Prophylactic or therapeutic agent for amyotrophic lateral sclerosis, Parkinson's disease, huntington's disease, spinal cerebellar ataxia, degenerative or neurological disease associated with aging, brain aging, or disease associated with brain aging

Abstract

[Problem] To provide a prophylactic or therapeutic agent for amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Huntington's disease (HD), cerebellar ataxia (SCA), degenerative diseases associated with aging or neurological diseases, brain aging, or diseases associated with brain aging, alzheimer's disease (AD), frontotemporal lobe degeneration (FTLD) and an antibody having a higher stability and exhibiting a prophylactic or therapeutic effect. A human monoclonal antibody (anti-human HMGB1 antibody) that specifically binds to human HMGB1 is used as a prophylactic or therapeutic agent for ALS, PD, HD, SCA, degenerative diseases associated with aging, neurological diseases, brain aging, or diseases associated with brain aging, said human monoclonal antibody comprising: a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 each comprising a specific amino acid sequence; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising a specific amino acid sequence. An antibody in which the light chain complementarity determining region (CDR) 3 of the anti-human HMGB1 antibody has been modified is used.

Description

Technical Field

[0001] The present invention relates to a preparation for preventing or treating amyotrophic lateral sclerosis (hereinafter, sometimes referred to as "ALS"), Parkinson's disease (hereinafter, sometimes referred to as "PD"), Huntington's disease (hereinafter, sometimes referred to as "HD"), spinocerebellar ataxia (hereinafter, sometimes referred to as "SCA"), degenerative diseases or neurological diseases associated with aging (more specifically, senescence), brain aging, or diseases accompanied by brain aging (hereinafter, these may be collectively referred to as "diseases, etc. of the present application"); an anti-human HMGB1 antibody; a preparation for preventing or treating Alzheimer's disease (hereinafter, sometimes referred to as "AD") or frontotemporal lobar degeneration (hereinafter, sometimes referred to as "FTLD"); and the like. Background Art

[0002] ALS is an adult-onset neurodegenerative disease that presents with systemic motor neuron disorders in the upper (cerebral cortical motor area) and lower (spinal cord anterior horn cells and brain stem motor nuclei). About 10% of ALS patients have familial diseases (gene mutations), while the remaining 90% have idiopathic (sporadic) diseases. The cause of ALS is still unclear. In addition, the effect of the ALS treatment drug riluzole (Patent Document 1) is limited to delaying the progression of symptoms by a few months. At present, there is no effective ALS treatment.

[0003] On the other hand, FTLD is the second to third most frequent early-onset neurodegenerative dementia after AD, and is considered to be highly associated with motor neuron disease. FTLD shows symptoms of significant behavioral and personality changes, often accompanied by language dysfunction, and gradually progresses to cognitive impairment and dementia. As causative genes of familial FTLD, VCP (Valosin-Containing Protein), PGRN (progranulin), CHMP2B (Charged Multivesicular Body Protein 2B), and TDP43 (Transactive Response DNA Binding Protein of 43kD) are known. Although research on FTLD is ongoing, as with ALS, the full picture of the pathogenesis is still unclear.

[0004] Frontotemporal dysfunction occurs in up to 50% of ALS patients, whereas motor neuron dysfunction occurs in 15% of FTLD patients. Thus, ALS and FTLD, although having significantly different symptoms, show important overlaps at the molecular level. Thus, ALS and FTLD occur due to a combination of various genetic and environmental factors, and more than 20 genes are thought to be associated with these diseases.

[0005] On the other hand, as one of the non-histone chromatin-related proteins related to the structural maintenance of DNA and transcriptional regulation, the HMGB1 (High Mobility Group Box 1) protein is known. Recently, the following properties of this HMGB1 have attracted attention: it not only functions in the nucleus, but also is released to the outside of the cell due to cell necrosis, or is actively secreted to the outside of the cell due to vascular inflammatory signal response, thereby functioning as the so-called DAMPs (damage-associated molecular patterns). The inventors of the present application have obtained the following insights: in AD, HMGB1 leaked from the cell due to neuronal necrosis induces the phosphorylation of serine (Ser46) at position 46 of MARCKS, which is a phosphorylase substrate, and induces the degeneration of neurites. The following insights have also been obtained: a mouse monoclonal antibody against HMGB1 was prepared, and the mouse monoclonal antibody inhibited the phosphorylation of Ser46 of MARCKS (Non-Patent Document 1). Furthermore, the inventors of the present application reported that the mouse monoclonal antibody and the human monoclonal antibody restored the cognitive impairment in Alzheimer's disease model mice (Patent Documents 2 and 3).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No. 2713384

[0009] Patent Document 2: International Publication No. 2018 / 030405 Pamphlet

[0010] Patent Document 3: International Publication No. 2020 / 059847 Pamphlet

[0011] Non-patent literature

[0012] Non-patent literature 1: SCIENTIFIC REPORT, 2016Aug 25; 6: 31895 Summary of the invention

[0013] Problems to be solved by the invention

[0014] The present invention aims to provide a preventive or therapeutic agent for ALS, PD, HD, SCA, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases associated with brain aging, and an antibody with higher stability that exerts a preventive or therapeutic effect against AD or FTLD.

[0015] Means for solving problems

[0016] The inventors of this application have continued to conduct in-depth research to solve the above problems. In the process, it was found that the human monoclonal antibody #129 prepared in the examples of International Publication No. 2020 / 059847 (Patent Document 3) is targeted at four gene mutations (VCP T262A Mutation, PGRN R504X Mutation, CHMP2B Q165X Mutations and TDP43 N267S Mutation) exerts a therapeutic effect, has the effect of inhibiting TRIAD (Transcriptional Repression-Induced Atypical cell Death) necrosis in nerve cells (also called "neurons"), and further inhibiting the synergistic amplification of TRIAD necrosis based on HMGB1-induced DNA damage. In addition, based on the prior art that TRIAD necrosis of nerve cells and DNA damage as the background occur in the conditions of HD, PD, SCA, and ALS, and the prior art that the above-mentioned four gene mutations are not only the cause of FTLD but also the cause of ALS, it can be said that the above-mentioned human monoclonal antibody #129 and HMGB1 antibodies having a common complementary determining region (CDR; Complementarity Determining Region) therewith generally have a therapeutic effect on neurodegenerative diseases including ALS, PD, HD, and SCA. In addition, it was confirmed that the above-mentioned human monoclonal antibody #129 reduced the number of senescent cells in the cerebral cortex of two AD model mice (5×FAD mice and APP-KI mice), and inhibited the cell aging of neurons, astrocytes, and microglia. Furthermore, since TRIAD necrosis was found to be age-related cell death itself, it can be said that human monoclonal antibody #129 and anti-HMGB1 antibodies having a common CDR therewith have therapeutic effects on degenerative diseases or neurological diseases associated with aging, brain aging itself, and diseases associated with brain aging.

[0017] In addition, in addition to human monoclonal antibody #129, human monoclonal antibody #129-L2, which is a modified antibody of the light chain CDR3 of human monoclonal antibody #129, was used to analyze the therapeutic effects on five model mice (HD model mice, PD model mice, ALS model mice, spinocerebellar ataxia [hereinafter, sometimes referred to as "SCA"] model mice, and aging model mice). The results confirmed that human monoclonal antibody #129 and human monoclonal antibody #129-L2 have therapeutic effects on HD, PD, ALS, SCA, and changes in cell properties, cell death, cognitive function (memory), and motor function associated with aging.

[0018] In addition, it was confirmed that human monoclonal antibody #129-L2 has the same degree of binding to human disulfide HMGB1 (ds-HMGB1) as human monoclonal antibody #129 and therapeutic effects on FTLD-ALS model mice and AD model mice, and the binding to human reduced HMGB1 is the same or weaker than that of human monoclonal antibody #129. In addition, it was confirmed that human monoclonal antibody #129-L2 is not modified with N-glycan chains, unlike human monoclonal antibody #129, and the stability of the antibody during storage is higher than that of human monoclonal antibody #129.

[0019] The present invention has been accomplished based on the above findings.

[0020] That is, the present invention is as follows.

[0021] [1] A preventive or therapeutic agent for amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases associated with brain aging, the preventive or therapeutic agent comprising a human monoclonal antibody that specifically binds to human HMGB1 and comprises:

[0022] A heavy chain complementary determining region (CDR) 1 comprising 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 into the amino acid sequence; a heavy chain CDR2 comprising 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 into the amino acid sequence; and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; and

[0023] A light chain CDR1 comprising the amino acid sequence shown in sequence number 4 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; a light chain CDR2 comprising the amino acid sequence shown in sequence number 5 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; and a light chain CDR3 comprising the amino acid sequence shown in sequence number 6, sequence number 20, or sequence number 17 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence.

[0024] [2] A preventive or therapeutic agent as described in [1] above, wherein the human monoclonal antibody comprises: a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 18.

[0025] [3] A preventive or therapeutic agent as described in [1] or [2] above, wherein the human monoclonal antibody comprises: a heavy chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; and a light chain comprising 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.

[0026] [4] The preventive or therapeutic agent according to any one of [1] to [3] above, which is administered intravenously.

[0027] [5] A human monoclonal antibody that specifically binds to human HMGB1 and comprises:

[0028] A heavy chain complementary determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0029] A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 17.

[0030] [6] A human monoclonal antibody as described in [5] above, comprising: a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 18.

[0031] [7] A human monoclonal antibody as described in [5] or [6] above, comprising: a heavy chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; and a light chain comprising 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.

[0032] [8] A preventive 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.

[0033] [9] The preventive or therapeutic agent according to [8] above, which is administered intravenously.

[0034]

[10] A preventive or therapeutic agent for amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, aging-related degenerative diseases or neurological diseases, brain aging, or diseases associated with brain aging, comprising the human monoclonal antibody described in any one of [5] to [7] above.

[0035]

[11] The preventive or therapeutic agent according to

[10] above, which is administered intravenously.

[0036] In addition, as another embodiment of the present invention, there can be mentioned:

[0037] A method for preventing or treating the disease of the present application, comprising the step of administering a human monoclonal antibody (hereinafter, sometimes referred to as "the human monoclonal antibody of the present application") to a subject in need of prevention or treatment of the disease of the present application,

[0038] The human monoclonal antibody specifically binds to human HMGB1 and comprises:

[0039] A heavy (H) chain complementary determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; an H chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; and an H chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; and

[0040] A light (L) chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; an L chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; and an L chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 20, or SEQ ID NO: 17 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence;

[0041] The human monoclonal antibody of the present application is used as a preventive or therapeutic agent for the disease of the present application;

[0042] The human monoclonal antibody of the present application, and its use in the prevention or treatment of the disease of the present application; and

[0043] The human monoclonal antibody of the present application is used for producing a preventive or therapeutic agent for the disease of the present application.

[0044] In addition, as another embodiment of the present invention, there can be mentioned:

[0045] A method for preventing or treating Alzheimer's disease, frontotemporal lobar degeneration, the disease of the present application, etc., comprising the step of administering a human monoclonal antibody (hereinafter, sometimes referred to as "human monoclonal antibody L1 / L2 of the present application") to a subject in need of prevention or treatment of Alzheimer's disease, frontotemporal lobar degeneration, the disease of the present application, etc.,

[0046] The human monoclonal antibody specifically binds to human HMGB1 and comprises:

[0047] H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; and H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; and

[0048] L chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; L chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and L chain CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 17;

[0049] The human monoclonal antibody L1 / L2 of the present application is used as a preventive or therapeutic agent for Alzheimer's disease, frontotemporal lobar degeneration, the disease of the present application, etc.;

[0050] The human monoclonal antibody L1 / L2 of the present application, for use in the prevention or treatment of Alzheimer's disease or frontotemporal lobar degeneration, the disease of the present application, etc.; and

[0051] The human monoclonal antibodies L1 / L2 of the present application are used for producing a preventive or therapeutic agent for Alzheimer's disease, frontotemporal lobar degeneration, the diseases of the present application, and the like.

[0052] Effects of the Invention

[0053] When the human monoclonal antibody of the present application is used, the disease of the present application can be prevented or treated without significant side effects. In addition, when the human monoclonal antibody L1 / L2 of the present application is used, Alzheimer's disease or frontotemporal lobar degeneration, the disease of the present application, etc. can be prevented or treated without significant side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] [ Figure 1 ] is to show the surface plasmon resonance (SPR) of two anti-human HMGB1 antibodies (human monoclonal antibody #129 of the present invention [ Figure 1 A], and mouse monoclonal antibody 2C8C[ Figure 1 B]) Diagram showing the results of analyzing the interaction with human HMGB1 protein.

[0055] [ Figure 2-1 ] is to show the immunohistochemical staining of four FTLD-ALS model mice (VCP T262A -KI mice Figure 2-1 A], PGRN R504X -KI mice Figure 2-1 B], CHMP2B Q165X -KI mice Figure 2-1 C], and TDP43 N267S -KI mice Figure 2-1 D]) is a graph showing the results of analyzing the pSer46-MARCKS level in the cerebral cortex of C57BL / 6J mice. It should be noted that when the pSer46-MARCKS level in C57BL / 6J mice was numerically expressed in the same manner, the result was 0 (zero).

[0056] [ Figure 2-2 ] is to show the Nissl staining of two FTLD-ALS model mice (VCP T262A -KI mice Figure 2-2 A], and PGRN R504X -KI mice Figure 2-2 B]) is a diagram showing the results of analyzing brain tissue of 100 μg / mL. The arrow in the left image indicates a vacuole. The right image is a high-magnification image of the area indicated by the black arrow in the left image.

[0057] [ Figure 3 ] Figure 3 A shows the expression of TDP43 against which human monoclonal antibody #129 of the present invention was administered N267S-KI mice ("KI Ab" in the figure [n=12]) or C57BL / 6J mice ("B6 Ab" in the figure [n=8]), TDP43 N267S -KI mice ("KI IgG" in the figure [n=11]) or C57BL / 6J mice ("B6 IgG" in the figure [n=6]), or TDP43 not administered N267S - A graph showing the results of Morris water maze test performed on KI mice ("KI nt" [n=10] in the figure) or C57BL / 6J mice ("B6 nt" [n=6] in the figure) at 10 months of age on days 1 to 5. Figure 3 B shows the PGRN to which the human monoclonal antibody #129 of the present invention was administered R504X -KI mice ("KIAb" in the figure [n=11]) or C57BL / 6J mice ("B6 Ab" in the figure [n=6]), PGRN administered with control human IgG R504X -KI mice ("KI IgG" in the figure [n=7]) or C57BL / 6J mice ("B6 IgG" in the figure [n=7]), or PGRN not administered R504X - A graph showing the results of Morris water maze test performed on KI mice ("KI nt" [n=11] in the figure) or C57BL / 6J mice ("B6 nt" [n=7] in the figure) on days 1 to 5 from 7 months of age. Figure 3 C shows the VCP against human monoclonal antibody #129 of the present invention administered T262A -KI mice ("KI Ab" in the figure [n=13]) or C57BL / 6J mice ("B6 Ab" in the figure [n=6]), VCP administered with control human IgG T262A -KI mice ("KI IgG" in the figure [n=5]) or C57BL / 6J mice ("B6 IgG" in the figure [n=7]), or VCP not administered T262A - Graphs showing the results of Morris water maze test performed on KI mice ("KI nt" [n=7] in the figure) or C57BL / 6J mice ("B6 nt" [n=6] in the figure) on days 1 to 5 from 10 months of age. Figure 3 D is a graph showing CHMP2B to which human monoclonal antibody #129 of the present invention was administered Q165X -KI mice ("KI Ab" in the figure [n=7]) or C57BL / 6J mice ("B6 Ab" in the figure [n=6]), CHMP2B administered with control human IgG Q165X-KI mice ("KI IgG" in the figure [n=5]) or C57BL / 6J mice ("B6 IgG" in the figure [n=7]), or CHMP2B not administered Q165X -KI mice ("KI nt" [n=5] in the figure) or C57BL / 6J mice ("B6n.t." [n=6] in the figure), the results of Morris water maze test performed on days 1 to 5 from 6 months of age. "*", "**", and "***" in the figure indicate that "KI nt" is statistically significantly different from "B6 nt" according to Tukey's HSD test (p<0.05, p<0.01, and p<0.001, respectively). In addition, "#", "##", and "###" in the figure indicate that "KIAb" is statistically significantly different from "KI nt" according to Tukey's HSD test (p<0.05, p<0.01, and p<0.001, respectively).

[0058] [ Figure 4 ] To show the effect of human monoclonal antibody #129 of the present invention on TDP43 N267S -KI mice ("KI Ab" in the figure [n = 12 ( Figure 4 A~C); n=9( Figure 4 D)]) or C57BL / 6J mice ("B6 Ab" in the figure [n=8 ( Figure 4 A to D)]), TDP43 treated with control human IgG N267S -KI mice ("KI IgG" in the figure [n=9 ( Figure 4 A~C); n=6( Figure 4 D)]) or C57BL / 6J mice ("B6IgG" in the figure [n=7 ( Figure 4 A~D)]), or no TDP43 N267S -KI mice ("KI nt" in the figure [n = 6 ( Figure 4 A); n = 10( Figure 4 B); n = 9 ( Figure 4 C); n = 7 ( Figure 4 D)]) or C57BL / 6J mice ("B6 nt" in the figure [n = 6 ( Figure 4 A); n = 12 ( Figure 4 B~D)]), at 7 months of age ( Figure 4 A) 8 months old ( Figure 4 B) 9 months old ( Figure 4 C), or 10 months old ( Figure 4D) A graph showing the results of the Y-maze test. The “*”, “**”, and “***” in the graph indicate that there are statistically significant differences (p<0.05, p<0.01, and p<0.001, respectively) according to Tukey's HSD test (hereinafter, Figures 5 to 16 (same as in ).

[0059] [ Figure 5 ] is to show the effect of human monoclonal antibody #129 of the present invention on PGRN R504X -KI mice ("KI Ab" in the figure [n = 15 ( Figure 5 A, B); n = 14 ( Figure 5 C)]) or C57BL / 6J mice ("B6 Ab" in the figure [n=7 ( Figure 5 A-C)]), PGRN treated with control human IgG R504X -KI mice ("KI IgG" in the figure [n=10 ( Figure 5 A to C)]) or C57BL / 6J mice ("B6 IgG" in the figure [n = 8 ( Figure 5 A~C)]), or PGRN not administered R504X -KI mice ("KIn.t." in the figure [n=11 ( Figure 5 A, C); n = 12 ( Figure 5 B)]) or C57BL / 6J mice ("B6 nt" in the figure [n = 7 ( Figure 5 A); n = 11 ( Figure 5 B, C)]), at 5 months of age ( Figure 5 A) 6 months old ( Figure 5 B), or 7 months old ( Figure 5 C) Graph showing the results of the Y-maze test.

[0060] [ Figure 6 ] is to show the VCP to which the human monoclonal antibody #129 of the present invention was administered T262A -KI mice ("KI Ab" in the figure [n = 15 ( Figure 6 A~C); n=12( Figure 6 D)]) or C57BL / 6J mice ("B6 Ab" in the figure [n=8 ( Figure 6 A~C); n=5( Figure 6 D)]), VCP administered with control human IgG T262A -KI mice ("KI IgG" in the figure [n=8 ( Figure 6 A); n = 7 ( Figure 6 B, C); n = 5 ( Figure 6D)]) or C57BL / 6J mice ("B6 IgG" in the figure [n=7 ( Figure 6 A~C); n=6( Figure 6 D)]), or VCP not administered T262A -KI mice ("KI nt" in the figure [n=11 ( Figure 6 A~C); n=5( Figure 6 D)]) or C57BL / 6J mice ("B6 nt" in the figure [n = 17 ( Figure 6 A); n = 16 ( Figure 6 B); n = 13 ( Figure 6 C); n = 12 ( Figure 6 D)]), at 7 months of age ( Figure 6 A) 8 months old ( Figure 6 B) 9 months old ( Figure 6 C), or 10 months old ( Figure 6 D) Graph showing the results of performing the Y-maze test.

[0061] [ Figure 7 ] is to show the CHMP2B to which the human monoclonal antibody #129 of the present invention was administered Q165X -KI mice ("KI Ab" in the figure [n = 8 ( Figure 7 A to C)]) or C57BL / 6J mice ("B6 Ab" in the figure [n = 8 ( Figure 7 A to C)]), CHMP2B treated with control human IgG Q165X -KI mice ("KI IgG" in the figure [n=8 ( Figure 7 A to C)]) or C57BL / 6J mice ("B6 IgG" in the figure [n = 8 ( Figure 7 A-C)]), or CHMP2B not administered Q165X -KI mice ("KI nt" in the figure [n = 8 ( Figure 7 A to C)]) or C57BL / 6J mice ("B6 nt" in the figure [n = 12 ( Figure 7 A); n = 10( Figure 7 B); n = 10 ( Figure 7 C)]), at 4 months of age ( Figure 7 A) 5 months old ( Figure 7 B), or 6 months old ( Figure 7 C) Graph showing the results of the Y-maze test.

[0062] [ Figure 8 ] Figure 8 A shows the expression of TDP43 against which human monoclonal antibody #129 of the present invention was administered N267S-KI mice ("KI Ab" in the figure [n=12]) or C57BL / 6J mice ("B6 Ab" in the figure [n=8]), TDP43 N267S -KI mice ("KI IgG" in the figure [n=9]) or C57BL / 6J mice ("B6 IgG" in the figure [n=7]), or TDP43 was not administered N267S - A graph showing the results of a fear-conditioning test performed on KI mice ("KI nt" [n=9] in the figure) or C57BL / 6J mice ("B6n.t." [n=7] in the figure) at 10 months of age. Figure 8 B shows the PGRN to which the human monoclonal antibody #129 of the present invention was administered R504X -KI mice ("KI Ab" in the figure [n=11]) or C57BL / 6J mice ("B6 Ab" in the figure [n=6]), PGRN administered with control human IgG R504X -KI mice ("KI IgG" in the figure [n=10]) or C57BL / 6J mice ("B6 IgG" in the figure [n=7]), or PGRN not administered R504X - A graph showing the results of a fear conditioning test performed on KI mice ("KI nt" in the figure [n=17]) or C57BL / 6J mice ("B6 nt" in the figure [n=11]) at 7 months of age. Figure 8 C is a VCP against human monoclonal antibody #129 of the present invention administered T262A -KI mice ("KI Ab" in the figure [n=15]) or C57BL / 6J mice ("B6 Ab" in the figure [n=4]), VCP administered with control human IgG T262A -KI mice ("KI IgG" in the figure [n=5]) or C57BL / 6J mice ("B6 IgG" in the figure [n=6]), or VCP not administered T262A - A graph showing the results of a fear conditioning test performed on KI mice ("KI nt" [n=9] in the graph) or C57BL / 6J mice ("B6n.t." [n=6] in the graph) at 10 months of age.

[0063] Figure 8 D is a CHMP2B protein to which human monoclonal antibody #129 of the present invention was administered Q165X -KI mice ("KIAb" in the figure [n=7]) or C57BL / 6J mice ("B6 Ab" in the figure [n=8]), CHMP2B administered with control human IgG Q165X-KI mice ("KI IgG" in the figure [n=5]) or C57BL / 6J mice ("B6 IgG" in the figure [n=6]), or CHMP2B not administered Q165X - A graph showing the results of a fear conditioning test performed on KI mice ("KI nt" in the figure [n=7]) or C57BL / 6J mice ("B6 nt" in the figure [n=8]) at 6 months of age.

[0064] [ Fig. 9 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig. 9 A], TDP43 N267S -KI mice Fig. 9 B], VCP T262A -KI mice Fig. 9 C], and CHMP2B Q165X -KI mice Fig. 9 D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6n.t." in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), and the results of analyzing the pSer46-MARCKS level in the cerebral cortex (n=3).

[0065] [ Fig.10 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig.10 A], TDP43 N267S -KI mice Fig.10 B], VCP T262A -KI mice Fig.10 C], and CHMP2B Q165X -KI mice Fig.10D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), and the results of analyzing the KDEL fluorescence level in the cerebral cortex (n=3).

[0066] [ Fig.11 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig.11 A], TDP43 N267S -KI mice Fig.11 B], VCP T262A -KI mice Fig.11 C], and CHMP2B Q165X -KI mice Fig.11 D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), and the results of analyzing the γH2AX fluorescence level in the cerebral cortex (n=3).

[0067] [ Fig.12 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig.12 A], TDP43 N267S -KI mice Fig.12 B], VCP T262A -KI mice Fig.12 C], and CHMP2B Q165X -KI mice Fig.12 D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), and the results of analyzing the 53BP1 fluorescence level in the cerebral cortex (n=3).

[0068] [ Fig.13 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig.13 A], TDP43 N267S -KI mice Fig.13 B], VCP T262A -KI mice Fig.13 C], and CHMP2B Q165X -KI mice Fig.13 D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), and the results of analyzing the pSer409 / 410-TDP43 fluorescence level in the cerebral cortex (n=3).

[0069] [ Fig.14] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig.14 A], TDP43 N267S -KI mice Fig.14 B], VCP T262A -KI mice Fig.14 C], and CHMP2B Q165X -KI mice Fig.14 D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), and the results of analyzing the p62 fluorescence level in the cerebral cortex (n=3).

[0070] [ Fig.15 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig.15 A], TDP43 N267S -KI mice Fig.15 B], VCP T262A -KI mice Fig.15 C], and CHMP2B Q165X -KI mice Fig.15 D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCPT262A -KI mice), and the results of analyzing the proportion of cells in which VAMP2 fluorescent signals and PSD95 fluorescent signals co-localized in the cerebral cortex (n=3).

[0071] [ Fig.16 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (PGRN R504X -KI mice Fig.16 A], TDP43 N267S -KI mice Fig.16 B], VCP T262A -KI mice Fig.16 C], and CHMP2B Q165X -KI mice Fig.16 D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by immunohistochemical staining at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), and the results of analyzing the proportion of cells in which the pSer203-tau fluorescence signal and the PSD95 fluorescence signal co-localized in the cerebral cortex (n=3).

[0072] [ Fig.17 ] is to show the effect of human monoclonal antibody #129 of the present invention on four FTLD-ALS model mice (TDP43 N267S -KI mice Fig.17 A], PGRN R504X -KI mice Fig.17 B], VCP T262A -KI mice Fig.17 C], and CHMP2B Q165X -KI mice Fig.17D]) ("KI Ab" in the figure); the four FTLD-ALS model mice administered with control human IgG ("KI IgG" in the figure); the four FTLD-ALS model mice not administered with control human IgG ("KI nt" in the figure); or C57BL / 6J mice not administered with control human IgG ("B6 nt" in the figure), each of which was analyzed by Western blotting at 6 months of age (C57BL / 6J mice and CHMP2B Q165X -KI mice) / 7-month-old (C57BL / 6J mice and PGRN R504X -KI mice) / 10 months old (C57BL / 6J mice, TDP43 N267S -KI mice, and VCP T262A -KI mice), the phosphorylation levels of four proteins involved in the core signaling of AD-FTLD in the cerebral cortex (pSer319-PKCα, pThr655-PKCγ, pSer643-PKCδ, and pSer729-PKCε) (lanes (1) to (4) respectively); the phosphorylation of a synaptic instability-related protein (pSer203-tau) (lane (5)) and the expression levels of two synapse-related proteins (PSD95 and VAMP2) (lanes (6) and (7) respectively); the phosphorylation of two DNA damage-related proteins ...6) and (7) respectively); the phosphorylation of two DNA damage-related proteins (pSer203-tau) (lane (7) and (8) respectively); the phosphorylation of two DNA damage-related proteins (pSer203-tau) (lane (7) and (8) respectively); the phosphorylation of two DNA damage-related proteins (pSer203-tau) (lane (7) and (8) respectively). The graph shows the results of the analysis of the expression levels of proteins (γH2AX and 53BP1) (lanes (8) and (9), respectively); the phosphorylation level of a cell death-related protein (pSer46-MARCKS) (lane (10)); the phosphorylation level of a DNA repair-related protein (pSer77 / 78-Ku70) (lane (11)); the level of a protein aggregation marker (pSer409 / 410-TDP43) (lane (12)); and the expression level of β-actin as an internal standard protein (lane (13)).

[0073] [ Figure 18a-b] Figure 18a is a graph showing the results of analyzing the level of DNA double-strand breaks in MAP2-positive neurons in the cerebral cortex using immunohistochemical staining with antibodies against two markers (γH2AX and 53BP1) in 4 groups of mice (non-transgenic sibling mice [B6 / SJL mice] to which the human monoclonal antibody #129 of the present invention was subcutaneously administered [hereinafter referred to as "subcutaneously administered"] to the neck and back region; B6 / SJL mice to which control IgG was subcutaneously administered; 5×FAD mice to which the human monoclonal antibody #129 of the present invention was subcutaneously administered; and 5×FAD mice to which control IgG was subcutaneously administered). The graph on the right shows the results of quantifying the fluorescence intensity detected by the antibody (N=3 mice, n=30 cells). Figure 18b is a graph showing the results of analyzing the entire cerebral cortex of the above-mentioned 4 groups of mice by Western blotting using anti-γH2AX antibodies and anti-53BP1 antibodies (n=3).

[0074] [ Figure 18c-d ] Figure 18c is a graph showing the results of analyzing the level of DNA double-strand breaks in MAP2-positive neurons in occipital cortical neurons by immunohistochemical staining using antibodies against two markers (γH2AX and 53BP1) in 4 groups of mice (B6 / SJL mice to which human monoclonal antibody #129 of the present invention was intravenously administered; B6 / SJL mice to which control IgG was intravenously administered; 5×FAD mice to which human monoclonal antibody #129 of the present invention was intravenously administered; and 5×FAD mice to which control IgG was intravenously administered). The graph on the right shows the results of quantifying the fluorescence intensity detected by the antibody (N=3 mice, n=30 cells). Figure 18d is a graph showing the results of analyzing the entire cerebral cortex of the above 4 groups of mice by Western blotting using anti-γH2AX antibodies and anti-53BP1 antibodies (n=3).

[0075] [ Fig.18e ] is a diagram showing the results of analyzing brain tissue samples (occipital lobe) of patients without neurological diseases and brain tissue samples (occipital lobe) of AD patients by immunohistochemical staining using an anti-γH2AX antibody.

[0076] [ Figure 18f-g] FIG. 18f is a graph showing the results of imaging analysis of the dynamical localization of Ku70 to DNA damage sites induced by microbeam irradiation, in which EGFP-Ku70 was transiently expressed in U2OS cells cultured in the presence (+) or absence (-) of 6 ng / mL (0.24 nM) of ds-HMGB1. FIG. 18g is a graph showing the results of quantitative analysis of the localization level of EGFP-Ku70 to DNA damage sites based on the time-lapse images of U2OS cells (n=5) obtained in FIG. 18f (n=5, mean±SEM).

[0077] [ Fig.19a ] is a diagram showing the results of analyzing neurons, astrocytes, and microglia in three types of mice (C57BL / 6J mice, 5×FAD mice, and APP-KI mice) by immunohistochemical staining using an anti-lamin B1 antibody.

[0078] [ Fig.19b ] is a diagram showing the results of analyzing neurons, astrocytes, and microglia in two AD model mice (5×FAD mice and APP-KI mice) to which human monoclonal antibody #129 of the present invention or control IgG was intravenously administered by immunohistochemical staining using anti-lamin B1 antibody and antibodies against three cell aging markers ("NeuN" in neurons, "GFAP" in astrocytes, and "Iba1" in microglia). The graph in the figure shows the results of measuring the ratio of cells positive for these cell aging markers and measuring the ratio of aging cells.

[0079] [ Fig. 20 ] Fig. 20 a is a diagram showing the results of analyzing the cerebral cortex of 8-month-old AD model mice (5×FAD mice) by immunohistochemical staining using three antibodies (anti-YAP antibody, anti-pSer46-MARCKS antibody, and anti-lamin B1 antibody). Fig. 20 b is a graph showing the results of analyzing the proportion of senescent cells (i.e., lamin B1 ring-negative cells) by immunohistochemical staining using an anti-lamin B1 antibody in the cerebral cortex of two types of AD model mice (5×FAD mice and APP-KI mice) to which human monoclonal antibody #129 of the present invention or control IgG was intravenously administered.

[0080] [ Fig.21a ] Fig.21aThe upper part of the figure shows the results of analyzing the nuclear localization level of NFκB in microglia by immunohistochemical staining using anti-NFκB antibodies for two normal mice (B6 / SJL mice or C57BL / 6 mice [B6]) or two AD model mice (5×FAD mice and APP-KI mice) at the age of 8 months. Fig.21a The lower part of FIG. 1 is a diagram showing the results of analyzing the nuclear localization level of NFκB in microglia by immunohistochemical staining using an anti-NFκB antibody in the two AD model mice to which human monoclonal antibody #129 of the present invention or control IgG was intravenously administered.

[0081] [ Figure 21b ] Figure 21b The upper part of the figure shows the results of analyzing the expression levels of MCP1 and IL6 around astrocytes and microglia in two normal mice (B6 / SJL mice or C57BL / 6 mice [B6]) or two AD model mice (5×FAD mice and APP-KI mice) at the age of 8 months by immunohistochemical staining using anti-MCP1 antibodies and anti-IL6 antibodies. Fig.21a The lower part is a graph showing the results of analyzing the expression levels of MCP1 and IL6 around astrocytes and microglia in the above two AD model mice to which the human monoclonal antibody #129 of the present invention or control IgG was intravenously administered, by immunohistochemical staining using anti-MCP1 antibody and anti-IL6 antibody.

[0082] [ Fig.22a ] is schematically shown Figure 22b Diagram of the experimental scheme for ~f.

[0083] [ Figure 22b ] shows that B6 / SJL mice or 5×FAD mice ( Fig.22a The left panel shows the results of analyzing the DNA double-strand break levels of MAP2-positive neurons in the occipital cortex neurons by immunohistochemical staining using antibodies against two markers (γH2AX and 53BP1). The right panel shows the results of quantifying the fluorescence intensity detected by the antibodies (N = 3 mice, n = 30 cells).

[0084] [ Fig.22c ] shows the effect of subcutaneous administration of PKC inhibitor (Go6976) on B6 / SJL mice or 5×FAD mice ( Fig.22aThe left figure shows the results of analyzing the expression levels of eight proteins (γH2AX, 53BP1, pSer77 / 78-Ku70, β-actin, pThr638-PKCα, pThr641-PKCβI, pSer660-PKCβII / δ, and pan-PKC) in the entire cerebral cortex using Western blotting.

[0085] [ Fig.22d ] shows the effect of subcutaneous administration of PKC inhibitor (Go6976) on B6 / SJL mice or 5×FAD mice ( Fig.22a (Left panel) is a diagram showing the results of the Y-maze test.

[0086] [ Fig.22e ] is to show the effect of subcutaneous administration of human monoclonal antibody #129 of the present invention or control human IgG on B6 / SJL mice or 5×FAD mice ( Fig.22a (center figure) is a graph showing the results of the Y-maze test.

[0087] [ Figure 22f ] shows the effect of subcutaneous administration of PKC inhibitor (Go6976) on B6 / SJL mice or 5×FAD mice ( Fig.22a ) of the cerebral cortex, B6 / SJL mice or 5×FAD mice ( Fig.22a ) of the cerebral cortex, B6 / SJL mice or 5×FAD mice ( Fig.22a The cerebral cortex of the brain (right figure) was subjected to immunohistochemical staining using anti-pSer46-MARCKS antibody and anti-amyloid protein (A) β antibody, and the proportions of active necrosis (Active Necrosis) (the set of 2 groups on the left in each graph), secondary necrosis (Secondary Necrosis) (the set of 2 groups in the center in each graph), and ghost of cell death (Ghost of Cell Death) (the set of 2 groups on the right in each graph) were analyzed.

[0088] [ Figure 23A-B] Figure 23A is a diagram schematically showing the scheme of the treatment experiment on Huntington's disease model mice (R6 / 2 mice). Figure 23B is a diagram showing the results of studying the therapeutic effect of human monoclonal antibody #129 on Huntington's disease model mice using the rotating rod test. In Figure 23B, the results (mean ± standard error) of three groups of normal mice administered with saline ("Normal + Saline" in the figure; the same in Figures 23 to 28), R6 / 2 mice administered with saline ("Tg + Saline" in the figure; the same in Figures 23 to 28), and R6 / 2 mice administered with human monoclonal antibody #129 ("Tg + Antibody" in the figure; the same in Figures 23 to 28) are shown (hereinafter, sometimes referred to as "3 groups for Huntington's disease model mouse analysis") for three consecutive days in each week. The values ​​in parentheses indicate the number of mice used in the measurement.

[0089] [ Fig.23C-D ] Figure 23C is a graph showing the results of studying the therapeutic effect of human monoclonal antibody #129 on Huntington's disease model mice using an open field test. Figure 23D is a graph showing the results of studying the therapeutic effect of human monoclonal antibody #129 on Huntington's disease model mice using a footprint test. In Figures 23C and 23D, representative examples and measurement results of three groups for analysis of Huntington's disease model mice are shown, and the box plots (box-plot) of the charts represent the maximum value, minimum value, median value, and quartiles (75% and 25%) (the same applies to the box plots in the following charts), and the values ​​in parentheses represent the number of mice used in the measurement. It should be noted that in Figure 23D, the stride (distance) was measured using three groups for analysis of Huntington's disease model mice.

[0090] [ Fig.24 ] Fig.24 A is a graph showing the results (n=4) of immunohistochemical staining of the cerebral cortex for detecting huntingtin (Htt), ubiquitin (Ub), and MAP2 in three groups of Huntington's disease model mice for analysis, and measuring the proportion (%) of cells with inclusion bodies positive for both Htt and Ub in MAP2-positive neurons. Fig.24 B is a graph showing the results of comparison of the number of MAP2-positive cells (number of neuronal cells) in the cerebral cortex among three groups for analysis of Huntington's disease model mice (n=4).

[0091] Fig.24 C is a graph showing the results of comparison of the number of Iba1-positive cells (number of microglial cells) in the cerebral cortex among three groups for analysis of Huntington's disease model mice (n=4). Fig.24D is a graph showing the results of comparing the number of GFAP-positive cells (number of astrocytes) in the cerebral cortex among the three groups for analysis of Huntington's disease model mice (n=4). "#", "##", and "###" in the figure indicate that there are statistically significant differences according to Tukey's HSD test (p<0.05, p<0.01, and p<0.001, respectively).

[0092] [ Fig.25A ] is a graph showing the results (n=4) of immunohistochemical staining for detecting Htt, Ub, and MAP2 in the striatum of three groups of Huntington's disease model mice for analysis, and measuring the proportion (%) of cells having inclusion bodies positive for both Htt and Ub among MAP2-positive neurons.

[0093] [ Figure 25B-D ] FIG. 25B is a graph showing the results of comparing the number of MAP2-positive cells (number of neuronal cells) in the striatum among the three groups for analysis of Huntington's disease model mice (n=4). FIG. 25C is a graph showing the results of comparing the number of Iba1-positive cells (number of microglial cells) in the striatum among the three groups for analysis of Huntington's disease model mice (n=4). FIG. 25D is a graph showing the results of comparing the number of GFAP-positive cells (number of astrocytes) in the striatum among the three groups for analysis of Huntington's disease model mice (n=4). "##" and "###" in the figure indicate that there are statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0094] [ Fig.26 ] Fig.26 A is a graph showing the results of measuring the proportion (%) of cells showing characteristics of TRIAD cell death (disappearance of nuclear YAP and pSer46-MARCKS positivity) by immunohistochemical staining for YAP and pSer46-MARCKS in the cerebral cortex of three groups of Huntington's disease model mice for analysis (n=4). Fig.26 B is a graph showing the results of comparison of the number of MAP2-positive neural cells (number of neuronal cells) showing abnormal KDEL staining images of the cerebral cortex among the three groups for analysis of Huntington's disease model mice (n=4). Fig.26C is a graph showing the results of comparing the signal intensity derived from 53BP1 (left graph) and the signal intensity derived from γH2AX (right graph) among three groups in the analysis of Huntington's disease model mice for 53BP1-positive, γH2AX-positive, and MAP2-positive nerve cells (neuronal cells) in the cerebral cortex. "#", "##", and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.05, p<0.01, and p<0.001, respectively).

[0095] [ Fig. 27 ] Fig. 27 A is a graph showing the results of measuring the proportion (%) of cells showing characteristics of TRIAD cell death (nuclear YAP disappearance and pSer46-MARCKS positivity) by immunohistochemical staining for YAP and pSer46-MARCKS in the striatum of three groups of Huntington's disease model mice for analysis (n=4). Fig. 27 B is a graph showing the results of comparison of the number of MAP2-positive nerve cells (number of neuronal cells) showing abnormal KDEL staining images of the striatum among the three groups for analysis of Huntington's disease model mice (n=4). Fig. 27 C is a graph showing the results of comparing the signal intensity derived from 53BP1 (left graph) and the signal intensity derived from γH2AX (right graph) among three groups in the analysis of Huntington's disease model mice for 53BP1-positive, γH2AX-positive, and MAP2-positive nerve cells (neuronal cells) in the striatum. "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0096] [ Fig.28 ] is a diagram showing the results of Western blotting of pSer46-MARCKS, 53BP1, pSer139-γH2AX, pSer319-PKCa, pSer203-Tau, Ub, and control β-actin in the whole brain including the basal ganglia in three groups for analysis of Huntington's disease model mice. Quantification and statistical comparison were performed for the bands of marker molecules other than Ub in the ladder or smear.

[0097] [ Figure 29A-B] FIG. 29A is a diagram schematically showing the scheme of the treatment experiment on Parkinson's disease model mice (α-synuclein A53T mice). FIG. 29B is a diagram showing the results of studying the therapeutic effect of human monoclonal antibody #129 on α-synuclein A53T mice using the rotating rod test. In FIG. 29B, the results of three groups (hereinafter sometimes referred to as "3 groups for Parkinson's disease model mouse analysis") of three consecutive tests on three days in each week are shown, including normal mice administered with saline ("Normal + Saline" in the figure; the same in FIGS. 29 to 32), α-synuclein A53T mice administered with saline ("Tg + Saline" in the figure; the same in FIGS. 29 to 32), and α-synuclein A53T mice administered with human monoclonal antibody #129 ("Tg + Antibody" in the figure; the same in FIGS. 29 to 32). The values ​​in parentheses indicate the number of mice used in the measurement.

[0098] [ Figure 29C-D ] Figure 29C is a diagram showing the results of studying the therapeutic effect of human monoclonal antibody #129 on α-synuclein A53T mice using an open field test. Figure 29D is a diagram showing the results of studying the therapeutic effect of human monoclonal antibody #129 on α-synuclein A53T mice using a footprint test. In Figures 29C and 29D, representative examples and measurement results of three groups for analysis of Parkinson's disease model mice are shown, and the values ​​in brackets represent the number of mice used in the measurement. It should be noted that in Figure 29D, the stride (distance) was measured using three groups for analysis of Parkinson's disease model mice.

[0099] [ Fig.30 ] Fig.30 A is a graph showing the results (n=4) of immunohistochemical staining for detecting pSer129-Syn (Ser129 phosphorylated synuclein), Ub, and MAP2 in the cerebral cortex of three groups of Parkinson's disease model mice for analysis, and measuring the proportion (%) of cells with inclusion bodies positive for both pSer129-Syn and Ub in MAP2-positive neurons. Fig.30 B is a graph showing the results of comparison of the number of MAP2-positive cells (number of neuronal cells) in the cerebral cortex among three groups for analysis of Parkinson's disease model mice (n=4). Fig.30 C is a graph showing the results of comparison of the number of Iba1-positive cells (number of microglial cells) in the cerebral cortex among three groups for analysis of Parkinson's disease model mice (n=4). Fig.30D is a graph showing the results of comparing the number of GFAP-positive cells (number of astrocytes) in the cerebral cortex among the three groups for analysis of Parkinson's disease model mice (n=4). "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0100] [ Fig.31 ] Fig.31 A is a graph showing the results of measuring the proportion (%) of cells showing characteristics of TRIAD cell death (nuclear YAP disappearance and pSer46-MARCKS positivity) by immunohistochemical staining for YAP and pSer46-MARCKS in the cerebral cortex of three groups of Parkinson's disease model mice for analysis (n=4). Fig.31 B is a graph showing the results of comparison of the number of MAP2-positive nerve cells (number of neuronal cells) showing abnormal KDEL staining images of the cerebral cortex among the three groups for Parkinson's disease model mouse analysis (n=4). Fig.31 C is a graph showing the results of comparing the signal intensity derived from 53BP1 (left graph) and the signal intensity derived from γH2AX (right graph) among the three groups for analysis of Parkinson's disease model mice for 53BP1-positive, γH2AX-positive, and MAP2-positive nerve cells (neuronal cells) in the cerebral cortex. "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0101] [ Fig.32 ] is a diagram showing the results of Western blotting of pSer46-MARCKS, 53BP1, pSer139-γH2AX, pSer319-PKCa, pSer203-Tau, Ub, and control β-actin in the whole brain including basal ganglia in three groups for analysis of Parkinson's disease model mice. Quantification and statistical comparison were performed for the bands of marker molecules other than ladder or tail Ub.

[0102] [ Figure 33A-B] FIG. 33A is a diagram schematically showing the scheme of the treatment experiment on the amyotrophic lateral sclerosis model mouse (SOD1-Tg mouse). FIG. 33B is a diagram showing the results of studying the therapeutic effect of human monoclonal antibody #129-L2 on SOD1-Tg mice using the rotating rod test. In FIG. 33B, the results (mean ± standard error) of three groups of normal mice administered with saline ("normal + saline" in the figure; the same in FIGS. 33 to 37), SOD1-Tg mice administered with saline ("Tg + saline" in the figure; the same in FIGS. 33 to 37), and SOD1-Tg mice administered with human monoclonal antibody #129-L2 ("Tg + antibody" in the figure; the same in FIGS. 33 to 37) are shown (hereinafter, sometimes referred to as "3 groups for analysis of amyotrophic lateral sclerosis model mice") for three consecutive days in each week. The numerical values ​​in parentheses indicate the number of mice used in the measurement.

[0103] [ Figure 33C-D ] Figure 33C is a graph showing the results of studying the therapeutic effect of human monoclonal antibody #129-L2 on SOD1-Tg mice using an open field test. Figure 33D is a graph showing the results of studying the therapeutic effect of human monoclonal antibody #129-L2 on SOD1-Tg mice using a footprint test. In Figures 33C and 33D, representative examples and measurement results of three groups for analysis of amyotrophic lateral sclerosis model mice are shown, and the numerical values ​​in parentheses represent the number of mice used in the measurement. It should be noted that in Figure 33D, the stride length (distance) was measured for the three groups for analysis of amyotrophic lateral sclerosis model mice.

[0104] [ Fig.34 ] Fig.34 A is a graph showing the results of measuring the ratio (%) of cells positive for both SOD1 and Ub in MAP2-positive neurons by immunohistochemical staining for SOD1, Ub, and MAP2 in the cerebral cortex of three groups of ALS model mice for analysis (n=4). Fig.34 B is a graph showing the results of immunohistochemical staining for detecting pSer386-Tau, Ub, and MAP2 in the cerebral cortex of three groups of amyotrophic lateral sclerosis model mice for analysis, and measuring the proportion (%) of cells positive for both pSer386-Tau and Ub among MAP2-positive neurons (n=4). Fig.34C is a graph showing the results of measuring the ratio (%) of cells positive for both TDP43 and Ub in MAP2-positive neurons by immunohistochemical staining for TDP43, Ub, and MAP2 in the cerebral cortex of three groups of ALS model mice for analysis (n=4). "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0105] [ Fig.35 ] Fig.35 A is a graph showing the results (n=4) of immunohistochemical staining for YAP and pSer46-MARCKS in the lumbar cord of three groups of amyotrophic lateral sclerosis model mice for analysis, and measuring the proportion (%) of cells showing characteristics of TRIAD cell death (disappearance of nuclear YAP and pSer46-MARCKS positivity). Fig.35 B is a graph showing the results of comparison of the number of MAP2-positive cells (number of neuronal cells) in the anterior horn of the lumbar spinal cord among three groups for analysis of amyotrophic lateral sclerosis model mice (n=4).

[0106] Fig.35 C is a graph showing the results of comparison of the number of Iba1-positive cells (the number of microglial cells) in the anterior horn of the lumbar spinal cord among three groups for analysis of amyotrophic lateral sclerosis model mice (n=4). Fig.35 D is a graph showing the results of comparing the number of GFAP-positive cells (number of astrocytes) in the anterior horn of the lumbar spinal cord among the three groups for analysis of amyotrophic lateral sclerosis model mice (n=4). "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0107] [ Fig.36 ] is a graph showing the results of comparing the signal intensity derived from 53BP1 (left graph) and the signal intensity derived from γH2AX (right graph) among three groups of ALS model mice for 53BP1-positive, γH2AX-positive, and MAP2-positive nerve cells (neuronal cells) in the cerebral cortex. The "###" in the figure indicates that there is a statistically significant difference (p<0.001) according to Tukey's HSD test.

[0108] [ Fig.37] is a diagram showing the results of Western blotting of pSer46-MARCKS, 53BP1, pSer139-γH2AX, pSer319-PKCa, pSer396-Tau, Ub, and β-actin as a control in the whole brain including the basal ganglia of three groups of amyotrophic lateral sclerosis model mice for analysis. Quantification and statistical comparison were performed for the bands of marker molecules other than ladder or tail Ub.

[0109] [ Fig.38 ] Fig.38 A is a diagram schematically showing the scheme of a therapeutic experiment on spinocerebellar ataxia model mice (mutant ataxin-1 gene knock-in mice). Fig.38 B is a graph showing the results of studying the therapeutic effect of human monoclonal antibody #129 on mutant ataxin-1 gene knock-in mice using a rotarod test. Figures 38 to 41 ) and mutant ataxin-1 knock-in mice administered with saline (“KI+saline” in the figure; Figures 38 to 41 ), and mutant ataxin-1 knock-in mice administered with human monoclonal antibody #129 (“KI+antibody” in the figure; Figures 38 to 41 The results of the three groups (hereinafter sometimes referred to as "three groups for spinocerebellar ataxia model mouse analysis") tested continuously for three days in each week (mean ± standard error). The values ​​in parentheses indicate the number of mice used in the measurement. Fig.38 C is a graph showing the results of studying the therapeutic effect of human monoclonal antibody #129 on mutant ataxin-1 gene knock-in mice using a footprint test. Representative examples and measurement results of three groups of spinocerebellar ataxia model mice are shown, and the values ​​in parentheses indicate the number of mice used in the measurement. Stride length (distance) was measured using three groups of spinocerebellar ataxia model mice.

[0110] [ Fig.39 ] Fig.39 A is a graph showing the results of quantification and comparison of pSer46-MARCKS staining signals in the granule cell layer of the cerebellar cortex of three groups of spinocerebellar ataxia model mice analyzed by immunohistochemistry using YAP, pSer46-MARCKS, and DAPI (n=4). Fig.39B is a graph showing the results of immunohistochemical staining for Atxn1, Ub, and MAP2 in the cerebral cortex of three groups of spinocerebellar ataxia model mice for analysis, and measuring the proportion (%) of cells positive for both Atxn1 and Ub among MAP2-positive neurons (n=4). Fig.39 C is a graph showing the results of comparing the signal intensity derived from 53BP1 in Purkinje cells (left graph) and the signal intensity derived from γH2AX (right graph) among the three groups for spinocerebellar ataxia model mouse analysis. "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0111] [ Fig.40 ] Fig.40 A is a graph showing the results of comparison of the number of calbindin-positive Purkinje cells showing abnormal KDEL staining images among three groups for analysis of spinocerebellar ataxia model mice (n=4). Fig.40 B is a graph showing the results of comparison of the number of Purkinje cells (number of neuronal cells) in the cerebellar cortex among three groups for analysis of spinocerebellar ataxia model mice (n=4). Fig.40 C shows the results of comparison of the number of Iba1-positive cells (the number of microglial cells) in the cerebellar cortex among the three groups for analysis of spinocerebellar ataxia model mice (n=4). Fig.40 D shows the results of comparison of the number of GFAP-positive cells (the number of astrocytes) in the cerebellar cortex among the three groups analyzed in the spinocerebellar ataxia model mouse (n=4).

[0112] [ Fig.41 ] is a diagram showing the results of Western blotting of pSer46-MARCKS, 53BP1, pSer139-γH2AX, pSer319-PKCa, pSer203-Tau, Ub, and control β-actin in the whole brain including the basal ganglia in three groups for analysis of spinocerebellar ataxia model mice. Quantification and statistical comparison were performed for the bands of marker molecules other than ladder or tail Ub.

[0113] [ Fig.42 ] Fig.42 A is a diagram schematically showing the scheme of a treatment experiment on aging model mice (12-month-old C57BL / 6 mice). Fig.42B is a diagram showing the results of using the Y-maze test to study the therapeutic effect of human monoclonal antibody #129-L2 on cognitive decline in aging model mice. The upper section shows the results of the Y-maze test of 12-month-old C57BL / 6 mice ("Aged + saline" in the figure) and 12-month-old C57BL / 6 mice ("Aged + Antibody" in the figure) given human monoclonal antibody #129-L2 at different ages. The lower section summarizes the results obtained in the upper section. The values ​​in brackets represent the number of mice used in the measurement.

[0114] [ Fig.43 ] in young mice (3-month-old C57BL / 6 mice) (“Young” in the figure; Figures 43-45 ), 20-month-old C57BL / 6 mice administered with saline (“Aged+Saline” in the figure; Figures 43-45 ), and 20-month-old C57BL / 6 mice administered with human monoclonal antibody #129-L2 (“Old+antibody” in the figure; Figures 43-45 In the 3 groups (hereinafter sometimes referred to as "3 groups for analysis of 20-month-old aging model mice"), YAP, pSer46-MARCKS, and lamin B1 immunohistochemical staining were performed on the cerebral cortex. Fig.43 A is a graph showing the results of measuring the ratio (%) of TRIAD necrotic cells determined to be cytoplasmic pSer46-MARCK positive and nuclear YAP absent (n=4). Fig.43 B is a graph showing the results of measuring the ratio (%) of aged cells in which the nuclear ring of lamin B1 disappeared (n=4). Fig.43 C is a graph showing the results of measuring the ratio (%) of cells having both TRIAD necrosis and senescence characteristics (n=4).

[0115] [ Fig.44 ] Fig.44 A is a graph showing the results of comparing the signal intensity derived from 53BP1 (left graph) and the signal intensity derived from γH2AX (right graph) among three groups of 20-month-old aging model mice analyzed for 53BP1-positive, γH2AX-positive, and MAP2-positive nerve cells (neuronal cells) in the cerebral cortex (n=4). Fig.44 B is a graph showing the results of comparison of the number of MAP2-positive cells (number of neuronal cells) in the cerebral cortex among the three groups for analysis of 20-month-old aging model mice (n=4).

[0116] Fig.44C is a graph showing the results of comparison of the number of Iba1-positive cells (number of microglial cells) in the cerebral cortex among the three groups for analysis of 20-month-old aging model mice (n=4). Fig.44 D is a graph showing the results of comparing the number of GFAP-positive cells (number of astrocytes) in the cerebral cortex among the three groups in the analysis of 20-month-old aging model mice (n=4). "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0117] [ Fig.45 ] is a diagram showing the results of Western blotting of pSer46-MARCKS, 53BP1, pSer139-γH2AX, pSer319-PKCa, pSer396-Tau, Ub, and control β-actin in the whole brain including basal ganglia in 3 groups for analysis of 20-month-old aging model mice. Quantification and statistical comparison were performed for the bands of marker molecules other than ladder or tail Ub.

[0118] [ Fig.46 ] Fig.46 A and B are diagrams showing the SPR analysis of two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2[ Fig.46 A] and human monoclonal antibody #129[ Fig.46 B]) Diagram showing the results of analyzing the interaction with human dsHMGB1. Fig.46 C and D show the SPR analysis of two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2[ Fig.46 C] and human monoclonal antibody #129[ Fig.46 D]) Diagram showing the results of analyzing the interaction with human reduced HMGB1.

[0119] [ Fig.47 ] Fig.47 A and B show the results of ELISA on two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2[ Fig.47 A] and human monoclonal antibody #129[ Fig.47 B]) Diagram showing the results of analyzing the interaction with human dsHMGB1. Fig.47 C and D show the results of ELISA on two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2[ Fig.47 C] and human monoclonal antibody #129[ Fig.47 D]) Diagram showing the results of analyzing the interaction with human reduced HMGB1.

[0120] [ Fig.48 ] is to show the effects of the above two anti-human HMGB1 antibodies of the present invention on three FTLD-ALS model mice (TDP43 N267S -KI mice Fig.48 A], PGRN R504X -KI mice Fig.48 B], or VCP T262A -KI mice Fig.48 C]) is a graph showing the results of the Y-maze test at 6 months of age, 7 months of age, or 8 months of age. The graphs show, from left to right, the results of normal mice (normal_saline), various FTLD-ALS model mice administered with saline (KI_saline), various FTLD-ALS model mice administered with human monoclonal antibody #129-L2 (KI_129-L2), and various FTLD-ALS model mice administered with human monoclonal antibody #129 (KI_129).

[0121] [ Fig.49 ] is to show that in three types of FTLD-ALS model mice (PGRN R504X -KI mice Fig.49 A], TDP43 N267S -KI mice Fig.49 B], or VCP T262A -KI mice Fig.49 C]), immunohistochemical staining for YAP and pSer46-MARCKS was performed on the cerebral cortex, and the proportion (%) of cells showing TRIAD cell death characteristics (nuclear YAP disappearance and pSer46-MARCKS positivity) was measured (n=3). The graphs show, from left to right, the results of normal mice ("B6-nT" in the figure), various FTLD-ALS model mice administered with saline ("KI-nT" in the figure), various FTLD-ALS model mice administered with control human IgG ("KI-IgG" in the figure), various FTLD-ALS model mice administered with human monoclonal antibody #129 ("KI-129" in the figure), and various FTLD-ALS model mice administered with human monoclonal antibody #129-L2 ("KI-129L2" in the figure). "##" and "###" in the figure indicate statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0122] [ Fig.50 ] is to show that the three types of FTLD-ALS model mice (PGRN R504X-KI mice Fig.50 A], TDP43 N267S -KI mice Fig.50 B], or VCP T262A -KI mice Fig.50 C]) shows the results of comparing the number of MAP2-positive neurons (number of neuronal cells) showing abnormal KDEL staining images of the cerebral cortex between normal mice ("B6-nT" in the figure), various FTLD-ALS model mice administered with physiological saline ("KI-nT" in the figure), various FTLD-ALS model mice administered with control human IgG ("KI-IgG" in the figure), various FTLD-ALS model mice administered with human monoclonal antibody #129 ("KI-129" in the figure), and various FTLD-ALS model mice administered with human monoclonal antibody #129-L2 ("KI-129L2" in the figure). The "###" in the figure indicates that there is a statistically significant difference according to Tukey's HSD test (p<0.001).

[0123] [ Fig.51 ] To show the number of γH2AX-positive and MAP2-positive neurons (neuronal cell number) in the cerebral cortex, three types of FTLD-ALS model mice (PGRN R504X -KI mice Fig.51 A], TDP43 N267S -KI mice Fig.51 B], or VCP T262A -KI mice Fig.51 C]) are compared between the signal intensity derived from γH2AX (n=3). The graphs show, from left to right, the results of normal mice ("B6-nT" in the figure), various FTLD-ALS model mice administered with physiological saline ("KI-nT" in the figure), various FTLD-ALS model mice administered with control human IgG ("KI-IgG" in the figure), various FTLD-ALS model mice administered with human monoclonal antibody #129 ("KI-129" in the figure), and various FTLD-ALS model mice administered with human monoclonal antibody #129-L2 ("KI-129L2" in the figure). The "##" and "###" in the figure indicate that there are statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0124] [ Fig.52] To show the number of 53BP1-positive and MAP2-positive nerve cells (neuronal cell number) in the cerebral cortex, three types of FTLD-ALS model mice (PGRN R504X -KI mice Fig.52 A], TDP43 N267S -KI mice Fig.52 B], or VCP T262A -KI mice Fig.52 C]) are compared between the signal intensity derived from 53BP1 between normal mice ("B6-nT" in the figure), various FTLD-ALS model mice administered with physiological saline ("KI-nT" in the figure), various FTLD-ALS model mice administered with control human IgG ("KI-IgG" in the figure), various FTLD-ALS model mice administered with human monoclonal antibody #129 ("KI-129" in the figure), and various FTLD-ALS model mice administered with human monoclonal antibody #129-L2 ("KI-129L2" in the figure). "##" and "###" in the figure indicate that there are statistically significant differences according to Tukey's HSD test (p<0.01 and p<0.001, respectively).

[0125] [ Fig.53 ] is to show that the three types of FTLD-ALS model mice (PGRN R504X -KI mice Fig.53 A], TDP43 N267S -KI mice Fig.53 B], or VCP T262A -KI mice Fig.53C]), immunohistochemical staining for detecting pSer409 / 410-TDP43 and Ub was performed on the cerebral cortex, and the proportion (%) of cells having inclusion bodies positive for both pSer409 / 410-TDP43 and Ub was measured (n=3). The graphs show, from left to right, the results of normal mice ("B6-nT" in the figure), various FTLD-ALS model mice administered with physiological saline ("KI-nT" in the figure), various FTLD-ALS model mice administered with control human IgG ("KI-IgG" in the figure), various FTLD-ALS model mice administered with human monoclonal antibody #129 ("KI-129" in the figure), and various FTLD-ALS model mice administered with human monoclonal antibody #129-L2 ("KI-129L2" in the figure). “#”, “##”, and “###” in the figures indicate statistically significant differences according to Tukey's HSD test (p<0.05, p<0.01, and p<0.001, respectively).

[0126] [ Fig.54 ] is a diagram showing the results of purification of human monoclonal antibody #129 from a CHO (Chinese Hamster Ovary) cell line that stably expresses human monoclonal antibody #129 and analysis by non-reducing capillary electrophoresis.

[0127] [ Fig.55 ] is a diagram showing the results of purification of human monoclonal antibody #129-L2 from a CHO cell line stably expressing human monoclonal antibody #129-L2 and analysis using non-reducing capillary electrophoresis.

[0128] [ Fig.56 ] is a graph showing the results of SDS-PAGE of human monoclonal antibody #129 purified from CHO cell line 2 clone stably expressing human monoclonal antibody #129 ("#129" in the figure), human monoclonal antibody #129-L1 purified from CHO cell line 1 clone stably expressing human monoclonal antibody #129-L1 ("#129L1" in the figure), and human monoclonal antibody #129-L2 purified from CHO cell line 1 clone stably expressing human monoclonal antibody #129-L2. "HC" in the figure indicates a band derived from the heavy chain of the antibody, and "LC" indicates a band derived from the light chain of the antibody.

[0129] [ Fig.57 ] is to show that when two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2[ Fig.57 A and B] and human monoclonal antibody #129[ Fig.57C and D]) Graphs showing the results of SPR analysis of the interaction with human dsHMGB1 before (left) or after (right) storage at 4°C for 6 months.

[0130] [ Fig.58 ] Fig.58 A is a diagram schematically showing the scheme of a treatment experiment on AD model mice (5×FAD mice). Fig.58 B is a graph showing the results of measuring the number of synapses in the cerebral cortex of AD model mice and examining the therapeutic effects of two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 or human monoclonal antibody #129). Fig.58 C is a graph showing the results of studying the therapeutic effects of the two anti-human HMGB1 antibodies of the present invention on cognitive impairment in AD model mice using a Y-maze test. Fig.58 The graphs in B and C show, from left to right, the results of normal mice administered with control human IgG ("ctrl B6 / SJL" in the figure), normal mice administered with human monoclonal antibody #129-L2 ("129L2 B6 / SJL" in the figure), normal mice administered with human monoclonal antibody #129 ("129B6 / SJL" in the figure), AD model mice administered with control human IgG ("ctrl5×FAD" in the figure), AD model mice administered with human 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).

[0131] [ Fig.59 ] is a graph showing the results of analyzing the ratio of active TRIAD necrosis, secondary TRIAD necrosis, and Aβ extracellular deposition (amyloid plaques) in the cerebral cortex of AD model mice (5×FAD mice) administered with two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 or human monoclonal antibody #129). The “-” in the graph indicates the result of administration of control human IgG, and the “+” indicates the result of administration of human monoclonal antibody #129-L2 (“129L2” in the graph) or human monoclonal antibody #129 (“129” in the graph). DETAILED DESCRIPTION

[0132] <Preventive or therapeutic agent of the present invention>

[0133] The first embodiment of the preventive or therapeutic agent of the present invention is a preparation containing the human monoclonal antibody of the present application (hereinafter sometimes referred to as "the preventive / therapeutic agent 1 of the present application"), which is specific to the use of "for the prevention or treatment of amyotrophic lateral sclerosis; Parkinson's disease; Huntington's disease; degenerative diseases or neurological diseases associated with aging; brain aging; and one or more of the diseases accompanied by brain aging". In addition, the second embodiment of the preventive or therapeutic agent of the present invention is a preparation containing the human monoclonal antibody L1 / L2 of the present application (hereinafter sometimes referred to as "the preventive / therapeutic agent 2 of the present application"), which is specific to the use of "for the prevention or treatment of Alzheimer's disease; frontotemporal lobar degeneration; amyotrophic lateral sclerosis; Parkinson's disease; Huntington's disease; degenerative diseases or neurological diseases associated with aging; brain aging; and one or more of the diseases accompanied by brain aging" (hereinafter sometimes referred to as "the preventive / therapeutic agent 1 of the present application" and "the preventive / therapeutic agent 2 of the present application"). The preventive / therapeutic agent of the present application can be used alone as a medicine (drug), or can be used in the form of a composition (pharmaceutical composition) by further mixing with an additive. It should be noted that "treatment of brain aging" is also referred to as "improvement of brain aging".

[0134] <Human monoclonal antibody L1 / L2 of the present application>

[0135] The human monoclonal antibody L1 / L2 of the present application is a human monoclonal antibody that specifically binds to human HMGB1 and comprises the following CDRs: an H chain CDR1 comprising the amino acid sequence of sequence number 1; an H chain CDR2 comprising the amino acid sequence of sequence number 2; and an H chain CDR3 comprising the amino acid sequence of sequence number 3; and an L chain CDR1 comprising the amino acid sequence of sequence number 4; an L chain CDR2 comprising the amino acid sequence of sequence number 5; and an L chain CDR3 comprising the amino acid sequence of sequence number 20 or sequence number 17, and the human monoclonal antibody L1 / L2 is included in the human monoclonal antibody of the present application.

[0136] In this specification, "prevention of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases accompanied by brain aging" includes not only inhibiting the onset and onset of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases accompanied by brain aging, but also delaying the onset and onset period of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases accompanied by brain aging. In addition, "treatment (improvement) of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases accompanied by brain aging" includes not only recovery and improvement of the lesions and symptoms of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases accompanied by brain aging, but also inhibiting their progression.

[0137] The human monoclonal antibody of the present application contained in the preventive / therapeutic agent 1 of the present application and the human monoclonal antibody L1 / L2 of the present application contained in the preventive / therapeutic agent 2 of the present application have high affinity for human HMGB1 protein as demonstrated in the Examples of the present application described later, and have the following effects: reducing and inhibiting the phosphorylation level of serine (Ser46) at position 46 in MARCKS (pSer46-MARCKS); restoring the repair function of DNA damage in nerve cells; inhibiting TRIAD necrosis or its amplification (also called "propagation"); and reducing the aggregates of proteins (e.g., TDP43) in cerebral cortical neurons. Therefore, as the prevention or treatment target of the preventive / therapeutic agent 1 of the present application, i.e., the disease of the present application, etc., and the prevention or treatment target of the preventive / therapeutic agent 2 of the present application, i.e., Alzheimer's disease or frontotemporal lobar degeneration, the disease of the present application, etc., preferably exemplified are diseases caused by one or more selected from the following: HMGB1 and / or pSer46-MARCKS; decreased repair function of DNA damage in nerve cells; TRIAD necrosis or its amplification; and protein (e.g., TDP43) aggregates in cerebral cortical neurons.

[0138] As the above-mentioned "degenerative diseases or neurological diseases associated with aging", for example, dementia with Lewy bodies, progressive supranuclear palsy, basal ganglia degeneration, striato-nigral degeneration, neuronal nuclear inclusion disease, multisystem degeneration, etc. can be cited. In addition, the above-mentioned "brain aging" refers to the reduction of cranial nerves, the increase of nerve cell death, and / or the decline of cognitive function with age. In addition, as the above-mentioned "diseases associated with brain aging", for example, multiple sclerosis, viral encephalitis, acute demyelinating encephalomyelitis, neuromyelitis optica, etc. can be cited.

[0139] In this specification, "TRIAD (necrosis)" refers to a slow necrotic cell death that occurs in nerve cells due to the specific inhibition of RNA polymerase II, which is a basic molecule of transcription, and is caused by the decrease in the function of YAP (Yes-associated protein) (literature "J Cell Biol (2006) 172 (4): 589-604."). TRIAD does not have the morphological and biochemical characteristics of apoptosis. In addition, unlike autophagic cell death, there is no expansion and increase of autophagosomes, and it has the morphological characteristics of endoplasmic reticulum expansion.

[0140] The disease targeted by the preventive / therapeutic agent of the present application may be a genetic disease (e.g., caused by VCP T262A Mutations such as VCP [Valosin-Containing Protein] mutations; PGRN R504X Mutations such as PGRN [progranulin] mutations; CHMP2B Q165X Mutations such as CHMP2B [Charged Multivesicular Body Protein 2B] mutations; TDP43 N267S Mutations such as TDP43 [Transactive Response DNA Binding Protein of 43kD] mutations; diseases caused by gene mutations such as these may also be sporadic diseases.

[0141] The human monoclonal antibody of the present application is preferably a human monoclonal antibody comprising an H chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 7, and an L chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 18. More preferably, it is a human monoclonal antibody comprising a heavy chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 9, and a light chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 22, or SEQ ID NO: 19. In addition, the human monoclonal antibody L1 / L2 of the present application is preferably a human monoclonal antibody comprising the following heavy chain variable region and the following light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 18, and more preferably a human monoclonal antibody comprising the following heavy chain and the following light chain, wherein the heavy chain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises 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.

[0142] In this specification, "HMGB1 (High Mobility Group Box 1)" is a protein also known as HMG1, HMG3, SBP-1, and HMG-1. HMGB1 derived from humans is typically a protein composed of an amino acid sequence determined based on the NCBI reference sequence: NP_002119.1 (a protein encoded by a nucleotide sequence determined by the NCBI reference sequence: NM_002128.5). However, the DNA sequence of a gene may mutate in nature (i.e., not artificially) due to mutations, etc., and the amino acid sequence of the protein encoded thereby may also be changed. Therefore, the HMGB1 bound by the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application includes, in addition to proteins composed of amino acid sequences determined based on the NCBI reference sequence: NP_002119.1, naturally occurring mutants of these proteins are also included.

[0143] In this specification, the so-called "antibody that specifically binds to human HMGB1" refers to an antibody that recognizes and binds to human HMGB1 through a highly specific recognition mechanism between antigens and antibodies. The human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application are preferably separated. Here, "separated" means that the antibody is removed from the environment in which it originally exists by artificial manipulation, or the antibody is expressed in an environment different from the environment in which it originally exists, so that the antibody exists in a state different from the state in which it originally exists. That is, the "isolated antibody" does not include: an antibody that is an antibody from a certain individual and is contained in the body of the individual or in tissues or body fluids (blood, plasma, serum, etc.) from the body without applying external manipulation (artificial manipulation). In addition, the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application are preferably antibodies produced by organisms or cells produced by artificial manipulation (for example, antibodies produced by hybridomas). The "antibodies produced by organisms or cells produced by artificial manipulation" do not include antibodies produced by naturally occurring organisms or B cells (without artificial manipulation).

[0144] In this specification, the so-called "monoclonal antibody" refers to an antibody (including functional fragments of antibodies) obtained from a substantially uniform antibody population. A monoclonal antibody is an antibody that recognizes a single determinant cluster on an antigen. The human monoclonal antibodies of the present application and the human monoclonal antibodies L1 / L2 of the present application include classes and subclasses of human immunoglobulins, and also include the forms of functional fragments of the antibodies. The classes and subclasses of the human monoclonal antibodies of the present application and the human monoclonal antibodies L1 / L2 of the present application include IgGs such as IgG1, IgG2, IgG3, and IgG4; IgAs such as IGA1 and IGA2; IgD; IgE; IgM; and so on.

[0145] The human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application 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 linked to the amino (N) terminus and carboxyl (C) terminus of each of these CDR1 to 3 regions, including a heavy chain variable region and a light chain variable region.

[0146] As the heavy chain FR in the above FR, there can be mentioned: heavy chain FR1 linked to the N-terminus of heavy chain CDR1; heavy chain FR2 linked between the C-terminus of heavy chain CDR1 and the N-terminus of heavy chain CDR2; heavy chain FR3 linked between the C-terminus of heavy chain CDR2 and the N-terminus of heavy chain CDR3; and heavy chain FR4 linked to the C-terminus of heavy chain CDR3. In addition, as the light chain FR in the above FR, there can be mentioned: light chain FR1 linked to the N-terminus of light chain CDR1; light chain FR2 linked 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.

[0147] Specific examples of the heavy chain FR1 include (HF1) a polypeptide comprising amino acid residues 1 to 30 of the amino acid sequence of SEQ ID NO: 7, or (HF1′) a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the polypeptide.

[0148] Specific examples of the heavy chain FR2 include (HF2) a polypeptide comprising amino acid residues 36 to 49 of the amino acid sequence of SEQ ID NO: 7, or (HF2′) a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the polypeptide.

[0149] Specific examples of the heavy chain FR3 include (HF3) a polypeptide comprising amino acid residues 67 to 98 of the amino acid sequence shown in SEQ ID NO: 7, a polypeptide comprising amino acid residues 67 to 98 of the amino acid sequence shown in SEQ ID NO: 11, or (HF3') a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the above polypeptide.

[0150] Specific examples of the heavy chain FR4 include (HF4) a polypeptide comprising amino acid residues 105 to 115 of the amino acid sequence shown in SEQ ID NO: 7, or (HF4') a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the polypeptide.

[0151] Specific examples of the light chain FR1 include (LF1) a polypeptide comprising amino acid residues 1 to 23 of the amino acid sequence shown in SEQ ID NO: 8, or (LF1′) a polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with the polypeptide.

[0152] Specifically, the light chain FR2 includes (LF2) a polypeptide comprising amino acid residues 35 to 49 of the amino acid sequence shown in SEQ ID NO: 8, or (LF2') a polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with the polypeptide.

[0153] Specifically, the light chain FR3 includes (LF3) a polypeptide comprising amino acid residues 57 to 88 of the amino acid sequence shown in SEQ ID NO: 8, or (LF3') a polypeptide comprising an amino acid sequence having a sequence identity of at least 80% with the polypeptide.

[0154] Specific examples of the light chain FR4 include (LF4) a polypeptide comprising amino acid residues 98 to 107 of the amino acid sequence shown in SEQ ID NO: 8, or (LF4') a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the polypeptide.

[0155] The human monoclonal antibodies of the present application and the human monoclonal antibodies L1 / L2 of the present application are human antibodies. Examples of the "human antibodies" include human chimeric antibodies, humanized antibodies, and fully human antibodies, and preferably humanized antibodies and fully human antibodies are exemplified.

[0156] In this specification, "human chimeric antibody" refers to an antibody obtained by linking the variable region of an antibody derived from a non-human animal (e.g., non-human mammals such as chicken, mouse, rat, and cow) to the constant region of an antibody derived from a human. Human chimeric antibodies can be obtained, for example, by immunizing a non-human animal (preferably a non-human mammal) with an antigen, cutting out the variable portion (variable region) of the antibody that binds to the antigen from the gene of the mouse monoclonal antibody, combining it with the gene of the antibody constant portion (constant region) derived from human bone marrow, assembling it in an expression vector, and introducing it into a host to produce human chimeric antibodies (e.g., Japanese Patent Publication No. 8-280387; U.S. Patent No. 4816397; U.S. Patent No. 4816567; U.S. Patent No. 5807715).

[0157] As human constant regions of human chimeric antibodies, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε can be mentioned in the heavy chain, and Cκ and Cλ can be mentioned in the light chain. The amino acid sequences of these constant regions and the base sequences encoding them are known. In addition, in order to improve the stability of the antibody itself or the stability of the production of the antibody, one or more amino acids in the constant region of the human antibody can be substituted, deleted, added, and / or inserted.

[0158] In this specification, the so-called "humanized antibody" refers to an antibody obtained by transplanting (CDR grafting) the gene sequence of the antigen binding site (CDR) of an antibody derived from a non-human animal (for example, non-human mammals such as chickens, mice, rats, and cattle) to an antibody gene derived from a human. The method for making it is overlap extension PCR (overlap extension PCR), etc., which is known (for example, European Patent Application Publication No. 239400, European Patent Application Publication No. 125023, International Publication No. 90 / 07861, International Publication No. 96 / 02576). The variable region of an antibody is usually composed of three CDRs sandwiched by four framework regions (framework region: FR). CDR is the region that essentially determines the binding specificity of the antibody. The amino acid sequence of CDR is rich in diversity, and on the other hand, the amino acid sequence constituting FR mostly shows high homology between antibodies with different binding specificities. Therefore, it is generally believed that by transplanting CDR, the binding specificity of a certain antibody can be transplanted to other antibodies. In addition, from the viewpoint of maintaining the function of CDR, when transplanting CDR derived from non-human to human FR, human FR with high homology to FR derived from the non-human animal can be selected. That is, the amino acids in CDR not only recognize antigens, but also coordinate with the amino acids of FR located near CDR and participate in maintaining the loop structure of CDR. Therefore, it is preferable to use the following human FR, which contains an amino acid sequence with high homology to the amino acid sequence of FR adjacent to the CDR to be transplanted.

[0159] For example, a search system (http: / / www.bioinf.org.uk / abysis / ) specialized for antibodies available on the Internet can be used to search for known human FRs with high homology to FRs derived from non-human animals. Mutations can be introduced into sequences other than the CDR of antibodies of non-human origin to be consistent with the sequence of human FRs obtained in this way. Alternatively, in the case where a gene (cDNA) encoding the amino acid sequence of human FRs obtained by retrieval can be obtained, non-human CDRs can also be introduced into their sequences. The introduction of mutations, etc. can be carried out using techniques known in the art such as nucleic acid synthesis and site-directed mutagenesis.

[0160] By qualitatively or quantitatively measuring and evaluating the affinity of the humanized antibody prepared in this way for the antigen, it is possible to appropriately select the FR of the human-derived antibody such that the CDR forms a good antigen-binding site when linked via the CDR. In addition, as needed, the amino acid residues of the FR can be substituted in such a way that the CDR of the humanized antibody forms a suitable antigen-binding site according to the method described in the document "Cancer Res, 1993, 53, 851-856" and the like, and then the affinity of the mutant antibody obtained by substituting the amino acid for the antigen can be measured and evaluated, so that the mutant FR sequence with the desired properties can be selected.

[0161] In this specification, "fully humanized antibody" refers to an antibody whose entire sequence is derived from a human sequence. Fully humanized antibodies can be produced, for example, in transgenic mice that are manipulated to express genes for human heavy and light chain antibodies. Transgenic mice that produce human antibodies can be prepared, for example, according to the methods described in International Publication No. 02 / 43478, U.S. Patent No. 6657103 (Abgenix), etc. Next, the hybridoma cell line obtained by fusion of B cells derived from transgenic mice producing the desired antibody can be prepared according to the method described in, for example, U.S. Patent No. 5,569,825; U.S. Patent No. 5,625,126; U.S. Patent No. 5,633,425; U.S. Patent No. 5,661,016; U.S. Patent No. 5,545,806; the document "Jakobovits, Adv. Drug Del. Rev. 31: 33-42 (1998)"; and the document "Green et al., J. Exp. Med. 188: 483-95 (1998)".

[0162] As described above, the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application include, in addition to the antibody consisting of the whole antibody, a functional fragment that is a part of the antibody (partial fragment) and specifically recognizes the HMGB1 protein. Examples of such functional fragments include Fab, Fab', F(ab')2, variable region fragment (Fv), disulfide bond Fv, single-chain Fv (scFv), sc(Fv)2, bispecific antibodies (diabody), multispecific antibodies, and polymers thereof.

[0163] Here, "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin comprising one light chain and a portion of a heavy chain. It can be obtained by papain digestion of an antibody, or by a recombinant method. "Fab'" contains one or more cysteines in the hinge region of an antibody, and differs from Fab by the addition of a small amount of residues at the carboxyl terminus of the heavy chain CH1 domain. "F(ab')2" refers to a divalent antigen-binding fragment of an immunoglobulin comprising portions of both light chains and both heavy chains.

[0164] "Variable region fragment (Fv)" is the smallest antibody fragment with complete antigen recognition and binding sites. Fv is a dimer formed by a strong non-covalent bond between the heavy chain variable region and the light chain variable region. "Single-chain Fv (scFv)" contains the heavy chain variable region and the light chain variable region of the antibody, and these regions exist in a single polypeptide chain. "sc(Fv)2" is formed by two heavy chain variable regions and two light chain variable regions combined by a linker or the like to form a single chain. "Bispecific antibody" refers to a small antibody fragment with two antigen binding sites, which contains a heavy chain variable region combined with a light chain variable region in the same polypeptide chain, and each region forms a pair with the complementary region of the other chain. "Multispecific antibody" is a monoclonal antibody with binding specificity to at least two different antigens. For example, it can be prepared by the simultaneous expression of two immunoglobulin heavy chain / light chain pairs with different specificities in two heavy chains.

[0165] The H chain CDR1 to 3 and L chain CDR1 to 3 of the human monoclonal antibody of the present application include the following modifications, wherein one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequences of sequence numbers 1 to 6 without reducing the desired activity (i.e., affinity for HMGB1). The human monoclonal antibody of the present application including the modified CDR can be prepared, for example, by introducing mutations into DNA encoding the antibody chain of human monoclonal antibody #129, or by peptide synthesis. Although it is believed that changes in amino acids in regions other than CDR of an antibody (e.g., FR, constant region) have relatively little effect on affinity for an antigen, methods for screening antibodies with improved affinity for an antigen by changing the amino acids in the CDR are currently known (e.g., reference "PNAS, 102: 8466-8471 (2005)"; reference "Protein Engineering, Design & Selection, 21: 485-493 (2008)"; International Publication No. 2002 / 051870; reference "J. Biol. Chem., 280: 24880-24887 (2005)"; reference "Protein Engineering, Design & Selection, 21: 345-351 (2008)"; reference "MAbs. Mar-Apr; 6(2): 437-45 (2014)"). Furthermore, currently, it is also possible to model antibodies with improved affinity for antigens by utilizing integrated computational chemistry systems (eg, MOE [Molecular Operating Environment], manufactured by CCG, Canada) (eg, http: / / www.rsi.co.jp / kagaku / cs / ccg / products / application / protein.html).

[0166] In this specification, the "multiple amino acids" in "replacement, deletion, addition, and / or insertion of one or more amino acids" refers to preferably within 10 amino acids, more preferably within 5 amino acids, and further preferably within 3 amino acids (for example, within 2 amino acids, 1 amino acid). As substitution, deletion, addition, and insertion of amino acids, conservative substitution is preferred. This "conservative substitution" refers to substitution with other amino acid residues having chemically identical side chains. The group of amino acid residues having chemically identical amino acid side chains is well known in the art. For example, they can be classified into acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine·arginine·histidine), neutral amino acids with hydrocarbon chains (glycine·alanine·valine·leucine·isoleucine·proline), amino acids with hydroxyl groups (serine·threonine), sulfur-containing amino acids (cysteine·methionine), amino acids with amide groups (asparagine·glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine·tyrosine·tryptophan).

[0167] As the H chain CDR1 in the human monoclonal antibody of the present application, since its effect is confirmed in the Examples of the present application described later, the H chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 is preferred. As the H chain CDR2 in the human monoclonal antibody of the present application, since its effect is confirmed in the Examples of the present application described later, the H chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2 is preferred. As the H chain CDR3 in the human monoclonal antibody of the present application, since its effect is confirmed in the Examples of the present application described later, the H chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 is preferred. As the H chain CDR4 in the human monoclonal antibody of the present application, since its effect is confirmed in the Examples of the present application described later, the H chain CDR4 comprising the amino acid sequence of SEQ ID NO: 4 is preferred. As for the L chain CDR1 in the monoclonal antibody, since its effect is confirmed in the Examples of the present application described later, the L chain CDR1 comprising the amino acid sequence of sequence number 4 is preferred. As for the L chain CDR2 in the human monoclonal antibody of the present application, since its effect is confirmed in the Examples of the present application described later, the L chain CDR2 comprising the amino acid sequence of sequence number 5 is preferred. As for the L chain CDR3 in the human monoclonal antibody of the present application, since its effect is confirmed in the Examples of the present application described later, the L chain CDR3 comprising the amino acid sequence of sequence number 6, sequence number 20, or sequence number 17 is preferred.

[0168] In the present specification, "at least 80% sequence identity" with a predetermined amino acid sequence preferably means at least 85% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95% (e.g., 96% or more, 97% or more, 98% or more, 99% or more, 100%) sequence identity. The homology of amino acid sequences can be determined using the BLASTP (amino acid level) program (e.g., document "J. Mol. Biol., 215: 403-410, 1990"). This program is based on the algorithm BLAST (e.g., document "Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990", document "Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993"). When analyzing an amino acid sequence using BLASTP, the parameters are, for example, score=50 and wordlength=3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed according to the method described in the document "Nucleic Acids Res. 25: 3389-3402, 1997". When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific methods of these analysis methods are known.

[0169] The human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application also include antibodies formed by post-translational processing of antibodies by changing the number and position of glycosylation sites. The modified body can improve the ADCC activity (antibody-dependent cellular cytotoxicity) of the antibody. Glycosylation of antibodies typically refers to N-linked or O-linked. Glycosylation of antibodies significantly depends on the host cell used to express the antibody. The change of glycosylation profile can be carried out by known methods such as the introduction or deletion of specific enzymes related to sugar production (Japanese Patent Publication No. 2008-113663, U.S. Patent No. 5047335, U.S. Patent No. 5510261, U.S. Patent No. 5278299, International Publication No. 99 / 54342). Furthermore, for the purpose of increasing the stability of the antibody, the human monoclonal antibody L1 / L2 of the present application can suppress deamidation by replacing an amino acid that can be deamidated or an amino acid adjacent to an amino acid that can be deamidated with other amino acids, or can increase the stability of the antibody by replacing glutamic acid with other amino acids.

[0170] The human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application can be produced by the known hybridoma method and the known recombinant DNA method. As a representative example of the hybridoma method, the method of Kohler and Milstein (document "Nature, 256: 495 (1975)") can be cited. The antibody-producing cells used in the cell fusion step in this method are spleen cells, lymphocytes, peripheral blood leukocytes, etc. of animals (e.g., mammals such as mice, rats, hamsters, rabbits, monkeys, and goats) immunized with antigens (HMGB1 protein, its partial peptides, proteins such as Fc protein fused thereto, or cells expressing them, etc.). Antibody-producing cells obtained by allowing the antigen to act on the above-mentioned cells or lymphocytes previously separated from animals that have not been immunized in a culture medium can also be used. As myeloma cells, various known cell strains can be used. As far as antibody-producing cells and myeloma cells are concerned, as long as they can be fused, they can also be derived from different animal species, but preferably they are derived 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 hybridomas producing monoclonal antibodies specific to the HMGB1 protein can be obtained by subsequent screening. Human monoclonal antibodies that specifically bind to the HMGB1 protein can be obtained by culturing hybridomas, or from the ascites of a mammal to which the hybridomas have been administered.

[0171] The recombinant DNA method is a method in which an antibody gene encoding the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application is cloned from a hybridoma, a B cell, etc., and then inserted into an appropriate vector, and introduced into a host cell (e.g., a mammalian cell line such as HEK cells, Escherichia coli, a yeast cell, an insect cell, a plant cell, etc.), so that the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application are produced as a recombinant antibody (e.g., document "Eur. J. Biochem. 192: 767-775 (1990)"). In the expression of the antibody gene encoding the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application, the antibody gene encoding the heavy chain or the light chain can be separately incorporated into an expression vector and the host cell can be transformed, or the antibody gene encoding the heavy chain and the light chain can be incorporated into a single expression vector and the host cell can be transformed (International Publication No. 94 / 11523). For the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application, the above-mentioned host cells can be cultured, separated and purified from the host cells or the culture medium, and obtained in a substantially pure and uniform form. The separation and purification of antibodies can use the methods used in the usual polypeptide purification. If a transgenic animal production technology is used to produce a transgenic animal (cow, goat, sheep, pig, etc.) into which an antibody gene is incorporated, human monoclonal antibodies derived from the human monoclonal antibody gene of the present application and the human monoclonal antibody L1 / L2 gene of the present application can also be obtained in large quantities from the milk of the transgenic animal.

[0172] In this specification, as additives, pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, isotonic agents, solubilizers, nutrients, etc. can be exemplified. As such pharmaceutically acceptable carriers, when the preventive / therapeutic agent of the present application is a powder, for example, mannitol, lactose, sucrose, human albumin, etc. can be cited, and when the preventive / therapeutic agent of the present application is a liquid, for example, physiological saline, water for injection, phosphate buffer, aluminum hydroxide, etc. can be cited.

[0173] As the administration object of the preventive / therapeutic agent 1 of the present application, any object (human) that needs to prevent or treat the disease of the present application, etc., can be cited, for example, objects diagnosed as having a high risk of onset or occurrence of the disease of the present application, etc., or patients who have already developed or developed the disease of the present application, etc. In addition, as the administration object of the preventive / therapeutic agent 2 of the present application, any object (human) that needs to prevent or treat Alzheimer's disease or frontotemporal lobar degeneration, the disease of the present application, etc. can be cited, for example, objects diagnosed as having a high risk of onset or occurrence of Alzheimer's disease or frontotemporal lobar degeneration, the disease of the present application, etc., or patients who have already developed or developed Alzheimer's disease or frontotemporal lobar degeneration, the disease of the present application, etc. can be cited.

[0174] As the administration method of the preventive / therapeutic agent of the present application, for example, an administration method based on injection (for example, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration), oral mucosal administration, rectal administration, local administration to the brain (intraventricular administration), transdermal administration, oral administration, etc., can be cited, and intravenous administration can be preferably exemplified. The administration amount of the human monoclonal antibody of the present application and the human monoclonal antibody L1 / L2 of the present application can be changed according to the age, weight, sex, health status, and degree of symptom progression of the subject to be administered. Generally, for adults, 0.1 to 1000 mg, preferably 1 to 100 mg, is administered per 1 kg of body weight per day. The preventive / therapeutic agent 1 of the present application can be used in combination with known drugs used in the prevention or treatment of the disease of the present application, etc. In addition, the preventive / therapeutic agent 2 of the present application can be used in combination with known drugs used in the prevention or treatment of Alzheimer's disease or frontotemporal lobar degeneration, the disease of the present application, etc.

[0175] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to these examples. It should be noted that this embodiment is implemented in strict accordance with the recommendations in the guidance outline for the management and use of experimental animals of the Japanese government and the National Institutes of Health. In addition, all experiments have been approved by the Gene Recombination Experiment Committee, the Ethics Committee, and the Animal Experiment Committee of the Tokyo Medical and Dental University.

[0176] Example 1

[0177] 1. Materials and Methods

[0178] [Anti-human HMGB1 antibody]

[0179] In this example, 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 an 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. It should be noted that human monoclonal antibody #129 was subcutaneously administered to mice, and the antibody concentration in the brain and serum was measured by ELISA. The results confirmed that 2% to 8% of the administered human monoclonal antibody #129 passed through the blood-brain barrier (BBB) ​​and was transferred to the brain.

[0180] [Table 1]

[0181]

[0182] The underlined region in the table represents the H chain V region (SEQ ID NO: 7). In addition, the three regions surrounded by the four corners in the table represent the H chain CDR1 (SEQ ID NO: 1), H chain CDR2 (SEQ ID NO: 2), and H chain CDR3 (SEQ ID NO: 3) in order from the amino terminal side.

[0183] [Table 2]

[0184]

[0185] The underlined region in the table represents the L chain V region (SEQ ID NO: 8). In addition, the three regions surrounded by the four corners in the table represent L chain CDR1 (SEQ ID NO: 4), L chain CDR2 (SEQ ID NO: 5), and L chain CDR3 (SEQ ID NO: 6) in order from the amino terminal side.

[0186] [Analysis of Antibody Affinity Based on Surface Plasmon Resonance (SPR)]

[0187] The interaction with human HMGB1 protein was analyzed using human monoclonal antibody #129 of the present invention and mouse monoclonal antibody 2C8C described in pamphlet No. 2018 / 030405 as a comparative control. Specifically, a sensor chip immobilized with human monoclonal antibody #129 of the present invention or mouse monoclonal antibody 2C8C and human HMGB1 protein as an analyte was used to measure the interaction over time using a surface plasmon resonance measuring device ("Biacore T100"; manufactured by GE Healthcare) to calculate the equilibrium dissociation constant (KD value) ( Figure 1 ).

[0188] [Creation of four types of FTLD-ALS model mice]

[0189] For the four FTLD-ALS model mice (VCP T262A -KI mice, PGRN R504X -KI mice, CHMP2B Q165X -KI mice and TDP43 N267S -KI mice) (sometimes referred to as "four types of FTLD-ALS model mice" in this specification) R504X -KI mice were produced according to the method described in the document "Nat. Commun., 2018 9, 433".

[0190] In addition, building VCP T262A-KI mouse production targeting vector. Specifically, the PCR product of the 5.4kb NotI-XhoI fragment amplified from the Bac clone (ID: RP23-111G9 or RP23-124L1) was subcloned into the PspOMI-XhoI site of pBS-DTA. Similarly, the 2.9kb BamHI-NotI fragment amplified from the same Bac clone was subcloned into pBS-LNL(-) with a Neo box (loxP-Neo-loxP). A 4.5kb fragment (1.6kb Neo box + 2.9kb fragment) was cut out using NotI treatment, and a 0.8kb XhoI-Smal fragment with a T262A mutation was amplified from the same Bac clone using PCR and treated with XhoI and SmaI. The 0.8kb and 4.5kb fragments were subcloned into the XhoI-NotI site of the targeting vector. After linearization of the targeting vector by NotI treatment, the fragment was electroporated into ES cells of C57BL / 6J background. VCP was screened from non-transgenic littermates by the following PCR: T262A -KI mice, and the PCR used a primer set for amplifying a DNA fragment containing a LoxP sequence (primer 1 [5'-ATATGCTCTCACTGTATGGTATTGC-3'; SEQ ID NO: 11]; and primer 2 [5'-TCCAGATGAGCTTAGAAGATTAGAA-3'; SEQ ID NO: 12]).

[0191] In addition, construct CHMP2B Q165X -KI mouse production targeting vector. Specifically, the PCR product of the 5.7kb ClaI-SalI fragment amplified from the Bac clone (ID: RP23-13H5 or RP23-273E18) was subcloned into the ClaI-SalI site of pBS-DTA (manufactured by Unitech). The 3.0kb SacII-NotI fragment was amplified from the Bac clone and subcloned into pBS-LNL(+) with a Neo box (loxP-Neo-loxP). SacII-NotI and a 4.6kb fragment (1.6kb Neo box + 3.0kb fragment) were subcloned into the SacII-ClaI site of the targeting vector. CHMP2B was screened out from non-transgenic littermates by the following PCR. Q165X -KI mice, and the PCR used a primer set for amplifying a DNA fragment containing a LoxP sequence (primer 3 [5'-TGGATTTTATTTATGTCTGAATGTG-3'; SEQ ID NO: 13] and primer 4 [5'-ATAAGCAACTTCACAAGGCATCTTA-3'; SEQ ID NO: 14]).

[0192] In addition, constructing TDP43 N267S -KI mouse production targeting vector. 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 (Unitech). A 2.7 kb SacII-NotI fragment was amplified from a Bac clone and subcloned into pA-LNL(+) with a Neo box (loxP-Neo-loxP). A 4.3 kb fragment (1.6 kb Neo box + 2.7 kb fragment) was subcloned into the SacII-ClaI site of the targeting vector. TDP43 was screened from non-transgenic littermates by the following PCR. N267S -KI mice, and the PCR used a primer set for amplifying a DNA fragment containing a LoxP sequence (primer 5 [5'-ACAGTTGGGTGTGATAGCAGGTACT-3'; SEQ ID NO: 15] and primer 6 [5'-TCGAGAATTACAGGAATGTATCATC-3'; SEQ ID NO: 16]).

[0193] [Immunohistochemical staining]

[0194] The brains were removed from four types of FTLD-ALS model mice or C57BL / 6J mice of various ages, fixed in PBS containing 4% paraformaldehyde for 12 hours, and then 5 μm thick brain tissue paraffin sections were prepared using a microtome (manufactured by Yamato Koki Kogyo Co., Ltd.). The prepared brain tissue paraffin sections were deparaffinized using xylene, rehydrated by immersion in 0.01 M citric acid buffer (pH 6.0), and then heated at 120°C for 15 minutes. Then, permeabilization was performed in PBS containing 0.5% triton-X100, and blocking was performed for 60 minutes in PBS containing 10% FBS. Then, antibody reaction was performed at 4°C for 12 hours using various primary antibodies for detecting 11 proteins (pSer46-MARCKS, γH2AX, 53BP1, phosphorylated TDP43 [Ser409 / 410] [pSer409 / 410-TDP43], ubiquitin, p62, KDEL, MAP2, PSD95, VAMP2, and phosphorylated tau [Ser203 (mouse) / Ser214 (human)] [pSer203-tau]) (Table 3). After washing with PBS three times, the secondary antibodies against various primary antibodies (Table 3) were used for antibody reaction at room temperature for 1 hour, and after washing with PBS three times, the cell nuclei were stained in PBS containing 0.2 μg / mL DAPI (4',6-diamidino-2-phenylindole), and the fluorescence images from the above 11 proteins and the fluorescence images from the cell nuclei were obtained using a confocal microscope (FV1200IX83, manufactured by Olympus). Then, based on the DAPI fluorescence image and the pSer46-MARCKS fluorescence image, the number of pSer46-MARCKS-positive cells (cells) per 143 μm×143 μm area was calculated ( Figure 2-1 and Fig. 9 In addition, based on the DAPI fluorescence image, KDEL fluorescence image, and MAP2 fluorescence image, the KDEL signal area ( Fig.10 In addition, based on the DAPI fluorescence image, the γH2AX fluorescence image, and the MAP2 fluorescence image, the γH2AX signal area ( Fig.11 In addition, based on the DAPI fluorescence image, 53BP1 fluorescence image, and MAP2 fluorescence image, the 53BP1 signal area ( Fig.12 In addition, based on the DAPI fluorescence image, pSer409 / 410-TDP43 fluorescence image, and ubiquitin fluorescence image, the pSer409 / 410-TDP43 signal area in the ubiquitin-positive inclusion body was measured ( Fig.13In addition, based on the DAPI fluorescence image and the p62 fluorescence image, the p62 signal area in the inclusion body was determined ( Fig.14 In addition, based on the DAPI fluorescence image, VAMP2 fluorescence image, and PSD95 fluorescence image, the proportion of cells in which VAMP2 signals and PSD95 signals were co-localized was determined ( Fig.15 In addition, based on the DAPI fluorescence image, pSer203-tau fluorescence image, and PSD95 fluorescence image, the proportion of cells in which the pSer203-tau signal and the PSD95 signal were co-localized was determined ( Fig.16 ).

[0195] [Table 3]

[0196]

[0197] [Nissl stain]

[0198] In the above-mentioned [Immunohistochemical staining], the paraffin sections of brain tissue that had been deparaffinized and rehydrated were washed with deionized water and immersed in a tar violet solution (300 mL of a liquid containing 0.1% tar violet and 5 drops of a 10% acetic acid solution) at 37°C for 15 minutes. Then, the sections were dehydrated with an ethanol solution and transparentized with xylene, and an electron microscope (H-7100, manufactured by Hitachi High-Tech) was used to obtain images of necrotic brain tissue ( Figure 2-2 ).

[0199] [Administration of anti-human HMGB1 antibody]

[0200] For the four FTLD-ALS model mice and their background mice (C57BL / 6J), 6 μg of human monoclonal antibody #129 or control human IgG (#12000C, manufactured by Thermo Fisher Scientific) per 30 g of body weight was administered intravenously once a month during the administration period shown in Table 4 ( Figure 3 ) The antibody administration start time for each FTLD-ALS model mouse was the age at which the onset of disease was confirmed by the Morris water maze test.

[0201] [Table 4]

[0202]

[0203] [Cognitive function test]

[0204] During each test period or test day shown in Table 4, three cognitive function tests (Morris water maze test, Y maze test, and fear conditioning test) were performed on four types of FTLD-ALS model mice according to the methods described in the literature "Hum Mol Genet 24, 540-558 (2015)" and the literature "Mol. Psychiatry 20, 459-471 (2015)". Specifically, in the Morris water maze test, mice were subjected to a Morris water maze test once a day for 5 days for a total of 4 times (60 seconds), and the average time (latency) to reach the platform was measured ( Figure 3 The Y-maze test used a Y-maze consisting of three identical arms with equal angles between the arms (manufactured by Ohara Medical Industry Co., Ltd.). The mouse was placed at the end of one arm and allowed to move freely in the maze for 8 minutes. The number of times it entered a new arm different from the previous arm was divided by the total number of times it moved from one arm to another, and the proportion of spontaneous alternation (expressed as alternation rate; Figures 4 to 7 In the fear conditioning test, 24 hours after the conditioning (65 dB white noise, 30 seconds + foot shock, 0.4 mA, 2 seconds), the freezing response (expressed as total freezing percentage [Total Freeze percent]; Figure 8 y axis).

[0205] [Western blotting]

[0206] Cerebral cortical tissues of four FTLD-ALS model mice or C57BL / 6J mice of various ages were solubilized in a solubilization buffer (100 mM Tris-HCl [pH 7.5], 2% SDS) containing a protease inhibitor cocktail (#539134, manufactured by CalBiochem, dilution ratio 1:100) at 4°C for 1 hour. After centrifugation (12,000 g × 10 minutes), the supernatant was recovered, and the same amount of sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% [w / v] SDS, 2.5% [v / v] 2-mercaptoethanol, 5% [v / v] glycerol, and 0.0025% [w / v] bromophenol blue) was added, and the protein amount in the sample was equalized using the BCA method (Pierce BCA protein assay kit, manufactured by Thermo Scientific). The protein samples were then separated by SDS-PAGE and transferred to a PVDF (polyvinylidene fluoride) membrane (Immobilon-P [Millipore]) by a semi-dry method. After blocking in a TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20) solution containing 5% skim milk, the samples were then incubated in a PBS solution containing 0.1% skim milk or Can Get The 13 proteins (phosphorylated PKCα[Ser319][pSer319-PKCα], phosphorylated PKCγ[Thr655][pThr655-PKCγ], phosphorylated PKCδ[Ser643][pSer643-PKCδ], phosphorylated PKCε[Ser729][pSer729-PKCε], phosphorylated tau[Ser203][pSer204-PKCε ... The membrane was washed three times with TBST solution, and then the secondary antibodies against the primary antibodies were used for 1 hour at room temperature.Then, the above 13 proteins were detected using ECL Prime Western blotting detection reagent (RPN2232, manufactured by GE Healthcare) and a luminescence imaging analyzer (ImageQuantLAS 500, manufactured by GE Healthcare). Fig.17 ).

[0207] [Table 5]

[0208]

[0209] 2. Results

[0210] [Analysis of Antibody Affinity Based on Surface Plasmon Resonance (SPR)]

[0211] The KD of mouse monoclonal antibody 2C8C is 1.95×10 -9 M, in contrast, the KD of the human monoclonal antibody #129 of the present invention was 3.79×10 -11 M. Considering that, except for the KD of mouse monoclonal antibody 2C8C, the KD of general antibody drugs is also around 10 -9 M~10 -10 M, which indicates that the human monoclonal antibody #129 of the present invention is an antibody with extremely high affinity for human HMGB1.

[0212] [Immunohistochemical staining]

[0213] pSer46-MARCKS is produced in the early stage before the onset of Alzheimer's disease. Due to neuronal necrosis, HMGB1 leaks from the cell, which induces phosphorylation of MARCKS and induces neurite degeneration (literature "SCIENTIFIC REPORT, 2016Aug25;6:31895"). Therefore, in the early stage of FTLD, four types of FTLD-ALS model mice aged 1 to 12 months were used to analyze whether MARCKS phosphorylation and neuronal necrosis occurred.

[0214] Results: In four FTLD-ALS model mice (VCP) at 1 month, 3 months, 6 months, and 12 months of age, T262A -KI mice, PGRN R504X -KI mice, CHMP2B Q165X -KI mice and TDP43 N267S -KI mice), the signal from pSer46-MARCKS was detected at a high level, and cells in which the DAPI signal from genomic DNA was reduced were observed ( Figure 2-1In addition, two FTLD-ALS model mice (VCP T262A -KI mice and PGRN R504X -KI mice), dilation of the endoplasmic reticulum (ER), a characteristic of necrosis, was observed. Figure 2-2 ).

[0215] These results indicate that necrosis occurs from the early stages of the development of FTLD-ALS.

[0216] [Therapeutic effect of human monoclonal antibody #129 on symptoms of FTLD-ALS model mice: cognitive function test]

[0217] Next, three cognitive function tests (Morris water maze test, Y maze test, and fear conditioning test) were used to analyze whether the symptoms of FTLD-ALS were improved when the human monoclonal antibody #129 of the present invention was administered to four types of FTLD-ALS model mice. First, in the Morris water maze test, the difference in cognitive function between the C57BL / 6J mice ( Figure 3 Compared with the "B6n.t." in 4 FTLD-ALS model mice ( Figure 3 The time to reach the platform increases with the increase of age ( Figure 3 ). On the other hand, when control human IgG was administered to four types of FTLD-ALS model mice ( Figure 3 In the case of "KI IgG" in the experiment, no change was observed in the extension of the time to reach the plateau. In contrast, when the human monoclonal antibody #129 of the present invention was administered to four types of FTLD-ALS model mice ( Figure 3 The results of the study showed that regardless of the type of FTLD mutant gene, the time from the early stage of FTLD onset to the plateau was significantly suppressed (shortened) to the same extent as that of C57BL / 6J mice in any FTLD-ALS model mice ( Figure 3 ).

[0218] This result shows that the human monoclonal antibody #129 of the present invention or the anti-HMGB1 antibody having the same CDR has the effect of preventing and improving the decline of cognitive function caused by FTLD-ALS. The same effect was also found in the Y maze test ( Figures 4 to 7 ), fear conditioning test ( Figure 8 ) was also confirmed.

[0219] [Therapeutic effect of human monoclonal antibody #129 on symptoms of FTLD-ALS model mice: immunohistochemical staining]

[0220] Next, the cognitive function improvement effect of the human monoclonal antibody #129 of the present invention on FTLD-ALS was analyzed using immunohistochemical staining. First, C57BL / 6J mice ( Figure 3 Compared with the "B6n.t." in 4 FTLD-ALS model mice ( Figure 3 The proportion of necrosis marker (pSer46-MARCKS) positive cells in the cerebral cortex of the rats with KI nt in the cerebral cortex increased ( Fig. 9 ), and the signal area from the ER marker (KDEL) increased ( Fig.10 ). On the other hand, when control human IgG was administered to four types of FTLD-ALS model mice ( Fig. 9 and 10 In the case of the "KIIgG" in the figure, no change was observed in the increase in the proportion of pSer46-MARCKS-positive cells or the increase in the signal area from KDEL. In contrast, when the human monoclonal antibody #129 of the present invention was administered to four types of FTLD-ALS model mice ( Fig. 9 and 10 The increase in the proportion of pSer46-MARCKS-positive cells and the increase in the signal area from KDEL were significantly suppressed to the same extent as in C57BL / 6J mice ( Fig. 9 and 10 ).

[0221] This result shows that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having a CDR common therewith have the effect of preventing and ameliorating necrosis and ER expansion caused by FTLD-ALS in the cerebral cortex.

[0222] In addition, it was shown that the signal areas derived from DNA damage markers (γH2AX and 53BP1) in the cerebral cortex of the four FTLD-ALS model mice increased compared with C57BL / 6J mice. In contrast, by administering the human monoclonal antibody #129 of the present invention, the increase in the signal areas derived from γH2AX and 53BP1 was significantly suppressed to the same extent as that of C57BL / 6J mice ( Fig.11 and 12 ).

[0223] This result shows that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having a CDR common therewith have the effect of preventing and ameliorating DNA damage caused by FTLD-ALS in the cerebral cortex.

[0224] In addition, it was shown that the signal areas from protein aggregation markers (pSer409 / 410-TDP43 and p62) in the cerebral cortex of the four FTLD-ALS model mice increased compared with C57BL / 6J mice. In contrast, the increase in the signal areas from pSer409 / 410-TDP43 and p62 was significantly suppressed by administering the human monoclonal antibody #129 of the present invention ( Fig.13 and 14 ).

[0225] This result shows that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having a CDR common therewith have the effect of preventing and improving protein aggregation caused by FTLD-ALS in the cerebral cortex.

[0226] In addition, the inventors of the present application predicted synaptic disorders by using the colocalization of VAMP2 and PSD95 and the colocalization of pSer203-tau and PSD95 as indicators through the analysis of phosphorylated proteomes. Therefore, synaptic disorders were analyzed using these colocalizations as indicators. The results showed that the colocalization of VAMP2 and PSD95 in the cerebral cortex of the four FTLD-ALS model mice was reduced compared with that of C57BL / 6J mice. In contrast, this reduction was significantly suppressed by administering the human monoclonal antibody #129 of the present invention ( Fig.15 ). In addition, it was shown that for the cerebral cortex of the four FTLD-ALS model mice, the co-localization of pSer203-tau and PSD95 in the cerebral cortex increased compared with that of C57BL / 6J mice, whereas this increase was significantly suppressed by administering the human monoclonal antibody #129 of the present invention ( Fig.16 ).

[0227] This result shows that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having a CDR common therewith have the effect of preventing and improving synaptic damage caused by FTLD-ALS in the cerebral cortex.

[0228] [Therapeutic effect of human monoclonal antibody #129 on symptoms of FTLD-ALS model mice: Western blotting]

[0229] In order to support the analysis results using immunohistochemical staining, Western blotting was used for analysis. First, the phosphorylation levels of four proteins involved in the core signal of AD-FTLD (pSer319-PKCα, pThr655-PKCγ, pSer643-PKCδ, and pSer729-PKCε) were analyzed. The results showed that the phosphorylation levels of the four FTLD-ALS model mice increased compared with C57BL / 6J mice based on the strict threshold of core node selection (centrality>2SD) The increase in the levels of pSer319-PKCα and pSer643-PKCδ was suppressed by administering the human monoclonal antibody #129 of the present invention to the four FTLD-ALS model mice ( Fig.17 On the other hand, no changes were observed between the above four FTLD-ALS model mice and C57BL / 6J mice in terms of the two phosphorylation levels (pThr655-PKCγ and pSer729-PKCε) predicted by dynamic network analysis under the relatively broad condition of core node selection (centrality > 0) ( Fig.17 Lanes (2) and (4)).

[0230] In addition, it was shown that the phosphorylation of synaptic instability-related proteins (pSer203-tau) increased in the four FTLD-ALS model mice compared with C57BL / 6J mice, whereas the increase in the pSer203-tau level was suppressed by administering the human monoclonal antibody #129 of the present invention to the four FTLD-ALS model mice ( Fig.17 Lane (5)). In addition, it is shown that the expression levels of synapse-related proteins (PSD95 and VAMP2) in the four FTLD-ALS model mice were reduced compared with those in C57BL / 6J mice. In contrast, by administering the human monoclonal antibody #129 of the present invention to the four FTLD-ALS model mice, the decrease in the expression levels of PSD95 and VAMP2 was suppressed ( Fig.17 Lanes (6) and (7)).

[0231] In addition, it was shown that the expression levels of DNA damage-related proteins (γH2AX and 53BP1) were increased in the four FTLD-ALS model mice compared with C57BL / 6J mice. In contrast, by administering the human monoclonal antibody #129 of the present invention to the four FTLD-ALS model mice, the increase in the expression levels of γH2AX and 53BP1 was suppressed ( Fig.17Lanes (8) and (9)).

[0232] In addition, it was shown that the phosphorylation level of the cell death-related protein (pSer46-MARCKS) was increased in the four FTLD-ALS model mice compared with the C57BL / 6J mice. In contrast, by administering the human monoclonal antibody #129 of the present invention to the four FTLD-ALS model mice, the increase in the pSer46-MARCKS level was suppressed ( Fig.17 ) in lane (10).

[0233] In addition, it was shown that the phosphorylation level of DNA repair-related proteins (pSer77 / 78-Ku70) was increased in the four FTLD-ALS model mice compared with C57BL / 6J mice. In contrast, by administering the human monoclonal antibody #129 of the present invention to the four FTLD-ALS model mice, the increase in the pSer77 / 78-Ku70 level was suppressed ( Fig.17 Lane (11)).

[0234] In addition, it was shown that the level of protein aggregation marker (pSer409 / 410-TDP43) was increased in the four types of FTLD-ALS model mice compared with C57BL / 6J mice, whereas the increase in the pSer409 / 410-TDP43 level was suppressed by administering the human monoclonal antibody #129 of the present invention to the four types of FTLD-ALS model mice ( Fig.17 Lane (12)).

[0235] The above results show that human monoclonal antibody #129 of the present invention and anti-HMGB1 antibodies having CDRs common therewith have the effect of preventing and ameliorating secondary neuronal damage and neurite damage induced by extracellular HMGB1 released from necrotic neurons due to FTLD-ALS.

[0236] [Side effects of human monoclonal antibody #129 of the present invention]

[0237] Furthermore, the side effects of the human monoclonal antibody #129 of the present invention were investigated. Figures 3 to 8 After the cognitive function test, the lungs, livers, kidneys, intestines, spleens, skeletal muscles, hearts and skins of the four FTLD-ALS model mice and C57BL / 6J mice to which the human monoclonal antibody #129 of the present invention was administered were cut open and pathological examinations were performed.

[0238] As a result, no abnormal phenomena (e.g., significant inflammation, cell death, tumors) were observed in any tissues due to administration of human monoclonal antibody #129 of the present invention, indicating that human monoclonal antibody #129 of the present invention and anti-HMGB1 antibodies having CDRs common therewith can prevent and improve the symptoms of FTLD-ALS model mice without significant side effects.

[0239] [Example 2]

[0240] 1. Materials and Methods

[0241] [AD model mice]

[0242] 5×FAD mice, i.e. transgenic mice overexpressing mutant human APP (770) having Swedish (KM670 / 671NL) mutation, Florida (I716V) mutation, and London (V717I) familial Alzheimer's disease (FAD) mutation, and human PS1 having two FAD mutations (M146L and L285V), were purchased from Jackson Laboratory. The genes encoding the above APP and PS1 of 5×FAD mice are under the control of mouse Thy1 promoter (literature "J Neurosci 26, 10129-10140 (2006)"). The background of 5×FAD mice is C57BL / SJL produced by mating C57BL / 6J females with SJL / J males. Using brain tissue samples of male 5×FAD mice at 1, 3, 6, and 12 months of age, comprehensive proteomic analysis was performed as described previously (reference “Hum Mol Genet 24, 540-558 (2015)”).

[0243] APP-KI(App NL-G-F / NL-G-F ) mice (literature "Nat. Neurosci. 17, 661-663 (2014)"), namely mice with Swedish (KM670 / 671NL) mutation, Beyreuther / Iberian (I716F) mutation, and Arctic (E693G) mutation of the humanized APP mouse gene were obtained from RIKEN BioResource Research Center.

[0244] [Administration of human monoclonal antibody #129 of the present invention]

[0245] For 5×FAD mice or B6 / SJL mice, 0.1 μg / kg of control IgG (human IgG isotype control, #12000C, manufactured by Thermo Fisher Scientific) or human monoclonal antibody #129 of the present invention was subcutaneously administered to the neck back region once a week from 6 months of age until 8 months of age, for a total of 9 times. When human monoclonal antibody #129 of the present invention was intravenously injected into various mice, human monoclonal antibody #129 of the present invention diluted with physiological saline to a final volume of 50 μL was injected into the tail vein once a month from 6 months after birth until 8 months (a total of 3 times).

[0246] [Immunohistochemical staining]

[0247] For immunohistochemical staining, mouse and human brain tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. Using a microtome (manufactured by Yamato Koki Kogyo Co., Ltd.), 5 μm thick sagittal sections or coronal sections were obtained. Immunohistochemical staining was performed using the following primary antibodies: rabbit anti-phosphorylated Ser46-MARCKS antibody (1:2000 [manufactured by GLBiochem (Shanghai) Co., Ltd.]); mouse anti-Aβ antibody (1:1000, clone 82E1, #10323, manufactured by IBL); mouse anti-MAP2 antibody (1:200, sc-32791, Santa Clara, CA); Cruz); rabbit anti-MAP2 antibody (1:2000, ab32454, Abcam); mouse anti-phospho-H2X (γH2X) antibody (1:300, JBW301, Millipore); rabbit anti-53BP1 antibody (1:5000, NB100-304, Novus); rabbit anti-YAP antibody (1:50, GTX129151, GeneTex); rabbit anti-NFκB antibody (1:100, #8242, CellSignaling Technology); mouse anti-IL6 antibody (1:50, ab208113, Abcam); rabbit anti-CCL2 / MCP1 antibody (1:100, NBP1-07035, Novus); rabbit anti-lamin B1 antibody (1:5000, ab16048, Abcam); goat anti-Iba1 antibody (1:500, #011-27991, Fujifilm Wako Pure Chemical Industries, Ltd.); rabbit anti-Iba1 antibody (1:100, #019-19741, Fujifilm Wako Pure Chemical Industries, Ltd.); mouse anti-GFAP antibody Cy3 conjugate (1:5000, #C9205, Sigma-Aldrich); mouse anti-NeuN antibody (1:1000, ab104224, Abcam); the reaction product was stained with Alexa Fluor-conjugated Visualization was performed with secondary antibodies against 488, 568, and 647 (1:1000, manufactured by Molecular Probes). Cell nuclei were stained with DAPI (0.2 μg / mL in PBS, #D523, manufactured by Dojindo Chemical Research Institute Co., Ltd.). Microscope images were all acquired using a confocal microscope (FV1200IX83, manufactured by Olympus).

[0248] [Western blotting]

[0249] Regarding the entire cerebral cortex of various mice, Western blotting was used to detect 10 proteins (γH2AX, 53BP1, γH2AX, 53BP1, pSer77 / 78-Ku70, β-actin, pThr638-PKCα, pThr641-PKCβI, pSer660-PKCβII / δ, and pan-PKC) using the antibodies in Tables 5 and 6 and according to the method described in the item [Western blotting] of Example 1 above.

[0250] [Table 6]

[0251]

[0252] [Time-lapse Ku70 imaging]

[0253] Laser microbeam irradiation and acquisition of signals from the injury site were performed according to known methods (references "Nat Commun 4, 1816 (2013)", "Nat Cell Biol 5, 255-260 (2003)"). U2OS cells were seeded in a 35 mm glass-bottomed dish (D11140H, Matsunami Glass IND) containing 2 mL of culture medium (DMEM, D5796, Sigma Aldrich, St. Louis, MO, USA). The next day, 2.5 g of EGFP-C1-FLAG-Ku70 plasmid (#46957, Addgene, Watertown, MA, USA) was transfected into U2OS cells using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA, USA). At 48 hours after transfection and 3 hours before laser microbeam irradiation and time-lapse imaging, ds-HMGB1 (6 ng / mL, 0.24 nM) and / or human monoclonal antibody #129 of the present invention (6 ng / mL, 0.04 nM) were added to the culture medium. U2OS cells were treated with 2 μM Hoechst33258 (DOJINDO, Kumamoto, Japan) for 20 minutes to sensitize DSB. Under live cell imaging conditions (37°C, 5% CO2) maintained in a Stage Top Incubator (Tokai Hit, Shizuoka, Japan), a confocal microscope (FV1200IX83, Olympus, Tokyo, Japan) was used to irradiate a rectangular area on the cell nucleus with a 405 nm laser diode (40× objective, 95%, 100 scans, 12.5 units / pixel). Time-lapse images were acquired every 30 seconds for 10 minutes.

[0254] [Y-maze test]

[0255] exist Fig.22d and Fig.22e The Y-maze test was performed according to the method described in the item [Cognitive function test] of Example 1 above.

[0256] 2. Results

[0257] [Blocking of HMGB1 signaling restores DNA damage repair function in neural cells]

[0258] The expression levels of γH2AX and 53BP1 in the cerebral cortex were analyzed by immunohistochemical staining and Western blotting. As a result, the expression levels of γH2AX and 53BP1 in 5×FAD mice subcutaneously administered with control IgG were significantly increased compared with B6 / SJL mice subcutaneously administered with human monoclonal antibody #129 of the present invention or control IgG (Figures 18a and b). On the other hand, the expression levels of γH2AX and 53BP1 in 5×FAD mice subcutaneously administered with human monoclonal antibody #129 of the present invention were significantly decreased compared with 5×FAD mice subcutaneously administered with control IgG (Figures 18a and b). In addition, the intravenous administration of human monoclonal antibody #129 of the present invention also significantly inhibited DNA damage in the cerebral cortical neurons of 5×FAD mice (Figures 18c and d).

[0259] The above results show that when the human monoclonal antibody #129 of the present invention or the anti-HMGB1 antibody having the same CDR as that of the present invention is administered to 5×FAD mice, DNA damage in cerebral cortical neurons can be suppressed. On the other hand, DNA damage in MAP2-positive neurons in the parietal cortex of human AD patients increases ( Fig.18e ), therefore, when the human monoclonal antibody #129 of the present invention or an anti-HMGB1 antibody having a CDR common therewith is administered to a human AD patient, it can be fully expected that it can also suppress DNA damage in cerebral cortical neurons.

[0260] It is known that Ku70 accumulates at DSB sites based on the non-homologous end joining (NHEJ) pathway and participates in DNA damage repair. Therefore, in order to verify the effect of the trigger signal based on extracellular HMGB1 on the accumulation of Ku70 at DSB sites in human U2OS cells that endogenously express TLR4 and RAGE receptors, time-lapse imaging analysis was performed on EGFP-Ku70 expressing cells after laser microbeam irradiation. First, in order to select the HMGB1 concentration used in U2OS cell culture, the HMGB1 concentration in the cerebrospinal fluid (CSF) of human AD patients (n=56) was evaluated by ELISA, and the results confirmed that the HMGB1 concentration in CSF was 11pg / mL to 13.7ng / mL (average=936pg / mL). Therefore, in this in vitro experiment, 6ng / mL (0.24nM) of ds-HMGB1 was used as a model of the HMGB1 concentration in the neuropipette space after neuronal cell damage.

[0261] When ds-HMGB1 was added to the culture medium under this condition, the accumulation of EGFP-Ku70 at the DNA damage site of U2OS cells was significantly delayed (Figures 18f and g). It is worth noting that after laser microbeam irradiation, the reservoir region of Ku70 was depleted (Figure 18f, arrow), while the recruitment of Ku70 from the retention region of Ku70 was suppressed by the addition of ds-HMGB1 (Figure 18f). On the other hand, when the human monoclonal antibody #129 of the present invention was added together with ds-HMGB1, the regionalization of EGFP-Ku70 to the DNA damage site was basically completely restored (Figures 18f and g).

[0262] These results show that in 5×FAD mice, the administration of human monoclonal antibody #129 of the present invention blocks HMGB1 signaling, thereby enabling the aggregation of Ku70 to DNA damage sites in neurons and restoring the DNA damage repair function in neurons, thereby inhibiting the amplification of TRIAD necrosis.

[0263] [TRIAD is a cell death phenotype of aging]

[0264] Aged glial cells and neurons have been identified in neurodegenerative brains and aged brains (literature “Aging Cell 17, (2018)”, “J. Clin. Invest. 128, 1208-1216 (2018)”, “Nat. Neurosci. 22, 719-728 (2019)”, “Nat. Neurosci. 22, 683-684 (2019)”, “Front. Cell. Neurosci. 14, 1-14 (2020)”, and “Nat. Rev. Neurosci. 21, 433-444 (2020)”). In terms of cell aging, multiple characteristics such as DNA damage, SASP / DAMP secretion (especially HMGB1 secretion), transcriptional changes, and cell apoptosis resistance (literature "Cell 179, 813-827 (2019)") are common to the characteristics of TRIAD necrosis (literature "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)").

[0265] Therefore, the identity between TRIAD and cell aging was verified. The nuclear membrane protein lamin B1, a marker of cell aging, was localized in a ring shape in neurons, astrocytes, and microglia of normal mice (C57BL / 6J mice). Fig.19a , arrows). In contrast, in some neurons and astrocytes of two AD model mice (5×FAD mice and APP-KI mice), the regional presence of ring-shaped lamin B1 was weakened or disappeared ( Fig.19a , arrows). In addition, although abnormal morphology of lamin B1 rings was observed in the microglia of the two AD model mice, most of the lamin B1 rings themselves remained ( Fig.19a ,arrow).

[0266] When the cerebral cortex of 5×FAD mice was analyzed by immunohistochemical staining using three antibodies (anti-YAP antibody, anti-pSer46-MARCKS antibody, and anti-lamin B1 antibody), it was found that most of the lamin B1 ring-negative senescent cells were nuclear YAP-negative, and the phenotypes of TRIAD and cellular aging overlapped ( Fig. 20a, thin open arrows). TRIAD-senescent cells surround larger pSer46-MARCKS-positive foci of early TRIAD necrosis ( Fig. 20 a, thick white arrows). On the other hand, nuclear YAP-positive cells maintained lamin B1 rings ( Fig. 20 a, arrow).

[0267] In addition, when the human monoclonal antibody #129 of the present invention was intravenously administered to two types of AD model mice (5×FAD mice and APP-KI mice), the number of senescent cells in the cerebral cortex was significantly reduced in all mice compared to the case where the control IgG was intravenously administered to the above two types of AD model mice ( Fig. 20 b), and the number of senescent cells in neurons, astrocytes, and microglia was significantly reduced ( Fig.19b ).

[0268] The above results show that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having a CDR common therewith have therapeutic effects on degenerative diseases or neurological diseases associated with aging, brain aging, and diseases associated with brain aging.

[0269] Furthermore, in order to study whether the human monoclonal antibody #129 of the present invention can treat inflammation around glial cells associated with cell aging, the transcription factor (NFκB) that regulates inflammation and the inflammatory cytokines (MCP1 and IL6) were used as indicators for analysis. In two AD model mice (5×FAD mice and APP-KI mice), the nuclear localization level of NFκB in microglia (based on anti-Iba1 antibody detection) was significantly increased compared with normal mice (B6 / SJL mice or C57BL / 6 mice) ( Fig.21a ), in contrast, when the human monoclonal antibody #129 of the present invention was administered to these AD model mice, the nuclear localization level of NFκB in microglia was significantly reduced ( Fig.21a In addition, in the above two AD model mice, compared with normal mice (B6 / SJL mice or C57BL / 6 mice), the expression levels of MCP1 and IL6 around astrocytes (based on anti-GFAP antibody detection) and microglia increased ( Figure 21b ), in contrast, when the human monoclonal antibody #129 of the present invention was administered to these AD model mice, the expression levels of MCP1 and IL6 around astrocytes and microglia were significantly reduced ( Figure 21b ).

[0270] The above results show that human monoclonal antibody #129 of the present invention and anti-HMGB1 antibodies having CDRs common therewith can treat inflammation around glial cells associated with cell aging.

[0271] [Blocking of HMGB1 signaling inhibits DNA damage and progression of Aβ pathology]

[0272] In order to verify in vivo whether the DNA damage repair function in neurons is restored when the activity of PKCα, a protein kinase of Ku70, is inhibited, PBS (control) or a PKCα inhibitor (Go6976) ( Fig.22a ), the expression levels of γH2AX and 53BP1 in the cerebral cortex were analyzed using immunohistochemistry and Western blotting, and a cognitive function test (Y-maze test) was used to analyze whether the symptoms of AD were improved.

[0273] Immunohistochemical staining and Western blotting showed that compared with B6 / SJL mice subcutaneously administered with Go6976 or PBS, 5×FAD mice subcutaneously administered with PBS had increased levels of three phosphorylation indicators of PKCα activation (pThr638-PKCα, pThr641-PKCβI, and pSer660-PKCβII / δ), increased levels of Ku70 phosphorylation (pSer77 / 78-Ku70), and significantly increased expression levels of γH2AX and 53BP1 ( Figure 22b On the other hand, compared with 5×FAD mice subcutaneously administered with PBS, 5×FAD mice subcutaneously administered with Go6976 showed a decrease in the phosphorylation level of PKCα, a decrease in the phosphorylation level of Ku70, and a significant decrease in the expression levels of γH2AX and 53BP1 ( Figure 22b and c).

[0274] In addition, a Y-maze test was performed, and the results showed that the cognitive function of 5×FAD mice subcutaneously administered with PBS was significantly decreased compared with B6 / SJL mice subcutaneously administered with Go6976 or PBS ( Fig.22d On the other hand, compared with 5×FAD mice subcutaneously administered with PBS, 5×FAD mice subcutaneously administered with Go6976 showed significantly improved cognitive function ( Fig.22d ).

[0275] The above results show that when the activity of PKCα, which is a protein kinase of Ku70, is inhibited in 5×FAD mice, DNA damage in cerebral cortical neurons is suppressed, and as a result, cognitive function is improved.

[0276] Next, in order to verify whether the cognitive function of 5×FAD mice is also improved when the human monoclonal antibody #129 of the present invention is administered instead of the PKCα inhibitor (Go6976), control IgG or human monoclonal antibody #129 of the present invention ( Fig.22a The central figure in Figure ) was used for analysis using a cognitive function test (Y-maze test).

[0277] As a result, the cognitive function of 5×FAD mice subcutaneously administered with control IgG was significantly decreased compared with that of B6 / SJL mice subcutaneously administered with control IgG ( Fig.22e On the other hand, the cognitive function of 5×FAD mice subcutaneously administered with the human monoclonal antibody #129 of the present invention was significantly improved compared with 5×FAD mice subcutaneously administered with the control IgG ( Fig.22e ).

[0278] This result shows that when human monoclonal antibody #129 of the present invention or an anti-HMGB1 antibody having a CDR common thereto is administered to 5×FAD mice, DNA damage in cerebral cortical neurons is suppressed, and as a result, cognitive function is improved.

[0279] Recently, we found that YAP-dependent active necrosis of cerebral cortical neurons in the ultra-early stage of AD (document "Nat. Commun. 11, 1-22 (2020)") is the same as TRIAD (document "J. Cell Biol. 172, 589-604 (2006)"), and then, in adjacent neurons, secondary necrosis with unknown characteristics and accompanied by extracellular Aβ aggregation as a remnant of cell death occurs (document "Nat. Commun. 11, 1-22 (2020)"). Therefore, in order to verify the effects of the PKCα inhibitor (Go6976) and the human monoclonal antibody #129 of the present invention on these two types of necrosis and extracellular Aβ aggregation in 5×FAD mice, PBS or the PKCα inhibitor (Go6976) ( Fig.22a ), and control IgG or human monoclonal antibody #129 of the present invention ( Fig.22a central figure in the figure) or intravenously administered control IgG or human monoclonal antibody #129 of the present invention ( Fig.22a The cells were analyzed by immunohistochemical staining using anti-pSer46-MARCKS antibodies and anti-Aβ antibodies.

[0280] As a result, when Go6976 and the human monoclonal antibody #129 of the present invention were administered to 5×FAD mice, secondary necrosis, clusters of necrotic neurons surrounding the central deposit of Aβ, and aggregation of extracellular Aβ were significantly reduced compared to the case of administration of PBS or control IgG as a control ( Figure 22f ).

[0281] This result shows that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having a CDR common therewith have an effect of inhibiting necrosis and an effect of inhibiting the synergistic amplification of TRIAD necrosis based on HMGB1-induced DNA damage.

[0282] [Investigation]

[0283] 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 preventing necrosis (TRIAD) in neurons accompanied by a decrease in YAP. In addition, the aforementioned present Example 1 shows that human monoclonal antibody #129 of the present invention has the effect of preventing and improving damage to neurites accompanied by MARCKS phosphorylation. Regarding the conditions that serve as the background for these therapeutic effects, there is amplification (or propagation) of TRIAD necrosis. After TRIAD necrosis occurs in neurons, neurons release HMGB1 containing dsHMGB1. dsHMGB1 activates the PKC-ERK system through binding to TLR4 and RAGE, and phosphorylates MARCKS (document "2016Aug 25; 6: 31895.doi: 10.1038 / srep31895."), and on this basis, it was clarified this time that activated PKC phosphorylates Ku70, reducing its DNA damage repair function (Figure 18 of this Example 2). Furthermore, it was revealed that the human monoclonal antibody #129 of the present invention inhibits TRIAD necrosis in neurons and blocks the synergistic amplification of TRIAD necrosis based on HMGB1-induced DNA damage ( FIGS. 18 and 22 of this Example 2).

[0284] This Example 1 shows that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having the same CDR as that of the present invention exert therapeutic effects on FTLD-ALS model mice. T262A Mutation, PGRN R504X Mutation, CHMP2B Q165X Mutations and TDP43 N267SMutations are not only the cause of FTLD, but also the cause of ALS, and FTLD and ALS can be understood in the art as the same disease spectrum (for example, the following documents 1 to 13). In addition, most patients with neurodegenerative diseases including FTLD and ALS (sporadic cases) are a heterogeneous group that does not have a single pathogenic gene mutation (or has multiple risk genes), and there is a problem that the effectiveness of existing therapeutic drugs also varies from patient to patient. As shown in this example, the human monoclonal antibody against HMGB1 in the present invention shows the same effect on conditions that may be caused by various reasons, and can be developed as a therapeutic drug for more patients including not only familial neurodegenerative diseases but also sporadic neurodegenerative diseases.

[0285] Document 1: Janssens J and Broeckhoven CV,Pathological mechanisms underlying TDP-43driven neurodegeneration in FTLD-ALS spectrumdisorders,HumanMolecular Genetics,Volume 22,Issue R1,15October 2013,Pages R77-R87,

[0286] Literature 2: Schymick JC, Yang Y, Andersen PM, et al., Progranulin mutations and amyotrophic lateral sclerosis or amyotrophic lateral sclerosis-frontotemporal dementia phenotypes. J Neurol Neurosurg Psychiatry. 2007; 78(7): 754-756. doi: 10.1136 / jnnp.2006.109553

[0287] Document 3: Sleegers K et al., Progranulin genetic variabilitycontributes toamyotrophic lateral sclerosis.Neurology 2008,71(4)253-259; DOI: 10.1212 / 01.wnl.0000289191.54852.75

[0288] Document 4: Scarian E et al., The Role of VCP Mutations in the Spectrum of Amyotrophic LateralSclerosis-Frontotemporal Dementia.2022Front.Neurol.doi: 10.3389 / fneur.2022.841394

[0289] Reference 5: Sreedharan J et al., TDP-43 Mutations in Familial and Sporadic Amyotrophic Lateral Sclerosis Science 2008 Vol 319, Issue 5870 pp. 1668-1672

[0290] Literature 6: Kabashi E et al., TARDBP mutations inindividuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008 May; 40(5): 572-4.

[0291] Document 7: Williams KL et al. A novel TARDBP mutation in an Australian amyotrophic lateral sclerosis kindred. J Neurol Neurosurg Psychiatry. 2009Nov; 80(11): 1286-8.

[0292] Document 8: Parkinson N et al., ALS phenotypes with mutations in CHMP2B (chargedmultivesicular bodyprotein 2B) Neurology 2006Sep 26;67(6):1074-7.

[0293] Literature 9: Cox LE et al. (2010) Mutations in CHMP2B in Lower Motor NeuronPredominant Amyotrophic Lateral Sclerosis(ALS).PLoS ONE5(3):e9872.https: / / doi.org / 10.1371 / journal.pone.0009872

[0294] Document 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. doi:10.1016 / j.nbd.2019.104710. Epub 2019 Dec 16. PMID: 31837425

[0295] Document 11: Vernay A et al., A transgenic mouse expressing CHMP2B intron5 mutant in neurons develops histological and behavioural features of amyotrophic lateral sclerosis and frontotemporal dementia. Hum Mol Genet. 2016 Aug 1;25(15):3341-3360. doi:10.1093 / hmg / ddw182. Epub 2016 Jun 21. PMID: 27329763

[0296] Document 12: Ugbode C, West RJH. Lessons learned from CHMP2B, implications for frontotemporal dementia and amyotrophic lateral sclerosis. Neurobiol Dis. 2021 Jan;147:105144. doi:10.1016 / j.nbd.2020.105144. Epub 2020 Nov 2. PMID: 33144171 Review.

[0297] Literature 13: Blair IP et al. FUS mutations in amyotrophic lateralsclerosis: clinical, pathological, neurophysiological and genetic analysis. J NeurolNeurosurg Psychiatry.2010Jun; 81(6): 639-45.doi: 10.1136 / jnnp.2009.194399.Epub2009Dec 3.PMID: 19965854

[0298] On the other hand, necrosis associated with decreased YAP (TRIAD) has been confirmed in addition to its presence in AD and FTLD, in Huntington's disease (HD) mice (Mao et al., Hum Mol Genet 2016), human HD patients (Yamanishi et al., Acta Neuropathol Commun 2017), and in mutant SOD1 transgenic mice, which are model mice for amyotrophic lateral sclerosis (ALS), a reduction in motor neurons has been confirmed (Morimoto et al., J Neurosci Res 2009). As for the increase in phosphorylated MARCKS, another characteristic of TRIAD, it has also been shown that the amount of phosphorylated MARCKS increases in the cerebrospinal fluid and serum of human ALS patients (Homma et al., LifeSci Alliance 2021). In addition, hyperphosphorylation of MARCKS was observed in Parkinson's disease (PD) model mice (A53T Bac Tg mice) (reference "Fujita et al., eNeuro 2018").

[0299] In addition, as shown in this Example 1, 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 can be fully expected to have an inhibitory effect on degenerative diseases such as ALS that are accompanied by TDP43 aggregation.

[0300] Furthermore, decreased DNA damage repair function is currently considered a common symptom of degenerative diseases (references “McKinnon Nature Neurosci 2013”, reference “Madabhushi et al., Neuron 2014”).

[0301] Based on the above, human monoclonal antibody #129 of the present invention and anti-HMGB1 antibodies having CDRs common therewith can be fully expected to generally exert therapeutic effects on degenerative diseases including ALS, PD, and HD.

[0302] In addition, DNA damage is known to be a cause of aging. The human monoclonal antibody #129 of the present invention reduces the number of senescent cells in the cerebral cortex of two AD model mice (5×FAD mice and APP-KI mice), and inhibits the cell aging of neurons, astrocytes, and microglia (Figures 19 and 20 of this Example 2). Therefore, it is confirmed that the human monoclonal antibody #129 of the present invention and the anti-HMGB1 antibody having a common CDR therewith have the effect of treating degenerative diseases or neurological diseases related to aging, brain aging and diseases accompanied by brain aging.

[0303] [Example 3]

[0304] 1. Materials and Methods

[0305] [Various disease model mice]

[0306] As Huntington's disease model mice, R6 / 2 mice were used (Document 1). As Parkinson's disease model mice, α-synuclein A53T mice were used (Document 2). As amyotrophic lateral sclerosis model mice, SOD1-Tg mice were used (Document 3). As spinocerebellar ataxia model mice, mutant ataxin-1 gene knock-in mice were used (Document 4). As aging model mice, 12-month-old C57BL / 6 mice were used.

[0307] [Antibody administration]

[0308] For various disease model mice, human monoclonal antibody #129 adjusted to 6 μg per 30 g body weight (equivalent to 0.2 mg / kg BW) using physiological saline or the same amount of physiological saline without antibody was administered intravenously once every two weeks during the administration period shown in Table 7. In addition, as a normal control group, the same amount of physiological saline was administered intravenously to normal genotype brother mice.

[0309] For Huntington's disease model mice and amyotrophic lateral sclerosis model mice in which motor function was not clearly improved after the second administration, the dose was increased to 60 μg per 30 g body weight (equivalent to 2 mg / kg BW) from the third administration onwards.

[0310] In an experiment on aging model mice, 6 μg per 30 g body weight (equivalent to 0.2 mg / kg BW) was intravenously administered once every 4 weeks.

[0311] [Table 7]

[0312]

[0313] [Modified Antibodies]

[0314] It is believed that there is an amino acid sequence in the light chain CDR3 of human monoclonal antibody #129 that is highly likely to be modified by a sugar chain. In the antibody administration experiment for amyotrophic lateral sclerosis model mice (SOD1-Tg mice) and aging model mice, the amino acid sequence modified form of the light chain CDR3 used in Example 4 described later, namely human monoclonal antibody #129-L2, was administered instead of human monoclonal antibody #129. The alignment of the amino acid sequences of the light chain variable regions of human monoclonal antibody #129 and human monoclonal antibody #129-L2 is shown in FIG25 . Using human monoclonal antibody #129 and human monoclonal antibody #129-L2, the binding to human dsHMGB1 was studied using the surface plasmon resonance method (SPR). As a result, the dissociation constants (K ) of human monoclonal antibody #129 and human monoclonal antibody #129-L2 for human dsHMGB1 were confirmed. D ) are 1.47×10 -11 and 1.44×10 -11 , both have basically the same binding force.

[0315] [Motor function test]

[0316] For various disease model mice and normal genotype sibling mice, during each test period shown in Table 7, three of the four motor function tests (rotarod test, open field test, footprint test, and Y maze test) were performed according to the methods described in the following documents 5 to 7. Specifically, in the rotarod test, the mouse was placed on a rotating rod (3.5 rpm), and the rotation speed was linearly increased to 35 rpm within 300 seconds, and continued at 35 rpm until 360 seconds. The mice were set with a rest interval of 30 to 60 minutes between tests and received 12 tests (4 tests per day for 3 consecutive days). In each test, the time until the mouse fell from the rotarod was recorded. The average time until the mouse fell from the rotarod was calculated for subsequent analysis. In addition, in the open field test, the mouse was placed in the center of the open field space (50 cm × 50 cm × 40 cm [height]) and allowed to explore for 10 minutes. At the center of the field, the light intensity was 70 lux. As an indicator, the moving distance (cm) is used. In addition, in the footprint test, in order to compare the walking pattern, the footprints during walking are recorded. The hind legs are covered with blue ink and the front legs are covered with red ink, and they are placed in a passage 40 cm long and 7.5 cm wide as a runway to the closed box. The white paper on the floor of the passage is replaced every time you walk. As for the footprint pattern, the parameters of the footprint pattern described in the following document 8 are measured: stride. In addition, regarding the Y-maze test, it is carried out according to the method described in Example 1.

[0317] [Immunohistochemical staining]

[0318] The brain was removed from each of the disease model mice or normal genotype sibling mice of various ages, fixed in PBS containing 4% paraformaldehyde for 12 hours, and then 5 μm thick brain tissue paraffin sections were made using a microtome (manufactured by Yamato Koki Kogyo Co., Ltd.). The prepared brain tissue paraffin sections were deparaffinized using xylene, rehydrated by immersion in 0.01M citric acid buffer (pH 6.0), and then heated at 120°C for 15 minutes. Then, permeabilization was performed in PBS containing 0.5% triton-X100, and blocking was performed in PBS containing 10% FBS for 60 minutes. Then, antibody reaction was performed at 4°C for 12 hours using various primary antibodies (Table 8) for detecting 17 proteins (pSer46-MARCKS, γH2AX, 53BP1, phosphorylated TDP43 [Ser409 / 410] [pSer409 / 410-TDP43], ubiquitin, KDEL, MAP2, phosphorylated tau [Ser396]), huntingtin, calbindin, α-synuclein, GFAP, Iba1, lamin B1, Ataxin-1, YAP, and SOD1). After washing with PBS three times, the antibody reaction was carried out at room temperature for 1 hour using a secondary antibody for various primary antibodies (Table 8), and after washing with PBS three times, the cell nuclei were stained in PBS containing 0.2 μg / mL of DAPI (4',6-diamidino-2-phenylindole), and a confocal microscope (FV1200IX83, manufactured by Olympus) was used to obtain fluorescence images from the above 17 proteins and fluorescence images from the cell nuclei. Then, based on the DAPI fluorescence image, for the cerebral cortex, the number of cells (individuals) that met the conditions described in the description of each figure in each 143 μm×143 μm area was calculated. In addition, for pSer46-MARCKS fluorescence images, γH2AX fluorescence images, 53BP1 fluorescence images, and KDEL fluorescence images, the signal values ​​of the regions of interest were measured.

[0319] [Table 8]

[0320]

[0321] [Western blotting]

[0322] Cerebral cortex tissues of disease model mice and normal genotype sibling mice of various ages, and cerebellar tissues of spinocerebellar ataxia model mice and their background mice were solubilized at 4°C for 1 hour in the presence of a solubilization buffer (100 mM Tris-HCl [pH 7.5], 2% SDS) containing a protease inhibitor cocktail (#539134, manufactured by CalBiochem, dilution ratio 1:100). After centrifugation (12,000 g × 10 minutes), the supernatant was recovered and the same amount of sample buffer (125 mM Tris-HCl, pH 6.8, 4% [w / v] SDS, 12% [v / v] 2-mercaptoethanol, 20% [v / v] glycerol, and 0.005% [w / v] bromophenol blue) was added. The protein samples were then separated by SDS-PAGE and transferred to a PVDF (polyvinylidene fluoride) membrane (Immobilon-P [Millipore]) by a semi-dry method. After blocking in a TBST (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20) solution containing 5% skim milk, the samples were then incubated in a PBS solution containing 0.1% skim milk or Can Get Antibody reaction was performed at 4°C for 12 hours in TBST solution containing Signal solution (Toyobo, Osaka, Japan) using various primary antibodies for detecting 9 proteins (phosphorylated PKCα[Ser319][pSer319-PKCα], phosphorylated tau[Ser203(mouse) / Ser214(human)][pSer203-tau], phosphorylated tau[Ser384(mouse) / Ser396(human)][pSer384-tau], phosphorylated γH2AX[Ser139][pSer139-γH2AX], 53BP1, phosphorylated TDP43[Ser409 / 410][pSer409 / 410-TDP43], pSer46-MARCKS, ubiquitin, and β-actin) (Table 9). After washing the membrane three times with TBST solution, antibody reaction was performed at room temperature for 1 hour using secondary antibodies against various primary antibodies (Table 9). Then, the above 9 proteins were detected using ECL Prime Western blotting detection reagent (RPN2235, manufactured by Cytiva) and luminescence imaging analyzer (ImageQuant LAS 500, manufactured by GE Healthcare).

[0323] [Table 9]

[0324]

[0325] 2. Results

[0326] [Huntington's disease model mouse]

[0327] As for the Huntington's disease model mice, as shown in the aforementioned "1. Materials and Methods", the experiment was conducted using R6 / 2 mice (Document 1) after the onset of the disease, and intravenous administration was performed once every 2 weeks (Figure 23A). At 6 weeks of age, when the administration began, the rotarod test showed that the motor function of R6 / 2 mice decreased. As shown in the aforementioned "1. Materials and Methods", in the rotarod test after 6 μg (equivalent to 0.2 mg / kg BW) of human monoclonal antibody #129 was administered twice, the improvement of motor function was not obvious, so from the third administration onwards, the amount was increased to 60 μg (equivalent to 2 mg / kg BW) per 30 g body weight. As a result, at the time point of 14 weeks of age after 2 intravenous administrations of 6 μg and 2 intravenous administrations of 60 μg, a total of 4 intravenous administrations, there was a significant difference in the residence time of the rotating rod test between normal mice and R6 / 2 mice administered with normal saline, while the significant difference disappeared between normal mice and R6 / 2 mice administered with human monoclonal antibody #129 (Figure 23B). However, no significant difference was observed between R6 / 2 mice administered with normal saline and R6 / 2 mice administered with human monoclonal antibody #129 (Figure 23B). On the other hand, at the time point of 14 weeks of age, in the open field test, in addition to the comparison between normal mice and R6 / 2 mice administered with normal saline, the total walking distance was also observed to be significantly different in the comparison between R6 / 2 mice administered with normal saline and R6 / 2 mice administered with human monoclonal antibody #129 (Figure 23C). Furthermore, at the 14-week-old time point, in the footprint test, in addition to the comparison between normal mice and R6 / 2 mice administered with saline, significant differences in stride were also observed in the comparison between R6 / 2 mice administered with saline and R6 / 2 mice administered with human monoclonal antibody #129 ( FIG. 23D ).

[0328] Next, at 14 weeks of age, brain tissues were sampled from three groups of mice (i.e., "Three groups for analysis of Huntington's disease model mice"), namely, normal mice administered with saline, R6 / 2 mice administered with saline, and R6 / 2 mice administered with human monoclonal antibody #129 after motor function analysis, and pathological analysis was performed. In the immunohistochemical staining method, triple staining of Htt, Ub, and MAP2 was performed. As a result, Ub-positive Htt nuclear inclusions ( Fig.24A). The same Htt nuclear inclusion bodies are a phenomenon that has been widely observed in R6 / 2 mice regardless of whether physiological saline is administered (Reference 1). On the other hand, it was confirmed that in R6 / 2 mice administered with human monoclonal antibody #129, Ub-positive Htt nuclear inclusion bodies were significantly reduced ( Fig.24 A). In addition, when the number of MAP2-positive neurons in the cerebral cortex was measured, a decrease was observed in R6 / 2 mice administered with saline compared to normal mice, whereas recovery was observed in R6 / 2 mice administered with human monoclonal antibody #129 compared to R6 / 2 mice administered with saline ( Fig.24 B). On the other hand, the number of microglia and astrocytes was studied by immunostaining of Iba1 and GFAP, but no significant difference was found between normal mice and R6 / 2 mice at the 14-week time point. In addition, no change was observed in the number of microglia and astrocytes in R6 / 2 mice by administering human monoclonal antibody #129 ( Fig.24 C and D).

[0329] Similar studies were conducted on the striatum. As a result, administration of human monoclonal antibody #129 significantly suppressed the formation of Ub-positive Htt nuclear inclusions in the striatum of R6 / 2 mice ( Fig.25A ). In addition, when the number of MAP2-positive neurons in the striatum was measured, the number of R6 / 2 mice administered with saline was reduced compared to normal mice. In contrast, in R6 / 2 mice administered with human monoclonal antibody #129, recovery was observed compared to R6 / 2 mice administered with saline (Figure 25B). On the other hand, the number of microglia and astrocytes was studied by immunostaining of Iba1 and GFAP, but at the 14-week time point, there was no significant difference between normal and R6 / 2 mice. In addition, no changes were observed in the number of microglia and astrocytes in R6 / 2 mice by administering human monoclonal antibody #129 (Figures 25C and D).

[0330] It is known that TRIAD neuronal necrosis releases HMGB1 (Documents 9 and 10), and the released HMGB1 induces TRIAD necrosis of surrounding neuronal cells (Document 11). On the other hand, as markers of TRIAD necrosis, cell body pSer46-MARCKS positivity (Documents 9 to 11) and a decrease in nuclear YAP (Documents 10 to 13) are known. Therefore, triple staining of YAP, pSer46-MARCKS, and DAPI was performed, and the results showed that in R6 / 2 mice administered with physiological saline, TRIAD necrosis with pSer46-MARCKS positivity and a decrease in nuclear YAP increased ( Fig.26 A). In contrast, in R6 / 2 mice administered with human monoclonal antibody #129, the number of TRIAD necrotic neurons was observed to be restored compared with that in R6 / 2 mice administered with saline ( Fig.26 A). It is also known that abnormal expansion of the endoplasmic reticulum occurs during TRIAD (References 10-13). Therefore, immunostaining was performed for KDEL, an endoplasmic reticulum marker, and the results showed that the number of neurons showing abnormal KDEL staining images increased in R6 / 2 mice administered with saline ( Fig.26 B) On the other hand, in R6 / 2 mice administered with human monoclonal antibody #129, the number of neurons showing abnormal KDEL staining images decreased ( Fig.26 B).

[0331] In addition, it is known that DNA damage of neurons increases in neurodegenerative diseases such as Huntington's disease (References 14 and 15). Therefore, using 53BP1 and γH2AX as DNA damage markers, DNA damage of neurons in the cerebral cortex was measured by quadruple staining with MAP2 and DAPI. The results showed that in R6 / 2 mice, DNA damage of neurons increased compared to normal mice, and the number of neurons with DNA damage was suppressed by administration of human monoclonal antibody #129 ( Fig.26 C).

[0332] Similar immunohistochemical studies were performed on the striatum, and the results confirmed that administration of human monoclonal antibody #129 also significantly inhibited the increase in TRIAD necrosis in R6 / 2 mice in the striatum ( Fig. 27 A), significantly inhibited the increase of KDEL abnormal staining images ( Fig. 27 B), significantly inhibited the increase of neurons containing DNA damage ( Fig. 27 C).

[0333] Next, in order to reconfirm the results of the pathological analysis, biochemical analysis was performed on the brain tissue samples of the three groups of 14-week-old Huntington's disease model mice. Specifically, when the protein levels of pSer46-MARCKS, 53BP1, γH2AX, Ub, and pSer203-Tau were compared by Western blotting, it was shown that these degenerative disease marker molecules were increased in R6 / 2 mice compared with normal mice, and by administering human monoclonal antibody #129, these increased degenerative disease marker molecules in R6 / 2 mice were reduced to normal levels ( Fig.28 ).

[0334] The above results show that human monoclonal antibody #129 has the effects of inhibiting the progression of Huntington's disease (HD) symptoms and cell death, and inhibiting the decline of motor function.

[0335] [Parkinson's disease model mouse]

[0336] For Parkinson's disease model mice, as shown in the aforementioned "1. Materials and Methods", experiments were conducted using α-synuclein A53T mice (Document 2) after onset of the disease, and intravenous administration was performed once every 2 weeks (Figure 29A). From 8 weeks to 18 weeks of age, 6 μg (equivalent to 0.2 mg / kg BW) of human monoclonal antibody #129 was administered for a total of 6 times (Figure 29A) per 30 g of body weight at intervals of once every 2 weeks. At 8 weeks of age at the beginning of administration, in the rotating rod test, α-synuclein A53T mice showed excessive activity (Figure 29B), indicating that Parkinson's disease had already occurred. At the time point of 12 weeks of age after a total of 3 administrations and at the time point of 20 weeks of age after a total of 6 administrations, the excessive activity of α-synuclein A53T mice was suppressed with significant differences on the second day (Day2) (Figure 29B). Similarly, in the open field test at 20 week-old time points, compared with normal mice, in the α-synuclein A53T mice given normal saline, the total moving distance increases, meaning the overactivity of α-synuclein A53T mice, but by giving human monoclonal antibody #129, the total moving distance is suppressed (Figure 29 C), showing the improvement of symptoms.On the other hand, in the footprint test at 20 week-old time points, in the comparison between the α-synuclein A53T mice given normal mice given normal saline, the α-synuclein A53T mice given normal saline and the α-synuclein A53T mice given human monoclonal antibody #129, there is no significant difference in stride (stride), meaning that there is no symptom of walking disorder (Figure 29 D).This means that the Parkinson's disease model mice used in the present embodiment are equivalent to the early stage after onset.

[0337] Next, at 20 weeks of age, brain tissues were sampled from the three groups of mice for Parkinson's disease model mouse analysis after motor function analysis, and pathological analysis was performed. In the immunohistochemical staining method, quadruple staining of pSer129-Syn, Ub, MAP2, and DAPI was performed, and the results showed that in α-synuclein A53T mice administered with saline, an increase in Ub-positive proteins in the cytoplasm of MAP2-positive brain neurons and an increase in pSer129-Syn in the nucleus were observed ( Fig.30 A). On the other hand, the positive phenomenon of pSer129-Syn and Ub was normalized by administering human monoclonal antibody #129 ( Fig.30 A). In MAP2 immunohistochemical staining, the number of neurons (neurons) in the cerebral cortex was reduced in α-synuclein A53T mice, but was normalized in α-synuclein A53T mice administered with human monoclonal antibody #129 ( Fig.30 B) On the other hand, in α-synuclein A53T mice, there was no change in the number of Iba1-positive microglia and GFAP-positive astrocytes, and there was no change in the number of cells even after administration of human monoclonal antibody #129 ( Fig.30 C and D).

[0338] Next, the neuronal necrosis known as TRIAD (Document 12) was studied. As mentioned above, it is known that TRIAD releases HMGB1 (Document 9, Document 10), and the released HMGB1 induces TRIAD necrosis of surrounding neuronal cells (Document 11). In addition, it has been reported that pSer46-MARCKS was stained in neurons in other Parkinson's disease model mice using immunohistochemical staining (Document 16). Therefore, triple staining of YAP, pSer46-MARCKS, and DAPI was performed, and the results showed that in the cerebral cortex of α-synuclein A53T mice administered with saline, TRIAD necrosis with pSer46-MARCKS positivity and reduced nuclear YAP increased compared to normal mice administered with saline ( Fig.31 A). In contrast, in α-synuclein A53T mice administered with human monoclonal antibody #129, the number of TRIAD necrotic neurons was significantly reduced compared with α-synuclein A53T mice administered with saline ( Fig.31 A). In addition, since TRIAD is known to be accompanied by abnormal expansion of the endoplasmic reticulum (References 12 and 13), immunostaining of KDEL, an endoplasmic reticulum marker, was performed, and the number of neurons showing abnormal KDEL staining images increased in α-synuclein A53T mice administered with saline ( Fig.30 B) On the other hand, in α-synuclein A53T mice administered with human monoclonal antibody #129, the neurons showing abnormal KDEL staining images were normalized ( Fig.30 B).

[0339] In addition, DNA damage in neurons was measured in the cerebral cortex using 53BP1 and γH2AX, which are DNA damage markers, by quadruple staining with MAP2 and DAPI. The results showed that in α-synuclein A53T mice, DNA damage in neurons increased compared to normal mice, and DNA damage was suppressed by administering human monoclonal antibody #129 ( Fig.30 C).

[0340] Next, in order to reconfirm the results of the pathological analysis, biochemical analysis of brain tissue samples of 3 groups of mice for analysis of Parkinson's disease model mice at 20 weeks of age was performed to reconfirm the results of the pathological analysis. When comparing 5 proteins (pSer46-MARCKS, 53BP1, γH2AX, Ub, and pSer203-Tau) by Western blotting, it was shown that in α-synuclein A53T mice, these 5 proteins were increased compared to normal mice. By administering human monoclonal antibody #129, the increased levels of these 5 proteins in α-synuclein A53T mice were reduced to normal levels ( Fig.32 ).

[0341] The above results show that human monoclonal antibody #129 has the effect of inhibiting the progression of Parkinson's disease (PD) symptoms and cell death, and inhibiting the decline of motor function.

[0342] [Amyotrophic lateral sclerosis model mouse]

[0343] As for the amyotrophic lateral sclerosis model mice, as shown in the aforementioned "1. Materials and Methods", experiments were conducted using SOD1-Tg mice (Document 3) after the onset of the disease, and intravenous administration was performed once every 2 weeks (Figure 33A). From 6 weeks to 9 weeks of age, 6 μg (equivalent to 0.2 mg / kg BW) of human monoclonal antibody #129-L2 was administered twice per 30 g of body weight at intervals of once every 2 weeks (Figure 33A). In the 6-week-old SOD1-Tg mice, the rotating rod test immediately before the administration showed a decrease in motor function (Figure 33B), indicating that amyotrophic lateral sclerosis in SOD1-Tg mice has already developed. Since no significant improvement was observed in the rotating rod test until 10 weeks of age, the administration was increased to 60 μg (equivalent to 2 mg / kg BW) per 30 g of body weight from the third administration at 10 weeks of age to the eighth administration at 20 weeks of age.

[0344] As a result of administering human monoclonal antibody #129-L2, although the rotarod residence time of SOD1-Tg mice was significantly reduced compared with normal mice at the 22-week-old time point, it was significantly suppressed by administering human monoclonal antibody #129-L2 ( Figure 33B ).

[0345] Similarly, in the open field test at the 22-week age time point, compared with normal mice, in the SOD1-Tg mice given saline, the total moving distance decreased, which means that motor function decreased, but by administering human monoclonal antibody #129-L2, the total moving distance increased (Figure 33C), showing improvement of symptoms. In addition, in the footprint test at the 22-week age time point, in the comparison between normal mice and SOD1-Tg mice given saline, a decrease in stride was observed, but by administering human monoclonal antibody #129-L2, the stride increased (Figure 33C), showing improvement of symptoms of motor function decreased (Figure 33D).

[0346] Next, at 22 weeks of age, brain tissue samples were collected from three groups of mice (i.e., "3 groups for analysis of amyotrophic lateral sclerosis model mice"), including normal mice given saline after motor function analysis, SOD1-Tg mice given saline, and SOD1-Tg mice given human monoclonal antibody #129-L2, for pathological analysis. In the immunohistochemical staining method, quadruple staining of Ub, SOD1, MAP2, and DAPI was performed, and the results showed that the staining signals of SOD1 and Ub increased in the large neurons of the anterior horn of the spinal cord of SOD1-Tg mice, and some showed inclusion body-like morphology. This disease morphology was basically eliminated by the administration of human monoclonal antibody #129-L2 ( Fig.34 A). In addition, in the quadruple staining of Ub, pSer396-Tau, MAP2, and DAPI, co-localization of Ub and pSer396-Tau in the cytoplasm was observed in large neurons of the anterior horn of the spinal cord of SOD1-Tg mice, but this was also almost eliminated by the administration of human monoclonal antibody #129-L2 ( Fig.34 B). In addition, in the quadruple staining of Ub, pTDP43, MAP2, and DAPI, almost no movement of pTDP43 to the cytoplasm was observed, but light staining of pTDP43 was observed in the cytoplasmic inclusion bodies of Ub ( Fig.34 C). The number of MAP2-positive nerve cells with such ubiquitin-positive cytoplasmic inclusions was measured in the lumbar cord. As a result, the number of such cells in SOD1-Tg mice was significantly increased compared with normal mice in any quadruple staining, but it was significantly inhibited by administering human monoclonal antibody #129-L2 ( Fig.34 A to C). There are few studies on TDP43 aggregates in SOD1-Tg mice, and no conclusions have been drawn. However, in the pathology of patients with amyotrophic lateral sclerosis (ALS) associated with human SOD mutations, few clear TDP43-positive inclusion bodies have been observed (Reference 17), which is consistent with the above phenomenon.

[0347] Next, the neuronal necrosis known as TRIAD (Document 12) was studied. As mentioned above, it is known that TRIAD releases HMGB1 (Document 9, Document 10), and the released HMGB1 induces TRIAD necrosis of surrounding neuronal cells (Document 11). In addition, in other immunohistochemical staining methods of SOD1-Tg mice, changes in YAP have been reported (Document 18). On the other hand, as markers of TRIAD necrosis, the positivity of cell body pSer46-MARCKS (Document 9) and the reduction of YAP in the nucleus (Document 10) are known. Therefore, triple staining of YAP, pSer46-MARCKS, and DAPI was performed, and as a result, in the large neurons (motor neurons) of the anterior horn of the spinal cord of SOD1-Tg mice administered with physiological saline, TRIAD necrosis with pSer46-MARCKS positivity and reduced YAP in the nucleus increased ( Fig.35 A). In contrast, in SOD1-Tg mice administered with human monoclonal antibody #129-L2, the number of neurons that underwent TRIAD necrosis was observed to be suppressed ( Fig.35 A).

[0348] Furthermore, when the number of MAP2-positive neurons in the lumbar spinal cord was measured, the number of MAP2-positive neurons in the SOD1-Tg mice administered with saline was reduced compared with that in the normal mice. In contrast, the number of MAP2-positive neurons in the SOD1-Tg mice administered with human monoclonal antibody #129-L2 was significantly reduced compared with that in the SOD1-Tg mice administered with saline ( Fig.35 B). In addition, in SOD1-Tg mice, a tendency toward decreased MAP2 staining in neuropil was observed, but this was also restored in SOD1-Tg mice administered with human monoclonal antibody #129-L2 ( Fig.35 B) Regarding the number of microglia in the spinal cord, there was no significant difference between normal mice and SOD1-Tg mice in Iba1 staining, and there was no change in the number of microglia in SOD1-Tg mice caused by the administration of human monoclonal antibody #129-L2 ( Fig.35 C). On the other hand, in GFAP staining, the number of astrocytes increased significantly in SOD1-Tg mice, but was significantly suppressed by administration of human monoclonal antibody #129-L2 ( Fig.35 D).

[0349] In addition, in neurodegenerative diseases represented by Huntington's disease, as mentioned above, it is known that DNA damage of neurons increases (References 14, 15). Therefore, using 53BP1 and γH2AX as DNA damage markers, 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 in SOD1-Tg mice, DNA damage of neurons increased compared with normal mice, and that DNA damage of neurons was suppressed by administration of human monoclonal antibody #129-L2 ( Fig.36 ).

[0350] Next, in order to reconfirm the results of pathological analysis, biochemical analysis was performed on the brain tissue samples of 3 groups of mice in the 22-week-old ALS model mice. Specifically, when the protein levels of pSer46-MARCKS, 53BP1, γH2AX, Ub, and pSer396-Tau were compared by Western blotting, it was shown that all of the above degenerative disease marker molecules increased in SOD1-Tg mice compared with normal mice, and by administering human monoclonal antibody #129-L2, these increased degenerative disease marker molecules in SOD1-Tg mice were reduced to normal levels ( Fig.37 ).

[0351] The above results show that human monoclonal antibody #129-L2 has the effects of inhibiting the progression of amyotrophic lateral sclerosis (ALS) and cell death, and inhibiting the decline of motor function.

[0352] [Spinocerebellar ataxia model mouse]

[0353] For the spinocerebellar ataxia model mice, as described in the above "1. Materials and Methods", experiments were conducted using mutant ataxin-1 gene knock-in mice (Reference 4) after the onset of the disease, and intravenous administration ( Fig.38 A). From 8 to 18 weeks of age, 6 μg (equivalent to 0.2 mg / kg BW) of human monoclonal antibody #129 was administered every 2 weeks for a total of 6 times ( Fig.38 A) At 8 weeks of age, the motor function of mutant ataxin-1 knock-in mice was found to be impaired in the rotarod test ( Fig.38 B), indicating that spinocerebellar ataxia has developed. At 20 weeks of age after a total of 6 administrations, the rotarod retention time of mutant ataxin-1 knock-in mice was significantly reduced compared to normal mice, but by administering human monoclonal antibody #129, the rotarod retention time of mutant ataxin-1 knock-in mice was significantly improved ( Fig.38B). Similarly, in the footprint test at the 20-week-old time point, a decrease in stride was observed in the comparison between normal mice and mutant ataxin-1 gene knock-in mice administered with saline, but the stride of mutant ataxin-1 gene knock-in mice increased significantly by administration of human monoclonal antibody #129 ( Fig.38 C), improvement of symptoms was observed.

[0354] Next, at 20 weeks of age, brain tissues were sampled from three groups of mice (i.e., "3 groups for analysis of spinocerebellar ataxia model mice"), including normal mice administered with saline, mutant ataxin-1 knock-in mice administered with saline, and mutant ataxin-1 knock-in mice administered with human monoclonal antibody #129 after motor function analysis, and pathological analysis was performed. First, TRIAD, a cell death target of human monoclonal antibody #129, was studied. It is known that neuronal necrosis called TRIAD (Document 12) releases HMGB1 as described above (Document 9, Document 10), and the released HMGB1 induces TRIAD necrosis of peripheral neuronal cells (Document 11) or neuronal degeneration (Document 9). On the other hand, as TRIAD necrosis markers, pSer46-MARCKS positivity of cell bodies or neurites (Documents 9 to 11) and a reduction in nuclear YAP (Documents 10 to 13) are known. In addition, it is known that in the case of spinocerebellar ataxia type 1 (SCA1), the expression level of YAP also affects the prognosis of mutant ataxin-1 knock-in mice (Reference 19). Therefore, triple staining of YAP, pSer46-MARCKS, and DAPI was performed, and the results showed that in mutant ataxin-1 knock-in mice administered with saline, the pSer46-MARCKS staining of the granule cells and their nerve fibers in the granule cell layer of the cerebellar cortex was significantly increased compared with normal mice administered with saline ( Fig.39 A). This is similar to the increase in pSer46-MARCKS staining observed in Alzheimer's disease when neurite degeneration is caused by HMGB1 release (Reference 9), indicating neurite degeneration in the granule cell layer. On the other hand, in mutant ataxin-1 knock-in mice administered with human monoclonal antibody #129, no such increase in pSer46-MARCKS staining was observed in granule cells and their nerve fibers, and it was normalized ( Fig.39 A).

[0355] Regarding inclusion bodies, as shown in a previous report (Reference 4), in mutant ataxin-1 gene knock-in mice, no significant nuclear inclusion bodies were observed in Purkinje cells, but colocalization of high signal Ub and Atxn1 was observed ( Fig.39 B) This phenomenon was not observed in normal mice, and it disappeared in mutant ataxin-1 gene knock-in mice by administering human monoclonal antibody #129 ( Fig.39 B).

[0356] In addition, it is known that DNA damage of nerve cells increases in neurodegenerative diseases represented by SCA1 (References 14, 15). Therefore, using 53BP1 and γH2AX as DNA damage markers, DNA damage of Purkinje cells was measured by quadruple staining with MAP2 and DAPI. The results showed that in mutant ataxin-1 gene knock-in mice, DNA damage of Purkinje cells increased compared with normal mice, and DNA damage of Purkinje cells was suppressed by administration of human monoclonal antibody #129 ( Fig.39 C).

[0357] Furthermore, in KDEL staining performed to detect endoplasmic reticulum abnormalities that are characteristic of TRIAD, KDEL signal values ​​in the Purkinje cell layer and granule cell layer were increased in mutant ataxin-1 gene knock-in mice compared to normal mice, confirming the endoplasmic reticulum instability observed in TRIAD ( Fig.40 A). MAP2, Iba1, and GFAP staining were used to confirm whether this phenomenon ultimately affects the number of Purkinje cells or the number of microglia and astrocytes in the cerebellar cortex ( Fig.40 B). As a result, in mutant ataxin-1 gene knock-in mice, the number of Purkinje cells decreased compared to normal mice, but cell shedding was inhibited by administration of human monoclonal antibody #129 ( Fig.40 B). On the other hand, microglia and astrocytes did not change ( Fig.40 B).

[0358] Next, in order to reconfirm the results of the pathological analysis, biochemical analysis was performed on the brain tissue samples of 3 groups of 20-week-old spinocerebellar ataxia model mice. Specifically, when the protein levels of pSer46-MARCKS, 53BP1, γH2AX, Ub, and pSer203-Tau were compared by Western blotting, it was shown that in mutant ataxin-1 gene knock-in mice, all of the above degenerative disease marker molecules increased compared to normal mice, and by administering human monoclonal antibody #129, these increased degenerative disease marker molecules in mutant ataxin-1 gene knock-in mice were reduced to normal levels ( Fig.41 ).

[0359] The above results show that human monoclonal antibody #129 has the effect of inhibiting the progression of disease and cell death in spinocerebellar ataxia and suppressing the decline of motor function.

[0360] [Aging model mouse]

[0361] As for the aging model mice, as described in the above "1. Materials and Methods", 12-month-old C57BL / 6 mice were used for the experiment, and intravenous administration was performed once every 4 weeks for a total of 8 times from 12 to 19 months of age ( Fig.42 A). From 12 months to 18 months of age, there was no significant difference in the Y-maze test between 12-month-old C57BL / 6 mice administered with saline and 12-month-old C57BL / 6 mice administered with human monoclonal antibody #129-L2. However, for 12-month-old C57BL / 6 mice administered with saline, a decrease in the Y-maze test score was observed at 19 months of age, and a significant difference was confirmed thereafter ( Fig.42 B).

[0362] Next, brain tissue was sampled from 3 groups of mice (i.e., "3 groups for analysis of 20-month-old aging model mice") after motor function analysis (3-month-old C57BL / 6 mice), 20-month-old C57BL / 6 mice given saline, and 20-month-old C57BL / 6 mice given human monoclonal antibody #129-L2, and pathological analysis was performed. Lamin B1 is a nuclear membrane supporting protein, and it is reported that it shows a nuclear ring staining pattern in immunohistochemical staining, but when the cell ages, the nuclear ring regionalization disappears (Document 20). In addition, in neurons with TRIAD necrosis, the nuclear ring regionalization of lamin B1 also disappears (Document 11), so there is a hypothesis that TRIAD is cell death in normal aging (Document 11). Therefore, we used three groups of mouse brain tissues from 20-month-old aging model mice for analysis, and performed triple immunostaining of two TRIAD markers (YAP and pSer46MARCKS) and lamin B1 to study whether TRIAD necrosis increases in normal aging and the effect of human monoclonal antibody #129-L2 on neuronal cell death in aging.

[0363] As a result, in the cerebral cortex of 20-month-old C57BL / 6 mice administered with saline, TRIAD necrosis, which showed disappearance of YAP nuclear staining and significant increase in pSer46-MARCKS cytoplasmic staining, was observed compared with 3-month-old C57BL / 6 mice ( Fig.43 ), and the number of aged neurons with disappearance of lamin B1 nuclear ring staining increased significantly ( Fig.43 ). In addition, the number of aged neurons that underwent TRIAD necrosis, which showed the three characteristics of disappearance of YAP nuclear staining, pSer46-MARCKS cytoplasmic staining, and disappearance of lamin B1 nuclear ring staining, also increased significantly ( Fig.43 On the other hand, in aged mice (20 months old) administered with human monoclonal antibody #129-L2, the proportion of cells showing these three characteristics was significantly suppressed ( Fig.43 ).

[0364] DNA damage in cerebral cortical neurons was measured by quadruple staining with MAP2 and DAPI using 53BP1 and γH2AX, which are DNA damage markers. The results showed that DNA damage in neurons increased in aged mice compared to young mice, and that DNA damage in cerebral cortical neurons in aged mice was suppressed by administration of human monoclonal antibody #129-L2 ( Fig.44 A). MAP2, Iba1, and GFAP staining were used to confirm whether this phenomenon ultimately affects the number of neurons, microglia, and astrocytes in the cerebral cortex ( Fig.44B). As a result, in 20-month-old C57BL / 6 mice administered with saline, the number of nerve cells decreased compared to young mice (3-month-old C57BL / 6 mice), but cell shedding was suppressed by administration of human monoclonal antibody #129-L2 ( Fig.44 B) On the other hand, no changes were observed in the number of microglia and astrocytes ( Fig.44 C and D).

[0365] Next, in order to reconfirm the results of pathological analysis, biochemical analysis was performed on the mouse brain tissue samples of the three groups of 20-month-old aging model mice. Specifically, when the protein levels of pSer46-MARCKS, 53BP1, γH2AX, Ub, and pSer203-Tau were compared by Western blotting, it was shown that all of the above degenerative disease marker molecules increased in old mice (20 months old) compared with young mice (3 months old), and by administering human monoclonal antibody #129-L2, these increased degenerative disease marker molecules in old mice were reduced to normal levels ( Fig.45 ).

[0366] The above results show that human monoclonal antibody #129-L2 has the effect of inhibiting changes in cell characteristics and cell death associated with aging and inhibiting the decline of cognitive function.

[0367] 3. Summary

[0368] The human monoclonal antibody of the present application was shown to have therapeutic effects on Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, spinocerebellar ataxia, and changes in cell characteristics, cell death, cognitive function (memory), and motor function associated with aging.

[0369] Document 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. Exon1 of the HD gene with an expanded CAG repeat is sufficient to cause aprogressive neurological phenotype in transgenic mice.Cell.87:493-506.

[0370] Literature 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. 2002 May 16;34(4):521-33.

[0371] Literature 3: Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, Caliendo J, Hentati A, Kwon YW, Deng HX, et al., Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science. 1994 264(5166):1772-1775. Erratum in: Science 1995 269(5221):149.

[0372] Literature 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.

[0373] Literature 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. 2009 Nov 15;18(22):4239-54.

[0374] Literature 6: Ito H, Shiwaku H, Yoshida C, Homma H, Luo H, Chen X, Fujita K, MusanteL, Fischer U, Frints SG, Romano C, Ikeuchi Y, Shimamura T, Imoto S, Miyano S, Muramatsu SI, Kawauchi T, Hoshino M, Sudol M, Arumughan A, Wanker EE, Rich T, Schwartz C, Matsuzaki F, Bonni A, Kalscheuer VM, Okazawa H. In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells. Mol Psychiatry. 2015 Apr;20(4):459-71.

[0375] Literature 7: Taniguchi JB, Kondo K, Fujita K, Chen X, Homma H, Sudo T, Mao Y, Watase K, Tanaka T, Tagawa K, Tamura T, Muramatsu SI, Okazawa H. RpA1 ameliorates symptoms of mutant ataxin-1 knock-in mice and enhances DNA damage repair. Hum Mol Genet. 2016 Oct 15;25(20):4432-4447.

[0376] Reference 8: Keiser, M.S., Boudreau, R.L. and Davidson, B.L. (2014) Broad Therapeutic Benefit After RNAi Expression Vector Delivery to Deep Cerebellar Nuclei: Implications for Spinocerebellar Ataxia Type 1 Therapy. Mol. Ther., 22, 588 - 595.

[0377] Reference 9: Fujita K, Motoki K, Tagawa K, Chen X, Hama H, Nakajima K, Homma H, Tamura T, Watanabe H, Katsuno M, Matsumi C, Kajikawa M, Saito T, Saido T, Sobue G, Miyawaki A, Okazawa H. HMGB1, a pathogenic molecule that induces neurite degeneration via TLR4 - MARCKS, is a potential therapeutic target for Alzheimer's disease. Sci Rep. 2016 Aug 25; 6: 31895.

[0378] Reference 10: Tanaka H, Homma H, Fujita K, Kondo K, Yamada S, Jin X, Waragai M, Ohtomo G, Iwata A, Tagawa K, Atsuta N, Katsuno M, Tomita N, Furukawa K, Saito Y, Saito T, Ichise A, Shibata S, Arai H, Saido T, Sudol M, Muramatsu SI, Okano H, Mufson EJ, Sobue G, Murayama S, Okazawa H. YAP - dependent necrosis occurs in early stages of Alzheimer's disease and regulates mouse model pathology. Nat Commun. 2020 Jan 24; 11(1): 507.

[0379] Figure 11:Tanaka H,Kondo K,Fujita K,Homma H,Tagawa K,Jin X,Jin M,Yoshioka Y,Takayama S,Masuda H,Tokuyama R,Nakazaki Y,Saito T,Saido T,MurayamaS,Ikura T,Ito N,Yamamori Y,Tomii K,Bianchi ME,Okazawa H.HMGB1 signaling phosphorylates Ku70 and impairs DNA damage repair in Alzheimer's disease pathology.Commun Biol.2021Oct 11:4(1):1

[0380] Figure 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 slowly progressive atypical neuronal death associated with changes in YAPisoforms and p73.J Cell Biol.2006Feb 13:172(4):589-604.

[0381] Figure 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.TargetingTEAD / YAP-transcription-dependent necrosis,TRIAD,ameliorates Huntington'sdisease pathology.Hum Mol Genet.2016Nov 125(21):4749-4770.

[0382] Reference 14: Qi ML, Tagawa K, Enokido Y, Yoshimura N, Wada Y, Watase K, Ishiura S, Kanazawa I, Botas J, Saitoe M, Wanker EE, Okazawa H. Proteome analysis of soluble nuclear proteins reveals that HMGB1 / 2 suppress genotoxic stress in polyglutamine diseases. Nat Cell Biol. 9(4): 402-14, 2007.

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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. Developmental YAPdeltaC determines adult pathology in a model of spinocerebellar ataxia type 1. Nat Commun. 2017 Nov 30;8(1):1864.

[0388] Reference 20: Freund A, Laberge RM, Demaria M, Campisi J. Lamin B1 loss is a senescence - associated biomarker. Mol Biol Cell. 2012 Jun;23(11):2066 - 75.

[0389] [Example 4]

[0390] As mentioned above, in the light chain CDR3 of human monoclonal antibody #129, it is believed that there is an amino acid sequence with a high possibility of accepting sugar chain modification, and two amino acid sequence modified forms of light chain CDR3 (human monoclonal antibody #129-L1 and human monoclonal antibody #129-L2) were prepared according to conventional methods (Table 10).

[0391] [Table 10]

[0392] Amino acid sequence of light chain CDR3 Human monoclonal antibody #129 LQHNSTPLT Human monoclonal antibody #129-L1 LQHNANPLT Human monoclonal antibody #129-L2 LQHASTPLT

[0393] The underlined amino acid sequences in the table indicate sites that have been altered relative to the amino acid sequence of the light chain CDR3 of human monoclonal antibody #129.

[0394] The antibody affinity of human monoclonal antibody #129 and human monoclonal antibody #129-L2 for human dsHMGB1 was analyzed by SPR described in Example 1. As a result, the dissociation constants (K D ) are 1.47×10 -11 and 1.44×10 -11 , showing that the two antibodies have essentially the same binding affinity to human dsHMGB1 ( Fig.46 B and A). In addition, the dissociation constants (K D ) were 2.07×10 -7 and 1.51×10 -5 , showing that compared with human monoclonal antibody #129, human monoclonal antibody #129-L2 has weaker binding ( Fig.46 D and C).

[0395] In addition, the dissociation constant (K D ) were not found to have any difference in binding ability to human dsHMGB1 between human monoclonal antibody #129 and human monoclonal antibody #129-L2 ( Fig.47 B and A). In addition, human monoclonal antibody #129 and human monoclonal antibody #129-L2 both had weak binding to human reduced HMGB1. In this case, no difference was observed in the binding ability of the two antibodies ( Fig.47 D and C).

[0396] Therefore, human monoclonal antibody #129 and human monoclonal antibody #129-L2 were used to study the effects of the Y-maze test described in Example 1 on the three FTLD-ALS model mice (TDP43 N267S -KI mice, PGRN R504X -KI mice, and VCPT262A -KI mice) were used to evaluate the therapeutic effect. Fig.48 The treatment experiment shown in the scheme is to administer physiological saline containing 0.2 mg / kg body weight (6 μg / mouse) of human monoclonal antibody #129 or human monoclonal antibody #129-L2 once every month. In addition, as a control group, various FTLD-ALS model mice or normal mice (brother mice without mutant genes of various FTLD-ALS model mice) are similarly administered with the same amount of physiological saline. It should be noted that administration begins after the onset of the disease is confirmed in the Y maze test.

[0397] As a result, in all FTLD-ALS model mice, improvement in cognitive function in the Y-maze test was observed from the first month after the administration of the two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 or human monoclonal antibody #129). Fig.48 ). Human monoclonal antibody #129 showed excellent therapeutic effects at several time points, but overall, no substantial difference in therapeutic effects was observed between human monoclonal antibody #129 and human monoclonal antibody #129-L2, and both observed statistically significant therapeutic effects ( Fig.48 ).

[0398] In addition, the two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129-L2 or human monoclonal antibody #129) were administered once a month twice more, and then the pathological analysis of brain tissue was performed according to the method described in Example 3. In the immunohistochemical staining method, pSer46-MARCKS ( Fig.49 ) and KDEL( Fig.50 ) staining to study TRIAD necrosis, and γH2AX ( Fig.51 ) and 53BP1( Fig.52 ) staining to study DNA damage, and pSer409 / 410-TDP43 ( Fig.53 ) were used to study inclusion bodies. As a result, overall, no substantial difference in therapeutic effect was observed between human monoclonal antibody #129 and human monoclonal antibody #129-L2, and statistically significant therapeutic effects were observed for both ( Figures 49 to 53 ).

[0399] On the other hand, differences were observed between human monoclonal antibody #129 and human monoclonal antibody #129-L2 in the following aspects. Specifically, human monoclonal antibody #129 was purified from multiple clones of a CHO cell line that stably expressed human monoclonal antibody #129 according to conventional methods, and analyzed by non-reducing capillary electrophoresis according to conventional methods. As a result, Fig.54 As shown in the figure, in all clones, a gentle peak was observed at a higher molecular weight side than the IgG peak (the position indicated by the arrow in the figure). On the other hand, human monoclonal antibody #129-L2 was purified from multiple clones of a CHO cell line stably expressing human monoclonal antibody #129-L2 and analyzed by non-reducing capillary electrophoresis. As shown in the figure, Fig.55 As shown, no gentle peak was observed on the high molecular weight side in any clone.

[0400] This result shows that human monoclonal antibody #129 is modified with N-glycan sugar chains, whereas human monoclonal antibody #129-L2 is not modified with N-glycan sugar chains.

[0401] In addition, using 5 μg of the three anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129, human monoclonal antibody #129-L1, and human monoclonal antibody #129-L2), SDS-PAGE was performed under normal reducing conditions, and the acrylamide gel was stained with Coomassie Brilliant Blue (CBB). As a result, a light band was observed above the band derived from the light chain of human monoclonal antibody #129, while no such band was observed above the band derived from the light chain of human monoclonal antibody #129-L1 or above the band derived from the light chain of human monoclonal antibody #129-L2 ( Fig.56 ).

[0402] This result shows that the light chain of human monoclonal antibody #129 is modified with N-glycans, whereas the light chains of human monoclonal antibody #129-L1 and human monoclonal antibody #129-L2 are not modified with N-glycans.

[0403] In addition, in order to study the stability of the two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129 and human monoclonal antibody #129-L2), the antibody affinity of the two anti-human HMGB1 antibodies of the present invention for HMGB1 was analyzed by SPR before and after storage at 4°C for 6 months. D For example, the value of 1.47×10 -11 ( Fig.57 C), whereas after 6 months storage at 4°C, the -10 ( Fig.57 D), increased by about 45 times. On the other hand, the K D For example, the value of 1.44×10 -11 (4℃)( Fig.57 A), on the other hand, after 6 months storage at 4°C, the-11 ( Fig.57 B), and stopped at a rise of about 2.5 times.

[0404] This result shows that compared with human monoclonal antibody #129, human monoclonal antibody #129-L2 has higher antibody stability.

[0405] In addition, two anti-human HMGB1 antibodies of the present invention (human monoclonal antibody #129 and human monoclonal antibody #129-L2) were used to study the therapeutic effect on Alzheimer's disease model mice (5×FAD mice). Specifically, a physiological saline solution containing 0.2 mg / kg body weight (6 μg / mouse) of human monoclonal antibody #129 or human monoclonal antibody #129-L2 was administered once a month for a total of 3 times (6 μg / mouse) to one Alzheimer's disease model mouse by intravenous injection from the tail vein. Fig.58 A). In addition, as a control group, physiological saline containing 0.2 mg / kg body weight (6 μg / head) of human IgG was administered in the same manner ( Fig.58 A). After administration, the number of synapses in the cerebral cortex was measured using two-photon microscopy ( Fig.58 B) Cognitive function test based on Y-maze test ( Fig.58 C), and immunohistochemical staining of mouse cerebral cortex using anti-pSer46-MARCKS antibody and anti-Aβ antibody to evaluate cell death and extracellular Aβ deposition ( Fig.59 ).

[0406] The results showed that human monoclonal antibody #129 and human monoclonal antibody #129-L2 equally normalized synaptic reduction in 8-month-old 5×FAD mice ( Fig.58 B), equally normalize cognitive decline ( Fig.58 C). It is also reported that in the pathological analysis of Alzheimer's disease, there are three stages: TRIAD necrosis of a single neuron due to intracellular amyloid accumulation (active TRIAD necrosis), TRIAD necrosis of a large number of surrounding neurons induced by HMGB1 released from active TRIAD necrosis (secondary TRIAD necrosis), and extracellular Aβ deposition (amyloid plaques) in which the cell debris of secondary TRIAD necrosis is removed ( Fig.59 A) Occurs sequentially in time (Reference "Tanaka et al., Nature Commun 2020"). It shows that human monoclonal antibody #129 and human monoclonal antibody #129-L2 do not affect active TRIAD necrosis, but significantly inhibit secondary TRIAD necrosis and amyloid plaques, and the effects of the two are equivalent ( Fig.59 B).

[0407] Industrial Applicability

[0408] The present invention contributes to the prevention and / or treatment of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, Alzheimer's disease, frontotemporal lobar degeneration, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases associated with brain aging.

Claims

1. A preventive or therapeutic agent for amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, degenerative diseases or neurological diseases associated with aging, brain aging, or diseases associated with brain aging, the preventive or therapeutic agent comprising a human monoclonal antibody that specifically binds to human HMGB1 and comprising: A heavy chain complementary determining region (CDR) 1 comprising 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 into the amino acid sequence; a heavy chain CDR2 comprising 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 into the amino acid sequence; and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; and A light chain CDR1 comprising the amino acid sequence shown in sequence number 4 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; a light chain CDR2 comprising the amino acid sequence shown in sequence number 5 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence; and a light chain CDR3 comprising the amino acid sequence shown in sequence number 6, sequence number 20, or sequence number 17 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence.

2. The preventive or therapeutic agent according to claim 1, wherein The human monoclonal antibody comprises: a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in sequence number 7; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in sequence number 8, sequence number 21, or sequence number 18.

3. The preventive or therapeutic agent according to claim 1 or 2, wherein The human monoclonal antibody comprises: a heavy chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in sequence number 9; and a light chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in sequence number 10, sequence number 22, or sequence number 19. The preventive or therapeutic agent according to claim 1 or 2, which is administered intravenously.

5. A human monoclonal antibody that specifically binds to human HMGB1 and comprises: A heavy chain complementary determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO:

17.

6. The human monoclonal antibody of claim 5, comprising: a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO:

18.

7. The human monoclonal antibody of claim 5, comprising: a heavy chain comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; and a light chain comprising 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. 8 . A preventive or therapeutic agent for Alzheimer's disease or frontotemporal lobar degeneration, comprising the human monoclonal antibody according to claim 5 .

9. The preventive or therapeutic agent according to claim 8, which is administered intravenously.

10. A preventive or therapeutic agent for amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, aging-related degenerative diseases or neurological diseases, brain aging, or diseases associated with brain aging, comprising the human monoclonal antibody according to any one of claims 5 to 7. The preventive or therapeutic agent according to claim 10 , which is administered intravenously.

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