Prophylactic or therapeutic agent for frontotemporal lobar degeneration

Abstract

The purpose of the present invention is to provide a preventative agent or a treatment agent for frontotemporal lobar degeneration, the agent exhibiting, without involving noticeable side effects, excellent preventive / treatment effects with respect to frontotemporal lobar degeneration occurring due to various causes.　In the present invention, a human monoclonal antibody that binds specifically to human HMGB1 can be used as a preventive agent or a treatment agent for frontotemporal lobar degeneration, the human monoclonal antibody including: a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 that comprise specific amino acid sequences; and a light chain CDR1, a light chain CDR2, and a light chain CDR3 that comprise specific amino acid sequences.

Description

[Technical Field]

[0001] The present invention relates to a formulation for preventing or treating frontotemporal lobar degeneration (FTLD). [Background technology]

[0002] Frontotemporal dementia (FILMS) is the second or third most common early-onset neurodegenerative dementia after Alzheimer's disease and is thought to be highly associated with motor neuron disease. FILMS presents with pronounced behavioral and personality changes, often accompanied by language impairment, which gradually progresses to cognitive impairment and dementia. Known causative genes for familial FILMS include VCP (Valosin-Containing Protein), PGRN (progranulin), CHMP2B (Charged Multivesicular Body Protein 2B), and TDP43 (Transactive Response DNA Binding Protein of 43 kD). Although research into FILMS is progressing, the complete mechanism of its onset remains unclear, similar to Alzheimer's disease.

[0003] Furthermore, research is also being conducted on therapeutic drugs for frontotemporal lobar degeneration. For example, it has been reported that inhibitors of the tyrosine kinase Src / c-Abl have the effect of improving the survival rate of cortical neurons derived from patients with frontotemporal lobar degeneration (Patent Document 1). In addition, the present inventors have developed an FTLD model mouse (PGRN) that has a stop codon at arginine (R) position 504 of PGRN. R504X When a serine / threonine kinase (b-RAF) inhibitor (vemurafenib) is administered to KI mice, PGRN R504X - It has been reported that behavioral abnormalities observed in KI mice are improved (Patent Document 2). However, at present, there is no cure for frontotemporal dementia, and unlike Alzheimer's disease, there are currently not enough therapeutic drugs to improve the symptoms of frontotemporal dementia.

[0004] On the other hand, the HMGB1 (High Mobility Group Box 1) protein is known as one of the non-histone chromatin-related proteins involved in DNA structure maintenance and transcriptional regulation. Recently, it has attracted attention that HMGB1 functions not only in the nucleus but also as a so-called DAMP (Damage-Associated Molecular Pattern), being released extracellularly due to cell necrosis or actively secreted extracellularly in response to vascular inflammatory signaling. The inventors of this invention have found that in Alzheimer's disease, HMGB1 leaked from cells due to neuronal necrosis induces phosphorylation of serine 46 (Ser46) of MARCKS, a substrate of phosphorylation enzymes, thereby inducing neurite degeneration. Based on this, they have produced a mouse monoclonal antibody against HMGB1 and found that this mouse monoclonal antibody inhibits the phosphorylation of Ser46 of MARCKS (Non-Patent Document 1). Furthermore, the inventors of this invention have reported that such mouse monoclonal antibodies and human monoclonal antibodies restore recognition impairment in Alzheimer's disease model mice (Patent Documents 3 and 4). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication 2018 / 216705 Brochure [Patent Document 2] International Publication 2015 / 099094 Brochure [Patent Document 3] International Publication No. 2018 / 030405 Brochure [Patent Document 4] International Publication No. 2020 / 059847 Brochure [Non-patent literature]

[0006] [Non-Patent Document 1] SCIENTIFIC REPORT, 2016 Aug 25;6:31895 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a preventive or therapeutic agent for frontotemporal lobar degeneration that exhibits excellent preventive and therapeutic effects against frontotemporal lobar degeneration caused by different factors, without significant side effects. [Means for solving the problem]

[0008] The inventors are diligently continuing their research to solve the above problems. In the process, they have developed FTLD model mice with four different causative genes, specifically mice in which the 262nd threonine (T) of VCP is replaced with alanine (A) (VCP T262A -KI mouse); A mouse with a stop codon at arginine(R) position 504 of PGRN (PGRN R504X -KI mouse); A mouse with a stop codon at glutamine (Q) position 165 of CHMP2B (CHMP2B Q165X -KI mice); and mice in which the 267th asparagine (N) of TDP43 is replaced with serine (S) (TDP43 N267S When human monoclonal antibody #129, prepared according to the examples in International Publication No. 2020 / 059847 (Patent Document 4), was administered to KI mice, it was found that cognitive decline and symptoms related to nerve damage caused by FTLD could be effectively prevented and improved in all FTLD model mice without significant side effects, thus completing the present invention.

[0009] In other words, the present invention is as follows. [1] Specifically binds to human HMGB1, and heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in said amino acid sequence; heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in said amino acid sequence; and heavy chain CDR3 consisting of 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 in said amino acid sequence; and light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in said amino acid sequence; light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in said amino acid sequence; and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in said amino acid sequence A prophylactic or therapeutic agent for frontotemporal lobar degeneration, comprising a human monoclonal antibody. [2] The prophylactic or therapeutic agent according to the above [1], wherein the human monoclonal antibody comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8. [3] The prophylactic or therapeutic agent according to the above [1] or [2], wherein the human monoclonal antibody comprises a heavy chain consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and a light chain consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10. [4] The prophylactic or therapeutic agent according to any one of the above [1] to [3], which is administered intravenously.

[0010] Moreover, as another embodiment of the present invention, It specifically binds to human HMGB1, and Heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in said amino acid sequence; heavy chain CDR 2 consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in said amino acid sequence; and heavy chain CDR 3 consisting of 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 in said amino acid sequence; Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted. A method for preventing or treating frontotemporal lobar degeneration, comprising the step of administering a human monoclonal antibody (hereinafter sometimes referred to as "the human monoclonal antibody") to a subject in need of prevention or treatment of frontotemporal lobar degeneration; or, The human monoclonal antibody in question, for use as a preventive or therapeutic agent for frontotemporal lobar degeneration; The human monoclonal antibody in question for use in the prevention or treatment of frontotemporal lobar degeneration; and, Use of the human monoclonal antibody in question for the manufacture of a preventive or therapeutic agent for frontotemporal lobar degeneration; One could list these: [Effects of the Invention]

[0011] Using this human monoclonal antibody, a different causative gene, specifically VCP, can be detected. T262A PGRN R504X CHMP2BQ165X , and TDP43 N267S can effectively prevent or treat the symptoms of frontotemporal lobar degeneration caused by the above without significant side effects. Therefore, the prophylactic or therapeutic agent for frontotemporal lobar degeneration containing the present human monoclonal antibody as an active ingredient is sufficiently expected to exert excellent prophylactic and therapeutic effects on frontotemporal lobar degeneration of various causes. [Brief Description of Drawings]

[0012] [Figure 1] This figure shows the results of analyzing the interaction between two types of anti-human HMGB1 antibodies (the human monoclonal antibody #129 of the present invention [Figure 1A], and the mouse monoclonal antibody 2C8C [Figure 1B]) and human HMGB1 protein by the surface plasmon resonance (SPR) method. [Figure 2-1] This figure shows the results of analyzing the pSer46-MARCKS levels in the cerebral cortex of four types of FTLD model mice (VCPT262A-KI mice [Figure 2-1A], PGRNR504X-KI mice [Figure 2-1B], CHMP2BQ165X-KI mice [Figure 2-1C], and TDP43N267S-KI mice [Figure 2-1D]) by an immunohistochemical staining method. When the pSer46-MARCKS levels in C57BL / 6J mice were similarly quantified, all were 0 (zero). [Figure 2-2] This figure shows the results of analyzing the brain tissues of two types of FTLD model mice (VCPT262A-KI mice [Figure 2-2A], and PGRNR504X-KI mice [Figure 2-2B]) by the Nissl staining method. The arrows in the left image indicate vacuoles. The right image is a high-magnification image of the region indicated by the black arrow in the left image. [Figure 3]Figure 3A shows the results of the Morris Water-maze test performed on TDP43N267S-KI mice (labeled "KI Ab" [n=12] in the figure) or C57BL / 6J mice (labeled "B6 Ab" [n=8] in the figure) administered with the human monoclonal antibody #129 of the present invention, TDP43N267S-KI mice (labeled "KI IgG" [n=11] in the figure) or C57BL / 6J mice (labeled "B6 IgG" [n=6] in the figure) administered with control human IgG, or untreated TDP43N267S-KI mice (labeled "KI nt" [n=10] in the figure) or C57BL / 6J mice (labeled "B6 nt" [n=6] in the figure) for 1 to 5 days starting at 10 months of age. Figure 3B shows the results of the Morris water maze test conducted for 1 to 5 days starting at 7 months of age on PGRNR504X-KI mice (labeled "KI Ab" in the figure [n=11]) or C57BL / 6J mice (labeled "B6 Ab" in the figure [n=6]) administered with the human monoclonal antibody #129 of the present invention, PGRNR504X-KI mice (labeled "KI IgG" in the figure [n=7]) or C57BL / 6J mice (labeled "B6 IgG" in the figure [n=7]) administered with control human IgG, or untreated PGRNR504X-KI mice (labeled "KI nt" in the figure [n=11]) or C57BL / 6J mice (labeled "B6 nt" in the figure [n=7]). Figure 3C shows the results of the Morris water maze test conducted for 1 to 5 days starting at 10 months of age on VCPT262A-KI mice (labeled "KI Ab" [n=13] in the figure) or C57BL / 6J mice (labeled "B6 Ab" [n=6] in the figure) administered with the human monoclonal antibody #129 of the present invention, VCPT262A-KI mice (labeled "KI IgG" [n=5] in the figure) or C57BL / 6J mice (labeled "B6 IgG" [n=7] in the figure) administered with control human IgG, or VCPT262A-KI mice (labeled "KI nt" [n=7] in the figure) or C57BL / 6J mice (labeled "B6 nt" [n=6] in the figure) that were not administered the antibody.Figure 3D shows the results of the Morris water maze test conducted for 1 to 5 days starting at 6 months of age on CHMP2BQ165X-KI mice (labeled "KI Ab" in the figure [n=7]) or C57BL / 6J mice (labeled "B6 Ab" in the figure [n=6]) administered with the human monoclonal antibody #129 of the present invention, CHMP2BQ165X-KI mice (labeled "KI IgG" in the figure [n=5]) or C57BL / 6J mice (labeled "B6 IgG" in the figure [n=7]) administered with control human IgG, or CHMP2BQ165X-KI mice (labeled "KI nt" in the figure [n=5]) or C57BL / 6J mice (labeled "B6 nt" in the figure [n=6]) that were not administered the antibody. In the figure, "*", "**", and "***" indicate that "KI nt" is statistically significant compared to "B6 nt" according to Tukey's HSD test (p<0.05, p<0.01, and p<0.001, respectively). Also, in the figure, "#", "##", and "###" indicate that "KI Ab" is statistically significant compared to "KI nt" according to Tukey's HSD test (p<0.05, p<0.01, and p<0.001, respectively). [Figure 4]TDP43N267S-KI mice administered with the human monoclonal antibody #129 of the present invention (labeled "KI Ab" in the figure [n=12 (Figures 4A-C); n=9 (Figure 4D)]) or C57BL / 6J mice (labeled "B6 Ab" in the figure [n=8 (Figures 4A-D)]), TDP43N267S-KI mice administered with control human IgG (labeled "KI IgG" in the figure [n=9 (Figures 4A-C); n=6 (Figure 4D)]) or C57BL / 6J mice (labeled "B6 IgG" in the figure [n=7 (Figures 4A-D)]), or untreated TDP43N267S-KI mice (labeled "KI nt" in the figure [n=6 (Figure 4A); n=10 (Figure 4B); n=9 (Figure 4C); n=7 (Figure 4D)]) or C57BL / 6J mice (labeled "B6 This figure shows the results of the Y-maze test performed on nt[n=6 (Figure 4A); n=12 (Figures 4B~D)]) at 7 months of age (Figure 4A), 8 months of age (Figure 4B), 9 months of age (Figure 4C), or 10 months of age (Figure 4D). The symbols "*", "**", 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) (the same applies to Figures 5 to 16 below). [Figure 5] PGRNR504X-KI mice administered with the human monoclonal antibody #129 of the present invention ("KI Ab" in the figure [n=15 (Figure 5A, B); n=14 (Figure 5C)]) or C57BL / 6J mice ("B6 Ab" in the figure [n=7 (Figure 5A~C)]), PGRNR504X-KI mice administered with control human IgG ("KI IgG" in the figure [n=10 (Figure 5A~C)]) or C57BL / 6J mice ("B6 IgG" in the figure [n=8 (Figure 5A~C)]), or untreated PGRNR504X-KI mice ("KI nt" in the figure [n=11 (Figure 5A, C); n=12 (Figure 5B)]) or C57BL / 6J mice ("B6 Ab" in the figure). This figure shows the results of the Y-maze test conducted on nt[n=7 (Figure 5A); n=11 (Figures 5B, C)]) at 5 months of age (Figure 5A), 6 months of age (Figure 5B), or 7 months of age (Figure 5C). [Figure 6]VCPT262A-KI mice administered with the human monoclonal antibody #129 of the present invention ("KI Ab" in the figure [n=15 (Figures 6A~C); n=12 (Figure 6D)]) or C57BL / 6J mice ("B6 Ab" in the figure [n=8 (Figures 6A~C); n=5 (Figure 6D)]), VCPT262A-KI mice administered with control human IgG ("KI IgG" in the figure [n=8 (Figure 6A); n=7 (Figures 6B, C); n=5 (Figure 6D)]) or C57BL / 6J mice ("B6 IgG" in the figure [n=7 (Figures 6A~C); n=6 (Figure 6D)]), or untreated VCPT262A-KI mice ("KI nt" in the figure [n=11 (Figures 6A~C); n=5 (Figure 6D)]) or C57BL / 6J mice ("B6 This figure shows the results of the Y-maze test performed on nt[n=17 (Figure 6A); n=16 (Figure 6B); n=13 (Figure 6C); n=12 (Figure 6D)]) at 7 months of age (Figure 6A), 8 months of age (Figure 6B), 9 months of age (Figure 6C), or 10 months of age (Figure 6D). [Figure 7] CHMP2BQ165X-KI mice (labeled "KI Ab" in the figure [n=8 (Figures 7A-C)]) or C57BL / 6J mice (labeled "B6 Ab" in the figure [n=8 (Figures 7A-C)]) administered with the human monoclonal antibody #129 of the present invention, CHMP2BQ165X-KI mice (labeled "KI IgG" in the figure [n=8 (Figures 7A-C)]) or C57BL / 6J mice (labeled "B6 IgG" in the figure [n=8 (Figures 7A-C)]) administered with control human IgG, or untreated CHMP2BQ165X-KI mice (labeled "KI nt" in the figure [n=8 (Figures 7A-C)]) or C57BL / 6J mice (labeled "B6 This figure shows the results of the Y-maze test performed on nt[n=12 (Figure 7A); n=10 (Figure 7B); n=10 (Figure 7C)]) at 4 months of age (Figure 7A), 5 months of age (Figure 7B), or 6 months of age (Figure 7C). [Figure 8]Figure 8A shows the results of a fear-conditioning test conducted at 10 months of age on TDP43N267S-KI mice (labeled "KI Ab" [n=12] in the figure) or C57BL / 6J mice (labeled "B6 Ab" [n=8] in the figure) administered with the human monoclonal antibody #129 of the present invention, TDP43N267S-KI mice (labeled "KI IgG" [n=9] in the figure) or C57BL / 6J mice (labeled "B6 IgG" [n=7] in the figure) administered with control human IgG, or untreated TDP43N267S-KI mice (labeled "KI nt" [n=9] in the figure) or C57BL / 6J mice (labeled "B6 nt" [n=7] in the figure). Figure 8B shows the results of fear conditioning tests conducted at 7 months of age on PGRNR504X-KI mice (labeled "KI Ab" [n=11] in the figure) or C57BL / 6J mice (labeled "B6 Ab" [n=6] in the figure) administered with the human monoclonal antibody #129 of the present invention, PGRNR504X-KI mice (labeled "KI IgG" [n=10] in the figure) or C57BL / 6J mice (labeled "B6 IgG" [n=7] in the figure) administered with control human IgG, or untreated PGRNR504X-KI mice (labeled "KI nt" [n=17] in the figure) or C57BL / 6J mice (labeled "B6 nt" [n=11] in the figure). Figure 8C shows the results of fear conditioning tests conducted at 10 months of age on VCPT262A-KI mice (labeled "KI Ab" [n=15] in the figure) or C57BL / 6J mice (labeled "B6 Ab" [n=4] in the figure) administered with the human monoclonal antibody #129 of the present invention, VCPT262A-KI mice (labeled "KI IgG" [n=5] in the figure) or C57BL / 6J mice (labeled "B6 IgG" [n=6] in the figure) administered with control human IgG, or VCPT262A-KI mice (labeled "KI nt" [n=9] in the figure) or C57BL / 6J mice (labeled "B6 nt" [n=6] in the figure) that were not administered the antibody.Figure 8D shows the results of fear conditioning tests conducted at 6 months of age on CHMP2BQ165X-KI mice (labeled "KI Ab" [n=7] in the figure) or C57BL / 6J mice (labeled "B6 Ab" [n=8] in the figure) administered with the human monoclonal antibody #129 of the present invention, CHMP2BQ165X-KI mice (labeled "KI IgG" [n=5] in the figure) or C57BL / 6J mice (labeled "B6 IgG" [n=6] in the figure) administered with control human IgG, or CHMP2BQ165X-KI mice (labeled "KI nt" [n=7] in the figure) or C57BL / 6J mice (labeled "B6 nt" [n=8] in the figure) that were not administered the antibody. [Figure 9] Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 9A], TDP43N267S-KI mouse [Figure 9B], VCPT262A-KI mouse [Figure 9C], and CHMP2BQ165X-KI mouse [Figure 9D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of immunohistochemical staining analysis of pSer46-MARCKS levels in the cerebral cortex of 6-month-old (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7-month-old (C57BL / 6J mice and PGRNR504X-KI mice), and 10-month-old (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice) mice for each of the following: [Figure 10]Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 10A], TDP43N267S-KI mouse [Figure 10B], VCPT262A-KI mouse [Figure 10C], and CHMP2BQ165X-KI mouse [Figure 10D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of analyzing the KDEL fluorescence level in the cerebral cortex by immunohistochemical staining for each of the following mice: 6 months old (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7 months old (C57BL / 6J mice and PGRNR504X-KI mice), and 10 months old (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice). [Figure 11] Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 11A], TDP43N267S-KI mouse [Figure 11B], VCPT262A-KI mouse [Figure 11C], and CHMP2BQ165X-KI mouse [Figure 11D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of immunohistochemical staining analysis of γH2AX fluorescence levels in the cerebral cortex for each of the following mice: 6 months old (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7 months old (C57BL / 6J mice and PGRNR504X-KI mice), and 10 months old (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice). [Figure 12]Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 12A], TDP43N267S-KI mouse [Figure 12B], VCPT262A-KI mouse [Figure 12C], and CHMP2BQ165X-KI mouse [Figure 12D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of immunohistochemical staining analysis of 53BP1 fluorescence levels in the cerebral cortex for each of the following mice: 6 months old (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7 months old (C57BL / 6J mice and PGRNR504X-KI mice), and 10 months old (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice). [Figure 13] Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 13A], TDP43N267S-KI mouse [Figure 13B], VCPT262A-KI mouse [Figure 13C], and CHMP2BQ165X-KI mouse [Figure 13D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of immunohistochemical staining analysis of pSer409 / 410-TDP43 fluorescence levels in the cerebral cortex of 6-month-old (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7-month-old (C57BL / 6J mice and PGRNR504X-KI mice), and 10-month-old (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice) mice for each of the following: [Figure 14]Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 14A], TDP43N267S-KI mouse [Figure 14B], VCPT262A-KI mouse [Figure 14C], and CHMP2BQ165X-KI mouse [Figure 14D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of analyzing the p62 fluorescence level in the cerebral cortex by immunohistochemical staining for each of the following mice: 6 months old (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7 months old (C57BL / 6J mice and PGRNR504X-KI mice), and 10 months old (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice). [Figure 15] Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 15A], TDP43N267S-KI mouse [Figure 15B], VCPT262A-KI mouse [Figure 15C], and CHMP2BQ165X-KI mouse [Figure 15D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of an analysis of the percentage of cells in the cerebral cortex where VAMP2 fluorescence signals and PSD95 fluorescence signals co-localize, performed by immunohistochemical staining, at 6 months of age (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7 months of age (C57BL / 6J mice and PGRNR504X-KI mice), and 10 months of age (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice) for each of the following conditions: "nt"); [Figure 16]Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (PGRNR504X-KI mouse [Figure 16A], TDP43N267S-KI mouse [Figure 16B], VCPT262A-KI mouse [Figure 16C], and CHMP2BQ165X-KI mouse [Figure 16D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). This figure shows the results (n=3) of an immunohistochemical staining analysis of the percentage of cells in the cerebral cortex that colocalize pSer203-tau fluorescence signals and PSD95 fluorescence signals at 6 months of age (C57BL / 6J mice and CHMP2BQ165X-KI mice), 7 months of age (C57BL / 6J mice and PGRNR504X-KI mice), and 10 months of age (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-KI mice) for each of the following mice: [Figure 17]Four types of FTLD model mice administered with the human monoclonal antibody #129 of the present invention (TDP43N267S-KI mouse [Figure 17A], PGRNR504X-KI mouse [Figure 17B], VCPT262A-KI mouse [Figure 17C], and CHMP2BQ165X-KI mouse [Figure 17D]) (labeled "KI Ab" in the figure); the same four types of FTLD model mice administered with control human IgG (labeled "KI IgG" in the figure); the same four types of FTLD model mice that were not administered (labeled "KI nt" in the figure); or the unadministered C57BL / 6J mouse (labeled "B6" in the figure). Regarding "nt"); at 6 months of age (C57BL / 6J mice and CHMP2BQ165X-KI mice) / 7 months of age (C57BL / 6J mice and PGRNR504X-KI mice) / 10 months of age (C57BL / 6J mice, TDP43N267S-KI mice, and VCPT262A-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); phosphorylation of one synaptic instability-related protein (pSer203-tau) (lane (5)); and two synaptic instability-related proteins. This figure shows the results of Western blotting analysis of the expression levels of naps-related proteins (PSD95 and VAMP2) (lanes (6) and (7), respectively); the expression levels of two DNA damage-related proteins (γH2AX and 53BP1) (lanes (8) and (9), respectively); the phosphorylation level of one cell death-related protein (pSer46-MARCKS) (lane (10)); the phosphorylation level of one DNA repair-related protein (pSer77-Ku70) (lane (11)); the level of one protein aggregation marker (pSer409 / 410-TDP43) (lane (12)); and the expression level of the internal standard protein, β-actin (lane (13)). [Modes for carrying out the invention]

[0013] The present invention provides a preventive or therapeutic agent for frontotemporal lobar degeneration, which is a preparation containing the human monoclonal antibody specified for the purpose of "preventing or treating frontotemporal lobar degeneration" (hereinafter sometimes referred to as "the preventive / therapeutic agent"). The preventive / therapeutic agent may be used alone as a pharmaceutical (formulation), or it may be mixed with additives and used in the form of a composition (pharmaceutical composition).

[0014] In this specification, "frontotemporal lobar degeneration (FTLD)" refers to a non-Alzheimer's disease type of neurodegenerative disorder that initially presents with atrophy of the frontal and temporal lobes of the human brain, and progresses to atrophy of the entire brain in the later stages. "Frontotemporal lobar degeneration" includes three clinically classified diseases: frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA), and semantic dementia (SD). Pathologically, it includes four diseases depending on the type of abnormal protein accumulated in the cells: FTLD-Tau, FTLD-TDP, FTLD-UPS, and FTLD-FUS. Furthermore, "frontotemporal lobar degeneration" also includes "familial frontotemporal lobar degeneration" and "hereditary frontotemporal lobar degeneration" caused by gene mutations, as well as "sporadic frontotemporal lobar degeneration" caused by environmental factors such as lifestyle and stress.

[0015] The above-mentioned "FTLD-Tau" is classified into "3R Tau" type, "4R Tau" type, and "3 / 4R Tau" type depending on the number of repeats in the microtubule-binding region of the tau protein that predominantly accumulates in cells. The "3R Tau" type includes FTLD (Pick's disease) with Pick globules, FTLD (FTLD-17) with MAPT (microtubule-associated protein tau) gene mutations, etc. The "4R Tau" type includes corticobasal degeneration, progressive supranuclear palsy, multiple system tauopathy with dementia, argyrophilic grain dementia (argyrophilic grain disease), FTLD (FTLD-17) with MAPT gene mutations, etc. The "3 / 4R Tau" type includes neurofibrillary tangle dementia, FTLD (FTLD-17) with MAPT gene mutations, etc. On the other hand, the FTLD group that is tau-negative and has ubiquitin-positive inclusions is called "FTLD-U," and includes the above-mentioned FTLD-TDP, FTLD-UPS, and FTLD-FUS.

[0016] The term "FTLD-TDP" above refers to a disease in which FTLD-U is positive for TDP43 (Transactive Response DNA Binding Protein of 43 kD), and such diseases are treated with PGRN. R504X FTLD with PGRN (progranulin) mutations such as: sporadic FTLD-TDP / FTLD-U; TDP43 N267S FTLD;VCP with TDP43 mutations such as these. T262A This includes FTLD with VCP (Valosin-Containing Protein) mutations, FTLD linked to chromosome 9, and others. Furthermore, "FTLD-FUS" refers to a type of FTLD-U in which TDP43 is negative and FUS (Fused In Sarcoma) is positive. Such diseases include neuronal intermediate filament inclusion disease, atypical FTLD-U, basophilic inclusion disease, and FTLD with FUS mutations.

[0017] Furthermore, the term "FTLD-UPS" refers to a disease that is a type of FTLD-U in which TDP43 is negative, and such diseases are classified under CHMP2B. Q165XThis includes FTLD and other conditions involving CHMP2B (Charged Multivesicular Body Protein 2B) mutations.

[0018] In this specification, "prevention of frontotemporal lobar degeneration" includes not only suppressing the onset or onset of frontotemporal lobar degeneration, but also delaying the onset and onset of frontotemporal lobar degeneration. Furthermore, "treatment of frontotemporal lobar degeneration" includes not only the recovery and improvement of lesions and symptoms of frontotemporal lobar degeneration, including the accumulation of abnormal proteins in cells, but also the suppression of its progression.

[0019] In this specification, "onset of frontotemporal lobar degeneration" means the appearance of symptoms such as memory impairment, higher brain dysfunction (aphasia, apraxia, agnosia, constructional apraxia), and personality changes, as determined by clinical diagnosis, and / or the appearance of brain atrophy as determined by imaging diagnosis. In addition, in this specification, "suffering from frontotemporal lobar degeneration" also means a state in which pathological changes specific to frontotemporal lobar degeneration (e.g., accumulation of abnormal proteins in cells) occur, even if such symptoms are not present.

[0020] The human monoclonal antibody contained in this preventive / therapeutic agent has high affinity for the human HMGB1 protein, as demonstrated in the embodiment described later, and has the effect of reducing and suppressing the phosphorylation level of serine 46 (Ser46) in MARCKS (pSer46-MARCKS). For this reason, frontotemporal lobar degeneration caused by HMGB1 and / or pSer46-MARCKS can be suitably exemplified as the target of prevention or treatment of frontotemporal lobar degeneration with this preventive / therapeutic agent.

[0021] The preferred human monoclonal antibody is one comprising a heavy (H) chain variable region consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a light (L) chain variable region consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. More preferably, the preferred human monoclonal antibody is one comprising a heavy chain consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and a light chain consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0022] In this specification, "HMGB1 (High Mobility Group Box 1)" is a protein also referred to as HMG1, HMG3, SBP-1, and HMG-1. Typically, as a human-derived protein, it consists of the amino acid sequence identified by the NCBI reference sequence: NP_002119.1 (a protein encoded by the nucleotide sequence identified by the NCBI reference sequence: NM_002128.5). However, the DNA sequence of a gene mutates naturally (i.e., unartificially) due to mutations, and the amino acid sequence of the protein it encodes is modified accordingly. Therefore, the HMGB1 to which the human monoclonal antibody in this case binds includes not only the protein consisting of the amino acid sequence identified by the NCBI reference sequence: NP_002119.1, but also naturally occurring variants of such protein.

[0023] In this specification, "antibody that specifically binds to human HMGB1" means an antibody that recognizes and binds to human HMGB1 through a highly specific antigen-antibody recognition mechanism. The human monoclonal antibody in question is preferably isolated. Here, "isolated" means that the antibody exists in a state different from its natural state, such as by being extracted from its natural environment through artificial manipulation, or by being expressed in an environment different from its natural environment. In other words, "isolated antibody" does not include antibodies derived from an individual that are present in the body of that individual or in tissues or bodily fluids (blood, plasma, serum, etc.) of that individual without external manipulation (artificial manipulation). Furthermore, the human monoclonal antibody in question is preferably an antibody produced from an organism or cell created through artificial manipulation (for example, an antibody produced from a hybridoma). Such "antibody produced from an organism or cell created through artificial manipulation" does not include antibodies produced from naturally occurring organisms or B cells (without artificial manipulation).

[0024] In this specification, “monoclonal antibody” means an antibody (including functional fragments of an antibody) obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies recognize a single determinant on an antigen. The human monoclonal antibodies in this specification include classes and subclasses of human immunoglobulins, as well as the form of functional fragments of such antibodies. The classes and subclasses of human monoclonal antibodies in this specification include IgG such as IgG1, IgG2, IgG3, IgG4; IgA such as IGA1, IGA2; IgD; IgE; IgM; and so on.

[0025] The human monoclonal antibody in question typically comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3, with framework regions (FRs) ligated to the amino (N) and carboxyl (C) terminals of each of these CDR1-3 regions.

[0026] Among the above FRs, the heavy chain FRs include heavy chain FR1, which is attached to the N-terminus of heavy chain CDR1; heavy chain FR2, which is attached between the C-terminus of heavy chain CDR1 and the N-terminus of heavy chain CDR2; heavy chain FR3, which is attached between the C-terminus of heavy chain CDR2 and the N-terminus of heavy chain CDR3; and heavy chain FR4, which is attached to the C-terminus of heavy chain CDR3. In addition, among the above FRs, the light chain FRs include light chain FR1, which is attached to the N-terminus of light chain CDR1; light chain FR2, which is attached between the C-terminus of light chain CDR1 and the N-terminus of light chain CDR2; light chain FR3, which is attached between the C-terminus of light chain CDR2 and the N-terminus of light chain CDR3; and light chain FR4, which is attached to the C-terminus of light chain CDR3.

[0027] Specifically, the heavy chain FR1 mentioned above can be (HF1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence shown in Sequence ID No. 7, or (HF1') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the said polypeptide. Specifically, the heavy chain FR2 mentioned above can be (HF2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence shown in Sequence ID No. 7, or (HF2') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the said polypeptide. Specifically, the heavy chain FR3 mentioned above can be (HF3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence shown in SEQ ID NO: 7, a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence shown in SEQ ID NO: 11, or (HF3') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the aforementioned polypeptide. Specifically, the heavy chain FR4 mentioned above can be (HF4) a polypeptide consisting of amino acid residues 105 to 115 of the amino acid sequence shown in Sequence ID No. 7, or (HF4') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the said polypeptide.

[0028] Specifically, the light chain FR1 mentioned above can be (LF1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence shown in SEQ ID NO: 8, or (LF1') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with such polypeptide. Specifically, the above light chain FR2 can be (LF2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence shown in Sequence ID No. 8, or (LF2') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the said polypeptide. Specifically, the above light chain FR3 can be (LF3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence shown in SEQ ID NO: 8, or (LF3') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with such polypeptide. Specifically, the light chain FR4 mentioned above can be (LF4) a polypeptide consisting of amino acid residues 98 to 107 of the amino acid sequence shown in Sequence ID No. 8, or (LF4') a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the aforementioned polypeptide.

[0029] The human monoclonal antibody in question is a human antibody. Examples of such "human antibodies" include human chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies and fully human antibodies being preferred examples.

[0030] 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., a non-human mammal such as a chicken, mouse, rat, or cow) with the constant region of a human antibody. A human chimeric antibody can be obtained, for example, by immunizing a non-human animal (preferably a non-human mammal) with an antigen, excising the antibody variable region that binds to the antigen from the gene of the mouse monoclonal antibody, linking it to the antibody constant region gene derived from human bone marrow, incorporating this into an expression vector, and introducing it into a host to produce the antibody (see, for example, Japanese Patent Publication No. 8-280387; U.S. Patent No. 4816397; U.S. Patent No. 4816567; U.S. Patent No. 5807715).

[0031] Examples of the constant regions of human chimeric antibodies include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε in the heavy chain, and Cκ and Cλ in the light chain. The amino acid sequences of these constant regions and the nucleotide sequences encoding them are publicly known. Furthermore, to improve the stability of the antibody itself or the stability of antibody production, one or more amino acids in the constant region of human-derived antibodies can be substituted, deleted, added, and / or inserted.

[0032] In this specification, "humanized antibody" refers to an antibody obtained by transplanting the gene sequence of the antigen-binding site (CDR) of an antibody derived from a non-human animal (e.g., a non-human mammal such as a chicken, mouse, rat, or cow) onto a human-derived antibody gene (CDR grafting). Methods for producing such antibodies are publicly known, such as overlap extension PCR (e.g., 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 flanked by four framework regions (FRs). The CDR is essentially the region that determines the binding specificity of the antibody. While the amino acid sequences of CDRs are highly diverse, the amino acid sequences constituting the FRs often show high homology even among antibodies with different binding specificities. Therefore, it is generally said that the binding specificity of one antibody can be transferred to another antibody by transplanting the CDR. Furthermore, from the perspective of maintaining the function of CDRs, when transplanting non-human derived CDRs into human FRs, human FRs with high homology to the non-human animal-derived FRs are selected. In other words, since the amino acids in CDRs not only recognize antigens but also coordinate with the amino acids of FRs adjacent to the CDRs and are involved in maintaining the loop structure of CDRs, it is preferable to use human FRs with amino acid sequences that have high homology to the amino acid sequences of FRs adjacent to the CDRs to be transplanted.

[0033] A search for known human FRs with high homology to non-human animal-derived FRs can be performed, for example, using an antibody-specific search system available on the internet (http: / / www.bioinf.org.uk / abysis / ). Mutations can then be introduced into sequences other than the CDR of the non-human antibody to match the human FR sequence obtained in this way. Alternatively, if the gene (cDNA) encoding the amino acid sequence of the human FR obtained through the search is available, a non-human CDR may be introduced into that sequence. Mutation introduction and other processes can be carried out using techniques known in the field, such as nucleic acid synthesis and site-directed mutagenesis.

[0034] By qualitatively or quantitatively measuring and evaluating the affinity of the humanized antibody produced in this manner to the antigen, a human-derived antibody FR that forms a good antigen-binding site for the CDR when linked via the CDR can be suitably selected. Furthermore, if necessary, amino acid residues of the FR can be substituted so that the humanized antibody's CDR forms an appropriate antigen-binding site, according to methods described in reference "Cancer Res, 1993, 53, 851-856," etc. By further measuring and evaluating the affinity of the mutant antibody with the substituted amino acids to the antigen, a mutant FR sequence with desired properties can be selected.

[0035] In this specification, "fully human antibody" means an antibody in which all sequences are derived from human sequences. Fully human antibodies can be produced, for example, in transgenic mice engineered to express human heavy chain and light chain antibody genes. Transgenic mice that produce human antibodies can be prepared, for example, according to methods described in International Publication No. 02 / 43478, U.S. Patent No. 6657103 (Abgenix), etc. Subsequently, hybridoma cell lines fused with B cells derived from transgenic mice that produce the desired antibody can be prepared, for example, by following the methods described in 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 reference "Jakobovits, Adv. Drug Del. Rev. 31:33-42 (1998)"; and the reference "Green, et al, J. Exp. Med. 188:483-95 (1998)".

[0036] As mentioned above, the human monoclonal antibody in question includes not only the entire antibody but also functional fragments that specifically recognize 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, diabodies, polyspecific antibodies, and polymers thereof.

[0037] Here, "Fab" refers to a monovalent antigen-binding fragment of immunoglobulin consisting of one light chain and a portion of the heavy chain. It can be obtained by papain digestion of the antibody or by recombinant methods. "Fab'" differs from Fab by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines in the hinge region of the antibody. "F(ab')2" refers to a bivalent antigen-binding fragment of immunoglobulin consisting of both light chains and portions of both heavy chains.

[0038] A "variable region fragment (Fv)" is the smallest antibody fragment that has complete antigen recognition and binding sites. Fv is a dimer in which the heavy chain variable region and the light chain variable region are strongly linked by non-covalent bonds. A "single-chain Fv (scFv)" contains the heavy chain variable region and the light chain variable region of an antibody, and these regions are located on a single polypeptide chain. "sc(Fv)2" is a single-chain formed by linking two heavy chain variable regions and two light chain variable regions with a linker or the like. A "diabody" is a small antibody fragment that has two antigen-binding sites. This fragment contains a heavy chain variable region bound to a light chain variable region within the same polypeptide chain, and each region forms a pair with a complementary region on another chain. A "polyspecific antibody" is a monoclonal antibody that has binding specificity to at least two different antigens. For example, it can be prepared by the simultaneous expression of two immunoglobulin heavy / light chain pairs in which the two heavy chains have different specificities.

[0039] The human monoclonal antibodies in question include modified versions of the H chain CDR1-3 and L chain CDR1-3 in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NOs. 1-6 without reducing the desired activity (i.e., affinity for HMGB1). Human monoclonal antibodies containing such modified CDRs can be produced, for example, by introducing mutations into the DNA encoding the antibody chain of human monoclonal antibody #129, or by peptide synthesis. While modifications to amino acids in regions of antibodies other than the CDR (e.g., FR, constant region) are thought to have relatively little effect on affinity to the antigen, methods for screening antibodies with enhanced affinity to the antigen by modifying the amino acids in the CDR are now publicly known (see, for example, "PNAS, 102:8466-8471 (2005)"; "Protein Engineering, Design & Selection, 21:485-493 (2008)"; International Publication No. 2002 / 051870; "J. Biol. Chem., 280:24880-24887 (2005)"; "Protein Engineering, Design & Selection, 21:345-351 (2008)"; and "MAbs. Mar-Apr; 6(2):437-45 (2014)"). Furthermore, it is now possible to model antibodies with enhanced affinity to antigens by utilizing integrated computational chemistry systems (for example, MOE [Molecular Operating Environment] [manufactured by CCG, Canada]) (see, for example, http: / / www.rsi.co.jp / kagaku / cs / ccg / products / application / protein.html).

[0040] In this specification, "multiple amino acids" in "substitution, deletion, addition, and / or insertion of one or more amino acids" means preferably no more than 10 amino acids, more preferably no more than 5 amino acids, and even more preferably no more than 3 amino acids (e.g., no more than 2 amino acids, 1 amino acid). Conservative substitutions are preferred among amino acid substitutions, deletions, additions, and insertions. Such "conservative substitutions" mean substitution with other amino acid residues having chemically similar side chains. Groups of amino acid residues having chemically similar amino acid side chains are well known in the art. For example, amino acids can be classified into acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), and neutral amino acids, which can be further classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxyl groups (serine, threonine), amino acids containing sulfur (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan).

[0041] In this specification, "at least 80% sequence identity" with respect to a given amino acid sequence preferably means at least 85% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95% sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, 100%). The homology of amino acid sequences can be determined using the BLASTP (amino acid level) program (see, for example, "J.Mol.Biol., 215:403-410, 1990"). This program is based on the BLAST algorithm (see, for example, "Proc.Natl.Acad.Sci.USA, 87:2264-2268, 1990" and "Proc.Natl.Acad.Sci.USA, 90:5873-5877, 1993"). When analyzing amino acid sequences using BLASTP, the parameters should be, for example, score=50 and wordlength=3. Alternatively, when analyzing amino acid sequences using the Gapped BLAST program, refer to the reference "Nucleic Acids Res. 25:3389-3402, 1997". This can be done according to the method described above. When using the BLAST and Gapped BLAST programs, use the default parameters of each program. The specific techniques for these analysis methods are publicly known.

[0042] The human monoclonal antibodies in this study include those in which the post-translational process of the antibody has been modified, such as by altering the number or location of glycosylation sites. Such modified antibodies may improve the ADCC activity (antibody-dependent cell-mediated cytotoxicity) of the antibody. Antibody glycosylation is typically N-linked or O-linked. Antibody glycosylation is highly dependent on the host cell used to express the antibody. Modification of the glycosylation pattern can be carried out by known methods such as the introduction or deletion of specific enzymes involved in 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, the human monoclonal antibodies in this study may have deamidation suppressed by substituting amino acids adjacent to deamidated amino acids with other amino acids for purposes such as increasing antibody stability, or by substituting glutamic acid with other amino acids to increase antibody stability.

[0043] The human monoclonal antibody in question can be produced by known hybridoma methods or known recombinant DNA methods. A typical example of a hybridoma method is the method of Kohler and Milstein (reference "Nature, 256:495 (1975)"). The antibody-producing cells used in the cell fusion step in this method are spleen cells, lymph node cells, peripheral blood leukocytes, etc., from animals (e.g., mammals such as mice, rats, hamsters, rabbits, monkeys, and goats) immunized with an antigen (HMGB1 protein, its partial peptide, proteins fused with Fc protein, etc., or cells expressing these, etc.). Antibody-producing cells obtained by reacting the above cells or lymphocytes, etc., previously isolated from unimmunized animals with the antigen in a culture medium can also be used. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be of different animal species origins, provided they are fused together, but preferably they are of the same animal species origin. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from antigen-immunized mice and mouse myeloma cells. Subsequent screening can then yield hybridomas that produce monoclonal antibodies specific to the HMGB1 protein. Human monoclonal antibodies that specifically bind to the HMGB1 protein can be obtained by culturing hybridomas or from the ascites fluid of mammals administered with hybridomas.

[0044] The recombinant DNA method involves cloning the antibody gene encoding the human monoclonal antibody in question from hybridomas or B cells, incorporating it into a suitable vector, and introducing it into host cells (e.g., mammalian cell lines such as HEK cells, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the human monoclonal antibody as a recombinant antibody (see, for example, reference "Eur.J.Biochem.192:767-775(1990)"). In the expression of the antibody gene encoding the human monoclonal antibody in question, the antibody genes encoding the heavy chain or light chain may be incorporated separately into expression vectors to transform the host cells, or the antibody genes encoding both the heavy chain and light chain may be incorporated into a single expression vector to transform the host cells (see International Publication No. 94 / 11523). The human monoclonal antibody in question can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cells, separating and purifying it from within the host cells or from the culture medium. The separation and purification of the antibody can be performed using methods commonly used for the purification of polypeptides. By using transgenic animal production technology to create transgenic animals (cows, goats, sheep, pigs, etc.) into which antibody genes have been incorporated, it is possible to obtain large quantities of human monoclonal antibodies derived from the human monoclonal antibody gene in question from the milk of these transgenic animals.

[0045] In this specification, examples of additives include pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, isotonic agents, solubilizers, and other compounding agents. Examples of such pharmaceutically acceptable carriers when the prophylactic / therapeutic agent is in powder form include mannitol, lactose, saccharose, and human albumin, and examples when the prophylactic / therapeutic agent is in liquid form include physiological saline, water for injection, phosphate buffer, and aluminum hydroxide.

[0046] The target population for this preventive / therapeutic agent is any human being who requires prevention or treatment of frontotemporal lobar degeneration. Examples include individuals diagnosed with a high risk of developing frontotemporal lobar degeneration, or patients who have already developed frontotemporal lobar degeneration.

[0047] Methods of administering this preventive / therapeutic agent include, for example, injection (e.g., subcutaneous, intramuscular, intravenous, intra-arterial), oral mucosal administration, rectal administration, local administration to the brain (intracerebroventricular administration), transdermal administration, and oral administration, with intravenous administration being a preferred example. The dosage of the human monoclonal antibody in this preventive / therapeutic agent may vary depending on the age, weight, sex, health status, and degree of symptom progression of the recipient, but is usually 0.1 to 1000 mg per kg of body weight per day for adults, preferably 1 to 100 mg. This preventive / therapeutic agent may be used in combination with known pharmaceuticals used for the prevention or treatment of frontotemporal lobar degeneration.

[0048] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. These examples were carried out in strict accordance with the recommendations in the guidelines for the management and use of laboratory animals issued by the Japanese government and the National Institutes of Health. Furthermore, all experiments were approved by the Genetic Engineering Experiment Committee, the Ethics Committee, and the Animal Experiment Committee of Tokyo Medical and Dental University. [Examples]

[0049] 1. Materials and Methods [Anti-human HMGB1 antibody] In this example, human monoclonal antibody (human-type IgG antibody) #129, prepared in the example of the international patent application (PCT-JP2019-36926 [International Publication 2020 / 059847 pamphlet]), was used as the anti-human HMGB1 antibody. The amino acid sequences of the H chain and L chain of human monoclonal antibody #129 are shown in Tables 1 and 2, respectively. When human monoclonal antibody #129 was subcutaneously administered to mice, the antibody concentrations in the brain and serum were measured by ELISA. The results confirmed that 2-8% of the administered human monoclonal antibody #129 crossed the blood-brain barrier (BBB) ​​and migrated into the brain.

[0050] [Table 1] The underlined region in the table represents the H chain V region (SEQ ID NO: 7). The three regions enclosed in squares in the table represent, from the amino terminus, H chain CDR1 (SEQ ID NO: 1), H chain CDR2 (SEQ ID NO: 2), and H chain CDR3 (SEQ ID NO: 3).

[0051] [Table 2] The underlined region in the table represents the L-chain V region (SEQ ID NO: 8). The three regions enclosed in squares in the table represent, from the amino-terminal end, L-chain CDR1 (SEQ ID NO: 4), L-chain CDR2 (SEQ ID NO: 5), and L-chain CDR3 (SEQ ID NO: 6).

[0052] [Analysis of antibody affinity using surface plasmon resonance (SPR)] The interaction between the human monoclonal antibody #129 of the present invention and the human HMGB1 protein was analyzed using the mouse monoclonal antibody 2C8C described in International Publication No. 2018 / 030405 as a comparative control. Specifically, a sensor chip immobilized with either the human monoclonal antibody #129 or the mouse monoclonal antibody 2C8C of the present invention was used, along with human HMGB1 protein as the analyte. The interaction was measured over time using a surface plasmon resonance analyzer ("Biacore T100"; GE Healthcare), and the equilibrium dissociation constant (KD value), an indicator of antibody affinity, was calculated (see Figure 1).

[0053] [Creation of 4 types of FTLD model mice] 4 types of FTLD model mice (VCP T262A -KI Mouse, PGRN R504X -KI mouse, CHMP2B Q165X -KI mouse, and TDP43 N267S -KI mice) (referred to as "four types of FTLD model mice" in this specification) - PGRN R504X -KI mice were prepared according to the method described in the literature "Nat. Commun., 2018 9, 433".

[0054] Also, VCP T262A- Targeting vectors for generating KI mice were constructed. Specifically, the PCR product of a 5.4kb NotI-XhoI fragment amplified from a Bac clone (ID: RP23-111G9 or RP23-124L1) was subcloned into the PspOMI-XhoI site of pBS-DTA. Similarly, a 2.9kb BamHI-NotI fragment amplified from the same Bac clone was subcloned into pBS-LNL(-) with a Neo cassette (loxP-Neo-loxP). A 4.5kb fragment (1.6kb Neo cassette + 2.9kb fragment) was excised by Notl treatment, and a 0.8kb XhoI-Smal fragment with the T262A mutation was amplified by PCR from the same Bac clone and subjected to XhoI and SmaI treatment. These 0.8kb and 4.5kb fragments were subcloned into the XhoI-NotI site of the targeting vector. After linearizing the targeting vector by NotI treatment, these fragments were electroporated into ES cells in a C57BL / 6J background. PCR using a primer set that amplifies DNA fragments containing the LoxP sequence (Primer 1 [5'-ATATGCTCTCACTGTATGGTATTGC-3'; SEQ ID NO: 11]; and Primer 2 [5'-TCCAGATGAGCTTAGAAGATTAGAA-3'; SEQ ID NO: 12]) was performed to extract VCP from non-transgenic littermates. T262A -KI mice were selected.

[0055] Also, CHMP2B Q165X- A targeting vector for generating KI mice was constructed. Specifically, the PCR product of a 5.7kb ClaI-SalI fragment amplified from a Bac clone (ID: RP23-13H5 or RP23-273E18) was subcloned into the ClaI-SalI region of pBS-DTA (Unitech). A 3.0kb SacII-NotI fragment was amplified from a Bac clone and subcloned into pBS-LNL(+) containing a Neo cassette (loxP-Neo-loxP). SacII-NotI and a 4.6kb fragment (1.6kb Neo cassette + 3.0kb fragment) were subcloned into the SacII-ClaI region of the targeting vector. By PCR using a primer set that amplifies DNA fragments containing the LoxP sequence (Primer 3 [5'-TGGATTTTATTTATGTCTGAATGTG-3'; SEQ ID NO: 13] and Primer 4 [5'-ATAAGCAACTTCACAAGGCATCTTA-3'; SEQ ID NO: 14]), CHMP2B was obtained from non-transgenic littermates. Q165X -KI mice were selected.

[0056] Also, TDP43 N267S - Targeting vectors for generating KI mice were constructed. Specifically, a 5.9kb ClaI-SalI fragment was amplified from a Bac clone (ID: RP23-364M1 or RP23-331P21) and subcloned into the ClaI-SalI region of pBS-TK (Unitech). A 2.7kb SacII-NotI fragment was amplified from a Bac clone and subcloned into pA-LNL(+) with a Neo cassette (loxP-Neo-loxP). A 4.3kb fragment (1.6kb Neo cassette + 2.7kb fragment) was subcloned into the SacII-ClaI region of the targeting vector. PCR using a primer set that amplifies DNA fragments containing the LoxP sequence (Primer 5 [5'-ACAGTTGGGTGTGATAGCAGGTACT-3'; SEQ ID NO: 15] and Primer 6 [5'-TCGAGAATTACAGGAATGTATCATC-3'; SEQ ID NO: 16] was performed to obtain TDP43 from non-transgenic littermates. N267S -KI mice were selected.

[0057] [Immunohistochemical staining method] Brains were extracted from four types of FTLD model mice or C57BL / 6J mice of various ages. After fixing in PBS containing 4% paraformaldehyde for 12 hours, 5 μm thick paraffin sections of brain tissue were prepared using a microtome (Daiwa Koki Kogyo Co., Ltd.). The prepared paraffin sections of brain tissue were deparaffinized with xylene, rehydrated in 0.01 M citrate buffer (pH 6.0), and then heat-treated at 120°C for 15 minutes. Subsequently, the sections were permeabilized in PBS containing 0.5% triton-X100 and blocked in PBS containing 10% FBS for 60 minutes. Subsequently, antibody reactions were carried out at 4°C for 12 hours using various primary antibodies (see Table 3) that detect 11 types of 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]). After washing three times with PBS, antibody reactions using secondary antibodies against various primary antibodies (see Table 3) were carried out at room temperature for 1 hour. After washing three times with PBS, the cell nuclei were stained in PBS containing 0.2 μg / mL of DAPI (4',6-diamidino-2-phenylindole), and fluorescence images from the 11 proteins and cell nuclei were obtained using a confocal microscope (FV1200IX83, Olympus). Subsequently, the number of pSer46-MARCKS-positive cells per 143 μm × 143 μm area was calculated based on the DAPI fluorescence images and pSer46-MARCKS fluorescence images (see Figures 2-1 and 9). In addition, the KDEL signaling region was measured in MAP2-positive cells, a marker of mature neurons, based on the DAPI fluorescence images, KDEL fluorescence images, and MAP2 fluorescence images (see Figure 10). Furthermore, the γH2AX signaling region was measured in MAP2-positive cells based on DAPI fluorescence images, γH2AX fluorescence images, and MAP2 fluorescence images (see Figure 11). In addition, the 53BP1 signaling region was measured in MAP2-positive cells based on DAPI fluorescence images, 53BP1 fluorescence images, and MAP2 fluorescence images (see Figure 12).Furthermore, the pSer409 / 410-TDP43 signaling region in ubiquitin-positive inclusions was measured based on DAPI fluorescence images, pSer409 / 410-TDP43 fluorescence images, and ubiquitin fluorescence images (see Figure 13). In addition, the p62 signaling region in inclusions was measured based on DAPI fluorescence images and p62 fluorescence images (see Figure 14). Furthermore, the percentage of cells in which VAMP2 signaling and PSD95 signaling co-localized was measured based on DAPI fluorescence images, VAMP2 fluorescence images, and PSD95 fluorescence images (see Figure 15). Furthermore, the percentage of cells in which pSer203-tau signaling and PSD95 signaling co-localized was measured based on DAPI fluorescence images, pSer203-tau fluorescence images, and PSD95 fluorescence images (see Figure 16).

[0058] [Table 3]

[0059] [Nissl staining method] In the section on [Immunohistochemical Staining Method] above, paraffin sections of brain tissue that had undergone deparaffinization and rehydration were washed with deionized water and then immersed in cresyl violet solution (300 mL of solution containing 0.1% cresyl violet and 5 drops of 10% acetic acid solution) at 37°C for 15 minutes. After that, the sections were dehydrated with ethanol solution and cleared with xylene, and then images of the necrotic brain tissue were obtained using an electron microscope (H-7100, Hitachi High-Tech Corporation) (see Figure 2-2).

[0060] [Administration of anti-human HMGB1 antibody] Four FTLD model mice and their background mice (C57BL / 6J) were administered via tail vein at a dose of 6 μg of human monoclonal antibody #129 or control human IgG (#12000C, Thermo Fisher Scientific) per 30 g of body weight, once a month, for the duration shown in Table 4 (Figure 3). The start of antibody administration in each FTLD model mouse was determined by the age at which the onset of the disease was confirmed by Maurice's water maze test.

[0061] [Table 4]

[0062] [Cognitive function test] During each test period or test day shown in Table 4, three types of cognitive function tests (Maurice's water maze test, Y-maze test, and fear conditioning test) were performed on four types of FTLD model mice according to the methods described in the literature "Hum Mol Genet 24, 540-558 (2015)" and "Mol. Psychiatry 20, 459-471 (2015)." Specifically, in the Morris's water maze test, mice were subjected to the Morris's water maze test four times (60 seconds) once a day for five days, and the average time (latency) to reach the platform (vertical axis in Figure 3) was measured. In the Y-maze test, a Y-maze (manufactured by Ohara Medical Industry Co., Ltd.) consisting of three identical arms with equal angles between each arm was used. A mouse was placed at the end of one arm and allowed to move freely through the maze for 8 minutes. The rate of spontaneous change was calculated by dividing the total number of times the mouse moved from one arm to another by the number of times it entered a new arm different from the previous one (shown as Alternation Rate; vertical axis in Figures 4-7). In the fear conditioning test, the freezing response was measured 24 hours after conditioning (65 dB white noise, 30 seconds + foot shock, 0.4 mA, 2 seconds) in the same chamber without a foot shock (shown as Total Freeze percent; vertical axis in Figure 8).

[0063] [Western blot method] Cerebral cortical tissue from four types of FTLD model mice or C57BL / 6J mice of various ages was dissolved at 4°C for 1 hour in the presence of a lysis buffer (100 mM Tris-HCl [pH 7.5], 2% SDS) containing a protease inhibitor cocktail (#539134, Calbiochem, dilution ratio 1:100). After centrifugation (12,000 g × 10 minutes), the supernatant was collected and the same volume 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. The protein content in the sample was then equalized using the BCA method (Pierce BCA protein assay kit, Thermo Scientific). Subsequently, the protein sample was separated by SDS-PAGE, then transferred to a PVDF (Poly Vinylidene Di-Fluoride) membrane (Immobilon-P [Millipore]) by a semi-dry method, blocked in a TBST solution (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20) containing 5% skim milk, and then treated with 0.1% skim milk or Can Get In a TBST solution containing Signal solution (Toyobo, Osaka, Japan), 13 types of proteins were found (phosphorylated PKCα [Ser319] [pSer319-PKCα], phosphorylated PKCγ [Thr655] [pThr655-PKCγ], phosphorylated PKCδ [Ser643] [pSer643-PKCδ], phosphorylated PKCε [Ser729] [pSer729-PKCε], phosphorylated tau [Ser203 (mouse)] / Antibody reactions using various primary antibodies (see Table 5) to detect Ser214 (human) [pSer203-tau], PSD95, VAMP2, γH2AX, 53BP1, phosphorylated TDP43 [Ser409 / 410] [pSer409 / 410-TDP43], pSer46-MARCKS, phosphorylated Ku70 [Ser77 / Ser78] [pSer77-Ku70], and β-actin were performed at 4°C for 12 hours.After washing the membrane three times with TBST solution, antibody reactions using secondary antibodies against various primary antibodies (see Table 5) were carried out at room temperature for 1 hour. Subsequently, the 13 proteins listed above were detected using ECL Prime Western blotting detection reagent (RPN2232, GE Healthcare) and a luminescence image analyzer (ImageQuantLAS 500, GE Healthcare) (see Figure 17).

[0064] [Table 5]

[0065] 2.Results [Analysis of antibody affinity using surface plasmon resonance (SPR)] The KD of the mouse monoclonal antibody 2C8C is 1.95 × 10⁶. -9 In contrast to M, the KD of human monoclonal antibody #129 of the present invention is 3.79 × 10⁻⁶. -11 It was M. In addition to the KD of the mouse monoclonal antibody 2C8C, the KD of general antibody drugs was also 10. -9 M~10 -10 Considering that it is within the M range, the human monoclonal antibody #129 of the present invention is an antibody with extremely high affinity for human HMGB1.

[0066] [Immunohistochemical staining method] pSer46-MARCKS is present from an early stage before the onset of Alzheimer's disease, and HMGB1 leaked from cells due to neuronal necrosis induces phosphorylation of MARCKS, leading to neurite degeneration (see "SCIENTIFIC REPORT, 2016 Aug 25;6:31895"). Therefore, we analyzed whether MARCKS phosphorylation and neuronal necrosis occur in the early stages of FTLD using four types of FTLD model mice aged 1 to 12 months.

[0067] As a result, four types of FTLD model mice (VCP) were found to be 1 month old, 3 months old, 6 months old, and 12 months old. T262A -KI Mouse, PGRN R504X -KI mouse, CHMP2B Q165X -KI mouse, and TDP43 N267S In the cerebral cortex of 1-KI mice, high levels of pSer46-MARCKS-derived signals were detected, and cells with reduced genomic DNA-derived DAPI signaling were observed (see Figure 2-1). In addition, two types of 1-month-old FTLD model mice (VCP T262A -KI mouse and PGRN R504X In the cerebral cortex of -KI mice, dilation of the endoplasmic reticulum (ER), a characteristic of necrosis, was observed (see Figure 2-2). These results indicate that necrosis occurs from the early stages of FTLD.

[0068] [Therapeutic effects of human monoclonal antibody #129 on symptoms in FTLD model mice: Cognitive function test] Next, we analyzed whether administering the human monoclonal antibody #129 of the present invention to four types of FTLD model mice improved the symptoms of FTLD using three types of cognitive function tests (Maurice's water maze test, Y-maze test, and fear conditioning test). First, in Morris's water maze test, it was confirmed that the time it took for the four types of FTLD model mice ("KI nt" in Figure 3) to reach the platform increased with age compared to normal C57BL / 6J mice ("B6 nt" in Figure 3) (see Figure 3). On the other hand, when control human IgG was administered to four types of FTLD model mice ("KI IgG" in Figure 3), no change was observed in the extension of the time to reach the platform. In contrast, when the human monoclonal antibody #129 of the present invention was administered to four types of FTLD model mice ("KI Ab" in Figure 3), regardless of the type of FTLD mutant gene, the extension of the time to reach the platform was significantly suppressed (shortened) from the early stages of FTLD onset to a level comparable to that of C57BL / 6J mice in all FTLD model mice (see Figure 3).

[0069] These results indicate that the human monoclonal antibody #129 of the present invention has the effect of preventing and improving cognitive decline caused by FTLD. Similar effects were also confirmed in the Y-maze test (see Figures 4-7) and the fear conditioning test (see Figure 8).

[0070] [Therapeutic effect of human monoclonal antibody #129 on symptoms in FTLD model mice: Immunohistochemical staining method] Next, the cognitive function-improving effect of the human monoclonal antibody #129 of the present invention on FTLD was analyzed using immunohistochemical staining. First, compared to normal C57BL / 6J mice ("KI nt" in Figure 3), the four FTLD model mice showed an increased proportion of necrosis marker (pSer46-MARCKS)-positive cells in the cerebral cortex (see Figure 9) and an enlargement of ER marker (KDEL)-derived signaling regions (see Figure 10). On the other hand, when control human IgG was administered to four types of FTLD model mice ("KI IgG" in Figures 9 and 10), no change was observed in the increase in the proportion of pSer46-MARCKS-positive cells or the increase in KDEL-derived signaling regions. In contrast, when the human monoclonal antibody #129 of the present invention was administered to four types of FTLD model mice ("KI Ab" in Figures 9 and 10), the increase in the proportion of pSer46-MARCKS-positive cells and the increase in KDEL-derived signaling regions were significantly suppressed to a level comparable to that of C57BL / 6J mice (see Figures 9 and 10).

[0071] These results indicate that the human monoclonal antibody #129 of the present invention has the effect of preventing and improving necrosis and ER dilation caused by FTLD in the cerebral cortex.

[0072] Furthermore, while the four FTLD model mice showed an increase in signal regions derived from DNA damage markers (γH2AX and 53BP1) in the cerebral cortex compared to C57BL / 6J mice, administration of the human monoclonal antibody #129 of the present invention significantly suppressed this increase in γH2AX and 53BP1-derived signal regions to a level comparable to that of C57BL / 6J mice (see Figures 11 and 12).

[0073] These results demonstrate that the human monoclonal antibody #129 of the present invention has the effect of preventing and improving DNA damage caused by FTLD in the cerebral cortex.

[0074] Furthermore, while the four FTLD model mice showed an increase in signal regions derived from protein aggregation markers (pSer409 / 410-TDP43 and p62) in the cerebral cortex compared to C57BL / 6J mice, administration of the human monoclonal antibody #129 of the present invention significantly suppressed this increase in pSer409 / 410-TDP43 and p62-derived signal regions (see Figures 13 and 14).

[0075] These results demonstrate that the human monoclonal antibody #129 of the present invention has the effect of preventing and improving protein aggregation caused by FTLD in the cerebral cortex.

[0076] Furthermore, the inventors predicted synaptic dysfunction using the co-localization of VAMP2 and PSD95, and the co-localization of pSer203-tau and PSD95, as indicators, through the analysis of phosphoproteome. Analyzing synaptic dysfunction using these co-localizations as indicators, the results showed that the co-localization of VAMP2 and PSD95 in the cerebral cortex of the four FTLD model mice was reduced compared to C57BL / 6J mice, while this reduction was significantly suppressed by administration of the human monoclonal antibody #129 of the present invention (see Figure 15). Additionally, the co-localization of pSer203-tau and PSD95 in the cerebral cortex of the four FTLD model mice was increased compared to C57BL / 6J mice, while this increase was significantly suppressed by administration of the human monoclonal antibody #129 of the present invention (see Figure 16).

[0077] These results demonstrate that the human monoclonal antibody #129 of the present invention has the effect of preventing and improving synaptic damage caused by FTLD in the cerebral cortex.

[0078] [Therapeutic effect of human monoclonal antibody #129 on symptoms in FTLD model mice: Western blot method] To support the results obtained using immunohistochemical staining, Western blotting analysis was performed. First, the phosphorylation levels of four proteins involved in the core signaling of AD-FTLD (pSer319-PKCα, pThr655-PKCγ, pSer643-PKCδ, and pSer729-PKCε) were analyzed. The results showed that the phosphorylation levels of two proteins (pSer319-PKCα and pSer643-PKCδ), which were predicted by dynamic network analysis with a strict threshold for core node selection (centrality > 2SD), were increased in the four-protein FTLD model mice compared to C57BL / 6J mice. However, administration of the human monoclonal antibody #129 of the present invention to the four-protein FTLD model mice suppressed the increase in these pSer319-PKCα and pSer643-PKCδ levels (see lanes (1) and (3) in Figure 17). On the other hand, under conditions of relatively loose core node selection (centrality > 0), no changes were observed in the two phosphorylation levels (pThr655-PKCγ and pSer729-PKCε) predicted by dynamic network analysis between the four FTLD model mice and the C57BL / 6J mouse (see lanes (2) and (4) in Figure 17).

[0079] Furthermore, while the phosphorylation of synaptic instability-related proteins (pSer203-tau) was increased in the four FTLD model mice compared to C57BL / 6J mice, administration of the human monoclonal antibody #129 of the present invention to the four FTLD model mice suppressed this increase in pSer203-tau levels (see lane (5) in Figure 17). In addition, while the expression levels of synapse-related proteins (PSD95 and VAMP2) were decreased in the four FTLD model mice compared to C57BL / 6J mice, administration of the human monoclonal antibody #129 of the present invention to the four FTLD model mice suppressed this decrease in the expression levels of PSD95 and VAMP2 (see lanes (6) and (7) in Figure 17).

[0080] Furthermore, while the expression levels of DNA damage-related proteins (γH2AX and 53BP1) were increased in the four FTLD model mice compared to C57BL / 6J mice, administration of the human monoclonal antibody #129 of the present invention to the four FTLD model mice suppressed this increase in the expression levels of γH2AX and 53BP1 (see lanes (8) and (9) in Figure 17).

[0081] Furthermore, while the phosphorylation level of cell death-related proteins (pSer46-MARCKS) was increased in the four FTLD model mice compared to C57BL / 6J mice, it was shown that administering the human monoclonal antibody #129 of the present invention to the four FTLD model mice suppressed this increase in pSer46-MARCKS levels (see lane (10) in Figure 17).

[0082] Furthermore, while the phosphorylation level of DNA repair-related proteins (pSer77-Ku70) was increased in the four FTLD model mice compared to C57BL / 6J mice, it was shown that administering the human monoclonal antibody #129 of the present invention to the four FTLD model mice suppressed this increase in pSer77-Ku70 levels (see lane (11) in Figure 17).

[0083] Furthermore, while the level of the protein aggregation marker (pSer409 / 410-TDP43) increased in the four FTLD model mice compared to the C57BL / 6J mice, it was shown that administering the human monoclonal antibody #129 of the present invention to the four FTLD model mice suppressed this increase in pSer409 / 410-TDP43 levels (see lane (12) in Figure 17).

[0084] The above results demonstrate that the human monoclonal antibody #129 of the present invention has the effect of preventing and improving secondary neuronal damage and neurite damage induced by extracellular HMGB1 released from necrotic neurons by FTLD.

[0085] [Side effects of human monoclonal antibody #129 of the present invention] Furthermore, the side effects of the human monoclonal antibody #129 of the present invention were investigated. After the completion of the cognitive function tests shown in Figures 3-8, the lungs, liver, kidneys, intestines, spleen, skeletal muscle, heart, and skin of four types of FTLD model mice and C57BL / 6J mice administered with the human monoclonal antibody #129 of the present invention were dissected and subjected to pathological examination.

[0086] As a result, no abnormal findings (e.g., obvious inflammation, cell death, tumors) were observed in any tissue following administration of the human monoclonal antibody #129 of the present invention. This indicates that the human monoclonal antibody #129 of the present invention can prevent or improve symptoms in FTLD model mice without significant side effects. [Industrial applicability]

[0087] This invention contributes to the prevention and / or treatment of FTLD.

Claims

1. It specifically binds to human HMGB1, and A heavy chain complementarity-determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1; a heavy chain CDR 2 consisting of the amino acid sequence shown in SEQ ID NO: 2; and a heavy chain CDR 3 consisting of the amino acid sequence shown in SEQ ID NO: 3; It comprises: light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4; light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5; and light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

6. A prophylactic or therapeutic agent for frontotemporal lobar degeneration, containing human monoclonal antibodies.

2. The preventive or therapeutic agent according to claim 1, wherein the human monoclonal antibody comprises a heavy chain variable region consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

3. The preventive or therapeutic agent according to claim 1 or 2, wherein the human monoclonal antibody comprises a heavy chain having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and a light chain having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

10.

4. A prophylactic or therapeutic agent according to any one of claims 1 to 3, which is administered intravenously.

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