WO2020100779A1 - Anticorps monoclonal se liant de manière spécifique à pser46-marcks - Google Patents

Anticorps monoclonal se liant de manière spécifique à pser46-marcks Download PDF

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WO2020100779A1
WO2020100779A1 PCT/JP2019/044042 JP2019044042W WO2020100779A1 WO 2020100779 A1 WO2020100779 A1 WO 2020100779A1 JP 2019044042 W JP2019044042 W JP 2019044042W WO 2020100779 A1 WO2020100779 A1 WO 2020100779A1
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amino acid
acid sequence
monoclonal antibody
seq
light chain
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岡澤　均
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国立大学法人 東京医科歯科大学
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—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

Definitions

  • the present invention provides a monoclonal antibody that specifically binds to pSer46-MARCKS, a diagnostic agent for neurite degenerative disease containing the monoclonal antibody or a labeled product thereof, a monoclonal antibody gene encoding the monoclonal antibody, a promoter, and the monoclonal antibody. And a host cell into which the vector has been introduced.
  • Alzheimer's disease and Parkinson's disease cause nerve cell death and eventually develop due to the formation of protein aggregates in the brain.
  • a ⁇ amyloid ⁇
  • tau lesion phosphorylation and polymerization
  • neurite degeneration is observed at the same time as or prior to the death of nerve cells (neurons). Degeneration of neurites in neurons is a very important phenomenon because it leads to the loss of proper networks between neurons, ie loss of neural function.
  • the present inventors performed a comprehensive proteome analysis of post-mortem brains of Alzheimer's disease model mice and Alzheimer's disease patients, and analyzed the abnormal phosphorylation signal network common to Alzheimer's disease. As a result, the present inventors have reported that phosphorylation of a substrate of a kinase called MARCKS occurs from an early stage before the onset of Alzheimer's disease (Patent Document 1 and Non-Patent Document 1). In addition, the present inventors have found that phosphorylation of the 46th serine (Ser46) in MARCKS occurs at an early stage before the onset of Alzheimer's disease, and that HMGB1 leaked from the cell due to necrosis of neurons causes the phosphorylation of MARCKS.
  • Ser46 46th serine
  • Non- Patent Document 2 a mouse monoclonal antibody against HMGB1 was produced, and it was reported that such mouse monoclonal antibody inhibits phosphorylation of Ser46 of MARCKS (non- Patent Document 2).
  • the present inventors have also found that such a mouse monoclonal antibody restores cognitive impairment in Alzheimer's disease model mice, reduces DNA damage in the cerebral cortex, and inhibits both A ⁇ and HMGB1 multimer formation. Is reported (Patent Document 2, Non-Patent Document 2).
  • Non-patent document 3 phosphorylation of Ser46 in MARCKS is detected not only in Alzheimer's disease but also in the early stage of onset of neurite degenerative diseases such as Parkinson's disease and dementia with Lewy bodies.
  • An object of the present invention is to provide a monoclonal antibody or the like which can specifically detect the presence or absence of phosphorylation of MARCKS protein in the brain in the early stage of the onset of neurite degenerative disease.
  • the inventors of the present invention continue to earnestly research to solve the above problems.
  • the 46th phosphorylated serine residue common to human and mouse ie, “pSer46-MARKKS”
  • pSer46-MARKKS 46th phosphorylated serine residue common to human and mouse
  • amino acid sequence around pSer46-MARKKS was human.
  • Cys cysteine residue for carrier protein conjugation is added to the amino (N) terminus of a phosphorylated MARCKS antigen peptide consisting of 14 amino acid sequences, and the carrier protein KLH is added to the Cys residue.
  • mice with (Keyhole limpet hemocyanin) conjugated (hereinafter sometimes referred to as “phosphorylated MARCKS antigen peptide / carrier protein complex”) does not bind to non-phosphorylated MARCKS antigen peptide. It was found that a monoclonal antibody that binds to a phosphorylated MARCKS antigen peptide can be obtained, and further, such a monoclonal antibody can detect pSer46-MARKKS in the brain of a neurite degeneration model animal, and completed the present invention. ..
  • a monoclonal antibody which binds to a phosphorylation epitope in MARCKS is an epitope consisting of an amino acid sequence in which the 12th serine residue is phosphorylated in the amino acid sequence shown in SEQ ID NO: 21,
  • the monoclonal antibody is (1-1) Heavy chain complementarity determining region (CDR) consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence ) 1; a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence; and shown in SEQ ID NO: 3 Or a heavy chain CDR3 comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted
  • a heavy chain CDR1 consisting of an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted; the amino acid sequence shown in SEQ ID NO: 8 or one or a plurality of amino acids substituted or deleted in the amino acid sequence , A heavy chain CDR2 consisting of an added and / or inserted amino acid sequence; and the amino acid sequence shown in SEQ ID NO: 9, or in which one or more amino acids have been substituted, deleted, added and / or inserted Heavy chain CDR3 consisting of the amino acid sequence A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence; the amino acid sequence represented by SEQ ID NO: 11, Or a light chain CDR2 consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence; and the amino acid sequence shown in SEQ ID NO
  • the monoclonal antibody of (1-1) further comprises (1-2) a heavy chain variable region consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, and a SEQ ID NO: A light chain variable region comprising an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence shown in 14,
  • the monoclonal antibody of (2-1) further comprises (2-2) a heavy chain variable region consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 15, and SEQ ID NO: 16.
  • the heavy chain comprising the monoclonal antibody of (1-1) or (1-2), further comprising (1-3) an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 17. And a light chain consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18,
  • the monoclonal antibody of (2-1) or (2-2) further comprises (2-3) a heavy chain consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, and a sequence
  • the monoclonal antibody according to [1] or [2] above which comprises the amino acid sequence represented by No. 20 and a light chain comprising an amino acid sequence having a sequence identity of 80% or more.
  • a diagnostic agent for neurite degenerative disease which comprises the monoclonal antibody according to any one of [1] to [3] above or a labeled product thereof.
  • the diagnostic agent according to [4] above, wherein the neurite degenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal lobar degeneration, or Lewy body dementia.
  • a monoclonal antibody gene which encodes the monoclonal antibody according to any one of [1] to [3] above.
  • a vector comprising a promoter and the monoclonal antibody gene according to the above [6] operably linked to the downstream of the promoter.
  • the step of administering the monoclonal antibody of the present invention or a labeled substance thereof to a subject and when pSer46-MARCKS is detected in the brain of the subject, the subject is diagnosed as likely to develop a neurite degenerative disease. However, when pSer46-MARCKS is not detected, the subject has a step of diagnosing that the subject is unlikely to develop a neurite degenerative disease.
  • a diagnostic method which optionally comprises a step of treating a neurite degenerative disease in a subject diagnosed with a high probability of developing a degenerative process; or
  • the monoclonal antibody of the present invention or a labeled product thereof for use in a method for diagnosing neurite degenerative disease; can be mentioned.
  • the monoclonal antibody of the present invention does not bind to the non-phosphorylated MARCKS protein, but specifically binds to the phosphorylated MARCKS protein pSer46-MARKKS.
  • pSer46-MARKKS is a biomarker of neurite degeneration disease in the early stage of onset, and therefore molecular imaging using the monoclonal antibody of the present invention (eg, PET [positron emission tomography] / SPECT [single photon emission computed tomography]) ),
  • the presence or absence of pSer46-MARCKS in the brain can be used as an index for diagnosing neurite degenerative disease at the early stage of onset, enabling early detection and early treatment of neurite degenerative disease, resulting in a quality of life (QOL). It is expected to have the effect of improving health care and reducing medical costs.
  • FIG. 3 shows the results of immunohistochemical staining of B6 / SJL mouse brain tissue using two types of monoclonal antibodies of the present invention (A4H7 and F8H5) and a polyclonal antibody against pSer46-MARCKS.
  • the lower Merge image is an image in which the upper pSer46-MARKKS image and the DAPI image are superimposed.
  • FIG. 3 shows the results of immunohistochemical staining of 5 ⁇ FAD mouse brain tissue using two types of monoclonal antibodies of the present invention (A4H7 and F8H5) and a polyclonal antibody against pSer46-MARCKS.
  • the lower Merge image is an image in which the upper pSer46-MARKKS image and the DAPI image are superimposed.
  • the monoclonal antibody of the present invention is a phosphorylation epitope in MARCKS, specifically, an epitope consisting of an amino acid sequence in which the 12th serine residue in the amino acid sequence of SEQ ID NO: 21 is phosphorylated (hereinafter, “phosphorylated MARCKS antigen”).
  • peptide contains the following heavy chain CDR1 to CDR3 (1-1) and light chain CDR1 to CDR3 (1-1) below, or 1) A heavy chain CDR1 to 3 of (1) and a light chain CDR1 to 3 of (2-1) below (hereinafter, sometimes referred to as “the present monoclonal antibody”), and preferably (1) A heavy chain variable region containing heavy chain CDR1 to 3 of 1) and a light chain variable region containing light chain CDR1 to 3 of (1-1) below, or a heavy chain of (2-1) below It comprises a heavy chain variable region containing CDR1 to 3 and a light chain variable region containing light chain CDR1 to 3 of (2-1) below.
  • Heavy chain complementarity determining region consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence
  • a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence
  • SEQ ID NO: 3 Or a heavy chain CDR3 comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence
  • a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence
  • the amino acid sequence represented by SEQ ID NO: 5 Or a light chain CDR2 consisting of an amino acid sequence in which one or more amino acids are substituted
  • the epitope of the monoclonal antibody of the present invention includes human MARCKS protein (specifically, protein having the amino acid sequence of SEQ ID NO: 22; the same applies hereinafter) and mouse MARCKS protein (specifically, protein having the amino acid sequence of SEQ ID NO: 23; In the following, the same), amino acid residues 35 to 48 correspond to the amino acid residue (ENGHVKVNGDA [pS] PA) in which the 46th serine residue of MARCKS is phosphorylated.
  • the present monoclonal antibody does not specifically bind to the non-phosphorylated MARCKS antigen peptide (ENGHVKVNGDASPA).
  • the present monoclonal antibody does not specifically bind to the human MARCKS protein and the mouse MARCKS protein in which the 46th serine residue is not phosphorylated, and the human MARCKS protein and the mouse in which the 46th serine residue is phosphorylated. It specifically binds to the MARCKS protein.
  • “specifically bind to MARCKS protein” means to recognize and bind to MARCKS protein by a recognition mechanism having high specificity between an antigen and an antibody.
  • the monoclonal antibody of the present invention is preferably isolated.
  • the term "separated” as used herein means that the antibody originally exists by artificially removing the antibody from the environment in which it originally exists, or expressing it in an environment different from the environment in which the antibody originally exists. It means that it exists in a state different from that in which it is operating. That is, the "separated antibody” is an antibody derived from a certain individual and is not subjected to any external manipulation (artificial manipulation), and the tissue or body fluid (blood or blood) in or inside the body of the individual.
  • the monoclonal antibody of the present invention is preferably an antibody produced by an organism or a cell produced by artificial manipulation (for example, an antibody produced by a hybridoma).
  • antibody produced by an organism or cell produced by artificial manipulation does not include an antibody produced by a naturally occurring organism (non-artificially manipulated) or B cell.
  • the term “monoclonal antibody” as used herein means a substantially homogeneous antibody (including a functional fragment of an antibody).
  • a monoclonal antibody recognizes a single determinant on the antigen.
  • the “antibody” in the present invention includes a class and a subclass of human immunoglobulin, and also includes a form of a functional fragment of such an antibody.
  • the class and subclass of the present monoclonal antibody include IgG such as IgG1, IgG2, IgG3, and IgG4; IgA such as IGA1 and IGA2; IgD; IgE; IgM; and the like, among which IgG and IgM are preferable examples. can do.
  • a framework region (FR) is usually linked to the amino (N) terminal and the carboxyl (C) terminal of each region of the heavy chain CDR1 to 3 and the light chain CDR1 to 3 in the monoclonal antibody of the present invention.
  • the heavy chain FR includes a heavy chain FR1 linked to the N terminus of heavy chain CDR1; a heavy chain FR2 linked between the C terminus of heavy chain CDR1 and the N terminus of heavy chain CDR2; The heavy chain FR3 linked between the C-terminus of the chain CDR2 and the N-terminus of the heavy chain CDR3; and the heavy chain FR4 linked to the C-terminus of the heavy chain CDR3;
  • the light chain FR includes a light chain FR1 linked to the N-terminus of the light chain CDR1; a light chain FR2 linked between the C-terminus of the light chain CDR1 and the N-terminus of the light chain CDR2.
  • the amino acid sequence and its length may be any as long as the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 in the monoclonal antibody of the present invention are capable of binding to the phosphorylated MARCKS antigen peptide due to their proximity to each other.
  • a heavy chain FR1 consisting of amino acid residues 1 to 30 of the amino acid sequence shown in SEQ ID NO: 13 or 15, or a sequence identity of 80% or more with said amino acid residue
  • a heavy chain FR1 consisting of an amino acid sequence having: a heavy chain FR2 consisting of amino acid residues 36 to 49 of the amino acid sequence shown in SEQ ID NO: 13 or 15, or an amino acid having 80% or more sequence identity with the amino acid residue
  • Heavy chain FR3 consisting of: a heavy chain FR4 consisting of amino acid residues 110 to 120 of the amino acid sequence shown in SEQ ID NO: 13 or 15, or a heavy chain consisting of an amino acid sequence having 80% or more sequence identity with said amino acid residue
  • Chain FR3 consist
  • those containing the heavy chain CDR1 to CDR3 of (1-1) above and the light chain CDR1 to CDR3 of (1-1) above are the heavy chain of (1-2) below.
  • a variable region and a light chain variable region of the following (1-2) and / or a heavy chain of the following (1-3) and a light chain of the following (1-3) are preferable.
  • those containing the heavy chain CDR1 to 3 of (2-1) and the light chain CDR1 to 3 of (2-1) above are further described in (2-2) below.
  • a heavy chain variable region and a light chain variable region of the following (2-2) and / or a heavy chain of the following (2-3) and a light chain of the following (2-3) are preferable. ..
  • the present monoclonal antibody includes human chimeric antibody or mouse chimeric antibody, humanized antibody or mouse antibody, fully human antibody or fully mouse antibody, and among them, humanized antibody and fully human antibody are preferable.
  • the “human chimeric antibody” links a variable region of an antibody derived from a non-human animal (eg, a non-human mammal such as chicken, mouse, rat, bovine) and a constant region of a human-derived antibody.
  • a non-human animal eg, a non-human mammal such as chicken, mouse, rat, bovine
  • the “mouse chimeric antibody” includes a variable region of an antibody derived from a non-mouse animal (for example, a non-mouse mammal such as chicken, rat, bovine) and a constant region of the antibody derived from a mouse. It means a linked antibody.
  • a human chimeric antibody immunizes a non-human animal (preferably a non-human mammal) with a phosphorylated MARCKS antigen peptide, and the antibody variable region (variable region) that binds to the phosphorylated MARCKS antigen peptide is derived from the gene of the obtained antibody. It can be obtained by excising and binding to an antibody constant region (constant region) gene derived from human bone marrow, incorporating this into an expression vector, introducing it into a host, and producing it (for example, JP-A-8-280387). , U.S. Pat. No. 4,816,397, U.S. Pat. No. 4,816,567, U.S. Pat. No. 5,807,715).
  • human constant regions of human chimeric antibodies include C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4, C ⁇ , C ⁇ , C ⁇ 1, C ⁇ 2, and C ⁇ in the heavy chain, and C ⁇ and C ⁇ in the light chain. ..
  • the amino acid sequences of these constant regions and the base sequences encoding them are known. Further, in order to improve the stability of the antibody itself or the stability of antibody production, one or more amino acids in the human-derived antibody constant region can be substituted, deleted, added and / or inserted. ..
  • humanized antibody means that the gene sequence of CDR of an antibody derived from a non-human animal (for example, a non-human mammal such as chicken, mouse, rat, cow, etc.) is transferred to a human-derived antibody gene ( CDR-grafted antibody.
  • human-derived antibody gene CDR-grafted antibody.
  • mouse antibody means that the gene sequence of CDR of an antibody derived from a non-mouse animal (for example, a non-mouse mammal such as chicken, rat, cow, etc.) is transferred to a mouse-derived antibody gene ( CDR-grafted antibody.
  • a method for producing a humanized antibody is publicly known, such as overlap extension PCR (eg, European Patent Application Publication No.
  • variable region of an antibody is usually composed of three CDRs sandwiched by four framework regions (FR).
  • CDRs are essentially the regions that determine the binding specificity of an antibody. While the CDR amino acid sequences are highly diverse, the FR-constituting amino acid sequences often show high homology even among antibodies having different binding specificities. Therefore, it is generally said that the grafting of CDR allows the binding specificity of one antibody to be transferred to another antibody.
  • a human FR having high homology with the FR derived from the non-human animal is selected. That is, the amino acids in the CDRs not only recognize the antigen but also coordinate with the amino acids of the FRs in the vicinity of the CDRs and are involved in maintaining the CDR loop structure. It is preferable to use a human FR having an amino acid sequence highly homologous to the amino acid sequence of the FR.
  • a search system For searching for known human FRs highly homologous to FRs derived from non-human animals, for example, a search system (http://www.bioinf.org.uk/abesis/) specialized for antibodies available on the Internet is used. It can be done using. A mutation can be introduced into a sequence other than the CDR of the non-human-derived antibody so as to match the sequence of the human FR thus obtained. Alternatively, if a gene (cDNA) encoding the amino acid sequence of human FR obtained by the search is available, a non-human CDR may be introduced into the sequence. Mutations can be introduced using techniques known in the art such as nucleic acid synthesis and site-directed mutagenesis.
  • the affinity of the humanized antibody thus produced for an antigen is qualitatively or quantitatively measured and evaluated, whereby the CDR forms a favorable antigen-binding site when linked via the CDR.
  • the FR of such a human-derived antibody can be suitably selected. If necessary, Sato, K .; et al. , Cancer Res, 1993, 53, 851-856, etc., the amino acid residue of FR can be substituted so that the CDR of the humanized antibody forms an appropriate antigen-binding site.
  • a mutant FR sequence having a desired property can be selected by measuring and evaluating the affinity of the mutant antibody having amino acid substitutions for the antigen.
  • fully human antibody means an antibody in which all amino acid sequences are human-derived amino acid sequences.
  • complete mouse antibody means an antibody in which all amino acid sequences are mouse-derived amino acid sequences.
  • fully human antibodies can be made in transgenic mice that have been engineered to express human heavy and light chain antibody genes.
  • a method for preparing a transgenic mouse producing a human antibody is described in, for example, WO 02/43478, US Pat. No. 6,657,103 (Abgenix), and the like.
  • B cells from transgenic mice that produce the desired antibody can then be fused to create a hybridoma cell line for continuous production of the antibody.
  • the present monoclonal antibody has a so-called “Y” -shaped four-chain structure (two polypeptide chains of a light chain and a heavy chain, two of which are linked by a disulfide bond in each constant region).
  • An antibody consisting of a basic structure having a disulfide-bonded four chain at each heavy chain hinge site hereinafter, may be referred to as “basic antibody” for convenience), and a part of the antibody (partial fragment)
  • a functional fragment that specifically binds to the phosphorylated MARCKS antigen peptide is also included.
  • Such functional fragments include Fab, F (ab ′) 2 , Fab ′, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2 , and polymers thereof. Can be mentioned.
  • the “Fab” means two fragments on the N-terminal side among the three fragments obtained by digesting a basic antibody with papain, and more specifically, a heavy chain fragment containing a variable region of a heavy chain,
  • the light chain fragment containing the variable region of the light chain means a double chain linked by a disulfide bond in each constant region.
  • Fab can also be obtained by a recombinant method.
  • F (ab ′) 2 means the N-terminal side fragment of the two fragments obtained by digesting the basic antibody with pepsin, and more specifically, the variable region of the heavy chain and the hinge.
  • a heavy chain fragment containing a site and a light chain fragment containing a variable region of a light chain are linked with a disulfide bond in each constant region.
  • Two of the two chains are linked with a disulfide bond at the heavy chain hinge site.
  • the “Fab ′” means a double chain obtained by cleaving the disulfide bond at the heavy chain hinge site of F (ab ′) 2 with a reducing agent.
  • Fv means the minimum antibody fragment having a complete antigen recognition and binding site.
  • Fv is a dimer in which a heavy chain variable region and a light chain variable region are strongly linked by a non-covalent bond.
  • Single-chain Fv (scFv)” comprises the heavy and light chain variable regions of an antibody, and these regions are present in a single polypeptide chain.
  • Sc (Fv) 2 is a single chain formed by linking two heavy chain variable regions and two light chain variable regions with a linker or the like.
  • the present monoclonal antibody has a modified (eg, glycosylation, acetylation, phosphorylation, lipid addition) or modification (eg, amino acid substitution, deletion) of its amino acid sequence without decreasing the affinity for the phosphorylated MARCKS antigen peptide. Can be lost, added and / or inserted).
  • modified or modified amino acid sequences are prepared, for example, by introducing mutations into the DNAs encoding the antibody chains of the two types of monoclonal antibodies (A4H7 or F8H5) shown in this Example, or by peptide synthesis. be able to.
  • the heavy chain of the antibody may be used, or the variable regions (FR and CDR) may be used, but the constant region is preferable.
  • modification or modification of amino acids other than CDR is considered to have relatively little effect on the affinity with phosphorylated MARCKS antigen peptide, currently, modification or modification of CDR amino acids results in higher affinity to the antigen.
  • the present monoclonal antibody may be an antibody containing at least one CDR of any one antibody selected from the group consisting of the two types of monoclonal antibodies (A4H7 or F8H5) shown in this Example.
  • the number of modified or altered amino acids is preferably 10 amino acids or less, more preferably 5 amino acids or less, and further preferably 3 amino acids or less (eg, 2 amino acids or less, 1 amino acid).
  • a plurality of amino acids in “a plurality of amino acids are substituted, deleted, added and / or inserted” means that the number of amino acid residues in CDR is several to ten or more. It is usually 3 or less amino acids, preferably 2 or less amino acids, more preferably 1 amino acid. Amino acid substitutions, deletions, additions and / or insertions (ie modifications) are preferably conservative substitutions. As used herein, “conservative substitution” means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the art to which the present invention pertains.
  • acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), and neutral amino acids having a hydrocarbon chain (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy group
  • amino acids serine / threonine
  • amino acids containing sulfur (cysteine / methionine)
  • amino acids with amide groups amino acids with imino groups (proline)
  • amino acids with aromatic groups phenylalanine / tyrosine / Tryptophan
  • 80% or more sequence identity means that the ratio of amino acid sequences identical to the amino acid sequence to be compared is at least 80%, preferably 85% or more, and more preferably 88. % Or more, more preferably 90% or more, even more preferably 93% or more, particularly preferably 95% or more, particularly preferably 98% or more, most preferably 99% sequence identity.
  • the identity of amino acid sequences can be determined using a BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl.
  • the subject monoclonal antibody can be produced by a known hybridoma method or a known recombinant DNA method.
  • Typical examples of the hybridoma method include Kohler and Milstein's method (Kohler & Milstein, Nature, 256: 495 (1975)).
  • a Cys residue for carrier protein conjugate is added, and the Cys residue is a carrier protein (eg, KLH, BSA [Bovine serum albumin]).
  • phosphorylated MARCKS antigen peptide / carrier protein complex (Ie, “phosphorylated MARCKS antigen peptide / carrier protein complex”) is immunized into a non-human mammal (eg, mouse, rat, hamster, rabbit, monkey, goat), and then Antibody-producing cells (eg, spleen cells, lymph node cells, peripheral blood leukocytes) isolated from the immunized non-human mammal can be used in the cell fusion step.
  • the antibody-producing cells used in the cell fusion step may be those isolated from a non-immunized non-human mammal and then cultured in the presence of a phosphorylated MARCKS antigen peptide / carrier protein complex.
  • the myeloma cells used in the cell fusion step various known cell lines can be used.
  • the antibody-producing cells and myeloma cells may be from different animal species, provided that they can be fused, but are preferably from the same animal species.
  • a hybridoma is produced by, for example, cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and is screened by ELISA using a plate on which phosphorylated MARCKS antigen peptide is immobilized.
  • a hybridoma producing a monoclonal antibody that specifically binds to the phosphorylated MARCKS antigen peptide can be obtained.
  • the human monoclonal antibody that specifically binds to the phosphorylated MARCKS antigen peptide can be obtained by culturing the hybridoma or from the ascites of the mammal to which the hybridoma is administered.
  • the hybridoma is preferably subcloned multiple times (at least once, preferably at least twice, more preferably at least three times).
  • an antibody gene encoding the monoclonal antibody of the present invention is cloned from a hybridoma, B cell or the like and incorporated into an appropriate vector, which is then used as a host cell (eg, mammalian cell line such as HEK cell, E. coli, yeast, etc. Cells, insect cells, plant cells, etc.) to produce the monoclonal antibody of the present invention as a recombinant antibody (eg, PJ Delves, Antibody Products: Essential Technologies, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem.
  • a host cell eg, mammalian cell line such as HEK cell, E. coli, yeast, etc. Cells, insect cells, plant cells, etc.
  • a recombinant antibody eg, PJ Delves, Antibody Products: Essential Technologies, 1997 WILEY, P.
  • the antibody gene encoding the present monoclonal antibody may be separately incorporated into an expression vector to transform a host cell.
  • the gene may be incorporated into a single expression vector to transform a host cell (see International Publication WO94 / 11523).
  • the monoclonal antibody of the present invention can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cell, separating and purifying from within the host cell or from the culture solution. For the separation and purification of the antibody, the method used in the purification of ordinary polypeptides can be used.
  • transgenic animals (cattle, goats, sheep, pigs, etc.) into which the subject monoclonal antibody gene is incorporated are produced using transgenic animal production technology
  • the subject monoclonal antibody gene can be obtained from the blood or milk of the transgenic animal. A large amount of the derived monoclonal antibody can also be obtained.
  • the prepared monoclonal antibody of the present invention does not specifically bind to the MARCKS protein in which the 46th serine residue is not phosphorylated in human or mouse, but specifically to the MARCKS protein in which the 46th serine residue is phosphorylated.
  • the binding can be confirmed using an immunological assay method such as immunohistochemical staining method, Western blotting method, and ELISA.
  • the diagnostic agent for neurite degenerative disease of the present invention includes the monoclonal antibody or its labeled product of the present invention, which is specified for the purpose of "for diagnosing neurite degenerative disease” (hereinafter, referred to as "the diagnostic agent of the present invention”).
  • the present diagnostic agent further includes a carrier; a pH buffering agent; a stabilizer; an instruction manual, for diagnosing neurite degenerative disease.
  • a diagnostic agent diagnostic kit
  • an adjunct such as an instruction sheet of
  • the monoclonal antibody of the present diagnostic agent is preferably a humanized antibody or a fully human antibody.
  • the monoclonal antibody of the present invention is capable of detecting pSer46-MARCKS in the brain of a neurite degeneration model animal, as shown in the Examples below.
  • the present inventors have found that pSer46-MARCKS is detected before extracellular accumulation of amyloid ⁇ in the brain of a neurite degeneration model animal, and therefore pSer46-MARCKS is an early diagnosis of neurite degeneration disease. It has been reported to be useful as a biomarker for medical use (Non-patent document 3).
  • the subject monoclonal antibody or its labeled substance was administered to a subject, and pSer46-MARCKS (in other words, "human MARCKS protein in which the 46th serine residue was phosphorylated") in the brain of the subject was examined.
  • pSer46-MARCKS in other words, "human MARCKS protein in which the 46th serine residue was phosphorylated"
  • the labeled substance of the present monoclonal antibody may be a substance in which a labeling substance is conjugated to the present monoclonal antibody (covalent bond or non-covalent bond).
  • labeling substance include peroxidase (eg, horseradish peroxidase), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malic acid.
  • Enzymes such as dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothiocyanate; 18 F, 15 O, 13 N, 11 C, 82 Rb, 68 Ga, 198 Au, 199 Au, 32 P, 33 P, 125 I, 131 I, 123 I, 90 Y, 186 Re, 188 Re, 62 Cu, 64 Cu, 67 Cu, 211 At , 47 Sc, 103 Pb, 109 Pb, 212 Pb, 71 Ge, 77 As, 105 Rh, 113 Ag, 119 Sb, 121 Sn, 131 Cs, 143 Pr, 161 Tb, 177 Lu , 191 Os, 193 Pt
  • neurite degenerative disease refers to degeneration (specifically, regression) of neurites (also referred to as “axons”) in nerve cells (neurons) of the central nervous system (ie, brain and spinal cord). , Loss). Degeneration of neuronal neurites leads to the loss of proper networks between neurons, ie loss of neural function (eg, sensory, memory, attention, executive functions). Since neurite degenerative disease has pSer46-MARCKS found in the early stage of development (Non-patent Document 3), it can be said to be a neurite degenerative disease accompanied by phosphorylation of MARCKS in the central nervous system (at least pSer46-MARCKS).
  • neurite degenerative disease examples include Alzheimer's disease, Parkinson's disease, Frontotemporal lobar degeneration (FTLD), Lewy body dementia, Huntington's disease, and amyotrophic lateral sclerosis (ALS). Amyotrophic lateral sclerosis), spinocerebellar ataxia (SCA), and the like.
  • FTLD Frontotemporal lobar degeneration
  • ALS amyotrophic lateral sclerosis
  • SCA spinocerebellar ataxia
  • Alzheimer's disease, Parkinson's disease, frontotemporal lobar degeneration, and Lewy body dementia are preferably exemplified. can do.
  • the method of administering the subject monoclonal antibody or its labeled product to a subject may be any method by which the subject monoclonal antibody or its labeled product reaches the brain of the subject, for example, a vein.
  • Internal administration, intraarterial administration, and local administration can be mentioned, and of these, intravenous administration can be preferably exemplified.
  • the dose of the monoclonal antibody or the labeled product of the present invention may vary depending on the age, weight, sex, health condition, etc. of the subject, but in general, for adults, 0.1 to 1000 mg / kg body weight per day, preferably 1 to 100 mg / day. Is.
  • pSer46-MARCKS When pSer46-MARCKS is detected in the brain of a subject administered with the present monoclonal antibody or its labeled product, the subject is diagnosed as likely to develop a neurite degenerative disease, and pSer46-MARKKS is detected. If not, the subject can be diagnosed as less likely to develop a neurite degenerative disease.
  • the presence or absence of pSer46-MARCKS is the presence or absence of an index (eg, enzyme activity level, color development level, luminescence [fluorescence] level, radioactivity level; the same applies hereinafter) derived from the labeling substance when the labeled product of the monoclonal antibody of the present invention is used. It can be diagnosed (determined) based on.
  • a labeled substance of a substance that binds to the monoclonal antibody of the present invention is administered to a subject before, at the same time as, or after the administration of the monoclonal antibody of the present invention, and the label of the labeled substance It is possible to make a diagnosis (determination) based on the presence or absence of an index derived from a substance.
  • the monoclonal antibody gene of the present invention is not particularly limited as long as it is an antibody gene encoding the present monoclonal antibody (hereinafter, sometimes referred to as “the present monoclonal antibody gene”), and the nucleotide sequence of the present monoclonal antibody gene is the present monoclonal antibody.
  • the amino acid sequence of the antibody and the known codon table those skilled in the art can specifically and clearly grasp the nucleotide sequence corresponding to the amino acid sequence.
  • the vector of the present invention is not particularly limited as long as it is a vector containing a promoter and the subject monoclonal antibody gene operably linked to the downstream of the promoter (hereinafter, sometimes referred to as “the subject vector”)
  • the host cell of the present invention is not particularly limited as long as it is a host cell into which the vector of the present invention has been introduced (hereinafter sometimes referred to as “the host cell of the present case”).
  • the subject vector can be appropriately selected depending on the type of the subject host cell (or host organism) to be introduced.
  • the term "gene operably linked to the downstream of a promoter” means that the promoter DNA and the gene DNA are functionally linked so that the promoter can initiate transcription of the gene. Means connected.
  • the promoter in the subject vector may be any region that initiates transcription of mRNA encoded by the subject monoclonal antibody gene located downstream of the promoter, and the promoter usually includes a transcription start point (TSS).
  • TSS transcription start point
  • the subject vectors include, for example, pcDNAI, pcDM8 (Funakoshi). Manufactured by K.K.), pAGE107 (JP-A-3-22979; Cytotechnology, 3,133, (1990)), pAS3-3 (JP-A-2-227075), pCDM8 (Nature, 329,840, (1987)), pcDNAI / Amp.
  • pAGE210 J. Biochemistry, 101, 1307 (1987)
  • vectors such as pAGE210, and those derived from such vectors
  • the promoter include the site. Examples include a promoter of an IE (immediate early) gene of megalovirus (CMV), an SV40 early promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, an SR ⁇ promoter and the like.
  • CMV immediate early gene of megalovirus
  • SV40 early promoter a retrovirus promoter
  • a metallothionein promoter a metallothionein promoter
  • heat shock promoter an SR ⁇ promoter and the like.
  • the vector of the present invention further contains a nucleotide sequence of an enhancer region or a ribosome binding site (RBS) in order to further enhance the gene expression efficiency, and the type of the host cell of the present invention for screening the host cell.
  • a drug resistance gene for example, spectinomycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, Those further containing a geneticin resistance gene etc.
  • the enhancer region is usually located upstream of the promoter, and the RBS is usually located between the promoter and the subject gene.
  • the nucleotide sequence of the subject antibody gene to be incorporated into the subject vector may have a codon sequence optimized for the subject host cell to be expressed.
  • the subject vector can be prepared by a known method using a gene recombination technique.
  • the biological species of the subject host cell may be any one as long as mRNA of the subject antibody gene is transcribed and the subject monoclonal antibody protein is expressed, and examples thereof include mammals (eg, human, mouse, rat, monkey, etc.), yeast. (For example, Saccharomyces Cerevisiae, Schizosaccharomyces Pombe, etc.) are mentioned, and among them, mammals can be preferably exemplified.
  • the subject host cell can be obtained by introducing (transfecting) the subject vector into the host cell by a method depending on the type of the host cell.
  • the method for introducing the vector of the present invention into the mammalian cells may be any method for introducing DNA into the mammalian cells, and examples thereof include electroporation (Cytotechnology, 3,133). (1990)), calcium phosphate method (JP-A-2-227075), lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)) and the like. ..
  • a method of producing the present monoclonal antibody which comprises culturing the present host cell and recovering the present monoclonal antibody, is also included in the embodiments of the present invention.
  • Example 1 Preparation of Monoclonal Antibody that Specifically Binds pSer46-MARCKS To ALB / C mice, an antigenic peptide containing pSer46-MARCKS and having an amino acid sequence around pSer46-MARCKS consisting of a full-length 14 amino acid sequence common to humans and mice was prepared. After immunization, spleen cells were prepared to prepare a hybridoma that produces a monoclonal antibody that specifically binds to pSer46-MARCKS. Then, a monoclonal antibody that specifically binds to pSer46-MARCKS was purified from the hybridoma.
  • a phosphorylated MARCKS antigen peptide (ENGHVKVNGDA [pS] PA) was used as an antigen to immunize mice. Cys residue for carrier protein conjugation was added to the N-terminal of phosphorylated MARCKS antigen peptide, and the carrier protein (KLH) was conjugated to such Cys residue (hereinafter referred to as “phosphorylated MARCKS antigen peptide / KLH complex”). 6) BALB / C mice (6 weeks old) were initially immunized with an emulsion obtained by mixing 50 to 100 ⁇ g of the “body” with an adjuvant (CFA; Complete Freund's Adjuvant) (manufactured by Sigma).
  • CFA Complete Freund's Adjuvant
  • emulsions prepared by mixing 50 to 100 ⁇ g of phosphorylated MARCKS antigen peptide / KLH complex and an adjuvant (IFA; Incomplete Freund's Adjuvant) (manufactured by Sigma) were respectively immunized. (2nd and 3rd immunization). Blood was collected 45 days after the first immunization, serum was prepared according to a standard method, and the serum antibody titer of the antiserum was confirmed according to the method described in the following items.
  • IFA Incomplete Freund's Adjuvant
  • Non-phosphorylated MARCKS peptide [ELISA] 2 ⁇ g / mL of phosphorylated MARCKS antigen peptide and 2 ⁇ g / mL of non-phosphorylated MARCKS antigen peptide (ENGHVKVNGDASPA; hereinafter simply referred to as “non-phosphorylated MARCKS peptide”) as a control, each in a 96-well microplate (manufactured by Nunc) 50 ⁇ L / well, and the mixture was incubated at 4 ° C. for 12 hours. After that, 2% Block Ace (manufactured by DS Pharma) was added at 200 ⁇ L / well to perform blocking treatment.
  • 2% Block Ace manufactured by DS Pharma
  • the serum prepared from the immunized mouse or the hybridoma culture supernatant was diluted 10000 times and 100 times with 0.1% Block Ace / PBS solution to prepare a serum sample or a hybridoma culture supernatant sample.
  • Each sample was added at 50 ⁇ L / well and incubated at room temperature for 2 hours to perform an antigen-antibody reaction treatment.
  • PBST Tween20-containing PBS
  • 2 ⁇ g / mL peroxidase-conjugated goat anti-mouse IgG manufactured by Jackson ImmunoResearch laboratories
  • TMB substrate manufactured by Thermo Fisher Scientific
  • 0.18 M sulfuric acid was added in an amount of 50 ⁇ L / well to stop the color reaction, and then the absorbance at 450 nm and 540 nm was measured by a plate reader (Bio-Rad). Quantitation was performed using a correction value obtained by subtracting the measurement value of 540 nm from the measurement value of 450 nm.
  • a positive control a positive antiserum collected before fusion was measured, and as a negative control, 3% skim milk was measured.
  • spleen cells were isolated from the spleen cells of the mouse having the highest antiserum serum antibody titer among the immunized mice, and polyethylene glycol 4000 was used to isolate P3U1 myeloma cells at a ratio of 1: 1. Fused. The fused cells were seeded in a 96-well plate at 5000 cells / well, and the cells were mixed with HAT selection medium (10% FCS-containing RPMI containing 0.1 mM hypoxanthine, 0.016 mM thymidine, 0.4 nM aminopterin) for 10 times.
  • HAT selection medium (10% FCS-containing RPMI containing 0.1 mM hypoxanthine, 0.016 mM thymidine, 0.4 nM aminopterin
  • Antibodies produced in the supernatants of hybridomas grown for a day and grown were detected according to the method described in the item of [ELISA] above.
  • Five kinds of hybridomas (2A9, 2H6, 4E6, 4F7, and 4F8) that showed positive results by ELISA were re-distributed to 0.2 cells / well, and the HT selective culture medium (0.1 mM hypoxanthine, 0.
  • the antibody produced in the supernatant of the hybridoma that had been cultured in 10% FCS-containing RPMI containing 016 mM thymidine) was detected according to the method described in the item of [ELISA] above.
  • hybridomas Three types of hybridomas (2A9, 2H6, and 4E6) that showed positive results by ELISA were cloned, and the antibody produced in the supernatant of the grown hybridomas was detected according to the method described in the item [ELISA] above.
  • two types of hybridoma clones (4E6E1 and 2H6C8) that produce an antibody that binds to the phosphorylated MARCKS antigen peptide without binding to the non-phosphorylated MARCKS peptide were obtained (see Table 1).
  • Total RNA was prepared from the above two types of hybridoma clones (A4H7 and F8H5) according to a standard method, RT-PCR was performed according to the standard method, and the amino acid sequences of the variable regions of the heavy chain and the light chain of the produced antibody were analyzed by DNA sequencing according to the standard method. Identified (see Tables 3 and 4). Also, antibody heavy chain CDRs 1, 2 and 3 are present at positions 31-35B, 50-65, and 95-102, respectively, according to kabat numbering, and antibody light chain CDRs 1, 2, and 3 are numbered according to kabat.
  • Each monoclonal antibody was purified from 10 mL of the culture supernatant of the above two types of hybridoma clones (A4H7 and F8H5) by affinity chromatography using protein G as a ligand (column: HiTrap protein G 1 mL).
  • affinity chromatography each monoclonal antibody was adsorbed to the column in the presence of 20 mM PBS (pH 7.2), the column was washed with 20 mM PBS (pH 7.2), and then 0.1 M glycine-HCl buffer (pH 2.7) was used. ) was used to elute the antibody.
  • the eluate was neutralized with 1 M Tris-HCl (pH 7.5) and then replaced with phosphate buffer (PBS) by ultrafiltration concentration operation to purify the monoclonal antibody.
  • PBS phosphate buffer
  • Example 2 Detection of pSer46-MARCKS in neurite degeneration disease model animal Since the present inventors have detected pSer46-MARCKS before extracellular amyloid ⁇ accumulation in the brain of the neurite degeneration model animal, pSer46- It has been reported that MARCKS is useful as a biomarker for early diagnosis of neurite degenerative disease (Non-patent Document 3). Therefore, it was confirmed whether or not the monoclonal antibody prepared in Example 1 above can detect pSer46-MARCKS in a neurite degeneration disease model animal.
  • a 5 ⁇ FAD (five Familial Alzheimer's Disease) transgenic mouse (hereinafter, referred to as “5 ⁇ FAD mouse”) that is an Alzheimer's disease model mouse (see “J Neurosci. 2006 Oct 4; 26 (40) 10129-10140”) was prepared. , Jackson Laboratory (Bar Harbor, Maine, USA) and used as a neurite degeneration disease model animal. The B6 / SJL mouse, which is the background of the model mouse, was used as a control. Since B6 / SJL mice are not sold, B6 (C57BL / 6) was purchased from Sankyo Lab Service Co., Ltd. and SJL from Oriental Yeast Co., Ltd., and B6 and SJL were crossed to produce B6 / SJL mice. Mice were created.
  • Brains were excised from 6-month 5 ⁇ FAD mice and B6 / SJL mice respectively, fixed with 0.1M PBS containing 4% paraformaldehyde for 24 hours, and then 0.1M PBS containing 15% sucrose. It was immersed in it for 2 days. After that, a 5 ⁇ m thick paraffin section was prepared using a microtome (manufactured by Daiwa Koki Kogyo Co., Ltd.), and the two kinds of monoclonal antibodies (A4H7 [1.4 mg / mL] prepared in Example 1 above, dilution ratio 1:50.
  • Alexa Fluor-568 fluorescence signal derived from pSer46-MARKKS is detected by staining the cell nucleus in PBS containing (4 ', 6-diamidino-2-phenylindole) and using a laser scanning microscope (FV1200, Olympus). Image (pSer46-MARKKS image) and an image for detecting the DAPI fluorescence signal derived from the cell nucleus (DAPI image) were acquired.
  • the monoclonal antibody of the present invention can be used to diagnose neurite degenerative diseases before extracellular amyloid ⁇ accumulation (that is, in the early stage) using the presence or absence of pSer46-MARCKS in brain tissue as an index. There is. Since the epitope of the monoclonal antibody of the present invention, that is, the amino acid sequence of the phosphorylated MARCKS antigen peptide is the same in human and mouse, the monoclonal antibody of the present invention is specific to pSer46-MARKKS even in human brain tissue. It is considered to be useful for early diagnosis of neurite degenerative disease.
  • the present invention contributes to early detection and early treatment of neurite degenerative diseases, improvement of QOL, reduction of medical costs, and the like.

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Abstract

Le but de la présente invention est de fournir un anticorps monoclonal, etc., capable de détecter de manière spécifique la présence/l'absence de phosphorylation de la protéine MARCKS dans le cerveau à l'étape initiale d'apparition d'une maladie neurodégénérative.　La présente invention permet d'utiliser un anticorps monoclonal qui se lie à un épitope phosphorylé spécifique dans MARCKS et qui comprend une CDR1 de chaîne lourde, une CDR2 de chaîne lourde et une CDR3 de chaîne lourde comprenant chacune une séquence d'acides aminés spécifique, et une CDR1 de chaîne légère, une CDR2 de chaîne légère et une CDR3 de chaîne légère comprenant chacune une séquence d'acides aminés spécifique, permettant ainsi de diagnostiquer une maladie neurodégénérative dans une étape précoce d'apparition de celle-ci par l'utilisation de la présence/l'absence de phosphorylation de la protéine MARCKS dans le cerveau en tant qu'indicateur.
PCT/JP2019/044042 2018-11-12 2019-11-11 Anticorps monoclonal se liant de manière spécifique à pser46-marcks WO2020100779A1 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FUJITA, K. ET AL.: "HMGB1, a pathogenic molecule that induces neurite degeneration via TLR4-MARCKS, is a potential therapeutic target for Alzheimer's disease", SCI. REP., vol. 6, 2016, pages 31895, XP055463477 *
FUJITA, K. ET AL.: "Ser46-Phosphorylated MARCKS Is a Marker of Neurite Degeneration at the Pre- aggregation Stage in PD/DLB Pathology", ENEURO, vol. 5, no. 4, July 2018 (2018-07-01), pages e 0217 - 18, XP055708480 *

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