ANTIBODY, POLYNUCLEOTIDE, PHARMACEUTICAL COMPOSITION, USES OF AN ANTIBODY, IN VITRO METHOD, METHOD O

专利摘要:
antibody, polynucleotide, pharmaceutical composition, in vitro method, detection methods, test kits, cell lineage and use of an antibody The present invention relates to methods and compositions for therapeutic and diagnostic use in the treatment of diseases and disorders that are caused by or are associated with neurofibrillary tangles. in particular, the invention relates to antibodies, which recognize and specifically bind to pathological phosphorylated protein tau conformers, and to methods and compositions involving said antibodies for therapeutic and diagnostic use in the treatment of tauopathies, including alzheimer (ad).
公开号:BR112014008202B1
申请号:R112014008202-2
申请日:2012-10-05
公开日:2022-01-04
发明作者:Andrea Pfeifer；Andreas Muhs；Fred Van Leuven；Maria Pihlgren；Oskar Adolfsson
申请人:Ac Immune S.A.；Katholieke Universiteit Leuven；
IPC主号:

专利说明:

[001] This application claims the benefit of European Patent Application No. EP12163319.2 filed on April 5, 2012 and International Patent Application No. PCT/EP2011/067604 filed on October 7, 2011, the contents of each one is hereby incorporated by reference thereto in its entirety.
[002] The present invention relates to methods and compositions for therapeutic and diagnostic use in the treatment of diseases and disorders that are caused by or are associated with neurofibrillary tangles. In particular, the invention relates to antibodies, which recognize and specifically bind to pathological phosphorylated protein tau conformers, and to methods and compositions involving said antibodies for therapeutic and diagnostic use in the treatment of tauopathies, including Alzheimer's disease. (GIVES).
[003] Neurofibrillary tangles and neuropil filaments (NTs) are the hallmark neuropathological features of Alzheimer's disease (AD). They are composed of the microtubule-associated protein tau that has undergone post-translational modifications, including phosphorylation, deamination, and isomerization to asparaginyl or aspartyl residues. They originate by the hyperphosphorylated aggregation of the protein tau and its conformers. AD shares this pathology with many neurodegenerative tauopathies, in particular with the specified types of frontotemporal dementia (FTD).
[004] Tau protein is a soluble, "naturally unfolded" protein that avidly binds to microtubules (MTs) to promote their assembly and stability. MTs are of great importance for the integrity of the cytoskeleton of neurons - and thus for the proper formation and functioning of neuronal circuits, hence for learning and memory. Binding of tau to MT is controlled by dynamic phosphorylation and dephosphorylation, as demonstrated primarily in vitro and in non-neuronal cells. Due to the large number of possible phosphorylation sites (>80), the exact contribution of each and the identity of responsible kinases remains largely undefined in vivo.
[005] In the AD brain, tau pathology develops later than and thus likely in response to amyloid pathology, which constitutes the essence of the amyloid cascade hypothesis. This is based on and indicated by studies in patients with AD and Down syndrome and is supported by studies in transgenic mice with combined amyloid and tau pathology (Lewis et al., 2001; Oddo et al., 2004; Ribe et al. , 2005; Muyllaert et al., 2006; 2008; Terwel et al., 2008).
[006] The exact timing of both pathologies in human AD patients, as well as mechanisms linking amyloid to tau pathology remain largely unknown, but are proposed to involve activation of neuronal signaling pathways that act on or by GSK3 and cdk5 as the main "tau-kinases" (reviewed by Muyllaert et al., 2006, 2008).
[007] The hypothesis that taupathy is not an innocent side effect but a major pathological executor in AD is based on genetic, pathological and experimental observations that fully corroborate one another:• In familial early AD cases that are due to mutations in amyloid precursor protein (APP) or presenilin, the obligatory pathogenicity cause is amyloid accumulation, but invariably the pathology comprises collateral tauopathy, identical in sporadic cases of late AD;• Severity of cognitive dysfunction and dementia correlates with tauopathy , not with amyloid pathology, most recently exemplified by several phase 1 & 2 clinical studies that include PIB-PET images for amyloid and identify many "false positives": cognitively normal individuals with high cerebral amyloid load;• In familial FTD , tauopathy is caused by mutant tau and causes neurodegeneration directly, without amyloid pathology;• In experimental mouse models, cognitive defects caused by amyloid pathology are almost completely alleviated by the absence of protein tau (Roberson et al., 2007).
[008] The combined arguments support the hypothesis that the protein tau plays an important role in cognitive disappearance in AD and related neurodegenerative tauopathies.
[009] A prominent emerging treatment of AD is by passive immunotherapy with specific mAbs, to clear amyloid peptides and their aggregates that are presumed to be neurotoxic or synaptotoxic.
[010] Immunotherapy targeted at tau pathology, as proposed here, is anticipated to neutralize pathological tau protein conformers that are known or postulated to cause synaptic dysfunction and neurodegeneration.
[011] Other therapeutic approaches that target tau protein are scarce and mainly comprise: • kinase inhibitors that are thought to increase tau phosphorylation to pathological levels • compounds that block the cytoplasmic aggregation of tau of hyperphosphorylated proteins.
[012] These approaches suffer from several specificity and efficacy retractions, a problem they share with attempts to modify APP and amyloid metabolism, all of which emphasize the importance of an ongoing search for additional treatment options, including immunotherapy against tau. Indeed, amyloid-targeted immunotherapy in a preclinical mouse model with combined AD-like pathology also demonstrated an effect on tau pathology although tau aggregates persisted (Oddo et al., 2004).
[013] Some doubts have been cast on the feasibility of addressing intracellular protein tau by immunotherapy. These have been countered by the most recent experimental study in a mouse model of taupathy (Asuni et al., 2007). They showed reduction in tangle pathology and functional improvements by vaccination with a phosphopeptide derived from protein tau. These data corroborate previous reports of immunotherapy targeting a-synuclein in models of Lewy bodies disease and Parkinson's disease (PD) (Masliah et al., 2005, 2011) and of superoxide dismutase in a model of amyotrophic lateral sclerosis ( ALS) (Urushitiani et al., 2007). These diseases are examples where intracellular proteins lead to synaptic defects and neurodegeneration through mechanisms not yet fully understood.
[014] There is an unmet need for passive AND/or active immunotherapies that work to neutralize pathological protein conformers that are known – or presumed – to cause neurodegenerative diseases, such as amyloid pathology in AD caused, for example, by intraneuronal aggregates of tau of the protein's hyperphosphorylated protein that are as typical for AD as amyloid.
[015] This unmet need is met by the present invention which provides for proteins recognizing and binding to the main pathological phospho-epitopes of the tau protein. In particular, the present invention provides specific antibodies against linear and conformational phospho-epitopes, both simple and complex on protein tau, particularly on aggregated protein tau which are believed to be responsible for synapto and neurotoxicity in tauopathies, including AD.
[016] In that sense, the present invention relates in one embodiment to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, such a binding protein or peptide or antibody recognizes and specifically binds to a phospho-epitope on a mammal, particularly on the human tau protein or a fragment thereof, particularly to a phospho-epitope on the aggregated tau protein, particularly on a pathological tau protein conformer, but, in one embodiment , does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein tau and is capable of detecting and/or modulating tau levels soluble, oligomeric and insoluble phosphorylated protein in vivo, particularly in the brain, particularly with a dissociation constant of at least 10 nM, particularly at least 8 nM, particularly at least 5 nM, particularly at least 2 nM, particularly at least 1 nM, particularly at least 500 pM, particularly at least 400 pM, particularly at least 300 pM , particularly at least 200 pM, particularly at least 100 pM, particularly at least 50 pM.
[017] In particular, the dissociation constant is in a range between 2 nM and 80 nM, particularly between 2 nM and 40 nM, particularly between 2 nM and 10 nM.
[018] In one aspect, provided herein is an antibody or functional fragment thereof, wherein said antibody or antibody fragment binds a phospho-epitope having, or within, the amino acid sequence VYKSPVVSGDTSPRHL (SEQ ID NO: 62) (Tau aa 393-408 of SEQ ID NO: 67, for example, as set forth in Table 1) comprising a Ser phosphorylated at position 396 (pS396) and at position 404 (pS404).
[019] In a second embodiment, the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein tau and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated protein tau in vivo and has an association constant rate of 104 M-1s-1 or greater, particularly from 3 - 5 x 10 4 M-1s-1 or greater, particularly 10 5 M-1s-1 or greater; particularly 2 - 9 x 10 5 M-1s-1 or greater; particularly 10 6 M-1s-1 or greater, particularly 1 - 4 x 10 6 M-1s-1 or greater, particularly 10 7 M-1s-1 or greater.
[020] In particular, the rate of association constant is in a range between 1.6 x 103 and 5 x 105, particularly between 2.4 x 104 and 5 x 105, between 3 x 103 and 5 x 105.
[021] In a third embodiment, the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and has a high affinity binding with a dissociation constant of at least minus 4 nM and an association constant rate of 105 M-1s-1 or greater, particularly a dissociation constant of at least 3 nM and an association constant rate of 106 M-1s-1 or greater, particularly a constant dissociation constant of at least 2 nM and an association constant rate of 104 M-1s-1 or greater, particularly a dissociation constant of at least 1 nM and an association constant rate of 105 M-1s-1 or greater, particularly a dissociation constant of at least 200 pM and an association constant rate of 105 M-1s-1 or greater, particularly a dissociation constant of at least 100 pM and an association constant rate of 106 M-1s-1 or larger.
[022] In particular, the dissociation constant is in a range between 2 nM and 80 nM and the association constant rate is in a range between 1.6 x 103 and 5 x 105, particularly the dissociation constant is in a range range between 2 nM and 40 nM and the rate of association constant is in a range between 2.4 x 104 and 5 x 105, particularly the dissociation constant is in a range between 2 nM and 10 nM and the rate constant of association is in a range between 3 x 103 and 5 x 105.
[023] An embodiment (4) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In any of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological Tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such a binding peptide or antibody binds to an epitope in a mammal, for particularly in the human Tau protein as shown in SEQ ID NO: 67, selected from the group consisting of Tau aa 393-401, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 396-401, comprising a Ser phosphorylated at position 396 (pS396 ), Tau aa 394-400, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 402-406, comprising a Ser phosphorylated at position 404 (pS404), and Tau aa 393-400, comprising a Ser phosphorylated at position 396 (pS396).
[024] An embodiment (5) refers to the binding peptide or antibody of any of the foregoing embodiments, wherein such peptide binds to an epitope in a mammal, particularly human Tau protein, but especially human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 393-401, comprising a phosphorylated Ser at position 396 (pS396).
[025] An embodiment (6) refers to the binding peptide or antibody of any of the foregoing embodiments, wherein such peptide binds to an epitope in a mammal, particularly to human Tau protein, but especially human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 396-401, comprising a phosphorylated Ser at position 396 (pS396).
[026] An embodiment (7) refers to the binding peptide or antibody of any of the foregoing embodiments, wherein such peptide binds to an epitope in a mammal, particularly human Tau protein, but especially human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 394-400, comprising a phosphorylated Ser at position 396 (pS396).
[027] An embodiment (8) refers to the binding peptide or antibody of any of the foregoing embodiments, wherein such peptide binds to an epitope in a mammal, particularly to human Tau protein, but especially human Tau protein. as shown in SEQ ID NO: 67, comprising Tau aa 402-406, comprising a Ser phosphorylated at position 404 (pS404).
[028] An embodiment (9) refers to the binding peptide or antibody of any of the foregoing embodiments, wherein such peptide binds to an epitope in a mammal, particularly to human Tau protein, but especially human Tau protein as shown in SEQ ID NO: 67, comprising Tau aa 393-400, comprising a phosphorylated Ser at position 396 (pS396).
[029] An embodiment (10) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising in sequence a CDR1 having the amino acid sequence shown in SEQ ID NO: 73, a CDR2 having the amino acid sequence shown in SEQ ID NO: 74, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence at least 60%, at least 70%, at least 80%, particularly at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% or 100% identical thereto and/or a second binding domain comprising in sequence a CDR1 having the amino acid sequence shown in SEQ ID NO: 70, or an amino acid sequence at least 95%, particularly 98%, particularly 99% identical thereto, a CDR2 having the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence at least 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 72, or an amino acid sequence of at least 60%, at least 70%, at least 80%, particularly at least 85%, particularly at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99 % or 100% identical to it.
[030] In one aspect, provided herein is an antibody or a functional fragment thereof, such antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or a fragment thereof, wherein the said antibody or fragment thereof comprises: (a) a first binding domain, comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 73, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 75; and/or (b) a second binding domain, comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 70, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 71, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 72.
[031] An embodiment (11) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of any of the embodiments above, such a binding peptide or antibody recognizes and specifically binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in one embodiment, does not bind to the corresponding non-phosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated protein Tau in vivo and wherein such binding peptide or antibody comprises a first binding domain comprises by sequencing a CDR1 with the amino acid sequence shown in SEQ ID NO: 81, a CDR2 with the amino acid sequence shown in SEQ ID NO: 82, and a CDR3 with the amino acid sequence shown in SEQ ID NO: 83, or a sequence of amino acids at least 60%, at least 70%, at least 80%, particularly at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, particularly at least 95 %, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% or 100% identical thereto and/or a second binding domain, comprising in sequence a CDR1 with the shown amino acid sequence in SEQ ID NO: 78, a CDR2 having the amino acid sequence shown in SEQ ID NO:79, and a CDR3 having the amino acid sequence shown in SEQ ID NO:80, or an amino acid sequence of at least 60%, at least 70 %, at least 80%, particularly at least 85%, particularly at least s90%, at least 91%, at least 92%, at least 93%, at least 94%, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% or 100% identical to it.
[032] In one aspect, provided herein is an antibody or a functional fragment thereof, such antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or a fragment thereof, wherein said antibody or fragment thereof comprises: (a) a first binding domain, comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 81, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 82, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 83; and/or (b) a second binding domain, comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 78, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 79, and a CDR3 comprising the amino acid sequence shown in SEQ IDNO: 80.
[033] An embodiment (12) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising in sequence a CDR1 having the amino acid sequence shown in SEQ ID NO: 93, a CDR2 having the amino acid sequence shown in SEQ ID NO: 94, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 95, or an amino acid sequence at least 60%, at least 70%, at least 80%, particularly at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% or 100% identical to any of the above CDRs and/or a second binding domain comprising , in sequence, a CDR1 with the amino acid sequence shown in SEQ ID NO: 12, a CDR2 with the amino acid sequence shown in SEQ ID NO: 90, and a CDR3 with the amino acid sequence shown in SEQ ID NO: 91, or an amino acid sequence of at least 60%, at least 70%, at least 80%, in particular at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, particularly at least 95%, particularly at least 96%, particularly at least 97% ,particularly at least 98%, particularly at least 99% or 100% identical to any of the above CDRs.
[034] In a particular aspect, provided herein is an antibody or a functional part thereof, such antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or a fragment thereof, wherein said antibody or fragment thereof comprises: (a) a first binding domain, comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 93, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 94 , and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 95; and/or (b) a second binding domain, comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 91.
[035] An embodiment (13) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising in sequence a CDR1 having the amino acid sequence shown in SEQ ID NO: 101, a CDR2 having the amino acid sequence shown in SEQ ID NO: 102, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 103, or an amino acid sequence at least 60%, at least 70%, at least 80%, particularly at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% or 100% identical to any of the above CDRs and/or a second binding domain comprising , in sequence, a CDR1 with the amino acid sequence shown in SEQ ID NO: 98, a CDR2 with the amino acid sequence shown in SEQ ID NO: 99, and a CDR3 with the amino acid sequence shown in SEQ ID NO: 100, or an amino acid sequence at least 60%, at least 70%, at least 80%, part particularly at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, particularly at least 95%, particularly at least 96%, particularly at least 97% ,particularly at least 98%, particularly at least 99% or 100% identical to any of the above CDRs.
[036] In one aspect, provided herein is an antibody or a functional fragment thereof, such antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or a fragment thereof, wherein the said antibody or fragment thereof comprises: (a) a first binding domain, comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 101, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 102, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 103; and/or (b) a second binding domain, comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 98, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 100.
[037] An embodiment (14) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising in sequence a CDR1 having the amino acid sequence shown in SEQ ID NO: 106, a CDR2 having the amino acid sequence shown in SEQ ID NO: 107, and a CDR3 having the amino acid sequence shown in SEQ ID NO: 108, or an amino acid sequence at least 60%, at least 70%, at least 80%, particularly at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% or 100% identical to any of the above CDRs and/or a second binding domain comprising , in sequence, a CDR1 with the amino acid sequence shown in SEQ ID NO: 89, a CDR2 with the amino acid sequence shown in SEQ ID NO: 115, and a CDR3 with the amino acid sequence shown in SEQ ID NO: 91, or an amino acid sequence at least 60%, at least 70%, at least 80%, part particularly at least 85%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, particularly at least 95%, particularly at least 96%, particularly at least 97% ,particularly at least 98%, particularly at least 99% or 100% identical to any of the above CDRs.
[038] In a specific aspect, provided herein is an antibody or a functional fragment thereof, such antibody or fragment thereof recognizes and specifically binds to a phospho-epitope on a mammalian Tau protein or a fragment thereof, wherein said antibody or fragment thereof comprises: (a) a first binding domain, comprising a CDR1 which comprises the amino acid sequence shown in SEQ ID NO: 106, a CDR2 which comprises the amino acid sequence shown in SEQ ID NO: 107, and a CDR3 which comprises the amino acid sequence shown in SEQ ID NO: 108; and/or (b) a second binding domain, comprising an amino acid sequence comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 89, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 115, and a CDR3 which comprises the amino acid sequence shown in SEQID NO:91.
[039] In one 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 a respective antibody or antibody fragment described herein is a heavy chain variable region.
[040] Another embodiment (15) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first li domain cation comprising the amino acid sequence shown in SEQ ID NO: 69, 77, 116/92, 118, 97, 105, or an amino acid sequence of particularly at least 85%, particularly at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% or 100% identical thereto, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 68, 76, 88, 96, 104, or an amino acid sequence of at least 80%, particularly at least 85%, particularly at least 86% , particularly at least 87%, particularly at least 88%, particularly at least 89%, particularly at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, particularly at least 95% ,particularly at least 96%, particularly at least 97%,particularly at least 98%, for specifically at least 99% or 100% identical to it.
[041] An embodiment (16) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence at least 98% or 99% identical thereto, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 68 , or an amino acid sequence at least 90%, 91%, 92%, or 93% identical thereto.
[042] An embodiment (17) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising the amino acid sequence shown 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 shown in SEQ ID NO: 76, or an amino acid sequence at least 88%, 89% or 90% identical thereto.
[043] An embodiment (18) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising the amino acid sequence shown in 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 comprising the amino acid sequence shown in SEQ ID NO: 88, or an amino acid sequence at least 90%, 91%, 92% or 93% identical thereto.
[044] An embodiment (19) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence at least 86%, 87%, 88% or 90% identical thereto.
[045] An embodiment (20) of the present invention relates to a protein or peptide binding of or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of In either of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in a embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of Soluble, oligomeric and insoluble phosphorylated protein tau in vivo and wherein such binding peptide or antibody comprises a first binding domain tion comprising the amino acid sequence shown in SEQ ID NO: 105, or an amino acid sequence at least 98% or 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 104, or an amino acid sequence at least 88%, 89% or 90% identical thereto.
[046] An embodiment (21) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of any one of the foregoing embodiments, wherein such a binding peptide or antibody comprises a first binding domain in the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence at least 93% identical thereto.
[047] An embodiment (22) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of any one of the foregoing embodiments, wherein such binding peptide or antibody comprises a first binding domain in the amino acid sequence shown 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 shown in SEQ ID NO: 76, or an amino acid sequence at least 90% identical thereto.
[048] An embodiment (23) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of any one of the foregoing embodiments, wherein such a binding peptide or antibody comprises a first binding domain in the amino acid sequence shown in SEQ ID NO: 116, 92 or 118 or an amino acid sequence at least 93-95% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88, or an amino acid sequence at least 93% identical thereto.
[049] An embodiment (24) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of any one of the foregoing embodiments, wherein such a binding peptide or antibody comprises a first binding domain in the amino acid sequence shown in SEQ ID NO: 97 or an amino acid sequence at least 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence at least 90% identical thereto.
[050] An embodiment (25) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or antibody of any one of the foregoing embodiments, wherein such a binding peptide or antibody comprises a first binding domain in the amino acid sequence shown in SEQ ID NO: 105 or an amino acid sequence at least 98% or 99% identical thereto; and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 104, or an amino acid sequence at least 90% identical thereto.
[051] An embodiment (26) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, according to embodiment (16), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 73-75 and such second binding domain contains the CDRs as shown in SEQ ID NOs: 70-72.
[052] An embodiment (27) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, according to embodiment (17), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 81-83 and such second binding domain contains the CDRs as shown in SEQ ID NOs: 78-80.
[053] An embodiment (28) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, according to embodiment (18), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 93-95 and such second binding domain contains the CDRs as shown in SEQ ID NOs: 12, 90 and 91.
[054] An embodiment (29) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, according to embodiment (19), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 101-103 and such second binding domain contains the CDRs as shown in SEQ ID NOs: 98-100.
[055] An embodiment (30) of the present invention relates to a binding protein or peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, according to embodiment (18), wherein said first binding domain contains the CDRs as shown in SEQ ID NOs: 89, 115, and 91 and such second binding domain contains the CDRs as shown in SEQ ID NOs: 106-108.
[056] In yet another embodiment (31) of the present invention relates to a protein or peptide binding or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or functional part thereof, particularly a binding peptide or a functional part thereof. antibody of any of the foregoing embodiments, such a binding peptide or antibody specifically recognizes and binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in one embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope, wherein said binding peptide or antibody has a high binding affinity for soluble, oligomeric and insoluble phosphorylated protein Tau and is capable of detecting and/or modulating levels of soluble, oligomeric and insoluble phosphorylated protein Tau in vivo and wherein such binding peptide or antibody comprises: a. a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 69, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 68; or a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 77, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 76; hear a first binding domain, comprising the amino acid sequence shown in SEQ ID NO: 116, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88; oud a first binding domain, comprising the amino acid sequence shown in SEQ ID NO: 92, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88; oue. a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 97, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 96; ouf. a first binding domain, comprising the amino acid sequence shown in SEQ ID NO: 105, and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 104.g. a first binding domain comprising the amino acid sequence shown in SEQ ID NO: 118 and/or a second binding domain comprising the amino acid sequence shown in SEQ ID NO: 88
[057] In one embodiment (32) of the invention, the binding peptide of any one of the foregoing embodiments is an antibody, particularly an antibody of the IgG2a, IgG2b or the IgG3 isotype, particularly a polyclonal antibody, a monoclonal antibody, an antibody chimeric, a humanized antibody or a fully human antibody.
[058] An embodiment (33) of the invention relates to a polynucleotide encoding the binding peptide of any of the foregoing embodiments.
[059] In one embodiment (34), such a 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 the amino acid sequence as depicted in SEQ ID NOs: 84-87, SEQ ID NOs: 109-114, 117 and 119;b. a nucleic acid molecule comprising a nucleotide sequence that has at least 85% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NOs: 109-114, 117 and 119;c. a nucleic acid molecule comprising a nucleotide sequence that has at least 90% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NOs: 109-114, 117 and 119;d. a nucleic acid molecule comprising a nucleotide sequence that has at least 95% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NOs: 109-114, 117 and 119;e. a nucleic acid molecule comprising a nucleotide sequence that has at least 98% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NOs: 109-114, 117 and 119;f. a nucleic acid molecule comprising a nucleotide sequence that has at least 99% sequence identity to the sequence shown in SEQ ID NOs: 84-87, SEQ ID NOs: 109-114, 117 and 119;g. a nucleic acid molecule comprising a nucleotide sequence of the complementary strand of which it hybridizes to the nucleic acid molecule of any one of a) - f);h. a nucleic acid molecule comprising a nucleotide sequence that deviates from the nucleotide sequence defined in any one of a) - g) by degeneracy of the genetic code: wherein such a nucleic acid molecule as defined in any one of a) - h) recognizes and specifically binds to a phospho-epitope in a mammal, particularly the human Tau protein or a fragment thereof, particularly the human Tau protein as per SEQ ID NO: 67, selected from the group consisting of Tau aa 393-401, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 396-401, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 394-400, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 402-406, comprising a Ser phosphorylated at position 404 (pS404) and aa Tau 393-400, comprising a Ser phosphorylated at position 396 (pS396), wherein, in one embodiment, such a binding peptide has a high binding affinity with a dissociation constant of at least 10 nM, particularly at least 8 nM, particularly at least 5 nM, particularly at least 2 nM, particularly at least 1 nM, particularly at least 500 pM, particularly at least 400 pM, particularly at least 300 pM, particularly at least 200 pM, particularly of at least 100 pM, particularly of at least 50 pM and/or has an association constant rate of 104 M-1s-1 or greater, particularly of between 3 - 5 x 104 M-1s-1 or greater, particularly 105 M-1s-1 major; particularly 6 - 9 x 10 5 M-1s-1 or greater; particularly of 106 M-1s-1 or greater, particularly of 1 - 4 x 106 M-1s-1 or greater, particularly of 107 M-1s-1 or greater, but, in one embodiment, does not bind to the corresponding non-phosphorylated epitope and/or unrelated.
[060] In various embodiments (35) of the invention, a binding peptide is provided or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide, according to any of the foregoing embodiments , or a combination thereof, which is capable of specifically recognizing and binding to a phospho-epitope in a mammal, particularly human Tau protein, particularly a microtubule-associated protein tau, particularly a hyperphosphorylated, microtubule-associated protein tau aggregated as that present in paired helical filaments (PHF), which are the predominant structures in neurofibrillary tangles, neuropil filaments, and dystrophic neurites, but, in one embodiment, does not bind to the corresponding unphosphorylated and/or unrelated epitope.
[061] In a specific embodiment (36) of the invention, the human tau protein is the human Tau protein as shown in SEQ ID NO: 67.
[062] Binding peptides and antibodies according to any of the foregoing modalities, therefore, can be used (37) for reducing levels of total soluble tau protein, particularly soluble phosphorylated tau protein, in the brain, particularly in the cerebral cortex. and/or hippocampus, from a mammal or a human containing increased levels of soluble tau protein and/or soluble phosphorylated tau protein.
[063] Binding peptides and antibodies according to any of the foregoing modalities can also be used (38) for reduction of paired helical filaments containing hyperphosphorylated tau protein (pTau PHF), in the brain, particularly in the cerebral cortex and/or hippocampus. , from a mammal or a human containing increased levels of such pTau or paired helical filaments.
[064] The reduction of the total soluble tau level of the protein and/or soluble phosphorylated tau protein and/or pTau paired helical filaments in the brain, particularly the cerebral cortex and/or hippocampus, of a mammal or a human containing increased levels of variants of such tau protein, which contribute to tau protein associated diseases, disorders or conditions in said mammal or human, can lead to an amelioration and/or alleviation of symptoms associated with such tau protein associated diseases, disorders or conditions (39).
[065] Binding peptides and antibodies according to any of the foregoing modalities can be used (40) in therapy, particularly human therapy, to slow or stop the progression of a tau protein-associated disease, disorder or condition.
[066] Binding peptides and antibodies according to any of the foregoing modalities may also be used (41) in therapy, particularly in human therapy, to ameliorate or alleviate symptoms associated with tau-associated diseases, disorders or conditions, such as, for example, impairment or loss of cognitive functions, including reasoning, situational judgment, memory capacity, learning, special navigation, etc.
[067] In one embodiment (42), the invention relates to binding peptides and antibodies according to any of the foregoing modalities for use in therapy, particularly for use in treating tauopathies, a group of protein-associated disorders and diseases. tau, or to alleviate symptoms associated with tauopathies.
[068] In one embodiment (43), the invention relates to binding peptides and antibodies according to any of the foregoing modalities for maintaining or enhancing cognitive memory capacity in a mammal suffering from a tauopathy.
[069] In another specific embodiment (44) of the invention, binding peptides and antibodies comprising at least one or all of the ACI-35-2A1-Ab1 antibody light chain CDRs; 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; as provided in SEQ ID NOs: 73-75, 81-83, 93-95, 101-103, 106-108 and/or at least one or all of the ACI-35-2A1-Ab1 antibody heavy chain CDRs; 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; as provided in SEQ ID NOs: 70-72, 78-80, (12, 90, 91), 98100, (89, 115, 91) are used in therapy, particularly in human therapy, to ameliorate or alleviate symptoms associated with diseases, disorders or conditions associated with tau protein, such as, for example, impairment or loss of cognitive functions, including reasoning, situational judgment, memory, learning, special navigation, etc.
[070] In another specific embodiment (45) of the invention, binding peptides and antibodies comprising at least one or all of the light chain CDRs of antibodies ACI-35-2 G 5-Ab2; Confirm ACI-35-2G5-Ab3 provided in SEQ ID NOs: 106-108 and/or at least one or all of the heavy chain CDRs of ACI-35-2G5-Ab2 antibodies; ACI-35-2G5-Ab3; as provided in SEQ ID NOs: 89, 115 and 91 are used in therapy, particularly in human therapy, to ameliorate or alleviate symptoms associated with diseases, disorders or conditions associated with tau protein, such as, for example, deficiency or loss of Cognitive functions including reasoning, situational judgment, memory capacity, learning, special navigation, etc.
[071] Binding of the peptides or antibodies, in accordance with the above modalities, to tangles of tau and pTau in the brain can be determined by applying protein immunoreactivity testing to selected brain sections and by Western blotting of brain homogenates , respectively, as described in the Examples.
[072] In another embodiment (46), the present invention provides a pharmaceutical composition, comprising a binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide, according to any of the foregoing embodiments, or a combination thereof, in a therapeutically effective amount together with a pharmaceutically acceptable carrier.
[073] In one embodiment (47), the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide, or a pharmaceutical composition, according to any one of the embodiments above, or a combination thereof, is used in therapy, particularly in human therapy for the treatment or alleviation of symptoms of tau protein associated diseases or disorders including neurodegenerative disorders such as tauopathies.
[074] Binding peptides, antibodies and/or pharmaceutical compositions according to any of the foregoing modalities, therefore, can be used (48) to slow or stop the progression of conditions, disorders or diseases associated with tau protein, upon administration of such binding peptides, antibodies and/or pharmaceutical compositions to an animal, particularly a mammal, particularly a human, suffering from such a disease or condition.
[075] Binding peptides, antibodies, and/or pharmaceutical compositions according to any of the foregoing embodiments, therefore, may also be used (49) to ameliorate or alleviate symptoms of tau-associated conditions, disorders, or diseases, such as such as impairment or loss of cognitive functions, including reasoning, situational judgment, memory capacity, learning, special navigation, etc. by administering such binding peptides, antibodies and/or pharmaceutical compositions to an animal, particularly a mammal, particularly a human, suffering from such a disease or condition.
[076] In one embodiment (50), the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments , or a combination thereof, is used in the treatment of diseases and disorders that are caused by or associated with the formation of neurofibrillary lesions, the predominant pathology of the brain in tauopathy comprising a heterogeneous group of neurodegenerative diseases or disorders, including diseases or disorders that manifest both amyloid tau pathologies, including, but not limited to, Alzheimer's disease, Creutzfeldt-Jakob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker syndrome, inclusion body myositis, and cerebral amyloid protein angiopathy prion, traumatic brain injury, and other diseases or disorders that do not show a pathology of the ami Distinct aloidosis including, but not limited to, amyotrophic lateral sclerosis-Parkinson-dementia of Guam complex, non-Chamorro motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with chromosome-linked parkinsonism 17, Hallevorden-Spatz syndrome, multiple system atrophy, Niemann-Pick disease, type C, pallid-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, post-encephalitis parkinsonism, myotonic dystrophy.
[077] In one embodiment (51), the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments , or a combination thereof, is used in the treatment of Alzheimer's disease.
[078] In one embodiment (52) of the invention, a method is provided for detecting and/or modulating levels of soluble and/or oligomeric and/or insoluble phosphorylated Tau protein, particularly in vivo, particularly in the brain, particularly in the cerebral cortex and /or hippocampus, from an animal, especially a mammal or a human, comprising administering to such an animal, particularly a mammal or human, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a part thereof functional thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments or a combination thereof.
[079] In one aspect, the modulation relates to the reduction of levels of total soluble tau protein, particularly soluble phosphorylated tau protein, in the brain, particularly in the cerebral cortex and/or hippocampus, of a mammal or a human containing levels increased levels of soluble tau protein and/or soluble phosphorylated tau protein.
[080] In one embodiment (53) of the invention, a method is provided for reducing levels of insoluble tau protein, particularly of paired helical filaments containing hyperphosphorylated tau protein (pTau PHF) in the brain, particularly in the cerebral cortex and/or hippocampus, from an animal, especially a mammal or a human, containing high levels of insoluble tau protein, particularly pTau paired helical filaments (pTau PHF) comprising administering to such an animal, particularly mammals or humans, the binding peptide or a moiety functional thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments or a combination thereof.
[081] In one embodiment (54) the present invention relates to a method for slowing or stopping the progression of a disease, disorder or condition associated with tau protein in an animal, particularly a mammal or a human, comprising administering to such an animal, particularly such a mammal or human, suffering from such a disease or condition, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition , according to any of the foregoing embodiments or a combination thereof.
[082] In one embodiment (55) the present invention relates to a method for ameliorating or alleviating symptoms associated with a tau-associated disease, disorder or condition, such as, for example, impairment or loss of cognitive functions, including reasoning, situational judgment, memory capacity, learning, special navigation, etc., in an animal, particularly a mammal or a human, comprising administering to such an animal, particularly such a mammal or human, suffering from such a disease or condition, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments or a combination thereof.
[083] In one embodiment (56), the present invention relates to a method for maintaining or increasing cognitive memory capacity in a mammal suffering from a tauopathy.
[084] In yet another embodiment (57) the present invention relates to a method provided for the treatment of a tau protein-associated disease or disorder, including a degenerative disorder or disease, such as tauopathy, comprising administering to such an animal, particularly such a mammal or human, suffering from such a disease or disorder, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any one of the foregoing modalities or a combination thereof.
[085] In one embodiment (58) of the invention, the method is provided for treating diseases and disorders that are caused by or associated with the formation of neurofibrillary lesions, the brain pathology predominant in tauopathy comprising a heterogeneous group of diseases or disorders neurodegenerative disorders, including diseases or disorders manifesting tau and amyloid pathologies, including, but not limited to, Alzheimer's disease, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, myositis with body of inclusion, and prion protein cerebral amyloid angiopathy, traumatic brain injury, and other diseases or disorders that do not show a distinct amyloid pathology, including, but not limited to, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, motor neuron disease non-Guamanian with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, neurofibrillary tangles diffuse iribrillar cells with calcification, frontotemporal dementia with chromosome 17-linked parkinsonism, frontotemporal dementia, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, pallid-pontine-nigral degeneration, Pick's disease, progressive subcortical gliosis , progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, post-encephalitic parkinsonism and myotonic dystrophy, which method comprises administering to such animal, particularly such mammal or human, suffering from such disease or disorder, the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments or a combination thereof
[086] In one embodiment (59) of the invention, a method is provided for inducing a passive immune response in an animal, especially a mammal or a human, suffering from a neurodegenerative disorder, such as a tauopathy, by administering to such an animal or being the binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments or a combination thereof.
[087] In yet another embodiment (60) of the invention, it relates to a method provided for diagnosing a tau protein-associated disease, disorder or condition in a patient, comprising detecting immunospecific binding to the binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any of the foregoing embodiments, to a tau protein epitope and a sample or in situ which includes the steps of: a. bringing the sample or a specific part of the body or area of the body suspected of containing the tau protein into contact with a binding peptide or a fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, in accordance with any one of the preceding claims, wherein such binding peptide or antibody or fragment thereof binds to an epitope of the tau;b protein. allowing such a binding peptide, particularly such an antibody, particularly such a monoclonal antibody or functional part thereof, to bind to tau protein to form an immune complex; c. detect the formation of the immune complex; ed. correlate the presence or absence of the immune complex with the presence or absence of tau protein in the sample or specific body part or area,
[088] In yet another embodiment (61) of the invention, a method is provided for diagnosing a predisposition to a tau protein-associated disease, disorder or condition in a patient, comprising detecting immunospecific binding to the binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any of the foregoing embodiments, to a tau protein epitope and a sample or in situ, which includes the steps of: a. bringing the sample or a specific part of the body or area of the body suspected of containing tau antigen into contact with a binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, in such a way as to according to any of the foregoing embodiments, wherein such peptide or fragment thereof binds to an epitope of the tau protein; b. allowing such a binding peptide, particularly such an antibody, particularly such a monoclonal antibody or functional part thereof, to bind to tau antigen to form an immune complex; c. detect the formation of the immune complex; ed. correlate the presence or absence of the immune complex with the presence or absence of tau antigen in the sample or specific body part or area, e. comparing the amount of such immune complexes to a normal control value; wherein an increase in the amount of said aggregate, compared to a normal control value, indicates that said patient is suffering from or at risk of developing a tau protein-associated disease or condition.
[089] In one embodiment (62) of the invention, a method is provided for monitoring minimal residual disease in a patient following treatment with the binding peptide or functional part thereof, namely an antibody, particularly a monoclonal antibody or a functional part thereof thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments, wherein such method comprises: a. bringing the sample or a specific part of the body or area of the body suspected of containing tau antigen into contact with the binding peptide or a fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, in accordance with any of the foregoing embodiments, wherein such binding peptide or antibody or fragment thereof binds to an epitope of the tau protein; b. allowing such a binding peptide, particularly such an antibody, particularly such a monoclonal antibody or functional part thereof, to bind to tau antigen to form an immune complex; c. detect the formation of the immune complex; ed. correlate the presence or absence of the immune complex with the presence or absence of tau antigen in the sample or specific part or area of the body, e. comparing the amount of such immune complexes to a normal control value, wherein an increase in the amount of said aggregate as compared to a normal control value indicates that said patient suffers from minimal residual disease.
[090] In one embodiment (63), a method is provided for predicting the responsiveness of a patient being treated with the binding peptide or functional part thereof, namely an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide or a pharmaceutical composition, according to any of the foregoing embodiments, comprising: a. bringing the sample or a specific part of the body or area of the body suspected of containing tau antigen into contact with a binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, accordingly with any of the foregoing embodiments, wherein such peptide or fragment thereof binds to an epitope of the tau;b protein. allowing such a binding peptide, particularly such an antibody, particularly such a monoclonal antibody or functional part thereof, to bind to tau antigen to form an immune complex; ç. detect the formation of the immune complex; ed. correlate the presence or absence of the immune complex with the presence or absence of tau antigen in the sample or specific part or area of the body, e. comparing the amount of said immune complex before and after initiation of treatment, wherein a decrease in the amount of said aggregate indicates that said patient has a high potential to be responsive to treatment.
[091] Anti-Tau antibodies and fragments thereof can be used in the above methods of the invention. In the above methods, the sample containing the antibody or fragment thereof can be immunologically enriched to increase the concentration of Tau protein in the sample by contacting the sample with an anti-Tau antibody or a fragment thereof attached to a solid support.
[092] Before step (a), the sample is immunologically enriched to increase the concentration of Tau protein in the sample by contacting the sample with an anti-Tau antibody or a fragment thereof attached to a solid support.
[093] In another embodiment (64), the invention relates to a test kit for the detection and diagnosis of diseases, disorders or conditions associated with tau protein comprising a binding peptide or an active fragment, particularly an antibody, particularly a monoclonal antibody or a functional moiety according to any of the foregoing embodiments.
[094] In one embodiment (65), said test kit comprises a container containing one or more binding peptides or active fragments thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of of the foregoing modalities and instructions for using the binding peptides for the purpose of binding to tau antigen to form an immune complex and detect immune complex formation such that the presence or absence of the immune complex correlates with the presence or absence of the tau antigen.
[095] In yet another embodiment (66), the present invention relates to an epitope from a mammal, particularly on the human Tau protein as in SEQ ID NO: 67, selected from the group consisting of Tau aa 393-401, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 396-401, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 394-400, comprising a Ser phosphorylated at position 396 (pS396), Tau aa 402-406, comprising a Ser phosphorylated at position 404 (pS404) and aa Tau 393-400, comprising a Ser phosphorylated at position 396 (pS396).
[096] In one embodiment (67), such an epitope consists of Tau aa 393-401, comprising a phosphorylated Ser at position 396 (pS396).
[097] In one embodiment (68), such an epitope consists of Tau aa 396-401, comprising a phosphorylated Ser at position 396 (pS396).
[098] In one embodiment (69), such an epitope consists of Tau aa394-400, comprising a phosphorylated Ser at position 396 (pS396).
[099] In one embodiment (70), such an epitope consists of Tau aa402-406, comprising a phosphorylated Ser at position 404 (pS404).
[0100] In one embodiment (71), such an epitope consists of Tau aa393-400, comprising a phosphorylated Ser at position 396 (pS396).
[0101] In another embodiment (72), the invention relates to a cell line producing a binding peptide or an active fragment thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any one of of the previous modalities.
[0102] In one embodiment (73), the invention relates to a cell line, which is A4-4A6-48 hybridoma cell line deposited on August 30, 2011 as DSM ACC3136.
[0103] In one embodiment (74), the invention relates to a cell line, which is A6-2G5-30 hybridoma cell line deposited on August 30, 2011 as DSM ACC3137.
[0104] In one embodiment (75), the invention relates to a cell line, which is A6-2G5-41 hybridoma cell line, deposited on August 30, 2011 as DSM ACC3138.
[0105] In one embodiment (76), the invention relates to a cell line, which is A4-2A1-18 hybridoma cell line deposited on August 30, 2011 as DSM ACC3139.
[0106] In one embodiment (77), the invention relates to a cell line, which is A4-2A1-40 hybridoma cell line deposited on August 30, 2011 as DSM ACC3140.
[0107] In one embodiment (78), the invention relates to a cell line, which is A6-1D2-12 hybridoma cell line deposited on September 6, 2011 as DSM ACC3141.
[0108] In one embodiment (79), the invention relates to a monoclonal antibody or a functional part thereof, composed of a light chain (VL) and/or a heavy chain domain (VH), which is encoded by a polynucleotide located in a nucleotide fragment obtainable by PCR amplification of A4-2A1-18 hybridoma cell line DNA deposited Aug 30, 2011 as DSM ACC3139 using:a. a primer pair, comprising a 5'-primer of SEQ ID NO: 149 and a 3'-primer of SEQ ID NO: 51 for amplifying a first binding domain; and/or b. a primer mixture, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 120, 123, 124, 136, 137, 138, 139 and 140 and a 3'-primer selected from the group consisting of SEQ ID NOs: 131 , 134 and 141-148, for amplifying a second binding domain.
[0109] In one embodiment (80), the invention relates to a monoclonal antibody or a functional part thereof, composed of a light chain (VL) and/or a heavy chain domain (VH), which is encoded by a polynucleotide located in a nucleotide fragment obtainable by PCR amplification of DNA from hybridoma cell line A6-2G5-30 deposited Aug 30, 2011 as DSM ACC3137 using:a. a primer mix, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 51 and 169-174 and a 3'-primer of SEQ ID NO: 51 for amplifying a first binding domain; and/or a primer mix, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 124, 127, and 150-158 and a 3'-primer selected from the group consisting of SEQ ID NOs: 130 and 159-168, for amplification of a second binding domain.
[0110] In one embodiment (81), the invention relates to a monoclonal antibody or a functional part thereof, composed of a light chain (VL) and/or a heavy chain domain (VH), which is encoded by a polynucleotide located in a nucleotide fragment obtainable by PCR amplification of A4-2A1-40 hybridoma cell line DNA deposited Aug 30, 2011 as DSM ACC3140 using:a. a primer mix comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 178, 179 and 180 and a 3'-primer of SEQ ID NO: 51 for amplifying a first binding domain; and/or a primer mixture, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 121, 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 a second binding domain.
[0111] In one embodiment (82), the invention relates to a monoclonal antibody or a functional part thereof, composed of a light chain (VL) and/or a heavy chain domain (VH), which is encoded by a polynucleotide located in a nucleotide fragment obtainable by PCR amplification of DNA from the hybridoma cell line A6-2G5-41 deposited August 30, 2011 as DSM ACC3138 using: a. a primer mix, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 51 and 188-192 and a 3'-primer of SEQ ID NO: 51 for amplifying a first binding domain; and/or a primer mixture, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 120, 124, 126, 181, 182 and 183 and a 3'-primer selected from the group consisting of SEQ ID NOs: 144, 145 and 184-187, for amplification of a second binding domain.
[0112] In one embodiment (83), the invention relates to a monoclonal antibody or a functional part thereof, composed of a light chain (VL) and/or a heavy chain domain (VH), which is encoded by a polynucleotides located in a nucleotide fragment obtainable by PCR amplification of DNA from hybridoma cell line A4-4A6-48 deposited Aug 30, 2011 as DSM ACC3136 using:a. a primer mix, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 50 and 201-204 and a 3'-primer of SEQ ID NO: 51 for amplifying a first binding domain; and/or a primer mixture, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 121, 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 a second binding domain.
[0113] In one embodiment (84), the invention relates to a monoclonal antibody or a functional part thereof, composed of a light chain (VL) and/or a heavy chain domain (VH), which is encoded by a polynucleotide located in a nucleotide fragment obtainable by PCR amplification of A6-1D2-12 hybridoma cell line DNA deposited September 6, 2011 as DSM ACC3141 using:a. a primer mix, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 209-214 and 219-221 and a 3'-primer of SEQ ID NO: 215, for amplifying a first binding domain; and/or a primer mixture, comprising a 5'-primer selected from the group consisting of SEQ ID NOs: 216, 217 and 218 and a 3'-primer of SEQ ID NO: 208, for amplification of a second binding domain.
[0114] In one embodiment (85), the antibody according to any of the foregoing embodiments can be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a camelid antibody, an antibody fragment dimer or a engineered or modified antibody.
[0115] In one embodiment (86), the invention provides a method for producing the binding peptides or antibodies of any of the foregoing modalities, the step comprising culturing the cell lineage of any of the foregoing modalities in a culture medium and, optionally, purifying the binding peptides or antibody from the culture medium or cell line.
[0116] In another embodiment (87), the present invention provides a method of detecting phospho-Tau (pTau) multimers in a brain sample comprising: a. contacting a sample with an antibody or a fragment thereof, according to any one of the preceding claims, which peptide or fragment binds to an epitope of the phospho-Tau;b protein. allowing the antibody to bind to the tau protein to form an immune complex; c. detect immune complex formation, particularly through the application of an ELISA assay.
[0117] In particular, the invention relates in a specific embodiment (88) to a method of post-mortem detection of phospho-Tau (pTau) multimers in brain homogenates of a subject suspected of suffering from a disease or disorders associated with tau and a health control subject comprising: a. contacting a sample of brain homogenates from both subjects with an antibody or a fragment thereof, according to any one of the preceding claims, which peptide or fragment binds to an epitope of the phospho-Tau;b protein. allowing the antibody to bind to the tau protein to form an immune complex; c. detect immune complex formation, particularly through the application of an ELISA ed. , comparing the amount or intensity of the immune complex in the sample obtained from the subject suspected of suffering from a disease associated with tau or from the control sample, in which an increase in the amount or intensity of said immune complex, compared to the control value, indicates that said patient suffered from minimal residual disease.
[0118] In one embodiment (89), the observed increase in the sample compared to the control samples is between 30% and 50%, particularly between 35% and 45%.
[0119] In one embodiment (90), the invention provides a binding peptide or protein or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, according to any of the foregoing embodiments, which antibody or fragment shows a favorable pK profile. In particular, such an antibody or fragment has a high serum concentration up to 10 days post administration, which indicates a pharmacokinetic (PK) profile favorably supports the use of such antibodies as a therapeutic antibody. (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. BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCESFIGURES
[0120] Figure 1 shows detection results of Tau multimers phosphorylated by ACI-35-2A1-Ab1 (left panel), ACI-35-2G5-Ab3 (middle panel) and a control antibody (HT7; right panel) in the brain homogenate of control and AD subjects.
[0121] Figure 2 (2A and 2B) shows the results of detection of total and p-Tau by commercial human brain homogenate antibodies.
[0122] Figure 3 (3A, 3B, 3C) shows the detection of phospho-Tau by ACI-35-2A1-Ab1 (A), ACI-35-1D2-Ab1 (B) and ACI-35-2G5-Ab3 (C) in human brain homogenate.
[0123] Figure 4 (4A, 4B, 4C) shows the results of detection of Tau-pS396 in human AD and control brain (Ctrl) by ACI-35-2A1-Ab1 (A), ACI-35-1D2- Ab1 (B) and ACI-35-2G5-Ab3 (C) antibodies using AlphaLISA. ****p<0.0001, **p<0.01 by Mann-Whitney test.
[0124] Figure 5 (5A and 5B) shows results of IHC (AT180) staining of Tau-pT231 in amygdala (A) and hippocampus (B), in the treatment of Tau transgenic mice with ACI-35-2G5-Ab3 .
[0125] Figure 6 (6A and 6B) shows results of IHC (HT7) staining of total Tau in the amygdala (A) and hippocampus (B) in the treatment of Tau transgenic mice with ACI-35-2G5-Ab3.
[0126] Figure 7 shows an SDS-PAGE for Tau-pS396 generated using different GSK3β conditions and the membrane stained using the ACI-35-2G5-Ab3 antibody.
[0127] Figure 8 shows specific AlphaLISA assay setup using ACI-35-2G5-Ab3-BT and Tau-13 antibodies.
[0128] Figure 9 shows the detection of Tau-pS396 in human brain fraction S1 from an AD donor; compared to the signal obtained from samples enriched with IP and non-IP samples.
[0129] Figure 10 shows results of detection of Tau-pS396 in human AD and control CSF (Ctrl) by ACI-35-2G5-Ab3 antibody using IP followed by AlphaLISA. *** p=0.0003 by Mann-Whitney test. SEQUENCES
[0130] SEQ ID NO: 46 - 57 depict the nucleotide sequences of forward and reverse VH/VK primers.
[0131] SEQ ID NO: 62 depicts the amino acid sequence of Tau antigen, T3 peptide (see Table 1).
[0132] SEQ ID NO: 67 depicts the amino acid sequence of the longest isoform of human tau (441 aa) also called Tau40.
[0133] SEQ ID NO: 68 depicts the amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-4A6-Ab1 produced by the A4-4A6-18 hybridoma cell line.
[0134] 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 A4-4A6-18 hybridoma cell line.
[0135] SEQ ID NO: 70 depicts the CDR1 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-4A6-Ab1.
[0136] SEQ ID NO: 71 depicts the CDR2 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-4A6-Ab1.
[0137] SEQ ID NO: 72 depicts the CDR3 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-4A6-Ab1.
[0138] SEQ ID NO: 73 depicts the amino acid sequence of the CDR1 of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1.
[0139] SEQ ID NO: 74 depicts the CDR2 amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1.
[0140] SEQ ID NO: 75 depicts the CDR3 amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-4A6-Ab1.
[0141] 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 A6-1D2-12 hybridoma cell line.
[0142] 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 A6-1D2-12 hybridoma cell line.
[0143] SEQ ID NO: 78 depicts the CDR1 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-1D2-Ab1.
[0144] SEQ ID NO: 79 depicts the CDR2 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-1D2-Ab1.
[0145] SEQ ID NO: 80 depicts the CDR3 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-1D2-Ab1.
[0146] SEQ ID NO: 81 depicts the CDR1 amino acid sequence of the light chain (VK) variable region of the monoclonal antibody ACI-35-1D2-Ab1.
[0147] SEQ ID NO: 82 depicts the CDR2 amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-1D2-Ab1.
[0148] SEQ ID NO: 83 depicts the CDR3 amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-1D2-Ab1.
[0149] SEQ ID NO: 84 depicts the nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.
[0150] SEQ ID NO: 85 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-4A6-Ab1 produced by hybridoma cell line A4-4A6-18.
[0151] 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 A6-1D2-12 hybridoma cell line.
[0152] 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 A6-1D2-12 hybridoma cell line.
[0153] SEQ ID NO: 88 depicts the amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody ACI-35-2A1-Ab1, ACI-35-2A1-Ab2 and ACI-35-4A6-Ab2, respectively , produced by hybridoma cell lines A4-2A1-18, A4-2A1-40 and A4-4A6-48, respectively.
[0154] SEQ ID NO: 89 depicts the CDR1 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
[0155] SEQ ID NO: 90 depicts the amino acid sequence of CDR2 of the variable region of the heavy chain (VH) of the monoclonal antibody ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, and ACI-35-4A6- Ab2, respectively.
[0156] SEQ ID NO: 91 depicts the amino acid sequence of CDR3 of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, ACI-35-4A6-Ab2 , ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
[0157] 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 A4-2A1-40 hybridoma cell line.
[0158] SEQ ID NO: 93 depicts the CDR1 amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab2.
[0159] SEQ ID NO: 94 depicts the CDR2 amino acid sequence of the light chain (VK) variable region of monoclonal antibody ACI-35-2A1-Ab2.
[0160] SEQ ID NO: 95 depicts the CDR3 amino acid sequence of the light chain (VK) variable region of the monoclonal antibody ACI-35-2A1-Ab2.
[0161] SEQ ID NO: 96 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-Ab1 produced by hybridoma cell line A6-2G5-08.
[0162] SEQ ID NO: 97 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-Ab1 produced by hybridoma cell line A6-2G5-08.
[0163] SEQ ID NO: 98 depicts the CDR1 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-2G5-Ab1.
[0164] SEQ ID NO: 99 depicts the CDR2 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-2G5-Ab1.
[0165] SEQ ID NO: 100 depicts the CDR3 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-2G5-Ab1.
[0166] SEQ ID NO: 101 depicts the CDR1 amino acid sequence of the light chain (VK) variable region of the monoclonal antibody ACI-35-2G5-Ab1.
[0167] SEQ ID NO: 102 depicts the CDR2 amino acid sequence of the light chain (VK) variable region of monoclonal antibody ACI-35-2G5-Ab1.
[0168] SEQ ID NO: 103 depicts the CDR3 amino acid sequence of the light chain (VK) variable region of the monoclonal antibody ACI-35-2G5-Ab1.
[0169] SEQ ID NO: 104 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3 respectively, produced by A6 hybridoma cell line. -2G5-30 and A6-2G5-41, respectively.
[0170] SEQ ID NO: 105 depicts the amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell line. A6-2G5-30 and A6-2G5-41, respectively.
[0171] SEQ ID NO: 106 depicts the CDR1 amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
[0172] SEQ ID NO: 107 depicts the CDR2 amino acid sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
[0173] SEQ ID NO: 108 depicts the CDR3 amino acid sequence of the light chain (VK) variable region of the monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively.
[0174] SEQ ID NO: 109 depicts the nucleotide sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, and ACI-35-4A6-Ab2 respectively , produced by hybridoma cell lines A4-2A1-18, A4-2A1-40 and A4-4A6-48, respectively.
[0175] 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 A4-2A1-40 hybridoma cell line.
[0176] SEQ ID NO: 111 depicts the nucleotide sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-AB1 produced by hybridoma cell line A6-2G5-08.
[0177] SEQ ID NO: 112 depicts the amino acid sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB1 produced by hybridoma cell line A6-2G5-08.
[0178] SEQ ID NO: 113 depicts the amino acid sequence of the heavy chain variable region (VH) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3 respectively, produced by A6 hybridoma cell line. -2G5-30 and A6-2G5-41, respectively.
[0179] SEQ ID NO: 114 depicts the nucleotide sequence of the light chain variable region (VK) of monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3, respectively, produced by hybridoma cell line A6-2G5-30 and A6-2G5-41, respectively.
[0180] SEQ ID NO: 115 depicts the CDR2 amino acid sequence of the heavy chain (VH) variable region of the monoclonal antibody ACI-35-2G5-AB2 and ACI-35-2G5-AB3.
[0181] 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 A4-2A1-18 hybridoma cell line.
[0182] SEQ ID NO: 117 depicts the nucleotide sequence of the light chain variable region (VK) of the monoclonal antibody ACI-35-2A1-Ab1 produced by the A4-2A1-18 hybridoma cell line.
[0183] SEQ ID NO: 118 depicts the amino acid sequence of the variable light chain (VK) region of the monoclonal antibody ACI-35-4A6-Ab2 produced by the A4-4A6-48 hybridoma cell line.
[0184] 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 A4-4A6-48 hybridoma cell line.
[0185] SEQ ID NO: 120 - 221 depict the denucleotide sequences of forward and reverse VH/VK primers. DEFINITIONS OF TERMS
[0186] The terms "polypeptide", "peptide" and "protein" as used herein are used interchangeably and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.
[0187] The term "polypeptide" or "linking peptide" is used interchangeably throughout this document and refers to chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group on the alpha carbon of one amino acid and the amino group on the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (that is, the terminal amino) has a free amino group, while the terminal amino acid at the other end of the chain chain (i.e. the terminal carboxy) has a free carboxyl group. As such, the term "amino terminal" (abbreviated N-terminal) refers to the free alpha-amino group on the amino acid at the amino terminus of the peptide, or the alpha group -amino (imine group when participating in a peptide bond) of an amino acid anywhere else within the peptide. Similarly, the term "carboxy terminal" (abbreviated C-terminal) refers to the free carboxyl group on the amino acid. to the carboxy terminus of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide. A binding peptide may constitute antibodies, such as polyclonal or monoclonal antibodies, human or humanized antibodies, diabodies, camelid antibodies, etc., or functional parts thereof, as defined herein.
[0188] The term "fragment of the same(s)" or "fragment", as used herein in the context of a peptide, refers to a functional peptide fragment that has essentially the same (biological) activity as an intact peptide defined herein. The terms, when used herein in the context of an antibody, refer to an antibody fragment that comprises a portion of an intact antibody that contains an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; single chain antibody molecules, including single chain Fv (scFv) molecules; and bispecific and multispecific antibodies and/or antibody fragments.
[0189] Normally, the amino acids that make up a peptide are numbered in order, starting at the amino terminus and increasing in the direction towards the carboxy terminus of the peptide. Thus, when one amino acid is said to "follow" the other, that amino acid is positioned closer to the carboxy terminus of the peptide than the previous amino acid.
[0190] The term "residue" is used in this document to refer to an amino acid that is incorporated into a peptide by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function similarly to natural amino acids (i.e. mimetic amino acid). In addition, an amide bond mimetic includes peptide backbone modifications known to those skilled in the art.
[0191] The phrase "consisting essentially of" is used in this document to exclude all elements that would substantially alter the essential properties of the peptides to which the phrase refers. Thus, the description of a peptide "consisting essentially of..." excludes any amino acid substitutions, additions or deletions that would substantially alter the biological activity of such peptide.
[0192] Furthermore, a person skilled in the art will recognize that, as mentioned above, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less 1%) in an encoded sequence are conservatively modified variations where the changes result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); e6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0193] The phrases "isolated" or "biologically pure" refer to material that is substantially or essentially free of components that normally accompany it as found in its native state. Thus, the peptides described herein do not contain materials normally associated with their in situ environment. Typically, the isolated, immunogenic peptides described herein are at least about 80% pure, generally at least about 90%, and preferably at least about 95%, as measured by band intensity on a silver colored gel.
[0194] Protein purity or homogeneity can be indicated by a number of methods known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization after staining. For certain purposes, high resolution will be required and HPLC or similar means for purification used.
[0195] When immunogenic peptides are relatively short in length (ie, less than about 50 amino acids), they are often synthesized using standard peptide chemical synthesis techniques.
[0196] Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence, is a preferred method for the chemical synthesis of the immunogenic peptides described herein. Solid phase synthesis techniques are known to those skilled in the art.
[0197] Alternatively, the immunogenic peptides described herein are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide, placing the nucleic acid in an expression cassette under the control of a specific promoter, expressing the peptide in a host, isolating the expressed peptide or polypeptide, and, if necessary, renaturing the peptide. Sufficient techniques to guide an individual skilled in the art through such procedures are found in the literature.
[0198] Once expressed, the recombinant peptides can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Substantially pure compositions of about 50% to 95% homogeneity are preferred, and homogeneity of 80% to 95% or greater is most preferred for use as therapeutic agents.
[0199] A person skilled in the art will recognize that, following chemical synthesis, biological expression or purification, the immunogenic peptides may have a substantially different conformation than the native conformations of the constituent peptides. In this case, it is often necessary to denature and reduce the antiproliferative peptide and then cause the peptide to refold into the preferred conformation. Methods of reducing and denaturing proteins and inducing refolding are well known to those skilled in the art.
[0200] The antigenicity of the purified protein can be confirmed, for example, by demonstrating a reaction with the immune serum or with antisera produced against the protein itself.
[0201] The terms "a", "a" and "the" in this document are defined to mean "one or more" and include the plural, unless the context is inappropriate.
[0202] The terms "detect" or "detected", as used herein, mean the use of known techniques for the detection of biological molecules, such as immunochemical or histological methods, and refer qualitatively or quantitatively to the determination of the presence or concentration of the biomolecule under investigation.
[0203] "Isolated" means a biological molecule free of at least some of the components with which it naturally occurs.
[0204] The terms "antibody" or "antibodies" or "functional parts thereof" as used herein are art-recognized terms and are understood to refer to active molecules or fragments of molecules that bind to known antigens, especially molecules of immunoglobulin and portions of immunoglobulin molecules that are immunologically active, i.e., that contain a binding site that immunospecifically binds an antigen. The immunoglobulin according to the invention can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of the immunoglobulin molecule.
[0205] "Antibodies" are intended, within the scope of the present invention, to include monoclonal, polyclonal, chimeric, single-chain, bispecific, simian, human and humanized antibodies, camelid antibodies, diabodies, as well as functional or active parts fragments of them. Examples of active fragments of molecules that bind to known antigens include Fab and F(ab')2 fragments, including the products of an immunoglobulin Fab expression library, and epitope-binding fragments of any of the antibodies and fragments mentioned above. .
[0206] Those active fragments can be derived from an antibody of the present 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 fraction containing Fab fragments can be collected and concentrated by membrane filtration and the like. For a more detailed description of general techniques for isolating active antibody fragments, 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).
[0207] A "humanized antibody" refers to a type of antibody engineered having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). (s).
[0208] A humanized antibody may further refer to an antibody having a variable region where one or more of its framework regions have human or primate amino acids. In addition, framework support residues may be altered to preserve 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)).
[0209] A "humanized antibody" can also be obtained by a novel genetic engineering approach that allows the production of polyclonal antibodies similar to those of human affinity matured in large animals such as rabbits (http://www .rctech.com/bioventures/therapeutic.php).
[0210] The term "fully human antibody" or "human" antibody is intended to refer to an antibody derived from transgenic mice carrying human antibody genes or from human cells. For the human immune system, however, the difference between "fully human", "human", and "humanized" antibodies may be negligible or non-existent, and as such, all three may be of equal efficacy and safety.
[0211] The term "monoclonal antibody" is also well recognized in the art and refers to an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically made by fusing a normally short-lived antibody-producing B cell into a rapidly growing cell, such as a cancer cell (sometimes referred to as an "immortal" cell). The resulting hybrid cell, or hybridoma, multiplies rapidly, creating a clone that produces large amounts of an antibody.
[0212] The term "antigen" refers to an entity or a fragment thereof that can induce an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions responsible for antigenicity or antigenic determinants.
[0213] As used herein, the term "soluble" means partially or completely dissolved in aqueous solution.
[0214] Also as used herein, the term "immunogenic" refers to substances that provoke or increase the production of antibodies, T cells, and other immune cells reactive against an immunogenic agent and contribute to an immune response in humans or animals. .
[0215] An immune response occurs when an individual produces sufficient antibodies, T cells and other immune cells reactive against administered immunogenic compositions of the present invention to moderate or attenuate the disease being treated.
[0216] The term "hybridoma" is art-recognized and is understood by those skilled in the art to refer to a cell produced by the fusion of an antibody-producing cell and an immortal cell, for example, a multiple myeloma cell. This hybrid cell is capable of producing a continuous supply of antibody. See the definition of "monoclonal antibody" above and the Examples below for a more detailed description of the fusion method.
[0217] The term "carrier", as used herein, means a structure into which the antigenic peptide or supramolecular construct can be incorporated or with which it can be associated, thereby presenting or exposing antigenic peptides or part of the peptide to the immune system. of a human or animal. Any particle that can be conveniently used in human or animal therapy, such as, for example, a vesicle, a particle or a body of particulate material can be used as a carrier within the context of the present invention.
[0218] The term "carrier" further comprises delivery methods in which supramolecular antigenic construct compositions comprising the antigenic peptide can be transported to desired sites by delivery mechanisms. An example of such a delivery system uses colloidal metals such as colloidal gold.
[0219] Carrier proteins that can be used in the supramolecular antigenic construct compositions of the present invention include, but are not limited to, maltose binding peptide "MBP"; bovine serum albumin "BSA"; hemocyanin from the snail Megathura crenulata "KLH"; ovalbumin; flagellin; thyroglobulin; serum albumin of any kind; gamma globulin of any kind; syngeneic cells; syngeneic cells with Ia antigens; and polymers of D- or L-amino acids.
[0220] Furthermore, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of binding peptide that, when administered to a human or animal, is sufficient to result in a therapeutic effect in said human or animal. animal. The effective amount is readily determined by a person skilled in the art following routine procedures.
[0221] "pTau PHF", "PHF", and "paired helical filaments" are used here interchangeably and refer to pairs of approximately 10 nm filaments wound in helices with a periodicity of 160 nm visible in electron microscopy. The width varies between 10 and 22 nm. PHFs are the predominant structures in Alzheimer's Disease (AD) neurofibrillary tangles and neuropil filaments. PHFs can also be seen in some but not all dystrophic neurites associated with neuritic plaques. PHF component is a hyperphosphorylated form of the microtubule-associated protein tau. PHFs are composed of disulfide-linked antiparallel hyperphosphorylated tau proteins. PHF tau can be truncated from its 20 C-terminal amino acid residues. The mechanisms underlying PHF formation are uncertain, but hyperphosphorylation of tau may disengage it from microtubules, increasing the soluble cluster of tau.
[0222] Within the scope of the present invention, it has been shown that the response induced by antibodies to the antigenic composition according to the invention is largely T cell independent. A nude mouse model was used in this regard, and nude mice were vaccinated and responses of antibodies measured to assess the Aβ-specific antibody response induced by the antigenic composition according to the invention in the immunized nude mice. Nude mice carry the Foxn1nu mutation and consequently have reduced T cell function due to the lack of an adequate thymus.
[0223] A "pharmaceutically effective amount", as used herein, refers to a dose of the active ingredient in a pharmaceutical composition suitable for curing, or at least partially containing, the symptoms of the disease, disorder or condition being treated or any associated complications.
[0224] The present invention provides binding peptides recognizing and binding to major pathological phospho-epitopes of tau protein. In particular, the present invention provides specific antibodies against linear and conformational phospho-epitopes, both simple and complex on protein tau, which are believed to be responsible for synapto and neurotoxicity in tauopathies, including AD.
[0225] In that sense, the present invention relates in one embodiment to a binding peptide or a functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, such a binding peptide or antibody recognizes and binds specifically to a phospho-epitope in a mammal, particularly to the human Tau protein or a fragment thereof, particularly to a pathological tau protein conformer, but, in one embodiment, does not bind to the corresponding non-phosphorylated epitope and/or epitopes unrelated, wherein said binding peptide or antibody has a high binding affinity with a dissociation constant of at least 10 nM, particularly of at least 8 nM, particularly of at least 5 nM, particularly of at least 2 nM, particularly of at least 2 nM at least 1 nM, particularly at least 500 pM, particularly at least 400 pM, particularly at least 300 pM, particularly at least 200 pM, particularly at least 100 pM, particularly at least 50 pM.
[0226] "Soluble Tau protein", as used herein, refers to proteins consisting of two fully solubilized Tau protein/peptide monomers or Tau-like peptides/proteins, or modified or truncated Tau peptides/proteins or other derivatives of Tau peptide/protein monomers and Tau protein oligomers. "Soluble tau" particularly excludes neurofibrillary tangles (NFT).
[0227] "Insoluble Tau", as used herein, refers to various aggregated monomers of the Tau peptides or proteins, or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or other peptide derivatives /Tau proteins forming oligomeric or polymeric structures that are insoluble in vitro in aqueous media and in vivo in mammals or the human body, more particularly in the brain, but particularly to various aggregated monomers of Tau or of modified or truncated Tau peptides/proteins or their derivatives, which are insoluble in mammals or in the human body, more particularly in the brain, respectively. "Insoluble Tau" particularly includes neurofibrillary tangles (NFT).
[0228] "Monomeric Tau" or "Tau Monomer" as used herein refers to fully solubilized Tau proteins without aggregated complexes in aqueous media.
[0229] "Aggregated Tau", "Oligomeric Tau" and "Tau Oligomer", as used herein, refer to various aggregated monomers of the Tau peptides or proteins, or of Tau-like peptides/proteins, or of peptides/proteins Modified or truncated Tau or other derivatives of Tau peptides/proteins forming oligomeric or polymeric structures which are insoluble or soluble in vitro in aqueous media and in vivo in mammals or the human body, more particularly in the brain, but particularly to various aggregated monomers of Tau or modified or truncated Tau peptides/proteins or derivatives thereof, which are insoluble or soluble in mammals or in the human body, more particularly in the brain, respectively.
[0230] A "modulating antibody" refers to an antibody or a functional fragment thereof, as described in the various embodiments, which may also up-regulate (e.g., activate or stimulate), to down-regulate (e.g., inhibit or suppress) or otherwise alter a functional property, biological activity or level of soluble and/or insoluble and/or oligomeric Tau protein, particularly soluble phosphorylated tau protein, in vivo, particularly in the brain, particularly in the cerebral cortex and/or hippocampus, from an animal, especially a mammal or a human, containing increasing levels of soluble tau protein and/or soluble phosphorylated tau protein. A modulating antibody or functional fragment thereof can act to modulate a tau protein or a polypeptide encoding such a tau protein directly or indirectly. In certain embodiments, a modulating antibody or functional fragment thereof reduces levels of soluble and/or insoluble and/or oligomeric tau protein, particularly soluble and insoluble tau protein, particularly soluble and insoluble oligomeric tau protein. In one aspect, the soluble and/or insoluble and/or oligomeric tau protein is phosphorylated tau protein, particularly soluble phosphorylated tau protein, in the brain, particularly the cerebral cortex and/or hippocampus, of a mammal or a human containing increased levels of protein soluble tau and/or phosphorylated tau protein, especially soluble tau protein and/or soluble phosphorylated tau protein.
[0231] In one embodiment, the present invention provides a pharmaceutical composition comprising a binding peptide or functional part thereof, particularly an antibody, particularly a monoclonal antibody or a functional part thereof, or a polynucleotide comprising a nucleic acid sequence encoding said binding peptide or antibody according to any of the embodiments described and claimed herein, or a combination thereof, in a therapeutically effective amount together with a pharmaceutically acceptable carrier.
[0232] Examples of pharmaceutically suitable carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffer saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, solutions sterile, etc.
[0233] The binding peptides according to the invention, including antibodies, particularly the monoclonal antibodies and active fragments thereof, may be prepared in a physiologically acceptable formulation and may include a pharmaceutically acceptable carrier, diluent and/or excipient using known techniques. . For example, the binding peptides according to the invention and as described herein, including any functionally equivalent binding peptides or functional parts thereof, in particular, the monoclonal antibodies of the invention including any functionally equivalent antibodies or functional parts thereof , are combined with a pharmaceutically acceptable carrier, diluent and/or excipient to form a therapeutic composition. Examples of pharmaceutically suitable carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffer saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. .
[0234] Formulation of the pharmaceutical composition according to the invention may be carried out according to standard methodology known to those skilled in the art.
[0235] The compositions of the present invention can be administered to a subject in the form of a solid, liquid or aerosol at the appropriate and pharmaceutically effective dose. Pills, creams and implantable dosage units are examples of solid compositions. Pills can be taken orally. Therapeutic creams can be administered topically. Implantable dosage units may be administered locally, for example at a tumor site, or may be implanted for systematic delivery of the therapeutic composition, for example subcutaneously. Formulations adapted for intramuscular, subcutaneous, intravenous, intra-arterial injection and formulations for topical and intraocular administration are examples of liquid compositions. Examples of aerosol formulations include inhaler formulations for administration to the lungs.
[0236] The compositions may be administered by standard routes of administration. In general, the composition can be administered topically, orally, rectally, nasally, intradermally, intraperitoneally, or parenterally (e.g., intravenously, subcutaneously, or intramuscularly).
[0237] In addition, the composition can be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the immediate vicinity where delivery is desired, for example at the site of a tumor. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals and administration sustained for a predetermined period of time.
[0238] The sustained release matrix, as used herein, is a matrix of materials, typically polymers degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is held by enzymes and body fluids. The sustained release matrix is desirably chosen for biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (glycolic acid polymer), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamine acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of a polylactide, polyglycolide or polylactide co-glycolide (copolymers of lactic acid and glycolic acid).
[0239] It is known to those skilled in the art that the dosage of the composition will depend on various factors such as, for example, the condition being treated, the specific composition used and other clinical factors such as weight, size, sex and general health status. of the patient, body surface area, the specific compound or composition to be administered, other drugs being administered simultaneously, and the route of administration.
[0240] The composition according to the invention can be administered in combination with other compositions comprising a biologically active substance or compound, such as, for example, a known compound used in the medication of tauopathies and/or amyloidosis, a group of diseases and disorders associated with amyloid or amyloid-like protein, such as the amyloid β protein involved in Alzheimer's disease.
[0241] The other biologically active substance or compound may exert its biological effect by the same or a similar mechanism as the therapeutic vaccine according to the invention, either by an independent mechanism of action or by a multiplicity of related mechanisms of action and /or independent.
[0242] Generally, the other biologically active compound may include neutron-transmission enhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepine tranquilizers, acetylcholine synthesis, enhancer storage or release, postsynaptic acetylcholine receptors, monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory drugs, antioxidants, and serotonergic receptor antagonists.
[0243] In particular, the biologically active agent or compound may include at least one compound selected from the group consisting of compounds against oxidative stress, anti-apoptotic compounds, metal chelators, DNA repair inhibitors such as pirenzepine and metabolites, acid 3 -amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS), secretase activators, [beta]- and 7-secretase inhibitors, tau proteins, neurotransmitter, /3-leaf breakers, anti -inflammatory drugs, "atypical antipsychotics", such as, for example, clozapine, ziprasidone, risperidone, aripiprazole or olanzapine or cholinesterase inhibitors (ChEIs) such as tacrine, rivastigmine, donepezil, and/or galanthamine and other drugs and nutritional supplements, such as e.g. vitamin B 12, cysteine, a precursor of acetylcholine, lecithin, choline, Ginkgo biloba, acetyl-L-carnitine, idebenone, propentofylline or a xanthine derivative, together with an acetyl-binding peptide in accordance with the invention, including antibodies, particularly the monoclonal antibodies and active fragments thereof, and, optionally, a carrier and/or a diluent and/or a pharmaceutically acceptable excipient and instructions for the treatment of diseases.
[0244] In another embodiment, the composition according to the invention may include niacin or memantine together with a binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies and active fragments thereof and, optionally, a carrier and/or a pharmaceutically acceptable diluent and/or excipient.
[0245] In yet another embodiment of the invention, compositions are provided comprising "atypical antipsychotics", such as, for example, clozapine, ziprasidone, risperidone, aripiprazole or olanzapine for treating positive and negative psychotic symptoms, including hallucinations, delusions, disorders (manifested by marked incoherence, derailment, tangential thinking), and bizarre or disorganized behavior, as well as anhedonia, flattening, apathy and social withdrawal, together with the binding peptide according to the invention, including antibodies, particularly antibodies monoclonal agents and active fragments thereof and, optionally, a pharmaceutically acceptable carrier and/or diluent and/or excipient.
[0246] Other compounds which can suitably be used in compositions in addition to the binding peptide according to the invention are those disclosed, for example, in WO 2004/058258 (see especially pages 16 and 17) including therapeutic drug targets ( page 36-39), alkanesulfonic acids and alkanolsulfuric acid (pages 3951), cholinesterase inhibitors (pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages 58-59), non-steroidal anti-inflammatory drugs inflammatory (pages 60-61), antioxidants (pages 61-62), peroxisome proliferator-activated receptor agonists (PPARs) (pages 63-67), cholesterol-lowering agents (pages 68-75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors (pages 77-78), metal chelators (pages 78-79), antipsychotics and antidepressants (pages 80-82), nutritional supplements (pages 83-89) and compounds, increasing the availability of biologically active substances in the brain (see pages 89-93) and prodrugs (pages 93 and 94), which document is incorporated herein by reference, but especially the compounds mentioned on the pages indicated above.
[0247] Pharmaceutically active protein substance may be present in amounts between 1 ng and 10 mg per dose. Generally, the administration regimen should be in the range of between 0.1 μg and 10 mg of the antibody according to the invention, particularly in a range of 1.0 μg to 1.0 mg, and more particularly in a range between 1 .0 μg and 100 μg, with all individual numbers within these ranges, also being part of the invention. If administration is by continuous infusion, a more suitable dosage may be in the range of between 0.01 µg and 10 mg units per kilogram of body weight per hour with all individual numbers within these ranges also being part of the invention.
[0248] Administration will generally be parenterally, for example, intravenously or subcutaneously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, emulsions and suspensions. Non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic injectable esters such as ethyl oleate. Aqueous solvents may be chosen from the group consisting of water, aqueous/alcohol solutions, emulsions or suspensions including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous transporters include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and others. Preservatives may also be present, such as antimicrobials, antioxidants, chelating agents, inert gases, etc.
[0249] The pharmaceutical composition may further comprise protein carriers, such as, for example, serum albumin or immunoglobulin, particularly of human origin. Additional biologically active agents may be present in the pharmaceutical composition of the invention depending on its intended use.
[0250] When the binding target is located in the brain, certain embodiments of the invention provide the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof, to cross the blood-brain barrier. Certain neurodegenerative diseases are associated with increased permeability of the blood-brain barrier, such that the binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies or active fragment thereof, can easily be introduced into the brain. When the blood-brain barrier remains intact, several approaches known in the art exist for transporting molecules across it, including, but not limited to, physical methods, lipid-based methods, and channel- and receptor-based methods.
[0251] Physical methods of transporting the binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies, or active fragment thereof, across the blood-brain barrier include, but are not limited to, bypassing the blood-brain barrier. entirely, or create openings in the blood-brain barrier. Contour methods include, but are not limited to, direct injection into the brain (See, for example, Papanastassiou et al., Gene Therapy 9: 398-406 (2002)) and implantation of a delivery device into the brain (see, for for example, Gill et al., Nature Med. 9: 589-595 (2003 ); and Gliadel Wafers(TM), Guildford Pharmaceutical ). Methods of creating openings in the barrier include, but are not limited to, ultrasound (see, for example, US Patent Publication No. 2002/0038086), osmotic pressure (for example, by administering hypertonic mannitol (Neuwelt, EA, Implication of the Blood-Brain Barrier and its Manipulation, VoIs 1 & 2, Plenum Press, NY (1989)), permeabilization by, for example, bradykinin or A-7 permeabilizer (see, for example, US Patent No. 5,112,596, 5,268,164, 5,506,206 and 5,686,416) and transfection of neurons that straddle the blood-brain barrier with vectors that contain genes encoding the binding peptide or antigen-binding fragment (see, for example, US Patent Publication No. 2003/0083299).
[0252] Lipid-based methods for transporting the binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies, or an active fragment thereof, across the blood-brain barrier include, among others, encapsulating the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies, or active fragment thereof, in liposomes that are coupled to active fragments thereof that bind to receptors on the vascular endothelium of the blood-brain barrier (see, for example, Publication No. 20020025313), and coating the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies, or active fragment thereof, on low-density lipoprotein particles (see, for example, Application Publication US Patent No. 20040204354) or apolipoprotein E (see, for example, US Patent Application Publication No. 20040131692).
[0253] Receptor and channel-based methods for transporting the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies, or active fragment thereof, across the blood-brain barrier include, among others, the use of glucocorticoid blockers to increase blood-brain barrier permeability (see, for example, US Patent Application Publication Nos. 2002/0065259, 2003/0162695 and 2005/0124533); activation of potassium channels (see, for example, US Patent Application Publication No. 2005/0089473), inhibition of ABC drug transporters (see, for example, US Patent Application Publication No. 2003/0073713); coating antibodies with a transferrin and modulating the activity of one or more transferrin receptors (see, for example, US Patent Application Publication No. 2003/0129186) and cationizing the antibodies (see, for example, US Patent No. 5,004 .697).
[0254] Single or repeated administrations of the binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies, or an active fragment thereof, or of a pharmaceutical composition according to the invention may be provided to a subject during a period of time. extended period of time. The duration of administration can be between 1 week and up to 12 months or more. During this time, the binding peptide, antibody, or pharmaceutical composition may be administered once a week, once every two weeks, three weeks, four weeks, etc., or at a greater or lesser frequency, depending on the subject's needs. to be treated.
[0255] In a further embodiment, the present invention provides methods and kits for the detection and diagnosis of diseases, disorders or conditions associated with tau protein, including neurodegenerative diseases or disorders such as tauopathy comprising a heterogeneous group of neurodegenerative diseases or disorders, including diseases or disorders that show the presence of tau and amyloid pathologies, including, but not limited to, Alzheimer's disease, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, myositis with inclusion body, and prion protein cerebral amyloid angiopathy, traumatic brain injury, and other diseases or disorders that do not show a distinct amyloid pathology, including, but not limited to, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, neuron disease non-Guamanian motor with neurofibrillary tangles, argyrophilic grain dementia, cortical degeneration obasal, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with chromosome 17-linked parkinsonism, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis , progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, post-encephalitic parkinsonism, and myotonic dystrophy. Pathological abnormalities may be caused by or associated with the formation of neurofibrillary lesions, the predominant pathology of the brain in tauopathy.
[0256] Furthermore, the present invention provides methods and kits for diagnosing a predisposition to diseases, disorders or conditions associated with tau protein, including neurodegenerative diseases or disorders such as tauopathies that comprise a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders which show the co-manifestation of tau and amyloid pathologies, or for monitoring minimal residual disease in a patient or for estimating a patient's responsiveness to a treatment with a binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof, or a composition according to the invention and as described herein. These methods include known immunological methods commonly used to detect or quantify substances in biological samples or under in situ conditions.
[0257] The diagnosis of a tau protein associated disease or condition, or a predisposition to a tau protein associated disease or condition in a subject in need thereof, particularly a mammal, more particularly a human, including neurodegenerative diseases or disorders such as tauopathies comprising a heterogeneous group of neurodegenerative diseases or disorders, including diseases or disorders manifesting tau and amyloid disorders, can be achieved by detecting the immunospecific binding of a binding peptide of the invention, particularly an antibody, particularly of a monoclonal antibody or an active fragment thereof, to an epitope of the tau protein in a sample or in situ, which includes contacting the sample or a particular body part or area suspected of containing the tau protein with an antibody that binds to an epitope of the tau protein, allowing the antibody to bind to the tau protein to form a comp immune complex, detecting immune complex formation 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 area, optionally comparing the amount of immune complex to a normal control value , wherein an increase in the amount of the immune complex compared to a normal control value indicates that the subject suffers from or is at risk of developing a tau protein-associated disease or condition.
[0258] The monitoring of minimal residual disease in a subject, particularly a mammal, more particularly a human, following treatment with a binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies and active fragments thereof, or a composition according to the invention can be achieved by detecting immunospecific binding of a binding peptide of the invention, particularly an antibody, particularly a monoclonal antibody or an active fragment thereof, to an epitope of tau protein in a sample or in situ, which includes contacting the sample or a specific body part or area of the body suspected of containing the tau protein with a binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof, which binds an epitope of the tau protein, allowing the binding peptide according to the invention, including antico rpos, particularly monoclonal antibodies and active fragments thereof, bind to tau protein to form an immune complex, detecting immune complex formation and correlating the presence or absence of the immune complex with the presence or absence of tau protein in the sample or part or specific body area, optionally comparing the amount of said immune complex to a normal control value, wherein an increase in the amount of said immune complex compared to a normal control value indicates that the subject may still be suffering from residual disease minimum.
[0259] Predicting the response of a subject, particularly a mammal, more particularly a human, to treatment with a binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies and active fragments thereof, or a composition of according to the invention may be achieved by detecting immunospecific binding of a binding peptide of the invention, particularly a monoclonal antibody or an active fragment thereof, to a tau protein epitope in a sample or in situ, which includes contacting the sample or a specific part of the body or area of the body suspected of containing the tau protein with a binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof, which binds an epitope of the tau protein, allowing the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and frag active components thereof, binds to tau protein to form an immune complex, detecting immune complex formation 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 area, optionally , comparing the amount of said immune complex before and after initiation of treatment, wherein a decrease in the amount of said immune complex indicates that said patient has a high potential to respond to treatment.
[0260] Biological samples that may be used in the diagnosis of a tau-associated disease or condition, for the diagnosis of a predisposition to a tau-associated disease or condition, including neurodegenerative diseases or disorders such as tauopathies that comprise a heterogeneous group of neurodegenerative diseases or disorders, including diseases or disorders manifesting tau and amyloid disorders, or for monitoring minimal residual disease in a patient or for estimating a patient's responsiveness to a treatment with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, or a composition according to the invention and as described herein are, for example, fluids such as serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymphatic fluid and the like or tissue or cell samples obtained from an organism such as tissue neural, cerebral, cardiac or vascular. To determine the presence or absence of tau protein in a sample, any immunoassay known to those skilled in the art can be used, such as assays using indirect detection methods using secondary detection reagents, ELISA and immunoprecipitation assays, and agglutination. A detailed description of these assays is, for example, given in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612, WO96/13590 to Maertens and Stuyver, Zrein et al. (1998) and WO96/29605.
[0261] For in situ diagnosis, the binding peptide according to the invention, including antibodies, particularly the monoclonal antibodies and active fragments thereof, of the invention or any active and functional part thereof can be administered to the body to be diagnosed by methods known in the art, such as, for example, intravenous, subcutaneous, intranasal, intraperitoneal, intracerebral, intraarterial injection, such that specific binding between an antibody according to the invention and an epitopic region on the amyloid protein can occur. The peptide/binding antigen complex may conveniently be detected by a tag attached to the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies, or a functional fragment thereof, or any other detection method known in the art. technique.
[0262] Immunoassays used in diagnostic applications or for diagnosing a predisposition to diseases or conditions associated with tau protein, including neurodegenerative diseases or disorders such as tauopathies that comprise a heterogeneous group of neurodegenerative diseases or disorders including diseases or disorders that manifest pathology of tau and amyloid, or for monitoring minimal residual disease in a patient or for estimating a patient's responsiveness to treatment with a binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof , or a composition according to the invention and as described herein, typically relies on labeled antigens, binding peptides or secondary reagents for detection. These proteins or reagents may be labeled with compounds generally known to those skilled in the art including enzymes, radioisotopes and fluorescent, luminescent and chromogenic substances, including but not limited to colored particles such as colloidal gold and latex granules. Of these, radioactive labeling can be used for almost all types of assays and with most variations. Enzyme-conjugated tags are particularly useful when radioactivity must be avoided or when rapid results are needed. Fluorochromes, while requiring expensive equipment to use, provide a very sensitive method of detection. Useful binding peptides for these assays are those disclosed as claimed herein, including antibodies, particularly monoclonal antibodies, polyclonal antibodies, and affinity purified polyclonal antibodies.
[0263] Alternatively, the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof, can be labeled indirectly by reaction with labeled substances that have an immunoglobulin affinity, such as protein A or G or second antibodies. The binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof, can be conjugated to a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the binding peptide according to the invention, including antibodies, particularly monoclonal antibodies and active fragments thereof, can be conjugated to biotin and the biotin/binding peptide conjugate detected using avidin or labeled streptavidin. Likewise, the binding peptide can be conjugated to a hapten and the hapten/binding peptide conjugate detected using labeled anti-hapten binding peptide.
[0264] Those skilled in the art are aware of these and other appropriate marks that may be employed in accordance with the present invention. The binding of such tags to binding peptides or fragments thereof can be accomplished using standard techniques commonly known to those skilled in the art. Typical techniques are described by 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). Coupling techniques mentioned above are the glutaraldehyde method, the periodate method, the dimaleimide method and others, all of which are incorporated by reference herein.
[0265] Current immunoassays utilize a dual antibody method to detect the presence of an analyte, where the antibody is labeled indirectly by reactivity with a second antibody that has been labeled with a detectable label. The second antibody is preferably one that binds to antibodies of the animal from which the monoclonal antibody is derived. In other words, if the monoclonal antibody is a mouse antibody, then the second labeled antibody is an anti-mouse antibody. In order for the antibody to be used in the assay described herein, this tag is preferably an antibody coated bead, particularly a magnetic bead. For the antibody to be used in the immunoassay described herein, the label is preferably a detectable molecule, such as a radioactive, fluorescent, or electrochemiluminescent substance.
[0266] An alternative dual antibody system, often referred to as quick format systems because they are adapted for rapid determinations of the presence of an analyte, may also be employed within the scope of the present invention. The system requires high affinity between the antibody and the analyte. In accordance with one embodiment of the present invention, the presence of amyloid protein is determined using a pair of antibodies, each specific for the amyloid protein. One of such antibody pairs is referred to herein as a "detector antibody" and the other of such antibody pair is referred to as a "capture antibody". The monoclonal antibody of the present invention can be used as a capture antibody or a detector antibody. The monoclonal antibody of the present invention can also be used as a detector and capture antibody together in a single assay. One embodiment of the present invention thus uses the double antibody sandwich method for the detection of amyloid protein in a biological fluid sample. In this method, the analyte (amyloid protein) is sandwiched between the detector antibody and the capture antibody, the capture antibody being irreversibly immobilized on a solid support. The detector antibody would contain a detectable tag in order to identify the presence of the antibody-analyte sandwich and therefore the presence of the analyte.
[0267] Exemplary solid phase substances include, but are not limited to, microtiter plates, polystyrene test tubes, magnetic, plastic or glass beads and sliders, which is known to those skilled in the radioimmunoassay and immunoassay art. enzyme. Methods for coupling antibodies to solid phases are also known to those skilled in the art. More recently, a number of porous materials, such as nylon, nitrocellulose, cellulose acetate, glass fibers and other porous polymers, have been employed as solid supports.
[0268] The present invention also relates to a diagnostic kit for the detection of tau protein in a biological sample comprising a composition as defined above. Furthermore, the present invention relates to the latter diagnostic kit which, in addition to a composition as defined above, also comprises 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 in Zrein et al. (1998).
[0269] It is yet another object of the present invention to provide novel immunoprobes and test kits for the detection and diagnosis of diseases and conditions associated with tau protein, comprising binding peptides in accordance with the present invention. For immunoprobes, the binding peptides are directly or indirectly attached to an appropriate reporter molecule, for example an enzyme or a radionuclide. The test kit includes a container containing one or more binding peptides according to the present invention and instructions for using the binding peptides for the purpose of binding to tau antigen to form an immune complex and detect immune complex formation, such that the presence or absence of the immune complex correlates with the presence or absence of the tau antigen. EXAMPLES
[0270] Example 1: Generation and screening of hybridomas and antibodies
[0271] The purpose of this study was to generate and screen for anti-Tau mAbs (monoclonal antibodies). Hybridomas were generated by fusing mouse spleen cells with a tau vaccine-immunized myeloma cell line. Hybridomas were evaluated for reactivity against phosphorylated and non-phosphorylated full-length Tau protein, as well as phosphorylated and non-phosphorylated Tau antigenic peptides used for vaccine preparation. Hybridoma screening was also performed for reactivity of hybridoma supernatant to tau tangles using immunochemistry on Tau transgenic mouse brain sections.1.1 METHODS1.1.1 FUSION
[0272] A wild type C57BL/6 mouse vaccinated with ACI-35 (Tau393-408 [S396, pS404])) was used for hybridoma production. The mouse was challenged with ACI-35 vaccine on day 0, then again on day 4, and the fusion was performed on day 7.
[0273] 6x107 (ACI-35), splenocytes from the immunized mouse were fused with 2x107 SP2-O-Ag14 myeloma cells at a ratio of 3 splenocytes/1 myeloma cell.
[0274] The fusions resulted in 8 x 96 well plates and the clones were named according to the plate (1-8) and then the row (AG) and finally the column (1-12).1.1.2 SCAN METHOD TO SELECT CLONES
[0275] The 8 x 96 well plates were first subjected to two scans for IgG expression. Positively expressing clones were transferred into 24-well plates and cell supernatants (=clones) from growing cells were tested in a Tau ELISA scan and a TAUPIR immunohistochemistry scan. ELISA and/or TAUPIR positive supernatants were transferred to T25 flasks, and the clones were rescanned for IgG expression in an ELISA and TAUPIR scan of Tau.1.1.3 IGG SCAN
[0276] ELISA plates (Costar; Sigma) were coated with 50 μl/well of anti-mouse IgG antibody (AbD Serotec, Düsseldorf, Germany) in coating buffer for 16 h at 4°C. After washing the plates with PBS/Tween, wells were blocked with 100 μl/well blocking solution for 1 hour at room temperature. Undiluted hybridoma supernatants (50 µl per well) were incubated for 1 hour at room temperature. After a wash, a mixture of anti-mouse IgG1, IgG2a, IgG2b, IgG3 or IgM (AbD Serotec) conjugated with horseradish peroxidase (HRP) was applied for 1 hour at room temperature. After a final wash, detection was performed with HRP substrate (TMB; 3-3',5,5'-tetramethylbenzidine), and the plates were read at 405 nm using a microplate reader. Results are expressed as optical density (O.D.).1.1.4 SCAN ELISA OF TAU HYBRIDOMAS
[0277] Hybridoma ELISA scan was performed on pTau peptide (ACI-35, T3.5: Tau393-408[S396/pS404; PolyPeptide Laboratories, Hiller0d, Denmark), the corresponding unphosphorylated Tau peptide (T3.6: Tau393-408, PolyPeptide Laboratories), phosphorylated full-length Tau protein (441aa) (pTau protein, Vandebroek et al, 2005) and full-length Tau protein (441aa) (Tau protein, SignalChem, Richmond, Canada). Finally, Bovine Serum Albumin (BSA) was used as a negative control.
[0278] Plates were coated with 10 µg/ml of the corresponding Tau peptide and 1 µg/ml of the corresponding Tau protein overnight at 4°C. After washing each well with PBS-0.05% Tween 20 and blocking with 1% BSA in PBS-0.05% Tween 20, undiluted hybridoma supernatant or negative control medium was added to the plates and incubated at 37°C. for 2 hours. After washing, the plates were incubated with an alkaline phosphatase (AP)-conjugated anti-mouse IgG total antibody (Jackson Laboratories, Baltimore, PA, USA) for 2 hours at 37°C. plates were incubated with pNPP (para-nitro-phenyl-phosphate), the phosphatase substrate for AP and read at 405 nm using an ELISA plate reader. Results are expressed in O.D. (Optical Density)
[0279] TAUPIR experiments were performed according to the protocol of EXAMPLE 3.1.2.1.1.6 IGG SCANNING IN T25 BOTTLES
[0280] Pacas ELISA were coated with 5ug/ml anti-mouse F(ab')2 IgG fragment specific antibody (Jackson Laboratories, Baltimore, PA, USA) in carbonate-bicarbonate coating buffer pH 9, 6 (Sigma, Buchs, Switzerland) overnight at 4°C. After washing the dishes, undiluted hybridoma supernatant, positive control IgG1 antibody (6E10 at 1ug/ml: Covance, Emeryville, CA, USA) or negative control (culture medium alone) was incubated for 1 h at room temperature. After a washing step, goat anti-mouse FcY IgG fragment-specific antibody (subclasses 1+2a+2b+3) conjugated with secondary AP (Jackson Laboratories, Baltimore, PA, USA) was incubated on the plates for 2 hours at 37°C. After a final wash, detection was performed with pNPP (para-nitro-phenyl-phosphate), the phosphatase substrate for AP, and plates were read at 405 nm using an ELISA plate reader. Results are expressed in O.D. (Optical Density). 1.2 RESULTS
[0281] Cell supernatants from the 8 x 96 well plates resulting from the fusion were screened for IgG production. Of 768 wells (8x96 wells) tested, 48 wells positive for IgG production were selected based on the best binding to vaccine phospho-peptide and full-length phospho-Tau. Selection was based on binding to peptide and full-length phospho-Tau protein by ELISA and also selectivity when compared to non-phospho peptide and full-length non-phospho Tau protein. 24 selected hybridomas were subcloned by seeding 2 plates per hybridoma at 1 cell/well and 1 plate at 0.5 cell/well. Supernatants were retested for phosphopeptide and phosphoprotein binding to verify the binding profile, after which stability was assessed in a 6-week culture. Eight stable clones were then selected and tested for isotyping and binding using ELISA and TAUPIR as described in Methods.1.3 CONCLUSION
[0282] The antibodies generated showed high specificity with pTau peptides with only marginal binding of non-phosphorylated peptides.
[0283] A total of 8 clones were selected for further subcloning and were sequenced (see Table 6 and Table 7) and 6 clones were deposited in the DSMZ (see Table 10).
[0284] The aforementioned positive mother clones were further cultured in 96-well plates, then 24-well plates, and finally T25 flasks. At each step, supernatants from hybridoma clones were selected by ELISA, Taupir and Western Blot. EXAMPLE 2 ANTIBODY LIGHT CHAIN AND HEAVY CHAIN VARIABLE REGIONS CLONING
[0285] Antibody heavy and light variable region genes from hybridoma cells are cloned and DNA sequences and location of complementarity determining regions (CDRs) determined, as well as antibody binding characteristics.
[0286] Total RNA was prepared from 3 x 10 6 hybridoma cells (1 vial) using the Qiagen RNeasy mini kit (CAT No: 74104). RNA was eluted in 50uL of water and verified on a 1.2% agarose gel.
[0287] VH and VK cDNAs were prepared using IgG reverse transcriptase and kappa constant region primers. First strand cDNAs were amplified by PCR using a large set of signal sequence primers. The amplified DNAs were gel purified and cloned into pGem® T Easy vector (Promega). The obtained VH and VK clones were scanned for inserts of the expected size. The DNA sequence of selected clones was determined in both directions by automated DNA sequencing. The locations of the complementarity determining regions (CDRs) in the sequences were determined with reference to other antibody sequences (Kabat EA et al., 1991). EXAMPLE 3 CONNECTION STUDIES I
[0288] The objective was to measure the phospho-Tau binding (pTau) of antibodies generated from subcloned hybridomas derived from mice immunized with the liposomal tau vaccines. To test this, an enzyme-linked immunosorbent assay (ELISA) was used to measure the binding of purified antibodies to phosphorylated and non-phosphorylated full-length Tau protein, as well as phosphorylated and non-phosphorylated Tau antigenic peptides used for liposomal vaccine preparation. .
[0289] The scan was completed by two other methods. Immunohistochemistry (IHC) was performed on brain sections from a transgenic Tau animal (TAUPIR) using an anti-tau antibody as the primary antibody. In addition, a western blot (WB) on brain protein homogenates from Tau transgenic mice was performed, using an anti-tau antibody as the antibody blotting.3.1 METHODS3.1.1. ELISAS: PHOSPHO-TAU BINDING ASSAY
[0290] An ELISA assay was used to test binding of the purified antibodies to Tau and pTau. Briefly, Nunc MaxiSorp 96-well plates (Nunc, Roskilde, Denmark) were coated with 1 µg/mL full-length Tau protein (441 aa) (SignalChem, Richmond, Canada) or phosphorylated full-length Tau protein (441 aa) (Vandebroek et al., 2005). In addition, plates were coated with 10 μg/ml of Tau-derived vaccine peptide, Tau393-408 (phosphorylated or not at S396 and S404). To test for cross-reactivity to Tau and pTau sequences from different pTau epitopes that were not used for vaccine preparation, plates were coated with 10 μg/mL of the following peptides: Tau393-408 (phosphorylated or not in S396 and S404), O Coating was done overnight in phosphate buffered saline (PBS) at 4°C. The plates were washed with 0.05% Tween20/PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween20/PBS for 1 hour at 37°C. The antibody being tested was then added in a double dilution series of 8 or 16 between 0 and 20 μg/mL, and allowed to incubate for 2 hours at 37°C. The plates were then washed as described above, and alkaline phosphatase (AP) conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, Suffolk, England) was added at a dilution of 1/6000 in 0.05% Tween20 /PBS for 2 hours at 37°C. After washing, the plates were incubated with disodium p-nitrophenyl phosphate hexahydrate (pNPP; Sigma-Aldrich, Buchs, Switzerland) phosphatase substrate solution and read at 405 nm after incubation times of 30 min, 1, 2 or 16 h. using an ELISA plate reader.3.1.2. TAUPIR AND WESTERN-BLOTS: ANTI-TAU ANTIBODY BINDING TO TAU TANGLES IN BRAIN SECTIONS OF A TAU TRANSGENIC ANIMAL (TAUPIR)
[0291] For TAUPIR staining, brain sections were from TPLH mice (transgenic mice expressing the longest isoform (441aa) of hTauP301L), old mice (> 18 months old) double biGT transgenic mice (transgenic GSK-3β transgenic mice with TPLH) and double transgenic biAT mice (hAPPV717I transgenic mice crossed with TPLH). As a negative control, sections from Tau knockout mice (TKO; 6 months old) were used. Brain sections were washed for 5 min in PBS, then incubated for 15 min at room temperature in 1.5% H2O2 in PBS:MeOH (1:1) to block endogenous peroxidase activity. After washing the sections 3 times in PBST (PBS/0.1% TritonX100), they were incubated for 30 min at room temperature in PBST+10% FCS (fetal calf serum) blocking solution. Incubation with the anti-Tau antibody being tested was done overnight at 4°C, using the following antibody concentrations: ACI-35-2A1-Ab1 at 0.0053 μg/mL, ACI-35-2A1-Ab2 at 0.0048 µg/ml, ACI-35-4A6-Ab1 at 0.015 µg/ml, ACI-35-1D2-Ab1 at 0.0047 µg/ml, ACI-35-2G5-Ab1 at 0.0055 µg/ml, and ACI-35-2G5-Ab2 and ACI-35-2G5-Ab3 at 0.01 µg/ml in PBST/10% FCS. Sections were then washed 3 times in PBST prior to incubation with HRP-conjugated goat anti-mouse secondary antibody (purchased from Dako, Glostrup, Denmark) in PBST/10% FCS for 1 hour at room temperature. were washed 3 times with PBST and incubated in 50 mM Tris/HCl pH 7.6 for 5 min. Detection was performed by incubating the sections for 3 min in diaminobenzidine (DAB: 1 tablet in 10 ml of 50 mM Tris.HCl + 3 μl 30% H2O2; MP Biomedicals, Solon, OH, USA). The reaction was stopped by washing the sections 3 times in PBST. Sections were then transferred to silanized glass plates and air-dried on a heated plate at 50°C for 2 hours. Contrast staining was performed using Mayer's hematoxylin incubation (Fluka Chemie, Buchs, Switzerland) for 1 min, followed by a 4 min wash step. in running water. Sections were dehydrated by passing through 50%, 70%, 90% and twice in 100% ethanol bath, then twice in xylene for 1 min. Finally the sections were mounted with DePeX (BDH Chemicals Ltd., Poole, England) on glass coverslips for imaging.
[0292] Additional staining (Western-blotting) was done on SDS-PAGE-separated brain homogenized proteins (10%) from wild-type (FVB) mice, Tau transgenic mice (TPLH and biGT), or knockout mice Tau (TKO). For Western blotting, antibodies were used at the following concentrations: ACI-35-2A1-Ab1 at 0.53 μg/mL, ACI-35-2A1-Ab2 at 0.48 μg/mL, ACI-35-4A6-Ab1 at 0. 5 µg/ml, ACI-35-1D2-Ab1 at 0.47 µg/ml, ACI-35-2G5-Ab1 at 0.55 µg/ml, ACI-35-2G5-Ab2 at 0.33 µg/ml, and ACI-35-2G5-Ab3 at 0.5 μg/mL.3.2 RESULTS
[0293]ACI-35-2A1-Ab1, ACI-35-2A1-Ab2, ACI-35-1D2-Ab1, ACI-35-2G5-Ab2 and ACI-35-2G5-Ab3 antibodies demonstrated high binding activity and specificity to the phosphorylated human Tau protein (Table 2), more specifically to the phospho-Tau antigenic peptide used in the corresponding vaccine. No cross-reactivity was observed to non-phosphorylated Tau, or to other peptides derived from phosphorylated and non-phosphorylated Tau tested. Antibody ACI-35-4A6-Ab1, according to its selection, exhibited high binding activity only to the phospho-Tau antigenic peptide used for the vaccine preparation. Low cross-reactivity was found with the non-phospho counterpart of the antigenic peptide used for vaccine preparation, which was expected based on clone selection. ACI-35-2G5-Ab1 antibody exhibited high binding activity only to the phospho-Tau antigenic peptide used for vaccine preparation. Small cross-reactivity was observed for the T4,5 phospho-peptide, which comprises the part of the antigenic peptide sequence used in the vaccine.
[0294] TAUPIR and WBs were used to look to visualize tangles of Tau in the brains of mice with advanced tauopathy (biGT > 18 months) and full-length Tau in denatured homogenates derived from these mice. Different regions of the brain were analyzed: the cortex and CA1 and CA3 and part of the dentate gyrus (DG) of the hippocampus. Antibodies ACI-35-2A1-Ab1 and ACI-35-2A1-Ab2 exhibited the best TAUPIR results with dense cytoplasmic staining and clear neuropil filaments, especially in the CA1 and CA3 regions of the hippocampus. ACI-35-4A6-Ab1 antibody was negative in TAUPIR with only faint sporadic tangle-like structures lightly stained. Antibody ACI-35-1D2-Ab1 showed good staining of cytoplasmic TAUPIR with neuropil filaments in the CA1 region. Antibody ACI-35-2G5-Ab1 was negative on TAUPIR with nuclear staining and only some tangle staining. Finally, ACI-35-2G5-Ab2 and ACI-35-2G5-Ab3 antibodies exhibited good cytoplasmic TAUPIR staining similar to neuropil filaments seen in hippocampal CA1 and CA3. Staining quality classification using + or - signs is shown in Table 2. Brain homogenates from Tau transgenic mice were stained, showing that all antibodies bound well to the expected Tau strands (Table 2, classified as +), with ACI-35-1D2-Ab1 and ACI-35-2G5-Ab1 also showing additional nonspecific binding (-/+).EXAMPLE 4BINDING STUDIES II 4.1 METHODS 4.1.1 SPR BINDING ASSAY
[0295] All SPR experiments were performed on a Biacore X instrument (GE Healthcare). SA sensor chip (streptavidin-derivatized carboxymethyl dextran) was purchased from GE Healthcare. Running buffer was PBS (Dulbecco's PBS, Sigma D8537). Non-covalently bound streptavidin was first removed from the sensor surface by injecting 8 pulses (~1 μL each) of 16 mM NaOH (aq). Phospho-tau peptide was then solubilized in PBS to give a final peptide concentration of 1 μM and then injected (35 μL) onto flow cell (fc) 2 of the sensor chip at 5 μl/min. After coupling, a final immobilization level of 130 RUs was obtained. To study the binding of antibodies to the chip surface, various concentrations of antibodies were prepared by serial two-fold dilutions with the running buffer. Injections were performed along both fc 1 and 2 with a flow rate of 50 μL/min. for 120 s. Flow cell 1 was not derivatized and fc 1 responses were subtracted from fc 2 to correct for mass refraction changes and instrument noise. After each injection, surfaces were immediately washed with running buffer for 100 s. To remove any remaining bound antibody from the chip, surface regeneration was performed by injecting 1 μL of 10 mM Glycine-HCl pH 1.7. Kinetic analyzes were performed using numerical integration algorithms and global analysis using BIAevaluation 3.0. The sensorgrams obtained for injections of antibodies at different concentrations were superimposed and the references set to zero. For curve fitting, all data were fitted simultaneously to a 1:1 homogeneous model (Langmuir).
[0296] Peptides used:
4.2 RESULTS
[0297] Binding of anti-tau antibodies to phosphorylated Tau peptide was monitored in real time using SPR. Analysis of the association and dissociation phases of antibody binding could be used to determine the association rate constant (ka), the dissociation rate constant (kd) as well as the dissociation constant KD.
[0298] It was concluded that all antibodies specifically bind to the T3.30 peptide on the surface of non-derivatized carboxymethyl dextran in the range of 46^734 nM of analyzed antibody (or 11.5^184 nM for ACI-35-4A6 -Ab1). Kinetic analyzes of the sensorgrams revealed that the KD dissociation constant for the binding interaction between the different antibodies and T3.30 is between 2 and 82 nM. This demonstrates, therefore, that the antibodies recognize the T3.30 phosphopeptide with very high affinity (Table 3). EXAMPLE 5BINDING STUDIES III ELISA SAMPLES IN THE HUMAN BRAIN (ELISA FOR THE DETECTION OF PHOSPHORYLATED TAU MULTIMERS)5.1 METHODS5.1.1 HUMAN SAMPLES: PREPARATION OF HUMAN BRAIN SAMPLES USED FOR THE ASSAYS DESCRIBED HERE
[0299] Post-mortem temporal cortex for ten Alzheimer's disease (AD) controls and ten age-matched controls were obtained from the University of Miami Brain Endowment Bank. The mean age of death for AD patients (seven females and three males) was 81.1 ± 7.3 years and for controls (free of neurological symptoms; nine females, one male) was 87.0 ± 5 years. .8 (not significantly different from AD patients by Student's t test). All samples were of Caucasian origin. AD samples were characterized by Braak disease stage (Braak and Braak (1991) Neuropathological staging of Alzheimer-related changes. Acta Neuropathol 82:239-259) as shown in Table 4.
[0300] Post-mortem temporal cortex for ten AD controls and ten age-matched controls were homogenized according to the following protocol. Brain fragments 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 inhibitors (30 mM NaF, 0.2 mM of Na3VO4, 1nM okadaic acid, 1mM PMSF, 5mM Na4P2O7) and protease inhibitors (Complete Mini; Roche, Switzerland). Homogenization was done on ice using a glass potter. This constitutes the Total Homogenized fraction (TH). Protein concentrations were measured using Bradford's reagent (Sigma).5.1.2 SETUP 1 ELISA: SETUP 1 ELISA ASSAY TO DETECT THE PRESENCE OF PHOSPHORYLATED TAU MULTIMERS IN POST-MORTEM HUMAN CORTICAL BRAIN HOMOGENIZES FROM INDIVIDUALS AFFECTED WITH DA AND CONTROLS COMBINED BY AGE
[0301] 96-well multititer plates were coated with antibodies overnight at 4°C at 5 µg/ml carbonate/bicarbonate buffer. After 4 washes in PBS-Tween, the plates were saturated with PBS-Tween 10% BSA for 1 hour at 37°C. Brain homogenates were then added to the wells at a concentration of 100 ng/μL in 50 μl of PBS and incubated for 2 hours at 37°C. After washing the plates, the same antibody as used for the coating, but biotinylated, was incubated for 1 hour at 37°C, at a final concentration of 5 μg/mL. The plates were washed and, after addition of avidin-peroxidase (Vectastain ABC kit, Vector Laboratories) and its substrate (ABTS, Roche 10881420), the plates were read at different time points. Values are expressed as mean OD ±SD for 10 DA and 10 control subjects.5.2 RESULTS
[0302] Antibodies ACI-35-2A1-Ab1 and ACI-35-2G5-Ab3 were tested for their ability to detect phosphoTau (pTau) multimers in brain homogenates from AD control subjects, using a Setup 1 a-specific ELISA. phospho multimer. We observed a highly significant difference (p < 0.001) between AD and age matched controls (n = 10) in this assay for both antibodies (Figure 1). Using post-mortem human AD brain cortical homogenates and age-matched controls, we have demonstrated the ability of anti-pTau antibodies ACI-35-2A1-Ab1 and ACI-35-2G5-Ab3 to detect Tau-pS396 multimers in post-mortem human brain samples.EXAMPLE 6LIGATION STUDIES IV - WESTERN BLOTS IN HUMAN BRAIN SAMPLES.6.1 METHODS6.1.1 HUMAN SAMPLES: THE SAME METHOD FOR HUMAN SAMPLE PREPARATION AS DESCRIBED IN METHOD 5.1.1.6.1.2 WESTERN-BLOTS : WESTERN-BLOT ASSAY TO DETECT THE PRESENCE OF PHOSPHORYLATED TAU MULTIMERS IN HUMAN POST-MORTEM CORTICAL BRAIN HOMOGENIZES FROM INDIVIDUALS AFFECTED BY DA AND CONTROLS MATCHED BY AGE
[0303] Anti-human Tau antibodies used in this study were mouse ACI-35-2A1-Ab1, ACI-35-1D2-Ab1, and ACI-35-2G5-Ab3, all directed against Tau-pS396. Mouse monoclonal antibody TAU-13 (Abcam ab24636) directed against total human Tau and rabbit monoclonal antibody E178 directed against Tau-pS396 (Abcam ab32057) were used as controls. 20 µg of each total homogenate was loaded per strip onto prefabricated 10% Bis-TRIS polyacrylamide gels (Nupage Novex 10% Bis-TRIS Midi Gel, Invitrogen). Proteins were resolved as recommended by the manufacturer in NuPAGE MOPS SDS running buffer (Invitrogen NP0001). Protein blotting was done for 3 h in 25 mM TRIS pH 8.6, 190 mM glycine buffer, 20% methanol, on ice on PVDF membranes (Immobilon-FL, Millipore IPFL00010). Membranes were blocked for 1 h in Licor blocking buffer (Odyssey) diluted 1/3 in PBS. Membranes were incubated overnight with the primary antibodies at the following concentrations: TAU-13 at 0.6 μg/mL, E178 diluted 1/5000, ACI-35-2A1-Ab1 at 0.53 μg/mL, ACI-35 -1D2-Ab1 at 0.47 μg/ml and ACI-35-2G5-Ab3 at 0.5 μg/ml, diluted 1/3 in Liquor buffer and 2/3 PBS with 0.1% Tween-20 (PBS-T). After 4 washes in PBS-T, membranes were incubated with goat anti-mouse antibody together with LICOR 800 dye (IRDye 800 CW goat anti-mouse, Odyssay) for 1 hour at room temperature, washed again 4 times with PBS. -T and scanned for image reproduction using the LICOR system.6.2 RESULTS
[0304]ACI-35-2A1-Ab1, ACI-35-1D2-Ab1, and ACI-35-2G5-Ab3 antibodies were tested for their ability to detect phosphoTau (pTau) in brain homogenates from AD control subjects, using Phospho-specific western blots. All post-mortem human cortex samples were first characterized using commercial antibodies against human Tau: anti-Total Tau (TAU-13) and anti-Tau pS396 (E178) antibodies. As shown in Figure 2A, using the TAU-13 antibody, we detected in all samples the characteristic Tau scale corresponding to different isoforms of Tau in the range of 50-70 kDa. Interestingly, in AD brain homogenates a relative change in Tau migration pattern was also observed as expected for the presence of hyperphosphorylated Tau in AD brains. Confirming this hypothesis, commercial anti-pS396 Tau antibody discriminated well against controls and DA (Figure 2B). Indeed, anti-pS396 Tau antibody revealed three main immunoreactive strands corresponding to (hyper)-phosphorylated isoforms of Tau in all AD brain homogenates and with very weak or absent intensity in healthy controls. In addition, AD samples exhibited a high molecular weight TAU-13 immunoreactive stain, likely reflecting the presence of Tau aggregates (Figure 2A).
[0305] Western blotting with ACI-35-2A1-Ab1 revealed the presence of two strands of immunoreactive proteins of the expected size for phospho-Tau in AD brain homogenates but not in controls (Figure 3A). Weak immunoreactions by western blot using ACI-35-2A1-Ab1 can be explained by the presence of two large nonspecific strands at ~35 and ~40 kDa. Western blotting with ACI-35-1D2-Ab1 revealed the presence of two strands of immunoreactive proteins of the expected size for phospho-Tau in AD brain homogenates but not in controls (Figure 3B). Weak immunoreactions by western blot using ACI-35-1-D2-Ab1 can be explained by the presence of unspecific strands at ~40 and ~50 kDa, as well as 4 unspecific photos between 80 kDa and 150 kDa. Western blotting with ACI-2G5-Ab3 revealed the presence of three major immunoreactive strands corresponding to (hyper)-phosphorylated isoforms of Tau in all AD brain homogenates and absent in healthy controls, except for one control subject (C22), who has a family history of AD (Figure 3C). This report demonstrated that ACI-35-2A1-Ab1, ACI-35-1D2-Ab1, and ACI-35-2G5-Ab3, can discriminate between AD and age-matched controls for the presence of pS396 Tau in human cortex post mortem. and, therefore, these monoclonal antibodies recognize pathological Tau isoforms associated with AD. EXAMPLE 7 CONNECTION STUDIES V - CONFIGURATION 1 (ELISA IN HUMAN BRAIN SAMPLES)7.1 METHODS7.1.1 HUMAN SAMPLES. THE SAME METHOD FOR PREPARATION OF HUMAN SAMPLES AS DESCRIBED IN METHOD 5.1.1., EXCEPT FOR THE LAST PART, PREPARATION OF S1.7.1.2 TAU PROTEIN FRACTION OF S1: SUBFRACTIONATION OF TOTAL HOMOGENATE FRACTIONS TO OBTAIN SOLUBLE PROTEINS FROM PHOSPHO-TAU AND TAU.
[0306] To prepare the Tau soluble fraction (S1) used for the AlphaLISA assay, half the volume of the TH fraction was split and stored at -80°C. The remainder of the TH fraction was subsequently processed by adding Triton X-100 to a final concentration of 0.4%. The samples were mixed well and vortexed several times before being centrifuged at 5'000 rpm for 5 min at 4°C. The supernatant constitutes the S1 fraction. Samples were split and stored at -80°C. Protein concentrations were measured using Bradford's reagent.7.1.3 ALPHALISA: ALPHALISA ASSAY TO DETECT THE PRESENCE OF PHOSPHORYLATED TAU IN HUMAN POST-MORTEM CORTICAL BRAIN HOMOGENATES FROM AD-AFFECTED INDIVIDUALS AND AGE-MATCHED CONTROLS
[0307]ACI-35-2A1-Ab1, ACI-35-1D2-Ab1, and ACI-35-2G5-Ab3 antibodies, all directed against Tau-pS396, were biotinylated using the EZ-Link Micro Sulfo-NHS-LC Kit - Biotinylation (Thermo Scientific), according to the manufacturer's instructions. Twenty-five times molar excess of Biotin over antibody was used in the labeling reaction. After biotinylation, excess free biotin was removed by dialysis against PBS using Slide-A-Lyzer MINI Dialysis Devices, 10K MWCO (Thermo Scientific). Biotinylated antibodies are designated ACI-35-2A1-Ab1-BT, ACI-35-1D2-Ab1-BT, and ACI-35-2G5-Ab3-BT. Tau-13 antibody was conjugated to activated Alpha Receptor beads (Perkin Elmer) using the following protocol: 0.1 mg of Tau-13 antibody solution (protein A column purified) was mixed with 1 mg of Bead pellets. of AlphaLISA Receptor and supplemented with 0.13 M phosphate buffer (pH 8.0) for a final reaction volume of 200 μL. Then, 1.25 μL of 10% Tween-20 and 10 μL of a 25 mg/mL solution of NaBH3CN was added and the tube was incubated for 48 h at 37°C, with light rotation (7 rpm). After the conjugation reaction, the active sites in the granules were blocked by the addition of 10 μL of a Carboxy-methoxylamine solution and additionally incubated at 37°C for 1 h. Finally, the granules were washed twice with 200 μL of 0.1 M Tris-HCl pH 8.0 and stored at 4°C in 200 μL of storage buffer (PBS with 0.05% Proclin-300) which resulted in a final AlphaLISA Receptor bead concentration of 5 mg/ml.
[0308] AlphaLISA is a homogeneous assay based on bead proximity chemiluminescence. If the Donor and Receptor Alpha granules are in close proximity, upon laser excitation, a cascade of chemical reactions produces an amplified signal. After excitation at 680 nm, the photosensitizer contained in the Donor granules converts ambient oxygen into a more reactive singlet oxygen species. These singlets diffuse (up to 200 nm, in 4 μseconds of a half-life) and produce a chemiluminescent reaction in Receptor granules, leading to the emission of light. The test setup was as follows:
[0309] Samples of S1 were previously diluted in Alpha Assay Buffer (PerkinElmer AL000C) to obtain a stock concentration of 20 μg/mL. The following reagents were added to a white 384-well OptiPlate (PerkinElmer) to a final volume of 50 μL: S1 Brain Homogenate (5 μL), 10 μL ACI-35-2A1-Ab1-BT, ACI-35- 1D2-Ab1-BT, or ACI-35-2G5-Ab3-BT for a final antibody concentration of 0.2 nM, 0.5 nM, or 0.5 nM, respectively, and 10 μL Receptor Bead Conjugate Tau13 to a final bead concentration of 2.5 μg/mL. The reaction mixture was incubated for 1 h at room temperature, and 25 µL of Streptavidin Donor beads were added and further incubated for 2 h at room temperature in the dark. Reading was done using the EnSpire Alpha instrument and analysis using EnSpire Workstation version 3.00. Statistical analysis of the data was performed using the GraphPad Prism software. Results are presented as Alpha units ±SD.7.2 RESULTS
[0310] An AlphaLISA assay was used to test ACI-35-2A1-Ab1, ACI-35-1D2-Ab1, and ACI-35-2G5-Ab3 antibodies for the ability to detect Tau-pS396 in post-menopausal human brain homogenates. death and discriminate AD from age-matched controls. All antibodies detected Tau-pS396 (Figure 4A, 4B, 4C). The difference in signal detection between AD and controls (n = 10) was also highly significant for all antibodies, showing increased signal in the brains of AD subjects; ACI-35-2A1-Ab1 (p<0.0001), ACI-35-1D2-Ab1 (p<0.0001), and ACI-35-2G5-Ab3 (p=0.002). In conclusion, 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 AD subject brain and to differentiate between AD and control donors.EXAMPLE 8 IN VIVO EFFICACY OF ACI-35-2G5-AB38.1 ANTIBODY METHODS8.1.1 STUDY SETUP: EFFECTS OF IN VIVO TREATMENT OF 2 ADMINISTRATIONS OF ANTI-PTAU ACI-35-2G5-AB3 ANTIBODY IN MICE STRANGENIC MICE TAU
[0311] Female and male transgenic Tau (TMHT) mice with a C57BL/6xDBA background, at an age of 6-7 months, were administered by i.p3 injection or 10 mg/kg of ACI-35-2G5-Ab3, or twice vehicle control (PBS), one week apart. On day 14, animals were sacrificed, brains were harvested and processed for immunohistochemistry (IHC). For the determination of Tau pathology in the hippocampus and amygdala, 5 slices (1 from each level) per brain were labeled using AT180 (for Tau-pT231) and HT7 antibodies (for total human Tau) and subsequently, immunoreactive areas were evaluated. using Image Pro Plus software (v. 6.2). Immunoreactive objects were measured above a size restriction (30 μm2 in the amygdala, 7 μm2 in the hippocampus) and above a dynamic intensity threshold. Total area and intensity of objects and individual threshold were automatically archived. If used, a dynamic threshold was defined as the mean intensity within the intensity area (AOI) plus a factor times the standard deviation of pixel intensities within the AOI. The size of the region was measured by manual definition of the hippocampus and amygdala. Data from the AT180 and HT7 IR areas were normalized to region (in hippocampus) or AOI size (in amygdala). 8.2 RESULTS
[0312] pTau AT180 antibody detects endogenous and human pTau (double phosphorylated at Thr231 and Ser235). For the Tau transgenic mice used in this study, AT180 histological measurements focused on amygdaloid and hippocampal neurons. Mice treated with ACI-35-2G5-Ab3 have a significant reduction in intensity of normalized sum and mean of AT180 somal staining, in the amygdala and hippocampus (Figure 5A and 5B), showing reduction of total somal pTau positive for AT180 in treated mice.
[0313] For total (transgenic) human Tau, HT7 antibody was used. HT7 recognizes normal human Tau between residue 159 and 163. Histological measurements focused on the immunoreactive somata of amygdaloid and hippocampal neurons. Mice treated with ACI-35-2G5-Ab3 had reduced HT7 immunoreactive area as well as being the sum and mean of HT7 intensity of immunoreactivity in the amygdala (Figure 6A). In the hippocampus, the same was observed for the mean intensity (figure 6B). However, there was an increase in HT7 labeling at the immunoreactive area and soma intensity in the hippocampus of mice treated with 10 mg/kg. This increase observed in the hippocampus was mainly due to the three mice out of the total of eight investigated mice.
[0314] Treatment of ACI-35-2G5-Ab3 significantly decreased levels of immunoreactive pTau AT180 in both investigated regions, as well as in somata of amygdaloid and hippocampal neurons. In the amygdala, the intensity of the staining sum was decreased for immunoreactive pTau AT180 and immunoreactive human total Tau HT7. Treatment with a dose of 3 mg/kg also significantly decreased mean HT7 intensity in both regions. However, at 10 mg/kg the mean HT7 immunoreactive area and amount area and intensity in the hippocampus were increased above those of the control treated mouse, suggesting that an ACI-35-2G5-Ab3 treatment leads to a pathological pTau transformation. .EXAMPLE 9 EPITOPE MAPPING OF ANTI-PTAU ANTIBODIES9.1 METHODS
[0315] Epitope mapping of anti-phospho Tau mouse monoclonal antibodies was performed by ELISA using different phospho and non-phospho peptide libraries. The amino acid sequences of the T3 peptide library used are shown in Table 11A. Each library consisted of short biotinylated peptides spanning phospho and non-phospho sequences present in the peptide vaccine. In addition, a peptide library was generated by replacing each residue of an antibody-binding peptide sequence with Alanine (Ala), as shown in Tables 11B and 11C. Each library consisted of short biotinylated peptides spanning phospho and non-phospho sequences present in the peptide vaccine. Peptide libraries were purchased from ANAWA Trading SA. Peptide libraries were purchased from ANAWA Trading SA. Epitope mapping was performed according to the manufacturer's instructions (Mimotopes). Briefly, streptavidin (NUNC) coated plates were blocked with 0.1% BSA in phosphate-buffered saline (PBS) overnight at 4°C. After washing with PBS-0.05% Tween 20, plates were coated for 1 h at room temperature with the different peptides from each library, diluted in 0.1% BSA, 0.1% sodium azide in PBS to a final concentration of 10 µM. After washing, the plates were incubated for 1 h at room temperature with the antibody to be tested diluted to 40 ng/ml in 2% BSA and 0.1% sodium azide in PBS. Plates were washed again and incubated with AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, Suffolk, England) at 1/6000 dilution for 1 h at room temperature. After a final wash, the plates were incubated with p-nitrophenyl phosphate disodium phosphate hexahydrate (pNPP; Sigma-Aldrich, Buchs, Switzerland) phosphatase substrate solution and read at 405 nm after 2 h incubation using an ELISA plate reader. . Binding was considered positive if the optical density (O.D.) was at least 2 times the O.D. background.9.2 RESULTS
[0316] As a result of the epitope mapping experiments, epitopes could be identified including the required phosphorylated amino acid residue (see table 5) to which the antibodies disclosed in this document specifically bind. • Tau aa 393-401, with requirement for pS396 (ACI-35-2A1-Ab1; ACI-35-2A1-Ab2)• Tau aa 396-401, with requirement for pS396 (ACI-35-4A6-Ab1)• Tau aa 394-400, with requirement for pS396 (ACI -35-1D2-Ab1)• Tau aa 402-406, with requirement for pS404 (ACI-35-2G5-Ab1)• Tau aa 393-400, with requirement for p396 (ACI-35-2G5-Ab2; ACI-35 -2G5-Ab3)EXAMPLE 10 PHOSPHORYLATION OF TAU IN SERINE 396 (PS396) USING GSK3 KINASE B AND SDS-PAGE ANALYSIS / WESTERN-BLOT10.1 METHODS
[0317]The longest isoform of full-length human Tau (TAU441; SignalChem) at a final concentration of 16 μM (20 μg Tau/25 μL reaction) was incubated with 0.018 U GSK3β/pmol Tau in buffer phosphorylation containing HEPES with pH 7.64 (40mM), EGTA (5 mM), MgCl2 (3 mM) and ATP (2 mM) for 1, 6 or 20 h at 4, 30 or 37°C. One unit of GSK3β is defined by the manufacturer (New England BioLabs) as the amount of enzyme that will transfer 1 pmol of phosphate from ATP to CREB phosphopeptide (KRREILSRRPpSYR) in 1 minute at 30°C.Tau phosphorylated with GSK3β (pTau-GSK3β) was probed with antibodies directed against serine phosphorylated Tau 202, 396, 404, 409, threonine 181, 205 and 231 and total Tau, performed in ELISA and direct Western-blots (WBs) to optimize and verify kinase activity and specificity ( not shown). In addition, blots were analyzed for the presence of GSK3β using an anti-GSK3α/β antibody (BioSource Invitrogen). For all WBs, pTau-GSK3β was diluted by adding an equal volume of sample buffer A (125 mM Tris-HCl pH 6.8, 4% [w/v] sodium dodecyl sulfate [SDS], 20% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol), and the samples were heated at 95°C for 10 min. 30 µg of sample was loaded onto a 4-12% Bis-Tris gel (Invitrogen) and run in MOPS SDS buffer (Invitrogen). Proteins were transferred to 0.45 µm of a 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 min. The membranes were then washed and blocked for 1 hour in blocking buffer (5% BSA in TBS [50 mM Tris-HCl, pH 7.6, 150 mM NaCl]). Membranes were stained overnight at 4°C, with the primary antibodies in blocking buffer and 0.1% Tween. Blotting with ACI-35-2G5-Ab3 was done with a 0.5 μg/mL antibody dilution. 10.2 RESULTS
[0318] Tau treated with GSK3β resulted in high presence of serine 396 phosphorylation of Tau (Tau-pS396), as verified using antibodies specific for different Tau phospho-serine and -threonine residues (not shown). Figure 7 shows an SDS-PAGE for Tau-pS396 generated using different GSK3β conditions and the membrane stained using the ACI-35-2G5-Ab3 antibody. The ACI-35-2G5-Ab3 antibody, specific for Taup-pS396, demonstrated a good signal for Tau-pS396, with strands also observed suggesting that it binds to dimers of Tau-pS396 (Figure 7, lanes 11 and 13). No strands were observed in the absence of GSK3β treatment (lanes 6-8 and 14-15).EXAMPLE 11 DETECTION OF TAU PHOSPHORYLATION (PSER396) IN HUMAN cerebrospinal fluid (CSF) SAMPLES11.1 METHODS11.1.1 HUMAN SAMPLES - BRAIN SAMPLES POST-MORTEM
[0319] Post-mortem temporal cortex from a donor with Alzheimer's disease (AD) and AD19 was obtained from the University of Miami Brain Endowment Bank5. Our kind acknowledgment to the University of Miami Brain Endowment Bank for providing the samples for this study. Demographic information about the donor is shown in Table 12 below, where the stage of Braak's disease (Braak and Braak (1991) Neuropathological staging of Alzheimer-related changes. Acta Neuropathol 82: 239-259) is also indicated.

[0320] Table 12: Description of the AD19 brain sample used in this study 11.1.2 PREPARATION OF THE POST-MORTEM BRAIN S1 HOMOGENATE FRACTION
[0321] Post-mortem temporal cortex of the AD19 donor was homogenized according to the following protocol. The brain fragment was 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 inhibitors (30 mM NaF, 0.2 mM Na3VO4, 1nM okadaic acid, 1mM PMSF, 5mM Na4P2O7) and protease inhibitors (Complete Mini, Roche 04 693124 001). Homogenization was done on ice using a glass potter. This constitutes the fraction of Homogenatototal (TH). Half volume of the TH fraction was divided and stored at -80°C. The remainder of the TH fraction was subsequently processed by adding Triton X-100 to a final concentration of 0.4%. The sample was mixed well and vortexed several times before being centrifuged at 5'000 rpm for 5 min at 4°C. The supernatant constitutes the S1 fraction. The sample was split and stored at -80°C. Protein concentration was measured using Bradford's reagent (Sigma B6916-500).11.1.3 HUMAN CSF SAMPLES
[0322] Cerebrospinal fluid (CSF) samples from patients with clinically confirmed mild to moderate Alzheimer's disease (AD) and healthy control volunteer donors (Ctrls) were provided by Charité School of Medicine Berlin. Our kind acknowledgment to Charité School of Medicine Berlin for providing samples for this study. Samples were split, stored at -80°C and used without further processing. Demographic and clinical information for CSF sample donors is shown in Table 13 below.

[0323] Table 13. Demographic and Clinical Information of CSF11.1.3 TAU Sample Donors CSF11.1.3.1 ANTIBODY COUPLING IMMUNO ENRICHMENT
[0324] For immuno-enrichment of CSF Tau, a commercial human Tau antibody (HT7 clone, Thermo Scientific MN 1000) was used. In order to couple HT7 to Protein G Dynabeads (Life Technologies 10004D), for each sample 1.5 mg (50 μL) of Protein G Dynabeads were resuspended by vortex mixing and transferred to a 1.7 mL (Axygen MCT-175-LC). The tubes were placed on a magnetic rack (DynaMag, Life Technologies 123.21D) to concentrate the beads on the side of the tube and remove the buffer. Binding of 1 μg of HT7 in 200 μL of PBS with Protein G Dynabeads was performed using a Hula Mixer (Life Technologies) at 10/20 rpm, 25°/10 tilt, 5°/2 vibrator for 10 min, after which the tubes were placed on the magnet, the buffer was removed and the tubes were washed once by gently pipetting with 200 μL of PBS/0.02% Tween 20 and twice with 200 μL of conjugation buffer (20 mM of Na Phosphate, 150 mM NaCl, freshly prepared). Wash plugs were always removed using the magnet. For crosslinking HT7 with Protein G Dynabeads, HT7 beads were resuspended in 250 μL of 5mM BS3 solution (Sigma-Aldrich S5799) dissolved in conjugation buffer and incubated with rotation (same settings as above) for 30 min at room temperature. (RT), the reaction was terminated by adding 12.5 μL of quenching buffer (1M Tris-HCl pH 7.5) for 15 min, followed by three washes with 200 μL of PBS/0.02% of Tween 20.11.1.3.1 CSF TAU IMMUNO-ENRICHMENT
[0325] The CSF was used diluted and 1 mL of CSF for each donor was transferred to the tube containing the cross-linked HT7 granules and incubated for 1 hour at 4°C under continuous rotation (10 rpm). After removing unbound material on the magnet, the granules were washed with 200 μL PBS/0.02% Tween 20 and Tau was eluted in 20 μL 1% sodium dodecyl sulfate (SDS) in PBS at 70° C for 10 min. In order to prevent the granules from settling, the tubes were immediately mixed (300 rpm in the heated horizontal mixer for 5 seconds every minute). After this incubation, the eluted samples were collected by placing the tubes on the magnet.
[0326] As a positive control, Tau was also enriched for human brain homogenates. For these serial dilutions of human brain fraction from donor S1 AD19 were prepared in PBS (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 eluted in 25 μL 1% SDS.11.1.3ALPHALISA.11.1.3.1 ALPHALISA ASSAY DESCRIPTION
[0327]AlphaLISA is a homogenous assay using Alpha bead based technology. AlphaLISA was selected as a technology platform based on sensitivity and minimum number of steps. Briefly, the test is based on the proximity of the bead. After excitation at 680 nm, the photosensitizer containing Donor granules converts ambient oxygen into singlet oxygen species, this diffusion function (up to 200 nm, within 4 μsec of a half-life) and produces a chemiluminescent reaction in the Receptor granules, leading to the emission of light.
[0328]The assay setup used in our experiments was as follows (see also Figure 8):• Pan-Tau Tau-13 antibody (Abcam ab24636), coupled with Alpha Receptor granules binds human Tau present in the sample and forms the complex " Receptor protein Tau beads-Tau13 antibody • Detection antibody ACI-35-2G5-Ab3-BT binds human Tau apS396 and allows binding of the pre-Streptavidin (SAv) coated Alpha Donor beads to the complex.
[0329] After bringing all the reagents into the reaction, the chemiluminescent signal is read using EnSpire Alpha reader 2390.11.1.5.2 BIOTHINYLATION OF ANTIBODY ACI-35-2G5-AB3To be used in the AlphaLISA assay, the antibody ACI-35-2G5 -Ab3 was biotinylated using the EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit (Thermo Scientific 21935), according to the manufacturer's instructions. Twenty-five times molar excess of Biotin over antibody was used in the labeling reaction. After biotinylation, excess free biotin was removed by washing the antibody four times in PBS using 50'000 MWCO Spin-X UF 500 Concentrator (Corning 431480). The biotinylated ACI-35-2G5-Ab3 antibody is indicated as ACI-35-2G5-Ab3-BT.11.1.5.3 COUPLING OF TAU-13 ANTIBODY TO RECEPTORALPHALISA GRANULES.
[0330]In order to be used in the AlphaLISA assay, the Tau-13 antibody was conjugated to activated Alpha Receptor beads (Perkin Elmer 6772001). The following conjugation protocol was used: 0.1 mg of Tau-13 antibody solution (purified on protein A column) was mixed with 1 mg of AlphaLISA Receptor Bead pellet and supplemented with 0.13 M phosphate buffer (pH 8.0) for a final reaction volume of 200 μL. Then, 1.25 μL of 10% Tween-20 and 10 μL of a 25 mg/mL solution of NaBH3CN were added and the tube was incubated for 48 h at 37°C, with light rotation (7 rpm). After the conjugation reaction, the active sites in the granules were blocked by the addition of 10 μL of a Carboxy-methoxylamine solution and additionally incubated at 37°C for 1 h. Finally, the granules were washed twice with 200 μL of 0.1 M Tris-HCl pH 8.0 and stored at 4°C in 200 μL of storage buffer (PBS with 0.05% Proclin-300) which resulted in a final AlphaLISA Receptor granule concentration of 5 mg/ml.11.1.5.3 DETECTION DETERMINATION LIMIT USING BRAINPS396-TAU
[0331] Tau immuno-enriched brain samples, S1 brain fraction samples and buffer blanks were used for this experiment. Each sample was analyzed in 50 μL final volume using a white 384-well OptiPlate (PerkinElmer 6007291). Dilutions of all reagents were made with Alpha Assay Buffer (PerkinElmer AL000C).• 5 μL of the sample (1/10 of the final volume, therefore, the final protein concentration in the assay corresponds to 1/10 of the sample concentration).• 10 μL of 0.5% SDS for S1 brain fraction samples or 10 μL of plain buffer for immuno-enriched Tau brain samples were added• 15 μL of ACI-35-2G5-Ab3-BT antibody (final concentration : 5 nM) mixed with Tau13 Receptor bead conjugate (final bead concentration: 2.5 μg/mL)• Incubation at room temperature for 1 hour • 20 μL of Streptavidin Donor beads (final bead concentration: 25 μg /mL)• Incubation at room temperature for 30 min (protected from light)• Reading using EnSpire Alpha instrument and analysis using EnSpire Workstation version 3.00.11.1.5.3 DETERMINATION OF IMMUNO-ENRICHED PS396-TAU IN CSF
[0332] Each sample was analyzed in a final volume of 50 μL using a white 384-well OptiPlate (PerkinElmer 6007291). Dilutions of all reagents were made with Alpha Assay buffer (PerkinElmer AL000C).• 5 μL of immunoprecipitated eluate from each donor • 20 μL of ACI-35-2G5-Ab3-BT antibody (final concentration: 5 nM) mixed with Tau13 Receptor beads (final bead concentration: 2.5 μg/mL)• Incubation at room temperature for 1 hour • 25 μL of Streptavidin Donor beads (final bead concentration: 25 μg/mL)• Incubation at room temperature for 30 min (protected from light)• Reading using EnSpire Alpha instrument and analysis using EnSpire Workstation version 3.00.11.1.6 STATISTICAL ANALYSIS
[0333] Statistical analysis of the data was performed using the GraphPad Prism software.11.1 RESULTS
[0334]Preliminary experiments indicated that the amount of pS396 present in human CSF was too low for detection. For this reason, an immunoenrichment protocol coupled with high sensitivity immunodetection was developed. The immunoenrichment protocol was first validated using post-mortem AD human brain material. Side-by-side comparison of homogenized untreated brain samples with immuno-enriched Tau samples revealed that, at corresponding concentrations, the AlphaLISA assay limit of detection of Tau13/ACI-35-2G5-Ab3 was reached at 0.5 μg/ mL for untreated samples and between 0.0020.006 μg/mL for immuno-enriched samples, indicating a 100-fold enrichment (Figure 9).
[0335] Next, the immunoenrichment protocol was applied to the live donor CSF samples (n=17 for patients with mild to moderate AD and n=16 for age-matched healthy volunteers). The data obtained (Figure 10) demonstrate that: a) following the Tau13/ACI-35-2G5-Ab3 immunoenrichment protocol, AlphaLISA detected pS396-Tau in all human CSF samples; and b) more importantly, a significant increase in the amount of pS396-Tau in CSF with AD was observed when compared to control (p = 0.0003, Mann-Whitney test).
[0336] In conclusion, an immuno-enrichment/immuno-detection protocol was developed, allowing detection of pS396-Tau in human CSF. Increase of pS396-Tau in the CSF of mild to moderate AD suggests that this method can be successfully used in clinical biomarker studies to assess disease progression, patient stratification and therapy efficacy. The ACI-35-2G5-Ab3 antibody detected pS396-Tau in all human CSF samples, and more importantly, the antibody was able to discriminate CSF with DA when compared to the control. TABLE 1
[0337] Description of Tau sequence, vaccine and antibodies
* Based on the longest isoform of human Tau (Tau441). p indicates phosphorylated residue.TABLE 2
[0338] Hybridoma scan for target binding


[0339] Binding intensity can be compared only within the same column, within the same assay (ELISA, or TAUPIR or WB).
[0340] - The connection is not good or there is no connection; + Good connection; ++ Very good connection; +++ Excellent binding (better than very good binding)TABLE 3
[0341] Anti-tau antibody binding affinity

[0342] a Analysis performed with a Phospho-peptide purity of 64% by HPLC.
[0343] b Analyzes performed with a Phospho-peptide purity of 87% by HPLC.TABLE 4
[0344] Description of subjects with AD used for this study.
TABLE 5
[0345] Tau amino acids and phospho-residues required for antibody binding.

[0346] *Based on the longest isoform of human Tau (Tau441)TABLE 6
[0347] Amino Acid Sequence of Heavy Chain and Light Chain Variable Regions (VH and VK) and CDRs








TABLE 7
[0348] Nucleotide Sequence of Heavy Chain and Light Chain Variable Regions (VH and VK)








[0349] Primers used for CDR sequencing of















[0350] Degenerate Codons: R = A or G S= C or G D = A or G or T B = C
[0351] or G or T
[0352] Y = C or T M = A or C H= A or C or T
[0353] K = G or T W = A or T V = A or G or CTABELA 9
[0354] Longest isoform of human Tau (441aa), also called Tau40
DEPOSITTABLE 10
[0355] The following hybridoma cell lines were deposited in the name of AC Immune SA, PSE-EPFL Building B, 1015 Lausanne/Switzerland and KatholiekeUniversiteit Leuven, Waaistraat 6 - Box 5105, 3000 Leuven/Belgium, with the “Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) at Braunschweig, Inhoffenstrasse 7 B, 38124 Braunschweig, in accordance with the provisions of the Treaty of Budapest:

TABLE 11A
[0356] Peptide library used for epitope mapping
TABLE 11B
[0357] Library of alanine replacement peptides (Ala) used for epitope mapping of pS396-specific antibodies
TABLE 11C
[0358] Alanine replacement peptide (Ala) library used for epitope mapping of pS404-specific antibodies
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权利要求:
Claims (17)
[0001]
1. ANTIBODY, or antigen-binding fragment thereof, which recognizes and specifically binds to a phospho-epitope on mammalian Tau protein or to a phospho-epitope on a fragment of mammalian Tau protein, characterized by said antibody or fragment of antibody having a binding affinity for phosphorylated, soluble, oligomeric and insoluble Tau protein with a dissociation constant ranging from at least 2 nM to 80 nM and wherein said antibody or fragment thereof comprises a light chain variable region, comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 106, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 107, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 108, and a heavy chain variable region, comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 89, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 115, i a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 91.
[0002]
2. ANTIBODY, according to claim 1, or an antigen-binding fragment thereof, characterized in that said antibody or antibody fragment: a) modulates soluble and insoluble Tau levels in the cerebral cortex and/or hippocampus; b) reduces total levels of soluble tau protein and/or soluble phosphorylated tau protein; and/orc) reduce levels of hyperphosphorylated tau protein containing paired helical filaments.
[0003]
3. ANTIBODY according to any one of claims 1 to 2, or an antigen-binding fragment thereof, characterized in that said antibody or antibody fragment has a dissociation constant of at least 10 nM.
[0004]
4. ANTIBODY according to any one of claims 1 to 3, or an antigen-binding fragment thereof, characterized in that it is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody or a binding fragment of the antigen of the same.
[0005]
5. ANTIBODY, according to any one of claims 1 to 4, or an antigen-binding fragment thereof, characterized by being of the IgG2b, IgG2a or IgG3 isotype.
[0006]
6. POLYNUCLEOTIDE, characterized in that it encodes the antibody as defined in any one of claims 1 to 5, or an antigen-binding fragment thereof, wherein the polynucleotide comprises a nucleic acid molecule comprising the sequence shown in SEQ ID NOs: 113 or 114.
[0007]
7. A PHARMACEUTICAL COMPOSITION, comprising an antibody or a functional fragment thereof, as defined in any one of claims 1 to 5, or a combination thereof, in a therapeutically effective amount together with a pharmaceutically acceptable carrier.
[0008]
8. USE OF AN ANTIBODY, or an antigen-binding fragment thereof, as defined in any one of claims 1 to 5, of a polynucleotide, as defined in claim 6, or of a pharmaceutical composition, as defined in claim 7, or a combination thereof, for the manufacture of a medicament for the treatment of a neurodegenerative disease or disorder, such as a tauopathy in a mammal, particularly a human in need of such treatment.
[0009]
9. USE OF AN ANTIBODY, or an antigen-binding fragment thereof, as defined in any one of claims 1 to 5, of a polynucleotide, as defined in claim 6, or of a pharmaceutical composition, as defined in claim 7, characterized for being for the manufacture of a medicament for treating or alleviating cognitive deficits in a mammal, particularly a human suffering from such a deficit, and optionally: (i) wherein the treatment or alleviation of cognitive deficits in a mammal, especially a human, leads to to a containment in the progression of cognitive deficits, or (ii) where treatment or alleviation of cognitive deficits in a mammal, particularly a human, leads to an increase in retention, particularly a complete restoration of cognitive memory capacity in the treated subject .
[0010]
10. USE OF AN ANTIBODY, or an antigen-binding fragment thereof, as defined in any one of claims 1 to 5, of a polynucleotide, as defined in claim 6, or of a pharmaceutical composition, as defined in claim 7, characterized for being for the manufacture of a medicament for treating diseases and disorders that are caused by or are associated with the formation of neurofibrillary lesions, the brain pathology predominant in tauopathy comprising a heterogeneous group of neurodegenerative diseases or disorders, including diseases or disorders that show the presence of tau and amyloid pathologies, including, but not limited to, Alzheimer's disease, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, and cerebral amyloid angiopathy prion protein, traumatic brain injury, and other diseases or disorders that do not show an amyloid pathology distinct oid including, but not limited to, amyotrophic lateral sclerosis/guam dementia-parkinsonism complex, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with linked parkinsonism to chromosome 17, frontotemporal dementia, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick type C disease, Pallido-ponto-nigral degeneration, Pick disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, dementia tangles only, post-encephalitic parkinsonism, and myotonic dystrophy.
[0011]
11. IN VITRO METHOD, for the diagnosis of a disease, disorder or condition associated with tau protein or a predisposition to a disease, disorder or condition associated with tau protein in a patient, characterized by comprising the detection of immunospecific binding of an antibody or of an active fragment thereof to a tau protein epitope in a sample, comprising the steps of: a) bringing the sample or a specific body part or area of the body suspected of containing the tau antigen in contact with an antibody or a fragment thereof, as defined in any one of claims 1 to 5, which antibody or fragment binds to an epitope of the tau protein; b) allowing the antibody to bind to the tau antigen to form an immune complex; c) detecting the formation of the immune complex; and d) correlate the presence or absence of the immune complex with the presence or absence of tau antigen in the sample or specific part or area of the body, wherein an increase in the amount of said aggregate, compared to a normal control value, indicates that said patient is suffering from or is at risk of developing a disease or condition associated with tau protein.
[0012]
12. POST-MORTEM DETECTION METHOD OF PHOSPHO-TAU MULTIMERS (pTau), in a sample of a subject suspected of suffering from a disease or disorder associated with tau, characterized by comprising: a) placing a sample of the subject in contact with an antibody or a fragment thereof as defined in any one of claims 1 to 5, which antibody or fragment binds to an epitope of the phospho-Tau protein; b) allowing the antibody to bind to the tau protein to form an immune complex; c) detect the formation of the immune complex; and d) comparing the amount or intensity of the immune complex in the sample obtained from the subject to the amount or intensity of the immune complex obtained from a healthy control subject, using the same conditions, where an increase in the amount or intensity of said immune complex, compared to the control value, indicates that said patient suffered from a disease or disorder associated with tau.
[0013]
13. METHOD OF DETECTION OF THE FORMATION OF AN IMMUNE COMPLEX, associated with a disease, disorder or condition associated with tau protein, or predisposition to a disease, disorder or condition associated with tau protein in a patient, characterized by comprising the detection of immunospecific binding of a tau protein. antibody or an active fragment thereof to a tau protein epitope in a sample or in situ, comprising the steps of: a) placing the sample or a specific part of the body or area of the body suspected of containing the tau antigen contacting an antibody or a fragment thereof as defined in one of claims 1 to 5, which antibody or fragment binds to an epitope of the tau protein; b) allowing the antibody to bind the tau antigen to form an immune complex; c) detect the formation of the immune complex; and d) correlate the presence or absence of the immune complex with the presence or absence of tau antigen in the sample or specific part or area of the body.
[0014]
14. METHOD according to any one of claims 11 to 13, characterized in that the sample is a cerebrospinal fluid sample or a brain sample.
[0015]
15. TEST KITS, for the detection and diagnosis of diseases, disorders or conditions associated with tau protein, characterized in that they comprise a container containing one or more antibodies, or antigen-binding fragments thereof, as defined in any one of claims 1 to 5 , and instructions for using the antibodies for the purpose of binding to tau antigen to form an immune complex and to detect immune complex formation, such that the presence or absence of the immune complex correlates with the presence or absence of the tau antigen.
[0016]
16. ISOLATED CELL LINE that produces an antibody, as defined in any one of claims 1 to 5, characterized in that it is the A6-2G5-30 hybridoma cell line, deposited on August 30, 2011 as DSM ACC3137 or the cell line A6-2G5-30 from hybridoma A6-2G5-41, deposited August 30, 2011 as DSM ACC3138.
[0017]
17. METHOD OF DETECTION OF DEFOPHOSPH-TAU MULTIMERS (pTau), in a brain sample from a subject suspected of suffering from a disease or disorder associated with tau protein, characterized by comprising: a) placing the sample in contact with an antibody or a fragment thereof as defined in one of claims 1 to 5, which antibody or fragment binds to an epitope of the phospho-Tau protein; b) allowing the antibody or fragment to bind to tau protein to form an immune complex; and c) detect the formation of the immune complex.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-16| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-10-06| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-06-08| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-07-20| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-08-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-11-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-04| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 05/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |

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申请号 | 申请日 | 专利标题

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