리학적 진단이 정상인 시료 또는 환자에 대하여， 대상 검사법 (ex . 본원 발명의 검사법)을 통해 정상 판정이 내려진 비율을 의미한다. 구체적으로 상기 민감도， 특이도， 양성 예측률 및 음성예측률로 이루어지는 군에서 선택되는 하나 이상이 80% 이상의 수준 (80% 내지 100¾>， 바람직하게는 85% 내지 99%， 더욱 바람직하게는 90 내지 98% 수준)을 나타내는 것이 특징이다. 상기 수준의 구체적 수치는 80 ), 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 및 100%로 이루어지는 군에서 선택되는 두 개의 숫자를 경계값으로 하는 범위값올 모두 포함한다. 본원 발명의 하나의 실시양태 (embodiment )에서， 상기 수치범위 중 구체적으로 80% 및 95%의 경계값이 선택될 수 있고， 이에 따라 80% 내지 95¾» 범위에 있는 모든 값들이 본 발명에서 의도됨은 당업자에 자명하다. 또 다른 하나의 실시양태 (embodiment )에서 바람직하게 상기 민감도， 특이도， 양성 예측률 및 /또는 음성예측률이 85% 이상의 수준 (85% 내지 100%， 바람직하게는 87% 내지 99%, 더욱 바람직하게는 90 내지 98% 수준)을 나타낼 수 있으나, 이에 제한되는 것은 아니다. In 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. Specifically, 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%. 98%, 99%, 100%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% And a range value including a boundary value of two numbers selected from the group consisting of " In an embodiment of the present invention, 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. In another embodiment, 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.
상기 민감도， 특이도, 양성 예측률 및 /또는 음성 예측률의 향상이 정확도의 향상으로 이어지는 것임은 당업자에게 자명하다. 따라서 본 발명의 방법은 정확도를 향상시키는 방법으로 이해될 수 있으며， 바람직하게 정확도가 90% 내지 100%, 더욱 바람직하게 정확도가 90% 내지 99% 수준을 나타내는 것일 수 있다. 상기 수준의 구체적 수치는 90%， 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5% , 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% 및 100%로 이루어지는 군에서 선택되는 두 개의 숫자를 경계값으로 하는 범위값을 모두 포함한다. 본원 발명의 하나의 실시양태 (embodiment )에서, 상기 수치범위 중 구체적으로 92.5% 및 99.5%의 경계값이 선택될 수 있고， 이에 따라 93.5% 내지 99.5% 범위에 있는 모든 값들이 본 발명에서 의도됨은 당업자에 자명하다. 또 다른 하나의 실시양태 (embodiment )에서 더욱 바람직하게 93¾> 내지 98%， 가장 바람직하게 95% 내지 98%수준을 나타내는 것일 수 있으나, 이에 제한되는 것은 아니다. It will be apparent to those skilled in the art that the improvement in sensitivity, specificity, positive predictive value, and / or negative predictive value leads to improvement in accuracy. Therefore, 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%. In an embodiment of the present invention, 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.
또한 본원 발명은， 췌장암에 대한 세포진 (cytodiagnosis) 검사 또는 조직검사에 있어서 , 형태학적 검사와 병용하여 The present invention also relates to a method of screening for cytodiagnosis or histologic examination of pancreatic cancer,
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS단백질의 발현수준을 측정하는 단계; 및  (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) 상기 (a) 단계에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 증가되었으면 췌장암 세포인 것으로 판단하는 단계를 포함하는 것을 특징으로 하는 췌장암세포 판별법을 추가로 수행하는 것을 특징으로 하는, 췌장암 진단에 필요한 정보를 제공하는 방법 (췌장암 진단 방법 )을 제공한다. (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 A method for diagnosing pancreatic cancer, a method for diagnosing pancreatic cancer, and a method for diagnosing pancreatic cancer.
상기 형태학적 검사란， 바람직한 일례로 전술한 ( i ) 및 ( i i ) 단계 과정을 포함하여 수행되는 검사를 포함하여， 이러한 방식에 준하는 다른 형태학적 검사 방식들도 모두 포함하는 의미이다. 이에 대한 설명은 전술한 바를 참조로 하여 당업자라면 그 방식을 적의 선택하여 사용 가능하다. . 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. .
상기 (a) 및 (b) 단계에 대한 구체적 설명은 전술한 바와 같으며， 이러한 단계가 보조적으로 (즉, 보조요법으로서) 수행되는 경우, 상기 형태학적 검사와 동시에 (simul taneous) , 별도로 (separate) 또는 순차적 (sequent ial )으로 수행될 수 있다. 또한， 상기 (a) 및 (b) 단계를 포함하는 판별법은 형태학적 검사 이전에， 동시에 또는 이후에 수행 가능하다. 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). In addition, the determination method including steps (a) and (b) above can be performed before, simultaneously, or after the morphological inspection.
【발명의 효과】 【Effects of the Invention】
MRS는 기존의 CEA와 같은 췌장암 마커보다 진단 정확도가 높으며， 특히 MRS의 발현과 더불어 키모트립신 (chymotrypsin)과 같은 선방세포 특이적 마커단백질을 추가의 이중마커 (dual marker)로 사용하면 췌장암 진단의 정확도가 현저히 상승된다. 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 >
【도면의 간단한 설명】 도 1은 si -MRS를 처리한 H460 세포의 세포 용출액을 사용하여， ant i-MRS 항체 (1E8 , 8A12)의 MRS 결합 강도 및 특이도를 공지의 BRIEF DESCRIPTION OF THE DRAWINGS 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,
항체 (Ab50793)와 비교한 웨스턴 블랏 결과이다. Western blot compared to antibody (Ab50793).
도 2는 PANC-1 세포주 (췌장암 세포주) 및 SCK 세포주 (비췌장암 세포주)의 세포 용출액을 사용하여， 본 발명 ant i-MRS 항체 ( 1E8 , 8A12)의 MRS 결합 강도 및 특이도를 공지의 시판 MRS항체 (Abl37105)와 비교한 웨스턴 블랏 결과이다. 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).
도 3은 1E8 항체의 다른 ARS(aminoacyl-tRNA synthetase) , AIMP 단백질에 대한 교차 활성을 확인하기 위하여 ELISA를 실시한 결과를 그래프로 나타낸 것이다. 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.
도 4는 8A12 항체의 다른 ARS(aminoacy卜 tRNA synthetase) , AIMP 단백질에 대한 교차 활성을 확인하기 위하여 ELISA를 실시한 결과를 그래프로 나타낸 것이다. 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.
도 5는 1E8 항체의 MRS+AIMP3 단백질에 대한 항체 친화력을 확인하기 위하여 수행한 SPR(Surface plasmon resonance) 실험 결과를 나타낸 것이다. FIG. 5 shows SPR (surface plasmon resonance) test results for confirming antibody affinity of 1E8 antibody against MRS + AIMP3 protein.
도 6은 1E8 항체가 AIMP3에 대해서는 반웅성이 없음을 확인한 SPR(Surface lasmon resonance)실험 결과를 나타낸 것이다. Fig. 6 shows the result of SPR (Surface lasmon resonance) test in which 1E8 antibody was confirmed to have no anti-maleicity against AIMP3.
도 7은 8A12 항체의 MRS+AIMP3 단백질에 대한 항체 친화력을 확인하기 위하여 수행한 SPR(Surface plasmon resonance) 실험 결과를 나타낸 것이다. FIG. 7 shows SPR (surface plasmon resonance) test results performed to confirm antibody affinity of 8A12 antibody against MRS + AIMP3 protein.
도 8은 8A12 항체가 AIMP3에 대해서는 반응성이 없음을 확인한 SPR(Surface lasmon resonance)실험 결과를 나타낸 것이다. Fig. 8 shows the result of SPR (surface lasmon resonance) test in which 8A12 antibody was confirmed to be non-reactive for AIMP3.
도 9는 PANC-1 세포주 (췌장암 세포주) 및 SCK 세포주 (비췌장암 세포주)에 대하여, 본 발명 ant i-MRS 항체 ( 1E8, 8A12)와 시판 MRS 항체 (Abl37105)를 이용하여 면역형광염색을 수행한 결과를 비교하여 나타낸다. 9 shows immunofluorescence staining of PANC-1 cell line (pancreatic cancer cell line) and SCK cell line (non-pancreatic cancer cell line) using the inventive ant i-MRS antibody (1E8, 8A12) and commercial MRS antibody (Abl37105) The results are compared and shown.
도 10은 임상현장에서 환자세포 시료 processing에 사용하는 Thinprep 장비 (Hologi c . Inc)를 활용하여， PANC-1 세포주로 임상조건과 유사하게 Thinprep 슬라이드를 제작하고 본 발명의 1E8 항체 및 8A12 항체의 면역형광염색을 수행한 결과를 나타낸다. 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.
도 11은 정상 환자로부터 분리한 췌장세포의 H&E 염색결과， MRS의 발현여부 관찰 결과 및 CEA의 발현여부 관찰 결과를 나타낸다 (각각의 슷자는 환자코드번호) . 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).
도 12는 췌장암 환자로부터 분리한 췌장세포에의 H&E 염색결과， MRS의 발현여부 관찰 결과 및 CEA의 발현여부 관찰 결과를 나타낸다 (각각의 슷자는 환자 코드번호) . FIG. 12 shows the results of H & E staining, MRS expression, and CEA expression in pancreatic cancer cells isolated from patients with pancreatic cancer.
도 13은 H&E 염색결과 비정형 세포로 판단되었고 추후 췌장암으로 확진된 환자의 췌장 세포진 시료에 대하여 MRS의 발현여부 관찰 결과 및 CEA의 발현여부 관찰 결과를 나타낸다 (각각의 슷자는 환자 코드번호) . 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.
도 14는 정상 췌장 조직 중 정상 선방세포 (acinar cel l )에서 MRS 발현 강도를 관찰한 결과를 나타낸다. FIG. 14 shows the results of observing MRS expression intensity in normal donor cells (acinar cells) in normal pancreatic tissues.
도 15는 정상 췌장 조직 중 선방세포 (acinar cel l )대한 H&E FIG. 15 shows the results of H & E for acinar cells in normal pancreatic tissues
보여준다. Show.
도 16은 정상 췌장 조직 중 선방세포 (acinar cel l )대한 MRS 및 키모트립신 단백질에 대한 이중 검출을 수행한 결과를 나타낸다. FIG. 16 shows the result of double detection of MRS and chymotrypsin protein for acinar cells in normal pancreatic tissues.
도 17은 췌장 선방세포 세포진 시료 대하여 본 발명의 이중염색법을 적용하였을 때 선방세포가 나타내는 대표적 염색 패턴 두 가지 증， MRS가 매우 미약하게 검출되고 상대적으로 키모트립신이 매우 강하게 검출되어 나타나는 패턴 양상을 보여준다. 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 .
도 18은 췌장 선방세포 세포진 시료 대하여 본 발명의 이중염색법을 적용하였을 때 선방세포가 나타내는 대표적 염색 패턴 두 가지 중, 키모트립신과 함께 MRS 검출도 상당히 나타나지만 merge 결과 상대적으로 키모트립신 검출이 강하게 나타나는 패턴 양상을 보여준다. 도 19는 기존 세포 병리검사상 (pap staining)으로 췌장암 확진되고， 최종적으로도 췌장암 확진된 환자의 췌장 세포 시료에 있어서， 본원 발명의 이중 염색법을 수행한 결과를 나타낸다. 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.
도 20은 기존 세포 병리검사 상 (pap staining)으로는 비정형세포 (atypical cel ls)로 분류되어 확진이 불가하였으나， 최종적으로 췌장암 확진된 환자의 췌장 세포 시료에 있어서， 본원 발명의 이중 염색법을 수행한 결과를 나타낸다. 20 shows that papain staining was classified as atypical cells and thus it was impossible to confirm the results. However, in a pancreatic cell sample of a patient diagnosed with pancreatic cancer finally, the double staining method of the present invention Results are shown.
【발명의 실시를 위한 형태】 이하본 발명을 상세히 설명한다. 단, 하기 실시예는 본 발명을 예 BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. However, the following examples illustrate the present invention in more detail.
실시예에 한정되는 것은 아니다. It is not limited to the embodiment.
실시예 1: 본원 발명의 췌장암 검사법에 대한 유용 항체 제작 (MRS에 대한 특이성이 높은 항체의 수득) 생체 내에서 MRS(methyonyl-tRNA synthetase)는 AIMP3(Aminoacyl tRNA synthetase com lex- interact ing mul t i funct ional protein 3)와 결합된 상태로 존재하며， UV 조사 등에 의해서 이러한 결합상태가 분리되는 것으로 알려졌다. 따라서 실질적으로 MRS의 정확한 검출을 위해서는 MRS가 AIMP3와 결합하고 있는 상태 등의 상황에서도 MRS만을 특이적으로 검출할 필요가 있으나， 현재 AIMP 종류 및 ARS 종류들에는 단백질 구조상 유사한 점이 많아서 시중 항체의 경우 다른 AIMP 및 ARS 종류들과 교차반웅성이 나타나는 문제점이 있다. 이에， 본 발명의 췌장암 검사법의 진단 정확도를 위하여, 본 발명자는 다른 단백질에는 교차반웅성이 없는 고감도의 MRS 항체를 다음과 같은 방법으로 제조하였다. 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.
1-1. MRS-AIMP3 단백질 제조 대장균 (E. col i ) 상에서 MRS-AIMP3 co-pur i f ied 단백질을 정제하였으며 구체적인 실험방법은 다음과 같다. BL21DE3 strain을 이용하여 MRS (서열번호 1)와 AIMP3서열번호 40， NCBI ref . 匪_004280.4)가 발현되도록 형질전환하고， LB 배지에서 배양한 뒤 단일 콜로니를 암피실린 (ampici l in)을 포함하는 5ml LB 액체 배지에서 0D 600값이 0.6 내지 0.8이 되도록 배양하였다. 이후 ImM의 IPTG를 넣어준 다음 37°C에서 3시간 동안 배양하였고， 그 다음 10분 동안 원심분리하여 세포만올 획득하였다. 세포액으로 SDS-PAGE를 실시하여 쿠마시 용액 (coomassie stain)을 이용하여 상기 단백질들의 발현을 확인하였다. 이 후， IPTG로 과발현을 유도하였던 세포액을 모아 원심 분리를 실시하여 세포를 획득하였다. imi DPBS로 세포를 풀어 준 후 초음파분쇄기를 이용하여 세포 용해하였고， 그 다음 용해된 세포로 원심분리를 실시하여 MRS-AIMP3 co-puri f ied 단백질을 분리하였다.. 1-1. Preparation of 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. Subsequently, 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.
1-2. MRS-AIMP3 단백질의 주입을 통한 마우스 면역화 하이브리도마 세포의 제조에 필요한 면역화된 마우스를 얻기 위하여， 상기 실시예 1-1에서 획득한 MRS-AIMP3 co-puri f ied 단백질을 8~10주령 마우스 4마리의 복강 내에 1차주사하였다. 몸무게 25 내지 30g의 10주령 BALB/c 마우스들을 Orient Bio Co. (Sungnam, KyungKiDo, Republ ic of Korea)로부터 구입하였고， 동물들은 일정한 조건 (온도: 20±2°C , 습도: 40~60%, 명암: 12시간 l ight/dark cycle)하에서 층분하게 적웅시킨 후에 본 연구에 이용하였다. 동물 실험은 서울대 학교의 대학 동물 관리 및 사용 위원회 지침을 준수하였다. 1차 면역화 후 마우스의 면역성을 높이기 위하여 2주 후에 동일한 용량의 MRS-AIMP3 co-puri f ied 단백질을 마우스의 복강 내에 2차 주사하였다. 그 후 1주일 후에， 세포융합 실험을 실시하기 3일 전 MRS-AIMP3 co-puri f ied 단백질을 마우스의 꼬리 정맥에 부스터 (booster) 주사하였다. 상기 면역화된 마우스를 에테르로 마취시킨 후 헤파린 처리된 주사기로 심장에서 채혈한 후， 혈액을 4°C에서 하룻밤 정치시키고 원심분리하여 혈청을 분리하였다. 분리된 혈청을 적당히 나누어 -80°C에 보관하였다. 1-2. Mouse immunization via injection of the MRS-AIMP3 protein To obtain the immunized mice necessary for the preparation of the hybridoma cells, the MRS-AIMP3 co-puri fi ed protein obtained in Example 1-1 was inoculated into mouse 8 The 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. In order to increase the immunity of the mice after the primary immunization, MRS-AIMP3 co-puri fi ed proteins of the same dose were injected into the abdominal cavity two weeks later. One week thereafter, 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.
1-3. 하이브리도마 세포 (hybridoma cel l ) 제조 먼저， 세포융합을 위해 골수종 세포 (myeloma cel l )를 준비하였다. 골수종 세포를 배양하고， 세포밀도를 2.5~5>< 10 ^11/111£로 하였다. 세포융합 24시간 전에 골수종 세포를 1/3로 회석하여 준비하였다. 상기 실시예 1-2에서 면역화된 마우스를 에테르로 마취시키고 비장을 채취하여 B 세포를 분리한 뒤， SF-DMEM2(DMEM + 2XAA)로 세척하고 세포를 용출시켰다. 세포 현탁액을 수거하여 튜브에 담고 정치시켜 무거운 덩어리들을 가라앉히고 상층액을 새 튜브로 옮긴 다음 1500rpm으로 5분 동안 원심분리하였다. 원심분리된 비장세포의 상층액을 제거하고 탭핑한 (tapping) 후 SF-DMEM2를 채웠다. B 세포와 골수종 세포를 각각 원심분리하고 세척한 다음, 세척 과정을 1회 더 반복하였다. 세척한 골수종 세포의 상층액올 제거하고 탭핑한 후 SF-DMEM2를 채웠다. 또한， 세척한 B 세포의 상충액을 제거하고 랩핑한 후, LBClysis buffer) Ιπ 에 적혈구 (RBC, red blood cell)를 넣어 처리한 후 SF-DMEM2를 채웠다. 그 다음， B 세포와 골수종 세포를 각각 원심분리하고， 원심분리된 B 세포와 골수종 세포의 상층액을 제거한 다음 탭핑하고 SF-DMEM2 10^를 채웠다. B 세포와 골수종 세포를 각각 e-튜브에서 100배로 회석하고 계수하여 농도를 결정하였다 [B 세포의 농도 (1X108, 8X107, 5X107), 골수종 세포의 농도 (1X107, 8X106, 5 X106)]. B 세포와 골수종 세포는 10:1의 비율로 결정하였다. 결정된 농도의 B 세포와 골수종 세포를 튜브에 함께 넣고 원심분리하였다. 원심분리된 세포의 상층액을 제거한 후 알콜솜 위에 엎어 놓고 30초〜 1분 동안 반건조시키고 탭핑하였다. 여기에 PEG(2 )를 1분 동안 천천히 넣으면서 피펫팅하여 반응시키고 SF-DMEM2를 넣으면서 튜브를 흔들어준 다음 원심분리하였다. 원심분리 후 상층액을 제거하고 탭핑하지 않은 상태에서 HT 배지 [HT50X(HT( sigma) 1 vial + SF-DMEM1 lOm^) l i, FBS lOm^, SF-DMEMKDMEM + lxAA) 30 ]를 방울방울 떨어뜨리고, 조금씩 속도를 올리면서 50 가 되도록 하였다. 이 현탁액을 다시 37 °C, 5% C02 배양기에서 3시간동안 배양하였다. 1-3. Preparation of Hybridoma Cells First, myeloma cells were prepared for cell fusion. Myeloma cells were cultured and the cell density was 2.5 to 5> 10 < 11 &gt; / 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. 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. After washing of the washed B cells, the cells were wrapped, and then red blood cells (RBCs) were added to the LBClysis buffer Ιπ, followed by filling with SF-DMEM2. Then, 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. After removing the supernatant from the centrifuged cells, 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.
1-4. MRS특이적 단일클론항체를 생산하는 하이브리도마 세포의 선별 상기 실시예 1-3에서 제조한 융합세포군 중에서 MRS를 잘 인식하면서， AIMP3를 인식하지 않는 세포를 선별하고， 항체의 생성여부를 확인하기 위하여， 다음과 같이 실험을 실시하였다. 먼저, 세포융합 후 8~9일째에 배지를 교환하고, 96 웰에서 배양시 및 24 웰에서 배양시， 잘 자랄 때까지 cDMEM2에서 배양하였다. 배지를 교환한 후 5~7일째에 색이 변한 웰의 상층액을 거두고 CDMEM2로 채운 후， 각 융합세포에서 생산된 항체와 MRS 및 AIMP3와의 결합성에 대한 ELISA 시험을 수행하였다. ELISA 시험 후 웰을 선택하여 24 웰로 옮겨 배양하였다. 24 웰에서 배양한 후 다시 ELISA 시험을 수행하였다. 구체적으로는， 24 웰의 융합세포 농도를 확인하고, 96 웰 플레이트에 0.5 cel l/wel l이 되도록 15 의 배양액에 융합세포를 회석하였다. 융합세포 희석액을 각 웰당 150 씩 분주하였다. 현미경으로 검경하여 1개의 세포가 들어있는 웰올 체크하였다. 세포가 어느 정도 자란 웰의 상층액을 거두어 ELISA 및 웨스턴 블랏으로 각 융합세포에서 생산된 항체와 MRS 및 AIMP3와의 결합성을 확인하여 1차 스크리닝을 수행하였다. 1차 스크리닝을 토대로 선택된 융합세포를 24 웰로 옮겨 배양하고 원심분리한 후 상층액을 거두어 ELISA 및 웨스턴 블랏으로 확인하여 2차 스크리닝을 수행하였다. 24 웰에서 키운 융합세포의 흡광도 (0.D 값)를 ELISA로 확인하고， 흡광도가 1.0이 넘는 융합세포만 선택하여 25T/C 배양 플라스크로 옮겨 배양하고 원심분리한 후 상층액을 거두어 ELISA 및 웨스턴 블랏으로 확인하여 3차 스크리닝을 수행하였다. 3차 스크리닝을 토대로 선택된 융합세포를 다시 75T/C 배양 플라스크로 옮겨 배양하고 ELISA로 흡광도를 확인하여 MRS를 잘 인식하면서 AIMP3를 인식하지 않는 세포를 선택하였고， 최종적으로 "1E8" 및 "8A12" 클론을 확보하였다. 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. 24 wells and then subjected to ELISA again. Specifically, the fusion cell concentration of 24 wells was checked, Fusion cells were seeded into 15 culture media to give a plate of 0.5 cel / well. Fusion cell dilutions were dispensed at 150 wells per well. The cells were examined with a microscope and checked with wells containing one cell. The supernatant of the wells to which the cells were grown to some extent was subjected to the first screening by confirming the binding between MRS and AIMP3 with the antibody produced in each fusion cell by ELISA and Western blotting. 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.
1-5. MRS 특이적 단일클론항체를 생산하는 하이브리도마 세포의 배양 및 항체 정제 1-5. Culturing of hybridoma cells producing MRS-specific monoclonal antibodies and antibody purification
MRS에 대한 단일클론항체는 상기 실시예 1-4에서 선택된 최종 융합세포 (하이브리.도마 세포 "1E8" 또는 "8A12" )로부터， 각각 다음의 두 가지 방법을 통해 수득될 수 있다. 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.
1) 7-8 주령의 암컷 마우스 복강 (abdominal cavi ty)에 프리스탄 (pri stane) 500 /^를 주사하였다. 75T/C 배양 플라스크에서 배양한 융합세포를 수거하여 원심분리한 다음, 상층액을 제거하고 인산염 완층액에 넣고 피펫팅하였다. 프리스탄 투여 7~10일 후 상기 실시예 1-4에서 선택한 융합세포를 각각 8 X 105~4X 107으로 마우스의 복강 내에 주사하였다. 1~2주 후에 마우스의 복강에 복수 (asci tes)가 가득찼을 때 18G 주사 바늘을 이용하여 복수를 뽑았다. 복수를 4°C에서 하룻밤 두었다가 다음날 원심분리하여 노란 지방층을 포함한 덩어리 물질을 제거하고 상층액만을 분리하였다. 분리한 상층액은 분주하여 -2CTC에 보관하였다. 상기 복수액으로부터 항체 정제를 위하예 저장액 (20% 에탄올)에 저장되어 있는 Protein A를 적당량 컬럼에 채우고 20% 에탄올을 홀려 내린 후， 5 Bed Volume의 결합 완층액 (20mM sodium phosphate , H 7.0)으로 세척하였다. 복수액을 인산염 완층액으로 적당량 회석시킨 후 Protein A 컬럼에 로딩하였다. 3 Bed Volume의 결합 완충액 (20mM sodium phosphate , pH 7.0)으로 결합한 후, 3 Bed Volume의 용출 완층액 (0.1M glycine buffer , H 3.0 2.5)으로 0.5mC씩 분획을 용출하였다. 각 분획을 의 중화 완층액 ( 1M Tris-HCl , pH 9.0)으로 중화시켰다. SDS-PAGE를 통해， 분획의 순도를 확인하였고, Ammersharm GE 컬럼으로 탈염 (desalt ing)하였다. 1) The female abdominal cavi ty of 7-8 weeks old was injected with 500 / ^ priestane. The fused cells cultured in a 75T / C culture flask were collected, centrifuged, and then the supernatant was removed, and the supernatant was added to the phosphate buffer solution and pipetted. 7-10 days after the administration of Pristane, the fusion cells selected in Examples 1-4 were injected into the abdominal cavity of mice at 8 × 10 5 to 4 × 10 7 , respectively. One or two weeks later, when the abdominal cavity of the mouse was filled with asci tes, the avalanche was removed using an 18G injection needle. 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. A 5-volume volume of 20 mM sodium phosphate (H 7.0) Lt; / RTI &gt; 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.
2) 상기 실시예 1-4에서 수득한 하이브리도마세포를 GlutaMAX(Gibco) (최종 5 mM)와 lx Cholesterol l ipid concentrate(Gibco)가 첨가된 무혈청 배지 (Thermo)에서 순응시켰다 (8A12 항체 생산 하이브리도마세포는 34-8F2로도 명명) . 이후 Cel lstack- 5 (Corning, Corning, NY)를 사용하여 최대 배양 부피 860 mL에서 배양을 수행하였다. 무혈청 배지 (Thermo)에 GlutaMAX(Gibco) (최종 5 mM)와 lx Cholesterol l ipid concentrate (Gibco)를 첨가하였으며， 초기 세포 농도는 1.4-2.0 X 105 cel l/mL로 접종하였다. 접종 4~5일 후， 2000 rpm에서 10분간 원심분리하여 세포를 제거하고 배양액 상층액을 회수하였다. 상층액의 pH를 확인 한 후 제조된 20X 결합 용액 (1M Potassium phosphate dibasic) (pH 9.0)을 사용하여 pH 7.6을 맞추었다. 이후 0.22um 필터를 사용하여 여과하여 중화된 항체배양액을 수득하였다. 수득한 항체배양액을 protein A 컬럼을 통해 정제를 하였다. protein A 컬럼에 10 컬럼 부피의 증류수를 흘려준 뒤 동량의 IX 결합용액 (50mM Potassium phosphate dibasic) (pH 9.0)을 홀려주었다. 이후， 수득한 항체배양액을 흘려주어 항체를 protein A에 결합시킨 뒤 IX 결합용액 (50mM Potassium phosphate dibasic) (pH 9.0)으로 washing하였다. 그 다음， protein A 에 결합된 항체를 용출하기 위해 2 컬럼 부피의 용출 용액 (0.2M Ci tric acid)(pH 3.0)을 홀려주어 용출액을 얻었다. 1M Tris 로 중화한 뒤 항체의 농도를 280nm 흡광도에서 측정하여 확인하였다. 그 후， GE PD-10 컬럼을 생리식염수 25 ml로 평형시킨 후， 원심분리 (1000g, 2분) 하였다. 이후 protein A 컬럼에서 얻어진 항체 용출액 2.5 ml를， 상기 GE PD- 10 컬럼에 넣고 원심분리 (lOOOg, 2분) 하여， 생리 식염수로 용액 교환이 된 항체를 수거하였다. 항체 농도를 280nm 흡광도에서 측정하여 확인하고 분주하여 -80°C에 보관하였다. 2) 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. Ten column volumes of distilled water were flowed through the protein A column and equilibrated with an equal volume of IX binding solution (50 mM Potassium phosphate dibasic) (pH 9.0). Then, the obtained antibody culture solution was poured and the antibody was bound to protein A, followed by washing with IX binding solution (50 mM Potassium phosphate dibasic) (pH 9.0). Then, two column volumes of elution solution (0.2 M Ci tric acid) (pH 3.0) were eluted to elute antibody bound to protein A to obtain an eluate. After neutralization with 1 M Tris, antibody concentration was determined by measuring absorbance at 280 nm. Thereafter, 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.
1-6. 항체의 서열정보 분석 및 클로닝 각각 1E8, 8A12 클론 발현 항체의 클로닝 및 서열 분석은 YBI0 inc . 및 앱클론 (Abclon Inc . Korea)에 의뢰하여 진행하였다. 간략하게, 먼저 1E8 또는 8A12 하이브리도마 세포에서 RNA를 추출하여 cDNA를 합성 하였다. 그 다음， 각각 VL, CL, VH 및 CHI에 특이적인 프라이머들을 사용하여 PCR을 수행하였다. 예상되는 크기의 PCR product를 agarose gel에서 정제하여 sequencing을 통해 서열을 확인하였고 Kabat numbering을 통해 CDR region올 확인하였다. 1E8 항체의 항원 결합부위 서열확인 결과를 표 1에 나타내었으며， 8A12 항체의 항원 결합부위 서열확인 결과를 표 2에 나타낸다. 확인된 서열로 Fab을 합성하여 MRS에 높은 결합력을 보임을 ELISA로 확인하였다. 또한 상기에서 확인된 서열은， 상기 실시예 1-5에서 하이브리도마 세포를 마우스 복강에 주입한 뒤 복수 정제를 통해 얻어진 항체의 단백질 서열 분석 결과 (mass spectrometry 결과)와도 일치됨을 확인하였다. 1-6. Sequence information analysis and cloning of antibodies The cloning and sequence analysis of the 1E8, 8A12 clone expressing antibodies, respectively, was performed using YBI0 inc. And Abclon Inc. (Korea). Briefly, cDNA was synthesized by first extracting RNA from 1E8 or 8A12 hybridoma cells. Then, VL, CL, PCR was performed using VH and CHI specific primers. The expected size of the PCR product was purified by agarose gel and sequencing was performed to confirm the sequence and confirmed the CDR region by Kabat numbering. 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.
수득된 1E8 Fab 또는 8A12 Fab 서열을 각각 mouse IgG heavy chain(pFUSE- mIgG2a-Fc, InvivoGen) 및 mouse l ight chain 서열 백터 (pFUSE2-CLIg-mK, InvivoGen)에 클로닝하였다. 그 다음 상기 백터를 freestyle 293F 세포에 PEKPolysciences , 23966-2)를 이용하여 공동형질전환시켜, 항체의 경쇄와 중쇄가 세포 안에서 함께 동시에 발현되도록 하였다. 형질전환된 293F 세포를 37°C , 8% C02조건에서 7일 동안 배양하였다. 그 다음， 세포를 수득하여 원심분리한 뒤 상층액을 수득하였다. 상층액의 pH를 확인 한 후， 제조된 20X 결합 용액 (1M Potassium phosphate dibasic) (pH .9.0)을 사용하여 상층액의 pH를 7.6으로 조정하였다. 이후 0.22 μ πι 필터로 상층액을 여과하여 중화된 항체배양액을 수득하였다. 항체배양액으로부터 상기 실시예 1-5의 2) 에서 기술된 방법으로 항체를 수득하 ¾다. 이렇게 수득된 1E8 IgG의 전체 항체는 서열번호 36의 아미노산 서열로 이루어지는 경쇄 및 서열번호 37의 아미노산 서열로 이루어지는 중쇄로 이루어지는 것을 확인하였다. 또한 8A12 IgG의 전체 항체는 서열번호 38의 아미노산 서열로 이루어지는 경쇄 및 서열번호 39의 아미노산 서열로 이루어지는 중쇄로 이루어지는 것을 확인하였다. 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. After confirming the pH of the supernatant, 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.
【표 1】 [Table 1]
Amino ac id sequence DNA sequence Amino ac id sequence DNA sequence
VH DVKLQESGPGLVNPSQSLSLTCTVTGYSIT VH DVKLQESGPGLVNPSQSLSLTCTVTGYSIT
FR1 Gatgtgaagct tcaggagtcgggacctggcctggtgaa FR1 Gatgtgaagct tcaggagtcgggacctggcctggtgaa
(서열번호 41) tcct ctcagtctctgtccctcacctgcactgtcactg gctat tcaatcacc  (SEQ ID NO: 41) tcct ctcagtctctgtccctcacctgcactgtcactg gctat tcaatcacc
(서열번호 42)
Figure imgf000044_0001
(SEQ ID NO: 42)
Figure imgf000044_0001
[Z 표】
Figure imgf000044_0002
[Z table]
Figure imgf000044_0002
99C0T0/8l0ZaM/X3d CZ.Z0S0/6T0Z OAV CDR-H2 YINYNGNTNLNPSLKS Tacataaactacaatggcaacactaacttaaatccat (서열번호 24) ctctcaaaagt 99C0T0 / 8l0ZaM / X3d CZ.Z0S0 / 6T0Z OAV CDR-H2 YINYNGNTNLNPSLKS Tacataaactacaatggcaacactaacttaaatccat (SEQ ID NO: 24) ctctcaaaagt
(서열번호 25)  (SEQ ID NO: 25)
RISI IRDTSKNQFFLQLNSVTTEDTATYYCAR RISI IRDTSKNQFFLQLNSVTTEDTATYYCAR
FR3 Cgaatctctatcattcgagacacatccaagaaccagt FR3 Cgaatctctatcattcgagacacatccaagaaccagt
(서열번호 61) tcttcctgcagttgaattctgtgacaactgaggacac agccacat at t actgtgcaaga  (SEQ ID NO: 61) tcttcctgcagttgaattctgtgacaactgaggacac agccacat at t actgtgcaaga
(서열번호 62)  (SEQ ID NO: 62)
CDR-H3 SLWPRGWFAY Tcact t tggcccaggggctggtttgcttac  CDR-H3 SLWPRGWFAY Tcact t tggcccaggggctggtttgcttac
(서열번호 26) (서열번호 27)  (SEQ ID NO: 26) (SEQ ID NO: 27)
FR4 WGQGTLVTVSA Tggggccaagggactctggtcactgtctctgca  FR4 WGQGTLVTVSA Tggggccaagggactctggtcactgtctctgca
(서열번호 63) (서열번호 64)  (SEQ ID NO: 63) (SEQ ID NO: 64)
VL FR1 DIQMTQSPSSMYASLGERVTITC gacat tCtgatgacccagtctccatcttccatgtatg  VL FR1 DIQMTQSPSSMYASLGERVTITC gacat tCtgatgacccagtctccatcttccatgtatg
(서열번호 65) catctct aggagagagagtcac tatcact tgc  (SEQ ID NO: 65) catctct aggagagagagtcac tatcact tgc
(서열번호 66)  (SEQ ID NO: 66)
CDR-L1 KASQDINSYLS Aaggcgagt caggacat taat age tattt aagc  CDR-L1 KASQDINSYLS Aaggcgagt caggacat taat age tattt aagc
(서열번호 16) (서열번호 17)  (SEQ ID NO: 16) (SEQ ID NO: 17)
FR2 WFQQKPG SP TLMY Tggt t ccagcagaaaccagggaaatctcct agaccc  FR2 WFQQKPG SP TLMY Tggt t ccagcagaaaccagggaaatctcct agaccc
(서열번호 67) tgatgtat  (SEQ ID NO: 67) tgatgtat
(서열번호 68)  (SEQ ID NO: 68)
CDR-L2 RANRLVD Cgtgcaaacagat t ggt agat  CDR-L2 RANRLVD Cgtgcaaacagat t ggt agat
(서열번호 18) (서열번호 19)  (SEQ ID NO: 18) (SEQ ID NO: 19)
FR3 GVPSRFSGSGSGQDYSLT I SSLEYEDMG I YYC Ggggtcccatcaaggt tcagtggcagtggatctggcc  FR3 GVPSRFSGSGSGQDYSLT I SSLEYEDMG I YYC Ggggtcccatcaaggt tcagtggcagtggatctggcc
(서열번호 69) aagat tat tctctcaccatcagcagcctggaatatga agatatgggaat ttattattgt  (SEQ ID NO: 69) aagat tat tctctcaccatcagcagcctggaatatga agatatgggaat ttattattgt
(서열번호 70)  (SEQ ID NO: 70)
CDR-L3 LQYDEFPRT Ctacagt atgatgagt t tcct cggacg  CDR-L3 LQYDEFPRT Ctacagt atgatgagt t tcct cggacg
(서열번호 20) (서열번호 21)  (SEQ ID NO: 20) (SEQ ID NO: 21)
FR4 FGGGTKLEI Ttcggtggaggcaccaagctggaaatcaaa  FR4 FGGGTKLEI Ttcggtggaggcaccaagctggaaatcaaa
(서열번호 71) (서열번호 72)  (SEQ ID NO: 71) (SEQ ID NO: 72)
1-7. MRS에 대한항체의 결합 특이성 비교 확인- 웨스턴 블랏 실험 상기 실시예에서 획득한 1E8 항체 및 8A12 항체의 MRS 결합능력을 확인하기 위하여 다음과 같이 웨스턴블랏 실험을 실시하였다. H460 세포를 10% FBSCFetal bovine serum, Hyclone , GE- 1 i fesciences) , 1% 페니실린 (Hyclone , GE l i fesciences)을 포함하는 DMEM (Hyclone , GE l i fesciences)배지에서 배양하였다. 각 세포는 5% C02 , 37°C의 조건에서 배양하였다. 상기 배양된 H460 세포에 si -MRS를 72시간 동안 처리하였다. 그 다음 H460 세포를 수득한 뒤， 용해시킨 후， H460 세포 용해물로 웨스턴 블랏올 실시하였다. 실험은 2번 반복되었다. 1차 항체로서 1E8 항체 또는 8A12 항체를 1 : 5000(0.2 yg/ml )으로 회석하여 사용하였고， 결합능력 비교를 위하여 시중에서 유통되고 있는 MRS 항체 (Abeam, Ab50793)을 동일한 방법으로 사용하였으며, 대조군으로는 튜블린 (Tubl in)을 사용하였다. 실험 결과 도 1에 나타난 바와 같이， 시중에 유통되고 있는 기존 MRS 항체는 si-MRS를 처리한 군에서 MRS를 전혀 검출하지 못하였고， si-MRS를 비 리한 군에서도 본원 발명의 8A12 항체 및 1E8 항체와 비교하여 현저히 낮은 수준의 검출능 (MRS와의 결합능)을 보였다. 반면 본원 발명의 1E8항체 및 8A12 항체는 기존 시중 MRS 항체보다 현저히 MRS 특이적 결합능 및 감도가 뛰어난 것을 확인하였으며， 특히 8A12 항체는 매우 우수한 결합능 및 감도를 보이는 것을 확인하였다. 1-7. Confirmation of Binding Specificity of Antibodies to MRS-Western Blot Experiment Western blotting experiments were carried out as follows to confirm the MRS binding ability of 1E8 antibody and 8A12 antibody obtained in the above Examples. 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. As a primary antibody, 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. On the other hand, 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.
또한 PANC-1 췌장암 세포주와 SCK (비췌장암 세포주) 세포주를 이용하여， 1E8 항체 및 8A12 항체의 MRS 결합능력을 추가 확인하였다. 이때 대조군으로는 시판되고 있는 MRS 항체 (Abeam, Abl37105)를 사용하였으며 (항체들은 1 : 1000로 회석하여 사용하였다. 0.137/ /πι1 ) , si -MRS를 처리하는 과정은 제외하고 전술한 바와 동일한 방식으로 웨스턴 블랏 실험을 수행하였다. 실험결과 도 2에서 보는 바와 같이, 본원 발명의 1E8 항체 및 8A12 항체는 MRS 특이적으로 검출을 수행하였으나, 동일 조건에서 기존 시판항체 Abl37105 항체는 비특이적 밴드가 많이 나타나는 것으로서 선택적 검출능이 매우 떨어지는 것으로 나타났다. 본 실험 조건하에서는 SCK 세포주 (비췌장암 세포주)에서 MRS는 거의 검출되지 않았다. In addition, 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. As a control, a commercially available MRS antibody (Abeam, Abl 37105) was used (antibodies were used at a ratio of 1: 1000, 0.137 / / πι1) Western blot experiment. As shown in FIG. 2, 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.
1-8. 다른 단백질에 대한 교차반응성 여부 확인 - ELISA 상기 실시예에서 획득한 8A12 항체가 다른 ARS(aminoacyl-tRNA synthetase) 단백질에 대한 교차 활성 (cross act ivity)이 있는지 여부를 확인하기 위하여 다음과 같이 실험을 실시하였다. 1-8. Determination of Cross-Reactivity to Other Proteins-ELISA The following experiment was conducted to confirm whether the 8A12 antibody obtained in the above example had crossactivity to other ARS (aminoacyl-tRNA synthetase) proteins Respectively.
96 웰 플레이트 (Corning 3690 f lat bottom, 96-wel l hal f-area plates)에 MRS 단백질 (His-MRS, MRS ful l )과 다른 ARS 단백질 (DX2 tag free , 34S-DX2, 34S- AIMP2, His-CRS, His-AIMPl, His-GRS, His.-WRS, His— KRS)을 각각 1 yg/ml의 농도로 코팅하였다. 1E8 항체 또는 8A12 항체를 500 ng/ml 농도로 각각의 ARS 단백질이 코팅된 96웰 플레이트에 넣은 뒤 1시간 동안 반응시켰다.. 그 후 HRP-conjugated ant i -mouse IgG 2차 항체를 첨가하여 1시간 동안 반응시켰고， ELISA를 실시하여 450nm에서 . 흡광도를 측정하였다. 기질로는 ΤΜΒ(3,3' ,5, 5' - MRS protein (His-MRS, MRS ful1) and other ARS proteins (DX2 tag free, 34S-DX2, 34S-AIMP2, His) were added to a 96 well plate (Corning 3690f lat bottom, 96- His-AIMPl, His-GRS, His.-WRS, His-KRS) were coated at a concentration of 1 yg / ml. 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, ΤΜΒ (3,3 ', 5,5'
Tetramethylbenzidine)을사용하였다. 그 결과 도 3에 나타난 바와 같이 , 1E8 항체는 MRS에만 결합하여 반응하고, 다른 ARS 단백질과 AIMP 단백질에는 반응하지 않는 것으로 나타났다. 또한 도 4에 나타난 바와 같이, 8A12 항체는 MRS에만 결합하여 반웅하고, 다른 ARS 단백질과 AIMP 단백질에는 반웅하지 않는 것으로 나타났다. 이를 통해， 1E8항체 및 8A12 항체는 다른 ARS 단백질과 AIMP 단백질에 대하여 교차 활성이 없으며， MRS만 특이적으로 검출하는 것을 확인할 수 있었다. Tetramethylbenzidine) was used. As a result, as shown in Fig. 3, the 1E8 antibody reacts only with MRS, But not to other ARS and AIMP proteins. In addition, as shown in Fig. 4, 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.
1-9. 표면 플라즈몬 공명을 이용하여 항체 친화성 확인 1-9. Confirm antibody affinity using surface plasmon resonance
1E8 항체 및 8A12 항체의 MRS 특이적 친화성을 확인하기 위해， MRS-AIMP3 co-puri f ied 단백질 (이하, MRS+AIMP3 단백질) 및 AIMP3 단백질을 이용하여 SPRCSurface plasmon resonance, 표면 플라즈몬 공명) 실험을 실시하였다. MRS+AIMP3 또는 AIMP3 단백질을 CM5 chip에 코팅하고， 1E8항체 또는 8A12 항체를 다양한 농도로 흘려보내면서 단백질과의 결합 반응 정도를 측정하였다. 분석시료나 버퍼는 30 ii l/min의 유속으로 8분 동안주입하였고， 20분 동안 세척하였다. 그 결과, 도 5 및 도 6에 나타난 바와 같이， 1E8 항체는 MRS+AIMP3 단백질에는 결합하나 AIMP3 단백질에는 결합하지 않는 것을 확인할 수 있었다. 또한 1E8항체는 MRS에 대하여 5.42nM KD value를 갖는 것을 확인할수 있었다 또한 도 7 및 도 8에 나타난 바와 같이， 8A12 항체는 MRS+AIMP3 단백질에는 결합하나 AIMP3 단백질에는 결합하지 않는 것을 확인할 수 있었다. 또한 8A12 항체는 MRS에 대하여 1.56nM KD value를 갖는 것을 확인할 수 있었다. To confirm the MRS-specific affinity of 1E8 antibody and 8A12 antibody, SPRCSurface plasmon resonance (SPR) experiments were performed using MRS-AIMP3 co-puried protein (hereinafter, MRS + AIMP3 protein) and AIMP3 protein Respectively. 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. 6, it was confirmed that the 1E8 antibody binds to the MRS + AIMP3 protein but does not bind to the AIMP3 protein. In addition, it was confirmed that the 1E8 antibody had a KD value of 5.42 nM for MRS. As shown in FIGS. 7 and 8, it was confirmed that the 8A12 antibody binds to the MRS + AIMP3 protein but not the AIMP3 protein. It was also confirmed that the 8A12 antibody had a KD value of 1.56 nM for MRS.
1-10. MRS항체의 결합부위 확인 상기 1E8 항체 또는 8A12 항체가 결합하는 부위 (에피토프, main binding si te)를 확인하기 위하여 다음과 같이 실험올 실시하였다. 먼저， MRS 전체 단백질에서 GST, catalyt ic domain, tRNA binding domain 부위들을 고려하여 각각 길이와 위치가 상이한 여러 개의 MRS 단편을 제작하였으며， pcDNA3 vector (EV)에 MRS 전체 단백질 또는 각각의 MRS 단편 (MRS fragment )을 클로닝 하였다. 각 MRS 단편의 위치는 서열번호 1의 MRS 전체 아미노산 서열 중에서, 1 ~ 266aa 단편， 267~597aa 단편 , l~598aa 단편 , 598~900aa 단편 , 660~860aa 단편 , 660-900 단편， 730-900 단편 등을 비롯하여 여러 가지 소단위 영역올 포함하는 위치에서 선정되었다. 이때， Myc 단백질을 각각의 펩타이드 N-말단에 결합시켰고， Myc 단백질을 대조군으로 사용하였다. 그 다음 H460 세포에， 클로닝한 백터 DNA 2 Ug을 제조사의 지침에 따라 Turbofect (Thermo)를 사용하여 트랜스펙션 (transfect ion) 시켰다. 24시간 후 세포를 수득하여 웨스턴 블랏을 실시하였다. 이때 1차 항체로 1E8 항체 및 8A12 항체를 1 : 5000(0.2Ug/mL)으로 회석하여 사용하였다. 상기 실험을 통하여 1E8 항체 및 8A12 항체는 서열번호 1의 MRS 단백질 중에서 최소한 598~900aa 영역쎄 epi tope가존재하는 것으로 나타났다. 1-10. Confirmation of binding site of MRS antibody To confirm the site (epitope, main binding si te) to which the 1E8 antibody or 8A12 antibody binds, the following experiment was conducted. First, several MRS fragments with different lengths and positions were prepared by considering the GST, catalytic ic domain and tRNA binding domain regions of the entire MRS protein. To the pcDNA3 vector (EV), the whole MRS protein or each MRS fragment ) Were cloned. 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. At this time, the Myc protein was bound to the N-terminal of each peptide, Myc protein was used as a control. Then 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. At this time, 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.
이에， 811-840aa 단편， 821_850aa 단편， 831-860aa 단편， 841-870aa 단편， 846-875aa 단편， 851-880aa 단편， 856-885aa 단편， 861-890aa 단편， 866-895aa 단편， 871-900aa 단편을 비롯하여 다양한 소단위 단편 펩타이드를 제작하고， 각각의 펩타이드를 300 ng/wel l씩 96 wel l EL ISA plate에 coat ing하여 통상의 프로토콜에 따라 ELISA를 수행하였다. 1차 항체로서 1E8 항체 또는 8A12 항체를 ΙΟηΜ 농도로 회석 ( lx PBST-Tween 0.05%)하여 사용하였으며， 2차 항체로 HRP conjugated Goat ant i -mouse IgG(Thermo)를 1 : 10000희석 ( lxPBST-TweenO .05%)하여 사용하였고， 450 nm에서 흡광도를 측정하였다. Thus, a fragment of 811-840aa fragment, 821_850aa fragment, 831-860aa fragment, 841-870aa fragment, 846-875aa fragment, 851-880aa fragment, 856-885aa fragment, 861-890aa fragment, 866-895aa fragment, 871-900aa fragment And various peptides were coated on a 96-well EL ISA plate at 300 ng / well in an ELISA according to a conventional protocol. As the primary antibody, 1E8 antibody or 8A12 antibody was used (1x PBST-Tween 0.05%) at a concentration of ΙOηηM, HRP conjugated Goat anti-mouse IgG (Thermo) was diluted 1: 10000 with a secondary antibody (lxPBST-TweenO .05%), and absorbance was measured at 450 nm.
실험결과， 1E8 항체와 8A12 항체는 MRS 단백질 중에서 861-900 aa 영역 (AQKADKNEVA AEVAKLLDLK KQLAVAEGKP PEAPKGKKKK, 서열번호 2)을 에피토프로 특이적으로 인식하는 것을 확인하였다. 이러한 실험 결과는 861-900 aa 영역을 에피토프로 인식하는 다른 결합분자 (다른 항체 및 이의 기능적 단편 등)들도， MRS 특이적 결합 및 MRS구분능이 뛰어날 것임을 시사한다. 이하에서， 본 발명자들이 신규하게 고안한 췌장암 검사법에 대하여， 상기에서 제작한 MRS 검출능이 뛰어난항체를 적용한 일 실시예를 보여준다. As a result, it was confirmed that the 1E8 antibody and the 8A12 antibody specifically recognize the 861-900 aa region (AQKADKNEVA AEVAKLLDLK KQLAVAEGKP PEAPKGKKKK, SEQ ID NO: 2) as an epitope among the MRS proteins. These results suggest that other binding molecules (such as other antibodies and functional fragments thereof) that recognize the 861-900 aa region as an epitope will also be able to distinguish between MRS-specific binding and MRS. Hereinafter, the present inventors have applied an antibody having excellent MRS detection capability to the pancreatic cancer test method newly devised by the present inventors.
1-11. 본원 발명의 ant i -MRS 항체를 이용한 췌장암 세포 염색 및 시판 항체와의 효과 대비 1-11. Staining of pancreatic cancer cells with the ant i -MRS antibody of the present invention and the effect of the antisera against commercial antibodies
PANC-1 췌장암 세포주와 SCK (비췌장암 세포주) 세포주에. 대하여， 각각 1E8 항체 또는 8A12 항체를 이용하여 MRS 형광염색을 수행하였다. 이때 대조군으로는 시판되고 있는 MRS 항체 (Abeam, Abl37105)를 사용하였다. 구체적으로 다음과 같은 과정으로 염색을 수행하였다. 슬라이드 상에 준비된 각각의 대상 세포에 0.2% tween 20을 포함하는 PBS를 처리하여 투과성.을 증가시킨 뒤 , 2% goat serum 으로 1 시간 동안 블록킹 (blocking)처리하였다. 1차 항체로서 본 발명의 항체들 (1E& 항체 또는 8A12 항체， Oncotag社 제작) 또는 Abl37105 항체 (Abeam)를 l y g/ml 37 °C 1시간동안 처리하고， 0.05% TBSTC500 μ ΐ )로 3회 세척하였다. 그 후 형광 물질이 결합되어있는 2차 ant ibody로서 Al exa-488-con j ugat ed secondary . ant i id ies (구입처: Molecular probes , Cat . No. A11001)를 1 : 100으로 회석하여 상온의 어두운 곳에서 1 시간 동안 처리하고， 0.05% TBSTC500 μ ΐ )로 3회 세척하였다. DAPI가 첨가되어있는 마운팅 솔루션 (Pro mg Gold ant i fade regent with DAPI/ Molecular probes , Cat . No. P36931)을 슬라이드 상의 조직에 20 μ 1 처리 후 커버슬립 (coversl ip)을 덮고， 공초점레이저 현미경 및 형광 현미경으로 관찰하였다. 도 9에서 보는 .바와 같이， 상기 실시예 1—7에서 MRS 특이성이 떨어지는 것으로 확인되었던 시판 Abl37105 항체는 PANC-1 췌장암 세포와 SCK 세포 (비췌장암 세포) 모두를 비특이적으로 염색하였으나, 본원 발명의 1E8 항체 및 8A12 항체는 MRS 만을 특이적으로 염색할 수 있음을 확인하였다. 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. At this time, a commercially available MRS antibody (Abeam, Abl 37105) was used as a control. Specifically, 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. As the primary antibody, 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) was diluted 1: 100, treated in the dark at room temperature for 1 hour, and washed three times with 0.05% TBSTC (500 μl). The mounting solution with DAPI (Pro mg Gold ant i fade regent with DAPI / Molecular probes, Cat. No. P36931) was treated with 20 μl 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.
또한 임상현장에서 환자세포 시료 processing에 사용하는 Thinprep 장비 (Hologic . Inc)를 활용하여， PANC-1 세포주로 임상조건과 유사하게 Thinprep 슬라이드를 제작하여 (하기 실시예 2 참조)， 본 발명의 1E8 항체 및 8A12 항체의 염색효과를 확인하였다. 그 결과 도 10에서 보는 바와 같이， 본원 발명의 1E8 항체 및 8A12 항체는 임상현장에서 흔히 사용되는 시료제공방법 (Thinprep 슬라이드 등)과 함께 사용될 수 있음을 확인하였다. 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.
실시예 2: 세포진 (cytodiagnosis) 검사법에 있어서, 췌장암세포 특이적 MRS 발현 검출법 (염색법)의 정립 및 효과 확인 Example 2: Identification and effect of pancreatic cancer cell-specific MRS expression detection (staining method) in cytodiagnosis assay
실험방법 Experimental Method
1) 검체로서 췌장 세포를， 통상적인 내시경초음파 세침홉입술 (EUS-FNA)에 따라 수득하였다. 먼저， 내시경초음파 기기 (a l inear array echoendoscope EUS, 제품명; GF-UCT140 또는 GF-UCT180 , 회사: Olympus , Japan)를 위나 십이지장으로 진행하고 내시경 선단에 장착된 초음파 장비를 이용하여 췌장 종괴를 초음파 영상으로 확인하였다. 초음파상으로 조준된 종괴에 대해 세침흡입을 위한 바늘 (제품명 : 22G Echo-ultraTM, 회사: Cook Medical , Cork, Ireland)을 진입하여 췌장 세포를 채취하였다. 그 후， 상기 채취된 췌장 세포를 Cellient Automated Cell Block System (Hologic)를 이용하는 통상적인 방법 (Antonio Ieni et al .， Cell-block procedure in endoscopic ultrasound一 guidedᅳ fineᅳ needle一 aspiration of gastrointestinal sol id neoplastic lesions, World J Gastrointest Endosc 2015 August 25; 7(11): 1014-1022)으로 파라핀 절편 (Cellient paraffin sections)으로서 슬라이드 상에 제공되었다. 또한 상기 췌장 세포 시료는 ThinPrep (Hologic. Inc)을 이용하여 통상적인 방법으로 ThinPrep 슬라이드에 도말하거나 direct smear하여 제공될 수 있다 (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.). 이들 세포 시료는 각각 하기의 검사법을 적용하여 결과가 비교되었다. 1) Pancreas cells as a sample were obtained according to a conventional endoscopic ultrasonic microhip lips (EUS-FNA). First, 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. Thereafter, the collected 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. 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.
2) 기존 세포학적 검사 방식에 의한 병리소견 (Conventional pathologic cytology)은 현재까지 흔히 사용되고 있는 H&E staining 방법을 이용한 염색 결과에 의하여 내려질 수 있다. 상기 H&E staining은 hematoxylin 및 eosin을 통상적 프로토콜에 따라 사용하여 수행되었다 (하기 실시예 3의 상세 프로토콜 참조). 또한 Pap staining 방법을 통한 염색 결과에 의하여 내려질 수 있다. 상기 Pap staining은 hematoxylin, 0G-6( Orange G—6), eosin azure를 통상적 프로토콜에 따라 사용하여 수행되었다 (하기 실시예 3의 상세 프로토콜 참조). 파라핀 절편 시료를 이용하는 경우에는， 통상적인 방법으로 파라핀 제거 및 수화 (hydration)를 진행한 후 염색 물질들을 처리하였다. 슬라이드 상에 세포가 한 겹으로 도말되어 있으며 세포핵 /세포질 비율 (N/C ratio)이 작고 핵막이 매끄러운 모양일 경우 양성 (benign, 정상) 세포로 판정하고， 세포가 3차원으로 도말되며 세포핵 /세포질 비율이 높고 염색질의 뭉침 현상이 보이며 핵막이 거친 모양이고 핵소체 및 유사분열이 출현할 경우 악성종양 세포로 판정하며， 세포의 변화가 악성 세포에는 미치지 못하나 양성 (benign)으로 판정할 수 없는 경우 비정형 세포 (atypical cell)로 판정한다. 3) 임상적 최종 진단 결과는 영상학적 검사 (복부 초음파， 복부전산화 단층촬영， 복부 자기공명영상， 내시경역행 담췌간조영술， 양전자방출 단층촬영)와 병리학적 검사 (세포진 검사， 조직검사)에 의하여 측정된 결과들에 의하여 의사가 종합적으로 최종 판단하였다. 2) Conventional pathologic cytology by conventional cytologic examination method can be obtained by the result of staining using H & E staining method which is commonly used so far. The 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). When 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. 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.
4) 본 발명자들은 췌장암 세포와 정상 췌장 세포 (암이 아닌 양성 (benign) 췌장염 세포 포함)에서의 MRS 발현도를 측정하기 위한 면역조직화학법 (특히， 면역형광염색)을 하기와 같이 개발하였다. 구체적으로， 상기 파라핀 절편을 사용하는 경우， 다음과 같이 처리하였다. 4) We developed immunohistochemistry (especially, immunofluorescence staining) for measuring MRS expression in pancreatic cancer cells and normal pancreatic cells (including non-cancerous benign pancreatic cells) as described below. Specifically, when the paraffin section was used, the following treatments were carried out.
① 파라핀 제거 : 60°C 오븐에서 파라핀을 녹임 ① Paraffin removal: Paraffin is dissolved in an oven at 60 ° C
② 수화 (Hydrat ion) : 자일렌 (Xylene)으로 5분씩 3번 세척， 100% 에탄올로 2분 세척， 95% 에탄을로 2분 세척， 90% 에탄을로 2분 세척， 70% 에탄을로 2분 세척， D.W로 2분 세척， PBS로 5분 세척 ② Hydration: Wash with xylene three times for 5 minutes, wash with 100% ethanol for 2 minutes, clean with 95% ethane for 2 minutes, wash with 90% ethane for 2 minutes, remove with 70% 2 minutes wash, 2 minutes DW wash, 5 minutes wash with PBS
③ 투과성 (peraeabi l ize) 증가 처리: 0.2% Triton X-100으로 30분간 처리하고 PBS로 5분 세척 (3) Increase in permeability (peraeabi l ize): Treatment with 0.2% Triton X-100 for 30 minutes and washing with PBS for 5 minutes
④ 전처리: 2% goat serum으로 1 시간 동안 블록킹 (blocking) ④ Pretreatment: Blocking with 2% goat serum for 1 hour,
⑤ 1차 항체 처리: ant i -MRS 항체 (대표적으로 본 발명의 8A12 항체 사용함, Oncotag社 제작)를 1 ： 300으로 희석하여 4°C에서 overnight 처리하고， PBS로 5분씩 3회 세척 (5) Primary antibody treatment: 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
⑥ 발색: 형광 물질이 결합되어있는 2차 ant ibody로서 Alexa-488-conjugated secondary ant ibodies (구입처: Molecular probes , Cat . No. A11001)를 1 :200 ~ 1 :300으로 회석하여 상온의 어두운 곳에서 1 시간 동안 처리하고, PBS로 5분씩 3회 세척 ⑥ Color development: 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
⑦ DAPI가 첨가되어있는 마운팅 솔루션 (ProLong Gold ant i fade regent with DAPI/ Molecular probes , Cat . No. P36931)을 슬라이드 상의 조직에 20 μ 1 처리 후 커버슬립 (coversl ip)을 덮었다. thinprep 슬라이드 시료의 경우에는, 파라핀 제거 과정 없이 상기 ③ 내지 ⑦의 과정을 포함하는 방법으로 처리되었으며, 상기 방법으로 준비된 표본 시료는 공초점 레이저 현미경 및 형광 현미경으로 관찰하였다. 상기 표본 시료에서 양성 세포 샘플 (PANC-1 , 도 15 참조) 및 음성 세포 샘플 (CT-26 , 도 16 참조)을 기준으로 MRS 염색 강도를 판단하였으며， 음성 대조군에 비하여 2배 이상 증가한 세포를 췌장암 세포인 것으로 판단하였다. 이러한 결과를 이러한 결과를 상기 검체에 대한 병리 소견 및 임상적 최종 진단결과와 비교하여 진단의 정확성 (민감도 및 특이도)을 확인하였다. ⑦ Covering slides (coversl ip) were covered with 20 μl of DAPI-added mounting solution (ProLong Gold ant i fade regent with DAPI / Molecular probes, Cat. No. P36931) on the slides. In the case of the thinprep slide samples, the samples were processed by a method including the above steps 3 to 7 without removing the paraffin. The sample prepared by the above method was observed with a confocal laser microscope and a fluorescence microscope. MRS staining intensity was determined based on positive cell samples (PANC-1, see FIG. 15) and negative cell samples (CT-26, see FIG. 16) in the sample above. Cells. These results were compared with pathologic findings of the specimens and clinical final diagnosis results to confirm the accuracy (sensitivity and specificity) of the diagnosis.
실험결과 구체적 실험결과는 하기 표 3, 표 4 및 표 5에서 보는 바와 같다. 정상 (Benign) 췌장 세포 시료 14개 및 췌장암 (Mai ignancy) 세포 시료 94개를 대상으로 본원 발명의 MRS 염색을 통한 췌장암 판별 방법을 적용한 결과， H&E staining 방식을 사용하는 기존 세포병리학적 검사 결과는 민감도가 75.5% 정도에 불과하였던 것과 대비하여 본원 발명의 MRS 염색은 민감도 (Sensi t ivi ty)가 92.6%로 나타났다. 특히, 기존에 세포학적 병리검사 방식으로는 비정형 (atypia)로 분류될 수밖에 없었던 세포 시료에 대해서도 췌장암 여부의 판별이 가능하였기 때문에， 기존 세포학적 병리검사로는 음성예측율 (NPV)이 36% 수준이었던데 반해서 MRS 염색올 통해서는 음성예측률이 현저히 높아지는 효과를 나타내었다. 이러한 결과는 췌장암에 있어 MRS를 마커로 이용하는 본 발명의 염색법은 세포 수준에서의 진단에서도 높은 판별능 (진단능)을 가짐을 보여주는 것으로, 후술하는 실시예 3에서 보이는 바와 같이 기존에 상용의 췌장암 마커들 (CEA와 같은 공지의 최장암 마커)이 세포 수준의 진단 (즉， cytodiagnosis)에서는 실효성을 나타내기 어려웠던 것과 명확히 구별되는 것이었다. Experimental Results The specific experimental results are shown in Table 3, Table 4 and Table 5 below. As a result of applying the MRS staining method for pancreatic cancer according to the present invention to 14 benign pancreatic cell samples and 94 pancreatic cancer cells (Mai ignancy), the results of the conventional cytopathological tests using the H & E staining method showed sensitivity The sensitivity of the MRS staining of the present invention was found to be 92.6%. In particular, the presence of pancreatic cancer was able to be identified in cell samples that were previously classified as atypia by cytopathologic examination. Thus, the conventional cytopathological examination revealed a negative predictive value (NPV) of 36% Whereas the negative predictive value was significantly increased by MRS stain. These results indicate that the staining method of the present invention using MRS as a marker in pancreatic cancer has a high discriminating ability in diagnosis at the cellular level. As shown in Example 3 described below, the conventional pancreatic cancer marker (Known as the longest cancer marker, such as CEA), was clearly indistinguishable from cell-level diagnosis (i. E., Cytodiagnosis).
【표 3】 비교 상세: 최종 임상병리학적 진단 결과 대비, 기존 세포학적 검사 결과 (convent ional pathologic cytology)와 본원 발명의 MRS 면역염색을 통한 검사 결과 비교 [Table 3] Comparison Details: Compared to the results of the final clinical pathologic diagnosis, results of conventional cytologic examination (convential pathologic cytology) and MRS immunostaining of the present invention
Final MRS immunostaining Final MRS immunostaining
Conventional pathologic  Conventional pathologic
clinicopathological  clinicopathological
cytology Posi t ive Negat ive  cytology Posi t ive Negat ive
diagnosis Malignancy (n=43) 42 1 diagnosis Malignancy (n = 43) 42 1
Malignancy  Malignancy
(n=43)  (n = 43)
Benign (n=0) 0 0  Benign (n = 0) 0 0
Malignancy (n=28) 26 2 Malignancy (n = 28) 26 2
Suspicious of malignancy  Suspicious of malignancy
(n=29)  (n = 29)
Benign (n=l) 1 0  Benign (n = 1) 1 0
Malignancy (n=18) 17 1 Malignancy (n = 18) 17 1
Atypical  Atypical
(n=21)  (n = 21)
Benign (n=3) 2 . 1  Benign (n = 3) 2. One
Malignancy (n=5) 2 3 Malignancy (n = 5) 2 3
Negative for malignancy  Negative for malignancy
(n=15)  (n = 15)
Benign (n=10) 2 8  Benign (n = 10) 2 8
【표 4] 비교 요약: 최종 임상병리학적 진단 결과 대비， 기존 세포학적 검사 결과 비교 (상기 표 3의 기존 세포학적 검사 결과에서 positive for mali nancy 판정 및 suspicious malignancy판정은 최종 positive로 분류하고， Atypia판정 및 Negative for malignancy판정은 최종 negative로 분류함) [Table 4] Comparison summary: Comparison of results between the final clinical pathological diagnosis results and the existing cytological test results (the positive cytokine test and the suspicious malignancy test were classified as the final positive result and the Atypia test And Negative for malignancy are classified as final negative)
Figure imgf000053_0001
Figure imgf000053_0001
PPV: positive predictive value, NPV: negative predictive value  PPV: positive predictive value, NPV: negative predictive value
【표 5] 비교 요약: 최종 임상병리학적 진단 결과 대비， 본원 발명의 MRS 면역염색을 통한 검사 결과 비교 Final cl inicopathological diagnosis [Table 5] Summary of comparison: Comparison of test results of MRS immuno-staining of present invention compared to final clinical pathological diagnosis result Final cl inicopathological diagnosis
Malignancy Benign  Malignancy Benign
Positive 87 5  Positive 87 5
MRS immunostaining  MRS immunostaining
Negative 7 9  Negative 7 9
Sensitivity: 92.6% Specificity: 64.3% Accuracy= 88.1% PPV: 94.6% NPV: 56.3%  Sensitivity: 92.6% Specificity: 64.3% Accuracy = 88.1% PPV: 94.6% NPV: 56.3%
PPV: positive predictive value, NPV: negative predictive value 실시예 3: 세포 수준에서, 상용 췌장암 마커 CEA와 본원 발명의 MRS의 췌장암판별능 비교 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
실험방법 Experimental Method
1) 환자군 및 세포진 (cytodiagnosis)을 위한 췌장 세포 시료의 수득 1) Obtaining pancreatic cell samples for patient and cytodiagnosis
26명의 췌장암 의심 환자에서 내시경 초음파하 세포흡입시술 (미세바늘흡입법， Fine needle aspiration)로 채취 및 수득된 췌장세포 시료 26례로 연구를 실행했으며 본 연구는 강남세브란스병원 연구윤리위원회의 승인을 받았다. 상기 환자들로부터 췌장 세포 시료는 상기 실시예 2와 동일한 방법으로, 내시경초음파 세침흡입술 (EUS— FNA)을 통해 수득되었다. 그 후 채취된 췌장 세포를 Cellient Automated Cell Block System Otologic)를 이용하는 통상적인 방법으로 파라핀 절편 (Cellient paraffin sections) 상태 혹은 Thinprep 방식으로 준비하였다 (실시예 2참조). In 26 suspected cases of pancreatic cancer, 26 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.
26명의 췌장암 의심환자는 추적 관찰을 통해 췌장암 (13례), 정상 췌장 (13례)으로 최종 진단되었다. 상기 췌장암으로 최종 진단된 13명의 환자들 중 병리학자에 의한 조직학적 관찰에 의해 췌장암 세포로 구분이 된 것은 7 례였고， 나머지 6 례는 조직학적 관찰에서는 비정형세포로 구분이 된 환자였으며 추적관찰을 통해 최종적으로는 췌장암으로 진단이 된 환자군이었다. 췌장세포 채취 시 환자로부터 서류에 의한 동의를 받았으며 췌장암 세포， 비정형세포, 정상세포는 병리학자에 의해 조직학적으로 확인되었다 (실시예 2의 판단기준 참조). 수득한 26례의 샘플 중 정상세포， 종양세포 및 비정형세포의 각 유형 별로 대표적인 진단사례를 각실험별 도면에서 보여준다. 2) 기존 세포진 염색 방법 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 염색: 파라핀 절편 시료는, Xylene으로 5분씩 3번 처리， 100% 에탄올 2분 처리, 95% 에탄을에서 2분 처리, 90% 에탄올에서 2분 처리， 70% 에탄올에서 2분 처리， 수듯물에서 10분간 처리를 각각 실시하여 Paraffin 제거와 Hydration올 실시하였다. 상온에서 hematoxyline을 30초 간 반웅시키고， 수듯물로 10분간 세척하였다. 상온에서 eosin올 1분간 반응시키고, 수듯물로 10분간 세척하였다. 70% 에탄올에서 1분, 90% 에탄을에서 1분， 95% 에탄을에서 1분， 100% 에탄을 1분， Xylene 5분씩 3번 처리를 실시하여 dehydration과 clearing을 실시하였다. Mounting solution을 슬라이드 조직에 떨어뜨린 후 coverslide로 세포를 덮은 다음 샘플을 광학 현미경으로 관찰하였다. 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.
pap 염색: Papanicolaou stain 은 Thermo Scientific의 Varistain 24-4 염색기를 사용하여 기기에 '내장돤 프로토콜에 따라 시행하였다. 프로토콜은 하기 표 6과 같다. pap staining: 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.
【표 6】 reagent programl program2 [Table 6] Reagent programl program2
1 water 10분 10분  1 water 10 min 10 min
2 Hema. 1분 30초 1분 30초  2 Hema. 1 minute 30 seconds 1 minute 30 seconds
3 water 30초 30초  3 water 30 seconds 30 seconds
4 water 30초 30초  4 water 30 seconds 30 seconds
5 0.5% Hcl. 7초 7초  5 0.5% HCl. 7 seconds 7 seconds
6 0.5% Amm. 5초 5초  6 0.5% Amm. 5 seconds 5 seconds
7 water 30초 30초  7 water 30 seconds 30 seconds
8 50% ale. 30초 30초  8 50% ale. 30 seconds 30 seconds
9 70% ale. 30초 30초  9 70% ale. 30 seconds 30 seconds
10 80% ale. 30초 30초  10 80% ale. 30 seconds 30 seconds
11 95% ale. 30초 30초 12 0G 6 1분 10초 11 95% ale. 30 seconds 30 seconds 12 0G 6 1 min 10 sec
13 95% ale . 30초 30초  13 95% ale. 30 seconds 30 seconds
14 95% ale . 30초 30초  14 95% ale. 30 seconds 30 seconds
15 EA 50 1분 10초  15 EA 50 1 minute 10 seconds
16 95% ale . 30초 30초  16 95% ale. 30 seconds 30 seconds
17 95% ale . 30초 30초  17 95% ale. 30 seconds 30 seconds
18 95% ale . 30초 30초  18 95% ale. 30 seconds 30 seconds
19 99% ale . 30초 30초  19 99% ale. 30 seconds 30 seconds
20 99%+xyl . 20초 20초  20 99% + xyl. 20 seconds 20 seconds
' 21 xyl . 30초 30초 '21 xyl. 30 seconds 30 seconds
22 xyl . 30초 30초  22 xyl. 30 seconds 30 seconds
23 xyl . 30초 30초  23 xyl. 30 seconds 30 seconds
24 xyl . 30초 30초  24 xyl. 30 seconds 30 seconds
25 end.  25 end.
3) 기존 염색법에 의한 형태학적 병리검사에 따른 세포진 (cytodiagnosis)에서 판별 기준 악성종양을 형태학적으로 진단하려 할 때 가장 결정적인 증거는 주변 정상 조직으로 침윤성 성장을 한다는 것이지만， 암조직과 주변 조직의 관계를 증명하기 용이한 조직검사와는 달리, 세포진 검사의 경우 낱개의 세포를 뽑아내어 도말한 것이므로 주변 조직과의 관계를 증명할 수 없다. 따라서 차선책으로 개개 세포의 비정형성 (atypia)을 평가하게 되는데 이 때 비정형성이라 함은 세포의 핵 (nucleus)이 커지고 세포질 (cytoplasm)은 줄어들어 세포핵 /세포질 비율 (Nuclear to cytoplasmic rat io, N/c rat io)이 커지는 것; 염색질 (chromat in)이 핵 내에 균등하게 분포되지 않고 부분적으로 뭉치는 현상 (clumping) , 핵 내 핵소체 (nucleolus)의 출현, 유사분열 (mitosis) 출현 등을 말한다. 이들 비정형성의 소견이 모두 나타나고 정도가 현저할 때 세포진 검사로도 악성종양을 진단하는 것이 가능하지만, 이들 소견이 부분적으로 나타나거나 정도가 미약할 경우에는 비정형세포 (atypical cel l )로 분류하여야 한다. 심한 염증 등 양성병변에서도 이와 같은 소견이 부분적으로 나타날 수 있기 때문이다. 반대로 이와 같은 소견이 어느 것도 보이지 않을 경우는 정상 세포로 판정할 수 있다. 물론 세포를 채취한 부위에서 관찰될 수 있는 세포라는 것이 전제되어야 한다. 3) Discriminatory criteria for cytodiagnosis by morphological pathologic examination by conventional staining method The most conclusive evidence for the morphological diagnosis of malignant tumors is the invasive growth of surrounding normal tissue, but the relationship between cancer tissue and surrounding tissues , It is impossible to prove the relationship with the surrounding tissues since the cells are extracted from individual cells in the case of cytological examination. Therefore, atypia of individual cells is assessed as a secondary measure. 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.
4) 공지의 상용 췌장암 마커인 CEA와 본원 발명의 MRS를 이용한 비교 염색 4) Comparative staining using CEA, a known commercial pancreatic cancer marker, and MRS of the present invention
MRS에 대한 면역염색은 상기 실시예 2에 기재된 방법과 동일하게 수행되었다 CEA에 대한 면역염색의 경우， 1차 항체로서 Carcinoembryonic Ant igen(CEA) ant ibody (구입처: Dako, Cat . No . M7072)를 이의 최적 처리 조건으로 사용한 것 이외에는 실시예 2와 동일한 과정으로 수행되었다. Immunostaining for MRS was carried out in the same manner as described in Example 2. For immunostaining for CEA, Carcinoembryonic Antigen (CEA) ant ibody (purchased from Dako, Cat. No. M7072) was used as a primary antibody The same procedure as in Example 2 was carried out except that the optimum treatment conditions were used.
5) 통계분석 실험결과는 최소 3회 이상의 독립적인 실험결과를 바탕으로 평균士 표준편차로 나타내었다. 통계적 유의성은 다수의 그룹 간의 차이 관찰을 위해 Student ' s t test를 활용하여 데이터를 분석하였다. P-value가 0.05 미만일 때의 실험결과가유의성이 있는 것으로 판단하였다. 5) Statistical analysis The experimental results are expressed as mean standard deviation based on at least 3 independent experiments. Data were analyzed using Student 's t test for statistical significance. The P-values of less than 0.05 were considered to be significant.
실험결과 Experiment result
3-1. 정상세포의 면역염색 정상환자 또는 췌장암 환자의 췌장으로부터 EUS-FNA 방법으로 분리한 세포를 H&E 염색을 통해 분석한 결과， 정상으로 판단된 세포를 MRS 또는 CEA 항체를 이용하여 면역형광염색을 수행하였다. 이에 대한 결과를 도 11에 나타내었다. 3-1. Immunofluorescence staining of normal cells Cells isolated from the pancreas of normal or pancreatic cancer patients by EUS-FNA method were analyzed by H & E staining, and normal cells were subjected to immunofluorescence staining using MRS or CEA antibody. The results are shown in Fig.
도 11에 나타낸 바와 같이， 네 명의 환자로부터 수득한 췌장세포는 H&E 염색에서 아무런 종양소견을 나타내지 않아 정상세포로 판단이 되었으며， 상기 정상으로 판단된 췌장세포에서는 MRS 및 CEA모두 염색이 되지 않는 것으로 확인되었다. As shown in Fig. 11, the 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 .
3-2. 종양세포의 면역염색 췌장암 환자의 췌장으로부터 분리한 세포를 H&E 염색을 통해 분석한 결과， 종양으로 판단된 세포를 MRS 또는 CEA항체를 이용하여 면역형광염색을 수행하였다ᅳ 이에 대한 결과를 도 12에 나타내었다. 3-2. Immunohistochemistry of tumor cells Cells isolated from pancreas of pancreatic cancer patients were analyzed by H & E staining. Immunofluorescent staining was performed using MRS or CEA antibody as the tumor cells The results are shown in Fig.
도 12에 나타낸 바와 같이， 네 명의 환자로부터 수득한 췌장세포는 H&E 염색에서 종양세포로 판단이 되었다. 네 명의 췌장암 환자 종양세포 모두에서 MRS 염색이 강하게 나타났으며， 이는 정상세포의 MRS 염색군과 비교해 통계학적으로 유의성 있는 결과로 나타났다 (p=0.019) . 그러나 CEA 염색은 두 명의 환자 샘플에서 만 약하게 염색이 되었으며， 이는 정상세포의 MRS 염색군과 비교해 통계학적인 유의성이 없었다 (ρ=0 · 187) . As shown in Fig. 12, pancreatic cells obtained from four patients were judged to be tumor cells in H & E staining. MRS staining was stronger in all four pancreatic cancer tumor cells, which was statistically significant (p = 0.019) as compared with MRS staining of normal cells. However, CEA staining was weakly stained only in two patient samples, which was not statistically significant (p = 0 · 187) compared with MRS staining in normal cells.
3-3. 비정형세포의 면역염색 3-3. Immunostaining of atypical cells
Η&Ε염색만으로는 종양세포인지 여부를 명확히 판단하기 어려운 비정형 (atypical )세포로서, 향후 환자의 예후를 추적 관찰한 결과 최종적으로 종양으로 판명이 되었던 환자의 췌장세포를 이용하여 MRS 또는 CEA염색을 수행하였다. 이에 대한 결과를 도 13에 나타내었다. 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.
도 13에 나타낸 바와 같이, H&E 염색을 통해서 비정형 세포 (atypical cel l )로 구분이 된 췌장세포는 종양인지 또는 정상세포인지의 구분이 명확하지 않다는 것을 알 수 있었다. 한편, 비정형 세포군에 포함된 환자들은 모두 향후 췌장암으로 확정 진단을 받은 환자들이다. 네 명의 비정형 췌장세포 모두에서 MRS 염색이 강하게 나타났으며， 이는 정상세포의 MRS 염색군과 비교해 통계학적으로 유의성 있는 결과로 나타났다 (p=0.020) . 그러나 네 명의 비정형 췌장세포 모두에서 CEA 염색이 전혀 ^타나지 않았다. 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. On the other hand, the patients included in the atypical cell group are all those who have been diagnosed as pancreatic cancer in the future. MRS staining was strongly observed in all four atypical pancreatic cells, which was statistically significant (p = 0.020) compared with MRS staining of normal cells. However, CEA staining was not observed in all four atypical pancreatic cells.
상기 실시예 3의 결과를 통해， 췌장암 의심 환자로부터 분리한 췌장세포에 H&E염색 및 MRS 염색을 함께 수행한다면, 다른 종양마커들보다 훨씬 높은 정확도로 췌장암올 진단할 수 있음을 알 수 있었다. 뿐만 아니라， H&E염색으로도 종양세포인지 정상세포인지 여부를 명확히 판단할 수 없는 비정형 (atypical ) 세포에서 기존의 종양마커들 (예를들어 , CEA)은 종양인지 여부를 명확하게 구분할 수 없었지만, MRS는 이러한 비정형 췌장세포 또한 종양세포인지 여부를 상당히 높은 정확도로 명확하게 구분할 수 있다는 점에서 매우 의미가 있는 췌장암 진단 마커라 할수 있다. The results of Example 3 above indicate that 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. In addition, conventional tumor markers (eg, CEA) in atypical cells, which can not be clearly distinguished from tumor cells or normal cells by H & E staining, can not be clearly distinguished from tumors, but MRS Is a very important diagnostic marker for pancreatic cancer in that these atypical pancreatic cells are also clearly distinguishable from tumor cells with a fairly high degree of accuracy can do.
실시예 4 : 정확도높은 췌장암 판별 방법 수립 _MRS 이증 염색법 Example 4: Establishment of accurate pancreatic cancer discrimination method _MRS staining
4-1. 위양성 결과의 원인 탐색 상기 실시예 3에서 보는 바와 같이 MRS는 CEA 등 기존 췌장암 마커 보다 높은 정확도로 췌장암의 진단이 가능한 것으로 입증되었지만， 전술한 실시예들에서의 실험은 이미 모든 확정 판단을 가진 시료에 대하여 수행한 것이므로， 췌장암 발병 의심 환자로부터 조직 채취 후 bl ind (미지) 상태.에서 어느 정도의 정확성을 가지고 진단가능한지 확인할 필요가 있다. 또한 상기 실시예 2에서 보는 바와 같이， MRS는 췌장암 진단에 있어서 매우 우수한 민감도를 나타내지만, MRS를 단독으로 췌장암을 판단할 시에 민감도에 비하여 특이도가 다소 낮은 경향을 보였다. 이에 본 연구자들은 새로운 환자들로부터 수득한 췌장 시료에서 MRS의 췌장암 판별능을 시험하던 중 세포조직병리검사상 negat ive (비췌장암)로 나타난 10건의 샘플 중에서 3건의 샘플에서 MRS 발현이 나타나는 듯한 모습올 관찰하였다. 즉， 정상 췌장 세포 시료에서 MRS 발현도가 높은 경우가 있어 위양성 (췌장암이 아닌 것을 췌장암이 발생한 것으로 판단할 수 있는 것) 결과가 포함되는 문제가 있음을 확인하였다. 4-1. Investigation of Causes of False Positive Results As shown in Example 3, MRS was proved to be able to diagnose pancreatic cancer with higher accuracy than existing pancreatic cancer markers such as CEA. However, 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. In addition, as shown in Example 2 above, 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. In this study, we investigated the ability of MRS to differentiate pancreatic cancer from pancreatic specimens obtained from new patients. Among the 10 samples that showed negat ive (non-pancreatic cancer) as a cytopathological test, three samples showed MRS expression Respectively. In other words, it was confirmed that the normal pancreatic cell sample had a high MRS expression level, and therefore, there was a problem that the result of false positives (which can be regarded as the occurrence of pancreatic cancer, which is not pancreatic cancer) is included.
이에 전체 검체들에 대해 H&E stain으로 암조직, 정상조직을 감별한 후 다시 MRS염색을 수행하는 것을 통해서 상기 위양성 판단의 원인을 파악한 결과， 도 14에 서 보는 바와 같이 정상 췌장 조직 중 정상인 선방세포 (acinar cel l , 췌관실질에 존재하는 세포)에서 MRS 발현이 높은 상태로 있는 것을 확인하였다. As a result, 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. As a result, as shown in FIG. 14, acinar cel l, pancreatic parenchymal cells) showed high expression of MRS.
4-2. MRS진단 정확도 향상 전략수립 상기 실시예 4-1. 항목의 결과에서 보는 바와 같이 정상인 선방세포 (acinar cel l )에서도 MRS가 높은 수준으로 발현되고 있어 이의 단독으로는 췌장암 판단의 위양성률에 기여하고 있기 때문에 (즉 MRS 단독으로만 판단할 시에는 진단의 특이도가 상대적으로 낮음)， 위양성률을 줄이기 위해 MRS와 선방세포 (acinar cel l ) 특이적 마커 단백질을 이용하여 이중 염색법을 신규하게 고안하였다. 본 발명자들은 선방세포 (acinar cell) 특이적 마커 후보들 중， 일례로 키모트립신을 사용하여 선방세포에서의 염색 양상을 확인하였다. 먼저, 선방세포를 포함하는 정상 조직에 대해서 구분 가능한지， 상기 본 발명의 이중 염색 전략을 ICC(I隱 unohistochemistry) 방법으로 검증한 결과를 도 16에서 보여준다. 도 16의 조직은 도 15의 H&E 염색 이미지에서 보여주듯이 정상인 조직이다. 도 16은 이러한 정상 조직에 본 발명의 이중 염색을 수행하였을 때 나타나는 선방세포 (acinar cell)의 모습과 주변의 다른 세포들의 모습을 각각 확대하여 나타낸다. acinar cell에서만 MRS와 키모트립신이 모두 강하게 발현되어 색이 진하게 겹쳐보였고, 정상 조직의 다른 부분에서는 MRS가 검출되지 않음을 확인하였다 (도 16). 이러한 염색양상을 기준으로， 선방세포 마커 (대표적으로 키모트립신)가 고강도로 검출되는 세포 및 조직 영역은 췌장암 판별 과정에서 배제가능하다. 4-2. 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. First, it is possible to distinguish the normal tissues including the secretory cells, and the result of the double staining strategy of the present invention is shown in FIG. 16 by ICC (Iunohistochemistry) method. 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.
4-3. 이중염색법 수립 파라핀 절편 시료를 이용하는 경우에는, 먼저 6(rc 오븐에서 파라핀을 녹이고， Xylene으로 5분씩 3번 세척， 100% 에탄을 2분 세척， 95% 에탄올에서 2분 세척， 90% 에탄을에서 2분 세척， 70% 에탄을에서 2분 세척， D.W에서 2분 세척하여 Paraffin 제거와 Hydration을 실하였다. 4-3. Double staining If paraffin slices are used, first rinse the paraffin in a 6 ° rc oven, wash with xylene three times for 5 min, clean 100% ethane for 2 min, clean with 95% ethanol for 2 min, Two minutes of washing, two minutes of washing in 70% ethane and two minutes of washing in DW were performed to remove paraffin and hydration.
MRS 및 키모트립신의 이중염색을 위하여 , MRS는 녹색 (Alexa Fluor 488)으로 검출되고 키모트립신은 붉은색 (Texas Red)으로 검출되도록 구체적 실험 프로토콜을 수립하였다. 먼저 0.2% Triton X-100를 처리하여 세포 투과성을 증가시켰다. blocking solution인 2% 염소 혈청으로 1 시간동안 반응시켰다. 그 후 췌장 세포 (검체)에 1차 항체인 Methionyl tRNA synthetase ant i body (1 ： 100) 및 Anti- Ant i -Chymot ryps i n ant i body (1 ： 100 회석， abeam, catalog no. abl55400)와 4°C에서 overnight 반웅 시켰다. 그 후 IX TBST로 30회 이상 dipping (1회로 간주) 하여 3회 이상 세척 후 각각 Alexa Fluor 488 및 Texas Red가 부착된 2차 항체 (1:200 ~1:300 회석 / ThermoFisher Scientific, catalog no. A-11001/ SANTA CRUZ BIOTECHNOLOGY, INC. , catalog no. sc-278)를 어두운 곳에서 상온에 1시간동안 처리하였다. IX TBST PBS로 30회 이상 dipping (1희로 간주) 하여 3회 이상 세척 후 DAPI가 첨가되어 있는 mounting solut ion(ProLong Gold anti fade regent with DAPI, Molecular probes, Cat. No. P36931)을 슬라이드 조직에 떨어뜨린 후 coverslide로 세포를 덮은 다음 샘플을 형광 현미경으로 관찰하였다. 췌장암 판별 기준은 하기 표 7와 같이 판단하였다. 키모트립신이 검출 (발현)되지 않고 MRS 단백질이 검출 (발현)되면 췌장암 세포로 판단하였다. 즉， 본 발명의 검사상 MRS (+) 및 ACT (-)인 경우에 췌장암 Posit ive로 분류하고， 나머지의 경우에는 Negat ive로 분류한다. For double staining of MRS and chymotrypsin, a specific experimental protocol was established to detect MRS as green (Alexa Fluor 488) and chymotrypsin as red (Texas Red). First, 0.2% Triton X-100 was treated to increase cell permeability. blocking solution, 2% goat serum for 1 hour. The pancreatic cells (specimens) were then incubated with a primary antibody, Methionyl tRNA synthetase ant i body (1: 100), and Anti- Ant-Chymotry in antibody (1: 100, abeam, catalog no. Abl55400) overnight it was in banung ° C. After dipping 30 times or more in IX TBST (counting 1 cycle), wash the plates three times or more with Alexa Fluor 488 and Texas Red attached secondary antibody (1: 200-1: 300 plates / ThermoFisher Scientific, catalog no. A -11001 / SANTA CRUZ BIOTECHNOLOGY, INC., Catalog no. Sc-278) were treated in the dark at room temperature for 1 hour. After 3 or more washes with 30 times or more dipping with IX TBST PBS, mounting solution (ProLong Gold anti-fade regent with DAPI, Molecular probes, Cat. No. P36931) with DAPI After plating, the cells were covered with coverslide and then the sample was observed with a fluorescence microscope. The criteria for discriminating pancreatic cancer were determined as shown in Table 7 below. When chymotrypsin was not detected (expressed) and the MRS protein was detected (expressed), it was judged to be a pancreatic cancer cell. That is, in the case of MRS (+) and ACT (-) of the present invention, pancreatic cancer is classified as Posit ive, and in the remaining cases, it is classified as Negat ive.
【표 7]
Figure imgf000061_0001
실시예 5: 세포진 (cytodiagnosis)에 있어서， 본 발명 이중염색법의 췌장암 판별능 평가 전술한 실시예들에서 사용된 것과는 다른 피검체들로부터 수득된 새로운 췌장 세포 시료 29개에 대하여 본 발명의 이중염색법을 적용하여 췌장암을 판별하였다. 이들 췌장암 세포는 전술한 바와 동일하게 EUS-FNA 방법으로 채취되었다. 본 발명의 이중염색법을 수행하는데 있어서， 병리학적 진단 결과나 최종 임상적 진단은 미지 (bl ind)인 상태로 진행되었다. [Table 7]
Figure imgf000061_0001
Example 5 Evaluation of Pancreatic Cancer Detecting Ability by the Double Staining Method of the Present Invention in Cytodiagnosis The 29 new pancreatic cell samples obtained from the subjects different from those used in the above Examples were subjected to the double staining method of the present invention To determine pancreatic cancer. These pancreatic cancer cells were collected by the EUS-FNA method as described above. In performing the double staining method of the present invention, the pathological diagnosis result or the final clinical diagnosis proceeded to be blind.
5-1. 정상 췌장선방세포 (acinar cel l )에서의 면역형광염색 양상 확인 췌장으로부터 EUS-FNA 방법으로 수득한 정상 선방세포 (acinar cel l )군 시료가 실제 본 발명의 이중 염색법으로 염색되었을 때 어떤 양상을 나타내는 지 확인하였다. 이에 대한 결과를 도 17 및 도 18에 나타내었다. 5-1. Examination of Immunofluorescence Staining Pattern in Normal Pancreatic Dendritic Cells (acinar cel 1) When a sample of acinar cel 1 group obtained from the pancreas by the EUS-FNA method was actually stained with the double staining method of the present invention, Respectively. The results are shown in Fig. 17 and Fig.
도 17 및 도 18에서 보는 바와 같이 선방세포의 경우 키모트립신이 강하게 검출 (발현)되어 붉은색이 매우 강하게 나타났다. 구체적으로 도 17에서 보는 바와 같이 MRS가 매우 미약하게 검출되고 상대적으로 키모트립신이 매우 강하게 검출되어 나타나는 패턴과 도 18에서 보는 바와 같이 키모트립신과 함께 MRS 검출도 상당히 나타나는 패턴이 있었다. 그러나 도 18과 같은 염색 패턴에서도 merge 이미지에서는 키모트립신을 나타내는 붉은 색이 높은 강도로 나타났으며， 이러한 염색 패턴은 선방세포 판단의 근거가 된다. As shown in Fig. 17 and Fig. 18, 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.
5-2. 기존 세포 병리검사상으로 췌장암 확진되고， 최종적으로도 췌장암 확진된 환자의 경우 염색 양상 췌장으로부터 분리한 세포를 pap staing올 통해 분석한 결과 종양세포로 판단된 세포 시료의 염색 양상을 본 발명의 이중 염색법으로 평가하였다. 5-2. In the case of 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.
도 19에서 보는 바와 같이， 본 실험에서 사용된 세포진 시료 (pap staing을 통해 분석한 결과 종양세포로 판단된 세포)는 붉은색은 거의 나타나지 않고 녹색이 매우 강한 발현을 띠고 있어, 즉 키모트립신이 거의 검출되지 않고 MRS의 발현이 매우 강하게 발현되고 있는 것으로서 췌장암으로 판단 가능함을 확인하였다. As shown in FIG. 19, the cytopenic samples used in this experiment (cells determined to be tumor cells by papa staining) 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.
5-3. 기존 세포 병리검사 상으로는 비정형세포 (atypical eel Is)로 분류되어 확진이 불가하였으나, 최종적으로 췌장암 확진된 환자의 경우 염색 양상 기존 pap staining상 비정형세포 (atypical eel Is)였으나 임상적으로 췌장암 확진된 환자의 세포진 (세포시료) 염색 양상을 본 발명의 이중 염색법으로 평가하였다. 5-3. The presence of 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.
그 결과 도 20에서 보는 바와 같이, MRS는 발현 강도가 매우 높게 나타났으며 키모트립신은 상대적으로 검출강도가 상당히 낮아 암세포임을 확인할 수 있었다 (merge 이미지 참조) . 이처럼 본 발명의 이중염색법은 비정형세포에 대해서도 암의 판정이 가능함을 확인하였다. As a result, as shown in FIG. 20, 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). As described above, the double staining method of the present invention confirmed that cancer can be judged also on non-specific cells.
5-4. 결과 종합 총 26개의 세포시료 검체에 대하여 본 발명의 이중 염색법을 수행하여 판단한 결과와， 최종적 임상 진단 결과를 비교하였으며, 그 결과를 하기 표 8에서 보여준다. 본 발명의 이중염색 결과, MRS (+) 및 선방세포 마커 (-) 인 경우에 췌장암 Posit ive로 분류하고， 나머지의 경우 (즉， MRS (+) 및 선방세포 마커 (+)로 나타나거나， MRS (-) 및 선방세포 마커 (-)로 나타나거나， MRS (-) 및 선방세포 마커 (+)로 나타나는 경우)에는 Negat ive로 분류한다. 5-4. 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.
【표 8] 최종 임상병리학적 진단 결과 대비, 본원 발명의 MRS 및 키모트립신 이중염색을 통한 검사 결과 비교 [Table 8] Comparison of test results of MRS and chymotrypsin double staining of the present invention as compared with the final clinical pathological diagnosis result
Figure imgf000063_0001
Figure imgf000063_0001
PPV: posit ive predict ive value, NPV: negat ive predict ive value 상기 표 8에서 보는 바와 같이， 본원 발명의 MRS 및 선방세포 마커 (대표적으로, 키모트립신) 이중 염색법을 통한 췌장암의 판정은 민감도 腦， 특이도 100%, 양성 예측치 (PPV) 100%, 음성 예측치 (NPV) 100%로 진단 정확도가 향상되었음을 확인하였다.  As shown in Table 8 above, the determination of 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%.
종합하여 상기 실시예 5에서 보는 바와 같이, 본 발명의 이중 염색법은 비정형 세포를 포함하여 기존 세포병리학적 판단방법 (pap-staining 또는 H&E stainig 등에 기반한 형태학적 판단 방법)으로는 확진 불가 및 미확진된 췌장세포 (특히 Atypia)에 대해서도 명확히 악성종양세포 여부를 판별 가능하여， 세포진에서의 진단효율올 현저히 상승시킬 수 있는 것으로 확인되었다. In addition, as shown in Example 5 above, 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.
【산업상 이용가능성】 이상 살펴본 바와 같이, 본 발명은 메티오닐 -티알엔에이 합성효소 및 선방세포 특이적 마커를 이용한 췌장암 진단 방법에 관한 것이다. MRS는 기존의 CEA와 같은 췌장암 마커보다 진단 정확도가 높으며, 특히 MRS의 발현과 더불어 키모트립신 (chymotrypsin)과 같은 선방세포 특이적 마커단백질을 추가의 이중마커 (dual marker)로 사용하면 췌장암 진단의 정확도가 현저히 상승되므로， 체외 진단산업 등의 분야에 있어서 산업상 이용가능성이 매우 우수하다. INDUSTRIAL APPLICABILITY As described above, 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. Especially, the use of 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.

Claims

【청구의 범위】 Claims:
【청구항 1】 메티오닐 티알엔에이 합성효소 (methionyl-tRNA synthetase , MRS) 단백질의 발현 수준을 측정하는 제제 및 선방세포 (acinar cel l ) 특이적 마커 단백질의 발현 수준을 측정하는 제제를 포함하는 췌장암 진단용 조성물.  The present invention relates to a method of measuring the expression level of methionyl-tRNA synthetase (MRS) protein and a method of measuring the expression level of acinar cell specific marker protein, Diagnostic composition.
【청구항 2】 거 U항에 . 있어서， 상기 선방세포 특이적 마커 단백질은 키모트립신 (Chymotrypsin) , 포스포리파아제 A2 그룹 IB(PLA2G1B, Phosphol ipase A2 group IB) 및 아밀라아제 A2(amylase 2A)로 이루어지는 군에서 선택되는 하나 이상의 것임올 특징으로 하는 췌장암 진단용 조성물. 2. Description of the Related Art Wherein the secretory cell specific marker protein is at least one selected from the group consisting of chymotrypsin, phospholipase A2 group IB (PLA2G1B, phospholipase A2 group IB) and amylase A2 (amylase 2A). / RTI &gt;
【청구항 3] 제 1항에 있어서, 상기 MRS 단백질은 서열번호 1로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 췌장암 진단용 조성물. [Claim 3] The composition for diagnosing pancreatic cancer according to claim 1, wherein the MRS protein comprises the amino acid sequence of SEQ ID NO: 1.
【청구항 4] 제 1항에 있어서， 상기 제제는 MRS 단백질 또는 선방세포 특이적 마커 단백질 각각에 대하여 특이적으로 결합하는 항체 또는 엘타머안 것을 특징으로 하는 췌장암 진단용 조성물. 4. The composition for diagnosing pancreatic cancer according to claim 1, wherein the agent is an antibody or an elastomer that specifically binds to each of the MRS protein or the secretory cell-specific marker protein.
【청구항 5】 제 4항에 있어서， 상기 MRS 단백질에 특이적으로 결합하는 항체는 MRS에서 서열번호 2로 표시되는 아미노산 서열을 포함하는 영역의 에피토프 (epi tope)에 특이적으로 결합하는 것을 특징으로 하는 항체 또는 이의 기능적 단편인 것을 특징으로 하는 조성물. 5. The method of claim 4, wherein the antibody specifically binding to the MRS protein is specifically bound to an epi tope of a region including the amino acid sequence represented by SEQ ID NO: 2 in MRS Lt; / RTI &gt; or a functional fragment thereof.
【청구항 6】 제 5항에 있어서, 상기 항체는 서열번호 28로 표시되는 아미노산서열을 포함하는 경쇄가변영역 (VL) ; 및 서열번호 30으로 표시되는 아미노산 서열을 포함하는 중쇄가변영역 (VH)을 포함하거나; 또는 서열번호 32로 표시되는 아미노산서열을 포함하는 경쇄가변영역 (VL) ; 및 서열번호 34로 표시되는 아미노산 서열을 포함하는 중쇄가변영역 (VH)을 포함하는 것을 특징으로 하는 조성물. [Claim 6] 6. The antibody of claim 5, wherein the antibody comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28; And a heavy chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 30; Or a light chain variable region (VL) comprising an amino acid sequence represented by SEQ ID NO: 32; And a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 34.
【청구항 7] 제 1항에 있어서， 상기 제제는 MRS 단백질 또는 선방세포 특이적 마커 단백질을 코딩하는 각각의 mRNA에 특이적으로 결합하는 프라이머 또는 프로브인 것을 특징으로 하는 조성물. 7. The composition according to claim 1, wherein the agent is a primer or a probe that specifically binds to each mRNA encoding the MRS protein or the secretory cell-specific marker protein.
【청구항 8】 메티오닐 티알엔에이 합성효소 단백질의 발현 수준을 측정하는 선방세포 특이적 마커 단백질의 발현 수준을 측정하는 제제를 포함하■ 진단용 키트. 8. The methionyl tial RNA synthetase, including agent measuring the expression level of the prior-cell specific protein markers for measuring the expression level of protein ■ diagnostic kit.
【청구항 9】 [Claim 9]
(a) 잠재 환자로부터 채취한 '췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (a) measuring the level of expression of acinar cell specific marker protein, and MRS proteins from "pancreatic samples collected from a potential patient; And
(b) 상기 (a) 단계의 측정 시료에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현되면 췌장암 세포인 것으로 판단하는 단계를 포함하는, 췌장암 진단 방법 . (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.
【청구항 10】 제 9항에 있어서, 상기 선방세포 특이적 마커 단백질은 키모트립신 (Chymotrypsin) , 포스포리파아제 A2 그룹 IB(PLA2G1B, Phosphol i ase A2 group IB) 및 아밀라아제 A2(amylase 2A)로 이루어지는 군에서 선택되는 하나 이상의 것임을 특징으로 하는 방법. 10. The method of claim 9, wherein the precursor cell-specific marker protein is Wherein the chymotrypsin is at least one selected from the group consisting of chymotrypsin, phospholipase A2 group IB (PLA2G1B, phospholipase A2 group IB) and amylase A2 (amylase 2A).
【청구항 11】 겨 19항에 있어서, 상기 방법은 11. The method of claim 9,
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계에서 측정한 메티오닐 티알엔에이 합성효소 수준을 음성 대조군과 비교하고, 음성 대조군과 비교하여 메티오닐 티알엔에이 합성효소 수준이 발현 증가되었고 선방'세포 특이적 마커 단백질이 발현되지 않았으면 췌장암 세포인 것으로판단하는 단계를 포함하는 방법 . (b) comparing a methicillin A synthetase levels O'Neill tial yen measured in the step (a) and the negative control group, as compared with the negative control methionyl tial RNA synthetase levels were increased expression prior "cell-specific marker proteins Is not expressed, it is determined to be a pancreatic cancer cell.
【청구항 12] 겨) 9항 내지 제 11항 중 어느 한 항에 있어서, 상기 시료는 췌장 세포인 특징으로 하는 방법 . 12. The method according to any one of claims 9 to 11, wherein the sample is a pancreatic cell.
【청구항 13] 제 12항에 있어서， 상기 췌장 세포는 미세바늘흡입법 (Fine needle aspirat ion)으로 분리하는 것을 특징으로 하는 방법. 13. The method of claim 12, wherein the pancreatic cells are separated by a fine needle aspiration method.
【청구항 14] 거 19항 또는 제 11항에 있어서， 상기 방법은 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하기 이전에， 동시에 또는 이후에 하기 단계를 추가적으로 포함하는 것을 특징으로 하는 방법 : 14. The method according to claim 19 or 11, characterized in that the method further comprises the step of simultaneously or successively measuring the expression level of the precursor cell-specific marker protein and the MRS protein :
( i ) 잠재 환자로부터 채취한 췌장 세포를 세포핵을 염색하는 DAPK4' ,6-diamidino-2-phenyl indole) , 메틸렌블루 (methylene blue) , 아세트산카민 (acetocarmine)， 를루이딘블루 (toluidine blue), 헤마톡실린 (hematoxylin) 및 훽스트 (Hoechst)로 이루어진 군에서 선택된 하나 이상의 염색용액， 및 세포질을 염색하는 에오신 (eosin), 크리스탈바이올렛 (crystal violet) 및 오렌지 G(orange G)로 이루어진 군에서 선택된 하나 이상의 염색 용액으로 세포 염색하는 단계; 및 (i) DAPK4 ', 6-diamidino-2-phenyl indole, methylene blue, acetocarmine, and toluidine to stain pancreatic cells from potential patients blue violet, blue, hematoxylin and Hoechst, and eosin, crystal violet and orange G staining cytoplasm. &Lt; / RTI &gt; staining the cell with at least one staining solution selected from; And
(ii) 상기 세포염색에 의해 췌장 세포를 악성종양세포， 비정형세포 (atypical cell) 또는 정상세포로 판단하는 단계. (ii) determining pancreatic cells as malignant tumor cells, atypical cells or normal cells by staining the cells.
【청구항 15】 제 14항에 있어서， 상기 (ii) 단계는 상기 (i) 단계의 세포 염색결과로부터 세포가 3차원으로 도말됨; 세포핵 /세포질 비율 (N/c ratio)이 높음; 염색질의 뭉침 현상 출현; 거친 모양의 핵막; 핵소체의 출현; 및 유사분열의 출현으로 이루어지는 군에서 선택된 두 가지 이상의 형태학적 이상을 보이는 경우에 악성 종양세포로 판정하며， 세포가 한 겹으로 도말되어 있으며 세포핵 /세포질 비율 (N/C ratio)이 작고 핵막이 매끄러운 모양일 경우에는 정상 세포로 판정하고， 세포의 변화가 악성 종양세포에는 미치지 못하나 정상으로 판정할 수 없는 경우 비정형 세포 (atypical cell)로 판정 하는 것을 특징으로 하는 방법 15. The method of claim 14, wherein step (ii) comprises: (i) laminating cells three-dimensionally from the cell staining result of step (i); High nuclear / cytoplasm ratio (N / c ratio); Appearance of chromatin aggregation; Rough-shaped nucleus; Emergence of nuclear bodies; And the appearance of mitosis, it is judged to be a malignant tumor cell. When the cells are laminated in one layer, the nucleus / cytoplasm ratio (N / C ratio) is small and the nuclear membrane is smooth Shaped cell is judged to be a normal cell, and when the change of the cell does not reach the malignant tumor cell but can not be judged as normal, the cell is judged to be an atypical cell
【청구항 16] 제 9항 또는 제 11항에 있어서， 상기 단백질 발현 수준을 측정하는 방법은 웨스턴 블랏， 효소면역분석법 (ELISA), 방사선면역분석， 방사선 면역 확산법, 오우크테로니 면역 확산법， 로케트면역전기영동, 면역염색법， 면역침전 분석법， 보체 고정 분석법， FACS(Fluorescence activated cell sorter) , SPR( surface plasmon resonance) 또는 단백질 칩 방법 중 어느 하나를 이용하는 것을 특징으로 하는 방법 . 16. The method according to claim 9 or 11, wherein the protein expression level is measured by Western blotting, enzyme immunoassay (ELISA), radioimmunoassay, radioimmunoassay, Ouchteroni immunodiffusion, Wherein the method comprises using any one of electrophoresis, immuno-staining, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), surface plasmon resonance (SPR) or protein chip methods.
【청구항 17] 췌장암에 대한 세포진 (cytodiagnosis) 검사 또는 조직 검사에 있어서, 하기 단계를 포함하는 것을 특징으로 하는 민감도 또는 특이도를 향상시키는 방법: 17. A method for improving sensitivity or specificity in cytodiagnosis or histology of pancreatic cancer comprising the steps of:
(a) 잠재 환자로부터 채취한 췌장 시료에서 선방세포 특이적 마커 단백질 및 MRS 단백질의 발현수준을 측정하는 단계; 및 (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) 상기 (a) 단계에서 선방세포 특이적 마커 단백질이 발현되지 않고 MRS 단백질이 발현 증가되었으면 췌장암 세포인 것으로 판단하는 단계. (b) determining that the precursor cell-specific marker protein is not expressed in the step (a) and that the expression of the MRS protein is increased, the cell is a pancreatic cancer cell.
【청구항 18] 제 17항에 있어서, 상기 방법은 민감도가 80% 이상인 것을 특징으로 하는 방법. 18. The method of claim 17, wherein the method has a sensitivity of at least 80%.
【청구항 19] 췌장암의 진단용 제제를 제조하기 위한 MRS 단백질 및 선방세포 특이적 마커 단백질의 발현 수준을 측정하는 제제의 용도. 19. Use of an agent for measuring the expression level of an MRS protein and a secretory cell-specific marker protein for producing a diagnostic agent for pancreatic cancer.
PCT/KR2018/010366 2017-09-05 2018-09-05 Method for diagnosing pancreatic cancer using methionyl-trna synthetase and acinar cell-specific marker WO2019050273A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2017-0113372 2017-09-05
KR20170113372 2017-09-05

Publications (1)

Publication Number Publication Date
WO2019050273A1 true WO2019050273A1 (en) 2019-03-14

Family

ID=65635014

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/010366 WO2019050273A1 (en) 2017-09-05 2018-09-05 Method for diagnosing pancreatic cancer using methionyl-trna synthetase and acinar cell-specific marker

Country Status (2)

Country Link
KR (1) KR102111681B1 (en)
WO (1) WO2019050273A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4067376A1 (en) * 2021-03-30 2022-10-05 Diaccurate Anti-pla2g1b monoclonal antibodies and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011072265A1 (en) * 2009-12-11 2011-06-16 Atyr Pharma, Inc. Aminoacyl trna synthetases for modulating 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 (en) * 2015-11-13 2017-08-24 재단법인 의약바이오컨버젼스연구단 Methods for screening anti-cancer agents inhibiting interactions between MRS and CDK4

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011072265A1 (en) * 2009-12-11 2011-06-16 Atyr Pharma, Inc. Aminoacyl trna synthetases for modulating 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 (en) * 2015-11-13 2017-08-24 재단법인 의약바이오컨버젼스연구단 Methods for screening anti-cancer agents inhibiting interactions between MRS and CDK4

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 (en) * 2021-03-30 2022-10-05 Diaccurate Anti-pla2g1b monoclonal antibodies and uses thereof
WO2022207674A1 (en) * 2021-03-30 2022-10-06 Diaccurate Anti-pla2g1b monoclonal antibodies and uses thereof

Also Published As

Publication number Publication date
KR102111681B1 (en) 2020-05-15
KR20190026632A (en) 2019-03-13

Similar Documents

Publication Publication Date Title
JP6666905B2 (en) PD-L1 antibody and use thereof
IL223874A (en) Antibody for the diagnosis and/or prognosis of cancer
JP6940505B2 (en) Composition and method for assessing the risk of developing cancer
US20170146541A1 (en) Detection of high-risk intraductal papillary mucinous neoplasm and pancreatic adenocarcinoma
US11561222B2 (en) Method for diagnosis of bile duct cancer using methionyl-tRNA synthetase in bile duct cell
KR102111699B1 (en) Method for diagnosing pancreatic cancer using methionyl-tRNA synthetase, and kit for diagnosing pancreatic cancer using thereof
WO2019050273A1 (en) Method for diagnosing pancreatic cancer using methionyl-trna synthetase and acinar cell-specific marker
EP2507628A1 (en) Improved methods and compositions for detecting and treating cea-expressing cancers
JP2021524576A (en) Compositions and Methods for Diagnosis and Treatment of Cancer
JP6959684B2 (en) Monoclonal antibody that specifically binds to MRS
KR20210020417A (en) Method for diagnosing lung cancer using methionyl-tRNA synthetase and pan-cytokeratin
WO2017053535A1 (en) Methods and diagnostics for cancer detection and treatment monitoring
JP2018516876A (en) IGF-1R antibody and its use for diagnosis of cancer
EA043033B1 (en) METHOD FOR CANCER RISK ASSESSMENT, CORRESPONDING SET, DATA STORAGE UNIT AND PROCESSING SYSTEM

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18853963

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18853963

Country of ref document: EP

Kind code of ref document: A1