Humanized antibody for treating or preventing cognitive disorders, process for producing the same, and agent for treating or preventing cognitive disorders using the same

Abstract

To provide methods for using and compositions of humanized antibodies that bind to a tau protein that is phosphorylated at the serine at a position 413.SOLUTION: The present invention provides an anti-pSer413 tau antibody comprising: a heavy chain variable domain consisting of a specific amino acid sequence; and a light chain variable domain consisting of a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of Japanese Patent Application No. 2017-035594 filed on February 27, 2017. The benefit of which is claimed and the contents of which are expressly incorporated herein by reference in their entirety.

[0002] II. SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety. ASCII Copy created on February 27, 2018. The name is 063785-06-5002-WO-SeqLstg-_ST25.txt and its size is 323,584 bytes.

[0003] III. Technology Field The present disclosure relates to humanized antibodies for use in the therapeutic or prophylactic treatment of cognitive disorders, A process for producing the humanized antibody, a therapeutic or preventive drug for cognitive impairment using the antibody, and The present invention relates to a method for treating or preventing cognitive impairment using the therapeutic agents disclosed herein. In particular, the present disclosure provides a novel humanized anti-phosphorylated tau antibody that has excellent effects on improving cognitive function. a method for producing the humanized antibody; a method for treating cognitive impairment comprising the humanized antibody; A preventive agent and a method for treating or preventing cognitive impairment using a humanized anti-phosphorylated tau antibody Regarding. [Background technology]

[0004] IV. Background of the Invention Cognitive impairment, or dementia, is a condition in which developed intelligence deteriorates due to some acquired cause. As a result, it is a pathological condition that impairs social adaptation. Cognitive impairment is caused by neurodegenerative diseases, vascular cognitive disorders, etc. diseases, prion diseases, infectious diseases, metabolic / endocrine disorders, traumatic and neurosurgical disorders, toxic disorders Classified as (Toshifumi Kishimoto & Shigeki Tak ahashi(Editor.), “STEP Series Seishinka”(Ja panese document), 2nd Edition, Kaibashobo, As of 2010, there are approximately 2.1 million people with HIV in Japan. There is dementia, with a prevalence of approximately 8-10% and >10% among those aged >65 years. This is recognized as a serious problem in the aging society worldwide (Taka Shi Asada, “Igaku no Ayumi” (Japanese docu ment),supplementary volume,“Cognitive di Soders”, Ishiyaku Publishing, 2011, pp.5-1 0) The majority of data on the underlying diseases of cognitive impairment are from Alzheimer's disease (AD) and pre- Neurodegenerative diseases such as temporal lobar degeneration (FTLD), of which about 35% are AD, and about 15% are % of patients had both AD and cerebrovascular disease, and 5% had FTLD (ibid.). Cognitive impairment associated with sexually transmitted diseases is characterized by progressive memory loss over a period of at least 6 months. The neurodegenerative process is characterized by an insidious onset of cognitive and / or personality changes. A consistent feature that is highly correlated with and includes the degree of damage is the presence of neurofibrillary tangles (NFTs). FT) (Alistair Burns et al. (Editor), D ementia,3rd Edition,2005,CRC Press,pp.40 8-464).

[0005] Tau protein is a member of the MAPT gene, which is located on chromosome 17 (17q21) of the human genome. Tau is a protein encoded by the tau gene. Tau protein is abundant in the central nervous system. Tau is one of the microtubule-associated proteins expressed in the brain. Tau is one of the most prominent neurodegenerative diseases. The main structures of the paired helical and straight fibrils that form NFTs in AD It has been found to be a structural protein and its intracellular expression is associated with various neuropathological conditions. Accumulation is shown.

[0006] Tau was initially identified as a mutation in the MAPT gene and a mutation in the parkinsonian protein linked to chromosome 17. Between tau accumulation in familial frontotemporal dementia with sonism (FTDP-17) Based on the association, FTDP-17 has been implicated in neurodegenerative diseases. More than 40 gene mutations have been reported in the gene (Tetsuaki Arai, “Sh inkei Naika”(Japanese document),Vol.72,s special number,(Suppl.6),2010,pp.46-51). child These genetic mutations change the ratio of tau isoforms or alter the structure, and and those that alter the interaction between mutant tau and microtubules, thus contributing to the development of pathology. However, unlike familial neurodegenerative diseases, MAPT mutations are It is rarely observed in sporadic neurodegenerative diseases such as D. Tau that accumulates in the brain is characterized by being highly modified through phosphorylation. Furthermore, in patients with mild cognitive impairment (MCI), the levels of phosphorylated tau in the spinal fluid and ptosis were significantly increased. A correlation was observed between the degree of tau atrophy and phosphorylated tau in tauopathy patients. (Wendy N oble et al.,Expert Opinion on Drug Disco Very, 2011, Vol. 6, No. 8, pp. 797-810). Based on this, we have been using quinine, an enzyme involved in phosphorylation, to inhibit the excessive phosphorylation of tau. Attempts to develop therapeutic approaches using inhibitors of GSK-3β, particularly GSK-3β inhibitors However, kinases such as GSK-3β are not involved in pathological conditions. They are also involved in the regulation of normal physiological processes, and therefore potential side effects Indeed, some of the sites on tau that are phosphorylated by GSK-3β have been implicated in Some of these correspond to sites of tau phosphorylation observed in fetal and normal human brain. (Burnes et al., supra), suggesting that this strategy may affect normal tau function. It is suggesting.

[0007] Extracellular tau is thought to originate from leakage from degenerating neurons as a result of cell death. However, recent studies have demonstrated that tau is processed after excessive intracellular phosphorylation and then actively transported into cells. It has been suggested that phosphorylated tau secreted from cells is secreted outside the cells. It is dephosphorylated at the phosphorylation site and then acts on the muscarinic receptors M1 and M3 of surrounding cells. It is believed that this has various effects, such as promoting intracellular tau phosphorylation and contributing to cell death. (Miguel Diaz-Hernandes et al., Journal of Biological Chemistry,2010,Vol.285,No. 42, pp.32539-32548 and Venessa Plouffe et al. .,PLoS ONE,2012,Vol.7,p.36873). In an attempt to achieve this goal, researchers have developed a method to treat tauopathies that targets the tau protein. Experiments on immunotherapy for HIV have been reported (Noble, supra, and Kishimo, 2003). see, Asada, supra, and Burns, supra).

[0008] The main symptoms of cognitive impairment in humans are memory impairment and cognitive dysfunction. The role of cognitive functions in memory-based judgment, communication, and performance On the other hand, motor function is important in Parkinson's disease, which is linked to chromosome 17. In familial frontotemporal dementia with dementia (FTDP-17) and end-stage Alzheimer's disease Although it is a symptom that can be seen in some cases, it is not necessarily the primary symptom of cognitive impairment. Therefore, the main issue to consider in treating cognitive disorders is the improvement of cognitive function. However, currently there are no suitable animal models to test tauopathy-associated cognitive impairment. The existence of a novel therapeutic agent that can be used to treat cognitive disorders allows for the identification of therapeutic or prophylactic agents. Furthermore, there are no drugs that have a specific and excellent effect on cognitive impairment. do not.

[0009] The role of phosphorylated tau in therapeutic and preventive applications against cognitive impairment in human subjects Not only does it have high binding affinity to the human antigen, but it also has a humanized structure that reduces antigenicity to the human body. There is a need for anti-phosphorylated tau antibodies. Summary of the Invention

[0010] V. Summary of the Invention Against this background, the present inventors have demonstrated that aberrant phosphorylation in the state of Alzheimer's disease Among the many phosphorylation sites available in the tau protein, the phospho- serine residue at position 413 We focused on the phosphorylation site (Ser413) and performed a polyclonal antibody specific to Ser413 phosphorylated tau. We have successfully produced rabbit and mouse monoclonal antibodies. A mouse model of cognitive impairment that showed abnormal expression of tau protein was administered monoclonal mouse antibodies. The authors confirmed that the administration of the compound resulted in improved cognitive function (WO2013 / 180238 A).

[0011] In some embodiments, the present disclosure provides a method for the preparation of a tautan that is phosphorylated at serine residue 413. Proteins, such as antibodies, that contain an antigen-binding portion that specifically binds to the protein (pS413-tau) In some embodiments, the present disclosure provides nucleic acids encoding those proteins. In some embodiments, the present disclosure provides nucleic acid sequences encoding these proteins. In some embodiments, the present disclosure provides a cell comprising such a nucleic acid. by administering to a patient in need thereof a protein, nucleic acid, or cell that exhibits the Methods for treating tauopathies (eg, Alzheimer's disease) are provided.

[0012] Thus, in some embodiments, the present invention provides a method for the preparation of a nucleotide sequence corresponding to at least Ser413 of SEQ ID NO:1. Tau protein or tau peptide phosphorylated at corresponding amino acid residues and antigen -Provides a humanized antibody or a fragment or derivative thereof that elicits an antibody response. In the case of 1.86×10 against Petite -8 It has an equilibrium dissociation constant of less than or equal to M.

[0013] In a further aspect, the humanized antibody or fragment or derivative thereof is a humanized antibody that binds to non-phosphorylated tautan. phosphorylated tau protein compared to tau protein (or tau peptide, e.g., SEQ ID NO: 69) The tau peptide has a higher selective affinity for the target substance (or tau peptide, e.g., SEQ ID NO:8).

[0014] In a further aspect, the humanized antibodies or fragments or derivatives thereof described herein are They have a greater ability to enter the brain when administered intravenously.

[0015] In a further embodiment, the humanized antibody or fragment or derivative thereof has the following CDRs: Contains: CDR-H1, an amino acid sequence having 80% or more homology to sequence 1M Sequence: CDR-H2 sequence has 84% ​​or more homology to sequence 2M or 2H1. The amino acid sequence that has 80% or more homology to sequence 3M as the CDR-H3 sequence. As a CDR-L1 sequence, the amino acid sequence is 87% or more of the sequence 4L1 or 4M. The amino acid sequence has a homology of 85% or more to the sequence 5M as the CDR-L2 sequence. and an amino acid sequence having a homology of 77% to sequence 6M as a CDR-L3 sequence. An amino acid sequence having the above homology.

[0016] In a further embodiment, the humanized antibody or fragment or derivative thereof has the following CDRs: As the CDR-H1 sequence, the amino acid sequence of SEQ ID NO:1M; as the CDR-H2 sequence, The amino acid sequence of sequence 2H1 or the substitution of Ala at position 15 with Asp. an amino acid sequence different from sequence 2H1 as the CDR-H3 sequence; an amino acid sequence of sequence 3M as the CDR-H3 sequence; As the CDR-L1 sequence, the amino acid sequence of sequence 4L1 or Ser at position 5 is Asn The amino acid sequence of sequence 4L1 that differs from sequence 4L1 in terms of substitutions; and as the CDR-L3 sequence, the amino acid sequence of sequence 6M.

[0017] In a further embodiment, the humanized antibody or fragment or derivative thereof has the following CDRs: (1) as the CDR-H1 sequence, an amino acid sequence of 1M; as the CDR-H2 sequence, The amino acid sequence of 2H1; the amino acid sequence of 3M; the CDR-L the amino acid sequence of 4L1 as the CDR-L1 sequence; the amino acid sequence of 5M as the CDR-L2 sequence; and as a CDR-L3 sequence, an amino acid sequence of 6M; or (2) as a CDR-H1 sequence, As the CDR-H2 sequence, the amino acid sequence of 2H1 is As the CDR-L1 sequence, the amino acid sequence of 3M is used; as the CDR-L2 sequence, the amino acid sequence of 4M is used; 5M amino acid sequence as CDR-L2 sequence; and 6M amino acid sequence as CDR-L3 sequence. or (3) a 1M amino acid sequence as a CDR-H1 sequence; As the sequence, the 2M amino acid sequence; as the CDR-H3 sequence, the 3M amino acid sequence; - As the CDR-L1 sequence, an amino acid sequence of 4M; as the CDR-L2 sequence, an amino acid sequence of 5M and as the CDR-L3 sequence, an amino acid sequence of 6M.

[0018] In a further aspect, the humanized antibody or fragment or derivative thereof comprises: as the heavy chain variable region , sequences VH11, VH12, VH47, VH61, VH62, VH64, and VH65 an amino acid sequence having 90% or more homology to a sequence selected from the group consisting of a light chain variable region and a and selecting from sequences VL15, VL36, VL46, VL47, VL48, and VL50. It has an amino acid sequence that is 90% or more identical to the sequence to be determined.

[0019] In a further aspect, the humanized antibody or fragment or derivative thereof comprises: as the heavy chain variable region , the amino acid sequence of sequence VH65, or the substitution of Ala to Thr at position 28 (Kabat numbering: H28), substitution of Asn to Ser at position 30 (Kabat numbering : H30), substitution of Val to Gly at position 49 (Kabat numbering: H49), Ala to Asp substitution at position 64 (Kabat numbering: H61) and Gln to Lys substitution (Kabat numbering: H75) an amino acid sequence that differs from sequence VH65 in that it contains one or more substitutions in The region is the amino acid sequence of VL47, or the substitution of Asp at position 17 with Glu ( Kabat numbering: L17), Ser to Asn substitution at position 28 (Kabat numbering: numbering: L27A), substitution of Gln to Leu at position 42 (Kabat numbering : L37), substitution of Gln to Arg at position 50 (Kabat numbering: L45), and a substitution of Arg to Leu at position 51 (Kabat numbering: L46) having an amino acid sequence that differs from sequence VL47 in that it contains one or more substitutions selected from .

[0020] In a further aspect, the humanized antibody or fragment or derivative thereof comprises a combination of the following sequences: (a) a combination of the sequence VH11 as the heavy chain variable region and the sequence VH12 as the light chain variable region; (b) sequence VH11 as the heavy chain variable region and sequence VL15 as the light chain variable region; (c) sequence VH11 as the heavy chain variable region and sequence VH12 as the light chain variable region (d) sequence VH11 as the heavy chain variable region and sequence VL 47; (e) sequence VH11 as the heavy chain variable region and sequence VL48 as the light chain variable region (f) sequence VH11 as the heavy chain variable region and sequence VL50 as the light chain variable region; g) the sequence VH12 as the heavy chain variable region and the sequence VL48 as the light chain variable region; (h) The sequence VH47 as the heavy chain variable region and the sequence VL48 as the light chain variable region; (i) heavy chain (j) sequence VH61 as the variable region and sequence VL48 as the light chain variable region; (k) heavy chain variable region, sequence VH62 as the light chain variable region and sequence VL48 as the light chain variable region; (l) the sequence VH64 as the light chain variable region and the sequence VL47 as the light chain variable region; (m) the sequence VH65 as the heavy chain variable region; and sequence VL47 as the light chain variable region.

[0021] In a further aspect, the present invention relates to a humanized antibody or fragment or derivative thereof as described herein. The present invention provides a drug for treating or preventing dementia, comprising a conductor.

[0022] In a further aspect, the present invention relates to a method for treating Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Tauopathy, Pick's disease, argyria, multisystem tauopathy with presenile dementia - (MSTD), familial frontotemporal dementia with parkinsonism linked to chromosome 17 Dementia with neurofibrillary tangles (FTDP-17), diffuse neurofibrillary tangles with calcification White matter tauopathy with globular glial inclusions (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), Dementia or tauopathy, including frontotemporal lobar degeneration with tau pathology (FTLD-tau) The present invention provides methods and agents for treating or preventing

[0023] In a further aspect, the present invention relates to a humanized antibody or fragment or derivative thereof as described herein. A pharmaceutical composition is provided that includes the conductor and a pharma- ceutically acceptable carrier.

[0024] In a further aspect, the present invention provides a method for the preparation of a polypeptide comprising: a) a vhCDR1 having SEQ ID NO: 86, a vhCDR2 having SEQ ID NO: 11 5, and vhCDR3 having SEQ ID NO: 88. and b): i) vlCDR1 having SEQ ID NO: 102, vlCDR2 having SEQ ID NO: 82 ii) vlCDR2 and vlCDR3 having SEQ ID NO: 91; CDR1, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO: 83 iii) a vlCDR1 having SEQ ID NO: 92, a vlCDR2 having SEQ ID NO: 82, and and vlCDR3 having SEQ ID NO: 83; iv) vlCDR1 having SEQ ID NO: 93, sequence v) vlCDR2 having the sequence number 82, and vlCDR3 having the sequence number 83; vlCDR1 having SEQ ID NO: 94, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO: 83. v) CDR1 having SEQ ID NO: 95, vCDR3 having SEQ ID NO: 82 v) CDR2 having SEQ ID NO: 83; and v) CDR3 having SEQ ID NO: 96. lCDR1 having SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83 3; viii) vlCDR1 having SEQ ID NO: 97, vlCDR2 having SEQ ID NO: 82 and vlCDR3 having SEQ ID NO: 83; ix) vlCDR1 having SEQ ID NO: 98, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO: 83; x) sequences vlCDR1 having the sequence number 99, vlCDR2 having the sequence number 82, and vlCDR3 having the sequence number 83 xi) vlCDR3 having SEQ ID NO: 100, vlCDR1 having SEQ ID NO: 82 and vlCDR2 having SEQ ID NO: 83; xii) vlCDR3 having SEQ ID NO: 101 vlCDR1 having SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83 a light chain variable domain comprising a set of vlCDRs selected from the group consisting of Anti-pSer413 tau antibodies are provided. In some cases, the antibodies are non-pSer413 tau antibodies of SEQ ID NO:69. Antibodies that bind to phosphorylated peptides of SEQ ID NO: 8 The body ratio is at least about 40.

[0025] In a further aspect, the present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: a) selecting from SEQ ID NO: 116 and SEQ ID NO: 117; and b) a heavy chain variable domain, No. 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: A light chain variable region selected from SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114. In some cases, the antibody comprises a sequence The phosphorylated peptide of SEQ ID NO:8 against an antibody that binds to the non-phosphorylated peptide of SEQ ID NO:69 The ratio of antibody that binds to the target polypeptide is at least about 40.

[0026] In a further embodiment, the antibody of the present invention is 7, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:14 2 and SEQ ID NO:144.

[0027] In a further aspect, the antibody of the invention comprises a light antibody selected from SEQ ID NO: 79 and SEQ ID NO: 80. The polypeptide chains contain constant domains.

[0028] In a further aspect, the antibody of the invention is selected from SEQ ID NO: 116 and SEQ ID NO: 117. The antibody comprises a heavy chain variable domain.

[0029] In a further aspect, the antibody of the invention is 5, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:11 0, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114. The antibody comprises a light chain variable domain.

[0030] In a further embodiment, the antibody comprises a LC of SEQ ID NO: 184 and a HC of SEQ ID NO: 144; LC of SEQ ID NO:184 and HC of SEQ ID NO:145; LC of SEQ ID NO:184 and HC of SEQ ID NO:146; LC of SEQ ID NO:184 and HC of SEQ ID NO:147; LC of SEQ ID NO:184 and HC of SEQ ID NO:148 HC of SEQ ID NO: 184 and LC of SEQ ID NO: 149; LC of SEQ ID NO: 184 and HC of SEQ ID NO: HC of No. 150; LC of SEQ ID NO: 184 and HC of SEQ ID NO: 151; LC of SEQ ID NO: 184 and HC of SEQ ID NO: 152; LC of SEQ ID NO: 184 and HC of SEQ ID NO: 153; LC of SEQ ID NO: 184 and HC of SEQ ID NO: 155; LC of SEQ ID NO:184 and HC of SEQ ID NO:156; LC of SEQ ID NO:184 and H of SEQ ID NO:157 C; LC of SEQ ID NO: 184 and HC of SEQ ID NO: 158; LC of SEQ ID NO: 184 and HC of SEQ ID NO: 1 59; LC of SEQ ID NO: 184 and HC of SEQ ID NO: 160; LC of SEQ ID NO: 184 HC of sequence number 161; LC of sequence number 185 and HC of sequence number 144; LC and HC of SEQ ID NO: 145; LC of SEQ ID NO: 185 and HC of SEQ ID NO: 146; SEQ ID NO: LC of SEQ ID NO:185 and HC of SEQ ID NO:147; LC of SEQ ID NO:185 and HC of SEQ ID NO:148; LC of SEQ ID NO:185 and HC of SEQ ID NO:149; LC of SEQ ID NO:185 and SEQ ID NO:150 HC of SEQ ID NO: 185 and LC of SEQ ID NO: 151; LC of SEQ ID NO: 185 and HC of SEQ ID NO: HC of No. 152; LC of SEQ ID NO: 185 and HC of SEQ ID NO: 153; LC of SEQ ID NO: 185 and HC of SEQ ID NO: 154; LC of SEQ ID NO: 185 and HC of SEQ ID NO: 155; LC of SEQ ID NO: 185 and HC of SEQ ID NO: 157; LC of SEQ ID NO:185 and HC of SEQ ID NO:158; LC of SEQ ID NO:185 and H of SEQ ID NO:159 C; LC of SEQ ID NO: 185 and HC of SEQ ID NO: 160; and LC of SEQ ID NO: 185 and HC of SEQ ID NO: No. 161.

[0031] In a further embodiment, the antibody of the present invention comprises a light antibody having or consisting of SEQ ID NO: 184. and a heavy chain having or consisting of SEQ ID NO:144.

[0032] In a further embodiment, the antibody of the present invention comprises a light antibody having or consisting of SEQ ID NO: 184. and a heavy chain having or consisting of SEQ ID NO:152.

[0033] In a further aspect, the present invention provides: a) SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164 , SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169 , SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174 , SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179 , SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184 and SEQ ID NO: 185; and b) a first nucleic acid encoding a light chain selected from SEQ ID NO: 144; SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:147; SEQ ID NO:148; SEQ ID NO: 149; SEQ ID NO:150; SEQ ID NO:151; SEQ ID NO:152; SEQ ID NO:153; SEQ ID NO: 154; SEQ ID NO:155; SEQ ID NO:156; SEQ ID NO:157; SEQ ID NO:158; SEQ ID NO: 159; a second nucleic acid encoding a heavy chain selected from SEQ ID NO:160 and SEQ ID NO:161; The present invention provides a nucleic acid composition comprising:

[0034] In a further aspect, the invention provides an expression vector composition comprising a first and a second nucleic acid. The first nucleic acid is contained in a first expression vector and the second nucleic acid is contained in a second expression vector. do.

[0035] In a further aspect, the invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: The present invention provides an expression vector composition.

[0036] In a further aspect, the invention relates to a host cell comprising the expression vector composition, and a method for producing said antibody. and culturing the host cell under conditions consistent with the present invention and recovering the antibody. Methods for producing antibodies are provided.

[0037] In a further aspect, the present invention provides a method for treating tauopathy in a subject comprising administering an antibody of the present invention. The present invention provides a method for treating

[0038] VI. Brief description of the drawings The invention is best understood from the following detailed description when read in conjunction with the accompanying drawing figures. The drawings include the following figures: [Brief description of the drawings]

[0039] [Figure 1A] An alignment of the various human isoforms of tau protein, generated using ClustalW, is shown. [Figure 1B] An alignment of the various human isoforms of tau protein, generated using ClustalW, is shown.

[0040] [Figure 2A] 1 shows an alignment of light chain variable region sequences according to some embodiments of the present disclosure, highlighting the CDR-L1, CDR-L2, and CDR-L3 sequences of several anti-pS413-tau binding proteins according to the Kabat numbering system.

[0041] [Figure 2B] 1 shows an alignment of heavy chain variable region sequences according to several embodiments of the present disclosure, highlighting the CDR-H1, CDR-H2, and CDR-H3 sequences of several anti-pS413-tau binding proteins according to the Kabat numbering system.

[0042] [Diagram 3] 1 shows the selective binding affinity of several anti-pS413-tau antibodies to phosphorylated peptide PD17P compared to non-phosphorylated peptide PD17, according to several embodiments of the present disclosure.

[0043] [Figure 4] 1 shows the binding affinity of several anti-pS413-tau antibodies to phosphorylated tau in brain homogenates from Alzheimer's disease patients, according to some embodiments of the present disclosure.

[0044] [Figure 5A]Pharmacokinetics of humanized antibody variants over time in mouse plasma, showing that five initial humanized antibody variants 2, 5, 6, 9, and 10 exhibited rapid elimination from mouse plasma. [Figure 5B] Figure 1 shows the pharmacokinetics of humanized antibody variants over time in mouse plasma, demonstrating that three subsequently developed humanized antibody variants, L15H11, L46H11, and L47H65, exhibited pharmacokinetic properties comparable to the chimeric antibody.

[0045] [Figure 6A] Figure 1 shows that different humanized antibody variants have different brain migration.Figure 2 shows the concentration of various humanized antibodies in brain homogenates. [Figure 6B] Figure 1 shows that different humanized antibody variants have different brain translocations.The ratio of the levels of a representative humanized antibody in the brain to the levels of the corresponding antibody in plasma is shown.

[0046] [Figure 7A] 1 shows an alignment of the amino acid sequences of the light chain variable regions of representative humanized antibody mutants with the parent mouse antibody. [Figure 7B] 1 shows an alignment of the amino acid sequences of the heavy chain variable regions of representative humanized antibody mutants with the parent mouse antibody.

[0047] [Figure 8] A to D show that the parental murine antibody (Figures 8A and 8B) and the chimeric antibody (Figures 8C and 8D) bind to the phosphorylated peptide (Figures 8A and 8C) but not to the non-phosphorylated peptide (Figures 8B and 8D).

[0048] [Figure 9] AC show binding of representative humanized antibody mutants to the pS413 peptide.

[0049] [Figure 10]A-E show comparable binding characteristics of the parent mouse antibody (FIG. 10A), the chimeric antibody (FIG. 10B), and selected humanized antibody variants (FIGS. 10C-10E) to S413 phosphorylated tau protein in brain homogenates from Alzheimer's disease patients.

[0050] [Figure 11A] 1 shows the heavy and light chain variable regions of several anti-pSer413 tau protein humanized antibodies of the present invention. [Figure 11B] 1 shows the heavy and light chain variable regions of several anti-pSer413 tau protein humanized antibodies of the present invention. [Figure 11C] 1 shows the heavy and light chain variable regions of several anti-pSer413 tau protein humanized antibodies of the present invention. [Figure 11D] 1 shows the heavy and light chain variable regions of several anti-pSer413 tau protein humanized antibodies of the present invention. [Figure 11E] 1 shows the heavy and light chain variable regions of several anti-pSer413 tau protein humanized antibodies of the present invention.

[0051] [Figure 12]A number of different human IgG constant domains are shown that are available for the combination of variable heavy and variable light chains of anti-pSer413 tau antibodies. "Human_IgG1_" is the constant domain of human wild type IgG1 (CH1-hinge-CH2-CH3). "Human IgG1_L234A_L235A" is the constant domain of human wild type IgG1 (CH1-hinge-CH2-CH3) with two amino acid substitutions L234A and L235A (sometimes referred to as "LALA" mutations) that reduce / eliminate effector function. "Human IgG1_L234A_L235A_D265S" is the constant domain of human wild type IgG1 (CH1-hinge-CH2-CH3) with three amino acid substitutions L234A, L235A and D265S that reduce / eliminate effector function. "Human IgG1_YTE_" or "Human IgG1_YTE(M252Y_S254T_T256E)" is the constant domain of human wild type IgG1 (CH1-hinge-CH2-CH3) with three amino acid substitutions M252Y, S254T, T256E that extend the half-life of the antibody in serum. "Human IgG1_N297A_" is the constant domain of human wild type IgG1 (CH1-hinge-CH2-CH3) with the amino acid substitution N297A, which eliminates a glycosylation site and reduces / eliminates effector function. "Human IgG1_N297Q_" is the constant domain of human wild type IgG1 (CH1-hinge-CH2-CH3) with the amino acid substitution N297Q, which eliminates a glycosylation site and reduces / eliminates effector function. "_human IgG2" is the constant domain of human wild type IgG2 (CH1-hinge-CH2-CH3). "_human IgG4" is the constant domain of human wild type IgG4 (CH1-hinge-CH2-CH3). "Human IgG4_S228P_" is the constant domain of human wild type IgG4 (CH1-hinge-CH2-CH3) with the amino acid substitution S228P to prevent arm exchange.

[0052] [Figure 13] The full-length heavy chains of the H11 and H65 variable regions are shown along with the main chain in Figure 12. A "slash" (" / ") indicates the junction between the variable and constant domains, and the CDRs are underlined.

[0053] [Figure 14] Full-length light chains containing either a human kappa or lambda light chain constant domain are shown for the 12 different variable light chain domains. A "slash" (" / ") indicates the junction between the variable and constant domains, and the CDRs are underlined.

[0054] [Figure 15] Figure 1 shows a matrix of possible combinations of heavy and light chains of the invention. The "A" in the box indicates that the heavy chain constant domain "human_IgG1_" is used. The "B" in the box indicates that the heavy chain constant domain "human_IgG1_L234A_L235A" is used. The "C" in the box indicates that the heavy chain constant domain "human_IgG1__L234A_L235A_D265S" is used. The "D" in the box means that the heavy chain constant domain "human_IgG1_YTE_" is used. The "E" in the box means that the heavy chain constant domain "human_IgG1_N297A_" is used. The "F" in the box means that the heavy chain constant domain "human_IgG1_N297Q_" is used. The "G" in the box means that the heavy chain constant domain "_human_IgG2" is used. The "H" in the box means that the heavy chain constant domain "_human_IgG4" is used. The "I" in the box means that the heavy chain constant domain "human_IgG4_S228P_" is used. The "J" in the box means that the human IgG1 constant domain with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions is used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] VII. Detailed Description A. Overview The present disclosure relates to a tau protein that is phosphorylated at serine residue 413 (pSer413- The present invention provides an antibody that contains an antigen-binding portion that specifically binds to tau. Also provided are nucleic acids that encode nucleic acids that encode the nucleic acids, and cells that contain such nucleic acids. The provided methods and compositions are used to treat tauopathies (e.g., Alzheimer's disease). .

[0056] The MAPT gene, which encodes tau, consists of 13 exons arranged in the genome. It has been identified as a novel protein that can be expressed through alternative splicing in several different protein entities. Tau protein can be expressed as multiple isoforms (see Arai, supra). Depending on the alternative splicing of exon 2 and exon 3, 0 to 2 sequences of 29 amino acids can be N-terminal acidic domain containing repeat sequences (N) (N0-N2), proline-rich intermediate domain , and three (3R) or four (4R) repeats (R) that contribute to microtubule binding. A C-terminal microtubule-binding domain (encoded by exons 9-12) (Burns et al. (ibid.) and Arai (ibid.). Thus, human tau contains 29 Six representative amplicons were selected according to the number of amino acid repeats (N) and microtubule-binding repeats (R). Has isoforms: 3R0N (352 amino acids), 3R1N (381 amino acids), 3 R2N (410 amino acids), 4R0N (383 amino acids), 4R1N (412 amino acids) Of these isoforms, only 3R0N is a foetal In the fetal brain, all six isoforms are present, with 4R being the most abundant. (Burns, supra). The difference is that exon 10 is either removed via alternative splicing (3R) or absent (4R). In both of these tau isoforms, amino To clearly identify the position of the acid residue, the longest isoform, i.e., 4R2N (sequence The amino acid numbers (1 to 441) of the sequence defined in column 1 are used as a reference. For example, "Ser413" refers to the 413th amino acid position of 4R2N (as defined in SEQ ID NO:1). This refers to the serine residue at position 384 of 4R1N (as defined in SEQ ID NO:2), 4R0 3R2N (as defined in SEQ ID NO: 4) 82, 3R1N (as defined in SEQ ID NO:5) and 353, 3R0N (as defined in SEQ ID NO:6) This corresponds to the serine at position 324 of the ribozyme (as defined above).

[0057] Tau is a nucleotide sequence consisting of a serine residue at position 413 (Ser413) of the amino acid sequence defined in SEQ ID NO:1. and when phosphorylated, referred to herein as "pSer413" The protein has a phosphorylated amino acid residue at the Ser413 position (thus, The tau protein is "pSer413 tau" or "pSer413-tau"). This is a site that is specifically phosphorylated in AD. As previously shown, tau is involved in a specific antigen-antibody reaction with PSer413-tau. The antibody was administered to transgenic mice that develop cognitive impairment during maturation, and the results showed that Cognitive function was restored to almost the same level as in the control group. Tautan having a phosphorylated amino acid residue at the position corresponding to Ser396 in the amino acid sequence A monoclonal antibody that has a stronger affinity for the protein than the above antibody for the equivalent antigen. Administration of the antibody at a similar concentration did not result in sufficient improvement in cognitive function. and function of the amino acid sequence defined in SEQ ID NO:1 at a position corresponding to Ser413. Since there was no specific information about the region that contains the amino acid residues, antibodies that bind to this region were It was a completely unexpected result that it had such a strong effect of improving cognitive function.

[0058] Thus, the present invention provides a method for treating cognitive disorders, such as tauopathies, in a human subject. and optimized anti-pSer413 tau antibodies useful as therapeutic or prophylactic agents for Regarding.

[0059] The humanized anti-phosphorylated tau antibody according to the present invention exhibits high binding affinity to phosphorylated tau. On the other hand, its antigenicity to the human body is significantly reduced, and it is expected to be effective in preventing tauopathy in human subjects. It can be effectively used as a therapeutic or preventive drug for cognitive disorders such as C. Amino acid modifications within the DRs reduce deamidation and increase stability compared to mouse CDRs ;See Examples 8, 9 and 10.

[0060] B. Definition As used herein, each of the following terms refers to the "Conditions" shall have the meaning set forth in the following paragraphs:

[0061] The articles "a" and "an" refer to one or more (i.e., at least one) of the grammatical object of the article. The term "an element" is used herein to refer to at least one of the elements. means one element or more than one element.

[0062] As used herein, "about" refers to a measurable value such as an amount, duration, etc. ±20% or ±10% from the specified value, more preferably ±5%, and even more preferably ±1 %, and even more preferably ±0.1%, but such variations are not intended to be construed as limiting the scope of the invention. The present invention relates to a method for producing a cellular endothelial cell, comprising:

[0063] As used herein, "elimination" refers to the reduction or elimination of activity. For example, "elimination of FcγR binding" refers to the elimination of FcγR binding by an Fc region that is more specific than an Fc region that does not contain a particular mutation. Amino acid variants have less than 50% initiation bonds and less than 70-80-90-95-98% Loss of activity is preferred, and generally activity is no longer detectable in a Biacore assay. It means below the level of binding.

[0064] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to Nonspecific cytotoxic cells expressing cγR recognize the bound antibody on the target cell and subsequently target ADCC refers to a cell-mediated response that results in cell lysis. increased binding to FcγRIIIa resulted in increased ADCC activity. As discussed herein, many embodiments of the invention completely eliminate ADCC activity. do.

[0065] As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to Fcγ Nonspecific cytotoxic cells expressing R recognize the bound antibody on the target cell and subsequently attack the target cell. It refers to a cell-mediated reaction that results in the phagocytosis of bacteria.

[0066] As used herein, an "antigen binding domain" or "ABD" refers to a portion of a polypeptide sequence. When present as a moiety, six phases that specifically bind to a target antigen as discussed herein Thus, an "antigen-binding domain" refers to a set of complementarity determining regions (CDRs) of the present invention. As is known in the art, these Cs bind to target antigens as outlined herein. The DR is usually a variable heavy chain CDR (vhCDR or V H CDR or CDR-HC) 1 and a set of variable light chain CDRs (vlCDRs or V L CDR or CDR-LC) 2 sets, each set consisting of three CDRs: vhCD for the heavy chain R1, vhCDR2, vhCDR3, and vlCDR1, vlCDR2, and vlCDR3 for the light chain. The CDRs are present in the variable heavy and variable light domains, respectively, and together Thus, in some cases, six of the antigen-binding domains are The variable heavy and variable light chains contribute 1 CDR to the Fab form. The variable heavy domain (vh or V H ;vhCDR1 , vhCDR2 and vhCDR3) and a variable light chain domain (vl or V L ;vl The C-terminus of the vh domain is heavily influenced by the CDR1, vlCDR2, and vlCDR3. The C-terminus of the vl domain binds to the N-terminus of the constant light chain domain. In the scFv format, the vh and vl domains are linked to the ends of the are typically linked to a single polypeptide sequence via the use of a linker as outlined herein. The VH-linker-vl or VL-linker-vl are covalently attached to the VH-linker-vl or VL-linker-vl. It can be either ka-vh, but the former is generally preferred (depending on the form used) , with an optional domain linker on each side. In addition, the CDRs are arranged in a framework in each of the variable heavy and light domains. For the light chain variable domain, these are FR1-vlC DR1-FR2-vlCDR2-FR3-vlCDR3-FR4, For the FR1-vhCDR1-FR2-vhCDR2-FR3-vhCDR1 DR3-FR4, and these framework regions are highly identical to the human germline sequence. The antigen-binding domain of the present invention includes Fab, Fv and scFv.

[0067] As used herein, "modification" refers to the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence. This means a deletion or modification to a portion of a protein that is chemically attached to the protein. For example, the modification may be an altered carbohydrate or a PEG structure attached to the protein. In the specification, "amino acid modification" refers to the substitution, insertion, and deletion of an amino acid in a polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications always refer to , DNA-encoded amino acids, e.g., 20 with codons in DNA and RNA This applies to individual amino acids.

[0068] As used herein, an "amino acid substitution" or "substitution" refers to a specific amino acid in a parent polypeptide sequence. This refers to replacing the amino acid at position with a different amino acid. In embodiments, the substitution does not occur naturally at the particular position and is not present in the organism or in any organism. For example, the substitution M252Y is It refers to a polypeptide, in this case an Fc variant, in which the methionine at position 252 is replaced by tyrosine. For clarity, the nucleic acid coding sequence is altered but not altered from the first amino acid. For example, CGG (which codes for arginine) is replaced by CGA (which codes for arginine) The arginine-encoding amino acid sequence is converted to an amino acid sequence that encodes arginine, thereby increasing the expression level in the host organism. amino acid substitutions; i.e., to create a new gene that encodes the same protein. Nevertheless, if the protein has the same amino acid as the first at a particular position, it is It is not an amino acid substitution.

[0069] As used herein, a "mutated protein" or "protein variant" or "mutated A "variant" is a protein that differs from the parent protein by at least one amino acid modification. A protein variant refers to a protein itself, a composition containing the protein, The term may refer to a protein variant or the amino acid sequence encoding the same. The organism may have at least one amino acid modification compared to the parent protein, e.g., compared to the parent It has about 1 to about 70 amino acid modifications, preferably about 1 to about 5 amino acid modifications. As described above, in some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, The peptide may be a F peptide derived from a human wild-type sequence, e.g., IgG1, IgG2, IgG3, or IgG4. The protein variant sequences herein are preferably identical to the parent protein sequence. at least about 80% identity to the sequence, most preferably at least about 90% identity; More preferably, the identity is at least about 95% to 98% to 99%. The quality may be determined by the mutant protein itself, a composition comprising the protein mutant, or a method for the production of the mutant protein. It may refer to a DNA sequence.

[0070] Thus, as used herein, an "antibody mutant" or a "mutant antibody" refers to at least As used herein, "I" refers to an antibody that differs from a parent antibody by at least one amino acid modification. "IgG variant" or "mutated IgG" refers to a variant of a parent IgG with at least one amino acid modification. G (again, often derived from human IgG sequences) and As used herein, the term "immunoglobulin variant" or "mutated immunoglobulin" refers to at least Immunoglobulin sequences that differ from the sequence of a parent immunoglobulin sequence by at least one amino acid modification As used herein, "Fc variant" or "variant Fc" refers to a variant of the Fc domain. The Fc variants of the present invention refer to proteins that contain amino acid modifications in their structure. Thus, for example, M252Y or 252Y is an Fc variant having a substituted tyrosine at position 252 relative to the parent Fc polypeptide, In that case, the numbering follows the EU index. Similarly, M252Y / S254T / T256E is a nucleotide sequence that is identical to the substitutions M252Y, S254T and T256 compared to the parent Fc polypeptide. E. The identity of the WT amino acid may not be specified, In that case, the aforementioned variant would be called 252Y / 254T / 256E. The order in which the substitutions are shown is: Note that this is arbitrary, i.e., for example, 252Y / 254T / 256E The same Fc variant as 254T / 252Y / 256E, and so on. For all positions contemplated herein, unless otherwise indicated, the amino acid position numbering is The numbering of variable domains follows Kabat, with the Fc domain included. For common domains, the EU Index should be followed. The EU index or EU numbering scheme in the bat refers to the numbering of EU antibodies. Refers to balling (Edelman et al., 1969, Proc Natl Ac Ad Sci USA 63:78-85, which is incorporated herein by reference in its entirety. The modification may be an addition, deletion, or substitution. Substitutions may include natural amino acids, and in some cases, , may contain synthetic amino acids.

[0071] As used herein, the term "protein" refers to a protein, polypeptide, or the like. The term "amino acids" refers to at least two covalently linked amino acids, including tides, oligopeptides and peptides. The peptidyl group may comprise natural amino acids and peptide bonds.

[0072] As used herein, "Fab" or "Fab region" refers to VH, CH1, VL, and Fab refers to a polypeptide that contains the CL and CL immunoglobulin domains. region, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein. There are cases where it is pointed out.

[0073] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to A polypeptide comprising a VL domain and a VH domain of a single antigen-binding domain (ABD). As will be appreciated by those skilled in the art, these are usually comprised of two chains or or in combination (typically using a linker as discussed herein) to produce scFvs. can be formed.

[0074] As used herein, "amino acid" and "amino acid identity" refer to the same amino acid sequence in DNA and RNA. It refers to one of the 20 naturally occurring amino acids encoded by

[0075] As used herein, "effector function" refers to the interaction between the Fc region of an antibody and an Fc receptor or Effector functions refer to the biochemical events that result from interaction with a ligand. These include, but are not limited to, CC, ADCP, and CDC.

[0076] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to A family of proteins that bind to the Fc region of IgG antibodies and are encoded by FcγR genes. In humans, this family includes the isoforms FcγRI (CD64), including FcγRIa, FcγRIb, and FcγRIc; Form FcγRIIa (including allotypes H131 and R131), FcγRIIb ( FcγRIIb-1 and FcγRIIb-2), and FcγRIIc RII (CD32); and isoform FcγRIIIa (allotype V158 and and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and Fc FcγRIII (CD16) (including Jefferies γRIIb-NA2) al., 2002, Immunol Lett 82:57-65, see the entirety. (incorporated herein by reference), as well as any undiscovered human FcγR or FcγR isoform or In some cases, the term "genetic variant" includes, but is not limited to, a specific variant or allotype. For example, the antibody may be modified to reduce or eliminate binding to one or more FcγR receptors, as outlined in Reducing binding to FcγRIIIa reduces ADCC, and in some cases, It is desirable to reduce binding to γRIIIa and FcγRIIb.

[0077] As used herein, "FcRn" or "neonatal Fc receptor" refers to the FcRn of IgG antibodies. c region and binds to the protein encoded, at least in part, by the FcRn gene. FcRn refers to FcRn from various antigens including, but not limited to, human, mouse, rat, rabbit, and monkey. As is known in the art, functional FcRn can be derived from any organism that does not express functional FcRn. A protein contains two polypeptides, often called heavy and light chains. The light chain is β-2-microglobulin, the heavy chain of which is encoded by the FcRn gene. Unless otherwise specified in the document, FcRn or FcRn protein refers to FcRn complex. It refers to the complex of the β-chain and β-2-microglobulin.

[0078] As used herein, a "parent polypeptide" refers to a polypeptide that is subsequently modified to produce a variant. A parent polypeptide refers to a naturally occurring polypeptide or a starting polypeptide from which a naturally occurring polypeptide is derived. The parent polypeptide may be a variant or modified form of the peptide. may refer to a composition that includes the parent polypeptide, or the amino acid sequence that encodes it. Thus, as used herein, a "parent immunoglobulin" refers to a gene that is used to generate variants. As used herein, "parent antibody" refers to an unmodified immunoglobulin polypeptide that is modified to By "antibody" is meant an unmodified antibody that is modified to generate a mutant antibody. Note that these include known, commercially available, recombinantly produced antibodies, as outlined below.

[0079] As used herein, the term "heavy chain constant region" refers generally to a heavy chain constant region of human IgG1, IgG2 or IgG 4. This refers to the CH1-hinge-CH2-CH3 portion of an antibody derived from SEQ ID NO:4.

[0080] As used herein, a "target antigen" refers to an antigen that is specifically bound by the variable region of a given antibody. In this case, the target antigen is a target antigen that is phosphorylated at Ser413. The first step is to identify tau protein ("pSer413-tau").

[0081] As used herein, "target cell" refers to a cell that expresses a target antigen.

[0082] As used herein, "variable region" refers to the kappa, lambda, and heavy chain immunoglobulins, respectively. Substantially any of the Vκ, Vλ, and / or VH genes that make up the locus By "immunoglobulin region" is meant a region of an immunoglobulin that contains one or more encoded Ig domains.

[0083] As used herein, "wild type or WT" refers to a gene found in nature, including allelic variations. The WT protein refers to an amino acid sequence or nucleotide sequence that is not intentionally modified. The amino acid sequence or nucleotide sequence is not shown.

[0084] As used herein, the term "tau protein" refers to an amino acid sequence defined in SEQ ID NOs: 1 to 6. The amino acid sequences are: 4R2N (as defined in SEQ ID NO:1), 4R1N (as defined in SEQ ID NO:2), 4R0N (defined in SEQ ID NO:3), 3R2N (defined in SEQ ID NO:4) ), 3R1N (defined in SEQ ID NO:5), and 3R0N (defined in SEQ ID NO:6) one of six isoforms of the human tau protein, as well as its genetic As explained in the "Background of the Invention" section above, More than 40 mutations are thought to be involved in the familial neurodegenerative disease FTDP-17. However, the site of the mutation in the tau protein has not been identified as FTDP-17. The amino acid sequence to be introduced into the amino acid sequence of SEQ ID NO: 1 to 6 should not be limited to the relevant site. The number of amino acid mutations should not be limited, but should be 1-50, particularly 1-30, and more particularly 1-10. However, the serine residue at position 413 (Ser413) of SEQ ID NO:1 may be Corresponding amino acid residues in the amino acid sequences of the present invention should preferably be conserved. Tau protein was also identified using the BLAST method (the default settings for PBLAST are NCBI ) of the amino acid sequence of human tau protein as defined in SEQ ID NO:1. Proteins with 80% or more similarity or identity to the sequence, and their isoforms Such tau proteins include those derived from chimpanzees, macaques, horses, pigs, The target animals include tau from non-human species such as dogs, mice, rabbits, and rats. Therapeutic agents targeting tau from such non-human animals for the purpose of improving cognitive function It is possible to produce a prophylactic agent.

[0085] As used herein, the term "tau peptide" refers to a portion of the amino acid sequence of the tau protein. The term "tau peptide" refers to a peptide having an amino acid sequence of tau peptide derived from tau protein. The position and length should not be limited, but preferably, at least the amino acid sequence corresponding to Ser413 is Derived from amino acid residues corresponding to amino acid numbers 410 to 421 of SEQ ID NO: 1, including amino acids for example a series of at least 3 consecutive amino acids, in particular at least 5 consecutive amino acids , and more particularly, should have at least 8 consecutive amino acids. The number of amino acids is also not limited, but is preferably 4 or more, particularly 6 or more, and more particularly 8 or more. It should have an acid length of 1.

[0086] As used herein, the term "tau" generally refers to tau protein or tau peptide. means.

[0087] The term "anti-pSer413 tau protein" refers to a tau protein that is an anti-pSer413 tau protein. The binding of tau protein with serine phosphorylated at position 413 was significantly increased compared to that of tau protein without serine phosphorylated at position 413. It means an antibody that preferentially binds to tau protein phosphorylated at Ser413. The tau protein phosphorylated at may be human and / or mouse.

[0088] As used herein, "position" refers to a location in the sequence of a protein. Sequentially or in established formats, e.g. the EU index for antibody numbering The numbering may be done according to the

[0089] As used herein, a "residue" refers to a position in a protein and its associated amino acid. For example, asparagine 297 (also called Asn297 or N297) (referred to as "residue 297") is the residue at position 297 of human antibody IgG1.

[0090] In the context of the present invention, the positions of amino acid residues in a tau protein or tau peptide are clearly defined. In order to achieve this, the amino acid numbers are identified based on the amino acid sequence defined in SEQ ID NO:1. For example, the amino acid residue corresponding to Ser413 in SEQ ID NO:1 is represented by the sequence Position 413 of SEQ ID NO:1 (4R2N), position 384 of SEQ ID NO:2 (4R1N), position 415 of SEQ ID NO:3 (4R 3R0N), 382nd position of SEQ ID NO:4 (3R2N), 35th position of SEQ ID NO:5 (3R1N) It refers to the serine residue at position 3, or position 324 of SEQ ID NO:6 (3R0N). The correspondence of amino acid residue positions between the forms is shown in Table 1 below. Table 1. Human tau protein isoforms [Table 1]

[0091] Table 1 shows these corresponding to positions 410 to 421 of the amino acid sequence defined in SEQ ID NO:1. While showing the positions of amino acid residues of isoforms of, for example, In addition, the corresponding positions of amino acid residues in other regions can be easily recognized. If you are a chemist, you may want to use the Needleman-Wunsch or Smith-Waterman Pairwise sequence alignments such as the ClustalW or PRRP methods Which multiple sequence alignments are used to identify amino acids in isoforms or homologs? As an example of the analysis of the corresponding positions, the corresponding positions of FIGS. B shows the amino acid sequence alignment of the six human isoforms based on ClustalW. These figures show the amino acid sequence as defined in SEQ ID NO:1. The structure surrounding the amino acid residue corresponding to Ser413 in the sequence is conserved among the six isoforms. This indicates that

[0092] The antibodies of the present invention are generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, they are expressed The present invention relates to a polypeptide that has been identified and isolated and / or recovered from a cell or cell culture that has been subjected to Ordinarily, an isolated polypeptide has been prepared by at least one purification step. An "isolated antibody" is one that is substantially free of other antibodies having different antigen specificities. "Recombinant" refers to an antibody that is produced using recombinant nucleic acid technology in an exogenous host cell. It means to create a body.

[0093] "Percent amino acid sequence identity" with respect to a protein sequence refers to the percentage of amino acid sequence identity between the alignment, and gapping if necessary to achieve the maximum percent sequence identity. and when any conservative substitutions are not considered part of the sequence identity. as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a given (parent) sequence Alignment for purposes of determining percent amino acid sequence identity may be performed, e.g. For example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) You may use publicly available computer software, such as the Software, to develop and This can be accomplished in a variety of ways that are within the skill of the art. Any alignment required to achieve maximum alignment over the entire length of the sequences It is possible to determine appropriate parameters for measuring alignment, including the algorithm. One particular program is described in U.S. Publication No. 2016, which is incorporated herein by reference. The ALIGN-2 process outlined in paragraphs

[0279] to

[0280] of Issue 0244525 Another approximate alignment for nucleic acid sequences is the Waterman,Advances in Applied Mathematics Sci., 2:482-489 (1981). The algorithm is based on the Dayhoff,Atlas of Protein Sequence ces and Structure,MODayhoff ed.,5 supp. l.3:353-358,National Biomedical Research Developed by the Foundation, Washington, DC, USA, Gribskov, Nucl. Acids Res. 14(6):6745-6763( By using a scoring matrix normalized by It can be applied to amino acid sequences.

[0094] An example of the implementation of this algorithm for determining percent sequence identity is "BestF "Genetics Computer Group (Madiso The default parameters for this method are provided by Wiscon sin Sequence Analysis Package Program Ma nual,Version 8(1995)(Genetics Computer G (available from Roup, Madison, WI). Another method for establishing sex percentages is described by John F. Collins and Shane S. Developed by Sturrok and owned by IntelliGenetics, Inc. (Mo Published by the University of Edinburgh and distributed by Untain View, CA The use of the MPSRCH program package, which is protected by copyright. If you want to use the default parameters for the scoring table, The Smith-Waterman algorithm can be used (e.g., Gap Open penalty 12, gap extension penalty 1, and gap 6). The "match" value of the data reflects the "sequence identity." or other suitable programs for calculating similarity are generally known in the art. Another alignment program is, for example, BLAST using default parameters. For example, BLASTN and BLASTP use the following default parameters: Can be used with: Genetic code = standard; Filter = none; Strand = both strands; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; Sort order = HIGH SCORE; Database = Non-redundant, GenBank+EMBL+ DDBJ+PDB+GenBank CDS translation+Swiss p rotein+Spupdate+PIR. For details of these programs, see blast. Put http: / / before ncbi.nlm.nih.gov / Blast.cgi The URL can be found at the internet address located by

[0095] The degree of identity between the amino acid sequences of the present invention ("sequences of the present invention") and the parent amino acid sequence is The number of exact matches in the alignment of two sequences is determined by the length of the "sequence of the invention" or or the length of the parent array, whichever is shorter. The result is Expressed as percent identity.

[0096] In some embodiments, the two or more amino acid sequences are at least 50%, 60%, 70%, In some embodiments, two or more amino acid sequences are 0%, 80% or 90% identical. The sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0097] "Specific binding" or "specifically binds" to a particular antigen or epitope By "specific" it is meant binding that is measurably different from non-specific interactions. A specific bond, for example, refers to the binding of a molecule to another molecule of generally similar structure but lacking binding activity. The binding of a specific molecule can be measured by determining the binding relative to that of a control molecule. Specific binding can be determined by competition with a control molecule that is similar to the target.

[0098] As used herein, the term "K assoc " or "K a " is a specific antibody-antigen interaction. While the term "K" is intended to refer to the rate of association of an action, dis "Also "K d " is intended to refer to the off-rate of a particular antibody-antigen interaction. The term "K" used in D " is K d Against K a (i.e., K d / K a ) and is obtained by mole It is intended to refer to the dissociation constant expressed as a concentration (M). K D Values ​​are in the art The determination can be made using methods well established in the art. So, the K of the antibody D A method for determining the amount of This is achieved through the use of biosensor systems such as the ORE® system. In certain embodiments, phosphorylated tau protein is used to determine K D Measure the value. In the form of K, phosphopeptides were usedD In some embodiments, the fixation Using phosphorylated antigens (e.g., phosphorylated tau protein or phosphorylated peptides), D value In other embodiments, the immobilized antibody (e.g., parent mouse antibody, chimeric antibody, or humanized antibody variants) to D In yet another embodiment, the analyte Using phosphorylated tau protein as K D In yet another embodiment, the concentration is measured. KD values ​​are measured using phosphorylated peptides as precipitates. In certain embodiments, bivalent binding In another embodiment, the KD value is measured in a monovalent binding mode.

[0099] "Disease" includes any condition in an animal, including a human, in which the animal is unable to maintain homeostasis. If this is not possible and the disease is not ameliorated, the animal's health will continue to deteriorate.

[0100] In contrast, a "disorder" in an animal, including a human, is one in which the animal is unable to maintain homeostasis. The health of the animal may be improved by the use of the animal's food, but the health of the animal may not be as favorable as the health of the animal would be if it were not disabled. If left untreated, disorders do not necessarily have adverse effects on the health of the animal. It does not make the condition worse.

[0101] The terms "treatment," "treating," "treat," and the like refer to a desired pharmacological and / or It refers to obtaining a physiological effect, which may be either completely or partially effective in treating a disease or its symptoms. prophylactic in that it specifically prevents or reduces the likelihood of a disease or its symptoms and / or may partially or completely alleviate the disease and / or adverse effects caused by the disease As used herein, "therapeutic" refers to a treatment that is completely curative. " includes any treatment of a disease in a mammal, particularly a human, in which: (a) a patient is predisposed to the disease, (b) preventing the onset of disease in a subject not yet diagnosed with the disease; (c) inhibiting, i.e. arresting the onset or progression of, a disease; "Treatment" also includes treating, preventing, and / or alleviating one or more symptoms of a disease. , including delivering an agent to produce a pharmacological effect in the absence of a disease or condition. For example, "treatment" refers to the alleviation of a disease state, such as in the case of a vaccine. By delivering a composition capable of eliciting an immune response or conferring immunity in the absence of Includes:

[0102] As used herein, the term "mammal" refers to any mammalian animal, including rodents, mice, and hamsters. Mammals of the order Entia, and Logomorpha such as rabbits, but In some embodiments, the mammal is a Felidae ( In some embodiments, the mammal is from the order Carnivora, which includes the families Cats (Felidae) and Canidae (Dogs). In the mammalian order Artiodactyla, which includes the genus Bovidae (cattle) and the genus Porcinea (pigs), or mammals of the order Perssodactyla, which includes the family Equidae (horses). Mammals are classified as belonging to the order Primates, Platyrrhine, or Simoidea (monkeys), or the order Apes (humans). Most preferably, the mammal is a human (e.g., ape or ape). In some embodiments, the mammal is a human. It is.

[0103] As used herein, the term "regression" and its derivatives do not necessarily mean that It does not mean 0% or complete regression, but rather that the skilled artisan has identified a potential benefit or cure. There are various degrees of regression that may be recognized as having a therapeutic effect. Any amount or level of regression of tauopathy in mammals can be provided. Furthermore, the regression provided by the methods of the present invention may be indicative of one or more pathologies of a disease, e.g., a tauopathy. For purposes of this specification, "regression" may include the progression of a disease or the regression of symptoms. delaying the onset of the disease, delaying the onset of symptoms, and / or delaying the onset of the condition. With respect to progressive diseases and disorders, "regression" refers to slowing the progression of the disease or disorder; Slowing the progression of symptoms of a disease or disorder and / or slowing the progression of the condition It may include.

[0104] An "effective amount" or "therapeutically effective amount" of a composition is an amount that provides a beneficial effect to a subject to which the composition is administered. An "effective amount" of a delivery vehicle includes an amount of a composition sufficient to effectively bind the composition. The compound may contain a sufficient amount to combine or deliver.

[0105] An "individual" or "host" or "subject" or "patient" refers to a subject or subject being treated for diagnosis, treatment, or therapy. It refers to any mammalian subject, particularly humans, for whom the administration of the drug is desired. Other subjects include bovine, canine, feline, and ovine serum albumin (OSA) and / or ovine serum albumin (OSA). , guinea pigs, rabbits, rats, mice, horses, and the like.

[0106] As used herein, the term "in conjunction with" refers to, for example, the entire course of administration of a second therapy. refers to the use of a first therapy during administration of a second therapy; The first and second therapies are administered over a period of time that overlaps with each other, e.g., administration of the first therapy is administered before administration of the second therapy. administration of the first therapy ends before administration of the second therapy ends; administration of the first therapy The administration of the second therapy begins before the administration of the first therapy and ends before the administration of the second therapy ends. administration of the first therapy begins before administration of the second therapy begins and ends after administration of the first therapy The administration of the second therapy ends before the administration of the first therapy begins; the administration of the second therapy ends before the administration of the first therapy begins The administration of the first therapy ends before the administration of the second therapy ends. In combination with" can also refer to a regimen that includes the administration of two or more therapies. "In combination with" means in the same or a different formulation, by the same or a different route, and in combination with It also refers to the administration of two or more therapies, which may be administered in the same or different dosage form types.

[0107] "Encode" means to encode a defined nucleotide sequence (i.e., rRNA, tRNA, and Either mRNA) or a defined amino acid sequence and the biological properties resulting therefrom as templates for the synthesis of other polymers and macromolecules in biological processes; A nucleotide in a polynucleotide, such as a gene, cDNA, or mRNA, that has a function Thus, for example, the transcription of mRNA corresponding to a gene is A gene is called a transcription factor when its transcription and translation produces a protein in a cell or other biological system. The coding strand (whose nucleotide sequence is identical to the mRNA sequence) The coding strand (often provided in the sequence listing) and the non-coding strand (of genes or cDNAs) A) is a template for transcribing the gene or It can be said to encode a protein or other product of the DNA.

[0108] The term "nucleic acid" includes single-stranded, double-stranded, oligonucleotides, or polynucleotides. The term "nucleoside" includes RNA or DNA molecules having multiple nucleotides of any form. The "opid sequence" refers to an oligonucleotide or polynucleotide in a single-stranded form of a nucleic acid. The sequence of nucleotides in the sequence.

[0109] A "nucleic acid construct" refers to one or more functional units that are not found together in nature. Examples of such sequences include circular, linear, double-stranded, chromosomal, External DNA molecules (plasmids), cosmids (plasmids containing COS sequences derived from lambda phage) mide), viral genomes containing non-naturally occurring nucleic acid sequences, etc.

[0110] As used herein, the term "operably linked" refers to a nucleic acid sequence that is in communication with a second polynucleotide. Polynucleotides in a functional relationship, e.g., at least one of two polynucleotides Both are capable of exerting a physiological effect on the other that is characterized by A single-stranded or double-stranded nucleic acid sequence comprising two polynucleotides arranged in a manner such that the nucleic acid sequence can be An example of such a nucleic acid sequence is a promoter operably linked to the coding region of a gene. The sequence is capable of promoting transcription of the coding region. For example, the phrase: "a promoter is operably linked to a nucleotide sequence "Linked to" and "the nucleotide sequence is operably linked to a promoter" are used herein as used herein. These terms are used interchangeably throughout the specification and are considered equivalent. In some cases, When the nucleic acid encoding the protein further comprises a promoter / regulatory sequence, the promoter / regulatory sequence is The regulatory sequence is a sequence encoding a desired protein that drives expression of the desired protein in a cell. Located at the 5' end of the sequence.

[0111] The terms "oligonucleotide" and "polynucleotide" are used interchangeably herein. and "nucleic acid molecule" refers to a nucleic acid molecule consisting of either ribonucleotides or deoxyribonucleotides. refers to a polymeric form of nucleotides of any length. Thus, the term includes single-stranded , double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA- RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural Natural, chemically or biochemically modified, non-natural, or derivatized nucleotides. The backbone of a polynucleotide includes, but is not limited to, sugars and phosphates. groups (such as may be commonly found in RNA or DNA), or modified or substituted The saccharide may contain a sugar or phosphate group.

[0112] The term "recombinant" as applied to a polynucleotide means that the polynucleotide is not naturally occurring. Such that they result in constructs that are distinct and / or different from the polynucleotides found in , production of various combinations of cloning, restriction or ligation steps, and other procedures. This term includes copies of the original polynucleotide construct as well as copies of the original. Each includes a progeny of the viral construct.

[0113] As used herein, the term "promoter" refers to a nucleic acid sequence to be transcribed, e.g., a desired A promoter generally comprises a DNA sequence operably linked to a nucleic acid sequence that encodes a gene. Typically, it is located upstream of the nucleic acid sequence to be transcribed and is involved in the transcription process by RNA polymerase and other transcription factors. Provides sites for specific binding.

[0114] A "vector" is capable of transferring gene sequences into a target cell. "Transducer construct," "expression vector," and "gene transfer vector" are used to express a gene of interest. Any nucleic acid construct capable of directing expression and transferring a genetic sequence into a target cell. introduction refers to the incorporation of all or part of a vector into a genome or chromosome. This can be accomplished by maintaining the vector as an exogenous element, either transiently or heritably. Thus, the term includes cloning and expression vehicles, as well as Includes integration vectors.

[0115] As used herein, the term "regulatory element" refers to a gene that regulates some aspect of the expression of a nucleic acid sequence. Examples of regulatory elements include nucleotide sequences that control the replication of a nucleic acid sequence. , enhancers, intracellular ribosomes that contribute to transcription and / or post-transcriptional processing Entry site (IRES), intron, replication origin, polyadenylation signal (pA), promoter These may include motors, enhancers, transcription termination sequences, and upstream regulatory domains. In this regard, regulatory elements also include cis-regulatory DNA elements as well as transposable elements (TEs). Those of skill in the art will be able to determine, with no more than routine experimentation, the presence of these and other regulatory elements in an expression construct. Expression constructs can be produced using recombinant approaches. The compounds can be prepared by conventional techniques or synthetically produced using well known methodologies.

[0116] "Control elements" or "control sequences" refer to any element or sequence that regulates the replication, copying, transcription, or spread of a polynucleotide. Molecules that contribute to the functional regulation of polynucleotides, including splicing, translation, or degradation Regulation is the process of increasing or decreasing the frequency, speed, or abundance of a given process. Specificity may be affected and may be stimulatory or inhibitory in nature. Regulatory elements known in the art include, for example, transcription factors such as promoters and enhancers. A promoter is a sequence that controls the transcription of a gene by binding RNA polymerase under certain conditions. In most cases, it initiates transcription of the coding region located downstream (3' direction) of the promoter. This is a region of DNA where

[0117] As used herein, an amino acid residue is "phosphorylated" to mean that a phosphate group has been attached to the amino acid residue. This means that the amino acid is bound to the side chain of the amino acid residue by an ester bond. Residues include serine (Ser), threonine (Thr), and tyrosine (Tyr). can be done.

[0118] VIII. Antibodies The present invention relates to an antibody and an antigen-binding Concerning fragments.

[0119] A traditional antibody structural unit generally comprises a tetramer. Each tetramer usually consists of two identical peptides. Each pair consists of two polypeptide chains, one "light" chain (usually with a molecular weight of about 25 kDa) They have one "heavy" chain (usually having a molecular weight of about 50-70 kDa) and one "heavy" chain (usually having a molecular weight of about 50-70 kDa). Human light chains are classified as κ and λ light chains. The present invention generally relates to antibodies based on the IgG class. This includes, but is not limited to, IgG1, IgG2, IgG3, and IgG4. There are several subclasses of IgG1, IgG2, and IgG4. is more frequently used than IgG3. IgG1 has 356 (D or E) and 358 It should be noted that there are different allotypes with polymorphisms at (L or M). The sequences shown in the specification use the 356D / 358L allotype, but other allotypes are also contemplated herein. That is, any of the antibodies containing an IgG1 Fc domain included herein. The sequence can have the 356E / 358M instead of the 356D / 358L allotype. Cut.

[0120] As will be appreciated by those skilled in the art, the exact numbering and arrangement of the CDRs may vary. However, the open-loop structure of the variable heavy and / or variable light chain sequences may vary between different ring systems. It should be understood that the disclosure includes the relevant (unique) CDRs. The disclosure of the variable heavy chain region includes vhCDRs (e.g., vhCDR1, vhCDR2 and vhCDR3). ), and the disclosure of each variable light chain region is a vlCDR (e.g., vlCDR1, vlCDR 2 and vlCDR3).

[0121] Antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and The method for identifying the sequences of CDR-L3 and CDR-L4 is the Kabat method ( t al.,The Journal of Immunology,1991,Vol. .147, No.5, pp.1709-1719) and Chothia's method (Al-L azikani et al., Journal of Molecular Biol. ogy, 1997, Vol. 273, No. 4, pp. 927-948). These methods are within the scope of common general knowledge of the skilled artisan and are outlined in, for example, Dr. Andrew C. Martin's group website (http: / / www.drewcmartin.com / ) The data are available at: w.bioinf.org.uk / abs / .

[0122] A useful comparison of CDR numbering is as follows (Lafranc et al. ,Dev.Comp.Immunol.27(1):55-77(2003)): Table 1 [Table 2]

[0123] Throughout this specification, residues in the variable domain (approximately residues 1 to 107 of the light chain variable region) are referred to as and heavy chain variable region residues 1 to 113) use the Kabat numbering system. For constant regions such as the Fc region, the EU numbering system is typically used (e.g. , Kabat et al., supra (1991).

[0124] The present invention provides many different sets of CDRs. In this case, a "complete set of CDRs" refers to Three variable light chain CDRs and three variable heavy chain CDRs, e.g., vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3, and may be part of a larger variable light or variable heavy chain domain. As more fully outlined in, the variable heavy and variable light chain domains are used to on separate polypeptide chains, when used together (e.g., when using Fab), or sc In the case of an Fv sequence, it can be on a single polypeptide chain.

[0125] CDRs contribute to forming the antigen-binding, or more specifically, epitope-binding, site of an antibody. An "epitope" is a specific antigen in the variable region of an antibody molecule known as the paratope. An epitope is a determinant that interacts with a binding site. A single antigen is a group of antigens that usually have specific structural features and specific charge characteristics. It may have multiple epitopes.

[0126] In this case, the epitope is not limited to the amino acid residues directly involved in the binding of the antibody of the present invention. , including phosphorylation at Ser413.

[0127] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected a large number of primary sequences of heavy and light chain variable regions. Based on the degree of conservation, each primary sequence is classified into CDR and framework, and the list is (The entirety of which is incorporated by reference, SEQUENCES OF IMMUNOLOGY ICAL INTEREST,5th edition,NIH publicatio n, No. 91-3242, EA Kabat et al.).

[0128] In the IgG subclass of immunoglobulins, the heavy chain contains several immunoglobulin domains. As used herein, "immunoglobulin (Ig) domain" refers to three distinct Ig domains. The term "constant heavy metal ion" refers to a region of an immunoglobulin having a constant secondary structure. The heavy chain domain includes the chain (CH) domain and the hinge domain. In contrast, IgG isotypes each have three CH regions. The "CH" domains are as follows: "CH1" is the EU Interleukin 1 (IE1) domain as described in Kabat. "CH2" refers to the EU index listed in Kabat. "CH3" refers to the EU index listed in Kabat. This refers to rankings 341 to 447 by ranking.

[0129] Another type of Ig domain in the heavy chain is the hinge region. "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" means A flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at position EU220 and the IgG The CH2 domain begins at residue EU237. Thus, for IgG, the antibody hin As used herein, the amino acid sequence is from position 221 (D221 in IgG1) to position 236 (G23 in IgG1). 6), in which case the numbering is based on the EU index as set out in Kabat. In some embodiments, for example with respect to the Fc region, a lower hinge is included, "Lower hinge" usually refers to position 226 or 230. pI mutants can also be generated in the range region.

[0130] Light chains generally consist of two domains: a variable light chain domain (containing the light chain CDRs) and a variable heavy chain domain (containing the light chain CDRs). together with the main domain forming the Fv region, and the constant light chain region (often called CL or Cκ (includes

[0131] Another region of interest for further substitutions, as outlined below, is the Fc region.

[0132] Thus, the present invention provides different antibody domains. As is known in the art, the antibodies of the present invention may contain different domains within the heavy and light chains. These domains include the Fc domain, the CH1 domain, and the , CH2 domain, CH3 domain, hinge domain, heavy chain constant domain (CH1-hinge di-Fc domain or CH1-hinge-CH2-CH3), variable heavy domain, variable light domain These include, but are not limited to, a constant chain domain, a light chain constant domain, a Fab domain, and an scFv domain. Not limited to these.

[0133] Thus, an "Fc domain" includes a -CH2-CH3 domain and optionally a hin The heavy chain contains a variable heavy domain and a constant domain. The light chain comprises a CH1-hinge-Fc domain, which comprises CH2-CH3. An scFv comprises a variable heavy chain, an scFv linker, and a variable light chain constant domain. Some embodiments of the invention comprise at least one scFv domain. It does not occur in nature, but generally consists of variable weight fragments linked by an scFv linker. As outlined herein, an scFv domain comprises a variable light chain domain and a variable light chain domain. Usually, the orientation from N-terminus to C-terminus is vh-scFv linker-vl, but Any scFv domain (or one constructed using vh and vl sequences from a Fab) ), this can be reversed to vl-scFv linker-vh.

[0134] As shown herein, recombinantly produced peptides containing traditional peptide bonds are There are many suitable scFv linkers that can be used, including: The linker peptide may include the amino acid residues: Gly, Ser, Ala, or Thr. to ensure that they adopt the correct conformation relative to each other so that they retain the desired activity. In one embodiment, the linker should be of sufficient length to link the two molecules together. In one embodiment, the amino acid sequence is about 1 to 50 amino acids long, preferably about 1 to 30 amino acids long. In some embodiments, linkers of from about 5 to about 20 amino acids in length may be used. Use 10 amino acids. Useful linkers include, for example, (GS)n, (GSGGS)n glycine-serine polymers including (GGGGS)n, and (GGGS)n, where n is , which is an integer of at least 1 (and typically 3-4), glycine-alanine polymer , alanine-serine polymers, and other flexible linkers. Ethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. A variety of non-proteinaceous polymers may be utilized as linkers, including, but not limited to, That is, it can be used as a linker.

[0135] Other linker sequences may be any sequence of the CL / CH1 domain of any length; e.g., CL The first 5 to 12 amino acid residues of the / CH1 domain may be included, but the first 5 to 12 amino acid residues of the CL / CH1 domain may be included. The linker may not include all residues. The linker can be derived from, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα 2, can be derived from immunoglobulin heavy chains of any isotype, including Cδ, Cε, and Cμ The linker sequence may also be used to bind other proteins such as Ig-like proteins (e.g., TCR, FcR, KIR). sequences derived from the proteins, hinge regions, and other naturally occurring sequences from other proteins. obtain.

[0136] In some embodiments, the linker may be any two domains as outlined herein. A "domain linker" is a "domain linker" that is used to link the domains together. can be used, but many embodiments use, for example, (GS), (GSGGS), Glycine-serine polymers including (GGGGS)n and (GGGS)n, where n is at least at least 1 (and typically an integer between 3-4-5) and of sufficient length and flexibility Each domain can be synthesized by using any peptide sequence that allows recombinant binding of the two domains. Allows the host to retain its biological function.

[0137] A. Chimeric and Humanized Antibodies In some embodiments, the antibodies herein are derived from a mixture of different species, For example, the antibody may be chimeric and / or humanized. Any "humanized antibody" refers to an antibody that combines regions from more than one species. For example, Chimeric antibodies are traditionally made up of a mixture of mouse (or in some cases rat) derived variable regions ( A "humanized antibody" generally comprises a variable domain and a constant domain(s) of human origin. It refers to a non-human antibody in which the main framework regions have been replaced with sequences found in a human antibody. Generally, in a humanized antibody, the entire antibody except for the CDRs is encoded by a polynucleotide sequence derived from a human source. or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acid derived from a non-human organism, may be used as a human antibody. The antibody is then generated by grafting the β-sheet framework of the antibody variable region (the specificity is determined by the grafted The preparation of such antibodies is described, for example, in WO92 / 1101. 8, Jones, 1986, Nature 321:522-525, Verhoeye N et al., 1988, Science 239:1534-1536 both of which are incorporated herein by reference in their entireties. To regain affinity lost due to the addition of the corresponding acceptor framework residues, "Backmutations" to donor residues that correspond to the nucleotide sequence are often required (US5530101; US5585089;US5693761;US5693762;US6180370; US5859205;US5821337;US6054297;US6407213, (Both are incorporated herein by reference in their entireties.) Humanized antibodies are also optimally At least a portion of an immunoglobulin constant region, typically a portion of the constant region of a human immunoglobulin. Humanized antibodies also contain at least a portion of a human Fc region. It can also be produced using mice with genetically modified immune systems. al., 2004, Biotechnol. Prog. 20:639-654, in its entirety. Various techniques and methods for humanizing and reshaping non-human antibodies are available. These methods are well known in the art (see Tsurube et al., all of which are incorporated herein by reference in their entireties). Ushita & Vasquez,2004,Humanization of Mo noclonal Antibodies,Molecular Biology of B Cells,533-545,Elsevier Science(USA), (See also the references cited therein.) For humanization methods, see the entire Jones et al., 1986, Nature 321:522- 525;Riechmann et al.,1988;Nature 332:323 -329;Verhoeyen et al.,1988,Science,239:1 534-1536;Queen et al.,1989,Proc Natl Aca d Sci,USA 86:10029-33;He et al.,1998,JI mmunol.160:1029-1035;Carter et al.,1992, Proc Natl Acad Sci USA 89:4285-9,Presta et al.,1997,Cancer Res.57(20):4593-9;Gor man et al.,1991,Proc.Natl.Acad.Sci.USA 8 8:4181-4185;O'Connor et al.,1998,Protein Eng 11:321-8, but are not limited to these. Humanization or other methods for reducing the immunogenicity of non-human antibody variable regions include, for example, Roguska et al., 1994, Proc. Nat. Surface reconstruction as described in Acad. Sci. USA 91:969-973 Examples of such methods include:

[0138] In certain embodiments, the antibodies of the invention are derived from a particular germline heavy chain immunoglobulin gene. and / or a light chain variable region derived from a particular germline light chain immunoglobulin gene. For example, such antibodies may be "the product of" or "derived from" a particular germline sequence. It may comprise or consist of a human antibody comprising a certain heavy or light chain variable region. A human antibody that is "the product of" or "derived from" human germline immunoglobulin sequences is a human antibody. The amino acid sequence of the human antibody was compared to that of human germline immunoglobulins. The human germline immunoglobulin sequence that is closest to (i.e., highest percent identity) By selecting specific human germline immunoglobulins, they can be identified. Human antibodies that are "the product of" or "derived from" a germline sequence have, as compared to the germline sequence, e.g. , amino acid substitutions resulting from spontaneous somatic mutations or deliberate site-directed mutagenesis However, humanized antibodies generally contain amino acid sequences identical to those of human antibodies. At least one amino acid sequence encoded by the germline immunoglobulin gene 80% identical to the germline immunoglobulin amino acid sequences of other species (e.g., mouse germline amino acid residues that identify the antibody as derived from a human sequence when compared to a human sequence In certain cases, humanized antibodies may contain a sequence similar to that of a germline immunoglobulin. At least 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identical Generally, a humanized antibody derived from a particular human germline sequence may be Amino acid sequences encoded by immunoglobulin genes and sequences of 10 to 20 amino acids or less In certain cases, humanized antibodies may exhibit only differences in the structure and structure of the germline immunoglobulin genes. The amino acid sequence encoded by the gene and up to 5, or even 4, 3, 2, or can represent one or fewer amino acid differences.

[0139] In one embodiment, the parent antibody is affinity matured, as known in the art. As described in US Pat. No. 7,657,380, structure-based methods for humanization and It may also be used for affinity maturation. Selection-based methods may be used to humanize antibody variable regions. and / or affinity maturation, methods of which are all incorporated herein by reference in their entireties. Wu et al., 1999, J. Mol. Biol. 294:1, incorporated herein by reference. 51-162;Baca et al.,1997,J.Biol.Chem.272( 16):10678-10684;Rosok et al., 1996, J. Biol .Chem.271(37):22611-22618;Rader et al.,1 998,Proc.Natl.Acad.Sci.USA 95:8910-8915; Krauss et al.,2003,Protein Engineering 1 6(10):753-759, but are not limited to these. Other humanization methods are described in detail by Tan et al., all of which are incorporated herein by reference in their entireties. l.,2002,J.Immunol.169:1119-1125;De Pasca lis et al., 2002, J. Immunol. 169:3076-3084 Grafting only a portion of the CDRs, including but not limited to the methods described herein. may include.

[0140] The term "antibody" is used in the broadest sense and includes, for example, an intact immunoglobulin or The antigen-binding portion may be produced by recombinant DNA techniques or as an intact They can be produced by enzymatic or chemical cleavage of antibodies. Hence, the term antibody includes , traditional tetrameric antibodies with two heavy chains and two light chains, as well as Fv, Fab and scFv. In some cases, the present invention includes any antigen-binding fragment thereof, as outlined herein. The present invention provides a bispecific antibody comprising at least one antigen-binding domain in

[0141] The humanized anti-phosphorylated tau antibodies according to the present invention should preferably exhibit improved plasma kinetics. In the present specification, the improvement of the plasma kinetics of a humanized antibody refers to the improvement of the kinetics of a humanized antibody in plasma. The parameters that represent the humanized antibody are altered compared to the corresponding non-human antibody. Examples of parameters that indicate plasma kinetics are: plasma half-life, mean residence time in plasma, Such parameters are "altered" when: The humanized antibody is a non-human antibody that is a "corresponding" antibody. A humanized antibody is a non-humanized antibody, e.g., a humanized antibody, that is used as a basis for producing a humanized antibody. By the term "antibody" is meant a mouse or chimeric antibody.

[0142] The term "chimeric antibody" refers to an antibody that combines one or more regions from one antibody with one or more regions from another antibody. The term "CDR-grafted antibody" refers to an antibody that contains one or more regions of a specific species or isoform. One or more CDRs from an antibody of the same or different species or isotype It is an antibody that comprises the framework of another antibody.

[0143] IX. Composition The present invention relates to a method for determining whether a phosphorylated Ser413 (pSer4 13) Many different antigen-binding domains (usually incorporated into antibodies) that bind to human tau protein (included).

[0144] The "antigen-antibody reaction" refers to a reaction against phosphorylated tau protein or phosphorylated tau peptide. The affinity of an antibody, i.e., the equilibrium dissociation constant (K D In certain embodiments, , using phosphorylated tau protein K D In some embodiments, the phosphate Using methylated tau peptides D In one embodiment, the phosphorylated tau peptide is 1 In another embodiment, the phosphorylated residue is the only one that is phosphorylated (e.g., SEQ ID NO: 75). The peptide is phosphorylated at multiple residues (e.g., SEQ ID NO:76 or SEQ ID NO:78 In some embodiments, the immobilized antigen (e.g., phosphorylated tau protein or Phosphorylated tau peptide) was used to D In this case, the affinity measurement is the avidity component. In other embodiments, the immobilized antibody (e.g., Mouse parent antibody, chimeric antibody, or humanized antibody variant) D Measure the value and In this case, the affinity measurement does not involve binding activity, i.e., it is a monovalent binding mode. In an embodiment, K is measured using immobilized antibodies and phosphorylated tau protein as the analyte. D Value In yet another embodiment, the immobilized antibody and the phosphopeptide as the analyte are Using K D In certain embodiments, the K value is measured in a bivalent binding manner. D Measure the value. In the embodiment, K D Measure the value.

[0145] Thus, in certain embodiments, K D The value is 5 x 10-8 M or less; some In the embodiment, K D The value is 5 x 10 -9 M or less; in other embodiments, K D The value is 5 x 10 -10 M or less; in yet other embodiments, K D The value is 1.86 x 10 -8 M or less In yet another embodiment, K D The value is 2 x 10 -9 M or less; and So, K. D The value is 2 x 10 -10 In certain embodiments, the amines disclosed herein are no greater than M. The antibody is a phosphorylated tau protein or phosphorylated tau peptide as an analyte. Measured using a peptide of 5 x 10 -8 K below M D Phosphorylated tau protein or ribonuclease In another specific embodiment, the antibodies disclosed herein bind to phosphorylated tau peptides. Immobilized phosphorylated tau protein or phosphorylated tau peptide and an antibody as an analyte Measured using 5 x 10 -9 K below M D phosphorylated tau protein or phosphorylated tau In yet another specific embodiment, the antibodies disclosed herein bind to a peptide. Using a fixed phosphorylated tau protein or phosphorylated tau peptide and an antibody as an analyte, 2×10 -9 K below M D phosphorylated tau protein or phosphorylated tau In another specific embodiment, the antibodies disclosed herein bind to an immobilized peptide. The phosphorylated tau protein or phosphorylated tau peptide was measured using an antibody as an analyte. The set value is 5×10 -10 K below M Dphosphorylated tau protein or phosphorylated tau peptide In another specific embodiment, the antibodies disclosed herein bind to immobilized phospholipids. Measured using oxidized tau protein or phosphorylated tau peptide and an antibody as an analyte 2×10 -10 K below M D to phosphorylated tau protein or peptide Combine.

[0146] Additionally, as outlined herein, the antibodies of the present invention do not contain a phosphate at position 413. Tau peptides or tau proteins phosphorylated at S413 were more likely to be phosphorylated than tau peptides or proteins. It preferentially binds to proteins.

[0147] The humanized anti-phosphorylated tau antibody according to the present invention is capable of inhibiting phosphorylated tau protein or is an antibody that can induce an antigen-antibody reaction with phosphorylated tau peptide.

[0148] The humanized anti-phosphorylated tau antibody according to the present invention is capable of inducing an antigen-antibody reaction. Phosphorylated tau protein and tau peptides are specifically linked to neurodegenerative diseases such as AD. Tau proteins and tau peptides that are phosphorylated at amino acid residues that are phosphorylated In particular, such tau proteins and tau peptides are as defined in SEQ ID NO:1. The amino acid sequence to be determined is phosphate-phosphorylated at least at serine residue 413 (Ser413) of the amino acid sequence to be determined. and one or more amino acid residues corresponding to amino acid numbers 410 to 421, That is, in the amino acid sequence defined in SEQ ID NO: 1, the serine residue at position 412 (Ser4 12), the threonine residue at position 414 (Thr414), and the serine residue at position 416 (Se r416) ​​may be phosphorylated at one or more amino acid residues corresponding to the An exemplary such phosphorylated peptide is one having the amino acid sequence defined in SEQ ID NO:7. pSer412 / pSer413(Cys-); the amino acid sequence defined in SEQ ID NO:8 pSer413(Cys-), having the amino acid sequence defined in SEQ ID NO:9 pSer409 / pSer412 / pSer413(Cys-) are included. Not limited to.

[0149] The phosphorylated tau peptide according to the present invention can be used in the production of the humanized anti-phosphorylated tau antibody according to the present invention. or as antigens to study reactivity.

[0150] The phosphorylated tau peptide according to the present invention can be prepared by those skilled in the art using suitable synthetic or genetic engineering methods. The phosphorylated peptide can be produced by the method of the present invention. As a method, the Boc method (Wakamiya T., Chemistry Letters , Vol. 22, p. 1401, 1993), Fmoc method (Perich, JW, I International Journal of Peptide and Prot ein Research, Vol. 40, pp. 134-140, 1992) and JP 3587390B and WO2013 / 180238, but those skilled in the art will recognize that Any other method can be used as needed. As an example of a method for producing the peptide, a nucleic acid encoding the peptide to be produced or its precursor is Genetic material (DNA or RNA) with a sequence is prepared, introduced into an expression vector, and processed. After any steps, such as the addition of a motor sequence, the vector is introduced into a suitable host for expression or In this case, even if the host is specific, A phosphorylation reaction is carried out in a host using an enzyme such as a kinase, which may be expressed by genetic engineering. or by recovering the desired peptide from the host and reacting with the target peptide by a kinase or the like. The peptide is phosphorylated at the desired site by inducing a phosphorylation reaction using the enzyme In the latter case, glycogen synthase kinase 3 (GSK3) or In the phosphorylation of tau, such as cyclin-dependent protein kinase 5 (CDK5), By exposing the target peptide to an enzyme known to play a role in Phosphorylation can be induced in vitro or in vivo using the methods described above. Among the peptides phosphorylated in vitro, the above-mentioned antibodies were selected through, for example, an antibody-antigen reaction. By selecting those that specifically bind to phosphorylated tau antibodies, the desired amino acid residues can be identified. The phosphorylated peptide can be recovered by the addition of .

[0151] The phosphorylated tau peptide according to the present invention has an N-terminal sequence and / or a C-terminal sequence which is The present invention can be modified with other functional substances suitable for the desired purpose. The phosphorylated tau peptide by or by adding a group, an acetyl group or a pyroglutamic acid to its N-terminus and / or C The termini can be modified, for example, with fluorescent substances. The N-terminus and / or C-terminus of the peptide may be modified, e.g., with a peptide or protein. Examples of peptides that can be used for such modifications include those with appropriate tag sequences (typically human stimulant tag or FLAG tag), T cell receptor (TCR) or major histocompatibility gene peptides having amino acid sequences that can be recognized by the MHC complex, such These include proteins derived from viruses or bacteria or peptides with sequences derived from proteins. Furthermore, the phosphorylated tau peptide according to the present invention may have amino acids other than the N-terminus and C-terminus. At least one amino acid residue in which the amino acid residue has been modified with any other compound or peptide. Those skilled in the art will recognize that such modifications to phosphorylated tau peptides include those having a phospho-Tau group. The resulting mixture may be subjected to any known method, for example, the method described in Hermanson et al., "Bioconjugation Ugate Techniques”,(US),Academic Press,19 This may be accomplished using the methods described in 96.

[0152] Those skilled in the art can easily determine the binding activity of the antibody by selecting an appropriate binding assay in a solid-phase or liquid-phase system. Thus, measurements of antigen-antibody reactions can be performed. Examples of such assays include: Enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), surface plasmon Fluorescence resonance energy transfer (FRETT), and luminescence resonance energy transfer ( Measurement of antigen-antibody binding affinity can be performed, for example, by For example, the antibody and / or antigen may be coupled to, for example, an enzyme, a fluorescent substance, a luminescent substance, or a radioisotope. Suitable for labeling with elements and measuring physical and / or chemical properties characteristic of the label used This can be done by detecting an antigen-antibody reaction using the above-mentioned method.

[0153] The humanized anti-phosphorylated tau antibody according to the present invention preferably binds to non-phosphorylated tau protein or and its binding affinity to phosphorylated tau protein or tau peptide. It should have a higher selective binding affinity to the peptide. The selective binding affinity of an antibody to a phosphorylated tau protein or tau peptide is improved by phosphorylating the The binding affinity of an antibody to a tau protein or tau peptide relative to the corresponding non-phosphorylated tau peptide. This means that the ratio of the binding affinity of a tau molecule to its binding affinity for a tau protein or tau peptide is increased. Such selective binding affinity can be achieved, for example, by binding phosphorylated tau protein or tau peptides. Immobilized plate and corresponding non-phosphorylated tau protein or tau peptide immobilized Using the plate, for example, ELISA (e.g., as the absorbance OD value of the emission wavelength) By measuring the binding affinity of the antibody to each of these proteins or peptides, and the binding affinities obtained for phosphorylated tau protein or tau peptides. The values ​​(e.g., absorbance OD values) were compared with the corresponding non-phosphorylated tau protein or tau peptide. The specific conditions for the ELISA are , as described in the Examples section below.

[0154] The humanized anti-phosphorylated tau antibody according to the present invention is selected according to the method described in Example 2 below. Insofar as specific binding affinity is required, phosphorylated tau protein or tau peptide is preferably used. Binding of selective binding affinity to non-phosphorylated tau protein or tau peptides The ratio of the affinity to the affinity of the target molecule is at least about 40 or more, particularly at least about 50 or more, and more particularly less than about 100 or more. In one embodiment, the phosphorylated peptide, such as SEQ ID NO:8, Binding affinity of anti-pSer413 tau antibody to the non-phosphorylated peptide of SEQ ID NO:69 The ratio is calculated by comparing the binding affinity of

[0155] The humanized anti-phosphorylated tau antibody according to the present invention preferably has a property of being capable of infecting the brain when administered into the blood. In this regard, the efficacy of antibodies administered into the blood should be improved. Improving the ability of an antibody to enter the human or any other mammalian host (e.g., mouse or rat) When administered into the blood, the ratio of antibody concentration in the brain to that in the plasma increases. The ability of an antibody to enter the brain can be achieved, for example, by administering the antibody via the blood to an animal (e.g. After a given period (e.g., one week), blood is collected and the brain Homogenates were prepared, and the antibodies of the obtained plasma samples and the obtained brain homogenate samples were analyzed. The concentration of the antibody was measured by a known method (e.g., sandwich ELISA using an anti-human polyclonal antibody). A) and the antibody concentration in the brain can be analyzed by dividing the antibody concentration in the plasma. Specific conditions for the ELISA may be those described in the Examples section below. The concentration of antibodies that enter the brain is generally about 0.1-0.3% of the concentration in plasma. It is known that...

[0156] The humanized anti-phosphorylated tau antibody according to the present invention is described in the following Example "[7] Example 7: Transplantation into the Brain." As long as the ratio is determined according to the method described in the "Analysis of the Brain" section, The antibody concentration in the blood is 0.30% or more, particularly 0.35% or more, more particularly 0.5% or more, based on the antibody concentration in plasma. Alternatively it should have a ratio of greater than or equal to 0.40%, and even more particularly greater than or equal to 0.45%.

[0157] A. Antigen-Binding Domains of the Invention The present invention relates to tauta that is not phosphorylated at Ser413, as described herein. Antigen binding preferentially to human pSer413 tau protein over binding to other proteins Provide the domain.

[0158] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising the polypeptide of the present invention, comprising administering to a mammalian subject the polypeptide of the present invention a polypeptide comprising the polypeptide of the present invention, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0159] In one embodiment, the present invention provides a method for the preparation of a nucleic acid molecule comprising the steps of: vlCDR1 having SEQ ID NO: 92; vlCDR2 having SEQ ID NO: 82; vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0160] In one embodiment, the present invention relates to a polypeptide having a vlCDR1 with SEQ ID NO: 93, a vlCDR2 with SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0161] In one embodiment, the present invention relates to a polypeptide having a vlCDR1 with SEQ ID NO: 94, a vlCDR2 with SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0162] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising the polypeptide of the present invention, comprising administering to a mammalian subject the polypeptide of the present invention a polypeptide comprising the polypeptide of the present invention, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0163] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising the polypeptide of the present invention, comprising administering to a mammalian subject the polypeptide of the present invention a polypeptide comprising the polypeptide of the present invention, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0164] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising the polypeptide of the present invention, comprising administering to a mammalian subject the polypeptide of the present invention a polypeptide comprising the polypeptide of the present invention, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0165] In one embodiment, the present invention relates to a polypeptide having a vlCDR1 with SEQ ID NO: 98, a vlCDR2 with SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0166] In one embodiment, the present invention provides a method for the preparation of a polypeptide comprising the polypeptide of the present invention, comprising administering to a mammalian subject the polypeptide of the present invention a polypeptide comprising the polypeptide of the present invention, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88. The present invention provides an antigen-binding domain comprising

[0167] In one embodiment, the present invention provides a polypeptide comprising a vlCDR1 having SEQ ID NO: 100, a vlCDR2 having SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 R1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88 An antigen-binding domain comprising:

[0168] In one embodiment, the present invention provides a polypeptide comprising a vlCDR1 having SEQ ID NO: 101, a vlCDR2 having SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 R1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88 An antigen-binding domain comprising:

[0169] In one embodiment, the present invention provides a polypeptide comprising a vlCDR1 having SEQ ID NO: 102, a vlCDR2 having SEQ ID NO: 82, vlCDR2 having SEQ ID NO: 83; vlCDR3 having SEQ ID NO: 86 R1; vhCDR2 having SEQ ID NO:115; and vhCDR3 having SEQ ID NO:88 An antigen-binding domain comprising:

[0170] The present invention provides an antigen-binding domain against human pSer413 tau protein. As shown in the Examples, a number of humanized variable heavy and variable light domains are provided, which include: They can be combined in any combination and can be combined with human IgG constant domains in any combination. It can be added to the

[0171] In one embodiment, the variable light domain Ta1505-VL46 (SEQ ID NO: 84) is pSer413 in combination with the chain domain Ta1505-VH11 (SEQ ID NO: 116) In this embodiment, the human heavy chain constant domain is SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, and 1 26, and the human light chain kappa constant domain, SEQ ID NO:52.

[0172] In one embodiment, the variable light domain Ta1505-VL46 (SEQ ID NO: 84) is In this embodiment, the VH domain Ta1505-VH65 (SEQ ID NO: 117) is combined with the VH domain Ta1505-VH65 (SEQ ID NO: 117). The human heavy chain constant domains are represented by SEQ ID NOs: 118, 119, 120, 121, 122, 1 23, 124, 125, and 126, and the human light chain kappa constant domain, SEQ ID NO:52 are selected.

[0173] In one embodiment, the variable light domain Ta1505-VL47 (SEQ ID NO: 104) is Combined with the heavy chain domain Ta1505-VH11 (SEQ ID NO: 116). In the human heavy chain constant domain, SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, SEQ ID NO:52 is selected from.

[0174] In one embodiment, the variable light domain Ta1505-VL47 (SEQ ID NO: 104) is Combined with the heavy chain domain Ta1505-VH65 (SEQ ID NO: 117). In the human heavy chain constant domain, SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, SEQ ID NO:52 is selected from.

[0175] In one embodiment, the variable light chain domain Ta1505-VL46_G34A (SEQ ID NO: 10 5) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0176] In one embodiment, the variable light chain domain Ta1505-VL46_G34A (SEQ ID NO: 10 5) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0177] In one embodiment, the variable light chain domain Ta1505-VL46_G34S (SEQ ID NO: 10 6) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0178] In one embodiment, the variable light chain domain Ta1505-VL46_G34S (SEQ ID NO: 10 6) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0179] In one embodiment, the variable light chain domain Ta1505-VL46_G34T (SEQ ID NO: 10 7) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0180] In one embodiment, the variable light chain domain Ta1505-VL46_G34T (SEQ ID NO: 10 7) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0181] In one embodiment, the variable light chain domain Ta1505-VL46_N33Q (SEQ ID NO: 10 8) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0182] In one embodiment, the variable light chain domain Ta1505-VL46_N33Q (SEQ ID NO: 10 8) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0183] In one embodiment, the variable light chain domain Ta1505-VL46_N33Q_G34A (sequence 109) in combination with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In this embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: 118, 119, 12 0, 121, 122, 123, 124, 125, and 126, and human light chain kappa constant domain The main sequence is selected from SEQ ID NO:52.

[0184] In one embodiment, the variable light chain domain Ta1505-VL46_N33Q_G34A (sequence 109) in combination with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In this embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: 118, 119, 12 0, 121, 122, 123, 124, 125, and 126, and human light chain kappa constant domain The main sequence is selected from SEQ ID NO:52.

[0185] In one embodiment, the variable light chain domain Ta1505-VL46_N33D (SEQ ID NO: 11 0) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0186] In one embodiment, the variable light chain domain Ta1505-VL46_N33D (SEQ ID NO: 11 0) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0187] In one embodiment, the variable light chain domain Ta1505-VL46_N33S (SEQ ID NO: 11 1) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0188] In one embodiment, the variable light chain domain Ta1505-VL46_N33S (SEQ ID NO: 11 1) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0189] In one embodiment, the variable light chain domain Ta1505-VL46_N33T (SEQ ID NO: 11 2) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0190] In one embodiment, the variable light chain domain Ta1505-VL46_N33T (SEQ ID NO: 11 2) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0191] In one embodiment, the variable light chain domain Ta1505-VL46_S28N (SEQ ID NO: 11 3) is combined with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0192] In one embodiment, the variable light chain domain Ta1505-VL46_S28N (SEQ ID NO: 11 3) is combined with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In one embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: s118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 13 , 122, 123, 124, 125, and 126, and the human light chain kappa constant domain, Selected from column number 52.

[0193] In one embodiment, the variable light chain domain Ta1505-VL46_G34A_S28N (sequence 114) in combination with the variable heavy domain Ta1505-VH11 (SEQ ID NO: 116). In this embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: 118, 119, 12 0, 121, 122, 123, 124, 125, and 126, and human light chain kappa constant domain The main sequence is selected from SEQ ID NO:52.

[0194] In one embodiment, the variable light chain domain Ta1505-VL46_G34A_S28N (sequence sequence number 114) in combination with the variable heavy domain Ta1505-VH65 (SEQ ID NO: 117). In this embodiment, the human heavy chain constant domain is selected from the group consisting of SEQ ID NOs: 118, 119, 12 0, 121, 122, 123, 124, 125, and 126, and human light chain kappa constant domain The main sequence is selected from SEQ ID NO:52.

[0195] The humanized anti-phosphorylated tau antibody of the present invention also preferably has a specific The antibody should have a heavy chain variable region and a light chain variable region having the amino acid sequence:

[0196] As a heavy chain variable region, the antibody of the invention comprises the sequence VH11 (defined in SEQ ID NO: 18), Sequence VH12 (defined in SEQ ID NO: 20), sequence VH47 (defined in SEQ ID NO: 22) ), VH61 (as defined in SEQ ID NO: 24), sequence VH62 (as defined in SEQ ID NO: 26) ), sequence VH64 (defined in SEQ ID NO:28), and sequence VH65 (defined in SEQ ID NO:30) and preferably 80% or more, in particular 85% or more, more particularly 90% or more, more particularly 95% or more, and even more particularly 100% identity to In one embodiment, the antibody of the invention should have an amino acid sequence having the heavy chain variable As a region, the amino acid sequence of the sequence VH65, or: Ala at position 28 (Kabat number: Asn at position 30 (Kabat number: H30) to Ser substitution of Val (Kabat number: H49) to Gly at position 49; substitution of Al at position 64 a (Kabat number: H61) to Asp substitution; and Gln at position 78 (Kabat number: Sequence V with one or more substitutions selected from the group consisting of substitutions of H75) to Lys It may comprise an amino acid sequence derived from H65.

[0197] As a light chain variable region, the antibody of the invention comprises the sequence VL15 (defined in SEQ ID NO: 32), Sequence VL36 (defined in SEQ ID NO: 34), sequence VL46 (defined in SEQ ID NO: 36) ), VL47 (defined in SEQ ID NO: 38), sequence VL48 (defined in SEQ ID NO: 40) ), and sequence VL50 (defined in SEQ ID NO: 42), and preferably , 80% or more, in particular 85% or more, more particularly 90% or more, even more particularly 95% or more More particularly, the amino acid sequence should have 100% identity. In an embodiment, the antibody of the present invention comprises, as a light chain variable region, the amino acid sequence of sequence VL47, or or: substitution of Asp (Kabat number: L17) at position 17 with Glu; (Kabat number: L27A) to Asn; Gln (Kabat number: L45) at position 50 to Arg and the group consisting of Arg (Kabat number: L46) at position 51 replaced by Leu. It may comprise an amino acid sequence derived from the sequence VL47 by one or more substitutions selected from:

[0198] In another embodiment, the humanized anti-phosphorylated tau antibody according to the invention is selected from the following: may include any combination of:

[0199] (1) a heavy chain variable region having the sequence VH11 (SEQ ID NO: 116) and a light chain variable region having the sequence String VL15 (SEQ ID NO:32);

[0200] (2) the heavy chain variable region has the sequence VH11 (SEQ ID NO: 116) and the light chain variable region has the sequence String VL36 (SEQ ID NO:34);

[0201] (3) the heavy chain variable region has the sequence VH11 (SEQ ID NO: 116) and the light chain variable region has the sequence String VL46 (SEQ ID NO:36);

[0202] (4) the heavy chain variable region has the sequence VH11 (SEQ ID NO: 116) and the light chain variable region has the sequence String VL47 (SEQ ID NO:38);

[0203] (5) the heavy chain variable region has the sequence VH11 (SEQ ID NO: 116) and the light chain variable region has the sequence String VL48 (SEQ ID NO:40);

[0204] (6) the heavy chain variable region has the sequence VH11 (SEQ ID NO: 116) and the light chain variable region has the sequence String VL50 (SEQ ID NO:42);

[0205] (7) the heavy chain variable region has the sequence VH12 (SEQ ID NO: 20) and the light chain variable region has the sequence VL48 (SEQ ID NO: 40);

[0206] (8) the heavy chain variable region has the sequence VH47 (SEQ ID NO: 22) and the light chain variable region has the sequence VL48 (SEQ ID NO: 40);

[0207] (9) the heavy chain variable region has the sequence VH61 (SEQ ID NO: 24) and the light chain variable region has the sequence VL48 (SEQ ID NO: 40);

[0208] (10) the heavy chain variable region has the sequence VH62 (SEQ ID NO: 26) and the light chain variable region has the sequence String VL48 (SEQ ID NO:40);

[0209] (11) The heavy chain variable region has the sequence VH64 (SEQ ID NO: 28) and the light chain variable region has the sequence String VL47 (SEQ ID NO:38);

[0210] (12) the heavy chain variable region has the sequence VH64 (SEQ ID NO: 28) and the light chain variable region has the sequence String VL48 (SEQ ID NO:40);

[0211] (13) the heavy chain variable region has the sequence VH65 (SEQ ID NO: 117) and the light chain variable region has the sequence Sequence VL47 (SEQ ID NO:37);

[0212] Selecting the amino acid sequences of the heavy and light chain CDRs and / or variable regions from the above, If necessary, the framework regions and / or constant regions of the heavy and light chains of a human antibody are By combining the amino acid sequence of the region with the humanized anti-ribosome antibody of the present invention, a person skilled in the art can easily produce the humanized anti-ribosome antibody of the present invention. It may be possible to design a phosphorylated tau antibody by using the framework regions of the heavy and light chains of a human antibody. The amino acid sequences of the constant regions and / or the amino acid sequences of the human IgG, IgA, IgM, Ig The amino acid sequences of the IgE, IgD, and IgE subtypes or variants thereof can be selected from the group consisting of do.

[0213] B. Specific anti-pSer413 tau antibody The present invention provides a number of specific antibodies that contain a pair of sequences, a heavy chain and a light chain, as follows: provide:

[0214] mAb-aPT1: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 144

[0215] mAb-aPT2: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 145

[0216] mAb-aPT3: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 146

[0217] mAb-aPT4: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 147

[0218] mAb-aPT5: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 148

[0219] mAb-aPT6: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 149

[0220] mAb-aPT7: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 150

[0221] mAb-aPT8: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 151

[0222] mAb-aPT9: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 152

[0223] mAb-aPT10: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 153

[0224] mAb-aPT11: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 154

[0225] mAb-aPT12: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 155

[0226] mAb-aPT13: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 156

[0227] mAb-aPT14: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 157

[0228] mAb-aPT15: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 158

[0229] mAb-aPT16: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 159

[0230] mAb-aPT17: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 160

[0231] mAb-aPT18: LC of SEQ ID NO: 184 and HC of SEQ ID NO: 161

[0232] mAb-aPT19: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 144

[0233] mAb-aPT20: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 145

[0234] mAb-aPT21: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 146

[0235] mAb-aPT22: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 147

[0236] mAb-aPT23: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 148

[0237] mAb-aPT24: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 149

[0238] mAb-aPT25: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 150

[0239] mAb-aPT26: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 151

[0240] mAb-aPT27: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 152

[0241] mAb-aPT28: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 153

[0242] mAb-aPT29: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 154

[0243] mAb-aPT30: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 155

[0244] mAb-aPT31: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 156

[0245] mAb-aPT32: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 157

[0246] mAb-aPT33: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 158

[0247] mAb-aPT34: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 159

[0248] mAb-aPT35: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 160

[0249] mAb-aPT36: LC of SEQ ID NO: 185 and HC of SEQ ID NO: 161

[0250] C. Antibodies that compete for binding Antigen binding to human tau protein phosphorylated at position 413 (pSer413) In addition to the domains and antibodies containing them, the present invention also relates to antibodies and methods for binding to the antibodies described herein. The present invention provides antibodies that compete with the IgG antibody.

[0251] The humanized anti-phosphorylated tau antibody according to the present invention is competitively bound to pSer413 tau. Humanized antibodies that are produced using the method are also included within the scope of the present invention. " means that when two or more monoclonal antibodies are present with an antigen, the antigen of one of the antibodies Competitive binding means that the binding of an antibody to an antigen is inhibited by the binding of another antibody to the antigen. In this case, for example, a certain amount (concentration) of a monoclonal antibody is usually used. The amount (concentration) of the monoclonal antibody is varied and the binding of the former monoclonal antibody is measured. The amount (concentration) of the latter monoclonal antibody is measured when the amount of the latter decreases. The degree of inhibition can be expressed in units of IC50 or Ki. A humanized antibody that causes competitive binding to the humanized anti-phosphorylated tau antibody of the present invention is The antigen-antibody binding of the present invention was measured using the humanized anti-phosphorylated tau antibody of the present invention at 10 nM. When the compound is tested, the IC50 is 1 μM or less, particularly 100 nM or less, and more particularly 10 nM or less. The present invention relates to an antibody which is

[0252] As known in the art, generally SPR / Biacore® or Oc Using the tet® binding assay, as well as ELISA and cell-based assays Competitive binding studies can be performed using

[0253] In some embodiments, the variable heavy and light domains of the competitive binding antibody are , at least 85%, 90%, 90%, or 100% for at least one of the variable regions outlined herein. 5%, 98% or 99% identity, and in the constant domain, as outlined in FIG. It has at least 99 or 100% identity to the human IgG sequence.

[0254] D. Amino acid substitutions

[0255] In addition to the sequences outlined herein, the present invention also provides sequences that have one or more amino acids compared to the parent starting sequence. The present invention provides antigen-binding domains which may have amino acid substitutions and antibodies containing them.

[0256] Thus, for example, for all of the variable heavy and light chain domains listed herein: Further variants can be made. As outlined herein, in some embodiments In one embodiment, the set of six CDRs has 0, 1, 2, 3, 4 or 5 amino acid modifications. In these embodiments, Generally, a single CDR has no more than one or two amino acid substitutions, and the mutated sequence is pSe r413 retains binding to tau protein. However, When amino acid mutations are generated in the CDRs, the resulting CDRs are SEQ ID NOs: 81, 82 and 83 (variable light chain CDRs) and SEQ ID NO: 8 6, 87 and 88 (variable heavy chain CDRs).

[0257] In some cases, the framework regions of the variable heavy and / or light chain domains In this embodiment, changes can be made in framework regions (e.g. Preferred variants in the CDRs (excluding CDRs) are at least about 80, 80, 90, 100, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 3 Thus, for example, the framework regions of The region shares at least 80, 85, 90 or 95% identity with the human germline sequence. As long as the CDRs described herein are identical, they may be combined with different frameworks derived from human germline sequences. It can be combined with a line sequence.

[0258] In some embodiments, for the humanized light chains outlined herein, the closest human light chain The germline is IGKV2-30, which has 81% identity to the sequences herein. Therefore, the identity to IGKV2-30 is at least 80%, 85%, 90% or Further variants may be made in the light chain framework regions as long as the identity is 95%. It is possible.

[0259] For the humanized heavy chains herein, the closest human germline for the heavy chain is The sequence is IGHV3-73, which has 85% identity to the sequence. at least 80%, 85%, 90% or 95% identical to GHV3-73 As long as the amino acid sequence is consistent with the amino acid sequence of the light chain framework region, further variants can be made in the light chain framework region.

[0260] Alternatively, the CDRs may be modified by amino acid modifications (e.g., 1, 2, 3, 4 or more of the set of CDRs). 5 amino acid modifications) (i.e., the CDRs can have variations within the set of six CDRs). Any combination of CDRs can be altered to produce a CDR with a total of less than six amino acid modifications. The DRs can be modified; for example, one in vlCDR1, two in vhCDR2, 0 in DR3, etc.), and framework regions with fewer bases than the human germline sequence. The framework regions may be altered as long as they retain at least 80, 85 or 90% identity. The resulting CDRs can be sequenced as outlined in PCT / JP13 / 65090, SEQ ID NO: SEQ ID NOs: 81, 82 and 83 (variable light chain CDRs) and SEQ ID NOs: 86, 87 and 88 (variable heavy chain CDRs) R) are different from the mouse CDRs shown.

[0261] Generally, the percent identity for comparison between anti-pSer413 tau antibodies is at least At least about 75%, at least 80%, at least 90%, and at least about 95, 96, 97 Percent identity is based on the total amino acid sequence. A sequence, e.g., across an entire heavy or light chain, or even across a portion of a chain. For example, included within the definition of the anti-pSer413 tau antibodies of the present invention are If the identity is 95% or 98% across the entire domain (e.g., across the variable region), or antibodies that share identity across their entire constant regions.

[0262] X. Nucleic Acids of the Invention Nucleic acid compositions encoding the anti-pSer413 tau antibodies of the present invention, as well as nucleic acid compositions comprising the same Expression vectors and host cells transformed with the nucleic acids and / or expression vector compositions are also provided. As will be appreciated by those of skill in the art, the protein sequences presented herein are provided in accordance with the principles of the genetic code. Due to degeneracy, any number of possible nucleic acid sequences can be encoded.

[0263] Thus, the present invention further provides nucleic acid compositions encoding antigen-binding domains and their As will be appreciated by those skilled in the art, in the case of an antigen-binding domain, a nucleic acid The composition generally comprises a first nucleic acid encoding a variable heavy chain domain and a second nucleic acid encoding a variable light chain domain. In the case of scFv, the nucleic acid sequence is separated by a proteinaceous linker. Thus, a single nucleic acid can be generated that encodes the variable heavy and light domains. For traditional antibodies, the nucleic acid composition generally comprises a first nucleic acid encoding a heavy chain and a second nucleic acid encoding a light chain. and a second nucleic acid encoding the two heavy chains and the two ribozymes, which spontaneously synthesize two heavy chains and two ribozymes when expressed in a cell. The light chain of the IgG1A is in the "traditional" tetrameric form.

[0264] As is known in the art, nucleic acids encoding the components of the present invention can be prepared by synthesis, as is known in the art. Depending on the host cell used to produce the heterodimeric antibody of the invention, expression may vary. As will be appreciated by those skilled in the art, these two nucleic acids can be integrated into a vector. They can be incorporated into a single expression vector or into two different expression vectors. The nucleic acid may be modified with any number of regulatory elements (promoters, origins of replication, selection markers, ribosomal The expression vector is operably linked to a gene encoding a chromosome-specific gene (e.g., a gene-binding site, an inducer, etc.). It may be an exogenous vector or an integrating vector.

[0265] The nucleic acids and / or expression vectors of the invention can then be used in mammalian cells, which can be used in a number of different embodiments. In addition to mammalian cells (e.g., CHO cells), bacterial cells, yeast cells, insect cells, and and / or transformed into any number of different types of host cells known in the art, including fungal cells. do.

[0266] The antibodies of the present invention can be prepared by injecting an expression vector(s) into the cells, as is well known in the art. Once produced, they are purified using traditional antibody purification processes. Do the following.

[0267] XI. Biological and Biochemical Assays The anti-pSer413 tau antibodies of the invention can be assayed for efficacy in several ways. As a preliminary step, ELISA technology (see, for example, Example 2(1)) Using techniques known in the art, such as Octet, Biacore or FACS, For binding to human tau protein or peptides phosphorylated at S413, Antibodies can be tested, in which case the phosphorylated peptide ( Binding to a non-phosphorylated peptide (e.g., SEQ ID NO: 69) and binding to a non-phosphorylated peptide (e.g., SEQ ID NO: 8). compare.

[0268] In some embodiments, the binding efficiency of the antibodies disclosed herein is determined using a Biacore assay. In a particular embodiment, phosphorylated tau protein is used to measure K D Measure the value In some embodiments, phosphorylated tau peptides are used to determine K D Measure the value. In embodiments, the phosphorylated tau peptide is phosphorylated at only one residue (e.g., the sequence In another embodiment, the phosphopeptide is phosphorylated at multiple residues ( For example, SEQ ID NO: 76 or SEQ ID NO: 78). In some embodiments, the immobilized antigen (e.g., phosphorylated tau protein or phosphorylated tau peptide) using K D Measure the value In this case, the affinity measurement involves binding activity, i.e., a bivalent binding mode. In embodiments, the immobilized antibody (e.g., a murine parent antibody, a chimeric antibody, or a humanized antibody) mutant) using K D value, where the affinity measurement does not include the binding active component. In yet another embodiment, the immobilized antibody and analyte are Using phosphorylated tau protein, D In yet another embodiment, the immobilization Using antibodies and phosphopeptides as analytes, D Specific embodiments So, in bivalent binding mode, K D In another embodiment, the K value is measured in a monovalent binding manner. D Measure the value Determine.

[0269] Furthermore, the anti-pSer413 tau antibody of the present invention also inhibited the expression of tau in human AD brain homogenates. Binding affinity can also be assayed, as well as HEC293 tau P301L agglutination. In neutralization tests of AD-derived autophagic seeds in collection assays and / or microfluidic In a model of AD-derived tau inoculation and propagation using human iPSC neurons in a mouse model The assay can be performed using the following methods:

[0270] Furthermore, the anti-pSer413 tau antibody of the present invention also demonstrated immunohistochemistry of brain samples from AD patients. The antibodies can also be used in quantitative assays.

[0271] Furthermore, the anti-pSer413 tau antibody of the present invention can be used to treat cognitive impairment, including Alzheimer's disease (AD). The compounds can be assayed for their efficacy in animal models of the disorders. Animal models for cognitive disorders include those for which therapeutic or preventive drugs for disorders are available. These include animal models of tauopathy, particularly animal models of tauopathy, which have been shown to express positive Animals expressing normal or mutant tau are animal models that exhibit cognitive impairment. Such animals expressing normal or mutant tau in vivo can be prepared by genetic engineering. A typical example is a transgenic mouse (Tg mouse). Animal models such as Tg mice expressing tau1 are useful as models of inherited familial tauopathies. However, there are currently no therapeutic or prophylactic drugs for sporadic tauopathies, which constitute the majority of human cases. For efficacy testing of preventive drugs, it is preferable to demonstrate the effect in Tg mice expressing normal tau. The most suitable Tg mouse expressing normal tau is the one produced in the production example of the present invention. The mice to be prepared are those that have been used in the production and use of transgenic mice. Kambe et al. (Neurobiol. ogy of Disease, Vol. 42, P. 404-414, 2011) and K imura et al.(The EMBO J.vol.26.P.5143-51 The Tg mice reported by (52, 2007) may be used. The impairment was observed in the mice of Kambe et al. and Kimura et al., but it was 1 Since the disease appears after 4 and 20 months of age, the patient is quite old at the time of onset, and the age effect is not known. The effects of long-term breeding may also be a factor, but the impact and effort involved are also issues.

[0272] The efficacy of therapeutic or prophylactic agents against cognitive impairment according to the present invention can be tested in animal models. A preferred method is a method for testing cognitive function, such as a memory and learning test. may be the Morris Water Maze test, the Step-Through Learning test or the Novel Object Recognition Test. However, in order to consider the amount of activity and the anxiety condition of the animals, it is preferable to use an open field test. It is a combination of behavioral measurement tests such as:

[0273] As a method for investigating the effect of the therapeutic or preventive agent for cognitive impairment according to the present invention, During administration to animal models, the levels of tau protein or phosphorylated tau were examined in brain tissue. In AD and other neurodegenerative diseases, the expression of tau protein can be Increased expression levels or abnormal phosphorylation of tau are associated with pathology (Khalid Iq bal et al.,Curr.Alzheimer Res.,Vol.7,p65 4-664, 2010). Tau expression and abnormal linkages were observed in several pathological animal models. It has also been shown that lowering phosphorylated tau levels leads to improved cognitive and motor function. It is well known (K. Santa Cruz et al., Science, 30, Vol.9,p.476-481,2005;Sylvie Le Corre et al.,Proc.Nat.Acad.Sci.USA,Vol.103,p.9673 -9678, 2006) as a method to examine changes in tau protein or phosphorylated tau. As described in the Examples, Western blotting and other methods were used to measure the expression of the IgG1-related protein using brain tissue homogenates. This can be achieved by a variety of methods, including ELISA (Xiyun Ch ai et al.,J.Biol.Chem.,Vol.286,p.34457-3 4467,2011) or immunohistochemical methods (David J. Irwin et al. al.,BRAIN,Vol.135,pp.807-818,2012) and other appropriate You can choose the method that suits you.

[0274] XII. Preparations Formulations of antibodies for use in accordance with the present invention may be prepared in any pharma- ceutically acceptable form to provide antibodies of the desired purity. In the form of a lyophilized formulation or an aqueous solution by mixing with a carrier, excipient or stabilizer. (Remington's Pharmaceutical Sc iences 16th edition, Osol, A. Ed.

[1980] as generally outlined).

[0275] The therapeutic or preventive agent for cognitive impairment according to the present invention comprises a humanized anti-phosphorylated tau antibody according to the present invention. In the form of a pharmaceutical composition containing, in addition to the body, a pharma- ceutically acceptable carrier and / or other excipients. The composition may be formulated in a pharma- ceutically acceptable carrier and / or other excipient. For example, University of the Sciences in Phila delphia,“Remington:The Science and Pract ice of Pharmacy,20th EDITION”, Lippincott This is carried out using the method described in Williams & Wilkins, 2000. Such therapeutic or prophylactic agents can be formulated by dissolving the components in a sterile aqueous or oily medium. as liquid formulations prepared by dissolving, suspending, or emulsifying in The vehicle or solvent for preparing such a formulation may be provided as a lyophilized formulation of the compound of formula (I). The medium may be an aqueous medium, examples of which include distilled water for injection and saline solution. These are optionally mixed with an osmotic pressure regulator (e.g., D-glucose, D-sorbitol, D-mannitol, and sodium chloride) may be added and / or used as appropriate. Suitable dissolving agents include alcohols (e.g., ethanol), polyalcohols (e.g., polyvinyl alcohols), propylene glycol or polyethylene glycol), or non-ionic surfactants (e.g. For example, it may be used in combination with polysorbate 80 or polyoxyethylene hydrogenated castor oil 50). Such formulations may also be prepared using an oily vehicle or solvent, examples of which include Examples of suitable oils include sesame oil and soybean oil, which may be optionally solubilized with a solubilizing agent such as benzoic acid. Benzyl and benzyl alcohol can be used in combination. Such liquid drugs are often In the case of Zarconium and procaine hydrochloride), stabilizers (e.g., human serum albumin and polyether glycol), preservatives (e.g., ascorbic acid, erythorbic acid, and their salt), colorants (e.g., copper chlorophyll, beta-carotene, red #2 and blue #1), preservatives (e.g. For example, paraoxybenzoic acid esters, phenol, benzethonium chloride, and benzyl chloride. ruthenium), thickeners (e.g., hydroxypropyl cellulose, carboxymethyl cellulose cellulose and their salts), stabilizers (e.g., human serum albumin, mannitol and sorbitol), and appropriate additions such as flavoring agents (e.g., menthol and citrus flavors). The agent can be prepared using various forms of therapeutic or prophylactic agents, such as powders, tablets, granules, etc. Oral dosage forms include solid dosage forms such as capsules, pills, suppositories, and lozenges. For solid formulations to be administered, excipients (e.g., microcrystalline cellulose, lactose, and denaturant) may be used. punch), lubricants (e.g. magnesium stearate and talc), binders (hydroxypropyl propylcellulose, hydroxypropylmethylcellulose, macrogol, etc.), and Additives such as disintegrants (e.g., starch and calcium carboxymethylcellulose) are used. If necessary, a preservative (e.g., benzyl alcohol, chlorobutanol) may be used. , methyl parahydroxybenzoate, propyl parahydroxybenzoate), antioxidants, colorants, sweeteners Additives such as flavorings may be used. As another alternative, the treatment may be applied onto the mucous membrane. Such formulations often have primarily mucosal adsorption or retention properties. Addition of pressure-sensitive adhesives, pressure-sensitive enhancers, viscosity adjusters, thickeners, etc. for the purpose of imparting properties (e.g., mucin, agar, gelatin, pectin, carrageenan, sodium alginate, Sodium, locust bean gum, xanthan gum, tragacanth gum, gum arabic, Tosan, pullulan, waxy starch, sucralfate, cellulose and its derivatives ( Hydroxypropyl methylcellulose, polyglycerol fatty acid ester, acrylic Acid alkyl (meth)acrylate copolymer, or its salt and polyglycerin fatty acid However, the form, solvent and additives of the therapeutic or prophylactic agent administered to the body may vary. The present invention is not limited to these, and may be appropriately selected by those skilled in the art.

[0276] The therapeutic or preventive agent for cognitive impairment of the present invention comprises, in addition to the humanized anti-phosphorylated tau antibody of the present invention, , other known drugs having the effect of treating or preventing cognitive impairment, particularly drugs having the effect of suppressing the progression of cognitive impairment Alternatively, the humanized antiphospholipid of the present invention may contain a specific drug having an effect of inhibiting the action of the humanized antiphospholipid. The therapeutic or preventive agent for cognitive impairment of the present invention containing a modified tau antibody is used for the treatment or prevention of cognitive impairment. The present invention contains a known drug having a preventive effect, particularly a drug having an effect of suppressing the progression of cognitive impairment. The compound may be combined with other therapeutic or prophylactic drugs and manufactured in the form of a kit. Ingredients that have the effect of suppressing ambulation include donepezil, galantamine, memantine, and rifabutin. These include, but are not limited to, bastigmine. The dosage of the component having the effect of suppressing the progression of cognitive impairment and the therapeutic agent or preventive agent containing the component having the effect of suppressing the progression of cognitive impairment The dose of the drug may be within the range of the dose used in normal treatment, but may be increased or decreased depending on the condition. It is possible to do so.

[0277] In WO2013 / 180238A (Patent Document 4), the present inventors have reported that Even when administered more peripherally, antibodies can act on the brain through the blood-brain barrier and exert their therapeutic effects. The experimental results shown in the figures are based on the results of the present invention, and the therapeutic or preventive agent for cognitive impairment of the present invention is It is also possible to prepare a formulation that efficiently delivers the modified anti-phosphorylated tau antibody to brain tissue. Such preparations are also encompassed in the therapeutic or prophylactic agents for cognitive disorders according to the present invention. A method for efficiently delivering antibodies or peptides to brain tissue through the blood-brain barrier, e.g., a method for targeting Methods of adding substances or encapsulating them in liposomes or nanoparticles are known. The targeting agents used include antibodies or those with the ability to bind to specific receptors or transporters. The charge characteristics of the peptides, proteins or other compounds may be changed by binding to the peptides, proteins or other compounds having the same charge characteristics. These include substances that partially change their binding properties to specific receptors or membrane proteins. Examples of peptides, proteins or other compounds having the structure include receptor ligands or membrane ligands. Ligands and their analogs that bind to proteins, as well as receptor ligands or membrane proteins Antibodies that bind to proteins, agonist compounds, antagonist compounds, and allosteric compounds These include schizophrenia drugs and their analogues. The property of binding to specific receptors or transporters. Receptor ligands or membrane targets for peptides, proteins or other compounds having Examples of proteins include the transferrin receptor (TfR) and the insulin receptor (IR). , insulin-like growth factor receptor (IGFR), LDL receptor-related protein (LRP) and and diphtheria toxin receptor (HB-EGF) (An gela R.Jones et al.,Pharm.Res.,2007,Vol. 24, No. 9, pp. 1759-1771). The target substance may be chemically added to the antibody used, and the method is known to those skilled in the art. For example, Hermanson et al., Bioconjugate technology The method described in Équipes, USA, Academic Press, 1996 was used. The target substance may also be an antibody or a peptide. Alternatively, the glycerol may be bound to liposomes or nanoparticles that are suitable for use in the treatment of inflammatory conditions (Sonu Bhaskar et al., 2013). l.,Particle and Fiber Toxicology,2010,7: 3) Furthermore, if the target substance is a peptide or protein, those skilled in the art can use genetic engineering techniques to The fusion peptides can be produced by chemical peptide synthesis or by the use of techniques such as A nucleic acid containing a nucleotide sequence that codes for the amino acid sequence of a protein or a nucleic acid sequence that encodes an antibody to be used. Combining nucleic acids containing nucleotide sequences encoding the amino acid sequences of the polypeptides or peptides The polypeptides can be produced as suitable fusion proteins, either by

[0278] The therapeutic or prophylactic agent according to the present invention is administered to a patient in need thereof for the purpose of improving symptoms. The drug may be administered orally or parenterally. For oral administration, the drug may be administered in the form of granules, powders, tablets, capsules, etc. The dosage form may be selected from the group consisting of cell, liquid, syrup, emulsion, suspension, or elixir. For parenteral administration, intranasal preparations may be prepared, and liquid, suspension or solid preparations may be used. Injections may be prepared as different forms of parenteral administration, and the injections may be subcutaneous, The injection may be selected as an intravenous injection, an infusion, an intramuscular injection, an intracerebroventricular injection or an intraperitoneal injection. Other preparations for parenteral administration include suppositories, sublingual preparations, transdermal preparations, and transnasal preparations other than mucosal preparations. In addition, the drug may be added to or coated on a stent or intravascular occlusion device. This allows for local intravascular administration via a method that involves administering the drug to the blood vessels.

[0279] The dosage of the therapeutic or prophylactic agent according to the present invention depends on the age, sex, weight and symptoms of the patient. Depending on the therapeutic effect, administration method, treatment time, or the type of active ingredient contained in the pharmaceutical composition, Although this may vary depending on the individual, the usual dosage for adults is 0.1 mg to 1 g, preferably 0.5 mg. The dosage may range from 100 mg to 200 mg of active compound. However, dosage may vary under various conditions. Since the dose varies depending on the patient, a dose less than the above may be sufficient, or the dose may be within these ranges. In some cases, a dose exceeding 100 mg / kg may be required. It can be effective in.

[0280] XIII. Administration of Antibodies

[0281] Once generated, the antibodies of the invention can be used to treat cognitive disorders such as tauopathies. As discussed herein, intracellular accumulation of tau protein is associated with a variety of neuropathologies. The disease caused by the intracellular accumulation of tau has been shown to be a pathological condition. These diseases are collectively referred to as "tauopathies" (herein incorporated by reference in their entirety). (See Arai, supra, for an overview of the disorders.) Diseases such as Alzheimer's disease (AD), corticobasal degeneration (CBD) or corticobasal degeneration (CBD) Progressive supranuclear palsy, Pick's disease, argyrophoric grain dementia (argyrophoric grain disease), Multisystem tauopathy with dementia (MSTD), linked to chromosome 17 and Parkinsonism Familial frontotemporal dementia with cerebrospinal fluid (FTDP-17), neurofibrillary tangle dementia, calcification dementia Diffuse neurofibrillary tangles associated with globular glial inclusions (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), and frontotemporal lobar degeneration with tau pathology (FTLD-tau). Tauopathies include: encephalitis lethargica sequelae, infectious diseases such as subacute sclerosing panencephalitis; Non-neurodegenerative diseases are also included, including traumatic conditions such as Sir disease.

[0282] Cognitive impairment is a type of intellectual disability that includes a decline in intellectual functions that were once developed or acquired. Although it is defined as harm ("New Psychiatry (Nankodo's Ess “Kunential Well-Advanced Series” (Japanese literature), Edited by Itoshi Kamijima and Shin-ichi Niwa, nkodo Co., Ltd., 2008, pp.69-70), in a broad sense, intellectual disabilities and Diagnosis of neurodegenerative diseases such as AD. In this study, whether a pathology is "neurodegenerative" can be determined, for example, by postmortem histological analysis. It is determined by analyzing whether neurofibrillary tangles (NFTs) are present. On the other hand, doctors use various methods, such as the Revised Hasegawa Dementia Scale (HD Neuropsychological testing by interview such as the Mini-Mental State Examination (MMSE) and Observational dementia assessment such as Clinical Dementia Rating (CDR) or Functional Assessment Staging (FAST) Trans-psychological testing; for example, increased levels of tau and phosphorylated tau, or A in cerebrospinal fluid Biochemical tests based on increased levels of β; as well as head CT, head MRI, SP ECT or imaging studies based on information obtained via PET can be used to assess cognitive impairment, especially The therapeutic or preventive agent for cognitive impairment of the present invention is a neurodegenerative disease. Patients diagnosed by a physician as suffering from cognitive impairment based on at least one of the following diagnostic indicators: The drug is administered to a patient to improve the patient's condition compared to the condition before administration, or to prevent the progression of the condition through administration. Efficacy of controlling the onset of symptoms, maintaining the pre-treatment pathology, or restoring the pre-treatment pathology It has results.

[0283] The therapeutic or preventive agent for cognitive impairment according to the present invention can be used in humans or non-human animals by Improve cognitive function, inhibit cognitive decline, or maintain cognitive function Examples of such non-human animals preferably have high homology to human tau. The animals should be those that express tau, which is a signaling molecule: chimpanzees, macaques, horses, pigs, dogs, These include, but are not limited to, mice, rabbits, rats and cats. EXAMPLES

[0284] XIV. Examples A. Example 1: Antibody Production 1. Mouse antibody (parent antibody) The mouse antibody Ta1505 was used as the parent antibody for the chimeric and humanized antibodies. Details of the antibody Ta1505 are described in the Examples of International Publication No. WO2013 / 180238A. The amino acid sequence of the light chain (L chain) of the mouse antibody Ta1505 is set forth in SEQ ID NO:44. The mouse antibody T is defined as having a nucleotide sequence encoding the same as SEQ ID NO: 45. The amino acid sequence of the heavy chain (H chain) of a1505 is defined in SEQ ID NO: 46, and the coding sequence thereof is The nucleotide sequence is defined in SEQ ID NO:47.

[0285] 2. Chimeric antibodies The light chain (L chain) and heavy chain (H chain) variable regions of the mouse antibody Ta1505 were transformed into human By combining with the constant regions of the L and H chains of immunoglobulin (Ig) G1 (κ), The amino acid sequence of the light chain (L chain) of the chimeric antibody was set forth in SEQ ID NO:48. The chimeric antibody is defined as having a nucleotide sequence encoding the same as SEQ ID NO: 49. The amino acid sequence of the heavy chain (H chain) is defined in SEQ ID NO:50, and the nucleotide sequence encoding it is The sequence is defined in SEQ ID NO: 51. Throughout this specification, this chimeric antibody is referred to as a "chimeric antibody " or "chimeric antibody Ta1505."

[0286] 3. Humanized Antibodies a. Design of VL and VH Select primary sequences with high homology from human germline and sequence the complementarity determining regions (CDRs). Humanization was performed by grafting, and a variety of humanized antibodies were engineered.

[0287] In particular, a human framework with high homology was selected from the database; The CDR regions of the antibody Ta1505 were inserted into each framework; e.g., while maintaining activity. To reduce immunogenicity, amino acid substitutions were made to the various light chain variable regions. The amino acid sequences of the heavy chain variable region (VL) and heavy chain variable region (VH) were designed. The sequence numbers and the SEQ ID NOs of the nucleotide sequences of the genes encoding them are shown in Table 2A. The amino acid sequence of H and the nucleotide sequence of the gene encoding it are shown in Table 2B. The alignment of the amino acid sequences of VL and VH is shown in Figures 2A and 2B, respectively. vinegar. Table 2A: Light chain variable region (VL) [Table 3] Table 2B: Heavy chain variable region (VH) [Table 4]

[0288] b. Selection of VL and VH combinations based on immunogenicity scores The VL and VH designed by the above procedure were arbitrarily combined to produce immunogens for each combination. The sex scores (DRB1 scores) were analyzed using the in vitro assay software Epibase ( The VL and VH combinations used in the following experiments were calculated using the immunoassay. The combination shows low immunogenicity with a specific immunogenicity score below a certain value (DRB1 score: 1500) The selected VL and VH combinations are shown in Table 3A, and the immunization status of these combinations was The pathogenicity scores (DRB1 scores) are shown in Table 3B. Table 3A: Selected VL and VH combinations [Table 5] Table 3B: Immunogenicity score (DRB1 score) [Table 6]

[0289] c. Expression culture and purification of humanized antibodies For each combination of VL and VH selected by the above procedure, The VL gene was transformed into a human immunoglobulin κ L chain constant gene. The amino acid sequence and nucleotide sequence of the CL gene are are defined in SEQ ID NO: 52 and SEQ ID NO: 53, respectively), It is fused in frame to the γ-1 heavy chain constant region (CH) gene (its amino acid sequence and and the nucleotide sequences are defined in SEQ ID NO:54 and SEQ ID NO:55), thereby There should be no mutations or deletions in the binding sites. The ends of the ribozymes were each connected to a signal sequence derived from the human immunoglobulin κ L chain gene (the amino acid sequence of which is The sequence and nucleotide sequence are defined in SEQ ID NO:56 and SEQ ID NO:57, respectively) and Signal sequence derived from the human immunoglobulin γ-1H chain gene (its amino acid sequence and nucleotide sequence) The octade sequences are defined in SEQ ID NO:58 and SEQ ID NO:59, respectively. The fusion should not result in mutations or deletions in the respective binding sites.

[0290] The nucleic acid constructs encoding the signal sequence-fused L chain and H chain designed by the above procedure were The vector was inserted into an expression vector for mammalian cells and expressed in ExpiCHO (Thermo Fish A standard primer for expressing antibodies was used (Protein Biosciences, Inc.). The cultures were cultured according to the protocol. Each culture was clarified by centrifugation, and the resulting culture supernatant was The humanized antibody was purified by applying it to a protein A affinity column. The fraction eluted with an acidic solution was quickly neutralized, concentrated, and subjected to a gel filtration column to remove aggregates. The buffer was replaced with phosphate-buffered saline (PBS) and the main fraction was collected. After diluting to an appropriate concentration with 100 mL of 10 ...

[0291] In the following description, each humanized antibody produced by the above procedure will be described with reference to its VL and VH For example, L15 for VL and L15 for VH. The humanized antibody produced using H11 as the target is called L15H11.

[0292] In the following tests, unless otherwise specified, 3% BSA / PBS was used to adjust the antibody concentration. was used as a solvent for

[0293] B. Example 2: ELISA Analysis of Antigenic Peptide Binding Affinity 1. ELISA analysis of binding affinity to antigen peptides Several humanized antibodies having the VL and VH combinations shown in Table 3A were subjected to enzyme-linked immunosorbent assay. The binding affinity to the antigen peptide was analyzed by enzyme-linked immunosorbent assay (ELISA).

[0294] Non-phosphorylated tau peptide PD17 (Ser413) and phosphorylated at Ser413 1 μg of phosphorylated tau peptide PD17P (pSer413) was added to cold PBS. The resulting solution was dispensed at 50 μL / well onto a plate and incubated at 4°C. After removing the solution, blocking buffer (3% bovine serum albumin (B SA)-PBS) was dispensed at 270 μL / well, and the plate was left to stand overnight at 4° C. After removing the antibody solution, the antibody solution was serially diluted with 3% BSA-PBS and then 50 μL / well was added. The non-phosphorylated tau peptide PD17 ( Amino acid sequences of phosphorylated tau peptide PD17P (pSer413) and phosphorylated tau peptide PD17P (pSer413) are defined in SEQ ID NO:60 and SEQ ID NO:8, respectively.

[0295] Tris-buffered saline containing 0.05% Tween 20 (TBS Each well was washed with PBS (-T). The plate was diluted 2000-fold with dilution buffer (3% BSA-PBS). Goat anti-human IgG-alkaline phosphatase conjugated (Sigma-Aldrich Co. T (manufactured by T.L. LLC) was added to the plate at 50 μL / well and reacted at room temperature for 60 minutes. After washing with BS-T, the plate was incubated with a color development solution (1 mg / mL p-nitrophenyl phosphate (pNPP ) solution) was added to the plate at 100 μL / well and allowed to develop for 40 minutes. The absorbance (optical density (OD) value) was measured at wavelength.

[0296] The ratio of the OD value of each antibody to the OD value of the 0.25 nM chimeric antibody was determined, and the resulting ratio was evaluated on the three-point scale described below to evaluate the reactivity.

[0297] +: Sufficient reactivity (0.6 ≤ OD ratio),

[0298] ±: Very weak reactivity (0.15 < OD ratio < 0.6), and

[0299] -: No reactivity (OD ratio ≤ 0.15).

[0300] Table 4A shows the evaluation results of the reactivity between the antibody and the phosphorylated tau peptide PD17P. Table 4 B shows the evaluation results of the reactivity between the antibody and the non-phosphorylated tau peptide PD17. Compared with the chimeric antibody all humanized antibodies showed higher reactivity with the phosphorylated tau peptide PD17P but lower reactivity with the non-phosphorylated tau peptide PD17. These results indicate that the humanized antibodies have selective reactivity with the phosphorylated tau peptide. Table 4A: ELISA analysis of reactivity with phosphorylated peptide PD17P [Table 7] Table 4B: ELISA analysis of reactivity with non-phosphorylated peptide PD17 [Table 8]

[0301] 2. ELISA analysis of selective binding affinity to phosphorylated tau peptide Some of the humanized antibodies having the combinations of VL and VH shown in Table 3A were subjected to enzyme-linked immunosorbent assay (ELISA) using the chimeric antibody Ta1505 as a reference antibody , the selective binding affinity of anti-tau humanized antibodies to phosphorylated tau peptides was analyzed. .

[0302] Contains 1 μg / mL of non-phosphorylated peptide PD17 or phosphorylated peptide PD17P The solution was dispensed at 50 μL / well onto the plate and left to stand overnight at 4° C. The solution was removed. Then, add 270 μL of blocking buffer (3% bovine serum albumin (BSA)-PBS) The plate was then left at 4°C overnight, allowing each well to receive the non-phosphorylated peptide. Plates on which the peptide PD17 or the phosphorylated peptide PD17P was immobilized were prepared.

[0303] A series of five concentrations of each antibody was incubated at 4 °C for 4 h, starting with an initial concentration of 0.6 μg / mL (4 nM). Each solution was added to the wells of the plate at 50 μL / well. The reaction was allowed to proceed at room temperature for 1 hour. After washing, goat anti-human IgG alkaline phosphatase-labeled was added. (Sigma-Aldrich Co. LLC.) was diluted 2000-fold and then added to 50 μl L / well was added to each well and the reaction was continued for another hour.

[0304] After a washing step, 1 mg / mL of p-nitrophenyl phosphate (pNPP) solution was added to each well. 100 μL / well was added and reacted for 20 minutes. The absorbance (OD) was measured at a wavelength of 405 nm. ) was measured.

[0305] The absorbance OD data for each antibody concentration was analyzed using SoftMax Pro software. Software Ver.6.5 (Molecular Devices Corporation n) and assigned to the following four parameter logistic regression curve: The following values ​​A to D were determined:

[0306]

number

[0307] Enter the absorbance OD value of the non-phosphorylated peptide PD17 at a concentration of 1 nM into the expression column and calculate the OD value. The concentration of each antibody corresponding to each peptide was calculated. Binding to phosphorylated tau peptide PD17P based on binding affinity to PD17 for The affinity scale factor was calculated as 1 / x, which is the ratio of the selective binding of the phosphorylated tau peptide to the was used as an index of compatibility.

[0308] The results are shown in the graph in Figure 3. Chimeric antibody Ta against phosphorylated tau peptide PD17P The binding affinity of 1505 was 30-fold higher than that for the non-phosphorylated peptide PD17. In contrast, the selective binding affinity of humanized antibodies shows high selectivity and scales well. The results showed that the humanized antibody reacted with the phosphorylated peptide in a 40-210-fold manner. It has been shown to have specific binding affinity.

[0309] 3.Selective binding affinity to the Ser413 phosphorylation site using phosphorylated tau peptide ELISA analysis The humanized antibodies having the VL and VH combinations shown in Table 3A were subjected to immunofluorescence assay using various phosphorylated peptides. ELISA using 100% phospho-Ser413 was detected among the major phosphorylation sites of the protein. Each was analyzed for its ability to selectively bind to the site.

[0310] Twelve phosphorylated peptides were used, as shown in Table 5. The columns indicate the phosphorylation site(s) of the 4R2N tau protein (e.g., Peptide number 1, “pS46”, indicates that the Ser46 site is a phosphorylation epitope. However, the description of multiple epitopes indicates that one peptide has two or three phosphorylation sites. With the exception of peptide number 12, which is an epitope containing the Ser413 site, but not phosphorylated (PD17). Table 5: Peptides for analyzing specific binding to the Ser413 phosphorylation site of tau protein. D [Table 9]

[0311] The 12 peptides shown in Table 5, namely peptide numbers 1 to 12, or and bovine serum albumin (BSA) or keyhole limpet hemocyanin (KL H) was diluted with PBS cooled to 4°C to 1 μg / mL. The solution was dispensed at 50 μL / well onto the plate and left to stand overnight at 4°C. After removing the solution, Blocking buffer (3% BSA-PBS) was dispensed at 270 μL / well and plated. The plate was left to stand overnight at 4°C. After removing the solution, the purified antibody was diluted to 150 ng with 3% BSA-PBS. 1 mL / mL, then added to the plate at 50 μL / well and incubated at room temperature for 90 minutes. Each well was washed with Tris-buffered saline containing 0.05% Tween 20 (TBS-T). Goat anti-human IgG (H+) diluted 2000-fold in dilution buffer (3% BSA-PBS) was used. L)-Alkaline phosphatase labeling (Sigma-Aldrich Co. LLC.) was added to the plate at 50 μL / well and reacted at room temperature for 60 minutes. Wash with TBS-T. Then, add 100 μL / well of color-developing solution (1 mg / mL pNPP solution) to the plate. The absorbance (OD value) was measured at a wavelength of 405 nm and a reference wavelength of 550 nm. ) was measured.

[0312] Responsiveness was assessed using a 3-point scale: +: sufficient reactivity (OD value ≦ 1.0); ±: Very slight reactivity (0.5≦OD value<1.0), and -: No reactivity (OD value <0.5).

[0313] The results are shown in Table 6. All humanized antibodies were 413:PRHLSNV(pS)(pS)TGSIDMVD), peptide number 10(pS er413:PRHLSNVS(pS)TGSIDMVD), and peptide number 11 (p Ser409 / pSer412 / pSer413:PRHL(pS)NV(pS)(pS )TGSIDMVD), but not Ser of tau protein. It did not bind to other peptides that were not phosphorylated at the site corresponding to 413. The pattern and intensity of the binding was virtually identical to that of the chimera (chimeric antibody Ta1505). These results show that the humanized antibody specifically targets tau protein containing the phosphorylated Ser413 site. These results indicate that the IgG4-dependent agonist has a significant binding affinity to the IgG4-dependent agonist. Table 6: Results of ELISA analysis of reactivity with various phosphorylated tau peptides. [Table 10]

[0314] C. Example 3: ELISA analysis of binding affinity to antigenic proteins Several humanized antibodies having the VL and VH combinations shown in Table 3A were analyzed by ELISA. The binding affinity to the antigen protein was analyzed.

[0315] To prepare phosphorylated tau protein pTau, we use the baculovirus system in insects. The tau protein 4R2N was expressed in cells. To generate au, we developed a recombinant vector capable of simultaneously expressing tau protein and GSK3β. A baculovirus was produced.

[0316] In the production of recombinant baculovirus, pFastBac1 and pFastBac Dual vector was used. For phosphorylated tau protein pTau, His tag The cDNA encoding the tau protein 4R2N with an additional C-terminus was cloned into pFastBac1 The highly phosphorylated tau protein hpTau was inserted into the His-tagged C-terminus. cDNA encoding the tau protein 4R2N and cDNA encoding GSK3β A was inserted into pFastBac Dual. His-tagged tau protein 4R2N The amino acid and nucleotide sequences are set forth in SEQ ID NOs: 70 and 71, respectively. The amino acid and nucleotide sequences of SK3β are set forth in SEQ ID NOs: 72 and 73, respectively. will be done.

[0317] The obtained vectors were each introduced into the cell line Sf9 by lipofection, and the cells were The recombinant baculovirus was then prepared by culturing the cells. The cell lines Sf9 or Tn5 were infected with tau, which resulted in the production of phosphorylated tau protein ( pTau) and hyperphosphorylated tau protein (hpTau) were expressed.

[0318] Cells expressing the desired protein are harvested and subjected to sonication and centrifugation to effect cell lysis. The product was then subjected to Ni-NTA column to purify tau protein. The protein concentration was measured using bovine serum albumin (BSA) as a standard. Determined by cholinesterase (BCA) assay.

[0319] The obtained highly phosphorylated tau protein (hpTau) and phosphorylated tau protein (pT au) was diluted to 1 μg / mL with chilled PBS and the diluted solution was placed in 50 μL / well. The plate was then dispensed and allowed to stand overnight at 4° C. After removing the solution, blocking buffer (3% 270 μL / well of a buffer solution (BSA-PBS) was dispensed, and the plate was left to stand overnight at 4° C. After removing the liquid, add 50 μL of a series of antibody solutions serially diluted with 3% BSA-PBS. The mixture was added to the plate at 100 μg / well and incubated at room temperature for 90 minutes.

[0320] The wells were then washed with Tris-buffered saline containing 0.05% Tween 20 (TBS-T). After washing, goat anti-human IgG- Alkaline phosphatase label (Sigma-Aldrich Co. LLC.) was added at 50 The plate was washed with TBS-T and incubated at room temperature for 60 minutes. , add 100 μL / well of color-developing solution (1 mg / mL pNPP solution) to the plate, The color was allowed to develop for 40 minutes, and the absorbance (OD value) was measured at a wavelength of 405 nm.

[0321] The ratio of the OD value of each antibody to the OD value of 0.25 nM chimeric antibody was determined, and the obtained ratio was expressed as follows: Responsiveness was assessed by rating on a 3-point scale: +: sufficient reactivity (OD ratio 0.6 or less); ±: Very low reactivity (0.15 < OD ratio < 0.6), and -: No reactivity (OD ratio ≤ 0.15).

[0322] Table 7A shows the results of evaluating the reactivity of the humanized antibodies with hyperphosphorylated tau protein hpTau. Table 7B shows the results of evaluating the reactivity of the humanized antibodies with phosphorylated tau protein pTau. The results indicate that all humanized antibodies have high reactivity with both hyperphosphorylated tau protein hpTau and phosphorylated tau protein pTau, compared to the reactivity of the chimeric antibodies. Table 7A: Results of ELISA analysis of reactivity with hyperphosphorylated tau protein hpTau [Table 11] Table 7B: Results of ELISA analysis of reactivity with phosphorylated tau protein pTau [Table 12]

[0323] D. Example 4: Analysis of binding affinity by SPR Several humanized antibodies having the combinations of VL and VH shown in Table 3A were subjected to analysis of binding affinity by surface plasmon resonance (SPR) using the chimeric antibody Ta1505 as a reference antibody.

[0324] Hyperphosphorylated tau protein 4R2N (hpTau: see Example 3) was used as the antigen protein, and non-phosphorylated tau protein (Tau: His-tagged non-phosphorylated tau protein 4R0N: ATGen Co., Ltd., catalog number: ATGP0795) produced by Escherichia coli was used as a negative control. The antigen peptide used ​​​​​​The peptides were monophosphorylated tau peptide (1×P), in which only Ser413 was phosphorylated, and S Triphosphorylation, in which Ser409 and Ser412 are phosphorylated in addition to Ser413 The amino acid sequences of these peptides are shown in Table 8. The negative control peptide used was a non-phosphorylated tau peptide (Non P), whose amino acid sequence was The columns are also shown in Table 8. The amino acid sequence of the His-tagged non-phosphorylated tau protein 4R0N is: Defined in SEQ ID NO: 74. Monophosphorylated tau peptide 1×P, triphosphorylated tau peptide The amino acid sequences of tau peptide 3×P and non-phosphorylated tau peptide Non P are shown in SEQ ID NOs: 75, 76, and 77. Table 8: Antigen peptides used for SPR [Table 13]

[0325] Hyperphosphorylated tau protein 4R2N (hpTau), non-phosphorylated tau protein 4R0 N, monophosphorylated tau peptide 1×P, triphosphorylated tau peptide 3×P, and nonphosphorylated tau peptide The binding affinity between the modified tau peptide Non P and each antibody was evaluated using the SPR system Biacore. Using T200 (GE Healthcare Japan), attached to the system The measurements were carried out in accordance with the evaluation instructions provided.

[0326] Binding affinity: NTA sensor chip (nitrilotriacetic acid (NTA) is already immobilized Prepare a layer of carboxymethyl dextran (Code No. BR-1005-32) Amine coupling kit (GE Healthcare Japan, code no. BR-1006-33) was used to fuse the His-tag with the amine coupling reaction. The above peptides and the antigen peptides shown in Table 8 were covalently bonded to the sensor chip. and measuring the binding kinetics of antibodies to the immobilized proteins and peptides. The measurements were performed by a method including:

[0327] HBS-N buffer solution (code number BR-1003-69) was used as the immobilization reaction solution. The nickel chloride solution was reacted with the NTA sensor chip to bind Ni to NTA. By using His-tagged hpTau or Tau protein as sensors, The antibody was then immobilized on the chip. Carbodiimide hydrochloride (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide The sensor was stimulated with a mixture of EDC (diethyl ether chloride) and N-hydroxysuccinimide (NHS). The 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( HEPES) buffer (0.01M HEPES, 150mM KCl, 2mM DTT, Each Hi was adjusted to a concentration of 100 to 500 ng / mL with 1 mM EDTA, pH 7.2. A solution of the s-tagged protein is applied to the sensor chip, thereby binding the protein to Ni The remaining active NHS was immobilized on the sensor chip via covalent binding with ethanol. The Ni ions were removed with EDTA solution (Ni-affinity amine). (See International Publication No. WO2005 / 022156) N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) hydrochloride The antigen peptide was activated with a mixture of IgG and N-hydroxysuccinimide (NHS). Each peptide (1xP, 3xP, and Non P) was immobilized on a sensor chip. Then, the antigen was diluted to 1-10 μM with 10 mM sodium acetate buffer (pH 4-4.5). A solution of peptides is applied to the sensor chip, thereby attaching the peptides to the sensor chip. The remaining active NHS was blocked with ethanolamine. A sensor chip was prepared on which each of the above proteins and peptides was immobilized.

[0328] CAPS buffer (pH 10.5) was applied to these sensor chips at 37°C. The surface of the sensor chip was stabilized by immobilizing a protein or peptide. Each antibody solution was prepared using HEPES buffer (pH 7.2) as the reaction solution. To achieve this, a 0.2–1 nM range was applied to the sensor chip surface for 150 seconds (uniform for the same evaluation). The binding rate was measured. The data was analyzed using Biacore analysis software (Biacore T200 evaluation software , version 3.0) were used for the analysis.

[0329] The binding rate of each antibody was measured by binding of the antigen protein 4R2N (hpTau) to the sensor chip. The binding rate of the non-phosphorylated protein 4R0N to the sensor chip as a negative control was calculated from the binding rate. The binding rate of each antigen peptide was corrected by subtracting the degree of binding of the antigen peptide 1 × P The binding rates of the non-phosphorylated peptide Non P as a negative control were The binding rate to the sensor chip was subtracted to correct the concentration of the sample. Within the range, to non-phosphorylated tau protein 4R0N or non-phosphorylated peptide Non P No binding of either antibody was observed.

[0330] Table 9A shows the binding of humanized antibodies to phosphorylated tau protein 4R2N (hpTau). Table 9B shows the activity of monophosphorylated tau peptide 1xP and triphosphorylated tau peptide The results show that each humanized antibody binds to 3×P. phosphorylated tau protein hpTau, monophosphorylated tau peptide 1xP, and triphosphorylated tau These results show that the peptide 3xP showed high binding activity to all of the peptides. Table 9A: Binding activity of humanized antibodies against phosphorylated tau protein (hpTau) (1:1 Bonding model) [Table 14] Table 9B: Binding activity of humanized antibodies to phosphorylated peptides (1xP, 3xP) (1:1 binding) Combined Model) [Table 15]

[0331] E. Example 5: Binding affinity analysis of pSer413-tau in AD patient brain homogenates The VL and VL sequences shown in Table 3A were determined using mouse antibody Ta1505 and a chimeric antibody as a reference antibody. Among the humanized antibodies having the combination of VH and VH, L15H11, L46H11, and L4 7H65 was used to detect Ser413 phosphate from clinical samples of Alzheimer's disease (AD) patients in the liquid phase. The binding affinity to purified tau protein (pSer413-tau) was evaluated.

[0332] Humanized, murine, and chimeric antibodies were purified using N-hydroxysuccinimide-LC- Biotin (NHS-LC-Biotin) (Thermo Fisher Scientific Each was biotinylated using ic KK) and then dialyzed against PBS.

[0333] Frozen hippocampal tissue (160 mg) from the brain of a patient with Braak stage V / VI AD was administered to 0.8 ml L of TBS-I (Tris-buffered saline, protease inhibitor cocktail, phosphatase inhibitor The solution was sonicated at 4°C for 3000 cycles in ice water. Centrifuge at 100,000 × g for 10 minutes, collect the supernatant, and then ultracentrifuge at 100,000 × g for 15 minutes at 4 °C. Brain homogenates were obtained using the Innotest pTau(pT181) kit (F ijirebio Inc.) was used as a tau protein phosphorylated at Ser413 (pSer413 The ELISA system used was the same as that used for the ELISA for β-tau. ONJ1) was replaced with the biotinylated antibody generated above.

[0334] Each of the biotinylated antibodies prepared above was dissolved at a concentration of 0.1 nM and diluted 1000 times. Add diluted brain homogenate to HT7 antibody-immobilized MT plate (included in the kit) The resulting samples were mixed and incubated overnight at 4°C. The next day, the plates were washed and Add HRP-conjugated streptavidin (CONJ2 in the kit) to the plate. After washing, the coloring reagent TMB was added and incubated at room temperature in the dark for 1 hour. Incubate for 30 minutes. Then, add the reaction stop solution (included in the kit as STOP solution). The reaction was stopped with 500 mM NaCl (containing 10 mM NaCl) and the absorbance was measured at a wavelength of 450 nm.

[0335] The results are shown in Figure 4. As shown in the graph in Figure 4, the humanized antibodies L15H11 and L46H1 1 and L47H65 all express Ser413 phosphorylated tautan derived from clinical samples of AD patients. It showed high binding affinity to the protein (pSer413-tau).

[0336] F. Example 6: Blood Rate Testing The five initial humanized antibody variants were compared for their pharmacokinetics in normal mice with a commercially available antibody. Herceptin (Genentech, south San Francisco, CA ) and the chimeric Ta1505 antibody. The light and heavy chains of each humanized antibody variant were are summarized in Table 10 below.

[0337] Table 10. Light and heavy chains of some early humanized antibody variants [Table 16]

[0338] Eleven-week-old male C57BL / 6J mice (Charles River Laboratories) were cultured at 10°C for 12 h. The antibody was intraperitoneally injected at a dose of 10 mg / kg into mice (Nippon Immunochemicals, Inc., Japan). Plasma was obtained from blood collected 1, 3, 8, 24, and 72 hours after dosing. was 2 animals per antibody.

[0339] Plasma antibody concentrations using sandwich ELISA with anti-human IgG polyclonal antibodies A calibration curve was plotted by serially diluting each antibody with mouse plasma. .

[0340] The plasma was diluted 1,000-fold and the antibody concentration was measured.

[0341] A 1 μg / mL solution of goat anti-human IgG (Fc) polyclonal antibody in PBS was placed in a 96-well plate. The mixture was transferred to a plate (MaxSorp (NUNC)) using a pipette and left to stand overnight at 4°C. After that, blocking was performed with 3% BSA / PBS, and the antibody was then immunolabeled with immobilized goat anti-human IgG (Fc) polyclonal antibody. Plates containing clonal antibodies were prepared. Antibodies were serially diluted using mouse plasma, and the standard Plasma and standard substances were diluted in 3% BSA / PBS and incubated with immobilized goat anti-human IgG (Fc) 50 μL / well was pipetted onto the plate containing the polyclonal antibody. The mixture was reacted at room temperature for 1.5 hours, and then the plate was washed with TBS-T (TBS, 0.05% After washing with Tween 20, alkaline phosphatase-labeled anti-human IgG (H+ L) Polyclonal antibody (Southern Biotech, Catalog No. 2087- 04) diluted 2000-fold with 3% BSA / PBS and pipetted at 50 μL per well. The plate was then washed with TBS-T and the colorimetric assay was performed. Drugs were added at 100 μL / well and the plates were incubated at room temperature for 1 hour. The absorbance was measured at 405 nm and 550 nm using a plate reader.

[0342] A calibration curve was plotted with the standard and used to calculate the plasma antibody concentration. The results are shown in Figure 5A. As shown in.

[0343] The first humanized variants tested (variants 2, 5, 6, 9, and 10) showed , which showed a rapid decrease in plasma concentration compared to the levels of Herceptin and the chimeric antibody. These early humanized antibodies were more potent than chimeric antibodies. The mutants showed shorter half-lives and poorer PK characteristics, which was attributed to their higher pI values. Further improvements were required to reach the PK of the chimeric antibody.

[0344]

[0345] Among the humanized antibodies having the combination of VL and VH shown in Table 3A, L15H11, L4 6H11 and L47H65 were subjected to a blood kinetics test. The reference antibodies used were or produced by recombinant expression based on amino acid sequence information disclosed in database information. The chimeric antibody Ta1505 and known humanized antibodies used in clinical trials for AD treatment were Specifically, the international ImMunoGeneTics in formation system (registered trademark) (IMGT) (http: / / www. imgt.org) database for solanezumab (anti-A antibody, Eli Lilly and Company); IPN007 (anti-tau N-terminal antibody, Bris Toll-Myers Squibb iPierian has issued WO2014 / 2009 21A1; and MAb3221 (Anti-tau-pS422 antibody, Roche Diagnostics KK) Uses the amino acid sequence of VH32VL21 disclosed in O2015 / 091656A1 did.

[0346] Each antibody was incubated with 11-week-old male C57BL / 6J mice (Charles River The animals were then intraperitoneally administered 10 mg / kg of Blood samples were collected at 1, 3, 8, and 24 hours after administration, as well as on the 3rd and 7th days. and plasma was collected. Each antibody was tested in triplicate (N=3).

[0347] The antibody concentration in plasma was measured by sandwich ELISA using anti-human polyclonal antibodies. Specifically, 1 μL of the mixture was placed in a 96-well plate (Max Sorp (NUNC)). Goat anti-human IgG (Fc) polyclonal antibody (Southern Biotech) at 100 μg / mL The cells were then fixed overnight at 4°C in a PBS solution containing 3% BSA-PBS, and blocked with 3% BSA-PBS. This resulted in the production of a goat anti-human IgG (Fc) polyclonal antibody-immobilized plate. did.

[0348] Separately, a series of standard antibody samples of known concentrations were collected from mice that did not receive the antibody. Each antibody was serially diluted with the collected plasma and measured to prepare a standard curve.

[0349] The plasma sample to be measured and the standard sample were each diluted 10,000 times and then probed with goat anti-human IgG. (Fc) Add 50 μL / well to polyclonal antibody-immobilized plate and place at room temperature for 1 hour The reaction was allowed to proceed for 30 minutes, after which the plate was washed with TBS-T (Tris-buffered saline, 0.05% Twe Then, the cells were washed with alkaline phosphatase diluted 2000 times with 3% BSA-PBS. Sphatase-labeled anti-human IgG (H+L) polyclonal antibody (Southern Biosciences, A solution of otech) was added to the plate at 50 μL / well and reacted at room temperature for 1 hour. After washing with TBS-T, add 100 μL of color-developing solution (1 mg / mL pNPP solution) per well. The plate was then incubated at room temperature for 1 hour. The absorbance was measured at a measurement wavelength of 405 nm and a reference wavelength of 550 nm. A calibration curve was prepared based on the results, and the antibody concentration in each plasma sample was calculated using this calibration curve. .

[0350] FIG. 5B is a graph showing the change in plasma antibody concentration over time. The humanized antibodies L15H11 (LC SEQ ID NO: 32, HC SEQ ID NO: 18), L46H11 ( L47H65 (LC SEQ ID NO: 38, HC SEQ ID NO: 18) and L47H65 (LC SEQ ID NO: 38, HC SEQ ID NO: 18). Row No. 30) (All human IgG1 (SEQ ID NO: 135) and human κ (SEQ ID NO: 79) ) showed essentially the same blood kinetics as the chimeric antibody Ta1505. The blood kinetics of 15H11, L46H11, and L47H65 were compared with those of the known humanized antibody IPN0. The blood kinetics of MAb07 and MAb3221 were comparable to those of the known humanized antibody solanezumab. was significantly better than

[0351] G. Example 7: Analysis of brain transport A study was conducted to analyze brain concentrations of humanized variants with improved plasma pharmacokinetics.

[0352] One week after injection of the antibody, the parent mouse antibody, the chimeric antibody, or the humanized antibody variant (L1 5H11, L36H11, L46H11, L48H12, L48H47, L48H64, Blood was collected from mice injected with either 100 or 150 μg / mL of 100% ... The rats were anesthetized with a triple anesthetic cocktail, the abdomen was opened, and blood was exsanguinated via the abdominal aorta. Brain tissue was collected. The collected brain tissue was divided into left and right hemispheres and placed in a dry ice / ethanol bath. The hemispheres were frozen at 4°C and stored at -80°C. Each frozen hemisphere was weighed and transferred to a 2 mL tube. Next, add 0.8 mL of TBS-I (Tris-buffered saline, protease inhibitor cocktail and Phosphatase inhibitor cocktail) was added and the mixture was sonicated in ice water. The mixture was centrifuged at 3000×g for 10 minutes at 4° C., and the supernatant was collected. Brain homogenates were obtained by centrifugation at 00,000×g and 4° C. for 15 minutes.

[0353] A 10 μg / mL solution of goat anti-human IgG (Fc) polyclonal antibody in PBS was added to 96 wells. The mixture was transferred to a well plate (MaxSorp (NUNC)) using a pipette and allowed to stand at 4°C overnight. Afterwards, blocking was performed with 3% BSA / PBS, and the antibody was incubated with immobilized goat anti-human IgG (Fc) polypeptide. Plates containing reclonal antibodies were prepared. The antibodies were serially diluted using homogenates to obtain standards.

[0354] Brain homogenates and standards were diluted 10-fold and immunolabeled with immobilized goat anti-human IgG (Fc) polyclonal antibody. 50 μL / well was pipetted into the plate containing the clonal antibody. The mixture was incubated at room temperature. The plate was then incubated with TBS-T (TBS, 0.025% Tween 20) for 2 hours. ) and then washed with alkaline phosphatase-labeled anti-human IgG (H+L) polyclonal antibody. The antibody (Sigma, Cat. No. SAB3701337-1MG) was diluted in 3% BSA / PB A solution diluted 2000-fold with S was pipetted at 50 μL / well, and the reaction was incubated at room temperature for 1 h. The plate was then washed with TBS-T and incubated with the color reagent pNPP (Sigma) Add 100 μL / well of 100 mM NaCl, Catalog No. P7998, and incubate the plate at room temperature for 1 hour. The absorbance was then measured at 405 nm and 550 nm using a plate reader. Measured.

[0355] A calibration curve was plotted using the standard substance and used to measure the antibody concentration in the brain homogenate. The antibody concentration per brain weight (total brain volume) was determined, and the ratio to the plasma concentration was calculated. .

[0356] FIG. 6A shows humanized antibody variants with good pharmacokinetics (e.g., L15H according to FIG. 5B). 11, L46H11, and L47H65) were increased in the brain.

[0357] Chimeric antibody Ta1505 and known humanized antibody solutions used in clinical trials for AD treatment Ranezumab, IPN007, and MAb3221 were used as reference antibodies to compare the humanized antibody L The brain transfer of each of 15H11, L46H11, and L47H65 was analyzed.

[0358] One week after administration of each antibody in Example 6, blood was collected from the mice, and then the mice were The animals underwent laparotomy under anesthesia with a mixture of three anesthetic drugs. The collected brain tissue was divided into left and right hemispheres, and then incubated with dry ice and ethanol for 1 h. The hemispheres were then frozen in ice cubes and stored at -80°C. Each frozen hemisphere was weighed and diluted with 2 mL of Transfer the mixture to a tube and add 0.8 mL of TBS-I (Tris-buffered saline, protease inhibitor). Each hemisphere was then subcutaneously incubated in ice water for 1 h. The sonicated solution was centrifuged at 3000×g for 10 minutes at 4°C, and the supernatant was collected. The mixture was then ultracentrifuged at 100,000×g for 15 minutes at 4° C. to obtain a brain homogenate.

[0359] Antibody levels in brain homogenates were measured by antigen ELISA using anti-human polyclonal antibodies. Specifically, PBS containing 10 μg / mL goat anti-human IgG Fc was used. The solution was added to a 96-well plate (Max Sorp (NUNC)) and fixed overnight at 4°C. After immobilization, the plate was blocked with 3% BSA-PBS to prepare an immobilized plate. Ta.

[0360] Separately, a series of antibody standards of known concentrations were prepared from mice that had not been treated with antibody. Each antibody was serially diluted with the collected brain homogenate and used to generate a standard curve. was measured.

[0361] Brain homogenate and the standard sample to be measured were each mixed in 0.1% skim milk-3% BSA- Dilute 10-fold with PBS, add 50 μL / well to PD17(P)-immobilized plate, and incubate at room temperature. The plate was then incubated with TBS-T (TBS, 0.05% Tween 20) for 2 hours. Then, the plate was washed with 0.1% skim milk-3% BSA-PBS at 2000 times dilution. Alkaline phosphatase-labeled anti-human IgG (H+L) polyclonal antibody (Sigma -Aldrich Co. LLC., Catalog No. SAB3701337: 1 mg) The solution was added to the plate at 50 μL / well and reacted at room temperature for 1 hour. After washing with -T, pNPP (Sigma-Aldrich Co. LLC, catalogue) was added. No. P7998: 100 mL) was added as a colorimetric substrate at 100 μL / well, followed by addition to the chamber. The plate was then incubated at room temperature for 1 hour at 405 nm. The absorbance was measured at 500 nm and 550 nm as a reference wavelength. The antibody level in each brain homogenate sample was calculated using the standard curve. It was rated as Bell.

[0362] Furthermore, the antibody level in plasma was measured in the same manner as in Example 6 using blood collected before laparotomy. The ratio of the antibody level in the brain to the antibody level in the plasma was calculated.

[0363] FIG. 6B shows the ratio of the levels of each antibody in the brain to the levels of the corresponding antibody in plasma. Graph showing plasma antibody titers of humanized antibodies L15H11, L46H11, and L47H65. The ratio of antibody concentration in the brain to the total body concentration was measured for chimeric antibody Ta1505 and the known humanized antibody I. The antibody concentration ratios were higher than those of PN007, MAb3221, and solanezumab. The results indicate that these humanized antibodies have high brain penetration.

[0364] Example 8: Further development of selective humanized antibodies Further mutations were introduced into the CDR1 sequence of VL46 to remove the deamidation hotspot. and / or the CDR sequences were reverted back to the parent mouse sequence. This is a summary of the mutations introduced. Table 11. Mutations introduced into VL46 CDR1 [Table 17]

[0365] Furthermore, in a newly generated humanized antibody variant, IgG carrying the S228P mutation Different IgG isotypes were tested, including IgG1 and IgG4. Table 12 shows the parental murine antibodies and The following summarizes the light and heavy chains of the newly generated humanized antibody variants, along with the chimeric and humanized antibodies. That is why. Table 12. Specific antibody light and heavy chains [Table 18]

[0366] H. Example 9: Biacore™ Binding Analysis of Newly Generated Humanized Antibody Variants The binding affinity of the newly generated humanized antibody variants was analyzed using the Biacore™ T200 and 4000 biosensors (GE Healthcare, Chicago, IL). The following running buffer was used: 10 mM HEPES (GE Healthcare) hcare, BR100671), 150mM NaCl (GE Healthcare , BR100671), 0.05%v / v surfactant P20 (GE Healthcar e, BR100671), 2mM DTT (Sigma, 10708984001, St Louis, MO), and 1 mM EDTA (GE Healthcare, 2899 5043) was used for fixation, sample dilution, and data collection unless otherwise noted below. .

[0367] The immobilized protein is recombinant human phosphorylated tau protein immobilized on the chip. The immobilized 1×P peptide refers to the phosphorylated peptide (TS) immobilized on the chip. PRHLSNVS(pS)TGSIDMVDSPC, SEQ ID NO: 75). Both methods have a binding activity component for affinity measurement. Antibody captured on the slide and 4× phosphorylated peptide (GAEIVYK(pS)P) in solution VVSGDT(pS)PRHLSNVS(pS)TGSIDMVD(pS)PQLATL ADEVSASLAKQGL, SEQ ID NO:78).

[0368] Binding of antibodies to immobilized phosphorylated tau protein was measured using a Series S SensorChip NT A (GE Healthcare, BR100034) was used for immobilization. The running buffer was 10 mM HEPES, 150 mM NaCl2, 0.05% v / v Surfactant P20, pH 7.4 (GE Healthcare, BR100671) The flow rate was 10 μL / min. 350 mM EDTA was injected for 1 min to stimulate the chip. Washing was followed by addition of 0.5 mM NiCl2 (GE Healthcare, NTA Reagent Kit). The chip was prepared for capturing his-tagged proteins by injecting 100 mM NaCl (pH 7.0) for 2 min. An amine coupling kit (GE Healthcare, BR100633) was used. Activate the chip according to the manufacturer's instructions with 500 μl of phosphate-phosporylated tau protein ( 80AWB) was injected until the desired amount of protein was immobilized. The chip was blocked with ethanolamine from the kit. Each experiment consisted of multiple levels of immobilized phosphorylation. Oxidized tau protein was used. The lowest level was 54 resonance units (RU) ~ 452 The range of RU varied, with the highest levels ranging from 463 RU to 1020 RU. To measure the affinity of the Ta1505 antibody binding to immobilized phosphorylated tau protein Prepare a five-point, 3-fold dilution series of each antibody, resulting in concentrations ranging from 0.37 nM to 30 nM. Each concentration and several buffer blanks were injected for 3 min at a flow rate of 45 μl / min. The dissociation of the β-aminobutyric acid was monitored for 15 min, and then 100 mM CAPS (Sigma, C6070 ), 1M KCl (Sigma, P9541), 1mM EDTA, 2mM DTT, p The surface was regenerated by injecting H10.5 for 30 seconds to 1 minute.

[0369] Antibody binding to the immobilized phosphorylated tau peptide was measured using a Series S sensor chip CM3 ( GE Healthcare, BR100536) was used for the measurement. The chip was activated using a binding kit and then incubated in 10 mM sodium acetate, pH 5.0. 30 μg / mL phosphorylated tau peptide (SEQ ID NO: 75) was added until 51 RU was immobilized. The chip was blocked with ethanolamine. PRHLSNVSSTGSIDMVDSPC, SEQ ID NO: 77) was used as a negative control reference. The surfaces were prepared similarly. Affinity of Ta1505 antibody binding to immobilized phosphorylated tau peptide To measure the potency of each antibody, a 5-point set of three antibodies was used, yielding concentrations from 0.37 nM to 30 nM. A 2-fold dilution series was prepared. Each concentration and several buffer blanks were diluted at a flow rate of 45 μL / min for 3 min. Dissociation of the antibody was monitored for 15 min. 100 mM hydrochloric acid (Fisher S Scientific SA56-1, Waltham, MA) for 30 seconds. The surface was regenerated.

[0370] Binding of phosphorylated tau peptides to immobilized antibodies was measured using a Series S sensor chip CM5 (G Measurements were performed using a amine coupling assay (E Healthcare, 29149603). The chip was activated using the kit and then incubated with 1 mM NaCl in 10 mM sodium acetate, pH 5.0. Antibody (GE Healthcare, BR100351) at 3 μg / ml was added to 280 9100RU of antibody was injected until immobilization. The chip was blocked with ethanolamine. Affinity of phosphorylated tau peptide (SEQ ID NO: 78) binding to immobilized Ta1505 antibody A six-point set of peptides was prepared to measure the activity of the peptides, with concentrations ranging from 5.1 nM to 500 nM. A 2.5-fold dilution series was prepared. Samples and several buffer blanks were diluted 3 times at 30-50 μL / min. The solution was injected for 1 min and dissociation was monitored for 15 min. The pH was adjusted to 3.0 with HCl or without adjustment. 5 and regenerate the surface by injecting 20 mM sodium acetate, pH 4.5 for 30 seconds. Ta.

[0371] Biacore™ T200 evaluation software version 2.0 or Biacore re(TM) 4000 Evaluation Software Version 1.1 (GE Healthcare The data was processed and fitted using the FTIR filter. The response was subtracted from the negative control flow cell. Subtract the response from the buffer injection or subtract the response from the results of two buffer injections. The data were "double referenced" by subtracting the mean. The data were then "1:1 matched". The association rate constant k a (M -1 s -1 where "M" is equal to the molar concentration where "s" is equal to seconds) and the dissociation rate constant k d (s -1 ) were determined. Using the constant, the equilibrium dissociation constant, K D (M)=k d / k a Table 13 shows the newly generated K of humanized antibody variants D The K values ​​were calculated for the parent mouse and chimeric antibodies. D Compared with the value This is what was stopped. Table 13. K of antibodies against phosphorylated tau protein or peptides D (nM) Value [Table 19]

[0372] I. Example 10: EL of parent mouse antibody, chimeric antibody, and several humanized antibody variants ISA binding analysis Phosphopeptides of parental murine antibodies, chimeric antibodies, and newly generated humanized antibody variants pS)TGSIDMVD (PRHLSNVS(pS)TGSIDMVD, SEQ ID NO: 79) and the corresponding non-phosphoryl Binding to the oxidized peptide (PRHLSNVSTGSIDMVD, SEQ ID NO: 80) was measured by ELISA. Analyzed by A.

[0373] Add 50 μL of 1 μg / ml phosphorylated or non-phosphorylated peptide in PBS to the ELISA plate. The plate was incubated overnight at 4°C. The plate was washed three times and blocked with 200 μL of superblock overnight at 4°C. On the day, plates were washed 3 times. Then, serial dilutions were added starting at 10 μg / mL, 1:3. Add 50 μL of antibody in diluted ELISA buffer to each well of the plate and incubate at room temperature for 1 hour. The plate was incubated and then washed three times. For the measurement of parental mouse antibodies, a dilution of 1:3000 was used. 50 μL of diluted goat anti-mouse HRP (Southern Biotech, 1030- 05) was added to the plate. For the measurement of chimeric and humanized antibodies, a 1:3000 dilution was used. 50 μL of goat anti-human HRP (Jackson Immunologics, 109-0 The ELISA plate was incubated at room temperature for 45 minutes and then incubated for 3 The plate was then washed twice and developed with ABTS at room temperature for 5 minutes. The absorbance at 405 nm was measured.

[0374] 8A and 8B show that the parent antibody produced by hybridoma or recombinant methods is phosphorylated. The IgG bound to the phosphorylated peptide (Figure 8A) but not to the non-phosphorylated peptide (Figure 8B). Similarly, chimeric antibodies (having either an IgG1 or IgG4 backbone) , bound to the phosphorylated peptide (Fig. 8C), but not to the non-phosphorylated peptide ( Figure 8D).

[0375] 9A-9C show that most of the newly generated humanized antibody variants were chimeric IgG4 antibodies. The phosphopeptide was bound with an affinity comparable to that of the human phospholipid, whereas some mutants were In particular, in Figure 9B, the amino acid N3 Human VL46 / VH11_N33D_IgG4 mimicking complete 3 to D deamidation The mutants significantly reduced binding to phosphorylated peptides, whereas the fully deamidated antibody This suggests that the binding affinity to phosphorylated tau protein would be dramatically lost. Therefore, reducing deamidation at N33 in humanized antibody variants is essential.

[0376] XV. Example 11: De novo generated human IgG in brain homogenates of Alzheimer's disease patients Antigen-binding analysis of engineered antibody mutants The parent mouse antibody, the chimeric antibody, and the selected humanized antibody variants were assayed for Alzheimer's disease in liquid phase. pSer413 phosphorylated tau protein (pSer413) from clinical samples of Alzheimer's disease (AD) patients -tau) were evaluated for their binding ability.

[0377] As shown in Figures 10A-10E, increasing concentrations of control human IgG (S Ink the AD brain samples with igma (Cat. No. I2511) and the selected antibody variants. After incubation, the total number of antibodies was detected using mouse biotinylated Ta1505 IgG2a antibody. The antibody occupancy on pSer413-tau was determined by the difference between antigen-bound and free antigen-bound. Ta.

[0378] Detection with the same mouse biotinylated Ta1505 IgG2a antibody confirmed the antibody variants Direct comparison of binding titers between N-hydroxysuccinimide-LC-biotin is possible. (NHS-LC-biotin) (Thermo Fisher Scientific K The mouse Ta1505 IgG2a antibody was biotinylated using 100 mM NaCl (K) and then incubated with PBS. Dialysis was performed.

[0379] Frozen prefrontal cortex tissue (100 mg) from the brain of an AD patient was analyzed by protease and phosphatase. Transcription inhibitor cocktail (Thermo Fisher Scientific, Cat. 1 mL of TBS-I (Tris-buffered saline, Catalog No. 1861281) No. BP2471-1, Thermo Fisher Scientific) The tissue was lysed on ice using the Qiagen Tissue Lyzer II. The lysed samples were then centrifuged in a Beckman Coulter Optima Max-XP ultracentrifuge. Centrifuge at 27,000 g (TLA-55 rotor) for 20 minutes at 4°C and collect the supernatant. and then centrifugation in a Beckman Coulter Optima Max-XP ultracentrifuge. Centrifuge at 150,000 × g (TLA-55 rotor) for 20 minutes at 4 °C to obtain the P2 fraction. The P2 fraction was homogenized by sonication and resuspended in TBS-I buffer to obtain a pellet called The protein concentration of the P2 fraction was determined using the Pierce™ BCA protein assay. Kit (Thermo Fisher Scientific, catalog number 23225 ) according to the manufacturer's instructions. Protein concentration was adjusted to 4 μg / mL. .

[0380] INNOTEST® pTau (pT181) Kit (Fijirebio (Inc., Cat. No. 81581) was used as a phospho-Ser413 tau protein (pSe r413-tau) was used as an ELISA system, but the biotin included in the kit The biotinylated antibody (designated CONJ1) was synthesized using the biotinylated mouse Ta1505 Ig It was replaced with G2a antibody.

[0381] Parental mouse antibodies, chimeric antibodies, humanized antibody variants, or control human IgG were incubated at 4°C for 1 h in a buffer containing 0.5% ethanol at 100°C for 2 h. 200 ml of stock in buffer (provided by INNOTEST® kit) The antibody solution was then serially diluted and pre-diluted at 1 / 1000 in the assay plate. The antibody-antigen mixture was then incubated with the diluted AD brain P2 fraction for 4 hours at room temperature. The mixture was diluted with biotinylated antibody mouse Ta1505 IgG2a (INNOTEST HT7 antibody with CONJ DIL1 (1 / 10 in CONJ DIL1 provided in the kit) The resulting samples were mixed and incubated at 4°C overnight. The next day, the plate was washed and incubated with HRP-labeled streptavidin (CON J2 (included in the kit) was added to the plate followed by incubation for 1 hour. After washing, the color-developing reagent TMB was added and incubated at room temperature for 30 minutes in the dark. The reaction was stopped using a stop solution (contained in the kit as STOP solution) and incubated for 450 n The absorbance was measured at a wavelength of 1000 nm.

[0382] Nonlinear analysis of the concentration-response curves shown in Figures 10A-10E reveals that the TA The concentration of each antibody required for 50% occupancy of pSer413 and the ratio of the maximum occupancy of each antibody 10A to 10E show the parental markers in brain homogenates of AD patients. In vitro characterization of murine antibodies, chimeric antibodies, and selected humanized antibody variants The binding characteristics, such as occupancy levels and maximum binding of pSer413 tau antigen, are comparable Shows.

[0383] XVI. Example 12: Stability and Purity Analysis of Newly Generated Humanized Antibody Variants By measuring the melting temperature (Tml) and aggregation temperature (Tagg) via nano-DSF The purity of each antibody was determined by SEC and non-reducing capillary column chromatography. Measured by SDS (NR-cSDS).

[0384] Tm and Tagg determination: PR.ThermControl v2.0.4 software A Prometheus NT.48 differential scanning fluorometer (Nano Using NanoDSF, Tm and Ta The excitation power was 40% and the temperature was ramped from 20 to 95 °C at a rate of 1 °C / min. The Tm and Tagg were measured automatically. The samples were diluted with 20 mM sodium acetate, p Prepare the solution by diluting it to 1 mg / mL in H5.5 buffer and allow it to wick by capillary action. The sample was then drawn into a Rometheus glass capillary tube (PR-L002).

[0385] Purity determination by SEC: S on an ACQUITY UPLC H-Class System EC was performed using a Waters (Milford, MA) ACQU column. ITY UPLC Protein BEH SEC Column (Part Number 18600522 The column temperature was 25°C. 10 μL of 1 mg / mL sample was injected at a system flow rate of 0.5 mL / min. The mobile phase was 00 mM sodium phosphate, 200 mM sodium chloride, and 0.02% sodium azide. The data were quantified at both 214 and 280 nm and Analysis was performed using BEH r3 software from Waters (Milford, MA). 10 μg of 200 SEC Protein Standard Mixture (Part No. 186006518) was injected USP resolution, theoretical plates, and tailing were measured.

[0386] Determination of purity by NR-cSDS: For the evaluation of purity by NR-cSDS, 1 mg Add 5 μL of each sample at 100 / mL to loading buffer containing 50 mM iodoacetamide. (HT Protein Express Sample Buffer(Perkin Elmer)) in a 96-well plate. The plate was incubated at 70°C for 20 minutes. After incubation for 1 h, 75 μL of water was added to each well. n Express Chip (Perkin Elmer) was used to create the LabChip The fluorescence of the samples was measured over time on a GXII system (Perkin Elmer). Electropherograms were collected by measuring the concentration of The integration was performed using version 4.1.1619.0 SP1 (Perkin Elmer).

[0387] Table 14 summarizes the stability and purity of the antibodies tested. Table 14. Stability and purity of various antibodies [Table 20]

[0388] All newly generated humanized antibody variants are safer than the parent mouse or chimeric antibodies. The quality and purity were maintained or improved. / VH65_IgG1 is stable as measured by Tml / Tagg The decline in sexuality was evident.

[0389] XVII. Example 13: Deamidation Analysis of Newly Generated Humanized Antibody Variants The deamidation level of amino acid N33 in the light chain CDR1 of various antibodies was measured. Stress conditions such as incubation at pH 10 or 4°C were tested as a control. Incubation was carried out at .

[0390] Incubation at 4°C and 50°C: Prepared in 20 mM sodium acetate, pH 5.5 The combined 2mg / ml samples were kept in a temperature-controlled stability chamber at 50°C for one week. For the comparative 4°C control, samples were kept at 4°C.

[0391] Incubation at pH 10: Formulated in 20 mM sodium acetate, pH 5.5 A 2 mg / mL sample was prepared by adjusting the pH to 10 with 0.5 M NaOH and storing in a temperature-controlled flask. The mixture was then stored in a constant temperature chamber at 25°C for 1 week. The sample was diluted with 2 mL of 1000 K MWCO, Thermo Fisher 89890. The mixture was buffer exchanged into 0 mM sodium acetate, pH 5.5 buffer.

[0392] Deamidation Analysis by Peptide Mapping: For Peptide Mapping by Mass Spectrometry 100 μg of each sample was added to 30 μL of 8 M guanidine / 1 M Tris hydrochloride solution (15:1 ), reduced with 2 μL of 1M DTT at 60°C for 30 min, and then diluted with 1M iodoacetate. The 7-kilodalton fragment was alkylated with 5 μL of 5-aminopropyl ether for 45 minutes in the dark. Samples were buffered in 50 mM ammonium bicarbonate using a molecular weight cutoff ZEBA cartridge. The samples were divided into different tubes and incubated with 2 μg of trypsin and chymotrypsin. The samples were digested in parallel at 37°C for 2 hours by adding 3 μL of 5M hydrochloride to each sample. The digestion was stopped by adding 1x50mm Peptide BEH-C18 at 40°C. Inject 2 μL of sample onto a Waters UPLC column (part number 186005592). The Dionex / QE plus MS was used to measure 2% to 36% amine in 0.1% formic acid. Data were acquired using a linear gradient of acetonitrile over 50 min. Database Search For more information, see PEAKS DB (Bioinformatics Solutions I nc.), and PepFinder (Thermo The samples were analyzed using the Fisher Scientific (Fisher Scientific) and manual verification. , which summarizes the percentage of deamidation of amino acid N33 in the light chain CDR1 of the tested antibodies. be. Table 15. Deamidation rate (%) of N33 in light chain CDR1 under various conditions [Table 21]

[0393] Parent mouse antibody and the original humanized variant (VL46 / VH11) IgG1 or IgG4 antibody Compared to the original humanized antibody variant, the newly generated humanized antibody variant has a deamidated N33 in the light chain CDR1. The results showed a significant decrease in the cytochrome P450 / 450 / 50 protein levels. The present application provides the following aspects of the invention. (Aspect 1) a) a heavy chain variable domain selected from SEQ ID NO:116 and SEQ ID NO:117; and b) SEQ ID NO: 114, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 1 07, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:1 12, an anti-p antibody comprising a light chain variable domain selected from SEQ ID NO: 113 and SEQ ID NO: 103. Ser413 tau antibody. (Aspect 2) a) a heavy chain variable domain selected from SEQ ID NO:116 and SEQ ID NO:117; and b) SEQ ID NO: 114, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 1 07, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:1 12, an anti-pS antibody comprising a light chain variable domain selected from SEQ ID NO: 113 and SEQ ID NO: 103 er413 tau antibody, the binding affinity of said antibody to the phosphorylated peptide of SEQ ID NO:8 to the binding affinity of said antibody to the non-phosphorylated peptide of SEQ ID NO: 69 is at least The anti-pSer413 tau antibody, (Aspect 3) The antibody is selected from the group consisting of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, From sequence number 139, sequence number 140, sequence number 141, sequence number 142 and sequence number 144 3. The anti-pSer413 tau antibody according to embodiment 1 or 2, comprising a heavy chain constant domain selected from . (Aspect 4) the antibody comprises a light chain constant domain selected from SEQ ID NO: 79 and SEQ ID NO: 80; An anti-pSer413 tau antibody according to any one of aspects 1 to 3. (Aspect 5) The variable light domain has SEQ ID NO: 114 and the variable heavy domain has SEQ ID NO: 11 6. The anti-pSer413 tau antibody according to any one of aspects 1 to 4, (Aspect 6) the light chain of the antibody has SEQ ID NO: 184 and the heavy chain of the antibody has SEQ ID NO: 144; An anti-pSer413 tau antibody according to embodiment 5. (Aspect 7) Aspect 5, wherein the light chain has SEQ ID NO: 184 and the heavy chain of the antibody has SEQ ID NO: 152. The anti-pSer413 tau antibody described in (Aspect 8) The heavy and light chains are LC SEQ ID NO: 184 and HC SEQ ID NO: 144; 184 and HC SEQ ID NO:145; LC SEQ ID NO:184 and HC SEQ ID NO:146; LC SEQ ID NO: No. 184 and HC SEQ ID NO: 147; LC SEQ ID NO: 184 and HC SEQ ID NO: 148; LC SEQ ID NO:184 and HC SEQ ID NO:149; LC SEQ ID NO:184 and HC SEQ ID NO:150; LC SEQ ID NO:184 and HC SEQ ID NO:151; LC SEQ ID NO:184 and HC SEQ ID NO:15 2; LC SEQ ID NO: 184 and HC SEQ ID NO: 153; LC SEQ ID NO: 184 and HC SEQ ID NO: 154; LC SEQ ID NO: 184 and HC SEQ ID NO: 155; LC SEQ ID NO: 184 and HC SEQ ID NO: No. 156; LC SEQ ID NO: 184 and HC SEQ ID NO: 157; LC SEQ ID NO: 184 and HC SEQ ID NO:158; LC SEQ ID NO:184 and HC SEQ ID NO:159; LC SEQ ID NO:184 and HC SEQ ID NO:160; LC SEQ ID NO:184 and HC SEQ ID NO:161; LC SEQ ID NO:185 and HC SEQ ID NO:144; LC SEQ ID NO:185 and HC SEQ ID NO:145; LC SEQ ID NO:1 85 and HC SEQ ID NO: 146; LC SEQ ID NO: 185 and HC SEQ ID NO: 147; LC SEQ ID NO: No. 185 and HC SEQ ID NO: 148; LC SEQ ID NO: 185 and HC SEQ ID NO: 149; LC SEQ ID NO: Column number 185 and HC sequence number 150; LC sequence number 185 and HC sequence number 151; L C SEQ ID NO: 185 and HC SEQ ID NO: 152; LC SEQ ID NO: 185 and HC SEQ ID NO: 153 LC SEQ ID NO:185 and HC SEQ ID NO:154; LC SEQ ID NO:185 and HC SEQ ID NO:1 55; LC SEQ ID NO: 185 and HC SEQ ID NO: 156; LC SEQ ID NO: 185 and HC SEQ ID NO: No. 157; LC SEQ ID NO: 185 and HC SEQ ID NO: 158; LC SEQ ID NO: 185 and HC SEQ ID NO: Row number 159; LC sequence number 185 and HC sequence number 160, and LC sequence number 18 8. The antibody according to any one of claims 1 to 7, wherein the antibody is selected from the pair of HC SEQ ID NO: 161 and HC SEQ ID NO: 5. pSer413 tau antibody. (Aspect 9) a) SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 1 66, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:1 71, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:1 76, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:1 81, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, and SEQ ID NO: 185. a first nucleic acid encoding a light chain to be expressed; and b) SEQ ID NO:144, SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:147; SEQ ID NO:1 48; SEQ ID NO:149; SEQ ID NO:150; SEQ ID NO:151; SEQ ID NO:152; SEQ ID NO:1 53; SEQ ID NO:154; SEQ ID NO:155; SEQ ID NO:156; SEQ ID NO:157; SEQ ID NO:1 58; SEQ ID NO: 159; SEQ ID NO: 160 and SEQ ID NO: 161 A nucleic acid composition comprising a second nucleic acid that binds to the nucleic acid. (Aspect 10) The first nucleic acid is contained in a first expression vector and the second nucleic acid is contained in a second expression vector. 10. An expression vector composition comprising the nucleic acid composition of embodiment 9, (Aspect 11) 10. The nucleic acid combination according to embodiment 9, wherein the first nucleic acid and the second nucleic acid are contained in an expression vector. An expression vector composition comprising the composition. (Aspect 12) 12. A host cell comprising the expression vector composition of embodiment 10 or 11. (Aspect 13) Culturing the host cell of embodiment 12 under conditions to express the antibody and to recover the antibody. A method for producing an anti-pSer413 tau antibody, comprising: (Aspect 14) The anti-pSer413 tau antibody according to any one of aspects 1 to 8 is administered to a subject. The method of claim 1, further comprising administering to said subject a therapeutically effective amount of a tauopathy. (Aspect 15) a) a vhCDR1 having SEQ ID NO: 86, a vhCDR2 having SEQ ID NO: 115, and A heavy chain variable domain comprising a vhCDR3 having SEQ ID NO:88; and b) i) vlCDR1 having SEQ ID NO: 102, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; ii) vlCDR1 having SEQ ID NO: 91, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; iii) vlCDR1 having SEQ ID NO: 92, vlCDR2 having SEQ ID NO: 82, and and vlCDR3 having SEQ ID NO: 83; iv) vlCDR1 having SEQ ID NO: 93, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; v) vlCDR1 having SEQ ID NO: 94, vlCDR2 having SEQ ID NO: 82, and vlCDR3 with sequence number 83; vi) vlCDR1 having SEQ ID NO: 95, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; vii) vlCDR1 having SEQ ID NO: 96, vlCDR2 having SEQ ID NO: 82, and and vlCDR3 having SEQ ID NO: 83; viii) vlCDR1 having SEQ ID NO: 97, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; ix) vlCDR1 having SEQ ID NO: 98, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; x) vlCDR1 having SEQ ID NO: 99, vlCDR2 having SEQ ID NO: 82, and vlCDR3 with sequence number 83; xi) vlCDR1 having SEQ ID NO: 100, vlCDR2 having SEQ ID NO: 82, and and vlCDR3 having SEQ ID NO: 83; and xii) vlCDR1 having SEQ ID NO: 101, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO: 83 an anti-pSer light chain variable domain comprising a set of vlCDRs selected from the group consisting of 413 Tau antibody. (Aspect 16) a) a vhCDR1 having SEQ ID NO: 86, a vhCDR2 having SEQ ID NO: 115, and A heavy chain variable domain comprising a vhCDR3 having SEQ ID NO:88; and b) i) vlCDR1 having SEQ ID NO: 102, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; ii) vlCDR1 having SEQ ID NO: 91, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; iii) vlCDR1 having SEQ ID NO: 92, vlCDR2 having SEQ ID NO: 82, and and vlCDR3 having SEQ ID NO: 83; iv) vlCDR1 having SEQ ID NO: 93, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; v) vlCDR1 having SEQ ID NO: 94, vlCDR2 having SEQ ID NO: 82, and vlCDR3 with sequence number 83; vi) vlCDR1 having SEQ ID NO: 95, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; vii) vlCDR1 having SEQ ID NO: 96, vlCDR2 having SEQ ID NO: 82, and and vlCDR3 having SEQ ID NO: 83; viii) vlCDR1 having SEQ ID NO: 97, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; ix) vlCDR1 having SEQ ID NO: 98, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO:83; x) vlCDR1 having SEQ ID NO: 99, vlCDR2 having SEQ ID NO: 82, and vlCDR3 with sequence number 83; xi) vlCDR1 having SEQ ID NO: 100, vlCDR2 having SEQ ID NO: 82, and and vlCDR3 having SEQ ID NO: 83; and xii) vlCDR1 having SEQ ID NO: 101, vlCDR2 having SEQ ID NO: 82, and vlCDR3 having SEQ ID NO: 83 an anti-pSer light chain variable domain comprising a set of vlCDRs selected from the group consisting of 413 tau antibody, The binding affinity of the antibody to the phosphorylated peptide of SEQ ID NO: 8 is The anti-pSe antibody has a binding affinity ratio of at least about 40 for the peptide. r413 tau antibody.

Claims

1. a) a heavy chain variable domain selected from SEQ ID NO: 116 and SEQ ID NO: 117; and b) SEQ ID NO: 114, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 1 07, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:1 12, an anti-p antibody comprising a light chain variable domain selected from SEQ ID NO: 113 and SEQ ID NO: 103; Ser413 tau antibody.

2. a) a heavy chain variable domain selected from SEQ ID NO: 116 and SEQ ID NO: 117; and b) SEQ ID NO: 114, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 1 07, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:1 12. Anti-pS comprising a light chain variable domain selected from SEQ ID NO: 113 and SEQ ID NO: 103 er413 tau antibody, the binding affinity of said antibody to the phosphorylated peptide of SEQ ID NO:8 to the binding affinity of said antibody to the non-phosphorylated peptide of SEQ ID NO:69 is at least The anti-pSer413 tau antibody,

3. The antibody is selected from the group consisting of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, From sequence number 139, sequence number 140, sequence number 141, sequence number 142 and sequence number 143 The anti-pSer413 tau antibody according to claim 1 or 2, comprising a heavy chain constant domain selected from the group consisting of: body.

4. the antibody comprises a light chain constant domain selected from SEQ ID NO: 79 and SEQ ID NO: 80; The anti-pSer413 tau antibody according to any one of claims 1 to 3.

5. The light chain variable domain has SEQ ID NO: 114 and the heavy chain variable domain has SEQ ID NO: 11 6. The anti-pSer413 tau antibody of claim 1 ,

6. the light chain of said antibody has SEQ ID NO: 184 and the heavy chain of said antibody has SEQ ID NO: 144; The anti-pSer413 tau antibody described in claim 5.

7. the light chain of said antibody has SEQ ID NO: 184 and the heavy chain of said antibody has SEQ ID NO: 152; The anti-pSer413 tau antibody described in claim 5.

8. The heavy and light chains are LC SEQ ID NO: 184 and HC SEQ ID NO: 144; 184 and HC SEQ ID NO: 145; LC SEQ ID NO: 184 and HC SEQ ID NO: 146; LC SEQ ID NO: No. 184 and HC SEQ ID NO: 147; LC SEQ ID NO: 184 and HC SEQ ID NO: 148; LC SEQ ID NO:184 and HC SEQ ID NO:149; LC SEQ ID NO:184 and HC SEQ ID NO:150; LC SEQ ID NO: 184 and HC SEQ ID NO: 151; LC SEQ ID NO: 184 and HC SEQ ID NO: 15 2; LC SEQ ID NO: 184 and HC SEQ ID NO: 153; LC SEQ ID NO: 184 and HC SEQ ID NO: 154; LC SEQ ID NO: 184 and HC SEQ ID NO: 155; LC SEQ ID NO: 184 and HC SEQ ID NO: No. 156; LC SEQ ID NO: 184 and HC SEQ ID NO: 157; LC SEQ ID NO: 184 and HC SEQ ID NO:158; LC SEQ ID NO:184 and HC SEQ ID NO:159; LC SEQ ID NO:184 and HC SEQ ID NO:160; LC SEQ ID NO:184 and HC SEQ ID NO:161; LC SEQ ID NO:185 and HC SEQ ID NO:144; LC SEQ ID NO:185 and HC SEQ ID NO:145; LC SEQ ID NO:1 85 and HC SEQ ID NO: 146; LC SEQ ID NO: 185 and HC SEQ ID NO: 147; LC SEQ ID NO: No. 185 and HC SEQ ID NO: 148; LC SEQ ID NO: 185 and HC SEQ ID NO: 149; LC SEQ ID NO: LC SEQ ID NO: 185 and HC SEQ ID NO: 150; LC SEQ ID NO: 185 and HC SEQ ID NO: 151; L C SEQ ID NO: 185 and HC SEQ ID NO: 152; LC SEQ ID NO: 185 and HC SEQ ID NO: 153 LC SEQ ID NO:185 and HC SEQ ID NO:154; LC SEQ ID NO:185 and HC SEQ ID NO:1 55; LC SEQ ID NO: 185 and HC SEQ ID NO: 156; LC SEQ ID NO: 185 and HC SEQ ID NO: No. 157; LC SEQ ID NO: 185 and HC SEQ ID NO: 158; LC SEQ ID NO: 185 and HC SEQ ID NO: Row number 159; LC SEQ ID NO: 185 and HC SEQ ID NO: 160, and LC SEQ ID NO: 18 5 and HC SEQ ID NO: 161 according to any one of claims 1 to 7. Anti-pSer413 tau antibody.

9. a) SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 1 66, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:1 71, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:1 76, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:1 81, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, and SEQ ID NO:

185. a first nucleic acid encoding a light chain to be expressed; and b) SEQ ID NO:144, SEQ ID NO:145; SEQ ID NO:146; SEQ ID NO:147; SEQ ID NO:1 48; SEQ ID NO:149; SEQ ID NO:150; SEQ ID NO:151; SEQ ID NO:152; SEQ ID NO:1 53; SEQ ID NO:154; SEQ ID NO:155; SEQ ID NO:156; SEQ ID NO:157; SEQ ID NO:1 58; SEQ ID NO:159; SEQ ID NO:160 and SEQ ID NO:161 A nucleic acid composition comprising a second nucleic acid that binds to the nucleic acid.

10. The first nucleic acid is contained in a first expression vector and the second nucleic acid is contained in a second expression vector.

10. An expression vector composition comprising the nucleic acid composition of claim 9.

11. The nucleic acid of claim 9, wherein the first nucleic acid and the second nucleic acid are contained in an expression vector. An expression vector composition comprising the composition.

12. A host cell comprising the expression vector composition of claim 10 or 11.

13. Culturing the host cell of claim 12 under conditions that allow expression of the antibody; and A method for producing an anti-pSer413 tau antibody, comprising recovering the antibody.

14. A pharmaceutical composition for treating a tauopathy in a subject, comprising any one of claims 1 to 8. The pharmaceutical composition comprising the anti-pSer413 tau antibody described in any one of claims 1 to 4.

15. The tauopathy is Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pittsburgh disease, or the like. M. nephropathy, argyroglobulin dementia (argyroglobulin disease), multisystem tauopathy with dementia in early old age (M STD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP) -17) Neurofibrillary tangle dementia, diffuse neurofibrillary tangle disease with calcification (DNTC) , white matter tauopathy with globular glial inclusions (WMT-GGI), frontal glial tau pathology Temporal Lobar Degeneration (FTLD-tau), Sequelae of Encephalitis Lethargica, Subacute Sclerosing Panencephalitis, and Boxer The pharmaceutical composition according to claim 14, wherein the therapeutic agent is selected from the group consisting of rheumatoid arthritis, ... and rheumatoid arthritis.

16. A compound according to any one of claims 1 to 8 in the manufacture of a medicament for treating a tauopathy in a subject. Use of an anti-pSer413 tau antibody according to claim 1.

17. The tauopathy is Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pittsburgh disease, or the like. M. nephropathy, argyroglobulin dementia (argyroglobulin disease), multisystem tauopathy with dementia in early old age (M STD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP) -17) Neurofibrillary tangle dementia, diffuse neurofibrillary tangle disease with calcification (DNTC) , white matter tauopathy with globular glial inclusions (WMT-GGI), frontal glial tau pathology Temporal Lobar Degeneration (FTLD-tau), Sequelae of Encephalitis Lethargica, Subacute Sclerosing Panencephalitis, and Boxer The use according to claim 16, wherein the disease is selected from the group consisting of:

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