WO2025192556A1 - Anticorps se liant à des agrégats de tau et son utilisation - Google Patents

Anticorps se liant à des agrégats de tau et son utilisation

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Publication number
WO2025192556A1
WO2025192556A1 PCT/JP2025/008900 JP2025008900W WO2025192556A1 WO 2025192556 A1 WO2025192556 A1 WO 2025192556A1 JP 2025008900 W JP2025008900 W JP 2025008900W WO 2025192556 A1 WO2025192556 A1 WO 2025192556A1
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WIPO (PCT)
Prior art keywords
tau
antibody
light chain
seq
heavy chain
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English (en)
Japanese (ja)
Inventor
義行 添田
診祐 石垣
太郎 立花
江美 林
彩霞 王
奈保子 中谷
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Cell Engineering Corp
Shiga University of Medical Science NUC
Gakushuin School Corp
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Cell Engineering Corp
Shiga University of Medical Science NUC
Gakushuin School Corp
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Publication of WO2025192556A1 publication Critical patent/WO2025192556A1/fr
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Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present disclosure relates to antibodies that bind to tau aggregates and uses thereof.
  • Tau a microtubule-associated protein, is highly soluble in healthy neurons.
  • Neurofibrillary tangles (NFTs), a pathological hallmark of Alzheimer's disease (AD), are primarily composed of highly phosphorylated, insoluble aggregated forms of tau.
  • Abnormal tau also accumulates in the brains of patients with other neurodegenerative disorders known as tauopathies, such as Pick's disease, chronic traumatic encephalopathy, corticobasal degeneration, and progressive supranuclear palsy.
  • Tau aggregation correlates with dementia severity1 and neuronal loss2 . Mutations in the MAPT gene cause the hereditary neurodegenerative tauopathy frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17).
  • tau-related toxicity such as neuronal loss and behavioral abnormalities
  • tau oligomers are mediated by the formation of tau oligomers, but not by monomeric tau or NFTs.
  • tau pathology and tau-related toxicity can be induced by extracellular, intracellular, or both types of tau oligomers.
  • 18,17,19-22 Exposure to recombinant tau oligomers prepared by polymerization impairs synaptic plasticity and neurotoxicity.
  • 23-26 Extracellular tau oligomers have been observed in vesicles in the brains of patients with AD, and these vesicular tau oligomers propagated tau pathology in vivo.
  • the present disclosure provides antibodies that bind to tau aggregates (particularly granular tau oligomers) and uses thereof.
  • the 3E7 antibody obtained using an isolated short peptide consisting of amino acids 422 to 435 of the amino acid sequence of human tau set forth in SEQ ID NO: 1 as an immunogen, also specifically bound to granular tau oligomers, similar to the 2D6-2C6 antibody.
  • the following inventions are provided. (1) (i) an 8-amino acid peptide having the amino acid sequence set forth in SEQ ID NO: 2; and (ii) a peptide that binds to tau aggregates, and An antibody or antigen-binding fragment thereof that binds more strongly to tau aggregates than to tau monomers. (2) The antibody or antigen-binding fragment thereof according to (1) above, wherein the tau aggregate comprises one or more selected from the group consisting of tau fibrils and granular tau oligomers.
  • tau fibrils and/or granular tau oligomers are tau fibrils and/or granular tau oligomers having phosphorylation of threonine in tau at a position corresponding to threonine (T) 427 in the amino acid sequence set forth in SEQ ID NO: 1, or the antibody or antigen-binding fragment thereof comprises tau fibrils and/or granular tau oligomers having said phosphorylation.
  • T threonine
  • the antibody or antigen-binding fragment thereof according to any one of (1) to (3) above, which does not exhibit significant binding to tau monomers.
  • An antibody or antigen-binding fragment thereof comprising a heavy chain variable region set forth in SEQ ID NO: 3 and a light chain variable region set forth in SEQ ID NO: 4, or comprising a heavy chain variable region comprising heavy chain CDR1 to heavy chain CDR3 of the heavy chain variable region and a light chain variable region comprising light chain CDR1 to light chain CDR3 of the light chain variable region.
  • An antibody or antigen-binding fragment thereof according to any one of (1) to (4) above comprising a heavy chain variable region set forth in SEQ ID NO: 40 and a light chain variable region set forth in SEQ ID NO: 41, or comprising a heavy chain variable region comprising heavy chain CDR1 to heavy chain CDR3 of the heavy chain variable region and a light chain variable region comprising light chain CDR1 to light chain CDR3 of the light chain variable region.
  • the antibody or antigen-binding fragment thereof according to any one of (1) to (6) above which is an isolated monoclonal antibody, a human chimeric antibody, or a humanized antibody.
  • a kit for use in detecting tau aggregates comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (8) above.
  • a composition comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (8) above.
  • the antibody of the present disclosure is capable of binding to, but is not limited to, tau fibrils and/or tau oligomers. In a preferred embodiment, the antibody of the present disclosure is capable of binding to, but is not limited to, tau oligomers, and may be useful for early diagnosis of Alzheimer's disease (AD) or for diagnosing the preclinical stage.
  • AD Alzheimer's disease
  • Figure 1 shows the binding ability of antibodies against granular tau oligomers to fractionated recombinant tau aggregates.
  • Recombinant human full-length (FL) tau was polymerized with heparin and fractionated into fractions (Fr) 1–6 by sucrose step gradient centrifugation. As the fraction number increased, higher density tau aggregates were observed. 28 For example, Fr. 1 contained monomeric and multimeric tau, while Fr. 3 contained abundant granular tau oligomers. Tau was detected in Fr.
  • Fraction 1 by dot blot analysis using the pan-tau rabbit polyclonal antibody JM (Panel A) and monoclonal antibodies 2D6-2C6 (Panel B), 2B2-1B6 (Panel C), and 8D6-1F7 (Panel D) derived from rats immunized with granular tau oligomers.
  • Fraction 1 contained tau at a concentration of 100 ⁇ g/ml.
  • Tau immunoreactivity was quantified by densitometry (ImageJ software), and levels of 2D6-2C6-, 2B2-1B6-, and 8D6-1F7-reactive tau were normalized to the corresponding JM-reactive tau. Quantitative data are presented as percentages of Fr.
  • TBS-soluble fractions were obtained from rTg4510 mice overexpressing human P301L mutant tau (0N4R) and 10-month-old non-transgenic mice (non-Tg). These fractions were subjected to dot blot (Panels A-D), immunohistochemistry (Panels E and F), and Western blot (Panel G) analyses. (Panels A-D) Dot blot analysis demonstrated the detection of tau in occipital cortex homogenates from rTg4510 mice.
  • MC1 Pan-tau mouse monoclonal antibody
  • tau aggregation antibodies such as 2D6-2C6 (Panel B), 2B2-1B6 (Panel C), and 8D6-1F7 (Panel D).
  • Tau immunoreactivity was quantified by densitometry.
  • MC-1-, 2D6-2C6-, 2B2-1B6-, and 8D6-1F7-reactive tau levels were normalized to the corresponding tau5-reactive tau levels. Quantitative data are presented as a percentage of non-Tg mice (mean ⁇ SD of 4-5 mice).
  • Full-length 2N4R tau consists of an N-terminal region containing a proline-rich region and an N-terminal insert, a microtubule-binding repeat region, and a C-terminal region.
  • the ⁇ N mutant deletes residues 1-243
  • the ⁇ R mutant deletes residues 244-369
  • the ⁇ C mutant deletes residues 370-441.
  • cDNAs encoding WT, ⁇ N, ⁇ R, and ⁇ C were transfected into cultured COS-7 cells. Tau protein in cell lysates was detected by SDS-PAGE Western blot analysis using the pan-tau antibody JM (Panel B, left) and 2D6-2C6 (Panel B, right).
  • Rats were immunized by injection with granular tau oligomers. 768 hybridomas were generated using the iliac lymph node assay. ELISA and dot blot assays showed that culture media from three hybridomas (2B2, 2D6, and 8D6) strongly bound to granular tau oligomers. From these, 191 clones were obtained by culturing single cells from each hybridoma. Repeated ELISA and dot blot assays identified rat monoclonal antibodies from hybridoma colonies 2B2-1B6, 2D6-2C6, and 8D6-1F7 as specific antibodies targeting granular tau oligomers. Figure 1 shows the binding ability of antibodies to recombinant tau aggregates fractionated at various concentrations.
  • Fraction 1 contained tau concentrations of 200 ⁇ g/ml (Panels A–D) and 50 ⁇ g/ml (Panels E–H).
  • Tau immunoreactivity was quantified using densitometry.
  • the levels of tau reactivity of 2D6-2C6, 2B2-1B6, and 8D6-1F7 were normalized to the corresponding JM-reactive tau.
  • Quantitative data are expressed as percentages relative to Fr. 1 (mean ⁇ SD from four experiments). P values were determined by one-way analysis of variance followed by Tukey's multiple comparison test. Significance levels are indicated by * (P ⁇ 0.05), ** (P ⁇ 0.01), *** (P ⁇ 0.001), and **** (P ⁇ 0.0001). "ns" indicates not significant. Significance is indicated by solid lines for comparisons of Fr. 1 with Fr. 2, 3, 4, 5, and 6, and by dotted lines for comparisons of Fr. 3 with Fr.
  • a "subject” refers to a mammal, including a human.
  • mammal refers to, for example, rodents such as mice and rats, livestock animals such as pigs and cows, pets such as rabbits, dogs and cats, and primates such as monkeys.
  • a human subject may be, but is not limited to, for example, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or 80 years of age or older.
  • treatment is used to encompass therapeutic treatment and prophylactic treatment.
  • treatment means curing, curing, preventing, or improving the remission of a disease or disorder, or reducing the rate of progression of a disease or disorder.
  • prevention means reducing the likelihood of developing a disease or condition, or delaying the onset of a disease or condition.
  • an antibody refers to a protein in which a pair of heterodimers formed by a heavy chain (H chain) and a light chain (L chain) stabilized by a disulfide bond further associate via disulfide bonds to form a heterotetramer structure.
  • An antibody can have specificity for an antigen. Specific binding means binding that is not a nonspecific adsorption. Specificity can be ensured by immunizing an animal with the antigen. Specificity can mean having a stronger affinity for the antigen than for at least one or several other proteins.
  • an antibody that has a strong binding affinity for a specific antigen is an antibody that can specifically bind to the antigen.
  • the heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, while the light chain consists of a light chain variable region VL and a light chain constant region CL.
  • the variable region fragment (Fv) consisting of VH and VL is directly involved in antigen binding and is the region that confers diversity to the antibody.
  • the antigen-binding region consisting of VL, CL, VH, and CH1 is called the Fab region, and the region consisting of the hinge region, CH2, and CH3 is called the Fc region.
  • the region that directly contacts the antigen has a high degree of diversity in amino acid sequence between antibodies and is called the complementarity-determining region (CDR).
  • CDR complementarity-determining region
  • Regions other than the CDR that have a nearly constant amino acid sequence between antibodies are called the framework region (FR).
  • the light chain and heavy chain variable regions each contain three CDRs, which are called heavy chain CDR1-3 and light chain CDR1-3, respectively, from the N-terminus.
  • the heavy chain variable region typically contains heavy chain FR1, heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3, and heavy chain FR4, in that order.
  • a light chain variable region typically comprises, in that order, light chain FR1, light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3, and light chain FR4.
  • the antibody may be a monoclonal or polyclonal antibody.
  • the antibody of the present invention may be of any isotype, including IgG, IgM, IgA, IgD, and IgE.
  • a non-human animal such as a mouse, rat, hamster, guinea pig, rabbit, or chicken
  • a chimeric antibody is an antibody in which antibody fragments from different species are linked.
  • IgG subclasses in humans include IgG1, IgG2, IgG3, and IgG4.
  • the antibody of the present invention may be of any subclass, but may also be one or more selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, such as IgG2, IgG3, or IgG4.
  • humanized antibody refers to an antibody in which the corresponding positions in a human antibody are replaced with amino acid sequences characteristic of antibodies of non-human origin (i.e., a CDR-grafted antibody).
  • CDR-grafted antibody examples include those having heavy chain CDRs 1-3 (HCDRs 1-3, respectively) and light chain CDRs 1-3 (LCDRs 1-3, respectively) of an antibody produced by immunizing a mouse or rat, with all other regions, including the four framework regions (FRs) of the heavy and light chains, derived from a human antibody.
  • Such antibodies are sometimes called CDR-grafted antibodies.
  • a fully humanized antibody is an antibody derived from a chromosomally transplanted non-human animal (e.g., a mouse) that has human immunoglobulin loci and has the same genetic sequence as a human.
  • the term "antigen-binding fragment" of an antibody refers to a fragment of an antibody that binds to an antigen.
  • the antibody may be an isolated monoclonal antibody (e.g., an isolated human chimeric antibody, an isolated humanized antibody, or an isolated human antibody).
  • isolated means at least separated from other components. “Isolated” is used in a sense that includes separation from contaminants to an extent that is compatible with a pharmaceutical product.
  • An isolated monoclonal antibody is an isolated monoclonal antibody even if it is mixed with another monoclonal antibody.
  • An isolated monoclonal antibody is an isolated monoclonal antibody even if it is mixed with pharmaceutical excipients and formulated.
  • tau refers to a protein that is abundant in central neurons and essential for the function of axons, which make up the brain's neural network. Tau is primarily localized in axons and regulates microtubule polymerization and stabilization. Tau has an N-terminal region (including N1 and N2), a proline-rich domain (PRD), a microtubule-binding region (including R1, R2, R3, and R4), and a C-terminal region. In tauopathies such as Alzheimer's disease, tau-mediated neurofibrillary tangles (NFTs) are formed.
  • NFTs neurofibrillary tangles
  • tau is hyperphosphorylated, detaches from microtubules, and aggregates to form paired helical filaments (PHFs) and NFTs.
  • PHFs paired helical filaments
  • NFTs paired helical filaments
  • tau is preferably human tau.
  • Human tau has, for example, the amino acid sequence (SEQ ID NO: 1) registered under NCBI Reference Sequence: NP_005901.2.
  • tau aggregates due to abnormal phosphorylation and other factors, forming tau aggregates.
  • tau aggregates include linear filaments (SFs), paired helical filaments (PHFs), amyloid fibrils (AFs), transient aggregates, and tau oligomers.
  • Linear filaments (SFs), paired helical filaments (PHFs), and amyloid fibrils (AFs) are sometimes collectively referred to as tau fibrils.
  • tau oligomers include granular tau oligomers, linear tau oligomers, and cyclic tau oligomers. Tau oligomers can be aggregates of several to several dozen tau proteins. Tau oligomers can bind to each other to form linear filaments.
  • Linear filaments can further aggregate to form helical filaments or amyloid fibrils.
  • Tau contained in tau aggregates is typically phosphorylated, but the degree of phosphorylation varies greatly depending on the type of aggregate.
  • Braak's stage classification of neurofibrillary tangles is as follows:
  • Alzheimer's disease or “Alzheimer's dementia” refers to a type of dementia that gradually leads to the loss of mental functions such as memory, thinking, judgment, and learning ability.
  • Alzheimer's dementia is characterized by neurodegeneration, including the loss of nerve cells, the accumulation of abnormal proteins called beta-amyloid, and neurofibrillary tangles.
  • Alzheimer's dementia accounts for approximately half of all dementia cases, and the onset and progression of symptoms are gradual and persistent.
  • Alzheimer's dementia can be diagnosed based on the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) or NINCDS-ADRDA published by the American Psychiatric Association.
  • MCI mimild cognitive impairment
  • GDS Global Deterioration Scale or Assessment of Primary Degenerative Dementia
  • GDS Global Deterioration Scale or Assessment of Primary Degenerative Dementia
  • a clinical dementia rating of 0.5 see Hughes CP et al., Br J Psychiatry 1982;140:566-572.
  • Antibodies of the present disclosure provides an antibody or antigen-binding fragment thereof that binds to tau aggregates.
  • the antibody that binds to tau aggregates may be a polyclonal antibody or a monoclonal antibody (preferably a recombinant antibody).
  • the monoclonal antibody may be a human chimeric antibody or a humanized antibody.
  • the antibodies of the present disclosure bind to tau aggregates but only weakly or not at all to tau monomers.
  • the antibodies of the present disclosure may bind to tau aggregates at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold stronger than to tau monomers.
  • the antibodies of the present disclosure may bind 3-10-fold, or 3-7-fold stronger than to tau aggregates.
  • the antibodies of the present disclosure bind to tau aggregates but do not exhibit substantial binding to tau monomers.
  • the tau aggregates are preferably obtained from a living brain and purified.
  • the tau monomers are preferably artificially translated or synthesized and purified. Binding activity can be assessed by a dot blot method (dot blot assay).
  • an antibody of the present disclosure binds more than 100 times, more than 200 times, more than 300 times, more than 400 times, more than 500 times, more than 600 times, more than 700 times, more than 800 times, more than 900 times, more than 1000 times, more than 1500 times, more than 2000 times, more than 2500 times, more than 3000 times, e.g., 100 to 5000 times more strongly (or more strongly)) to a neuronal homogenate derived from an rTg4510 transgenic mouse overexpressing human P301L mutant tau than to a neuronal homogenate derived from a non-transgenic mouse (non-Tg mouse). Binding activity can be assessed by a dot blot method (dot blot assay).
  • the antibody of the present disclosure exhibits the property of co-localizing with tau aggregates bound by the AT8 antibody in paraffin-embedded sections of the medial temporal cortex obtained from autopsy brains of subjects with Braak stage V-VI AD.
  • the AT8 antibody is a mouse IgG1 antibody capable of binding to human tau phosphorylated at S202 and T205, and is registered under PRID: AB_223647.
  • the AT8 antibody is commercially available and has been used in numerous academic publications.
  • the AT8 antibody can be purchased, for example, from Thermo Fisher Scientific (e.g., Catalog Number: MN1020).
  • the antibodies of the present disclosure have higher selectivity for tau aggregates than the MC1 antibody (RRID: AB_2314773). In certain aspects, the antibodies of the present disclosure bind to tau aggregates more strongly than the MC1 antibody (RRID: AB_2314773) by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1000 times, for example, 100 to 2000 times (e.g., 1000 to 2000 times) stronger.
  • 100 to 2000 times e.g., 1000 to 2000 times
  • the antibodies of the present disclosure can bind to a peptide (particularly a linear peptide) consisting of an amino acid sequence of 8 to 15 amino acids in length (preferably 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length), which is a partial sequence of the amino acid sequence of human tau (SEQ ID NO: 1) registered under NCBI Reference Sequence: NP_005901.2, including the amino acid sequence at amino acid positions 423-430 (PQLATLAD; SEQ ID NO: 2).
  • the peptide region is inaccessible to antibodies in human tau monomers, but may become accessible to antibodies upon oligomerization. Therefore, an antibody that binds to the peptide region can be selective for tau oligomers rather than tau monomers.
  • an antibody that binds to tau oligomers and the peptide region (or the peptide).
  • the antibodies of the present disclosure can also bind to the phosphorylated form of the peptide (i.e., phosphorylated peptide).
  • the above phosphorylated peptide is preferably phosphorylated at the threonine (T) corresponding to position 427 of SEQ ID NO: 1.
  • the antibody of the present disclosure comprises an amino acid sequence of 8 to 15 amino acids in length (preferably 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length) that is a partial sequence of the amino acid sequence of human tau registered under NCBI Reference Sequence: NP_005901.2 (SEQ ID NO: 1), including the amino acid sequence at amino acid positions 423-430 (PQLATLAD; SEQ ID NO: 2), and binds to peptides phosphorylated at amino acid position 427, but does not bind to peptides at amino acid position 427 that are not phosphorylated, and is capable of binding to tau aggregates.
  • the antibody of the present disclosure consists of an amino acid sequence of 8 to 15 amino acids in length (preferably 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length) that is a partial sequence of the amino acid sequence of human tau registered under NCBI Reference Sequence: NP_005901.2 (SEQ ID NO: 1), including the amino acid sequence at amino acid positions 423-430 (PQLATLAD; SEQ ID NO: 2), and is capable of binding to peptides in which amino acid position 427 is phosphorylated and peptides in which amino acid position 427 is not phosphorylated, and is capable of binding to tau aggregates.
  • SEQ ID NO: 1 amino acid sequence of 8 to 15 amino acids in length
  • PQLATLAD amino acid sequence at amino acid positions 423-430
  • the antibody of the present disclosure binds to the C-terminal region of tau.
  • the C-terminal region of tau is, for example, positions 370 to 441, preferably positions 417 to 441, and more preferably positions 423 to 430 of the amino acid sequence set forth in SEQ ID NO: 1.
  • Antibodies of the present disclosure are preferably capable of binding to tau pretangles. Pretangles cause less damage to neurons, but are an early form of tau aggregates and may be an indicator for the early diagnosis of Alzheimer's disease. Antibodies of the present disclosure are also preferably capable of binding to mature tau tangles. Mature tau tangles are the final form of tau aggregates and may be an indicator of diseases closely related to the progression of Alzheimer's disease. Mature tangles may serve as a therapeutic target. Antibodies of the present disclosure are capable of binding to both tau pretangles and mature tau tangles.
  • the antibody of the present disclosure a heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO:5, a heavy chain CDR2 set forth in SEQ ID NO:6, and a heavy chain CDR3 set forth in SEQ ID NO:7; an antibody comprising a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO:8, a light chain CDR2 set forth in SEQ ID NO:9, and a light chain CDR3 set forth in SEQ ID NO:10; a heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 11, a heavy chain CDR2 set forth in SEQ ID NO: 12, and a heavy chain CDR3 set forth in SEQ ID NO: 13; an antibody comprising a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 14, a light chain CDR2 set forth in SEQ ID NO: 15, and a light chain CDR3 set forth in SEQ ID NO: 16;
  • the antibody of the disclosure comprises a heavy chain variable region set forth in SEQ ID NO: 3 and a light chain variable region set forth in SEQ ID NO: 4; an antibody having an amino acid sequence that differs by one amino acid from the amino acid sequence of the antibody; an antibody having an amino acid sequence differing by 2 to 5 amino acids from the amino acid sequence of the antibody; an antibody having an amino acid sequence that has 90% or more, or 95% or more sequence identity with the amino acid sequence of the antibody; an antibody comprising a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the above antibody and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the above antibody; or an antibody that competes with the above antibody for binding to the peptide region (or the above peptide).
  • the antibody of this embodiment may be capable of binding to tau aggregates (particularly tau oligomers) and may not show substantial binding to tau monomers.
  • the antibody of the present disclosure a heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 42, a heavy chain CDR2 set forth in SEQ ID NO: 43, and a heavy chain CDR3 set forth in SEQ ID NO: 44; an antibody comprising a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 60, a light chain CDR2 set forth in SEQ ID NO: 61, and a light chain CDR3 set forth in SEQ ID NO: 62; a heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 45, a heavy chain CDR2 set forth in SEQ ID NO: 46, and a heavy chain CDR3 set forth in SEQ ID NO: 47; an antibody comprising a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 63, a light chain CDR2 set forth in SEQ ID NO: 64, and a light chain CDR3 set forth in SEQ ID NO
  • the antibody of the disclosure comprises an antibody comprising a heavy chain variable region set forth in SEQ ID NO: 40 and a light chain variable region set forth in SEQ ID NO: 41; an antibody having an amino acid sequence that differs by one amino acid from the amino acid sequence of the antibody; an antibody having an amino acid sequence differing by 2 to 5 amino acids from the amino acid sequence of the antibody; an antibody having an amino acid sequence that has 90% or more, or 95% or more sequence identity with the amino acid sequence of the antibody; an antibody comprising a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the above antibody and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the above antibody; or an antibody that competes with the above antibody for binding to the peptide region (or the above peptide).
  • the antibody of this embodiment may be capable of binding to tau aggregates (particularly tau oligomers) and may not show substantial binding to tau monomers.
  • the antibody disclosed herein is an antibody that binds to an isolated short peptide consisting of amino acids 422 to 435 of the amino acid sequence of human tau set forth in SEQ ID NO: 1, and exhibits selectivity for granular tau oligomers that is equal to or greater than that of an antibody (reference antibody) comprising a heavy chain variable region set forth in SEQ ID NO: 3 and a light chain variable region set forth in SEQ ID NO: 4.
  • the selectivity can be confirmed by dot blot using granular tau oligomers and tau monomers (see, for example, Figures 9 and 10).
  • the antibody may be a monoclonal antibody, and preferably a humanized antibody or a human antibody.
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 5 to 7, respectively, and the light chain variable region including the light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 8 to 10, respectively;
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 11 to 13, respectively, and the light chain variable region including the light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 14 to 16, respectively;
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 17 to 19, respectively, and the light chain variable region including the light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody have the amino acid sequences set forth in
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 42 to 44, respectively, and the light chain variable region including the light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 60 to 62, respectively;
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 45 to 47, respectively, and the light chain variable region including the light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 63 to 65, respectively;
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the antibody have the amino acid sequences set forth in SEQ ID NOs: 48 to 50, respectively, and the light chain variable region including the light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody have the
  • the antibody is a human chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody has an Fc region derived from IgG1, IgG2, IgG3, or IgG4.
  • Antibodies used for diagnostic purposes may be polyclonal or monoclonal.
  • the antibody may be a non-human chimeric antibody or a non-humanized antibody, or a human chimeric antibody or a humanized antibody.
  • the primary antibody may be a non-human animal antibody (e.g., a mouse antibody, a rat antibody, a rabbit antibody, a chicken antibody, a camel antibody, a shark antibody, etc.).
  • the non-human animal antibody bound to a human sample can be suitably detected by a labeled secondary antibody that binds to the non-human animal antibody or its Fc region.
  • Antibodies used for therapeutic purposes are preferably monoclonal antibodies, and when used for human therapeutic purposes, they are preferably human chimeric antibodies, more preferably humanized antibodies, and even more preferably human antibodies.
  • the subclass of the antibody Fc region can be, for example, IgG1, IgG2, or IgG4.
  • the present disclosure provides a method for producing the antibody of the present disclosure.
  • the antibody of the present disclosure can be obtained by selecting, from antibodies obtained by immunization with a tau oligomer as an immunogen, antibodies that bind to a peptide (particularly a linear peptide) consisting of an amino acid sequence of 8 to 15 amino acids in length (preferably 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length), which has a sequence that is a partial sequence of the amino acid sequence of human tau (SEQ ID NO: 1) registered under NCBI Reference Sequence: NP_005901.2, including the amino acid sequence at amino acid positions 423-430 (PQLATLAD; SEQ ID NO: 2).
  • the antibody of the present disclosure can also be obtained by immunizing an animal with the above-described peptide as an immunogen.
  • the peptide can be directly fused to a carrier protein (e.g., keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA)) with or without a spacer peptide, and then used to immunize an animal (e.g., rats or mice).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the antibodies of the present invention can be prepared as an immunogen by combining a synthetic human tau (422-435) peptide (SEQ ID NO: 39) obtained by linking a cysteine to the C-terminus thereof with KLH-mmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS).
  • the immunogen can be mixed with an adjuvant and then administered to an animal.
  • Booster immunizations can be performed as needed.
  • Antibody-producing cells can be collected from the spleen or iliac lymph nodes.
  • An example of the iliac lymph node method in mice is described in JP 4098796B.
  • Hybridomas can be obtained by cell fusion of antibody-producing cells with myelomas.
  • the binding properties of antibodies produced from hybridoma clones can be examined, and clones with the desired binding properties can be selected. For example, culture supernatants of hybridoma clones can be collected, and antibodies contained in the culture supernatants can be screened for reactivity with tau oligomers. Screening can be easily performed using methods such as ELISA or dot blot.
  • the antibodies of the present disclosure can also be obtained using, as an immunogen, a peptide (particularly a linear peptide) consisting of an amino acid sequence 8 to 15 amino acids in length (preferably 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length), which is a partial sequence of the amino acid sequence of human tau registered under NCBI Reference Sequence: NP_005901.2 (SEQ ID NO: 1), including the amino acid sequence at amino acid positions 423-430 (PQLATLAD; SEQ ID NO: 2). Binding to tau oligomers can be ensured by selecting an antibody that binds to the peptide from antibodies that bind to the peptide.
  • the antibodies of the present disclosure may also be obtained by phage display.
  • Phage display libraries are suitable for producing human antibodies, for example, by constructing them using antibody gene fragments derived from humans.
  • Phage display libraries may be commercially available (e.g., Morphosys HuCAL TM , Dyax libraries, Tripeptide libraries, etc.), or may be constructed independently.
  • Phage display libraries may also be constructed by obtaining antibody-producing cells from immunized animals or humans and obtaining antibody gene fragments.
  • Specific antibodies can be obtained by binding the phage display library to tau oligomers or the above-mentioned peptides, removing unbound phages by washing, extracting and growing phages that showed binding, and analyzing the antibody gene fragments contained in the obtained phages.
  • Dot blotting is a protein detection technique.
  • proteins separated after gel electrophoresis are transferred to a membrane and then detected by reacting the transferred proteins with antibodies.
  • dot blotting is performed without electrophoresis.
  • a sample containing a protein preferably purified tau monomer, purified tau oligomer, purified tau aggregate, etc.
  • PVDF polyvinylidene fluoride
  • HRP horseradish peroxidase
  • alkaline phosphatase are commonly used as labels. When incubated in the presence of a chemiluminescent substrate, these labels emit a signal proportional to the amount of secondary antibody binding (i.e., the amount of primary antibody binding, i.e., the amount of antigen protein).
  • a radioisotope (RI) can also be used as a label, in which case a signal proportional to the amount of RI is emitted.
  • Antibodies that compete with one another for binding to an antigen can be obtained by competitive assays well known to those skilled in the art.
  • Antibodies that can block the binding of a desired antibody by, for example, at least 20%, preferably at least 20-50%, more preferably at least 50%, more preferably 60%, more preferably 70%, more preferably 80%, and especially preferably 90% or more in a competitive assay can be considered to be competing antibodies for binding to the same antigen.
  • Competing antibodies can be confirmed by a cross-blocking assay, preferably a competitive ELISA assay.
  • the antigen is coated on, for example, a microtiter plate, and a candidate competing antibody is added and incubated to allow binding between the antigen and the candidate antibody.
  • the desired antibody is then labeled and added to the well, incubated, and washed. The amount of binding of the desired antibody is quantified to determine whether the antibodies competed. If there is competition, the amount of label remaining in the well should be reduced.
  • antibody A dissociates the binding between antibody B and an antigen does not necessarily mean that antibody B will also dissociate the binding between antibody A and an antigen.
  • antibody A exhibits significantly stronger binding to an antigen than antibody B.
  • antibody B dissociates the binding between antibody A and an antigen; in this specification, this competitive state is referred to as "antibody A and antibody B competing with each other for binding to an antigen.”
  • Amino acid mutations in antibodies can be performed by those skilled in the art using site-directed mutagenesis techniques. The binding properties of antibodies with amino acid mutations can be confirmed as described above. Antibodies with amino acid mutations can fully or partially retain the binding properties of the original antibody, but in some cases may even enhance the binding properties of the original antibody.
  • monoclonal antibodies can be obtained by obtaining a gene encoding an antibody directly from antibody-producing cells and introducing it into antibody-producing cells (e.g., Cheney hamster ovary cells (CHO cells)), or by obtaining a gene encoding an antibody from a hybridoma and introducing it into antibody-producing cells.
  • antibody-producing cells e.g., Cheney hamster ovary cells (CHO cells)
  • the obtained antibody can be isolated or purified and mixed with pharmaceutically acceptable additives.
  • Tau monomers can be prepared, for example, by expressing tau in cultured cells. This preparation can be performed appropriately by those skilled in the art. For example, cDNA encoding tau can be introduced into E. coli, causing the E. coli to synthesize tau protein, and then the tau protein can be recovered and purified to obtain tau monomers.
  • Tau oligomers can be obtained by polymerizing tau monomers in the presence of heparin. The obtained tau oligomers can be separated by sucrose density gradient centrifugation to obtain fractions containing granular tau oligomers and fractions containing tau fibrils.
  • sucrose gradient solution consisting of multiple layers, preferably four layers (e.g., 20%, 30%, 40%, 50%), can be prepared in a buffer, and polymerized tau can be applied to the solution followed by centrifugation to obtain fractions containing granular tau oligomers and fractions containing tau fibrils.
  • a method for selecting an antibody of the present disclosure includes, but is not limited to, obtaining an antibody (preferably a monoclonal antibody) that binds to tau; and obtaining from the monoclonal antibodies antibodies that bind to the peptide (preferably a synthetic short peptide) set forth in SEQ ID NO: 2.
  • the method may further comprise selecting antibodies that have higher selectivity for granular tau oligomers than for tau monomers.
  • a method for producing an antibody of the present disclosure includes, but is not limited to, obtaining an antibody (preferably a monoclonal antibody) that binds to tau; Obtaining an antibody that binds to the peptide set forth in SEQ ID NO: 2 (preferably a synthetic short-chain peptide) from the monoclonal antibody; The method may further comprise selecting an antibody having higher selectivity for granular tau oligomers than for tau monomers.
  • a method for producing an antibody of the present disclosure includes, but is not limited to, immunizing a non-human animal with a partial peptide of human tau to obtain a monoclonal antibody (wherein the partial peptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 39, and is preferably linked to a carrier protein); and selecting from the obtained monoclonal antibodies antibodies having higher selectivity for granular tau oligomers than for tau monomers.
  • the antibodies of the present disclosure bind to tau aggregates, preferably only weakly or substantially not to tau monomers, and can be used to detect tau aggregates in a sample.
  • Brain tissue samples can be derived from any one or more patients or healthy individuals selected from the group consisting of patients with Alzheimer's dementia (mild, moderate, and severe), patients with mild cognitive impairment (MCI), healthy individuals, healthy individuals suspected of having Alzheimer's dementia, and healthy individuals suspected of having MCI (including healthy individuals or patients in the borderline stage).
  • Brain tissue samples can be derived from, for example, a sample (e.g., prefrontal cortex) of a patient in Braak stage I or II, a presymptomatic stage of Alzheimer's dementia. Samples can be homogenized or processed into tissue sections.
  • Tau aggregates can be determined to have been detected when signals characteristic of tau aggregates (e.g., distributed along the axons and dendrites of neuronal cells and measuring several micrometers to several tens of micrometers) are observed on the tissue sections. Signals characteristic of tau aggregates are readily understood by those skilled in the art.
  • the sample containing tau aggregates may also be a bodily fluid sample.
  • the sample is derived from a living or cadaveric organism.
  • the bodily fluid sample contains, for example, phosphorylated tau or tau aggregates, which may be bound by the antibodies of the present disclosure.
  • a method for detecting tau aggregates comprising: contacting the isolated sample with an antibody of the present disclosure; washing away unreacted antibodies of the present disclosure; wherein the presence of antibody bound to the sample indicates the presence of tau aggregates.
  • a method for diagnosing whether a subject from which a sample is derived has Alzheimer's disease or mild cognitive impairment comprising: contacting the isolated sample with an antibody of the present disclosure; washing away unreacted antibodies of the present disclosure; and the presence of the antibody bound to the sample indicates that the subject has Alzheimer's disease or mild cognitive impairment, or is at risk of developing these diseases.
  • the term "method for diagnosing” can be interpreted as "a method for diagnosing” or "a method for obtaining preliminary information for diagnosis,” etc. This method may be, for example, an industrially applicable method, or may not include, for example, a step of diagnosis by a physician.
  • Methods for detecting tau aggregates and diagnosing whether a subject from which a sample is derived has Alzheimer's disease or mild cognitive impairment may be performed in vitro, ex vivo, or in vitro.
  • Samples can be used that are derived from tissues that are more appropriate for the suspected stage.
  • the site to be isolated as a sample can be determined based on the Braak stage classification shown in Table 1.
  • the Braak stage classification of the tissue can include one or more selected from the group consisting of stage I, stage II, stage III, stage IV, stage V, and stage VI.
  • the tissue comprises tissue with a Braak stage classification of stage I or stage II.
  • the tissue comprises tissue with a Braak stage classification of stage III.
  • the tissue comprises tissue with a Braak stage classification of stage IV or stage V. It is important to observe tissues in which NFT is present.
  • tissue containing one or more regions selected from the group consisting of the transentorhinal cortex, entorhinal cortex, fusiform (occipitotemporal) association area, medial temporal gyrus, occipital lobe, parastriate area, striate area, hippocampus, cerebral neocortex, subcortical nuclei, and brainstem region can be used as a sample.
  • the presence of an antibody bound to a sample can typically be detected by a labeled secondary antibody that binds to the antibody.
  • the antibody that is brought into contact with the sample can be derived from a different species than the sample. This makes it easier to specifically detect only that antibody with the secondary antibody.
  • Horseradish peroxidase (HRP) and alkaline phosphatase are commonly used labels. When incubated in the presence of a chemiluminescent substrate, these labels emit a signal that corresponds to the amount of secondary antibody bound (i.e., the amount of primary antibody bound, i.e., the amount of antigen protein). Quantitation with a certain degree of accuracy is possible by preparing a calibration curve using a standard substance.
  • the present disclosure provides a kit for use in the above method.
  • the kit may include an antibody of the present disclosure.
  • the kit may further include a labeled secondary antibody for detecting the antibody of the present disclosure.
  • Commonly used labels include peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase, and ⁇ -glucuronidase, and the kit may further include substrates for these labels.
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -glucuronidase substrates for these labels.
  • peroxidase substrates examples include 3,3'-diaminobenzidine (DAB), orthophenylenediamine (OPD), tetramethylbenzidine (TMB), 2,2'-aminoazobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), luminol, and acridinium ester.
  • DAB 3,3'-diaminobenzidine
  • OPD orthophenylenediamine
  • TMB tetramethylbenzidine
  • ABTS 2,2'-aminoazobis(3-ethylbenzothiazoline-6-sulfonic acid)
  • luminol examples include 3,3'-diaminobenzidine (DAB), orthophenylenediamine (OPD), tetramethylbenzidine (TMB), 2,2'-aminoazobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), luminol, and acridinium este
  • Substrates for alkaline phosphatase include, for example, 5-bromo-4-chloro-3-indolyl phosphate tosyl salt (BCIP), nitroblue tetrazolium hydrochloride (NBT), 1-naphthol phosphate tosyl salt (NPT), 4-methylumbelliferyl phosphate (MUP), and 7-hydroxycoumarin phosphate (HCP).
  • Substrates for ⁇ -glucuronidase include, for example, 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronide (X-Gluc).
  • the antibodies of the present disclosure can bind to tau aggregates. Accordingly, the antibodies of the present disclosure can be used to remove tau aggregates.
  • the antibodies have the ability to bind to Fc ⁇ receptors (e.g., Fc ⁇ RI, such as Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII, such as Fc ⁇ RIIa, Fc ⁇ RIIb, and Fc ⁇ RIIc; and Fc ⁇ RIII, such as Fc ⁇ RIIIa and Fc ⁇ RIIIb), thereby recruiting immune cells such as macrophages and microglia to phagocytose tau aggregates or destroy tau aggregates bound to the antibodies.
  • Fc ⁇ receptors e.g., Fc ⁇ RI, such as Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc
  • Fc ⁇ RII such as Fc ⁇ RIIa, Fc ⁇ RIIb, and Fc ⁇ RIIc
  • Fc ⁇ RIII such as Fc ⁇ RIIIa and Fc
  • the present disclosure provides a method for removing tau aggregates in a subject having tau aggregates, comprising administering to the subject an effective amount of an antibody of the present disclosure, thereby causing the antibody to bind to and remove tau aggregates.
  • Removal of tau aggregates can be effective in preventing or treating Alzheimer's disease.
  • the subject may be a subject who has not yet developed Alzheimer's dementia.
  • the subject may be a subject who has developed Alzheimer's dementia.
  • the subject may also be a subject with MCI.
  • the antibodies of the present disclosure can also be used to treat tauopathy.
  • the present disclosure provides a method of treating a tauopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody of the present disclosure.
  • the tauopathy may be selected, for example, from the group consisting of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia.
  • the antibodies of the present disclosure can be used to treat MCI accompanied by accumulation of abnormal tau protein in the brain. Therefore, the present disclosure provides a method of treating MCI accompanied by accumulation of abnormal tau protein in the brain in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody of the present disclosure.
  • the present disclosure provides an antibody of the present disclosure for use in the above method.
  • the present disclosure provides a composition comprising an antibody of the present disclosure for use in the above method.
  • the present disclosure provides use of an antibody of the present disclosure in the manufacture of a medicament or composition for use in the above method.
  • the present disclosure provides use of an antibody of the present disclosure in the above method.
  • Materials Recombinant tau was prepared by inserting full-length human 2N4R tau cDNA into the pRK172 vector.
  • 28 Wild-type 2N4R and mutant 2N4R tau with a deletion of the repeat region from 244 to 369 aa ( ⁇ R), a deletion of the N-terminal region from 1 to 243 aa ( ⁇ N), and a deletion of the C-terminal region from 370 to 441 aa ( ⁇ C) were inserted into the pCI-neo vector (Promega) and transiently transfected (Fig. 3a).
  • Peptides purity ⁇ 50%) were obtained from Cosmo Bio Co., Ltd. Chemical reagents were purchased from Nacalai Tesque, Inc., Fujifilm Wako Pure Chemical Industries, Ltd., and Sigma-Aldrich.
  • Sucrose step gradient centrifugation Sucrose density gradient centrifugation was performed based on a previously described method with minor modifications. 28 Briefly, a sucrose gradient solution consisting of four layers (20%, 30%, 40%, and 50%) was prepared in a buffer containing HEPES (10 mM, pH 7.4) and NaCl (100 mM). Polymerized tau was layered on top of the sucrose gradient solution. After centrifugation at 50,000 rpm for 2 hours in an MLS50 rotor (Beckman Coulter) or a TLS55 rotor (Beckman Coulter), the layers were collected as fractions 1–5, and the pellet was collected as fraction 6.
  • HEPES 10 mM, pH 7.4
  • NaCl 100 mM
  • AFM Atomic force microscope
  • mice Animals: rTg4510 mice (Jackson Laboratory, 024854) expressing human P301L mutant tau and non-transgenic mice were housed under standard conditions at approximately 25°C. At 10 months of age, mice were anesthetized with isoflurane and euthanized. The left hemisphere was placed in 10% formalin and paraffin blocks were prepared from it. The occipital cortex was extracted from the right hemisphere for biochemical assays and stored in a deep freezer.
  • TBS-soluble fraction from mouse brain: The occipital cortex of rTg4510 mice was homogenized in cold TBS buffer containing protease and phosphatase inhibitors. After homogenization, the mixture was centrifuged at 23,000 rpm for 15 min at 4°C in a TLA55 rotor (Beckman Coulter). The supernatant was stored in a deep freezer prior to biochemical experiments.
  • Dot blot analysis was used to detect tau in samples. These samples included fractions 1–6 from sucrose gradient step centrifugation and the TBS-soluble fraction from mouse occipital cortex. One to two microliters of sample was loaded onto a nitrocellulose membrane (Cytiva, Tokyo, Japan).
  • the membrane was then blocked with 5% milk for 1 hour at room temperature and incubated overnight at 4°C with antibodies against granular tau oligomers and pan-tau antibodies, such as JM (immunogen, 2N4R-tau; 36 ), tau5 (epitope, tau 210-241 AA residues; Invitrogen, AHB0042), RTM38 (immunogen, 417-441 AA residues; Fujifilm Wako Pure Chemical Industries, Ltd., 017-26893), and Tau46 (epitope, 404-441 AA residues; Invitrogen, 13-6400).
  • Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) were added to the membrane for 2 hours at room temperature. Chemiluminescence was detected with an Amersham Imager 600 (Cytiva) using a Chemi-Lumi One series (Nacalai Tesque, Kyoto, Japan).
  • Paraffin sections of the medial temporal cortex were obtained from autopsy brains of Braak stage V-VI AD patients.
  • Immunohistochemistry Tau in paraffin sections of mouse and human brain was detected using the AT8 antibody (phospho-Ser202, Thr205, Ser208) and antibodies against granular tau oligomers according to previously described protocols74,75 .
  • tau fibrils During tau aggregation, monomeric tau polymerizes, primarily forming intermediate aggregates called tau oligomers and granular tau oligomers, before finally forming fibrillar tau (tau fibrils).
  • Sucrose density gradient centrifugation can separate aggregated tau samples from recombinant tau, 28 mouse brain, 35 and human brain, 31 into six fractions based on size and density. Each of the six fractions is composed of the following tau species: Fraction 1: no apparent aggregates, Fraction 2: small tau granules, Fraction 3: granular tau oligomers, and Fractions 4–6: short and long tau fibrils.
  • the binding efficiencies of the three antibodies to these tau fractions were examined by dot blot analysis.
  • the control pan-tau antibody (JM; derived from the immunogen 2N4R-tau) 36 showed similar signal intensities for tau in fractions 1 to 6 (Fig. 1A).
  • the signal generated by the 2D6-2C6 antibody was increased 3.9-fold, 5.4-fold, and 6.0-fold in fractions 3, 4, and 5-6, respectively, compared to fraction 1 (monomer) (Fig. 1B).
  • the 2B2-1B6 antibody showed a dotted signal increase in fractions 2 (2.0-fold), 3 (3.3-fold), 4 (2.3-fold), 5 (2.3-fold), and 6 (2.8-fold) compared to fraction 1 (Fig. 1D).
  • rTg4510 transgenic mice overexpressing human P301L mutant tau which is genetically associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), exhibit increased neurofibrillary tangle (NFT) pathology with age. 37
  • the binding of three rat monoclonal antibodies to samples from rTg4510 mice was assessed by dot blot analysis.
  • the pan-tau antibody (tau5) detects both human and mouse tau. 5 Indeed, tau5 was able to react with cell homogenates from rTg4510 and non-Tg mice ( Figure 2A).
  • the cDNA of the 2D6-2C6 antibody was obtained from the hybridoma clone producing the antibody, and the amino acid sequences of the heavy chain variable region and light chain variable region of the 2D6-2C6 antibody were determined.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.
  • the amino acid sequences of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 were predicted from the heavy chain variable region and light chain variable region of the 2D6-2C6 antibody using each prediction algorithm (see Tables 5 and 6).
  • the A11 polyclonal antibody recognizes a conformation common to the oligomerization of amyloidogenic proteins, regardless of the AA sequence. 43 Using A11 in dot blot analysis, recombinant tau fibrils were detected, but not oligomers, including granular tau oligomers. 28 The mouse monoclonal antibody MC1 recognizes pathological conformations of tau in vitro, in vivo, and in AD human subjects. 44,45 In vitro experiments, MC1 gave positive signals from tau oligomers to tau fibrils.
  • TOC1 is a mouse monoclonal antibody immunogenized against cross-linked dimeric tau. 46 Its epitope is located between 209-224 aa on tau. 47 TOC1 binds to oligomeric tau, but to my knowledge, there have been no reports of TOC1 reacting with tau fibrils.
  • TauRD ⁇ K mutant tau repeat region lacking lysine 280
  • T22 polyclonal antibody and the TORC1 monoclonal antibody were identified as tau oligomer antibodies by Kayed et al. These antibodies detect oligomeric tau by SDS-PAGE Western blot analysis, suggesting that the conformation of antibody-positive tau oligomers is resistant to high concentrations of detergent. However, the epitope of either antibody has not been reported. 49,50 TNT2 binds to the phosphatase activation domain (7-12 AA sequence) on tau and detects early pretangle tau pathology in AD human subjects.
  • the C-terminal region containing calpain-cleaved Tau-CTF24 (residues L243-L441) accelerated heparin-induced tau fibrillogenesis compared with full-length tau.57
  • Overexpression of caspase-cleaved ⁇ D421-tau also promoted oligomerization in mouse brain.
  • TauC3 antibody-positive ⁇ D421 tau has also been observed in the brains of AD patients.53,59 However, tauC3 recognizes mature tangles, not pretangles, suggesting that tau is cleaved after the formation of tau fibrils.
  • a recent report showed that aggregation of 0N3R recombinant tau exposes the C-terminal region outside the repeat domain.61
  • 2D6-2C6 immunosignals colocalized with AT8 immunosignals, which can detect phosphorylated tau aggregates (see Figure 3), indicating that the 423-430 AA residues of tau recognized by 2D6-2C6 are externally exposed during tau aggregation in human samples.
  • 2D6-2C6 binds to all fractions 3 and above, antibodies that bind to the 423-430 AA residues of tau (e.g., 2D6-2C6) can bind to both pretangles and mature tangles.
  • tau aggregation antibodies with a novel mechanism may be derived from immunization with ⁇ -sheet-positive granular tau oligomers. This approach is in contrast to previous reports that used ⁇ -sheet-negative oligomers, including cross-linked tau dimers, 46 and low-n oligomers, 48 as immunogens.
  • the 3A7 antibody-producing clone was further subjected to a single-cell cloning process to obtain the 3A7-1E9 antibody.
  • the affinity of the obtained antibody for granular tau oligomers was confirmed in the same manner as above.
  • the obtained 3A7-1E9 antibody exhibited superior selectivity for granular tau oligomers compared to the 2D6-2C6 antibody.
  • cDNA for the 3A7-1E9 antibody was obtained from the hybridoma clone producing the antibody, and the amino acid sequences of the heavy chain variable region and light chain variable region of the 3A7-1E9 antibody were determined.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 40
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41.
  • the amino acid sequences of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 were predicted from the heavy chain variable region and light chain variable region of the 3A7-1E9 antibody using each prediction algorithm (see Tables 7 and 8).
  • Tau antibodies are essential tools for biomarker analysis of AD. For example, ELISA analysis showed that tau phosphorylation at T217 and T181 in plasma and cerebrospinal fluid (CSF) was increased in AD and preclinical AD patients compared with control groups. 69,70 Goedert et al. reported that ring-shaped tau aggregates obtained from 0N4R P301S tau transgenic mice by sucrose gradient centrifugation increased tau seed assembly in cultured cells. 35 Furthermore, spherical vesicular tau oligomers from AD patients expanded tau pathology in vitro and in vivo.
  • CSF cerebrospinal fluid
  • OptoTau a tau protein fused with CRY2olig
  • 71 In our previous report, we used biochemical techniques to demonstrate that granular tau oligomers are observed at Braak stage I. 31 Therefore, antibodies that bind to tau 423-430 AA residues (e.g., 2D6-2C6 antibody against granular tau oligomers) may be useful for detecting preclinical biomarkers of AD.
  • 2D6-2C6 was produced by immunization with recombinant tau aggregates.
  • 2D6-2C6 colocalized with pretangles and neurons stained with the tau phosphorylation antibody AT8, which recognizes NFTs, indicating that 2D6-2C6 reacts with phosphorylated tau aggregates.
  • Tau phosphorylation at the T427AA residue was more strongly stained in AD and FTLD-tau patients than in healthy controls.
  • the T427AA residue is located within the epitope (423-430AA sequence) of 2D6-2C6.
  • a new TAO kinase inhibitor reduces tau phosphorylation at sites associated with neurodegeneration in human tauopathies. Acta Neuropathol Commun 6, 37, doi:10.1186/s40478-018-0539-8 (2016). 73 Soeda, Y. et al. Toxic tau oligomer formation blocked by capping of cysteine residues with 1,2-dihydroxybenzene groups. Nat Commun 6, 10216, doi:10.1038/ncomms10216 (2015). 74 Ishigaki, S. et al. Altered Tau Isoform Ratio Caused by Loss of FUS and SFPQ Function Leads to FTLD-like Phenotypes. Cell Rep.

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Abstract

La présente divulgation concerne un anticorps qui se lie à des agrégats de tau et un fragment de liaison à l'antigène dudit anticorps. De préférence, l'anticorps et le fragment de liaison à l'antigène de celui-ci peuvent se lier sélectivement à des agrégats de tau plutôt qu'à des monomères de tau.
PCT/JP2025/008900 2024-03-12 2025-03-11 Anticorps se liant à des agrégats de tau et son utilisation Pending WO2025192556A1 (fr)

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