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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/41—Glycosylation, sialylation, or fucosylation
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present invention relates to amyotrophic lateral sclerosis (hereinafter sometimes referred to as "ALS”), Parkinson's disease (hereinafter sometimes referred to as “PD”), and Huntington's disease (hereinafter sometimes referred to as “HD”). , spinocerebellar ataxia (hereinafter sometimes referred to as “SCA”), degenerative or neurological diseases associated with aging (more specifically, cellular senescence), brain aging, or diseases accompanied by brain aging ( Preparations for the prevention or treatment of Alzheimer's disease (hereinafter sometimes referred to as "AD”) or frontotemporal lobe
• the present invention relates to preparations for preventing or treating degenerative diseases (hereinafter sometimes referred to as "FTLD”); and the like.
• ALS is an adult-onset neurodegenerative disease that exhibits systematic motor neuron damage in the upper (cerebral cortex motor cortex) and lower (spinal cord ventral horn cells and brainstem motor nucleus). Approximately 10% of ALS patients have a familial disease (genetic mutation), whereas the remaining 90% have an idiopathic (sporadic) disease. The cause of ALS is still unknown, and the effectiveness of the ALS treatment drug riluzole (Patent Document 1) is limited to delaying the progression of symptoms for several months, so there is currently no effective treatment for ALS. .
• FTLD is the second to third most common early-onset neurodegenerative dementia after AD, and is thought to be highly related to motor neuron disease.
• FTLD exhibits symptoms of marked behavioral and personality changes, often accompanied by language impairment, which gradually progresses to cognitive impairment and dementia.
• VCP Valosin-Containing Protein
• PGRN progranulin
• CHMP2B Charge Multisicular Body Protein 2B
• TDP43 Transactive Response DNA Binding Protein of 43 kD
• ALS and FTLD develop due to a combination of various genetic and environmental factors, and more than 20 genes are thought to be involved in these diseases.
• HMGB1 High Mobility Group Box 1
• HMGB1 High Mobility Group Box 1
• HMGB1 has been shown not only to function in the nucleus, but also to be released to the outside of cells due to cell necrosis, or to be actively secreted outside of cells in response to vascular inflammatory signals. ) is also attracting attention.
• Ser46 46th serine
• Non-Patent Document 1 we produced a mouse monoclonal antibody against HMGB1, and found that this mouse monoclonal antibody inhibits the phosphorylation of Ser46 of MARCKS (Non-Patent Document 1). The present inventors have also reported that such mouse monoclonal antibodies and human monoclonal antibodies recover cognitive impairment in Alzheimer's disease model mice (Patent Documents 2 and 3).
• Patent No. 2713384 International Publication No. 2018/030405 pamphlet International Publication No. 2020/059847 pamphlet
• the object of the present invention is to provide a prophylactic or therapeutic agent for ALS, PD, HD, SCA, aging-related degenerative diseases or neurological diseases, brain aging, or diseases accompanied by brain aging, and preventive or therapeutic agents for AD or FTLD.
• the objective is to provide antibodies that are effective and have higher stability.
• human monoclonal antibody #129 reduces the number of senescent cells in the cerebral cortex of two types of AD model mice (5xFAD mice and APP-KI mice), and also reduces cell senescence in neurons, astrocytes, and microglia. It was confirmed that it suppresses Furthermore, since we discovered that TRIAD necrosis is age-related cell death itself, human monoclonal antibody #129 and an anti-HMGB1 antibody that has a common CDR with this antibody can be used to treat aging-related degenerative diseases or neurological diseases, as well as brain diseases. It can be said that it has the effect of treating aging itself and diseases associated with brain aging.
• human monoclonal antibody #129-L2 which is a modified antibody of the light chain CDR3 of human monoclonal antibody #129, was used to test five types of mouse models (HD model mouse, PD model mouse, ALS model mouse).
• HD model mouse PD model mouse
• ALS model mouse a modified antibody of the light chain CDR3 of human monoclonal antibody #129
• SCA spinocerebellar ataxia
• human monoclonal antibody #129 and human monoclonal antibody #129-L2 were found to be HD It was confirmed that the drug exerts therapeutic effects on PD, ALS, SCA, as well as aging-associated cellular changes, cell death, cognitive function (memory), and motor function.
• human monoclonal antibody #129-L2 has the same binding strength for human disulfide HMGB1 (ds-HMGB1) as human monoclonal antibody #129 and a therapeutic effect on FTLD-ALS model mice and AD model mice, It was confirmed that the binding strength to human reduced HMGB1 was as weak as or weaker than that of human monoclonal antibody #129. Furthermore, unlike human monoclonal antibody #129, human monoclonal antibody #129-L2 does not undergo N-glycan sugar chain modification, and the stability of the antibody when stored is actually lower than that of human monoclonal antibody #129. I also confirmed that it was expensive.
• ds-HMGB1 human disulfide HMGB1
• the present invention has been completed based on these findings.
• the present invention is as follows. [1] specifically binds to human HMGB1, and Heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted; SEQ ID NO: 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; and the amino acid sequence shown in SEQ ID NO: 3 or A heavy chain CDR3 consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted; Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted; the amino acid sequence shown in SEQ ID NO: 5, or A light chain CDR2 consisting of an amino acid sequence in which one or more amino acid sequence
• a human monoclonal antibody has a heavy chain variable region consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 18.
• a human monoclonal antibody has a heavy chain consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and a heavy chain shown in SEQ ID NO: 10, SEQ ID NO: 22, or SEQ ID NO: 19.
• the prophylactic or therapeutic agent according to [1] or [2] above which comprises a light chain consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence.
• the prophylactic or therapeutic agent according to any one of [1] to [3] above, which is administered intravenously.
• [5] specifically binds to human HMGB1, and Heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1; heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2; and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and, Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4; light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5; and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 17.
• Human monoclonal antibody Human monoclonal antibody.
• a heavy chain variable region consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and at least 80% or more with the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 18.
• a prophylactic or therapeutic agent for Alzheimer's disease or frontotemporal lobar degeneration comprising the human monoclonal antibody according to any one of [5] to [7] above.
• the prophylactic or therapeutic agent according to [8] above which is administered intravenously.
• Amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, aging-related degenerative diseases or neurological diseases including the human monoclonal antibody according to any one of [5] to [7] above.
• the prophylactic or therapeutic agent according to [10] above which is administered intravenously.
• H chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted
• H 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
• the amino acid shown in SEQ ID NO: 3 H chain CDR3 consisting of a sequence or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted
• Light (L) chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted
• SEQ ID NO: 5 L chain CDR2 consisting of an amino acid sequence or an amino acid sequence in which one or more amino acids
• a human monoclonal antibody (hereinafter referred to as "the subject human monoclonal antibody") comprising an amino acid sequence consisting of an amino acid sequence or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and/or inserted;
• a method for preventing or treating the subject disease, etc. which includes the step of administering a substance (sometimes referred to as "") to a subject in need of prevention or treatment of the subject disease, etc.;
• the human monoclonal antibody for use as a preventive or therapeutic agent for the disease;
• the present human monoclonal antibody for use in the prevention or treatment of the present disease, etc.; and the use of the present human monoclonal antibody for producing a prophylactic or therapeutic agent for the present disease.
• H chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1
• H chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2
• H chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3
• L chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4
• L chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5
• L chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 17.
• the step of administering a human monoclonal antibody (hereinafter sometimes referred to as "the subject human monoclonal antibody L1/L2") to a subject in need of prevention or treatment of Alzheimer's disease, frontotemporal lobar degeneration, the subject disease, etc.
• a method for preventing or treating Alzheimer's disease, frontotemporal lobar degeneration, the subject disease, etc. The present human monoclonal antibody L1/L2 for use as a preventive or therapeutic agent for Alzheimer's disease, frontotemporal lobar degeneration, the present disease, etc.; The present human monoclonal antibody L1/L2 for use in the prevention or treatment of Alzheimer's disease or frontotemporal lobar degeneration, the subject disease, etc.; and a prophylactic or therapeutic agent for Alzheimer's disease, frontotemporal lobar degeneration, the subject disease, etc.
• Use of the subject human monoclonal antibody L1/L2 to produce; can be mentioned.
• the subject disease, etc. can be prevented or treated without significant side effects.
• the present human monoclonal antibody L1/L2 Alzheimer's disease, frontotemporal lobar degeneration, the present disease, etc. can be prevented or treated without significant side effects.
• FIG. 2 is a diagram showing the results of analyzing pSer46-MARCKS levels in the cerebral cortex of mice [FIG.
• FIG. 2 is a diagram showing the results of a Morris Water-maze test conducted for 1 to 5 days from the age of 10 months.
• FIG. 6 shows the results of Morris' water maze test conducted for 1 to 5 days from the age of 7 months.
• FIG. 1 shows the results of Morris' water maze test conducted for 1 to 5 days from the age of 7 months.
• This figure shows the results of Morris' water maze test conducted for 1 to 5 days from the age of 6 months. "*", "**”, and "***" in the figure indicate that there is a statistically significant difference between "KI n.t.” and "B6 n.t.” by Tukey's HSD test.
• "B6 Ab” in the figure [n 7 ( Figures 5A to C)])
• FIG. 6 is a diagram showing the results of a Y-maze test performed on mice at 7 months old (FIG. 6A), 8 months old (FIG. 6B), 9 months old (FIG. 6C), or 10 months old (FIG. 6D).
• FIG. 3 is a diagram showing the results of a fear conditioning test conducted at 7 months of age.
• FIG. 2 is a diagram showing the results of a fear conditioning test conducted at 10 months of age.
• FIG. 3 is a diagram showing the results of a fear conditioning test conducted at 6 months of age.
• Four types of FTLD-ALS model mice PGRN R504X -KI mouse [Figure 9A], TDP43 N267S -KI mouse [Figure 9B], VCP T262A -KI mouse [Figure 9C], and CHMP2B Q165X -KI mice [FIG.
• Four types of FTLD-ALS model mice PGRN R504X -KI mouse [Figure 10A], TDP43 N267S -KI mouse [Figure 10B], VCP T262A -KI mouse [Figure 10C], and CHMP2B Q165X -KI mice [FIG.
• FIG. 10D ("KI Ab” in the figure); such four types of FTLD-ALS model mice administered with control human IgG ("KI IgG” in the figure); Four types of FTLD-ALS model mice ("KI n.t.” in the figure); or untreated C57BL/6J mice ("B6 n.t.” in the figure); C57BL/6J mice and CHMP2B Q165X -KI mice) / 7 months old (C57BL/6J mice and PGRN R504X -KI mice) / 10 months old (C57BL/6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice) FIG.
• Four types of FTLD-ALS model mice PGRN R504X -KI mouse [Figure 11A], TDP43 N267S -KI mouse [Figure 11B], VCP T262A -KI mouse [Figure 11C], and CHMP2B Q165X -KI mice [FIG.
• FIG. 11D ("KI Ab” in the figure); such four types of FTLD-ALS model mice administered with control human IgG ("KI IgG” in the figure); Four types of FTLD-ALS model mice ("KI n.t.” in the figure); or untreated C57BL/6J mice ("B6 n.t.” in the figure); C57BL/6J mice and CHMP2B Q165X -KI mice) / 7 months old (C57BL/6J mice and PGRN R504X -KI mice) / 10 months old (C57BL/6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice) FIG.
• Four types of FTLD-ALS model mice were administered with human monoclonal antibody #129 of the present invention (PGRN R504X -KI mouse [Figure 12A], TDP43 N267S -KI mouse [ Figure 12B], VCP T262A -KI mouse [Figure 12C], and CHMP2B Q165X -KI mice [FIG.
• FIG. 12D ("KI Ab” in the figure); such four types of FTLD-ALS model mice administered with control human IgG ("KI IgG” in the figure); Four types of FTLD-ALS model mice ("KI n.t.” in the figure); or untreated C57BL/6J mice ("B6 n.t.” in the figure); C57BL/6J mice and CHMP2B Q165X -KI mice) / 7 months old (C57BL/6J mice and PGRN R504X -KI mice) / 10 months old (C57BL/6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice) FIG.
• Four types of FTLD-ALS model mice PGRN R504X -KI mouse [Figure 13A], TDP43 N267S -KI mouse [Figure 13B], VCP T262A -KI mouse [Figure 13C], and CHMP2B Q165X -KI mice [FIG.
• FIG. 13D ("KI Ab” in the figure); such four types of FTLD-ALS model mice administered with control human IgG ("KI IgG” in the figure); Four types of FTLD-ALS model mice ("KI n.t.” in the figure); or untreated C57BL/6J mice ("B6 n.t.” in the figure); C57BL/6J mice and CHMP2B Q165X -KI mice) / 7 months old (C57BL/6J mice and PGRN R504X -KI mice) / 10 months old (C57BL/6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice) FIG.
• Four types of FTLD-ALS model mice PGRN R504X -KI mouse [Figure 14A], TDP43 N267S -KI mouse [ Figure 14B], VCP T262A -KI mouse [ Figure 14C], and CHMP2B Q165X -KI mice [FIG.
• Four types of FTLD-ALS model mice PGRN R504X -KI mouse [Figure 15A], TDP43 N267S -KI mouse [Figure 15B], VCP T262A -KI mouse [Figure 15C], and CHMP2B Q165X -KI mice [FIG.
• KI Ab ("KI Ab” in the figure); such four types of FTLD-ALS model mice administered with control human IgG ("KI IgG” in the figure); Four types of FTLD-ALS model mice ("KI n.t.” in the figure); or untreated C57BL/6J mice ("B6 n.t.” in the figure); C57BL/6J mice and CHMP2B Q165X -KI mice) / 7 months old (C57BL/6J mice and PGRN R504X -KI mice) / 10 months old (C57BL/6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice) FIG.
• Four types of FTLD-ALS model mice PGRN R504X -KI mouse [Figure 16A], TDP43 N267S -KI mouse [Figure 16B], VCP T262A -KI mouse [Figure 16C], and CHMP2B Q165X -KI mice [FIG.
• FIG. 16D ("KI Ab” in the figure); such four types of FTLD-ALS model mice administered with control human IgG ("KI IgG” in the figure); Four types of FTLD-ALS model mice ("KI n.t.” in the figure); or untreated C57BL/6J mice ("B6 n.t.” in the figure); C57BL/6J mice and CHMP2B Q165X -KI mice) / 7 months old (C57BL/6J mice and PGRN R504X -KI mice) / 10 months old (C57BL/6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice) FIG.
• Four types of FTLD-ALS model mice TDP43 N267S -KI mouse [Figure 17A], PGRN R504X -KI mouse [Figure 17B], VCP T262A -KI mouse [Figure 17C], and CHMP2B Q165X -KI mice [FIG.
• FIG. 18a shows four groups of mice (non-transgenic sibling mice [B6/SJL mice] to which human monoclonal antibody #129 of the present invention was subcutaneously administered to the dorsal neck region [hereinafter simply referred to as "subcutaneous administration"]; control IgG In B6/SJL mice subcutaneously administered; 5xFAD mice subcutaneously administered human monoclonal antibody #129 of the present invention; and 5xFAD mice subcutaneously administered control IgG, two DNAs of MAP2-positive neurons in the cerebral cortex were detected.
• FIG. 2 is a diagram showing the results of analyzing the strand scission level by immunohistochemical staining using antibodies against two types of markers ( ⁇ H2AX and 53BP1).
• Figure 18c shows four groups of mice (B6/SJL mice administered intravenously with human monoclonal antibody #129 of the present invention; B6/SJL mice administered intravenously with control IgG; human monoclonal antibody #129 of the present invention administered intravenously).
• FIG. 2 is a diagram showing the results of analyzing a brain tissue sample (occipital lobe) of a patient without neurological disease and a brain tissue sample (occipital lobe) of an AD patient by immunohistochemical staining using an anti- ⁇ H2AX antibody.
• Figure 18f shows that EGFP-Ku70 was transiently expressed in U2OS cells cultured in the presence (+) or absence (-) of 6 ng/mL (0.24 nM) ds-HMGB1.
• FIG. 3 is a diagram showing the results of imaging analysis of the dynamic localization of Ku70 to DNA damage sites induced by irradiation.
• FIG. 3 is a diagram showing the results of analysis by immunohistochemical staining using antibodies against different cell aging markers (“NeuN” in nerve cells, “GFAP” in astrocytes, and “Iba1” in microglia). The graph in the figure shows the results of measuring the percentage of cells positive for these cellular senescence markers and measuring the percentage of senescent cells.
• FIG. 20a shows immunohistochemical staining of the cerebral cortex of an 8-month-old AD model mouse (5xFAD mouse) using three types of antibodies (anti-YAP antibody, anti-pSer46-MARCKS antibody, and anti-lamin B1 antibody).
• FIG. Figure 20b shows immunization using anti-lamin B1 antibody on the cerebral cortex of two types of AD model mice (5xFAD mouse and APP-KI mouse) to which human monoclonal antibody #129 of the present invention or control IgG was intravenously administered.
• FIG. 2 is a diagram showing the results of analyzing the percentage of senescent cells (ie, lamin B1 ring-negative cells) by performing a histochemical staining method. The upper panel of FIG.
• FIG. 21a shows microglia in two types of normal mice (B6/SJL mice or C57BL/6 mice [B6]) or two types of AD model mice (5 ⁇ FAD mice and APP-KI mice) at the age of 8 months.
• FIG. 2 is a diagram showing the results of analysis of the nuclear translocation level of NF ⁇ B in 2008 by immunohistochemical staining using an anti-NF ⁇ B antibody.
• the lower part of FIG. 21a shows the immunohistochemical staining using an anti-NF ⁇ B antibody of the nuclear translocation level of NF ⁇ B in microglia in the above two types of AD model mice to which human monoclonal antibody #129 of the present invention or control IgG was intravenously administered.
• FIG. 3 is a diagram showing the results of analysis of the expression levels of MCP1 and IL6 in sites and around microglia by immunohistochemical staining using an anti-MCP1 antibody and an anti-IL6 antibody.
• the lower part of FIG. 21a shows the expression levels of MCP1 and IL6 around astrocytes and microglia in the two types of AD model mice intravenously administered with human monoclonal antibody #129 of the present invention or control IgG.
• FIG. 22 is a diagram schematically showing the experimental protocol of FIGS. 22b-f.
• a PKC inhibitor Go6976
• FIG. 22 is a diagram showing the results of a Y-maze test performed on B6/SJL mice or 5 ⁇ FAD mice (left panel of FIG. 22a) to which a PKC inhibitor (Go6976) was subcutaneously administered.
• FIG. 22 is a diagram showing the results of a Y-maze test performed on B6/SJL mice or 5 ⁇ FAD mice (left panel of FIG. 22a) to which a PKC inhibitor (Go6976) was subcutaneously administered.
• FIG. 22 is a diagram showing the results of a Y-maze test performed on B6/SJL mice or 5 ⁇ FAD mice (center image in FIG. 22a) to which human monoclonal antibody #129 of the present invention or control human IgG was subcutaneously administered.
• Cerebral cortex in B6/SJL mice or 5 ⁇ FAD mice (left panel of Figure 22a) to which PKC inhibitor (Go6976) was subcutaneously administered and B6/SJL mice to which human monoclonal antibody #129 of the present invention or control human IgG was subcutaneously administered.
• Cerebral cortex in mice or 5 ⁇ FAD mice center image in FIG.
• FIG. 23A is a diagram schematically showing a protocol for a therapeutic experiment on a Huntington's disease model mouse (R6/2 mouse).
• FIG. 23B is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on Huntington's disease model mice using the Rotarod test.
• normal mice administered with physiological saline ("Normal+saline” in the figure; same in Figures 23-28)
• R6/2 mice administered with physiological saline (“Tg+saline” in the figure; Figures 23-28) 3 groups (hereinafter referred to as ⁇ 3 groups for Huntington's disease model mouse analysis'')
• ⁇ 3 groups for Huntington's disease model mouse analysis'' 3 groups
• human monoclonal antibody #129-administered R6/2 mice (“Tg+antibody” in the figure; the same in Figures 23 to 28)
• the results (average value ⁇ standard error) of three consecutive days of testing in each week are shown.
• FIG. 23C is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on Huntington's disease model mice using an open field test.
• FIG. 23D is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on Huntington's disease model mice using a footprint test.
• FIGS. 23C and 23D representative examples and measurement results of the three groups for Huntington's disease model mouse analysis are shown, and the box plots of the graphs show the maximum value, minimum value, median value, and quartiles (75% and 25%). ) (the same applies to the box plots in the graph below), and the numbers in parentheses indicate the number of animals used in the measurement.
• step length was measured in three groups for analysis of Huntington's disease model mice.
• Figure 24A shows that immunohistochemical staining to detect huntingtin (Htt), ubiquitin (Ub), and MAP2 was performed on the cerebral cortex of three groups of Huntington's disease model mice for analysis.
• Figure 26A shows the characteristics of TRIAD cell death (disappearance of nuclear YAP and pSer46-MARCKS) performed on the cerebral cortex of three groups of Huntington's disease model mice for analysis.
• FIG. 26C shows the signal intensity derived from 53BP1 (left figure) and the signal intensity derived from ⁇ H2AX (right figure) for 53BP1-positive, ⁇ H2AX-positive, and MAP2-positive neurons (neuron cells) in the cerebral cortex.
• FIG. 3 is a diagram showing the results of comparison among three groups for disease model mouse analysis. "#", “##”, and “###” in the figure are statistically significant differences according to Tukey's HSD test (p ⁇ 0.05, p ⁇ 0.01, and p ⁇ 0.001).
• FIG. 27C shows the signal intensity derived from 53BP1 (left figure) and the signal intensity derived from ⁇ H2AX (right figure) for 53BP1-positive, ⁇ H2AX-positive, and MAP2-positive neurons (neuron cells) in the striatum. It is a figure showing the results of comparison among three groups for Huntington's disease model mouse analysis.
• FIG. 29A is a diagram schematically showing a protocol for a therapeutic experiment on Parkinson's disease model mice ( ⁇ -synuclein A53T mice).
• FIG. 29B is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on ⁇ -synuclein A53T mice using the rotarod test.
• FIG. 29C is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on ⁇ -synuclein A53T mice using an open field test.
• FIG. 29D is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on ⁇ -synuclein A53T mice using a footprint test.
• FIGS. 29C and 29D representative examples and measurement results of three groups of Parkinson's disease model mice for analysis are shown, and the numbers in parentheses indicate the number of mice used for the measurements.
• step length (distance) was measured in three groups of Parkinson's disease model mice for analysis.
• FIG. 30A shows immunohistochemical staining to detect pSer129-Syn (Ser129 phosphorylated synuclein), Ub, and MAP2 on the cerebral cortex of three groups of Parkinson's disease model mice.
• FIG. 3 shows immunohistochemical staining to detect pSer129-Syn (Ser129 phosphorylated synuclein), Ub, and MAP2 on the cerebral cortex of three groups of Parkinson's disease model mice.
• Figure 31A shows the characteristics of TRIAD cell death (disappearance of nuclear YAP and pSer46-MARCKS) performed on the cerebral cortex of three groups of Parkinson's disease model mice for analysis.
• FIG. 3 is a diagram showing the results of comparison among three groups for disease model mouse analysis. "##" and "###” in the figure indicate that there is a statistically significant difference according to Tukey's HSD test (p ⁇ 0.01 and p ⁇ 0.001, respectively).
• FIG. 33A is a diagram schematically showing a protocol for a therapeutic experiment on amyotrophic lateral sclerosis model mice (SOD1-Tg mice).
• SOD1-Tg mice amyotrophic lateral sclerosis model mice
• FIG. 33B is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129-L2 on SOD1-Tg mice using the rotarod test.
• normal mice administered with physiological saline ("Normal+saline” in the figure; same in Figures 33 to 37)
• SOD1-Tg mice administered with physiological saline (“Tg+saline” in the figure; Figures 33 to 37) 3 groups (hereinafter referred to as "amyotrophic lateral sclerosis model mice”
• the results (mean value ⁇ standard error) of three consecutive days of testing in each week of the three analytical groups are shown.
• the numbers in parentheses indicate the number of animals used in the measurements.
• FIG. 33C is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129-L2 on SOD1-Tg mice using an open field test.
• FIG. 33D is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129-L2 on SOD1-Tg mice using a footprint test.
• FIGS. 33C and 33D representative examples and measurement results of three groups of amyotrophic lateral sclerosis model mice for analysis are shown, and the numbers in parentheses indicate the number of mice used for the measurements.
• step length (Distance) was measured in three groups for analysis of amyotrophic lateral sclerosis model mice.
• Figure 34A shows that immunohistochemical staining to detect SOD1, Ub, and MAP2 was performed on the cerebral cortex of three groups of amyotrophic lateral sclerosis model mice for analysis.
• Figure 34B shows that immunohistochemical staining to detect pSer386-Tau, Ub, and MAP2 was performed on the cerebral cortex of three groups of amyotrophic lateral sclerosis model mice for analysis.
• Figure 35A shows the characteristics of TRIAD cell death (nuclear YAP Fig.
• FIG. 35C is a diagram showing the results of comparing the number of Iba1-positive cells (number of microglial cells) in the anterior horn of the lumbar spinal cord among three groups for analysis of am
• GFAP-positive cells number of astrocytes
• MAP2-positive neurons neuroneuron cells
• FIG. 3 is a diagram showing the results of Western blotting to detect actin.
• FIG. 38A is a diagram schematically showing a protocol for a therapeutic experiment on spinocerebellar ataxia model mice (mutant ataxin-1 knock-in mice).
• FIG. 38B is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on mutant ataxin-1 knockin mice using the rotarod test.
• mice administered with physiological saline (“Normal+saline” in the figure; same in Figures 38 to 41), mutant ataxin-1 knock-in mice administered with physiological saline (“KI+saline” in the figure; same in Figures 38 to 41) ), and 3 groups of mutant ataxin-1 knock-in mice administered with human monoclonal antibody #129 ("KI+antibody” in the figure; the same in Figures 38 to 41) (hereinafter, "3 groups for spinocerebellar ataxia model mouse analysis”)
• 3 groups for spinocerebellar ataxia model mouse analysis The results (mean value ⁇ standard error) of three consecutive days of testing in each week are shown.
• the numbers in parentheses indicate the number of animals used in the measurements.
• 38C is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129 on mutant ataxin-1 knockin mice using a footprint test. Representative examples and measurement results of three groups of spinocerebellar ataxia model mice for analysis are shown, and the numbers in parentheses indicate the number of mice used for the measurements. Step length (distance) was measured in three groups for analysis of spinocerebellar ataxia model mice.
• Figure 39A shows the signal of pSer46-MARCKS staining in the granule cell layer of the cerebellar cortex in three groups of spinocerebellar ataxia model mice whose cerebellar cortex was subjected to immunohistochemistry using YAP, pSer46-MARCKS, and DAPI.
• FIG. 39B shows that immunohistochemical staining to detect Ataxin-1 (Atxn1), Ub, and MAP2 was performed on the cerebral cortex of three groups of spinocerebellar ataxia model mice for analysis.
• FIG. 39C is a diagram showing the results of comparing the signal intensity derived from 53BP1 (left figure) and the signal intensity derived from ⁇ H2AX (right figure) in Purkinje cells among three groups for spinocerebellar ataxia model mouse analysis.
• FIG. 2 is a diagram showing the results of Western blotting. Quantification and statistical comparison were performed for the marker molecule bands excluding Ub, which form a ladder or smear.
• FIG. 42A is a diagram schematically showing a protocol for a treatment experiment on aging model mice (12-month-old C57BL/6 mice).
• 42B is a diagram showing the results of examining the therapeutic effect of human monoclonal antibody #129-L2 on cognitive decline in aging model mice using a Y-maze test.
• the upper row shows 12-month-old C57BL/6 mice administered with physiological saline ("Aged+saline” in the figure) and 12-month-old C57BL/6 mice administered with human monoclonal antibody #129-L2 ("Aged+antibody” in the figure).
• Aged+saline in the figure
• human monoclonal antibody #129-L2 (“Aged+antibody” in the figure).
• the lower row summarizes the results obtained in the upper row.
• the numbers in parentheses indicate the number of animals used in the measurements.
• Young mice (3-month-old C57BL/6 mice) (“Young” in the figure; same in Figures 43 to 45), 20-month-old C57BL/6 mice administered with physiological saline (“Aged+saline” in the figure; 43 to 45), and human monoclonal antibody #129-L2-administered 20-month-old C57BL/6 mice ("Aged+antibody” in the figure; the same in Figures 43-45).
• Figure 44A shows the signal intensity derived from 53BP1 (left figure) and the signal intensity derived from ⁇ H2AX (right figure) for 53BP1-positive, ⁇ H2AX-positive, and MAP2-positive neurons (neuron cells) in the cerebral cortex.
• FIG. 2 is a diagram showing the results of Western blotting. Quantification and statistical comparison were performed for the marker molecule bands excluding Ub, which form a ladder or smear.
• Figures 46A and B show the interaction between two types of anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 [ Figure 46A] and human monoclonal antibody #129 [ Figure 46B]) and human dsHMGB1 using SPR. It is a figure showing the result of analysis by.
• Figures 46C and D show the interaction between two types of anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 [ Figure 46C] and human monoclonal antibody #129 [Figure 46D]) and human reduced HMGB1.
• SPR is a diagram showing the results of analysis by SPR.
• Figures 47A and B show the interaction between two types of anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 [ Figure 47A] and human monoclonal antibody #129 [Figure 47B]) and human dsHMGB1 using ELISA.
• FIG. Figures 47C and D show the interaction between two types of anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 [ Figure 47C] and human monoclonal antibody #129 [Figure 47D]) and human reduced HMGB1.
• FIG. Figures 47C and D show the interaction between two types of anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 [ Figure 47C] and human monoclonal antibody #129 [Figure 47D]) and human reduced HMGB1.
• ELISA method is a diagram showing the results of analysis by ELISA method.
• FIG. 48C is a diagram showing the results of a Y-maze test conducted at 6, 7, or 8 months of age.
• mice normal mice (normal saline), various FTLD-ALS model mice administered with physiological saline (KI_saline), various FTLD-ALS model mice administered with human monoclonal antibody #129-L2 (KI_129-L2) ) and various FTLD-ALS model mice (KI_129) administered with human monoclonal antibody #129.
• Three types of FTLD-ALS model mice (PGRN R504X -KI mouse [Figure 49A], TDP43 N267S -KI mouse [ Figure 49B], or VCP T262A -KI mouse [ Figure 49B]) were administered with the above two types of anti-human HMGB1 antibodies of the present invention.
• Each graph shows, from left to right, normal mice ("B6-nT” in the diagram), various FTLD-ALS model mice administered with physiological saline (“KI-nT” in the diagram), and control human IgG administered.
• the number of MAP2-positive neurons (neuron cell number) showing abnormal KDEL staining images in the cerebral cortex was measured in three types of FTLD-ALS model mice (PGRN R504X -KI mice) administered with the above two types of anti-human HMGB1 antibodies of the present invention.
• PGRN R504X -KI mice TDP43 N267S -KI mice
• VCP T262A -KI mice VCP T262A -KI mice
• Each graph shows, from left to right, normal mice ("B6-nT" in the diagram), various FTLD-ALS model mice administered with physiological saline (“KI-nT" in the diagram), and control human IgG administered.
• Each graph shows, from left to right, normal mice ("B6-nT” in the diagram), various FTLD-ALS model mice administered with physiological saline (“KI-nT” in the diagram), and control human IgG administered.
• Various FTLD-ALS model mice (“KI-IgG” in the figure), various FTLD-ALS model mice administered with human monoclonal antibody #129 (“KI-129” in the figure), and human monoclonal antibody #129-L2
• the results are shown for various FTLD-ALS model mice ("KI-129L2" in the figure) administered with.
• "#", "##”, and "###" in the figure have statistically significant differences according to Tukey's HSD test (p ⁇ 0.05, p ⁇ 0.01, and p ⁇ 0.001).
• FIG. 2 is a diagram showing the results of purifying human monoclonal antibody #129 from a CHO (Chinese Hamster Ovary) cell line that stably expresses human monoclonal antibody #129 and analyzing it by non-reducing capillary electrophoresis.
• FIG. 2 is a diagram showing the results of purifying human monoclonal antibody #129-L2 from a CHO cell line that stably expresses human monoclonal antibody #129-L2 and analyzing it by non-reducing capillary electrophoresis.
• FIG. 3 is a diagram showing the results of SDS-PAGE.
• "HC” indicates a band derived from the antibody heavy chain
• "LC” indicates a band derived from the antibody light chain.
• FIG. 58A is a diagram schematically showing a protocol for a therapeutic experiment on AD model mice (5 ⁇ FAD mice).
• FIG. 58B shows the results of measuring the number of synapses in the cerebral cortex in AD model mice and examining the therapeutic effects of two types of anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 or human monoclonal antibody #129).
• FIG. FIG. 58C is a diagram showing the results of examining the therapeutic effects of the above two types of anti-human HMGB1 antibodies of the present invention on cognitive decline in AD model mice using a Y-maze test. The graphs in FIGS.
• 58B and C show, from left to right, a normal mouse administered with control human IgG ("ctrl B6/SJL” in the figure), a normal mouse administered with human monoclonal antibody #129-L2 ("129L2” in the figure), and a normal mouse administered with human monoclonal antibody #129-L2 ("129L2" B6/SJL”), normal mice administered with human monoclonal antibody #129 ("129 B6/SJL” in the figure), AD model mice administered with control human IgG (“ctrl 5xFAD” in the figure), human The results of AD model mice administered with monoclonal antibody #129-L2 (“129L2 5 ⁇ FAD” in the figure) and AD model mice administered with human monoclonal antibody #129 (“129 5 ⁇ FAD” in the figure) are shown below.
• FIG. 3 is a diagram showing the results of analyzing the proportions of primary TRIAD necrosis, secondary TRIAD necrosis, and A ⁇ extracellular deposits (amyloid plaques) by performing immunohistochemical staining using antibodies and anti-A ⁇ antibodies.
• a first aspect of the preventive or therapeutic agent of the present invention is “amyotrophic lateral sclerosis; Parkinson's disease; Huntington's disease; degenerative diseases or neurological diseases associated with aging; brain aging; and diseases accompanied by brain aging.
• a second aspect of the preventive or therapeutic agent of the present invention is “Alzheimer's disease; frontotemporal lobar degeneration; amyotrophic lateral sclerosis; Parkinson's disease; Huntington's disease; degenerative diseases related to aging or An agent containing the present human monoclonal antibodies L1/L2 specified for the use of "for the prevention or treatment of one or more of neurological diseases; brain aging; and diseases accompanied by brain aging” (hereinafter referred to as "the present preventive agent”).
• prophylactic agent/therapeutic agent 1 and “prophylactic agent/therapeutic agent 2” may be collectively referred to as the “prophylactic agent/therapeutic agent 2").
• the prophylactic/therapeutic agent may be used alone as a pharmaceutical (preparation), or may be mixed with additives and used in the form of a composition (pharmaceutical composition).
• “Treatment of brain aging” is also referred to as “improvement of brain aging.”
• H chain CDR1 consists of the amino acid sequence of SEQ ID NO: 1
• H chain CDR2 consists of the amino acid sequence of SEQ ID NO: 2
• H chain CDR3 consisting of the sequence; and L chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4; L chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and L chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 17; It is a human monoclonal antibody containing the following, and is included in the present human monoclonal antibody.
• prevention of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, aging-related degenerative diseases or neurological diseases, brain aging, or diseases accompanied by brain aging includes amyotrophic lateral sclerosis In addition to suppressing the incidence and onset of sclerosis, Parkinson's disease, Huntington's disease, degenerative diseases or neurological diseases related to aging, brain aging, or diseases accompanied by brain aging, it also helps prevent the onset of diseases such as amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. It also includes delays in the timing of onset or onset of diseases, degenerative diseases or neurological diseases related to aging, brain aging, or diseases accompanied by brain aging.
• treatment (improvement) of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, degenerative or neurological diseases related to aging, brain aging, or diseases accompanied by brain aging includes amyotrophic lateral sclerosis. It includes not only recovery and improvement of lesions and symptoms of sclerosis, Parkinson's disease, Huntington's disease, aging-related degenerative diseases or neurological diseases, brain aging, or diseases accompanied by brain aging, but also suppression of their progression.
• the present human monoclonal antibody contained in the present prophylactic/therapeutic agent 1 and the present human monoclonal antibody L1/L2 contained in the present prophylactic/therapeutic agent 2 are based on the human HMGB1 protein, as demonstrated in the present example described below.
• TRIAD It has the effect of inhibiting necrosis or its amplification (also referred to as "propagation”); the effect of reducing protein (eg, TDP43) aggregates in cerebral cortical neurons; Therefore, this disease, etc., which is the target of prevention or treatment with the present prophylactic/therapeutic agent 1, Alzheimer's disease, frontotemporal lobar degeneration, or the present disease, etc., which is the target of prevention or treatment with the present preventive/therapeutic agent 2, etc.
• HMGB1 and/or pSer46-MARCKS are one or more selected from HMGB1 and/or pSer46-MARCKS; a decrease in the repair function of DNA damage in neurons; TRIAD necrosis or its amplification; and protein (e.g., TDP43) aggregates in cerebral cortical neurons;
• the causes can be suitably exemplified.
• Examples of the above-mentioned "ageing-related degenerative diseases or neurological diseases” include Lewy body dementia, progressive supranuclear palsy, basal ganglia degeneration, striatonigral degeneration, and intranuclear inclusion disease. , multisystem degeneration, etc.
• the above-mentioned “brain aging” means a decrease in cranial nerves, an increase in nerve cell death, and/or a decline in cognitive function due to aging.
• examples of the above-mentioned “diseases associated with brain aging” include multiple sclerosis, viral encephalitis, acute demyelinating encephalomyelitis, neuromyelitis optica, and the like.
• TRIAD necrosis
• RNA polymerase II a basic molecule of transcription. It is caused by a decline in the function of the associated protein (Reference ⁇ J CellBiol (2006) 172 (4): 589-604.'').
• TRIAD does not have the morphological and biochemical features of apoptosis, and unlike autophagic cell death, there is no expansion or increase in autophagosomes, but the morphological feature is that the endoplasmic reticulum swells.
• Target diseases of the present preventive/therapeutic agent include genetic diseases (for example, VCP [Valosin-Containing Protein] mutations such as VCP T262A mutation; PGRN [progranulin] mutations such as PGRN R504X mutation; CHMP2B [such as CHMP2B Q165X mutation)] [Charged Multivesicular Body Protein 2B] mutation; TDP43 [Transactive Response DNA Binding Protein of 43 kD] mutation such as TDP43 N267S mutation; or a sporadic disease.
• VCP Valosin-Containing Protein
• PGRN progranulin] mutations such as PGRN R504X mutation
• CHMP2B such as CHMP2B Q165X mutation
• TDP43 Transactive Response DNA Binding Protein of 43 kD
• TDP43 N267S mutation or a sporadic disease.
• the subject human monoclonal antibody has a heavy chain variable region consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a heavy chain variable region shown in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 18.
• a human monoclonal antibody comprising an L chain variable region consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 is preferable, and has at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9.
• a human monoclonal antibody comprising a heavy chain consisting of an amino acid sequence and a light chain consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 22, or SEQ ID NO: 19. preferable.
• the present human monoclonal antibody L1/L2 has a heavy chain variable region consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a heavy chain variable region shown in SEQ ID NO: 21 or SEQ ID NO: 18.
• a human monoclonal antibody comprising a light chain variable region consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 is preferred, and has at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9. More preferred is a human monoclonal antibody comprising a heavy chain consisting of an amino acid sequence and a light chain consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 19.
• HMGB1 High Mobility Group Box 1
• HMG1, HMG3, SBP-1, and HMG-1 are proteins also referred to as HMG1, HMG3, SBP-1, and HMG-1.
• a human-derived protein is typically a protein consisting of the amino acid sequence specified by the NCBI reference sequence: NP_002119.1 (a protein encoded by the nucleotide sequence specified by the NCBI reference sequence: NM_002128.5).
• NP_002119.1 a protein encoded by the nucleotide sequence specified by the NCBI reference sequence: NM_002128.5
• the DNA sequence of a gene is mutated in the natural world (ie, non-artificially) due to its mutation, and the amino acid sequence of the protein encoded by it is also modified accordingly.
• HMGB1 to which the present human monoclonal antibody and the present human monoclonal antibodies L1/L2 bind includes proteins consisting of the amino acid sequence specified by NCBI reference sequence: NP_002119.1, as well as naturally occurring variants of such proteins. is also included.
• an antibody that specifically binds to human HMGB1 refers to an antibody that recognizes and binds to human HMGB1 through a highly specific antigen-antibody recognition mechanism.
• the subject human monoclonal antibodies and the subject human monoclonal antibodies L1/L2 are preferably separated.
• “separated” means that the antibody is removed from its original environment through artificial manipulation, or expressed in an environment different from that in which the antibody originally exists. It means existing in a state different from the state in which it is.
• an "isolated antibody” is an antibody that is derived from a certain individual and that has not been subjected to external manipulation (artificial manipulation) and that has been isolated from the body of the individual or from tissues or body fluids (blood) derived from the body. , plasma, serum, etc.) is not included.
• the present human monoclonal antibody and the present human monoclonal antibody L1/L2 are preferably antibodies produced from organisms or cells produced by artificial manipulation (for example, antibodies produced from hybridomas).
• Such "antibodies produced from organisms or cells produced by artificial manipulation” do not include antibodies produced from naturally occurring organisms or B cells (that have not been subjected to artificial manipulation).
• monoclonal antibody means an antibody (including functional fragments of antibodies) obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies are those that recognize a single determinant on an antigen.
• the present human monoclonal antibodies and the present human monoclonal antibodies L1/L2 include classes and subclasses of human immunoglobulins, and also include forms of functional fragments of such antibodies. Classes and subclasses of the present human monoclonal antibody and the present human monoclonal antibody L1/L2 include IgG such as IgG1, IgG2, IgG3, and IgG4; IgA such as IGA1 and IGA2; IgD; IgE; IgM; and the like.
• the subject human monoclonal antibody and the subject human monoclonal antibody L1/L2 typically include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3, and A framework region (FR) is connected to the amino (N) terminus and carboxyl (C) terminus of each of these CDR1-3 regions, and includes a heavy chain variable region and a light chain variable region.
• FR framework region
• the heavy chain FRs include: heavy chain FR1 connected to the N-terminus of heavy chain CDR1; heavy chain FR2 connected between the C-terminus of heavy chain CDR1 and the N-terminus of heavy chain CDR2; Heavy chain FR3 is linked between the C-terminus of chain CDR2 and the N-terminus of heavy chain CDR3; and heavy chain FR4 is linked to the C-terminus of heavy chain CDR3.
• the light chain FRs include: light chain FR1 connected to the N-terminus of light chain CDR1; light chain FR2 connected between the C-terminus of light chain CDR1 and the N-terminus of light chain CDR2; ; light chain FR3 linked between the C-terminus of light chain CDR2 and the N-terminus of light chain CDR3; and light chain FR4 linked to the C-terminus of light chain CDR3.
• the heavy chain FR1 is (HF1) a polypeptide consisting of the 1st to 30th amino acid residues of the amino acid sequence shown by SEQ ID NO: 7, or (HF1') at least 80% or more of such a polypeptide.
• a polypeptide consisting of an amino acid sequence having sequence identity of Specifically, the heavy chain FR2 is (HF2) a polypeptide consisting of the 36th to 49th amino acid residues of the amino acid sequence shown by SEQ ID NO: 7, or (HF2') at least 80% or more of such a polypeptide.
• the heavy chain FR3 includes (HF3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence shown by SEQ ID NO: 7, and a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence shown by SEQ ID NO: 11. or (HF3') consisting of an amino acid sequence having at least 80% or more sequence identity with such a polypeptide,
• the heavy chain FR4 is (HF4) a polypeptide consisting of the 105th to 115th amino acid residues of the amino acid sequence shown by SEQ ID NO: 7, or (HF4') at least 80% or more of such a polypeptide.
• a polypeptide consisting of an amino acid sequence having sequence identity can be mentioned.
• the light chain FR1 is (LF1) a polypeptide consisting of the 1st to 23rd amino acid residues of the amino acid sequence shown by SEQ ID NO: 8, or (LF1') at least 80% or more of such a polypeptide.
• a polypeptide consisting of an amino acid sequence having sequence identity of Specifically, the light chain FR2 is (LF2) a polypeptide consisting of the 35th to 49th amino acid residues of the amino acid sequence shown by SEQ ID NO: 8, or (LF2') at least 80% or more of such a polypeptide.
• a polypeptide consisting of an amino acid sequence having sequence identity of Specifically, the light chain FR3 is (LF3) a polypeptide consisting of the 57th to 88th amino acid residues of the amino acid sequence shown by SEQ ID NO: 8, or (LF3') at least 80% or more of such a polypeptide.
• a polypeptide consisting of an amino acid sequence having sequence identity of Specifically, the light chain FR4 is (LF4) a polypeptide consisting of the 98th to 107th amino acid residues of the amino acid sequence shown by SEQ ID NO: 8, or (LF4') at least 80% or more of such a polypeptide.
• LF4 a polypeptide consisting of an amino acid sequence having sequence identity can be mentioned.
• the present human monoclonal antibody and the present human monoclonal antibodies L1/L2 are human antibodies.
• Such "human antibodies” include, for example, human chimeric antibodies, humanized antibodies, fully human antibodies, etc., and preferred examples include humanized antibodies and fully human antibodies.
• human chimeric antibody refers to a linkage between the variable region of an antibody derived from a non-human animal (e.g., a non-human mammal such as chicken, mouse, rat, cow, etc.) and the constant region of a human-derived antibody. This is an antibody that has been tested.
• Human chimeric antibodies can be produced, for example, by immunizing a non-human animal (preferably a non-human mammal) with an antigen and cutting out the antibody variable region (variable region) that binds to the antigen from the gene of the mouse monoclonal antibody.
• Human constant regions of human chimeric antibodies include, for example, 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. be able to.
• the amino acid sequences of these constant regions and the base sequences encoding them are known.
• one or more amino acids in the human-derived antibody constant region may be substituted, deleted, added, and/or inserted. can.
• humanized antibody refers to the gene sequence of the antigen-binding region (CDR) of an antibody derived from a non-human animal (e.g., a non-human mammal such as a chicken, mouse, rat, or cow) derived from a human. It is an antibody grafted onto an antibody gene (CDR grafting), and its production method is known such as overlap extension PCR (for example, European Patent Application Publication No. 239400, European Patent Application Publication No. 125023). , WO 90/07861, WO 96/02576).
• the variable region of an antibody is usually composed of three CDRs sandwiched between four framework regions (FR). CDRs are the regions that essentially determine the binding specificity of an antibody.
• the amino acid sequences of CDRs are highly diverse, the amino acid sequences constituting FRs often show high homology even between antibodies with different binding specificities. Therefore, it is generally said that the binding specificity of one antibody can be transplanted to another antibody by transplanting CDRs. Furthermore, from the viewpoint of maintaining CDR function, when transplanting a non-human-derived CDR to a human FR, a human FR that is highly homologous to the non-human animal-derived FR is selected. In other words, amino acids in CDRs not only recognize antigens but also coordinate with amino acids in FRs near the CDRs and are involved in maintaining the loop structure of the CDRs. It is preferable to use a human FR consisting of an amino acid sequence highly homologous to the amino acid sequence of the FR.
• Search for known human FRs that are highly homologous to FRs derived from non-human animals can be performed using, for example, a search system specialized for antibodies available on the Internet (http://www.bioinf.org.uk/abysis/). This can be done by using Mutations can be introduced into sequences other than the CDRs of an antibody derived from a non-human 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 through the search is available, non-human-derived CDRs may be introduced into the sequence. Introduction of mutations can be performed using techniques known in the art, such as nucleic acid synthesis and site-directed mutagenesis.
• the CDRs form a good antigen-binding site when linked through the CDRs.
• FRs of human-derived antibodies can be suitably selected.
• amino acid residues in the FRs can be substituted so that the CDRs of the humanized antibody form appropriate antigen-binding sites, according to the method described in the literature "Cancer Res, 1993, 53, 851-856.”
• a mutant FR sequence having desired properties can be selected by measuring and evaluating the affinity of the mutant antibody with the amino acid substitution for the antigen.
• Fully human antibody refers to an antibody in which all sequences of the antibody are derived from humans.
• Fully human antibodies can be produced, for example, in transgenic mice engineered to express human heavy and light chain antibody genes.
• Transgenic mice that produce human antibodies can be prepared, for example, according to the methods described in WO 02/43478 pamphlet, US Patent No. 6,657,103 (Abgenix), and the like.
• a hybridoma cell line fused with B cells derived from a transgenic mouse that produces the desired antibody can be obtained using, for example, U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,625,126; U.S. Pat. Patent No. 5661016; U.S. Patent No. 5545806; Document "Jakobovits, Adv. Drug Del. Rev. 31:33-42 (1998)”; Document “Green, et al, J. Exp. Med. 188 :483-95 (1998).
• the subject human monoclonal antibody and the subject human monoclonal antibody L1/L2 include not only the whole antibody but also a part (partial fragment) of the antibody that has a functional ability to specifically recognize the HMGB1 protein. Also includes fragments. Such functional fragments include Fab, Fab', F(ab') 2 , variable region fragment (Fv), disulfide bonded Fv, single chain Fv (scFv), sc(Fv) 2 , diabody, multispecific. Examples include antibodies, polymers thereof, and the like.
• Fab refers to a monovalent antigen-binding fragment of an immunoglobulin consisting of one light chain and part of a heavy chain. It can be obtained by papain digestion of antibodies and also by recombinant methods. "Fab'” differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the antibody's hinge region. "F(ab')2" means a bivalent antigen-binding fragment of an immunoglobulin consisting of portions of both light chains and both heavy chains.
• a “variable region fragment (Fv)” is the smallest antibody fragment that has 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 tightly linked by non-covalent bonds.
• a “single chain Fv (scFv)” includes the heavy and light chain variable regions of an antibody, which regions are present in a single polypeptide chain.
• sc(Fv)2 is a single chain made by linking two heavy chain variable regions and two light chain variable regions with a linker or the like.
• a “diabody” is a small antibody fragment with two antigen-binding sites, which contains a heavy chain variable region joined to a light chain variable region in the same polypeptide chain, each region having a distinct It forms a pair with the complementary region of the strand.
• a “multispecific antibody” is a monoclonal antibody that has binding specificity for at least two different antigens. For example, two heavy chains can be prepared by co-expression of two immunoglobulin heavy/light chain pairs with different specificities.
• the H chain CDRs 1 to 3 and the L chain CDRs 1 to 3 of the present human monoclonal antibody contain one or more amino acids in the amino acid sequences of SEQ ID NOs: 1 to 6 without decreasing the desired activity (i.e., affinity for HMGB1). Modifications in which are substituted, deleted, added, and/or inserted are included.
• the subject human monoclonal antibody containing such modified CDRs can be produced, for example, by introducing mutations into DNA encoding the antibody chain of human monoclonal antibody #129 or by peptide synthesis.
• a plurality of amino acids in “substitution, deletion, addition, and/or insertion of one or more amino acids” is preferably within 10 amino acids, more preferably within 5 amino acids, and even more preferably within 3 amino acids. It means within an amino acid (for example, within 2 amino acids, 1 amino acid).
• conservative substitutions are preferred.
• Such “conservative substitutions” refer to substitutions with other amino acid residues having chemically similar side chains. Groups of amino acid residues having chemically similar amino acid side chains are well known in the art.
• acidic amino acids aspartic acid and glutamic acid
• basic amino acids lysine, arginine, histidine
• neutral amino acids include amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), and hydroxy groups.
• serine, threonine sulfur-containing amino acids
• sulfur-containing amino acids cyste, methionine
• amide group-containing amino acids asparagine, glutamine
• imino group-containing amino acids proline
• aromatic group-containing amino acids phenylalanine, tyrosine, tryptophan
• the H chain CDR1 in the subject human monoclonal antibody the H chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1 is preferable, as its effect has been demonstrated in the present example described below, and as the H chain CDR2 in the subject human monoclonal antibody, The H chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2 is preferable because its effect has been demonstrated in the present example described below.
• the H chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3 is preferred because it has been demonstrated, and the L chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4 is preferable as the L chain CDR1 of the present human monoclonal antibody, since its effect has been demonstrated in the present example described later.
• L chain CDR1 consisting of the amino acid sequence is preferred, and L chain CDR2 in the human monoclonal antibody of the present invention is preferably L chain CDR2 consisting of the amino acid sequence SEQ ID NO: 5, as its effect has been demonstrated in the present example described below.
• L chain CDR3 in the present human monoclonal antibody the L chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 20, or SEQ ID NO: 17 is preferable because its effect has been demonstrated in the present example described below.
• amino acid sequence homology can be determined using the BLASTP (amino acid level) program (for example, literature “J. Mol. Biol., 215:403-410, 1990").
• BLASTP amino acid level
• the program uses the algorithm BLAST (for example, the document “Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990", the document "Proc. Natl.
• the subject human monoclonal antibodies and the subject human monoclonal antibodies L1/L2 include those in which post-translational processes of antibodies are modified, such as by changing the number or position of glycosylation sites. Such variants can improve the ADCC activity (antibody-dependent cytotoxic activity) of antibodies.
• Glycosylation of antibodies is typically N-linked or O-linked. Glycosylation of antibodies is highly dependent on the host cell used to express the antibody. Modification of the glycosylation pattern can be performed by known methods such as introduction or deletion of specific enzymes involved in sugar production (Japanese Patent Application Laid-open No. 2008-113663, US Pat. No. 5,047,335, US Pat. No. 5,510,261, US Pat. No.
• amino acids that are deamidated for the purpose of increasing antibody stability, and amino acids adjacent to the amino acids that are deamidated may be substituted with other amino acids.
• Deamidation may be suppressed by substituting with glutamic acid, or glutamic acid may be substituted with another amino acid to increase the stability of the antibody.
• the subject human monoclonal antibody and the subject human monoclonal antibody L1/L2 can be produced by a known hybridoma method or a known recombinant DNA method.
• a typical example of the hybridoma method is the method of Kohler and Milstein (Reference "Nature, 256:495 (1975)").
• the antibody-producing cells used in the cell fusion step in this method are animals ( Examples include spleen cells, lymph node cells, and peripheral blood leukocytes of mammals such as mice, rats, hamsters, rabbits, monkeys, and goats. It is also possible to use antibody-producing cells obtained by allowing an antigen to act in a medium on the above-mentioned cells or lymphocytes previously isolated from non-immunized animals.
• Antibody-producing cells and myeloma cells may originate from different animal species as long as they can fuse, but preferably they originate from the same animal species.
• Hybridomas are produced, for example, by cell fusion between spleen cells obtained from mice immunized with an antigen and mouse myeloma cells, and a subsequent screening identifies hybridomas that produce monoclonal antibodies specific to the HMGB1 protein.
• a human monoclonal antibody that specifically binds to the HMGB1 protein can be obtained by culturing a hybridoma or from the ascites of a mammal to which the hybridoma has been administered.
• the recombinant DNA method involves cloning the antibody genes encoding the subject human monoclonal antibodies and the subject human monoclonal antibodies L1/L2 from hybridomas, B cells, etc., inserting them into an appropriate vector, and injecting them into host cells (e.g., HEK cells, etc.). (e.g. mammalian cell lines, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the subject human monoclonal antibodies and the subject human monoclonal antibodies L1/L2 as recombinant antibodies (for example, as described in the document "Eur. J.Biochem.192:767-775(1990)”).
• host cells e.g., HEK cells, etc.
• host cells e.g., HEK cells, etc.
• mammalian cell lines, Escherichia coli, yeast cells, insect cells, plant cells, etc. e.g. mammalian cell lines, Escherichia coli, yeast
• the antibody genes encoding heavy chains or light chains may be separately incorporated into expression vectors and host cells may be transformed.
• Antibody genes encoding heavy and light chains may be incorporated into a single expression vector to transform host cells (WO 94/11523).
• the present human monoclonal antibody and the present human monoclonal antibody L1/L2 can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cells and separating and purifying them within the host cells or from the culture solution. For isolation and purification of antibodies, methods commonly used for purification of polypeptides can be used.
• transgenic animal cow, goat, sheep, pig, etc.
• the subject human monoclonal antibody gene and the subject human monoclonal antibody can be produced from the milk of the transgenic animal. It is also possible to obtain large quantities of human monoclonal antibodies derived from antibody L1/L2 genes.
• additives include pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, isotonic agents, solubilizing agents, nutrients, and other ingredients.
• pharmaceutically acceptable carriers include, for example, mannitol, lactose, sucrose, human albumin, etc. when the present prophylactic/therapeutic agent is a powder, and when the present prophylactic/therapeutic agent is a liquid.
• examples include physiological saline, water for injection, phosphate buffer, aluminum hydroxide, and the like.
• Subjects to whom the present prophylactic/therapeutic agent 1 may be administered may be subjects (humans) who require prevention or treatment of the subject disease, etc., for example, those who have been diagnosed as having a high risk of developing or developing the subject disease, etc. Examples include subjects and patients who have developed or are developing the subject disease.
• the subject to whom the present preventive/therapeutic agent 2 is administered may be any subject (human) who requires prevention or treatment of Alzheimer's disease, frontotemporal lobar degeneration, the subject disease, etc., for example, Alzheimer's disease or Targets who have been diagnosed with or are at high risk of developing frontotemporal lobar degeneration, the disease, etc., and patients who have developed or are developing Alzheimer's disease, frontotemporal lobar degeneration, the disease, etc. can be mentioned.
• the methods of administering the prophylactic/therapeutic agent include, for example, injection (e.g., subcutaneous, intramuscular, intravenous, intraarterial), oral mucosal administration, intrarectal administration, and local administration to the brain. (intracerebroventricular administration), transdermal administration, oral administration, etc., and intravenous administration is preferably exemplified.
• the dosage of the subject human monoclonal antibody and the subject human monoclonal antibody L1/L2 may vary depending on the age, weight, sex, health condition, and degree of progression of the symptoms of the subject, but it is usually administered per 1 kg of body weight per day for adults. The amount is 0.1 to 1000 mg, preferably 1 to 100 mg.
• the present preventive/therapeutic agent 1 may be used in combination with known pharmaceuticals used for the prevention or treatment of the present disease. Further, the present preventive/therapeutic agent 2 may be used in combination with known pharmaceuticals used for the prevention or treatment of Alzheimer's disease, frontotemporal lobar degeneration, the present disease, and the like.
• human monoclonal antibody (human IgG antibody) #129 prepared in the example of the international patent application (PCT-JP2019-36926 [International Publication No. 2020/059847 pamphlet]) was used as the anti-human HMGB1 antibody. .
• the amino acid sequences of the H chain and L chain of human monoclonal antibody #129 are shown in Tables 1 and 2, respectively.
• the underlined region in the table represents the H chain V region (SEQ ID NO: 7).
• the three regions surrounded by squares in the table indicate, in order from the amino terminal side, H chain CDR1 (SEQ ID NO: 1), H chain CDR2 (SEQ ID NO: 2), and H chain CDR3 (SEQ ID NO: 3).
• the underlined region in the table indicates the L chain V region (SEQ ID NO: 8). Furthermore, the three regions surrounded by squares in the table indicate, in order from the amino terminal side, L chain CDR1 (SEQ ID NO: 4), L chain CDR2 (SEQ ID NO: 5), and L chain CDR3 (SEQ ID NO: 6).
• FTLD-ALS model mice Four types of FTLD-ALS model mice (VCP T262A -KI mouse, PGRN R504X -KI mouse, CHMP2B Q165X -KI mouse, and TDP43 N267S -KI mouse) (herein referred to as "four types of FTLD-ALS model mice") PGRN R504X -KI mice were produced according to the method described in the literature "Nat. Commun., 2018 9, 433".
• a targeting vector for producing VCP T262A -KI mice was constructed. Specifically, a PCR product of a 5.4 kb NotI-XhoI fragment amplified from a Bac clone (ID: RP23-111G9 or RP23-124L1) was subcloned into the PspOMI-XhoI site of pBS-DTA. Similarly, a 2.9 kb BamHI-NotI fragment amplified from the same Bac clone was subcloned into pBS-LNL(-) carrying the Neo cassette (loxP-Neo-loxP).
• a 4.5 kb fragment (1.6 kb Neo cassette + 2.9 kb fragment) was cut out by Notl treatment, and a 0.8 kb XhoI-Smal fragment with the T262A mutation was amplified by PCR from the same Bac clone. SmaI treatment was performed. The 0.8 kb and 4.5 kb fragments were subcloned into the XhoI-NotI sites of the targeting vector. After linearizing the targeting vector by NotI treatment, the fragment was electroporated into ES cells in a C57BL/6J background.
• VCP T262A -KI mice were selected from non-transgenic littermates.
• a targeting vector for producing CHMP2B Q165X -KI mice was constructed. Specifically, a PCR product of a 5.7 kb ClaI-SalI fragment amplified from a Bac clone (ID: RP23-13H5 or RP23-273E18) was subcloned into the ClaI-SalI site of pBS-DTA (manufactured by Unitech). did. A 3.0 kb SacII-NotI fragment was amplified from the Bac clone and subcloned into pBS-LNL(+) containing the Neo cassette (loxP-Neo-loxP).
• SacII-NotI and the 4.6 kb fragment were subcloned into the SacII-ClaI site of the targeting vector.
• primer set primer 3 [5'-TGGATTTTATTTATGTCTGAATGTG-3'; SEQ ID NO: 13] and primer 4 [5'-ATAAGCAACTTCACAAGGCATCTTA-3'; SEQ ID NO: 14]
• CHMP2B Q165X -KI mice were selected from non-transgenic littermates.
• a targeting vector for producing TDP43 N267S -KI mice was constructed. Specifically, a 5.9 kb ClaI-SalI fragment was amplified from a Bac clone (ID: RP23-364M1 or RP23-331P21) and subcloned into the ClaI-SalI site of pBS-TK (manufactured by Unitech). A 2.7 kb SacII-NotI fragment was amplified from the Bac clone and subcloned into pA-LNL(+) containing the Neo cassette (loxP-Neo-loxP).
• the 4.3 kb fragment (1.6 kb Neo cassette + 2.7 kb fragment) was subcloned into the SacII-ClaI site of the targeting vector.
• a primer set primer 5 [5'-ACAGTTGGGTGTGATAGCAGGTACT-3'; SEQ ID NO: 15] and primer 6 [5'-TCGAGAATTACAGGAATGTATCATC-3'; SEQ ID NO: 16]
• primer 6 [5'-TCGAGAATTACAGGAATGTATCATC-3'; SEQ ID NO: 16]
• the number of pSer46-MARCKS-positive cells per 143 ⁇ m x 143 ⁇ m area was calculated based on the DAPI fluorescence image and the pSer46-MARCKS fluorescence image (FIG. 2-1 and FIG. 9). Furthermore, based on the DAPI fluorescence image, KDEL fluorescence image, and MAP2 fluorescence image, the KDEL signal area was measured for MAP2-positive cells, which are markers of mature neurons (FIG. 10). Furthermore, the ⁇ H2AX signal region was measured for MAP2-positive cells based on the DAPI fluorescence image, ⁇ H2AX fluorescence image, and MAP2 fluorescence image (FIG. 11).
• the 53BP1 signal region was measured for MAP2-positive cells based on the DAPI fluorescence image, 53BP1 fluorescence image, and MAP2 fluorescence image (FIG. 12). Furthermore, pSer409/410-TDP43 signal regions in ubiquitin-positive inclusion bodies were measured based on DAPI fluorescence images, pSer409/410-TDP43 fluorescence images, and ubiquitin fluorescence images (FIG. 13). Furthermore, the p62 signal region in the inclusion body was measured based on the DAPI fluorescence image and the p62 fluorescence image (FIG. 14).
• the percentage of cells in which the VAMP2 signal and the PSD95 signal were colocalized was measured (FIG. 15). Furthermore, based on the DAPI fluorescence image, pSer203-tau fluorescence image, and PSD95 fluorescence image, the percentage of cells in which pSer203-tau signal and PSD95 signal were colocalized was measured (FIG. 16).
• Y-maze test a Y-maze (manufactured by Ohara Medical Sangyo Co., Ltd.) consisting of three identical arms with equal angles between the arms was used. The mouse was placed at the end of one arm and allowed to move freely through the maze for 8 minutes, and the total number of times it moved from one arm to another was calculated as the number of times it entered a new arm that was different from the previous one. The rate of spontaneous change was calculated by dividing (expressed as Alternation Rate; vertical axis in Figures 4 to 7).
• the freezing response was measured in the same chamber without foot shock (total freezing) 24 hours after conditioning (65 dB white noise, 30 seconds + foot shock, 0.4 mA, 2 seconds). Displayed as percentage [Total Freeze percent; vertical axis in Figure 8).
• sample buffer 62.5 mM Tris-HCl, pH 6.8, 2% [w/v] SDS, 2.5%
• BCA method PulierceBCA protein assay kit, Thermo (manufactured by Scientific) to equalize the amount of protein in the sample.
• the protein sample was separated by SDS-PAGE, transferred to a PVDF (Poly Vinylidene Di-Fluoride) membrane (Immobilon-P [manufactured by Millipore]) by a semi-dry method, and then added to TBST (10 mM Tris) containing 5% skim milk.
• PVDF Poly Vinylidene Di-Fluoride
• TBST 5% skim milk
• pSer46-MARCKS is generated at an early stage before the onset of Alzheimer's disease, and HMGB1 leaked from cells due to neuronal necrosis induces phosphorylation of MARCKS and degeneration of neurites (Reference ⁇ SCIENTIFIC REPORT, 2016 Aug 25;6:31895”). Therefore, we analyzed whether MARCKS phosphorylation and neuronal necrosis occur even in the early stages of FTLD onset using four types of FTLD-ALS model mice aged 1 to 12 months.
• the present inventors predicted synaptic disorders using colocalization of VAMP2 and PSD95 and colocalization of pSer203-tau and PSD95 as indicators through phosphoproteome analysis. Therefore, as a result of analyzing synaptic disorders using these colocalizations as an indicator, we found that the colocalization of VAMP2 and PSD95 in the cerebral cortex was decreased in the four types of FTLD-ALS model mice compared to C57BL/6J mice. In contrast, administration of human monoclonal antibody #129 of the present invention showed that such a decrease was significantly suppressed (FIG. 15).
• pSer203-tau synaptic instability-related protein
• pSer77/78-Ku70 phosphorylation (pSer77/78-Ku70) level of DNA repair-related proteins was increased in the four types of FTLD-ALS model mice compared to C57BL/6J mice, whereas It was shown that the increase in pSer77/78-Ku70 levels was suppressed by administering 129 to four types of FTLD-ALS model mice (lane (11) in FIG. 17).
• pSer409/410-TDP43 protein aggregation marker
• the background of the 5xFAD mice was C57BL/SJL, which was created by crossing C57BL/6J females and SJL/J males.
• Comprehensive proteomic analysis was performed using brain tissue samples from male 5 ⁇ FAD mice aged 1, 3, 6, and 12 months as previously described (Reference “Hum Mol Genet 24, 540-558 ( 2015).
• APP-KI App NL-GF/NL-GF ) mouse (Reference “Nat. Neurosci. 17, 661-663 (2014).”), that is, the Swedish (KM670/671NL) mutation of the humanized APP mouse gene, Beyreuther Mice with /Iberian (I716F) mutation and Arctic (E693G) mutation were obtained from RIKEN BioResource Center.
• 0.1 ⁇ g/kg of control IgG (human IgG isotype control, #12000C, manufactured by Thermo Fisher Scientific) or human monoclonal antibody #129 of the present invention was administered to 5 ⁇ FAD mice or B6/SJL mice for 6 to 8 months.
• Subcutaneous administration (hereinafter simply referred to as "subcutaneous administration") was administered once a week to the cervical dorsal region (9 times in total) until the child reached the age of 50 years.
• the human monoclonal antibody #129 of the present invention When the human monoclonal antibody #129 of the present invention is intravenously injected into various types of mice, the human monoclonal antibody #129 of the present invention diluted with physiological saline to a final volume of 50 ⁇ L is administered once a month into the tail vein from 6 to 8 months of age. (3 times in total).
• mouse and human brain tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. Sagittal sections or coronal sections with a thickness of 5 ⁇ m were obtained using a microtome (manufactured by Yamato Koki Kogyo Co., Ltd.).
• Immunohistochemical staining was performed using the following primary antibodies: rabbit anti-phosphorylated Ser46-MARCKS antibody (1:2000 [GL Biochem (Shanghai) Co., Ltd.]); mouse anti-A ⁇ antibody (1:1000, Clone 82E1, #10323, manufactured by IBL); Mouse anti-MAP2 antibody (1:200, sc-32791, manufactured by Santa Cruz); Rabbit anti-MAP2 antibody (1:2000, ab32454, manufactured by Abcam); Mouse anti-phosphorylation H2X ( ⁇ H2X) antibody (1:300, JBW301, manufactured by Millipore); Rabbit anti-53BP1 antibody (1:5000, NB100-304, manufactured by Novus); Rabbit anti-YAP antibody (1:50, GTX129151, manufactured by GeneTex) ;Rabbit anti-NF ⁇ B antibody (1:100, #8242, manufactured by CellSignaling Technology);Mouse anti-IL6 antibody (1:50, ab208113, manufactured by Abcam);Rabbit anti-CCL2/MCP1 antibody (1:
• DAPI 0.2 ⁇ g/mL in PBS, #D523, manufactured by Dojindo Laboratories. All microscopic images were obtained using a confocal microscope (FV1200IX83, manufactured by Olympus).
• Lipofectamine LTX (Thermo Fisher Scientific, U2OS cells were transfected with 2.5 g of EGFP-C1-FLAG-Ku70 plasmid (#46957, Addgene, Watertown, MA, USA) using the EGFP-C1-FLAG-Ku70 plasmid (#46957, Addgene, Watertown, MA, USA).
• ds-HMGB1 (6 ng/mL, 0.24 nM
• human monoclonal antibody #129 of the invention (6 ng/mL, 0.04 nM) was added to the medium.
• U2OS cells were treated with 2 ⁇ M Hoechst33258 (DOJINDO, Kumamoto, Japan) for 20 minutes to sensitize them to DSB.
• a 405 nm laser diode 40 ⁇
• a rectangular area on the cell nucleus was irradiated with a laser using an objective lens (95%, 100 scans, 12.5 units/pixel). Time-lapse images were acquired every 30 seconds for 10 minutes.
• CSF cerebrospinal fluid
• Cellular senescence is characterized by multiple characteristics of TRIAD necrosis, including DNA damage, SASP/DAMP secretion (particularly HMGB1 secretion), transcriptional changes, and apoptosis resistance (Reference “Cell 179, 813-827 (2019).”). Common (References “Nat. Commun. 11, 1-22 (2020).”, “Sci Rep 6, 31895 (2016).”, “J. Cell Biol. 172, 589-604 (2006).” , “Hum Mol Genet 25, 4749-4770 (2016).” and “Cell Death Dis 7, e2207 (2016).”).
• Lamin B1 a nuclear membrane protein that is a cell aging marker, was localized in a ring shape in neurons, astrocytes, and microglia of normal mice (C57BL/6J mice) (Fig. 19a, arrow), but there were two types. In some neurons and astrocytes of AD model mice (5 ⁇ FAD mice and APP-KI mice), the localization of ring-shaped lamin B1 became thinner or disappeared (Fig. 19a, arrow). . In addition, although an abnormal morphology of the lamin B1 ring was observed in the microglia of these two types of AD model mice, most of the lamin B1 ring itself remained (FIG. 19a, arrow).
• NF ⁇ B transcription factors
• MCP1 and IL6 inflammatory cytokines
• 5xFAD mice subcutaneously administered PBS showed higher levels of three types of PKC ⁇ activation indicators than B6/SJL mice subcutaneously administered Go6976 or PBS.
• B6/SJL Control IgG or human monoclonal antibody #129 of the present invention was subcutaneously administered to mice or 5 ⁇ FAD mice (center image in FIG. 22a), and analyzed using a cognitive function test (Y-maze test).
• PBS or PKC ⁇ inhibitor (Go6976) was subcutaneously administered to 6-week-old (1.5-month) B6/SJL mice or 5 ⁇ FAD mice (left panel in Fig. 22a), and Control IgG or human monoclonal antibody #129 of the present invention was administered subcutaneously (middle image in FIG. 22a) or intravenously (right image in FIG. 22a) to B6/SJL mice or 5 ⁇ FAD mice, and anti-pSer46-MARCKS antibody was administered to B6/SJL mice or 5 ⁇ FAD mice. and analyzed by immunohistochemical staining using anti-A ⁇ antibody.
• Patent Document 3 shows that human monoclonal antibody #129 of the present invention, which is an anti-dsHMGB1 human monoclonal antibody, has the effect of inhibiting necrosis in neurons accompanied by YAP decrease (TRIAD). Furthermore, the aforementioned Example 1 shows that the human monoclonal antibody #129 of the present invention has the effect of preventing and improving neurite damage accompanied by MARCKS phosphorylation. The pathological condition underlying these therapeutic effects is the amplification (or spread) of TRIAD necrosis. When neurons undergo TRIAD necrosis, they release HMGB1, including dsHMGB1.
• Example 1 it was shown that human monoclonal antibody #129 of the present invention and an anti-HMGB1 antibody having a common CDR with this antibody exert therapeutic effects on FTLD-ALS model mice.
• the VCP T262A mutation, PGRN R504X mutation, CHMP2B Q165X mutation, and TDP43 N267S mutation that this FTLD-ALS model mouse has are known to cause not only FTLD but also ALS, and FTLD and ALS are the same. It is understood in this field as a disease spectrum (eg, references 1 to 13 below).
• the human monoclonal antibody against HMGB1 of the present invention has shown uniform effects on pathological conditions that can be caused by various causes, and has been shown to be effective against not only familial neurodegenerative diseases but also sporadic neurodegenerative diseases. It can be developed as a therapeutic agent for more patients, including patients.
• Reference 8 Parkinson Net al, ALS phenotypes with mutations in CHMP2B (charged multivesicular bodyprotein 2B) Neurology 2006 Sep 26;67(6):1074-7.
• Reference 9 Cox LE et al. (2010) Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS).
• Reference 10 Waegaert R et al. Longitudinal transcriptomic analysis of altered pathways in a CHMP2B intron5 -based model of ALS-FTD. Neurobiol Dis. 2020 Mar;136:104710.
• necrosis associated with decreased YAP is also observed in Huntington's disease (HD) mice (Reference ⁇ Mao et al, Hum Mol Genet 2016'') and human HD patients (Reference ⁇ Yamanishie et al, Acta 2016''). Its existence has also been confirmed in ⁇ Neuropathol Commun 2017''), and a decrease in motor neurons has been confirmed in mutant SOD1 transgenic mice, which are model mice for amyotrophic lateral sclerosis (ALS) (Reference ⁇ Morimoto et al, J Neurosci Res 2009).
• ALS amyotrophic lateral sclerosis
• human monoclonal antibody #129 of the present invention has the effect of reducing TDP43 aggregates in cerebral cortical neurons in four types of FTLD-ALS model mice. Therefore, human monoclonal antibody #129 of the present invention is fully expected to have an inhibitory effect on degenerative diseases such as ALS accompanied by TDP43 aggregation.
• human monoclonal antibody #129 of the present invention and anti-HMGB1 antibodies having common CDRs are fully expected to exhibit therapeutic effects on degenerative diseases in general, including ALS, PD, and HD.
• human monoclonal antibody #129 of the present invention has been shown to increase the number of senescent cells in the cerebral cortex of two types of AD model mice (5xFAD mice and APP-KI mice).
• Human monoclonal antibody #129 of the present invention and anti-HMGB1 having a common CDR with this antibody inhibit cell aging of neurons, astrocytes, and microglia ( Figures 19 and 20 of Example 2). It has been confirmed that the antibody has the effect of treating degenerative diseases or neurological diseases associated with aging, brain aging, and diseases associated with brain aging.
• R6/2 mice were used as Huntington's disease model mice.
• ⁇ -synuclein A53T mice were used as Parkinson's disease model mice.
• SOD1-Tg mice were used as amyotrophic lateral sclerosis model mice.
• Mutant ataxin-1 knock-in mice were used as spinocerebellar ataxia model mice.
• a 12-month-old C57BL/6 mouse was used as an aging model mouse.
• mice were placed on a rotating rod (3.5 rpm) and the rotation speed was increased linearly to 35 rpm for 300 seconds and continued at 35 rpm until 360 seconds. Mice received 12 trials (4 trials per day for 3 consecutive days) with a 30-60 minute rest interval between trials. The time taken for the mouse to fall from the rod was recorded on each trial.
• the average time to fall from the rotarod was calculated and used for subsequent analysis.
• the mouse was placed in the center of an open field space (50 cm x 50 cm x 40 cm [height]) and allowed to explore for 10 minutes.
• the light intensity was 70 lux at the center of the field.
• the moving distance (cm) was used as the index.
• the footprint test footprints were recorded while walking to compare walking patterns.
• the hind paws were covered with blue ink and the front paws were covered with red ink, and the animals were placed on a 40 cm long and 7.5 cm wide walkway that served as a runway to the closed box.
• the blank paper on the aisle floor was replaced after each run.
• the footprint pattern the footprint pattern parameters described in Document 8 below were measured: stride.
• the Y-maze test was conducted according to the method described in Example 1.
• Brains were removed from disease model mice or normal genotype sibling mice at various ages, fixed in PBS containing 4% paraformaldehyde for 12 hours, and then sliced into 5 ⁇ m sections using a microtome (manufactured by Daiwa Koki Kogyo Co., Ltd.). Thick paraffin sections of brain tissue were prepared. The prepared brain tissue paraffin sections were deparaffinized using xylene, immersed in 0.01M citrate buffer (pH 6.0) for rehydration, and then heated at 120°C for 15 minutes. I did it.
• sample buffer 125 mM Tris-HCl, pH 6.8, 4% [w/v] SDS, 12% [v/v] ] of 2-mercaptoethanol, 20% [v/v] glycerol, and 0.005% [w/v] bromophenol blue
• sample buffer 125 mM Tris-HCl, pH 6.8, 4% [w/v] SDS, 12% [v/v] ] of 2-mercaptoethanol, 20% [v/v] glycerol, and 0.005% [w/v] bromophenol blue
• Htington's disease model Brain tissues were sampled from mice in Group 3 for Mouse Analysis and subjected to pathological analysis.
• immunohistochemical staining triple staining of Htt, Ub, and MAP2 was performed, and Ub-positive Htt intranuclear inclusions were observed in MAP2-positive cerebral neurons in saline-treated R6/2 mice.
• Figure 24A Similar Htt intranuclear inclusions are a finding that has been widely observed in R6/2 mice with or without saline administration (Reference 1).
• human monoclonal antibody #129 has the effect of suppressing pathological changes and cell death in the pathology of Huntington's disease (HD), and suppressing motor function decline.
• Parkinson's disease model mice As shown in "1. Materials and Methods" above, Parkinson's disease model mice were tested using post-onset ⁇ -synuclein A53T mice (Reference 2), and intravenous administration was performed once every two weeks. (Figure 29A). Human monoclonal antibody #129 at a dose of 6 ⁇ g per 30 g body weight (corresponding to 0.2 mg/kg BW) was administered six times in total at intervals of two weeks from 8 weeks of age to 18 weeks of age (FIG. 29A). At 8 weeks of age, at the start of administration, the rotarod test showed that ⁇ -synuclein A53T mice were hyperactive (FIG. 29B), indicating that they had already developed Parkinson's disease.
• Hyperactivity of ⁇ -synuclein A53T mice was significant on day 2 (Day 2) at 12 weeks of age after a total of 3 administrations and at 20 weeks of age after a total of 6 administrations. differentially suppressed ( Figure 29B). Similarly, in an open field test at 20 weeks of age, the total distance traveled by ⁇ -synuclein A53T mice increased compared to normal mice, indicating hyperactivity in ⁇ -synuclein A53T mice. , administration of human monoclonal antibody #129 suppressed the total distance traveled (FIG. 29C), indicating improvement in symptoms.
• TRIAD neuronal necrosis
• TRIAD necrosis which indicates decreased nuclear YAP, was increased (FIG. 31A).
• ⁇ -synuclein A53T mice treated with human monoclonal antibody #129 the number of TRIAD necrotic neurons was significantly decreased compared to ⁇ -synuclein A53T mice treated with physiological saline (FIG. 31A).
• TRIAD is known to be accompanied by abnormal endoplasmic reticulum expansion (Reference 12, Reference 13)
• immunostaining for KDEL an endoplasmic reticulum marker
• was performed in ⁇ -synuclein A53T mice administered with saline The number of neurons showing abnormal KDEL staining was increased (FIG. 30B).
• nerve cells showing abnormal KDEL staining were normalized (FIG. 30B).
• DNA damage of neurons was measured in the cerebral cortex by quadruple staining with MAP2 and DAPI using DNA damage markers 53BP1 and ⁇ H2AX.
• the results showed that DNA damage in nerve cells was increased in ⁇ -synuclein A53T mice compared to normal mice, and that DNA damage was suppressed by administration of human monoclonal antibody #129 (FIG. 30C).
• human monoclonal antibody #129 has the effect of suppressing pathological changes and cell death in the pathological condition of Parkinson's disease (PD), and suppressing motor function decline.
• Amyotrophic lateral sclerosis model mouse Amyotrophic lateral sclerosis model mice were tested using SOD1-Tg mice (Reference 3) after the onset of the disease, as described in "1. Materials and Methods" above, and intravenous injection was performed once every two weeks. intravenous administration was performed ( Figure 33A). Human monoclonal antibody #129-L2 was administered twice every 2 weeks from 6 weeks of age to 9 weeks of age at a dose of 6 ⁇ g per 30 g of body weight (corresponding to 0.2 mg/kg BW) ( Figure 33A ).
• mice administered with physiological saline were sampled from mice of "Atrophic Lateral Sclerosis Model Mouse Analysis Group 3" and pathological analysis was performed.
• human monoclonal antibody #129-L2 i.e., “muscular Brain tissues were sampled from mice of "Atrophic Lateral Sclerosis Model Mouse Analysis Group 3”
• pathological analysis was performed.
• the immunohistochemical staining method when quadruple staining of Ub, SOD1, MAP2, and DAPI was performed, there was an increase in the staining signals of SOD1 and Ub in the spinal ventral horn large neurons of SOD1-Tg mice, and some of them were found to be inclusion bodies. It showed similar morphology.
• TRIAD neuronal necrosis
• DNA damage in nerve cells increases in the pathology of neurodegenerative diseases such as Huntington's disease (References 14 and 15). Therefore, using DNA damage markers 53BP1 and ⁇ H2AX, DNA damage of neurons was measured in the anterior horn of the lumbar spinal cord by quadruple staining with MAP2 and DAPI. The results showed that neuronal DNA damage was increased in SOD1-Tg mice compared to normal mice, and that neuronal DNA damage was suppressed by administration of human monoclonal antibody #129-L2 ( Figure 36).
• human monoclonal antibody #129-L2 has the effect of suppressing pathological changes and cell death in the pathological state of amyotrophic lateral sclerosis (ALS), and suppressing motor function decline.
• ALS amyotrophic lateral sclerosis
• TRIAD which is a cell death target of human monoclonal antibody #129.
• TRIAD neuronal necrosis
• DNA damage in nerve cells increases (References 14 and 15). Therefore, DNA damage in Purkinje cells was measured by quadruple staining with MAP2 and DAPI using DNA damage markers 53BP1 and ⁇ H2AX. The results showed that DNA damage in Purkinje cells was increased in mutant ataxin-1 knock-in mice compared to normal mice, and that DNA damage in Purkinje cells was suppressed by administration of human monoclonal antibody #129 ( Figure 39C).
• KDEL staining performed to detect endoplasmic reticulum abnormalities which is a characteristic of TRIAD, showed that KDEL signal values in the Purkinje cell layer and granule cell layer were increased in mutant ataxin-1 knock-in mice compared to normal mice. Endoplasmic reticulum instability observed with TRIAD was observed ( Figure 40A). Whether such findings ultimately affected the number of Purkinje cells or the number of microglia and astrocytes in the cerebellar cortex was confirmed by staining for MAP2, Iba1, and GFAP (FIG. 40B).
• human monoclonal antibody #129 has the effect of suppressing pathological changes and cell death in spinocerebellar ataxia, and suppressing motor function decline.
• LaminB1 is a nuclear membrane lining protein and shows a nuclear ring-shaped staining pattern in immunohistochemical staining, but it is said that the nuclear ring-shaped localization disappears during cell aging (Reference 20).
• DNA damage in cerebral cortical neurons was measured by quadruple staining with MAP2 and DAPI. The results showed that DNA damage in neurons increased in older mice compared to younger mice, and that administration of human monoclonal antibody #129-L2 suppressed DNA damage in cerebral cortical neurons in older mice. was shown (FIG. 44A). Whether such findings ultimately affected the number of neurons, microglia, and astrocytes in the cerebral cortex was confirmed by staining for MAP2, Iba1, and GFAP (FIG. 44B).
• human monoclonal antibody #129-L2 has the effect of suppressing cell changes and cell death associated with aging, and suppressing cognitive function decline.
• This human monoclonal antibody is effective against Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, spinocerebellar ataxia, as well as aging-associated cellular changes, cell death, cognitive function (memory), and motor function. It has been shown to have therapeutic effects.
• Reference 1 Mangiarini, L., K. Sathasivam, M. Seller, B. Cozens, A. Harper, C. Hetherington, M. Lawton, Y. Trottier, H. Lehrach, S. Davies, and G. Bates. 1996 . Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell. 87:493-506.
• Reference 2 Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VM. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron.
• Reference 4 Watase K, Weeber EJ, Xu B, Antalffy B, Yuva-Paylor L, Hashimoto K, Kano M, Atkinson R, Sun Y, Armstrong DL, Sweatt JD, Orr HT, Paylor R, Zoghbi HY.
• a long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration. Neuron. 2002 Jun 13;34(6):905-19.
• Reference 5 Ito H, Yoshimura N, Kurosawa M, Ishii S, Nukina N, Okazawa H. Knock-down of PQBP1 impairs anxiety-related cognition in mouse. Hum Mol Genet.
• Reference 12 Hoshino M, Qi ML, Yoshimura N, Miyashita T, Tagawa K, Wada Y, Enokido Y, Marubuchi S, Harjes P, Arai N, Oyanagi K, Blandino G, Sudol M, Rich T, Kanazawa I, Wanker EE , Saitoe M, Okazawa H. Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73. J Cell Biol. 2006 Feb 13;172(4):589-604.
• Reference 13 Mao Y, Chen X, Xu M, Fujita K, Motoki K, Sasabe T, Homma H, Murata M, Tagawa K, Tamura T, Kaye J, Finkbeiner S, Blandino G, Sudol M, Okazawa H. Targeting TEAD /YAP-transcription-dependent necrosis, TRIAD, ameliorates Huntington's disease pathology. Hum Mol Genet. 2016 Nov 1;25(21):4749-4770.
• Reference 15 Enokido Y, Tamura T, Ito H, Arumughan A, Komuro A, Shiwaku H, Sone M, Foulle R, Sawada H, Ishiguro H, Ono T, Murata M, Kanazawa I, Tomilin N, Tagawa K, Wanker EE , Okazawa H. Mutant huntingtin impairs Ku70-mediated DNA repair. J Cell Biol. 2010 189(3):425-43, 2010.
• Reference 16 Fujita K, Homma H, Kondo K, Ikuno M, Yamakado H, Tagawa K, Murayama S, Takahashi R, Okazawa H.
• Ser46-Phosphorylated MARCKS Is a Marker of Neurite Degeneration at the Pre-aggregation Stage in PD/DLB Pathology. eNeuro. 2018 Sep 4;5(4):ENEURO.0217-18.2018. Reference 17: Forsberg KM, Graffmo KS, Stenvall E, Tabikh N, Marklund SL, Brannstrom T, Andersen PM. Widespread CNS pathology in amyotrophic lateral sclerosis homozygous for the D90A SOD1 mutation. Acta Neuropathol. 2023 Jan;145(1):13 -28.
• Reference 18 Morimoto N, Nagai M, Miyazaki K, Kurata T, Takehisa Y, Ikeda Y, Kamiya T, Okazawa H, Abe K. Progressive decrease in the level of YAPdeltaCs, prosurvival isoforms of YAP, in the spinal cord of transgenic mouse Carrying a mutant SOD1 gene. J Neurosci Res. 2009 Mar;87(4):928-36.
• Reference 19 Fujita K, Mao Y, Uchida S, Chen X, Shiwaku H, Tamura T, Ito H, Watase K, Homma H, Tagawa K, Sudol M, Okazawa H.
• the underlined amino acid sequence in the table indicates the modified site of the light chain CDR3 amino acid sequence of human monoclonal antibody #129.
• the antibody affinity of human monoclonal antibody #129 and human monoclonal antibody #129-L2 for human dsHMGB1 was analyzed by SPR as described in Example 1.
• the dissociation constants (K D ) of human monoclonal antibody #129 and human monoclonal antibody #129-L2 for human dsHMGB1 were 1.47 ⁇ 10 ⁇ 11 and 1.44 ⁇ 10 ⁇ 11 , respectively, and both antibodies , was shown to have almost equivalent binding strength to human dsHMGB1 ( Figures 46B and A).
• dissociation constants (K D ) of human monoclonal antibody #129 and human monoclonal antibody #129-L2 for human reduced HMGB1 are 2.07 x 10 -7 and 1.51 x 10 -5 , respectively.
• Antibody #129-L2 was shown to bind weaker than human monoclonal antibody #129 ( Figures 46D and C).
• human monoclonal antibody #129 and human monoclonal antibody #129-L2 we investigated the therapeutic effects on three types of FTLD-ALS model mice (TDP43 N267S -KI mouse, PGRN R504X -KI mouse, and VCP T262A -KI mouse). was investigated using the Y-maze test described in Example 1. Physiological saline containing 0.2 mg/kg body weight (6 ⁇ g/mouse) of human monoclonal antibody #129 or human monoclonal antibody #129-L2 was intravenously injected into each FTLD-ALS model mouse through the tail vein. Administration was carried out once a month according to the protocol of the therapeutic experiment described in 48.
• HMGB1 antibodies of the present invention were administered once a month twice, and then pathological analysis of brain tissue was performed. It was carried out according to the method described in Example 3.
• TRIAD necrosis was examined by staining for pSer46-MARCKS ( Figure 49) and KDEL ( Figure 50), and DNA damage was examined by staining for ⁇ H2AX ( Figure 51) and 53BP1 ( Figure 52).
• human monoclonal antibody #129 was purified according to a standard method from multiple clones of a CHO cell line that stably expresses human monoclonal antibody #129, and analyzed by non-reducing capillary electrophoresis according to a standard method. As shown in Figure 2, a gentle peak (indicated by an arrow in the figure) was observed on the higher molecular weight side than the IgG peak in all clones.
• human monoclonal antibody #129-L2 was purified from multiple clones of the CHO cell line stably expressing human monoclonal antibody #129-L2 and analyzed by non-reducing capillary electrophoresis, as shown in Figure 55. No gentle peak on the high molecular weight side was observed in any of the clones. This result shows that human monoclonal antibody #129 underwent N-glycan sugar chain modification, whereas human monoclonal antibody #129-L2 did not undergo N-glycan sugar chain modification.
• Alzheimer's disease model mice (5 ⁇ FAD mice) were investigated using two types of anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129 and human monoclonal antibody #129-L2).
• physiological saline containing human monoclonal antibody #129 or human monoclonal antibody #129-L2 at 0.2 mg/kg body weight (6 ⁇ g/mouse) was administered intravenously to one Alzheimer's disease model mouse from the tail vein. Injections were administered once a month for a total of three times starting from the age of 6 months after the onset of symptoms (FIG. 58A).
• human monoclonal antibody #129 and human monoclonal antibody #129-L2 both have no effect on primary TRIAD necrosis, they significantly suppress secondary TRIAD necrosis and amyloid plaques, and the effects of both are equivalent. was shown (FIG. 59B).
• the present invention treats amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, Alzheimer's disease, frontotemporal lobar degeneration, degenerative or neurological diseases associated with aging, brain aging, or brain aging. It contributes to the prevention and/or treatment of accompanying diseases.

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