Antibodies that bind a conformation-specific epitope of TAU protein

Abstract

The invention concerns isolated, monoclonal anti-Tau antibodies and antibody fragments that recognize a phosphorylated amino acid stretch in R3 that overlaps with the hexapeptide PHF6, in particular antibodies and antibody fragments that bind to a conformation-specific epitope in Tau comprising said phosphorylated amino acid stretch and an amino acid stretch in the R1 repeat, for use in the prevention or treatment of a Tauopathy in a subject, as well as use of these antibodies and antibody fragments for in vitro or ex vivo diagnosis of a Tauopathy. The invention further provides nucleic acids encoding these antibodies and antibody fragments, vectors comprising said nucleic acids, and (pharmaceutical) compositions comprising said antibody and antibody fragments, nucleic acids or vectors, as well as their use in the uses and methods of the invention.

Description

[0001] ANTIBODIES THAT BIND A CONFORMATION-SPECIFIC EPITOPE OF TAU PROTEIN

[0002] FIELD OF THE INVENTION

[0003] The present invention relates broadly to the field of medicine, more particularly to the field of Tan-related disorders and diseases or Tauopathies. In particular, the invention provides monoclonal antibodies and antibody fragments targeting a conformation-specific epitope of Tau, nucleic acids encoding said antibodies and antibody fragments, and vectors comprising said nucleic acids, for use in the prevention or treatment and diagnosis of a Tauopathy in a subject.

[0004] BACKGROUND OF THE INVENTION

[0005] The increase in aging of the population comes with an enormous socio-economic challenge related to the increasing incidence of age-related neurodegenerative disorders such as Alzheimer’s disease (AD). Predictions indicate that more than 70 million people will suffer from neurodegenerative diseases by 2030 and surpass 130 million by 2050 since no efficacious therapies are currently available.

[0006] Hallmark lesions of AD and other neurodegenerative diseases in the brain often involve an accumulation of protein aggregates. Tau pathologies or Tauopathies, including AD, Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Argyrophilic grain disease (AGD), Pick’s disease (PiD) and primary age-related tauopathy (PART), are associated with misfolding and aggregation of Tau. The abundance of Tau inclusions in the brain correlates well with disease severity. Tau is a microtubule-associated protein required to stabilize and separate the microtubules in axons that serve as tracks for intracellular transport. The protein contains a N-terminal projection domain and a C-terminal microtubule binding domain (MTBD) of which the boundary is formed by the proline-rich segments Pl and P2. Up to six Tau isoforms can be found in the human brain that are generated by alternative splicing of the MAPT gene transcript. The different isoforms differ in the number of aminoterminal inserts (indicated as N1 and N2) in the projection domain and in the number of repeats (indicated as R1 tot R4) in the MTBD (Fig. 1). In case of neurodegeneration, mutations in the MAPT gene, changes in metabolism and alterations in posttranslational modifications, such as hyperphosphorylation, affect the conformation of Tau, thereby triggering its oligomerization and assembly into paired helical filaments (PHF) and neurofibrillary tangles (NFT). Upon assembly, Tau is no longer able to stabilize the microtubules, which results in disturbed intracellular transport and eventually in cell death. Three aggregation-prone regions (APRs), i.e. PHF6* in R2, PHF6 in R3 and PAM4 in R4, were shown to drive the self-assembly of Tau by enhancing the formation of betastructure. These beta-sheet enriched pre-fibrillar Tau species can induce a particular pathological structure to its physiological counterpart, a necessary process for the pathology development and progression of Tauopathy, known as seeding. It is hypothesized that the unique structural compositionand maturation status of Tau aggregates are the driving forces of pathology initiation. However, the initial sequence of structural events that triggers the pathological shift of the Tau protein and the generation of different end-state structures, is poorly understood.

[0007] Several approaches have been proposed for therapeutically interfering with progression of Tau pathology and preventing the subsequent molecular and cellular consequences. Given that NFT are composed of hyperphosphorylated, misfolded and aggregated forms of Tau, interference at each of these stages provides targets that can be pursued. Introducing agents that limit phosphorylation, block misfolding or prevent aggregation appear promising strategies. For example, passive immunization with anti-phospho-Tau antibodies in mouse models have led to dramatic decreases in Tau aggregation and improvements in cognitive parameters.

[0008] However, the gap of knowledge on early pathological Tau structures and the late-stage diagnosis are some of the main reasons for the low efficacy of current Tau-targeting therapies. Indeed, at present we are lacking diagnostic tests to recognize and distinguish Tauopathy patients at early disease stage, prior to the extensive occurrence of neuronal damage.

[0009] Moreover, recent studies also reported the accumulation of phosphorylated Tau and pathological Tau conformers in the retina. In this so-called primary retinal tauopathy (PReT) four stages (0-3) can be distinguished depending on the distribution of phosphorylated Tau throughout the retinal layers and, interestingly, these stages slightly correlated with the presence of ocular inflammation, the neuropathological diagnosis of AD and primary Tauopathies, as well as with age. However, albeit phosphorylated Tau accumulates in a cytoplasmic and synaptic pattern, no fibrillar Tau structures have been observed in PReT and a more detailed biochemical analyses confirmed the molecular Tau composition of PReT to be different from that found in AD and other brain.

[0010] WO 2015 / 035190 Al discloses the “MCI” antibody, which binds to a discontinuous epitope of Tau which contains both a sequence in the N-terminus and a sequence in the R3 repeat. WO 2014 / 008404 Al discloses different anti-Tau monoclonal antibodies. These antibodies were shown to block Tau aggregation directly or indirectly.

[0011] However, there remains a need for agents capable of detecting and interfering with early pathological conformational changes of Tau for the diagnosis and treatment of Tauopathies at an earlier stage. There is also a need for agents capable of detecting Tau conformers in non-cerebral Tauopathies.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention addresses these needs in the art with antibodies and antibody fragments and related nucleic acids, vectors, compositions and kits, and uses and methods provided herein.In particular, provided herein are isolated, e.g., recombinant, monoclonal antibodies and antibody fragments which bind to a conformation-specific epitope in Tan (e.g., human Tau). In particular, antibodies and antibody fragments are disclosed herein that bind to a phosphorylated amino acid stretch in R3 that overlaps with the hexapeptide PHF6, and to an amino acid stretch in the R1 repeat.

[0014] As shown in the experimental section, it was found that such molecules have the capacity to prevent the formation and propagation of Tau seeds, rendering these molecules particularly suitable for the prevention or treatment of a Tauopathy in a subject. Also advantageously, these antibodies and antibody fragments showed intracellular safety. Moreover, these antibodies and antibody fragments demonstrated high sensitivity in binding to early Tau conformers and structures in Alzheimer’s disease (AD) brain tissue, to Tau aggregates in brain tissue of other Tauopathies, including Corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), primary age-related tauopathy (PART), and Pick’s disease (PiD), as well as to Tau conformers in primary retinal tauopathy (PReT). Antibodies and antibody fragments provided herein capable of binding to a discontinuous epitope between the R1 and R3 repeats (e.g. the monoclonal antibody 16B12) provide for improved Tau biomarkers and allow for improved therapeutic interventions at an earlier pathological stage. Indeed, the present inventors identified an interaction of R1 repeat region with the APR PHF6 of the R3 region of Tau protein as an early and crucial event for maturation of Tau pathology and fibril formation. They found incorporation of the R1 region in the fibrillar Tau core as a primary event in Tau maturation post phosphorylation, critical for fibril maturation and seeding competence.

[0015] Accordingly, a first aspect of the invention provides an isolated monoclonal antibody, or an epitopebinding fragment of an antibody, capable of specifically binding to Tau, wherein the antibody or antibody fragments is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO: 2 having phosphorylation at Ser316, Ser320 and Ser324 and optionally the amino acid sequence set forth in SEQ ID NO: 3, a nucleic acid encoding the antibody or the antibody fragment, or a vector comprising said nucleic acid, for use in the prevention or treatment of a Tauopathy in a subject. In particular embodiments, the antibody is capable of specifically binding to both the amino acid sequence set forth in SEQ ID NO: 2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO: 3.

[0016] In particular embodiments, the invention provides an isolated monoclonal antibody, or an epitopebinding fragment of an antibody, capable of specifically binding to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324, a nucleic acid encoding the antibody or the antibody fragment, or a vector comprising said nucleic acid, for use in the prevention or treatment of a Tauopathy in a subject.

[0017] In particular embodiments, the antibody or antibody fragment is capable of specifically binding to a conformational Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:31(VYKPVDLSKVTSKCGSLG) and having phosphorylation at one or more of Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. Indeed, it is expected that the epitope need not be phosphorylated at all three positions, for the antibody or antibody fragment to ensure the technical effect.

[0018] In preferred embodiments, the antibody or antibody fragment is capable of specifically binding to a conformational Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In particular embodiments, the epitope is determined by ELISA-based peptide array scanning.

[0019] In embodiments, the antibody or antibody fragment is a humanized antibody, an affinity matured antibody, or a combination thereof.

[0020] In particular embodiments the antibody or antibody fragment is an antibody or antibody fragment comprising a light chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:4, a CDR2 with the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:7, a CDR2 with the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:9.

[0021] In particular embodiments, the antibody or antibody fragment is an antibody or antibody fragment comprising a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence which has at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence which has at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11, or a humanized variant thereof.

[0022] In particular embodiments, the antibody fragment is an scFv fragment such as an scFv fragment comprising the amino acid sequence set forth in SEQ ID NO: 14, or a humanized variant thereof. In particular embodiments, the antibody or antibody fragment is an intrabody.

[0023] In embodiments, the Tauopathy is selected from the group consisting of: Alzheimer’s disease (AD), Pick’s disease (PiD), Corticobasal Degeneration (CBD), Chronic Traumatic Encephalopathy (CTE), Progressive Supranuclear Palsy (PSP), primary age-related tauopathy (PART), Argyrophilic Grain Disease (AGD), and primary retinal tauopathy (PReT).

[0024] Another aspect is directed to a method for in vitro or ex vivo diagnosis of Braak stage I or II Alzheimer’s disease or primary age-related tauopathy (PART), or primary retinal tauopathy (PReT) stage 0 or 1 in a subject, comprising the following steps:

[0025] contacting an isolated monoclonal antibody, or an epitope-binding fragment of an antibody, capable of specifically binding to Tau, wherein the antibody or antibody fragment is capable ofspecifically binding to the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 with a biological sample of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and

[0026] - detecting the formation of said complex.

[0027] In particular embodiments, the method comprises:

[0028] - contacting an isolated monoclonal antibody, or an epitope-binding fragment of an antibody, capable of specifically binding to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 with a biological sample of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and

[0029] - detecting the formation of said complex.

[0030] In embodiments, the antibody or antibody fragment is capable of specifically binding to a conformational Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:31 and having phosphorylation at one or more of Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In embodiments, the antibody or antibody fragment is capable of specifically binding to a conformational Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 as determined by ELISA-based peptide array scanning.

[0031] Yet another aspect is directed to a method for in vitro or ex vivo diagnosis of a non-cerebral Tauopathy, particularly a retinal Tauopathy such as primary retinal tauopathy (PReT), in a subject, comprising the following steps:

[0032] - contacting an isolated monoclonal antibody, or an epitope-binding fragment of an antibody, capable of specifically binding to Tau, wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 with a biological sample, in particular a non-cerebral biological sample such as retinal sample, of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and

[0033] - detecting the formation of said complex.

[0034] In particular embodiments, the method comprises:

[0035] - contacting an isolated monoclonal antibody, or an epitope-binding fragment of an antibody, capable of specifically binding to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence setforth in SEQ ID NO:3 with the biological sample of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and

[0036] - detecting the formation of said complex.

[0037] In embodiments, the antibody or antibody fragment is capable of specifically binding to a conformational Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:31 and having phosphorylation at one or more of Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In embodiments, the antibody or antibody fragment is capable of specifically binding to a conformational Tau epitope comprising the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 as determined by ELISA-based peptide array scanning.

[0038] A further aspect is directed to an isolated monoclonal antibody or an epitope-binding fragment of an antibody, comprising a light chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:4, a CDR2 with the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:7, a CDR2 with the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:9.

[0039] In particular embodiments, the antibody or antibody fragment comprises a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 11, or a humanized variant thereof.

[0040] In particular embodiments, the antibody or antibody fragment comprises a light chain with the amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain with the amino acid sequence set forth in SEQ ID NO: 13, or a humanized variant thereof.

[0041] In particular embodiments, the antibody fragment is an scFv fragment such as an scFv fragment comprising the amino acid sequence set forth in SEQ ID NO: 14, or a humanized variant thereof. Further aspects are directed to:

[0042] - an isolated nucleic acid which encodes the aforementioned antibody or antibody fragment;

[0043] - a vector comprising the aforementioned nucleic acid;

[0044] - a host cell comprising the aforementioned nucleic acid or vector; and

[0045] - a pharmaceutical composition comprising the aforementioned antibody or antibody fragment, nucleic acid or vector; and further comprising a pharmaceutically acceptable carrier.

[0046] A further aspect is directed to the aforementioned antibody or antibody fragment, nucleic acid, vector or pharmaceutical composition for use in medicine.Particular embodiments are directed to the aforementioned antibody or antibody fragment, nucleic acid, vector or pharmaceutical composition for use in the prevention or treatment of a Tauopathy in a subject. Yet a further aspect is directed to a method for in vitro or ex vivo diagnosis of a Tauopathy in a subject, comprising the following steps:

[0047] - contacting the aforementioned antibody or antibody fragment with a biological sample of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and

[0048] - detecting the formation of said complex.

[0049] The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby specifically incorporated in this specification.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1. A) Graphical representation of the longest Tau2N4R isoform with the N-terminal inserts N1 and N2, the proline-rich segments Pl and P2 and the microtubule binding repeats R1 to R4 including the previously validated aggregation-prone regions PHF6* in R2 and PHF6 in R3. The epitope sequences for the monoclonal antibodies ADx215 (SEQ ID NO: 17), 15A10 (SEQ ID NO: 19), 16B12 (SEQ ID NO:2 and 3), 20G10 (SEQ ID NO:21), 18F12 (SEQ ID NO: 18), ADx201 / 202 (SEQ ID NO:20), 11E12E10 (SEQ ID NO:22) and 9H6F2 (SEQ ID NO:23, 24 and 25) are shown. Full lines represent the core epitopes while dashed lines refer to the phospho-peptides recognized by 9H6F2 and 16B12 for which the sequences are shown in italic. B) Results of the peptide array scans determining the discontinuous epitope of 16B12 with strong affinity for peptides containing the R1 sequence 249PMPDLKN255 (SEQ ID NO:3) independent of neighbouring phosphorylation sites as well as moderate affinity for the R3-localized phosphopeptide 309VYKPVDLpSKVTpSKCGpSLG326 (SEQ ID NO:2). 16B12 had no affinity for any other peptide. C) Comparative ELISA with the monoclonal antibodies when used to capture or detect protein Tau in various combinations. Protein Tau was obtained from three different sources: recombinant human Tau purified from E. coli (bTau, nonphosphorylated, left panel), recombinant human Tau purified from yeast (yTau, phosphorylated, middle panel), and E. coli purified and heparin-induced fibrillar Tau (fTau, right panel). For each, a gradient scale based on the percentile range from 5 to 95 is shown. C) Determination of the monoclonal antibody stability by monitoring the Tagg and Tm.

[0052] Figure 2. Validation of the conformational nature of the 16B12 epitope. A) Pre-incubation of the antibodies and a peptide containing its R1 epitope (R1 peptide) significantly reduced, but did not abrogate, 16B12 staining on AD brain sections (case 15, Table 3), while MCI and AT8 staining were unaffected and clearly stained pretangles and neurofibrillary tangles. In contrast, pre-treatment of thesections with Proteinase K (PK treatment) reduced staining by 16B12 and MCI butnot AT8, confirming that, similar as MCI, the 16B12 antibody detects a conformation-dependent Tan structure. Pretangle s / neurofibrillary tangles (NFTs) are indicated with arrows. B) Competitive IHC was performed on human AD brain (H area-case 15, Table 3) tissue. The tissue was first stained with MCI antibody, then with either biotinylated 16B 12 (16B12-bt) or biotinylated ADx201 (ADx201-bt). Singlestained sections with 16B12-bt and ADX201-bt served as positive controls. Pretangles / neurofibrillary tangles (NFTs) are indicated with arrows and neuritic plaques (NP) with circles.

[0053] Figure 3. Immunohistochemical detection of the Tau maturation states with the monoclonal antibody 16B12 as well as phosphorylation- (AT8, pS203 / T205) and conformation-specific (MCI) antibodies in different Braak stages (TMAs). Pretangle s / NFTs are marked with dense arrows and neuropil threads (NT) with dashed arrows.

[0054] Figure 4. Diagnostic performance of anti-Tau monoclonal antibodies and isotype controls in Alzheimer’s disease. A) Immunohistochemical detection of Tau NT (dashed arrow), NP (circles) and pretangles / NFTs (dense arrow) in the hippocampus (H) of an AD case (15). B) Representative images of tissue microarray cores indicating the grading (0-4) for Tau pathology evaluation using AT8.

[0055] Figure 5. Diagnostic comparison of the monoclonal antibodies 16B12 and MCI in different Tauopathies (TMAs). Representative images of the Immunohistochemical detection of the different Tau pathological lesions (PT = Pretangles; OCB = Oligodendroglial coiled bodies; PB = Pick bodies; AP = Astrocytic plaques; TPA = Tau-positive astrocytes) in different Tauopathies, including Argyrophilic grain disease (AGD), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), and Pick’s disease (PiD).

[0056] Figure 6. Detection of Tau 16B12 conformation in primary retinal tauopathy (PReT). In the retina of a PReT stage 0 case (case 37) with negative MCI staining (MCI “-”), the monoclonal antibody 16B12 detected its Tau conformers in the photoreceptor cell layer (PRL) (dashed arrows). In the retina of case 38, 16B 12-positive material was seen in the outer (OPL) and inner plexiform (IPL), the inner nuclear layer (INL) and the photoreceptor cell layer (PRL). This case was earlier staged as PReT stage 1 and was also MCl-negative (MCI “-”). In PReT stage 3 cases (cases 39, 40 and 41), which all exhibit the MCI conformation (MCI “+”), the 16B12 Tau conformation was abundantly detected in all the layers (lower panel; dense and dashed arrows). In the Alzheimer’s disease case 41 even retinal ganglion cells (dense arrows) exhibited the 16B12 epitope, which was less obvious in non-demented PReT-stage 3 cases (cases 38 and 40). PRL=photoreceptor layer, ONL = outer nuclear layer; OPL=outer plexiform layer, INL=inner nuclear layer, IPL=inner plexiform layer, GCL=ganglion cell layer, RNFL= retinal nerve fiber layer.Figure 7. Analysis of anti-Tau monoclonal antibody anti-seeding capacity. A) Dose-response curves of the Tan seeding in Tan biosensor cells with 3 different human cases (cases 16, 17 and 18, Table 3). B) Unpaired t-test (n=4) analysis of the seeding-preventing effect of the indicated anti-Tau monoclonal antibodies when compared to control MA-VillinlEl. Treatment with 16B12 shows significant reduction of the seeding effect in all the cases. The values of spot / cell are normalized to the MA-VillinlEl.

[0057] Figure 8. Analysis of anti-seeding capacity of ScFv-16B12 and ScFv-MCl intrabody constructs. A-B) One-way Anova (n=6) analysis of the seeding -preventing effect of ScFv-16B12 and ScFv-MCl intrabodies in the Tan biosensor cells upon seeding with brain-extracted tan aggregates of different patients: Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Pick’s disease (PiD) and argyrophilic grain disease (AGD) patients . An off-target ScFv served as the negative control (Ctrl). C) Transfection efficiency and cellular viability are lower in MCI expressing cells.

[0058] DETAILED DESCRIPTION

[0059] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0060] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

[0061] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from... to... ” or the expression “between... and... ” or another expression.

[0062] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0063] Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses interalia a reference to any one of said members, or to any two or more of said members, such as, e.g. any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0064] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0065] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0066] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

[0067] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0068] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understoodby those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

[0069] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein..

[0070] The inventors have found that monoclonal antibodies and antibody fragments capable of binding to a specifically defined conformational epitope of Tau in fact recognize pathologic Tau conformers in various cerebral tauopathies, including, e.g., Alzheimer’s disease, primary age-related tauopathy (PART), progressive supranuclear palsy, Corticobasal degeneration, Pick’s disease, and argyrophilic grain disease. Moreover, these antibodies and antibody fragments were shown to be capable of detecting pathological Tau conformers in non-brain tissue such as in retinal tissue of primary retinal tauopathy (PReT) patients. Thus these antibodies have important diagnostic applications. Moreover, these antibodies are capable of recognizing a pathological conformation of Tau in an early stage, such as in PART Braak stage I or II patients or in PReT stage 0 or 1 patients. Finally, the inventors have found that these antibodies are capable of directly interfering with Tau seeding, and this at an early pathological state of a Tauopathy, making them particularly useful in therapy.

[0071] Accordingly, provided herein are isolated monoclonal antibodies and antibody fragments capable of specifically binding to Tau, wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence in Tau set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence in Tau set forth in SEQ ID NO:3, for use in the prevention or treatment of a Tauopathy in a subject.

[0072] Binding or specific binding to the amino acid sequences in Tau set forth in SEQ ID NO:2 or 3 may be determined by assessing binding or specific binding to a Tau peptide comprising SEQ ID NO:2 or 3, such as a Tau peptide of 20 amino acids or less comprising SEQ ID NO:2 or 3, for example a Tau peptide consisting of SEQ ID NO:2 or a Tau peptide consisting of SEQ ID NO:3, 30 or 32. In certain embodiments, specific binding to an amino acid sequence in Tau or a Tau peptide may be determined by ELISA-based peptide array scanning, e.g. as described in the Examples

[0073] In embodiments, isolated monoclonal antibodies and antibody fragments capable of specifically binding to a Tau epitope comprising, consisting of or within the amino acid sequence set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324, for use in the prevention or treatment of a Tauopathy in a subject, are provided.

[0074] The term "antibody" as used herein refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen. The antibodies describedherein can be of any isotype, including, e.g., IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. The term encompasses naturally, recombinantly, semi-synthetically or synthetically produced antibodies. Hence, for example, an antibody can be present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom; or an antibody can be recombinant, and / or can be, partly or entirely, chemically or biochemically synthesised.

[0075] The terms "epitope-binding fragment", "fragment," and "antibody fragment" are used interchangeably to refer to any molecule comprising a fragment of a traditional antibody as described herein that has the epitope-binding capacity as detailed herein. Antibody fragments of the invention may be obtained by enzymatic or chemical proteolysis, or by recombinant DNA technology techniques well known to the skilled person. Antibody fragments of the invention may also be chemically coupled, non-covalently bound, ortranslationally fused to other proteins or peptides. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, mini-antibodies, scFv, scFv-Fc, minibodies, diabodies, etc. For instance, an “Fab” refers to a molecule which contains a monovalent antigen-binding fragment of an antibody molecule, i.e. a light chain and the variable and first constant region of the heavy chain; it can be produced by digestion of whole antibody with the enzyme papain , or can recombinantly expressed in a suitable host cell. A “Fab”’ fragment s different from a Fab fragment in that a Fab' fragment also has several residues derived from the carboxyl terminus of a heavy chain CHI region, which contains one or more cysteine residues from the hinge region of an antibody.. A “F(ab’)2” is a dimer of two Fab' fragments held together by two disulfide bonds. It can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction. Alternatively, Fab’ fragments can be recombinantly expressed in bacteria and then dimerized (i.e. chemically coupled) to generate F(ab')2 molecules or generated directly in specialized cells. . An “Fv” fragment refers to a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains. A “single chain Fv fragment” or “scFv” is molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Mini -antibodies comprise variable regions of the VH and VL chains connected by a flexible linker and further include parts of the constant region (like CHI and CL domains). Mini -antibodies can be obtained by various methods starting from natural materials or recombinant technology. Methods of making these epitope -binding fragments and molecules comprising them are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). The epitope -binding fragments can also be included in larger molecules such as but not limited to diabodies, triabodies, tetrabodies, dual variable domain immunoglobulin, tandem diabodies, scFv3, etc.Antibodies and antibody fragments described herein are primarily amino acid-based molecules but may also include one or more modifications, including, but not limited to, the addition of fluorescent moieties, chemical tags, etc.

[0076] A “monoclonal antibody” refers to a population of substantially homogeneous antibodies derived from a population of substantially homogenous cells (or clones). The individual antibodies making up the population are identical in amino acid sequence and / or bind the same epitope, except for possible naturally occurring mutations or variants that may arise during production, which may be present in minor amounts. Monoclonal antibodies can be human, murine, rat, or of any other origin (including, e.g., chimeric or humanized antibodies). A monoclonal antibody can be produced by a single clone of B-lymphocytes by hybridoma technologies as known to the skilled person. Monoclonal antibodies can also be made by recombinant DNA technology, phage display technologies, synthetic technologies, e.g., CDR-grafting, or combinations of such or other technologies known in the art. For example, DNA encoding the monoclonal antibodies can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of e.g. murine antibodies). Hybridoma cells can serve as a source of such DNA. To synthesize monoclonal antibodies, the isolated DNA can be placed into expression vectors as described elsewhere herein, which can then be transfected into host cells as described elsewhere herein. Methods of expressing antibodies and antibody fragments in host cells of interest are known in the art and such embodiments are within the scope of the disclosure.

[0077] As used herein, the term "isolated" or "purified" in association with a polypeptide or nucleic acid, such as an antibody or antigen-binding fragment, means that the polypeptide or nucleic acid is substantially or essentially free from components that normally accompany it in its native state. Thus, the term "isolated" includes polypeptides or nucleic acids taken from the original environment, for example, if it is naturally occurring. Isolated polypeptides include the naturally produced polypeptides contained in cell lysates, the polypeptides in purified or partially purified form, recombinant polypeptides, the polypeptides expressed or secreted by cells, and in heterologous host cells or cultures of the polypeptide. In connection with nucleic acids, the term isolated or purified indicates that the nucleic acid is not in its natural genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

[0078] The term “recombinant” when referring to antibodies or antibody fragments refers to such molecules created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology as described in more detail elsewhere herein. Recombinant molecules may be expressed in a human cell (e.g., CHO cells) expression system, or a non-human cell expression system, or isolated from a recombinant combinatorial libraries. A “recombinant” molecule may share a sequence with a molecule isolated from an organism (e.g., a mouse), but differs therefrom at least inthat it has been expressed via recombinant DNA technology. Such molecules may have post-translational modifications (e.g., glycosylation) that differ from the molecules isolated from the organism.

[0079] The antibodies and antibody fragments as described herein include “humanized” antibodies and antibody fragments. “Humanized” antibodies or antibody fragments refer to chimeric antibodies that contain minimal sequences derived from non-human antibodies in order to avoid immunological reactions upon use in humans. Accordingly such molecules are of particular interest for in vivo use. For example, humanized antibodies include human antibodies (recipient antibodies) in which one or more regions of an antibody of a mouse, rat, or rabbit having the desired specificity, affinity and / or capacity is introduced, replacing the corresponding sections thereof . Typically, the regions ensuring binding to the antigen are the CDR regions. In some instances, Fv framework (FR) residues of the human antibodies can also be replaced by corresponding non-human residues if these are found to be relevant for epitope binding. Humanized antibodies may also comprise residues which are found neither in the human antibody nor in the imported CDR or FR sequences. Humanization is particularly relevant for antibodies comprising an immunoglobulin constant region (Fc). However also antibody fragments not comprising Fc regions such as scFvs, diabodies and Fabs from rodent origin can cause immunogenicity problems which can be avoided by grafting CDRs into properly selected human antibody frameworks. Methods for humanizing non-human antibodies are well known in the art.

[0080] The above also applies to antibody and epitope -binding fragments developed for use in other species, i.e. the application also envisages molecules, in particular antibodies, which have been adjusted to minimize reactivity in any particular species by combining the epitope -binding residues of an antibody obtained in a given species with a background antibody structure of the species to which the fragment is to be administered.

[0081] In embodiments, the antibody or antibody fragment is a humanized antibody or antibody fragment. In particular embodiments, the antibody or antibody fragment is a humanized antibody or antibody fragment comprising a LCVR with an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or between about 70% and about 90% identical to the sequence of SEQ ID NO: 10 and a HCVR with an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or between about 70% and about 90% identical to the sequence of SEQ ID NO: 11. In particular embodiments, the antibody or antibody fragment is a humanized antibody or antibody fragment comprising a LCVR with an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or between about 70% and about 90% identical to the sequence of SEQ ID NO: 10 and a HCVR with an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or between about 70% and about 90% identical tothe sequence of SEQ ID NO: 11, wherein said LCVR comprises the LCDR1 of SEQ ID NON, the LCDR2 of SEQ ID NO:5 and the LCDR3 of SEQ ID NO:6 and wherein said HCVR comprises the HCDR1 of SEQ ID NO:7, the HCDR2 of SEQ ID NO:8 and the HCDR3 of SEQ ID NO:9. In particular embodiments, the antibody or antibody fragment is a humanized antibody or antibody fragment comprising a LCVR with the amino acid sequence set forth in SEQ ID NO: 10, except for at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or between about 10 and about 35 mutations in SEQ ID NO: 10 and / or a HCVR with the amino acid sequence set forth in SEQ ID NO: 11, except for except for at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or between about 10 and about 35 mutations in SEQ ID NO: 11. In particular embodiments, the antibody or antibody fragment is a humanized antibody or antibody fragment comprising a LCVR with the amino acid sequence set forth in SEQ ID NO: 10, except for at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or between about 10 and about 35 mutations in SEQ ID NO: 10 and / or a HCVR with the amino acid sequence set forth in SEQ ID NO: 11, except for at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or between about 10 and about 35 mutations in SEQ ID NO: 11, wherein said LCVR comprises the LCDR1 of SEQ ID NON, the LCDR2 of SEQ ID NON and the LCDR3 of SEQ ID NON and wherein said HCVR comprises the HCDR1 of SEQ ID NO:7, the HCDR2 of SEQ ID NON and the HCDR3 of SEQ ID NO: 9.

[0082] The antibodies and antibody fragments as described herein include “affinity matured” antibodies and antibody fragments. An “affinity matured” antibody or antibody fragment is used herein to refer to an antibody or antibody fragment comprising one or more alterations in one or more CDRs, which result in an improvement in the affinity (i.e. KD, kd or ka) of the antibody or antibody fragment for its epitope compared to a parent antibody, which does not possess the alteration(s). A variety of procedures for producing affinity matured antibodies are known in the art. For example, a (parent) antibody or antibody fragments can be mutated and selected for increased epitope affinity. Altered versions or variants of an antibody or antibody fragment can be generated by changing the sequences of the encoding genes in the CDR1, CDR2, CDR3, or framework regions, using methods such as oligonucleotide -mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E. coli as known to the skilled person. Measuring binding affinity of an antibody or antibody-fragment to an antigen is known to the skilled person and includes, e.g., solution-affinity ELISA, real-time, label free bio-layer interferometry assay, e.g., an Octet® RED96 system (ForteBio), or surface plasmon resonance (SPR), e.g., BIACORE™.

[0083] In embodiments, the antibody or antibody fragment is an affinity matured antibody or antibody fragment. In particular embodiments, the antibody or antibody fragment is an affinity matured antibody or antibody fragment comprising a light chain variable region comprising a CDR1 with the amino acidsequence set forth in SEQ ID NO:4, a CDR2 with the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:7, a CDR2 with the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:9, except for 1 to 5, 2 to 5, 3 to 5, 1 to 4, 1 to 3, or 1 or 2 mutations in the CDRs. In particular embodiments, the antibody or antibody fragment is an affinity matured antibody or antibody fragment comprising a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 11, except for 1 to 5, 2 to 5, 3 to 5, 1 to 4, 1 to 3, or 1 or 2 mutations in the CDRs. In particular embodiments, the antibody or antibody fragment is an affinity matured antibody or antibody fragment comprising a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 11, except for 1 to 15, 1 to 12, 1 to 10, 2 to 15, 5 to 15 mutations in said sequences. In particular embodiments, the antibody or antibody fragment is an affinity matured antibody or antibody fragment comprising a LCVR with an amino acid sequence that is at least about 85%, at least about 90% or at least about 95% identical to the sequence set forth in SEQ ID NO: 10 and a HCVR with an amino acid sequence that is at least about 85%, at least about 90% or at least about 95% identical to the sequence of SEQ ID NO: 11. In particular embodiments, the affinity matured antibody or antibody fragment binds to an epitope as described herein with at least 20%, or increasing order of preference, at least 30%, at least 40%, or at least 50% greater affinity than the parental antibody binds to the epitope, for example as measured by the Kd.

[0084] Also disclosed herein is a method of preparing an antibody or antibody fragment capable of specifically binding to a Tau epitope as described herein comprising providing an (parental) antibody or antibody fragment capable of specifically binding to said Tau epitope as described herein (e.g. 16B12 monoclonal antibody), and subjecting said (parental) antibody or antibody fragment to affinity maturation, wherein the antibody or antibody fragment produced has a greater affinity to the Tau epitope than the parental antibody or antibody fragment.

[0085] Further disclosed herein is an (affinity matured) antibody or antibody fragment obtained by the aforementioned method.

[0086] The terms “Tau” or “Tau protein” as used herein refers to a microtubule-associated protein that stabilizes and separates the microtubules in axons, which serve as tracks for intracellular transport. Human Tau contains a N-terminal projection domain and a C-terminal microtubule binding domain (MTBD) of which the boundary is formed by the proline-rich segments Pl and P2. Up to six Tau isoforms can be found in the human brain that are generated by alternative splicing of the Tau gene (MAPI) gene transcript. The different isoforms differ in the number of amino-terminal inserts (indicated as N1 and N2) in the projection domain and in the number of repeats (indicated as R1 tot R4) in theMTBD. The term Tau as used herein encompasses all Tan isoforms. In particular embodiments, the antibodies and antibody fragments are capable of binding to human Tau comprising the amino acid sequence set forth in SEQ ID NO: 1.

[0087] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDA KSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDG TGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSP GTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGG KVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKS EKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSN VSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:1)

[0088] SEQ ID NO:1 provided herein represents the sequence of 2N / 4R human Tau. Throughout this disclosure, specific residues and positions are represented with respect to SEQ ID NO:1. If in this disclosure an amino acid residue is indicated by a residue position, such description corresponds to the specific position within SEQ ID NO: 1. For example, recitation of “Ser316” or “S316” indicates that the identified residue corresponds to the serine residue at 316th position in SEQ ID NO:1. Such nomenclature applies even if a fragment of SEQ ID NO: 1 is identified. For example, the phrase “Ser316 within SEQ ID NO: 2” indicates that the identified residue corresponds to the serine residue at 316th position in SEQ ID NO: 1 even if SEQ ID NO:2 contains only 18 amino acids. Sequence alignment of SEQ ID NO: 1 with the identified sequence can be performed to identify the referenced position. As used herein, the term “epitope” refers to a surface or region on one or more entities (e.g., Tau protein) that is capable of interacting with an antibody or antigen-binding fragment or other binding biomolecule, in particular with a specific antigen-binding site of the antibody or the antibody fragment e.g., a variable region of an antibody molecule, known as a paratope. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond, i.e. the site that elicits an immune response. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as sugar side chains, phosphoryl groups, or sulfonyl groups. For example, a protein epitope may contain one or more amino acids and / or post-translational modifications (e.g., phosphorylated residues) which interact with an antibody or antibody fragment. A single antigen may have more than one epitope. Thus, different antibodies and antibody fragments may bind to different areas on an antigen and may have different biological effects. Epitopes may be linear or conformational. A conformational epitope refers an epitope involving a specific three-dimensional arrangement of the entity(ies) having or forming the epitope. A conformational protein epitope is typically composed of non-linear stretches of amino acids. Methods for determining the epitope of an antibody or antibody fragment are well known to the skilled person and include, for example, ELISA-based peptide array scanning, alanine scanning mutational analysis, peptide blot analysis, peptide cleavage analysis,crystallographic studies and NMR analysis. In certain embodiments, the epitope is determined by ELISA-based peptide array scanning, e.g. as described in the Examples.

[0089] “Specificity of binding” or “binding specificity” or “specifically binding” refers to the situation in which a molecule A is, at a certain concentration binding to a target of interest (e .g . protein) with higher affinity (e.g. at least 2-fold, 5 -fold, or at least 10-fold higher affinity, e.g. at least 20-, 50- or 100-fold or more higher affinity) than the affinity with which it is possibly (if at all) binding to other targets (targets not of interest). Specific binding does not mean exclusive binding. However, specific binding does mean that a binder has a certain increased affinity or preference for one or a few of its targets. Exclusivity of binding refers to the situation in which a binder is binding only to the target of interest. As used herein in embodiments, the expression “specifically bind” refers to measurable and reproducible binding between an epitope and an antibody or antibody fragment that is determinative of the presence of the epitope. For example, antibodies and antibody fragments thereof as described herein are capable of specifically or preferentially binding to specific Tau epitopes with greater affinity, avidity, more readily, and / or with greater duration than to other Tau epitopes. An antibody or antibody fragment that is capable of specifically or preferentially binding to a first epitope may or may not be capable of specifically or preferentially binding to a second epitope. As such “specific binding” does not necessarily require (although it can include) exclusive binding.

[0090] The antibodies and antibody fragments disclosed herein are capable of specifically binding to the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324, which has been found to correspond to (part of) a (discontinuous) conformational Tau epitope.

[0091] More particularly this implies that, the antibodies and antibody fragments disclosed herein are capable of specifically binding to a Tau epitope comprising a specific phosphorylation pattern. In embodiments, the antibodies and antibody fragments described herein are capable of specifically binding to a Tau epitope, in particular a phosphorylated Tau epitope, formed by, comprising, consisting of or within, the amino acid sequence set forth in SEQ ID NO:31 and comprising or having phosphorylation at one or more of Ser316, Ser320 and Ser324. In embodiments, the antibodies and antibody fragments are capable of specifically binding to a Tau epitope formed by, comprising, consisting of, or within amino acid residues 309 to 326 of Tau, wherein one or more of Ser316, Ser320 and Ser324 are phosphorylated and wherein the amino acid sequence of Tau is as set forth in SEQ ID NO: 1. In embodiments, the antibodies and antibody fragments described herein are capable of specifically binding to a Tau epitope, in particular a phosphorylated Tau epitope, formed by, comprising, consisting of or within, the amino acid sequence set forth in SEQ ID NO:2 comprising or having phosphorylation at Ser316, Ser320 and Ser324. In embodiments, the antibodies and antibody fragments are capable of specifically binding to a Tau epitope formed by, comprising, consisting of, or within amino acid residues 309 to 326 of Tau, wherein Ser316, Ser320 and Ser324 are phosphorylated and wherein the amino acid sequence of Tau isas set forth in SEQ ID NO: 1. In certain embodiments, the antibody or antigen binding fragment thereof does not specifically bind to the epitope when the epitope is not phosphorylated at the corresponding positions.

[0092] Antibodies and antibody fragments that are capable of specifically binding to the aforementioned phosphorylated epitope were shown to be capable of inhibiting seeding of Tan protein. In embodiments, the antibodies and antibody fragments described herein are capable of inhibiting seeding of Tan protein, optionally as determined in a seeding assay, e.g. as described in the Examples. Briefly such assay is based on the Tan MTBD P301S FRET Biosensor HEK-293 cell line constitutively expressing the Microtubule Binding Domain (MTBD) of Tau with a P301S mutation, fused to either CFP (Cyan Fluorescent Protein) or YFP (Y ellow Fluorescent Protein) that together generate a FRET (Forster Resonance Energy Transfer) signal upon induction of Tau-P301S MTBD aggregation. The intracellular aggregation of Tau-P301S MTBD protein is induced in the presence of Tau seeds such as Tau seeds extracted from brain of AD patients, leading to a FRET signal.

[0093] In further embodiments, the antibodies and antibody fragments disclosed herein are also capable of specifically binding to the amino acid sequence set forth in SEQ ID NO:3, which was found, together with SEQ ID NO:2 to be part of a (discontinuous) conformational Tau epitope.

[0094] Accordingly, in embodiments, the antibody and antibody fragments disclosed herein are capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, formed by, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:31 and comprising or having phosphorylation at one or more of Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In embodiments, the antibody and antibody fragments are capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, formed by, comprising, consisting of, or within amino acid residues 309 to 326 and 249 to 255 of Tau, wherein one or more of Ser316, Ser320 and Ser324 are phosphorylated and wherein the amino acid sequence of Tau is as set forth in SEQ ID NO:1. In particular embodiments, the antibody and antibody fragments disclosed herein are capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, formed by, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:2 comprising or having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In embodiments, the antibody and antibody fragments are capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, formed by, comprising, consisting of, or within amino acid residues 309 to 326 and 249 to 255 of Tau, wherein Ser316, Ser320 and Ser324 are phosphorylated and wherein the amino acid sequence of Tau is as set forth in SEQ ID NO: 1. In certain embodiments, the antibody or antigen binding fragment thereof does not specifically bind to the epitope when the epitope is not phosphorylated at the corresponding positions.Advantageously, antibodies and antibody fragments that are capable of specifically binding to the aforementioned conformational epitope can recognize pathologic Tan conformers in various cerebral tauopathies, including, e.g., Alzheimer’s disease, primary age-related tauopathy (PART), progressive supranuclear palsy, Corticobasal degeneration, Pick’s disease, and argyrophilic grain disease. Also advantageously, these antibodies and antibody fragments were shown to be capable of detecting pathological Tau conformers in non-brain tissue such as in retinal tissue of primary retinal tauopathy (PReT) patients. These antibodies and antibody fragments are capable of binding a pathologic Tau conformer in a non-cerebral Tauopathy such as PReT.

[0095] Further advantageously, antibodies and antibody fragments that are capable of specifically binding to the aforementioned conformational epitope can recognize a pathological conformation of Tau in an early stage. In embodiments, the antibodies and antibody fragments are capable of binding to a conformation of Tau characterized by a backfold of the R1 repeat onto the R3 repeat. In embodiments, the antibody and antibody fragments are capable of binding pathologic Tau conformers in an early stage of Alzheimer’s disease, primary age-related tauopathy (PART), progressive supranuclear palsy, Corticobasal degeneration, Pick’s disease, argyrophilic grain disease and primary retinal tauopathy (PReT). In particular embodiments, the antibody and antibody fragments are capable of binding pathologic Tau conformers in Alzheimer’s disease or PART Braak stage I or II patients. In particular embodiments, the antibody and antibody fragments are capable of binding pathologic Tau conformers in PReT stage 0 or 1 patients. A further advantageous is that these antibodies and antibody fragments are capable of interfering with Tau seeding at an early pathological stage. In embodiments, the antibodies and antibody fragments described herein are capable of inhibiting seeding of Tau protein, optionally as determined in a seeding assay, at an early pathological state of a Tauopathy. The term "Tau conformer" refers to a phosphorylated Tau molecule that has the propensity to self-assemble. "Tau aggregate" and "aggregated Tau", are used interchangeably to denote protein structures comprising more than one Tau molecule or Tau conformer, as opposed to non-pathologic "monomeric Tau" and "Tau monomers". As such, the terms "Tau aggregate" and "aggregated Tau" include but are not limited to dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, enneamers, decamers, dodecamers, icosamers, triacontamers, tetracontamers, or higher-order oligomers and multimers of Tau, non-limiting examples of which are granular aggregates, paired helical fragments (PHF), straight filaments, neurofibrillary tangles (NFT), pretangles (PT), neuropil threads (NT), oligodendroglial coiled bodies (OCB), pick bodies (PB), Tau positive astrocytes (TPA), tufted astrocytes (TA), argyrophilic grains, balloon neurons (BN) and astrocytic plaques (AP). The monomers in Tau aggregates may be covalently linked to each other, or non-covalently by weak intermolecular forces, including but not limited to hydrophobic or hydrophilic interactions, hydrogen bonding, salt bridges, or van der Waals forces.In embodiments, the antibodies comprise four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., "full-length antibody"). Each heavy chain comprises a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (comprised of domains CHI, CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). The light or heavy chain variable region is composed of three hypervariable regions called "complementarity determining regions" or "CDRs" and a framework region (FR) that separates them. The framework region of the antibody, that is, the framework region that constitutes the combination of the light chain and the heavy chain, plays a role of locating and aligning CDRs, which are mainly responsible for binding to the antigen. Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Heavy chain CDRs can also be referred to as HCDRs or CDR-Hs, and numbered HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3). Likewise, light chain CDRs can be referred to as LCDRs or CDR-Ls, and numbered LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3).

[0096] The assignment of amino acids to each domain may be in accordance with the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institute of Health, Bethesda, Maryland (1991)). However, it is well known to those skilled in the art that the domains of an antibody can be defined in various ways in the art, such as the IMGT domain delineation system for variable (V) domains and V-like domains (Lefranc et al. 2003 Dev Comp Immunol. 27:55-77) and Chothia definition rules based on the location of structural loop regions (see JMol Biol273:927-48, 1997). It should be understood by those skilled in the art that, although the amino acid sequences of CDRs and FRs of a given antibody or antibody fragment, or its region (e.g., variable region), described herein are based on the Kabat definitions rules, corresponding amino acid sequences based on other definition rules are also envisaged herein.

[0097] Table 1 refers to amino acid sequences of an exemplary anti-Tau antibody as described herein.

[0098] Table 1. Exemplary anti-Tau antibody

[0099]

[0100]

[0101] In embodiments, the antibody or antibody fragment comprises a light chain variable region comprising a CDR1 (LCCDR1) with the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 (LCCDR2) with the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 (LCCDR3) with the amino acid sequence set forth in SEQ ID NO: 6. In embodiments, the antibody or antibody fragment comprises a heavy chain variable region comprising a CDR1 (HCCDR1) with the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 (HCCDR2) with the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 (HCCDR3) with the amino acid sequence set forth in SEQ ID NO: 9. In particular embodiments, the antibody or antibody fragment comprises the LCCDR1 of SEQ ID NON, the LCCDR2 of SEQ ID NO:5, the LCCDR3 of SEQ ID NO:6, and the HCCDR1 of SEQ ID NO:7, the HCCDR2 of SEQ ID NO:8 and the HCCDR3 of SEQ ID NO:9. In further embodiments, the antibody or antibody fragment comprises a light chain variable region (LCVR) with the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 11. In particular embodiments, the antibody or antibody fragment can be the antibody 16B12 comprising a light chain with the amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain with the amino acid sequence set forth in SEQ ID NO: 13. In particular embodiments, the antibody fragment is an scFv fragment comprising the amino acid sequence of SEQ ID NO: 14.

[0102] Also provided herein are antibodies and antibody fragments (e.g. humanized antibodies and antibody fragments or affinity matured antibody and antibody fragments) comprising a LCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the LCVR of SEQ ID NO: 10 and antibodies and antibody fragments (e.g. humanized antibodies and antibody fragments or affinity matured antibody and antibody fragments) comprising a HCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the LCVR of SEQ ID NO: 11. In embodiments, the antibody or antibody fragment (e.g. humanized antibodies and antibodyfragments or affinity matured antibody and antibody fragments) comprises a LCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the LCVR of SEQ ID NO: 10 and / or a HCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the HCVR of SEQ ID NO: 11. The variation can be in one or more CDR and / or one or more FR.

[0103] Also disclosed herein are antibodies and antibody fragments comprising an LCVR and / or an HCVR amino acid sequence that is set forth herein, e.g. the LCVR and HCVR amino acid sequences set forth in SEQ ID NO: 10 and 11, respectively, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) mutations such as, for example, missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. In embodiments, the antibody or antibody fragment (e.g. humanized antibodies and antibody fragments or affinity matured antibody and antibody fragments) comprises a LCVR with the amino acid sequence set forth in SEQ ID NO: 10, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mutations in SEQ ID NOTO and / or a HCVR with the amino acid sequence set forth in SEQ ID NO: 11, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mutations in SEQ ID NO: 11. Mutations can be in one or more CDR and / or one or more FR.

[0104] In embodiments, the LCVR and / or HCVR of the anti-Tau antibody or antibody fragment can have a sequence variation as described herein but the antibody or antibody fragment comprises three or more, in particular six, of the CDRs set forth in SEQ ID NO:4-9 (e.g., LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5 and LCDR3 of SEQ ID NO:6; and / or HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8 and HCDR3 of SEQ ID NOV). Accordingly, in embodiments, an antibody or antibody fragment (e.g. a humanized antibody or antibody fragment) comprises a LCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10, wherein said LCVR comprises the LCDR1 of SEQ ID NO:4, the LCDR2 of SEQ ID NO:5 and the LCDR3 of SEQ ID NO:6. In embodiments, an antibody or antigen-binding fragment of the invention (e.g. a humanized antibody or antibody fragment) comprises a HCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11, wherein said HCVR comprises the HCDR1 of SEQ ID NO: 7, the HCDR2 of SEQ ID NO: 8 and the HCDR3 of SEQ ID NO: 9. In embodiments, the antibody or antibody fragment (e.g. a humanized antibody or antibody fragment) comprises a LCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10 and a HCVR with an amino acid sequence that is at least about 75%, preferably at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11, wherein said LCVR comprises the LCDR1 of SEQ ID NO:4, the LCDR2 of SEQ ID NO:5 and the LCDR3 of SEQ ID NO:6 and wherein said HCVR comprises theHCDR1 of SEQ ID NO:7, the HCDR2 of SEQ ID NO:8 and the HCDR3 of SEQ ID NO:9.

[0105] Also disclosed herein are antibodies and antibody fragments (e.g. affinity matured antibodies or antibody fragments) comprising one or more variant CDRs, e.g., any one or more variant LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. In embodiments, the antibody or antibody fragment comprises one or more of a LCDR1, a LCDR2, a LCDR3, a HCDR1, a HCDR2 and a HCDR3 with at least 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity to SEQ ID NO:4-9, respectively. In embodiments, the antibody or antibody fragment comprises one or more of a LCDR1, a LCDR2, a LCDR3, a HCDR1, a HCDR2 and a HCDR3 with 1, 2 or 3 mutations in the amino acid sequence set forth in SEQ ID NO:4-9, respectively.

[0106] It is appreciated that a skilled person is capable of assessing sequence identity between sequences. Since methods and tools to verify sequence identity between different sequences of amino acids or nucleic acids are well known. Such tools include (Protein) BLAST, ClustalW2, SIM alignment tool, TranslatorX, and T-COFFEE. The percentage of identity between two sequences may show minor differences depending on the algorithm choice and parameters. The term “sequence identity” as used herein refers to the relationship between sequences at the amino acid (or nucleotide) level. The expression “% identical” is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions and / or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. A reference window is chosen and the “% identity” is then calculated by determining the number of amino acids (or nucleotides) that are identical between the sequences in the window, dividing the number of identical amino acids (or nucleotides) by the number of amino acids (or nucleotides) in the window and multiplying by 100. Unless indicated otherwise, the sequence identity is calculated over the whole length of the reference sequence. An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide will entail aligning the two amino acid sequences using the Blast 2 sequences (B12seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters. An example of suitable algorithm parameters include: matrix = Blosum62, cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3). A skilled person readily appreciates that any sequences represented in sequence databases or in the present specification may be of precursors of peptides, polypeptides, proteins, or nucleic acids and may include parts which are processed away from mature molecules. Antibodies and antibody fragments as described herein may encompass conservatively modified antibody and antibody fragment variants. A “conservatively modified variant” or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in apolypeptide with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu. Hence in embodiments, a sequence having a given percentage sequence identity as given herein before may be a sequence having one or more conservative amino acid substitutions as compared to the reference sequence.

[0107] Changes such as substitutions may also be made to the residues, including residues in the CDRs, of the antibodies and antibody fragments described herein that do not interfere with the ability of the antibody or antibody fragment to recognize and bind to its cognate epitope. For example, changes that do not affect epitope recognition, yet increase the binding affinity of the antibody or antibody fragment for the epitope may be made (e.g. affinity matured antibodies or antibody fragments). Methods suitable for determining whether a variant antibody or antibody fragment retains binding to the epitope on human Tau have been described elsewhere herein.

[0108] Variants of the antibodies and antibody fragments as described herein are preferably functional variants. “Functional variants,” as used herein, refers to variants of the antibodies and antibody fragments described herein in which one or more amino acid residues have been changed without significantly altering one or more functional properties of the antibody or antibody fragment, such as, e.g., its antiseeding ability.

[0109] Further disclosed herein are methods of producing the Tau antibodies or antibody fragments thereof as disclosed herein. For example, a nucleotide sequence encoding a Tau antibody or an antigen binding fragment thereof as described herein can be incorporated into a suitable expression vector as described herein. Such vector can be introduced into a host cell as described herein and the host cell can be cultured under conditions that are appropriate for the expression, preferably, a high level expression, of the Tau antibody or the antibody fragment thereof encoded by the vector.Also disclosed herein are methods for screening for antibodies or antibody fragments that are capable of inhibiting seeding of Tan protein. In particular embodiments, the method comprises screening a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tan, for an antibody or antibody fragment capable of specifically binding to Tan, wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In further embodiments, the method comprises screening a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tau, for an antibody or antibody fragment capable of specifically binding to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324, preferably to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In particular embodiments the method comprises determining whether an antibody or antibody fragment is capable of competing with binding of an antibody or antibody fragment as described herein to Tau. In particular embodiments, said method further comprises improving the affinity of said antibody or antibody for said epitope by affinity maturation

[0110] Also disclosed herein are methods for screening for antibodies or antibody fragments that are capable of binding a pathological Tau conformer of an early stage Tauopathy and / or a non-cerebral Tauopathy such as PReT. In particular embodiments the method comprises screening a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tau, for an antibody or antibody fragment capable of specifically binding to Tau, wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In further embodiments, the method comprises screening a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tau, for an antibody or antibody fragment capable of specifically binding to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In particular embodiments the method comprises determining whether an antibody or antibody fragment is capable of competing with the binding of an antibody or antibody fragment as described herein to Tau. In particular embodiments, said method further comprises improving the affinity of said antibody or antibody fragment for said epitope by affinity maturation.

[0111] Further disclosed herein is a method for obtaining antibodies or antibody fragments that are capable of inhibiting seeding of Tau protein. In particular embodiments, the method comprises providing an antibody or antibody fragment capable of specifically binding to Tau, wherein the antibody or antibodyfragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In further embodiments, the method comprises providing an antibody or antibody fragment that is capable of specifically binding to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324, preferably to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. Also disclosed herein is a method for obtaining antibodies or antibody fragments that are capable of binding a pathological Tau conformer of an early stage Tauopathy and / or a non-cerebral Tauopathy such as PReT. In particular embodiments the method comprises providing an antibody or antibody fragment capable of specifically binding to Tau, wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In further embodiments, the method comprising providing an antibody or antibody fragment that is capable of specifically binding to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3. In particular embodiments, said methods further comprise improving the affinity of said antibody or antibody fragment for said epitope by affinity maturation.

[0112] In particular embodiments the methods for generating antibodies or antibody fragments according to the invention comprise:

[0113] generating a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tau, and

[0114] selecting from said library an antibody or antibody fragment that is capable of specifically binding to the amino acid sequence in Tau set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence in Tau set forth in SEQ ID NO:3.

[0115] In certain embodiments, said selection an antibody or antibody fragment that is capable of specifically binding to the amino acid sequence in Tau set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence in Tau set forth in SEQ ID NO:3 may comprise selecting an antibody or antibody fragment that is capable of specifically binding to a Tau peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 (such as a Tau peptide consisting of SEQ ID NO:2) and to a Tau peptide comprising the amino acid sequence set forth in SEQ ID NO:3 (such as. a Tau peptide consisting of SEQ ID NO:30 (PMPDLKNVKSKIGSTENL) or 32 (PVPMPDLKNVKSKIGSTE)), for example by ELISA-based peptide array scanning e.g. as described in the Examples.In particular embodiments the methods comprise:

[0116] generating a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tan, and

[0117] selecting from said library an antibody or antibody fragment that is capable of specifically binding to a Tan epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324 or to a Tau epitope comprising the amino acid sequence set forth in SEQ ID NO: 2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO: 3.

[0118] In particular embodiments, the method comprises:

[0119] generating a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tau, and

[0120] determining whether said antibody or antibody fragment is capable of competing with binding of an antibody or antibody fragment as described herein to Tau.

[0121] In particular embodiments, the method comprises:

[0122] generating a library of antibodies or antibody fragments, preferably a library of antibody or antibody fragments specifically binding to Tau, and

[0123] determining whether said antibody or antibody fragment is capable of capable of competing with binding of an antibody or antibody fragment as described herein to binding to the amino acid sequence set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 and.

[0124] In particular embodiments, the library is obtained by immunizing a test animal with Tau, in particular (hyper)phosphorylated Tau such as recombinant Tau produced in the BY4741 pho85AadhlA yeast strain or Tau isolated from a biological sample of a subject with a Tauopathy such as from brain tissue of an AD patient, and optionally boosting the immunized test animal with a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 and having phosphorylation at Ser316, Ser320 and Ser324 and a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3. In further particular embodiments, the library is a synthetic library.

[0125] In a further aspect, the invention provides a nucleic acid such as an isolated nucleic acid or a nucleic acid construct comprising a polynucleotide sequence, such as a coding sequence, that is encoding an antibody or antibody fragment as described herein. It is particularly envisaged that the nucleic acid sequences encode the antibody or antibody fragment with all the features and variations described herein, mutatis mutandis. Thus, the encoded antibody or antibody fragment is in essence as described herein..“Nucleic acid(s)” or “nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides; the sequential linear arrangement of the nucleotides together resulting in / forming the “nucleotide sequence”, “DNA sequence”, or “RNA sequence”. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single -stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, “caps”, and substitution of one or more of the naturally occurring nucleotides with an analog. Modifications to nucleic acids can be introduced at one or more levels: phosphate linkage modification (e.g. introduction of one or more of phosphodiester, phosphoramidate or phosphorothioate bonds), sugar modification (e.g. introduction of one or more of LNA (locked nucleic acids), 2'-O-methyl, 2'-O-methoxy-ethyl, 2’ -fluoro, S-constrained ethyl or tricyclo-DNA) and / or nonribose modifications (e.g. introduction of one or more of phosphorodiamidate morpholines or peptide nucleic acids).

[0126] By "nucleic acid construct" is meant a nucleic acid molecule that has been constructed in order to comprise one or more functional units not found together in nature, thus having a nucleotide sequence not found in nature (non-native nucleotide sequence). Examples include circular, linear, doublestranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, RNA or mRNA constructs, and the like.

[0127] An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest, which is operably linked to a (gene) promoter. Expression cassettes are generally DNA constructs preferably including (5’ to 3’ in the direction of transcription): a (gene) promoter region, a polynucleotide sequence of interest with a transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal; all these elements being operably or operatively linked meaning that all of these regions should be capable of operating (being expressed) in a cell, such as prokaryotic (e.g. bacterial) or eukaryotic (e.g. mammalian, yeast, insect, fungal, plant, algal) cells, when transformed into that cell. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the cell to be transformed, may be derived from an alternative source, or may be synthetic, as long as it is functional in the cell. Such expression cassettes can be constructed in e.g. a “vector” or “expression vector” as described herein.

[0128] The nucleic acids described herein comprise a coding sequence encoding an antibody or antibody fragment as described herein. In particular embodiments, nucleic acids are provided comprising nucleic acid sequences encoding a light and / or a heavy immunoglobulin chain, or a HCVR and / or a LCVR of an antibody or antibody fragment as described herein. A “coding sequence” is a nucleotide sequence that can be transcribed into mRNA and / or translated into a polypeptide when placed under the controlof appropriate (gene) regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5 '-terminus and a translation stop codon at the 3 '-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.

[0129] The present invention also provides a vector including a herein described nucleic acid molecule inserted therein. Accordingly, a further aspect relates to a vector comprising a nucleic acid as described herein. The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of carrying another nucleic acid molecule to which it has been linked. Said vectors may include a cloning or expression vector, as well as a delivery vehicle such as a viral, lentiviral or adenoviral vector.

[0130] “Expression vectors” may comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. In particular, an expression vector as described herein may comprise a nucleic acid molecule as described herein comprising a nucleic acid sequence encoding an antibody or an antibody fragment as described herein operably linked to at least one regulatory sequence. Regulatory sequences are selected to direct the expression of the protein of interest, in particular the antibody or antibody fragment, in a suitable host cell, and include promoters, enhancers, and other expression control elements as known to the skilled person. In particular, an expression vector as described herein may comprises a nucleic acid expression cassette as described herein. Hence, in embodiments, the vector includes a promoter for driving expression of the nucleic acid of interest, optionally a nucleic acid sequence encoding a signal peptide that secretes the antibody or antigen-binding fragment, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is manipulated in a production strain or cell line, the vector may or may not be integrated into the genome of the host cell when introduced into the host cell. Any of a variety of expression vectors known to those skilled in the art can be used in the present invention, and the choice of the expression vector is dependent on the nature of the host cell of choice.

[0131] “Cloning vectors” are generally used to engineer and amplify a certain desired DNA fragment. Thus, a cloning vectors may contain origin of replication that matches the cell type specified by the cloning vector, and may lack functional sequences needed for expression of the desired DNA fragments. Preferably, the vector contains one or more selection markers. The choice of the selection markers may depend on the host cells of choice, although this is not critical to the present invention as is well known to persons skilled in the art.

[0132] The construction of (expression) vectors for use in transfecting cells is also well known in the art, and thus can be accomplished via standard techniques.Non-limiting examples of vectors useful in the methods of the disclosure include plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors such as a lentiviral, adenoviral, adeno-associated viral (AAV) or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). The choice of the vector may bee dependent amongst others on the nature of the host cell of choice.

[0133] One further aspect provides for a host cell comprising an antibody or antibody fragment thereof as described herein. The host cell may therefore comprise a nucleic acid molecule encoding the antibody or antibody fragment or a vector comprising the nucleic acid molecule. Host cells can be either prokaryotic or eukaryotic. Non-limiting examples of host cells that may be used to produce an antibody or antibody fragment as described herein include, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for production of antibodies and antibody fragments include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Suitable yeast host cells include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pasioris). Hansenula (e.g. Hansenula polymorpha). Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are non-limiting examples of yeast hosts, and are convenient fungal hosts. Animal host cells suitable for use with the invention include insect cells and mammalian cells (e.g. derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems. Alternatively, the host cells may also be transgenic animals or plants. Introduction of a vector in a host cell can be effected by, e.g., calcium phosphate transfection, virus infection, DEAE-dextran-mediated transfection, lipofectamin transfection or electroporation, and any person skilled in the art can select and use an introduction method suitable for the expression vector and host cell used.

[0134] As noted above, the Tau antibodies and antibody fragments disclosed herein are useful for treating or preventing Tauopathies. Accordingly, in an aspect, the invention provides an antibody or antibody fragment, a nucleic acid encoding the antibody or antibody fragment, or a vector comprising the nucleic acid as described herein for use in the prevention or treatment of a Tauopathy in a subject. Related aspects are directed to a method of treating or preventing a Tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antibody fragment, a nucleic acid encoding the antibody or antibody fragment, or a vector comprising the nucleic acid as described herein. Yet another aspect is directed to use of an antibody or antibody fragment, a nucleic acid encoding the antibody or antibody fragment, or a vector comprising the nucleic acid as described herein for the manufacture of a medicament for the prevention or treatment of a Tauopathy in a subject.As used herein, the terms “therapy” or “treatment” refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder, in particular a Tauopathy. Measurable lessening includes any statistically significant decline in a measurable symptom or marker. Generally, the terms encompass both curative treatments and treatments directed to reduce symptoms and / or slow progression of the disease. The terms encompass both the therapeutic treatment of an already developed pathological condition, in particular a Tauopathy, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition, in particular a Tauopathy. Beneficial or desired clinical results include, but are not limited to, prevention of a disease, reduction of the incidence of a disease, alleviation of symptoms associated with a disease, diminishment of extent of a disease, stabilisation of the disease, delay or slowing of the progression of a disease, amelioration or palliation of a disease, or combinations thereof. In certain embodiments, the terms may relate to therapeutic treatments. In certain other embodiments, the terms may relate to preventative treatments.

[0135] As used herein, a “Tauopathy” refers to a neurodegenerative disease or disorder characterized by misfolding and / or aggregation of the microtubule associated protein Tau. Tauopathies are highly heterogeneous, with the main differences residing in, e.g., the unique lesion morphology, the affected cells and brain regions, the Tau isoforms involved and the structures of end-state Tau filaments. Tau is normally a very soluble protein known to associate with microtubules based on the extent of its phosphorylation. Tau is considered a critical component of intracellular trafficking processes, particularly in neuronal cells, given their unique and extended structure. Abnormal phosphorylation of Tau leads to misfolding and depresses its binding to microtubules and microtubule assembly activity. Further, hyperphosphorylation of Tau renders it prone to aggregation. Especially in brain Tauopathies, the Tau may become hyperphosphorylated and aggregates as neurofibrillary tangles (NFT) of paired helical filaments (PHF), twisted ribbons or straight filaments. Pathological Tau impacts on neuronal death through multiple mechanisms and, as such, NFTs are thought to contribute to widespread neuronal cell loss, leading to a variety of behavioural and cognitive deficits.

[0136] Non-limiting examples of Tauopathies that can be treated according to the methods of the disclosure include Alzheimer’s disease (AD), Pick disease (PiD), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), frontotemporal labor degeneration, primary age-related tauopathy (PART), Argyrophilic Grain Disease (AGD) or primary retinal tauopathy (PReT). In particular embodiments, the Tauopathy is selected from the group comprising or consisting of: Alzheimer’s disease (AD), primary age-related tauopathy (PART), Pick disease (PiD), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), frontotemporal labor degeneration, Argyrophilic Grain Disease (AGD) and primary retinal tauopathy (PReT). In particular embodiment, the Tauopathy is selected fromthe group comprising or consisting of: Alzheimer’s disease (AD), primary age-related tauopathy (PART), Pick disease (PiD), progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), and Argyrophilic Grain Disease (AGD). In particular embodiments, the Tauopathy is Alzheimer’s disease (AD), PART or Pick disease (PiD). In particular embodiments, the Tauopathy is AD or PART. As shown in the examples section, the antibodies and antibody fragments disclosed herein where able to interfere with, in particular to inhibit or prevent, the seeding effect of Tau, rendering these antibodies and antibody fragments suitable for the treatment and prevention of Tau-related diseases and disorders. Accordingly, in embodiments, the antibody or antibody fragment may be used to reduce Tau seeding, prevent Tau seeding and / or prevent the propagation of Tau seeds in a subject. As used herein "Tau seeding" refers to the process by which misfolded and / or aggregated Tau proteins propagate their abnormal conformation to normal Tau, leading to the spread of Tau pathology across the brain. Without wishing to be bound by any theory, this seeding and spreading mechanism is thought to be central to the progression of several Tauopathies. Three aggregation-prone regions (APRs) of Tau, i.e. PHF6* in R2, PHF6 in R3 and PAM4 in R4, were previously shown to drive the self-assembly of Tau by enhancing the formation of beta-structure. These beta-sheet enriched pre-fibrillar Tau species can induce a particular pathological structure to its physiological counterpart, a necessary process for the pathology progression of Tauopathy, known as seeding. Tau seeding activity may be assessed in vitro or in cellulo in cellular Tau models as described elsewhere herein or in vivo in mouse models. Tau seeds for efficacy studies may be variant Tau fibrils such as, but not limited to, hTau40, P301S, P301L, and KI 8. Tau seeds may also be prepared from brain lysates of subjects with Alzheimer’s disease, for example, as described in the Examples.

[0137] As used herein, the term “subject” refers to a mammal (e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit), preferably a human, for example, in need of treatment of a disease or disorder such as a Tauopathy. In particular embodiments, the subject is a subject, such as a human subject, who has a Tauopathy, or is predisposed to developing a Tauopathy. The aforementioned terms do not imply that symptoms are present.

[0138] Administration of the antibody or antibody fragment, nucleic acid or vector, may be carried out using known procedures, at dosages and for periods of time effective to treating or preventing a disease or disorder, in particular a Tauopathy, contemplated in the disclosure.

[0139] The terms "therapeutically effective amount" and "therapeutically effective dose" as used herein, generally denote an amount sufficient to elicit the pharmacological effect or medicinal response in a subject that is being sought by a medical practitioner such as a medical doctor, clinician, surgeon, veterinarian, or researcher, which may include inter alia alleviation of the symptoms of the disease being treated, in either a single or multiple doses.The term “prophylactically effective amount” generally denotes an amount sufficient to elicit the preventative effect, such as inhibition or delay of the onset of a disease or disorder, in a subject that is being sought by the medical practitioner, in either a single or multiple doses.

[0140] An effective amount of a nucleic acid encoding an anti-Tau antibody or antibody fragment as described herein or a vector comprising a nucleic acid encoding an anti-Tau antibody or antibody fragment, may denote an amount of nucleic acid or vector sufficient for expression of an effective amount of the antibody or antibody fragment encoded by the nucleic acid or the vector. Non-limiting examples of factors that may determine an effective amount of a nucleic acid or a vector include, for example, copy number of the coding sequence encoding the antibody or antibody fragment, the promoter, regulatory sequences, etc.

[0141] An effective amount of the active compound necessary for adequate disease or disorder treatment and / or prevention may vary according to factors such as the state of a disease or disorder in the patient; the age, sex, and weight of the subject. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the antibody or antibody fragment, the nucleic acid encoding the antibody or antibody fragment, or the vector comprising the nucleic acid, without undue experimentation. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is therapeutically or prophylactically effective for a particular patient, composition, and mode of administration, without being toxic to the subject or patient.

[0142] In particular embodiments, the antibodies and fragments of the invention are “intrabodies”. An “intrabody” refers to an antibody or antibody fragment that is not secreted from a cell in which it is produced and works within a cell to bind to an intracellular protein, e.g. Tau. Methods of preparing an intrabody are known in the art. Intrabodies are often single chain variable fragments (scFvs) expressed from a recombinant nucleic acid molecule. A single chain variable fragment (scFv) can be prepared by linking a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody as known in the art. In such scFvs, the HCVR and the LCVR may be linked together via a linker such as a (648)3 linker of SEQ ID NO: 15. Intrabodies maybe expressed within a target cell. Such expression can be accomplished by introduction into the target cell of a nucleic acid construct encoding an intrabody. Intrabodies can also be modified for intracellular localization when produced in cells other than target cells, such as prokaryotes or other suitable host cells. For example, lipid moieties may be joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies may also be engineered to be retained intracellularly. For example, as is known in the art, an intrabody can be engineered into a nucleic acid construct or vector to encode sub-cellular trafficking signals at its N or C terminus to allow expression at high concentrations in a target sub-cellularcompartment. For example, intrabodies intended to exert activity in the nucleus may be engineered to include a nuclear localization signal.

[0143] A nucleic acid encoding an antibody or antibody fragment or a (recombinant) vector comprising such nucleic acid as described herein can be used in e.g. gene therapy setting. “Gene therapy” as used herein refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. For such applications, the nucleic acid molecule or vector as described herein allow for production of the antibody or antibody fragment within a cell. A large set of methods for gene therapy are available in the art and include, for instance (adeno-associated) virus-mediated gene therapy. A plethora of delivery methods are well known to those of skill in the art and include but are not limited to viral delivery systems, microinjection of DNA plasmids, biolistics of naked nucleic acids, use of a liposome or an artificial exosome, administration of the nucleic acid or vector formulated in a nanoparticle or lipid or lipid-comprising particle. In vivo delivery by administration to an individual patient may occur by systemic administration (e.g., intravenous, intraperitoneal infusion or brain injection). In particular embodiments, a nucleic acid encoding an antibody or antibody fragment as described herein, or a vector comprising a nucleic acid encoding an antibody or antibody fragment as described herein, may be administered to a target cell, tissue, or organ by any one of the aforementioned delivery methods. In particular embodiments, a nucleic acid encoding an antibody or antibody fragment as described herein, in particular an intrabody, may be administered to a target cell, tissue, or organ, in the form of a recombinant adeno-associated virus (AAV) particle, wherein said AAV particle has a viral genome engineered to encode the antibody or antibody fragment as described herein, in particular the intrabody. The AAV particle, or a plurality of AAV particles, may be provided, e.g., delivered, via any of several routes of administration to the target cell, tissue, or organ, as known to the skilled person.

[0144] Adeno-associated viruses (AAV) are small non-enveloped icosahedral capsid viruses of the Parvoviridae family characterized by a single stranded DNA viral genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. The Parvoviridae family includes the Dependovirus genus which includes AAV, capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species. AAV have proven to be useful as a biological tool due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profde. The genome of the virus may be modified using methods known in the art to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to express or deliver a desired nucleic acid construct or payload, e.g., a nucleic acid encoding an antibody or antibody fragment, in particular an intrabody, as described herein.The anti-Tau antibodies and epitope -binding fragments disclosed herein, nucleic acids encoding said antibodies or antibody fragments or vectors comprising nucleic acids encoding said antibodies or antibody fragments, when used for treating or preventing Tauopathies, may be administered to subjects in the form of pharmaceutical compositions. As used herein the term “pharmaceutical composition” refers to compositions including at least one active ingredient and, most often, a pharmaceutically acceptable excipient.

[0145] Accordingly, a further aspect provides pharmaceutical compositions comprising an antibody or antibody fragment disclosed herein, a nucleic acid encoding said antibody or antibody fragment or a vector comprising said nucleic acid as described herein, and a pharmaceutically acceptable excipient The term "pharmaceutically acceptable", as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the active ingredient, in particular the antibody or antibody fragment, the nucleic acid or the vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0146] As used herein, the terms “carrier” or “excipient” or “diluent” are used interchangeably and broadly include any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCI, acetate or phosphate buffers), solubilisers (such as, e.g., Tween® 80, Polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives (such as, e.g., ThimerosalTM, benzyl alcohol), antioxidants (such as, e.g., ascorbic acid, sodium metabisulfite), tonicity controlling agents, absorption delaying agents, adjuvants, bulking agents (such as, e.g., lactose, mannitol) and the like. The use of such media and agents for the formulation of pharmaceutical compositions is well known in the art. The antibody or antibody fragment, the nucleic acid encoding it, or the vector comprising the nucleic acid can be administered systemically or directly to a target cell, tissue or organ, in particular a cell, tissue or organ where pathological Tau is observed or thought to be present. In some embodiments, the target cell is a central nervous system (CNS) cell. In some embodiments, the target tissue is a CNS tissue. The target CNS tissue may be brain tissue. In some embodiments, the brain target comprises caudate, putamen, thalamus, superior colliculus, cortex, and corpus collosum.

[0147] In a non-limiting example, the antibody or antibody fragment, the nucleic acid or the vector can be administered by injection into a blood vessel supplying the brain or into the brain itself.

[0148] The anti-Tau antibodies and antibody fragments provided herein may also serve as diagnostic tools for the detection of unique phosphorylation and / or conformational states of Tau. Accordingly, furtherdisclosed herein are methods and kits for diagnosing or detecting a Tauopathy as defined elsewhere herein in a subject using or comprising an antibody or antibody fragment as described herein.

[0149] A further aspect is related to use of an antibody or antibody fragment as described herein in the in vitro or ex vivo diagnosis of a Tauopathy in a subject.

[0150] Provided herein is a method, in particular an in vitro or ex vivo method, for detecting Tau (e.g., human Tau) comprising: (a) contacting a sample (e.g., a biological sample such as a tissue section) with an anti-Tau antibody or antibody fragment described herein for a time sufficient to allow specific binding of the antibody or antibody fragment to Tau in the sample, and (b) contacting the sample with a detection reagent, e.g., an (secondary) antibody, that specifically binds to the anti-Tau antibody or antibody fragment, to thereby detect Tau bound by the anti-Tau antibody.

[0151] Also provided are methods of detecting the presence of Tau (e.g., human Tau) in a sample, or measuring the amount of Tau in a sample, comprising contacting a sample (e.g., biological sample such as a tissue sample) with an anti-Tau antibody or antibody fragment described herein under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau, and detecting the formation of a complex. In some embodiments, the method may also include contacting the sample with a control antibody (e.g., an isotype control antibody) in parallel, wherein the difference in complex formation between the anti-Tau antibody or antibody fragment and sample and control antibody and sample is indicative of the presence of Tau in the sample.

[0152] A further aspect is related to use of an antibody or antibody fragment as described herein in the in vitro or ex vivo diagnosis of a Tauopathy in a subject.

[0153] Further provided herein is a method, in particular an in vitro or ex vivo method, for the diagnosis of a Tauopathy or of a predisposition to a Tauopathy in a subject in need thereof, comprising detecting the presence of Tau in a biological sample from the subject according to the method as described above, wherein the presence of Tau in the biological sample indicates that the subject is suffering from or is at risk of developing a Tauopathy. In certain embodiments, the method may further comprise measuring the amount of Tau in the biological sample from the subject, and comparing the amount of Tau to a normal control value, wherein an increase in the amount of the Tau compared to a normal control value indicates that the subject is suffering from or is at risk of developing a Tauopathy.

[0154] Advantageously, antibody and antibody fragments disclosed herein which are capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3, can recognize a pathological conformation of Tau in an early stage, rendering these antibodies and antibody fragments particularly suitable for the diagnosis of an early stage Tauopathy, such as Braak stage I or II Alzheimer’s diseaseor primary age-related tauopathy (PART), or primary retinal tauopathy (PReT) stage 0 or 1. In particular embodiments, the Tauopathy is an early stage Tauopathy, particularly Braak stage I or II AD or PART, or PReT stage 0 or 1.

[0155] Also advantageously, antibody and antibody fragments disclosed herein which are capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3, can recognize a pathological conformation of Tau in non-cerebral tissue such as in retinal tissue of a PReT patient. In particular embodiments, the Tauopathy is a non-cerebral Tauopathy, particularly primary retinal tauopathy (PReT).

[0156] A further aspect is directed to use of an antibody or antibody fragment capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 as described herein, for the in vitro or ex vivo diagnosis of an early stage Tauopathy such as Braak stage I or II Alzheimer’s disease or primary age-related tauopathy (PART) or primary retinal tauopathy (PReT) stage 0 or 1 in a subject in need thereof.

[0157] A related aspect is directed to a method, in particular an in vitro or ex vivo method, for the diagnosis of an early stage Tauopathy such as Braak stage I or II Alzheimer’s disease or primary age-related tauopathy (PART) or primary retinal tauopathy (PReT) stage 0 or 1 in a subject in need thereof, comprising detecting the presence of Tau in a biological sample from the subject using an antibody or antibody fragment capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 as described herein according to a method as described above.

[0158] A further aspect is directed to use of an antibody or antibody fragment capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 as described herein, for the in vitro or ex vivo diagnosis of a non-cerebral Tauopathy such as primary retinal tauopathy (PReT) in a subject in need thereof.

[0159] A related aspect is directed to a method, in particular an in vitro or ex vivo method, for the diagnosis of a non-cerebral Tauopathy such as primary retinal tauopathy (PReT) in a subject in need thereof, comprising detecting the presence of Tau in a biological sample, in particular a non-cerebral biologicalsample such as a retinal sample, from the subject using an antibody or antibody fragment capable of specifically binding to a Tau epitope, in particular a conformational Tau epitope, comprising, consisting of, or within the amino acid sequences set forth in SEQ ID NO:2 comprising phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 as described herein according to a method as described above.

[0160] The diagnostic methods disclosed herein may rely on known immunological methods commonly used for detecting and / or quantifying substances in biological samples. Such method may include, but are not limited to immunohistochemical techniques, mass spectrometry, array analysis (e.g., antibody array or protein array), Western blotting, flow cytometry, immunoprecipitation, surface plasmon resonance analysis and ELISA. Immunohistochemical methods may include staining tissue sections to determine the presence and / or level of one or more Tau proteins. Tissue sections may come from formalin-fixed or unfixed fresh frozen tissues, or from formalin fixed paraffin-embedded (FFPE) tissues.

[0161] These immunological methods may rely on the use of labelled antibody or antibody fragments as described herein. The antibodies or antibody fragments can be labelled with compounds generally known to those of ordinary skill in the art including enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances including, but not limited to coloured particles, such as colloidal gold and latex beads. Alternatively, the antibody or antibody fragments as described herein may be labelled indirectly by reaction with a labelled substance that has affinity for the antibody or antibody fragment, such as protein A or G or a secondary antibody. The antibody or antibody fragment may also be conjugated with a second substance and detected with a labelled third substance having an affinity for the second substance conjugated to the antibody or antibody fragment. For example, the antibody or antibody fragment as described herein may be conjugated to biotin and the antibody / antibody fragment-biotin conjugate detected using labelled avidin or streptavidin. Those of ordinary skill in the art will know of these and other suitable labels which may be employed in accordance with the present invention. The binding of these labels to binding peptides or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art.

[0162] Biological samples that may be used in the diagnostic method are, for example, tissue or cell samples obtained from a subject such as neural, brain, retinal, cardiac or vascular tissue or fluids such as serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymphatic fluid and the like. In embodiments, the biological sample is a cerebral biological sample. In certain embodiments, the biological sample comprises a tissue section derived from subject CNS tissue. In other embodiments, the biological sample is a non-cerebral biological sample such as a retinal biological sample. In particular embodiments, the biological sample may comprise retinal tissue derived from the subject.The present invention also relates to a diagnostic kit for detecting Tan in a biological sample comprising an antibody or antibody fragment as described herein. In embodiments, the kit may also comprise one or more of a buffer, a secondary antibody or a detection reagent. The kit may also include instructions for employing kit components.

[0163] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.

[0164] EXAMPLES

[0165] Materials and methods

[0166] Anti-Tau monoclonal antibodies

[0167] Table 2. Anti-Tau according to embodiments of the invention and comparative anti-Tau antibodies.

[0168]

[0169] Human autopsy cases

[0170] A total of 36 human autopsy cases were used in this study. These cases were used for biochemical analyses and in vitro experiments.

[0171] The brains were collected at the university hospitals in Ulm (Germany) and Leuven (Belgium). Autopsies were performed in accordance with German / Belgian law after approval by the ethical committees from Ulm (Germany, study number 54 / 08) and UZ Leuven (Belgium, study numbersS59292, S52791, S66705, S63759). In the context of biobank characterization, phases of A plaque deposition were assessed as described previously, based on anti-A i7-24-stained brain sections (Thai et al. (2002) Neurology 58, 1791-1800). Braak stages for neurofibrillary tangles (NFT) spread in the brain were determined as previously described based on anti-p-Tau202 / 205immunostained sections (Braak et al. (2006) Acta Neuropathol 112, 389-404). The neuropathological diagnosis was performed as published by the National Institute of Aging and Alzheimer Association working group (NIA-AA criteria) (Hyman et al. (2012) Alzheimers Dement 8, 1-13). Global Clinical dementia rating (CDR) scores were retrospectively assessed according to previously established guidelines (Morris (1993) Neurology 43, 2412-2414). Primary age-related tauopathy (PART) was determined when the definition of definite or probable PART was fulfilled (Crary et al. (2014) Acta Neuropathol. 128: 755-66). Retinal samples were obtained from UZ Leuven Biobank. The project was performed following Belgian law and was approved by the local ethical committee in Leuven / Belgium (S64492). Primary retinal tauopathy and the stages thereof were determined as described in Walkiewicz et al. (2024) Alzheimer’s Dement. 20:330-340.Table 3. Overview of human autopsy and biopsy (eye enucleation 37-41) samples used herein.

[0172]

[0173]

[0174] Epitope screening assays

[0175] Mapping of the monoclonal antibodies epitopes was described in detail in Rosseels et. al. (2015; J Biol Chem 290:4059-4074) and Verelst et. al. (2020; Front Mol Biosci 7:48). For most antibodies, including 16B12, 9H6F2 and 11E12E10, epitope determination was performed using a library of overlapping synthetic peptides (Pepscan, Lelystad, The Netherlands), as described in Langedijk et al. (2011, Anal Biochem 417, 149-155). Two peptide arrays, one non-phosphorylated and one phosphorylated, were designed covering the full human Tau2N4R sequence. Each peptide was 18 amino acids long and had 16 amino acids overlap with the subsequent peptide. The phosphorylated peptides were designed based on the possible phosphorylation sites described by Sergeant et al. (2008, Expert Rev Proteomics 5: 207-224). The binding capacity of the antibodies to the generated peptides was determined via a Pepscan-based ELISA. In short, the peptide array was incubated overnight at 4 °C with the monoclonal antibody followed by several washing cycles. Afterwards, the peptide arrays were incubated with a rabbit antimouse IgG HRP conjugate (Southern Biotech, Uden, The Netherlands) for 1 h at 25 °C. After several wash cycles, a solution of 20 pL / mL of peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 3% H2O2 were added. After 1 h of incubation, the colorimetric reaction was quantified.

[0176] Epitope binning sandwich ELISA assays

[0177] Polystyrene 96-well flat-bottom microtiter plates were coated with 4 pg / mL of one of the capture antibodies (ADx215, 18F12, 15A10, ADx201, ADx202, 9H6F2, 11E12E10, 16B12 or 20G10; see Fig.

[0178] 1 for the epitopes) in PBS (100 pL / well) and incubated overnight at 4 °C. Afterwards, plates were blocked for 2 h at RT with 200 pL PBS + 1% bovine serum albumin (BSA). The plates were washed three times with washing buffer (PBS + 0.002% Tween 80) and either 30 ng / mL of human Tau2N4R purified from E. coli (rPeptide, Watkinsville, GA, USA), 30 ng / mL of human Tau2N4R purified from yeast as described in Verelst et al. (2020. Front. Mol. Biosci. 7:48), or 30 ng / mL preformed filaments of bacterial expressed human Tau2N4R (rPeptide, Watkinsville, GA, USA) in PTA buffer (PBS + 0.1% BSA + 0.002% Tween 80) was added to the plates and incubated for 2 h at room temperature. Afterwards, the plates were washed and incubated for 1 h at room temperature with 100 pL / well biotin-conjugated detection antibody (antibody-bt) diluted in PTA buffer (1 / 1000 dilution for 11E12E10 and 16B12; 1 / 2000 dilution for 9H6F2, ADx202 and 20G10; 1 / 4000 dilution for ADx215, 18F12, 15A10 and ADx201). Biotinylation of the antibodies was performed with the Sulfo-NHS-LC-Biotin kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Biotinylated antibodies that were able to bind the Tau antigen were detected with 1 / 10000 poly-HRP-conjugated streptavidin (Sanquin-Guilde Healthcare, Frankfurt, Germany) diluted in PTA buffer (100 pL / well) and incubated for an additional 30 min at room temperature. After a final washing step, 100 pL of o-phenylenediamine dihydrochloride (OPD) substrate in citrate buffer was added to the plates. After30 min incubation at room temperature in the dark, the colour reaction was stopped with 50 pL / well of 4M H2SO4. Optical density (OD) was measured at 492 nm with an ELx808 ELISA reader (Agilent-BioTek, Santa Clara, CA, USA). Epitope binning graphs were made in Microsoft Excel usingconditional formatting with color scales of the average values obtained with bTau, ytau or fTau to calculate and depict the percentiles as color-gradient (ranging from 5 to 95).

[0179] Temperature of melting (T m) and aggregation (T agg)

[0180] To measure the Tm and Tagg of the monoclonal antibodies, an Uncle instrument (Unchained Labs, Gent, Belgium) was used. This instrument measures the intrinsic fluorescence while the temperature is changing, causing the protein unfolding. For the measurement, the monoclonal antibodies concentration was 1 mg / mL, diluted in PBS. The temperature range was from 15 to 95 °C with a changing rate of 0.3 °C / min.

[0181] Immunohistochemistry (IHC) and tissue microarrays (TMA)

[0182] Paraffin-embedded human brain sections of 5 pm thickness were deparaffinized and processed with citrate buffer for epitope retrieval (pH=6, EnvisionTM Flex Target Retrieval Solution K8005, Agilent-Dako, Santa Clara, CA, USA). Endogenous mouse peroxidase was blocked (peroxidase blocking solution S2023, Agilent-Dako, Santa Clara, CA, USA) for 5 minutes to avoid unspecific reactions in all slides. All the monoclonal antibodies for characterization, including 3 isotype controls, were used as primary antibodies in different dilutions (Table 2) for overnight incubation. The controls were MA-Villin9Hll (IgG2b kappa) and MA-VillinlEl (IgGl kappa) that recognize human villin protein and MA-COV4F11 (IgG2a kappa) that recognizes SARS-CoV-2 spike / RBD protein (PharmAbs). A secondary goat anti-mouse HRP antibody was applied for 30 minutes (Vector Laboratories, Newark, NJ, USA). Peroxidase-conjugated anti-mouse EnVision+ Single Reagent (K4001, Agilent-Dako, Santa Clara, CA, USA) was used as secondary antibody for 1 h incubation. Liquid DAB+ kit (K3468, Agilent-Dako, Santa Clara, CA, USA) was used as a chromogen to yield brown reaction products. Envision Flex Wash buffer (K8000 / 8002, Agilent-Dako, Santa Clara, CA, USA) was used for washing steps. Finally, sections were hematoxylin counterstained, dehydrated and mounted using a Leica Autostainer XL (Leica, Deer Park, IL, USA).

[0183] TMAs were used to evaluate the binding specificity of anti-Tau monoclonal antibodies in fresh frozen tissue. Sections from Entorhinal Region (ER), Hippocampus (H), Superior Temporal cortex (ST), Basal Ganglia (BG), Frontal cortex (F), Nucleus Basalis of Meynert (NBM) of different Braak stages of AD pathology (Table 3), were also used for immunohistochemical analysis. For the fresh-frozen TMA tissue, 5 pm thick tissue sections were rehydrated in wash buffer for 10 min, followed by the peroxidase blocker. No further pretreatment or fixation step was performed. After incubation with the primary antibody the subsequent steps were similar as described above in IHC assay for paraffin embeddedtissue sections. The AT8 antibody (p-TauS202 / T205, MN1020, ThermoFisher Scientific, Waltham, MA, USA), ATI 80 (pTauT231, Thermo Fisher cat#MN1040) and PHF1 antibody were used to detect Tau phosphorylated at different epitopes, while the MCI antibody (gift from Dr. P. Davies, New York) was used for the detection of an AD-specific Tau conformation (amino acid residues 7-9; 313-322) (Table 2).

[0184] For the peptide pre-absorption assay, we pre-incubated 16B12 (1 mg / mL, 1:200), MCI (1 mg / mL, 1:50), and AT8 (0.2 mg / mL, 1:200) with the R1 peptide (PMPDLKNVKSKIGSTENL (SEQ ID NO:30), 50 pM) for 30 minutes before performing IHC on human AD brain sections (Table 3, case 15), as described above. For the proteinase K (PK) digestion, human AD brain sections (Table 3, case 15) were treated with PK (Agilent-Dako, CA, USA) for 1 minute, following the manufacturer's instructions, prior to the IHC staining.

[0185] For screening of the Tau antibodies in other Tauopathies besides Alzheimer’s disease (AD), sections were used from argyrophilic grain disease (AGD), Pick’s disease (PiD), Corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) cases (Table 3).

[0186] For histological analysis of retinal tissues, eyes were fixed in 4% buffered paraformaldehyde solution (PFA) for approximately 24 hours before dissection into slices of 4 mm and embedded in paraffin. Paraffin blocks were microtomed at 10 pm. Paraffin sections were deparaffinized and heat-pretreated using EnvisionTM Flex Target Retrieval Solution Low pH (Dako). Treatment with 98% formic acid for 3 minutes at room temperature was performed before the immunohistochemical analysis.

[0187] Extraction of Tau aggregates

[0188] Sarkosyl -insoluble material was extracted from cortex tissue of four individuals (cases 16-19, Table 3), as described in Goedert et al. (1992) Neuron 8, 159-168). Briefly, tissue homogenisation was performed with a FastPrep (MP Biomedicals, Fisher Scientific, Merelbeke, Belgium) in 10 volumes (w / v) cold buffer (10 mM Tris-HCl pH 7.4, 0.8 M NaCl, 1 mM EGTA and 10% sucrose). Universal Nuclease (Pierce, ThermoFisher Scientific, Waltham, MA, USA) was added to the supernatant, followed by a 30 min incubation at room temperature (RT). Subsequently, the sample was brought to 1% Sarkosyl (Sigma- Aldrich, St. Louis, Missouri, USA) and incubated for 1 h at RT while shaking (400 rpm), followed by centrifugation at 350.000 x g for 1 h at 4 °C. The pellet was washed and centrifuged at 350.000 x g for 30 min at 4 °C and resuspended in 50 mM Tris-HCl pH 7.4 (175 mg of brain tissue per 100 pL, stock concentration). Finally, the human tissue homogenates were stored at -80 °C until further use.

[0189] Tau concentration determination by ELISA

[0190] To monitor the concentration of total Tau in brain homogenates, a commercially available kit (Total Tau human ELISA Kit KHB0041, ThermoFisher Scientific, Waltham, MA, USA) was used accordingto the manufacturer instructions. The optical density (OD) was measured at 492 nm with an ELx808 ELISA reader (Agilent-BioTek, Santa Clara, CA, USA). The final concentrations of total Tau were determined by interpolating the values within the polynomial range of the standard curve, using GraphPad Prism software (Graphpad Software, Boston, MA, USA). To ensure accuracy, duplicates of all samples were analyzed.

[0191] Cell seeding

[0192] The Tau MTBD P301S FRET Biosensor HEK-293 cell line (CRL-3275, ATCC- LGC Standards, Molsheim Cedex, France) stably expresses constructs where the microtubule binding domain (MTBD) of Tau-P301S is fused to CFP and YFP. Transfection with Tau seeds nucleates the aggregation of these Tau reporter proteins, thereby producing a FRET signal. For seeding experiments, the cells were cultured in DMEM medium, supplemented with 10% FBS, 1 mM sodium pyruvate and non-essential amino acids (Gibco, Thermofisher Scientific, Waltham, MA, USA), under an atmosphere of 5% CO2 at 37 °C. Cells were plated at 20000 cells / well in poly-L-Ly sine -coated 96-well PhenoPlates (PerkinElmer, Mechelen, Belgium). After 5 h, the monoclonal antibodies (5 ng / mL) and the isotype controls were pre-incubated with sonicated (30 s on, 30 s off at 10 °C for 15 min with a Bioruptor Pico (Diagenode, Seraing, Belgium) extracted aggregates from diseased brain (5 ng / mL) for 1 h at room temperature. Subsequently, the cells were transfected with the mAb-aggregate complex using Lipofectamine 3000 (Thermofisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Each complex was mixed with Lipofectamine 3000 reagent and added to a mixture of Opti-MEM medium (Gibco, Thermofisher Scientific, Waltham, MA, USA) with Lipofectamine 3000. After a 15 min incubation at room temperature, 10 pL of mixture was added per well in a total volume of 100 pL. After 48 h, cell medium was replaced with 40 pL 4% formaldehyde and cells were incubated for 5 mins, then washed three times with PBS. Nuclear staining was performed with DAPI (DI 306, Thermofisher Scientific, Waltham, MA, USA) diluted (1:5000 from a stock of 5 mg / mL) in 1% BSA in PBS for 30 min. Four individual plate preparations were performed per complex as independent experiments (n = 4). High -content screening was performed at the VIB Imaging Core (Leuven, Belgium), using an Operetta (PerkinElmer, Mechelen, Belgium) equipped with proper filter channels to track Tau aggregation through the FRET signal. Image storage (15 fields in 6 planes at a 40x magnification were acquired per well) and segmentation analysis was performed using the Columbus Plus digital platform (PerkinElmer, Mechelen, Belgium). For the dose-response curves of the human-extracted Tau aggregates (Fig. 7A), the cells were transfected with a series of dilutions of the stock (see extraction of Tau aggregates methodology) and followed similar transfection steps as above.

[0193] Tau purification, immunization and antibody selection

[0194] The longest human Tau isoform (441 amino acids) containing an amino-terminal polyhistidine (His6) tag (SEQ ID NO:29) and the AK280 mutation (Tau2N4R-AK280) was used as antigen. The protein wasconstitutively expressed in the BY4741 pho85AadhlA yeast strain. Tan phosphorylation is enhanced in this strain and the purified preparations contain a small fraction of hyperphosphorylated Tau that is in the MCl / Alz50 conformation and is capable to spontaneously seed Tau fibrillization.

[0195] Female SJL / J Rj mice (ethical approval P049 / 2020) were used for immunization, which was started by two subsequent subcutaneous administration of 10 pg of yeast-purified human Tau2N4R-lK280. To generate high affine antibodies, three extra immunizations and two boosters were given. Plasma obtained through eye bleeding was used to determine the anti -Tau titers via an ELISA containing coated human Tau2N4R-AK280 (2 pg / mL) and using a rabbit anti-mouse antibody conjugated with horseradish peroxidase (HRP) (Nordic MUBio, Susteren, The Netherlands) as secondary antibody. Once high titers were obtained, the spleen cells were fused with SP2 / 0 myeloma cells using polyethylene glycol (PEG), as described in Greenfield (2018, Cold Spring Harb Protoc).

[0196] After selection of the resulting hybridoma cells in hypoxanthine-aminopterin-thymidine medium, a screening was performed based on ELISA assays using either human wild-type Tau2N4R purified from the pho85AadhlA yeast strain (yTau) or from Escherichia coli (bTau) (rPeptide, Watkinsville, GA, USA) as coated antigen (2 pg / mL) and a rabbit HRP -conjugated anti-mouse antibody for detection. Generation and expression ofScFv-16B12 and ScFv-MCl

[0197] The monoclonal antibody 16B12 was sequenced by Absolute Antibody (Redcar, Cleveland, TS104RF, UK) (Table 1) and based on these data, the sequences of the heavy (VH) and light chains (VL) were used for the intrabody design. The CDRs of 16B12 were identified using the Kabat definition. The nucleotide sequence was codon optimized for human and CHO cells, followed by gene synthesis of an insert in which the variable regions of the heavy (HCVR; SEQ ID NO: 11) and light chains (LCVR; SEQ ID NO: 10) were separated by a (G4S)3-linker (SEQ ID NO: 15). Gene synthesis was performed by GenScript Biotech B.V. (Rijswijk, 2288 EG, The Netherlands). The insert was cloned into the pcDNA3. l(+)-C-DYK plasmid for expression under control of the CMV promoter and providing a C-terminal FLAG (DYK)-tag for visualization and purification. An ScFv intrabody version of MCI (ScFv-MCl; SEQ ID NO: 16) was constructed using the same framework as ScFv-16B12 based on antibody sequences described in Hayashi et al. 2016 (US 2016 / 0251420 Al).

[0198] Western blot analysis

[0199] For SDS-PAGE and Western blot analysis, wild-type Tau2N4R purified from the pho85AadhlA yeast strain (yTau) or from Escherichia coli (bTau) (rPeptide, Watkinsville, GA, USA) and sarkosyl-insoluble homogenates of a non-AD human case (Case 19, Table 3) and an AD case (Case 16, Table 3) were used. The total protein content for all the samples was quantified using the Pierce® BCA Protein assay Kit (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. For electrophoresis, the different samples (2 pg of bTau or yTau, 20 pg of non-AD or AD) were loadedinto Mini-PROTEAN TGX Precast Gels (BioRad, Temse, Belgium). The proteins were transferred into 0.2 um nitrocellulose membranes (Trans-Blot Turbo, BioRad, Temse, Belgium). The membranes were then blocked and incubated overnight at 4 °C with 11E12E10 (1:1000), 9H6F2 (1:3000), PHF1 (pS396 / pS404, gift from Dr. P. Davies, New York; 1:3000) and Tau5 (Total Tau, ThermoFisher, Scientific, Waltham, MA, USA, 1:3000). Membranes were washed and incubated with 1:3000 antimouse secondary antibody (Aglient Technologies, Santa Clara, CA, USA) for 1 hour at room temperature. Immunoblots were washed and detected using the SuperSignal Maximum Sensitive Substrate Kit (ThermoFisher, Scientific, Waltham, MA, USA) with ChemiDoc MP Imaging System (BioRad).

[0200] Statistical Analysis

[0201] Statistical analysis was performed with Prism 10.2.0-10.2.2, using unpaired student’s t-tests to determine the statistical significance of differences between samples unless otherwise indicated. Example 1: Epitope binning analysis of anti-Tau monoclonal antibodies

[0202] A comparative sandwich ELISA assay was performed using three different sources of Tau, including yTau, bTau, and heparin-induced filaments of bacterially expressed human Tau2N4R (fTau), and the indicated anti-Tau monoclonal antibodies to capture or to detect protein Tau (Fig. 1A; Table 2). The epitopes of the monoclonal antibodies are shown in Fig. 1A.

[0203] With recombinant bTau, all monoclonal antibodies, except the phospho-specific monoclonal antibody 15A10, captured protein Tau fairly well, but when used as detection antibodies 16B12 and 11E12E10 particularly had difficulty in accessing their epitopes in the R1 repeat (Fig. 1C). This suggests that these epitopes are partially shielded due to the intermolecular interactions and the compaction of the MTBD. In heparin-induced filaments, this shielding appeared to be enhanced and extended to the C-terminus since not only 16B12 and 11E12E10, but also 20G10 failed to properly capture or detect fibrillar fTau (Fig. 1C).

[0204] A similar trend was seen when soluble phosphorylated yTau was used. Indeed, also here the monoclonal antibodies 16B12, 11E12E10, and 20G10 were hindered to capture Tau (Fig. 1C), though detection of the protein with these monoclonal antibodies was still possible. In fact, as compared to the data obtained for bTau or fTau, the detection of phosphorylated yTau with 16B12 and 11E12E10 markedly improved when protein Tau was captured using 9H6F2, ADx201 or ADx202. Interestingly, these three monoclonal antibodies show affinity for the sequence 219PTREPK224 (SEQ ID NO:26), which is part of a polyproline helix in P2 that was previously reported to interact with the N-terminal insert N2, with sequences at the C-terminal borders of R2 and R4, as well as with an amphiphatic helix in the C terminus of Tau (Mukrasch et al. (2009) PLoS Biol 7:e34). Hence, it is likely that capturing yTau at this polyproline helix may hamper one or more of these interactions, which otherwise would contribute tothe observed shielding of the R1 region. A more moderate improvement of the detection of phosphorylated Tan with 16B12 and 11E12E10 was observed when yTau was captured with ADx215. Apart from being engaged in strong interactions at the very N-terminus, the sequence corresponding to the ADx215 core epitope was shown to engage in contacts with amino acid stretches that include the epitope sequences of 16B12 and 11E12E10 in the R1 and R3 repeats (Mukrash et al. 2009 PLoS Biol 7:e34). Thus, while our data suggest that capturing Tau with 9H6F2, ADx201 or ADx202 mainly affects interactions of the P2 segment with the repeat region and C-terminus, the capture of Tau by ADx215 appears to prevent the backfold of the N-terminus and the repeat region. This rather limited effect of ADx215 on shielding of the repeat region may not be surprising when considering that the yTau preparations only contain a small fraction of hyperphosphorylated Tau that is in the aggregation-prone conformation recognized by MCl / Alz50. The latter has a discontinuous epitope formed by the amino acid residues 7EFE9 (SEQ ID NO:27) in the N-terminus and 313VDLSKVTSKC322 (SEQ ID NO:28) in the R3 repeat and this sequence in R3 is indeed part of the aforementioned phospho-peptide (309VYKPVDLpSKVTpSKCGpSLG326 (SEQ ID NO:2)) recognized by 16B12. Finally, the monoclonal antibodies 18F12 and 15A10 had no major effect on the detection of yTau by 16B12 and 11E12E10. These monoclonal antibodies recognize the loops formed by backfold of the N-terminal insert N1 on N2 and of the proline-rich segments Pl on P2, respectively, suggesting that their binding could even stabilize the compacted Tau structure.

[0205] For 15A10, the data confirmed its phosphorylation-dependency since the monoclonal antibody detected yTau but failed to recognize bTau and fFau.

[0206] To rule out that failure to capture or detect protein Tau with some monoclonal antibodies would be due to differences in their stability, the aggregation and meting temperature (Tagg and Tm, respectively) for each antibody was monitored. As shown in Fig. ID, the Tagg of all the monoclonal antibodies was between 60 °C and 80 °C. Comparable values were obtained when measuring the Tm, with the only exception being 20G10 for which the Tm was even higher than the Tagg. These Tagg and Tm values indicate that all monoclonal antibodies were stable under the conditions used for the aforementioned smFRET and ELISA assays.

[0207] Example 2: Epitope Specificity and Early Pathology Detection by the 16B12 monoclonal antibody The 16B12 mAb, recognizes a discontinuous epitope in Tau protein including a sequence in R1 (249PMPDLKN255 (SEQ ID NO: 3)) and in R3 (309VYKPVDLpSKVTpSKCGpSLG326 (SEQ ID NO:2)), as shown in an ELISA-based peptide array scanning (Fig. IB). To demonstrate that this epitope is indeed conformational, two distinct experiments were performed.

[0208] In the pre-absorption experiment, a peptide sequence containing the partial epitope of 16B12 in Rl, thus without including the R3 region, was used. An excess (50 pM) of this Rl peptide(PMPDLKNVKSKIGSTENL (SEQ ID NO:30)) was pre-incubated with 16B12, as well as with MCI or AT8 as epitope controls, and subsequently immunohistochemistry (IHC) was performed on AD tissue sections (Table 3, case 15) (Fig. 2A; black arrows: Pretangles and neurofibrillary tangles (NFT)). Pre-treatment with the R1 peptide reduced epitope availability only for 16B12, but did not completely abolish its detection of Tau pathology. This indicates that even when R1 is partially blocked, the epitope formed by the R1-R3 interaction remains at least partially accessible for 16B12 binding.

[0209] In addition, another IHC experiment was conducted in which human brain sections (Table 3, case 15) were treated with proteinase K (PK). Along with 16B12, MCI was included as another conformational antibody and AT8 as a non-conformational control to assess staining specificity (Fig. 2A). Treatment with PK had a strong effect in 16B12 and MCI, that are both conformational and no effect in AT8, that detects phosphorylated Tau.

[0210] Finally, to confirm that the 16B12 antibody has a partially overlapping epitope with the MCI antibody in the R3 region, a competitive IHC on human AD brain (Table 3, case 15) tissue was conducted. Initially, the tissue was stained with the MCI antibody, followed by staining with either biotinylated 16B12 (16B12-bt) or biotinylated ADx201 (ADX201-bt), the latter targeting the proline-rich segment P2 of Tau (Fig. 1A). Single-stained sections with ADx201-bt and 16B12-bt served as positive controls. As illustrated in Fig. 2B (black arrows: Pretangles / NFTs; circles: Neuritic plaques (NP)), competition between 16B12 and MCl completely abolished the binding of 16B 12, showing that the 16B12 antibody has a partially overlapping epitope with the MCI antibody. The binding of ADx201 was only slightly affected, as anticipated, since MC 1 occupies space on the fibrils core and / or obscures part of the PHF epitope that is adjacent to the PRD (ADx201 epitope) when Tau obtains the fibrillar form.

[0211] When comparing the detection of Tau pathology with the 16B12 and MCI monoclonal antibodies, 16B12 was able to detect the characteristic lesions of AD prior than MCI, indicating that 16B12 Tau conformation is obtained earlier than the MC 1 one (Fig. 3, black arrows: Neurofibrillary tangles (NFT); dashed arrows: Neuropil threads (NT), Example 3).

[0212] Example 3: 16B12 monoclonal antibody detects early structural maturation of AD Tau pathology An immunohistochemical analysis (IHC) of tissue sections obtained from post-mortem human hippocampus of a Braak stage VI AD patient (case 15 in Table 3) was conducted to determine whether the monoclonal antibody epitopes are detectable in AD brain, and if so, at which stages and lesions. As gold standard for Tau IHC, the diagnostic antibody AT8 was used, which specifically binds to phosphorylated Tau at Ser202 and Thr205 32 (Table 2) as previously recommended by the BrainNetEurope.

[0213] All monoclonal antibodies tested were capable of detecting neuropil Tau and neurofibrillary tangles (NFTs, arrows) in the hippocampus of a Braak stage VI AD case to a similar extent as AT8 (Fig. 4A).Neuritic plaques (NP, circles) were detected in sections stained with ADx215, 9H6F2, 15A10, ADx201, ADx202, 16B12, 20G10 (Fig. 4A). The different isotype control monoclonal antibodies, i.e. MA-VillinlEl, MA-Villin9Hl 1 and MA-C0V4F11, did not show any reactivity towards Tau pathological species.

[0214] The performance of the monoclonal antibodies was also investigated in sections from different brain areas and with varying degrees of pathology, according to the Braak staging (cases 1-15, Table 3). IHC was performed using fresh-frozen tissue microarrays (TMAs) and the antibody binding was analyzed in multiple tissue samples simultaneously mounted on a single slide. The monoclonal antibody was evaluated by scoring their performance from 0 (for no pathology detection) to grade 4 (for major neuropil-like / NFT Tau pathology). Fig. 4B shows representative images of tissue microarray cores indicating the grading (0-4) for Tau pathology evaluation using the antibody AT8. Grade 1-2 is characterized by a low presence of NFTs (< 4) and grade 3-4 by higher amount of pathological Tau (NFTs > 5). Neuropil threads (NT) were detected in the grades 1-4 and their increased presence also determined the difference in grades of pathology. We observed increased detection sensitivity of all 9 monoclonal antibodies candidates, with ADX215, 9H6F2, 15A10, ADx201, 16B12 and 20G10 being more sensitive in detecting Tau species in early stages of pathology (Braak I-III) compared to the rest of the candidates (Table 4). The monoclonal antibodies were also tested on TMAs of a non-AD case; no tangle-like pathology was detected, providing evidence of the specificity of our monoclonal antibodies. The isotype monoclonal antibody controls also did not recognize Tau pathology in the different TMAs used.

[0215] Table 4. Tau pathology burden in tissue from different areas at different Braak stages based on a grade from 0 (for no pathology detection) to stage 4 (for major neuropil-like Tau pathology). Entorhinal Region (ER), Hippocampus (H), Superior Temporal cortex (ST) Basal Ganglia (BG), Frontal cortex (F), Nucleus Basalis of Meynert (NBM).

[0216]

[0217] The monoclonal antibodies demonstrating better performance in recognizing Tan lesions in the early stages of AD, were compared with antibodies known for identifying Tan pathology, i.e. AT180, AT8, PHF1, and MCI. The presence of ATI 80, AT8, PHF1, and MCI in the entorhinal or frontal cortex was evaluated (Table 5), indicating that the Tan phosphorylation-specific monoclonal antibodies ATI 80 and AT8 recognized NFTs, pretangles (PT), and thread-like Tan pathology in all cases, including Braak stage 0 and a non-AD primary age-related Tauopathy (PART) case (case 23, Table 3). PHF1, also targeting phosphorylated Tau, exhibited performance nearly equivalent to ATI 80 and AT8, starting from Braak stage I. From Braak stages I to III, the monoclonal antibodies 9H6F2, 15A10, 16B12, and 20G10 performed comparably to PHF1 and surpassed the detection with the conformational-specific monoclonal antibody MCI (Table 5). It is known that the MCI conformation, recognizing the discontinuous epitope of N-terminal and R3 regions, precedes the appearance of mature neurofibrillary tangles (Weaver et al. (2000) Neurobiol Aging 21:719-727). The conformational-specific 16B12 targeting sequences in R1 and the phosphorylated APR PHF6 in R3, detected Tau pathology, especially PT and NFT, in earlier stages of AD than MCI. This indicates that the formation of the fibrillar core incorporates part of the fuzzy coat carrying the R1 region in early stages of Tau pathology, before the MCI conformation is obtained, but after the appearance of phosphorylation-specific epitopes such as AT180 and AT8. Along with the 16B12-specific conformation, the 9H6F2 conformation, indicative for phosphorylation and structural changes of the proline rich region, was also detected in early stages of AD pathology. ADx201 performed similarly to MCI. In summary, albeit the antibodies 9H6F2 and 16B12 did not stain NFT or PT in AT8 or ATI 80-positive neurons of Braak stage 0 cases, they showed detection of pathological Tau as early as in Braak stage I of AD cases , further suggesting that changes in the Rl, R3 and the proline-rich regions appear to be crucial for developing fibrillar Tau aggregates. Table 5. Comparison of anti -Tau monoclonal antibodies (9H6F2, 15A10, ADx201, 16B12, 20G10) in immunohistochemical detection of the different Tau maturations states with known phosphorylation-(AT180, AT8, PHF1) and conformation-specific (MCI) antibodies in different Braak stages (TMAs). PT: pretangles; NFT: neurofibrillary tangles; NT: neuropil threads. F: Frontal cortex; E: Entorhinal cortex.

[0218]

[0219] Example 4: Diagnostic potential of anti-Tau monoclonal antibodies in other Tauopathies than AD Samples from AGD, PSP, CBD, and PiD cases (Table 6) were stained with the indicated monoclonal antibodies, including 9H6F2, 15A10, ADx210, 16B12 and 20G10 (Fig. 5). In AGD, all monoclonal antibodies demonstrated similar pathological detection capabilities, recognizing PT, NT, and oligodendroglial coiled bodies (OCB). In PSP, 16B 12 detected OCB in only one of the two human cases tested, whereas the other candidates showed higher sensitivity in detecting oligodendroglial pathology. In CBD, all monoclonal antibodies performed similarly, recognizing all the main characteristic lesions, including PT, NT, OCB, balloon neurons, and astrocytic plaques. For PiD, most monoclonal antibodies recognized NT, OCB, Pick bodies (PB), and Tau-positive astrocytes (TP A). 15A10 and 20G10 failed to detect Pick bodies, but 15A10 was effective in detecting neuronal -related Tau pathology, such as PT and NT, with high sensitivity in the other tested Tauopathies. 9H6F2, targeting the discontinuous epitope formed by interaction between the P 1 and P2 segments, was able to detect all the characteristic lesions with increased sensitivity in all the Tauopathies tested, indicating that the interaction between these Pl and P2 proline-rich segments may be a shared common feature in Tau fibrils among the different Tauopathies.

[0220] Table 6. Detection of pathological Tau lesions by anti-Tau monoclonal antibodies in different Tauopathies (Argyrophilic grain disease (AGD), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Pick’s disease (PiD)), including pretangles (PT), neuropil threads (NT), oligodendroglial coiled bodies (OCB), pick bodies (PB), Tau positive astrocytes (TPA), balloon neurons (BN) and astrocytic plaques (AP) depending on the disease.

[0221]

[0222] The monoclonal antibody 16B12 showed comparable detection capabilities as MCI antibody for advanced pathological lesions in other Tauopathies, as assessed by immunohistochemistry on tissue microarrays representing argyrophilic grain disease (AGD), PSP, CBD, and PiD (Fig. 5 and Table 7). Table 7. Diagnostic comparison of monoclonal antibody 16B12 and MCI in different Tauopathies (TMAs) (Argyrophilic grain disease (AGD), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Pick’s disease (PiD)). Detection of pathological Tau lesions, including pretangles (PT), neuropil threads (NT), oligodendroglial coiled bodies (OCB), tufted astrocytes (TA), balloonneurons (BN), astrocytic plaques (AP), pick bodies (PB), and Tau-positive astrocytes (TP A) depending on the disease.

[0223]

[0224] In retinal biopsies of cases with primary retinal tauopathy (PReT), the monoclonal antibody 16B12 detected its Tau conformers in the photoreceptor cell layer in a PReT stage 0 case and in the outer and inner plexiform layers, the inner nuclear layer and photoreceptor cell layer of a PReT stage 1 case (Fig.

[0225] 6; Photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), retinal nerve fiber layer (RNFL)). Both cases were previously shown to be negative for MCI staining (Walkiewicz et al. (2024) Alzheimer’s Dement. 20:330-340). In PReT stage 3 cases, which stain positive for the MCI conformation, the 16B12 Tau conformation is abundantly detected in all retinal cell layers and in one of these cases that was also diagnosed with Alzheimer’s disease (case 41, Fig. 6) even retinal ganglion cells displayed the 16B12 epitope.

[0226] Example 5: Seeding-preventing effect of anti-Tau monoclonal antibodies in AD

[0227] It was tested whether the anti-Tau monoclonal antibodies could prevent the elongation of human AD-derived Tau seeds, a process that initiates the Tau pathology. To investigate his seeding-preventing effect of the monoclonal antibodies, the ultrasensitive Tau biosensor cell line was used, which forms aggregates when treated with Tau seeds extracted from human AD brain (Holmes et al. (2014) Proc Natl Acad Sci U S A lll:E4376-4385). Initially, sarkosyl-insoluble Tau aggregates from the brain of three individuals diagnosed with AD was used (case 16, 17 and 18, Table 3) to determine the percentage of biosensor cells containing fluorescent puncta and dose-response curves were generated of seeding efficiency (Fig. 7A). Starting from these dose-response curves, the final concentration of Tau seeds that was needed to test the seeding-preventing effect of the monoclonal antibodies in cellulo was determined by selecting a concentration for which approximately 50-70% of the cells were positive for puncta (5 ng / mL of total Tau) in all the 3 cases (Fig. 7A). To ensure the binding and formation of Tau-monoclonal antibody complexes, a pre-incubation step of the human-extracted Tau seeds with the monoclonal antibodies was included. The effect for seed elongation of the anti-Tau monoclonal antibodies was compared to that of the respective isotype antibody controls. Only 16B12 was able to significantlyprevent the elongation of the Tan seeds in all three samples analyzed (Fig. 7B). 16B12 has its core epitope in the microtubule binding repeat Rl, but when Tau is phosphorylated it also recognizes a sequence that overlaps with the hexapeptide PHF6 and the C-terminal part of the MCl / Alz50 epitope (Fig. 1 A, B). This sequence corresponds to the first two P-sheets in the core of the PHF filaments found in AD. Without wishing to be bound by any theory, these data do underscore that Tau seeds are formed by phosphorylated Tau and that the P-sheets in the repeat region are essential for their elongation into filaments. Further, without wishing to be bound by any theory, 16B12 may hamper Tau seed elongation by stabilizing a phosphorylation-dependent backfold of Rl onto R3. 11E12E10 failed to reduce the elongation of Tau seeds. The core epitope of 11E12E10 partially overlaps with the core epitope of 16B12 in Rl but 11E12E10 does not have the affinity for sequence in R3 (Fig. 1A). Targeting other early structural alterations, such as the 9H6F2 conformation, also did not show inhibition of Tau seeding (Fig. 7B), indicating the necessity of the repeat region for seeding and propagation of Tau pathology.

[0228] Example 6: Anti-Seeding Activity of ScFv-16B12

[0229] ScFv-16B12 was expressed from a vector system and assessed for its anti-seeding activity using the Tau biosensor cell line of Example 5. The ScFv was tested against brain-derived Tau aggregates from patients with various Tauopathies, including Alzheimer’s disease (AD), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Argyrophilic grain disease (AGD) and Pick’s disease (PiD). Additionally, recombinant Tau produced in Escherichia coli was used. An off-target ScFv served as the negative control. Quantitative analysis revealed that ScFv-16B12 significantly reduced the number of fluorescent spots (normalized as spot area per cell area) compared to the ScFv-control, across cells treated with recombinant Tau or Tau aggregates from AD (Fig. 8A), PSP, CBD, AGD and PiD patients(Fig. 8B), showing robust anti-seeding capability of ScFv-16B12.

[0230] The anti-seeding activity of ScFv-16B12 was also compared with that of ScFv-MCl in case of Tau aggregates derived AD patients. An ScFv intrabody version of MCI (ScFv-MCl; SEQ ID NO: 16) was constructed using the same framework as ScFv-16B12 (Fig. 8A). Notably, when expressed in the Tau biosensor cells, ScFv-MCl exhibited reduced abundance and caused cellular toxicity (Fig. 8C).

[0231] Statistical analyses of anti-seeding performance demonstrated that ScFv-16B12 significantly inhibited Tau seeding, whereas ScFv-MCl was ineffective (Fig. 8A).

Claims

1. 58CLAIMS1. An isolated monoclonal antibody, or an epitope -binding fragment of an antibody, capable of specifically binding to Tan , wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO: 2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3, a nucleic acid encoding the antibody or the antibody fragment, or a vector comprising said nucleic acid, for use in the prevention or treatment of a Tauopathy in a subject.

2. The antibody or antibody fragment, the nucleic acid or the vector for use according to claim 1, wherein the antibody or antibody fragment is capable of specifically binding to a conformational Tau epitope formed by the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3, wherein the epitope is determined by ELISA-based peptide array scanning.

3. The antibody or antibody fragment, the nucleic acid or the vector for use according to claim 1 or 2, wherein the antibody or antibody fragment is an antibody or antibody fragment comprising a light chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:4, a CDR2 with the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:7, a CDR2 with the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:9.

4. The antibody or antibody fragment, the nucleic acid or the vector for use according to any one of claims 1 to 3, wherein the antibody or antibody fragment is an antibody or antibody fragment comprising a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence which has at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence which has at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11, or a humanized variant thereof.

5. The antibody fragment, the nucleic acid encoding the antibody fragment or the vector comprising said nucleic acid encoding the antibody fragment for use according to any one of claims 1 to 4, wherein the antibody fragment is an scFv fragment such as an scFv fragment comprising the amino acid sequence set forth in SEQ ID NO: 14, or a humanized variant thereof.

6. The antibody or antibody fragment, the nucleic acid or the vector for use according to any one of claims 1 to 5, wherein the antibody or antibody fragment is an intrabody.

597. The antibody or antibody fragment, the nucleic acid or the vector for use according to any one of claims 1 to 6, wherein said Tauopathy is selected from the group consisting of: Alzheimer’s disease (AD), Pick’s disease (PiD), Corticobasal Degeneration (CBD), Chronic Traumatic Encephalopathy (CTE), Progressive Supranuclear Palsy (PSP), primary age-related tauopathy (PART), Argyrophilic Grain Disease (AGD), and primary retinal tauopathy (PReT).

8. A method for in vitro or ex vivo diagnosis of Braak stage I or II Alzheimer’s disease or PART, or primary retinal tauopathy (PReT) stage 0 or 1 in a subject, comprising the following steps:- contacting an isolated monoclonal antibody, or an epitope-binding fragment of an antibody, capable of specifically binding to Tau , wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 with a biological sample of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and- detecting the formation of said complex.

9. A method for in vitro or ex vivo diagnosis of a non-cerebral Tauopathy, particularly a retinal Tauopathy such as primary retinal tauopathy (PReT), in a subject, comprising the following steps:- contacting an isolated monoclonal antibody, or an epitope-binding fragment of an antibody, capable of specifically binding to Tau, wherein the antibody or antibody fragment is capable of specifically binding to the amino acid sequence set forth in SEQ ID NO:2 having phosphorylation at Ser316, Ser320 and Ser324 and the amino acid sequence set forth in SEQ ID NO:3 with a non-cerebral biological sample of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and- detecting the formation of said complex.

10. An isolated monoclonal antibody or an epitope-binding fragment of an antibody, comprising a light chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 with the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 with the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain variable region comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO:7, a CDR2 with the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 with the amino acid sequence set forth in SEQ ID NO: 9.

11. The antibody or antibody fragment according to claim 10, comprising a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 11, or a humanized variant thereof, optionally comprising60a light chain with the amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain with the amino acid sequence set forth in SEQ ID NO: 13, or a humanized variant thereof.

12. The antibody fragment according to any one of claims 10 to 11, which is an scFv fragment such as an scFv fragment comprising the amino acid sequence set forth in SEQ ID NO: 14, or a humanized variant thereof.

13. The antibody or antibody fragment according to any one of claims 10 to 12, wherein the antibody or antibody fragment is an intrabody.

14. An isolated nucleic acid which encodes the antibody or antibody fragment according to any one of claims 10 to 13; a vector comprising said nucleic acid according; a host cell comprising said nucleic acid or said vector; or a pharmaceutical composition comprising the antibody or antibody fragment according to any one of claims 10 to 13, said nucleic acid or said vector, and further comprising a pharmaceutically acceptable carrier.

15. An antibody or antibody fragment according to any one of claims 10 to 13, a nucleic acid, a vector, or a pharmaceutical composition according to claim 14, for use in medicine, preferably for use in the prevention or treatment of a Tauopathy in a subject, optionally wherein said Tauopathy is selected from the group consisting of: Alzheimer’s disease (AD), Pick’s disease (PiD), Corticobasal Degeneration (CBD), Chronic Traumatic Encephalopathy (CTE), Progressive Supranuclear Palsy (PSP), primary age-related tauopathy (PART), Argyrophilic Grain Disease (AGD), and primary retinal tauopathy (PReT).

16. A method for in vitro or ex vivo diagnosis of a Tauopathy in a subject, comprising the following steps:- contacting an antibody or antibody fragment according to any one of claims 10 to 13 with a biological sample of the subject under conditions that allow for formation of a complex between the antibody or antibody fragment and Tau; and- detecting the formation of said complex,optionally wherein said Tauopathy is selected from Braak stage I or II Alzheimer’s disease, or PART, or primary retinal tauopathy (PReT) stage 0 or 1, or wherein said Tauopathy is a non-cerebral Tauopathy, particularly a retinal Tauopathy such as primary retinal tauopathy (PReT).

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