WO2020100779A1 - Monoclonal antibody specifically binding to pser46-marcks - Google Patents

Monoclonal antibody specifically binding to 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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PCT/JP2019/044042
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Japanese (ja)
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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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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.

Abstract

The purpose of the present invention is to provide a monoclonal antibody, etc., capable of specifically detecting the presence/absence of phosphorylation of MARCKS protein in the brain in the initial stage of onset of a neurodegenerative disease.　Through the present invention, when a monoclonal antibody is used that binds to a specific phosphorylated epitope in MARCKS and that includes a heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each comprising a specific amino acid sequence, and a light chain CDR1, light chain CDR2, and light chain CDR3 each comprising a specific amino acid sequence, a neurodegenerative disease in the early stage of onset thereof can be diagnosed using the presence/absence of phosphorylation of MARCKS protein in the brain as an indicator.

Description

ｐＳｅｒ４６－ＭＡＲＣＫＳに特異的に結合するモノクローナル抗体Monoclonal antibody that specifically binds to pSer46-MARKKS
　本発明は、ｐＳｅｒ４６－ＭＡＲＣＫＳに特異的に結合するモノクローナル抗体、前記モノクローナル抗体又はその標識物を含む、神経突起変性疾患の診断剤、前記モノクローナル抗体をコードするモノクローナル抗体遺伝子、プロモーターと、前記モノクローナル抗体遺伝子とを含むベクター、及び前記ベクターが導入されている宿主細胞に関する。 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.
　アルツハイマー病やパーキンソン病に代表される神経変性疾患は、脳内においてタンパク質の凝集が形成され、神経細胞死を引き起こし、最終的に発症すると考えられている。例えば、アルツハイマーの病因及び発症機構については、アミロイドβ（Ａβ）の凝集（アミロイド病変）が生じ、Ａβの凝集によって微小管結合タンパク質であるタウ（ｔａｕ）のリン酸化及び重合（ｔａｕ病変）が促進され、ひいては神経細胞死等に至るという機構（アミロイドカスケード仮説）が有力視されている。しかしながら、Ａβの凝集がどのようなメカニズムで形成され、神経細胞死がどのようなメカニズムで誘導されるかについては解明されておらず、アルツハイマー病に限らず、神経変性疾患全般において、根治薬の提供に至っていないのが現状である。 It is thought that neurodegenerative diseases represented by Alzheimer's disease and Parkinson's disease cause nerve cell death and eventually develop due to the formation of protein aggregates in the brain. For example, regarding the etiology and pathogenic mechanism of Alzheimer's disease, aggregation of amyloid β (Aβ) (amyloid lesion) occurs, and the aggregation of Aβ promotes phosphorylation and polymerization (tau lesion) of the microtubule-binding protein tau. The mechanism (amyloid cascade hypothesis) that leads to neuronal cell death and the like is considered to be promising. However, the mechanism by which Aβ aggregation is formed and the mechanism by which neuronal cell death is induced have not been elucidated, and not only Alzheimer's disease but also neurodegenerative diseases in general can be used as radical medicines. The current situation is that it has not been provided.
　多くの神経変性疾患においては、神経細胞（ニューロン）死と同時に、あるいはそれに先行して神経突起の変性が観察される。ニューロンにおける神経突起の変性は、ニューロン間の適切なネットワークの喪失、すなわち、神経機能の喪失につながることから極めて重要な現象である。 In many neurodegenerative diseases, 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.
　本発明者らは、アルツハイマー病モデルマウス及びアルツハイマー病患者の死後脳を対象に網羅的なプロテオーム解析を行い、アルツハイマー病に共通する異常リン酸化シグナルネットワークの分析を行った。その結果、本発明者らは、ＭＡＲＣＫＳというリン酸化酵素の基質のリン酸化が、アルツハイマー病発症前の早い段階から生じていることを報告している（特許文献１、非特許文献１）。また、本発明者らは、ＭＡＲＣＫＳにおける４６番目のセリン（Ｓｅｒ４６）のリン酸化が、アルツハイマー病発症前の早い段階から生じていること、ニューロンのネクローシスにより細胞内から漏出したＨＭＧＢ１が、ＭＡＲＣＫＳのリン酸化を誘導し、神経突起の変性を誘導するという知見を得て、ＨＭＧＢ１に対するマウスモノクローナル抗体を作製し、かかるマウスモノクローナル抗体が、ＭＡＲＣＫＳのＳｅｒ４６のリン酸化を阻害することを報告している（非特許文献２）。また、本発明者らは、かかるマウスモノクローナル抗体が、アルツハイマー病モデルマウスにおける認識障害を回復すること、大脳皮質におけるＤＮＡ損傷を減少させること、及び、Ａβ及びＨＭＧＢ１双方の多量体形成を阻害することを報告している（特許文献２、非特許文献２）。さらに、本発明者らは、ＭＡＲＣＫＳにおけるＳｅｒ４６のリン酸化は、アルツハイマー病に限らず、パーキンソン病やＬｅｗｙ小体型認知症等の神経突起変性疾患の発症初期においても検出されることを報告している（非特許文献３）。 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. Based on the finding that it induces oxidation and degeneration of neurites, 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 Aβ and HMGB1 multimer formation. Is reported (Patent Document 2, Non-Patent Document 2). Furthermore, the present inventors have reported that 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. (Non-patent document 3).
国際公開第２０１５／０９９０９４号パンフレットInternational Publication No. 2015/099094 Pamphlet 国際公開第２０１８／０３０４０５号パンフレットInternational Publication No. 2018/030405 Pamphlet
　本発明の課題は、神経突起変性疾患の発症初期において、脳内のＭＡＲＣＫＳタンパク質のリン酸化の有無を特異的に検出できるモノクローナル抗体等を提供することにある。 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.
　本発明者らは、上記課題を解決すべく鋭意研究を続けている。その過程において、ＭＡＲＣＫＳタンパク質におけるリン酸化部位のうち、ヒトとマウスで共通する４６番目のリン酸化セリン残基（すなわち、「ｐＳｅｒ４６－ＭＡＲＣＫＳ」）を含み、かつ、ｐＳｅｒ４６－ＭＡＲＣＫＳ周辺のアミノ酸配列がヒトとマウスで共通する全長１４アミノ酸配列からなるリン酸化ＭＡＲＣＫＳ抗原ペプチドのアミノ（Ｎ）末端に、キャリアタンパク質コンジュゲート用のシステイン（Ｃｙｓ）残基を付加させ、そのＣｙｓ残基にキャリアタンパク質であるＫＬＨ（Keyhole limpet hemocyanin）をコンジュゲートしたもの（以下、「リン酸化ＭＡＲＣＫＳ抗原ペプチド・キャリアタンパク質複合体」ということがある）をマウスに免疫すると、非リン酸化型のＭＡＲＣＫＳ抗原ペプチドには結合せずに、リン酸化ＭＡＲＣＫＳ抗原ペプチドに結合するモノクローナル抗体を得ることができ、さらに、かかるモノクローナル抗体は、神経突起変性モデル動物の脳内のｐＳｅｒ４６－ＭＡＲＣＫＳを検出できることを見出し、本発明を完成するに至った。 The inventors of the present invention continue to earnestly research to solve the above problems. In the process, of the phosphorylation site in the MARCKS protein, the 46th phosphorylated serine residue common to human and mouse (ie, “pSer46-MARKKS”) was contained, and the amino acid sequence around pSer46-MARKKS was human. Common to mouse and mouse, a cysteine (Cys) 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. Immunization of 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. ..
　すなわち、本発明は以下のとおりである。
〔１〕ＭＡＲＣＫＳにおけるリン酸化エピトープに結合するモノクローナル抗体であって、
前記ＭＡＲＣＫＳにおけるリン酸化エピトープが、配列番号２１に示されるアミノ酸配列において、１２番目のセリン残基がリン酸化されたアミノ酸配列からなるエピトープであり、
前記モノクローナル抗体が、
（１－１）配列番号１に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖相補性決定領域（ＣＤＲ）１；配列番号２に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ２；及び配列番号３に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ３；と、
配列番号４に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ１；配列番号５に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ２；及び配列番号６に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ３；とを含むか、或いは
（２－１）配列番号７に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ１；配列番号８に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ２；及び配列番号９に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ３；と、
配列番号１０に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ１；配列番号１１に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ２；及び配列番号１２に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ３；とを含む；
ことを特徴とする前記モノクローナル抗体。
〔２〕（１－１）のモノクローナル抗体が、さらに、（１－２）配列番号１３に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域と、配列番号１４に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域とを含み、
（２－１）のモノクローナル抗体が、さらに、（２－２）配列番号１５に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域と、配列番号１６に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域とを含む
ことを特徴とする請求項１に記載のモノクローナル抗体。
〔３〕（１－１）又は（１－２）のモノクローナル抗体が、さらに、（１－３）配列番号１７に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖と、配列番号１８に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖とを含み、
（２－１）又は（２－２）のモノクローナル抗体が、さらに、（２－３）配列番号１９に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖と、配列番号２０に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖とを含む
ことを特徴とする上記〔１〕又は〔２〕に記載のモノクローナル抗体。
〔４〕上記〔１〕～〔３〕のいずれかに記載のモノクローナル抗体又はその標識物を含むことを特徴とする神経突起変性疾患の診断剤。
〔５〕神経突起変性疾患が、アルツハイマー病、パーキンソン病、前頭側頭葉変性症、又はＬｅｗｙ小体型認知症であることを特徴とする上記〔４〕に記載の診断剤。
〔６〕上記〔１〕～〔３〕のいずれかに記載のモノクローナル抗体をコードすることを特徴とするモノクローナル抗体遺伝子。
〔７〕プロモーターと、該プロモーターの下流に作動可能に連結されている上記〔６〕に記載のモノクローナル抗体遺伝子とを含むことを特徴とするベクター。
〔８〕上記〔７〕に記載のベクターが導入されていることを特徴とする宿主細胞。 That is, the present invention is as follows.
[1] A monoclonal antibody which binds to a phosphorylation epitope in MARCKS,
The 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 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, deleted, added and / or inserted in the amino acid sequence; and the amino acid sequence shown in SEQ ID NO: 6, or one or more in the amino acid sequence Or a light chain CDR3 consisting of an amino acid sequence in which the amino acid of is substituted, deleted, added and / or inserted, or (2-1) the amino acid sequence shown in SEQ ID NO: 7, or 1 in the amino acid sequence. Or 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: 12, or one or more in the amino acid sequence A light chain CDR3 consisting of an amino acid sequence in which the amino acid of is substituted, deleted, added and / or inserted;
The monoclonal antibody as described above.
[2] 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. 2. The monoclonal antibody according to claim 1, which comprises a light chain variable region consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence described above.
[3] 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.
[4] 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.
[5] 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.
[6] A monoclonal antibody gene, which encodes the monoclonal antibody according to any one of [1] to [3] above.
[7] A vector comprising a promoter and the monoclonal antibody gene according to the above [6] operably linked to the downstream of the promoter.
[8] A host cell into which the vector according to [7] above has been introduced.
　また本発明の実施の他の形態として、
　本発明のモノクローナル抗体又はその標識物を被験者に投与するステップと、前記被験者の脳において、ｐＳｅｒ４６－ＭＡＲＣＫＳが検出された場合、前記被験者は神経突起変性疾患を発症している可能性が高いと診断し、ｐＳｅｒ４６－ＭＡＲＣＫＳが検出されなかった場合、前記被験者は神経突起変性疾患を発症している可能性が低いと診断するステップとを含む、神経突起変性疾患の診断方法であって、さらに、神経突起変性疾患を発症している可能性が高いと診断された被験者に対して、神経突起変性疾患を治療する処置を施すステップを任意で含む、前記診断方法；や、
　神経突起変性疾患の診断方法におけるバイオマーカーとして使用するための本発明のモノクローナル抗体又はその標識物；や、
　神経突起変性疾患の診断方法における使用のための本発明のモノクローナル抗体又はその標識物；
を挙げることができる。 As another embodiment of the present invention,
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 as a biomarker in a method for diagnosing neurite degenerative disease;
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.
　本発明のモノクローナル抗体は、非リン酸化型のＭＡＲＣＫＳタンパク質には結合せずに、リン酸化型のＭＡＲＣＫＳタンパク質のｐＳｅｒ４６－ＭＡＲＣＫＳに特異的に結合する。また、ｐＳｅｒ４６－ＭＡＲＣＫＳは、発症初期の神経突起変性疾患のバイオマーカーであることから、本発明のモノクローナル抗体を用いた分子イメージング（例えば、ＰＥＴ［positron emission tomography］／ＳＰＥＣＴ［single photon emission computed tomography］）を行い、脳内のｐＳｅｒ４６－ＭＡＲＣＫＳの有無を指標として、発症初期の神経突起変性疾患を診断することができ、神経突起変性疾患の早期発見及び早期治療が可能となり、ＱＯＬ（Quality of Life）の向上や医療費削減等の効果が期待される。 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. In addition, 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.
Ｂ６／ＳＪＬマウスの脳組織について、２種類の本発明のモノクローナル抗体（Ａ４Ｈ７及びＦ８Ｈ５）と、ｐＳｅｒ４６－ＭＡＲＣＫＳに対するポリクローナル抗体とを用いた免疫組織化学染色法を行った結果を示す図である。下段のＭｅｒｇｅ画像は、上段のｐＳｅｒ４６－ＭＡＲＣＫＳ画像と、ＤＡＰＩ画像とを重ねた画像である。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. ５×ＦＡＤマウスの脳組織について、２種類の本発明のモノクローナル抗体（Ａ４Ｈ７及びＦ８Ｈ５）と、ｐＳｅｒ４６－ＭＡＲＣＫＳに対するポリクローナル抗体とを用いた免疫組織化学染色法を行った結果を示す図である。下段のＭｅｒｇｅ画像は、上段のｐＳｅｒ４６－ＭＡＲＣＫＳ画像と、ＤＡＰＩ画像とを重ねた画像である。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.
＜本発明のモノクローナル抗体＞
　本発明のモノクローナル抗体は、ＭＡＲＣＫＳにおけるリン酸化エピトープ、具体的には、配列番号２１のアミノ酸配列において、１２番目のセリン残基がリン酸化されたアミノ酸配列からなるエピトープ（以下、「リン酸化ＭＡＲＣＫＳ抗原ペプチド」ということがある）に結合し、かつ、下記（１－１）の重鎖ＣＤＲ１～３と、下記（１－１）の軽鎖ＣＤＲ１～３とを含むか、或いは、下記（２－１）の重鎖ＣＤＲ１～３と、下記（２－１）の軽鎖ＣＤＲ１～３とを含むモノクローナル抗体（以下、「本件モノクローナル抗体」ということがある）であり、好ましくは、下記（１－１）の重鎖ＣＤＲ１～３を含む重鎖可変領域と、下記（１－１）の軽鎖ＣＤＲ１～３を含む軽鎖可変領域とを含むか、或いは、下記（２－１）の重鎖ＣＤＲ１～３を含む重鎖可変領域と、下記（２－１）の軽鎖ＣＤＲ１～３を含む軽鎖可変領域とを含むものである。
（１－１）配列番号１に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖相補性決定領域（ＣＤＲ）１；配列番号２に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ２；及び配列番号３に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ３；
配列番号４に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ１；配列番号５に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ２；及び配列番号６に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ３；
（２－１）配列番号７に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ１；配列番号８に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ２；及び配列番号９に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ３；
配列番号１０に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ１；配列番号１１に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ２；及び配列番号１２に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ３； <The monoclonal antibody of the present invention>
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”). (Hereinafter sometimes referred to as “peptide”) and 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.
(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 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, deleted, added and / or inserted in the amino acid sequence; and the amino acid sequence shown in SEQ ID NO: 6, or one or more in the amino acid sequence Light chain CDR3 consisting of an amino acid sequence in which the amino acids of are substituted, deleted, added and / or inserted;
(2-1) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7 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 heavy chain CDR2 consisting of the amino acid sequence shown, 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 the amino acid sequence shown in SEQ ID NO: 9, or the amino acid A heavy chain CDR3 consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added and / or inserted in the 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: 12, or one or more in the amino acid sequence Light chain CDR3 consisting of an amino acid sequence in which the amino acids of are substituted, deleted, added and / or inserted;
　本件モノクローナル抗体のエピトープは、ヒトＭＡＲＣＫＳタンパク質（具体的には、配列番号２２のアミノ酸配列を有するタンパク質；以下、同じ）及びマウスＭＡＲＣＫＳタンパク質（具体的には、配列番号２３のアミノ酸配列を有するタンパク質；以下、同じ）における３５～４８番目のアミノ酸残基において、前記ＭＡＲＣＫＳの４６番目のセリン残基がリン酸化されたアミノ酸残基（ＥＮＧＨＶＫＶＮＧＤＡ［ｐＳ］ＰＡ）に相当する。一方、本件モノクローナル抗体は、非リン酸化型のＭＡＲＣＫＳ抗原ペプチド（ＥＮＧＨＶＫＶＮＧＤＡＳＰＡ）には特異的に結合しない。このため、本件モノクローナル抗体は、４６番目のセリン残基がリン酸化していないヒトＭＡＲＣＫＳタンパク質及びマウスＭＡＲＣＫＳタンパク質に特異的に結合せず、４６番目のセリン残基がリン酸化したヒトＭＡＲＣＫＳタンパク質及びマウスＭＡＲＣＫＳタンパク質に特異的に結合する。 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. On the other hand, the present monoclonal antibody does not specifically bind to the non-phosphorylated MARCKS antigen peptide (ENGHVKVNGDASPA). Therefore, 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.
　本明細書において、「ＭＡＲＣＫＳタンパク質に特異的に結合する」とは、抗原－抗体間の特異性の高い認識機構によって、ＭＡＲＣＫＳタンパク質を認識し結合することを意味する。本件モノクローナル抗体は、分離されているものが好ましい。ここで「分離されている」とは、人為的操作によって、抗体を、本来存在する環境から取り出したり、抗体が本来存在する環境とは別の環境下で発現させる等して、抗体が本来存在している状態とは異なった状態で存在していることを意味する。すなわち、「分離されている抗体」には、ある個体由来の抗体であって、かつ外的操作（人為的操作）が施されずに、当該個体の体内中又は体内由来の組織若しくは体液（血液、血漿、血清等）中に含まれる状態の抗体は含まれない。また、本件モノクローナル抗体は、人為的操作によって作製した生物又は細胞から産生される抗体（例えば、ハイブリドーマから産生される抗体）が好ましい。かかる「人為的操作によって作製した生物又は細胞から産生される抗体」には、（人為的操作の施されていない）天然に存在する生物又はＢ細胞から産生される抗体は含まれない。 In the present specification, “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. , Plasma, serum, etc.) are not included. Further, 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). Such "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.
　本明細書において「モノクローナル抗体」とは、実質的に均一な抗体（抗体の機能的断片を含む）を意味する。モノクローナル抗体は、抗原上の単一の決定基を認識するものである。本発明における「抗体」は、ヒトの免疫グロブリンのクラス、サブクラスを含み、また、かかる抗体の機能的断片の形態も含む意である。本件モノクローナル抗体のクラス、サブクラスには、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４等のＩｇＧ；ＩＧＡ１、ＩＧＡ２等のＩｇＡ；ＩｇＤ；ＩｇＥ；ＩｇＭ；などが含まれ、これらの中でも、ＩｇＧやＩｇＭを好適に例示することができる。 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.
　本件モノクローナル抗体における重鎖ＣＤＲ１～３及び軽鎖ＣＤＲ１～３の各領域のアミノ（Ｎ）末端及びカルボキシル（Ｃ）末端には、通常、フレームワーク領域（ＦＲ）が連結されている。かかるＦＲのうち重鎖ＦＲとしては、重鎖ＣＤＲ１のＮ末端に連結されている重鎖ＦＲ１；重鎖ＣＤＲ１のＣ末端と重鎖ＣＤＲ２のＮ末端の間に連結されている重鎖ＦＲ２；重鎖ＣＤＲ２のＣ末端と重鎖ＣＤＲ３のＮ末端の間に連結されている重鎖ＦＲ３；及び、重鎖ＣＤＲ３のＣ末端に連結されている重鎖ＦＲ４；を挙げることができる。また、上記ＦＲのうち軽鎖ＦＲとしては、軽鎖ＣＤＲ１のＮ末端に連結されている軽鎖ＦＲ１；軽鎖ＣＤＲ１のＣ末端と軽鎖ＣＤＲ２のＮ末端の間に連結されている軽鎖ＦＲ２；軽鎖ＣＤＲ２のＣ末端と軽鎖ＣＤＲ３のＮ末端の間に連結されている軽鎖ＦＲ３；及び、軽鎖ＣＤＲ３のＣ末端に連結されている軽鎖ＦＲ４；を挙げることができる。 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. Among such FRs, 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; Further, among the above FRs, 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. A light chain FR3 linked between the C terminus of the light chain CDR2 and the N terminus of the light chain CDR3; and a light chain FR4 linked to the C terminus of the light chain CDR3.
　上記ＦＲとしては、本件モノクローナル抗体における重鎖ＣＤＲ１～３及び軽鎖ＣＤＲ１～３が、相互に近接することにより、リン酸化ＭＡＲＣＫＳ抗原ペプチドに対する結合性が生じるものであれば、アミノ酸配列やその長さは特に制限されず、具体的には、配列番号１３又は１５に示されるアミノ酸配列の１～３０番目のアミノ酸残基からなる重鎖ＦＲ１、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖ＦＲ１；配列番号１３又は１５に示されるアミノ酸配列の３６～４９番目のアミノ酸残基からなる重鎖ＦＲ２、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖ＦＲ２；配列番号１３又は１５に示されるアミノ酸配列の６７～９８番目のアミノ酸残基からなる重鎖ＦＲ３、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖ＦＲ３；配列番号１３又は１５に示されるアミノ酸配列の１１０～１２０番目のアミノ酸残基からなる重鎖ＦＲ４、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖ＦＲ４；配列番号１４又は１６に示されるアミノ酸配列の１～２３番目のアミノ酸残基からなる軽鎖ＦＲ１、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖ＦＲ１；配列番号１４又は１６に示されるアミノ酸配列の３５～４９番目のアミノ酸残基からなる軽鎖ＦＲ２、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖ＦＲ２；配列番号１４又は１６に示されるアミノ酸配列の５７～８８番目のアミノ酸残基からなる軽鎖ＦＲ３、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖ＦＲ３；配列番号１４又は１６に示されるアミノ酸配列の９８～１０７番目のアミノ酸残基からなる軽鎖ＦＲ４、又は前記アミノ酸残基と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖ＦＲ４；を挙げることができる。 As the above-mentioned FR, 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. Is not particularly limited, and specifically, 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 A heavy chain FR2 consisting of a sequence; a heavy chain FR3 consisting of the 67th to 98th amino acid residues of the amino acid sequence shown in SEQ ID NO: 13 or 15, or an amino acid sequence 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 FR4; light chain FR1 consisting of amino acid residues 1 to 23 of the amino acid sequence shown in SEQ ID NO: 14 or 16, or light chain FR1 consisting of an amino acid sequence having 80% or more sequence identity with said amino acid residue A light chain FR2 consisting of amino acid residues 35 to 49 of the amino acid sequence shown in SEQ ID NO: 14 or 16, or a light chain FR2 consisting of an amino acid sequence having 80% or more sequence identity with the amino acid residue; Light chain FR3 consisting of the 57th to 88th amino acid residues of the amino acid sequence shown in No. 14 or 16, or light chain FR3 consisting of an amino acid sequence having 80% or more sequence identity with the above amino acid residue; SEQ ID NO: 14 Or a light chain FR4 consisting of the 98th to 107th amino acid residues of the amino acid sequence shown in 16 or a light chain FR4 consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid residue. ..
　本件モノクローナル抗体のうち、上記（１－１）の重鎖ＣＤＲ１～３と、上記（１－１）の軽鎖ＣＤＲ１～３とを含むものとしては、さらに、下記（１－２）の重鎖可変領域と、下記（１－２）の軽鎖可変領域を含むもの、及び／又は、下記（１－３）の重鎖と、下記（１－３）の軽鎖を含むものが好ましい。また、本件モノクローナル抗体のうち、上記（２－１）の重鎖ＣＤＲ１～３と、上記（２－１）の軽鎖ＣＤＲ１～３とを含むものとしては、さらに、下記（２－２）の重鎖可変領域と、下記（２－２）の軽鎖可変領域を含むもの、及び／又は、下記（２－３）の重鎖と、下記（２－３）の軽鎖を含むものが好ましい。
（１－２）配列番号１３に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域；配列番号１４に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域；
（２－２）配列番号１５に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域；配列番号１６に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域；
（１－３）配列番号１７に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖；配列番号１８に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖；
（２－３）配列番号１９に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖；配列番号２０に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖； Among the monoclonal antibodies of the present invention, 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. Further, among the monoclonal antibodies of the present invention, 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. ..
(1-2) Heavy chain variable region consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13; having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14 Light chain variable region consisting of an amino acid sequence;
(2-2) Heavy chain variable region consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 15; having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 16 Light chain variable region consisting of an amino acid sequence;
(1-3) A heavy chain consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 17; an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 18. A light chain consisting of;
(2-3) Heavy chain consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19; an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 20 A light chain consisting of;
　本件モノクローナル抗体には、ヒトキメラ抗体又はマウスキメラ抗体、ヒト化抗体又はマウス化抗体、完全ヒト抗体又は完全マウス抗体等が含まれ、中でも、ヒト化抗体、完全ヒト抗体が好ましい。 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.
　本明細書において、「ヒトキメラ抗体」とは、非ヒト動物（例えば、ニワトリ、マウス、ラット、ウシ等の非ヒト哺乳動物）由来の抗体の可変領域と、ヒト由来の抗体の定常領域とを連結した抗体を意味する。また、本明細書において、「マウスキメラ抗体」とは、非マウス動物（例えば、ニワトリ、ラット、ウシ等の非マウス哺乳動物）由来の抗体の可変領域と、マウス由来の抗体の定常領域とを連結した抗体を意味する。例えば、ヒトキメラ抗体は、リン酸化ＭＡＲＣＫＳ抗原ペプチドを非ヒト動物（好ましくは非ヒト哺乳動物）に免疫し、得られた抗体の遺伝子からリン酸化ＭＡＲＣＫＳ抗原ペプチドと結合する抗体可変部（可変領域）を切り出して、ヒト骨髄由来の抗体定常部（定常領域）遺伝子と結合し、これを発現ベクターに組み込んで宿主に導入して産生させることにより取得することができる（例えば、特開平８－２８０３８７号公報、米国特許第４８１６３９７号公報、米国特許第４８１６５６７号公報、米国特許第５８０７７１５号公報）。 In the present specification, 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. Antibody. In addition, in the present specification, 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. For example, 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).
　ヒトキメラ抗体のヒトの定常領域としては、例えば重鎖においては、Ｃγ１、Ｃγ２、Ｃγ３、Ｃγ４、Ｃμ、Ｃδ、Ｃα１、Ｃα２、及びＣεが挙げられ、また軽鎖においては、ＣκやＣλが挙げられる。これらの定常領域のアミノ酸配列、並びにそれをコードする塩基配列は公知である。また、抗体自体の安定性、又は抗体の産生の安定性を改善するために、ヒト由来の抗体定常領域中の１又は複数のアミノ酸を置換、欠失、付加及び／又は挿入をすることができる。 Examples of human constant regions of human chimeric antibodies include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε in the heavy chain, and Cκ and Cλ 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. ..
　本明細書において、「ヒト化抗体」とは、非ヒト動物（例えば、ニワトリ、マウス、ラット、ウシ等の非ヒト哺乳動物）由来の抗体のＣＤＲの遺伝子配列をヒト由来の抗体遺伝子に移植（ＣＤＲグラフティング）した抗体を意味する。また、本明細書において、「マウス化抗体」とは、非マウス動物（例えば、ニワトリ、ラット、ウシ等の非マウス哺乳動物）由来の抗体のＣＤＲの遺伝子配列をマウス由来の抗体遺伝子に移植（ＣＤＲグラフティング）した抗体を意味する。例えば、ヒト化抗体の作製方法は、オーバーラップエクステンションＰＣＲ等、公知である（例えば、欧州特許出願公開第２３９４００号明細書、欧州特許出願公開第１２５０２３号明細書、国際公開第９０／０７８６１号、国際公開第９６／０２５７６号）。抗体の可変領域は、通常、４つのフレームワーク領域（framework region:ＦＲ）にはさまれた３つのＣＤＲで構成されている。ＣＤＲは、実質的に、抗体の結合特異性を決定している領域である。ＣＤＲのアミノ酸配列は多様性に富む一方、ＦＲを構成するアミノ酸配列は、異なる結合特異性を有する抗体の間でも、高い相同性を示すことが多い。そのため、一般に、ＣＤＲの移植によって、ある抗体の結合特異性を、他の抗体に移植することができるといわれている。また、ＣＤＲの機能の維持の観点から、非ヒト由来ＣＤＲのヒトＦＲへの移植においては、その非ヒト動物由来のＦＲと相同性の高いヒトＦＲが選択される。すなわち、ＣＤＲ内のアミノ酸は、抗原を認識するばかりでなく、ＣＤＲの近傍にあるＦＲのアミノ酸と配位し、ＣＤＲのループ構造の維持にも関与しているため、移植すべきＣＤＲに隣接しているＦＲのアミノ酸配列と相同性の高いアミノ酸配列からなるヒトＦＲを利用することが好ましい。 As used herein, the term "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. In the present specification, the term “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. For example, a method for producing a humanized antibody is publicly known, such as overlap extension PCR (eg, European Patent Application Publication No. 239400, European Patent Application Publication No. 125023, International Publication No. 90/07861, International Publication No. 96/02576). The 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. From the viewpoint of maintaining the function of CDR, when transplanting a non-human CDR to a human FR, 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.
　非ヒト動物由来のＦＲと相同性の高い公知のヒトＦＲの検索を、例えば、インターネットで利用可能な抗体に特化した検索システム（ｈｔｔｐ：／／ｗｗｗ．ｂｉｏｉｎｆ．ｏｒｇ．ｕｋ／ａｂｙｓｉｓ／）を利用して行うことができる。このようにして得られたヒトＦＲの配列と一致するように、非ヒト由来の抗体のＣＤＲ以外の配列に変異を導入することができる。あるいは、検索によって得られたヒトＦＲのアミノ酸配列をコードする遺伝子（ｃＤＮＡ）が入手可能な場合は、その配列中に非ヒト由来ＣＤＲを導入してもよい。変異の導入等は核酸合成、部位特異的変異誘発などの当該分野で公知の技術を用いて行うことができる。 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.
　このようにして作製されたヒト化抗体の抗原への親和性を定性的又は定量的に測定し、評価することによって、ＣＤＲを介して連結されたときに該ＣＤＲが良好な抗原結合部位を形成するようなヒト由来の抗体のＦＲが好適に選択できる。また必要に応じ、Ｓａｔｏ，Ｋ．ｅｔ　ａｌ．，Ｃａｎｃｅｒ　Ｒｅｓ，１９９３，５３，８５１－８５６等に記載の方法に沿って、ヒト化抗体のＣＤＲが適切な抗原結合部位を形成するようにＦＲのアミノ酸残基を置換することもでき、さらに当該アミノ酸を置換した変異型抗体の抗原への親和性を測定して評価することによって、所望の性質を有する変異ＦＲ配列を選択することができる。 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.
　本明細書において、「完全ヒト抗体」とは、すべてのアミノ酸配列がヒト由来のアミノ酸配列からなる抗体を意味する。また、本明細書において、「完全マウス抗体」とは、すべてのアミノ酸配列がマウス由来のアミノ酸配列からなる抗体を意味する。例えば、完全ヒト抗体は、ヒト重鎖及び軽鎖抗体の遺伝子を発現するように操作されたトランスジェニックマウスにおいて作製することができる。ヒト抗体を産生するトランスジェニックマウスの調製方法は、例えば、国際公開第０２／４３４７８号パンフレット、米国特許第６６５７１０３号明細書（Ａｂｇｅｎｉｘ）等に記載されている。次いで、所望の抗体を産生するトランスジェニックマウス由来のＢ細胞が融合されて、抗体の連続産生用のハイブリドーマ細胞株が作製され得る。例えば、米国特許第５５６９８２５号明細書；米国特許第５６２５１２６号明細書；米国特許第５６３３４２５号明細書；米国特許第５６６１０１６号明細書；及び米国特許第５５４５８０６号明細書；並びにＪａｋｏｂｏｖｉｔｓ，Ａｄｖ．Ｄｒｕｇ Ｄｅｌ．Ｒｅｖ．３１：３３－４２（１９９８）；Ｇｒｅｅｎ，ｅｔ ａｌ，Ｊ．Ｅｘｐ．Ｍｅｄ．１８８：４８３－９５（１９９８）参照。 In the present specification, “fully human antibody” means an antibody in which all amino acid sequences are human-derived amino acid sequences. Further, in the present specification, the “complete mouse antibody” means an antibody in which all amino acid sequences are mouse-derived amino acid sequences. For example, 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. For example, US Pat. No. 5,569,825; US Pat. No. 5,625,126; US Pat. No. 5,633,425; US Pat. No. 5,661,016; and US Pat. No. 5,545,806; and Jakobovits, Adv. Drug Del. Rev. 31: 33-42 (1998); Green, et al. Exp. Med. 188: 483-95 (1998).
　また、本件モノクローナル抗体には、いわゆる、“Ｙ”字型の４本鎖構造（軽鎖及び重鎖の２つのポリペプチド鎖が、それぞれの定常領域においてジスルフィド結合で結合した２本鎖の２つが、それぞれの重鎖ヒンジ部位においてジスルフィド結合で結合した４本鎖）を有する基本構造からなる抗体（以下、便宜上「基本抗体」ということがある）の他、抗体の一部分（部分断片）であって、リン酸化ＭＡＲＣＫＳ抗原ペプチドに特異的に結合する機能的断片も含まれる。かかる機能的断片としては、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、可変領域断片（Ｆｖ）、ジスルフィド結合Ｆｖ、一本鎖Ｆｖ（ｓｃＦｖ）、ｓｃ（Ｆｖ）２、及びこれらの重合体等を挙げることができる。 In addition, 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. Examples of 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.
　上記「Ｆａｂ」とは、基本抗体をパパイン消化によって得られる３つの断片のうち、Ｎ末端側の２つの断片を意味し、より具体的には、重鎖の可変領域を含む重鎖断片と、軽鎖の可変領域を含む軽鎖断片とがそれぞれの定常領域においてジスルフィド結合で結合した２本鎖を意味する。なお、Ｆａｂは、組換え方法によって得ることもできる。また、上記「Ｆ（ａｂ’）２」とは、基本抗体をペプシン消化によって得られる２つの断片のうち、Ｎ末端側の断片を意味し、より具体的には、重鎖の可変領域とヒンジ部位を含む重鎖断片と、軽鎖の可変領域を含む軽鎖断片とがそれぞれの定常領域においてジスルフィド結合で結合した２本鎖の２つが、重鎖ヒンジ部位においてジスルフィド結合で結合した４本鎖を意味する。また、上記「Ｆａｂ’」とは、Ｆ（ａｂ’）２の重鎖ヒンジ部位におけるジスルフィド結合を、還元剤処理で切断することにより得られる２本鎖を意味する。 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. Further, the above “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. Means Further, 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.
　上記「Ｆｖ」とは、完全な抗原認識及び結合部位を有する最少の抗体断片を意味する。Ｆｖは、重鎖可変領域及び軽鎖可変領域が非共有結合により強く連結されたダイマーである。「一本鎖Ｆｖ（ｓｃＦｖ）」は、抗体の重鎖可変領域及び軽鎖可変領域を含み、これらの領域は、単一のポリペプチド鎖に存在する。「ｓｃ（Ｆｖ）２」は、２つの重鎖可変領域及び２つの軽鎖可変領域をリンカー等で結合して一本鎖にしたものである。 The "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.
　本件モノクローナル抗体は、リン酸化ＭＡＲＣＫＳ抗原ペプチドに対する親和性を減少させることなく、そのアミノ酸配列を修飾（例えば、糖鎖付加、アセチル化、リン酸化、脂質付加）又は改変（例えば、アミノ酸の置換、欠失、付加及び／又は挿入）することができる。このようなアミノ酸配列の修飾体又は改変体は、例えば、本実施例に示す２種類のモノクローナル抗体（Ａ４Ｈ７又はＦ８Ｈ５）の抗体鎖をコードするＤＮＡへの変異導入によって、あるいは、ペプチド合成によって作製することができる。本件モノクローナル抗体のアミノ酸配列が修飾又は改変される部位は、修飾又は改変される前の本件モノクローナル抗体と同等のエピトープ（すなわち、リン酸化ＭＡＲＣＫＳ抗原ペプチド）に対する結合レベル活性を有する限り、抗体の重鎖又は軽鎖の定常領域であってもよく、また、可変領域（ＦＲ及びＣＤＲ）であってもよいが、定常領域であることが好ましい。ＣＤＲ以外のアミノ酸の修飾又は改変は、リン酸化ＭＡＲＣＫＳ抗原ペプチドとの親和性への影響が相対的に少ないと考えられるが、現在では、ＣＤＲのアミノ酸を修飾又は改変し、抗原へのアフィニティーが高められた抗体をスクリーニングする手法が公知である（ＰＮＡＳ，１０２：８４６６－８４７１（２００５）、Ｐｒｏｔｅｉｎ　Ｅｎｇｉｎｅｅｒｉｎｇ，Ｄｅｓｉｇｎ＆Ｓｅｌｅｃｔｉｏｎ，２１：４８５－４９３（２００８）、国際公開第２００２／０５１８７０号、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２８０：２４８８０－２４８８７（２００５）、Ｐｒｏｔｅｉｎ　Ｅｎｇｉｎｅｅｒｉｎｇ，Ｄｅｓｉｇｎ＆Ｓｅｌｅｃｔｉｏｎ，２１：３４５－３５１（２００８）、ＭＡｂｓ．Ｍａｒ－Ａｐｒ；６（２）：４３７－４５（２０１４））。また、現在では、統合計算化学システム等（例えば、Ｍｏｌｅｃｕｌａｒ　Ｏｐｅｒａｔｉｎｇ　Ｅｎｖｉｒｏｍｅｎｔ、カナダＣＣＧ社製）を利用することにより、抗原へのアフィニティーが高められた抗体をモデリングすることもできる（例えば、ｈｔｔｐ：／／ｗｗｗ．ｒｓｉ．ｃｏ．ｊｐ／ｋａｇａｋｕ／ｃｓ／ｃｃｇ／ｐｒｏｄｕｃｔｓ／ａｐｐｌｉｃａｔｉｏｎ／ｐｒｏｔｅｉｎ．ｈｔｍｌ　参照）。さらに、Ｐｒｏｔｅｉｎ　Ｅｎｇ　Ｄｅｓ　Ｓｅｌ．２０１０　Ａｕｇ；２３（８）：６４３－５１に記載のとおり、抗原へのアフィニティーにおいて、重鎖可変領域のＣＤＲ１及び軽鎖可変領域のＣＤＲ３は関与していない例が知られている、また同様に、Ｍｏｌｅｃｕｌａｒ　Ｉｍｍｕｎｏｌｏｇｙ　４４：１０７５－１０８４（２００７））には、大抵の抗体においては、軽鎖可変領域のＣＤＲ２は抗原へのアフィニティーに関与していないということが報告されている。このように、抗体の抗原へのアフィニティーにおいては、重鎖可変領域及び軽鎖可変領域各々のＣＤＲ１～３の全てを要せずとも、同等の活性を発揮し得る。実際に、Ｂｉｏｃｈｅｍ　Ｂｉｏｐｈｙｓ　Ｒｅｓ　Ｃｏｍｍｕｎ．２００３　Ｊｕｌ　１８；３０７（１）：１９８－２０５、Ｊ　Ｍｏｌ　Ｂｉｏｌ．　２００４　Ｊｕｌ　９；３４０（３）：５２５－４２、Ｊ　Ｍｏｌ　Ｂｉｏｌ．　２００３　Ａｕｇ　２９；３３１（５）：１１０９－２０においては、元の抗体の少なくとも１のＣＤＲを保持することによって、抗原へのアフィニティーが維持された例が報告されている。したがって、本件モノクローナル抗体は、本実施例に示す２種類のモノクローナル抗体（Ａ４Ｈ７又はＦ８Ｈ５）からなる群から選択されるいずれか１つの抗体の少なくとも１のＣＤＲを含む抗体でもあり得る。本件モノクローナル抗体に関し、修飾又は改変されるアミノ酸数は、好ましくは、１０アミノ酸以内、より好ましくは５アミノ酸以内、さらに好ましくは３アミノ酸以内（例えば、２アミノ酸以内、１アミノ酸）である。 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). Such 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. As long as the site where the amino acid sequence of the subject monoclonal antibody is modified or altered has a binding level activity to an epitope (that is, phosphorylated MARCKS antigen peptide) equivalent to that of the subject monoclonal antibody before the modification or alteration, the heavy chain of the antibody Alternatively, the constant region of the light chain may be used, or the variable regions (FR and CDR) may be used, but the constant region is preferable. Although 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. Techniques for screening the obtained antibodies are known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-593 (2008), International Publication No. 2002/051870, J. Biol. Chem. , 280: 24880-24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008), MAbs. Mar-Apr; 6 (2): 437-45 (2014)). Further, at present, it is also possible to model an antibody having an increased affinity for an antigen by using an integrated computational chemistry system or the like (for example, Molecular Operating Environment, manufactured by CCG of Canada) (for example, http: /// www.rsi.co.jp/kagaku/cs/ccg/products/application/protein.html). Furthermore, Protein Eng Des Sel. As described in 2010 Aug; 23 (8): 643-51, it is known that CDR1 of the heavy chain variable region and CDR3 of the light chain variable region are not involved in the affinity to the antigen. , Molecular Immunology 44: 1075-1084 (2007)), it has been reported that in most antibodies, the CDR2 of the light chain variable region is not involved in the affinity for the antigen. Thus, in the affinity of the antibody to the antigen, equivalent activity can be exhibited without requiring all CDR1 to CDR3 of each of the heavy chain variable region and the light chain variable region. In fact, Biochem Biophys Res Commun. 2003 Jul 18; 307 (1): 198-205, J Mol Biol. 2004 Jul 9; 340 (3): 525-42, J Mol Biol. In 2003 Aug 29; 331 (5): 1109-20, it is reported that the affinity for the antigen was maintained by retaining at least one CDR of the original antibody. Therefore, 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. Regarding the monoclonal antibody of the present invention, 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).
　本明細書において、「複数のアミノ酸が置換、欠失、付加及び／又は挿入されている」における「複数のアミノ酸」は、ＣＤＲのアミノ酸残基が数～十数個であることを考慮すると、通常３個以下のアミノ酸であり、好ましくは２個以下のアミノ酸、より好ましくは１個のアミノ酸である。アミノ酸の置換、欠失、付加及び／又は挿入（すなわち、改変）は、好ましくは、保存的な置換である。本明細書において、「保存的な置換」とは、化学的に同様な側鎖を有する他のアミノ酸残基で置換することを意味する。化学的に同様なアミノ酸側鎖を有するアミノ酸残基のグループは、本発明の属する技術分野でよく知られている。例えば、酸性アミノ酸（アスパラギン酸及びグルタミン酸）、塩基性アミノ酸（リシン・アルギニン・ヒスチジン）、中性アミノ酸においては、炭化水素鎖を持つアミノ酸（グリシン・アラニン・バリン・ロイシン・イソロイシン・プロリン）、ヒドロキシ基を持つアミノ酸（セリン・トレオニン）、硫黄を含むアミノ酸（システイン・メチオニン）、アミド基を持つアミノ酸（アスパラギン・グルタミン）、イミノ基を持つアミノ酸（プロリン）、芳香族基を持つアミノ酸（フェニルアラニン・チロシン・トリプトファン）で分類することができる。 In the present specification, “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. For example, 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 With amino acids (serine / threonine), amino acids containing sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / Tryptophan) can be classified.
　本明細書において、「８０％以上の配列同一性」とは、比較対象のアミノ酸配列と同一のアミノ酸配列の割合が少なくとも８０％であることを意味し、好ましくは８５％以上、より好ましくは８８％以上、さらに好ましくは９０％以上、さらにより好ましくは９３％以上、特に好ましくは９５％以上、特により好ましくは９８％以上、最も好ましくは９９％の配列同一性を意味する。アミノ酸配列の同一性は、ＢＬＡＳＴＰ（アミノ酸レベル）のプログラム（Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２１５：４０３－４１０，１９９０）を利用して決定することができる。該プログラムは、Ｋａｒｌｉｎ及びＡｌｔｓｃｈｕｌによるアルゴリズムＢＬＡＳＴ（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８７：２２６４－２２６８，１９９０，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９０：５８７３－５８７７，１９９３）に基づいている。ＢＬＡＳＴＰによってアミノ酸配列を解析する場合には、パラメーターは、例えばｓｃｏｒｅ＝５０、ｗｏｒｄｌｅｎｇｔｈ＝３とする。また、Ｇａｐｐｅｄ　ＢＬＡＳＴプログラムを用いて、アミノ酸配列を解析する場合は、Ａｌｔｓｃｈｕｌら（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．２５：３３８９－３４０２，１９９７）に記載されているように行うことができる。ＢＬＡＳＴとＧａｐｐｅｄ　ＢＬＡＳＴプログラムを用いる場合には、各プログラムのデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は公知である。 In the present specification, “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. Acad. Sci. USA, 90: 5873-5877, 1993) by Karlin and Altschul. There is. When the amino acid sequence is analyzed by BLASTP, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known.
　本件モノクローナル抗体は、公知のハイブリドーマ法や、公知の組換えＤＮＡ法によって作製することができる。ハイブリドーマ法としては、代表的には、コーラー及びミルスタインの方法（Ｋｏｈｌｅｒ＆Ｍｉｌｓｔｅｉｎ，Ｎａｔｕｒｅ，２５６：４９５（１９７５））を挙げることができる。ハイブリドーマ法において、例えば、リン酸化ＭＡＲＣＫＳ抗原ペプチドのＮ末端又はＣ末端に、キャリアタンパク質コンジュゲート用のＣｙｓ残基を付加させ、そのＣｙｓ残基にキャリアタンパク質（例えば、ＫＬＨ、ＢＳＡ［Bovine serum albumin］）をコンジュゲートしたもの（すなわち、「リン酸化ＭＡＲＣＫＳ抗原ペプチド・キャリアタンパク質複合体」）を、非ヒト哺乳動物（例えば、マウス、ラット、ハムスター、ウサギ、サル、ヤギ）へ免疫し、その後、当該免疫した非ヒト哺乳動物から単離した抗体産生細胞（例えば、脾臓細胞、リンパ節細胞、末梢血白血球）を、細胞融合工程に用いることができる。また、細胞融合工程に用いる抗体産生細胞は、免疫されていない非ヒト哺乳動物から単離され、その後、リン酸化ＭＡＲＣＫＳ抗原ペプチド・キャリアタンパク質複合体の存在下で培養したものを用いてもよい。細胞融合工程に用いるミエローマ細胞は、公知の種々の細胞株を使用することが可能である。抗体産生細胞及びミエローマ細胞は、それらが融合可能であれば、異なる動物種起源のものでもよいが、好ましくは、同一の動物種起源のものである。ハイブリドーマは、例えば、抗原で免疫されたマウスから得られた脾臓細胞と、マウスミエローマ細胞との間の細胞融合により産生され、リン酸化ＭＡＲＣＫＳ抗原ペプチドを固相化したプレートを用いたＥＬＩＳＡによるスクリーニングにより、リン酸化ＭＡＲＣＫＳ抗原ペプチドに特異的に結合するモノクローナル抗体を産生するハイブリドーマを得ることができる。リン酸化ＭＡＲＣＫＳ抗原ペプチドに特異的に結合するヒトモノクローナル抗体は、ハイブリドーマを培養することにより、また、ハイブリドーマを投与した哺乳動物の腹水から、取得することができる。ハイブリドーマは、複数回（少なくとも１回、好ましくは少なくとも２回、より好ましくは少なくとも３回）サブクローニングすることが好ましい。 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)). In the hybridoma method, for example, at the N-terminal or C-terminal of the phosphorylated MARCKS antigen peptide, a Cys residue for carrier protein conjugate is added, and the Cys residue is a carrier protein (eg, KLH, BSA [Bovine serum albumin]). )) (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. As 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).
　上記組換えＤＮＡ法は、本件モノクローナル抗体をコードする抗体遺伝子をハイブリドーマやＢ細胞等からクローニングし、適当なベクターに組み込んで、これを宿主細胞（例えば、ＨＥＫ細胞等の哺乳類細胞株、大腸菌、酵母細胞、昆虫細胞、植物細胞等）に導入し、本件モノクローナル抗体を組換え抗体として産生させる手法である（例えば、Ｐ．Ｊ．Ｄｅｌｖｅｓ，Ａｎｔｉｂｏｄｙ　Ｐｒｏｄｕｃｔｉｏｎ：Ｅｓｓｅｎｔｉａｌ　Ｔｅｃｈｎｉｑｕｅｓ，１９９７　ＷＩＬＥＹ、Ｐ．Ｓｈｅｐｈｅｒｄ　ａｎｄ　Ｃ．Ｄｅａｎ　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ，２０００　ＯＸＦＯＲＤ　ＵＮＩＶＥＲＳＩＴＹ　ＰＲＥＳＳ、Ｖａｎｄａｍｍｅ　Ａ．Ｍ．ｅｔ　ａｌ．，Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．１９２：７６７－７７５（１９９０））。本件モノクローナル抗体をコードする抗体遺伝子の発現においては、重鎖又は軽鎖をコードする抗体遺伝子を別々に発現ベクターに組み込んで宿主細胞を形質転換してもよく、重鎖及び軽鎖をコードする抗体遺伝子を単一の発現ベクターに組み込んで宿主細胞を形質転換してもよい（国際公開第９４／１１５２３号公報　参照）。本件モノクローナル抗体は、上記宿主細胞を培養し、宿主細胞内又は培養液から分離・精製し、実質的に純粋で均一な形態で取得することができる。抗体の分離・精製は、通常のポリペプチドの精製で使用されている方法を使用することができる。トランスジェニック動物作製技術を用いて、本件モノクローナル抗体遺伝子が組み込まれたトランスジェニック動物（ウシ、ヤギ、ヒツジ、ブタ等）を作製すれば、そのトランスジェニック動物の血液やミルクから、本件モノクローナル抗体遺伝子に由来するモノクローナル抗体を大量に取得することもできる。 In the above recombinant DNA method, 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. 192: 767-775 (1990). In the expression of the antibody gene encoding the present monoclonal antibody, the antibody gene encoding the heavy chain or the light chain 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. If 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.
　作製した本件モノクローナル抗体が、ヒト又はマウスにおいて、４６番目のセリン残基がリン酸化していないＭＡＲＣＫＳタンパク質に特異的に結合せず、４６番目のセリン残基がリン酸化したＭＡＲＣＫＳタンパク質に特異的に結合することは、免疫組織化学染色法、ウェスタンブロッティング法、ＥＬＩＳＡ等の免疫学的測定法を用いて確認することができる。 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.
＜本発明の神経突起変性疾患の診断剤＞
　本発明の神経突起変性疾患の診断剤は、「神経突起変性疾患を診断するための」という用途に特定された、本件モノクローナル抗体又はその標識物を含むもの（以下、「本件診断剤」ということがある）であり、本件診断剤には、本件モノクローナル抗体又はその標識物単独からなる診断剤の他、さらに、担体；ｐＨ緩衝剤；安定剤；取扱説明書、神経突起変性疾患を診断するための説明書等の添付文書；などの付加物を含む診断剤（診断用キット）が含まれる。本件診断剤における本件モノクローナル抗体としては、ヒト化抗体や完全ヒト抗体が好ましい。 <Diagnostic agent for neurite degenerative disease of the present invention>
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"). In addition to the diagnostic agent consisting of the monoclonal antibody or its labeled substance alone, the present diagnostic agent further includes a carrier; a pH buffering agent; a stabilizer; an instruction manual, for diagnosing neurite degenerative disease. Included is a diagnostic agent (diagnostic kit) containing 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.
　本件モノクローナル抗体は、後述の実施例で示すとおり、神経突起変性モデル動物の脳内のｐＳｅｒ４６－ＭＡＲＣＫＳを検出できるものである。また、本発明者らは、神経突起変性モデル動物の脳において、細胞外にアミロイドβが蓄積する前に、ｐＳｅｒ４６－ＭＡＲＣＫＳが検出されることから、ｐＳｅｒ４６－ＭＡＲＣＫＳは、神経突起変性疾患の早期診断用バイオマーカーとして有用であることを報告している（非特許文献３）。これらの知見を考慮すると、本件モノクローナル抗体又はその標識物を被験者に投与し、当該被験者の脳内におけるｐＳｅｒ４６－ＭＡＲＣＫＳ（換言すると、「４６番目のセリン残基がリン酸化したヒトＭＡＲＣＫＳタンパク質」）の有無を、イメージング技術等により検出することにより、神経突起変性疾患の診断、具体的には、神経突起変性疾患の発症（好ましくは発症早期）の有無の判定することができる。 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. In addition, the present inventors have found that pSer46-MARCKS is detected before extracellular accumulation of amyloid β 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). Considering these findings, 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. By detecting the presence or absence by an imaging technique or the like, it is possible to diagnose a neurite degenerative disease, specifically, to determine whether or not the neurite degenerative disease has developed (preferably the onset).
　本件モノクローナル抗体の標識物としては、本件モノクローナル抗体に標識物質がコンジュゲート（共有結合又は非共有結合）したものであればよい。本明細書において、「標識物質」としては、例えば、ペルオキシダーゼ（例えば、horseradish peroxidase）、アルカリフォスファターゼ、β－Ｄ－ガラクトシダーゼ、グルコースオキシダーゼ、グルコ－ス－６－ホスフェートデヒドロゲナーゼ、アルコール脱水素酵素、リンゴ酸脱水素酵素、ペニシリナーゼ、カタラーゼ、アポグルコースオキシダーゼ、ウレアーゼ、ルシフェラーゼ若しくはアセチルコリンエステラーゼ等の酵素；フルオレスセインイソチオシアネート、フィコビリタンパク、希土類金属キレート、ダンシルクロライド若しくはテトラメチルローダミンイソチオシアネート等の蛍光物質；１８Ｆ、１５Ｏ、１３Ｎ、１１Ｃ、８２Ｒｂ、６８Ｇａ、１９８Ａｕ、１９９Ａｕ、３２Ｐ、３３Ｐ、１２５Ｉ、１３１Ｉ、１２３Ｉ、９０Ｙ、１８６Ｒｅ、１８８Ｒｅ、６２Ｃｕ、６４Ｃｕ、６７Ｃｕ、２１１Ａｔ、４７Ｓｃ、１０３Ｐｂ、１０９Ｐｂ、２１２Ｐｂ、７１Ｇｅ、７７Ａｓ、１０５Ｒｈ、１１３Ａｇ、１１９Ｓｂ、１２１Ｓｎ、１３１Ｃｓ、１４３Ｐｒ、１６１Ｔｂ、１７７Ｌｕ、１９１Ｏｓ、１９３Ｐｔ、１９７Ｈｇ等の放射性同位元素；を挙げることができる。 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). In the present specification, examples of the “labeling substance” include peroxidase (eg, horseradish peroxidase), alkaline phosphatase, β-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, 197 Hg, and other radioactive isotopes;
　本明細書において、「神経突起変性疾患」とは、中枢神経系（すなわち、脳や脊髄）の神経細胞（ニューロン）における神経突起（「軸索」ともいう）が変性（具体的には、退縮、喪失）する疾患を意味する。ニューロンの神経突起が変性すると、ニューロン間の適切なネットワークが喪失、すなわち神経機能（例えば、知覚機能、記憶機能、注意機能、実行機能）の喪失につながる。神経突起変性疾患は、発症初期においてｐＳｅｒ４６－ＭＡＲＣＫＳが認められることから（非特許文献３）、中枢神経系におけるＭＡＲＣＫＳのリン酸化（少なくとも、ｐＳｅｒ４６－ＭＡＲＣＫＳ）を伴う神経突起変性疾患ということができる。神経突起変性疾患としては、具体的には、アルツハイマー病、パーキンソン病、前頭側頭葉変性症（ＦＴＬＤ；Frontotemporal lobar degeneration）、Ｌｅｗｙ小体型認知症、ハンチントン病、筋委縮性側索硬化症（ＡＬＳ；Amyotrophic lateral sclerosis）、脊髄小脳失調症（ＳＣＡ；Spinocerebellar ataxia）等を挙げることができ、これらの中でも、アルツハイマー病、パーキンソン病、前頭側頭葉変性症、及びＬｅｗｙ小体型認知症を好適に例示することができる。 As used herein, the term “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). Specific examples of the neurite degenerative disease 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. Among these, Alzheimer's disease, Parkinson's disease, frontotemporal lobar degeneration, and Lewy body dementia are preferably exemplified. can do.
　神経突起変性疾患を診断するために、本件モノクローナル抗体又はその標識物を被験者へ投与する方法としては、本件モノクローナル抗体又はその標識物が被験者の脳内に到達する方法であればよく、例えば、静脈内投与、動脈内投与、局所投与を挙げることができ、これらの中でも静脈内投与を好適に例示することができる。本件モノクローナル抗体又はその標識物の投与量は、被験者の年齢、体重、性別、健康状態等により変動しうるが、通常、成人には体重１ｋｇ当たり１日０．１～１０００ｍｇ、好ましくは１～１００ｍｇである。 In order to diagnose a neurite degenerative disease, 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.
　本件モノクローナル抗体又はその標識物を投与した被験者の脳において、ｐＳｅｒ４６－ＭＡＲＣＫＳが検出された場合、前記被験者は神経突起変性疾患を発症している可能性が高いと診断し、ｐＳｅｒ４６－ＭＡＲＣＫＳが検出されなかった場合、前記被験者は神経突起変性疾患を発症している可能性が低いと診断することができる。ｐＳｅｒ４６－ＭＡＲＣＫＳの有無は、本件モノクローナル抗体の標識物を用いる場合、その標識物質に由来する指標（例えば、酵素活性レベル、発色レベル、発光［蛍光］レベル、放射能レベル；以下、同じ）の有無を基に診断（判定）することができる。一方、本件モノクローナル抗体を用いる場合、本件モノクローナル抗体に結合する物質（例えば、抗体）の標識物を、本件モノクローナル抗体の投与前、投与と同時に、又は投与後に被験者に投与し、前記標識物における標識物質に由来する指標の有無を基に診断（判定）することができる。 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. On the other hand, when the monoclonal antibody of the present invention is used, a labeled substance of a substance that binds to the monoclonal antibody of the present invention (for example, an antibody) 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.
＜本発明のモノクローナル抗体遺伝子＞
　本発明のモノクローナル抗体遺伝子としては、本件モノクローナル抗体をコードする抗体遺伝子（以下、「本件モノクローナル抗体遺伝子」ということがある）であれば特に制限されず、本件モノクローナル抗体遺伝子のヌクレオチド配列は、本件モノクローナル抗体のアミノ酸配列と、公知のコドン表を参照することにより、当業者はかかるアミノ酸配列に対応するヌクレオチド配列を具体的かつ明確に把握することができる。 <Monoclonal antibody gene of the present invention>
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. By referring to 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.
＜本発明のベクターや宿主細胞＞
　本発明のベクターとしては、プロモーターと、該プロモーターの下流に作動可能に連結されている本件モノクローナル抗体遺伝子とを含むベクターで（以下、「本件ベクター」ということがある）あれば特に制限されず、また、本発明の宿主細胞としては、本件ベクターが導入されている宿主細胞（以下、「本件宿主細胞」ということがある）であれば特に制限されない。本件ベクターは、導入する本件宿主細胞（又は宿主生物）の種類に応じて適宜選択することができる。 <Vector and host cell of the present invention>
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.
　本明細書において、「プロモーターの下流に作動可能に連結されている・・・遺伝子」とは、プロモーターが遺伝子の転写を開始することができるように、プロモーターＤＮＡと、遺伝子ＤＮＡとが機能的に連結されていることを意味する。 As used herein, 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.
　本件ベクターにおけるプロモーターとしては、プロモーターの下流に位置する本件モノクローナル抗体遺伝子がコードするｍＲＮＡの転写を開始させる領域であればよく、プロモーターには、通常転写開始点（ＴＳＳ）が含まれる。 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).
　本件宿主細胞として哺乳動物細胞（例えば、ヒト由来のナマルバ［Namalwa］細胞、サル由来のＣＯＳ細胞、チャイニーズハムスター卵巣由来のＣＨＯ細胞等）を用いる場合、本件ベクターとしては、例えば、ｐcDNAＩ、ｐｃＤＭ８（フナコシ社製）、ｐＡＧＥ１０７（特開平３－２２９７９号公報；Cytotechnology,3,133,(1990)）、ｐＡＳ３－３（特開平２－２２７０７５号公報）、ｐＣＤＭ８（Nature,329,840,(1987)）、ｐcDNAＩ／Ａｍｐ（Invitrogen社製）、ｐＲＥＰ４（Invitrogen社製）、ｐＡＧＥ１０３（J.Biochemistry,101,1307（1987））、ｐＡＧＥ２１０等のベクター又はかかるベクター由来のものを挙げることができ、プロモーターとしては、例えば、サイトメガロウイルス（ＣＭＶ）のＩＥ（immediate early）遺伝子のプロモーター、ＳＶ４０の初期プロモーター、レトロウイルスのプロモーター、メタロチオネインプロモーター、ヒートショックプロモーター、ＳＲαプロモーター等を挙げることができる。 When mammalian cells (for example, human-derived Namalwa [Namalwa] cells, monkey-derived COS cells, Chinese hamster ovary-derived CHO cells, etc.) are used as the subject host cells, 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. (Manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 (J. Biochemistry, 101, 1307 (1987)), vectors such as pAGE210, and those derived from such vectors can be mentioned. Examples of 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 SRα promoter and the like.
　本件ベクターとしては、遺伝子発現効率をさらに高めるために、エンハンサー領域やリボソーム結合領域（ＲＢＳ；ribosome binding site）の塩基配列をさらに含むものや、本件宿主細胞のスクリーニングのために、本件宿主細胞の種類に応じた薬剤耐性遺伝子（例えば、スペクチノマイシン耐性遺伝子、クロラムフェニコール耐性遺伝子、テトラサイクリン耐性遺伝子、カナマイシン耐性遺伝子、アンピシリン耐性遺伝子、ピューロマイシン耐性遺伝子、ハイグロマイシン耐性遺伝子、ブラストサイジン耐性遺伝子、ジェネティシン耐性遺伝子等）をさらに含むものが好ましい。エンハンサー領域は、通常プロモーターの上流に配置され、ＲＢＳは、通常プロモーターと本件遺伝子の間に配置される。本件ベクターに組み込む本件抗体遺伝子のヌクレオチド配列は、発現させる本件宿主細胞に合わせてコドン配列の最適化がされていてもよい。本件ベクターは、遺伝子組み換え技術を用いて公知の方法により作製することができる。 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. Depending on the 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.) are preferable. 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.
　本件宿主細胞の生物種としては、本件抗体遺伝子のｍＲＮＡが転写され、本件モノクローナル抗体タンパク質が発現されるものであればよく、例えば、哺乳動物（例えば、ヒト、マウス、ラット、サル等）、酵母（例えば、Saccharomyces Cerevisiae、Schizosaccharomyces Pombe等）が挙げられ、中でも、哺乳動物を好適に例示することができる。 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.
　本件宿主細胞は、本件ベクターを、宿主細胞の種類に応じた方法により、宿主細胞へ導入（トランスフェクション）することにより得ることができる。 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.
　宿主細胞として上記哺乳動物細胞を用いる場合、本件ベクターの哺乳動物細胞への導入方法としては、哺乳動物細胞にＤＮＡを導入する方法であればよく、例えば、エレクトロポレーション法（Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０））、リン酸カルシウム法（特開平２－２２７０７５号公報）、リポフェクション法（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．，８４，７４１３（１９８７））等の方法を挙げることができる。 When the above-mentioned mammalian cells are used as the host cells, 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. ..
　本件宿主細胞を培養し、本件モノクローナル抗体を回収することを含む、本件モノクローナル抗体の生産方法；も、本発明の態様に含まれる。 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.
　以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to Examples, but the technical scope of the present invention is not limited to these exemplifications.
実施例１．ｐＳｅｒ４６－ＭＡＲＣＫＳに特異的に結合するモノクローナル抗体の作製
　ｐＳｅｒ４６－ＭＡＲＣＫＳを含み、かつ、ｐＳｅｒ４６－ＭＡＲＣＫＳ周辺のアミノ酸配列がヒトとマウスで共通する全長１４アミノ酸配列からなる抗原ペプチドを、ＡＬＢ／Ｃマウスに免疫した後、脾臓細胞を調製し、ｐＳｅｒ４６－ＭＡＲＣＫＳに特異的に結合するモノクローナル抗体を産生するハイブリドーマを作製した。その後、かかるハイブリドーマからｐＳｅｒ４６－ＭＡＲＣＫＳに特異的に結合するモノクローナル抗体を精製した。 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.
［抗原ペプチドとマウスへの免疫］
　マウスに免疫する抗原として、リン酸化ＭＡＲＣＫＳ抗原ペプチド（ＥＮＧＨＶＫＶＮＧＤＡ［ｐＳ］ＰＡ）を使用した。リン酸化ＭＡＲＣＫＳ抗原ペプチドのＮ末端に、キャリアタンパク質コンジュゲート用のＣｙｓ残基を付加させ、かかるＣｙｓ残基にキャリアタンパク質（ＫＬＨ）をコンジュゲートしたもの（以下、「リン酸化ＭＡＲＣＫＳ抗原ペプチド・ＫＬＨ複合体」という）５０～１００μｇと、アジュバント（ＣＦＡ；Complete Freund's Adjuvant）（シグマ社製）とを混合して得られたエマルジョンを、ＢＡＬＢ／Ｃマウス（６週齢）６匹にそれぞれ初回免疫した。初回免疫後２１日目及び３５日目に、リン酸化ＭＡＲＣＫＳ抗原ペプチド・ＫＬＨ複合体５０～１００μｇと、アジュバント（ＩＦＡ；Incomplete Freund's Adjuvant）（シグマ社製）を混合して調製したエマルジョンを、それぞれ免疫した（２回目及び３回目免疫）。初回免疫後４５日目に採血し、定法にしたがって血清を調製後、以下の項目に記載の方法に従って、抗血清の血清抗体価を確認した。 [Immunity of antigen peptide and mouse]
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 μg of the “body” with an adjuvant (CFA; Complete Freund's Adjuvant) (manufactured by Sigma). On days 21 and 35 after the first immunization, emulsions prepared by mixing 50 to 100 μg 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.
［ＥＬＩＳＡ］
　リン酸化ＭＡＲＣＫＳ抗原ペプチド２μｇ／ｍＬと、コントロールとして非リン酸化型のＭＡＲＣＫＳ抗原ペプチド（ＥＮＧＨＶＫＶＮＧＤＡＳＰＡ；以下、単に「非リン酸化ＭＡＲＣＫＳペプチド」という）２μｇ／ｍＬとを、それぞれ９６ウェルマイクロプレート（Nunc社製）に５０μＬ／ウェルずつ添加して４℃で１２時間インキュベートした。その後、２％ ブロックエース（DSファーマ社製）を、２００μＬ／ウェルずつ添加してブロッキング処理を行った。次に、免疫マウスから調製した血清、又はハイブリドーマ培養上清を、０．１％ ブロックエース／ＰＢＳ溶液にて、それぞれ１００００倍及び１００倍に希釈し、血清サンプル又はハイブリドーマ培養上清サンプルを調整後、各サンプルを、５０μＬ／ウェルずつ添加して、２時間、室温にてインキュベートし、抗原抗体反応処理を行った。Tween20含有ＰＢＳ（ＰＢＳＴ）溶液でウェルを洗浄した後、２μｇ／ｍＬ ペルオキシダーゼがコンジュゲートしたヤギ抗マウスＩｇＧ（Jackson ImmunoResearch laboratories社製）を添加して２時間、室温にてインキュベートし、２次抗体反応処理を行った。ＰＢＳＴ溶液で５回ウェルを洗浄後、水分を除去した後にＴＭＢ基質（Thermo Fisher Scientific社製）を５０μＬ／ウェルずつ添加し、発色反応を行った。１５分後に０．１８Ｍ 硫酸を５０μＬ／ウェルずつ添加して、発色反応を停止させた後、４５０ｎｍと５４０ｎｍの吸光度をプレートリーダー（Bio-Rad社製）で測定した。４５０ｎｍの測定値から、５４０ｎｍの測定値を差し引いた補正値を用いて定量を行った。なお、ポジティブコントロールとして、融合前採血した陽性の抗血清を測定し、ネガティブコントロールとして３％スキムミルクを測定した。 [ELISA]
2 μg / mL of phosphorylated MARCKS antigen peptide and 2 μg / 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 μL / 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 μL / well to perform blocking treatment. Next, 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 μL / well and incubated at room temperature for 2 hours to perform an antigen-antibody reaction treatment. After washing the wells with a Tween20-containing PBS (PBST) solution, 2 μg / mL peroxidase-conjugated goat anti-mouse IgG (manufactured by Jackson ImmunoResearch laboratories) was added and incubated at room temperature for 2 hours, followed by secondary antibody reaction. Processed. After washing the wells 5 times with a PBST solution, water was removed, and then 50 μL / well of TMB substrate (manufactured by Thermo Fisher Scientific) was added to carry out color reaction. After 15 minutes, 0.18 M sulfuric acid was added in an amount of 50 μL / 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. As a positive control, a positive antiserum collected before fusion was measured, and as a negative control, 3% skim milk was measured.
［ハイブリドーマの作製］
　最終免疫から２０日後、免疫マウスのうち、抗血清の血清抗体価が１番高かったマウスの脾臓細胞から脾臓細胞を単離し、ポリエチレングリコール４０００を用いて、Ｐ３Ｕ１ミエローマ細胞と１：１の割合で融合した。融合した細胞は、９６穴プレートに５０００個／ウェルになるよう撒き、ＨＡＴ選択培養液（０．１ｍＭヒポキサンチン、０．０１６ｍＭチミジン、０．４ｎＭアミノプテリンを含む、１０％ＦＣＳ含有ＲＰＭＩ）で１０日間培養し、増殖したハイブリドーマの上清中に産生された抗体を、上記［ＥＬＩＳＡ］の項目に記載の方法に従って検出した。ＥＬＩＳＡにより陽性を示した５種類のハイブリドーマ（２Ａ９、２Ｈ６、４Ｅ６、４Ｆ７、及び４Ｆ８）を、０．２個／ウェルに撒き直し、さらに１０日間ＨＴ選択培養液（０．１ｍＭヒポキサンチン、０．０１６ｍＭチミジンを含む、１０％ＦＣＳ含有ＲＰＭＩ）で培養し、増殖したハイブリドーマの上清中に産生された抗体を、上記［ＥＬＩＳＡ］の項目に記載の方法に従って検出した。ＥＬＩＳＡにより陽性を示した３種類のハイブリドーマ（２Ａ９、２Ｈ６、及び４Ｅ６）をクローン化し、増殖したハイブリドーマの上清中に産生された抗体を、上記［ＥＬＩＳＡ］の項目に記載の方法に従って検出した。その結果、非リン酸化ＭＡＲＣＫＳペプチドに結合せずに、リン酸化ＭＡＲＣＫＳ抗原ペプチドに結合する抗体を産生する２種類のハイブリドーマクローン（４Ｅ６Ｅ１及び２Ｈ６Ｃ８）が得られた（表１参照）。 [Preparation of hybridoma]
Twenty days after the final immunization, 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. 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. 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. As a result, 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).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　上記２種類のハイブリドーマクローン（４Ｅ６Ｅ１及び２Ｈ６Ｃ８）を基に、それぞれ２回サブクローニングを行い、２種類のハイブリドーマクローン（４Ｅ６Ｅ１Ａ４Ｈ７［以下、単に「Ａ４Ｈ７」という］及び２Ｈ６Ｃ８Ｆ８Ｈ５［以下、単に「Ｆ８Ｈ５」という］）を得た（表２参照）。 Based on the above-mentioned two kinds of hybridoma clones (4E6E1 and 2H6C8), subcloning was carried out twice, and two kinds of hybridoma clones (4E6E1A4H7 [hereinafter, simply referred to as "A4H7"] and 2H6C8F8H5 [hereinafter, simply referred to as "F8H5"]) Was obtained (see Table 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　上記２種類のハイブリドーマクローン（Ａ４Ｈ７及びＦ８Ｈ５）から定法に従って全ＲＮＡを調製し、定法に従ってＲＴ－ＰＣＲを行い、産生抗体の重鎖及び軽鎖の可変領域のアミノ酸配列を、定法に従ってＤＮＡシーケンシングにより同定した（表３及び４参照）。また、抗体重鎖ＣＤＲ１、２及び３は、kabatによる番号付けでそれぞれ３１～３５Ｂ、５０～６５、及び９５～１０２の位置に存在し、抗体軽鎖ＣＤＲ１、２、及び３は、kabatによる番号付けでそれぞれ２４～３４、５０～５６、及び８９～９７に存在することが知られている（文献「kabat, E.A. et al., (1991) NIH Publication No. 91-3242, sequences of proteins of immunological interest」参照）。かかる点を考慮し、同定した重鎖及び軽鎖の可変領域のアミノ酸配列を基に、kabatによる番号付けを行い、産生抗体の重鎖及び軽鎖のＣＤＲ１～３のアミノ酸配列を推定した（表３及び４参照）。 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. In addition, it is known to exist in 24-34, 50-56, and 89-97, respectively (reference "kabat, EA et al., (1991) NIH Publication No. 91-3242, sequences of immunological. interest ”). In consideration of this point, numbering by kabat was performed based on the identified amino acid sequences of the heavy and light chain variable regions to deduce the amino acid sequences of CDR1 to 3 of the heavy and light chains of the produced antibody (Table 3 and 4).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
［モノクローナル抗体の精製］
　上記２種類のハイブリドーマクローン（Ａ４Ｈ７及びＦ８Ｈ５）の培養上清１０ｍＬから、プロテインＧをリガンドとしたアフィニティークロマトグラフィー（カラム：HiTrap protein G 1 mL）により、各モノクローナル抗体を精製した。アフィニティークロマトグラフィーに際しては、各モノクローナル抗体を２０ｍＭ ＰＢＳ(ｐＨ７．２)存在下でカラムに吸着させ、２０ｍＭ ＰＢＳ(ｐＨ７．２)でカラムを洗浄後、０．１Ｍ グリシン－ＨＣｌ緩衝液（ｐＨ２．７)を用いて抗体を溶出した。溶出後の液は、１Ｍ Ｔｒｉｓ－ＨＣｌ(ｐＨ７．５)を用いて中和した後、限外濾過濃縮操作によりリン酸緩衝液（ＰＢＳ）に置換し、モノクローナル抗体を精製した。その結果、Ａ４Ｈ７のハイブリドーマクローンからは、１．４ｍｇ／ｍＬのモノクローナル抗体が５ｍＬ得られ、Ｆ８Ｈ５のハイブリドーマクローンからは、１．２ｍｇ／ｍＬのモノクローナル抗体が５ｍＬ得られた。 [Purification of monoclonal antibody]
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). In the 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. As a result, 5 mL of 1.4 mg / mL monoclonal antibody was obtained from the A4H7 hybridoma clone, and 5 mL of 1.2 mg / mL monoclonal antibody was obtained from the F8H5 hybridoma clone.
実施例２．神経突起変性疾患モデル動物におけるｐＳｅｒ４６－ＭＡＲＣＫＳの検出
　本発明者らは、神経突起変性モデル動物の脳において、細胞外にアミロイドβが蓄積する前に、ｐＳｅｒ４６－ＭＡＲＣＫＳが検出されることから、ｐＳｅｒ４６－ＭＡＲＣＫＳは、神経突起変性疾患の早期診断用バイオマーカーとして有用であることを報告している（非特許文献３）。そこで、上記実施例１で作製したモノクローナル抗体が、神経突起変性疾患モデル動物におけるｐＳｅｒ４６－ＭＡＲＣＫＳを検出できるものであるかどうかを確認した。 Example 2. Detection of pSer46-MARCKS in neurite degeneration disease model animal Since the present inventors have detected pSer46-MARCKS before extracellular amyloid β 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.
［神経突起変性疾患モデル動物］
　アルツハイマー病モデルマウスである５×ＦＡＤ（five Familial Alzheimer’s Disease）トランスジェニックマウス（以下、「５×ＦＡＤマウス」という）（文献「J Neurosci. 2006 Oct 4;26(40) 10129-10140」参照）を、Jackson Laboratory（Bar Harbor, Maine, USA）から購入し、神経突起変性疾患モデル動物として用いた。また、かかるモデルマウスのバックグランドであるＢ６／ＳＪＬマウスをコントロールとして用いた。なお、Ｂ６／ＳＪＬマウスは販売されていないので、Ｂ６(C57BL/6)を三協ラボサービス株式会社から、ＳＪＬをオリエンタル酵母工業株式会社からそれぞれ購入し、Ｂ６とＳＪＬを交配してＢ６／ＳＪＬマウスを作製した。 [Model animal for neurite degeneration disease]
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.
［免疫組織化学染色法］
　６か月の５×ＦＡＤマウス及びＢ６／ＳＪＬマウスからそれぞれ脳を摘出し、４％パラホルムアルデヒドを含む０．１Ｍ ＰＢＳで２４時間固定処理を行った後、１５％ ショ糖を含む０．１Ｍ ＰＢＳ中に２日間浸漬した。その後、ミクロトーム（大和光機工業社製）を用いて５μｍ厚のパラフィン切片を作製し、上記実施例１で作製した２種類のモノクローナル抗体（Ａ４Ｈ７［１．４ｍｇ／ｍＬ］、希釈倍率１：５０；及びＦ８Ｈ５［１．２ｍｇ／ｍＬ］、希釈倍率１：１００）と、コントロールとしてｐＳｅｒ４６－ＭＡＲＣＫＳに対するポリクローナル抗体（ＲＢ５８８１、バイオロジカ社委託製造、希釈倍率１：１０００）（非特許文献２の３頁、下から２９～２７行目参照）とを用いた１次抗体反応処理を定法に従って行った。その後、それぞれの１次抗体を検出するために、Alexa Fluor-568がコンジュゲートした２次抗体（Molecular Probes社製）を用いた２次抗体反応処理を定法に従って行い、０．２μｇ／ｍＬのＤＡＰＩ（4',6-diamidino-2-phenylindole）を含むＰＢＳ中で、細胞核を染色し、レーザー走査型顕微鏡（FV1200、オリンパス社製）を用い、ｐＳｅｒ４６－ＭＡＲＣＫＳ由来のAlexa Fluor-568蛍光シグナルを検出する画像（ｐＳｅｒ４６－ＭＡＲＣＫＳ画像）と、細胞核由来のＤＡＰＩ蛍光シグナルを検出する画像（ＤＡＰＩ画像）を取得した。 [Immunohistochemical staining method]
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 μm 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. And F8H5 [1.2 mg / mL], dilution ratio 1: 100, and a polyclonal antibody against pSer46-MARCKS as control (RB5881, manufactured by Biologica, dilution ratio 1: 1000) (Non-patent document 2, page 3, (See lines 29 to 27 from the bottom) and a primary antibody reaction treatment was performed according to a standard method. Then, in order to detect each primary antibody, secondary antibody reaction treatment using a secondary antibody conjugated with Alexa Fluor-568 (Molecular Probes) was performed according to a standard method, and 0.2 μg / mL DAPI was used. 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.
　その結果、上記実施例１で作製した２種類のモノクローナル抗体（Ａ４Ｈ７及びＦ８Ｈ５）を用いた場合、ｐＳｅｒ４６－ＭＡＲＣＫＳに対するポリクローナル抗体を用いた場合と同様に、コントロールであるＢ６／ＳＪＬマウスの脳組織（具体的には大脳皮質）においては、ｐＳｅｒ４６－ＭＡＲＣＫＳに特異的なシグナルは検出されなかったのに対して（図１参照）、５×ＦＡＤマウスの脳組織（具体的には大脳皮質）においては、ｐＳｅｒ４６－ＭＡＲＣＫＳに特異的なシグナルが検出された（図２参照）。
　この結果は、本発明のモノクローナル抗体を用いると、脳組織におけるｐＳｅｒ４６－ＭＡＲＣＫＳの有無を指標として、細胞外にアミロイドβが蓄積する前（すなわち、早期）の神経突起変性疾患を診断できることを示している。なお、本発明のモノクローナル抗体のエピトープ、すなわち、リン酸化ＭＡＲＣＫＳ抗原ペプチドのアミノ酸配列は、ヒトとマウスで同じであることから、本発明のモノクローナル抗体は、ヒト脳組織においても、ｐＳｅｒ４６－ＭＡＲＣＫＳに特異的に結合することができ、神経突起変性疾患の早期診断に有用であると考えられる。 As a result, when the two types of monoclonal antibodies (A4H7 and F8H5) produced in Example 1 above were used, as in the case of using the polyclonal antibody against pSer46-MARCKS, the brain tissue of the control B6 / SJL mouse ( Specifically, no signal specific to pSer46-MARCKS was detected in the cerebral cortex (see FIG. 1), whereas in 5 × FAD mouse brain tissue (specifically, cerebral cortex). , PSer46-MARCKS specific signal was detected (see FIG. 2).
This result shows that the monoclonal antibody of the present invention can be used to diagnose neurite degenerative diseases before extracellular amyloid β 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.
　本発明は、神経突起変性疾患の早期発見及び早期治療の他、ＱＯＬ向上や医療費削減等に資するものである。 The present invention contributes to early detection and early treatment of neurite degenerative diseases, improvement of QOL, reduction of medical costs, and the like.

Claims (8)

  1. 　ＭＡＲＣＫＳにおけるリン酸化エピトープに結合するモノクローナル抗体であって、
    前記ＭＡＲＣＫＳにおけるリン酸化エピトープが、配列番号２１に示されるアミノ酸配列において、１２番目のセリン残基がリン酸化されたアミノ酸配列からなるエピトープであり、
    前記モノクローナル抗体が、
    （１－１）配列番号１に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖相補性決定領域（ＣＤＲ）１；配列番号２に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ２；及び配列番号３に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ３；と、
    配列番号４に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ１；配列番号５に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ２；及び配列番号６に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ３；とを含むか、或いは
    （２－１）配列番号７に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ１；配列番号８に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ２；及び配列番号９に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる重鎖ＣＤＲ３；と、
    配列番号１０に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ１；配列番号１１に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ２；及び配列番号１２に示されるアミノ酸配列、又は該アミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加及び／又は挿入されているアミノ酸配列からなる軽鎖ＣＤＲ３；とを含む；
    ことを特徴とする前記モノクローナル抗体。     A monoclonal antibody that binds to a phosphorylated epitope on MARCKS, comprising:
    The phosphorylation epitope in MARCKS is an epitope consisting of an amino acid sequence in which the 12th serine residue in the amino acid sequence shown in SEQ ID NO: 21 is phosphorylated,
    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 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, deleted, added and / or inserted in the amino acid sequence; and the amino acid sequence shown in SEQ ID NO: 6, or one or more in the amino acid sequence Or a light chain CDR3 consisting of an amino acid sequence in which the amino acid of is substituted, deleted, added and / or inserted, or (2-1) the amino acid sequence shown in SEQ ID NO: 7, or 1 in the amino acid sequence. Or 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 more 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: 12, or one or more in the amino acid sequence Light chain CDR3 consisting of an amino acid sequence in which the amino acids of are substituted, deleted, added and / or inserted;
    The monoclonal antibody as described above.
  2. 　（１－１）のモノクローナル抗体が、さらに、（１－２）配列番号１３に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域と、配列番号１４に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域とを含み、
    （２－１）のモノクローナル抗体が、さらに、（２－２）配列番号１５に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖可変領域と、配列番号１６に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖可変領域とを含む
    ことを特徴とする請求項１に記載のモノクローナル抗体。     The monoclonal antibody of (1-1) further comprises (1-2) a heavy chain variable region comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 13, and SEQ ID NO: 14. A light chain variable region comprising an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence described above,
    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. 2. The monoclonal antibody according to claim 1, which comprises a light chain variable region consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence described above.
  3. 　（１－１）又は（１－２）のモノクローナル抗体が、さらに、（１－３）配列番号１７に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖と、配列番号１８に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖とを含み、
    （２－１）又は（２－２）のモノクローナル抗体が、さらに、（２－３）配列番号１９に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる重鎖と、配列番号２０に示されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなる軽鎖とを含む
    ことを特徴とする請求項１又は２に記載のモノクローナル抗体。     The monoclonal antibody of (1-1) or (1-2) further comprises (1-3) a heavy chain comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, and a sequence A light chain comprising an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by 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 claim 1 or 2, 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.
  4. 　請求項１～３のいずれかに記載のモノクローナル抗体又はその標識物を含むことを特徴とする神経突起変性疾患の診断剤。 A diagnostic agent for neurite degeneration disease, which comprises the monoclonal antibody according to any one of claims 1 to 3 or a labeled product thereof.
  5. 　神経突起変性疾患が、アルツハイマー病、パーキンソン病、前頭側頭葉変性症、又はＬｅｗｙ小体型認知症であることを特徴とする請求項４に記載の診断剤。 The diagnostic agent according to claim 4, wherein the neurite degenerative disease is Alzheimer's disease, Parkinson's disease, frontotemporal lobar degeneration, or dementia with Lewy bodies.
  6. 　請求項１～３のいずれかに記載のモノクローナル抗体をコードすることを特徴とするモノクローナル抗体遺伝子。 A monoclonal antibody gene characterized by encoding the monoclonal antibody according to any one of claims 1 to 3.
  7. 　プロモーターと、該プロモーターの下流に作動可能に連結されている請求項６に記載のモノクローナル抗体遺伝子とを含むことを特徴とするベクター。 A vector comprising a promoter and the monoclonal antibody gene according to claim 6, which is operably linked to the downstream of the promoter.
  8. 　請求項７に記載のベクターが導入されていることを特徴とする宿主細胞。 A host cell into which the vector according to claim 7 has been introduced.
PCT/JP2019/044042 2018-11-12 2019-11-11 Monoclonal antibody specifically binding to pser46-marcks WO2020100779A1 (en)

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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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