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WO2011046309A2 - Marqueur de diagnostic pour carcinome hépatocellulaire comprenant des auto-anticorps anti-fasn et composition de diagnostic pour carcinome hépatocellulaire comprenant des antigènes de celui-ci - Google Patents

Marqueur de diagnostic pour carcinome hépatocellulaire comprenant des auto-anticorps anti-fasn et composition de diagnostic pour carcinome hépatocellulaire comprenant des antigènes de celui-ci Download PDF

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WO2011046309A2
WO2011046309A2 PCT/KR2010/006727 KR2010006727W WO2011046309A2 WO 2011046309 A2 WO2011046309 A2 WO 2011046309A2 KR 2010006727 W KR2010006727 W KR 2010006727W WO 2011046309 A2 WO2011046309 A2 WO 2011046309A2
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Prior art keywords
autoantibody
hepatocellular carcinoma
antigen
fasn
antibody
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WO2011046309A3 (fr
Inventor
Eun-Wie Cho
Chang-Kyu Heo
Mi-Kyung Woo
Hai Min Hwang
Hyang-Sook Yoo
Jeong-Heun Ko
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Priority claimed from KR1020100018205A external-priority patent/KR101138460B1/ko
Application filed by Korea Research Institute of Bioscience and Biotechnology KRIBB filed Critical Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority to US13/501,426 priority Critical patent/US8658769B2/en
Publication of WO2011046309A2 publication Critical patent/WO2011046309A2/fr
Publication of WO2011046309A3 publication Critical patent/WO2011046309A3/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • the present invention relates to a diagnostic method for hepatocellular carcinoma comprising an agent capable of assessing the expression level of anti-FASN autoantibody, more particularly, to a diagnostic composition for hepatocellular carcinoma comprising an agent capable of assessing the expression level of the autoantibody, a hybridoma cell line producing the autoantibody, a diagnostic kit for hepatocellular carcinoma comprising the composition, a method for detecting the autoantibody of hepatocellular carcinoma patient using the composition, and a method for screening a therapeutic agent for hepatocellular carcinoma by administering candidate materials for hepatocellular carcinoma treatment to confirm a reduction in the expression level of the autoantibody.
  • Liver diseases including hepatitis, liver cirrhosis, hepatocellular carcinoma or the like are the most common diseases in Korea, Japan, Taiwan, China, and most of Southeast Asian countries.
  • hepatocellular carcinoma is the fourth leading cause of death worldwide (five hundred and five thousand people) (World Health Organization, 1997).
  • World Health Organization 1997
  • the incidence of hepatocellular carcinoma ranks third (11.5%) among causes of cancer (Cancer Incidence in Korea, 2002).
  • AFP hepatocellular carcinoma
  • biomarkers have been suggested for the diagnosis, prognosis, or evaluation of treatment efficacy.
  • AFP and PIVKA-II are the most well-known biomarkers.
  • there is still a weak point in their specificity and sensitivity With recent advances in genomics and proteomics, several candidate proteins and genes as hepatocellular carcinoma markers have been reported. However, the reported genes are mainly used to target the tissue, and there is still no evidence for their secretion to the blood and feasibility of using as serological diagnostic markers.
  • tumor markers have been discovered from the blood, tissue, or discharge, but many tumor markers are detected or their expression increased even though cancer development is not observed. Therefore, these markers used for cancer diagnosis are only incidental tools and have not become independent diagnostic tools.
  • an individual has an immune system which is unique in its ability to distinguish between self and non-self molecules, whereby antigen-antibody reaction (humoral immune response) and cellular immune response are normally induced in response to only foreign antigens exposed to the immune system.
  • antigen-antibody reaction humidity immune response
  • cellular immune response cellular immune response
  • production of antibodies against self-antigens is observed in certain diseases.
  • localization of antigen expression is different from that in normal cells, leading to secretion of the intracellular proteins from the cell, or the antigens undergo a conformational change or other abnormal properties are manifested.
  • tumor-associated antigens HER-2/neu oncoprotein was reported to be a receptor protein located on the cell membrane and induce autoantibodies.
  • a tumor suppressor protein p53 was also reported to induce autoantibodies.
  • cell proliferation-associated proteins, cyclin B1 and CENP-F (centromere protein F), and onconeurological proteins, Hu and Yo were also known to induce autoantibodies.
  • SEREX selological analysis of recombinant cDNA expression libraries of human tumors with autologous serum
  • PTM posttranslational modifications
  • proteomic technologies tumor-derived proteins are separated in 2D-PAGE, protein spots are visualized showing a reactivity to the blood plasma of cancer patients as an autoantibody sample , and then the proteins are identified by mass spectrometry.
  • This method is also called SERPA (serological proteome analysis).
  • MAPPing Multiple affinity protein profiling
  • a protein chip is manufactured by separation of tumor cell lysate into several thousand fractions, and then the reactivity of a patient's blood thereto is analyzed to detect autoantibodies.
  • proteomic technologies have the advantage of directly analyzing the antibody reactivity to tumor cell-derived proteins retaining PTM properties, thereby detecting various autoantibodies, which could not be detected by SEREX.
  • these proteomic technologies also have drawbacks.
  • the serum of a patient is a mixture of numerous autoantibodies, and thus the analytical range is determined by differences in their quantity and affinity to antigens, resulting in the failure of analysis of the desired autoantibody.
  • Another problem is that patient-dependency on an autoantibody to be analyzed impairs a systematic analysis on the production of autoantibody in cancer development. In addition, it is very hard to collect an excessive amount of blood from a patient, and therefore, further studies cannot be conducted.
  • the other problem is the conservation of the epitope recognized by an antibody.
  • the epitope of an antibody can be divided into two types: a protein sequence-dependent epitope (sequential epitope) and a protein structure-dependent epitope (conformational epitope).
  • a protein sequence-dependent epitope sequence-dependent epitope
  • a protein structure-dependent epitope formational epitope
  • 2D electrophoresis is performed for analysis of the protein mixed solution, in which proteins to be analyzed are denatured using SDS and urea, and the linearized proteins are reacted with antibodies.
  • the epitope is a sequential epitope, the antibody-antigen reaction can be detected, but if the epitope is a conformational epitope, the antibody-antigen reaction cannot be detected.
  • the present inventors have developed an effective identification method for autoantibodies, and they investigated an autoantibody that is significantly increased in hepatocellular carcinoma by using the method, completing the present invention.
  • FASN fatty acid synthase
  • hepatocellular carcinoma can be diagnosed with a high specificity and sensitivity using a non-invasive biological sample such as blood, blood plasma, serum, and lymphatic fluid, without performing invasive diagnosis such as tissue biopsy.
  • a non-invasive biological sample such as blood, blood plasma, serum, and lymphatic fluid
  • hepatocellular carcinoma can be easily diagnosed using the identified amino acid sequence only, without need of designing reactive materials to identify the marker, thereby being effective for the development of a diagnostic kit for hepatocellular carcinoma.
  • FIG. 1 is an overview of the method of obtaining hepatocellular carcinoma-associated autoantibody from hepatocellular carcinoma mouse model.
  • H-ras12V transgenic HCC mouse model (characterized by the occurrence of hepatocellular carcinoma at 8-10 months of age) was acquired, and splenocytes from H-ras12V transgenic mice at 10 months or older were fused with the mouse myeloma cells Sp2/0, and selection of B-cell hybridomas producing HCC-associated autoantibodies was performed.
  • FIG. 2 is the result of analyzing the reactivity of autoantibodies against hepatocellular carcinoma cells, in which the autoantibodies are produced by B cell hybridoma clones derived from H-ras12V transgenic HCC mouse model.
  • the hepatocellular carcinoma cell line HepG2 was fixed with paraformaldehyde and permeabilized with a permeabilization reagent, and then treated with autoantibodies. After treatment with a primary antibody, the residual antibodies were washed out, and the cells were treated with a fluorescent-labeled secondary antibody, followed by flow cytometric analysis.
  • B-cell hybridoma clones being highly reactive to HepG2 cells were observed in F or M mouse showing a high development of hepatocellular carcinoma, whereas fewer B-cell hybridoma clones and lower reactivity of autoantibodies produced therefrom were observed in B or D mouse which did not develop hepatocellular carcinoma despite being transgenic.
  • FIG. 3 is the result of analyzing a monoclonal antibody K1, which is produced from a K1 clone that is an autoantibody-producing B cell derived from K mouse among H-ras12V HCC mouse models.
  • FIG. 3a is the result of selecting ten antibodies, which are highly reactive to HCC cell line, from the B-cell hybridoma clones from K mouse. For selection, the human HCC cell line HepG2 and mouse HCC cell line Hepa-1c1c7 were subjected to intracellular staining, followed by flow cytometric analysis. Thereafter, the highly reactive K1 antibody was first analyzed.
  • FIG. 3 is the result of analyzing a monoclonal antibody K1, which is produced from a K1 clone that is an autoantibody-producing B cell derived from K mouse among H-ras12V HCC mouse models.
  • FIG. 3a is the result of selecting ten antibodies, which are highly reactive to HCC cell line, from the B-cell hybridoma
  • FIG. 3B is the result of analyzing the reactivity of the K1 antibody to other cancer cells, in which it was highly reactive to most of the cancer cell lines.
  • FIG. 3c is the result of Western blotting to detect the target antigen of the K1 autoantibody. Total cell lysates of various cancer cell lines were separated on 8-10% SDS-PAGE gel, followed by Western blotting and immunostaining with K1 autoantibody. The target antigen of K1 antibody was detected as a protein of a high molecular weight (>200 KD: indicated by an arrow).
  • FIG. 3d is the result of immunohistochemical staining to examine the intracellular localization of K1 autoantigen. Its expression was localized mainly in the cytoplasm of three liver cell lines (Hepa-1c1c7, Hep3B, Chang), and localized mainly in the membrane of HepG2 cells.
  • FIG. 4 is the result of panning of the phages against K1 autoantibody to define the epitope sequence of K1 autoantibody using the phage peptide library.
  • FIG. 4a After four rounds of panning (FIG. 4a), five phages with different insert peptide sequences were selected (the sequences are represented in FIG. 5), and their reactivity against K1 antibody was analyzed by ELISA. The K1-p7 phage showed the highest reactivity (FIG. 4b).
  • these epitopes have a cyclic form maintained by two cysteines, and to analyze the conformation-dependency of antibody binding, the cyclic form was reduced and the reactivity was compared (FIG. 4c).
  • FIG. 5 shows the peptide sequences of the cyclic epitopes, of which the reactivity against K1 antibody was analyzed.
  • FIG. 6 is the result of identification of K1 autoantigen.
  • FIG. 6a is the result of purifying the antigen protein using K1 antibody in order to identify K1 autoantigen.
  • the purified protein was treated with trypsin, and cleaved to peptides, followed by mass spectrometric analysis for protein identification.
  • the protein band corresponding to K1 autoantigen was identified as FASN (fatty acid synthase) (the result of sequence analysis is shown in FIG. 7).
  • FIG. 7 HepG2 cells were transfected with siRNA against FASN to suppress the expression of FASN, and the reactivity of K1 antibody was analyzed. As shown in FIG.
  • FIG. 6c shows the expression of FASN in the liver tissue of H-ras12V HCC mouse model. FASN expression was remarkably increased in the HCC tissue (R7-1, R7-2), compared to the normal liver tissue of a 7 month-old mouse (W7-1, W7-2).
  • FIG. 7 is the result of showing that K1 antigen identified by mass spectrometric analysis in FIG. 6a is FASN.
  • FIG. 8 is the result of detecting autoantibody in the sera from HCC patient and healthy person by ELISA using a mimotope against K1 autoantibody, K1-p7 phage as a coating antigen. As a result, it was found that the sensitivity of this ELISA was 96.55% and specificity was 100% when the cutoff value was 0.114.
  • FIG. 9 is the result of 1% agarose gel electrophoresis of variable regions of heavy and light chains (V H , V L ) obtained by RT-PCR, in order to determine the base sequence of the antigen biding site of K1 monoclonal antibody.
  • the amplified DNA was cloned into a pCR2.1TOPO vector, and transformed into E coli DH5a, followed by DNA extraction and sequence analysis.
  • FIG. 10 is the result of sequence analysis of variable region of heavy chain (V H ) of K1 antibody.
  • V H variable region of heavy chain
  • the sequence of 381 bases from the 5'-end were analyzed, and the sequence of amino acid expressed therefrom was marked.
  • CDR Complementarity Determining Region
  • Kabat sequence database Sequences of Proteins of Immunological Interest , 5th Ed.,(1991) U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD).
  • FIG. 11 is the result of sequence analysis of variable region of light chain (V L ) of K1 antibody.
  • V L variable region of light chain
  • the present invention relates to an autoantibody recognizing FASN (fatty acid synthase) or a fragment comprising an antigen-binding site thereof.
  • FASN fatty acid synthase
  • autoantibody refers to an antibody formed in response to one of the individual's own body constituents, and is also called “self antibody”.
  • individuals do not generate an immune response to their own body constituents, and thus do not produce antibodies against them.
  • individuals recognize their own body constituents as antigens to produce antibodies, which causes autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. In other cases, they may produce antibodies against tumor-associated antigens (TAA).
  • TAA tumor-associated antigens
  • anti-FASN autoantibody means an autoantibody induced against FASN in patients with hepatocellular carcinoma.
  • the present inventors identified autoantibody-expressing B cells from patients with hepatocellular carcinoma, and confirmed expression of a specific autoantibody having a high reactivity with cancer cells, the corresponding antibody is designated as K1 antibody (hereinbelow, referred to as 'anti-FASN antibody', 'K1 antibody' 'K1 autoantibody' or 'autoantibody K1'). Thereafter, they identified an autoimmune antigen for the corresponding antibody, and demonstrated that the autoantibody is an autoantibody against FASN (anti-FASN autoantibody).
  • the present inventors examined binding sites of the autoantibody in order to identify its specific epitope sequence. As a result, it was confirmed that the autoantibody specifically binds with one or more sequences selected from the group consisting of RMSRRSN (Arg-Met-Ser-Arg-Arg-Ser-Asn; SEQ ID NO. 1), RRRLNRT (Arg-Arg-Arg-Leu-Asn-Arg-Thr; SEQ ID NO. 2), RMRIRRN (Arg-Met-Arg-Ile-Arg-Arg-Asn; SEQ ID NO. 3), HPHPRPR (His-Pro-His-Pro-Arg-Pro-Arg; SEQ ID NO.
  • MRNRPKR Metal-Arg-Asn-Arg-Pro-Lys-Arg; SEQ ID NO. 5
  • the autoantibody specifically binds with MRNRPKR (Met-Arg-Asn-Arg-Pro-Lys-Arg).
  • one antibody molecule has two heavy chains and two light chains, and each heavy and light chain contains an N-terminal variable region.
  • Each variable region consists of three complementarity determining regions (CDR) and four framework regions (FRs), and the complementarity determining region exists as a relatively short peptide sequence, which determines the antigen-binding specificity of an antibody and is maintained by the framework regions of variable region.
  • the antibody of the present invention comprises a fragment including an essential sequence which is directly involved in the antigen FASN binding, and more preferably, a fragment directly involved in the binding with a mimotope which mimics the structure of FASN epitope.
  • the autoantibody of the present invention is a part of the variable region of a heavy chain, and may include an autoantibody consisting of the CDR1 sequence of SEQ ID NO. 8, the CDR2 sequence of SEQ ID NO. 9, and the CDR3 sequence of SEQ ID NO. 12, or a fragment including an antigen-binding site thereof.
  • the autoantibody of the present invention may be an autoantibody including all of the sequences of CDR1, CDR2 and CDR3 or a fragment thereof.
  • the autoantibody of the present invention is a part of the variable region of light chain, and may include an autoantibody consisting of the CDR1 sequence of SEQ ID NO. 14, the CDR2 sequence of SEQ ID NO. 16, and the CDR3 sequence of SEQ ID NO. 18, or a fragment including an antigen-binding site thereof.
  • a nucleic acid sequence encoding the sequence is also included in the present invention.
  • the autoantibody of the present invention is a sequence of the variable region of heavy chain, and may be an autoantibody consisting of an amino acid sequence of SEQ ID NO. 20 or a fragment including an antigen-binding site thereof.
  • the autoantibody of the present invention is a sequence of the variable region of light chain, and may be an autoantibody consisting of an amino acid sequence of SEQ ID NO. 22 or a fragment including an antigen-binding site thereof.
  • the heavy and light chains may be used alone or together depending on the purpose, and any combination of a plurality of CDR sequences and light chains and heavy chains can be made by a conventional genetic engineering method depending on the purpose of those skilled in the art.
  • the FASN fatty acid synthase
  • the FASN is an enzyme that functions to produce long-chain saturated fatty acids from acetyl-CoA and malonyl-CoA and is involved in energy metabolism, appetite regulation or the like.
  • the autoantibody of the present invention may be an intact form consisting of two full-length light chains and two full-length heavy chains as well as a functional fragment of the antibody molecule which is capable of achieving antibody-antigen binding.
  • the functional fragment of the antibody molecule means a fragment having an antigenic binding function, and its length or shape is not limited, exemplified by Fab, F(ab'), F(ab') 2 and Fv.
  • the functional fragment of the autoantibody of the present invention is not limited to these examples.
  • the anti-FASN autoantibodies or the fragment comprising an antigen-binding site thereof of the present invention is an antibody that specifically recognizes the amino acid sequence consisting of RMSRRSN, RRRLNRT, RMRIRRN, HPHPRPR or MRNRPKR, and preferably an antibody that specifically recognizes the amino acid region consisting of the MRNRPKR sequence.
  • the present invention relates to a diagnostic composition for hepatocellular carcinoma, comprising an agent capable of assessing the expression level of anti-FASN autoantibody.
  • diagnosis means confirmation of a pathological state or characteristic.
  • diagnosis is to confirm the development of hepatocellular carcinoma.
  • the anti-FASN autoantibody of the present invention is used as a diagnostic marker for hepatocellular carcinoma, the development of hepatocellular carcinoma is confirmed by assessing the expression level of the anti-FASN autoantibody of the present invention in the sample of a subject.
  • a diagnostic marker means a material capable of distinguishing hepatocellular carcinoma cells from normal cells, and may include an organic biomolecule such as a polypeptide, a nucleic acid (e.g., mRNA etc.), a lipid, a glycolipid, a glycoprotein, and a sugar (monosaccharide, disaccharide, oligosaccharide etc.), which is expressed at a higher or lower level, as compared to its level in normal cells.
  • a nucleic acid e.g., mRNA etc.
  • lipid e.g., a lipid, a glycolipid, a glycoprotein, and a sugar (monosaccharide, disaccharide, oligosaccharide etc.)
  • the diagnostic marker for hepatocellular carcinoma of the present invention is an anti-FASN autoantibody, which is highly expressed in the whole blood, blood, serum, or blood plasma of a subject with hepatocellular carcinoma, as compared to the whole blood, blood, serum, or blood plasma of a subject with normal liver.
  • hepatocellular carcinoma means a cancer derived from hepatic cells, and encompasses primary hepatocellular carcinoma that begins within the liver and metastatic hepatocellular carcinoma that spread to the liver from other sites.
  • the cause of hepatocellular carcinoma is unclear, but it has been revealed that most of hepatocellular carcinoma patients have liver cirrhosis, and hepatocellular carcinoma frequently occurs in patients with liver cirrhosis or chronic active hepatitis B, or hepatitis B carriers.
  • the present inventors confirmed that the development of hepatocellular carcinoma in a subject can be diagnosed using the autoantibody marker of the present invention with high sensitivity and specificity.
  • the term "agent capable of assessing the expression level of anti-FASN autoantibody” means a molecule used for the detection of markers by assessing the expression level of anti-FASN autoantibodies which is increased in the whole blood, serum and blood plasma, lymphatic fluid, and interstitial fluid of a subject, and preferably an antigen protein that specifically binds to the autoantibody.
  • the analysis method may include Western blotting, ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip, but are not limited thereto.
  • the diagnosis of hepatocellular carcinoma can be achieved by an antibody-antigen reaction between the anti-FASN autoantibody of the present invention and the specific antigen thereof, and the term "antigen-antibody complex", as used herein, refers to binding products of a hepatocellular carcinoma marker, autoantibody to the antigen specific thereto.
  • the amount of formed antigen-antibody complexes may be quantitatively determined by measuring the signal size of a detection label.
  • Such a detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive isotopes, but the present invention is not limited to the examples.
  • enzymes available as detection labels include, but are not limited to, ⁇ -glucuronidase, ⁇ -glucosidase, ⁇ -galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphenolpyruvate decarboxylase, and ⁇ -latamase.
  • fluorescent substances include, but are not limited to, fluorescin, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamin.
  • ligands include, but are not limited to, biotin derivatives.
  • luminescent substances include, but are not limited to, acridinium esters, luciferin and luciferase.
  • microparticles include, but are not limited to, colloidal gold and colored latex.
  • redox molecules examples include, but are not limited to, ferrocene, ruthenium complexes, viologen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, hydroquinone, K 4 W(CN) 8 , [Os(bpy) 3 ] 2+ , [RU(bpy) 3 ] 2+ , and [MO(CN) 8 ] 4- .
  • radioactive isotopes include, but are not limited to, 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re.
  • peptide library refers to collections of several hundred to several thousand peptides, of which amino acid sequences are artificially changed by combinatorial chemistry.
  • the peptide library technique is used to develop potential drug candidates having biological activity by exploring peptides having amino acid sequences of useful functions using the peptide library, represented by PS-SPCL (Positional-Scanning Synthetic Peptide Combinatorial Library) and OBOPL (One-Bead-One-Peptide Library).
  • PS-SPCL Pural-Scanning Synthetic Peptide Combinatorial Library
  • OBOPL One-Bead-One-Peptide Library
  • the type of peptide library available in the present invention is not limited to the examples.
  • PS-SPCL is a mixture made of fixing 19 amino acid except cysteine and connecting 19 amino acid mixture to the rest of the region.
  • PS-SPCL is a method of exploring 47 million different peptides by screening 114 pools.
  • OBOPL is a tool of attaching a specific sequence (e.g., pentapeptide) to a resin having polystyrene as a substrate and polyethylene glycol (PEG) as a linker so as to explore the attached sequence.
  • a method of identifying the epitope sequence for the autoantibody of the present invention includes the above described identification methods, and any method of identifying epitopes available in the art can be used without limitation.
  • the present inventors had employed a cyclic peptide phage display library system which has a cyclic structure formed by 7 amino acids. Preferably, they selected phages that specifically bind with anti-FASN antibody. From the selected phages, only the groups showing a high reactivity with anti-FASN antibody of the present invention were purified, and then treated with a coating antigen. Subsequently, the reactivity with immune antibodies binding to the antigen was confirmed to identify amino acid sequences thereof.
  • the present inventors investigated anti-FASN antibody as a diagnostic marker for hepatocellular carcinoma by the following demonstration.
  • spleen cells were obtained from a hepatocellular carcinoma mouse model as parent cells, and fused with myeloma cells to produce B cell hybridomas.
  • the antibodies secreted from the produced hybridomas the antibodies showing reactivity with hepatocellular carcinoma cells were selected to isolate autoantibodies available as a marker. Subsequently, the presence of antigens being reactive with the isolated autoantibodies was examined (identification of macromolecular protein of 200 KD or more).
  • the isolated autoantibody K1 was purified in a large amount, and affinity chromatography was performed to identify the antigen protein, and a phage display-peptide library consisting of 7 amino acids was used to identify the epitope sequence binding with the purified autoantibody, thereby confirming the specific epitope sequence. Accordingly, it was demonstrated that the specific antigen protein is FASN, leading to the investigating of a sequence of the structural mimic of the epitope that binds with the autoantibody of the present invention.
  • the autoantigen may have a new antigenicity by post translational modification (PTM).
  • PTM post translational modification
  • autoantigens are expressed in normal cells.
  • the neo-antigenic site may have a PTM property upon the production of autoantibodies.
  • recombinant proteins of the identified autoantigens are prepared without analysis and reproduction of PTM to perform the detection method, it is impossible to detect the neo-antigenic site having a PTM property.
  • the present inventors determined the epitope using a phage peptide library.
  • a phage peptide library In view of the fact that the antigenicity of autoantigens is restricted to one or two epitopes of a target protein, they assumed that presentation of single epitopes identified from the peptide library is sufficient for the detection of autoantibody. Consequently, an experiment was performed to identify the autoantibody recognizing the epitope identified from the peptide library.
  • a structure having the highest reactivity with the antibody can be selected by the determination of epitopes with the peptide library, and therefore, a protein sequence of the antigen itself and a modified structure after post-transcriptional modification can be also identified.
  • the selected antigen peptide-expressing phages can be readily proliferated, it is very useful for the development of diagnostic methods such as design of proteins chips.
  • the present inventors identified the epitopes from the peptide library, leading to identification of the specific epitope sequence which binds with the autoantibody of the present invention. They also confirmed that the identified amino acid fragment can be used in a diagnostic kit for hepatocellular carcinoma.
  • the present invention relates to a hybridoma cell line producing an anti-FASN autoantibody.
  • hybridoma cell means a cell prepared by artificial fusion of two different types of cells.
  • a hybridoma cell line is prepared by fusion of two or more of homogeneous or heterogeneous cells using a material such as polyethylene glycol or a specific species of virus, whereby different functions of the different cells are integrated into one cell.
  • These hybridoma cells can be prepared by fusion of cancer cells proliferated in vitro and any differentiated large cells extracted from the living body.
  • the hybridoma may be represented by lymphocyte hybridomas, including a hybridoma cell generated by fusion of myeloma cell and B cell, a hybridoma cell generated by fusion of T cell and tumor cell thereof, and a hybridoma cell generated by fusion of lymphokine (biologically active material)-producing T cell and tumor cell thereof.
  • lymphokine biologically active material
  • the fused cells were cultured, and then a B lymphocyte hybridoma producing hepatocellular carcinoma cell-reactive antibody was only selected, which was designated as TAB-K1.
  • TAB-K1 a B lymphocyte hybridoma producing hepatocellular carcinoma cell-reactive antibody was only selected, which was designated as TAB-K1.
  • the hybridoma was deposited at the Biological Resource Center in the Korea Research Institute of Bioscience and Biotechnology on Jan. 5, 2010 under the Accession No. KCTC 11612BP.
  • the present invention relates to a diagnostic kit for hepatocellular carcinoma, comprising an antigen which specifically binds to anti-FASN autoantibody.
  • the antigen which specifically binds to anti-FASN autoantibody (or polypeptide which specifically binds to the autoantibody) of the present invention encompasses all of the proteins capable of specifically binding to the autoantibody, but is not limited to particular proteins or polypeptides.
  • the antigen may include all fragments thereof or variants thereof as long as they can be recognized by the anti-FASN autoantibody.
  • the antigen may consist of at least 7 amino acids, preferably 9 amino acids, and more preferably 12 amino acids or more.
  • the antigen may be an epitope sequence which can be recognized by the autoantibody marker of the present invention.
  • the size or type of the epitope sequence is not limited, as long as it can be recognized by the autoantibody of the present invention.
  • the epitope sequence may be a polypeptide sequence consisting of 7 amino acids.
  • the sequence consisting of 7 amino acids may be a sequence including any one or more polypeptides selected from the group consisting of RMSRRSN (Arg-Met-Ser-Arg-Arg-Ser-Asn), RRRLNRT (Arg-Arg-Arg-Leu-Asn-Arg-Thr), RMRIRRN (Arg-Met-Arg-Ile-Arg-Arg-Asn), HPHPRPR(His-Pro-His-Pro-Arg-Pro-Arg) and MRNRPKR (Met-Arg-Asn-Arg-Pro-Lys-Arg), but the type of the sequence to be recognized by the autoantibody of the present invention is not limited to these examples.
  • the present inventors Using a 7 peptide phage library, the present inventors identified a sequence that specifically recognizes the autoantibody of the present invention. As a result, it was found that the autoantibody of the present invention shows particularly high reactivity to the sequence of MRNRPKR (Met-Arg-Asn-Arg-Pro-Lys-Arg).
  • the present invention relates to a method for detecting anti-FASN autoantibody in the sample, taking from a hepatocellular carcinoma patient, using the diagnostic composition for hepatocellular carcinoma.
  • the detection method of anti-FASN autoantibody of the present invention may comprise the steps of (a) assessing the expression level of anti-FASN autoantibody in the sample of a subject suspected of having hepatocellular carcinoma; and (b) comparing the expression level of anti-FASN autoantibody with that of a normal control group.
  • sample includes, but is not limited to, samples displaying a difference in expression levels of the hepatocellular carcinoma marker anti-FASN autoantibody, such as whole blood, serum, blood, blood plasma, saliva, urine, sputum, lymphatic fluid, cerebrospinal fluid, and interstitial fluid.
  • the method for assessing the expression level of the autoantibody of the present invention may include Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip, but is not limited thereto.
  • the detection method of the anti-FASN autoantibody of the present invention may be performed by comparing the expression level of autoantibody in a subject suspected of having hepatocellular carcinoma with that in a healthy subject. If the subject suspected of having hepatocellular carcinoma displays a higher level of the autoantibody than the normal control group, the corresponding subject can be diagnosed as a patient with hepatocellular carcinoma.
  • any one or more selected from the above methods may be employed, and if necessary, hepatocellular carcinoma can be diagnosed by the two steps of a primary test and a confirmatory test.
  • an allogenic sera screening method can be used as the primary test and Western analysis can be used as the confirmatory test to distinguish consistently between positive and false positive reactions.
  • the detection or diagnosis method of the present invention shows a high consistency of results, even though any of the above methods is adopted to analyze the samples obtained from an identical subject. Therefore, an accurate detection of anti-FASN autoantibody can be achieved, and the development of hepatocellular carcinoma can be diagnosed.
  • the allogenic sera screening method can be performed as follows.
  • the proteins are transferred to a membrane from a microorganism capable of expressing an amino acid sequence recognized by the autoantibody of the present invention. Subsequently, the membrane is blocked, and reacted with a biological sample such as blood, blood plasma, and serum obtained from a subject suspected of having hepatocellular carcinoma, and the binding between antigens immobilized on the membrane and the secondary antibody of the autoantibody of the present invention is confirmed by a typical method, thereby performing a screening.
  • a biological sample such as blood, blood plasma, and serum obtained from a subject suspected of having hepatocellular carcinoma
  • the 'microorganism capable of expressing an amino acid sequence' used in the above step may be a bacteria transformed with an expression vector having a gene encoding the antigen for anti-FASN autoantibody or a bacteria transformed with a general expression vector, and preferably, the antigen includes FASN or a fragment thereof that can be recognized by the autoantibody of the present invention. More preferably, the antigen is an epitope for the autoantibody of the present invention, and may be RMSRRSN, RRRLNRT, RMRIRRN, HPHPRPR or MRNRPKR identified by the present inventors, and most preferably, MRNRPKR (Met-Arg-Asn-Arg-Pro-Lys-Arg).
  • the membrane, to which proteins are transferred may be any membrane having the capability of attaching proteins, such as NC (nitrocellulose), nylon, and PVDF, and the type of the membrane is not limited to these examples.
  • proteins can be transferred to the membrane as follows.
  • the proteins can be transferred to the membrane by contacting the membrane on the medium and separating therefrom after plaque formation.
  • the membrane is contacted on the medium and separated therefrom, and then frozen and thawed to rupture the cell wall, thereby exposing the proteins on the membrane.
  • the dilution ratio is controlled depending on properties of the blood (viscosity), and also properly determined by those skilled in the art.
  • the blood may be diluted at a ratio of 1:200.
  • reaction may be examined by a colorimetric method using an enzyme or a fluorescent substance, a radiometric assay, and immunohistochemistry without limitation, and preferably a rapid and simple colorimetric method using an enzyme.
  • an antigen-antibody complex is bound with a secondary antibody that is labeled with an enzyme catalyzing a colorimetric reaction of a substrate, and then a colorimetric substrate is added, followed by confirmation of the color development.
  • an enzyme available in the diagnostic method of the present invention, any enzyme used in the conventional enzyme-colorimetric method can be adopted without limitation, such as alkaline phosphatase, peroxidase, and glucose oxidase.
  • Western blot may be adopted to detect the autoantibody of the present invention or to diagnose hepatocellular carcinoma.
  • a gene encoding the amino acids recognized by anti-FASN autoantibody of the present invention is cloned to an expression vector that is prepared to produce fusion proteins, and then E.coli was transformed with the recombinant plasmid.
  • the proteins extracted from E.coli is subjected to SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis), and then the proteins are transferred to a proper membrane. Thereafter, as in the allogenic sera screening method, the membrane is reacted with the blood of a subject suspected of having hepatocellular carcinoma, so as to detect the antibody.
  • the expression vector to be used in the cloning of the gene encoding the antigen for the autoantibody of the present invention may include a gene of GST (glutathione-S-transferase) or ⁇ -galactosidase, and preferably a vector that can be stably expressed in E.coli.
  • ELISA may be adopted to detect the autoantibody of the present invention or to diagnose hepatocellular carcinoma, in which an ELISA plate is coated with the identified antigen sequence, and treated with a primary antibody, washed, and then treated with a secondary antibody, so as to detect the reaction between the antigen and the primary antibody.
  • the epitope sequence reacting with the autoantibody of the present invention was identified using a 7 peptide phage library, and the identified sequence was used to react with a primary antibody, followed by reaction with IgGAM-HRP. Then, the formation of antigen-antibody complex and the amount thereof were examined. As a result, a distinct pattern was observed between the sera of the healthy subject and the hepatocellular carcinoma subject.
  • hepatocellular carcinoma can be diagnosed with a high specificity and sensitivity.
  • hepatocellular carcinoma patients can be diagnosed and distinguished from healthy subjects to the level of about 97% sensitivity and 100% sensitivity by ELISA (Enzyme-linked immunosorbent assay).
  • the present invention relates to a method for screening a therapeutic agent for hepatocellular carcinoma, in which materials expected to treat hepatocellular carcinoma are administered, and the expression levels of anti-FASN autoantibody are assessed before and after administration of the candidate materials, whereby the candidate material reducing the expression level is determined as a therapeutic agent.
  • the method for screening a therapeutic agent for hepatocellular carcinoma of the present invention may include the steps of (a) assessing the expression level of anti-FASN autoantibody; (b) administering candidate materials expected to treat hepatocellular carcinoma; and (c) confirming a reduction in the expression level of anti-FASN autoantibody, compared to step (a).
  • the step of assessing the expression level of anti-FASN autoantibody may be performed by the above-described method of assessing the expression level that is typically employed in the art, without limitation, and exemplified by Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip.
  • the materials expected to treat hepatocellular carcinoma are also called candidate materials, and the materials are not particularly limited, as long as they are expected to directly or indirectly ameliorate or improve hepatocellular carcinoma.
  • the material includes all materials expected to show therapeutic effects, such as compounds, genes, and proteins.
  • the expression levels of anti-FASN autoantibody are assessed before and after administration of the candidate materials, and the candidate material showing a reduction in the expression level can be determined as a therapeutic agent for hepatocellular carcinoma.
  • FIG. 1 is an overview of the method of obtaining hepatocellular carcinoma-associated autoantibody from hepatocellular carcinoma mouse model.
  • H-ras12V transgenic HCC mouse model characterized by the occurrence of hepatocellular carcinoma at 8-10 months of age, was acquired, and splenocytes from the mouse models that developed hepatocellular carcinoma were fused with the mouse myeloma cells Sp2/0. The fused cells were primarily selected using HAT medium, and cells forming clones were separately cultured.
  • B-cell hybridomas producing HCC-associated autoantibodies were only selected from the cell media, and maintained. The reactivity of the B-cell hybridoma medium to HCC cells was examined by the following method in Example 2.
  • FIG. 2 and FIGs. 3a and 3b are the results of detecting autoantibody by intracellular staining as described above.
  • the subject cells were collected and lysed in RIPA buffer (PBS containing 0.1% SDS, 0.1% sodium deoxycholate, 1.0% NP40, protease inhibitor cocktail (Roche)), or lysed in SDS-PAGE sample buffer and heated.
  • Bradford assay was performed for protein quantification.
  • the prepared protein sample was run on 8% ⁇ 10% reduced SDS-PAGE, and transferred onto a PVDF membrane. Thereafter, the membrane was blocked with 5% skim milk (TBS), and treated with a primary antibody.
  • TBS skim milk
  • the purified K1 antibody was used at a concentration of 10 ⁇ g/ml as a primary antibody.
  • the membrane was washed with TBST (0.02% Tween-20 containing TBS), and treated with secondary antibody (anti-mouse IgGAM-HRP). Then, the protein band corresponding to antibody reaction was confirmed by ECL. ⁇ -actin was used as a protein loading control for result comparison. As shown in FIG. 3c, it was found that the K1 antigen is a protein of a high molecular weight (>200 KD) and overexpressed in most cancer cells lines.
  • K1 autoantibody-specific antigen For analysis of K1 autoantibody-specific antigen, purified monoclonal autoantibodies are needed. Therefore, in the present invention, a cell culture medium where K1 autoantibody-producing clones were mass-produced, or a K1 autoantibody-producing cell, was peritoneally injected into mice. The ascites fluid was obtained, and used for the purification of K1 autoantibody. The isotype of K1 autoantibody was found to be IgM. For the purification of IgM, a MBP (Mannose-binding protein)-immobilized agarose (Pierce) or Protein L agarose was used. The purified antibody was confirmed by SDS-PAGE and Coomassie staining, followed by Bradford assay for protein quantification.
  • MBP Mannose-binding protein
  • K1 antibody-specific antigen protein To examine the intracellular localization of the K1 antibody-specific antigen protein, cells were cultured on a coverslip, and then washed with PBS, followed by staining in the same manner as in the intracellular staining method. Confocal laser microscopy (Zeiss) was performed. In this connection, the purified K1 antibody was used as a primary antibody at a concentration of 5 ⁇ g/ml, and anti-mouse IgGAM-FITC (Sigma) was used as a secondary antibody. The stained coverslip was treated with a mounting solution containing DAPI, and placed on a slide. As shown in FIG.
  • phage display cyclic peptide library system (Ph.D.-C7C Phage Display Peptide Library kit; New England Biolabs Inc.), which is a cyclic random hepta-peptide phage library formed by cysteine residues at both ends, was used, performed in accordance with manufacturer's instructions.
  • 2x10 11 phage virions were incubated with 300 ng of K1 antibody in 200 ⁇ l of TBST solution at room temperature for 20 min, and reacted with 25 ⁇ l of protein L-agarose beads that were treated with a blocking solution (0.1 M NaHCO 3 (pH 8.6), 5 mg/ml BSA, 0.02% NaN 3 ) at room temperature for 15 min.
  • a blocking solution 0.1 M NaHCO 3 (pH 8.6), 5 mg/ml BSA, 0.02% NaN 3
  • An antibody-virion-protein L-agarose complex was formed, and washed with TBST, followed by elution of the virions bound to antibody in 1 ml of buffer (0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA).
  • the phage was used as a coating antigen, and K1 antibody was used as a primary antibody to perform ELISA.
  • the 96-well Maxisorp plate was coated with purified phage (10 10 pfu/well) in 100 ul of a coating buffer (0.1 M sodium carbonate buffer (pH 8.6)).
  • a coating buffer 0.1 M sodium carbonate buffer (pH 8.6)
  • the phage-coated plate was stored at 4°C for 16 hrs or longer. After phage coating, 300 ⁇ l of 5% skim milk solution was added, and incubated at room temperature for 1 hr for blocking the plate.
  • the plate was washed with TBST (TBS containing 0.2% Tween-20) five times, and 100 ng/100 ⁇ l of K1 antibody was added thereto, followed by incubation at room temperature for 1 hr and 30 min. After incubation, the plate was washed with TBST five times, and treated with a secondary antibody, anti-mouse IgGAM-HRP (Sigma) diluted at a ratio of 1:2000, followed by incubation at room temperature for 1 hr and 30 min.
  • TBST TBS containing 0.2% Tween-20
  • the phage peptide display library utilized in the present invention is characterized by a cyclic hepta-peptide phage library. In this case, a conformation-dependent epitope is generally obtained.
  • the cyclic structure of the phage epitope was converted into a linear form by treatment of DTT, and then the reactivity of treated phages against the antibody was examined (FIG. 4c).
  • 10 10 phages were diluted in 20 ⁇ l of TBS, and reacted with 0.25 ⁇ l of 1 M DTT at 55°C for 20 min, and then reacted with 0.25 ⁇ l of 2.5 M iodoacetamide at room temperature for 20 min.
  • Example 8 Competitive inhibition of antibody reaction to confirm whether the phage mimics the epitope structure of a specific autoantigen
  • K1-p7 phage was found to competitively inhibit 50% or more of the binding of K1 antibody to HepG2 cells under the above described conditions, which demonstrates that K1p7 phage properly mimics the epitope structure of K1 antibody-specific target antigen.
  • Example 9 Purification of antigen protein for autoantigen identification and protein identification by mass spectrometric analysis
  • K1 antibody was conjugated to a resin, and used for immunoprecipitation of K1 antibody-binding protein in the HepG2 cell lysate.
  • an aminolink resin Pierce
  • sodium cyanoborohydride were used for conjugation of K1 antibody to the resin.
  • the resin was reacted with Hepa-1c1p cell lysate, and washed with PBS.
  • Specific elution of antigen proteins by competitive inhibitor was performed using 10 12 K1-p7 phages dissolved in 100 ⁇ l of PBS. The eluted antigen protein solution was concentrated using speedvac, and separated on an 8-10% SDS-PAGE.
  • liver tissues (R7-1, R7-2, W7-1, W7-2) were obtained from two mice of seven month-old H-ras12V and normal mice each, and total RNA was extracted therefrom using a RNA purification column (Qiagene). 1 ⁇ g of the total RNA was reacted with reverse transcriptase (Gibco) to synthesize cDNA, and FASN expression was analyzed using the primer set used in Example 10. As shown in FIG. 6c, FASN expression in the liver tissue from H-ras12V mice developing hepatocellular carcinoma was increased about 2-fold more than in the normal tissues.
  • Example 12 ELISA for diagnosis of hepatocellular carcinoma by use of K1-specific phage, K1-p7
  • the human serum is a mixture of various proteins and antibodies, and induces non-specific reaction. Therefore, in the present invention, 20 ⁇ g of the cell lysate protein of ER2780 which is a host cell used for phage amplification and 10 11 M13 bacteriophages (empty phage; Eph) without a peptide library sequence was reacted with 0.1 ⁇ l of each serum sample for 90 min for pre-immunoadsorption, and diluted with a protein-free blocking buffer to prepare 100 ⁇ l of solution, and used as a primary antibody for phage ELISA. The primary antibody reaction was performed at room temperature for 1 hr, and the unreacted antibodies were washed with a washing solution five times.
  • Anti-human IgGAM-HRP (Abcam) was diluted in the protein-free blocking buffer at a ratio of 1:2000, and 100 ⁇ l thereof was used as a secondary antibody, followed by incubation at room temperature for 1 hr. Thereafter, washing was performed five times, and 100 ⁇ l of TMB solution was added for HRP reaction. Then, absorbance was measured at 450 nm. As shown in FIG. 8, the serum of a patient with hepatocellular carcinoma was clearly distinguished from that of normal subjects, and 96.55% sensitivity and 100% specificity were observed by ROC curve. These results indicate that ELISA composed of a mimotope against anti-FASN autoantibody, K1-p7 phage can be used as an efficient diagnostic method of hepatocellular carcinoma.
  • K1 antibody is IgM
  • the amplified DNA was examined by agarose gel electrophoresis, and then cloned into a pCR2.1TOPO vector (Invitrogen), followed by transformation into E. coli DH5a.
  • the vector DNA was extracted from the transformed cells, and the corresponding sequence was analyzed.

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Abstract

La présente invention porte sur des auto-anticorps reconnaissant spécifiquement la séquence d'épitope de FASN (synthase d'acide gras), plus particulièrement, sur l'auto-anticorps ou sur un fragment comprenant un site de liaison d'antigène de celui-ci, sur une composition de diagnostic pour le carcinome hépatocellulaire comprenant un agent apte à évaluer le niveau d'expression de l'auto-anticorps, sur une ligne de cellule d'hybridome produisant l'auto-anticorps, sur un ensemble de diagnostic pour le carcinome hépatocellulaire comprenant la composition, sur un procédé pour détecter l'auto-anticorps d'un patient souffrant d'un carcinome hépatocellulaire à l'aide de la composition, et sur un procédé pour évaluer un agent thérapeutique pour le carcinome hépatocellulaire par administration de matériaux candidats pour le traitement du carcinome hépatocellulaire afin de confirmer une réduction du niveau d'expression d'auto-anticorps.
PCT/KR2010/006727 2009-10-12 2010-10-01 Marqueur de diagnostic pour carcinome hépatocellulaire comprenant des auto-anticorps anti-fasn et composition de diagnostic pour carcinome hépatocellulaire comprenant des antigènes de celui-ci Ceased WO2011046309A2 (fr)

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WO2013123152A3 (fr) * 2012-02-17 2014-11-13 Seattle Genetics, Inc. Anticorps dirigés contre l'intégrine αvβ6 et leur utilisation pour le traitement du cancer
US9234893B2 (en) 2011-02-18 2016-01-12 Korea Research Institute Of Bioscience And Biotechnology Marker comprising anti-CK8/18 complex autoantibody and its use for diagnosing cancer
WO2018005904A3 (fr) * 2016-07-01 2018-03-01 Ludwig Institute For Cancer Research Ltd Procédés et compositions pour l'inhibition du pdgf-cc
EP4075138A4 (fr) * 2019-12-10 2023-11-08 Ajou University Industry-Academic Cooperation Foundation Composition faisant intervenir un auto-anticorps wasf2 pour le diagnostic précoce du carcinome hépatocellulaire
US11827709B2 (en) 2019-12-05 2023-11-28 Seagen Inc. Anti-AVB6 antibodies and antibody-drug conjugates

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AU2003226370A1 (en) * 2002-04-17 2003-11-03 The Burnham Institute Inhibition of fatty acid synthase by beta-lactones and other compounds for inhibition of cellular proliferation
EP1565180A4 (fr) * 2002-10-31 2008-02-27 Fasgen Llc Procede d'inhibition du developpement du cancer par des inhibiteurs de la synthase des acides gras

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US9234893B2 (en) 2011-02-18 2016-01-12 Korea Research Institute Of Bioscience And Biotechnology Marker comprising anti-CK8/18 complex autoantibody and its use for diagnosing cancer
CN103732627A (zh) * 2011-06-02 2014-04-16 韩国生命工学研究院 包含抗-atic自身免疫抗体的肝癌诊断标记物以及包含其抗原的肝癌诊断用组合物
CN103732627B (zh) * 2011-06-02 2016-08-17 韩国生命工学研究院 包含抗-atic自身免疫抗体的肝癌诊断标记物以及包含其抗原的肝癌诊断用组合物
US9493566B2 (en) 2012-02-17 2016-11-15 Seattle Genetics, Inc. Antibodies to integrin AVB6 and use of same to treat cancer
CN105017420A (zh) * 2012-02-17 2015-11-04 西雅图基因公司 针对整联蛋白αvβ6的抗体和使用该抗体治疗癌症
CN104220094A (zh) * 2012-02-17 2014-12-17 西雅图基因公司 针对整联蛋白αvβ6的抗体和使用该抗体治疗癌症
WO2013123152A3 (fr) * 2012-02-17 2014-11-13 Seattle Genetics, Inc. Anticorps dirigés contre l'intégrine αvβ6 et leur utilisation pour le traitement du cancer
EA031069B1 (ru) * 2012-02-17 2018-11-30 Сиэтл Дженетикс, Инк. АНТИТЕЛА ПРОТИВ ИНТЕГРИНОВ αVβ6 И ИХ ПРИМЕНЕНИЕ ДЛЯ ЛЕЧЕНИЯ ЗЛОКАЧЕСТВЕННЫХ НОВООБРАЗОВАНИЙ
WO2018005904A3 (fr) * 2016-07-01 2018-03-01 Ludwig Institute For Cancer Research Ltd Procédés et compositions pour l'inhibition du pdgf-cc
US11352420B2 (en) 2016-07-01 2022-06-07 Paracrine Therapeutics Ab Methods and compositions for PDGF-CC inhibition
US11827709B2 (en) 2019-12-05 2023-11-28 Seagen Inc. Anti-AVB6 antibodies and antibody-drug conjugates
US12268751B2 (en) 2019-12-05 2025-04-08 Seagen Inc. Anti-αVβ6 antibodies and antibody-drug conjugates
EP4075138A4 (fr) * 2019-12-10 2023-11-08 Ajou University Industry-Academic Cooperation Foundation Composition faisant intervenir un auto-anticorps wasf2 pour le diagnostic précoce du carcinome hépatocellulaire

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