HK1210031B - Therapeutic agent or prophylactic agent for dementia - Google Patents

Description

Dementia treatment or prevention drugs

Technical Field

The present invention relates to a therapeutic or preventive drug for dementia. Specifically, the present invention relates to a novel therapeutic or preventive drug for dementia comprising an anti-phosphoprotein or peptide antibody and an anti-phospho-tau antibody or an antigen capable of inducing the anti-phospho-tau antibody, which has an excellent effect of improving cognitive function.

Background Art

Dementia refers to a state in which once-developed intelligence declines due to some acquired reasons, resulting in difficulty in social adaptation. Dementia diseases are divided into neurodegenerative diseases, vascular dementia, prion diseases, infectious diseases, metabolic and endocrine diseases, traumatic and brain surgical diseases, toxic diseases, etc. (Non-patent Document 1). In Japan, there were approximately 2.1 million dementia patients in 2010. It is reported that the prevalence rate among the elderly over 65 years old is about 8-10% or more, which is considered a major problem in the global aging trend (Non-patent Document 2). Looking at the underlying diseases of dementia, it can be seen that: about 35% are Alzheimer's disease (AD), about 15% are mixed types of AD and cerebrovascular diseases, and 5% are frontotemporal lobar degeneration (FTLD). Neurodegenerative diseases such as AD or FTLD, which are degenerative diseases, account for the majority of the proportions (Non-patent Document 2). Dementia caused by neurodegenerative diseases is a disease characterized by the latent onset of memory impairment and/or personality changes that lasts for at least 6 months. A consistent feature of the neurodegenerative process that is highly correlated with the degree of cognitive impairment is neurofibrillary changes (neurofibrillary tangles, NFTs) (Non-Patent Document 3).

In the human body, tau (protein) refers to the protein encoded by the MAPT gene present in chromosome 17 (17q21), and is one of the microtubule-associated proteins expressed in a large number in the central nervous system. It has been found that tau is the main constituent protein of the double-stranded helical fibers (paired helical filament) and straight fibers (straightfilament) of the NFT in AD, a representative of neurodegenerative diseases, and it has been further clarified that tau accumulates in cells in various neurological diseases. This disease in which tau accumulates in cells is collectively referred to as Tauopathy (Tau disease, Tauopathy) (non-patent literature 4). Neurodegenerative diseases included in tauopathies include Alzheimer's disease (AD), corticobasal degeneration (CBD or CBS), progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multiple system tauopathy with dementia (MSTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), neurofibrillary dementia, diffuse neurofibrillary changes with calcification (DNTC), white matter tauopathies with globular glial inclusions (WMT-GGI), and frontotemporal lobar degeneration with tau-positive inclusions (FTLD-tau). In addition to neurodegenerative diseases, tauopathies also include diseases caused by infectious diseases such as post-encephalitis lethargica and subacute sclerosing panencephalitis, and diseases caused by trauma such as pugilistic encephalopathy (Non-Patent Document 4).

The genomic structure of the MAPT gene contains 13 exons, which express multiple isoforms of the protein through alternative splicing (Non-Patent Document 4). In addition, as a structural feature of tau, the 29 amino acid repeat sequence (N) based on the alternative splicing of exon 2 and exon 3 is composed of 0 to 2 (N0 to N2) N-terminal acidic domains, an intermediate domain containing a large number of prolines, and a C-terminal microtubule binding domain (encoded by exons 9 to 12) containing 3 (3R) or 4 (4R) repeat sequences (R) involved in microtubule binding (Non-Patent Documents 3 and 4). Therefore, based on the 29-amino acid repeat sequence (N) and the number of repeat sequences (R) involved in microtubule binding, tau has six representative isoforms: 3R0N (352 amino acids), 3R1N (381 amino acids), 3R2N (410 amino acids), 4R0N (383 amino acids), 4R1N (412 amino acids), and 4R2N (441 amino acids). Of these isoforms, only 3R0N is present in the fetal brain. In contrast, all six isoforms are present in the adult human brain, but the 4R isoform is predominant (Non-Patent Document 3). The difference between the 3R and 4R isoforms is determined by the fact that the 3R isoform (4R) contains exon 10 removed by alternative splicing. Thus, although there are several isoforms in tau, in order to identify the amino acid numbers at the corresponding positions, the isoform is represented by the amino acid numbers (1 to 441) in 4R2N (SEQ ID NO: 1), which is the longest isoform. For example, when Ser413 is represented, it represents the 413th amino acid residue, which is serine, in 4R2N (SEQ ID NO: 1), the 384th amino acid residue, which is serine, in 4R1N (SEQ ID NO: 2), the 355th amino acid residue, which is serine, in 4RON (SEQ ID NO: 3), the 382nd amino acid residue, which is serine, in 3R2N (SEQ ID NO: 4), the 353rd amino acid residue, which is serine, in 3R1N (SEQ ID NO: 5), and the 324th amino acid residue, which is serine, in 3RON (SEQ ID NO: 6).

The participation of tau in neurodegenerative diseases exists in the frontotemporal dementia (FTDP-17) with chain in initial chromosome 17 companion parkinsonism by the relation explanation of MAPT gene mutation and tau accumulation, in FTDP-17, report the gene mutation (non-patent literature 4) of more than 40 kinds in MAPT gene.Such gene mutation can cause the ratio change of tau isoform or the change of the interaction of sudden change tau and microtubule, and hints at the possibility of participating in morbid formation.But, be different from familial neurodegenerative diseases, in sporadic neurodegenerative diseases such as AD, often can not see the sudden change of MAPT.In addition, as the feature of tau accumulated in neurodegenerative diseases, there is the feature of height accepting phosphorylation modification.In addition, in the patient (MCI) that demonstrates mild cognitive impairment, between the phosphorylation tau amount in bone marrow fluid and pituitary atrophy, find correlation, think that phosphorylation tau is the high reliability biomarker (non-patent literature 5) about the neurodegeneration in Tau disease patient. Therefore, in order to suppress the excessive phosphorylation of the phosphoric acid of tau, attempt to use in treatment for the kinase as the enzyme relevant with phosphorylation, wherein particularly the enzyme inhibitor of GSK-3 β , and developed (non-patent literature 5).But think that the kinases such as GSK-3 β are not only relevant with function control under morbid state, in normal physiological effect, therefore worry about side effect etc..In fact, in the site of tau by GSK-3 β phosphorylation, there are several sites consistent with the phosphorylation site of tau seen in fetus or normal person's brain (non-patent literature 3), thus also consider its possibility of affecting normal tau function.

In addition, according to previous considerations, it is believed that the tau present in the extracellular space leaks out of the cell due to the cell death of the degenerated nerve cells, but according to recent studies, it is believed that after being hyperphosphorylated in the cell, tau undergoes processing and is actively secreted outside the cell. It is believed that the phosphorylated tau secreted outside the cell undergoes dephosphorylation at some of the phosphorylation sites, and then interacts with the M1 and M3 of the muscarinic receptors of the surrounding cells, participating in the effects of hyperphosphorylation of tau or induction of cell death in the cell (non-patent document 6, non-patent document 7). In this way, it is believed that tau plays the role of a factor acting outside the cell, so the possibility of using high molecules such as antibodies that are difficult to play a role inside the cell as a therapeutic ingredient is gradually being taken seriously. However, as mentioned above, a part of the tau secreted outside the cell undergoes processing and also undergoes dephosphorylation, etc. Based on the structural information of the hyperphosphorylated tau that has been targeted so far, there is the possibility of further changes. Moreover, it is also considered that the possibility of drugs that act on the dephosphorylated tau part also affects the normal tau function. When targeting pathological tau with antibodies, it is important to select a pathologically specific site, that is, a site that interacts with the phosphorylated epitope of tau. However, the presence of such complex information makes it more difficult to select such an epitope.

As an attempt to point to the specific effect of tau, the invention (non-patent literature 5, patent literature 1, patent literature 2, patent literature 3) of the immunological treatment of Tau disease with tau protein as the target has been reported. Immunological treatment refers to a therapy carried out for the purpose of inducing the production of specific antibodies by administering peptide vaccines, etc., and due to the high specificity for the target protein or peptide, it is expected to reduce side effects. As such a combination, it has been reported that an animal model expressing mutant tau is immunized by using a vaccine of a partial peptide of phosphorylated tau (a peptide equivalent to a phosphorylated amino acid residue of Ser396 and Ser404 and a peptide equivalent to a phosphorylated amino acid residue of Ser262), thereby improving the motor function of the model animal and improving the like. But, in these reports, be to use the research of the transgenic mouse (tg mouse) of gene mutation imported P301L (as the mutation that the proline of the 301st amino acid residue of tau is changed into leucine), although this tg mouse is about the gene mutation model of FTDP-17 as one of familial neurodegenerative disease, but may not show the morbidity of most, particularly sporadic neurodegenerative disease in the neurodegenerative disease that is not accompanied by the tau gene mutation comprised by Tau protein disease.In addition, P301Ltg mouse is the model of motor dysfunction, is not the model (non-patent literature 8) of cognitive dysfunction that becomes a problem in people's dementia etc., therefore, according to the result in this animal model, it is difficult to know whether it can be suitable for the treatment of people's dementia. In patent documentation 4, give the antibody that antigen-antibody reaction occurs with the phosphorylated tau peptide of Ser409, observe the effect about Tau protein disease treatment.But, as expensive peptide vaccine, total dosage is many and long until the period that embodies effect. Furthermore, the immune response of administered humans or animals varies depending on their genetic background, and peptide vaccines may not effectively induce antibody production in all individuals. Therefore, immunotherapy using passive immunization with antibodies is considered, but there are numerous phosphorylation sites in tau, and there is little information on which phosphorylation sites antibodies are effective against. Furthermore, even if existing antibodies are shown to be effective in animal models, they are not considered to be sufficiently potent for therapeutic use.

Furthermore, when using antibodies as the main agent of therapeutic or preventive drugs, the dosage of the antibody used for treatment must be carefully considered to avoid side effects and medical economic issues. Dosage is particularly important for chronic diseases or genetic disorders. For example, Actemra (registered trademark) (tocilizumab), a human anti-IL-6R antibody, is used in treatment at a dosage of 8 mg/kg body weight, administered at intervals of 1 to 4 weeks; Soliris (registered trademark) (eculizumab), a humanized anti-complement C5 antibody, is used in treatment at a dosage of 600 to 900 mg per adult, administered every 2 to 4 weeks. These antibodies are excellent antibodies selected from a wide range of antibodies and their dosages are among the highest among currently used antibody drugs. Therefore, future antibody drugs will also need to achieve the same or lower efficacy at dosages. Furthermore, the brain is the target organ for treatment of dementias such as AD. However, when administering systemically, such as intravenously or subcutaneously, the blood-brain barrier has been reported to limit the transfer rate of antibodies from the blood to the brain. Therefore, compared to diseases targeting other organs, it is difficult to expect pharmacological effects from antibodies used to treat dementia, presenting a major challenge.

In people's dementia, cardinal symptoms are memory impairment and cognitive dysfunction, and particularly cognitive function is to play judgment, communication, behavioral function according to memory, and is therefore an important function, so it occupies an important position as the symptom of dementia. On the other hand, about motor function, although this function is the symptom confirmed in the frontotemporal dementia (FTDP-17) or terminal Alzheimer's disease of the companion parkinsonism syndrome of chain 17 chromosomes, it is not necessarily the cardinal symptom shown in dementia. Therefore, when considering the treatment of dementia, improving cognitive function becomes a major issue. However, under the current situation, in order to solve such a problem, it has not yet been shown that the appropriate animal model of the necessary cognitive dysfunction caused by Tau disease can be used to obtain the dementia therapeutic drug or the method for the preventive drug demonstrating excellent cognitive function improvement effect, in addition, the dementia therapeutic drug or the preventive drug demonstrating specific excellent effect do not exist to dementia.

Therefore, under the circumstances described above, there is a demand for the development of therapeutic or preventive drugs having a strong effect on improving cognitive function, in particular.

Prior art literature

Patent Literature

Patent Document 1: US8012936;

Patent Document 2: WO2010/142423;

Patent Document 3: WO2010/144711;

Non-patent literature

Non-patent literature 1: Kishimoto Toshifumi and Takahashi Shigeki (eds.), STEP Series Psychiatry, 2nd edition, pp. 103-104, Kaiba Shobo, 2008;

Non-patent literature 2: Asada Takashi, "Dementia," Supplementary Journal of Medical Advances, pp. 5-10, Ichiyaku Publishing, 2011;

Non-Patent Document 3: Alistair Burns, John O’Brien, and David Ames, eds., Dementia. 3rd edition, pp. 408–464, 2005;

Non-patent document 4: Tetsuaki Arai, Neurology, Vol. 72, Special Supplement No. 6, pp. 46-51, 2010;

Non-Patent Literature 5: Wendy Noble et al., Expert Opin. Drug Discov., Vol. 6, No. 8, pp. 797-810, 2011;

Non-Patent Literature 6: Miguel Diaz-Hernandes et al., Journal of Biological Chemistry, Vol. 285, pp. 32539-32548, 2010;

Non-patent document 7: Venessa Plouffe et al., PLoS ONE, Vol. 7, p. 36873, 2012;

Non-Patent Literature 8: Alistair Burns, John O’Brien, and David Ames, eds., Dementia. 3rd edition, page 459, 2005.

Summary of the Invention

Problems to be solved by the invention

The present invention aims to provide a therapeutic or preventive drug for dementia by focusing on tau phosphorylation in tauopathies.

In addition, the present invention also aims to provide a therapeutic or preventive drug for dementia containing as an active ingredient an antibody that specifically reacts with tau corresponding to phosphorylated amino acid residues around Ser413, or a peptide having an amino acid sequence around Ser413 and at least one phosphorylated amino acid residue.

Furthermore, the present invention aims to provide a monoclonal antibody having a high cognitive function-improving effect, and further provides a method for producing an antibody more suitable for the treatment of dementia, such as a humanized antibody, based on structural analysis of the monoclonal antibody.

Means for solving problems

The present invention is as follows. It should be noted that the tau protein in the present invention not only includes 4R2N, but also includes all 6 isoforms. In addition, the position of the amino acid residue in the present invention is conveniently specified according to the amino acid residue shown in SEQ ID NO: 1. For example, when expressed as an amino acid residue equivalent to Ser413 of SEQ ID NO: 1, it means the serine at position 413 of SEQ ID NO: 1 (4R2N), the serine at position 384 of SEQ ID NO: 2 (4R1N), the serine at position 355 of SEQ ID NO: 3 (4R0N), the serine at position 382 of SEQ ID NO: 4 (3R2N), the serine at position 353 of SEQ ID NO: 5 (3R1N), or the serine at position 324 of SEQ ID NO: 6 (3R0N).

(1) A dementia treatment or prevention drug comprising as an active ingredient an antibody that reacts with a tau protein corresponding to at least one of the amino acid residues 410 to 421 of the tau protein represented by SEQ ID NO: 1 and undergoes an antigen-antibody reaction.

(2) The therapeutic or preventive drug for dementia according to (1), wherein the antibody is an antibody that undergoes an antigen-antibody reaction with phosphorylated tau protein, which is characteristic of dementia.

(3) The dementia therapeutic or preventive drug according to (1) or (2), wherein the antibody undergoes an antigen-antibody reaction with tau protein phosphorylated at one or more sites selected from Ser412, Ser413, Thr414, and Ser416.

(4) The dementia therapeutic or preventive drug according to any one of (1) to (3), wherein the antibody competes for binding to tau protein with an antibody comprising a VH composed of the amino acids described in SEQ ID NO: 20 and a VL composed of the amino acid sequence described in SEQ ID NO: 26.

(5) The dementia therapeutic or preventive drug according to any one of (1) to (3), wherein the antibody comprises a VH consisting of the amino acid sequence described in SEQ ID NO: 20 and a VL consisting of the amino acid sequence described in SEQ ID NO: 26.

(6) The therapeutic or preventive drug for dementia according to any one of (1) to (4), wherein the antibody is an antibody that undergoes an antigen-antibody reaction with a tau protein in which the amino acid residue corresponding to Ser413 is phosphorylated.

(7) The dementia therapeutic or preventive drug according to any one of (1) to (6), wherein the antibody comprises an H-chain CDR sequence represented by SEQ ID NOs: 7 to 13, an H-chain CDR sequence represented by at least one of SEQ ID NOs: 7 to 13, or an H-chain CDR sequence having 85% or more identity with at least one of the H-chain CDR sequences represented by SEQ ID NOs: 7 to 13; and/or an L-chain CDR sequence represented by SEQ ID NOs: 14 to 17, an L-chain CDR sequence represented by at least one of SEQ ID NOs: 14 to 17, or an L-chain CDR sequence having 85% or more identity with at least one of the L-chain CDR sequences represented by SEQ ID NOs: 14 to 17.

(8) The dementia therapeutic or preventive drug according to any one of (1) to (7), wherein the antibody comprises an H chain variable region represented by any one of SEQ ID NOs: 18 to 24, or an H chain variable region having a sequence having 85% or more homology with any one of SEQ ID NOs: 18 to 24; and/or an L chain variable region represented by any one of SEQ ID NOs: 25 to 30, or an L chain variable region having a sequence having 85% or more homology with any one of SEQ ID NOs: 25 to 30.

(9) The therapeutic or preventive drug for dementia according to any one of (1) to (8), wherein the antibody is a humanized antibody or a chimeric antibody.

(10) A dementia treatment or prevention drug containing a peptide as an active ingredient, wherein the peptide is a peptide comprising at least 8 consecutive amino acid sequences in an amino acid sequence consisting of amino acid residues corresponding to amino acid numbers 410 to 421 of SEQ ID NO: 1, and at least one amino acid residue contained in the peptide is phosphorylated.

(11) The dementia therapeutic or preventive drug according to (10), wherein at least one of the phosphorylated amino acid residues contained in the peptide is an amino acid residue corresponding to Ser412, Ser413, Thr414 or Ser416 of SEQ ID NO: 1.

(12) The dementia therapeutic or preventive drug according to (10) or (11), wherein the phosphorylated amino acid residue contained in the peptide is at least an amino acid residue corresponding to Ser413 of SEQ ID NO: 1.

(13) The therapeutic or preventive drug for dementia according to any one of (1) to (12), wherein the dementia is tauopathy.

(14) The dementia therapeutic or preventive drug described in (13), wherein the tauopathy is Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multisystem tauopathy in dementia patients (MSTD), frontotemporal dementia with parkinsonian syndrome linked to chromosome 17 (FTDP-17), neurofibrillary dementia, diffuse neurofibrillary changes with calcium deposition (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), or frontotemporal lobar degeneration with tau-positive inclusions (FTLD-tau).

(15) A monoclonal antibody that undergoes an antigen-antibody reaction with a peptide having at least eight consecutive amino acids in an amino acid sequence consisting of amino acid numbers 410 to 421 of SEQ ID NO: 1, wherein the amino acid residue corresponding to Ser413 of SEQ ID NO: 1 contained in the peptide is phosphorylated.

(16) An antibody directed against phosphorylated tau protein, wherein binding to the antigen is competitive between the antibody and an antibody comprising a VH composed of the amino acids set forth in SEQ ID NO: 20 and a VL composed of the amino acid sequence set forth in SEQ ID NO: 26.

(17) An antibody directed against phosphorylated tau protein, comprising a VH consisting of the amino acids set forth in SEQ ID NO: 20 and a VL consisting of the amino acid sequence set forth in SEQ ID NO: 26.

(18) A monoclonal antibody comprising an H chain and/or an L chain, wherein the H chain has an H chain CDR sequence represented by SEQ ID NOs: 7 to 13, an H chain CDR sequence represented by at least one of SEQ ID NOs: 7 to 13, or a sequence having 85% or more homology to at least one of the H chain CDR sequences represented by SEQ ID NOs: 7 to 13, and the L chain has an L chain CDR sequence represented by SEQ ID NOs: 14 to 17, an L chain CDR sequence represented by at least one of SEQ ID NOs: 14 to 17, or a sequence having 85% or more homology to at least one of the L chain CDR sequences represented by SEQ ID NOs: 14 to 17.

(19) A monoclonal antibody comprising an H chain variable region represented by any one of SEQ ID NOs: 18 to 24, or an H chain variable region having 85% or more homology to any one of SEQ ID NOs: 18 to 24, and/or an L chain variable region represented by any one of SEQ ID NOs: 25 to 30, or an L chain variable region having 85% or more homology to any one of SEQ ID NOs: 25 to 30.

(20) The monoclonal antibody according to any one of (15) to (19), wherein the antibody is a humanized antibody or a chimeric antibody.

(21) A peptide comprising at least 8 consecutive amino acid sequences in an amino acid sequence consisting of amino acid residues corresponding to amino acid numbers 410 to 421 of SEQ ID NO: 1, wherein at least one of the amino acid residues contained in the peptide is phosphorylated.

(22) The peptide described in (21) is characterized in that at least one of the amino acid residues corresponding to Ser412, Ser413, Thr414 and Ser416 of SEQ ID NO: 1 is phosphorylated.

(23) The peptide according to (21) or (22), wherein the phosphorylated amino acid residue is an amino acid residue corresponding to Ser413 of SEQ ID NO: 1.

Effects of the Invention

The present invention can provide a therapeutic or preventive drug for dementia by containing as an active ingredient an antibody that specifically reacts with tau phosphorylated around the amino acid residue corresponding to Ser413 of SEQ ID NO: 1, or a peptide having an amino acid sequence around Ser413 of SEQ ID NO: 1 and at least one amino acid residue phosphorylated.

The present invention also provides a monoclonal antibody having a high cognitive function-improving effect, and further provides a method for producing an antibody more suitable for the treatment of dementia, such as a humanized antibody, based on structural analysis of the monoclonal antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the results of aligning the first half of the amino acid sequences of human tau protein isoforms using ClustalW.

FIG2 is a diagram showing the results of aligning the second half of the amino acid sequences of human tau protein isoforms using ClustalW.

FIG3 is a graph showing the specificity of rabbit polyclonal antibodies against pSer413 peptide.

FIG. 4 is a graph showing the results of a trial experiment in model mice administered with a pSer413-recognizing rabbit polyclonal antibody.

FIG5 is a graph showing the results of a probe experiment in model mice administered with a pSer413-recognizing rabbit polyclonal antibody.

FIG6 is a line graph showing the results of an open field test in model mice administered with a pSer413-recognizing rabbit polyclonal antibody.

FIG7 is a bar graph showing the results of an open field test in model mice administered with a pSer413-recognizing rabbit polyclonal antibody.

FIG8 is a graph showing the results of a trial experiment in model mice administered with a pSer413-recognizing mouse monoclonal antibody (Ta1505).

FIG. 9 is a graph showing the results of a probing experiment using model mice administered with a pSer413-recognizing mouse monoclonal antibody (Ta1505).

FIG10 is a line graph showing the results of an open field test in model mice administered with a pSer413-recognizing mouse monoclonal antibody (Ta1505).

FIG11 is a bar graph showing the results of an open field test in model mice administered with a pSer413-recognizing mouse monoclonal antibody (Ta1505).

FIG. 12 is a diagram showing the results of a trial experiment in model mice administered with a pSer396-recognizing mouse monoclonal antibody (Ta9).

FIG. 13 is a graph showing the results of a probing experiment using model mice administered with a pSer396-recognizing mouse monoclonal antibody (Ta9).

FIG14 is a line graph showing the results of an open field test in model mice administered with a pSer396-recognizing mouse monoclonal antibody (Ta9).

FIG15 is a bar graph showing the results of an open field test in model mice administered with a pSer396-recognizing mouse monoclonal antibody (Ta9).

FIG. 16 is a graph showing the amount of synaptophysin in the hippocampus of model mice to which Ta1505 antibody was administered.

FIG. 17 is a diagram showing gene fragments contained in the tau gene.

FIG. 18 is a graph showing the results of a water maze trial of learning-impaired mice (Tau-Tg) administered with the Ta1505 antibody.

FIG. 19 is a graph showing the results of a water maze exploration test in learning-impaired mice (Tau-Tg) to which Ta1505 antibody was administered.

FIG20 is a graph showing the results of a water maze trial of learning-impaired mice (Tau-Tg) administered with the Ta9 antibody.

FIG21 is a graph showing the results of a water maze exploration test in mice with learning impairment (Tau-Tg) to which Ta9 antibodies were administered.

FIG22 presents photographs showing immunohistochemical staining of the hippocampal CA3 and CA23 regions using Ta1505 antibody in memory-impaired mice (Tau-Tg) administered with control IgG (1 mg/mouse) or Ta1505 antibody (1 mg/mouse).

FIG23 presents photographs showing immunohistochemical staining of the parahippocampus using the Ta1505 antibody in memory-learning-impaired mice (Tau-Tg) administered with control IgG (1 mg/mouse).

FIG. 24 presents photographs showing immunohistochemical staining of the parahippocampus region using the Ta1505 antibody in memory-learning-impaired mice (Tau-Tg) to which the Ta1505 antibody (1 mg/mouse) had been administered.

FIG25 presents photographs showing immunohistochemical staining of the hippocampal CA3 and CA23 regions using AT8 in learning-impaired mice (Tau-Tg) administered with control IgG (1 mg/mouse) or Ta1505 antibody (1 mg/mouse).

FIG26 shows photographs of immunohistostaining of the parahippocampus with AT8 in memory-learning-impaired mice (Tau-Tg) to which a control antibody (1 mg/mouse) had been administered.

FIG27 shows photographs showing immunohistochemical staining of the parahippocampus region using AT8 in memory-learning-impaired mice (Tau-Tg) to which Ta1505 (1 mg/mouse) had been administered.

FIG28 is a graph showing the amounts of G2, AT8, PHF1, and Ta1505-reactive Tau in TBS-soluble brain fractions of memory-learning-impaired mice (Tau-Tg) to which Ta1505 (1 mg/mouse) was administered.

FIG29 is a graph showing the amounts of G2, AT8, PHF1, and Ta1505-reactive Tau in sarkosyl-soluble brain fractions of memory-learning-impaired mice (Tau-Tg) to which Ta1505 (1 mg/mouse) was administered.

DETAILED DESCRIPTION

The present inventors made antibodies that specifically reacted with tau phosphorylated at the amino acid residue Ser413 of SEQ ID NO: 1, which is a specific phosphorylation site in AD, and gave them to tg mice that developed cognitive dysfunction as they matured. They found that cognitive function could be restored to almost the same level as in the control group. On the other hand, when compared with antibodies to tau phosphorylated at the amino acid residue Ser396 of SEQ ID NO: 1, which had a stronger affinity than the antibodies of the present invention for the same antigen at the same concentration, no sufficient improvement in cognitive function was found. The portion surrounding the amino acid residue Ser413 of SEQ ID NO: 1 is a region with no information about the relationship between the structure and function of tau, and the fact that antibodies that reacted specifically with this portion had such a strong effect on improving cognitive function was a completely unexpected result. Therefore, the portion surrounding the amino acid residue Ser413 of SEQ ID NO: 1, which had not been paid attention to so far, is an important site for the onset of cognitive dysfunction in Tau protein disease. This was clarified for the first time by the present invention, thereby completing the present invention.

[Anti-phospho-tau antibody]

Tau (protein) in the present invention includes not only the human tau proteins set forth in SEQ ID NOs: 1-6, but also their genetic variants. As described in the aforementioned background art, FTDP-17, a familial neurodegenerative disease associated with dementia, is known to have over 40 mutations, but the mutation sites are not limited to these. Furthermore, tau proteins with 1-50, preferably 1-30, and more preferably 1-10 amino acid mutations relative to SEQ ID NOs: 1-6 are also considered tau in the present invention. Furthermore, proteins and isoforms thereof that exhibit 80% or greater identity to the human tau protein set forth in SEQ ID NO: 1 according to the BLAST method (using the default conditions of NCBI's PBLAST) are also included. Such tau includes tau from species other than humans, such as chimpanzees, rhesus monkeys, horses, pigs, dogs, mice, rabbits, and rats. To improve cognitive function in such animals, therapeutic or preventive drugs targeting such tau can also be developed.

In addition, the amino acid numbering, i.e., the position of the amino acid residue in the present invention is conveniently specified according to the numbering shown in SEQ ID NO: 1. Thus, for example, when it is expressed as an amino acid residue equivalent to Ser413 of SEQ ID NO: 1, it refers to a serine as the amino acid residue at position 413 of SEQ ID NO: 1 (4R2N), position 384 of SEQ ID NO: 2 (4R1N), position 355 of SEQ ID NO: 3 (4R0N), position 382 of SEQ ID NO: 4 (3R2N), position 353 of SEQ ID NO: 5 (3R1N), or position 324 of SEQ ID NO: 6 (3R0N). Table 1 shows the positions of amino acid residues that are located at the same position in each isoform. It should be noted that although Table 1 shows the positions of amino acid residues in each isoform equivalent to SEQ ID NO: 410 to 421 of 1, the positional relationship between amino acid residues located at other positions can be easily understood, for example, based on Figures 1 and 2.

[Table 1]

Anti-phosphorylated tau antibodies refer to antibodies that react with tau obtained by phosphorylating more than one amino acid residue in the amino acid sequence of tau shown above. As phosphorylated amino acid residues, serine (Ser), threonine (Thr), tyrosine (Tyr) and the like can be cited. In addition, the site of phosphorylated tau for which the anti-phosphorylated tau antibodies of the present invention react with antigens and antibodies is preferably a site that is specifically phosphorylated in neurodegenerative diseases such as AD. In addition, another preferred embodiment of the site in phosphorylated tau at which the anti-phospho-tau antibody of the present invention undergoes an antigen-antibody reaction is an antibody that undergoes an antigen-antibody reaction with tau phosphorylated at one or more sites selected from the group consisting of amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1. More preferably, it is an antibody that undergoes an antigen-antibody reaction with tau phosphorylated at amino acid residues corresponding to Ser412, Ser413, or both of SEQ ID NO: 1. Even more preferably, it is an antibody that undergoes an antigen-antibody reaction with tau phosphorylated at amino acid residues corresponding to Ser413 of SEQ ID NO: 1. Here, the amino acid residues equivalent to Ser412, Ser413, Thr414 and Ser416 of SEQ ID NO: 1 refer to sites corresponding to the amino acid numbering in human 4R2Ntau (SEQ ID NO: 1), but as described in the above-mentioned background technology, the corresponding sites in the isoforms and the sites corresponding to the homologues outside the human are treated in the same manner, regardless of the amino acid numbering attached to the amino acid sequence. In order to understand the corresponding sites in such isoforms or homologues, those skilled in the art can appropriately analyze and understand by methods such as Pairwise sequence alignments such as the Needleman-Wunsch method or the Smith-Waterman method, or multiple sequence alignments such as the ClustalW method or the PRRP method. As an example of an analysis method for corresponding sites, Figures 1 and 2 show the results obtained by arranging the amino acid sequences (single letter representation) of 6 isoforms in humans using ClustalW. When observing these results, the structures around the amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1 are conserved in the six isoforms, and it can be easily understood whether the corresponding amino acid is any one of them.

The anti-phosphorylated tau antibody that can be used in the therapeutic or preventive agent of the present invention is an antibody that specifically reacts with a tau protein in which at least one of the amino acid residues 410 to 421 present in the tau protein of SEQ ID NO: 1 is phosphorylated. It is an antibody that specifically reacts with a tau protein in which one or more sites selected from the group consisting of amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1 are phosphorylated. More preferably, it is an antibody that competes for binding with an antibody having a VH amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence of SEQ ID NO: 26 (hereinafter also referred to as "1505 antibody"). More preferably, it is an antibody that specifically reacts with a tau protein phosphorylated at Ser413. As described above, tau protein in which at least one of the amino acid residues corresponding to positions 410 to 421 of SEQ ID NO: 1 is phosphorylated, tau protein in which one or more sites selected from the group consisting of amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1 are phosphorylated, or tau protein in which a site corresponding to the amino acid residue corresponding to Ser413 of SEQ ID NO: 1 is phosphorylated means tau protein in which the corresponding sites are phosphorylated, including isoforms in humans or other species, but is more preferably human tau protein, and more preferably any one of the six human isoforms.

Furthermore, in the present invention, a tau protein in which at least one of the amino acid residues corresponding to positions 410 to 421 of SEQ ID NO: 1 is phosphorylated, a tau protein in which one or more sites selected from the group consisting of amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1 are phosphorylated, or a tau protein in which a site corresponding to the amino acid residue corresponding to Ser413 of SEQ ID NO: 1 is phosphorylated, and a peptide having an amino acid sequence that is completely identical or has a sequence identity of 80% or more with a portion of the amino acid residues corresponding to positions 410 to 421 of the tau protein, and in which the amino acid residues are phosphorylated, is also included in such phosphorylated tau proteins. Antibodies that specifically react with such peptides are also anti-phosphorylated tau antibodies of the present invention.

In the present invention, "performing an antigen-antibody reaction" means binding to a tau protein that is phosphorylated at least one of the amino acid residues corresponding to positions 410 to 421 of SEQ ID NO: 1, a tau protein that is phosphorylated at one or more sites selected from the group consisting of amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1, and/or a tau protein that is phosphorylated at a site corresponding to the amino acid residue Ser413 of SEQ ID NO: 1 with an affinity of 1× ^10-6 M or greater, preferably binding with an affinity of 1× ^10-7 M or greater, and further preferably binding with an affinity of 1× ^10-8 M or greater.

Furthermore, "specifically" means that the binding to tau protein corresponding to at least one of the amino acid residues 410 to 421 of SEQ ID NO: 1 is phosphorylated, tau protein phosphorylated at one or more sites selected from the group consisting of amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1, and/or tau protein phosphorylated at the site corresponding to the amino acid residue Ser413 of SEQ ID NO: 1 is 10 times or more, preferably 30 times or more, and even more preferably 100 times or more stronger than the binding to tau protein not phosphorylated at the site (including a peptide having an amino acid sequence identical to a portion of tau protein or having a sequence identity of 80% or more).

Furthermore, "competing for binding" with an antibody having a VH amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence of SEQ ID NO: 26 (antibody 1505) means that when the monoclonal antibody binds to an antigen in the presence of another antibody, the binding of the monoclonal antibody is inhibited. This is generally measured by measuring the amount (concentration) at which the amount of the monoclonal antibody binding to the antigen decreases when the amount (concentration) of the other antibody added is varied relative to a certain amount (concentration) of the monoclonal antibody. The degree of inhibition can be expressed as an _IC50 or Ki value. In the present invention, an antibody that competes for binding with an antibody having a VH amino acid sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 26 (antibody 1505) means an antibody having an IC50 value of less than 1 μM, more preferably an _IC50 value of less than 100 nM, and even more preferably an _IC50 value of less than 10 nM, when the monoclonal antibody is used at ₁₀ nM to detect antigen-antibody binding.

Regarding the antibody-antigen binding of such an antibody to phosphorylated tau protein, those skilled in the art can appropriately select a binding assay in a solid phase or liquid phase system to conduct the assay. Examples of such methods include, but are not limited to, ELISA, EIA, surface plasmon resonance, FRET, and LRET. Furthermore, when measuring such antigen-antibody binding, the antibody and/or antigen (phosphorylated tau protein or tau protein) can be labeled with an enzyme, a fluorescent substance, a luminescent substance, a radioisotope, or the like, and the antigen-antibody reaction can be detected using a method suitable for measuring the physical and/or chemical properties of the labeled substance.

In addition, the anti-phospho-tau antibody of the present invention also includes the following antibody, wherein the H chain variable region of the antibody comprises the amino acid sequence of CDR-H1 consisting of SEQ ID NO: 7 or 8, the amino acid sequence of CDRH-H2 consisting of any one selected from SEQ ID NO: 9, 10, 11 and 12, and the amino acid sequence of CDRH-H3 consisting of SEQ ID NO: 13, and the L chain variable region comprises the amino acid sequence of CDR-L1 consisting of SEQ ID NO: 14 or 15, the amino acid sequence of CDR-L2 consisting of SEQ ID NO: 16, and the amino acid sequence of CDR-L3 consisting of SEQ ID NO: 17. Preferably, the group of CDR-H1, CDR-H2 and CDR-H3 contained in the H chain variable region is selected from the following combinations, namely SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 13; SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 13; and any one of SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 13, and the group of CDR-L1, CDR-L2 and CDR-L3 contained in the L chain variable region is SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17; or SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17; more preferably, an antibody wherein the combination of the group of CDR-H1, CDR-H2 and CDR-H3 contained in the H chain variable region, and the group of CDR-L1, CDR-L2 and CDR-L3 contained in the L chain variable region is any one selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17; NO: 16 and SEQ ID NO: 17; and SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17. Antibodies in which at least one of the corresponding CDR sequences in the amino acid sequences of these CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 shows 85% or more identity, preferably 90% or more identity, to any of SEQ ID NOs: 7 to 17 according to the BLAST method (default conditions of NCBI's PBLAST) are also included in the antibodies of the present invention.

Here, as a method for identifying the sequence of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 in an antibody, those skilled in the art can identify the sequence of the portion corresponding to each CDR by using, for example, the method of Kabat or the method of Chothia. The methods of Kabat (Kabat, E.A. and Wu, T.T., J. Immunol., 147, 1709-1719, 1991) or Chothia (Al-Lazikani, B., Lesk, A.M. and Chothia, C., J. Mol. Biol., 273, 927-948, 1997) are common technical knowledge to those skilled in the art, and an overview thereof can be found, for example, on the homepage of Dr. Andrew C.R. Martin's Group (http://www.bioinf.org.uk/abs/).

Another aspect of the antibody of the present invention is an antibody having an amino acid sequence of the H chain variable region (VH) selected from any one of SEQ ID NOs: 18 to 24 and an amino acid sequence of the L chain variable region (VL) selected from any one of SEQ ID NOs: 25 to 30. Preferably, the combination of the amino acid sequences of VH and VL is any one selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 25, SEQ ID NO: 19 and SEQ ID NO: 26, SEQ ID NO: 20 and SEQ ID NO: 26, SEQ ID NO: 21 and SEQ ID NO: 27, SEQ ID NO: 22 and SEQ ID NO: 28, SEQ ID NO: 23 and SEQ ID NO: 29, and SEQ ID NO: 24 and SEQ ID NO: 30. Here, at least one of the VH and VL amino acid sequences is any one of the VH amino acid sequences shown in SEQ ID NOs: 18 to 24 or any one of the VL amino acid sequences shown in SEQ ID NOs: 25 to 30, and antibodies whose sequences show 85% or more identity (identities), preferably 90% or more identity, to SEQ ID NOs: 18 to 30 by the BLAST method (default conditions of NCBI's PBLAST) are also included in the antibodies of the present invention.

In this antibody, the amino acid sequence of the constant region is selected from mammalian IgG, IgA, IgM, IgE, IgD subtypes or mutants thereof.

Based on the information of the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 and/or the nucleotide sequences of the genes encoding them, and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 and/or the nucleotide sequences of the genes encoding them, those skilled in the art can design recombinant antibodies such as humanized antibodies suitable for administration to the target species according to therapeutic purposes. Based on the information of the amino acid sequences of the H chain variable regions and/or the nucleotide sequences of the genes encoding them, and the amino acid sequences of the L chain variable regions and/or the nucleotide sequences of the genes encoding them, those skilled in the art can design chimeric antibodies and the like that meet the purpose. In addition, those skilled in the art can also use known techniques to appropriately prepare low-molecular-weight antibodies or scaffold antibodies based on information on the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 and/or the nucleotide sequences of the genes encoding them, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 and/or the nucleotide sequences of the genes encoding them, or the amino acid sequences of the H chain variable regions and/or the nucleotide sequences of the genes encoding them, and the amino acid sequences of the L chain variable regions and/or the nucleotide sequences of the genes encoding them.

The antibodies of the present invention may be composed of two H chains and two L chains, or may be composed of only two H chains. Furthermore, the antibodies of the present invention may be antibody fragments, and examples of such antibody fragments include structures such as F(ab')2, Fab, and Fv.

The antibodies of the present invention can be obtained by methods well known to those skilled in the art. The antibodies of the present invention may be polyclonal antibodies or monoclonal antibodies (Milstein et al., Nature (England) October 6, 1983, Vol. 305, No. 5934, pp. 537-540). For example, a tau protein corresponding to at least one of the amino acid residues 410 to 421 of SEQ ID NO: 1 is phosphorylated, a tau protein selected from one or more sites of Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1 is phosphorylated, a tau protein phosphorylated at Ser413 of SEQ ID NO: 1, or a peptide that is completely identical to or has a sequence homology of 80% or more to at least a portion of the amino acid sequence of tau protein 410 to 421 of SEQ ID NO: 1 and the amino acid is phosphorylated can be used as an antigen to sensitize a mammal, and polyclonal antibodies can be recovered from the serum of the animal. In addition, when using a peptide as an antigen, an antigen in a form bound to a carrier protein such as BSA or KLH or polylysine can be used. In addition, as a specific example of a peptide used as an antigen, as described in Examples 1 and 2, a peptide of SEQ ID NO: 31 or 32 in which the site of Ser413 of SEQ ID NO: 1 is phosphorylated can be used, but it is not limited thereto. A hybridoma obtained by removing immune cells from an antigen-sensitized mammal and fusing them with myeloma cells or the like can be cloned, and the monoclonal antibody of the present invention can be recovered from its culture. As a method for obtaining such a monoclonal antibody, as described in Example 2, as the monoclonal antibody thus obtained, monoclonal antibodies (Ta1501, 1502, 1505-1509) having VH amino acid sequences of SEQ ID NO: 18 to 24 and VL sequences of SEQ ID NO: 25 to 30 can be listed, but it is not limited thereto.

The monoclonal antibody obtained can be obtained as a nucleic acid molecule having a gene sequence encoding the amino acids of the antibody protein, or such a nucleic acid molecule can be used to make an antibody by genetic engineering methods. Using the gene information of the antibody, such as the H chain, L chain, and the CDR sequence of their variable regions, it is possible to make changes to improve the binding or specificity of the antibody, or to make antibodies suitable for use in therapeutic drugs by changing the antibodies of animals such as mice into human antibodies. This is a technology well known to those skilled in the art. In addition, human monoclonal antibodies can also be obtained by using non-human transgenic animals into which human antibody genes have been introduced as animals for antigen sensitization. In addition, as a method that does not require sensitization of animals, those skilled in the art can appropriately adopt the following technology: using a phage library expressing the antigen binding region of a human antibody or a portion thereof (human antibody phage display), obtain antibodies that specifically bind to the corresponding antigen or phage clones consisting of a specific amino acid sequence, and make human antibodies based on this information. (For example, refer to the review by Taketo Tanaka et al., Keio J. Med., Vol. 60, pp. 37-46.)

In addition, as a method for preparing the above-mentioned monoclonal antibody, the hybridoma producing the antibody you want to obtain can be cultivated respectively, and the antibody can be purified from the culture supernatant obtained according to a conventional method. In addition, as other preparation methods, the gene encoding the antibody can also be obtained by producing the hybridoma producing the antibody you want to obtain or the phage clone obtained by human antibody phage display, etc., more specifically, the gene encoding the heavy chain and/or light chain of the immunoglobulin, and a vector for expressing the gene is made, which is introduced into a host cell (mammalian cell, insect cell, microorganism, etc.) to produce the antibody. At this time, for the gene encoding the heavy chain and/or light chain of the immunoglobulin, a gene change for importing the desired characteristic or the structural information of the variable region or CDR region of the immunoglobulin is used to make humanized antibodies, antibody chimeric proteins, low molecular weight antibodies or scaffold antibodies, which can be implemented by those skilled in the art by adopting known technology. In addition, in order to improve the performance of the antibody or avoid side effects, changes are introduced into the structure of the antibody constant region or changes in the sugar chain portion are carried out, which can also be appropriately carried out using technology well known to those skilled in the art.

[Phosphorylated peptide having an amino acid sequence derived from tau]

The present invention also includes peptides comprising a portion of the amino acid sequence of tau, wherein one or more amino acid residues are phosphorylated. Phosphorylated amino acid residues refer to a state in which a phosphate group is ester-bonded to the side chain of the amino acid residue. Representative examples of phosphorylated amino acid residues include serine, threonine, and tyrosine. Such phosphorylated peptides are peptides comprising at least three consecutive amino acids and having a length of eight or more amino acids, preferably at least five consecutive amino acids and having a length of eight or more amino acids, and more preferably at least eight consecutive amino acids and having a length of eight or more amino acids, in an amino acid sequence consisting of amino acid residues corresponding to amino acid residues 410 to 421 of SEQ ID NO: 1. Furthermore, the phosphorylated amino acid residue in such phosphorylated peptides is any one of amino acid residues corresponding to amino acid residues 410 to 421 of SEQ ID NO: 1, preferably any one of amino acid residues corresponding to Ser412, Ser413, Thr414, and Ser416 of SEQ ID NO: 1, and more preferably the amino acid residue corresponding to Ser413 of SEQ ID NO: 1. Representative examples of such phosphorylated peptides include the phosphorylated peptide (SEQ ID NO: 31) used for antibody preparation in Example 1, but are not limited thereto.

The phosphorylated peptides of the present invention having an amino acid sequence derived from tau can be used in dementia therapeutic or preventive drugs characterized by comprising an antigen or peptide used in the preparation of an anti-phospho-tau antibody according to the present invention. Depending on the intended purpose, the N-terminus and/or C-terminus of the phosphorylated peptide can also be modified with substances having other functions. For example, the N-terminus of the phosphorylated peptide may be modified with a methionine residue, an acetyl group, or pyroglutamic acid, or with a fluorescent substance. In addition, the substance that modifies the N-terminus and/or C-terminus of the phosphorylated peptide can be a peptide or protein. Examples of such substances include: peptides with an appropriate tag sequence (typically a histidine tag or FLAG tag) added to the N-terminus or C-terminus, peptides with amino acid sequences for recognition by T cell receptors (TCRs) or major histocompatibility antigens (MHCs), viral or bacterial proteins, or peptides with sequences derived from such proteins. In addition, the phosphorylated peptides of the present invention also include peptides in which at least one amino acid residue other than the N-terminus and C-terminus is modified with a compound or peptide. Such modification of phosphorylated peptides can be carried out by those skilled in the art using known methods, for example, the method described in Bioconjugate Techniques by Hermanson et al. (USA, published in 1996, Academic Press).

The phosphorylated peptides of the present invention can be prepared by those skilled in the art using appropriate synthesis methods or genetic engineering methods. As methods for preparing phosphorylated peptides by synthesis, examples include: a method using the Boc method (Wakamiya T. et al., Chemistry Letters, Vol. 22, p. 1401, 1993), a method using the Fmoc method (PERICH, J. W., International Journal of Peptide and Protein Research, Vol. 40, p. 134-140, 1992), or a method described in Japanese Patent No. 3587390, but those skilled in the art can also implement using other appropriate methods. In addition, in the preparation method using the genetic engineering method, a genetic material (DNA, RNA) having a nucleotide sequence encoding the peptide to be prepared or its precursor is modulated, inserted into an appropriate expression vector or a promoter sequence is added, and then introduced into an appropriate host to be expressed, or prepared by a method using a cell-free protein synthesis system, etc. In this case, to obtain a phosphorylated peptide at the desired site, a phosphorylation reaction can be carried out in the host by appropriately utilizing kinases possessed by the host or induced to express through genetic engineering, or the target peptide can be recovered and then subjected to a phosphorylation reaction in vitro by a kinase or the like. To carry out this phosphorylation reaction in vitro, enzymes known to function in tau phosphorylation of the target peptide, such as GSK3 or CDK5, can be utilized. Thus, to obtain a phosphorylated peptide at the target amino acid residue from among the phosphorylated peptides phosphorylated in the host or in vitro, a method can be employed, such as recovering the phosphorylated peptide that has specifically bound to the antibody through an antibody-antigen reaction against the above-mentioned anti-phospho-tau antibody.

[Dementia therapeutic or preventive drugs containing anti-phospho-tau antibodies or phosphorylated tau peptides]

Dementia in humans refers to a state of decreased cognitive function that has developed or been acquired, and is considered a type of intellectual disability (Kunitoshi Ueshima and Shinichi Niwa, eds., NANKODO's ESSENTIALWELL-ADVANCED SERIES New Psychiatry, pp. 69-70, 2008). However, it is generally considered to be a disease that presents with intellectual disability and/or memory impairment. In neurodegenerative diseases such as AD, "neurodegeneration" can be determined by, for example, confirming the presence of neurofibrillary changes (NFTs) through postmortem histological analysis. However, to diagnose neurodegenerative diseases, doctors diagnose dementia, particularly dementia as a neurodegenerative disease, based on neuropsychological tests such as the interview test/Hasegawa's Short-Form Scale-Revised (HDS-R) or the Mini-Mental State Examination (MMSE), observational tests such as the Clinical Dementia Rating (CDR) or the Functional Assessment Staging (FAST), biochemical tests such as increased levels of tau or phosphorylated tau or increased levels of Aβ in cerebrospinal fluid, and imaging tests such as head CT, neck MRI, SPECT, or PET. Therefore, when the dementia therapeutic or preventive drug of the present invention is administered to a patient diagnosed with dementia by a doctor, the dementia therapeutic or preventive drug of the present invention has an effect of improving the condition compared to before administration in one or more diagnostic items of the above-mentioned neurodegenerative diseases, or has an effect of suppressing the progression of the disease or maintaining or restoring the condition to the state before administration.

Patients to whom the therapeutic or preventive drug for dementia of the present invention is administered include patients with dementia, particularly patients with tauopathy, including patients with Alzheimer's disease (AD), corticobasal degeneration (CBD or CBS), progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multisystem tauopathy (MSTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), neurofibrillary dementia, diffuse neurofibrillary changes with calcification (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), frontotemporal lobar degeneration with tau-positive inclusions (FTLD-tau), sequelae of encephalitis lethargica, subacute sclerosing panencephalitis, or pugilistic encephalopathy. Therefore, the dementia therapeutic agent of the present invention is a tauopathy therapeutic agent or preventive agent, and from another point of view, it can also be said to be a therapeutic agent or preventive agent for Alzheimer's disease (AD), corticobasal degeneration (CBD or CBS), progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multisystem tauopathy in dementia (MSTD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), neurofibrillary dementia, diffuse neurofibrillary changes with calcification (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), frontotemporal lobar degeneration with tau-positive inclusions (FTLD-tau), sequelae of encephalitis lethargica, subacute sclerosing panencephalitis, or pugilistic encephalopathy.

Furthermore, the dementia therapeutic or preventive drugs of the present invention can also be described as drugs having the effect of improving cognitive function, inhibiting cognitive decline, or maintaining cognitive function in non-human animals. Examples of such non-human animals include, but are not limited to, animals expressing tau highly homologous to human tau, chimpanzees, rhesus monkeys, horses, pigs, dogs, mice, rabbits, rats, and cats.

[Model Animals of Dementia]

As the animal for studying the dementia therapeutic drug of the present invention or preventive drug, the model animal of dementia can be enumerated, wherein the model animal of Tau disease is enumerated especially.In addition, as the animal model of Tau disease, it is the animal expressing normal type or gene mutation type tau in the brain, particularly the animal model of cognitive dysfunction. This animal expressing normal type or gene mutation type tau in the brain can be made by genetic manipulation, and as its representative animal, transgenic mice (Tg mice) can be enumerated. Animal models such as Tg mice expressing gene mutation type tau are effective as the model of hereditary familial Tau disease, but in order to observe the effect of therapeutic drug or preventive drug for the sporadic Tau disease that accounts for the majority in people, it is hoped that in the Tg mice expressing normal type tau, effect is demonstrated. The mice produced in the preparation examples of the present invention are most suitable as such normal-tau-expressing Tg mice. However, Tg mice reported by Kobe et al. (Neurobiology of Disease, Vol. 42, pp. 404-414, 2011) or Kimura et al. (The EMBO J., Vol. 26, pp. 5143-5152, 2007) can also be used. However, while cognitive impairment was confirmed in the mice of Kobe et al. and Kimura et al., these mice were over 14 and 20 months old, respectively, indicating that the onset of the disease occurred after reaching a certain age. This raises the possibility of age-related effects, the effects of long-term care, and the labor required. In contrast, the mice produced in the preparation examples of the present invention express normal human tau and develop cognitive impairment at a relatively early age, around 6 months of age. Therefore, they are the most suitable dementia model, eliminating factors such as aging. Using such a model allows for more precise evaluation of therapeutic or preventive drugs for dementia that improve cognitive function.

As a method for studying the effects of the dementia therapeutic or preventive drugs of the present invention in animal models, a method for testing cognitive functions such as memory learning tests is preferred. Such methods can be used, such as the Morris water maze test, the step-through learning test, and the novel substance recognition test. However, in order to confirm conditions such as the amount of behavior or the animal's anxiety, it is desirable to combine them with a behavioral measurement test such as the open field test.

In addition, as a method for studying the effects of the dementia therapeutic or preventive drugs of the present invention, a method can also be used to study the increase or decrease of tau protein or phosphorylated tau in brain tissue when administering the drug to dementia model animals. It has been reported that in AD and other neurodegenerative diseases, increased tau protein expression or abnormally phosphorylated tau is associated with pathological conditions (Khalid Iqbal et al., Curr. Alzheimer Res., Vol. 7, pp. 654-664, 2010). Furthermore, it is also known that in several disease model animals, reducing tau expression or the amount of abnormally phosphorylated tau can improve cognitive or motor function (K. SantaCruz et al., Science, Vol. 309, pp. 476-481, 2005; Sylvie LeCorre et al., Porc. Nat. Acad. Sci. USA, Vol. 103, pp. 9673-9678, 2006). As a method for studying the increase or decrease of tau protein or phosphorylated tau, as described in the Examples, brain tissue homogenate can be used and performed by methods such as Western blotting. In addition, those skilled in the art can also appropriately select ELISA methods (Xiyun Chai et al., J. Biol. Chem., Vol. 286, pp. 34457-34467, 2011) or tissue immunochemistry methods (David J. Irwin et al., BRAIN, Vol. 135, pp. 807-818, 2012) for implementation.

The effects of the therapeutic or preventive drug for dementia of the present invention in model animals can be used as data on pharmacological effects in the development of therapeutic or preventive drugs for humans and the like.

[Dementia treatment or prevention drugs]

As an embodiment of the therapeutic or preventive drug for dementia of the present invention, there is an embodiment comprising an anti-phosphorylated tau antibody, preferably an antibody that specifically reacts with tau protein in which at least one of the amino acid residues 410 to 421 corresponding to SEQ ID NO: 1 is phosphorylated, and an antibody that reacts with tau protein phosphorylated at one or more sites selected from the group consisting of amino acid residues Ser412, Ser413, Thr414, and Ser416 corresponding to SEQ ID NO: 1. More preferably, it is an antibody that competes for binding with the (1505) antibody having a VH amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence of SEQ ID NO: 26. Even more preferably, it is an antibody that reacts with tau protein phosphorylated at the amino acid residue Ser413 corresponding to SEQ ID NO: 1. Description of such antibodies is described in the above-mentioned "Anti-phosphorylated tau antibodies" section.

Another embodiment of the dementia therapeutic or preventive drug of the present invention comprises a peptide comprising a portion of a tau sequence in which one or more amino acid residues are phosphorylated. Such a phosphorylated peptide is a peptide comprising at least three consecutive amino acids and having a length of eight or more amino acids, preferably a peptide comprising at least five consecutive amino acids and having a length of eight or more amino acids, and more preferably a peptide comprising at least eight consecutive amino acids and having a length of eight or more amino acids, within an amino acid sequence consisting of amino acid residues corresponding to amino acid residues 410 to 421 of SEQ ID NO: 1. Furthermore, the phosphorylated amino acid residue in the phosphorylated peptide is any one of amino acid residues corresponding to amino acid residues 410 to 421 of SEQ ID NO: 1, preferably any one of amino acid residues corresponding to Ser412, Ser413, Thr414, or Ser416 of SEQ ID NO: 1, and more preferably an amino acid residue corresponding to Ser413 of SEQ ID NO: 1. A description of such a peptide is provided in the "Phosphorylated Peptide Having an Amino Acid Sequence Derived from Tau" section above.

The dementia therapeutic or preventive drug of the present invention may contain any pharmaceutically acceptable additive. Formulations using pharmaceutically acceptable additives may also be prepared according to the methods described in "REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20th EDITION, University of the Sciences in Philadelphia, Williams & Wilkins, December 15, 2000." One form of such a therapeutic or preventive drug is provided as a liquid formulation prepared by dissolving, suspending, or emulsifying in a sterile aqueous or oily liquid. Examples of such solvents include aqueous liquids such as distilled water for injection and physiological saline. In addition, osmotic pressure regulators (e.g., D-glucose, D-sorbitol, D-mannitol, sodium chloride, etc.) may also be added, or appropriate cosolvents such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, polyoxyethylene hydrogenated castor oil 50) may be used in combination. In addition, an oily liquid may be used as a solvent. Examples of such oily liquids include sesame oil and soybean oil. Benzyl benzoate, benzyl alcohol, and the like may also be used as a solubilizing agent. Such liquid preparations may also contain additives such as buffers (e.g., phosphate buffers, acetate buffers), analgesics (e.g., benzalkonium chloride, procaine hydrochloride), stabilizers (e.g., human serum albumin, polyethylene glycol), preservatives (e.g., ascorbic acid, erythorbic acid, and their salts), colorants (e.g., copper chlorophyllin, β -carotene, Red No. 2, Blue No. 1), preservatives (e.g., parabens, phenol, benzethonium chloride, benzalkonium chloride), thickeners (e.g., hydroxypropylcellulose, carboxymethylcellulose, and their salts), stabilizers (e.g., human serum albumin, mannitol, sorbitol), and flavoring agents (e.g., menthol, citrus flavoring). In addition, as other forms of therapeutic or preventive medicines, solid dosage forms such as powders, tablets, granules, capsules, pills, suppositories, lozenges can be enumerated. When being a solid dosage form administered as a form of oral preparation, as additives, excipients (such as crystalline cellulose, lactose, starch etc.), lubricants (such as magnesium stearate, talc etc.), binders (hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyethylene glycol etc.), disintegrants (such as starch, carboxymethyl cellulose calcium etc.) etc. can be used. In addition, as required, additives such as preservatives (such as benzyl alcohol, chlorobutanol, methylparaben, propylparaben etc.), antioxidants, coloring agents, sweeteners can be used. Other forms include therapeutic or prophylactic drugs for mucosal application. In such preparations, with the primary purpose of imparting adsorption or retention to the mucosa, adhesives, adhesion enhancers, thickeners, thickening agents, and the like (e.g., mucin, agar, gelatin, pectin, carrageenan, sodium alginate, locust bean gum, xanthan gum, tragacanth gum, gum arabic, chitosan, pullulan, waxy starch, sucralfate, cellulose and its derivatives (e.g., hydroxypropyl methylcellulose, polyglycerol fatty acid esters, acrylic acid (meth) alkyl ester copolymers or their salts, polyglycerol fatty acid esters, etc.) may be contained as additives. However, the form, solvent, or additives of the therapeutic or prophylactic drug administered to a living body are not limited to these, and those skilled in the art can appropriately select them.

The dementia treatment or preventive drug of the present invention also includes: in addition to the aforementioned anti-phospho-tau antibody or phosphorylated peptide, a therapeutic or preventive drug that also contains an existing substance that has an effect of inhibiting the progression of dementia. Furthermore, the dementia treatment or preventive drug of the present invention also includes: a kit for combining the aforementioned therapeutic or preventive drug containing the anti-phospho-tau antibody or phosphorylated peptide with an existing therapeutic or preventive drug containing a substance that has an effect of inhibiting the progression of dementia. Examples of substances that inhibit the progression of dementia include, but are not limited to, donepezil, galantamine, memantine, or rivastigmine. The dosage of the substance that has an effect of inhibiting the progression of dementia or the therapeutic or preventive drug containing a substance that has an effect of inhibiting the progression of dementia can be based on the dosage typically used for treatment, but can also be increased or decreased depending on the situation.

In addition, the antibodies used in the embodiments of the present invention can pass through the blood-brain barrier even through the peripheral administration such as intraperitoneal administration, and exert their effects in the brain, demonstrating their efficacy. This is demonstrated by the results of the embodiments. However, it is also possible to prepare a formulation for effectively supplying the anti-phospho-tau antibodies used in the dementia treatment or prevention drugs of the present invention to brain tissue, and such a formulation is also included in the dementia treatment or prevention drugs of the present invention. As methods for effectively supplying antibodies or peptides to brain tissue through the blood-brain barrier, there are known methods of adding targeting substances or encapsulating them in liposomes or nanoparticles. As substances used for targeting, substances that change the charge characteristics of the whole or part by binding to the antibody can be used, or peptides, proteins, or compounds that have the characteristics of binding to specific receptors or transport proteins can be used. Examples of peptides, proteins, or compounds that have the characteristics of binding to specific receptors or membrane proteins include ligands or analogs thereof that bind to receptor ligands or membrane proteins, antibodies that bind to receptor ligands or membrane proteins, agonist compounds, antagonist compounds, allosteric modulators, or similar compounds thereof. As the example of the ligand or membrane protein of the target receptor with the characteristic peptide or protein or compound combined with specific receptor or transporter etc., transferrin receptor (TfR), insulin receptor (IR), insulin-like growth factor receptor (IGFR), LDL receptor-related protein (LRP) or diphtheria toxin receptor (HB-EGF) etc. can be enumerated, but it is not limited to this (Angela R. Jones etc., Pharm. Res., 24 volumes, 1759-1771 pages, 2007). The substance for targeting can also be chemically added in the antibody used in the dementia therapeutic drug of the present invention or the preventive drug, and as its method, those skilled in the art can refer to known methods, such as Hermanson etc. Bioconjugate techniques (USA) issued in 1996 and the method of recording in Academic Press is suitably carried out. In addition, the substance for targeting can be combined with liposome or nanoparticle comprising antibody or peptide (Sonu Bhaskar etc., Particle and Fibre Toxicology, 7 volumes No. 3, 2010). Moreover, when the substance used for targeting is a peptide or protein, those skilled in the art can prepare it in the form of a fusion peptide synthesized by peptide chemical synthesis, or combine a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of the peptide or protein with a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of the antibody or peptide used, and prepare it in the form of an appropriate fusion protein using genetic engineering technology.

The therapeutic agent or preventive agent of the present invention can be administered orally or parenterally for the purpose of improving symptoms. When orally administering, dosage forms such as granules, powders, tablets, capsules, liquids, syrups, emulsions or suspensions, elixirs can be selected. When administering parenterally, for example, nasal preparations can be made, and liquids, suspensions, solid preparations, etc. can be selected. In addition, as other parenteral administration methods, injections can be made, and as injections, subcutaneous injections, intravenous injections, drip injections, intramuscular injections, intraventricular injections, or intraperitoneal injections, etc. can be selected. In addition, as other preparations for parenteral administration, suppositories, sublingual preparations, transdermal preparations, and mucosal administration of medicines other than nasal preparations can also be enumerated. Moreover, topical administration within a blood vessel can also be carried out by comprising or coating a stent or intravascular embolic agent.

The dosage of the therapeutic or preventive agent of the present invention varies depending on the patient's age, sex, weight, symptoms, therapeutic effect, administration method, treatment duration, or the type of active ingredient contained in the pharmaceutical composition. However, generally, the main agent can be administered to an adult in a range of 0.1 mg to 1 g, preferably 0.5 mg to 200 mg, per dose. However, since the dosage varies depending on various conditions, an amount less than the above dosage may be sufficient, while a dosage exceeding the above range may be necessary.

Furthermore, the therapeutic or preventive drug of the present invention can achieve its effect within a short administration period.

Example

The present invention will be described in further detail below based on examples, but these examples are not intended to limit the present invention. The experiments were performed with the approval of the Animal Experiment Committee of Abeno District, Osaka City University.

[Example 1] Preparation of rabbit polyclonal antibodies against pSer413 peptide

To prepare anti-pSer413 peptide antibodies, a synthetic peptide (SEQ ID NO: 31, synthesized by Medical Biology Laboratory [MBL] Co., Ltd.) in which Ser at position 413 of the amino acid sequence corresponding to SEQ ID NO: 1 of human tau protein is phosphorylated and Cys is added to the C-terminus of the amino acid sequence corresponding to Asn at positions 410 to Ser at positions 416 (hereinafter referred to as pSer413(S) peptide) was used as an antigen.

pSer413(S) peptide: N-AsnValSer(pSer)ThrGlySerCys-C (SEQ ID NO: 31)

After the synthesis of the pSer413 (S) peptide, it was purified by HPLC and covalently bonded to keyhole limpet hemocyanin (KLH). 0.1 mg of the resulting conjugate was used to immunize one rabbit once. The first immunization was performed by mixing 0.2 mL of the conjugate solution (conjugate concentration was 0.5 mg/mL) with an equal amount of Freund's complete adjuvant and injecting 50 μL intradermally into eight sites on the back of shaved Japanese white rabbits. For the second and subsequent immunizations, Freund's incomplete adjuvant was used, and the same immunization was repeated two more times every two weeks. Two weeks after the last immunization, a portion of blood was collected, and the antibody titer in the serum was confirmed by ELISA using the immunizing peptide conjugate. One week later, the animal was slaughtered and whole blood was collected. Serum was prepared from the obtained blood, and antibodies were purified from the serum using an affinity column for the pSer413 (Af) peptide (SEQ ID NO: 32).

It should be noted that the confirmation of serum antibody values was carried out according to the following method. After the pSer413 (S) peptide was diluted to 5 μg / mL with PBS, 100 μL / well was injected into the plate and allowed to stand at 4 ° C overnight. After removing the solution, 250 μL / well of blocking buffer (MBL) was injected and allowed to stand at 4 ° C overnight. The dilution series of pre-immune rabbit serum and post-immune rabbit serum was set to 100, 500, 2,500, 12,500, 62,500 times and a blank. Rabbit serum diluted with PBS was added at 100 μL / well and reacted at 25 ° C for 60 minutes. After washing, an 8,000-fold dilution of anti-rabbit IgG-peroxidase label (MBL) with dilution buffer (MBL) was added at 100 μL / well and reacted at 25 ° C for 60 minutes. After washing, add 100 μL/well of color development solution (MBL) and allow color to develop for 3-10 minutes. Then, add 100 μL/well of 2N sulfuric acid to stop the reaction. After the reaction is stopped, measure the absorbance at a measurement wavelength of 450 nm and a reference wavelength of 620 nm.

The antibody reactivity of the purified antibodies was confirmed as follows. The pSer413(L) peptide [a peptide obtained by further extending the pSer413(S) peptide (underlined portion) in the N- and C-terminal directions (SEQ ID NO: 33, synthesized by Biosynthesis): N-ProArgHisLeuSerAsnValSer (pSer) ThrGlySerIleAspMetValAsp -C] or the NonP(L) peptide (SEQ ID NO: 34, synthesized by Biosynthesis) with the same amino acid sequence but without phosphorylation at the site corresponding to Ser413 was diluted to 1 μg/mL in PBS. 50 μL/well was then dispensed into the plate and incubated at 4°C overnight. After removing the solution, 250 μL/well of blocking buffer (3% BSA-PBS) was dispensed into the plate and incubated at 37°C for at least 1 hour. Purified antibodies were diluted with PBS, and serially diluted antibodies were added at 50 μL/well, with the cells reacting at 25°C for 90 minutes. After washing, goat anti-rabbit IgG-alkaline phosphatase label (Bioscience) diluted 2,000-fold in dilution buffer (3% BSA-PBS) was added at 50 μL/well, with the cells reacting at 25°C for 60 minutes. After washing, a color development solution (a solution of 1 mg/mL PNPP (p-nitrophenyl phosphate) in 0.1 M diethanolamine buffer (pH 9.8) containing 2.5 _mM MgCl₂) was added at 100 μL/well, and color was developed for 30 minutes. The absorbance was measured at a measurement wavelength of 405 nm and a reference wavelength of 550 nm.

Results

As shown in Figure 3, the purified antibody has high specificity for the pSer413(L) peptide and shows little reaction with the nonP(L) peptide. Therefore, this antibody is an antibody that specifically reacts with pSer413(L) (in this specification, sometimes referred to as "pSer413-recognizing rabbit polyclonal antibody" or "pS413AB").

[Example 2] Preparation of anti-pSer413 peptide monoclonal antibodies (Ta15 series)

The synthetic peptide pSer413(Im) peptide (SEQ ID NO: 35) was used as an antigen, in which the amino acid residue corresponding to Ser at position 413 of the amino acid sequence of human tau protein shown in SEQ ID NO: 1 was phosphorylated and GlyCysSerGly was added to the N-terminus of the sequence corresponding to Thr at positions 403 to Pro at positions 423. After synthesis, the pSer413(Im) peptide was purified by HPLC and covalently bonded to keyhole limpet hemocyanin (KLH). 0.04 mg of the resulting conjugate was used to immunize one mouse once. 0.4 ml of the conjugate solution (1.1 mg/ml in terms of peptide) was mixed with 0.7 mL of physiological saline and 1.1 mL of Freund's complete adjuvant, and 200 μL was injected into the peritoneal cavity of 10 mice for immunization. The same immunization was repeated three times every two weeks (with the same immunization site and antigen amount, and Freund's incomplete adjuvant). For the only mouse among 10 mice with an increased serum antibody titer against the antigen, 100 μL of an antigen solution (dissolved in physiological saline) at 0.5 mg/mL in terms of peptide was injected into the tail vein one month after the last immunization. Three days later, the animal was slaughtered and the spleen was collected. After cutting the spleen using two 18G injection needles, the cut spleen was gently crushed with the surface of a rubber stopper. The crushed spleen cells were suspended in about 10 mL of cold RPMI1640 medium, the supernatant was filtered through a 40 μm cell strainer, and the filtrate was collected into a 50 mL centrifuge tube. The spleen cell fragments were crushed again with the surface of a rubber stopper, and the fragments were also suspended and filtered with cold RPMI1640 medium, and the filtrate was collected. Cold RPMI1640 medium (or cold PBS) was added to make the spleen cell suspension finally reach 40 mL. The lymphocyte concentration in the suspension was measured using a hemocytometer, and finally the lymphocytes equivalent to 2×10 ⁷ cells were transferred to a 50 mL centrifuge tube. To this, 4× ¹⁰⁷ logarithmically growing mouse myeloma cells, P3X63Ag8·U1 strain (abbreviated as P3·U1), cultured in medium B (RPMI1640 + 10% fetal bovine serum + 2mM glutamine + 100μg/mL streptomycin + 100 units/mL penicillin), were added. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant discarded. Cell clumps were thoroughly dispersed by tapping the tube. 0.5mL of polyethylene glycol solution (composed of 2.3mL RPMI1640 + 1.4mL polyethylene glycol 2000 + 0.3mL dimethyl sulfoxide; hereinafter referred to as "PEG solution") was added to the suspension to gently suspend the cells. After one minute, 0.5mL of PEG solution was slowly added dropwise over one minute, followed by 1.0mL of RPMI1640 over another minute, and then 2mL of RPMI1640 over two minutes. After that, 4 mL of HAT/GIT culture medium (GIT culture medium [Nippon Pharmaceutical] + 5% fetal bovine serum + 100 μg/mL streptomycin + 100 units/mL penicillin + 95 μM hypoxanthine + 0.4 μM aminopterin + 16 μM thymidine) was added dropwise over 2 minutes, and 4 mL of HAT/GIT culture medium was added dropwise over 2 minutes. Finally, HAT/GIT culture medium was added to obtain 40-50 mL of cell suspension. After culturing in a thermostatic bath at 37°C for 30 minutes, it was inoculated into 7 96-well culture plates. It should be noted that as a culture plate, a 96-well plate (feeder plate) after culturing peritoneal macrophages (feeder cells) of mice (ICR line) for several days (>1×10 ⁵ / well) was used. Afterwards, the plate was cultured at 37°C and 5% CO ₂ for 7 to 10 days.

Hybridomas were obtained by replacing half of the culture medium with fresh HT culture medium (a culture medium obtained by removing aminopterin from the HAT/GIT culture medium) every week.

Antibody screening was performed as follows. The pSer413(L) peptide was diluted to 1 μg/mL in PBS and injected into a 96-well plate at 50 μL/well. The plates were then incubated at 4°C overnight. After removing the solution, 250 μL/well of blocking buffer (3% BSA-PBS) was injected and incubated at room temperature for at least 1 hour. The blocking buffer (3% BSA-PBS) was aspirated and hybridoma supernatant was added at 30-50 μL/well. The plates were reacted at 25°C for 60 minutes. After washing with phosphate-buffered saline (PBS-Tween) containing 0.05% Tween 20, 100 μL/well of secondary antibody (alkaline phosphatase-labeled goat anti-mouse IgG-Fc antibody (Southern Biotech) diluted 2000-fold in blocking buffer) was added. The plates were incubated at 25°C for 60 minutes. After washing, a substrate solution (a solution prepared by adding 0.7-1.2 mg/mL of PNPP [p-nitrophenyl phosphate] to 0.1 M diethanolamine buffer (pH 9.8) containing 2.5 _mM MgCl2) was added at 100 μL/well. After color development at 25°C for 60 minutes, the absorbance was measured at a measurement wavelength of 405 nm.

After this screening, cells were removed from wells that showed positive results, counted using a hemocytometer, and seeded into 96-well plates (previously feeder plates) at 1 to 3 cells per well. Cloning was performed using the limiting dilution method (Ta1501, Ta1502, Ta1505-1509). For subsequent analysis, clones were cultured in large quantities and antibodies were purified using a protein G column. Ta1505 and Ta1505-2 are identical. Isotype determination was performed using a kit from AbD serotec.

The purified antibodies were reacted with ELISA plates coated with partial peptides targeting various phosphorylation sites on tau to investigate phospho-specificity. The method is as follows.

Various tau peptides (pS46 [SEQ ID NO: 36], pS199 [SEQ ID NO: 37], pS202 [SEQ ID NO: 38], pT212 [SEQ ID NO: 39], pS214 [SEQ ID NO: 40], pT212/pS214 [SEQ ID NO: 41], pT217 [SEQ ID NO: 42], pS413 [SEQ ID NO: 33], non pS413 [SEQ ID NO: 34]) in 10% DMSO or peptide BSA conjugates (pS400 [SEQ ID NO: 43]-BSA, pS412 [SEQ ID NO: 44]-BSA, pS400 [SEQ ID NO: 44]-BSA, pS412 [SEQ ID NO: 45]-BSA, pS412 [SEQ ID NO: 46]-BSA, pS412 [SEQ ID NO: 47]-BSA, pS412 [SEQ ID NO: 48]-BSA, pS412 [SEQ ID NO: 49]-BSA, pS412 [SEQ ID NO: 50]-BSA, pS412 [SEQ ID NO: 51]-BSA, pS412 [SEQ ID NO: 52]-BSA, pS412 [SEQ ID NO: 53]-BSA, pS412 [SEQ ID NO: 54]-BSA, pS412 [SEQ ID NO: 55]-BSA, pS412 [SEQ ID NO: 56]-BSA, pS412 [SEQ ID NO: 57]-BSA, pS214 [SEQ ID NO: 40], pT212/pS214 [SEQ ID NO: 41], pT217 [SEQ ID NO: 42] A PBS solution of 44-BSA was diluted to 1 μg/mL and added to a 96-well plate (MaxiSorp: Nunc) at 50 μL/well. The plates were incubated overnight at 4°C for fixation. The peptide solution was then removed, and the plates were washed three times with TBS containing 0.05% Tween 20. Then, 280 μL/well of 3% BSA/PBS was added, and the plates were blocked at 37°C for 1 hour.

The blocking solution was removed by aspiration, and the cells were washed three times with TBS containing 0.05% Tween 20. Then, 50 μL/well of various Ta15 series monoclonal antibody solutions (1 μg/mL, diluted in 3% BSA-PBS) were added and allowed to react at room temperature for 1 hour. After washing three times with TBS containing 0.05% Tween 20, 50 μL of alkaline phosphatase-conjugated goat anti-mouse IgG antibody (ThermoScience) diluted 2000-fold in PBS containing 3% BSA was added and allowed to react at room temperature for 1 hour. After washing three times with TBS containing 0.05% Tween 20, 100 μL of a 1 mg/mL solution of PNPP (p-nitrophenyl phosphate) dissolved in 0.1 M diethanolamine (pH 10) was added and allowed to react at room temperature for 1 hour. The absorbance at 405 nm was measured. The reactivity of each antibody with various peptides is evaluated in Table 2. Reactivity was rated on a three-level scale. +: Reactivity; -: No reactivity; ±: Some reactivity was observed, but very weak.

[Table 2]

Among the peptides studied, these antibodies (hereinafter referred to as "Ta15 series") reacted only with pSer413 and had extremely high specificity for the phosphate group.

[Example 3] Binding affinity measurement of Ta15 series antibodies

To evaluate the binding affinity of the Ta15 series antibodies for an antigenic peptide (pSer413 peptide (Im): see Example 2), measurements were performed using the surface plasmon resonance (SPR) system Biacore (GE Healthcare Japan, BIACORE 3000, code #BR-1100-45) and the respective Biacore products according to the manufacturer's instructions. One measurement method involved covalently immobilizing an anti-mouse antibody (code #BR-1008-38) on a CM5 chip (a chip with a carboxymethyl dextran layer, code #BR-1100-14) (code #BR-1006-33) via amine coupling. The Ta15 series antibodies were then allowed to bind to the immobilized anti-mouse antibody, and the binding kinetics of the bound Ta15 series antibodies to the antigenic peptide were measured.

The CM5 chip was activated using HBS-EP buffer (code #BR-T-1001-88) as the specific binding reaction solution, and a mixed solution of N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Anti-mouse antibodies were diluted to 0.001 mg/mL in 10 mM sodium acetate buffer (pH 5.0) and reacted with the four flow cells on the chip to covalently bond the anti-mouse antibodies to the CM5 chip. The chip was then blocked with ethanolamine. Of the four flow cells immobilized with anti-mouse antibodies, a negative antibody (mouse IgG2a isotype control, R&D Systems, code #MAB003) was captured in one flow cell at a density of approximately 1000 RU, while the Ta15 series antibodies were captured in the remaining flow cells at a density of approximately 1000 RU. The peptide was reacted for 3 minutes in HBS-EP buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v Surfactant P20) supplemented with 1M NaCl to remove nonspecifically adsorbed antibodies. The HBS-EP buffer was then reacted for 10 minutes at a flow rate of 50uL/min to stabilize the baseline. The peptide was diluted in HBS-EP buffer in stages between 100pM and 5mM (estimated to be around the binding dissociation constant [KD]). The resulting peptide solutions (five concentrations) were reacted continuously for 4 to 6 minutes at 6-minute intervals, starting from the lower concentration, to measure binding kinetics.

As the specific binding kinetic data of the antigen peptide, in order to eliminate the influence of noise generated by the operation, the binding kinetic data of the non-phosphorylated peptide (non pSer413 peptide) as a negative peptide was obtained and subtracted from the binding kinetic data of the antigen peptide. It should be noted that no binding of the non-phosphorylated peptide to the antibody was confirmed in terms of the measured concentration. Using Biacore analysis software (BIAevaluation: Single cycle kinetics analysis, code#AP-4000-01), the specific binding kinetic data were fitted by single cycle kinetics 1:1 binding with drift model, and the kinetic association rate (Ka) and dissociation rate (Kd) were obtained at the same time (Karlsson. R., Katsamba, P. S., Nordin, H., Pol, E. and Myszka, D. G. (2006). "Analyzing a kinetic titration series using affinity biosensors." Anal. Biochem. 349(1): 136-47). The equilibrium dissociation constant (KD) value, which is the affinity measurement value of the Ta15 series antibodies, was calculated from Kd/Ka.

Results

[Table 3]

Among the Ta15 series of antibodies, the Ta1505 antibody, which has the strongest affinity for the antigen peptide, was used in behavioral experiments using memory-impaired mice.

[Example 4] Preparation of monoclonal antibodies recognizing pSer396 and/or pSer404 peptides

A synthetic peptide (SEQ ID NO: 47; synthesized by Biosynthesis) in which GlyCys was added to the N-terminus of the sequence from Arg 379 to Leu 408, corresponding to the amino acid residues Ser at positions 396 and 404 of the amino acid sequence of human tau protein shown in SEQ ID NO: 1, in which amino acid residues are phosphorylated (hereinafter referred to as pSer396/pSer404 peptide).

pSer396/pSer404 peptide: N-GlyCys-ArgGluAsnAlaLysAlaLysThrAspHisGlyAlaGluIleValTyr Lys(pSer)ProValValSerGlyAspThr(pSer)ProArgHisLeu-C (SEQ ID NO: 47)

After synthesis, the pSer396/pSer404 peptide was purified by HPLC. Approximately 0.04 mg of the conjugate, covalently bonded to maleimide-activated KLH (keyhole limpet hemocyanin) (Thermo Scientific), was used to immunize one Balb/c mouse. Immunizations were performed by mixing 0.3 mL (0.77 mg/mL, peptide equivalent) of the conjugate solution with 0.3 mL of Freund's complete adjuvant and injecting 100 μL intraperitoneally into each of four mice. Two of these mice were immunized intraperitoneally (with the same amount of antigen in Freund's incomplete adjuvant) and intraplantar (with antigen + Titer Max adjuvant) two weeks apart. The remaining two mice were immunized intraperitoneally two more times two weeks apart. For mice with high serum antibody titers against the antigen and that have been immunized intraperitoneally and in the plantar, the antigen solution (dissolved in physiological saline) is injected into the tail vein 15 days after the last immunization. The animals are slaughtered 3 days later and the spleen is removed. After cutting the spleen with two 18G injection needles, the cut spleen is slowly crushed with the surface of a rubber stopper. The crushed spleen cells are suspended in about 10 mL of cold RPMI1640 medium, the supernatant is filtered with a 40μm cell strainer, and the filtrate is collected into a 50 mL centrifuge tube. The spleen cell fragments are crushed again with the surface of a rubber stopper, and they are also suspended and filtered with cold RPMI1640 medium, and the filtrate is collected. Cold RPMI1640 medium (or cold PBS) is added to make the spleen cell suspension finally reach 40 mL. The lymphocyte concentration in the suspension is measured using a hemocytometer, and finally lymphocytes equivalent to 2×10 ⁷ cells are transferred to a 50 mL centrifuge tube. To this, add 4× ¹⁰⁷ logarithmically growing mouse myeloma P3/U1 cells cultured in medium B (RPMI1640 + 10% fetal bovine serum + 2mM glutamine + 100μg/mL streptomycin + 100 units/mL penicillin). Centrifuge at 1500rpm for 5 minutes, and discard the supernatant. Tap the test tube to thoroughly disperse cell clumps. Add 0.5mL of PEG solution to gently suspend the cells. After 1 minute, slowly add 0.5mL of PEG solution dropwise over 1 minute, then slowly add 1.0mL of RPMI1640 dropwise over 1 minute, and then slowly add 2mL of RPMI1640 dropwise over 2 minutes. After that, 4 mL of HAT/GIT culture medium (GIT culture medium [Nippon Pharmaceutical] + 5% fetal bovine serum + 100 μg/mL streptomycin + 100 units/mL penicillin + 95 μM hypoxanthine + 0.4 μM aminopterin + 16 μM thymidine) was added dropwise over 2 minutes, and 4 mL of HAT/GIT culture medium was added dropwise over 2 minutes. Finally, HAT/GIT culture medium was added to form a 40-50 mL cell suspension. After culturing in a thermostatic bath at 37°C for 30 minutes, it was inoculated onto 7 96-well culture plates. It should be noted that as a culture plate, a 96-well plate (feeder plate) in which peritoneal macrophages (feeder cells) of mice (ICR line) were cultured for several days (>1×10 ⁵ / well) was used. Afterwards, the plate was cultured at 37°C and 5% CO ₂ for 7 to 10 days.

Half the volume of the culture medium was replaced with new HT culture medium (a culture medium obtained by removing aminopterin from the HAT/GIT culture medium) every week to obtain hybridomas.

Screening of monoclonal antibodies was performed as follows. As screening antigens, pSer396/pSer404 peptide-BSA, in which BSA was conjugated to the N-terminal Cys of the pSer396/pSer404 peptide (N-GlyCys-ArgGluAsnAlaLysAlaLysThrAspHisGlyAlaGluIleValTyrLys(pSer)ProValValSerGlyAsp Thr(pSer)ProArgHisLeu-C) (SEQ ID NO: 47), and non-phosphorylated peptide-BSA, in which BSA was conjugated to the N-terminal Cys of the non-phosphorylated peptide (N-GlyCys-ArgGluAsnAlaLysAlaLysThrAspHisGlyAlaGluIleValTyrLysSerProValValSerGlyAspThrSerProArgHisLeu-C) (SEQ ID NO: 48), were used. Non-phosphorylated peptide-BSA or pSer396/pSer404 peptide-BSA was diluted to 1 μg/mL in PBS and dispensed into a 96-well plate at 50 μL/well. The plate was then incubated at 4°C overnight. After removing the solution, 250 μL/well of blocking buffer (3% BSA-PBS) was dispensed and incubated at room temperature for at least 1 hour. The blocking buffer (3% BSA-PBS) was aspirated and hybridoma supernatant was added at 30-50 μL/well. The plate was reacted at 25°C for 60 minutes. After washing with phosphate-buffered saline (PBS-Tween) containing 0.05% Tween 20, 100 μL/well of detection reagent (a solution prepared by adding alkaline phosphatase-labeled protein A diluted 2,000-fold to blocking buffer) was added. The plate was incubated at 25°C for 60 minutes. After washing, a substrate solution (a solution prepared by adding 0.7-1.2 mg/mL of PNPP [p-nitrophenyl phosphate] to 0.1 M diethanolamine buffer (pH 9.8) containing 2.5 _mM MgCl2) was added at 100 μL/well. After color development at 25°C for 60 minutes, the absorbance was measured at a measurement wavelength of 405 nm.

Cells were removed from wells showing low reactivity with non-phosphorylated peptide-BSA but high reactivity with pSer396/pSer404 peptide-BSA, seeded at 1-3 cells/well in a 96-well plate (previously a feeder plate), and cloned using limiting dilution. Clones producing Ta9 antibodies (IgG3/κ) were selected. For subsequent analysis, clones were cultured in large quantities and the antibodies were purified using a protein G column.

The KD value of the Ta9 antibody for the pSer396/pSer404 peptide was measured according to the method described in Example 3. The KD value was 1.08×10 ⁻¹⁰ , indicating a higher affinity for the peptide than that of the Ta15 series antibodies.

[Example 5] Behavioral test: Effects of acquired antibodies on mice with memory and learning impairment

The effects of administration of the following three antibodies on memory and learning-impaired mice (Tau-Tg) were studied.

(1) pSer413-recognizing rabbit polyclonal antibody: 9-11 month old Tau-Tg (609 strain) mice or non-Tg mice (normal control) were used, n = 9-10/group;

(2) Ta1505 (pSer413-recognizing monoclonal antibody): 14-month-old Tau-Tg (784 strain) mice or non-Tg mice were used, n = 9-10/group;

(3) 14-month-old Ta9 (pSer396-recognizing monoclonal antibody), Tau-Tg (609 strain) or non-Tg mice were used, n = 9-10/group.

The experiment used male heterologous mutant Tau-Tg mice (609 strain or 784 strain) aged 9-14 months and their non-Tg littermates. The groups were divided in a way that there was no difference in average body weight between the groups. For heterologous mutant Tau-Tg mice, 1 mg of antibody diluted with PBS or citrate buffer (pH 5) was administered intraperitoneally to each mouse once a week for a total of 5 times. As a negative control group, the same amount of buffer used in antibody preparation or a mouse IgG monoclonal antibody that is not reactive to tau was administered intraperitoneally. As a positive control, the same amount of buffer used in antibody preparation was administered intraperitoneally to non-Tg littermates.

The group structures of (1) to (3) are as follows.

(1) Mice used: Mutant Tau-Tg (609 strain), 9-11 months old

＜Group composition＞

Evaluation antibody group: 1.6 mg/mL anti-tau pSer413 rabbit polyclonal antibody dissolved in PBS (n=10);

Control antibody group: PBS (n=9);

non-Tg group: PBS (n=9);

(2) Mice used: Mutant Tau-Tg (784 strain), 14 months old

＜Group composition＞

Evaluation antibody group: 3.84 mg/ml anti-tau pSer413 mouse monoclonal antibody Ta1505 dissolved in 0.1 M citrate buffer (pH 5) (n=10);

Control antibody group: 4.28 mg/ml anti-Pseudomonas aeruginosa mouse monoclonal antibody 4C10F4 dissolved in 0.1 M citrate buffer (pH 5) (n=9);

Non-Tg group: 0.1 M citrate buffer (pH 5) (n=9).

(3) ＜Mice used＞: Mutant Tau-Tg (609 strain), 14 months old

＜Group composition＞

Evaluation antibody group: 2.66 mg/ml anti-tau pSer396 mouse monoclonal antibody Ta9 dissolved in 0.02 M citrate buffer (pH 6) (n=10);

Control antibody group: 4.50 mg/ml anti-Pseudomonas aeruginosa monoclonal antibody 6F11 dissolved in 0.02 M citrate buffer (pH 6) (n=9);

Non-Tg group: 0.02 M citrate buffer (pH 6) (n=9).

Starting from the Monday following the final administration, a spatial reference memory test (water maze test) was performed using the Morris water maze. From the day after the water maze test, the spontaneous movement of the mice was measured using an open field apparatus (open field test).

Water maze test: (1) to (3) common

Preparation: A black pool with an inner diameter of 100 cm and a height of 45 cm was filled with 16 cm of water. The water temperature was adjusted to 21-23°C and was maintained colorless and transparent without coloring with titanium oxide or other additives. No chlorine was added. Feces was removed after each trial day, and approximately 10 L of water was changed each time. Trial (Acquisition) Test: A 15 cm high transparent platform was sunken 20 cm from the wall (30 cm from the center). The area, including the sunken platform, was divided into four quadrants. Mice were randomly introduced from any of the three quadrants without the platform. Each attempt was limited to 60 seconds, and five attempts were conducted per day. The interval between attempts was approximately 5 minutes. The time it took for the mouse to finally reach the platform (escape time) was recorded. Mice that did not escape within 60 seconds were manually guided toward the platform by the operator, and the escape time was counted as 60 seconds. Mice on the platform were removed from the pool after 10 seconds and allowed to move freely and dry before the next attempt. The daily performance of the mice was calculated as the average escape time in seconds from 5 attempts.

The acquisition test was terminated when the performance of the control group (non-Tg) stabilized, and the probe test was performed the next day.

Probe Test: On the day following the final day, the platform was removed from the pool and 60 seconds of free swimming were recorded using a digital camera. The recorded video was then used to measure the time the mouse spent swimming in the target quadrant (of the four quadrants) containing the platform. This time, the percentage of the 60-second period was expressed. The swimming period up to 30 seconds after placement in the pool was also analyzed.

The significant difference test of the trial (acquisition) test was performed by repeated measures, Fisher's PLSD, while the significant difference test of the probe test was performed by Fisher's PLSD.

<Open field test: (1) to (3) common>

Apparatus: A 30×30×30 cm square box was constructed using 5 mm thick transparent acrylic sheets and stored in a soundproof environment. A 40 W incandescent lamp was placed above the storage environment, illuminating the floor surface at 110 lux.

Behavioral measurement: (i) Infrared light beams were placed on the outside of the acrylic box, 1.5 cm from the board surface, and at intervals of 10 cm. The number of times the mouse blocked two consecutive light beams was detected and counted as locomotion.

(ii) Place infrared beams on two opposing sides, 3.5 cm from the plate surface. Detect and count mice when they partially block the beams, which is considered rearing.

Experiment: The mice were given a 20-minute activity session. The mice were placed in the center of the acrylic box, and the door was quickly closed to create a soundproof environment. The activity session then began.

Free behavior was measured under light conditions for the first 10 minutes of the activity period, and under dark conditions for the last 10 minutes after the lights were removed.

Analysis: The minute-by-minute behavioral volume of mice is displayed in line graphs in terms of exercise and feeding.

When the mouse responded to the change of the environment to dark conditions 10 minutes after the start of the activity period and a change in the amount of behavior of the mouse was confirmed, the vision was defined as normal.

The total amount of behavior in each of the light condition and the dark condition, as well as the total amount of behavior over 20 minutes, are displayed as bar graphs.

The significant differences among the groups were tested on the total amount of movement and feeding behaviors.

Although it is explained that movement is mainly spontaneous behavior and feeding is mainly exploratory behavior, in fact, the two indicators affect each other (for example, even if movement seems to be decreasing, if feeding is actually increasing at this time, it cannot be said that the behavior has decreased).

＜Immunohistochemistry test; only performed (2)＞

After the behavioral test, five rats were selected from each group and fixed with formalin by reflux. After paraffin embedding, thin sections were made from the brain using a microtome and deparaffinized by treating with xylene and ethanol for 10 minutes each, four times. The sections were boiled in citrate buffer (pH 6) for 30 minutes (antigen activation treatment), returned to room temperature, and then washed twice with Tris-buffered saline (TS) for 10 minutes. After blocking with TS containing 20% bovine serum at room temperature for 60 minutes, mouse anti-human synaptophysin antibody (Sigma's SVP-38 diluted 200-fold with TS containing 10% bovine serum) was mounted and incubated overnight at 4°C. After washing with TS for 10 minutes, two times, FITC-labeled anti-mouse IgG antibody (VECTOR's anti-mouse IgG diluted 20-fold with TS containing 10% bovine serum) was mounted and incubated for 60 minutes at room temperature. After washing with TS for 10 minutes twice, the cells were embedded in VECTASHIELD (VECTOR) and observed under a microscope. Fluorescence intensity was quantified and digitized using NIH Image J and expressed in arbitrary units.

Results

1. Effects of each antibody administration on memory impairment

(1) pSer413 rabbit polyclonal antibody

The results of (1-1) to (1-3) are shown in Figures 4 to 7. Overall, passive immunization with anti-pSer413 polyclonal antibodies significantly improved memory impairment in the Tau-Tg model mice, reaching levels comparable to those of non-Tg mice. However, in (1-3), no significant differences were observed in spontaneous locomotion or visual acuity between the groups.

(2) The results of pSer413 mouse monoclonal antibody (1505 antibody) (2-1) to (2-3) are shown in Figures 8 to 11. Overall, passive immunization with anti-pSer413 monoclonal antibody significantly improved memory impairment in model mice, reaching levels comparable to those observed in non-Tg mice. However, no significant differences were observed between the groups in spontaneous locomotion and visual acuity in (2-3).

Based on the results of (2-1) and (2-2), the pSer413 epitope is considered to be a good epitope as a target for passive immunotherapy for both polyclonal antibodies and monoclonal antibodies.

(3) pSer396 mouse monoclonal antibody (Ta9 antibody)

The results of (3-1) to (3-3) are shown in Figures 12 to 15. Overall, Ta9 administration significantly improved memory impairment, reaching levels comparable to those observed in non-Tg mice. However, in (3-3), no significant differences were observed in spontaneous movement or visual acuity between the groups.

However, Ta9 has a weaker efficacy than Ta1505. Although the antigen affinity is Ta9>Ta1505 (see Examples 2 and 3), the efficacy is Ta9<Ta1505 (Example 5), suggesting that the difference in efficacy is caused by the difference in phosphorylated epitopes.

[Example 6] Effects of Ta1505 Antibody Administration on Neurological Function

The effect of Ta1505 antibody administration on the level of synaptophysin, a known marker for neural function, was examined by immunostaining the hippocampal CA3 region, a memory center, with an anti-synaptophysin antibody. Fluorescence intensity was quantified using NIH-Image J.

The results are shown in Figure 16. Administration of the Ta1505 antibody restored the amount of synaptophysin in the hippocampus, but the recovery was not significant.

[Example 7] Determination of the nucleotide sequence of Ta15 series monoclonal antibody cDNA

(1) Purification of hybridoma total RNA

Cultivate various hybridomas that produce monoclonal antibodies, and use 1 mL of ISOGEN per well of a 6-well plate to dissolve the cells. Add 0.2 mL of chloroform to the lysate, mix by vortexing, and let it stand at room temperature for 2 to 3 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes, and transfer the upper layer to a new tube. Add 0.5 mL of isopropanol, mix, and let it stand at room temperature for 10 minutes. Centrifuge it at 15,000 rpm and 4°C for 15 minutes to precipitate the total RNA. Add 1 mL of 75% ethanol to the pellet, mix thoroughly, and centrifuge it at 10,000 rpm and 4°C for 5 minutes. Air-dry the pellet, dissolve it in water that does not contain DNase or RNase, and store it at -80°C.

(2) Obtaining H-chain and L-chain cDNAs by 5’-RACE and 3’-RACE and determining the nucleotide sequences of these cDNAs

Primers for 5'-RACE (Rapid Amplification of cDNA Ends) and 3'-RACE were synthesized based on the known cDNA sequences of the H chain constant regions of mouse IgG2a and IgG2b. Similarly, primers for 5'-RACE (Rapid Amplification of cDNA Ends) and 3'-RACE were synthesized based on the cDNA sequences of the mouse L chain constant regions.

Separately, 1 μg of total RNA collected from hybridomas was used to synthesize cDNA for 5'-RACE and 3'-RACE using the SMART-RACE kit (Clonetech). PCR reactions were performed using Advantage 2 polymerase mix (Clonetech) according to the accompanying protocol. The PCR products obtained by 5'-RACE and 3'-RACE were subjected to agarose gel electrophoresis. The major amplified DNA fragment was excised from the agarose gel, inserted into a TA cloning vector (Invitrogen), and transformed into E. coli to obtain multiple clones. Plasmids were prepared from these transformants according to conventional methods, and the nucleotide sequence of the inserted DNA fragment was determined.

(3) Obtaining full-length cDNAs for the H and L chains and determining their nucleotide sequences

Based on the nucleotide sequence information, the cDNA sequences encoding the N- and C-termini of the H and L chains were determined, and forward and reverse primers were designed to amplify the full-length sequences. Using these primers, the full-length H and L chains were amplified using Prime STAR MaxPCR (manufactured by TaKaRa). The resulting PCR fragments were cloned into the pEF4 vector. Using these products, the full-length cDNA sequences were finally determined.

The obtained nucleotide sequence information was translated into amino acid sequences, and the CDR regions were identified using IgBLAST (NCBI, http://www.ncbi.nlm.nih.gov/igblast/) according to the KABAT method (Kabat, E.A. and Wu, T.T., J. Immunol., 147, 1709-1719, 1991).

The resulting sequence of the CDR region is shown in SEQ ID NO: 14.

Based on the homology of these sequences, information on the CDR sequences of antibodies that specifically recognize pSer413 was obtained (Tables 4 to 6).

[Table 4]

Amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of each monoclonal antibody

Monoclonal antibody cloning CDR-H1 CDR-H2 CDR-H3 Ta1501 SEQ ID NO: 7 SEQ ID NO: 9 SEQ ID NO: 13 Ta1502 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 13 Ta1505 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 13 Ta1506 SEQ ID NO: 7 SEQ ID NO: 10 SEQ ID NO: 13 Ta1507 SEQ ID NO: 8 SEQ ID NO: 12 SEQ ID NO: 13 Ta1508 SEQ ID NO: 7 SEQ ID NO: 10 SEQ ID NO: 13 Ta1509 SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 13

[Table 5]

Amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of each monoclonal antibody

Monoclonal antibody cloning CDR-L1 CDR-L2 CDR-L3 Ta1501 SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 17 Ta1502 SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 17 Ta1505 SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 17 Ta1506 SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 17 Ta1507 SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 17 Ta1508 SEQ ID NO: 14 SEQ ID NO: 16 SEQ ID NO: 17 Ta1509 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17

[Table 6]

Amino acid sequences of VH and VL of each monoclonal antibody

Monoclonal antibody cloning VH VL Ta1501 SEQ ID NO: 18 SEQ ID NO: 25 Ta1502 SEQ ID NO: 19 SEQ ID NO: 26 Ta1505 SEQ ID NO: 20 SEQ ID NO: 26 Ta1506 SEQ ID NO: 21 SEQ ID NO: 27 Ta1507 SEQ ID NO: 22 SEQ ID NO: 28 Ta1508 SEQ ID NO: 23 SEQ ID NO: 29 Ta1509 SEQ ID NO: 24 SEQ ID NO: 30

[Example 8] Behavioral test: Effects of acquired antibodies on mice with memory and learning impairment

The effects of 0.1 mg of the following two antibodies on mice with memory and learning impairment were examined.

(4) Ta1505 (pSer413-recognizing monoclonal antibody): 10-month-old Tau-Tg (784 strain) mice or non-Tg mice were used, n = 9-10/group;

(5) Ta9 (monoclonal antibody recognizing pSer396): 11-month-old Tau-Tg (784 strain) mice or non-Tg mice were used, n=8-10/group.

The experiment used 10-11 month old male heterologous mutant Tau-Tg mice (strain 784) and their non-Tg littermates. The groups were divided in such a way that there was no difference in average body weight between the groups. For heterologous mutant Tau-Tg mice, 0.1 mg of antibody diluted in PBS was administered intraperitoneally to each mouse once a week for a total of 5 times. As a negative control group, the same amount of PBS used in the antibody preparation or a mouse IgG monoclonal antibody that is not reactive to tau was administered intraperitoneally. As a positive control, the same amount of PBS used in the antibody preparation was administered intraperitoneally to non-Tg littermates.

The group structures of (4) to (5) are as follows.

(4)＜Mice used＞：Tau-Tg mutant (784 strain), 10mo

＜Group composition＞

Evaluation antibody group: 0.25 mg/ml anti-tau pSer413 mouse monoclonal antibody Ta1505 dissolved in PBS (n=10);

Control antibody group: 0.25 mg/ml anti-Pseudomonas aeruginosa mouse monoclonal antibody 4C10F4 dissolved in PBS (n=10);

Non-Tg group: PBS (n=9).

(5)＜Mice used＞：Tau-Tg mutant (784 strain), 11mo

＜Group composition＞

Evaluation antibody group: 0.25 mg/ml anti-tau pSer396 mouse monoclonal antibody Ta9 dissolved in PBS (n=10);

Control antibody group: 0.25 mg/ml anti-Pseudomonas aeruginosa monoclonal antibody 6F11 dissolved in PBS (n=8);

Non-Tg group: PBS (n=8).

Starting from the week following the final administration, a spatial reference memory test (water maze test) was performed using the Morris water maze. The water maze test was performed in the same manner as in Example 5.

Results

1. Effects of each antibody administration on memory impairment

(4) pSer413 mouse monoclonal antibody (1505 antibody)

The results of the trial test (4-1) and the detection test (4-2) are shown in Figures 18 and 19. Overall, passive immunization with anti-pSer413 monoclonal antibodies improved memory impairment in model mice by more than 50% compared to non-Tg mice.

According to the results of (4-1) and (4-2), the efficacy of the pSer413 epitope monoclonal antibody was confirmed at a dose of 0.1 mg.

(5) pSer396 mouse monoclonal antibody (Ta9 antibody)

The results of the trial test (5-1) and the probe test (5-2) are shown in Figures 20 and 21. In general, the memory impairment was not improved in the administration of Ta9 at a dose of 0.1 mg.

These experiments further clearly suggested that the difference in pharmacodynamics due to the difference in phosphorylated epitopes was caused in the 0.1 mg administration.

It should be noted that 0.1 mg of antibody was administered to each mouse, which is equivalent to a dosage of approximately 2.5 mg/kg.

[Example 9] Changes in the amount of phosphorylated Tau in the brain caused by administration of Ta1505 antibody

Regarding the hippocampus, a memory center, immunohistochemical staining with the pSer413-recognizing Ta1505 antibody and the AT8 antibody (PHF recognizes the pSer202/pThr205 epitope) was performed to investigate the effect of Ta1505 antibody administration on the amount of phosphorylated Tau in the brains of Tau-Tg mice.

After the behavioral test, 5 rats were selected from each group and fixed with 4% paraformaldehyde/PBS by reflux. The brain was removed, embedded in paraffin, and 5 μm thin sections were made using a microtome. The brain was treated with xylene and ethanol for 10 minutes each, 4 times, and then deparaffinized. The sections were boiled at pH 2 and room temperature for 10 minutes (antigen activation treatment). After returning to room temperature, the sections were washed with Tris-buffered saline (TS) for 10 minutes, and washed twice. The sections were blocked with TS containing 20% bovine serum at room temperature for 60 minutes.

Anti-tau antibodies (Ta1505, AT8) diluted to 1 μg/mL in TS containing 10% bovine serum were embedded and treated overnight at 4°C. After washing with TS for 10 minutes and twice, the cells were treated with biotin-labeled anti-mouse antibodies (VECTOR) diluted 500-fold in TS containing 10% bovine serum for 60 minutes at room temperature.

After washing with TS for 10 minutes and twice, the plate was embedded with HRP-labeled ABC solution (VECTOR), reacted at room temperature for 30 minutes, washed again, developed with diaminobenzidine (DAB), sealed with Entellan (Merck), and observed and photographed.

The results of immunohistochemical staining using Ta1505 are shown in Figures 22 to 24 .

In Figure 22, Ta1505-positive Tau staining was confirmed in the hippocampal CA3 area (first column from the left) and the hippocampal CA23 area (second column from the left) of the 5 individuals in the control IgG administration group, but the staining levels of the hippocampal CA3 area (third column from the left) and the hippocampal CA23 area (fourth column from the left) of the 5 individuals in the Ta1505 administration group were significantly lower than those in the control IgG administration group. It can be seen from this that by administering Ta1505, a reduction in Ta1505-positive Tau, i.e., Ser413 phosphorylated Tau, was confirmed in the hippocampal CA3 area and the hippocampal CA23 area. In more detail, in the control IgG group, brown fine dots gathered and stained into a thick line from left to right in CA3. In CA23, it was stained into a thick curve from the upper right to the left. In the Ta1505 administration group, very light brown fine dots gathered and stained into a thick line from the upper left to the lower right in CA3. CA23 was stained in a thick horizontal curve from the upper right to the left. Almost no staining was observed in 4 individuals of CA3 and 3 individuals of CA23.

In Figures 23 and 24 , AT8-positive Tau staining was confirmed in the cortical regions (perirhinal cortex (first column from the left in Figure 23 ), lateral enthrone cortex (second column from the left in Figure 23 ), and medial enthrone cortex (third column from the left in Figure 23 )) of the five individuals in the control IgG-administered group. (Staining appears as brown spots in Figure 23 )

The staining levels in the cortical areas (Perirhinal Cortex (first column from the left in Figure 24), Lateral Entrhinal Cortex (second column from the left in Figure 24), and Medial Entrhinal Cortex (third column from the left in Figure 24)) of the five individuals in the Ta1505-administered group were significantly lower than those in the control IgG-administered group, confirming that the staining did not reach a detectable level.

This revealed that administration of Ta1505 reduced Ta1505-positive Tau, namely, Ser413 phosphorylated Tau, in the cortical regions (perirhinal cortex, lateral enthrone cortex, and medial enthrone cortex).

In FIG23 , the surface is dyed in a brown spotty manner, but in FIG24 , the brown spotty dyeing is hardly confirmed.

In the brain (= hippocampal CA3 region, hippocampal CA23 region, PRh, Ent (Lateral, Medial)), administration of the Ta1505 antibody was confirmed to reduce the amount of Ser413 phosphorylated Tau.

PRh＝Perirhinal Cortex

Ent＝Entrhinal Cortex

The results of immunohistochemical staining using AT8 are shown in Figures 25 to 27 .

In Figure 25, AT8-positive Tau staining was confirmed in the CA3 region (first column from the left) and CA23 region (second column from the left) of the hippocampus of 5 individuals in the control IgG administration group, but the staining levels in the CA3 region (third column from the left) and CA23 region (fourth column from the left) of the hippocampus of 5 individuals in the Ta1505 administration group were significantly lower than those in the control IgG administration group. It can be seen from this that by administering Ta1505, a reduction in AT8-positive Tau, i.e., Ser202/Thr205 phosphorylated Tau, was confirmed in the CA3 region and CA23 region of the hippocampus. Specifically, in Figure 25, in the control IgG group, fine brown dots gathered and were stained into a thick line from the upper left to the lower right in CA3. In CA23, they were stained into a thick curve from the upper right to the left. In the Ta1505 administration group, very light brown dots gathered and were stained into a thick line from the upper left to the lower right in CA3. In CA23, it is stained horizontally in a thick curved line from the upper right to the left.

In Figure 26 , AT8-positive Tau staining was confirmed in the cortical regions (perirhinal cortex (first column from the left in Figure 26 ), lateral enthrone cortex (second column from the left in Figure 26 ), and medial enthrone cortex (third column from the left in Figure 26 )) of five individuals in the control IgG-administered group. (In Figure 26 , staining appears as brown spots.)

In Figure 27, the staining levels of the cortical regions (perirhinal cortex (first column from the left in Figure 27), lateral enthrone cortex (second column from the left in Figure 27), and medial enthrone cortex (third column from the left in Figure 27)) of the five individuals in the Ta1505-administered group were significantly lower than those in the control IgG-administered group. (Staining was performed as light brown spots in Figure 27.)

This indicates that administration of Ta1505 reduced AT8-positive Tau, specifically Ser202/Thr205 phosphorylated Tau, in the cortical regions (perirhinal cortex, lateral enthrone cortex, and medial enthrone cortex). In the brain (hippocampal CA3 and CA23 regions, PRh, Ent (Lateral, Medial)), administration of the Ta1505 antibody demonstrated a trend toward a decrease in the amount of Ser202/Thr205 phosphorylated Tau, which is recognized by AT8.

The results showed that the antibody administration improved the pathological condition in the brain, confirming the aforementioned memory impairment improvement effect. In addition, the results showed that the intraperitoneally administered antibody acted in the brain.

[Example 10] Effect of Ta1505 Antibody Administration on Tau Levels in the Brain

Using AT8 antibodies (pSer202/pThr205 recognition mouse monoclonal antibody, manufactured by INNAX), which are believed to recognize hyperphosphorylated Tau that constitutes PHF, G2 (anti-human specific N-terminal region recognition antibody: rabbit polyclonal antibody), PHF1 (pSer396/pSer404 recognition mouse monoclonal antibody, which are believed to recognize hyperphosphorylated Tau that constitutes PHF), and Ta1505, the effect of 1505 antibody administration on the amount of Tau and hyperphosphorylated Tau in brain homogenates of Tau-Tg mice after antibody administration was studied by Western blotting.

100-200 mg of mouse cerebral hemisphere was sonicated with 5-fold volume of TBS (containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail), centrifuged at 100,000 g for 15 minutes at 4°C, and the supernatant was recovered as a TBS-soluble fraction.

The precipitate was then suspended in 1% sarkosyl/TBS (containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail), incubated at room temperature for 1 hour, and centrifuged at 100,000 g for 15 minutes at room temperature. The supernatant was used as the sarkosyl-soluble fraction.

The TBS-soluble and sarkosyl-soluble fractions were separated by electrophoresis using a 7% Tris-acetate gel, transferred to a PVDF membrane, and blocked overnight at room temperature with 1% BSA/3% skim milk/0.05% Tween 20/TBS. The membrane was then reacted with an antibody solution using an HRP-conjugated antibody as a secondary antibody, and analyzed and quantified using a LAS3000 image analyzer.

The results are shown in Figures 28 and 29.

In the TBS-soluble fraction, administration of the Ta1505 antibody confirmed a significant reduction in human Tau (recognized by the G2 antibody), hyperphosphorylated Tau (recognized by AT8 (pS202/pT205 epitopes) and PHF1 (pS396/pS404 epitopes), and pS413Tau (recognized by Ta1505) in the brains of Tau-Tg mice.

In the sarkosyl-soluble fraction, administration of the Ta1505 antibody confirmed a significant decrease in hyperphosphorylated Tau (recognized by AT8) in the brain of Tau-Tg mice.

The results showed that the antibody administration improved the pathological condition in the brain, confirming the aforementioned memory impairment improvement effect. In addition, the results showed that the intraperitoneally administered antibody acted in the brain.

[Preparation Example 1] Tg mice expressing normal human tau (Tau-Tg mice) as a model of cognitive impairment

In studies of the pharmacological effects of the antibodies of the present invention on improving cognitive function, Tg mice, characterized by the development of cognitive impairment around six months of age, were used, following the same expression pattern observed in humans with normal tau, specifically, those expressing only 3R tau during fetal development and expressing both 3R and 4R tau with growth. These Tg mice were generated by the following method.

The gene structure used to produce Tg mice uses the following nucleic acid: downstream of the α-calmodulin kinase IIα (CaMKII) promoter (8.5 kb), the simian virus 40 (SV40) 5' intron (0.3 kb), the tau gene (Tau; 7.3 kb), the SV40 3' intron (0.8 kb), and the SV40 polyA signal (0.3 kb) are arranged in this order, and the tau gene supply nucleic acid has the structure shown in Figure 17. The tau gene used was obtained by the same method as described in Yamashita T. et al. (FEBS Letter, Vol. 579, P241-244, 2005). It is a 7.3 kb long gene that includes, starting from the cDNA encoding human tau, the nucleotide sequence corresponding to the portion of exons 1 to 9, the nucleotide sequence of the first 18 nucleotides and the last 3 kb portion of intron 9, the nucleotide sequence of exon 10, the first 3 kb and the last 38 nucleotides of intron 10 (wherein the 16th nucleotide cytosine from the 5' end of intron 10 is substituted with thymine), and the nucleotide sequence of the cDNA corresponding to exons 11 to 13. The tau gene was cloned into the EcoRV site of the vector pNN265 (Choi T et al., Mol. Cell. Biol., Vol. 11, pp. 3070-3074, 1991), which contains the SV40 5' intron, 3' intron, and poly(A) signal sequence. A DNA fragment containing the SV40 5' intron, tau gene, SV40 3' intron, and poly(A) signal sequence was excised from the resulting plasmid using the restriction enzymes XhoI and NotI. This DNA fragment was then cloned into the pMM403 vector (Mayford M et al., Cell, Vol. 81, pp. 891-904, 1995), which contains the CaMKII promoter. The resulting plasmid was then excised using the restriction enzyme Sfil, which contained a 17.2 kb gene fragment with the structure shown in Figure 17 . This fragment was separated by agarose electrophoresis and purified from the corresponding gel portion using the QIAGEN(R) QIAquick Gel Extraction Kit (Cat. No. 28704). The resulting gene (DNA fragment) was injected into pronuclear embryos obtained by mating C57BL/6 mice according to a known method [Hogan, B et al., "Manipulating the mouse embryo. A Laboratory Manual," Cold Spring Harbor Laboratory (1986)]. The fragment was injected into the oviduct of a pseudopregnant female C57BL mouse that had been injected. The tails of the resulting pups were partially cut off, and PCR was used to confirm the insertion of the introduced gene. Female or male mice carrying the introduced gene were mated with normal female or male mice to maintain Tg mice carrying the introduced tau gene and used in experiments. It should be noted that Tau-Tg mice designated as strain 609 and strain 784 described in the Examples were produced by the same method and exhibited the same characteristics (or the description of the strains was deleted in the Examples).

Claims (19)

1. A therapeutic or preventive medicine for treating or preventing Tau proteinosis, comprising an antibody, wherein said antibody is characterized by being selected from one of the following (a)-(e): (a) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 8, the CDR-H2 sequence shown in SEQ ID NO: 9, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; (b) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 7, the CDR-H2 sequence shown in SEQ ID NO: 9, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; (c) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 7, the CDR-H2 sequence shown in SEQ ID NO: 10, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; (d) The antibody contains The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 8, the CDR-H2 sequence shown in SEQ ID NO: 12, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; and (e) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 7, the CDR-H2 sequence shown in SEQ ID NO: 11, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 15, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; and The antibody thereunder binds to tau protein that is phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 with greater affinity than the tau protein that is not phosphorylated at position 413. 2. The therapeutic or preventive medicine of claim 1, wherein the antibody is characterized by being selected from one of (i)-(vii): (i) The antibody comprises VH having the sequence shown in SEQ ID NO: 20 and VL having the sequence shown in SEQ ID NO: 26; (ii) The antibody comprises VH having the sequence shown in SEQ ID NO: 18 and VL having the sequence shown in SEQ ID NO: 25; (iii) The antibody comprises VH having the sequence shown in SEQ ID NO: 19 and VL having the sequence shown in SEQ ID NO: 26; (iv) The antibody comprises VH having the sequence shown in SEQ ID NO: 21 and VL having the sequence shown in SEQ ID NO: 27; (v) The antibody comprises VH having the sequence shown in SEQ ID NO: 22 and VL having the sequence shown in SEQ ID NO: 28; (vi) The antibody comprises VH having the sequence shown in SEQ ID NO: 23 and VL having the sequence shown in SEQ ID NO: 29; and (vii) The antibody comprises VH having the sequence shown in SEQ ID NO: 24 and VL having the sequence shown in SEQ ID NO: 30. 3. The therapeutic or preventive agent of claim 1, wherein the antibody has an affinity for tau protein phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 that is at least 10 times greater than that for tau protein unphosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1. 4. The therapeutic or preventive agent of claim 1, wherein the antibody has an affinity for tau protein phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 that is at least 30 times greater than that for tau protein unphosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1. 5. The therapeutic or preventive agent of claim 1, wherein the antibody has an affinity for tau protein phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 that is at least 100 times greater than that for tau protein unphosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1. 6. The therapeutic or preventative medicine of claim 1, wherein the antibody is a humanized antibody or a chimeric antibody. 7. The therapeutic or preventive medicine of any one of claims 1-6, wherein the tau protein disease is selected from Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, aerophilic granular dementia (aristocratic granular disease), multisystem tau protein disease in patients with dementia (MSTD), frontotemporal dementia linked to chromosome 17 with Parkinson's syndrome (FTDP-17), neurofibrillary tangles dementia, diffuse neurofibrillary tangles with calcium deposition (DNTC), white matter tau protein disease with globular glial inclusions (WMT-GGI), and frontotemporal degeneration with tau-positive inclusions (FTLD-tau). 8. A monoclonal antibody, characterized in that it is selected from one of the following (a)-(e), wherein (a) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 8, the CDR-H2 sequence shown in SEQ ID NO: 9, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; (b) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 7, the CDR-H2 sequence shown in SEQ ID NO: 9, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; (c) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 7, the CDR-H2 sequence shown in SEQ ID NO: 10, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; (d) The antibody contains The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 8, the CDR-H2 sequence shown in SEQ ID NO: 12, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 14, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; and (e) The antibody comprises The heavy chain variable region has three CDRs, namely the CDR-H1 sequence shown in SEQ ID NO: 7, the CDR-H2 sequence shown in SEQ ID NO: 11, and the CDR-H3 sequence shown in SEQ ID NO: 13. The light chain variable region has three CDRs, namely the CDR-L1 sequence shown in SEQ ID NO: 15, the CDR-L2 sequence shown in SEQ ID NO: 16, and the CDR-L3 sequence shown in SEQ ID NO: 17; and The monoclonal antibody thereunder binds to tau protein that is phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 with greater affinity than the tau protein that is not phosphorylated at position 413. 9. The monoclonal antibody of claim 8, wherein the binding between the monoclonal antibody and the antigen is competitive with the binding between the antigen and an antibody characterized by being selected from one of (i)-(vii): (i) The antibody comprises VH having the sequence shown in SEQ ID NO: 20 and VL having the sequence shown in SEQ ID NO: 26; (ii) The antibody comprises VH having the sequence shown in SEQ ID NO: 18 and VL having the sequence shown in SEQ ID NO: 25; (iii) The antibody comprises VH having the sequence shown in SEQ ID NO: 19 and VL having the sequence shown in SEQ ID NO: 26; (iv) The antibody comprises VH having the sequence shown in SEQ ID NO: 21 and VL having the sequence shown in SEQ ID NO: 27; (v) The antibody comprises VH having the sequence shown in SEQ ID NO: 22 and VL having the sequence shown in SEQ ID NO: 28; (vi) The antibody comprises VH having the sequence shown in SEQ ID NO: 23 and VL having the sequence shown in SEQ ID NO: 29; and (vii) The antibody comprises VH having the sequence shown in SEQ ID NO: 24 and VL having the sequence shown in SEQ ID NO: 30. 10. The monoclonal antibody of claim 8, wherein the antibody is characterized by being selected from one of (i)-(vii): (i) The antibody comprises VH having the sequence shown in SEQ ID NO: 20 and VL having the sequence shown in SEQ ID NO: 26; (ii) The antibody comprises VH having the sequence shown in SEQ ID NO: 18 and VL having the sequence shown in SEQ ID NO: 25; (iii) The antibody comprises VH having the sequence shown in SEQ ID NO: 19 and VL having the sequence shown in SEQ ID NO: 26; (iv) The antibody comprises VH having the sequence shown in SEQ ID NO: 21 and VL having the sequence shown in SEQ ID NO: 27; (v) The antibody comprises VH having the sequence shown in SEQ ID NO: 22 and VL having the sequence shown in SEQ ID NO: 28; (vi) The antibody comprises VH having the sequence shown in SEQ ID NO: 23 and VL having the sequence shown in SEQ ID NO: 29; and (vii) The antibody comprises VH having the sequence shown in SEQ ID NO: 24 and VL having the sequence shown in SEQ ID NO: 30. 11. The monoclonal antibody of claim 8, wherein the affinity of the monoclonal antibody for tau protein phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 is at least 10 times that for tau protein unphosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1. 12. The monoclonal antibody of claim 8, wherein the affinity of the monoclonal antibody for tau protein phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 is at least 30 times that for tau protein unphosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1. 13. The monoclonal antibody of claim 8, wherein the affinity of the monoclonal antibody for tau protein phosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1 is at least 100 times that for tau protein unphosphorylated at position 413 of the tau protein shown in SEQ ID NO: 1. 14. The monoclonal antibody of claim 8, wherein the antibody is a humanized antibody or a chimeric antibody. 15. Use of the monoclonal antibody of any one of claims 8-14 in the preparation of a medicament for treating tau proteinosis. 16. The use of claim 15, wherein the tau disease is selected from Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, aerophilic granular dementia (aristocratic granular disease), multisystem tau disease in patients with dementia (MSTD), frontotemporal dementia linked to chromosome 17 with Parkinson's syndrome (FTDP-17), neurofibrillary tangles dementia, diffuse neurofibrillary tangles with calcium deposits (DNTC), white matter tau disease with globular glial inclusions (WMT-GGI), and frontotemporal degeneration with tau-positive inclusions (FTLD-tau). 17. A peptide consisting of SEQ ID NO: 35, wherein the amino acid residue of Ser at position 413 of the amino acid sequence of the human tau protein shown in SEQ ID NO: 1 is phosphorylated. 18. Use of the peptide of claim 17 in the preparation of a monoclonal antibody for the treatment or prevention of Tau proteinosis. 19. The use of claim 18, wherein the tau disease is selected from Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, aerophilic granular dementia (aristocratic granular disease), multisystem tau disease in patients with dementia (MSTD), frontotemporal dementia linked to chromosome 17 with Parkinson's syndrome (FTDP-17), neurofibrillary tangles dementia, diffuse neurofibrillary tangles with calcium deposits (DNTC), white matter tau disease with globular glial inclusions (WMT-GGI), and frontotemporal degeneration with tau-positive inclusions (FTLD-tau).