TW201321409A - Pharmaceutical composition - Google Patents

Abstract

The present invention relates to methods and compositions for the therapeutic and diagnostic use in the treatment of diseases and disorders which are caused by or associated with neurofibrillary tangles. In particular, the invention relates to antibodies, which specifically recognize and bind to phosphorylated pathological protein tau-conformers and to methods and compositions involving said antibodies for the therapeutic and diagnostic use in the treatment of tauopathies including Alzheimer's Disease (AD).

Description

Pharmaceutical composition

The present invention relates to methods and compositions for the treatment and diagnosis of diseases and conditions associated with or associated with neurofibrillary tangles. In particular, the present invention relates to antibodies that specifically recognize and bind to phosphorylated pathological protein τ conformers, and for treatment of τ lesions including Alzheimer's Disease (AD) and Methods and compositions for the diagnosis of such antibodies.

The present application claims the benefit of the European Patent Application No. EP12163319.2 filed on Apr. 5, 2012, and the International Patent Application No. PCT/EP2011/067604, filed on Oct. 7, 2011, each of which The content of this is hereby incorporated by reference in its entirety.

Neurofibrillary tangles and neurofibrillar filaments (NT) are the major neuropathological markers of Alzheimer's disease (AD). It consists of a microtubule-associated protein τ that has been post-translationally modified, including phosphorylation, deamination, and isomerization of anthraquinone oxime or aspartame sulfhydryl residues. It is produced by the aggregation of hyperphosphorylated protein τ and its conformational isomers. AD shares this pathology with many neurodegenerative tauopathy, in particular with the specified type of frontotemporal dementia (FTD).

Protein τ is a readily soluble "natural unfolded" protein that binds affinity to microtubules (MT) to facilitate its assembly and stability. MT is critical to the cytoskeletal integrity of neurons, and thus is essential for the proper formation and functioning of neuronal circuits and thus for learning and memory. τ and MT Binding is controlled by dynamic phosphorylation and dephosphorylation, as shown primarily in in vitro and non-neuronal cells. Since the number of possible phosphorylation sites is large (>80), the exact contribution of each phosphorylation site in vivo and the identity of the responsible kinase are still largely unclear.

In the AD brain, the formation of tauopathy is later than amyloidosis, and thus may be a response to amyloidosis, thereby constituting the essence of the amyloid cascade hypothesis. This is based on studies of patients with AD and Down syndrome and is indicated by these studies and confirmed by studies of transgenic mice with combined amyloidosis and tauopathy ( Lewis et al, 2001; Oddo et al, 2004; Ribe et al, 2005; Muyllaert et al, 2006, 2008; Terwel et al, 2008).

The exact timing of the two lesions in human AD patients and the mechanisms by which amyloid is associated with tauopathy are largely unknown, but have been proposed to act on the major "τ kinases" GSK3 and cdk5 or by the major "τ Activation of the neuronal signaling pathway by kinases GSK3 and cdk5 (reviewed by Muyllaert et al., 2006, 2008).

The hypothesis that τ lesions are not the only side effects in AD and is the primary pathological performer is based on reliable genetic, pathological, and experimental observations that are well-confirmed with each other: ‧ in amyloid precursor (APP) or presenilin In the early episodes of familial AD caused by mutations in (presenilin), the underlying cause is amyloid accumulation, but the lesions always contain parallel tau lesions, consistent with the case of late-onset sporadic AD cases; The severity of dysfunction and dementia is associated with tauopathy, and with starch It is not related to protein-like lesions. Recently, PIB-PET including amyloid imaging and identification of many "false positives" (normal cognitive individuals with higher brain amyloid load) have been illustrated in several clinical Phase 1 and Phase 2 studies; ‧In familial FTD, tauopathy is induced by mutant tau protein and directly causes neurodegeneration without amyloidosis; ‧in the experimental mouse model, cognitive deficit caused by amyloidosis due to protein τ It does not exist and is almost completely relieved (Roberson et al., 2007).

The combination argument supports the hypothesis that protein τ plays a major role in the cognitive decline of AD and related neurodegenerative tauopathy.

One of the emerging well-known therapies for AD is passive immunotherapy, which uses specific mAbs to remove amyloid peptides and their aggregates that are assumed to be neurotoxic or synaptotoxic.

The immunotherapy targeting the tauopathy proposed herein is expected to counteract the pathological protein t conformational isomer known or presumed to cause synaptic dysfunction and neurodegeneration.

Other therapeutic methods for targeting protein tau are rare and mainly include: ‧ kinase inhibitors, which are thought to enhance the phosphorylation of tau protein to a pathological extent; ‧ compounds that block the accumulation of hyperphosphorylated protein tau in the cytoplasm .

These methods have a number of shortcomings in terms of specificity and efficacy, and are common problems in their attempts to alter the metabolism of APP and amyloid, all of which emphasize continued search for other treatment options (including immunotherapy for tau protein). The importance of it. Indeed, in preclinical mouse models with combined AD-like lesions, although tau protein aggregates persist, immunotherapies targeting amyloid also show effects on tauopathy (Oddo et al., 2004).

There has been some suspicion about the feasibility of immunotherapy to treat intracellular protein τ. Recent experimental studies of mouse models of τ lesions have dispelled this suspicion (Asuni et al., 2007). This study shows that vaccination by phosphorylated peptides derived from protein tau reduces tangles and improves function. These data confirm the immunotherapy and muscle atrophy targeting α-synuclein in Parkinson's Disease (PD) and Lewy body disease models (Masliah et al., 2005, 2011). Previous reports of superoxide dismutase in the lateral lateral sclerosis (ALS) model (Urushitiani et al., 2007). These diseases are examples of intracellular proteins that cause synaptic deficits and neurodegeneration by mechanisms that have not been fully understood to date.

There is still an unmet need for passive and/or active immunotherapy with an effect against pathological protein conformers that are known or presumed to cause neurodegenerative disorders, such as in AD, for example Amyloid lesions caused by aggregates of neurons that over-phosphorylate protein tau, which are as typical for amyloid as for AD.

The present invention satisfies this unmet need, and the present invention provides a binding protein that recognizes and binds to the major pathological phosphorylation epitope of tau protein. In particular, the present invention provides for protein tau, in particular, tau aggregates believed to cause synaptic toxicity and neurotoxicity in tau lesions (including AD) Specific antibodies for linear and conformational, simple and complex phosphorylation epitopes.

Thus, in one embodiment, the invention relates to a binding peptide or protein or a functional part thereof, in particular to an antibody, in particular, to a monoclonal antibody or a functional part thereof, the binding peptide or A protein or antibody recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope on a human tau protein or a fragment thereof, specifically, a phosphorylation antigen on a tau protein aggregate In particular, a phosphorylation epitope on a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein The binding peptide or antibody has high binding affinity to soluble, oligomeric and insoluble phosphorylated tau protein and is capable of detecting and/or regulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo, especially in the brain. The dissociation constant is in particular at least 10 nM, in particular at least 8 nM, in particular at least 5 nM, in particular at least 2 nM, in particular at least 1 nM, in particular at least 500 pM, in particular at least 400 pM, in particular at least 300 pM, in particular at least 200 pM, in particular at least 100 pM, in particular at least 50 pM.

In particular, the dissociation constant ranges from 2 nM to 80 nM, especially from 2 nM to 40 nM, especially from 2 nM to 10 nM.

In one aspect, provided herein is an antibody or a functional fragment thereof, wherein the antibody or antibody fragment binds to an amino acid sequence VYKSPVVSGDTSPRHL (SEQ ID NO: 62) (SEQ ID NO: 67 of τ aa 393-408 , for example, listed in Table 1 or phosphoric acid in the amino acid sequence The epitope is ligated, and the amino acid sequence comprises a phosphorylated Ser at position 396 (pS396) and at position 404 (pS404).

In a second embodiment, the invention relates to a binding peptide or protein or a functional part thereof, in particular to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein tau conformer, but in one embodiment, does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody pair is soluble, Oligomeric and insoluble phosphorylated tau proteins have high binding affinity and are capable of detecting and/or modulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and having a 10 ⁴ M ^-1 s ^-1 or greater than 10 ⁴ M ^-1 s ^-1 association rate constant, especially 3 x 10 ⁴ M ^-1 s ^-1 to 5 × 10 ⁴ M ^-1 s ^-1 or greater than 5 × 10 ⁴ M ^-1 s ^-1 , especially 10 ⁵ M ^-1 s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 , especially 2 × 10 ⁵ M ^-1 s ^-1 Up to 9×10 ⁵ M ^-1 s ^-1 or greater than 9×10 ⁵ M ^-1 s ^-1 , especially 10 ⁶ M ^-1 s ^-1 or greater than 10 ⁶ M ^-1 s ^-1 , especially 1 × 10 ⁶ to 4 × 10 ⁶ M ^-1 s ^-1 or greater than 4 × 10 ⁶ M ^-1 s ^-1 , especially 10 ⁷ M ^-1 s ^-1 or an association rate constant greater than 10 ⁷ M ^-1 s ^-1 .

In particular, the association rate constant ranges from 1.6 x 10 ³ to 5 x 10 ⁵ , especially from 2.4 x 10 ⁴ to 5 x 10 ⁵ , from 3 x 10 ³ to 5 x 10 ⁵ .

In a third embodiment, the invention relates to a binding peptide or protein or a functional part thereof, in particular to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligo Poly- and insoluble phosphorylated tau protein has high binding affinity and is capable of detecting and/or regulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and has high binding affinity with a dissociation constant of at least 4 nM and association The rate constant is 10 ⁵ M ^-1 s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 , in particular, the dissociation constant is at least 3 nM and the association rate constant is 10 ⁶ M ^-1 s ^-1 or greater than 10 ⁶ M ^-1 s ^-1 , in particular, the dissociation constant is at least 2 nM and the association rate constant is 10 ⁴ M ^-1 s ^-1 or greater than 10 ⁴ M ^-1 s ^-1 , in particular, the dissociation constant is at least 1 nM and the association rate constant is 10 ⁵ M ^-1 s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 , in particular, the dissociation constant is at least 200 pM and the association rate constant is 10 ⁵ M ^-1 s ^-1 Or greater than 10 ⁵ M ^-1 s ^-1 , in particular, the dissociation constant is at least 100 pM and the association rate constant is 10 ⁶ M ^-1 s ^-1 or greater than 10 ⁶ M ^-1 s ^-1 .

In particular, the dissociation constant is in the range of 2 nM to 80 nM and the association rate constant is in the range of 1.6 × 10 ³ to 5 × 10 ⁵ , in particular, the dissociation constant is in the range of 2 nM to 40 nM and the association rate The constant is in the range of 2.4 × 10 ⁴ to 5 × 10 ⁵ , specifically, the dissociation constant is in the range of 2 nM to 10 nM and the association rate constant is in the range of 3 × 10 ³ to 5 × 10 ⁵ .

One embodiment (4) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, one A monoclonal antibody or a functional portion thereof, in particular, a binding peptide or antibody of any of the above embodiments, which recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, human tau protein a phosphorylation epitope on a fragment or a fragment thereof, specifically binding to a pathological protein tau conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated antigen a peptide, wherein the binding peptide or antibody has high binding affinity for soluble, oligomeric and insoluble phosphorylated tau protein and is capable of detecting and/or modulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding The peptide or antibody binds to a mammalian epitope, in particular, an epitope on the human tau protein as set forth in SEQ ID NO: 67, selected from the group consisting of: a phosphorylated Ser at position 396 τ aa 393-401 of pS396); τ aa 396-401 comprising phosphorylated Ser (pS396) at position 396; τ aa 394-400 comprising phosphorylated Ser (pS396) at position 396; phosphorus at position 404 Of Ser (pS404) of τ aa 402-406: and phosphorylation at position 396 comprising Ser (pS396) of τ aa 393-400.

An embodiment (5) is the binding peptide or antibody of any of the above embodiments, wherein the peptide binds to a mammalian epitope, in particular, an epitope on a human tau protein, but particularly as SEQ ID NO :67, the epitope on human tau protein, SEQ ID NO: 67 comprising τ aa 393-401 comprising phosphorylated Ser (pS396) at position 396.

An embodiment (6) is the binding peptide or antibody of any of the above embodiments, wherein the peptide binds to a mammalian epitope, in particular, an epitope on a human tau protein, but in particular as SEQ ID NO :67 The epitope on human tau protein, SEQ ID NO: 67 comprises τ aa 396-401 comprising a phosphorylated Ser (pS396) at position 396.

An embodiment (7) is the binding peptide or antibody of any of the above embodiments, wherein the peptide binds to a mammalian epitope, in particular, an epitope on a human tau protein, but particularly as SEQ ID NO :67 The epitope on human tau protein, SEQ ID NO: 67 comprises τ aa 394-400 comprising phosphorylated Ser (pS396) at position 396.

An embodiment (8) is the binding peptide or antibody of any of the above embodiments, wherein the peptide binds to a mammalian epitope, in particular, an epitope on a human tau protein, but particularly as SEQ ID NO An epitope on human tau protein as indicated at 67, comprising τ aa 402-406 containing phosphorylated Ser (pS404) at position 404.

An embodiment (9) is the binding peptide or antibody of any of the above embodiments, wherein the peptide binds to a mammalian epitope, in particular, an epitope on a human tau protein, but particularly as SEQ ID NO :67 The epitope on human tau protein, SEQ ID NO: 67 comprises τ aa 393-400 comprising phosphorylated Ser (pS396) at position 396.

An embodiment (10) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to the pathological protein τ conformer, but in one embodiment does not bind to the corresponding Phosphorylating epitopes and/or not binding to an unrelated epitope, wherein the binding peptide or antibody has high binding affinity for soluble, oligomeric and insoluble phosphorylated tau proteins and is capable of detecting and/or modulating solubility, oligomerization and The insoluble phosphorylated tau protein is in vivo and wherein the binding peptide or antibody comprises: a first binding domain comprising CDR1 having the amino acid sequence of SEQ ID NO: 73 in sequence, having SEQ CDR2 of the amino acid sequence of ID NO: 74 and CDR3 having the amino acid sequence of SEQ ID NO: 75, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, especially at least 96%, especially at least 97%, especially at least 98%, especially at least 99% or 100% identical amine a base acid sequence; and/or a second binding domain comprising, in sequence, a CDR1 having the amino acid sequence set forth in SEQ ID NO: 70 or at least 95%, especially 98%, especially 99% identical thereto An amino acid sequence having the CDR2 of the amino acid sequence set forth in SEQ ID NO: 71 or a 94%, 95%, 96%, 97%, 98% or 99% less amino acid sequence, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 72 or at least 60%, at least 70% thereof At least 80%, in particular at least 85%, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98% In particular, at least 99% or 100% identical amino acid sequence.

In one aspect, provided herein is an antibody or a functional fragment thereof, which antibody recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: (a) a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and comprising SEQ ID NO: 75 CDR3 of the amino acid sequence shown; and/or (b) a second binding domain comprising an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 70, comprising SEQ ID NO: CDR2 of the amino acid sequence shown at 71 and CDR3 comprising the amino acid sequence of SEQ ID NO:72.

An embodiment (11) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, specifically, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligo The poly- and insoluble phosphorylated tau protein has high binding affinity and is capable of detecting and/or regulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises: a first binding domain, the first a binding domain comprising, in sequence, a CDR1 having the amino acid sequence set forth in SEQ ID NO: 81, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 82, and having SEQ ID NO: 83 CDR3 of the amino acid sequence shown, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% identical amino acid sequence; and/or a second binding domain, The second binding domain comprises, in sequence, a CDR1 having the amino acid sequence set forth in SEQ ID NO: 78, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 79, and an amine group having the SEQ ID NO: 80 CDR3 of the acid sequence, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, In particular, at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100%, of the amino acid sequence is identical.

In one aspect, provided herein is an antibody or a functional fragment thereof, which antibody recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: (a) a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and comprising SEQ ID NO: 83 a CDR3 comprising an amino acid sequence; and/or (b) comprising a second binding domain of an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, comprising SEQ ID NO: 79 CDR2 of the amino acid sequence shown and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:80.

An embodiment (12) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above Example knot a peptide or antibody that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, in particular, binds To a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligomeric, and insoluble The phosphorylated tau protein has high binding affinity and is capable of detecting and/or modulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises: a first binding domain, the first binding domain CDR1 having the amino acid sequence shown in SEQ ID NO: 93, CDR2 having the amino acid sequence shown in SEQ ID NO: 94, and CDR3 having the amino acid sequence shown in SEQ ID NO: 95 are sequentially included. Or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, with any of the above CDRs, Especially at least 96%, especially at least 97%, especially at least 98%, especially at least 99% or 100% identical amino acid sequence; and/or a second binding domain comprising the amino acid of SEQ ID NO: 89 in sequence CDR1 of the sequence, CDR2 having the amino acid sequence set forth in SEQ ID NO: 90, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 91, or at least 60%, at least 70%, with any of the above CDRs, At least 80%, in particular at least 85%, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, Especially at least 99% or 100% identical amino acid sequence.

In a certain aspect, an antibody or a functional portion thereof is provided herein, The antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: (a) a first binding domain comprising SEQ ID NO: 93 CDR1 of the amino acid sequence shown, comprising CDR2 of the amino acid sequence set forth in SEQ ID NO: 94 and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95; and/or (b) comprising an amine A second binding domain of a base acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and comprising SEQ ID NO: 91 The CDR3 of the amino acid sequence shown.

An embodiment (13) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligo The poly- and insoluble phosphorylated tau protein has high binding affinity and is capable of detecting and/or regulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises: a first binding domain, the first a binding domain comprising, in sequence, a CDR1 having the amino acid sequence set forth in SEQ ID NO: 101, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 102, and having SEQ ID NO: 103 Shown CDR3 of the amino acid sequence, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94% with any of the above CDRs a semi-amino acid sequence, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100%; and/or a second binding domain, the second binding domain being sequentially CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98, CDR2 having the amino acid sequence set forth in SEQ ID NO: 99, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 100, or At least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, especially at least with any of the above CDRs 96%, especially at least 97%, especially at least 98%, especially at least 99% or 100% identical amino acid sequence.

In one aspect, provided herein is an antibody or a functional fragment thereof, which antibody recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: (a) a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 101, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 102, and comprising SEQ ID NO: 103 a CDR3 comprising an amino acid sequence; and/or (b) comprising a second binding domain of an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98, comprising SEQ ID NO: CDR2 of the amino acid sequence shown at 99 and CDR3 comprising the amino acid sequence of SEQ ID NO: 100.

One embodiment (14) of the present invention relates to a binding peptide or protein or The functional part, in particular, relates to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, a binding peptide or antibody of any of the above examples, which recognizes and specifically binds Phosphorylation epitopes to mammals, in particular, phosphorylated epitopes that bind to human tau proteins or fragments thereof, in particular, to pathological protein t conformational isomers, but in one embodiment Does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody has high binding affinity for soluble, oligomeric and insoluble phosphorylated tau protein and is capable of detecting and/or Modulating the amount of soluble, oligomeric, and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises: a first binding domain comprising the amino acid of SEQ ID NO: 106 in sequence CDR1 of the sequence, CDR2 having the amino acid sequence set forth in SEQ ID NO: 107, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 108, or at least 60% to any of the above CDRs, 70% less, at least 80%, in particular at least 85%, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular At least 98%, especially at least 99% or 100% identical amino acid sequence; and/or a second binding domain comprising, in sequence, CDR1 having the amino acid sequence set forth in SEQ ID NO:89 CDR2 having the amino acid sequence set forth in SEQ ID NO: 115 and CDR3 having the amino acid sequence set forth in SEQ ID NO: 91, or at least 60%, at least 70%, at least 80%, or any of the above CDRs In particular at least 85%, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular At least 98%, especially at least 99% or 100% identical amino acid sequence.

In a particular aspect, provided herein is an antibody or a functional fragment thereof, which antibody recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises (a) a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 106, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 107, and comprising SEQ ID NO: 108 a CDR3 of the amino acid sequence shown; and/or (b) a second binding domain comprising an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, comprising SEQ ID NO CDR2 of the amino acid sequence shown by 115 and CDR3 comprising the amino acid sequence shown by SEQ ID NO:91.

In a certain embodiment, the first binding domain of an antibody or antibody fragment thereof described herein is a light chain variable region, and the second binding domain of an antibody or antibody fragment thereof described herein is a heavy chain variable region.

In another embodiment (15), the present invention relates to a binding peptide or protein or a functional portion thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional portion thereof, in particular, A binding peptide or antibody according to any of the above embodiments, wherein the binding peptide or antibody recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof , in particular, binds to a pathological protein tau conformer, but in one embodiment does not bind to the corresponding unphosphorylated epitope and/or does not bind to an unrelated antigen a peptide, wherein the binding peptide or antibody has high binding affinity for soluble, oligomeric and insoluble phosphorylated tau protein and is capable of detecting and/or modulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding The peptide or antibody comprises a first binding domain comprising an amino acid sequence as set forth in SEQ ID NO: 69, 77, 116/92, 118, 97, 105, or in particular at least 85%, in particular at least 90 thereto %, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, especially at least 96%, especially at least 97%, especially at least 98%, especially at least 99% or 100% identical amino acids a sequence; and/or a second binding domain comprising an amino acid sequence of SEQ ID NO: 68, 76, 88, 96, 104, or at least 80%, especially at least 85%, especially at least 86%, in particular at least 87%, in particular at least 88%, in particular at least 89%, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular At least 97%, especially at least 98%, especially at least 99% or 100% identical amino acid sequence.

An embodiment (16) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, specifically, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligo Poly- and insoluble phosphorylated tau protein Having high binding affinity and capable of detecting and/or modulating the amount of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises a first binding domain comprising SEQ ID NO: The amino acid sequence shown at 69, or an amino acid sequence at least 98% or 99% identical thereto; and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO:68 Or an amino acid sequence that is at least 90%, 91%, 92% or 93% identical thereto.

An embodiment (17) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligo The poly- and insoluble phosphorylated tau protein has high binding affinity and is capable of detecting and/or modulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises a first binding domain, the first The binding domain comprises an amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence at least 93%, 94% or 95% identical thereto; and/or a second binding domain, the second binding Comprising SEQ ID NO: 76 of the amino acid sequence shown in, or in connection with at least 88%, 89% or 90% identical to the amino acid sequence.

One embodiment (18) of the present invention relates to a binding peptide or protein or The functional part, in particular, relates to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, a binding peptide or antibody of any of the above examples, which recognizes and specifically binds Phosphorylation epitopes to mammals, in particular, phosphorylated epitopes that bind to human tau proteins or fragments thereof, in particular, to pathological protein t conformational isomers, but in one embodiment Does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody has high binding affinity for soluble, oligomeric and insoluble phosphorylated tau protein and is capable of detecting and/or Regulating the amount of soluble, oligomeric, and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises a first binding domain comprising an amino acid of SEQ ID NO: 116, 92 or 118 a sequence, or an amino acid sequence at least 93%, 94% or 95% identical thereto; and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 88, or At least 90%, 91%, 92% or 93% identical amino acid sequence.

An embodiment (19) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, specifically, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligo Poly- and insoluble phosphorylated tau protein Having high binding affinity and capable of detecting and/or modulating the amount of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises a first binding domain comprising SEQ ID NO: The amino acid sequence shown at 97, or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 96, or At least 86%, 87%, 88% or 90% identical amino acid sequence.

An embodiment (20) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above A binding peptide or antibody of an embodiment that recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically Binding to a pathological protein τ conformer, but in one embodiment does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody is soluble, oligo The poly- and insoluble phosphorylated tau protein has high binding affinity and is capable of detecting and/or modulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding peptide or antibody comprises a first binding domain, the first The binding domain comprises an amino acid sequence set forth in SEQ ID NO: 105, or an amino acid sequence at least 98% or 99% identical thereto; and/or a second binding domain, the second binding domain comprising SEQ ID NO: 104 of the amino acid sequence shown in, or in connection with at least 88%, 89% or 90% identical to the amino acid sequence.

One embodiment (21) of the present invention relates to a binding peptide or protein or The functional part, in particular, relates to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, a binding peptide or antibody of any of the above embodiments, wherein the binding peptide or antibody comprises a first binding a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 69, or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising SEQ ID NO The amino acid sequence shown at 68, or an amino acid sequence which is at least 93% identical thereto.

An embodiment (22) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above The binding peptide or antibody of the embodiment, wherein the binding peptide or antibody comprises a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence at least 95% identical thereto And/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence at least 90% identical thereto.

An embodiment (23) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above The binding peptide or antibody of the embodiment, wherein the binding peptide or antibody comprises a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 116, 92 or 118, or at least 93%, 95 thereto a % identical amino acid sequence; and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 88, or an amino acid sequence at least 93% identical thereto.

One embodiment (24) of the present invention relates to a binding peptide or protein or The functional part, in particular, relates to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, a binding peptide or antibody of any of the above embodiments, wherein the binding peptide or antibody comprises a first binding a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 97, or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising SEQ ID NO The amino acid sequence shown in 96, or an amino acid sequence which is at least 90% identical thereto.

An embodiment (25) of the present invention relates to a binding peptide or protein or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, any of the above A binding peptide or antibody according to the embodiment, wherein the binding peptide or antibody comprises a first binding domain comprising an amino acid sequence set forth in SEQ ID NO: 105, or an amine at least 98% or 99% identical thereto a base acid sequence; and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 104, or an amino acid sequence at least 90% identical thereto.

An embodiment (26) of the present invention relates to a binding peptide or protein according to embodiment (16) or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, Wherein the first binding domain comprises a CDR as set forth in SEQ ID NOs: 73-75 and the second binding domain comprises a CDR as set forth in SEQ ID NOs: 70-72.

One embodiment (27) of the present invention relates to a binding peptide or protein according to embodiment (17) or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, Wherein the first binding domain comprises a CDR as set forth in SEQ ID NOs: 81-83, and the second junction The domain contains the CDRs as set forth in SEQ ID NOs: 78-80.

An embodiment (28) of the present invention relates to a binding peptide or protein according to embodiment (18) or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, Wherein the first binding domain comprises a CDR as set forth in SEQ ID NOs: 93-95 and the second binding domain comprises a CDR as set forth in SEQ ID NOs: 89-91.

An embodiment (29) of the present invention relates to a binding peptide or protein according to embodiment (19) or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, Wherein the first binding domain comprises a CDR as set forth in SEQ ID NOs: 101-103 and the second binding domain comprises a CDR as set forth in SEQ ID NOs: 98-100.

An embodiment (30) of the present invention relates to a binding peptide or protein according to embodiment (18) or a functional part thereof, in particular, to an antibody, in particular, a monoclonal antibody or a functional part thereof, Wherein the first binding domain comprises the CDRs set forth in SEQ ID NOs: 89, 115 and 91 and the second binding domain comprises the CDRs set forth in SEQ ID NOs: 106-108.

In another embodiment (31), the invention relates to a binding peptide or protein or a functional part thereof, in particular to an antibody, in particular, a monoclonal antibody or a functional part thereof, in particular, A binding peptide or antibody according to any of the above embodiments, wherein the binding peptide or antibody recognizes and specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof , in particular, binds to a pathological protein tau conformer, but in one embodiment does not bind to the corresponding unphosphorylated epitope and/or does not bind to an unrelated antigen a peptide, wherein the binding peptide or antibody has high binding affinity for soluble, oligomeric and insoluble phosphorylated tau protein and is capable of detecting and/or modulating the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo and wherein the binding The peptide or antibody comprises: a. a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 69 and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 68; or b. a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 77 and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 76; or c. comprising SEQ ID NO: 116 a first binding domain of the amino acid sequence shown and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 88; or d. comprising the amino acid sequence set forth in SEQ ID NO: 92 a first binding domain and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 88; or e. a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 97 and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 96; or f. a first binding comprising the amino acid sequence set forth in SEQ ID NO: 105 a domain and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 104; g. a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 118 and/or comprising SEQ ID NO Second binding of the amino acid sequence shown in :88 area.

In one embodiment (32) of the present invention, the binding peptide of any of the above embodiments is an antibody, specifically, an antibody having the same type of IgG2a, IgG2b or IgG3, in particular, a plurality of antibodies, monoclonal antibodies, and embedded antibodies Antibody, humanized antibody or fully human antibody.

One embodiment (33) of the invention pertains to a polynucleotide encoding a binding peptide of any of the preceding embodiments.

In one embodiment (34), the polynucleotide comprises a nucleic acid molecule selected from the group consisting of: a. a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising SEQ ID NO: 84-87 , amino acid sequences of SEQ ID NOS: 109-114, 117 and 119; b. comprising at least 85 of the sequences set forth in SEQ ID NO: 84-87, SEQ ID NOs: 109-114, 117 and 119 Nucleic acid molecule having a nucleotide sequence of % sequence identity; c. a nucleoside comprising at least 90% sequence identity to the sequences set forth in SEQ ID NO: 84-87, SEQ ID NOs: 109-114, 117 and 119 a nucleic acid molecule of an acid sequence; d. a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the sequences set forth in SEQ ID NO: 84-87, SEQ ID NOs: 109-114, 117 and 119; a nucleic acid molecule comprising a nucleotide sequence having at least 98% sequence identity to the sequences set forth in SEQ ID NO: 84-87, SEQ ID NOs: 109-114, 117 and 119; f. comprising and SEQ ID NO: a nucleic acid molecule having a nucleotide sequence of at least 99% sequence identity as shown in SEQ ID NOS: 109-114, 117 and 119; g. a nucleic acid molecule comprising a nucleotide sequence a complementary strand of the nucleotide sequence that hybridizes to any of a)-f); h. a nucleic acid molecule comprising a nucleotide sequence as defined by any of a)-g) The nucleotide sequence differs in the degeneracy of the genetic code, wherein the nucleic acid molecule as defined in any one of a)-h) recognizes and specifically binds to a mammalian phosphorylation epitope, in particular a phosphorylation epitope on human tau protein or a fragment thereof, in particular, a phosphorylation epitope on human tau protein as set forth in SEQ ID NO: 67, selected from the group consisting of: at position 396 τ aa 393-401 comprising phosphorylated Ser (pS396); τ aa 396-401 comprising phosphorylated Ser (pS396) at position 396; τ aa 394-400 comprising phosphorylated Ser (pS396) at position 396; 404 comprising τ aa 402-406 phosphorylated Ser (pS404); and τ aa 393-400 comprising phosphorylated Ser (pS396) at position 396, wherein, in one embodiment, the binding peptide has high binding affinity, The dissociation constant is at least 10 nM, in particular, at least 8 nM, in particular, at least 5 nM, in particular, at least 2 nM In particular, at least 1 nM, in particular, at least 500 pM, in particular, at least 400 pM, in particular, at least 300 pM, in particular, at least 200 pM, in particular, at least 100 pM, specific words , at least 50 pM, and/or its association rate constant is 10 ⁴ M ^-1 s ^-1 or greater than 10 ⁴ M ^-1 s ^-1 , in particular, 3 × 10 ⁴ to 5 × 10 ⁴ M ^-1 s ^-1 or greater than 5 × 10 ⁴ M ^-1 s ^-1 , in particular, 10 ⁵ M ^-1 s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 , in particular, 6 × 10 ⁵ to 9 × 10 ⁵ M ^-1 s- ¹ or greater than 9×10 ⁵ M ^-1 s ^-1 , in particular, 10 ⁶ M ^-1 s ^-1 or greater than 10 ⁶ M ^-1 s ^-1 , in particular, 1 × 10 ⁶ to 4 × 10 ⁶ M ^-1 s ^-1 or greater than 4 × 10 ⁶ M ^-1 s ^-1 , in particular, 10 ⁷ M ^-1 s ^-1 or greater than 10 ⁷ M ^-1 s ^-1 , but In one embodiment, the corresponding unphosphorylated epitope is not bound and/or does not bind to an unrelated epitope.

In various embodiments (35) of the present invention, there is provided a binding peptide according to any of the above embodiments, or a functional part thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional part thereof, or a poly Nucleotide, or a combination thereof, capable of specifically recognizing and binding to a mammalian phosphorylation epitope, in particular, human tau protein, in particular, microtubule-associated protein τ, in particular, microtubule-associated Hyperphosphorylation of protein τ aggregates, such as in the form of paired helical filaments (PHF), which are neurofibrillary tangles, neurofibrillar filaments, and major structures in dystrophic neurites An epitope, but in one embodiment, does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope.

In a particular embodiment (36) of the invention, the human tau protein is the human tau protein as set forth in SEQ ID NO:67.

The binding peptides and antibodies according to any of the above embodiments are therefore useful for reducing total soluble tau protein, in particular soluble phosphorylated tau protein, in mammals or humans containing higher levels of soluble tau protein and/or soluble phosphorylated tau protein The content of the brain, specifically in the cerebral cortex and / or hippocampus (37).

The binding peptides and antibodies according to any of the above examples can also be used to reduce the inclusion of a pair of helical filaments containing hyperphosphorylated tau protein (pTau PHF). The amount of such pTau-paired helical filaments in the brain of a mammal or human, specifically in the cerebral cortex and/or hippocampus (38).

Decreasing total soluble tau protein and/or soluble phosphorylated tau protein and/or pTau paired helical filaments in the brain of a mammal or human containing a higher level of such tau protein variants, specifically in the cerebral cortex And/or the content in the hippocampus may ameliorate and/or alleviate symptoms associated with a tau protein-related disease, disorder or condition (39), which result in such tau protein-related diseases in the mammal or human , illness or condition.

The binding peptides and antibodies according to any of the above embodiments are therefore useful in therapy, in particular, in human therapy to slow or prevent progression of a tau protein related disease, disorder or condition (40).

The binding peptides and antibodies according to any of the above embodiments may further be used in therapy, in particular, in human therapy to ameliorate or alleviate symptoms associated with a tau protein-related disease, disorder or condition (41), such as cognition Function (including reasoning, situational judgment, memory ability, learning, special navigation, etc.) obstacles or loss.

In one embodiment (42), the invention relates to a binding peptide and antibody according to any of the above embodiments for use in therapy, in particular for the treatment of tauopathy (a group of tau protein related diseases and disorders) Or relieve symptoms associated with tauopathy.

In one embodiment (43), the invention relates to a binding peptide and antibody according to any of the above embodiments for use in maintaining or enhancing the cognitive memory capacity of a mammal suffering from a tauopathy.

In another specific embodiment (44) of the invention, the binding peptide and antibody package At least one or all of the light chain CDRs comprising: ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, ACI-35-4A6-Ab1, ACI-35-4A6-Ab2, ACI-35 -1D2-Ab1, ACI-35-2G5-Ab1, ACI-35-2G5-Ab2, ACI-35-2G5-Ab3 (eg SEQ ID NOs: 73-75, 81-83, 93-95, 101-103, And at least one or all of the heavy chain CDRs of the following antibodies: ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, ACI-35-4A6-Ab1, ACI- 35-4A6-Ab2, ACI-35-1D2-Ab1, ACI-35-2G5-Ab1, ACI-35-2G5-Ab2, ACI-35-2G5-Ab3 (SEQ ID NOs: 70-72, 78-80, 89-91, 98-100 (89, 115, 91)), such binding peptides and antibodies are used in therapy, in particular, in human therapy to ameliorate or alleviate diseases, disorders or conditions associated with tau protein Related symptoms, such as cognitive function (reasoning, situational judgment, memory, learning, special navigation, etc.) are obstacles or loss.

In another specific embodiment (45) of the invention, the binding peptide and antibody comprise the light chain CDRs of the antibodies ACI-35-2G5-Ab2, ACI-35-2G5-Ab3 as set forth in SEQ ID NOs: 106-108 At least one or all of and/or at least one or all of the heavy chain CDRs of the antibodies ACI-35-2G5-Ab2, ACI-35-2G5-Ab3 as set forth in SEQ ID NOs: 89, 115 and 91 The binding peptides and antibodies are used in therapy, in particular, in human therapy to ameliorate or alleviate symptoms associated with a tau protein-related disease, disorder or condition, such as cognitive function (reasoning, situational judgment, memory, Learning, special navigation, etc.) obstacles or loss.

The tau protein entanglement and pTau of the peptide or antibody according to the above examples are bound to the brain by applying a protein immunoreactivity test to the selected brain slice, respectively. And determined by Western blot analysis of brain tissue homogenates, as described in the examples.

In another embodiment (46), the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a binding peptide according to any of the above embodiments, or a functional portion thereof, specifically an antibody, specifically a monoclonal antibody or A functional portion thereof, or a polynucleotide or a combination thereof, and a pharmaceutically acceptable carrier.

In one embodiment (47), a binding peptide according to any of the above embodiments, or a functional portion thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional portion thereof, or a polynucleotide, or a pharmaceutical combination , or a combination thereof, for use in therapy, in particular, in human therapy to treat or ameliorate symptoms of a disease or condition associated with tau protein, including neurodegenerative disorders, such as tauopathy.

The binding peptide, antibody and/or pharmaceutical composition according to any of the above embodiments can therefore be used to slow or prevent the onset of tau protein after administration to an animal suffering from the disease or condition (specifically, mammals, especially humans) Progress in related diseases, disorders or conditions (48).

The binding peptide, antibody and/or pharmaceutical composition according to any of the above embodiments may be further used for improvement after administration to an animal (in particular, a mammal, especially a human) suffering from a disease, condition or condition associated with tau protein Or relieve symptoms or symptoms associated with the disease or condition (49), such as cognitive function (including reasoning, situational judgment, memory ability, learning, specific navigation, etc.).

In one embodiment (50), a binding peptide according to any of the above embodiments, or a functional portion thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional portion thereof, or a polynucleotide, or a pharmaceutical combination Object, or a combination thereof, for Treatment of diseases and conditions caused by or formation of neurofibrillary lesions, neurofibrillary lesions are the main brain lesions in tauopathy, and tauopathy contains a group of heterogeneous neurodegenerative diseases or conditions, including the appearance of τ Diseases or conditions of lesions and amyloidosis, including but not limited to Alzheimer's disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down syndrome, Gustman - Gerstmann-Sträussler-Scheinker disease, inclusion body myositis and prion protein cerebral amyloid angiopathy, traumatic brain injury and other diseases that do not show different amyloidosis or Symptoms, including but not limited to, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, non-Guam motoneuron disease with neurofibrillary tangles, Argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification , associated with chromosome 17 related Parkinson's disease, temporal lobe dementia, Halleworden-Spatz disease, multiple systemic atrophy, type C Niemann-Pick disease (Niemann- Pick disease, type C), Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical neuroglial proliferative disease, progressive supranuclear palsy, subacute Sclerosing panencephalitis, only Tangle-only dementia, post-encephalopathy Parkinson's disease (Postencephalitic Parkinsonism), atrophic muscle rigidity.

In one embodiment (51), a binding peptide according to any of the above embodiments or Its functional part, in particular, an antibody, in particular, a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, or a combination thereof, is used for the treatment of Alzheimer's disease.

In one embodiment (52) of the present invention, there is provided for detecting and/or modulating soluble and/or oligomeric and/or insoluble phosphorylated tau protein, especially in vivo, especially in animals (specifically, breastfeeding Method for the content of the brain, especially in the cerebral cortex and/or hippocampus, of the animal or human), comprising administering to the animal, in particular, to the mammal or human, a binding peptide according to any of the above examples or Its functional part, in particular, an antibody, in particular, a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, or a combination thereof.

In one aspect, regulation refers to the reduction of soluble tau protein, particularly soluble phosphorylated tau protein, in animals (in particular, mammals or humans) that contain higher levels of soluble tau protein and/or soluble phosphorylated tau protein. The content of the brain (specifically, the cerebral cortex and/or the hippocampus).

In one embodiment (53) of the present invention, there is provided a method for reducing insoluble tau protein (specifically, a pair of helical filaments (pTau PHF) containing a hyperphosphorylated tau protein) containing a higher content of insoluble tau Method for the content of the brain (specifically, the cortex and/or hippocampus) of an animal (specifically, pTau-paired filament filament (pTau PHF)) (specifically, mammalian or human) Including administering to the animal (specifically, to the mammal or human) a binding peptide according to any of the above embodiments, or a functional portion thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional portion thereof Or a polynucleotide or pharmaceutical composition, or a combination thereof.

In one embodiment (54), the invention relates to a method of slowing or preventing the progression of a tau protein-related disease, disorder or condition in an animal, in particular, a mammal or a human, comprising administering to the disease or disease The animal (specifically, the mammal or human) is administered a binding peptide according to any of the above embodiments or a functional portion thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional portion thereof, or A polynucleotide or pharmaceutical composition, or a combination thereof.

In one embodiment (55), the present invention relates to a symptom (such as cognitive, (including reasoning,) for improving or alleviating an animal (in particular, a mammal or a human) associated with a tau protein-related disease, disorder, or condition. A method of conditional judgment, memory ability, learning, special navigation, etc.), comprising administering to the animal (in particular, the mammal or human) suffering from the disease or condition according to any of the above embodiments A binding peptide or a functional portion thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional portion thereof, or a polynucleotide or pharmaceutical composition, or a combination thereof.

In one embodiment (56), the invention relates to a method of maintaining or enhancing the cognitive memory capacity of a mammal suffering from a tauopathy.

In another embodiment (57) of the present invention, there is provided a method of treating a tau protein-related disease or condition, including a neurodegenerative disease or condition, such as a tauopathy, comprising administering to an animal suffering from the disease or condition (specifically stated) Administering to a mammal, but especially to a human, a binding peptide according to any of the above embodiments, or a functional part thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional part thereof, or a polynucleotide Or a pharmaceutical composition, or a combination thereof.

In one embodiment (58) of the invention, a treatment is provided by a neuron A method of causing or causing diseases and conditions associated with the formation of neurofibrillary lesions, neurofibrillary lesions are the major brain lesions in tauopathy, and tauopathy includes a group of heterogeneous neurodegenerative diseases or conditions, including the appearance of tauopathy and A disease or condition of amyloidosis, including (but not limited to) Alzheimer's disease, CJD, boxer dementia, Down's syndrome, Gustman-Stossler-Syck disease, inclusion Myositis and prion protein brain amyloid angiopathy, traumatic brain injury and other diseases or conditions that do not show different amyloidosis, including but not limited to amyotrophic lateral sclerosis/Guam-type Parkinson's Syndrome-dementia complex, non-Guam motoneuron disease with neurofibrillary tangles, argyrophilic dementia, cortical basal ganglia degeneration, diffuse neurofibrillary tangles with calcification, associated with chromosome 17 Parkinson's disease, temporal lobe dementia, Hallerwalden-Schappz disease, multiple systemic atrophy, type C Niemann-Pick disease, globus pallidus-ponsio-denebral degeneration, Pick's disease, Enter Subcortical neuroglial proliferative disease, progressive nuclear paralysis, subacute sclerosing panencephalitis, only tangled dementia, Parkinson's disease after encephalitis, atrophic myotonia, the method involves the disease or condition The animal (specifically, to a mammal, but especially to a human) is administered a binding peptide according to any of the above embodiments or a functional part thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional part thereof, Or a polynucleotide or pharmaceutical composition, or a combination thereof.

In another embodiment (59) of the present invention, there is provided a method of inducing a passive immune response in an animal (in particular, a mammal or a human) suffering from a neurodegenerative disorder (such as a tauopathy) by The animal or human is administered a binding peptide according to any of the above embodiments or a functional portion thereof, specific In other words, an antibody, in particular, a monoclonal antibody or a functional portion thereof, or a polynucleotide or pharmaceutical composition, or a combination thereof.

In another embodiment (60) of the present invention, a method of diagnosing a tau protein-related disease, disorder or condition in a patient, comprising detecting a binding peptide or an active fragment thereof according to any of the above embodiments (specifically) , the antibody, in particular, the monoclonal antibody or a functional portion thereof, immunospecific binding to the epitope of the tau protein in the sample or in situ, comprising the steps of: a. making a sample suspected of containing tau protein or specific A body part or a body part is contacted with a binding peptide according to any of the above technical means or a fragment thereof (specifically, an antibody, in particular, a monoclonal antibody or a functional part thereof), wherein the binding peptide or antibody or fragment thereof binds to τ An antigenic determinant; b. binding the binding peptide (specifically, the antibody, in particular, the monoclonal antibody or a functional portion thereof) to the tau protein to form an immune complex; c. detecting the immune complex The formation of a substance; and d. obtaining the correlation of the presence or absence of the immune complex with the presence or absence of tau protein in the sample or a particular body part or region.

In another embodiment (61) of the present invention, a method of diagnosing a predisposition of a tau protein-related disease, disorder, or condition in a patient, comprising detecting a binding peptide according to any of the above embodiments or An immunospecific binding of an active fragment (specifically, an antibody, in particular, a monoclonal antibody or a functional portion thereof) to an epitope of a tau protein in a sample or in situ, comprising the steps of: a. causing a suspected τ a sample or specific body part or body region of an antigen and a binding peptide or active fragment thereof according to any of the above embodiments (specifically , the antibody, in particular, the monoclonal antibody or a functional portion thereof, is contacted, the peptide or fragment thereof binds to the epitope of the tau protein; b. makes the binding peptide (specifically, the antibody, specifically, the a monoclonal antibody or a functional portion thereof) binds to a tau antigen to form an immune complex; c. detects the formation of the immune complex; and d. obtains the presence or absence of the immune complex with a sample or a specific body part or region Correlation of the presence or absence of a tau antigen; e. Comparison of the amount of the immune complex with a normal control value; wherein the amount of the aggregate is greater than the normal control value, indicating that the patient is suffering from a tau protein related disease Or the condition or at risk of developing a disease or condition associated with tau protein.

In another embodiment (62) of the present invention, there is provided a binding peptide according to any of the above embodiments, or a functional portion thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional portion thereof, Or a method of monitoring a microscopic residual disease after treatment with a polynucleotide, or a pharmaceutical composition, wherein the method comprises: a. combining a sample or a specific body part or body region suspected of containing a tau antigen with any of the above embodiments The peptide or a functional part thereof (specifically, an antibody, in particular, a monoclonal antibody or a functional part thereof) is contacted, and the peptide or a fragment thereof binds to an epitope of tau protein; b. makes the binding peptide (specifically, , the antibody, in particular, the monoclonal antibody or a functional portion thereof, binds to a tau antigen to form an immune complex; c. detects the formation of the immune complex; and d. obtains the presence or absence of the immune complex Existence with samples or specific body parts Correlation of the presence or absence of τ antigen in a sub-region or region; e. comparing the amount of the immune complex with a normal control value; wherein the amount of the aggregate is greater than the normal control value, indicating that the patient is still suffering from a small amount Residual disease.

In an embodiment (63), a method for predicting the use of a binding peptide according to any of the above embodiments or a functional portion thereof (specifically, an antibody, in particular, a monoclonal antibody or a functional portion thereof, or a polynucleoside) A method of treating a patient's reactivity with an acid or a pharmaceutical composition comprising:

a. contacting a sample or a specific body part or body region suspected of containing a tau antigen with a binding peptide according to any of the above embodiments or an active fragment thereof (specifically, an antibody, in particular, a monoclonal antibody or a functional portion thereof) The peptide or a fragment thereof binds to an epitope of tau protein;

b. binding the binding peptide (specifically, the antibody, in particular, the monoclonal antibody or a functional portion thereof) to the tau antigen to form an immune complex;

c. detecting the formation of the immune complex; and

d. Obtaining the correlation of the presence or absence of the immune complex with the presence or absence of a tau antigen in the sample or a particular body part or region.

e. Comparing the amount of the immune complex before and after the start of treatment, wherein a decrease in the amount of the aggregate indicates that the patient is more likely to respond to the treatment.

Anti-tau protein antibodies and fragments thereof can be used in the above methods of the invention. In the above method, a sample containing an antibody or a fragment thereof can be immunoblotted by contacting the sample with an anti-tau protein antibody or a fragment thereof attached to a solid support to increase the concentration of tau protein in the sample.

Prior to step (a), the sample is immunostained by contacting the sample with an anti-tau protein antibody or fragment thereof attached to a solid support to increase the concentration of tau protein in the sample.

In another embodiment (64), the invention relates to a test kit for detecting and diagnosing a tau protein-related disease, disorder or condition comprising a binding peptide according to any of the above embodiments or an active fragment thereof, specific In other words, antibodies, in particular, monoclonal antibodies or functional parts thereof.

In one embodiment (65), the test kit comprises a container containing one or more binding peptides according to any of the above embodiments, or an active fragment thereof (specifically, an antibody, particularly a monoclonal antibody or a functional portion thereof), And relating to the use of a binding peptide or antibody to bind to a tau antigen to form an immune complex and to detect the formation of the immune complex to obtain the correlation between the presence or absence of the immune complex and the presence or absence of a tau antigen Instructions.

In another embodiment (66), the invention relates to a mammalian epitope, in particular, an epitope on a human tau protein as set forth in SEQ ID NO: 67, selected from the group consisting of : τ aa 393-401 comprising phosphorylated Ser (pS396) at position 396; τ aa 396-401 comprising phosphorylated Ser (pS396) at position 396; τ aa 394- comprising phosphorylated Ser (pS396) at position 396 400; τ aa 402-406 (pS404) comprising phosphorylated Ser at position 404 and τ aa 393-400 comprising phosphorylated Ser (pS396) at position 396.

In one embodiment (67), the epitope is comprised of τ aa 393-401 comprising a phosphorylated Ser (pS396) at position 396.

In one embodiment (68), the epitope is comprised of 396 at position 396. Composition of τ aa 396-401 containing phosphorylated Ser (pS396).

In one embodiment (69), the epitope is comprised of τ aa 394-400 comprising phosphorylated Ser (pS396) at position 396.

In one embodiment (70), the epitope is comprised of τ aa 402-406 comprising phosphorylated Ser (pS404) at position 404.

In one embodiment (71), the epitope is comprised of τ aa 393-400 comprising phosphorylated Ser (pS396) at position 396.

In another embodiment (72), the invention relates to a cell which produces a binding peptide according to any of the above embodiments or an active fragment thereof (specifically, an antibody, in particular, a monoclonal antibody or a functional portion thereof) Strain.

In one embodiment (73), the present invention relates to a cell line which is a hybridoma cell line A4-4A6-48 deposited with DSM ACC3136 on August 30, 2011.

In one embodiment (74), the present invention relates to a cell line which is a hybridoma cell line A6-2G5-30 deposited with DSM ACC3137 on August 30, 2011.

In one embodiment (75), the present invention relates to a cell line which is a hybridoma cell line A6-2G5-41 deposited with DSM ACC3138 on August 30, 2011.

In one embodiment (76), the present invention relates to a cell line which is a hybridoma cell line A4-2A1-18 deposited with DSM ACC3139 on August 30, 2011.

In one embodiment (77), the present invention relates to a cell line which is a hybridoma cell line A4- deposited with DSM ACC3140 on August 30, 2011. 2A1-40.

In one embodiment (78), the present invention relates to a cell line which is a hybridoma cell line A6-1D2-12 deposited with DSM ACC3141 on September 6, 2011.

In one embodiment (79), the invention relates to a monoclonal antibody or a functional portion thereof comprising a light chain (VL) and/or heavy chain (VH) domain, which comprises a polynucleoside located on a nucleotide fragment Acid-encoded, the nucleotide fragment can be obtained by PCR amplification using the following hybridoma cell line A4-2A1-18 deposited with DSM ACC3139 on August 30, 2011. a primer pair comprising the 5' primer of SEQ ID NO: 149 and the 3' primer of SEQ ID NO: 51 for amplification of the first binding domain; and/or b. A primer mix comprising a 5' primer selected from the group consisting of SEQ ID NOs: 120, 123, 124, 136, 137, 138, 139 and 140 and selected from the group consisting of SEQ ID NOs: 131, 134 and 141-148 The 3' primer of the population is used to amplify the second binding domain.

In one embodiment (80), the invention relates to a monoclonal antibody or a functional portion thereof comprising a light chain (VL) and/or heavy chain (VH) domain, which comprises a polynucleoside located on a nucleotide fragment Acid-encoded, the nucleotide fragment can be obtained by PCR amplification using the following hybridoma cell line A6-2G5-30 deposited with DSM ACC3137 on August 30, 2011 to obtain a mixture of primers selected from the group consisting of SEQ ID NO: a 5' primer of the group consisting of 51 and 169-174 and a 3' primer of SEQ ID NO: 51 for amplifying the first binding domain; and/or b. a primer mixture comprising SEQ ID NO: 124 , 127 and 150- A 5' primer of the group consisting of 158 and a 3' primer selected from the group consisting of SEQ ID NO: 130 and 159-168 are used to amplify the second binding domain.

In one embodiment (81), the invention relates to a monoclonal antibody or a functional portion thereof comprising a light chain (VL) and/or heavy chain (VH) domain, which comprises a polynucleoside located on a nucleotide fragment Acid-encoded, the nucleotide fragment can be obtained by PCR amplification using the following hybridoma cell line A4-2A1-40 deposited with DSM ACC3140 on August 30, 2011 to obtain a mixture of primers selected from the group consisting of SEQ ID NO: a 5' primer of the group consisting of 178, 179 and 180 and a 3' primer of SEQ ID NO: 51 for amplifying the first binding domain; and/or b. a primer mixture comprising a fragment selected from SEQ ID NO: 121 a 5' primer of the group consisting of 127, 139, 154, 155 and 175, and a 3' primer selected from the group consisting of SEQ ID NOS: 128, 129, 147, 176 and 177 for amplifying the second binding domain .

In one embodiment (82), the invention relates to a monoclonal antibody or a functional portion thereof comprising a light chain (VL) and/or heavy chain (VH) domain, which comprises a polynucleoside located on a nucleotide fragment Acid-encoded, the nucleotide fragment can be obtained by PCR amplification using the following hybridoma cell line A6-2G5-41 deposited with DSM ACC3138 on August 30, 2011 to obtain a mixture of primers selected from the group consisting of SEQ ID a 5' primer of the group consisting of 51 and 188-192 and a 3' primer of SEQ ID NO: 51 for amplifying the first binding domain; and/or b. a primer mixture comprising SEQ ID NO: 120 a 5' primer of the group consisting of 124, 126, 181, 182, and 183 and selected from the group consisting of SEQ ID NO: A 3' primer of the group consisting of 144, 145 and 184-187 is used to amplify the second binding domain.

In one embodiment (83), the invention relates to a monoclonal antibody or a functional portion thereof comprising a light chain (VL) and/or heavy chain (VH) domain, which comprises a polynucleoside located on a nucleotide fragment Acid-encoded, the nucleotide fragment can be obtained by PCR amplification using the following hybridoma cell line A4-4A6-48 deposited with DSM ACC3136 on August 30, 2011 to obtain a mixture of primers selected from the group consisting of SEQ ID NO: a 5' primer of the group consisting of 50 and 201-204 and a 3' primer of SEQ ID NO: 51 for amplifying the first binding domain; and/or b. a primer mixture comprising SEQ ID NO: 121 a 5' primer of the group consisting of 137, 151 and 193-197 and a 3' primer selected from the group consisting of SEQ ID NOS: 131, 141, 144, 166, 198, 199 and 200 for amplifying the second binding area.

In one embodiment (84), the invention relates to a monoclonal antibody or a functional portion thereof comprising a light chain (VL) and/or heavy chain (VH) domain, which comprises a polynucleoside located on a nucleotide fragment Acid-encoded, the nucleotide fragment can be obtained by PCR amplification using the following hybridoma cell line A6-1D2-12 deposited with DSM ACC3141 on September 6, 2011 to obtain a mixture of primers selected from the group consisting of SEQ ID NO: a 5' primer of the group consisting of 209-214 and 219-221 and a 3' primer of SEQ ID NO: 215 for amplifying the first binding domain; and/or b. a primer mixture comprising a fragment selected from SEQ ID NO : 5' primer of the group consisting of 216, 217 and 218 and 3' primer of SEQ ID NO: 208 for amplification Second binding domain.

In one embodiment (85), the antibody according to any of the above embodiments may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a camelid antibody, a bifunctional antibody, or a modified or Engineered antibodies.

In one embodiment (86), the invention provides a method of preparing a binding peptide or antibody of any of the above embodiments, comprising the steps of culturing a cell line of any of the above embodiments in a suitable culture medium, and optionally The cell strain or medium is purified to bind a peptide or antibody.

In another embodiment (87), the invention provides a method of detecting phosphorylated tau protein (pTau) in a brain sample, comprising: a. contacting a sample with an antibody or fragment thereof according to any of the above technical solutions, The peptide or fragment thereof binds to an epitope of phosphorylated tau protein; b. binds the antibody to tau protein to form an immune complex; c. detects the formation of an immune complex, in particular, by applying an ELISA assay Measurement.

In particular, the invention in a particular embodiment (88) relates to phosphorylated tau protein (pTau) multimers in brain tissue homogenates of individuals and healthy control individuals suspected of having a tau protein-related disease or condition. The method for detecting post-mortem comprises: a. contacting a brain tissue homogenate sample of two individuals with an antibody or a fragment thereof of any of the above technical means, the peptide or a fragment thereof binding to an epitope of phosphorylated tau protein; b. The antibody binds to tau protein to form an immune complex; c. detecting the formation of immune complexes, in particular by applying an ELISA assay, and d. the amount or intensity of immune complexes in a sample obtained from an individual suspected of having a tau protein-related disease and a control When the sample is compared, wherein the amount or intensity of the immune complex is increased compared to the control value, it indicates that the patient has suffered from a small residual disease.

In one embodiment (89), the increase observed in the test sample was between 30% and 50% compared to the control sample, in particular between 35% and 45%.

In one embodiment (90), the invention provides a binding peptide or protein according to any of the above embodiments, or a functional portion thereof, in particular, an antibody, in particular, a monoclonal antibody or a functional portion thereof, the antibody or The fragment shows a favorable pK curve. In particular, the antibody or fragment has a high serum concentration up to 10 days after administration, indicating that the pharmacokinetic (PK) curve advantageously supports the use of such antibodies as therapeutic antibodies. (Deng et al, Expert Opin Drug Metab Toxicol, 2012, 8(2) 141-60; Putman et al, Trends Biotech, 2010 (28) 509-516; Bai, S. Clin Pharmacokinet 2012; 51(2): 119 -135).

sequence

SEQ ID NOS: 46-57 depict the nucleotide sequences of the VH/VK forward and reverse primers.

SEQ ID NO: 62 depicts the amino acid sequence of the tau antigen peptide T3 (see Table 1).

SEQ ID NO: 67 depicts the longest human τ (441 aa) (also known as Tau 40) The amino acid sequence of the isoform.

SEQ ID NO: 68 depicts the amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.

SEQ ID NO: 69 depicts the amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1 produced by the hybridoma cell line A4-4A6-18.

SEQ ID NO: 70 depicts the amino acid sequence of CDR1 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-4A6-Ab1.

SEQ ID NO: 71 depicts the amino acid sequence of CDR2 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-4A6-Ab1.

SEQ ID NO: 72 depicts the amino acid sequence of CDR3 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-4A6-Ab1.

SEQ ID NO:73 depicts the amino acid sequence of CDR1 of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1.

SEQ ID NO: 74 depicts the amino acid sequence of CDR2 of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1.

SEQ ID NO: 75 depicts the amino acid sequence of CDR3 of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1.

SEQ ID NO: 76 depicts the amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-1D2-Ab1 produced by the hybridoma cell line A6-1D2-12.

SEQ ID NO:77 depicts the amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-1D2-Ab1 produced by the hybridoma cell line A6-1D2-12.

SEQ ID NO: 78 depicts the amino acid sequence of CDR1 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-1D2-Ab1.

SEQ ID NO:79 depicts the heavy chain of the monoclonal antibody ACI-35-1D2-Ab1 The amino acid sequence of the CDR2 of the variable region (VH).

SEQ ID NO: 80 depicts the amino acid sequence of CDR3 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-1D2-Ab1.

SEQ ID NO: 81 depicts the amino acid sequence of CDR1 of the light chain variable region (VK) of the monoclonal antibody ACI-35-1D2-Ab1.

SEQ ID NO: 82 depicts the amino acid sequence of CDR2 of the light chain variable region (VK) of the monoclonal antibody ACI-35-1D2-Ab1.

SEQ ID NO:83 depicts the amino acid sequence of CDR3 of the light chain variable region (VK) of the monoclonal antibody ACI-35-1D2-Ab1.

SEQ ID NO: 84 depicts the nucleotide sequence of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.

SEQ ID NO: 85 depicts the nucleotide sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1 produced by the hybridoma cell line A4-4A6-18.

SEQ ID NO: 86 depicts the nucleotide sequence of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-1D2-Ab1 produced by the hybridoma cell line A6-1D2-12.

SEQ ID NO: 87 depicts the nucleotide sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-1D2-Ab1 produced by the hybridoma cell line A6-1D2-12.

SEQ ID NO: 88 depicts monoclonal antibodies ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, respectively, produced by hybridoma cell lines A4-2A1-18, A4-2A1-40, and A4-4A6-48, respectively. And the amino acid sequence of the heavy chain variable region (VH) of ACI-35-4A6-Ab2.

SEQ ID NO:89 depicts monoclonal antibodies ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, ACI-35-4A6-Ab2, ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively. Amino acid sequence of CDR1 of heavy chain variable region (VH) Column.

SEQ ID NO: 90 depicts the amino acid sequence of CDR2 of the heavy chain variable region (VH) of the monoclonal antibodies ACI-35-2A1-Ab1, ACI-35-2A1-Ab2 and ACI-35-4A6-Ab2, respectively.

SEQ ID NO: 91 depicts monoclonal antibodies ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, ACI-35-4A6-Ab2, ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively. Amino acid sequence of CDR3 of the heavy chain variable region (VH).

SEQ ID NO: 92 depicts the amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab2 produced by the hybridoma cell line A4-2A1-40.

SEQ ID NO:93 depicts the amino acid sequence of CDR1 of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab2.

SEQ ID NO: 94 depicts the amino acid sequence of CDR2 of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab2.

SEQ ID NO: 95 depicts the amino acid sequence of CDR3 of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab2.

SEQ ID NO: 96 depicts the amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-2G5-Ab1 produced by the hybridoma cell line A6-2G5-08.

SEQ ID NO: 97 depicts the amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-Ab1 produced by the hybridoma cell line A6-2G5-08.

SEQ ID NO: 98 depicts the amino acid sequence of CDR1 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 99 depicts the amino acid sequence of CDR2 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 100 depicts the amino acid sequence of CDR3 of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 101 depicts the amino acid sequence of CDR1 of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 102 depicts the amino acid sequence of CDR2 of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 103 depicts the amino acid sequence of CDR3 of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-Ab1.

SEQ ID NO: 104 depicts the heavy chain variable regions of monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell lines A6-2G5-30 and A6-2G5-41, respectively. The amino acid sequence of (VH).

SEQ ID NO: 105 depicts the light chain variable regions of monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell lines A6-2G5-30 and A6-2G5-41, respectively. The amino acid sequence of (VK).

SEQ ID NO: 106 depicts the amino acid sequence of CDR1 of the light chain variable region (VK) of the monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.

SEQ ID NO: 107 depicts the amino acid sequence of CDR2 of the light chain variable region (VK) of monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.

SEQ ID NO: 108 depicts the amino acid sequence of CDR3 of the light chain variable region (VK) of the monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.

SEQ ID NO: 109 is depicted by hybridoma cell line A4-2A1- 18. Heavy chain variable region (VH) of monoclonal antibodies ACI-35-2A1-Ab1, ACI-35-2A1-Ab2 and ACI-35-4A6-Ab2 produced by A4-2A1-40 and A4-4A6-48 Nucleotide sequence.

SEQ ID NO: 110 depicts the nucleotide sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab2 produced by the hybridoma cell line A4-2A1-40.

SEQ ID NO: 111 depicts the nucleotide sequence of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-2G5-AB1 produced by the hybridoma cell line A6-2G5-08.

SEQ ID NO: 112 depicts the nucleotide sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-AB1 produced by the hybridoma cell line A6-2G5-08.

SEQ ID NO: 113 depicts the heavy chain variable regions of monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell lines A6-2G5-30 and A6-2G5-41, respectively. Nucleotide sequence of (VH).

SEQ ID NO: 114 depicts the light chain variable regions of monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell lines A6-2G5-30 and A6-2G5-41, respectively. The nucleotide sequence of (VK).

SEQ ID NO: 115 depicts the amino acid sequence of CDR2 of the heavy chain variable region (VH) of the monoclonal antibodies ACI-35-2G5-AB2 and ACI-35-2G5-AB3.

SEQ ID NO: 116 depicts the amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab1 produced by the hybridoma cell line A4-2A1-18.

SEQ ID NO: 117 depicts the hybridoma cell line A4-2A1-18 produced Nucleotide sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab1.

SEQ ID NO: 118 depicts the amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab2 produced by hybridoma cell line A4-4A6-48.

SEQ ID NO: 119 depicts the nucleotide sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab2 produced by the hybridoma cell line A4-4A6-48.

SEQ ID NOs: 120-221 depict the nucleotide sequences of the VH/VK forward and reverse primers.

Definition of Terms

The terms "polypeptide", "peptide" and "protein" as used herein are interchangeable and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

The terms "peptide" or "binding peptide" are used interchangeably herein and refer to an amino acid (usually an L-amino acid) chain of alpha carbon via a peptide bond, which is composed of one amino acid. It is formed by a condensation reaction between a carboxyl group of the α carbon and an amine group of the α carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amine end) has a free amine group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminus) has a free carboxyl group. Thus, the term "amine end" (abbreviated as N-terminus) refers to a free alpha amine group on the amino acid at the amino terminus of the peptide, or an alpha amine group of an amino acid located at any other position within the peptide. (Imino group when participating in peptide bonds). Similarly, the term "carboxy terminus" (abbreviated as C-terminus) refers to a free carboxyl group on the amino acid at the carboxy terminus of the peptide, or a carboxyl group of an amino acid located at any other position within the peptide. Binding peptides can constitute antibodies, such as multiple strains Or a monoclonal antibody, a human or humanized antibody, a bifunctional antibody, a camelid antibody, or the like, or a functional portion thereof as defined herein.

The term "fragment" or "fragment" as used herein in the context of a peptide refers to a functional peptide fragment having substantially the same (biological) activity as the intact peptide as defined herein. When the term is used herein in the context of an antibody, it is meant an antibody fragment comprising a portion of an intact antibody that contains the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; bifunctional antibodies; single-chain antibody molecules, including single-chain Fv (scFv) molecules; and bispecific and multispecific antibodies and/or Antibody fragment.

Typically, the amino acids that make up the peptide are numbered sequentially, starting at the amine end and increasing in the direction toward the carboxy terminus of the peptide. Therefore, if an amino acid is said to "follow" another amino acid, the position of the amino acid at the carboxy terminus of the peptide is closer than that of the previous amino acid.

The term "residue" is used herein to mean an amino acid that is incorporated into a peptide by a guanamine bond. Thus, the amino acid can be a naturally occurring amino acid or, unless otherwise limited, a known analog of a native amino acid (ie, an amino acid mimetic) that acts with the naturally occurring amino acid. similar. In addition, the guanamine linkage mimetic includes peptide backbone modifications well known to those skilled in the art.

The phrase "consisting essentially of" is used herein to exclude any element that substantially alters the principal characteristics of the peptides referred to in the phrase. Thus, the description of a peptide "consisting essentially of" excludes any amino acid substitution, addition or deletion that would substantially alter the biological activity of the peptide.

In addition, those skilled in the art will recognize the coding sequence as mentioned above. Individual substitutions, deletions or additions in a column that alter, add or delete a single amino acid or a small percentage of amino acid (typically less than 5%, more typically less than 1%) are conservative modifications, wherein the change results in an amine The base acid is substituted with a chemically similar amino acid. It is well known in the art to provide a conservative representation of functions similar to amino acids. The following six groups each contain an amino acid that is conservatively substituted for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), Gluten (E); 3) aspartic acid (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I) ), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The phrase "isolated" or "biologically pure" means that the substance is substantially or substantially free of components normally associated with it as found in its natural state. Thus, the peptides described herein do not contain materials normally associated with their in situ environment. Typically, the isolated immunogenic peptide described herein has a purity of at least about 80%, typically at least about 90%, and preferably at least about 95%, as measured by the intensity of the strip on the silver stained gel.

Protein purity or homogeneity can be indicated by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of protein samples followed by visual inspection after staining. For some purposes, high resolution will be required and HPLC or similar purification means will be utilized.

When immunogenic peptides are relatively short in length (i.e., less than about 50 amino acids), they are often synthesized using standard chemical peptide synthesis techniques.

The C-terminal amino acid of the sequence is linked to an insoluble support, followed by solid phase synthesis of the remaining amino acids in the sequential addition sequence to a preferred method of chemically synthesizing the immunogenic peptides described herein. Solid phase synthesis techniques are known to those skilled in the art.

Alternatively, the immunogenic peptides described herein are synthesized using recombinant nucleic acid methods. In general, the method comprises establishing a nucleic acid sequence encoding a peptide, placing the nucleic acid in a performance cassette under the control of a specific promoter, expressing the peptide in a host, isolating the expressed peptide or polypeptide, and, if necessary, Peptide renaturation. Techniques sufficient to guide those skilled in the art to understand such procedures are found in the literature.

Once expressed, the recombinant peptide can be purified according to standard procedures including, but not limited to, ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, and the like. For use as a therapeutic agent, the homogeneity of the substantially pure composition is preferably from about 50% to 95%, and most preferably from 80% to 95% or more.

Those skilled in the art will recognize that after chemical synthesis, biological expression or purification, the immunogenic peptide can have a configuration that is substantially different from the natural configuration of the constituent peptides. In this case, it is often necessary to denature and reduce the antiproliferative peptide and then to refold the peptide into a preferred configuration. Methods for reducing and denaturation of proteins and inducing refolding are well known to those skilled in the art.

The antigenicity of the purified protein can be verified, for example, by displaying reactivity with immune sera or with antisera raised against the protein itself.

The terms "a" and "the", as used herein, are defined to mean "one or more" and include plural.

The term "detecting" as used herein means using known techniques (such as exemption) Epidemiological or histological methods) detect biomolecules and specify or quantify the presence or concentration of biomolecules in the study.

"Isolation" means that the biomolecule does not contain at least some of the components of the natural component with which it is present.

The term "antibody" or "functional portion thereof" as used herein is a term that has been recognized in the art and is understood to mean a molecule or a molecularly active fragment that binds to a known antigen, in particular, an immunoglobulin. The immunologically active portion of a molecule and an immunoglobulin molecule, that is, a molecule containing a binding site for immunospecific binding to an antigen. The immunoglobulin of the present invention may be an immunoglobulin molecule of any type (IgG, IgM, IgD, IgE, IgA, and IgY) and classes (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses.

Within the scope of the present invention, "antibody" is intended to include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, bispecific antibodies, monkey antibodies, human antibodies and human antibodies, camelid antibodies, dual functions. An antibody and a functional part or active fragment thereof. Examples of molecularly active fragments that bind to known antigens include Fab and F(ab') ₂ fragments (including products of Fab immunoglobulin expression libraries) and epitope-binding fragments of any of the above-mentioned antibodies and fragments.

Such active fragments can be produced from the antibodies of the invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. The appropriate fractions containing the Fab fragments can then be collected and concentrated by membrane filtration and similar techniques. For a further description of the general techniques for isolating active fragments of antibodies, see, for example, Khaw, B. A. et al., J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al, Methods Enzymology, 121: 663-69, Academic Press, (1986).

"Humanized antibody" refers to an engineered type of antibody whose CDRs are derived from a non-human donor immunoglobulin and the remaining immunoglobulin source of the molecule is derived from one (or more) human immunoglobulin.

A humanized antibody can further refer to an antibody having one or more of the framework regions of a human or primate amino acid. In addition, the framework support residues can be altered to retain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, for example, Queen et al, Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al, Bio/Technoloy, 9:421 (1991)).

"Humanized antibodies" can also be obtained by novel genetic engineering methods that produce affinity-mature human-like antibodies in large animals such as rabbits (http://www.rctech.com/bioventures/therapeutic. Php).

The term "fully human antibody" or "human" antibody means an antibody derived from a transgenic mouse carrying a human antibody gene or derived from a human cell. However, for the human immune system, the differences between "fully human" antibodies, "human" antibodies, and "humanized" antibodies are negligible or non-existent and thus all three can be equally effective and safe.

The term "monoclonal antibody" is also well-known in the art and refers to an antibody that is produced in large quantities in a laboratory from a single pure line and that recognizes only one antigen. Individual antibodies are usually made by fusing a common short-lived B cell that produces antibodies with fast-growing cells, such as cancer cells (sometimes called "immortal" cells). Ready. The resulting hybrid cells or fusion tumors rapidly multiply to form a pure line that produces a large amount of antibodies.

The term "antigen" refers to an entity or fragment thereof that is capable of inducing an immune response in an organism, in particular, an animal, more specifically, a mammal, including a human. The term includes immunogens and regions or antigenic determinants that result in antigenicity.

As used herein, the term "soluble" means partially or completely dissolved in an aqueous solution.

Furthermore, the term "immunogenicity" as used herein refers to a substance that elicits or enhances the production of antibodies, T cells, and other reactive immune cells against an immunogenic agent and that promotes an immune response in a human or animal body.

An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells against the immunogenic composition of the invention administered to alleviate or alleviate the condition being treated.

The term "hybridomas" is known in the art and is known to those of ordinary skill in the art and refers to cells produced by the fusion of cells producing antibodies with immortalized cells (e.g., multiple myeloma cells). This fused cell is capable of producing a continuous supply of antibodies. See the definition of "monoclonal antibody" above and examples of fusion methods in more detail below.

The term "vector," as used herein, means a structure into which an antigenic peptide or supramolecular construct can be incorporated or can be bound, whereby a portion of an antigenic peptide or peptide is presented or exposed to the immune system of a human or animal. Any particle suitable for use in animal or human therapy, such as vesicles, particles or microsomes, can be used as a carrier in the context of the present invention.

The term "vector" further encompasses delivery methods in which a supramolecular antigenic construct composition comprising an antigenic peptide can be transported to a desired site by a delivery mechanism. An example of such a delivery system utilizes a colloidal metal such as colloidal gold.

Carrier proteins useful in the supramolecular antigenic construct compositions of the present invention include, but are not limited to, the maltose binding peptide "MBP"; bovine serum albumin "BSA"; keyhole lympet hemocyanin "KLH "ovalbumin"; flagellin; thyroglobulin; serum albumin of any species; gamma globulin of any species; syngeneic cells; homologous cells carrying the Ia antigen; and D-amino A polymer of an acid and/or an L-amino acid.

Additionally, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a binding peptide sufficient to produce a therapeutic effect in a human or animal when administered to a human or animal. It is easy for a person of ordinary skill to determine the effective amount in accordance with conventional procedures.

"pTau PHF", "PHF" and "paired helical filaments" are used synonymously herein and refer to a pair of filaments of about 10 nm which are wound into a helical shape with a periodicity of 160 nm visible under an electron microscope. . The width varies between 10 nm and 22 nm. PHF is the main structure in the neurofibrillary tangles of Alzheimer's disease (AD) and in the filaments of the nerve fiber network. PHF can also be found in some, but not all, dystrophic neurites associated with neuritic plaques. The main component of PHF is the hyperphosphorylated form of microtubule-associated protein τ. PHF consists of a disulfide-linked anti-parallel hyperphosphorylated tau protein. The 20 amino acid residues at the C-terminus of the PHF tau protein can be truncated. The mechanism underlying the formation of PHF has not yet been determined, but hyperphosphorylation of tau protein can cause it to detach from microtubules and increase the pool of soluble tau.

Within the scope of the invention, it has been demonstrated that the response induced by an antibody against an antigenic composition of the invention is substantially independent of T cells. In this regard, a nude mouse model was used and nude mice were vaccinated and antibody responses were measured to evaluate the Aβ-specific antibody response induced by the antigen composition of the present invention in immunized nude mice. Nude mice carry the Foxnlnu mutation and the result has reduced T cell function due to the lack of an appropriate thymus.

"Pharmaceutically effective amount" as used herein means a dose of the active ingredient in a pharmaceutical composition sufficient to cure or at least partially arrest the symptoms of, or associated with, the disease, condition or condition being treated.

The present invention provides a binding peptide that recognizes and binds to the major pathological phosphorylation epitope of tau protein. In particular, the present invention provides specific antibodies directed against linear and conformational, simple and complex phosphorylation epitopes on protein τ, which are believed to result in tauopathy (including AD) on such protein τ. Synaptic toxicity and neurotoxicity.

Thus, in one embodiment, the invention relates to a binding peptide or protein or a functional part thereof, in particular to an antibody, in particular, a monoclonal antibody or a functional part thereof, which recognition peptide or antibody recognition And specifically binds to a mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically, to a pathological protein τ conformer, but In one embodiment, it does not bind to a corresponding unphosphorylated epitope and/or does not bind to an unrelated epitope, wherein the binding peptide or antibody has a high binding affinity with a dissociation constant of at least 10 nM, in particular, at least 8 nM, in particular, at least 5 nM, in particular, at least 2 nM, in particular, at least 1 nM, in particular, at least 500 pM, in particular at least 400 pM, in particular at least 300 pM, in particular at least 200 pM, in particular at least 100 pM, in particular at least 50 pM.

As used herein, a "soluble tau" protein refers to a protein consisting of a fully dissolved tau/peptide monomer, or a tau-like peptide/protein, a modified or truncated tau peptide/protein, or a tau peptide/protein. Other derivatives of monomers, and tau oligomers. "Soluble τ" specifically excludes neurofibrillary tangles (NFT).

As used herein, "insoluble tau" refers to the accumulation of tau peptides or proteins or tau-like peptides/proteins that are insoluble in the aqueous medium and in vivo in mammals or humans, and more specifically in the brain. a monomeric, or modified or truncated taupeptide/protein or a multi-aggregating monomer that forms an oligomer or a polymer structure of a taupeptide/protein derivative, but specifically, refers to a mammal or human More specifically, a poly-aggregating monomer that is insoluble in the brain or a modified or truncated taupeptide/protein or a derivative thereof. "Insoluble tau" specifically includes neurofibrillary tangles (NFT).

As used herein, "monomer τ" or "τ monomer" refers to a fully dissolved tau protein, while an aqueous medium has no aggregated complex.

""aggregation τ", "oligomeric τ" and "τ oligomer" mean in vitro in an aqueous medium and in vivo in a mammal or human body, more specifically, in the brain, or insoluble in the brain. Multi-aggregation of dissolved tau peptides or protein or τ-like peptides/proteins, or multi-aggregation of modified or truncated taupeptides/proteins or other derivatives of tau peptides/proteins that form oligomers or polymer structures Monomer, but specifically, refers to the amount of tau or modified or truncated tau peptide/protein or its derivatives in mammals or humans, more specifically in the brain, insoluble or soluble in the brain. Aggregate the monomer. "

"Modulating antibody" refers to an antibody or functional fragment thereof as described herein in various embodiments which may be up-regulated (eg, activated or stimulated), down-regulated (eg, inhibited or suppressed), or otherwise altered in solubility and/or insoluble and / or oligomeric tau protein (specifically, soluble phosphorylated tau protein) in vivo, in particular, in animals (specifically, containing higher levels of soluble tau protein and / or soluble phosphorylated tau protein Functional properties, biological activity or content in the brain (specifically, cerebral cortex and/or hippocampus) of mammals or humans. The modulatory antibody or functional fragment thereof can have the effect of directly or indirectly modulating the tau protein or a polypeptide encoding the tau protein. In certain embodiments, the modulatory antibody or functional fragment thereof reduces the amount of soluble and/or insoluble and/or oligomeric (especially soluble and insoluble) tau protein, in particular, soluble and insoluble and oligomeric tau protein. In one aspect, the soluble and/or insoluble and/or oligomeric tau protein is an animal (specifically, contains a higher amount of tau protein and/or phosphorylated tau protein (specifically, soluble tau protein and/or Phosphorylated tau protein in the brain (specifically, cerebral cortex and/or hippocampus) of soluble or phosphorylated tau protein), in particular, soluble phosphorylated tau protein.

In one embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a binding peptide or a functional portion thereof according to any of the embodiments described and claimed herein, in particular, an antibody, in particular, a single A strain antibody or a functional portion thereof, or a polynucleotide comprising a nucleic acid sequence encoding the binding peptide or antibody, or a combination thereof, and a pharmaceutically acceptable carrier.

Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solution, water, emulsions. (such as oil/water emulsions), various types of humectants, sterile solutions, and the like.

The binding peptides of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) can be prepared in a physiologically acceptable formulation using known techniques and can comprise a pharmaceutically acceptable carrier, diluted And/or excipients. For example, a binding peptide according to the invention and as described herein, including any functionally equivalent binding peptide or a functional portion thereof, in detail, includes any functionally equivalent antibody or a functional portion thereof, the monoclonal antibody of the invention and a pharmaceutical A combination of a carrier, a diluent, and/or an excipient that is acceptable to form a therapeutic composition. Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions (such as oil/water emulsions), various types of humectants, sterile solutions. Wait.

Formulations of the pharmaceutical compositions of the present invention can be made according to standard methods known to those of ordinary skill in the art.

The compositions of the present invention can be administered to a subject in a solid, liquid or aerosol form at a suitable dosage that is pharmaceutically effective. Examples of solid compositions include pills, creams, and implantable dosage units. Pills can be administered orally. The therapeutic ointment can be administered topically. The implantable dosage unit can be administered topically (e.g., at a tumor site) or can be implanted for systemic release of a therapeutic composition (e.g., subcutaneously). Examples of liquid compositions include formulations suitable for intramuscular, subcutaneous, intravenous, intraarterial injection and formulations for topical and intraocular administration. Examples of aerosol formulations include inhalation formulations for administration to the lung.

The composition can be administered by standard routes of administration. In general, the compositions can be administered by topical, oral, rectal, nasal, intradermal, intraperitoneal or parenteral (e.g., intravenous, subcutaneous or intramuscular) routes.

In addition, the compositions can be incorporated into sustained release matrices, such as biodegradable polymers, which are implanted near where delivery is desired (eg, a tumor site). The method includes single-dose administration, repeated dose administration at predetermined time intervals, and continuous administration for a predetermined period of time.

Sustained release matrices as used herein are matrices made from materials which are typically polymeric which are degradable by enzymatic or acid/base hydrolysis or by dissolution. After insertion into the body, the matrix is affected by enzymes and body fluids. Sustained release matrix should be selected from biocompatible substances such as liposome, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (lactic acid and glycolic acid) Copolymers), polyanhydrides, polyorthoesters, peptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids (such as phenylalanine, tyrosine) , isoleucine), polynucleotide, polyvinyl propylene, polyvinylpyrrolidone and polyfluorene. Preferably, the biodegradable matrix is a matrix of one of polylactide, polyglycolide or polylactide co-glycolide (copolymer of lactic acid and glycolic acid).

It is well known to those skilled in the relevant art that the dosage of the composition will depend on various factors such as the condition being treated, the particular composition employed, and other clinical factors such as the weight, size, sex, and general health of the patient; a specific compound or composition to be administered; other drugs being administered at the same time; and a route of administration.

The compositions of the present invention may be administered in combination with other compositions comprising biologically active substances or compounds, such as proteins for the treatment of tauopathy and/or amyloidosis, amyloid or amyloid-like proteins (such as A known compound relating to a group of diseases and disorders involving amyloid beta protein of Alzheimer's disease.

Other biologically active substances or compounds may exert their biological effects by the same or similar mechanisms as the therapeutic vaccines of the invention or by unrelated mechanisms of action or by multiple related and/or unrelated mechanisms of action.

In general, other biologically active compounds may include neutron transmission enhancers, psychotropic drugs, acetylcholinesterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepine tranquilizers, acetylcholine Synthetic, storage or release enhancer, acetylcholine post-synaptic receptor agonist, monoamine oxidase A or monoamine oxidase B inhibitor, N-methyl-D-aspartate glutamate receptor antagonist, non- Steroid anti-inflammatory drugs, antioxidants, and serotonergic receptor antagonists.

In particular, the bioactive agent or biologically active compound may comprise at least one compound selected from the group consisting of oxidative stress compounds, anti-apoptotic compounds, metal chelators, DNA repair inhibitors (such as pirenzepine). (pirenzepin)) and metabolites (3-amino-1-propanesulfonic acid (3APS), 1,3-propane disulfonate (1,3PDS)), secreted enzyme activator, [β]-secretase and 7-secretase inhibitor, tau protein, neurotransmitter, beta tablet disruptor, anti-inflammatory molecule, "atypical antipsychotic" (such as clozapine, ziprasidone, risperidone) (risperidone), aripiprazole or olanzapine or cholinesterase inhibitor (ChEI) (such as tacrine, rivastigmine, winternipazole) (donepezil) and/or galantamine and other drugs and nutritional supplements Such as vitamin B12, cysteine, acetylcholine precursor, lecithin, choline, Ginkgo biloba, acetyl-L-carnitine, idebenone, valproate ( Propentofylline), or a xanthine derivative), and a binding peptide of the invention, including antibodies, in particular, monoclonal antibodies and active fragments thereof, and optionally pharmaceutically acceptable carriers and/or diluents And/or excipients and instructions for treating the disease.

In another embodiment, the composition of the invention may comprise niacin or memantine and a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof), and optionally A pharmaceutically acceptable carrier and/or diluent and/or excipient is selected.

In another embodiment of the invention, the provided compositions comprise for the treatment of positive and negative psychotic symptoms, including hallucinations, delusions, thought disorders (from apparent inconsistency, derailment, tangentiality) and behavior "atypical antipsychotics" such as clozapine, ziprasidone, risperidone, aripiprazole or singularity or confusion and lack of pleasure, flattened affect, apathy and social withdrawal Olanzapine), and the binding peptides of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof), and optionally pharmaceutically acceptable carriers and/or diluents and/or agents Shape agent.

Other compounds which may be suitable for use in the compositions in addition to the binding peptides of the invention are compounds disclosed, for example, in WO 2004/058258 (see especially pages 16 and 17), including therapeutic drug targets (pp. 36-39). ), alkanesulfonic acid and alkanol sulfate (pages 39-51), cholinesterase inhibitors (pages 51-56), NMDA receptor antagonists (pages 56-58), estrogen (58- Page 59), non-steroidal elimination Inflammatory Drugs (pages 60-61), Antioxidants (pp. 61-62), Peroxisome Proliferator-Accepting Receptor (PPAR) Agonists (pages 63-67), Cholesterol-Reducing Agents (68-75) Page), amyloid inhibitors (pages 75-77), amyloid formation inhibitors (pages 77-78), metal chelators (pages 78-79), antipsychotics and antidepressants (p. Pages 80-82), nutritional supplements (pp. 83-89) and compounds that increase the availability of biologically active substances in the brain (see pages 89-93) and prodrugs (pp. 93 and 94), but In particular, the compounds mentioned on the page numbers of the above indications are incorporated herein by reference.

The protein pharmaceutically active substance may be present in an amount between 1 ng and 10 mg per dose. In general, the administration regimen should be in the range between 0.1 μg and 10 mg of the antibody of the invention, in particular in the range of 1.0 μg to 1.0 mg, and more specifically between 1.0 μg and 100 μg. All individual values within such ranges are also within the scope of the invention. If the administration is carried out via continuous infusion, a more appropriate dose may be in the range between 0.01 μg and 10 mg per kilogram of body weight per hour, with all individual values within such ranges being part of the invention.

Administration will generally be parenteral, such as intravenous or subcutaneous. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The aqueous solvent can be selected from the group consisting of water, alcohol/aqueous solutions, emulsions or suspensions (including physiological saline and buffered media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluids and Nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose) and other substances. Preservatives such as antibacterial agents, antioxidants, chelating agents, inert gases and the like may also be present.

The pharmaceutical composition may additionally comprise a protein carrier, such as serum albumin or immunoglobulin, in particular, a protein carrier of human origin. Depending on the intended use of the pharmaceutical composition of the invention, other bioactive agents may be present.

Certain embodiments of the invention provide a binding peptide of the invention, including antibodies, in particular, monoclonal antibodies and active fragments thereof, that can cross the blood brain barrier when the binding target is in the brain. Certain neurodegenerative diseases are associated with increased permeability of the blood-brain barrier, and thus the binding peptides of the present invention, including antibodies, in particular, monoclonal antibodies or active fragments thereof, can be easily introduced into the brain. Several methods of transporting molecules across the blood-brain barrier are known in the art, including, but not limited to, physical methods, lipid-based methods, and receptor- and channel-based methods, as the blood-brain barrier remains intact.

Physical means for transporting a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies or active fragments thereof) across the blood-brain barrier include, but are not limited to, completely bypassing the blood-brain barrier or by producing in the blood-brain barrier Opening. Bypass methods include, but are not limited to, direct injection into the brain (see, eg, Papanastassiou et al, Gene Therapy 9:398-406 (2002)) and implantation of a delivery device into the brain (see, eg, Gill et al, Nature Med. 9). : 589-595 (2003); and Gliadel Wafers (TM), Guildford Pharmaceutical). Methods of forming openings in the barrier include, but are not limited to, ultrasonic waves (e.g., U.S. Patent Publication No. 2002/0038086), osmotic pressure (e.g., by administration of high tensile mannitol (Neuwelt, EA, Implication of the Blood-Brain Barrier and its Manipulation, Vol. 1 and 2, Plenum Press, NY (1989)), permeation by, for example, bradykinin or penetrant A-7 (see, e.g., U.S. Patent Nos. 5,112,596, 5,268,164, 5,506,206 And 5,686,416), and transfecting a neuron across the blood-brain barrier with a vector containing a gene encoding a binding peptide or antigen-binding fragment (see, e.g., U.S. Patent Publication No. 2003/0083299).

Lipid-based methods of delivering a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies or active fragments thereof) across the blood-brain barrier include, but are not limited to, binding peptides of the invention (including antibodies, in particular, A monoclonal antibody or an active fragment thereof is encapsulated in a liposome that is coupled to an active fragment of a receptor that binds to the vascular endothelium of the blood-brain barrier of the binding peptide (see, for example, US Patent Application Publication No. 20020225313 And the binding peptide of the present invention (including an antibody, in particular, a monoclonal antibody or an active fragment thereof) to a low-density lipoprotein particle (see, for example, US Patent Application Publication No. 20040204354) or a lipoprotein E (apolipoprotein E) (see, for example, U.S. Patent Application Publication No. 20040131692).

Receptor- and channel-based methods of delivering a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies or active fragments thereof) across the blood-brain barrier include, but are not limited to, the use of glucocorticoid blockers to enhance blood-brain Permeability of the barrier (see, for example, U.S. Patent Application Publication Nos. 2002/0065259, 2003/0162695, and 2005/0124533); activated potassium channels (see, e.g., U.S. Patent Application Publication No. 2005/0089473), Inhibiting ABC drug delivery agents (see, e.g., U.S. Patent Application Publication No. 2003/0073713); coating antibodies with transferrin and modulating one or more transferrin receptors The activity (see, e.g., U.S. Patent Application Publication No. 2003/0129186), and the cationization of antibodies (see, e.g., U.S. Patent No. 5,004,697).

A binding peptide (including an antibody, in particular, a monoclonal antibody or an active fragment thereof) of the present invention or a pharmaceutical composition of the present invention may be administered to an individual repeatedly or over an extended period of time. The duration of administration can range from 1 week to as many as 12 months or more. During this time, the binding peptide, antibody or pharmaceutical composition may be administered once a week; every two weeks, three weeks, four weeks, etc.; or at a higher or lower frequency, depending on the needs of the individual being treated.

In another embodiment, the invention provides methods and kits for detecting and diagnosing a tau protein-related disease, disorder, or condition, including a neurodegenerative disease or disorder, such as a tauopathy, wherein the tauopathy comprises a heterogeneous neurodegeneration Sexual disease or condition, including diseases or conditions that develop tauopathy and amyloidosis, including (but not limited to) Alzheimer's disease, CJD, boxer dementia, Down syndrome, Gustman-S Desler-Stoker's disease, inclusion body myositis and prion protein brain amyloid angiopathy, traumatic brain injury and other diseases or conditions that do not show different amyloidosis, including but not limited to muscle atrophy Lateral lateral sclerosis/Guam-type Parkinson's disease-dementia complex, non-Guam-type motoneuron disease with neurofibrillary tangles, argyrophilic dementia, cortical basal ganglia degeneration, diffuse nerve with calcification Fibrillar tangles, frontotemporal dementia associated with chromosome 17 Parkinson's disease, Hallerwalden-Sparts disease, multiple systemic atrophy, type C Niemann-Pick disease, globus pallidus-pons - substantia nigra, Pick's disease, progressive subcortical neuroglial proliferative disease, progressive nuclear paralysis, subacute sclerosing panencephalitis, only Entangled dementia, Parkinson's disease after encephalitis, atrophic muscle rigidity. Pathological abnormalities may be caused by the formation of neurofibrillary lesions or with the formation of neurofibrillary lesions, which are the main brain lesions in τ lesions.

In addition, the methods and kits provided herein are useful for diagnosing tau protein-related diseases, disorders, or conditions, including neurodegenerative diseases or conditions, such as tauopathy (including a group of heterogeneous neurodegenerative diseases or conditions, including showing tauopathy) a predisposition to a disease or condition coexisting with amyloidosis, or for monitoring a small residual disease in a patient, or for predicting a patient's use of a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies and The active fragment) or the reactivity of the composition according to the invention and as described herein. Such methods include known immunological methods commonly used to detect or quantify biological samples or substances in situ conditions.

A disease or condition in which a tau protein-associated disease or condition is diagnosed (specifically, mammals, more specifically, humans) suffering from a tau protein-related disease or condition (including a neurodegenerative disease or condition, such as a tauopathy, including The propensity of a group of heterogeneous neurodegenerative diseases or conditions, including diseases or conditions that exhibit tauopathy and amyloidosis, can be detected by detecting a binding peptide of the invention (specifically, antibodies, in particular, monoclonal antibodies or The active fragment thereof is achieved by immunospecific binding of the epitope of the tau protein in the sample or in situ, including an antibody that suspects a sample containing tau protein or a specific body part or body region and an epitope binding to tau protein. Contacting, binding the antibody to tau protein to form an immune complex, detecting the formation of the immune complex and obtaining the presence or absence of the immune complex with the presence or absence of tau protein in the sample or specific body part or region Correlation, compare the immune complexes as appropriate The amount of the compound and the normal control value, wherein the amount of the immune complex is greater than the normal control value indicates that the individual is suffering from a tau protein-related disease or condition or is at risk of developing a tau protein-related disease or condition.

Monitoring of the individual (specifically, mammals, more specifically humans) after treatment with the binding peptides of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) or compositions of the invention Residual disease can be detected by detecting the immunospecific binding of a binding peptide of the present invention (specifically, an antibody, in particular, a monoclonal antibody or an active fragment thereof) to an epitope of a tau protein in a sample or in situ. Achieving, comprising contacting a peptide (including an antibody, in particular, a monoclonal antibody and an active fragment thereof) of the present invention with a sample or a specific body part or body region suspected of containing tau protein and an epitope binding to tau protein The binding peptides of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) bind to tau protein to form an immune complex, detect the formation of the immune complex and obtain the presence or absence of the immune complex Correlation between the presence or absence of tau protein in a sample or a specific body part or region, and comparing the amount of the immune complex to a normal control value as appropriate, wherein the ratio of the immune complex is Often increases the control value, indicating that the individual may still be suffering from slight residual disease.

Predicting the responsiveness of an individual (specifically, mammalian, more specifically, human) to a therapeutic peptide of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) or compositions of the invention may be detected by Measuring the binding specificity of a binding peptide (specifically, a monoclonal antibody or an active fragment thereof) to an epitope of a tau protein in a sample or in situ, including a sample or a specific body part suspected of containing tau protein or Body area and resistance to tau protein The binding of the peptides of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) to the original antibody, allows binding of the binding peptides of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) to Tau protein to form an immune complex, detecting the formation of the immune complex and correlating the presence or absence of the immune complex with the presence or absence of tau protein in the sample or specific body part or region, as appropriate The amount of the immune complex before and after the start, wherein a decrease in the amount of the immune complex indicates that the patient is more likely to respond to the treatment.

It can be used for the diagnosis of tau protein-related diseases or conditions, for the diagnosis of tau protein-related diseases or conditions (including neurodegenerative diseases or conditions, such as tauopathy, including a group of heterogeneous neurodegenerative diseases or conditions, including the appearance of tauopathy a disease or condition associated with amyloid), or for monitoring a microscopic residual disease in a patient or for predicting a patient's binding to a peptide of the invention, including an antibody, in particular, a monoclonal antibody and an active fragment thereof, or as described herein The biological sample of the reactivity of the composition of the invention is, for example, a body fluid such as serum, plasma, saliva, gastric secretion, mucus, cerebrospinal fluid, lymph, and the like, or a tissue or cell sample obtained from an organism, such as a nerve. , brain, heart or vascular tissue. To determine the presence or absence of tau protein in a sample, any immunoassay known to those of ordinary skill can be used, such as analysis using an inter-detection method using a secondary detection reagent, ELISA, and immunoprecipitation and agglutination analysis. Detailed descriptions of such analyses are provided, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612; Maertens and Stuyver WO 96/13590; Zrein et al. (1998) and WO 96/ 29605.

At the time of on-site diagnosis, the binding peptides of the invention (including antibodies of the invention, in particular, monoclonal antibodies and active fragments thereof, or any active and functional portions thereof) may be by methods known in the art (such as intravenous The organism to be diagnosed is administered by subcutaneous, intranasal, intraperitoneal, intracerebral, intraarterial injection to cause specific binding between the antibody of the present invention and the epitope of the amyloid protein. The binding peptide/antigen complex is preferably detected via a label linked to a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies or functional fragments thereof) or any other detection method known in the art.

For diagnostic applications, or for the diagnosis of tau protein-related diseases or conditions (including neurodegenerative diseases or conditions, such as tauopathy, including a group of heterogeneous neurodegenerative diseases or conditions, including the appearance of tauopathy and amyloidosis) a predisposition to a disease or condition, or for monitoring a microscopic residual disease in a patient, or for predicting a patient's binding to a peptide of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) or as described herein Immunoassays for the reactivity of the compositions of the invention are typically dependent on the labeled antigen, binding peptide or secondary detection reagent. Such proteins or agents can be labeled with compounds commonly known to those of ordinary skill in the art, including enzymes, radioisotopes, and fluorescent, luminescent, and chromogenic materials including, but not limited to, dyed particles such as colloidal gold and latex beads. Among them, radioactive labels can be used for almost all types of analysis with the most changes. Labels that bind to enzymes are especially useful when radioactivity must be avoided or when rapid results are required. Although fluorescent dyes require expensive equipment in use, they provide a very sensitive detection method. Binding peptides suitable for use in such assays are the binding peptides disclosed and claimed herein, including antibodies, in particular, monoclonal antibodies, polyclonal antibodies, and affinity-purified strains. antibody.

Alternatively, a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) can be reacted by a labeling substance (such as protein A or G or a secondary antibody) having affinity for an immunoglobulin. Indirect tagging. The binding peptides of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) can be combined with a second substance and detected with a labeled third substance having an affinity for a second substance that binds to the antibody. For example, a binding peptide of the invention (including antibodies, in particular, monoclonal antibodies and active fragments thereof) can be bound to biotin and detected using labeled avidin or streptavidin. Binding peptide/biotin conjugate. Similarly, the binding peptide can bind to the hapten and the labeled anti-hapten binding peptide is used to detect the binding peptide/hapten conjugate.

Those of ordinary skill in the art will recognize such and other suitable indicia that can be employed in accordance with the present invention. The binding of such markers to the binding peptide or fragment thereof can be accomplished using standard techniques generally known to those of ordinary skill in the art. Typical techniques are described in Kennedy, J. H., et al, 1976 (Clin. Chim. Acta 70: 1-31), and Schurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 57: 1-40). The coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimethyleneimine method and others, all of which are incorporated herein by reference.

Current immunoassays utilize a diabody assay that detects the presence of an analyte, wherein the antibody is indirectly labeled by reactivity with a second antibody that has been labeled with a detectable label. The secondary antibody is preferably an antibody that binds to an antibody of an animal from which the monoclonal antibody is derived. In other words, if the monoclonal antibody is a mouse antibody, it is labeled. The secondary antibody is an anti-mouse antibody. For antibodies to be used in the assays described herein, the label is preferably an antibody coated bead, in particular, a magnetic bead. For antibodies to be employed in the immunoassays described herein, the label is preferably a detectable molecule such as a radioactive, fluorescent or electrochemiluminescent substance.

Alternative diabody systems (commonly referred to as rapid format systems) can also be used within the scope of the present invention because of their suitability for rapid determination of the presence of an analyte. This system requires high affinity between the antibody and the analyte. According to one embodiment of the invention, a pair of antibodies specific for amyloid proteins are used to determine the presence of amyloid proteins. One of these antibody pairs is referred to herein as a "detection antibody" and the other of these antibody pairs is referred to herein as a "capture antibody." The monoclonal antibodies of the present invention can be used as a capture antibody or a detection antibody. The monoclonal antibodies of the present invention can also be used as both capture and detection antibodies in a single assay. Thus, one embodiment of the invention uses a double antibody sandwich method to detect amyloid proteins in biological fluid samples. In this method, an analyte (amyloid protein) is sandwiched between a detection antibody and a capture antibody, and the capture antibody is irreversibly immobilized on a solid support. The detection antibody contains a detectable label to identify the presence of the antibody-analyte core and thereby identify the presence of the analyte.

Exemplary solid phase materials include, but are not limited to, microtiter plates, polystyrene test tubes, magnetic, plastic or glass beads, and slides well known in the art of radioimmunoassay and enzyme immunoassays. Methods for coupling antibodies to a solid phase are also well known to those of ordinary skill in the art. Recently, a variety of porous materials such as nylon, nitrocellulose, cellulose acetate, glass fibers, and other porous polymers have been used as solid supports.

The invention also relates to a diagnostic kit for detecting tau protein in a biological sample comprising a composition as defined above. Furthermore, the invention relates to a latter diagnostic kit comprising, in addition to the composition as defined above, a detection reagent as defined above. The term "diagnostic kit" generally refers to any diagnostic kit known in the art. More specifically, the latter term refers to a diagnostic kit as described by Zrein et al. (1998).

Another object of the present invention is to provide novel immunological probes and test kits for detecting and diagnosing tau protein-related diseases and conditions comprising the binding peptides of the present invention. For immunological probes, the binding peptide is linked directly or indirectly to a suitable reporter molecule, such as an enzyme or radionuclide. The test kit includes a container containing one or more binding peptides of the invention and a binding peptide for binding to a tau antigen to form an immune complex and detecting the formation of the immune complex to obtain the presence or absence of the immune complex A specification of the correlation of the presence or absence of tau protein.

Instance Example 1: Production and screening of fusion tumors and antibodies

The goal of this study was to generate and screen anti-τ mAbs (monoclonal antibodies). Hybridomas are produced by fusion of spleen cells immunized with tau vaccine to myeloma cell lines. The reactivity of the fusion tumor against phosphorylated and non-phosphorylated full-length tau protein, as well as phosphorylated and non-phosphorylated tau antigen peptides used in vaccine formulations, was evaluated. The fusion tumor was also screened by immunochemical analysis of brain slices of tau protein transgenic mice according to the reactivity of the fusion tumor supernatant to tau protein entanglement.

1.1 method 1.1.1 Fusion

The fusion tumor producing line used wild type C57BL/6 mice inoculated with ACI-35 (Tau393-408 [pS396, pS404]). Mice were given an ACI-35 vaccine on day 0, followed by a second episode on day 4 and fusion on day 7.

6×10 ⁷ (ACI-35) splenocytes of the immunized mice were fused with 2×10 ⁷ SP2-O-Ag14 myeloma cells at a ratio of 3 spleen cells/1 myeloma cells.

The fusion results were generated in 8x96 well plates and the pure lines were named according to the culture plates (1-8), then according to the columns (A-G) and finally according to rows (1-12).

1.1.2 Select pure screening method

The 8x96 well plate was first screened twice for IgG. The positive expression was then transferred to a 24-well plate and the cell supernatant (= pure line) of the growing cells was tested in a τ ELISA screen and an immunohistochemical TAUPIR screen. Positive supernatants in ELISA and/or TAUPIR were transferred to T25 flasks and screened for pure IgG performance in τ ELISA screening and TAUPIR screening.

1.1.3 IgG screening

ELISA plates (Costar; Sigma) were coated with 50 μL of coating buffer containing anti-mouse IgG antibody (AbD Serotec, Düsseldorf, Germany) for 16 hours at 4 °C per well. After washing the dishes with PBS/Tween, each well was blocked with 100 μL of blocking solution for 1 hour at ambient temperature. Undiluted fusion tumor supernatant (50 μl/well) was incubated for 1 hour at ambient temperature. After washing, a mixture of anti-mouse IgG1, IgG2a, IgG2b, IgG3 or IgM (AbD Serotec) combined with horseradish peroxidase (HRP) was applied at ambient temperature. Time. After the final wash, detection was performed with HRP substrate (TMB; 3-3', 5, 5'-tetramethylbenzidine) and the plate was read at 405 nm using a micro-disc reader. The results are expressed in terms of optical density (O.D.).

1.1.4 fusion tumor τ ELISA screening

Fusion tumor ELISA screening was performed on the following: pTau peptide (ACI-35, T3.5: Tau393-408 [pS396/pS404; PolyPeptide Laboratories, Hiller d, Denmark), corresponding non-phosphorylated tau protein peptide (T3.6: Tau393-408, PolyPeptide Laboratories), phosphorylated full-length (441 aa) tau protein (pTau protein, Vandebroek et al, 2005) and full-length (441 aa) tau protein (τ protein, SignalChem, Richmond, Canada). Finally, bovine serum albumin (BSA) was used as a negative control.

Each plate was coated overnight at 4 °C with 10 μg/ml of the corresponding tau peptide and 1 μg/ml of the corresponding tau protein. After each well was washed with PBS-0.05% Tween 20 and blocked with PBS-0.05% Tween 20 containing 1% BSA, undiluted fusion tumor supernatant or medium negative control was added to each dish at 37 °C. Cultivate for 2 hours. After washing, the plates were incubated with alkaline phosphatase (AP)-conjugated anti-mouse IgG total antibody (Jackson Laboratories, Baltimore, PA, USA) for 2 hours at 37 °C. After washing, the plates were incubated with pNPP (p-nitrophenyl phosphonate) (AP phosphatase substrate) and read at 405 nm using an ELISA disc reader. The results are expressed in O.D. (optical density).

1.1.5 Fusion tumor IHC screening: binding of anti-tau protein antibody to tangles in brain slices of transgenic mice (TAUPIR)

The TAUPIR experiment was carried out according to the protocol of Example 3.1.2.

1.1.6 T25 flask IgG screening

The ELISA plate was coated with a 5 ug/ml anti-mouse IgG F(ab')2 fragment-specific antibody (Jackson Laboratories, Baltimore, PA, USA) with a carbonate-bicarbonate coating buffer pH 9.6 (Sigma) at 4 °C. , Buchs, Switzerland) coated overnight. After washing the dishes, undiluted fusion tumor supernatant, positive control IgG1 antibody (6E10, 1 ug/ml: Covance, Emeryville, CA, USA) or negative control (single medium) were incubated for 1 hour at room temperature. After the washing step, secondary AP-conjugated goat anti-mouse IgG (subclass 1+2a+2b+3) Fcγ fragment-specific antibody (Jackson Laboratories, Baltimore, PA, USA) was incubated on tray for 2 hours at 37 °C. After the final wash, pNPP (p-nitrophenyl phosphate) (AP phosphatase substrate) was detected and read at 405 nm using an ELISA reader. The results are expressed in O.D. (optical density).

1.2 results

Cell supernatants produced by fusion in an 8x96 well plate were screened for IgG production. Of the 768 wells tested (8 x 96 wells), 48 wells positive for IgG production were selected based on optimal binding to the vaccine phosphorylated peptide and optimal binding to full length phosphorylated tau protein. Selection is based on ELISA, based on binding to peptides and full-length phosphorylated tau protein, and selectivity when compared to non-phosphorylated peptides and non-phosphorylated full-length tau protein. Twenty-four selected fusion tumors were subcultured by inoculating 2 dishes (1 cell/well) and 1 disk (0.5 cells/well) per fusion tumor. The supernatant was again tested for binding to phosphorylated and phosphorylated proteins to examine the binding profile, followed by assessment of stability in 6 week culture. Eight stable lines were then selected and tested for isotypes and combined using ELISA and TAUPIR assays as described in the methods.

1.3. Conclusion

The antibodies produced have shown high specificity for p-peptides with only minor binding to non-phosphorylated peptides.

A total of 8 pure lines were selected for further colonization and sequencing (see Tables 6 and 7) and 6 pure lines were deposited in DSMZ (see Table 10).

The positive mothers above were purely in 96-well plates, then further cultured in 24-well plates and finally in T25 flasks. At each stage, the supernatant of the fusion tumor was screened by ELISA, Taupir, and Western blotting.

Example 2: Selection of antibody light chain and heavy chain variable regions

The antibody heavy and light chain variable region genes from the fusion tumor cells are selected and the position of the DNA sequence and the complementarity determining region (CDR) and antibody binding characteristics are determined.

Using the Qiagen RNeasy mini kit (Cat No: 74104), using a 3 × 10 ⁶ th (1 vial) Preparation of hybridoma cells total RNA. RNA was eluted in 50 uL of water and tested on a 1.2% agarose gel.

Using reverse transcriptase and primers prepared V _H and V _K cDNA IgG and κ constant region. The first strand of cDNA was amplified by PCR using a large set of signal sequence primers. The amplified DNA was gel purified and cloned into the vector pGem ^® T Easy (Promega). Homogenous expected size of the insert for screening the resulting V _H and V _K. The DNA sequence of the selected pure line is determined in both directions by automated DNA sequencing. The position of the complementarity determining region (CDR) in the sequence is determined with reference to other antibody sequences (Kabat EA et al, 1991).

Example 3: Combining Research I

The goal is to measure the secondary selection of mice immunized with τ liposome vaccine. Binding of antibodies produced by tumors to phosphorylated tau protein (pTau). To test this binding, the binding of purified antibodies to phosphorylated and non-phosphorylated full-length tau proteins was measured using enzyme-linked immunosorbent assay (ELISA), as well as phosphorylated and non-phosphorylated tau protein antigens used to prepare liposome vaccines. Binding of peptides.

Screening is done by two other methods. Immunohistochemistry (IHC) analysis (TAUPIR) was performed on brain sections of tau protein transgenic animals using anti-tau protein antibody as primary antibody. In addition, Western blot (WB) analysis was performed on the brain protein homogenate of tau protein transgenic mice using anti-tau protein antibody as a dot ink antibody.

3.1 method 3.1.1. ELISA: Phosphorylated tau protein binding assay

To test the binding of purified antibodies to τ and pTau, ELISA analysis was used. Briefly, Nunc MaxiSorp 96-well plates (Nunc, Roskilde, Denmark) used 1 μg/mL full-length (441 aa) tau protein (SignalChem, Richmond, Canada) or phosphorylated full-length (441 aa) tau protein (Vandebroek et al., 2005) Coating. In addition, the plates were coated with 10 μg/mL of the tau derivative vaccine peptide Tau393-408 (S396 and S404 phosphorylated or not unphosphorylated). To test for cross-reactivity with the tau sequence and the pTau sequence of the different pTau epitopes not used to prepare the vaccine, the plates were coated with 10 μg/mL of peptide: Tau393-408 (S396 and S404 phosphorylated or unphosphorylated) . Coating overnight in phosphate buffered saline (PBS) at 4 °C. The plates were washed extensively with 0.05% Tween 20/PBS and then blocked with 0.05% Tween 20/PBS containing 1% bovine serum albumin (BSA) for 1 hour at 37 °C. The antibody tested was then added in 8 or 16 double serial dilutions between 20 μg/mL and 0 μg/mL, and allowed in Incubate at 37 ° C for 2 hours. Plates were then washed as described above and 1/6000 dilutions of AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, Suffolk, England) in 0.05% Tween 20/PBS were added for 2 hours at 37 °C. After washing, the plate was incubated with p-nitrophenyl phosphate disodium hexahydrate (pNPP; Sigma-Aldrich, Buchs, Switzerland) phosphatase substrate and incubated at 30 minutes, 1 hour, 2 hours or 16 hours. After that, it was read at 405 nm using an ELISA disc reader.

3.1.2. TAUPIR and Western blot analysis: the binding of anti-tau protein antibody to tau protein entanglement in brain slices of tau protein transgenic animals (TAUPIR)

For TAUPIR staining, brain sections were obtained from TPLH mice (transgenic mice expressing the longest isoform of hTau ^P301L (441 aa)), older (>18 months old) double transgenic gene biGT mice (with TPLH hybridized GSK-3β transgenic mouse) and double transgenic gene biAT mouse (hAPP ^V717I transgenic mouse hybridized with TPLH). As a negative control, sections of τ gene knockout mice (TKO; 6 months old) were used. Brain sections were washed in PBS for 5 minutes, followed by incubation in PBS:MeOH (1:1) containing 1.5% H ₂ O ₂ for 15 minutes at room temperature to block endogenous peroxidase activity. After washing the sections 3 times in PBST (PBS/0.1% Triton X100), they were incubated in PBST + 10% FCS (fetal calf serum) blocking solution for 30 minutes at room temperature. Incubation overnight at 4 °C with the anti-tau protein antibody tested using the following antibody concentrations in PBST/10% FCS: 0.0053 μg/mL of ACI-35-2A1-Ab1, 0.0048 μg/mL of ACI-35-2A1 -Ab2, 0.015 μg/mL of ACI-35-4A6-Ab1, 0.0047 μg/mL of ACI-35-1D2-Ab1, 0.0055 μg/mL of ACI-35-2G5-Ab1, and 0.01 μg/mL of ACI- 35-2G5-Ab2 and ACI-35-2G5-Ab3. Sections were then washed 3 times in PBST and subsequently incubated with HRP-conjugated goat anti-mouse (purchased from Dako, Glostrup, Denmark) secondary antibody in PBST/10% FCS for 1 hour at room temperature. Prior to detection, sections were washed 3 times with PBST and incubated for 5 minutes in 50 mM Tris/HCl pH 7.6. This was carried out by incubating the sections in diaminobenzidine (DAB: 1 spindle in 10 ml of 50 mM Tris.HCl + 3 μL H ₂ O ₂ 30%; MP Biomedicals, Solon, OH, USA) for 3 minutes. Detection. The reaction was stopped by washing the sections 3 times in PBST. The sections were then transferred to a decadiated glass dish and air dried on a 50 ° C hot plate for 2 hours. Comparative staining was performed using Mayers hematoxylin (Fluka Chemie, Buchs, Switzerland) for 1 minute, followed by washing with running tap water for 4 minutes by a washing step. Sections were dehydrated by passage in 50%, 70%, 90%, and 100% (twice) ethanol baths followed by 2 passes in xylene for 1 minute. Finally, the sections were mounted under a cover glass for imaging using DePeX (BDH Chemicals Ltd., Poole, England).

Brain tissue homogenate protein isolated from wild-type mouse (FVB) tau protein transgenic mice (TPLH and biGT) or tau knockout mice (TKO) by SDS-PAGE (10%) was further stained (Western ink) Point method). For Western blotting, the following concentrations of antibody were used: 0.53 μg/mL of ACI-35-2A1-Ab1, 0.48 μg/mL of ACI-35-2A1-Ab2, 0.5 μg/mL of ACI-35-4A6-Ab1 0.47 μg/mL of ACI-35-1D2-Ab1, 0.55 μg/mL of ACI-35-2G5-Ab1, 0.33 μg/mL of ACI-35-2G5-Ab2, and 0.5 μg/mL of ACI-35-2G5 -Ab3.

3.2 Results

Antibodies ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, ACI-35-1D2-Ab1, ACI-35-2G5-Ab2 and ACI-35-2G5-Ab3 phosphorylate human tau protein (Table 2) More specifically, it shows high binding activity and specificity for the antigenic phosphorylated tau protein peptide used in the corresponding vaccine. No cross-reactivity with non-phosphorylated tau protein or with other phosphorylated and non-phosphorylated tau protein derived peptides was observed. The antibody ACI-35-4A6-Ab1 showed high binding activity only to the antigenic phosphorylated tau protein peptide used for the preparation of the vaccine according to its selection. The cross-reactivity with the non-phosphorylated counterpart of the antigenic peptide used to prepare the vaccine was found to be low, and the results were estimated based on the pure line selection. The antibody ACI-35-2G5-Ab1 showed high binding activity only to the antigenic phosphorylated tau protein peptide used for the preparation of the vaccine. The cross-reactivity with the T4.5 phosphorylated peptide was observed to be small, and the T4.5 phosphorylated peptide contained a portion of the antigenic peptide sequence used in the vaccine.

TAUPIR and WB are used to examine the binding of tau protein entanglement in the brain of mice with advanced tauopathy (biGT > 18 months), and the full length of the homogenate from the denatured tissue derived from these mice. The combination of τ. Different brain regions were analyzed: the CA1, CA3 and dentate gyrus (DG) parts of the cortex and hippocampus. The antibodies ACI-35-2A1-Ab1 and ACI-35-2A1-Ab2 showed the best TAUPIR results: the cytoplasmic staining was dense and the nerve fiber filaments were transparent, especially in the CA1 and CA3 regions of the hippocampus. The antibody ACI-35-4A6-Ab1 was negative in TAUPIR, which showed only a slightly stained dim tangled structure. The antibody ACI-35-1D2-Ab1 showed good cytoplasmic TAUPIR staining and the presence of neurofibrillar filaments in the CA1 region. Antibody ACI-35-2G5-Ab1 at Negative in TAUPIR: nuclear staining and only some tangles staining. Finally, ACI-35-2G5-Ab2 showed similar good cytoplasmic TAUPIR staining with ACI-35-2G5-Ab3 antibody, and nerve fiber filaments were observed in both CA1 and CA3 of hippocampus. The dye quality ratings using the + or - symbols are shown in Table 2. Ink dot analysis of brain tissue homogenate of tau protein transgenic mice showed that all antibodies were well bound to the expected τ band (Table 2, rated +), among which ACI-35-1D2-Ab1 and ACI-35 -2G5-Ab1 also showed additional non-specific binding (-/+).

Example 4: Binding Study II 4.1 method 4.1.1. SPR combination analysis

All SPR experiments were performed on a Biacore X instrument (GE Healthcare). The sensor wafer SA (streptavidin-derived carboxymethyl polydextrose) was purchased from GE Healthcare. The running buffer was PBS (Dulbecco's PBS, Sigma D8537). The non-covalently bound streptavidin on the surface of the sensor was first removed by injecting 8 pulses (about 1 μL each) of 16 mM NaOH (aqueous solution). The phosphorylated tau protein peptide was then dissolved in PBS to give a final peptide concentration of 1 μM and then injected (35 μL) at 5 μL/min onto the flow cell (fc) 2 of the sensor wafer. After coupling, a final degree of fixation of 130 RU was obtained. To study the binding of the antibody to the surface of the wafer, several concentrations of antibody were prepared by serial 2-fold dilution with running buffer. Injections were performed on fc 1 and fc 2 for 120 seconds at a flow rate of 50 μL/min. Flow cell 1 is underived and reacts fc 2 from fc 1 to correct for instrument noise and bulk refractive index changes. Wash with running buffer immediately after each injection The surface is 100 seconds. To remove any residual binding antibodies on the wafer, surface regeneration was performed by injecting 1 μL of 10 mM glycine hydrochloride pH 1.7. Kinetic analysis was performed using a numerical integration algorithm and overall analysis was performed using BIAevaluation 3.0. The sensor profiles of the different concentrations of antibody injections obtained were superimposed and the baseline was adjusted to zero. For curve fitting, all data were fitted simultaneously with respect to the 1:1 homogeneous Langmuir model.

Peptide used

4.2 Results

The binding of the anti-tau protein antibody to the phosphorylated tau protein peptide was immediately monitored using SPR. Analysis of the binding and dissociation phases of antibody binding can be used to determine the association rate constant ( k _a ), the dissociation rate constant ( k _d ), and the dissociation constant K _D .

In the range of 46→734 nM of the antibody analyzed (or 11.5→184 nM for ACI-35-4A6-Ab1), all antibodies were found to specifically bind to the peptide T3 on the surface of the underivatized carboxymethyl polydextrose. 30. Kinetic analysis of the sensor map revealed that the dissociation constant K _D of the binding interaction between different antibodies and T3.30 was between 2 nM and 82 nM. Therefore, it was demonstrated that the antibody recognized the phosphorylated peptide T3.30 with a very high affinity (Table 3).

Example 5: ELISA for human brain samples in combination with Study III (ELISA for detection of phosphorylated tau protein multimers) 5.1 method 5.1.1. Human samples: preparation of human brain samples for analysis as described herein

Brain Endowment Bank, from the University of Miami The University of Miami received postmortem cortex from 10 patients with Alzheimer's disease (AD) and 10 age-matched controls. The mean age of death in AD patients (7 females, 3 males) was 81.1 ± 7.3 years, and the mean age of death in controls (no neurological symptoms; 9 females, 1 male) was 87.0 ± 5.8 (based on history Student t-test (student t-test), no significant difference with AD patients). All samples were from Caucasian (Caucasian). AD samples were characterized by Braak disease stage (Braak and Braak (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82: 239-259), as shown in Table 4.

The post-mortem cortex of 10 AD patients and 10 age-matched controls were homogenized according to the following protocol. Brain sections were weighed and homogenized in 9 volumes of 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA containing phosphatase inhibitor (30 mM NaF, 0.2 mM Na ₃ VO ₄ , 1 nM Okada Well, 1 mM PMSF, 5 mM Na ₄ P ₂ O ₇ ) and protease inhibitor (completely small; Roche, Switzerland). Homogenization on ice using glassware. This constitutes the total tissue homogenate fraction (TH). Protein concentration was measured using Bradford reagent (Sigma).

5.1.2. Configuration 1 ELISA: Configuration 1 ELISA analysis can detect the presence of phosphorylated tau protein multimers in human cerebral cortical tissue homogenate after death in AD individuals and age-matched controls

A multi-titrator 96-well plate was coated overnight at 4 °C with a carbonate/bicarbonate buffer containing 5 μg/ml of antibody. After washing 4 times in PBS-Tween, the plates were saturated with PBS-Tween 10% BSA for 1 hour at 37 °C. Then add to the hole Brain tissue homogenate in 50 μL PBS at a concentration of 100 ng/μL was incubated at 37 ° C for 2 hours. After washing the dishes, the same biotin-labeled antibody as used for coating was incubated at 37 ° C for 1 hour at a final concentration of 5 μg/mL. The plates were washed and after the addition of avidin-peroxidase (Vectastain ABC kit, Vector Laboratories) and its substrate (ABTS, Roche 10881420), the plates were read at different times. Values are expressed as the mean OD±SD of 10 AD individuals and 10 control individuals.

5.2 results

The antibodies ACI-35-2A1-Ab1 and ACI-35-2G5-Ab3 were tested for phosphorylation and multimer specific configuration 1 ELISA to detect phosphorylated tau protein (pTau) in brain tissue homogenates of AD and control individuals. The ability of the polymer. In this analysis of the two antibodies, a very significant (p < 0.001) difference between AD patients and age-matched controls (n = 10) was observed (Figure 1). ACI-35-2A1-Ab1 and ACI-35-2G5-Ab3 anti-pTau antibodies were able to detect τ-pS396 multimerization in human brain samples after death using AD patients and age-matched controls. body.

Example 6: Binding Study IV - Western blot analysis of human brain samples 6.1 method 6.1.1. Human sample: Same as the human sample preparation method described in Method 5.1.1. 6.1.2. Western blot analysis: Western blot analysis can detect the presence of phosphorylated tau protein in the cerebral cortex homogenate of individuals with AD and age-matched controls

The anti-human tau antibody used in this study was mouse ACI-35-2A1-Ab1, ACI-35-1D2-Ab1 and ACI-35-2G5-Ab3, all of which were directed against τ-pS396. A mouse monoclonal tau-13 antibody (Abeam ab24636) against total human tau protein and a rabbit monoclonal antibody E178 (Abeam ab32057) against tau-pS396 were used as controls. A total of 20 μg of various tissue homogenates were loaded onto a 10% polyacrylamide Bis-TRIS pre-cast gel (Nupage Novex 10% Bis-TRIS Midi gel, Invitrogen) per lane. Proteins were resolved according to the manufacturer's recommendations for NuPAGE MOPS SDS running buffer (Invitrogen NP0001). Protein dot analysis was performed on a PVDF membrane (Immobilon-FL, Millipore IPFL00010) on ice in 25 mM TRIS pH 8.6, 190 mM glycine buffer, 20% methanol on ice. The membrane was blocked for 1 hour in Licor blocking buffer (Odyssey) which had been diluted in 1/3 in PBS. The membrane was incubated overnight with the following concentrations of primary antibody: 0.6 μg/mL of TAU-13, 1/5000 diluted E178, 0.53 μg/mL of ACI-35-2A1-Ab1, 0.47 μg/mL of ACI-35-1D2 -Ab1, and 0.5 μg/mL of ACI-35-2G5-Ab3, these antibodies are in Licor buffer Dilution and PBS + 0.1% Tween-20 (PBS-T) dilution. After washing 4 times with PBS-T, the membrane was incubated with goat anti-mouse antibody-coupled LICOR 800 dye (goat anti-mouse IRDye 800 CW, Odyssay) for 1 hour at room temperature, washed again 4 times with PBS-T, and used The LICOR system scans for image reproduction.

6.2 Results

Detection of antibodies ACI-35-2A1-Ab1, ACI-35-1D2-Ab1 and ACI-35-2G5-Ab3 using phosphorylation-specific Western blotting methods to detect phosphorylation in brain tissue homogenates of AD and control individuals The ability of tau protein (pTau). First make All post-mortem human cortical samples were characterized with commercial antibodies against human tau protein (anti-total tau protein (TAU-13) and anti-pS396[tau] (E178) antibodies). As shown in Figure 2A, characteristic τ ladders corresponding to different τ isoforms in the range of 50-70 kDa were detected in all samples using the TAU-13 antibody. Interestingly, in the AD brain homogenate, a relative change in the mode of τ migration was also observed, as was the presence of hyperphosphorylated tau protein in the AD brain. By demonstrating this hypothesis, the commercial anti-pS396 tau antibody was able to discriminate between controls and AD (Fig. 2B). In fact, the anti-pS396 tau antibody revealed that there are three major immunoreactive bands corresponding to the (over)phosphorylated isoforms of tau protein in all AD brain homogenates, whereas the intensity in healthy controls is weak or does not exist. In addition, AD samples showed high molecular weight TAU-13 immunoreactive smears, possibly indicating the presence of aggregated τ (Fig. 2A).

Western blotting with ACI-35-2A1-Ab1 revealed that there were two immunoreactive protein bands in the AD brain homogenate for the expected size of phosphorylated tau protein, but not in the control (Fig. 3A). The presence of two major non-specific bands at approximately 35 kDa and approximately 40 kDa illustrates the weak immune response as demonstrated by Western blotting using ACI-35-2A1-Ab1. Western blotting using ACI-35-1D2-Ab1 revealed that there were two immunoreactive protein bands in the AD brain homogenate for the expected size of phosphorylated tau protein, but not in the control (Fig. 3B). The presence of a non-specific band at approximately 40 kDa and approximately 50 kDa and the presence of four non-specific bands between 80 kDa and 150 kDa may indicate the use of ACI-35-1D2-Ab1 as indicated by the Western blot method Weak immune response. Western blotting method using ACI-2G5-Ab3 revealed that there are three (per)phosphoric acids corresponding to tau protein in all AD brain homogenates. The primary immune response zone of the isoform was not present in healthy controls except for one control individual with a family history of AD (C22) (Fig. 3C). This report demonstrates that ACI-35-2A1-Ab1, ACI-35-1D2-Ab1, and ACI-35-2G5-Ab3 can distinguish between AD patients and age-matched controls for the presence of pS396 tau protein in human post-mortem cortex, and therefore The monoclonal antibodies recognize AD-related pathological τ isoforms.

Example 7: Binding Study V-Configuration 1 (ELISA for human brain samples) 7.1 method 7.1.1. Human samples. The same procedure as the human sample preparation method as described in Method 5.1.1. except for the last part (preparation of the S1 separation portion). 7.1.2. Separation of S1 tau protein: Secondary separation of the fraction of the total tissue homogenate to obtain soluble tau and phosphorylated tau protein.

To prepare a soluble tau (S1) fraction for AlphaLISA analysis, half of the volume of TH was aliquoted and stored at -80 °C. The remaining TH fraction was further processed by the addition of Triton X-100 until a final concentration of 0.4%. The samples were thoroughly mixed and vortexed several times, followed by centrifugation at 5,000 rpm for 5 minutes at 4 °C. The supernatant constitutes the S1 separation portion. Samples were aliquoted and stored at -80 °C. Protein concentration was measured using Bradford reagent.

7.1.3. AlphaLISA: AlphaLISA analysis detects the presence of phosphorylated tau protein in the cerebral cortical tissue homogenate of AD individuals and age-matched controls.

The antibodies ACI-35-2A1-Ab1, ACI-35-1D2-Ab1 and ACI- against τ-pS396 were used in the EZ-Link Micro Sulfo-NHS-LC-Biotinylation kit (Thermo Scientific) according to the manufacturer's instructions. 35- 2G5-Ab3 was biotinylated. Biotin was used in the labeling reaction in an excess of 25 times the molar concentration of the antibody. After biotin labeling, excess free biotin was removed by dialysis against PBS using a Slide-A-Lyzer MINI dialysis apparatus 10K MWCO (Thermo Scientific). The biotinylated antibodies were named ACI-35-2A1-Ab1-BT, ACI-35-1D2-Ab1-BT and ACI-35-2G5-Ab3-BT. Antibody Tau-13 was bound to activated alpha acceptor beads (Perkin Elmer) using the following protocol: 0.1 mg of Tau-13 antibody solution (purified by Protein A column) mixed with 1 mg of AlphaLISA acceptor beads and supplemented 0.13 M phosphate buffer (pH 8.0) until the final reaction volume is 200 μL. Next, 1.25 μL of 10% Tween-20 and 10 μL of 25 mg/mL NaBH ₃ CN solution were added and the tubes were incubated at 37 ° C for 48 hours under gentle rotation (7 rpm). After the binding reaction, the active sites on the beads were blocked by the addition of 10 μL of a carboxymethoxyamine solution and further incubated at 37 ° C for 1 hour. Finally, the beads were washed twice with 200 μL of 0.1 M Tris-HCl pH 8.0 and in 200 μL of storage buffer (PBS with 0.05% Proclin-300) (making the final concentration of AlphaLISA receptor beads 5 mg/mL) ) Store at 4 ° C.

AlphaLISA is a homogeneous analysis based on bead proximity chemiluminescence. If the alpha donor and acceptor beads are in close proximity, the chemical reaction cascade produces an amplification signal upon laser excitation. Upon excitation at 680 nm, the sensitizer contained in the donor beads converts the surrounding oxygen into a more reactive singlet oxygen species. These singlet diffusions (up to 200 nm, in the 4 microsecond half-life) and produce a chemiluminescent reaction in the acceptor beads, causing luminescence. The analysis configuration is as follows: S1 sample pre-thinned in Alpha Assay Buffer (PerkinElmer AL000C) A 20 μg/mL stock concentration was obtained. Add the following reagent to a 384-well white OptiPlate (PerkinElmer) to a final volume of 50 μL: S1 brain homogenate (5 μL), 10 μL of ACI-35-2A1-Ab1-BT, ACI-35-1D2-Ab1- BT or ACI-35-2G5-Ab3-BT (final antibody concentration 0.2 nM, 0.5 nM or 0.5 nM, respectively), and 10 μL of Tau13-receptor bead conjugate (final bead concentration 2.5 μg/mL) . The reaction mixture was incubated for 1 hour at room temperature, and 25 μL of streptavidin donor beads were added and further incubated for 2 hours at room temperature in the dark. Values were read using an EnSpire Alpha instrument and analyzed using EnSpire Workstation version 3.00. Statistical analysis of the data was performed using the GraphPad Prism software. Results are presented in alpha units ± SD.

7.2 Results

AlphaLISA assay was used to test antibodies ACI-35-2A1-Ab1, ACI-35-1D2-Ab1 and ACI-35-2G5-Ab3 to detect τ-pS396 in human brain tissue homogenate and to discriminate between AD and age-matched controls Ability. All antibodies detected τ-pS396 (Figs. 4A, 4B, 4C). For all antibodies, the difference in signal detection between AD and controls (n=10) was also significantly higher, showing increased signal in the brain of AD individuals; ACI-35-2A1-Ab1 (p<0.0001), ACI-35-1D2-Ab1 (p<0.0001), and ACI-35-2G5-Ab3 (p=0.002). In summary, AlphaLISA technology was used to demonstrate the ability of ACI-35-2A1-Ab1, ACI-35-1D2-Ab1 and ACI-35-2G5-Ab3 to detect pS396-Tau in the brain of AD individuals, and to distinguish between AD and control. The ability of the person.

Example 8: In vivo efficacy of ACI-35-2G5-Ab3 antibody 8.1 method 8.1.1. Study configuration: anti-pTau antibody ACI-35-2G5-Ab3 was administered to tau protein transgenic mice for 2 times in vivo treatment or fruit

Females with a C57BL/6xDBA background of 6-7 months of age were administered by intraperitoneal injection of 3 or 10 mg/kg of ACI-35-2G5-Ab3 or vehicle control (PBS) twice (one week apart) Male tau protein transgenic mice (TMHT). On the 14th day, the animals were euthanized, the brain was harvested and subjected to immunohistochemistry (IHC) treatment. To determine tauopathy in the hippocampus and tonsils, AT180 (for Tau-pT231) and HT7 (for total human tau) antibodies were used, with 5 sections per brain (one for each) and then Image Pro Plus (for each) V6.2) Software assesses the area of immunoreactivity. The immunoreactivity target was measured by exceeding the size limit (30 μm ^{2 in the} tonsil, 7 μm ^{2 in the} hippocampus) and exceeding the dynamic intensity threshold threshold. The total area and intensity of the target and individual thresholds are automatically filed. If used, the dynamic threshold is defined as the average intensity (AOI) plus factor in the intensity region multiplied by the pixel intensity standard deviation within the AOI. The area size of the hippocampus and amygdala was measured by hand. The AT180 and HT7 IR area data were corrected for the area (in the hippocampus) or the AOI size (in the tonsil).

8.2 results

The AT180 pTau antibody detects endogenous and human pTau (double phosphorylation at Thr231 and Ser235). For the tau protein transgenic mice used in this study, AT180 histological measurements were concentrated in hippocampus and tonsil neurons. Mice treated with ACI-35-2G5-Ab3 showed a significant decrease in AT180 mean and corrected neuronal somatic labeling in the tonsils and hippocampus (Figures 5A and 5B), showing overall neurons in treated mice. Somatic cell AT180- Positive pTau is reduced.

For total human (transgenic gene) τ, HT7 antibody was used. HT7 recognizes normal human τ between residues 159 and 163. Histological measurements focused on immunoreactive somatic cells of hippocampus and tonsil neurons. The HT7 immunoreactive area and the total intensity and average intensity of HT7 immunoreactivity in the tonsillar of mice treated with ACI-35-2G5-Ab3 were reduced (Fig. 6A). In the hippocampus, the average intensity was also observed to decrease (Fig. 6B). However, an increase in the HT7-labeled immunoreactive area and total intensity in the hippocampus of mice treated with 10 mg/kg was observed. This enhancement observed in the hippocampus was mainly due to three of the total eight mice studied.

ACI-35-2G5-Ab3 treatment resulted in a significant reduction in AT180 immunoreactive pTau levels in hippocampus and tonsil neuronal somatic cells in both studied areas. In the tonsil, the total marker intensity of AT180 immunoreactive pTau and HT7 immunoreactive total human tau protein was reduced. The 3 mg/kg dose treatment also significantly reduced the mean HT7 strength in both regions. However, at 10 mg/kg, the average HT7 immunoreactive area and total intensity in the hippocampus increased beyond the control-treated mice, indicating that ACI-35-2G5-Ab3 treatment caused pathological pTau changes.

Example 9: epitope mapping of anti-pTau antibodies 9.1 method

Antigenic epitopes were determined by anti-phosphating tau protein mouse monoclonal antibodies by ELISA using different phosphorylated and unphosphorylated peptide libraries. The amino acid sequence of the peptide library T3 used is shown in Table 11A. Each library is a biotinylated short peptide in the range of phosphorylated and unphosphorylated sequences present in the peptide vaccine composition. Further, as shown in Tables 11B and 11C, a peptide library was produced by substituting each residue in the peptide sequence of the antibody with alanine (Ala). Each library consists of a biotinylated short peptide present within the range of phosphorylated and unphosphorylated sequences present in the peptide vaccine. The peptide library was purchased from ANAWA Trading SA. The peptide library was purchased from ANAWA Trading SA. The epitope localization was performed according to the manufacturer's instructions (Mimotopes). Briefly, streptavidin coated disks (NUNC) were blocked overnight at 4 °C with phosphate buffered saline (PBS) containing 0.1% BSA. After washing with PBS-0.05% Tween-20, each plate was coated with different peptides from each library for 1 hour at room temperature and diluted to a final concentration of 10 in PBS containing 0.1% BSA, 0.1% sodium azide. μM. After washing, each plate was incubated with the tested antibody diluted to 40 ng/ml in PBS containing 2% BSA and 0.1% sodium azide for 1 hour at room temperature. The plates were washed again and incubated with 1/6000 diluted AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, Suffolk, England) for 1 hour at room temperature. After the final wash, each plate was incubated with p-nitrophenyl phosphate disodium hexahydrate (pNPP; Sigma-Aldrich, Buchs, Switzerland) phosphatase substrate, and after 2 hours incubation time, an ELISA reader was used. Read at 405 nm. If the optical density (O.D.) exceeds the background O.D by at least 2 times, the binding is considered positive.

9.2 results

As a result of the epitope-localization experiments, identifiable epitopes include the essential phosphorylated amino acid residues to which the antibodies disclosed herein specifically bind (see Table 5).

‧τ aa 393-401, requires pS396 (ACI-35-2A1-Ab1; ACI-35- 2A1-Ab2)

‧τ aa 396-401, need pS396 (ACI-35-4A6-Ab1)

‧τ aa 394-400, need pS396 (ACI-35-1D2-Ab1)

‧τ aa 402-406, requires pS404 (ACI-35-2G5-Ab1)

‧τ aa 393-400, requires p396 (ACI-35-2G5-Ab2; ACI-35-2G5-Ab3)

Example 10: Phosphorylation of tau in serine 396 (pS396) using GSK3β kinase and SDS-PAGE/Western blot analysis 10.1 method

The longest isoform of human full-length tau (TAU441; SignalChem) with a final concentration of 16 μM (20 μg τ/25 μL of reactant) was combined with 0.018 U GSK 3β/pmol of tau in phosphorylation buffer at 4 The incubation was carried out at ° C, 30 ° C or 37 ° C for 1 hour, 6 hours or 20 hours. The phosphorylation buffer contained HEPES pH 7.64 (40 mM), EGTA (5 mM), MgCl ₂ (3 mM) and ATP (2 mM). One unit of GSK3β is defined by the manufacturer as the amount of enzyme that transfers 1 pmol of phosphate from ATP to CREB phosphorylated peptide (KRREILSRRPpSYR) in 1 minute at 30 °C. Detection of GSK3β phosphorylated tau (pTau-GSK3β) by antibodies against tau amino acids 202, 396, 404, 409, threonine 181, 205 and 231 phosphorylated tau and total tau protein, using direct ELISA Western blotting (WB) analysis to optimize and validate kinase activity and specificity (not shown). In addition, the anti-GSK3α/β antibody (BioSource Invitrogen) was used to detect the ink spots for the presence of GSK3β. For all WB, by adding an equal volume of sample buffer A (125 mM Tris-HCl pH 6.8, 4% [w/v] sodium lauryl sulfate [SDS], 20% glycerol, 0.01% bromophenol blue ( Bromophenol blue), 5% β-mercaptoethanol) was used to dilute pTau-GSK3β, and the sample was heated to 95 ° C for 10 minutes. 30 μg of the sample was loaded onto a 4-12% Bis-Tris gel (Invitrogen) and run in MOPS SDS buffer (Invitrogen). The protein was transferred to a 0.45 μm PVDF membrane in transfer buffer (25 mM Tris (pH 8.6), 190 mM glycine, 20% methanol). To verify protein transfer, membranes were stained with Ponceau S for 5 minutes. The membrane was then washed and blocked in blocking buffer (5% BSA in TBS [50 mM Tris-HCl (pH 7.6), 150 mM NaCl]) for 1 hour. The membrane was incubated overnight at 4 °C with a blocking buffer containing primary antibody and 0.1% Tween. ACI-35-2G5-Ab3 dot ink was performed at a dilution concentration of 0.5 μg/mL antibody.

10.2 Results

Treatment of τ with GSK3β resulted in the presence of hyperphosphorylation in tausine 396 (τ-pS396) as verified using antibodies specific for different tau phosphorylated serine and threonine residues (not shown). Fig. 7 shows the case of SDS-PAGE on τ-pS396 produced using different GSK3β conditions, and a film in which ink was spotted using the ACI-35-2G5-Ab3 antibody. The ACI-35-2G5-Ab3 antibody specific for τ-pS396 showed a good signal for τ-pS396, and a colored band was also observed, indicating its binding to the τ-pS396 dimer (Fig. 7, lanes 11 and 13). In the absence of GSK3β treatment, no colored bands were observed (lanes 6-8 and 14-15).

Example 11: Detection of tau phosphorylation in human cerebrospinal fluid (CSF) samples (pSer396) 11.1 method 11.1.1 Human sample - post-mortem brain sample

After the death of an Alzheimer's disease (AD) donor, AD19, the sacral cortex was obtained from the Brain Donation Library of the University of Miami. The inventor provided a sample of the Brain Donation Library of the University of Miami for this study. Demographic information about the donors is shown in Table 12 below, which also indicates the Braak disease stage (Braak and Braak (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82: 239-259).

11.1.2 Using the post-mortem brain to prepare tissue homogenate to separate part S1

The post-mortem cortex of the AD19 donor was homogenized according to the following protocol. Brain sections were weighed and homogenized in 9 volumes of 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA containing phosphatase inhibitor (30 mM NaF, 0.2 mM Na ₃ VO ₄ , 1 nM Okada Well, 1 mM PMSF, 5 mM Na ₄ P ₂ O ₇ ) and protease inhibitor (completely small; Roche 04 693 124 001). Homogenization on ice using glassware. This constitutes the total tissue homogenate fraction (TH). A half volume of the TH fraction was aliquoted and stored at -80 °C. The remaining TH fraction was further processed by the addition of Triton X-100 until a final concentration of 0.4%. The samples were thoroughly mixed and vortexed several times, followed by centrifugation at 5,000 rpm for 5 minutes at 4 °C. The supernatant constitutes the S1 separation portion. Samples were aliquoted and stored at -80 °C. Protein concentration was measured using Bradford reagent (Sigma B6916-500).

11.1.3 Human CSF samples

Cerebrospinal fluid (CSF) samples of clinically confirmed mild to moderate Alzheimer's disease (AD) patients and healthy voluntary control donors (Ctrl) were provided by Charité School of Medicine Berlin. The inventor provided samples for Charité School of Medicine Berlin for this study. Aliquots of the samples were stored at -80 ° C and used without further processing. Demographic and clinical information on CSF sample donors is shown in Table 13 below.

11.1.4 Immunoenhancement of CSF tau protein 11.1.4.1 Antibody coupling

For immunoprecipitation of CSF tau protein, a commercial human tau protein antibody (pure HT7, Thermo Scientific MN 1000) was used. To couple HT7 to the protein G Dynabeads (Life Technologies 10004D), 1.5 mg (50 μL) of protein G Dynabeads was resuspended by vortexing and transferred to a 1.7 mL maximum recovery tube (Axygen MCT-175-LC) for each sample. in. The tube was placed on a magnetic support (DynaMag, Life Technologies 123.21D) to concentrate the beads on the tube side and remove the buffer. 1 μg of HT7 in 200 μL PBS was combined with protein G Dynabeads for 10 minutes using a Hula mixer (Life Technologies) at 10/20 rpm, 25°/10 tilt, 5°/2 vibration, and then the tube was placed on the magnet. The buffer was removed and the tube was washed twice by pipetting 200 μL PBS/0.02% Tween 20 once and 200 μL of binding buffer (20 mM sodium phosphate, 150 mM NaCl, freshly prepared) twice. Always use a magnet to remove the wash buffer. To make HT7 was cross-linked with protein G Dynabeads, and HT7-beads were resuspended in 250 μL of 5 mM BS3 solution (Sigma-Aldrich S5799) dissolved in binding buffer and spun at room temperature (RT) (configured above) The same) incubation was carried out for 30 minutes, and the reaction was terminated by adding 12.5 μL of quenching buffer (1 M Tris-HCl pH 7.5) for 15 minutes, followed by washing three times with 200 μL of PBS/0.02% Tween 20.

11.1.4.2 Immunoenhancement of CSF tau protein

CSF was used without dilution and 1 mL of CSF from each donor was transferred to a tube containing HT7 cross-linked with beads and incubated for 1 hour at 4 °C under continuous rotation (10 rpm). After removing the unbound material on the magnet, the beads were washed with 200 μL PBS/0.02% Tween 20 and the τ was dissolved in 20 μL of PBS containing 1% sodium dodecyl sulfate (SDS) at 70 ° C. minute. To avoid bead settling, the tubes were briefly mixed (300 rpm in a heated horizontal mixer for 5 seconds per minute). After this incubation, the dissolved sample was collected by placing the tube on a magnet.

As a positive control, the τ in the human brain tissue homogenate was also enriched. Serial dilutions of human brain S1 isolated from PBS in preparation of AD19 donor (0.5 μg/mL, 0.17 μg/mL, 0.056 μg/mL, 0.019 μg/mL, 0.006 μg/mL, 0.002 μg/mL, 0.0007 μg) /mL). Each sample (1 mL) was then treated as described above and dissolved in 25 μL of 1% SDS.

11.1.5 AlphaLISA. 11.1.5.1 AlphaLISA analysis description

AlphaLISA is a homogeneous analysis using bead-based Alpha technology. AlphaLISA is selected as a technology platform based on the least sensitive and few steps use. In short, the analysis is based on bead proximity. At 680 nm excitation, the donor beads containing the photosensitizer convert the surrounding oxygen into a singlet oxygen species that diffuse (up to 200 nm, within a half-life of 4 microseconds) and produce chemistry in the acceptor beads. Luminescence reaction, causing luminescence.

The analytical configuration used in the experiments of the present invention is as follows (see also Figure 8):

‧Pan-Tau antibody Tau-13 (Abcam ab24636) coupled with Alpha acceptor beads binds to human tau present in the sample and forms a "tau protein-Tau-13 antibody-receptor bead" complex

Detecting antibody ACI-35-2G5-Ab3-BT binds to human tau protein pS396 and binds streptavidin (SAV) coated Alpha donor beads to the complex.

After all reagents were reacted, the chemiluminescent signal was read using an EnSpire Alpha 2390 reader.

11.1.5.2 Biotin labeling of ACI-35-2G5-Ab3 antibody

Antibody ACI-35-2G5-Ab3 was biotinylated using the EZ-Link Micro Sulfo-NHS-LC-Biotin Labeling Kit (Thermo Scientific 21935) according to the manufacturer's instructions for use in AlphaLISA assays. Biotin was used in the labeling reaction in an excess of 25 times the molar concentration of the antibody. After biotin labeling, excess free biotin was removed by washing the antibody four times with PBS using a 50,000 MWCO Spin-X UF 500 concentrator (Corning 431480). The biotinylated ACI-35-2G5-Ab3 antibody was named ACI-35-2G5-Ab3-BT.

11.1.5.3 Coupling the Tau-13 antibody to the AlphaLISA acceptor beads.

Antibody Tau-13 was bound to activated Alpha receptor beads (Perkin Elmer 6772001) for use in AlphaLISA assays. The following combination protocol was used: 0.1 mg of Tau-13 antibody solution (purified by Protein A column) was mixed with 1 mg of AlphaLISA acceptor beads and supplemented with 0.13 M phosphate buffer (pH 8.0) until the final reaction volume was 200. μL. Next, 1.25 μL of 10% Tween-20 and 10 μL of 25 mg/mL NaBH ₃ CN solution were added and the tubes were incubated at 37 ° C for 48 hours under gentle rotation (7 rpm). After the binding reaction, the active site on the beads was blocked by adding 10 μL of a carboxymethoxyamine solution and further incubated at 37 ° C for 1 hour. Finally, the beads were washed twice with 200 μL of 0.1 M Tris-HCl pH 8.0 and in 200 μL of storage buffer (PBS containing 0.05% Proclin-300) (making the final concentration of AlphaLISA receptor beads 5 mg/mL) ), stored at 4 ° C.

11.1.5.4 Limitation of detection using brain pS396-Tau

In this experiment, an immunoenhanced tau brain sample, an S1 brain separation fraction sample, and a blank buffer were used. Each sample was analyzed in a final volume of 50 μL using a 384-well white OptiPlate (PerkinElmer 6007291). All reagents were diluted using Alpha Assay Buffer (PerkinElmer AL000C).

‧5 μL sample (1/10 final volume, so the final protein concentration in the analysis corresponds to a sample concentration of 1/10)

‧ Add 10 μL of 0.5% SDS (for S1 brain separation of some samples) or 10 μL of normal buffer (for immunoenhanced τ brain samples)

‧ Mix 15 μL of ACI-35-2G5-Ab3-BT antibody (final concentration: 5 nM) with Tau13-receptor bead conjugate (final bead concentration: 2.5 μg/mL)

‧ Incubate for 1 hour at room temperature

‧20 μL of streptavidin donor beads (final bead concentration: 25 Gg/mL)

‧ Incubate for 30 minutes at room temperature (protected from light)

‧Use EnSpire Alpha instrument readings and analyze with EnSpire Workstation version 3.00

11.1.5.5 Determination of immunoconcentration in CSF pS396-Tau

Each sample was analyzed in a final volume of 50 μL using a 384-well white OptiPlate (PerkinElmer 6007291). All reagents were diluted using Alpha Assay Buffer (PerkinElmer AL000C).

‧5 μL of immunoprecipitate solution from each donor

‧ Mix 20 μL of ACI-35-2G5-Ab3-BT antibody (final concentration: 5 nM) with Tau13-receptor bead conjugate (final bead concentration: 2.5 μg/mL)

‧ Incubate for 1 hour at room temperature

‧25 μL of streptavidin donor beads (final bead concentration: 25 μg/mL)

‧ Incubate for 30 minutes at room temperature (protected from light)

‧Use EnSpire Alpha instrument readings and analyze with EnSpire Workstation version 3.00

11.1.6 Statistical analysis

Statistical analysis of the data was performed using the GraphPad Prism software.

11.2 Results

Preliminary experiments have shown that the amount of pS396 present in human CSF is too low for detection. For this reason, a protocol for immunoconcentration combined with high-sensitivity immunodetection has been developed. First, use the brain tissue of human AD to verify the immunoenhancement program. Parallel ratio of untreated brain tissue homogenate sample to τ immunoconcentration sample It is revealed that at the corresponding concentration, the detection limit of the Tau13/ACI-35-2G5-Ab3 AlphaLISA analysis is reached at 0.5 μg/mL for the untreated sample, and between 0.002 and 0.006 for the immunoprecipitated sample. Gg/mL reached the detection limit of the Tau13/ACI-35-2G5-Ab3 AlphaLISA assay, indicating a 100-fold increase (Figure 9).

Next, an immunoenhancement regimen was applied to live donor CSF samples (n=17 for mild to moderate AD patients and n=16 for age-matched healthy volunteers). The data obtained (Figure 10) showed: a) following the immunoenhancement protocol, Tau13/ACI-35-2G5-Ab3 AlphaLISA detected pS396-Tau in all human CSF samples; and b) more importantly, with the control group (p=0.0003, Mann-Whitney test) A significant increase in the amount of pS396-Tau in AD CSF was observed.

In summary, an immunoenhancement/immunoassay protocol was developed to detect pS396-Tau in human CSF. An increase in pS396-Tau in CSF of mild to moderate AD indicates that this method can be successfully used in clinical biomarker studies to assess disease progression, patient grading, and therapeutic efficacy. The ACI-35-2G5-Ab3 antibody detected pS396-Tau in all human CSF samples and, more importantly, the antibody was able to discriminate AD CSF (compared to the control group).

*Based on the longest isoform of human τ (Tau441). p indicates a phosphorylated residue.

In the same assay (ELISA, or TAUPIR, or WB), only the binding strength within the same column can be compared.

- poor or lack of binding; + good combination; ++ combination is very good; +++ combination is great (better than good combination)

^a was analyzed by HPLC using 64% phosphorylated peptide purity.

^b Analysis was performed by HPLC using 87% phosphorylated peptide purity.

*Based on the longest isoform of human τ (Tau441)

Degenerate codon: R=A or G S=C or G D=A or G or T B=C or G or T

Y=C or T M=A or CH=A or C or T

K=G or T W=A or T V=A or G or C

Hosting:

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Figure 1 shows the detection of human brain homogenate in control individuals and AD individuals by ACI-35-2A1-Ab1 (left panel), ACI-35-2G5-Ab3 (middle panel) and control antibody (HT7; right panel) The result of phosphorylated tau protein multimers.

Figure 2 (2A and 2B) shows the results of detection of total tau protein and p-Tau in human brain tissue homogenate by commercial antibodies.

Figure 3 (3A, 3B, 3C) shows the detection of human brain tissue by ACI-35-2A1-Ab1 (A), ACI-35-1D2-Ab1 (B) and ACI-35-2G5-Ab3 (C) The case of phosphorylated tau protein in the slurry.

Figure 4 (4A, 4B, 4C) shows the detection of humans by ACI-35-2A1-Ab1 (A), ACI-35-1D2-Ab1 (B) and ACI-35-2G5-Ab3 (C) antibodies using AlphaLISA AD and control (Ctrl) results of τ-pS396 in the brain. According to the Mann-Whitney test, **** p < 0.0001, ** p < 0.01.

Figure 5 (5A and 5B) shows the results of τ-pT231 (AT180) IHC staining in tonsil (A) and hippocampus (B) after treatment with ACI-35-2G5-Ab3 in tau protein transgenic mice.

Figure 6 (6A and 6B) shows the results of total tau protein (HT7) IHC staining in tonsil (A) and hippocampus (B) after treatment with ACI-35-2G5-Ab3 in tau protein transgenic mice.

Figure 7 shows the SDS-PAGE of τ-pS396 produced using different GSK3β conditions, and the film stained with ACI-35-2G5-Ab3 antibody.

Figure 8 shows a specific AlphaLISA assay configuration using ACI-35-2G5-Ab3-BT and a tau-13 antibody.

Figure 9 shows the detection of τ-pS396 in the human S1 brain segregation portion of an AD donor; comparison of signals obtained from IP-enriched samples and non-IP samples.

Figure 10 shows the results of detecting τ-pS396 in human AD and control (Ctrl) CSF by ACI-35-2G5-Ab3 antibody using IP followed by AlphaLISA. According to the Mann-Whitney test, *** p=0.0003.

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<210> 17

<211> 11

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..11

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 17

<210> 18

<211> 10

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..10

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 18

<210> 19

<211> 19

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..19

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 19

<210> 20

<211> 8

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..8

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 20

<210> 21

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 21

<210> 22

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 22

<210> 23

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 23

<210> 24

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> SOURCE

<222> 1..16

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 24

<210> 25

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 25

<210> 26

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 26

<210> 27

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 27

<210> 28

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 28

<210> 29

<211> 11

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..11

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 29

<210> 30

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 30

<210> 31

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 31

<210> 32

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 32

<210> 33

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 33

<210> 34

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 34

<210> 35

<211> 345

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..345

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 35

<210> 36

<211> 345

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..345

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 36

<210> 37

<211> 345

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..345

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 37

<210> 38

<211> 360

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..360

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 38

<210> 39

<211> 357

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..357

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 39

<210> 40

<211> 339

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..339

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 40

<210> 41

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 41

<210> 42

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 42

<210> 43

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 43

<210> 44

<211> 321

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..321

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 44

<210> 45

<211> 339

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..339

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 45

<210> 46

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 46

<210> 47

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<220>

<221> variation

<222> 30..30

<223> / Replacement = "n=i"

<400> 47

<210> 48

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..32

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 48

<210> 49

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 49

<210> 50

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..39

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 50

<210> 51

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..30

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 51

<210> 52

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 52

<210> 53

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..33

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 53

<210> 54

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..39

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 54

<210> 55

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..33

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 55

<210> 56

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..39

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<220>

<221> misc_feature

<222> 27..27

<223> / Replacement = "n=i"

<400> 56

<210> 57

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..37

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 57

<210> 58

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..30

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-37" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 18..18

<223> Phosphorylated serine

<220>

<221> VARIANT

<222> 26..26

<223> Phosphorylated serine

<400> 58

<210> 59

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-33" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 14..14

<223> Phosphorylated serine

<400> 59

<210> 60

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-39" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 7..7

<223> Phosphorylated sulphate

<220>

<221> VARIANT

<222> 9..9

<223> Phosphorylated serine

<400> 60

<210> 61

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-39" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 7..7

<223> Phosphorylated serine

<220>

<221> VARIANT

<222> 10..10

<223> Phosphorylated sulphate

<400> 61

<210> 62

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-35" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 4..4

<223> Phosphorylated serine

<220>

<221> VARIANT

<222> 12..12

<223> Phosphorylated serine

<400> 62

<210> 63

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..18

<223> / Molecular Type = "Protein" / Comment = "Description of manual sequence: ACI-36" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 4..4

<223> Phosphorylated serine

<220>

<221> VARIANT

<222> 9..9

<223> Phosphorylated serine

<400> 63

<210> 64

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-34" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 3..3

<223> Phosphorylated serine

<220>

<221> VARIANT

<222> 6..6

<223> Phosphorylated sulphate

<220>

<221> VARIANT

<222> 13..13

<223> Phosphorylated sulphate

<220>

<221> VARIANT

<222> 15..15

<223> Phosphorylated serine

<400> 64

<210> 65

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..12

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-42" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 3..3

<223> Phosphorylated serine

<400> 65

<210> 66

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<222> 1..10

<223> / Molecular Type = "Protein" / Comment = "Description of Artificial Sequence: ACI-43" /organism="Artificial sequence"

<220>

<221> VARIANT

<222> 6..6

<223> Phosphorylated serine

<400> 66

<210> 67

<211> 441

<212> PRT

<213> Homo sapiens

<220>

<221> Source

<222> 1..441

<223> / Molecular Type = "Protein" /Biology = "Homo sapiens"

<400> 67

<210> 68

<211> 117

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..117

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 68

<210> 69

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..112

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 69

<210> 70

<211> 10

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..10

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 70

<210> 71

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 71

<210> 72

<211> 8

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..8

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 72

<210> 73

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 73

<210> 74

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 74

<210> 75

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 75

<210> 76

<211> 119

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..119

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 76

<210> 77

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..112

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 77

<210> 78

<211> 12

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..12

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 78

<210> 79

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 79

<210> 80

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 80

<210> 81

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 81

<210> 82

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 82

<210> 83

<211> 8

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..8

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 83

<210> 84

<211> 351

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..351

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 84

<210> 85

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 85

<210> 86

<211> 357

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..357

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 86

<210> 87

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 87

<210> 88

<211> 115

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..115

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 88

<210> 89

<211> 10

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..10

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 89

<210> 90

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 90

<210> 91

<211> 6

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..6

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 91

<210> 92

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> SITE

<222> 3..3

<223> Xaa is Met or Val

<220>

<221> Source

<222> 1..112

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 92

<210> 93

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 93

<210> 94

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 94

<210> 95

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 95

<210> 96

<211> 115

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..115

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 96

<210> 97

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..112

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 97

<210> 98

<211> 10

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..10

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 98

<210> 99

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 99

<210> 100

<211> 6

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..6

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 100

<210> 101

<211> 16

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..16

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 101

<210> 102

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 102

<210> 103

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 103

<210> 104

<211> 115

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..115

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 104

<210> 105

<211> 107

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..107

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 105

<210> 106

<211> 11

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..11

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 106

<210> 107

<211> 7

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..7

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 107

<210> 108

<211> 9

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..9

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 108

<210> 109

<211> 345

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..345

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 109

<210> 110

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 110

<210> 111

<211> 345

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..345

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 111

<210> 112

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 112

<210> 113

<211> 345

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..345

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 113

<210> 114

<211> 321

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..321

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 114

<210> 115

<211> 17

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..17

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 115

<210> 116

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..112

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 116

<210> 117

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 117

<210> 118

<211> 112

<212> PRT

<213> Mus musculus

<220>

<221> Source

<222> 1..112

<223> / Molecular Type = "Protein" /Biology = "Mature Mouse"

<400> 118

<210> 119

<211> 336

<212> DNA

<213> Mus musculus

<220>

<221> Source

<222> 1..336

<223> / Molecular type = "DNA" /Biology = "Mature Mouse"

<400> 119

<210> 120

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 120

<210> 121

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 121

<210> 122

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 122

<210> 123

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 123

<210> 124

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 124

<210> 125

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 125

<210> 126

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 126

<210> 127

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 127

<210> 128

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 128

<210> 129

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 129

<210> 130

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 130

<210> 131

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 131

<210> 132

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 132

<210> 133

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 133

<210> 134

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 134

<210> 135

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 135

<210> 136

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 136

<210> 137

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 137

<210> 138

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 138

<210> 139

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 139

<210> 140

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 140

<210> 141

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..33

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 141

<210> 142

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 142

<210> 143

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 143

<210> 144

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 144

<210> 145

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 145

<210> 146

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 146

<210> 147

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 147

<210> 148

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 148

<210> 149

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 149

<210> 150

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 150

<210> 151

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 151

<210> 152

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 152

<210> 153

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 153

<210> 154

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 154

<210> 155

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 155

<210> 156

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 156

<210> 157

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 157

<210> 158

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..33

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 158

<210> 159

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 159

<210> 160

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 160

<210> 161

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 161

<210> 162

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 162

<210> 163

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 163

<210> 164

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..33

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 164

<210> 165

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 165

<210> 166

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 166

<210> 167

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..33

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 167

<210> 168

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 168

<210> 169

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 169

<210> 170

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..40

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 170

<210> 171

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..38

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 171

<210> 172

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 172

<210> 173

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 173

<210> 174

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 174

<210> 175

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 175

<210> 176

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 176

<210> 177

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 177

<210> 178

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 178

<210> 179

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 179

<210> 180

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 180

<210> 181

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 181

<210> 182

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 182

<210> 183

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..33

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 183

<210> 184

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 184

<210> 185

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..31

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 185

<210> 186

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 186

<210> 187

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 187

<210> 188

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..32

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 188

<210> 189

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..32

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 189

<210> 190

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 190

<210> 191

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 191

<210> 192

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..41

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 192

<210> 193

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 193

<210> 194

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 194

<210> 195

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 195

<210> 196

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "- Description of artificial sequence: primer" /organism="Artificial sequence"

<400> 196

<210> 197

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 197

<210> 198

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 198

<210> 199

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..35

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 199

<210> 200

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..34

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 200

<210> 201

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 201

<210> 202

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..36

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 202

<210> 203

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..39

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 203

<210> 204

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..39

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 204

<210> 205

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..26

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 205

<210> 206

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..23

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 206

<210> 207

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..25

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<220>

<221> variation

<222> 15

<223> / Replacement = "n=i"

<400> 207

<210> 208

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..26

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<220>

<221> variation

<222> 21

<223> / Replacement = "n=i"

<400> 208

<210> 209

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..25

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 209

<210> 210

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..29

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 210

<210> 211

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..29

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 211

<210> 212

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..29

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 212

<210> 213

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..27

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 213

<210> 214

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..27

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 214

<210> 215

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..21

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 215

<210> 216

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..26

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 216

<210> 217

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..26

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 217

<210> 218

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..26

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 218

<210> 219

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..24

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 219

<210> 220

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..32

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<220>

<221> variation

<222> 27

<223> / Replacement = "n=i"

<400> 220

<210> 221

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<222> 1..31

<223> / Molecular type = "DNA" /Comment = "Description of Artificial Sequence: Intro" /organism="Artificial sequence"

<400> 221

Claims (91)

An antibody or a functional fragment thereof that recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or antibody fragment has high binding affinity for soluble, oligomeric, and insoluble phosphorylated tau protein And can detect and / or regulate the content of soluble, oligomeric and insoluble phosphorylated tau protein in vivo. An antibody or a functional fragment thereof, wherein the antibody or antibody fragment binds to or within the amino acid sequence VYKSPVVSGDTSPRHL (SEQ ID NO: 62) (τ aa 393-408 of SEQ ID NO: 67) Phosphorylation of an epitope comprising a phosphorylated Ser at position 396 (pS396) and position 404 (pS404). The antibody or functional fragment thereof of claim 2, wherein the antibody or antibody fragment is a regulatory antibody that modulates the soluble and/or insoluble tau protein content in the brain of the mammal. The antibody of claim 3, or a functional fragment thereof, wherein the antibody or antibody fragment modulates a soluble and/or insoluble tau protein content in the cerebral cortex and/or hippocampus. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment reduces the total soluble and/or insoluble tau protein content. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment reduces the amount of soluble and/or insoluble phosphorylated tau protein. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment reduces the paired helical filaments containing hyperphosphorylated tau protein The content. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment reduces the content of total soluble tau protein, soluble phosphorylated tau protein and pTau paired helical filaments. The antibody or functional fragment thereof according to any of the preceding claims, wherein the mammal is a human. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 10 nM. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 82 nM, 5 nM, at least 2 nM, at least 1 nM, at least 500 pM, at least 300 pM, at least 200 pM, at least 100 pM. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has an association rate constant of 10 ⁴ M ^-1 s ^-1 or greater than 10 ⁴ M ^-1 s ^-1 , 10 ⁵ M ^{- 1} s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 , 10 ⁶ M ^-1 s ^-1 or greater than 10 ⁶ M ^-1 s ^-1 . The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 4 nM and an association rate constant of 10 ⁵ M ^-1 s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 high binding affinity. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 3 nM and an association rate constant of 10 ⁶ M ^-1 s ^-1 or greater than 10 ⁶ M ^-1 s ^-1 high binding affinity. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 2 nM and an association rate constant of 10 ⁴ M ^-1 s ^-1 or greater than 10 ⁴ M ^-1 s ^-1 high binding affinity. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 1 nM and an association rate constant of 10 ⁵ M ^-1 s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 high binding affinity. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 200 pM and an association rate constant of 10 ⁵ M ^-1 s ^-1 or greater than 10 ⁵ M ^-1 s ^-1 high binding affinity. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment has a dissociation constant of at least 100 pM and an association rate constant of 10 ⁶ M ^-1 s ^-1 or greater than 10 ⁶ M ^-1 s ^-1 high binding affinity. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or antibody fragment binds to an epitope selected from the group consisting of: a. VYKSPVVSG (τ aa 393-401 of SEQ ID NO: 67) , which comprises a phosphorylated Ser (pS396) at position 396; b. SPVVSG (τ aa 396-401 of SEQ ID NO: 67), which comprises a phosphorylated Ser (pS396) at position 396; c. YKSPVVS (SEQ ID NO: 67 τ aa 394-400), which comprises a phosphorylated Ser (pS396) at position 396; d. DTSPR (τ aa 402-406 of SEQ ID NO: 67), which comprises a phosphorylated Ser (pS404) at position 404; And e. VYKSPVVS (τ aa 393-400 of SEQ ID NO: 67), which comprises a phosphorylated Ser (pS396) at position 396. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody The fragment or a fragment thereof binds to an epitope on a mammalian tau protein (specifically, a human tau protein, but particularly as shown in SEQ ID NO: 67), which comprises a phosphorylated Ser at position 396 ( τ aa 393-401 (VYKSPVVSG) of pS396). The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof binds to a mammalian tau protein (specifically, a human tau protein, but in particular a human tau protein as set forth in SEQ ID NO: 67 An antigenic determinant comprising τ aa 396-401 (SPVVSG) comprising a phosphorylated Ser (pS396) at position 396. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof binds to a mammalian tau protein (specifically, a human tau protein, but in particular a human tau protein as set forth in SEQ ID NO: 67 An antigenic determinant comprising τ aa 394-400 (YKSPVVS) comprising a phosphorylated Ser (pS396) at position 396. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof binds to a mammalian tau protein (specifically, a human tau protein, but in particular a human tau protein as set forth in SEQ ID NO: 67 An epitope above which comprises a τ aa 402-406 (DTSPR) comprising a phosphorylated Ser (pS404) at position 404. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof binds to a mammalian tau protein (specifically, a human tau protein, but in particular a human tau protein as set forth in SEQ ID NO: 67 An antigenic determinant comprising a τ aa 393-400 (VYKSPVVS) comprising a phosphorylated Ser (pS396) at position 396. The antibody or a functional fragment thereof according to any one of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises the first a binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and comprising SEQ ID NO: 75 CDR3 of the amino acid sequence, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% identical amino acid sequence; and/or a second binding domain comprising a SEQ ID comprising The CDR1 of the amino acid sequence represented by NO: 70 or an amino acid sequence thereof which is at least 95%, especially 98%, especially 99% identical thereto, comprising or at least the CDR2 of the amino acid sequence shown by SEQ ID NO: 71 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and comprising the amine group of SEQ ID NO: 72 CDR3 of the sequence or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, especially at least 96%, especially at least 97%, especially at least 98%, especially at least 99% or 100% identical amino acid sequence. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: a a first binding domain comprising an amine comprising SEQ ID NO: 73 CDR1 of the acid sequence, CDR2 comprising the amino acid sequence of SEQ ID NO: 74 and CDR3 comprising the amino acid sequence of SEQ ID NO: 75; and/or b. comprising the amino acid sequence a second binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 70, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 71, and an amino group comprising SEQ ID NO: 72 CDR3 of the acid sequence. The antibody or a functional fragment thereof according to any one of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises the first a first binding domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 81, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 82, and having the SEQ ID NO: 83 CDR3 of the amino acid sequence, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95 %, especially at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% identical amino acid sequence; and/or a second binding domain comprising SEQ ID NO: CDR1 of the amino acid sequence shown at 78, CDR2 having the amino acid sequence of SEQ ID NO: 79, and CDR3 having the amino acid sequence of SEQ ID NO: 80, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, In particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% identical amino acid sequence. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: a a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and comprising the SEQ ID NO: 83 a CDR3 comprising an amino acid sequence; and/or b. a second binding domain comprising an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, comprising SEQ ID NO: 79 CDR2 of the amino acid sequence and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:80. The antibody or a functional fragment thereof according to any one of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises the first a first binding domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 93, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 94, and having the SEQ ID NO: 95 CDR3 of the amino acid sequence, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94% with any of the above CDRs a semi-amino acid sequence, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100%; and/or a second binding domain, the second binding domain comprising CDR1 of the amino acid sequence shown by SEQ ID NO: 89 has SEQ ID CDR2 of the amino acid sequence represented by NO: 90 and CDR3 having the amino acid sequence of SEQ ID NO: 91, or at least 60%, at least 70%, at least 80%, especially at least 85 with any of the above CDRs %, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% Consistent amino acid sequence. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: a a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94, and comprising the SEQ ID NO: 95 a CDR3 comprising an amino acid sequence; and/or b. a second binding domain comprising an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, comprising SEQ ID NO: 90 CDR2 of the amino acid sequence and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91. The antibody or a functional fragment thereof according to any one of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises the first a first binding domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 101, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 102, and having the SEQ ID NO: 103 CDR3 of the amino acid sequence, or at least 60%, at least 70%, at least with any of the above CDRs 80%, in particular at least 85%, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular At least 99% or 100% identical amino acid sequence; and/or a second binding domain comprising, in sequence, CDR1 having the amino acid sequence set forth in SEQ ID NO: 98, having SEQ ID NO: CDR2 of the amino acid sequence shown at 99 and CDR3 having the amino acid sequence of SEQ ID NO: 100, or at least 60%, at least 70%, at least 80%, especially at least 85%, with any of the above CDRs, In particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% identical Amino acid sequence. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: a a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 101, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 102, and comprising the SEQ ID NO: 103 a CDR3 comprising an amino acid sequence; and/or b. a second binding domain comprising an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98, comprising SEQ ID NO: 99 CDR2 of the amino acid sequence and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:100. An antibody, or a functional fragment thereof, according to any of the preceding claims, a fragment thereof that recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises a first binding domain comprising the gene set forth in SEQ ID NO:106 CDR1 of the amino acid sequence, CDR2 having the amino acid sequence of SEQ ID NO: 107, and CDR3 having the amino acid sequence of SEQ ID NO: 108, or at least 60%, at least 60% of any of the above CDRs 70%, at least 80%, in particular at least 85%, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, especially at least 99% or 100% identical amino acid sequence; and/or a second binding domain comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 89 with SEQ ID CDR2 of the amino acid sequence represented by NO: 115 and CDR3 having the amino acid sequence of SEQ ID NO: 91, or at least 60%, at least 70%, at least 80%, especially at least 85 with any of the above CDRs %, in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, in particular at least 95%, in particular at least 96%, in particular Less 97%, especially at least 98%, especially at least 99% or 100% identical to the amino acid sequence. The antibody or a functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or fragment thereof comprises: a a first binding domain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 106, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 107, and comprising the SEQ ID NO: 108 Amino acid sequence CDR3; and/or b. a second binding domain comprising an amino acid sequence comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89 comprising the amino acid of SEQ ID NO: 115 CDR2 of the sequence and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91. The antibody or functional fragment thereof according to any of the preceding claims, comprising a first binding domain comprising a plurality selected from the group consisting of SEQ ID NO: 69, 77, 116, 92, 118, 97, 105 Amino acid sequence, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, especially at least 95%, Especially at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% identical amino acid sequence; and/or a second binding domain comprising SEQ ID NO: 68, 76 , an amino acid sequence of 88, 96, 104, or at least 60%, at least 70%, at least 80%, especially at least 85%, especially at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% or 100% identical amino acid sequence. An antibody, or a functional fragment thereof, according to any of the preceding claims, which recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or functional fragment thereof comprises a first binding domain, The first binding domain comprises an amino acid sequence set forth in SEQ ID NO: 69 or an amino acid sequence at least 98% or 99% identical thereto; and/or a second binding domain comprising SEQ ID NO The amino acid sequence shown at 68 or At least 90%, 91%, 92% or 93% identical amino acid sequence. An antibody, or a functional fragment thereof, according to any of the preceding claims, which recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or functional fragment thereof comprises a first binding domain, The first binding domain comprises an amino acid sequence set forth in SEQ ID NO: 77 or an amino acid sequence at least 93%, 94% or 95% identical thereto; and/or a second binding domain comprising The amino acid sequence set forth in SEQ ID NO: 76 or an amino acid sequence which is at least 88%, 89% or 90% identical thereto. An antibody, or a functional fragment thereof, according to any of the preceding claims, which recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or functional fragment thereof comprises a first binding domain, The first binding domain comprises an amino acid sequence of SEQ ID NO: 116, 92 or 118 or an amino acid sequence at least 93%, 94% or 95% identical thereto; and/or a second binding domain, the The di-binding domain comprises an amino acid sequence of SEQ ID NO: 88 or an amino acid sequence which is at least 90%, 91%, 92% or 93% identical thereto. An antibody, or a functional fragment thereof, according to any of the preceding claims, which recognizes and specifically binds to a phosphorylation epitope on a mammalian tau protein or a fragment thereof, wherein the antibody or functional fragment thereof comprises a first binding domain, The first binding domain comprises an amino acid sequence of SEQ ID NO: 97 or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising SEQ ID NO: 96 The amino acid sequence shown or an amino acid sequence which is at least 86%, 87%, 88% or 90% identical thereto. An antibody, or a functional fragment thereof, according to any of the preceding claims, which A phosphorylation epitope that specifically binds to a mammalian tau protein or a fragment thereof, wherein the antibody or functional fragment thereof comprises a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 105 Or an amino acid sequence that is at least 98% or 99% identical thereto; and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 104 or at least 88%, 89% or 90% identical amino acid sequence. The antibody or functional fragment thereof according to any of the preceding claims, comprising: a. a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 69 and/or comprising the SEQ ID NO: 68 a second binding domain of the amino acid sequence; or b. a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 77 and/or a second comprising the amino acid sequence set forth in SEQ ID NO: 76 a binding domain; or c. a first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 116 and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 88; or d. a first binding domain of the amino acid sequence set forth in SEQ ID NO: 92 and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 88; or e. comprising SEQ ID NO: 97 a first binding domain of the amino acid sequence and/or a second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 96; or f. comprising the amino acid sequence of SEQ ID NO: 105 a binding domain and/or a second junction comprising the amino acid sequence set forth in SEQ ID NO: 104 The first binding domain comprising the amino acid sequence set forth in SEQ ID NO: 118 and/or the second binding domain comprising the amino acid sequence set forth in SEQ ID NO: 88. The antibody or functional fragment thereof according to any one of the preceding claims, which is a multi-strain antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody, or a functional fragment thereof. The antibody or functional fragment thereof according to any of the preceding claims, which has an IgG2b, IgG2a or IgG3 isotype. The antibody or functional fragment thereof according to any of the preceding claims, wherein the mammalian tau protein is a human tau protein. The antibody or functional fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof binds to a pathological protein τ conformer, but does not bind to a corresponding unphosphorylated epitope and/or an unrelated epitope . A polynucleotide encoding an antibody or a functional fragment thereof according to any of the preceding claims. The polynucleotide of claim 46, which comprises a nucleic acid molecule selected from the group consisting of: a. a nucleotide sequence comprising the same as SEQ ID NO: 35-45, SEQ ID NO: 84-87, SEQ ID NO a nucleic acid molecule having a sequence of at least 85% sequence identity as shown in 109-112 and 117-119; b. a nucleotide sequence comprising the same as SEQ ID NO: 35-45, SEQ ID NO: 84-87, SEQ ID NO: 109-112 and 117 a nucleic acid molecule having a sequence identity of at least 90%; c. A nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the sequences set forth in SEQ ID NO: 35-45, SEQ ID NO: 84-87, SEQ ID NO: 109-112, and 117-119 ; d. a nucleic acid comprising a nucleotide sequence having at least 98% sequence identity to the sequences set forth in SEQ ID NO: 35-45, SEQ ID NO: 84-87, SEQ ID NO: 109-112, and 117-119; a nucleic acid having a nucleotide sequence of 100% sequence identity to the sequence of SEQ ID NO: 35-45, SEQ ID NO: 84-87, SEQ ID NO: 109-112, and 117-119 a nucleic acid molecule in which the complementary strand of the nucleotide sequence contained is hybridized with the nucleic acid molecule of any of a) to d); or g. the nucleotide sequence contained in any one of a) to e) The defined nucleotide sequence differs in a nucleic acid molecule degenerate in the genetic code: wherein the antibody or functional fragment thereof encoded by the nucleic acid molecule as defined in any of a) to g) recognizes and specifically binds to A mammalian phosphorylation epitope, in particular, a phosphorylation epitope that binds to a human tau protein or a fragment thereof, specifically, to a pathological protein τ conformer, but not A corresponding unphosphorylated epitope and/or an unrelated epitope, wherein the antibody or antibody fragment has high binding affinity for soluble, oligomeric, and insoluble phosphorylated tau protein and is capable of detecting and/or modulating in vivo solubility, The content of oligomeric and insoluble phosphorylated tau protein. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or a functional fragment thereof according to any one of the preceding claims, or a combination thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 48, comprising at least two antibodies or functional fragments thereof according to any one of the preceding claims, wherein the antibodies or functional fragments thereof recognize and bind to two different phosphoric acids on the mammalian tau protein The epitope is determined. A pharmaceutical composition according to claim 48 or 49, which additionally comprises another antibody or a functional fragment thereof which recognizes and binds to an amyloidogenic protein or peptide. An antibody, or a functional fragment thereof, a polynucleotide or a pharmaceutical composition thereof, or a combination thereof, according to any of the preceding claims, for use in therapy, in particular for the treatment of a mammal in need of such treatment, In particular, a neurodegenerative disease or condition in humans, such as a tauopathy. An antibody, or a functional fragment thereof, a polynucleotide, a pharmaceutical composition, or a combination thereof, according to claim 51, for use in the treatment of a neurodegenerative disease or condition in a mammal, wherein the neurodegenerative disease or condition is a tauopathy. An antibody, or a functional fragment thereof, a polynucleotide, a pharmaceutical composition, or a combination thereof, according to claim 51 or 52, for use in the treatment of a neurodegenerative disease or condition in a mammal, such as a tauopathy, wherein the mammal is a human . An antibody, or a functional fragment, polynucleotide or pharmaceutical composition thereof, according to any of the preceding claims, for use in the treatment or amelioration of a mammal suffering from a cognitive deficit, in particular a human suffering from such a defect. The antibody or functional fragment thereof, polynucleotide or pharmaceutical composition of claim 54, wherein the treatment or amelioration of a cognitive deficit in a mammal, particularly a human, can arrest the progression of such cognitive deficits. An antibody, or a functional fragment thereof, a polynucleotide or a pharmaceutical combination thereof, according to claim 54 The treatment, or the amelioration of cognitive deficits in a mammal, especially a human, results in enhanced retention of the cognitive memory capacity of the treated individual, particularly complete recovery. The antibody of claim 52, or a functional fragment thereof, a polynucleotide or a pharmaceutical composition, wherein the tauopathy is selected from the group consisting of: Alzheimer's Disease, Creutzfeldt-Jacob Disease), boxing dementia (Dementia pugilistica), Down's Syndrome, Gerstmann-Sträussler-Scheinker disease, amyotrophic lateral sclerosis/Guam Type of amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, non-Guam-type motoneuron disease with neurofibrillary tangles, argyrophilic grain dementia, cortical base Corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with chromosomal 17-associated Parkinson's disease, frontotemporal dementia, Hall Hadden-Spartz Disease (Hallevorden-Spatz disease), multiple system atrophy, type N Niemann-Pick disease (type C), globus pallidus-pond-deno-nigral degenerat (Pallido-ponto-nigral degenerat Ion), Pick's disease, progressive subcortical neurogliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, only tangled dementia (Tangle-only dementia), after encephalitis Parkinsonitic Parkinsonism and atrophic muscle rigidity. An antibody, or a functional fragment thereof, or a polynucleotide thereof, according to any one of the preceding claims Or a pharmaceutical composition for treating Alzheimer's disease or frontotemporal dementia. The antibody or functional fragment thereof, polynucleotide or pharmaceutical composition of claim 51, wherein the neurodegenerative disease or condition exhibits both tauopathy and amyloidosis. An antibody, or a functional fragment thereof, a polynucleotide or a pharmaceutical composition thereof, according to any of the preceding claims, for use in the treatment of a disease caused by the formation of a neurofibrillary lesion or associated with the formation of a neurofibrillary lesion and A condition in which the neurofibrillary lesion is a major brain lesion in a tauopathy comprising a group of heterogeneous neurodegenerative diseases or conditions, including diseases or conditions that exhibit both tauopathy and amyloidosis, including (but not limited to) Alzheimer's disease, CJD, boxer dementia, Down syndrome, Gustman-Stossler-Syck disease, inclusion body myositis and prion protein brain amyloid angiopathy, Traumatic brain injury, and other diseases or conditions that do not show different amyloidosis, including but not limited to amyotrophic lateral sclerosis/Guam-type Parkinson's disease-dementia complex with neurofibrillary tangles Non-Guam motoneuron disease, argyrophilic granule dementia, cortical basal ganglia degeneration, diffuse neurofibrillary tangles with calcification, and sacral leaf associated with chromosome 17 associated Parkinson's disease Dementia, frontotemporal dementia, Hallerwalden-Schappz disease, multiple systemic atrophy, type C Niemann-Pick disease, globus pallidus-ponsema - substantia nigra degeneration, Pick's disease, progressive cortex Lower glial proliferative disease, progressive nuclear paralysis, subacute sclerosing panencephalitis, only tangled dementia, Parkinson's disease after encephalitis, and atrophic myotonia. The antibody, or a functional fragment thereof, polynucleotide or pharmaceutical composition of claim 54, 55, 56 or 58, wherein the mammal is a human. A method of treating, ameliorating or preventing a neurodegenerative disease or condition, such as a tauopathy, comprising administering to a mammal suffering from the disease or condition an antibody or a functional fragment thereof, a polynuclear antibody according to any one of the preceding claims Glyceric acid or pharmaceutical composition, or a combination thereof. The method of claim 62, wherein the neurodegenerative disease or condition is a tauopathy. The method of claim 63, wherein the τ lesion is selected from the group consisting of: Alzheimer's disease, CJD, boxer dementia, Down syndrome, Gustman-Stosler-Shi Yin Crohn's disease, inclusion body myositis and prion protein brain amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis / Guam-type Parkinson's disease-dementia complex, with neurofibrillary tangles Guam-type motor neuron disease, argyrophilic dementia, cortical basal ganglia degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with chromosomal 17-associated Parkinson's disease, frontotemporal lobe Dementia, Hallerwalden-Schappz disease, multiple systemic atrophy, type C Niemann-Pick disease, globus pallidus-ponsius-substantia nigra degeneration, Pick's disease, progressive subcortical neuroglial proliferative disease, Progressive nuclear paralysis, subacute sclerosing panencephalitis, only tangled dementia, Parkinson's disease after encephalitis, atrophic myotonia. The method of claim 62, 63 or 64 wherein the mammal is a human. A method of inducing a passive immune response in an animal (especially a mammal or a human) suffering from a neurodegenerative disorder such as a tauopathy to the animal Or human, an antibody, or a functional fragment or pharmaceutical composition thereof, or a combination thereof, according to any one of the preceding claims. A method of diagnosing a patient's tau protein-related disease, disorder, or condition or having a tendency to develop a tau protein-related disease, disorder, or condition, comprising detecting an antibody or an active fragment thereof and determining an antigen in the sample or in situ tau protein The immunospecific binding of the group, the method comprising the steps of: a. contacting the sample or a specific body part or body region suspected of containing a tau antigen with an antibody or fragment thereof according to any one of the preceding claims, the antibody or a fragment thereof binds to an epitope of the tau protein; b. binds the antibody to the tau antigen to form an immune complex; c. detects formation of the immune complex; and d. obtains the presence or absence of the immune complex There is a correlation with the presence or absence of a tau antigen in the sample or a particular body part or region, wherein an increase in the amount of the aggregate compared to the normal control value indicates that the patient is suffering from a tau protein-related disease or condition or is presenting At risk of a disease or condition associated with tau protein. A method of monitoring a minimally residual disease in a patient after treatment with an antibody or pharmaceutical composition according to any of the preceding claims, wherein the method comprises: a. causing a sample or a specific body part or body region suspected of containing a tau antigen The antibody or fragment thereof according to any one of the preceding claims, wherein the peptide or fragment thereof binds to an epitope of the tau protein; b. binds the antibody to the tau antigen to form an immune complex; c. detects the Formation of immune complexes; and d. obtaining a correlation between the presence or absence of the immune complex and the presence or absence of a tau antigen in the sample or a particular body part or region, wherein the amount of the immune complex is greater than the normal control value, indicating the patient Still suffering from tiny residual diseases. A method of predicting the reactivity of a patient treated with an antibody or pharmaceutical composition according to any one of the preceding claims, comprising: a. making a sample suspected of containing a tau antigen or a specific body part or body area as claimed in claim 1 Contacting an antibody or a fragment thereof according to any one of 45, wherein the peptide or fragment thereof binds to an epitope of the tau protein; b. binding the antibody to the tau antigen to form an immune complex; c. detecting the immune complex And/or d. obtaining the correlation between the presence or absence of the immune complex and the presence or absence of a tau antigen in the sample or a particular body part or region, e. comparing the immunization before and after the start of the treatment The amount of complex, wherein the amount of the immune complex is reduced, indicates that the patient is more likely to respond to the treatment. The method of any one of claims 67 to 69, wherein the step of obtaining the presence or absence of a correlation of the presence or absence of the immune complex with the presence or absence of a tau antigen in the sample or specific body part or region comprises comparing the immunization The step of the amount of the complex and the normal control value. The method of any one of clauses 67 to 70, wherein the sample is brain tissue homogenate or cerebrospinal fluid. A method for detecting post-mortem detection of a phosphorylated tau protein (pTau) polymer in a brain sample of an individual suspected of suffering from a tau protein-related disease or disorder Containing: a. contacting a brain sample of the individual with an antibody or fragment thereof according to any one of the preceding claims, the antibody or fragment thereof binding to an epitope of the phosphorylated tau protein; b. binding the antibody to the a tau protein to form an immune complex; c. detecting the formation of the immune complex, and d. obtaining the amount or intensity of the immune complex in the sample obtained from the individual from the healthy control individual using the same conditions The amount or intensity of the immune complex is compared, wherein an increase in the amount or intensity of the immune complex compared to the control value indicates that the patient has developed a tau protein related disease or condition. The method of claim 72, wherein the increase observed in the test sample is between 30% and 100% compared to the control sample. The method for diagnosing a tau protein-related disease according to any one of claims 67 to 73, wherein, prior to step (a), the sample is increased by contacting the anti-tau protein antibody or a fragment thereof attached to the solid support The sample is incubated with an immune response to increase the concentration of tau protein in the sample. A test kit for detecting and diagnosing a disease, disorder or condition associated with tau protein comprising an antibody as claimed in claims 1 to 45. A test kit according to the preceding claims, comprising a container containing one or more of the antibodies of claim 1 or a functional fragment thereof, and the use of such antibodies for binding to a tau antigen to form an immune complex And detecting the formation of the immune complex to obtain the presence or absence of a correlation of the presence or absence of the immune complex with the presence or absence of a tau antigen. An epitope selected from the group consisting of VYKSPVVSG (SEQ ID NO: 67, τ aa 393-401) comprising phosphorylated Ser (pS396) at position 396 and phosphorylated Ser (pS396) at position 396 SPVVSG (τ aa 396-401 of SEQ ID NO: 67), YKSPVVS comprising phosphorylated Ser (pS396) at position 396 (τ aa 394-400 of SEQ ID NO: 67), and phosphorylated Ser at position 404 ( DTSPR of pS404) (τ aa 402-406 of SEQ ID NO: 67), and VYKSPVVS of phosphorylated Ser (pS396) at position 396 (τ aa 393-400 of SEQ ID NO: 67). A cell line which produces the antibody of any of the preceding claims. The cell line of claim 78, which is a fusion tumor cell line A4-4A6-48 deposited with DSM ACC3136 on August 30, 2011. The cell line of claim 78, which is a fusion tumor cell line A6-2G5-30 deposited with DSM ACC3137 on August 30, 2011. The cell line of claim 78, which is a fusion tumor cell line A6-2G5-41 deposited with DSM ACC3138 on August 30, 2011. The cell line according to claim 78, which is a fusion tumor cell line A4-2A1-18 deposited with DSM ACC3139 on August 30, 2011. The cell line of claim 78, which is a fusion tumor cell line A4-2A1-40 deposited with DSM ACC3140 on August 30, 2011. The cell line of claim 78, which is a fusion tumor cell line A6-1D2-12 deposited with DSM ACC3141 on September 6, 2011. A vector comprising the polynucleotide of claim 46 or 47, wherein the polynucleotide is operably linked. A host cell comprising the vector of claim 85. The host cell of claim 86, which produces the antibody of any one of claims 1 to 45, or a functional fragment thereof. A host cell as claimed in claim 86 or 87 which is a mammalian cell. The host cell of claim 86 or 87 which is a Chinese hamster ovary cell. A method of producing an antibody, or a functional fragment thereof, according to any one of the preceding claims, comprising cultivating a host cell according to any one of claims 86 to 89 under conditions suitable for expression of the antibody or fragment, and recovering the Antibody or fragment. A method for detecting a phosphorylated tau protein (pTau) multimer in a brain sample, comprising: a. contacting the sample with an antibody or fragment thereof according to any one of claims 1 to 45, the antibody or fragment thereof Binding the epitope of the phosphorylated tau protein; b. binding the antibody or fragment thereof to the tau protein to form an immune complex; and c. detecting the formation of the immune complex.