WO2006050667A1 - ANTICORPS MONOCLONAUX DIRIGES CONTRE L’ACETYLCHOLINESTERASE LIEE A L’APOPTOSE (AR-AChE) ET LEUR USAGE - Google Patents

ANTICORPS MONOCLONAUX DIRIGES CONTRE L’ACETYLCHOLINESTERASE LIEE A L’APOPTOSE (AR-AChE) ET LEUR USAGE Download PDF

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WO2006050667A1
WO2006050667A1 PCT/CN2005/001888 CN2005001888W WO2006050667A1 WO 2006050667 A1 WO2006050667 A1 WO 2006050667A1 CN 2005001888 W CN2005001888 W CN 2005001888W WO 2006050667 A1 WO2006050667 A1 WO 2006050667A1
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ache
monoclonal antibody
antibody
apoptosis
cells
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PCT/CN2005/001888
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Chinese (zh)
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Xuejun Zhang
Jun Wu
Hua Jiang
Anchun Xiang
Bao Zhang
Weirong Wu
Weiyuan Ye
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Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/53—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
    • 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
    • G01N33/57496—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 involving intracellular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a monoclonal antibody against apoptosis-associated acetylcholinesterase (AR-AChE) and uses thereof.
  • AR-AChE apoptosis-associated acetylcholinesterase
  • Acetylcholinesterase (EC 3. 1. 17, acetylcholinesterase, AChE) is a hydrolase of the neurotransmitter acetylcholine and belongs to glycoproteins. Mainly found in electrical organs of electric sputum, mammalian cholinergic nerves, neuromuscular junctions, erythrocyte membranes (Zakut-H; et al: J-Clin-Invest. 1990, 86 (3): 900-8) Rodent megakaryocyte platelets ( ⁇ Shafferman and B. Velan, Multidisplinary approaches to cholinesterase functions. 1992 Plenum Press, New York). AChE can be divided into neuromuscular and red blood cell types.
  • the molecular structure it can be divided into different subunits (heteromeric class) and the same subunit multimer type (homomeric class;).
  • the catalytic subunits of the different subunit multimeric types are linked to or linked to the triple helix collagen subunit.
  • the same subunit multimer type is subdivided into subtypes such as hydrophilic and lipophilic-linked.
  • the AChE tetramer on the erythrocyte membrane has a molecular weight of about 260 KDa.
  • the function of AChE, in the cholinergic nervous system, is to hydrolyze the neurotransmitter acetylcholine to produce choline and acetic acid.
  • AChE has a hydrolyzed protein activity of a serine protease.
  • AChE m NA transcription has been detected in in vitro cultured tumor cell lines and AChE activity has been detected in the serum of ovarian cancer patients after chemotherapy.
  • the inventors first discovered that mammalian cells express AChE during apoptosis (Zhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF., Induction of Acetylcholinesterase Expression Cell Apoptosis in Various Cell Types. Cell Death and Differentiation 9 (8) : 790-800, 2002. ), Neural-derived Cell Lines (Yang L, Heng-Yi He, Xue-Jun Zhang Increased expression of intranuclear AChE involved in Apoptosis of SK-N-SH cells. Neuroscience research 42 (4) : 261-268, 2002. ) The same phenomenon occurs.
  • AChE expressed by apoptotic cells as apoptosis-associated acetylcholinesterase (AR-AChE) (Xue-Jun Zhang, Lei Yang, Qi-) Huang Jin, Yu-Fang Shi*, Hua Jiang, Heng-Yi He, Kelvin NG and Zi-Qing Jiang.
  • apoptotic mammalian cells express apoptosis-related acetylcholinesterase (AR- AChE) . Acta biochimica et biophysica sinica 35 (2): Pp213 ( 2003.).
  • apoptotic cells expressing AR-AChE Using the characteristics of apoptotic cells expressing AR-AChE, the inventors can screen anti-tumor drugs by detecting the activity of the enzyme ("Anti-tumor drug screening method", CN 1186859 has been authorized. Patent No.: ZL 97 1 25220. 3) and used as a marker for apoptotic cells.
  • the inventors found that cells that do not express AChE express AChE in a large amount during apoptosis, mainly in the nucleus, and AChE is present in apoptotic bodies as the nucleus is formed into apoptotic bodies.
  • the inventors established a method for detecting apoptotic cells and determining the effect of chemotherapy using a method for detecting AChE activity (a method for screening anti-tumor drugs and judging clinical efficacy, Chinese Patent No. 97 1 25220. 3). Due to the activity assay, only qualitative analysis of the protein can be performed, and the presence of butyrylcholinesterase (BuChE) in the serum interferes with the activity of AChE and affects the activity assay of AChE.
  • BuChE butyrylcholinesterase
  • AR-AChE can be distinguished from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptosis, basic research, theoretical and clinical studies of Alzheimer's disease (AD), AD-assisted diagnosis and treatment, judgment of chemotherapy effects, etc. Semi-quantitative and quantitative analysis is available. Therefore, it is necessary to develop a specific monoclonal antibody against AR-AChE.
  • An object of the present invention is to provide a monoclonal antibody against apoptosis-associated acetylcholinesterase (AR-AChE) and a mouse hybridoma cell line producing the same.
  • AR-AChE apoptosis-associated acetylcholinesterase
  • Another object of the present invention is to provide the use of a monoclonal antibody against AR-AChE, which can be used to determine the activity and expression level of AR-AChE upon apoptosis.
  • the antibody can be prepared into a kit, screening anti-tumor drugs, measuring the effect of anti-tumor drugs, assisting in the diagnosis of neurodegenerative diseases such as AD, and detecting urgency.
  • the expression level of apoptosis in sexual organ damage provides doctors with reference data on the onset and recovery of patients.
  • a further object of the present invention is to provide a method for screening an antitumor chemotherapeutic drug and an immunoassay for detecting apoptosis-associated acetylcholinesterase.
  • a first aspect of the invention provides a monoclonal antibody which specifically recognizes apoptosis-associated acetylcholinesterase.
  • the above monoclonal antibody is secreted by a mouse hybridoma cell line having the accession number CCTCC NO: - C200413, CCTCC NO: - C200414 or CCTCC NO: - C200415.
  • the above monoclonal antibodies include humanized antibodies.
  • the above antibody is a chimeric monoclonal antibody comprising a non-human variable region and a human light chain and heavy chain constant region.
  • a second aspect of the invention provides a hybridoma cell line, characterized in that said hybridoma cell line produces said monoclonal antibody.
  • the hybridoma cell line is a mouse hybridoma cell line.
  • the above hybridoma cell line is a mouse hybridoma cell line deposited under CCTCC NO: - C200413, CCTCC NO: - C200414, CCTCC NO: -C200415.
  • the present invention also provides a monoclonal antibody secreted by the above hybridoma cell line.
  • a third aspect of the invention provides an immunoassay which is carried out using at least one of the above monoclonal antibodies.
  • the specimen in the above immunoassay is blood, tissue or cerebrospinal fluid, preferably blood supernatant.
  • the above immunoassay can be carried out using an ELISA technique or an immunochromatography technique.
  • the above monoclonal antibody is a monoclonal antibody of one or more subtypes secreted by a mouse hybridoma cell line of accession numbers CCTCC NO: -C200413, CCTCC NO: - C200414, CCTCC N0: - C200415.
  • a fourth aspect of the invention provides a kit comprising at least one of the above monoclonal antibodies.
  • the monoclonal antibody is selected from the group consisting of ARA-Ml, ARA-M5, ARA-M7 (hybridoma cell line, see description on page 7).
  • the kit of the invention can be used for screening anti-tumor drugs, judging the therapeutic effect of anti-tumor drugs, diagnosing neurodegenerative diseases and detecting the expression level of apoptosis-associated acetylcholinesterase, especially the expression level of AR-AChE in acute organ damage.
  • a fifth aspect of the invention provides a composition comprising at least one of the above monoclonal antibodies and a pharmaceutically acceptable carrier and/or excipient.
  • the above composition comprises a monoclonal antibody of one or more subtypes secreted by a mouse hybridoma cell line of accession number CCTCC NO: - C200413, CCTCC NO: ⁇ C200414, CCTCC NO: - C200415.
  • the composition of the present invention is useful for treating diseases caused by abnormal expression of apoptosis-associated acetylcholinesterase, including neurodegenerative diseases.
  • a fifth aspect of the invention provides the use of the above monoclonal antibody for screening an antitumor drug.
  • a sixth aspect of the invention provides a method of screening an antitumor drug, which comprises the following steps -
  • a drug candidate is a drug that promotes tumor apoptosis, and conversely, the drug candidate is ineffective.
  • the monoclonal antibody is selected from any one of ARA-M1, ARA-M5, and ARA-M7.
  • AR-AChE is detected within 18-24 hours after the drug candidate is added.
  • a seventh aspect of the present invention provides the use of the above monoclonal antibody for determining the therapeutic effect of an antitumor drug.
  • An eighth aspect of the present invention provides a method for determining the therapeutic effect of an antitumor drug, comprising the steps of: detecting the expression level of apoptosis-associated acetylcholinesterase in peripheral blood of a tumor patient after treatment with an antitumor drug, 8 to 168 hours later, And compared with before treatment; the expression of apoptosis-associated acetylcholinesterase increased, indicating that the anti-tumor drug is effective, no change or the expression amount does not increase, then the anti-tumor drug is ineffective.
  • the preferred detection time is 12-120 hours.
  • a ninth aspect of the present invention provides the use of the above monoclonal antibody for the preparation of a medicament for diagnosing or treating a neurodegenerative disease.
  • a tenth aspect of the present invention provides the use of the above monoclonal antibody for the preparation of a kit for detecting apoptosis-related acetylcholinesterase expression levels in acute organ damage.
  • the inventors have found an isoform of acetylcholinesterase (AChE) from apoptotic cells, named AR-AChE, and applied for a patent (a human acetylcholinesterase isoform protein (AR-AChE) And gene coding sequence.
  • the inventors found that many cell lines express AChE during apoptosis (Zhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF., Induction of Acetylcholinesterase Expression during Apoptosis in Various Cell Types.
  • AChE on synaptic and motor endplates AChE on erythrocyte membrane, and AChE in megakaryocytes. Therefore, the inventors referred to AChE expressed by apoptotic cells as apoptosis-related acetylcholinesterase (AR). -AChE).
  • AR apoptosis-related acetylcholinesterase
  • -AChE an antibody against AR-AChE expressed by apoptotic cells, which is called an anti- apoptosis-related acetylcholinesterase antibody.
  • AR-AChE the inventors were able to prepare monoclonal and polyclonal antibodies.
  • Anti-neurite AChE antibodies on the market today are also different from antibodies against AChE on erythrocyte membranes.
  • the antibody of the present invention recognizes only AChE expressed by apoptotic cells, which the inventors call (apoptosis-related acetylcholinesterase, abbreviation AR-AChE), while anti-neurite AChE antibodies on the market may recognize AR-AChE.
  • AR-AChE apoptosis-related acetylcholinesterase
  • the inventors have obtained apoptosis-associated acetylcholinesterase (AR-AChE) as an antigen (Xue-Jun Zhang, Lei Yang, Qi-Huang Jin, Yu-Fang Shi*, Hua Jiang, Heng-Yi He, Kelvin NG) And Zi-Qing Jiang.
  • Various apoptotic mammalian cells express apoptosis-related acetylcholinesterase (AR-AChE) . Acta biochimica et biophysica sinica— 35 (2): pp213, 2003. On this basis, the inventors prepared monoclonal antibodies against AR-AChE.
  • AR-AChEo With the specific production of anti-AR-AChE monoclonal antibodies, AR-AChEo can be distinguished from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptosis, and for basic research. AD research Study, AD assisted diagnosis and treatment; judgment of chemotherapy effect and so on. This is the first monoclonal antibody in the world to specifically recognize AR-AChE expressed by apoptotic cells.
  • the inventors isolated AR-AChE from apoptotic cells using a Chinese patent (ZL 97 1 25216. 5, a method for obtaining brain-type AChE from mammalian cells), (Qi-Huang JIN, Yu-Fang SHI, Heng -Yi HE, Kelvin W Ng, Hua JIANG, Lei YANG, Zi-Qing JIANG and Xue-Jun Zhang Isolation of AChE from Apoptotic Human Lung Fibroblast Cells by Antibody Affinity Chromatography. BioTechniques 33 (4): S92 - S97, 2002. ) . It was used to immunize BaLb/c mice, and three clones were screened to specifically produce monoclonal antibodies against apoptosis-associated acetylcholinesterase (AR-AChE).
  • AR-AChE apoptosis-associated acetylcholinesterase
  • the monoclonal antibody of the present invention can be produced by the hybridoma of the present invention, and thus can be obtained from the culture solution for culturing the hybridoma of the present invention.
  • the method of producing the monoclonal antibody of the present invention is not particularly limited, but if the genetically engineered antibody specifically binds to the AR-AChE protein, it is also within the scope of the present invention.
  • the hybridoma of the present invention produces a monoclonal antibody recognizing AR-AChE, and can be obtained by fusing spleen cells or lymph node cells of an animal immunized with AR-AChE protein with myeloma cells.
  • the hybridoma cell lines AM-Ml, ARA-M5, ARA-M7 of the present invention have been deposited with the China Center for Type Culture Collection (Wuhan) on September 21, 2004, in accordance with the Budapest Convention for the Conservation of Microorganisms for Patent Procedures.
  • the deposit numbers are in turn CCTCC NO: - C200413, CCTCC NO. - -C200414, CCTCC NO : - C200415.
  • Hybridomas of the invention can be produced using cell fusion techniques known in the art. Therefore, AR-AChE is used as an immunogen to immunize animals other than human, and the spleen cells or lymph node cells of the animal are fused with myeloma cells to produce a hybridoma, and a hybrid which produces a monoclonal antibody recognizing AR-AChE is selected therefrom. Tumor, thereby obtaining a hybridoma of the present invention.
  • the immunogen for preparing the hybridoma described above is not particularly limited and may be recombinant AR-AChE, purified AR-AChE protein or tumor cells containing AR-AChE.
  • an engineered strain expressing an AR-AChE protein or a fusion protein containing an AR-AChE protein can be cultured on a suitable medium.
  • the invention also encompasses the use of humanized antibodies, as well as humanized antibodies, in the manufacture of a medicament for the treatment of a neurodegenerative disease, such as AD.
  • a neurodegenerative disease such as AD.
  • One skilled in the art can prepare humanized antibodies using the monoclonal antibodies of the present invention.
  • Chimeric monoclonal antibodies can be made using recombinant DNA techniques known in the art. For example, a gene encoding a constant region of a non-human antibody molecule is replaced with a gene encoding a human constant region (see, Robinson et al, PCT Patent Publication PCT / US86/02262; Akira, et al, European Patent Application 184, 187; or Taniguchi, M . European Patent Application 171, 496). It is possible to replace the variable region portion that does not involve binding to the antigen by replacing the equivalent portion of the human variable region with The chimeric antibody is "humanized" in one step.
  • the field of application of the monoclonal antibody of the present invention is not particularly limited, and can be used for immunoassays, including quantitative or semi-quantitative detection.
  • the monoclonal antibody of the present invention can detect the presence or absence of AR-AChE as a basis for judging the tumor chemotherapy results.
  • An antigen-binding fragment of an antibody of the present invention refers to an antibody fragment which binds to an epitope of an apoptosis-associated acetylcholinesterase.
  • the animal which is immunized to produce the hybridoma of the present invention is not particularly limited and includes, for example, goat, sheep, guinea pig, mouse, rat, and rabbit. Preferred among them are mice.
  • the immunoassay of the present invention is carried out using at least one monoclonal antibody of the present invention, and the method can be carried out using only one antibody, or in combination with an AR-AChE polyclonal antibody, or simultaneously using two or more monoclonal antibodies (e.g., ARA - Ml and ARA - M5).
  • monoclonal antibodies e.g., ARA - Ml and ARA - M5
  • enzyme immunoassay EIA:
  • enzyme-linked immunosorbent assay ELISA
  • RIA radioimmunoassay
  • FIA fluorescent immunoassay
  • Western blot immunochromatography and the like.
  • the various immunoassays described above can be used to measure target antigens or antibodies by labeling antigens or antibodies by competition or sandwich methods.
  • ELISA and immunochromatography techniques are preferred.
  • the above sandwich method includes, for example, sandwiching the AR-AChE between the monoclonal antibody of the present invention in a fixed subtype and the monoclonal antibody of the present invention or the anti-AR-AChE polyclonal antibody in another subtype labeled with the labeling agent. Then, a substrate or the like against a label (for example, an enzyme) is added, a color or the like is displayed, thereby detecting AR-AChE in the specimen.
  • Monoclonal antibodies with high sensitivity are generally considered as preferred labeled antibodies because of their high specificity and low background noise, which makes detection more accurate, compared to polyclonal antibodies with low specificity and high background noise. '
  • the above competitive methods are based, for example, on the quantitative competitive binding reaction of AR-AChE in a test specimen and a known amount of labeled AR-AChE to a monoclonal antibody of the present invention.
  • a predetermined amount of the antibody immobilized on the carrier and a predetermined amount of AR-AChE labeled with the labeling agent are added to the sample solution containing AR-AChE. Then, the activity of the labeling agent remaining on the carrier or the activity of the labeling agent not remaining on the carrier is determined. In this regard, it is preferred to add the antibody and the labeled antigen almost simultaneously.
  • a radioactive isotope such as 125 1
  • an enzyme an enzyme substrate, a phosphorescent substance, a fluorescent substance, a biotin, and a coloring matter
  • the maleimide method can be used (J. Biochem. (1976), 79, 233), activated biotin method (J. Am. Chem. SOC. (1978) , 100, 3585) or hydrophobic bond method.
  • a substrate suitable for the enzyme used in the case can be selected, including, for example: ABTS, luminol _3 ⁇ 40 2 , 0 -phenylenediamine- 3 ⁇ 40 2 (for peroxidase), phosphoric acid p-Nitrophenyl ester, methyl formate phosphate, 3-(2'-spirocyclobutane)-4-methoxy-4-one (3'-phosphoryloxy)phenyl- 1,2- Dioxetane (for alkaline phosphatase), p-nitrophenyl-BETA-D-galactose (for beta-galactosidase).
  • Radiolabeling of the above antigens or antibodies can be conveniently carried out using a commercially available Bolton-Hunter reagent.
  • a Bolton-Hunter reagent can be added to a solution (prepared by dissolving an antigen or an antibody in 0.1 aqueous sodium hydrogencarbonate solution), followed by 1-2 hours, using a G-25 desalting column, etc. The unreacted Bolton-Hunter reagent fraction was removed.
  • the 125 1 radiolabel can be conveniently carried out using the Chlorphenamine T method, the iodine method, or the like.
  • the above fluorescent substances such as fluorescein and rhodamine, can be conveniently labeled using an activated ester method or an isocyanate method ("Enzyme Immunoassay", published by Igaku Shoin, 1987).
  • an activated ester method or an isocyanate method Enzyme Immunoassay, published by Igaku Shoin, 1987.
  • colored latex particles and colloidal gold can be used.
  • the immunoassay of the present invention uses the monoclonal antibody of the present invention, and thus has outstanding characteristics, and recognizes only AR-AChE present in all apoptotic cells (including tumors or nerve cells) without erroneous detection due to cross-reaction Other substances and normal AChE allow for very specific assays.
  • the kit of the present invention can be used.
  • the kit of the present invention contains at least one monoclonal antibody of the present invention and may contain only one monoclonal antibody.
  • the monoclonal antibody of the present invention to be used in the kit of the present invention is not particularly limited, but may be any monoclonal antibody recognizing AR-AChE, and may also be a decomposition product of the monoclonal antibody of the present invention or an antigen-binding fragment thereof.
  • the monoclonal antibody of the present invention can be immobilized on a solid phase in advance, and the monoclonal antibody of the present invention can also be labeled with the above-mentioned labeling agent.
  • the kit of the present invention can be used to detect a sample containing 'AR-AChE, such as blood, tissue or cerebrospinal fluid, by immunological methods, so that the presence or absence of AR-AChE and the change in expression amount can be judged.
  • a sample containing 'AR-AChE such as blood, tissue or cerebrospinal fluid
  • the solid phase of the kit used in the present invention is not particularly limited, but includes, for example, a polymer such as polystyrene, glass beads, magnetic particles, a microtiter plate, a filter paper for immunochromatography, a glass filter or other insoluble carrier.
  • a polymer such as polystyrene, glass beads, magnetic particles, a microtiter plate, a filter paper for immunochromatography, a glass filter or other insoluble carrier.
  • Kits of the invention may also contain other components.
  • the above other components are not particularly limited, but include, for example, an enzyme for labeling, a substrate thereof, a radioactive isotope, a phosphor, a fluorescent substance, a coloring matter, a buffer, and a petri dish, and the above-mentioned components.
  • the kit of the present invention may be a kit for carrying out the above competitive method or the above-described sandwich method.
  • a kit using a sandwich method is preferred.
  • the above sandwich method has the advantages of high sensitivity, short reaction time required, high accuracy, and convenient operation.
  • a kit can be prepared which makes the test method convenient and simple, and which can be easily observed by visual observation.
  • the formation of the enzyme reaction product is determined by the amount of the target substance to be tested, so that a system can be established which can easily determine the presence or absence of AR-AChE by visual observation through color development or the like. , can also be used to test its 0. D value, quantitative and semi-quantitative detection.
  • Kit of the present invention is used is not particularly limited samples, but includes, for example: blood and cerebrospinal fluid or tissue, preferably blood.
  • the monoclonal antibody of the invention and the kit of the invention can be used for screening anti-tumor drugs, and assisting in judging anti-tumor drugs Therapeutic effects, preparation of drugs for the diagnosis or treatment of neurodegenerative diseases, detection of apoptosis in acute organ damage
  • the above method for screening an antitumor drug comprises the following steps:
  • step (2) adding a candidate drug to the cell culture medium in the step (1), detecting the expression level of apoptosis-associated acetylcholinesterase using the above monoclonal antibody or the above kit; promoting the apoptosis-associated acetylcholinesterase or
  • a drug candidate with an increased expression amount is a drug that promotes apoptosis of a tumor cell, and conversely, the drug candidate is ineffective.
  • the recognition of AR-AChE by a monoclonal antibody can eliminate the influence of other substances present in the sample, thereby detecting the presence of AR-AChE with very high sensitivity and specificity. Since hybridomas that produce monoclonal antibodies against AR-AChE have been established, the same monoclonal antibodies can now be produced almost permanently.
  • the diagnostic kit of the present invention uses a monoclonal antibody with a high level of detection sensitivity and does not cause false negative or false positive problems, and there is no difference between batch and batch. If the labeled antibody is preferably a monoclonal antibody different from the monoclonal antibody on the solid phase, it is easier to control the quality of the product and to establish a uniform standard.
  • the AR-AChE detection kit detects the principle of using anti-AR-AChE monoclonal antibody and anti-AR-AChE polyclonal antibody or another subtype anti-AR-AChE monoclonal antibody as the immobilized antibody and the enzyme-labeled antibody. If AR-AChE is present in the sample to be tested, an antibody-antigen-enzyme-labeled antibody complex is formed, and a substrate of TMB (enzyme-free color-developing series) is added to produce a color reaction, and vice versa, no color reaction occurs.
  • Double antibody sandwich ELISA is a very sensitive detection technique, and the ELISA is highly sensitive and easy to operate. The development of supporting instruments and equipment has standardized and automated the operating procedures, thus further improving stability.
  • the present invention provides a hybridoma cell line which specifically produces an anti-AR-AChE monoclonal antibody, and an anti-human AR-AChE monoclonal antibody can be produced from these cell lines, and a kit can be prepared using the antibody for basic research, AD research, AD Auxiliary diagnosis and treatment; judgment of the effect of chemotherapy, etc. - So far, only the M30 antibody has been used to determine the effect of chemotherapy by serological methods.
  • ALEXIS Biochemicals Corporation and PolyPhqrma Group Inc., which launched the test kit U30-ApoptosenseTM ELISA Kite.
  • the main component of this kit is M30 antibody, a mouse hybridoma monoclonal antibody, subtype As IgG2b, it recognizes a small fragment of the C-terminus of keratin (Cytokeratin) 18 (CK18).
  • CK18 is a medium fiber of epithelial cells.
  • the activated kinase (Caspa S e) 3, 6, 7, 9 cuts the C-terminus of CK18 into a small fragment. Since this small fragment appears only in apoptotic cells, it becomes a specific marker for epithelial cell apoptosis.
  • M30 antibody apoptosis of tissue cells and cultured cells can be recognized, and changes in serum M30 can also be detected by ELISA to determine the effect of chemotherapy. But it is only for epithelial-related tumors and is expensive.
  • the method of the invention can be used to screen new anti-tumor drugs in vitro.
  • the human tumor cell strain cultured in vitro is added with different doses of the anti-tumor drug candidate to be detected, and after 18-20 hours, the cells or the culture solution are detected and passed.
  • the presence and amount of AR-AChE changes to determine whether the drug being screened is effective or not. If the AR-AChE is increased, indicating that the drug has induced apoptosis, it is an effective anti-tumor drug.
  • the method of the invention can help clinicians to selectively select tumor chemotherapy drugs, aim at finding the best drugs in the shortest time and bring the gospel to the patients.
  • Chemotherapy drugs are mainly aimed at tumor cells (also normal cells), causing apoptosis to achieve therapeutic goals.
  • the invention is based on new discoveries in scientific research. The principle is that cells that do not normally express AChE express AR-AChE in a large amount during apoptosis, and this enzyme is stably present in apoptotic cells. In normal people or tumor patients, although there is no apoptosis in the body, but the number is small, the apoptotic cells are quickly phagocytosed by the surrounding macrophages, and the AR-AChE in the apoptotic cells is not released into the bloodstream. in.
  • AChE accelerates the precipitation of amyloid beta peptide, which is independent of classical enzyme catalysis (Inestrosa NC, Alvarez A, Perez CA, Moreno RD, Vicente M, Linker C, Casanueva 01, Soto C, Garrido J, Acetylchol inesterase Accelerate assembly of amyloid-beta-protoss into Alzheimer's fibrils : possible role of the peripheral site of the enzyme, Neuron, 1996 16 (4); 881-891.
  • AChE antisense nucleic acids directly indicate that AChE plays a role in neuronal loss ( Shohami E, Kaufer D, Chen Y, Seidman S, Cohen 0, Ginzberg D, Melamed-Book N, Yirmiya R, Soreq H, Antisense prevention of neuronal damages following head injury in mice, J Mol Med, 2000 78 (4); 228-236.
  • This monoclonal antibody in addition to being used to identify evidence of apoptosis in brain tissue sections and cerebrospinal fluid, may also be used as a diagnostic reagent (especially after preparation of adult-derived antibodies), such as to prevent AChE acceleration. The precipitation of amyloid peptides, thereby achieving the effect of preventing the progressive development of AD.
  • the medicament includes a monoclonal antibody of the invention and a pharmaceutically acceptable carrier and/or excipient.
  • the method of the present invention detects apoptosis or apoptosis by detecting the AR-AChE activity expressed by the cells after the anti-tumor drug is administered to cells which do not express AChE in vitro, or determining the serum AR-AChE content.
  • the cell AChE reaction is positive, indicating that the drug has induced apoptosis, and is an effective anti-tumor drug, which can conveniently and effectively screen anti-tumor drugs, and can also help doctors select chemotherapy drugs that are particularly sensitive to patients to improve the therapeutic effect.
  • the results of detecting the AR-AChE content by the method of the present invention to identify apoptotic cells cultured in vitro are consistent with the classical method.
  • A. is immunofluorescence
  • 1 antibody is mouse anti-human AR-AChE monoclonal antibody
  • 2 antibody is goat anti-mouse IgG antibody
  • Rhodamine label and 2 apoptotic cells are positive.
  • B. is Hoechst No. 33258 staining showing all nuclei.
  • C. is the overlap of A and B.
  • D. is a visible light photo. This photograph shows that two apoptotic cells show positive and others are not.
  • A. is immunofluorescence
  • 1 antibody is mouse anti-human AR-AChE monoclonal antibody
  • 2 antibody is goat anti-mouse IgG antibody
  • Rhodamin label apoptotic nucleated cells show positive, red blood cells are negative;
  • B. is Hoechst No. 33258 staining, showing nuclear material, only one nucleated cell in the whole field is red blood cells;
  • the apoptotic HELA lysate a. No substrate, but replaced with an equal amount of PBS; b. No antibody, substrate reaction, positive result; c. and d. Antibody, forming a complex, centrifuging, taking supernatant (c) and Precipitation (d) was used as a substrate reaction, and both were negative.
  • the cultured HELA cells the antibody is a mouse anti-human AR-AChE monoclonal antibody (ARA-M5) prepared by the inventors, 1 antibody is directly linked to HRP, DAB reaction, normal HELA cells are negative, and apoptotic cells are positive.
  • ARA-M5 mouse anti-human AR-AChE monoclonal antibody
  • the antigen is derived from apoptotic HELA cells;
  • the antigen is derived from apoptotic HELA cells
  • the antigen is derived from electroporation acetylcholinesterase (Sigma).
  • black arrows point to apoptotic cells
  • white arrows point to normal cells
  • C. is immunofluorescence
  • 1 antibody is mouse anti-human AR-AChE monoclonal antibody
  • 2 antibody is goat anti-mouse IgG antibody
  • Rhodamin label only apoptotic bodies show positive cells, while normal cells are negative
  • This monoclonal antibody specifically recognizes apoptotic cells.
  • the antibody is a mouse anti-human AR-AChE monoclonal antibody (ARA-M1) prepared by the inventors, 1 antibody is directly linked to Rhodamine, red is positive cells (cross-reactive with rat AR-AChE); large pieces of nerve tissue There was no positive, there was cholinergic nerves present, and synaptic acetylcholinesterase did not show positive.
  • ARA-M1 mouse anti-human AR-AChE monoclonal antibody
  • A. is Hoechst No. 33258 staining, showing the nucleus
  • B. is cytochemical Ache staining (Konovsky and Root method), brownish yellow (dark gray in the figure) is AChE positive, can be seen more uniform and does not show the outline of cells or nuclei, indicating that the product is not concentrated in the cytoplasm or nucleus, This is completely different from the distribution found in apoptosis by the inventors;
  • C. is the result of simultaneous photographing of visible light photographs with Hoechst No. 33258 staining, further demonstrating that AChE positivity is not in the nucleus.
  • the photo is divided into five parts, A, B, C, D, and E, all of which are in the same field of view.
  • B. is a photo of visible light, and there is a condensed cell with no cell protrusion on the lower right side of the cell mass;
  • C is immunofluorescence, 1 antibody is mouse anti-human AR-AChE monoclonal antibody, 2 antibody is goat anti-mouse IgG antibody, Rhodamin label, and one apoptotic cell in the lower right shows positive;
  • D. is hoechest staining, showing the nucleus
  • E. is the overlap of A, C, and D.
  • Human SK-N-SH cells cultured in vitro can express a small amount of synaptic AChE normally. This photo shows that one apoptotic cell is positive and the other is not positive. TUNEL staining agrees well with anti-AR-AChE positive. , indicating that the monoclonal antibody specifically recognizes the AR-AChE of apoptotic cells, but does not recognize the synaptic AChE expressed by SK-N-SH cells.
  • FIG. 1 Serum AR-AChE in 30 normal newborn cord blood and one tumor patient.
  • mice mouse: BaLb I c mice, 3 males 6 to 10 weeks old.
  • Human AR-AChE Garfield's complete adjuvant (limited to the first use) or Freund's incomplete adjuvant, mixed together in equal volumes, with two syringes, through a three-way valve, repeated pushes to make the two into a fuel pack The chyle of water.
  • Second immunization dose is the same as above, Jiafu's incomplete adjuvant subcutaneous
  • the third dose is the same as above, without adjuvant, intraperitoneal injection (ip)
  • the neck is killed, soaked in 75% alcohol, disinfected for 3 to 5 minutes.
  • the myeloma cells were resuscitated two weeks before the preparation for fusion.
  • the inventors used the myeloma SP2/0 cell line, RPMI1640 medium, the concentration of calf serum was 20%, and the maximum density of cells did not exceed 10 s / ml. 1: 10 dilution passage, pass every 3 to 5 days.
  • Preparation of spleen cell suspension The spleen was taken out under aseptic conditions, washed once with an incomplete culture solution, placed on a stainless steel mesh in a dish, and ground to a cell suspension with a syringe needle and counted.
  • the fused cell suspension was added to a 96-well plate containing feeder cells, 100 ⁇ l / ⁇ , 37 ° C 5 C0 2 incubator.
  • a 96-well plate contains 1 X 10 7 spleen cells.
  • the HAT was selected by adding HAT.
  • the HT medium was used instead, and the culture was maintained for two weeks, and the general culture solution was used instead.
  • the inventors used an ELISA screening method. IV. Cloning and cryopreservation of hybridomas
  • the hybridoma cells contain 1 X 10 6 or more per ampule.
  • the ascites was prepared by intraperitoneal injection of 0.5 ml of Pristane or planting paraffin in BaLb / c mice. After 1 to 2 weeks, IX 10 6 hybridoma cells were intraperitoneally injected. After inoculation for 7 to 10 days, the mice were sacrificed. , collect ascites with a dropper.
  • AR-AChE antibody normal HELA cells were negative and apoptotic cells were positive (the results of the product are shown in the next paragraph, and the monoclonal antibody is shown in Figure 4).
  • the AR-AChE antibody recognizes the same band as the commercial AChE antibody (see Figure 5).
  • the inventors have reported (Zhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF., Induction of Acetylcholinesterase Expression during Apoptosis in Various Cell Types. Cell Death and Differentiation 9 (8): 790-800, 2002.), normal live HELA cells do not express AChE, whereas apoptotic HELA cells express AChE instead of butyrylcholinesterase (BuChE).
  • the inventors used two affinity columns (concanavalin A - Sepharose and edrophonium - Sepharose) (Ji Young Son, Sook Shin, Kwang Ho Choi, and In Kook Park.
  • AChE activity of apoptotic HELA can be blocked by the inventor's monoclonal antibody (identification of McAb neutralization activity, Figure 3), the inventors will perform supernatant and precipitation as WESTERN BLOT, showing no in the supernatant AChE, while in the pellet, there is an AChE positive band.
  • the antibody of the present invention recognizes only AR-AChE expressed by apoptotic cells, and is different from the commercially available antibody, ⁇ JS immunoassay, which recognizes only AR-AChE expressed by apoptotic cells, and specific evidence (Fig. 1, 6).
  • Figure 6 shows that some nucleated cells isolated from blood were placed at room temperature for 18 hours, and some cells were apoptotic. The photos are all the same field of view.
  • FIG. 1 Human and rat synaptic (brain) AChE are not recognized (Fig. 1, Fig. 7).
  • Figure 7 shows rat brain tissue sections, AR-AChE immunohistochemistry and TUNEL double staining.
  • the rat is subjected to ischemic treatment of the middle cerebral artery (Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989 Jan; 20 (l): 84- 91.) From the damage surrounding group ⁇ only. photo A, B, and C are all the same field of view.
  • Anti-neuronal AChE antibodies on the market today are also different from antibodies against AChE on erythrocyte membranes.
  • the antibody of the present invention recognizes only AChE expressed by apoptotic cells, which the inventors call (apoptosis-related acetylcholinesterase, abbreviation AR-AChE), and the anti-neuronal AChE antibody on the market can recognize AR-AChE.
  • AR-AChE apoptosis-related acetylcholinesterase
  • the inventors have obtained apoptosis-associated acetylcholinesterase (AR-AChE) as an antigen (Xue-Jun Zhang, Lei Yang, Qi-Huang Jin, Yu-Fang Shi*, Hua Jiang, Heng-Yi He, Kelvin NG) And Zi-Qing Jiang.
  • Various apoptotic mammalian cells express apoptosis-related acetylcholinesterase (AR-AChE) . Acta biochimica et biophysica sinica 35 (2): pp213, 2003. On this basis, the inventors prepared monoclonal antibodies against AR-AChE.
  • AR-AChE can be distinguished from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptotic phenomena, for basic research, AD research, AD-assisted diagnosis and treatment, and the judgment of chemotherapy effects. This is the first monoclonal antibody in the world to specifically recognize AR-AChE expressed by apoptotic cells.
  • Example 4 Observing the distribution of normal brain type AChE and the relationship between enzyme and nucleus
  • acetylcholinesterase substrate reaction solution 0.1 M phosphate buffer Ph 6. 0 150 ml, acetylthiocholine 100 mg / 200 ml, 0.1 M sodium citrate llml, 30 mM copper sulfate 20 ml, 5 mM ferric cyanide Potassium (20 ml), the enzyme reaction was carried out.
  • the five-month human fetal brain tissue was inoculated, frozen in 4% paraformaldehyde, immunohistochemical reaction and Hoechst staining, showing nuclear and AR-AChE positive cells, respectively (see Figure 1), indicating that the antibody of the present invention does not recognize large pieces.
  • the presence of acetylcholinesterase recognizes only apoptosis-associated AChE.
  • Example 6 Using human SK-N-SH cells cultured in vitro, it was confirmed that the monoclonal antibody of the present invention specifically utilizes human SK-N-SH cells cultured in vitro, indicating that the antibody of the present invention recognizes only apoptotic cells, but does not recognize normal cells.
  • C-1 antibody is a mouse anti-human AR-AChE monoclonal antibody (ARA-M7), 2 anti-mouse anti-mouse IgG antibody (1:100 dilution, Rhodamine conjugated anti-mouse IgG-, Santa Cruz), and an apoptotic cell on the lower right side showed positive.
  • ARA-M7 mouse anti-human AR-AChE monoclonal antibody
  • 2 anti-mouse anti-mouse IgG antibody (1:100 dilution, Rhodamine conjugated anti-mouse IgG-, Santa Cruz
  • an apoptotic cell on the lower right side showed positive.
  • Human SK-N-SH cells cultured in vitro can express a small amount of synaptic AChE normally.
  • the semi-quantitative analysis of acetylcholinesterase in human serum using the antibody of the present invention can be used to judge the effect of tumor chemotherapy (including leukemia).

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Abstract

Sur base de la découverte de l’acétylcholinestérase liée à l’apoptose (AR-AChE), l’invention utilise de l’AR-AChE humaine purifiée en tant qu’antigène afin d’immuniser les souris, fusionner les cellules, obtenir des hybridomes, spécifiquement exprimant et secrétant des anticorps monoclonaux dirigés contre l’acétylcholinestérase liée à l’apoptose et des lignées cellulaires d’hybridomes ARA-M1, ARA-M5 et ARA-M7, et produire des anticorps. Les anticorps peuvent être utiles pour l’analyse des médicaments anti-tumoraux et la détermination des chimiothérapies cliniques, la préparation de médicaments pour le diagnostic et le traitement des troubles neuro-régressifs, tels que le dosage AD, la recherche clinique et basale et le diagnostic de cellules apoptotiques, et la mesure des niveaux d’expression de l’apoptose dans les organes endommagés. L’invention concerne également les procédés d’immuno-dosage et les kits à utiliser lors de dosages semi-quantitatifs et/ou quantitatifs pour l’AR-AChE et autres.
PCT/CN2005/001888 2004-11-12 2005-11-10 ANTICORPS MONOCLONAUX DIRIGES CONTRE L’ACETYLCHOLINESTERASE LIEE A L’APOPTOSE (AR-AChE) ET LEUR USAGE WO2006050667A1 (fr)

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US8895004B2 (en) 2007-02-27 2014-11-25 AbbVie Deutschland GmbH & Co. KG Method for the treatment of amyloidoses
US8987419B2 (en) 2010-04-15 2015-03-24 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9822171B2 (en) 2010-04-15 2017-11-21 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US10047121B2 (en) 2010-08-14 2018-08-14 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9062101B2 (en) 2010-08-14 2015-06-23 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins

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